Structural combination of established 5-HT2A receptor ligands: New aspects of the binding mode

Article abstract of DOI:10.1111/j.1747-0285.2010.01011.x

MH.MZ, MDL 100907, and altanserin are structurally similar 4-benzoyl-piperidine derivatives and are well accommodated to receptor interaction models. We combined structural elements of different high-affinity and selective 5-HT_2A antagonists, as MH.MZ, altanserin, and SR 46349B, to improve the binding properties of new compounds. Three new derivatives were synthesized with a 4-benzoyl-piperidine moiety as the lead structure. The in vitro affinity of the novel compounds was determined by a [³H]MDL 100907 competition binding assay. The combination of MH.MZ and SR 46349B resulted in a compound (8) with a moderate affinity toward the 5-HT_2A receptor (K_i = 57 nm). The remarkably reduced affinity of other compounds (4a), (4b), and (4c) (K_i = 411, 360 and 356 nm respectively) indicates that MH.MZ can only bind to the 5-HT_2A receptor with the p-fluorophenylethyl residue in a sterically restricted hydrophobic binding pocket.

Full text of DOI:10.1111/j.1747-0285.2010.01011.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01011.x
Chem Biol Drug Des 2010; 76: 361–366
Research Letter
Structural Combination of Established 5-HT2A
Receptor Ligands: New Aspects of the Binding
Mode
Vasko Kramer1,*,†, Matthias M. Herth1,†
Martin A. Santini2, Mikael Palner2, Gitte
,
alcoholism, disorder, and anxiety disorders) are thought to act, at
least partially, through serotoninergic mechanisms (2). Most if not
all clinically approved atypical antipsychotic drugs are potent
5-HT2A receptor antagonists (3,4).
2
1
¨
M. Knudsen and Frank Rosch
1Institute of Nuclear Chemistry, University of Mainz, Fritz-
Strassmann-Weg 2, D-55128 Mainz, Germany
2Center for Integrated Molecular Brain Imaging and University of
Copenhagen, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen
Ø, Denmark
In particular, it was shown by [18F]altanserin and positron emission
tomography (PET) that 5-HT2A receptor binding in patients with mild
cognitive impairment is reduced and hippocampal 5-HT2A receptor
availability is decreased in major depressive patients (5).
*Corresponding author: Vasko Kramer, kramerv@uni-mainz.de
Both authors are equally contributed.
To date, high-affinity and selective 5-HT2A receptor antagonists are
well known, such as MDL 100907, MH.MZ, altanserin, and SR
46349B (Figure 1) (6,7). MH.MZ, MDL 100907, and altanserin are
structurally similar 4-benzoyl-piperidine derivatives, whereas SR
46349B contains no piperidine but a dimethylaminoethyl oxime
ether moiety.
MH.MZ, MDL 100907, and altanserin are structur-
ally similar 4-benzoyl-piperidine derivatives and
are well accommodated to receptor interaction
models. We combined structural elements of dif-
ferent high-affinity and selective 5-HT2A antago-
nists, as MH.MZ, altanserin, and SR 46349B, to
improve the binding properties of new com-
pounds. Three new derivatives were synthesized
Especially, MH.MZ, MDL 100907 and altanserin are in accordance
regarding their structural body with a rudimentary pharmacophore
model published by Andersen et al. (1) Two aryl substituents, sepa-
rated by distance a, located distances b and c from an amine moi-
ety (Figure 2) are required in this model for effective 5-HT2A
receptor ligands. Distances suggested by Anderson et al. for a, b,
and c are 5.1, 7.5, and 8.1 ꢀ, respectively.
with
structure. The in vitro affinity of the novel com-
pounds was determined by
[3H]MDL 100907
a 4-benzoyl-piperidine moiety as the lead
a
competition binding assay. The combination of
MH.MZ and SR 46349B resulted in a compound (8)
with a moderate affinity toward the 5-HT2A recep-
tor (Ki = 57 nM). The remarkably reduced affinity
of other compounds (4a), (4b), and (4c) (Ki = 411,
360 and 356 nM respectively) indicates that
MH.MZ can only bind to the 5-HT2A receptor with
Recently, Herth et al. (6,8,9) described 18F-labelable MDL 100907
derivatives, MH.MZ, and the corresponding ketone derivative DD-1,
retaining the favorable characteristics of the parent compound
(Table 1). This is surprising because of the fact that the affinity is
not retained by changing the ketone body of altanserin to the sec-
ondary alcohol altanserinol as shown by Tan et al. (10).
the p-fluorophenylethyl residue in
a sterically
restricted hydrophobic binding pocket.
