Ring expansions of tetrahydroprotoberberines and related dibenzo[c,g]azecines modulate the dopamine receptor subtype affinity and selectivity

Article abstract of DOI:10.1002/ardp.200900267

The affinities of tetrahydroprotoberberines for dopamine receptors dramatically decrease after cleaving the central C-N bond to the analogous ten-membered dibenzo[c,g]azecines [1]. In the present work, we also synthesized eleven-membered homologues of the

Full text of DOI:10.1002/ardp.200900267

Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
M. Schulze et al.
207
Full Paper
Ring Expansions of Tetrahydroprotoberberines and Related
Dibenzo[c,g]azecines Modulate the Dopamine Receptor
Subtype Affinity and Selectivity
Maria Schulze1, Oliver Siol2, Werner Meise3, Jochen Lehmann1, and Christoph Enzensperger1
1 Institut fꢀr Pharmazie, Lehrstuhl fꢀr Pharmazeutische/Medizinische Chemie, Friedrich-Schiller-Universitꢁt
Jena, Jena, Germany
2 Institut fꢀr Pharmazie, Lehrstuhl fꢀr Pharmazeutische Biologie, Friedrich-Schiller-Universitꢁt Jena, Jena,
Germany
3 Institut fꢀr Pharmazie, Rheinische Friedrich-Wilhelms-Universitꢁt Bonn, Bonn, Germany
The affinities of tetrahydroprotoberberines for dopamine receptors dramatically decrease after
cleaving the central C-N bond to the analogous ten-membered dibenzo[c,g]azecines [1]. In the
present work, we also synthesized eleven-membered homologues of these heterocycles and
measured the affinities of the resulting dibenzazaundecenes and their underlying homoberber-
ines for human dopamine receptors as well as the cytotoxic effects of all target compounds on
human glia cells. The tetracyclic iso-C-homoberberine-derivatives revealed to be D4-selective
antagonists, while all other active compounds showed a significant D1/D5 selectivity. Distances
in energy-minimized conformations were measured in order to explain our findings.
Keywords: Azecines / Cytotoxicity / Dopamine receptor / SAR / Scaffold /
Received: November 3, 2009; Accepted: December 2, 2009
DOI 10.1002/ardp.200900267
Introduction
Dopamine receptors play a key role in many psychiatric,
motoric, or endocrinologic disorders. Even though there
are many compounds targeting the dopamine system, it
is not clarified yet if a certain selectivity profile at the
dopamine receptor subtypes might be advantageous for
clinical use. Antipsychotic drugs used in therapy exert
different selectivity profiles (e. g. haloperidol and cloza-
pine). While haloperidol has the highest affinities for D2
and D3 receptors, clozapine preferably binds to D1 and D4
receptors and causes less extrapyramidal side effects [2].
Thus, it is still attractive to look for new scaffolds with
novel selectivity profiles to investigate the therapeutic
value of subtype-selective compounds and to perform
Correspondence: Christoph Enzensperger, Institut fꢀr Pharmazie, Lehr-
stuhl fꢀr Pharmazeutische/Medizinische Chemie, Friedrich-Schiller-Uni-
versitꢁt Jena, Philosophenweg 14, D-07743 Jena, Germany.
E-mail: ch.enzensperger@uni-jena.de
Figure 1. Scaffolds of the test compounds: the underlying tetra-
cyclic structures and ring-opened azecine- and azacyclounde-
cene scaffolds.
Fax: +49 3641 949-802
Abbreviation: tetrahydroprotoberberines (THPBs)
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208
M. Schulze et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
liq
Reagents and Conditions: (a) i) toluene reflux; ii) POCl3, MeCN; iii) NaBH4/MeOH; (b) methyl iodide, MeCN; (c) Na/NH3
.
Scheme 1. Synthesis of compounds 1–6.
cross-target SAR-studies within the dopamine receptors. affinity and selectivity profiles for all dopamine receptor
In former studies, we investigated tetrahydroprotober- subtypes (radioligand binding and functional calcium
berines (THPBs) 1 along with their ring-opened deriv- assay).
atives 2 and found that ring opening of 1a to 2a decreases
Low general cytotoxicity of candidate compounds is a
dopamine receptor affinities dramatically [1]. Further- basic requirement for drug development. Accordingly,
more, ring expansion goes along with a shift of selectivity we measured the cytotoxicity of our target compounds
from similar antagonist activities at all receptors for 1a, up to 250 lM by an in-vitro cell-culture-based MTT assay.
toward D1/D5 selectivity for 2a.
