Design, synthesis and biological evaluation of nitric oxide releasing derivatives of dapagliflozin as potential anti-diabetic and anti-thrombotic agents

Article abstract of DOI:10.1016/j.bmc.2018.06.017

The cardiovascular complications were highly prevalent in type 2 diabetes mellitus (T2DM), even at the early stage of T2DM or the state of intensive glycemic control. Therefore, there is an urgent need for the intervention of cardiovascular complications in T2DM. Herein, the new hybrids of NO donor and SGLT2 inhibitor were design to achieve dual effects of anti-hyperglycemic and anti-thrombosis. As expected, the preferred hybrid 2 exhibited moderate SGLT2 inhibitory effects and anti-platelet aggregation activities, and its anti-platelet effect mediated by NO was also confirmed in the presence of NO scavenger. Moreover, compound 2 revealed significantly hypoglycemic effects and excretion of urinary glucose during an oral glucose tolerance test in mice. Potent and multifunctional hybrid, such as compound 2, is expected as a potential candidate for the intervention of cardiovascular complications in T2DM.

Full text of DOI:10.1016/j.bmc.2018.06.017

Accepted Manuscript
Design, synthesis and biological evaluation of nitric oxide releasing derivatives
of dapagliflozin as potential anti-diabetic and anti-thrombotic agents
Zheng Li, Xue Xu, Liming Deng, Ruoxian Liao, Ruiying Liang, Bo Zhang,
Luyong Zhang
PII:
S0968-0896(18)30938-6
https://doi.org/10.1016/j.bmc.2018.06.017
BMC 14409
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 May 2018
10 June 2018
13 June 2018
Please cite this article as: Li, Z., Xu, X., Deng, L., Liao, R., Liang, R., Zhang, B., Zhang, L., Design, synthesis and
biological evaluation of nitric oxide releasing derivatives of dapagliflozin as potential anti-diabetic and anti-
thrombotic agents, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.017
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Design, synthesis and biological evaluation of nitric oxide releasing derivatives of
dapagliflozin as potential anti-diabetic and anti-thrombotic agents
1
2
1
1
1
3, *
1,
Zheng Li , Xue Xu , Liming Deng , Ruoxian Liao , Ruiying Liang , Bo Zhang , Luyong Zhang
4, 5*
1
2
3
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
Guangzhou General Pharmaceutical Research Institute Co., Ltd., Guangzhou 510240, PR China.
Department of Clinical Laboratory Science, Wuxi People’s Hospital of Nanjing Medical
University, 299 Qingyang Road, Wuxi, Jiangsu 214023, China
4
Guangzhou key laboratory of construction and application of new drug screening model systems,
Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
5
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,
PR China.
Abstract
The cardiovascular complications were highly prevalent in type 2 diabetes mellitus (T2DM), even
at the early stage of T2DM or the state of intensive glycemic control. Therefore, there is an urgent
need for the intervention of cardiovascular complications in T2DM. Herein, the new hybrids of
NO donor and SGLT2 inhibitor were design to achieve dual effects of anti-hyperglycemic and
anti-thrombosis. As expected, the preferred hybrid 2 exhibited moderate SGLT2 inhibitory effects
and anti-platelet aggregation activities, and its anti-platelet effect mediated by NO was also
confirmed in the presence of NO scavenger. Moreover, compound 2 revealed significantly
hypoglycemic effects and excretion of urinary glucose during an oral glucose tolerance test in
*
Corresponding author.
E-mail addresses: zhangbo_cpu1713@163.com (B. Zhang), lyzhang@cpu.edu.cn (L. Zhang).
1

