Organic & Biomolecular Chemistry
Paper
1
5
2
03.5; HRMS (ESI) exact mass calcd for C30
77.1294 found m/z [M + H] 577.1300; HPLC (system QC): t = thioureido)ethoxy)ethoxy)ethyl)-4-(3-methyl-5-oxo-4,5-dihydro-
7.433 min (95% purity).
H
21
N
6
O
5
S m/z
N-(2-(2-(2-(3-(4-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)phenyl)
+
R
1H-pyrazol-1-yl)benzamide (8).
tert-Butyl 4-((((3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
-yl)methylene)hydrazono)methyl)benzylcarbamate (4). To
4
a solution of tert-butyl(4-(1,2,4,5-tetrazin-3-yl)benzyl)carbamate
(
(
106 mg, 0.369 mmol, 5 equiv.) in a mixture of ACN/PBS
7.6 mL, 4.8 : 2.8, v/v) was added 5-methyl-2-phenyl-2,4-
dihydro-3H-pyrazol-3-one (13 mg, 0.074 mmol, 1 equiv.). The
reaction mixture was stirred for 2 h at room temperature. Then
the crude product was diluted with AcOEt and washed with de-
4
ionized water. The organic layer was dried over MgSO and
concentrated under vacuum. The crude product was purified
by preparative TLC (2 mm, AcOEt/cyclohexane, 1 : 1, v/v) to give
the product (30.1 mg, 0.069 mmol, yield 94%) as a pale yellow
−
1
amorphous powder. M.p. 96 °C; IR (neat cm ) 3473, 3333,
2
1
973, 2913, 2860, 1669, 1597, 1525, 1497, 1268, 1248, 1162,
085, 1055, 956; H NMR (300 MHz, CDCl ) δ 8.08 (d, J = 15.3 Step 1: To a solution of 4-(3-methyl-5-oxo-4,5-dihydro-1H-
3
1
3
2
Hz, 1H), 8.02–7.92 (m, 3H), 7.63 (d, J = 8.1 Hz, 2H), 7.38 (dt, J = pyrazol-1-yl)benzoic acid (1.0 g, 4.58 mmol, 1 equiv.) in dry
1
1
3.0, 6.8 Hz, 4H), 7.15 (t, J = 7.3 Hz, 1H), 4.97 (t, J = 5.6 Hz, DMF (25 mL) was added N-hydroxysuccinimide (580 mg,
1
3
H), 4.34 (d, J = 5.5 Hz, 2H), 2.27 (s, 13H), 1.47 (s, 9H);
C
5.04 mmol, 1.1 equiv.) and EDCI·HCl (965 mg, 5.04 mmol,
NMR (75 MHz, CDCl
3
) δ 165.4, 156.0, 149.9, 148.3, 146.6, 1.1 equiv.). The reaction mixture was stirred for 8 h at room
1
7
C
42.7, 138.9, 131.8, 128.9, 128.1, 127.9, 124.6, 118.9, 101.2, temperature. Then, the crude product was diluted with AcOEt
9.9, 44.5, 28.5, 12.8; HRMS (ESI) exact mass calcd for and washed with a solution of saturated aq NH
4
Cl (×3) and
brine. The organic layer was concentrated under vacuum to
E)-4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl- give 1.276 g (4.033 mmol, 88% raw yield) of the product as a
+
+
24
28 5 3
H N O m/z 434.2192 found m/z 434.2197 [M + H ] .
