Synthesis, molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective

Article abstract of DOI:10.1016/j.ejmech.2009.10.046

The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromophenyl; the chiral carbons swing from R,R to R,S, S,R and S,S; the piperazinedione ring is maintained or reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP) selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma cells. The R absolute configuration of C-5 in the β-carboline-hydantoin and C-6 in the β-carboline-piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil analogues that were synthesized from l-tryptophan were more active than those derived from d-tryptophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5 inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE isozymes or PDE5 splice variants may be involved.

Full text of DOI:10.1016/j.ejmech.2009.10.046

European Journal of Medicinal Chemistry 45 (2010) 1278–1286
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, molecular modeling and biological evaluation of novel tadalafil
analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors,
new stereochemical perspective
,
b
b
,
,
Ashraf H. Abadi a , Bernard D. Gary , Heather N. Tinsley c, Gary A. Piazza b c, Mohammad Abdel-Halim a
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo 11835, Egypt
b Division of Drug Discovery, Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham AL 35205, USA
c Department of Pharmacology and Toxicology, The University of Alabama at Birmingham, 2000 Ninth Avenue South, Birmingham, AL 35205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromo-
phenyl; the chiral carbons swing from R,R to R,S, S,R and S,S; the piperazinedione ring is maintained or
reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared
analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP)
selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma
Received 11 July 2009
Received in revised form
25 September 2009
Accepted 22 October 2009
cells. The R absolute configuration of C-5 in the
piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil
analogues that were synthesized from -tryptophan were more active than those derived from -tryp-
tophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5
inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no
apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE
isozymes or PDE5 splice variants may be involved.
b-carboline-hydantoin and C-6 in the b-carboline-
Keywords:
b-Carboline
L
D
PDE5 inhibition
Stereochemistry
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
apoptosis in cell lines derived from many cancers including colon,
bladder, prostate, breast and lung. They maintained similar rank
Phosphodiesterase type 5 (PDE5) is a key enzyme involved in the
regulation of cGMP-specific signaling pathways by catalyzing the
hydrolysis of the cyclic nucleotide cGMP into 50 guanosine mono-
phosphates. It’s an essential regulator in normal physiological
processes such as smooth muscle contraction and relaxation and it is
the major PDE isozyme in penile corpus cavernosum tissue that plays
a key role in the control of penile erection [1,2]. Tadalafil (Fig. 1) is an
orders of apoptosis induction, growth inhibition and PDE5 and PDE2
inhibition. They also caused sustained intracellular cGMP increase
in the colon tumor cells; thus it is proposed that the cGMP mediated
the mechanism underlying the actions of exisulind and its
analogues on apoptosis in neoplastic cells [4–6]. These effects in
neoplastic cells are not solely dependent on the specific inhibition of
PDE5, rather, it is related to non-selective inhibition of the cGMP-
PDEs, which may explain why highly selective PDE5 inhibitors do
not induce apoptosis in tumor cell lines. Accordingly, it is apparently
important to maintain cross-reactivity among PDE isoforms [7].
In the present work we report the synthesis of novel tadalafil
related analogues, evaluate the activity of these compounds as
PDE5 inhibitors as well as growth inhibitory agents and determine
the correlation between these two activities.
orally active tetrahydro-b-carboline (THBC) derivative with PDE5
inhibitory properties that is marketed for the treatment of male
erectile dysfunction. Its local vasodilatation action is mediated
through high levels of cGMP in male corpus cavernosum [3].
Recent studies showed that PDE5 and other cGMP-PDEs are over
expressed in a variety of cancers like colon, pancreatic, lung and
bladder cancers relative to normal tissues. Moreover, inhibition and
consequent high levels of cGMP may be associated with anticancer
and apoptotic activities [4,5]. Exisulind (Fig. 1) and its analogues
(CP78, CP 461, CP 248) have been shown to selectively induce
2. Chemistry
The general synthesis of the target
-thiohydantoin derivatives is illustrated in Schemes 1 and 2;
meanwhile, the synthesis of the -carbolines-piperazinedione
derivatives is illustrated in Scheme 3.
b-carboline-hydantoin and
b
* Corresponding author. Tel.: þ202 27590716; fax: þ202 27581041.
E-mail address: ashraf.abadi@guc.edu.eg (A.H. Abadi).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.046

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
1279
O
methylamine, ethylamine, butylamine and benzylamine in refluxing
methanol.
HOOC
H3C
F
N
The cis/trans stereochemistry for the THBCs (3a–d) was assigned
depending on the comprehensive study of 13C NMR spectroscopy
data established in previous literature, the signals for C-1 and C-3 in
the trans-diastereomers are clearly upfield from those of the cor-
responding cis-isomers in the carbon spectrum. This is probably
due to the 1,3 interactions present in the trans-isomer [9–11]. For
the THBCs (3a–d), only the signal of C-1 in the trans-isomer
H3C
N
N
H
O
O
O
appeared at higher field compared to that of the cis-isomer, with
z2–3, no analogous difference was detected with C-3. Regarding
the 1H NMR signals for the proton at C-1 of 3a–d, they appeared at
5.5–5.7. On cyclization to the hydantoin, thiohydantoin or piper-
azinedione derivatives the same proton makes a huge downfield
shift to z 6.7. This deshielding effect can be explained from the
D
S
O
d
O
CH3
Fig. 1. Chemical structure of the specific PDE5 inhibitor tadalafil (left) and the non-
d
specific PDE5 inhibitor exisulind (right).
d
Both
synthesized by a general synthetic procedure for amino acid esters
[8]. The - and -Tryptophan methyl esters and 2-bromobenzalde-
D
-tryptophan and
L-tryptophan methyl esters were
energy minimized form of the particular THBC and its respective
hydantoin derivative where in the former case the 2-bromo
substituent on the phenyl ring is at the same direction and close to
the proton at C-1 (distance z2.6 Å), formation of the new ring
causes the pendant phenyl to rotate around its axis orienting the 2-
bromo substituent in different plane far from the respective proton
(distance z4.2 Å), Fig. 2.
