Please cite this article in press as: Skwarska et al., Development and pre-clinical testing of a novel hypoxia-activated KDAC inhibitor, Cell Chemical
Biology (2021), https://doi.org/10.1016/j.chembiol.2021.04.004
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(
20 mL) and extracted with ethyl acetate (2 3 50 mL). The organic components were combined and washed with water (50 mL), brine
(
4
50 mL) then dried (MgSO ), filtered, and concentrated in vacuo. Purification using column chromatography, eluting with 0–60% ethyl
acetate:petroleum ether, yielded the title compound (4) (0.62 g, 82%) as a colorless oil. Rf 0.71 (50% ethyl acetate:petroleum ether);
ꢀ1
1
nmax (thin film)/cm 2976, 1722, 1690, 1478, 1368, 1323, 1254, 1168, 1137; H NMR at 363 K (500 MHz, (D
6 H
-DMSO) d 8.00 (1H, d, J
8
.1), 7.66–7.57 (3H, m), 7.42 (1H, d, J 7.2), 7.25 (2H, d, J 7.8), 7.23–7.13 (2H, m), 6.53 (1H, d, J 16.1), 4.42 (2H, s), 3.74 (3H, s), 3.33 (2H,
13
t, J 7.1), 2.84 (2H, t, J 7.1), 2.46 (3H, s), 1.64 (9H, s), 1.34 (9H, s); C NMR at 363 K (126 MHz, (D
6
-DMSO) d
C
166.0, 154.5, 149.6, 143.5,
1
1
40.8, 134.9, 132.9, 132.6, 129.1, 127.8, 127.4, 122.7, 121.8, 117.3, 117.2, 114.8, 114.4, 83.1, 78.5, 50.8, 49.6, 46.1, 27.5, 27.4, 22.1,
+
+
+
2+
41 2 6
2.7; HRMS m/z (ESI ) Found: 549.2953, C32H N O requires [M+H] 549.2959; LRMS (ESI ) 338 ([M+2Na+2MeCN] , 23%), 225
3
+
(
[M+2Na+H+2MeCN] , 100%), 202 (26%), 123 (71%); HPLC Method C, Retention time - 15.0 min, 99%.
E)-3-(4-[(tert-Butyloxycarbonyl-[2-(1-[tert-butyloxycarbonyl]-2-methyl-1H-indol-3-yl)ethyl]amino)methyl]phenyl)
(
prop-2-enoic acid (5)
A solution of lithium hydroxide (2.05 g, 85.6 mmol, 10.0 eq) in water (57 mL) was slowly added to a stirred solution of 4 (4.70 g,
ꢁ
8
.56 mmol, 1.0 eq) in tetrahydrofuran (57 mL) and methanol (57 mL) at 0 C. The reaction mixture was stirred at rt for 6 h then diluted
with ethyl acetate (250 mL) and quenched with aqueous 1 M hydrochloric acid (200 mL). The reaction was extracted with ethyl acetate
3 3 200 mL) and the organic components were combined and washed with water (300 mL), brine (300 mL) then dried (MgSO ),
filtered, and concentrated in vacuo to yield the title compound (5) (4.54 g, 99%) as a colorless fluffy, foamy solid. R 0.61 (100% ethyl
acetate); mp 94–96 C (from THF); nmax (thin film)/cm 2976, 2930, 1726, 1687, 1410, 1366, 1258, 1136, 1116; H NMR at 363 K (500
MHz, (D -DMSO) d 8.00 (1H, d, J 8.0), 7.57 (2H, d, J 7.7), 7.55 (1H, d, J 15.9), 7.42 (1H, d, J 7.4), 7.24 (2H, d, J 7.7), 7.23–7.13 (2H, m),
.44 (1H, d, J 15.9), 4.41 (2H, s), 3.33 (2H, t, J 7.2), 2.84 (2H, t, J 7.2), 2.46 (3H, s), 1.64 (9H, s), 1.34 (9H, s); C NMR at 363 K (126 MHz,
-DMSO) d 166.8, 154.5, 149.6, 142.7, 140.4, 134.9, 132.94, 132.93, 129.1, 127.6, 127.4, 122.7, 121.8, 118.9, 117.2, 114.8, 114.4,
(
4
f
ꢁ
ꢀ1
1
6
H
13
6
(
D
6
C
-
ꢀ
ꢀ
8
3.2, 78.5, 49.6, 46.1, 27.5, 27.4, 22.1, 12.8; HRMS m/z (ESI ) Found: 533.2648, C31
H
37
N
2
O
6
requires [MꢀH] 533.2657; LRMS (ESI )
ꢀ
5
33 ([MꢀH] , 100%); HPLC Method A, Retention time - 13.1 min, 99%.
