Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.09.029

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-β-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.

Full text of DOI:10.1016/j.ejmech.2012.09.029

European Journal of Medicinal Chemistry 57 (2012) 329e343
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the PDE5 H-pocket by ensemble docking and structure-based design
and synthesis of novel -carboline derivatives
b
Nermin S. Ahmed a, Amal H. Ali a, Shreen M. El-Nashar a, Bernard D. Gary b, Alexandra M. Fajardo b,
,
Heather N. Tinsley c, Gary A. Piazza b, Matthias Negri d, Ashraf H. Abadi a
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Main Entrance, Al-Tagmoaa Al-Khames, Cairo 11835, Egypt
b Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Suite 3029, Mobile, AL 36604-1405, USA
c Department of Biology, Chemistry and Mathematics, University of Montevallo, Montevallo, AL, USA
d Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tet-
rahydro-b-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some
Received 11 April 2012
Received in revised form
20 September 2012
Accepted 24 September 2012
Available online 29 September 2012
compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly
versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of
selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its
occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-
alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of
hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configu-
ration has been revealed through molecular docking.
Keywords:
PDE5 inhibitors
Tadalafil
Ó 2012 Elsevier Masson SAS. All rights reserved.
Carbolines
Ensemble docking
1. Introduction
treatment of MED and pulmonary hypertension [9,10]. Ongoing
clinical studies with PDE5 inhibitors have highlighted the potential
The cyclic nucleotides adenosine monophosphate (cAMP) and
cyclic guanosine monophosphate (cGMP) play important regula-
tory roles in signal transduction processes. cGMP is recognized as
a second messenger for atrial naturetic peptide and nitric oxide
signalling resulting in the relaxation of blood vessels [1,2]. Phos-
phodiesterase (PDE) enzymes play an important regulatory role in
both the cAMP and cGMP transduction processes through degra-
dation of these second messengers to the corresponding mono-
use of PDE5 inhibitors in range of additional indications such as
cardioprotection [11], enhancement of memory and cognitive
functions [12] and the treatment of diabetic peripheral neuropathy
[13]. Also, recent studies suggest their possible therapeutic appli-
cation of PDE5 inhibitors in cancer therapy [14,15].
The high degree of similarity within the PDE enzyme family [16]
is the main reason for selectivity issues affecting many known PDE5
inhibitors. The active site of PDE5 is placed in the center of the C-
terminal helical bundle domain and it includes five subsites: while
the metal-binding site (M site) and the core pocket (Q-pocket; lined
by Gln817, Phe820, Val782, Tyr612) are common to most of the
PDE family members, the hydrophobic pocket (H-pocket), the
Q2-pocket (lined by Phe786, Phe787, Leu804, Ile813, Met816) and
the lid region (L region) show significant differences with respect to
other PDE [17e19]. Therefore targeting at least one of these last
three regions represents a promising strategy to reduce the
promiscuity affecting many PDE5 inhibitors.
nucleotides,
adenosine
monophosphate
and
guanosine
monophosphate [3e5]. The PDE superfamily consists of 11 family
members, coded for by 21 genes with multiple splice variants.
PDE4, PDE7 and PDE8 specifically hydrolyze cAMP; PDE5, PDE6 and
PDE9 are cGMP specific, whilst PDE1, PDE2, PDE 3, PDE10 and
PDE11 show dual specificity and hydrolyze both nucleotides. PDE5
is widely distributed, throughout the body, being found in smooth
muscle throughout the body and also found in platelets and lung
[6e8]. This has led to the approval of sildenafil and tadalafil for the
Several crystal structures of PDE5 exist, either co-crystallized
with different inhibitors or substrates or as apoform (Table SI1).
Interestingly, they present the largest structural diversity in the
characteristic regions of PDE5, in particular in the H-loop (residues
* Corresponding author. Tel.: þ20 227590716; fax: þ20 227581041.
E-mail address: ashraf.abadi@guc.edu.eg (A.H. Abadi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.029

330
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
660e683) and the M-loop (residues 788e811) [20]. H-loop and M-
loop are often not resolved at all or, alternatively, they are mutated
into chimeric PDE5/PDE4 or PDE5/PDE6 hybrid forms (Table SI1).
Remarkably, the conformational ensemble exists not only by virtue
of the presence of different ligands, but also with the very same
inhibitor. This suggests for PDE5 the existence of a multitude of
metastable states and that approaching ligands will bind into pre-
existing PDE5 conformations following a selected-fit mechanism.
However, this also means that a larger sampling of the conforma-
tional space of PDE5 might be required for the search of the
“correct” PDE5 conformer to best reproduce binding mode and
structureeactivity relationships at least within one inhibitor series.
In the two crystal structures with tadalafil I the H-loop is either
only partially resolved (PDB 1UDU [19]; dimer form; residues
I665eL675 are missing) or it is replaced by the PDE4 counterpart
(PDB 1XOZ [18]; chimeric PDE5-PDE4H-loop construct). In the
chimeric crystal 1XOZ the H-loop assumes a bi-helical conforma-
tion with access to the binding site widely open. In 1UDU, on the
contrary, the H-loop is resolved only until Tyr664, but with the
existing segment supportive of the existence of a closed-conformer
as highlighted by the close proximity of the N-Met group of tada-
set of inhibitors (1, 29, 30) indicated the occluded/closed conformer
as the most appropriate to resemble the binding behaviour of these
inhibitor-series, but also as capable to host larger N-alkyl chains.
Due to these results our laboratory designed a novel compound
series which maintains the tetracyclic-b-carboline moiety of
compound I and its active analogues IIeIV (Table 1) [23e25], but
replaced the pendant benzodioxole of I with a 4-bromophenyl ring
in order to exploit the PDE5 characteristic Q2-pocket in terms of
activity and selectivity. Further, we exploited the stereoisomers and
varied the N-alkyl substituents on both hydantoin and piper-
azinedione rings in length, branching, and bulkiness (ethyl, butyl,
sec-butyl and tert-butyl instead of methyl). Finally, we retrospec-
tively analyzed an enlarged set of inhibitors in multiple single-
conformer docking runs with Autodock Vina with the N-alkyl
substituents acting as probes in the exploration of H-loop and L-
loop regions. This allowed insights into the influence of H-loop on
the inhibitory potency, the identification of the closed PDE5
conformer as the preferred one for binding of the
b-carbolines,
notably in agreement with the folding of the PDE5-I co-crystal
1UDU, and finally the determination of two stereospecific binding
modes, one for 5/6R and one for 5/6S-derivatives.
ꢀ
lafil within 4 A of the backbone of Asn662-Ser663-Tyr664.
Tadalafil is mainly stabilized by a single monodentate hydrogen
2. Results
bond with Gln817 and by p-stacking with the hydrophobic p-clamp
forming residues Phe820 and Val782 as well as with the Q2-pocket
lining Phe786 and Leu804. Point mutations confirmed the impor-
tance of these interactions, but also suggested an important role in
tadalafil binding to His613 and Tyr612 [21]. On the contrary no clear
interaction pattern could be determined for the piperazinedione
ring and its N-methyl substituent due to the conformational vari-
2.1. Chemistry
The general synthesis of hydantoin compounds are depicted in
Schemes 1 and 2. Pure D- or L-tryptophan methyl ester [24] and p-
bromobenzaldehyde were subjected to a PicteteSpengler reaction.
Since we initially desired access to both the cis- and trans-isomers
(1e4), the reaction was carried under non stereospecific conditions.
ability of the H-loop. As most of the b-carboline derivatives re-
ported in the last years bear structural variations in this region of I
[9,10,22e26], it is important to solve the role played by the H-loop
in inhibitor binding to further improve the drug design.
The cis- and trans-1,3-disubstituted tetrahydro-b-carbolines (1e4)
were separated by column chromatography, each of the pure dia-
stereomers was allowed to react with commercially available
isocyanates to yield the desired cis- and trans-hydantoins.
Piperazinedione derivatives were prepared by the chlor-
The aim of this study was the development of a series of novel b-
carboline derivatives, the elucidation of their binding mode and
their preference for the PDE5 conformation. A particular focus was
set on the investigation of different H-loop conformers as well as on
the identification of the best PDE5 conformer best resembling the
structureeactivity profile for this class of inhibitors. Thereby, we
combined molecular modelling with chemical synthesis and bio-
logical evaluation. Results of a first ensemble docking with a small
oacetylation of pure cis- and trans-1,3-disubstituted tetrahydro-b-
carbolines (1e4) in the presence of NaHCO3. This step provided, the
respective chloroethanone derivative in an excellent yield. The
piperazinedione diastereomers were then obtained by ring closure
of the corresponding chloroacetyl derivatives in the presence of
primary amines, namely ethylamine and tert-butylamine.
Table 1
% Inhibition and IC50 values of tadalafil and some analogues.
O
O
R
N
O
O
R
N
N
N
N
N
N
N
O
O
O
O
N
N
H
Br
N
H
H
N
H
O
Cl
O
Cl
I
II
III
IV
Code
R
Absolute stereo-chemistry
% PDE5 inhibition at 10
mM
IC50 mM
I
II
III
IV
Methyl
Ethyl
Ethyl
Ethyl
(6R,12aR)
(5R,11aR)
(6R, 12aR)
(6R,12aR)
100
85
98
0.003
1.1
0.32
0.003
105

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
331
l
l
l
l
l
Scheme 1. Synthesis of hydantoin series.
The assignment of cis-/trans-stereochemistry for the tetrahydro-
-carbolines (1e4) was based on detailed study of 13C NMR spec-
difference in Rf values are not as significant in the piperazinedione
series as it is in the hydantoin series. In mass spectrometry, the
molecular ion peaks appeared at Mþ and Mþ þ 2 due to the isotopic
nature of the bromine atom.
b
troscopy data as established from previous studies [9,22]. Signals
for C-1 and C-3 in the trans-isomers appear at higher field in the
carbon spectrum than the analogous carbons of the corresponding
cis-isomer, probably due to the 1, 3-interactions present in the
trans-tetrahydro-
proton at C-1, of the 1,3-disubstituted-tetrahydro-
4) appeared at about 5.20e5.35 ppm, a remarkable downfield
b
-carboline isomer. The 1H NMR signals for the
2.2. Molecular modelling
b-carbolines (1e
d
2.2.1. Structural analysis
shift is noticed upon cyclization to the hydantoin and piper-
azinedione derivatives, the introduced carbonyl group deshields
the same proton which is now attached to C-5 and C-6, respectively.
Notably, regarding the tetrahydro-b-carbolines a significant
difference has been noted between the 2 isomers regarding their Rf
values. A correlation exists between Rf value on TLC and the
All crystal structures of PDE5 were retrieved from the PDB
database [27] and superimposed using software package MOE
(MOE 2010.10; Molecular Operating Environment; http://www.
chemcomp.com) with PDB 2H42 (chain A) [20] as reference
structure. Only two out of twenty-three crystal structures are fully
solved and with wild-type sequence (PDB 2H42, 2H44). Therefore,
the PDE5 crystals were clustered using the Consensus module of
MOE including only those with a resolved H-loop and with
sequence variability as additional criterion (i.e. PDE5, PDE4 and
stereochemistry of the 1, 3-disubstituted tetrahydro-b-carbolines.
The cis-isomer is systematically less polar than the trans-isomer;
however, in the hydantoin series, the polarity is reversed, thus, the
cis-isomer becomes more polar than the trans-isomer. The
ꢀ
PDE6). Clustering with an RMSD cut-off >1 A yielded into three

