Synthesis of Polyethylene Glycol-Based Compounds

General method A. PEG (1 eq.) was solubilised in dioxane anhydrous and NaH (2 eq.) was added under stirring. The resulting mixture was stirred at r.t. for 3h. The mixture was cooled down to 0 °C using ice bath and tert-butylbromo acetate (2 eq.) was added drop by drop. The resulting mixture was stirred at r.t O/N. The precipitate was filtered off and the organic phase evaporated to dryness. The resulting oil was taken up with ethyl acetate, washed with water, dried over MgSO₄ and evaporated to dryness. The resulting oil was purified by column chromatography using a gradient of ethyl acetate from 50% to 100% v/v in heptane.
General method B. tert-butyl esters 1, 2, 3 or 12 were dissolved in a solution of 50% v/v trifluroacetic acid in DCM. The resulting solution was stirred for 1 h or until complete conversion of starting material. The solvent was removed under high vacuum. The resulting carboxylic acid was used as crude in the next step without any further purification. To a solution of carboxylic acid in 1 ml DMF were added HATU (1 eq.) and HOBT (1 eq.) and the pH of the reaction mixture was adjusted to > 9 by addition of DIPEA (3 eq.). The resulting solution was stirred at room temperature for 5 min and then amine 7 or 8 was added. The mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. Water was added and the mixture was extracted with ethyl acetate (×3). The combined organic phases were washed with brine (×2), dried over MgSO₄ and evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 20% to 95% v/v acetonitrile in 0.1% aqueous solution of ammonia to yield the desired compound.
di‐tert‐butyl 3, 6, 9, 12 – tetraoxatetradecanedioate (1)
Following general method A, from triethylene glycol (1.125 g, 1 ml, 7.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (595.75 mg, 14.9 mmol, 2 eq.) and tert-Butyl bromoacetate (2.905 g, 2.19 ml, 14.9 mmol, 2 eq.), compound 1 was obtained as an oil after high vacuum. Yield: 538 mg, 1.42 mmol (19%). ¹H NMR (500 MHz, CDCl₃): δ 3.81 (s, 4H), 3.51–3.46 (m, 12H), 1.26 (s,18H). ¹³C NMR (126 MHz, CDCl₃): δ 169.1, 80.9, 70.1, 70.0, 68.5, 27.5. Analytical data matched those previously reported^{73 (link)}.
di‐tert‐butyl 3,6,9,12,15-pentaoxaheptadecanedioate (2)
Following general method A, from tetrathylene glycol (1.125 g, 1 ml, 5.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (463 mg, 11.5 mmol, 2 eq.) and tert-Butyl bromoacetate (2.25 g, 1.7 ml, 11.5 mmol, 2 eq.), compound 2 was obtained as an oil after high vacuum. Yield: 500 mg, 1.18 mmol (10%). ¹H NMR (500 MHz, CDCl₃): δ 3.86 (s, 4H), 3.55–3.49 (m, 16H), 1.31 (s, 9H). Analytical data matched those previously reported^{73 (link)}.
di‐tert‐butyl 3,6,9,12,15,18-hexaoxaicosanedioate (3)
Following general method A, from pentaethylene glycol (1.126 g, 1 ml, 4.72 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral oil (377 mg, 9.45 mmol, 2 eq.) and tert-Butyl bromoacetate (1.872 g, 1.7 ml, 11.5 mmol, 2 eq.), compound 3 was obtained as an oil after high vacuum. Yield: 300 mg, 0,641 mmol (14%). ¹H NMR (400 MHz, CDCl₃): δ 3.94 (s, 4H), 3.66–3.56 (m, 20H), 1.40 (s, 18H). Analytical data matched those previously reported^{73 (link)}.
N¹,N¹⁴-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxatetradecanediamide (CM09)
Following general method B, from compound 1 (6.80 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol) and DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM09 was obtained as a white solid. Yield: 8 mg, 0.007 mmol (40%). ¹H NMR (400 MHz, CDCl₃): δ 8.61 (s, 2H), 7.48–7.45 (m, 2H), 7.31–7.27 (m, 8H), 7.23 (d, J = 10.2 Hz, 2H), 4.64–4.59 (m, 2H), 4.52–4.46 (m, 4H), 4.41–4.38 (m, 2H), 4.31–4.25 (m, 2H), 4.01–3.94 (m, 4H), 3.82 (d, J = 15.7 Hz, 2H), 3.62–3.52 (m, 12H), 2.45 (s, 6H), 2.42–2.34 (m, 2H), 2.12–2.06 (m, 2H), 1.19 (s, 2H), 0.89 (s, 18H); ¹³C NMR (101 MHz, CDCl₃): δ 170.2, 169.9, 169.6, 149.3, 147.5, 137.3, 130.6, 129.9, 128.4, 127.1, 69.9, 69.5, 69.3, 69.1, 57.6, 56.1, 55.9, 42.2, 35.5, 34.6, 25.4, 15.1. HRMS (ESI) m/z: [M + H]⁺ calculated for: C₅₄H₇₄N₈O₁₂S₂: 1090.49; observed: 1091.5093.
