Synthesis and pharmacological evaluation of 1,2,3,4-tetrahydropyrazino[1,2-a]indole and 2-[(phenylmethylamino)methyl]-1H-indole analogues as novel melatoninergic ligands

Article abstract of DOI:10.1016/j.bmc.2009.04.068

Two novel series of melatonin-derived compounds have been synthesized and pharmacologically evaluated at the MT₁ and MT₂ subtypes of melatonin receptors. Compounds 12b-c are non-selective high-affinity MT₁ and MT₂

Full text of DOI:10.1016/j.bmc.2009.04.068

Bioorganic & Medicinal Chemistry 17 (2009) 4583–4594
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of 1,2,3,4-tetrahydropyrazino
[1,2-a]indole and 2-[(phenylmethylamino)methyl]-1H-indole analogues
as novel melatoninergic ligands
Christian Markl a, Mohamed I. Attia a, Justin Julius b, Shalini Sethi b, Paula A. Witt-Enderby b,
Darius P. Zlotos a,
*
a Institute of Pharmacy and Food Chemistry, Pharmaceutical Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
b Division of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, 421 Mellon Hall, Pittsburgh, PA 15282, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel series of melatonin-derived compounds have been synthesized and pharmacologically evalu-
ated at the MT1 and MT2 subtypes of melatonin receptors. Compounds 12b–c are non-selective high-
affinity MT1 and MT2 receptor ligands (Ki = 7–11 nM). Compound 12b had little intrinsic activity at the
MT1 receptor and no intrinsic activity at the MT2 receptor. Compound 20d displayed the highest MT2
binding affinity (Ki = 2 nM) and moderate selectivity toward the MT2 subtype (Ki MT1/MT2 ratio = 8)
behaving as MT2 antagonist and MT1 agonist (IC50 = 112 pM). The findings help define SARs around the
positions 1 and 2 of melatonin with respect to binding affinity, MT2 selectivity, and intrinsic activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 21 November 2008
Revised 27 April 2009
Accepted 30 April 2009
Available online 5 May 2009
Keyword:
Melatonin receptor ligands
O
1. Introduction
CH3
N
H
The neurohormone melatonin 1 exerts its diverse physiological
actions mostly through activation of the two high affinity G-pro-
tein-coupled MT1 and MT2 receptors1 (Fig. 1).
MeO
N
H
An accurate characterization of melatonin receptor-mediated
functions requires MT1 and MT2 selective ligands. However, pro-
nounced subtype selectivity is still a challenge, and only recently a
limited number of selective compounds have been identified.2–4
The majority of subtype selective ligands behave as MT2 receptor
antagonists. A common structural feature in most of the of MT2-
selective antagonists is the presence of a lipophilic substituent
located out-of-the plane of their core nucleus in a position corre-
sponding to positions 1 and 2 in melatonin.5 The most recently
published series include N-(3,3-diphenylpropenyl)alkanamides,6
N-substituted (anilinoethyl)amides,7 3-phenylnaphthalenic deriv-
atives,8 and 2-[(2,3-dihydro-1H-indol-1-yl)methyl]melatonin ana-
logues,9 However, there are only limited studies exploring the
steric and electronic properties of the hydrophobic binding pocket
accommodating this lipophilic group.8–13 In the course of our stud-
ies on novel ligands for melatonin receptors, we have recently syn-
thesized rigid pentacyclic compounds 2a–c (Fig. 2) possessing an
indoline moiety attached to the positions 1 and 2 of melatonin.14,15
While the non-methoxy derivative 2a displayed only micromolar
1
Figure 1.
Ki = 1.7
affinity for MT2 receptors (Ki = 410 nM) being 4.4-fold higher than
for the MT1 subtype (Ki = 1.8 M). Removal of the methoxy group
lM), the dimethoxy analogue 2b exhibited nanomolar
l
from the indoline moiety of 2b led to a sixfold increase in binding
affinity at both receptor subtypes (compound 2c MT1: Ki = 320 nM;
MT2: Ki = 65 nM).15 The rather poor selectivity and moderate bind-
ing of 2b–c can be most likely explained by the bulkiness and/or
the unfavorable spatial orientation of the indoline moiety, which
is, due to the nearly planar geometry of the dihydropyrazino-diin-
dole ring system, not able to occupy the lipophilic binding pocket
of the MT2 receptors. In this paper, we report the synthesis of
two novel series of melatoninergic ligands formally derived from
compound 2c by a removal of the indoline benzene ring and by a
simultaneous cleavage of the central piperazine ring and the indo-
line moiety, respectively, as shown in Figure 2. Additionally, the
piperazine ring of the tricyclic tetrahydro[1,2-a]indole scaffold
was expanded to the seven-membered 1,4-diazepane skeleton
and cleaved to give the corresponding ethyl(methyl)-aminomethyl
binding affinity for both receptor subtypes (MT1: Ki = 1 lM; MT2:
* Corresponding author. Tel.: +49 931 888 5489; fax: +49 931 888 5494.
E-mail address: zlotos@pzlc.uni-wuerzburg.de (D.P. Zlotos).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.068

4584
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
MeO
R
R
R1
O
ii
O
i
N
H
CH3
N
H
CH3
N
N
CO2Me
N
H
CO2Me
N
N
3a,b
CH3
4a,b
CN
N
R
MeO
R
O
R2
iii
iv
N
H
CH3
2a R1 = R2 = H
2b R1 = R2 = OMe
2c R1 = OMe, R2 = H
N
O
N
H
N
NH
N
NH
5a,b
H3C
6a,b
R
R
R
8
9
Figure 2.
7
10
1
v
vi
6
N
N
NCH3
substituted analogue. The novel melatonin-derived compounds
help define SARs around the positions 1 and 2 of melatonin with
respect to binding affinity, MT2 subtype selectivity, and intrinsic
activity.
4
NCbz
3
7a,b
8b
vi
R
R
2. Results and discussion
2.1. Chemistry
NMe2
CN
vii
viii
N
N
We chose the non-methoxy tricyclic compound 12a as our ini-
tial target in order to establish a viable route to the more biologi-
cally relevant melatonin-derived molecules 12b,c. The synthetic
route applied for the preparation of 12a is shown in Scheme 1.
The key intermediate is the unsubstituted tetrahydropyrazi-
no[1,2-a]indole 6a. Guandalini et al. obtained 6a by N-cyanome-
thylation of indole-2-carboxylic acid ethyl ester and subsequent
reductive cyclization using LiAlH4 in only 24%.16 The low yield
was due to the formation of the corresponding aminoalcohol
resulting from simultaneous reduction of both cyano and ester
moieties. In order to avoid the formation of this undesired by-
product, we applied catalytic hydrogenation for the crucial cycliza-
tion step. Thus, the cyanomethylated indole-2-carboxylic acid
methyl ester 4a was selectively reduced to the corresponding pri-
1
1
NR
NR
10a: R1=Cbz
10b: R1=CH3
9a: R1=Cbz
9b: R1=CH3
R
R
O
N
H
NH2
ix
3
R
N
N
NCH3
NCH3
12a: R3 = nPr
12b: R3 = nPr
11a,b
mary ammonium salt using H2 (25 hPa) Pd/C in methanol/HClconcd
.
12c: R3 = CH3, R = OCH3
The spontaneous ring closure proceeded by adding aqueous
ammonia to yield lactam 5a (76%) which was reduced to the key
intermediate 6a (95%) using LiAlH4. For the introduction of the
melatonin-like ethylamine side chain our standard procedure
involving a sequence of a Mannich reaction, quaternization of the
Mannich base, substitution of the trimethylamine moiety by a cya-
nide, and a final reduction of the cyanomethyl group to the ethyl-
amine moiety was applied.14,15 In order to avoid methylation of the
secondary amino group of the piperazine ring of 6a, the latter was
protected by a Cbz group prior to the aminomethylation step. Man-
nich reaction of the Cbz-protected tricycle 7a using dimethylme-
thyleniminium iodide, quaternization of the Mannich base 9a
with methyl iodide, and subsequent substitution of the trimethy-
lammonium group with potassium cyanide provided nitrile 10a.
The simultaneous reduction of the cyano and the Cbz groups using
LiAlH4 yielded the crude amine 11a which was subjected to N-acyl-
ation using butyric anhydride to give butyramide 12a.
a: R = H
b: R = OCH3
Scheme 1. Reagents and conditions: (i) (1) KOtBu, DMF; (2) chloroacetonitrile
65 °C–rt; (ii) 5a: (1) 25 hPa H2, 10% Pd/C, MeOH, HClconcd, rt; (2) 25% NH3; 5b:
NaBH4, CoCl2, MeOH/THF, rt; (iii) LiAlH4, Et2O, THF, 0 °C–reflux; (iv) CbzCl, 2 M
NaOH, dioxane, 2 M Na2CO3, 0 °C; (v) LiAlH4, Et2O, THF, reflux; (vi) (CH2@NMe2)+Iꢀ,
CH2Cl2, reflux; (vii) (1) MeI, CH2Cl2, rt; (2) KCN, dicyclohexyl-[18]-crown-[6], MeCN,
reflux; (viii) 11a: LiAlH4, Et2O, THF, 0 °C–reflux; 11b: NaBH4, CoCl2, MeOH/THF, rt;
(ix) butyric anhydride or acetic anhydride, Et3N, CH2Cl2, 0 °C–rt.
Cyanomethylation of the latter provided the nitrile 4b. The reduc-
tive cyclization of 4b applying similar reaction conditions as for the
synthesis of 4a (25 hPa H2, 10% Pd/C) yielded the lactam 5b in only
49%. A better yield of 5b (60%) could be achieved by using cobalt-
(II)-chloride/sodium borohydride as reducing agent.17 After LiAlH4
reduction, the resulting amine 6b was N-protected to give the car-
bamate 7b. Unfortunately, after treatment of 7b with Eschenmo-
ser’s salt, we were unable to isolate the desired pure Mannich
For the synthesis of the 8-methoxy-tetrahydropyrazino[1,2-
a]indole analogs 12b,c, 5-methoxy-indole-2-carboxylic acid
methyl ester 3b was used as starting material (see Scheme 1). N-

C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
4585
product. An alternative approach involving reduction of the Cbz
MeO
MeO
moiety and subsequent Mannich reaction proved to be successful.
Thus, treatment of 7b with LiAlH4 and aminomethylation of the
resulting amine 8b gave the Mannich base 9b. The subsequent
quaternization with methyl iodide proceeded regioselectively
affecting exclusively the sterically less hindered N,N-dimethyl-
methylamine side chain. Nucleophilic substitution of the resulting
trimethyl ammonium group by potassium cyanide provided the
cyanomethyl analogue 10b which was converted to the corre-
sponding ethylamine 11b by means of NaBH4/CoCl2 reduction. Fi-
nally, the crude 11b was acylated using butyric and acetic
anhydride to give the respective melatoninergic ligands 12b and
12c. Another approach toward the target compound 12c starting
from 2-[(N-benzyl-N-methyl)aminomethyl]melatonin 20f is dis-
cussed later (see Scheme 3).
i
NMe2
N
CO2H
N
CO2Me
H
14
H
13
ii
iii
MeO
MeO
CN
O
N
N
CO2H
H
N
R
H
15
16a-c
H3C
For the preparation of 2-[(phenylmethylamino)-methyl]-indole
analogues 20a–f we applied two different synthetic routes dis-
played in Scheme 2. Starting from the commercially available
5-methoxyindole-2-carboxylic acid 13, the target compounds
20a–c were prepared in five steps commencing with the amide for-
mation using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDCIꢁHCl) as a coupling reagent and the appropriate N-substituted
methylamine to give N-ethyl (16a), N-p-chlorophenyl (16b), and
N-p-methoxyphenyl (16c) amides. For the subsequent Mannich–
Eschenmoser aminomethylation, more vigorous reaction condi-
tions were required due to the electron-withdrawing effect of the
amide function in position 2 of the indole ring. Satisfactory yields
were achieved using Eschenmoser’s salt in refluxing chloroform.
The Mannich bases 17a–c were converted to the cyanomethyl ana-
logues 18a–c using our standard procedure (1. MeI; 2. KCN, CH3CN,
dicylohexyl-[18]-crown[6]). For the synthesis of the cyanomethyl
derivatives 18d–f, we applied another reaction sequence using
our key intermediate 15 which was prepared via the aminomethy-
lated 5-methoxyindole-2-carboxylic acid methyl ester 14 as previ-
ously reported.9 Thus, condensation of 15 with N-methylaniline,
p-CF3-N-methylaniline, and N-methylbenzylamine provided the
amides 18d–f. Simultaneous nitrile and amide reduction using
LiAlH4 afforded the ethylamines 19a–f which were converted to
the desired melatoninergic ligands 20a–f by N-acylations using
acetic anhydride.
Starting from the benzylamine analogue 20f, we developed an-
other approach toward the 1,2,3,4-tetrahydropyrazino[1,2-a]in-
dole melatonin derivative 12c based on reductive ring closure of
the N-cyanomethyl analogue 21 outlined in Scheme 3.18
Thus, compound 21, obtained by cyanomethylation of 20f, was
subjected to catalytic hydrogenation to give 12c via partial hydroge-
nation of a nitrile function to an iminium ion followed by an intramo-
lecularnucleophilicsubstitution withthedebenzylated methylamine
acting as a nucleophile and ammonia as a leaving group.
The same strategy was applied to build the seven-membered
ring of the 2,3,4,5-tetrahydro-1H-[1,4]-diazepino[1,2-a]indole ana-
logue 23. Thus, N-cyanoethylation of compound 20f using acrylo-
nitrile provided the nitrile 22. Following the reductive cyclization
procedure, 22 was converted to the desired target compound 23
by catalytic hydrogenation.
iii
iv
MeO
MeO
NMe2
CN
vi
v
O
O
N
N
H
H
N
N
R
R
H3C
17a-c
H3C
18a-c (v) 18d-f (iii)
MeO
MeO
NH2
H
CH3
N
vii
N
O
H
N
N
H
R
N
H3C
R
H3C
19a-f
20a-f
R
a
b
c
d
e
f
Et
Cl
OMe
CF3
Scheme 2. Reagents and conditions: (i) (1) MeOH, H2SO4, reflux; (2) (CH2@NMe2)+Iꢀ,
CHCl3, reflux; (ii) (1) MeI, CH2Cl2, rt; (2) KCN, dicyclohexyl-[18]-crown-[6], MeCN,
reflux; (3) LiOH, THF, rt; (4) 2 M HClaq; (iii) HN(CH3)R, EDCIꢁHCl, CH2Cl2, rt; (iv)
(CH2@NMe2)+Iꢀ, CHCl3, reflux; (v) (1) MeI, CH2Cl2, rt; (2) KCN, dicyclohexyl-[18]-
crown-[6], MeCN, reflux; (vi) LiAlH4, Et2O, THF, 0 °C–rt; (vii) Ac2O, Et3N, CH2Cl2, 0 °C–
rt.
2.2. Pharmacology
The affinity of the target compounds for human MT1 or MT2
melatonin receptors expressed in CHO cells was measured by com-
petition binding analysis using the radioligand, 2-[125I]-iodomela-
tonin. Melatonin competition assays were run in parallel and the
affinity of melatonin for the MT1 or MT2 melatonin receptors is
in the range of the reported literature. The results are compiled
in Table 1. Compounds having the highest binding affinity and/or
selectivity (12b and 20d) were subjected to functional studies
using cyclic AMP assay in CHO cells expressing human MT1 or
MT2 receptors. The results are shown in Figure 3.
2.3. Discussion
The tricyclic 1,2,3,4-tetrahydropyrazino[1,2-a]indole analogues
12a–c derived from the pentacyclic ligands 2a–c by removal of