332
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
l
l
l
l
l
Scheme 2. Synthesis of piperazinedione series.
distinct clusters resembling the open, occluded and closed
conformers of PDE5 (Fig. 1; Table SI1).
compared to 1XOZ and 1UDU) and it was stabilized in both X-rays
by the same interactions: 1) single monodentate hydrogen bond
Superimposition of the two crystal structures with I, namely
PDB 1XOZ [18] and 1UDU [19], resulted in a backbone RMSD of
w1 A for the whole protein (2 A binding site residues). Compound I
between the eNHe of the tetracyclic-
Gln817, highly conserved within the PDE family; 2) CHe
action between the eCHe (R isomer) in 6 of I and Phe820 of the P-
clamp; 3) -stacking and van der Waals interactions between the
b
-carboline moiety and
p
inter-
ꢀ
ꢀ
superimposed well although in 1UDU it results slightly displaced
p

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
333
performed [35] The number of compounds were increased up to
twenty-five, taking care to include different stereoisomers with N-
alkyl substituents of different length, shape and size, and docked
them in each of the four PDE5 conformers used in the ensemble.
Plotting the predicted binding affinities of the top ranked pose of
each compound against the pIC50 values resulted in the closed
conformer (2H44, r ¼ 0.83; Fig. 2) clearly outperforming the open
(1XOZ, r ¼ 0.52, 3BJC, r ¼ 0.40) and the occluded (2H42, r ¼ 0.51)
(Fig. SI1) conformers both in terms of affinity prediction and
discrimination between potent and inactive/weak inhibitors.
2.3. Biology
Compounds were evaluated in an in vitro assay for inhibitory
activity against human PDE5; each compound was evaluated in two
steps. The first step was the determination of the percentage of
inhibition at 10 mM performed in triplicate. For compounds dis-
playing a percentage of inhibition greater than 60%, the IC50 was
determined from a concentrationeresponse curve using a range of
8 concentrations (1 nMe10 mM) with at least two replicates per
concentration. The results are shown in Tables 2e5. Moreover, for
compound 8, 25, 28, 29 and 33 the selectivity towards other PDEs
(PDE1A, PDE2A, PDE3A, PDE3B, PDE9A, PDE10A and PDE11A) were
evaluated were evaluated at a concentration of 50 mM, the results
are shown in Table 6. For compounds that displayed greater than
50% inhibition, IC50 values were determined.
Fig. 1. Superimposition of PDE5 structures resembling six distinct H-loop conformers.
H-loops are rendered as cartoons (PDB 1RKP green, 3BJC blue, 1XOZ yellow, 2H40 red,
2H42 cyan, 2H44 magenta) whereas the rest of the PDE5 structure as thin ribbons. The
surface of the binding pocket of tadalafil is shown as lines. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
3. Discussion
pendant benzodioxole of I and the Q2-pocket lining residues
Ala783, Phe786, Phe787, Leu804, Ile813 and Met816 as well as
between the b-carboline moiety and Tyr612, Leu765, Ala767, Ile768,
Val782, Gln775, Ile778 and Ala779; 4) water-mediated hydrogen
bond with His613/Asn661, residues involved in metal ions ligation
(Zn2þ and Mg2þ).
Ensemble docking of multiple protein structures has proven
successful as tool in drug design and enzyme dynamics issues [28e
32]. The strong advantages of this method are the inclusion of
protein plasticity and the significantly reduced risk of inadvertently
choosing an unsuitable protein model. Further an ensemble con-
sisting of a reduced yet significant amount of conformations is often
enough to improve the docking performance [33]. This study
systematically investigated the effect of different N-alkyl substitu-
ents on their inhibitory potency, making use of four representative
PDE5 conformations.
2.2.2. Ensemble docking
For these studies there was a need to identify which PDE5
conformer best reflects the binding behaviour of tadalafil and
congeners. Based on the results of the Consensus Scoring our
conformational ensemble was built including four most represen-
tative structures, namely PDB 2H44 (closed) [20], 2H42 (occluded)
[20], 3BJC [16] and 1XOZ (open) [19]. For the open state two
structures were included in order to consider both the wild type
form (3BJC) as well as the original tadalafil co-crystal structure
(1XOZ), which is a hybrid form. As 3BJC lacks part of the M-loop
a homology model using the M-loop of 2H42 as template was built.
This PDE5 conformer collection was explored with a small sub-set
of inhibitors in an ensemble docking using GOLDv5.0 with GOLD-
SCORE (GS) [33,34] fitness function. The inhibitor set comprised
compound I (with known IC50), and the newly designed 29 and 30,
which differ in stereochemistry. According to the GS scoring values
and the number of poses found per conformer a preference for the
closed (2H44)/occluded (2H42) crystals emerged for compound 29,
whereas for I no clear preference could be determined with the
poses equally divided in open (1XOZ) and occluded (2H42)
conformers. Conversely, for the 6S-isomer 30 a clear preference for
the open conformers resulted, with the N-alkyl substituent point-
ing into the buried wall of the Q-pocket (Table SI2).
2.2.3. Multiple single-conformer docking
In order to validate the results of the ensemble approach on
a larger dataset and to retrospectively derive a precise structuree
activity relationships for this inhibitor class we performed
a multiple single conformer docking with AutoDock Vina v.1.1.2 was
Fig. 2. Binding affinities (in kcal/mol) plotted versus the pIC50 of 25 compounds as
output of the closed PDE5 conformer docking run with Autodock Vina 1.1.2 with the
regression coefficient r.

334
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
Table 2
Table 4
% Inhibition and IC50 values for tetrahydro-
b
-carbolines.
% Inhibition and IC50 values for chloroethanone derivatives.
O
O
O
Cl
N
O
NH
O
N
H
N
H
Br
Br
Code
Absolute stereo-chemistry
% PDE5 inhibition at 10
m
M
IC50
mM
Code
Absolute stereo-chemistry
% PDE5 inhibition at 10
mM
IC50 (mM)
1
2
3
4
(1R,3R)
(1S,3R)
(1S,3S)
(1R,3S)
10
10
17
75
ND*
ND
ND
4.2
21
22
23
24
(1R,3R)
(1S,3R)
(1S,3S)
(1R,3S)
25
17
19
21
>10
>10
>10
>10
The introduction of a 4-bromophenyl instead of the pendant
benzodioxole ring of compound I was guided by several consider-
ations: first, the size of Q2-pocket strongly diverges within the PDE
family making it an ideal targeting for improved selectivity and
potency of novel inhibitors; second, as bromine is an isostere of
chlorine and as the activity of compound IV (p-chlorophenyl) is
almost 100 times the activity of the o-bromine analogue III,
studying the impact of positional isomerism is of great importance;
third, bromine is bulkier and more lipophilic than chlorine and may
fill the prevalently lipophilic Q2-pocket. In fact, potent compounds
were obtained as evidenced by the compound 29 (IC50 ¼ 3 nM),
which is as active as tadalafil I and analogue IV outperforming the
o-bromophenyl analogue III (IC50 ¼ 320 nM).
All short tetrahydro-b-carboline derivatives (1e4; 21e24), with
the exception of compound 4 (IC50 ¼ 4.2
mM), possess no PDE5
inhibitory activity indicating the crucial need for a fused tetracyclic
ring. Introduction of carbonyls is not sufficient to yield inhibition
suggesting that a rigidification of the carbonyls must occur in order
to interact via water-bridging with His613 and with the backbone
carbonyl of Met816 as seen for I in 1XOZ [21,36]. Still, given an
appropriate stereochemistry (i.e. 4 e 1R,3S) some potency might be
Table 5
Table 3
% Inhibition and IC50 values for piperazinedione derivatives.
% Inhibition and IC50 values for hydantoin derivatives.
O
R
N
O
R
N
N
O
N
O
N
H
N
H
Br
Br
Code
R
Absolute
% PDE5
IC50 m
M
Code
R
Absolute
% PDE5 inhibition
IC50 m
M
stereo-chemistry
inhibition at 10
m
M
stereo-chemistry
at 10 mM
5
6
7
8
Ethyl
Ethyl
Ethyl
Ethyl
Butyl
Butyl
Butyl
Butyl
sec-Butyl
sec-Butyl
sec-Butyl
sec-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
(5R,11aR)
(5S,11aR)
(5S,11aS)
(5R,11aS)
(5R,11aR)
(5S,11aR)
(5S,11aS)
(5R,11aS)
(5R,11aR)
(5S,11aR)
(5S,11aS)
(5R,11aS)
(5R,11aR)
(5S,11aR)
(5S,11aS)
(5R,11aS)
94.4
89.3
79.8
100.9
98.7
72.1
81.5
106.0
83
67
72
92
91
2.8
3.9
>10
0.051
0.230
>10
>10
0.04
0.37
6.0
3.3
0.15
0.27
2.2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Methyl
Methyl
Methyl
Methyl
Ethyl
Ethyl
Ethyl
Ethyl
Butyl
Butyl
Butyl
Butyl
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
(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,12aR)
(6S,12aS)
(6R,12aS)
98
94
87
91
96
97.9
83
98.7
87
81.5
80
0.019
6.6
1.9
0.012
0.003
1.9
0.22
<0.01
0.009
6.6
5.9
0.01
ND
ND
ND
9
10
11
12
13
14
15
16
17
18
19
20
92
30
27
12
78
38
94
ND
0.27
21
ND

