Synthesis of eudistomin D analogues and its effects on adenosine receptors

Article abstract of DOI:10.1016/j.bmc.2008.01.041

Six analogues (1-6) of eudistomin D, a β-carboline alkaloid from a marine tunicate Eudistoma olivaceum, were synthesized, and their affinity and selectivity for adenosine receptors A₁, A_2A, and A₃ were examined. All the synthetic compounds 1-6 did not show affinity to the adenosine A₁ receptor. δ-Carboline 3 exhibited the most potent affinity to the adenosine receptor A₃ among compounds 1-6. δ-Carbolines 3 and 4 showed better affinity than the corresponding β-carbolines 1 and 2, respectively, while N-methylation (2, 4, and 6, respectively) of the pyrrole ring in 1, 3, and 5 resulted in the reduced affinity to the adenosin A₃ receptor. On the other hand, an eudistomin D derivative, BED, exhibited modest affinity to all the receptors A₁, A_2A, and A₃ but no selectivity.

Full text of DOI:10.1016/j.bmc.2008.01.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3825–3830
Synthesis of eudistomin D analogues
and its effects on adenosine receptors
Haruaki Ishiyama,a Kengo Ohshita,a Tetsuro Abe,a
Hiroyasu Nakatab and Jun’ichi Kobayashia,*
aGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
bDepartment of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan
Received 29 December 2007; revised 21 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—Six analogues (1–6) of eudistomin D, a b-carboline alkaloid from a marine tunicate Eudistoma olivaceum, were synthe-
sized, and their affinity and selectivity for adenosine receptors A1, A2A, and A3 were examined. All the synthetic compounds 1–6 did
not show affinity to the adenosine A1 receptor. d-Carboline 3 exhibited the most potent affinity to the adenosine receptor A3 among
compounds 1–6. d-Carbolines 3 and 4 showed better affinity than the corresponding b-carbolines 1 and 2, respectively, while
N-methylation (2, 4, and 6, respectively) of the pyrrole ring in 1, 3, and 5 resulted in the reduced affinity to the adenosin A3 receptor.
On the other hand, an eudistomin D derivative, BED, exhibited modest affinity to all the receptors A1, A2A, and A3 but no
selectivity.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and 4 were also synthesized. The 2-N-methyl derivatives
(5 and 6, respectively) of 1 and 2 were also prepared to
examine effects of the 2-N-methyl group on the activity
of adenosine receptors.
During our continuing search for bioactive metabolites
from marine organisms, we have found that eudistomin
D, a b-carboline alkaloid from a marine tunicate Eudis-
toma olivaceum,1 and its analogues such as 7-bromoeudis-
tomin D (BED)2 are potent inducers of Ca2+-release from
sarcoplasmic reticulum (SR)3 as well as inhibitors of
phosphodiesterase,4 and are more potent than caffeine.
Caffeine exhibits a variety of physiological activities (or
action) including regulation of the blood pressure, respi-
ratory functioning, gastric and colonic activity, urine vol-
ume, and exercise performance.5 The mechanism of
actions of caffeine is reported to be competitive antago-
nism to A1 and A2A adenosine receptors,6 induction of
Ca2+-release from SR, inhibition of phosphodiesterase,
and so on. Previously, our group has found that a hybrid
molecule of caffeine and eudistomin D showed a potent
affinity for adenosine receptor A3 subtype.7 In the present
study, the methylated analogues (1 and 2, respectively) of
eudistomoin D and BED in place of the bromine atom
were designed and synthesized. d-Carboline analogues 3
2. Results and discussion
2.1. Chemistry
The synthesis of b-carboline analogues 1 and 2 is sum-
marized in Scheme 1. Coupling of 2,6-dimethyl-4-
bromoanisole (7)8 and 3-aminopyridine (8) in DMF
with Pd2(dba)3, Xphos,9 and NaOtBu afforded 9 in
88% yield. Photocyclization of 9 in toluene gave 10
and 11 in 31% and 45% yield, respectively, and treat-
ment of 10 with BBr3 in CH2Cl2 furnished 1 in 78%
yield. Methylation of 10 with MeI and NaH in DMF
provided 12 in 57% yield, which was treated with BBr3
in CH2Cl2 to afford 2 in 78% yield.