Moreover, a correlation exists between Rf value on TLC and the
stereochemistry of the 1,3-disubstituted THBC 3a–d, where cis-
isomer is systematically less polar than the trans-isomer while in
the hydantoin series, the polarity is reversed, thus, the cis-isomer
becomes more polar than the trans-isomer [12]. This is clear from
the Rf values of the 1R, 3R; 1S, 3R; 1S, 3S and 1R, 3S of the THBC
derivatives 3a–d equal to 0.38; 0.15; 0.39 and 0.14, respectively,
meanwhile the Rf value of the corresponding hydantoins 4a–d were
0.38, 0.46, 0.39 and 0.45, respectively. In addition, it was found that
D
L
hyde were subjected to Pictet–Spengler reaction under non-stereo
specific conditions to give the corresponding cis- and trans-1,3-
disubstituted THBCs (3a–d). The produced cis- and trans-diastereo-
mers of the 1,3-disubistituted THBC were separated by column
chromatography using CH2Cl2 as an eluent. The respective pure cis-
or trans-isomer was reacted with commercially available ethyl
isocyanate to produce the desired cis- and trans-hydantoins (4a–d)
meanwhile reaction with ethyl isothiocyanate gave only the corre-
sponding thiohydantoins of the trans-configuration (5a, 5b). Reac-
tion of the respective THBC with chloroacetyl chloride provided the
corresponding amide derivative (6a–d). The
b-carboline-piper-
azinedione derivatives with the respective N-alkyl or N-benzyl
substituents (7a–10d) were obtained by ring closure of the open
amides 6a–d in the presence of primary amines, namely
the Rf values of
b-carboline-piperazinedione derivatives increase
HOOC
H2N
H3COOC
H2N
a
CH3OH
+
N
H
N
H
D-tryptophan
b
O
N
H3COOC
HN
O
N
H3COOC
HN
N
N
O
c
c
O
+
N
H
N
H
N
H
3a
N
H
4a
Br
Br
Br
3b
4b
Br
d
d
O
O
N
S
N
N
N
S
N
N
H
H
Br
Br
5b
5a
Scheme 1. Conditions: (a) CH3COC l, reflux, neutralization; (b) 2-bromobenzaldehyde, CF3COOH, Room temperature; (c)C2H5NCO, 2-butanone, reflux; (d)C2H5NCS, 2-butanoe,
reflux.

1280
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
HOOC
H2N
H3COOC
a
H2N
+
CH3OH
N
N
H
H
L-tryptophan
b
O
O
N
H3COOC
HN
H3COOC
HN
N
N
N
O
c
c
O
+
N
H
N
H
N
H
N
H
Br
Br
Br
Br
4d
3d
3c
4c
d
d
O
O
N
N
S
N
N
S
N
H
N
H
Br
Br
5a
5b
Scheme 2. Conditions: (a) CH3COC l, reflux, neutralization; (b) 2-bromobenzaldehyde, CF3COOH, Room temperature; (c) C2H5NCO, 2-butanone, reflux; (c) C2H5NCS, 2-butanone,
reflux.
with increasing of size of the N-substituent, this may be due to
increased lipophilicity.
During the attempt to synthesize the thiohydantoin series, only
the trans-isomers were obtained (5a, 5b). The 13C NMR, 1H NMR
spectra, Rf and m.p. for the thiohydantoins obtained on treating the
The infrared spectra of all derivatives showed bands at
z3400 cmꢀ1 for the indole N–H stretching. Compounds 3 a–
d showed peaks at z1750 cmꢀ1 for the ester carbonyl stretching. On
the other hand, the b-carboline-hydantoin derivatives 4a–d showed
2 carbonyl stretching peaks at z1760 and 1700 Cmꢀ1, as one of the
cis- and trans-THBC derived from
thiocyanate, were completely matching with those derived from the
trans- and cis-THBCs isomers derived from -Tryptophan. This
D
-tryptophan with ethyl iso-
carbonyls is flanked between 2 nitrogen atoms, meanwhile the
other is flanked between an N and a C, respectively. The b-carboline-
L
piperazinedione derivatives showed 2 carbonyl stretching peaks at
z1660 and 1650 cmꢀ1. The relatively lower stretching values of the
carbonyls of the 6 membered derivatives relative to the 5-
membered derivatives may be explained by the higher ring strain of
the smaller ring, leading to higher double bond character of the
carbonyl groups.
observation has been previously reported by others and demon-
strates the enantiomeric nature of the two products [11,13,14].
Mass spectrometry to all derivatives showed the molecular ion
peaks at m/z Mþ and Mþ þ 2 due to the isotopic nature of bromine
atom. Moreover, the 1.3-disubstituted THBC derivatives 3a–
d showed molecular ion peaks that were also the base peaks
indicating their stable nature. Mass spectrometry for most hydan-
toin, thiohydantoin and piperazinedione derivatives showed a base
peak at m/z Mþ ꢀ80 indicating that the bromine atom was the most
liable fragment to be lost on electron bombardment.
3. Biological results and discussion
All compounds and their intermediates were tested for their in
vitro tumor cell growth inhibitory activity using the human HT-29
R
H3COOC
O
3
H3COOC
Cl CH2
N
12a
HN
N
b
a
O
N
1
O
N
N
H
H
6
Br
N
H
Br
Br
3
6
7-10
Cpd. #
7a
R
Me
Me
Me
Me
Et
Stereochemistry
6R,12aR
6S,12aR
Cpd. #
9a
R
Stereochemistry
6R,12aR
6S,12aR
Bu
Bu
Bu
Bu
Benzyl
Benzyl
Benzyl
Benzyl
7b
9b
7c
9c
6S,12aS
6S,12aS
7d
9d
6R,12aS
6R,12aS
8a
10a
10b
10c
10d
6R,12aR
6S,12aR
6R,12aR
6S,12aR
8b
Et
8c
Et
Et
6S,12aS
6S,12aS
8d
6R,12aS
6R,12aS
Scheme 3. Conditions: (a) ClCH2COCl, NaHCO3, Room temperature; (b) R-NH2, MeOH, Reflux.

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
1281
Fig. 2. The energy minimized forms of 3d (left) and 4d (right) showing orientations of the bromine substituent relative to the hydrogen on the chiral carbon derived from the
aldehyde.
colon tumor cell line as well as their inhibitory properties vs
recombinant human PDE5 enzyme.
HT-29 tumor cell line is also known to express PDE5 and other
cGMP degrading PDE isozymes. Most of compounds were evaluated
in 2 steps, first, the percentage inhibition at a screening dose of
from amino acid with the R configuration (
suggests the feasibility of synthesizing active compounds derived
from -tryptophan rather than from -tryptophan and offers a highly
-tryptophan is much cheaper than D-
D-tryptophan). This
L
D
economic advantage as
tryptophan.
L
100
m
M was performed in triplicate, then for compounds displaying
b-Carboline-thiohydantoins are markedly less potent than their
a percentage of inhibition >70% were evaluated by testing a range
of 10 concentrations with at least two replicates per concentration
to calculate an IC50 value. For a few compounds, the IC50 was
initially determined. The results are summarized in Table 1.
From these data we conclude that the stereochemistry of C-5 of
corresponding hydantoin congeners, which may be due to the
lower electro-negativity of sulfur relative to oxygen, 5a vs 4b and
5b vs 4d.
For the impact of the terminal ring size, viz hydantoins vs
piperazinediones, compounds are almost of the same activity. It is
apparent that a wide range of N-alkyl or N-aryl groups and possibly
other lipophilic functions can be tolerated on the piperazinedione,
given that their impact upon activity is almost the same.
