Increased bioavailability of tricin-amino acid derivatives via a prodrug approach

Article abstract of DOI:10.1021/jm1015457

Tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone) has demonstrated diverse biological activities. This compound has a high anti-human cytomegalovirus (HCMV) activity; however, its oral availability is low. To improve its bioavailability, we synthesized tricin-amino acid derivatives as prodrugs and investigated their cell permeability, stability in vitro, and oral availability in vivo. The results demonstrated that the tricin-alanine-glutamic acid conjugate exhibited enhanced permeability, stability in MDCK cells, and excellent bioavailability after oral administration in Crl:CD (SD) male rats. Tricin-alanine-glutamic acid conjugate is a potential new anti-HCMV drug.

Full text of DOI:10.1021/jm1015457

ARTICLE
pubs.acs.org/jmc
Increased Bioavailability of Tricin-Amino Acid Derivatives via a
Prodrug Approach
||
Masayuki Ninomiya,† Kaori Tanaka,§, Yuzo Tsuchida,^ Yoshinori Muto,‡ Mamoru Koketsu,*,† and
||
Kunitomo Watanabe*,§,
†Department of Materials Science and Technology, Faculty of Engineering, ‡Department of Functional Bioscience, School of Medicine,
§Division of Anaerobe Research, Life Science Research Center, and United Graduate School of Drug Discovery and Medical
Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
^Hououdou Co., Ltd., 4-3-2 Ebara, Shinagawa, Tokyo 142-0063 Japan
ABSTRACT: Tricin (40,5,7-trihydroxy-30,50-dimethoxyflavone)
has demonstrated diverse biological activities. This compound
has a high anti-human cytomegalovirus (HCMV) activity; how-
ever, its oral availability is low. To improve its bioavailability, we
synthesized tricin-amino acid derivatives as prodrugs and
investigated their cell permeability, stability in vitro, and oral
availability in vivo. The results demonstrated that the tricin-
alanine-glutamic acid conjugate exhibited enhanced permeabil-
ity, stability in MDCK cells, and excellent bioavailability after oral administration in Crl:CD (SD) male rats. Tricin-alanine-
glutamic acid conjugate is a potential new anti-HCMV drug.
’ INTRODUCTION
growth of the host cells. Thus, tricin was determined to be
sufficiently safe for clinical development as a candidate anti-
HCMV drug.
Flavonoids constitute a large family of natural products that
can be found in nearly all plant species. At present, flavonoids
comprise more than 6500 natural compounds.1 The study of
flavonoids is becoming increasingly interesting because of their
important metabolic roles in plants and animals and their potent
and diversified biological activities.2 In particular, flavones, a
subclass of flavonoids, are of interest due to having biological
activities such as inhibition of retroviral reverse transcriptases,3,4
protein-tyrosine kinases,5,6 and serine-threonine kinases.5
They possess anticancer7,8 and chemopreventive effects8,9 and
inhibit HIV-induced syncytium formation.10
In this study, we first prepared a large quantity of tricin and
evaluated its bioavailability after oral administration in Crl:CD
(SD) rats in vivo; however, the plasma tricin concentration was
quite low, as is typically the case for other flavonoids. To date, the
clinical use of flavonoids has been limited by their poor bioavail-
ability. The bioavailability of tricin is similar to that of quercetin,
which also has a wide range of biological activities.24 To over-
come this limitation, a variety of modifications of quercetin, such
as glycosylation,25 acylation,26 and liposomalization,27 have been
performed. Similarly, Cai et al. recently attempted to improve the
oral bioavailability of tricin via a methylation approach.28 In
addition, Kim et al. reported that quercetin-amino acid deriva-
tives demonstrate favorable cell permeability in vitro through
interaction with the peptide transporter PepT1.29 In the present
study, we endeavored to improve the oral bioavailability of tricin
via a prodrug approach. To that end, we synthesized several
amino acid derivatives of tricin as prodrugs and evaluated the
relative cell permeabilities and half-lives of the tricin-amino acid
derivatives in Madin-Darby canine kidney (MDCK) cells in
vitro and associated plasma tricin concentrations in male rats in
vivo. The results demonstrated that the tricin-alanine-glutamic
acid conjugate (T-Ala-Glu) exhibited excellent bioavailability
after oral administration, which was clearly confirmed by an
increase in cellular permeability.
The flavone tricin (40,5,7-trihydroxy-30,50-dimethoxyflavone)11
occurs in its glycoside form in Poaceae (formerly Gramineae)
plants including rice, oats, barley, and wheat.12,13 This compound
has previously been reported to possess antioxidant activity,14
assessed using the 2,20-azobis(2,4-dimethylvaleronitrile) (AMVN)-
initiated oxidation assay system with methyl linolate.15 It has
been reported that tricin shows antiviral,16 anti-inflammatory,17
antihistamic,18 and anticancer activities.19 Its role in cancer
prevention is also an active area of research.20,21
Recently, our phytochemical research led to the isolation of
tricin from a hot water extract of Sasa albo-marginata leaves. The
isolated tricin showed anti-human cytomegalovirus (HCMV) acti-
vity in studies of the replication of HCMV in MRC-5 cells.22
Tricin exhibited a distinct anti-HCMV effect with an EC50 of
0.17 μg mL-1; in contrast, ganciclovir (GCV), which is an anti-
HCMV drug in the marketplace, had an EC50 of 1.03 μg mL-1 23
.
This result demonstrates that the antiviral activity of tricin is
stronger than that of GCV. Tricin greatly reduced produc-
tion of HCMV virions in MRC-5 cells but did not inhibit
Received: December 3, 2010
Published: February 14, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Tricin 4a
a Reagents and conditions: (a) LiHMDS, THF, -78 ꢀC f room temperature, 3 days. (b) 0.5% H2SO4 in AcOH, 100 ꢀC, overnight.
’ RESULTS AND DISCUSSION
extensively metabolized in the liver and excreted via bile.37 It has
also been reported that liver microsomes from immature and adult
female rats catalyzed glucuronidation of estrone and estradiol
more rapidly than did liver microsomes from age-matched male
rats.38 Thus, the differing results for the male and female groups
shown in Figure 1 may be due to immediate metabolism of tricin,
similar to estrogens, in the female groups but not in the male
groups. The results demonstrated that tricin was not orally avail-
able in its native form. Therefore, modifications were necessary
to improve tricin bioavailability.
