Synthesis and aldose reductase inhibitory activity of botryllazine A derivatives

Article abstract of DOI:10.1248/cpb.c19-00003

Aldose reductase (AR) is associated with the onset of diabetic complications. Botryllazine A and its analogues were synthesized and evaluated for human AR inhibitory activity. Analogues possessing aromatic bicyclic systems at the C5 position of the centra

Full text of DOI:10.1248/cpb.c19-00003

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Chem. Pharm. Bull. 67, 556–565 (2019)
Vol. 67, No. 6
Regular Article
Synthesis and Aldose Reductase Inhibitory Activity of Botryllazine A
Derivatives
,a,b
Ryota Saito,* Kana Ishibashi,a Maiko Noumi,a Sota Uno,a Shoko Higashi,a Masaru Goto,c
Shunsuke Kuwahara,a,b and Toshiya Komatsud
a Department of Chemistry, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: b Research Center
for Materials with Integrated Properties, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan:
c Department of Biomolecular Science, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: and
d Faculty of Pharmaceutical Sciences, Teikyo Heisei University; 4–21–2 Nakano, Nakano-ku, Tokyo 164–8530, Japan.
Received January 4, 2019; accepted March 26, 2019
Aldose reductase (AR) is associated with the onset of diabetic complications. Botryllazine A and its
analogues were synthesized and evaluated for human AR inhibitory activity. Analogues possessing aromatic
bicyclic systems at the C5 position of the central pyrazine ring exhibited superior AR inhibiting activity
relative to the parent botryllazine A. In addition, the benzoyl groups at positions C2 and C3 of the pyrazine
ring were dispensable for this improved inhibitory activity. Conversely, a benzoyl group—containing pheno-
lic hydroxyl groups—at either position C2 or C3 of the pyrazine ring was essential for attainment of high
inhibitory activity approaching that of sorbinil (a highly effective AR inhibitor).
Key words aldose reductase (AR); aldose reductase inhibitor (ARI); diabetic complication; botryllazine A;
pyrazine; docking study
tual development of diabetic complications.2,3)
Inhibition of AR has thus been considered a useful strategy
Introduction
Diabetes mellitus (DM) is a metabolic abnormality char-
acterized by a steady hyperglycemic state, which can cause for attenuating development of diabetic complications. Various
severe complications including neuropathy, nephropathy, and AR inhibitors (ARIs) have been developed, and most highly
retinopathy.1) Under normoglycemic conditions, most circu- potent ARIs contain either a glycine unit (–NHCH2COOH) or
lating glucose molecules are metabolized via glycolysis to a spirohydantoin skeleton as key structures.4) Under conditions
produce ATP, while only approximately 5% are metabolized of physiological pH, protons dissociate from such functional
via the polyol pathway (in which glucose is reduced to sorbi- groups, and resulting corresponding conjugate bases tightly
tol by reduced nicotinamide adenine dinucleotide phosphate bind the AR active site via hydrogen-bonding interactions.4)
(NADPH)-requiring aldose reductase (AR), followed by con- However, unfavorable adverse effect profiles, low efficacy,
version of sorbitol to fructose by NAD+-requiring sorbitol and toxicity have resulted in most compounds exhibiting such
dehydrogenase (SDH))2,3) (Fig. 1). Hyperglycemic conditions, architecture being inappropriate for clinical use.5–9) Thus, de-
however, overwhelm the glycolytic system and activate AR velopment of alternate high-potency ARIs is desirable. In the
to convert excess glucose into sorbitol, elevating flux through present study, we focused on the phenolic hydroxyl groups of
this alternate pathway (under hyperglycemic conditions, 11% botryllazines A and B derived from Botryllus leachi.10) These
of glucose is metabolized via the polyol pathway in human compounds possess electron-withdrawing pyrazine or carbon-
erythrocytes).3) Because the sorbitol to fructose conversion yl groups at the p-position of the phenolic hydroxyl groups,
rate remains unchanged (i.e., lags behind sorbitol formation), favoring proton dissociation and formation of phenolate anions
and highly hydrophilic sorbitol cannot easily diffuse across capable of interacting with the AR active site at physiological
cell membranes, an abnormal accumulation of intracellular pH. Based on this idea, we have previously synthesized and
sorbitol leads to osmotic stress. In addition, chronic accel- evaluated the AR inhibitory activity of botryllazine B and its
eration of the polyol pathway results in an increased level of derivatives.11) However, no ARI structure–activity relationship
advanced glycation end products (AGEs), formed by reaction (SAR) study of botryllazine A has been conducted. In the
of proteins with accumulating fructose, thereby favoring even- present study, we have thus synthesized several botryllazine
Fig. 1. Polyol Pathway of Glucose Metabolism
*To whom correspondence should be addressed. e-mail: saito@chem.sci.toho-u.ac.jp
© 2019 The Pharmaceutical Society of Japan

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A derivatives (Fig. 2) and evaluated their inhibitory activities 1a–c) were started from 2,3-dichoropyrazine, which was lithi-
against recombinant human AR, thereby providing the first ated using lithium tetramethylpiperidide (LiTMP), followed
SAR data for this family of compounds.
by reaction with anisaldehyde to produce the corresponding
alcohol 3, as shown in Chart 1.12) These steps were repeated
to produce a diol 4,12) which was oxidized using 2-iodoxyben-
Results and Discussion
The synthesis of botryllazine A and its derivatives (Fig. 2: zoic acid (IBX) to produce an unpurified intermediate 5. A
Suzuki–Miyaura coupling reaction using appropriate boronic
acids produced further intermediates 6a–c.
Initially, reductive dechlorination of 6a was attempted using
the method reported by Buron et al.12) However, this approach
produced a low yield (4.3%) of desired product 7a, while un-
desired pyrazinone derivative 8a was the major product (Table
1, entry 1). The formation of 8a was apparently due to partici-
pation of a small volume of water—added to the catalyst Pd/C
to avoid spontaneous combustion—in the reaction. It thus ap-
pears that the quality or condition of the catalyst significantly
Fig. 2. Structures of Botryllazine A (1a) and Derivatives (1b, 1c, and
2a–c) Examined during the Present Study
influences the reaction. In order to minimize participation of
water in the reaction, a dried matrix-embedded Pd/C catalyst
Chart 1. Synthesis of Key Intermediates 6a–c en Route to Production of Botryllazine A and Its Derivatives
Table 1. Optimizing Reductive Dechlorination of 6a–c
Entry
Chloride
Equivalent of Pd/C
Solventa)
Hydrogen source
Additive
Yield
1
2
3
4
5
6
7
8
6a
6a
6a
6a
6a
6a
6b
6c
0.1
Acetone
Acetone
Acetone
THF
HCOOH
HCOOH
HCOOH
HCOOH
HCOOH
HCOOH
HCOOH
HCOOH
—
—
7a (4.3%)
7a (42%)
—
8a (61%)
8a (37%)
—
0.1b)
0.1b)
0.1b)
0.1b)
0.05
0.05
0.05
—
—
—
—
Acetone
Acetone
Acetone
Acetone
MS3A
—
7a (29%)
7a (20%)
7b (—)
7c (54%)
—
8a (19%)
8b (54%)
8c (37%)
—
—
a) All solvents were dehydrated. b) Matrix carbon, dry-support (purchased from Sigma-Aldrich).

