Inhibitory effect of ursolic acid derivatives on recombinant human aldose reductase

Article abstract of DOI:10.1134/S1068162011050050

Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives with ursane skeleton were tested for recombinant

Full text of DOI:10.1134/S1068162011050050

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 5, pp. 569–577. © Pleiades Publishing, Ltd., 2011.
Inhibitory Effect of Ursolic Acid Derivatives
on Recombinant Human Aldose Reductase1
, 2
, 2
Eun Ha Leea , S. A. Popovb , Joo Young Leea, A. V. Shpatovb, T. P. Kukinab, Suk Woo Kanga,
, 3
CheolꢀHo Pana, Byung Hun Uma, and Sang Hoon Junga
aNatural Products Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute,
Gangneung 210ꢀ340, Gangwonꢀdo, Republic of Korea
bNovosibirsk Institute of Organic Chemistry, Novosibirsk, Russian Federation
Received January 14, 2011; in final form March 14, 2011
Abstract—Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an
important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives
with ursane skeleton were tested for recombinant human aldose reductase (rhAR) inhibitory activity for
development of diabetic complications. Among them,
acid (XV) showed most potent rhAR inhibitory activity in vitro. Inhibition mode of
enꢀ28ꢀoyl)ꢀ4ꢀaminobutyric acid (XV) was tested uncompetitively by kinetic analysis using the Lineweaverꢀ
Burk plots. Ursolic acid derivative ꢀ(3 ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoyl)ꢀ4ꢀaminobutyric acid is able to inhibit
rhAR uncompetitively and could be offered as a lead compound for AR inhibition.
N
ꢀ(3
β
ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoyl)ꢀ4ꢀaminobutyric
N
ꢀ(3 ꢀhydroxyursꢀ12ꢀ
β
N
β
Keywords: aldose reductase, Nꢀ(3
diabetic complication
βꢀhydroxyursꢀ12ꢀenꢀ28ꢀoyl)ꢀ4ꢀaminobutyric acid, ursolic acid derivatives,
s
DOI: 10.1134/S1068162011050050
3 2 1
INTRODUCTION
plants. Ursolic acid (I) and its derivatives have been
reported to possess antioxidant [12], antiꢀinflammaꢀ
tory [13], antiꢀHIV, and antiꢀtumor activities [14, 15].
In addition, these compounds are expected to be
potent bioactive compounds, such as antiꢀcancer
agents. Although ursolic acid derivatives have demonꢀ
strated antiꢀdiabetic activity [16], there have been no
reports on inhibitory potency for AR.
Diabetes is a group of metabolic disorders characꢀ
terized by hyperglycemia and insufficiency of secreꢀ
tion or action of endogenous insulin [1]. Prolonged
diabetes may develop diabetic complications such as
retinopathy, nephropathy, and neuropathy.
The mechanism underlying complications of diaꢀ
betes still remains unclear, but increased AR activity,
increased advanced glycation endproducts (AGEs)
formation, activation of protein kinase C isoforms and
increased hexosamine pathway flux have been known
as important factors [2].
The purpose of the present study was therefore to
investigate the inhibitory effects of ursolic acid (
I) and
its derivatives against rhAR. Ursolic acid ( ) and its
I
synthetic derivatives were tested as possible rhAR
inhibitors for treatment for diabetic complications.
AR (EC 1.1.1.21) is the first enzyme of the polyol
pathway which reduces excess Dꢀglucose into Dꢀgluciꢀ
tol with concomitant conversion of NADPH to
NADP+ [3, 4], which results in progressive irreversible
complications for lens, kidneys or nerves [5–8].
RESULTS AND DISCUSSION
A large variety of structurally diverse compounds
have been reported to have potent aldose reductase
inhibitory potency in vitro and in vivo studies. Several
studies of naturally occurring compounds have also
been reported [9–11].
