Z. Li et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
7
(
2
.49–7.42 (m, 2H), 7.18 (s, 1H), 7.07 (d, J = 8.8 Hz, 1H), 7.02–6.96
m, 2H), 6.77 (t, d, J = 9.0 Hz, 1H), 6.69 (s, 2H), 5.09 (s, 2H), 4.74 (s,
H), 4.66 (t, J = 6.0 Hz, 2H), 4.43 (t, J = 6.0 Hz, 2H), 1.91(s, 6H); 13
NMR (75 MHz, DMSO‑d
4.4. Animals and statistical analysis of the data
C
Rabbits and mice were purchased from Guangdong Medical
Laboratory Animal Center (Guangdong, China). All animals were ac-
climatized for one week before the experiments, and fed standard pel-
lets and water ad libitum. The animal room was maintained under a
controlled temperature (23 ± 2 °C) with constant light/black cycle
(12 h) and humidity (50 ± 10%). All animal experimental protocols
were approved by the ethical committee at Guangdong Pharmaceutical
University and conducted according to the Laboratory Animal
Management Regulations in China and adhered to the Guide for the
Care and Use of Laboratory Animals published by the National
Institutes of Health (NIH Publication NO. 85-23, revised 2011).
Statistical analyses were performed using GraphPad InStat version
6
) δ: 170.3, 157.5, 153.7, 152.3, 140.5, 139.7,
1
7
36.4, 133.5, 128.6, 128.8, 128.3, 125.9, 115.8, 112.7, 110.5, 104.2,
3.6, 69.7, 66.8, 65.7, 20.6; ESI-MS m/z: 484.1 [M−H] ; Anal. calcd.
−
For C25
N, 2.88.
8
H24FNO : C, 61.85; H, 4.98; N, 2.89; Found: C, 61.81; H, 4.97;
4.1.8. 2-(4-((2′,6′-Dimethyl-4′-(3-(nitrooxy)propoxy)-[1,1′-biphenyl]-3-
yl)methoxy)-2-fluorophenoxy)acetic acid (3)
Yield: 27%; m.p. 105–107 °C; 1H NMR (300 MHz, DMSO‑d
) δ:
6
7.48–7.42 (m, 2H), 7.19 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.02–6.97
(
m, 2H), 6.78 (t, d, J = 9.0 Hz, 1H), 6.69 (s, 2H), 5.09 (s, 2H), 4.74 (s,
5.00 (San Diego, CA, USA). General effects were analyzed by using a
2
2
1
1
4
2
H), 4.58 (t, J = 6.1 Hz, 2H), 4.33 (t, J = 6.1 Hz, 2H), 2.17–2.13 (m,
H), 1.91(s, 6H); C NMR (75 MHz, DMSO‑d ) δ: 170.1, 157.9, 153.5,
52.5, 140.2, 139.7, 136.4, 133.5, 128.6, 128.8, 128.3, 125.9, 115.8,
12.7, 110.5, 104.2, 71.5, 69.7, 65.7, 64.3, 26.7, 20.8; ESI-MS m/z:
8
98.1 [M−H] ; Anal. calcd. For C26H26FNO : C, 62.52; H, 5.25; N,
.80; Found: C, 62.56; H, 5.24; N, 2.81.
one-way ANOVA with Tukey’s multiple-comparison post hoc test.
1
3
6
4.5. Anti-platelet aggregation
−
Blood were collected from rabbit carotid artery into heparin-con-
taining tubes. After centrifugation at 500 rpm for 10 min to obtain
platelet-rich plasma (PRP), the remaining blood was further centrifuged
at 3000 rpm for another 10 min to obtain platelet-poor plasma (PPP).
PRP was pre-incubated in duplicate for 5 min at 37 °C with vehicle, the
tested compounds, or positive control (Ticlopidine) at the concentration
of 0.1 mM. Carboxy-PTIO (20 μM) was added and incubated with the
drug-platelet suspension in the indicated group. The platelet aggrega-
tion in individual PPR samples was induced by ADP (10 μM), followed
by recording of light transmission at maximal aggregation within 5 min.
