Synthesis and pharmacological evaluation of 6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a′]diindole analogs as melatonin receptor ligands

Article abstract of DOI:10.1016/j.tet.2006.10.081

The synthesis of two melatonin-derived analogs of the novel 6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a′]diindole ring system is described. The non-methoxy and methoxy analogs, 4a and 4b were prepared in seven steps starting from indoline-2-carboxylic acid 5a and 5-methoxyindoline-2-carboxylic acid 5b, respectively. While 4a exhibited micromolar affinities for both melatonin receptors, the methoxy analog 4b displayed moderate affinity for MT₂ receptors (K_i=0.41 μM) being 4.4-fold higher than for the MT₁ subtype.

Full text of DOI:10.1016/j.tet.2006.10.081

Tetrahedron 63 (2007) 754–760
Synthesis and pharmacological evaluation of 6a,7-
dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindole
analogs as melatonin receptor ligands
Mohamed I. Attia,a Justin Julius,b Paula A. Witt-Enderbyb and Darius P. Zlotosa,
*
aPharmaceutical Institute, University of Wu€rzburg, Am Hubland, 97074 Wu€rzburg, Germany
bDivision of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, 421 Mellon Hall, Pittsburgh, PA 15282, USA
Received 28 August 2006; revised 26 October 2006; accepted 29 October 2006
Available online 16 November 2006
Abstract—The synthesis of two melatonin-derived analogs of the novel 6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindole ring system is
described. The non-methoxy and methoxy analogs, 4a and 4b were prepared in seven steps starting from indoline-2-carboxylic acid 5a and
5-methoxyindoline-2-carboxylic acid 5b, respectively. While 4a exhibited micromolar affinities for both melatonin receptors, the methoxy
analog 4b displayed moderate affinity for MT2 receptors (Ki¼0.41 mM) being 4.4-fold higher than for the MT1 subtype.
Ó 2006 Elsevier Ltd. All rights reserved.
O
O
1. Introduction
N
H
CH3
H3CO
H3CO
Melatonin (1) is a hormone exerting its multiple pharma-
cological actions through two G-protein-coupled receptors
MT1 and MT2.1 Exploring the physiological role of each
of these subtypes requires subtype selective MT1 and MT2
ligands. Although several MT1 and MT2 selective agonists
and antagonists are known to date,2 conformationally re-
stricted melatoninergic agents are still needed in order to
explore the structural differences between MT1 and MT2
binding pockets. 3D-QSAR analysis of subtype selective
melatoninergic ligands revealed that MT2 binding affinity
could be enhanced by occupying the out-of-plane region sur-
rounding positions 1 and 2 of melatonin as well as the area
corresponding to the methoxy substituent.3 This pharmaco-
phore hypothesis could be exemplified by the rigid tetra-
cyclic indole analogs (2) and (3), respectively, which are
among the most MT2 selective melatoninergic ligands de-
scribed to date (Fig. 1).4 In this paper, we report the synthesis
of two novel pentacyclic non-methoxy and methoxy melato-
nin analogs (4a) and (4b), respectively, in which an indoline
moiety is attached to the positions 1 and 2 of melatonin.
These novel agents could help probing the existing pharma-
cophore for potent MT2 selective ligands.
1
N
H
N
H
C3H7
N
N
2
O
HN
C3H7
H3CO
3
Figure 1.
2. Results and discussion
2.1. Synthesis of the non-methoxy compound 4a
We chose the non-methoxy compound 4a as our initial target
in order to establish a viable synthetic route to the more
biologically relevant molecule 4b. In the course of our
studies on allosteric ligands of muscarinic M2 receptors, we
recently reported the synthesis of 6H,13H-pyrazino[1,2-
a;4,5-a0]diindole (6aS,13aS-7a).5 The synthetic sequence
Keywords: 6a,7-Dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindole; Melato-
ninergic ligands; Nitrogen heterocycles; Dehydrogenation.
* Corresponding author. Tel.: +49 931 8885489; fax: +49 931 8885494;
e-mail: zlotos@pzlc.uni-wuerzburg.de
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.10.081

M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
755
involved the self-condensation of (2S)-(ꢀ)-indoline-2-car-
boxylic using DCC,6 followed by reduction of the resulting
dilactam with borane. Starting from the commercially avail-
able racemic indoline-2-carboxylic acid (5a), we now
applied the same strategy to afford 6H,13H-pyrazino[1,2-
a;4,5-a0]diindole (7a) (Scheme 1). Thus, self-coupling of
5a using DCC in THF afforded dilactam (6a) in 41% yield.
Reduction of both carbonyl groups of 6a could be achieved
by heating at reflux in THF with borane to afford the tetra-
hydro-6H,13H-pyrazino[1,2-a;4-5-a0]diindole 7a in excellent
yield (98%). In the next critical step, one of the two indoline
moieties of 7a should be selectively dehydrogenated. Keep-
ing in mind that by refluxing 6aS,13aS-7a in toluene with an
excess of Pd/C 10%, introduction of both C6a–C7 and
C13a–C14 double bonds occured,5 more gentle reaction
conditions should be applied. We were pleased to find that
heating of 7a in toluene at 80 ꢁC with reduced amount of
Pd/C 10% provided the monodehydrogenation product (8a)
in 48% yield. Under these conditions, both starting material
and the didehydrogenation product were present in the reac-
tion mixture, as indicated by TLC. We were gratified to find
that the desired compound 8a could be separated from the
aforementioned side products by means of silica gel chroma-
tography.
With this key intermediate in hand, wewere now prepared for
introduction of the ethylamine side chain. In our first attempt
we applied the glyoxyamide method involving reaction with
(COCl)2 followed by treatment with ammonia and subse-
quent reduction of both carbonyl groups. Thus, 8a could be
converted to the corresponding 14-glyoxyamide by treatment
with (COCl)2 in diethyl ether and subsequent introduction of
ammonia gas in 91% yield. Unfortunately, all reduction at-
temptsto transform the 8a-glyoxyamide to the corresponding
ethylamine analog using LiAlH4 and borane failed. We were
pleased to observe that another approach involving a se-
quence of a Mannich reaction, quaternization of the Mannich
base, substitution of the trimethylamine moiety by a cyanide,
and a final reduction of the cyanomethyl group to the ethyl-
amine moiety, proved to be successful. Thus, aminomethyla-
tion of 8a using dimethylmethyleneammonium iodide
(Eschenmoser’s salt) in dichloromethane afforded the
Mannich base (9a) in 77% yield. Treatment of 9awith methyl
iodide in dichloromethane and refluxing of the resulting
trimethylammonium iodide with potassium cyanide and
dicyclohexyl-[18]-crown-[6] afforded the nitrile (10a) in
69% overall yield. Reduction of 10a using LiAlH4 in diethyl
ether and toluene provided the ethylamine (11a), which
could be easily converted to the desired melatoninergic
ligand 4a by acetylation using acetic anhydride in dichloro-
methane in 88% yield.