Key words: chemical structure, drug design, G-protein coupled
receptor, receptor and ligands (agonist ⁄ antagonist)
Therefore, the purpose of this study was to combine structural ele-
ments of MH.MZ, MDL 100907, altanserin, and SR 46349B to study
their structure–activity relationship (SAR), and thereby the influence
on the 5-HT2A affinity of different structural substitutions on the
carbonyl moiety of altanserin (Figure 3).
Received 7 October 2009, revised 1 May 2010 and accepted for publica-
tion 2 May 2010
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter impli-
cated in almost every conceivable physiologic or behavioral function
as affect, aggression, appetite, cognition, endocrine function, gas-
trointestinal function, motor function, neurotrophism, sex, sleep, and
vascular function (1). Moreover, most drugs that are currently used
for the treatment of psychiatric disorders (e.g., Alzheimer's disease,
depression, mania, schizophrenia, autism, obsessive compulsive,
For 3-(2-(4-(3-(2-fluoroethoxy)-2-methoxybenzoyl)-piperidin-1-yl)-ethyl)-
2-thioxo-2,3-dihydro-1H-quinazolin-4-on (4a) the p-fluorophenyl ring
of altanserin was replaced by the 3-fluoroethoxy-2-methoxyphenyl
ring of MH.MZ. 3-(2-(4-(3-(2-Fluoroethoxy)-2-methoxy-a-hydroxyben-
zyl)-piperidin-1-yl)-ethyl)-2-thioxo-2,3-dihydro-1H-quinazolin-4-on (4b)
contains a quinazolinone ring instead of the p-fluorophenyl substitu-
ent of MH.MZ. To investigate SAR about the size of the aromatic
361

Kramer et al.
O
O
OH
O
O
N
F
N
N
S
N
H
Altanserin Ki = 0.7 nM
F
MDL 100907 Ki = 0.3 nM
N
O
OH
O
F
O
N
N
MH.MZ Ki = 9 nM
F
Figure 1: Structure and affini-
ties of selective 5-HT2A
antagonists.
OH
F
SR 46349B Ki = 1.2 nM
to that reported by Tan et al. (12) Therefore, reaction with boc-pro-
tected bromoethyl-amine, deprotection, and ring closure with
methyl-2-isothiocyanatobenzoatea resulted in compounds (4a), (4b),
and (4c) (Figure 4).
a
The 4-(3-(2-fluoroethoxy)-2-methoxybenzyl)-piperidine moiety of
MH.MZ and the dimethylaminoethyl oxime ether residue of SR
46349B were combined following a condensation reaction of ketone
DD-1 and amine (7).b The necessary educts were produced as
reported by Herth et al. (6) and Villani et al. (13), respectively. Fig-
ure 5 illustrates the synthetic route to (8).
c
b
N
Figure 2: A general pharmacophore model to account for the
binding of 5-HT2A antagonists (1).
To study whether the new compounds are full antagonists as MDL
100907 and altanserin, their activity toward the 5-HT2A receptor
was determined by a functional assay that assesses the activation
of the receptor by measuring the accumulation of formed inositol-
phosphate (IP).c The results (Figure 6) demonstrate that all new
compounds, as well as MH.MZ, and DD-1 are full antagonists.
