Synthesis and molecular modeling of novel tetrahydro-β-carboline derivatives with phosphodiesterase 5 inhibitory and anticancer properties

Article abstract of DOI:10.1021/jm100842v

New derivatives based upon the tetrahydro-β-carboline-hydantoin and tetrahydro-β-carboline-piperazinedione scaffolds were synthesized. All compounds were evaluated for their ability to inhibit PDE5 in vitro, and numerous compounds with IC₅₀ values in the low nanomolar range were identified including compounds derived from l-tryptophan. Compounds with high potency versus PDE5 were then evaluated for inhibitory activity against other PDEs to assess isozyme selectivity. Compound 5R,11aS-5-(3,4-dichlorophenyl)-2- ethyl-5,6,11,11a-tetrahydro-1H-imidazo[1′,5′:1,6]pyrido[3,4-b] indole-1,3(2H)dione 14 showed a selectivity index of >200 for cGMP hydrolysis by PDE5 versus PDE11. Meanwhile, 6R,12aR-6-(2,4-dichlorophenyl)-2-ethyl-2,3,6, 7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4dione 45 demonstrated strong potency for inhibition of PDE11 with an IC₅₀ value of 11 nM, representing the most potent PDE11 inhibitor thus far reported. Docking experiments differentiated between active and inactive analogues and revealing the conformational, steric, and lipophilic necessities for potent PDE5 inhibition. Many derivatives, including potent PDE5 inhibitors, were able to inhibit the growth of the MDA-MB-231 breast tumor cell line with low micromolar potency.

Full text of DOI:10.1021/jm100842v

J. Med. Chem. 2011, 54, 495–509 495
DOI: 10.1021/jm100842v
Synthesis and Molecular Modeling of Novel Tetrahydro-β-carboline Derivatives with
Phosphodiesterase 5 Inhibitory and Anticancer Properties
Heba A. Mohamed,†, Nancy M. R. Girgis,†, Rainer Wilcken,‡ Matthias R. Bauer,‡ Heather N. Tinsley,§ Bernard D. Gary,§
Gary A. Piazza,§ Frank M. Boeckler,‡ and Ashraf H. Abadi*,†
†Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt,
‡Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of Pharmaceutical and Medicinal Chemistry,
Institute of Pharmacy, Eberhard-Karls-University, Tu€bingen, Auf der Morgenstelle 8, 72076 Tu€bingen, Germany, and
§Division of Drug Discovery, Department of Biochemistry and Molecular Biology, Southern Research Institute,
The University of Alabama at Birmingham, 2000 Ninth Avenue South, Birmingham, Alabama 35205, United States.
The first and second author contributed equally to this work
Received July 7, 2010
New derivatives based upon the tetrahydro-β-carboline-hydantoin and tetrahydro-β-carboline-piper-
azinedione scaffolds were synthesized. All compounds were evaluated for their ability to inhibit PDE5 in
vitro, and numerous compounds with IC50 values in the low nanomolar range were identified including
compounds derived from L-tryptophan. Compounds with high potency versus PDE5 were then evaluated
for inhibitory activity against other PDEs to assess isozyme selectivity. Compound 5R,11aS-5-(3,4-
dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)dione
14 showed a selectivity index of >200 for cGMP hydrolysis by PDE5 versus PDE11. Meanwhile,
6R,12aR-6-(2,4-dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-
1,4dione 45 demonstrated strong potency for inhibition of PDE11 with an IC50 value of 11 nM,
representing the most potent PDE11 inhibitor thus far reported. Docking experiments differentiated
between active and inactive analogues and revealing the conformational, steric, and lipophilic necessities for
potent PDE5 inhibition. Many derivatives, including potent PDE5 inhibitors, were able to inhibit the
growth of the MDA-MB-231 breast tumor cell line with low micromolar potency.
Introduction
PDE5 has received considerable attention over the years for
having been successfully developed as a therapeutic target for
erectile dysfunction. PE5 inhibitors may also have benefits for
pulmonary hypertension, cardioprotection, and enhancing
cognitive function.3-5
Cyclic adenosine monophosphate (cAMPa) and cyclic gua-
nosine monophosphate (cGMP) are important second mes-
sengers, responsible for transducing various extracellular
signals to the intracellular milieu. The intracellular levels of
cyclic nucleotides are controlled by the activities of cyclases,
which catalyze the formation of cyclic nucleotides from their
nucleotide triphosphate precursors and phosphodiesterases
(PDEs), which hydrolyze the cyclic nucleotide bond to termi-
nate the signal.1 On the basis of sequence homology and
regulatory properties, the PDEs can be classified into 11
distinct isozyme families. Often, there is more than one gene
product within each family and each gene generates multiple
splice variants.2 PDE5 is a cGMP specific PDE that is expressed
in vascular smooth muscle and lung platelets, although recent
reports demonstrate that it is also overexpressed in various
carcinomas including breast, colon, lung, and bladder cancers.
Recent studies have demonstrated that PDE5 may serve as
a novel target for the cancer chemopreventive drug, sulindac.6
Moreover, certain tadalafil related analogues have shown tumor
cell growth inhibitory properties versus colorectal HT-29
cancer cell line that appear to be associated with their ability
to inhibit PDE5.7 Other reports have revealed anticancer
activities to various tetrahydro-β-carbolines through different
mechanisms, including inhibition of topoisomerase II,8 cyclin
dependent kinases,9 and mitotic kinesin Eg5.10 These obser-
vations suggest thattadalafilcan serveas a good starting point
for the discovery of novel anticancer compounds. However,
traditional PDE5 inhibitors are unable to induce cGMP
signaling in tumor cells and thus lack anticancer activity.
Three PDE5 inhibitors, tadalafil (Figure 1), sildenafil, and
vardenafil, have received Food and Drug Administration
(FDA) approval for the treatment of erectile dysfunction and
pulmonary hypertension. Although these drugs share a com-
mon mechanism of action, they have different pharmaco-
kinetic profiles, PDE isozyme selectivities, and interaction
mechanism with PDE5.11-13 For example, tadalafil has a long
half-life of 17.5 h and can also inhibit PDE11. The interaction
of tadalafil with PDE5 involves hydrogen bonding with the
conserved Gln817, which is essential for nucleotide recognition
*To whom correspondence should be addressed. Phone:
þ20227590716. Fax: þ20227581041. E-mail: ashraf.abadi@guc.edu.eg.
a Abbreviations: cAMP, cyclic adenosine monophosphate; cGMP,
cyclic guanosine monophosphate; PDEs, phosphodiesterases; PDE5,
phosphodiesterase5; PDE3B, phosphodiesterase3B; PDE4, phospho-
diesterase4; FDA, Food and Drug Administration; t-butyl, tertiary-
butyl; CH2Cl2, methylene chloride; NOESY, nuclear Overhauser effect
spectroscopy; Rf, retardation factor; μM, micromolar; PDB, Protein
Data Bank; TMS, tetramethylsilane; TLC, thin layer chromatography;
IMAP, immobilized metal affinity for phosphochemicals; FP, fluores-
cence polarization; TAMRA-cGMP, tetramethylrhodamine-cGMP.
r
2010 American Chemical Society
Published on Web 12/28/2010
pubs.acs.org/jmc

496 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
and selectivity, and π-π stacking with Phe820. Unlike silde-
nafil and vardenafil, the rigid chemical structure of tadalafil,
which has only one rotatable bond, contributes to its binding
affinity because entropy is lost upon protein binding. Addi-
tionally, tadalafil has no direct or water mediated interaction
with the metal ions that are present in the binding pocket
of PDE5.14-16
Chemically, tadalafil (Figure 1) is a tetrahydro-β-carboline-
piperazinedione with two chiral carbons that are both of the R
configuration, a methyl substituent on the piperazinedione
nitrogen, and a pendant 1,3-benzodioxol substituent attached
to C-6.
To expand the scope of structure-activity relationships
with this class of compounds, herein we report the discovery
of novel tadalafil analogues with the tetrahydro-β-carboline-
imidazoline and the tetrahydro-β-carboline-piperazinedione
scaffolds; the chiral carbons are of the R,R, R,S, S,R, or S,S
configuration, the terminal ring nitrogen substituent is methyl,
ethyl, butyl, or t-butyl, and the pendant aryl is 2,4-dichloro-
phenyl, 3,4-dichlorophenyl, or 2,6-dichlorophenyl.
To evaluate the biochemical and biological relevance of
these derivatives, they were assayed for their ability to inhibit
the recombinant human PDE5 enzyme and to inhibit tumor cell
growth usingthehumanMDA-MB-231breast tumorcell line.
acid esters.17 The respective ester was subjected to Pictet-
Spengler reaction with 2,4-dichlorobenzaldehyde, 3,4-dichloro-
benzadehyde, and 2,6-dichlorobenzaldehyde. The reaction of
the ester with 2,4-dichlorobenzaldehyde and 3,4-dichloroben-
zadehyde gave both the cis and trans isomers as expected.
Because the cis and trans isomers are diastereomers, they were
separated by column chromatography using CH2Cl2 as an
eluent. The reaction of the ester with the 2,6-dichlorobenzal-
dehyde yielded exclusively the cis isomer. Previously reported
data indicated that the trans isomer is the thermodynamically
favored isomer while the cis-isomer is kinetically favored.18-21
A bulky tryptophan ester substituent, particularly benzyl,
favors the formation of the corresponding trans isomer.
Conversely, an allyl ester favors the formation of the cis
isomer upon reaction with an aldehyde.18-21 We found that
an aromatic aldehyde with bulky substituents at positions 2
and 6 of the benzene ring favors the formation of the cis
isomer upon reaction with tryptophan methyl ester, probably
due to restricted rotation. The Pictet-Spengler reaction was
carried out at room temperature under nonstereospecific
conditions. As expected, the kinetics were in favor of the cis
isomer.18-21 Thus, the formation of diastereomers 3, 5, and 7
exceeded the formation of diastereomers 4, 6, and 8, respec-
tively, with % de=11%, 27%, and 25%, respectively, mean-
while, for the isomers 1 and 9, there was no preferentiality
relative to 2 and 10.
Chemistry
Reaction of the respective pure cis- or trans-isomers of 1,3-
disubstituted tetrahydro-β-carboline (1-10) with commer-
cially available ethyl- and t-butyl isocyanate produced the
corresponding cis- and trans-hydantoins (11-30).
Treatment of (1-10) with chloroacetyl chloride provided
the corresponding chloroacetyl derivative(31-40). The β-car-
boline-piperazinedione derivatives with the respective N-alkyl
substituents were obtained by reaction of the respective
chloroethanone derivative with different primary amines,
The syntheses of the 1,3-disubstituted tetrahydro-β-carbo-
lines (Scheme 1), the tetrahydro-β-carboline-hydantoins
(Scheme 2), and the tetrahydro-β-carboline-piperazinediones
(Scheme 3) are shown. Both D- and L-tryptophan methyl ester
were synthesized by a general synthetic procedure for amino
Scheme 2a
a Conditions: (a) ethyl or t-butylisocyanate/2-butanone/reflux; R =
-C2H5 or -C-(CH3)3, X,Y = 2,4-dichloro, 3,4-dichloro, or 2,6-di-
chloro. For exact individual assignment, see Table 2. Stereocenters are
labeled.
Figure 1. Chemical structure of tadalafil with the points of mod-
ifications encircled.
Scheme 1. D or L-Tryptophan, D- or L-Tryptophan Methyl Ester, and 1,3-Disubstituted Tetrahydro-β-arbolinea
a Conditions: (a) CH3COCl/methanol/reflux/6 h, basify and extract with CH2Cl2; (b) 2,4-dichlorobenzaldehyde, 3,4-dichlorobenzadehyde, or 2,6-
dichlorobenzaldehyde/TDA/4 days/RT. For exact indivividual assignment, see Table 1. Stereocenters are labeled.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 497
Scheme 3
Conditions: (a) Cl-CO-CH2-Cl/chloroform/NaHCO3/RT/1 h; (b) methyl, ethyl, butyl or t-butyl amine/methanol/reflux/16hrs. For exact individual
assignment, see Tables 3 and 4. Stereocenters are labeled
Table 1. Chemical Structure, Growth Inhibition of MDA-MB-231 Breast Tumor Cell Line, and PDE5 Inhibitory Activity of 1,3-Disubstituted-
tetrahydro-β-carbolines (1-10)
% growth
inhibition
at 10 μMa
growth inhibition
IC50 (μM) and
CI at p < 0.05b
% PDE 5
inhibition at
10 μMa
PDE 5 inhibition
IC50 (μM) and CI
at p < 0.05b
compd
stereochemistry
X,Y
1
2
1R,3R
1S,3R
1S,3S
1R,3S
1R,3R
1S,3R
1S,3S
1R,3S
1R,3R
1S,3S
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
29
78
50
28
43
100
62
48
25
52
ND
6.73 (2.21-12.5)
ND
65
40
55
82
78
50
18
24
25
15
7.2 (5.51-9.01)
ND
3
ND
0.57 (0.55-0.77)
1.61 (1.30-1.98)
ND
ND
ND
ND
ND
4
ND
ND
5
6
4.62 (4.09-5.23)
6.98 (5.93-8.22)
ND
ND
ND
7
8
9
10
a Calculated from duplicate values. b Calculated from at least 8 concentrations, each with quadruple replicates, p < 0.05.
including methylamine, ethylamine, butylamine, and t-butyl-
amine in refluxing methanol (41-68).
Mass spectrometry of all derivatives showed molecular ion
peaks at Mþ, Mþ þ 2, and Mþ þ 4 due to the isotopic nature
and number of chlorine atoms in the molecule. Most of the
1,3-disubstituted-tetrahydro-β-carboline derivatives deriva-
tives (1-10) showed the base peaks as molecular ion peaks,
indicating their stable nature.
The infrared spectra of (1-10) showed bands at a stretching
frequency around 3400 cm-1 for the N-H stretching and a
band around 1730 cm-1 for the ester carbonyl stretching. On
the other hand, the β-carboline-hydantoin derivatives (11-30)
showed two carbonyl stretching bands because the hydantoin
carbonyls are not equivalent as one of the carbonyls is flanked
between two nitrogen atoms and the other between carbon
and nitrogen.
The stretching vibrations of the two carbonyls of the
piperazinedione derivatives (41-68) were generally at lower
wave numbers relative to their hydantoin analogues. This may
be due to the higher ring strain of the five-membered
ring relative to the six-membered ring, producing higher
double bond characteristics of the carbonyls in the hydantoin
series.
The optical rotations of derivatives (5-8) showed that they
are optically active, with those derived from D-tryptophan
dextrorotatory and those derived from L-tryptophan levoro-
tatory. The optical rotation angle of a 0.1% ethanolic solution of
derivative 5 relative to 7 and compound 6 relative to 8 showed
equivalent values but opposite signs, indicating their enantio-
meric nature.
The assignment of cis/trans-stereochemistry for the tetra-
hydro-β-carbolines (1-10) was based on a detailed study of
13C NMR spectroscopic data well established in previous
literature.22,23 The 13C NMR signals for C-1 and C-3 are
more shielded in the trans-isomer compared to cis-isomer with
Δδ 3-4 ppm, which may be due to 1,3-diaxial spatial inter-
actions present in the trans-isomer. The tetrahydropyridine
ring exists in half-chair conformation where the ester at C-3 is
equatorially located. No analogous difference was detected
with C-3 in compounds (1-10). Moreover, a 500 MHz 2D
NOESY experiment that was carried out for cis-1 and trans-2
showed a clear correlation between the C-1 and C3-hydrogens
as expected for a cis-relationship with a coupling constant of
4.1 Hz (Supporting Information).
Moreover, a correlation exists between the Rf value on TLC
and the stereochemistry of the 1,3-disubstituted tetrahydro-β-
carboline (1-10) and their hydantoin analogues (11-30) where
the cis-isomer issystematicallylesspolar than thetrans-isomer
for the tetrahydro-β-carboline. However, in the hydantoin
series, the polarity is reversed, thus the cis-isomer becomes
more polar than the trans-isomer. For example, the Rf values
of the 1R,3R, 1S,3R, 1S,3S, and 1R,3S isomers of the (1-4)
and theircorrespondingN-ethylhydantoinanalogues(11-14)
using CH2Cl2 as the mobile phase were 0.19, 0.12, 0.19, and
0.12 and 0.16, 0.28, 0.16, and 0.28, respectively.
The 1H NMR signals for the proton at C-1 of derivatives
1-10 appeared at δ 5.2-6.2. Cyclization to the corresponding
hydantoin (11-30) or piperazinedione derivatives (41-68)
causes a large downfield shift to δ 5.8-7.0; this is mostly due
to the electron withdrawing effect of the carbonyl in the
neighboring ring.
Results and Discussion
All of the final compounds and intermediates synthesized
were screened for in vitro tumor cell growth inhibition activity
using the human MDA-MB-231 breast tumor cell line and for