mice. Potent and multifunctional hybrid, such as compound 2, is expected as a potential candidate
for the intervention of cardiovascular complications in T2DM.
Keywords: Cardiovascular complications; Diabetes; SGLT2; Hybrid; NO donor.
1. Introduction
Type 2 diabetes mellitus (T2DM) is a prevalent life-long disease characterized by insulin
deficiency and peripheral insulin resistance.[1, 2] There is an inspiring array of anti-diabetic drugs
for maintaining the ideal level of blood glucose.[3-6] However, many observational and clinical
researches indicated cardiovascular complications were highly prevalent in diagnosed and
undiagnosed T2DM, even at the early stage of T2DM or the state of intensive glycemic control.[7,
8
] Thus, a novel anti-diabetic drug with intervention for cardiovascular complications is an unmet
clinical need for T2DM.
Currently, the sodium-dependent glucose co-transporter 2 (SGLT2), one of the most
successfully anti-diabetic targets in the last few years, plays a key role in the reabsorption of
glucose from kidney.[9-12] Several SGLT2 inhibitors derived from C-aryl glucosides have been
approved for the treatment of T2DM,[13-16] and the potential chemical space of SGLT2 inhibitors
has also been explored in our laboratory (Figure 1).[17, 18] Moreover, clinical researches
revealed that the administration of SGLT2 inhibitors such as canagliflozin and empagliflozin is
associated with the cardiovascular benefits,[19, 20] which might attributed to the osmotic diuresis
induced by inhibition of SGLT2.[21]
2

Figure 1: Several representative SGLT2 inhibitors approved for the treatment of T2DM.
Nitric oxide (NO), a well-known signaling molecule, plays an important role in a variety of
physiological processes in cardiovascular system, including the inhibition of platelet aggregation,
relaxation of vascular smooth muscle, reduced adherence and activation of neutrophil.[22-24]
Accordingly, we hypothesize that the conjugation of SGLT2 inhibitor with NO donor may provide
novel hybrids with dual effects on anti-hyperglycemic and anti-thrombosis, and thereby reducing
the morbidity of cardiovascular complications.
In this study, the first-in-class SGLT2 inhibitor dapagliflozin was selected due to its high
potency and known modifiable 4-position (green mark in Figure 2) identified by the previous
structure-activity relationship study.[18, 25] The new hybrids of NO donor and dapagliflozin were
synthesized to verify our hypothesis above (Figure 2). Indeed, these hybrids exhibited the dual
effects on anti-hyperglycemic and anti-thrombosis despite a decreased inhibition against SGLT2.
Figure 2: The hybrid strategy of NO donor and dapagliflozin.
3

2. Results and Discussion
2.1. Chemistry
The synthesis of compounds 1-3 is depicted in Scheme 1. To a mixture of gluconolactone 1a in
anhydrous tetrahydrofuran was added N-methylmorpholine and trimethylsilyl chloride, providing
the persilylated gluconolactone 2a in a high yield.[26] The 5-bromo-2-chlorobenzoyl chloride was
obtained by treating 5-bromo-2-chlorobenzoic acid 3a with oxalyl chloride in the presence of
catalytic amount of DMF. The intermediate 4a was generated by Friedel–Crafts acylating reaction
of starting material phenetole with 5-bromo-2-chlorobenzoyl chloride, followed by
3 2
recrystallization from ethanol. Reduction of 4a by treating with triethylsilane and BF ·OEt
generated the desired intermediate 5a. Lithium halogen exchange in intermediate 5a, followed by
adding the nascent lithiated intermediate to the persilylated gluconolactone 2a, provide the lactols,
which were further converted to the desilylated 6a in presence of methanesulfonic acid. Reduction
of the intermediate 6a using triethylsilane and BF
with acetic anhydride, furnished the β-anomer 8a after recrystallization from ethanol. The
β-anomer 8a was treated with BBr to give phenol 9a, which was condensed with bromoalkane,
3 2
·OEt at -10 °C, followed by peracetylation
3
N
followed by hydrolysis, given intermediate 10a. The S 2 reaction of silver nitrate with
intermediate 10a in acetonitrile afforded the target compounds 1-3.
4