N¹,N¹⁷-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaheptadecanediamide (CM10)
Following general method B, from compound 2 (7.60 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol) and DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM10 was obtained as a white solid. Yield: 6 mg, 0.005 mmol (30%). ¹H NMR (500 MHz, CDCl₃): δ p.p.m., 7.58–7.55 (m, 2H), 7.35–7.30 (m, 10H), 4.72–4.67 (m, 2H), 4.57–4.47 (m, 6H), 4.38–4.33 (m, 2H), 4.15–3.91 (m, 8H), 3.68–3.56 (m, 20H), 2.51 (s, 6H), 2.45–2.38 (m, 2H), 2.20–2.14 (m, 2H), 0.95 (s, 18H). ¹³C NMR (126 MHz, CDCl₃): δ 170.3, 169.8, 169.6, 149.6, 146.9, 137.5, 129.5, 128.47, 128.40, 127.1, 70.0, 69.56, 69.52, 69.5, 69.2, 69.1, 57.7, 56.03, 55.98, 42.1, 35.6, 34.6, 25.4, 14.8. HRMS (ESI)m/z: [M + H]⁺ calculated for: C₅₆H₇₈N₈O₁₃S₂: 1134.51; observed: 1135.5538.
N¹,N²⁰-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide (CM11)
Following general method B, from compound 3 (8.39 mg, 0.018 mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg, 0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98 mg, 0.054 mmol, 3 eq) compound CM11 was obtained as a white solid. Yield: 11.74 mg, 0.0099 mmol (55%). ¹H NMR (400 MHz, CDCl₃): δ 8.61 (s, 2H), 7.41–7.38 (m, 2H), 7.29 (t, J = 7.6 Hz, 10H), 4.66–4.61 (m, 2H), 4.49–4.41 (m, 6H), 4.35–4.29 (m, 2H), 3.98–3.91 (m, 6H), 3.62–3.50 (m, 24H), 2.45 (s, 6H), 2.42–2.35 (m, 2H), 2.11–2.06 (m, 2H), 0.88 (s, 18H); ¹³C NMR (101 MHz, CDCl₃): δ 171.2, 170.9, 170.4, 150.3, 148.5, 138.3, 131.6, 130.9, 129.5, 128.1, 71.2, 70.61, 70.59, 70.5, 70.4, 70.3, 58.6, 57.0, 43.2, 36.5, 35.6, 26.4, 16.1. HRMS (ESI) m/z: [M + H]⁺ calculated for: C₅₈H₈₂N₈O₁₄S₂: 1178.54; observed: 1179.6015.
N¹,N²⁰-bis((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide (CMP98)
Following general method B, from compound 3 (7.12 mg, 0.028 mmol, 1 eq.), compound 8 (18.06, 0.040 mmol, 2.1 eq.), HATU (15.2 mg, 0.040 mmol, 2 eq.), HOAT (5.44 mg, 0.040 mmol, 2 eq.), DIPEA (7.45 mg, 0.0010 ml, 3 eq.), compound CMP98 was obtained as a white solid. Yield: 10.58 mg, 0.0089 mmol (45%). ¹H NMR (400 MHz, CDCl₃): δ 9.09 (s, 2H), 8.02 (s, 2H), 7.31 (d, J = 8.5 Hz, 4H), 7.22 (d, J = 8.0 Hz, 4H), 7.16 (d, J = 9.2 Hz, 2H), 4.75–4.64 (m, 4H), 4.51 (d, J = 8.9 Hz, 2H,), 4.41–4.37 (m, 2H), 4.24–4.17 (m, 2H), 3.94 (d, J = 3.2 Hz, 4H), 3.84–3.81 (m, 4H), 3.62–3.54 (m, 20H), 2.49–2.47 (m, 2H), 2.44 (s, 6H), 2.26–2.17 (m, 4H), 0.93 (s, 18H); ¹³C NMR (101 MHz, CDCl₃): δ 173.2, 171.5, 169.7, 151.8, 138.8, 132.9, 129.5, 129.2, 128.3, 71.2, 71.1, 70.6, 70.48, 70.45, 70.4, 70.3, 59.9, 58.5, 56.5, 43.2, 35.6, 35.2, 26.4, 15.0. HRMS (ESI)m/z: [M + H]⁺ calculated for: C₅₈H₈₂N₈O₁₄S₂: 1178.54; observed: 1179.6087.