Bromine – a bulky and electronegative substituent – at C-2 of
the pendant phenyl still produces highly active compounds in the
contrary to what has been previously reported for the 2-methoxy
the
tives and C-6 of the
most crucial factor for the PDE5 inhibitory activity. In the
b
-carboline-hydantoin and
-carboline-piperazinedione derivatives are the
-carbo-
b-carboline-thiohydantoin deriva-
b
b
line-hydantoins 4a–d, the order of activity is 5R, 11aS (4d) > 5R, 11aR
(4a) > 5S, 11aS (4c) >5S, 11aR (4b) and for the piperazinediones, the
order of activity is 6R, 12aS > 6R, 12aR > 6S, 12aS > 6S, 12aR. This can
be observed by the THBC precursors (3a–d) where the order of their
PDE5 inhibitory potency is matched with this stereochemistry. These
results are in marked contrast to the results obtained with tadalafil
isomers whereby the order of activity is 6R, 12aR > 6R, 12aS > 6S,
12aR > 6S, 12aS [15]. This leads to the assumption that the scope and
limitations of such class of compounds are still to be explored.
Meanwhile, the effect of the stereochemistry of the chiral carbon
derived from the amino acid is of lower impact. Interestingly, the
substituent in the
b-carboline-hydantoin derivatives [15]. This
confirms the necessity to explore the scope and limitations of this
class of compounds.
Molecular modeling studies were performed to dock compound
4d to the human PDE5 using the MOE software [16]. Detailed view
showing compounds 4d is able to dock to the active pocket of PDE5
and interact with the side chain of Gln 817 which forms a single, not
bidentate, hydrogen bond with the indole NH group of the
respective compound. Compounds 4d did not interact with the
L-region of the protein and the H pocket is filled with the
chiral center derived from amino acid with the S configuration (L-
tryptophan) may lead to more active isomers than those derived
Table 1
Inhibitory effect of the synthesized compounds on HT29 cells and PDE5.
Cpd.#
% Growth
Inhibition
Growth
inhibition
%PDE5 inhibition
PDE5 inhibition
Cpd.#
% Growth Inhibition
Growth inhibition
%PDE5 inhibition
PDE5 inhibition
IC50 mM
at 100
m
M
IC50
m
M
at 100
m
M
IC50 m
M
at 100
m
M
at 100
m
M
IC50 mM
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
6a
6b
6c
6d
7a
89.04
85.02
92.75
57.46
79.54
65.14
77.86
69.76
ND
ND
ND
ND
ND
48
78
26
ND
21
ND
24
20
>100
22.5
1.6
>100
4.1
85.52
21.50
45.97
91.13
98.65
93.01
89.51
99.462
82.80
93.55
86.02
47.85
35.75
47.85
95.97
5.6
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
Tadalafil
61.73
76.14
ND
ND
65.61
ND
47.25
55.74
50.20
ND
ND
34.20
ND
ND
27
25.5
>100
ND
32
ND
ND
ND
>100
>100
ND
>100
>100
>100
>100
75.54
92.74
97.58
97.04
91.67
90.32
89.25
99.73
66.66
69.09
93.81
91.94
69.36
48.66
99.99
99.99
88
2.8
0.06
0.32
41
NDa
ND
3.1
0.29
4.0
0.64
0.038
74
6.1
29
ND
ND
ND
0.1
32
0.05
0.26
ND
ND
0.05
3.4
ND
ND
0.04
ND
50.40
1.6
ND
ND
ND
ND
0.004
a
ND ¼ not determined.

1282
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
Fig. 3. Detailed view showing the docking and interaction of tadalfil (left), compound 4d (right) with human PDE5(1UDU).
2-bromphenyl moiety. The more extensive interactions with the H
pocket may be one of the reasons that compound 4d could main-
tain high affinity to PDE5 without binding to the L region. Inter-
estingly, tadalafil interacts in a very similar fashion, Fig. 3.
Science, Cairo University; and are within 0.4% of the theoretical
value, unless stated otherwise.
5.1.1. Methyl 1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
As per the anticancer activity, compounds (6a, 6c and 6d) out of
the four open chain amides obtained by the acylation of the NH of
the THBC derivatives with chloroacetyl chloride were found to be
among the most active growth inhibitory agents with IC50 values of
1.6, 4.1 and 1.6 mM, respectively. However, this activity may be due
to the alkyl halide nature and expected toxicity of the respective
compounds.
For this class of compounds, there does not appear to be a corre-
lation between the PDE5 inhibition and tumor cell growth inhibitory
activity, as compound 4d the most potent PDE5 inhibitor is not the
most potent as compound 6d, which was the most potent compound
that inhibited HT-29 tumor cell growth, but did not show PDE5
inhibitory activity. This leads us to hypothesize that other PDE
isozymes or possibly PDE5 splice variants may be involved.
carboline-3-carboxylate (3a–d)
The appropriate tryptophan methyl ester (3.42 g, 15.7 mmol)
and 2-bromobenzaldehyde (3.19 g, 17.25 mmol) were dissolved in
CH2Cl2 (10 mL) and cooled to 0 ꢁC in an ice bath. To this solution
was added dropwise TFA (1 mL), and the mixture was stirred at
room temperature for 4 days under N2 atmosphere. The reaction
mixture was then basified with dilute NH4OH solution and
extracted with CH2Cl2 (3 ꢂ 10 mL). The organic layer was washed
with water, brine, dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure. The residue was purified and
the isomers were separated by column chromatography on silica
gel eluting with CH2Cl2, to give first the appropriate cis-isomer
followed by the trans-one.
5.1.2. (1R,3R) Methyl 1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
4. Conclusion
carboline-3-carboxylate (3a)
White solid; 33%; mp 105–106 ꢁC; 1H NMR (DMSO-d6):
d 10.46
Compound 4d derived from
L
-Tryptophan was the most potent
(s, 1H, NH), 7.7–7.68 (d, 1H, Ar), 7.46–7.43 (d, 1H, Ar), 7.33–7.20
(m, 4H, Ar), 7.04–6.93 (m, 2H, Ar), 5.64(s, 1H, CHPh), 3.95–3.91(m,
1H, CHCOOCH3), 3.68 (s, 3H, OCH3), 3.07–3.03 (dd, 1H, CHaHb),
PDE5 inhibitor (IC50 ¼ 0.038 M) in a series of tadalafil analogues
m
that were synthesized. The results suggest a new direction for
further research to synthesize new PDE5 inhibitors with lower cost.
The correlation between the PDE5 inhibitory activity and their
tumor cell growth inhibitory activity needs further study on other
PDE isozymes and splice variants.
2.89–2,75 (m, 1H, CHaHb); 13C NMR (DMSO-d6):
d 172.88, 140.75,
136.36, 134.16, 132.69, 130.74, 128.02, 126.44, 124.02, 120.94, 118.54,
117.70, 111.32, 107.65, 56.03 (C1), 51.89(C3), 51.85, 25.26; m/z 386
(Mþ þ 2), m/z 384 (Mþ, 100%); Anal. (C19H17BrN2O2) C: 59.23,
H:4.45, N: 7.27; found: C:59.75, H:4.51, N:7.10.
