Design of novel β-carboline derivatives with pendant 5-bromothienyl and their evaluation as phosphodiesterase-5 inhibitors

Article abstract of DOI:10.1002/ardp.201200334

New derivatives with the tetrahydro-β-carboline-imidazolidinedione and tetrahydro-β-carboline-piperazinedione scaffolds and a pendant bromothienyl moiety at C-5/C-6 were synthesized and tested for their ability to inhibit PDE5 in vitro. The following SAR can be concluded: The tetracyclic scaffold is essential for PDE5 inhibition; the ethyl group is the most suitable among the adopted N-substituents on the terminal ring (hydantoin/ piperazinedione); the appropriate stereochemistry of C-5/C-6 derived from the aldehyde rather than C-11a/C-12a derived from tryptophan appears crucial for inhibition of PDE5; surprisingly, derivatives with the hydantoin terminal ring are more active than their analogs with the piperazinedione ring; the selectivity versus PDE5 relative to PDE11 with cGMP as a substrate is mainly a function of the substitution and stereochemistry pattern of the external ring, in other words of the interaction with the H-loop residues of the isozymes. Thirteen derivatives showed PDE5 inhibitory activity with IC₅₀ values in the range of 0.16-5.4 μm. Compound 8 was the most potent PDE5 inhibitor and showed selectivity towards PDE5 versus other PDEs, with a selectivity index of 49 towards PDE5 rather than PDE11 with cGMP as the substrate. New derivatives with the tetrahydro-β-carboline-imidazolidinedione and tetrahydro-β-carboline-piperazinedione scaffolds and a pendant bromothienyl moiety at C-5/C-6 were synthesized and tested for their ability to inhibit PDE5 in vitro. Of these, 13 derivatives showed PDE5 inhibitory activity with IC ₅₀ values in the range of 0.16-5.4 μm and with compound 8 being the most active derivative. The tetracyclic scaffold was shown to be essential for PDE5 inhibition. Copyright

Full text of DOI:10.1002/ardp.201200334

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
23
Full Paper
Design of Novel b-Carboline Derivatives with Pendant
5-Bromothienyl and Their Evaluation as
Phosphodiesterase-5 Inhibitors
Dalia S. El-Gamil1, Nermin S. Ahmed1, Bernard D. Gary2, Gary A. Piazza2, Matthias Engel3,
Rolf W. Hartmann3, and Ashraf H. Abadi1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in
Cairo, Cairo, Egypt
2 Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
3 Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS), Saarbru¨cken, Germany
New derivatives with the tetrahydro-b-carboline-imidazolidinedione and tetrahydro-b-carboline-
piperazinedione scaffolds and a pendant bromothienyl moiety at C-5/C-6 were synthesized and
tested for their ability to inhibit PDE5 in vitro. The following SAR can be concluded: The
tetracyclic scaffold is essential for PDE5 inhibition; the ethyl group is the most suitable among
the adopted N-substituents on the terminal ring (hydantoin/piperazinedione); the appropriate
stereochemistry of C-5/C-6 derived from the aldehyde rather than C-11a/C-12a derived from
tryptophan appears crucial for inhibition of PDE5; surprisingly, derivatives with the hydantoin
terminal ring are more active than their analogs with the piperazinedione ring; the selectivity
versus PDE5 relative to PDE11 with cGMP as a substrate is mainly a function of the substitution
and stereochemistry pattern of the external ring, in other words of the interaction with the H-loop
residues of the isozymes. Thirteen derivatives showed PDE5 inhibitory activity with IC50 values in the
range of 0.16–5.4 mm. Compound 8 was the most potent PDE5 inhibitor and showed selectivity
towards PDE5 versus other PDEs, with a selectivity index of 49 towards PDE5 rather than PDE11 with
cGMP as the substrate.
Keywords: b-Carboline / Growth inhibition / Phosphodiesterase-5 inhibitors / Stereochemistry
Received: August 21, 2012; Revised: September 8, 2012; Accepted: September 19, 2012
DOI 10.1002/ardp.201200334
Introduction
ficity, and mode of regulation [1–5]. PDE5 is a cGMP-specific
phosphodiesterase found in vascular and tracheal smooth
muscles, platelets, gastrointestinal epithelial cells, spleen,
kidney, prostate gland, and in the Purkinje cells of the
cerebellum [1, 6, 7]. Three PDE5 inhibitors, sildenafil, varde-
nafil, and tadalafil, have been marketed for the treatment of
male erectile dysfunction (MED) [8], and were recently
approved for treatment of pulmonary arterial hypertension
(PAH) [9]. In addition, PDE5 inhibitors have gained attention
as potential therapeutic agents for cardiac hypertrophy
associated with heart failure [10, 11], Raynaud’s disease
[12], stroke [13], Alzheimer’s disease [14, 15], anticancer
agents [16, 17], and reversal of multiple drug resistance [18].
The superposition of all reported PDE5A inhibitors has
revealed a conserved binding mode with the enzyme that
compromise two major interaction types: one or two hydro-
Cyclic adenosine monophosphate (cAMP) and cyclic guano-
sine monophosphate (cGMP) are critical intracellular second
messengers, which through activation of protein kinase A
(PKA) and protein kinase G (PKG), respectively, mediate bio-
logical responses to a variety of cellular stimuli. Catabolism
of cAMP and cGMP is directed by phosphodiesterases (PDEs), a
complex superfamily containing 11 highly related gene fam-
ilies and over 100 distinct isoforms. PDE isoforms differ in
amino acid sequence, tissue distribution, substrate speci-
Correspondence: Prof. Ashraf H. Abadi, Faculty of Pharmacy and
Biotechnology, Department of Pharmaceutical Chemistry, German
University in Cairo, Cairo 11835, Egypt.
E-mail: ashraf.abadi@guc.edu.eg
Fax: þ202-27581041
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

24
D. S. El-Gamil et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Results and discussion
Chemistry
The synthesis of the 1,3-disubstituted tetrahydro-b-carbolines
(Scheme 1), tetrahydro-b-carboline-hydantoin derivatives
(Scheme 2) and tetrahydro-b-carboline-piperazinedione
derivatives (Scheme 3) are shown. Both D-tryptophan and
L-tryptophan methyl esters were synthesized by a general
synthetic procedure for amino acid esters [23]. The respective
ester and 5-bromo-2-thiophenecarbaldehyde were subjected
to a Pictet–Spengler reaction. The reaction was carried out at
room temperature under non-stereoselective conditions,
since both the cis-and trans-isomers (1–4) were required.
The trans-isomer precipitated throughout the reaction and
was obtained by filtration. The filtrate contained cis- and trans-
diastereomers of the 1,3-disubstituted tetrahydro-b-carboline
(1–4) that were separated by flash column chromatography
using CH2Cl2 as an eluent.
Figure 1. Detailed mode view showing the docking and interaction
The pure cis- and trans-diastereomers were reacted with
commercially available ethyl, butyl, tert-butyl and allyl iso-
cyanates to yield the desired cis- and trans-hydantoin deriva-
tives (5–20). The cis-tetrahydro-b-carbolines needed 70 h for
reaction completion compared to the respective trans-isomers
which needed only 16 h. This can be attributed to the fact
that cis- and trans-isomers are diastereomers that show differ-
ent rates in chemical reactions.
of tadalafil with human PDE5.
gen bond interactions with Gln 817 and hydrophobic p–p
stacking interactions between the planar ring portion of the
inhibitors and a hydrophobic P clamp that anchors these
inhibitors in the active site (PDB: 1UDU, PDB: 1XOZ, PDB:
2H44; Fig. 1) [19–21]. Recently, reported tadalafil analogs have
revealed two further hydrophobic interactions: interaction
between the alkyl side chain on the nitrogen of the terminal
ring and Ile665 and hydrophobic interaction of the pendant
aryl with Met816 in the Q2 pocket [22].
Treatment of the tetrahydro-b-carbolines (1, 4), having the
S configuration at C-1, with chloroacetyl chloride provided
the corresponding chloroethanone derivatives (21, 22). The
piperazinedione derivatives with the respective N-ethyl sub-
stituent were obtained by ring closure of the chloroethanone
derivative in the presence of ethyl amine in refluxing meth-
anol (23, 24).
Based on previously reported interactions, our laboratory
designed novel tadalafil analogs based upon the tetrahydro-b-
carboline-imidazolidinedione and tetrahydro-b-carboline-
piperazinedione scaffolds with 5-bromo-2-thienyl substituent
as the pendant aryl to increase the potential hydrophobic
interaction with residues lining the Q2 pocket. The effects of
size, branching, and unsaturation of substituents on the
nitrogen of the terminal ring, and the configuration of the
two chiral carbons upon activity were studied.
Refluxing the cis-tetrahydro-b-carboline 1 in butanone
under oxygen atmosphere led to its full aromatization into
the b-carboline 25 (Scheme 4). Analogous conditions failed to
oxidize the trans-tetrahydro-b-carboline 2 suggesting that
the cis-isomers are more susceptible to oxidation than the
trans-isomers.
R,S
H3COOC
H3COOC
HOOC R,S
R,S
HN
H2N
H2N
N
H
b
a
R,S
S
N
H
N
H
Br
_
1 4
Scheme 1. Synthesis of the 1,3-disubstituted tetrahydro-betacarbolines. Reagents and conditions: (a) CH3COCl, methanol, reflux, 5 h,
basify with dilute NH4OH and extract with CH2Cl2; (b) 5-bromothiophene-2-carboxaldehyde, TFA, RT, 4 days, 11–50.3%. For exact
individual assignment, see Table 1.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Tetrahydro-b-Carbolines as PDE5 Inhibitors
25
H3COOC
O
R,S
N
N
H
S
R
N
O
N
S
a
R,S NH
a
1
_
1 4
Br
Br
_
25
5 20
Scheme 4. Synthesis of fully aromatic betacarboline. Reagents
and conditions: (a) 2-butanone, reflux, under O2 atmosphere,
16 h, 72.8%.