(
E)-tert-Butyl-3-(2-((4-(3-((benzyloxy)amino)-3-oxoprop-1-en-1-yl)benzyl)(tert-butoxycarbonyl)amino)ethyl)-2-methyl-
1
H-indole-1-carboxylate (6)
PyBOP (0.11 g, 0.21 mmol, 1.1 eq) was added to a solution of 5 (0.10 g, 0.19 mmol, 1.0 eq) and triethylamine (0.10 mL, 0.75 mmol, 4.0
eq) in dry tetrahydrofuran (1.9 mL). The reaction mixture was stirred for 15 min at rt before O-(benzyl)hydroxylamine hydrochloride
(
(
(
(
36 mg, 0.22 mmol, 1.2 eq) was added. Stirring was continued at rt for 18 h, the reaction mixture was diluted with ethyl acetate
20 mL) and quenched with aqueous 1 M solution of hydrochloric acid (10 mL). The reaction mixture was extracted with ethyl acetate
20 mL), the organic components were washed with a saturated solution of sodium hydrogen carbonate (40 mL), water (40 mL), brine
4
40 mL), dried (MgSO ), filtered, and concentrated in vacuo. Purification using column chromatography, eluting with 0–5% ethanol:-
chloroform, then a second purification using column chromatography, eluting with 0–50% ethyl acetate:petroleum ether, yielded the
ꢁ
title compound (6) (87 mg, 73%) as a colorless solid. R
f
0.45 (50% ethyl acetate:petroleum ether); mp 71–74 C (from dichlorometha-
1
ꢀ1
ne:hexane); nmax (thin film)/cm 3192, 2976, 1728, 1687, 1660, 1514, 1459, 1366, 1322, 1252, 1158, 1136, 1117, 1046; H NMR at
3
63 K (500 MHz, D
.0), 8.02 (1H, ddd, J 7.8, 7.3, 1.1), 7.98 (1H, ddd, J 7.5, 7.3, 1.3), 7.30 (1H, d, J 15.8), 5.71 (2H, s), 5.22 (2H, s), 4.15 (2H, t, J 7.2), 3.65
6 H
-DMSO ) d 11.71 (1H, s), 8.82 (1H, d, J 7.8), 8.30 (2H, d, J 8.4), 8.30 (1H, d, J 15.8), 8.29–8.13 (6H, m), 8.06 (2H, d, J
8
1
3
(
6 C
2H, t, J 7.2), 3.27 (3H, s), 2.45 (9H, s), 2.16 (9H, s); C NMR at 363 K (126 MHz, D -DMSO) d 163.3,* 154.4, 149.5, 139.9, 138.9,
1
4
6
35.7, 134.9, 133.3, 132.9, 129.1, 128.2, 127.7, 127.6, 127.4, 127.1, 122.7, 121.7, 118.1, 117.1, 114.8, 114.4, 83.1, 78.5, 76.9,
+
+
requires [M+H] 640.33811; LRMS m/z (ESI )
+
9.5, 46.1, 27.5, 27.4, 22.1, 12.7; HRMS m/z (ESI ) Found: 640.33788, C38
H
46
N
3
O
6
+
62 ([M+Na] , 16%), 606 (34%), 562 (20%), 506 (38%), 461 (44%), 423 (38%), 405 (13%), 361 (100%), 317 (26%), 300 (25%), 282
1
13
(
39%), 266 (10%); HPLC Method A, Retention time - 13.3 min, >99%. *Signal at 163.3 observed by H- C HMBC correlation see
supplemental information for details.
