In vitro solubility, stability and permeability of novel quercetin-amino acid conjugates

Article abstract of DOI:10.1016/j.bmc.2008.12.043

In order to discover a quercetin prodrug with improved bioavailability, we synthesized nine quercetin-amion acid conjugates and estimated their pharmacokinetic properties including water solubility, stability against chemical or enzymatic hydrolysis, and

Full text of DOI:10.1016/j.bmc.2008.12.043

Bioorganic & Medicinal Chemistry 17 (2009) 1164–1171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro solubility, stability and permeability of novel quercetin–amino
acid conjugates
Mi Kyoung Kim a, Kwang-su Park a, Woon-seok Yeo a, Hyunah Choo b, Youhoon Chong a,
*
a Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
b Life Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to discover a quercetin prodrug with improved bioavailability, we synthesized nine quercetin–
amion acid conjugates and estimated their pharmacokinetic properties including water solubility, stabil-
ity against chemical or enzymatic hydrolysis, and cell permeability. Among the synthesized quercetin
prodrugs, quercetin–glutamic acid conjugate Qu-E (4g/5g) showed remarkable increases in water solubil-
ity, stability, and cell permeability compared with quercetin, which warrants further development as a
quercetin prodrug.
Received 17 November 2008
Revised 11 December 2008
Accepted 13 December 2008
Available online 25 December 2008
Keywords:
Quercetin
Prodrug
Ó 2008 Elsevier Ltd. All rights reserved.
Solubility
Stability
Permeability
1. Introduction
ited due to the low bioavailability presumably conferred by the fast
metabolism and excretion of the prodrug.
Quercetin (30,40,3,5,7-pentahydroxyflavone) is a member of the
class of flavonol (Fig. 1), which is abundant in many common foods
including apples, onions, grapes, red wine and green tea, at an
average level of 10 mg/kg.
The quercetin–glucose conjugate, which is the natural form of
the quercetin obtained from the plant source, is absorbed into
the apical membrane of the enterocyte. Once absorbed into the
apical membrane of the enterocyte, quercetin–glucose conjugate
is known to be hydrolyzed to quercetin which is then exhaus-
tively metabolized to the methylated, sulfonylated, and glucu-
ronidated quercetins by the enterocytic transferases.6 These
quercetin metabolites are then easily transported out to the
intestinal lumen by ABS/MDR translocator.6 Analogous conjuga-
tion reactions also take place in the liver.6,7 As a result, only very
low levels of quercetin are found in blood or plasma even after a
quercetin-rich meal.8–12 Studies with everted intestine segments
and analysis of blood and lymph samples have led to the identi-
fication of up to 23 different quercetin conjugates13–19 all arising
via single or multiple glucuronation, sulfation, and methylation
of the hydroxyls, but the metabolites may not themselves have
biological activities.20,21
Taken together, results from the Q12 study indicates that a good
quercetin prodrug should have increased water solubility as well
as decreased rate of hydrolysis to prevent the deactivating metab-
olism. In this study, as a part of our ongoing efforts to discover a
quercetin prodrug with improved bioavailability, we designed nine
quercetin–amino acid conjugates. Herein we report syntheses and
evaluation of the pharmacokinetic properties of the quercetin pro-
drugs including water solubility, chemical stability in the buffer
solution, enzymatic stability, and cell permeability across the
MDCK (Madin–Darby canine kidney) cell.
Quercetin has many biological activities including inhibition of
a number of tyrosine kinases.1,2 Clinical trials exploring different
schedules of administration of quercetin have been held back by
its extreme water insolubility, requiring formulation in dimethyl-
sulfoxide (DMSO) where 150 mg quercetin is soluble in 1 mL of
DMSO.3 To overcome these limitations, QC12 (Fig. 1), a water-sol-
uble glycine carbamate prodrug of quercetin, was synthesized and
evaluated in a clinical study to investigate its pharmacokinetics
following oral administrations to cancer patients.4 The glycine car-
bamate moiety of QC12 was designed to be lost in the blood stream
by hydrolysis to give the active ingredient, quercetin.4 In vitro
hydrolysis of QC12 to quercetin at 37 °C in water showed the pro-
drug to be stable with a half-life of 16.9 h while the half-life in
whole blood was only 0.39 h. Also, following oral administration
neither QC12 nor quercetin was detected in the serum, demon-
strating that QC12, like quercetin is not orally bioavailable.5 Thus,
the advantage presented by QC12 is its high aqueous solubility
compared to that of quercetin, eliminating the need for formula-
tion in DMSO. However, the utility of QC12 for oral dosage is lim-
* Corresponding author.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.043

M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
1165
Figure 1. Structures of quercetin and Q12.
down-field shifted (0.3 ppm) with other protons’ chemical shifts
unchanged (Fig. 2).24
2. Results and discussion
In addition to quercetin–amino acid conjugates
4 and 5
2.1. Syntheses of the Q12 analogues
(Scheme 1), two quercetin–dipeptide conjugates such as querce-
tin-alanine-aspartic acid (Qu-AD) and quercetin-alanine-glutamic
acid (Qu-AE) were also prepared. Usually, amino acids are trans-
ported into the intestine by help of human peptide transporter,
hPepT1, which is involved in absorption of various peptides across
the intestinal epithelium. As hPepT1 is known to recognize amino
acids attached to a drug, it can be hijacked to transport the drug-
amino acid conjugates.25–27 Thus, the peptide transporter may be
exploited to increase oral absorption of drug with poor essence per-
meability by attaching them to an enzymatically stable peptide,
which is recognized by hPepT1. Also, recent studies on the hPepT1
revealed that it is more selective for dipeptides such as Asp-Ala
and Glu-Ala.28 Therefore we attached these two dipeptides to quer-
cetin to improve absorption by help of the peptide transpoter
(Scheme 2).
