Residues at the indole-NH of LE300 modulate affinities and selectivities for dopamine receptors

Article abstract of DOI:10.1002/ardp.201000121

To further investigate SAR in the class of azecine-type dopamine receptor antagonists, we synthesized a series of derivatives, substituted at the indole-NH of the lead compound LE300 by different alkyl chains in addition to phenylpropyl, allyl, propargyl, and acetyl residues. The affinities of the target compounds for all human dopamine receptors (D₁-D₅) were investigated by radioligand binding assay and their functionality by a calcium assay. Both the affinities and selectivities for the dopamine receptors were found to be affected by the nature of the substituent. The N14-methylated derivative displayed the highest affinities for all D-receptors. In general, the affinities decreased with increasing chain length of the N-alkyl. Different substituents, partly led to altered affinity, and selectivity profile when compared with our lead LE300. Benz[d]indolo[2,3-g]azecines, based on the lead compound LE 300, represent a novel class of potent dopamine receptor antagonists. A series of LE 300 derivatives, substituted at the indole-NH by different groups, was synthesized and the affinities for all human cloned dopamine receptors was tested.

Full text of DOI:10.1002/ardp.201000121

28
Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Full Paper
Residues at the Indole-NH of LE300 Modulate Affinities and
Selectivities for Dopamine Receptors
Dina Robaa1, Robert Kretschmer1, Oliver Siol2, Shams ElDin AbulAzm3, ElSayeda ElKhawass3,
Jochen Lehmann1,4, and Christoph Enzensperger1
1
¨
Institut fu¨r Pharmazie, Lehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Friedrich Schiller Universitat
Jena, Germany
¨
Institut fu¨r Pharmazie, Lehrstuhl fu¨r Pharmazeutische Biologie, Friedrich Schiller Universitat Jena,
2
Germany
3 Department of Medicinal Chemistry, Faculty of Pharmacy, University of Alexandria, Egypt
4 College of Pharmacy, King Saud University Riyadh, Saudi Arabia
To further investigate SAR in the class of azecine-type dopamine receptor antagonists, we synthesized
a series of derivatives, substituted at the indole-NH of the lead compound LE300 by different alkyl
chains in addition to phenylpropyl, allyl, propargyl, and acetyl residues. The affinities of the target
compounds for all human dopamine receptors (D1–D5) were investigated by radioligand binding assay
and their functionality by a calcium assay. Both the affinities and selectivities for the dopamine
receptors were found to be affected by the nature of the substituent. The N14-methylated derivative
displayed the highest affinities for all D-receptors. In general, the affinities decreased with increasing
chain length of the N-alkyl. Different substituents, partly led to altered affinity, and selectivity profile
when compared with our lead LE300.
Keywords: azecines / dopamine receptors / indole-NH / SAR
Received: April 29, 2010; Revised: June 7, 2010; Accepted: June 11, 2010
DOI 10.1002/ardp.201000121
Introduction
the methoxylated derivative (2) led to increased affinities for
all dopamine receptors except for D1 [5]. The N14-methylated
derivative of 1 was subsequently synthesized and as expected
showed similar promising affinities. Hence, we synthesized a
series of derivatives of 1, substituted at the indole-NH with
different alkyl chains, in order to study the effect of the
substituent’s length and bulk on the affinities. Moreover,
N-allyl-, propargyl-, and acyl derivatives were prepared, which
possess different electronic as well as steric properties com-
pared with the alkyl groups. Finally, 1 was substituted with a
phenylpropyl group, to examine the effect of both an
additional aromatic group and a bulky moiety. The affinities
of the prepared compounds for the human dopamine recep-
tors (D1–D5) were investigated by radioligand binding exper-
iments, and their functionality as agonists or antagonists was
determined by a functional Calcium assay at the D1-, D2-,
and D5-receptors.
Dopamine is a key neurotransmitter in the brain, playing a
regulatory role in locomotion, cognition, emotion and event
prediction. Dysfunctions of the dopaminergic system have
been linked to several neuropsychiatric diseases; hence
dopamine receptors have long served as attractive targets
for drug development [1]. Dopamine receptor antagonists,
for instance, have been implemented as antipsychotics.
The azecine-type dopamine receptor antagonists, based on
the lead compound 1 (LE 300), represent a novel class of
dopamine receptor ligands [2–4]. They were found to
possess high affinities for dopamine receptors and a unique
selectivity profile for the D1-like receptors.
Some previous studies have shown that substitution of the
indole-NH might be advantageous; indole-NH-methylation of
Chemistry
Correspondence: Christoph Enzensperger, Institut fu¨r Pharmazie,
Lehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Friedrich Schiller
¨
Universitat Jena, Germany.
For the synthesis of the quinolizine 3, we simplified previous
E-mail: ch.enzensperger@uni-jena.de
Fax: þ49-3641-949802
methods to a one-step reaction, which we already adopted for
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Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Residues at the Indole-NH of LE300
29
the synthesis of analogous quinolizines [6]. Tryptamine was
refluxed with 2-(2-bromoethyl)benzaldehyde in dioxane in
the presence of an equimolar amount of trifluoroacetic acid.
This one-pot Pictet-Spengler procedure, showed obvious
advantages over the previously applied one, where trypt-
amine was first reacted with 1-isochromanone and the
resulting amide cyclised (Bischler-Napieralski) using POCl3
followed by NaBH4 reduction [4, 7]. The obtained quinolizine
was then quaternized with methyl iodide, and the quater-
nary salt was treated under Birch conditions with sodium
in liquid ammonia to afford the indolobenzazecine 1.
