Chemical Linkers

Abstract

Provided are heterobifunctional chemical linkers that are useful in protein degradation agents and other medicinal chemical applications.Heterobifunctional linkers include alkyne-piperidine and alkane-piperidine linkers; alkane-piperazine linkers; alkane amino PEGylation linkers; dipiperazine linkers; piperazine-pyridine / pyrimidine-alkyne linkers; pyridine-PEG-piperazine linkers and pyrimidine-piperazine-PEG linkers.Also provided are combinations of provided linkers with ligands that target degradation pathways, such as E3 ubiquitin ligase enzymes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 265,717, filed December 20, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Protein degraders are heterobifunctional molecules that contain at one end a ligand that targets a protein of interest (A); at the other end a ligand that targets a ligase or other enzyme useful for degrading the protein of interest (C); and a linker (B) that connects the protein-targeting ligand and the enzyme-targeting ligand. Much of the research in such protein degraders has focused on ligands that target ligases, particularly ligands that target E3 ubiquitin ligase enzymes. Another focus of research is the combination of protein-targeting ligands and ligase-targeting ligands. However, the choice of linker has been little studied.

[0003] The choice of linker is important for these heterobifunctional proteolytic agents, although it has been little studied. The length and polarity of the linker affect, for example, cell permeability and solubility. There is a need for new linkers that can provide properties tailored to heterobifunctional proteolytic agents. Due to the myriad of potential applications, there is a need for new linkers that can provide specific properties, such as lipophilicity or hydrophilicity, high solubility, and rigidity or flexibility. Summary of the Invention

[0004] summary Chemical linkers useful in proteolytic and other applications are provided. In a first embodiment, the chemical linker is represented by formula I [ka] wherein R1 is optional and, when present, is selected from piperidine and piperazine; R2 is selected from C1-C8 alkanes and C2-C8 alkynes; and PG is an amine protecting group. is a chemical linker.

[0005] A second embodiment is a compound of formula II [ka] (wherein X is C or N; m is 1 to 6; Y is -O- or -CH2-, n is 0 to 4, o is 0 or 1, and p is 0 to 9.) Provide a linker for:

[0006] A third embodiment is a compound of formula III [ka] wherein Y is -O- or -CH2-; R3 is C1-C6 alkyl, C3-C6 cycloalkyl, or cycloalkyl-substituted alkyl; and p is 1 or 2. Provide a linker for:

[0007] A fourth embodiment is a compound of formula IV [ka] wherein PG is an amine protecting group; m is 1 to 4; each X is independently C or N; and Z is selected from -C(O)-, -C(S)-, -CH2-, or -CF2-. Provide a linker for:

[0008] A fifth embodiment is a compound of formula V [ka] (wherein PG is an amine protecting group, X is N or C, and p is 1 to 3.) Provide a linker for:

[0009] A sixth embodiment is a compound of formula VI [ka] where PG is an amine protecting group; each X is independently C or N; and n is 0 to 2. Provide a linker for:

[0010] A seventh embodiment is a compound of formula VII [ka] where PG is an amine protecting group; X is C or N; Y is N, O or C; m is 1 to 3; and n is 1 to 3. Provide a linker for: [Brief description of the drawings]

[0011] [Figure 1] The figure is an illustration showing a proteolytic agent that includes (A) a ligand that targets a protein of interest, (B) a connecting linker, and (C) an enzyme-targeting ligand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description A proteolytic agent is a combination of three components: A, a ligand for a target protein, B, a linker molecule, and C, a handle or ligand for an enzyme, such as an E3 ubiquitin ligase enzyme. Novel heterobifunctional chemical linkers of formulas I-VII are provided. The disclosed linkers are particularly useful in the fields of targeted protein degradation and targeted drug delivery. One exemplary use is in proteolytic agents, i.e., in proteolytic targeting chimeras (PROTACs). Such proteolytic agents include a ligand specific for a target protein (A), a handle for an E3 ubiquitin ligase enzyme (C), and a linker (B) that connects the target protein-specific ligand with the E3 ubiquitin ligase enzyme, as illustrated in the figures. The linkers disclosed herein are designed to have hydrophobic or hydrophilic functional groups, high solubility, and various degrees of rigidity and / or flexibility, making these linkers suitable for a wide range of applications. Also provided are proteolytic agent components that include a chemical linker that is attached to a handle for an E3 ubiquitin ligase enzyme. Such a proteolytic agent component is in a state where it binds to a target protein ligand.The linkers provided herein are also useful in other applications, including targeted drug delivery and discovery.

[0013] In a first embodiment, the chemical linker has formula I [ka] [wherein R1 is optional and, when present, is selected from piperidine and piperazine; R2 is a C1-C8 alkane, i.e., -CH2- to -C8H 16 - and C2-C8 alkynes, e.g. -C≡C- or -C2- to -C8H 12 -, and PG is an amine protecting group. In some embodiments, R2 is selected from ethane (-C2H4-) and ethyne (-C≡C-). In some embodiments, the protecting group PG is tert-butoxycarbonyl (tert-butyloxycarbonyl, Boc).

[0014] In some preferred embodiments, the linker of formula I is [ka] It is one of the following.

[0015] In a second embodiment, the compound of formula II [ka] (wherein X is selected from C and N; m is 1 to 6; Y is selected from -O- and -CH2-, n is 0 to 4, o is 0 or 1, and p is 0 to 9.) A linker of the formula:

[0016] In some embodiments, the linker of formula II is of formula IIa [ka] wherein Y is selected from -CH2- and -O; n is 0 to 5, and p is 1 to 5. In some embodiments of Formula IIa, n is 0, 1, or 4. In some embodiments of Formula IIa, p is 1 or 5.

[0017] In some embodiments, the linker of formula II is [ka] wherein X is selected from C and N, Y is selected from -CH2- and -O-; m is 0 or 1, and n is 0, 1, 2, or 3. is a linker.

[0018] In yet other embodiments, the linker of formula II is of formula IIc [ka] wherein X is selected from C and N; Z is selected from -CH2-, -CF2-, -C(O)-, and -C(S)-; m is 2 to 5; p is 1 or 2; and PG is an amine protecting group. In some embodiments, m is 2, 3, or 5. In some embodiments, the preferred amine protecting group, PG, is Boc.

[0019] Also, Formula III [ka] wherein Y is selected from -O- and -CH2-; R3 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, and cycloalkyl-substituted alkyl; and p is 1 or 2. In various embodiments, R3 is selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and methylenecyclobutyl. [ka] is selected from.

[0020] Also, Formula IV [ka] where PG is an amine protecting group; m is 1 to 4; each X is independently selected from C and N; and Z is selected from -C(O)-, -C(S)-, -CH2-, and -CF2-. In some embodiments, the preferred amine protecting group, PG, is Boc. In some embodiments, both X are C. In other embodiments, both X are N. In yet other embodiments, one X is C and one X is N.

[0021] Also, formula V [ka] (wherein PG is an amine protecting group, X is N or C, and p is 1 to 3.) In some embodiments, the preferred amine protecting group, PG, is Boc.

[0022] Also, Formula VI [ka] (wherein PG is an amine protecting group; X is C or N; and n is 0 to 2.) In some embodiments, the preferred amine protecting group, PG, is Boc.

[0023] Also, Formula VII [ka] wherein PG is an amine protecting group; X is C or N; Y is selected from N, O and C; m is 1 to 3; and n is 1 to 3. In some embodiments, the preferred amine protecting group, PG, is Boc.

[0024] The linkers of formulas I-VII are useful in a myriad of applications, for example in protein degraders, as illustrated in the figures. The length and polar composition of the linker of the protein degrader influences properties such as cell permeability and solubility. The linkers provided herein provide a mixture of hydrophobic and hydrophilic functional groups, balancing the hydrophobic-hydrophilic properties of the resulting hybrid compounds. These linkers, when combined with various ligands for the E3 ubiquitin ligase enzyme, can be used to generate a wide variety of degrader components (B+C). Using these degrader components and combining them with ligands (A) for the target protein, a large library of protein degraders for each purpose can be created, which allows for screening a large number of compounds to be screened, potentially identifying new drugs.