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Chart 2. Dehydroxylation Reactions of 8b
Table 2. Demethylation Reactions of 7a–c and 8a–c
Fig. 3. An ORTEP Diagram of 1b
(Color figure can be accessed in the online version.)
was employed (Table 1, entry 2). While this improved the
yield (42%) of 7a, a significant quantity of the hydroxylated
product 8a was still formed. When examining the effects of
solvent-mediated drying (Table 1, entries 3 and 4) the reac-
tion failed to proceed altogether. Instead, addition of a 3Ǻ
molecular sieve to the reaction mixture successfully enhanced
anhydrous conditions and suppressed formation of pyrazinone
8a, but also depressed the yield (29%) of 7a (Table 1, entry 5).
Another dechlorination condition—using triethylsilane as the
hydrogen source, in combination with palladium acetate13)— chloride-mediated deprotection to produce botryllazine A and
yielded no improvement (data not shown). Using the original its analogues 1a–c (Table 2). Pyrazinones 8a–c were also sub-
condition in conjunction with a lower Pd/C concentration jected to the same deprotection reactios to produce 2a–c, the
(0.05eq instead of 0.1eq) (Table 1, entry 6), 6c conversion corresponding C6-hydroxylated derivatives of 1a–c (Table 2).
still resulted in the hydroxylated product, but the yield of 7c
We also synthesized compound 10, which is a position iso-
improved (54%) (Table 1, entry 8). We thus concluded that, mer of botryllazine B, to investigate the effect of the C3 sub-
during palladium-catalyzed dechlorination of structure 6-type stituent in botryllazine A (1a). The synthetic route is shown
chloropyrazines, formation of the hydroxylation product in Chart 3. 5-(4-Methoxyphenyl)pyrazine-2-carboxylic acid
could be only partially attenuated, and only an unsatisfactory (11)18) was first converted into amide 12, which was reacted
improvement in yield of the desired reaction products was with 4-methoxyphenylmagnisiumbromide to give 13. Then,
achievable.
methyl protecting groups in 13 were deprotected with pyri-
We therefore adopted a new synthetic strategy (conversion dinium chloride to afford 10.
of the hydroxylated products) to obtain 7b (Chart 2). This ap-
The AR inhibitory activities of 1a–c, 2a–c, and 10 were
proach first converted the pyrazinone 8b into the correspond- evaluated in vitro, by measuring inhibition of human AR-
ing triflate 9b, which were then successfully reduced via the mediated D,L-glyceraldehyde reduction in the presence of
triethylsilane-Pd(dppf)2Cl2 system,14–17) providing a satisfac- cofactor NADPH.19) Consumption of NADPH—a validated
tory yield of 7b (Chart 2). Fortunately, 2-naphthyl derivative and reliable proxy for progress of this reaction—was spectro-
1b provided single crystals suitable for X-ray crystallographic photometrically monitored.11) Inhibitory activities were quan-
analysis. The structure was successfully solved (Fig. 3), unam- titatively expressed as IC50 values. Results are shown in Table
biguously confirming successful dehydroxylation of the C6- 3, accompanied by reference IC50 values of botryllazine B and
hydroxylated byproducts.
epalrestat.
Obtained products 7a–c were then subjected to pyridinium
The IC50 value of botryllazine A (1a) was estimated at

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559
Reagents and conditions: (i) thionyl chloride, reflux; (ii) morpholine, CH2Cl2, 0°C to r.t.; (iii) 4-(OMe)C6H4MgBr, THF, −100°C to r.t., then H3O+; (iv) pyridinium chlo-
ride, 210°C.
Chart 3. Synthesis of Compound 10
Table 3. In Vitro AR Inhibitory Activities of Botryllazine
A Derivatives (1a–c and 2a–c), Botryllazine B, 10, and
Epalrestat
the hydroxybenzoyl group at the C2 position resulted in an
increment in inhibitory activity (botryllazine B11) versus 1a),
suggesting that a second benzoyl group (in addition to the one
present at the C3 position) is dispensable for enhanced AR
inhibitory activity. Moreover, removal of the hydroxybenzoyl
group from the C3 position of 1a also resulted in an increment
of the inhibitory activity (10 vs. 1a), and the IC50 value was
almost the same as that of botryllazine B,11) suggesting that
two benzoyl groups on C2 and C3 are unnecessary and only
one benzoyl group on either C2 or C3 is effective for gaining
high AR inhibitory activity.
To develop an understanding of the relationship between
botryllazine A analogue structure and AR inhibitory activ-
ity, docking studies were carried out using known human
AR X-ray crystal structures. Reported AR crystal structures
demonstrate that the active site can be divided into three sub-
regions: catalytic, hydrophobic, and specificity pockets. The
catalytic pocket, also referred to as an anion-binding pocket,
is composed of Tyr48, His110, Trp20, and Trp111 side chains,
and is positioned in proximity to cofactor NADPH. Key struc-
tures (glycine units and spirohydantoins) of the highly potent
ARIs bind this pocket in their conjugate base forms at physi-
ological pH. The adjacent hydrophobic pocket is composed of
Trp20, Phe122, and Trp219 residues; therefore, a requirement
of highly potent ARIs is the presence of a hydrophobic moi-
ety in addition to their acidic groups.4,9,22–24) The specificity
pocket is adjacent to the other two pockets,4) and has a flex-
ible structure to accommodate various ligands with diverse
structures, whereas architecture of the catalytic pocket is
near-identical in most reported crystal structures. This ligand-
induced-fit function is achieved by movement of the Cys298–
6.47 µM, an approximately 3-fold higher level of inhibitory Cys303 loop region, such that AR is able to assume two dis-
activity than previously-reported (19.4µM).20) Such disagree- tinct specificity pocket states (based on the position of these
ment likely results from differences in experimental condi- residues): “open” and “closed.”24) While hundreds of crystal
tions. In the present study, the bioassay was carried out in a structures of human AR containing various kinds of inhibitors
1cm quartz cell (total volume 1.0mL) at 25°C, while previ- have been registered with the Protein Data Bank (PDB), AR
ous studies employed 96-well microtiter plates (total volume ligand-binding modes can be classified into five types (PDB
0.2mL) at 37°C.20) Furthermore, enzyme concentration in the structures 1PWM, 1Z3N, 2FZD, 2NVC, and 2NVD) based on
present study (1.0–1.2U/mL) differed from that in the previous specificity pocket state (open or closed) and ligand orientation
study (0.5mU/mL). However, since the IC50 value of positive in the active site.25,26) These PDB structures are frequently
control epalrestat estimated by the present study agrees with used in AR docking studies,24,27,28) and were thus employed
previously-reported values, this verifies that current study by the present study as well. The present study employed one
experimental data represent absolute values.