Preparation of Tricin. Previously, we isolated tricin from
S. albo-marginata and confirmed its anti-HCMV activity.22,30 In
this study, we used a chemical synthesis approach because
tricin was required in gram quantities. We prepared tricin
through a condensation reaction of methyl benzoate 1 with
acetophenone 2, followed by acid cyclodehydration.31 The
required methyl 4-O-tert-butyldimethylsilyl-3,5-dimethoxy-
benzoate 1 and 20,40-O-bis(tert-butyldimethylsilyl)-60-hydro-
xyacetophenone 2 were prepared by protection of suitable
starting materials with tert-butyldimethylsilyl chloride in tetra-
hydrofuran (THF) in the presence of N,N-diisopropylethyla-
mine (DIPEA). The condensation reaction of 1 with 8 equiv of
lithium bis(trimethylsilyl)amide (LiHMDS) and 1.5 equiv of 2
in THF at -78 ꢀC raised to room temperature over 3 days gave
intermediate 3 as a mixture of tautomers. These were subjected
to acid cyclodehydration and deprotection with 0.5% H2SO4 in
acetic acid at 100 ꢀC overnight. These reaction conditions
afforded the corresponding tricin 4 at an overall yield of 68%
(105.0 g). The purity of the tricin was checked using reverse-
phase high-performance liquid chromatography (RP-HPLC)
and was confirmed to be 99% (Scheme 1).
Synthesis of Tricin Prodrugs. A significant problem in tricin
oral delivery is its low bioavailability after oral administration, as
demonstrated by our data. As discussed above, poor absorption
and rapid depredation may contribute to this problem. There-
fore, it is necessary to increase the absorption rate of tricin as
well to decrease the rate of metabolism. Previously, QC12
[30-(N-carboxymethyl)carbamoyl-3,40,5,7-tetra-hydroxyflavone;
quercetin 30-glycine conjugate], a quercetin prodrug, had been
designed and evaluated for preclinical and clinical pharmacoki-
netic properties.39 Several quercetin-amino acid derivatives
were prepared, and their solubility, stability, and permeability
in vitro were estimated.29 The results of these studies suggested
that conjugation with amino acids was the most promising
approach. To identify a tricin prodrug with improved drug
availability, we synthesized amino acid derivatives from tricin.
A series of tricin-amino acid conjugates were synthesized. Six
amino acids, including four neutral amino acids (glycine, Gly;
alanine, Ala; valine, Val; and phenylalanine, Phe) and two acidic
amino acids (aspartic acid, Asp; and glutamic acid, Glu), were
attached to the 40-position of the tricin B ring. First, treatment of
the amino acid tert-butyl esters with bis(4-nitrophenyl)carbonate
was carried out in the presence of DIPEA for 12 h, and then, tricin
and the additional amine were added to the reaction mixture. The
reaction was carried out successfully at room temperature, and
the desired products 6a-f were obtained after the usual workup
of the reaction mixtures. The tert-butyl groups of the products
were deprotected with trifluoroacetic acid (TFA) to yield the
tricin-amino acid conjugates 7a-f (Scheme 2).
Plasma Tricin Concentration after Oral Administration in
Rats. Tricin was orally administered to male and female Crl:CD
(SD) rats at doses of 100, 300, and 1000 mg kg-1. Blood was col-
lected 0, 0.5, 2, and 4 h after administration of tricin. Quantitation
of tricin in plasma was conducted by RP-HPLC.32
For male rats, a small amount of tricin was detected in the
plasma 0.5, 2, and 4 h after tricin administration (Figure 1). In the
300 and 1000 mg kg-1 groups, the plasma tricin concentrations
after 2 and 4 h were nearly the same. The results for female rats
were different from those for male rats. Inthe 100 and 300 mgkg-1
groups of females, tricin was not detected in the plasma after 0.5,
2, or 4 h. In addition, in the 1000 mg kg-1 group, tricin was
detected in the plasma after administration but gradually de-
creased after peaking at 0.5 h. Collectively, our in vivo measure-
ments indicated that plasma tricin concentrations at all dosages
in both males and females were quite low, less than 60 ng mL-1
.
In addition, we synthesized the tricin-dipeptide conjugates
T-Ala-Asp and T-Ala-Glu from 7b using the peptide-coupling
reagent water-soluble carbodiimide (WSC) and 1-hydroxyben-
zotriazole (HOBt).40 Deprotections were carried out using TFA
To date, tricin has been investigated as a putative cancer
chemopreventive agent, and Cai et al. performed an experiment
in which it was administered orally to C57BL/6J mice.33 They
also reported low concentrations of orally administered tricin in
the mouse plasma. It is generally assumed that tricin is poorly
absorbed from the gastrointestinal tract (GIT) in its native form.
A substantial portion of tricin is metabolized by UDP-glucurono-
syltransferases34 in the gut mucosal cells, and approximately half
of the glucuronidates are excreted back into the gut lumen, mainly
through the multidrug resistance protein multidrug resistance
protein 2 (MRP2) pump.35,36 The unmetabolized tricin is then
(Scheme 3). Structures were elucidated through IR, 1H and 13
C
NMR, correlation spectroscopy, heteronuclear multiple quan-
tum coherence, heteronuclear multiple bond correlation, MS,
and elemental analysis.
Cell Permeability. We used MDCK cells to investigate trans-
epithelial transport of the synthesized tricin prodrugs (7a-f and
9a,b) across cell monolayers. MDCK cells have properties
resembling Caco-2 cells, which are known to express the peptide
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ARTICLE
Figure 1. Plasma tricin concentration after oral administration of tricin in Crl:CD (SD) rats. (A) Males and (B) females (means ( SEMs, n = 3).
Scheme 2. Synthesis of Tricin-Amino Acid Conjugates 7a-fa
a Reagents and conditions: (a) (4-NO2-PhO)2CO, DIPEA, 0 ꢀC f room temperature, 12 h. (b) Tricin 4, DIPEA, room temperature, 12 h. (c) TFA,
DCM, 0 ꢀC f room temperature, 4 h.
Scheme 3. Synthesis of Tricin-Dipeptide Conjugates 9a and 9a
a Reagents and conditions: (a) Amino acid tert-butyl ester, WSC, HOBt, DMF, 0 ꢀC, 3 days. (b) TFA, DCM, 0 ꢀC f room temperature, 4 h.
transporter PepT1 and form tight layers on membrane filters of
transport analysis plates.41,42 The prepared MDCK monolayers
were checked by measuring Lucifer yellow rejection after the
transport experiments.
quercetin. These observations strongly suggest that introduction
of Ala-Glu dipeptide into tricin may improve tricin permeability
into cells and that T-Ala-Glu is a strong prodrug candidate for
tricin.
