R. Wang et al.
BioorganicChemistry83(2019)461–467
was raised to 40 °C for 30 min. With the termination of heating, the
mixture was poured into 50 ml ice-water under stirring. Extraction with
20 ml DCM/ether (1:5, V/V) for 4 times. All the extractions were
combined. The organic layer was then washed water, 0.1 mol/L HCl
solution, water, 0.1 mol/L NaOH solution, and water, respectively.
Then it was dried over anhydrous MgSO4. Excess solvents were re-
moved by rotative evaporation under reduced pressure. The residues
were purified by flash column liquid chromatography with EtOAc/
petroleum ether (1:5, V/V) as eluant, led to white solid 289.8 mg, yield
88.9%. 1H NMR (300 MHz, CDCl3) δ: 7.67 (d, J = 3.0 Hz, 1H), 7.64 (d,
J = 3.0 Hz, 1H), 7.21 (d, J = 3.0 Hz, 1H), 7.19 (d, J = 3.0 Hz, 1H),
4.22–4. 33 (m, 1H), 2.38 (s, 3H), 1.26 (s, 3H), 1.24 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ: 166.6, 141.5, 132.1, 129.1, 129.1, 126.8, 126.8,
DCM with 0.5 ml DIPEA. This solution was then added to the activated
body dropwise. Afterwards, the reaction mixture was warmed up to
room temperature and kept stirring for 3–4 h. Then, 100 ml saturated
NaCl solution was added to quench the reaction. The mixture was ex-
tracted by DCM for 5 times (5 × 60 ml). All the extractions were
combined. The organic layer was dried over anhydrous Na2SO4. Excess
solvents were removed by rotative evaporation under reduced pressure.
The residues were purified by flash column liquid chromatography with
CHCl3/methanl (10:1, V/V) as eluant, following further purification by
RP-HPLC with methanol/water (50:50, V/V) as eluant, led to slight
yellow sticky oil 690.1 mg, yield 69.0%, purity 98.5%. 1H NMR
(300 MHz, CD3OD) δ: 7.83–7.78 (m, 2H), 7.52–7.47 (m, 2H), 7.38–7.30
(m, 5H), 5.10 (d, J = 6.6 Hz, 2H), 4.29–3.96 (m, 5H), 3.57 (m, 2H),
3.22 (s, 1H), 3.17 (s, 2H), 2.05 (m, 3H), 1.87–1.66 (m, 2H), 1.26 (d,
J = 6.7 Hz, 6H); 13C NMR (75 MHz, CD3OD) δ: 173.58, 166.38, 165.55,
156.31, 140.79, 137.39, 134.46, 129.11, 128.77(2), 128.31, 127.75,
127.71, 127.48, 127.27(2), 65.88, 65.84, 56.59, 52.32, 48.91, 45.92,
41.43, 29.76, 24.29, 21.89 (2); ESI-MS (m/z): 532.5[M+Na]+; HRMS
(m/z): calc. for [C27H35N5O5 + Na]+ 532.2536, found 532.2533.
41.7, 22.8, 21.3, 21.3. ESI-MS m/z: 178.2[M+H]+, 200.3[M+Na]+
5.3.4. Synthesis of N-isopropyl-4-formylbenzamide (3)
.
To a 20-ml pressure-proof tube, N-isopropyl-4-methylbenzamide
(177.2 mg, 1.0 mmol) and 2.0 ml of 3.5 mol/L HNO3 solution were
added, and well-distributed by ultrasonic. A solution of ceric ammo-
nium nitrate (2.19 g, 4.0 mmol) in 8.0 ml 3.5 mol/L HNO3 was added
dropwise to the tube with stirring. At the end of addition, the sealed
tube was put over an oil-bath at 100 °C to react for 24 h. Afterwards, the
tube was cooled down and the reaction mixture was poured into 100 ml
of saturated NaCl solution. Then the mixture was extracted by DCM for
3 times (3 × 100 ml). All the extractions were combined. The organic
layer was dried over anhydrous Na2SO4. Excess solvents were removed
by rotative evaporation under reduced pressure. The residues were
purified by flash column liquid chromatography with EtOAc/petroleum
5.4. Biological section
5.4.1. Cell culture
All the cell lines were grown in specific media supplemented with
10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin and 100 µg/
ml streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were grown
in a 5% CO2 humidified atmosphere in incubators maintained at 37 °C.