Substitution of the indole-NH was achieved by the reaction
of 1 with the corresponding halide derivative after deproto-
nation with sodium hydride in DMF. For this purpose a
twofold excess of NaH and only an equimolar amount of
the halide derivative were used, to ensure that no quaterni-
zation took place at the basic, central alicylic N. The N7,N14-
dimethylated compound 5 was obtained by treating the
quinolizine with excess of methyl iodide in the presence
of sodium hydride, which led to the simultaneous N14-alky-
lation and N7-quaternisation. The formed quaternary salt 4
was then cleaved under Birch conditions (Scheme 1).
Figure 1. Alkyne/allene rearrangement of compound 11.
rearrangement (Fig. 1), which has been described for acety-
lenes [8]. A mixture of the 2-propynyl, 1-propynyl, and allenyl
derivatives was obtained, but could not be separated. The 13C-
NMR spectrum also showed characteristic peaks for the three
predicted products. Therefore, only an equimolar amount of
NaH was used. This did not affect a complete reaction, hence
accounting for the low yield. However it led to the propargyl
derivative as a sole product.
However, when trying to synthesize the propargyl deriva-
tive 11, three different products were obtained. The GC-MS
spectrum showed 3 peaks of the same mass (m/z 328), beside
the peak of the starting material. Even when the reaction Pharmacology
temperature was decreased to 08C and the reaction time was
reduced from 24 h to 2 h, a mixture of the three products in
addition to the starting material was obtained. We assume
that the excess of NaH led to a base-catalyzed alkyne/allene
All the target compounds were screened for their affinities
for the human cloned dopamine receptor subtypes D1, D2L,
D3, D4.4, and D5. These receptors were stably expressed in
Scheme 1. Synthesis of N14-alkylated
LE300. Reagents and conditions: (a) Dioxane,
TFA, reflux, 18 h; (b) NaOH; (c) NaH, THF, xss
methyl iodide, 08C–r.t., 24 h; (d) Na0, liq. NH3,
S408C, 10 min; (e) Methyl iodide, acetone, r.t.,
4 h; (f) NaH, DMF, RX, 08C–r.t., 24 h (warm to
408C with acetyl chloride).
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HEK293 or CHO cells; [3H]SCH 23390 and [3H]spiperone
were used as radioligands at the D1-like and D2-like receptors,
respectively. Ki values are given in nanomolar units (Tab. 1).
The detailed protocol has been previously described [9].
The functionality at the D1-, D2-, and D5-receptors was
tested in an intracellular calcium fluorescence assay [10],
where all prepared compounds were found to possess
antagonistic properties.
serves as H-donor. Moreover, compared with the other 3-
carbon chains (N-allyl- (10) and N-propyl- (8)), the N-prop-
argyl-derivative 11 has a straight shape and less confor-
mational flexibility.
The derivative substituted with a phenylpropyl group (13)
also showed significantly reduced affinities to all subtypes.
The longer alkyl chains like pentyl- (14) and octyl- (15)
showed an interesting effect on the binding to the D1-family:
For the D1-receptor the pentyl derivative (14) has a clearly
higher affinity than the octyl-derivative (15). This is in line
with all other receptor subtypes except the D5 receptor,
where the affinity of octyl-derivative (15) is higher than that
of the pentyl-derivative 14. The phenylpropyl-derivative (13),
which might display similar space filling like 15, has a higher
affinity for the D5-receptor than the much smaller pentyl-
derivative (14).
As some of the prepared compounds displayed interestingly
high affinities for the dopamine receptors, the cytotoxicity
of some selected compounds was investigated, in order to test
out their applicability in an in-vivo assay as antipsychotics.
Moreover, it was of interest to study the effect of the different
substituents on the cytotoxicity of the compounds. This was
tested in an MTT cell proliferation assay, the protocol has been
described [11]. (Cc50-values are given in Table 1).
The fact that substitution of indole-NH with small groups
(Me and Et) did not decrease the affinities, proves that the
indole-NH does not play a role as H donor in the interaction
with the receptor. We assume that the enhancing effect of
methyl substitution on the affinities is rather caused by steric
effects.
Results and discussion
With regard to the binding profile (Table 1), all N14-alkylated
derivatives showed a higher affinity for the D1-like receptors.
The N14-methylated derivative 5 displayed the highest affin-
ities among all prepared derivatives. Compared with the lead
1, compound 5 showed a 5–15 fold increase in affinities for all
D-receptor subtypes, except for D1. The ethylated derivative 6
displayed lower affinities than the methylated one (5), but
they were nearly equal to those of 1. It is quite evident, that
the affinities decreased with increasing alkyl chain length.
This was most pronounced in the N14-octyl derivative 15,
which showed the lowest affinities among all tested com-
pounds. The only exception was the pentyl derivative 14,
which displayed lower affinities for D5 than 15; in fact it
showed the highest D1/D5-selectivity (1:10).
It is apparent that increased alkyl chain length goes along
with increased cytotoxicity. The methylated derivative 5
exhibited a low cytotoxicity, whereas the octyl derivative
15 displayed a sixfold increase in cytotoxicity. The high
affinities together with the low cytotoxicity of the methyl-
ated derivative, rendered it suitable further in-vivo studies,
the results are reported in a separate paper.
In conclusion: To elucidate the effect of indole-NH substi-
tution, eleven new N14-substituted benzindoloazecines were
synthesized and their affinities for all dopamine receptors
were determined by radioligand binding experiments.
The affinities and even selectivity were seriously affected
by the nature and bulk of the substituent. Interesting is also
that many N14-substituted derivatives showed an increase
in the affinity for D4. All in all, a clear correlation (Fig. 2) can
be seen between the chain length and the affinities; an
optimal binding affinity was obtained with a methyl group.
Longer alkyl chains and bulky substituents were unfavorable.