Among 20 amino acids, 4 nonpolar (Ala, Val, Phe, and Met), 1
positively charged (Lys), and 2 negatively charged (Asp, Glu) amino
acids were attached to the B-ring of quercetin via a carbamate link-
age (Scheme 1).
Syntheses of amino acid carbamate derivatives of quercetin
from partly protected quercetin and amino acid ester isocyanates
was previously reported by Wu et al.22 In this study, instead of
using amino acid ester isocyantes, we prepared a series of querce-
tin–amino acid conjugates through conversion of amino acids (1,
Scheme 1) to the corresponding activated urethanes23 followed
by alcoholysis with quercetin. The reaction smoothly underwent
to provide the quercetin–amino acid conjugates (2 and 3, Scheme
1) as inseparable mixtures of two regioisomers in an approxi-
mately 3:1 ratio. Deprotection of the tert-butyl group by treatment
with TFA provided the desired quercetin prodrugs (4 and 5,
Scheme 1). Biasuto et al. also reported that esterification on the
quercetin B-ring catechol moiety gave inseparable regioisomeric
mixtures of quercetin-30-ester and 40-ester due to the rapid intra-
molecular transesterification reaction.24 Thus, the two isomers
would be expected to have indistinguishable physicochemical
properties. Investigation of the 1H NMR spectra of the final
products revealed that the major product was 30-O-substituted
A regioisomeric mixture of the quercetin-alanine conjugates 4a
and 5a was coupled with H-Glu(OtBu)-OtBuꢀHCl [or H-Asp(OtBu)-
OtBuꢀHCl], and then treated with TFA to provide the corresponding
quercetin–dipeptide conjugates 6h and 7h (6i and 7i) as an insep-
arable mixture in 45% yield.
2.2. Pharmacokinetic properties of the quercetin conjugates
0
2.2.1. Solubility
quercetin, which showed significant down-field shifts of dH6
0
The quercetin prodrugs synthesized in this study showed dra-
matic increases in water solubility compared with the parent drug,
quercetin. The solubilities of the quercetin-amino acid conjugates
in PBS buffer are summarized in Table 1.
(0.3 ppm) and dH2 (0.23 ppm) due to the introduction of the carba-
mate group at the 30-OH (para- and ortho- position to H20 and H60,
respectively, Fig. 2).24 By the same token, the minor product was
assigned to be 40-O-substituted quercetin of which dH5 was
0
Scheme 1. Syntheses of the Q12 analogues. Reagents and conditions: (a) (4-NO2-PhO)2CO, DIPEA, THF/DMF, 0 °C to rt; (b) quercetin, DIPEA, DMF, 0 °C to rt; (c) TFA, CH2Cl2,
0 °C to rt.

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M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
1
Figure 2. H NMR chemical shifts of quercetin, quercetin-30-O-amino acid, and quercetin-40-O-amino acid.
Scheme 2. Syntheses of quercetin–dipeptide conjugates. Reagents and conditions: (a) EDC, H-Asp(OtBu)-OtBuꢀHCl or H-Glu(OtBu)-OtBuꢀHCl, HOBt, DMF; (b) TFA, CH2Cl2.
Table 1
tin-glutamic acid (Qu-E, 4g/5g), quercetin-alanine-glutamic acid
(Qu-AE, 6i/7i), quercetin-aspartic acid (Qu-D, 4e/5e) and querce-
tin-alanine-aspartic acid (Qu-AD, 6h/7h) had two carboxylic acid
groups, of which interaction with water must have increased their
solubilities in water. In contrast, lysine conjugate (Qu-K, 4c/5c)
with the positively charged side chain showed lowest increase in
solubility compared with quercetin.
Solubility of quercetin and quercetin–amino acid conjugates in PBS buffer
Compound
Water solubility (
l
M)
Fold increasea
Qu
50
1290
767
1.0
Qu-A (4a/5a)
Qu-V (4b/5b)
Qu-K (4c/5c)
Qu-F (4d/5d)
Qu-D (4e/5e)
Qu-M (4f/5f)
Qu-E (4g/5g)
Qu-AD (6h/7h)
Qu-AE (6i/7i)
25.8
15.3
6.8
35.3
45.2
13.5
53.0
41.9
52.6
338
1766
2262
675
2649
2093
2628
2.2.2. Stability
Unlike QC12 (t1/2 = 16.9 h)4, no quercetin prodrug synthesized
in this study was hydrolyzed in PBS buffer up to 17 h at 37 °C (data
not shown). Presumably, the amino acid side chains provided addi-
tional stabilities to the synthesized QC12 analogues (4, 5, 6, and 7)
against hydrolysis by blocking the approach of water molecules to
the carbamate linkage. On the other hand, in the presence of
hydrolyzing enzymes drug-amino acid conjugates become easily
hydrolyzed and the short half-life (0.39 h) of QC12 in whole blood4
can also be attributed to the fast enzymatic hydrolysis by various
endogenous hydrolyzing enzymes. In order to estimate the rate
of enzymatic hydrolysis, the QC12 analogues were treated with
whole cell lysates. Thus, MDCK cell was decomposed with lysis
buffer (pH 7.6) to give cell lysate, in which many enzymes were
activated at once to hydrolyze the quercetin prodrugs. In cell ly-
a
Fold increase in water solubility relative to quercetin (Qu).