Scheme 2. Synthesis of tetrahydro-betacarboline-hydantoin
derivatives. Reagents and conditions: (a) ethyl or butyl or t-butyl
or allyl isocyanate, 2-butanone, reflux, under N2 atmosphere,
16–70 h, 25–87.2%; R ¼ –C2H5 or –(CH2)3CH3 or –C–(CH3)3 or
–
–CH –CH CH . For exact individual assignment, see Table 2.
–
2
2
gave positive peaks, while signals arising from –CH2 and –C
groups gave negative peaks.
The assignment of cis/trans configuration for the tetrahy-
dro-b-carbolines (1–4) was based on a detailed study of
13C NMR spectroscopy data well-established in previous
literature. The 13C NMR signals for C1 and C3 are more
shielded in the trans-isomer compared to the cis-isomer with
Dd ꢀ 4 ppm, which may be due to 13C g-gauche effect. The
tetrahydropyridine ring exists in half-chair conformation
where the C-3 ester is equatorially located, leading to 1,3-
diaxial spatial interactions in the trans-isomer [24]. Moreover,
a correlation exists between the Rf value on TLC and the
configuration of the 1,3-disubstituted tetrahydro-b-carbo-
lines (1–4). The cis-isomer appears to be 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. For example, the Rf values of the 1S, 3R
and 1R, 3R isomers 1, 2 and their corresponding hydantoin
analogs 5, 6 using CH2Cl2 as the mobile phase were 0.53, 0.22,
and 0.26, 0.49, respectively.
Concerning the 1H NMR signals for the proton at C-5 of
hydantoin and C-6 of piperazinedione derivatives, they
appeared at d 6.16–7.00 which is greatly downshifted from
the same proton in the respective tetrahydro-b-carbolines (1–
4) that appeared at d 5.51–5.54. This can be attributed to the
electron-withdrawing effect of carbonyl functional groups in
hydantoin and piperazinedione rings.
Mass spectrometry of all derivatives showed a molecular
ion peak at Mþ and Mþþ2 with relative ratio of approxi-
mately 1:1 due to the isotopic nature of the bromine atom.
The infrared spectra of all derivatives showed bands at
ꢀ3300 cmꢁ1 for the N–H stretching. All the tetrahydro-b-
carbolines (1–4) showed peaks at ꢀ1740–1700 cmꢁ1 for the
carbonyl stretching (ester carbonyl). On the other hand the
hydantoins showed two carbonyl stretching peaks at ꢀ1760
and 1700 cmꢁ1, as one of the carbonyls is flanked between a
N and a C, meanwhile the other is flanked between two
nitrogen atoms leading to higher single bond character
of the carbonyl group. The piperazinediones showed two
carbonyl stretching peaks at ꢀ1670 and 1640 cmꢁ1. The
relatively lower stretching values of the carbonyls of the
six-membered derivatives relative to the five-membered
derivatives may be explained by the higher ring strain of
the hydantoin ring, leading to higher double bond characters
of the carbonyl groups.
Interpretation of 13C NMR signals was based on both the
chemical shifts in 13C NMR spectra and DEPT-135 spectra
(distortionless enhancement by polarization transfer). All
carbons that have an attached proton provided a signal
in DEPT spectra but the phase of the signal differed based
on whether the number of attached hydrogens is an odd or
an even number. Signals arising from –CH or –CH3 groups
O
O
R,S
R,S
H3COOC
N
N
N
S
NH
a
b
N
H
Cl
S
Scheme 3. Synthesis of tetrahydro-betacar-
boline-piperazinedione derivatives. Reagents
and conditions: (a) Cl–CO–CH2–Cl, chloro-
form, NaHCO3, RT, 2 h; (b) ethylamine,
methanol, reflux, 16 h, 12–52.1%. For exact
individual assignment, see Table 3.
S
1 , 4
O
S
Br
23 , 24
Br
21 , 22
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

26
D. S. El-Gamil et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Table 1. PDE5 inhibitory activity of 1, 3-disubstituted tetrahydro-b-
carbolines (1–4) and the fully aromatic b-carboline 25.
Table 2. PDE5 inhibitory activity of tetrahydro-b-carboline-
hydantoins (5–20).
Compound Chirality
PDE5 %
inhibition
PDE5
inhibition
IC50 (mm)
Compd
R
Chirality
PDE5 % PDE5
inhibition inhibition
at 10 mma)
at 10 mma) IC50 (mm)b)
1
2
3
4
25
1S, 3R
1R, 3R
1R, 3S
1S, 3S
NA
<60
<60
<60
<60
<60
ND
ND
ND
ND
ND
5
6
7
8
–C2H5
–C2H5
–C2H5
5S, 11aR
5R, 11aR
5R, 11aS
5S, 11aS
5S, 11aR
5R, 11aR
5R, 11aS
5S, 11aS
5S, 11aR
5R, 11aR
5R, 11aS
5S, 11aS
5S, 11aR
5R, 11aR
5R, 11aS
5S, 11aS
99
66
78
99
0.2
4.8
2
–C2H5
0.16
0.17
ND
ND
0.47
0.39
ND
ND
1
0.29
5.4
ND
0.32
9
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–C–(CH3)3
–C–(CH3)3
–C–(CH3)3
–C–(CH3)3
–CH2–CH¼CH2
–CH2–CH¼CH2
–CH2–CH¼CH2
–CH2–CH¼CH2
99
10
11
12
13
14
15
16
17
18
19
20
<60
<60
92
a)
Calculated from triplicate values.
97
<60
<60
90
96
67
Biology
All the final compounds and tetrahydro-b-carboline inter-
mediates synthesized were tested for in vitro inhibition of
recombinant human PDE5 at screening doses of 10 mM; for
compounds displaying a percentage of inhibition >60%, the
IC50 was then determined by testing a range of 10 concen-
trations, each with double replicates. The results are shown
in Tables 1–3. Moreover, the two most active compounds 8, 9
were tested versus an array of other PDEs (PDE1A, PDE2A,
PDE3A, PDE9A, PDE10A, and PDE11A) to decide about their
selectivity profile using tadalafil as a positive control; results
are shown in Table 4.
<60
97
a)
Calculated from triplicate values.
Calculated from 10 concentrations, each with double replicates.
b)
Table 3. PDE5 inhibitory activity of tetrahydro-b-carboline-
piperazinediones (23, 24).
Compound
R
Chirality
PDE5 %
inhibition
PDE5
inhibition
Based on the introduced structural modifications, the
following SAR for PDE5 inhibitors can be concluded:
The tetrahydro-b-carboline derivatives 1–4 and the fully
aromatic b-carboline 25 showed no PDE5 inhibition,
suggesting that the tetracyclic scaffold is an essential feature
for PDE5 inhibition. Moreover, lacking of an N-alkyl substitu-
ent and thus the lack of possible hydrophobic interaction
with Ile665 of the H-loop could be another factor contribu-
ting to their lack of activity. The importance of this type of
interaction has been reported before. Moreover, a docking
experiment showed the involvement of one of the amide
carbonyls in indirect interaction through a water molecule
with Met816 (Fig. 2). This confirms the importance of
the external ring in modulating the inhibition of PDE5.
Replacing the 1,3-benzodioxole group of tadalafil with a
5-bromo-2-thienyl group still produces active candidates with
appreciable PDE5 inhibitory activity, compound 8 showed
at 10 mma)
IC50 (mm)b)
23
24
C2H5
C2H5
6S, 12aR
6S, 12aS
81
87
1.71
1
a)
Calculated from triplicate values.
Calculated from 10 concentrations, each with double replicates.
b)
the highest activity with an IC50 of 0.16 mm. The relatively
smaller size of the 5-bromo-2-thienyl group seems to allow its
fitting in the relatively narrow Q2 pocket of the PDE5 enzyme
whose size is believed to govern the binding of the ligands to
the enzyme [19, 25]. In addition, the lipophilicity of the
5-bromo-2-thienyl group was calculated to be higher than that
of the 1,3-benzodioxole group of tadalafil (clog p ¼ 2.693 and
2.107, respectively), which may improve the possible hydro-
phobic interaction with Met816 in the Q2 pocket of the
Table 4. IC50 (mm)a) of the most active tetrahydro-b-carboline-hydantoins and tadalafil against an array of PDEs.
Compound PDE1A
cGMP
PDE2A
cGMP
PDE3A
cGMP
PDE5A
cGMP
PDE9A
cGMP
PDE10A
cGMP
PDE11A
cGMP
PDE11A
cAMP
Selectivity index IC50
(PDE11A/PDE5A) cGMP/cGMP
8
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.16
0.17
0.004
>50
>50
>50
>50
>50
>50
7.8
2.3
0.05
11.3
4
0.3
49
14
13
9
Tadalafil
a)
Calculated from 10 concentrations, each with double replicates.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Tetrahydro-b-Carbolines as PDE5 Inhibitors
27
This suggestion was further encouraged by a quantum
mechanics-based study which proved that benzene-thio-
phene heterodimers are more stable p–p complexes than
benzene–benzene homodimers with interaction energies of
ꢁ2.774 and ꢁ0.38 kcal/mol, respectively [28]. On the other
hand, the lipophilicity of the 5-bromo-2-thienyl group was
calculated to be relatively lower than that of the 2-bromo-
phenyl group (clog p ¼ 2.693 and 3.005, respectively), which
may have caused lower hydrophobic interaction with resi-
dues in the Q2 pocket and thus the unexpected marked
decrease in potency. This may imply that the relatively lower
lipophilic properties of thiophene compared to benzene
had more influence on interaction and potency than its
polarizability. Accordingly, in future studies, it would be
beneficial to use lipophilic carboaryl substituents rather
than the polarizable thienyl in order to maximize possible
hydrophobic interaction with the enzyme.
