Improving metabolic stability with deuterium: The discovery of HWL-066, a potent and long-acting free fatty acid receptor 1 agonists

Article abstract of DOI:10.1111/cbdd.13321

The free fatty acid receptor 1 (FFA1) is a potential target due to its function in enhancing of glucose-stimulated insulin secretion. The FFA1 agonist GW9508 has great potential for the treatment of type 2 diabetes mellitus, but it has been suffering from high plasma clearance probably because the phenylpropanoic acid is vulnerable to β-oxidation. To identify orally available analog without influence on the unique pharmacological mechanism of GW9508, we tried to interdict the metabolically labile group by incorporating two deuterium atoms at the α-position of phenylpropionic acid affording compound 4 (HWL-066). As expected, HWL-066 revealed a lower clearance (CL?=?0.23?L^?1?hr^?1?kg^?1), higher maximum concentration (C_max?=?5907.47?μg/L), and longer half-life (T_1/2?=?3.50?hr), resulting in a 2.8-fold higher exposure than GW9508. Moreover, the glucose-lowering effect of HWL-066 was far better than that of GW9508 and comparable with TAK-875. Different from glibenclamide, no side-effect of hypoglycemia was observed in mice after oral administrating HWL-066 (80?mg/kg).

Full text of DOI:10.1111/cbdd.13321

Received: 2 October 2017
DOI: 10.1111/cbdd.13321
Revised: 21 February 2018
Accepted: 11 March 2018
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R E S E A R C H A R T I C L E
Improving metabolic stability with deuterium: The discovery
of HWL-066, a potent and long-acting free fatty acid receptor 1
agonists
Chunxia Liu1
Yuxuan Dai1
Zheng Li2
Wei Shi1
Wenlong Huang
Huilan Li
1
Nasi Wang
1
|
|
|
|
|
Chen Liao1
1,3
Hai Qian
3
|
|
|
1Center of Drug Discovery, State Key
Laboratory of Natural Medicines, China
Pharmaceutical University, Nanjing, China
2School of Pharmacy, Guangdong
Pharmaceutical University, Guangzhou,
China
3Jiangsu Key Laboratory of Drug
The free fatty acid receptor 1 (FFA1) is a potential target due to its function in en-
hancing of glucose-stimulated insulin secretion. The FFA1 agonist GW9508 has
great potential for the treatment of type 2 diabetes mellitus, but it has been suffering
from high plasma clearance probably because the phenylpropanoic acid is vulnerable
to β-oxidation. To identify orally available analog without influence on the unique
pharmacological mechanism of GW9508, we tried to interdict the metabolically la-
bile group by incorporating two deuterium atoms at the α-position of phenylpropi-
onic acid affording compound 4 (HWL-066). As expected, HWL-066 revealed a
Discovery for Metabolic Disease, China
Pharmaceutical University, Nanjing, China
Correspondence
−
1
−1
−1
Wenlong Huang and Hai Qian, Centre of
Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical
University, Nanjing, China.
lower clearance (CL = 0.23 L hr kg ), higher maximum concentration
Cmax = 5907.47 μg/L), and longer half-life (T1/2 = 3.50 hr), resulting in a 2.8-fold
(
higher exposure than GW9508. Moreover, the glucose-lowering effect of HWL-066
was far better than that of GW9508 and comparable with TAK-875. Different from
glibenclamide, no side-effect of hypoglycemia was observed in mice after oral ad-
ministrating HWL-066 (80 mg/kg).
Emails: ydhuangwenlong@126.com,
qianhai24@163.com
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 81273376
and 81673299
K E Y W O R D S
deuterium, diabetes, free fatty acid receptor, pharmacokinetic profiles, phenylpropanoic acid
secretagogues with low risk of hypoglycemia would be ad-
vantageous for the management of T2DM. The free fatty acid
receptor 1 (FFA1) has emerged as a promising antidiabetic
1
INTRODUCTION
|
Type 2 diabetes mellitus (T2DM) is a progressive metabolic
[5]
target. FFA1 is mainly expressed in pancreas where it stim-
syndrome characterized by insufficient insulin secretion
ulates glucose-dependent insulin secretion, providing a huge
advantage of decreasing the risk of hypoglycemia compared
[1,2]
and/or insulin resistance.
