Targeting type 2 diabetes with c-glucosyl dihydrochalcones as selective sodium glucose co-transporter 2 (sglt2) inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1021/acs.jmedchem.6b01134

Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC₅₀ = 9.23 nM) were considerably weaker inhibitors of SGLT1 (IC₅₀ = 10.19 μM). They showed no effect on the sodium independent GLUT family of glucose transporters, and the most potent ones were not acutely toxic to cultured cells. The interaction of a C-glucosyl dihydrochalcone with a POPC membrane was modeled computationally, providing evidence that it is not a pan-assay interference compound. These results point toward the discovery of structures that are potent and highly selective inhibitors of SGLT2.

Full text of DOI:10.1021/acs.jmedchem.6b01134

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Article
Targeting Type 2 Diabetes with C-Glucosyl Dihydrochalcones
as Selective Sodium Glucose Co-Transporter 2 (SGLT2)
Inhibitors: Synthesis and Biological Evaluation
Ana Rita Jesus, Diogo Vila-Viçosa, Miguel Machuqueiro, Ana Patrícia
dos Santos Marques, Timothy Michael Dore, and Amelia Pilar Rauter
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01134 • Publication Date (Web): 28 Nov 2016
Downloaded from http://pubs.acs.org on December 4, 2016
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Targeting Type 2 Diabetes with CꢀGlucosyl
Dihydrochalcones as Selective Sodium Glucose Coꢀ
Transporter 2 (SGLT2) Inhibitors: Synthesis and
Biological Evaluation
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Ana R. Jesus,†,§ Diogo Vila-Viçosa,† Miguel Machuqueiro,† Ana P. Marques,† Timothy M.
Dore,*,§ Amélia P. Rauter*,†
†Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Ed C8, Piso
5, Campo Grande, 1749ꢀ016 Lisboa, Portugal.
§New York University Abu Dhabi, PO Box 129188, Abu Dhabi, United Arab Emirates
ABSTRACT: Inhibiting glucose reabsorption by sodium glucose coꢀtransporter proteins
(SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective
inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with
SGLT1 inhibition. A library of Cꢀglucosyl dihydrochalcones and their dihydrochalcone and
chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cellꢀbased
fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC50 = 9ꢀ23 nM)
were considerably weaker inhibitors of SGLT1 (IC50 = 10ꢀ19 ꢀM). They showed no effect on the
sodium independent GLUT family of glucose transporters, and the most potent ones were not
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acutely toxic to cultured cells. The interaction of a Cꢀglucosyl dihydrochalcone with a POPC
membrane was modelled computationally, providing evidence that it is not panꢀassay
interference compound (PAINS). These results point toward the discovery of structures that are
potent and highly selective inhibitors of SGLT2.
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Keywords:
sodium glucose coꢀtransporter, SGLT2 inhibition; selectivity; Cꢀglucosyl
dihydrochalcones; synthesis; umbrella sampling
INTRODUCTION
Type 2 diabetes is a chronic metabolic disease in which hyperglycemia results mainly from the
malfunction of insulin produced by βꢀcells in the pancreas.1ꢀ3 In diabetic subjects, hyperglycemia
can lead to hyperfiltration of glucose, resulting in glucosuria (excretion of glucose through
urine).4 Food digestion, glycogen breakdown, and gluconeogenesis provide glucose to the human
body. Inhibition of glucose absorption in the intestine to treat type 2 diabetes is the mechanism
of action of the wellꢀknown αꢀglucosidase inhibitors acarbose, miglitol, and voglibose.5ꢀ7 More
recently, inhibition of glucose reabsorption in the kidneys has become a viable strategy for
lowering blood sugar levels patients with type 2 diabetes.8ꢀ11 On a daily basis, the renal
glomerulus filters approximately 180 g of glucose, which is reabsorbed by the kidneys with
>99% sugar reabsorption primarily in the proximal tubules12ꢀ14 by sodium glucose coꢀtransporter
proteins (SGLTs). There are 6 isoforms of SGLTs;4, 15 SGLT1 and SGLT2 are the most wellꢀ
known.16, 17 SGLT1 is located in the small intestine, heart, trachea, and kidney and has a high
affinity to both glucose and galactose, while the single substrate for SGLT2, located only in the
kidneys, is glucose.4, 18ꢀ20
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Phlorizin (1), a natural glucosylated dihydrochalcone found in the bark of apple trees, was the
first SGLT inhibitor reported in literature (Figure 1).21 However, it was found to cause severe
side effects in the gastrointestinal tract because it inhibits both SGLT1 and SGLT2, and its
metabolite, phloretin, inhibits the glucose transporter GLUT1. Inhibition of the GLUT family of
transporters is another possible source of adverse side effects. For these reasons and phlorizin’s
low oral bioavailability, it did not proceed to clinical trials.22, 23 Pharmaceutical companies have
modified phlorizin structure to increase selectivity. For example, the methyl carbonate of 3ꢀ
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(benzofuranꢀ5ꢀyl)ꢀ1ꢀ[2ꢀ(βꢀ
D
ꢀglucopyranosyloxy)ꢀ6ꢀhydroxyꢀ4ꢀmethylphenyl]propanꢀ1ꢀone
Tꢀ
1095A, 2)24 is absorbed and subsequently metabolized into its active form. This compound has a
4ꢀfold increase in the SGLT2/SGLT1 selectivity, yet similarly to phlorizin it also has poor
bioavailability. The replacement of the Oꢀglucosides by Cꢀglucosyl analogs,23ꢀ29 which are stable
to hydrolysis and resistant to endogenous hydrolases, leads to a longer halfꢀlife and duration of
action.28 Among those approved as drugs, canagliflozin (3), dapagliflozin (4a), and empaglifozin
(4b) have high selectivity of SGLT2 over SGLT1, whereas others are still in clinical trials.4, 13ꢀ15,
20, 25 Studies on the safety and efficacy of 3 and 4a show that these two drugs are safe in the short
term, but longꢀterm safety remains undetermined.26, 30 Both drugs cause renalꢀrelated side effects,
namely hypoglycemic episodes, urinary tract infections, female genital mycotic infections, and
nasopharyngitis.30 Most of these side effects are related to SGLT1 inhibition. Although some of
these drugs are highly selective for SGLT2 over SGLT1 (>2000ꢀfold), new selective drugs
toward SGLT2 over SGLT1 and GLUT transporters with fewer side effects could possibly be
created by exploiting other synthetically accessible compound families.
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Figure 1. Structure of SGLT2 inhibitors phlorizin (1), phlorizin analogue 2, canagliflozin (3),
dapagliflozin (4a), and empaglifozin (4b).
CꢀGlucosyl dihydrochalcones are found in green rooibos (Aspalathus linearis) extracts, for
which there is evidence of antidiabetic activity.31, 32 To further investigate the possible molecular
mechanisms for this observation, we synthesized a small library of Cꢀglucosyl dihydrochalcones
(12a,cꢀh), their chalcone precursors (8aꢀh) and dihydrochalcone aglycones (9a,cꢀh) (Schemes 1ꢀ
We also describe the first and successful use of triethylsilane and palladium on carbon for the
selective alkene hydrogenation of the reported chalcones, in the presence of the carbonyl group.