In the present study, we applied this ring-opening
approach on ring-enlarged THPBs to obtain B- (3) and C-
homologues 5, which were synthesized by Meise et al. [3,
Results and discussion
4]. Compounds 3 and 5 were quaternized with methyl Chemistry
iodide, followed by cleavage of the central C-N bond with The synthesis of compounds 1–6 is outlined in Scheme 1.
Na in liq. NH3. The resulting eleven-membered homo- Ring expanded THPB homologues 3 and 5 were synthe-
logues 4 and 6 represent derivatives of novel heterocyclic sized according to previously described procedures [3, 4],
ring systems (Fig. 1). They were investigated together starting with substituted phenylethyl- or phenylpropyl-
with the THPB-homologues 3 and 5 with respect to their amines 7 and a suitable lactone 8, or its corresponding
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Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
Ring-Enlarged Tetrahydroberberines and Dibenzo[c,g]azecines
209
Table 1. Affinities (Ki) for dopamine D1–D5 receptor subtypes determined by radioligand-binding experiments.
Compound
(Radioligand binding studies) Ki (nM)
D3
D1
D2L
D4.4
D5
5.9 {
974 {
30 (rat) {
3748 {
4.4 {
1a
183.5 € 27.5 #
320 € 159 §
251 € 56 #
2565 € 1673 §
185 € 97 #
1b
2a
474 € 156 §
1093 € 61.5 #
>10000
1122 € 30.5 #
>10000
>10000
>10000
398 € 150.5 #
1508 € 136 #
1727 € 238 §
3a
3b
4a
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
4b
5a
2343 € 43 #
662 € 172 #
>10000
>10000
>10000
>10000
>10000
>10000
224 € 117 #
1174 € 221 #
5c
1446 € 176 #
1482 € 329 #
4475 € 14.5 #
>10000
>10000
>10000
427 € 230 #
282 € 115 §
626 € 230 §
843 € 243 #
5d
6a
6c
119 € 74 §
118 € 57 §
1502 € 443 #
917 € 151 #
3120 € 1085 #
3045 € 22 #
726 € 131 #
475 € 254 #
492 € 293 §
97.5 € 30 #
§ Experiments were performed in triplicate, values are given as mean € SD; # experiments were performed twice, values are given
as mean € SEM; { values are from the PDSP database [5].
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Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
Table 2. MTT test results measured on human glia cells.
hydroxy ester 9. Ring opening was performed after qua-
ternization with methyl iodide under Birch conditions
(liq. NH3/Na; Scheme 1).
Compound
Viability at 100 lM (%)
cc50 (lM)
1a
2a
3a
3b
4a
4b
5a
5c
5d
6a
6c
97.8 € 5.2
77.3 € 22.6
94.2 € 3.3
91.8 € 4.2
115.8 € 9.9
78.9 §
63.7 € 1.4
99.3 € 11.0
93.2 € 8.0
71.2 € 8.4
91.7 € 17.3
>250
158.7 € 14.7
>250
Pharmacology
The affinities of the tetracyclic and tricyclic compounds
were determined by radioligand-binding experiments at
all human cloned dopamine receptors according to a pre-
viously described method [1]. The Ki values of the binding
assay are given in nM units. Their functionalities were
defined by an intracellular calcium assay [1] (Table 1).
The investigated tetracyclic THPBs 1a, b showed affin-
ities for all dopamine receptors, but preferable for D1/D5.
The lowest affinities were detected for the D4 receptor.
Stepholidine, a well studied representative of the THPBs
is a ligand for dopamine receptors, which has agonistic
activities at D1 and antagonist properties at D2 receptors
[6]. Yet, all our compounds turned out to be antagonists
for D1 and D2 receptors. This may be explained by the
different substitution pattern compared to stepholidine,
whose phenolic group in position 2 is similar to the
intrinsic agonist dopamine. At all our molecules, this
OH-group is missing. It can be speculated that this partic-
ular phenolic group is crucial for the functionality at
dopamine receptors. Ring opening of 1a to dibenzo[c,g]-
azecine 2a lead to a nearly complete loss of affinities.
