Dopamine/serotonin receptor ligands. Part 15: Oxygenation of the benz-indolo-azecine LE 300 leads to novel subnanomolar dopamine D1/D5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.11.093

Relying on the high affinities of the benz-indolo-azecine LE 300 (1) and the hydroxylated dibenz-azecine LE 404 (2b) for the D₁/D₅ receptor subtypes, we synthesized methoxylated, hydroxylated and an indole-N methylated derivatives of 1 (Fig. 1). Hydroxylation of azecine derivatives is beneficial with regard to the affinities and selectivities for all the dopamine receptor subtypes. The 'serotonin-derived' 3-oxygenated target compounds but not the 11-oxygenated analogues were superior to the unsubstituted LE 300. 11-Methoxy-7,14-dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]benzazecine (3e) was found to be the most potent antagonist at D₂/D₃/D₄ and D₅ receptor subtypes (K_i for D₅ = 0.23 nmol) of all known benz-indolo-azecines.

Full text of DOI:10.1016/j.bmcl.2006.11.093

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1399–1402
Dopamine/serotonin receptor ligands. Part 15: Oxygenation
of the benz-indolo-azecine LE 300 leads to novel
subnanomolar dopamine D1/D5 antagonistsq
Christoph Enzensperger, Susann Kilian, Marit Ackermann, Anne Koch,
Kristin Kelch and Jochen Lehmann*
Institut fu¨r Pharmazie, Lehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Friedrich-Schiller-Universita¨t Jena,
Philosophenweg 14, D-07743 Jena, Germany
Received 21 September 2006; revised 28 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—Relying on the high affinities of the benz-indolo-azecine LE 300 (1) and the hydroxylated dibenz-azecine LE 404 (2b) for
the D1/D5 receptor subtypes, we synthesized methoxylated, hydroxylated and an indole-N methylated derivatives of 1 (Fig. 1).
Hydroxylation of azecine derivatives is beneficial with regard to the affinities and selectivities for all the dopamine receptor subtypes.
The ‘serotonin-derived’ 3-oxygenated target compounds but not the 11-oxygenated analogues were superior to the unsubstituted LE
300. 11-Methoxy-7,14-dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]benzazecine (3e) was found to be the most potent
antagonist at D2/D3/D4 and D5 receptor subtypes (Ki for D5 = 0.23 nmol) of all known benz-indolo-azecines.
Ó 2006 Elsevier Ltd. All rights reserved.
Locomotion, emotion, cognition and endocrinal secre-
tion are linked with dopaminergic transmission. Most
of the dopamine receptor antagonists used as antipsy-
chotics inhibit the D2-family (D2, D3 and D4), ‘aze-
cine-styled’ dopamine antagonists like 1 (LE 300)5,2–4
or its dibenzo-analogues4 2 showed selectivity primarily
for the D1 family (D1 and D5) (Fig. 1).
R
R=OCH ; R'=H
a:
3
CH3
CH3
N
N
b: R=OH; R'=H
N
H
1
c:
R=R'=H
2
Figure 1. The lead compounds LE 300 (1) and dibenzazecines 2a–d.
Previous investigations of this novel class of antidopam-
inergic drugs revealed that replacing the indole moiety in
the lead 1 with benzene is tolerated without a loss in
affinity for most of the dopamine receptor subtypes.3
(compare 1 and 2c in Table 1). But the affinities of 2c
were increased by substituents in the aromatic system
(compare 2c–b in Table 1). The phenolic 2b showed a
73-fold higher affinity for the D1 receptor compared to
the MeO-congener 2a, and its affinity for D5 was also
higher (Table 1). On the other hand, the affinity for
D2 was slightly higher for the MeO-compound 2a than
for 2b (Table 1).