In Scheme 2 is shown the synthesis of d-carboline ana-
logues 3 and 4. Treatment of 11 with BBr3 in CH2Cl2
furnished 3 in 70% yield. Methylation of 11 with MeI
and NaH in DMF provided 13 in 85% yield, which
was treated with BBr3 in CH2Cl2 to afford 4 in 83%
yield.
Keywords: Eudistomin D analogues; Caffeine; Adenosine receptors;
SAR.
*
Corresponding author. Tel.: +81 11 706 3239; fax: +81 11 706
4989; e-mail: jkobay@pharm.hokudai.ac.jp
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.041

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H. Ishiyama et al. / Bioorg. Med. Chem. 16 (2008) 3825–3830
Me
Me
Br
Me
HO
HO
Me
N
HO
HO
N
N
N
Me
N
N
H
N
H
Me
N
H
Me
eudistomin D
1
2
3
Me
Me
Br
Me
HO
Me
HO
Me
HO
Br
HO
Me
N
N+
N+
N
N
H
Me
N
Me
N
H
N
Me
Me
BED
4
5
6
The synthesis of analogues 5 and 6 is summarized in
Scheme 3. While methylation of 10 with MeI in acetone
followed by treatment with BBr3 in CH2Cl2 furnished 5
in 35% yield for 2 steps, methylation of 10 with MeI and
NaH in THF followed by treatment with BBr3 in
CH2Cl2 provided 6 in 8% yield for 2 steps.
was shown. Affinities of reference ligands, that is, the non-
selective caffeine, the A1-selective xanthine amine conge-
ner (XAC), the A2A-selective CGS21680, and the A3-
selective 50-(N-ethylcarboxamido)adenosine (NECA),
were also shown for comparison. These compounds
showed reasonable affinity for each receptor confirming
the validity of our assay.
2.2. Biological evaluation
All the synthetic compounds 1–6 did not show affinity to
the adenosine A1 receptor (Table 1). d-Carboline 3
exhibited the most potent affinity to the adenosine
receptor A3 among compounds 1–6. d-Carbolines 3
and 4 showed better affinity than the corresponding b-
carbolines 1 and 2, respectively, while N-methylation
(2, 4, and 6, respectively) of the pyrrole ring in 1, 3,
and 5 resulted in the reduced affinity to the adenosine
The potency of these synthetic compounds as adenosine
receptor ligands was investigated in radioligand binding
assays at human recombinant adenosine A1, A2A, and
A3 receptors expressed in membranes of HEK293T cell
as previously reported.7 The results expressed as Ki values
are presented in Table 1. When the affinity of the test com-
pounds was very low, percentage of inhibition at 100 lM
Me
Me
a
MeO
Me
b
MeO
+
N
N
H2N
N
H
Me
Br
7
8
9
Me
Me
MeO
MeO
Me
N
+
N
Me
N
N
H
H
10
11
(31%)
(45%)
Me
Me
c
MeO
Me
HO
Me
N
N
N
H
N
H
10
1
Me
Me
Me
MeO
Me
HO
Me
MeO
Me
d
e
N
N
N
N
N
N
H
Me
Me
10
12
2
Scheme 1. Synthesis of compounds 1 and 2. Reagents and conditions: (a) Pd2(dba)3, XPhos, NaOtBu/DMF, 120 °C, 20 h (88%); (b) hv/toluene, rt,
15 h; (c) BBr3/CH2Cl2, rt, 3 h (78%); (d) MeI, NaH/THF, rt, 3 h (57%); (e) BBr3/CH2Cl2, rt, 3 h (78%).

H. Ishiyama et al. / Bioorg. Med. Chem. 16 (2008) 3825–3830
3827
A3 receptor. Likely to this, it has been found for hybrid
molecules of caffeine and eudistomin D, in which com-
pounds having a nitrogen at the b-position of the pyri-
dine ring (b-N type) showed lower affinity than the
corresponding d-N type compounds, while N-methyla-
tion of a pyrrole ring significantly lowered the potency
as adenosine receptor ligands.7 An eudistomin D deriv-
ative, BED, exhibited modest affinity to all the receptors
A1, A2A, and A3 but no selectivity (Table 1).