However, against this trend, longer alkyl chains seem to
influence the binding to the D1- and the D5-receptor in
opposing directions. The pentyl-derivative (14) has the
lowest D5-affinity and longer chains seem to be better toler-
ated at the D5-receptor than at the D1-receptor. This effect
can be further exploited in order to gain D1- or D5-selective
compounds, which might be valuable tools but are not yet
available [12].
Modification of the alkyl chains did not lead to significant
alterations in the affinities for the different D-receptor. The
fluoroethylated derivative 7 exhibited affinities similar to the
ethylated one. The cyclopropylmethyl derivative 9 generally
showed affinities similar to the N14-propyl derivative 8,
except at D5, where they were much lower.
Compared with 1, the affinities of the N-acetylated com-
pound 12 for the D1- and D3-receptors were lower, those for D4
and D5 almost equal, while a slight enhancement in the
affinity for D2 could be observed.
The allylated derivative 10 displayed an unexpected
selectivity profile; showing the highest affinity for D4-subtype
among all synthesized compounds. A similar effect was
not noticed in compound 11, where the indole-NH is
substituted with a propargyl group. In spite of its bulk,
the propargyl derivative exhibited rather high affinities for
the D1- and the D5-receptor. This might be explained in
different ways: Either the p-bonds enable further interactions
with the receptors (like stacking) or the terminal alkyne-H
Furthermore, the cytotoxicity of the synthesized deriva-
tives increased with increasing chain length. On the whole,
the N14-methylated derivative showed the most interesting
affinities together with lowest cytotoxicity, which rendered it
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Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Residues at the Indole-NH of LE300
31
Table 1. Affinities (Ki, nM) for human D1–D5-receptors, determined by radioligand binding experiments and cytotoxicity (Cc50, mM)
Compounds
Ki, [nM]
Cytotoxicity
Cc50, [mM]c
HEK D1
HEK D2L
HEK D3
CHO D4.4
HEK D5
CH
3
N
1.9 (ꢀ0.9)ꢁ
44.7 (ꢀ15.8)ꢁ
40.3 (ꢀ14.4)ꢁꢁ
109 (ꢀ39)ꢁ
7.5 (ꢀ0.3)ꢁ
N
H
1 (LE300)
O
C
H3
CH
3
N
2
2.0(ꢀ1.6)ꢁꢁ
1.7 (ꢀ0.6) ꢁꢁ
3.78 (ꢀ2.4) ꢁꢁ
21.55 (ꢀ3.5) ꢁꢁ
0.23 (ꢀ0.06)ꢁꢁ
N
CH
3
CH
3
3
3
N
N
N
5
6
7
2.0 (ꢀ0.5) a
3.2 (ꢀ0.8) a
12.6 (ꢀ3.6) a
8.2 (ꢀ5.8) b
51.7 (ꢀ12.4) a
54 (ꢀ5) b
7.2 (ꢀ2.8) b
181 (ꢀ66) b
119 (ꢀ24) b
13.2 (ꢀ3) b
65.5 (ꢀ28.9) a
11.9
0.5 (ꢀ0.1) b
7.5 (ꢀ1.5) b
3.8 (ꢀ0.8) b
139.6 (ꢀ22.1)
N
CH
3
CH
CH
N
N
92.1 (ꢀ7.8)
C
H3
F
CH
3
N
22.3 (ꢀ7.3) a
197 (ꢀ5.5) b
307 (ꢀ15) b
84 (ꢀ11) a
9.8 (ꢀ2.6) b
N
8
CH
3
CH
CH
3
N
N
14.3 (ꢀ0.2) b
241 (ꢀ17) b
305 (ꢀ68) b
39 (ꢀ24) b
57.1 (ꢀ40) a
N
9
3
27 (ꢀ15) b
28.9 (ꢀ 9.3) b
99.3 (ꢀ15) b
9.1 (ꢀ3.6) a
5.5 (ꢀ2) a
85.5 (ꢀ8.2)
N
10
CH
2
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Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Table 1. (continued )
Compounds
Ki, [nM]
Cytotoxicity
Cc50, [mM]c
HEK D1
HEK D2L
HEK D3
CHO D4.4
HEK D5
CH
3
N
N
11
12
13
6.9 (ꢀ0.2) b
128 (ꢀ 45) a
411 (ꢀ81) b
158 (ꢀ 46) b
896 (ꢀ169) a
36.5
1.2 (ꢀ0.3) b
HC
CH
3
N
22.7 (ꢀ 9.7) b
14.2 (ꢀ1.3) b
126 (ꢀ0) b
9.9 (ꢀ1.2) b
N
H C
3
O
CH
3
N
116 (ꢀ30) a
356 (ꢀ16) b
1627 (ꢀ535) a
25.6 (ꢀ5.3) b
N
CH
3
N
23.2 (ꢀ12) a
773 (ꢀ160) b
390 (ꢀ67.5) b
455 (ꢀ8.0) b
261 (ꢀ65) a
N
14
CH
3
CH
3
N
184 (ꢀ60) a
3383(ꢀ694) b
1760 (ꢀ532) a
1414 (ꢀ531) a
77.3 (ꢀ3) b
24.6 (ꢀ5.0)
N
15
4
CH
3
ꢁ
ꢁꢁValues taken from [3]
Values taken from [5]
Ki-values are the means of three experiments; performed in triplicate, SEM is given in parantheses
Ki-values are the means of two; experiments; performed in triplicate ꢀ SEM
a
b
c
Cc50 values are means of three experiments, standard deviation is given in parentheses
1H- and 13C-NMR spectral data were obtained from a Bruker
Advance 250 spectrometer (250 MHz) and Advance 400 spec-
trometer (400 MHz). TLC was performed on silica gel F254 plates
(Merck). MS data were determined by GC/MS, using a Hewlett
Packard GCD-Plus (G1800C) apparatus (HP-5MS column; J&W
Scientific). Purities of the compounds were determined by
elemental analysis, performed on a Hereaus Vario EL apparatus.