R
R
O
(ii)
(i)
2.2. Synthesis of the methoxy compound 4b
N
N
CO2H
N
H
O
With a viable synthetic route in hand, we were now prepared
for the synthesis of the melatonin-derived agent 4b. The
synthesis of the commercially unavailable starting material,
5-methoxyindoline-2-carboxylic acid (5b) was reported in
the patent literature.7 Stanton et al. obtained 5b$HCl in three
steps involving N-acetylation of 5-methoxyindole-2-carb-
oxylic acid (12) followed by catalytic hydrogenation using
platinum oxide and a subsequent deacetylation using 2 M
aqueous HCl. Unfortunately, by adopting this literature strat-
egy we were unable to obtain 5b$HCl, after the deacetyla-
tion step, in any detectable amount. However, we were
fortunate to develop a practical and an efficient alternative
route to 5b$HCl (Scheme 2). Our approach commenced
with the acid catalyzed esterification of the free carboxylic
acid functionality of 12 to give the known methyl ester
(13) in 96% yield. Subsequent protection of the indole nitro-
gen of 13 by introduction of tert-butoxycarbonyl (Boc)
group in acetonitrile in the presence of DMAP furnished
14 in 97% yield. The Boc protected ester was reduced to
the corresponding dihydroindole derivative (15) by catalytic
hydrogenation at 20 bar over Pd/C 10% in methanol. Hydro-
lysis of the ester functionality of 15 using LiOH in THF/H2O
followed by N-deprotection in HCl/ethyl acetate mixture af-
forded the desired compound 5b$HCl mp 175–178 ꢁC (lit.7
90–92 ꢁC) in an overall yield of 91% based on 12. Such
a huge difference of melting points indicates that the com-
pound described in the patent literature can impossibly be
5b$HCl. It should be mentioned that attempts to hydrolyze
the ester functionality of 15 to the corresponding carboxylic
acid 5b without protection of the indolic nitrogen gave
decomposition products or non-separable reaction mixture.
5a, 5b HCl
6a,b
·
R
1
R
R
2
14a
14
3
13a
(iii)
13
(iv)
4a
N
4
N
N 11a 11
N
6a
6
10
R
7a,b
8a,b
7
7a
9
8
R
R
R
CN
N(CH3 2
)
(vi)
(v)
N
N
N
N
9a,b
10a,b
R
R
R
R
CH3
NH2
N
H
O
(vii)
R
N
N
N
N
11a,b
4a,b
R
a: R = H
b: R = OCH3
Scheme 1. Reagents: (i) 6a: DCC, THF; 6b: DCC, Et3N, CH2Cl2; (ii) borane–
THF complex, THF; (iii) 8a: Pd/C 10%, toluene; 8b: Pd/C 10%; (iv) CH2]
NMe+2Iꢀ, CH2Cl2; (v) 1. MeI, CH2Cl2, 2. KCN, dicyclohexyl-[18]-crown-
[6], MeCN; (vi) LiALH4, Et2O; and (vii) acetic anhydride, Et3N, CH2Cl2.
The next two reaction steps proceeded similarly to those
in the non-methoxy series. Thus, condensation of 5b using

756
M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
Table 1. Competition of 4a, 4b, and melatonin for 2-[125I]-iodomelatonin
H3CO
H3CO
binding to human MT1 and MT2 melatonin receptors expressed in CHO
cells
(i)
(ii)
CO2CH3
CO2H
N
H
N
H
Ki (range of SEM)
12
13
Compound MT1
MT2
N
4a
Melatonin
1.0 mM (1.0–1.1 mM) 1.7 mM (1.5–2.0 mM)
0.45 nM (0.39–0.53 nM) 1.0 nM (0.88–1.2 nM)
5
5
H3CO
H3CO
(iv)
(iii)
4b
Melatonin
1.8 mM (1.1–2.9 mM)
0.44 nM (0.12–1.1 nM)
0.41 mM (0.22–0.77 mM)
6.0 nM (1.9–19 nM)
7
6
CO2CH3
CO2CH3
N
N
Boc
All competition binding experiments were performed on CHO whole cell
lysates using 90–125 pM 2-[125I]-iodomelatonin at 25 ꢁC. The affinity (KD)
of 2-[125I]-iodomelatonin for MT1 and MT2 receptors expressed in CHO
cells is 80 pM and 150 pM, respectively; N: number of experiments.
Boc
15
14
H3CO
H3CO
Cl
CO2H
(v)
CO2H
N
N
affinity for the receptors when compared to melatonin. How-
ever, compound 4b displays nanomolar affinity for MT2 re-
ceptors and, in fact, has 4.4-fold higher affinity for MT2
receptors than MT1 suggesting that 4b may be more
selective for MT2 than MT1 receptors. Also, compound 4b
has a better affinity profile for the MT2 receptors when com-
pared against melatonin with it having 68 times less affinity
for the MT2 receptors compared to 4000 times less affinity
for the MT1 receptors.
H2
Boc
16
5b HCl
Scheme 2. Reagents: (i) H2SO4, CH3OH; (ii) (Boc)2O, DMAP, MeCN;
(iii) H2, Pd/C 10%; MeOH; (iv) LiOH, THF, H2O; and (v) HCl/ethyl acetate.
DCC in dichloromethane afforded dilactam (6b) in 45%
yield. Reduction of both carbonyl groups of 6b could be
achieved by heating at reflux in THF with borane to afford
the dimethoxytetrahydro-6H,13H-pyrazino[1,2-a;4-5-a0]di-
indole (7b) in excellent yield (95%). Introduction of the
double bond in one of the indoline moieties of 7b proved
to be extremely difficult. While in the non-methoxy series,
refluxing of 7a in toluene with Pd/C 10% led to dehydroge-
nation of both indoline moieties,5 attempts to introduce at
least one double bond in 7b with Pd/C 10% in refluxing tol-
uene or xylene were unsuccessful. Furthermore, g-MnO2,8
TCCA,9 DDQ,10 palladium dichloride,11 sulfur,12 and Swern
oxidation13 also failed to give the desired product, although
each of these reagents had been reported to oxidize indoline
to indole. Ultimately, we were pleased to find that dehydro-
genation of 7b to give 8b could be achieved by heating 7b at
150 ꢁC without solvent in the presence of Pd/C 10% in 50%
yield. For the introduction of the ethylamine side chain, we
applied the same procedure as in the non-methoxy series.
Thus, aminomethylation of 8b using dimethylmethylene-
ammonium iodide (Eschenmoser’s salt) in dichloromethane
afforded the Mannich base (9b) in 98% yield. Quaternization
of 9b with methyl iodide in dichloromethane and refluxing
of the resulting trimethylammonium iodide with potassium
cyanide and dicyclohexyl-[18]-crown-[6] afforded the nitrile
(10b) in 65% overall yield. Reduction of 10b using LiAlH4
in diethyl ether and THF provided the ethylamine (11b)
(95%), which could be converted to the desired melatonin-
derived agent 4b by acetylation using acetic anhydride in
dichloromethane in 60% yield.
