Communications
ArH), 7.44 (1H, dd, J=2 Hz, ArH), 6.67–6.62 (1H, m, ArH), 5.99 (1H,
108.1 (ArC), 63.4 (CH), 61.6 (CH), 56.9 (CH ), 41.05, 41.0, 40.9, 40.2,
2
bs, NH), 4.62 (1H, bs, NHBoc), 3.44–3.39 (2H, q, J=8 Hz, CH ), 3.15
36.8, 30.11, 30.07, 29.7, 29.4, 26.9, 25.3 ppm; MS m/z (+ES): 967
2
+
+
+
(
2H, m, CH ), 1.67–1.46 (6H, m, CH ), 1.45 ppm (9H, s, NHBoc);
[2M+Na ], 495 [M+Na] , 473 [M+H] ; HRMS (+ES) found: [M+
2
2
1
3
+
C NMR (101 MHz, CDCl ): d =162.7 (C=O), 156.2 (C=O), 144.7
Na] , 495.2025 (C H N O SNa requires 495.2042).
3
C
24 32
4
4
(
(
[
ArC), 143.7 (ArC), 122.6 (ipso-ArC), 108.3 (ArC), 39.3 (CH ), 29.8
2
6
-(Hydroxymethyl)-2-oxo-2H-chromene-3-carboxylic acid 16: 2-
CH ), 29.2 (CH ), 28.4 (Boc), 23.9 ppm (CH ); MS m/z (+ES): 615.2
2
2
2
+
+
+
Hydroxy-5-(hydroxymethyl)benzaldehyde (1.2 g, 7.9 mmol) was dis-
solved in CH Cl2 and treated with 2,2-dimethyl-1,3-dioxane-4,6-
2M+Na] , 319.6 [M+Na] , 297.2 [M+H] .
2
tert-Butyl 5-(benzofuran-5-carboxamido)pentylcarbamate 13:
Following the standard procedure, benzofuran-5-carboxylic acid 12
dione (1.4 g, 9.5 mmol) and pyridine (1.3 mL, 15.8 mmol). The solu-
tion was held at reflux for 2 h; then, after cooling, the precipitate
was collected by filtration and washed with CH Cl to afford the
(
100 mg, 0.62 mmol) was transformed into a cream-coloured solid
3 (73 mg, 34%); R =0.8 (n-hexane/EtOAc 1:1); IR (ATR): n˜ =3372
2
2
1
1
title compound as a pale-yellow solid (1.2 g, 70%); H NMR
f
(
NH), 3328 (NH), 2932, 2870, 1685 (C=O), 1628 (C=O), 1522, 1473,
(400 MHz, DMSO): d =8.43 (1H, s, ArH), 7.73 (1H, s, ArH), 7.58 (1H,
H
1
8
7
365, 1164, 1136, 1115, 1044, 1023, 1012, 949, 908, 883, 847,
dd, J=9, 2 Hz, ArH), 7.34 (1H, s, ArH), 4.55 ppm (2H, s, ArCH ); MS
2
À1
1
+
+
21 cm ; H NMR (700 MHz, CDCl ): d =8.06 (1H, d, J=2 Hz, ArH),
m/z (+ES): 243 [M+Na] , 221 [M+H] ; all data agree with those
3
H
[23]
.72 (1H, dd, J=9, 2 Hz, ArH), 7.68 (1H, d, J=2 Hz, ArH), 7.52 (1H,
reported previously.