All values for C, H, and N were found to be within ꢀ0.4. All
compounds showed >95% purity.
suitable for further investigation in in-vivo studies as a poten-
tial antipsychotic
Experimental
Chemistry
Melting points are uncorrected and were measured in open
capillary tubes, using a Gallenkamp melting point apparatus.
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Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Residues at the Indole-NH of LE300
33
Figure 2. The effect of the substituent on the affinities for the different D-receptors.
3.10–4.23 (m, 8H, 5, 6, 8, 9), 6.34 (s, 1H, 14b), 6.79–6.82 (d, J ¼ 7,
1H, Ar), 7.10–7.16 (t, J ¼ 7, 1H, Ar), 7.23–7.61 (m, 6H, Ar). 13C-NMR
250 MHz (DMSO-d6): d 17.7 (9), 24.3 (5), 31.1 (indole N-Me), 49.3
(Nþ-Me), 53.4 (8), 62.4 (6), 64.4 (14b), 104.0 (13), 110.7 (9a), 119.3
(1a), 120.2 (11), 123.0 (12), 125.7 (14c), 127.5 (2), 128.2 (3), 129.0
(9b), 129.3 (4), 129.4 (1), 131.4 (14a), 132.2 (4a), 138.3 (13a).
5,6,8,9,14,14b-Hexahydroindolo[2’,3’:3,4]pyrido[2,1-a]-
isoquinoline (3)
A solution of tryptamine (4.2 g, 26.45 mmol), 2-(2-bromoethyl)-
benzaldehyde (6.9 g, 32.4 mmol), and trifluoroacetic acid
(2.99 g, 26.25 mmol) in 150 mL dioxane, kept under nitrogen,
was refluxed and stirred vigorously for 18 h. The separated
yellow solid was filtered off, washed with dioxane and then
repeatedly with diethyl ether. The obtained solid was then alkali-
nized using 1 N NaOH, and the formed base was extracted with
dichloromethane. Finally, the organic phase was dried over
Na2SO4, and evaporated under reduced pressure leaving a beige
solid. (4 g, yield 55.6%). M.p.: 117–1198C, (Lit. 167–1688C [7], 95–
988C and 125–1278C [13]). 1H-NMR 400 MHz (CDCl3): d 2.69–2.81,
2.85–3.14, 3.42–3.55 (m, 8H, 5, 6, 8, 9), 5.39 (s, 1H, 14b), 7.11–7.18
(m, 2H, 11, 12), 7.22–7.23 (d, J ¼ 7, 1H, 4), 7.27–7.31 (m, 2H, 2, 13),
7.34–7.37 (t, J ¼ 7, 1H, 3), 7.41–7.42 (d, J ¼ 7, 1H, 10), 7.52–7.54 (d,
J ¼ 7, 1H, 1) 7.74 (s, 1H, NH). GC-MS: m/z: 273 (10%), 245 (6%), 230
(8%), 143 (3%), 130 (12%), 115 (17%), 89 (8%), 77 (17%), 63 (11%), 51
(16%), 42 (100%).
7,14-Dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]-
benzazecine (5)
Purified by column chromatography (EtOAc). Yellow powder.
Yield 20%. M.p. 103–1058C. 1H-NMR 400 MHz (CDCl3): d 2.35 (s,
3H, N-Me), 2.45–2.48 (t, J ¼ 6, 2H, 5), 2.70–2.73 (t, J ¼ 6, 2H, 9),
2.76–2.81 (m, 4H, 6, 8), 3.56 (s, 3H, indole N-Me), 4.54 (s, 2H, 15),
7.04–7.08 (ddd, J ¼ 5/2/0.6, 1H, Ar), 7.10–7.12 (dd, J ¼ 7/1, 1H,
Ar), 7.16–7.21 (m, 3H, Ar), 7.24–7.26 (d, J ¼ 8, 1H, Ar), 7.32–7.33
(dd, J ¼ 8/3, 1H, Ar), 7.50–7.52 (d, J ¼ 8, 1H, Ar). GC-MS: m/z: 304
(21%), 260 (9%), 246 (53%), 232 (100%), 217 (63%), 200 (17%), 168
(10%), 157 (47%), 144 (55%), 128 (38%), 115 (88%), 104 (61%), 91
(41%), 77 (78%), 63 (34%). Anal. calcd. for C21H24N2 ꢂ 0.2 EtOAc:
C, 81.30%; H, 8.01%; N, 8.70%. Found: C, 81.56%; H, 8.30%; N,
8.89%.
7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]-
benzazecine (1)
Evaporation of the solvent yielded a creamy white powder,
which was crystallized from methanol, giving white cubic
crystals. Yield 69%. M.p. 90–928C (Lit. 56–588C [4]). 1H-NMR
250 MHz (CDCl3): d 2.28 (s, 3H, N-Me), 2.61–2.90 (m, 8H, 5, 6,
8, 9), 4.35 (s, 2H, 15), 7.07–7.36 (m, 7H, Ar), 7.46–7.49 (d, J ¼ 7,
1H, Ar), 7.82 (s, 1H, NH). GC-MS: m/z: 290 (21%), 245 (22%), 230
(49%), 217 (100%), 204 (10%), 189 (15%), 177 (12%), 154 (16%),
143 (100%), 130 (48%), 115 (94%), 104 (71%), 89 (65%), 77 (71%),
63 (58%).