The insulin secretagogues such
as sulfonylureas are widely used for the administration of
[6–8]
to sulfonylureas.
Moreover, the limited tissue distribution
[
3]
T2DM. However, sulfonylureas are often associated with
of FFA1 reduces the possibility of “on-target” side-effects in
[4]
the side-effect of hypoglycemia. Therefore, novel insulin
[9]
other tissues.
[10]
As summarized in our review,
many synthetic ago-
[
11–18]
Abbreviations: AUC, area under curve; CL, plasma clearance; Cmax, maxi-
mum plasma concentration; DMF, N,N-dimethylformamide; FFA1, free
fatty acid receptor 1; FLIPR, fluorometric imaging plate reader; NMR, nu-
clear magnetic resonance; OGTT, oral glucose tolerance test; PDB, Protein
Data Bank; SD rats, Sprague Dawley rats; SD, standard deviation; T1/2
plasma half-life time; T2DM, type
tetrahydrofuran.
nists have been reported in different state (Figure 1).
Among them, the candidates TAK-875, LY2881835, and
[5,10]
AMG-837 have reached clinical trials.
Besides, the
chemical space of FFA1 agonists with different scaffolds has
,
[19–25]
also been comprehensively explored in our laboratory.
On the whole, GW9508, one of the first reported FFA1
2
diabetes mellitus; THF,
Chem Biol Drug Des. 2018;1–8.
wileyonlinelibrary.com/journal/cbdd
© 2018 John Wiley & Sons A/S
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1

2
LIU et aL.
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FIGURE 1 Reported FFA1 agonists and its phenylpropanoic acid moiety (red mark)
FIGURE 2 The route of β-oxidation and the deuterated strategy to decrease the β-oxidation of GW9508
agonists, was developed by GlaxoSmithKline as a widely
used pharmacological tool to explore the physiological
half-life than GW9508. Although in-vitro activity difference
between GW9508 and HWL-066 is quite small, the glucose-
lowering effect of HWL-066 was far better than that of
GW9508. Besides, the induced-fit docking study and the risk
of hypoglycemia were also explored.
[
26]
function of FFA1.
Moreover, GW9508 have multimech-
anisms to regulate insulin sensitivity and glucose homeosta-
sis by reducing fetuin-A levels and activating Akt/GSK-3β
[
27]
pathway.
Therefore, GW9508 has great potential for the
treatment of T2DM. However, GW9508 has been suffered
2
2
METHODS AND MATERIALS
General chemistry
−1
−1
−1
|
from high plasma clearance (CL = 24.9 ml min kg ) in
rats probably because the phenylpropanoic acid is vulnerable
.1
[
11,17]
|
to β-oxidation.
As summarized in Figure 2, the path-
way of β-oxidation is derived from the cinnamic acid 2 and
All commercially available materials and reagents were used
without purification unless otherwise indicated. Purification
by column chromatography was carried out using silica gel
(200–300 mesh). Melting points were determined on a RY-1
melting point apparatus and were uncorrected. We detected
[11,28]
ultimately afford the benzoic acid 3,
and the pathway
can be blocked by structural optimizations such as α- and/or
[
29]
β-disubstitution. Therefore, we tried to decrease the gen-
eration of cinnamic acid 2 by incorporating two deuterium
atoms at the α-position of phenylpropionic acid (compound
1
13
the NMR spectra (300 MHz for H NMR, 75 MHz for
C
4
, HWL-066), because the kinetic isotope effect of deute-
NMR spectra) on Bruker ACF-300Q instrument. Chemical
shifts are showed as values relative to internal standard (tetra-
methylsilane), and coupling constants (J values) were given
in hertz (Hz). LC/MS spectra were determined on Waters
rium at sites of metabolism is the rate-determining step can
[
30–32]
reduce metabolism.
As expected, HWL-066 revealed a
lower clearance, higher maximum concentration, and longer

LIU et aL.