Only at the D1 and D5 receptor micromolar affinities
could be detected. Neither ring expansion of ring B of the
THPBs 1a, b to isochino[3,2-a][2]benzazepines 3a, b nor
their ring opening to the respective dibenzo[c,g]azacy-
cloundecenes 4a, b led to ligands with noteworthy affin-
ities for dopamine receptors. But on the contrary, when
THPBs 1a, b were expanded at the central C-ring, we
obtained antagonists with Ki values comparable with
those of the parent THPBs. In contrast to THPBs, the
homo-C congeners 5a, c, d exhibited a distinct D4 selectiv-
ity and almost no affinity for D3 receptors. This might be
interesting, since Seeman et al. observed that D4 receptors
were elevated in the brains (post mortem) of schizophrenic
patients [8]. Furthermore, the atypical antipsychotic
drug clozapine was found to bind more tightly to D4
receptors than to the other dopamine receptor subtypes
[2]. Clinical evidence for the influence of D4 receptors in
schizophrenia was proved by D4 selective antagonists [9].
Interestingly, ring opening of homo-C THPB derivatives
5a, c to compounds 6a, c increased the affinities for D1,
D2, D3, and D5 receptors. Only at D4 receptors, affinities
revealed to be the same or were even decreased. Thus,
compounds 6a, c are D1- or in case of 6c D1/D5-selective,
with the weakest affinity for D3. The increase of affinities
by providing higher conformational flexibility was dis-
cussed in a previous paper [1]. Conclusively, we can de-
>250
165.4 € 9.8
poor solubility
poor solubility
>250
>250
125.1 € 15.7
177.5 € 18.0
§ Due to poor solubility, all values except one were discarded.
monstrate that ring opening, going along with higher
conformational flexibility, maintains or even increases
the affinity of the already active homo-C THPBs but does
not turn the inactive homo-B derivatives into compounds
with distinct affinity.
It is an essential feature for all CNS drugs not to be
toxic. During screening investigations, we observed for
the dibenz[c,g]azecine 2a cytotoxic effects on glia cells.
Therefore, we screened all target compounds using an in-
vitro MTT-assay on human U87-MG glia cells at 100 lM
and determined cc50 values up to 250 lM (see Table 2). Sol-
ubility of all compounds in all concentrations was moni-
tored by microscopy. Values of incompletely dissolved
compounds were neglected.
Ring expansion of THPBs 1 to C- and B-homologues, 3
and 5, respectively, and their ring-opened derivatives, 4
and 6, respectively, did not decrease cytotoxicity in gen-
eral. The results are rather inconsistent. Neither a specific
scaffoldnoroneofthesubstitutionpatternscouldbeiden-
tified as responsible for toxicity. In general, ring opening
led to higher toxicity. Almost all ring-closed compounds
showed no toxicity up to 250 lM. At 100 lM, the methoxy-
lated dibenzo[c,g]azecine 2a, the C-ring-expanded mono-
methoxylated compounds 5a and 6a and the dimethoxy-
substituted B-homologue 4b displayed cytotoxic effects
(viability of cells compared to control a90%). Unfortu-
nately, the poor solubility of compounds 4b and 5a pre-
cluded us from determining their exact cc50 values. Focus-
ing on the monomethoxy series, C-ring expansion led to
higher toxicity, while the B-homologues showed no (3a) or
less (4a) toxic effects. The unsubstituted homo-C-deriv-
atives 5c and 6c showed significant lower cytotoxicity
than theiraromatic-substituted congeners.
In-silico studies
Structure-based modeling is difficult for the dopamine
receptors, since the X-ray structure or a reliable homol-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
Ring-Enlarged Tetrahydroberberines and Dibenzo[c,g]azecines
211
Table 3. Distances in energy-minimized conformations of the
tetracycles 1, 3, 5 between the centroids of the two aromatic
moieties (d1), from the nitrogen to the substituted aromatic moi-
ety (d2) and to the unsubstituted one (d3), and between the
nitrogen and the center between the two aromatic moieties (d4).