[0025] Further provided are compounds having a linker of formula I-VII and a ligand that triggers a degradation pathway. In some embodiments, the ligand that triggers a degradation pathway is an E3 ubiquitin ligase enzyme. In some embodiments, the ligand for the E3 ubiquitin ligase enzyme is a derivative of an immunomodulatory drug (IMiD). In some embodiments, the derivative of an IMiD is selected from a pomalidomide derivative, a thalidomide derivative, a lenalidomide derivative, and a VH032 derivative.

[0026] The linkers provided herein are also useful to researchers working in other therapeutic areas and medicinal chemistry projects. The linkers provided are useful in the design and function of biologically active molecules and in targeted drug delivery and discovery. EXAMPLES

[0027] Example 1. Preparation of Representative Alkyne-Piperidine Linkers [ka] Step 1: Synthesis of tert-butyl 4-(2,2-dibromoethenyl)piperidine-1-carboxylate Triphenylphosphine (123.0 g, 468.8 mmol, 4.0 equiv.) and tetrabromomethane (77.76 g, 234.42 mmol, 2.0 equiv.) were placed in a 3 L four-neck round-bottom (RB) flask equipped with a rotor, thermometer, and a 500 mL pressure-equalizing addition funnel topped with a N2 inlet adapter. The reaction was then cooled to -30°C (± 5°C) with a dry ice-isopropyl alcohol (IPA) mixture while stirring under a nitrogen atmosphere. Dichloromethane (DCM) (500 ml) was added slowly through the addition funnel over 45 min with vigorous stirring under a nitrogen atmosphere. The reaction was stirred at -20 °C for 15 min, followed by the slow addition of a solution of 1-boc piperidine-4-carboxaldehyde 1 (25.0 g, 117.21 mmol, 1.0 equiv) in DCM (125 ml) over a period of 2 h under vigorous stirring at a temperature of -20 °C (± 5 °C). The reaction was then stirred at room temperature (RT) under nitrogen for 18 h. After completion of the reaction (as confirmed by TLC in 10% ethyl acetate / hexane; R f The reaction was filtered through a sintered funnel (pH 7.5:0.5) and the residue was washed with DCM (100 ml). The filtrate was concentrated to dryness. The concentrated fraction was diluted with hexane (1 L) to give a suspension. The suspension was filtered through a sintered funnel and the residue was washed with hexane (200 ml). The filtrate was concentrated at 40 °C. The suspension preparation, filtration, and subsequent filtrate concentration were repeated twice to remove the unwanted polar impurity (triphenylphosphine oxide). Finally, the compound was dried under vacuum to give the dibromoalkene 2 (24 g, 79% yield) as an off-white solid. 1 H NMR (CDCl3, 400 MHz): δ 6.23(d, J = 8.8 Hz, 2H), 4.05(brs, 2H), 2.79(t, J = 11.6 Hz, 2H), 2.45-2.39(m, 1H), 1.70-1.66(m, 2H), 1.46(s, 9H), 1.35-1.28(m, 2H).

[0028] Step 2: Synthesis of tert-butyl 4-(3-ethoxy-3-oxoprop-1-yn-1-yl)piperidine-1-carboxylate A 1 L 4-neck RB flask equipped with a rotor, cooling bath, thermometer, and two 500 ml pressure equalizing addition funnels topped with N2 inlet adapters was charged with tert-butyl 4-(2,2-dibromoethenyl)piperidine-1-carboxylate 2 (15.0 g, 40.64 mmol, 1.0 equiv.), and tetrahydrofuran (THF) (300 ml). The reaction was then cooled to -78 °C via a dry ice acetone bath under nitrogen atmosphere, followed by the dropwise addition of 1.4 M n-butyllithium (84 ml, 117.86 mmol, 2.9 equiv.) over 1 h at -78 °C through the liquid addition funnel under nitrogen atmosphere. The reaction was then stirred at -78 °C for 1 h and then at 0 °C for an additional 1 h under nitrogen atmosphere. The reaction was cooled to -78°C and ethyl chloroformate (14.4 ml, 150.37 mmol, 3.7 equiv) was added dropwise to the reaction over 30 minutes under nitrogen. The reaction was allowed to warm to 25°C over 2 hours and completion of the reaction was confirmed by thin layer chromatography (TLC) (30% ethyl acetate in hexanes, R f =0.6, visualized after charging with phosphomolybdic acid (PMA) TLC staining). The reaction mixture was carefully quenched by dropwise addition of ice-cold water (15 ml) at -78°C (±2°C) and carefully diluted by dropwise addition of saturated NaCl solution (200 ml) at -78°C (±2°C) and extracted with ethyl acetate (250 mL X 3). The organic layers were combined, dried over anhydrous sodium sulfate (50 g), filtered, and concentrated to dryness under reduced pressure at 42°C to give the crude product. The crude material was purified by silica gel (60-120 mesh) column chromatography with 4% ethyl acetate-hexane. The pure fractions were combined and concentrated to dryness under reduced pressure at 42°C to give ester 3 (6.5 g, 57% yield) as a light brown oily liquid. 1H NMR (CDCl3, 400 MHz): δ 4.20-4.15(m, 2H), 3.69-3.65(m, 2H), 3.17-3.11(m, 2H), 2.69-2.65(m, 1 H), 1.81-1.75(m, 2H), 1.64-1.62(m, 2H), 1.40(s,9H), 1.30-1.25(m, 3H).

[0029] Step 3: Synthesis of 1-piperidinecarboxylic acid, 4-(2-carboxyethynyl)-, 1-(1,1-dimethylethyl) ester A 500 mL four-neck RB flask was equipped with a rotor, a thermometer, and a N2 inlet adapter. tert-Butyl 4-(3-ethoxy-3-oxoprop-1-yn-1-yl)piperidine-1-carboxylate 3 (6.50 g, 23.10 mmol, 1 equiv.), THF (65 mL) and water (26 mL) were added to it. The reaction was then cooled to 0°-5°C in an ice bath. One lot of lithium hydroxide monohydrate (1.94 g, 46.23 mmol, 2 equiv.) was added. The reaction was then stirred at 25°C for 15 h. After completion of the reaction (confirmed by TLC in 10% methanol / DCM; R f After 10 min at 37° C. (pH 7.0:0.1), the reaction was concentrated to remove volatile solvents and the residue was washed with ethyl acetate (3× 100 ml). The ethyl acetate fraction was discarded and the aqueous fraction was acidified with saturated aqueous citric acid to pH 3. The aqueous fraction was then extracted with ethyl acetate (3×250 ml). The combined organics were washed with water (3×100 ml) and brine (2×100 ml). The organic layer was dried over anhydrous sodium sulfate (50 g), filtered and concentrated to dryness under reduced pressure at 42° C. to give the crude product. The crude material was washed with n-hexane (200 ml), filtered and dried in vacuum to give acid 4 (4.50 g, 77% yield) as an off-white solid. 1 H NMR (CDCl3, 400 MHz): δ 8.50(bs, 1H), 3.71-3.68(m, 2H), 3.23-3.16(m, 2H), 2.74-2.72(m, 1H), 1.84-1.81(m, 2H), 1.68-1.63(m, 2H), 1.44(s,9H).

[0030] Example 2. Synthesis of Representative Alkane-Piperazine Linkers [ka] Step 1: Synthesis of tert-butyl 3-(2-hydroxyethoxy)propanoate To the reaction flask was added ethylene glycol (125.0 g, 2.01 mol, 1.0 equiv) in 1000 ml dry THF with stirring. Sodium metal (1.38 g, 60.0 mmol, 0.03 equiv) was then added portionwise to the reaction flask and the reaction mixture was heated at 50°C for 4 h to obtain a clear solution. The reaction mixture was cooled to 0°C and a solution of tert-butyl acrylate (206.1 g, 1.61 mol, 0.8 equiv) in 300 ml dry THF was added dropwise over 1 h. The reaction mixture was then stirred at room temperature for 15-16 h. The progress of the reaction was monitored by TLC (30% ethyl acetate in hexanes; Rf: 0.25). The reaction mixture was diluted with 1.5 L of ethyl acetate and the organic layer was washed with ice-cold water, saturated NaHCO3 and brine. The organic layer was concentrated on a rotary evaporator to give the crude product as a pale green gel, which was then purified via column chromatography on 100-200 mesh silica gel, eluting the product with 10-15% ethyl acetate in hexanes. The fractions were combined and evaporated to give pure tert-butyl 3-(2-hydroxyethoxy)propanoate 5 (68.0 g, 18% yield) as a pale greenish liquid. 1 H NMR (CDCl3, 400 MHz): δ 3.75(t, 4H), 3.62(t, 2H), 2.52(t, 2H), 1.43(s, 9H).