3). Whereas Cꢀglucosylation of small phenols has been quite well investigated,33ꢀ42 we describe
here a new procedure for the Cꢀglucosylation of unprotected dihydrochalcones that uses
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a catalyst. The compounds were assessed
for their ability to inhibit glucose uptake by human SGLT1 and SGLT2 in vitro using HEK293
cells stably expressing one or the other of the two proteins and for their impact on cell viability
using an assay of metabolic capacity and on GLUTꢀbased glucose transport. To ascertain the risk
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that the Cꢀglucosyl dihydrochalcones were pan assay interference compounds (PAINS),43
especially those that modulate membranes and muddle the response of membrane receptors, we
adapted a known computational protocol44 to quantify the effects of one of the most active Cꢀ
glucosyl dihydrochalcones (nothofagin, 12h) in a {(2R)ꢀ3ꢀhexadecanoyloxyꢀ2ꢀ[(Z)ꢀoctadecꢀ9ꢀ
enoyl]oxypropyl} 2ꢀ(trimethylazaniumyl)ethyl phosphate (POPC) model membrane.
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RESULTS AND DISCUSSION
Chemistry. CꢀGlucosyl dihydrochalcones 12a,cꢀg (Scheme 2) were prepared from the
corresponding dihydrochalcones 9a,cꢀg (Scheme 1) and 2,3,4,6ꢀtetraꢀOꢀbenzylꢀDꢀglucopyranose
(10). Nothofagin45 (12h) was synthesized by a variation on the strategy (Scheme 3).
Dihydrochalcones 9a,cꢀh were synthesized starting from 1ꢀ[2,4ꢀbis(ethoxymethoxy)ꢀ6ꢀ
hydroxyphenyl]ethanꢀ1ꢀone (5) and paraꢀsubstituted benzaldehydes 6aꢀh in three steps. Aldol
condensation of 5 with aromatic aldehydes 6aꢀh under basic conditions provided 2′,4′ꢀ
bis(ethoxymethoxy)ꢀ6′ꢀhydroxychalcones 7aꢀh in good yields (85ꢀ95%). Deprotection of 7aꢀh in
the presence of the Lewis acid FeCl3⋅6H2O afforded the 2′,4′,6′ꢀtrihydroxychalcones 8aꢀh in 67ꢀ
96% yield. These two steps were carried out with both microwave and conventional heating. The
results (Table 1) show that microwave heating did not change the yield substantially, but the
reaction time was decreased in both steps.
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Scheme 1. Synthesis of 2′,4′,6′ꢀtrihydroxychalcones and 2′,4′,6′ꢀtrihydroxydihydrochalcones.a
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aReagents and conditions: (i) 50% aq. NaOH (w/v), EtOH, rt, 24 h; (ii) 50% aq. NaOH (w/v),
EtOH, 120 ºC, 250 W, 1 h; (iii) FeCl3·6H2O, MeOH, reflux, 2ꢀ3 h; (iv) FeCl3·6H2O, MeOH, 80
°C, 7ꢀ10 min; (v) Et3SiH, Pd/C, EtOAc, MeOH, rt, 10 min.
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Table 1. Reaction yields of chalcone and dihydrochalcone synthesis.
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Yield (%)a
Compound
Conventional
Microwave
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9h
aIsolated yield
Addition of triethylsilane to a palladiumꢀcharcoal catalyst generated molecular hydrogen in
situ, resulting in a rapid and highꢀyielding reduction of the chalcone double bond in 8aꢀh to
afford the corresponding dihydrochalcones 9a,cꢀh (Scheme 1) in high yield (Table 1), except for
the bromine derivative 8b, which underwent reduction of the bromoaryl moiety, affording
compound 9c instead of 9b. This bromineꢀhydrogen exchange has been observed in other
hydrosilaneꢀmediated reductions.46 Nevertheless, this method efficiently generated molecular
hydrogen in situ for the reduction of the carbonꢀcarbon double bond in α,βꢀunsaturated
compounds, avoiding the handling of H2 bottles.
CꢀGlucosylation of dihydrochalcone 9a using 2,3,4,6ꢀtetraꢀOꢀbenzylꢀDꢀglucopyranose (10) as
the glucosyl donor in the presence of 0.25 equiv of TMSOTf, the Cꢀglucosyl dihydrochalcone
11a was obtained in 12% yield (Scheme 2). Increasing the amount of the catalyst up to 0.50
equiv improved the yield of 11a to 43%, whereas using 0.75 equiv led to the formation of
secondary products. The C-glucosylation of dihydrochalcones 9cꢀg was carried out using 0.50
equiv. of TMSOTf affording the protected Cꢀglucosyl dihydrochalcones 11cꢀg in moderate yield
(Scheme 2). Removal of benzyl groups from the glucosyl moiety led to the formation of the
target Cꢀglucosyl dihydrochalcones 12a,cꢀg in 28ꢀ35% overall yield for the 5 steps.
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Scheme 2. Synthesis of Cꢀglucosyl dihydrochalcones.a,b
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R
R
OH
O
HO
O
OH
O
HO
BnO
BnO
BnO
BnO
BnO
O
i)
BnO
+
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OH
OBn
OBn
OH
OH
11a
11c R = H
11d R = Me 52.1%
11e R = OMe 39.3%
11f R = OEt 46.7%
R = F
42.8%
41.8%
9a R = F
9c
9d R = Me
9e R = OMe
9f R = OEt
9g R = OPr
10
R = H
11g
R = OPr 45.9%
9h
R = OH
R
OH
O
HO
HO
HO
O
HO
OH
ii)
OH
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R = F
R = H
84.0%
82.0%
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12d R = Me 98.0%
12e R = OMe 84.0%
12f R = OEt 89.0%
12g R = OPr 90.6%
12hb R = OH
aReagents and conditions: (i) TMSOTf, CH2Cl2, CH3CN, drierite, 0 ºC then rt, 2ꢀ5 h; (ii)
Et3SiH, Pd/C, EtOAc, MeOH, rt, 5 h; bFor reagents and conditions, see Scheme 3.
Nothofagin (12h) was prepared following an approach based on our previous work,42 starting
with the Cꢀglucosylation of acetophloroglucinol (13) under the same conditions as described
above, followed by benzylation, aldol condensation, and deprotection (Scheme 3). The yield for
the 5ꢀstep synthesis of 12h was 36%, which is an improvement over the yield of 28% obtained in
the 8ꢀstep synthesis by Minehan et al.47
~
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Scheme 3. Synthesis of nothofagin (12h).a
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aReagents and conditions: (i) TMSOTf, CH2Cl2, CH3CN, drierite, 0 ºC then rt, 5 h, 56%; (ii)
BnBr, K2CO3, DMF, rt, 1.5 h, 74%; (iii) 50% aq. NaOH (w/v), EtOH, reflux, 24 h, 63%; (iv)
Et3SiH, Pd/C, EtOAc, MeOH, rt, 5 h, 79%
Biological evaluation. The ability of compounds 1, 4, 8aꢀh, 9a,cꢀh, and 12a,cꢀh to inhibit
glucose uptake in vitro was evaluated in HEK293 cells stably expressing the human SGLT1 or
SGLT2 proteins. HEK293 cells were transfected with either a pCMV6ꢀNeoꢀSGLT1 or a
pCMV6ꢀNeoꢀSGLT2 plasmid, and stably transfected clones were selected using G418, an analog
of the antibiotic neomycin sulfate. The expression of SGLT1 or SGLT2 genes in stably
transfected HEK293 cells was confirmed by reverse transcription PCR (RTꢀPCR) (Figure 2).
Strong bands corresponding to the PCR products 328 bp for SGLT1 (lane 3) and 321 bp for
SGLT2 (lane 5) in both cell lines were observed. No expression of SGLT1 and SGLT2 was
observed in nonꢀtransfected HEK293 cells (lanes 2 and 5) used as negative control.
