Chiral indolo[3,2-f][3]benzazecine-type dopamine receptor antagonists: Synthesis and activity of racemic and enantiopure derivatives

Article abstract of DOI:10.1021/jm200676f

Racemic and enantiopure 8-substituted derivatives of the lead dopamine receptor antagonist LE 300 (1) were prepared, and their affinities for the dopamine receptors (D₁-D₅) were tested. The separate enantiomers showed significantly different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the substituents point below the reference plane of the indolo[3,2-f][3]benzazecine scaffold were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy derivative was shown to be selective for the D₅-receptor, even against D₁.

Full text of DOI:10.1021/jm200676f

BRIEF ARTICLE
pubs.acs.org/jmc
Chiral Indolo[3,2-f][3]benzazecine-Type Dopamine Receptor
Antagonists: Synthesis and Activity of Racemic and Enantiopure
Derivatives
Dina Robaa,|| Christoph Enzensperger,† Shams ElDin AbulAzm,‡ Mohamed M. Hefnawy,§
Hussein I. El-Subbagh,z Tanveer A. Wani,§ and Jochen Lehmann*,†,§
Institut f€ur Pharmazie, Abteilung Medizinische Chemie, Martin Luther Universit€at Halle-Wittenberg, Germany
†Institut f€ur Pharmazie, Lehrstuhl f€ur Pharmazeutische/Medizinische Chemie, Friedrich Schiller Universit€at Jena, Jena, Germany
‡Department of Medicinal Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
§Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
zDepartment of Pharmaceutical Chemistry, College of Pharmaceutical Sciences & Pharmaceutical Industries, Future University,
Cairo, Egypt
S Supporting Information
b
ABSTRACT: Racemic and enantiopure 8-substituted derivatives of the lead dopamine receptor antagonist LE 300 (1) were
prepared, and their affinities for the dopamine receptors (D1ꢀD5) were tested. The separate enantiomers showed significantly
different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the substituents point below the reference plane of the
indolo[3,2-f][3]benzazecine scaffold were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy
derivative was shown to be selective for the D5-receptor, even against D1.
Chart 1. Substitution of the Lead Indolobenzazecine Deri-
vative 1 at Position 8
’ INTRODUCTION
Dysfunctions of the dopaminergic system have been linked
with several neurological and psychiatric disorders, mainly
Parkinson’s disease,1 schizophrenia,2,3 depression,4 attention-
deficit hyperactivity disorder,5 and alcohol dependence.6
Partially hydrogenated indolo[3,2-f][3]benzazecines7,8 constitute a
structurally novel class of dopamine receptor antagonists distinguished
by their nanomolar to subnanomolar affinities for all dopamine
receptors and a general preference for the receptors of the D1 family.
Since the discovery of the lead compound 7-methyl-6,7,8,9,14,15-
hexahydro-5H-indolo[3,2-f][3]benzazecine (1, LE300),8 con-
siderable work has been accomplished in developing the SAR
of this class of compounds.7,9,10 This revealed that the hetero-
cyclic backbone scaffold is optimally made up of two aromatic
rings (indole and benzene, or two benzene rings) surrounding a
central hydrogenated azecine moiety. Both aromatic rings should
be separated by a methylene group. Further studies showed that
small substituents at the indole nitrogen are well tolerated or may
even result in increased affinities.11
The aim of the present work was to investigate the first
indolo[3,2-f][3]benzazecine derivatives substituted at a C-atom
of the azecine ring next to the central nitrogen. Since this type of
substitution results in the formation of a chiral center, we wanted
to investigate not only the racemic but also the enantiopure
derivatives (Chart 1).
Our primary target compounds were indolobenzazecine deri-
vatives substituted at position 8 with a small residue such as
methyl, hydroxymethyl, and carboxyl groups. The assignment of
the configuration of these substituents differs from the hydro-
xymethyl and carboxy to the methyl derivatives. This is due to the
change in the priorities of the groups involved at the chiral center.
According the CahnꢀIngoldꢀPrelog priority rules, a hydroxy-
methyl or a carboxyl group has a different priority than a methyl
group. To allow better presentation of the results, those en-
antiomers where the substituents point below the reference plane
will be defined as α-isomers. Those bearing substituents pointing
above the reference plane will be defined as β-isomers (Chart 1).
