PI3k inhibitors and use thereof

Abstract

The present intention relates to compound comprising the general structure P-L-J-E, wherein P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds class 1 phosphoinositide 3-kinase (PI3K), L is a linker with a linker length of 12 to 21 atoms, J is a junction selected from (formula I), (formula II) and (formula III), wherein R7 is selected from H and the group consisting of -C1-C3 alkyl; E is an E3 ubiquitin ligase binding moiety that binds cereblon, J is a junction selected from (formula IV), (formula I), (formula II) and (formula III), R7 is selected from H and the group consisting of -C1-C3 alkyl; and SP is chosen in such way that the length of the linker L is 12 to 21 atoms.

Description

[0001] PI3K inhibitors and use thereof

[0002] The PIK3CA locus, frequently mutated in a diverse range of tumors, encodes the druggable kinase p110α. Despite the apparent promise of this target, drug development against it has been challenging. Most p110α inhibitors have been hampered by a lack of isoform specificity, often affecting other isoforms such as p110β, p110δ, and p110γ, as well as, to a lesser extent, mTOR and DNA-PK, especially at biologically meaningful concentrations. Even the most specific inhibitors, like alpelisib, have shown on-target metabolic adverse effects, notably hyperglycemia and hyperinsulinemia. This is primarily due to the inhibition of glucose import upon pan-PI3K inhibition, which, in turn, prompts insulin secretion. This counteractive response not only undermines the drug's efficacy but can even fuel tumor growth. A recent report has shown that high affinity engagement of p110α can lead to protein degradation in a limited number of cell lines expressing mutated p110α, while sparing the wild-type. While this pioneering study established that mutant p110α could suffer greater instability than wild-type under certain conditions, the scope of the degradation was limited. Whether isoform selectivity in p110α degradation could be achieved with other protein degradation techniques remains unstudied.

[0003] Proteolysis targeting chimeras (PROTACs) are bispecific small molecules that can hijack E3 ubiquitin ligases and steer them to novel targets. After several processive ubiquitin-transfers to a protein (typically a 4-6-mer ubiquitin chain linked at K48), it is recognized and degraded by the 26S proteasome. The ability of small molecules to behave as effectors that adapt substrate selection of E3 ligases has generated great excitement in drug discovery. The approach is fundamentally different in mechanism and consequence than conventional reversible inhibitors, offering catalytic turnover and durable inactivation of the target protein. The durability arises because the target protein must be resynthesized for it to regain its function, meaning the therapeutic effect can endure even after the drug is gone. Indeed, protein degradation can even be optimized to achieve selectivity amongst closely related targets, such as kinase isoforms. Despite the importance of p110α in oncology and challenges of targeting it selectively, PROTACs have not been investigated as a means to improve selectivity for specific class I PI3K isoforms. This is primarily because previous efforts could not identify potent PROTACs. One study with a cereblon-directing PROTAC (1) and another one with a VHL-directing PROTAC (2) have shown only moderate degradation of p110α at high concentrations - isoform selectivity and mechanism of action were not studied. Here, we investigate whether E3 ligase specificity could be exμloited to promote isoform-selective degradation of class I PI3Ks. Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to selective and potent PROTAC for degradation of kinases . This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examμles, figures and general description of this specification. of the Invention

[0004] A first aspect of the invention relates to compound comprising the general structure

[0005] P-L-J-E, wherein

[0006] P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds class 1 phosphoinositide 3-kinase (PI3K),

[0007] L is a linker with a linker length of 12 to 21 atoms,

[0008] J is a junction selected from, , , , wherein R7is selected from

[0009] H and the group consisting of -C1-C3alkyl;

[0010] E is an E3 ubiquitin ligase binding moiety that binds cereblon,

[0011] J is a junction selected from , R7is selected from

[0012] H and the group consisting of -C1-C3alkyl; and SP is chosen in such way that the length of the linker L is 12 to 21 atoms.

[0013] A second aspect of the invention relates to a use of the compound according to the first aspect of the invention as a medicament.

[0014] A third aspect of the invention relates to a use of the compound according to the first aspect of the invention for the treatment of a disease, wherein the disease is cancer.

[0015] Terms and definitions

[0016] General

[0017] For purposes of interpreting this specification, the following definitions will apμly and whenever appropriate, terms used in the singular will also include the μlural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control. The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For examμle, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For examμle, description referring to “about X” includes description of “X.”

[0020] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include μlural referents unless the context clearly dictates otherwise.

[0021] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0023] The term phosphoinositide 3-kinases (PI3K) in the context of the present specification relates to a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (Ptdlns). The pathway, with oncogene PIK3CA and tumor suppressor gene PTEN, is imμlicated in the sensitivity of cancer tumors to insulin and IGF1 , and in calorie restriction.

[0024] The term E3 ubiquitin ligase in the context of the present specification relates to a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognises a protein substrate, and assists or directly catalyses the transfer of ubiquitin from the E2 to the protein substrate and thus enables movement of ubiquitin from a ubiquitin carrier to a substrate.

[0025] The term cereblon or CRBN in the context of the present specification relates to a protein which forms an E3 ubiquitin ligase comμlex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1), which ubiquitinates other proteins. CRBN is recruited by proteolysis targeting chimeras (PROTACS) or protein degraders to a protein of interest, leading to its ubiquitination and subsequent degradation.

[0026] The term linker in the context of the present specification relates to a chemical structure comprising different chemical moieties, particularly selected from secondary amines, tertiary amines, carbonyl moieties, linear alkyl moieties, cyclic alkyl moieties, aryl moieties and / or heteroaryl moieties comprising one or more heteroatoms as specified herein, and connects two binding moieties, particularly a phosphoinositide 3-kinase binding moiety and an E3 ubiquitin ligase binding moiety.

[0027] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0028] Cancer Immunotherapy

[0029] The term cancer as used in the context of the present specification relates to malignant neoμlastic disease; the terms “cancer” and “malignant neoμlastic disease” are used synonymously herein.

[0030] As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow. Detailed Description of the Invention A first aspect of the invention relates to compound comprising the general structure P-L-J-E, wherein P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds class 1 phosphoinositide 3-kinase (PI3K), L is a linker with a linker length of 12 to 21 atoms, J is a junction selected from, , and 7 , wherein R is selected from H and the group consisting of -C1-C3alkyl; E is an E3 ubiquitin ligase binding moiety that binds cereblon, Jis a junction selected from , and ,R7is selected fromH and the group consisting of -C1-C3alkyl; and SP is chosen in such way that the length of the linker L is 12 to 21 atoms. A linker length of 12 to 21 allows for the two proteins to come into contact and form a ternary complex, while shorter linkers are dictated by their geometry hindering the two proteins to come into contact. Additionally, longer linkers tend to fail to fulfil the Lipinski rule of 5 which generalises properties aimed for in drug molecules. The length of the linker or linker length is counted according to the following examples: In certain embodiments, P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds PI3K-alpha and / or PI3K-beta. In certain embodiments, P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds

[0031] PI3K-alpha.

[0032] In certain embodiments, P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds PI3K-alpha and PI3K-beta.

[0033] In certain embodiments, E has the chemical structure wherein wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0034] X2is CH or N,

[0035] X3and X4are each selected from CH2, C, N, and SO2, particularly X3and X4are selected from CH2, C and N,

[0036] X5, X6, X7and X8are each selected from C and N,

[0037] X9is CH or N,

[0038] R2is selected from the group of halogens, and a is 0, 1 or 2.

[0039] The chemical formulae of E were extracted from WO 2019 / 195609 A2; 2019 (4). In certain embodiments, p is 0 or 1.

[0040] In certain embodiments, E has the chemical structure wherein

[0041] X is CH, wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0042] X2is CH or N,

[0043] X3and X4are each selected from CH2, C, N, and SO2,

[0044] X5, X6, X7and X8are each selected from C and N,

[0045] R2is selected from the group of halogens, and a is 0, 1 or 2. In certain embodiments, E has the chemical structure wherein

[0046] X is CH or N, wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0047] X2is CH or N,

[0048] X3and X4are each selected from CH2, C, and N, X5, X6, X7and X8are each selected from C and N,

[0049] X9is CH or N,

[0050] R2is selected from the group of halogens, and a is 0, 1 or 2.

[0051] In certain embodiments, E has the chemical structure wherein

[0052] X is CH or N, wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0053] X2is CH or N,

[0054] X3and X4are each selected from CH2, C, N, and SO2, two, three or all of X5, X6, X7and X8are C,

[0055] X9is CH or N, R2is selected from the group of halogens, and a is 0, 1 or 2.

[0056] In certain embodiments, E has the chemical structure wherein

[0057] X is CH or N,

[0058] wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0059] X2is CH or N,

[0060] X3and X4are selected from CH2, C, N, and SO2, all of X5, X6, X7and X8are C.

[0061] X9is CH or N,

[0062] R2is selected from the group of halogens, and a is 0, 1 or 2.

[0063] In certain embodiments, E has the chemical structure wherein

[0064] X is CH or N, wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0065] X2is CH or N,

[0066] X3and X4are each selected from CH2, C, N, and SO2,

[0067] X5, X6, X7and X8are each selected from C and N, X9is N;

[0068] R2is selected from the group of halogens, and a is 0, 1 or 2.

[0069] In certain embodiments, wherein E has the chemical structure wherein

[0070] X is CH or N,

[0071] R1has the chemical structure wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0072] X2is CH or N,

[0073] X3and X4are each selected from CH2, C, N, and SO2,

[0074] X5, X6, X7and X8are each selected from C and N,

[0075] X9is CH or N,

[0076] R2is F, and a is 0, 1 or 2, In certain embodiments, E has the chemical structure wherein

[0077] X is CH or N,

[0078] wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1 ,

[0079] X2is CH or N,

[0080] X3and X4are selected from CH2, C and N,

[0081] X5, X6, X7and X8are selected from C and N, particularly two, three or all of X5, X6, X7and X8are C, more particularly all of X5, X6, X7and X8are C.

[0082] X9is CH or N, particularly N;

[0083] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0084] In certain embodiments, E has the chemical structure wherein

[0085] X is CH, wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1 ,

[0086] X2is CH or N,

[0087] X3and X4are selected from CH2, C and N,

[0088] X5, X6, X7and X8are selected from C and N, particularly two, three or all of X5, X6, X7and X8are C, more particularly all of X5, X6, X7and X8are C.

[0089] X9is CH or N, particularly N;

[0090] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0091] In certain embodiments, E has the chemical structure wherein

[0092] X is CH or N,

[0093] R1has the chemical structure wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1 ,

[0094] X2is CH or N,

[0095] X3and X4are selected from CH2, C and N,

[0096] X5, X6, X7and X8are selected from C and N, particularly two, three or all of X5, X6, X7and X8are C, more particularly all of X5, X6, X7and X8are C.

[0097] X9is CH or N, particularly N;

[0098] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1. In certain embodiments, E has the chemical structure wherein

[0099] X is CH, wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1 ,

[0100] X2is CH or N,

[0101] X3and X4are selected from CH2, C and N,

[0102] X5, X6, X7and X8are selected from C and N, particularly two, three or all of X5, X6, X7and X8are C, more particularly all of X5, X6, X7and X8are C.

[0103] X9is CH or N, particularly N;

[0104] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0105] In certain embodiments, a is 0 or 1.

[0106] In certain embodiments R1has the chemical structure wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0107] X2is CH or N,

[0108] X3and X4are each selected from CH2, C, N, and SO2,

[0109] X5, X6, X7and X8are each selected from C and N,

[0110] X9is CH or N,

[0111] R2is selected from the group of halogens, and a is 0, 1 or 2. wherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0112] In certain embodiments, if E is , the length of the linker L is 13 to 21.

[0113] In certain embodiments, if E is , the length of the linker L is 14 to 21. the lenth of the linker L is 16 to 21.

[0114] In certain embodiments, E is selected from

[0115] In certain embodiments, E is selected from wherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0116] In certain embodiments, E is selected from

[0117] wherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1. In certain embodiments, R2is selected from the group of halogens and a is 0 or 1.

[0118] In certain embodiments, R2is F and a is 0 or 1.

[0119] In certain embodiments, E is selected from

[0120] In certain embodiments, E is selected from

[0121] In certain embodiments, E is selected from

[0122] In certain embodiments, P has the chemical structure wherein X11, X12and X13are each selected from CH and N;

[0123] X14is N or CH; R6is selected from one of the moieties of wherein X15is N or 0,

[0124] R10is selected from the group consisting of -C1-C3alkyl, f is 0,1 or 2, and X16and X17are selected from CH and N; R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and -CH2F; h is 0 or 1 ; R12is selected from the group consisting of -NH2, - NHC(=O)C1-3alkyl, -NHOC1-3alkyl; X18and X19are each selected from N and C;

[0125] R13is selected from the moieties

[0126] R14is -C1-C3alkyl and j is 0 or 1.

[0127] Several of the possible chemical formulae of P were extracted from Borsari et al. (3).

[0128] In certain embodiments, P has the chemical structure wherein

[0129] X11, X12and X13N,

[0130] X14is N or CH;

[0131] R6is selected from one of the moieties of wherein X15is N or 0,

[0132] R10is selected from the group consisting of -C1-C3alkyl, f is 0,1 or 2, and X16and X17are selected from CH and N;

[0133] R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and -CH2F; h is 0 or 1 ; R12is selected from the group consisting of -NH2, - NHC(=O)C1-3alkyl, -NHOC1-3alkyl; X18and X19are each selected from N and C;

[0134] R13is selected from the moieties ;

[0135] R14is -C1-C3alkyl and j is 0 or 1.

[0136] In certain embodiments, P has the chemical structure

[0137] X11is N;

[0138] X12and X13are selected from CH and N;

[0139] X14is N or CH;

[0140] R6is wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16;

[0141] R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and -CH2F; R12is selected from the group consisting of -NH2, -NHC(=O)C1-3alkyl; X18and X19are each selected from N and C.

[0142] In certain embodiments, P has the chemical structure wherein

[0143] X11is N;

[0144] X12and X13are selected from CH and N;

[0145] X14is N or CH;

[0146] R6is wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16; wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and -CH2F; R12is selected from the group consisting of -NH2, -NHC(=O)C1-3alkyl; X18and X19are each selected from N and C.

[0147] In certain embodiments, P is selected from any one of the moieties

[0148] wherein X14is CH or N.

[0149] In certain embodiments, P is selected from any one of the moieties wherein X14is CH or N.

[0150] In certain embodiments, P is selected from any one of the moieties In certain embodiments, P is selected from any one of the moieties

[0151] In certain embodiments, wherein X14is N.

[0152] A piperazine provides a validated exit vector for the creation of chimeric molecules.

[0153] In certain embodiments, P is selected from In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8comprises different moieties, of which two of the different moieties are -C(=O)- moieties, wherein the two

[0154] -C(=O)- moieties are separated from one another by one or more -CH2- moiety, and wherein

[0155] SP is chosen in such way that the sum of R8dand SP is 12 to 21 atoms.

[0156] The embodiment is to be understood in the way that if R8comprises 5 atoms, SP comprises 7 to 16 atoms to reach a sum of 12 to 21 which refers to the full length of the linker, i.e. if the length of the linker is 15, and R8comprises 5 atoms, then SP comprises 10 atoms. In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8comprises different moieties, of which two of the different moieties are -C(=O)- moieties, wherein the two -C(=O)- moieties are separated from one another by two or more -CH2- moieties, and wherein SP is chosen in such way that the sum of R8dand SP is 12 to 21 atoms.

[0157] In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8comprises different moieties, of which two of the different moieties are -C(=O)- moieties, wherein the two -C(=O)- moieties are separated from one another by two or more -CH2- moieties, and wherein SP is chosen in such way that the sum of R8dand SP is 12 to 21 atoms, and wherein SP is chosen in such way that the sum of R8dand SP is 12 to 21 atoms.

[0158] In certain embodiments, R8has a length of 1 to 10 atoms.

[0159] In certain embodiments, R8has a length of 1 to 8 atoms.

[0160] In certain embodiments, R8has a length of 4 to 8 atoms.

[0161] In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from one of the moieties

[0162] Y6, Y7, Y8, Y9, Y10, Y11, Y12and Y13are selected from CH and N, and SP is chosen in such way that the length of the linker L is 12 to 21 atoms.

[0163] In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from any one of the moieties and SP is chosen in such way that the length of the linker

[0164] L is 12 to 21 atoms.

[0165] In certain embodiments, L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from any one of the moieties and SP is chosen in such way that the length of the linker L is 12 to 21 atoms.

[0166] In certain embodiments, R8is selected from any one of the moieties

[0167] In certain embodiments, R8is selected from any one of the moieties

[0168] In certain embodiments, the compound comprises the general formula (I) wherein

[0169] P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds class 1 phosphoinositide 3-kinase (PI3K),

[0170] L is a linker with a linker length of 12 to 21 atoms,

[0171] J is a junction selected from, wherein R7is selected from

[0172] H and the group consisting of -C1-C3alkyl;

[0173] E is an E3 ubiquitin ligase binding moiety that binds cereblon, r is an integer in the range of 1 to 6,

[0174] SP is chosen in such way that the sum of atoms of -C(=O)-(CH2)r-C(=O)-, r and SP is 12 to 21 atoms.

[0175] In certain embodiments, r is an integer in the range of 1 to 4.

[0176] In certain embodiments, r is an integer in the range of 1 to 3.

[0177] In certain embodiments, the compound comprises the general formula (I) wherein

[0178] P is a phosphoinositide 3-kinase (PI3K) binding moiety that binds PI3K-alpha and / or PI3K- beta,

[0179] L is a linker with a linker length of 12 to 21 atoms,

[0180] J is a junction selected from, wherein R7is selected from

[0181] H and the group consisting of -C1-C3alkyl;

[0182] E is an E3 ubiquitin ligase binding moiety that binds cereblon, r is an integer in the range of 1 to 6,

[0183] SP is chosen in such way that the sum of atoms of -C(=O)-(CH2)r-C(=O)-, r and SP is 12 to 21 atoms.

[0184] The -C(=O)-(CH2)r-C(=O)- part of the linker provides an additional handle for potency and selectivity in degradation and induces additional interactions when binding the kinase isoforms p110alpha and p110beta.

[0185] In certain embodiments, the compound comprises the general formula (II) wherein r is an integer in the range of 1 to 6,

[0186] P has the chemical structure wherein

[0187] X11, X12and X13are each selected from CH and N;

[0188] X14is N or CH;

[0189] R6is selected from one of the moieties of wherein X15is N or O,

[0190] R10is selected from the group consisting of -C1-C3alkyl, f is 0,1 or 2, and X16and X17are selected from CH and N;

[0191] R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and -CH2F; h is 0 or 1 ; R12is selected from the group consisting of -NH2, - NHC(=O)C1-3alkyl, -NHOC1-3alkyl; X18and X19are each selected from N and

[0192] C;

[0193] R13is selected from the moieties

[0194] R14is -C1-C3alkyl and j is 0 or 1 ; E has the chemical structure wherein

[0195] X is CH or N, particularly CH, wherein R3and R4are each selected from =0 and -CH3, and b and c are 0 or 1 ,

[0196] X2is CH or N,

[0197] X3and X4are each selected from CH2, C, N, and SO2,

[0198] X5, X6, X7and X8are each selected from C and N,

[0199] X9is CH or N,

[0200] R2is selected from the group of halogens, and a is 0, 1 or 2,

[0201] J is a junction selected from , , , R7is selected from

[0202] H and the group consisting of -C1-C3alkyl; and SP is chosen in such way that the length of the linker L is 12 to 21 atoms.

[0203] In certain embodiments, r is an integer in the range of 1 to 4.

[0204] In certain embodiments, r is an integer in the range of 1 to 3.

[0205] In certain embodiments, R7is H.

[0206] In certain embodiment, the compound comprises the general structure

[0207] P-L-J-E, wherein P has the chemical structure wherein

[0208] X11is N;

[0209] X12and X13are selected from CH and N;

[0210] X14is N or CH;

[0211] R6is wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16;

[0212] R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and - CH2F; R12is selected from the group consisting of -NH2, -NHC(=O)C1-3alkyl; X18and X19are each selected from N and C;

[0213] E has the chemical structure wherein

[0214] X is CH or N, particularly CH, R1has the chemical structure wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1

[0215] X2is CH or N,

[0216] X4is selected from CH2, C and N,

[0217] X9is CH or N, particularly N;

[0218] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0219] X9is CH or N, particularly N;

[0220] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1 and

[0221] J is a junction selected from, , wherein R7is selected from H and the group consisting of -C1-C3alkyl;

[0222] L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from one of the moieties and SP is selected from a. -(CH2)n-; wherein n is an integer in the range of 4 and 14, and wherein n is chosen in such way that the length of the linker L is 12 to 21 atoms; b. -(CH2)m-B-; wherein m is an integer in the range of 0 and 9, wherein B and m are chosen in such way that the length of the linker L is 12 to 21 atoms; c. -(CH2)2-NR9-(CH2)3-; wherein R9is selected from H and -C(=O)CH3and wherein

[0223] , wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4 and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms, R8, m and B are chosen in such way that the length of the linker L has particularly 12 to 18 atoms.

[0224] In certain embodiments, the compound comprises the general structure

[0225] P-L-J-E, wherein

[0226] P has the chemical structure wherein

[0227] X11is N;

[0228] X12and X13are selected from CH and N;

[0229] X14is N or CH;

[0230] R6is , wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16;

[0231] R9is selected from one of the moieties of wherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and - CH2F; R12is selected from the group consisting of -NH2, -NHC(=O)C1-3alkyl; X18and X19are each selected from N and C

[0232] E has the chemical structure wherein

[0233] X is CH or N, particularly CH,

[0234] R1has the chemical structure wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1

[0235] X2is CH or N,

[0236] X4is selected from CH2, C and N,

[0237] X9is CH or N, particularly N;

[0238] R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

[0239] X9is CH or N, particularly N; R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1 ; and

[0240] J is a junction selected from, wherein R7is selected from

[0241] H and the group consisting of -C1-C3alkyl;

[0242] L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from one of the moieties and SP is selected from a. -(CH2)n-; wherein n is an integer in the range of 4 and 14, and wherein n is chosen in such way that the length of the linker L is 12 to 21 atoms; b. -(CH2)m-B-; wherein m is an integer in the range of 0 and 9, wherein B and m are chosen in such way that the length of the linker L is 12 to 21 atoms, in particular R8, m and B are chosen in such way that the length of the linker L is 12 to 18 atoms; and wherein , wherein Y1, Y2and Y3are selected from CH and

[0243] N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4 and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0244] In certain embodiments, SP is selected from a. -(CH2)n-; wherein n is an integer in the range of 4 and 14, and wherein n is chosen in such way that the length of the linker L is 12 to 21 atoms; b. -(CH2)m-B-; wherein m is an integer in the range of 0 and 9, wherein B and m are chosen in such way that the length of the linker L is 12 to 21 atoms; and c. -C-; C is chosen in such way that the length of the linker L is 12 to 21 atoms wherein

[0245] B is selected from -C(=O)-, wherein Y1, Y2and Y3are selected from CH and

[0246] N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4;

[0247] C is selected from wherein Z2, Z3, and Z4are selected from -CH and N.

[0248] In certain embodiments, Z1is O.

[0249] In certain embodiments, wherein in case Z2is N, Z3is CH or in case Z3is N, Z2is CH.

[0250] In certain embodiments, L is -R8d-(CH2)n-J-; wherein n is an integer between 4 and 12, particularly wherein n is an integer between 6 and 12, and wherein

[0251] R8is selected from one of the moieties

[0252] wherein Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12and Y13are selected from CH and N; and J is selected from In certain embodiments, L is

[0253] -R8d-(CH2)n-J-; wherein n is an integer between 4 and 12, particularly wherein n is an integer between 6 and 12, and wherein

[0254] R8is selected from one of the moieties and J is selected from

[0255] In certain embodiments, L is -R8d-(CH2)n-J-; wherein n is an integer between 4 and 12, particularly wherein n is an integer between 6 and 12, and wherein

[0256] R8is selected from one of the moieties

[0257]

[0258] In certain embodiments, L is -R8d-(CH2)n-J-; wherein n is an integer between 4 and 12, particularly wherein n is an integer between 6 and 12, and wherein

[0259] R8is selected from one of the moieties

[0260] In certain embodiments, n is an integer between 6 and 12.

[0261] In certain embodiments, L is

[0262] -R8d-(CH2)n-J-; wherein R8dis selected from ; d is 1 ; J is , and n is an integer in the range of 4 to 12.

[0263] In certain embodiments, L is -R8d-(CH2)n-J-; wherein R8dis selected from ° integer in the range of 4 to 12.

[0264] In certain embodiments, L is -R8d-(CH2)n-J-; wherein R8dis selected from ° integer in the range of 5 to 9.

[0265] In certain embodiments, R8is selected from any one of the moieties In certain embodiments, R8is selected from any one of the moieties

[0266] In certain embodiments, R8is selected from is an integer in the range of 6 to 12.

[0267] In certain embodiments, R8is selected from and n is an integer in the range of 6 to 12.

[0268] In certain embodiments, wherein in case R8is and J is NH, n is an integer in the range of 8 to 12.

[0269] In certain embodiments, wherein in case R8is and J is NH, n is an integer in the range of 6 to 9.

[0270] In certain embodiments, R8dis and n is an integer in the range of 4 to 10.

[0271] Compounds containing the piperazine unit in R8are selective towards PI3Kalpha which has a glutamine (Q853) which allows this part of the linker to interact with the protein. In turn, PI3Kbeta does not have this amino acid residue.

[0272] In certain embodiments, R8dis d is 1 ; J is and n is an integer in the range of 4 to 10.

[0273] In certain embodiments, R8dis ; d is 1 ; J is and n is an integer in the range of 5 to 9.

[0274] In certain embodiments, R8dis d is 1 ; J is and n is an integer in the range of 5 to 9. In certain embodiments, L is

[0275] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties

[0276] Y6, Y7, Y8, Y9, Y10, Y11, Y12and Y13are selected from CH and N; and J is selected from and B is selected from -C(=O)-, , wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0277] In certain embodiments, L is

[0278] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from and B is selected from -C(=O)- wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0279] In certain embodiments, L is -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from , and B is selected from selected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms. In certain embodiments, L is -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from and B is selected from wherein Y1and Y3are selected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0280] In certain embodiments, -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from and B is selected from wherein Y1is selected from CH and N, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0281] In certain embodiments, L is

[0282] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties

[0283] and J is selected from , and and B is selected from -C(=O)-, , wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0284] In certain embodiments, L is

[0285] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from and B is selected from -C(=O)-, , wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0286] In certain embodiments, L is

[0287] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from , and B is selected from -C(=O)-, , wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0288] In certain embodiments, L is

[0289] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from an d , and B is selected from , wherein Y1and Y3are selected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms. In certain embodiments, L is

[0290] -R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moieties and J is selected from , and B is selected from wherein Y1is selected from CH and N, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

[0291] In certain embodiments, R8is selected from any one of the moieties

[0292] In certain embodiments, B is selected from any one of the moieties -C=O-, , -NHC(=O)-

[0293] In certain embodiments, B is selected from any one of the moieties

[0294] In certain embodiments, B is selected from any one of the moieties , and

[0295] In certain embodiments, wherein in case R8is m is an integer in the range of 7 to 9. In certain embodiments, wherein in case R8is , or

[0296] In certain embodiments, L is

[0297] -R8d-(CH2)2-NR9-(CH2)3-J; wherein R9is selected from H and -C(=O)CH3and wherein

[0298] In certain embodiments, the length of the linker L is 12 to 18 atoms.