Ursolic acid (I) is a pentacyclic triterpene acid,
present in many food, medicinal herbs and other
We synthesized ursolic acid esters and amides (III)–
(
3
VII), (XII)–(XV) starting from ursolic acid (
I) and
β
ꢀacetoxyꢀursꢀ12ꢀenꢀ28ꢀoic acid (II). Synthesized
compounds (III)–(VII), (XII)–(XV) together with
accessible ursonic acid (IX), benzyl ursonate (VIII), and
methyl corosolate (X) were tested for rhAR inhibition.
Alkylation of ursolic acid (
I) with alkyl chlorides in
DMF in the presence of inorganic base (K2CO3) was
found to be the convenient route to the ursolic acid
esters (III)–(VII) (Scheme 1, yields 84–86%). Reacꢀ
1
The article is published in the original.
These authors contributed equally to this work.
Corresponding author: phone: +82ꢀ33ꢀ650ꢀ3653; fax: +82ꢀ33ꢀ
650ꢀ3679; eꢀmail: shjung507@gmail.com.
2
3
tion of ursolic acid (I) with racemic epichlorohydrine
569

570
EUN HA LEE et al.
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INHIBITORY EFFECT OF URSOLIC ACID DERIVATIVES
571
O
(
(
(
XIII
XIV
XV
)
)
n
n
= 1
= 2
H
H
1.
H2N
n
OAlk
Cl
)
n
= 3
R4
2. KOH
SOCl
2
(II)
R3
R1
H
N
O
O
(XI)
(XII)
H
H
N
O
R2
H
H
(XI)
O
1'
N
O
O
3'
2'
N
(
XIII) R4 =
HN
(
XII) R4 =
(
XIV) R4 = HN
OH
1'
3'
1'
2'
OH
2'
R1 = OAc, R2, R3 = H
O
3'
(
XV) R4 = HN
R1 = OH, R2, R3 = H
1'
OH
2'
4'
Scheme 2. Synthesis of amide derivatives of ursolic acid.
resulted in glycidyl ursolate (
diastereomers. Reactions of pharmacophore amines itive, which implies that compound (XV) can bind to
(imidazole and esters of amino acids) with ꢀacetoxyꢀ neither substrate binding region nor the nucleotide
ursꢀ12ꢀenꢀ28ꢀoyl chloride (XI) were used to prepare binding region of rhAR.
V) as a 1 : 1 mixture of two Inhibition of rhAR by compound (XV) was uncompetꢀ
3β
the amides of ursolic acid 3ꢀacetate (Scheme 2).
ꢀ(3 ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoyl) aminoacids (XIII)–
XV) were obtained by oneꢀpot deprotection of carꢀ
Thus, compound (XV) could be offered as a lead
compound for AR inhibition.
N
(
β
boxyꢀgroup of acylated aminoacid and 3ꢀhydroxyꢀ
group in the ursane moiety with ethanolic alkali
(Scheme 2). The same method of hydrolysis was used
to prepare ursolic acid derivative (IV) from 2ꢀethoxyꢀ
carbonylmethyl ursolate (III) (Scheme 1). 3ꢀOxoꢀ
derivatives: ursonic acid (IX) and benzyl ursonate
Table 1. Inhibitory effects of the ursolic acid derivatives on rhAR
Compound Concentration, µM % inhibition IC50, µM
0.78 2.1
(
(
(
(
(
(
(
(
(
(
(
(
(
I
)
20
20
20
20
20
20
20
20
20
20
20
20
20
>20
II
III
IV
)
4.69 4.11
3.09 3.57
3.56 4.07
5.64 2.45
3.56 1.56
0.36 4.11
5.77 2.05
3.90 5.02
11.14 2.88
8.31 4.80
3.34 1.76
20.31 4.59
81.27 1.54
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
(
VIII), were prepared by oxidation of ursolic acid (I)
)
and benzyl ursolate (VI) with CrO3 in acetoneꢀCH2Cl2
)
(Scheme 1).