’ CHEMISTRY
To obtain the R and S enantiomers (α and β isomers,
respectively) of the target 8-methylated benzinodoloazecines
((+)-5 and (ꢀ)-5), racemic α-methyltryptamine (2) was first
converted to the quinolizine derivative 3 by reaction with 2-(2-
bromoethyl)benzaldehyde following a previously established pro-
cedure for the synthesis of analogous quinolizines.12 Subsequent
quaternization with methyl iodide and reduction of the obtained
quaternary salts under Birch conditions (Na, liq NH3) yielded the
Received: May 27, 2011
Published: September 05, 2011
r
2011 American Chemical Society
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BRIEF ARTICLE
target azecine rac-5 (Scheme 1). Resolution of the racemate to
obtain the enantiomers(+)-5 and (ꢀ)-5 was achieved by means of
preparative chiral HPLC on a cellulose-based chiral stationary
phase. Attempts to crystallize the enantiopure derivatives to assign
the absolute configuration by X-ray were not successful. To
determine the configurations of these separated enantiomers, an
alternative synthetic route was adopted (Scheme 3), where we
started from an enantiopure quinolizine derivative. This will be
described in more detail below.
The quinolizine ester derivatives 8 and 9, obtained from
the enantiomerically pure methyl esters of D- and L-tryptophan
(6 and 7), served as starting materials for the preparation of
further 8-substituted indolobenzazecine derivatives. Conversion
of the quinolizine esters 8 and 9 into the corresponding azecines,
however, failed at the last step, namely, the ring cleavage of the
quaternary salts 10 and 11 under Birch conditions. As an
alternative route, the ester function of the quaternary salts 10
and 11 was first hydrolyzed, and the resulting carboxylic acid
derivatives 12 were reduced under Birch conditions, yielding
the amino acid azecine derivatives rac-13. These were in turn
reduced using lithium aluminum hydride to give the hydroxymethyl
derivatives rac-14 (Scheme 2). However, investigations of the
enantiomeric purity of the hydroxymethyl derivatives rac-14
by means of chiral HPLC (enantiomeric excess (ee) 0.12% and
0.52%, respectively) and measurement of their optical rotation
revealed that these derivatives were obtained in a racemized
form, despite the fact that we started from enantiopure com-
pounds. It can be presumed that this racemization took place by
deprotonation/reprotonation due to the basic conditions used
for the hydrolysis of the ester function of the quaternary salts 10
and 11 to the corresponding carboxylic acid derivative 12,
despite the mild conditions applied (1 N sodium hydroxide,
0 °C for 2 h).
Scheme 3. Synthetic Route B: 8-Hydroxymethylquinolizine
as starting Material for Enantiopure 8-Hydroxymethyl- and
8-Methylindolobenzazecinesa
Scheme 1. Synthesis of the 8-Methylbenzinodoloazecine
Derivative 5 and Its Enantiomeric Separationa
a Reagents and conditions: (a) LiAlH4, dry THF, room temp, 90 min;
(b) MeI, acetone, 24 h; (c) Na0, liq NH3, ꢀ40 °C, 10 min; (d) MsCl,
TEA, DCM, 12 h, room temp; (e) LiAlH4, dry THF, reflux, 2 h.
a Reagents and conditions: (a) TFA, dioxane, reflux, 6 h; (b) MeI,
acetone, 24 h; (c) Na0, liq NH3, ꢀ40 °C, 10 min.
Scheme 2. Synthetic Route A for the Preparation of Benzinodoloazecines Derived from D- and L-Tryptophana
a Reagents and conditions: (a) TFA, dioxane, reflux, 3 h; (b) MeI, acetone, 35 °C, 24 h; (c) Na0, liq NH3, ꢀ40 °C, 10 min; (d) 1 N NaOH, MeOH, 0 °C,
2 h; (e) LiAlH4, dry THF, reflux, 5 h.
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Table 1. Affinities (Ki, nM) for Human D1ꢀD5 Receptors, Determined by Radioligand Binding Experimentsa
a Ki values are the mean of two to three experiments, performed in triplicate ((SEM). The asterisk (/) indicates values taken from ref 10. CHO cell lines
were used for D4.4. HEK cell lines were used for D1, D2L, D3, and D5.