Me
Me
a
MeO
Me
N
HO
Me
N
N
H
N
H
11
3
b
Me
Me
MeO
Me
N
HO
Me
N
c
N
N
3. Experimental
3.1. Chemistry
Me
Me
13
4
Scheme 2. Synthesis of compounds 3 and 4. Reagents and conditions:
(a) BBr3/CH2Cl2, rt, 4 h (70%); (b) MeI, NaH/THF, rt, 3 h (85%); (c)
BBr3/CH2Cl2, rt, 4 h (83%).
3.1.1. Instruments and analyses. The IR spectrum was re-
corded on a JASCO FT/IR-5300 spectrometer. UV
spectra were recorded on a Shimadzu UV1600PC spec-
trophotometer. Proton and carbon NMR spectra were
recorded on Bruker 500 and/or 600 MHz and JEOL
400 MHz spectrometers. EI mass spectra were obtained
on a DX-303 mass spectrometer.
Me
Me
MeO
Me
a, b
HO
Me
+
Me
N
N
N
H
3.1.2. N-(4-Methoxy-3,5-dimethylphenyl)pyridin-3-amine
(9). DMF (1.5 mL) was added to an oven-dried Schlenk
tube charged with 3-aminopyridine (8) (47.7 mg,
0.51 mmol), Pd2(dba)3 (23 mg, 25 lmol), 2-dicyclohexyl-
phosphino-20,40,60-triisopropylbiphenyl (21 mg, 43 lmol),
and NaOtBu (67 mg, 0.7 mmol). The mixture was stirred
for 10 min at ambient temperature. 2,6-Dimethyl-4-
bromoanisole (7) (80 lL, 0.51 mmol) was added and then
stirred for 20 h at 120 °C. The reaction mixture was
quenchedwith H2O,andextractedwithCHCl3. Theextract
was washed with brine, dried over MgSO4, filtered and con-
centrated under reduced pressure. Purification with a silica
gel column chromatography (hexane/EtOAc = 9:1 ! 2:1)
provided 9 (101.5 mg, 0.45 mmol) in 88% yield.
N
H
5
10
Me
Me
MeO
Me
HO
Me
c, d
+
Me
N
N
N
H
N
Me
6
10
Scheme 3. Synthesis of compounds 5 and 6. Reagents and conditions:
(a) MeI, acetone, rt, 2 h; (b) BBr3/CH2Cl2, rt, 8 h (35% for 2 steps); (c)
MeI, NaH/THF, rt, 1 h; (d) BBr3/CH2Cl2, rt, 8 h (8% for 2 steps).
1
Compound 9: pale yellow amorphous solid; H NMR
(400 MHz, CDCl3) d 2.24 (6H, s), 3.69 (3H, d,
J = 1.9 Hz), 6.03 (1H, br s), 6.75 (2H, s), 7.11 (1H, dt,
J = 1.8 and 5.3 Hz, 1H), 7.30 (1H, dt, J = 8.3 and
1.4 Hz), 8.30 (1H, s); 13C NMR (100 MHz, CDCl3) d
16.17, 59.78, 119.57, 122.15, 123.54, 131.69, 137.06,
138.85, 137.06, 138.85, 138.91, 140.58, 140.80, 152.12;
EI-MS m/z 228 (M+); HREIMS Calcd for C14H16N2O
(M+) m/z 228.1262, found m/z 228.1263.
Table 1. Affinities of caffeine and eudistomin D analogues (1–6) and
BED at human adenosine A1, A2A, and A3 receptors
Compound
Kia,b (lM)
A2A
A1
A3
1
>100 lM
>100 lM
>100 lM
>100 lM
>100 lM
>100 lM
>100 lM
80.6 10
24.9 1.2
44.1 10.6
>100 lM
>100 lM
17.7 3.0
43.4 1.4
4.43 2.2
13.3 3.9
29.7 7.4
>100 lM
2.05 0.69
2
3
4
5
3.1.3. 8-Methoxy-7,9-dimethyl-d-carboline (10) and 6-
Methoxy-5,7-dimethyl-b-carboline (11). A solution of 9
(120 mg, 0.53 mmol) in toluene (200 mL) was irradiated
with tungsten lamp 15 h at ambient temperature. The
mixture was concentrated in vacuo and purified by a flash
column chromatography on silica gel (hexane/ace-
tone = 7:1 ! 3:1 ! CHCl3/MeOH = 20:1 ! 10:1 ! 5:1)
to yield 10 (37 mg, 0.16 mmol, 31%) and 11 (53 mg,
0.24 mmol, 45%).