[0031] Step 2: Synthesis of 3-(2-hydroxyethoxy)propanoic acid In a 1 L RB flask, tert-butyl 3-(2-hydroxyethoxy)propanoate 5 (68.0 g, 357.4 mmol, 1.0 equiv) was placed in 400 ml DCM. The solution was cooled to 0° C. and then a solution of TFA (108 ml) in 200 ml DCM was added through an addition funnel over 30 min. The reaction mixture was then stirred at room temperature for 15-16 h. The progress of the reaction was monitored by TLC (5% methanol in dichloromethane; Rf: 0.1). After completion, the reaction mixture was evaporated to dryness using a rotary evaporator to obtain a thick gel. The crude material was dissolved in 20 mL of 10% methanol in DCM and triturated with n-hexane (250 ml X 2). The hexane layer was then decanted and the residue was evaporated to obtain a light brown gel as tert-butyl 3-(2-hydroxyethoxy)propanoate 6 (44.0 g, 92% yield). 1 H NMR (CDCl3, 400 MHz): δ 3.75(t, 4H), 3.62(t, 2H), 2.32(t, 2H), 11.2(brs, 1H).

[0032] Step 3: Synthesis of ethyl 3-(2-hydroxyethoxy)propanoate A 1000 ml four-neck RB flask was fitted with a reflux condenser and a N2 inlet adapter, 3-(2-hydroxyethoxy)propanoic acid 6 (40.0 g, 198.2 mmol, 1.0 equiv.) was dissolved in 600 ml ethanol and stirred into the reaction flask. Concentrated H2SO4 (12.5 ml) was then added dropwise to the reaction mixture and the reaction mixture was refluxed at 75°C for 4 h. After completion (checked by TLC in 5% methanol in dichloromethane; Rf: 0.6), the reaction mixture was cooled to 30°C (±2) and diluted with 1.5 L of DCM and the organic layer was washed with cold water (1000 ml X 2) and saturated NaHCO3 solution (1000 ml). The organic layer was evaporated to dryness using a rotary evaporator to obtain a pale yellow liquid. The crude material was purified by silica gel (100-200 mesh) column chromatography using 2% methanol in DCM as eluent. The fractions containing the pure product were combined and evaporated to dryness under reduced pressure at 40 °C to give ethyl 3-(2-hydroxyethoxy)propanoate 7 as a pale yellow liquid (25 g, 52% yield). The crude product was carried on to the next step without any purification. 1 H NMR (CDCl3, 400 MHz): δ 4.02(q, 2H),3.75(t, 4H), 3.62(t, 2H), 2.52(t, 2H), 1.23(t, 3H), LCMS(ESI, m / z): 163.0(M+H) + .

[0033] Step 4: Synthesis of ethyl 3-(2-bromoethoxy)propanoate A 2 L 4-neck RB flask was fitted with a rotor, a 500 ml addition funnel and a N2 inlet adapter, and ethyl 3-(2-hydroxyethoxy)propanoate 7 (25.0 g, 154.1 mmol, 1.0 equiv.) in 600 ml dry THF was added to the reaction flask with stirring. Triphenylphosphine (60.6 g, 231.2 mmol, 1.5 equiv.) was added to the reaction mixture in one portion, and the reaction mixture was stirred at room temperature for 10-15 min to obtain a clear solution. The reaction mixture was cooled to 0 °C, and a solution of CBr4 (76.7 g, 231.2 mmol, 1.5 equiv.) in 400 ml dry THF was added dropwise to the reaction mixture over 30 min. The reaction mixture was then stirred at room temperature for 2-3 h. The progress of the reaction was monitored by TLC (30% ethyl acetate in hexane; Rf: 0.7). The white precipitate was filtered through filter paper, washed with THF (100 ml), and the filtrate was evaporated to give a sticky precipitate. The crude material was then purified using silica gel (60-120 mesh) column chromatography eluting with 30% ethyl acetate in hexane. The pure fractions containing the pure product were combined and evaporated to dryness under reduced pressure at 40 °C to give ethyl 3-(2-bromoethoxy)propanoate 8 (21 g, 61% yield) as a gummy liquid. 1 H NMR (CDCl3, 400 MHz): δ 4.02(q, 2H), 3.75(t, 4H), 3.43(t, 2H), 2.62(t, 2H), 1.43(s, 9H), 1.23(t, 3H)

[0034] Step 5: Synthesis of tert-butyl 4-(2-(3-ethoxy-3-oxopropoxy)ethyl)piperazine-1-carboxylate A 1000 mL four-neck RB flask was fitted with a rotor, reflux condenser and N2 inlet adapter, and ethyl 3-(2-bromoethoxy)propanoate 8 (14.6 g, 64.9 mmol, 1.10 equiv.) was stirred into 600 ml acetonitrile in the reaction flask. To this reaction mixture was then added Boc-piperazine (11.0 g, 58.9 mmol, 1.0 equiv.) in one portion, followed by potassium carbonate (20.4 g, 147.3 mmol, 2.5 equiv.). KI (2.4 g, 14.7 mmol, 0.25 equiv.) was then added and the reaction mixture was refluxed at 82 ± 2 °C for 18 h. The progress of the reaction was monitored by TLC (5% methanol in dichloromethane; Rf: 0.3), the reaction mixture was cooled to room temperature and the white precipitate was filtered through a cellite bed and washed with acetonitrile (100 ml). The filtrate was evaporated to dryness and the crude compound was purified via column chromatography (60-120 mesh silica gel) using 2-3% methanol in dichloromethane as eluent. The organic fractions containing the pure product were combined and evaporated to dryness under reduced pressure at 40 °C to give tert-butyl 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-1-carboxylate 9 (15 g, 77% yield) as a pale yellow solid. 1 H NMR (CDCl3, 400 MHz): δ 4.02(q, 2H), 3.65(t, 4H), 3.43(t, 4H), 2.72(t, 2H), 2.52(t, 4H), 2.45(t, 2H), 1.43(s, 9H), 1.23(t, 3H).

[0035] Step 6: Synthesis of 3-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethoxy)propanoic acid tert-Butyl 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-1-carboxylate 9 (15.0 g, 45.4 mmol, 1.0 equiv.) was dissolved in a mixture of THF-water (1:1, 400 ml). Lithium hydroxide monohydrate (5.7 g, 136.2 mmol, 3.0 equiv.) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 4-5 h. Completion of the reaction was confirmed by TLC monitoring (20% methanol in dichloromethane; Rf; 0.015). The reaction mixture was diluted with 500 ml ethyl acetate and the separated organic layer was discarded. The aqueous layer was acidified to pH approx. 6 (± 0.2) using a pH meter with 1N citric acid solution and the aqueous layer was lyophilized for 18 h to give an off-white solid. The crude material was then subjected to silica gel (60-120 mesh size) column chromatography using 8-10% methanol in dichloromethane as eluent. Fractions containing pure product were combined and evaporated to dryness under reduced pressure to give 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-1-carboxylic acid 10 (6.6 g, 48% yield) as white solid flakes. 1 H NMR (D6-DMSO, 400 MHz): δ 10.5-13.10(brs, 1H), 3.65(t, 4H), 3.43(t, 4H), 2.72(t, 2H), 2.52(t, 4H), 2.45(t, 2H), 1.43(s, 9H).

[0036] Example 3. Preparation of Representative Alkanamino PEGylated Linkers [ka] Step 1: Synthesis of tert-butyl N-[2-(2-hydroxyethoxy)ethyl]carbamate A solution of di-tert-butyl dicarbonate (32.0 g, 148.0 mmol, 1.1 equiv) in methylene chloride (70 ml) was added dropwise to a solution of 3-amino-1-propanol 11 (10.1 g, 134.0 mmol, 1.0 equiv) in methylene chloride (100 ml). The reaction mixture was stirred overnight and washed with saturated aqueous sodium bicarbonate solution, water and then brine. The organic layer was dried (Na2SO4) and the organic layer was rotary evaporated to give 3-(N-tert-butoxycarbonylamino)-1-propanol 12 (23 g, 98% yield) as a colorless oil. 1 H NMR (CDCl3, 400 MHz): δ 4.78 (brs, 1H), 3.65 (m, 2H), 3.30 (m, 2H), 2.90 (brs, 1H), 1.68 (m, 2H), 1.48 (s, 9H).