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Figure 2. Expression of SGLT1 and SGLT2 in HEK293 cells by RTꢀPCR. Two bands of 328
and 321 bp are observed corresponding to SGLT1 and SGLT2 PCR products, respectively. Lane
1: 2.5 Kbp ladder; lane 2: nonꢀtransfected HEK293 cells; lane 3: pCMV6ꢀNeoꢀSGLT1
transfected HEK293 cells; lane 4: pCMV6ꢀNeoꢀSGLT2 transfected HEK293 cells; lane 5: nonꢀ
transfected HEK293 cells
The overexpression of SGLT1 and SGLT2 proteins in the two stable HEK293 cell lines was
confirmed by SDSꢀPAGE (Figure 3). Stronger bands in SGLT1 and SGLT2 stably transfected
cells are observed (lanes 2 and 4, respectively) relative to nonꢀtransfected HEK293 cells (lanes 1
and 3).
B
A
3
4
1
2
SGLT1
βꢀactin
SGLT2
βꢀactin
Figure 3. Immunoblot of SGLT1 and SGLT2 expression in stably transfected HEK293 cells. (A)
Lane 1: SGLT1 expression in nonꢀtransfected HEK293 cells, lane 2: SGLT1 expression in stable
HEK293ꢀSGLT1 cells; (B) Lane 3: SGLT2 expression in nonꢀtransfected HEK293 cells, lane 4:
SGLT2 expression in stable HEK293ꢀSGLT2 cells. βꢀActin was used as a loading control.
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The acute toxicity of compounds 1, 8aꢀh, 9a,cꢀh, and 12a,cꢀh was evaluated using a cell
viability assay that assesses the metabolic capacity of cells. Nonꢀviable cells lack metabolic
capacity. After 20ꢀ24 hours of incubation with the compounds at a concentration of 100 ꢀM, the
compounds showed no significant toxicity to HEK293 cells relative to untreated cells (Figure 4).
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80.0
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Compound
Figure 4. Cell viability assay applied to HEK293 cells using compounds 1, 8aꢀh, 9a,cꢀh, and
12a,cꢀh. Data are the mean ± SD values from three independent experiments. The column
labeled UT is untreated cells. DMSO was used as negative control.
The glucose uptake in HEK293 cells stably expressing SGLT1 or SGLT2 proteins was
measured by the methods described by Petty48 and Minokoshi49 with minor modifications. The
method is based on the phosphorylation of 2ꢀdeoxyglucose (2DG) by endogenous hexokinase
forming 2ꢀdeoxyglucoseꢀ6ꢀphosphate (2DG6P), which is later oxidized by G6PDH in the
presence of NADP+. The stoichiometrically generated NADPH is then amplified by the
diaphoraseꢀresazurin cycling system to produce a highly fluorescent molecule, resofurin. The
amount of resofurin formed is proportional to the amount of 2DG taken up by the cells and can
be quantified using fluorescence spectroscopy. To confirm the viability of this assay for our
system, the amount of 2DG taken up by HEK293 stably overexpressing either SGLT1 or
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SGLT2, and nonꢀtransfected HEK293 cells was measured (Figure 5). We observed a greater than
2ꢀfold increase in 2DG uptake in both stable cell lines compared to the control HEK293 cells.
The 2DG uptake in the presence of compounds 1, 4, 8aꢀh, 9a,cꢀh, and 12a,cꢀh at a concentration
of 100 ꢀM was measured in both stable cell lines and showed a complete inhibition of both
SGLT1 and SGLT2 (Figure 6).
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3.000
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HEK293-SGLT2
Figure 5. 2ꢀDeoxyglucose uptake assay using HEK293 stably overexpressing either SGLT1 or
SGLT2 and nonꢀtransfected HEK293 cells. Data are the mean ± SD values from three
independent experiments.
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140.0
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1
4
8a 8b 8c 8d 8e 8f 8g 8h 9a 9c 9d 9e 9f 9g 9h 12a 12c 12d 12e 12f 12g 12h
SGLT1
SGLT2
Figure 6. SGLT1 and SGLT2 inhibition at 100 ꢀM of compounds 1, 4, 8aꢀh, 9a,cꢀh, and 12a,cꢀ
h. Data are mean ± SD values from three independent experiments.
The IC50 values for the inhibition of glucose uptake by HEK293 cells stably transfected with
SGLT1 or SGLT2 were measured independently (Table 2). Cells were treated with
concentrations of compounds 1, 4, 8aꢀh, 9a,cꢀh, and 12a,cꢀh ranging from 0.1 nM to 200 ꢀM
and the level of glucose uptake was measured by the 2ꢀDG uptake assay. Phlorizin (1) and
dapagliflozin (4a) were used as nonꢀselective and selective SGLT2 inhibitors, respectively. Their
IC50 values are 0.5 and 1.8 ꢀM, respectively, for SGLT1 and 67.3 and 0.8 nM, respectively, for
SGLT2.
All of the synthesized Cꢀglucosyl dihydrochalcones were potent inhibitors of SGLTs with at
least 500ꢀfold selectivity for SGLT2 over SGLT1. Inhibition of SGLTs by 12c, might explain its
antihyperglycemic properties observed in rats.50 The most potent compounds against SGLT2
were 12a (9.1 nM) and nothofagin (12h, 11.9 nM), the latter of which corroborates the results of
computational docking studies using a homology model of SGLT2 that suggested nothofagin
was an inhibitor of SGLT2.51 Compounds 12a and 12h were also the most selective for SGLT2
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over SGLT1 (1065 and 1597ꢀfold, respectively). The change from the Oꢀglucoside, found in the
nonꢀselective SGLT inhibitor phlorizin (1), to the corresponding Cꢀglucosyl derivative,
nothofagin (12h), resulted in a 40ꢀfold decrease in activity against SGLT1 and a nearly 6ꢀfold
increase in activity against SGLT2. Overall, 12h is less active than dapagliflozin (4a) against
SGLT2, and it is 10ꢀfold less active against SGLT1 than 4.
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Table 2. IC50 values of compounds 1, 4, 8aꢀh, 9a,cꢀh and 12a,cꢀh for SGLT1 and SGLT2
5
6
7
8
9
IC50 (ꢀM) a
SGLT1
Compound
Selectivity
SGLT1/SGLT2
SGLT2
0.0673 ±
0.0051
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1
4
7.4
0.499 ± 0.120
2250
1.80 ± 0.33
0.0008 ±
0.0001
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
8a
8b
8c
8d
8e
8f
25.60 ± 1.73 51.70 ± 1.90
34.60 ± 0.98 23.50 ± 1.20
25.40± 1.98
10.40± 0.70
50.90± 1.37
33.90± 1.00
31.00± 1.30
33.30 ± 0.99
34.10 ± 1.70
18.20 ± 0.24
26.00 ± 1.40
26.20 ± 2.40
8g
8h
9a
9c
9d
9e
9f
52.60 ± 0.39 22.70 ± 0.73
9.60± 0.87
7.90 ± 1.00
9.90 ± 0.78
15.80 ± 1.10
14.70 ± 0.63
18.00 ± 0.95
13.30 ± 0.65 18.70 ± 1.40
15.10 ± 0.61 16.70 ± 1.70
11.60 ± 0.60 16.40 ± 1.00
9g
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ꢀ
9h
12.40± 1.40
9.70 ± 0.89
18.30 ± 0.59
12a
1065
500
582
648
673
661
1597
0.0091 ±
0.0057
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12c
12d
12e
12f
9.90 ± 0.43
10.60 ± 0.89
13.80 ± 0.57
14.60 ± 0.10
15.60 ± 0.92
19.00 ± 1.3
0.0198 ±
0.0024
0.0182 ±
0.0020
0.0213 ±
0.0031
0.0217 ±
0.0044
12g
12h
0.0227 ±
0.0039
0.0119 ±
0.0026
aData are mean ± SD values from three independent experiments.