6
BED
7.37 2.13 4.92 1.17
(7-bromoeudistomin D)
Caffeine
XAC
49.0 19.6 18.1 5.9
0.009 0.001 nd
>100 lM
nd
CGS21680
NECA
nd
nd
0.0462 0.0084 nd
nd
0.020 0.009
nd, not determined.
a The Ki values are means SEM of two or three separate assays, each
performed in duplicate.
b The binding of each radioactive ligand to membranes prepared from
HEK293T cells expressing human adenosine A1, A2A, or A3 recep-
tors was best-fitted to a one-site model of binding with estimated Kd
(dissociation constant) values of 5, 52, and 6.5 nM, respectively, and
Bmax values of 8600, 7000, and 310 fmol/mg protein, respectively.
Compound 10: pale yellow amorphous solid; UV
(MeOH) kmax 309 (e 27400), 266 (28100), 239 (30100),
217 (55700) nm; IR (KBr) 3360, 2920, 2860, 1630 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6) d 2.39 (3H, s), 2.88
(3H, s), 3.69 (3H, s), 7.17 (1H, s), 7.30 (1H, dd, J = 8.2

3828
H. Ishiyama et al. / Bioorg. Med. Chem. 16 (2008) 3825–3830
and 5.0 Hz), 7.78 (1H, dd, J = 8.2 and 1.4 Hz), 8.40 (1H,
dd, J = 5.0 and 1.4 Hz), 11.18 (1H, s); 13C NMR
(125 MHz, CDCl3) d 12.73, 17.33, 60.53, 109.65, 116.90,
118.96, 126.52, 127.97, 131.91, 133.28, 137.00, 141.56,
151.10, 165.70; EI-MS m/z 226 (M+); HREIMS Calcd
for C14H14N2O (M+) m/z 226.1106, found m/z 226.1106.
Compound 2: brown amorphous solid; UV (MeOH)
kmax 426 (e 700), 326 (3000), 273 (4400), 203
1
(8500) nm; IR (KBr) 3360, 3190, 2930, 1640 cmꢀ1; H
NMR (400 MHz, DMSO-d6) d 2.46 (3H, s), 2.77 (3H,
s), 4.00 (3H, s), 7.55, (1H, s), 8.51 (1H, d, J = 6.3 Hz),
8.61 (1H, d, J = 6.3 Hz), 9.52 (1H, s); EI-MS m/z 226
(M+); HREIMS Calcd for C14H14N2O (M+) m/z
226.1106, found m/z 226.1095.
Compound 11: brown amorphous solid; UV (MeOH)
kmax 294 (e 8000), 240 (15500), 205 (16800) nm; IR
(KBr) 3330, 2930, 2860, 1640, 1460 cmꢀ1
;
1H NMR
3.1.7. 8-Hydroxy-7,9-dimethyl-d-carboline (3). To a solu-
tion of methylether 11 (10 mg, 0.044 mmol) in CH2Cl2
(1 mL) was added BBr3 (1 M in CH2Cl2, 5 mL, 5 mmol)
and the reaction mixture was stirred for 4 h at ambient
temperature. The mixture was concentrated in vacuo
and purified by a flash column chromatography on silica
gel (CHCl3/MeOH = 5:1) to yield 3 (6.6 mg, 0.031 mmol)
in 70% yield.
(400 MHz, DMSO-d6) d 2.43 (3H, s), 2.73 (3H, s), 3.70
(3H, s), 7.35 (1H, s), 8.27 (1H, s), 8.36 (1H, s), 8.99 (1H,
s), 11.93 (1H, s); 13C NMR (125 MHz, DMSO-d6) d
13.15, 17.33, 60.59, 110.75, 116.04, 118.64, 125.41,
128.23, 132.22, 133.86, 136.59, 137.40, 137.98, 150.08;
EI-MS m/z 226 (M+); HREIMS Calcd for C14H14N2O
(M+) m/z 226.1106, found m/z 226.1094.