[0037] Step 2: Synthesis of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-methylbenzenesulfonate To a solution of tert-butyl N-[2-(2-hydroxyethoxy)ethyl]carbamate 12 (20.0 g, 114.0 mmol, 1.0 equiv.) in methylene chloride was added triethylamine (31.8 ml, 228.0 mmol, 2.0 equiv.) at 0° C. using an ice-cooled bath. p-Toluenesulfonyl chloride (32.6 g, 171.0 mmol, 1.5 equiv.) was added in portions over 15 min. The reaction mixture was then stirred overnight and then quenched with cold water (100 ml). The organic layer was dried (Na2SO4) and the volatiles removed by evaporation to give a gummy residue which was purified by column chromatography using silica 60-120 mesh and the compound was eluted with 10% ethyl acetate / hexane to give 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-methylbenzenesulfonate 13 (12 g, 32% yield) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3): δ 1H NMR (400 MHz, CDCl3): δ 3.58(t, J = 5.09 Hz, 2H), 3.51(t, J = 5.24 Hz, 2H), 3.34-3.27(m, 2H), 2.78(t, J = 4.89 Hz, 2H), 1.44(s, 9H).

[0038] Step 3: Synthesis of 1,1-dimethylethyl N-[2-[2-(methylamino)ethoxy]ethyl]carbamate To a solution of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-methylbenzenesulfonate 13 (8.0 g, 24.3 mmol, 1.0 equiv.) in EtOH (40 ml) in a sealed tube, methylamine (10 mL, 33 wt.% in absolute ethanol) was added at room temperature, and the sealed tube was sealed and placed in an oil bath. The reaction mixture was heated to 110° C., and the reaction mixture was stirred overnight. The reaction mixture was then evaporated under reduced pressure to give a gummy residue, which was purified by silica gel column chromatography eluting with 70% ethyl acetate / hexane to give 1,1-dimethylethyl N-[2-[2-(methylamino)ethoxy]ethyl]carbamate 14 (4.3 g, 98% yield) as a colorless liquid. 1 H NMR (400 MHz, CDCl3): δ 4.10-4.08(m, 2H), 3.57-3.54(m, 2H), 3.3 3.37(m, 2H), 3.17-3.16(m, 2H), 2.38(s, 3H), 1.37(s, 9H).

[0039] Step 4: Synthesis of ethyl 3-{[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethyl](methyl)amino}propanoate To a solution of 1,1-dimethylethyl N-[2-[2-(methylamino)ethoxy]ethyl]carbamate 14 (1.0 g, 5.31 mmol, 1.0) in THF (10 ml) was added N,N-diisopropylethylamine (2.8 ml, 15.9 mmol, 3.0) and ethyl acrylate (1.0 g, 10.6 mmol, 2.0) at 0 °C. The reaction was stirred at 0 °C for 10 min and then slowly heated to 105 °C and heated at the same temperature for 3 h. The reaction was quenched with water and extracted with DCM three times. The organic layer was dried (Na2SO4) and the volatiles removed by evaporation to give crude 4-benzyl 7-ethyl 8-oxo-4-azaspiro[2.5]octane-4,7-dicarboxylate which was purified by column chromatography using silica 100-200 mesh and the compound was eluted in 5% MeOH / DCM to give pure ethyl 3-{[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethyl](methyl)amino}propanoate 15 (0.48 g, 31% yield) as a colorless liquid. 1 H NMR (400 MHz, CDCl3): δ 4.07(q, J = 7.17 Hz, 2H), 3.52-3.41(m, 4H), 3.26-3.19(m, 2H), 2.71(t, J = 7.46 Hz, 2H), 2.53(t, J = 5.45 Hz, 2H), 2.42(t, J = 7.26 Hz, 2H), 2.22(s, 3H), 1.37(s, 9H), 1.18(t, J = 7.34, 3H).

[0040] Step 5: Synthesis of 3-{[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethyl](methyl)amino}propanoic acid To a solution of ethyl 3-{[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethyl](methyl)amino}propanoate 15 (10 g, 34.7 mmol, 1.0 equiv.) in MeOH-THF-H2O (1:1:1, 30 ml) was added LiOH.H2O (1.7 g, 69.4 mmol, 2.0 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 18 h. The solvent was evaporated under reduced pressure and extracted three times with DCM. The organic layer was dried (Na2SO4) and the organic layer was rotary evaporated to give the crude product as the lithium salt of the carboxylic acid. This salt was then redissolved in distilled water and further treated with Amberlyst-15 hydrogen ion resin. The pH of the aqueous layer was adjusted to pH ∼5 using a pH meter. The resin was removed by filtration and the aqueous layer was lyophilized to give 3-{[2-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)ethyl](methyl)amino}propanoic acid 16 (3.8 g, 42% yield) as a white gummy hygroscopic solid. 1 H NMR (400 MHz, CDCl3): δ 3.57-3.51(m, 4H), 3.32-3.30(m, 2H), 2.69-2.65(m, 2H), 2.60-2.56(m, 2H), 2.38-2.34(m, 2H), 2.16(s, 3H), 1.37(s, 9H).

[0041] Example 4. Preparation of Representative Dipiperazine Linkers [ka] Step 1: Synthesis of tert-butyl (2-(piperazin-1-yl)ethyl)carbamate A 2000 mL three-neck RB flask was fitted with a rotor, reflux condenser and N2 inlet adapter. tert-Butyl (2-bromoethyl)carbamate 17 (24.4 g, 108.9 mmol, 1.0 equiv) was added to 1200 mL of acetonitrile. To the reaction flask, under stirring under N2 atmosphere, piperazine (37.5 g, 435.7 mmol, 4.0 equiv) was added followed by K2CO3 (45.2 g, 326.7 mmol, 3.0 equiv) and KI (18.1 g, 108.9 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 20 min. The reaction mixture was stirred at 80 °C for 10 h. The progress of the reaction was monitored by TLC (R in 10% MeOH:DCM, f The reaction mixture was monitored by HPLC (HPLC:0.2). After completion, the reaction mixture was cooled to room temperature, acetonitrile was evaporated and the residue was diluted with DCM (1 L). The organic layer was washed with water (3 X 500 mL). The organic layer was dried over sodium sulfate and evaporated to dryness using a rotary evaporator to give the crude product. The crude material was purified by silica gel (60-120 mesh) column chromatography with 4% methanol-DCM. The pure fractions were combined and concentrated to dryness under reduced pressure at 42 °C to give tert-butyl (2-(piperazin-1-yl)ethyl)carbamate 18 (12.0 g, 48% yield) as a pale yellow oily liquid. 1 H NMR (CDCl3, 400 MHz): δ 3.31-3.21(m, 2H), 2.65(m, 4H), 2.46(m, 2H), 2.34(m, 4H), 1.40(s, 9H).

[0042] Step 2: Synthesis of tert-butyl (2-(4-(piperazine-1-carbonyl)piperazin-1-yl)ethyl)carbamate A 2000 ml three-neck RB flask was fitted with a rotor, a 250 ml addition funnel and a N2 inlet adapter. tert-Butyl (2-(piperazin-1-yl)ethyl)carbamate 18 (12 g, 52.3 mmol, 1.0 equiv) in DCM (800 ml) was stirred for 10 min and cooled to 0 °C. DIPEA (14.0 ml, 78.5 mmol, 1.5 equiv) was then added, followed by triphosgene (4.7 g, 15.7 mmol, 0.30 equiv) (dissolved in 100 ml DCM) dropwise through the addition funnel and the resulting reaction mixture was stirred at 0 °C under N2 atmosphere for 1 h. The progress of the reaction was monitored by TLC (10% methanol in dichloromethane). After complete consumption of tert-butyl (2-(piperazin-1-yl)ethyl)carbamate by TLC, N,N-diisopropylethylamine (14.0 ml, 78.5 mmol, 1.5 equiv.) was added followed by piperazine (13.5 g, 157.0 mmol, 3.0 equiv.) in one portion and stirred at room temperature for 3 h. The reaction mixture was poured into ice water (500 ml) and extracted with DCM (3×300 ml). The organic layer was separated and washed with water (3×1 L). The organic layer was dried over sodium sulfate and the reaction mixture was evaporated to dryness using a rotary evaporator to give the crude product. The crude material was purified using C18 (120 g) column chromatography using 40% acetonitrile in water as eluent followed by combining and lyophilizing fractions containing pure product to give a light brown solid which was triturated in diethyl ether:hexane (2:1). The precipitated solid was separated by decanting the solvent layer and evaporated to dryness under reduced pressure at 40 °C to give tert-butyl (2-(4-(piperazine-1-carbonyl)piperazin-1-yl)ethyl)carbamate 19 (2.8 g, 16% yield) as a pale yellow solid. 1 H NMR(D 6- DMSO, 400 MHz): δ 9.31(brs, 1H), 6.73(brs, 1H), 2.89-3.65(m, 14H), 2.65-2.23(m, 6H), 1.40(s, 9H). LCMS(ESI, m / z): 342.1(M+H) + .