14-Ethyl-7-methyl-6,7,8,9,14,15-hexahydro-5H-indolo-
[3,2-f][3]benzazecine (6)
Evaporation of the solvent gave a yellow powder. Crystallized
from ethanol/H2O giving yellow crystals. Yield 56%. M.p. 103–
1048C. 1H-NMR 250 MHz (CDCl3): d 1.01–1.07 (t, J ¼ 7, 3H, 2’), 2.37
(s, 3H, N-Me), 2.43–2.48 (t, J ¼ 6, 2H, 5), 2.71–2.77 (m, 6H, 6, 8, 9),
4.02–4.11 (q, J ¼ 7, 2H, 1’), 4.58 (s, 2H, 15), 7.02–7.33 (m, 7H, Ar),
7.50–7.53 (d, J ¼ 7, 1H, Ar). 13C-NMR 250 MHz (CDCl3): 14.8 (2’),
23.3 (9), 32.8 (15), 33.23 (5), 38.2 (1’), 45.4 (N-Me), 59.0 (8), 59.4 (6),
108.8 (13), 112.8 (9a), 117.9 (10), 118.6 (11), 121.5 (12), 126.3 (3),
126.7 (2), 127.9 (9b), 131.0 (4), 131.7 (1), 134.9 (13a), 135.7 (14a),
138.1 (4a), 142.2 (15a). GC-MS: m/z: 318 (32%), 274 (9%), 260 (38%),
246 (98%), 230 (100%), 217 (76%), 202 (21%), 171 (19%), 158 (39%),
143 (44%), 130 (37%), 115 (68%), 104 (28%), 91 (33%), 77 (49%), 65
7,14-Dimethyl-5,6,8,9,14,14b-hexahydroindolo-
[2’,3’:3,4]pyrido[2,1-a]isoquinolin-7-ium iodide (4)
Yellow powder. Yield 82%. M.p. 254–2558C. 1H-NMR 250 MHz
(DMSO-d6): d 3.25 (s, 3H, Nþ-Me), 3.78 (s, 3H, indole N-Me),
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D. Robaa et al.
Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
(22%). Anal. calcd. for C22H26N2: C, 82.97%; H, 8.23%; N, 8.80%.
Found: C, 83.05%; H, 8.44%; N, 8.70%.
202 (14%), 182 (13%), 167 (35%), 156 (17%), 144 (19%), 130 (29%),
115 (82%), 103 (34%), 91 (43%), 77 (67%), 63 (30%). Anal. calcd.
for C23H26N2 ꢂ 0.25EtOAc: C, 81.78%; H, 8.01%; N, 7.95%. Found:
C, 81.44%; H, 8.26%; N, 8.01%.
14-(2-Fluoroethyl)-7-methyl-6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine (7)
7-Methyl-14-prop-2-ynyl-6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine hydrochloride (11)
Crystallized from Methanol then recrystallized from hexane
giving white crystals. Yield 38%. M.p. 107–1088C. 1H-NMR
250 MHz (CDCl3): 2.34 (s, 3H, N-Me), 2.38–2.42 (t, J ¼ 6, 2H, 5),
2.67–2.72 (m, 6H, 6, 8, 9), 4.21 (dd, 10/4, 2H, 1’), 4.35 (dd, J ¼ 6/4,
2H, 2’), 4.61 (s, 2H, 15), 7.03–7.33 (m, 7H, Ar), 7.52 (d, J ¼ 8, 1H,
Ar). 13C-NMR 250 MHz (CDCl3): 23.4 (9), 32.9 (15), 33.3 (5), 43.7 (d
(23.3Hz)) (1’), 45.3 (N-Me), 58.8 (8), 59.6 (6), 82.2 (d (171.7 Hz)) (2’),
108.8 (13), 113.6 (9a), 118.90 (10), 119.1 (11), 121.1 (12), 126.4 (3),
126.9 (2), 128.0 (9b), 131.2 (4), 131.9 (1), 135.3 (13a), 136.5 (14a),
137.8 (4a), 142.3 (15a). GC-MS: m/z: 336 (15%), 316 (9%), 278 (24%),
264 (51%), 230 (56%), 217 (36%), 156 (30%), 143 (33%), 128 (37%),
115 (100%), 104 (84%), 91 (44%), 78 (100%), 65 (47%). Anal. calcd.
for C22H25FN2: C, 78.54%; H, 7.49%; N, 8.33%. Found: 78.22%; H,
7.48%; N, 8.14%.
The base was purified by column chromatography (DCM/MeOH,
20:1), to separate the product from the unreacted starting
material. The obtained product was converted to the HCl salt,
and crystallized twice from methanol/ether yielding a white
solid. Yield 11%. M.p. 171–1748C. 1H-NMR 250 MHz (MeOD): d
2.58–2.60 (t, 1H, 3’), 2.89 (s, 3H, N-Me), 2.91–2.98 (t, J ¼ 8, 2H, 5),
3.30–3.35 and 3.54–3.65 (m, 6H, 6, 8, 9), 4.52 (s, 2H, 1’), 4.86 (s, 2H,
15, under MeOD), 7.03–7.30 (m, 7H, Ar), 7.50–7.53 (dd, J ¼ 8/1,
1H, Ar). 13C-NMR 250 MHz (MeOD): 18.3 (9), 28.1 (5), 30.1 (15), 32.2
(1’), 43.6 (N-Me), 52.8 (8), 56.5 (6), 72.2 (3’), 77.8 (2’), 109.2 (13),
109.6 (9a), 117.7 (10), 119.8(11), 122.0 (12), 126.9 (3), 127.6 (2),
127.9 (9b), 131.8 (4), 131.4 (1), 133.4 (13a), 135.4 (14a), 135.8 (4a),
136.8 (15a). Anal. calcd. for C23H24N2 ꢂ HCl ꢂ 0.25 H2O: C, 74.78%;
H, 6.96%; N, 7.58%; Cl, 9.60%. Found: C, 74.81%; H, 7.29%; N,
7.54%; Cl, 9.94%.