498 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
Table 2. Chemical Structure, Growth Inhibition of MDA-MB-231 Breast Tumor Cell Line, and PDE5 Inhibitory Activity of Tetrahydro-β-carboline-
hydantoins (11-30)
% growth
inhibition at
10 μMa
growth inhibition
IC50 (μM) and CI
at p < 0.05b
% PDE 5
inhibition at
10 μMa
PDE 5 inhibition
IC50 μM and CI at
p < 0.05b
compd
stereochemistry
X,Y
R
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5R,11aR
5S,11aR
5S,11aS
5R,11aS
5R,11aR
5S,11aR
5S,11aS
5R,11aS
5R,11aR
5S,11aS
5R,11aR
5S,11aR
5S,11aS
5R,11aS
5R,11aR
5S,11aR
5S,11aS
5R,11aS
5R,11aR
5S,11aS
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
39
54
29
68
50
60
78
18
55
52
68
65
22
69
99
58
70
60
38
15
ND
ND
95
90
85
95
95
78
60
90
18
37
80
73
75
95
82
65
80
88
52
25
0.010 (0.005-0.024)
0.090 (0.08-0.11)
0.85 (0.67-1.07)
0.007 (0.004-0.009)
0.15 (0.10-0.24)
2.86 (2.40-3.40)
8.75 (6.90-10.64)
0.30 (0.23-0.40)
ND
ND
5.37 (4.71-6.11)
ND
ND
4.78 (1.03-12.12)
ND
-C2H5
-C2H5
ND
ND
ND
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
2.26 (1.52-3.33)
0.64 (0.50-0.80)
ND
0.59 (0.41-0.85)
1.48 (1.19-1.84)
4.14 (3.27-5.23)
0.070 (0.06-0.08)
1.87 (1.53-2.28)
6.38 (5.01-8.12)
2.31 (1.79-2.97)
0.012 (0.005-0.024)
ND
1.26 (1.04-1.53)
3.25 (1.46-4.0)
ND
1.43 (0.94-3.79)
ND
ND
ND
ND
a Calculated from duplicate values. b Calculated from at least 8 concentrations, each with quadruple replicates, p < 0.05.
Table 3. Chemical Structure, Growth Inhibition of MDA-MB-231 Breast Tumor Cell Line, and PDE5 Inhibitory Activity of 1,2,3-Trisubstituted-
tetrahydro-β-carbolines (31-40)
% growth
inhibition at
10 μMa
growth inhibition
IC50 (μM) and CI
at p < 0.05b
% PDE 5
inhibition at
10 μMa
PDE 5 inhibition
IC50 μM and CI
at p < 0.05b
compd
stereochemistry
X,Y
31
32
33
34
35
36
37
38
39
40
1R,3R
1S,3R
1S,3S
1R,3S
1R,3R
1S,3R
1S,3S
1R,3S
1R,3R
1S,3S
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
98
98
55
98
40
50
43
55
15
30
0.56 (0.52-0.61)
0.07 (0.05-0.11)
ND
66
25
99
38
40
30
20
40
27
67
2.30 (1.71-3.08)
ND
0.38 (0.25-0.44)
ND
1.24 (1.05-1.48)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.73 (0.64-0.83)
a Calculated from duplicate values. b Calculated from at least 8 concentrations, each with quadruple replicates, p < 0.05.
inhibition of recombinant human PDE5 at a single concentra-
tion of 10 μM. For compounds showing >60% inhibition, the
IC50 was determined by testing a range of eight concentrations
with quadruple replicates per concentration, tadalafil used as
a positive control. The results are shown in Tables 1-5.
For the 1,3-disubstituted tetrahydro-β-carbolines (1-10),
most of the derivatives were inactive against PDE5 at 10 μM,
with only 3 compounds (1, 4, and 5) showing activity below
the screening dose. Two of the active compounds have a 2,4-
dichlorophenyl substituent while one has a 3,4-dichlorophenyl
substituent. Conversely, all derivatives with the 2,6-dichlor-
ophenyl substituent (9 and 10) were inactive. This indicates
that PDE5 inhibitory activity is dependent on the positional
isomerization state of the compounds.
For the hydantoins (11-30), only derivatives with 2,4-
dichloro or 3,4-dichloro substitution at C-5 were able to inhibit
PDE5. Again, all derivatives with the 2,6-dichloro substitution
were inactive, confirming the importance of positional isomers
for PDE5 inhibitory activity. The most active compounds
were either 5R,11aS or 5R,11aR configuration. Additionally,
all compounds with the S configuration at the C-5 were
inactive or less active than their R congeners, indicating that
the stereochemical configuration of the carbon derived from
the aldehyde and not the stereochemical aspects of the carbon
derived from the tryptophan amino acid is an important
determinant of PDE5 inhibitory activity. This observation
expands the chemical space from which potent PDE5 inhibi-
tors may be discovered and presents an important economic
advantage, as the natural L-tryptophan is much more
widely available and cheaper than the non-natural D-isomer.
Additionally, the compounds derived from L-tryptophan
were more active than those derived from D-tryptophan
(e.g., 14 > 11, 24 > 21 and 28 > 25), further supporting
these conclusions.
The nature of the alkyl side chain group on the hydantoin
ring does not significantly influence the potency. It seems that
a wide range of substituents on the hydantoin nitrogen appear
to be tolerated without affecting PDE5 inhibitory activity.
Most of the 1,2,3-trisubstituted tetrahydro-β-carboline de-
rivatives 31-40 were inactive as PDE5 inhibitors. However,
three compounds (31, 33, and 40) did show moderate activity
with IC50 values of 0.38-2.3 μM. Compounds 33 and 40 were

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 499
Table 4. Chemical Structure, Growth Inhibition of MDA-MB-231 Breast Tumor Cell Line, and PDE5 Inhibitory Activity of Tetrahydro-β-carboline-
piperazinedione Derivatives (41-68)
% growth
inhibition at
10 μMa
growth inhibition
IC50 (μM)b and
CI at p < 0.05
% PDE 5
inhibition at
10 μMa
PDE 5 inhibition
IC50 μMb and CI
at p < 0.05
compd
stereochemistry
X,Y
R
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R12aR
6S,12aS
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R,12aR
6S,12aR
6S,12aS
6R,12aS
6R,12aR
6S,12aS
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
2,6-dichloro
2,6-dichloro
-CH3
-CH3
-CH3
-CH3
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C2H5
-C4H9
-C4H9
10
65
70
23
30
66
52
20
30
44
35
50
31
24
45
65
82
42
15
70
71
30
36
40
58
50
16
20
ND
3.26 (2.52-4.20)
7.10 (4.70-9.33)
ND
ND
95
90
82
95
91
90
85
95
80
45
67
78
40
28
95
69
78
89
94
70
78
70
35
30
35
85
21
21
0.050 (0.03-0.068)
1.20 (0.82-2.34)
0.85 (0.70-1.03)
0.050 (0.041-0.067)
0.002 (0.0006-0.008)
0.26 (0.14-0.48)
0.99 (0.87-1.13)
0.010 (0.006-0.022)
1.34 (0.60-2.87)
ND
1.54 (1.21-1.95)
ND
ND
ND
ND
ND
3.30 (1.11-4.60)
2.04 (1.86-2.22)
ND
ND
ND
ND
ND
ND
0.27 (0.19-0.37)
1.11 (0.56-2.21)
4.46 (3.70-5.36)
0.29 (0.23-0.35)
0.19 (0.09-0.43)
1.26 (0.70-2.35)
1.35 (1.08-1.69)
0.26 (0.20-0.32)
ND
1.20 (0.66-2.18)
3.28 (2.75-3.86)
ND
-C4H9
-C4H9
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
-C-(CH3)3
ND
2.92 (1.61-5.29)
2.59 (1.48-4.56)
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.27 (1.11-1.45)
ND
ND
a Calculated from duplicate values. b Calculated from at least 8 concentrations, each with quadruple replicates, p < 0.05.
Table 5. PDE Isozyme Selectivity of the Most Active Tetrahydro-β-carboline-hydantoins and Piperazinedione Derivatives
PDE3B
inhibition IC50
PDE4B
inhibition IC50
PDE5A inhibition
PDE3B
PDE11A inhibition
PDE11A inhibition
IC50 (μM,cGMP)b inhibition IC50 (μM,cGMP)b and (μM,cAMP)b and IC50 (μM, cAMP)b and IC50, (μM, (cGMP)b selectivity
compd
11
and CI at p < 0.05
(μM,cAMP)b
CI at p < 0.05
CI at p < 0.05
CI at p < 0.05
and CI at p < 0.05
indexa
0.010 (0.007-0.013)
0.007 (0.005-0.011)
0.002 (0.0009-0.006)
>50
>50
>50
>50
>50
>50
>50
>50
34 (24.8-47.1)
3.60 (2.80-4.60) 13.00 (10.9-15.6)
>50
>50
2.00 (1.4-2.90)
0.31 (0.25-0.38)
1.50 (1.28-1.80)
0.011 (0.008-0.015)
0.05 (0.044-0.055)
31.0
208.3
5.50
14
45
0.104 (0.09-0.14)
0.30 (0.23-0.38)
tadalafil 0.003 (0.0007-0.005)
16.60
a IC50 of PDE11A (cGMP)/IC50 of PDE5A (cGMP). b Calculated from at least 8 concentrations, each with quadruple replicates, p < 0.05.
of the S,S configuration and compound 40 was the only
derivative with the pendant 2,6-dichlorophenyl that showed
PDE5 inhibitory activity. This demonstrates that these deri-
vatives may be following a different trend, which could be due
to their being alkyl halides and, therefore, having an alternate
means of interacting with PDE5.
We then expanded the hydantoin ring to a six-membered
piperazinedione system. Again, PDE5 inhibitory activity was
limited to derivatives with pendant 2,4-dichlorophenyl or 3,4-
dichlorophenyl, whereas those derivatives with the 2,6-di-
chlorophenyl substituent were inactive, confirming that the
potency for PDE5 inhibition is related to the nature of the
positional isomers. Furthermore, these observations indicate
that those derivatives with the 2,6-dichlorophenyl moiety
may be sterically locked in a conformation that prevents
binding to PDE5.
derived from the amino acid. In the piperazinediones, we intro-
duced four substituents on the nitrogen of the piperazinedione
ring with 2,4-dichlorophenyl, viz methyl, ethyl, butyl, and
tert-butyl. The order of PDE inhibitory activity was ethyl >
methyl > tert-butyl > butyl as shown by compounds 41 and
44 versus 45 and 48 versus 59 and 62 versus 55 and 58,
respectively. This indicates that there is an optimum size for
the N substitution and steric parameters are of importance in
this regard.
Compounds 11, 14, and 45 were the most potent of these
derivatives and showed comparable potency to tadalafil for
PDE5 inhibition. Therefore, we determined their isozyme
selectivity by comparing their potency for PDE5 inhibition
to their potency for inhibition of PDE3, PDE4, and PDE11.
As summarized in Table 5, all three compounds as well as the
reference compound tadalafil lacked the ability to inhibit
either cAMP or cGMP hydrolysis by PDE3B at concentra-
tions up to 50 μM. Interestingly, compounds 11 and 14
showed mild to moderate inhibition of the cAMP selective
PDE4B despite tadalafil having little effect on the activity of
The 6R,12aR or 6R,12aS rather than the 6S,12aR or
6S,12aS were more potent for PDE5 inhibition, confirming
that PDE5 inhibition is predominantly determined by the
stereochemical state of C-6 rather than the chiral carbon

500 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
˚
Figure 3. Docked pose of compound 11 (orange) in the cavity of
1UDU superimposed on tadalafil, white, featuring van der Waals
spheres on the chlorine atoms illustrating the steric requirements
of 11.
Figure 2. Side view of the crystal structure 1UDU with 2.83 A
resolution (yellow) containing a tadalafil molecule (green) in the
active side. Another PDE5 structure from the PDB with a higher
˚
resolution of 2.30 A that was cocrystallized with sildenafil, 2H42
(light blue), is superposed onto 1UDU to account for unresolved
amino acids in the flexible loop region of 1UDU.
this PDE isoform. The effects of the compounds on cAMP
and cGMP hydrolysis by PDE11A showed that the piperazi-
nedione derivative 45 and tadalafil were the most efficacious
and potent for PDE11 inhibition. Furthermore, compound 45
represents the most potent PDE11 inhibitor reported to date
withanIC50 value of11nM. Thehydantoinderivatives11and
14 were more selective for inhibition of cGMP hydrolysis by
PDE5 compared to inhibition of cGMP hydrolysis by PDE11A
with selectivity indices of 31 and 208, respectively. Conversely,
tadalafil possessed a selectivity index of just 16.6 for PDE5
versus PDE11, suggesting thatcompounds11and14possess a
lower potential for side effects due to their reduced effect upon
PDE11. Additionally, these studies demonstrate that the
PDE5/PDE11 selectivity of these compounds is primarily
influenced by the size and interactions of the tetracycle terminal
ring and not the pendant aryl.
We performed in silico docking studies in order to elucidate
the structural basis for the binding of the compounds.
Tadalafil has been cocrystallized with human PDE5 (PDB
1UDU) at a low resolution of 2.8 A, while sildenafil has been
cocrystallized with human PDE5 (PDB 2H42) at a better
˚
resolution of 2.3 A. Analysis of the crystal structures showed
Figure 4. Docked pose of compound 11 (orange) in the cavity of
1UDU (yellow). The flexible loop from 2H42 containing Ile665 is
superimposed for comparison (light blue).
ꢀ
˚
with the central scaffold of tadalafil. The 4-chloro substituent
is buried deeply within the central hydrophobic cavity. The
geometry of the hydrogen bond with Gln817 as well as the π-
stacking interaction with Phe820, as observed in tadalafil, is
also conserved.The interaction with Gln817 is believed to
restrict the rotational freedom of the side chain of Gln817
which is critical for cGMP binding.24
Compound 12 differs from 11 only in one stereocenter. The
inversion of the 5R center to 5S induces a 180° flip of the
central scaffold. This has consequences both on the proposed
binding mode (i.e., the docking pose possessing the optimal
consensus score) and on the measured affinity. The hydrogen
bond between Gln817 and the NH-group of the indole moiety
present in both tadalafil and all potent novel compounds of R
configuration is lost. Additionally, the added carbonyl group
clashes with the carbonyl of Gln817 (red spheres), leading to
significantly reduced binding affinity. However, the π-π stack-
ing interaction with Phe820 is conserved as shown in Figure 5.
The biological data revealed the 2,6-dichloro analogues to
be substantially impaired PDE5 inhibitors. The docked pose
that some residues in close proximity to the binding site are
not resolved in 1UDU. Superimposing the two crystal struc-
tures highlights the difference between 1UDU and 2H42:
following Tyr664, which is a part of the flexible loop, Ile665
is present in 2H42 but not in 1UDU. From this structural
comparison, it seems reasonable to assume that this isoleucine
residue could form substantial hydrophobic interactions with
the N-alkyl substituent of tadalafil or its analogues as shown
in Figure 2.
Compound 11, which was found to inhibit PDE5 with low
nanomolar affinity, docked into the PDE5 active site in
1UDU in a very similar manner to that of tadalafil as shown
in Figure 3. However, the effects of the hydrophobic side
chains at the five-membered ring can not be reproduced due
to the missing hydrophobic loop region containing Ile665
(Figure 4). Compound 11 has the same stereoconfiguration as
tadalafil at the carbon where the dichlorobenzene ring is
attached, leading to a rather close match of the docked pose