To probe whether or not the Cꢀglucosyl dihydrochalcone inhibitors of SGLT2 might also
interfere with glucose transport through the GLUT family of transporters, the 2ꢀDG uptake assay
was performed on the SGLT1 and SGLT2 stable cell lines in buffer lacking sodium by
substituting choline chloride for sodium chloride. GLUT transporters are sodium independent,
whereas SGLTs are sodium dependent. In the absence of sodium, GLUT transporters remain
functional, whereas SGLTs do not transport glucose. When the 2ꢀDG uptake assay was
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performed in buffer lacking sodium, the Cꢀglucosyl dihydrochalcones 12a,cꢀh did not inhibit the
uptake of glucose unless sodium was present, whereas all of the aglycones (chalcones and
dihydrochalcones) inhibited glucose uptake regardless of whether sodium was present or not
(Figure 7). In control experiments, cytochalasin B, a known inhibitor of GLUT transporters,52ꢀ54
inhibited glucose uptake in the absence and presence of sodium, whereas phlorizin (1), which is
not known to inhibit GLUT transporters, did not inhibit glucose uptake in the absence of sodium.
These results suggest that the Cꢀglucosyl dihydrochalcones 12a,cꢀh do not interfere with GLUT
transporters.
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120.0
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80.0
SGLT1_sodium
60.0
SGLT2_sodium
SGLT1_choline
SGLT2_choline
40.0
20.0
0.0
-20.0
Figure 7. 2DG assay performed in sodium and choline buffer. The concentration of chalcones
8aꢀh, dihydrochalcones 9a,cꢀh, and Cꢀglucosyl dihydrochalcones 12a,cꢀh was 100 ꢀM. Phlorizin
(PHLO) and cytochalasin B (CytocB) were used as negative and positive controls, respectively.
There are online services that identify PAINS,55, 56 however, their false discovery rate is
significant because these tools are more oriented toward highꢀthroughput screening. Andersen
and coꢀworkers developed a computational protocol to quantify the effects of several
phytochemicals in a POPC membrane model.44 Their approach was based on potential of mean
force (PMF) calculations in the absence and presence of the phytochemicals. By moving a 0.9
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nm radius spherical probe across the different systems, the authors were able to estimate how the
compounds alter the energy required to perturb the bilayer. In their results, curcumin, a known
PAINS compound,43 was found to have a strong effect, while resveratrol only had a mild effect.44
We adapted this protocol,44 and performed the PMF calculations with an atomistic force field
(GROMOS 54A7),57 instead of the MARTINI force field,44 which is coarse grained and less
detailed. Figure 8 shows the PMFs for translocating a probe of radius ~0.6 nm across a pure
POPC bilayer and binary mixtures of 12h or resveratrol in POPC. In these PMF profiles, we
observe a minimum of energy in the lipid tail region, which differs from the energy maximum
that Andersen and coꢀworkers observed.44 This deviation probably results from the differing
detail levels of the force fields used. Our profiles are in good agreement with the PMF reported
for a methaneꢀlike sphere across a bilayer using an atomistic model.58 Resveratrol, which is a
mild PAINS compound according to a similar method,44 seems to alter the energetics of
membrane permeation in both the head group and center of the bilayer regions. Nothofagin (12h)
only has a minor effect on the head group region, where it is concentrated, with no apparent
perturbation of the remaining membrane. Therefore, it is unlikely that 12h can act as a
membrane modulator and present itself as a false positive in membrane protein assays, like other
known PAINS.
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Figure 8. Symmetrized PMF for translocating a probe of radius ~0.6 nm across a POPC
bilayer. The bilayer normal is set to the Xꢀaxis with zero at the center of the bilayer. POPC is the
pure lipid bilayer, 12h is nothofagin, and Res is resveratrol. For profiles with error bars, see
Supporting Information.
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CONCLUSION
CꢀGlucosyl dihydrochalcones were prepared by direct Cꢀglucosylation of a series of
unprotected dihydrochalcones, which were synthesized in high yield from 1ꢀ[2,4ꢀ
bis(ethoxymethoxy)ꢀ6ꢀhydroxyphenyl]ethanꢀ1ꢀone (5) and paraꢀsubstituted benzaldehydes (6aꢀ
h). The chalcones, dihydrochalcones, and Cꢀglucosyl dihydrochalcones showed little toxicity to
HEK293 cells in culture. At high concentration (100 ꢀM), all of the compounds completely
inhibited the glucose uptake in HEK293 cells stably expressing SGLT1 or SGLT2 proteins, but
the Cꢀglucosyl dihydrochalcones were particularly potent against SGLT2 (IC50 = 9ꢀ23 nM). The
Cꢀglucosyl dihydrochalcones demonstrated significantly high selectivity toward SGLT2 over
SGLT1, but they were not more selective than dapagliflozin (4a), which is currently on the
market for the treatment of type 2 diabetes. Nevertheless, the Cꢀglucosyl dihydrochalcones were
all less active than dapagliflozin (4a) toward SGLT1. Inhibition of SGLT1 is thought to be the
cause of some adverse side effects of SGLT inhibitors, including dapagliflozin (4a). The Cꢀ
glucosyl dihydrochalcones 12a,cꢀh appear to be inactive against the nonꢀsodium dependent
GLUT family of glucose transporters, another potential source of adverse side effects. The
computational PMF profile of 12h provides evidence that the Cꢀglucosyl dihydrochalcones are
not PAINS and suggests that the glucosyl group plays an important role in preventing deep
membrane insertion. These results also show the importance of Cꢀglucosylation to achieve
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higher selectivity for SGLT2 over SGLT1 and GLUT, by comparison to those obtained for
5
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phlorizin, the Oꢀglucosyl analog.
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EXPERIMENTAL SECTION
Chemistry. Solvents and reagents were obtained from commercial sources and used without
further purification. Solutions were concentrated below 50 ºC under vacuum on rotary
evaporators. Qualitative TLC was performed on preꢀcoated 0.50ꢀꢀm silica gel 60 plates with a
UV indicator; compounds were detected by UV light (254 nm) and spraying with a 10%
methanol solution of sulfuric acid or with a 5% ethanol solution of iron(III) chloride hexahydrate
followed by heating. Column chromatography was carried out on silica gel (40ꢀ60 ꢀm). NMR
1
13
spectra were recorded at 400.13 MHz for HꢀNMR and 100.62 MHz for CꢀNMR. Chemical
shifts are given in ppm relative to tetramethylsilane. Assignments were made, when needed, with
the help of COSY, HMQC, and HMBC experiments. Protons and carbons in ring A (aromatic
ring nearest to the glucosyl group) are assigned as H′, C′; in ring B (aromatic ring furthest from
the glucosyl group) as H′′, C′′; and those belonging to the glucosyl moiety as H′′′, C′′′ to
facilitate the description of the corresponding chemical shifts, while those belonging to the
propanone moiety are assigned as C, H. HRMS spectra were acquired in an FTICR mass
spectrometer equipped with a dual ESI/MALDI ion source and a 7ꢀT actively shielded magnet.