7-Methyl-14-propyl -6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine (8)
Evaporation of the solvent gave a yellow solid. Crystallized from
isopropanol/H2O giving yellow crystals. Yield 56%. M.p. 73–748C.
1H-NMR 250 MHz (CDCl3): d 0.75–0.81 (t, J ¼ 7, 3H, 3’), 1.43–1.52
(sextet, J ¼ 7, 2H, 2’), 2.34 (s, 3H, N-Me), 2.42–2.47 (t, J ¼ 6, 2H, 5),
2.69–2.73 (m, 6H, 6, 8, 9), 3.93–3.97 (t, J ¼ 7, 2H, 1’), 4.55 (s, 2H,
15), 7.02–7.37 (m, 7H, Ar), 7.51–7.53 (d, J ¼ 8, 1H, Ar). GC-MS: m/z:
332 (37%), 274 (23%), 260 (64%), 244 (35%), 230 (100%), 217 (67%),
202 (16%), 154 (15%), 143 (21%), 130 (24%), 115 (30%), 103 (17%),
91 (22%), 71 (30%). Anal. calcd. for C23H28N2: C, 83.09%; H, 8.49%;
N, 8.43%. Found: C, 83.09%; H, 8.62%; N, 8.34%.
14-Acetyl-7-methyl-6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine (12)
Purified by column chromatography (EtOAc/MeOH, 16:5), yield-
ing a brown oil, which solidifies to a yellowish brown solid on
standing. Yield 63%. M.p. 99–1008C. 1H-NMR 250 MHz (CDCl3): d
2.28 (s, 3H, N-Me), 2.40–2.44 (t, J ¼ 6, 2H, 5), 2.62–2.73 (m, 9H, 6, 8,
9, COMe), 4.75 (s, 2H, 15), 7.00–7.46 (m, 7H, Ar), 7.78–7.81 (dd,
J ¼ 8/2, 1H, Ar). 13C-NMR 250 MHz (CDCl3): 23.9 (9), 27.6 (COMe)
33.6 (5), 33.7 (15), 45.8 (N-Me), 58.3 (8), 60.5 (6), 114.3 (13), 118.3
(10), 121.2 (9a), 122.6 (11), 123.8 (12), 126.1 (3), 126.5 (2), 130.3
(9b), 131.1 (4), 131.7 (1), 135.9 (13a), 136.9 (14a), 138.1 (4a), 141.4
(15a), 170.5 (CO). GC-MS: m/z: 332 (12%), 289 (11%), 246 (39%), 230
(199%), 217 (91%), 202 (14%), 170 (16%), 160 (74%), 144 (31%), 130
(42%), 115 (56%), 103 (21%), 91 (24%), 77 (41%), 63(22%). Anal.
calcd. for C22H24N2O ꢂ 0.25 EtOAc: C, 77.93%; H, 7.39%; N, 7.90%.
Found: C, 77.98%; H, 7.49%; N, 7.95%.
14-(Cyclopropylmethyl)-7-methyl-6,7,8,9,14,15-
hexahydro-5H-indolo[3,2-f][3]benzazecine (9)
Purified by column chromatography (DCM/MeOH, 9:1), yielding a
creamy white oil. Yield 63%. 1H-NMR 250 MHz (CDCl3): d 0.18–
0.24 (m, 2H, 3’), 0.40–0.47 (m, 2H, 4’), 0.84–1.08 (m, 1H, 2’), 2.36 (s,
3H, N-Me), 2.48–2.53 (t, J ¼ 6, 2H, 5), 2.71–2.78 (m, 6H, 6, 8, 9),
3.91–3.94 (d, J ¼ 6, 2H, 1’), 4.55 (s, 2H, 15), 7.03–7.31 (m, 7H, Ar),
7.47–7.50 (d, J ¼ 7, 1H, Ar). GC-MS: m/z: 344 (27%), 286 (13%), 272
(27%), 239 (45%), 230 (100%), 217 (60%), 202 (19%), 160 (30%), 144
(324%), 130 (44%), 115 (72%), 103 (22%), 91 (29%), 71 (34%). Anal.
calcd. for C24H28N2 ꢂ 0.25 H2O: C, 82.60%; H, 8.23%; N, 8.03%.
Found: C, 82.48%; H, 8.32%; N, 7.91%.
7-Methyl-14-(3-phenylpropyl)-6,7,8,9,14,15-hexahydro-
5H-indolo[3,2-f][3]benzazecine (13)
The base was converted to the HCl salt, and the aqueous solution
was washed twice with EtOAc. The aqueous solution was then
alkalinized, and the formed base was extracted with chloroform.
Evaporation of the solvent gave a brown oil that did not crystal-
lize. Yield 57%. 1H-NMR 250 MHz (CDCl3): d 1.70–1.81 (q, J ¼ 8,
2H, indole N-CH2 CH2CH2Ph), 2.35 (s, 3H, N-Me), 2.38–2.52 (m, 4H,
5, indole N-CH2CH2 CH2Ph), 2.68–2.76 (m, 6H, 6, 8, 9), 3.95–4.02 (t,
J ¼ 8, 2H, indole N-CH2CH2CH2Ph), 4.53 (s, 2H, 15), 7.03–7.32 (m,
12H, Ar), 7.50–7.53 (d, J ¼ 7, 1H, Ar). GC-MS: m/z: 408 (3%), 244
(4%) 230 (8%), 217 (6%), 143 (9%), 128 (7%), 117 (17%), 104 (13%), 91
(100%), 77 (23%), 65 (22%). Anal. calcd. for C29H32N2 ꢂ 0.75 H2O):
C, 82.52%; H, 8.00%; N, 6.64%. Found: C, 82.88%; H, 8.30%; N,
6.46%.