4586
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
MeO
MeO
H
N
CH3
H
N
CH3
O
O
N
i
ii
N
H
12c
N
CH3
Ph
N
H3C
20f
Ph
CN
21
iii
MeO
MeO
1 0
H
N
CH3
H
CH3
8
N
O
O
7
iv
1
N
N
CH3
Ph
N
N
5
CH3
3
4
23
22
CN
Scheme 3. Reagents and conditions: (i) (1) KOtBu, DMF; (2) bromoacetonitrile 65 °C–rt; (ii) H2 (7 hPa), 10% Pd/C, AcOH; (iii) H2C@CH–CN, Triton-BÒ, THF, dioxane, 0 °C–rt;
(iv) H2 (7 hPa), 10% Pd/C, AcOH.
Table 1
maximum inhibition at 10 nM. By contrast, 12b displayed no
intrinsic activity at MT2 receptors as no concentration-dependent
Binding affinitya of the target compounds for the human MT1 and MT2 receptors
expressed in CHO cells obtained in competition radioligand binding assays using 2-
inhibition of forskolin-induced cAMP accumulation occurred over
a wide range of concentrations (Fig. 3). Interestingly, the tetrahy-
dropyrido[1,2-a]indole analogue of 12b, formally obtained by
replacement of the NCH3 moiety with a CH2 group, was reported
to be a full melatoninergic agonist in the Xenopus laevis melano-
phore assay.19
Expansion of the six-membered piperazine ring of the acetam-
ide 12c to the seven-membered 1,4-diazepane ring produced a
dramatical decrease of binding affinity at both receptor subtypes.
[
125-I]-iodomelatonin
pKi MT1 SEM
pKi MT2 SEM
Melatonin
2a10
2b10
2c11
12a
12b
12c
20a
20b
20c
20d
20e
20f
23
9.34 0.10
6.00 0.01
5.74 0.20
6.50 0.03
6.59 0.03
8.18 0.03
7.93 0.04
6.00 0.20
6.59 0.12
8.09 0.06
7.81 0.19
6.96 0.02
5.89 0.04
5.25 08
9.02 0.09
5.77 0.06
6.39 0.27
7.19 0.03
7.07 0.11
8.16 0.07
8.11 0.05
6.65 0.08
7.37 0.05
8.03 0.12
8.64 0.12
7.20 0.01
6.18 0.06
6.05 0.02
The resulting compound 23 (MT1: Ki = 5.62 lM; MT2: Ki = 883 nM)
showed 480-fold and 110-fold higher binding constants at MT1 and
MT2 receptors, respectively, than the parent ligand 12c. In contrast
to the non-selective agent 12c, compound 23 displayed slight
selectivity (sixfold) for the MT2 receptors.
In order to examine whether the lacking MT2 selectivity of the
tricyclic compounds 12b and 12c is caused by the non-flexibility
of the piperazine ring, we synthesized a ring-opened analogue of
12c by formally breaking one of the C–N bonds of the piperazine
ring. The resulting compound 20a showed dramatically reduced
binding affinities for both receptor subtypes (85-fold for MT2 and
30-fold for MT1) when compared to the parent ligand 12c. Replace-
ment of the N-ethyl group of 20a by a phenyl ring generated a
compound having the best pharmacological profile of the whole
series. The resulting ligand 20d exhibited an excellent MT2 binding
affinity (Ki = 2.3 nM) being 7-times more selective for the MT2 than
for the MT1 receptors (MT1: Ki = 15.6 nM). The findings are in
agreement with the structures of the most MT2-selective ligands,
all of them possessing an aromatic ring in a position topologically
equivalent to N1–C2 region of melatonin.
Functional analysis of 20d revealed that it displayed strong ago-
nist activity at MT1 receptors with a potency value IC50 = 112 pM
and maximal (60%) inhibition at 1 lM. By contrast, 20d did not
possess intrinsic activity at MT2 receptors as no concentration-
dependent inhibition of forskolin-induced cAMP accumulation oc-
curred over wide range of concentrations (Fig. 3).
In the further optimization attempt, we substituted the benzene
ring of 20d with groups that are present in some of the most MT2-
selective agents such as chloro10 and methoxy12 groups. Unfortu-
nately, the binding affinity of the p-chloro-substituted analogue
a
pKi values were calculated from IC50 values obtained from competitive curves
according to the method of Cheng and Prusoff and are the mean of at least three
determinations performed in duplicate.
the indoline benzene ring exhibited considerably higher binding
affinities for both MT1 and MT2 receptor subtypes than the parent
compounds. Within the series, the non-methoxy butyramide 12a
displayed much lower affinities for melatonin receptors (MT1:
Ki = 256 nM; MT2: Ki = 86 nM) than the methoxy analogues 12b,c
which is in agreement with findings observed for other melatonin-
ergic ligands and confirms the importance of the methoxy group
for binding at both receptor subtypes.2 The methoxy butyramide
12b (MT1: Ki = 6.6 nM; MT2: Ki = 6.9 nM) and acetamide 12c
(MT1: Ki = 11.7 nM; MT2: Ki = 7.8 nM) are nearly equipotent high-
affinity ligands. However, compared to the slightly MT2-selective
pentacyclic ligand 2c, 12b, and 12c are not able to differentiate be-
tween MT1 and MT2 receptors indicating that the N-methylpiperi-
dine ring attached to positions 1 and 2 of melatonin is not able to
reach the lipophilic binding pocket of MT2 receptors in the region
corresponding to N1–C2 of melatonin. Even though affinity values
of 12b for binding to each of the receptors is similar, functional
analysis reveals that 12b has partial agonist activity at MT1
receptors with a potency (IC50) value of 3.14 nM and about 12%

C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
4587
Figure 3. Functional analysis of 10 nM melatonin (top graphs) to inhibit forskolin-induced cAMP accumulation in MT1-CHO cells (left graph, top) or MT2-CHO cells (right
graph, top). Bottom graphs show the results from the functional analysis for compounds 12b or 20d at MT1 receptors (left graph, bottom) or MT2 receptors (right graph,
bottom) expressed in CHO cells. Each data point represents the mean standard error of two to three independent experiments. Where appropriate, curves were fit by non-
linear regression analysis by 1-site fit to obtain potency (IC50) values using GraphPad Prism software.
20b for both receptor subtypes were reduced by a factor of approx.
17 (MT1: Ki = 257 nM; MT2: Ki = 42 nM) when compared to the
unsubstituted ligand 20d while the moderate selectivity ratio was
maintained (Ki MT1/MT2 = 6). Introduction of the more lipophilic
p-CF3 substituent further worsened the pharmacological profile
yielding a non-selective ligand 20e (MT1: Ki = 111 nM; MT2:
Ki = 63 nM). Interestingly, the p-OMe analogue 20c is a high-affinity
non-selective agent (MT1: Ki = 8 nM; MT2: Ki = 9 nM). This is in
agreement with our previous findings in the 2-[(2,3-dihydro-1H-in-
dol-1-yl)methyl]-melatonin series with the p-OMe-indoline ana-
logue showing also no subtype specificity.9 The non-selective
binding behavior of 20c is probably caused by the competition of
both methoxy groups for binding at the MT2 receptor region accom-
modating the methoxy group of melatonin inducing the unfavorable
ligand orientation. In order to probe the sterical requirements of the
hydrophobic binding pocket, we modified the structure of our most
MT2-selective agent 20d by replacing the phenyl group with a longer
benzyl substituent. The resulting ligand 20f showed dramatically
reduced binding affinities for both receptor subtypes (80-fold for
MT1, 285-fold for MT2) indicating a sterically restricted MT2-binding
pocket.
tonin derivatives 20b–f, the unsubstituted phenyl analogue 20d
had the best pharmacological profile showing high binding affinity
(Ki = 2 nM) and moderate selectivity (Ki MT1/MT2 ratio = 8) toward
the MT2 subtype. Interestingly, 20d showed strong agonist proper-
ties at the MT1 receptor with no intrinsic activity at the MT2 recep-
tor. The novel compounds help defining SARs around the positions
1 and 2 of melatonin with respect to binding affinity, MT2 subtype
selectivity, and intrinsic activity.
4. Experimental
4.1. Chemistry
Melting points were determined using a capillary melting point
apparatus (Gallenkamp, Sanyo) and are uncorrected. Column chro-
matography was carried out on Silica Gel 60 (0.063–0.200 mm) ob-
tained from Merck. Bruker AV-400 spectrometer was used to
obtain 1H NMR and 13C NMR spectra. Proton chemical shifts are re-
ferred to CHCl3 (7.24 ppm). Carbon chemical shifts are referred to
CDCl3 (77.00 ppm). The NMR resonances were assigned by means
of HH-COSY, HMQC, and HMBC experiments. EI mass spectra were
determined on a Finnigan MAT 8200, Finnigan MAT 90, and on a
ESI-microTOF spectrometers. IR spectra, recorded as ATR, were ob-
tained by using a Biorad PharmalyzIR FT-IR instrument. Elemental
analyses were performed by the microanalytical section of the
Institute of Inorganic Chemistry, University of Würzburg. All reac-
tions were carried out under an argon atmosphere.
3. Conclusions
In summary, we report the synthesis and pharmacological eval-
uation of two novel series of melatonin-derived ligands at the MT1
and MT2 subtypes of melatonin receptors. The 1,2,3,4-tetrahydro-
pyrazino[1,2-a]indole analogues 12b,c are high-affinity MT1 and
MT2 receptor ligands displaying no subtype selectivity (Ki = 7–
11 nM). Functional analysis of 12b showed that it was a partial
agonist at MT1 receptors and possessed no intrinsic activity at
MT2 receptors. From the 2-[(phenyl-methylamino)methyl]-mela-
4.1.1. General procedure for the synthesis of 4a,b
To a solution of the respective methyl ester 3a,b (6.34 mmol) in
anhydrous DMF (15 mL) potassium tert-butoxide (1.06 g,
9.45 mmol) was added at room temperature. After 40 min,