[0299] In certain embodiments, the compound is selected from

[0300]

[0301] In certain embodiments, the compound is selected from

[0302] In certain embodiments, the compound is selected from and

[0303] A second aspect of the invention relates to a use of the compound according to the first aspect of the invention as a medicament.

[0304] A third aspect of the invention relates to a use of the compound according to the first aspect of the invention for the treatment of a disease, wherein the disease is cancer.

[0305] A fourth aspect of the invention relates to the use of the compound according to the first aspect of the invention for the manufacture of a medicament for the treatment of cancer.

[0306] Medical treatment

[0307] Similarly, within the scope of the present invention is a method or treating cancer in a patient in need thereof, comprising administering to the patient a compound according to the above description.

[0308] Pharmaceutical Compositions, Administration / Dosage Forms and Salts

[0309] According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0310] The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examμles of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examμles of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

[0311] In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.

[0312] Similarly, a dosage form for the prevention or treatment of cancer is provided, comprising a compound according to any of the above aspects or embodiments of the invention.

[0313] Certain embodiments of the invention relate to a dosage form for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

[0314] Certain embodiments of the invention relate to a dosage form for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0315] Certain embodiments of the invention relate to a dosage form for topical administration. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemμlified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Princiμles and Practice (1st Edition, Wiley 2011 , ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2ndEd., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1stEd. CRC Press 1989; ISBN-13: 978-0824781835). In embodiments of the invention relating to topical uses of the compounds of the invention, the pharmaceutical composition is formulated in a way that is suitable for topical administration such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like, comprising the active ingredient together with one or more of solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those skilled in the art.

[0316] The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0317] In certain embodiments, the pharmaceutical composition of the present invention can be in unit dosage of about 1-5000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

[0318] Method of Manufacture and Method of Treatment according to the invention

[0319] The invention further encompasses, as an additional aspect, the use of a compound as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of a cancer.

[0320] Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a disease associated with cancer. This method entails administering to the patient an effective amount of a compound as identified herein, or its pharmaceutically acceptable salt.

[0321] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0322] The invention is further illustrated by the following examμles and figures, from which further embodiments and advantages can be drawn. These examμles are meant to illustrate the invention but not to limit its scope.

[0323] Description of the Figures

[0324] Fig. 1 shows integrated western blot signals of PI3K normalised to DMSO and alpha- tubulin of the degraders. MCF7 cells were treated for6h. The standard deviation was calculated for WJ112-14 at all 4 concentrations and for WJ213-14 at the optimal concentration of 10 nM. This analysis points to a sweet spot in hydrophobic linker length between 14-16 atoms, where molecules were especially potent degraders (except for VHL targeting molecule WJ203-14, which was inactive and is not part of the present invention). A rigid bis-piperidyl linker (WJ204-12), with a different exit vector from the imide scaffold and a 12- atom linker, also led to potent degradation. The tolerance of a shorter linker when connected at the 3-position in the isoindoline (WJ204-12) scaffold is consistent with structural data μlacing this position further out of the CRBN binding pocket. WJ117-16 is not part of the invention. WJ112-14Meis a negative control.

[0325] Fig. 2 shows rapid protein degradation of p110alpha which is consistent with a protein level effect. The protein degradation is comμlete at 6 h with WJ112-14.

[0326] Fig. 3 shows the degradation recovery after deμletion establishes pHOalpha as a valid degradation target. At 48h with WJ112-14, approximately 20-30 % recovery occurs, which is consistent with the general concept of potent degradation, where the treatment effect is still visible after the compound has been removed from the medium. This shows that the protein was fully removed and the cells need to re-synthesize the protein.

[0327] Fig. 4 shows a comparison to state-of-the-art degraders. A) structures of taselisib and CRBN-targeted pHOalpha degrader “D” disclose in Li et al. (1). The data indicates that even in taselisib-responsive cell lines the PROTAC degrader WJ112-14 outperforms taselisib and is active in cells where taselisib is inactive, such as in MCF7 and T47D cell lines. Similarly, WJ2112-14 and WJ213-14 further outperform “D” from Li et al (1) in MCF7 cell lines where “D” shows little detectable degradation at 6 h or 24 h.

[0328] Fig. 5 shows selective degradation of pHOalpha over p110beta in different cell lines by WJ112-14.

[0329] Fig. 6 shows the tumour volume kinetics in mice on tumour model Cal-33 with WJ213- 14 treatment over 26 days, after which the mice were sacrificed to see ensure maximal protein degradation in the tumour cells. WJ213-14 shows ideal protein degradation in Cal-33 cell line.

[0330] Fig. 7 shows a structural overview of PI3K PROTACs series 1. The left side disμlays alkyl-linker structures ranging from 11 to 17 atoms. Centrally located are lead compound WJ112 and its optimized variant WJ213. WJ214 and WJ204 feature modified linker designs. Control compounds derived from WJ112 are shown in purμle: WJ203 (VHL binder), WJ157 (non-functional CRBN binder), and WJ117 (PEG-linker).

[0331] Fig. 8 shows measured inhibition constants Ki of the PROTAC library against purified PI3K α from TR-FRET assay. The dashed line indicates PQR514 parent inhibitor potency. All compounds containing the parent core maintained binding activity. Data represent the mean with 95 % confidence intervals (Cl) from three reμlicates. Fig. 9 shows calculated IC50 values for AKTpS473 in MCF-7 cells at the 2 h time point. The dashed line indicates PQR514 parent inhibitor potency. Data represent the mean with 95 % Cl from three reμlicates.

[0332] Fig. 10 shows PROTACs grouped as series 2, investigating various linker properties. Compounds from series 1 exμloring linker features are also included. For each PROTAC, the linker structure is presented above a graph showing dose- response effects on PI3Kα levels from both PRM (blue) and western blot (red) data. All PROTACs incorporate the same PQR514-based PI3K binder (yellow circle). Variable cereblon binders are denoted by red, blue, or purμle circles, with WJ710 and WJ799 featuring distinct CRBN binders shown in comμlete structural detail. PRM data (previously presented) includes all peptides at a cut- off dotP >0.7, with means and standard errors indicated by bars and error bars, respectively. PRM measurements were performed in biological triμlicates. For western blot data, individual dots represent independent biological experiments, with means and standard errors shown by bars and error bars. The y-axis shows the normalised PI3K signal. The x-axis shows the concentrations of 10 μM, 1 μM, 0.1 μM, 10 nM and 1 nM. Compounds WJ112, WJ204, WJ213, WJ214, and WJ748 show degradation of more than 80% at a concentration of 100 nM. WJ204, WJ213 and WJ748 still show degradation of more than 80% at a concentration of only 10 nM. Compounds WJ112 and WJ214 still show degradation of 50 to 79% at 10 nM.

[0333] Fig. 11 shows PROTAC series 3: Comparison of PI3Kα degradation by ten WJ213- based PROTACs differing only in their CRBN binder, a) WJ213 core structure shared by all presented compounds, b) Structures of I Mi Ds pomalidomide, thalidomide, and lenalidomide. Exit vector position numbering shown on thalidomide, c) TPS-normalized PI3Kα signal from western blotting following treatment with indicated PROTACs at specified concentrations. Experiments were conducted in MCF-7 cells for 6 h. All derivatives except WJ709 - where insufficient linker length is suspected - demonstrate strong and comparable PI3Kα degradation, indicating broad tolerability of CRBN binder modifications. Individual dots represent independent biological experiments. Mean and standard error are shown for WJ213. The y-axis shows the normalised PI3K signal. The x-axis shows the concentrations of 10 μM, 1 μM, 0.1 μM, 10 nM and 1 nM. Compounds WJ701 , WJ703, WJ704, and WJ706 show degradation of more than 80% at a concentration of 100 nM. WJ701 , and WJ706 still show degradation of more than 80% at a concentration of only 10 nM. WJ703, and WJ704 still show degradation of 50 to 79% at 10 nM.

[0334] Examμles

[0335] Biological and biochemical assays

[0336] Example 1: Cell culture

[0337] MCF7 cells (RRID: CVCL_0031 , human, female) were obtained from DSMZ (ACC 115) and grown in DMEM (Sigma Aldrich, D5796) supμlemented with 10 % FCS (BioConcept, 2-01 F00- I) and 1 % Penicillin-Streptomycin (BioConcept, 4-01 F00-H). MCF7 cells were passaged by trypsination with 0.05 % trypsin (Gibco, 25300053). Cells were kept in a humidified incubator at 37 °C and 5 % CO2.

[0338] Example 2: Western Blotting

[0339] Procedure 1

[0340] Cells were seeded at 800 k in 2 ml comμlete growth medium in 6 well μlates and grown for 24 h. Cells were treated from DMSO stocks at indicated concentrations. Final DMSO concentration was kept below 1 %. Cells were treated for the indicated time after which cells were washed twice with PBS (Sigma-Aldrich, D8537) and then harvested by scraping (Cell Scrapers, Sarstedt, 83.3951) and resuspended in PBS. Cells were collected by centrifugation (15 min, 8.8 krcf, 4 °C) and the supernatant was removed by suction. Cell pellets were stored at -80 °C until further processing.

[0341] Cell pellets were resuspended in RIPA lysis buffer (50 mM Tris at pH 8, 150 mM NaCI, 1 % Triton X-100, 0.25 % sodium deoxycholate, 0.1 % SDS supμlemented with comμlete Mini Protease Inhibitor (Roche) and 1 mM sodium orthovanadate (Sigma Aldrich)), left on ice for 30 min and centrifuged at 16 krcf and 4 °C for 15 min. The supernatant was collected, and protein concentration was measured (DC Protein Assay, Bio-Rad). Aliquots of 50 pg of protein were combined with 3X loading dye (New England BioLabs, B7703S) and heated to 98 °C for 5 min shaking at 300 rpm. The samμles were separated by 8% SDS-PAGE apμlying 80 - 120 Volt. Analytes were transferred onto nitrocellulose membranes (#10600021 , Amersham, GE Healthcare Life Sciences, Chalfont St. Giles, UK) using a semi-dry blotting apparatus (Trans- Blot Turbo with corresponding transfer packs, BioRad). Membranes were blocked with Intercept® (TBS) Blocking Buffer (LI-COR) for 1 h at room temperature while shaking. Primary antibodies used for incubation at 4 °C overnight: anti-p110a (ref: Hu Q, Klippel A, Muslin A J, Fantl W J, Williams L T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995; 268: 100-10), anti-p110b (#3011 , CST), anti- mTOR (#2983, CST). The blots incubated with secondary antibodies using anti-Mouse (Licor, IRDye® 680RD Goat anti-Mouse IgG, 1 :10000 dilution) or anti-Rabbit secondary antibody (Licor, IRDye® 800CW Goat anti-Rabbit IgG, 1 :10000 dilution) and the signals analyzed using LI-COR Odyssey CLx infrared scanner and processed using LI-COR image Studio software.

[0342] Procedure 2

[0343] Unless indicated differently, inhibitor treatments were performed in 6-well dishes and on cell cultures that had reached a final density of no more but 80 - 90 % confluence at the time of harvest. For harvest, wells were first rinsed with 2x 2 mL of cold D-PBS buffer and cells detached from the growth support by scaping at cold. Cells were centrifuged (5 min, 100 ref, 4 °C) and the resultant pellet flash-frozen and stored at minus 80 °C for later use. For Western analyses, pellets were disrupted in 1x cold Lysis Buffer (#9803, Cell Signaling Technologies (CST), Danvers, MA, USA) supμlemented with Protease / Phosphatase Inhibitor Cocktail (#78442, Thermo Fisher, Waltham, MA, USA) and cleared by centrifugation (15 min, 21 000 ref, 4 °C). Protein content was determined by Bradford using a Coomassie Plus Protein Assay Reagent (Thermo Scientific, #1856210) and following the manufacturers’ instructions. Cleared protein lysates were denatured with Laemmli buffer and an equivalent of 10 pg total protein per lane separated over 8 - 12 % (depending on the analyte’s size) bis-acrylamide (#161- 0148, BioRad, Hercules, CA, USA) gels by discontinuous SDS-PAGE. Analytes were transferred onto nitrocellulose membranes (#10600021 , Amersham, GE Healthcare Life Sciences, Chalfont St. Giles, UK) using a semi-dry blotting apparatus (Trans-Blot Turbo with corresponding transfer packs, BioRad). Membranes were blocked for 1 h at RT with 10 % w / v nonfat dry milk (#9999S, Cell Signaling Technology (CST), Danvers, MA, US) diluted in TBS buffer supμlemented with 0.1 % Tween-20 (p1379, Sigma), and proteins stained with the following primary antibodies (o / n at 4 °C): anti-p110a (ref: Hu Q, Klippel A, Muslin A J, Fantl W J, Williams L T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995; 268: 100-10), anti-p110b (#3011 , CST), anti- mTOR (#2983, CST), anti-p85a (ab133595, Abeam, Cambridge, UK), anti-p85b, (ab28356, Abeam), anti-pAKT_T308 (#13038, CST), anti-pAKT_S473 (#4058, CST), anti- pRPS6_S240 / 244 (#5364, CST), or anti-tubulin a (T9046, Sigma). Proteins were standardly detected by ECL reaction (immobilon HRP substrate solution, Millipore, Burlington, MA) involving HRP-linked anti-rabbit (#7074, CST) or anti-mouse (#7076, CST) secondary reagents. Where necessary, membranes were cleared of primary antibodies using the Restore Western Blot Stripping Buffer (#21059, Thermo Fisher) and re-cycled for iterative detection. Chemiluminescence was recorded on a Fusion FX image reader (Vilber, Collegien, France) set to autoexposure at high sensitivity. Example 3: Plasmid and Stable Cell Line Generation

[0344] Stable EGFP_PI3CA cell line was created by integration of the pcDNA5 / FRT expression vector containing EGFP-PI3CA sequence into the genome of Flp-ln-293 cells (Invitrogen) via FLP recombinase-mediated DNA recombination at the FRT site (Flp-ln system, Invitrogen). Cells were transfected with the expression vector and selected in presence of hygromycin.

[0345] The pcDNA5 / FRT / EGFP_PI3CA expression vector was generated by insertion of a PI3CA coding sequence fused to the C-terminus of EGFP (using GGGGSGGGGS linker) into pcDNA5 / FRT / TO between Aflll and Xhol restriction sites.

[0346] PI3CA coding sequence was obtained from the μlasmid PIK3CA-WT (Addgene μlasmid # 16643). PIK3CA-WT (Addgene μlasmid # 16643; http: / / n2t.net / addgene: 16643 ; RRID:Addgene_16643).

[0347] Example 4: Flow Cytometry

[0348] 800 x 103 cells were seeded in a 6 well-μlate format in 1 ml growing medium. After 24 h the cells were treated. After 24 h, the growing medium was removed and 0.05 % Trypsin was added to detach the cells. The trypsin was neutralized by the addition of growing medium and the cells collected via centrifugation (8000 ref at 4 °C for 5 min). The pellets were resuspended in 300 pL cold PBS (2.5 % FCS). Centrifugation and resuspension were repeated two more times and then measured on a BD LSR Fortessa Analyzer. The data was analyzed with Cytoflow 1.2. Blue Laser: 488 nm, LP Mirror 505 nm, BP Filter 512 nm / 25 nm.

[0349] Example 5: TR-FRET-Assay

[0350] The kinetic constant (Ki) of compounds for p110a were determined by LanthaScreen Technology Ell Kinase Binding Assay (Life Technologies). An N-terminally His6-tagged p110a recombinant protein was combined with a biotinylated anti-His6-tag antibody (2 nM, #6089), an Europium-labeled Streptavidin comμlex (2 nM, #5899) and an AlexaFluor647-labelled kinase Tracer (Tracer 314, #6087, Kd of 2.26 for p110a at 20 nM) in TR-FRET assay buffer (50 mM N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) pH 7.5, 10 mM MgCh, 1 mM ethylene glycol-bis(β-aminoethyl ether)- N,N,N',N'- tetraacetic acid (EGTA), and 0.01 % (v / v) Brij-35). The prepared recombinant protein-antibody-tracer mix was dispensed in a 384- well μlate (5 μl per well). The μlate was then centrifuged (1700 rpm for 5 min) and incubated in dark at RT for 45 min. Subsequently, compounds were diluted in TR-FRET assay buffer and then dispensed into a 384-well μlate (in triμlicate) at a final concentration range between 10 μM and 0.2 nM using Dispendix l-DOT One and incubated in the dark at RT for 60 min. Time resolved FRET was measured with a Synergy Neo2 μlate reader. (BioTek instruments; Emission filters: 665 / 8 nm and 620 / 10 nm; excitation with 337 nm TRF laser; 100 ps delay, 200 ps data collection; 37 °C; dichroic mirror 400 nm).(5) The emission ratio of 665 / 620 was normalized to a non-inhibitor control and subsequently, the data was fitted to the “log(inhibitor) vs. normalized response - variable slope” curve using Graphpad Prism software (Version 9.5.1).

[0351] The Ki values were calculated according to the following formula:

[0352] Table 1: Mean Ki values with SD for TR-FRET

[0353] Compound Ki [nM] SD

[0354] WJ111-11 1.849 0.083

[0355] WJ200-12 1.837 0.018

[0356] WJ201-13 1.850 0.138

[0357] WJ112-14 1.662 0.034

[0358] WJ202-15 3.432 0.086

[0359] WJ208-16 3.985 0.413

[0360] WJ209-17 9.836 0.783

[0361] WJ204-12 1.163 0.110

[0362] WJ213-14 0.851 0.013

[0363] WJ203-14 2.809 0.021

[0364] WJ214-14 2.452 0.093

[0365] WJ112-14-Me 3.471 1.141

[0366] WJ117-16 4.990 0.109

[0367] Li_2018_D 19.448 0.007

[0368] PQR514 2.840 0.130

[0369] Example 6: In-Cell Western

[0370] 1800 MCF7 cells / well were seeded in a 96 well-μlate format pCIear® CELLSTAR® (Greiner Bio-One, Cat. #7.655 090) and grown for 24 h in 100 μl DMEM low glucose (Sigma, Cat. #D6046-1 L) containing 50 ml (10 %) of heat inactivated FCS (Sigma, Cat. #F7524), 5 ml (1 %) of 200 mM L-glutamine (Sigma, Cat. #G7513) and 5 ml (1 %) of penicillin - streptomycin (Sigma, Cat. #P4333). The compounds for treatment were diluted from 2 mM stock solutions in DMSO into 150 μM, 15 μM, and 0.15 μM dilutions in 1X PBS (pH 7.40, 0.22 mm filtered, without Ca / CI / Mg, Sigma, Cat. #D8537-500ML) in l-DOT PURE S Plates 100 (Art. Nr. D1610021800) and subsequently transferred via IDOT dispenser into the wells of the cell culture μlate. The final treatment concentrations for each compound were 3000, 1024, 512, 256, 128, 64, 32, 16, 8, 4, and 0.8 nM. The cells were incubated with the compounds for 2 h. 10 % (w / v) PFA (Paraformaldehyde) in PBS (Sigma-Aldrich, Cat. #158127-500G) was filtered (0.22 pm filter), dispensed into the wells and incubated for fixation at room temperature for 30 min. The fixation-buffer was removed and the cells were washed with 150 μl 1 x PBS (3 times). For permeabilization 60 μl per well of 0.1 % (v / v) Triton X-100, 1 % (w / v) BSA (Bovine Serum Albumin), (Sigma, Cat. #T8787-250ML) in PBS (Sigma, Cat. #A3912-100G) were dispensed and incubated for 30 min at room temperature. The buffer was removed and 60 μl per well of a prepared buffer of 5 % Goat Serum (Sigma, Cat. #G6767), 1 % BSA, 0.1 % Triton in PBS was dispensed for blocking by incubating for 30 min. The buffer was removed and the μlates were incubated overnight at 4 °C (sealed and shaking) with 50 μl per well of a mixture of rabbit anti-pPKB S473 antibodies (Cell Signaling Technology, Cat. #4058L) diluted 1 :500 in PBS (containing 5 % Goat Serum, 1 % BSA, 0.1 % Triton) and Mouse anti-aTubulin antibodies (Sigma, Cat. #T9026) diluted 1 :2000 in PBS (containing 5 % Goat Serum, 1 % BSA, 0.1 % T riton). The μlates were washed with PBS three times. Secondary antibodies (Goat anti-mouse IRDye680 (LICOR, #D00115-03) and Goat anti-rabbit IRDye800 (LICOR, #D00304-15)) were diluted and dispensed the same way as the primary antibodies in the previous step, then incubated for 90 min at room temperature, protected from light and shaking. The buffer was removed and the μlates were washed three times with PBS 1X. The μlates were analyzed with the LICOR Infrared scanning system Odyssey CLx and the software image Studio. Images were acquired with the auto setting, scan control 84 pm, quality medium and focus distance 40 pm.

[0371] Table 2: Mean IC50values with SD for In-cell western blots

[0372] While TR-FRET data enabled the calculation of Ki-values, the ICW assays provided insights about their cellular inhibitory activity quantified as IC50-values based on the phosphorylation levels (Ser473) of the first PI3Ka downstream target protein kinase B (PKB / Akt). Comparing the data across this compound series revealed some key trends. First, all molecules with the PQR514 core have Ki-values below 10 nM, many of them even lower than PQR514 ( itself, suggesting that linker interactions in or around the active site can further stabilize binding - a fact reiterated by the 10-fold increase in affinity for the hydrophobic versus hydrophilic linker ((WJ208-16 vs WJ117-16). Additionally, the TR-FRET data and the ICWs exhibit the same potency trends indicating strong PI3Ka inhibition by WJ213-14 and WJ204-12 in both a biochemical and cellular context.

[0373] Example 7: In-vivo tumour model Cal-33 with WJ213-14 treatment

[0374] Cell culture

[0375] CAL-33 cells were originally purchased from ATCC (USA). Cells were cultured in comμlete medium containing Dulbecco’s modified Eagle medium (DMEM, Sigma Aldrich, #D6429) supμlemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, #1050064) and penicillin (100 units / ml) / streptomycin (100 μg / ml) (Gibco, #15140122) in a humidified atmosphere at 37°C with 5% CO2. The cells were passaged twice a week according to confluence.

[0376] Cell line-derived xenografts (CDX)

[0377] A total of 1 x 106of CAL-33 cells were suspended in 100 μl of DMEM and subcutaneously injected into the left and right flank of male NOD.Cg-Prkdcscid / J mice. Three weeks after the imμlantation of cells, when the tumor sizes reached 100 - 250 mm3, the mice were divided in two groups and they started receiving treatment. The treatment scheme in our study consisted of a 30 mg / kg WJ213-14 intraperitoneal (i.p.) injection 3 times per week for three weeks. The Vehicle group (control) were given an i.p. injection 20 % (wv) beta captisol in NaCI aq. 0.9 % 3 times per week. T umor sizes were measured by caliper twice a week. T umor volumes were calculated by using the following formula: (L x W2) / 2, where L was the longest axis and W was the shortest axis of the tumor. All animal experiments were performed according to national and international regulations and were approved by the BRFAA (Biomedical Research Foundation of the Academy of Athens, Athens Greece) ethical committee. Male NOD.Cg- Prkdcscid / J mice were purchased from the Jax repository (Bar Harbor, ME USA) and bred in- house.

[0378] Western blotting

[0379] Tissue samμles were lysed in RIPA buffer (EMD Millipore, #20188), and the total protein concentration was determined by using the bicinchoninic acid (BCA) kit (Pierce, #23227). The samμles were loaded into 10-15% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS- PAGE) gels (20 μg of protein per lane) for electrophoresis, and then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were washed two times with 0.1% TBST and blocked with 5% BSA for 1 h. The membranes were then incubated with primary antibody anti-PIK3CA (Cell Signaling Technology, #4254) overnight at 4°C. After three washes, the membranes were incubated with secondary goat anti- rabbit (Cell Signaling Technology, #7074) or goat anti-mouse (Cell Signaling Technology, #7076) antibodies couμled to horseradish peroxidase (HRP) at room temperature for 2 h. Finally, the immune comμlexes were detected using an enhanced chemiluminescence (ECL) HRP assay kit (Pierce, #32109). Beta-actin (Cell Signaling Technology, #3700) was used as the internal control for all experiments. Chemical synthesis

[0380] Example 1: General procedure A Synthesis of intermediate 3

[0381] To a mixture of 2-(2, 6-dioxo-3-piperidyl) -4-fluoro-isoindoline-1 , 3-dione (1 , 200 mg, 724 μmol, 1 eq), DIEA (187 mg, 1.45 mmol, 252 μl, 2 eq) in NMP (2 ml) was added 2 (1.1 eq), then the mixture was stirred at 90 °C for 12 h. LC-MS showed 2-(2,6-dioxo-3-piperidyl)-4-fluoro- isoindoline-1 ,3-dione consumed comμletely and one main peak with desired m / z was detected. TLC (Petroluem / EtOAc = 1 :1) indicated the starting material was consumed comμletely and a major spot observed. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (3x 20 ml). The organic layer was dried over anhydrous Na2SC>4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 to 50 % Ethyl acetate / Petroleum ethergradient @ 40 ml min-1) to give compounds 3 (50 - 100 % yield) as yellow solids. Synthesis of intermediate 4

[0382] To a solution of 3 (180 mg, 349 μmol, 1 eq) in DCM (0.5 ml) was added TFA (693 mg, 6.08 mmol, 450 μL, 17.4 eq), then the mixture was stirred at 10 °C for 0.5 h. LC-MS showed tert-butyl N-[9-[[2-(2, 6-dioxo-3-piperidyl) -1 , 3-dioxo-isoindolin-4-yl]amino]nonyl]carbamate consumed comμletely and one main peak with desired mass was detected. The mixture was concentrated to give crude 4 as yellow solids which were used in the next step without further purification.