3.1.4. 6-Hydroxy-5,7-dimethyl-b-carboline (1). To a solu-
tion of methylether 10 (60 mg, 0.265 mmol) in CH2Cl2
(30 mL) was added BBr3 (1 M in CH2Cl2, 5 mL, 5 mmol)
and the reaction mixture was stirred for 3 h at ambient
temperature. The mixture was concentrated in vacuo
and purified by a flash column chromatography on silica
gel (CHCl3/MeOH = 5:1) afforded 1 (45 mg, 0.212 mmol)
in 78% yield.
Compound 3: yellow amorphous solid; UV (MeOH) kmax
313 (e 4800), 272 (5100), 241, (5500), 211 (11400) nm; IR
(KBr) 3400, 3180, 1620 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6) d 2.40 (3H, s), 2.80 (3H, s), 7.35 (1H, s), 7.80
(1H, dd, J = 8.0 and 5.2 Hz), 8.49 (1H, d, J = 8.0 Hz),
8.57 (1H, d, J = 5.2 Hz), 12.25 (1H, s); 13C NMR
(150 MHz, DMSO-d6) d 14.33, 19.45, 112.94, 114.73,
119.94, 121.08, 128.21, 133.15, 134.27, 137.10, 138.52,
140.11, 150.27; EI-MS m/z 212 (M+); HREIMS
Calcd for C13H12N2O (M+) m/z 212.0950, found m/z
212.0934.
Compound 1: brown amorphous solid; 1H NMR
(500 MHz, DMSO-d6) d 2.42 (3H, s), 2.74 (3H, s), 7.42,
(1H, s), 8.44 (1H, d, J = 6.0 Hz), 8.58 (1H, d,
J = 6.0 Hz), 9.17 (1H, s), 12.42 (1H, s); 13C NMR
(125 MHz, CD3OD) d 13.28, 18.68, 111.17, 117.69,
117.80, 119.67, 133.30, 134.29, 134.62, 139.01, 148.00;
EI-MS m/z 212 (M+); HREIMS Calcd for C13H12N2O
(M+) m/z 212.0949, found m/z 212.0945.
3.1.8. 8-Methoxy-5,7,9-trimethyl-d-carboline (13). To a
solution of 11 (20 mg, 0.088 mmol) in THF (5 mL) was
added NaH (45 mg, 1.8 mmol) and the reaction mixture
was stirred for 30 min at ambient temperature. To the
reaction mixture was added MeI (0.5 mL, 8.0 mmol)
and stirred for 3 h. The reaction mixture was concentrated
under reduced pressure. Purification with a silica gel col-
umn chromatography (hexane/acetone = 8:1 ! 6:1) pro-
vided 13 (18 mg, 0.075 mmol) in 85% yield.
3.1.5. 6-Methoxy-5,7,9-trimethyl-b-carboline (12). To a
solution of 10 (10 mg, 0.044 mmol) in THF (5 mL) was
added NaH (25 mg, 1.0 mmol) and the reaction mixture
was stirred for 30 min at ambient temperature. To the
reaction mixture was added MeI (50 lL, 0.8 mmol) and
stirred for 3 h. The reaction mixture was concentrated un-
der reduced pressure. Purification with a silica gel column
chromatography (CHCl3/MeOH = 20:1 ! 15:1) pro-
vided 12 (6.0 mg, 25 lmol) in 57% yield.
Compound 13: yellow amorphous solid; UV (MeOH)
kmax 312 (e 2600), 276 (3100), 217 (7500), 204
1
(8500) nm; IR (KBr) 2930, 2850, 1630, 1600 cmꢀ1; H
NMR (400 MHz, DMSO-d6) d 2.43 (3H, s), 2.88 (3H,
s), 3.67 (3H, s), 3.80 (3H, s), 7.29 (1H, s), 7.36 (1H,
dd, J = 8.0 and 4.3 Hz), 7.91 (1H, d, J = 8.0 Hz), 8.42
(1H, d, J = 4.3 Hz); 13C NMR (125 MHz, CDCl3) d
12.76, 17.54, 29.67, 60.55, 107.42, 114.64, 118.70,
119.16, 126.64, 131.61, 134.66, 138.49, 140.84, 143.35,
150.78; EI-MS m/z 240 (M+); HREIMS Calcd for
C15H16N2O (M+) m/z 240.1263, found m/z 240.1263.