(
ene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (5) and tert- pale brown amorphous powder. The crude product was directly
butyl 4-(bis(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol- used without further purification. To a solution of 2,5-dioxo-
4
-yl)methyl)benzylcarbamate (6). To a solution of tert-butyl(4- pyrrolidin-1-yl-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)
3
0
(1,2,4,5-tetrazin-3-yl)benzyl)carbamate
3
(25.6
mg, benzoate (200 mg, 0.634 mmol, 1 equiv.) in dry DMF (5 mL)
added 2,2′-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine)
0
.089 mmol, 1 equiv.) in a mixture of ACN/PBS (1.7 mL; 1 : 07, were
v/v) was added commercially available 5-methyl-2-phenyl-2,4- (465 μL, 3.17 mmol, 5 equiv.) and DIEA (439 μL, 2.536 mmol,
dihydro-3H-pyrazol-3-one (77.6 mg, 0.445 mmol, 5 equiv.). The 4 equiv.). The reaction mixture was stirred for 4 h at room temp-
reaction mixture was stirred for 3 h at room temperature. erature. After the reaction was completed, the crude product was
Then, the crude product was diluted with AcOEt and washed evaporated under vacuum and purified by flash-column chrom-
with deionized water. The organic layer was dried over MgSO
4
atography (silica gel, DCM/MeOH, from 1 : 0 to 1 : 1, v/v). The
and concentrated under vacuum. The crude product was puri- product was obtained as an orange oil (217.7 mg, 6.24 mmol,
−1
fied by flash-column chromatography (silica gel, AcOEt/cyclo- 99% yield). IR (neat cm ) 2966, 2919, 1723, 1595, 1490, 1130,
1
hexane from 1 : 9 to 7 : 3, v/v). The product 5 was obtained as a 730; H NMR (300 MHz, MeOD) δ 7.95 (d, J = 8.9 Hz, 2H),
13
pale yellow amorphous powder (42.7 mg, 0.076 mmol, yield 7.86–7.71 (d, J = 8.9 Hz, 2H), 3.70–3.42 (m, 14H), 2.09 (s, 3H);
5%) and compound 6 was isolated as a yellow amorphous NMR (75 MHz, MeOD) δ 169.9, 165.7, 163.9, 152.2, 144.8, 129.6,
powder (11.4 mg, 0.032 mmol, yield 36%). Compound 5: 128.8, 120.5, 71.2, 70.6, 41.1, 40.7, 14.6; HRMS (ESI) exact mass
C
8
−
1
+
m.p. 137 °C; IR (neat cm ): 2921, 1705, 1596, 1499, 1365, calcd for C17
1
(
H
25
N
4
O
4
m/z 349.1876; found m/z 349.1888 [M + H] .
Step 2: To a solution of commercially available fluorescein
d, J = 7.6 Hz, 4H), 7.25 (t, J = 9.4 Hz, 4H), 7.10 (m, 6H), 4.88 (s, isothiocyanate (40 mg, 0.103 mmol, 1 equiv.) in a 2 mL solu-
1
412, 1272, 1164, 1023, 751. H NMR (300 MHz, CDCl ) δ 7.56
3
1
9
1
3
H), 4.73 (s, 1H), 4.16 (d, J = 5.9 Hz, 2H), 2.09 (s, 6H), 1.41 (s, tion of dry MeOH/THF (1 : 1, v/v) were added N-(2-(2-(2-amino-
1
3
H); C NMR (75 MHz, CDCl ) δ 157.9, 156.2, 146.6, 140.2, ethoxy)ethoxy)ethyl)-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-
3
37.2, 136.9, 129.0, 127.6, 127.5, 126.3, 121.4, 105.9, 79.7, 44.4, 1-yl)benzamide (39.4 mg, 0.113 mmol, 1.1 equiv.) and DIEA
3.6, 28.5, 11.8; HRMS (ESI) exact mass calcd for C33
H
36
N
5
O
4
(72 μL, 0.412 mmol, 4 equiv.). The reaction mixture was stirred
for 2 h at room temperature. After the reaction was completed
+
m/z 566.2767 found m/z 566.2770 [M + H] .
Compound 6: m.p. 173 °C; IR (neat cm−1): 2921, 2855, (monitored by RP-HPLC, system QC), the crude product was
31
1
8
7
61, 1584, 1547, 1488, 1324, 1172, 1091, 1059, 1005, 891, evaporated under vacuum and the resulting residue was
1
14, 750. H NMR (300 MHz, CDCl ) δ 7.96–7.83 (m, 4H), diluted with aq 0.1% TFA and purified by semi-preparative
3
.50–7.38 (m, 4H), 7.32–7.26 (m, 3H), 2.38 (s, 6H); exact RP-HPLC (system B). The product-containing fractions were
mass calcd for C21
H
19
N
4
O
2
m/z 359.1508 found m/z 359.1501 lyophilized to give 28.3 mg (37% yield) of the product as an
+
[M + H] .
orange amorphous powder. M.p. 169 °C (degradation); IR
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem., 2019, 17, 388–396 | 393