1H NMR data confirmed compound purity ≥95%.
General Procedure for the Synthesis of Protected C-Glucosyl Dihydrochalcones. Under a
N2 atmosphere, a solution of dihydrochalcone 9 (2 equiv) in acetonitrile (2 mL) was added to a
solution of 2,3,4,6ꢀtetraꢀOꢀbenzylꢀDꢀglucopyranose (10) (1.23 mmol) in dichloromethane (10
mL) at room temperature. Drierite (200 mg) was added and the reaction vessel was maintained
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under N2. The mixture was cooled to ꢀ30 ºC and TMSOTf (0.5 equiv) was added dropꢀwise.
After 20ꢀ30 min the reaction was allowed to reach room temperature. The total consumption of
the glycosyl donor was confirmed by TLC (1:1 hexane/acetone) and after 2ꢀ5 h the reaction was
quenched with a saturated aqueous solution of NaHCO3. Drierite was removed by filtration
through celite and the residue was extracted with dichloromethane. The extracts were washed
with brine, dried over anhydrous MgSO4, and concentrated to an oil, which was purified by
column chromatography (7:1 hexane/acetone).
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3-(4-Fluorophenyl)-1-[3-(2,3,4,6-tetra-O-benzyl-β- -glucopyranosyl)-2,4,6-tri-
D
1
hydroxyphenyl]propan-1-one (11a). Yield 43%; Rf = 0.58 (3:1 hexane/EtOAc); H NMR
(CDCl3, 400 MHz, δ): 14.30 (2H, s, OH2′, OH6′); 9.72 (1H, s, OH4′); 7.37ꢀ6.95 (24H, m, ArH,
H2′′, H3′′, H5′′, H6′′); 6.01 (1H, s, H5′); 4.98 (2H, s, ꢀOCH2Ph); 4.88ꢀ4.86 (3H, m, ꢀOCH2Ph,
H1′′′); 4.73, 4.29 (2H, parts A and B of AB system, J = 10.7 Hz, ꢀOCH2Ph); 4.57, 4.48 (2H,
parts A and B of AB system, J = 9.2 Hz, ꢀOCH2Ph); 4.57, 4.48 (2H, parts A and B of AB system,
J = 9.2 Hz, ꢀOCH2Ph); 3.90ꢀ3.60 (6H, m, H2′′′, H3′′′, H4′′′, H5′′′, H6a′′′, H6b′′′); 3.28ꢀ4.24 (2H,
13
m, H2); 2.95 (2H, t, J = 7.21 Hz, H3); C NMR (CDCl3, 100 MHz, δ): 204.8 (C1); 162.5
(C2′,C6′); 160.1 (C4′, C4′′); 136.8 (C1′′); 138.2, 137.7, 137.3, 137.2 (CqꢀPh); 129.8 (C2′′, C6′′,
JC,F = 7.8 Hz); 128.7, 128.6, 128.5, 128.3, 128.0, 127.8, 127.6 (CHꢀPh); 115.0 (C3′′, C5′′, JC.F
=
21.4 Hz); 86.2 (C2′′′); 78.7 (C3′′′); 76.2 (C4′′′); 75.7 (C5′′′); 75.3 (ꢀOCH2Ph); 75.1 (C1′′′); 75.0,
74.6, 73.4 (ꢀOCH2Ph); 67.7 (C6′′′); 45.9 (C2); 29.6 (C3); HRMSꢀESI (m/z): [M + Na]+ calcd for
C49H47FO9 821.30963; found, 821.30943.
3-Phenyl-1-[3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2,4,6-trihydroxyphenyl]-
1
propan-1-one (11c). Yield 42%; Rf = 0.19 (3:1 hexane/acetone); H NMR (CDCl3, 400 MHz,
δ): 7.35ꢀ6.98 (25H, m, PhH, H2′′, H3′′, H4′′, H5′′, H6′′); 5.96 (1H, s, 1H, H5′); 4.95 (2H, s, ꢀ
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OCH2Ph); 4.85 (1H, d, J =8.7 Hz, H1′′′); 4.83, 4.51 (2H, parts A and B of AB system, J =11.0
Hz, ꢀOCH2Ph); 4.69, 4.27 (2H parts A and B of AB system, J =10.0 Hz, ꢀOCH2Ph); 4.55, 4.46
(2H, parts A and B of AB system, J =11.3 Hz, ꢀOCH2Ph); 3.86ꢀ3.58 (6H, m, H2′′′, H3′′′, H4′′′,
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H5′′′, H6a′′′, H6b′′′); 3.30ꢀ3.26 (2H, m, H2); 2.97 (2H, t, J = 8.9 Hz, Hꢀ3); C NMR (CDCl3,
100 MHz, δ): 205.0 (C1); 141.8 (C1′′); 138.2, 137.7, 137.3, 136.2 (CqꢀPh); 128.7, 128.5, 128.4,
128.3, 128.0, 127.9, 127.8, 127.6 (CHꢀPh, C2′′, C3′′, C5′′, C6′′); 125.9 (C4′′); 106.0 (C3′); 102.8
(C1′); 99.9 (C5′); 86.2 (C2′′′); 78.7 (C3′′′); 77.2 (C4′′′); 76.3 (C5′′′); 75.7 (ꢀOCH2Ph); 75.3
(C1′′′); 75.2, 75.0, 73.4 (ꢀOCH2Ph); 67.5 (C6′′′); 45.9 (C2); 30.5 (C3); HRMSꢀESI (m/z): [M +
Na]+ calcd for C49H48O9 803.31905; found 803.32129.
3-(4-Methylphenyl)-1-[3-(2,3,4,6-tetra-O-benzyl-β-
D
-glucopyranosyl)-2,4,6-trihydroxy-
1
phenyl]propan-1-one (11d). Yield 52%; Rf = 0.60 (3:1 hexane/ EtOAc); H NMR (CDCl3, 400
MHz, δ): 14.03 (2H, s, OH2′, OH6′); 9.87 (1H, s, 1H, OH4′); 7.34ꢀ6.97 (24H, m, ArH, H2′′,
H3′′, H5′′, H6′′); 5.89 (1H, s, H5′); 4.95, 4.91 (2H, parts A and B of AB system, J = 10.0 Hz, ꢀ
OCH2Ph); 4.87 (1H, d, J = 10.0 Hz, H1′′′); 4.83 (1H, J = 8.5 Hz, part A of AB system AB of, ꢀ
OCH2Ph); 4.62, 4.21 (2H, parts A and B of AB system, J =10.6 Hz, ꢀOCH2Ph); 4.57ꢀ4.47 (3H,
m, part B system AB of ꢀOCH2Ph, parts A and B of AB system of ꢀOCH2Ph); 3.80ꢀ3.59 (6H, m,
H2′′′, H3′′′, H4′′′, H5′′′, H6a′′′, H6b′′′); 3.27 (2H, t, J = 7.4 Hz, H2); 2.97 (2H, t, H3); 2.92 (3H,
s, ꢀCH34′′); 13C NMR (CDCl3, 100 MHz, δ): 205.2 (C1); 138.8 (C1′′); 138.3, 137.8, 137.1, 136.4
(CqꢀPh); 135.4 (C4′′); 129.1, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7 (CHꢀ
Ph, C2′′, C3′′, C5′′, C6′′); 105.7 (C3′); 102.5 (C1′); 97.5 (C5′); 86.2 (C2′′′); 81.7 (C3′′′); 78.3
(C4′′′); 77.3 (C5′′′); 76.1 (C1′′′); 75.7, 75.3, 74.9, 73.5 (ꢀOCH2Ph); 67.9 (C6′′′); 46.1 (C2); 30.1
(C3); HRMSꢀESI (m/z): [M + Na]+ calcd for C50H50O9 817.33478; found 817.33552.