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 501
Figure 6. Effects of 2,6-dichloro substitution at the aromatic ring
system exemplified by the docked pose of 53 (orange) superimposed
on tadalafil, white.
Figure 5. Docked pose of compound 12, orange, superimposed on
tadalafil, white.
active among all of the compounds presented here for growth
inhibition with a submicromolar IC50 value. Again, none of the
active compounds were of the 2,6-dichlorophenyl derivatives.
In addition to PDE5 inhibitors, this series of compounds were
alkylating agents and this may contribute to their activity.
For the piperazinedione derivatives 41-68, seven com-
pounds showed growth inhibitory activity, with IC50 values
in the range of 1.2-7.1 μM. As with PDE5 inhibition, none of
the active growth inhibitors were of 2,6-dichlorphenyl sub-
stituent, confirming that such an isomer is deleterious to both
PDE5 and growth inhibitory activity. Additionally, all active
compounds were of the S configuration at the C-6 position,
indicating stereochemical preferentiality for this series of
compounds. The active derivatives had different substituents
on the terminal piperazinedione N, demonstrating that a wide
variety of substituents can be tolerated at this residue. Finally,
all of the active derivatives demonstrated appreciable potency
for PDE5 inhibition. Together, these data suggest that mod-
ifications to the terminal ring of the tetracycle in tetrahydro-β-
carboline-hydantoin and tetrahydro-β-carboline-piperazine-
dione skeletons improves the PDE5 selectivity and tumor cell
growth inhibitory properties of this group of compounds.
of compound 53 demonstrates that the steric requirements of
the compound (chloro substituents shown as green spheres)
exceed the size of the binding pocket (shown as a transparent
surface). These steric hindrances likely reduce binding affinity.
Additionally, the missing 4-chloro substituent in 53 leaves the
deep hydrophobic cavity unaddressed, as shown in Figure 6.
Because of previous reports that PDE5 may serve as a novel
targetfor the discovery of new anticancer drugs, the tumorcell
growth inhibitory activity of these derivatives was measured
versus the human MDA-MB-231 breast tumor cell line, a cell
line that has been shown to rely on PDE5 for growth and
survival.6 Among the 1,3-disubstituted tetrahydro-β-carbo-
lines 1-10, only compounds 2, 6, and 7 showed IC50 values
of <10 μM. The three compounds share the 1S chiral carbon,
and they all possess 2,4-dichlorophenyl or 3,4-dichlorophenyl
pendant aryls. This stereochemistry preference is opposite to
that observed with PDE5 inhibition, whereas the pendant aryl
positional isomer preference is the same.
Only seven compounds of the hydantoin derivatives 11-30
showed growth inhibitory activity, with IC50 values of 0.64-
5.37 μM. Again, all the active compounds had either 2,4-
dichlorophenyl or 3,4-dichlorophenyl substitution but none
of them are 2,6-dichlorophenyl derivatives. This supports the
dependence of the growth inhibitory activity on the substitu-
tion patterns at the aromatic ring. Conversely, the compounds
were of variable stereochemistry at the chiral carbons, indi-
cating insensitivity to chirality issues within this series of
compounds. Interestingly, all growth inhibitory compounds
in this series were also PDE5 inhibitors, although potency
for PDE5 inhibition did not correlate with growth inhibition
and may reflect additional factors when comparing activities
within cells compared with effect observed in isolated
enzymes.
Conclusions
The present structure-activity relationship study revealed
the following first-time essential findings for interaction be-
tween tetrahydro-β-carboline-hydantoins and tetrahydro-β-
carboline-piperazinedione derivatives and PDE5 including:
the interaction between the alkyl side chain on the N of the
terminal ring and Ile665 and hydrophobic interaction of the
pendant aryl with Met816, this being in addition to the known
H-bonding of the indole NH with Gln817 and π-π stacking
between the indole ring and the phenyl of Phe820. The
stereochemistry of the chiral carbon derived from the alde-
hyde, not the one derived from the amino acid, is the most
crucial feature for PDE5 inhibition; this expands the chemical
space from which novel PDE5 inhibitors might be explored.
The size and lipophilicity of the aryl attached to the stereo-
carbon derived from the aldehyde modulates activity as well.
The cross interaction with PDE11 can be modulated by the
nature of the terminal ring; this may evoke new studies to
improve PDE5/PDE11 selectivity or to bend toward one of
For the chloroethanone derivatives 31-40, only three com-
pounds showed appreciable growth inhibitory effect, namely
31, 32, and 34. Compound 32 differs from 31 by one stereo-
center, and its growth inhibitory potency is almost 10 times
that of 31. This indicates that the potency of these compounds
for growth inhibition is related to the stereochemistry of the
C-1 derived from the aldehyde. Compound 32 was the most