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
335
Table 6
IC50 M) of compound 25 and tadalafil versus an array of PDEs.
(
m
Cpd PDE1A inhibition PDE2A inhibition PDE3A inhibition
PDE3B inhibition
IC50
PDE5A
PDE9A
PDE10A inhibition
PDE11A inhibition
IC50 mM
IC50 IC50
m
M
mM
IC50 m
M
m
M
inhibition inhibition IC50
mM
IC50 m
M
IC50 mM
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
cGMP
cGMP
cAMP
cGMP
cAMP
cGMP
I
8
25
28
29
33
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.003
0.051
0.019
0.012
0.003
0.009
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
26.1
>50
>50
>50
41.7
>50
14.7
>50
0.295
44.00
0.6
1.30
0.30
1.20
0.05
35.70
0.270
1.13
0.27
0.60
retained with the
bond with Gln817 and
(Phe820 as well), eventually reinforced by catione
between the protonated secondary amine (protonation state at pH
7 predicted with MOE) and Phe786, as suggested by docking poses
(Fig. SI3). This hypothesis is strengthen by the fact the insertion of
a chloroethanone group on the nitrogen depletes all inhibitory
potency.
b
-carboline moiety being able to form a hydrogen
-stacking with the P-clamp residue Val782
interactions
outer wall of the Q2-pocket, delimiting it and contributing to
narrow the access path to the binding site. H-loop and M-loop are
the most flexible regions of the enzyme and both are partially not
resolved in the PDE5 crystal ensemble. Considering the labile
nature of the M-loop 3.10 helix it is possible that its unfolding could
affect both the M-loop and the tightly interconnected H-loop [37],
thus eventually leading to solvent exposure of the Q-pocket.
p
p
3.2. 5/6 S-isomers
3.1. 5/6 R-isomers
All 5/6 S derivatives were docked rotated of 180ꢀ compared to
the 5/6 R isomers with the N-alkyl pointing towards the buried Q-
pocket with the 4-bromophenyl ring fitted into the Q2-pocket.
Among the most potent analogues with C5/6 are of the S configu-
ration (18, 30), they are stabilized by hydrogen bonds with Gln817
The best scored docking poses of the 5/6 R derivatives all
resemble the crystallographic binding mode of I and its interactions
with the Q-pocket, P-clamp and Q2-pocket residues. Accordingly,
the reasons for the marked differences in terms of potency have to
be identified in the final five or six membered rings and their N-
alkyl substituents. The last consideration fits also for the 5/6 S
analogues, albeit in that case the residues involved in favourable or
repulsive interactions are placed on the opposite and buried part of
the Q-pocket.
The most active 5R-hydantoin analogue is 12 (IC50 ¼ 40 nM)
bearing an n-butyl group, whereas the most active piperazinedione
analogue 29 (IC50 ¼ 3 nM) has an ethyl on the N. Flexible and non-
branched rather than bulky and rigid alkyl chains seems to ensure
a higher potency [24]. For the hydantoin series the activity
increases with the length (n-butyl > ethyl), whereas for the
piperazinediones ethyl was the best substituent followed by n-
butyl and methyl. For both series the activity decreases with the
bulkiness (n-Bu > sec-Bu > tert-Bu) of the N-substituent, with
stronger effects for the piperazinediones, whereas a tert-butyl (37e
40) depletes the inhibitory potency in contrast to their hydantoin
analogues.
However, comparing hydantoin and piperazinedione congeners
the length of the N-alkyl chain seems to play a subordinated role
compared to the size of the fused ring, as evidenced by the 1000
times improved activity of the six member ring compound 29
compared to its five member congener 5 (IC50 of 3 and 2800 nM,
respectively). As seen in the docking poses (Fig. 3) this might be due
to the facts that while 29 can interact with both the crystallographic
waters (and thus with His613 and Met816), the small hydantoin
ring of 5 can only catch one water molecule. Furthermore, the
methylene at the bridgehead between the two nitrogen atoms in
29, missing in the hydantoin series, is close to the L-region residues
Ile665, Leu725, Leu804 and Met816 ensuring a better anchoring
inside the binding pocket in virtue of favourable van der Waals
interactions. The N on the terminal piperazinedione ring placed
closer to the H-loop (Fig. 4) explains the diverse SAR observed for
the R substituent in the two series, in particular the preference for
n-butyl (12) and ethyl (29) as well as the moderate potency of tert-
via one of their carbonyl oxygen on the terminal ring and
p-
stacking with the P-clamp residue Phe820 (Fig. 5). The alkyl groups,
such as tert-butyl in the hydantoin derivative 18 and ethyl in the
piperazinedione derivatives 30 and 31, are buried in a hydrophobic
pocket formed by Tyr612, Leu765, Ala767, Ile768, Gln775, Ile778
and Val782, but their stabilization is strongly dependent on size,
and bulkiness otherwise it results in severe steric clashes. The
carboline moiety is placed into the H-pocket, where it interacts via
p-stacking with Phe786 and van der Waals interactions with
Ser663, Ile665, Leu725 and His613. Notably, in this pose the car-
boline would displace the important water molecule linking I with
His613 thus explaining (paired with the stringent requirements for
the n-alkyl substituent) the overall weaker inhibitory potency of
the 5/6 S isomers with respect to their 5/6 R congeners.
The close vicinity of the N-alkyl groups to the H-loop residues
Asn661, Ser663 and Ile665 (H-loop) for the 5/6 R-isomers as well as
to Tyr612 and Gln775 for the 5/6 S-isomers suggests that replacing
the N-alkyl group with polar acceptor atoms containing groups or
with halogen atoms in analogy to the recent study of Xu et al. [38],
might lead to novel
and selectivity.
b-carboline derivatives with improved potency
3.3. Selectivity
Compounds 8, 25, 28, 29 and 33 showed inhibitory activities
versus PDE5 and PDE11 with cGMP as the substrate with selectivity
profiles of 700,14, 94, 90, and 67, respectively (Table 6). Interestingly,
four out of the five compounds showed selectivity profile better than
tadalafil (selectivity index; 17). Increasing the bulkiness of the
terminal ring N-alkyl substituent and reducing the size of the
terminal ring are the main determinant favouring selectivity. Since
these N-alkyl substituents are involved in hydrophobic interaction
with ILE665 of the H-loop, thus, it seems that there are crucial
differences between PDE5 and PDE11 in the size and nature of the
residues in those particular corresponding pockets.
butyl (20; IC50 0.27 mM) versus an IC50 more than IC50 > 10 mM (37).
Leu804 is placed at the top of a 3.10 helix within the M-loop in
close vicinity to the H-loop residues S663-Y664. Here it forms the
Differences in the selectivity of 8 versus 25 may also indicate
differences in the occurrence of water molecules in the pocket.

336
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
Fig. 3. Binding modes of compounds 5 (rosa) and 29 (cyan). The reduced size of the hydantoin ring of 5 allows interaction with only one water molecule (sphere representation) in
contrast to 29, which is more tightly anchored into the active side by two water-mediated bridges with His613/Asn661 and Met816. H-bonds are rendered as green dots, whereas
CHe
acids in magenta. H-bonds and all
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
p
and other
p-stacking interactions as yellow ones. In the two 2D-ligand-PDE5 interactions plots hydrophobic/aromatic residues are coloured in green, whereas polar amino
p-stacking interactions are shown as green dotted lines, whereas intra-residue interactions in green-yellow. The active site contour is also shown.
4. Conclusion
disclose the modulating effects of N-alkyl substituents on the
inhibitory potency.
In this study, a novel series of hydantoin and piperazinedione
b
-
Compounds with high activity and selectivity profile towards
cGMP PDE5 versus cGMP PDE11 were obtained e.g. 8, 28, 29, and 33.
Thus, carbolines with different terminal rings and different terminal
N-substituents might be proposed to modulate selectivity. Although
the catalytic domain is highly conserved between PDE5 and PDE11
the functional role of PDE11 is controversial therefore the identifi-
cation of selective PDE5 analogues is required. Future design of PDE5
inhibitors should consider the use of variable polar N-substituents
rather than the classical non-polar alkyl groups this modification
would reveal novel H-loop interactions which potentially could
enhance potency and selectivity of novel inhibitors.
carbolines were presented as selective PDE5 inhibitors. Via ensemble
docking the effects of different N-alkyl substituents as well as of
different stereoisomers on PDE5 inhibition were tested. For these
inhibitor series a preference for the closed/occluded PDE5 conformer
was determined, which closely resembled the existing part of the
truncated PDE5-compound I co-crystal complex 1UDU. Furthermore,
an appreciable correlation between the predicted binding affinityand
the pIC50 values of the investigated compounds was observed for the
closed conformer, whereas with the open conformer there was no
distinction betweenpotentand inactive compounds. Finally, we could
show the necessity of R-absolute configuration at C-5 and C-6 was
demonstrated and regardless to the absolute configuration at C-11a
and C-12, and the existence of two stereospecific binding modes for 5/
6R and 5/6S-iosmers, respectively.
5. Experimental
5.1. Chemistry
The largest differences in potency prediction were observed for
the n-butyl-piperazinedione series which were all underestimated.
This leads to the conclusion that the closed/occluded conformer
still might not be adequate and further conformational studies of
the diverse H-loop conformers and the importance of Ile665,
Leu804 and Met816 sites need to be further investigated to fully
All starting materials were commercially available and of pure
analytical grade. All reactions were carried out under inert gas
(nitrogen). Organic extracts were dried over anhydrous Na2SO4.
Solvents were removed under reduced pressure using a rotavap.
Reaction progress was monitored by TLC, performed on precoated

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
337
Fig. 4. Binding poses of compounds 12 (green), 20 (yellow), 33 (violet) and 37 (rosa) and crystallographic pose of I (brown). Steric clashes (energy contribution >1 kcal/mol)
between the t-butyl-bearing compounds 20 and 37 and the amino acid residues Tyr612, Ile665, Leu725, and F786 are shown as cyan dots and discs, and might explain the reduced
inhibitory potency. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
silica gel plates (ALUGRAM SIL G/UV254) and detection of the
components was made by short UV light. Column chromatography
in deuterated chloroform (CDCl3) and dimethylsulfoxide (DMSO).
Chemical shifts ( ) were reported in parts per million (ppm)
d
was performed using silica-gel (70e200
m
m). Melting points were
downfield from TMS; multiplicities are abbreviated as: s: singlet; d:
doublet; t: triplet; q: quartet; m: multiplet; dd: doublet of doublet;
brs: broad. Mass spectra were made on Focus GC/Polaris MS, model
5890; series II at an ionization potential of 70 eV. Elemental analysis
determined on Buchi Melting Point apparatus and are uncorrected.
FTIR spectra were recorded on Nicolet Avatar 380 spectrometer. 1H
spectra were run at 300 MHz and 13C spectra were run at 75.46 MHz
Fig. 5. Binding modes of the best ranked poses of the 5/6 S-isomer compounds 15 (magenta; clashes with Ile778, Leu765 as orange disks), 18 (green) and 30 (yellow). Steric clashes
with Leu765, Ile778 and F820 are rendered as cyan dots and discs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