1‐phenyl‐2,5,8,11,14-pentaoxahexadecan-16-ol (9)
Pentaethylene glycol (9.53 g, 50 mmol, 5 eq.) was added dropwise to a suspension of NaH 60% in mineral oil (800 mg, 20 mmol, 2.5 eq.) in 20 ml of DMF at 0 °C. The resulting mixture was stirred at r.t for 1 h. The reaction mixture was cooled to 0^oC, benzyl chloride (1 ml, 1.1 g, 8.72 mmol, 1 eq.) was added. The resulting mixture was stirred O/N at r.t. The reaction was quenched with a saturated solution of NH₄Cl and the aqueous phase was extracted with ethyl acetate (×3). The combined organic phases were dried over MgSO₄ and evaporated to dryness. The resulting oil was purified by column chromatography (from 0 to 60% of ethyl acetate in heptane) to afford the title compound as a oil. Yield: 2.055 g, 6.25 mmol (71%). ¹H NMR (400 MHz, CDCl₃): δ 7.28–7.19 (m, 5H), 4.50 (s, 2H), 3.66–3.52 (m, 20H), 2.50 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 138.2, 128.3, 127.8, 127.6, 73.2, 72.7, 70.61, 70.58, 70.53, 70.51, 70.2, 69.4, 61.7
tert-butyl 1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-oate (10)
To a stirred solution of 9 (2.055 g, 6.25 mmol, 1 eq.) in 12.8 ml of DCM was added 37% solution of NaOH (12.8 ml), followed by tert-butylbromo acetate (4.882 g, 25 mmol, 4 eq.) and TBABr (2118 mg, 6.37 mmol, 1.02 eq.). The resulting solution was stired O/N at r.t. The reaction mixture was extracted with ethyl acetate (×3). The organic phases were combined and washed with brine (×1), dried over MgSO₄ and concentrate in vacuo. The resulting brow oil was purified by column chromatography (from 0 to 30% of ethyl acetate in petroleum) to afford the titled compound as colorless oil. Yield: 2.216 g, 5 mmol (80%). ¹H NMR (500 MHz, CDCl₃): δ 7.28–7.20 (m, 5H), 4.50 (s, 2H), 3.95 (s, 2H), 3.65–3.55 (m, 20H), 1.40 (s, 9H). ¹³C NMR (126 MHz, CDCl₃): δ169.7, 128.4, 127.7, 127.6, 81.5, 73.2, 70.7, 70.7, 70.6, 70.6, 69.4, 69.1, 28.1. MS (ESI)m/z: [(M-tBu) + H]⁺ calculated for: C₂₃H₃₈O₈: 442.26; observed: 387.20.
19,19-dimethyl-17-oxo-3,6,9,12,15,18-hexaoxaicosanoic acid (11)
10 (2.216 g, 5 mmol, 1 eq.) was dissolved in 75 ml of ethanol, Pd/C (10 wt%) was added and the resulting mixture was place under hydrogen and stirred at r.t. until complete conversion of the starting material. The reaction mixture was filtered through celite, the celite pad was washed few times using ethanol. The filtrate was concentrated in vacuum to give an oil that was used for the next step without further purification. Yield: 1764 g, 5 mmol (quantitative). BAIB (3.546 g, 11.01 mmol, 2.2 eq.) and TEMPO (171.87 mg, 1.10 mmol, 0.22 eq.) were added to a solution of ACN/H₂O 1:1 containing previous obtained oil (1.764 g, 5 mmol, 1 eq.). The resulting mixture was stirred at r.t until complete conversion of the starting material. The crude was purified using ISOLUTE® PE-AX anion exchange column. The column was equilibrate with methanol, the reaction mixture poured in the column and let it adsorbed in the pad. The column was washed with methanol (×3) to elute all the unbound material. Then, the titled product was eluted using a 50% solution of formic acid in methanol. The organic phase was evaporated to dryness to afford the title compound as oil. Yield: 1.200 g, 3.27 mmol (65%). ¹H NMR (400 MHz, CDCl₃): δ 4.12 (s, 2H), 3.98 (s, 2H), 3.72–3.60 (m, 16H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃): δ 172.6, 169.7, 81.6, 71.0, 70.59, 70.56, 70.54, 70.46, 70.38, 70.35, 70.30, 68.9, 68.8, 28.1.