5. Experimental section
5.1.3. (1S,3R) Methyl 1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
5.1. Chemistry
carboline-3-carboxylate (3b)
White solid; 41%; mp 104–105 ꢁC; 1H NMR (DMSO-d6):
d 10.73
All starting materials were commercially available and used
without further purification. All reactions were carried out with the
use of standard techniques under an inert atmosphere (N2). The
analytical thin-layer chromatography (TLC) was carried out on
E. Merck 60-F254 precoated silica gel plates and components were
usually visualized using UV light, Flash column chromatography
was performed on silica gel 60 (E. Merck, 230–400 mesh). Melting
points were determined on Buchi Melting Point apparatus and are
uncorrected. Proton NMR (1H NMR) and carbon NMR (13C NMR)
spectra were recorded at ambient temperature on Varian Mercury
VX-300 MHz spectrometer using tetramethylsilane as internal
standard, and proton chemical shifts are expressed in ppm in the
indicated solvent. The following abbreviations are used for multi-
plicity of NMR signals: (s) singlet, (d) doublet, (t) triplet, (q)
quadruplet, (dd) double doublet, (m) multiplet. The elemental
analyses were performed by the Microanalytical Unit, Faculty of
(s,1H, NH), 7.69–7.62 (m,1H, Ar), 7.49–7.47 (d,1H, Ar), 7.25–7.22 (m,
4H, Ar), 7.05–6.98 (m, 2H, Ar), 5.67(s, 1H, CHPh), 3.68–3.63 (m, 4H,
CHCOOCH3 þ OCH3), 3.14–3.08 (m, 1H, CHaHb), 2.90–2,87 (dd, 1H,
CHaHb); 13C NMR (DMSO-d6):
d 173.53, 141.11, 136.2, 132.93, 130.37,
129.53, 127.47, 126.35, 124.09, 121.12, 118.51, 117.81, 111.19, 107.86,
53.69 (C1), 51.83(C3), 51.36, 24.89; m/z 386 (Mþ þ 2), m/z 384 (Mþ,
100%); Anal. (C19H17BrN2O2) C: 59.23, H:4.45, N: 7.27; found;
C:59.50, H:4.20, N:7.05.
5.1.4. (1S,3S) Methyl 1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylate (3c)
White solid; 41%; mp 104–105 ꢁC; 1H NMR (DMSO-d6):
d 10.46
(s, 1H, NH),7.70–7.67 (d, 1H, Ar), 7.46–7.43 (d, 1H, Ar),7.33–7.20 (m,
5H, Ar þ NH),7.04–6.93 (m, 2H, Ar),5.64 (s,1H, CHPh), 3.93(m, 1H,
CHCOOCH3), 3.68 (s, 3H, OCH3), 3.07–3.03 (dd, 1H, CHaHb), 2.90–
2.80 (m, 1H, CHaHb); 13C NMR (DMSO-d6):
d 172.80, 136.36, 134.07,

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
1283
132.64, 130.74, 129.83, 128.01,126.43, 124.02, 120.93, 118.52, 117.67,
5.1.11. 5-(2-Bromophenyl)-2-ethyl-1-oxo-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6] pyrido[3,4-b]indole-3(2H)-thione (5a, 5b)
Excess Ethyl isothiocyanate (135 l, 1.5 mmol) was added to
115.92, 111.30, 107.63, 56.61(C1), 51.85(C3), 25.20; m/z 386
(Mþ þ 2), m/z 384.13 (Mþ, 100%); Anal. (C19H17BrN2O2) C: 59.23,
H:4.45, N: 7.27; found: C:59.02, H:4.50, N:7.09.
m
a well stirred solution of the appropriate beta-carboline 3a–
d (0.38 g, 1 mmol) in methyl ethyl ketone (10 mL) and the mixture
was stirred at reflux for 16 h under a nitrogen atmosphere. The
solvent was evaporated under reduced pressure, the residue was
purified using preparative TLC and developed with CH2Cl2, the
required band was stripped and dissolved in CH2Cl2 The solution
was then filtered to remove the undesired silica, the filtrate was
evaporated under reduced pressure to obtain pure powder of
desired product.
5.1.5. (1R,3S) Methyl 1-(2-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylate (3d)
White solid; 28%; mp 193–195 ꢁC; 1H NMR (DMSO-d6):
d 10.71
(s, 1H, NH),7.69–7.67 (t, 1H, Ar), 7.48–7.46 (d, 1H, Ar), 7.24–7.21 (d,
3H, Ar), 7.07–6.95(m, 2H, Ar), 6.76–6.5(m,1H, Ar), 5.66(s,1H, CHPh),
3.67–3.62(m, 4H, CHCOOCH3 þ OCH3), 3.13–3.07 (m, 1H, CHaHb),
2.89–2.80 (dd, 1H, CHaHb); 13C NMR (DMSO-d6):
d 173.51, 141.10,
136.19, 132.91, 130.35, 129.50, 127.45, 126.34, 124.06, 121.09, 118.48,
117.77, 111.16, 107.83, 53.64(C1), 51.79(C3), 51.35, 24.89; m/z 386
(Mþ þ 2), m/z 384 (Mþ, 100%); Anal. (C19H17BrN2O2) C: 59.23,
H:4.45, N: 7.27; found: C: 58.94, H:4.58, N:7.11.
5.1.12. (5S,11aR)-5-(2-Bromophenyl)-2-ethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo [10,50:1,6]pyrido[3,4-b]indole-3(2H)-
thione (5a)
Yellow white solid; 56%; mp 228–229 ꢁC; 1H NMR (CDCl3):
d 8.10
5.1.6. 5-(2-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-1H-
(s, 1H, NH), 7.72–7.70 (d, 1H, Ar), 7.54–7.51 (d, 1H, Ar), 7.33–7.13 (m,
7H, Ar þ CHPh), 4.82–4.76 (dd, 1H, CHC(O)N), 4.01–3.94 (m, 2H,
NCH2), 3.59–3.53 (m,1H CHaHb), 2.95–2.91 (m, 1H, CHaHb), 1.32–
imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (4a–d)
Excess Ethyl isocyanate (135 ml, 1.6 mmol) was added to a well
stirred solution of the appropriate beta-carboline 3a–d (0.38 g,
1 mmol) in methyl ethyl ketone (10 mL) and the mixture was
stirred at reflux for 16 h under a nitrogen atmosphere. The
solvent was evaporated under reduced pressure, the residue was
purified using preparative TLC and developed with CH2Cl2, the
required band was stripped and dissolved in CH2Cl2 The solution
was then filtered to remove the undesired silica, the filtrate was
evaporated under reduced pressure to obtain pure powder of
desired product.