Scheme 1. Synthesis of target compounds 1-3. Reagents and conditions: (a) TMSCl, NMM, THF, 35 °C; (b)
COCl) , CH Cl , DMF, then phenetole, AlCl , 0 °C; (c) Et SiH, BF ·OEt , CH Cl , CH CN, 25 °C; (d) n-BuLi,
THF, toluene, -78 °C, then 2a followed by MeOH, CH SO H; (e) Et SiH, BF ·OEt , CH Cl , CH CN, -10 °C; (f)
, -78 °C; (h) bromoalkane, K CO , CH CN, reflux, 5 h, then
CN, reflux, 2 h.
(
2
2
2
3
3
3
2
2
2
3
3
3
3
3
2
2
2
3
Ac
2
O, pyridine, CH
2
Cl
2
, DMAP; (g) BBr
3 2
, CH Cl
2
2
3
3
2 2
LiOH·H O, THF, H O, MeOH; (i) AgNO
3
, CH
3
2.2. The inhibitory effects of hybrids on human SGLT
Aiming to relieve the cardiovascular complications of T2DM, we hypothesize that the hybrids of
dapagliflozin and NO donor might have dual effects on anti-hyperglycemic and anti-thrombosis.
The in vitro SGLT inhibitory effects (IC50) of synthesized hybrids was evaluated by determining
the inhibition of methyl-a-D-glucopyranoside (AMG) uptake in HEK293 cells expressing human
SGLT2 or SGLT1. As shown in Table 1, the direct hybrid (compound 1) of dapagliflozin and NO
donor nitrate revealed a significantly decreased inhibitory effect on SGLT1 and SGLT2 compared
with dapagliflozin, which probably attributed to the hydrophily of nitrate. Therefore, the
methylene was introduced to improve the lipophilicity in this position, and indeed, the obtained
analogs 2 (IC50 = 125.7 nM) and 3 (IC50 = 183.9 nM) exhibited dramatic improvement on
inhibitory activity against SGLT2 in comparison to the parent compound 1 (IC50 = 246.3 nM).
5

Table 1: In vitro inhibitory activity and selectivity for hSGLT
a
b
Compd.
n
hSGLT2 IC50 (nM)
1.4
hSGLT1 IC50 (nM)
1830
Selectivity versus hSGLT1
1307
Dapagliflozin
1
2
3
0
1
2
246.3
125.7
183.9
> 10000
> 10000
> 40
> 80
> 54
> 10000
a
b
IC50 values for hSGLT2 are averages of three independent determinations. IC50 values for hSGLT1 are averages
of three independent determinations.
2.3. Anti-platelet aggregation activity
The anti-platelet aggregation activities of tested compounds (0.1 mM) were performed on the
adenosine diphosphate (ADP)-induced platelet aggregation in rabbit platelet rich plasma by using
Born's turbidimetric method.[27] Ticlopidine was selected as positive control. As shown in Figure
3
, compounds 1, 2 and 3 inhibited the ADP-induced platelet aggregation by 11.61%, 14.43% and
14.12%, respectively, which were much stronger than that of dapagliflozin. This result suggested
that the incorporation of NO donor may improve the inhibitory effect on the ADP-induced platelet
aggregation. To confirm this speculation above, the carboxy-PTIO (a NO scavenger) was then
incubated with the drug-platelet suspension and tested for inhibitory activity. Indeed, the
anti-platelet effect of compound 2 was decreased from 14.43% to 3.64% by treatment with
carboxy-PTIO (20 μM). These results further clarified the important role of NO donor for the
anti-platelet effect, and support our hypothesis on the hybrids with dual effects on
anti-hyperglycemic and anti-thrombosis.
6

Figure 3: In vitro inhibitory effects for ADP-induced rabbit platelet aggregation. Rabbit platelet suspensions were
pre-incubated with tested compounds (0.1 mM) at 37 °C for 5 min followed by addition of ADP (10 μM).
Carboxy-PTIO (20 μM) was added and incubated with the drug-platelet suspension in the indicated group. Results
are expressed as means (%) ± SD of each group from three independent experiments. **p≤0.01 and ***p≤0.001
were analyzed using a one-way ANOVA with Tukey’s multiple-comparison post hoc test.
2
.4. Oral glucose tolerance test of compound 2
Based on the desirable anti-platelet aggregation and inhibitory activity against SGLT2, compound
was selected to assess its glucose-lowering effects in mice (Figure 4). Gratifyingly, compound 2
20 mg/kg) significantly suppressed the excursion of blood glucose levels by 26.3%, and the
2
(
glucose-lowering effect was in proximity to that of dapagliflozin (- 34.7%) despite a 10-fold dose
of compound 2 was used in this study. Actually, the relatively high dose might provide a better
anti-thrombotic effect based on NO donor, and thereby get a better balance between hypoglycemic
effect and cardiovascular benefits.
7