502 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
them. Evaluation of the new 68 derivatives in a cancer cell line
screening showed that the antiproliferative activity is not
necessarily parallel to the PDE5 inhibition; however, the latter
may be among the contributing factors to the antiproliferative
activity and thus it is important to strike the right balance
between selectivity/nonselectivity of cGMP-PDEs inhibition.
The new emerging indications for PDE5 inhibitors confirm
the need to the discovery of more inhibitors of PDE5 for novel
indications.
Ar), 7.35 (d, 1H, J=7.0 Hz, Ar), 7.26-7.15 (m, 4H, Ar þ NH),
7.04-6.93 (m, 2H, Ar), 5.82 (s, 1H, CHPh), 4.03-4.00 (m, 1H,
CHCOOCH3), 3.83 (s, 3H, OCH3), 3.25 (dd, 1H, J=4.3/7.0 Hz,
CHaHb), 3.23-3.04 (m, 1H, CHaHb). 13C NMR: δ 172.82, 136.16,
135.01, 134.11, 132.10, 131.23, 130.02, 128.06, 126.81, 123.40,
122.26, 119.83, 118.28, 110.97, 109.33, 56.55 (C1), 54.53, 52.40
(C3), 25.28. MS: m/z 378 (Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374
(Mþ), m/z 217 (100%). Elemental analysis: calculated for (C19H16-
Cl2N2O2) C, H, N.
(1S,3R)Methyl-1-(2,4-Dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (2). Yield 34%; yellow powder; mp
130-133 °C. Rf = 0.12 (CH2Cl2). IR (cm-1): 3388 (-NH-),
1735(-CO-).1HNMR:δ7.64 (s, 1H, NH), 7.56 (d, 1H, J=7.0 Hz,
Ar), 7.47 (s, 1H, NH), 7.00 (d, 1H, J = 7.0 Hz, Ar), 7.27-7.12
(m, 5H, Ar), 5.97 (s, 1H, CHPh), 3.92-3.88 (m, 1H, CHCOO-
CH3), 3.74 (s, 3H, OCH3), 3.30 (dd, 1H, J=4.6/7.6 Hz, CHaHb),
3.17-3.15 (m, 1H, CHaHb). 13C NMR: δ 173.09, 137.15, 136.29,
134.73, 134.51, 131.16, 130.83, 129.78, 127.27, 126.63, 122.48,
119.82, 118.38, 111.08, 109.43, 52.40(C1), 52.27, 51.15(C3), 24.39.
MS: m/z 378 (Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%).
Elemental analysis: calculated for (C19H16Cl2N2O2) C, H, N.
(1S,3S)Methyl-1-(2,4-Dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (3). Yield 45%; white powder; mp
90-92 °C; Rf=0.19 (CH2Cl2). IR (cm-1): 3433 (-NH-), 1728
(-CO-). 1H NMR: δ 7.90 (s, 1H, NH), 7.54-7.43 (m, 2H, Ar),
7.38 (d, 1H, J=7.2 Hz, Ar), 7.26-7.10 (m, 5H, Ar þ NH), 5.8 (s,
1H, CHPh), 3.90 -3.87 (m, 1H, CHCOOCH3), 3.82 (s, 3H,
OCH3), 3.25 (dd, 1H, J=4.3/7.6 Hz, CHaHb), 3.01-2.90 (m,
1H, CHaHb). 13C NMR: δ 172.90, 136.15, 135.10, 134.65, 133.15,
132.12, 131.45. 130.0, 129.38, 128.04, 126.85, 122.01, 119.8, 115.92,
110.96, 109.37, 56.57 (C1), 51.36 (C3), 25.38. MS: m/z 378
(Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%). Elemental
analysis: calculated for (C19H16Cl2N2O2) C, H, N.
Experimental Section
Chemistry. Mass spectra were measured using a Jeol MSta-
tion, double-focusing magnetic sector (MS 700). Melting points
were determined on a Buchi melting point apparatus and are
uncorrected. IR spectra were recorded on Perkin-Elmer Paragon
1
1000 using KBr-KCl plates. H NMR and 13C NMR spectra
were recorded on Jeol Eclipse, 400 and 500 MHz device using
CDCl3 as a solvent and TMS as a standard, chemical shifts (δ)
were reported in parts per million (ppm) downfield from TMS,
and J is expressed in Hz. The following abbreviations are used
for multiplicity of NMR signals: s=singlet, d=doublet, q =
quartet, m = multiplet, dd = doublet of doublet, br = broad.
Elemental analyses were performed by the Microanalytical
Unit, Faculty of Science, Cairo University; found values were
within (0.4% of the theoretical ones unless otherwise indicated.
Column chromatography was performed using silica-gel 70-
230 mesh. Reaction progress was monitored by TLC, performed
on precoated silica gel plates (ALUGRAM SIL G/UV254), and
detection of the components was made by short UV light. All
reactions were carried out under nitrogen or argon. All chemi-
cals and solvents were obtained from Sigma-Aldrich and were
used without further purification. All the described compounds
had >95% purity as determined by GC/MS and the areas under
the peaks.
(1R,3S)Methyl-1-(2,4-Dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (4). Yield 30%; white powder; mp
128-130 °C; Rf = 0.12 (CH2Cl2). IR (cm-1): 3396 (-NH-),
1739 (-CO-). 1H NMR: δ 7.80 (s, 1H, NH), 7.49 (s, 1H, NH),
7.46 (d, 1H, J=7.2 Hz, Ar), 7.19-7.11 (m, 5H, Ar), 6.94 (d, 1H,
J=7.0 Hz, Ar), 5.30 (s, 1H, CHPh), 3.74 (s, 3H, OCH3), 3.68-
3.62 (m, 1H, CHCOOCH3), 3.30 (dd, 1H, J=4.6/7.6 Hz, CHaHb),
3.16-3.14 (m, 1H, CHaHb). 13C NMR: δ 173.17, 136.27, 134.70,
134.50, 131.12. 131.0, 129.77, 127.27, 126.65, 122.46, 120.01,
119.82, 118.39, 111.06, 109.46, 52.38 (C1), 51.28 (C3), 51.14.
MS: m/z 378 (Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%).
Elemental analysis: calculated for (C19H16Cl2N2O2) C, H, N.
(1R,3R)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (5). Yield 35%; yellow powder; mp
90-92 °C; Rf = 0.15 (CH2Cl2). IR (cm-1): 3396 (-NH-), 1734
(-CO-). 1H NMR: δ 9.0 (s, 1H, NH), 7.51-7.44 (m, 5H, Ar þ
NH), 7.25 (d, 1H, J=7.2 Hz, Ar), 7.20-7.10 (m, 2H, Ar), 5.23 (s,
1H, CHPh), 3.96 (dd, J=4.0/7.6 Hz, 1H, CHaHb,), 3.82 (s, 3H,
OCH3), 3.05-3.01 (m, 1H, CHCOOCH3), 3.00-2.95 (m, 1H,
CHaHb). 13C NMR: δ 172.88, 140.98, 136.25, 133.30, 133.12,
132.74, 130.92, 130.58, 127.94, 126.90, 122.35, 119.89, 118.37,
111.03, 109.34, 57.73 (C1), 56.64, 52.42 (C3), 25.43. MS:m/z 378
(Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%). Elemental
Procedure for Preparation of D-andL-Tryptophan Methyl Ester.13
A 250 mL round-bottomed flask containing methanol (100 mL)
was cooled in an ice-water bath. Acetyl chloride are (8 mL) was
then added dropwise to the flask over a period of 8 min using a
graduated dropping funnel while the flask was still in the ice bath.
The solution was stirred for 5 min before adding D or L-tryptophan
(15 g, 68.77 mmol) in one portion. The solution was then heated
to reflux and left for 5 h. It was then left to cool to room
temperature, and the solvent was removed under reduced pressure
to give tryptophan methyl ester hydrochloride. The free base was
obtained by adding dilute NH4OH and extracting the base using
CH2Cl2 (5 ꢀ 50 mL). The organic layer was dried over anhy-
drous Na2SO4 and put under reduced pressure to give a yellow-
ish solid. This solid was then used without further purification.
General Procedures for the Preparation of Methyl 1-(Aryl)-
2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate. The appropri-
ate tryptophan methyl ester (15.52 g, 60.93 mmol) was added to
the appropriate dichlorobenzaldehyde (11.72 g, 66.95 mmol)
and dissolved in CH2Cl2 (100 mL). The solution was cooled to
0 °C in an ice bath. Trifluoroacetic acid (3 mL) was then added
to the solution dropwise while still in the ice bath, and the
mixture was left to stir for 4 days at room temperature and under
N2 atmosphere. The reaction mixture was made basic by adding
dilute NH4OH solution and then extracted with CH2Cl2 (3 ꢀ
100 mL). The organic layer was washed with water and brine
and dried over anhydrous Na2SO4. It was then filtered and
evaporated under reduced pressure.
analysis: calculated for (C19H16Cl2N2O2), C, H, N. [R]20
þ58.35 (c=0.1, EtOH)
=
D
(1S,3R)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (6). Yield 20%; yellow powder; mp
140-143 °C; Rf=0.10 (CH2Cl2). IR (cm-1):3345 (-NH-), 1710
1
(-CO-). HNMR: δ 7.55 (d, 1H, J=7.2 Hz,, Ar), 7.50-7.02
(m, 3H, Ar), 7.20-7.10 (m, 5H, Ar), 5.49 (s, 1H, CHPh),
3.98-3.95 (m, 1H, CHCOOCH3), 3.73 (s, 3H, OCH3), 3.37 (d,
1H, J=8.0 Hz, CHaHb), 3.29 (dd, 1H, J=4.6/8.0 Hz, CHaHb,).
13C NMR: δ 172.87, 148.02, 136.27, 135.02, 132.95, 132.55, 130.72,
130.57, 128.02, 126.69, 122.51, 119.85, 118.45, 111.09, 108.57,
53.94 (C1), 52.59 (C3), 52.40, 24.17. MS: m/z 378 (Mþ þ 4), m/z
376 (Mþ þ 2), m/z 374 (Mþ, 100%). Elemental analysis:
The residue was purified and the isomers were separated by
column chromatography on silica gel eluting with CH2Cl2 to
give the respective cis-isomer first followed by the trans-isomer.
(1R,3R)Methyl-1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (1). Yield 35%; yellow powder; mp
88-92 °C; Rf=0.19 (CH2Cl2). IR (cm-1): 3406 (-NH-), 1734
(-CO-). 1H NMR: δ 7.48 (s, 1H, NH), 7.51 (d, 1H, J=7.0 Hz,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 503
calculated for (C19H16Cl2N2O2) C, H, N. [R]20D=þ69.5 (c =
0.1, EtOH).
4.40 (dd, 1H, J=4.3/7.0 Hz, CHC(O)N), 3.58-3.55 (m, 3H,
NCH2 þ CHaHb), 3.08-3.05 (m, 1H, CHaHb), 1.23 (t, 3H, J =
7.2 Hz, CH3). 13C NMR: δ 171.29, 154.39, 136.67, 135.06, 134.68,
133.98, 132.21, 129.44, 129.37, 128.16, 125.95, 123.14, 120.37,
118.50, 111.31, 107.61, 57.93 (C1), 51.46 (C3), 33.76, 22.46,
13.46. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ 2), m/z 413 (Mþ),
m/z 252 (100%). Elemental analysis: calculated for (C21H17Cl2-
N3O2) C, H, N.
(1S,3S)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (7). Yield 38%; yellow powder; mp
88-91 °C; Rf=0.15 (CH2Cl2). IR (cm-1): 3395 (-NH-), 1734
(-CO-). 1H NMR: δ 7.51-7.44 (m, 5H, Ar þ NH), 7.25 (d, 1H,
J=7.2 Hz, Ar), 7.20-7.10 (m, 2H, Ar), 5.24 (s, 1H, CHPh), 3.95
(dd, 1H, J=4.0/8.0 Hz, CHCOOCH3), 3.82 (s, 3H, OCH3), 3.25
(dd, 1H, J=4.6/7.6 Hz, CHaHb), 3.05-2.95 (m, 1H, CHaHb).
13C NMR: δ 172.78, 140.81, 136.27, 133.16, 133.12, 132.78,
130.919, 130.62, 127.99, 127.51, 126.88, 122.37, 119.90, 118.37,
111.04, 109.32, 57.70 (C1), 52.42 (C3), 25.36. MS: m/z 378
(Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%). Elemental
analysis: calculated for (C19H16Cl2N2O2) C, H, N. [R]20D=-58.35
(c=0.1, EtOH)
(1R,3S)Methyl-1-(3,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (8). Yield 25%; yellow powder; mp
139-142 °C; Rf = 0.10 (CH2Cl2). IR (cm-1): 3386 (-NH-),
1731 (-CO-). 1H NMR: δ 7.55 (d, 2H, J=7.0 Hz, Ar þ NH),
7.40-7.35 (m, 3H, Ar þ NH), 7.27 (d, 2H, J = 7.0 Hz, Ar),
7.21-7.10 (m, 2H, Ar), 5.45 (s, 1H, CHPh), 3.97-3.93 (m, 1H,
CHCOOCH3), 3.73 (s, 3H, OCH3), 3.31-2.26 (m, 1H, CHaHb),
3.18 (dd, 1H, J=4.6/8.0 Hz, CHaHb,). 13C NMR: δ 173.13, 139.96,
136.04, 135.80, 132.57, 130.45, 130.22, 129.07, 128.84, 126.98,
124.06, 121.91, 119.77, 118.01, 110.99, 108.63, 53.20 (C1), 52.36
(C3), 25.72. MS: m/z 378 (Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374
(Mþ, 100%). Elemental analysis: calculated for (C19H16Cl2-
N2O2) C, H, N. [R]20D=-69.5 (c=0.1, EtOH)
(1R,3R)Methyl-1-(2,6-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3 carboxylate (9). Yield 55%; red powder; mp 102-
105 °C; Rf=0.13 (100% CH2Cl2). IR (cm-1): 3389 (-NH-),
1729 (-CO-). 1H NMR: δ 7.58-7.52 (m, 3H, Ar þ NH), 7.28-
7.27 (m, 3H, Ar þ NH), 7.26-7.25 (m, 3H, Ar), 6.29 (s, 1H,
CHPh), 4.14-4.06 (m, 1H, CHCOOCH3), 3.85 (s, 3H, OCH3),
3.32 (dd, 1H, J=4.0/8.0 Hz, CHaHb), 3.03-2.99 (m, 1H, CHaHb).
13C NMR: δ 172.25, 138.07, 136.20, 135.88, 134.37, 130.47, 129.40,
127.84, 126.60, 121.82, 119.66, 118.07, 112.06, 111.02, 56.83
(C1), 54.07, 52.21 (C3), 37.04, 27.15. MS: m/z 378 (Mþ þ 4), m/z
376 (Mþ þ 2), m/z 374 (Mþ), m/z 217 (100%). Elemental analysis:
calculated for (C19H16Cl2N2O2) C, H, N.
(1S,3S)Methyl-1-(2,6-dichlorophenyl)-2,3,4,9-tetrahydro-1H-
β-carboline-3-carboxylate (10). Yield 57%; red powder; mp 101-
103 °C; Rf=0.13 (100% CH2Cl2). IR (cm-1): 3386 (-NH-), 1730
(-CO-). 1H NMR: δ 7.51-7.50 (m, 3H, Ar þ NH), 7.43 (d, 1H,
J=7.2 Hz, Ar), 7.26-7.25 (m, 3H, Ar þ NH), 7.13-7.11 (m, 2H,
Ar), 6.21(s, 1H, CHPh), 4.07-4.04 (m, 1H, CHCOOCH3), 3.83(s,
3H, OCH3), 3.25 (dd, 1H, J=4.0/8.0 Hz, CHaHb), 2.99-2.96 (m,
1H, CHaHb). 13C NMR: δ 173.13, 136.96, 135.80, 134.3, 132.57,
130.46, 129.08, 128.84, 126.98, 121.91, 119.77, 118.01, 110.99,
108.64, 57.21 (C1), 54.21, 52.36 (C3), 36.88, 25.72. MS: m/z 378
(Mþ þ 4), m/z 376 (Mþ þ 2), m/z 374 (Mþ, 100%). Elemental
analysis: calculated for (C19H16Cl2N2O2) C, H, N.
(5S,11aR) 5-(2,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo [10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (12).
Yield 57%; yellow powder; mp 126-129 °C; Rf=0.28 (CH2Cl2).
1
IR (cm-1): 3312 (-NH-), 1767, 1710 (-CO-). H NMR: δ
7.87 (s, 1H, NH), 7.53 (d, 1H, J=7.0 Hz, Ar), 7.31-7.12 (m, 6H,
Ar), 6.68 (s, 1H, CHPh), 4.51 (dd, 1H, J=4.3/7.6 Hz, CHC-
(O)N), 3.63-3.61 (m, 3H, NCH2 þ CHaHb), 2.92-2.89 (m, 1H,
CHaHb), 1.24 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ 172.51,
155.24, 136.56, 135.80, 135.28, 133.57, 130.29, 130.17, 129.37,
128.10, 125.92, 123.11, 120.34, 118.46, 111.31, 107.61, 54.71
(C1), 49.13 (C3), 33.76, 22.46, 13.46. MS: m/z 417 (Mþ þ 4), m/z
415 (Mþ þ 2), m/z 413 (Mþ), m/z 252 (100%). Elemental
analysis: calculated for (C21H17Cl2N3O2) C, H, N.
(5S,11aS) 5-(2,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo [10,50:1,6] pyrido[3,4-b]indole-1,3(2H)-dione (13).
Yield 68%; yellow powder; mp 226-228 °C; Rf=0.16 (CH2Cl2).
1
IR (cm-1): 3331 (-NH-), 1706 (-CO-), 1624 (-CO-). H
NMR: δ 7.90 (s, 1H, NH), 7.57 (d, 2H, J=7.8 Hz, Ar), 7.25-
7.18 (m, 5H, Ar), 6.41 (s, 1H, CHPh), 4.38 (dd, 1H, J=4.6/7.8 Hz,
CHC(O)N), 3.56-3.49 (m, 3H, NCH2 þ CHaHb), 3.07-3.04
(m, 1H, CHaHb), 1.20 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ
171.27, 154.33, 136.65, 135.04, 134.62, 133.98, 132.17, 129.37,
129.35, 128.10, 125.30, 123.08, 120.31, 118.45, 111.30, 107.54,
57.89 (C1), 51.41 (C3), 33.72, 22.42, 13.42. MS: m/z 418 (Mþ þ
4), m/z 416 (Mþ þ 2), m/z 414 (Mþ, 100%). Elemental analysis:
calculated for (C21H17Cl2N3O2) C, H, N.
(5R,11aS) 5-(2,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo [10,50:1,6] pyrido[3,4-b]indole-1,3(2H)-dione (14).
Yield 45%; yellow powder; mp 125-128 °C; Rf=0.28 (CH2Cl2).
1
IR (cm-1): 3331 (-NH-), 1704 (-CO-), 1623 (-CO-). H
NMR: δ 7.96 (s, 1H, NH), 7.51 (d, 2H, 8.0 J=Hz, Ar), 7.26-
7.12 (m, 5H, Ar), 6.21 (s, 1H, CHPh), 4.51-4.48 (m, 1H,
CHC(O)N), 3.63-3.60 (m, 3H, NCH2 þ CHaHb), 2.92-2.88
(m, 1H, CHaHb), 1.24 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ
172.50, 155.20, 136.55, 135.78, 135.23, 133.57, 130.22, 130.17,
130.13, 128.03, 125.89, 123.06, 120.28, 118.42, 111.27, 107.08,
54.64 (C1), 49.09 (C3), 33.91, 23.33, 13.46. MS: m/z 418 (Mþ þ
4), m/z 416 (Mþ þ 2), m/z 414 (Mþ), m/z 378 (100%). Elemental
analysis: calculated for C21H17Cl2N3O2 C, H, N.
(5R,11aR) 5-(3,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (15).
Yield 52%; white powder; mp 229-232 °C; Rf = 0.18 (CH2Cl2).
1
IR (cm-1): 3332 (-NH-), 1706 (-CO-), 1623 (-CO-). H
NMR: δ 7.88 (s, 1H, NH), 7.58 (d, 1H, J=7.0 Hz, Ar), 7.38 (s,
1H, Ar), 7.26-7.12 (m, 5H, Ar), 5.77 (s, 1H, CHPh), 4.35 (dd,
1H, J=4.6/9.7 Hz, CHC(O)N), 3.57-3.50 (m, 3H, NCH2 þ
CHaHb), 3.08-3.06 (m, 1H, CHaHb), 1.15 (t, 3H, J=7.2 Hz, CH3).
13C NMR: δ 171.19, 154.79, 139.06, 136.86, 132.99, 132.73,
132.19, 130.88, 129.72, 127.13, 126.04, 123.16, 120.38, 118.61,
111.32, 107.52, 57.90 (C1), 55.60, 53.45 (C3), 33.74, 13.46. MS:
m/z 418 (Mþ þ 4), m/z 416 (Mþ þ 2), m/z 414 (Mþ, 100%).
Elemental analysis: calculated for (C21H17Cl2N3O2) C, H, N.
(5S,11aR) 5-(3,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (16).
Yield 49%; yellow powder; mp 100-102 °C; Rf =0.36 (CH2-
Cl2). IR (cm-1): 3389 (-NH-), 1704 (-CO-), 1624 (-CO-).
1H NMR: δ 7.94 (s, 1H, NH), 7.57 (d, 1H, J = 8.0 Hz, Ar),
7.44-7.41 (m, 2H, Ar), 7.30 (d, 1H, J=8.0 Hz, Ar), 7.26-7.21
(m, 4H, Ar), 6.26 (s, 1H, CHPh), 4.28-4.23 (m, 1H, CHC(O)N),
3.93 (q, 2H, J=7.2 Hz, NCH2), 3.61-3.58 (m, 1H, CHaHb),
General Procedures for the Preparation of 5-(Aryl)-2-alkyl-
5,6,11,11a-tetrahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione. An appropriate amount of 1,3-disubstituted tetra-
hydro-β-carbolines 1-10 (0.38 g, 1 mmol) was dissolved in methyl
ethyl ketone (10 mL) and stirred. Excess amount of ethyl or tert-
butyl isocyanate (1.6 mmol) was added, and the mixture was then
stirred and left to reflux under N2 atmosphere for 16 h. The solvent
was evaporated under reduced pressure, and the residue was
purified by column chromatography on silica gel eluting with
CH2Cl2. The solution containing the desired product from the
column was evaporated under reduced pressure to give the pure
powder of the desired product.
(5R,11aR) 5-(2,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (11).
Yield 58%; yellow powder; mp 223-226 °C; Rf=0.16 (100%
1
CH2Cl2). IR (cm-1): 3321 (-NH-), 1764, 1705 (-CO-). H
NMR: δ 7.79 (s, 1H, NH), 7.57 (d, 1H, J=7.0 Hz, Ar), 7.50-
7.49 (m, 1H, Ar), 7.30-7.12 (m, 5H, Ar), 6.45 (s, 1H, CHPh),
2.92-2.89 (m, 1H, CHaHb), 1.14 (t, 3H, J=7.2 Hz, CH3). 13
NMR: δ 172.28, 154.84, 139.21, 136.68, 133.42, 133.25, 131.15,
C