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3-(4-Methoxyphenyl)-1-[3-(2,3,4,6-tetra-O-benzyl-β- -glucopyranosyl)-2,4,6-tri-
D
5
6
7
8
1
hydroxyphenyl]propan-1-one (11e). Yield 39%; Rf = 0.19 (1:1 hexane/acetone); H NMR
(CDCl3, 400 MHz, δ): 14.43 (2H, s, OH2′, OH6′); 7.33ꢀ7.12 (20H, m, ArH); 6.98 (2H, d, J =7.8
Hz, H2′′, H6′′); 6.82 (2H, d, H3′′, H5′′); 5.89 (1H, s, H5′); 4.96ꢀ4.88 (3H, m, parts A and B of
system AB of ꢀOCH2Ph, H1′′′); 4.82 (1H, part A of system AB of ꢀOCH2Ph), 4.59, 4.20 (2H,
parts A and B of AB system, J =8.9 Hz, ꢀOCH2Ph); 4.55ꢀ4.46 (3H, m, part B of system AB of ꢀ
OCH2Ph, parts A and B of AB system); 3.80ꢀ3.59 (10H, m, H2′′′, H3′′′, H4′′′, H5′′′, H6a′′′,
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H6b′′′, ꢀOCH34′′); 3.27 (2H, t, J =7.3 Hz, H2); 2.93 (2H, t, H3); C NMR (CDCl3, 100 MHz,
δ): 205.2 (C1); 138.4, 137.7, 137.1, 136.6 (CqꢀPh); 133.9 (C1′′); 129.5 (C2′′, C6′′); 128.7, 128.6,
128.5, 128.1, 128.0, 127.7 (CHꢀPh); 113.8 (C3′′, C5′′); 105.6 (C3′); 102.5 (C1′); 97.3 (C5′); 86.2
(C2′′′); 81.1 (C3′′′); 78.8 (C4′′′); 77.4 (C5′′′); 76.0 (C1′′′); 75.8, 75.3, 74.8, 73.5 (ꢀOCH2Ph);
68.0 (C6′′′); 55.3 (ꢀOCH34′′); 46.2 (C2); 31.9 (C3); HRMSꢀESI (m/z): [M + Na]+ calcd for
C50H50O10 833.32962; found 833.33521.
3-(4-Ethoxyphenyl)-1-[3-(2,3,4,6-tetra-O-benzyl-β-
D
-glucopyranosyl)-2,4,6-trihydroxy-
1
phenyl]propan-1-one (11f). Yield 47%; Rf = 0.16 (1:1 hexane/acetone); H NMR (CDCl3, 400
MHz, δ): 7.32ꢀ7.11 (20H, m, ArH); 6.98 (2H, d, J = 7.6 Hz, H2′′, H6′′); 6.81 (2H, d, H3′′, H5′′);
5.92 (1H, s, H5′); 4.97ꢀ4.82 (4H, m, parts A and B of system AB of ꢀOCH2Ph, part A of system
AB of ꢀOCH2Ph, H1′′′); 4.63, 4.22 (2H, parts A and B of system A, J =10.3 Hz, ꢀOCH2Ph); 4.56ꢀ
4.55 (3H, m, part B of system AB of ꢀOCH2Ph, parts A and B of system AB of ꢀOCH2Ph); 4.00
(2H, q, J = 7.4 Hz, J = 13.1 Hz, ꢀOCH2CH34′′); 3.81ꢀ3.59 (6H, m, H2′′′, H3′′′, H4′′′, H5′′′,
H6a′′′, H6b′′′); 3.28ꢀ3.24 (2H, m, H2); 2.91 (2H, t, J = 7.6 Hz, H3); 1.40 (3H, t, ꢀOCH2CH34′′);
13C NMR (CDCl3, 100 MHz, δ): 205.2 (C1); 157.1 (C4′′); 138.3, 137.7, 137.2, 136.4 (CqꢀPh);
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133.8 (C1′′); 129.4 (C2′′, C6′′); 128.7, 128.5, 128.4, 128.1, 128.0, 127.8, 127.6 (CHꢀPh); 114.4
(C3′′, C5′′); 105.7 (C3′); 102.6 (C1′); 97.5 (C5′); 86.2 (C2′′′); 81.9 (C3′′′); 78.7 (C4′′′); 77.2
(C5′′′); 76.1 (C1′′′); 67.8 (C6′′′); 75.7, 75.3, 74.9, 73.4 (ꢀOCH2Ph); 63.4 (ꢀOCH2CH34′′); 46.2
(C2); 29.7 (C3); 14.9 (ꢀOCH2CH34′′); HRMSꢀESI (m/z): [M + Na]+ calcd for C51H52O10
847.34527; found 847.34662.
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3-(4-Propyloxyphenyl)-1-[3-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2,4,6-tri-
1
hydroxyphenyl]propan-1-one (11g). Yield 46%; Rf = 0.18 (1:1 hexane/acetone); H NMR
(CDCl3, 400 MHz, δ): 14.03 (s, 2H, OH2′, OH6′); 7.34ꢀ7.12 (20H, m, ArH); 6.98 (2H, d, J = 6.8
Hz, H2′′, H6′′); 6.82 (2H, d, H3′′, H5′′); 5.93 (1H, s, H5′); 4.96, 4.92 (2H, parts A and B of
system AB, ꢀOCH2Ph); 4.88 (1H, d, J = 9.6 Hz, H1′′′); 4.83 (1H, part A of system AB, J = 10.5
Hz, ꢀOCH2Ph); 4.65, 4.24 (2H, parts A and B of system AB, J =10.3 Hz, ꢀOCH2Ph); 4.56ꢀ4.45
(3H, m, parts A and B of system AB, part B of system AB of ꢀOCH2Ph); 3.89 (2H, t, J = 6.9 Hz,
ꢀOCH2CH2CH34′′); 3.82ꢀ3.60 (6H, m, H2′′′, H3′′′, H4′′′, H5′′′, H6a′′′, H6b′′′); 3.30ꢀ3.18 (2H, m,
H2); 2.91 (2H, t, J = 7.1 Hz, H3); 1.79 (2H, q, J = 14.1 Hz, J = 20.4 Hz, ꢀOCH2CH2CH34′′); 1.03
13
(3H, t, ꢀOCH2CH2CH34′′); C NMR (CDCl3, 100 MHz, δ): 205.2 (C1); 157.3 (C4′′); 138.3,
137.7, 137.3, 136.7 (CqꢀPh); 133.7 (C1′′); 129.4 (C2′′, C6′′); 128.7, 128.5, 128.4, 128.2, 128.1,
128.0, 127.9, 127.8, 127.6 (CHꢀPh); 114.4 (C3′′, C5′′); 105.8 (C3′); 102.7 (C1′); 97.7 (C5′); 86.2
(C2′′′); 81.7 (C3′′′); 78.7 (C4′′′); 77.2 (C5′′′); 76.2 (C1′′′); 69.5 (C6′′′); 75.7, 75.3, 74.9, 73.4 (ꢀ
OCH2Ph); 67.7 (ꢀOCH2CH2CH34′′); 46.2 (C2); 29.7 (C3); 22.7 (ꢀOCH2CH2CH34′′); 10.6 (ꢀ
OCH2CH2CH34′′); HRMSꢀESI (m/z): [M + Na]+ calcd for C52H54O10 861.36092; found
861.36124.