Regarding the hydantoin analogues (5–20), the substituent
on the nitrogen of the terminal ring was modified from
N-methyl of tadalafil to N-ethyl, butyl, tert-butyl, and allyl in
hydantoin derivatives with 11 compounds showing PDE5
inhibitory activity. N-Substitution seems to have slight influ-
ence on activity, with the ethyl substituent being the most
active. All N-ethyl derivatives were active as PDE5 inhibitors
with IC50 ranging from 0.16 to 4.84 mm where the most active
compound 8 had an N-ethyl substituent. This may encourage
the use of less bulky groups in the future. However, none of the
other bulkier or unsaturated side chains had deleterious effect
on activity and they possessed comparable PDE5 inhibitory IC50
values, indicating the ability of the nitrogen of the hydantoin
ring to tolerate various types of substituents. These N-alkyl
derivatives are expected to have hydrophobic interaction with
residue Ile665 of the H-loop.
Figure 2. Detailed mode view showing the docking and interaction
of 8 with human PDE5.
enzyme. However, looking for the thiophene ring as an
isosteric replacement of pendant phenyl led to pronounced
reduction in PDE5 inhibitory activity. The most potent com-
pound 8 showed four times lower potency than a previously
reported classical isostere, shown in Fig. 3 [26]. The two
isosteres, thiophene, and benzene rings were compared.
The thiophene ring has higher electron-rich properties, com-
pared to benzene, which arises from the fact that the lone
pair of electrons in the p orbital of the sulfur atom contrib-
utes to the aromatic sextet and pushes high electron density
toward the ring carbons that accordingly acquire partial
negative charge. Accordingly, it was suggested that the large
atomic polarizability of the sulfur atom would provide
higher dispersion forces compared to benzene, which may
lead to better p–p stacking and/or Van der Waals forces with
hydrophobic residues lining the Q2 hydrophobic pocket [27].
Expansion of the terminal ring from hydantoin in
compounds 5, 8 to piperazinedione in compounds 23, 24
led to active candidates but, surprisingly, showed a six- to
ninefold decrease in IC50 of PDE5 inhibitory activity com-
pared to the respective hydantoins. This implies that the
increase in the size of the fused ring has an impact on
PDE5 inhibitory activity in this series of compounds.
Regarding the absolute stereo-chemical requirement for
PDE5 inhibition, the stereochemistry of C5 of the hydantoins
and C6 of the piperazinediones appeared to be critical for
activity. The results showed that all potent compounds with
IC50 < 1 mM possess an absolute S configuration at C-5 or C-6
regardless of the absolute configuration at C-11a or C-12a.
This suggests that activity is mainly set by the stereochem-
istry of the chiral carbon derived from the aldehyde rather
than that derived from the amino acid. This may double the
chemical space from which PDE5 inhibitors might be discov-
ered and open a new horizon towards economical synthesis
of active compounds using the cheaper L-tryptophan.
Figure 3. Chemical structure of the bromophenyl isostere of
compound 8, PDE5 inhibition IC50 ¼ 0.038 mm [26].
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

28
D. S. El-Gamil et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
detector (Thermo Finnigan, San Jose, CA). The MS detection
was carried out at a spray voltage of 4.2 kV, a nitrogen sheath
gas pressure of 4.0 ꢂ 105 Pa, an auxiliary gas pressure of
1.0 ꢂ 105 Pa, a capillary temperature of 4008C, capillary voltage
of 35 V, and source CID of 10 V. All samples were injected by
autosampler (Surveyor, Thermo Finnigan) with an injection vol-
ume of 10 mL. A RP C18 NUCLEODUR 100-3 (125 mm ꢂ 3 mm)
column (Macherey-Nagel) was used as stationary phase. The sol-
vent system consisted of water containing 0.1% TFA in aceto-
nitrile. HPLC method: flow rate 400 mL/min. The percentage of
the solvent system started at an initial of 5%, was increased up to
100% during 16 min, kept at 100% for 2 min, and flushed back to
the 5% in 2 min. Melting points were determined on a Mettler
FP1 melting point apparatus and are uncorrected. FTIR spectra
were recorded on a Nicolet Avatar 380 spectrometer. 1H NMR
spectra were recorded at 500 MHz while 13C NMR and DEPT
spectra were recorded at 125 Hz using a Bruker DRX-500 spec-
trometer. 1H shifts are referenced to the residual protonated
solvent signal (d 2.50 for DMSO-d6 and d 7.26 for CDCl3) and
13C shifts are referenced to the deuterated solvent signal
(d 39.5 for DMSO-d6 and d 77.2 for CDCl3). Chemical shifts are
given in parts per million (ppm), and all coupling constants (J) are
given in Hz. The following abbreviations are used for multiplicity
of NMR signals: s, singlet; d, doublet; t, triplet; q, quartret; m,
multiplet; dd, doublet of doublet; dt, doublet of triplet; qd,
quartet of doublet; ddd, doublet of doublet of doublet. Flash
column chromatography was carried out using silica gel 60 (40–
63 mM). The reaction progress was determined by TLC analysis on
Alugram SIL G/UV254 (Macherey-Nagel). Visualization was
accomplished with UV light. All reactions were carried out under
nitrogen or argon. All chemicals and solvents were obtained
from Sigma–Aldrich and were used without further purification.
All the described compounds had >95% purity as determined by
HPLC/MS and the areas under the peaks.
Compounds 8, 9 were the most potent PDE5 inhibitors.
Therefore, we determined their isozyme selectivity by com-
paring their potency for PDE5 inhibition to their potency for
inhibition of PDE1A, PDE2A, PDE3A, PDE9A, PDE10A using
cGMP as a substrate and inhibition of PDE11A using both
cAMP and cGMP as substrates; the results are summarized in
Table 4. Interestingly, our most potent compound 8 showed
49 times selectivity towards PDE5 rather than PDE11 with
cGMP as substrate. Meanwhile, tadalafil was only 13 times as
selective. Moreover, compound 9 showed 14 times selectivity
towards PDE5 rather than PDE11 which is slightly higher
compared to tadalafil. This indicates that the selectivity to
PDE5 versus PDE11 is mainly a function of the size of the
terminal ring and its terminal nitrogen substituent, the
smaller is the better. This indicates that there are potential
differences between PDE5 and PDE11 in the H-loop region.
Moreover, compounds 8, 9 as well as the reference compound
tadalafil lacked the ability to inhibit cGMP hydrolysis by
PDE1A, PDE2A, PDE3A, PDE9A, PDE10A at concentrations
up to 50 mM, showing that PDE5 and PDE11 are of the highest
catalytic pocket similarity among different PDEs. This
indicates that compounds 8, 9 might be free from those
side effects attributed to cross-reactivity with other PDEs,
particularly PDE11.
In silico binding mode
To further investigate the binding mode of our compounds
compared to tadalafil, in silico docking experiment was imple-
mented to dock tadalafil and the most active compound 8 to
the PDE5 crystal structure (1XOZ) using the MOE 2009.10
software. A 2D view of the interaction between tadalafil and
the human PDE5 binding pocket was compared to the inter-
action of 8 with the residues lining the binding pocket of
PDE5 as shown in Figs. 1 and 2. The results showed that 8
interacts with human PDE5 in a similar manner to tadalafil,
where both can form a single H-bond with Gln817, the
protein residue involved in nucleotide recognition, through
the NH of the indole ring of the tetrahydro-b-carboline
moiety. The indole ring in both compounds showed p–p
stacking with the hydrophobic residues lining the cavity
of the active sites, namely Phe820. Moreover, compound 8
showed a water-mediated interaction with Met816. Finally,
the bromine atom seems to occupy the relatively narrow Q2
pocket, occupied by the 1,3-benzodioxole group of tadalafil,
allowing some kind of hydrophobic interaction with Met816.
Procedure for the preparation of D- and L-tryptophan methyl
ester [23]
A 250 mL round bottom flask containing methanol (150 mL) was
cooled in an ice-water bath, then acetyl chloride (10 mL)
was added dropwise. Solid D- or L-tryptophan (15 g, 73.45 mmol)
was added to the well-stirred solution. The solution was then
heated to reflux for 5 h, the solvent was removed under reduced
pressure to give tryptophan methyl ester hydrochloride. The free
base was obtained by adding dilute NH4OH (50 mL) and extrac-
tion with CH2Cl2 (5 ꢂ 50 mL). The organic layer was dried over
anhydrous Na2SO4, evaporated under reduced pressure to give a
yellow oil which solidifies on cooling. The product was used
without further purification.
General procedure for the preparation of methyl 1-(5-
bromo-2-thienyl)-2,3,4,9-tetrahydro-1H-b-carboline-3-
carboxylates 1–4
The appropriate tryptophan methyl ester (10 g, 45.82 mmol)
was added to 5-bromothiophene-2-carboxaldehyde (9.5 g,
49.73 mmol) and dissolved in CH2Cl2 (20 mL). The solution
was cooled to 08C in an ice bath. To this solution trifluoroacetic
acid (2 mL) was added dropwise, and the mixture was stirred at
room temperature for 4 days under nitrogen atmosphere. The
reaction mixture was then basified with dilute NH4OH solution.
The trans-isomer was precipitated in the organic layer and
obtained by vacuum filtration, washed with CH2Cl2 and ether.
Experimental
Chemistry
Mass spectrometric analysis (HPLC–ESI-MS) was performed on
a TSQ quantum (Thermo Electron Corporation) instrument
equipped with an ESI source and a triple quadrupole mass
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Tetrahydro-b-Carbolines as PDE5 Inhibitors
29
The filtrate, containing a mixture of cis- and trans-isomers, was
then extracted with CH2Cl2 (3 ꢂ 50 mL). The organic layer was
washed with water, dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure. The residue was purified and
the isomers were separated by flash column chromatography
on silica gel eluting with CH2Cl2, to give first the appropriate
cis-isomer followed by the trans-isomer.