338
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
were performed by the Microanalytical Unit, Faculty of Science,
Ar), 7.29e7.23 (t, 3H, Ar), 7.08e6.96 (m, 2H, Ar), 5.35 (s, 1H,
CHPh), 3.83e3.78 (t, 1H, CHCOOCH3), 3.64 (s, 3H, OCH3), 3.13e
Cairo University; the found values were within ꢁ0.4% of the theo-
retical ones, unless otherwise indicated.
D
- &
L-Tryptophan methyl
3.06 (dd,
J
¼
15.18, 5.12 Hz, 1H, CHaHb), 2.97e2.90 (dd,
ester were prepared by a known procedure [22e24].
J ¼ 15.18, 7.38 Hz, 1H, CHaHb). 13C NMR:
d 173.66, 142.39, 136.18,
133.76, 130.97, 130.45, 126.48, 120.90, 120.34, 118.40, 117.60,
111.05, 106.70, 53.44 (C1), 51.85 (C3), 51.57, 24.68. MS: m/z 386
(Mþ þ 2), m/z 384 (Mþ, 100%). Elemental analysis: calculated for
(C19H17BrN2O2) C, H, N.
5.2. Methods
5.2.1. General procedures for the preparation of methyl-1-(4-
bromophrnyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylate
(1e4)
5.2.1.5. General procedures for the preparation of: 2-alkyl-5-(4-
The appropriate tryptophan methyl ester (11.8 g, 54.16 mmol)
was added to 4-bromobenzaldehyde (11 g, 59.48 mmol) and dis-
solved in CH2Cl2 (10 mL). The solution was cooled to 0 ꢀC in an ice
bath. To this solution TFA (3 mL) was added dropwise, 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 50 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 chromatog-
raphy on silica gel eluting with CH2Cl2, to give first the appropriate
cis-isomer followed by the trans-one.
bromophenyl)-5,6,11,11a-tetrahydro-1H-imidazo
[3,4-b]indole-1,3(2H)-dione (5e20). Excess alkyl isocyanate
(1.6 mmol) was added to well stirred solution of the
[10,50:1,6]pyrido
a
appropriate beta carboline 1e4 (0.38 g, 1 mmol) in methyl ethyl
ketone (10 mL). The mixture was then stirred at reflux for 16 h
under N2 atmosphere. The solvent was evaporated under reduced
pressure, the residue was purified using column chromatography
on silica gel, eluting with CH2Cl2.
5.2.1.6. (5R,11aR)-5-(4-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(5). Yield
20%; yellowish white powder; mp 250e253 ꢀC; Rf ¼ 0.51 (CH2Cl2/
MeOH 99.5:0.5); IR (cmꢃ1): 3240 (eNHe), 1703, 1692 (eCOe). 1H
5.2.1.1. Methyl (1R,3R)-1-(4-bromophenyl)-2,3,4,9-tetrahydro-1H-
b
-
NMR: d 7.85 (s, 1H, NH), 7.66e7.63 (m, 1H, Ar), 7.57e7.54 (m, 1H, Ar),
carboline-3-carboxylate (1). Yield 34%; yellow powder; mp 202e
7.25e7.06 (m, 6H, Ar), 6.49 (s, 1H, CHPh), 4.41e4.37 (m, 1H, CHC(O)
N), 3.57e3.51 (m, 3H, NCH2 þ CHaHb), 3.10e3.01 (m, 1H, CHaHb),
1.23e1.18 (t, 3H, CH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 218
(100%). Elemental analysis: calculated for (C21H18BrN3O2) C, H, N.
205 ꢀC; Rf ¼ 0.72 (CH2Cl2/MeOH 99:1); IR (cmꢃ1): 3375 (eNHe),
1737 (eCOe). 1H NMR:
d 10.3 (s, 1H, NH), 7.56e7.53 (m, 2H, Ar),
7.46e7.43 (d, 1H, Ar), 7.34e7.31 (m, 2H, Ar), 7.24e7.21 (m, 1H, Ar),
7.05e6.94 (m, 2H, Ar), 5.23 (s, 1H, CHPh), 3.91e3.86 (dd, J ¼ 10.93,
4.16 Hz, 1H, CHCOOCH3), 3.72 (s, 3H, OCH3), 3.08e3.2 (m, 1H,
5.2.1.7. (5S,11aR)-5-(4-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (6). Yield 27%;
yellow powder; mp 110e113 ꢀC; Rf ¼ 0.6 (CH2Cl2/MeOH 99.5:0.5); IR
CHaHb), 2.91e2.82 (m, 1H, CHaHb). 13C NMR:
d 172.52, 141.31, 136.29,
134.66, 130.96, 130.66, 126.34, 120.70, 120.60, 118.27, 117.37, 110.99,
106.91, 57.12 (C1), 56.06 (C3), 51.50, 25.16. MS: m/z 386 (Mþ þ 2), m/
z 384 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C19H17BrN2O2) C, H, N.
(cmꢃ1): 3311 (eNHe),1763,1691 (eCOe).1H NMR:
d 7.84 (s,1H, NH),
7.59e7.56 (m, 1H, Ar), 7.51e7.48 (m, 1H, Ar), 7.33e7.16 (m, 6H, Ar),
6.30 (s, 1H, CHPh), 4.30e4.24 (dd, J ¼ 11.1, 5.1 Hz, 1H, CHC(O)N),
3.66e3.58 (m, 2H, NCH2), 3.54e3.47 (dd, J ¼ 15.45, 5.4 Hz, 1H,
CHaHb), 2.94e2.85 (ddd, J ¼ 15.38, 11.01 Hz, 1H, CHaHb), 1.28e1.26 (t,
3H, CH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 218 (100%).
Elemental analysis: calculated for (C21H18BrN3O2) C, H, N.
5.2.1.2. Methyl (1S,3R)-1-(4-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylate (2). Yield 36%; yellow powder; mp 177e
180 ꢀC; Rf ¼ 0.43 (CH2Cl2/MeOH 99:1); IR (cmꢃ1): 3334 (eNHe),
1709 (eCOe). 1H NMR:
d 10.61 (s, 1H, NH), 7.53e7.44 (dd, 3H,
Ar), 7.24 (t, 3H, Ar), 7.06e6.97 (m, 2H, Ar), 5.32 (s, 1H, CHPh), 3.80e
3.75 (dd, J ¼ 7.09, 5.39 Hz, 1H, CHCOOCH3), 3.63 (s, 3H, OCH3),
3.10e3.04 (dd, J ¼ 15.17, 5.14 Hz, 1H, CHaHb), 2.95e2.87 (dd,
5.2.1.8. (5S,11aS)-5-(4-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(7). Yield
23%; yellowish white powder; mp 251e253 ꢀC; Rf ¼ 0.51 (CH2Cl2/
J ¼ 15.21, 7.32 Hz, 1H, CHaHb). 13C NMR:
d 173.47, 142.26, 136.05,
MeOH 99.5:0.5); IR (cmꢃ1): 3241 (eNHe), 1703, 1692 (eCOe). 1H
133.62, 130.80, 130.26, 126.35, 120.72, 120.14, 118.22, 118.43, 110.88,
106.56, 53.29 (C1), 51.71 (C3), 51.37, 24.52. MS: m/z 386 (Mþ þ 2),
m/z 384 (Mþ, 100%). Elemental analysis: calculated for
(C19H17BrN2O2) C, H, N.
NMR: d 7.86 (s, 1H, NH), 7.63e7.54 (m, 2H, Ar), 7.27e7.03 (m, 6H, Ar),
6.49 (s, 1H, CHPh), 4.41e4.37 (m, 1H, CHC(O)N), 3.56e3.49 (m, 3H,
NCH2 þ CHaHb), 3.10e3.01 (m,1H, CHaHb),1.23e1.18 (t, 3H, CH3). MS:
m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C21H18BrN3O2) C, H, N.
5.2.1.3. Methyl (1S,3S)-1-(4-bromophenyl)-2,3,4,9-tetrahydro-1H-b-
carboline-3-carboxylate (3). Yield 33%; yellow powder; mp 205e
5.2.1.9. (5R,11aS)-5-(4-Bromophenyl)-2-ethyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (8). Yield 25%;
yellow powder; mp 115e118 ꢀC; Rf ¼ 0.6 (CH2Cl2/MeOH 99.5:0.5); IR
207 ꢀC; Rf ¼ 0.73 (CH2Cl2/MeOH 99:1); IR (cmꢃ1): 3382 (eNHe),
1726 (eCOe). 1H NMR:
d 10.32 (s, 1H, NH), 7.54e7.42 (dd, 2H, Ar),
7.32e7.21 (m, 4H, Ar), 7.03e6.95 (m, 2H, Ar), 5.20 (s, 1H, CHPh), (dd,
J ¼ 10.93, 4.16 Hz,1H, CHCOOCH3), 3.70 (s, 3H, OCH3), 3.08e3.04 (m,
(cmꢃ1): 3313 (eNHe),1763,1692 (eCOe).1H NMR:
d 7.76 (s,1H, NH),
7.59e7.56 (d,1H, Ar), 7.51e7.49(m, 2H,Ar), 7.33e7.24 (m, 5H, Ar), 6.29
(s, 1H, CHPh), 4.30e4.24 (dd, J ¼ 11.00, 5.51 Hz, 1H, CHC(O)N), 3.62e
3.59 (m, 2H, NCH2), 3.55e3.48 (dd, J ¼ 15.38, 5.55 Hz, 1H, CHaHb),
2.95e2.85 (ddd, J ¼ 15.38,11.00,1.80 Hz,1H, CHaHb),1.26e1.23 (t, 3H,
CH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C21H18BrN3O2) C, H, N.
1H, CHaHb), 2.90e2.82 (m, 1H, CHaHb). 13C NMR:
d 172.63, 141.12,
136.45, 134.64, 131.21, 130.98, 126.45, 120.95, 120.91, 118.54, 117.65,
111.24, 107.03, 57.16 (C1), 56.15 (C3), 51.86, 25.21. MS: m/z 386
(Mþ þ 2), m/z 384 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C19H17BrN2O2) C, H, N.
5.2.1.4. Methyl (1R,3S)-1-(4-bromophenyl)-2,3,4,9-tetrahydro-1H-
b
-
5.2.1.10. (5R,11aR)-5-(4-Bromophenyl)-2-butyl-5,6,11,11a-tetrahydro-
carboline-3-carboxylate (4). Yield 42%; yellow powder; mp 179e
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(9). Yield
182 ꢀC; Rf ¼ 0.43 (CH2Cl2/MeOH 99:1); IR (cmꢃ1): 3333 (eNHe
38%; yellowish white powder; mp 225e228 ꢀC; Rf ¼ 0.48 (CH2Cl2/
), 1709 (eCOe). 1H NMR:
d 10.59 (s, 1H, NH), 7.53e7.45 (dd, 3H,
MeOH 99.5:0.5); IR (cmꢃ1): 3298 (eNHe), 1766, 1692.3 (eCOe). 1H