V
)
The majority of the tested compounds manifested
no inhibitory effect on rhAR. However compound
VI
VII
VIII
IX
)
)
(
XV) exhibited 81.27% inhibition (at 20 μM concenꢀ
tration), which surpassed the positive control value
(Table 1). The rhAR inhibition for 4ꢀaminobutyric
acid derivative (XV) is superior over the same for
)
)
X
)
βꢀalanine derivative (XIV) (20.3% at 20
μM concenꢀ
XII
XIII
XIV
)
tration) and glycine derivative (XIII) (3.3% at 20
μM
)
concentration). It seems probable, that carbon chain
elongation in the tested series of aminoacids derivaꢀ
tives results in rhAR inhibition enhancing.
)
20
10
The inhibitory potency of compound (XV) on
71.20 2.57
54.50 2.14
34.88 1.88
68.40 1.48
(
XV
)
4.44
8.75
rhAR as expressed by IC50 values, was 4.44
μM, which
5.0
2.5
20
10
5.0
2.5
exceeded significantly the same for the soꢀcalled tetꢀ
ramethyleneglutaric acid (TMG) (IC50 = 8.75 μM
) as
a positive control.
53.12 0.81
37.08 3.20
22.51 2.54
To determine kinetic mode of action, the inhibitory
activity of compound (XV) against rhAR using the
LineweaverꢀBurk plots was tested as shown in Figure.
TMG
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

572
EUN HA LEE et al.
(
VII) (Scheme 1). To a suspension of ursolic acid (I)
100
90
80
70
60
50
40
30
20
10
(1.0 g, 2 mmol) in DMF (10 ml) anhydrous powdered
K2CO3 (0.45 g, 3.3 mM) and corresponding alkyl
chloride (2.0–2.1 mmol) were added. The mixture was
stirred at ambient temperature for 3–7 h (TLC moniꢀ
toring). The reaction mixture was poured in water and
the precipitate was washed with water (3 × 20 ml) to
0
4
6
8
µ
µ
µ
µ
M
M
M
M
remove residual solvent and salts. The precipitate was
dried on open air and percolated through a short aluꢀ
mina column to give after concentration the ursolic
acid ester as a white powder, which was crystallized
from an appropriate solvent.
0
5
10
15
20
25
Method B
ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoyl]ꢀ
XV (Scheme 2). To a suspension of amino acid ester
hydrochloride (4.3–4.5 mmol), pyridine 0.6
(7.4 mmol) and triethylamine (0.6 g, 6 mmol) in
CH2Cl2 (10 ml), ꢀacetoxyꢀursꢀ12ꢀenꢀ28ꢀoyl chloꢀ
.
General procedure for the synthesis of Nꢀ
1/[S], mM–1
[3
(
β
ωꢀamino acids (XIII)–
)
LineweaverꢀBurk plots from the kinetic studies of rhAR in
the presence of compound (XV). Results are the represenꢀ
tatives of three independent experiments.
g
3β
ride (XI) (1 g, 1.9 mmol) was added. The mixture was
kept at ambient temperature for 5 h and evaporated in
EXPERIMENTAL
vacuum to give esters of
oyl] ꢀamino acid. A solution of KOH (0.4 g,
7.2 mM) in ethanol (20 ml) was added to the obtained
residue. The mixture was refluxed for 1 h, cooled to
ambient temperature and concentated in vacuum. The
residue was washed with 1 N aqueous HCl (30 ml),
Nꢀ[3βꢀacetoxyursꢀ12ꢀenꢀ28ꢀ
1H and 13C NMR spectra were registered on a
ꢀω
Bruker AVꢀ300 (300.13 MHz for 1Н, 75.48 MHz for
13C) and Bruker AMꢀ400 (400.13 MHz for 1Н,
100.62 MHz for 13C) instruments at room temperature
in CDCl3 or pyridineꢀd5. The chemical shifts are given
in ppm relative to signals of the solvents used as interꢀ
nal standards: in 1H NMR spectra δH 7.24 (CHCl3) or
δН 7.15 (pyridine) and in 13C NMR spectra δC 76.90
water (
hydroxyursꢀ12ꢀenꢀ28ꢀoyl]ꢀ
which were crystallized from an appropriate solvent.