(d, J ¼ 7.8 Hz, 1H, Ar), 7.27 (d, J ¼ 8.0 Hz, 1H, Ar), 7.06–7.01
(m, 2H, Ar), 6.95 (td, J ¼ 7.5, 1.0 Hz, 1H, Ar), 6.85 (dd, J ¼ 3.8,
1.0 Hz, 1H, Ar), 5.51 (s, 1H, NHCHCS), 3.83 (dd, J ¼ 7.7, 5.2 Hz,
1H, CHCOOCH3), 3.63 (s, 3H, OCH3), 2.98 (dd, J ¼ 15.3, 4.8 Hz, 1H,
CHaHb), 2.83 ppm (ddd, J ¼ 15.2, 7.8, 1.2 Hz, 1H, CHaHb). 13C NMR
(125 MHz, DMSO-d6): d 173.61 (COOCH3), 149.93, 136.00, 133.33,
129.82, 126.29, 125.88, 121.12, 118.57, 117.84, 111.17, 110.42,
106.00, 52.12 (C1), 51.72 (C3), 50.08 (CH3), 24.67 ppm (CHaHb).
IR: 3319.24 (–NH–), 1699.41 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity
98.5%, for C17H15BrN2O2S: m/z (%) 392 [Mþþ2], 390 [Mþ], 303
(100%).
(1S,3R) Methyl 1-(5-bromo-2-thienyl)-2,3,4,9-tetrahydro-
1H-b-carboline-3-carboxylate (1)
Yellow powder (2.17 g, 12.1%): Rf ¼ 0.53 (CH2Cl2); mp: 157–
1598C; 1H NMR (500 MHz, DMSO-d6): d 10.49 (s, 1H, NH), 7.41
(d, J ¼ 7.8 Hz, 1H, Ar), 7.25–7.22 (m, 1H, Ar), 7.14–7.08 (m, 2H,
Ar), 7.04–6.98 (m, 1H, Ar), 6.97–6.92 (m, 1H, Ar), 5.54 (s, 1H,
NHCHCS), 3.84 (dd, J ¼ 11.1, 4.0 Hz, 1H, CHCOOCH3), 3.70
(s, 3H, OCH3), 3.02–2.97 (m, 1H, CHaHb), 2.79–2.71 ppm
General procedure for the preparation of 5-(5-bromo-2-
thienyl)-2-alkyl-5,6,11,11a-tetrahydro-1H-imidazo-
[10,5 0:1,6]pyrido[3,4-b]indole-1,3(2H)-diones 5–20
Excess ethyl or butyl or tert-butyl or allyl isocyanate (1.6 mmol)
was added to a well-stirred solution of the appropriate tetra-
hydro-b-carbolines 1–4 (0.39 g, 1 mmol) in methyl ethyl ketone
(10 mL). The mixture was then refluxed under nitrogen atmos-
phere for 70 h in case of cis-tetrahydro-b-carbolines 1, 3 while
the trans-tetrahydro-b-carbolines 2, 4 were refluxed for 16 h.
The solvent was evaporated to dryness under reduced pressure
and the product was then purified using flash column chroma-
tography on silica gel, eluting with CH2Cl2.
(m, 1H, CHaHb). 13C NMR (125 MHz, DMSO-d6):
d 172.40
(COOCH3), 148.23, 136.30, 134.15, 129.59, 126.66, 126.33,
121.01, 118.57, 117.79, 111.28, 110.95, 106.29, 55.97 (C1),
53.38 (C3), 51.85 (CH3), 24.94 ppm (CHaHb). IR: 3394.39,
3328.32 (–NH–), 1737.67 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity
98.37%, for C17H15BrN2O2S: m/z (%): 392 [Mþþ2], 390 [Mþ], 305
(100%).
(1R,3R) Methyl 1-(5-bromo-2-thienyl)-2,3,4,9-tetrahydro-
1H-b-carboline-3-carboxylate (2)
(5S,11aR) 5-(5-Bromo-2-thienyl)-2-ethyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (5)
Pale yellow powder (9.02 g, 50.3%): Rf ¼ 0.22 (CH2Cl2); mp: 205–
2088C; 1H NMR (500 MHz, DMSO-d6): d 10.81 (s, 1H, NH), 7.41
(d, J ¼ 7.8 Hz, 1H, Ar), 7.27 (d, J ¼ 8.0 Hz, 1H, Ar), 7.06–7.02
(m, 2H, Ar), 6.96–6.93 (m, 1H, Ar), 6.85 (dd, J ¼ 3.8, 0.9 Hz,
1H, Ar), 5.51 (s, 1H, NHCHCS), 3.83 (dd, J ¼ 7.7, 5.2 Hz,
1H, CHCOOCH3), 3.63 (s, 3H, OCH3), 2.98 (dd, J ¼ 15.2, 5.1 Hz,
1H, CHaHb), 2.83 ppm (dd, J ¼ 14.7, 7.3 Hz, 1H, CHaHb). 13C NMR
(125 MHz, DMSO-d6): d 173.56 (COOCH3), 149.93, 135.94, 133.46,
129.88, 126.23, 125.88, 121.16, 118.56, 117.93, 111.17, 110.48,
105.97, 52.17 (C1), 51.72 (C3), 50.08 (CH3), 24.61 ppm (CHaHb).
IR: 3317.89 (–NH–), 1697.84 cmꢁ1 (–CO–). HPLC/MS (ESI):
Purity 98%, for C17H15BrN2O2S: m/z (%): 392 [Mþþ2], 390 [Mþ],
303 (100%).
Yellow powder (0.13 g, 30%): Rf ¼ 0.26 (CH2Cl2); mp: 189–1918C;
1H NMR (500 MHz, DMSO-d6): d 10.90 (s, 1H, NH), 7.57–7.54 (m,
1H, Ar), 7.29 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.12–7.07 (m, 3H, Ar),
7.02 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.23 (s, 1H, NCHCS),
4.50 (dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N), 3.42 (qd, J ¼ 7.3,
2.9 Hz, 2H, CH2CH3), 3.29 (dd, J ¼ 14.5, 4.3 Hz, 1H, NCHCHaHb),
2.85 (ddd, J ¼ 14.6, 11.6, 1.8 Hz, 1H, NCHCHaHb), 1.10 ppm
(t, J ¼ 7.2 Hz, 3H, CH2CH3). 13C NMR (125 MHz, DMSO-d6):
d 171.03, 154.20 (NCO), 146.13, 136.69, 133.52, 129.53, 127.46,
125.69, 121.78, 118.95, 118.40, 111.50, 111.12, 105.43, 57.76 (C5),
51.26 (C11a), 32.78 (CH2CH3), 21.67 (CHaHb), 13.28 ppm (CH2CH3).
IR: 3275.82 (–NH–), 1764.63, 1698.05 cmꢁ1 (–CO–). HPLC/MS (ESI):
Purity 100%, for C19H16BrN3O2S: m/z (%):431 [Mþþ2], 429 [Mþ],
242 (100%).
(1R,3S) Methyl 1-(5-bromo-2-thienyl)-2,3,4,9-tetrahydro-
1H-b-carboline-3-carboxylate (3)
Yellow powder (1.97 g, 11%): Rf ¼ 0.53 (CH2Cl2); mp: 157–1588C;
1H NMR (500 MHz, DMSO-d6):
d 10.49 (s, 1H, NH), 7.41
(5R,11aR) 5-(5-Bromo-2-thienyl)-2-ethyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (6)
(d, J ¼ 7.8 Hz, 1H, Ar), 7.25–7.23 (m, 1H, Ar), 7.14–7.08 (m, 2H,
Ar), 7.04–6.99 (m, 1H, Ar), 6.94 (ddd, J ¼ 8.0, 7.1, 1.0 Hz, 1H, Ar),
5.53 (s, 1H, NHCHCS), 3.84 (dd, J ¼ 11.1, 4.0 Hz, 1H, CHCOOCH3),
3.70 (s, 3H, OCH3), 2.99 (ddd, J ¼ 14.7, 4.0, 1.6 Hz, 1H, CHaHb),
2.75 ppm (ddd, J ¼ 14.7, 11.1, 2.3 Hz, 1H, CHaHb). 13C NMR
White powder (0.11 g, 25%): Rf ¼ 0.49 (CH2Cl2); mp: 132–1358C;
1H NMR (500 MHz, DMSO-d6): d 11.10 (s, 1H, NH), 7.54 (d, J ¼ 7.8,
1H, Ar), 7.34 (dt, J ¼ 8.2, 0.9 Hz, 1H, Ar), 7.17–7.07 (m, 2H, Ar),
7.03 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.92 (dd, J ¼ 3.8, 0.7 Hz,
1H, Ar), 6.45 (s, 1H, NCHCS), 4.48 (dd, J ¼ 10.9, 5.6 Hz,
1H, CHC(O)N), 3.46 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 3.38–3.30 (dd,
J ¼ 15.0, 5.5 Hz, 1H, NCHCHaHb), 2.80 (ddd, J ¼ 15.1, 11.0, 1.7 Hz,
1H, NCHCHaHb), 1.12 ppm (t, J ¼ 7.2 Hz, 3H, CH2CH3). 13C NMR
(125 MHz, DMSO-d6): d 171.99, 153.94 (NCO), 144.65, 136.58,
130.16, 130.05, 127.54, 125.54, 122.01, 118.95, 118.41, 111.58,
111.48, 106.25, 52.43 (C5), 46.70 (C11a), 33.14 (CH2CH3),
22.31 (CHaHb), 13.24 ppm (CH2CH3). IR: 3323.84 (–NH–),
1765.72, 1697.64 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%, for
C19H16BrN3O2S: m/z (%): 431 [Mþþ2], 429 [Mþ], 242 (100%).
(125 MHz, DMSO-d6):
d 172.49 (COOCH3), 148.31, 136.29,
134.21, 129.58, 126.61, 126.34, 121.00, 118.56, 117.79, 111.27,
110.91, 106.29, 55.97 (C1), 53.38 (C3), 51.84 (CH3), 24.97 ppm
(CHaHb). IR: 3393.8, 3328.06 (–NH–), 1737.5 cmꢁ1 (–CO–). HPLC/
MS (ESI): Purity 100%, for C17H15BrN2O2S: m/z (%) 392 [Mþþ2], 390
[Mþ], 305 (100%).