Figure 4: Effects of compound 2 during OGTT in fasting ICR mice. (A) represented time-dependent changes of
blood glucose levels after oral administration of test compounds, followed by oral glucose load (3 g/kg),
respectively. (B) represented AUC0−120min of plasma glucose levels. Results are mean ± SD (n = 6 per group). **p
≤
0.01 and ***p≤0.001 were analyzed using a one-way ANOVA with Tukey’s multiple-comparison post hoc test.
To verify the SGLT2-mediated mechanism of compound 2, the urine of each group were
collected to evaluate the level of urinary glucose (Table 2). Dapagliflozin and compound 2, which
exhibited robustly glucose-lowering effect, markedly increased the level of urinary glucose while
the control group could not. Moreover, the level of urinary glucose was consistent with the
changes of plasma glucose at the same time, namely, the more urinary glucose excreted, the more
reduction of corresponding blood glucose (Table 2 and Figure 4). These results suggested that the
glucose-lowering effect of compound 2 was attributed to the decreased glucose reabsorption by
inhibiting SGLT2 in kidney.
Table 2: Urine glucose excretion effects of dapagliflozin and compound 2 in mice
Groups
-30min
0min
-
15min
-
30min
-
60min
-
120min
Control
-
-
-
Dapagliflozin (2 mg/kg)
++
+++
++++
++++
+++
Compound 2 (20 mg/kg)
-
+
++
++++
+++
++
8

-1
Symbol represents the mean urine glucose levels of each group (n=6). -: no glucose; ±: 100 mg· dL ; +: 250
-1
-1
-1
-1
mg· dL ; ++：500 mg·dL ; +++: 1000 mg· dL ; ++++: 2000 mg· dL .
3. Conclusion
Aiming to relieve the cardiovascular complications of T2DM, the design strategy of incorporating
dapagliflozin with NO donor was developed to get a dual efficacy of anti-hyperglycemic and
anti-thrombosis. Indeed, these hybrids of dapagliflozin and nitrate revealed moderate SGLT2
inhibitory effects and anti-platelet aggregation activities in vitro. Among them, the preferred
compound 2 exhibited better potency both in anti-platelet aggregation (14.43%) and SGLT2
inhibition (IC50 = 125.7 nM). Moreover, the anti-platelet effect of NO donor in compound 2 was
clarified by adding the NO scavenger carboxy-PTIO. In subsequently in vivo study, compound 2
revealed significantly glucose-lowering effects and excretion of urinary glucose despite a 10-fold
higher dose compared with dapagliflozin. However, it's not always a bad thing for the higher dose.
Actually, the high dose of compound 2 might provide a better anti-thrombotic effect in vivo, and
thereby get a more appropriate balance both in glucose-lowering effect and cardiovascular benefits.
Altogether, compound 2 appears as a promising candidate for further evaluation, and more
advanced studies based on the hybrid strategy will provide us more competitive anti-diabetic drug
with cardiovascular benefits.
4. Experimental section
4.1. Chemistry
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).
9

Melting points were determined on a RY-1 melting-point apparatus and were uncorrected. The
1
13
NMR spectra (300 MHz for H NMR, 75 MHz for C NMR spectra) were detected on Bruker
ACF-300Q instrument. Chemical shifts are showed as values relative to internal standard
(
tetramethylsilane), and coupling constants (J values) were given in hertz (Hz). LC/MS spectra
were determined on Waters liquid chromatography-mass spectrometer system (ESI). Elemental
analyses were carried out by Heraeus CHN-O-Rapid analyzer. The positive control dapagliflozin
was synthesized as previously reported.[26]
2,3,4,6-tetra-O-Trimethylsilyl-β-D-glucolactone (2a).
To a stirred solution of gluconolactone 1a ( 2.0 g, 11.2 mmol) and NMM (10.0 mL, 89.6 mmol) in
anhydrous THF (30 mL) at -10 °C was added TMSCl (9.0 mL, 67.2 mmol) via a dropping funnel
at a rate such that the temperature did not exceed 0 °C. After 1 h, the reaction was heated to 35 °C
and stirred overnight. After dilution with toluene (60 mL), the mixture was cooled to -10 °C prior
2
to cautiously adding H O (60 mL) at a rate such that the temperature did not exceed 10 °C. The
2 4 2
organic layer was separated and washed with aqueous NaH PO , H O, and brine, and then dried
and filtered. The filtrate was concentrated under reduced pressure. The resultant light yellow oil
was twice taken up with anhydrous toluene and concentrated to yield 2,3,4,6-tetra-O-trimethylsilyl
1
-
β-D-glucolactone 2a (5.2 g, 99% crude yield) as a colorless oil. H NMR (300 MHz, CDCl
3
) δ:
4
9
.02-3.98 (m, 1H), 3.85 (d, J = 7.9 Hz, 1H), 3.79 (t, J = 7.4 Hz, 1H), 3.71-3.55 (m, 3H), 0.08 (s,
H), 0.06 (s, 9H), 0.05 (s, 9H), 0.01 (s, 9H).
5
-Bromo-2-chloro-4’-ethoxybenzophenone (4a)
10