[0043] Example 5. Preparation of Representative Piperazine-Pyridine / Pyrimidine-Alkyne Linkers [ka] Step 1: Synthesis of tert-butyl (3-(6-fluoropyridin-3-yl)prop-2-yn-1-yl)carbamate A 2 L RB flask was fitted with a rotor, condenser and an argon inlet adapter. 1-Propanol (500 mL) was charged and degassed with argon for 60 min. N-Boc-propargylamine (44.2 g, 283.9 mmol, 1.0 equiv.) was added and degassed with Ar for 60 min. Copper(I) iodide (5.4 g, 28.4 mmol, 0.1 equiv.) was then added and degassed with Ar for 30 min. Tetrakis(triphenylphosphine)palladium(0) (16.4 g, 14.2 mmol, 0.05 equiv.) was then added and degassed with Ar for 30 min and stirred under Ar.

[0044] Preparation of sodium carbonate solution A 250 mL three-neck RB flask was fitted with a rotor and an Ar inlet adapter. Demineralized water (150 mL) was taken in the RB flask and degassed with Ar for 30 min. Sodium carbonate (39.11 g, 369.1 mmol, 1.3 equiv.) was then added to DM water, degassed with Ar for 30 min, and stirred under Ar atmosphere to prepare an aqueous solution of sodium carbonate. The aqueous solution of sodium carbonate was added to the reaction mixture in a 2 L RB flask and stirred under Ar atmosphere.

[0045] Preparation of 5-bromo-2-fluoropuridine in 1-propanol A 250 mL three-necked RB flask was equipped with a rotor and an Ar inlet adapter. 1-Propanol (100 mL) was placed in the RB flask and degassed with Ar for 30 min. 5-Bromo-2-fluoropuridine 20 (50 g, 283.9 mmol, 1.0 equiv.) was added, degassed with Ar for 30 min, and stirred for 30 min to prepare a solution.

[0046] The prepared solution was added to the reaction in a 2 L RB flask and degassed with Ar for 30 min. The reaction was then refluxed under Ar atmosphere for 18 h. The progress of the reaction was monitored by TLC in 10% ethyl acetate in hexane. It was cooled to room temperature and concentrated to dryness on a rotary evaporator at 40-45 °C under reduced pressure. The residue was dissolved in 1 L of DCM and passed through a bed of anhydrous sodium sulfate. The bed was washed with DCM (500 mL). The filtrate was concentrated on a rotary evaporator at 45 °C under reduced pressure to obtain the crude material. The crude material was purified by silica gel (100-200 mesh) column chromatography using 5% ethyl acetate in hexane as eluent. The pure fraction was collected in 3.5-5% ethyl acetate in hexane. Pure fractions were combined and evaporated at 45° C. to give tert-butyl-{3-(6-fluoropyridin-3-yl)prop-2-yn-1-yl}carbamate 21 (36 g, 50% yield) as a pale yellow liquid. 1 H NMR (CDCl3, 400 MHz): δ 8.62(s, 1H), 8.36(m, 1H), 8.20(brs, 1H), 7.23(m, 1H), 3.97(s, 2H), 1.40(s, 9H).

[0047] Step 2: Synthesis of tert-butyl 4-(4-ethoxy-4-oxobutyl)piperazine-1-carboxylate A 1 L 4-neck RB flask was fitted with a rotor, an addition funnel and a N2 inlet adapter. 1-Boc piperazine (38.0 g, 204.0 mmol, 1.0 equiv) in 380 ml of THF was added at room temperature. Triethylamine (34.5 ml, 244.8 mmol, 1.2 equiv) was added dropwise over 20 min using an addition funnel and then stirred for 30 min. Ethyl 4-bromobutyrate (41.8 g, 214.2 mmol, 1.05 equiv) was added dropwise to the reaction mixture and stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC using 50% ethyl acetate in hexanes. The reaction was diluted with ethyl acetate (1 L). To the above material was added saturated NaHCO3 solution and the layers were separated. The aqueous layer was back extracted with ethyl acetate. The organic layers were combined and washed with brine solution (500 ml). The organic layer was dried over sodium sulfate and evaporated at 40-45 °C to give the crude material. The crude compound was purified by column chromatography on silica gel (100-200 mesh) with ethyl acetate in hexane as eluent. Pure fractions eluted with 35-50% ethyl acetate in hexane were collected and evaporated to give tert-butyl 4-(4-ethoxy-4-oxobutyl)piperazine-1-carboxylate 22 (37 g, 61% yield) as a light brown oily liquid. 1 H NMR (CDCl3, 400 MHz): δ 4.07(q, J = 7.17 Hz, 2H), 3.21(m, 4H), 2.48(m, 8H), 1.89(m, 2H), 1.40(s, 9H), 1.18(t, J = 7.34, 3H).

[0048] Step 3: Synthesis of ethyl 4-(piperazin-1-yl)butanoate hydrochloride A 2 L 4-neck RB flask was fitted with a rotor, a 500 mL addition funnel and a nitrogen inlet adapter. At room temperature, tert-butyl 4-(4-ethoxy-4-oxobutyl)piperazine-1-carboxylate 22 (37.5 g, 125.0 mmol) was added followed by 1,4-dioxane (375 ml). The material was cooled to 10-15°C. Using an addition funnel, 4M HCl in 1,4-dioxane (300 mL) was added dropwise over 45 minutes. It was allowed to warm to room temperature and stirred for 5 hours. The progress of the reaction was monitored by TLC in 10% methanol in dichloromethane. After completion of the reaction, the material was transferred to a 2 L Buchi flask and evaporated to dryness. To the resulting solid was added diethyl ether (2 X 300 mL) and evaporated to dryness. To the resulting solid was added 500 mL diethyl ether and filtered under nitrogen. The solid was washed with diethyl ether (2×200 mL). The off-white solid ethyl 4-(piperazin-1-yl)butanoate hydrochloride 23 (31 g, 99% yield) was collected under nitrogen and dried under reduced pressure at 45° C. The crude compound was carried on to the next step without further characterization.

[0049] Step 4: Synthesis of ethyl 4-(4-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)pyridin-2-yl)piperazin-1-yl)butanoate A 1 L three-necked RB flask was fitted with a rotor, dropping funnel and N2 inlet adapter. Ethyl 4-(piperazin-1-yl)butanoate hydrochloride 23 (25 g, 105.5 mmol, 1.0 equiv) was added to DMSO (26 ml) at room temperature. DIPEA (90.0 ml, 527.5 mmol, 5.0 equiv) was added to this and stirred for 20 min. tert-Butyl (3-(6-fluoropyridin-3-yl)prep-2-yn-1yl)carbamate 21 (26.4 g, 105.5 mmol, 1.0 equiv) was added to the reaction mixture. The resulting clear solution was heated to 110 °C and maintained overnight (16 h). The progress of the reaction was monitored by TLC in 50% ethyl acetate:hexane. The reaction was poured into ice-cold water (750 ml) and extracted with ethyl acetate (3×1000 ml). The combined organic layers were washed with water (1000 ml), saturated NaHCO3 (500 ml) and brine (1000 ml), dried over sodium sulfate and evaporated under reduced pressure to give the crude product. The crude material was purified using silica gel (100-200 mesh) column chromatography with 50% ethyl acetate in hexane as eluent to give ethyl 4-(4-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)pyridin-2-yl)piperazin-1-yl)butanoate 24 (28 g, 62% yield). 1 H NMR (CDCl3, 400 MHz): δ 8.62(s, 1H), 8.36(m, 1H), 8.20(brs, 1H), 7.23(m, 1H), 4.07(q, J = 7.17 Hz, 2H), 3.97(s, 2H), 3.21(m, 4H), 2.48(m, 8H), 1.89(m, 2H), 1.40(s, 9H), 1.18(t, J = 7.34, 3H).