Figure 2 indicates that the MDCK cell permeabilities of all of
the tested prodrugs [T-Gly (7a), T-Ala (7b), T-Val (7c), T-Phe
(7d), T-Asp (7e), T-Glu (7f), T-Ala-Asp (9a), and T-Ala-Glu
(9b)] increased after attaching amino acids and dipeptides to
tricin. Among the prodrugs, the tricin-dipeptide conjugate 9b
(T-Ala-Glu) showed an excellent relative permeation rate of 6.3.
On the basis of a previous report that the quercetin-dipeptide
conjugate (Q-Ala-Glu as a regioisomeric mixture of the 30-ester
and 40-ester of the quercetin B ring) had good cell permeability,29
we prepared and evaluated Q-Ala-Glu also. The MDCK cell
permeability of Q-Ala-Glu was 4.35 times higher than that of
In mammals, the peptide transporter PepT1 belongs to the
proton-coupled oligopeptide transporter (POT) family, which is
a high-capacity low-affinity transporter. PepT1 has nutritional
importance because of its role in intestinal absorption of small
peptides from the diet and in reabsorption of peptide-bound
amino nitrogen from glomerular filtrate in the kidneys.43 PepT1
also has significance due to its ability to transport therapeutic
agents (e.g., β-lactam antibiotics, antiviral nucleoside prodrugs)
and peptidomimetics (e.g., 5-aminolevulinic acid) into cells.
Researchers have previously reported that modified Ala-Asp-
and Ala-Glu-benzyl esters as model prodrugs have an affinity for
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ARTICLE
Figure 2. Relative permeabilities of tricin and quercetin prodrugs in
MDCK cells (means ( SEMs, n = 3).
Figure 3. Plasma tricin concentration after oral administration of T-Ala-
Glu in Crl:CD (SD) male rats (means ( SEMs, n = 3).
Table 1. Half-Lives (t1/2) of Tricin and Quercetin Prodrugs
in MDCK Cell Lysate (Means ( SEMs, n = 3)
Table 2. Pharmacokinetics Parameters of Tricin after Oral
Administration of T-Ala-Glu in Crl:CD (SD) Male Rats
compound
half-life (t1/2) (min)
dose (mg/kg) Cmax 1 (ng/mL) Cmax 2 (ng/mL) AUCa0-8 (ng h/mL)
T-Ala-Glu (9b)
165
110
Q-Ala-Glu
100
300
692
2932
4448
2497
2869
2876
10261
15077
21525
PepT1 and efficient permeabilities.44,45 Thus, this peptide trans-
porter likely improves the oral absorption of T-Ala-Glu.
Stability. Release of the model drugs by hydrolysis of the
carbamate linkages in the model prodrugs was examined by using
various endogenous hydrolysis enzymes in cells. The time
required for 50% release of the model drugs (t1/2; half-life) after
treatment with cell lysate was determined.
We lysed MDCK cells with lysis buffer and prepared whole
MDCK cell lysates. T-Ala-Glu had a half-life of 165 min, which
was longer than that of Q-Ala-Glu (Table 1). This was likely
caused by the low affinity of T-Ala-Glu for the enzyme active site.
T-Ala-Glu has greater steric hindrance due to the existence of
bulky functional groups (methoxy groups)37 around the carba-
mate linkage as compared to Q-Ala-Glu. This in vitro metabolism
experiment indicates that T-Ala-Glu exhibits strong resistance
against enzymatic hydrolysis and is more stable than Q-Ala-Glu
in the cell lysate. The longer half-life and the higher stability of
T-Ala-Glu in the cell lysate may ensure sufficient stability during
the transport process in vivo.
Bioavailability of the T-Ala-Glu after Oral Administration
in Vivo. On the basis of the results described above, we
evaluated tricin in plasma after oral administration of T-Ala-
Glu in male rats. Crl:CD (SD) male rats received an oral bolus of
100, 300, or 1000 mg kg-1 of T-Ala-Glu in vivo. Blood was
collected at 0, 0.5, 2, 4, and 8 h after T-Ala-Glu administration.
Tricin derived from parent T-Ala-Glu in plasma was analyzed by
RP-HPLC.32
1000
a AUC, area under the concentration curve.
vivo absorption experiment revealed the existence of double-site
absorption. This phenomenon in Figure 3 implies the possibility
of reabsorption by enterohepatic circulation and intestinal
absorption, because PepT1 is expressed in the GIT and kidney.43
However, details of this mechanism are still ambiguous, and
further in-depth studies are required.
Recently, Cai et al. reported a study of the administration of
30,40,5,50,7-pentamethoxyflavone (40,5,7-tri-O-methylated tricin)
orally to mice at a single dose, 300 mg kg-1 by gavage.28 The
maximum concentration in plasma was 1452 ng mL-1 (4.4 nmol
mL-1). In contrast, T-Ala-Glu in our study reached approxi-
mately twice this concentration.
In addition, we examined the oral toxicity effects of T-Ala-Glu
over 14 days with repeated single doses of 300 mg kg-1 day-1 in
Crlj:CD1 (ICR) mice. No abnormal findings were evident in
general appearance, body weight, food consumption, hematol-
ogy, blood chemistry, or autopsy tests (data not shown). Thus,
we conclude that T-Ala-Glu is safe for oral administration.
In the present study, we endeavored to improve the oral
bioavailability of tricin via a prodrug approach. Our work high-
lighted the importance of tricin-amino acid derivatives as oral
administration forms of tricin. Among them, T-Ala-Glu appears
to be a good candidate for development of a new anti-HCMV
drug.
In all groups, plasma tricin concentrations after administration
of T-Ala-Glu were substantially higher than after administration
of intact tricin (Figures 1 and 3 and Table 2). Even in the 100 mg
kg-1 group, the maximum concentration of tricin was 2497 ng
mL-1 (7.2 nmol mL-1). In the 300 and 1000 mg kg-1 groups,
the tricin concentrations exceeded 2800 ng mL-1 (8.5 nmol
mL-1), about 45 times higher than after administration of intact
tricin. These results suggest that T-Ala-Glu was actively absorbed
through interaction with PepT1 and that inactivation by various
metabolic enzymes was prevented. In addition, T-Ala-Glu de-
monstrated a bimodal time course in the plasma profile; the in
’ CONCLUSIONS
Because tricin was not found to be orally available in its native
form, tricin-amino acid derivatives were prepared as prodrugs,
and their bioavailabilities were evaluated in vitro and in vivo.
Among the prodrugs, the T-Ala-Glu exhibited enhanced perme-
ability, stability in MDCK cells, and excellent bioavailability after
oral administration in Crl:CD (SD) male rats, with good safety.