The inhibition rate was calculated as following: inhibition rate
4
.1.9. 2-(4-((2′,6′-Dimethyl-4′-(4-(nitrooxy)butoxy)-[1,1′-biphenyl]-3-
yl)methoxy)-2-fluorophenoxy)acetic acid (4)
Yield: 39%; m.p. 109–111 °C; 1H NMR (300 MHz, DMSO‑d
.47–7.42 (m, 2H), 7.16 (s, 1H), 7.08 (d, J = 8.8 Hz, 1H), 7.01–6.95
) δ:
6
7
(
m, 2H), 6.76 (t, d, J = 9.1 Hz, 1H), 6.69 (s, 2H), 5.09 (s, 2H), 4.74 (s,
2
1
1
1
2
H), 4.38 (t, J = 6.0 Hz, 2H), 4.12 (t, J = 6.0 Hz, 2H), 1.93 (s, 6H),
1
3
.87–1.82 (m, 2H), 1.65–1.62 (m, 2H); C NMR (75 MHz, DMSO‑d ) δ:
6
70.3, 157.9, 153.3, 152.5, 140.2, 139.7, 136.7, 133.5, 128.6, 128.5,
28.1, 125.9, 115.8, 112.7, 110.5, 104.5, 74.6, 69.7, 68.3, 65.7, 27.5,
(
%) = (A − B)/A × 100%, A = the platelet aggregation of vehicle
−
8
3.4, 20.6; ESI-MS m/z: 512.1 [M−H] ; Anal. calcd. For C27H28FNO :
group, B = the platelet aggregation of compound group.
C, 63.15; H, 5.50; N, 2.73; Found: C, 63.18; H, 5.51; N, 2.72.
4.6. NO release assay
4.2. Molecular modeling
NO release of compound 3 was determined by Griess assay. Briefly,
MOE (version 2014.0901, The Chemical Computing Group,
compound 3 (0.03, 0.1, 0.2, and 0.3 mM) in phosphate buffer solution
containing 2% dimethyl sulfoxide and 5.0 mM L-cysteine at pH 7.4 was
incubated at 37 °C for 15 to 300 min and were sampled every 15 min for
120 min and then every 30 min for the remaining time. The collected
samples (2 mL) were mixed with 0.5 mL of Griess reagent and incubated
at 37 °C for 10 min, followed by measuring at 550 nm. The different
concentrations of nitrite were used as standards to calculate the con-
centrations of NO.
the reported crystal structure of hFFA1 (PDB ID: 4PHU). Prior to mo-
lecular docking, other crystallized ligands and water were removed,
and the obtained protein was performed by Protonate 3D prior to the
Gaussian Contact surface was draw around the binding pocket of TAK-
8
75. Subsequently, the binding pocket with Gaussian Contact surface
was isolated. Compound 3 was induced-fit docked into the binding
pocket and ranked with London dG scoring function. For energy
minimization in binding pocket, MOE Forcefield Refinement was per-
formed and ranked with London dG scoring function.
4.7. Oral glucose tolerance test in mice
Normal male ICR mice (Comparative Medicine Centre of Yangzhou
.3. Ca2+ influx activity of CHO cells stably expressing human FFA1
FLIPR Assay)
University) after adaptation one week were fasted for 12 h, weighted,
bled via the tail tip, and randomized into 4 groups (n = 6 per group).
Compound 3 (20 mg/kg), TAK-875 (20 mg/kg), or vehicle (0.5% me-
thylcellulose aqueous solution) was orally administered 30 min before
oral glucose load (3 g/kg). Blood samples were collected immediately at
4
(
CHO cells stably expressing human FFA1 were plated at a density of
2
15 K cells/well and incubated 12 h in 5% CO at 37 °C. Subsequently,
−
30 min (just before drug administration), 0 min (just before glucose
culture medium was removed and washed with Hank’s Balanced Salt
Solution (100 μL). Then, cells were incubated in loading buffer (re-
cording medium containing 2.5 μg/mL fluorescent calcium indicator
Fluo 4-AM, 0.1% fatty acid-free BSA and 2.5 mmol/L probenecid) for
challenge), and 15, 30, 60 and 90 min after glucose load. The blood
glucose was measured by plasma glucose test strips (SanNuo ChangSha,
China).
1
h at 37 °C. Various concentrations of test compounds or γ-linolenic
2+
acid (Sigma) were added into the cells, and the intracellular Ca flux
signals after addition were monitored by FLIPR Tetra system (Molecular
Devices) for 90 s. The agonistic activities of test compounds on human
FFA1 were expressed as [(A − B)/(C − B)] × 100 (increase of the in-
tracellular calcium concentration (A) in the test compounds-treated
cells and (B) in vehicle-treated cells, and (C) in 10 μM γ-linolenic acid-
treated cells).
Acknowledgements
This study was supported by grants from the National Natural
Science Foundation of China (Grants 81320108029 and 81773995), the
Starting Foundation for Ph.D., Guangdong Pharmaceutical University
(Grant 43540073) and Guangdong Province Medical Science and
Technology Research Fund (Grant A2018273).
6