Compound
Measured distances (ꢀ)
d1
d2
d3
d4
1a cis
1a trans
3a cis
3a trans
5a cis
5a trans
4.678
6.612
6.535
6.592
4.504
6.311
3.844
3.806
3.944
3.940
3.821
3.842
3.774
3.755
3.697
3.773
4.478
3.747
3.007
1.834
1.984
2.005
3.501
2.108
Figure 2. Measured distances of energy-minimized conforma-
tion of compound 5a.
picture, the flexed structure of 1 and 5 might be responsi-
ble for their affinity.
ogy model is not available so far. Nevertheless, we wanted
to compare the conformational space of the pharmaco-
phors in order to correlate distances with the affinities
for dopamine receptors.
Conclusion
Though our affinities gains were, in general, only moder-
Therefore, we measured the following distances: ate, we could observe some interesting changes in the
Between the centroids of the two aromatic moieties (d1), selectivity at the dopamine receptor subtypes by ring
from the nitrogen to the substituted aromatic moiety enlargement of tetrahydroprotoberberines (THPBs) and
(d2) and the unsubstituted one (d3), respectively. More- their corresponding dibenz[c,g]azecines. Ring expansion
over, we determined the distance between the nitrogen of the C-ring in the THPBs to isochino[1,2-b][3]benzaze-
and the center between the two aromatic moieties, pines (5a, c, d) yielded dopamine antagonists with dis-
which is a parameter for the angulations of the whole tinct D4 selectivity, while the expansion of the other cen-
molecule (d4). In Fig. 2, as an example, these distances tral ring (B) led to compounds with much lower affinities
are demonstrated for compound 5a.
for all dopamine receptor subtypes. We assume that the
In order to select a reasonable conformation for the highly flexed conformation might be responsible for the
minimization, we relied on the observations reported for affinities of the homo-C derivatives 5 and the missing
(l)-stepholidine before [10]. Herein it is assumed that (l)- affinities of homo-B derivatives 3. By ring opening of the
stepholidine binds in its cis-protonated conformation to enlarged, but still active tetracycles 5 to compounds 6,
the binding pocket. Hence, we used the cis-protonated (l)- the D4 selectivity of 5 changed into a D1/D5 preference,
stepholidine as template for our tetracyclic compounds going along with increased affinities. Cytotoxic effects
(1, 3, and 5) prior to energy minimization. To comple- were found preferable for the ring-opened compounds.
ment our investigations, we also measured the respective Substitution at the aromatic ring systems seems to be dis-
trans-isomers (1a, 3a, 5a) (Table 3). Due to their high flexi- advantageous regarding cytotoxicity. The unsubstituted
bility, it is difficult to include the ring-opened derivatives compound 5c displays the lowest cytotoxicity of all active
(2, 4, and 6) to this investigation. Computational exami- compounds. Furthermore, 5c shows a new selectivity pro-
nations on this special topic are ongoing.
file and, hence, might be used for the further develop-
The active compounds within our investigated set are ment of atypical antipsychotics.
1a-cis and 5a-cis. It is obvious that the distances between
the two aromatics (d1) of these two compounds are signif-
icantly shorter compared to all other molecules we exam-
ined. The other way around can be observed for the dis-
Experimental
Chemistry
tance between the nitrogen and the center between the
aromatic rings (d4) where the distances are significantly
longer. These two parameters reflect the strongly angu-
lated shape of compounds 1 and 5. Since the homo-B
derivative 3a, which lacks affinity, does not show such a
Syntheses were performed under nitrogen with solvents and
reagents of commercial availability with no further purification.
Melting points are uncorrected and were measured in open
capillary tubes, using a Gallenkamp melting point apparatus
(Weiss-Gallemkamp, London, UK). 1H- and 13C-NMR spectral data
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M. Schulze et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
were obtained from a Bruker Advance 250 spectrometer (250
MHz) and Advance 400 spectrometer (400 MHz), (Bruker, Rhein-
statten, Germany) respectively. Elemental analyses were per-
formed on a Heraeus Vario EL apparatus (Heraeus, Hanau, Ger-
many) for all test compounds. MS data were determined by GC/
MS, using a Hewlett–Packard GCD-Plus (G1800C) apparatus (HP-
5MS column; J&W Scientific, Folsom, CA, USA). Synthesis and
analytical data of compounds 1 and 2 were described previously
[1].
and drying in vacuo resulted in the pure products with almost
100% yield.