[0383] Synthesis of compound 6 (n=1, 2, 3, 4, 5, 6)

[0384] To a solution of 4 (170 mg, 410 μmol, 1 eq) and 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin- 5-yl]-6-morpholino-1 ,3,5-triazin-2-yl]piperazin-1-yl]-4-oxo-butanoic acid (255 mg, 517 μmol, 1.3 eq) in DMF (5 ml) was added HATU (340 mg, 894 μmol, 2.2 eq) and DIEA (255 mg, 1.97 mmol, 344 μl, 4.8 eq), then the resulting mixture was stirred at 20 °C for 2 h. LCMS showed full consumption of the starting material and the desired MS was detected. The reaction mixture was partitioned between ethyl acetate (60 ml) and brine (50 ml). The aqueous layer was extracted with ethyl acetate (100 ml) twice. The combined organic layers were dried over Na2SO4, filtrated and evaporated to give a crude material. The crude material was purified by Prep-HPLC (column: llnisil 3-100 C18 Ultra 150 x 50 mm x 3 μm; mobile phase: [water(FA)-ACN];B%: 48 %-78 %,7 min). The eluent solution was lyophilized to give final compounds 6 (20 - 60 % yield) as yellow solids.

[0385] 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2- yl]piperazin-1-yl]-N-[7-[[2-(2, 6-dioxo-3-piperidyl) -1, 3-dioxo-isoindolin-4- yl]amino]heptyl]-4-oxo-butanamide (WJ200- 12)

[0386] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.08 (s, 1 H), 9.10 (s, 1 H), 7.80 - 7.78 (m, 1 H), 7.63 (t, J = 54.0 Hz, 1 H ), 7.62 - 7.50 (m, 3H), 7.08 (d, J = 8.6 Hz, 1 H), 7.01 (d, J = 7.0 Hz, 1 H), 6.51 (br t, J = 5.8 Hz, 1 H), 5.09 - 4.97 (m, 1 H), 3.87 - 3.72 (m, 8H), 3.67 - 3.61 (m, 4H), 3.58 - 3.48 (m, 4H), 3.29 - 3.25 (m, 2H), 3.05 - 2.97 (m, 2H), 2.95 - 2.82 (m, 1 H), 2.63-2.53 (m, 4H), 2.36 - 2.28 (m, 2H), 2.06 - 1.97 (m, 1 H), 1.63 - 1.51 (m, 2H), 1.39 - 1.22 (m, 8H).

[0387] ESI-HRMS (m / z): [M]+H+calcd. for C40H50F2N13O7: 862.3916; found: 862.3919 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2- yl]piperazin-1-yl]-N-[8-[[2-(2, 6-dioxo-3-piperidyl) -1, 3-dioxo-isoindolin-4- yl]amino]octyl]-4-oxo-butanamide (WJ201- 13)

[0388] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.08 (s, 1 H), 9.11 (s, 1 H), 7.77 (d, J = 5.6 Hz, 1 H), 7.63 (t, J = 54.0 Hz, 1 H ), 7.62-7.51 (m, 3H), 7.08 (d, J = 8.6 Hz, 1 H), 7.01 (d, J = 7.0 Hz, 1 H), 6.51 (t, J = 5.8 Hz, 1 H), 5.11 - 4.96 (m, 1 H), 3.86 - 3.71 (m, 8H), 3.68 - 3.62 (m, 4H), 3.58 - 3.48 (m, 4H), 3.30 - 3.25 (m, 2H), 3.06 - 2.95 (m, 2H), 2.93 - 2.82 (m, 1 H), 2.63 - 2.53 (m, 4H), 2.37 - 2.29 (m, 2H), 2.08 - 1.96 (m, 1 H), 1.63 - 1.51 (m, 2H), 1.39 - 1.23 (m, 10H).

[0389] ESI-HRMS (m / z): [M]+H+calcd. for C41H52F2N13O7: 876.4070; found: 876.4075

[0390] 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6-morpholino- 1, 3, 5-triazin-2- yl]pi perazin- 1-yl]-N-[9-[[2-(2, 6-dioxo-3-piperidyl)- 1, 3-dioxo-isoindolin-4-yl] amino]nonyl]-4-oxo-butanamide (WJ112-14)

[0391] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.08 (s, 1 H), 9.11 (s, 1 H), 7.79-7.75 (m, 1 H), 7.63 (t, J = 54.0 Hz, 1 H ), 7.61-7.52 (m, 3H), 7.08 (d, J = 8.6 Hz, 1 H), 7.01 (d, J = 7.0 Hz, 1 H), 6.57- 6.46 (m, 1 H), 5.09-4.97 (m, 1 H), 3.87-3.71 (m, 8H), 3.67-3.61 (m, 4H), 3.57-3.48 (m, 4H), 3.29- 3.23 (m, 2H), 3.05-2.96 (m, 2H), 2.93-2.81 (m, 1 H), 2.65-2.54 (m, 4H), 2.38-2.26 (m, 2H), 2.08- 1.97 (m, 1 H), 1.61-1.51 (m, 2H), 1.37-1.21 (m, 12H).

[0392] ESI-HRMS (m / z): [M]+H+calcd. for C42H54F2N13O7: 890.4228; found: 890.4232 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl] -6-morpholino- 1, 3, 5-triazin-2- yl]piperazin-1-yl]-N-[10-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4- yl]amino]decyl]-4-oxo-butanamide (WJ202-15)

[0393] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.09 (s, 1H), 9.12 (s, 1H), 7.80-7.77 (m, 1H), 7.63 (t, J = 54.0 Hz, 1H ), 7.61-7.51 (m, 3H), 7.09 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.52 (t, J = 5.9 Hz, 1H), 5.13 - 4.97 (m, 1H), 3.89 - 3.71 (m, 8H), 3.69 - 3.62 (m, 4H), 3.59 - 3.49 (m, 4H), 3.31 - 3.24 (m, 2H), 3.06 - 2.97 (m, 2H), 2.95 - 2.83 (m, 1H), 2.64 - 2.54 (m, 4H), 2.39 - 2.29 (m, 2H), 2.09- 1.97 (m, 1H), 1.62- 1.51 (m, 2H), 1.43- 1.21 (m, 14H).

[0394] ESI-HRMS (m / z): [M]+H+calcd. for C43H56F2N13O7: 904.4382; found: 904.4388

[0395] 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2- yl]piperazin-1-yl]-N-[11-[[2-(2, 6-dioxo-3-piperidyl) -1, 3-dioxo-isoindolin-4- yl]amino]undecyl]-4-oxo-butanamide (WJ208- 16)

[0396] 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.05 (brs, 1H), 9.11 (s, 1H), 7.80 - 7.76 (m, 1H),7.63 (t, J= 54 Hz, 1H), 7.61 - 7.52 (m, 3H), 7.07 (d, J= 8.7 Hz, 1H), 7.01 (d, J= 7.0 Hz, 1H), 6.51 (brt, J= 5.8 Hz, 1H), 5.04 (dd, J= 5.4, 12.8 Hz, 1H), 3.85-3.70 (m, 8H), 3.67-3.62 (m, 4H), 3.56-3.49 (m, 4H), 3.29-3.24 (m, 2H), 3.04-2.96 (m, 2H), 2.93 - 2.82 (m, 1H), 2.63 - 2.54 (m, 4H), 2.36-2.28 (m, 2H), 2.07- 1.97 (m, 1H), 1.60- 1.51 (m, 2H), 1.36- 1.22 (m, 16H).

[0397] ESI-HRMS (m / z): [M]+H+calcd. for C44H58F2N13O7: 918.4539; found: 918.4545 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6-morpholino- 1, 3, 5-triazin-2- yl]piperazin-1-yl]-N-[12-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4- yl]amino]dodecyl]-4-oxo-butanamide (WJ209- 17)

[0398] 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.07 (s, 1 H), 9.10 (s, 1 H), 7.80 - 7.73 (m, 1 H), 7.63 (t, J = 54 Hz, 1 H), 7.61 - 7.51 (m, 3H), 7.07 (d, J = 8.6 Hz, 1 H), 7.01 (d, J = 6.9 Hz, 1 H), 6.50 (t, J = 5.2 Hz, 1 H), 5.08-5.01 (m, 1 H), 3.85 - 3.72 (m, 8H), 3.66-3.62 (m, 4H), 3.54-3.50 (m, 4H), 3.30 - 3.25 (m, 2H), 3.03 - 2.97 (m, 2H), 2.90 - 2.83 (m, 1 H), 2.61 - 2.55 (m, 4H), 2.35 - 2.30 (m, 2H), 2.05 - 1.98 (m, 1 H), 1.59-1.52 (m, 2H), 1.35 - 1.21 (m, 18H).

[0399] ESI-HRMS (m / z): [M]+H+calcd. for C45H60F2N13O7: 932.4697; found: 932.4701

[0400] Synthesis of compound 1

[0401] 2-(2, 6-Dioxopiperidin-3-yl) -4-fluoroisoindoline- 1, 3-dione (7)

[0402] A round bottom flask was charged with 3-fluorophthalic anhydride (18.2 mmol, 3.02 g, 1 eq), 3-aminopiperidine-2, 6-dione hydrochloride (3.00 g, 18.2 mmol, 1 eq) and sodium acetate (2.24 g, 27.3 mmol, 1.5 eq) in acetic acid (60 ml). The mixture was heated to reflux and stirred for 6 h. The reaction was allowed to cool to room temperature and the acetic acid was removed in vacuo. H2O (80 ml) was added to the residue and subsequently extracted with EtOAc (4 x 80 ml). The combined organic layers were concentrated in vacuo and DCM was added to the crude and the insoluble solid was filtered off and washed with DCM to yield 2-(2,6- dioxopiperidin-3-yl)-4-fluoroisoindoline-1 , 3-dione (1 , 4.32 g, 15.6 mmol, 86 %) as a grey powder.

[0403] 1H-NMR (400 MHz, 298 K, DMSO-d6, d / ppm): 11.14 (s, 1 H), 7.97-7.92 (m, 1 H), 7.80-7.71 (m, 2H), 5.18-5.14 (m, 1 H), 2.94-2.85 (m, 1 H), 2.64-2.53 (m, 2H), 2.10-2.03 (m, 1 H). Synthesis of compound 5

[0404] 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2- yl]piperazin-1-yl]-4-oxo-butanoic acid (5)

[0405] To a solution of 4-(difluoromethyl) -5-(4-morpholino-6-piperazin-1-yl-1 , 3, 5-triazin-2-yl) pyrimidin-2-amine (7, 2.2 g, 4.34 mmol, 1 eq) and tetrahydrofuran-2, 5-dione (8, 500 mg, 5.00 mmol, 1.15 eq) in DCM (50 ml) was added TEA (1.45 g, 14.37 mmol, 2 ml, 3.31 eq) at 20 °C, then the reaction mixture was stirred at 20 °C for 12 h. LCMS showed some of 4- (difluoromethyl) -5-(4-morpholino-6-piperazin-1-yl-1 , 3, 5-triazin-2-yl) pyrimidin-2-amine was consumed and desired MS was detected. The residue mixture was concentrated to give crude material. The crude product was triturated with 20 ml (PE / EtOAC=5 / 1), filtered to give 4-[4-[4- [2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2-yl]piperazin- 1 -yl]-4- oxo-butanoic acid (5, 3.00 g, crude) as a white solid.1H NMR (400 MHz, CDCI3) δ ppm: 8.93 (s, 1 H), 7.63 (t, J = 54 Hz, 1 H), 7.56 (s, 2H), 3.85 - 3.74 (m, 8H), 3.67-3.63 (m, 4H), 3.57-3.52 (m, 4H), 2.61 - 2.56 (m, 2H), 2.47 - 2.43 (m, 2H).

[0406] UHPLC-ESIMS (m / z): 494.2 [M+H]+.

[0407] Synthesis of WJ204- 12 according to the general procedure 5-[4-[[1-[4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]- 6-morpholino-1, 3, 5- triazin-2-yl]piperazin- 1-yl]-4-oxo-butanoyl]-4-piperidyl]methyl] - 1-piperidyl]-2-(2, 6- dioxo-3-piperidyl) -6-fluoro-isoindoline-1 , 3-dione (WJ204-12)1H NMR (400 MHz, DMSO-d6) δ ppm: 11.09 (s, 1 H), 9.11 (s, 1 H), 7.69 (d, J = 11.4 Hz, 1 H), 7.63 (t, J = 54.0 Hz, 1 H ), 7.57 (br s, 2H), 7.42 (d, J = 7.4 Hz, 1 H), 5.13 - 5.05 (m, 1 H), 4.40 - 4.30 (m, 1 H), 3.95 - 3.73 (m, 8H), 3.71 - 3.62 (m, 4H), 3.62 - 3.54 (m, 4H), 3.54 - 3.48 (m, 2H), 3.05-2.89 (m, 2H), 2.88 - 2.80 (m, 2H), 2.68 - 2.52 (m, 6H), 2.08 - 1.97 (m, 1 H), 1.82 - 1 .72 (m, 2H), 1.72 - 1.49 (m, 4H), 1.32 - 1.21 (m, 2H), 1.20 - 1.12 (m, 2H), 1.10 - 0.98 (m, 1 H), 0.97 - 0.82 (m, 1 H).

[0408] Synthesis of WJ213-14 according to the general procedure

[0409] 4-[ 6-[ 4-[ 4-[ 4-[ 4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]- 6-morpholino- 1,3,5- triazin-2-yl]piperazin- 1-yl]-4-oxo-butanoyl]piperazin- 1-yl]hexylamino]-2-(2, 6-dioxo-

[0410] 3-piperidyl)isoindoline- 1, 3-dione (WJ213- 14)

[0411] 1H NMR (400 MHz, DMSO-d6) 5 = 11.09 (s, 1 H), 9.10 (s, 1 H), 7.50 (m, 5H), 7.17 - 6.91 (m, 2H), 6.51 (m, 1 H), 5.04 (m, 1 H), 3.75 (m, 12H), 3.64 (m, 12H), 3.29 (m, 2H), 2.97 - 2.78 (m, 2H), 2.71 - 2.59 (m, 3H), 2.38 - 2.22 (m, 6H), 2.08 - 1.97 (m, 1 H), 1.57 (m, 2H), 1.49 - 1.26 (m, 6H).

[0412] ESI-HRMS (m / z): [M]+H+calcd. for C43H55F2N14O7: 917.4344; found: 917.4341

[0413] Synthesis of linker 2h

[0414] To a mixture of 2-(6-bromohexyl)isoindoline-1 , 3-dione (8, 1 g, 3.22 mmol, 1 eq), Nal (500 mg, 3.34 mmol, 1.03 eq) , TEA (650 mg, 6.42 mmol, 1 ml, 1.99 eq) in THF (10 ml) was added tert- butyl piperazine- 1 -carboxylate (9, 600 mg, 3.22 mmol, 1 eq), and then the mixture was stirred at 70 °C for 12 h. LC-MS indicated the mass of desired product was detected. TLC (Petroluem / EtOAc=1 :2) indicated a major spot observed. The reaction mixture was diluted with water (80 ml), extracted with EtOAc (3x 60 ml). The organic layer was washed with brine (2x 60 ml), dried over Na2SC>4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-20% Ethyl acetate / Petroleum ethergradient @ 30 ml min-1) to give tert-butyl 4-[6- (1 ,3-dioxoisoindolin-2-yl)hexyl]piperazine-1-carboxylate (10, 960 mg, 2.31 mmol, 72 % yield) as a white solid.

[0415] UHPLC-ESIMS (m / z): 416.2 [M+H]+

[0416] 1H NMR (400 MHz, DMSO-d6) δ = 7.90 - 7.80 (m, 4H), 3.55 (t, J = 7.1 Hz, 2H), 3.26 (d, J = 4.1 Hz, 4H), 2.27 - 2.21 (m, 6H), 1.59 (m, 2H), 1.43 - 1.34 (m, 11 H), 1.32 - 1.24 (m, 4H). tert-butyl 4-(6-aminohexyl) piperazine-1-carboxylate (2h)

[0417] To a mixture of tert-butyl 4-[6-(1 , 3-dioxoisoindolin-2-yl) hexyl]piperazine-1 -carboxylate (900 mg, 2.17 mmol) in EtOH (10 ml) was added (600 mg, 11.9 mmol), then the mixture was stirred at 90 °C for 1 h. The mixture was cooled to 20 °C and filtered. The filtrate was concentrated to give the crude product. The crude product was triturated with DCM (30 ml), filtered to give filtrate. The filtrate was concentrated to give tert-butyl 4-(6-aminohexyl) piperazine-1 -carboxylate (2h, 400 mg, 1.40 mmol, 64.7 % yield) as yellow oil.

[0418] 1H NMR (400 MHz, CHCI3-Cd) δ = 3.48-3.34 (m, 4 H), 2.68 (t, J=7.2 Hz, 2 H), 2.43-2.27 (m, 6 H), 1.56-1.39 (m, 17 H). Synthesis of WJ214-14

[0419] 2-(2,6-dioxo-3-piperidyl)-4-(10-hydroxydec-1-ynyl)isoindoline-1, 3-dione (13)

[0420] To a mixture of 4-bromo-2-(2,6-dioxo-3-piperidyl)isoindoline-1 , 3-dione (11 , 2 g, 5.9 mmol, 1 eq), dec-9-yn-1-ol (12, 1.00 g, 6.48 mmol, 1.1 eq), TEA (7.27 g, 71.9 mmol, 10 ml, 12 eq) in DMF (10 ml) was added Pd(PPh3)2CI2(400 mg, 569 μmol, 0.096 eq) and Cui (120 mg, 630 μmol, 0.1 eq), then the mixture was stirred at 80 °C for 12 h under N2 atmosphere. LC-MS indicated desired MS was detected. TLC (Petroluem / EtOAc=1 :2) indicated a major spot observed. The mixture was poured into water (60 ml) and extracted with EtOAc (3x 60 ml). The organic layer was washed with NH4CI (2x 60 ml), brine (3x 60 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-20% Ethyl acetate / Petroleum ethergradient @ 30 ml min-1) to give 2-(2,6-dioxo-3-piperidyl)- 4-(10-hydroxydec-1-ynyl)isoindoline-1 , 3-dione (13, 2.3 g, 5.60 mmol, 94.5 % yield) as a white solid.

[0421] 1H NMR (400 MHz, DMSO-d6) δ = 11.14 (s, 1 H), 7.92 - 7.77 (m, 3H), 5.15 (dd, J = 5.3, 12.7 Hz, 1 H), 4.33 (t, J = 5.1 Hz, 1 H), 3.40 - 3.36 (m, 3H), 3.00 - 2.81 (m, 1 H), 2.57 (br d, J = 11.9 Hz, 2H), 2.12 - 2.00 (m, 2H), 1.66 - 1.55 (m, 2H), 1.51 - 1.37 (m, 4H), 1.29 (br s, 6H).

[0422] 10-[2-(2, 6-dioxo-3-piperidyl)- 1, 3-dioxo-isoindolin-4-yl]dec-9-ynyl-4- methylbenzene-sulfonate (14)

[0423] To a mixture of 2-(2,6-dioxo-3-piperidyl)-4-(10-hydroxydec-1-ynyl)isoindoline-1 , 3-dione (13, 1 g, 2.44 mmol, 1 eq), DMAP (30 mg, 246 μmol, 0.1 eq), TEA (1.24 g, 12.2 mmol, 1.7 ml, 5 eq) in DCM (10 ml) was added 4-methylbenzenesulfonyl chloride (650 mg, 3.41 mmol, 1.4 eq), then the mixture was stirred at 0-20 °C for 12 h. TLC (Petroluem / EtOAc=1 :1) indicated the starting material was consumed comμletely and a major spot observed. The mixture was poured into water (60 ml) and extracted with EtOAc (3x 60 ml). The organic layer was washed with NH4CI (3x 60 ml), brine (3x 60 ml). The organic layer was dried over Na2SO4, filtered and concentrated to give 10-[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-4-yl]dec-9-ynyl 4- methylbenzenesulfonate (14, 1.1 g, crude) as a colorless oil.

[0424] 4-(10-azidodec-1-ynyl) -2-(2, 6-dioxo-3-piperidyl) isoindoline-1, 3-dione (15)

[0425] To a solution of 10-[2-(2, 6-dioxo-3-piperidyl) -1 , 3-dioxo-isoindolin-4-yl]dec-9-ynyl 4- methylbenzenesulfonate (14, 1.1 g, 1.95 mmol) in DMF (20 ml) was added Na N3(210 mg, 3.23 mmol), then the mixture was stirred at 60 °C for 2 h. TLC (Petroluem / EtOAc=5:1) indicated a major spot observed. The mixture was poured into sat. NaHCO3(60 ml) and extracted with EtOAc (3x 60 ml). The organic layers were washed with brine (3x 100 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 - 30 % Ethyl acetate / Petroleum ethergradient @ 30 ml min-1) to give 4-(10-azidodec-1-ynyl) -2- (2, 6-dioxo-3-piperidyl) isoindoline-1 , 3-dione (15, 500 mg, 1.15 mmol, 58.9 % yield) as a white solid.

[0426] 4-(10-aminodec-1-ynyl) -2-(2, 6-dioxo-3-piperidyl) isoindoline-1, 3-dione (16)

[0427] To a solution of 4-(10-azidodec-1-ynyl) -2-(2, 6-dioxo-3-piperidyl) isoindoline-1 , 3-dione (15, 500 mg, 1.15 mmol, 1 eq) in THF (5 ml) and H2O (2 ml) was added PPh3(364 mg, 1.39 mmol, 1.2 eq). The mixture was stirred at 60 °C for 2 h. TLC (Petroluem / EtOAc=2:1) indicated a major spot observed. The mixture was poured into NaHCO3(60 ml) and extracted with EtOAc (3x 60 ml). The organic layer was washed with brine (3x 60 ml). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 - 20 % Ethyl acetate / Petroleum ethergradient @ 40 ml min-1) to give 4-(10-aminodec-1-ynyl) - 2-(2, 6-dioxo-3-piperidyl) isoindoline-1 , 3-dione (16, 560 mg, 172 μmol, 15 % yield, 12.6 % purity) as a white solid.

[0428] UHPLC-ESIMS (m / z): 410.3 [M+H]+

[0429] 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin-2- yl]piperazin-1-yl]-N-[10-[2-(2, 6-dioxo-3-piperidyl) -1, 3-dioxo-isoindolin-4-yl]dec-9- ynyl]-4-oxo-butanamide (WJ214- 14)

[0430] To a solution of 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6-morpholino-1 , 3, 5-triazin- 2-yl]piperazin-1-yl]-4-oxo-butanoic acid (200 mg, 405.30 μmol, 1.11 eq), HATU (190.00 mg, 499.70 μmol, 1.36 eq), DIEA (148.40 mg, 1.15 mmol, 200.00 pL, 3.13 eq) in DMF (5 mL) was added 4-(10-aminodec-1-ynyl) -2-(2, 6-dioxo-3-piperidyl) isoindoline-1 , 3-dione (300 mg, 366.32 μmol, 50% purity, 1 eq). The mixture was stirred at 20 °C for 2 hr. LC-MS (EW35368- 137-P1A) indicated the Ms of desired product was detected. The mixture was poured into water (60 mL) and extracted with EtOAc (60 mL*3). The organic layer was washed with NaHCOs (60 mL*2), NH4CI (60 mL*2), brine (60 mL*3), dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by prep- HPLC (column: Phenomenex Luna C18 150*25mm*10um;mobile phase: [water (FA) -ACN];B%: 39%-69%, 10min). After Prep- Fl PLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give 4-[4-[4-[2-amino-4-(difluoromethyl) pyrimidin-5-yl]-6- morpholino-1 , 3, 5-triazin-2-yl]piperazin-1 -yl]-N-[10-[2-(2, 6-dioxo-3-piperidyl) -1 , 3-dioxo- isoindolin-4-yl]dec-9-ynyl]-4-oxo-butanamide (WJ214-14, 40 mg, 44.87 μmol, 12.25% yield, 99.26% purity) was obtained as a white solid

[0431] 1H NMR (400 MHz, DMSO) δ(ppm): 11.31 (s, 1 H), 9.11 (s, 1 H), 7.87 - 7.47 (m, 7H), 5.13 (dd, J = 5.4, 12.7 Hz, 1 H), 3.91 - 3.47 (m, 16H), 3.01 (m, 2H), 2.94 - 2.82 (m, 1 H), 2.65 - 2.54 (m, 6H), 2.36 - 2.30 (m, 2H), 2.10 - 2.02 (m, 1 H), 1.63 - 1.52 (m, 2H), 1.47 (br d, J = 5.1 Hz, 2H), 1.41 - 1.34 (m, 2H), 1.32 - 1.23 (m, 6H).

[0432] ESI-HRMS (m / z): [M]+H+calcd. for C43H51F2N12O7: 885.3952; found: 885.3966

[0433] Synthesis of WJ111-11 2-(6-Bromohexyl)isoindoline- 1,3-dione (26)

[0434] A round-bottom flask was charged with phthalimide (25, 2.65 g, 18 mmol, 1.0 eq.), 1 ,6- dibromohexane (24, 8.3 mL, 54 mmol, 3.0 eq.), potassium carbonate (9.95 g, 72 mmol, 4.0 eq.) and acetonitrile (60 mL). The mixture was then heated to reflux overnight. Precipitated solids were filtered off and the filtrate was concentrated in vacuo. The crude was purified by column chromatography (SIO2, cyclohexane+0.1 % TFA / EtOAc+O.1% TFA) to yield 2-(6- bromohexyl)isoindoline-1 , 3-dione (26, 4.75 g, 15.3 mmol, 85%) as a colourless oil.

[0435] 1H-NMR (400 MHz, 298 K, CD CI3, d / ppm): 7.86-7.82 (m, 2H), 7.73-7.69 (m, 2H), 3.70-3.67 (m, 2H), 3.41-3.37 (m, 2H), 1.89-1.82 (m, 2H), 1.73-1.65 (m, 2H), 1.52-1.45 (m, 2H), 1.40-1.33 (m, 2H).

[0436] 2-(6-Azidohexyl)isoindoline- 1,3-dione (27)

[0437] Sodium azide (2.30 g, 35.3 mmol, 4.0 eq.) was added to a solution of 2-(6- bromohexyl)isoindoline-1 , 3-dione (26, 2.74 g, 8.83 mmol, 1.0 eq.) in DMF (30 mL). The reaction was heated to 70 °C under reflux until no starting material was detected by TLC. The heating was removed and the reaction was quenched by the addition of water (30 mL). The aqueous layer was then extracted with DCM (3 x 30 mL) and the combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo to yield 2-(6- azidohexyl)isoindoline-1 , 3-dione (27, 2.35 g, 8.63 mmol, 98%) as a pale yellow oil. The product was used without further purification.

[0438] 1H-NMR (400 MHz, 298 K, CD CI3, d / ppm): 7.85-7.83 (m, 2H), 7.72-7.70 (m, 2H), 3.70-3.66 (m, 2H), 3.27-3.23 (m, 2H), 1.72-1.65 (m, 2H), 1.63-1.55 (m, 2H), 1.45-1.32 (m, 4H).