The lead compound 1 of all azecine-type dopamine
receptor ligands has not been substituted up to now in
the aromatic rings or in the indole nitrogen. To investi-
gate if the effects of methoxylation and hydroxylation of
azecine derivatives with regard to the affinities and selec-
tivities for all of the dopamine receptor subtypes are
beneficial, we attached MeO-/HO-groups to the benz-
indolo-azecine scaffold of 1 at the indole moiety (3a
and b) and at the benzene part (3c and d), respectively
(Fig. 2). Furthermore, we methylated the indole-N of
3a, which yielded 3e. Finally, we characterized the inter-
action of these target compounds with the human-
cloned D1–D5 receptor subtypes by radioligand-binding
experiments and by a functional calcium assay.
Keywords: Dopamine receptor ligands; Azecines; LE 300; Oxygena-
tion; N-methylation.
q
To synthesize the 11-methoxy-hexahydro-5H-indolo[3,2-
f][3]benzazecine 3a, the corresponding indolo-benzaze-
cin-11-ol 3b and the 7,14-dimethyl derivative 3e, a
See Ref. 1.
Corresponding author. Tel.: +49 3641 949803; fax: +49 3641
949802; e-mail: j.lehmann@uni-jena.de
*
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.093

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C. Enzensperger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1399–1402
Table 1. Affinities (Ki, nM) for dopamine receptor D1–D5 subtypes determined by radioligand-binding experiments
CH3
H3CO
HO
CH3
H3CO
CH3
N
N
CH3
CH3
N
N
N
N
H
N
H
N
H
N
H
N
CH3
3a
3b
3c
3d
3e
OCH3
Ki (nM)
OH
Compound
HEK D1
CHO D2L
44.5 15.8
CHO D3
CHO D4.4
HEK D5
1 (LE-300)7
1.9 0,9
28.5 9.7
0.39 0.22
4.5 2.1
40.3 14.4a
75.7 7.3b
47 24
109 39
43.4 13.2b
7.5 0.3
38.3 24a
1.5 0.2
2a
2b3
2c3
3a
3b
3c
13
9
17.5 2.1
56.5 9.0
11.9 5.6a
38.4 14a
22.8 11.1a
74.7 34a
1.70 0.6a
11.3
1
52.5 6.4
475 48.5b
944 171b
1135 237b
2070 776b
3.78 2.4a
134 15
266 22b
398 288a
92.6 5.3a
1359 926a
21.55 3.5b
11.2 1.8
3.6 0.6a
0.39 0.16a
31.5 14.7b
5.4 3.6a
0.23 0.06a
0.82 0.056b
0.56 0.06b
19.0 2.3b
3.7 1.1b
3d
3e
2.00 1.6a
a Ki values are means of three experiments, performed in triplicate SEM.
b Ki values are means of two experiments, performed in triplicate SEM.
R1
H3CO
Cl
10
Cl
H3CO
9
8
R1=OCH , R2=H, R3=H
R1=OH, R2=H, R3=H
a:
3
CH3
6
NH
O
Cl
a
NH2
N
12
b:
c:
+
O
N
H
13
N
R3
5
N
H
R1=H, R2=OCH , R3=H
3
4
(68%)
5
3
1
R1=H, R2=OH, R3=H
4
b, c
d:
e:
2
R1=OCH , R2=H, R3=CH
R2
H3CO
H3CO
3
3
CH3
+
N
N
Figure 2. Novel derivatives of LE 300 (1) with numbering of the ring
system.
d
N
H
N
H
(72%)
7
(50%)
6
mixture of equimolar amounts of 5-methoxy-tryptamine
and chloroethylbenzoylchloride6 (4) was reacted togeth-
er with 3 mol of triethylamine in dichloromethane. The
resulting benzamide derivative 5 was cyclized with POCl3
in acetonitrile. NaBH4 reduction of the intermediate in
methanol yielded the pentacyclic benzindolo-quinolizine
6. After quaternization with methyl iodide in acetone, the
resulting compound 7 was cleaved using elemental sodi-
um in liquid ammonia to yield 3a.11 The phenolic ana-
logue 3b12 was obtained from 3a by ether cleavage with
BBr3 in dichloromethane.