Ethyl (3 ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoylꢀoxy)acetate
(III) was prepared in reaction of ursolic acid ( ) with
ethyl 2ꢀchloroacetate (Method A). Yield 0.92 g (84%),
white powder, mp 176–177 (MeCN), +68
α 25
1.0, CHCl3). 1H NMR and 13C NMR data are given
in the Tables 2 and 5.
Potassium 2ꢀ(3 ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoylꢀoxy)aceꢀ
tate (IV). To a solution of KOH (0.5 g) in EtOH
(20 ml), ethyl(3 ꢀhydroxyursꢀ12ꢀenꢀ28ꢀoylꢀoxy)aceꢀ
3
×
20 ml) and dried on open air to give
Nꢀ[3βꢀ
ωꢀamino acid as powders,
(CDCl3) or δC 123.50 (pyridineꢀ
Hertz. Signals in the NMR spectra were assigned by
correlation with those of ursolic acid ) [17, 18],
ꢀacetoxyursꢀ12ꢀenꢀ28ꢀoic acid (II) [19], benzyl
d5). J values are given in
β
I
(I
3
β
°C
[ ]
ursolate (VI) [20], and methyl 3ꢀoxoursꢀ12ꢀenꢀ28ꢀ
oate [21].
(c
Melting points were measured on a Kofler melting
point apparatus and are uncorrected. Optical rotation
was measured on a PolAAR 3005 polarimeter (Great
Britain) at 589 nm in a 5 cm long tube. TLC was carꢀ
ried out on Sorbfil plates (Russia). Purity of the tested
β
β
tate (III) (1.0 g, 1.8 mM) was added. The mixture was
refluxed for 0.5 h, and left to cool to ambient temperꢀ
ature. The needles of K salt (IV) were filtered and
washed with cold EtOH.
compounds was ≥
90% (by 1H NMR).
Reagents. DLꢀGlyceraldehyde, NADPH were purꢀ
chased from Sigma Chemical (USA). Ampicillin,
IPTG and imidazole were purchased from USB Corꢀ
poration (USA). Human ALR2 cDNA clone was purꢀ
chased from 21C Frontier Human Gene Bank (Rep.
(3βꢀHydroxyursꢀ12ꢀenꢀ28ꢀoylꢀoxy)acetic acid (IVa).
Washing of (IV) with 10% HCl and distilled water and
drying on air resulted in acid (IVa). Yield 73%, white
powder, mp 175°C (dec.), [ ] +52 (c 1.0, MeOH).
α 25
of Korea). Ursolic acid (
extracts of lingonberry (Vaccinium vitisꢀidaea) fruits
peels as described [22]. ꢀAcetoxyursꢀ12ꢀenꢀ28ꢀoic
acid (II), benzyl ursolate ( ), ursonic acid (IX), and
ꢀacetoxyursꢀ12ꢀenꢀ28ꢀoyl chloride (XI) were preꢀ
pared from ursolic acid ( ) according to the published
methods [23–25]. Methyl corosolate ( ) was isolated
I) was isolated from the
1H NMR and 13C NMR data are given in the Tables 2
and 5.
3β
V
Glycidyl 3
prepared as diastereomeric mixture (1 : 1) in reaction
of ursolic acid ( ) and racemic epichlorohydrine
(3.02 g, 23.8 mM) (Method A). Yield 0.92 g (84%),
white powder, mp 102–103 (MeCN), +44
α 25
1.0, CHCl3). 1H NMR and 13C NMR data are given
βꢀhydroxyursꢀ12ꢀenꢀ28ꢀoates (V) were
3β
I
I
X
from sea buckthorn (Hippophae rhamnoides) leave
methanolic extract as described [26]. All other chemiꢀ
cals were of analytical grade. All the solvents used were
reagent quality.
°C
[ ]
(c
in the Tables 2 and 5.