[0439] 6-Azidohexan-1 -amine (28)

[0440] 2-(6-Azidohexyl)isoindoline-1 , 3-dione (27, 1.28 g, 4.7 mmol, 1.0 eq.) and hydrazine monohydrate (1.4 mL, 28.2 mmol, 6.0 eq,) were dissolved in EtOH (15 mL) and stirred under reflux for 3h. The reaction mixture was then filtered and concentrated in vacuo. The residue was redissolved in EtOAc and left at room temperature overnight whereupon a solid precipitated. The solid was filtered off and the filtrate was concentrated in vacuo to yield 6- azidohexan-1 -amine (28, 611 mg, 4.30 mmol, 91%) as a yellow oil. The product was used without further purification.

[0441] 1H-NMR (400 MHz, 298 K, CD CI3, d / ppm): 3.28-3.24 (m, 2H), 2.72-2.68 (m, 2H), 1 .97-1.96 (m, 2H), 1.64-1.57 (m, 2H), 1.48-1.43 (m, 2H), 1.40-1.34 (m, 4H). 4-((6-Azidohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (29)

[0442] DI PEA (0.8 mL, 4.64 mmol, 5.5 eq.) was added to a solution of 2-(2,6-dioxopiperidin-3-yl)-4- fluoroisoindoline-1 , 3-dione (1 , 233 mg, 0.84 mmol, 1.0 eq.) and 6-azidohexan-1 -amine (28, 300 mg, 2.11 mmol, 2.5 eq.) in dioxane (3.0 mL). The reaction was then stirred at 100 °C overnight. The solvent was removed in vacuo and the crude was purified by column chromatography (SiC>2, cyclohexane+0.1% TFA / EtOAc+O.1 % TFA) to yield 4-((6- azidohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (29, 57.4 mg, 0.14 mmol, 17%) as a green solid.

[0443] 1H-NMR (400 MHz, 298 K, CDCI3, d / ppm): 7.93 (s, 1 H), 7.52-7.48 (m, 1 H), 7.11-7.09 (m, 1 H), 6.89-6.87 (m, 1 H), 6.23 (s, 1 H), 4.94-4.89 (m, 1 H), 3.30-3.27 (m, 3H), 2.92-2.72 (m, 4H), 2.16- 2.11 (m, 1 H), 1.71-1.61 (m, 4H), 1.46-1.44 (m, 4H).

[0444] 4-((6-Aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione (30)

[0445] 4-((6-Azidohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (29, 57.4 mg,

[0446] 144 μmol, 1.0 eq.) was dissolved in methanol (2.0 mL) and treated with Pd / C (10 wt % on activated carbon, 5.80 mg). The mixture was stirred under a hydrogen atmosphere for 3 h and subsequently filtered over celite. The filtrate was concentrated in vacuo to yield 4-((6- aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (30, 9.40 mg, 25.0 μmol, 18%) as a yellow solid. The product was used without further purification.

[0447] 1H-NMR (400 MHz, 298 K, DMSO-d6, d / ppm): 7.61-7.53 (m, 1 H), 7.11-6.98 (m, 1 H), 6.55-6.50 (m, 1 H), 5.07-5.03 (m, 1 H), 3.31 (s, 4H), 2.78-2.61 (m, 3H), 2.34-2.26 (m, 2H), 2.04-2.01 (m, 1 H), 1.57-1.52 (m, 2H), 1.40-1.14 (m, 4H).

[0448] 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2- yl) piperazin- 1-yl)-N-( 6-( (2-(2, 6-dioxopiperidin-3-yl)- 1, 3-dioxoisoindolin-4- yl)amino)hexyl)-4-oxobutanamide (WJ111-11)

[0449] To a solution of 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6-morpholino-1 ,3,5-triazin-2- yl]piperazin-1-yl]-4-oxo-butanoic acid (13.3 mg, 26.9 μmol, 1.0 eq.) and DIEA (9.19 pL, 53.7 μmol, 2.0 eq.) in DMF (1.0 mL) was added TBTU (12.9 mg, 40.3 μmol, 1.50 eq.). The solution was stirred at rt. for 30 min. 4-((6-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1 , 3-dione (30, 10.0 mg, 26.9 μmol, 1.0 eq) was added to the reaction mixture and stirred for 24h. LC-MS showed full consumption of the starting material and the desired MS was detected. The solvent was removed in vacuo and the precipitate was dissolved in water / MeCN (1 :1). The crude material was purified by Prep-HPLC [water(FA)-ACN];B%: 0 - 99%). The eluent solution was lyophilized to give final compounds 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-1 ,3,5-triazin-2-yl)piperazin-1-yl)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1 ,3- dioxoisoindolin-4-yl)amino)hexyl)-4-oxobutanamide (WJ111-11 , 2.30 mg, 10.1% yield) as yellow solid.

[0450] 1H {19F} NMR (cryo-600 MHz, DMSO-d6) δ ppm: 11.11 (s, 1 H), 9.11 (s, 1 H), 7.80 (t, J = 5.7 Hz, 1 H), 7.67 - 7.55 (m, 3H), 7.09 (d, J = 8.6 Hz, 1 H), 7.02 (d, J = 7.0 Hz, 1 H), 6.55 (br s, 1 H), 5.07 - 5.04 (m, 1 H), 3.79 - 3.71 (m, 8H), 3.67 - 3.63 (m, 4H), 3.54 - 3.52 (m, 4H), 3.29 (t, J = 6.3 Hz, 2H), 3.02 (q, J = 6.5 Hz, 2H), 2.90 - 2.84 (m, 1 H), 2.63 - 2.55 (m, 4H), 2.33 (t, J = 7.0 Hz, 2H), 2.03 - 2.01 (m, 1 H), 1.57 - 1.54 (m, 2H), 1.40 - 1.38 (m, 2H), 1.34 - 1.29 (m, 8H).

[0451] ESI-HRMS (m / z): [M]+H+calcd. for C39H47F2Ni3NaO7: 870.3581 ; found: 870.3582.

[0452] Synthesis of PQR514 (P)

[0453] 4,4 ' -(6-chloro-1,3,5-triazine-2,4-diyl)dimorpholine

[0454] To a solution of morpholine (b, 4.72 g, 54.2 mmol, 4.77 ml, 2 eq ) in DCM (30 ml) was added 2,4,6-trichloro-1 ,3,5-tri- azine (a, 5 g, 27.1 mmol, 1 eq ) at 0 °C, then a solution of NaHCO3 (4.56 g, 54.2 mmol, 2 eq ) in water (5 ml) was added into the mixture at 0 °C. Then the resulting mixture was stirred at 25 °C for 3 h. The reaction mixture was partitioned between DCM (40 ml) and water (20 ml), the aqueous layer was extracted with DCM (2x20 ml). The combined or- ganic layers were washed with brine (2x20 ml), dried over anhydrous Na2SO4, filtered and evaporated to give the title compound (c, 7 g, crude) as white solid which was used in next step without purification.

[0455] MS: [M+H]+ 286.2 / 288.2 m / z. tert-butyl(tert-butoxycarbonyl) (4-(difluoromethyl) -5-(4, 6-dimorpholino- 1, 3, 5-triazin- 2-yl) pyrimidin-2-yl) carbamate

[0456] To a solution of tert-butyl (tert-butoxycarbonyl)(4-(1-fluoroethyl)-5-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)pyrim- idin-2-yl)carbamate (b, 1.1 g, 2.33 mmol, 1 eq ) and 4,4'-(6-chloro- 1 ,3,5-triazine-2,4-diyl)dimorpholine (a, 730 mg, 2.55 mmol, 1.1 eq ) in dioxane (10 ml) / THF (1 ml) was added Pd(OAc)2 (20 mg, 89 μmol, 0.038 eq ), K2CO3 (970 mg, 7.02 mmol, 3 eq ) and PPh3 (80 mg, 305 μmol, 0.13 eq ) in turns, then the mixture was stirred at 60 °C for 12 h under N2. The reaction mixture was filtered and the filtrate was concentrated to give crude title compound (c, 1 .4 g, crude) as black oil which was used in next step directly.

[0457] MS: [M+H]+ 595.4 m / z.

[0458] 4-(difluoromethyl)-5-(4,6-dimorpholino- 1,3,5-triazin-2-yl)pyrimidin-2-amine

[0459] To a solution of tert-butyl (tert-butoxycarbonyl)(4-(difluoromethyl)-5-(4,6-dimorpholino-1 ,3,5- triazin-2-yl)pyrimidin- 2-yl)carbamate (a, 1.4 g, 2.35 mmol, 1 eq ) in dioxane (5 ml) was added HCI / dioxane (2 mol dm-3, 8 ml), then the mixture was stirred at 60 °C for 2 h. The reaction mixture was adjusted to pH 7-8 with NaHCO3 (aq.), then the resulting mixture was partitioned between EtOAc (40 ml) and water (20 ml). The aqueous layer was extracted with EtOAc (2x20 ml), the combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material. The crude material was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, Eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 20 ml min-1) to afford the crude title compound. Then the crude com- pound was triturated with DMSO at 25 °C for 30 min to give the title compound (b, PQR514, 47.6 mg, 121 μmol, 5.1 % yield, 100 % purity) as white solid.

[0460] MS: [M+H]+ 395.3 m / z.

[0461] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 9.24 (s, 1 H), 7.75, 7.62, 7.49 (each s, 1 H), 5.56 (s, 2 H), 3.86 (br s,8 H), 3.79 - 3.73 (m, 8 H). tert-butyl-4-( ( 1-(2-(2, 6-dioxopiperidin-3-yl)-6-fluoro- 1, 3-dioxoisoindolin-5- yl)piperidin-4-yl)methyl) piperazine- 1- carboxylate

[0462] To a solution of 2-(2,6-dioxo-3-piperidyl)-5,6-difluoro-isoindoline-1 , 3-dione (a, 50 mg, 170 μmol, 1 eq ) in DMSO (1 ml) was added DIEA (65 mg, 503 μmol, 88 μl, 3 eq ) and tert-butyl 4- (4-piperidylmethyl) piperazine- 1 -carboxylate (b, 50 mg, 176 μmol, 1 eq ), then the mixture was stirred at 140 °C for 1 h. The mixture was poured into water (10 ml) and extracted with EtOAc (3x10 ml). The organic layer was washed with brine (3x10 ml). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep- TLC (Petroluem / EtOAc=1 :2) to give the title compound (c, 90 mg, 161.40 μmol, 95 % yield) as white solid.

[0463] MS: [M+H]+ 558.2 m / z.

[0464] 2- (2, 6-dioxopiperidin-3-yl)-5-fluoro-6-(4-(piperazin- 1-ylmethyl)piperidin- 1- yl)isoindoline- 1, 3-dione

[0465] A solution of tert-butyl 4-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1 ,3-dioxoisoindolin-5- yl)piperidin-4-yl)methyl)piper- azine-1 -carboxylate (a, 90 mg, 161 μmol, 1 eq ) in DCM (1 ml) was added TFA (307 mg, 2.69 mmol, 0.2 ml, 17 eq ), then the mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated to give the title compound (b, 70 mg, TFA salt) as white solids.

[0466] MS: [M+H]+ 458.2 m / z. Synthesis of WJ254

[0467] 5-(4-((4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl) methyl) pi peridin- 1-yl)-2- (2, 6- dioxopiperidin-3-yl)-6-fluoroisoindoline- 1, 3-dione

[0468] To a solution of 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6-morpholino-1 ,3,5-triazin-2- yl]piperazin-1-yl]- 4-oxo-butanoic acid (b, 80 mg, 162 μmol, 1.3 eq ), HATU (70 mg, 184 μmol, 1.5 eq ) and DIEA (50 mg, 387 μmol, 67 μl, 3.2 eq ) in DMF (2 ml) was added 2-(2,6- dioxopiperidin-3-yl)-5-fluoro-6-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindo- line-1 , 3-dione (a, 70 mg, 122 μmol, 1 eq , TFA salt), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was purified by prep-HPLC (column: Waters Xbridge C18 150x50 mmx10 μm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 28 %-58 % B over 10 min). After Prep- HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ254, 65.5 mg, 68.2 μmol, 56 % yield, 97 % purity) as yellow solid.

[0469] MS: [M+H]+ 933.4 m / z.

[0470] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.22 - 10.92 (m, 1 H), 9.11 (s, 1 H), 7.77, 7.63, 7.50 (each s, 1 H), 7.70 (d, J = 11.5 Hz, 1 H), 7.63 - 7.51 (m, 2 H), 7.43 (d, J = 7.6 Hz, 1 H), 5.10 (dd, J = 5.3 Hz, J = 12.9 Hz, 1 H), 3.88 - 3.72 (m,8 H), 3.69 - 3.39 (m, 15 H), 2.88 (br t, J = 12.3 Hz, 3 H), 2.63 - 2.55 (m, 5 H), 2.37 (br s, 2 H), 2.29 (br s, 2 H), 2.20 (br dj = 6.6 Hz, 2 H), 2.07 - 2.00 (m, 1 H), 1.87 - 1 .80 (m, 2 H), 1 .78 - 1.70 (m, 1 H), 1.32 - 1 .22 (m, 2 H).

[0471] 2-(2, 6-dioxopiperidin-3-yl)-4-( (3-hydroxypropyl)amino)isoindoline- 1, 3-dione

[0472] To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1 , 3-dione (a, 500 mg, 1.81 mmol), DIEA (700 mg, 5.42 mmol, 943 μl) in DMSO (5 ml) was added 3-aminopropan-1-ol (b, 170 mg, 2.26 mmol, 175 μl), then the mixture was stirred at 130 °C for 12 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (3x30 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the crude prod- uct which was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 600 mg, crude) as a yellow solid.

[0473] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.09 (s, 1 H), 7.61 - 7.56 (m, 1 H), 7.10 (d, / = 8.8 Hz, 1 H), 7.02 (d, J = 7.2 Hz, 1 H), 6.70 (t, J = 5.6 Hz, 1 H), 5.08 - 5.01 (m, 1 H), 4.66 - 4.63 (m, 1 H), 3.52 - 3.50 (m, 2 H), 3.37 - 3.35 (m, 2 H), 2.94 -2.82 (m, 1 H), 2.62 - 2.55 (m, 2 H), 2.06 - 2.00 (m, 1 H), 1.73 - 1.71 (m, 2 H).

[0474] 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanal

[0475] To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-((3-hydroxypropyl)amino)isoindoline-1 , 3-dione (a, 300 mg, 905 μmol) in DCM (1 ml) was added Dess-Martin (420 mg, 990 μmol, 307 μl), then the mixture was stirred at 20 °C for 12 h. The mixture was concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phe- nomenex luna C18 150x25 mmx 10 pm; mobile phase: [water (FA)-ACN]; gradient: 19 %-49 % B over 10 min). After Prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (b, 150 mg, 456 μmol, 50 % yield) as yellow solid.

[0476] MS: [M+H]+ 330.1 m / z. tert-butyl (2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-

[0477] 1,3,5-triazin-2-yl)piperazin-1- yl)-4-oxobutanoyl)piperazin-1-yl)ethyl)carbamate To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (a, 100 mg, 203 μmol), DIEA (100 mg, 774 μmol, 135 μl) in DMF (2 ml) was added HATU (100 mg, 263 μmol) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h. tert-butyl N-(2-piperazin-1-ylethyl)carbamate (b, 60 mg, 262 μmol) was added and the mixture was stirred at 20 °C for another 12 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (3x30 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the title compound (c, 120 mg, crude) as yellow solid.

[0478] MS: [M+H]+ 705.3 m / z.

[0479] 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin-2- yl)piperazin-1-yl)-4-(4-(2- aminoethyl) piperazin-1 -yl)butane-1, 4-dione

[0480] To a solution of tert-butyl (2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-1 ,3,5-triazin-2-yl)- piperazin-1-yl)-4-oxobutanoyl)piperazin-1-yl)ethyl)carbamate (a, 100 mg, 142 μmol) in DCM (1 ml) was added TFA (0.2 ml, 2.69 mmol), then the mixture was stirred at 20 °C for 12 h. The mixture was concentrated to give the title compound (b, 100 mg, TFA salt) as yellow solid.

[0481] MS: [M+H]+ 605.4 m / z.

[0482] Synthesis of WJ255

[0483] 4-((3-((2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-

[0484] 1, 3, 5-triazin-2-yl) piperazin-1 -yl)-4-oxobutanoyl)piperazin-1-yl)ethyl)amino)propyl)- amino)-2-(2, 6-dioxopiperidin-3-yl)isoindoline- 1, 3-dione

[0485] To a mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin-

[0486] 2-yl)piperazin-1-yl)- 4-(4-(2-aminoethyl)piperazin-1-yl)butane-1 , 4-dione (a, 50 mg, 83 μmol),

[0487] 3-((2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoin- dolin-4-yl)amino)propanal (b, 30 mg, 91 μmol) in MeOH (1 ml) was added NaBH(OAc)3 (17.5 mg, 83 μmol), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the crude product. The mixture was pu- rified by prep-HPLC (column: Phenomenex luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 13 %-43 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the crude product. The mixture was purified by prep-HPLC (column: Phenomenex luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 14 %-34 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ255, 6.2 mg, 6.75 μmol, 8.2 % yield, 100 % purity) as yellow solid.

[0488] MS: [M+H]+ 918.3 m / z. 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11 .07 (s, 1 H), 9.11 (s, 1 H), 8.28 (s, 1 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H), 7.62- 7.52 (m, 3 H), 7.50 (s, 0.25 H), 7.11 (d, J = 8.8 Hz, 1 H), 7.02 (d, J = 6.8 Hz, 1 H), 6.85 (t, J = 4.8 Hz, 1 H), 5.07 - 5.01 (m,1 H), 3.89 - 3.71 (m, 10 H), 3.67 - 3.50 (m, 12 H), 2.92 - 2.84 (m, 1 H), 2.75 - 2.56 (m, 9 H), 2.40 - 2.28 (m, 7 H), 2.05 - 1.99(m, 1 H), 1.76 - 1.70 (m, 2 H). tert-butyl 4-(6-( 1 ,3-dioxoisoindolin-2-yl) hexyl) piperazine- 1 -carboxylate

[0489] A mixture of tert-butyl piperazine- 1 -carboxylate (a, 5 g, 26.9 mmol), 2-(6- bromohexyl)isoindoline-1 , 3-dione (b, 9 g, 29.0 mmol), Nal (4.02 g, 26.9 mmol), TEA (12 ml, 86.2 mmol) in THF (50 ml) was stirred at 70 °C for 12 h. The mixture was concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 %-40 % EtOAc / Petroleum ether gradient at 80 ml min-1) to give the title compound (c, 10 g, 24.1 mmol, 90 % yield) as a white solid. tert-butyl 4-(6-aminohexyl) piperazine- 1 -carboxylate

[0490] To a mixture of tert-butyl 4-[6-(1 ,3-dioxoisoindolin-2-yl)hexyl]piperazine-1-carboxylate (a, 10 g, 24.1 mmol) in EtOH (100 ml) was added NH2NH2 ■ H2O (7.53 g, 120 mmol, 7.31 ml, 80 % purity), then the mixture was stirred at 90 °C for 12 h. The mixture was cooled to 20 °C and filtered, the filtrate was concentrated to give the crude product. The crude product was triturated with DCM (30 ml) to give the title compound (b, 6 g, 21.0 mmol, 87 % yield) as yellow oil.

[0491] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 3.40 - 3.32 (t, J = 7.2 Hz, 4 H), 2.61 (t, J = 6.8 Hz, 2 H), 2.33 - 2.23(m, 6 H), 1.47 - 1.35 (m, 13 H), 1.31 - 1.21 (m, 4 H). tert-butyl 4-(6-((2-(2, 6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-5- yl)amino)hexyl)piperazine-1-carboxylate

[0492] A mixture of 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1 , 3-dione (a, 200 mg, 724 μmol, 1 eq ), tert-butyl 4-(6-amino- hexyl)piperazine-1 -carboxylate (b, 206 mg, 724 μmol, 1 eq ), KF (200 mg, 3.44 mmol, 270 μl, 4.75 eq ) in DMSO (2 ml) was stirred at 120 °C for 1 h. The mixture was diluted with EtOAc (50 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep-HPLC (column: Waters Xbridge 150x25 mmx5 pm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 38 %-58 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, 100 mg, 185 μmol, 26 % yield) as a white solid.

[0493] MS: [M+H]+ 542.3 m / z.

[0494] 2-(2,6-dioxopiperidin-3-yl)-5-((6-(piperazin-1-yl)hexyl)amino)isoindoline-1, 3-dione

[0495] To a solution of tert-butyl 4-[6-[[2-(2,6-dioxo-3-piperidyl)-1 ,3-dioxo-isoindolin-5- yl]amino]hexyl]piperazine-1-car- boxylate (a, 100 mg, 185 μmol, 1 eq ) in DCM (1 ml) was added TFA (0.2 ml, 2.69 mmol, 14.6 eq ), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the title compound (b, 80 mg, 181 μmol, 98 % yield) as yellow solid.

[0496] MS: [M+H]+ 442.4 m / z.

[0497] Synthesis of WJ701

[0498] 5-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)hexyl)amino)-2-(2, 6- dioxopiperidin-3-yl)isoindoline- 1, 3-dione

[0499] To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 90 mg, 182 μmol, 1.01 eq ), DIEA (74.2 mg, 574 μmol, 0.1 ml, 3.17 eq ) in DMF (1 ml) was added HATU (90 mg, 237 μmol, 1.31 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h. 2-(2,6-dioxopiperidin-3-yl)-5-((6- (piperazin-1- yl)hexyl)amino)isoindoline-1 , 3-dione (a, 80 mg, 181 μmol, 1 eq ) was added and the resulting mixture was stirred at 20 °C for another 0.5 h. The mixture was diluted with EtOAc (50 ml). The mixture was washed with brine (3x30 ml). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Waters xbridge 150x25 mmx10 pm; mobile phase: [water (NH4HCO3)- ACN]; gradient: 37 %-57 % B over 8 min). After prep-HPLC purification, the eluent was concentrated to remove or- ganic solvents. The residual aqueous solution was lyophilized to give the crude product which was purified by prep- HPLC (column: Phenomenex luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 10 %-40 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ701 , 38.3 mg, 41.8 μmol, 23 % yield, 100 % purity) as a white solid.

[0500] MS: [M+H]+ 917.4 m / z.

[0501] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.05 (s, 1 H), 9.11 (s, 1 H), 8.14 (s, 0.25 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H),7.60 - 7.53 (m, 3 H), 7.50 (s, 0.25 H), 7.10 (t, J = 5.2 Hz, 1 H), 6.94 (d, / = 1.6 Hz, 1 H), 6.84 (dd, J = 2.0 Hz, J = 8.4 Hz,1 H), 5.05 - 4.98 (m, 1 H), 3.86 - 3.71 (m, 8 H), 3.67 - 3.62 (m, 4 H), 3.58 - 3.50 (m, 4 H), 3.45 - 3.40 (m, 4 H), 3.17 - 3.12(m, 2 H), 2.92 - 2.82 (m, 1 H), 2.62 - 2.52 (m, 6 H), 2.36 - 2.32 (m, 2 H), 2.31 - 2.24 (m, 4 H), 2.02 - 1.95 (m, 1 H), 1.62 -1.53 (m, 2 H), 1 .47 - 1.29 (m, 6 H). tert-butyl-4-( 6-( (2-(2, 6-dioxopiperidin-3-yl)-6-fluoro- 1, 3-dioxoisoindolin-5- yl)amino)hexyl)piperazine-1-carboxylate

[0502] A mixture of 2-(2,6-dioxo-3-piperidyl)-5,6-difluoro-isoindoline-1 , 3-dione (a, 50 mg, 170 μmol, 1 eq ), tert-butyl 4-(6- aminohexyl)piperazine-1 -carboxylate (b, 50 mg, 175 μmol, 1.03 eq ), DIEA (70 mg, 539 μmol, 94 μl, 3.17 eq ) in DMSO (2 ml) was stirred at 80 °C for 1 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (3x30 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 20 ml min-1) to give the title compound (c, 60 mg, 107 μmol, 63 % yield) as yellow solid.

[0503] MS: [M+H]+ 560.4 m / z.

[0504] 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-((6-(piperazin-1-yl)hexyl)amino)isoindoline- 1, 3-dione

[0505] To a solution of tert-butyl 4-[6-[[2-(2,6-dioxo-3-piperidyl)-6-fluoro-1 ,3-dioxo-isoindolin-5- yl]amino]hexyl]piperazine- 1 -carboxylate (a, 60 mg, 107 μmol, 1 eq ) in DCM (5 ml) was added TFA (0.5 ml), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 50 mg, TFA salt) as yellow oil.

[0506] MS: [M+H]+ 460.2 m / z.

[0507] Synthesis of WJ702

[0508] 5-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)hexyl)amino)-2-(2, 6- dioxopiperidin-3-yl)-6-fluoroisoindoline- 1, 3-dione To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 40.0 mg, 81.1 μmol, 0.93 eq ), DIEA (40.0 mg, 310 μmol, 54 μl, 3.56 eq ) in DMF (2 ml) was added HATLI (40.0 mg, 105 μmol, 1.21 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h. 2-(2,6-dioxopiperidin-3-yl)-5- fluoro-6-((6- (piperazin-1-yl)hexyl)amino)isoindoline-1 , 3-dione (a, 40.0 mg, 87.1 μmol, 1 eq ) was added and the resulting mixture was stirred at 20 °C for another 12 h. The mixture was diluted in EtOAc (50 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 16 %- 46 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the crude product. The crude product was purified by prep-HPLC (column: Waters xbridge 150x25 mmx10 μm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 38 %-58 % B over 8 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ702, 30.1 mg, 30.8 μmol, 35 % yield, 96 % purity) as yellow solid.

[0509] MS: [M+H]+ 935.3 m / z.

[0510] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.07 (s, 1 H), 9.11 (s, 1 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H), 7.59 - 7.54 (m,2 H), 7.50 (s, 0.25 H), 7.09 (d, J = 7.2 Hz, 1 H), 6.95 - 6.86 (m, 1 H), 5.09 - 5.00 (m, 1 H), 3.87 - 3.70 (m, 8 H), 3.68 - 3.62 (m, 4 H), 3.60 - 3.50 (m, 4 H), 3.47 - 3.38 (m, 4 H), 3.28 - 3.23 (m, 2 H), 2.94 - 2.80 (m, 1 H), 2.61 - 2.54 (m, 4 H), 2.36 - 2.31 (m, 2 H), 2.29 - 2.21 (m, 4 H), 2.06 - 1.96 (m, 1 H), 1.63 - 1.53 (m, 2 H), 1.48 - 1.39 (m, 2 H), 1.39 - 1.27 (m, 4 H). tert-butyl 4-(6-hydroxyhexyl) piperazine- 1 -carboxylate

[0511] To a mixture of tert-butyl piperazine-1 -carboxylate (a, 2.00 g, 10.7 mmol, 0.97 eq ), K2CO3 (3.00 g, 21.7 mmol, 2.0 eq ) in MeCN (40 ml) was added 6-bromohexan-1-ol (b, 2.00 g, 11.1 mmol, 1.45 ml, 1.0 eq ), then the mixture was stirred at 60 °C for 2 h. The mixture was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluent of 0 %-100 % ethyl acetate / petroleum ether gradient at 50 ml min-1) to give the title compound (c, 2.00 g, 7.0 mmol, 63 % yield) as colorless oil.

