3
616 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Rook et al.
(
HRMS) data were determined on a TSQ Quantum AM spectro-
(BTC) were obtained from Fluka (Buchs, Switzerland). Galan-
tamine hydrobromide, which was used as reference substance
for cholinesterase inhibition, was purchased from JANSSEN-
CILAG GmbH (Neuss, Germany).
Stock solutions of test compounds were prepared in water or
ethanol. At least five different concentrations of the test com-
pound were measured at 25 °C at 412 nm, and each concentra-
tion was tested in triplicate. The inhibition curves were obtained
by plotting percentage enzyme activity (100% for the reference)
versus logarithm of the test compound concentration.
meter (Therma Electron Corporation). IR data were obtained
from a Magna-IR FT-IR spectrometer system 550 by Nicolet
(
WI).
The preparation of the following derivatives was described
5
earlier: 6-methoxy-1,2,3,4-tetrahydro-β-carboline (2), 6-meth-
oxy-β-carboline (5), 6-hydroxy-β-carboline (6), 6-methoxy-1-
methyl-β-carboline (7), 6-bromo-β-carboline (8), and 3,4-
dihydro-6-methoxy-β-carboline (3).
carboline (1) and β-carboline (4) were prepared analogously to
the 6-methoxy and 6-hydroxy derivatives. The insertion of
substituents at position 9 (19a-b) was achieved as previously
described.
5
5
5
21
2
0
1,2,3,4-Tetrahydro-β-
5
Assay of Glutamate-Induced Excitotoxicity. L12-G10 and
L13-E6 cells are derivatives of L(tk-) cells that express cDNAs
of the human NR1-1a/NR2A and NR1-1a/NR2B subunits of
5,29
Protocols for the preparation of the target com-
3
3
pounds and intermediates together with their physical and
spectral data (NMR, elemental analysis, HRMS) are given in
the Supporting Information.
the NMDA receptor (NR), respectively.
Untransfected
L(tk-) cells were purchased from ECACC (Salisbury, UK).
The cells were cultured in Minimal Essential Medium (MEM)
supplemented with 10% fetal bovine serum, a mixture of peni-
cillin and streptomycin (100U/100 μg/mL; Biochrom,
Germany), and 100 μM ketamine to reduce excitotoxicity
generated by background NR expression prior to induction.
Ketamine and memantine were provided by Sigma and pre-
pared as 100 mM stock solutions in PBS. Stock solutions of test
compounds were prepared at 50 mM in DMSO. Glutamate-
2
Synthesis of the N -Bivalent Compounds (11-15): General
Procedure. A solution of 8.2 mmol β-carboline (4, 5, 6) and
3
4
.7 mmol R,ω-dibromoalkane in 10 mL DMF was refluxed for
22
-6 h under nitrogen as previously described. After cooling to
room temperature, 40 mL of acetone was added and the
precipitated solids were isolated by filtration and dried in vacuo.
Synthesis of the Quaternary Salts (9, 10, 20, 22): General
Procedure. A 10-fold molar excess of alkyl iodide was added to a
solution of the respective β-carboline derivative (4, 19, 21) in
acetone. Under nitrogen, the mixture was stirred at room
temperature for 24 h. The precipitated solids were isolated by
filtration and dried in vacuo.
For Example: 2-Methyl-9-[9-(2-methyl-β-carboline-9-yl)nonyl]-
β-carboline-2-ium Diiodide (22b). From 21b and methyl iodide;
yield 98% slightly yellow crystals; mp 250-251 °C, H NMR: 400
MHz (DMSO-d ) δ = 1.17-1.30 (m, 10H, NCH CH (CH ) ),
3
3
induced excitotoxicity was measured as described with
modifications. Cells were seeded into 96-well microtiter plates
4
at 1 ꢀ 10 /well. After 24 h of cultivation at 37 °C and 5% CO
2
,
the expression of NR was induced by the addition of 4 μM
dexamethasone. After additional 16 h of incubation, cells were
washed three times with MEM containing 1% bovine albumin
to remove residual ketamine. The cells were preincubated for
30 min with 200 μL of phenol-red-free MEM containing the
test compounds (final DMSO content 0.1%). NR-mediated
excitotoxicity was then triggered by adding 10 μM of both
(S)-glutamate and glycine. The LDH assay was performed 4 h
after the addition of glutamate/glycine according to the manual
provided with Roche’s Cytotoxicity Detection Kit (LDH). All
tests were done in triplicate and are expressed as mean values (
SD. Baseline excitotoxicity (0%) was defined as the LDH
release after addition of glutamate and glycine but in the
presence of 100 μM ketamine. Conversely, 100% excitotoxicity
was defined as the LDH release induced by glutamate/glycine
in the absence of ketamine. IC50 values were calculated using
the GraphPad Prism 5.0 software package.