Table 1: Affinities toward 5-HT2A receptors of various antago-
nists and the new compounds. Data presented as Ki [nM] € SD
(n = 4) (5, 7, 10)
Compound
Ki [nM]
Altanserin
Altanserinol
MDL 100907
MH.MZ
DD-1
SR 46349B
(4a)
(4b)
0.74 € 0.88
186
0.3 € 0.1
9.02 € 2.11
3.23 € 0.18
1.2
411 € 347
390 € 60
356 € 56
57 € 18
5-HT2A receptor affinity (Ki) was determined by a radioligand com-
petition binding assay with GF-62 cells, a clonal cell line expressing
high amounts (5–7 pmol ⁄ mg) of the 5-HT2A receptor. The test-tubes
contained [3H]MDL 100907 (0.3 nM) and seven different concentra-
tions of the test compounds (1 lM–1 pM) in a total of 1 mL assay
buffer. Ketanserin (1 lM) was added to determine non-specific bind-
ing. Binding affinities of the tested and reference compounds are
summarized in Table 1.d
(4c)
(8)
The combination of structural elements of MH.MZ and SR 46349B
led to the medium affine compound E-dimethylaminoethoxy-[3-(2-
fluoroethoxy)-2-methoxy-phenyl]-1-[2-(p-fluorophenyl)-ethyl]-pipe-ridin-
4-yl-methanonoxim (8). It has a ꢀ6 fold lower Ki-value (57 nM)
compared to MH.MZ. This probably is attributed to the additional
space required by the dimethylaminoethyl oxime residue. However,
the retained affinity indicates that substitution with larger substitu-
ents in that position is tolerated to a certain point. There might
also be a positive hydrogen bond interaction between the oxime
nitrogen and a serine residue. The same interaction was also
observed for the carbonyl residue of altanserin (14,15). Therefore,
compound (8) could be used as a lead structure for further
ether substituents, the 2-fluoroethyl substituent of compound
(4b) was replaced by a methoxy group in 3-(2-(4-(2,3-dimethoxy
benzoyl)-piperidin-1-yl)-ethyl)-2-thioxo-2,3-dihydro-1H-quinazolin-4-on
(4c). Moreover, the influence of the ketone, the secondary hydroxyl
or the dimethylaminoethyl oxime ether moiety of SR 46349B,
toward the affinity profile of these compounds is determined by the
comparison of compound (8), MH.MZ and DD-1 (Figure 3).
Organic syntheses of key intermediates (1a), (1b), and (1c) were
carried out similar to a route described by Ullrich et al. (11) Synthe-
sis of the quinazolinone derivatives was achieved by a route similar
362
Chem Biol Drug Des 2010; 76: 361–366

Structural Combination of Established 5-HT2A Receptor Ligands
A
C
O
O
O
O
R
R4
F
O
R
N
R4
R
B
N
S
N
H
F
F
O
N
OH
O
R1
R2
R1
R3
R1
R3
Figure 3: A: Variation of the benzoyl moiety: compound (4a) and (4b) (black), compound (4c) (green), and altanserin (red); B: Variation of
the a-benzyl position: compound (8) (black), MH.MZ (green) and DD-1 (red); C: Variation of the phenethyl group of MH.MZ: compound (4b)
(black) and MH.MZ (red).
R2
R2
O
O
O
O
ii)
i)
R1
R1
O
NH
N
N
O
H
(2a-c)
(1a-c)
R2
R2
O
O
iii)
O
O
R1
O
R1
N
N
N
NH2
Figure 4: Synthesis of (4a),
(4b), and (4c): reagents and
conditions: (i) tertiary-butyl-2-brom-
ethylcarbamate, KI, K2CO3, DMF, rt,
51–77%; (ii) TFA, rt, 83–95%; (iii)
methyl-2-isothiocyanatobenzoate,
THF, rt, 30–32%.
(3a-c)
(4a-c)
S
N
H
(a)
(b)
(c)
R1 = Me; R2 = = O
R1 = Me; R2 = -OH
R1 = Fluorethyl; R2 = -OH
optimization. Changing the secondary hydroxyl group of MH.MZ and
compound (4b) to a carbonyl group in DD-1 and (4a) showed no
dramatic effects on the binding profile. The affinity of (4a), (4b),
and (4c) is 411, 390, and 356 nM, respectively (Table 1). The
descending affinity in the order (4a) > (4b) > (4c) is in accordance
with the increasing bulk by the fluoroethoxy group.
group and the methoxy group is not tolerated by the receptor binding
pocket. We suggest that the benzyl alcohol of MH.MZ and the ben-
zoyl ring of altanserin cannot bind to the same site at the 5-HT2A
receptor. Two different binding modes might be possible for antago-
nists with a 4-benzoylpiperidin lead structure. MH.MZ may bind to
the 5-HT2A receptor with the p-fluorophenylethyl residue at the same
hydrophobic binding pocket as the benzoyl ring of altanserin.
