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chromatography. 1H NMR (300 MHz, CDCl3) d 8.80 (d, J ¼
5.1 Hz, 1H), 8.15 (d, J ¼ 9.0 Hz, 1H), 8.09 (s, 1H), 8.06 (d, J ¼
1.8 Hz, 1H), 7.74 (dd, J ¼ 9.0, 4.5 Hz, 2H), 7.45 (dd, J ¼ 9.0,
1.8 Hz, 1H), 7.29–7.23 (m, 2H), 7.01 (d, J ¼ 5.4 Hz, 1H), 5.54 (s,
2H). 13C NMR (75 MHz, CDCl3) d 175.35, 161.23, 152.10, 149.03,
143.35, 136.34, 133.05, 127.22, 126.98, 123.43, 122.78, 122.66,
121.59, 119.66, 117.05, 116.74, 101.55, 62.30. HRMS m/z: calcd
4.3. Procedures for biological assays
4.3.1. Recombinant FP2 protein preparation. Recombinant
FP2 was prepared according to the method described by Shenai,
et al.62 and Kumar, et al.63 with slight modication. Briey,
bacteria containing the PRSET-A FP2 plasmid were grown to
mid-log phase and induced with isopropyl-1-thio-b-D-gal-
actopyranoside (IPTG, 1 mM) for 5 h at 37 ꢁC. IPTG induced
pellet were suspended in ice-cold native buffer (50 mM
NaH2PO4 and 100 mM NaCl). Added lysozyme (10 mL/1 mL; 1 mg
mLꢀ1 (FC); stock 100 mg mLꢀ1), mixed well and keep in ice for
30 min. Sonicated and centrifuged at 15 000 rpm for 45 min at
4 ꢁC. The pellet was washed twice with native buffer. Finally, the
pellet was solubilized in UB [8 M urea, 20 mM Tris, 250 mM
NaCl, pH 8.0]; (5 mL gꢀ1 of inclusion body pellet) at room
temperature for 60 min with gentle stirring. The insoluble
material was separated by centrifuging at 15 000 rpm for 60 min
C
18H12ClFN4O [M + H]+ 354.0684 found 355.0793 elemental
anal.: calcd: C, 60.85; H, 3.32; Cl, 9.99; F, 5.63; N, 15.97; O, 4.42.
4.2.3.7. 7-Chloro-4-{[1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl]
methoxy} quinoline (T7). Light yellow solid, (Rf ¼ 0.3 in pure
ethyl acetate) mp 166–168 ꢁC, 72% yield obtained aer column
chromatography. 1H NMR (300 MHz, CDCl3) d 8.79 (d, J ¼
5.1 Hz, 1H), 8.53 (s, 1H), 8.44 (d, J ¼ 8.7 Hz, 1H), 8.18 (d, J ¼
8.7 Hz, 1H), 8.10 (d, J ¼ 9.0 Hz, 2H), 8.02 (s, 1H), 7.46 (d, J ¼
9.0 Hz, 1H), 7.38 (s, 1H), 7.03 (d, J ¼ 5.1 Hz, 1H), 5.56 (s, 2H). 13
C
NMR (75 MHz, CDCl3) d 165.53, 157.39, 148.65, 145.87, 140.32,
132.44, 131.29, 130.23, 128.52, 127.44, 125.49, 106.60, 66.82,
45.41, 44.85, 44.30. HRMS m/z: calcd C18H12ClN5O3 [M + H]+
381.0629 found 382.0733 elemental anal.: calcd: C, 57.00; H,
3.35; Cl, 9.36; N, 18.43; O, 13.01.
ꢁ
at 4 C. Added imidazole to 10 mM, Triton X-100 to 1% super-
natant. In order to purify of recombinant protein, supernatant
was incubated overnight at a temperature of 4 ꢁC with a nickel-
nitrilotriacetic acid (Ni-NTA) resin. The resin was loaded on the
column and it was washed with 10 bed volumes each of UB.
Finally, the bound protein was eluted in UB with 250 mM
imidazole and measured by the bicinchoninic acid assay. For
refolding, the fractions containing FP2 protein were pooled in
ice-cold refolding buffer and diluted 100-fold using Tris–HCl
(100 mM), 1 mM EDTA, 20% glycerol, 250 mM L-arginine, 1 mM
GSH, 1 mM GSSG, pH 8.0. The mixture was incubated with
4.2.3.8. 7-Chloro-4-{[1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-
yl]methoxy} quinoline (T8). Yellow solid powder, (Rf ¼ 0.5 in pure
ethyl acetate) mp 179–181 ꢁC, 76% yield obtained aer column
chromatography. 1H NMR (300 MHz, CDCl3) d 8.80 (d, J ¼
5.4 Hz, 1H), 8.15 (d, J ¼ 9.0 Hz, 1H), 8.06 (s, 2H), 7.65 (d, J ¼
9.0 Hz, 2H), 7.45 (d, J ¼ 9.0 Hz, 1H), 7.27 (s, 1H), 7.04 (d, J ¼
8.7 Hz, 2H), 5.53 (s, 2H), 3.88 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 175.36, 161.39, 160.10, 151.97, 148.75, 142.87, 136.33, 130.13,
126.93, 123.52, 122.29, 121.68, 119.65, 114.86, 101.57, 62.37,
55.64. HRMS m/z: calcd C19H15ClN4O2 [M + H]+ 366.0884 found
367.0927 elemental anal.: calcd: C, 62.03; H, 4.02; Cl, 10.00; N,
15.55; O, 8.54.