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
339
NMR:
d
7.89 (s, 1H, NH), 7.57e7.56 (d, 2H, Ar), 7.48e7.44 (d, 1H, Ar),
5.2.1.16. (5S,11aS)-5-(4-Bromophenyl)-2-sec-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (15).
Yield 40%; yellow powder; mp 240e243 ꢀC; Rf ¼ 0.5 (CH2Cl2); IR
7.27e7.17 (m, 5H, Ar), 5.77 (s, 1H, CHPh), 4.42e4.30 (dd, J ¼ 11.4,
4.5 Hz, 1H, CHC(O)N), 3.51e3.46 (m, 3H, NCH2 þ CHaHb), 3.11e2.94
(dd, 1H, CHaHb), 1.62e1.54 (m, 2H, CH2CH2CH3), 1.33e1.27 (m, 2H,
CH2CH3), 0.94e0.89 (t, 3H, CH3). MS: m/z 453 (Mþ þ 2), m/z 451
(Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.
(cmꢃ1): 3292 (eNHe), 1762, 1692 (eCOe). 1H NMR:
d 7.85 (s, 1H,
NH), 7.57e7.45 (m, 3H, Ar), 7.21e7.17 (m, 5H, Ar), 5.76 (s, 1H, CHPh),
4.36e4.30 (m, 1H, CHC(O)N), 4.07e3.98 (m, 1H, NCH), 3.54e3.47
(dd, 1H, CHaHb), 3.08e2.95 (m, 1H, CHaHb), 2.01e1.86 (m, 1H,
NCHCHaHb), 1.76e1.62 (m, 1H, NCHCHaHb), 1.40e1.37 (dd, 3H,
NCHCH3), 0.88e0.83 (t, 3H, NCHCH2CH3). MS: m/z 453 (Mþ þ 2), m/
z 451 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.
5.2.1.11. (5S,11aR)-5-(4-Bromophenyl)-2-butyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(10).
Yield 45%; yellow powder; mp 188e191 ꢀC; Rf ¼ 0.7 (CH2Cl2/MeOH
99.5:0.5); IR (cmꢃ1): 3303 (eNHe), 1760, 1692 (eCOe). 1H NMR:
d
7.85 (s,1H, NH), 7.58e7.56 (m,1H, Ar), 7.50e7.48 (m,1H, Ar), 7.32e
5.2.1.17. (5R,11aS)-5-(4-Bromophenyl)-2-sec-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (16).
Yield 34%; yellowish white powder; mp 230e233 ꢀC; Rf ¼ 0.43
(CH2Cl2); IR (cmꢃ1): 3342 (eNHe), 1759, 1692 (eCOe). 1H NMR:
7.19 (m, 6H, Ar), 6.28 (s,1H, CHPh), 4.29e4.24 (dd, J ¼ 11.00, 5.50 Hz,
1H, CHC(O)N), 3.55e3.48 (m, 3H, NCH2 þ CHaHb), 2.93e2.85 (m, 1H,
CHaHb), 1.64e1.59 (m, 2H, CH2CH2CH3), 1.27e1.24 (m, 2H, CH2CH3),
0.96e0.93 (t, 3H, CH3). MS: m/z 453 (Mþ þ 2), m/z 451 (Mþ), m/z 218
(100%). Elemental analysis: calculated for (C23H22BrN3O2) C, H, N.
d
7.83 (s, 1H, NH), 7.58e7.56 (d, 1H, Ar), 7.51e7.47 (m, 2H, Ar),
7.32e7.18 (m, 5H, Ar), 6.26 (s, 1H, CHPh), 4.26e4.19 (m, 1H,
CHC(O)N), 4.13e4.02 (m, 1H, NCH), 3.54e3.46 (dd, 1H, CHaHb),
2.92e2.83 (dd, 1H, CHaHb), 2.04e1.91 (m, 1H, NCHCHaHb), 1.79e
1.67 (m, 1H, NCHCHaHb), 1.42e1.39 (d, 3H, NCHCH3), 0.89e0.84
(t, 3H, NCHCH2CH3). MS: m/z 453 (Mþ þ 2), m/z 451 (Mþ), m/z
218 (100%). Elemental analysis: calculated for (C23H22BrN3O2)
C, H, N.
5.2.1.12. (5S,11aS)-5-(4-Bromophenyl)-2-butyl-5,6,11,11a-tetrahy-
dro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(11).
Yield 35%; yellowish white powder; mp 227e228 ꢀC; Rf ¼ 0.48
(CH2Cl2/MeOH 99.5:0.5); IR (cmꢃ1): 3294 (eNHe), 1766, 1692 (e
COe). 1H NMR:
d 7.90 (s, 1H, NH), 7.59e7.56 (m, 2H, Ar), 7.48e
7.46 (d, 1H, Ar), 7.23e7.16 (m, 5H, Ar), 5.78 (s, 1H, CHPh), 4.41e
4.31 (dd, J ¼ 11.4, 4.5 Hz, 1H, CHC(O)N), 3.55e3.44 (m, 3H,
NCH2 þ CHaHb), 3.09e3.00 (m, 1H, CHaHb), 1.62e1.54 (m, 2H,
CH2CH2CH3), 1.33e1.27 (m, 2H, CH2CH3), 0.94e0.89 (t, 3H, CH3).
MS: m/z 453 (Mþ þ 2), m/z 451 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C23H22BrN3O2) C, H, N.
5.2.1.18. (5R,11aR)-5-(4-Bromophenyl)-2-tert-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (17).
Yield 33%; yellowish white powder; mp 269e270 ꢀC; Rf ¼ 0.29
(CH2Cl2); IR (cmꢃ1): 3411 (eNHe), 1764, 1703 (eCOe). 1H NMR:
d
7.8 (s, 1H, NH), 7.48e7.45 (d, 2H, Ar), 7.21e7.16 (m, 6H, Ar), 5.7 (s,
1H, CHPh), 4.24e4.20 (dd, 1H, CHC(O)N), 3.49e3.42 (dd,1H, CHaHb),
3.05e2.94 (m, 1H, CHaHb), 1.59 (s, 9H, CH3). MS: m/z 453 (Mþ þ 2),
m/z 451 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.
5.2.1.13. (5R,11aS)-5-(4-Bromophenyl)-2-butyl-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione
(12). Yield
47%; yellow powder; mp 186e188 ꢀC; Rf ¼ 0.7 (CH2Cl2/MeOH
99.5:0.5); IR (cmꢃ1): 3315 (eNHe), 1763, 1692 (eCOe). 1H NMR:
d
7.85 (s, 1H, NH), 7.58e7.47 (dd, 3H, Ar), 7.32e7.19 (m, 5H, Ar), 6.27
5.2.1.19. (5S,11aR)-5-(4-Bromophenyl)-2-tert-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (18).
Yield 53%; yellow powder; mp 137e140 ꢀC; Rf ¼ 0.49 (CH2Cl2); IR
(s, 1H, CHPh), 4.29e4.24 (dd, J ¼ 11.00, 5.50 Hz, 1H, CHC(O)N), 3.56e
3.48 (m, 3H, NCH2 þ CHaHb), 3.94e3.84 (m,1H, CHaHb),1.64e1.59 (m,
2H, CH2CH2CH3), 1.35e1.27 (m, 2H, CH2CH3), 0.96e0.91 (t, 3H, CH3).
MS: m/z 453 (Mþ þ 2), m/z 451 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C23H22BrN3O2) C, H, N.
(cmꢃ1): 3327 (eNHe), 1760, 1692 (eCOe). 1H NMR:
d 7.74 (s, 1H,
NH), 7.51e7.48 (m, 2H, Ar), 7.27e7.21 (m, 6H, Ar), 6.2 (s, 1H, CHPh),
4.17e4.12 (dd, 1H, CHC(O)N), 3.50e3.43 (dd,1H, CHaHb), 2.90e2.81
(m, 1H, CHaHb), 1.62 (s, 9H, CH3). MS: m/z 453 (Mþ þ 2), m/z 451
(Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.
5.2.1.14. (5R,11aR)-5-(4-Bromophenyl)-2-sec-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (13).
Yield 50%; yellow powder; mp 233e235 ꢀC; Rf ¼ 0.27 (CH2Cl2); IR
(cmꢃ1): 3291 (eNHe), 1762, 1692 (eCOe). 1H NMR:
d
7.89 (s, 1H,
5.2.1.20. (5S,11aS)-5-(4-Bromophenyl)-2-tert-butyl-5-(4-
bromophenyl)-5,6,11,11a-tetrahydro-1H-imidazo[10,50:1,6]pyrido[3,4-
b]indole-1,3(2H)-dione (19). Yield 50%; yellowish white powder;
mp 265e268 ꢀC; Rf ¼ 0.27 (CH2Cl2); IR (cmꢃ1): 3295 (eNHe),
NH), 7.58e7.56 (d, 2H, Ar), 7.48e7.45 (d, 2H, Ar), 7.21e7.15 (m, 4H,
Ar), 5.75 (s, 1H, CHPh), 4.35e4.30 (m, 1H, CHC(O)N), 4.05e4.00 (m,
1H, NCH), 3.54e3.48 (m, 1H, CHaHb), 3.08e2.98 (m, 1H, CHaHb),
2.01e1.88 (m, 1H, NCHCHaHb), 1.78e1.64 (m, 1H, NCHCHaHb), 1.40e
1.37 (dd, 3H, CHCH3), 0.88e0.83 (t, 3H, CH2CH3). MS: m/z 453
(Mþ þ 2), m/z 451 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C23H22BrN3O2) C, H, N.
1762, 1692.4 (eCOe). 1H NMR:
d 7.73 (s, 1H, NH), 7.50e7.45 (m,
2H, Ar), 7.21e7.17 (m, 6H, Ar), 5.71 (s, 1H, CHPh), 4.25e4.20 (dd,
1H, CHC(O)N), 3.49e3.42 (dd,1H, CHaHb), 3.05e2.95 (m, 1H,
CHaHb), 1.59 (s, 9H, CH3). MS: m/z 453 (Mþ þ 2), m/z 451 (Mþ),
m/z 218 (100%). Elemental analysis: calculated for (C23H22BrN3O2)
C, H, N.
5.2.1.15. (5S,11aR)-5-(4-Bromophenyl)-2-sec-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (14).
Yield 48%; yellow powder; mp 227e230 ꢀC; Rf ¼ 0.45 (CH2Cl2); IR
5.2.1.21. (5R,11aS)-5-(4-Bromophenyl)-2-tert-butyl-5,6,11,11a-tetra-
hydro-1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-1,3(2H)-dione (20).
Yield 42%; yellow powder; mp 139e142 ꢀC; Rf ¼ 0.48 (CH2Cl2); IR
(cmꢃ1): 3343 (eNHe), 1760, 1692 (eCOe). 1H NMR:
d 7.81 (s, 1H,
NH), 7.58e7.48 (dd, 3H, Ar), 7.32e7.21 (m, 5H, Ar), 6.26 (s, 1H,
CHPh), 4.26e4.19 (m, 1H, CHC(O)N), 4.13e4.03 (m, 1H, NCH), 3.54e
3.47 (dd, 1H, CHaHb), 2.92e2.83 (dd, 1H, CHaHb), 2.04e1.92 (m, 1H,
NCHCHaHb), 1.79e1.67 (m, 1H, NCHCHaHb), 1.42e1.39 (d, 3H,
CHCH3), 0.89e0.84 (t, 3H, CH2CH3). MS: m/z 453 (Mþ þ 2), m/z 451
(Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.
(cmꢃ1): 3326 (eNHe), 1761, 1691 (eCOe). 1H NMR:
d 7.74 (s, 1H,
NH), 7.51e7.49 (m, 2H, Ar), 7.27e7.20 (m, 6H, Ar), 6.23 (s, 1H, CHPh),
4.17e4.10 (m, 1H, CHC(O)N), 3.50e3.42 (dd,1H, CHaHb), 2.90e2.80
(m, 1H, CHaHb), 1.63 (s, 9H, CH3). MS: m/z 453 (Mþ þ 2), m/z 451
(Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C23H22BrN3O2) C, H, N.