Compound 12: brown amorphous solid; UV (MeOH)
kmax 398 (e 1800), 322 (9200), 266 (14400), 215
(24800) nm; IR (KBr) 3420, 2920, 2850, 1720,
1640 cmꢀ1; 1H NMR (400 MHz, DMSO-d6) d 2.52 (3H,
s), 2.79 (3H, s), 3.74 (3H, s), 4.02 (3H, s), 7.63 (1H, s),
8.58 (1H, d, J = 6.3 Hz), 8.65 (1H, d, J = 6.3 Hz), 9.56
(1H, s); EI-MS m/z 240 (M+); HREIMS Calcd for
C15H16N2O (M+) m/z 240.1263, found m/z 240.1257.
3.1.9. 8-Hydroxy-5,7,9-trimethyl-d-carboline (4). To a
solution of methylether 11 (10 mg, 0.042 mmol) in
CH2Cl2 (1 mL) was added BBr3 (1 M in CH2Cl2, 5 mL,
5 mmol) and the reaction mixture was stirred for 4 h at
ambient temperature. The mixture was concentrated in
vacuo and purified by a flash column chromatography
on silica gel (CHCl3/MeOH = 5:1) to provide 4 (7.8 mg,
0.035 mmol) in 83% yield.
3.1.6. 6-Hydroxy-5,7,9-trimethyl-b-carboline (2). To a
solution of methylether 12 (5.5 mg, 0.023 mmol) in
CH2Cl2 (1 mL) was added BBr3 (1 M in CH2Cl2,
5 mL, 5 mmol) and the reaction mixture was stirred
for 4 h at ambient temperature. The mixture was con-
centrated in vacuo and purified by a flash column chro-
matography on silica gel (CHCl3/MeOH = 5:1) to
provide 2 (4.2 mg, 0.018 mmol) in 78%.
Compound 4: yellow amorphous solid; UV (MeOH) kmax
351 (e 560), 313 (2500), 277 (3100), 245 (3000), 217 (5600)

H. Ishiyama et al. / Bioorg. Med. Chem. 16 (2008) 3825–3830
3829
202 (5400) nm; IR (KBr) 3390, 2930, 1650 cmꢀ1
;
1H
Center (Rolla, MO, USA) were transiently transfected
into HEK293T cells using Effectene (Quiagen). Cells were
maintained at 37 °C in humidified air containing 5% CO2
in Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum, 100 lg/mL kanamycin for
48 h. The cells were harvested and homogenized in lysis
buffer containing 50 mM Tris–HCl buffer (pH 7.4) with
a protease-inhibitor mixture (Roche Diagnostics) and
subjected to low-speed centrifugation to remove organ-
elles and nuclei. The resulting supernatant was subjected
to centrifugation at 30,000g for 20 min, and precipitated
cell membranes were collected, washed twice, resus-
pended in the lysis buffer, and stored at ꢀ80 °C until
use.
NMR (400 MHz,CD3OD) d 2.58 (3H, s), 2.93 (3H, s),
4.09 (3H, s), 7.54 (1H, s), 7.97 (1H, dd, J = 8.6 and
5.9 Hz), 8.56 (1H, dd, J = 5.9 and 0.9 Hz), 8.73 (1H, dd,
J = 8.6 and 0.9 Hz); 13C NMR; d 12.50, 18.17, 28.72,
107.68, 115.36, 118.66, 119.07, 127.17, 134.11, 136.15,
139,91, 146.82; EI-MS m/z 226 (M+); HREIMS Calcd
for C14H14N2O (M+) m/z 226.1106, found m/z 226.1099.
3.1.10. 8-Hydroxy-2,5,7-trimethyl-b-carboline (5). To a
solution of 8 (4.8 mg, 21 lmol) in acetone (1 mL) was
added MeI (0.1 mL, 1.6 mmol) and stirred for 1 h. The
reaction mixture was concentrated and subjected to the
next reaction without further purification. To the crude
methylether was added BBr3 (1 M in CH2Cl2, 3 mL,
3 mmol) and the reaction mixture was stirred for 8 h at
ambient temperature. The mixture was concentrated in
vacuo and purified by a flash column chromatography
on amino silica gel (CHCl3/MeOH = 2:1) to yield 5
(2.3 mg, 7.5 lmol) in 35% yield.