Accordingly, the hydrolysis step was circumvented, and the ester
function of the quinolizines 8 and 9 was first reduced using lithium
aluminum hydride. The resulting hydroxymethylquinolizines 15
and 16 were subsequently quaternized, and the quaternary salts 17
and 18 were finally reduced with sodium in liquid ammonia to give
the target azecines (ꢀ)-14 and (+)-14 (Scheme 3). The enantio-
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 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 (Table 1). A detailed protocol has been elsewhere described.15
Additionally, the compounds were tested in an intracellular Ca2+
assay to determine their functionality (agonist or antagonist) at
the D1 and D2 receptors. HEK293 cells stably expressing the
respective D-receptor were loaded with a fluorescent dye, and
after preincubation with rising concentrations of the test com-
pound, an agonist (SKF 38393 for D1 and quinpirole for D2) was
added and the Ca2+-induced fluorescence was measured with a
microplate reader. The ability of the test compound to suppress
the agonist-induced Ca2+ influx is an indication of antagonistic or
inverse agonistic properties at the receptor.13
purity of the obtained azecines was ascertained by measuring their
rt
496
specific rotation ([α] of ꢀ158.4° and +161°, respectively) and
by chiral HPLC. These findings substantiate our earlier speculations
that in the previously adopted synthetic procedure racemization
took place at a step prior to the synthesis of the hydroxymethyl
derivative rac-14 and that it most probably occurred during hydro-
lysis. It could also be concluded that the amino acid derivative rac-13
is not enantiomerically pure and that it racemized to the same extent
as the hydroxymethyl derivative rac-14.
In order to identify the configuration of the aforementioned sepa-
rated S and R enantiomers of 8-methylindolobenzazecine ((+)-5 and
(ꢀ)-5), Scheme 1), we adopted an alternative route for the synthesis
of the (8S)-enantiomer (+)-5 (α-isomer) starting from an enantio-
merically pure precursory substance, namely, the (8R)-hydroxy-
methylquinolizine 15 (α-isomer). This was first converted to the
corresponding mesylate, which was directly reduced with lithium
aluminum hydride to give the methylquinolizine 19. Reduction
with sodium in liquid ammonia yielded the corresponding (8S)-
azecine derivative (+)-5 (α- isomer) (Scheme 3). Comparison
of the respective specific rotation of the herein obtained
azecine with the previously obtained ones (Scheme 1, (+)-5
and (ꢀ)-5)) enabled us to assign the respective configuration.
’ RESULTS AND DISCUSSION
Indolobenzazecine derivatives substituted at position 8 with
three different residues (methyl, hydroxymethyl, and carboxylic
acid) were prepared. The 8-methyl and 8-hydroxymethyl deri-
vatives were obtained as the separated α and β isomers ((+)-5,
(ꢀ)-5, (ꢀ)-14, and (+)-14, respectively), while the amino acid
derivative could only be obtained in a racemized form (rac-13).
All tested compounds showed antagonistic properties at the
investigated dopamine receptors in the functional Ca2+ assay.
Compared with many other indolobenzazecine derivatives,
the racemized amino acid derivative rac-13 showed a pro-
nounced decrease in the affinities for all dopamine receptors.
Interestingly however, rac-13 displayed practically no affinities
for all dopamine receptors (Ki > 10 000 nM) except for the D5
receptor, where it displayed submicromolar Ki (∼657 nM).
’ PHARMACOLOGY
All the enantiopure target compounds ((+)-5, (ꢀ)-5, (+)-14,
and (ꢀ)-14) and the racemized amino acid derivative rac-13
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BRIEF ARTICLE
Hitherto, compounds with a significant selectivity for D5 over D1
have not been achieved, with the exception of some other
azecine-type ligands,14,15 which renders the compound highly
attractive as a potential D5-selective pharmacological tool. Se-
paration of the enantiomers of compound rac-13 could not be
accomplished, but it would be of great value, as one of the
enantiomers would presumably show higher affinities or more
pronounced selectivity.
The separated α and β enantiomers of 8-methylbenzindoloa-
zecine derivatives ((+)-5 and (ꢀ)-5) displayed a high discre-
pancy in their affinities. The α-enantiomer (+)-5 was almost as
active as the lead indolobenzazecine 1. The corresponding β-
isomer (ꢀ)-5 exhibited at least 100-fold reduction in affinities for
all dopamine receptors, showing only micromolar affinities for
D1-like receptors (D1 and D5), and was practically devoid of
affinities for D2-like receptors (D2, D3, and D4).
A similar but less pronounced difference was observed be-
tween the α and β enantiomers of 8-hydroxymethylindoloben-
zazecine ((ꢀ)-14 and (+)-14). The α-enantiomer (ꢀ)-14 was
also more active than the β-enantiomer (+)-14. However, only a
10-fold difference in affinities between both enantiomers was
found. While the α-isomer of 8-hydroxymethylindolobenzaze-
cine was relatively less active than its 8-methylated counterpart,
the β-isomer of the hydroxymethyl derivative was more active
than the methylated one.
NaOH. The resulting quinolizine base was extracted with dichloro-
methane and recrystallized or purified chromatographically.
(8RS,14bRS)-8-Methyl-5,6,8,9,14,14b-hexahydroindolo-
[20,30:3,4]pyrido[2,1-a]isoquinoline (3). Crystallization from
isopropanol/hexane yielded pale yellow needle-shaped crystals. Yield
70%. Mp: 75ꢀ77 °C (for analytical data, see Supporting Information).