340
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
5.2.1.22. General procedures for the preparation of methyl-1-(4-
bromophenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H- -carboline-
3-carboxylate (21e24). Chloroacetyl chloride (0.74 mL, 9.34 mmol)
was added dropwise to a well stirred solution of the appropriate
carboline (1e4) I (1.5 g, 3.89 mmol) and NaHCO3 (0.39 g, 4.7 mmol)
in CHCl3 (40 mL) 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.
5.2.1.28. (6R,12aR)-6-(4-Bromophenyl)-2-methyl-2,3,6,7,12,12a-hex-
b
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(25).
Yield 37%; yellowish white powder; mp 266e269 ꢀC; Rf ¼ 0.55
b
-
(CH2Cl2/MeOH 97:3); IR (cmꢃ1): 3248 (eNHe), 1677, 1642 (eCOe).
1H NMR:
d 7.92 (s, 1H, NH), 7.63e7.60 (m, 1H, Ar), 7.38e7.36 (d, 2H,
Ar), 7.28e7.25 (m, 1H, Ar), 7.22e7.16 (m, 4H, Ar), 6.17 (s, 1H, CHPh),
4.35e4.29 (dd,1H, CHC(O)N), 4.14e4.08 (d, 1H, CHaHbC(O)N), 3.96e
3.90 (d, 1H, CHaHbC(O)N), 3.84e3.78 (dd,1H, CHaHb), 3.26e3.17 (dd,
1H, CHaHb), 3.05 (s, 3H, NCH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ),
m/z 218 (100%). Elemental analysis: calculated for (C21H18BrN3O2)
C, H, N.
5.2.1.23. Methyl
(1R,3R)-1-(4-bromophenyl)-2-(chloroacetyl)-
2,3,4,9-tetrahydro-1H-
b
-carboline-3-carboxylate (21). Yield 85%;
5.2.1.29. (6S,12aR)-6-(4-Bromophenyl)-2-methyl-2,3,6,7,12,12a-hex-
yellow powder; mp 237e240 ꢀC; Rf ¼ 0.41 (CH2Cl2); IR (cmꢃ1):
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(26).
3244 (eNHe), 1729, 1657 (eCOe). 1H NMR:
d
8.00 (brs, 1H, NH),
Yield 25%; white powder; mp 287e290 ꢀC; Rf ¼ 0.52 (CH2Cl2/
7.88e7.85 (d, 1H, Ar), 7.57e7.54 (d, 1H, Ar), 7.31e7.14 (m, 6H, Ar),
6.73 (s, 1H, CHPh), 5.18e5.14 (m, 1H, CHCOOCH3), 4.19e4.15 (m, 2H,
COCH2Cl), 3.64 (s, 3H, OCH3), 3.37e3.33 (dd,1H, CHaCHb), 3.14e3.06
(dd, 1H, CHaHb). MS: m/z 464 (Mþ þ 4), m/z 462 (Mþ þ 2), m/z 460
(Mþ), m/z 383 (100%). Elemental analysis: calculated for
(C21H18BrClN2O3) C, H, N.
MeOH 97:3); IR (cmꢃ1): 3271 (eNHe), 1657, 1641 (eCOe). 1H
NMR:
d 8.03 (s, 1H, NH), 7.56e7.53 (d, 1H, Ar), 7.44e7.41 (m, 2H,
Ar), 7.33e7.14 (m, 5H, Ar), 6.90 (s, 1H, CHPh), 4.30e4.25 (dd, 1H,
CHC(O)N), 4.17e4.11 (d, 1H, CHaHbC(O)N), 4.02e3.90 (d, 1H,
CHaHbC(O)N), 3.60e3.54 (dd, 1H, CHaHb), 3.01e2.92 (m,
4H, CHaHb þ NCH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ), m/z
218 (100%). Elemental analysis: calculated for (C21H18BrN3O2)
C, H, N.
5.2.1.24. Methyl (1S,3R)-1-(4-chlorophenyl)-2-(chloroacetyl)-2,3,4,9-
tetrahydro-1H-b-carboline-3-carboxylate (22). Yield 90%; yellowish
green powder; mp 110e113 ꢀC; Rf ¼ 0.17 (CH2Cl2); IR (cmꢃ1): 3323
5.2.1.30. (6S,12aS)-6-(4-Bromophenyl)-2-methyl-2,3,6,7,12,12a-hex-
(eNHe),1736,1658 (eCOe). 1H NMR:
d
22 (s,1H, NH), 7.61e7.50 (m,
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(27).
2H, Ar), 7.28e7.08 (m, 6H, Ar), 6.65 (s, 1H, CHPh), 5.31e5.28 (m, 1H,
CHCOOCH3), 4.19e4.06 (m, 2H, COCH2Cl), 3.82 (s, 3H, OCH3), 3.38e
3.29 (dd, 1H, CHaHb), 3.14e3.07 (dd, 1H, CHaHb). MS: m/z 464
(Mþ þ 4), m/z 462 (Mþ þ 2), m/z 460 (Mþ), m/z 385 (100%).
Elemental analysis: calculated for (C21H18BrClN2O3) C, H, N.
Yield 31%; yellow powder; mp 264e266 ꢀC; Rf ¼ 0.52 (CH2Cl2/
MeOH 97:3); IR (cmꢃ1): 3234 (eNHe), 1677, 1642 (eCOe). 1H NMR:
d
7.92 (s, 1H, NH), 7.39e7.36 (m, 2H, Ar), 7.28e7.16 (m, 6H, Ar), 6.17
(s, 1H, CHPh), 4.35e4.28 (dd, 1H, CHC(O)N), 4.14e4.08 (d, 1H,
CHaHbC(O)N), 3.96e3.90 (d, 1H, CHaHbC(O)N), 3.84e3.78 (dd, 1H,
CHaHb), 3.26e3.17 (dd,1H, CHaHb), 3.05 (s, 3H, NCH3). MS: m/z 425
(Mþ þ 2), m/z 423 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C21H18BrN3O2) C, H, N.
5.2.1.25. Methyl
(1S,3S)-1-(4-bromophenyl)-2-(2-chloroacetyl)-
2,3,4,9-tetrahydro-1H-b-carboline-3-carboxylate (23). Yield 82%;
yellowish green powder; mp 227e230 ꢀC; Rf ¼ 0.41 (CH2Cl2); IR
(cmꢃ1): 3345 (eNHe), 1730, 1657 (eCOe). 1H NMR:
d
7.97 (brs, 1H,
5.2.1.31. (6R,12aS)-6-(4-Bromophenyl)-2-methyl-2,3,6,7,12,12a-hex-
NH), 7.68e7.65 (d, 1H, Ar), 7.57e7.54 (d, 1H, Ar), 7.31e7.12 (m, 6H,
Ar), 6.73 (s, 1H, CHPh), 5.18e5.14 (m, 1H, CHCOOCH3), 4.19e4.15 (m,
2H, COCH2Cl), 3.64 (brs, 3H, OCH3), 3.37e3.33 (dd, 1H, CHaHb),
3.14e3.07 (dd,1H, CHaHb). MS: m/z 464 (Mþ þ 4), m/z 462 (Mþ þ 2),
m/z 460 (Mþ), m/z 383 (100%). Elemental analysis: calculated for
(C21H18BrClN2O3) C, H, N.
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(28).
Yield 20%; yellow powder; mp 190e194 ꢀC; Rf ¼ 0.5 (CH2Cl2/
MeOH 97:3); IR (cmꢃ1): 3303 (eNHe), 1658, 1649 (eCOe). 1H
NMR:
d 7.82 (s, 1H, NH), 7.55e7.52 (m, 1H, Ar), 7.39e7.37 (d, 3H,
Ar), 7.22e7.18 (m, 4H, Ar), 6.18 (s, 1H, CHPhAr), 4.34e4.30 (dd, 1H,
CHC(O)N), 4.14e4.08 (d, 1H, CHaHbC(O)N), 3.96e3.90 (d, 1H,
CHaHbC(O)N), 3.84e3.78 (dd, 1H, CHaHb), 3.26e3.17 (m, 1H,
CHaHb), 3.05 (s, 3H, NCH3). MS: m/z 425 (Mþ þ 2), m/z 423 (Mþ),
m/z 218 (100%). Elemental analysis: calculated for (C21H18BrN3O2)
C, H, N.
5.2.1.26. Methyl (1R,3S)-1-(4-chlorophenyl)-2-(chloroacetyl)-2,3,4,9-
tetrahydro-1H-b-carboline-3-carboxylate (24). Yield 93%; yellowish
green powder; mp 110e114 ꢀC; Rf ¼ 0.17 (CH2Cl2); IR (cmꢃ1): 3324
(eNHe), 1727, 1658 (eCOe). 1H NMR:
d 8.22 (s, 1H, NH), 7.62e7.50
(m, 2H, Ar), 7.29e7.08 (m, 6H, Ar), 6.65 (s, 1H, CHPh), 5.31e5.27 (m,
1H, CHCOOCH3), 4.19e4.06 (m, 2H, COCH2Cl), 3.64 (s, 1H, OCH3),
3.35e3.32 (dd, 1H, CHaHb), 3.15e3.07 (dd, 1H, CHaHb). MS: m/z 464
(Mþ þ 4), m/z 462 (Mþ þ 2), m/z 460 (Mþ), m/z 385 (100%).
Elemental analysis: calculated for (C21H18BrClN2O3) C, H, N.
5.2.1.32. (6R,12aR)-2-Ethyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(29).
Yield 39%; yellow powder; mp 276e279 ꢀC; Rf ¼ 0.63 (CH2Cl2/
MeOH 97:3); IR (cmꢃ1): 3185 (eNHe), 1657, 1650 (eCOe). 1H NMR:
d
7.92 (s, 1H, NH), 7.62e7.60 (m, 1H, Ar), 7.39e7.36 (d, 2H, Ar), 7.29e
7.24 (m, 1H, Ar), 7.21e7.17 (m, 4H, Ar), 6.19 (s, 1H, CHPh), 4.33e4.28
(dd, J ¼ 11.25, 4.44 Hz, 1H, CHC(O)N), 4.13e4.07 (d, J ¼ 17.51 Hz, 1H,
CHaHbC(O)N), 3.95e3.89 (d, J ¼ 17.45 Hz, 1H, CHaHbC(O)N), 3.83e
3.76 (dd, J ¼ 16.11, 4.50 Hz, 1H, CHaHb), 3.72e3.65 (m, 1H,
NCHbHa), 3.42e3.35 (m, 1H, NCHbHa), 3.26e3.17 (dd, J ¼ 16.09,
11.47 Hz, 1H, CHaHb), 1.23e1.18 (t, 3H, CH3). MS: m/z 439 (Mþ þ 2),
m/z 437 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C22H20BrN3O2) C, H, N.
5.2.1.27. General procedures for the preparation of 2-alkyl-6-(4-
bromophenyl)-2,3,6,7,12,12a-hexahydropyrazino
[10,20:1,6]pyrido
[3,4-b]indole-1,4-dione (25e40). A solution of the appropriate
chloroacetyl derivative (21e24) (1.4 mmol, 1 equiv, 0.646 g) and
the appropriate alkylamine (2.8 mmol, 2 equiv) in methanol
(25 mL) was refluxed 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.
5.2.1.33. (6S,12aR)-2-Ethyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(30).
Yield 30%; yellowish white powder; mp 260e263 ꢀC; Rf ¼ 0.62
(CH2Cl2/MeOH 97:3); IR (cmꢃ1): 3270 (eNHe), 1658, 1649 (eCOe).