[0050] Step 5: Synthesis of 4-(4-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)pyridin-2-yl)piperazin-1-yl)butanoic acid A 500 ml three-neck RB flask was fitted with a rotor, an addition funnel and a N2 inlet adapter. 4-(4-(5-(3-((tert-butoxycarbonyl)amino) prop-1-yn-1-yl)pyridin-2-yl)piperazin-1-yl)butanoate 24 (18.0 g, 41.8 mmol, 1.0 equiv), THF (144 ml) and methanol (36 ml) were added to the reaction flask at room temperature. To the above solution was added an aqueous solution of lithium hydroxide monohydrate (3.5 g LiOH in 72 mL water, 83.6 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 4 h. The progress of the reaction was monitored by TLC using 20% ​​methanol in dichloromethane. The reaction was diluted with ethyl acetate (250 ml) and the layers were separated. The aqueous layer was washed with ethyl acetate (250 mL). The organic layer was discarded. The pH of the aqueous layer was adjusted to 5.5-5.7 using Amberlyst acidic resin (Amberlyst IR 120 hydrogen form). A solid precipitated during acidification. The solid was filtered and washed with acetone (100 mL). The solid was then purified by reverse-phase column chromatography using acetonitrile in water as eluent. The pure fractions were concentrated by lyophilization to give 4-(4-(5-(3-((tert-butoxycarbonyl)amino)prop-1-yn-1-yl)pyridin-2-yl)piperazin-1-yl)butanoic acid 25 (10.9 g, 65%) as an off-white solid. 1 H NMR (D6-DMSO, 400 MHz): δ 8.64(s, 1H), 8.37(m, 1H), 8.23(brs, 1H), 7.21(m, 1H), 3.97(s, 2H), 3.21(m, 4H), 2.46(m, 8H), 1.88(m, 2H), 1.41(s, 9H).

[0051] Example 6. Preparation of Representative Pyridine-PEG-Piperazine Linkers [ka] Step 1: Synthesis of 1,1-dimethylethyl N-[2-(2-hydroxyethoxy)ethyl]carbamate 2-(2-aminoethoxy)ethanol 26 (20.0 g, 190.22 mmol, 1.0 equiv.) was dissolved in 100 mL of methylene chloride and triethylamine (53.0 ml, 380.44 mol, 2.0 equiv.) was added. The solution was cooled in an ice bath and di-tert-butyl dicarbonate (62.27 g, 285.33 mol, 1.5 equiv.) in 60 mL of methylene chloride was added dropwise through an addition funnel. The reaction was allowed to warm slowly to room temperature and stirred overnight at room temperature. Upon completion (as confirmed by TLC in 80% EtOAc in hexanes), R f = 0.30), the reaction was washed with water and the organic layer was washed with dilute HCl solution (0.5 N), water (100 ml), brine and dried over sodium sulfate. The solvent was evaporated and the crude product was purified by column chromatography to give 1,1-dimethylethyl N-[2-(2-hydroxyethoxy)ethyl]carbamate 27 (25 g, 64% yield) as an oily liquid. 1H (400 MHz, CDC13) δ 5.28(brs, 1H), 3.74(m, 2H), 3.59-3.54(m, 4H), 3.32- 3.28(m, 2H), 3.08(brs, 1H), 1.44(s, 9H).

[0052] Step 2: Synthesis of 2-[2-[(tert-butoxycarbonyl)amino]ethoxy]ethyl methanesulfonate To a stirred solution of 1,1-dimethylethyl N-[2-(2-hydroxyethoxy)ethyl]carbamate 27 (10.0 g, 48.7 mmol, 1.0 equiv.) in DCM (100 ml) was added triethylamine (10.2 ml, 73.0 mmol, 1.5 equiv.) and methanesulfonyl chloride (5.6 ml, 73.0 mmol, 1.5 equiv.) under inert atmosphere at 0° C. The reaction mixture was allowed to warm to ambient temperature and the progress of the reaction was monitored by TLC (30% EtOAc-Hexane). Upon completion, the reaction mixture was diluted with DCM (200 ml) and washed with water (100 ml X 2), brine, and sodium sulfate. The organic layer was concentrated under reduced pressure to give 2-[2-[(tert-butoxycarbonyl)amino]ethoxy]ethyl methanesulfonate 28 as an oily liquid (13.5 g, 98% yield), which was used in the next step without further purification. 1 H NMR (CDCl3, 400 MHz): δ 4.83(brs, 1H), 4.30(m, 2H), 3.66(m, 2H), 3.49(m, 2H), 3.25(m, 2H), 2.99(s, 3H), 1.37(s, 9H).

[0053] Step 3: Synthesis of 5-bromo-2-pyrimidinecarboxylic acid methyl ester To a stirred suspension of 5-bromopyrimidine-2-carboxylic acid 29 (25.0 g, 123.2 mmol, 1.0 equiv) in DCM (250 ml) was added oxalyl chloride (15.6 ml, 184.73 mmol, 1.5 equiv) dropwise over 20 min at 0–5 °C. The reaction mixture was stirred at 0–5 °C for 10 min followed by the addition of DMF (0.2 mL). The reaction mixture was gradually warmed to ambient temperature and stirred for 5 h. The reaction mixture was cooled again to 0–5 °C and methanol (200 ml) was added over 20 min. The reaction mixture was warmed to room temperature and stirred overnight. The reaction progress was monitored by TLC (5% MeOH in DCM, visualized by UV) and concentrated under reduced pressure after complete conversion. The crude reaction mixture was diluted with DCM (1 L) and washed with saturated sodium bicarbonate solution (500 mL), water (500 ml), and brine, and the organic layer was dried over Na2SO4, filtered, and concentrated to give 5-bromo-2-pyrimidinecarboxylic acid methyl ester 30 (17.0 g, 63% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz): δ 8.91(s, 2H), 4.00(s, 3H).

[0054] Step 4: Synthesis of methyl 5-[4-[(1,1-dimethylethoxy)carbonyl]-1-piperazinyl]-2-pyrimidinecarboxylate A 2 L four-necked round bottom flask equipped with an argon inlet, thermopocket, and condenser was charged with toluene (1350 ml) and purged with a gentle stream of Ar for 1 h. 5-Bromo-2-pyrimidine carboxylic acid methyl ester 30 (27.0 g, 124.42 mmol, 1.0 equiv.), 1-Boc-piperazine (29.0 g, 155.53 mmol, 1.25 equiv.), and Cs2CO3 (121.6 g, 373.26 mmol, 3.0 equiv.) were added and purged with Ar for 1 h. RuPhos (5.80 g, 12.44 mmol, 0.1 equiv.) and tris(dibenzylideneacetone)dipalladium(0) (2.85 g, 3.11 mmol, 0.025 equiv.) were added at room temperature and purged with Ar for 1 h. The reaction mixture was stirred at 115 °C for 16 h. After completion of the reaction (monitored by TLC in 5% MeOH in DCM, visualized by UV), the reaction mixture was filtered through a pad of Celite and washed with 10% MeOH in EtOAc (1 L). The filtrate was concentrated under reduced pressure, and the residue was dissolved again in ethyl acetate (1 L). The organic layer was washed with saturated sodium bicarbonate solution (500 mL), water (500 ml), and brine. The organic layer was dried over Na2SO4, filtered, and then concentrated under reduced pressure. The crude compound was purified by silica gel chromatography to give methyl 5-[4-[(1,1-dimethylethoxy)carbonyl]-1-piperazinyl]-2-pyrimidinecarboxylate 31 (17.0 g, 42% yield) as a white solid. 1 H NMR (D6-DMSO, 400 MHz): δ 8.46(s, 2H), 3.76(m, 2H), 3.54(m, 2H), 3.23(m, 4H), 2.65(s, 3H), 1.86(s, 9H).