2.4. Discussion
According to the existing pharmacophore model, potent
MT2 selective ligands include a methoxy group and a bulky
lipophilic substituent in positions corresponding to N1 or C2
of melatonin that is located out of the plane of the indole
ring.3 Both ligands 4a and 4b are derived from desmethoxy-
melatonin and melatonin, respectively, by attaching an
indoline moiety to the positions 1 and 2 of desmethoxy-
melatonin and melatonin, respectively, via methylene groups.
Compound 4a exhibited rather poor affinity for both MT1
and MT2 receptors (Ki¼1.0 and 1.7 mM, respectively)
when compared to melatonin and to other potent melato-
ninergic ligands.2 Interestingly, while the introduction of
the methoxy group in the indole ring of 4a to give the
melatonin-derived compound 4b did not affect the MT1
binding (Ki¼1.8 mM), the affinity for the MT2 receptors
was increased 4.4-times confirming the importance of the
methoxy group for binding at the MT2 receptors. However,
the MT2 binding constant of 4b (Ki¼0.41 mM) indicates
only a moderate affinity for MT2 receptors when compared
to other melatoninergic ligands.2 The most likely explana-
tion for the poor selectivity and moderate binding affinity
of 4b is the unfavorable spatial orientation of the indoline
moiety, which is, due to its rigidity and bulkiness, not able
to occupy the lipophilic binding pocket of the MT2 recep-
tors. Therefore, synthetic work on more flexible analogs of
4b is continued in our laboratory.
2.3. Pharmacological studies
The affinity of compounds 4a and 4b for the human MT1 or
MT2 melatonin receptors was measured by competition
binding analysis using the radioligand 2-[125I]-iodomelato-
nin. Melatonin competition assays were run in parallel and
the affinity of melatonin for the MT1 or MT2 melatonin re-
ceptors is in the range of the reported literature. As shown
in Table 1, compound 4a displays micromolar affinity for
both melatonin receptors and has 1700–2000 times less
3. Conclusions
In summary, in search for subtype selective ligands
of melatonin receptors, we prepared two derivatives of
thenovel6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindole
ring system. The non-methoxy and methoxy analogs, 4a
and 4b, respectively, were prepared in seven steps starting
from the corresponding indoline-2-carboxylic acids 5a and

M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
757
5b, respectively. For the commercially unavailable 5-me-
thoxy-indoline-2-carboxylic acid 5b, a novel and an efficient
synthesis was developed. While 4a exhibited similar micro-
molar affinities for both melatonin receptors, the methoxy
analog 4b displayed moderate affinity for MT2 receptors
(Ki¼0.41 mM) being 4.4-fold higher than for the MT1 sub-
type.
solvent was evaporated in vacuo to give 15 as a white solid,
which was recrystallized from methanol to yield 3.82 g
(86%) of pure 15 as colorless crystals mp 88–90 ꢁC. FTIR
(ATR) n¼1737, 1704, 1491, 1021, 629 cmꢀ1
.
1H NMR
(400 MHz, CDCl3): d 1.46 (s, 9H, –C(CH3)3), 3.05 (dd,
1H, J¼16.7, 4.3 Hz, Ha-3), 3.45 (dd, 1H, J¼16.7, 12.1 Hz,
Hb-3), 3.70 (s, 3H, CO2CH3), 3.73 (s, 3H, OCH3), 4.81–
4.89 (m, 1H, H-2), 6.67–6.71 (m, 2H, Harom.), 6.75–7.78
(m, 1H, Harom.). 13C NMR (100 MHz, CDCl3): d 28.3
(C(CH3)3), 32.8 (C-3), 52.3 (CO2CH3), 55.7 (OCH3),
60.6 (C-2), 81.1 (C(CH3)3), 110.9, 112.5, 115.1 (Carom.),
129.2 (C-3a), 136.2 (C-7a), 151.6 (urethane), 155.7 (C-5),
172.5 (ester). MS (EI): m/z (%)¼307 (M+, 5), 207 (28),
148 (100). Anal. Calcd for C16H21NO5: C, 62.51; H, 6.89;
N, 4.56. Found: C, 62.50; H, 6.71; N, 4.49.
4. Experimental
4.1. General
Melting points were determined using a capillary melting
point apparatus (Gallenkamp, Sanyo) and are uncorrected.
Column chromatography was carried out on silica gel 60
(0.063–0.200 mm) obtained from Merck. A Bruker AV-400
4.1.3. 5-Methoxy-2,3-dihydro-1H-indole-2-carboxylic
acid hydrochloride (5b$HCl). Lithium hydroxide solution
2 M (30 ml) was added to a stirred solution of 15 (4.40 g,
14.3 mmol) in THF (80 ml). The resulting reaction mixture
was stirred at room temperature for 18 h and the solvent
was evaporated under reduced pressure. The residue was
diluted with water (20 ml) and extracted with diethyl ether
(2ꢂ25 ml). The aqueous layer was acidified using 0.5 M
potassium hydrogen sulfate solution and extracted with di-
ethyl ether (3ꢂ30 ml). The organic extracts were combined,
dried (Na2SO4), and evaporated under vacuum to give 4.19 g
(100%) of 5-methoxy-2,3-dihydro-1H-indole-1,2-dicarbox-
ylic acid 1-tert-butyl ester 16 as a pale yellow powder mp
104–105 ꢁC. The crude 16 was dissolved in approximately
2.5 M dry hydrogen chloride/ethyl acetate solution (33 ml)
and the reaction mixture was stirred at ambient temperature
for 1 h. The precipitated solid was filtered off, washed with
diethyl ether (20 ml), and dried to give 2.81 g (85%) of
5b$HCl as a white solid mp 175–178 ꢁC (lit.7 90–92 ꢁC).
1
spectrometer was used to obtain H NMR (400 MHz) and
13C NMR (100 MHz) spectra. Proton chemical shifts are
referred to CHCl3 (7.24 ppm) and DMSO-d6 (2.55 ppm).
Carbon chemical shifts are referred to CDCl3 (77.00 ppm)
and DMSO-d6 (39.50 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 and on a ESI-microTOF spectrometers. IR
spectra, recorded as ATR, were obtained by using a Biorad
PharmalyzIR FT-IR instrument. Elemental analyses were
performed by the microanalytical section of the Institute of
€
Inorganic Chemistry, University of Wurzburg. All reactions
were carried out under an argon atmosphere.
4.1.1. 5-Methoxy-1H-indole-1,2-dicarboxylic acid 1-tert-
butyl ester 2-methyl ester (14). A solution of 13 (4.70 g,
22.9 mmol), di-tert-butyl dicarbonate (5.50 g, 25.2 mmol),
and a catalytical amount of 4-(dimethylamino)-pyridine
(0.55 g) in acetonitrile (60 ml) was stirred at ambient tem-
perature for 72 h. The volatiles were removed under reduced
pressure and the residue was dissolved in diethyl ether
(100 ml). The resulted solution was washed with 1 M HCl
(2ꢂ20 ml), water (2ꢂ20 ml), dried (Na2SO4), and evapo-
rated under vacuum to give 6.76 g (97%) of 14 as a pale
yellow solid, which was pure enough to be used in the
next step without further purification. Analytical sample
was obtained by silica gel chromatography (n-pentane/
diethyl ether, 1:1) to afford 14 as a colorless crystalline solid
mp 65–66 ꢁC. FTIR (ATR) n¼1740, 1715, 1321, 1067,
FTIR (ATR) n¼2967–2523, 1741, 1499, 1026, 663 cmꢀ1
.