N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
341
1H NMR:
d
7.84 (s, 1H, NH), 7.64e7.60 (m, 1H, Ar), 7.39e7.37 (d, 2H,
5.2.1.38. (6S,12aS)-2-Butyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hexahy-
dropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione (35). Yield 28%,
yellowish brown powder; mp 135e130 ꢀC; Rf ¼ 0.77 (CH2Cl2/MeOH
Ar), 7.21e7.18 (m, 5H, Ar), 6.19 (s, 1H, CHPh), 4.34e4.29 (dd,
J ¼ 10.65, 4.2 Hz, 1H, CHC(O)N), 4.13e4.07 (d, J ¼ 17.7 Hz, 1H,
CHaHbC(O)N), 3.95e3.89 (d, J ¼ 17.4 Hz, 1H, CHaHbC(O)N), 3.83e
3.76 (dd, J ¼ 16.20, 4.50 Hz, 1H, CHaHb), 3.72e3.65 (m, 1H,
NCHbHa), 3.42e3.35 (m, 1H, NCHbHa), 3.26e3.17 (dd, J ¼ 16.45,
11.7 Hz,1H, CHaHb), 1.23e1.19 (t, 3H, CH3). MS: m/z 439 (Mþ þ 2), m/
z 437 (Mþ), m/z 217 (100%). Elemental analysis: calculated for
(C22H20BrN3O2) C, H, N.
97:3); IR (cmꢃ1): 3278 (eNHe), 1658, 1650 (eCOe). 1H NMR:
d 7.85
(s, 1H, NH), 7.63e7.60 (d, 1H, Ar), 7.39e7.37 (d, 2H, Ar), 7.21e7.16 (m,
5H, Ar), 6.20 (s,1H, CHPh), 4.34e4.29 (dd, J ¼ 10.8, 4.5 Hz,1H, CHC(O)
N), 4.12e4.07 (d, J ¼ 17.4 Hz, 1H, CHaHbC(O)N), 3.95e3.89 (d,
J ¼ 17.4 Hz, 1H, CHaHbC(O)N), 3.81e3.74 (dd, J ¼ 16.2, 4.5 Hz, 1H,
CHaHb), 3.62e3.55 (m, 1H, NCHaHb), 3.40e3.30 (m, 1H, NCHaHb),
3.27e3.17 (dd, J ¼ 16.2, 11.4 Hz, 1H, CHaHb), 1.57e1.54 (m, 2H,
CH2CH2CH3), 1.36e1.31 (m, 2H, CH2CH3), 0.98e0.93 (t, 3H, CH3). MS:
m/z 467 (Mþ þ 2), m/z 465 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C24H24BrN3O2) C, H, N.
5.2.1.34. (6S,12aS)-2-Ethyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(31).
Yield 20%; yellow powder; mp 270e273 ꢀC; Rf ¼ 0.62 (CH2Cl2/
MeOH 97:3); IR (cmꢃ1): 3187 (eNHe), 1658, 1651 (eCOe). 1H NMR:
d
7.97 (s, 1H, NH), 7.61e7.60 (m, 1H, Ar), 7.39e7.36 (d, 2H, Ar), 7.29e
5.2.1.39. (6R,12aS)-2-Butyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
7.24 (m, 1H, Ar), 7.21e7.17 (m, 4H, Ar), 6.20 (s, 1H, CHPh), 4.35e4.30
(dd, J ¼ 11.32, 4.10 Hz, 1H, CHC(O)N), 4.14e4.08 (d, J ¼ 17.51 Hz, 1H,
CHaHbC(O)N), 3.95e3.89 (d, J ¼ 17.45 Hz, 1H, CHaHbC(O)N), 3.83e
3.76 (dd, J ¼ 16.11, 4.0 Hz, 1H, CHaHb), 3.71e3.64 (m, 1H, NCHaHb),
3.43e3.35 (m, 1H, NCHaHb), 3.26e3.17 (dd, J ¼ 16.09, 11.47 Hz, 1H,
CHaHb), 1.22e1.18 (t, 3H, CH3). MS: m/z 439 (Mþ þ 2), m/z 437 (Mþ),
m/z 217 (100%). Elemental analysis: calculated for (C22H20BrN3O2)
C, H, N.
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(36).
Yield 20%, whitish yellow powder; mp 203e206 ꢀC; Rf ¼ 0.77
(CH2Cl2/MeOH 97:3); IR (cmꢃ1): 3257 (eNHe), 1657, 1650 (eCOe).
1H NMR:
d 7.83 (s, 1H, NH), 7.63e7.53 (m, 1H, Ar), 7.40e7.37 (m, 1H,
Ar), 7.26e7.16 (m, 6H, Ar), 6.19 (s, 1H, CHPh), 4.35e4.26 (dd, J ¼ 11.7,
4.5 Hz, 1H, CHC(O)N), 4.13e4.08 (d, J ¼ 17.4 Hz, 1H, CHaHbC(O)N),
3.95e3.89 (d, J ¼ 17.4 Hz, 1H, CHaHbC(O)N), 3.82e3.76 (dd, J ¼ 15.9,
4.5 Hz, 1H, CHaHb), 3.72e3.63 (m, 1H, NCHaHb), 3.44e3.32 (m, 1H,
NCHaHb), 3.26e3.17 (dd, J ¼ 15.6, 11.7 Hz, 1H, CHaHb), 1.62e1.53 (m,
2H, CH2CH2CH3), 1.37e1.27 (m, 2H, CH2CH3), 0.98e0.93 (t, 3H, CH3).
MS: m/z 467 (Mþ þ 2), m/z 465 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C24H24BrN3O2) C, H, N.
5.2.1.35. (6R,12aS)-2-Ethyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
ahydropyrazino
[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(32).
Yield 23%; yellow powder; mp 265e268 ꢀC; Rf ¼ 0. 63 (CH2Cl2/
MeOH 97:3); IR (cmꢃ1): 3282 (eNHe), 1658. 1649 (eCOe). 1H NMR:
d
7.87 (s, 1H, NH), 7.66e7.61 (m, 1H, Ar), 7.39e7.36 (d, 2H, Ar), 7.21e
5.2.1.40. (6R,12aR)-6-(4-Bromophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (37).
Yield 30%, yellowish white powder; mp 117e120 ꢀC; Rf ¼ 0.72
(CH2Cl2/MeOH 90:10); IR (cmꢃ1): 3278 (eNHe), 1730, 1657 (eCOe
7.17 (m, 5H, Ar), 6.19 (s, 1H, CHPh), 4.34e4.29 (dd, 1H, CHC(O)N),
4.13e3.08 (d, J ¼ 17.48 Hz, 1H, CHaHbC(O)N), 3.95e3.89 (d,
J ¼ 17.45 Hz, 1H, CHaHbC(O)N), 3.83e3.76 (dd, 1H, CHaHb), 3.73e
3.66 (m, 1H, NCHaHb), 3.44e3.33 (m, 1H, NCHaHb), 3.26e3.17 (dd,
1H, CHaHb), 1.23e1.18 (t, 3H, CH3). MS: m/z 439 (Mþ þ 2), m/z 437
(Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C22H20BrN3O2) C, H, N.
). 1H NMR:
d 8.14 (s, 1H, NH), 7.56e7.54 (d, 2H, Ar), 7.34e7.33 (d, 2H,
Ar), 7.20e7.12 (m, 4H, Ar), 6.91 (s, 1H, CHPh), 4.92e4.90 (dd, 1H,
CHC(O)N), 4.73e4.71 (d, 1H, CHaHbC(O)N), 3.69e3.58 (m, 2H,
CHaHbC(O)N þ CHaHb), 2.94e2.89 (dd, 1H, CHaHb), 1.14 (s, 9H, CH3).
MS: m/z 467 (Mþ þ 2), m/z 465 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C24H24BrN3O2) C, H, N.
5.2.1.36. (6R,12aR)-2-Butyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hex-
ahydropyrazino[10,20:1,6]
pyrido[3,4-b]-b]indole-1,4-dione
(33).
Yield 30%, yellow brown powder; mp 130e133 ꢀC; Rf ¼ 0.76
5.2.1.41. (6S,12aR)-6-(4-Bromophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (38).
Yield 28%, yellowish white powder; mp 133e136 ꢀC; Rf ¼ 0.73
(CH2Cl2/MeOH 90:10); IR (cmꢃ1): 3170 (eNHe), 1728, 1665 (e
(CH2Cl2/MeOH 97:3); IR (cmꢃ1): 3273 (eNHe), 1658, 1650 (eCOe).
1H NMR:
d 7.96 (s, 1H, NH), 7.62e7.60 (m, 1H, Ar), 7.38e7.35 (d, 2H,
Ar), 7.29e7.26 (m, 1H, Ar), 7.22e7.16 (m, 4H, Ar), 6.20 (s, 1H, CHPh),
4.33e4.28 (dd, J ¼ 11.40, 4.18 Hz, 1H, CHC(O)N), 4.12e4.06 (d,
J ¼ 17.38 Hz, 1H, CHaHbC(O)N), 3.94e3.88 (d, J ¼ 17.50 Hz, 1H,
CHaHbC(O)N), 3.81e3.74 (dd, J ¼ 16.12, 4.57 Hz, 1H, CHaHb), 3.62e
3.33 (m, 1H, NCHaHb), 3.62e3.55 (m, 1H, NCHaHb), 3.40e3.30 (dd,
J ¼ 16.05, 11.5, 1H, CHaHb), 1.62e1.52 (m, 2H, CH2CH2CH3), 1.39e1.31
(m, 2H, CH2CH3), 0.98e0.93 (t, 3H, CH3). MS: m/z 467 (Mþ þ 2), m/z
465 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C24H24BrN3O2) C, H, N.
COe). 1H NMR:
d 8.5 (s, 1H, NH), 7.50e7.45 (d, 2H, Ar), 7.36e
7.25 (m, 4H, Ar), 7.15e7.08 (m, 2H, Ar), 6.44 (s, 1H, CHPh), 4.94e
4.92 (m, 1H, CHC(O)N), 4.63e4.61 (d, 1H, CHaHbC(O)N), 4.12e
4.07 (d, 1H, CHaHbC(O)N), 3.83e3.63 (m, 1H, CHaHb), 3.14e2.90
(m, 1H, CHaHb), 1.24 (s, 9H, CH3). MS: m/z 467 (Mþ þ 2), m/z
465 (Mþ), m/z 218 (100%). Elemental analysis: calculated for
(C24H24BrN3O2) C, H, N.
5.2.1.42. (6S,12aS)-6-(4-Bromophenyl)-2-tert-butyl-2,3,6,7,12,12a-
5.2.1.37. (6S,12aR)-2-Butyl-6-(4-bromophenyl)-2,3,6,7,12,12a-hexahy-
dropyrazino [10,20:1,6]pyrido[3,4-b]indole-1,4-dione (34). Yield 25%,
yellowish white powder; mp 207e210 ꢀC; Rf ¼ 0.76 (CH2Cl2/MeOH
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(39).
Yield 34%, yellowish white powder; mp 120e123 ꢀC; Rf ¼ 0.72
(CH2Cl2/MeOH 90:10); IR (cmꢃ1): 3291 (eNHe), 1730,1657 (eCOe).
97:3); IR (cmꢃ1): 3273 (eNHe), 1658,1649.7 (eCOe). 1H NMR:
d
7.88
1H NMR:
d 8.1 (s, 1H, NH), 7.57e7.56 (d, 2H, Ar), 7.35e7.33 (d, 2H,
(s, 1H, NH), 7.63e7.60 (d, 1H, Ar), 7.39e7.36 (m, 2H, Ar) 7.20e7.17 (m,
5H, Ar), 6.20 (s, 1H, CHPh), 4.35e4.26 (dd, J ¼ 11.7, 4.2 Hz,1H, CHC(O)
N), 4.13e4.07 (d, J ¼ 17.7 Hz, 1H, CHaHbC(O)N), 3.94e3.89 (d,
J ¼ 17.4 Hz, 1H, CHaHbC(O)N), 3.81e3.74 (dd, J ¼ 15.9, 4.5 Hz, 1H,
CHaHb), 3.59e3.53 (m, 1H, NCHaHb), 3.43e3.41 (m, 1H, NCHaHb),
3.26e3.17 (dd, J ¼ 15.6, 11.7 Hz, 1H, CHaHb), 1.60e1.50 (m, 2H,
CH2CH2CH3), 1.36e1.29 (m, 2H, CH2CH3), 0.93e0.83 (t, 3H, CH3). MS:
m/z 467 (Mþ þ 2), m/z 465 (Mþ), m/z 218 (100%). Elemental analysis:
calculated for (C24H24BrN3O2) C, H, N.
Ar), 7.21e7.11 (m, 4H, Ar), 6.92 (s, 1H, CHPh), 4.95e4.94 (m, 1H,
CHC(O)N), 4.25e4.19 (d, 1H, CHaHbC(O)N), 3.67e3.58 (m, 2H,
CHaHbC(O)N þ CHaHb), 2.99e2.94 (m, 1H, CHaHb), 1.14 (s, 9H, CH3).
MS: m/z 467 (Mþ þ 2), m/z 465 (Mþ), m/z 218 (100%). Elemental
analysis: calculated for (C24H24BrN3O2) C, H, N.
5.2.1.43. (6R,12aS)-6-(4-Bromophenyl)-2-tert-butyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(40).
Yield 20%, yellowish white powder; mp 135e138 ꢀC; Rf ¼ 0.73