[0512] 1 H-NMR (400 MHz, CHLOROFORM d): δ [[pm] 3.64 (t, J = 6.6 Hz, 2 H), 3.49 - 3.38 (m, 4 H), 2.45 - 2.27 (m, 6 H), 1.62 - 1 .49 (m, 4 H), 1 .46 (s, 9 H), 1.42 - 1 .30 (m, 4 H). tert-butyl 4-(6-oxohexyl)piperazine- 1 -carboxylate

[0513] To a solution of (COCI)2 (435 mg, 3.43 mmol, 0.30 ml, 2.0 eq ) in DCM (10 ml) was added a solution of DMSO (330 mg, 4.22 mmol, 0.33 ml, 2.4 eq ) in DCM (2 ml) dropwise at -60 °C under N2. Then the mixture was stirred at -60 °C for 10 min. A solution of tert-butyl 4-(6- hydroxyhexyl) piperazine- 1 -carboxylate (a, 500 mg, 1.75 mmol, 1.0 eq ) in DCM (2 ml) was added dropwise and the mixture was stirred at -60 °C for 2 h. NEt3 (1.19 g, 11.8 mmol, 1.64 ml, 6.8 eq ) was added dropwise. Then the mixture was warmed up to 20 °C and stirred at 20 °C for 2 h. The mixture was poured into sat. K2CO3 (20 ml) and extracted with DCM (3x20 ml). The organic layer was dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-10 % DCM / MeOH gradient at 20 ml min-1) to give the title compound (b, 250 mg, 879 μmol, 50 % yield) as colorless oil.

[0514] 1 H-NMR (400 MHz, CHLOROFORM d): δ [ppm] 9.77 (t, J = 1.6 Hz, 1 H), 3.44 (t, J = 4.8 Hz, 4 H), 2.47 - 2.42 (m, 2 H), 2.40 - 2.34 (m, 6 H), 1 .69 - 1.63 (m, 2 H), 1.55 - 1.49 (m, 2 H), 1.46 (s, 9 H), 1.38 - 1.32 (m, 2 H). tert-butyl 4-(6-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)amino)hexyl) piperazine- 1 -carboxylate A mixture of 3-(6-amino-1-oxo-isoindolin-2-yl) piperidine-2, 6-dione (a, 100 mg, 386 μmol, 1.0 eq ), tert-butyl 4-(6- oxohexyl) piperazine- 1 -carboxylate (b, 120 mg, 422 μmol, 1.1 eq ), AcOH (5 mg, 83 μmol, 4.77 μl, 0.22 eq ) in MeOH (1 ml) was stirred at 20 °C for 1 h. NaBH3CN (30 mg, 477 μmol, 1.2 eq ) was added and the mixture was stirred at 20 °C for another 12 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (2x30 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %- 100 % ethyl acetate / petroleum ether gradient at 30 ml min-1) to give the title compound (c, 100 mg, 190 μmol, 49 % yield) as a white solid.

[0515] MS: [M+H]+ 528.3 m / z.

[0516] 3-( 1-oxo-6-((6-(piperazin- 1-yl)hexyl)amino)isoindolin-2-yl) piperidine-2, 6-dione

[0517] To a solution of tert-butyl 4-(6-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5- yl)amino)hexyl)piperazine-1-carboxy- late (a, 100 mg, 190 μmol, 1.0 eq ) in DCM (2 ml) was added CF3COOH (0.5 ml), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the title compound (b, 100 mg, 185 μmol, 97 % yield, CF3COOH salt) as yellow oil.

[0518] MS: [M+H]+ 428.3 m / z.

[0519] Synthesis of WJ703

[0520] 3-(6-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl) piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)hexyl)amino)- 1- oxoisoindolin-2-yl) piperidine-2, 6-dione To a solution of 4-[4-[4-[2-amino-4-(difluoromethyl)pyrimidin-5-yl]-6-morpholino-1 ,3,5-triazin- 2-yl]piperazin-1 -yl]- 4-oxo-butanoic acid (b, 90 mg, 182 μmol, 0.98 eq ), DIEA (74.2 mg, 574 μmol, 0.10 ml, 3.1 eq ) in DMF (2 ml) was added HATLI (90 mg, 237 μmol, 1.3 eq ), then the mixture was stirred at 0 °C for 0.5 h. 3-[1-oxo-6-(6-piperazin-1-ylhexylamino)- isoindolin-2- yl]piperidine-2, 6-dione (a, 80 mg, 187 μmol, 1.0 eq ) was added and the resulting mixture was stirred at 20 °C for another 1 h. The mixture was diluted in EtOAc (50 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-MeCN]; gradient: 10 %-40 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ703, 73.2 mg, 81.1 μmol, 43 % yield, 100 % purity) as a white solid.

[0521] MS: [M+H]+ 903.5 m / z.

[0522] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.96 (s, 1 H), 9.12 (s, 1 H), 8.14 (s, 0.4 H), 7.78 (s, 0.25 H), 7.64 (s, 0.5 H), 7.58 (s, 2 H), 7.51 (s, 0.25 H), 7.26 (d, J = 8.4 Hz, 1 H), 6.88 - 6.82 (m, 1 H), 6.79 (d, J = 2.0 Hz, 1 H), 5.88 (s, 1 H), 5.11 - 5.00 (m, 1 H), 4.31 - 4.23 (m, 1 H), 4.19 - 4.10 (m, 1 H), 3.86 - 3.73 (m, 8 H), 3.68 - 3.63 (m, 4 H), 3.60 - 3.46 (m, 8 H), 3.07 - 3.01 (m, 2 H), 2.94 - 2.86 (m, 1 H), 2.62 - 2.56 (m, 4 H), 2.45 - 2.31 (m, 6 H), 2.03 - 1.95 (m, 1 H), 1 .60 - 1 .53 (m, 2 H), 1.50- 1.30 (m, 6 H). tert-butyl 4-(6-((2-(2, 6-dioxopiperidin-3-yl)-1-oxoisoindolin-4- yl)amino)hexyl)piperazine-1-carboxylate

[0523] A mixture of 3-(4-amino-1-oxo-isoindolin-2-yl)piperidine-2, 6-dione (a, 100.0 mg, 386 μmol, 1.0 eq ), tert-butyl 4-(6- oxohexyl)piperazine-1-carboxylate (b, 120.0 mg, 422 μmol, 1.1 eq ), AcOH (5.00 mg, 83 μmol, 4.77 μl, 0.22 eq ) in MeOH (2 ml) was stirred at 20 °C for 1 h. NaBH3CN (30.0 mg, 477 μmol, 1.2 eq ) was added and the mixture was stirred at 20 °C for another 12 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (2x30 ml). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-100 % ethyl acetate / petroleum ether gradient at 30 ml min-1) to give the title compound (c, 100 mg, 190 μmol, 49 % yield) as a white solid.

[0524] MS: [M+H]+ 528.3 m / z.

[0525] 3-( 1-oxo-4-((6-(piperazin- 1-yl)hexyl)amino)isoindolin-2-yl)piperidine-2, 6-dione

[0526] To a solution of tert-butyl 4-(6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4- yl)amino)hexyl)piperazine-1-carboxy- late (a, 100 mg, 190 μmol, 1.0 eq ) in DCM (5 ml) was added CF3COOH (1.54 g, 13.5 mmol, 1.00 ml, 71 eq ), then the mixture was stirred at 20 °C for 0.5 h. The reaction mixture was concentrated to give the title compound (b, 100 mg, 185 μmol, 97 % yield, CF3COOH) as yellow oil.

[0527] Synthesis of WJ704

[0528] 3-(4-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)hexyl)amino)- 1- oxoisoindolin-2-yl) piperidine-2, 6-dione

[0529] To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 90 mg, 182 μmol, 0.98 eq ), DIEA (72.6 mg, 561 μmol, 97.8 μl, 3.0 eq ) in DMF (2 ml) was added HATU (90 mg, 237 μmol, 1.3 eq ), then the mixture was stirred at 0 °C for 0.5 h. 3-(1-oxo-4-((6-(piperazin-1-yl)hexyl)- amino)isoindolin-2- yl)piperidine-2, 6-dione (a, 80 mg, 187 μmol, 1.0 eq ) was added and the resulting mixture was stirred at 20 °C for another 1 h. The mixture was diluted in EtOAc (50 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give a crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-MeCN]; gradient: 14 %-44 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ704, 40.1 mg, 43.9 μmol, 23 % yield, 99 % purity, formate) as white solid.

[0530] MS: [M+H]+ 903.5 m / z.

[0531] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.01 (s, 1 H), 9.11 (s, 1 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H), 7.57 (s, 2 H), 7.50 (s, 0.25 H), 7.28 (t, J = 7.6 Hz, 1 H), 6.92 (d, J = 8.0 Hz, 1 H), 6.74 (d, J = 8.0 Hz, 1 H), 5.55 (t, J = 5.6 Hz, 1 H), 5.15 - 5.06 (m, 1 H), 4.26 - 4.18 (m, 1 H), 4.17 - 4.08 (m, 1 H), 3.85 - 3.71 (m, 8 H), 3.67 - 3.63 (m, 4 H), 3.58 - 3.51 (m, 4 H), 3.45 - 3.41 (m, 4 H), 3.14 - 3.09 (m, 2 H), 2.97 - 2.89 (m, 1 H), 2.61 - 2.54 (m, 4 H), 2.37 - 2.32 (m, 2 H), 2.31 - 2.25 (m, 4 H), 2.07 - 1.99 (m, 1 H), 1.64 - 1.53 (m, 2 H), 1.47 - 1.29 (m, 6 H). tert-butyl 4-(6-((2-fluoro-4-nitrophenyl)amino)hexyl)piperazine-1 -carboxylate

[0532] A mixture of 1 ,2-difluoro-4-nitro-benzene (a, 400 mg, 2.51 mmol, 278 μl, 1.0 eq ), tert-butyl 4- (6-aminohexyl)piper- azine- 1 -carboxylate (b, 700 mg, 2.45 mmol, 0.98 eq ), K2CO3 (700 mg, 5.06 mmol, 2.0 eq ) in MeCN (5 ml) was stirred at 20 °C for 12 h. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Col- umn, eluent of 0 %- 100 % ethyl acetate / petroleum ether gradient at 30 ml min-1) to give the title compound (c, 900 mg, 2.12 mmol, 84 % yield) as a yellow oil.

[0533] MS: [M+H]+ 425.3 m / z. tert-butyl 4-(6-((tert-butoxycarbonyl)(2-fluoro-4- nitrophenyl)amino)hexyl)piperazine-1 -carboxylate To a mixture of tert-butyl 4-(6-((2-fluoro-4-nitrophenyl)amino)hexyl)piperazine-1 -carboxylate (a, 0.900 g, 2.12 mmol, 1.0 eq ), NEt3 (2.70 g, 26.7 mmol, 3.71 ml, 13 eq ), DMAP (27.0 mg, 221 μmol, 0.10 eq ) in DCM (10 ml) was added Boc2O (720 mg, 3.30 mmol, 758 μl, 1.6 eq ), then the mixture was stirred at 20 °C for 2 h. The mixture was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Col- umn, eluent of 0 %-60 % ethyl acetate / petroleum ether gradient at 30 ml min-1) to give the title compound (b, 800 mg, 1.52 mmol, 72 % yield) as colorless oil.

[0534] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 8.07 - 8.02 (m, 1 H), 8.01 - 7.96 (m, 1 H), 7.43 - 7.35 (m, 1 H), 3.69 - 3.59 (m, 2 H), 3.48 - 3.39 (m, 4 H), 2.44 - 2.25 (m, 6 H), 1.58 - 1.45 (m, 13 H), 1.42 (s, 9 H), 1.32 - 1.28 (m, 4 H). tert-butyl 4-(6-((4-amino-2-fluorophenyl)(tert-butoxycarbonyl)amino) hexyl)piperazine- 1 -carboxylate

[0535] To a solution of Pd / C (0.200 g, 188 μmol, 10 % purity, 0.14 eq ) in MeOH (20 ml) was added tert-butyl 4-(6-((tert-butoxy- carbonyl)(2-fluoro-4-nitrophenyl)amino)hexyl)piperazine-1- carboxylate (a, 700 mg, 1.33 mmol, 1.0 eq ) under N2, then the mixture was stirred at 20 °C under H2 (40 psi) for 12 h. The mixture was filtered and the filtrate was concentrated to give the title compound (b, 500 mg, 1.01 mmol, 76 % yield) as colorless oil.

[0536] MS: [M+H]+ 495.3 m / z. tert-butyl 4-(6-((4-((2,6-bis(benzyloxy)pyridin-3-yl)amino)-2-fluorophenyl)(tert- butoxycarbonyl)amino) hexyl) - piperazine-1 -carboxylate

[0537] A mixture of tert-butyl 4-(6-((4-amino-2-fluorophenyl)(tert- butoxycarbonyl)amino)hexyl) piperazine-1 -carboxylate (a, 200 mg, 404 μmol, 1.0 eq ), 2,6-dibenzyloxy-3-bromo-pyridine (b, 200 mg, 540 μmol, 1.3 eq ), XPhos (20 mg, 40.4 μmol, 0.10 eq ), Pd2(dba)3 (40 mg, 43.7 μmol, 0.11 eq ), Cs2CO3 (400.0 mg, 1 .23 mmol, 3.0 eq ) in dioxane (2 ml) was stirred at 100 °C under N2 for 12 h. The mixture was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %- 100 % ethyl acetate / petroleum ether gradient at 40 ml min-1) to give the title compound (c, 120 mg, 118 μmol, 29 % yield, 77 % purity) as brown solid.

[0538] MS: [M+H]+ 784.4 m / z. tert-butyl4-(6-((tert-butoxycarbonyl)(4-((2,6-dioxopiperidin-3-yl)amino)-2- fluorophenyl)amino) hexyl)- piperazine- 1 -carboxylate

[0539] To a mixture of Pd / C (100 mg, 94.0 μmol, 10 % purity, 0.74 eq ) in MeOH (10 ml) was added tert-butyl 4-(6-((4-(- (2,6-bis(benzyloxy)pyridin-3-yl)amino)-2-fluorophenyl)(tert- butoxycarbonyl)amino) hexyl)piperazine-1 -carboxylate (a, 100 mg, 128 μmol, 1.0 eq ) under N2, then the mixture was stirred at 20 °C under H2 (40 [)) for 12 h. The mixture was filtered and the filtrate was concentrated to give the title compound (b, 80 mg, crude) as yellow solid.

[0540] MS: [M+H]+ 606.5 m / z.

[0541] 3-((3-fluoro-4-((6-(piperazin-1-yl)hexyl)amino)phenyl)amino)piperidine-2, 6-dione

[0542] To a solution of tert-butyl 4-(6-((tert-butoxycarbonyl)(4-((2,6-dioxopiperidin-3-yl)amino)-2- fluorophenyl)amino)hexyl)- piperazine-1 -carboxylate (a, 80 mg, 132 μmol, 1.0 eq ) in DCM (2 ml) was added CF3COOH (307 mg, 2.69 mmol, 0.200 ml, 20 eq ), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the crude title compound (b, 50 mg, crude) as yellow solid. Synthesis of WJ705

[0543] 3-((4-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperazin-1 -yl)-4-oxobutanoyl)piperazin-1-yl)hexyl)amino)-3- fluorophenyl)amino)piperidine-2, 6-dione

[0544] To a mixture of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 60 mg, 122 μmol, 0.99 eq ), DIEA (50 mg, 387 μmol, 67.4 μl, 3.1 eq ) in DMF (1 ml) was added HATLI (60 mg, 158 μmol, 1.3 eq ), then the mixture was stirred at 20 °C for 0.5 h. 3-((3-fluoro-4-((6-(piperazin-1-yl)hexyl)amino)- phenyl)amino)piperidine-2, 6-dione (a, 50 mg, 123 μmol, 1.0 eq ) was added and the resulting mixture was stirred at 20 °C for 1 h. The mixture was diluted in EtOAc (50 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concen- trated to give a crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-MeCN]; gradient: 5 %-35 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ705, 23.1 mg, 26.2 μmol, 21 % yield, 100 % purity) as brown solid.

[0545] MS: [M+H]+ 881.4 m / z.

[0546] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.75 (s, 1 H), 9.11 (s, 1 H), 8.17 (s, 1 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H), 7.58 (m, 2 H), 7.50 (s, 0.25 H), 6.56 - 6.48 (m, 2 H), 6.41 - 6.35 (m, 1 H), 5.35 (d, J = 7.6 Hz, 1 H), 4.20 - 4.10 (m, 1 H), 3.85 - 3.72 (m, 8 H), 3.67 - 3.63 (m, 4 H), 3.58 - 3.51 (m, 4 H), 3.45 - 3.40 (m, 4 H), 2.95 (t, J = 6.8 Hz, 2 H), 2.74 - 2.66 (m, 1 H), 2.58 - 2.55 (m, 3 H), 2.35 - 2.32 (m, 2 H), 2.29 - 2.24 (m, 4 H), 2.12 - 2.06 (m, 1 H), 1.86 - 1 .77 (m, 1 H), 1.55 - 1.48 (m, 2 H), 1.45 - 1.39 (m, 2 H), 1.35 - 1.27 (m, 4 H).

[0547] 2-(methylamino)-3-nitrobenzoic acid

[0548] A mixture of 2-fluoro-3-nitro-benzoic acid (a, 5.00 g, 27.0 mmol, 1.0 eq ), CH3NH2 ■ HCI (b, 9.12 g, 135 mmol, 5.0 eq ), DIEA (34.9 g, 270 mmol, 47.1 ml, 10 eq ) in EtOH (50 ml) was stirred at 80 °C for 2 h. The mixture was concentrated to give the crude product. Water (50 ml) was added and the mixture was adjusted to pH=3-4 with 1 mol 1-1 HCI (aq). Then the mixture was extracted with EtOAc (3x50 ml). The organic layers were dried over Na2SO4, filtered and concentrated to give the title compound (c, 5.00 g, 25.5 mmol, 94 % yield) as yellow solid.

[0549] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 8.12 - 8.06 (m, 1 H), 8.02 - 7.94 (m, 1 H), 6.76 (tj = 8.0 Hz, 1 H), 2.73 (s, 3 H).

[0550] 1 -methyl- 7-nitro- 1, 3-dihydro-2H-benzo[d]imidazol-2-one

[0551] To a solution of 2-(methylamino)-3-nitro-benzoic acid (a, 5.00 g, 25.5 mmol, 1.0 eq ) in tBuOH (50 ml) was added DPPA (7.71 g, 28.0 mmol, 6.05 ml, 1.1 eq ) and DIEA (6.46 g, 50.0 mmol, 8.70 ml, 2.0 eq ), then the mixture was stirred at 90 °C for 12 h. The mixture was concentrated to remove solvent and the residual was poured into water (50 ml), a lot of precipitate was formed. Then the mixture was filtered and the filter cake was dried under vacuum to give the crude product which was triturated with EtOAc (30 ml) to give the title compound (b, 4.50 g, 23.3 mmol, 91 % yield) as yellow solid.

[0552] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.62 (s, 1 H), 7.59 (d, J = 8.4 Hz, 1 H), 7.31 (d, J = 8.0 Hz, 1 H), 7.14 (t, J = 8.0 Hz, 1 H), 3.34 (s, 3 H).

[0553] 3-(3-methyl-4-nitro-2-oxo-2, 3-di hydro- 1 H-benzo[ d]imidazol- 1 -yl) piperidine-2, 6- dione To a mixture of 3-methyl-4-nitro-1 H-benzimidazol-2-one (a, 1.00 g, 5.18 mmol, 1.0 eq ) in THF (30 ml) was added NaH (260 mg, 6.50 mmol, 60 % purity, 1.3 eq ) at 0 °C under N2, then the mixture was stirred at 0 °C for 0.5 h. 3-bromopiperi- dine-2, 6-dione (b, 1.99 g, 10.4 mmol, 2.0 eq ) was added and the resulting mixture was stirred at 80 °C for another 2 h. The mixture was cooled to 20 °C and then poured into sat. NH4CI (30 ml), extracted with EtOAc (3x30 ml). The organic layer was washed with brine (2x30 ml), dried over Na2SO4, filtered and concentrated to give the title compound (c, 500 mg, crude) as yellow solid.

[0554] MS: [M+H]+ 305.0 m / z.

[0555] 3-(4-amino-3-methyl-2-oxo-2, 3-dihydro- 1 H-benzo[d]imidazol- 1-yl)piperidine-2, 6- dione

[0556] To a mixture of Pd / C (200 mg, 188 μmol, 10 % purity, 0.29 eq ) in MeOH (20 ml) and THF (20 ml) was added 3-(3- methyl-4-nitro-2-oxo-benzimidazol-1-yl)piperidine-2, 6-dione (a, 200 mg, 657 μmol, 1 eq ) under N2. Then the mixture was stirred at 20 °C under H2 (15 psi) for 12 h. The mixture was filtered and the filtrate was concentrated to give the title compound (b, 150 mg, 547 μmol, 83 % yield) as yellow solid. tert-butyl 4-(6-((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-4-yl)amino)hexyl)- piperazine-1 -carboxylate

[0557] A mixture of 3-(4-amino-3-methyl-2-oxo-benzimidazol-1-yl)piperidine-2, 6-dione (a, 100 mg, 365 μmol, 1 eq ), tert- butyl 4-(6-oxohexyl)piperazine-1-carboxylate (b, 100 mg, 352 μmol, 0.96 eq ), Ti(0Et)4 (200 mg, 877 μmol, 182 μl, 2.4 eq ) in DMF (2 ml) was stirred at 30 °C for 2 h. Then NaBH3CN (30 mg, 477 μmol, 1.31 eq ) was added and the resulting mixture was stirred at 20 °C for 12 h. The mixture was diluted in EtOAc (30 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give a crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 150 mg, 276 μmol, 76 % yield) as yellow oil. MS: [M+H]+ 543.3 m / z.

[0558] 3-(3-methyl-2-oxo-4-((6-(piperazin-1-yl)hexyl)amino)-2,3-dihydro-1H- benzo[d]imidazol- 1-yl)piperidine-2, 6- dione

[0559] To a solution of tert-butyl 4-(6-((1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1 H- benzo[d]imidazol-4-yl)- amino)hexyl)piperazine-1-carboxylate (a, 120 mg, 221 μmol, 1 eq ) in DCM (5 ml) was added TFA (0.4 ml), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the title compound (b, 100 mg, crude) as yellow oil.

[0560] Synthesis of WJ706

[0561] 3-(4-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5- triazin-2-yl)piperazin-1-yl)-4-oxobutanoyl)piperazin-1-yl)hexyl)amino)-3-methyl-2- oxo-2, 3-dihydro- 1 H-benzo[d]imidazol- 1-yl) pi peridi ne-2, 6-dione

[0562] To a mixture of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 100 mg, 203 μmol, 0.90 eq ), DIEA (100 mg, 774 μmol, 135 μl, 3.4 eq ) in DMF (1 ml) was added HATU (103 mg, 271 μmol, 1.2 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h. 3-(3-methyl-2-oxo-4-((6-(piper- azin-1 - yl)hexyl)amino)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)piperidine-2, 6-dione (a, 100 mg, 226 μmol, 1 eq ) was added and the resulting mixture was stirred at 20 °C for another 1 h. The mixture was diluted in EtOAc (50 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep- HPLC (column: Phenomenex Luna C18 150x25 mmx10 μm; mobile phase: [water (FA)-ACN]; gradient: 11 %- 41 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ706, 26.2 mg, 28.5 μmol, 13 % yield, 100 % purity, formate) as a white solid.

[0563] MS: [M+H]+ 918.4 m / z.

[0564] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.05 (s, 1 H), 9.11 (s, 1 H), 7.77 (s, 0.25 H), 7.64 (s, 0.5 H), 7.57 (s, 2 H), 7.50 (s, 0.25 H), 6.86 (t, J = 8.4 Hz, 1 H), 6.49 (d, J = 8.0 Hz, 1 H), 6.41 (d, J = 8.4 Hz, 1 H), 5.32 - 5.22 (m, 1 H), 5.04 - 4.95 (m, 1 H), 3.85 - 3.74 (m, 8 H), 3.67 - 3.63 (m, 4 H), 3.61 (s, 3 H), 3.58 - 3.51 (m, 4 H), 3.45 - 3.40 (m, 4 H), 3.06 - 3.01 (m, 2 H), 2.92 - 2.82 (m, 1 H), 2.74 - 2.65 (m, 1 H), 2.60 - 2.53 (m, 5 H), 2.36 - 2.33 (m, 2 H), 2.30 - 2.25 (m, 4 H), 2.01 - 1.92 (m, 1 H), 1.65 - 1.58 (m, 2 H), 1.46 - 1.32 (m, 6 H).

[0565] 6-bromo-5-fluoro- 1 -methyl- 1H-indazol-3-amine

[0566] To a solution of 4-bromo-2,5-difluoro-benzonitrile (a, 5 g, 22.9 mmol, 1 eq ) in EtOH (100 ml) was added TEA (9.31 g, 92.0 mmol, 12.8 ml, 4.01 eq ) and methylhydrazine sulfuric acid (b, 9.92 g, 68.8 mmol, 3.00 eq ). The mixture was stirred at 80 °C for 12 h. The reaction mixture was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluent of 35 % EtOAc / Petroleum ether gradient at 100 ml min-1) to give the title compound (c, 2.6 g, 10.7 mmol, 46 % yield) as yellow solid.

[0567] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 7.81 (d, J = 5.6 Hz, 1 H), 7.62 (d, J = 8.9 Hz, 1 H), 5.51 (s, 2 H), 3.71 (s, 3 H).

[0568] 3-((6-bromo-5-fluoro-1-methyl-1H-indazol-3-yl)amino) propanoic acid

[0569] To a solution of 6-bromo-5-fluoro-1-methyl-indazol-3-amine (a, 2.6 g, 10.7 mmol, 1 eq ) in HCI (26 ml) was added TBAB (3.43 g, 10.7 mmol, 1 eq ) and acrylic acid (b, 1.15 g, 16.0 mmol, 1.10 ml, 1.50 eq ), then the mixture was stirred at 100 °C for 12 h. The reaction mixture was adjusted to pH=8 with NaHCO3 (aq.), then the mixture was acidified with acetic acid to pH=5. A lot of precipitate was formed, then the mixture was filtered, the filter cake was washed with water (50 ml) and dried under vacuum to give the title compound (c, 1.1 g, crude) as white solid.