1.28 (t, 3H, CH3); 13C NMR (DMSO-d6):
d 180.65, 172.82, 138.27,
137.03, 132.96, 131.00, 129.90, 129.57, 128.23, 125.48, 123.53, 121.75,
119.01, 118.20, 111.76, 105.09, 58.03, 55.91, 35.84, 22.47, 12.71; m/z
441 (Mþ þ 2), 439 (Mþ), m/z 360 (100%); Anal. (C21H18BrN3OS)
C:57.28, H:4.12, N:9.54; found: C:57.19, H:4.22, N:9.46.
5.1.13. (5R-11aS)-5-(2-Bromophenyl)-2-ethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo [10,50:1,6]pyrido[3,4-b]indole-3(2H)-
thione (5b)
Yellowish white solid; 45%; mp 226–227 ꢁC; 1H NMR (CDCl3):
5.1.7. (5R,11aR)-5-(2-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
d 8.09 (s,1H, NH), 7.72–7.70 (d,1H, Ar), 7.54–7.52 (d,1H, Ar), 7.30–7.13
1H-imidazo[10,50:1,6] pyrido[3,4-b]indole-1, 3(2H)-dione (4a)
(m, 7H, Ar þ CHPh), 4.82–4.77 (dd, 1H, CHC(O)N), 4.01–3.94 (m, 2H,
Buff solid; 55%; mp 210–213 ꢁC; 1H NMR (CDCl3):
d 7.85 (s, 1H,
NCH2), 3.61–3.49 (m,1H, CHaHb), 2.95–2.91 (m,1H, CHaHb),1.32–1,27
NH), 7.66–7.63 (m, 1H, Ar), 7.57–7.54 (m, 1H, Ar),7.25–7.06 (m, 6H,
Ar), 6.49(s,1H, CHPh), 4.41–4.37(dd,1H, CHC(O)N), 3.57–3.51 (m, 3H,
NCH2 þ CHaHb), 3.10–3.01(m, 1H, CHaHb), 1.23–1.18 (t, 3H, CH3); m/z
425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.69, H:4.34, N:9.96.
(t, 3H, CH3); 13C NMR (DMSO-d6):
d d 180.65, 172.82, 138.29, 137.04,
132.95, 131.00, 129.89, 129.58, 128.22, 125.48, 123.54, 121.74, 119.00,
118.20, 111.76, 105.09, 58.03, 55.91, 35.84, 22.48, 12.72; m/z 441
(Mþ þ 2), 439.04 (Mþ), m/z 360 (100%); Anal. (C21H18BrN3OS)
C:57.28, H:4.12,N: 9.54; found: C:57.18, H:4.20, N:9.50.
5.1.8. (5S,11aR)-5-(2-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
5.1.14. General procedure for methyl 1-(2-Bromophenyl)-2-(2-
1H-imidazo[10,50:1,6] pyrido[3,4-b]indole-1,3(2H)-dione (4b)
chloroacetyl)-2,3,4,9-tetrahydro-1H-
b
-carboline-3-carboxylate
yellow solid; 52%; mp 137–138 ꢁC; 1H NMR (CDCl3):
d
7.96 (s,1H,
(6a–d)
NH), 7.67–7.65 (m, 1H, Ar), 7.64–7.55(m, 1H, Ar), 7.28–7.14(m, 6H,
Ar), 6.74(s,1H, CHPh), 4.60–4.55(dd,1H, CHC(O)N), 3.63–3.51(m, 3H,
NCH2 þ CHaHb), 2.93–2.83 (m,1H, CHaHb),1.26–1,21 (t, 3H, CH3); m/z
425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.19, H:4.34, N:9.92.
To a stirred solution of the appropriate b-carboline 3a–d (2.195 g,
5.7 mmol) and NaHCO3 (0.575 g, 6.89 mmol) in CHCl3 (40 mL) was
added dropwise chloroacetyl chloride (1.09 mL, 13.69 mmol) under
ice cooling. The mixture was then stirred at room temperature
under a nitrogen atmosphere for 1 h. The mixture was diluted with
CH2Cl2, washed with a solution of NaHCO3, brine, dried over Na2SO4,
and evaporated under reduced pressure. The residue was then
crystallized from diethyl ether.
5.1.9. (5S,11aS)-5-(2-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6] pyrido[3,4-b]indole-1, 3(2H)-dione (4c)
Yellowish white solid; 63%; mp 213–214 ꢁC; 1H NMR (CDCl3):
d
7.86 (s, 1H, NH), 7.63–7.54 (m, 2H, Ar), 7.27–7.03(m, 6H, Ar), 6.49(s,
5.1.15. Methyl (1R,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-
1H, CHPh), 4.41–4.37(dd, 1H, CHC(O)N), 3.57–3.49(m, 3H,
NCH2 þ CHaHb), 3.10–3.01(m, 1H, CHaHb), 1.23–1,18 (t, 3H, CH3); m/z
425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.35, H:4.34, N:10.01.
2,3,4,9-tetrahydro-1H-
b-carboline-3-carboxylate (6a)
White solid; 88%; mp 113–115 ꢁC; 1H NMR (CDCl3):
d
8.00 (br s,
1H, NH), 7.88–7.85 (d, 1H, Ar), 7.57–7.54 (d, 1H, Ar), 7.31–7.14 (m,
6H, Ar), 6.73 (s,1H, CHPh), 5.18–5.14 (m, 1H, CHCOOCH3), 4.19–
4.15(m, 2H, COCH2Cl), 3.64 (br s, 3H, OCH3), 3.37–3.28 (m, 2H, CH2);
m/z 462 (Mþ þ 2), m/z 460(Mþ), m/z 385 (100%); Anal.
(C21H18BrClN2O3). C:54.63, H:3.93, N:6.07; found: C: 54.16, H:4.02,
N:6.06.
5.1.10. (5R,11aS)-5-(2-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6] pyrido[3,4-b]indole-1, 3(2H)-dione (4d)
White solid; 50%; mp 140–141 ꢁC; 1H NMR (CDCl3):
d 7.90 (s, 1H,
NH), 7.68–7.66 (m,1H, Ar), 7.53–7.52 (m,1H, Ar),7.28–7.14 (m, 6H, Ar),
6.74 (s, 1H, CHPh), 4.61–4.56(dd, 1H, CHC(O)N), 3.63–3.52 (m, 3H,
NCH2 þ CHaHb), 2.93–2.84 (m,1H, CHaHb),1.27–1,22 (t, 3H, CH3); m/z
425 (Mþ þ 2), m/z 423 (Mþ), m/z, 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.39, H:4.19, N:9.88.