4588
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
chloroacetonitrile (0.79 mL, 12.6 mmol) was added dropwise and
the solution was heated at 65 °C for 30 min and left stirring at
room temperature for 20 h. The reaction mixture was poured onto
ice (20 g) and the mixture was extracted with ethyl acetate
(5 ꢂ 30 mL). The combined organic layers were washed with satu-
rated NaHCO3 solution (2 ꢂ 20 mL), saturated sodium chloride
solution (2 ꢂ 20 mL) and water (20 mL). The extracts were dried
(Na2SO4) and evaporated under vacuum. Recrystallization from
methanol gave 4a,b.
were washed with brine (2 ꢂ 20 mL) and dried (Na2SO4). Removal
of solvent at reduced pressure afforded a yellow solid that was
purified by silica gel chromatography (methanol–ethyl acetate–n-
pentane 1:2:2) to afford 5b (1.76 g, 60%). The spectral data of 5b
were identical to those previously reported.21
4.1.4. General procedure for the synthesis of 6a,b
A solution of the respective lactam 5a,b (8.16 mmol) in absolute
THF (50 mL) was added to a stirred suspension of LiAlH4 (625 mg,
16.5 mmol) in absolute diethyl ether (50 mL) at 0–5 °C. The reaction
mixture was refluxed for 4 h. The reaction was quenched by a slow
addition of water at 0–5 °C. The formed precipitate was filtered off
through a pad of CeliteÒ545 and washed with ethyl acetate
(300 mL). The combined organic layers were extracted with 2 M
hydrochloric acid (3 ꢂ 100 mL). The aqueous layer was separated
and basified with 25% ammonia. The resulting suspension was ex-
tracted with ethyl acetate (4 ꢂ 50 mL). Drying of the organic layer
over Na2SO4 andevaporation of thesolventaffordedthe crudeamines
6a,b which were used for the next step without further purification.
4.1.1.1. Methyl 1-(cyanomethyl)-1H-indole-2-carboxylate (4a).
Compound 4a (1.15 g, 85%) was obtained from 3a (1.11 g) as pale
yellow needles; mp: 117 °C; 1H NMR (400 MHz, CDCl3) d = 3.92 (s,
3H, OCH3), 5.56 (s, 2H, CH2CN), 7.22 (ddd, J = 1.4, 6.6, 7.9 Hz, 1H, H-
6), 7.35 (d, J = 1.0 Hz, 1H, H-3), 7.38–7.40 (m, 1H, H-7), 7.41–7.45
(m, 1H, H-5), 7.69 ppm (d, J = 7.3, 1H, H-4); 13C NMR (100 MHz,
CDCl3): d = 32.28 (CH2CN), 52.07 (OCH3), 109.59 (C-3), 112.79 (C-
7), 114.85 (CN), 122.05 (C-4), 123.23 (C-6), 126.26 (C-2), 126.35
(C-3a), 126.47 (C-5), 138.72 (C-7a), 162.26 ppm (C@O); IR:
m
= 2950, 2836, 1703, 1520, 1437 cmꢀ1; MS (EI, 70 eV) m/z (%): 214
(83) [M+], 199 (100), 183 (23), 182 (35), 154 (39), 143 (26), 128
(20), 127 (16), 115 (21). Anal. Calcd for C12H10N2O2: C, 67.28; H,
4.71; N, 13.08. Found: C, 67.62; H, 4.71; N, 13.03.
4.1.4.1. 1,2,3,4-Tetrahydropyrazino[1,2-a]indole (6a). Compound
6a (1.35 g, 95%) was obtained from 5a (1.52 g) as a colorless viscous
oil that crystallized after 5 min. The spectral data of 6a were identical
to those previously reported.22
4.1.1.2. Methyl 1-(cyanomethyl)-5-methoxy-1H-indole-2-carboxyl-
ate (4b). Compound 4b (1.29 g, 83%) was obtained from 3b
(1.11 g) as pale brown needles; mp: 131–132 °C; 1H NMR
(400 MHz, CDCl3): d = 3.81 (s, 3H, C-5-OCH3), 3.90 (s, 3H, CO2CH3),
5.51 (s, 2H, CH2CN), 7.06–7.07 (m, 1H, H-3), 7.06–7.07 (m, 1H, H-4),
7.23 (d, J = 8.8 Hz, 1H, H-7), 7.27 ppm (d, J = 8.8 Hz, 1H, H-6); 13C
NMR (100 MHz, CDCl3): d = 32.38 (CH2CN), 51.99 (CO2CH3), 55.69
(OCH3), 109.59 (C-3), 103.36 (C-4), 110.35 (C-7), 112.21 (C-6),
114.89 (CN), 126.53 (C-2), 126.80 (C-3a), 134.06 (C-7a), 155.55
4.1.4.2. 8-Methoxy-1,2,3,4-tetrahydropyrazino[1,2-a]indole (6b).
Compound 6b (1.55 g, 94%) was obtained from 5b (1.77 g) as a
pale brown solid. The spectral data of 6b were identical to those
previously reported.22
4.1.5. General procedure for the synthesis of 7a,b
The respective amine 6a,b (16.4 mmol) was dissolved in a 4:1
mixture of 2 M Na2CO3/1,4-dioxane (83 mL) and the solutions of
benzyl chloroformate (5.63 mL, 40.1 mmol) in 1,4-dioxane (34 mL)
and 2 M aqueous NaOH (20.7 mL, 41.4 mmol) were added dropwise
and simultaneously under ice-cooling. After 3.5 h of stirring, the or-
ganic solvent was removed under reduced pressure and the mixture
was extracted with CH2Cl2 (4 ꢂ 100 mL). Drying of the combined or-
ganic layers (Na2SO4) and removal of the solvent gave 7a,b. Analyt-
ical samples were obtained by recrystallization from methanol.
(C-5), 162.19 ppm (C@O); IR:
m = 2999, 2924, 2824, 2853, 1626,
1526, 1439 cmꢀ1; MS (EI, 70 eV) m/z (%): 244 (98) [M+], 229
(100), 201 (14), 184 (20), 173 (12), 158 (14), 142 (11), 141 (15).
Anal. Calcd for C13H12N2O3: C, 62.93; H, 4.95; N, 11.47. Found: C,
62.81; H, 4.96; N, 11.40.
4.1.2. 3,4-Dihydropyrazino[1,2-a]indol-1(2H)-one (5a)
To a solution of 4a (1.34 g, 6.26 mmol) in MeOH (140 mL) and
concentrated hydrochloric acid (3.1 mL) were added 10% Pd/C
(310 mg) in one portion. The mixture was hydrogenated (25 hPa)
at room temperature for 24 h. The catalyst was removed by filtra-
tion and washed with methanol. The resulting green solution was
evaporated and the resulting green solid was dissolved in water
(168 mL). The solution was extracted with CH2Cl2 (3 ꢂ 50 mL)
and the aqueous layer was made basic with aqueous ammonia
(pH >10). The mixture was stirred for 80 min to accomplish ring
closure, extracted with CHCl3 (5 ꢂ 50 mL) and the extract was
dried over Na2SO4. Evaporation of the solvent afforded 5a
(882 mg, 76%) as colorless crystals. The spectral data of 5a were
identical to those previously reported.20
4.1.5.1. Benzyl 3,4-dihydropyrazino[1,2-a]indole-2(1H)-carbox-
ylate (7a). Compound 7a (4.16 g, 83%) was obtained from 6a
(2.82 g) as a pale yellow viscous oil, that crystallized after 5 h. The
spectral data of 7a were identical to those previously reported.16
4.1.5.2. Benzyl 8-methoxy-3,4-dihydropyrazino[1,2-a]indole-
2(1H)-carboxylate (7b). Compound 7b (4.41 g, 80%) was ob-
tained from 6b (3.32 g) as colorless crystals: mp 122 °C; 1H NMR
(400 MHz, CDCl3): d = 3.84 (s, 3H, OCH3), 3.96–4.11 (m, 4H, H-3,
H-4), 4.87 (s, 2H, H-1), 5.20 (s, 2H, CH2Ph), 6.21 (s, 1H, H-10),
6.84 (dd, J = 8.6, 2.4 Hz, 1H, H-7), 7.04 (d, J = 2.4 Hz, 1H, H-9),
7.14 (d, J = 8.6 Hz, 1H, H-6), 7.32–7.34 ppm (m, 5H, Ph-H); 13C
NMR (100 MHz, CDCl3): d = 41.44 (C-1, C-4), 42.30 (C-3), 55.78
(OCH3), 67.58 (CH2Ph), 96.60 (C-9), 102.20 (C-10), 109.19 (C-6),
110.98 (C-7), 128.02, 128.29, 128.51 (C-Ph), 131.25 (C-5a), 132.21
(C-9a), 132.40 (C-10a), 136.25 (C-10), 154.49 (C@O), 155.13 (C-8)
4.1.3. 8-Methoxy-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one
(5b)
To a solution of 4b (3.32 g, 13.6 mmol) in absolute methanol
(160 mL) and absolute THF (80 mL) was added anhydrous cobalt
chloride (3.60 g, 28.0 mmol) followed by sodium borohydride
(2.56 g, 68.0 mmol) in small portions. Evolution of hydrogen gas
was observed and black precipitate appeared. When the addition
was complete, stirring was continued for 1 h at room temperature.
Hydrochloric acid (10%, 80 mL) was added to dissolve the black
precipitate and methanol was removed under reduced pressure.
25% Aqueous ammonia (3.4 mL) was added and the mixture was
extracted with ethyl acetate (6 ꢂ 40 mL). The combined extracts
ppm; IR:
m = 3015, 2959, 2937, 2893, 2838, 1689, 1484,
1417 cmꢀ1; MS (EI, 70 eV) m/z (%): 336 (14) [M+], 245 (100), 201
(31), 158 (10), 91 (27), 65 (7). Anal. Calcd for C20H20N2O3: C,
71.41; H, 5.99; N, 8.33. Found: C, 71.32; H, 6.02; N, 8.27.
4.1.6. 8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indole (8b)
A solution 7b (415 mg, 1.23 mmol) in absolute diethyl ether
(6.5 mL) and absolute THF (2 mL) was added dropwise to a stirred