2 2
To a stirred suspension of 5-bromo-2-chlorobenzoic acid 3a (10.0 g, 42.0 mmol) in CH Cl (100
mL) was added oxalyl chloride (14.4 mL, 168.0 mmol) and DMF (0.5 mL). After stirring at room
temperature overnight, the reaction was concentrated under reduced pressure. Following
2 2
dissolution of the obtained 5-bromo-2-chlorobenzoyl chloride in CH Cl (100 mL), the resulting
yellow solution was transferred to a 250 mL 3-neck flask equipped with a mechanical stirrer, and
an internal thermometer. This mixture was cooled to −5 °C prior to adding phenetole (5.3 mL, 42
mmol). Subsequently, AlCl
to ensure that the temperature did not exceed 0 °C. After 2 h, the reaction was quenched by
2
pouring over ice. The resulting suspension was diluted with H O and extracted with CH Cl . The
3
(6.2 g, 46.2 mmol) was added in small portions over 30 min at a rate
2
2
2
combined organic extracts were washed with HCl (1 N), H O, NaOH (1 N), and brine prior to
2 4
drying over Na SO . Filtration, concentration under reduced pressure and recrystallization twice
from absolute EtOH yielded 5-bromo-2-chloro-4’-ethoxybenzophenone 4a (11.6 g, 80%) as a
1
white solid. mp 60-62 °C. H NMR (300 MHz, CDCl
3
) δ: 7.79-7.75 (m, 2H), 7.55-7.48 (m, 1H),
7.33-7.26 (m, 2H), 6.95 (d, J = 8.73 Hz, 2H), 4.15 (q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 2H).
5
-Bromo-2-chloro-4’-ethoxydiphenylmethane (5a)
SiH (10.0 mL, 62.8 mmol) and 4a (10.0 g, 1.2 mmol) in 1:1
To a stirred solution of Et
3
2 2 3 2
CH Cl /MeCN (80 mL) at -10 °C was added BF ·OEt (6.0 mL, 47.6 mmol) at such a rate that the
temperature did not exceed 0 °C. The reaction mixture was stirred overnight at room temperature.
Upon cooling, the reaction was quenched with aqueous 2 N KOH (100 mL). The aqueous layer
2 2 2
was extracted with CH Cl and the combined organic layers were washed with KOH (2 N), H O
2 4
and brine prior to drying over Na SO . After filtration and removal of the volatiles under reduced
11

pressure, the residue was recrystallized from absolute EtOH to give the desired product 5a (8.12 g,
1
8
5%) as a white solid. mp 40-41 °C. H NMR (300 MHz, CDCl
3
) δ: 7.28-7.20 (m, 3H), 7.09 (d, J
=
8.6 Hz, 2H,), 6.85 (d, J = 8.6 Hz, 2H), 3.98 (q, J = 7.0 Hz, 2H), 3.94 (s, 2H), 1.40 (t, J = 7.0 Hz,
2H).
(
2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(4-chloro-3-(4-ethoxybenzyl)phenyl)tetrahydro-2H-pyra
n-3,4,5-triyl triacetate (8a)
To a stirred -78°C solution of 5a (5.0 g, 15.4 mmol) in 60 mL of 1:2 anhydrous THF/toluene
2
under N was added n-BuLi (10.6 mL, 1.6 M) in hexane dropwise to insure the temperature
remained below -70 °C. After stirring for 30 minutes following the addition, this solution was
transferred by cannula to a stirred -78 °C solution of 2,3,4,6-tetra-O-Trimethylsilyl-β-D
-
-
glucolactone (7.9 g, 16.9 mmol) in 20 mL of toluene at a rate that maintained the reaction below
70 °C. The solution was stirred for 30 min at -78 °C prior to quenching by addition of 10 mL of
MeOH containing methanesulfonic acid (3.0 mL, 46.2 mmol). The reaction stirred overnight as
the temperature rose to 20 °C. The reaction, once complete, was quenched by the addition of
3 2
aqueous NaHCO until the pH was weakly basic. The mixture was diluted with H O and extracted
2 4
with EtOAc. The combined organic layers were washed with brine and dried over Na SO . After
concentration using a rotary evaporator, the residue was dissolved in hot toluene (50 mL). The
resulting solution was poured into 250 mL of stirred hexane. The precipitate was collected by
vacuum filtration, the resulting filter cake was washed with hexane and then air dried to give a
crude product, which was used for the next reaction without further purification.
2 2
To a solution of the obtained crude 6a (3.0 g, 6.84 mmol) in 60 mL of 1:1 CH Cl /MeCN at
12