[0570] 1-(6-bromo-5-fluoro- 1 -methyl- 1H-indazol-3-yl)dihydropyrimidine-2,4( 1H,3H)-dione

[0571] To a solution of 3-((6-bromo-5-fluoro-1 -methyl- 1H-indazol-3-yl)amino)propanoic acid (a, 800 mg, 2.53 mmol, 1 eq ) in AcOH (8 ml) was added sodium cyanate (330 mg, 5.08 mmol, 2.01 eq ), the mixture was stirred at 60 °C for 12 h. Then HCI (2 mol dm-3, 8.00 ml, 6.32 eq ) was added and the resulting mixture was stirred at 60 °C for 3 h. The mixture was filtered, the filter cake was washed with water and dried under vacuum to give the title compound (b, 250 mg, crude) as a white solid.

[0572] 1-(6-((diphenylmethylene)amino)-5-fluoro-1-methyl-1H-indazol-3- yl)dihydropyrimidine-2,4( 1H, 3H)-dione

[0573] To a solution of 1-(6-bromo-5-fluoro-1-methyl-indazol-3-yl)hexahydropyrimidine-2, 4-dione (a, 180 mg, 528 μmol, 1 eq ), diphenylmethanimine (b, 180 mg, 993 μmol, 167 μl, 1.88 eq ) in dioxane (5 ml) was added Xantphos Pd G4 (54.0 mg, 56.1 μmol, 0.11 eq ) and tBuONa (150 mg, 1.56 mmol, 2.96 eq ) in turns, then the mixture was stirred at 110 °C for 12 h under N2. The reaction mixture was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Col- umn, eluent of 55 % EtOAc / Petroleum ether gradient at 40 ml min-1) to give the title compound (c, 150 mg, 340 μmol, 64 % yield) as white solid.

[0574] MS: [M+H]+ 442.1 m / z. 1-(6-amino-5-fluoro- 1 -methyl- 1H-indazol-3-yl)dihydropyrimidine-2,4( 1H,3H)-dione

[0575] To a solution of 1-(6-((diphenylmethylene)amino)-5-fluoro-1-methyl-1 H-indazol-3- yl)dihydropyrimidine-2,4(1 H,3H)- dione (a, 120 mg, 272 μmol, 1 eq ) in DCM (4 ml) was added TFA (1.23 g, 10.8 mmol, 0.8 ml, 39.6 eq ), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmxio pm; mobile phase: [water (FA)-ACN]; gradient: 5 %-35 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (b, 60 mg, TFA salt) as a white solid. tert-butyl 4-( 6-( (3-(2, 4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro- 1 -methyl- 1H- indazol-6-yl)amino) hexyl)- piperazine- 1 -carboxylate

[0576] To a solution of 1-(6-amino-5-fluoro-1-methyl-1 H-indazol-3-yl)dihydropyrimidine-2,4(1 H,3H)- dione (a, 50 mg, 180 μmol, 1 eq ), tert-butyl 4-(6-oxohexyl)piperazine-1-carboxylate (b, 60 mg, 211 μmol, 1.17 eq ) in DMF (1 ml) was added NaBH3CN (20 mg, 318 μmol, 1.76 eq ) and AcOH (20 mg, 333 μmol, 19.1 μl, 1 .85 eq ) in turns, then the mixture was stirred at 25 °C for 2 h. The mixture was poured into water (10 ml) and extracted with EtOAc (3x10 ml). The organic layer was washed with NaHCO3 (2x10 ml), brine (3x10 ml), dried over Na2SO4, filtered and concentrated to give a crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 16 % MeOH / EtOAc at 18 ml min-1) to give the title compound (c, 42 mg, 77.0 μmol, 43 % yield) as a colorless oil. MS: [M+H]+ 546.3 m / z. 1-(5-fluoro-1-methyl-6-((6-(piperazin-1-yl)hexyl)amino)-1H-indazol-3- yl)dihydropyrimidine-2,4( 1H, 3H)-dione

[0577] To a solution of tert-butyl 4-(6-((3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl- 1 H-indazol-6-yl)amino)- hexyl)piperazine-1 -carboxylate (a, 40 mg, 73.3 μmol, 1 eq ) in DCM (2 ml) was added TFA (307 mg, 2.69 mmol, 0.2 ml, 36.7 eq ), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to give the title com- pound (b, 40 mg, TFA salt) as colorless oil.

[0578] MS: [M+H]+ 446.3 m / z.

[0579] Synthesis of WJ707

[0580] 1-(6-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)hexyl)amino)-5-fluoro- 1- methyl-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione

[0581] To a solution of 1-(5-fluoro-1-methyl-6-((6-(piperazin-1-yl)hexyl)amino)-1 H-indazol-3- yl)dihydropyrimidine-2,4(1 H,3H)- dione (a, 40 mg, 89.8 μmol, 1 eq ), 4-(4-(4-(2-amino-4- (difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin-2-yl)- piperazin-1 -yl)-4-oxobutanoic acid (b, 45 mg, 91.2 μmol, 1.02 eq ) in DMF (1 ml) was added HATU (42 mg, 110 μmol, 1.23 eq ) and DIEA (40 mg, 310 μmol, 53.9 μl, 3.45 eq ) in turns, then the mixture was stirred at 25 °C for 2 h. The mix- ture was poured into water (10 ml) and extracted with EtOAc (3x10 ml). The organic layer was washed with brine (3x10 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 14 %-44 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ707, 27.5 mg, 29.4 μmol, 33 % yield, 98.6 % purity) as a white solid.

[0582] MS: [M+H]+ 921.4 m / z.

[0583] 1 H-NMR (400 MHz, DMSO d6): δ [ppm] 10.47 (s, 1 H), 9.11 (s, 1 H), 8.13 (s, 1 H), 7.77, 7.63, 7.50 (each s, 1 H), 7.62 - 7.52 (m, 2 H), 7.21 (d, J = 12.0 Hz, 1 H), 6.55 (d, J = 7.2 Hz, 1 H), 5.71 (br d, J = 1.8 Hz, 1 H), 3.90 - 3.82 (m, 7 H), 3.76 (br s, 7 H), 3.65 (br d, J = 4.5 Hz, 4 H), 3.60 - 3.44 (m, 9 H), 3.17 - 3.12 (m, 2 H), 2.72 (t, J = 6.7 Hz, 2 H), 2.57 (br s, 4 H), 2.42 - 2.30 (m, 4 H), 1.69 - 1.61 (m, 2 H), 1.51 - 1.43 (m, 2 H), 1.43 - 1.31 (m, 4 H).

[0584] (R)-4-amino-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide

[0585] To a 4-amino-2-fluoro-benzoic acid (a, 1 g, 6.45 mmol, 1 eq ), (3S)-3-aminopiperidine-2,6- dione (b, 1.25 g, 7.59 mmol, 1.18 eq , HCI), DIEA (2.97 g, 23.0 mmol, 4.00 ml, 3.56 eq ) in THF (20 ml) was added T4P (5.57 g, 7.74 mmol, 5.21 ml, 50 % purity, 1.20 eq ) at 0 °C, then the mixture was stirred at 20 °C for 12 h. The mixture was diluted in EtOAc (30 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give a crude product which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-70 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 400 mg, 1.51 mmol, 23 % yield) as yellow solid.

[0586] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.82 (s, 1 H), 7.89 - 7.79 (m, 1 H), 7.53 - 7.47 (m, 1 H), 6.45 - 6.38 (m, 1 H), 6.34 - 6.28 (m, 1 H), 6.00 (s, 2 H), 4.76 - 4.65 (m, 1 H), 2.82 - 2.69 (m, 1 H), 2.54 - 2.51 (m, 1 H), 2.17 - 1.94 (m, 2 H). tert-butyl-(R)-4-(6-((4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3- fluorophenyl)amino)hexyl)piperazine-1-carboxylate

[0587] A mixture of (R)-4-amino-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide (a, 100 mg, 377 μmol, 1 eq ), tert-butyl 4-(6- oxohexyl)piperazine-1 -carboxylate (b, 100 mg, 352 μmol, 0.93 eq ), AcOH (10 mg, 167 μmol, 9.53 μl, 0.44 eq ) in MeOH (1 ml) was stirred at 20 °C for 0.5 h. NaBH3CN (30 mg, 477 μmol, 1.27 eq ) was added and the mixture was stirred at 20 °C for another 12 h. The mixture was diluted in EtOAc (30 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 18 ml min-1) to give the title compound (c, 150 mg, 270 μmol, 72 % yield, 96 % purity) as yellow solid.

[0588] MS: [M+H]+ 534.3 m / z.

[0589] (R)-N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-((6-(piperazin-1- yl)hexyl)amino)benzamide

[0590] To a solution of tert-butyl (R)-4-(6-((4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3- fluorophenyl)amino)hexyl)piperazine- 1 -carboxylate (a, 150 mg, 281 μmol, 1 eq ) in DCM (5 ml) was added TFA (1 ml), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 120 mg, 277 μmol, 98 % yield) as yellow oil.

[0591] MS: [M+H]+ 434.3 m / z.

[0592] Synthesis of WJ708

[0593] (R)-4-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperazin-1 -yl)-4-oxobutanoyl)piperazin-1-yl)hexyl)amino)-N- (2, 6- dioxopiperidin-3-yl) -2-fluorobenzamide

[0594] To a mixture of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (a, 140 mg, 284 μmol, 1.02 eq ), DIEA (150 mg, 1.16 mmol, 202 μl, 4.19 eq ) in DMF (2 ml) was added HATU (140 mg, 368 μmol, 1.33 eq ) at 0 °C, then the mixture was stirred at 20 °C for 0.5 h, (R)-N-(2,6-dioxopiperidin-3- yl)-2-fluoro-4-((6- (piperazin-1-yl)hexyl)amino)benzamide (b, 120 mg, 277 μmol, 1 eq ) was added and the mixture was stirred at 20 °C for 1 h. The mixture was diluted in EtOAc (50 ml). The mixture was washed with brine (2x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmxio pm; mobile phase: [water (FA)-ACN]; gradient: 12 %-42 % B over 10 min). After prep- HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ708, 63.2 mg, 69.5 μmol, 25 % yield, 100 % purity) as a white solid.

[0595] MS: [M+H]+ 909.4 m / z.

[0596] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.83 (s, 1 H), 9.12 (s, 1 H), 8.15 (s, 1 H), 7.88 - 7.81 (m, 1 H), 7.78 (s, 1 H), 7.64 (s, 1 H), 7.62 - 7.52 (m, 3 H), 7.51 (s, 1 H), 6.58 - 6.51 (m, 1 H), 6.48 - 6.42 (m, 1 H), 6.35 - 6.28 (m, 1 H), 4.76 - 4.66 (m, 1 H), 3.87 - 3.73 (m, 8 H), 3.68 - 3.63 (m, 4 H), 3.59 - 3.51 (m, 4 H), 3.46 - 3.41 (m, 4 H), 3.08 - 3.02 (m, 2 H), 2.80 -

[0597] 2.72 (m, 1 H), 2.63 - 2.55 (m, 5 H), 2.38 - 2.33 (m, 2 H), 2.31 - 2.25 (m, 4 H), 2.18 - 2.08 (m, 1 H), 2.07 - 1.99 (m, 1 H), 1.58 - 1.50 (m, 2 H), 1.48 - 1.41 (m, 2 H), 1.40 - 1.29 (m, 4 H). tert-butyl 4-(6-(4-(2,4-dioxotetrahydropyrimidin-1(2H)- yl) phenoxy) hexyl) piperazine- 1 -carboxylate

[0598] To a mixture of 1-(4-hydroxyphenyl)hexahydropyrimidine-2, 4-dione (a, 100 mg, 485 μmol, 1 eq ), tert-butyl 4-(6-hy- droxyhexyl)piperazine-1 -carboxylate (b, 160 mg, 559 μmol, 1.15 eq ), PPh3 (150 mg, 572 μmol, 1.18 eq ) in THF (1 ml) and DMSO (1 ml) was added DIAD (120 mg, 593 μmol, 115 μl, 1 .22 eq ) at 0 °C under N2. Then the mixture was stirred at 20 °C for 12 h. The mixture was diluted in EtOAc (30 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-100 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 140 mg, 274 μmol, 57 % yield, 93 % purity) as colorless oil.

[0599] MS: [M+H]+ 475.3 m / z.

[0600] 1-(4-((6-(piperazin-1-yl) hexyl)oxy)phenyl)dihydropyrimidine-2, 4(1 H, 3H)-dione

[0601] To a solution of tert-butyl 4-(6-(4-(2,4-dioxotetrahydropyrimidin-1 (2H)- yl)phenoxy)hexyl)piperazine-1-carboxylate (a, 140 mg, 295 μmol, 1 eq ) in DCM (5 ml) was added TFA (1 ml), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the title compound (b, 110 mg, 294 μmol, 100 % yield) as yellow oil.

[0602] MS: [M+H]+ 375.3 m / z.

[0603] Synthesis of WJ709

[0604] 1-(4-((6-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl) piperazin- 1 -yl)-4-oxobutanoyl) piperazin- 1 -yl) hexyl) oxy) phenyl) - dihydropyrimidine-2, 4(1 H, 3H) -dione

[0605] To a mixture of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 130 mg, 263 μmol, 0.99 eq ), DIEA (104 mg, 801 μmol, 140 μl, 3 eq ) in DMF (2 ml) was added HATLI (120 mg, 316 μmol, 1.18 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h, 1-(4-((6-(piperazin-1-yl)hexyl)- oxy)phenyl)dihydropyrimidine-2,4(1 H,3H)-dione (a, 100 mg, 267 μmol, 1 eq ) was added and the resulting mixture was stirred at 20 °C for another 1 h. The mixture was diluted in EtOAc (50 ml), washed with brine (2x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmxio pm; mobile phase: [water (FA)-ACN]; gradient: 12 %-42 % B over 10 min). After prep- HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ709, 32.3 mg, 36.0 μmol, 13 % yield, 95 % purity) as a white solid.

[0606] MS: [M+H]+ 850.4 m / z.

[0607] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.30 (s, 1 H), 9.12 (s, 1 H), 7.78, 7.64, 7.51 (each s, 1 H), 7.58 (s, 2 H), 7.25 - 7.19 (m, 2 H), 6.97 - 6.90 (m, 2 H), 4.00 - 3.94 (m, 2 H), 3.77 (s, 8 H), 3.73 - 3.70 (m, 2 H), 3.67 - 3.64 (m, 4 H), 3.63 - 3.44 (m, 8 H), 2.71 - 2.67 (m, 2 H), 2.58 (s, 6 H), 1 .77 - 1.68 (m, 2 H), 1.53 - 1.39 (m, 4 H), 1.39 - 1 .32 (m, 2 H). 1-(4-bromophenyl)dihydropyrimidine-2,4(1H,3H)-dione

[0608] A mixture of 4-bromoaniline (a, 5 g, 29.1 mmol, 1 eq ) in acrylic acid (8.38 g, 116 mmol, 7.97 ml, 4.00 eq ) was stirred at 110 °C for 3 h. Urea (10 g, 167 mmol, 8.95 ml, 5.73 eq ) and AcOH (50 ml) was added and the resulting mixture was stirred at 120 °C for 12 h. The mixture was poured into water (100 ml) and filtered. The filter cake was dried under vacuum to give the title compound (b, 6 g, 22.3 mmol, 77 % yield) as a white solid.

[0609] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.42 (s, 1 H), 7.65 - 7.53 (m, 2 H), 7.35 - 7.26 (m, 2 H), 3.78 (t, J = 6.4 Hz, 2 H), 2.70 (t, J = 6.4 Hz, 2 H). tert-butyl-4-((4-(4-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperazin-1- yl)methyl)piperidine-1-carboxylate

[0610] A mixture of 1-(4-bromophenyl)hexahydropyrimidine-2, 4-dione (a, 200 mg, 743 μmol, 1 eq ), tert-butyl 4-(piperazin- 1-ylmethyl)piperidine-1-carboxylate (b, 240 mg, 847 μmol, 1.14 eq ), Xphos Pd G4 (70.0 mg, 81.4 μmol, 0.11 eq ), Cs2CO3 (800 mg, 2.46 mmol, 3.30 eq ) in dioxane (2 ml) was stirred at 100 °C under N2 for 12 h. The mixture was concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmxio pm; mobile phase: [water (FA)-ACN]; gradient: 8 %-38 % B over 10 min). After prep- HPLC purification, the eluent was concen- trated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, 15 mg, 31.8 μmol, 4 % yield) as white solid. 1-(4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)phenyl)dihydropyrimidine-2,4(1H,3H)- dione

[0611] To a solution of tert-butyl 4-((4-(4-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenyl)piperazin-1- yl)methyl)piperidine- 1 -carboxylate (a, 15 mg, 31.8 μmol, 1 eq ) in DCM (1 ml) was added TFA (0.3 ml), then the mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated to give the title compound (b, 12 mg, crude) as a yellow oil.

[0612] Synthesis of WJ710

[0613] 1-(4-(4-((1-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl) piperazin- 1 -yl)-4-oxobutanoyl) pi peridi n-4-yl) methyl) piperazin- 1-yl)- phenyl)dihydropyrimidine-2, 4( 1H, 3H)-dione

[0614] To a mixture of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 20 mg, 40.5 μmol, 1.25 eq ), DIEA (12.5 mg, 96.9 μmol, 16.9 μl, 3 eq ) in DMF (0.5 ml) was added HATU (20 mg, 52.6 μmol, 1.63 eq ) at 0 °C, then the mixture was stirred at 20 °C for 0.5 h, 1-(4-(4-(piperidin-4-ylmethyl)- piperazin-1- yl)phenyl)dihydropyrimidine-2,4(1 H,3H)-dione (a, 12 mg, 32.3 μmol, 1 eq ) was added and the resulting mix- ture was stirred at 20 °C for 1 h. The mixture was diluted in EtOAc (50 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmxio pm; mobile phase: [water (FA)-ACN]; gradient: 8 %-38 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ710, 15.6 mg, 18.4 μmol, 57 % yield, 100 % purity) as a white solid.

[0615] MS: [M+H]+ 847.4 m / z. 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.26 (s, 1 H), 9.11 (s, 1 H), 7.77 (s, 0.25 H), 7.64 (s, 0.5 H), 7.62 - 7.52 (m, 2 H), 7.50 (s, 0.25 H), 7.17 - 7.12 (m, 2 H), 6.98 - 6.89 (m, 2 H),

[0616] 4.41 - 4.29 (m, 1 H), 3.95 - 3.73 (m, 10 H), 3.69 (t, J = 6.8 Hz,

[0617] 2 H), 3.66 - 3.63 (m, 4 H), 3.59 - 3.51 (m, 4 H), 3.36 - 3.33 (m, 4 H), 3.15 - 3.08 (m, 4 H),

[0618] 3.07 - 2.92 (m, 2 H), 2.68 (t, J = 6.8 Hz, 2 H), 2.57 - 2.54 (m, 4 H), 2.18 (d, J = 6.8 Hz, 2 H),

[0619] 1.80 - 1.66 (m, 3 H), 1.10 - 1.00 (m, 1 H), 0.95 - 0.86 (m, 1 H). tert-butyl (9-oxononyl)carbamate

[0620] To a mixture of tert-butyl N-(9-hydroxynonyl)carbamate (a, 500 mg, 1.93 mmol, 1 eq ) in DCM (10 ml) was added DMP (980 mg, 2.31 mmol, 716 μl, 1.2 eq ), then the mixture was stirred at 25 °C for 2 h. The reaction mixture was filtered and the filtrate was concentrated to give a residue which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-30 % EtOAc / Petroleum ether gradient at 18 ml min-1) to afford the title compound (b, 380 mg, 1.48 mmol, 77 % yield) as yellow oil.

[0621] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 9.65 (s, 1 H), 6.74 (br t, J = 4.5 Hz, 1 H), 2.88 (q, J = 6.7 Hz, 2 H), 2.40 (dt, J = 1 .6 Hz, J = 7.3 Hz, 2 H), 1.56 - 1.46 (m, 2 H), 1.39 - 1.31 (m, 11 H), 1.23 (br s, 8 H). tert-butyl (9-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl)piperazin- 1-yl)- nonyl)carbamate

[0622] To a mixture of tert-butyl N-(9-oxononyl)carbamate (a, 50 mg, 194 μmol, 1 eq ) in DMF (2 ml) was added 4-(diflu- oromethyl)-5-(4-morpholino-6-piperazin-1-yl-1 , 3, 5-triazin-2-yl)pyrimidin-2- amine (b, 100 mg, 197 μmol, 1.01 eq , TFA), NaBH(OAc)3 (80 mg, 377 μmol, 1.94 eq ) and AcONa (30 mg, 366 μmol, 1.88 eq ) in turns, then the mixture was stirred at 25 °C for 12 h. The residue was partitioned between ethyl acetate (10 ml) and water (5 ml). The aqueous layer was extracted with ethyl acetate (10 ml) twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material which was triturated with EtOAc at 25 °C for 30 min to give the title compound (c, 70 mg, 110 μmol, 57 % yield) as a white solid.

[0623] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 9.11 (s, 1 H), 7.74 - 7.47 (m, 3 H), 6.79 - 6.72 (m, 1 H), 3.82 - 3.60 (m, 10 H), 3.39 - 3.24 (m, 8 H), 2.88 (br d, J = 6.5 Hz, 2 H), 1.36 (s, 9 H), 1.32 - 1.18 (m, 14 H).

[0624] 5-(4-(4-(9-aminononyl)piperazin-1-yl)-6-morpholino- 1,3,5-triazin-2-yl)-4- (difluoromethyl)pyrimidin-2-amine

[0625] A mixture of tert-butyl (9-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5- triazin-2-yl)piperazin- 1-yl)nonyl)carbamate (a, 70 mg, 110 μmol, 1 eq ) in HCI / dioxane (2 mol dm-3, 1 ml, 18.1 eq ) was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated to give the title compound (b, 60 mg, HCI salt) as white solid which was used in next step directly.

[0626] MS: [M+H]+ 535.4 m / z.

[0627] 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-4-oxobutanoic acid

[0628] To a mixture of 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1 , 3-dione (a, 1 g, 3.66 mmol, 1 eq ) in AcOH (18 ml) was added tetrahydrofuran-2, 5-dione (b, 1.83 g, 18.3 mmol, 5 eq ) and AcOK (1.44 g, 14.6 mmol, 4 eq ), then the mixture was stirred at 100 °C for 6 h. EtOAc (30 ml) and H2O (30 ml) was added into the reaction mixture, a lot of precipitate was formed. The precipitate was collected and triturated with EtOAc / H2O (10 ml, 1 / 1) at 25 °C for 30 min to give the title compound (c, 420 mg, 1.13 mmol, 31 % yield) as white solid.

[0629] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 12.35 - 12.09 (m, 1 H), 11.22 - 11.10 (m, 1 H), 9.76 (s, 1 H), 8.46 (d, J = 8.4 Hz, 1 H), 7.83 (t, J = 7.9 Hz, 1 H), 7.61 (d, J = 7.3 Hz, 1 H), 5.15 (dd, J = 5.4 Hz, J = 12.7 Hz, 1 H), 2.96 - 2.84 (m, 1 H), 2.74- 2.67 (m, 2 H), 2.65 - 2.54 (m, 4 H), 2.12 - 2.03 (m, 1 H). Synthesis of WJ740

[0630] N1-(9-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin- 2-yl)piperazin- 1-yl)nonyl)-N4-(2-(2, 6-dioxopiperidin-3-yl)- 1, 3-dioxoisoindolin-4-yl)- succinamide

[0631] To a mixture of 5-(4-(4-(9-aminononyl)piperazin-1-yl)-6-morpholino-1 ,3,5-triazin-2-yl)-4- (difluoromethyl)pyrimidin- 2-amine (a, 60 mg, 112 μmol, 1 eq ) in DMF (2 ml) was added 4-((2- (2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)- amino)-4-oxobutanoic acid (b, 50 mg, 134 μmol, 1.19 eq ), HATU (70 mg, 184 μmol, 1.64 eq ) and DIEA (45 mg, 348 μmol, 60.7 μl, 3.10 eq ) in turns, then the mixture was stirred at 25 °C for 12 h. The reaction mixture was adjusted to pH=7 with HCOOH and then purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 16 %-46 % B over 10 min). The eluate was directly lyophilized to give a crude product which was purified by prep-HPLC (column: Waters Xbridge C18 150x25 mmx10 pm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 46 %-66 % B over 8 min). The eluate was directly lyophilized to give a crude product which was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mm*5 pm; mobile phase: [water (FA)-ACN]; gradient: 15 %-45 % B over 10 min). The eluate was directly lyophilized to give the title compound (c, WJ740, 9.5 mg, 10.7 μmol, 9.5 % yield, 100 % purity) as white solid.

[0632] MS: [M+H]+ 890.4 m / z.

[0633] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.15 (s, 1 H), 9.72 (s, 1 H), 9.11 - 9.06 (m, 1 H), 8.47 (d, J = 8.4 Hz, 1 H), 7.88 - 7.79 (m, 2 H), 7.76 - 7.49 (m, 4 H), 5.18 - 5.11 (m, 1 H), 3.74 (br s, 8 H), 3.64 (br d, J = 4.6 Hz, 4 H), 3.02 (q, J = 6.4 Hz, 2 H), 2.95 - 2.87 (m, 1 H), 2.67 (br t, / = 7.0 Hz, 3 H), 2.45 - 2.24 (m, 9 H), 2.10 - 2.02 (m, 1 H), 1.45 - 1.34 (m, 4 H), 1.22 (br s, 10 H).

[0634] (5-((tert-butoxycarbonyl)amino)pentyl)triphenylphosphonium bromide To a mixture of tert-butyl N-(5-bromopentyl)carbamate (a, 2 g, 7.51 mmol, 1 eq ) in toluene (20 ml) was added PPh3 (2 g, 7.63 mmol, 1.01 eq ), then the mixture was stirred at 110 °C for 12 h. The reaction mixture was filtered and the filtrate was dried under vacuum to give the title compound (b, 2.5 g, 4.73 mmol, 63 % yield) as white solid which was used in next step directly.