(1S,3S) Methyl 1-(5-bromo-2-thienyl)-2,3,4,9-tetrahydro-
1H-b-carboline-3-carboxylate (4)
Pale yellow powder (7.3 g, 40.7%): Rf ¼ 0.22 (CH2Cl2); mp: 205–
2078C; 1H NMR (500 MHz, DMSO-d6): d 10.81 (s, 1H, NH), 7.41
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

30
D. S. El-Gamil et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
7.01 (m, 1H, Ar), 6.92 (dt, J ¼ 2.3, 1.4 Hz, 1H, Ar), 6.45 (s, 1H,
NCHCS), 4.49 (dd, J ¼ 11.0, 5.5 Hz, 1H, CHC(O)N), 3.42 (t, J ¼ 7.1,
2H, CH2 CH2CH2CH3), 3.35 (dd, J ¼ 15.0, 5.5 Hz, 1H, NCHCHaHb),
2.79 (ddd, J ¼ 15.1, 11.0, 1.8 Hz, 1H, NCHCHaHb), 1.56–1.48
(m, 2H, CH2 CH2CH2CH3), 1.29–1.23 (m, 2H, CH2 CH2CH2CH3),
0.87 ppm (t, J ¼ 7.5 Hz, 3H, CH2CH2CH2CH3). 13C NMR (125 MHz,
DMSO-d6): d 172.23, 154.12 (NCO), 144.65, 136.62, 130.17, 130.05,
127.46, 125.54, 122.01, 118.95, 118.42, 111.56, 111.48, 106.25,
52.40 (C5), 46.64 (C11a), 37.71, 29.59, 22.46, 19.33, 13.46 ppm
(CH3). IR: 3317.76 (–NH–), 1764.58, 1695.60 cmꢁ1 (–CO–). HPLC/
MS (ESI): Purity 100%, for C21H20BrN3O2S: m/z (%): 459 [Mþþ2],
457 [Mþ] (100%).
(5R,11aS) 5-(5-Bromo-2-thienyl)-2-ethyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (7)
Dark yellow powder (0.14 g, 32.9%): Rf ¼ 0.26 (CH2Cl2); mp: 193–
1968C; 1H NMR (500 MHz, DMSO-d6): d 10.90 (s, 1H, NH), 7.57–7.54
(m,1H, Ar), 7.29 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.13–7.07 (m, 3H, Ar),
7.02 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.23 (s, 1H, NCHCS), 4.50
(dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N), 3.42 (qd, J ¼ 7.2, 3.3 Hz,
2H, CH2CH3), 3.32–3.28 (m, 1H, NCHCHaHb), 2.85 (ddd, J ¼ 14.8,
11.6, 1.8 Hz, 1H, NCHCHaHb), 1.11 ppm (t, J ¼ 7.2 Hz, 3H,
CH2CH3). 13C NMR (125 MHz, DMSO-d6): d 171.05, 154.23 (NCO),
146.08, 136.63, 133.51, 129.52, 127.45, 125.72, 121.77, 118.94,
118.39, 111.43, 111.11, 105.19, 57.57 (C5), 51.47 (C11a), 32.67
(CH2CH3), 21.68 (CHaHb), 13.28 ppm (CH2CH3). IR: 3328.56 (–NH–),
1764.73, 1697.84 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%,
for C19H16BrN3O2S: m/z (%) 431 [Mþþ2], 429 [Mþ], 242 (100%).
(5R,11aS) 5-(5-Bromo-2-thienyl)-2-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (11)
Yellow powder (0.27 g, 58.7%): Rf ¼ 0.35 (CH2Cl2); mp: 181–
1838C; 1H NMR (500 MHz, DMSO-d6): d 10.90 (s, 1H, NH), 7.56–
7.53 (m, 1H, Ar), 7.29 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.13–7.07
(m, 3H, Ar), 7.02 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.23 (s, 1H,
NCHCS), 4.52 (dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N), 3.39 (t, J ¼ 7.1,
2H, CH2 CH2CH2CH3), 3.34–3.29 (m, 1H, NCHCHaHb), 2.84
(ddd, J ¼ 14.8, 11.6, 1.8 Hz, 1H, NCHCHaHb), 1.52–1.46 (m, 2H,
CH2 CH2CH2CH3), 1.29–1.24 (m, 2H, CH2 CH2CH2CH3), 0.88 ppm
(t, J ¼ 7.3 Hz, 3H, CH2CH2CH2CH3). 13C NMR (125 MHz, DMSO-d6):
d 171.28, 154.34 (NCO), 146.13, 136.62, 133.52, 129.52, 127.40,
125.69, 121.77, 118.94, 118.39, 111.43, 111.08, 105.36, 57.65 (C5),
51.26 (C11a), 37.45, 29.58, 21.78, 19.28, 13.45 ppm (CH3).
IR: 3303.53 (–NH–), 1755.54, 1697.13 cmꢁ1 (–CO–). HPLC/MS
(ESI): Purity 100%, for C21H20BrN3O2S: m/z (%): 459 [Mþþ2], 457
[Mþ] (100%).
(5S,11aS) 5-(5-Bromo-2-thienyl)-2-ethyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (8)
Yellow powder (0.25 g, 58.4%): Rf ¼ 0.48 (CH2Cl2); mp: 135–
1388C; 1H NMR (500 MHz, DMSO-d6): d 11.10 (s, 1H, NH), 7.54
(m, 1H, Ar), 7.34 (dt, J ¼ 8.2, 0.9 Hz, 1H, Ar), 7.15–7.10 (m, 2H, Ar),
7.03 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.92 (dd, J ¼ 3.8, 0.7 Hz,
1H, Ar), 6.45 (s, 1H, NCHCS), 4.48 (dd, J ¼ 10.9, 5.7 Hz,
1H, CHC(O)N), 3.46 (q, J ¼ 7.2, 2H, CH2CH3), 3.37–3.32
(dd, J ¼ 15.0, 5.5 Hz, 1H, NCHCHaHb), 2.80 (ddd, J ¼ 15.1, 10.9,
1.7 Hz, 1H, NCHCHaHb), 1.12 ppm (t, J ¼ 7.2 Hz, 3H, CH2CH3).
13C NMR (125 MHz, DMSO-d6): d 171.98, 153.94 (NCO), 144.65,
136.62, 130.16, 130.05, 127.50, 125.54, 122.00, 118.94, 118.41,
111.60, 111.54, 106.25, 52.43 (C5), 46.61 (C11a), 33.05 (CH2CH3),
22.31 (CHaHb), 13.24 ppm (CH2CH3). IR: 3316.6 (–NH–),
1765.74, 1695.22 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 97.62%,
for C19H16BrN3O2S: m/z (%): 431 [Mþþ2], 429 [Mþ] (100%).
(5S,11aS) 5-(5-Bromo-2-thienyl)-2-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (12)
Yellow powder (0.31 g, 67.4%): Rf ¼ 0.62 (CH2Cl2); mp: 98–1018C;
1H NMR (500 MHz, DMSO-d6): d 11.11 (s, 1H, NH), 7.56–7.53
(m, 1H, Ar), 7.34 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.15–7.10 (m, 2H,
Ar), 7.03 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.91 (dd, J ¼ 3.8,
0.8 Hz, 1H, Ar), 6.45 (s, 1H, NCHCS), 4.49 (dd, J ¼ 11.0, 5.6 Hz,
1H, CHC(O)N), 3.42 (t, J ¼ 7.1, 2H, CH2 CH2CH2CH3), 3.36
(dd, J ¼ 15.0, 5.5 Hz, 1H, NCHCHaHb), 2.79 (ddd, J ¼ 15.1, 11.0,
1.8 Hz, 1H, NCHCHaHb), 1.56–1.49 (m, 2H, CH2 CH2CH2CH3), 1.29–
1.24 (m, 2H, CH2 CH2CH2CH3), 0.88 ppm (t, J ¼ 7.4 Hz, 3H,
(5S,11aR) 5-(5-Bromo-2-thienyl)-2-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (9)
Yellow powder (0.2 g, 43.4%): Rf ¼ 0.35 (CH2Cl2); mp: 178–1818C;
1H NMR (500 MHz, DMSO-d6): d 10.90 (s, 1H, NH), 7.57–7.54
(m, 1H, Ar), 7.29 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.13–7.07 (m, 3H,
Ar), 7.02 (ddd, J ¼ 8.0, 7.1, 1.1 Hz, 1H, Ar), 6.23 (s, 1H, NCHCS),
4.52 (dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N), 3.39 (t, J ¼ 7.1, 2H, CH2
CH2CH2CH3), 3.34–3.28 (m, 1H, NCHCHaHb), 2.84 (ddd, J ¼ 14.8,
11.6, 1.8 Hz, 1H, NCHCHaHb), 1.52–1.46 (m, 2H, CH2 CH2CH2CH3),
1.29–1.24 (m, 2H, CH2 CH2CH2CH3), 0.88 ppm (t, J ¼ 7.4 Hz, 3H,
CH2CH2CH2CH3). 13C NMR (125 MHz, DMSO-d6): d 171.28, 154.48
(NCO), 146.11, 136.74, 133.46, 129.49, 127.51, 125.75, 121.77,
119.05, 118.39, 111.43, 111.10, 105.42, 57.71 (C5), 51.31 (C11a),
37.45, 29.64, 21.82, 19.31, 13.45 ppm (CH3). IR: 3304.49 (–NH–),
1755.60, 1698.53 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 99.5%,
for C21H20BrN3O2S: m/z (%): 459 [Mþþ2], 457 [Mþ] (100%).
CH2CH2CH2CH3). 13C NMR (125 MHz, DMSO-d6):
d 172.23,
154.12 (NCO), 144.65, 136.62, 130.17, 130.05, 127.46, 125.54,
122.01, 118.95, 118.42, 111.56, 111.48, 106.24, 52.40 (C5),
46.63 (C11a), 37.80, 29.67, 22.46, 19.33, 13.46 ppm (CH3).
