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V. Zima et al. / European Journal of Medicinal Chemistry 208 (2020) 112754
under an inert atmosphere. Consumption of the starting material
was monitored by TLC. Once the reaction was complete, the pH of
the reaction was neutralized by addition of amberlite IR 120.
Amberlite was filtered out and the beads were rinsed with meth-
anol several times. The filtrate was then evaporated to dryness
under reduced pressure, and the remaining product was purified by
flash chromatography to afford the desired free acid 7 (0.90 g, 97%).
4.8.5. tert-Butyl (Z)-5-(4-(4-(tert-butoxy)-3-hydroxy-4-oxobut-2-
enoyl)-4-(4-chlorobenzyl)- piperidine-1-carbonyl)-2-((2,2-
dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)oxy)
benzoate (11)
Compound 10 (0.065 g, 0.136 mmol) was dissolved in DCM
(1 mL), and the resulting solution was cooled down in an ice bath.
Trifluoroacetic acid (100%, 0.2 mL) was added, and the reaction was
stirred for 15 min at room temperature. Once TLC showed total
consumption of the starting material, the solvents were evaporated
under reduced pressure, and the resulting oil was dried under high
vacuum for 2 h. The dried mixture was then dissolved in DCM
(1.5 mL) followed by addition of triethylamine (0.3 mL) and 9
(0.082 g, 0.136 mmol). The reaction mixture was stirred overnight
at room temperature. The solvents were removed under reduced
pressure, and the residue was purified by flash chromatography
(DCM/MeOH 20:1) to yield the desired amide 11 (0.080 g, 68%). 1H
1H NMR (300 MHz, CDCl3)
d
8.38 (d, J ¼ 2.3 Hz, 1H), 8.11 (dd, J ¼ 8.8,
2.3 Hz, 1H), 6.98 (d, J ¼ 8.9 Hz, 1H), 4.24 (t, J ¼ 4.9 Hz, 2H), 3.90 (dd,
J ¼ 5.6, 4.2 Hz, 2H), 3.73 (dt, J ¼ 5.2, 2.0 Hz, 2H), 3.69e3.58 (m, 8H),
3.35 (t, J ¼ 5.0 Hz, 2H), 1.57 (s, 9H). 13C NMR (75 MHz, CDCl3)
d 170.7,
164.9, 161.9, 134.8, 133.6, 123.0, 121.5, 112.7, 81.7, 71.0, 70.8, 70.7,
70.7, 70.1, 69.4, 68.7, 50.7, 28.3. HRMS (ESI) m/z calcd for
C
20H29N3O8Na [MþNa]þ 462.1847, found 462.1848.
NMR (400 MHz, CDCl3)
d
14.76 (s, 1H), 7.73 (d, J ¼ 2.3 Hz, 1H), 7.44
(dd, J ¼ 8.6, 2.3 Hz, 1H), 7.20 (d, J ¼ 8.4 Hz, 2H), 6.95 (d, J ¼ 8.6 Hz,
1H), 6.93e6.86 (m, 2H), 6.37 (s, 1H), 5.02 (s, 1H), 4.20 (t, J ¼ 5.0 Hz,
2H), 3.90 (t, J ¼ 5.0 Hz, 2H), 3.78e3.69 (m, 2H), 3.69e3.57 (m, 8H),
3.53 (t, J ¼ 5.1 Hz, 2H), 3.34e3.25 (m, 2H), 3.05 (bs, 2H), 2.84 (s, 2H),
2.25e2.02 (bs, 2H), 1.72 (bs, 2H), 1.56 (s, 9H), 1.56 (s, 9H), 1.43 (s,
4.8.3. 3-(tert-Butoxycarbonyl)-4-((2,2-dimethyl-4-oxo-3,8,11,14-
tetraoxa-5-azahexadecan-16-yl)oxy)benzoic acid (8)
Compound 7 (0.64 g, 1.46 mmol) and Lindlar catalyst (0.19 g,
30 wt%) were suspended in methanol (15 mL). The flask was first
purged with argon, and hydrogen gas was bubbled through the