General procedure for the synthesis of C-glucosyl dihydrochalcones. Compound 11 or 1642
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(0.5 mmol) was dissolved in EtOAc (1 mL) and methanol (2 mL). The 5% Pd/C catalyst was
added under a N2 atmosphere followed by dropwise addition of triethylsilane (10 equiv);
effervescence was observed, indicating that hydrogen was being formed in situ. The reaction was
maintained at room temperature until TLC confirmed completion (5 h). The catalyst was
removed by filtering the reaction mixture through celite. The volume of solvent in the filtrate
was reduced to oneꢀthird under vacuum, and most of the triethylsilane was removed by
extraction (three times) with acetonitrile/hexane (1:1). The final product was purified by column
chromatography (EtOAc only).
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3-(4-Fluorophenyl)-1-[3-(β-D-glucopyranosyl)-2,4,6-trihydroxyphenyl]propan-1-one
1
(12a). Yield 84%; mp 80.2ꢀ80.7 ºC; Rf = 0.36 (7:1 CH2Cl2/MeOH); H NMR (acetoneꢀd6, 400
MHz, δ): 7.33 (2H, dd, JH,H =8.37 Hz, JH,F = 5.2 Hz, H2′′, H6′′); 7.05 (2H, t, JH,F = 8.7 Hz, H3′′,
H5′′); 5.95 (1H, s, H5′); 4.94 (1H, d, J = 9.6 Hz, H1′′′); 3.91ꢀ3.83 (2H, m, H6a′′′, H6b′′′); 3.67
(2H, t, J = 9.0 Hz, H2′′′, H3′′′); 3.58 (1H, t, J = 9.2 Hz, H4′′′); 3.50 (1H, ddd, J = 9.6 Hz, J = 6.1
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Hz, J = 3.0 Hz, H5′′′); 3.39 (2H, t, J = 7.3 Hz, H2); 2.98 (2H, t, H3); C NMR (acetoneꢀd6, 100
MHz, δ): 204.5 (C1′); 163.0 (C4′); 162.4 (C2′, C6′); 160.0 (C4′′); 138.0, 137.9 (C1′′); 130.2,
130.1 (C2′′, C6′′); 114.9, 114.7 (C3′′, C5′′); 104.5 (C3′); 103.4 (C1′); 95.6 (C5′); 81.1 (C5′′′);
78.3 (C4′′′); 75.4 (C1′′′); 73.3 (C3′′′); 69.6 (C2′′′); 60.9 (C6′′′); 45.9 (C2); 29.7 (C3); HRMSꢀESI
(m/z): [M + Na]+ calcd for C21H23FO9 461.12183; found 461.12238.
1-[3-(β-D-Glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-phenylpropan-1-one (12c). Yield
1
82.0%; mp 80.0ꢀ80.1 ºC; Rf = 0.40 (7:1 CH2Cl2/MeOH); H NMR (acetoneꢀd6, 400 MHz, δ):
12.20 (1H, s, OH6′); 11.50 (1H, s, OH2′); 9.10 (1H, s, OH4′); 7.30ꢀ7.26 (4H, m, H2′′, H3′′, H5′′,
H6′′); 7.21ꢀ7.17 (1H, m, H4′′); 5.95 (1H, s, H5′); 4.94 (1H, d, J = 9.7 Hz, H1′′′); 3.91ꢀ3.81 (2H,
m, H6a′′′, H6b′′′); 3.70ꢀ3.62 (2H, m, H2′′′, H3′′′); 3.58ꢀ3.46 (2H, m, H4′′′, H5′′′); 3.40 (2H, t, J =
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7.7 Hz, H2); 2.99 (2H, t, H3); C NMR (acetoneꢀd6, 100 MHz, δ): 205.5 (C1); 163.0 (C2′, C4′,
C6′); 141.9 (C1′′); 128.5 (C2′′, C6′′); 128.3 (C3′′, C5′′); 125.8 (C4′′); 104.5 (C3′); 103.8 (C1′);
95.6 (C5′); 81.1 (C5′′′); 78.7 (C4′′′); 75.4 (C1′′′); 73.3 (C3′′′); 69.9 (C2′′′); 60.7 (C6′′′); 45.8
(C2); 30.4 (C3); HRMSꢀESI (m/z): [M + Na]+ calcd for C21H24O9 443.13125; found 443.13169.
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1-[3-(β-
D
-Glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-(4-methylphenyl)propan-1-one
1
(12d). Yield 82.0%; mp 117.0ꢀ117.5 ºC; Rf = 0.38 (7:1 CH2Cl2/MeOH); H NMR (acetoneꢀd6,
400 MHz, δ): 7.19 (2H, d, J = 7.2 Hz, H2′′, H6′′); 7.11 (2H, d, H3′′, H5′′); 5.96 (1H, s, H5′);
4.95 (1H, d, J = 9.1 Hz, H1′′′); 3.91ꢀ3.84 (6H, m, H6a′′′, H6b′′′); 3.67 (2H, t, J = 9.1 Hz, H2′′′,
H3′′′); 3.58 (1H, t, J = 9.0 Hz, H4′′′); 3.50 (1H, ddd, J = 9.5 Hz, J = 6.6 Hz, J = 2.9 Hz, H5′′′);
3.38 (2H, t, J = 6.9 Hz, H2); 2.95 (2H, t, H3); 2.30 (3H, s, ꢀCH34′′); (acetoneꢀd6, 100 MHz, δ):
204.8 (C1); 163.2 (C2′, C6′); 163.0 (C4′); 138.9 (C1′′); 134.9 (C4′′); 128.9 (C2′′, C6′′); 128.2
(C3′′, C5′′); 104.5 (C3′); 103.4 (C1′); 95.6 (C5′); 81.1 (C5′′′); 78.4 (C4′′′); 75.4 (C1′′′); 73.4
(C3′′′); 69.6 (C2′′′); 60.7 (C6′′′); 45.9 (C2); 30.0 (C3); 20.5 (ꢀCH34′′); HRMSꢀESI (m/z): [M +
Na]+ calcd for C22H26O9 457.14690; found 457.14758.
1-[3-(β- -Glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-(4-methoxyphenyl)propan-1-one
D
(12e). Yield 84.0%; mp 90.4ꢀ90.7 ºC; Rf = 0.31 (7:1 CH2Cl2/MeOH); 1H NMR (acetoneꢀd63, 400
MHz, δ): 12.29 (2H, s, OH2′, OH6′); 11.57 (1H, s, OH4′); 9.17 (1H, s, OHꢀ4′′); 7.21 (2H, d, J =
7.7 Hz, H2′′, H6′′); 6.85 (2H, d, H3′′, H5′′); 5.94 (1H, s, H5′); 4.94 (1H, d, J = 9.7 Hz, H1′′′);
3.91ꢀ3.87 (6H, m, H6a′′′, H6b′′′); 3.77 (3H, s, ꢀOCH34′′); 3.66 (2H, t, J = 9.0 Hz, H2′′′, H3′′′);
3.56 (1H, t, J = 8.7 Hz, H4′′′); 3.50 (1H, ddd, J = 9.9 Hz, J = 6.7 Hz, J = 3.3 Hz, H5′′′); 3.36 (2H,
t, J = 7.6 Hz, H2); 2.92 (2H, t, H3); 13C NMR (acetoneꢀd6, 100 MHz, δ): 205.3 (C1); 162.9 (C2′,
C4′, C6′); 158.0 (C4′′); 133.8 (C1′′); 129.4 (C2′′, C6′′); 113.7 (C3′′, C5′′); 104.5 (C3′); 104.5
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(C1′); 95.6 (C5′); 81.1 (C5′′′); 78.4 (C4′′′); 75.4 (C1′′′); 73.4 (C3′′′); 69.6 (C2′′′); 60.6 (C6′′′);
54.5 (ꢀOCH34′′); 46.2 (C2); 29.5 (C3); HRMSꢀESI (m/z): [M + Na]+ calcd for C22H26O10
473.14182; found 473.14245.