The fold increase in solubility was as low as 6.8 fold in lysine
conjugate (Qu-K, 4c/5c) and as high as 53.0 fold in glutamic acid
conjugate (Qu-E, 4g/5g). Among the mono-amino acid conjugates,
phenylalanine (Qu-F, 4d/5d), aspartic acid (Qu-D, 4e/5e), and glu-
tamic acid (Qu-E, 4g/5g) showed remarkable increases in solubility
and the dipeptide conjugates such as alanine–aspartic acid (Qu-AD,
6h/7h) and alanine–glutamic acid conjugates (Qu-AE, 6i/7i)
showed 41.9-fold and 52.6-fold increases, respectively. Querce-

M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
1167
Table 2
prodrugs did not show significant increases in cell permeabilities
except aspartic acid [Qu-D (4e/5e)], glutamic acid [Qu-E (4g/5g)]
and alanine–glutamic acid [Qu-AE (6i/7i)] conjugates (Fig. 3). In
particular, the dipeptide Qu-AE (6i/7i) showed remarkable increase
in cell permeability, which indicates that the human peptide trans-
porter hPepT1 might be at work in recognition as well as transport
of the quercetin–dipeptide conjugates.
Half-lifes (t1/2) of quercetin prodrugs in cell lysate
Compound
Half-life (t1/2) (min)
Qu-A (4a/5a)
Qu-V (4b/5b)
Qu-K (4c/5c)
Qu-F (4d/5d)
Qu-D (4e/5e)
Qu-M (4f/5f)
Qu-E (4g/5g)
Qu-AD (6h/7h)
Qu-AE (6i/7i)
30
<30
180
<30
85
90
180
90
3. Conclusion
100
Quercetin has many physiological activities. However, querce-
tin has pharmacokinetic problems such as low solubility in water
and fast metabolism as well as excretion, which results in its low
bioavailability. In this study, we synthesized new quercetin pro-
drugs such as quercetin-amino acid conjugates and quercetin–
dipeptide conjugates to improve pharmacokinetic properties of
quercetin. The quercetin prodrugs showed remarkable increases
in water solubilities (6.8–53.0 fold increase relative to quercetin).
In particular, amino acids such as aspartic acid and glutamic acid
increased the water solubilities of their corresponding quercetin
conjugates Qu-D (4e/5e) and Qu-E (4g/5g) by 45.2 and 53.0 folds,
respectively. Like QC12, quercetin prodrugs were stable in PBS buf-
fer (t1/2 > 17 h) but susceptible in enzymatic hydrolysis in cell ly-
sate which contained various activated hydrolyzing enzymes.
However, quercetin–amino acid conjugates such as Qu-K (4c/5c)
and Qu-E (4g/5g) showed strong resistance against hydrolases to
show significantly extended half-lives (180 min) compared with
that (0.39 h) of QC12.
sate, the quercetin prodrugs were hydrolyzed to quercetin but the
rates of hydrolysis were dependent upon the amino acid attached
to quercetin (Table 2). Amino acids with hydrophobic side chains
such as alanine, valine, and phenylalanine were easily hydrolyzed
from the corresponding conjugates [Qu-A (4a/5a), Qu-V (4b/5b),
and Qu-F (4d/5d)] whereas quercetin conjugates with attached
amino acids such as lysine [Qu-K (4c/5c)], aspartic acid [Qu-D
(4e/5e)], methionine [Qu-M (4f/5f)], and glutamic acid [Qu-E (4g/
5g)] showed extended half-lifes of more than 85 min. It is worth
to note that polar amino acid side chains with more than two
methylene units (lysine and glutamic acid) show strong resistance
against enzymatic hydrolysis and dipeptide conjugates Qu-AD (6h/
7h) and Qu-AE (6i/7i) are not as stable as the corresponding mono-
amino acids Qu-D (4e/5e) and Qu-E (4g/5g).
2.2.3. Cell permeability
The quercetin prodrugs Qu-D (4e/5e), Qu-E (4g/5g) and Qu-AE
(6i/7i) also showed significantly increased intestinal permeability
at MDCK cell in comparison with quercetin.
Taken together, through synthesis and In vitro pharmacokinetic
analyses of quercetin–amino acid conjugates, we have identified a
novel quercetin-amino acid conjugate, Qu-E (4g/5g), of which
remarkably increased water solubility, stability, and cell perme-
ability compared with quercetin and QC12 (Table 3) warrants fur-
ther development as a quercetin prodrug.
The passive transports of quercetin and its prodrugs were esti-
mated by non-cell-based permeability assay, first. An artificial cell
membrane made of hexadecane, however, did not allow transport
of neither quercetin nor its amino acid conjugates (data not
shown).