Our findings suggest that T-Ala-Glu may be beneficial as a
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ARTICLE
substantially improved oral administration form and useful in
future research toward development of a new anti-HCMV drug.
6.23 (1H, d, J = 2.3 Hz, H-6), 4.14 (1H, quint, J = 7.3 Hz, H-200), 3.92
(6H, s, 30- and 50-OMe), 1.46 (9H, s, tBu), 1.41 (3H, d, J = 7.3 Hz,
H-300). 13C NMR (100 MHz; acetone-d6): δ 183.2, 172.5, 165.0, 164.3,
163.3, 158.8, 154.5 (2C), 153.9, 133.3, 129.8, 106.4, 105.5, 104.5 (2C),
99.8, 95.0, 81.6, 56.9 (2C), 51.6, 28.1 (3C), 18.0. FABMS: m/z = 502
[M þ H]þ. Anal. calcd for C25H27NO10: C, 59.88; H, 5.43; N, 2.79.
Found: C, 59.92; H, 5.41; N, 2.83.
’ EXPERIMENTAL SECTION
General Chemistry Methods. All solvents and reagents were
purchased from the suppliers and used without further purification. IR
spectra were recorded on a JASCO FT/IR-460 Plus spectrophotometer
(Tokyo, Japan). 1H (600 and 400 MHz) and 13C (150 and 100 MHz)
NMR spectra were recorded with a JEOL ECA 600 spectrometer and a
JEOL ECX 400 spectrometer (Tokyo, Japan) with tetramethylsilane as
an internal standard. MS spectra were obtained using a JEOL JMS-700/
GI spectrometer. UV-vis absorption was measured using a PerkinElmer
EnVision microplate-reader (Waltham, MA). The used HPLC and
microrefrigerated centrifuge were manufactured by Shimadzu Corp.
(Kyoto, Japan) and Kubota Co., Ltd. (Tokyo, Japan), respectively. Silica
gel column chromatography (CC) was performed on silica gel N-60
(40-50 μm). Thin-layer chromatography (TLC) spots on plates pre-
coated with silica gel 60 F254 were detected with a UV lamp (254 nm).
Fractionations for all CCs were based on TLC analyses. The purity of
tricin was checked using RP-HPLC32 method and confirmed to be 99%.
Tricin prodrugs were checked by combustion analysis, and the purity of
each compound was g95%.
40-O-CO-(Val-OtBu)-tricin (6c). Yellow amorphous powder, 52%
1
yield. IR (film): 3343, 1654, 1625, 1595, 13637, 1163, 694 cm-1. H
NMR (400 MHz; acetone-d6): δ 7.36 (2H, s, H-20 and H-60), 6.87-6.83
(1H, m, NH), 6.80 (1H, s, H-3), 6.53 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H,
d, J = 2.0 Hz, H-6), 4.10-4.04 (1H, m, H-200), 3.92 (6H, s, 30- and 50-
OMe), 2.26-2.14 (1H, m, H-300), 1.48 (9H, s, tBu), 1.04-1.00 (6H, m,
2Me of Val). 13C NMR (100 MHz; acetone-d6): δ 182.3, 170.6, 164.2,
163.4, 162.5, 158.0, 153.6 (3C), 132.5, 128.9, 105.5, 104.7, 103.7 (2C),
99.0, 94.2, 81.0, 60.5, 56.0 (2C), 31.1, 27.4 (3C), 18.6, 17.3. FABMS: m/
z = 530 [M þ H]þ. Anal. calcd for C27H31NO10: C, 61.24; H, 5.90; N,
2.65. Found: C, 61.31; H, 5.95; N, 2.56.
40-O-CO-(Phe-OtBu)-tricin (6d). Yellow amorphous powder, 51%
1
yield. IR (film): 3388, 1654, 1617, 1594, 1364, 1162, 697 cm-1. H
NMR (400 MHz; acetone-d6): δ 7.36 (2H, s, H-20 and H-60), 7.36-7.30
(4H, m, Ar of Phe), 7.28-7.23 (1H, m, Ar of Phe), 6.92-6.88 (1H, m,
NH), 6.81 (1H, s, H-3), 6.55 (1H, d, J = 1.9 Hz, H-8), 6.24 (1H, d, J = 1.9
Hz, H-6), 4.40-4.34 (1H, m, H-200), 3.90 (6H, s, 30- and 50-OMe), 3.19
(1H, dd, J = 13.7, 6.0 Hz, H-300), 3.08 (1H, dd, J = 13.7, 6.0 Hz, H-300),
1.42 (9H, s, tBu). 13C NMR (100 MHz; acetone-d6): δ 183.2, 171.2,
165.8, 164.4, 163.4, 158.9, 154.5 (2C), 153.8, 138.1, 133.2, 130.4 (2C),
129.2 (2C), 127.5, 126.9, 106.4, 104.5 (3C), 99.9, 95.0, 82.0, 57.2, 56.9
(2C), 38.3, 28.1 (3C). FABMS: m/z = 578 [M þ H]þ. Anal. calcd for
C31H31NO10: C, 64.46; H, 5.41; N, 2.43. Found: C, 64.34; H, 5.30; N,
2.55.
Tricin (4). Reaction of 4-O-tert-butyldimethylsilyl-3,5-dimethoxy-
benzoate 1 (152.5 g, 0.47 mol) with LiHMDS (1.0 M solution in THF;
3.76 L, 3.76 mol) and 20,40-O-bis(tert-butyldimethylsilyl)-60-hydroxya-
cetophenone 2 (279.7 g, 0.94 mol) in THF (2.0 L) at -78 ꢀC and
brought to room temperature for 3 days yielded intermediate 3. The
reaction mixture was partitioned with EtOAc and washed three times
with 5% aqueous HCl and once with brine. The organic phase was dried
over Na2SO4, filtered, and evaporated in vacuo. The residue was dried
under vacuum overnight, and the completely dry residue was stirred with
0.5% H2SO4 in acetic acid (1.0 L) at 100 ꢀC overnight. The reaction
mixture was evaporated, MeOH was added, and the insoluble matter was
filtered and washed with MeOH to afford tricin 4 (105.0 g, yield: 68%) as
a yellow amorphous powder. 1H NMR (400 MHz; acetone-d6): δ 7.39
(2H, s, H-20 and H-60), 6.74 (1H, s, H-3), 6.56 (1H, d, J = 2.0 Hz, H-8),
6.26 (1H, d, J = 2.0 Hz, H-6), 3.97 (6H, s, 30- and 50-OMe). 13C NMR
(100 MHz; acetone-d6): δ 183.1, 165.1, 164.9, 163.3, 158.8, 149.1 (2C),
140.9, 122.3, 105.4 (2C), 105.2, 104.7, 99.7, 94.9, 56.9 (2C). FABMS:
m/z = 331 [M þ H]þ.