5.1.16. Methyl (1S,3R)-1-(2-bromophenyl)-2-(2-chloroacetyl)-
2,3,4,9-tetrahydro-1H-
White solid; 74%; mp 132–135 ꢁC; 1H NMR (CDCl3):
NH), 7.61–7.50 (m, 2H, Ar), 7.28–7.08 (m, 6H, Ar), 6.65 (s,1H, CHPh),
b-carboline-3-carboxylate (6b)
d
8.22 (s, 1H,

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A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
5.31 (br s, 1H, CHCOOCH3), 4.19–4.06 (m, 2H, COCH2Cl), 3.82–3.72
(m, 5H, OCH3 þ CH2); m/z 462 (Mþ þ 2), m/z 460(Mþ), m/z 383
(100%); Anal. (C21H18BrClN2O3). C:54.63, H:3.93, N:6.07; found:
C:54.33, H:3.92, N:5.99.
5.1.23. (6R,12aS)-6-(2-Bromophenyl)-2-methyl -2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (7d)
Greenish white solid; 37%; mp 201–204 ꢁC; 1H NMR (CDCl3):
d
8.07 (s, 1H, NH), 7.63–7.52 (m, 2H, Ar), 7.27–7.14 (m,6H, Ar), 6.85
(brs,1H, CHPhAr), 4.37–4.32 (dd,1H, CHC(O)N), 4.14–4.01 (m, 3H,
CH2C(O)N þ CHaHb), 3.53–3.46 (dd,1H, CHaHb), 3.01 (s, 3H, NCH3); m/
z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.31, H:4.32, N:9.96.
5.1.17. Methyl (1S,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-
2,3,4,9-tetrahydro-1H-
b-carboline-3-carboxylate (6c)
White solid; 83%; mp 112–113 ꢁC; 1H NMR (CDCl3):
d
7.97 (br s,
1H, NH), 7.68–7.65 (d, 1H, Ar), 7.57–7.54 (d, 1H, Ar),7.31–7.12 (m, 6H,
Ar), 6.73 (s,1H, CHPh), 5.18–5.14(m,1H, CHCOOCH3), 4.19–4.15 (m,
2H, COCH2Cl), 3.64 (br s, 3H, OCH3), 3.37–3.26 (m, 2H, CH2); m/z 462
(Mþ þ 2), m/z 460(Mþ), m/z 383 (100%); Anal. (C21H18BrClN2O3).
C:54.63, H:3.93, N:6.07; found: C:54.66, H:3.89, N:6.03.
5.1.24. (6R,12aR)-6-(2-Bromophenyl)-2-ethyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (8a)
Buff solid; 30%; mp 121–122 ꢁC; 1H NMR (CDCl3):
d 8.28 (s, 1H,
NH), 7.58–7.55 (m, 2H, Ar), 7.31–7.05 (m,6H, Ar), 6.73 (s, 1H, CHPh),
4.42–4.32 (m, 1H, CHC(O)N), 4.16–3.96 (m, 3H, CH2C(O)N þ CHaHb),
3.46–3.32 (m, 1H, CHaHb), 3.03–2.93 (m, 2H, NCH2), 1.26–1.21 (t, 3H,
CH3); m/z 439 (Mþ þ 2), m/z 437 (Mþ), m/z 358 (100%) m/z 437; Anal.
(C22H20BrN3O2) C: 60.28, H:4.60, N:9.59; found: C:60.40,H:4.57,
N:9.48.
5.1.18. Methyl (1R,3S)-1-(2-bromophenyl)-2-(2-chloroacetyl)-
2,3,4,9-tetrahydro-1H-
b-carboline-3-carboxylate (6d)
White solid; 81%; mp 132–133 ꢁC; 1H NMR (CDCl3):
d
8.22 (s, 1H,
NH), 7.62–7.50 (m, 2H, Ar), 7.29–7.08 (m, 6H, Ar), 6.65 (s,1H, CHPh),
5.31 (br s,1H, CHCOOCH3), 4.19–4.06 (m, 2H, COCH2Cl), 3.74–3.65
(m, 5H, OCH3 þ CH2); m/z 462 (Mþ þ 2), m/z 460(Mþ), m/z 385
(100%); Anal. (C21H18BrClN2O3). C:54.63, H:3.93, N:6.07; found: C:
54.55, H:3.90, N:6.13.
5.1.25. (6S,12aR)-6-(2-Bromophenyl)-2-ethyl -2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (8b)
White solid; 28%; mp 180–182 ꢁC; 1H NMR (DMSO-d6):
d 7.95
(s, 1H, NH), 7.68–7.65 (m, 1H, Ar), 7.55–7.53 (d, 1H, Ar), 7.34–7.15 (m,
6H, Ar), 6.85 (s, 1H, CHPh), 4.37–4.32 (dd, 1H, CHC(O)N), 4.15–4.01
(m, 3H, CH2C(O)N þ CHaHb), 3.52–3.47 (dd, 1H, CHaHb), 3.03–2.93
(m, 2H, NCH2), 1.23–1.19 (t, 3H, CH3); m/z 439 (Mþ þ 2), m/z 437
(Mþ), m/z 358 (100%); Anal. (C22H20BrN3O2). C: 60.28, H:4.60,
N:9.59; found: C:60.15, H:4.50, N:9.62.
5.1.19. Preparation of 2-alkyl or 2-benzyl-6-(2-Bromophenyl)-
2,3,6,7,12,12a-hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-
dione (7a–10d)
A solution of the of the appropriate chloroacetyl derivative 6a–
d (1.4 mmol, 1 equiv) and the appropriate amine, namely methyl-
amine (7a–d); ethylamine (8a–d), butylamine (9a–d); benzylamine
(10a–d) (2.8 mmol, 2 equiv) in methanol (25 mL) was heated to
reflux under a nitrogen atmosphere for 16 h. The reaction mixture
was cooled to room temperature and evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2, and the
organic layer was washed with water, dried over Na2SO4, filtered,
and concentrated to dryness. The crude product was then purified
using column chromatography, using CH2Cl2 as an eluent.
5.1.26. (6S,12aS)-6-(2-Bromophenyl)-2-ethyl -2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (8c)
Greenish white solid; 25%; mp 125–127 ꢁC; 1H NMR (CDCl3):
d
8.25 (s, 1H, NH), 7.64–7.52(m, 2H, Ar), 7.25–7.11(m,6H, Ar),6.72
(s,1H, CHPh),4.41–4.35 (dd,1H, CHC(O)N),4.15–4.01 (m,3H,
CH2C(O)N þ CHaHb),3.52–3.47 (dd, 1H, CHaHb), 3.03–2.93 (m, 2H,
NCH2), 1.24–1.19 (t, 3H, CH3); m/z 439 (Mþ þ 2), m/z 437 (Mþ), m/z
358 (100%) m/z 437; Anal. (C22H20BrN3O2). C: 60.28, H:4.60, N:9.59;
found: C:60.51, H:4.61, N:9.56.