Methyl(8R)-5,6,8,9,14,14b-Hexahydroindolo[20,30:3,4]pyrido-
[2,1-a]isoquinoline-8-carboxylate (8). 8 was purified by column
chromatography, dichloromethane/methanol (100:1). Yield 55%. Mp
84ꢀ87 °C (for analytical data, see Supporting Information).
Methyl (8S)-5,6,8,9,14,14b-Hexahydroindolo[20,30:3,4]pyrido-
[2,1-a]isoquinoline-8-carboxylate (9). 9 was purified by column
chromatography, dichloromethane/methanol (100:1). Yield 59%. Mp
84ꢀ86 °C (for analytical data, see Supporting Information).
General Procedure for the Ring-Opening. Ammonia was
condensed in a three-necked 100 mL flask, which was equipped with a
balloon and a stopper and cooled in a liquid nitrogen bath. After filling 3/4 of
the flask’s volume, the cooling bath was removed and ammonia was allowed
to liquefy. The respective quaternary salts were then added to the stirred liquid
ammonia. This was followed by gradual addition of small pieces of sodium
metal until the blue color remained for 10 min. A few drops of saturated
ammonium chloride solution were added to terminate the reaction, and the
mixture was stirred under nitrogen until the ammonia completely evaporated.
An amount of 10 mL of water was added to the residue, and the mixture was
then extracted with 30 mL of diethyl ether. The organic phase was dried over
Na2SO4, and the solvent was removed under reduced pressure.
(8RS)-7,8-Dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-
f][3]benzazecine (rac-5) and Its Separated R and S Enantio-
mers (+)-5 and (ꢀ)-5. Creamy white solid. Yield 89%. Mp 82ꢀ84 °C
(for analytical data, see Supporting Information).
’ EXPERIMENTAL SECTION
General Methods. Melting points are uncorrected and were
measured in open capillary tubes using a Gallenkamp melting point
apparatus. 1H and 13C NMR spectral data were obtained from a Bruker
Advance 250 spectrometer (250 MHz) and Advance 400 spectrometer
(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
Heraeus Vario EL apparatus, or by HPLC. All values for C, H, and N
were found to be within (0.4. All compounds showed >95% purity. The
HPLC system for the preparative resolution of (+)-5 and (ꢀ)-5
consisted of a Labomatic-HD-200 pump and UV/vis filterꢀphotometer
(Knauer, Berlin) detector (220 nm). Separation was performed on a
Chiralcel OD column [cellulose tris(3,5-dimethylphenylcarbamate)]
(50 cm ꢁ 5 cm) with a cooling mantle. Analytical separation of rac-5
and analysis of the separated (+)-5 and (ꢀ)-5 was performed on HPLC
system consisting of a system controller SCL10-Avp, autoinjector SIL-
10A, 50 μL sample loop, UV detector SPD-10A, two pumps LC-10A
(Shimadzu, Duisburg), and a Chiralcel OD column (10 μm, 250 mm ꢁ
4.6 mm) (Daicel Chemical Industries, Tokyo). The ee values were
calculated from peak areas. Chiral HPLC analyses of rac-14, (+)-14, and
(ꢀ)-14 were performed on HPLC instrument (Water, U.S.) equipped
with a pump (model PU-980), a UV/vis detector (model UV-975), and
injection valve with 20 μL sample loop. The chiral stationary phase
(CSP) used in this separation was the macrolide-type antibiotic teico-
planin, known as Teicoplanin T (150 mm ꢁ 4.6 mm i.d.) purchased
from Advanced Separation Technologies (Whippany, NJ, U.S.) in the
polar-organic separation mode.
The enantiomers were separated using a preparative Chiracel OD
column (cellulose tris(3,5-dimethylphenylcarbamate): eluent, hexane/
ethanol9:1;flowrate, 194 mL/min;temp, 25°C;UV detectorλ =220 nm.
The isolation was accomplished by 1 mL injection of a solution of rac-5
(75 mg in 1 mL of n-hexane/ethanol 1:1). Enantiomeric purity was assessed
by analytical chiral HPLC (column, Chiralcel OD; eluent n-hexane/
ethanol 9:1; flow rate, 1.3 mL/min; UV detector λ = 220 nm), where
the first eluted enantiomer ((+)-5) showed 99.4% ee (reten-
tion time, 11.8 min) and the second eluted compound ((ꢀ)-5) showed
98.5% ee (retention time, 22.8 min). Specific rotation of (+)-5 was
rt
rt
[α] 132° (c 1, CHCl3), and for (ꢀ)-5 was [α] ꢀ128° (c 1, CHCl3).