14-Allyl-7-methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f]-
[3]benzazecine (10)
Purified by column chromatography (EtOAc/MeOH, 2:1), yielding
a colorless oil, which solidifies to a waxy white solid on standing.
Yield 25%. M.p. 78–808C. 1H-NMR 250 MHz (CDCl3): d 2.27 (s, 3H,
N-Me), 2.45–2.50 (t, J ¼ 6, 2H, 5), 2.72–2.77 (m, 6H, 6, 8, 9), 4.50 (s,
2H, 15), 4.60–4.62 (m, 2H, 1’), 4.75–4.82 (dd, J ¼ 17/1, 1H, 3’),
4.99–5.04 (dd, J ¼ 10/1, 1H, 3’), 5.62–5.77 (m, 1H, 2’), 7.03–7.30
(m, 7H, Ar), 7.50–7.53 (dd, J ¼ 8/1, 1H, Ar). 13C-NMR 250 MHz
(CDCl3): 23.1 (9), 32.5 (5), 33.1 (15), 45.3 (N-Me), 45.7 (1’), 58.8 (8),
59.0 (6), 109.1 (13), 113.0 (9a), 115.9 (2’), 117.8 (10), 118.8 (11),
120.8 (12), 126.3 (3), 126.7 (2), 127.8 (9b), 131.1 (4), 131.7 (1), 133.3
(3’), 135.3 (13a), 136.4 (14a), 137.8 (4a), 141.8 (15a). GC-MS: m/z:
330 (13%), 272 (22%), 258 (37%), 244 (39%), 230 (100%), 217 (60%),
7-Methyl-14-pentyl-6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine (14)
Purified by column chromatography (DCM/MeOH, 9.5:0.5), yield-
ing creamy white oil. Yield 41%. 1H-NMR 250 MHz (CDCl3): d
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
Residues at the Indole-NH of LE300
35
filtermat was moistened with 50 mL Microscint 20TM scin-
tillation cocktail. The radioactivity retained on the filters
was counted using a Top Count NXTTM microplate scintil-
lation counter (Packard, Ct., USA). For the determination of
the Ki-values at least two independent experiments each in
triplicate were performed.
0.81–0.87 (t, J ¼ 7, 3H, 5’), 1.41–1.49 (m, 6H, 2’– 4’), 2.37 (s, 3H, N-
Me), 2.44–2.48 (t, J ¼ 6, 2H, 5), 2.72–2.77 (m, 6H, 6, 8, 9), 3.92–3.99
(t, J ¼ 8, 2H, 1’), 4.56 (s, 2H, 15), 7.03–7.33 (m, 7H, Ar), 7.50–7.53
(d, J ¼ 8, 1H, Ar). 13C-NMR 250 MHz (CDCl3): 13.9 (5’), 22.4 (4’),
23.2 (9), 29.1 (3’), 29.6 (2’), 32.8 (5), 33.1 (15), 43.8 (1’), 45.2 (N-
Me), 58.9 (8), 59.6 (6), 109.0 (13), 112.7 (9a), 117.8 (10), 118.5
(11), 120.6 (12), 126.3 (3), 126.7 (2), 127.7 (9b), 131.0 (4), 131.7
(1), 135.1 (13a), 136.1 (14a), 138.0 (4a), 142.0 (15a). GC-MS: m/z:
360 (13%), 302 (10%), 288 (22%), 255 (50%), 244 (32%), 230
(100%), 217 (69%), 202 (19%), 160 (22%), 144 (48%), 130
(63%), 115 (90%), 105 (40%), 91 (47%), 71 (47%). Anal. calcd.
for C25H32N2 ꢂ HCl ꢂ 0.75 H2O: C, 73.15%; H, 8.47%; N, 6.82%.
Found: C, 72.90%; H, 8.74%; N, 6.64%.
The competition binding data were analyzed with
GraphPad PrismTM software using nonlinear regression with
sigmoidal dose response equation. Microsoft ExcelTM was
used to calculate the mean and the standard error of the
mean. Ki-values were calculated from IC50-values applying the
equation of Cheng and Prusoff [14].
7-Methyl-14-octyl-6,7,8,9,14,15-hexahydro-5H-indolo-
[3,2-f][3]benzazecine (15)
Functional assay
Measuring intracellular Ca2þ with a fluorescence microplate
reader [3, 10]. Human D1- and D2L-receptors were stably
expressed in human embryonic kidney cells (HEK293) and
cultured as mentioned above. Human D1- and D2L-receptor
cell lines were grown on T 175-culture dishes (Greiner bio-
one, Frickenhausen) to 85–90% confluence. The medium was
removed via a suction apparatus and the cells were rinsed
twice with 6 mL Krebs-HEPES buffer (118 mM NaCl, 4.7 mM
KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.2 mM NaHCO3,
11.7 mM D-Glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4).
After these two washes, cells were loaded with 3 mL of a 0.5 M
Oregon GreenTM 488 BAPTA-1/AM-solution (Molecular
Probes, Eugene, OR) (in DMSO) in 6 mL Krebs-HEPES buffer
containing 3 mL of a 20% pluronic F-127-solution (Sigma
Aldrich) (in DMSO) for 45 min at 378C. After 35 min
incubation, the culture dish was rapped slightly in order
to remove all cells from the dish for further incubation.