[0635] MS: [M+H]+ 448.4 m / z. benzyl 4-(6-((tert-butoxycarbonyl)amino)hex- 1-en- 1-yl)piperidine- 1 -carboxylate

[0636] To a mixture of 5-(tert-butoxycarbonylamino)pentyl-triphenyl-phosphonium bromide (a, 2 g, 3.78 mmol, 1.17 eq ) and benzyl 4-formylpiperidine-1 -carboxylate (b, 800 mg, 3.24 mmol, 1 eq ) in DCM (20 ml) was added NaOH (aq., 1.60 g, 20.0 mmol, 50 % purity, 6.18 eq ), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was adjusted to pH=7 with AcOH and then partitioned between DCM (40 ml) and water (20 ml). The aqueous layer was extracted with DCM (20 ml) twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-30 % EtOAc / Petroleum ether gradient at 30 ml min-1) to afford the title compound (c, 260 mg, 624 μmol, 19 % yield) as yellow oil. tert-butyl (6-(piperidin-4-yl)hexyl)carbamate

[0637] To a mixture of Pd / C (100 mg, 624 μmol, 10 % purity, 1.00 eq ) in MeOH (10 ml) was added benzyl 4-(6-((tert-butoxy- carbonyl)amino)hex-1-en-1-yl)piperidine-1 -carboxylate (a, 260 mg, 624 μmol, 1 eq ), then the mixture was stirred at 30 °C for 12 h under H2 (balloon). The reaction mixture was filtered and the filtrate was concentrated to give the title compound (b, 150 mg, 527 μmol, 84 % yield) as off-white oil which was used in next step directly.

[0638] MS: [M+H]+ 285.2 m / z. tert-butyl ( 6-( 1-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin-2-yl)piperazin-1- yl)-4-oxobutanoyl)piperidin-4-yl)hexyl)carbamate

[0639] To a mixture of tert-butyl (6-(piperidin-4-yl)hexyl)carbamate (a, 150 mg, 527 μmol, 1.30 eq ) in DMF (5 ml) was added 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5- triazin-2-yl)piperazin-1-yl)-4-oxobutanoic acid (b, 200 mg, 405 μmol, 1 eq ), HATLI (240 mg, 631 μmol, 1.56 eq ) and DIEA (160 mg, 1.24 mmol, 216 μl, 3.05 eq ) in turns, then the mixture was stirred at 25 °C for 12 h. The residue was partitioned between EtOAc (20 ml) and brine (20 ml). The aqueous layer was extracted with EtOAc (10 ml) twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material which was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %-80 % EtOAc / Petroleum ether gradient at 20 ml min-1) to afford the title compound (c, 300 mg, 395 μmol, 97 % yield, 100 % purity) as yellow oil.

[0640] MS: [M+H]+ 760.4 m / z.

[0641] 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2- yl)piperazin-1-yl)-4-(4-(6- aminohexyl)piperidin-1-yl)butane-1, 4-dione

[0642] To a solution of tert-butyl (6-(1-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-1 ,3,5-triazin-2-yl)- piperazin-1-yl)-4-oxobutanoyl)piperidin-4-yl)hexyl)carbamate (a, 300 mg, 395 μmol, 1 eq ) in dioxane (1 ml) was added HCI / dioxane (2 mol dm-3, 1 ml, 5.07 eq ), then the mixture was stirred at 25 °C for 1 h. The reaction mixture was con- centrated to give the title compound (b, 400 mg, crude) as yellow solid which was used in next step directly.

[0643] MS: [M+H]+ 660.5 m / z. Synthesis of WJ748

[0644] 4-((6-(1-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin-1-yl)-4-oxobutanoyl)piperidin-4-yl)hexyl)amino)-2-(2,6- dioxopiperidin-3-yl)isoindoline- 1, 3-dione

[0645] To a mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-(4-(6-aminohexyl)piperidin-1-yl)butane-1 , 4-dione (a, 100 mg, 152 μmol, 1 eq ) in DMSO (2 ml) was added 2-(2,6-diox- opiperidin-3-yl)-4-fluoroisoindoline-1 , 3-dione (b, 50 mg, 181 μmol, 1.19 eq ) and DIEA (50 mg, 387 μmol, 67.4 μl, 2.55 eq ), then the mixture was stirred at 80 °C for 2 h. The reaction mixture was partitioned between EtOAc (10 ml) and brine (10 ml). The aqueous layer was extracted with EtOAc (10 ml) twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material. The crude material was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mm*5 pm; mobile phase: [water (FA)-ACN]; gradient: 46 %-76 % B over 10 min). The eluate was lyophilized directly to give the title compound (c, WJ748, 3.0 mg, 3.28 μmol, 2.2 % yield, 100 % purity) as yellow solid.

[0646] MS: [M+H]+ 916.4 m / z.

[0647] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.16 - 10.97 (m, 1 H), 9.11 (s, 1 H), 7.78 - 7.49 (m, 4 H), 7.09 (d, J = 8.7 Hz, 1 H), 7.02 (d, J = 7.1 Hz, 1 H), 6.56 - 6.49 (m, 1 H), 5.04 (dd, J = 5.4 Hz, J = 12.8 Hz, 1 H), 4.37 - 4.28 (m, 1 H), 3.91 - 3.72 (m, 10 H), 3.65 (br d, J = 4.4 Hz, 4 H), 3.60 - 3.48 (m, 4 H), 2.99 - 2.82 (m, 2 H), 2.56 (br d, J = 4.3 Hz, 4 H), 2.05 -1.99 (m, 1 H), 1.69 - 1.54 (m, 3 H), 1.70 - 1.16 (m, 10 H), 1.06 - 0.98 (m, 1 H), 0.92 - 0.84 (m, 1 H). tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-5-yl)-2, 7- diazaspiro[3.5]nonane- 7-carboxylate

[0648] A mixture of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1 , 3-dione (a, 200 mg, 724 μmol, 1 eq ), tert-butyl 2,7-di- azaspiro[3.5]nonane-7-carboxylate (b, 200 mg, 884 μmol, 1.22 eq ), KF (100 mg, 1.72 mmol, 2.38 eq ) in DMSO (0.2 ml) was stirred at 120 °C for 1 h. The reaction mixture was diluted with EtOAc (30 ml) and the resulting mixture was washed with brine (3x30 ml), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product which was puri- fied by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %-50 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 200 mg, 414 μmol, 57 % yield, 100 % purity) as yellow solid.

[0649] MS: [M+H]+ 383.1 m / z.

[0650] 2- (2, 6-dioxopiperidin-3-yl)-5-(2, 7-diazaspiro[3.5]nonan-2-yl)isoindoline- 1, 3-dione

[0651] To a solution of tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-5-yl)-2,7- diazaspiro[3.5]nonane-7-car- boxylate (a, 200 mg, 414 μmol, 1 eq ) in DCM (5 ml) was added TFA (0.5 ml), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 200 mg, 403 μmol, 97 % yield, TFA salt) as yellow solid.

[0652] MS: [M+H]+ 383.2 m / z. tert-butyl(3-(2-(2-(2, 6-dioxopiperidin-3-yl)- 1 ,3-dioxoisoindolin-5-yl)-2, 7- diazaspiro[3.5]nonan-7-yl)propyl)carbamate To a mixture of 2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[3.5]nonan-2-yl)isoindoline-1,3- dione (a, 200 mg, 523 μmol, 1 eq ), tert-butyl (3-oxopropyl)carbamate (b, 100 mg, 577 μmol, 1.10 eq ), AcONa (80.0 mg, 975 μmol, 1.86 eq ) in MeOH (10 ml) was stirred at 20 °C for 0.5 h. NaBH3CN (50.0 mg, 796 μmol, 1.52 eq ) was added and the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the crude product which was purified by flash silica gel chromatog- raphy (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %-10 % MeOH / EtOAc gradient at 30 ml min-1) to give the title compound (c, 150 mg, 256 μmol, 49 % yield, 92 % purity) as yellow solid.

[0653] MS: [M+H]+ 540.4 m / z.

[0654] 5-(7-(3-aminopropyl)-2, 7-diazaspiro[3.5]nonan-2-yl)-2-(2, 6-dioxopiperidin-3- yl)isoindoline- 1, 3-dione

[0655] To a solution of tert-butyl (3-(2-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-5-yl)-2,7- diazaspiro[3.5]nonan-7-yl)- propyl)carbamate (a, 100 mg, 185 μmol, 1 eq ) in DCM (1 ml) was added TFA (307 mg, 2.69 mmol, 0.2 ml, 14.5 eq ), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 80 mg, 182 μmol, 98 % yield) as yellow solid.

[0656] MS: [M+H]+ 440.2 m / z.

[0657] Synthesis of WJ761

[0658] 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2- yl)piperazin-1-yl)-N-(3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7- diazaspiro[3.5]nonan-7-yl)propyl)-4-oxobutanamide

[0659] To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 80 mg, 162 μmol, 0.89 eq ), DIEA (74.2 mg, 574 μmol, 0.1 ml, 3.15 eq ) in DMF (1 ml) was added HATU (100 mg, 263 μmol, 1.44 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h, 5-(7-(3-aminopropyl)-2,7-di- azaspiro[3.5]nonan- 2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (a, 80 mg, 182 μmol, 1 eq ) was added and the resulting mixture was stirred at 20 °C for another 1 h. The mixture was concentrated to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 11 %-41 % B over 10 min). After prep-HPLC purification, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the crude product. The crude product was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 12 %-42 % B over 10 min). After prep-HPLC purification, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ761 , 28.1 mg, 30.7 μmol, 17 % yield, 100 % purity, formate) as a white solid.

[0660] MS: [M+H]+ 915.6 m / z.

[0661] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.06 (s, 1 H), 9.11 (s, 1 H), 7.82 (t, J = 4.8 Hz, 1 H), 7.77 (s, 0.25 H), 7.64 (s, 1 H), 7.62 (s, 0.5 H), 7.57 (s, 2 H), 7.50 (s, 0.25 H), 6.76 (d, J = 2.0 Hz, 1 H), 6.65 - 6.60 (m, 1 H), 5.11 - 4.97 (m, 1 H), 3.85 - 3.70 (m, 12 H), 3.66 - 3.62 (m, 4 H), 3.57 - 3.51 (m, 4 H), 3.08 - 3.02 (m, 2 H), 2.92 - 2.81 (m, 1 H), 2.62 - 2.52 (m, 4 H), 2.41 - 2.20 (m, 8 H), 2.05 - 1.96 (m, 1 H), 1.80 - 1.69 (m, 4 H), 1.60 - 1.51 (m, 2 H). tert-butyl 3-(2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 , 3, 5-triazin-2-yl) piperazin- 1-yl)-4-oxobutanoyl)piperazin-1-yl)ethyl)pyrrolidine-1- carboxylate

[0662] A mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin-2- yl)piperazin-1-yl)-4- (piperazin-1 -yl)butane-1 , 4-dione (a, 100 mg, 167 μmol, 1 eq ), tert-butyl 3-(2-bromoethyl)pyrrolidine-1-carboxylate (b, 50.0 mg, 180 μmol, 1.07 eq ), K2CO3 (50.0 mg, 362 μmol, 2.16 eq ), Nal (50.0 mg, 334 μmol, 1.99 eq ) in DMF (1 ml) was stirred at 50 °C for 12 h. The reaction mixture was diluted with EtOAc (30 ml), washed with brine (3x30 ml), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-20 % MeOH / DCM gradient at 30 ml min-1) to give the title compound (c, 60 mg, 79.1 μmol, 47 % yield, 100 % purity) as a white solid.

[0663] MS: [M+H]+ 759.5 m / z.

[0664] 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2- yl)piperazin-1-yl)-4-(4-(2- (pyrrolidin-3-yl)ethyl)piperazin-1-yl)butane-1, 4-dione

[0665] A mixture of tert-butyl 3-(2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-1 ,3,5-triazin-2-yl)- piperazin-1 -yl)-4-oxobutanoyl)piperazin-1-yl)ethyl)pyrrolidine-1- carboxylate (a, 60 mg, 79.1 μmol, 1 eq ) in HCI / diox- ane (2 mol dm-3, 1 ml, 25.3 eq ) was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 60 mg, crude, HCI salt) as a white solid.

[0666] MS: [M+H]+ 659.5 m / z. Synthesis of WJ765

[0667] 5-(3-(2-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5- triazin-2-yl)piperazin- 1-yl)-4-oxobutanoyl)piperazin- 1-yl)ethyl)pyrrolidin- 1-yl)-2- (2, 6- dioxopiperidin-3-yl)isoindoline- 1,3-dione

[0668] A mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin-2- yl)piperazin-1-yl)-4- (4-(2-(pyrrolidin-3-yl)ethyl)piperazin-1-yl)butane-1 , 4-dione (b, 50 mg, 71.9 μmol, 0.99 eq , HCI salt), 2-(2,6-dioxopiperidin- 3-yl)-5-fluoroisoindoline-1 , 3-dione (a, 20 mg, 72.4 μmol, 1 eq ), KF (12.0 mg, 207 μmol, 2.85 eq ) in DMSO (1 ml) was stirred at 120 °C for 1 h. The mixture was filtered and the filtrate was concentrated to give the crude product which was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 15 %-45 % B over 10 min). After prep-HPLC purification, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ765, 35.1 mg, 38.4 μmol, 53 % yield, 100 % purity) as yellow solid.

[0669] MS: [M+H]+ 915.5 m / z.

[0670] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.06 (s, 1 H), 9.11 (s, 1 H), 7.77 (s, 0.25 H), 7.65 (s, 0.5 H), 7.63 (d, J = 3.6 Hz, 1 H), 7.57 (s, 2 H), 7.50 (s, 0.25 H), 6.93 - 6.88 (s, 1 H), 6.83 - 6.77 (m, 1 H), 5.08 - 5.01 (m, 1 H), 3.87 - 3.71 (m, 8 H), 3.68 - 3.63 (m, 4 H), 3.62 - 3.54 (m, 4 H), 3.53 - 3.42 (m, 6 H), 3.07 - 3.00 (m, 1 H), 2.92 - 2.83 (m, 1 H), 2.64 - 2.52 (m, 8 H), 2.46 - 2.30 (m, 6 H), 2.22 - 2.13 (m, 1 H), 2.05 - 1.94 (m, 1 H), 1.74 - 1.58 (m, 3 H). tert-butyl (4-(4-cyano- 1H-imidazol- 1-yl)butyl)carbamate

[0671] To a mixture of 1 H-imidazole-4-carbonitrile (a, 350 mg, 3.76 mmol, 1.09 eq ) in MeCN (10 ml) was added K2CO3 (1.2 g, 8.68 mmol, 2.52 eq ) and tert-butyl (4-bromobutyl)carbamate (b, 870 mg, 3.45 mmol, 708 μl, 1 eq ), then the resulting mix- ture was stirred at 60 °C and stirred for 11 h. The reaction mixture was concentrated to give a residue which was puri- tied by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-30 % EtOAc / Petroleum ether gradient at 20 ml min-1) to afford the title compound (c, 900 mg, 3.40 mmol, 99 % yield) as off-white oil.

[0672] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 7.52 (s, 1 H), 7.49 (s, 1 H), 4.04 (t, J = 7.3 Hz, 2 H), 3.18 (br d, J = 6.3 Hz, 2 H), 1.87 - 1.80 (m, 2 H), 1.51 (br d, J = 7.8 Hz, 2 H), 1.45 (s, 9 H). tert-butyl (4-(4-(aminomethyl)-1H-imidazol- 1-yl)butyl)carbamate

[0673] To a mixture of Raney-Ni (100 mg) in MeOH (8 ml) was added tert-butyl (4-(4-cyano-1 H- imidazol-1-yl)butyl)carba- mate (a, 450 mg, 1.70 mmol, 1 eq ) and NH3 ■ H2O (220 mg, 1.76 mmol, 242 μl, 28 % purity, 1.03 eq ), then the mixture was stirred at 25 °C under H2 for 12 h. The reaction mixture was filtered and the filtrate was concentrated to give the title compound (b, 430 mg, crude) as off-white oil which was used in next step without purification.

[0674] MS: [M+H]+ 269.2 m / z. tert-butyl(4-(4-( ((2-(2, 6-dioxopiperidin-3-yl) - 1, 3-dioxoisoindolin-5- yl)amino)methyl)-1 H-imidazol- 1-yl)butyl)carbamate

[0675] To a mixture of tert-butyl (4-(4-(aminomethyl)-1 H-imidazol-1-yl)butyl)carbamate (a, 400 mg, 1.49 mmol, 1 eq ) in DMSO (10 ml) was added 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline- 1 , 3-dione (b, 530 mg, 1.92 mmol, 1.29 eq ) and KF (260 mg, 4.48 mmol, 3.00 eq ), then the mixture was stirred at 110 °C for 2 h. The reaction mixture was partitioned between EtOAc (30 ml) and brine (20 ml). The aqueous layer was extracted with EtOAc (20 ml) twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated to give a crude material which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-10 % EtOAc / MeOH gradient at 30 ml min-1) to afford the title compound (c, 130 mg, 248 μmol, 17 % yield, 100 % purity) as yellow oil.

[0676] MS: [M+H]+ 525.2 m / z. 5-(((1-(4-aminobutyl)- 1H-imidazol-4-yl)methyl)amino)-2-(2, 6-dioxopiperidin-3- yl)isoindoline- 1, 3-dione

[0677] To a mixture of tert-butyl (4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-5- yl)amino)methyl)-1 H-imidazol-1- yl)butyl)carbamate (a, 130 mg, 248 μmol, 1 eq ) in DCM (1 ml) was added TFA (768 mg, 6.73 mmol, 0.5 ml, 27.2 eq ), then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated to give the title compound (b, 100 mg, crude) as yellow oil.

[0678] MS: [M+H]+ 425.3 m / z.

[0679] Synthesis of WJ771

[0680] 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin-2- yl)piperazin-1-yl)-N-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5- yl)amino)- methyl)-1 H-imidazol- 1-yl)butyl)-4-oxobutanamide

[0681] To a mixture of 5-(((1-(4-aminobutyl)-1 H-imidazol-4-yl)methyl)amino)-2-(2,6-dioxopiperidin-3- yl)isoindoline-1 , 3-dione (a, 80 mg, 188 μmol, 1 eq ) in DMF (2 ml) was added 4-(4-(4-(2-amino- 4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3,5-triazin-2-yl)piperazin-1-yl)-4-oxobutanoic acid (b, 70 mg, 142 μmol, 0.75 eq ), EDCI (60 mg, 313 μmol, 1.66 eq ), HOBt (40 mg, 296 μmol, 1.57 eq ) and DIEA (80 mg, 619 μmol, 108 μl, 3.28 eq ) in turns, then the mixture was stirred at 25 °C for 12 h. The reaction mixture was partitioned between EtOAc (10 ml) and water (10 ml), a lot of residue precipitate was formed. The precipitate was collected and then purified by prep-HPLC (column: Welch Xtimate C18 150x25 mm*5 pm; mobile phase: [water (FA)-ACN]; gradient: 14 %-44 % B over 10 min) to afford the title compound (c, WJ771 , 25.5 mg, 28.3 μmol, 15 % yield, 100 % purity) as yellow solid.

[0682] MS: [M+H]+ 900.4 m / z. 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.05 (s, 1 H), 9.11 (s, 1 H), 8.19 - 7.63 (m, 2 H),

[0683] 7.60 - 7.55 (m, 3 H), 7.49 (s, 1 H), 7.41 (br t, J = 5.6 Hz, 1 H), 7.09 - 7.02 (m, 2 H), 6.92 (dd, / = 2.1 Hz, J = 8.4 Hz, 1 H), 5.06 - 4.98 (m, 1 H), 4.23 (br d, J = 5.4 Hz, 2 H), 3.90 (br t, / = 7.1 Hz, 2 H), 3.76 (br s, 8 H), 3.65 (br d, J = 4.0 Hz, 4 H), 3.52 (br s, 4 H), 3.03 (q, J = 6.5 Hz, 2 H), 2.92 - 2.82 (m, 1 H), 2.60 - 2.55 (m, 4 H), 2.34 - 2.30 (m, 2 H), 2.02 - 1 .95 (m, 1 H), 1.70 - 1.63 (m, 2 H), 1.36 - 1 .27 (m, 2 H). tert-butyl (8-oxooctyl) carbamate

[0684] To a solution of tert-butyl (8-hydroxyoctyl)carbamate (a, 2 g, 8.15 mmol, 1 eq ) in DCM (20 ml) was added Dess- Martin (3.80 g, 8.97 mmol, 2.78 ml, 1.1 eq ), then the mixture was stirred at 20 °C for 12 h. The mixture was filtered and the filtrate was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %-20 % EtOAc / Petroleum ether gradient at 40 ml min-1) to give the title compound (b, 1 g, 4.11 mmol, 50 % yield) as colorless oil.

[0685] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 9.77 (t, J = 1.7 Hz, 1 H), 4.52 (s, 1 H), 3.17 - 3.05 (m, 2 H), 2.47 - 2.38 (m, 2 H), 1.67 - 1.60 (m, 2 H), 1.54 - 1.38 (m, 13 H), 1.35 - 1.29 (m, 6 H). tert-butyl non-8-yn-1-ylcarbamate

[0686] To a mixture of tert-butyl (8-oxooctyl)carbamate (a, 1 g, 4.11 mmol, 1 eq ), K2CO3 (2.27 g, 16.4 mmol, 4 eq ) in MeOH (20 ml) was added dimethyl (1-diazo-2-oxopropyl)phosphonate (b, 1 g, 5.21 mmol, 1.27 eq ) at 0 °C, then the mixture was stirred at 20 °C for 12 h. The mixture was filtered and the filtrate was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0 %- 10 % EtOAc / Petroleum ether gradient at 40 ml min-1) to give the title compound (c, 500 mg, 2.09 mmol, 51 % yield) as color- less oil.

[0687] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 4.50 (s, 1 H), 3.20 - 3.02 (m, 2 H), 2.24 - 2.13 (m, 2 H), 1.94 (t, J = 2.4 Hz, 1 H), 1.57 - 1.37 (m, 15 H), 1.36 - 1.29 (m, 4 H). methyl 3-azidopropanoate

[0688] To a solution of methyl 3-bromopropanoate (a, 500 mg, 2.99 mmol, 328 μl, 1 eq ) in DMSO (5 ml) was added NaN3 (260 mg, 4.00 mmol, 1 .34 eq ), then the mixture was stirred at 50 °C for 12 h. The reaction was poured into sat. NaHCO3 (20 ml) and extracted with MTBE (3x20 ml). The organic layer was washed with brine (3x30 ml). The organic layer was dried over Na2SO4, filtered and concentrated to 10 ml, then added DCE (20 ml) and concentrated to 5 ml. The title compound (b, 1 g, crude) was dissolved in DCE and was used for next step directly. methyl 3-(4-(7-((tert-butoxycarbonyl)amino)heptyl)-1H- 1,2,3-triazol- 1- yl)propanoate

[0689] To a mixture of tert-butyl non-8-yn-1-ylcarbamate (a, 250 mg, 1.04 mmol, 1 eq ), methyl 3- azidopropanoate (b, 130 mg, 1.01 mmol, 0.96 eq ), sodium (2R)-2-((1S)-1 ,2-dihydroxyethyl)- 4-hydroxy-5-oxo-2H-furan-3-olate (100 mg, 505 μmol, 0.48 eq ) in DCM (2 ml) and H2O (2 ml) was added CuSO4 ■ 5H2O (50 mg, 200 μmol, 0.19 eq ), then the mixture was stirred at 20 °C for 12 h. The mixture was diluted in DCM (50 ml). The mixture was washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %- 70 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 200 mg, 543 μmol, 52 % yield) as a white solid.

[0690] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 7.37 (s, 1 H), 4.74 - 4.58 (m, 2 H), 4.56 - 4.40 (m, 1 H), 3.71 (s, 3 H), 3.15 - 3.05 (m, 2 H), 2.98 (t, J = 6.4 Hz, 2 H), 2.77 - 2.61 (m, 2 H), 1.73 - 1.63 (m, 2 H), 1.53 - 1 .42 (m, 11 H), 1.39 - 1.28, (m, 6 H).

[0691] 3-(4-(7-((tert-butoxycarbonyl)amino)heptyl)-1H-1,2,3-triazol-1-yl)propanoic acid

[0692] To a solution of methyl 3-(4-(7-((tert-butoxycarbonyl)amino)heptyl)-1 H-1 ,2,3-triazol-1- yl)propanoate (a, 200 mg, 543 μmol, 1 eq ) in MeOH (5 ml) was added LiOH (2 mol dm-3, 1 ml, 3.68 eq ), then the mixture was stirred at 20 °C for 1 h. Water (10 ml) was added and the mixture was adjusted to pH=5-6 with 1 n HCI (aq). Then the mixture was extracted with EtOAc (3x20 ml), dried over Na2SO4, filtered and concentrated to give the title compound (b, 200 mg, crude) as a white solid. tert-butyl (7-(1-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperazin- 1- yl) -3-oxopropyl) -IH-1,2, 3-t n azol-4- yl)heptyl)carbamate

[0693] To a solution of 3-(4-(7-((tert-butoxycarbonyl)amino)heptyl)-1 H-1 ,2,3-triazol-1-yl)propanoic acid (a, 200 mg, 564 μmol, 1 eq ), DIEA (219 mg, 1 .69 mmol, 295 μl, 3 eq ) in DMF (1 ml) was added HATLI (240 mg, 631 μmol, 1.12 eq ), then the mix- ture was stirred at 0 °C for 0.5 h. 4- (difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1 ,3,5-triazin-2-yl)pyrimidin-2- amine (b, 240 mg, 610 μmol, 1.08 eq ) was added and the mixture was stirred at 20 °C for another 1 h. The reaction mixture was diluted with EtOAc (50 ml), washed with brine (3x30 ml), dried over anhydrous Na2SO4, filtered and con- centrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %- 100 % EtOAc / Petroleum, then EtOAc / MeOH=10 / 1 gradient at 30 ml min-1) to give the title compound (c, 300 mg, 411 μmol, 73 % yield) as a white solid.

[0694] MS: [M+H]+ 730.6 m / z.

[0695] 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin-2- yl) piperazin- 1-yl)-3-(4-(7- aminoheptyl)- 1H- 1, 2, 3-triazol- 1-yl) propan- 1-one

[0696] To a solution of tert-butyl (7-(1-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6- morpholino-1 ,3, 5-triazin-2-yl)- piperazin- 1-yl)-3-oxopropyl)-1 H-1 ,2,3-triazol-4- yl)heptyl)carbamate (a, 130 mg, 178 μmol, 1 eq ) in DCM (5 ml) was added TFA (650 μl), then the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the title compound (b, 130 mg, crude, TFA salt) as yellow solid. MS: [M+H]+ 630.5 m / z.

[0697] Synthesis of WJ780

[0698] 5-((7-(1-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin- 1-yl)-3-oxopropyl)- 1H- 1, 2, 3-triazol-4-yl)heptyl)amino)-2-(2, 6- dioxopiperidin-3-yl)isoindoline- 1, 3-dione

[0699] To a mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 3-(4-(7-aminoheptyl)-1 H-1 ,2,3-triazol-1-yl)propan-1-one (a, 130 mg, 175 μmol, 1 eq , TFA salt), 2-(2,6-dioxopiperidin-3- yl)-5-fluoroisoindoline-1 , 3-dione (b, 60 mg, 217 μmol, 1.24 eq ), KF (30 mg, 516 μmol, 2.95 eq ) in DMSO (1 ml) was stirred at 120 °C for 1 h. The mixture was filtered and the filtrate was concentrated to give the crude product which was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mm*5 pm; mobile phase: [water (FA)-ACN]; gradient: 38 %-68 % B over 10 min). After prep-HPLC purification, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ780, 15.3 mg, 17.3 μmol, 9.9 % yield, 100 % purity, formate) as yellow solid.