For cell-based permeability assay, the MDCK cell is used which
is a common model for studying cell growth regulation, metabo-
lism, and transport mechanisms in distal renal epithelia.29–34 Like
Caco-2 cells, MDCK cells have been shown to differentiate into
columnar epithelium and to form tight junctions when cultured
on semipermeable membranes.35,36 Before the transepithelial
transport experiments, the MDCK monolayers were checked by
measuring TEER (transepithelial electrical resistance). For the
transport experiments, only monolayers displaying TEER values
4. Experimental
4.1. Materials and general methods
Quercetin (30,40,3,5,7-pentahydroxyflavone)-dihydrate, 1-hydro-
xybenzotriazole (HOBt), trifluoroacetic acid (TFA), dimethyl sulfox-
991–1055
X
were chosen.37,38 Compared with quercetin, quercetin
Figure 3. MDCK cell permeabilities of quercetin prodrugs relative to quercetin.

1168
M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
Table 3
(400 MHz, acetone-d6) d (ppm) 12.48 (s, 1H), 8.00–8.24 (m, 2H),
7.12 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 6.28 (s, 1H), 4.03–4.12 (m,
1H), 2.20–2.28 (m, 1H), 1.50 (s, 9H), 1.04 (s, 6H); For 3b, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.12 (s, 1H), 7.89 (d, J = 1.6 Hz,
1H), 7.74 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 6.52(s, 1H),
6.28(s, 1H), 4.03–4.12 (m, 1H), 2.20–2.28 (m, 1H), 1.50 (s, 9H),
1.04 (s, 6H).
Water solubility, stability, and permeability of quercetin (Qu), QC12, and Qu-E (4g/5g)
Compound
Qu
50
QC12
NDa
Qu-E (4g/5g)
Water solubility (
Stability (t1/2, h)
PBS buffer
Cell lysate
Relative permeability (MDCK cell)
lM)
2649
—
—
1.0
16.9b
0.39b
NDa
>17
3.0
5.2
a
4.2.1.3. 40-O-CO-[Lys(Boc)-OtBu]-quercetin (2c) and 30-O-CO-
[Lys(Boc)-OtBu]-quercetin (3c) (50% yield). LC/MS (ESI) m/z
Found: 630.4 [M+H]+; Calcd for C31H38N2O12: 630.24; For 2c, 1H
NMR (400 MHz, acetone-d6) d (ppm) 12.14 (s, 1H), 8.03–8.10 (m,
2H), 7.12 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H), 6.27 (s, 1H), 4.16 (d,
J = 4.9 Hz, 1H), 3.12 (d, J = 4.8 Hz, 2H), 2.88 (s, 4H), 1.79–1.93 (m,
2H), 1.49 (s, 9H), 1.41 (s, 9H); For 3c, 1H NMR (400 MHz, ace-
tone-d6) d (ppm) 12.09 (s, 1H), 7.89 (s, 1H), 7.76 (d, J = 8.5 Hz,
1H), 7.30 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H), 6.27 (s, 1H), 4.16 (d,
J = 4.9 Hz, 1H), 3.12 (d, J = 4.8 Hz, 2H), 2.88 (s, 4H), 1.79–1.93 (m,
2H), 1.49 (s, 9H), 1.41 (s, 9H).
Not determined.
Taken from Ref. 3.
b
ide (DMSO), and RIPA buffer were purchased from Sigma–Aldrich.
Various amino acids were obtained from Bachem. Bis(4-nitro-
phenyl) carbonate and 1-ethyl-3-(30-dimethylaminopropyl)carbo-
diimide (EDC) were purchased from TCI Int. Phenylmethane-
sulfonyl fluoride (PMSF) was purchased from Fluka. Dulbecco0s
Modified Eagle Medium (DMEM), penicillin, streptomycin and fetal
bovine serum (FBS) were purchased from Invitrogen. The 96-well
MultiScreen Caco-2 plate (PSHT004S5) and MillicellÒ-ERS system
ohm meter (MERS 000 01) were purchased from Millipore Corp.
The STX-100M electrode was purchased from World Precision
Instruments, Sarasota, FL. The 100 mm cell culture plate was pur-
chased from Dishes BiousingTM.
Nuclear magnetic resonance spectra were recorded on a Bruker
400 AMX spectrometer (Karlsruhe, Germany) at 400 MHz for 1H
NMR and 100 MHz for 13C NMR with tetramethylsilane as the
internal standard. Chemical shifts (d) are reported as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or br s (broad sin-
glet). Coupling constants are reported in hertz. The chemical shifts
are reported as parts per million (d) relative to the solvent peak.
TLC was performed on silica gel-60 F254 purchased from Merck.
Column chromatography was performed using either silica gel-60
(220–440 mesh) for flash chromatography. Mass spectrometric
data (MS) were obtained by electronspray ionization (ESI).