solution for 1 h. The catalyst was filtered out and the filtrate was
evaporated to dryness under reduced pressure. The resulting crude
product was dissolved in DCM (15 mL), the reaction was cooled
down in an ice bath, and triethylamine (0.4 mL, 2.92 mmol) and di-
tert-butyl dicarbonate (0.38 g, 1.75 mmol) were added. After stir-
ring the reaction for 24 h at room temperature, the mixture was
diluted with DCM (20 mL) and the organic phase was washed with
a saturated solution of ammonium chloride (2 ꢂ 5 mL) and brine
(5 mL). The organic layer was then evaporated to dryness and pu-
rified by flash chromatography (DCM/MeOH 20:1) to yield 8 (0.48 g,
9H). 13C NMR (100 MHz, CDCl3)
d 205.2, 169.6, 169.1, 165.1, 161.0,
159.1,134.6,133.8,133.4,133.2,132.0,131.3 ( ꢂ 2),130.7,128.6 ( ꢂ 2),
127.6, 122.8, 113.3, 99.0, 84.3, 81.7, 71.1, 70.8, 70.7, 70.3, 70.3, 69.6,
68.8, 49.7 ( ꢂ 2), 45.7, 40.5, 38.7 ( ꢂ 2), 32.9, 28.6 ( ꢂ 3), 28.4 ( ꢂ 3),
27.9 ( ꢂ 3).
4.8.6. 5-(4-((Z)-3-Carboxy-3-hydroxyacryloyl)-4-(4-chlorobenzyl)
piperidine-1-carbonyl)-2-((13,35-dioxo-39-((3aR,4R,6aS)-2-
oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)-
3,6,9,16,19,22,25,28,31-nonaoxa-12,34-diazanonatriacontyl)oxy)
benzoic acid (12)
64%). 1H NMR (300 MHz, CDCl3)
d
8.36 (d, J ¼ 2.3 Hz, 1H), 8.09 (dd,
J ¼ 8.8, 2.3 Hz, 1H), 6.96 (d, J ¼ 8.9 Hz, 1H), 5.11 (s, 1H), 4.24 (dd,
J ¼ 5.7, 4.2 Hz, 2H), 3.90 (dd, J ¼ 5.5, 4.3 Hz, 2H), 3.74 (dt, J ¼ 4.5,
3.1 Hz, 2H), 3.68e3.55 (m, 6H), 3.52 (t, J ¼ 5.2 Hz, 2H), 3.36e3.19 (m,
Compound 11 (0.020 g, 0.023 mmol) was dissolved in DCM
(1 mL), and the solution was cooled down in an ice bath. Neat tri-
fluoroacetic acid (0.2 mL) was added dropwise, and the reaction
was stirred for 15 min. Once TLC showed complete consumption of
the starting material, the solvents were evaporated under reduced
pressure, and the remaining oil was dried for 3 h under high vac-
uum. The dried product was dissolved in DCM (1 mL) followed by
addition of triethylamine (0.3 mL) and biotin-PEG6-NHS ester
(0.016 g, 0.023 mmol). After stirring the reaction for 24 h at room
temperature, the solvent was removed under reduced pressure and
the remaining mixture dried under high vacuum for 2 h. The crude
mixture was dissolved in DCM (1 mL), trifluoroacetic acid (0.5 mL)
was added, and the reaction was stirred for 1 h at room tempera-
ture. After removing the solvents under reduced pressure, the
mixture was purified by preparative HPLC to yield the desired
compound 12 (0.004 g, 15%). 1H NMR (600 MHz, Methanol-d4)
2H),1.57 (s, 9H),1.42 (s, 9H). 13C NMR (75 MHz, CDCl3)
d 170.1,165.0,
161.7, 149.5, 134.8, 133.5, 122.9, 122.0, 112.6, 81.7, 79.3, 71.0, 70.7,
70.6, 70.3 ( ꢂ 2), 69.5, 68.7, 40.5, 28.5 ( ꢂ 3), 28.3 ( ꢂ 3). HRMS (ESI)
m/z calcd for C25H40NO10 [MþH]þ 514.2647, found 514.2646.
