W. Duan et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4
.1.7.1. 4-((3-Carboxypropoxy)carbonyl)benzo[c][1,2,5]oxadi-
4.1.8.3. 4-(((6-(Hydroxyamino)-6-oxohexyl)oxy)carbonyl)benzo-
1
azole 1-oxide (23a). Weak yellow solid, yield: 68%. H NMR
600 MHz, d -DMSO) d 8.07 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 7.9 Hz,
H), 7.38 (t, J = 7.8 Hz, 1H), 4.46 (d, J = 5.9 Hz, 2H), 2.55
[c][1,2,5]oxadiazole 1-oxide (24c). Weak yellow solid, yield:
1
(
1
6
77%. H NMR (500 MHz, d
6
-DMSO) d10.36 (s, 1H), 8.70 (s, 1H),
8.20 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.1 Hz,
1H), 4.34 (t, J = 9.7 Hz, 2H), 1.93 (t, J = 11.2 Hz, 2H), 1.78 (tt,
13
(
t, J = 6.5 Hz, 2H), 2.23–2.08 (m, 2H).
C
NMR (151 MHz,
1
3
d
6
-DMSO) d 173.6, 159.2, 130.3, 128.9, 123.5, 122.8, 120.9, 60.5,
J = 14.2, 9.7 Hz, 2H), 1.58–1.22 (m, 4H). C NMR (151 MHz, d
6
-
+
2
5.7, 18.9. ESI-MS m/z 267.2 [M+H] .
DMSO) d 169.5, 164.4, 145.1, 135.2, 132.6, 128.7, 127.5, 126.9,
6
C
6.4, 32.7, 28.9, 25.7, 25.5. HRMS (AP-ESI) m/z calcd for
+
4
.1.7.2. 4-((4-Carboxybutoxy)carbonyl)benzo[c][1,2,5]oxadiazole
13
15
H N
3
O
6
Na [M+Na] : 332.0853; found: 332.0856.
1
1
-oxide (23b). Weak yellow solid, yield: 72%. H NMR (600 MHz,
CDCl
3
) d 8.06 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.38
4
.1.8.4. 4-(((7-(Hydroxyamino)-7-oxoheptyl)oxy)carbonyl)benzo-
(
t, J = 7.9 Hz, 1H), 4.40 (t, J = 6.2 Hz, 2H), 2.39 (t, J = 7.1 Hz, 2H),
[c][1,2,5]oxadiazole 1-oxide (24d). Weak yellow solid, yield:
1
2
1
.82 (dd, J = 13.3, 6.6 Hz, 2H), 1.75–1.66 (m, 2H), 1.55–1.45 (m,
1
7
8
1
2
6
1%. H NMR (600 MHz, d -DMSO) d 10.33 (s, 1H), 8.65 (s, 1H),
.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.3 Hz, 1H), 7.57 (t, J = 8.0 Hz,
H), 4.34 (t, J = 6.5 Hz, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.78–1.68 (m,
H). 1 C NMR (151 MHz, CDCl
3
) d 179.7, 164.3, 145.3, 135.2,
3
+
28.2, 125.8, 66.2, 33.9, 28.3, 25.5, 24.3. ESI-MS m/z 281.2 [M+H] .
H), 1.55–1.48 (m, 2H), 1.40 (dt, J = 14.8, 7.5 Hz, 2H), 1.30 (dt,
4.1.7.3. 4-(((5-Carboxypentyl)oxy)carbonyl)benzo[c][1,2,5]oxadi-
13
6
J = 14.6, 7.4 Hz, 2H). C NMR (151 MHz, d -DMSO) d 169.5, 164.4,
1
azole 1-oxide (23c). Weak yellow solid, yield: 77%. H NMR
1
2
3
45.1, 135.3, 132.6, 128.7, 127.5, 126.9, 66.3, 32.7, 28.7, 28.4, 25.6,
5.4. HRMS (AP-ESI) m/z calcd for
46.1010; found: 346.1040.