546
546
Alternatively, (8S)-7,8-dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo-
[3,2-f][3]benzazecine ((+)-5) was prepared starting from 20 (Scheme 3).
HPLCretentiontime:11.64min (column, ChiralcelOD; eluentn-hexane/
ethanol 9:1; flow rate, 1.3 mL/min; UV detector λ 220 nm). [α]r5t46 136°
(c 1, CHCl3). This revealed that the first eluted enantiomer (compound
(+)-5) bears an S configuration at position 8.
7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]-
benzazecine-8-carboxylic Acid (rac-13). The same procedure for
ring-opening was applied. After evaporation, water was added and
the obtained solution was carefully treated with 1 N HCl until the target
amino acid precipitated as a creamy white solid (pH 6), which was filtered
off and dried. White solid. Yield 60ꢀ67%. Compound chars without
melting at 220 °C (for analytical data, see Supporting Information).
[(8R)-7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f]-
[3]benzazecin-8-yl]methanol ((ꢀ)-14). (ꢀ)-14 was purified by
column chromatography, dichloromethane/methanol (11:1). Yellowish
white solid. Yield 66.5%. Mp 83ꢀ85 °C (for analytical data, see
Supporting Information).
[(8S)-7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f]-
[3]benzazecin-8-yl]methanol ((+)-14). (+)-14 was purified by
column chromatography, dichloromethane/methanol (11:1). Yellowish
white solid. Yield 62.6%. Mp 83ꢀ85 °C (for analytical data, see
Supporting Information).
General Procedure for the Preparation of the Quinolizine
Derivatives 3, 8, and 9 Starting from the Respective Amines. A
solution of the respective arylethylamine (α-methyltryptamine, methyl D-
or L-tryptophanate) (1 mmol), 2-(2-bromoethyl)benzaldehyde (1.2 mmol),
and trifluoroacetic acid (1 mmol) in dioxane was refluxed under nitrogen
for 3 (tryptophanate esters) to 6 h (methyltryptamine). The solvent was
then evaporated under reduced pressure, the residue basified with 2 N
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General Procedure for the Reduction of the Ester Function.
To a suspension of lithium aluminum hydride (2 mmol) in dry THF was
added a solution of the respective ester (1 mmol) in dry THF, and the
mixture was stirred at room temperature for 90 min (heated under reflux for
3 h for compound rac-14). Excess lithium aluminum hydride was carefully
quenched with methanol. The mixture was filtered off, and the filtrate was
evaporated under reduced pressure.
pharmacological assays. Furthermore, we thank the DFG for
the financial support of the project (Grant EN 875/1-1).
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[(8RS)-7-Methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f]-
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antagonist. J. Med. Chem. 2006, 49, 2110–2116.
(15) Wittig, T. W.; Decker, M.; Lehmann, J. Dopamine/serotonin
receptor ligands. 9. Oxygen-containing midsized heterocyclic ring
systems and nonrigidized analogues. A step toward dopamine D5
receptor selectivity. J. Med. Chem. 2004, 47, 4155–4158.
(8S,14bRS)-8-Methyl-5,6,8,9,14,14b-hexahydroindolo-
[20,30:3,4]pyrido[2,1-a]isoquinoline (19). To an ice-cold solution
of 15 (1.7 mmol) and triethylamine (2 mmol) in 30 mL of dichloro-
methane was slowly added mesyl chloride (2.5 mmol). The mixture was
then stirred for 12 h, allowing it to reach room temperature. The mixture
was then washed with 100 mL of 0.1 N HCl, then with 100 mL of dilute
K2CO3 solution, and finally with 100 mL of brine. The organic phase was
evaporated under reduced pressure leaving a yellow solid, which was
used without further purification.
To a suspension of 100 mg of lithium aluminum hydride was added a
solution of the crude mesylate derivative in 20 mL of dry THF. The
mixture was heated under reflux for 2 h. Excess lithium aluminum
hydride was carefully quenched with a saturated solution of potassium
sodium tartarate. The mixture was filtered off and the filtrate was
evaporated under reduced pressure, leaving a creamy white solid.
Crystallization from isopropanol/hexane yielded a pale yellow com-
pound. Yield 88%. Mp: 75ꢀ77 °C.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
physical and spectral data for some target and intermediary
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +49 3641 949803. Fax: +49 3641 949802. E-mail:
j.lehmann@uni-jena.de.
’ ACKNOWLEDGMENT
We thank B€arbel Schmalwasser, Petra Wiecha, and Heidi
Traber for skillful technical assistance in performing the
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