1H NMR (400 MHz, D2O): d 3.39 (dd, 1H, J¼16.7,
6.6 Hz, Ha-3), 3.60 (dd, 1H, J¼16.7, 9.6 Hz, Hb-3), 3.73
(s, 3H, OCH3), 4.95 (dd, 1H, J¼9.6, 6.6 Hz, H-2), 6.89
(dd, 1H, J¼8.6, 1.76 Hz, H-6), 6.93 (s, 1H, H-4), 7.32 (d,
1H, J¼8.6 Hz, H-7). 13C NMR (100 MHz, D2O): d 33.0
(C-3), 55.8 (OCH3), 60.6 (C-2), 110.9 (C-4), 114.8 (C-6),
120.0 (C-7), 127.3 (C-3a), 135.4 (C-7a), 160.6 (C-5),
171.4 (ester). MS (EI): m/z (%)¼193 (M+, 31), 148 (100),
133 (53). Anal. Calcd for C10H12ClNO3: C, 52.39; H,
5.28; N, 6.11. Found: C, 52.02; H, 5.22; N, 5.99.
666 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.59 (s, 9H,
.
–C(CH3)3), 3.81 (s, 3H, OCH3), 3.89 (s, 3H, CO2CH3),
6.99–7.02 (m, 3H, H-3, H-4, H-6), 7.95 (d, 1H, J¼9.6 Hz,
H-7). 13C NMR (100 MHz, CDCl3): d 27.7 (C(CH3)3),
52.2 (CO2CH3), 55.6 (OCH3), 84.3 (C(CH3)3), 103.6
(C-3), 114.5 (C-4), 115.7 (C-6), 116.3 (C-7), 128.1 (C-2),
130.8 (C-3a), 132.6 (C-7a), 149.2 (urethane), 156.2 (C-5),
162.2 (ester). MS (EI): m/z (%)¼305 (M+, 8), 205 (46),
173 (100). Anal. Calcd for C16H19NO5: C, 62.93; H, 6.28;
N, 4.59. Found: C, 63.05; H, 6.31; N, 4.57.
4.1.4. 6a,7,13a,14-Tetrahydropyrazino[1,2-a;4,5-a0]di-
indole-6,13-dione (6a). DCC (6.0 g, 29.1 mmol) was added
to the solution of the racemic 2,3-dihydro-1H-indole-2-
carboxylic acid 5a (2.5 g, 15.3 mmol) in anhydrous THF
and the reaction mixture was stirred at room temperature
for 2 h. Precipitates were filtered off and the filtrate was
evaporated under reduced pressure. The oily residue was
purified by silica gel chromatography (CH2Cl2) to give 6a
(0.92 g, 42%) as a colorless solid mp 264 ꢁC. FTIR (ATR)
1
n¼2918, 2851, 1674, 1599, 1483, 1458, 1413 cmꢀ1. H
4.1.2. 5-Methoxy-2,3-dihydro-1H-indole-1,2-dicarbox-
ylic acid 1-tert-butyl ester 2-methyl ester (15). Pd/C 10%
(0.60 g) was added to a stirred solution of 14 (4.41 g,
14.4 mmol) in methanol (160 ml). The reaction mixture
was hydrogenated under 20 bar of H2 for 18 h at room tem-
perature. The catalyst was removed by filtration and the
NMR (400 MHz, CDCl3): d 3.43 (dd, 2H, J¼16.7,
10.4 Hz, Ha-7, Ha-14), 3.79 (dd, 2H, J¼16.7, 8.8 Hz, Hb-7,
Hb-14), 4.96 (dd, 2H, J¼10.4, 8.8 Hz, H-6a, H-13a), 7.09
(m, 2H, H-2, H-9), 7.24 (m, 4H, H-1, H-8, H-3, H-10),
8.09 (d, 2H, J¼7.8 Hz, H-4, H-11). 13C NMR (100 MHz,
CDCl3): d 30.1 (C-7, C-14), 61.7 (C-6a, C-13a), 115.8

758
M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
(C-4, C-11), 125.0 (C-3, C-10, C-2, C-9), 127.9 (C-1, C-8),
129.7 (C-7a, C-14a), 140.5 (C-4a, C-11a), 164.2 (2ꢂC]O).
MS (EI): m/z (%)¼290.1 (M+, 100%), 143.1 (10), 117.1
(84). Anal. Calcd for C18H14N2O2: C, 74.47; H, 4.86;
N, 9.65. Found: C, 74.52; H, 4.91; N, 9.44.
purification. Analytical sample was obtained by recrystalli-
zation from ethyl acetate to give 7b as colorless crystals
mp 129–131 ꢁC. FTIR (ATR) n¼1490, 1215, 1136,
1
636 cmꢀ1. H NMR (400 MHz, CDCl3): d 2.69 (dd, 2H,
J¼16.2, 2.6 Hz, Ha-7, Ha-14), 2.88 (dd, 2H, J¼12.1,
10.4 Hz, Ha-6, Ha-13), 3.25 (dd, 2H, J¼16.2, 9.6 Hz, Hb-7,
Hb-14), 3.39 (dd, 2H, J¼10.1, 3.0 Hz, Hb-6, Hb-13), 3.72
(s, 6H, 2ꢂOCH3), 4.35–4.42 (m, 2H, H-6a, H-13a), 6.34
(d, 2H, J¼8.3 Hz, H-4, H-11), 6.62 (dd, 2H, J¼8.3, 2.5 Hz,
H-3, H-10), 6.70–6.72 (m, 2H, H-1, H-8). 13C NMR
(100 MHz, CDCl3): d 33.2 (C-7, C-14), 51.8 (C-6, C-13),
55.7 (2ꢂOCH3), 57.0 (C-6a, C-13a), 108.3 (C-4, C-11),
111.8 (C-1, C-8), 112.1 (C-3, C-10), 129.7 (C-7a, C-14a),
146.1 (C-4a, C-11a), 153.4 (C-2, C-9). HRMS (ESI, pos.)
C20H20N2Oꢃ2H+: m/z calcd 323.1759, m/z found 323.1755.