4ꢀNitrobenzyl
3βꢀhydroxyursꢀ12ꢀenꢀ28ꢀoate
Syntheses. Method A. General procedure for the synꢀ (4ꢀnitrobenzyl ursolate) (VII) was prepared in reaction
thesis of alkyl 3βꢀhydroxyursꢀ12ꢀenꢀ28ꢀoates (III)– of ursolic acid (I) with 4ꢀnitrobenzyl chloride
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

INHIBITORY EFFECT OF URSOLIC ACID DERIVATIVES
Table 2. 1H NMR data for compounds (II), (III), (IVa
573
)
,
(V) (δH
(J))
Atom No.
3
(
II) (pyridineꢀd5
)
(
III
)
(
CDCl3)
(
IVa) (pyridineꢀd5
)
(
V
)
(
CDCl3)
3.18 dd (10.2, 4.5); 3.18 dd
(10.2, 4.5)
4.61 dd (10.7, 5.0)
1.81 dd (8.4, 2.7) (2H)
5.39 dd (3.1, 3.1)
3.19 dd (10.9, 5.2)
1.87 dd (8.8, 3.6) (2Í)
5.22 dd (3.6, 3.6)
3.39 dd (9.3, 6.8)
11
12
1.88 dd (8.6, 3.7); 1.88 dd (8.6,
3.7) (2H)
5.24 dd (3.8, 3.8); 5.24 dd (3.8,
3.8)
5.36 dd (3.5, 3.5)
2.06§ ddd (13.2, 13.2, 3.3) (Hax)
2.23§ ddd (13.2, 13.2, 4.4) (Hax)
15
16
18
23
2.55 d (11.4)
0.84# s
0.81# s
0.94 s
0.76# s
1.16 s
0.94‡ d (6.4)
0.92‡ d (6.4)
2.23 d (11.3)
0.96§ s
0.75§ s
0.89§ s
0.71§ s
1.05§ s
0.83‡ d (6.5)
0.91‡ d (AB3,
2.47 d (11.3)
1.13§ s
0.88§ s
0.97§ s
0.87§ s
1.18§ s
0.89# d (6.4)
2.22 d (11.7); 2.21 d (10.9)
0.89§ s; 0.89§ s (3H)
0.75§ s; 0.75§ s (3H)
0.96§ s; 0.96§ s (3H)
0.73§ s; 0.72§ s (3H)
1.06 s; 1.06 s (3H)
24
25
26
27
29
30
0.83# d (6.4); 0.83# d (6.4) (3H)
J
BA = 6.0 0.87# m
0.91# d (AB3,
J
BA = 5.8); 0.91#
(3H))
d (AB3, BA = 5.8 (3H))
J
1'
2'
–
–
–
2.80 dd (4.9, 2.9); 2.78 dd (5.0,
2.9) (H1'trans); 2.59 dd (4.9, 2.6);
2.61 dd (5.0, 2.6) (H1'
)
cis
–
4.49 (AB, ΔνAB = 19.6 4.95 (AB, ΔνAB = 29.4 3.15 m; 3.13 m
Hz, AB = 15.7 (2H)) Hz, AB = 15.6 (2H))
J
J
1.99 br.s (OC(O)CH3)
4.16 q (7.2) (OCH2CH3)
4.31 dd (12.2, 3.3); 4.29 dd
(12.2, 3.3) (H3'a); 3.82 dd (12.2,
6.0); 3.80 dd (12.2, 6.3) (H3'b)
1.24 t (7.2)
(OCH2CH3)
§, #, ‡
Signals marked with the same symbols may be exchanged within the column.
(Method A). Yield 1.03 g (86%), white powder, mp added. The mixture was kept at ambient temperature
25
214–215
°
C
(MeCN),
[α]
+36 (c 1.0, CHCl3). for 3 h, washed with water (
2 × 50 ml), dried
1H NMR and 13C NMR data are given in the Tables 3
(
(
Na2SO4), and concentrated to give after percolation
and 5.
Al2O3, CCl4) imidazolide derivative (XII) (0.82 g,
25
87%), mp 204–205
°
C
(EtOH),
[α]
+18 (c 1.0,
Benzyl 3ꢀoxoursꢀ12ꢀenꢀ28ꢀoate (benzyl ursonate)
(VIII). To a stirred suspension of benzyl ursolate (VI)
(5 g, 9.1 mmol) in CH2Cl2 (20 ml) and acetone
(20 ml), a solution of Jones reagent (9 ml, 3 equiv.) was
CHCl3). 1H NMR and 13C NMR data are given in the
Tables 3 and 5.