C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
4589
suspension of LiAlH4 (94 mg, 2.48 mmol) in absolute diethyl ether
(8 mL) at 0–5 °C. The reaction mixture was refluxed for 4 h. The
reaction was quenched by a slow addition of water at 0–5 °C. The
formed precipitate was filtered off through a pad of CeliteÒ545
and washed with ethyl acetate (120 mL). The organic layer was
separated and the aqueous layer extracted with ethyl acetate
(3 ꢂ 30 mL). Drying of the combined organic layers over Na2SO4
and evaporation of the solvent afforded 8b (255 mg, 96%) as a
white solid. The spectral data of 8b were identical to those previ-
ously reported.23
dicyclohexyl-[18]-crown-[6] (2.70 g, 7.25 mmol) and potassium
cyanide (5.29 g, 8.12 mmol) were added. The resulting reaction
mixture was heated at reflux for 2.5 h. The solvent was evaporated
under reduced pressure and the residue was extracted with ethyl
acetate (3 ꢂ 100 mL). The organic layer was washed with water
(2 ꢂ 100 mL), dried (Na2SO4), and evaporated in vacuo. The residue
was purified by silica gel chromatography (CHCl3/methanol/25%
ammonia, 100:10:1) to afford 10a (2.09 g, 96%) as a red-brown vis-
cous oil. 1H NMR (400 MHz, CDCl3): d = 3.73 (s, 2H, CH2CN), 3.96–
4.01 (m, 2H, H-3), 4.04–4.13 (m, 2H, H-4), 4.88 (s, 2H, H-1), 5.20 (s,
2H, CH2Ph), 7.16–7.38 (m, 3H, H-6, H-7, H-8), 7.24 (m, 5H, Ph-H),
7.57 (d, J = 7.6 Hz, 1H, H-9) ppm; 13C NMR (100 MHz, CDCl3):
d = 12.58 (CH2CN), 29.67 (C-1), 40.93 (C-4), 41.47 (C-3), 67.87
(CH2Ph), 108.90 (C-6), 117.43 (CN), 117.73 (C-9), 120.63 (C-7),
121.92 (C-8), 126.49 (C-9a), 128.15, 128.33, 128.60 (C-Ph),
135.66 (C-10a), 136.06 (C-5a, C-10), 155.05 (C@O) ppm; IR:
4.1.7. Benzyl 10-[(dimethylamino)methyl]-3,4-dihydro pyraz-
ino[1,2-a]indole-2(1H)-carboxylate (9a)
Dimethylmethyleniminium iodide (2.09 g, 13.6 mmol) was
added to a solution of compound 7a (2.00 g, 6.53 mmol) in abso-
lute CH2Cl2 (105 mL). After heating for 2.5 h at reflux, the reaction
mixture was made basic with 25% ammonia. The organic layer was
separated, washed with water and dried over Na2SO4. The solvent
was removed in vacuo and the residue purified by silica gel chro-
matography (CHCl3/methanol/25% ammonia, 100:10:1) to give 9a
(2.29 g, 96%) as a yellow viscous oil. 1H NMR (400 MHz, CDCl3):
d = 2.26 (s, 6H, N(CH3)2), 3.56 (s, 2H, CH2N(CH3)2), 3.90–4.14 (m,
4H, H-3, H-4), 4.91 (s, 2H, H-1), 5.21 (s, 2H, CH2Ph), 7.11–7.23
(m, 2H, H-7, H-8), 7.25 (m, 5H, Ph-H), 7.32–7.42 (m, 1H, H-6),
7.65 (d, J = 7.8 Hz, 1H, H-9) ppm; 13C NMR (100 MHz, CDCl3):
d = 30.83 (C-1), 41.44 (C-4), 41.66 (C-3), 45.24 (N(CH3)2), 53.05
(CH2N(CH3)2), 67.57 (CH2Ph), 106.85 (C-10), 108.42 (C-6), 118.88
(C-9), 119.97 (C-8), 121.09 (C-7), 128.16, 128.37, 128.51 (Car),
130.04 (C-9a), 136.03 (C-10a), 136.30 (C-5a, C.-10), 155.14 (C@O)
m
= 2922, 2853, 2361, 1734, 1704, 1458 cmꢀ1; HRMS-ESI m/z
[M+Na]+ calcd for C21H19N3O2Na: 368.1354, found: 368.1369.
4.1.10. (8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indol-10-yl)acetonitrile (10b)
Methyl iodide (0.09 mL, 1.46 mmol) was added to a solution of
9b (200 mg, 0.732 mmol) in absolute CH2Cl2 (6 mL). The reaction
mixture was stirred at room temperature for 2 h. The volatiles
were removed in vacuo to afford 9b methoiodide. This crude
ammonium salt was suspended in absolute acetonitrile (49 mL),
dicyclohexyl-[18]-crown-[6] (314 mg, 0.843 mmol) and potassium
cyanide (615 mg, 9.44 mmol) were added. The resulting reaction
mixture was heated at reflux for 2.5 h. The solvent was evaporated
under reduced pressure and the residue was dissolved in ethyl ace-
tate (50 mL). The organic layer was washed with water
(2 ꢂ 15 mL), dried (Na2SO4), and evaporated in vacuo. The residue
was purified by silica gel chromatography (CHCl3/methanol/25%
ammonia, 100:10:1) to afford 10b (155 mg, 83%) as a yellow vis-
cous oil. 1H NMR (400 MHz, CDCl3): d = 2.50 (s, 3H, NCH3), 2.89
(t, J = 5.6 Hz, 2H, H-3), 3.67 (s, 2H, CH2CN), 3.73 (s, 2H, H-1), 3.85
(s, 3H, OCH3), 3.94 (d, J = 5.6 Hz, 2H, H-4), 6.83 (dd, J = 8.8, 2.4 Hz,
1H, H-7), 6.97 (d, J = 2.4 Hz, 1H, H-9), 7.14 (d, J = 8.8 Hz, 1H, H-6)
ppm; 13C NMR (100 MHz, CDCl3): d = 12.51 (CH2CN), 41.85 (C-4),
45.83 (NCH3), 51.57 (C-1), 51.97 (C-3), 55.85 (OCH3), 96.15 (C-
10), 99.59 (C-9), 109.62 (C-6), 111.33 (C-7), 117.72 (CN), 127.07
(C-9a), 130.83 (C-5a), 132.41 (C-10a), 154.62 (C-8) ppm; IR:
ppm; IR:
m ;
= 3032, 2940, 2855, 2810, 1699, 1455, 1418 cmꢀ1
HRMS-ESI m/z [M+H]+ calcd for C22H26N3O2: 364.2025, found:
364.2012.
4.1.8. 1-(8-Methoxy-2-methyl-1,2,3,4-tetrahydro-pyrazino[1,2-
a]indol-10-yl)-N,N-dimethylmethanamine (9b)
Dimethylmethyleniminium iodide (185 mg, 1.00 mmol) was
added to a solution of 8b (108 mg, 0.499 mmol) in absolute CH2Cl2
(12 mL). After heating for 2.5 h at reflux the resulting white precip-
itate was filtered off, dissolved in water (150 mL) and the solution
was made basic with 25% ammonia. The resulting suspension was
extracted with CH2Cl2 (5 ꢂ 25 mL). The filtrate of the original reac-
tion mixture was basified with 25% ammonia, the organic layer
was separated and the aqueous layer was extracted with CH2Cl2
(2 ꢂ 50 mL). The combined organic layers were washed with
water, dried over Na2SO4 and evaporated in vacuo to give 9b
(103 mg, 76%) as a yellow viscous oil. The crude product was used
for the next step without further purification. 1H NMR (400 MHz,
CDCl3): d = 2.24 (s, 6H, N(CH3)2), 2.50 (s, 3H, N(CH3)), 2.88 (t,
J = 5.6 Hz, 2H, H-3) 3.47 (s, 2H, CH2N(CH3)2), 3.73 (s, 2H, H-1),
3.85 (s, 3H, OCH3) 4.03 (t, J = 5.6 Hz, 2H, H-4), 6.79 (dd, J = 8.6,
2.3 Hz, 1H, H-7), 7.08 (d, J = 2.3 Hz, 1H, H-9), 7.11 (d, J = 8.6 Hz,
1H, H-6) ppm; 13C NMR (100 MHz, CDCl3): d = 41.87 (C-4), 45.33
(N(CH3)2), 45.96 (NCH3), 52.22 (C-1), 52.38 (C-3), 53.22
(CH2N(CH3)2), 55.98 (OCH3), 101.20 (C-9), 105.65 (C-10), 109.03
(C-6), 110.31 (C-7), 129.18 (C-9a), 131.17 (C-5a), 133.40 (C-10a),
m
= 2930, 2858, 1583, 1483, 1452 cmꢀ1; HRMS-ESI m/z [M+H]+
calcd for C15H18N3O: 256.1444, found: 256.1448.
4.1.11. 2-(2-Methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indol-10-
yl)ethylamine (11a)
A solution of 10a (2.09 g, 6.05 mmol) in absolute THF (174 mL)
was added to a stirred suspension of LiAlH4 (2.94 g, 78.7 mmol) in
absolute diethyl ether (192 mL) at 0–5 °C. The reaction mixture
was refluxed for 3 h and quenched by a slow addition of water at
0–5 °C. The formed precipitate was filtered off through a pad of
CeliteÒ545 and washed with diethyl ether (500 mL). The organic
layer was separated, washed with water (2 ꢂ 60 mL) and dried
over Na2SO4. The volatiles were removed under vacuum to afford
11a as a yellow viscous oil that was used in the next step without
further purification.
154.27 (C-8) ppm; IR:
m ;
= 2930, 2858, 1583, 1483, 1452 cmꢀ1
HRMS-ESI m/z [MꢀNH(CH3)2]+ calcd for C14H17N2O: 229.1335,
found: 229.1332.
4.1.12. 2-(8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indol-10-yl)ethylamine (11b)
4.1.9. Benzyl 10-(cyanomethyl)-3,4-dihydropyrazino[1,2-a]indole-
2(1H)-carboxylate (10a)
To a solution of 10b (295 mg, 1.16 mmol) in absolute methanol
(13.5 mL) was added anhydrous cobalt chloride (306 mg,
2.38 mmol) followed by sodium borohydride (217 mg, 5.78 mmol)
in small portions. Evolution of hydrogen gas was observed and
black precipitate appeared. When the addition was completed,
stirring was continued for 1 h at room temperature. Hydrochloric
Methyl iodide (3.82 mL, 60.3 mmol) was added to a solution of
9a (2.29 g, 6.30 mmol) in absolute CH2Cl2 (20 mL). The reaction
mixture was stirred at room temperature for 15 h. The volatiles
were removed in vacuo to afford 9a methoiodide. This crude
ammonium salt was suspended in absolute acetonitrile (420 mL),