4.1.5. 2,9-Dimethoxy-6a,7,13a,14-tetrahydropyrazino[1,2-
a;4,5-a0]diindole-6,13-dione (6b). Triethylamine (2.5 ml,
17.8 mmol) and DCC (8.00 g, 38.8 mmol) were added to
a stirred suspension of 5b$HCl (4.00 g, 17.4 mmol) in dry
CH2Cl2 (80 ml) at ambient temperature. The resulting reaction
mixture was stirred at room temperature for 18 h. The precip-
itated solids were filtered off and washed with CH2Cl2
(20 ml). The combined filtrate and washing were washed with
1 N HCl (2ꢂ15 ml), water (2ꢂ20 ml), and cooled at ꢀ30 ꢁC
for 18 h. The precipitates were filtered off and the organic
solvent was removed under vacuum. The residue was recrys-
tallized from methanol to afford 1.37 g (45%) of 6b as
a pale yellow solid mp 254–255 ꢁC. FTIR (ATR) n¼1660,
4.1.8. 6a,7-Dihydro-6H,13H-pyrazino[1,2-a;5-a0]diindole
(8a). Pd/C 10% (0.25 g) was added to a solution of 7a (0.65 g,
2.5 mmol) in toluene (100 mL). The reaction mixture was
heated for 3 h at 80 ꢁC. Pd/C was filtered off through
a pad of CeliteÒ545 and washed with toluene. The solvent
was removed in vacuo and the residue purified by silica
gel chromatography (CH2Cl2/hexane, 1:1) to give 8a
(0.31 g, 48%) as a yellow crystalline solid mp 170 ꢁC.
FTIR (ATR) n¼2833, 2787, 1609, 1476, 1450, 1371, 1336,
1
1600, 1488, 1400, 822 cmꢀ1. H NMR (400 MHz, CDCl3):
d 3.37 (dd, 2H, J¼16.9, 10.4 Hz, Ha-7, Ha-14), 3.72 (dd,
2H, J¼16.9, 8.9 Hz, Hb-7, Hb-14), 3.77 (s, 6H, 2ꢂOCH3),
4.94 (t, 2H, J¼9.4 Hz, H-6a, H-13a), 6.75 (dd, 2H, J¼8.6,
2.5 Hz, H-3, H-10), 6.79–6.82 (m, 2H, H-1, H-8), 7.98 (d,
2H, J¼8.6 Hz, H-4, H-11). 13C NMR (100 MHz, CDCl3):
d 30.5 (C-7, C-14), 55.6 (2ꢂOCH3), 61.8 (C-6a, C-13a),
111.0 (C-1, C-8), 112.6 (C-3, C-10), 116.4 (C-4, C11),
131.4, 134.2 (C-4a, C-11a, C-7a, C-14a), 157.0 (C-2, C-9),
163.4 (2ꢂlactam). MS (EI): m/z (%)¼350 (M+, 41), 147
(100), 132 (56). Anal. Calcd for C20H18N2O4: C, 68.55; H,
5.18; N, 8.00. Found: C, 68.39; H, 5.27; N, 7.90.
1240, 1173, 734 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d 2.89 (dd, 1H, J¼14.9, 10.9 Hz, Ha-7), 3.24 (dd, 1H,
J¼14.9, 7.6 Hz, Hb-7), 3.83 (dddd, 1H, J¼10.9, 10.9, 7.6,
3.8 Hz, H-6a), 3.97 (t, 1H, J¼10.9 Hz, Ha-6), 4.19 (dd,
1H, J¼14.9, 1.2 Hz, Ha-13), 4.52 (dd, 1H, J¼10.9, 3.8 Hz,
Hb-6), 4.90 (d, 1H, J¼14.9 Hz, Hb-13), 6.35 (s, 1H, H-14),
6.63 (d, 1H, J¼7.6 Hz, H-11), 6.79 (m, 1H, H-9), 7.09–
7.20 (m, 4H, H-2, H-10, H-3, H-8), 7.31 (d, 1H, J¼8.1 Hz,
H-4), 7.57 (d, 1H, J¼7.8 Hz, H-1). 13C NMR (100 MHz,
CDCl3): d 33.2 (C-7), 44.6 (C-13), 47.2 (C-6), 61.6 (C-6a),
97.2 (C-14), 107.4 (C-11), 108.6 (C-4), 119.4 (C-9), 120.0,
120.2, 120.9 (C-2, C-1, C-8), 124.7 (C-3), 127.8 (C-10),
128.9 (C-14a), 132.6 (C-7a), 135.9 (C-4a), 150.9 (C-11a).
MS (EI): m/z (%)¼260.1 (M+, 35), 143.1 (100), 115.1
(28). Anal. Calcd for C18H16N2: C, 83.05; H, 6.19; N,
10.76. Found: C, 82.82; H, 6.52; N, 10.42.
4.1.6. 6a,7,13a,14-Tetrahydro-6H,13H-pyrazino[1,2-
a;4,5-a0]diindole (7a). Borane–THF complex 1 M (15 ml)
was added dropwise to a stirred solution of 6a (0.90 g,
3.1 mmol) in dry THF (100 ml) at room temperature. The
reaction mixture was refluxed for 17 h, allowed to cool
and 2 M HCl (8 ml) was added dropwise under ice-cooling.
After heating for ½ h at reflux, the reaction mixture was
basified with 25% ammonia under ice-cooling and extracted
with CH2Cl2 (3ꢂ100 ml). The combined organic layers
were washed with water (3ꢂ15 ml), dried (MgSO4), and
evaporated in vacuo to yield 0.80 g (98%) of 7a as a white
solid, which was pure enough to be used in the next step
without further purification. Analytical sample was obtained
by recrystallization from ethyl acatate to give 7a as colorless
crystals mp 140 ꢁC. The IR, NMR, and MS data of 7a are
identical with the corresponding data of the (6aS,13aS)-7a
stereomer.5
4.1.9. 2,9-Dimethoxy-6a,7-dihydro-6H,13H-pyrazino[1,2-
a;5-a0]diindole (8b). A mixture of 7b (0.6 g, 1.86 mmol)
and Pd/C 10% (0.10 g) was heated at 150 ꢁC for 1 h. The
reaction mixture was allowed to cool, CH2Cl2 (2ꢂ25 ml)
was added and the catalyst was removed by filtration. The
organic extracts were combined, dried (Na2SO4), and evapo-
rated under reduced pressure. The residue was recrystallized
from chloroform to afford 0.3 g (50%) of 8b as a light brown
solid mp 256–257 ꢁC. FTIR (ATR) n¼2831, 2360, 1485,
4.1.7. 2,9-Dimethoxy-6a,7,13a,14-tetrahydro-6H,13H-
pyrazino[1,2-a;4,5-a0]diindole (7b). Borane–THF complex
1 M (8.5 ml) was added dropwise to a stirred suspension of
6b (0.63 g, 1.8 mmol) in dry THF (40 ml) at room tempera-
ture. The reaction mixture was refluxed for 17 h, allowed
to cool and 2 M HCl (8.5 ml) was added dropwise under
ice-cooling. The resulting reaction mixture was refluxed
for 1 h, allowed to cool, basified with 25% ammonium
hydroxide solution and extracted with ethyl acetate
(3ꢂ20 ml). The combined organic layers were washed with
water (2ꢂ15 ml), dried (Na2SO4), and evaporated in vacuo
to yield 0.55 g (95%) of 7b as a white solid, which was
pure enough to be used in the next step without further
1
1238, 1026, 844, 735 cmꢀ1. H NMR (400 MHz, CDCl3):
d 2.85 (dd, 1H, J¼14.6, 7.3 Hz, Ha-7), 3.17 (dd, 1H,
J¼14.6, 7.3 Hz, Hb-7), 3.69–3.73 (m, 1H, H-6a), 3.75 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 3.91–3.97 (m, 1H, Ha-6),
4.05 (d, 1H, J¼14.4 Hz, Ha-13), 4.44 (dd, 1H, J¼10.6,
3.8 Hz, Hb-6), 4.81 (d, 1H, J¼14.4 Hz, Hb-13), 6.26 (s, 1H,
H-14), 6.54 (d, 1H, J¼8.3 Hz, H-11), 6.71 (dd, 1H, J¼8.3,
2.5 Hz, H-10), 6.82–6.84 (m, 2H, H-3, H-8), 7.04 (d, 1H,
J¼2.3 Hz, H-1), 7.18 (d, 1H, J¼8.6 Hz, H-4). 13C NMR
(100 MHz, CDCl3): d 33.5 (C-7), 45.6 (C-13), 47.2 (C-6),
55.9, 56.0 (2ꢂOCH3), 62.4 (C-6a), 96.9 (C-1), 102.3
(C-14), 107.8 (C-11), 109.2 (C-4), 110.8 (C-3), 111.9 (C-10),

M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
759
112.4 (C-8), 128.9, 130.6, 131.3, 133.5, 145.1, (C-4a, C-7a,
C-11a, C-13a, C-14a), 153.9, 154.5 (C-2, C-9). HRMS (ESI,
pos.) C20H20N2Oꢃ2H+-2: m/z calcd 319.1447, m/z found
319.1441.