Compound 11a
Yield: 98.2%, white powder, m.p.: 2158C; 1H-NMR 250 MHz
(CDCl3) d: 1.97–2.13 (m, 2H), 2.76–2.91 (m, 1H), 3.11–3.29 (m, 2H),
3.39 (s, 3H, NMe), 3.53–3.82 (m, 2H), 3.78 (s, 3H, OMe), 3.91–3.96
(d, 1H, J = 13.9 Hz), 5.18–5.24 (d, 1H, J = 15.2 Hz), 5.59–5.69 (m,
2H), 6.72–6.78 (m, 2H), 7.13–7.29 (m, 4H), 7.74–7.77 (d, 1H, J = 8.3
Hz); 13C-NMR, dept 250 MHz (CDCl3) d: 23.1, 30.5, 34.0, 48.6 (NMe),
55.5 (MeO), 59.8, 67.5, 73.6 (14a), 112.0, 117.6, 123.5, 126.6,
127.4, 127.8, 128.4, 128.8, 129.6, 134.4, 141.4; 160.6. Anal. calcd.
for C20H24INO N 0.25 H2O: C 56.41%, H 5.80%, N 3.29%; found: C
56.21%, H 5.75%, N 3.11%.
3-Methoxy-5,6,7,9,14,14a-hexahydroisoquino[3,2-a]
[2]benzazepine 3a and 2,3-dimethoxy-5,6,7,9,14,14a-
hexahydroisoquino[3,2-a][2]benzazepine 3b
Isochroman-3-one (25 mmol, 3.7 g) and a suitable phenethyl-
amine/phenpropylamine, respectively (25 mmol) were refluxed
in toluene (100 mL) for 6 h. After cooling to room temperature,
the amide precipitated as a white solid, which could be filtered
off. The solid was washed twice with toluene and dried in vacuo
to remove the starting material. The product could be identified
by the typical two singlets in 1H-NMR spectrum at 4.65 and 3.59
ppm. The amide was refluxed in 50 mL of a mixture of phos-
phoryl chloride in acetonitrile (1:8) for 18 h. After evaporation of
the solvents, the resulting brown oil was washed twice with
petroleum ether and the residue was solved in methanol. Cooled
by an ice bath, a ten-fold excess of sodium boron hydride was
added in small portions. The suspension was refluxed for half an
hour. After evaporation of the solvent, the residue was dissolved
in water and extracted with ethyl acetate. The organic layer was
dried over MgSO4 and evaporated in vacuo. The resulting yellow
oil was crystallized from methanol to yield the respective tetra-
cycle. Analytical data is given in reference [4]. Yields and melting
points: for compound 3a: yield 29%, white crystals, m.p. 1298C;
for compound 3b: yield 31%, white crystals, m. p. 91–938C.
Compound 11b
Yield: 96.5%, white powder, m.p.: 2208C; 1H-NMR 250 MHz
(DMSO-d6) d: 1.92–2.22 (m, 2H), 2.71–2.81 (m, 2H), 3.05 (s, 3H,
NMe), 3.24–3.52 (m, 2H), 3.75–3.84 (m, 8H), 4.73–5.05 (dd, 2H, J =
64.6; 15.5 Hz), 5.10–5.17 (dd, 1H, J = 13.2; 4.8 Hz); 6.92 (s, 1H);
7.04 (s, 1H), 7.24–7.32 (m, 4H); 13C-NMR 250 MHz (DMSO-d6) d:
23.1, 29.9, 32.5, 48.9 (NMe), 56.1, 56.7, 59.2, 67.8, 73.7, 115.6,
116.4, 124.3, 127.2, 127.5, 127.7, 128.6, 129.2, 131.2, 133.8,
147.3, 149.4. Anal. calcd. for C21H26INO N 0.10 H2O: C 55.30%, H
6.03%, N 2.79%.
Compound 12a
Yield: 94.0%, white solid, m.p.: 2558C; 1H-NMR 250 MHz (metha-
nol-d4) d: 3.27 (s, 3H, NMe), 3.13–3.35 (m, 4H), 3.41–3.57 (m, 2H),
3.78 (s, 3H, MeO), 3.65–3.97 (m, 3H), 4.15–4.32 (m, 1H), 4.94–4.98
(d, 1H, J = 9.8 Hz), 6.85 (s, 1H), 6.90–6.94 (d, 1H, J = 8.7 Hz), 7.22–
7.39 (m, 5H). Anal. calcd. for C20H24INO N 0.25 H2O: C 56.13, H
6.01, N 2.98.