e
g
f
(30%)
(82%)
(85%)
3b
3e
3a
Scheme 1. Synthesis of benz-indolo-azecines 3a and 3b. Reagents and
conditions: (a) 5-methoxy-tryptamine, 3 mol NEt3, CH2Cl2, 0 °C–rt;
(b) POCl3/acetonitrile 1:20, 5 h, 30 °C, under nitrogen; (c) NaBH4,
MeOH, 0 °C, 1 h, reflux; (d) methyl iodide, acetone; (e) Na, liq NH3;
(f) BBr3, CH2Cl2, 5 h, reflux; (g) Na, liq NH3, methyl iodide.
Interestingly, the dimethylated benz-indolo-acezine 3e
could be obtained in a one-pot reaction out of 7 just
by adding 1.5 mol of methyl iodide to the solution of
7 in liquid ammonia after adding the sodium which is
needed to cleave the central C–N bond. Purification
was performed by preparing the hydrochloride salt of
3e15 and recrystallization from isopropanole (Scheme 1).
derivative 10. Quaternization with methyl iodide and
the subsequent ring cleavage of the quaternary salt 11
resulted in 3c.13 The O-demethylation was conducted
with HBr in acetic acid and produced the phenolic qua-
ternary salt 12. The target compound 3d14 was obtained
by ring cleavage (Scheme 2).
All of the target compounds were screened for their
binding affinities for human-cloned D1, D2L, D3, D4.4
and D5 receptors, stably expressed in HEK 293 or
CHO cells, by radioligand-binding experiments. The
protocol has been described.8,16
3-Methoxyphenethylamine and the indololactone 87
were used for synthesizing the 3-methoxy-7-methyl-
hexahydro-indolo[3,2-f][3]-benzazecine 3c and the corre-
sponding indolo[3,2-f][3]-benzazecin-3-ol 3d. Applying
the cyclization/reduction sequence with POCl3 in aceto-
nitrile and NaBH4 in methanol to the hydroxyethyl-
benzamide 9 yielded the corresponding quinolizine
Additionally, the functionality of the target compounds
was investigated for the D1, D2 and D5 receptors using a

C. Enzensperger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1399–1402
1401
H3CO
is quite relevant. The compounds 3c and d, which may
be considered a combination of 1 and 2a, b rather than
3a, b, exhibited lower affinities than their indole-substi-
tuted counterparts 3a, b and e. Compounds 3c and d
do maintain the oxygenated benzene ring of 2a and b,
consequently the introduction of the indole for the
unsubstituted benzene ring is disfavoured. Perhaps the
indole—rather than the benzene-moiety is involved in
receptor binding, and hydroxylation or—to a lesser ex-
tent—methoxylation at the indole, yielding ‘serotonin-
derived’ structures, facilitates the receptor binding,
whereas these additional substituents in the benzene part
do not enhance or lower the protein binding significant-
ly. The substituents on benzene ring effect steric or elec-
tronic interaction, that causes a decrease in affinity. The
selectivity profile is characterized by a preference for the
D1/D5 family and for 3e there is a remarkable 9-fold D5
selectivity over the D1 receptor.
O
H3CO
OH
NH
O
O
NH2
a
HN
+
HN
(47%)
9
8
b, c
HO
H3CO
H3CO
CH
3
CH
3
N+
N+
N
d
e
HN
HN
HN
(42%)
10
(32%)
12
(57%)
11
f
f
(76%)
(87%)
3c
3d
Scheme 2. Synthesis of benz-indolo-azecines 3c and 3d. Reagents and
conditions: (a) 1.3 mol 3-methoxyphenethylamine, toluene, 24 h,
reflux; (b) POCl3/acetonitrile 1:1, 24 h, reflux, under nitrogen; (c)
NaBH4, MeOH, 0°, 1 h, reflux; (d) methyl iodide, acetonitrile; (e) HBr/
HOAc, 3 h, reflux; (f) Na, liq NH3.