1
6
2
2
2 5
þ
1
4
2
.78-1.85 (mc, 4H, 2 ꢀ NCH
2
CH
2
), 4.51 (s, 6H, 2 ꢀ N CH
3
),
.55-4.59 (t, J = 7.3, 4H, 2 ꢀ NCH
2
), 7.47-7.51 (dt, J = 0.6, 7.8,
H, 2 ꢀ 6), 7.83-7.87 (dt, J = 1.0, 8.2, 2H, 2 ꢀ 7), 7.92-7.95 (d,
J = 8.5, 2H, 2 ꢀ 8), 8.52-8.53 (d, J = 7.9, 2H, 2 ꢀ 5), 8.67-8.68
(
9
dd, J = 0.9, 6.5, 2H, 2 ꢀ 4), 8.82-8.84 (d, J = 6.4, 2H, 2 ꢀ 3),
1
3
.69 (s, 2H, 2 ꢀ 1). C NMR: 100 MHz (DMSO-d
6
) δ = 26.70
þ
(
4
N(CH ) (CH ) ), 29.16 (2 ꢀ NCH CH ), 44.07 (2 ꢀ N CH ),
2
2
2 5
2
2
3
8.31 (2 ꢀ NCH ), 111.84 (aromatic), 118.10 (aromatic), 119.45
2
(
1
(
aromatic), 122.15 (aromatic), 124.24 (aromatic), 130.01 (aromatic),
31.85 (aromatic), 132.47 (aromatic), 133.95 (aromatic), 135.78
aromatic), 144.50 (aromatic). Anal. (C H I N ) C,H,N.
3
3
38 2
4
9
To exclude direct inhibition of LDH by the test compounds,
cells were incubated with test compounds as described above but
in the absence of NR agonists. Then all cells were lysed by
addition of 1% triton X-100 and LDH activity was determined
Synthesis of the N -Bivalent Compounds (21a-c): General
Procedure. According to ref 23, 85 mmol of comminuted NaOH
and 1.5 mmol R,ω-dibromoalkane were added to a solution of
3
stirred at room temperature for 4 h, and 10 mL of water was
added and the aqueous layer was extracted with chloroform.
The combined organic phases were washed with 10% NaOH
.0 mmol β-carboline 4 in 7.5 mL of DMSO. The mixture was
(
see Table S2, Supporting Information).
Acknowledgment. We thank Dr. C. Enzensperger for
solution, dried over MgSO
purified by column chromatography with chloroform/methanol
4
, and evaporated. The product was
helpful discussions. We are grateful to the expert technical
assistance of Petra Wiecha and Heidemarie Graf. We thank
Dr. D. Steinhilber (University of Frankfurt) for the generous
gift of L12-G10 and L13-E6 cells.
(
12/1) as the mobile phase.
Synthesis of the Partially Reduced Compounds (16a-b, 23):
General Procedure. According to refs 30,31, a solution of
0.35 mmol of the respective quaternary β-carboline (11d,11i,22b)
in 50 mL of methanol was treated with 10 mmol of NaBH slowly
Supporting Information Available: Synthetic procedures and
spectral data for intermediates and target compounds; details of
the AChE and BChE assays; specificity controls assay of
glutamate-induced excitotoxicity. This material is available free
of charge via the Internet at http://pubs.acs.org.
4
under nitrogen. The mixture was stirred for 2-4 h at room
temperature, the organic phase was evaporated, and 10 mL of
water was added. The aqueous phase was then extracted with
dichloromethane. The organic phase was dried over MgSO4,
evaporated, and dried in vacuo.
Pharmacological Studies. AChE and BChE Assays. Inhibi-
tion of AChE and BChE by test compounds was analyzed using
References
5
,32
the Ellman assay as described previously.
AChE (EC3.1.1.7,
(1) Al-Shamma, A.; Drake, S.; Flynn, D. L.; Mitscher, L. A.; Park,
Y. H.; Rao, G. S.; Simpson, A.; Swayze, J. K.; Veysoglu, T.; Wu,
S. T. Antimicrobial agents from higher plants. Antimicrobial agents
from Peganum harmala seeds. J. Nat. Prod. 1981, 44, 745–747.
Type V-S, from electric eel) and BChE (EC3.1.1.8, from equine
serum) were purchased from Sigma-Aldrich (Steinheim,
Germany) with a purity of g60% protein by biuret. 5,5 -
Dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB),
acetylthiocholine (ATC), and butyrylthiocholine iodides
0
(
2) Airaksinen, M. M.; Kari, I. β-Carbolines, psychoactive com-
pounds in the mammalian body. Part I: occurance, origin and
metabolism. Med. Biol. 1981, 59, 21–34.
Rook, Yvonne