4590
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
acid (10%, 7 mL) was added to dissolve the black precipitate and
methanol was removed in vacuo. 25% Ammonia was added and
the aqueous mixture was extracted with ethyl acetate
(7 ꢂ 20 mL). The combined extracts were dried (Na2SO4) and the
volatiles were removed under vacuum to afford 11b as a yellow
viscous oil that was used in the next step without further
purification.
2363, 1641, 1483, 1452, 1432, 1229, 1164, 1040, 795 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C17H24N3O2: 302.1863, found:
302.1866.
4.1.14. General procedure for the synthesis of 16a–c
The appropriate N-methylamine (1 equiv) was added to a solu-
tion of the 5-methoxy-1H-indole-2-carboxylic acid (13) (1 equiv)
and EDCIꢁHCl (1.5 equiv) in absolute CH2Cl2 (25 mL). The mixture
was stirred for 20 h and the participated solid was collected by fil-
tration. The filtrate was concentrated under vacuum yielding some
more precipitate that was again collected by filtration. The com-
bined precipitates were washed with 2 M hydrochloric acid, satu-
rated NaHCO3 solution, and water and dried in vacuo to give the
crude amides 16a–c which were used in the next step without fur-
ther purification. Analytical samples of 16a–c were obtained by
recrystallization from isopropanol.
4.1.13. General procedure for the synthesis of 12a–c
A stirred solution of the respective amine 11a,b (1 equiv) in abso-
lute CH2Cl2 (90 mL) was treated with triethylamine (3.5 equiv) and
the respective acylation agent (2.5 equiv) at 0–5 °C. The reaction mix-
ture wasstirred at ambienttemperature for7 h. Thesolventwasevap-
orated under reduced pressure and the residue was purified by silica
gel chromatography (CHCl3–MeOH–25% NH3 100:10:1).
4.1.13.1. N-[2-(2-Methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indol-10-yl)ethyl]butanamide (12a). Compound 12a (107 mg,
10%) was obtained from 11a (810 mg) and butyric anhydride
(1.72 mL) as yellow crystals. Mp 177–178 °C (diethyl ether); 1H
NMR (400 MHz, CDCl3): d = 0.89 (t, J = 7.6 Hz, 3H, CH2CH2CH3),
1.50–1.67 (m, 2H, CH2CH2CH3), 2.02 (t, J = 7.1 Hz, 2H, CH2CH2CH3),
2.48 (s, 3H, NCH3), 2.86 (t, J = 6.5 Hz, 2H, CH2CH2N), 2.87 (t,
J = 5.3 Hz, 2H, H-3), 3.47 (q, J = 6.5 Hz, 2H, CH2CH2N), 3.68 (s, 2H, H-
1), 4.05 (t, J = 5.3 Hz, 2H, H-4), 5.59 (s br, 1H, C(O)NH), 7.06–7.18 (m,
2H, H-7, H-8), 7.22–7.26 (m, 1H, H-6), 7.15 (d, J = 7.6 Hz, 1H, H-9)
ppm; 13C NMR (100 MHz, CDCl3): d = 13.73 (CH2CH2CH3), 18.97
(CH2CH2CH3), 23.91 (CH2CH2N), 38.66 (CH2CH2N), 39.86
(CH2CH2CH3), 41.86 (C-4), 46.00 (NCH3), 52.11 (C-1), 52.46 (C-3),
105.55 (C-10), 108.64 (C-6), 118.03 (C-9), 119.48 (C-8), 120.80 (C-7),
128.03 (C-9a), 131.36 (C-10a), 135.91 (C-5a), 172.89 (C@O) ppm; IR:
4.1.14.1. N-Ethyl-5-methoxy-N-methyl-1H-indole-2-carboxam-
ide (16a). Compound 16a (0.900 g, 74%) was obtained from 13
(1.0 g) as a pale yellow solid; mp 128–130 °C; 1H NMR (400 MHz,
CDCl3): d = 1.30 (t, J = 7.1 Hz, 3H, CH3–CH2), 3.31 (s, 3H, N–CH3),
3.72 (q, J = 7.1 Hz, 2H, CH3–CH2), 3.83 (s, 3H, OCH3), 6.74 (s, 1H,
H-3), 6.93 (dd, J = 2.3, 8.8 Hz, 1H, H-6), 7.05 (d, J = 2.3 Hz, 1H, H-
4), 7.33 (d, J = 8.8 Hz, 1H, H-7), 9.88 (br, 1H, NH) ppm; 13C NMR
(100 MHz, CDCl3): d = 12.87 (CH3–CH2), 35.38 (N–CH3), 44.57
(CH3–CH2), 55.60 (OCH3), 102.13 (C-4), 104.77 (C-3), 112.71 (C-
7), 115.44 (C-6), 128.05, 130.26, 130.99 (C-2, C-3a, C-7a), 154.39
(C-5), 162.72 ppm (C@O); IR:
m ;
= 3254, 2963, 1596, 1401 cmꢀ1
MS (EI, 70 eV) m/z (%): 232 [M]+, 173 (100), 158 (21), 119 (17).
Anal. Calcd for C13H16N2O2: C, 67.15; H, 7.02; N, 11.89. Found: C,
67.22; H, 6.94; N, 12.06.
m
= 3308, 3053, 2961, 2930, 2873, 2340, 1634, 1460 cmꢀ1; HRMS-
ESI m/z [M+H]+ calcd for C18H26N3O 300.2070, found: 300.2070.
4.1.14.2. N-(4-Chlorophenyl)-5-methoxy-N-methyl-1H-indole-
2-carboxamide (16b). Compound 16b (1.00 g, 61.0%) was ob-
tained from 13 (1.00 g) as a pale yellow solid; mp 245–246 °C;
1H NMR (400 MHz, DMSO-d6): d = 3.41 (s, 3H, N–CH3), 3.71 (s,
3H, OCH3), 5.43 (d, J = 1.5 Hz, 1H, H-3), 6.84 (dd, J = 2.5, 8.8 Hz,
1H, H-6), 6.89 (d, J = 2.5 Hz, 1H, H-4), 7.34 (d, J = 8.8 Hz, 1H, H-7),
7.45 (m, 2H, Har), 7.57 (m, 2H, Har), 11.52 (br, 1H, NH) ppm; 13C
NMR (100 MHz, DMSO-d6): d = 38.34 (N–CH3), 55.11 (OCH3),
101.69 (C-4), 105.45 (C-3), 112.98 (C-7), 115.09 (C-6), 129.53,
129.63 (Car), 126.95, 130.10, 130.94 (C-2, C-3a, C-7a), 132.06,
4.1.13.2. N-[2-(8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazi-
no[1,2-a]indol-10-yl)ethyl]butanamide (12b). Compound 12b
(446 mg, 38%) was obtained from 11b (923 mg) and butyric anhy-
dride (1.54 mL) as yellow viscous oil; 1H NMR (400 MHz, CDCl3):
d = 0.81 (t, J = 7.3 Hz, 3H, CH2CH2CH3), 1.51–1.63 (m, 2H,
CH2CH2CH3), 2.03 (t, J = 7.6 Hz, 2H, CH2CH2CH3), 2.46 (s, 3H,
NCH3), 2.75–2.87 (m, 4H, CH2CH2N, H-3), 3.46 (q, J = 6.9 Hz, 2H,
CH2CH2N), 3.63 (s, 2H, H-1), 3.81 (s, 3H, OCH3), 3.98 (t, J = 5.5 Hz,
2H, H-4), 5.63 (br s, 1H, NH), 6.79 (dd, J = 8.8, 2.3 Hz, 1H, H-7),
6.95 (d, J = 2.2 Hz, 1H, H-9), 7.10 (d, J = 8.8 Hz, 1H, H-6) ppm; 13C
NMR (100 MHz, CDCl3): d = 3.69 (CH2CH2CH3), 18.97 (CH2CH2CH3),
23.89 (CH2CH2N), 38.63 (CH2CH2N), 39.53 (CH2CH2CH3), 41.87 (C-
4), 45.92 (NCH3), 52.04 (C-1), 52.38 (C-3), 55.91 (OCH3), 100.38
(C-9), 105.23 (C-10), 109.28 (C-6), 110.49 (C-7), 128.38 (C-9a),
131.26 (C-5a), 132.08 (C-10a), 154.19 (C-8), 172.81 (C@O) ppm;
IR: 3283, 2931, 2853, 2791, 1643, 1549, 1483 cmꢀ1; HRMS-ESI m/
z [M+H]+ calcd for C19H28N3O2: 330.2176, found: 330.2175.
143.31 (Car), 153.67 (C-5), 161.43 (C@O) ppm; IR:
m = 3330, 2950,
1610, 1586, 1390 cmꢀ1; MS (EI, 70 eV) m/z (%):314 (66 [M]+, 174
(100), 173 (95), 158 (21), 141 (45). Anal. Calcd for C17H15ClN2O2:
C, 64.52; H, 4.89; N, 8.90. Found: C, 64.87; H, 4.80; N, 8.89.
4.1.14.3. 5-Methoxy-N-(4-methoxyphenyl)-N-methyl-1H-
indole-2-carboxamide (16c). Compound 16c (2.92 g, 94%) was
obtained from 13 (1.91 g) as a pale yellow solid; mp: 211–212 °C;
1H NMR (400 MHz, CDCl3): d = 3.45 (s, 3H, NCH3), 3.74 (s, 3H, C-40-
OCH3), 3.88 (s, 3H, C-5-OCH3), 6.78 (d, J = 2.3 Hz, 1H, H-4), 6.82
(dd, J = 8.8, 2.3 Hz, 1H, H-6), 6.95–7.01 (m, 2H, H-30, H-50), 7.16–
7.25 (m, 2H, H-20, H-60), 7.24 (s, 1H, H-3), 7.26 (d, J = 8.8 Hz, 1H, H-
7), 9.49 (s, 1H, NH); 13C NMR (100 MHz, CDCl3): d = 39.13 (NCH3),
56.57 (C-5-OCH3), 56.60 (C-40-OCH3), 102.30 (C-4), 106.61 (C-6),
112.45 (C-7), 115.03 (C-30), 115.96 (C-3), 127.26 (C-10), 129.06 (C-
20), 130.09 (C-3a), 130.65 (C-7a), 136.92 (C-2), 154.28 (C-5), 159.38
4.1.13.3. N-[2-(8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazi-
no[1,2-a]indol-10-yl)ethyl]acetamide (12c). Compound 12c
(397 mg, 37%) was obtained from 11b (923 mg) and acetic anhy-
dride (0.89 mL) as a yellow viscous oil; 1H NMR (400 MHz, CDCl3):
d = 1.87 (s, 3H, C(O)CH3), 2.47 (s, 3H, NCH3), 2.81 (t, J = 6.5 Hz, 2H,
CH2CH2N), 2.84 (t, J = 5.6 Hz, 2H, H-3), 3.42 (q, J = 6.5 Hz, 2H,
CH2CH2N), 3.64 (s, 2H, H-1), 3.82 (s, 3H, OCH3), 3.99 (t, J = 5.6 Hz,
2H, H-4), 5.67 (br s, 1H, NH), 6.79 (dd, J = 8.6, 2.3 Hz, 1H, H-7),
6.96 (d, J = 2.3 Hz, 1H, H-9), 7.11 (d, J = 8.6 Hz, 1H, H-6) ppm; 13C
NMR (100 MHz, CDCl3): d = 23.29 (C(O)CH3), 23.77 (CH2CH2N),
39.90 (CH2CH2N2), 41.89 (C-4), 45.94 (NCH3), 52.05 (C-1), 52.40
(C-3), 55.98 (OCH3), 100.34 (C-9), 105.24 (C-10), 109.37 (C-6),
110.56 (C-7), 128.41 (C-9a), 131.28 (C-5a), 132.06 (C-10a),
(C-40), 162.10 ppm (C@O); IR:
m = 3273, 1618, 1597, 1509,
1390 cmꢀ1; MS (EI, 70 eV) m/z (%): 310 (26) [M]+, 174 (11), 137
(100), 122 (21), 119 (11). Anal. Calcd for C20H19N3O2: C, 69.66; H,
5.85; N, 9.03. Found: C, 69.47; H, 5.81; N, 8.97.
4.1.15. General procedure for the synthesis of 17a–c
N,N-Dimethylmethyleniminium iodide (2.5 equiv) was added to
a solution of the respective amide 16a–c (1 equiv) in absolute
154.23 (C-8), 170.02 (C@O) ppm; IR:
m = 3286, 2933, 2849, 2730,

C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
4591
CHCl3 (50 mL). After heating for 16 h at reflux, the reaction mixture
was made basic with 25% ammonia. The organic layer was sepa-
rated, washed with water and dried over MgSO4. The solvent was
removed in vacuo to give the crude products 17a–c which were
pure enough to be used in the next step without further purifica-
tion as indicated by 1H NMR.
CH3–CH2), 2.94 (s, 3H, N–CH3), 3.39 (q, J = 7.1 Hz, 2H, CH3–CH2),
3.76 (s, 2H, CH2–CN), 3.77 (s, 3H, OCH3), 6.82 (dd, J = 2.3, 8.8 Hz,
1H, H-6), 6.94 (d, J = 2.3 Hz, 1H, H-4), 7.12 (d, J = 8.8 Hz, 1H, H-7),
9.39 ppm (br, 1H, NH); 13C NMR (100 MHz, CDCl3): d = 12.88
(CH3–CH2), 13.78 (CH2–CN), 34.79 (N–CH3), 44.15 (CH3–CH2),
55.92 (OCH3), 100.20 (C-4), 104.07 (CN), 113.15 (C-7), 115.39 (C-
6), 117.49 (C-3), 126.58, 129.44, 130.86 (C-2, C-3a, C-7a), 154.99
(C-5), 164.30 ppm (C@O); MS (EI, 70 eV) m/z (%): 271 (61) [M]+,
4.1.15.1. 3-[(Dimethylamino)methyl]-N-ethyl-5-methoxy-N-meth-
yl-1H-indole-2-carboxamide (17a). Compound 17a (0.60 g, 75%)
212 (100), 184 (31); IR:
Anal. Calcd for C15H17N3O2: C, 66.40; H, 6.32; N, 15.49. Found: C,
m .
= 3263, 2965, 2221, 1611, 1548 cmꢀ1
1
was obtained from 16a (0.64 g) as a pale yellow viscous oil; H NMR
(400 MHz, CDCl3): d = 1.15 (t, J = 6.7 Hz, 3H, CH3–CH2), 2.19 (s, 6H,
NMe2), 3.00 (s, 3H, N–CH3), 3.48 (q, J = 6.7 Hz, 2H, CH3–CH2), 3.59 (s,
2H, CH2–NMe2), 3.81 (s, 3H, OCH3), 6.83 (dd, J = 2.3, 8.8 Hz, 1H, H-6),
7.17–7.19 (m, 2H, H-4, H-7), 9.29 ppm (br, 1H, NH); 13C NMR
(100 MHz, CDCl3): d = 12.66 (CH3–CH2), 35.17 (N–CH3), 44.36 (CH3–
CH2), 45.22 (NMe2), 54.23 (CH2–NMe2), 55.77 (OCH3), 101.80 (C-4),
111.84 (C-3), 112.19 (C-7), 113.78 (C-6), 127.97, 130.14, 130.94 (C-2,
C-3a, C-7a), 154.12 (C-5), 165.39 ppm (C@O).
66.01; H, 5.95; N, 15.19.
4.1.16.2. N-(4-Chlorophenyl)-3-(cyanomethyl)-5-methoxy-N-
methyl-1H-indole-2-carboxamide
(18b). Compound
18b
(0.16 g, 56%) was obtained from 17b (0.30 g) after purification by
silica gel chromatography (ethyl acetate) as a light red viscous
oil. 1H NMR (400 MHz, CDCl3): d = 3.48 (s, 3H, N–CH3), 3.78 (s,
2H, CH2–CN), 3.81 (s, 3H, OCH3), 6.89 (dd, J = 2.3, 8.8 Hz, 1H, H-
6), 6.94 (d, J = 2.3 Hz, 1H, H-4), 7.06 (m, 1H, H-7), 7.09 (m, 2H,
Har), 7.28 (m, 2H, Har), 8.17 ppm (br, 1H, NH); 13C NMR
(100 MHz, CDCl3): d = 3.79 (CH2–CN), 38.37 (N–CH3), 55.69
(OCH3), 99.64 (C-4), 107.88 (CN), 112.86 (C-7), 116.58 (C-6),
117.62 (C-3), 126.25, 128.0, 130.51 (C-2, C-3a, C-7a), 127.15,
129.23, 133.04, 142.02 (Car), 154.95 (C-5), 162.92 (C@O) ppm; m/
4.1.15.2. N-(4-Chlorophenyl)-3-[(dimethylamino)methyl]-5-meth-
oxy-N-methyl-1H-indole-2-carboxamide (17b). Compound 17b
(0.42 g, 89.0%) was obtained from 16b (0.40 g) as a light brown viscous
oil; 1H NMR (400 MHz, CDCl3): d = 2.14 (s, 6H, NMe2), 3.27 (s, 2H, CH2–
NMe2), 3.43 (s, 3H, N–CH3), 3.76 (s, 3H, OCH3), 6.79 (dd, J = 2.5, 8.8 Hz,
1H, H-6), 7.02 (ddd, J = 2.0, 3.0, 8.6 Hz, 2H, Har), 7.06 (m, 2H, H-4, H-7),
7.15 (ddd, J = 2.0, 3.0, 8.6 Hz, 2H, Har), 8.62 ppm (br, 1H, NH); 13C NMR
(100 MHz, CDCl3): d = 38.03 (N–CH3), 45.36 (NMe2), 53.79 (CH2–
NMe2), 55.71 (OCH3), 101.81 (C-4), 112.19 (C-7), 114.82 (C-3), 114.92
(C-6), 126.87 (ArCH), 127.97, 129.11, 130.96, (C-2, C-3a, C-7a), 129.23
(Car), 131.94, 142.53 (Car), 154.19 (C-5), 164.91 ppm (C@O).
z (EI): 353 (60, M+), 212 (63), 141 (100); IR:
m = 3283, 3059,
2939, 2198, 1625, 1458 cmꢀ1; MS (EI, 70 eV) m/z (%): 353 (60)
[M]+, 212 (63), 141 (100). Anal. Calcd for C19H16ClN3O2: C, 64.50;
H, 4.56; N, 11.88. Found: C, 63.89; H, 4.14; N, 11.78.
4.1.16.3. 3-(Cyanomethyl)-5-methoxy-N-(4-methoxyphenyl)-N-
methyl-1H-indole-2-carboxamide (18c). Compound 18c (1.34 g,
90%) was obtained from 17c (1.56 g) after purification by silica gel
chromatography (CH2Cl2–MeOH–25% NH3 160:10:1) as colorless
crystals. Analytical sample was obtained by recrystallization from
t-butyl methyl ether; mp: 126 °C; 1H NMR (400 MHz, CDCl3):
d = 3.44 (s, 3H, NCH3), 3.77 (s, 3H, C-40-OCH3), 3.81 (s, 3H, C-5-
OCH3), 3.94 (s, 2H, CH2CN), 6.82–6.90 (m, 2H, H-30, H-50), 6.95–
7.02 (m, 3H, H-4, H-6, H-7), 7.08–7.15 (m, 2H, H-20, H-60),
7.72 ppm (br s, 1H, NH); 13C NMR (100 MHz, CDCl3): d = 13.86
(CH2CN), 38.73 (NCH3), 55.53 (C-40-OCH3), 55.70 (C-5-OCH3),
99.65 (C-4), 108.73 (CN), 112.70 (C-7), 115.23 (C-30), 116.34 (C-
6), 117.95 (C-2), 126.23 (C-3), 127.54 (C-20), 128.14 (C-3a),
130.30 (C-7a), 136.15 (C-10), 154.79 (C-5), 156.63 (C-40),
4.1.15.3. 3-[(Dimethylamino)methyl]-5-methoxy-N-(4-methoxy-
phenyl)-N-methyl-1H-indole-2-carboxamide (17c). Compound
17c (572 mg, 78%) was obtained from 16c (300 mg) as a colorless
viscous oil; dH 1H NMR (400 MHz, CDCl3): d = 2.52 (s, 6H, N(CH3)2),
3.94 (s, 3H, NCH3), 3.76 (s, 3H, C-40-OCH3), 3.85 (s, 3H, C-5-OCH3),
3.97 (s, 2H, CH2N), 6.81–6.87 (m, 3H, H-6, H-30, H-50), 7.00 (d,
J = 9.1 Hz, 1H, H-7), 7.10–7.17 (m, 2H, H-20, H-60), 7.30 (d,
J = 2.0 Hz, 1H, H-4), 7.83 ppm (br s, 1H, NH); 13C NMR (100 MHz,
CDCl3): d = 38.92 (NCH3), 44.16 (N(CH3)2), 53.07 (CH2N), 55.07 (C-
5-OCH3), 56.23 (C-40-OCH3), 101.37 (C-4), 112.33 (C-6), 113.27 (C-
3), 115.02 (C-30), 116.05 (C-7), 127.57 (C-10, C-20), 129.39 (C-2),
130.43 (C-3a), 136.16 (C-7a), 154.83 (C-5), 158.66 (C-40),
163.49 ppm(C@O); IR:
m
= 3230, 2934, 2832, 3770, 1619, 1510,
158.73 ppm (C@O); IR: m = 3287 2359, 1654, 1591, 1580, 1510,
1457, 1435 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C21H26N3O3:
1456, 1436 cmꢀ1; MS (EI, 70 eV) m/z (%): 349 (19) [M]+, 137
(100), 122 (27). Anal. Calcd for C20H19N3O3: C, 68.75; H, 5.48; N,
12.03. Found: C, 68.64; H, 5.31; N, 11.93.
368.1969, found 368.1969.
4.1.16. General procedure for the synthesis of 18a–c
Methyl iodide (1.59 mL, 25.5 mmol) was added to a solution of
the appropriate Mannich base 17a–c (4.25 mmol) in absolute
CH2Cl2 (150 mL). The reaction mixture was stirred at room temper-
ature for 3 h. The volatiles were removed in vacuo to afford 17a–c
methoiodides. This crude ammonium salt was suspended in abso-
lute acetonitrile (170 mL). Dicyclohexyl-[18]-crown-[6] (2.03 g,
5.45 mmol) and potassium cyanide (3.98 g, 61.1 mmol) were
added and the resulting reaction mixture was heated at reflux for
3 h. The solvent was evaporated under reduced pressure and the
residue was extracted with ethyl acetate (2 ꢂ 50 mL). The organic
layer was washed with water (2 ꢂ 85 mL), dried (Na2SO4) and
evaporated in vacuo.
4.1.17. General procedure for the synthesis of 18d–f
The appropriate N-methyl amine (1.00 mmol) was added to a
solution of 15 (230 mg, 1.00 mmol) and EDCIꢁHCl (288 mg,
1.50 mmol) in absolute CH2Cl2 (1.5 mL). The mixture was stirred
for 14 h and poured into 2 M hydrochloric acid (8 mL). The phases
were separated und the aqueous layer was extracted with CH2Cl2
(3 ꢂ 8 mL). The combined organic layers were washed with NaH-
CO3 solution and water. After drying (Na2SO4) the solvent was
evaporated under reduced pressure and the residue was subjected
to silica gel chromatography (ethyl acetate for 18d, ethyl acetate/
n-pentane 3:2 for 18e. 18f was used without further purification).
Analytical samples of 18d–f were obtained by recrystallization
from t-butyl methyl ether.
4.1.16.1. 3-(Cyanomethyl)-N-ethyl-5-methoxy-N-methyl-1H-
indole-2-carboxamide (18a). Compound 18a (0.27 g, 60%) was
obtained from 17a (0.48 g) after purification by silica gel chroma-
tography (CHCl3/ethyl acetate, 1:1) as a light brown solid, mp:
133–135 °C. 1H NMR (400 MHz, CDCl3): d = 1.09 (t, J = 7.1 Hz, 3H,
4.1.17.1. 3-(Cyanomethyl)-5-methoxy-N-methyl-N-phenyl-1H-
indole-2-carboxamide (18d). Compound 18d (192 mg, 60%) was
obtained from 15 (230 mg) and methylphenylamine (107 mg) as
pale red crystals; mp: 126–127 °C; 1H NMR (400 MHz, CDCl3):