Selecting one of the more attractive 11-oxygenated
derivatives (3a), we methylated the indole nitrogen,
and the resulting compound 3e showed an increase in
all of the affinities compared to the desmethyl derivative
3a with the exception of that for D1. The affinity for the
D1 receptor decreased by a factor of 2.4, whereas the
affinity for D5 increased by factor 15 yielding a 9-fold
D5-selectivity. Furthermore, a surprising increase was
observed for D2 and D3: the affinity rose from 12 to
1.7 nM and from 474 to 3.8 nM, respectively. For D4
the affinity improved more than 10-fold. Thus, com-
pound 3e, showing the same high affinity for the D1
and D2 receptors, a very high affinity for D5 and consid-
erable affinities for D3 and D4, features a receptor profile
which is quite unusual for the azecine-like dopamine
receptor antagonists and different from those being
investigated so far by our group.
calcium fluorescence assay which has been developed
and established in our group.9
None of the reported compounds increased the intramo-
lecular calcium concentration of any type of cell. This
excludes agonistic activity. Instead, the compounds sup-
pressed the calcium signal which was induced by stan-
dard dopamine agonists (D1/D5 receptors: SKF 38393,
D2: quinpirol) in a concentration-dependent manner
and produced sigmoidal curves which characterize the
inhibition of the respective agonists’ action (data not
shown). These findings confirmed that the compounds
are either antagonists or inverse agonists in both of
the dopamine receptor families. Affinity data (Table 1)
were generated from radioligand-binding experiments.
Since the affinity of 3e is not reduced in comparison to
3a, it can be excluded that the indole NH of the benz-
indolo-azecines interacts in the same way as the OH in
2b. The influence on the affinity profile caused by the
indole N-methyl group must rather be a steric than an
electronic effect.
SAR with regard to the influence of the HO-/MeO-sub-
stitution of the benz-indolo-azecines on the affinity for
the D1 receptor are similar to those found for the di-
benz-azecines: Of all of the new indolic compounds
3a–d, the phenolic derivatives 3b, d are superior to their
methoxy analogues 3a, c in binding to the D1 receptor.
The same was found for the D5 receptor. In the affinity
for the D2 receptor, we observed a marginally higher
affinity for the methoxy-substituted 2a in the dibenzo-
azecines 2a, b (Table 1). This tendency could be seen
in former investigations,4 is observed for several unpub-
lished compounds and was affirmed for the new benz-
indolo-azecines: in both series of compounds—the ones
with an oxygenated indole moiety (3a and b) and also
the ones with the, respectively, substituted benzene moi-
ety (3c and d)—the methoxy-derivatives 3a, c exhibited a
3-fold higher affinity for the D2 receptor compared to
their phenolic congeners 3b and d (Table 1).
In general the SAR regarding the interaction of the HO-
and the MeO-compounds with the D1 receptor are sim-
ilar for the dibenzo- and the benzindolo-derivatives. But
the significant loss in affinity that occurs in the unsubsti-
tuted dibenzazecine 2c by introducing a MeO-substitu-
ent (2a) was not found for the benz-indolo-azecine.
Here the compound with a MeO-substitution at the
indole moiety (3a) shows higher affinity than the unsub-
stituted 1.
In conclusion: to investigate the influence of methoxyla-
tion, hydroxylation and indole-N-methylation, we syn-
thesized five appropriate derivatives of the dopamine
antagonist LE 300, measured the affinities for all hu-
man-cloned dopamine receptors and characterized
them as antagonists or inverse agonists in a fluorescence
calcium assay. These structural modifications partial-
ly improved affinities and changed selectivities.