3
|
liquid chromatography–mass spectrometer system (ESI). We
carried out elemental analyses by Heraeus CHN-O-Rapid
analyzer. The positive control (TAK-875 and GW9508) was
synthesized as previously reported procedures.[
the residual white solid was used in the next step without
further purification. To a solution of the obtained interme-
diate 5a (0.8 g, 4.0 mmol) in dichloromethane (20 ml) was
slowly added thionyl chloride (2.9 g, 24 mmol) and a cata-
lytic amount of DMF at room temperature. After stirring at
12,26]
4
0°C for 4 hr, the reaction was concentrated under reduced
2
.1.1
Methyl 3-(4-hydroxyphenyl)
|
pressure. The residue was purified by silica gel column chro-
matography using a mixture of petroleum ether/ethyl acetate
propanoate-2,2-d (3a)
2
A mixture of Meldrum’s acid (1.3 g, 9.1 mmol) and
(20:1, v/v) as eluent to afford intermediate 6a (0.75 g, 86%)
1
4
-hydroxybenzaldehyde (1.0 g, 8.2 mmol) in water (10 ml)
as a solid. H NMR (300 MHz, DMSO-d ) δ: 7.45–7.37 (m,
6
was stirred at 75°C for 2 hr. After cooling to room temper-
ature, the solid product was filtered on Buchner and dried
under vacuum. The purification from traces of starting alde-
hyde, when present, was performed by crystallization with
ethanol. The obtained intermediate (1.8 g, 7.2 mmol) was
3H), 7.21 (d, J = 7.6 Hz, 1H), 7.16–7.13 (m, 1H), 7.09–7.06
(m, 1H), 7.01 (d, J = 7.9 Hz, 2H), 6.96–6.93 (m, 1H), 4.86
(s, 2H).
2
.1.3
3-(4-((3-phenoxybenzyl)oxy)phenyl)
|
stirred in methanol (20 ml), and NaBH was added slowly
4
propanoic-2,2-d acid (4)
2
to keep the temperature between 23 and 28°C. After stir-
ring for another 15 min, the reaction mixture was acidified
To a solution of 6a (0.5 g, 2.3 mmol) and intermediate 3a
(
PH: 5–6) with 1N hydrochloric acid solution, and extracted
(0.4 g, 2.3 mmol) in acetonitrile was added K CO (0.6 g,
2
3
with ethyl acetate (4 × 25 ml), the organic fractions were
combined, washed with saturated brine (2 × 15 ml) prior to
drying over anhydrous sodium sulfate. After filtration and
concentrating with a rotary evaporator, the residue was used
in the next step without further purification. To a solution of
4.6 mmol) at room temperature. The reaction mixture was
heated to reflux with stirring for 8 hr. Then, the reaction mix-
ture was cooled to room temperature followed by filtration,
and the filtrate was concentrated under vacuum. The residue
was purified by silica gel column chromatography using a
mixture of petroleum ether/ethyl acetate (4:1, v/v) as eluent to
afford a white solid. To a solution of the obtained solid (0.6 g,
the obtained solid in DMF (10 ml) was added 0.5 ml D O
2
at room temperature. After stirring at 100°C for 12 hr, the
mixture was diluted with water (60 ml), extracted with ethyl
acetate (4 × 25 ml), washed with saturated brine (2 × 15 ml)
prior to drying over anhydrous sodium sulfate, and concen-
trated in vacuo. The residue was dissolved in MeOH (20 ml),
and then conc. H SO (2 ml) was added slowly at ambient
1.7 mmol) in 2:3:1 THF/MeOH/H O (18 ml) was added
2
LiOH·H O (0.2 g, 6.9 mmol). After stirring at room tempera-
2
ture for 4 hr, the volatiles were removed under reduced pres-
sure. The residue was acidified with 1N hydrochloric acid
solution and then filtered, and the filter cake was washed with
5 ml of water, dried in vacuum to afford a white powder. The
white powder was purified by column chromatography using
a mixture of petroleum ether/ethyl acetate (2:1–1:2, v/v)
2
4
temperature. After stirring at reflux for 3 hr, the volatiles
were removed under reduced pressure. The residue was
extracted with ethyl acetate (3 × 25 ml), and the combined
organic phases were washed with brine (2 × 15 ml), dried,
and filtered. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl
as eluent to afford the target compound 4 (0.45 g, 56%) as
1
white solid, m.p. 85–87°C. H NMR (300 MHz, DMSO-d )
6
δ: 12.13 (s, 1H), 7.46–7.38 (m, 3H), 7.20 (d, J = 7.5 Hz, 1H),
7.17–7.10 (m, 3H), 7.08–7.05 (m, 1H), 7.01 (d, J = 7.9 Hz,
2H), 6.96–6.93 (m, 1H), 6.89 (d, J = 8.3 Hz, 2H), 5.05 (s,
acetate (10:1, v/v) as eluent to afford intermediate 3a (0.84 g,
1
5
1
(
6%) as a solid. H NMR (300 MHz, DMSO-d ) δ: 9.32 (s,
6
1
3
H), 7.16 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 7.9 Hz, 2H), 3.63
s, 3H), 2.74 (s, 2H).