-
3 3 2
10 °C was added Et SiH (2.2 g, 13.7 mmol) followed by addition of BF ·Et O (1.3 g, 10.3 mmol)
at a rate such that the temperature was maintained between -5 °C to -10 °C. The mixture was
warmed to 0 °C and stirred for 5 h. When HPLC analysis revealed that the reaction was complete,
3
the reaction was quenched by the addition of saturated aqueous NaHCO and then extracted with
2 2 2 2 4
CH Cl . The combined organic layers were washed with H O, brine, dried over Na SO , filtered,
and concentrated under reduced pressure. The residue was purified by column chromatography to
give the desired product 7a (1.9 g, 30% of two steps) as a white solid.
2 2
To a solution of 7a (1.48 g, 3.6 mmol) and pyridine (2.92 mL, 36.2 mmol) in CH Cl at 0 °C
was added Ac
temperature from 25 °C to 32 °C had subsided, DMAP (22 mg, 0.18 mmol) was added. The
reaction was quenched after 1.5 h by the addition of H O once TLC analysis indicated the reaction
2
O (3.72 mL, 36.2 mmol) in one portion. After the resulting exotherm raising the
2
2 2
to be complete and extracted with CH Cl . The combined organic layers were washed with HCl (1
2 4
N), brine, dried over Na SO , filtered, and concentrated under reduced pressure. The residue was
recrystallized from absolute EtOH to give the desired product 8a (1.49 g, 71%) as a white solid.
The mother liquors contained the corresponding α-C-glucoside as well as a more polar furanose
1
isomer. mp 124-126 °C. H NMR (300 MHz, DMSO-d
6
) δ: 7.43 (d, J = 8.1 Hz, 1H), 7.29-7.25 (m,
2
H), 7.07 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 5.37 (t, J = 9.5 Hz, 1H), 5.09 ( t, J = 9.5
Hz, 1H), 4.97 ( t, J = 9.7 Hz, 1H), 4.67 (d, J = 9.7 Hz, 1H), 4.15-4.03 (m, 3H), 4.00-3.91 (m, 4H),
.01 (s, 3H), 1.99 (s, 3H), 1.92 (s, 3H), 1.68 (s, 3H), 1.31 (t, J = 6.9 Hz, 3H).
2
(
2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(4-chloro-3-(4-hydroxybenzyl)phenyl)tetrahydro-2H-pyr
an-3,4,5-triyl triacetate (9a)
13

