Discovery of tetrahydro-?-carboline derivatives as a new class of phosphodiesterase 4 inhibitors

Article abstract of DOI:10.1007/s00044-017-2011-x

Abstract: Phosphodiesterase 4 is the primary enzyme responsible for degradation of the second messenger cAMP in many of the cells releasing proinflammatory mediators. Inhibition of this enzyme could help in the management of various inflammatory conditions such as asthma, chronic obstructive pulmonary disorder, arthritis, and psoriasis. In this study, two novel series of tetrahydro-β-carbolines were designed by combining the pharmacophoric features of both tadalafil and piclamilast. Twenty-two compounds were synthesized and assessed for Phosphodiesterase 4 inhibition, four of them showed superior activity to the reference compound IBMX. Docking studies showed that the prepared compounds interact with the crucial Gln443 with variable interactions with the hydrophobic pocket Q2. This is the first report of tetrahydro-β-carbolines as a scaffold for Phosphodiesterase 4 inhibition. Currently, further optimization of the substituents is carried out to fine-tune the hydrophobic interactions and enhance the potency of this novel series of inhibitors.

Full text of DOI:10.1007/s00044-017-2011-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2011-x
ORIGINAL RESEARCH
Discovery of tetrahydro-ß-carboline derivatives as a new class of
phosphodiesterase 4 inhibitors
Ahmad Abdelwaly1 Ismail Salama1 Mohamed S. Gomaa1 Mohamed A. Helal1
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Received: 26 April 2017 / Accepted: 27 July 2017
© Springer Science+Business Media, LLC 2017
Graphical abstract
Abstract Phosphodiesterase 4 is the primary enzyme
responsible for degradation of the second messenger cAMP
in many of the cells releasing proinflammatory mediators.
Inhibition of this enzyme could help in the management of
various inflammatory conditions such as asthma, chronic
obstructive pulmonary disorder, arthritis, and psoriasis. In
this study, two novel series of tetrahydro-β-carbolines were
designed by combining the pharmacophoric features of both
tadalafil and piclamilast. Twenty-two compounds were
synthesized and assessed for Phosphodiesterase 4 inhibi-
tion, four of them showed superior activity to the reference
compound IBMX. Docking studies showed that the pre-
pared compounds interact with the crucial Gln443 with
variable interactions with the hydrophobic pocket Q2. This
is the first report of tetrahydro-β-carbolines as a scaffold for
Phosphodiesterase 4 inhibition. Currently, further optimi-
zation of the substituents is carried out to fine-tune the
hydrophobic interactions and enhance the potency of this
novel series of inhibitors.
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Keywords COPD Asthma PDE4 β-Carboline
Docking
Introduction
The cyclic nucleotide Phosphodiesterases (PDE) represent a
family of related phosphohydrolases responsible for the
hydrolysis and degradation of 3ʹ cyclic phosphate bonds of
adenosine and/or guanosine 3ʹ,5-cyclic monophosphate
(Sunden et al. 2016). Till date, 11 families of mammalian
PDEs have been identified, and they have been targeted for
the treatment of various diseases including asthma, chronic
obstructive pulmonary disease (COPD), cancer, Alzhei-
mer’s disease, Parkinson’s disease, and depression
(Krymskaya and Panettieri 2007; Garcia-Osta et al. 2012;
Otero et al. 2014). Of these families, PDE4 is specific to
cAMP and is highly expressed in immune and inflammatory
cells such as eosinophils, macrophages, T-lymphocytes, and
neutrophils. Inhibition of PDE4 was found to increase the
levels of intracellular cAMP, thus activating protein kinase
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2011-x) contains supplementary material,
which is available to authorized users.
* Mohamed A. Helal
mohamed_hilal@pharm.suez.edu.eg
1
Medicinal Chemistry Department, Faculty of Pharmacy, Suez
Canal University, Ismailia 41522, Egypt

Med Chem Res
A (also known as cAMP-dependent protein kinase) (Hei-
mann et al. 2010). This eventually results in a wide range of
anti-inflammatory effects in leukocytes. Therefore, PDE4
inhibitors are expected to be effective in the treatment of
certain inflammatory disorders such as asthma and chronic
obstructive pulmonary disease (COPD) (Parikh and Chak-
raborti 2016; Richmond et al. 2016).
within the enzyme. In this study, twenty-two diverse com-
pounds were prepared and their inhibitory profile on pur-
ified human PDE4B isoenzyme was determined.