This result totally reflects the slightly reduced affinity for compounds
with larger substituents at the phenyl ring of MH.MZ (Table 2) (5).
The binding properties in the receptor–altanserin complex are
described in a 3D-QSAR study published by Dezi et al. (16) Therein,
altanserin binds to the 5-HT2A receptor with the p-fluorobenzoyl
moiety in a hydrophobic binding pocket. This result is in good
agreement with experimental data from previously published muta-
genesis experiments (14,15). The size of the binding pocket is
restricted by the amino acids Trp6.48, Phe6.51, and Phe6.52 from
which Phe6.52 participates in p–p interactions with the aromatic
ring of the ligands (15).
The two different binding modes of altanserin and MH.MZ also
account for the retained affinity of MH.MZ compared to altanseri-
nol. By varying size and hydrophobic properties of the substituent in
position 4 of the phenylethyl substituent, it should be possible to
improve the binding characteristics of MH.MZ.
The lipophilicities of the reference compounds were determined
using the HPLC method according to Krass et al. (17) Soerensen
buffer was used as eluent, and log p values were calculated from
The remarkably reduced affinity of compounds (4a), (4b), and (4c)
indicates that the additional space required by the fluoroethoxy
Chem Biol Drug Des 2010; 76: 361–366
363

Kramer et al.
i)
ii)
N
N
N
H2N
N
OH
O
O
(6)
(7)
O
O
O
F
N
iii)
DD-1
F
N
O
O
N
O
F
N
(8)
F
Figure 5: Synthesis of (8): reagents and conditions: (i) dimethylamino-ethylchloride hydrochloride, K2CO3, benzene, reflux, 54%; (ii) hydro-
chloric acid 10%, reflux, 74%; (iii) 1.25 M hydrochloric acid in ethanol, reflux, 23%.
1500
Table 2: Affinities toward human 5-HT2A receptor of different
substituted antagonists (5)
O
OH
1000
500
0
O
F
N
R
Compound
R
Ki [nM]
MH.MZ
Nitro-MH.MZ
Methoxy-MH.MZ
F
9.02 € 2.11
26 € 6,7
59 € 57
-NO2
-OMe
Basal
5-HT
MH.MZ DD-1
4a
4b
4c
8
Figure 6: Functional properties of the new compounds toward
the 5-HT2A receptor. The full agonist 5-HT activates 5-HT2A robustly
(p < 0.01, one-way ANOVA with Tukeys post-hoc test), while the new
compounds all exert antagonistic properties. Data presented as %
of basal € SD (n = 3–6).
trate the blood–brain barrier. The increased lipophilicity of (8) can
be explained by the dimethylaminoethyl oxime residue. Compounds
(4a), (4b), and (4c), in contrast, are more hydrophilic than altanser-
in because of the two additional aromatic ether functions. These
derivatives are more polar and may not be able to cross the blood–
brain barrier.
retention times of the respective substances.e The calculated log
p values are displayed in Table 3.
In conclusion, four new 5-HT2A antagonists based on a 4-(benzoyl)-
piperidine moiety were synthesized in good chemical yields. The
agonistic activity was determined by a functional assay, and the
affinity to the 5-HT2A receptor was determined in a [3H]MDL 100907
The lipophilicities of altanserin, MDL 100907, and MH.MZ are in
the range of log p = 2–3 and indicate a good possibility to pene-
364
Chem Biol Drug Des 2010; 76: 361–366

Structural Combination of Established 5-HT2A Receptor Ligands
Table 3: Lipophilicities ⁄ log p values of altanserin, MDL 100907,
6. Herth M.M., Kramer V., Piel M., Palner M., Riss P.J., Knudsen
G.M., Rçsch F. (2009) Synthesis and in vitro affinities of various
MDL 100907 derivatives as potential 18F-radioligands for 5-HT2A
receptor imaging with PET. Bioorg Med Chem;17:2989–3002.
7. Rinaldi-Carmona M., Congy C., Santucci V., Simiand J., Gautret
B., Neliat G., Labeeuw B., Le Fur G., Breliere J.C. (1992) Bio-
chemical and pharmacological properties of SR 46349B, a new
potent and selective 5-hydroxytryptamine2 receptor antagonist.