additional crystal structure recently reported by Klebe and
All examined compounds exhibited high inhibitory activity, colleagues (PDB ID: 4IGS),29) given that the binding moiety of
with IC50 values in the range of 1.34–9.64µM (i.e., comparable the ligand in this structure is a phenolic hydroxy group (con-
to that of sorbinil, a highly-effective AR inhibitor).9,21) Intro- sidered a key structure by the present study).
ducing a bicyclic heterocycle substituent at the C5 position
Prior to carrying out docking simulations, ligand optimized
marginally enhanced inhibitory activity (1b, 1c, 2b, and 2c), structures and atom charges were calculated using the PM7
but the presence of a hydroxy group at the C6 position did method.30–32) All docking experiments were performed using
not markedly alter inhibitory activity (activity comparison GOLD Suite software.33,34) The ChemPLP scoring function35)
of 2a–c—which possess a C6 hydroxy group—with corre- was used to predict docking poses for each of the six specific-
sponding C6-unhydroxylated 1a–c). Furthermore, removal of ity pocket states, and these were then rescored using the Astex

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Table 4. Astex Statistical Potential (ASP) Fitness Scores for Docking of 1a–c, 2a–c, and Botryllazine B with Six Different Protein Data Bank (PDB)
Protein Structures, Including Their Correlation (r2) with Empirical pIC50 Values
Protein structure (state of the specificity pocket)
Compound
1PWM (closed)
1Z3N (open)
2FZD (open)
2NVC (closed)
2NVD (closed)
4IGS (closed)
1a
115.5816
117.2774
121.7395
115.1591
121.0703
125.6161
108.2904
100.8451
115.0713
109.9514
109.3466
115.0403
117.0648
97.7533
110.3716
104.9774
107.417
107.1515
109.2310
111.3878
110.6786
110.1408
112.6584
100.7796
91.8687
99.4169
96.1964
93.6458
98.1937
100.4004
105.9115
105.4701
107.2104
109.1394
104.3201
108.4239
113.1883
94.6767
1b
1c
2a
109.7031
107.5076
107.4877
105.9444
2b
2c
Botryllazine B
r
0.761
0.306
0.169
0.373
0.510
0.910
Fig. 4. Predicted Binding Modes for 1a–c and 2a–c Docked into 4IGS
(Color figure can be accessed in the online version.)
Statistical Potential (ASP)36) scoring function, validated for between docking scores and experimental pIC50 values was
protein-ligand specific hydrogen bond scoring.37) The combi- poor (r=0.761), whereas a strong correlation was observed
nation of these scores was used to determine the optimal pose for the 4IGS AR structure (r=0.910). A possible criticism is
of each ligand (i.e., for which both scoring functions yielded that the GOLD analysis should consider “flexible” dockings
positive, high scores) (Table 4).
using the software’s Side Chain Flexibility function, but we
Botryllazine B, which exhibited high AR inhibitory activ- previously demonstrated that the better method for predicting
ity, yielded lower docking scores than the other ligands. This an appropriate protein structure that accounts for experimental
is attributed to the smaller molecular size of botryllazine results is assessment of the docking score-pIC50 correlation.38)
B (producing fewer points of interaction with the AR ac-
Docking positions for 1a–c and 2a–c with 4IGS demon-
tive site) relative to the botryllazine A derivatives (1a–c and strated that 2a, which exhibited the lowest inhibitory activity
2a–c), making direct comparison of their docking scores of the examined botryllazine A derivatives, was predicted to
meaningless. However, comparison of botryllazine A deriva- assume a different position relative to the other derivatives
tives’ docking scores is of utility, given their strong structural (Fig. 4). The hydroxy group of the C5 phenol of 2a forms a
resemblances. Closer examination of 1a–c and 2a–c docking hydrogen bond with His110 of the catalytic pocket, while the
scores demonstrated that almost all compounds exhibited op- other derivatives form hydrogen bonds with His110 via the
timal fit with the 1PWM AR structure. However, correlation hydroxy group of the C2 benzoyl groups (reinforced by C5

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561
aryl scaffolds adjacent to Trp219 of the hydrophobic pocket).
Synthesis
of
2,3-Dichloro-5,6-bis(1-oxo-4-methoxy-
According to our previous pharmacophore modeling analysis phenylmethyl)pyrazine (5) A solution of n-BuLi in hexane
of 4IGS, a lipophilic interaction with Trp219 is an important (2.6M, 18.0mL, 46.8mmol) was added to stirred anhydrous
feature conferring additional inhibitory activity.38) Indeed, the tetrahydrofuran (THF) (30mL) at −40°C under an atmosphere
quinolin-3-yl residue of compound 1c, the most potent among of Ar. Then, 2,2,6,6-tetramethylpiperidine (8.2mL, 48.2mmol)
1a–c, forms a π–π interaction with the indole ring of Trp219. was added to the solution. The mixture was warmed to 0°C.