4.1.12. (6a,7-Dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]-
diindol-14-yl)-acetonitrile (10a). Methyl iodide (0.5 ml)
was added to a solution of 9a (0.17 g, 0.54 mmol) in dry
CH2Cl2 (50 ml). The reaction mixture was stirred at room
temperature for 1 h. The volatiles were removed under vac-
uum and the residual ammonium salt was dissolved in dry
acetonitrile (150 ml). Dicyclohexyl-[18]-crown-[6] (0.30 g)
and potassium cyanide (0.50 g) were added and resulting
reaction mixture was heated at reflux for 2 h. The solvent
was evaporated under reduced pressure and the residue
was subjected to silica gel chromatography (CHCl3) to
afford 10a (0.11 g, 69%) as a brown solid mp 154 ꢁC.
FTIR (ATR) n¼2881, 2830, 1607, 1474, 1454, 1340, 1250,
1H, J¼14.9, 10.9 Hz, Ha-7), 3.25 (dd, 1H, J¼14.9, 7.6 Hz,
Hb-7), 3.75–3.86 (m, 3H, H-6a, –CH2CN), 3.96 (d, 1H,
J¼10.9 Hz, Ha-6), 4.15 (d, 1H, J¼14.9 Hz, Ha-13), 4.51
(dd, 1H, J¼10.9, 3.8 Hz, Hb-6), 4.93 (d, 1H, J¼15.1 Hz,
Hb-13), 6.68 (d, 1H, J¼7.8 Hz, H-11), 6.81 (m, 1H, H-9),
7.15–7.27 (m, 4H, H-2, H-10, H-3, H-8), 7.32 (d, 1H,
J¼7.8 Hz, H-4), 7.59 (d, 1H, J¼7.8 Hz, H-1). 13C NMR
(100 MHz, CDCl3): d 12.9 (–CH2CN), 33.3 (C-7), 43.3
(C-13), 47.3 (C-6), 61.3 (C-6a), 97.5 (–CN), 107.6 (C-11),
109.0 (C-4), 117.6 (C-14), 117.7 (C-1), 119.8 (C-9), 120.6
(C-2), 121.9 (C-3), 124.8 (C-10), 127.9 (C-8), 126.9 (C-14a),
128.8 (C-7a), 130.4 (C-13a), 135.7 (C-4a), 150.6 (C-11a).
MS (EI): m/z (%)¼299.1 (M+, 63), 272.1 (14), 259.1 (14),
182.1 (100). Anal. Calcd for C20H17N3: C, 80.24; H, 5.72;
N, 14.04. Found: C, 79.81; H, 5.75; N, 13.64.
4.1.10. (13a,14-Dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]-
diindol-7-yl)-dimethylamine (9a). Dimethylmethylenim-
minium iodide (0.40 g, 2.2 mmol) was added to a solution of
compound 8a (0.34 g, 1.3 mmol) in dry CH2Cl2 (100 mL).
After heating for 1 h at reflux, the reaction mixture was
made basic with 25% ammonia. The organic layer was
separated, washed with water and dried over MgSO4. The
solvent was removed in vacuo and the residue purified by
silica gel chromatography (CHCl3/methanol/25% ammonia,
100:10:1) to give 9a (0.32 g, 77%) as a colorless crystalline
solid mp 132 ꢁC. FTIR (ATR) n¼2957, 2932, 2814, 1605,
1
754, 736 cmꢀ1. H NMR (400 MHz, CDCl3): d 2.90 (dd,
1457, 1334, 1244, 757, 740 cmꢀ1 1H NMR (400 MHz,
.
CDCl3): d 2.27 (s, 6H, NMe2), 2.89 (dd, 1H, J¼14.6,
10.9 Hz, Ha-7), 3.24 (dd, 1H, J¼14.6, 7.6 Hz, Hb-7),
3.55 (d, 1H, J¼13.1 Hz, –HCH–NMe2), 3.61 (d, 1H,
J¼13.1 Hz, –HCH–NMe2), 3.82 (dddd, 1H, J¼10.9, 10.9,
7.6, 3.8 Hz, H-6a), 3.96 (t, 1H, J¼10.9 Hz, Ha-6), 4.12 (d,
1H, J¼15.4 Hz, Ha-13), 4.51 (dd, 1H, J¼10.9, 3.8 Hz,
Hb-6), 4.95 (d, 1H, J¼15.1 Hz, Hb-13), 6.68 (d, 1H,
J¼7.6 Hz, H-11), 6.79 (m, 1H, H-9), 7.11–7.21 (m, 4H,
H-2, H-10, H-3, H-8), 7.29 (d, 1H, J¼8.1 Hz, H-4), 7.67 (d,
1H, J¼7.8 Hz, H-1). 13C NMR (100 MHz, CDCl3): d 33.3
(C-7), 43.1 (C-13), 45.8 (–NMe2), 47.2 (C-6), 53.5 (–CH2–
NMe2), 61.5 (C-6a), 107.0 (C-14), 107.5 (C-11), 108.6
(C-4), 118.9 (C-1), 119.4 (C-9), 119.8 (C-2), 120.1 (C-3),
124.8 (C-10), 127.8 (C-8), 128.8, 128.9 (C-7a, C-14a),
130.9 (C-13a), 135.7 (C-4a), 151.0 (C-11a). HRMS (ESI,
pos.) C21H23Nꢃ3H+: m/z calcd 318.1970, m/z found 318.1965.