Pharmacol Exp Ther;262:759–768.
and new MDL 100907 derivatives. Data presented as Ki [nM] € SD
(n = 3)
Compound
Log p-values
Altanserin
MDL 100907
MH.MZ
DD-1
2.15 € 0.01
2.98 € 0.01
2.80 € 0.01
3.08 € 0.1
(4a)
(4b)
(4c)
(8)
1.65 € 0.02
1.31 € 0.01
1.53 € 0.01
3.80 € 0.01
8. Herth M.M., Debus F., Piel M., Palner M., Knudsen G.M., Lꢁd-
dens H., Rçsch F. (2008) Total synthesis and evaluation of
[18F]MHMZ. Bioorg Med Chem Lett;18:1515–1519.
9. Herth M.M., Piel M., Debus P., Schmitt U., Lꢁddens H., Rçsch F.
(2009) Preliminary in vivo and ex vivo evaluation of the 5-HT2A
imaging probe [18F]MH.MZ. Nucl Med Biol;36:447–454.
10. Tan P.Z., Baldwin R.M., Van dyck C.H., Al-Tikriti M., Roth B.,
Khan N., Charney D.S., Innis R.B. (1999) Characterization of
radioactive metabolites of 5-HT2A receptor PET ligand
[18F]altanserin in human and rodent. Nucl Med Biol;26:601–608.
11. Ullrich T., Rice K.C. (2000) A Practical Synthesis of the Serotonin
5-HT2A Receptor Antagonist MDL 100907, its Enantiomer and
their 3-Phenolic Derivatives as Precursors for [11C]Labeled PET
Ligands. Bioorg Med Chem;8:2427–2432.
binding assay. The combination of MH.MZ and SR 46349B repre-
sents a compound (8) with a moderate affinity toward the 5-HT2A
receptor (Ki = 57 nM). This is possible because of the increased
bulky group required by the dimethylaminoethyl oxime residue. The
affinity of (4a), (4b), and (4c) in contrast is reduced significantly.
This indicates that MH.MZ binds to the 5-HT2A receptor with the
p-fluorophenylethyl residue in a sterically restricted hydrophobic
binding pocket. Herth et al. (6) could already demonstrate that an
improved binding characteristic of MH.MZ is accessible by variation
of the substituent in the para-position. Small lipophilic residues such
as a methyl group or a hydrogen may lead to higher affinities,
whereas larger substituents lead to medium affine compounds.
12. Tan P.Z., Baldwin R.M., Fu T., Charney D.S., Innis R.B. (1999)
Rapid synthesis of F-18 and H-2 dual-labeled altanserin, a meta-
bolically resistant PET ligand for 5-HT2A receptors. J Lab Comp
Radiopharm;42:457–467.
13. Villani F.J., Tavares R.F., Ellis C.A. (1969) Oximino ethers: dialkyl-
aminoalkyl derivatives. J Pharm Sci;58:138.
Acknowledgments
14. Braden M.R., Parrish J.C., Naylor J.C., Nichols D.E. (2006) Mole-
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Phe339(6.51) and Phe340(6.52) with superpotent N-benzyl phe-
nethylamine agonists. Mol Pharmacol;70:1956–1964.
15. Choudhary M.S., Craigo S., Roth B.L. (1993) A single point muta-
tion (Phe340>Leu340) of a conserved phenylalanine abolishes
4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]mesu-
lergine but not [3H]ketanserin binding to 5-hydroxytryptamine2
receptors. Mol Pharmacol;43:755–761.
Financial support by Friedrich-Naumann-Stiftung and the European
Network of Excellence (EMIL) is gratefully acknowledged. We also
thank the VCI (Verband der chemischen Industrie e.V.) for the dona-
tion of solvents. Financial support by Friedrich-Naumann-Stiftung
and the Deutsche Forschungsgemeinschaft (DFG) is gratefully
acknowledged.