5.1.20. (6R,12aR)-6-(2-Bromophenyl)-2-methyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (7a)
Yellowish white solid; 34%; mp 175–178 ꢁC; 1H NMR (CDCl3):
5.1.27. (6R,12aS)-6-(2-Bromophenyl)-2-ethyl -2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (8d)
Yellowish white solid; 30%; mp 179–180 ꢁC; 1H NMR (CDCl3):
d
8.27 (s, 1H, NH), 7.59–7.56 (m, 2H, Ar), 7.31–7.13 (m, 6H, Ar),
6.72(s, 1H, CHPh), 4.43–4.34(m, 1H, CHC(O)N), 4.11–3.92 (m, 3H,
CH2C(O)N þ CHaHb), 3.32–3.22(m, 1H, CHaHb), 3.09(s,3H, NCH3);
m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%) m/z 423; Anal.
(C21H18BrN3O2). C:59.45, H:4.28, N:9.90; found: C:59.44, H:4.19,
N:9.82.
d
7.99 (s, 1H, NH), 7.66–7.51 (m, 2H, Ar), 7.32–7.13 (m, 6H, Ar), 6.86
(s,1H, CHPh), 4.35–4.29 (dd, 1H, CHC(O)N), 4.12–3.98 (m, 3H,
CH2C(O)N þ CHaHb), 3.51–3.44 (dd, 1H, CHaHb), 3.00–2.91 (m, 2H,
NCH2), 1.23–1.17 (t, 3H, CH3); m/z 439 (Mþ þ 2), m/z 437 (Mþ), m/z
358 (100%) m/z 437; Anal. (C22H20BrN3O2) C: 60.28, H:4.60, N:9.59;
found: C:60.40,H:4.62, N:9.60.
5.1.21. (6S,12aR)-6-(2-Bromophenyl)-2-methyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (7b)
White solid; 29%; mp 202–204 ꢁC; 1H NMR (CDCl3):
d
8.15 (s, 1H,
5.1.28. (6R,12aR)-6-(2-Bromophenyl)-2-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (9a)
Greenish white solid; 37%; mp 129–131 ꢁC; 1H NMR (CDCl3):
NH), 7.66–7.63 (m, 1H, Ar), 7.54–7.52 (d, 1H, Ar), 7.33–7.13 (m, 6H,
Ar), 6.82 (brs,1H, CHPhAr), 4.36–4.31 (dd, 1H, CHC(O)N), 4.14–4.01
(m, 3H, CH2C(O)N þ CHaHb), 3.52–3.46 (dd, 1H, CHaHb), 3.01 (s, 3H,
NCH3); m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal.
(C21H18BrN3O2) C:59.45, H:4.28, N:9.90; found: C:59.61, H:4.29,
N:9.82.
d
8.27 (s, 1H, NH), 7.56–7.53 (m, 2H, Ar), 7.30–7.11(m, 6H, Ar), 6.72
(s, 1H, CHPh), 4.41–4.36 (dd,1H, CHC(O)N), 4.14–4.04 (m, 2H,
CH2C(O)N), 3.81–3.74 (dd, 1H, CHaHb), 3.55–3.46 (m, 1H, CHaHb),
3.31–3.22 (t, 2H, NCH2), 1.64–1.52 (m, 2H, CH2CH2CH3), 1.42–1.33
(m, 2H, CH2CH3), 0.98–0.94 (t, 3H, CH3); m/z 467 (Mþ þ 2), m/z 465
(Mþ), m/z 386 (100%) m/z 465; Anal. (C24H24BrN3O2) C:61.81,
H:5.19, N:9.01; found: C:62.01, H:5.20, N:9.04.
5.1.22. (6S,12aS)-6-(2-Bromophenyl)-2-methyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (7c)
White powder; 32%; mp 177–178 ꢁC; 1H NMR (CDCl3):
d 8.24
(s, 1H, NH), 7.58–7.54 (m, 2H, Ar), 7.30–7.04 (m, 6H, Ar), 6.70 (s, 1H,
CHPh), 4.41–4.36 (m, 1H, CHC(O)N), 4.10–3.91 (m, 3H,
CH2C(O)N þ CHaHb), 3.31–3.21 (m,1H, CHaHb), 3.08 (s, 3H, NCH3); m/
z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 344 (100%); Anal. (C21H18BrN3O2)
C:59.45, H:4.28, N:9.90; found: C:59.44, H:4.27, N:9.79.
5.1.29. (6S,12aR)-2-Butyl-6-(2-bromophenyl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (9b)
Yellowish white solid; 36%; mp 135–136 ꢁC; 1H NMR (CDCl3):
d
7.99 (s,1H, NH), 7.65–7.62 (m,1H, Ar), 7.53–7.51 (d,1H, Ar) 7.32–7.13
(m, 6H, Ar), 6.82 (s, 1H, CHPh), 4.33–4.39 (dd, 1H, CHC(O)N), 4.12–

A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
1285
3.98 (m, 2H, CH2C(O)N), 3.54–3.44 (m, 2H, NCH2), 3.32–3.22 (m, 1H,
5.2. Biology
CHaHb), 3.00–2.91 (m, 1H, CHaHb), 1.60–1.52 (m, 2H, CH2CH2CH3),
1.38–1.25 (m, 2H, CH2CH3), 0.97–0.93 (t, 3H, CH3); m/z 467(Mþ þ 2),
m/z 465(Mþ), m/z 217 (100%); Anal. (C24H24BrN3O2) C:61.81, H:5.19,
N:9.01; found: C:61.91, H:5.19, N:9.06.
5.2.1. Cell cultures
HT-29 tumor cells were obtained from ATCC. They were grown
under standard cell culture conditions at 37 ꢁC in a humidified
atmosphere with 5% CO2. Cells were grown in RPMI 1640 supple-
mented with 5% fetal bovine serum. 2 mM glutamine, 100 units/mL
5.1.30. (6S,12aS)-6-(2-Bromophenyl)-2-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (9c)
Yellowish white solid; 39%; mp127–130 ꢁC; 1H NMR (CDCl3):
penicillin, 100 units/mL streptomycin, and 0.25 mg/mL amphoter-
icin. 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 [6].
d
8.27 (s,1H, NH), 7.57–7.54 (m, 2H, Ar), 7.28–7.11 (m, 6H, Ar),6.72 (s,
1H, CHPh), 4.41–4.38 (dd, 1H, CHC(O)N), 4.14–3.94 (m, 2H,
CH2C(O)N), 3.81–3.44(m, 2H, NCH2), 3.46–3.36(m,1H, CHaHb),3.28–
3.20 (m, 1H, CHaHb), 1.64–1.54 (m, 2H, CH2CH2CH3), 1.40–1.33 (m,
2H, CH2CH3), 0.99–0.94 (t, 3H, CH3); m/z 467(Mþ þ 2), m/z 465(Mþ),
m/z 217 (100%); Anal. (C24H24BrN3O2) C:61.81, H:5.19, N:9.01;
found: C:61.80, H:5.14, N:8.97.