Compound 12c
3-Methoxy-5,6,8,9,14,14a-hexahydroisoquino[1,2-b]
[3]benzazepine 5a and 5,6,8,9,14,14a-
Yield: 91.8%, white solid, m.p: 2578C; 1H-NMR 250 MHz (metha-
nol-d4) d: 3.27 (s, 3H, NMe), 3.30–3.49 (m, 4H), 3.55–4.01 (m, 5H),
4.20–4.41 (m, 1H), 5.01–5.05 (d, 1H, J = 9.9 Hz), 7.24–7.39 (m, 8H).
Anal. calcd, for C19H22IN: C 58.32%, H 5.67%, N 3.58%.
hexahydroisoquino[1,2-b][3]benzazepine 5c
The synthesis was performed according to
a procedures
described before [3], starting with a phenethylamine (2.4 mmol)
and methyl[2-(2-hydroxyethyl)phenyl]acetate (2 mmol). The
crude products were crystallized from toluene. Analytical data is
given in reference [3]. Yields and melting points: for compound
5a: yield 32%, white crystals, m. p. 80–828C; for compound 5c:
yield 22%, white crystals, m.p. 61–628C.
11-Methoxy-6-methyl-6,7,8,9,14,15-hexahydro-5H-
dibenzo[c,g]azacycloundecene hydrochloride 4a,
11,12-dimethoxy-6-methyl-6,7,8,9,14,15-hexahydro-5H-
dibenzo[c,g]azacycloundecene hydrochloride 4b,
3-methoxy-7-methyl-6,7,8,9,14,15-hexahydro-5H-
dibenzo[d,h]azacycloundecene hydrochloride 6a, and
7-methyl-6,7,8,9,14,15-hexahydro-5H-
3-Methoxy-8-methyl-5,6,7,9,14,14a-
hexahydroisoquino[3,2-a][2]benzazepinium iodide 11a,
2,3-dimethoxy-8-methyl-5,6,7,9,14,14a-
dibenzo[d,h]azacycloundecene hydrochloride 6c
In a 100-mL three-neck flask equipped with a balloon as an over-
flow tank and cooled in a liquid nitrogen bath, ammonia was
condensed until it was 3/4 filled. The cooling bath was removed
and the ammonia was allowed to liquefy. After suspending 1
mmol of the quaternary ammonium salts (11, 12) in the liquid
ammonia, small pieces of sodium were added to the stirred mix-
ture until the developing blue colour remained for 10–15 min.
The mixture was quenched with 1–2 drops of saturated aqueous
NH4Cl. The ammonia was evaporated under nitrogen and the
residue portioned between ether and water. The aqueous phase
was extracted with ether (3–15 mL) and the pooled organic
hexahydroisoquino[3,2-a][2]benzazepinium iodide 11b,
3-methoxy-7-methyl-5,6,8,9,14,14a-
hexahydroisoquino[1,2-b][3]benzazepinium iodide 12a,
and 7-methyl-5,6,8,9,14,14a-hexahydroisoquino[1,2-b]
[3]benzazepinium iodide 12c
10 mmol of the tetracyclic compounds (3, 5) were solved in aceto-
nitrile (20 mL). A 20-fold excess of methyl iodide was added and
the mixture stirred overnight. The quaternary ammonium salt
precipitated as white to light yellow powder and could be iso-
lated by filtration. Washing with small amounts of acetonitrile
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Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
Ring-Enlarged Tetrahydroberberines and Dibenzo[c,g]azecines
213
phases were dried over MgSO4 and evaporated. Products were
purified by column chromatography (CH3OH/CHCl3; 1:3). The
product fractions were merged and the solvent removed in
vacuo. The resulting oil was dissolved in little methanol, fol-
lowed by the addition of a few drops of etheric HCl and crystalli-
zation from methanol/ether.
Radioligand binding experiments
Cells were grown at 378C under a humidified atmosphere of 5%
CO2:95% air in HAM/F12-medium (Sigma-Aldrich, Germany) for
CHO cells and Dulbecco's modified Eagles Medium Nutrient mix-
ture F-12 Ham for HEK293 cells, each supplemented with 10%
fetal bovine serum, 1 mM L-glutamine and 0.2 lg/mL of G 418 (all
by Sigma-Aldrich).