ether (1:5, V/V) as eluant, led to white solid 136.2 mg, yield 71.2%. 1
H
5.4.2. MTT assay
NMR (300 MHz, CD3OD) δ: 10.03 (s, 1H), 7.95 (d, J = 9.0 Hz, 2H), 7.58
(d, J = 9.0 Hz, 2H), 4.10–4.30 (m, 1H), 1.26 (d, J = 6.0 Hz, 3H), 1.24
(d, J = 6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ: 193.5, 168.4, 130.6,
3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay was used to detect inhibition of cellular proliferation
mediated by the drugs. This assay was applied to all cell lines. The
process was described below: Cells in suspension were plated in 96-well
plates at a density of 1 × 104 cells/well and cultured for 24 h. Then the
medium was replaced with the respective medium containing drugs at
different concentrations and incubated for 48 h. The final DMSO con-
centration in all experiments was less than 0.1% in medium. The con-
centration range of tested samples was 0–50 μM and two-fold serial
dilutions were applied. Afterwards, 10 μL MTT solutions (5 mg/ml)
were added to each well, and the plate was incubated for an additional
4 h. The absorbance of the converted dye in living cells was measured at
a wavelength of 570 nm using a microplate reader (Bio-Rad; Hercules,
CA, USA) after 100 μL of DMSO added. IC50 values were determined by
the nonlinear multipurpose curve fitting program GraphPad Prism. All
of the tests were repeated at least 3 times.
129.0, 128.1(2), 127.2(2), 43.4, 22.5(2); ESI-MS (m/z): 192.3[M+H]+
,
224.5[M+Na]+
.
5.3.5. Synthesis of procarbazine (4)
To 250-ml flask, N-isopropyl-4-formylbenzamide (192.3 mg,
a
1.0 mmol), methyl hydrazine hydrochloride (505.0 mg, 3.5 mmol), and
20 ml absolute ethanol were added. After stirred for 20 min, 1.0 ml
triethylamine was added. The sealed flask was kept over an oil-bath at
60 °C to react for 6 h. Afterwards, excess solvents were removed. The
residues were re-dissolved in 10.0 ml DMF, following the addition of
NaCNBH3 (126.0 mg, 2.0 mmol) slowly at 0 °C. The mixture was then
warmed up to room temperature and kept stirring overnight. The re-
action was quenched by the addition of 200 ml of saturated NaCl so-
lution. Then the mixture was extracted by EtOAc for
5 times
(5 × 40 ml). All the extractions were combined. The organic layer was
dried over anhydrous Na2SO4. Excess solvents were removed by rota-
tive evaporation under reduced pressure. The residues were purified by
flash column liquid chromatography with EtOAc/petroleum ether (1:3,
V/V) as eluant, led to white solid 161.9 mg, yield 74.0%, purity 97.3%.
1H NMR (300 MHz, CDCl3) δ: 7.47 (d, J = 7.0 Hz, 1H), 7.43 (d,
J = 7.0 Hz, 1H), 7. 31 (d, J = 7.0 Hz, 1H), 7. 24 (d, J = 7.0 Hz, 1H),
3.88–3.91 (m, 1H), 3.93 (s, 2H), 2.47 (s, 3H), 1.25 (d, J = 6.0 Hz, 3H),
1.23 (d, J = 6. 0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ: 166.2, 144.4,
130.1, 125.8, 125.8, 125.7, 125.7, 55.2, 40.4, 33.7, 22.3, 22.3; ESI-MS
5.4.3. Chromatography for detection of purity and concentration
High-performance liquid chromatography (HPLC) assay for clea-
vage of Z-GP-Pcb by FAPα was performed with Agilent 1200 series
(Karlsruhe, Germany); UV-detection at 214/245 nm; column: ZirChrom
Separations Inc. (Anoka, MN), reverse-phase C18 (4.6 × 250 mm) with
precolumn; conditions: flow 1.0 ml/min; mobile phase: water/me-
thanol (40/60, V/V); time: 0–15 min; injection volume:10 μL. All of the
tests were repeated at least 3 times.
5.4.4. Cleavage of Z-GP-Pcb by rhFAPα
(m/z): 222.3[M+H]+, 244.3[M+Na]+
.
Z-GP-Pcb was incubated in Tris-buffer (pH7.4) with rhFAPα (2 μg/
mL, and 5 μg/mL, respectively) at a final concentration of 30 μM at
37 °C for 1, 2, 4, 8, 16, and 24 h. Subsequently, the sample of each time
point was collected and analyzed by RP-HPLC. All of the tests were
repeated at least 3 times.
5.3.6. Synthesis of carbobenzoxy glycylprolyl procarbazine (Z-GP-Pcb,
5α/5β)
Z-GP-OH (612.0 mg, 2.0 mmol), HOBt (283.0 mg, 2.1 mmol), HATU
(798.0 mg, 2.1 mmol), and 10.0 ml dried DCM were added to a 50-ml
flask. The flask was cooled over an ice-bath. Then the addition of 0.3 ml
DIPEA was followed under stirring. The mixture was stirred for 30 min
to activate the carboxyl group, which was called activated body.
Meanwhile, procarbazine (442.6 mg, 2.0 mmol) was dissolved in 5.0 ml
5.4.5. Cleavage of Z-GP-Pcb with tumor homogenate
Tumor tissue was obtained from model mice bearing H22 tumor and
cut into small pieces. Every 200 mg of tumor tissue was added to 800 μL
of homogenates buffer [50 mM Tris-HCl buffer (pH 7.4)] to prepare
466