2H), 2.75 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 174.28,
6
157.25, 156.94, 140.03, 133.58, 130.48, 129.64, 123.98,
1
22.82, 119.16, 118.17, 117.87, 115.13, 69.13, 29.89.
−
ESI-MS m/z: 349.1 [M–H] . Anal. calcd. For C H D O :
2
2
18
2
4
2
.1.2
1-(chloromethyl)-3-phenoxybenzene
|
C, 75.41; H, 6.33; Found: C, 75.38; H, 6.32.
(
6a)
To a stirred mixture of NaBH (0.5 g, 13.2 mmol) and methyl
4
2
2
.2
Biological studies
Ca2+ influx activity (FLIPR assay)
|
3
-phenoxybenzoate (1.0 g, 4.4 mmol) in reflux anhydrous
THF (20 ml) was added MeOH (1 ml). After refluxing for
further 30 min, the reaction mixture was poured into ice
water (10 ml), and extracted with ethyl acetate (3 × 15 ml),
the organic fractions were combined, washed with saturated
brine (2 × 15 ml) prior to drying over anhydrous Na SO .
.2.1
|
CHO cells expressing hFFA1 were seeded on 96-well tissue
culture plates (15K cells per well) and incubated at 37°C for
16 hr under 5% CO prior to the assay. The medium in cul-
2
4
2
After filtration and concentrating with a rotary evaporator,
ture wells was removed and washed with Hank’s balanced

4
LIU et aL.
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salt solution (100 μl per well). Then, cells were incubated
in buffer containing 2.5 μg/ml Fluo 4-AM (fluorescent cal-
cium indicator), 0.1% fatty acid-free BSA, and 2.5 mmol/L
probenecid for 1 hr at 37°C. Test compounds or γ-linolenic
acid (Sigma) with various concentrations were added into
2
.2.5
Pharmacokinetic analysis in rats
|
Eight-week-old male SD rats (Comparative Medicine Centre
of Yangzhou University) after 1-week adaptation were fasted
for 12 hr, weighted, and randomized into two groups (n = 4
per group). The pharmacokinetic profiles of GW9508 and
HWL-066 were evaluated in fasted SD rats following sin-
gle oral dosing (3 mg/kg suspended in 0.5% methylcellulose
aqueous solution). At 5, 15, 30, and 45 min and 1, 2, 4, 6,
8, 12, 18, and 24 hr after oral administration, blood samples
were collected from tail vein into heparin-containing micro-
centrifuge tubes. The plasma was separated by centrifug-
ing at 10000 rpm for 10 min and then stored at −80°C until
analysis. Plasma proteins were precipitated with acetonitrile
containing an internal standard, mixed by vortexing, and cen-
trifuged at 17,530 g for 14 min. The supernatant was diluted
and centrifuged again, and 5 μl of supernatant was analyzed
by LC-PDA-MS/MS (Waters) to measure drug levels. We
performed pharmacokinetic profiles using das 2.1.1 software.