‘Serotonin-derived’ compounds were superior to their
In addition to these general relations, it is noteworthy
that the structural area where oxygenation takes place

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C. Enzensperger et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1399–1402
1
175–180 °C. H NMR (250 MHz; methanol d4): d 2.2 (s,
3H, N–CH3) 2.9–2.6 (m, 8H, H-5, 6, 8, 9), 4.2 (s, 2H, H-
15), 6.65 (dd, 1H, J = 2.3, 8.6 Hz, H-12), 6.8 (d, 1H,
J = 2.3 Hz, H-10), 7.1 (d, 1H, J = 8.6 Hz, H-13), 7.25 (m,
4H, H-2,3,4), 7.3 (mc, 1H, H-1). ppm. GC/MS: M = 306
(85%), M = 262, 261 (37%), M = 234 (50%), M = 207
(30%), M = 160 (29%), M = 146 (29%), M = 58 (100%).
Anal. (C20H22N2O): C, N, H.
‘tryptamine-derived’ isomers. The one-pot reaction for
both the indole-N-methylation and the ring expansion
procedure under Birch conditions represents a novel
synthetic strategy that might be useful for the preparation
of further N-alkylated indolo-azecines. It might also be
used for alkylating phenolic groups simultaneously.
13. 3-Methoxy-7-methyl-6,7,8,9,14,15-hexahydro-5H-indo-
lo[3,2-f][3]benzazecine (3c): white powder, mp 252 °C
(HCl salt). 1H NMR (250 MHz; CDCl3): (free base) d
2.6 (s, 3H, N–CH3) 2.5 (mc, 2H, H-5 or H-9), 2.6 (mc,
4H, H-6 or H-8 and 5 or 9), 2.7 (mc, 2H, H-6 or H-8),
2.8 (s, 3H, –OCH3), 4.2 (s, 2H, H-15), 6.5 (d, 1H,
J = 2.7 Hz, H-4), 6.6 (dd, 1H, J = 2.9; 8.4 Hz, H-2), 7.0
(mc, 2H, H-11 and H-12), 7.1 (d, 1H, J = 2.7 Hz, H-10),
7.2 (d, 1H, J = 1.5 Hz, H-13),7.3 (mc, 1H, H-1), 7.6 (s,
1H, –NH) ppm. GC/MS: M = 320 (100%); M = 275
(35%); M = 262 (31%) M = 248 (35%); M = 217 (25%);
M = 190 (28%); M = 143 (26%). Anal. (C21H25N2OCl):
C, N, H.
Acknowledgements
We thank Ba¨rbel Schmalwasser, Petra Wiecha and Hei-
di Traber for skillful technical assistance in performing
the pharmacological assays.
References and notes
1. Hamacher, A.; Weigt, M.; Wiese, M.; Hoefgen, B.;
Lehmann, J.; Kassack, M. U. BMC Pharmacol. 2006, 6,
11.
2. Enzensperger, C.; Lehmann, J. J. Med. Chem. 2006, 49,
6408.
14. 7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]ben-
1
zazecin-3-ol (3d): off-white powder, mp 135 °C. H NMR
3. Hoefgen, B.; Decker, M.; Mohr, P.; Schramm, A. M.;
Rostom, S. A. F.; El Subbagh, H.; Schweikert, P. M.;
Rudolf, D. R.; Kassack, M. U.; Lehmann, J. J. Med.
Chem. 2006, 49, 760.
4. Mohr, P.; Decker, M.; Enzensperger, C.; Lehmann, J.
J. Med. Chem. 2006, 49, 2110.
5. Decker, M.; Schleifer, K.; Nieger, M.; Lehmann, J. Eur.
J. Med. Chem. 2004, 39, 481.
6. Odasso, G.; Winters, G.; Schiatti, P.; Selva, D.; Nathan-
sohn, G. Farmaco 1977, 32, 159.