E)-tert-Butyl-3-(2-((tert-butoxycarbonyl)(4-(3-(((4-nitrobenzyl)oxy)amino)-3-oxoprop-1-en-1-yl)benzyl)amino)ethyl)-
-methyl-1H-indole-1-carboxylate (7)
(
2
PyBOP (48 mg, 0.093 mmol, 1.1 eq) was added to a solution of 5 (45 mg, 0.084 mmol, 1.0 eq) and triethyl amine (35 mL, 0.25 mmol, 3.0
eq) in dry tetrahydrofuran (1.0 mL). The reaction mixture was stirred for 15 min at rt before O-(4-nitrobenzyl)-hydroxylamine (S7)
(
(
17 mg, 0.10 mmol, 1.2 eq) was added. Stirring was continued at rt for 18 h, the reaction mixture was diluted with ethyl acetate
20 mL) and quenched with an aqueous 1 M solution of hydrochloric acid (10 mL). The reaction mixture was extracted with ethyl ac-
etate (20 mL), the organic components were washed with a saturated aqueous solution of sodium hydrogen carbonate (40 mL), water
(
40 mL), brine (40 mL), dried (MgSO
4
), filtered, and concentrated in vacuo. Purification using column chromatography, eluting with 0–
ꢁ
1
0% ethanol:chloroform, yielded the title compound (7) (54 mg, 92%) as a pale-yellow solid. R
f
; mp 90–92 C (from DMSO:H
2
O); nmax
ꢀ
1
thin film)/cm 3199, 2978, 1728, 1688, 1523, 1460, 1346, 1323, 1159, 1137, 1117; H NMR at 363 K (500 MHz, (D
1
(
6 H
-DMSO) d 11.04
(1H, br s), 8.22 (2H, d, J 8.3), 8.00 (1H, d, J 8.1), 7.72 (2H, d, J 8.3), 7.50 (2H, d, J 7.9), 7.50 (1H, d, J 15.9) 7.41 (1H, d, J 7.6), 7.24 (2H, d,
J 7.9), 7.23–7.13 (2H, m), 6.46 (1H, d, J 15.9), 5.05 (2H, s), 4.41 (2H, s), 3.33 (2H, t, J 7.4), 2.84 (2H, t, J 7.4), 2.46 (3H, s), 1.64 (9H, s),
1
3
.35 (9H, s); C NMR at 363 K (126 MHz, D
1
6 C
-DMSO) d 163.5,* 154.4, 149.5, 147.1, 143.4, 140.0, 139.2, 134.9, 133.2, 132.9, 129.1,
1
28.9, 127.4, 127.2, 122.8, 122.7, 121.7, 117.8, 117.1, 114.8, 114.4, 83.1, 78.5, 75.6, 49.6,* 46.1, 27.5, 27.4, 22.1, 12.7; HRMS m/z
ꢀ
ꢀ
ꢀ
ꢀ
(
ESI ) Found: 683.3083, C38
13.3 min, 98%. *Signal at 163.5 observed by H- C HMBC correlation, signal at 49.6 observed by H- C HSQC correlation see
supplemental information for details.
H
43
N
4
O
8
requires [MꢀH] 683.3086; LRMS (ESI ) 683 ([MꢀH] , 100%); HPLC Method B, Retention time
1 13 1 13
-
e9 Cell Chemical Biology 28, 1–13.e1–e13, September 16, 2021