IR: 3321.19 (–NH–), 1765.08, 1696.47 cmꢁ1 (–CO–). HPLC/MS
(ESI): Purity 97.62%, for C21H20BrN3O2S: m/z (%): 459 [Mþþ2],
457 [Mþ], 242 (100%).
(5S,11aR) 5-(5-Bromo-2-thienyl)-2-tert-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (13)
Yellow powder (0.23 g, 50%): Rf ¼ 0.42 (CH2Cl2); mp: 196–1998C;
1H NMR (500 MHz, DMSO-d6): d 10.88 (s, 1H, NH), 7.56–7.53
(m, 1H, Ar), 7.29 (dt, J ¼ 8.2, 1.0 Hz, 1H, Ar), 7.12–7.06 (m, 3H,
Ar), 7.01 (ddd, J ¼ 8.0, 7.1, 1.1 Hz, 1H, Ar), 6.17 (s, 1H, NCHCS),
4.36 (dd, J ¼ 11.4, 4.2 Hz, 1H, CHC(O)N), 3.23 (dd, J ¼ 14.9, 4.3 Hz,
(5R,11aR) 5-(5-Bromo-2-thienyl)-2-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (10)
Yellow powder (0.26 g, 57.6%): Rf ¼ 0.62 (CH2Cl2); mp: 100–
1028C; 1H NMR (500 MHz, DMSO-d6): d 11.11 (s, 1H, NH), 7.56–
7.53 (m, 1H, Ar), 7.36–7.31 (m, 1H, Ar), 7.15–7.11 (m, 2H, Ar), 7.06–
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Tetrahydro-b-Carbolines as PDE5 Inhibitors
31
1H, CHaHb), 2.77 (ddd, J ¼ 14.8, 11.6, 1.8 Hz, 1H, CHaHb),
1.53 ppm (s, 9H, CH3). 13C NMR (125 MHz, DMSO-d6): d 172.09,
155.06 (NCO), 146.78, 136.60, 133.65, 129.58, 126.84, 125.72,
121.72, 118.92, 118.37, 111.41, 110.74, 105.39, 57.00 (C5),
56.86 (C(CH3)3), 51.21 (C11a), 28.41 ((CH3)3), 21.99 ppm (CHaHb).
IR: 3336.41 (–NH–), 1762.7, 1701.88 cmꢁ1 (–CO–). HPLC/MS (ESI):
Purity 100%, for C21H20BrN3O2S: m/z (%): 459 [Mþþ2], 457 [Mþ],
242 (100%).
(5S,11aR) 5-(5-Bromo-2-thienyl)-2-allyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (17)
Yellow powder (0.24 g, 53.2%): Rf ¼ 0.27 (CH2Cl2); mp: 116–
1198C; 1H NMR (500 MHz, DMSO-d6): d 10.91 (s, 1H, NH), 7.56
(d, J ¼ 8 Hz, 1H, Ar), 7.29 (ddd, J ¼ 8.1, 1.8, 0.8 Hz, 1H, Ar), 7.13–
7.07 (m, 3H, Ar), 7.04–7.00 (m, 1H, Ar), 6.25 (s, 1H, NCHCS), 5.83–
–
–
5.77 (m, 1H, CH CH H ), 5.15–5.12 (m, 1H, CH CH H ), 5.11–5.08
a
–
–
b
a b
–
(m, 1H, CH CH H ), 4.58 (dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N),
–
a
b
(5R,11aR) 5-(5-Bromo-2-thienyl)-2-tert-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (14)
4.00 (dt, J ¼ 5.0, 1.5 Hz, 2H, NCH2), 3.36–3.30 (m, 1H, CHaHb),
2.87 ppm (ddd, J ¼ 13.4, 11.6, 1.8 Hz, 1H, CHaHb). 13C NMR
(125 MHz, DMSO-d6): d 170.90, 153.98 (NCO), 145.99, 136.63,
133.48, 132.05, 129.57, 127.54, 125.68, 121.79, 118.96, 118.44,
Dark yellow powder (0.16 g, 34.6%): Rf ¼ 0.67 (CH2Cl2); mp: 206–
2078C; 1H NMR (500 MHz, DMSO-d6): d 11.09 (s, 1H, NH), 7.56–7.53
(m, 1H, Ar), 7.33 (dt, J ¼ 8.2, 0.9 Hz, 1H, Ar), 7.15–7.10 (m, 2H, Ar),
7.03 (ddd, J ¼ 7.9, 7.0, 1.0 Hz, 1H, Ar), 6.90 (dd, J ¼ 3.8, 0.7 Hz,
1H, Ar), 6.41 (s, 1H, NCHCS), 4.33 (dd, J ¼ 10.9, 5.7 Hz,
1H, CHC(O)N), 3.3 (dd, J ¼ 15.2, 5.7 Hz, 1H, CHaHb), 2.75 (ddd,
J ¼ 15.1, 10.8, 1.8 Hz, 1H, CHaHb), 1.56 ppm (s, 9H, CH3). 13C NMR
(125 MHz, DMSO-d6): d 173.06, 154.66 (NCO), 144.60, 136.67,
130.17, 130.13, 127.48, 125.58, 121.99, 118.95, 118.40, 111.57,
111.47, 106.11, 56.92 (C(CH3)3), 51.46 (C5), 46.37 (C11a),
28.13 ((CH3)3), 22.70 ppm (CHaHb). IR: 3333.69 (–NH–),
1761.99, 1693.57 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 99%, for
C21H20BrN3O2S: m/z (%): 459 [Mþþ2] (100%), 457 [Mþ].
–
116.30 (CH CH H ), 111.50, 111.13, 105.39, 57.80 (C5), 51.37
a
–
b
(C11a), 39.85 (NCH2), 21.85 ppm (CHaHb). IR: 3303.75 (–NH–),
1766.58, 1701.92 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%,
for C20H16BrN3O2S: m/z (%): 443 [Mþþ2], 441 [Mþ], 383 (100%).
(5R,11aR) 5-(5-Bromo-2-thienyl)-2-allyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (18)
Yellow powder (0.39 g, 87.2%): Rf ¼ 0.53 (CH2Cl2); mp: 80–838C;
1H NMR (500 MHz, DMSO-d6): d 11.11 (s, 1H, NH), 7.55 (dd, J ¼ 7.9,
2.7 Hz, 1H, Ar), 7.36–7.33 (m, 1H, Ar), 7.16–7.11 (m, 2H, Ar), 7.04
(ddt, J ¼ 7.9, 7.0, 0.9 Hz, 1H, Ar), 6.92 (dd, J ¼ 3.8, 0.7 Hz, 1H, Ar),
–
6.47 (s, 1H, NCHCS), 5.87–5.79 (m, 1H, CH CH H ), 5.14–5.12
–
a
b
–
–
(m, 1H, CH CH H ), 5.11–5.08 (m, 1H, CH CH H ), 4.54
a b
–
–
a
b
(5R,11aS) 5-(5-Bromo-2-thienyl)-2-tert-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6] pyrido[3,4-b]indole-
1,3(2H)-dione (15)
(dd, J ¼ 10.8, 5.3 Hz, 1H, CHC(O)N), 4.04 (dt, J ¼ 5.1, 1.6 Hz, 2H,
NCH2), 3.36 (dd, J¼ 15,5.5 Hz, 1H, CHaHb), 2.82 ppm (ddd,
J ¼ 15.1, 11.0, 1.7 Hz, 1H, CHaHb). 13C NMR (125 MHz, DMSO-d6):
d 171.85, 153.71 (NCO), 144.58, 136.63, 132.09, 130.17, 130.02, 127.50,
Pale yellow powder (0.25 g, 54.8%): Rf ¼ 0.42 (CH2Cl2); mp: 200–
2038C; 1H NMR (500 MHz, DMSO-d6): d 10.88 (s, 1H, NH), 7.55–7.53
(m, 1H, Ar), 7.2 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.11–7.06 (m, 3H, Ar),
7.01 (ddd, J ¼ 8.0, 7.1, 1.1 Hz, 1H, Ar), 6.16 (s, 1H, NCHCS),
4.36 (dd, J ¼ 11.4, 4.2 Hz, 1H, CHC(O)N), 3.25 (ddd, J ¼ 14.7,
4.3, 1.3 Hz, 1H, CHaHb), 2.77 (ddd, J ¼ 14.8, 11.5, 1.8 Hz,
1H, CHaHb), 1.53 ppm (s, 9H, CH3). 13C NMR (125 MHz, DMSO-
d6): d 172.01, 155.03 (NCO), 146.83, 136.57, 133.64, 129.58, 126.84,
125.72, 121.72, 118.92, 118.37, 111.41, 110.74, 105.39, 57.00 (C5),
56.86 (C(CH3)3), 51.21 (C11a), 28.41 ((CH3)3), 21.90 ppm (CHaHb).
IR: 3336.12 (–NH–), 1762.37, 1702.56 cmꢁ1 (–CO–). HPLC/MS (ESI):
Purity 96.34%, for C21H20BrN3O2S: m/z (%): 459 [Mþþ2], 457 [Mþ],
242 (100%).
–
–
125.54, 122.02, 118.96, 118.43, 116.45 (CH CH H ), 111.58, 111.49,
b
a
106.24, 52.55 (C5), 46.71 (C11a), 40.05 (NCH2), 22.44 (CHaHb).
IR: 3322.87 (–NH–), 1767.37, 1698.06 cmꢁ1 (–CO–). HPLC/MS (ESI):
Purity 100%, for C20H16BrN3O2S: m/z (%): 443 [Mþþ2], 441 [Mþ] (100%).