d, J=9 Hz, ArH), 6.82 (1H, dd, J=2 Hz, 1, ArH), 6.24 (1H, bs, NH),
.57 (1H, bs, NH), 3.49 (2H, q, J=6 Hz, CH ), 3.15–3.12 (2H, m,
6
1
-(Chloromethyl)-2-oxo-N-(5-(5-((3aS,4S,6aR)-2-oxohexahydro-
H-thieno[3,4-d]imidazol-4-yl)pentanamido)pentyl)-2H-chro-
4
2
CH ), 1.69–1.64 (2H, m, CH ), 1.58–1.51 (4H, m, CH ), 1.45–1.41 ppm
2
2
2
1
3
mene-3-carboxamide 20: 6-(Hydroxymethyl)-2-oxo-2H-chromene-
-carboxylic acid 16 (50 mg, 023 mmol) was dissolved in thionyl
(
(
9H, s, NHBoc); C NMR (700 MHz, CDCl ): d =167.0 (C=O), 156.5
3 C
3
ipso-ArC), 156.1 (ipso-ArC), 146.2 (ArC), 130.0 (ipso-ArC), 127.5 (ipso-
chloride (4 mL) and held at reflux for 2 h. At the same time, tert-
butyl 5-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido)pentylcarbamate 18 (97 mg, 0.23 mmol) was dis-
ArC), 123.3 (ArC), 120.6 (ArC), 111.4 (ArC), 107.0 (ArC), 40.2 (CH2),
0.0 (CH ), 29.8 (CH ), 29.3(CH ), 28.4 (Boc), 24.0 ppm (CH ); MS m/z
4
2
2
2
2
[24]
+
+
(
+ES): 369 [M+Na] , 244 [MÀBoc] ; HRMS (+ES) found: [M+
+
solved in CH Cl (2 mL) and treated with TFA (2 mL). Evaporation of
Na] , 369.1803 (C H N O Na requires 369.1790).
2
2
1
9
26
2
4
the two reactions to remove the thionyl chloride and TFA was fol-
Standard procedure the formation of 11 and 14: The N-Boc-pro-
tected furancarboxamide 10 or 13 (0.2–0.4 mmol) was dissolved in
CH Cl (5 mL) and treated with excess TFA (~1 mL). After stirring
lowed by dissolution of the two reactions in CH Cl2 (2 mL). The
2
crude 6-(chloromethyl)-2-oxo-2H-chromene-3-carbonyl chloride 17
was then added via cannula to a mixture of crude N-(5-aminopen-
tyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
2
2
for the reported time and temperature, the solvent was removed
in vacuo, and the resultant material was dissolved and evaporated
with CH Cl (310 mL) to ensure removal of excess TFA. The resul-
pentanamide 19 and Et N (95 mL, 0.7 mmol) at room temperature.
3
The reaction was stirred for 2 h and then evaporated. Flash chro-
matography (CH Cl , CH Cl /MeOH [99:1], [98:2], [95:5], [9:1]) fol-
2
2
tant material was then dissolved in DMF, treated with biotin–NHS
1.1 equiv) and Et N (2.1 equiv) and stirred for 4 h. The solvent was
2
2
2
2
(
lowed by trituration with MeOH afforded the title compound as
3
then removed in vacuo, and the resultant material was subjected
to flash chromatography (CH Cl /MeOH). Subsequent trituration
a white gummy solid (55 mg, 44%); R =0.3 (CH Cl /MeOH 9:1); IR
f
2
2
(ATR): n˜ =3512–3150, 2928, 2854, 1697 (C=O), 1653 (C=O), 1614
2
2
À1
1
with CH Cl (5 mL) produced the desired product.
(C=O), 1573, 1536, 1419, 1246, 1168, 1021, 827 cm
; H NMR
2
2
(
8
500 MHz, DMSO): d =8.80 (1H, s, ArH), 8.67 (1H, t, J=6 Hz, NH),
.02 (1H, d, J=2 Hz, ArH), 7.80 (1H, dd, J=9, 2 Hz, ArH), 7.52 (1H,
H
N-[5-(Furan-3-ylformamido)pentyl]-5-{2-oxohexahydro-1H-
thieno[3,4-d]imidazolidin-4-yl}pentanamide 11: Following the
standard procedure, N-Boc-protected furancarboxamide 10
d, J=9 Hz, ArH), 6.42 (1H, s, NH), 6.35 (1H, s, NH), 4.84 (2H, s,
ArCH ), 4.29 (1H, m, CH), 4.15 (1H, m, CH), 3.09–3.00 (4H, m, CH ),
2
2
2
(
(
100 mg, 0.4 mmol) was transformed into a yellow solid 11
.80 (2H, dd, J=12 Hz, 5, CH ), 2.64 (2H, m, CH ), 2.02 (2H, t, J=
2 2
1
113 mg, 79%); R =0.3 (CH Cl /MeOH 9:1); H NMR (400 MHz,
f
2
2
6 Hz, CH ), 1.62–1.23 (6H, m, CH ), 1.15 ppm (2H, t, J=6 Hz, CH );
2 2 2
CD OD): d =8.06 (1H, dd, J=1 Hz, ArH), 7.58 (1H, dd, J=2 Hz,
13
3
H
C NMR (500 MHz, DMSO): d =172.6 (C=O), 163.4 (ipso-ArC), 161.6
C
ArH), 6.81 (1H, dd, J=2, 1 Hz, ArH), 4.53 (1H, ddd, J=8, 5, 1 Hz,
(C=O), 161.0 (C=O), 154.2 (C=O), 147.5 (ArC), 135.39 (ipso-ArC),
CHH), 4.32 (1H, dd, J=8, 4 Hz, (CHH), 3.26–3.18 (4H, m, CH ), 2.72
2
135.20 (ArC), 130.9 (ArC), 120.4 (ipso-ArC), 119.2 (ipso-ArC), 117.4
(ArC), 61.7, 59.9, 56.1, 46.4, 45.7, 38.9, 35.9, 29.5, 29.4, 28.9, 28.7,
26.0, 24.5, 9.3 ppm; MS m/z (+ES): 549 [( Cl)M+H] ; HRMS (+ES)
(
2H, m, CH ), 2.21 (2H, t, J=7 Hz, CH ), 1.82–1.39 ppm (12H, m,
2 2
+
+
CH ); MS m/z (+ES): 445 [M+Na] , 424 [M+H] .