504 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
130.00, 129.12, 127.62, 126.20, 125.98, 123.28, 120.40, 118.58,
111.35, 108.62, 53.11 (C1), 50.93 (C3), 33.88, 13.51. MS: m/z 418
(Mþ þ 4), m/z 416 (Mþ þ 2), m/z 414 (Mþ, 100%). Elemental
analysis: calculated for (C21H17Cl2N3O2) C, H, N.
111.26, 107.40, 58.26, 57.26 (C1), 51.33 (C3), 29.70, 28.74, 22.53.
MS: m/z 445 (Mþ þ 4), m/z 443 (Mþ þ 2), m/z 441 (Mþ), m/z 252
(100%). Elemental analysis: calculated (C23H21Cl2N3O2) C, H, N.
(5S,11aR) 5-(2,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(22). Yield 47%; yellowish powder; mp 205-208 °C; Rf=0.60
(5S,11aS) 5-(3,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (17).
Yield 67%; yellow powder; mp 230-234 °C; Rf=0.17 (CH2Cl2).
IR (cm-1): 3363 (-NH-), 1708 (-CO-), 1699(-CO-). 1H
NMR: δ 7.74 (s, 1H, NH), 7.58 (dd, 1H, J=2.0/8.0 Hz, Ar), 7.38
(d, 1H, 8.0 Hz, Ar), 7.26-7.19 (m, 5H, Ar), 5.75 (s, 1H, CHPh),
4.36 (dd, 1H, J=4.3/6.8 Hz, CHC(O)N), 3.55-3.50 (m, 3H,
NCH2 þ CHaHb), 3.09-3.02 (m, 1H, CHaHb), 1.21 (t, 3H, J=
7.2 Hz, CH3). 13C NMR: δ 171.14, 154.78, 139.00, 136.82, 133.02,
132.75, 132.16, 130.89, 129.67, 127.10, 126.03, 123.16, 120.39,
118.61, 111.32, 107.52, 57.90(C1), 55.60(C3), 33.74, 22.379,
13.46. MS: m/z 418 (Mþ þ 4), m/z 416 (Mþ þ 2), m/z 414
(Mþ, 100%). Elemental analysis: calculated for (C21H17Cl2-
N3O2) C, H, N.
1
(CH2Cl2). IR (cm-1): 3312 (-NH-), 1764, 1698 (-CO-). H
NMR: δ 7.86 (s, 1H, NH), 7.53-7.50 (m, 2H, Ar), 7.28-7.15 (m,
5H, Ar), 6.64 (s, 1H, CHPh), 4.39 (dd, 1H, J = 4.3/7.0 Hz,
CHC(O)N), 3.51 (dd, 1H, J=4.3/7.0 Hz, CHaHb), 2.88-2.85
(m, 1H, CHaHb), 1.61 (s, 9H, C(CH3)3). 13C NMR: δ 173.67,
156.09, 136.13, 135.08, 133.35, 131.36, 130.40, 130.20, 130.07,
128.10, 125.97, 122.97, 120.24, 118.44, 111.21, 106.96, 58.27,
54.14 (C1), 48.94 (C3), 28.68, 23.61. MS: m/z 445 (Mþ þ 4), m/z
443 (Mþ þ 2), m/z 441 (Mþ), m/z 57.1 (100%). Elemental analysis:
calculated (C23H21Cl2N3O2) C, H, N.
(5S,11aS) 5-(2,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(23). Yield 58%; white powder; mp 288-290 °C; Rf=0.42 (CH2-
Cl2). IR (cm-1): 3309 (-NH-), 1702 (-CO-), 1624 (-CO-).
1H NMR: δ 7.81 (s, 1H, NH), 7.53 (d, 1H, J=7.2 Hz, Ar), 7.45
(d, 1H, J=7.2 Hz, Ar), 7.25-7.16 (m, 4H, Ar), 6.99-7.01 (m,
1H, Ar), 6.42 (s, 1H, CHPh), 4.24 (q, 1H, J=7.6 Hz, CHC(O)N),
3.43 (dd, 1H, J=4.0/7.6 Hz, CHaHb), 2.99-2.98 (m, 1H, CHaHb),
1.63 (s, 9H, C(CH3)3). 13C NMR: δ 172.32, 155.32, 136.58,
135.93, 134.22, 132.93, 132.51, 131.5, 129.53, 128.71, 128.15,
125.98, 122.99, 121.7, 120.26, 118.49, 111.25, 107.36, 58.23,
57.23 (C1), 51.31 (C3), 28.72, 22.50. MS:m/z 445 (Mþ þ 4), m/
z 443 (Mþ þ 2), m/z 441 (Mþ, 100%).Elemental analysis:
calculated (C23H21Cl2N3O2) C, H, N.
(5R,11aS) 5-(3,4-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (18).
Yield 50%; yellow powder; mp 105-107 °C; Rf=0.36 (CH2Cl2).
1
IR (cm-1): 3389 (-NH-), 1702 (-CO-), 1624 (-CO-). H
NMR: δ 8.10 (s, 1H, NH), 7.54 (d, 1H, J=7.4 Hz, Ar), 7.42 (d,
1H, J=7.4 Hz, Ar), 7.32-7.31 (m, 1H, Ar), 7.26-7.22 (m, 4H,
Ar), 6.26 (s, 1H, CHPh), 4.27-4.25 (m, 1H, CHC(O)N), 3.93-
3.93 (m, 3H, NCH2 þ CHaHb), 2.92-2.88 (m, 1H, CHaHb), 1.21
(t, 3H, J=7.2 Hz, CH3). 13C NMR: δ 172.30, 154.84, 139.28,
136.71, 133.39, 131.12, 129.99, 129.15, 127.61, 126.03, 125.98,
123.23, 120.35, 118.56, 111.36, 108.54, 53.10 (C1), 50.92 (C3),
33.88, 22.36, 13.50. MS: m/z 418 (Mþ þ 4), m/z 416 (Mþ þ 2), m/
z 414 (Mþ), m/z 213 (100%). Elemental analysis: calculated for
(C21H17Cl2N3O2) C, H, N.
(5R,11aS) 5-(2,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(24). Yield 54%, white powder; mp 204-207 °C; Rf = 0.59
(5R,11aR) 5-(2,6-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (19).
Yield 56%; yellow powder; mp 84-86 °C; Rf=0.21 (CH2Cl2).
1
(CH2Cl2). IR (cm-1): 3311 (-NH-), 1698, 1622 (-CO-). H
NMR: δ 7.92 (s, 1H, NH), 7.52 (d, 1H, J=7.2 Hz, Ar), 7.47 (d,
1H, J=7.2 Hz, Ar), 7.25 (d, 1H, J=7.0 Hz, Ar), 7.25-7.13 (m,
4H, Ar), 6.71 (s, 1H, CHPh), 4.37 (q, 1H, J=7.6 Hz, CHC(O)N),
3.49 (dd, 1H, 4.0/7.6 Hz, CHaHb), 2.87-2.84 (m, 1H, CHaHb),
1.62 (s, 9H, C(CH3)3). 13C NMR: δ 73.67, 156.06, 136.51,
136.11, 135.05, 133.37, 130.37, 130.16, 130.09, 128.06, 125.96,
122.94, 120.21, 118.42, 111.21, 106.94, 58.26, 57.23, 54.09 (C1),
48.92 (C3), 28.67, 23.60. MS: m/z 445 (Mþ þ 4), m/z 443 (Mþ þ
2), m/z 441 (Mþ), 442 (100%). Elemental analysis: calculated for
(C23H21Cl2N3O2) C, H, N.
1
IR (cm-1): 3338 (-NH-), 1768, 1707 (-CO-). H NMR: δ
7.65 (s, 1H, NH), 7.68 (d, 1H, J=8.0 Hz, Ar), 7.48-7.47 (m, 1H,
Ar), 7.30-7.17 (m, 5H, Ar), 6.84 (s, 1H, CHPh), 4.43 (dd, 1H,
J=4.0/8.4 Hz, CHC(O)N), 3.96-3.92 (m, 2H, NCH2), 3.57-
3.54 (m, 1H, CHaHb) 3.11-3.07 (m, 1H, CHaHb), 1.22 (t, 3H,
J=7.2 Hz, CH3). 13C NMR: δ 171.61, 153.90, 136.48, 135.47,
134.88, 132.00, 130.27, 129.92, 129.19, 128.75, 125.88, 122.86,
120.24, 118.48, 111.14, 108.49, 57.78 (C1), 51.56 (C3), 33.63,
21.82, 13.48. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ 2), m/z 413
(Mþ), m/z 214 (100%). Elemental analysis: calculated for
(C21H17Cl2N3O2) C, H, N.
(5R,11aR) 5-(3,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(25). Yield 53%; yellow powder; mp 175-177 °C; Rf = 0.38
(CH2Cl2). IR (cm-1): 3278 (-NH-), 1706 (-CO-), 1647
(-CO-). 1H NMR: δ 8.88 (s, 1H, NH), 7.54-7.43 (m, 2H,
Ar), 7.26-7.17 (m, 5H, Ar), 6.42 (s, 1H, CHPh), 4.25 (dd, 1H,
J=3.6/7.6 Hz, CHC(O)N), 3.56-3.53 (m, 1H, CHaHb), 3.48-
3.41 (m, 1H, CHaHb), 1.63 (s, 9H, C(CH3)3). 13C NMR: δ 173.06,
157.86, 143.22, 136.94, 133.83, 132.57, 132.33, 131.60, 128.31,
126.21, 126.58, 125.60, 122.54, 119.89, 118.60, 111.10, 105.99,
56.61 (C1), 54.64, 52.37, 51.36(C3), 28.69, 23.37. MS:m/z 445
(Mþ þ 4), m/z 443 (Mþ þ 2), m/z 441 (Mþ), m/z 442 (100%).
Elemental analysis: calculated for (C23H21Cl2N3O2) C, H, N.
(5S,11aR) 5-(3,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(26). Yield 56%; yellow powder; mp 275-277 °C; Rf = 0.66
(CH2Cl2). IR (cm-1): 3329 (-NH-), 1701 (-CO-), 1624
(5S,11aS) 5-(2,6-Dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahy-
dro1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (20).
Yield 52%; yellow powder; mp 78-80 °C; Rf=0.22 (CH2Cl2).
1
IR (cm-1): 3338 (-NH-), 1768, 1707 (-CO-). H NMR: δ
7.63 (s, 1H, NH), 7.56 (d, 1H, J=8.0 Hz, Ar), 7.48-7.47 (m, 1H,
Ar), 7.30-7.14 (m, 5H, Ar), 6.84 (s, 1H, CHPh), 4.42 (dd, 1H,
J=4.0/8.6 Hz, CHC(O)N), 3.96-3.93 (m, 2H, NCH2), 3.57-
3.54 (m, 1H, CHaHb) 3.10-3.07(m, 1H, CHaHb), 1.23 (t, 3H,
J=7.2 Hz, CH3). 13C NMR: δ 171.61, 153.90, 136.48, 135.48,
134.88, 132.01, 130.27, 129.93, 129.19, 128.75, 125.87, 122.85,
120.24, 118.48, 111.15, 108.49, 57.78 (C1), 51.57 (C3), 33.63,
21.82, 13.48. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ 2), m/z 413
(Mþ), m/z 214 (100%). Elemental analysis: calculated for
(C21H17Cl2N3O2) C, H, N.
(5R,11aR) 5-(2,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(21). Yield 50%; yellowish powder; mp 280-282 °C; Rf =
0.41(CH2Cl2). IR (cm-1): 3308 (-NH-), 1756, 1699 (-CO-).
1H NMR: δ 7.80 (s, 1H, NH), 7.53 (d, 1H, J=8.0 Hz, Ar), 7.47 (s,
1H, Ar), 7.30-7.17 (m, 5H, Ar), 6.37 (s, 1H, CHPh), 4.24 (dd,
1H, J=4.3/7.0 Hz, CHC(O)N), 3.43 (dd, 1H, J=4.2/7.0 Hz,
1
(-CO-). H NMR: δ 7.94 (s, 1H, NH), 7.58 (d, 2H, J=8.0
Hz, Ar), 7.42-7.37 (m, 4H, Ar), 7.29-7.21 (m, 1H, Ar), 6.22 (s,
1H, CHPh), 4.13 (q, 1H, J = 7.6 Hz, CHC(O)N), 3.47-3.43 (m,
1H, CHaHb), 2.87-2.84 (m, 1H, CHaHb), 1.59 (s, 9H, C(CH3)3).
13C NMR: δ 173.47, 155.63, 139.41, 136.64, 133.33, 133.09,
131.10, 129.98, 129.41, 127.65, 126.05, 123.14, 120.30, 118.58,
111.28, 108.52, 58.21, 53.44, 52.40 (C1), 50.68 (C3), 29.65, 28.68,
23.52. MS: m/z 445 (Mþ þ 4), m/z 443 (Mþ þ 2), m/z 441 (Mþ,
100%). Elemental analysis: calculated (C23H21Cl2N3O2) C, H, N.
CHaHb), 2.98-2.97 (m, 1H, CHaHb), 1.58 (s, 9H, C(CH3)3). 13
C
NMR: δ 172.34, 155.35, 136.58, 135.94, 134.27, 132.91, 132.54,
131.23, 129.58, 128.72, 128.18, 126.02, 123.04, 120.30, 118.53,