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a3-(2-(4-(3-(2-Fluoroethoxy)-2-methoxybenzo-yl)-piperidin-1-yl)-ethyl)-2-
thioxo-2,3-dihydro-1H-quinazolin-4-on (4a); 3-(2-(4-(3-(2-Fluoroeth-
oxy)-2-methoxy-a-hydroxybenzyl)-piperidin-1-yl)-ethyl)-2-thioxo-2,3-
dihydro-1H-quinazolin-4-on (4b) and 3-(2-(4-(2,3-dimethoxy-a-hydro-
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Chem Biol Drug Des 2010; 76: 361–366
365

Kramer et al.
(4a): (0.18 mmol; 30%) 1H-NMR (300 MHZ, DMSO-d6)
d
Kindom) in labeling medium [inositol-free DMEM (US Biological)
containing 10% dFBS and P ⁄ S] overnight at 37 ꢀC and 5% CO2.
The cells were washed once with incubation buffer (20 mM HEPES,
pH 7.4; 20 lM LiCl, 1 mM MgCl2, 1 mM CaCl2; all from Sigma) and
incubated at 37 ꢀC in the same buffer for 30 min. The solutions
were removed, and test compounds diluted in incubation buffer
were added to the wells for 30 min at 37 ꢀC. The formed inositol
phosphates were extracted with 10 mM ice-cold formic acid (Sigma)
for 30 min at 4 ꢀC. Supernatants were transferred to columns with
AG 1-X8 anion exchange resin (Bio-Rad, Hercules, California, USA)
and eluted directly into Ultima-FLO AF scintillation liquid (Packard,
[ppm] = 12.908 (bs, 1H); 7.939 (d, 1H); 7.722 (t, 1H); 7.399-7.292 (m,
2H); 7.206 (d, 1H); 7.100 (t, 1H); 6.952 (d, 1H); 4.851 (t, 1H); 4.695
(t, 1H); 4.510 (t, 2H); 4.338 (t, 1H); 4.238 (t, 1H); 3.802 (s, 3H); 3.321
(s, 1H); 3.025-2.881 (m, 3H); 2.601 (t, 2H); 2.107 (t, 2H); 1.709 (d,
2H); 1.541-1.377 (m, 2H); MS (FD) m ⁄ z (% rel Int.): 485.4 (100 [M]+);
486.4 (69.5 [M + 1]+); 487.4 (24.3 [M + 2]+);
(4b): (0.19 mmol; 32%) 1H-NMR: (300 MHZ, DMSO-d6)
d
[ppm] = 12.875 (bs, 1H); 7.932 (d, 1H); 7.717 (t, 1H); 7.391-7.275 (m,
2H); 7.031-6.872 (m, 3H); 4.938 (d, 1H); 4.832 (t, 1H); 4.675 (t, 1H);
4.582 (t, 1H); 4.487 (t, 2H); 4.272 (t, 1H); 4.172 (t, 1H); 3.722 (s, 3H);
3.320 (bs, 1H); 3.001-2.829 (m, 2H); 2.548 (t, 2H); 1.879 (q, 2H); 1.781
(d, 1H); 1.411 (bs, 1H); 1.314-1.130 (m, 2H); MS (FD) m ⁄ z (% rel Int.):
487.4 (43.6 [M]+); 488.4 (100 [M + 1]+); 489.4 (25.9 [M + 2]+); (4c):
(0.26 mmol; 43%) 1H-NMR: (300 MHZ, DMSO-d6) d [ppm] = 7.641
(m, 1H); 7.521 (d, 1H); 7.432 (m, 1H); 7.377-7.277 (m, 2H); 6.921-
6.728 (m, 2H); 5.007 (d, 1H); 4.769 (m, 2H); 4.172 (t, 1H); 3.829 (s,
3H); 3.806 (s, 3H); 3.320 (bs, 1H); 3.262 (m, 2H); 2.799-2.648 (m, 2H);
2.104 (m, 2H); 1.859 (m, 2H); 1.555-1.253 (m, 2H); MS (ESI) m ⁄ z (%
rel Int.): 455.6 (32.4 [M]+); 456.6 (100 [M + 1]+); 457.6 (21.2
[M + 2]+).