(5R,11aS) 5-(5-Bromo-2-thienyl)-2-allyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (19)
Yellow powder (0.37 g, 83.6%): Rf ¼ 0.28 (CH2Cl2); mp: 121–1238C;
1H NMR (500 MHz, DMSO-d6): d 10.91 (s, 1H, NH), 7.56 (d, J ¼ 8 Hz,
1H, Ar), 7.29 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.13–7.07 (m, 3H, Ar), 7.02
(ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.25 (s, 1H, NCHCS), 5.83–5.77
–
–
–
–
(m, 1H, CH CH H ), 5.16–5.14 (m, 1H, CH CH H ), 5.13–5.10
a
b
a b
(5S,11aS) 5-(5-Bromo-2-thienyl)-2-tert-butyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (16)
–
–
(m, 1H, CH CH H ), 4.58 (dd, J ¼ 11.5, 4.3 Hz, 1H, CHC(O)N),
a
b
4.00 (dt, J ¼ 5.0, 1.6 Hz, 2H, NCH2), 3.36–3.30 (m, 1H, CHaHb),
2.88 ppm (ddd, J ¼ 14.7, 11.6, 1.8 Hz, 1H, CHaHb). 13C NMR
(125 MHz, DMSO-d6): d 170.90, 153.93 (NCO), 146.04, 136.64,
133.49, 132.01, 129.53, 127.48, 125.68, 121.79, 118.96, 118.40,
Pale yellow powder (0.17 g, 37%): Rf ¼ 0.67 (CH2Cl2); mp: 206–
2078C; 1H NMR (500 MHz, DMSO-d6): d 11.09 (s, 1H, NH), 7.55–7.52
(m, 1H, Ar), 7.33 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.15–7.10 (m, 2H, Ar),
7.03 (ddd, J ¼ 7.9, 7.1, 1.0 Hz, 1H, Ar), 6.90 (dd, J ¼ 3.8, 0.7 Hz,
1H, Ar), 6.40 (s, 1H, NCHCS), 4.33 (dd, J ¼ 10.5, 5.5 Hz,
1H, CHC(O)N), 3.30 (dd, J ¼ 15.2, 5.7 Hz, 1H, CHaHb), 2.75
(ddd, J ¼ 15.2, 10.7, 1.8 Hz, 1H, CHaHb), 1.56 ppm (s, 9H, CH3).
13C NMR (125 MHz, DMSO-d6): d 173.05, 154.60 (NCO), 144.63,
136.59, 130.16, 130.12, 127.47, 125.57, 121.98, 118.94, 118.39,
111.56, 111.46, 106.21, 56.98 (C(CH3)3), 51.56 (C5), 46.37
(C11a), 28.31 ((CH3)3), 22.74 ppm (CHaHb). IR: 3333.69 (–NH–),
1761.99, 1693.57 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%,
for C21H20BrN3O2S: m/z (%): 459 [Mþþ2] (100%), 457 [Mþ].
–
–
116.31 (CH CH H ), 111.43, 111.18, 105.36, 57.80 (C5), 51.30
a
b
(C11a), 39.76 (NCH2), 21.77 ppm (CHaHb). IR: 3286.42 (–NH–),
1764.54, 1700.95 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%,
for C20H16BrN3O2S: m/z (%): 443 [Mþþ2], 441 [Mþ], 383 (100%).
(5S,11aS) 5-(5-Bromo-2-thienyl)-2-allyl-5,6,11,11a-
tetrahydro-1H-imidazo[10,5 0:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione (20)
Yellow powder (0.36 g, 82.5%); Rf ¼ 0.52 (CH2Cl2); mp 75–788C;
1H NMR (500 MHz, DMSO-d6): d 11.11 (s, 1H, NH), 7.55 (d, J ¼ 8 Hz,
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

32
D. S. El-Gamil et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
1H, Ar), 7.34 (dt, J ¼ 8.2, 0.9 Hz, 1H, Ar), 7.15–7.11 (m, 2H, Ar),
7.04 (ddd, J ¼ 7.9, 7.0, 1.0 Hz, 1H, Ar), 6.92 (dd, J ¼ 3.8, 0.7 Hz,
(6S,12aS) 6-(5-Bromo-2-thienyl)-2-ethyl-2,3,6,7,12,12a-
hexahydropyrazino[10,2 0:1,6]pyrido[3,4-b]indole-1,4-dione
(24)
–
1H, Ar), 6.47 (s, 1H, NCHCS), 5.87–5.79 (m, 1H, CH CH H ), 5.14–
a
–
b
–
–
–
5.12 (m, 1H, CH CH H ), 5.11–5.09 (m, 1H, CH CH H ), 4.55
a
–
b
a b
Yellow powder (0.32 g, 52.1%): Rf ¼ 0.26 (CH2Cl2/MeOH 99:1);
mp: 191–1948C; 1H NMR (500 MHz, CDCl3): d 11.15 (s, 1H, NH),
7.54–7.51 (m, 1H, Ar), 7.34 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.15–7.09
(m, 2H, Ar), 7.03 (ddd, J ¼ 8.0, 7.0, 1.0 Hz, 1H, Ar), 6.93 (s, 1H,
NCHCS) 6.70 (dd, J ¼ 3.8, 0.9 Hz, 1H, Ar), 4.28 (d, J ¼ 17.8 Hz,
1H, NC(O)CHaHb), 4.18 (dd, J ¼ 11.8, 4.3 Hz, 1H, CHC(O)N), 4.06
(d, J ¼ 17.8 Hz, 1H, NC(O)CHaHb), 3.46–3.39 (m, 1H, NCHCHaHb),
3.33–3.26 (m, 2H, NCH2CH3), 2.95 (ddd, J ¼ 15.4, 11.8, 1.5 Hz, 1H,
NCHCHaHb), 1.08 ppm (t, J ¼ 7.1 Hz, 3H, NCH2CH3). 13C NMR
(125 MHz, CDCl3): d 163.84, 162.46 (NCO), 143.23, 136.27,
129.98, 129.20, 128.21, 125.66, 121.93, 118.96, 118.30, 111.88,
111.42, 107.52, 52.27 (C6), 48.20 (NC(O)CHaHb), 47.04 (C12a),
40.14 (NCH2CH3), 26.53 (NCHCHaHb), 11.40 ppm (NCH2CH3).
IR: 3261.47 (–NH–), 1673.06, 1644.96 cmꢁ1 (–CO–). HPLC/MS
(ESI): Purity 100%, for C20H18BrN3O2S: m/z (%) 445 [Mþþ2], 443
[Mþ], 282 (100%).
(dd, J ¼ 11.1, 5.6 Hz, 1H, CHC(O)N), 4.04 (dt, J ¼ 5.3, 1.7 Hz,
2H, NCH2), 3.37 (dd, J ¼ 15, 5.5 Hz, 1H, CHaHb), 2.83 ppm
(ddd, J ¼ 15.1, 11.0, 1.7 Hz, 1H, CHaHb). 13C NMR (125 MHz,
DMSO-d6): d 171.85, 153.76 (NCO), 144.58, 136.62, 132.10,
130.17, 130.05, 127.50, 125.58, 122.03, 118.96, 118.43, 116.44
–
(CH CH H ), 111.62, 111.49, 106.24, 52.55 (C5), 46.71 (C11a),
a
–
b
40.5 (NCH2), 22.44 ppm (CHaHb). IR: 3319.27 (–NH–),
1766.89, 1697.98 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%,
for C20H16BrN3O2S: m/z (%): 443 [Mþþ2], 441 [Mþ], 242 (100%).
General procedure for the preparation of methyl 1-(5-
bromo-2-thienyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-
1H-b-carboline-3-carboxylates 21, 22
Chloroacetyl chloride (1 mL, 12.5 mmol) was added dropwise to
a well-stirred solution of the appropriate b-carboline 1, 4 (2 g,
5.13 mmol) and NaHCO3 (0.52 g, 6.19 mmol) in CHCl3 (40 mL)
under ice cooling. The mixture was then stirred at room tempera-
ture under a nitrogen atmosphere for 2 h. The mixture was
diluted with CH2Cl2, washed with a solution of NaHCO3, dried
over Na2SO4, and evaporated under reduced pressure. The resi-
due was then crystallized from diethyl ether. The product was
then used without further purification.
Methyl 1-(5-bromo-2-thienyl)-b-carboline-3-carboxylate
(25)
A well-stirred solution of the tetrahydro-b-carboline 1 (0.39 g,
1 mmol) in methyl ethyl ketone (10 mL) was refluxed under
oxygen atmosphere for 20 h. The solvent was evaporated to
dryness under reduced pressure and the product was then puri-
fied using flash column chromatography on silica gel, eluting
with CH2Cl2.
Yellow powder (0.28 g, 72.8%): Rf ¼ 0.65 (CH2Cl2); mp: 223–
2258C; 1H NMR (500 MHz, DMSO-d6): d 11.91 (s, 1H, NH), 8.86
(s, 1H, CHCN), 8.41 (dt, J ¼ 8.0, 1.0 Hz, 1H, Ar), 7.98 (d, J ¼ 4.0 Hz,
1H, Ar), 7.75 (dt, J ¼ 8.2, 0.9 Hz, 1H, Ar), 7.64 (ddd, J ¼ 8.2, 7.1,
1.2 Hz, 1H, Ar), 7.47 (d, J ¼ 4.0 Hz, 1H, Ar), 7.35 (ddd, J ¼ 8.0, 7.1,
1.0 Hz, 1H, Ar), 3.94 ppm (s, 3H, OCH3). 13C NMR (125 MHz,
DMSO-d6): d 165.35 (COOCH3), 144.55, 141.61, 136.13, 135.11,
131.90, 131.79, 130.14, 129.13, 127.15, 122.00, 120.92, 120.81,
116.75, 115.09, 112.91, 52.16 ppm (CH3). IR: 3326.68 (–NH–),
1739.34 cmꢁ1 (–CO–). HPLC/MS (ESI): Purity 100%, for
C17H11BrN2O2S: m/z (%): 388 [Mþþ2] (100%), 386 [Mþ].