342
N.S. Ahmed et al. / European Journal of Medicinal Chemistry 57 (2012) 329e343
(CH2Cl2/MeOH 90:10); IR (cmꢃ1): 3255 (eNHe), 1729, 1658 (e
COe). 1H NMR:
8.54 (s, 1H, NH), 7.79e7.75 (d, 2H, Ar), 7.55e
were merged from 1XOZ into PDB 3BJC, 2H42 and 2H44 after
superimposition. These newly generated complexes were refined
using the LigX module of MOE. A force constant of 1 kcal/mol was
used to restrain the Ca atoms of the protein, the ligand and the
water molecules. The refined and superposed complexes were used
in the ensemble docking.
d
7.53 (d, 2H, Ar), 7.34e7.14 (m, 4H, Ar), 6.81 (s, 1H, CHPh), 4.92e
4.86 (dd, 1H, CHC(O)N), 4.12e4.02 (d, 1H, CHaHbC(O)N), 3.82e
3.76 (d, 1H, CHaHbC(O)N), 3.65e3.59 (dd, 1H, CHaHb), 3.10e3.08
(dd, 1H, CHaHb), 1.27 (s, 9H, CH3). MS: m/z 467 (Mþ þ 2), m/z
465 (Mþ), m/z 218 (100%). Elemental analysis: calculated
for (C24H24BrN3O2) C, H, N.
Ensemble docking was performed using the docking software
GOLDv5.0 and the GOLDSCORE (GS) fitness function [34,35]. The
refined structures 1XOZ, 3BJC, 2H42 and 2H44 were used as
representative ensemble. The active site was set by including all
5.3. Biological evaluation
ꢀ
residues within 9 A of compound I. Each compound of the small
All the synthesized compounds were evaluated for their inhib-
itory properties versus recombinant PDE5. Compounds showing
PDE5 for compounds showing a cGMP percentage of inhibition
>65%, their in vitro ability to inhibit the growth of breast cancer cell
line MDA-MB-231 were determined. For all assays, tadalafil (PDE5/
PDE11 inhibitor) was used for comparison. Compound 25, was
further tested against PDE3A cAMP, PDE3B cGMP, PDE4B, PDE11A
cAMP, PDE11A cGMP to further prove its selectivity.
subset (I, 29, 30) was docked 50 times and the top 10 ranked poses
retained.
Multiple single conformer docking was performed using Auto-
Dock Vina v1.1.2 [36]. The same four complexes as for the ensemble
docking were used with a set of 25 compounds (I, 1, 4, 5, 8, 9, 11, 12,
13, 14, 15, 16, 17, 18, 20, 21, 25, 26, 29, 30, 33, 34, 35, 36, 37), however
with stripped metal ions Zn2þ and Mg2þ. The docking parameters
were kept to their default values. However, exhaustiveness was set
ꢀ
to 30. The size of the docking grid was 27 Å ꢂ 25 Å ꢂ 27 A, which
5.3.1. Phosphodiesterase inhibitory activity
encompassed the entire PDE5 active site, and centred close to
Phe820.
PDE activity was measured using an adaptation of the IMAP1
fluorescence polarization phosphodiesterase assay (Molecular
Devices, Sunnyvale, CA, USA). PDE hydrolysis of the fluorescent-
labelled substrate allows it to bind the IMAP1 reagent, which
increases fluorescence polarization (FP). The assay used fluorescein
(Fl)-cAMP and tetramethylrhodamine (TAMRA)ecGMP as
substrates. The different excitation and emission spectra of the
substrates (485e530 nm for Fl and 530e590 nm for TAMRA)
allowed for simultaneous measurement of cAMP and cGMP
hydrolysis in the same well. The assays were performed in 96-well
microtiter plates using a reaction buffer containing 10 mM TriseHCl
(pH 7.2), 10 mM MgCl2, 0.05% NaN3 and 0.1% phosphate-free bovine
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.029.
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