tert-butyl (S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosanoate (12)
To a solution of PEG linker 11 (78.8 mg, 0.215 mmol, 1 eq.) in 1.5 ml DMF was added HATU (81.74 mg, 0.215 mmol, 1 eq.), HOAT (29.26 mg, 0.215 mmol, 1 eq.), DIPEA (80.13 mg, 0.106 ml, 0.645 mmol, 3 eq.) and the solution was stirred at room temperature for 5 min. Compound 7 (100 mg, 0.215 mmol, 1 eq.) was added and the pH of the reaction mixture was adjusted to > 9 by addition of DIPEA(80.13 mg, 0.106 ml, 0.645 mmol, 3 eq.). The mixture was stirred at room temperature until no presence of the starting materials was detected by LC-MS. The solvent was evaporated under reduced pressure to give the corresponding crude, which was purified by HPLC using a gradient of 20–95% v/v acetonitrile in 0.1% aqueous solution of ammonia to yield the titled compound as white solid. Yield: 75.6 mg, 0.094 mmol (44%). ¹H NMR (400 MHz, CDCl₃): δ 9.00 (s, 1H), 7.45 (t, J = 5.9 Hz, 1H), 7.39–7.33 (m, 4H), 7.29 (d, J = 8.9 Hz, 1H), 4.71 (t, J = 8.0 Hz, 1H), 4.59–4.48 (m, 3H), 4.34 (dd, J = 5.2, 14.6 Hz, 1H), 4.08–3.92 (m, 5H), 3.69–3.61 (m, 18H), 2.52 (s, 3H), 2.47–2.41 (m, 1H), 2.19–2.11 (m, 1H), 1.46 (s, 9H), 0.97 (s, 9H). ¹³C NMR (101 MHz, CDCl₃): δ 171.3, 171.1, 170.5, 170.0, 151.7, 139.1, 129.4, 128.3, 82.0, 71.1, 70.6, 70.4, 70.4, 70.3, 70.3, 70.2, 70.2, 68.9, 58.7, 57.3, 56.8, 43.1, 36.3, 35.1, 28.1, 26.4, 15.1. MS (ESI)m/z: [M + H]⁺ calculated for: C₃₈H₅₈N₄O₁₁S₂: 778.38; observed: 779.4.
N¹-((R)-1-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-N¹⁷-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaheptadecanediamide (CMP99)
Following general method B, from compound 12 (75.6 mg, 0.094 mmol, 1 eq.) and trifluoroacetic acid (1 ml in 1 ml of DCM), the carboxylic acid derivative was obtained as oil. The crude was used for the next step without further purification. Yield: 70 mg, 0.094 mmol (quantitative). MS (ESI) m/z: [M + H]⁺ calculated for: C₃₄H₅₀N₄O₁₁S: 722.32; observed: 723.3. Following general method B, from compound 13 (5.5 mg, 0.006 mmol, 1 eq.), compound 8 (2.77 mg, 0.006 mmol, 1 eq.), HATU (2.28 mg, 0.0.006 mmol, 1 eq.), HOAT (1 mg, 0.0.006 mmol, 1 eq.), DIPEA (2.23 mg, 0.002 ml, 0.018 mmol, 3 eq.), CMP99 was obtained as a white solid. Yield: 4.5 mg, 0.004 mmol (66%). ¹H NMR (400 MHz, CDCl3): δ 8.74 (s, 1H), 8.73 (s, 1H), 7.37–7.34 (m, 9H), 7.18 (d, J = 8.9 Hz, 1H), 4.76–4.64 (m, 3H), 4.59–4.44 (m, 5H), 4.37–4.26 (m, 2H), 4.05–3.59 (m, 27H), 2.52 (s, 6H), 2.31–2.11 (m, 4H), 0.96 (s, 9H), 0.95 (s, 9H). ¹³C NMR (101 MHz, CDCl₃): δ 173.0, 171.3, 170.0, 150.7, 145.4, 138.4, 129.53, 129.49, 128.2, 128.16, 71.2, 71.0, 70.54, 70.48, 70.4, 70.3, 58.4, 57.0, 56.7, 43.4, 35.2, 35.0, 26.4, 26.35, 15.9. HRMS (ESI)m/z: [M + H]⁺ calculated for: C₅₆H₇₈N₈O₁₃S₂: 1134.51; observed: 1135.5814.