The naphthalen-2-yl group of 1b also occurs in proximity to After 30-min stirring at this temperature, the mixture was
Trp219, and the same trend is observed for 2b and 2c. Based cooled to −80°C and a solution of 3 (2.69g, 9.43mmol) in
on these results, we conclude that an aromatic face-to-face in- THF (5.0mL) was added. After 30min at −80°C, p-anisal-
teraction between the C5 aromatic ring of a botryllazine A de- dehyde (1.3mL, 10.7mmol) dissolved in THF (0.5mL) was
rivative and the hydrophobic pocket Trp219 residue—as well added and the stirring was continued for 4h at −80°C. The
as hydrogen bonding between one hydroxybenzoyl group and crude product was treated with a solution of saturated sodium
the catalytic pocket His110—enhances AR inhibitory activity hydrogenosulfite (20mL). The mixture was filtrated and the
of botryllazine A-based compounds.
residue was washed with dichloromethane (DCM). After sepa-
rating the organic and aqueous phases, the aqueous layer was
extracted with DCM (3×20mL). The combined organic ex-
Conclusion
In the present study, botryllazine A derivatives were tracts were then dried over sodium sulfate and evaporated. To
synthesized and evaluated for AR inhibitory activity. The the crude product in MeCN (45.0mL) was added IBX (6.60g,
previously-reported botryllazine A preparation method proved 23.6mmol) at room temperature. After 17h, the reaction mix-
irreproducible, and thus an alternative synthetic route involv- ture was filtered through a pad of celite using DCM as the
ing dehydroxylation was established. Botryllazine A was eluent, and concentrated. The crude product was purified by
originally reported to exhibit higher AR inhibitory activity column chromatography on silica gel (eluent, CHCl3) to afford
1
than botryllazine B.20) In disagreement with this, the present 5 as yellow solid (1.90g, 48%). H-NMR (CDCl3, 400MHz)
study demonstrated that botryllazine A derivatives are less δ: 7.94 (4H, d, J=8.7Hz), 6.95 (4H, d, J=8.7Hz), 3.85 (6H,
potent than corresponding botryllazine B derivatives, but s). 13C-NMR (CDCl3, 100MHz) δ: 188.3, 164.7, 150.5, 146.4,
they do exhibit inhibitory activities comparable to that of the 133.2, 127.6, 114.1, 55.7. FAB-MS (matrix: DTT/TG=1/2) m/z:
highly-effective AR inhibitor sorbinil. Furthermore, introduc- 417 ([M+H]+). These spectral data were in good accordance
ing a quinoline ring at the C5 position enhances AR inhibitory with those reported previously.12)
activity. As indicated by the higher IC50 values obtained for
Synthesis of 2-Chloro-3-(naphthalen-2-yl)-5,6-bis(1-oxo-
botryllazine B than botryllazine A derivatives, having two 4-methoxyphenylmethyl)pyrazine (6b) A mixture of 5b
para-hydroxybenzoyl groups (i.e., at both the C2 and C3 posi- (1.50g, 3.60mmol), 2-naphtaleneboronic acid (620mg,
tions of the central pyrazine ring) is dispensable for high AR 361mmol), Pd(PPh3)4 (282mg, 180µmol), aqueous 2M po-
inhibitory activity. The docking study suggests that the botryl- tassium carbonate (3.4mL, 6.80mmol) and ethanol (1.1mL)
lazine A derivative series molecules enter the AR active site in toluene (17mL) was heated to reflux under Ar for 17h.
when the specificity pocket is in its closed state. Furthermore, After cooling to room temperature, the reaction mixture was
it appears that under such circumstances a π–π interaction— diluted with 3mL of a mixture of water and CHCl3 (1:1)
between inhibitor bicyclic systems at the C5 position and the and the organic layer was separated. The aqueous layer was
hydrophobic pocket Trp219 residue—plays an important role extracted with CHCl3 (3 ×20mL). The combined organic
in conferring enhanced AR inhibitory activity. These results extracts were dried over sodium sulfate and evaporated. The
provide important insights which will contribute towards the crude product was purified by column chromatography on
design of novel botryllazine-based AR inhibitors.
silica gel (eluent; hexane–AcOEt=7:3) to afford 6b as yellow
1
solid (1.19g, 65%). H-NMR (CDCl3, 400MHz) δ: 8.48 (1H,
s), 8.07 (2H, d, J=8.8Hz), 8.03 (2H, d, J=8.8Hz), 7.99–7.89
Experimental
General NMR spectra were recorded on a JNM-ECP400 (4H, m), 7.60–7.54 (2H, m), 6.99 (2H, d, J=8.8Hz), 6.94 (2H,
spectrometer (JEOL Ltd., Japan). Chemical shifts (δ) are d, J=8.8Hz), 3.89 (3H, s), 3.86 (3H, s). 13C-NMR (CDCl3,
reported in ppm using tetramethylsilane or an undeuterated 100 MHz) δ: 189.9, 189.2, 164.5, 164.4, 151.6, 149.9, 145.5,
solvent as internal standards in the deuterated solvent used. 134.0, 133.4, 133.3, 132.8, 132.4, 130.5, 129.0, 128.3, 128.3,
Coupling constants (J) are given in Hz. Chemical shift mul- 128.1, 127.8, 126.4, 114.1, 114.0, 77.3, 55.7, 55.64. ESI-MS m/z:
tiplicities are reported as s=singlet, d=doublet, t=triplet, 531.1073 (Calcd for C30H21ClN2NaO4 [M+Na]+: 531.1082).
m=multiplet. FAB mass spectra were taken on a JMS-600-H
Synthesis of 2-Chloro-3-(quinolin-3-yl)-5,6-bis(1-oxo-4-
mass spectrometer (JEOL Ltd., Japan). Xenon was used as methoxyphenylmethyl)pyrazine (6c) This material was
a bombardment gas, and all analyses were carried out in prepared from 5c (202.3mg, 485µmol) with 3-quinolinebo-
positive mode with the ionization energy and the accelerating ronic acid (74.8mg, 432µmol) in a procedure analogous to
voltage set at 70eV and 3kV, respectively. A mixture of di- that for obtaining 6b. The product was obtained as yellow
1
thiothreitol (DTT) and α-thioglycerol (TG) (1:1) was used as solid (90.6mg, 37%). H-NMR (CDCl3, 400MHz) δ: 9.45 (1H,
a liquid matrix. High-resolution electrospray ionization (HR- d, J=2.0Hz), 8.81 (1H, s), 8.19 (1H, d, J=8.4Hz), 8.08–8.02
ESI)-MS were measured on a JEOL Model JMS-T100CS mass (4H, m), 7.96 (1H, d, J=8.0Hz), 7.85 (1H, t, J=7.4Hz),
spectrometer. All conventional chemicals used in the present 7.66 (1H, t, J=7.4Hz), 7.02–6.95 (4H, m), 3.91 (3H, s), 3.88
study are commercially available and were used as received. (3H, s). 13C-NMR (CDCl3, 100MHz) δ: 189.6, 188.9, 164.6,
Column chromatography was carried out on silica gel (particle 164.5, 151.1, 150.9, 150.3, 148.9, 148.4, 145.7, 137.9, 133.3,
size; 46–50µm; Fuji Silysia Chemical Ltd.).
133.2, 131.3, 129.5, 128.8, 128.1, 127.6, 114.1, 114.0, 55.7. ESI-

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MS m/z: 532.1046 (Calcd for C29H20ClN3NaO4 [M+Na]+: J=8.8Hz), 6.87 (2H, d, J=8.8Hz), 3.89 (3H, s), 3.82 (3H, s).
532.1040).
13C-NMR (DMSO-d6, 100MHz) δ: 188.7, 187.4, 164.5, 163.6,
A General Procedure for the Reductive Dechlorination 156.4, 150.5, 148.0, 136.7, 133.4, 132.3, 131.9, 131.3, 129.8,
Reactions of Chloropyrazines 6 In a flask was placed a 129.4, 128.8, 128.7, 127.8, 127.3, 114.9, 114.8, 114.4, 114.1,
solution of 6 (2.19mmol), triethylamine (20.8mmol), formic 56.3, 56.1. ESI-MS m/z: 513.1387 (Calcd for C29H21N3NaO5
acid (10.6mmol) and Pd/C (10%) (0.1 or 0.05eq) in an anhy- [M+Na]+: 514.1373).
drous solvent (23mL) and the mixture was heated at refluxing
Synthesis of 2,3-Bis(1-oxo-4-methoxyphenylmethyl)-
temperature for 3h. After cooling to room temperature, the 5-(naphthalen-2-yl)-6-trifluoromethanesulfonylpyrazine
reaction mixture was filtrated by celite, then water (20mL) (9b) Triethylamine (0.13mL, 933µmol) and trifluorometh-
and CHCl3 (20mL) were added to the reaction mixture and anesulfonic anhydride (0.14mL, 832µmol) were added to a
separated. The aqueous layer was extracted with CHCl3 DCM solution (3.8mL) of 8b (400mg, 865µmol) and stirred
(3 ×20mL). The combined organic extracts were then dried for 2h. Then water (15mL) and CHCl3 (15mL) were added
over sodium sulfate and evaporated. The crude product was to the reaction mixture and separated. The aqueous layer was
purified by column chromatography on silica gel (eluent; extracted with CHCl3 (3 ×15mL). The combined organic
CHCl3–AcOEt=49:1) to afford 7 and 8 with yields shown in extracts were then dried over sodium sulfate and evaporated.
Table 1.
The crude product was purified by column chromatography
2,3-Bis(1-oxo-4-methoxyphenylmethyl)-5-(4-methoxy- on silica gel (eluent; chloroform) to afford 9b as yellow solid
1
1
phenyl)pyrazine (7a) was obtained as yellow solid; H-NMR (502mg, 93%). H-NMR (CDCl3, 400MHz) δ: 8.61 (1H, s),
(CDCl3, 400MHz) δ: 9.09 (1H, s), 8.04 (6H, m), 7.00 (6H, 8.15–8.11 (3H, m), 8.04–7.96 (4H, m), 7.91 (1H, d, J=7.6Hz),
m), 3.88 (9H, s). 13C-NMR (CDCl3, 100MHz) δ: 191.3, 7.60 (2H, quint, J=7.6Hz), 6.99 (4H, t, J=8.6Hz), 3.90 (3H,
190.8, 164.1, 162.1, 153.2, 150.9, 149.2, 139.0, 133.3, 129.1, s), 3.87 (3H, s). 13C-NMR (CDCl3, 100MHz) δ: 189.7, 188.1,
128.6, 127.5, 114.8, 113.9, 113.8, 55.6. FAB-MS (matrix: 164.7, 164.5, 153.6, 147.6, 147.3, 144.6, 134.6, 133.6, 133.2,
DTT/TG=1/1) m/z: 455 ([M+H]+). These data were in good 133.0, 130.8, 129.3, 129.3, 129.0, 128.4, 128.2, 127.9, 127.6,
accordance with those reported previously.12)
127.1, 125.6, 123.24, 120.0, 116.9, 113.7, 55.7. ESI-MS m/z:
3-(4-Methoxyphenyl)-5,6-bis(1-oxo-4-methoxyphenyl- 645.0901 (Calcd for C31H21F3N2NaO7S [M+Na]+: 645.0914).
methyl)pyrazin-2(1H)-one (8a) was obtained as yellow solid; Synthesis of 2,3-Bis(1-oxo-4-methoxyphenylmethyl)-5-
1H-NMR (CDCl3, 400MHz) δ: 8.49 (2H, d, J=8.8Hz), 8.01 (naphthalen-2-yl)pyrazine (7b) To a mixture of 9b (400mg,
(2H, d, J=8.8Hz), 7.79 (2H, d, J=8.4Hz), 6.97–6.92 (4H, m), 643 µmol), Pd(dppf)2Cl2 (24.1mg, 32.9µmol) in 1.5mL of
6.85 (2H, d, J=8.8Hz), 3.90 (3H, s), 3.89 (3H, s), 3.83 (3H, anhydrous DMF at 60°C was added triethylsilane (0.25mL,
s). 13C-NMR (CDCl3, 100MHz) δ: 188.4, 186.5, 164.6, 163.7, 1.57mmol). After 6h, the mixture was successively washed
162.0, 155.0, 150.4, 137.5, 133.2, 132.5, 131.2, 129.2, 128.3, with water (30mL). The aqueous layer was extracted with
127.524, 114.326, 113.6, 113.5, 77.4, 77.1, 76.8. ESI-MS m/z: CHCl3 (3 ×30mL). The combined organic extracts were then
471.1573 (Calcd for C27H23N3N2O6 [M+H]+: 471.1556).
dried over sodium sulfate and evaporated. The crude product
3-(Naphthalen-2-yl)-5,6-bis(1-oxo-4-methoxyphenyl- was purified by column chromatography on silica gel (eluent;
1
methyl)pyrazin-6(1H)-one (8b) was obtained as yellow solid; CHCl3) to afford 7b as yellow solid (183mg, 60%). H-NMR
1H-NMR (CDCl3–methanol-d4 =4:1, 400MHz) δ: 9.06 (1H, (CDCl3, 400MHz) δ: 9.28 (1H, s), 8.61 (1H, s), 8.20 (1H, d,
s), 8.20 (1H, d, J=8.4Hz), 7.99 (6H, d, J=8.8Hz), 7.80 (1H, J=8.8Hz), 8.10–8.06 (4H, m), 7.97–7.94 (2H, m), 7.87 (1H, d,
d, J=8.8Hz), 7.76–7.70 (4H, m), 7.41–7.35 (2H, m), 6.82 (2H, J=6.8Hz), 7.57–7.54 (2H, m), 6.98–6.96 (4H, m), 3.86 (3H,
d, J=8.8Hz), 6.79 (2H, d, J=8.4Hz), 3.75 (3H, s), 3.70 (3H, s). 13C-NMR (CDCl3, 100MHz) δ: 191.2, 190.8, 164.2, 153.3,
s). 13C-NMR (CDCl3, 100MHz) δ: 188.7, 187.1, 164.5, 163.6, 151.1, 150.2, 140.0, 134.5, 133.4, 133.2, 132.3, 129.2, 129.1,
155.3, 141.1, 134.2, 133.2, 132.8, 132.2, 131.6, 130.2, 129.3, 128.6, 128.6, 127.9, 127.9, 127.8, 127.0, 124.1, 114.0, 113.9,
128.9, 128.0,127.8, 127.5, 126.2, 125.1, 114.2, 113.4, 77.4, 77.4, 55.6. ESI-MS m/z: 497.1460 (Calcd for C30H22N2NaO4
55.4, 55.3. ESI-MS m/z: 513.1432 (Calcd for C30H22N2NaO5 [M+Na]+: 497.1472).
[M+Na]+: 513.1421).
Synthesis of Botryllazine A (1a)12) This material was
2,3-Bis(1-oxo-4-methoxyphenylmethyl)-5-(quinolin-3- prepared by the literature method, and all the spectral data
yl)pyrazine (7c) was obtained as yellow solid; 1H-NMR were in good accordance with those reported previously.12)
(CDCl3, 400MHz) δ: 9.64 (1H, d, J=1.8Hz), 9.33 (1H, s), 1H-NMR (methanol-d4, 400MHz) δ: 9.21 (1H, s), 8.08 (2H, d,
8.90 (1H, d, J=1.8Hz), 8.18 (2H, d, J=8.0Hz), 8.07 (2H, d, J=8.4Hz), 7.89 (2H, d, J=8.8Hz), 7.85 (2H, d, J=8.8Hz),
J=8.0Hz), 8.06 (2H, d, J=8.0Hz), 7.96 (1H, d, J=8.0Hz), 6.94 (2H, d, J=8.4Hz), 6.88–6.83 (4H, m). 13C-NMR (meth-
7.82 (1H, t, J=8.0Hz), 7.64 (1H, t, J=8.0Hz), 6.97 (4H, anol-d4, 100MHz) δ: 191.9, 191.4, 163.2, 163.1, 160.4, 152.4,
d, J=8.8Hz), 3.89 (6H, s). 13C-NMR (CDCl3, 100MHz) δ: 151.5, 148.5, 139.3, 133.2, 133.1, 129.0, 127.4, 127.3, 126.1,
190.8, 190.6, 164.3, 153.5, 150.9, 151.2, 148.9, 148.6, 139.9, 115.8, 115.0. Anal. Calcd for C21H16N2O5·0.8H2O: C, 69.90; H,
135.2, 133.3, 133.2, 131.3, 129.5, 128.9, 128.4, 127.8, 127.7, 3.91; N, 6.79. Found: C, 67.50; H, 4.29; N, 6.37.
127.6, 114,0, 113.9, 55.7. ESI-MS m/z: 498.1404 (Calcd for
Synthesis of 5-(Naphthalen-2-yl)-2,3-bis(1-oxo-4-hy-
droxyphenylmethyl)pyrazine (1b) The deprotection reac-
C29H20N3NaO4 [M+Na]+: 498.1430).
5,6-Bis(1-oxo-4-methoxyphenylmethyl)-3-(quinolon-3- tion for 7b was performed using the same procedure as that
yl)pyrazin-2(1H)-one (8c) was obtained as yellow solid; for obtaining 1a.12) After recrystallization from isopropanol/
1H-NMR (DMSO-d6, 400MHz) δ: 9.75 (1H, d, J=2.4Hz), hexane mixed solvent system, 1b was obtained as yellow solid
1
9.62 (1H, d, J=2.4Hz), 8.12 (1H, d, J=8.0Hz), 8.08 (2H, d, (56.7mg, 66%). H-NMR (DMSO-d6, 400MHz) δ: 9.65 (1H,
J=8.8Hz), 7.97 (1H, d, J=8.0Hz), 7.84 (2H, d, J=8.8Hz), s), 8.85 (1H, s), 8.31 (1H, d, J=8.8Hz), 8.13–8.09 (2H, m),
7.83 (1H, t, J=8.0Hz), 7.61 (1H, t, J=8.0Hz), 6.96 (2H, d, 8.01 (2H, d, J=7.6Hz), 7.86 (4H, dd, J=8.8Hz and 2.4Hz),

Vol. 67, No. 6 (2019)
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563
7.66–7.60 (2H, m). 13C-NMR (DMSO-d6, 100MHz) δ: 191.6, pyrazine-2-carboxylic acid (11,18) 202mg, 869µmol) in SOCl2
191.3, 163.4, 163.3, 152.5, 151.2, 150.0, 140.5, 134.6, 133.5, (5.0mL) was refluxed for 3h. The excess SOCl2 was removed
133.2, 133.1, 132.4, 128.8, 127.5, 127.5, 127.4, 127.3, 127.3, under reduced pressure, and the resulting crude product was
126.6, 123.7, 115.1, 115.0. Anal. Calcd for C28H18N2O4·0.4H2O: once dried in vacuo, and then dissolved in 3.0mL of dichlo-
C, 75.33; H, 4.06; N, 6.27. Found: C, 74.13; H, 4.20; N, 6.06.
romethane. To this dichloromethane solution, morpholine
Synthesis of 2,3-Bis(1-oxo-4-hydroxyphenylmethyl)-5- (0.30mL, 3.44mmol) in 1.3mL of dichloromethane was added
(quinolin-3-yl)pyrazine (1c) The deprotection reaction at 0°C. The mixture was stirred at 0°C to room temperature
for 7c was performed using the same procedure as that for at 15h, Aqueous Na2CO3 solution (2%, 2.5mL) was added
obtaining 1a.12) After recrystallization from isopropanol/hex- to the reaction mixture, and the organic layer was separated,
ane mixed solvent system, 1c was obtained as yellow solid washed with 3mL of water, and dried over Na2SO4. Removal
1
(15.2mg, 16%). H-NMR (DMSO-d6, 400MHz) δ: 9.65 (1H, of the solvent gave the product as orange solids (241mg, 81%).
d, J=2.0Hz), 9.57 (1H, s), 9.21 (1H, d, J=1.2Hz), 8.13 (2H, This material was used directly in the next step without fur-
1
t, J=7.4Hz), 7.93–7.87 (5H, m), 7.72 (1H, t, J=7.4Hz), 6.88 ther purification. H-NMR (CDCl3, 400MHz) δ: 8.99 (1H, s),
(2H, t, J=8.0Hz). 13C-NMR (DMSO-d6, 100MHz) δ: 191.3, 8.91 (1H, s), 8.03 (2H, d, J=8.8Hz), 7.05 (2H, d, J=8.8Hz),
191.1, 163.3, 152.9, 151.1, 148.8, 148.3, 148.1, 140.3, 136.0, 3.89 (3H, s), 3.84 (4H, s), 3.76-3.75 (8H, m). 13C-NMR (CDCl3,
133.5, 133.4, 131.6, 131.1, 129.0, 128.7, 128.2, 128.0, 127.9, 100 MHz) δ: 165.6, 161.8, 153.0, 145.7, 145.4, 138.8, 128.8,
127.8, 127.1, 115,4. Anal. Calcd for C27H17N3O4·0.9H2O: C, 128.2, 114.8, 67.2, 67.0, 55.6, 47.9, 43.1. ESI-MS m/z: 322.1173
72.48; H, 3.83; N, 9.39. Found: C, 70.00; H, 3.94; N, 8.96.
Synthesis of 3-(4-Hydroxyphenyl)-5,6-bis(1-oxo-4-hy-
(Calcd for C16H17N3NaO3 [M+Na]+: 322.1168).
Synthesis of (4-Methoxyphenyl)(5-(4-methoxyphenyl)-
droxyphenylmethyl)pyrazin-2(1H)-one (2a) The deprotec- pyarzin-2-yl)methanone (13) Under argon atmosphere,
tion reaction for 8a was performed using the same procedure magnesium (1.05mg, 43.2mmol) was placed in a 30-mL
as that for obtaining 1a.12) After recrystallization from iso- 2-necked flask equipped with a dropping funnel. THF (5.0mL)
propanol/hexane mixed solvent system, 2a was obtained as was added to the flask, and the suspension was cooled to
1
yellow solid (35.9mg, 20%). H-NMR (methanol-d4, 400MHz) –15°C. Then, p-bromoanisol (1.0mL, 7.99mmol) in THF
δ: 10.57 (1H, s), 10.42 (1H, s), 9.98 (1H, s), 8.24 (2H, d, (7.0mL) was added from the dropping funnel over 20min, and
J=8.8Hz), 7.90 (2H, d, J=8.8Hz), 7.77 (2H, d, J=8.4Hz), then the mixture was stirred for 1h at 70°C. After cooling to
6.87–6.82 (6H, m). 13C-NMR (methanol-d4, 100MHz) δ: room temperature, the resulting Grignard reagent was added
188.8, 187.3, 163.4, 162.4, 160.0, 155.7, 133.6, 132.6, 131.1, to a solution of 12 (102mg, 0.34mmol) in THF (8.0mL) at
128.2, 127.6, 127.0, 116.0, 115.5, 115.4. Anal. Calcd for −100°C. The mixture was then allowed to warm up to room
C24H16N2O6·H2O: C, 67.29; H, 3.76; N, 6.54. Found: C, 64.57; temperature with stirring in a period of 36h. After completion
H, 3.97; N, 6.18.
of the reaction, the reaction mixture was neutralized with 6M
Synthesis of 3-(Naphthalen-2-yl)-5,6-bis(1-oxo-4-hy- HCl (0.3mL), and separated between water and chloroform.
droxyphenylmethyl)pyrazin-2(1H)-one (2b) The deprotec- The organic layer was dried over Na2SO4. Removed of the sol-
1
tion reaction for 8b was performed using the same procedure vent gave 13 as yellow solid (61.5mg, 56%). H-NMR (CDCl3,
as that for obtaining 1a.12) After recrystallization from isopro- 400MHz) δ: 9.23 (1H, s), 9.04 (1H, s), 8.18 (2H, d, J=8.8Hz),
panol/hexane mixed solvent system, 2b was obtained as yel- 8.10 (2H, d, J=8.8Hz), 7.07 (2H, d, J=8.8Hz), 7.01 (2H,
1
low solid (32.3mg, 34%). H-NMR (DMSO-d6, 400MHz) δ: d, J=8.8Hz), 3.91 (6H, s). 13C-NMR (CDCl3, 100MHz) δ:
9.08 (1H, s), 8.20 (1H, d, J=8.8Hz), 7.99 (2H, d, J=8.8Hz), 190.6, 164.0, 162.1, 153.8, 147.6, 145.7, 139.2, 133.5, 129.1,
7.99 (1H, d, J=8.0Hz), 7.76–7.70 (4H, m), 7.38 (2H, quint, 129.0, 128.3, 114.8, 113.8, 55.6. ESI-MS m/z: 343.1052 (Calcd
J=6.8Hz), 6.82 (2H, d, J=8.8Hz), 6.79 (2H, d, J=8.4Hz). for C19H16N2NaO3 [M+Na]+: 343.1059). Anal. Calcd for
13C-NMR (DMSO-d6, 100MHz) δ: 188.7, 187.1, 164.5, 163.6, C19H16N2O3: C, 71.24; H, 5.03; N, 8.74. Found: C, 70.86; H,
155.3, 141.1, 134.2, 133.2, 132.84, 132.2, 131.6, 130.2, 129.3, 5.06; N, 8.64.
128.9, 128.0, 127.8, 127.5, 126.2, 125.1, 114.2, 113.35, 77.4,
55.4, 55.3. Anal. Calcd for C28H18N2O5·0.6H2O: C, 72.72; H, azin-2-yl)methanone (10) Pyridine hydrochloride (4.37g,
3.92; N, 6.06. Found: C, 71.08; H, 4.14; N, 5.88. 37.8mmol) was heated to 210°C, 13 (61.3mg, 0.191mmol) was
Synthesis of (4-Hydroxyphenyl)(5-(4-hydroxypheny)pyr-
Synthesis of 5,6-Bis(1-oxo-4-hydroxyphenylmethyl)-3- added, and the mixture was kept at this temperature for 2h.
(quinolin-3-yl)pyrazin-2(1H)-one (2c) The deprotection Then, the reaction mixture was poured on to ice, and organic
reaction for 8c was performed using the same procedure as materials were extracted with AcOEt. The combined organic
that for obtaining 1a.12) After recrystallization from isopropa- extracts were dried over Na2SO4, and evaporated. The product
nol/hexane mixed solvent system, 2c was obtained as yellow was obtained as orange solid (69.7mg, quant.). For data collec-
1
1
solid (11.2mg, 13%). H-NMR (DMSO-d6, 400MHz) δ: 10.59 tion, this material was recrystallized from methanol. H-NMR
(1H, s), 10.43 (1H, s), 9.58 (1H, s), 9.40 (2H, s), 8.13 (1H, d, (DMSO-d6, 400MHz) δ: 10.55 (1H, s), 10.13 (1H, s), 9.25 (1H,
J=8.0Hz), 8.07 (1H, d, J=8.4Hz), 7.96 (2H, d, J=8.4Hz), d, J=1.0Hz), 9.05 (1H, d, J=1.0Hz), 8.13 (2H, d, J=8.6Hz),
7.86–7.77 (3H, m), 7.68 (1H, t, J=7.4Hz), 6.89 (2H, d, 7.99 (2H, d, J=8.6Hz), 6.95 (2H, d, J=8.6Hz), 6.90 (2H, d,
J=8.8Hz), 6.85 (2H, d, J=8.8Hz). 13C-NMR (DMSO-d6, J=8.6Hz). 13C-NMR (DMSO-d6, 100MHz) δ: 189.8, 162.6,
100 MHz) δ: 188.6, 163.6, 162.6, 150.5, 148.0, 136.7, 133.7, 160.2, 152.8, 147.3, 144.6, 139.0, 133.4, 129.0, 127.0, 125.9,
132.7, 132.1, 131.3, 129.8, 129.2, 128.7, 128.0, 127.8, 127.3, 116.1, 115.1. Anal. Calcd for C17H12N2O3·0.7CH3OH: C, 67.55;
116.2, 115.7, 115.5. Anal. Calcd for C27H17N3O5·1.3H2O: C, H, 4.74; N, 8.90. Found: C, 67.76; H, 4.60; N, 8.66.
69.97; H, 3.70; N, 9.07. Found: C, 66.51; H, 3.80; N, 8.31.
X-Ray Structure Determination A single crystal of 1b
Synthesis of (5-(4-Methoxyphenyl)pyrazin-2-yl)(morpho- was mounted on the top of a glass fiber, and the data col-
lino)methanone (12) A solution of 5-(4-methoxyphenyl)- lection was carried out on a Bruker SMART diffractometer

564
Chem. Pharm. Bull.
Vol. 67, No. 6 (2019)
Table 5. The Crystallographic Data of 1b
Empirical formula
Formula weight
Crystal system
C29H22N2O5
478.49
Triclinic
P-1
Space group
Unit cell dimensions
a=7.3554(6)Å, b=11.6050(10)Å, c=14.4957(13)Å
α=73.0552(15)°, β=77.1608(16)°, γ=84.8397(17)°
Volume
1153.61(17)Å3
Z
4
Density (calculated)
Absorption coefficient
F(000)
1.377Mg/m3
0.095mm−1
500
Theta range for data collection
Index ranges
1.84 to 27.48°
−8<=h<=9, −13<=k<=15, −18<=l<=18
Reflections collected
Independent reflections
Completeness to theta=27.48°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
Largest diff. peak and hole
7350
5245 [R(int)=0.0262]
98.9%
Empirical
0.9964 and 0.9834
Full-matrix least-squares on F2
5245/0/329
1.045
R1 =0.0890, wR2 =0.2242
R1 =0.1380, wR2 =0.2584
1.220 and −0.495e·Å−3
R1 =Σ(|F0|−|Fc|)/Σ|F0|; wR2 =[Σw(|F0|−|Fc|)2/Σw F0
]
.
2
1/2
equipped with a CCD area detector at 100K. The data were formed using the GOLD Suite software package (ver. 5.6.1).
corrected for Lorentz and polarization effects, and absorp- ChemPLP scoring function was used to predict the docking
tion corrections were applied with the SADABS program.39) poses, and the obtained poses were rescored with Astex Sta-
The structure was solved by direct methods and subsequent tistical Potential (ASP),36) known as a good scoring function
difference Fourier syntheses using the program SHELXTL.40) for proteins making specific hydrogen bonds with ligands.37)
All non-H atoms were refined anisotropically, and H atoms The best pose for each ligand was determined by consider-
were placed in calculated positions and thereafter refined with ing the results obtained with both of these scoring functions
Uiso(H)=1.2Ueq(C). The obtained cystallographic data are (both ChemPLP and ASP scores give positive values, with
summarized in Table 5.
the higher score indicating the more likely pose). The protein
In Vitro AR Inhibitory Assay11) Enzyme activity was structures were obtained from the Protein Data Bank (PDB).
measured by monitoring the decay in absorbance at 340nm, The optimized structures and atom charges of the selected
which accompanies the oxidation of NADPH catalyzed by ligands for the docking simulations were calculated by the
recombinant human AR. The reaction mixture contained semi-empirical PM7 calculation method.30) All docking poses
0.2M phosphate buffer (pH 6.2), 1.5mM NADPH, 100mM were visualized by the Hermes software package (ver. 1.9.1)
D,L-glyceraldehyde, and 3.6µU/mL AR, in a total volume of from the Cambridge Crystallographic Data Centre (CCDC).
1.0mL. All the above reagents, except D,L-glyceraldehyde,
were incubated at 25°C for 3min. The reaction was initiated
Acknowledgments This research was supported by JSPS
by the addition of D,L-glyceraldehyde, and it was monitored KAKENHI Grant Number JP25410179. Partial financial sup-
spectrophotometrically for 3min at the same temperature. port to RS by the Faculty of Science Special Grant for Pro-
Inhibitory activity was tested in the above assay conditions by moting Scientific Research at Toho University is also grate-
including the in the inhibitors dissolved in dimethyl sulfoxide fully appreciated.
(DMSO) at desired concentrations in the reaction mixture.
The final concentration of DMSO in the reaction mixture was
Conflict of Interest The authors declare no conflict of
kept at a constant concentration of 0.3%. Reference blank interest.
assay, lacking either the substrate or enzyme, were routinely
included; the rates were subtracted from the reaction rates, to
Supplementary Materials The online version of this ar-
correct for the nonenzymatic oxidation of NADPH. IC50 val- ticle contains supplementary materials.
ues, which express the inhibitor concentrations that produce
the 50% inhibition on the oxidation of NADPH catalyzed by
the AR, were calculated using a log linear regression analysis
of the log dose-inhibition plots. Each plot was provided using
at least three concentrations of inhibitor with two replicates at
each concentration.
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