4592
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
d = 3.50 (s, 3H, NCH3), 3.80 (s, 3H, OCH3), 3.88 (s, 2H, CH2CN), 6.86
(dd, J = 2.5, 8.8 Hz, 1H, H-6), 6.95–7.00 (m, 2H, H-4, H-7), 7.15–7.38
(m, 5H, Har), 7.75 ppm (br s, 1H, NH); 13C NMR (100 MHz, CDCl3):
d = 13.82 (CH2CN), 38.46 (NCH3), 55.71 (OCH3), 99.69 (C-4),
112.67 (C-7), 116.48 (C-6), 117.83 (CN), 126.24 (C-3), 126.26 (C-
20, C-60), 127.60 (C-40), 128.09 (C-2), 130.06 (C-30, C-50), 130.35
(C-3a), 132.54 (C-7a), 143.51 (C-10), 154.85 (C-5), 162.57 ppm
and acetic anhydride (2.5 equiv) at 0–5 °C. The reaction mixture
was stirred at ambient temperature for 3 h. The solvent was evap-
orated and the residue was purified by silica gel chromatography.
4.1.19.1. N-[2-(2-{[Ethyl(methyl)amino]methyl}-5-methoxy-
1H-indol-3-yl)ethyl]acetamide (20a). Compound 20a (0.10 g,
41%, elution with CHCl3–MeOH–25% NH3, 100:10:1) was obtained
from 19a (0.21 g) as a light brown viscous oil; 1H NMR (400 MHz,
CDCl3): d = 1.13 (t, J = 7.1 Hz, 3H, CH3–CH2), 1.84 (s, 3H, C(O)CH3),
2.20 (s, 3H, N–CH3), 2.53 (q, J = 7.1 Hz, 2H, CH3–CH2), 2.89 (t,
J = 6.3 Hz, 2H, CH2–CH2–N), 3.44–3.48 (m, 2H, CH2–CH2–N), 3.56
(s, 2H, CH2–N–CH3), 3.81 (s, 3H, OCH3), 6.79 (dd, J = 2.3, 8.8 Hz,
1H, H-6), 6.93 (br, 1H, NH), 6.95 (d, J = 2.3 Hz, 1H, H-4), 7.19 (d,
J = 8.8 Hz, 1H, H-7), 8.63 ppm (br, 1H, NH); 13C NMR (100 MHz,
CDCl3): d = 11.99 (CH3–CH2), 22.99 (C(O)CH3), 23.41 (CH2–CH2–
N), 40.31 (CH2–N–CH3), 41.54 (N–CH3), 51.62 (CH3–CH2), 52.26
(CH2–CH2–N), 55.90 (OCH3), 100.25 (C-4), 110.50 (C-3), 111.64
(C-7), 111.96 (C-6), 128.44, 130.51, 133.33 (C-2, C-3a, C-7a),
(C@O); IR:
m = 3269, 2360, 2330, 2623, 2604, 1623, 1604, 1582,
1538, 1493, 1456, 1434 cmꢀ1; MS (EI, 70 eV) m/z (%): 319 (49)
[M]+, 213 (28), 212 (48), 108 (19), 107 (100), 106 (19), 73 (15),
44 (16). Anal. Calcd for C19H17N3O2: C, 71.46; H, 5.37; N, 13.16.
Found: C, 71.19; H, 5.41; N, 12.95.
4.1.17.2. 3-(Cyanomethyl)-5-methoxy-N-methyl-N-[4-(trifluo-
romethyl)phenyl]-1H-indole-2-carboxamide (18e). Compound
18e (217 mg, 56%) was obtained from 15 (230 mg) and methyl-
[4-(trifluoromethyl)phenyl)] amine (175 mg) as colorless crystals;
mp: 166–167 °C; 1H NMR (400 MHz, CDCl3): d = 3.53 (s, 3H,
NCH3), 3.70 (s, 2H, CH2CN), 3.80 (s, 3H, OCH3), 6.86–6.95 (m, 2H,
H-4, H-6), 7.08 (d, J = 8.6 Hz, 1H, H-7), 7.25 (d, J = 8.3 Hz, 2H, H-
30, H-50), 7.56 (d, J = 8.3 Hz, 2H, H-20, H-60), 8.35 ppm (br s, 1H,
NH); 13C NMR (100 MHz, CDCl3): d = 13.75 (CH2CN), 38.15
(NCH3), 55.69 (OCH3), 99.64 (C-4), 112.93 (C-7), 116.79 (C-6),
117.43 (CN), 122.15 (CF3) 124.85 (C-3), 125.81 (C-20, C-60), 126.30
(C-2), 126.92 (C-30, C-50), 127.93 (C-40), 129.05 (C-3a), 130.86 (C-
153.99 (C-5), 170.27 ppm (C@O); IR:
m ;
= 3841, 2934, 1645 cmꢀ1
HRMS-ESI m/z [M+Na]+ calcd for C17H25N3O2Na: 326.1846, found:
326.1842.
4.1.19.2. N-[2-(2-{[(4-Chlorophenyl)(methyl)amino]methyl}-5-
methoxy-1H-indol-3-yl)ethyl]acetamide (20b). Compound 20b
(520 mg, 48%, elution with ethyl acetate) was obtained from 19b
(0.10 g) as a yellow powder; mp: 61–63 °C; 1H NMR (400 MHz,
CDCl3): d = 1.88 (s, 3H, C(O)CH3), 2.87 (s, 3H, N–CH3), 2.93–2.95
(m, 2H, CH2–CH2–N), 3.45–3.50 (m, 2H, CH2–CH2–N), 3.83 (s, 3H,
OCH3), 4.49 (s, 2H, CH2–N–CH3), 5.69 (br, 1H, NH), 6.76 (m, 2H,
Har), 6.79 (dd, J = 2.3, 8.8 Hz, 1H, H-6), 6.99 (d, J = 2.3 Hz, 1H, H-
4), 7.14 (m, 1H, H-7), 7.17 (m, 2H, Har), 8.05 ppm (br, 1H, NH);
13C NMR (100 MHz, CDCl3): d = 23.31 (C(O)CH3), 24.13 (CH2–CH2–
N), 38.74 (N–CH3), 40.21 (CH2–N–CH3), 49.61 (CH2–CH2–N),
55.95 (OCH3), 100.35 (C-4), 109.31 (C-3), 111.67 (C-6), 116.89 (C-
7), 115.0, 123.19, 128.94, 130.44 (C-2, C-3a, C-7a), 129.19,
133.25, 148.76 (Car), 154.20 (C-5), 170.10 (C@O) ppm; IR:
7a), 146.68 (C-10), 155.03 (C-5), 163.20 ppm (C@O); IR:
m = 3364,
2359, 2342, 1630, 1611 cmꢀ1; MS (EI, 70 eV) m/z (%): 388 (10),
387 (40) [M]+, 213 (52), 212 (100), 184 (10), 170 (12). Anal. Calcd
for C20H16F3N3O2: C, 62.01; H, 4.16; N, 10.8. Found: C, 61.97; H,
4.42; N, 10.57.
4.1.17.3. N-Benzyl-3-(cyanomethyl)-5-methoxy-N-methyl-1H-
indole-2-carboxamide (18f). Compound 18f (293 mg, 88%) was
obtained from 15 (230 mg) and benzylmethylamine (121 mg) as
colorless crystals; mp: 177–178 °C; 1H NMR (400 MHz, CDCl3):
d = 3.04 (3H, s, NCH3), 3.85 (s, 3H, OCH3), 3.92 (s, 2H, CH2CN),
4.68 (s, 2H, CH2Ph), 6.92 (dd, J = 2.3, 8.8 Hz, 1H, H-6), 7.06 (d,
J = 2.3 Hz, 1H, H-4), 7.16 (d, J = 8.8, 1H, H-7), 7.22–7.40 (m, 5H,
Har), 8.63 ppm (br s, 1H, NH); 13C NMR (100 MHz, CDCl3):
d = 13.74 (CH2CN), 35.34 (NCH3), 54.66 (CH2Ph), 55.81 (OCH3),
99.90 (C-4), 104.09 (CN), 112.86 (C-7), 115.89 (C-6), 117.37 (C-3),
126.68 (C-2), 127.54 (C-40), 128.04 (C-20, C-60), 128.74 (C-3a),
128.83 (C-30, C-50), 130.50 (C-10), 136.18 (C-7a), 155.07 (C-5),
m
= 3269, 2926, 1645 cmꢀ1; HRMS-ESI m/z [M+Na]+ calcd for
C21H24ClN3O2Na: 408.1455, found: 408.1449.
4.1.19.3. N-[2-(5-Methoxy-2-{[(4-methoxy-
phenyl)(methyl)amino]methyl}-1H-indol-3-yl)-ethyl]acetam-
ide (20c). Compound 20c (46 mg, 12%, elution with ethyl acetate)
was obtained from 19c (349 mg) as colorless foam; mp: 45–49 °C;
1H NMR (400 MHz, CDCl3): d = 1.84 (s, 3H, C(O)CH3), 2.78 (s, 3H,
NCH3), 2.93 (t, J = 6.5 Hz, 2H, CH2CH2N), 3.48 (q, J = 6.5 Hz, 2H,
CH2CH2N), 3.75 (s, 3H, C-40-OCH3), 3.83 (s, 3H, C-5-OCH3), 4.37 (s,
2H, CH2N), 5.95 (br s, 1H, C(O)NH), 6.78–6.94 (m, 5H, H-6, H-20,
H-30, H-50, H-60), 6.99 (d, J = 2.3 Hz, 1H, H-4), 7.16 (d, J = 8.8 Hz,
1H, H-7), 8.25 ppm (br, 1H, N-1-H); 13C NMR (100 MHz, CDCl3):
d = 23.24 (C(O)CH3), 23.96 (CH2CH2N), 39.92 (NCH3), 40.23
(CH2CH2N), 51.56 (CH2N), 55.62 (C-40-OCH3), 55.93 (C-5-OCH3),
100.31 (C-4), 109.62 (C-3), 111.68 (C-7), 111.90 (C-6), 114.58 (C-
30, C-50), 117.38 (C-20, C-60), 128.10 (C-3a), 128.81 (C-7a), 130.44
(C-2), 133.07 (C-10), 153.76 (C-40), 154.12 (C-5), 170.11 ppm
164.36 ppm (C@O); IR:
m = 3256, 3244, 1602, 1549, 1485, 1457,
1412 cmꢀ1; MS (EI, 70 eV) m/z (%): 333 (26) [M]+, 242 (100), 212
(23), 211 (47), 186 (15), 120 (25), 91 (100). Anal. Calcd for
C20H19N3O2: C, 72.05; H, 5.74; N, 12.60. Found: C, 72.11; H, 5.35;
N, 12.72.
4.1.18. General procedure for the synthesis of 19a–f
A solution of the appropriate amide 18a–f (1 equiv) in absolute
THF (25 mL) was added to a stirred suspension of LiAlH4 (11 equiv)
in absolute diethyl ether (30 mL) at 0–5 °C. The reaction mixture
was refluxed for 3 h. The reaction was quenched by a slow addition
of water at 0–5 °C. The formed precipitate was filtered off through
a pad of CeliteÒ545 and washed with ethyl acetate (150 mL). The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (3 ꢂ 75 mL). The combined organic layers were
washed with water (2 ꢂ 75 mL) and dried over Na2SO4. The vola-
tiles were removed under vacuum to afford the respective amines
19a–f as viscous oils that were used in the next step without fur-
ther purification.
(C@O); IR:
m = 3277, 3063, 2995, 2934, 2833, 1639, 1510, 1485,
1452, 1437 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C22H28N3O3:
382.2125, found 382.2133. Anal. Calcd for C22H27N3O3: C, 69.27;
H, 7.13; N, 11.02. Found: C, 68.90; H, 7.10; N, 10.92.
4.1.19.4. N-[2-(5-Methoxy-2-{[(methyl(phenyl)amino]methyl}-
1H-indol-3-yl)ethyl]acetamide (20d). Compound 20d (49 mg,
14%, elution with CHCl3–MeOH–25% NH3 100:10:1) was obtained
from 19d (319 mg) as a yellow viscous oil; 1H NMR (400 MHz,
CDCl3): d = 1.84 (s, 3H, C(O)CH3), 2.90 (t, J = 6.5 Hz, 2H, CH2CH2N),
2.92 (s, 3H, NCH3), 3.45 (q, J = 6.5 Hz, 2H, CH2CH2N), 3.83 (s, 3H,
OCH3), 4.49 (s, 2H, CH2N), 5.84 (br s, 1H, C(O)NH), 6.75–6.84 (m,
4.1.19. General procedure for the synthesis of 20a–f
A stirred solution of the respective amine 19a–f (1 equiv) in
absolute CH2Cl2 (25 mL) was treated with triethylamine (3.5 equiv)