4.1.13. (2,9-Dimethoxy-6a,7-dihydro-6H,13H-pyr-
azino[1,2-a;4,5-a0]diindol-14-yl)-acetonitrile (10b). Methyl
iodide (0.1 ml) was added to a solution of 9b (0.21 g,
0.56 mmol) in dry CH2Cl2 (20 ml). The reaction mixture
was stirred at room temperature for 1 h. The volatiles
were removed under vacuum to afford 9b methoiodide mp
134–136 ꢁC. This crude ammonium salt was suspended in
dry acetonitrile (20 ml), dicyclohexyl-[18]-crown-[6]
(0.24 g) and potassium cyanide (0.47 g) were added. The
resulting reaction mixture was heated at reflux for 3 h. The
solvent was evaporated under reduced pressure and the res-
idue was dissolved in ethyl acetate (40 ml). The organic
layer was washed with water (3ꢂ15 ml), dried (Na2SO4),
and evaporated in vacuo. The residue was purified by silica
gel chromatography (ethyl acetate/chloroform, 8:2) to
afford 10b (0.13 g, 65%) as a dark red viscous oil that
solidified on cooling in the freezer. FTIR (ATR) n¼2918,
4.1.11. 2,9-Dimethoxy-14-[(dimethylamino)methyl]-
6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindole
(9b).
Dimethylmethylenimminium
iodide
(0.27 g,
1.46 mmol) was added to a solution of 8b (0.38 g,
1.19 mmol), in dry CH2Cl2 (150 ml). The reaction mixture
was refluxed for 1 h, allowed to cool and basified with 25%
ammonia. The organic layer was separated, washed with
water (2ꢂ30 ml), dried (Na2SO4), and evaporated under
reduced pressure to give 0.44 g (98%) of 9b as a dark red
solid, which was used in the next step without further purifi-
cation. Analytical sample was obtained by recrystallization
from methanol to give 9b as a light red solid mp 180–
182 ꢁC. FTIR (ATR) n¼2941, 2360, 1487, 1236, 1030,
794, 609 cmꢀ1. 1H NMR (400 MHz, DMSO-d6): d 2.27 (s,
6H, NMe2), 2.83 (dd, 1H, J¼14.6, 11.4 Hz, Ha-7), 3.15
(dd, 1H, J¼14.6, 7.3 Hz, Hb-7), 3.63–3.70 (m, 1H, H-6a),
3.76 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.89 (s, 2H, CH2–
NMe2), 3.91–3.94 (m, 1H, Ha-6), 3.97 (d, 1H, J¼14.9 Hz,
Hb-6), 4.41 (dd, 1H, J¼10.6, 3.8 Hz, Hb-6), 4.87 (d, 1H,
J¼14.9 Hz, Hb-13), 6.60 (d, 1H, J¼8.6 Hz, H-11), 6.72
(dd, 1H, J¼8.6, 2.5 Hz, H-10), 6.82–6.85 (m, 2H, H-3,
H-8), 7.12 (d, 1H, J¼2.3 Hz, H-1), 7.16 (d, 1H, J¼8.6 Hz,
H-4). 13C NMR (100 MHz, DMSO-d6): d 33.5 (C-7), 44.5
(C-13), 45.5 (NMe2), 47.2 (C-6), 53.5 (CH2–NMe2), 56.01,
56.03 (2ꢂOCH3), 62.3 (C-6a), 101.3 (C-1), 107.9 (C-11),
109.1 (C-4), 110.7 (C-3), 111.9 (C-10), 112.4 (C-8), 128.2,
130.6, 130.8, 132.7, 145.0 (C-4a, C-7a, C-11a, C-13a,
C-14a), 153.4, 153.9 (C-2, C-9). HRMS (ESI, pos.)
C23H27N3Oꢃ2H+-2: m/z calcd 376.2025, m/z found 376.2008.
2850, 2361, 1485, 1230, 799, 735 cmꢀ1
.
1H NMR
(400 MHz, CDCl3): d 2.82 (dd, 1H, J¼14.9, 11.6 Hz,
Ha-7), 3.15 (dd, 1H, J¼14.9, 7.3 Hz, Hb-7), 3.69–3.71
(m, 1H, H-6a), 3.73 (s, 2H, CH2–CN), 3.75 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 3.90 (d, 1H, J¼10.9 Hz,
Ha-6), 3.98 (d, 1H, J¼14.9 Hz, Ha-13), 4.40 (dd, 1H,
J¼10.9, 3.8 Hz, Hb-6), 4.82 (d, 1H, J¼14.9 Hz, Hb-13),
6.57 (d, 1H, J¼8.3 Hz, H-11), 6.71 (dd, 1H, J¼8.3,
2.5 Hz, H-10), 6.82 (d, 1H, J¼2.2 Hz, H-8), 6.87 (dd, 1H,
J¼8.8, 2.2 Hz, H-3), 6.99 (d, 1H, J¼2.3 Hz, H-1), 7.17 (d,
1H, J¼8.8 Hz, H-4). 13C NMR (100 MHz, CDCl3): d 12.8
(CH2CN), 33.4 (C-7), 44.1 (C-13), 47.2 (C-6), 55.9, 56.0
(2ꢂOCH3), 62.1 (C-6a), 97.1 (CN), 99.7 (C-1), 107.9
(C-11), 109.7 (C-4), 111.8 (C-3), 111.9 (C-10), 112.5
(C-8), 106.1, 117.7, 127.2, 128.8, 130.5, 144.7 (C-4a,
C-7a, C-11a, C-13a, C-14, C-14a), 154.2, 154.8 (C-2,

760
M. I. Attia et al. / Tetrahedron 63 (2007) 754–760
C-9). HRMS (ESI, pos.) C23H27N3Oꢃ2H+-2: m/z calcd
358.1555, m/z found 358.1550.
gel chromatography (ethyl acetate/chloroform, 8:2) to yield
4b (0.190 g, 60%) as an orange solid mp 101–104 ꢁC. FTIR
(ATR) n¼3480–3360, 2361, 1639, 1488, 1228, 742 cmꢀ1
.