2
+
the cells, and the Ca flux signals after addition were re-
corded by the FLIPR Tetra system (Molecular Devices). The
hFFA1 agonistic activities of test compounds were expressed
2
+
as [(A − B)/(C − B)] × 100 (the increase in Ca flux signals
(
A) in test compounds and (B) in vehicle, and (C) in 10 μM γ-
linolenic acid). EC value of test compound was calculated
5
0
by GraphPad InStat version 5.00 (GraphPad Software, San
Diego, CA, USA).
2
.2.2
Molecular modeling
|
moe (version 2014.0901, The Chemical Computing Group,
Montreal, Canada) was used to perform the docking modeling
based on the reported crystal structure of hFFA1 (PDB ID:
4
PHU). Prior to molecular docking, other crystallized ligands
and water were removed, and the obtained protein was performed
by Protonate 3D prior to the Gaussian Contact or Gaussian con-
tact surface was drawn around the binding pocket of TAK-875.
Later, the binding pocket with Gaussian Contact surface was
isolated. HWL-066 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
performed and ranked with London dG scoring function.
2
.2.6
Oral glucose tolerance test in mice
|
Normal male ICR mice (Comparative Medicine Centre of
Yangzhou University) after one-week adaptation were fasted
for 12 hr, weighted, bled via the tail tip, and randomized
into four groups (n = 6 per group). HWL-066 (20 mg/kg),
GW9508 (20 mg/kg), TAK-875 (20 mg/kg), or vehicle (0.5%
methylcellulose aqueous solution) was orally administered
3
0 min before oral glucose load (3 g/kg). Blood samples
were collected immediately at −30 min (just before drug ad-
ministration), 0 min (just before glucose challenge), and 15,
30, 60, and 90 min after glucose load. The blood glucose was
measured by plasma glucose test strips (SanNuo ChangSha,
China).
2
.2.3
Animals
|
We purchased 8-week-old male SD rats (180–200 g) and
1
0-week-old male ICR mice (18–22 g) from Comparative
Medicine Centre of Yangzhou University (Jiangsu, China, an-
imal batch number for rats 201711823, for mice 201704423).
All mice and rats were acclimatized for 1 week before the ex-
periments and fed standard pellets and water ad libitum. We
maintained the animal room under a controlled temperature
2
.3
The risk of hypoglycemia in mice
|
Ten-week-old male normal ICR mice were fasted overnight
and randomized into three groups (n = 6). HWL-066 (80 mg/
kg), glibenclamide (15 mg/kg), or vehicle was orally admin-
istered, and blood was collected from tail vein immediately
before administration (0 hr) and at 0.5, 1, 1.5, 2, 2.5, and 3 hr
after administration and measure blood glucose as described
above.
(
23 ± 2°C) with constant light/black cycle (12 hr) and hu-
midity (50 ± 10%). All animal experimental protocols were
approved by the ethical committee at China 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 pub-
lished by the National Institutes of Health (NIH Publication
NO. 85-23, revised 2011).
3
RESULTS AND DISCUSSION
Chemistry
|
2
.2.4
Statistical analysis of the data
3.1
|
|
We performed statistical analyses using GraphPad InStat
version 5.00 (San Diego, CA, USA). We analyzed general
effects by Student’s t test or using a one-way ANOVA with
Tukey’s multiple-comparison post-hoc test.
The target compound 4 (HWL-066) was prepared accord-
ing to the synthetic route depicted in Scheme 1. The key
intermediate 2a was prepared by Knoevenagel conden-
sation of Meldrum’s acid and 4-hydroxybenzaldehyde,

LIU et aL.