Materials and methods
Chemistry
The first generation PDE4 inhibitor, rolipram, provided
the prototype molecule for a number of second generation
inhibitors such as roflumilast (Fig. 1) which has gained the
FDA approval for reducing COPD exacerbation in 2011
(Field 2011; Wang et al. 2012). Despite the extensive work
in this field, the therapeutic usefulness of many PDE4
inhibitors was limited owing to their adverse effects such as
nausea, vomiting, and vasculopathy. Originally, it was
assumed that the gastrointestinal side effects might be due
either to binding of the inhibitor at a high-affinity allosteric
binding site of PDE4, the so called high-affinity rolipram
binding site, or to the activation of emetic centers within the
central nervous system (Salari-Sharif and Abdollahi 2010;
Ulrik and Calverley 2010; Lahu et al. 2011). However,
recent research is highlighting the role of the receptor
subtypes where PDE4B seems to be the main subtype
mediating the anti-inflammatory activity, while PDE4D
mediates the gastrointestinal adverse effects through the
stimulation of neurons in the vomiting center of the area
postrema (De Savi et al. 2014; Fox et al. 2014). These
observations highlight the need for the development of
PDE4 inhibitors possessing a pronounced anti-inflammatory
activity with safer therapeutic window.
All solvents and reagents were used as received from the
chemical suppliers (Aldrich and Fisher). For air and
moisture sensitive reactions, solvents dried over molecular
sieves 4 Å and kept under argon atmosphere. Flash column
chromatography was performed using LobaChemie silica
gel (230–400 mesh) and was used as received. Melting
points (MP) were determined in open capillaries on an
Electrothermal IA9100 melting point apparatus and were
uncorrected.
Low-resolution mass spectra (MS) were recorded with a
Shimadzu QP2010-Plus gas chromatograph/mass spectro-
meter (GC/MS), electron impact (EI+) 70 eV maintained at
250 °C, equipped with Shimadzu workstation or with
electrospray ionization (ESI+) in positive ion mode with a
Waters Multi-mode ESCi mass detector. 1H- and 13C-NMR
were recorded at 400 MHz on a Bruker Ascend Aeon 400
(400 MHz) at room temperature within the range (20–25 °C).
Chemical shifts (δ) are expressed as parts per million (ppm),
using tetramethylsilane as the internal standard. For Signal
multiplicities, the following abbreviations are used as fol-
lows; s (singlet), d (doublet), dd (doublets of doublet), t
(triplet), q (quartet), br s (broad singlet) and m (multiplet).
Coupling constants (J values) are given in hertz (Hz).
Analytical high-performance liquid chromatography
(HPLC) was used for determination of the purity of the
prepared compounds using Shimadzu LC-20AD equipped
with a SHIM-PACK CLC-ODS column (4.6 mm × 250
mm) and a Shimadzu SPD-M20A. Two methods were
employed; method A: acetonitrile/water = 70/30 and
Herein, we report the design approach and synthesis of 2-
and 6-substituted 1,2,3,4-tertahydro-β-carbolines as novel
PDE4 inhibitors. The design was inspired by the polycyclic
scaffold of the potent PDE5 inhibitor tadalafil. We were
encouraged by the accessibility of the tetrahydro-β-
carboline nucleus via the well-studied Pictet-Spengler
reaction. Moreover, molecular docking simulation was uti-
lized to understand the mode of binding of these molecules
Fig. 1 Chemical Structures of
selected PDE4 inhibitors,
tadalafil (PDE5 inhibitor), and
the two designed series of
compounds

Med Chem Res
method B: methanol/water = 70/30. Isocratic HPLC mode
was used and the flow rate was 1.0 mL/min.
purification. MP: 113–116 °C. 1H-NMR (400 MHz,
CDCl3): δ 2.93–2.98 (t, 2H) 3.05–3.11 (t, 2H) 7.03 (s, 1H,
Ar–H) 7.13–7.19 (td, J = 7.52, 1.10 Hz, 1H, Ar–H) 7.23
(td, J = 7.52, 1.10 Hz, 1H, Ar–H) 7.36–7.40 (d, J = 7.82
Hz, Ar–H) 7.66 (d, J = 7.82 Hz, 1H, Ar–H) 8.56 (br. s., 1H,
NH). MS (EI+) m/z: 190 [M+].