C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
4593
3H, H-30, H-40, H-50), 6.86 (d, J = 8.8 Hz, 1H, H-6), 6.98 (d, J = 2.2 Hz,
1H, H-4), 7.12 (d, J = 8.8 Hz, 1H, H-7); 7.20–7.28 (m, 2H, H-20,H-60),
8.27 ppm (br, 1H, N-1-H); 13C NMR (100 MHz, CDCl3): d = 23.19
(C(O)CH3), 24.03 (CH2CH2N), 38.56 (NCH3), 40.16 (CH2CH2N),
49.67 (CH2N), 55.88 (OCH3), 100.28 (C-4), 109.13 (C-3), 111.64
(C-6), 111.69 (C-7), 114.02 (C-20, C-60), 118.57 (C-40), 128.24 (C-
3a), 128.90 (C-7a), 129.39 (C-30, C-50), 130.43 (C-2), 150.02 (C-10),
154.06 (C-5), 170.14 ppm (C@O); IR: 3270, 3063, 2934, 2831,
1633, 1595, 1487, 1450, 1435 cmꢀ1; HRMS-ESI m/z [M+Na]+ calcd
for C21H25N3O2Na: 374.1839, found: 374.1836.
3.43 (q, J = 6.4 Hz, 2H, CH2CH2N), 3.53 (s, 4H, CH2N, CH2Ph), 3.83
(s, 3H, OCH3), 5.16 (s, 2H, CH2CN), 5.78 (br, 1H, C(O)NH), 6.90
(dd, J = 8.8, 2.2 Hz, 1H, H-6), 7.01 (d, J = 2.2 Hz, 1H, H-4), 7.12–
7.40 ppm (m, 6H, H-7, H-20, H-30, H-40, H-50, H-60); 13C NMR
(100 MHz, CDCl3): d = 22.59 (C(O)CH3), 23.28 (CH2CN), 23.38
(CH2CH2N), 40.14 (CH2CH2N), 42.01 (NCH3), 44.01 (CH2N), 55.95
(OCH3), 62.39 (CH2Ph), 101.34 (C-4), 109.29 (C-6), 112.61 (C-7),
113.09 (C-3), 115.18 (CN), 127.53 (C-40), 128.46 (C-3a), 128.52
(C-30, C-50), 129.12 (C-20, C-60), 131.40 (C-7a), 133.01 (C-2),
138.11 (C-10), 154.87 (C-5), 170.16 ppm (C@O); IR:
3028, 2929, 2834, 2794, 2342, 1632, 1484, 1454, 1432,
1364 cmꢀ1 HRMS-ESI m/z [M+Na]+ calcd for C24H28N4O2Na:
m = 3299,
4.1.19.5. N-{2-(5-Methoxy-2-({[methyl[4-(trifluoromethyl)phe-
nyl]amino}]-methyl)-1H-indol-3-yl)]ethyl}acetamide (20e).
Compound 20e (78 mg, 21%, elution with CHCl3–MeOH–25%
NH3 100:10:1) was obtained from 19e (350 mg) as colorless crys-
tals; mp:165–167 °C. Analytical sample was obtained by recrystal-
lization from CHCl3. 1H NMR (400 MHz, CDCl3): d = 1.90 (s, 3H,
C(O)CH3), 2.95 (t, J = 6.5 Hz, 2H, CH2CH2N), 2.98 (s, 3H, NCH3),
3.48 (q, J = 6.5 Hz, 2H, CH2CH2N), 3.83 (s, 3H, OCH3), 4.62 (s, 2H,
CH2N), 5.65 (br s, 1H, C(O)NH), 6.77–6.87 (m, 3H, H-6, H-20, H-60),
7.00 (d, J = 2.3 Hz, 1H, H-4), 7.15 (d, J = 8.8 Hz, 1H, H-7); 7.46 (d,
J = 8.6 Hz, 2H, H-30,H-50), 8.01 ppm (br, 1H, N-1-H); 13C NMR
(100 MHz, CDCl3): d = 23.34 (C(O)CH3), 24.22 (CH2CH2N), 38.30
(NCH3), 40.16 (CH2CH2N), 48.24 (CH2N), 55.95 (OCH3), 100.39 (C-
4), 109.52 (C-3), 111.72 (C-6), 112.01 (C-20, C-60), 112.13 (C-7),
123.47 (C-40), 126.15 (CF3), 126.71 (C-30, C-50), 128.94 (C-3a),
130.52 (C-7a), 132.68 (C-2), 150.02 (C-10), 151.96 (C-5),
;
427.2104, found: 427.2113.
4.1.19.8. N-[2-(2-{[(Benzyl(methyl)amino]methyl}-1-[2-(cya-
no)ethyl]-5-methoxy-1H-indol-3-yl)ethyl]acetamide (22). To a
stirred solution of acetamide 20f (410 mg, 1.12 mmol) and acrylo-
nitrile (0.16 mL, 2.24 mmol) in dioxane–THF (6.7 mL, 1:1) at 0 °C
was added dropwise a catalytical amount of Triton-BÒ (40% ben-
zyltrimethylammonium hydroxide solution in MeOH, 0.06 mL).
The cooling bath was removed and reaction mixture was stirred
for 2 h. The mixture was acidified with 2 M hydrochloric acid
(4 mL) and the volatiles were evaporated in vacuo. The residue
was purified by column chromatography on silica gel (CHCl3–
MeOH–25% NH3 100:10:1) to yield 22 (288 mg, 61%) as a yellow
viscous oil; 1H NMR (400 MHz, CDCl3): d = 1.90 (s, 3H, C(O)CH3),
2.14 (s, 3H, NCH3), 2.72 (t, J = 7.2 Hz, 2H, CH2CH2CN), 2.88–2.94
(m, 2H, CH2CH2N), 3.44 (q, J = 6.4 Hz, 2H, CH2CH2N), 3.55 (s, 2H,
CH2Ph), 3.58 (s, 2H, CH2N), 3.83 (s, 3H, OCH3), 4.37 (t, J = 7.2 Hz,
2H, CH2CH2CN), 5.88 (br, 1H, C(O)NH), 6.86 (dd, J = 8.8, 2.3 Hz,
1H, H-6), 7.01 (d, J = 2.3 Hz, 1H, H-4), 7.12 (d, J = 8.8 Hz, 1H, H-7),
7.23–7.39 ppm (m, 5H, H-20, H-30, H-40, H-50, H-60); 13C NMR
(100 MHz, CDCl3): d = 23.16 (C(O)CH3), 18.19 (CH2CH2CN), 24.15
(CH2CH2N), 39.17 (CH2CH2CN), 40.02 (CH2CH2N), 41.91 (NCH3),
50.22 (CH2N), 55.90 (OCH3), 62.81 (CH2Ph), 101.04 (C-4), 109.27
(C-6), 112.06 (C-3), 112.19 (C-7), 117.56 (CN), 127.50 (C-40),
128.08 (C-3a), 128.52 (C-30, C-50), 129.29 (C-20, C-60), 130.93 (C-
7a), 133.61 (C-2), 138.21 (C-10), 154.36 (C-5), 170.14 ppm (C@O);
170.11 ppm (C@O); IR:
m ;
= 3206, 2937, 2846, 1659, 1613 cmꢀ1
HRMS-ESI m/z [M+Na]+ calcd for: C22H24F3N3O2Na: 442.1713,
found: 442.1710. Anal. Calcd for C22H24F3N3O2: C, 63.00; H, 5.77;
N, 10.02. Found: C, 62.65; H, 5.81; N, 9.82.
4.1.19.6. N-[2-(2-{[(Benzyl(methyl)amino]methyl}-5-methoxy-
1H-indol-3-yl)ethyl]acetamide (20f). Compound 20f (350 mg,
96% , elution with CHCl3–MeOH–25 NH3 100:10:1) was obtained
from 19f (333 mg) as
a
light brown viscous oil; 1H NMR
(400 MHz, CDCl3): d = 1.76 (s, 3H, C(O)CH3), 2.19 (s, 3H, NCH3),
2.90 (t, J = 6.3 Hz, 2H, CH2CH2N), 3.48 (q, J = 5.8 Hz, 2H, CH2CH2N),
3.56 (s, 2H, CH2N), 3.82 (s, 3H, OCH3), 6.56 (br s, 1H, C(O)NH), 6.81
(d, J = 8.8, 1.8 Hz, 1H, H-6), 6.96 (d, J = 1.8 Hz, 1H, H-4), 7.20 (d,
J = 8.6 Hz, 1H, H-7), 7.24–7.34 (m, 5H, H-20, H-30, H-40, H-50, H-60),
8.38 (br, 1H, N-1-H); 13C NMR (100 MHz, CDCl3): d = 23.05
(C(O)CH3), 23.38 (CH2CH2N), 40.22 (CH2CH2N), 42.33 (NCH3),
52.54 (CH2N), 55.91 (OCH3), 62.70 (CH2Ph), 100.28 (C-4), 110.23
(C-3), 111.52 (C-6), 111.87 (C-7), 127.45 (C-40), 128.41 (C-30, C-
50), 128.62 (C-3a), 129.25 (C-20, C-60), 130.43 (C-7a), 133.82 (C-2),
IR:
m
= 3287, 2927, 2832, 2361, 2340, 1649, 1542, 1482, 1454,
1423 cmꢀ1
;
HRMS-ESI m/z [M+Na]+ calcd for C25H30N4O2Na:
441.2266, found: 441.2261.
4.1.20. General procedure for the synthesis of 12c, 23
To a solution of the respective acetamide 21, 22 (0.717 mmol) in
glacial acetic acid (1.45 mL) was added 10% Pd/C (37 mg) in one
portion. The mixture was hydrogenated (7 hPa) at room tempera-
ture for 48 h. The catalyst was removed by filtration through a
pad of CeliteÒ545 and washed with CHCl3 (50 mL). The resulting
solution was made basic with 25% aqueous NH3 and extracted with
CHCl3. The combined CHCl3 solutions were dried over Na2SO4 and
the solvent was evaporated under vacuum. The residue was puri-
fied by silica gel chromatography (CHCl3–MeOH–25% NH3
100:10:1).
137.90 (C-10), 154.00 (C-5), 170.14 ppm (C@O); IR:
m = 3242,
3061, 3029, 2935, 2876, 2832, 2795, 2358, 2340, 1649, 1556,
1485, 1453 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C22H28N3O2:
366.2176, found: 366.2173.
4.1.19.7. N-[2-(2-{[(Benzyl(methyl)amino]methyl}-1-(cyano)-
methyl-5-methoxy-1H-indol-3-yl)ethyl]acetamide (21). To
a
solution of the acetamide 20f (365 mg, 1.00 mmol) in absolute
DMF (2.5 mL) potassium tert-butoxide (134 mg, 1.20 mmol) was
added at room temperature. After 40 min, bromoacetonitrile
(0.07 mL, 1.03 mmol) was added dropwise and the solution was
heated at 65 °C for 30 min and left stirring at room temperature
for 5 h. Another portion of bromoacetonitrile (0.05 mL) was added
and the reaction was stirred for a further hour. The reaction mix-
ture was poured onto ice (20 g) and the mixture was extracted
with CHCl3 (5 ꢂ 15 mL). The extracts were dried (Na2SO4), evapo-
rated under vacuum and the residue was purified by silica gel chro-
matography (CH2Cl2/THF 1:1) to afford 21 as yellow viscous oil
(183 mg, 45%). 1H NMR (400 MHz, CDCl3): d = 1.90 (s, 3H,
C(O)CH3), 2.48 (s, 3H, NCH3), 2.91 (t, J = 6.4 Hz, 2H, CH2CH2N),
4.1.20.1. N-[2-(8-Methoxy-2-methyl-1,2,3,4-tetrahydropyrazi-
no[1,2-a]indol-10-yl)ethyl]acetamide (12c). Compound 12c
(108 mg, 50%) was obtained from 21 (290 mg) as yellow viscous oil.
4.1.20.2. N-[2-(9-Methoxy-2-methyl-2,3,4,5-tetrahydro-1H-
[1,4]diazepino[1,2-a]indol-11-yl)ethyl]acetamide (23). Com-
pound 23 (92 mg, 41%) was obtained from 22 (300 mg) as colorless
foam; mp: 45–49 °C; 1H NMR (400 MHz, CDCl3): d = 1.77–1.84 (m,
2H, H-4), 1.86 (s, 3H, C(O)CH3), 2.36 (s, 3H, NCH3), 2.85–2.94 (m,
4H, CH2CH2N, H-3), 3.44 (q, J = 6.1 Hz, 2H, CH2CH2N), 3.73 (s, 2H,
H-1), 3.83 (s, 3H, OCH3), 4.16 (t, J = 4.3 Hz, 2H, H-5), 5.99 (br 1H,
C(O)NH), 6.83 (dd, J = 8.8, 2.5 Hz, 1H, H-8), 6.95 (d, J = 2.5 Hz, 1H,