4.1.14. N-[2-(6a,7-Dihydro-6H,13H-pyrazino[1,2-a;4,5-
a0]diindol-14-yl)-ethyl]-acetamide (4a). A solution of 10a
(0.100 g, 0.334 mmol) in dry toluene (30 ml) was added to
a stirred suspension of LiAlH4 (0.30 g) in dry diethyl ether
(30 ml) at 0–5 ꢁC. The reaction mixture was heated at
40 ꢁC for 2 h. Water (3 ml) was added dropwise under ice-
cooling and the reaction mixture was stirred for 1 h at
room temperature. The precipitate was filtered off and
washed with diethyl ether. The combined filtrate and wash-
ings were dried (Na2SO4), filtered, and evaporated under
vacuum to afford 0.100 g (99%) of 2-(6a,7-dihydro-
6H,13H-pyrazino[1,2-a;4,5-a0]diindol-14-yl)-ethylamine 11a
as a pale yellow viscous oil. A stirred solution of 11a
(0.100 g, 0.329 mmol) in dry CH2Cl2 (10 ml) was treated
with triethylamine (0.30 ml) and acetic anhydride (0.20 ml,
2.11 mmol) at 0–5 ꢁC. The reaction mixture was stirred at
ambient temperature for 3 h. The solvent was evaporated
and the residue was purified by silica gel chromatography
(chloroform/ methanol/25% ammonia 100:10:1) to afford
4a (0.110 g, 88%) as a yellow solid mp 88 ꢁC. FTIR
(ATR) n¼2925, 1648, 1608, 1551, 1477, 1454, 1373,
1H NMR (400 MHz, CDCl3): d 1.90 (s, 3H, CH3), 2.82–
2.96 (m, 3H, Ha-7, CH2–CH2–N), 3.17 (dd, 1H, J¼
14.9 Hz, Hb-7), 3.45–3.58 (m, 2H, CH2–CH2–N), 3.62–
3.70 (m, 1H, H-6a), 3.75 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 3.88–3.94 (m, 2H, Ha-6, Ha-13), 4.43 (dd, 1H,
J¼10.9, 3.8 Hz, Hb-6), 4.77 (d, 1H, J¼14.7 Hz, Hb-13),
5.57 (br, 1H, NH), 6.58 (d, 1H, J¼8.3 Hz, H-11), 6.71 (dd,
1H, J¼8.3, 1.9 Hz, H-10), 6.82 (s, 1H, H-8), 6.85 (dd, 1H,
J¼8.8, 2.0 Hz, H-3), 7.00 (d, 1H, J¼2.0 Hz, H-1), 7.18
(d, 1H, J¼8.8 Hz, H-4). 13C NMR (100 MHz, CDCl3):
d 23.5 (CH3), 24.1 (CH2–CH2–N), 33.5 (C-7), 39.8 (CH2–
CH2–N), 44.4 (C-13), 47.2 (C-6), 55.99, 56.03 (2ꢂOCH3),
62.5 (C-6a), 100.4 (C-1), 107.9 (C-11), 109.4 (C-4), 111.1
(C-3), 111.5 (C-10), 112.5 (C-8), 106.1, 128.5, 130.6,
130.7, 131.2, 145.0 (C-4a, C-7a, C-11a, C-13a, C-14,
C-14a), 154.1, 154.4 (C-2, C-9), 170.1 (C]O). HRMS
(ESI, pos.) C24H27N3Oꢃ3H+-2: m/z calcd 404.1974, m/z found
404.1970.
4.2. Binding assays
1339, 1298, 1247, 741 cmꢀ1
.
1H NMR (400 MHz,
To assess the affinity, competition binding assays were per-
formed on compounds 4a and 4b. Chinese hamster ovary
(CHO) cells stably transfected with the human MT1 or
MT2 melatonin receptors weregrown to confluence on 10-cm
diameter culture dishes and then resuspended in 50 mM
ice-cold Tris–HCl (pH 7.4 containing protease inhibitors).
CHO whole cell lysates were then added to tubes containing
Tris–HCl (50 mM containing protease inhibitors) and
2-[125I]-iodomelatonin (90–125 pM) in the absence or pres-
ence of compound 4a or 4b (1 pM–100 mM). All reactions
were incubated for 1 h at 25 ꢁC, rapidly filtered and counted
in a gamma counter.
CDCl3): d 1.90 (s, 3H, CH3), 2.87 (dd, 1H, J¼14.8,
10.9 Hz, Ha-7), 2.91–3.03 (m, 2H, –CH2–CH2–NH–), 3.24
(dd, 1H, J¼14.9, 7.6 Hz, Hb-7), 3.46–3.62 (m, 2H, –CH2–
CH2–NH–), 3.79 (dddd, 1H, J¼11.0, 11.0, 7.6, 3.6 Hz,
H-6a), 3.94 (d, 1H, J¼10.9 Hz, Ha-6), 4.05 (d, 1H,
J¼14.9 Hz, Ha-13), 4.51 (dd, 1H, J¼10.9, 3.6 Hz, Hb-6),
4.86 (d, 1H, J¼14.9 Hz, Hb-13), 5.52 (br, 1H, NH), 6.66
(d, 1H, J¼7.8 Hz, H-11), 6.80 (m, 1H, H-9), 7.11–7.23 (m,
4H, H-2, H-10, H-3, H-8), 7.31 (d, 1H, J¼8.0 Hz, H-4),
7.55 (d, 1H, J¼7.8 Hz, H-1). 13C NMR (100 MHz,
CDCl3): d 23.4 (CH3), 24.1 (–CH2–CH2–NH–), 33.3
(C-7), 39.9 (–CH2–CH2–NH–), 43.5 (C-13), 47.2 (C-6),
61.7 (C-6a), 106.5 (C-14), 107.6 (C-11), 108.8 (C-4),
118.2 (C-1), 119.6 (C-9), 119.8 (C-2), 121.2 (C-3), 124.7
(C-10), 127.8 (C-8), 128.1 (C-14a), 128.9 (C-7a), 129.8
(C-13a), 136.0 (C-4a), 150.8 (C-11a), 170.1 (C]O). MS
(EI): m/z (%)¼345.2 (M+, 24), 259.1 (100), 259.1 (14),
156.1 (42). Anal. Calcd for C22H23N3O: C, 76.49; H, 6.71;
N, 12.16. Found: C, 76.10; H, 6.70; N, 11.80.
References and notes
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4.1.15. N-[2-(2,9-Dimethoxy-6a,7-dihydro-6H,13H-pyra-
zino[1,2-a;4,5-a0]diindol-14-yl)-ethyl]-acetamide (4b). A
solution of 10b (0.300 g, 0.83 mmol) in dry THF (30 ml)
was added to a stirred suspension of LiAlH4 (0.650 g,
17.00 mmol) in dry diethyl ether (20 ml) at 0–5 ꢁC. The
reaction mixture was refluxed for 3 h. The reaction was
quenched by a slow addition of saturated sodium sulfate
solution at 0–5 ꢁC. The formed precipitate was filtered off
and washed with THF (10 ml). The combined filtrates and
washings were dried (Na2SO4), filtered and evaporated
under vacuum to afford 0.290 g (96%) of 2-(2,9-dime-
thoxy-6a,7-dihydro-6H,13H-pyrazino[1,2-a;4,5-a0]diindol-
14-yl)-ethylamine 11b as a pale yellow viscous oil. A stirred
solution of 11b (0.290 g, 0.80 mmol) in dry CH2Cl2 (20 ml)
was treated with triethylamine (0.39 ml, 2.80 mmol) and
acetic anhydride (0.20 ml, 2.11 mmol) at 0–5 ꢁC. The reac-
tion mixture was stirred at ambient temperature for 18 h. The
solvent was evaporated and the residue was purified by silica
€
€
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