2 2
To a solution of 8a (1.0 g, 1.7 mmol) in CH Cl at -78 °C was added dropwise a 1.0 M solution of
BBr
The reaction mixture was quenched by the addition of saturated aqueous sodium bicarbonate and
extracted with CH Cl . The combined organic layers were washed with NaHCO (1 N), brine,
3 2 2
(0.48 mmol, 5.2 mmol) in CH Cl , and the resulting solution was stirred at -10 °C for 3 h.
2
2
3
2 4
dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by
2 2
medium-pressure column chromatography (CH Cl : MeOH = 30 : 1, v/v) to give the desired
1
product 9a as a white solid (0.6 g, 63%). H NMR (300 MHz, DMSO-d
6
) δ: 7.43 (d, J = 8.3 Hz,
1
1
3
5
H), 7.26-7.20 (m, 2H), 6.97 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 5.35 (t, J = 9.4 Hz,
H), 5.06 (t, J = 9.5 Hz, 1H), 4.94 (t, J = 9.6 Hz, 1H), 4.67 (d, J = 9.7 Hz, 1H), 4.16-4.05 (m, 3H),
.98-3.86 (m, 2H), 2.01 (s, 3H), 2.00 (s, 3H), 1.92 (s, 3H), 1.69 (s, 3H); MS (ESI, 40 eV) m/z (%):
+
49.1 (100) [M+H] .
General procedure for synthesis of compounds 1-3
To a solution of 9a (1.0 equiv) in acetonitrile was added the corresponding bromoalkane (1.0
2 3
equiv) and K CO (2.0 equiv). The reaction mixture was reflux for 5 h, and then cooled to room
temperature and filtered. The filtrate was concentrated under reduced pressure and the resultant
residue was redissolved in ethyl acetate and washed with brine. The organic layer was dried over
2 4 2
Na SO , filtered, and concentrated under reduced pressure. The residue in THF/MeOH/H O (2: 3:
1
2
) was added LiOH·H
h. The reaction mixture was concentrated under reduced pressure and the residue redissolved in
and brine. The organic layer was dried over
2
O (1.2 equiv), and the resulting mixture was stirred at room temperature for
ethyl acetate and washed with 5% aqueous KHSO
4
2 4
Na SO , filtered, and concentrated under reduced pressure. The residue was purified by column
chromatography to give the desired products 10a. To a solution of 10a (1.0 equiv) in acetonitrile
14

was added silver nitrate (1.5 equiv). The reaction mixture was stirred at reflux for 2 h under dark
condition, and then cooled to room temperature and filtered. The filtrate was concentrated under
2 2
reduced pressure and the resultant residue was purified by column chromatography (CH Cl :
MeOH = 30 : 1, v/v) to give the target compounds 1-3 as glassy off-white amorphous solid.
2
-(4-(2-chloro-5-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)benzyl)phenoxy)ethyl nitrate (1)
1
Yield 35%; mp: 75-77 °C; H NMR (300 MHz, DMSO-d
6
) δ: 7.38 (d, J = 8.2 Hz, 1H), 7.26-7.20
(
m, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 4.96 (d, J = 5.5 Hz, 2H), 4.81 (d, J =
9
.6 Hz, 1H), 4.66 (t, J = 6.0 Hz, 2H), 4.50 (t, J = 5.7 Hz, 1H), 4.43 (t, J = 6.0 Hz, 2H), 4.08-3.99
13
(
6
m, 3H), 3.73-3.68 (m, 1H), 3.49-3.42 (m, 1H), 3.31-3.08 (m, 4H). C NMR (75 MHz, DMSO-d )
δ: 153.7, 143.5, 137.6, 133.9, 130.8, 129.6, 128.9, 125.5, 115.1, 83.1, 78.2, 75.6, 72.3, 71.6, 70.2,
+
6
6.2, 61.8, 37.4. ESI-MS m/z: 492.1 [M+Na] . Anal. calcd. For C21
H
24ClNO
9
: C, 53.68; H, 5.15;
N, 2.98; Found: C, 53.56; H, 5.14; N, 2.98.
3
-(4-(2-chloro-5-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)benzyl)phenoxy)propyl nitrate (2)
1
Yield 28%; mp: 64-66 °C; H NMR (300 MHz, DMSO-d
6
) δ: 7.41 (d, J = 8.3 Hz, 1H), 7.28-7.22
(
m, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 4.95 (d, J = 5.5 Hz, 2H), 4.83 (d, J =
9
.5 Hz, 1H), 4.58 (t, J = 6.1 Hz, 2H), 4.52 (t, J = 5.6 Hz, 1H), 4.31 (t, J = 6.1 Hz, 2H), 4.06-3.97
13
(
(
m, 3H), 3.71-3.66 (m, 1H), 3.47-3.41 (m, 1H), 3.32-3.09 (m, 4H), 2.16-2.12 (m, 2H). C NMR
75 MHz, DMSO-d ) δ: 156.1, 144.3, 137.8, 134.2, 131.5, 129.8, 128.4, 125.6, 115.4, 84.6, 78.6,
6
15