1H-Indole-3-carbaldehyde (2)
In an oven dried two-neck round bottom flask purged with
argon at 0 °C, N,N-dimethylformamide (DMF) (1.5 ml) was
added, followed by POCl3 (0.5 ml, 5.65 mmol). The mix-
ture was stirred for 10 min before a solution of indole (0.5 g,
4.7 mmol) in DMF (3 ml) was added dropwise under argon.
After stirring for 2 h, 1N NaOH (35 ml) was added slowly,
upon which a white precipitate was formed. The precipitate
was washed with water and EtOAc yielding 0.6 g (97%).
This compound was used directly in the next reaction
without further purification. MP: 193–198 °C. 1H-NMR
(400 MHz, DMSO-d6): δ 7.19–7.30 (m, 2H), 7.52 (d, J =
8.07 Hz, 1H), 8.12 (d, J = 7.82 Hz, 1H), 8.29 (s, 1H),
9.92–9.99 (m, 1H, CHO), 12.14 (br. s., 1H, NH). MS (EI+)
m/z: 145 [M+]
2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indole (tetrehydro-
β-carboline) (5)
Formaldehyde solution 40% (0.2 ml) was added to a solu-
tion of compound 4 (0.2 g, 1.24 mmol) in 6 ml glacial acetic
acid and the mixture was then refluxed for 3 h. The reaction
mixture is then allowed to cool before aqueous ammonia
was added until the pH turned alkaline, upon which a pre-
cipitate was formed. The precipitate was extracted 3 times
with EtOAc. The solution is then washed with water (3 ×
30 ml) and once with brine (1 × 30 ml), the filtrate is dried
on anhydrous Na2SO4, evaporated under vacuum and pur-
ified by flash column chromatography on silica gel by
eluting with a mixture of DCM/methanol (80:20) to yield
3-(2-Nitrovinyl)-1H-indole (3)
1
89% a brown solid. MP: 198–203 °C; H-NMR (400 MHz,
In an oven dried round bottom flask, a mixture of 2 (0.6 g,
4.13 mmol), ammonium acetate (0.94 g, 15.9 mmol) and
nitromethane (12 ml) was refluxed for 1 h. The solvent was
then removed in vacuo and the residue was purified by flash
chromatography on silica gel eluting with a mixture of
DCM/hexane (90:10) to yield 0.73 g (94%) as a yellow
solid. MP: 155–158 °C. 1H-NMR (400 MHz, CDCl3): δ
7.33–7.41 (m, 2H, Ar–H) 7.50 (d, J = 6.36 Hz, 1H, Ar–H)
7.71 (d, J = 2.45 Hz, 1H, Ar–H) 7.80–7.86 (m, 2H, Ar–H)
8.32 (d, J = 13.45 Hz, 1H, Alkene-H). 13C-NMR (400
MHz, CDCl3): δ 109.78, 112.27, 120.57 (2 C), 122.67,
124.33, 132.27, 133.08 (1 C) 133.39 (2 C). MS (EI+) m/z:
188 [M+].
DMSO-d6): δ 2.61 (t, J = 5.50 Hz, 2H) 3.00 (t, J = 5.50 Hz,
2H) 3.25 (s., 2H) 6.90–6.96 (m, 1H, Ar–H) 6.97–7.03 (m,
1H, Ar–H) 7.27 (d, J = 7.82 Hz, 1H, Ar–H) 7.36 (d, J =
7.82 Hz, 1H, Ar–H) 10.67 (br. s, 1H, NH). 13C-NMR (400
MHz, DMSO-d6): δ 22.40, 42.96, 43.72, 107.34, 111.26,
117.64, 118.63, 120.72, 127.65, 134.25, 135.99. MS (EI+)
m/z: 172 [M+].