Article
(5S,11aS)-5-(3,4-Dichlorophenyl)-2-t-butyl-5,6,11,11a-tetra-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 505
3.43-3.39 (m, 2H, CH2). MS: m/z 454 (Mþ þ 4), m/z 452
(Mþ þ 2), m/z 450 (Mþ), m/z 373 (100%). Elemental analysis:
calculated for (C21H17Cl3N2O3) C, H, N.
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (27).
Yield 48%; yellow powder; mp 174-175 °C; Rf=0.36 (CH2Cl2).
IR (cm-1):3361 (-NH-), 1704 (-CO-), 1624 (-CO-). 1H
NMR: δ 8.89 (s, 1H, NH), 7.61-7.36 (m, 4H, Ar), 7.26-7.22 (m,
3H, Ar), 6.32 (s, 1H, CHPh), 4.21 (dd, 1H, J = 3.6/7.6 Hz,
CHC(O)N), 3.43 (dd, 1H, J=3.6/7.8 Hz, CHaHb), 3.03-3.00
(m, 1H, CHaHb), 1.58 (s, 9H, C(CH3)3). 13C NMR: δ 172.21,
155.78, 139.73, 136.80, 133.06, 132.42, 130.97, 129.12, 126.58,
126.12, 123.06, 120.33, 118.64, 111.24, 107.55, 58.26 (C1), 57.21,
55.55, 53.42 (C3), 52.37, 29.68, 28.71, 23.37. MS: m/z 445 (Mþ þ
4), m/z 443 (Mþ þ 2), m/z 441 (Mþ), m/z 442 (100%). Elemental
analysis: calculated for (C23H21Cl2N3O2) C, H, N.
(1S,3R)Methyl 1-(2,4-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (32). Yield 80%; green
powder; mp 106-109 °C; Rf=0.37 (100% CH2Cl2). IR (cm-1):
3370 (-NH-), 1739, 1668 (-CO-). 1HNMR:δ8.08 (s, 1H, NH),
7.51 (d, 1H, J = 7.2, Ar), 7.28-7.11 (m, 6H, Ar), 6.57 (s, 1H,
CHPh), 5.30 (br s, 1H, CHCOOCH3), 4.18-4.10 (m, 2H, COC-
H2Cl), 3.64 (s, 3H, OCH3), 3.53-3.49 (m, 2H, CH2). MS: m/z 454
(Mþ þ 4), m/z 452 (Mþ þ 2), m/z 450 (Mþ), m/z 373 (100%).
Elemental analysis: calculated for (C21H17Cl3N2O3) C, H, N.
(1S,3S)Methyl 1-(2,4-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (33). Yield 88%; white
powder; mp 215-218 °C; Rf = 0.52 (CH2Cl2). IR (cm-1): 3315
(5R,11aS) 5-(3,4-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(28). Yield 55%; yellow powder; mp 273-275 °C; Rf = 0.68
(CH2Cl2). IR (cm-1): 3320 (-NH-), 1702 (-CO-), 1624
1
(-NH-), 1737 (-CO-), 1664 (-CO-). H NMR: δ 7.50 (brs,
1H, NH), 7.26-7.15 (m, 7H, Ar), 6.57 (s, 1H, CHPh), 4.52 (s, 1H,
CHCOOCH3), 4.20-4.15 (m, 2H, COCH2Cl), 3.63 (br s, 3H,
OCH3), 3.49-3.48 (m, 2H, CH2). MS: m/z 454 (Mþ þ 4), m/z 452
(Mþ þ 2), m/z 450 (Mþ, 100%). Elemental analysis: calculated for
(C21H17Cl3N2O3) C, H, N.
1
(-CO-). H NMR: δ 7.80 (s, 1H, NH), 7.59 (d, 2H, J=8.2
Hz, Ar), 7.44-7.39 (m, 4H, Ar), 7.29-7.20 (m, 1H, Ar), 6.30 (s,
1H, CHPh), 4.14 (q, 1H, J=8.0 Hz, CHC(O)N), 3.48-3.44 (m,
1H, CHaHb), 2.88-2.85 (m, 1H, CHaHb), 1.61 (s, 9H, C(CH3)3).
13C NMR: δ 173.43, 155.61, 139.36, 136.61, 133.35, 133.12,
131.12, 129.99, 129.40, 127.67, 126.03, 123.16, 120.33, 118.57,
111.26 108.59, 58.19, 52.40 (C1), 50.70 (C3), 28.66, 23.51.; MS:
m/z 445 (Mþ þ 4), m/z 443 (Mþ þ 2), m/z 441 (Mþ, 100%), m/z
442 (100%). Elemental analysis: calculated for (C23H21Cl2-
N3O2) C, H, N.
(1R,3S)Methyl 1-(2,4-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (34). Yield 75%; white
powder; mp 109-112 °C; Rf =0.37 (CH2Cl2). IR (cm-1): 3347
1
(-NH-), 1739 (-CO-), 1668 (-CO-). H NMR: δ 7.80 (brs,
1H, NH), 7.39 (d, 1H, J=8.0 Hz, Ar), 7.18-7.12 (m, 6H, Ar), 6.54
(s, 1H, CHPh), 5.56 (d, 1H, J=7.0 Hz, CHCOOCH3), 3.95-3.93
(m, 2H, COCH2Cl), 3.72 (s, 3H, OCH3), 3.31 (dd, 1H, J = 3.6/7.6
Hz, CHaCHb), 3.18 (dd, 1H, J=3.6/7.6 Hz, CHaCHb). MS: m/z
454 (Mþ þ 4), m/z 452 (Mþ þ 2), m/z 450 (Mþ), m/z 373 (100%).
Elemental analysis: calculated for (C21H17Cl3N2O3) C, H, N.
(1R,3R)Methyl 1-(3,4-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (35). Yield 86%; white
powder; mp 283-285 °C; Rf =0.47 (CH2Cl2). IR (cm-1): 3283
(-NH-), 1737 (-CO-), 1662 (-CO-). 1H NMR: δ 8.9 (s, 1H,
NH), 7.60 (d, 2H, J=7.6 Hz, Ar), 7.32-7.16 (m, 5H, Ar), 6.83 (s,
1H, CHPh), 4.99 (d, 1H, J = 7.0 Hz, CHCOOCH3), 4.35 (m,
COCH2Cl), 4.22 (dd, 1H, J=3.6/7.6 Hz, CHaCHb), 3.70 (dd, 1H,
J=3.6/7.6 Hz, CHaCHb), 3.49 (s, 3H, OCH3). MS:m/z 454 (Mþ þ
4), m/z 452 (Mþ þ 2), m/z 450 (Mþ, 100%). Elemental analysis:
calculated for (C21H17Cl3N2O3) C, H, N.
(5R,11aR) 5-(2,6-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1H-imidazo [10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(29). Yield 48%; orange-yellow powder; mp 245-248 °C; Rf =
0.46 (CH2Cl2). IR (cm-1): 3356 (-NH-), 1762, 1698 (-CO-).
1H NMR: δ 7.62 (s, 1H, NH), 7.55 (d, 1H, J=8.2 Hz, Ar), 7.48-
7.47 (m, 1H, Ar), 7.26-7.19 (m, 5H, Ar), 6.79 (s, 1H, CHPh),
4.27 (dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 3.42-3.41 (dd, J =3.6/
7.6 Hz, 1H, CHaHb), 3.08-3.05 (m, 1H, CHaHb), 1.61 (s, 9H,
C(CH3)3). 13C NMR: δ 172.68, 154.73, 136.49, 134.80, 134.39,
132.58, 131.45, 130.29, 129.56, 129.28, 128.88, 125.93, 122.80,
120.16, 118.54, 111.09, 108.54, 58.03, 56.93 (C1), 53.44, 51.30
(C3), 28.76, 21.85. MS: m/z 445 (Mþ þ 4), m/z 443 (Mþ þ 2), m/z
441 (Mþ), m/z 385 (100%). Elemental analysis: calculated for
(C23H21Cl2N3O2) C, H, N.
(5S,11aS) 5-(2,6-Dichlorophenyl)-2-tert-butyl-5,6,11,11a-tet-
rahydro-1Himidazo [10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(30). Yield 45%; yellowish powder; mp 243-245 °C; Rf=0.44
(CH2Cl2). 1H NMR: δ 7.64 (s, 1H, NH), 7.56 (d, 1H, J=8.2 Hz,
Ar), 7.49-7.48 (m, 1H, Ar), 7.30-7.17 (m, 5H, Ar), 6.80 (s, 1H,
CHPh), 4.27 (dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 3.41 (dd, 1H,
J=3.6/7.6 Hz, CHaHb), 3.09-3.06 (m, 1H, CHaHb), 1.62 (s, 9H,
C(CH3)3). 13C NMR: δ 172.67, 154.73, 136.47, 135.43, 134.79,
134.38, 132.58, 131.47, 130.29, 129.57, 129.29, 128.89, 122.81,
120.17, 118.54, 111.09, 108.55, 58.03, 56.93 (C1), 51.30 (C3),
29.04, 28.75, 21.84. MS: m/z 445 (Mþ þ 4), m/z 443 (Mþ þ 2), m/
z 441 (Mþ), m/z 385 (100%). Elemental analysis: valculated for
(C23H21Cl2N3O2) C, H, N.
General Procedures for the Preparation of Methyl 1-(2,6-
Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-β-
carboline-3-carboxylate. Chloroacetyl chloride (6.41 mmol) was
added dropwise to a well stirred solution of an appropriate
amount of β-carboline 1-10 (2.67 mmol) and NaHCO3 (0.271 g,
3.23 mmol) in CHCl3 (40 mL) under ice cooling. The mixture
was then left to stir under nitrogen atmosphere at room tem-
perature for 1 h. The mixture was diluted with CH2Cl2, washed
with a solution of NaHCO3 and brine, dried over Na2SO4, and
evaporated under reduced pressure.
(1S,3R)Methyl 1-(3,4-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (36). Yield 79%; white
powder; mp 163-165 °C; Rf = 0.22 (CH2Cl2). IR (cm-1):3387
(-NH-), 1734(-CO-), 1663 (-CO-). 1H NMR: δ 8.9 (s, 1H,
NH), 7.77 (d, 2H, J = 8.2 Hz, Ar), 7.62-7.42 (m, 2H, Ar), 7.26-
7.23 (m, 3H, Ar), 6.72 (s, 1H, CHPh), 4.99-4.97 (m, 1H, CHC-
OOCH3), 4.36-4.34 (m, 2H, COCH2Cl), 4.10 (s, 1H, CHaCHb),
3.75 (dd, 1H, J=3.6/7.6 Hz, CHaCHb), 3.49 (s, 3H, OCH3). MS:
m/z 454 (Mþ þ 4), m/z 452 (Mþ þ 2), m/z 450 (Mþ, 100%).
Elemental analysis: calculated for (C21H17Cl3N2O3) C, H, N.
(1S,3S)Methyl 1-(3,4-Dichlorophenyl)-2-(2chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (37). Yield 84%; white
powder; mp 283-284 °C; Rf =0.45 (CH2Cl2). IR (cm-1): 3281
1
(-NH-), 1736(-CO-), 1662 (-CO-). H NMR:δ 8.9 (s, 1H,
NH), 7.77 (d, 2H, J=8.0 Hz, Ar), 7.61(d, 2H,J=8.0 Hz, Ar), 7.32-
7.17 (m, 3H, Ar), 6.83 (s, 1H, CHPh), 4.98 (d, 1H, J=7.0 Hz, CHC-
OOCH3), 4.36-4.34 (m, 2H, COCH2Cl),4.21(d,1H,J =3.4/7.6 Hz,
CHaCHb), 3.70 (dd, 1H, J=3.4/7.6 Hz, CHaCHb), 3.46 (s, 3H,
OCH3). MS: m/z 454 (Mþ þ 4), m/z 452 (Mþ þ 2), m/z 450 (Mþ,
100%). Elemental analysis: calculated for (C21H17Cl3N2O3) C,H,N.
(1R,3S)Methyl 1-(3,4-Dichlorophenyl)-2(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (38). Yield 77%; white
powder; mp 162-165 °C; Rf =0.24 (CH2Cl2). IR (cm-1): 3275
(-NH-), 1738, 1663 (-CO-). 1H NMR: δ 7.9 (s, 1H, NH), 7.75
(d, 2H, J=7.6 Hz, Ar), 7.22-7.12 (m, 5H, Ar), 6.03 (s, 1H, CHPh),
4.16 (d, 1H, J = 7.0 Hz, CHCOOCH3), 4.13-4.06 (m, 2H,
COCH2Cl), 3.61-3.58 (m, 2H, CH2), 3.47 (s, 3H, OCH3). MS:
m/z 454 (Mþ þ 4), m/z 452 (Mþ þ 2), m/z 450 (Mþ, 100%).
Elemental analysis: calculated for (C21H17Cl3N2O3) C, H, N.
(1R,3R)Methyl
1-(2,4-Dichlorophenyl)-2-(2-chloroacetyl)-
2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate (31). Yield 78%;
orange powder; mp 215-218 °C; Rf=0.53 (CH2Cl2). IR (cm-1):
3294 (-NH-), 1731, 1666 (-CO-). 1HNMR:δ7.50 (s, 1H, NH),
7.30-7.16 (m, 7H, Ar), 6.63 (s, 1H, CHPh), 4.53 (s, 1H, CHCO-
OCH3), 4.20-4.15 (m, 2H, COCH2Cl), 3.64 (s, 3H, OCH3),

506 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
(1R,3R)Methyl 1-(2,6-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxylate (39). Yield 76%; brown
powder; mp 147-150 °C; Rf=0.44 (100% CH2Cl2). IR (cm-1):
3304 (-NH-), 1739, 1666 (-CO-). 1H NMR: δ 8.98 (s, 1H, NH),
7.75-7.15 (m, 7H, Ar), 6.90 (s, 1H, CHPh), 4.99 (s 1H,
CHCOOCH3), 4.25-4.22 (m, 2H, COCH2Cl), 3.67 (s, 3H, OCH3),
3.37-3.16 (m, 2H, CH2).MS:m/z454 (Mþ þ 4), m/z452 (Mþ þ 2),
m/z 450 (Mþ), m/z 375 (100%). Elemental analysis: calculated for
(C21H17Cl3N2O3) C,H,N.
30%; white powder; mp 301-303 °C; Rf=0.23 (CH2Cl2/MeOH
98:2). IR (cm-1): 3305 (-NH-), 1665 (-CO-), 1624 (-CO-).
1H NMR: δ 8.09 (s, 1H, NH), 7.54 (d, 2H, J=8.0 Hz, Ar), 7.31
(d, 2H, J =8.0 Hz, Ar), 7.26-7.08 (m, 3H, Ar), 6.32 (s, 1H,
CHPh), 4.24 (dd, 1H, J=3.6/7.2 Hz, CHaHb), 4.14 (d, 1H, J=
7 Hz, CHC(O)N), 4.07 (d, 2H, J=7 Hz, CH2C(O)N), 3.47 (dd,
1H, J=3.6/7.2 Hz, CHaHb), 3.01 (s,3H, NCH3). 13C NMR: δ
165.86, 162.97, 135.61, 135.01, 133.70, 131.75, 130.25, 129.04,
128.15, 127.15, 126.14, 123.08, 120.31, 118.43, 111.33, 109.23,
52.95 (C1), 51.39, 49.34 (C3), 33.76, 27.16. MS: m/z 417 (Mþ þ
4), m/z 415 (Mþ þ 2), m/z 413 (Mþ), m/z 414 (100%). Elemental
analysis: calculated for (C21H17Cl2N3O2) C, H, N.
(1S,3S)Methyl 1-(2,6-Dichlorophenyl)-2-(2-chloroacetyl)-2,3,4,
9-tetrahydro-1H-β-carboline-3-carboxyl (40). 78%; brown pow-
der; mp 153-156 °C. Rf=0.42 (100% CH2Cl2). IR (cm-1): 3309
1
(6R,12aR) 6-(2,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (45). Yield
34%; white powder; mp 203-207 °C; Rf=0.36 (CH2Cl2/MeOH
98:2). IR (cm-1): 3444 (-NH-), 1663, 1628 (-CO-). 1H NMR:
δ 8.12 (s, 1H, NH), 7.59-7.11 (m,7H, Ar), 6.66 (s, 1H, CHPh),
4.38 (dd, 1H, J=3.6/7.2 Hz, CHC(O)N), 4.27-3.78 (m, 3H,
CH2C(O)N þ CHaHb), 3.44-3.40 (m, 1H, CHaHb), 3.25-3.23
(m, 2H, NCH2), 1.23 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ
166.57, 165.53, 165.35, 138.83, 136.26, 133.61, 132.04, 129.42,
128.25, 127.20, 122.83, 120.21, 118.56, 111.30, 106.58, 56.27
(C1), 53.42 (C3), 49.56, 41.30, 27.00, 24.12, 12.00. MS: m/z 431
(Mþ þ 4), m/z 429 (Mþ þ 2), m/z 427 (Mþ), m/z 57 (100%).
Elemental analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6S,12aR) 6-(2,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (46). Yield
33%; white powder; mp 227-230 °C; Rf=0.37 (CH2Cl2/MeOH
98:2). IR (cm-1): 3394(-NH-), 1663, 1627 (-CO-). 1H NMR:
δ 8.32 (s, 1H, NH), 7.54-7.09 (m,7H, Ar), 6.72(s, 1H, CHPh),
4.21 (dd, 1H, J=3.6/7.2 Hz, CHC(O)N), 4.12-3.99 (m, 3H,
CH2C(O)N þ CHaHb), 3.57-3.49 (m, 1H, CHaHb), 3.47-3.37
(m, 2H, NCH2), 1.22 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ
165.39, 163.36, 136.36, 135.57, 135.03, 133.68, 131.84, 130.20,
129.12, 127.14, 126.15, 122.99, 120.23, 118.40, 111.38, 109.11,
52.99 (C1), 49.38 (C3), 48.80, 40.87, 27.04, 11.71. MS: m/z 431
(Mþ þ 4), m/z 429 (Mþ þ 2), m/z 427 (Mþ), m/z 57 (100%).
Elemental analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6S,12aS) 6-(2,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (47). Yield
33%; white powder; mp 205-208 °C; Rf = 0.35 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3316 (-NH-), 1670 (-CO-), 1663
(-CO-). 1H NMR: δ 8.21 (s, 1H, NH), 7.57 (d, 2H, J = 7.6 Hz,
Ar), 7.52 (d, 2H, J = 7.6 Hz, Ar), 7.39-7.10 (m, 3H, Ar), 6.62 (s,
1H, CHPh), 4.37 (dd, 1H, J = 3.6/7.2 Hz, CHC(O)N),
4.11-4.04 (m, 3H, CH2C(O)N þ CHaHb), 3.41-3.39 (m, 1H,
CHaHb), 2.99-2.96 (m, 2H, NCH2), 1.22 (t, 3H, J = 7.2 Hz,
CH3). 13C NMR: δ 165.56, 163.36, 156.59, 131.84, 131.28,
130.21, 129.42, 128.26, 127.24, 122.83, 120.25, 120.21, 118.57,
111.37, 109.16, 106.59, 56.29 (C1), 53.57 (C3), 40.96, 30.95,
27.02. MS: m/z 430 (Mþ þ 4), m/z 428 (Mþ þ 2, 100%), m/z 426
(Mþ). Elemental analysis: calculated for (C22H19Cl2N3O2)
C, H, N.
(-NH-), 1737, 1666 (-CO-). H NMR: δ 8.98 (s, 1H, NH),
7.30-7.15 (m, 7H, Ar), 6.90 (s, 1H, CHPh), 4.99 (s, 1H, CHC-
OOCH3), 4.25-4.21 (m, 2H, COCH2Cl), 3.68 (s, 3H, OCH3),
3.22-3.17 (m, 2H, CH2). MS: m/z 454 (Mþ þ 4), m/z 452 (Mþ þ
2), m/z 450 (Mþ), m/z 375 (100%). Elemental analysis: calcu-
lated for (C21H17Cl3N2O3) C, H, N.
General Procedures for the Preparation of 2-Alkyl 6-(Aryl)-
2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-
1,4-dione. An appropriate amount of methyl amine (2.8 mmol)
was added to a solution of the appropriate amount of chloro-
acetyl derivative (1.4 mmol) in methanol (25 mL). The mixture
was heated to reflux under nitrogen atmosphere for 16 h. It was
then cooled to room temperature and evaporated to dryness under
reduced pressure. The residue was then dissolved in CH2Cl2, the
organic layer was washed with water and dried over Na2SO4,
filtered, and evaporated under reduced pressure. The crude prod-
uct was then purified using column chromatography.
(6R,12aR) 6-(2,4-Dichlorophenyl)-2-methyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (41). Yield
38%; white powder; mp 222-225 °C; Rf=0.24 (CH2Cl2/MeOH
98:2). IR (cm-1): 3307 (-NH-), 1665, 1624 (-CO-). 1H NMR:
δ 8.07 (s, 1H, NH), 7.58-7.17 (m, 7H, Ar), 6.64 (s, 1H, CHPh),
4.25 (dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 4.23-4.07 (m, 3H,
CH2C(O)N þ CHaHb), 3.47 (dd, 1H, J=3.4/7.6 Hz, CHaHb),
3.01 (s,3H, NCH3). 13C NMR: δ 165.86, 162.96, 136.31, 135.61,
135.01, 131.76, 130.25, 129.05, 127.16, 126.14, 123.08, 120.31,
118.43, 111.33, 109.24, 56.45 (C1), 52.94, 51.39 (C3), 49.33,
33.34, 27.16. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ 2), m/z
413 (Mþ), m/z 57 (100%). Elemental analysis: calculated for
(C21H17Cl2N3O2) C, H, N.
(6S,12aR) 6-(2,4-Dichlorophenyl)-2-methyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (42). Yield
35%; white powder; mp 305-308 °C; Rf=0.24 (CH2Cl2/MeOH
98:2). IR (cm-1): 3305 (-NH-), 1665, 1624 (-CO-). 1H NMR:
δ 8.27 (s, 1H, NH), 7.51-7.06 (m, 7H, Ar), 6.75 (s, 1H, CHPh),
4.23 (dd, 1H, J=3.4/7.2 Hz, CHC(O)N), 4.13 -4.01 (m, 3H,
CH2C(O)N þ CHaHb), 3.47 (dd, 1H, J=3.4/7.2 Hz, CHaHb),
3.00 (s,3H, NCH3). 13C NMR: δ 165.88, 162.99, 136.35, 135.59,
135.00, 133.71, 131.79, 130.21, 129.06, 127.15, 123.03, 120.26,
118.41, 111.37, 109.14,53.44, 52.92 (C1), 51.38, 49.37 (C3),
33.33, 27.17. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ 2), m/z
413 (Mþ), m/z 57 (100%). Elemental analysis: calculated for
(C21H17Cl2N3O2) C, H, N.
(6R,12aS) 6-(2,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (48). Yield
30%; white powder; mp 225-228 °C; Rf=0.34 (CH2Cl2/MeOH
98:2). IR (cm-1): 3287 (-NH-), 1665 (-CO-), 1658 (-CO-).
1H NMR: δ 8.34 (s, 1H, NH), 7.52 (d, 2H, J=7.6 Hz, Ar), 7.43
(d, 2H, J =7.6 Hz, Ar), 7.30-7.16 (m, 3H, Ar), 6.72 (s, 1H,
CHPh), 4.21 (dd, 1H, J=3.6/7.2 Hz, CHC(O)N), 4.07-3.99 (m,
3H, CH2C(O)N þ CHaHb), 3.48-3.43 (m, 1H, CHaHb), 2.99-
2.96 (m, 2H, NCH2), 1.18 (t, 3H, J=7.2 Hz, CH3). 13C NMR: δ
165.38, 136.35, 136.34, 135.57, 135.02, 133.66, 131.82, 130.19,
129.10, 127.13, 122.99, 121.30, 120.23, 118.40, 111.36, 109.09,
52.98 (C1), 49.37 (C3), 48.80, 33.80, 27.03. MS: m/z 430 (Mþ þ
4), m/z 428 (Mþ þ 2, 100%), m/z 426 (Mþ). Elemental analysis:
calculated for (C22H19Cl2N3O2) C, H, N.
(6S,12aS) 6-(2,4-Dichlorophenyl)-2-methyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (43). Yield
33%; white powder; mp 223-225 °C; Rf=0.22 (CH2Cl2/MeOH
98:2). IR (cm-1): 3314 (-NH-), 1702 (-CO-), 1664 (-CO-).
1H NMR: δ 8.12 (s, 1H, NH), 7.55 (d, 2H, J=7 Hz, Ar), 7.40 (d,
2H, J=7 Hz, Ar), 7.28-7.10 (m, 3H, Ar), 6.34 (s, 1H, CHPh),
4.09-4.02 (m, 1H, CHC(O)N), 4.12-3.94 (m, 2H, CH2C(O)N),
3.81 (dd, 1H, J = 3.4/7.2 Hz, CHaHb), 3.25-3.22 (m, 1H,
CHaHb), 3.09 (s,3H, NCH3). 13C NMR δ 166.13, 165.99,
138.84, 136.30, 133.61, 131.27, 129.43, 128.25, 127.24, 125.84,
122.87, 120.23, 118.59, 111.33, 111.26, 106.62, 56.23 (C1), 53.63,
53.45 (C3), 51.89, 33.76. MS: m/z 417 (Mþ þ 4), m/z 415 (Mþ þ
2), m/z 413 (Mþ),
(6R,12aR) 6-(3,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (49). Yield
35%; white powder; mp 298-300 °C; Rf=0.33 (CH2Cl2/MeOH
(6R,12aS) 6-(2,4-Dichlorophenyl)-2-methyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (44). Yield