Radioligand binding assays with a whole-cell-suspension [1]
were carried out in triplicate in a volume of 550 lL in 96-well
plates (Greiner bio-one, Frickenhausen, Germany), containing:
TRIS-Mg2+-buffer (345 lL), [3H]-ligand (50 lL), whole-cell-suspen-
sion (100 lL), and appropriate drug dilutions (55 lL). [3H]
SCH23390 was used as radioligand for D1 and D5, and [3H] spiper-
one for D2-D3. Non-specific binding was determined using flu-
phenazine (100 lM) for D1 and D5 tests and haloperidol (10 lM)
for D2, D3, and D4 tests. For determining the Ki values at least two
independent experiments, each in triplicate, were performed.
The competition binding data were analyzed with Graph Pad
PrismTM 3.0. Ki values were calculated from IC50 values applying
the equation of Cheng and Prusoff [11].
Compound 4a
Yield: 32%, white powder, m.p.: 2318C;1H-NMR 400 MHz (metha-
nol-d4) d: 2.05–2.47 (m, 4H), 2.76–2.90 (m, 2H), 3.01–3.15 (m, 5H),
3.41 (s, 1H, 5), 3.34–3.49 (m, 2H), 3.77 (s, 3H, OMe), 4.16 (s, 1H, 5),
6.75–6.82 (m, 2H), 7.23–7.25 (d, J = 8.3 Hz, 1H), 7.33–7.38 (t, J = 7.5
Hz, 1H), 7.48–7.55 (m, 3H); 13C-NMR 400 MHz (methanol-d4) d:
23.9, 24.8, 28.3, 33.3, 33.6, 42.2, 54.4, 55.5, 111.9, 115.2, 126.7,
130.2, 130.5, 130.5, 130.8, 131.4, 132.5, 140.4, 142.8, 158.4. Anal.
calcd. for C20H26ClNO N 0.20 H2O: C 71.61%, H 8.21%, 3.96%.
Compound 4b
Yield: 43%, white crystals, m.p.: 2188C;1H-NMR 250 MHz (metha-
nol-d4) base d: 1.58–1.90 (m, 4H), 2.16 (s, 3H, NMe), 2.37–2.51 (m,
2H), 2.59–2.90 (m, 4H), 2.92–3.50 (m, 2H), 3.86 (s, 3H, OMe), 3.92
(s, 3H, OMe), 6.70 (s, 1H, 1), 6.80 (s, 1H, 4), 7.15–7.34 (m, 4H); 13C-
NMR 250 MHz (methanol-d4) d: 23.8, 31.7, 32.6, 32.9, 47.6, 55.1,
55.1, 55.2, 114.7, 116.4, 125.7, 126.2, 126.8, 126.9, 129.1, 134.0,
136.5, 139.9, 146.9, 149.5; GC-MS (m/z): 265 (4%), 250 (2%), 234
(4%), 219 (1%), 207 (2%), 193 (5%), 178 (8%), 160 (14%), 146 (29%),
130 (13%), 115 (66%), 104 (100%), 91 (53%), 78 (87%), 65 (28%).
Anal. calcd. for C21H28ClNO2 N 2 H2O: C 63.53%, H 7.85%, N 3.26%.
Functional calcium assay
Screening for agonistic and antagonist activity was performed
in an intracellular calcium assay using a NOVOstar microplate
readerTM (BMG LabTechnologies, Offenburg, Germany) with a
pipettor system. Agonistic activities were tested by injecting 20
lL buffer alone as negative control, standard agonist in buffer as
positive control (final concentration: 1 lM), and test compounds
in buffer in rising concentrations, respectively, each into sepa-
rate wells. Screening for antagonist activities was performed by
pre-incubating the cells with 20 lL of the test compound dilu-
tions (final concentrations: 100 lM, 50 lM, 10 lM, 5 lM, 1 lM,
500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM) at 378C 30 min
prior to injection of 20 lL standard agonist per well. Fluores-
cence measurement started simultaneously to the automatic
injection. SKF 38393 was used as standard agonist for D1 recep-
tors and quinpirole for D2 receptors. At least two independent
experiments each in four or six replications were performed.
Fluorescence intensity was measured at 520 nm (bandwidth 25
nm) for 30 s at 0.4-s intervals. Excitation wavelength was 485 nm
(bandwidth 20 nm). Agonistic or antagonist activities were
assessed by a dose-response curve obtained by determination of
the maximum fluorescence intensity of each data set and non-
linear regression with sigmoidal dose-response equation using
Graph Pad PrismTM 3.0.