5.2.2. 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. Cell viability was measured 72 h after
treatment by the Cell Titer Glo Assay (Promega), which is a lumi-
nescent 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 VictorÒ multi-label plate reader [6].
5.1.31. (6R,12aS)-6-(2-Bromophenyl)-2-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (9d)
Yellowish white solid; 31%; mp 135–137 ꢁC; 1H NMR (CDCl3):
d
7.98 (s, 1H, NH), 7.71–7.53 (m, 2H, Ar), 7.29–7.13 (m, 6H, Ar), 6.82
(s,1H, CHPh), 4.34–4.29 (dd, 1H, CHC(O)N), 4.12–3.97 (m, 2H,
CH2C(O)N), 3.61–3.44 (m, 2H, NCH2), 3.34–3.21 (m, 1H, CHaHb),
3.00–2.90 (m, 1H, CHaHb), 1.57–1.47 (m, 2H, CH2CH2CH3), 1.35–1.32
(m, 2H, CH2CH3), 0.98–0.91 (t, 3H, CH3); m/z 467 (Mþ þ 2), m/z
465(Mþ), m/z 386 (100%); Anal. (C24H24BrN3O2) C:61.81, H:5.19,
N:9.01; found: C:61.77, H:5.23, N:9.06.
5.2.3. Phosphodiesterase assay
5 m/mL of purified PDE 5 (BPS Biosciences) was added to the
wells of black 96-well non-binding plates. Immediately, the protein
was treated with compound or vehicle control and 50 nM TAMRA-
cGMP (Molecular Devices) were added to each assay well. The
plates were incubated for 1.5 h at 30 ꢁC.After incubation, IMAP 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 using a Biotek Synergy 4 plate reader.
5.1.32. (6R,12aR)-2-Benzyl-6-(2-bromophenyl)-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (10a)
White solid; 23%; mp 135–137 ꢁC; 1H NMR (CDCl3):
d 8.26 (s, 1H,
NH), 7.59–7.56 (m, 2H, Ar), 7.56–7.21 (m,11H, Ar), 6.71 (s,1H, CHPh),
4.91–4.83 (m, 1H, CHC(O)N), 4.55–4.50 (m, 2H, CH2Ph), 3.95–3.81
(m, 3H, CH2C(O)N þ CHaHb), 3.38–3.28 (m, 1H, CHaHb); m/z 501
(Mþ þ 2), m/z 499(Mþ), m/z 420 (100%); Anal. (C27H22BrN3O2)
C:64.81, H:4.43, N:8.40; found: C:64.98, H:4.52, N:8.49.
5.2.4. Experimental design and data analysis
5.1.33. (6S,12aR)-2-Benzyl-6-(2-bromophenyl)-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (10b)
Yellowish white solid; 34%; mp 124–127 ꢁC; 1H NMR (CDCl3):
Drug effects on tumor cell growth and PDE activity were
measured and potency was expressed by an IC50 value (50% inhibi-
tory concentration).For growth assays, the IC50 value was deter-
mined by testing a range of 8 concentrations with at least four
replicates per concentration. For enzyme assays, the IC50 value was
determined by testing a range of 10 concentrations with at least two
replicates per concentration. Dose–response curves were analyzed
using PrismTM 4 software (GraphPad) to calculate IC50 values using
a four parameter logistic equation. All in vitro experiments involved
dose–response analysis, which was repeated at least twice to
confirm reproducibility of IC50 values.
d
8.03 (s, 1H, NH), 7.67–7.54 (m, 2H, Ar), 7.38–7.14(m, 11H, Ar), 6.84
(s, 1H, CHPh), 4.90–4.85 (m, 1H, CHC(O)N), 4.43–4.38 (m, 2H,
CH2Ph), 3.95–3.94 (m, 2H, CH2–C(O)N), 3.57–3.51 (dd, 1H, CHaHb),
3.04–2.94 (m, 1H, CHaHb); MS: m/z 501 (Mþ þ 2), m/z 499 (Mþ), m/z
420 (100%); Anal. (C27H22BrN3O2). C:64.81, H:4.43, N:8.40; found:
C:64.98, H:4.57, N:8.38.
5.1.34. (6S,12aS)-2-Benzyl-6-(2-bromophenyl)-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (10c)
Yellowish white solid; 32%; mp 138–140 ꢁC; 1H NMR (CDCl3):
5.3. Molecular modeling
d
8.26(s,1H, NH), 7.59–7.50 (m, 2H, Ar), 7.16–7.03 (m,11H, Ar), 6.70 (s,
1H, CHPh), 4.93–4.86 (m, 1H, CHC(O)N), 4.55–4.42(m, 2H, CH2Ph),
4.01–3.81 (m, 3H, CH2C(O)N þ CHaHb), 3.38–3.28 (m,1H, CHaHb); m/
z 501 (Mþ þ 2), m/z 499 (Mþ), m/z 420 (100%); Anal. (C27H22BrN3O2).
C:64.81, H:4.43, N:8.40; found: C:65.08, H:4.32, N:8.40.
Compound 4d was drawn and subjected to energy minimization
by the AM1 procedure, the partial charges are also calculated. The
human PDE5 X-ray crystal structure complexed with tadalafil (PDB
code: 1UDU) was used for docking experiments. One of the protein
chain, metals and the old ligands were deleted. The docking was
done with the default settings of the MOE-DOCK [16] as follow: The
option: Rotate Bonds was selected to give flexible ligand-rigid
receptor docking. The scoring function was London dG with
a replacement of Alpha Triangle.
5.1.35. (6R,12aS)-2-Benzyl-6-(2-bromophenyl)-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (10d)
Yellowish white solid; 25%; mp 128–130 ꢁC; 1H NMR (CDCl3):
d
7.95 (s, 1H, NH), 7.63–7.60 (m, 2H, Ar), 7.34–7.13 (m, 11H, Ar), 6.82
(s, 1H, CHPh), 4.89–4.84 (m, 1H, CHC(O)N), 4.56–4.33 (m, 2H,
CH2Ph), 4.01–3.87 (m, 2H, CH2C(O)N), 3.71–3.67 (dd, 1H, CHaHb),
3.35–3.28 (m, 1H, CHaHb); m/z 501 (Mþ þ 2), m/z 499 (Mþ), m/z 420
(100%); Anal. (C27H22BrN3O2) C:64.81, H:4.43, N:8.40; found:
C:64.78, H:4.42, N:8.44.
Acknowledgment
This work benefited from partial financial support from the
Faculty of Postgraduate Studies, the German University in Cairo.

1286
A.H. Abadi et al. / European Journal of Medicinal Chemistry 45 (2010) 1278–1286
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The first author is grateful to the Alexander von Humboldt foun-
dation, Germany for sponsoring a postdoctoral fellowship that
helps in accomplishing this work.
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