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 507
98:2). IR (cm-1):3317 (-NH-), 1673 (-CO-), 1662 (-CO-).
1H NMR: δ 7.93 (s, 1H, NH), 7.57-7.54 (m, 2H, Ar), 7.42-7.38
(m, 2H, Ar), 7.27-7.16 (m, 3H, Ar), 7.01 (s, 1H, CHPh), 4.26
(dd, 1H, J=3.6/7.2 Hz, CHC(O)N), 4.12-3.98 (m, 3H, CH2C-
(O)N þ CHaHb), 3.60 (dd, 1H, J=3.6/7.2 Hz, CHaHb), 3.01-
2.96 (m, 2H, NCH2), 1.20 (t, 3H, J=7.2 Hz, CH3). MS:m/z 430
(Mþ þ 4), m/z 428 (Mþ þ 2, 100%), m/z 426 (Mþ). Elemental
analysis: calculated (C22H19Cl2N3O2) C, H, N.
(6S,12aR) 6-(3,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (50). Yield
28%; yellow powder; mp 92-95 °C; Rf=0.33 (CH2Cl2/MeOH
98:2). IR (cm-1): 3330 (-NH-), 1659 (-CO-), 1663 (-CO-).
1H NMR: δ 8.52 (s, 1H, NH), 7.57-7.55 (m, 2H, Ar), 7.43-7.40
(m, 2H, Ar), 7.28-7.12 (m, 3H, Ar), 6.72 (s, 1H, CHPh), 3.97-
3.93 (m, 3H, CH2C(O)N þ CHaHb), 3.25 (dd, 1H, J=3.6/7.2 Hz,
CHC(O)N), 3.17 (dd, 1H, J=3.6/7.2 Hz, CHaHb), 3.01-2.96
(m, 2H, NCH2), 1.24 (t, 3H, J=7.2 Hz, CH3). MS: m/z 430
(Mþ þ 4), m/z 428 (Mþ þ 2, 100%), m/z 426 (Mþ). Elemental
analysis: calculated for (C22H19Cl2N3O2) C, H, N.
130.24, 129.14, 127.16, 126.17, 123.05, 120.28, 118.42, 111.34,
109.23, 56.44 (C1), 53.07 (C3), 49.36, 45.74, 28.58, 27.07, 19.93,
13.79. MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455 (Mþ),
m/z 57 (100%).Elemental analysis: calculated for (C24H23Cl2-
N3O2) C, H, N.
(6S,12aR) 6-(2,4-Dichlorophenyl)-2-butyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (56). Yield
38%; white powder; mp 203-206 °C; Rf=0.52 (CH2Cl2/MeOH
98:2). IR (cm-1): 3326 (-NH-), 1668, 1651 (-CO-). 1H NMR:
δ 8.28 (s, 1H, NH), 7.52-7.07 (m, 7H, Ar), 6.73 (s, 1H, CHPh),
4.22-4.20 (dd, 1H, CHC(O)N), 4.11-4.03 (m, 2H, CH2C-
(O)N), 3.45-3.43 (dd, 1H, CHaHb), 3.28-3.24 (m, 1H, CHaHb),
2.99-2.96 (t, 2H, NCH2), 1.59-1.51 (m, 2H, CH2CH2CH3),
1.37-1.32 (m, 2H, CH2CH3), 0.97-0.94 (t, 3H, CH3). 13C
NMR: δ 165.57, 163.37, 136.31, 135.57, 135.01, 133.64, 131.81,
130.20, 129.11, 127.13, 126.14, 123.00, 120.24, 118.39, 111.35,
109.16, 53.03 (C1), 49.37 (C3), 49.33, 45.72, 28.55, 27.05, 19.90,
13.77. MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455 (Mþ),
m/z 57.1 (100%). Elemental analysis: calculated for (C24H23Cl2-
N3O2) C, H, N.
(6S,12aS) 6-(2,4-Dichlorophenyl)-2-butyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (57). Yield
34%; white powder; mp 140-143 °C; Rf=0.51 (CH2Cl2/MeOH
98:2). IR (cm-1): 3366 (-NH-), 1654 (-CO-), 1633 (-CO-).
1H NMR: δ 8.07 (s, 1H, NH), 7.57-7.55 (m, 2H, Ar), 7.32-7.13
(m, 5H, Ar), 6.87 (s, 1H, CHPh), 4.30-4.25 (d, 1H, CHC(O)N),
3.55-3.53 (m, 2H, CH2C(O)N), 3.32-3.29 (dd, 1H, CHaHb),
2.91-2.87 (m, 1H, CHaHb), 2.17-2.16 (t, 2H, NCH2), 1.50-
1.46 (m, 2H, CH2CH2CH3), 1.36-1.30 (m, 2H, CH2CH3),
0.93-0.89 (t, 3H, CH3). 13C NMR: δ 171.22, 169.44, 136.34,
135.77, 132.78, 130.58, 130.50, 128.06, 126.82, 122.61, 121.70,
119.89, 118.89, 111.11, 110.60, 57.44 (C1), 54.44 (C3), 52.29,
39.02, 31.55, 30.95, 24.61, 20.10, 13.77. MS: m/z 458 (Mþ þ 4),
m/z 456 (Mþ þ 2, 100%), m/z 454 (Mþ). Elemental analysis:
calculated for (C24H23Cl2N3O2) C, H, N.
(6R,12aS) 6-(2,4-Dichlorophenyl)-2-butyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (58). Yield
35%; yellow powder; mp 207-210 °C; Rf=0.51 (CH2Cl2/MeOH
98:2). IR (cm-1): 3399 (-NH-), 1699 (-CO-), 1653 (-CO-).
1H NMR: δ 8.44-8.41 (d, 1H, NH), 7.52-7.50 (d, 2H, Ar),
7.42-7.45 (d, 1H, Ar), 7.35-7.12 (m, 3H, Ar), 7.06-7.01 (d, 1H,
Ar), 6.70 (d, 1H, CHPh), 5.31 (s, 1H, CHC(O)N), 4.21-4.19
(dd, 1H, CHaHb), 4.08-3.98 (m, 2H, CH2C(O)N), 2.99-2.95
(m, 1H, CHaHb), 2.17-2.16 (t, 2H, NCH2), 1.57-1.54 (m, 2H,
CH2CH2CH3), 1.36-1.32 (m, 2H, CH2CH3), 0.97-0.93 (t, 3H,
CH3). 13C NMR: δ 171.21, 165.59, 136.35, 135.00, 133.66,
131.84, 130.18, 129.13, 127.13, 126.15, 122.96, 120.20, 118.39,
111.37, 109.09, 53.45, 53.02 (C1), 49.39 (C3), 49.34, 45.73, 30.95,
29.70, 19.93, 13.79. MS: m/z 458 (Mþ þ 4), m/z 456 (Mþ þ 2,
100%), m/z 454 (Mþ). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
(6R,12aR) 6-(2,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (59).
Yield 32%; white powder; mp 140-142 °C; Rf=0.65 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3309 (-NH-), 1724, 1666(-CO-). 1H
NMR: δ 8.15 (s, 1H, NH), 7.54-7.12 (m, 7H, Ar), 6.62 (s, 1H,
CHPh), 4.29-4.25 (dd, 1H, CHC(O)N), 4.21-3.96 (m, 3H,
CH2C(O)N þ CHaHb), 3.71-3.69 (dd, 1H, CHaHb), 1.52
(s,9H, C(CH3)3). 13C NMR: δ 167.55, 167.12, 164.12, 138.84,
136.00, 133.66, 129.43, 128.28, 127.35, 127.15, 122.79, 120.24,
118.53, 111.32, 109.39, 106.67, 58.15 (C1), 57.12, 53.90 (C3),
53.25, 47.19, 27.96, 26.82, 24.11. MS: m/z 459 (Mþ þ 4), m/z 457
(Mþ þ 2), m/z 455 (Mþ), m/z 57 (100%). Elemental analysis:
calculated for (C24H23Cl2N3O2) C, H, N.
(6S,12aS) 6-(3,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (51). Yield
28%; yellow powder; mp 298-301 °C; Rf = 0.34 (CH2Cl2/
MeOH 98.2). IR (cm-1):3325 (-NH-), 1712 (-CO-), 1663
1
(-CO-). H NMR: δ 8.19 (brs, 1H, NH), 7.54-7.53 (m, 2H,
Ar), 7.40-7.32 (m, 2H, Ar), 7.26-7.19 (m, 3H, Ar), 6.70 (s, 1H,
CHPh), 4.24 (dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 4.16-3.98 (m,
3H, CH2C(O)N þ CHaHb), 3.55 (dd, 1H, J=3.6/7.6 Hz, CHaHb),
2.95-2.94 (m, 2H, NCH2), 1.19 (t, 3H, J=7.2 Hz, CH3). MS:m/z
430 (Mþ þ 4), m/z 428 (Mþ þ 2, 100%), m/z 426 (Mþ).
Elemental analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6R,12aS) 6-(3,4-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (52). Yield
34%; white powder; mp 94-96 °C; Rf = 0.35 (CH2Cl2/MeOH
98:2). IR (cm-1): 3337 (-NH-), 1710 (-CO-), 1665 (-CO-).
1H NMR: δ 7.93 (s, 1H, NH), 7.57-7.54 (m, 2H, Ar), 7.42-7.38
(m, 2H, Ar), 7.28-7.19 (m, 3H, Ar), 7.01 (s, 1H, CHPh), 4.26
(dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 4.12-3.98 (m, 3H, CH2C-
(O)N þ CHaHb), 3.55 (dd, 1H, J=3.6/7.6 Hz, CHaHb), 3.01-
2.96 (m, 2H, NCH2), 1.22 (t, 3H, J=7.2 Hz, CH3). MS: m/z 430
(Mþ þ 4), m/z 428 (Mþ þ 2, 100%), m/z 426 (Mþ). Elemental
analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6R,12aR) 6-(2,6-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (53). Yield
30%; yellowish white powder; mp 110-112 °C; Rf=0.36 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3446 (-NH-), 1670, 1630(-CO-). 1H
NMR: δ 9.00 (s, 1H, NH), 7.57-7.37 (m,7H, Ar), 6.97 (s, 1H,
CHPh), 4.22 (dd, 1H, J=3.6/7.6 Hz, CHC(O)N), 3.78-3.65 (m,
3H, CH2C(O)N þ CHaHb), 3.59-3.55 (m, 1H, CHaHb), 3.25-
3.23 (m, 2H, NCH2), 1.25 (t, 3H, J=7.2 Hz, CH3). MS: m/z 431
(Mþ þ 4), m/z 429 (Mþ þ 2), m/z 427 (Mþ), m/z 57 (100%).
Elemental analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6S,12aS) 6-(2,6-Dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (54). Yield
28%; yellowish white powder; mp 112-115 °C; Rf=0.34 (CH2Cl2/
MeOH 98:2). 1H NMR: δ 8.99 (s, 1H, NH), 7.52-7.12 (m,7H,
Ar), 6.20 (s, 1H, CHPh), 4.18-4.15 (m, 1H, CHC(O)N), 4.07-
4.02 (m, 3H, CH2C(O)N þ CHaHb), 3.83-3.73 (m, 1H, CHaHb),
3.28-3.24 (m, 2H, NCH2), 1.32 (t, 3H, J=7.2 Hz, CH3). MS: m/
z 431 (Mþ þ 4), m/z 429 (Mþ þ 2), m/z 427 (Mþ), m/z 57 (100%).
Elemental analysis: calculated for (C22H19Cl2N3O2) C, H, N.
(6R,12aR) 6-(2,4-Dichlorophenyl)-2-butyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (55). Yield
37%; white powder; mp 140-142 °C; Rf=0.52 (CH2Cl2/MeOH
98:2). IR (cm-1): 3285 (-NH-), 1662, 1654 (-CO-). 1H NMR:
δ 8.15 (s, 1H, NH), 7.54-7.08 (m, 7H, Ar), 6.73 (s, 1H, CHPh),
4.23-4.21 (dd, 1H, CHC(O)N), 4.12-4.04 (m, 2H, CH2C-
(O)N), 3.48-3.46 (dd, 1H, CHaHb), 3.29-3.25 (m, 1H, CHaHb),
2.99-2.96 (t, 2H, NCH2), 1.59-1.55 (m, 2H, CH2CH2CH3),
1.38-1.34 (m, 2H, CH2CH3), 0.97-0.94 (t, 3H, CH3). 13C
NMR: δ 165.58, 163.39, 136.32, 135.87, 135.04, 133.66, 131.82,
(6S,12aR) 6-(2,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (60).
Yield 30%; white powder; mp 200-202 °C; Rf=0.64 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3399 (-NH-), 1741, 1663 (-CO-).
1H NMR: δ 8.27(s, 1H, NH), 7.52-7.06 (m, 7H, Ar), 6.70 (s, 1H,
CHPh), 4.24-4.20 (m, 1H, CHC(O)N), 4.15-3.97 (m, 3H,