Waltham, Massachusetts, USA) using
a 2 M ammonium for-
mate ⁄ 0.1 M formic acid solution. Accumulated [3H]inositol phos-
phates were measured with a Tri-Carb 2900TR liquid scintillation
counter (Packard Instruments) after 1-h incubation at room tempera-
ture. Statistics were performed with GRAPHPAD PRISM 5 (GraphPad
Software, San Diego, California, USA).
dCompetition binding experiments were carried out in test-tubes
containing [3H]MDL 100907 (0.2 nM), seven different concentrations
of the test compound (1 lM–1 pM) and 10–20 lg GF-62 clonal cells
in a total of 1 mL assay buffer (50 mM Tris-Base, 120 mM NaCl,
50 mM KCl, 1% bovine serum albumine, 0.1% ascorbic acid, pH 7.4,
37 ꢀC). Ketanserin (1 lM) was used to determine non-specific bind-
ing. Incubation was carried out for 1 h at 37 ꢀC and terminated by
rapid filtration over glass fiber GF ⁄ C filters presoaked in 1% poly-
ethyleneimine, using a Brandel cell harvester. Filters were washed
with 300 mL cold assay buffer (titrated to pH 7.4 at 4 ꢀC). Filters
were placed in scintillation vials, and 2.5 mL Ultima Gold scintilla-
tion fluid was added. The scintillation cocktails were placed in cold
and dark overnight and counted for 4 min in a Tri-Carb 2900TR
Liquid Scintillation Analyser from Packard. Ki and error values were
calculated with Graphpad Prism 5.
bE-Dimethylaminoethoxy-[3-(2-fluoroethoxy)-2-methoxy-phenyl]-1-[2-(p-
fluorophenyl)-ethyl]-pipe-ridin-4-yl-methanonoxim (8): 2 mmol MA-1
and 2 mmol dimethylaminoethoxy-amine (7) were dissolved in etha-
nol containing 1.25 M HCl and heated under reflux for 20 h. After
evaporation of the solvent and extraction with chloroform, the resi-
due was taken up in concentrated ammonia solution. Extraction
with chloroform, evaporation and CC (chloroform ⁄ methanol 5:1)
gave the product as colorless crystals (0.5 mmol; 25%). 1H-NMR
(300 MHZ, CDCl3) d [ppm] = 7.148-7.072 (m, 3H); 7.026-6.829 (m,
4H); 6.581 (dd, 1H); 4.841 (t, 1H); 4.683 (t, 1H); 4.274 (t, 1H); 4.181
(t, 1H); 4.142 (t, 2H); 3.806 (s, 3H); 3.048-2.951 (m, 3H); 2.771-2.688
(m, 2H); 2.628 (t, 2H); 2.580-2.483 (m, 2H); 2.240 (s, 6H); 2.085 (q,
2H); 1.909-1.792 (m, 2H); 1.778-1.596 (m, 2H); MS (FD) m ⁄ z (% rel
Int.): 490.5 (100 [M + 1]+); 491.5 (27.9 [M + 2]+).
eLipophilicities were determined using the HPLC system described
previously with a LiChrospher 100 RP 18 EC-5l (250 · 7.8 mm) and
a 20 lL loop. Soerensen buffer was used as eluent with a flow
rate of 4 mL ⁄ min. Retention times for all tested compounds and for
reference substances (ascorbic acid, benzaldehyde, anisol, toluene,
4-bromoanisol, and 4-iodoanisol) of known log p were assessed and
enabled the calculation of the capacity factor k. A plot of these
reference values against their known log p values gave a reference
curve that was used to calculate log p values for synthesized
compounds.
cPI hydrolysis assay: GF-62 cells (1.5 · 106 cells ⁄ mL) were cultured
overnight in Dulbecco's modified Eagle's medium (DMEM; Invitro-
gen, Carlsbad, California, USA) supplemented with 10% fetal bovine
serum (FBS; Invitrogen), 1 mM sodium pyruvate (Sigma-Aldrich,
St. Louis, Missouri, USA), penicillin (P: 100 units ⁄ mL; Invitrogen),
and streptomycin (S: 100 lg ⁄ mL; Invitrogen) at 37 ꢀC and 5% CO2.
Subsequently, cells were incubated with 4 lCi ⁄ well of myo-(1,2)-
[3H]-inositol (Amersham, Little Chalfont, Buckinghamshire, United
366
Chem Biol Drug Des 2010; 76: 361–366