4594
C. Markl et al. / Bioorg. Med. Chem. 17 (2009) 4583–4594
H-10), 7.13 ppm (d, J = 8.8 Hz, 1H, H-7); 13C NMR (100 MHz,
CDCl3): d = 23.05 (C(O)CH3), 23.74 (CH2CH2N), 40.02 (CH2CH2N 2),
28.32 (C-4), 44.84 (NCH3), 44.18 (C-1), 51.55 (C-5), 60.49 (C-3),
55.95 (OCH3), 100.49 (C-10), 109.60 (C-11), 111.68 (C-7), 109.53
(C-8), 127.20 (C-10a), 131.53 (C-6a), 136.60 (C-11a), 153.75 (C-9),
170.16 (C@O) ppm; HRMS-ESI m/z [M+H]+ calcd for: C18H26N3O2:
316.2019, found: 316.2023.
and potency (IC50) values were calculated using the commercially
available software (GraphPad PRISMÒ).
Acknowledgment
Thanks are due to Anita Betz, University of Würzburg, for her
skilful assistance in synthesizing compound 15.
4.2. Pharmacology
References and notes
4.2.1. Competition binding analysis
1. Reppert, S. M.; Weaver, D. R.; Godson, C. Trends Pharmacol. Sci. 1996, 17, 100.
2. Zlotos, D. P. Arch. Pharm. Chem. Life Sci. 2005, 338, 229.
3. Spadoni, G.; Bedini, A. In From Molecules to Therapy; Pandi-Perumal, S. R.,
Cardinali, D. P., Eds.; Nova Science, 2007; pp 33–45.
4. Garratt, P. J.; Tsotinis, A. Mini-Rev. Med. Chem. 2007, 10, 1075.
5. Rivara, S.; Mor, M.; Lorenzi, S.; Lodola, A.; Plazzi, P. V.; Spadoni, G.; Bedini, A.;
Tarzia, G. Arkivoc 2006, VIII, 8.
6. Bedini, A.; Spadoni, G.; Gatti, G.; Lucarini, S.; Tarzia, G.; Rivara, S.; Lorenzi, S.;
Lodola, A.; Mor, M.; Lucini, V.; Pannacci, M.; Scaglione, F. J. Med. Chem. 2006, 49,
7393.
7. Rivara, S.; Lodola, A.; Mor, M.; Bedini, A.; Spadoni, G.; Lucini, V.; Pannacci, M.;
Fraschini, F.; Scaglione, F.; Sanchez, R. O.; Gobbi, G.; Tarzia, G. J. Med. Chem.
2007, 50, 6618.
All compounds were tested for their binding affinity and selec-
tivity for each of the melatonin receptor subtypes, MT1 and MT2
using competition binding analysis. Briefly, cells expressing the hu-
man MT1 or MT2 melatonin receptor (MT1-CHO, MT2-CHO) were
grown to confluence on 10 cm cell culture plates until they reach
approximately 80% confluence. Next, cells were washed, lifted,
and added to tubes containing 80–100 pM 2-[125I]-iodomelatonin
in the absence (total binding) or presence of melatonin (1 pM to
1 lM) or the test compounds (1 pM to 1 lM). The reactions were
incubated for 1 h at 25 °C, terminated following the addition of
cold Tris-HCl solution (50 mM, pH 7.4) and then filtered through
glass fiber filters (Schleicher and Schuell, Keene, NH) saturated in
polyethylenimine 0.5% solution (v/v). Radioactivity was deter-
mined using a gamma counter. Data points were fit by site non-lin-
ear regression analysis based upon the lowest residual sum of
squares (GraphPad Prism) and affinity constants (Ki) values
calculated.
8. Poissonnier-Durieux, S.; Ettaoussi, M.; Pérès, B.; Boutin, JA.; Audinot, V.;
Bennejean, C.; Delagrange, P.; Caignard, DH.; Renard, P.; Berthelot, P.; Lesieur,
D.; Yous, S. Bioorg. Med. Chem. 2008, 16, 8339.
9. Zlotos, D. P.; Attia, M. I.; Julius, J.; Shalini, S.; Witt-Enderby, P. A. J. Med. Chem.
2009, 52, 826.
10. Rivara, S.; Lorenzi, S.; Mor, M.; Plazzi, P. V.; Spadoni, G.; Bedini, A.; Tarzia, G. J.
Med. Chem. 2005, 48, 4049.
11. Faust, R.; Garratt, P. J.; Jones, R.; Yeh, L.-K.; Tsotinis, A.; Panoussopoulou, M.;
Calogeropoulou, T.; Teh, M.-T.; Sugden, D. J. Med. Chem. 2000, 43, 1050.
12. Wallez, V.; Durieux-Poissonier, S.; Chavatte, P.; Boutin, J. A.; Audinot, V.;
Nicolas, J.-P.; Bennejean, C.; Delagrange, P.; Renard, P.; Lesieur, D. J. Med. Chem.
2002, 45, 2788.
13. Karageorge, G. N.; Bertenshaw, S.; Iben, L.; Xu, C.; Sarbin, N.; Gentile, A.;
Dubowchik, G. M. Bioorg. Med. Chem. Lett. 2004, 14, 5881.
14. Attia, M. I.; Julius, J.; Witt-Enderby, P. A.; Zlotos, D. P. Tetrahedron 2007, 63, 754.
15. Attia, M. I.; Witt-Enderby, P. A.; Julius, J. Bioorg. Med. Chem. 2008, 16, 7654.
16. Guandalini, L.; Martini, E.; Gualtieri, F.; Romanelli, M. N.; Varani, K. Arkivoc
2004(V), 286.
17. Brimble, M. A.; Brimble, M. T.; Hodges, R.; Lane, G. Aust. J. Chem. 1988, 41, 1583.
18. Diker, K.; El Biach, K.; Döé de Maindreville, M.; Lévy, J. J. Nat. Prod. 1997, 60,
791.
19. Tsotinis, A.; Panoussopoulou, M.; Sivananthan, S.; Sugden, D. Il Farmaco 2001,
56, 725.
20. Campiani, G.; Butini, S.; Trotta, F.; Fattorusso, C.; Catalanotti, B.; Aiello, F.;
Gemma, S.; Nacci, V.; Novellino, E.; Stark, J. A.; Cagnotto, A.; Fumagalli, E.;
Carnovali, F.; Cervo, L.; Mennini, T. J. Med. Chem. 2003, 46, 3822.
21. Zaitsev, S. A.; Glushkov, R. G.; Mashkovskii, M. D.; Andreeva, N. I. Khim.-Farm.
Zh. 1989, 23, 1201.
22. Rajur, S. B.; Merwade, A.-Y.; Hendi, S. B.; Basanagoudar, L. D. Indian J. Chem.,
Sect. B 1989, 28B, 1065.
23. Yamada, Y.; Takai, H.; Hatano, K.; Sakakibara, M.; Matsui, M. Agric. Biol. Chem.
1972, 36, 106.
4.2.2. Cyclic AMP assays
The cAMP accumulation assays were carried out by Enzyme Im-
muno Antibody (EIA) kit according to manufacturer’s directions.
Stable CHO cell lines expressing human MT1 or human MT2 recep-
tors were cultured on 10 cm plates in F-12 media containing 10%
FBS and 1% pen/strep until they were 70–80% confluent, after
which the cells were lifted and plated in 24- well plates. The fol-
lowing day, the cells were incubated in serum-free media contain-
ing one of the following treatment groups for 20 minutes at 37 °C—
(a) 30
forskolin (maximal accumulation), (c) 30
skolin, and 10 nM melatonin, or (d) 30
l
M rolipram alone (basal), (b) 30
l
l
M rolipram and 100
M rolipram, 100 M for-
M rolipram, 100 M for-
lM
l
l
l
skolin, and 12b or 20d (in concentrations ranging from 10ꢀ11 M
to 10ꢀ6 M). Cyclic AMP accumulation was expressed as a percent-
age of forskolin response within each group. Where appropriate,
curves were fit using non-linear regression analysis (one-site)