3.2.3. Adenosine receptor binding assays. Radioligand
binding experiments to adenosine A1, A2A, and A3 recep-
tors were carried out by using [3H]DPCPX,
[3H]CGS21680, and [3H]NECA, respectively. Cell mem-
branes expressing adenosine A1, A2A, and A3 receptors
were incubated with 4 nM [3H]DPCPX, 15 nM
[3H]CGS21680, or 32 nM [3H]NECA, respectively, in
the presence of 9 to 10 different concentrations of test
compounds in 250 lL of assay buffer containing 50 mM
Tris–acetate buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA
and 1 U/mL adenosine deaminase for 60 min at 25°C.
The incubated mixture was harvested on Whatman GF/
B filters pre-soaked in 0.1% polyethyleneimine by a cell
harvester, and washed three times with 50 mM Tris–
HCl buffer (pH 7.4). The radioactivity on the filter was
measured by a scintillation counter. All experiments were
carried out two or three times in duplicate. The nonspe-
cific binding for adenosine A1, A2A, and A3 receptors
was defined as the binding activity in the presence of
XAC, CGS21680, and NECA, respectively, at 10 lM
each. Kd and Bmax values in saturation and inhibition
studies were determined using one-site binding model by
nonlinear regression analysis (GraphPad Prism 4; Graph-
Pad, San Diego, CA, USA).
Compound 5: brown amorphous solid; UV (MeOH) kmax
325 (e 8200), 277 (10000) nm; IR (KBr) 3400, 1640 cmꢀ1
;
1H NMR (400 MHz, CD3OD) d 2.52 (3H, s), 2.83 (3H, s),
4.50 (3H, s), 7.40 (1H, s), 8.35 (1H, d, J = 6.3 Hz), 8.56
(1H, d, J = 6.3 Hz), 9.04 (1H, s).
3.1.11. 8-Hydroxy-2,5,7,9-tetramethyl-b-carboline (6). To
a solution of 8 (10 mg, 47 lmol) in THF (1 mL) was added
NaH (6.3 mg, 160 lmol) and the reaction mixture was
stirred for 10 min at ambient temperature. To the reaction
mixture was added MeI (50 lL, 400 lmol) and stirred for
1 h. The reaction mixture was filtered with SiO2 pad and
subjected to the next reaction without further purifica-
tion. To the crude methylether was added BBr3 (1 M in
CH2Cl2, 2 mL, 2 mmol) and the reaction mixture was stir-
red for 8 h at ambient temperature. The mixture was con-
centrated in vacuo and purified by a flash column
chromatography on amino silica gel (CHCl3/
MeOH = 2:1) to afford 6 (1.2 mg, 3.7 lmol) in 8% yield.
Compound 6: brown amorphous solid; UV (MeOH) kmax
Acknowledgments
327 (e 2500), 280 (3600) nm; IR (KBr) 3400, 1645 cmꢀ1
;
1H NMR (400 MHz,CD3OD) d 2.58 (3H, s), 2.85 (3H,
s), 4.09 (3H, s), 4.56 (3H, s), 7.58 (1 H, s), 8.43 (1H, d,
J = 6.3 Hz), 8.62 (1H, d, J = 6.3 Hz), 9.31 (1H, s).
We thank S. Oka, Center for Instrumental Analysis,
Hokkaido University, for EI-MS measurements. This
work was partly supported by a Grant-in-Aid from
the Uehara Memorial Foundation and Grant-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
3.2. Biological assays
3.2.1. Radioligand materials. [3H]-8-Cyclopentyl-1,3-
dipropylxanthine ([3H]DPCPX), [3H]-2-[4-(2-carboxy-
ethyl)-phenethylamino]-50-N-ethylcarboxamidoadenosine
([3H]CGS21680), and [3H]-50-N-ethylcarboxamidoadeno-
sine([3H]NECA)werepurchasedfrom Perkin-Elmer(Bos-
ton, MA, USA). Unless otherwise stated, all other
materials used for ligand binding assay were purchased
from Sigma to Aldrich (St. Louis, MO, USA).
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