508 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Mohamed et al.
CH2C(O)N þ CHaHb), 3.44-3.40 (dd, 1H, CHaHb), 1.51 (s,9H,
C(CH3)3). 13C NMR: δ 166.73, 164.12, 136.36, 135.54, 133.65,
131.81, 130.19, 129.11, 127.14, 126.22, 122.95, 120.20, 118.40,
111.35, 109.30, 55.05(C1), 53.89, 53.45 (C3), 52.84, 49.25, 46.63,
27.96, 27.68, 26.83. MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/
z 455 (Mþ), m/z 57 (100%). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
(6S,12aS) 6-(2,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (61).
Yield 32%; yellow powder; mp 145-148 °C; Rf=0.62 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3320 (-NH-), 1710 (-CO-), 1665
(-CO-). 1H NMR: δ 8.95 (s, 1H, NH), 7.53-7.51 (m, 1H, Ar),
7.46-7.42 (d, 2H, Ar), 7.27-7.12 (m, 4H, Ar), 6.62 (s, 1H,
CHPh), 4.31-4.25 (dd, 1H,CHC(O)N), 3.99-3.95 (dd, 1H,
CHaHb), 3.46-3.42 (m, 2H, CH2C(O)N), 2.96-2.92 (m, 1H,
CHaHb), 1.48 (s, 9H, C(CH3)3). 13C NMR: δ 167.55, 167.12,
166.72, 164.12, 131.81, 130.21, 129.42, 128.27, 127.14, 120.23,
118.52, 118.40, 111.25, 109.36, 106.67, 58.15 (C1), 57.12, 53.89
(C3), 53.26, 47.19, 46.63, 27.96, 24.10. MS: m/z 458 (Mþ þ 4), m/
z 456 (Mþ þ 2, 100%), m/z 454 (Mþ). Elemental analysis:
calculated for (C24H23Cl2N3O2) C, H, N.
Yield 36%; yellow powder; mp 95-97 °C; Rf=0.61 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3300 (-NH-), 1695 (-CO-), 1662
(-CO-). 1H NMR: δ 8.89 (s, 1H, NH), 7.70-7.65 (d, 1H, Ar),
7.45-7.42 (d, 1H, Ar), 7.29-7.14 (m, 5H, Ar), 6.80 (s, 1H,
CHPh), 3.97-3.93 (dd, 1H, CHC(O)N), 3.73-3.26 (m, 3H,
CH2C(O)N þ CHaHb), 3.26-3.25 (dd, 1H, CHaHb), 1.26 (s,
9H, C(CH3)3). MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455
(Mþ), m/z 375 (100%). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
(6R,12aR) 6-(2,6-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (67).
Yield 26%; yellowish white powder; mp 99-103 °C; Rf=0.61
(CH2Cl2/MeOH 98:2). IR (cm-1): 3317 (-NH-), 1730, 1685
(-CO-). 1H NMR: δ 8.99 (s, 1H, NH), 7.58-7.15 (m, 7H, Ar),
6.31 (s, 1H, CHPh), 4.32-4.31 (m, 1H, CHC(O)N), 4.17-4.07
(m, 3H, CH2C(O)N þ CHaHb), 3.92-3.85 (dd, 1H, CHaHb),
1.27 (s,9H, C(CH3)3). MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2),
m/z 455 (Mþ), m/z 57 (100%). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
(6S,12aS) 6-(2,6-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (68).
Yield 23%; yellowish-white powder; mp 90-92 °C; Rf=0.63
(CH2Cl2/MeOH 98:2). IR (cm-1): 3320 (-NH-), 1731, 1690
(-CO-). 1H NMR: δ 8.99 (s, 1H, NH), 7.60-7.13 (m, 7H, Ar),
6.24 (s, 1H, CHPh), 4.11-4.09 (m, 1H, CHC(O)N), 4.07-3.80
(m, 3H, CH2C(O)N þ CHaHb), 3.39-3.32 (dd, 1H, CHaHb),
1.26 (s,9H, C(CH3)3). MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2),
m/z 455 (Mþ), m/z 57 (100%). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
Biology. Phosphodiesterase Assay. Human recombinant PDE
5A (12.5 units/mL), 10 units/mL PDE3B, 400 units/mL PDE4B,
or 50 units/mL PDE11A (BPS Biosciences) were added to the
wells of black 96-well nonbinding plates. Immediately, the pro-
tein was treated with compound or vehicle control. Enzyme was
incubated with drug for 30 min prior to addition of 50 nM
TAMRA-cGMP and/or 50 nM fluorescein-cAMP (Molecular
Devices) wereaddedtoeach assay well. The plates wereincubated
for 1.5h at 30 °C. After incubation, immobilizedmetal affinity for
phosphochemicals (IMAP)-based fluorescence polarization (FP)
phosphodiesterase evaluation assay (Molecular Devices) binding
reagent was added to each well, and the plates were incubated for
an additional 30 min at 30 °C. FP was measured according to
manufacturer’s specifications usingaBiotekSynergy4platereader.
Cell Cultures. MDA-MB-231 breast tumor cells were obtained
from ATCC. They were grown under standard cell culture condi-
tions at 37 °C in a humidified atmosphere with 5% CO2. Cells were
grown in RPMI 1640 supplemented with 5% fetal bovine serum,
2 mM glutamine, 100 units/mL penicillin, 100 units/mL strepto-
mycin, and 0.25 μg/mL amphotericin. Cells were harvested at 70-
90% confluence with trypsin/EDTA and used immediately. Cell
count and viability was determined by Trypan blue staining
followed by hemocytometry. Only cultures displaying >95% cell
viability were used for experiments.
(6R,12aS) 6-(2,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a
exahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (62).
Yield 25%; yellow powder; mp 198-200 °C; Rf=0.61 (CH2Cl2/
MeOH 99.5:0.5). IR (cm-1): 3387 (-NH-), 1741 (-CO-), 1663
1
(-CO-). H NMR: δ 8.11-8.07 (d, 1H, NH), 7.51-7.49 (m,
2H, Ar), 7.27-7.09 (m, 5H, Ar), 6.58 (s, 1H, CHPh), 5.29-5.28
(dd, 1H, CHC(O)N), 3.66-3.61 (dd, 1H, CHaHb), 3.45-3.42
(m, 2H, CH2C(O)N), 2.96-2.91 (dd, 1H, CHaHb), 1.07 (s, 9H,
C(CH3)3). 13C NMR: δ 173.39, 171.76, 167.34, 131.56, 129.61,
129.42, 128.93, 128.88, 128.01, 127.04, 125.92, 120.05, 119.91,
118.32, 111.24, 105.97, 104.65, 55.95 (C1), 53.44 (C3), 50.51,
46.00, 28.63, 24.05. MS: m/z 458 (Mþ þ 4), m/z 456 (Mþ þ 2,
100%), m/z 454 (Mþ). Elemental analysis: calculated for
(C24H23Cl2N3O2) C, H, N.
(6R,12aR) 6-(3,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido [3,4-b]indole-1,4-dione (63).
Yield 36%; white powder; mp 299-303 °C; Rf=0.62 (CH2Cl2/
MeOH 98:2). IR (cm-1):3387 (-NH-), 1695 (-CO-), 1652
(-CO-). 1H NMR: δ 7.96 (s, 1H, NH), 7.55-7.53 (d, 2H, Ar),
7.43-7.31 (d, 2H, Ar), 7.28-7.00 (m, 3H, Ar), 6.71 (s, 1H,
CHPh), 4.35-4.28 (dd, 1H, CHC(O)N), 4.66-4.60 (dd, 1H,
CHaHb), 4.14-4.00 (m, 3H, CH2C(O)N þ CHaHb), 1.27 (s, 9H,
C(CH3)3). MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455
(Mþ, 100%). Elemental analysis: calculated for (C24H23Cl2-
N3O2) C, H, N.
(6S,12aR) 6-(3,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (64).
Yield 35%; yellow powder; mp 93-96 °C; Rf=0.62 (CH2Cl2/
MeOH 98:2). IR (cm-1):3377 (-NH-), 1662 (-CO-), 1653
(-CO-). 1H NMR: δ 8.01 (s, 1H, NH), 7.57-7.55 (d, 2H, Ar),
7.43-7.41 (d, 2H, Ar), 7.29-7.14 (m, 3H, Ar), 6.78 (s, 1H,
CHPh), 3.97-3.93 (dd, 1H, CHC(O)N), 3.73-3.26 (m, 3H,
CH2C(O)N þ CHaHb), 3.26-3.25 (dd, 1H, CHaHb), 1.27 (s,
9H, C(CH3)3). MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455
(Mþ, 100%). Elemental analysis: calculated for (C24H23Cl2-
N3O2) C, H, N.
(6S,12aS) 6-(3,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (65).
Yield 38%; yellow powder; mp 298-301 °C; Rf=0.62 (CH2Cl2/
MeOH 98:2). IR (cm-1): 3322 (-NH-), 1715, 1663 (-CO-).
1H NMR: δ 7.87 (s, 1H, NH), 7.55-7.54 (d, 2H, Ar), 7.40-7.33
(d, 2H, Ar), 7.27-7.01 (m, 3H, Ar), 6.61 (s, 1H, CHPh), 4.30-
4.25 (dd, 1H, CHC(O)N), 4.14-4.00 (m, 3H, CH2C(O)N þ
CHaHb), 4.60-4.57 (dd, 1H, CHaHb), 1.29 (s, 9H, C(CH3)3).
MS: m/z 459 (Mþ þ 4), m/z 457 (Mþ þ 2), m/z 455 (Mþ), m/z
375 (100%). Elemental analysis: calculated for (C24H23Cl2-
N3O2) C, H, N.
Growth Assays. Tissue culture treated microtiter 96-well plates
were seeded at a density of 5000 cells/well. The plates were
incubated for 18-24 h prior to any treatment. All test compounds
were solubilized in 100% DMSO and diluted with media to obtain
a final DMSO concentration of 0.1%. Cells were dosed, incubated
at 37 °C, and cell viability measured 72 h after treatment by the Cell
Titer Glo Assay (Promega), which is a luminescent assay that is an
indicator of live cells as a function of metabolic activity and ATP
content. The assay was performed according to manufacturer’s
specifications. Luminescence was measured by a Perkin-Elmer
Victor multilabel plate reader.
Experimental Design and Data Analysis. Drug effects on tumor
cell growth and PDE activity were measured and potency was
expressed by an IC50 value (50% inhibitory concentration). For
growth assays, the IC50 value was determined by testing a range of
eight concentrations with quadruple replicates per concentration.
For enzyme assays, the IC50 value was determined by testing a
(6R,12aS) 6-(3,4-Dichlorophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (66).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 509
range of 10 concentrations with quadruple replicates per concen-
tration. Dose response curves were analyzed using PrismTM 4
software (GraphPad) to calculate IC50 values using a four para-
meter logistic equation.
inhibits growth and induces apoptosis of human breast tumor cells
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Design, synthesis and biological evaluation of new tryptamine and
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Molecular Modeling. The crystal structure of tadalafil in
complex with PDE5 (PDB 1UDU) was prepared for docking
using Molecular Operating Environment software (MOE)
2009.10 (CCG). Addition of hydrogens and assignment
of correct protonation states of all residues at physiological
pH = 7.4 was performed using the Protonate3D module in
MOE. The compound structures were drawn using ChemSketch.
We employed a Pipeline Pilot (SciTegic Inc.) workflow to
systematically name and ionize the compounds (pH=7.4). In
the same workflow, we set a chiral flag for each compound
using the ChemAxon Standardizer component to keep the
molecules in their correct stereoconfigurations during later
steps. We then employed the wash and minimization procedures in
MOE to prepare the final compound library for docking.
The docking procedure was performed with GOLD v3.2
(CCDC). For all docking runs, we used default parameters.
The genetic algorithm was set to automatic mode at 200%
search effiency. The binding site was defined by specifying the
coordinates of the cocrystallized ligand tadalafil. The active site
˚
radius setting remained at the default value of 10 A with cavity
detection enabled.
Three independent docking experiments were done for all
compounds, employing the three implemented scoring functions
in GOLD (GoldScore, ChemScore, and ASP) to drive the
genetic algorithm (GA). In each docking experiment, a total
of 10 GA runs per compound were conducted, resulting in 10
binding poses for each compound per experiment. Thus, we
obtained a total of 30 poses per compound.
All poses generated with one scoring function were then
rescored in GOLD utilizing the remaining two scoring functions.
Further rescoring of all poses was performed with DrugScoreCSD
and DrugScorePDB using the DrugScore Online service v0.9
(http://pc1664.pharmazie.uni - marburg.de/drugscore/). Finally,
a consensus score was calculated amounting to the arithmetic
mean of the normalized Goldscore, Chemscore, ASP, DrugScor-
eCSD, and DrugScorePDB values. The depicted binding modes
represent the docking pose with the optimal consensus score.
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Supporting Information Available: Results from elemental
analyses for compounds 1-68 and the NOESY spectra for
compounds 1 and 2. This material is available free of charge
via the Internet at http://pubs.acs.org.
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