40-O-CO-[Asp(OtBu)-OtBu]-tricin (6e). Yellow amorphous powder,
53% yield. IR (film): 3343, 1654, 1616, 1593, 1365, 1161, 694 cm-1. 1H
NMR (400 MHz; acetone-d6): δ 7.37 (2H, s, H-20 and H-60), 6.98-6.93
(1H, m, NH), 6.81 (1H, s, H-3), 6.54 (1H, d, J = 2.0 Hz, H-8), 6.24 (1H,
d, J = 2.0 Hz, H-6), 4.49-4.44 (1H, m, H-200), 3.93 (6H, s, 30- and 50-
OMe), 2.83 (1H, dd, J = 16.0, 6.0 Hz, H-300), 2.75 (1H, dd, J = 16.0, 6.0
Hz, H-300), 1.47 (9H, s, tBu), 1.46 (9H, s, tBu). 13C NMR (100 MHz;
acetone-d6): δ 182.3, 169.6, 169.5, 164.2, 163.5, 163.4, 162.5, 158.0,
153.7 (2C), 132.4, 129.1, 105.6, 104.7, 103.7 (2C), 99.0, 94.2, 81.4, 80.7,
56.1 (2C), 51.9, 37.5, 27.4 (3C), 27.3 (3C). FABMS: m/z = 602 [M þ
H]þ. Anal. calcd for C30H35NO12: C, 59.89; H, 5.86; N, 2.33. Found: C,
60.03; H, 5.95; N, 2.45.
Amino Acid tert-Butyl Esters of Tricin 6a-f. DIPEA (0.66
mmol) was added to a stirred solution of amino acid tert-butyl esters
5a-f (0.33 mmol) and bis(4-nitrophenyl)carbonate (0.33 mmol) in
THF (5 mL) at 0 ꢀC, and stirring was continued for 12 h at room
temperature. Tricin 4 (0.30 mmol) and additional DIPEA (0.66 mmol)
were added to the reaction mixture at room temperature. After the
mixture was stirred for 12 h, the reaction mixture was evaporated, and
the residue was purified by silica gel CC eluted with CHCl3/MeOH
(50/1) to yield amino acid tert-butyl esters of tricin 6a-f.
40-O-CO-[Glu(OtBu)-OtBu]-tricin (6f). Yellow amorphous powder,
61% yield. IR (film): 3419, 1649, 1619, 1605, 1366, 1159, 695 cm-1. 1H
NMR (400 MHz; acetone-d6): δ 7.36 (2H, s, H-20 and H-60), 7.01-6.97
(1H, m, NH), 6.80 (1H, s, H-3), 6.53 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H,
d, J = 2.0 Hz, H-6), 4.24-4.18 (1H, m, H-200), 3.93 (6H, s, 30- and 50-
OMe), 2.45-2.41 (2H, m, H-400), 2.20-2.11 (1H, m, H-300), 1.98-1.89
(1H, m, H-300), 1.47 (9H, s, tBu), 1.44 (9H, s, tBu). 13C NMR (100
MHz; acetone-d6): δ 182.3, 171.6, 170.8, 164.2, 163.5, 163.4, 162.5,
158.0, 153.6 (2C), 153.4, 132.5, 129.0, 105.5, 104.7, 103.7 (2C), 99.0,
94.2, 81.1, 79.8, 56.1 (2C), 54.4, 31.1, 27.5 (3C), 27.3 (3C). FABMS: m/
z = 616 [M þ H]þ. Anal. calcd for C31H37NO12: C, 60.48; H, 6.06; N,
2.28. Found: C, 60.61; H, 6.01; N, 2.35.
40-O-CO-(Gly-OtBu)-tricin (6a). Yellow amorphous powder, 63%
1
yield. IR (film): 3435, 1650, 1621, 1603, 1365, 1159, 645 cm-1. H
NMR (400 MHz; acetone-d6): δ 7.37 (2H, s, H-20 and H-60), 7.00 (1H,
br t, J = 6.0 Hz, NH), 6.82 (1H, s, H-3), 6.54 (1H, d, J = 1.8 Hz, H-8),
6.24 (1H, d, J = 1.8 Hz, H-6), 3.97 (6H, s, 30- and 50-OMe), 3.84 (2H, d, J
= 6.0 Hz, H-200), 1.46 (9H, s, tBu). 13C NMR (100 MHz; acetone-d6): δ
182.3, 168.8, 164.2, 163.4, 162.5, 158.0, 153.9, 153.7 (2C), 132.4, 129.0,
105.5, 104.7, 103.6 (2C), 99.0, 94.2, 80.9, 56.1 (2C), 43.5, 28.8 (3C).
FABMS: m/z = 488 [M þ H]þ. Anal. calcd for C24H25NO10: C, 59.13;
H, 5.17; N, 2.87. Found: C, 59.15; H, 5.18; N, 2.88.
Amino Acid Conjugates of Tricin 7a-f. TFA (2 mL) was
added to a stirred solution of amino acid tert-butyl esters of tricin 6a-f
(0.082 mmol) in dichloromethane (DCM; 5 mL) at 0 ꢀC, and stirring
was continued for 4 h at room temperature. The reaction mixture was
evaporated, and the residue was suspended in DCM (5 mL) and filtered,
and the filtrate was washed with DCM (5 mL) to yield the amino acid
conjugates of tricin 7a-f.
40-O-CO-(Ala-OtBu)-tricin (6b). Yellow amorphous powder, 55%
1
yield. IR (film): 3433, 1650, 1619, 1602, 1363, 1162, 644 cm-1. H
NMR (400 MHz; acetone-d6): δ 7.36 (2H, s, H-20 and H-60), 7.00 (1H,
br d, J = 9.2 Hz, NH), 6.81 (1H, s, H-3), 6.53 (1H, d, J = 2.3 Hz, H-8),
40-O-CO-Gly-tricin (7a). Yellow amorphous powder, 94% yield. IR
(film): 3294, 1715, 1656, 1639, 1612, 1354, 1166, 663 cm-1. 1H NMR
1533
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Journal of Medicinal Chemistry
ARTICLE
(400 MHz; DMSO-d6): δ 10.86 (1H, br s, COOH), 8.05 (1H, t, J = 6.0
Hz, NH), 7.34 (2H, s, H-20 and H-60), 7.09 (1H, s, H-3), 6.56 (1H, d, J =
1.9 Hz, H-8), 6.20 (1H, d, J = 1.9 Hz, H-6), 3.85 (6H, s, 30- and 50-OMe),
3.72 (2H, d, J = 6.0 Hz, H-200). 13C NMR (100 MHz; DMSO-d6): δ
182.5, 171.7, 164.9, 163.3, 161.9, 158.0, 154.1, 153.5 (2C), 132.1, 128.9,
106.1, 104.5, 104.2 (2C), 99.5, 94.9, 56.9 (2C), 42.9. FABMS: m/z = 432
[M þ H]þ. Anal. calcd for C20H17NO10: C, 55.69; H, 3.97; N, 3.25.