4.2.1.4. 40-O-CO-(Phe–OtBu)-quercetin (2d) and 30-O-CO-(Phe–
OtBu)-quercetin (3d) (55% yield). LC/MS (ESI) m/z Found: 549.3
[M+H]+; Calcd for C29H27NO10: 549.16; For 2d, 1H NMR (400 MHz,
acetone-d6) d (ppm) 12.14 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.34–7.39
(m, 5H), 7.11 (d, J = 8.1 Hz. 1H), 6.56 (s, 1H), 6.27 (s, 1H), 4.41–4.45
(m, 1H), 3.12–3.22 (m, 2H), 1.46 (s, 9H); For 3d, 1H NMR (400 MHz,
acetone-d6) d (ppm) 12.09 (s, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.72 (d,
J = 8.6 Hz, 1H), 7.10–7.29 (m, 6H), 6.56 (s, 1H), 6.27 (s, 1H), 4.41–
4.45 (m, 1H), 3.12–3.22 (m, 2H), 1.46 (s, 9H).
4.2.1.5. 40-O-CO-[Asp(OtBu)-OtBu]-quercetin (2e) and 30-O-CO-
[Ala(OtBu)-OtBu]-quercetin (3e) (60% yield). LC/MS (ESI) m/z
Found: 573.3 [M+H]+; Calcd for C28H31NO12: 573.18; For 2e, 1H
NMR (400 MHz, acetone-d6) d (ppm) 12.14 (s, 1H), 8.08 (d,
J = 1.7 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H),
6.54 (s, 1H), 6.28 (s, 1H), 4.49–4.54 (t, J = 2.2 Hz, 1H), 2.04–2.06
(m, 2H), 1.48 (s, 6H); For 3e, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.09 (s, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.76 (d, J = 8.6 Hz,
1H), 7.29 (d, J = 8.6 Hz, 1H), 6.54 (s, 1H), 6.28 (s, 1H), 4.49–4.54
(t, J = 2.2 Hz, 1H), 2.04–2.06 (m, 2H), 1.48 (s, 6H).
4.2. Syntheses of quercetin conjugates
4.2.1. tert-Butyl-amino acid carbamate derivatives of quercetin
(2 and 3)
Amino acids [H-Ala-OtBuꢀHCl (1a), H-Val-OtBuꢀHCl (1b), H-
Lys(Boc)-OtBuꢀHCl (1c), H-Phe-OtBuꢀHCl (1d), H-Asp(OtBu)-Ot-
BuꢀHCl (1e), H-Met-OtBuꢀHCl (1f) and H-Glu(OtBu)-OtBuꢀHCl
(1g)] (3 mmol) were dissolved in anhydrous THF (20 mL). Bis(4-
nitrophenyl) carbonate (3 mmol) and N,N-diisopropylethylamine
(6 mmol) were added to this solution, and the reaction mixture
was stirred at room temperature. After 12 h, quercetin (3 mmol)
was added, and the mixture was stirred for 12 h at room temper-
ature. The resulting solution was concentrated under reduced pres-
sure, and the residue was purified by column chromatography on
silica gel (dichloromethane/acetone = 6:1) to afford inseparable
mixtures of the regioisomers (2 and 3) as yellow solids.
4.2.1.6. 40-O-CO-(Met-OtBu)-quercetin (2f) and 30-O-CO-(Met-
OtBu)-quercetin (3f) (55% yield). LC/MS (ESI) m/z Found: 533.2
[M+H]+; Calcd for C25H27NO10S: 533.14; For 2f, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.08 (s, 1H), 7.99–8.10 (m, 2H),
7.12 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 6.27 (s, 1H), 4.33–4.37 (m,
1H), 3.03–3.12 (m, 2H), 2.63–2.72 (m, 2H), 2.12 (s, 3H), 1.48 (s,
9H); For 3f, 1H NMR (400 MHz, acetone-d6) d (ppm) 12.05(s, 1H),
7.90 (d, J = 1.9 Hz, 1H), 7.75 (dd, J = 6.6, 5.0 Hz, 1H), 7.29 (d,
J = 8.5 Hz, 1H), 6.53 (s, 1H), 6.27 (s, 1H), 4.33–4.37 (m, 1H), 3.03–
3.12 (m, 2H), 2.63–2.72 (m, 2H), 2.12 (s, 3H), 1.48 (s, 9H).
4.2.1.7. 40-O-CO-[Glu(OtBu)-OtBu]-quercetin (2g) and 30-O-CO-
[Glu(OtBu)-OtBu]-quercetin (3g) (60% yield). LC/MS (ESI) m/z
Found: 587.3 [M+H]+; Calcd for C29H33NO12: 587.20; For 2g, 1H
NMR (400 MHz, acetone-d6) d (ppm) 12.14 (s, 1H), 8.00–8.10 (m,
2H), 7.12 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H), 6.28 (s, 1H), 4.38–4.44
(m, 2H), 2.35–2.59 (m, 4H), 1.48 (s, 6H); For 3g, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.08 (s, 1H), 7.94 (d, J = 1.6 Hz,
1H), 7.80 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H),
6.28 (s, 1H), 4.38–4.44 (m, 2H), 2.35–2.59 (m, 4H), 1.48 (s, 6H).