To this cell suspension 5 mL Krebs-HEPES buffer were
added again to rinse all cells from the plate. The resulting
suspension was portioned into ten 1.5 mL Eppendorf
caps and centrifuged at 10640 RCF for 10 s. The supernatant
buffer was removed and the resulting ten pellets were
Evaporation of the solvent gave a white oily substance, which
did not crystallize. Yield 81%. 1H-NMR 400 MHz (CDCl3): d 0.90–
0.94 (t, J ¼ 7, 3H, 8’), 1.20–1.45 (m, 12 H, 2’–7’), 2.40 (s, 3H, N-Me),
2.45–2.48 (t, J ¼ 6, 2H, 5), 2.70–2.73 (t, J ¼ 6, 2H, 9), 2.76–2.81
(m, 4H, 6, 8), 3.56 (s, 2H, 1’), 4.58 (s, 2H, 15), 7.05–7.21 (m, 5H, Ar),
7.26–7.28 (d, J ¼ 8, 1H, Ar), 7.31–7.33 (dd, J ¼ 8/2, 1H, Ar), 7.51–
7.53 (d, J ¼ 8, 1H, Ar). 13C-NMR 400 MHz (CDCl3): 14.1 (8’), 22.6
(7’), 23.4 (9), 27.0 (3’), 29.2 (5’), 29.3 (2’), 30.0 (4’), 31.8 (6’), 32.9 (5),
33.4 (15), 43.8 (1’), 45.3 (N-Me), 59.1 (8), 59.6 (6), 109.0 (13), 112.7
(9a), 117.8 (10), 118.5 (11), 120.6 (12), 126.3 (3), 126.7 (2), 127.8
(9b), 131.0 (4), 131.7 (1), 135.2 (13a), 136.1 (14a), 138.1 (4a), 142.3
(15a). GC-MS: m/z: 402 (23%), 344 (14%), 297 (28%), 260 (9%), 246
(38%), 230 (73%), 217 (37%), 200 (6%), 171 (7%), 158 (26%), 144
(33%), 129 (30%), 115 (64%), 104 (41%), 91 (32%), 77 (100%). Anal.
calcd. for C28H38N2: C, 83.53%, H, 9.51%, N, 6.96%. Found: C,
83.38%, H, 9.18%, N, 6.46%.
Pharmacology
Radioligand binding assay
The binding studies were performed following the protocol
previously described but in 96-well format [3]. The assays,
using the whole-cell-suspension, were carried out in tripli-
cate in a volume of 550 mL (final concentration): TRIS-Mg2þ
-
buffer (345 mL), [3H]-ligand (50 mL), whole-cell-suspension
(100 mL) and appropriate drugs (55 mL). Non-specific binding
was determined using fluphenazine (100 mM) for D1- and
D5-tests and haloperidol (10 mM) for D2-, D3-, and D4-tests.
The incubation was initiated by addition of the
radioligand and was carried out in 96 deep well plates
(Greiner bio-one, Frickenhausen) using a thermocycler
(Thermocycler comfort1, Eppendorf, Wessling) at 278C.
The incubation was terminated after 90 min by rapid filtra-
tion with a PerkinElmer Mach III HarvesterTM using a
PerkinElmer Filtermat A, previously treated with a 0.25%
polyethyleneimine-solution (Sigma-Aldrich) and washed
with water. The filtermat was dried for 3 min at 400 W
using a microwave oven (MW 21, Clatronic, Kempen). The
dry filtermat was placed on a filter plate (Omni filter
platesTM, PerkinElmer Life Sciences) and each field of the
`
divided into two portions a 5 pellets, each portion was
suspended in 1 mL Krebs-HEPES buffer. The two suspensions
were centrifuged again for 10 s. After removing the
buffer, the two pellets were combined and re-suspended
in 1 mL buffer, diluted with 17 mL Krebs-HEPES buffer
and plated into 96-well plates (OptiPlate HTRF-96TM
,
Packard, Meriden, CT; Cellstar, Tissue Culture Plate, 96W,
Greiner bio-one, Frickenhausen). The microplates were kept
at 378C under an atmosphere containing 5% CO2 for 30 min
before they were used for the assay.
Screening for agonistic and antagonistic activity was
performed using a NOVOstar microplate readerTM (BMG
LabTechnologies) with
a
pipettor system. Agonistic
activities were tested by injecting 20 mL buffer alone as
negative control, standard agonist in buffer as positive
control, and rising concentrations of the test compounds
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

36
D. Robaa et al.
Arch. Pharm. Chem. Life Sci. 2011, 1, 28–36
TM
in buffer, each into separate wells. Fluorescence measure-
ment started simultaneously with the automatic injection.
SKF 38393 was used as standard agonist for D1-receptors
and quinpirole for D2-receptors (final concentration: 1 mM).
Screening for antagonistic activities was performed by
pre-incubating the cells with 20 mL of the test compound
dilutions (final concentrations: 100 mM, 50 mM, 10 mM, 5 mM,
1 mM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0,1 nM) at 378C
30 min prior to injection of 20 mL standard agonist per well.
As standard agonists, we used as described for the agonist
screening SKF38393 for D1- and quinpirole for D2-receptors,
respectively. Fluorescence measurement also started simul-
taneously with the automatic injection. At least two inde-
pendent experiments, each carried out in four or six
replications, were performed.
Graph Pad Prism
least three times.
3.0. All experiments were repeated at
We thank Ba¨rbel Schmalwasser, Petra Wiecha, and Heidi Traber for skillful
technical assistance in performing the pharmacological assays. Dina
Robaa thanks the Egyptian Cultural Affairs and Mission Section for her
stipend. Furthermore we would like to thank the DFG for the financial
support of the project (EN 875/1-1). Robert Kretschmer thanks the Hans-
Bo¨ckler-Stiftung for a scholarship.
The authors have declared no conflict of interest.
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ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com