Found: C, 55.39; H, 3.94; N, 3.32.
Dipeptide tert-Butyl Esters of Tricin 8a and 8b. 1-Ethyl-
3-(3-dimethylaminopropyl) carbodiimide (0.34 mmol) and 1-hydroxy-
1H-benzotriazole (0.62 mmol) were added to a stirred solution of the
alanine conjugate of tricin 7b (0.31 mmol) and amino acid tert-butyl
esters 5e and 5f (0.31 mmol) in N,N-dimethylformamide (DMF; 5 mL)
at 0 ꢀC, and stirring was continued for 3 days. The reaction mixture was
evaporated, and the residue was purified by silica gel column CC eluted
with CHCl3 to yield dipeptide tert-butyl esters of tricin 8a and 8b.
40-O-CO-Ala-[Asp(OtBu)-OtBu]-tricin (8a). Yellow amorphous powder,
32% yield. IR (film): 3406, 1656, 1619, 1603, 1366, 1160, 633 cm-1. 1H
NMR (400 MHz; acetone-d6): δ 7.51 (1H, br d, J = 8.2 Hz, NH), 7.34
(2H, s, H-20 and H-60), 7.00 (1H, br d, J = 7.3 Hz, NH), 6.80 (1H, s,
H-3), 6.53 (1H, d, J = 1.9 Hz, H-8), 6.23 (1H, d, J = 1.9 Hz, H-6), 4.68-
4.63 (1H, m, H-2000), 4.30 (1H, quint, J = 6.8 Hz, H-200), 3.91 (6H, s, 30-
and 50-OMe), 2.74 (2H, d, J = 5.5 Hz, H-3000), 1.44 (9H, s, tBu), 1.43
(3H, d, J = 6.8 Hz, H-300), 1.40 (9H, s, tBu). 13C NMR (100 MHz;
acetone-d6): δ 182.3, 171.9, 169.6 (3C), 164.3, 163.4, 162.5, 158.0,
153.6 (2C), 131.9, 129.0, 105.5, 104.7, 103.5 (2C), 99.0, 94.3, 81.3, 80.6,
56.0 (2C), 50.8, 49.5, 37.3, 27.4 (3C), 27.3 (3C), 18.1. FABMS: m/z =
673 [M þ H]þ. Anal. calcd for C33H40N2O13: C, 58.92; H, 5.99; N, 4.16.
Found: C, 58.91; H, 5.92; N, 4.12.
40-O-CO-Ala-tricin (7b). Yellow amorphous powder, 81% yield. IR
(film): 3306, 1714, 1649, 1616, 1599, 1361, 1132, 639 cm-1. 1H NMR
(400 MHz; DMSO-d6): δ 10.86 (1H, br s, COOH), 8.11 (1H, d, J = 7.8
Hz, NH), 7.33 (2H, s, H-20 and H-60), 7.08 (1H, s, H-3), 6.56 (1H, d, J =
1.9 Hz, H-8), 6.20 (1H, d, J = 1.9 Hz, H-6), 4.03 (1H, quint, J = 7.3 Hz,
H-200), 3.84 (6H, s, 30- and 50-OMe), 1.29 (3H, d, J = 7.3 Hz, H-300). 13C
NMR (100 MHz; DMSO-d6): δ 182.5, 174.5, 164.9, 163.3, 161.9, 158.0,
153.5 (3C), 132.2, 128.8, 106.1, 104.5, 104.2 (2C), 99.5, 94.9, 57.0 (2C),
50.0, 17.8. FABMS: m/z = 446 [M þ H]þ. Anal. calcd for C21H19NO10:
C, 56.63; H, 4.30; N, 3.14. Found: C, 56.94; H, 4.24; N, 3.12.
40-O-CO-Val-tricin (7c). Yellow amorphous powder, 90% yield. IR
(film): 3320, 1714, 1656, 1619, 1600, 1364, 1162, 642 cm-1. 1H NMR
(400 MHz; DMSO-d6): δ 10.91 (1H, br s, COOH), 8.06 (1H, d, J = 9.2
Hz, NH), 7.38 (2H, s, H-20 and H-60), 7.12 (1H, s, H-3), 6.59 (1H, d, J =
1.9 Hz, H-8), 6.23 (1H, d, J = 1.9 Hz, H-6), 3.95-3.88 (1H, m, H-200),
3.88 (6H, s, 30- and 50-OMe), 2.15-2.08 (1H, m, H-300), 0.96 (3H, d, J =
3.8 Hz, 300-Me), 0.94 (3H, d, J = 3.8 Hz, 300-Me). 13C NMR (100 MHz;
DMSO-d6): δ 182.0, 172.9, 164.4, 162.9, 161.4, 157.5, 153.7, 153.0
(2C), 131.8, 128.3, 105.6, 104.0, 103.7 (2C), 99.0, 94.4, 59.8, 56.4 (2C),
30.0, 19.1, 18.0. FABMS: m/z = 474 [M þ H]þ. Anal. calcd for
C23H23NO10: C, 58.35; H, 4.90; N, 2.96. Found: C, 58.23; H, 4.82;
N, 2.87.