5
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SCHEME 1 Synthesis of target compounds 4. Reagents and conditions: (a) Meldrum’s acid, water, 75°C, 2 hr; (b) NaBH , r.t.; (c) D O,
4
2
DMF, 100°C, 12 hr, then MeOH, H SO , reflux, 3 hr; (d) NaBH , CH OH, THF, reflux, 1 hr; (e) SOCl , CH Cl , DMF, 40°C, 4 hr; (f) K CO ,
2
4
4
3
2
2
2
2
3
acetonitrile, reflux, 8 hr; (g) LiOH·H O, THF/MeOH/H O, r.t., 4 hr
2
2
TABLE 1 Agonistic activities and PK parameters for GW9508 and HWL-066 in SD rats
CL
EC50 (nm)a
Dose (po)b
(L hr−1 kg−1
−1
)
Cmax (μg/L)
AUC0–24 hr (μg/L hr)
T1/2 (hr)
Compd.
GW9508
HWL-066
65.9
70.8
3 mg/kg
3 mg/kg
1.97 ± 0.45
0.23 ± 0.16
1,587.14 ± 345.72
5,907.47 ± 1,253.26
16,185.36 ± 1,241.65
45,387.33 ± 5,046.32
2.19 ± 0.61
3.50 ± 0.57
a
EC50 values are averages of three independent determinations.
b
po: oral administration; GW9508 and HWL-066 were suspended in 0.5% methylcellulose aqueous solution. Results are expressed as mean ± SD for four SD rats (fasted
for 12 hr) in each group.
followed by reduction with sodium borohydride. The in-
termediate 2a was further converted to deuterated inter-
mediate 3a by decarboxylation and esterification. The key
intermediate 5a was synthesized by reduction in commer-
cially available 4a with sodium borohydride, followed by
chlorinating provide intermediate 6a. The intermediate 6a
was condensed with 3a, followed by hydrolysis, furnished
HWL-066.
the corresponding metabolite derived from β-oxidation
was not detected after treatment with HWL-066 (3 mg/kg).
These results demonstrated our strategy has successfully re-
duced β-oxidation by incorporating two deuterium atoms at
the α-position of phenylpropionic acid, leading to the sig-
nificantly improved PK properties.
3
.3
Docking study
|
We tried induced-fit docking study of HWL-066 using
3
.2
In-vitro activity and pharmacokinetic
|
moe based on the crystal complex of receptor ligated with
evaluation of HWL-066
[
33]
TAK-875 (PDB code: 4PHU).
As shown in Figure 3,
The agonistic activities of GW9508 and HWL-066 were
HWL-066 fit very well into the binding pocket of TAK-
875 with slightly rotated side chain of Tyr91 and Trp174.
Moreover, the acidic scaffold in HWL-066 formed strong
hydrogen-bonding interaction with residues Arg2258,
Arg183, and Tyr91. In addition, the edge-on interaction
was formed between Trp174 and the right-hand benzene of
HWL-066. Notably, the terminal benzene ring of HWL-066
has a hydrophobic interaction with hydrophobic residues at
the outside of binding pocket.
2
+
investigated by monitoring Ca signal using a fluoro-
metric imaging plate reader (FLIPR) assay. As shown in
Table 1, the EC values of GW9508 and HWL-066 were
5
0
6
5.9 nm and 70.8 nm, respectively. This positive result in-
dicated that deuterium at the α-position of phenylpropionic
acid has little effect on FFA1 agonistic activity. Based on
its favorable in-vitro activity, HWL-066 was chosen and
assessed for its oral pharmacokinetic (PK) parameters
(
Table 1). As expected, excellent PK profiles were observed
in HWL-066 (3 mg/kg), in particular, a lower clearance
3
0
.4
Oral glucose tolerance test of HWL-
−
1
−1
−1
|
(
CL = 0.23 L hr kg ), higher maximum plasma con-
66
centration (C = 5907.47 μg/L), and longer half-life
max
(
(
T
= 3.50 hr), leading to a 2.8-fold higher exposure
Encouraged by the positive in-vitro potency and PK pro-
files, the glucose-lowering effect of HWL-066 was evalu-
ated in ICR mice during an OGTT (Figure 4). As shown in
1
/2
AUC0–24 hr = 45387.33 μg/L hr) than GW9508 (3 mg/kg,
AUC
= 16,185.36 μg/L hr). Unlike GW9508, indeed,
0
–24 hr

6
LIU et aL.