N
ꢀ(3
β
ꢀHydroxyursꢀ12ꢀenꢀ28ꢀoyl)glycine (XIII)
added at 5°C in 0.5 h. Reaction was monitored by
was prepared by reaction of
3βꢀacetoxyursꢀ12ꢀenꢀ28ꢀ
TLC and after 1 h a mixture of isopropyl alcohol
(20 ml) and water (20 ml) was added. The mixture was
stirred for 1 h, diluted with water (100 ml), and
oyl chloride (XI) with ethyl glycinate hydrochloride
and subsequent hydrolysis (Method B). Yield 0.72 g
(82%), white powder, mp 192–194°C (EtOH), [192–
extracted with CH2Cl2 (3
extract was concentrated, percolated (Al2O3, CCl4
and evaporated to give benzyl ursonate (VIII) .6 g
(83%), white crystals, mp 158–160 (MeCN),
]25 +76 ( 1.0, CHCl3). 1H NMR and 13C NMR data
are given in the Tables 3 and 5.
ꢀ(3 ꢀAcetoxyursꢀ12ꢀenꢀ28ꢀoyl)imidazole (XII). ride and subsequent hydrolysis (Method B). Yield
To a solution of imidazole 0.2 g (2.9 mmol) and pyriꢀ 0.8 g (78%), white amorphous powder, mp 182–204
dine 0.1 g (1.2 mmol) in CH2Cl2 (20 ml), ꢀacetoxyꢀ (dec.),
α]25 +53 ( 1.0, EtOH). 1H NMR and
ursꢀ12ꢀenꢀ28ꢀoyl chloride
XI) 1 g (1.9 mmol) was 13C NMR data are given in the Tables 4 and 5.
× 50 ml). The organic
194
13C NMR data are given in the Tables 4 and 5.
° c
C (EtOH), [α]25 +53 ( 1.0, CHCl3). 1H NMR and
)
4
°С
N
ꢀ(3 ꢀHydroxyursꢀ12ꢀenꢀ28ꢀoyl)ꢀ ꢀalaninate (XIV)
β
β
[α
c
was prepared by reaction of ꢀacetoxyursꢀ12ꢀenꢀ28ꢀ
3β
oyl chloride (XI) with methyl ꢀalaninate hydrochloꢀ
β
N
β
°
C
3β
[
c
(
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574
EUN HA LEE et al.
Table 3. 1H NMR data for compounds (VII), (VIII), (XII) (δH
(
J
), CDCl3)
VIII
AB = 16.0,
(H2ax)); 2.39 ddd (ABXY,
BÀ = 16.0,
BY = 3.8 (H2eq))
Atom No.
2
(VII
)
(
)
(XII)
2.56 ddd (ABXY,
J
J
AX = 10.9,
JAY = 7.2
J
JBX = 6.9,
J
3
11
12
16
3.18 dd (11.0, 4.7)
5.22 dd (3.3, 3.3)
–
4.45 dd (10.0, 6.0)
5.20 dd (3.5, 3.5)
1.93 dd (8.3, 3.6)
5.27 dd (3.6, 3.6)
2.29 ddd (14.2, 14.2, 4.2) (H16ax
)
18
23
2.23 d (11.1)
0.95§ s (3H)
0.84§ s (3H)
0.55§ s (3H)
0.74§ s (3H)
1.05§ s (3H)
0.84# d (6.0) (3H)
2.30 d (11.2)
1.10 s (3H)
2.43 d (11.1)
0.82§ s (3H)
0.81§ s (3H)
1.06§ s (3H)
1.04§ s (3H)
24
25
26
27
29
0.90 s (3H)
0.70 s (3H)
0.65 s (3H)
1.07 s (3H)
1.10 s (3H)
0.98# d (AB3,
JBA = 6.3 (3H))
0.87 d (6.4) (3H)
0.92# d (AB3,
J
BA = 5.3 (3H))
0.89# d (6.3) (3H)
8.23 br.s
'
0.95 d (A'B3, JB'A' = 5.8 (3H))
30
1'
5.07 (A''B'', ΔνA''B'' = 35.5 Hz,
J
A''B'' = 12.4 (2H))
5.10 (A'B', ΔνA'B = 46.0 Hz,
'
JA'B' = 13.4 (2H))
2'
3'
4'
5'
–
7.48 d (8.6) (2H)
8.19 d (8.6) (2H)
–
–
7.36 m (2H)
7.02 br.s
7.52 br.s
7.36 m (2H)
–
7.36 m (1H)
–
2.02 s (3H) (OC(O)CH3)
§, #
Signals marked with the same symbols may be exchanged within the column.