(
600 MHz, CDCl
H), 7.42 (t, J = 9.0 Hz, 1H), 4.33 (t, J = 9.5 Hz, 2H), 2.21 (t,
J = 16.1 Hz, 2H), 1.95–1.71 (m, 2H), 1.68–1.44 (m, 2H), 1.33 (m,
3
) d 8.04 (d, J = 10.0 Hz, 1H), 7.70 (d, J = 9.9 Hz,
+
14 17 3 6
C H N O Na [M+Na] :
1
H). 1 C NMR (151 MHz, CDCl
31.0, 130.3, 116.4, 66.7, 34.5, 29.3, 25.9, 24.9. ESI-MS m/z 295.3
3
2
1
3
) d 177.2, 167.2, 166.5, 133.4,
4
.1.8.5. 4-(((8-(Hydroxyamino)-8-oxooctyl)oxy)carbonyl)benzo-
+
[c][1,2,5]oxadiazole 1-oxide (24e). Weak yellow solid, yield:
[
M+H] .
1
79%. H NMR (600 MHz, d
6
-DMSO) d 10.30 (s, 1H), 8.61 (s, 1H),
8
.20 (dd, J = 8.1, 1.4 Hz, 1H), 8.12 (dd, J = 7.8, 1.4 Hz, 1H), 7.57
4
.1.7.4. 4-(((6-Carboxyhexyl)oxy)carbonyl)benzo[c][1,2,5]oxadi-
1
(t, J = 8.0 Hz, 1H), 4.35 (t, J = 6.6 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H),
.76–1.71 (m, 2H), 1.49 (dd, J = 14.7, 7.3 Hz, 2H), 1.43–1.37
azole 1-oxide (23d). Weak yellow solid, yield: 71%. H NMR
600 MHz, CDCl ) d 11.18 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.94 (d,
J = 7.9 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 4.40 (t, J = 6.4 Hz, 2H), 2.37
1
(
3
13
(
(
m, 2H), 1.34–1.30 (m, 2H), 1.26 (dt, J = 13.5, 6.8 Hz, 2H). C NMR
151 MHz, d -DMSO) d 169.6, 164.4, 145.1, 135.2, 132.6, 128.7,
6
(
t, J = 7.2 Hz, 2H), 2.08–1.80 (m, 2H), 1.76–1.62 (m, 2H), 1.58–
1
3
127.0, 66.4, 32.7, 28.9, 28.8, 28.4, 25.7, 25.5. HRMS (AP-ESI) m/z
calcd for C15
1
.32 (m, 4H). C NMR (151 MHz, CDCl
3
) d 179.4, 164.2, 145.3,
+
18 3 6
H N O Na [M+Na] : 360.1166; found: 360.3301.
1
35.0, 133.6, 128.0, 125.6, 66.3, 33.8, 28.6, 28.4, 25.6, 24.5. ESI-
+
MS m/z 309.3 [M+H] .
4
.2. Biological materials and methods
4
.1.7.5. 4-(((7-Carboxyheptyl)oxy)carbonyl)benzo[c][1,2,5]oxadi-
1
azole 1-oxide (23e). Weak yellow solid, yield: 79%. H NMR
600 MHz, CDCl ) d 11.01 (s, 1H), 8.08 (dd, J = 7.8, 1.5 Hz, 1H),
.95 (dd, J = 8.1, 1.5 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 4.41
t, J = 6.7 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1.85–1.79 (m, 2H),
.70–1.63 (m, 2H), 1.47 (dd, J = 14.1, 6.6 Hz, 2H), 1.41 (dd,
4.2.1. In vitro inhibition of Hela cells extracts fluorescence assay
(
7
(
1
3
In vitro HDACs inhibition assays were conducted as previously
1
9
described in our group. In brief, 10
was mixed with 0.0097, 0.039, 0.39, 1.56, 6.25, 25
compounds (50 L) and SAHA, and then they were incubated for
l
L of Hela nuclear extracts
l
M of target
l
13
J = 12.0, 8.3 Hz, 4H). C NMR (151 MHz, CDCl
3
) d 179.7, 164.2,
30 min, using 100% and none HDACs groups as control group.