|
(
a)
(b)
FIGURE 3 The induced-fit docking study of HWL-066 in the reported complex of FFA1 (PDB code: 4PHU). Overlay of TAK-875 (green)
and HWL-066 (blue) bound to FFA1, and the key residues ((a)Trp174, Arg2258, Arg183, Tyr2240, (b) Arg2258, Arg183, and Tyr91,Tyr2240) are
labeled in white. Hydrogen bonds are represented by yellow dashed lines
FIGURE 4 Effects of HWL-066 during OGTT in fasting ICR mice. (a) represented time-dependent changes of blood glucose levels after oral
administration of HWL-066, followed by oral glucose load (3 g/kg), respectively. (b) represented AUC
of plasma glucose levels. Results are
–90 min
0
mean ± SD (n = 6 per group). **p ≤ 0.01 was analyzed using a one-way ANOVA with Tukey’s multiple-comparison post-hoc test
Figure 4b, HWL-066 (20 mg/kg) significantly lowered the
plasma glucose levels, and the AUC of blood glu-
a relatively safe hypoglycemic agent without the side-effect of
hypoglycemia.
0
–90 min
cose was compared to that of TAK-875. However, GW9508
(
20 mg/kg) only slightly decreased glucose levels, and there
was no statistical difference between control and GW9508-
treated group. These results are in accordance with the phar-
macokinetic difference between GW9508 and HWL-066,
which indicated that our strategy to incorporate deuterium at
the α-position of phenylpropionic acid indeed improved the
glucose-lowering effect in mice.
3
.5
The risk of hypoglycemia in mice
|
The risk of hypoglycemia in fasting ICR mice was evaluated
by administrating HWL-066 (80 mg/kg) in comparison with
glibenclamide. As shown in Figure 5, the glucose levels of
glibenclamide group (15 mg/kg) were far below that of nor-
mal fasting levels in mice. In contrast, HWL-066 (80 mg/kg)
only slightly reduced glucose levels in mice, and there was no
statistical difference between control and HWL-066 groups.
Therefore, these results demonstrated that HWL-066 might be
FIGURE 5 Effects of HWL-066 on fasting glucose of normal
mice. Results are expressed as mean ± SD for six mice per group.
*
p ≤ 0.05 and **p ≤ 0.01 compared to control group by Student’s t test

LIU et aL.
7
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[
9] G. V. Rayasam, V. K. Tulasi, J. A. Davis, V. S. Bansal, Expert
Opin. Ther. Targets 2007, 11, 661.
3
.6
Conclusions and future directions
|
[
10] Z. Li, X. Xu, W. Huang, H. Qian, Med. Res. Rev. 2017, https://doi.
Aiming to improving the PK properties of GW9508, the de-
sign strategy of incorporating two deuterium atoms at the
α-position of phenylpropionic acid was developed to block
the initial dehydrogenation reaction of β-oxidation. Indeed,
the obtained HWL-066 revealed superior PK profiles, espe-
cially with respect to plasma clearance and plasma exposure.
Moreover, the glucose level was significantly suppressed in
org/10.1002/med.21441.
[
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HWL-066-treated group, and the AUC
of blood glu-
–90 min
0
cose was compared to that of TAK-875. In addition, HWL-066
revealed a lower risk of hypoglycemia than glibenclamide.
Therefore, HWL-066 will be a better pharmacological tool to
explore the physiological function of FFA1 and provided a lot
of useful information for us to develop FFA1 agonists.
1
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ACKNOWLEDGMENTS
This study was supported by grants from the National Natural
Science Foundation of China (Grants 81673299 and 81273376).
[
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
ORCID
[
16] J. B. Houze, L. Zhu, Y. Sun, M. Akerman, W. Qiu, A. J. Zhang, R.
Sharma, M. Schmitt, Y. Wang, J. Liu, J. Liu, J. C. Medina, J. D.
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Lin, Bioorg. Med. Chem. Lett. 2012, 22, 1267.
Hai Qian
http://orcid.org/0000-0002-3827-0992
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How to cite this article: Liu C, Li Z, Shi W, et al.
Improving metabolic stability with deuterium: The
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