4.2.3.9. 7-Chloro-4-{[1-(2-methylphenyl)-1H-1,2,3-triazol-4-yl]
methoxy} quinoline (T9). Snow white solid, (Rf ¼ 0.5 in pure ethyl
acetate) mp 180–182 ꢁC, 73% yield obtained aer column
chromatography. 1H NMR (300 MHz, CDCl3) d 8.80 (d, J ¼
5.1 Hz, 1H), 8.12 (dd, J ¼ 16.5, 9.0 Hz, 3H), 7.62 (d, J ¼ 8.1 Hz,
2H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.33 (d, J ¼ 7.9 Hz, 2H), 7.01 (d, J ¼
4.8 Hz, 1H), 5.53 (s, 2H), 2.43 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 175.33, 161.35, 152.04, 148.90, 142.98, 139.39, 136.30, 134.47,
130.34, 127.06, 126.94, 123.50, 121.47, 120.56, 119.75, 119.67,
101.56, 62.38, 21.05. HRMS m/z: calcd C19H15ClN4O [M + H]+
350.0934 found 351.0924 elemental anal.: calcd: C, 65.05; H,
4.30; Cl, 10.11; N, 16.00; O, 5.01.
ꢁ
moderate stirring at 4 C for 24 h, and concentrated to 25 mL
using a stirred cell withꢁa 10 kDa cut-off membrane (Pellicon XL
device, Millipore) at 4 C. The sample was then ltered using
a 0.22 mm syringe lter. The puried and concentrated protein
was quantied using bicinchoninic acid assay.
4.3.2. Enzyme assay and kinetic analysis. FP2 activity was
carried out as described previously by Kumar, et al.63 Briey, in
96 well plate 200 mL of assay buffer (100 mM sodium acetate pH
5.5, 10 mM DTT) containing 15 mM FP2 enzyme, 10 mM uo-
rogenic
substrate
benzyloxycarbonyl-Phe-Arg-7-amino-4-
methylcoumarin hydrochloride (ZFR-AMC) was added and the
release of 7-amino-4-methyl coumarin (AMC) was monitored
(excitation 355 nm; emission 460 nm) over 30 min at RT using
a LS50B PerkinElmer uorimeter. To analyze the effect of
inhibitors on enzyme activity, recombinant FP2 was pre-
incubated with different concentration of each inhibitor for
10 min at room temperature. Remaining activity was deter-
mined using the uorogenic substrate. Kinetic constants km,
Vmax, and IC50 values were determined using PRISM soware
(Graph Pad, San Diego).
4.3.3. P. falciparum culture and inhibition assay. P. falci-
parum strain 3D7 was cultured with human erythrocytes (4%
hematocrit) in RPMI media (Invitrogen) supplemented with
0.5% albumax and 4% hematocrit using a protocol described
previously.69 Cultures were synchronized by repeated sorbitol
treatment following Lambros and Vanderberg.70 Each growth
inhibition assay was performed in triplicate, and the experi-
ment was repeated twice. Each well contained 0.2 mL of
complete media (RPMI (Invitrogen) with 0.5% albumax), 2%
4.2.3.10. 7-Chloro-4-[(1-phenyl-1H-1,2,3-triazol-4-yl)methoxy]
quinoline (T10). White, (Rf ¼ 0.3 in pure ethyl acetate) mp 168–
1
ꢁ
170 C, 71% yield obtained aer column chromatography. H
NMR (300 MHz, CDCl3) d 8.77 (d, J ¼ 5.1 Hz, 1H), 8.15 (d, J ¼
5.7 Hz, 1H), 8.03 (s, 1H), 7.76 (d, J ¼ 7.8 Hz, 2H), 7.57–7.43 (m,
4H), 7.27 (s, 1H), 6.99 (d, J ¼ 5.4 Hz, 1H), 5.53 (s, 2H). 13C NMR
(75 MHz, CDCl3) d 160.87, 152.44, 149.69, 143.31, 136.78,
135.91, 129.86, 129.15, 127.82, 126.70, 123.43, 121.44, 120.64,
119.70, 101.51, 62.27. HRMS m/z: calcd C18H13ClN4O [M + H]+
336.0778 found 337.0790 elemental anal.: calcd: C, 64.00; H,
4.00; Cl, 10.35; N, 17.00; O, 4.90.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 39410–39421 | 39419