35
+
2
35
+
35
found: [( Cl)M+H] , 549.1928 (C H ClN O S requires 549.1938).
26
34
4
5
N-(5-(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-
4
-yl)pentanamido)pentyl)benzofuran-5-carboxamide 14: Follow-
Baculosome CYPs: Baculosome CYP1A2 and CYP3A4 were pur-
chased from Thermo Fisher Scientific and were used for the Vivid
assays and streptavidin affinity enrichment.
ing the standard procedure, tert-butyl 5-(benzofuran-5-carboxami-
do)pentylcarbamate 13 (100 mg, 0.29 mmol) was transformed into
a white gummy solid 14 (100 mg, 73%); R =0.3 (CH Cl /MeOH
f
2
2
9
1
:1); IR (ATR): n˜ =3584–3072, 2940, 2868, 1697 (C=O), 1676 (C=O),
Cytochrome P450 fluorescence assays: The Vividꢁ CYP3A4 assay
(Life Technologies, Carlsbad, CA, USA) was run according to the
manufacturer’s instructions. A master pre-mix was prepared, com-
prising 100 mm potassium phosphate buffer (4500 mL; pH 8.0), re-
À1
635 (C=O), 1524, 1461, 1316, 1262, 1179, 1104, 1025, 826 cm
;
1
H NMR (700 MHz, CD OD): d =8.11 (1H, dd, J=2 Hz, 1, ArH), 7.84
3
H
(
1H, d, J=2 Hz, ArH), 7.78 (1H, dd, J=9, 2 Hz, ArH), 7.56 (1H, dt,
J=9, 1 Hz, ArH), 6.93 (1H, dd, J=2, 1 Hz, ArH), 4.47 (1H, ddd, J=8,
, 1 Hz, CH), 4.27 (1H, dd, J=8, 5 Hz, CH), 3.42–3.39 (3H, t, J=8 Hz,
CH ), 3.21–3.15 (4H, m, CH ), 2.90 (1H, dd, J=13, 5 Hz, CH ), 2.68
generation system (100 mL; 333 mm glucose-6-phosphate (G6P)
À1
5
and 30 UmL
glucose-6-phosphate dehydrogenase (G6PD) in
100 mm potassium phosphate buffer, pH 8.0), and CYP3A4 BACU-
2
2
2
ꢁ
(
1H, d, J=13 Hz, CH ), 2.17 (2H, m, CH ), 1.70–1.55 (4H, m, CH ),
LOSOMES (400 mL; microsomes from baculovirus-infected cells co-
2
2
2
1
3
1
.45–1.38 ppm (4H, m, CH2); C NMR (700 MHz, CD OD): d =176.1
expressing human CYP3A4, NADPH-cytochrome P450 reductase,
and human cytochrome b5). An aliquot of master pre-mix (50 mL)
was pre-incubated with 40 mL of either 10% MeOH (control) or
3
C
(
(
C=O), 170.5 (C=O), 166.1 (C=O), 158.0 (ipso-ArC), 147.9 (ArC), 130.9
ArC), 128.9 (ipso-ArC), 124.8 (ArC), 121.9 (ipso-ArC), 112.1 (ArC),
ChemMedChem 2016, 11, 1122 – 1128
1126
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim