Neuroprotective principles from Gastrodia elata

Article abstract of DOI:10.1021/np0605182

Serum deprivation-induced neuronal-like PC12 cell apoptosis was used as an ischemic/hypoxic model to screen neuroprotective compounds from the rhizomes of Gastrodia elata, a traditional Chinese medicine. Two active compounds, bis(4-hydroxybenzyl)sulfide (

Full text of DOI:10.1021/np0605182

J. Nat. Prod. 2007, 70, 571-574
571
Neuroprotective Principles from Gastrodia elata
Nai-Kuei Huang,† Yijuang Chern,‡ Jim-Min Fang,§ Chia-I Lin,§ Wan-Ping Chen,† and Yun-Lian Lin*,†
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China, Institute of Biomedical Sciences, Academia Sinica,
Nankang, Taipei 115, Taiwan, Republic of China, and Department of Chemistry, National Taiwan UniVersity, Taipei 106, Taiwan,
Republic of China
ReceiVed October 19, 2006
Serum deprivation-induced neuronal-like PC12 cell apoptosis was used as an ischemic/hypoxic model to screen
neuroprotective compounds from the rhizomes of Gastrodia elata, a traditional Chinese medicine. Two active compounds,
bis(4-hydroxybenzyl)sulfide (1) and N6-(4-hydroxybenzyl)adenine riboside (2), together with 15 known compounds
were obtained from the active fraction. Compound 2 was further elucidated by chemical synthesis. Compounds 1 and
2 potently prevented PC12 cell apoptosis in concentration-dependent manners with EC50 values of 7.20 µM and 3.7 ×
10-8 M, respectively, and IC50 values of 42.90 µM (Ki 24.10 µM) and 4.660 µM (Ki 2.620 µM), respectively, in an
adenosine A2A receptor binding assay.
Neuronal cell death induced by apoptosis is a normal aspect of
development in which the death program is triggered by failure of
a given neuron to receive limited supplies of target-derived
neurotrophic factors. Apoptosis can be triggered in a rat phechro-
mocytoma (PC12) cell culture system by deprivation of either
serum1 or trophic factor/nerve growth factor,2 and thus these are
commonly used to study neuronal differentiation and cell death. In
Huntington’s disease, GABAergic striopallidal neurons selectively
degenerate in sync with the progression of the disease.3 Mutant
huntingtin is known to bind to transcriptional factors and as a
consequence to reduce acetylated histone levels, which causes
reduced expressions of genes essential for neuronal survival.4 In
the brain, the adenosine A2A receptor (A2A-R) gene is heavily
expressed in GABAergic striopallidal neurons.3 During neuronal
development, expression of A2A-R is transiently regulated in various
areas of the developing rat brain,5 suggesting that adenosine may
play an important role in brain development.6 Additionally, an A2A
-
R-specific agonist was shown to specifically ameliorate several
major symptoms of neural degenerative disease.7
were then subjected to further chromatography using Sephadex LH-
20 and RP-18 columns. Among 17 purified compounds, bis(4-
hydroxybenzyl) sulfide (1)22 and N6-(4-hydroxybenzyl)adenine
riboside (2) were identified as the active components in the PC12
assay system. The remaining compounds were 4-hydroxybenzal-
dehyde,16 4-hydroxybenzyl alcohol,16 4-hydroxybenzyl methyl
ether,16 4-hydroxybenzyl ethyl ether,23 1,4-benzenediol,24 gastro-
din,16 4-(â-D-glucopyranosyloxy)benzyl methyl ether,16 parishin,16
bis(4-hydroxybenzyl) ether,17 trimethyl citrate,25 bis(4-hydroxy-
benzyl) sulfoxide,20 bis(4-hydroxybenzyl),19 5-(hydroxymethyl)-2-
furfuraldehyde,26 uridine,27 and adenosine.27 The structures of the
aforementioned compounds were elucidated via their 1D and 2D
NMR and MS analyses and by comparisons with published data.
Of the 15 compounds, trimethyl citrate was likely an artifact.
Compound 2 was isolated as colorless needles with an mp of
216-219 °C. Its molecular formula of C17H20O5N5 was deduced
from the HRFABMS ion at m/z 374.1361 [M + H]+ and the 13C
NMR data. The IR spectrum suggested the presence of hydroxy
(3327, 1125, and 1057, cm-1) and aromatic moieties (1630 and
1514 cm-1). The 1H and 13C NMR spectra showed a pattern similar
to those of adenosine [δH 3.57 and 3.64 (1H each, m), 3.95 (1H, t,
J ) 1.8 Hz), 4.13 (1H, m), 4.60 (1H, m), 5.88 (1H, d, J ) 5.5 Hz),
8.20 and 8.35 (1H each, s), 8.28 and 9.19 (1H each, br s); δC 61.7
(t), 70.7 (d), 73.5 (d), 85.9 (d), 88.0(d), 120.4 (s), 139.8 (d), 148.4
(s), 152.3 (d), and 154.4 (s)] except for a p-hydroxybenzylamine
moiety [δH 4.60 (2H, m, J ) 6.0 Hz), 6.66 and 7.14 (2H each, d,
J ) 8.5 Hz); δC 42.4 (t), 114.9 (d), 114.9 (d), 128.6 (d), 128.6
(d), 130.8 (s, C-1′′), and 156.1 (s, C-4′′)] in compound 2,
The rhizomes of Gastrodia elata have been used in traditional
Chinese medicine for the treatment of headaches, dizziness, vertigo,
and convulsive illnesses, such as epilepsy and tetanus.8 In line with
these medicinal uses, many studies have been performed to evaluate
the effects of G. elata on the prevention of neuronal damage,9-15
which have been supplemented by a series of phytochemical
studies.16-23 Additionally, we previously reported that an aqueous
ethanolic extract of G. elata exhibited activity in preventing PC12
cell apoptosis induced by serum deprivation through suppression
of the JNK pathway.15 In a continuation of this investigation, we
herein report the isolation of two active compounds, bis(4-
hydroxybenzyl)sulfide (1) and N6-(4-hydroxybenzyl)adenine ribo-
side (2), which demonstrated the ability to prevent serum deprivation-
induced apoptosis in PC12 cells and to bind A2A-R. Notably,
compound 2 was first found from a natural source, and its structure
was verified through synthesis.
Results and Discussion
An aqueous ethanolic extract of G. elata was first concentrated
in Vacuo and then fractionated using a Diaion HP-20 column with
an elution gradient from H2O to MeOH. Fractions showing
protection of PC12 cells from serum deprivation-induced apoptosis
* Corresponding author. Tel: +886-2-28201999, ext. 6531. Fax: +886-
2-28250743. E-mail: yllin@nricm.edu.tw.
†National Research Institute of Chinese Medicine.
‡Institute of Biomedical Sciences, Academia Sinica.
§ Department of Chemistry, National Taiwan University.
10.1021/np0605182 CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/24/2007

572 Journal of Natural Products, 2007, Vol. 70, No. 4
Scheme 1. Semisynthesis of Compound 2
Huang et al.
Extraction and Isolation. Slices of GE were extracted at 60 °C
using 80% EtOH overnight (3 × 60 L). The crude extract was
concentrated under reduced pressure. The dried sample (which had
substituting the amino group in adenosine. The linkage of the
p-hydroxybenzylamino group to C-7 was supported by HMBC
correlations of H-7′′ with C-1′′, C-2′′ (6′′), and C-6 and by
1
comparison of the H and 13C NMR data with those of N6-(m-
hydroxybenzyl)adenine, a plant growth regulator that was reported
by Strnad et al.28 The m-hydroxybenzyl group was replaced by a
p-hydroxybenzyl in 2. This is the first report of the presence of
compound 2 in a natural source. Its structure was further confirmed
by semisynthesis via the substitution reaction of 4-hydroxybenzy-
lamine hydrochloric salt (4) and 6-chloropurine ribonucleoside (5)
in the presence of diisopropylethylamine (Scheme 1). The synthetic
sample of compound 2 showed identical MS and 1H and 13C NMR
spectroscopic profiles to those of the natural product.
All of the aforementioned compounds were subjected to the
protection assay for the prevention of PC12 cell apoptosis induced
by serum deprivation. Both compounds 1 and 2 showed significant
efficacy in a concentration-dependent manner (Figure 1A), with
EC50 values of 7.20 ( 0.20 µM and (3.7 ( 0.3) × 10-8 M,
respectively. In the A2A receptor binding assay compound 1 had
an IC50 of 42.90 ( 3.35 µM and a Ki value of 24.10 ( 1.88 µM,
whereas those for compound 2 were 4.660 ( 0.051 µM and 2.620
( 0.029 µM, respectively. However, the major components, parishin
and gastrodin, showed no protective effect even at a concentration
higher than 1 × 10-4 M, whereas the protective effect of adenosine
was about 80% at 1 × 10-4 M (Figure 1B). In this study, the A2A
R
activity level of compound 2 was similar to those reported for N6-
substituted adenosines (such as N6-phenyladenosine) and N6-
substituted 2-chloroadenosines for rat brain striatum A2 adenosine
receptor (A2AR).29
In summary, two neuroprotive compounds (1 and 2) were isolated
from the active fraction of G. elata. Compound 2 has not previously
been reported from natural sources. Both 1 and 2 prevent serum-
deprived PC12 cell apoptosis, suggesting a therapeutic potential in
treating neurotoxicity or as neuroprotectants and are candidates for
further development in the therapeutic use against neural degenera-
tive disease.
Experimental Section
General Experimental Procedures. Melting points were determined
on a Yanagimoto micromelting point apparatus and are uncorrected.
IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectropho-
tometer. UV spectra were measured on a Hitachi U-3310 spectropho-
tometer. NMR spectra were run on Varian unity INOVA-500 and
Bruker AVANCE 400 spectrometers. Mass spectra (EIMS, HREIMS,
and HRFABMS) were recorded on a JEOL JMS-100 and a JEOL SX-
102A instrument, respectively.
Figure 1. Prevention of serum deprivation-induced PC12 cell
apoptosis by (A) compounds 1 and 2 and (B) adenosine, gastrodin,
and parishin from G. elata. Serum-deprived PC12 cells were treated
with or without the indicated drug at the desired concentration for
24 h. Cell viability was monitored by the MTT assay and is
expressed as a percentage of the MTT activity measured in the
serum-containing group. Data points represent the mean ( SEM
of at least three independent experiments (n ) 3-6). (#: p < 0.05,
serum-deprived group compared with control; *: p < 0.05,
compounds 1- and 2-treated groups compared with serum-deprived
group).
Plant Material. Dried rhizomes of G. elata (GE) (12 kg) were
purchased from a local herbal store in Taipei. The specimen was
identified by comparison with voucher specimens deposited earlier at
the Herbarium of National Research Institute of Chinese Medicine.

NeuroprotectiVe Principles from Gastrodia elata
Journal of Natural Products, 2007, Vol. 70, No. 4 573
about a 15% yield based on the dried rhizome weight) was subjected
to Diaion HP-20 column chromatography with elution using a gradient
from H2O to MeOH. The active fractions of 50%-75% MeOH/H2O
were repeatedly purified by passage over a Sephadex LH-20 column
and elution with MeOH or 80% MeOH to yield 1 (1.85 g), 2 (167
mg), 4-hydroxybenzaldehyde (78 mg), 4-hydroxybenzyl alcohol (315
mg), 4-hydroxybenzyl methyl ether (85 mg), 4-hydroxybenzyl ethyl
ether (93 mg), 1,4-benzenediol (63 mg), gastrodin (25 g), 4-(â-D-
glucopyranosyloxy)benzyl methyl ether (35 mg), parishin (55 g), bis-
(4-hydroxybenzyl) ether (48 mg), trimethyl citrate (3.6 g), bis(4-
hydroxybenzyl) sulfoxide (36 mg), bis(4-hydroxybenzyl) (45 mg),
5-(hydroxymethyl)-2-furfuraldehydealdehyde (26 mg), uridine (32 mg),
and adenosine (43 mg).
system (Millipore MultiScreen MAFB) was used for the binding
experiments. Before use, the filters in the plates were presoaked in 20
µL of binding buffer. For the saturation binding experiments, increasing
concentrations of the radioligands for the A2A-R ([3H]SCH 58261) were
incubated with the membrane protein in a final volume of 300 µL in
binding buffer. The incubation mixture contained 22.5 µg of protein
for [3H]DPCPX binding and 60 µg of protein for [3H]SCH 58261
binding. GTP at a final concentration of 500 µM was used where
indicated. Nonspecific binding was defined as the binding in the
presence of 500 µM 2-chloroadenosine. Samples were incubated at
room temperature for either 2 h for [3H]DPCPX binding or 1 h for
[3H]SCH 58261 binding, filtered through the built-in filter at the bottom
of the wells, and washed three times with 300 µL of ice-cold binding
buffer. After addition of 30 µL of scintillation fluid to the dried filter,
the plates were incubated overnight at room temperature and counted.
IC50 values were determined by a nonlinear, least-squares regression
analysis using Data Analysis Toolbox (MDL Information System, San
Leandro, CA).34 The inhibition constants (Ki) were calculated using
the equation of Cheng and Prusoff.35
Compound 1: pale yellow needles from EtOH; mp 136-138 °C;
IR (KBr) νmax 3285 (OH), 1604, 1600, 1509 (CdC), 1214, 1089 (OH)
1
cm-1; EIMS m/z (%) 246 (M+, 35), 200 (15), 107 (100); H and 13C
NMR data are in good agreement with the published data.22
Compound 2: colorless needles from EtOH; mp 216-219 °C; [R]25
D
-87 (c 0.1, MeOH); IR (KBr) νmax 3327, 3164, 2927, 1630, 1514,
1
1125, 1057, 815 cm-1; H NMR (DMSO-d6, 500 MHz) δ 3.57 and
Acknowledgment. We are grateful to the National Science Council,
Republic of China, for support of this research under grant NSC 95-
2323-B-077-002.
3.64 (1H each, m, H-5′), 3.95 (1H, t, J ) 1.8 Hz, H-4′), 4.13 (1H, m,
H-3′), 4.60 (3H, m, H-2′, H-7′′), 5.88 (1H, d, J ) 6.5 Hz, H-1′)), 6.66
and 7.14 (2H each, d, J ) 8.5 Hz, H-3′′(5′′), H-2′′(6′′)), 8.20 and 8.35
(1H each, s, H-8 and H-2), 8.28 and 9.19 (1H each, br s, NH, OH);
13C NMR (DMSO-d6, 125 MHz) δ 42.4 (t, C-7′′), 61.7 (t, C-5′), 70.7
(d, C-3′), 73.5 (d, C-2′), 85.9 (d, C-4′), 88.0(d, C-1′), 114.9 (d, C-3′′-
(5′′)), 120.4 (s, C-5), 128.6 (d, C-2′′(6′′)), 130.8 (s, C-1′′), 139.8 (d,
C-8), 148.4 (s, C-4), 152.3 (d, C-2), 154.5 (s, C-6), and 156.1 (s, C-4′′);
HMBC correlations H-2/C-4, C-6; H-8/C-4, C-5; H-7′′/C-6, C-1′′, C-2′′-
(6′′); FABMS m/z (%) 374 [(M + H)+, 28], 242 (15), 154 (95), 136
(83), 56 (100); HRFABMS m/z 374.1361 (calcd for C17H20O5N5,
374.1388).
Synthesis of Compound 2. Hydroxylamine hydrochloride (1.29 g,
18.6 mmol) and NaOAc (1.67 g, 20.4 mmol) were added to a solution
of 4-hydroxybenzaldehyde (1.25 g, 10.2 mmol) in EtOH (20 mL). The
reaction mixture was stirred at room temperature for 6 h. EtOH was
removed under reduced pressure. H2O was added to the residue and
then extracted with Et2O (3×). The combined organic layer was dried
over MgSO4. After the volatiles were removed by rotary evaporation
under reduced pressure, the residue was recrystallized from CH2Cl2 to
give oxime 3 (1.3 g, 93%):30,31 C7H7NO2; light yellow solid; mp 92.0-
93.6 °C; ESIMS m/z 138.0620 (M+ + H) (calcd for C7H8NO2,
138.0600).
A solution of 3 (342 mg, 2.5 mmol) and concentrated HCl (1 mL)
in EtOH (20 mL) was subjected to hydrogenation at atmospheric
pressure in the presence of 10% Pd/C (80 mg) for 4.5 h. The reaction
mixture was filtered through Celite. The filtrate was concentrated to
yield the hydrochloric salt of amine 4 as a light yellow solid, which
was used for the next step without further purification.31,32
A mixture of amine 4 (395 mg, as the hydrochloric salt), 6-chloro-
purine ribonucleoside (5) (143 mg, 0.5 mmol), and diisopropylethy-
lamine (2 mL, 12 mmol) in PrOH (25 mL) was heated to 70 °C for 6
h. After evaporation, the mixture was triturated with H2O to give a
white precipitate, which was filtered to yield the desired product 2 (151
mg, 81%).32,33 The physical data (including the mp, MS, Rf value of
TLC, 1H and 13C NMR) of the synthesized product were in good
agreement with compound 2 isolated from the rhizomes of G. elata.
Cell Culture. PC12 cells purchased from ATCC (Manassas, VA)
were maintained in DMEM supplemented with 10% horse serum and
5% fetal bovine serum and incubated in a CO2 incubator (5%) at 37
°C.
References and Notes
(1) Batistatou, A.; Greene, L. A. J. Cell. Biol. 1991, 115, 461-471.
(2) Rukenstein, A.; Rydel, R. E.; Greene, L. A. J. Neurosci. 1991, 11,
2552-2563.
(3) Glass, M.; Dragunow, M.; Faull, R. L. Neuroscience 2000, 97, 505-
19.
(4) Gardian, G.; Vecsei, L. J. Neural Transm. 2004, 111, 1485-
1494.
(5) Weaver, D. R. Brain Res. Mol. Brain Res. 1993, 20, 313-27.
(6) Lee, Y. C.; Chien, C. L.; Sun, C. N.; Huang, C. L.; Huang, N. K.;
Chiang, M. C.; Lai, H. L.; Lin, Y. S.; Chou, S. Y.; Wang, C. K. L.;
Tai, M. H.; Liao, W. L.;Lin, T. N.; Liu, F. C.; Chern, Y. Eur. J.
Neurosci. 2003, 18, 1786-1796.
(7) Chou, S. Y.; Lee, Y. C.; Chen, H. M.; Chiang, M. C.; Lai, H. L.;
Chang, H. H.; Wu, Y. C.; Sun, C. N.; Chieh, C. L.; Lin, Y. S.; Wang,
S. C.; Tung, Y. Y.; Chang, C.; Chern. Y. J. Neurochem. 2005, 93,
310-320.
(8) Hus, H. Y.; Chen, Y. P.; Hsu, S. J.; Hsu, C. S.; Chen, C. C.; Chang,
H. C. Oriental Materia Medica: A Concise Guide; Oriental Health
Art Institute: California, 1986; p 662.
(9) An, S. J.; Park, S. K.; Hwang, I. K.; Choi, S. Y.; Kim, S. K.; Kwon,
O. S.; Jung, S. J.; Baek, N. I.; Lee, H. Y.; Won, M. H.; Kang, T. C.
J. Neurosci. Res. 2003, 71, 534-543.
(10) Kim, H. J.; Moon, K. D.; Oh, S. Y.; Kim, S. P.; Lee, S. R. Neurosci.
Lett. 2001, 314, 65-68.
(11) Kim, H. J.; Lee, S. R.; Moon, K. D. Phytother. Res. 2003, 17, 909-
912.
(12) Kim, H. J.; Moon, K. D.; Lee, D. S.; Lee, S. H. J. Ethnopharmacol.
2003, 84, 95-98.
(13) Hsieh, C. L.; Chiang, S. Y.; Cheng, K. S.; Lin, Y. H.; Tang, N. Y.;
Lee, C. J.; Pon, C. Z.; Hsieh, C. T. Am. J. Chin. Med. 2001, 29,
331-341.
(14) Hsieh, C. L.; Chang, C. H.; Chiang, S. Y.; Li, T. C.; Tang, N. Y.;
Pon, C. Z.; Hsieh, C. T.; Lin, J. G. Life Sci. 2000, 67, 1185-
1195.
(15) Huang, N. K.; Lin, Y. L.; Cheng, J. J.; Lai, W. L. Life Sci. 2004, 75,
1649-1657.
(16) Taguchi, H.; Yoshioka, I.; Yamasak, K.; Kim, I. H. Chem. Pharm.
Bull. 1981, 29, 55-62.
(17) Noda, N.; Kobayashi, Y.; Miyahara, K.; Fukahori, S. Phytochemistry
1995, 39, 1247-1248.
(18) Lin, J. H.; Liu, Y. C.; Hau, J. P.; Wen, K. C. Phytochemistry 1996,
42, 549-551.
MTT Assay. PC12 cells were serum-deprived by three washes of
PBS and resuspended in DMEM. The suspended cells were plated on
96-well plates (1 × 104 cells/well) and treated with the indicated
reagent(s). After treatment for 21 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) was added to the medium (0.5
mg/mL), and the mixture was incubated at 37 °C for another 3 h. After
discarding the medium, 100 µL of DMSO was then applied to the well
to dissolve the formazan crystals, and the absorbances at 570 and 630
nm in each well were measured on a micro-ELISA reader.
A2A Receptor Binding Assay. Membranes were thawed and
preincubated for 60 min at 3 °C at a protein concentration of 0.8 µg/
mL in binding buffer with adenosine deaminase (2 U/mL). In one set
of experiments we also incubated the membranes without adenosine
deaminase for [3H]DPCPX binding. A 96-well microplate filtration
(19) Hayashi, J.; Sekine, T.; Deguchi, S.; Lin, Q.; Horie, S.; Tsuchiya,
S.; Yano, S.; Watanabe, K.; Ikegami, F. Phytochemistry 2002, 59,
513-519.
(20) Yun-Choi, H. S.; Pyo, M. K.; Park, K. M. Arch. Pharm. Res. 1997,
20, 91-92.
(21) Huang, Z. B.; Song, D. M.; Chen, F. K. Chin. J. Med. Chem. 2005,
15, 227-229.
(22) Xiao, Y. Q.; Li, L.; You, X. L. China J. Chin. Materia Med. 2002,
27, 35-36.
(23) Wang, L.; Xiao, H. B.; Liang, X. M. Chin. Trad. Herbal Drugs 2003,
34, 584-585.
(24) Coll, J.; Bowden, B. J. Nat. Prod. 1986, 49, 934-936.
(25) Sagner, S.; Kneer, R.; Wanner, G.; Cosson, J. P.; Deus-Neumann,
B.; Zenk, M. H. Phytochemistry 1998, 47, 339-347.

574 Journal of Natural Products, 2007, Vol. 70, No. 4
Huang et al.
(26) Shimizu, M.; Zenko, Y.; Tanaka, R.; Matsuzawa, T.; Morita, N.
Chem. Pharm. Bull. 1993, 41, 1469-1471.
(32) Trivedi, B. K.; Blankley, C. J.; Bristol, J. A.; Hamilton, H. W.; Patt,
W. C.; Kramer, W. J.; Johnson, S. A.; Bruns, R. F.; Cohen, D. M.;
Ryan, M. J. J. Med. Chem. 1991, 34, 1043-1049.
(33) Golisade, A.; Wiesner, J.; Herforth, C.; Jomaab, H.; Linka, A. Bioorg.
Med. Chem. 2002, 10, 769-777.
(34) Uhlen, S.; Dambrova, M.; Nasman, J.; Schioth, H. B.; Gu, Y.;
Wikberg-Matsson, A.; Wikberg, J. E. Eur. J. Pharmacol. 1998, 343,
93-101.
(27) Lin, Y. L.; Wang, W. Y.; Kuo, Y. H.; Chen, C. F. J. Chin. Chem.
Soc. 2000, 47, 247-251.
(28) Strnad, M.; Hanus, J.; Vanek, T.; Kaminek, M.; Ballantine, J. A.;
Fussell, B.; Hanke, D. E. Phytochemistry 1997, 45, 213-218.
(29) Thompson, R. D.; Secunda, S.; Daly, J. W.; Olsson, R. A. J. Med.
Chem. 1991, 34, 3388-3390.
(30) Koyama, H.; Boueres, J. K.; Han, W.; Metzger, E. J.; Bergman, J.
P.; Gratale, D. F.; Miller, D. J.; Tolman, R. L.; MacNaul, K. L.;
Berger, J. P.; Doebber, T. W.; Leung, K.; Moller, D. E.; Hecka, J.
V.; Sahooa, S. P. Bioorg. Med. Chem. Lett. 2003, 13, 1795-1799.
(31) Gannett, P. M.; Nagel, D. L.; Reilly, P. J.; Lawson, T.; Sharpe, J.;
Toth, B. J. Org. Chem. 1988, 53, 1064-1071.
(35) Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099-
3108.
NP0605182