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D
-glucopyranosyl)-2,4,6-trihydroxyphenyl]propan-1-one
1
(12f). Yield 89.0%; mp 91.4ꢀ92.0 ºC; Rf = 0.10 (EtOAc); H NMR (acetoneꢀd6, 400 MHz, δ):
12.36 (1H, s, OH6′); 11.45 (1H, s, OH2′); 9.11 (1H, s, OH4′); 7.17 (2H, d, J = 8.2 Hz, H2′′,
H6′′); 6.82 (2H, d, H3′′, H5′′); 5.94 (1H, s, H5′); 4.93 (1H, d, J = 9.7 Hz, H1′′′); 3.99 (2H, q, J =
7.2 Hz, J = 13.3 Hz, ꢀOCH2CH34′′); 3.89ꢀ3.86 (6H, m, H6a′′′, H6b′′′); 3.72ꢀ3.47 (4H, m, H2′′′,
H3′′′, H4′′′, H5′′′); 3.33 (2H, t, J = 9.4 Hz, H2); 2.89 (2H, t, H3); 1.34 (3H, t, ꢀOCH2CH34′′); 13C
NMR (acetoneꢀd6, 100 MHz, δ): 204.9 (C1); 163.1 (C2′, C6′); 163.0 (C4′′); 157.3 (C4′′); 133.7
(C1′′); 129.4 (C2′′, C6′′); 114.3 (C3′′, C5′′); 104.5 (C3′); 103.1 (C1′); 95.6 (C5′); 81.1 (C5′′′);
78.4 (C4′′′); 75.3 (C1′′′); 73.3 (C3′′′); 69.7 (C2′′′); 62.9 (ꢀOCH2CH34′′); 60.8 (C6′′′); 46.1 (C2);
29.6 (C3); 14.3 (ꢀOCH2CH34′′); HRMSꢀESI (m/z): [M + Na]+ calcd for C23H28O10 487.15747;
found 487.15789.
1-[3-(β-D-Glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-(4-propyloxyphenyl)propan-1-one
1
(12g). Yield 90.6%; mp 80.0ꢀ80.2 ºC; Rf = 0.10 (EtOAc); H NMR (acetoneꢀd6, 400 MHz, δ):
7.11 (2H, d, J = 7.6 Hz, H2′′, H6′′); 6.85 (2H, d, H3′′, H5′′); 5.95 (1H, s, H5′); 4.94 (1H, d, J =
8.8 Hz, H1′′′); 3.94ꢀ3.90 (2H, m, ꢀOCH2CH2CH34′′); 3.89ꢀ3.83 (6H, m, H6a′′′, H6b′′′); 3.66 (2H,
t, J = 9.3 Hz, H2′′′, H3′′′); 3.58ꢀ3.49 (2H, m, H4′′′, H5′′′); 3.36 (2H, t, J = 7.6 Hz, H2); 2.92 (2H,
t, H3); 1.81ꢀ1.73 (2H, m, ꢀOCH2CH2CH34′′); 1.02 (3H, t, ꢀOCH2CH2CH34′′); 13C NMR
(acetoneꢀd6, 100 MHz, δ): 204.8 (C1); 162.9 (C2′, C4′, C6′); 157.5 (C4′′); 133.7 (C1′′); 129.3
(C2′′, C6′′); 114.3 (C3′′, C5′′); 104.5 (C3′); 103.4 (C1′); 95.6 (C5′); 81.1 (C5′′′); 78.5 (C4′′′);
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75.4 (C1′′′); 73.3 (C3′′′); 69.6 (C2′′′); 69.1 (ꢀCH2CH2CH34′′); 60.6 (C6′′′); 40.2 (C2); 29.7 (C3);
22.4 (ꢀCH2CH2CH34′′); 9.9 (ꢀCH2CH2CH34′′); HRMSꢀESI (m/z): [M + Na]+ calcd for C24H30O10
501.17312; found 501.17373.
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-Glucopyranosyl)-2,4,6-trihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one
1
(12h). Yield 78.8%; mp 133.3ꢀ133.4 ºC; Rf = 0.26 (7:1 CH2Cl2/MeOH); H NMR (acetoneꢀd6,
400 MHz, δ): 7.11 (2H, d, J = 8.0 Hz, H2′′, H6′′); 6.75 (2H, d, H3′′, H5′′); 5.96 (1H, s, H5′);
4.94 (1H, d, J = 9.6 Hz, H1′′′); 3.90ꢀ3.83 (2H, m, H6a′′′, H6b′′′); 3.30ꢀ3.64 (2H, m, H2′′′, H3′′′);
3.56 (1H, t, J = 8.9 Hz, H4′′′); 3.50 (1H, ddd, J = 9.6 Hz, J = 6.6 Hz, J = 3.1 Hz, H5′′′); 3.34 (2H,
t, J = 7.3 Hz, H2); 2.88 (2H, t, H3); 13C NMR (acetoneꢀd6, 100 MHz, δ): 204.9 (C1′); 163.1 (C2′,
C4′); 162.9 (C6′); 155.5 (C4′′); 132.5 (C1′′); 129.4 (C2′′, C6′′); 115.1 (C3′′, C5′′); 104.5 (C3′);
103.4 (C1′); 81.1 (C5′′′); 78.5 (C4′′′); 75.4 (C1′′′); 73.3 (C3′′′); 69.6 (C2′′′); 60.6 (C6′′′); 46.3
(C2); 29.7 (C3); HRMSꢀESI (m/z): [M + Na]+ calcd for C21H24O10 459.12617; found 459.12664.
Biological Evaluation. 2ꢀDeoxyꢀDꢀglucose (2DG), 2ꢀdeoxyꢀDꢀglucose 6ꢀphosphate sodium
salt, hexokinase (from Saccharomyces cerevisae), glucoseꢀ6ꢀphosphate dehydrogenase (G6PDH,
from Leuconostoc mesenteroides), resazurin sodium salt, triethanolamine (TEA) buffer, HEPES,
βꢀnicotinamide adenine dinucleotide phosphate (βꢀNADP+), adenosine 5′ꢀtriphosphate disodium
salt (ATP), phlorizin, cytochalasin B, diaphorase (from Clostridium kluyveri type IIꢀL), bovine
serum albumin (BSA), potassium chloride, sodium deoxycholate, choline chloride, fetal bovine
serum (FBS), Dulbeco’s modified Eagle’s medium (DMEM), Geneticin® (G418), phosphate
buffered saline, 2ꢀ[Nꢀ(7ꢀnitrobenzꢀ2ꢀoxaꢀ1,3ꢀdiazolꢀ4ꢀyl)amino]ꢀ2ꢀdeoxyglucose (2ꢀNBDG), and
dapagliflozin (4a) were purchased from commercial sources.
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