40-O-CO-Ala-[Glu(OtBu)-OtBu]-tricin (8b). Yellow amorphous powder,
33% yield. IR (film): 3343, 1657, 1627, 1601, 1366, 1160, 633 cm-1. 1H
NMR (400 MHz; acetone-d6): δ 7.46 (1H, br d, J = 7.8 Hz, NH), 7.34
(2H, s, H-20 and H-60), 6.97 (1H, br d, J = 7.8 Hz, NH), 6.80 (1H, s,
H-3), 6.53 (1H, d, J = 1.9 Hz, H-8), 6.24 (1H, d, J = 1.9 Hz, H-6), 4.42-
4.36 (1H, m, H-2000), 4.29 (1H, quint, J = 7.3 Hz, H-200), 3.90 (6H, s, 30-
and 50-OMe), 2.34-2.30 (2H, m, H-4000), 2.14-2.05 (1H, m, H-3000),
1.92-1.82 (1H, m, H-3000), 1.45 (9H, s, tBu), 1.43 (3H, d, J = 7.3 Hz,
H-300), 1.38 (9H, s, tBu). 13C NMR (100 MHz; acetone-d6): δ 182.3,
172.1, 171.5, 170.7, 164.3, 163.4, 162.5, 158.0, 153.6 (2C), 153.1, 132.0,
128.9, 105.4, 104.7, 103.5 (2C), 99.0, 94.3, 81.2, 79.7, 56.0 (2C), 52.3,
50.7, 31.2, 27.4 (3C), 27.3 (3C), 27.2, 18.0. FABMS: m/z = 687 [M þ
H]þ. Anal. calcd for C34H42N2O13: C, 59.47; H, 6.16; N, 4.08. Found:
C, 59.61; H, 6.11; N, 4.10.
40-O-CO-Phe-tricin (7d). Yellow amorphous powder, 80% yield. IR
(film): 3310, 1713, 1655, 1612, 1602, 1358, 1164, 646 cm-1. 1H NMR
(400 MHz; DMSO-d6): δ 10.86 (1H, br s, COOH), 8.15 (1H, d, J = 8.2
Hz, NH), 7.30 (2H, s, H-20 and H-60), 7.29-7.20 (5H, m, Ar of Phe),
7.07 (1H, s, H-3), 6.55 (1H, d, J = 1.9 Hz, H-8), 6.19 (1H, d, J = 1.9 Hz,
H-6), 4.20-4.13 (1H, m, H-200), 3.78 (6H, s, 30- and 50-OMe), 3.08 (1H,
dd, J = 13.7, 4.6 Hz, H-300), 2.87 (1H, dd, J = 13.7, 4.6 Hz, H-300). 13C
NMR (100 MHz; DMSO-d6): δ 182.5, 173.5, 164.9, 163.3, 161.9, 158.0,
153.7, 153.4 (2C), 138.2, 132.2, 129.7 (2C), 128.8 (3C), 127.0, 106.1,
104.5, 104.2 (2C), 99.5, 94.9, 56.9 (2C), 56.2, 37.0. FABMS: m/z = 522
[M þ H]þ, 504 [M þ H - H2O]þ. Anal. calcd for C27H19NO10: C,
62.19; H, 4.45; N, 2.69. Found: C, 62.33; H, 4.31; N, 2.60.
Dipeptide Conjugates of Tricin 9a and 9b. TFA (2 mL) was
added to a stirred solution of the dipeptide tert-butyl esters of tricin 8a
and 8b (0.095 mmol) in DCM (5 mL) at 0 ꢀC, and stirring was conti-
nued for 4 h at room temperature. The reaction mixture was evaporated,
and the residue was suspended in DCM (5 mL) and filtered, and the
filtrate was washed with DCM (5 mL) to yield the dipeptide conjugates
of tricin 9a and 9b.
40-O-CO-Ala-Asp-tricin (9a). Yellow amorphous powder, 91% yield.
40-O-CO-Asp-tricin (7e). Yellow amorphous powder, 90% yield. IR
(film): 3309, 1721, 1654, 1610, 1601, 1359, 1165, 645 cm-1. 1H NMR
(400 MHz; DMSO-d6): δ 10.90 (2H, br s, COOH), 8.15 (1H, d, J = 8.2
Hz, NH), 7.37 (2H, s, H-20 and H-60), 7.12 (1H, s, H-3), 6.59 (1H, d, J =
1.9 Hz, H-8), 6.23 (1H, d, J = 1.9 Hz, H-6), 4.35 (1H, q, J = 7.8 Hz,
H-200), 3.88 (6H, s, 30- and 50-OMe), 2.77 (1H, dd, J = 16.5, 6.7 Hz,
H-300), 2.63 (1H, dd, J = 16.5, 6.7 Hz, H-300). 13C NMR (100 MHz;
DMSO-d6): δ 182.0, 172.3, 171.6, 164.4, 162.8, 161.4, 157.5, 153.0,
152.9 (2C), 131.7, 128.3, 105.6, 104.0, 103.8 (2C), 99.0, 94.4, 56.4 (2C),
55.8, 29.6. FABMS: m/z = 490 [M þ H]þ. Anal. calcd for C22H19NO12:
C, 53.99; H, 3.91; N, 2.86. Found: C, 53.69; H, 3.89; N, 2.86.
1
IR (film): 3372, 1722, 1655, 1611, 1601, 1359, 1164, 663 cm-1. H
NMR (600 MHz; DMSO-d6): δ 10.91 (2H, br s, COOH), 8.15 (1H, d, J
= 8.2 Hz, NH), 7.99 (1H, d, J = 7.8 Hz, NH), 7.37 (2H, s, H-20 and H-60),
7.12 (1H, s, H-3), 6.59 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H, d, J = 2.0 Hz,
H-6), 4.56 (1H, quint, J = 7.8 Hz, H-2000), 4.13 (1H, quint, J = 6.8 Hz,
H-200), 3.88 (6H, s, 30- and 50-OMe), 2.69 (1H, dd, J = 16.5, 6.0 Hz,
H-3000), 2.62 (1H, dd, J = 16.5, 6.0 Hz, H-3000), 1.29 (3H, d, J = 6.8 Hz,
H-300). 13C NMR (150 MHz; DMSO-d6): δ 182.0, 172.3, 172.0, 171.6,
164.4, 162.8, 161.4, 157.5, 153.0 (2C), 152.7, 131.7, 128.3, 105.6, 104.0,
103.7 (2C), 99.0, 94.4, 56.4 (2C), 50.1, 48.5, 36.0, 18.4. FABMS: m/z =
561 [M þ H]þ. Anal. calcd for C25H24N2O13: C, 53.57; H, 4.32; N, 5.00.
Found: C, 53.44; H, 4.38; N, 4.95.