Table 4. 1H NMR data for compounds (XIII), (XIV), (XV) (δH
(J
), pyridineꢀd5
)
Atom No.
(
XIII
)
(
XIV
)
(XV)
3
12
18
23
3.39 dd (7.8, 7.8)
5.50 dd (3.0, 3.0)
2.34 d (10.7)
3.37 dd (9.6, 6.3)
5.42 dd (3.2, 3.2)
2.21 d (10.7)
3.38 dd (10.0, 6.0)
5.42 dd (3.0, 3.0)
2.31 d (10.8)
1.14§ s
1.13§ s
1.13§ s
0.93§ s
0.96§ s
0.85§ s
1.17§ s
0.89# d (6.4)
0.85# m
0.95§ s
0.96§ s
0.87§ s
1.17§ s
0.88# d (5.8)
0.87# m
0.95§ s
0.95§ s
0.90§ s
1.17§ s
0.88# d (6.5)
0.87# m
24
25
26
27
29
30
2'
4.37 (AXY, ΔνXY = 73.4 Hz,
YA = 3.9 (2H))
JXY = 17.7,
J
XA = 5.1, 2.75 ddd ((6.2, 6.2, 1.9) (2H)) 2.54 t (7.3 (2H))
J
3'
4'
–
–
3.75 m (2H)
–
3.49 m (2H)
NH
7.70 (AXY, JAX
=
J
AY = 4.6)
7.33 dd (5.6, 5.6)
7.28 dd (4.6, 4.6)
§, #
Signals marked with the same symbols may be exchanged within the column.
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INHIBITORY EFFECT OF URSOLIC ACID DERIVATIVES
575
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576
EUN HA LEE et al.
Nꢀ(3β
ꢀHydroxyursꢀ12ꢀenꢀ28ꢀoyl)ꢀ4ꢀaminobutyric ted against inhibitory activity. TMG (tetramethylene
acid (XV) was prepared by reaction of 3βꢀacetoxyꢀursꢀ glutaric acid) was used as positive control.
12ꢀenꢀ28ꢀoyl chloride (XI) with methyl 4ꢀaminobuꢀ
tanoate hydrochloride and subsequent hydrolysis
(Method B). Yield 0.79 g (75%), white powder, mp
ACKNOWLEDGMENTS
167–170°C (EtOH), [ c
α]25 +60 ( 1.0, EtOH). 1H NMR
This research was financially supported by the
Ministry of Education, Science Technology (MEST),
Gangwon Province, Gangneung City, Gangneung
Science Industry Foundation (GSIF) as the R&D
Project for Gangneung science park promoting proꢀ
gram.
and 13C NMR data are given in the Tables 4 and 5.
Preparation of rhAR. Open reading frame of
human AR was amplified by PCR and inserted into
E. coli expression vector pETꢀ23b (Merck, Darmsꢀ
tadt, Germany). Then the recombinant expression
plasmid was transformed into E. coli BL21(DE3) host
strain. The transformant was grown in LB broth conꢀ
taining 50 μg/ml of ampicillin at 37°C, 200 rpm.
When the absorbance at 600 nm reached 0.8, the
expression of recombinant AR was induced by IPTG
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