Compound 6a
Yield: 73%, white powder, m.p.: 968C; 1H-NMR 250 MHz (metha-
nol-d4) d: 2.87–2.91 (m, 7H), 3.02–3.10 (m, 4H), 3.47–3.51 (m, 4H),
3.76 (s, 3H, OMe), 6.79–6.82 (m, 2H), 7.12–7.38 (m, 5H); 13C-NMR,
dept 250 MHz (Methanol-d4) d: 26.4, 26.6, 33.4, 34. 3, 40.7 (N-Me),
53.7, 54.4 (O-Me), 112.8, 115.4, 126.3, 127.4, 129.9, 130.1, 131.0,
131.9, 134.9, 136.1, 140.1, 158.3; GC-MS (m/z): 325 (2%), 292 (2%),
283 (2%), 265 (8%), 253 (4%), 239 (5%), 220 (12%), 206 (4%), 189
(1%), 178 (15%), 165 (7%), 154 (11%), 146 (24%), 131 (20%), 117
(61%), 104 (100%), 91 (67%), 78 (82%), 65 (32%). Anal. calcd. for
C20H26ClNO N H2O: C 69.39%, H 8.19%, N 3.73%.
Compound 6c
Yield: 62%, white powder, m.p.: 1458C; 1H-NMR 450 MHz (metha-
nol-d4) d: 2.91–2.98 (m, 7H), 3.09–3.13 (t, J = 7.4 Hz, 4H), 3.47–3.51
(t, J = 7.5 Hz, 4H), 7.16–7.21 (m, 2H), 7.24–7.28 (m, 4H), 7.39–7.41
(m, 2H); 13C-NMR, dept 400 MHz (methanol-d4) d: 26.3, 34.1, 40.6
(N-Me), 53.8, 126.4, 127.4, 130.0, 130.2, 134.8, 140.0; GC-MS (m/z):
295 (1%), 280 (2%), 264 (3%), 249 (1%), 235 (1%), 223 (3%), 204
(1%), 190 (4%), 178 (5%), 160 (5%), 147 (24%), 132 (14%), 117 (47%),
104 (100%), 91 (72%), 78 (79%), 65 (32%). Anal. calcd. for
C19H24ClN N 0.30 H2O: C 72.83%, H 8.33%, N 4.24%.
MTT-test
U87-MG glia cells (ATCC HTB-14) were cultured at 378C, and 5%
CO2 in DMEM (PAA, E15-883) + 10% FCS (Thermo Scientific
HyClone. Logan UT, USA). In 96-well plates, 15 000 cells were dis-
persed in 200 lL into each well. After 24 h, the medium was
replaced by DMEM + FCS containing the test compound solved
in a final concentration of 0.25% DMSO. To minimize edge-
effects as mentioned by Rasmussen [12], each concentration of
each compound was measured in six wells distributed across the
96-well plate and only the inner 6 6 10 wells of the plate were
analyzed. The outer wells were filled with 200 lL PBS-buffer. As
positive control, six wells with cells and medium containing
0.25% DMSO were analyzed. After 24 h of incubation, the
medium was removed and 100 lL MTT (Fluka), dissolved in phe-
nol-red-free DMEM (without FCS) at 0.5 mg/mL, were added to
Pharmacology
Radioligand binding experiments and the functional calcium
assay were performed as described in detail in a former publica-
tion [1]. Human D1, D2L, D3, and D5 receptors were expressed in
HEK cells and D4.4 receptors were expressed in CHO cells, respec-
tively.
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

214
M. Schulze et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 207–214
each well. The plates were incubated for 4 h. Cells were killed
and formazan crystals were solubilized by addition of 100 lL of
20% sodium dodecylsulfate (SDS) in H2O followed by incubation
overnight at 378C. Optical density was measured at 544 nm
using a microplate reader (Galaxy FluoStar, BMG Labtechnolo-
gies) with background substraction (compounds in the same
preparation without cells). Cytotoxicity values were calculated
as percentage of positive control (= 100%). Cc50 values were calcu-
lated with Graph Pad PrismTM 3.0. All experiments were repeated
at least three times.
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i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com