[0055] Step 5: Synthesis of methyl 5-(piperazin-1-yl)pyrimidine-2-carboxylate hydrochloride To a stirring suspension of methyl 5-[4-[(1,1-dimethylethoxy)carbonyl]-1-piperazinyl]-2-pyrimidinecarboxylate 31 (15 g, 46.53 mmol, 1.0 equiv.) in dichloromethane (300 ml) at 0° C. under inert atmosphere was added 4 N HCl in 1,4-dioxane (150 ml). The reaction was allowed to warm to room temperature and stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM, UV visualization) and after completion of the reaction, the reaction was concentrated under reduced pressure. The residue was triturated with diethyl ether (200 ml X 2) to give methyl 5-(piperazin-1-yl)pyrimidine-2-carboxylate hydrochloride 32 (12 g, 99% crude yield) as a greenish solid. The compound was carried on to the next step without further characterization.

[0056] Step 6: Synthesis of methyl 5-(4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)pyrimidine-2-carboxylate To a stirring suspension of methyl 5-(piperazin-1-yl)pyrimidine-2-carboxylate hydrochloride 32 (12.0 g, 46.4 mmol, 1.0 equiv.) in DMF (200 ml) was added 2-[2-[(tert-butoxycarbonyl)amino]ethoxy]ethyl methanesulfonate 28 (19.7 g, 69.6 mmol, 1.5 equiv.), K2CO3 (16.0 g, 116.2 mmol, 2.5 equiv.) and KI (0.8 g, 4.6 mmol, 0.1 equiv.) at room temperature under inert atmosphere. The reaction mixture was heated at 85° C. for 16 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM, UV visualization) and after completion, the reaction was concentrated under reduced pressure. Water (200 ml) was added to the residue and the mixture was extracted with ethyl acetate (200 ml X 2). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to give methyl 5-(piperazin-1-yl)pyrimidine-2-carboxylate hydrochloride 33 (12 g, 99% crude yield) as a green solid, which was carried on to the next step without further characterization.

[0057] Step 7: Synthesis of methyl 5-(4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)pyrimidine-2-carboxylate A 500 ml three-neck RB flask was fitted with a rotor, an addition funnel and a N2 inlet adapter. Methyl 5-(piperazin-1-yl)pyrimidine-2-carboxylate hydrochloride 33 (4.0 g, 9.8 mmol, 1.0 equiv.), THF (14.4 ml) and methanol (3.6 ml) were added to the reaction flask at room temperature. LiOH solution (820 mg LiOH, 19.6 mmol, 2.0 equiv. in 7.2 mL water) was added dropwise to the above solution. The reaction mixture was stirred at room temperature for 4 h. The progress of the reaction was monitored by TLC using 20% ​​methanol in dichloromethane. The reaction was diluted with ethyl acetate (250 ml) and the two layers were separated. The aqueous layer was washed with ethyl acetate (250 mL). The pH of the aqueous layer was adjusted to 5.5-5.7 using Amberlyst acidic resin (Amberlyst IR 120 hydrogen form). The solid that precipitated during acidification was filtered and washed thoroughly with acetone (100 mL). The solid was purified by reverse phase column chromatography to give methyl 5-(4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)pyrimidine-2-carboxylate 34 (3.3 g, 85%) as an off-white solid. 1 H NMR (D6-DMSO, 400 MHz): δ 11.96(brs, 1H), 8.76(s, 2H), 6.76(brs, 1H), 3.61-3.40(m, 8H), 3.09(m, 6H), 2.76(m, 2H), 1.86(s, 9H).

[0058] Example 7. Preparation of Representative Pyrimidine-Piperazine-PEG Linkers [ka] Step 1: Synthesis of tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate Under inert atmosphere, 1-Boc-piperazine (25.0 g, 134.2 mmol, 1.0 equiv.), K2CO3 (46.3 g, 335.5 mmol, 2.5 equiv.), KI (2.2 g, 13.4 mmol, 0.1 equiv.) in acetonitrile (250 ml) were taken in a 500 ml three-necked RB flask and 3-bromo-1-propanol (20.5 g, 147.6 mmol, 1.1 equiv.) in 100 ml acetonitrile was added to it. The reaction mixture was refluxed overnight and completion of the reaction was confirmed by TLC (5% MeOH-DCM, visualization: PMA, R f 0.25). The reaction mixture was concentrated and then extracted with ethyl acetate (500 ml). The organic layer was washed with water and brine. The ethyl acetate layer was dried over Na2SO4, filtered and then concentrated under reduced pressure. The crude material was purified by column chromatography (silica gel, 2% methanol in DCM) to give tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate 36 (25 g, 76% yield) as an off-white solid. 1 H NMR (CDCl3, 400 MHz): δ 3.50 (m, 2H), 3.19 (m, 4H), 2.43 (m, 6H), 1.59 (m, 2H), 1.81 (s, 9H).

[0059] Step 2: Synthesis of tert-butyl 4-(3-((5-bromopyridin-2-yl)oxy)propyl)piperazine-1-carboxylate Under an inert atmosphere, tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate 36 (20.0 g, 113.9 mmol, 1.0 equiv) was placed in THF (300 ml) and NaH (60% dispersion in mineral oil, 5.4 g, 134.4 mmol, 1.18 equiv) was added in portions. The reaction mixture was stirred for 0.5 h and 5-bromo-2-fluoropyridine (23.0 g, 131.0 mmol, 1.15 equiv) in THF (50 ml) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was allowed to warm to room temperature and completion of the reaction was confirmed by TLC (40% ethyl acetate in hexanes, R f0.5). The reaction mixture was quenched with cold saturated NH4Cl solution. The solvent and volatiles were removed under reduced pressure. The residue was extracted with ethyl acetate (500 ml X 1). The organic layer was washed with water, brine, and then dried over Na2SO4. The solvent was concentrated under reduced pressure. The crude material was purified by column chromatography (silica gel, ethyl acetate-hexane) to give tert-butyl 4-(3-((5-bromopyridin-2-yl)oxy)propyl)piperazine-1-carboxylate 37 (25 g, 76% yield) as an off-white solid. 1 H NMR (CDCl3, 400 MHz): δ 8.21(s, 1H), 7.53(d, 1H), 6.57(d, 1H), 4.30(m, 2H), 3.43(m, 4H), 2.51(m, 2H), 2.39(m, 4H), 1.89(m, 2H), 1.43(s, 9H).

[0060] Step 3: Synthesis of diethyl 2-(6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)pyridin-3-yl)malonate Under an inert atmosphere, tert-butyl 4-(3-((5-bromopyridin-2-yl)oxy)propyl)piperazine-1-carboxylate 37 (33.0 g, 82.4 mmol, 1.0 equiv) was placed in DMSO (330 ml) and K3PO4 (52.5 g, 247.2 mmol, 3.0 equiv), CuI (1.56 g, 8.2 mmol, 0.1 equiv), benzoxazole (1.95 g, 16.4 mmol, 0.2 equiv) were added and the solution was purged with Ar for 0.5 h. Diethyl malonate (18.8 mL, 123.6 mmol, 1.5 equiv) was added and the reaction mixture was stirred at 85 °C for 18 h. The reaction was complete by TLC (70% EtOAc-hexane, R f0.4). The reaction mixture was poured into ice-cold water and extracted with EtOAc (500 ml). The organic layer was washed with water, brine and dried over Na2SO4. The solvent was concentrated and purified by column chromatography (silica gel, EtOAc-hexane) to give diethyl 2-(6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)pyridin-3-yl)malonate 38 (24 g, 61% yield) as a pale yellow liquid. 1 H NMR (CDCl3, 400 MHz): δ 8.21(s, 1H), 7.53(d, 1H), 6.57(d, 1H), 4.57(s, 1H), 4.30(m, 2H), 4.23(q, 4H), 3.43(m, 4H), 2.51(m, 2H), 2.39(m, 4H), 1.89(m, 2H), 1.43(s, 9H), 1.30(t, 6H).

[0061] Step 4: Synthesis of 2-(6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)pyridin-3-yl)acetic acid Diethyl 2-(6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)pyridin-3-yl)malonate 38 (24.0 g, 50.1 mmol, 1.0 equiv.) was placed in DMF (190 ml) and KOH (28.1 g, 501.0 mmol, 10.0 equiv., dissolved in 48 ml of water) was added to it. The reaction mixture was stirred at 115° C. for 18 h. The reaction was complete by TLC (5% MeOH-DCM, R f 0.2). The reaction mixture was poured into ice-cold water and extracted with EtOAc (500 ml X 2). The organic layer was washed with water, brine and dried over Na2SO4. The solvent was concentrated and the residue was purified by column chromatography (silica gel, EtOAc-hexane) to give 2-(6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)pyridin-3-yl)acetic acid 39 (15 g, 78% yield) as an off-white solid. 1H NMR (D6-DMSO, 400 MHz): δ 8.21(s, 1H), 7.53(d, 1H), 6.57(d, 1H), 4.30(t, 2H), 3.67-3.43(m, 6H), 2.51(m, 2H), 2.39(m, 4H), 1.89(m, 2H), 1.43(s, 9H).