1
2
34.9, 133.3, 128.1, 127.9, 125.6, 66.4, 33.9, 28.8, 28.7, 28.5, 25.7,
4.5. ESI-MS m/z 323.3 [M+H] .
Five minutes later, fluorogenic substrate Boc-Lys (acetyl)-AMC
+
(40
l
L) was added, and the mixture was incubated at 37 °C for
L of developer con-
3
0 min and then stopped by addition of 100
l
4
1
4
.1.8. Synthesis of compound 24
The method is as the same as the preparation of compound
9a–19e.
taining trypsin and TSA. After incubation at 37 °C for 20 min, fluo-
rescence intensity was measured using a microplate reader at
excitation and emission wavelengths of 390 nm and 460 nm,
respectively. The inhibition ratios were calculated from the fluo-
rescence intensity readings of tested wells relative to those of con-
trol wells, and the IC50 values were calculated using a regression
analysis of the concentration/inhibition data.
.1.8.1. 4-((4-(Hydroxyamino)-4-oxobutoxy)carbonyl)benzo[c]-
[
1,2,5]oxadiazole 1-oxide (24a). Weak yellow solid, yield: 68%.
1
H NMR (600 MHz, d
6
-DMSO) d 10.46 (s, 1H), 8.77 (s, 1H), 8.19
(
d, J = 8.0 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H),
4
.33 (t, J = 6.1 Hz, 2H), 2.14 (t, J = 7.0 Hz, 2H), 2.00–1.94 (m, 2H).
4.2.2. In vitro antiproliferative assay
1
3
C NMR (151 MHz, d
28.7, 126.9, 65.8, 29.2, 24.6. HRMS (AP-ESI) m/z calcd for
6
-DMSO) d 168.9, 164.3, 145.1, 135.4, 132.6,
In vitro antiproliferative assays were determined by the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
1
C
+
17
11 3 6
H N O
Na [M+Na] : 304.0540; found: 304.0563.
method as previously described. Briefly, all cell lines were main-
11
tained in RPMI1640 medium containing 10% PBS at 37 °C in 5% CO
2
4
.1.8.2. 4-(((5-(Hydroxyamino)-5-oxopentyl)oxy)carbonyl)benzo-
humidified incubator. Cells were passaged the day before dosing
into a 96-well cell plate and allowed to grow for a minimum of
4 h prior to addition of compounds. These tested compounds were
all dissolved in DMSO and then diluted in culture medium so that
the effective DMSO concentration did not exceed 0.2%. After com-
pounds addition, the plates were incubated for an additional 48 h,
and then 0.5% MTT solution was added to each well. After further
incubation for 4 h, formazan formed from MTT was extracted by
[c][1,2,5]oxadiazole 1-oxide (24b). Weak yellow solid, yield:
1
7
8
1
2
1
6
2%. H NMR (600 MHz, d -DMSO) d 10.40 (s, 1H), 8.71 (s, 1H),
.12 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.4 Hz,
H), 4.32 (t, J = 9.9 Hz, 2H), 2.14 (t, J = 16.1 Hz, 2H), 2.00–1.84 (m,
H), 1.58–1.29 (m, 2H). 13C NMR (151 MHz, d
-DMSO) d 168.8,
64.2, 145.1, 135.4, 132.5, 128.8, 126.9, 65.3, 34.4, 28.8, 23.1.
6
+
13 3 6
HRMS (AP-ESI) m/z calcd for C12H N O Na [M+Na] : 318.0697;
found: 318.0946.
adding 200 lL of DMSO for 15 min. Absorbance was then