General procedure for the preparation of 2-ethyl-6-(5-
bromo-2-thienyl)-2,3,6,7,12,12a-hexahydropyrazino-
[10,2 0:1,6]pyrido[3,4-b]indole-1,4-diones 23, 24
A
solution of the appropriate chloroethanone derivative
(1.4 mmol, 1 equiv, 0.65 g) and ethylamine (2.8 mmol, 2 equiv)
in methanol (25 mL) was heated to reflux under nitrogen atmos-
phere 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 flash
column chromatography eluting with CH2Cl2/MeOH (99.5:0.5).
Biology
Phosphodiesterase assay
(6S,12aR) 6-(5-Bromo-2-thienyl)-2-ethyl-2,3,6,7,12,12a-
hexahydropyrazino[10,2 0:1,6]pyrido[3,4-b]indole-1,4-dione
(23)
Five units per milliliter of purified PDE5 (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 and 50 nM fluorescein-cAMP
(Molecular Devices) were added to each assay well. The plates
were incubated for 1.5 h at 308C. 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 308C. FP was measured according to the
manufacturer’s specifications using a Biotek Synergy 4 plate
reader.
White powder (75 mg, 12%): Rf ¼ 0.27 (CH2Cl2/MeOH 99:1);
mp: 173–1758C; 1H NMR (500 MHz, CDCl3): d 11.09 (s, 1H, NH),
7.51 (d, J ¼ 7.9 Hz, 1H, Ar), 7.30 (dt, J ¼ 8.1, 0.9 Hz, 1H, Ar), 7.19
(ddd, J ¼ 8.2, 7.1, 1.2 Hz, 1H, Ar), 7.13 (ddd, J ¼ 8.0, 7.2, 1.0 Hz,
1H, Ar), 7.00 (s, 1H, NCHCS), 6.84 (d, J ¼ 3.8, 1H, Ar), 6.61
(dd, J ¼ 3.8, 0.8 Hz, 1H, Ar), 4.36 (dd, J ¼ 11.8, 4.2 Hz,
1H, CHC(O)N), 4.11 (d, J ¼ 17.8 Hz, 1H, NC(O)CHaHb), 3.99
(d, J ¼ 17.8 Hz, 1H, NC(O)CHaHb), 3.58–3.50 (m, 2H, NCH2CH3),
3.43–3.36 (m, 1H, NCHCHaHb), 2.91 (ddd, J ¼ 15.6, 11.8, 1.6 Hz,
1H, NCHCHaHb), 1.18 ppm (t, J ¼ 7.2 Hz, 3H, NCH2CH3). 13C NMR
(125 MHz, CDCl3): d 164.48, 161.84 (NCO), 142.24, 136.36,
129.47, 128.45, 128.31, 126.04, 123.06, 120.19, 118.64, 113.89,
111.27, 109.24, 57.60 (C6), 51.30 (C12a), 48.78 (NC(O)CHaHb),
41.05 (NCH2CH3), 27.45 (NCHCHaHb), 11.59 ppm (NCH2CH3).
IR: 3245.67 (–NH–), 1663.53, 1646.86 cmꢁ1 (–CO–). HPLC/MS
(ESI): Purity 100%, for C20H18BrN3O2S: m/z (%): 445 [Mþþ2], 443
[Mþ], 242 (100%).
Experimental design and data analysis
The IC50 value was determined by testing a range of ten concen-
trations 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 logis-
tic equation. All in vitro experiments involved dose–response
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 23–33
Tetrahydro-b-Carbolines as PDE5 Inhibitors
33
analysis, which was repeated at least twice to confirm reprodu-
cibility of IC50 values.
[12] R. R. Freedman, R. Girgis, M. D. Mayes, Lancet 1999, 354, 739.
[13] L. Zhang, R. L. Zhang, Y. Wang, C. Zhang, Z. G. Zhang,
H. Meng, M. Chopp, Stroke 2005, 36, 847–852.
Molecular modeling
[14] D. Puzzo, A. Staniszewski, S. X. Deng, L. Privitera, E. Leznik,
S. Liu, H. Zhang, Y. Feng, A. Palmeri, D. W. Landry,
O. Arancio, J. Neurosci. 2009, 29, 8075–8086.
To reveal the mode of interaction of 8 with the residues lining
the binding pocket of PDE5, a docking experiment was imple-
mented to dock compound 8 into the active site of PDE5 with the
program MOE version 2009.10. 8 was constructed into the MOE
window, the energy of the compound was minimized and the
minimized conformer was saved. The Protein Data Bank crystal
structure of PDE5 co-crystallized with tadalafil (1XOZ) was
imported into MOE. The structure was protonated and the bind-
[15] I. G. Ko, M. S. Shin, B. K. Kim, S. E. Kim, Y. H. Sung, T. S. Kim,
M. C. Shin, H. J. Cho, S. C. Kim, S. H. Kim, K. H. Kim, K. H. D.
H. Shin, C. J. Kim, Pharmacol. Biochem. Behav. 2009, 91, 629–
635.
[16] K. Han, N. Arber, H. Yamamoto, J. T. Lim, T. Delohery,
R. Pamukcu, G. A. Piazza, W. Q. Xing, I. B. Weinstein,
Breast Cancer Res. Treat. 1998, 48, 195–203.
˚
ing pocket was selected and extended 4.5 A around the pocket.
The tadalafil molecule was then removed and the ligand was
loaded into the MOE, where docking into the pocket was
launched. The poses from the ligand conformation were gener-
ated using alpha triangle, the scoring function used was London
dG with no refinement. To ensure more accurate docking pro-
cedures, tadalafil was redocked to the binding pocket using
the same MOE settings as for compound 8.
[17] J. D. Whitt, N. Li, H. N. Tinsley, X. Chen, W. Zhang, Y. Li, B. D.
Gary, A. B. Keeton, Y. Xi, A. H. Abadi, W. E. Grizzle, G. A.
Piazza, Cancer Prev. Res. (Phila.) 2012, 5, 822–833.
[18] P. R. Ding, A. K. Tiwari, S. Ohnuma, J. W. Lee, X. An, C. L. Dai,
Q. S. Lu, S. Singh, D. H. Yang, T. T. Talele, S. V. Ambudkar,
Z. S. Chen, PLoS ONE 2011, 6, e19329.
[19] G. L. Card, B. P. England, Y. Suzuki, D. Fong, B. Powell, B. Lee,
C. Luu, M. Tabrizizad, S. Gillette, P. N. Ibrahim, D. R. Artis,
G. Bollag, M. V. Milburn, S. H. Kim, J. Schlessinger, K. Y.
Zhang, Structure 2004, 12, 2233–2247.
The authors have declared no conflict of interest.
References
[20] R. Zoraghi, J. D. Corbin, S. H. Francis, J. Biol. Chem. 2006, 281,
5553–5558.
[1] S. H. Francis, I. V. Turko, J. D. Corbin, Prog. Nucleic Acid Res.
Mol. Biol. 2001, 65, 1–52.
[21] R. Zoraghi, S. H. Francis, J. D. Corbin, Biochemistry 2007, 46,
13554–13563.
[2] C. Mehats, C. B. Andersen, M. Filopanti, S. L. Jin, M. Conti,
Trends Endocrinol. Metab. 2002, 13, 29–35.
[22] H. A. Mohamed, N. M. Girgis, R. Wilcken, M. R. Bauer, H. N.
Tinsley, B. D. Gary, G. A. Piazza, F. M. Boeckler, A. H. Abadi,
J. Med. Chem. 2011, 54, 495–509.
[3] M. Conti, S. L. Jin, Prog. Nucleic Acid Res. Mol. Biol. 1999, 63, 1–38.
[4] S. H. Soderling, J. A. Beavo, Curr. Opin. Cell Biol. 2000, 12, 174–179.
[5] M. Conti, J. Beavo, Annu. Rev. Biochem. 2007, 76, 481–511.
[23] A. Dondoni, D. Perrone, Org. Syn. Coll. 2000, 10, 140.
[24] F. Ungemach, D. Soerens, R. Weber, M. DiPierro, O. Campos,
P. Mokry, J. M. Cook, J. V. Silverton, J. Am. Chem. Soc. 1980, 102,
6976–6984.
[6] J. Kotera, K. Fujishige, H. Akatsuka, Y. Imai, N. Yanaka,
K. Omori, J. Biol. Chem. 1998, 273, 26982–26990.
[7] A. T. Bender, J. A. Beavo, Neuochem. Int. 2004, 45, 853–857.
[25] J. Sung, K. Y. Hwang, Y. H. Jeon, J. I. Lee, Y. S. Heo, J. H. Kim,
J. Moon, J. M. Yoon, Y. L. Hyun, E. Kim, S. J. Eum, S. Y. Park,
J. O. Lee, T. G. Lee, S. Ro, J. M. Cho, Nature 2003, 425, 98–102.
[8] A. Briganti, A. Salonia, A. Gallina, A. Sacca, P. Montorsi,
P. Rigatti, F. Montorsi, Nat. Clin. Pract. Urol. 2005, 2, 239–247.
[26] A. H. Abadi, B. D. Gary, H. N. Tinsley, G. A. Piazza, M. Abdel-
Halim, Eur. J. Med. Chem. 2010, 45, 1278–1286.
[9] A. Sebkhi, J. W. Strange, S. C. Phillips, J. Wharton, M. R.
Wilkins, Circulation 2003, 107, 3230–3235.
[27] S. Tsuzuki, K. Honda, R. Azumi, J. Am. Chem. Soc. 2002, 124,
12200–12209.
[10] J. T. Shin, M. J. Semigran, Heart Fail. Clin. 2010, 6, 215–222.
[11] E. Takimoto, H. C. Champion, M. Li, D. Belardi, S. Ren, E. R.
Rodriguez, D. Bedja, K. L. Gabrielson, Y. Wang, D. A. Kass,
Nat. Med. 2005, 11, 214–222.
[28] O. Castellano, R. Gimon, H. Soscun, Energy Fuels 2011, 25,
2526–2541.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com