4.2.1.1. 40-O-CO-(Ala-OtBu)-quercetin (2a) and 30-O-CO-(Ala-
OtBu)-quercetin (3a) (60% yield). LC/MS (ESI) m/z Found: 473.3
[M+H]+; Calcd for C23H23NO10
:
473.13; For 2a, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.14 (s, 1H), 8.02–8.16 (m, 2H),
7.12 (d, J = 8.7 Hz, 1H), 6.54 (s, 1H), 6.27 (s, 1H), 4.16–4.23 (m,
1H), 1.46 (s, 12H); For 3a, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.09 (s, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.76 (dd, J = 6.8,
1.8 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 6.27 (s, 1H),
4.16–4.23 (m, 1H), 1.46 (s, 12H).
4.2.2. Amino acid carbamate derivatives of quercetin (4 and 5)
The tert-butyl-amino acid quercetin carbamates (2 and 3, 2.2
mmol) obtained above were dissolved in anhydrous CH2Cl2 (5
mL), and the resulting solution was treated with trifluoroacetic
4.2.1.2. 40-O-CO-(Val-OtBu)-quercetin (2b) and 30-O-CO-(Val-
OtBu)-quercetin (3b) (55% yield). LC/MS (ESI) m/z Found: 501.3
[M+H]+; Calcd for C25H27NO10
:
501.16; For 2b, 1H NMR

M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
1169
acid (2 mL) at 0 °C. The reaction mixture was stirred for 4 h at room
temperature, and concentrated under reduced pressure. The resi-
due was recrystallized from a mixture of acetone (1 mL) and
J = 8.7 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 6.54 (s, 1H), 6.27 (s, 1H),
4.52–4.56(m, 1H), 2.63–2.72 (m, 2H), 2.02–2.05 (m, 2H), 1.69 (s, 3H).
CH2Cl2 (10 mL). The mixture was filtered through a Buchner or
4.2.2.7. 40-O-CO-Glu-quercetin (4g) and 30-O-CO-Glu-quercetin
(5g) (88% yield). LC/MS (ESI) m/z Found: 476.2 [M+H]+; Calcd for
C21H17NO12: 475.08; For 4g, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.14 (s, 1H), 8.00–8.10 (m, 2H), 7.12 (d, J = 8.6 Hz, 1H),
6.56 (s, 1H), 6.28 (s, 1H), 4.38–4.44 (m, 2H), 2.35–2.59 (m, 4H);
For 5g, 1H NMR (400 MHz, acetone-d6) d (ppm) 12.08 (s, 1H),
7.94 (d, J = 1.6 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.6 Hz,
1H), 6.56 (s, 1H), 6.28 (s, 1H), 4.38–4.44 (m, 2H), 2.35–2.59 (m, 4H).
}
Hirsch funnel, and the filter cake was washed with CH2Cl2 to give
inseparable mixtures of the regioisomers (4 and 5) as yellow solids.
4.2.2.1. 40-O-CO-Ala-quercetin (4a) and 30-O-CO-Ala-quercetin
(5a) (95% yield). LC/MS (ESI) m/z Found: 418.2 [M+H]+; Calcd for
C19H15NO10: 417.07; For 4a, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.14 (s, 1H), 8.00–8.06 (m, 2H), 7.11 (d, J = 8.4 Hz, 1H),
6.54 (s, 1H), 6.27 (s,1H), 4.33–4.38 (m, 1H), 1.51 (s, 3H); For 5a,
1H NMR (400 MHz, acetone-d6) d (ppm) 12.09 (s, 1H), 7.89 (s,
1H), 7.74 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.54 (s, 1H),
6.27 (s, 1H), 4.16–4.23 (m, 1H), 1.46 (s, 12H).
4.2.3. Dipeptide carbamate derivatives of quercetin (6 and 7)
Quercetin–alanine conjugate (4a and 5a) (2 mmol) was dis-
solved in dimethylformamide (DMF) (4 mL), and 1-hydroxybenzo-
triazole (HOBT) (4 mmol) and H-Glu(OtBu)-OtBuꢀHCl (2 mmol) or
H-Asp(OtBu)-OtBuꢀHCl (2 mmol) was added to the solution. And
4.2.2.2. 40-O-CO-Val-quercetin (4b) and 30-O-CO-Val-quercetin
(5b) (89% yield). LC/MS (ESI) m/z Found: 446.2 [M+H]+; Calcd for
C21H19NO10: 445.10; For 4b, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.48 (s, 1H), 8.00–8.24 (m, 2H), 7.12 (d, J = 8.6 Hz, 1H),
6.53 (s, 1H), 6.28 (s, 1H), 4.03–4.12 (m, 1H), 2.20–2.31 (m, 1H),
1.04 (s, 6H); For 5b, 1H NMR (400 MHz, acetone-d6) d (ppm)
12.12 (s, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.31
(d, J = 8.6 Hz, 1H), 6.53(s, 1H), 6.28(s, 1H), 4.03–4.12 (m, 1H),
2.20–2.31 (m, 1H), 1.04 (s, 6H).