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(3-
(trifluoromethyl) phenyl) methanone (6a)
Under an inert argon atmosphere and at 0 °C, 3-(tri-
fluoromethyl)benzoyl chloride (0.21 ml, 1.44 mmol) is
added dropwise to a mixture of compound 5 (0.2 g, 1.16
mmol) and N,N-diisopropylethylamine (DIPEA) (0.3 ml,
1.72 mmol) in chloroform (6 ml). After stirring at room
temperature for 2 h, the reaction mixture was extracted
using chloroform (1 × 30 ml), saturated solution of ammo-
nium chloride (1 × 30 ml), and brine (1 × 30 ml). The
organic phase was dried over Na2SO4, concentrated in
vacou, and purified by flash column chromatography on
silica gel by eluting with a mixture of DCM/methanol
(98:2) to give the desired products (6a) as golden yellow
2-(1H-Indol-3-yl)ethenamine (tryptamine) (4)
Under an inert nitrogen atmosphere and at 0 °C, compound
3 (0.73 g, 3.88 mmol) was dissolved in 8 ml tetrahydrofuran
(THF) followed by slow addition of 1 M LiAlH4 solution in
THF (4.8 ml, 23.3 mmol). The mixture was allowed to
warm to room temperature and stirred for 36 h. The reaction
was then quenched by dropwise addition of a saturated
aqueous solution of Rochelle’s salt and the resultant
biphasic mixture was stirred for another 24 h. The two
layers were separated and the organic one was extracted
with 1M aqueous hydrochloric acid, while the aqueous
phase was basified with 3M aqueous potassium hydroxide
and extracted with diethyl ether, dried over Na2SO4, filtered
and concentrated under vacuo to furnish the desired com-
pound, as a brown crystalline powder with 79% yield,
which was used for the next step without further
1
solid. Yield: 75%. H-NMR(400 MHz, CDCl3): δ 2.89 (br.
s., 2H) 2.99 (br. s., 2H) 4.99 (br. s., 2H) 7.12–7.25 (m, 2H)
7.38 (d, 1H, Ar–H) 7.47–7.56 (m, 1H, Ar–H) 7.63 (d, 1H,
Ar–H) 7.70 (d, 1H, Ar–H) 7.73–7.83 (m, 2H, Ar–H) 8.08
(br. s., 1H, NH).13C-NMR (400 MHz, CDCl3): δ 22.24,
41.08, 45.29, 107.06, 110.85, 114.46, 115.78, 115.68,
115.87, 118.56, 122.13, 127.02, 129.2, 129.33, 132.31
132.12, 132.84, 136.31, 168.46. MS (EI+) m/z: 344 [M+].

Med Chem Res
HPLC purity: method A: 99.5%, UV 275 nm, retention time
2.15 min, method B: 99.0%, UV 275 nm, retention time
4.74 min.
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(3-
nitrophenyl)methanone (6e)
Compound 6e (0.33 g) was synthesized from 5 (0.2 g, 1.16
mmol) and benzoyl chloride (0.26 ml, 1.4 mmol) according
to the procedure used to synthesize 6a as yellow solid.
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(3-
fluorophenyl)methanone (6b)
1
Yield: 89%. H-NMR (400 MHz, CDCl3): δ 2.92 (br. s.,
2H) 3.76 (br. s., 2H) 5.00 (br. s., 2H) 7.16 (td, J = 7.40,
1.10 Hz, 1H) 7.22 (t, J = 7.40 Hz, 1H) 7.34–7.44 (m, 1H)
7.51 (d, J = 7.34 Hz, 1H) 7.69 (t, J = 7.58 Hz, 1H) 7.85 (t,
J = 7.58 Hz, 1H) 8.04–8.12 (m, 1H) 8.32–8.46 (m, 2H).
13C-NMR (400 MHz, CDCl3): δ 22.14, 41.19, 46.17,
111.07, 117.99, 119.92, 122.19, 124.77, 126.71, 129.99,
132.93, 136.27, 137.58, 148.04, 175.72.MS (EI+) m/z: 321
[M+].
Compound 6b (0.307 g) was synthesized from 5 (0.2 g,
1.16 mmol) and 3-fluorobenzoyl chloride (0.17 ml, 1.4
mmol) according to the procedure used to prepare 6a as off-
1
white solid. Yield: 89.7%. H-NMR (400 MHz, CDCl3): δ
2.88 (br. s., 2H) 3.75 (br. s., 2H) 4.97 (br. s., 2H) 7.13–7.17
(m, 2H) 7.19 (br. s., 1H) 7.21 (br. s., 1H) 7.23 (br. s., 1H)
7.35–7.39 (m, 2H) 7.42 (d, 1H), 8.14 (br. s., 1H, NH). 13C-
NMR (400 MHz, CDCl3): δ 22.09, 41.10, 46.01, 107.91,
111.08, 112.88, 114.14, 116.92, 118.24, 122.00, 122.57,
126.78, 129.70, 130.55, 136.48, 138.09, 163.86, 170.05.MS
(EI+) m/z: 294 [M+].