(250 MHz; CDCl3): d 2.3 (s, 3H, N–CH3), 2.5 (dd, 2H,
J = 5.4 Hz, H-5 or H-9), 2.6 (dd, 2H, J = 5.2 Hz, H-5 or
H-9), 2.7 (mc, 2H, H-6 or H-8), 2.8 (mc, 2H, H-6 or H-8),
4.2 (s, 2H, H-15), 6.5 (d, 1H, J = 2.7 Hz, H-4), 6.6 (dd, 1H,
J = 2.9; 8.2 Hz, H-2), 7.0 (mc, 2H, H-11 and H-12), 7.05
(mc, 1H, H-10 or H-13), 7.1 (mc, 1H, H-10 or H-13), 7.3
(mc, 1H, H-1), 7.7 (s, 1H, –NH) ppm. GC/MS: M = 306
(100%); M = 261 (45%); M = 248 (41%); M = 234 (60%);
M = 176 (48%); M = 143 (53%). Anal. (C20H22N2O): C,
N, H.
7. Witt, T.; Hock, F. J.; Lehmann, J. J. Med. Chem. 2000, 43,
2079.
8. Decker, M.; Lehmann, J. Arch. Pharm. Life Sci. 2003, 336,
466.
9. Kassack, M. U.; Hoefgen, B.; Lehmann, J.; Eckstein, N.;
Quillan, J. M.; Sadee, W. J. Biomol. Screen. 2002, 7, 233.
10. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
11. 11-Methoxy-7-methyl-6,7,8,9,14,15-hexahydro-5H-indo-
lo[3,2-f][3]benzazecine (3a): white foam, mp 160 °C. 1H
NMR (250 MHz; CDCl3): d 2.6 (s, 3H, N–CH3) 2.9–2.6
(m, 8H, H-5, 6, 8, 9), 3.8 (s, 3H, –OCH3), 4.4 (s, 2H, H-
15), 6.8 (dd, 1H, J = 2.5, 8.7 Hz, H-12), 6.95 (d, 1H,
J = 2.5 Hz, H-10), 7.2 (m, 4H, H-2,3,4,13), 7.3 (mc, 1H, H-
1), 7.7 (s, 1H, –NH) ppm. GC/MS: M = 320 (79%),
M = 275, 276 (32%), M = 262 (27%), M = 248 (32%),
M = 173 (16%), M = 160 (53%), M = 58 (100%). Anal.
(C21H24N2O): C, N, H.
15. 11-Methoxy-7,14-dimethyl-6,7,8,9,14,15-hexahydro-5H-
indolo[3,2-f][3]benzazecine hydrochloride (3e): off-white
solid, mp 226–242 °C (slow decomposition). 1H NMR
(250 MHz; CDCl3): d 2.36 (s, 3H, N–CH3), 2.53–2.46 (m,
4H, H-6 and H-8), 2.8–2.73 (m, 4H, H-5 and H-9), 3.51 (s,
3H, indole-N–CH3), 3.87 (s, 3H, –OCH3), 4.4 (s, 2H, H-
15), 6.85–6.8 (dd, 1H, J = 8.7, 2.4 Hz, H-12), 6.93–6.94 (d,
1H, J = 2.4 Hz, H-10), 7.02–7.33 (m, 5H, H-1, H-4), 7.1
(d, 1H, J = 8.7 Hz, H-13) ppm. GC/MS: M = 334 (100%);
M = 290 (18%); M = 276 (57%); M = 262 (82%); M = 229
(80%); M = 187 (34%); M = 174 (65%); M = 160 (34%).
Anal. (C22H27N2OCl): C, N, H.
16. [3H]SCH 23390 and [3H]spiperone were used as radioli-
gands at the D1-like and D2-like receptor family, respec-
tively. Incubation at 27 °C was terminated after 90 min by
rapid filtration with a Perkin-Elmer Mach III harvester.
At least two independent experiments were carried out,
each in triplicate. Ki values were calculated from IC50
values, applying the equation of Cheng and Prusoff,10 and
are given in nanomolar units (Table 1).
12. 7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]ben-
zazecin-11-ol (3b): from isopropanol grey powder, mp