40-O-CO-Glu-tricin (7f). Yellow amorphous powder, 83% yield. IR
(film): 3299, 1719, 1650, 1614, 1601, 1358, 1165, 645 cm-1. 1H NMR
(400 MHz; DMSO-d6): δ 10.93 (2H, br s, COOH), 8.15 (1H, d, J = 8.2
Hz, NH), 7.38 (2H, s, H-20 and H-60), 7.12 (1H, s, H-3), 6.60 (1H, d, J =
2.0 Hz, H-8), 6.24 (1H, d, J = 2.0 Hz, H-6), 4.09-4.02 (1H, m, H-200),
3.89 (6H, s, 30- and 50-OMe), 2.40-2.35 (2H, m, H-400), 2.09-2.00 (1H,
m, H-300), 1.88-1.78 (1H, m, H-300). 13C NMR (100 MHz; DMSO-d6):
δ 182.5, 174.3, 173.6, 164.9, 163.3, 161.9, 158.0, 153.9, 153.4 (2C), 132.3,
128.8, 106.1, 104.5, 104.2 (2C), 94.9 (2C), 56.9 (2C), 53.7, 30.4, 26.9.
FABMS: m/z = 504 [M þ H]þ. Anal. calcd forC23H21NO12: C, 54.87; H,
4.20; N, 2.78. Found: C, 54.62; H, 4.28; N, 2.84.
40-O-CO-Ala-Glu-tricin (9b). Yellow amorphous powder, 96% yield.
1
IR (film): 3296, 1711, 1650, 1619, 1602, 1362, 1161, 645 cm-1. H
NMR (600 MHz; DMSO-d6): δ 10.90 (2H, br s, COOH), 8.09 (1H, d,
J = 7.8 Hz, NH), 7.98 (1H, d, J = 7.8 Hz, NH), 7.37 (2H, s, H-20 and
H-60), 7.12 (1H, s, H-3), 6.59 (1H, d, J = 2.0 Hz, H-8), 6.24 (1H, d, J =
2.0 Hz, H-6), 4.28-4.22 (1H, m, H-2000), 4.12 (1H, quint, J = 7.3 Hz,
H-200), 3.89 (6H, s, 30- and 50-OMe), 2.31-2.23 (2H, m, H-4000), 2.03-
1.94 (1H, m, H-3000), 1.83-1.74 (1H, m, H-3000), 1.27 (1H, d, J = 7.3 Hz,
H-300). 13C NMR (150 MHz; DMSO-d6): δ 182.0, 173.7, 173.1,
1534
dx.doi.org/10.1021/jm1015457 |J. Med. Chem. 2011, 54, 1529–1536

Journal of Medicinal Chemistry
ARTICLE
172.2, 164.4, 162.8, 161.4, 157.5, 153.0 (2C), 152.7, 131.7, 128.3, 105.5,
104.0, 103.7 (2C), 99.0, 94.4, 56.4 (3C), 51.1, 29.9, 26.4, 18.2. FABMS:
m/z = 575 [M þ H]þ. Anal. calcd for C26H26N2O13: C, 54.36; H, 4.56;
N, 4.88. Found: C, 54.40; H, 4.48; N, 4.92.
performance liquid chromatography; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; PBS, phosphate-buffered saline; PepT,
peptide transporter; POT, proton-coupled oligopeptide trans-
porter; WSC, water-soluble carbodiimide
Biological Evaluation Methods. Cell Permeability Evalua-
tions. MDCK cells were seeded in culture flasks and passaged in
minimum essential medium (MEM) supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin (100 U mL-1). Cells
were seeded onto tissue culture-treated transwells (1.12 cm2, 0.4 μm
pore size) at a density of 2.7 ꢀ 105 cm-2 and grown at 37 ꢀC in a
humidified incubator under an atmosphere of 5% CO2. Transport
experiments were performed 2 days after seeding. To evaluate the cell
permeability of the prodrugs, the rate of drug transport from apical to
basolateral solution was determined. The concentrations of the tested
prodrugs were 10, 20, 60, and 100 μM. The prodrugs were diluted with
phosphate-buffered saline (PBS) buffer, and 300 μL was added to the
filter well of an apical plate. The basolateral medium was 500 μL of
MEM. The 12-well plates were incubated under 5% CO2 atmosphere at
37 ꢀC. After 1 h, 150 μL samples were collected from the apical and
basolateral solutions and measured using a UV-vis spectrometer at
330 nm.
Stability Evaluations. MDCK cell lysates were prepared using
1.33 ꢀ 106 cells in accordance with the manual.46 Stability evaluation
procedures were carried out as described previously.32
Animal Experiments. The experimental and study design were
approved by the Committee of Safety Research Institute for Chemical
Compounds Co., Ltd. under the Institutional Animal Care Guidelines.
All handling and procedures were carried out in accordance with the
appropriate Institutional Animal Care Guidelines.
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Three week old male and female Crl:CD (SD) rats were purchased
from Charles River Laboratories, Inc. (Wilmington, MA). All animals
were housed in metal cages (2-3 rats/cage) and had free access to tap
water and a basal diet, Charles River Formula-1 (Oriental Yeast Co.,
Ltd., Tokyo, Japan). The animals were kept in an experimental animal
room under controlled conditions of humidity (50 ( 20%), light (12/12
h light/dark cycle), and temperature (22 ( 3 ꢀC). After 2 weeks of
quarantine, the animals were divided into three experimental groups. All
handing and procedures were carried out in accordance with appropriate
Institutional Animal Care Guidelines. Tricin 4 was mixed with 1%
aqueous methylcellulose solution (Shin-Etsu Chemical Co., Ltd., To-
kyo, Japan) and sonicated to produce a visually homogeneous solution
(10 mL kg-1) for gavage. The animals received a single oral dose (100,
300, or 1,000 mg kg-1) by gavage. Blood was collected at 0, 0.5, 2, 4, and
8 h after administration by syringe from the tail of the rat. Plasma was
obtained by centrifugation (6000 rpm, 10 min, 4 ꢀC). Biomatrices were
held at -25 ꢀC until analysis, no longer than 19 days. Plasma tricin
concentrations were analyzed as described previously.32
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’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ81-58-293-2619. Fax: þ81-58-293-2794. E-mail:
koketsu@gifu-u.ac.jp (M.K.). Tel: þ81-58-230-6555. Fax:
þ81-58-230-6551. E-mail: kuni@gifu-u.ac.jp (K.W.).
’ ABBREVIATIONS USED
AMVN, 2,20-azobis(2,4-dimethylvaleronitrile); DCM, dichloro-
methane; DIPEA, N,N-diisopropylethylamine; DMF, N,N-di-
methylformamide; GCV, ganciclovir; GIT, gastrointestinal tract;
HCMV, human cytomegalovirus; HOBt, 1-hydroxybenzotria-
zole; LiHMDS, lithium bis(trimethylsilyl)amide; MDCK, Madin-
Darby canine kidney; MEM, minimum essential medium;MRP2,
multidrug resistance protein 2; RP-HPLC, reverse-phase high-
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