[0062] Example 8. First representative example of linking linker B to E3 ligase ligand C [ka] Step 1: Synthesis of 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione-5-carboxamide An oven-dried 250 mL three-neck RB flask was equipped with a rotor, thermometer, and N2 inlet adapter. The system was flushed with N2 for 10 min, and 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-1-carboxylic acid 10 (4.19 g, 13.8 mmol, 1.2 equiv.) and DMF (75 ml) were added with stirring. To this solution, HATU (8.80 g, 23.0 mmol, 2.0 equiv.) was then added and stirred at room temperature for 10–15 min. The reaction mixture was cooled to 0 °C using an ice bath. C5-lenalidomide 42 (3.0 g, 11.5 mmol, 1.0 equiv.) and N-ethyl-diisopropylamine (8.0 ml, 46.0 mmol, 4.0 equiv.) were added and the reaction mixture was stirred at room temperature for 16 h. Completion of the reaction was confirmed by TLC (5% MeOH in, R f= 0.3). The reaction mixture was poured into ice-cold water and extracted with ethyl acetate (600 ml). The organic layer was washed with saturated NaHCO3 solution (250 ml) and brine solution (300 ml), dried over Na2SO4 and evaporated on a rotary evaporator to give the crude product as a gummy liquid. The crude was purified on a silica gel (10-200 mesh) column with 10% MeOH in dichloromethane to give 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione-5-carboxamide as a light brown solid. The column pure material was repurified through reverse phase column chromatography using a C18 column with water:acetonitrile as eluent to give 1.2 g of pure 4-(2-(2-(ethoxycarbonyl)ethoxy)ethyl)piperazine-3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione-5-carboxamide (29% yield). 1 H NMR (D6-DMSO, 400 MHz): δ 11.00(s, 1H), 10.43(s, 1H), 9.01(brs, 1H), 8.05(s, 1H), 7.67(s, 1H), 5.01(m, 1H), 4.41(d, 1H) ), 4.29(m, 1H), 3.65(t, 4H), 3.43(t, 4H), 2.72(t, 2H), 2.52(t, 4H), 2.45(t, 2H), 1.43(s, 9H).

[0063] Example 9. A second representative example of linking linker B to E3 ligase ligand C [ka] Step 1: tert-Butyl (2-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carbonyl)piperazin-1-yl)ethyl)carbamate A 250 mL three-neck RB flask was fitted with a rotor, reflux condenser and N2 inlet adapter. 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione 40 (3.0 g, 10.0 mmol, 1.0 equiv.) and tert-butyl (2-(4-(piperazine-1-carbonyl)piperazin-1-yl)ethyl)carbamate 19 (4.45 g, 13.0 mmol, 1.3 equiv.) in 50 ml of NMP were added to the reaction flask with stirring, followed by DIPEA (5.6 ml, 32.0 mmol, 3.2 equiv.). The reaction mixture was stirred at room temperature for 20 min, then at 80 °C for 16 h. The progress of the reaction was monitored by TLC in 5% MeOH:DCM. The reaction mixture was cooled to room temperature, poured into ice water (250 ml) and extracted with ethyl acetate (3 x 200 ml). The organic layer was dried over sodium sulfate and rotary evaporated to dryness to give the crude product. The crude material was purified using silica gel (230-400 mesh size) column chromatography with 60% acetone in hexane as eluent. Evaporation of pure fractions gave a yellow solid, tert-butyl (2-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carbonyl)piperazin-1-yl)ethyl)carbamate 41 (1.2 g, 18% yield). 1H NMR (CDCl3, 400 MHz): δ 11.31(brs, 1H), 11.01(s, 1H), 8.53(brs, 1H), 7.87(m, 1H), 7.37(m, 2H), 5.05( m, 1H), 3.76-3.57(m, 4H), 2.89-3.65(m, 14H), 2.65-2.23(m, 6H), 1.40(s, 9H).

[0064] The examples herein are for illustrative purposes and are not meant to limit the scope of the invention, which is recited in the claims.

Claims

1. Formula I 【Chemistry 1】 [During the ceremony, R 1 is optional and when present, R 1 is selected from piperidine and piperazine; R 2 is C 1 -C 8 Alkanes and C 2 -C 8 is selected from the group consisting of alkynes, and PG is an amine protecting group. Linker.

2. R 2 2. The linker of claim 1, wherein is selected from the group consisting of ethane and ethyne.

3. 2. The linker of claim 1, wherein PG is tert-butoxycarbonyl (Boc).

4. The linker 【Chemistry 2】 2. The linker of claim 1, selected from:

5. Formula II 【Transformation 3】 [During the ceremony, X is selected from C and N; m is 1 to 6; Y is -O- and -CH 2 - selected from n is 0 to 4, o is 0 or 1, and p is 0 to 9. Linker.

6. The linker is of formula IIa 【Chemistry 4】 [During the ceremony, Y is -CH 2 - and -O-; n is 0 to 5, and p is 1 to 5. The linker of claim 5,

7. 7. The linker of claim 6, wherein n is 0, 1, or 4.

8. 7. The linker of claim 6, wherein p is 1 or 5.

9. The linker is of formula IIb 【Transformation 5】 [During the ceremony, X is selected from C and N; Y is -CH 2 - and -O-; m is 0 or 1, and n is 0, 1, 2, or 3. The linker of claim 5,

10. Formula IIc 【Transformation 6】 [During the ceremony, X is selected from the group consisting of C and N; Z is -CH 2 -, -CF 2 selected from the group consisting of -, -C(O)-, and -C(S)-; m is 2 to 5; p is 1 or 2; and PG is an amine protecting group. Linker.

11. 11. The linker of claim 10, wherein m is 2, 3, or 5.

12. 11. The linker of claim 10, wherein PG is Boc.

13. Formula III 【Transformation 7】 [During the ceremony, Y is -O- and -CH 2 - selected from; R 3 is C 1 -C 6 Alkyl, C 3 -C 6 is selected from the group consisting of cycloalkyl, and cycloalkyl-substituted alkyl, and p is 1 or 2. Linker.

14. R 3 is methyl, ethyl, isopropyl, tert-butyl, n-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and methylenecyclobutyl 【Transformation 8】 14. The linker of claim 13, selected from the group consisting of:

15. Formula IV 【Chemistry 9】 [During the ceremony, PG is an amine protecting group; m is 1 to 4; each X is independently selected from C and N; and Z is -C(O)-, -C(S)-, or -CH 2 -, and -CF 2 - is selected from the group consisting of Linker.

16. 16. The linker of claim 15, wherein PG is Boc.

17. Formula V 【Chemistry 10】 [During the ceremony, PG is an amine protecting group, X is selected from N and C, and p is 1 to 3. Linker.

18. 18. The linker of claim 17, wherein PG is Boc.

19. Equation VI 【Chemistry 11】 [During the ceremony, PG is an amine protecting group; each X is independently selected from C and N; and n is 0 to 2. Linker.

20. 20. The linker of claim 19, wherein PG is Boc.

21. Formula VII 【Chemistry 12】 [During the ceremony, PG is an amine protecting group; X is selected from C and N; Y is selected from N, O and C; m is 1 to 3; and n is 1 to 3. Linker.

22. 22. The linker of claim 21, wherein PG is Boc.

23. A compound comprising a linker according to any one of claims 1 to 22 and a ligand that triggers a degradation pathway.

24. 24. The compound of claim 23, wherein the ligand that triggers the degradation pathway is a ligand for an E3 ubiquitin ligase enzyme.

25. 25. The compound of claim 24, wherein the ligand for the E3 ubiquitin ligase enzyme is a derivative of an immunomodulatory drug (IMiD).

26. 26. The compound of claim 25, wherein the derivative of IMiD is selected from the group consisting of pomalidomide derivatives, thalidomide derivatives, lenalidomide derivatives, and VH032 derivatives.

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