4.8.4. 3-(tert-Butyl) 1-(2,5-dioxopyrrolidin-1-yl) 4-((2,2-dimethyl-
4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)oxy)isophthalate
(9)
Compound 8 (0.48 g, 0.94 mmol) was dissolved in THF (17 mL)
under an inert atmosphere followed by addition of O-(benzotriazol-
1-yl)-N,N,N0,N0-tetramethyl-uronium tetrafluoroborate (0.39 g,
1.22 mmol) and triethylamine (0.26 mL, 0.22 mmol). The reaction
was stirred for 10 min at room temperature, and N-hydrox-
ysuccinimide (0.016 g, 1.88 mmol) was added. After stirring for
16 h at room temperature, the solvent was evaporated under
reduced pressure and the resulting mixture was purified by flash
chromatography (DCM/MeOH 20:1) to afford the activated ester 9
d
7.87 (d, J ¼ 2.3 Hz, 1H), 7.59 (dd, J ¼ 8.6, 2.3 Hz, 1H), 7.29e7.20 (m,
4H), 7.07e6.99 (m, 2H), 4.49 (ddd, J ¼ 7.9, 5.0, 1.0 Hz, 1H), 4.39e4.25
(m, 4H), 3.94e3.88 (m, 2H), 3.79e3.73 (m, 2H), 3.71 (t, J ¼ 6.2 Hz,
3H), 3.69e3.55 (m, 26H), 3.53 (td, J ¼ 5.5, 4.1 Hz, 4H), 3.35 (td,
J ¼ 5.5, 2.5 Hz, 4H), 3.20 (ddd, J ¼ 8.9, 6.0, 4.6 Hz, 1H), 3.06 (bs, 2H),
2.96 (s, 2H), 2.92 (dd, J ¼ 12.7, 5.0 Hz, 1H), 2.70 (d, J ¼ 12.7 Hz, 1H),
2.44 (t, J ¼ 6.2 Hz, 2H), 2.27e2.12 (m, 4H), 1.78e1.55 (m, 6H),
(0.47 g, 81%). 1H NMR (300 MHz, CDCl3)
d
8.40 (d, J ¼ 2.3 Hz, 1H),
8.13 (dd, J ¼ 8.8, 2.2 Hz, 1H), 7.02 (d, J ¼ 8.9 Hz, 1H), 5.01 (bs, 1H),
4.24 (t, J ¼ 5.0 Hz, 2H), 3.88 (q, J ¼ 5.1 Hz, 2H), 3.71 (dd, J ¼ 6.1,
3.5 Hz, 2H), 3.60 (dd, J ¼ 14.6, 3.6 Hz, 6H), 3.49 (t, J ¼ 5.3 Hz, 2H),
3.26 (q, J ¼ 5.5 Hz, 2H), 2.86 (s, 4H),1.54 (s, 9H),1.39 (s, 9H). 13C NMR
1.49e1.38 (m, 2H). 13C NMR (150 MHz, Methanol-d4)
d 207.3, 176.1,
174.0, 171.2, 168.6, 167.7, 166.1, 164.7, 160.7, 135.9, 133.9, 133.9, 133.1,
132.7 ( ꢂ 2), 132.0, 129.2 ( ꢂ 2), 129.2, 129.0, 121.9, 114.9, 71.9, 71.6,
71.6 ( ꢂ 7), 71.5, 71.5, 71.4, 71.3, 71.3, 70.6, 70.5, 70.4, 70.3, 68.3, 63.4,
61.6, 57.0, 51.1, 46.6, 45.7, 41.1, 40.4, 40.4, 37.6, 36.7, 34.0, 33.4, 29.8,
29.5, 26.9. HRMS (ESI) m/z calcd for C57H82ClN5O20S [MþH]þ
1224.5035, found 1224.5035.
(75 MHz, CDCl3)
d 169.4 (x2), 164.3, 162.8, 161.0, 156.0, 135.2, 134.0,
123.4, 116.8, 113.0, 81.9, 79.1, 71.0, 70.6, 70.6, 70.2, 70.2, 69.3, 68.8,
40.4, 28.45 ( ꢂ 3), 28.2 ( ꢂ 3), 25.7 ( ꢂ 2). HRMS (ESI) m/z calcd for
C
29H42N2O12Na [MþNa]þ 633.2630, found 633.2630.