then
1-ethyl-3-(30-dimethylaminopropyl)carbodiimide
(EDC)
(2 mmol) was added and the solution was stirred for 3 days at
0 °C. The resulting solution was concentrated under reduced pres-
sure, and the residue was filtered through a short silica gel column
washing with a mixture of CH2Cl2 and acetone (6:1). After concen-
tration of the filtrate under reduced pressure, the residue was used
for the next step without further purification. The tert-butyl-dipep-
tide quercetin carbamates obtained above (1.8 mmol) were dis-
solved in CH2Cl2 (5 mL). To this solution, trifluoroacetic acid
(2 mL) was added at 0 °C, and the solution was stirred for 4 h at
room temperature. The resulting solution was concentrated under
reduced pressure, and the residue was recrystallized from a mix-
ture of acetone (1 mL) and CH2Cl2 (10 mL). The mixture was fil-
4.2.2.3. 40-O-CO-Lys-quercetin (4c) and 30-O-CO-Lys-quercetin
(5c) (90% yield). LC/MS (ESI) m/z Found: 475.3 [M+H]+; Calcd for
C22H22N2O10: 474.13; For 4c, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.14 (s, 1H), 8.00–8.03 (m, 2H), 7.15 (d, J = 9.1 Hz, 1H),
6.58 (d, J = 1.6 Hz, 1H), 6.26 (d, J = 1.8 Hz, 1H), 4.34 (d, J = 4.1 Hz,
1H), 3.77–3.90 (m, 2H), 1.84–2.00 (m, 4H), 1.67 (q, J = 7.06 Hz,
2H); For 5c, 1H NMR (400 MHz, acetone-d6) d (ppm) 12.09 (s,
1H), 7.88 (s, 1H), 7.73 (dd, J = 6.8, 1.8 Hz, 1H), 7.27 (d, J = 8.5 Hz,
1H), 6.56 (d, J = 1.8 Hz, 1H), 6.29 (d, J = 1.8 Hz, 1H), 4.34 (d,
J = 4.1 Hz, 1H), 3.77–3.90 (m, 2H), 1.84–2.00 (m, 4H), 1.67 (q,
J = 7.06 Hz, 2H).
}
tered through a Buchner or Hirsch funnel, and the filter cake was
washed with CH2Cl2 to give mixtures of the regioisomers (6 and
7) as yellow solids.
4.2.3.1. 40-O-CO-Ala-Asp-quercetin (6h) and 30-O-CO-Ala-Asp-
quercetin (7h) (90% yield). LC/MS (ESI) m/z Found: 547.3
[M+H]+; Calcd for C24H22N2O13
:
546.11; For 6h, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.16 (s, 1H), 7.96–8.00 (m, 2H),
7.11 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 6.27 (s, 1H), 4.50–4.58 (m,
1H), 4.30–4.36 (m, 1H), 2.40–2.50 (m, 4H), 1.46 (s, 3H); For 7h,
1H NMR (400 MHz, acetone-d6) d (ppm) 12.09 (s, 1H), 7.88 (d,
J = 1.6 Hz, 2H), 7.73 (d, J = 8.7 Hz. 1H), 7.31 (d, J = 8.7 Hz, 1H),
6.56 (s, 1H), 6.27 (s, 1H), 4.50–4.58 (m, 1H), 4.30–4.36 (m, 1H),
2.40–2.50 (m, 4H), 1.46 (s, 3H).
4.2.2.4. 40-O-CO-Phe-quercetin (4d) and 30-O-CO-Phe-quercetin
(5d) (90% yield). LC/MS (ESI) m/z Found: 494.3 [M+H]+; Calcd for
C25H19NO10: 493.10; For 4d, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.14 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.34–7.37 (m, 5H),
7.11 (d, J = 8.1 Hz, 1H), 6.57 (s, 1H), 6.28 (s, 1H), 4.51–4.59 (m,
1H), 3.12–3.33 (m, 2H); For 5d, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.09 (s, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.72 (d, J = 8.6 Hz,
1H), 7.10–7.29 (m, 6H), 6.57 (s, 1H), 6.28 (s, 1H), 4.51–4.59 (m,
1H), 3.12–3.33 (m, 2H).
4.2.3.2. 40-O-CO-Ala-Glu-quercetin (6i) and 30-O-CO-Met-quer-
cetin (7i) (92% yield). LC/MS (ESI) m/z Found: 533.3 [M+H]+;
Calcd for C23H20N2O13: 532.10; For 6i, 1H NMR (400 MHz, ace-
tone-d6) d (ppm) 12.14 (s, 1H), 8.02–8.06 (m, 2H), 7.11 (d,
J = 8.6 Hz, 1H), 6.55 (s, 1H), 6.28 (s, 1H), 4.33–4.42 (m, 2H), 2.35–
2.61 (m, 2H); For 7i, 1H NMR (400 MHz, acetone-d6) d (ppm)
12.08 (s, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.35
(d, J = 8.6 Hz, 1H), 6.55 (s, 1H), 6.28 (s, 1H), 4.33–4.42 (m, 2H),
2.35–2.61 (m, 2H).
4.2.2.5. 40-O-CO-Asp-quercetin (4e) and 30-O-CO-Asp-quercetin
(5e) (92% yield). LC/MS (ESI) m/z Found: 462.2 [M+H]+; Calcd for
C20H15NO12: 461.06; For 4e, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.14 (s, 1H), 8.08 (d, J = 1.7 Hz, 1H), 8.04 (d, J = 8.8 Hz,
1H), 7.13 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.28 (s, 1H), 4.68–4.69
(t, J = 2.2 Hz, 1H), 3.00 (d, J = 5.4 Hz, 2H); For 5e, 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.09 (s, 1H), 7.90 (d, J = 1.9 Hz,
1H), 7.77 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 6.57 (s, 1H),
6.28 (s, 1H), 4.68–4.69 (t, J = 2.2 Hz, 1H), 3.00 (d, J = 5.4 Hz, 2H).