[0700] MS: [M+H]+ 886.7 m / z.

[0701] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.06 (s, 1 H), 9.10 (s, 1 H), 7.82 (s, 1 H), 7.75 (s, 0.25 H), 7.62 (s, 0.5 H), 7.61 - 7.53 (m, 2 H), 7.48 (s, 0.25 H), 7.10 (t, J = 4.8 Hz, 1 H), 6.93 (d, J = 1.2 Hz, 1 H), 6.86 - 6.80 (m, 1 H), 5.07 - 4.97 (m, 1 H), 4.53 (t, J = 6.8 Hz, 2 H), 3.81 - 3.72 (m, 8 H), 3.66 - 3.62 (m, 4 H), 3.56 - 3.48 (m, 4 H), 3.16 - 3.09 (m, 2 H), 3.02 (t, J = 6.8 Hz, 2 H), 2.93 - 2.79 (m, 1 H), 2.60 - 2.57 (m, 2 H), 2.56 - 2.52 (m, 2 H), 2.02 - 1 .95 (m, 1 H),

[0702] 1.60 - 1 .52 (m, 4 H), 1 .36 - 1.28 (br s, 6 H).

[0703] 1 H-NMR (400 MHz, DMSO - d6 = D2O): δ [ppm] 9.08 (s, 1 H), 7.79 (s, 1 H), 7.73 (s, 0.25 H),

[0704] 7.60 (s, 0.5 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.46 (s, 0.25 H), 6.91 (d, J = 1.2 Hz, 1 H), 6.83 - 6.77 (m, 1 H), 5.03 - 4.97 (m, 1 H), 4.52 (t, J = 6.8 Hz, 2 H), 3.76 - 3.69 (m, 8 H), 3.64 - 3.61 (m, 4 H), 3.54 - 3.45 (m, 4 H), 3.10 (t, J = 6.8 Hz, 2 H), 3.00 (t, J = 6.8 Hz, 2 H), 2.90 - 2.78 (m, 1 H), 2.61 - 2.56 (m, 2 H), 2.55 - 2.51 (m, 2 H), 2.02 - 1.94 (m, 1 H), 1.58 - 1.49 (m, 4 H), 1.32 - 1.24 (m, 6 H). tert-butyl (7-azidoheptyl) carbamate

[0705] To a solution of tert-butyl (7-bromoheptyl)carbamate (a, 2 g, 6.80 mmol, 1 eq ) in DMF (20 ml) was added NaN3 (560 mg, 8.61 mmol, 1.27 eq ), then the mixture was stirred at 80 °C for 12 h. The reaction was poured into sat. NaHCO3 (50 ml) and extracted with EtOAc (3x50 ml). The organic layer was washed with brine (3x50 ml), dried over Na2SO4, filtered and concentrated to give the crude title compound (b, 1.5 g, crude) as colorless oil.

[0706] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 4.52 (s, 1 H), 3.28 (t, J = 7.2 Hz, 2 H), 3.20 - 3.06 (m, 2 H), 1.69 - 1.54 (m, 2 H), 1.54 - 1.32 (m, 11 H), 1.31 - 1 .22 (m, 6 H). methyl 3-(1-(7-((tert-butoxycarbonyl)amino)heptyl)-1H-1,2,3-triazol-4- yl)propanoate

[0707] To a mixture of tert-butyl (7-azidoheptyl)carbamate (a, 500 mg, 1.95 mmol, 1 eq ), methyl pent-

[0708] 4-ynoate (b, 220 mg, 1.96 mmol, 1.01 eq ), sodium (2R)-2-[(1S)-1 ,2-dihydroxyethyl]-4-hydroxy-

[0709] 5-oxo-2H-furan-3-olate (150 mg, 757 μmol, 0.39 eq ) in DCM (5 ml) and water (2 ml) was added CuSO4 ■ 5H2O (100 mg, 401 μmol, 0.21 eq ), then the mixture was stirred at 20 °C for 12 h. The mixture was diluted in DCM (50 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 %-70 % EtOAc / Petroleum ether gradient at 30 ml min-1) to give the title compound (c, 500 mg, 1.36 mmol, 70 % yield) as white solid.

[0710] 1 H-NMR (400 MHz, CHLOROFORM > d): δ [ppm] 7.32 (s, 1 H), 4.51 (s, 1 H), 4.30 (s, 2 H), 3.68 (s, 3 H), 3.15 - 3.01 (m, 4 H), 2.75 (s, 2 H), 1.94 - 1.83 (m, 2 H), 1.44 (s, 11 H), 1.33 (s, 6 H).

[0711] 3-( 1-(7-((tert-butoxycarbonyl)amino) heptyl) -1H-1,2, 3-triazol-4-yl) propanoic acid To a solution of methyl 3-(1-(7-((tert-butoxycarbonyl)amino)heptyl)-1 H-1 ,2,3-triazol-4- yl)propanoate (a, 200 mg, 543 μmol, 1 eq ) in MeOH (5 ml) was added LiOH (2 mol dm-3, 1 ml, 3.68 eq ), then the mixture was stirred at 20 °C for 12 h. Water (10 ml) was added and the mixture was adjusted to pH=5-6 with 1 n HCI (aq). Then the mixture was extracted with EtOAc (3x20 ml). The organic layers were dried over Na2SO4, filtered and concentrated to give the crude title compound (b, 200 mg, crude) as white solid. tert-butyl (7-(4-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperazin- 1- yl) -3-oxopropyl) -1H-1,2, 3-triazol- 1 - yl)heptyl)carbamate

[0712] To a mixture of 3-(1-(7-((tert-butoxycarbonyl)amino)heptyl)-1 H-1 ,2,3-triazol-4-yl)propanoic acid (a, 200 mg, 564 μmol, 1 eq ), DIEA (200 mg, 1.55 mmol, 270 μl, 2.74 eq ) in DMF (2 ml) was added HATLI (260 mg, 684 μmol, 1.21 eq ) at 0 °C, then the mixture was stirred at 0 °C for 0.5 h. 4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1 ,3,5-triazin-2-yl)- pyrimidine- amine (b, 230 mg, 585 μmol, 1.04 eq ) was added and the mixture was stirred at 20 °C for 1 h. The reaction mixture was diluted with EtOAc (50 ml), washed with brine (3x30 ml), dried over anhydrous Na2SO4, filtered and con- centrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, eluent of 0 %-100 % EtOAc / Petroleum, then EtOAc / MeOH=10 / 1 gradient at 30 ml min-1) to give the title compound (c, 300 mg, 394 μmol, 70 % yield, 96 % purity) as white solid.

[0713] MS: [M+H]+ 730.6 m / z.

[0714] 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5-triazin-2- yl) piperazin- 1-yl)-3-( 1-(7- aminoheptyl)- 1H- 1, 2, 3-triazol-4-yl)propan- 1-one

[0715] A solution of tert-butyl (7-(4-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1 ,3,5-triazin-2-yl)piper- azin-1-yl)-3-oxopropyl)-1 H-1 ,2,3-triazol-1-yl)heptyl)carbamate (a, 300 mg, 411 μmol, 1 eq ) in HCI / dioxane (2 mol dm-3, 5 ml, 24.3 eq ) was stirred at 20 °C for 12 h. The mixture was concentrated to give the title compound (b, 270 mg, 405 μmol, 99 % yield, HCI salt) as white solid.

[0716] MS: [M+H]+ 630.5 m / z.

[0717] Synthesis of WJ781

[0718] 5-((7-(4-(3-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1,3,5- triazin-2-yl)piperazin- 1-yl)-3-oxopropyl)- 1H- 1, 2, 3-triazol- 1-yl)heptyl)amino)-2-(2, 6- dioxopiperidin-3-yl)isoindoline- 1, 3-dione

[0719] To a mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 3-(1-(7-aminoheptyl)-1 H-1 ,2,3-triazol-4-yl)propan-1-one (a, 100 mg, 150 μmol, 1 eq , HCI salt), 2-(2,6-dioxopiperidin-3- yl)-5-fluoroisoindoline-1 , 3-dione (b, 50.0 mg, 181 μmol, 1.21 eq ), KF (30 mg, 516 μmol, 3.44 eq ) in DMSO (1 ml) was stirred at 110 °C for 1 h. The mixture filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 38 %-58 % B over 10 min). After prep-HPLC purifi- cation, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the crude product which was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 35 %-65 % B over 10 min). After prep-HPLC purification, the eluate was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ781 , 11.2 mg, 12.6 μmol, 8.4 % yield, 100 % purity) as white solid.

[0720] MS: [M+H]+ 886.7 m / z.

[0721] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.06 (s, 1 H), 9.10 (s, 1 H), 7.84 (s, 1 H), 7.76 - 7.49 (m, 4 H), 7.12 - 7.07 (m, 1 H), 6.94 - 6.91 (m, 1 H), 6.85 - 6.80 (m, 1 H), 5.07 - 4.98 (m,

[0722] 1 H), 4.31 - 4.25 (m, 2 H), 3.76 (br s, 8 H), 3.66 - 3.62 (m, 4 H), 3.53 (s, 4 H), 3.15 - 3.08 (m,

[0723] 2 H), 2.90 - 2.80 (m, 3 H), 2.76 - 2.69 (m, 2 H), 2.01 - 1.96 (m, 1 H), 1.82 - 1.74 (m, 2 H), 1.59 - 1 .49 (m, 2 H), 1.38 - 1.29 (m, 4 H), 1.27 - 1 .20 (m, 4 H). 4-(((2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)oxy)methyl)benzaldehyde

[0724] To a mixture of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1 , 3-dione (a, 200 mg, 729 μmol, 1 eq ), 4-(bromomethyl)- benzaldehyde (b, 150 mg, 754 μmol, 1 eq ), K2CO3 (200 mg, 1.45 mmol, 2 eq ) in DMF (2 ml) was stirred at 20 °C for 2 h.

[0725] The mixture was filtered and purified by prep-HPLC (column: Phenomenex luna C18 150x40 mmx15 pm; mobile phase: [water (FA)-ACN]; gradient: 35 %-65 % B over 10 min). After prep- HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, 150 mg, 382 μmol, 52 % yield) as a white solid.

[0726] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.12 (s, 1 H), 10.02 (s, 1 H), 7.97 (dj = 8.4 Hz, 2 H), 7.89 - 7.81 (m, 1 H), 7.74 (d, J = 8.0 Hz, 2 H), 7.59 (d, J = 8.4 Hz, 1 H), 7.50 (d, J = 7.2 Hz, 1 H), 5.51 (s, 2 H), 5.15 - 5.06 (m, 1 H), 2.96 - 2.83 (m, 1 H), 2.65 - 2.54 (m, 2 H), 2.07 - 2.00 (m, 1 H).

[0727] Synthesis of WJ792

[0728] 4-((4-((4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1,3,5- triazin-2-yl) piperazin- 1 -yl)-4-oxobutanoyl) piperazin- 1 -yl) methyl) benzyl) oxy) -2- (2, 6- dioxopiperidin-3-yl)isoindoline- 1,3-dione To a mixture of 1-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-(piperazin-1-yl)butane-1 , 4-dione (b, 150 mg, 251 μmol, 0.98 eq , HCI salt), 4-(((2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoin- dolin-4-yl)oxy)methyl)benzaldehyde (a, 100 mg, 255 μmol, 1 eq ), AcOH (10 mg, 167 μmol, 9.5 μl, 0.65 eq ) in DMF (1 ml) was stirred at 20 °C for 0.5 h. NaBH3CN (20 mg, 318 μmol, 1.25 eq ) was added and the mixture was stirred at 20 °C for another 12 h. The mixture was filtered and the filtrate was concentrated to give the crude product which was pu- rified by prep-HPLC (column: Waters xbridge 150x25 mmx10 pm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 25 %-55 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the crude product which was purified by prep- HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 14 %-44 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ792, 13.2 mg, 14.1 μmol, 5.5 % yield, 100 % purity) as a white solid.

[0729] MS: [M+H]+ 938.5 m / z.

[0730] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 11.11 (s, 1 H), 9.11 (s, 1 H), 7.86 - 7.80 (m, 1 H), 7.77 (s, 0.25 H), 7.63 (s, 0.5 H), 7.61 (s, 1 H), 7.59 - 7.52 (m, 2 H), 7.50 (s, 0.25 H), 7.49 - 7.45 (m, 3 H), 7.39 - 7.34 (m, 2 H), 5.35 (s, 2 H), 5.12 - 5.06 (m, 1 H), 3.87 - 3.70 (m, 8 H), 3.67 - 3.62 (m, 4 H), 3.60 - 3.55 (m, 2 H), 3.54 - 3.49 (m, 4 H), 3.49 - 3.41 (m, 4 H), 2.94 - 2.83 (m, 1 H), 2.62 - 2.53 (m, 6 H), 2.41 - 2.35 (m, 2 H), 2.33 - 2.28 (m, 2 H), 2.06 - 1.98 (m, 1 H). methyl 4-(4-(4-((tert-butoxycarbonyl)amino)butyl)piperazin-1-yl)-2-fluorobenzoate

[0731] A mixture of methyl 2,4-difluorobenzoate (a, 200 mg, 1.16 mmol, 1.5 eq ), tert-butyl (4- (piperazin-1-yl)butyl)carba- mate (b, 200 mg, 777 μmol, 1 eq ), K2CO3 (268 mg, 1.94 mmol, 2.5 eq ) in DMSO (1 ml) was stirred at 80 °C for 1 h. The mixture was diluted in EtOAc (50 ml), washed with brine (3x30 ml), dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by prep-HPLC (column: Phenomenex luna C18 150x25 mmx10 pm; mobile phase: [water (FA)-ACN]; gradient: 18 %-38 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, 150 mg, 329 μmol, 42 % yield, formate) as a white solid. 1 H-NMR (400 MHz, METHANOL > d4): δ [ppm] 8.37 (s, 1 H), 7.84 (t, J = 8.8 Hz, 1 H), 6.86 - 6.81 (m, 1 H), 6.79 - 6.70 (m, 1 H), 3.86 (s, 3 H), 3.63 - 3.55 (m, 4 H), 3.16 - 3.06 (m, 6 H), 3.00 - 2.85 (m, 2 H), 1.78 - 1.67 (m, 2 H), 1.61 - 1 .52 (m, 2 H), 1.45 (s, 9 H).

[0732] 4-(4-(4-((tert-butoxycarbonyl)amino)butyl)piperazin-1-yl)-2-fluorobenzoic acid

[0733] To a solution of methyl 4-(4-(4-((tert-butoxycarbonyl)amino)butyl)piperazin-1-yl)-2- fluorobenzoate (a, 50 mg, 122 μmol, 1 eq ) in MeOH (5 ml) was added LiOH (1 mol dm-3, 1 ml), then the mixture was stirred at 20 °C for 12 h. The mixture was concentrated to give the aqueous solution. The residual aqueous solution was lyophilized to give the title compound (b, 50 mg, crude) as a white solid.

[0734] MS: [M+H]+ 396.2 m / z. tert-butyl (R)-(4-(4-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3- fluorophenyl)piperazin-1-yl)butyl)carbamate

[0735] To a solution of 4-(4-(4-((tert-butoxycarbonyl)amino)butyl)piperazin-1-yl)-2-fluorobenzoic acid (a, 150 mg, 379 μmol, 1 eq ), DIEA (148 mg, 1.15 mmol, 0.2 ml, 3 eq ) in THF (2 ml) was added T4P (144 mg, 400 μmol, 1.1 eq ), then the mixture was stirred at 0 °C for 0.5 h. (R)-3- aminopiperidine-2, 6-dione (b, 100 mg, 608 μmol, 1.6 eq , HCI) was added and the mixture was stirred at 20 °C for 1 h. The mixture was concentrated to give the crude product which was purified by flash silica gel chromatography (ISCO; 4 g SepaFlash Silica Flash Column, Eluent of 0 %-100 % EtOAc / Petroleum, then EtOAc / MeOH=10 / 1 gradient at 30 ml min-1) to give the title compound (c, 80 mg, 158 μmol, 42 % yield) as a white solid.

[0736] MS: [M+H]+ 506.3 m / z. (R)-4-(4-(4-aminobutyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2- fluorobenzamide

[0737] To a solution of tert-butyl (R)-(4-(4-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3- fluorophenyl)piperazin-1-yl)butyl)carbamate (a, 80 mg, 158 μmol, 1 eq ) in DCM (0.5 ml) was added TFA (0.1 ml), then the mixture was stirred at 20 °C for 12 h. The mixture was concentrated to give the title compound (b, 80 mg, crude, TFA salt) as yellow oil.

[0738] MS: [M+H]+ 406.3 m / z.

[0739] Synthesis of WJ999

[0740] (R)-4-(4-(4-(4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino- 1, 3, 5-triazin-2-yl) piperazin-1 -yl)-4-oxobutanamido)butyl)piperazin-1-yl)-N-(2, 6- dioxopiperidin-3-yl)-2-fluorobenzamide

[0741] To a solution of 4-(4-(4-(2-amino-4-(difluoromethyl)pyrimidin-5-yl)-6-morpholino-1 ,3,5-triazin- 2-yl)piperazin-1-yl)- 4-oxobutanoic acid (b, 50 mg, 101 μmol, 1.05 eq ), DIEA (50 mg, 387 μmol, 67 μl, 4 eq ) in DCM (1 ml) was added EDCI (20 mg, 104 μmol, 1.1 eq ) and HOBt (20 mg, 148 μmol, 1.5 eq ), then the mixture was stirred at 20 °C for 0.5 h. (R)-4-(4- (4- aminobutyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide (a, 50 mg, 96 μmol, 1 eq , TFA salt) was added and the mixture was stirred at 20 °C for 12 h. The mixture was poured into water (10 ml) and DCM (5 ml). The mixture was filtered and the filter cake was dried under vacuum to give the crude product. The crude product was purified by prep-HPLC (column: Welch Xtimate C18 150x25 mmx5 pm; mobile phase: [water (FA)-ACN]; gradient: 12 %-42 % B over 10 min). After prep-HPLC purification, the eluent was concentrated to remove organic solvents. The residual aqueous solution was lyophilized to give the title compound (c, WJ999, 13.1 mg, 14.9 μmol, 15 % yield, 100 % purity, formate) as a white solid. MS: [M+H]+ 881.7 m / z.

[0742] 1 H-NMR (400 MHz, DMSO - d6): δ [ppm] 10.86 (s, 1 H), 9.11 (s, 1 H), 8.05 (t, J = 6.8 Hz, 1 H), 7.83 (t, J = 4.8 Hz, 1 H), 7.77 (s, 0.25 H), 7.67 - 7.53 (m, 3 H), 7.63 (s, 0.5 H), 7.50 (s, 0.25 H), 6.86 - 6.72 (m, 2 H), 4.80 - 4.67 (m, 1 H), 3.86 - 3.72 (m, 8 H), 3.67 - 3.63 (m, 4 H), 3.56 - 3.52 (m, 4 H), 3.29 - 3.25 (m, 4 H), 3.09 - 3.03 (m, 2 H), 2.81 - 2.72 (m, 1 H), 2.59 (t, J = 6.8 Hz, 2 H), 2.55 - 2.52 (m, 1 H), 2.48 - 2.42 (m, 4 H), 2.37 - 2.27 (m, 4 H), 2.20 - 2.06 (m, 1 H), 2.05 - 1.96 (m, 1 H), 1.51 - 1.35 (m, 4 H).

[0743] Cited prior art documents:

[0744] 1. W. Li, C. Gao, L. Zhao, Z. Yuan, Y. Chen, Y. Jiang, Eur. J. Med. Chem. 2018, 151, 237- 247.

[0745] 2. H. Wang, C. Li, X. Liu, M. Ma, Bioorg. Med. Chem. 2022, 61, 116707.

[0746] 3. Borsari, C., Rageot, C., Beaufils, F, Bohnacker, T., Keles, E., Buslov, I., Melone, A., Sele, A.M., Hebeisen, P., Fabbro, D., Hillmann.vP., and Wymann, M.P., ACS Medicinal Chemistry Letters 2019 10 (10), 1473-1479.

[0747] 4. WO 2019 / 195609 A2; 2019.

[0748] 5. Bohnacker, T.; Prota, A. E.; Beaufils, F.; Burke, J. E.; Melone, A.; Inglis, A. J.; Rageot, D.; Sele, A. M.; Cmiljanovic, V.; Cmiljanovic, N.; Bargsten, K.; Aher, A.; Akhmanova, A.; Diaz, J. F.; Fabbro, D.; Zvelebil, M.; Williams, R. L.; Steinmetz, M. O.; Wymann, M. P. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 2017, 8, 14683

[0749] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

Claims

Claims1. A compound comprising the general structureP-L-J-E, whereinP is a phosphoinositide 3-kinase (PI3K) binding moiety that binds class 1 phosphoinositide 3-kinase (PI3K), particularly a phosphoinositide 3-kinase (PI3K) binding moiety that binds PI3K-alpha and / or PI3K-beta,L is a linker with a linker length of 12 to 21 atoms, in particular with a linker length of 12 to 18 atoms,J is a junction selected from,, and, wherein R7is selected fromH and the group consisting of -C1-C3alkyl;E is an E3 ubiquitin ligase binding moiety that binds cereblon.

2. The compound according to claim 1 , comprising the general structureP-L-J-E, whereinP has the chemical structurewhereinX11is N;X12and X13are selected from CH and N;X14is N or CH;R6is» / wvI, wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16;R9is selected from one of the moieties ofwherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and - CH2F; R12is selected from the group consisting of -NH2, -NH2(=O)C1-3alkyl; X18and X19are each selected from N and C;E has the chemical structurewhereinX is CH or N, particularly CH,wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1X2is CH or N,X4is selected from CH2, C and N,X9is CH or N, particularly N;R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.X9is CH or N, particularly N;R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1 andJ is a junction selected from, , and, wherein R7isselected from H and the group consisting of -C1-C3alkyl;L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from one of the moieties andand SP is selected from a. -(CH2)n-; wherein n is an integer in the range of 4 and 14, and wherein n is chosen in such way that the length of the linker L is 12 to 21 atoms; b. -(CH2)m-B-; wherein m is an integer in the range of 0 and 9, wherein B and m are chosen in such way that the length of the linker L is 12 to 21 atoms; c. -(CH2)2-NR9-(CH2)3-; wherein R9is selected from H and -C(=O)CH3and whereinB is selected from -C(=O)-,NHC(=O)-(CH2)q-C(=O)-,, wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, andNH, and q is an integer in the range of 1 to 4 and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

3. The compound according to claim 1 , comprising the general structureP-L-J-E, whereinP has the chemical structurewhereinX11is N;X12and X13are selected from CH and N;X14is N or CH;R6is, wherein X15is N or O, R10is selected from the group consisting of - C1-C3alkyl, f is 0,1 or 2, and X16;R9is selected from one of the moieties ofwherein R11is selected from the group consisting of -C1-C3alkyl, -CF3, -CHF2and - CH2F; R12is selected from the group consisting of -NH2, -NHC(=O)C1-3alkyl; X18and X19are each selected from N and CE has the chemical structurewhereinX is CH or N, particularly CH,wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1X2is CH or N,X4is selected from CH2, C and N,X9is CH or N, particularly N;R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.X9is CH or N, particularly N;R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1 ; andJ is a junction selected from,, and, wherein R7is selected fromH and the group consisting of -C1-C3alkyl;L is of the formula R8dSP, wherein d is 0 or 1 and R8is selected from one of the moietiesand SP is selected from a. -(CH2)n-; wherein n is an integer in the range of 4 and 14, and wherein n is chosen in such way that the length of the linker L is 12 to 21 atoms;b. -(CH2)m-B-; wherein m is an integer in the range of 0 and 9, wherein B and m are chosen in such way that the length of the linker L is 12 to 21 atoms; and whereinwherein Y1, Y2and Y3are selected from CH andN, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4 and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

4. The compound according to any of the preceding claims, wherein E has the chemical structurewhereinX is CH or N, particularly CH,wherein R3and R4are =0 or -CH3, and b and c are 0 or 1 , p is 0 or 1 ,X2is CH or N,X4is selected from CH2, C and N,X9is CH or N, particularly N;R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

5. The compound according to any of the preceding claims, wherein E is selected fromwherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

6. The compound according to any of the preceding claims, wherein E is selected fromwherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

7. The compound according to any of the preceding claims, wherein E is selected fromwherein R2is selected from the group of halogens, particularly F, and a is 0, 1 or 2, particularly 0 or 1.

8. The compound according to any of the preceding claims, wherein E is selected from9. The compound according to any of the preceding claims, wherein E is selected from10. The compound according to any of the preceding claims, wherein E is selected from11. The compound according to any of the preceding claims, wherein P is selected from any one of the moieties12. The compound according to any of the preceding claims, wherein P is13. The compound according to any of the preceding claims, wherein L is -R8d-(CH2)n-J-; wherein n is an integer between 4 and 12, particularly wherein n is an integer between 6 and 12, and wherein R8is selected from one of the moietiesand J is selected from14. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)n-J-; wherein R8dis selected from; d is 1 ; J is, and n is an integer in the range of 4 to 12, particularly wherein R8dis selected fromand n is an integer in therange of 4 to 12, more particularly R8dn is an integer in the range of 5 to 9.

15. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesand J is selected from, and B is selected from -C(=O)-wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

16. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietieswherein Y1and Y3areselected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

17. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesand J is selected fromand, and B is selected fromselected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

18. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesand J is selected from, and B is selected from,wherein Y1is selected from CH and N, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

19. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesand J is selected from, and B is selected from -C(=O)-,-NHC(=O)-(CH2)q-C(=O)-,, wherein Y1, Y2and Y3are selected from CH and N, and Z1is selected from O, and NH, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.

20. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesand J is selected fromand and B is selected fromandwherein Y1and Y3are selected from CH and N, and q is an integer in the range of 1 to 4, and wherein R8, m and B are chosen in such way that the length of the linker L has 12 to 21 atoms.21 . The compound according to any of the preceding claims, wherein L is-R8d-(CH2)m-B-J-; wherein m is an integer between 0 and 9, and wherein R8is selected from one of the moietiesare chosen in such way that the length of the linker L has 12 to 21 atoms.

22. The compound according to any of the preceding claims, wherein L is-R8d-(CH2)2-NR9-(CH2)3-J; wherein R9is selected from H and -C(=O)CH3and wherein23. The compound according to any of the preceding claims, wherein the compound is selected from24. The compound according to any of the preceding claims, wherein the compound is selected from25. A compound according to claims 1 to 24 for use as a medicament.

26. A compound according to claims 1 to 24 for use in the treatment of a disease, wherein the disease is cancer.

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