+
7
6.3, 71.3, 70.6, 69.3, 64.8, 62.3, 36.5, 26.4. ESI-MS m/z: 506.1 [M+Na] . Anal. calcd. For
22 9
C H26ClNO : C, 54.61; H, 5.42; N, 2.89; Found: C, 54.75; H, 5.43; N, 2.89.
4
-(4-(2-chloro-5-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)benzyl)phenoxy)butyl nitrate (3)
1
Yield 37%; mp: 61-63 °C; H NMR (300 MHz, DMSO-d
6
) δ: 7.44 (d, J = 8.1 Hz, 1H), 7.25-7.20
(
m, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.96 (d, J = 5.6 Hz, 2H), 4.85 (d, J =
9
.6 Hz, 1H), 4.50 (t, J = 5.7 Hz, 1H), 4.35 (t, J = 6.2 Hz, 2H), 4.26 (t, J = 6.2 Hz, 2H), 4.05-3.96
(
(
m, 3H), 3.70-3.65 (m, 1H), 3.47-3.41 (m, 1H), 3.32-3.09 (m, 4H), 1.92-1.88 (m, 2H), 1.65-1.62
13
m, 2H). C NMR (75 MHz, DMSO-d
6
) δ: 154.7, 142.6, 137.5, 133.8, 131.2, 129.2, 128.1, 125.9,
+
1
14.8, 83.8, 78.3, 76.7, 71.5, 70.2, 68.5, 63.4, 62.6, 36.5, 27.5, 23.4. ESI-MS m/z: 520.1 [M+Na] .
9
Anal. calcd. For C23H28ClNO : C, 55.48; H, 5.67; N, 2.81; Found: C, 55.36; H, 5.66; N, 2.81.
4.2. The Inhibitory Effects on hSGLT1 and hSGLT2.
HEK293 cells expressing hSGLT2 or hSGLT1 were seeded on 96-well plates and washed twice
with the pretreatment buffer (10 mmol/L HEPES, 5 mmol/L Tris, 140 mmol/L choline chloride, 2
2 2
mmol/L KCl, 1 mmol/L CaCl , and 1 mmol/L MgCl , pH 7.4), then incubated with the uptake
2
buffer (10 mmol/L HEPES, 5 mmol/L Tris, 140 mmol/L NaCl, 2 mmol/L KCl, 1 mmol/L CaCl ,
and 1 mmol/L MgCl
2
, pH 7.4) containing different concentrations of tested compounds for 15 min,
14
followed by the addition of C-labeled (4 µmol/L) and non-labeled (10 µmol/L)
α-methyl-D-glucopyranoside (AMG) for 2 h at 37 °C. The cells were washed twice with ice-cold
washing buffer (pretreatment buffer containing 100 µmol/L phlorizin) and then solubilized in
16

NaOH (200 mmol/L). The radioactivity was measured using a liquid scintillation counter. IC50
value of test compound was calculated by GraphPad InStat version 5.00 (GraphPad software, San
Diego, CA, USA).
4
.3. Animals and statistical analysis.
Rabbits and mice were purchased from Guangdong Medical Laboratory Animal Center
Guangdong, China). All animals were acclimatized for one week before the experiments, and fed
(
standard pellets 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 5.00 (San Diego, CA,
USA). General effects were analyzed by using a one-way ANOVA with Tukey’s
multiple-comparison post hoc test.
4.4. Anti-platelet aggregation
Blood were collected from rabbit carotid artery into heparin-containing 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
17

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 aggregation 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 (%) = (A-B)/A × 100%, A = the platelet aggregation of vehicle group, B = the
platelet aggregation of compound group.
4.5. Oral glucose tolerance test and glucosuria effects of compound 2
Normal male ICR mice after one week adaptation were fasted for 12 h, weighted, bled via the tail
tip, and randomized into 3 groups (n = 6 per group). Compound 2 (20 mg/kg), dapagliflozin (2
mg/kg), or vehicle (0.5% methylcellulose aqueous solution) was orally administered 30 min
before oral glucose load (3 g/kg). Blood samples and urine were collected immediately at -30 min
(
just before drug administration), 0 min (just before glucose challenge), and 15, 30, 60 and 120
min after glucose load. The blood glucose was measured by plasma glucose test strips (SanNuo
ChangSha, China), and the urinary glucose was determined by Tes-Tape (Gaoerbao GuangZhou,
GuangZhou, China).
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).
18

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Graphical abstract
Graphical Abstract
21