5. Solubility test
Stock solution of the quercetin prodrugs were prepared at
10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM and 250 mM
in 1% DMSO, and then serially diluted in 99% phosphate buffered
saline (PBS, pH 7.4) buffer. As a result, the diluted compounds have
4.2.2.6. 40-O-CO-Met-quercetin (4f) and 30-O-CO-Met-quercetin
(5f) (90% yield). LC/MS (ESI) m/z Found: 478.3 [M+H]+; Calcd for
C21H19NO10S: 477.07; For 4f, 1H NMR (400 MHz, acetone-d6) d
(ppm) 12.08 (s, 1H), 7.98–8.10 (m, 2H), 7.13 (d, J = 8.5 Hz, 1H),
6.54 (s, 1H), 6.27 (s, 1H), 4.52–4.56 (m, 1H), 2.63–2.72 (m, 2H),
2.02–2.05 (m, 2H), 1.69 (s, 3H); For 5f, 1H NMR (400 MHz, ace-
tone-d6) d (ppm) 12.05 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.76 (d,
a
final concentration of 100
1500 M, 2000 M and 2500
pound in each 96-well plate was set to be 250
was measured by the NEPHELOstar laser based microplate nephe-
l
M, 250
M. The volume of the test com-
l, and the solubility
lM, 500 lM, 1000 lM,
l
l
l
l

1170
M. K. Kim et al. / Bioorg. Med. Chem. 17 (2009) 1164–1171
lometer which checks the solubility of compounds by measuring
forward light scatter in microplates. All raw data were processed
using the BMG LABTECH NEPHELOstar Galaxy Evaluation software.
ated, the MultiScreen Caco-2 plate was removed from the incuba-
tor and the electrical resistance for each well was determined. The
TEER values across the cell monolayers were measured using a Mil-
licell-ERS voltohmmeter. The electrical resistance of MDCK cell
monolayers at the densities chosen ranged from 991 to 1055
X.
6. Stability test
And then, the monolayer formed by exchanging with the sterile
PBS buffer, pH 7.4, in the filter plate was washed three times the
volume of the culture medium. To determine the rate of drug
Lysis buffer [RIPA buffer with PMSF (1 mM)] (60 ll, 10 mM)
were added to 2.5 ꢁ 104 cells/well MDCK cells. This could break
transport in the apical to basolateral direction, 75
tin prodrug solution, diluted with PBS buffer, pH 7.4, to adjust drug
concentration ranging from 0.05 M to 10 M, was added to the
filter well (donor, apical plate). The transport analysis plate (accep-
tor, basolateral plate) was filled with 250 L of PBS buffer. On the
other hand, to determine transport rates from the basolateral to
apical direction, 250 L of the quercetin prodrugs was added to
the transport analysis plate wells and filled the filter wells (apical
compartment) were filled with 75 L of the PBS buffer. The 96-well
lL of the querce-
the cell membrane of the cells and release the cytoplasmic en-
zymes. PBS buffer (180
ium to the optimum pH value (pH 7) for the enzymes. The
quercetin prodrugs (60 M) were added into the reaction mixture
and incubated at 37 °C. At different time points (0, 30, 60, 90, 120,
150, 180, 210, 240, 300, and 360 min), an aliquot (300 l) of the
ll) was added which neutralized the med-
l
l
l
l
l
l
reaction mixture was taken out, filtered, and injected into the HPLC
equipped with a C-18 reverse phase column; flow rate, 1 mL/min;
detection, UV 340 nm; mobile phase, 0–8 min (10% aqueous aceto-
nitrile and 0.1% formic acid), 8–12 min (40% aqueous acetonitrile
and 0.1% formic acid), 12–15 min (90% aqueous acetonitrile and
0.1% formic acid) and 15–20 min (10% aqueous acetonitrile and
0.1% formic acid).
l
MultiScreen Caco-2 plates were incubated at 37 °C, 5% CO2, 95%
relative humidity and typical incubation times were 1–2 h. After
incubation, UV–vis absorption of the 96-well filter plates and 96-
well transport analysis plates were measured by UV–vis
spectrophotometer.
7. Cell permeability assay
Acknowledgments
7.1. Non-cell-based permeability
This work was supported by grant KRF-2007-313-C00476 from
the Korea Research Foundation, Republic of Korea (MOEHRD, Basic
Research Promotion Fund) and by grants from Biogreen 21 (Korea
Ministry of Agriculture and Forestry), and the second Brain Korea
21 (Korea Ministry of Education). KSP is supported by the second
Brain Korea 21.
For non-cell-based permeability assay, a 5% solution (v/v) of
hexadecane in hexane was prepared. This mixture was pipetted
15l006C each in the 96-well MultiScreen permeability donor
plate. The plates were allowed to dry for 1 h in a fume hood to en-
sure complete evaporation of the hexane resulting in a uniform
layer of hexadecane, and added 300 ll of 1% DMSO in PBS buffer
References and notes
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TEER (transepithelial electrical resistance) values were measured
using a Millicell-ERS voltohmmeter. Inserts with TEER values
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>25 k
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lL of cell dilu-
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