Cyclopropyl(3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-
yl)methanone (6f)
Compound 6f (0.24 g) was synthesized from 5 (0.2 g, 1.16
mmol) and cyclopropanecarbonyl chloride (0.126 ml, 1.38
mmol) according to the procedure used to synthesize 6a as
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(3,4-
dimethoxyphenyl) methanone (6c)
1
yellow solid. Yield: 85%. H-NMR (400 MHz, CDCl3): δ
0.86–0.97 (m, 2H) 1.05–1.17 (m, 2H) 1.89–2.02 (m, 1H)
2.93 (t, J = 5.62 Hz, 2H) 4.03 (t, J = 5.62 Hz, 2H)
4.82–4.92 (m, 2H) 7.08–7.16 (m, 1H) 7.17–7.23 (m, 1H)
7.34 (d, J = 7.82 Hz, 1H) 7.50 (d, J = 7.82 Hz, 1H) 8.91
(br. s., 1H, NH).13C-NMR (400 MHz, CDCl3): δ 7.09, 7.61,
8.82, 14.82, 22.22, 40.01, 44.18, 107.66, 111.13, 119.32,
122.11, 126.83, 127.43, 130.68, 136.46, 173.76. MS (EI+)
m/z: 240 [M+].
Compound 6c (0.36 g) was synthesized from 5 (0.2 g,
1.16 mmol) and 3,4-dimethoxybenzoyl chloride (0.28 g,
1.4 mmol) according to the procedure used to synthesize 6a
as off-white solid. Yield: 91.1%. 1H-NMR (400 MHz,
CDCl3): δ 2.91 (br. s., 2H) 3.89–3.97 (m, 8H, 2
O–CH3 and 2 CH2) 4.89 (br. s., 2H) 6.94 (d, 1H, Ar–H)
7.06–7.20 (m, 5H, Ar–H) 7.50 (d, 1H, Ar–H).13
C-NMR (400 MHz, CDCl3): δ 55.97, 55.98, 56.03,
56.04, 110.48, 111.08, 117.91, 119.39, 119.52, 119.59,
120.07, 121.84, 122.24, 126.82, 128.35, 130.15, 136.31,
149.01, 150.54, 167.15.MS (EI+)m/z: 336 [M+]. HPLC
purity: method A: 99.2%, UV 275 nm, retention time
1.92 min, method B: 99.1%, UV 254 nm, retention time
4.66 min.
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(4-
fluorophenyl)methanone (6g)
Compound 6g (0.300 g) was synthesized from 5 (0.2 g,
1.16 mmol) and 4-fluorobenzoyl chloride (0.17 ml, 1.4
mmol) according to the procedure used to synthesize 6a as
off-white solid. Yield: 88%. 1H-NMR (400 MHz, CDCl3): δ
2.90 (br. s., 2H) 3.77 (br. s., 2H) 4.95 (br. s., 2H) 7.12–7.16
(m, 2H, Ar–H) 7.16–7.18 (m, 2H, Ar–H) 7.19 (br. s., 1H,
Ar–H) 7.20 (s, 1H, Ar–H) 7.52 (br. s., 2H, Ar–H). 13C-
NMR (400 MHz, CDCl3): δ 22.14, 41.31, 46.21, 107.82,
111.10, 115.46, 115.68, 117.91, 119.67, 121.95, 126.79,
129.33, 129.92, 131.98, 132.60, 136.31, 162.38, 168.46.MS
(EI+) m/z: 294 [M+].
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(phenyl)
methanone (6d)
Compound 6d (0.278 g) was synthesized from 5 (0.2 g,
1.16 mmol) and 3,4-dimethoxybenzoyl chloride (0.16 ml,
1.4 mmol) according to the procedure used to synthesize 6a
as off-white solid. Yield: 86.72%. 1H-NMR (400 MHz,
CDCl3): δ 2.88 (br. s., 2H) 3.76 (br. s., 2H) 4.98 (br. s., 2H)
7.08–7.22 (m, 3H, Ar–H) 7.49 (br. s., 6H, Ar–H). 13C-NMR
(400 MHz, CDCl3): δ 23.84, 29.71, 41.52, 112.58, 120.95,
121.41, 125.59, 126.85, 127.50, 128.44, 128.60, 128.63,
129.96, 131.58, 132.78, 134.24, 137.61, 180.75. MS (EI+)
m/z: 276 [M+].
1-(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)
ethanone (6h)
Compound 6h (0.228 g) was synthesized from 5 (0.2 g,
1.16 mmol) and acetyl chloride (0.11 g, 1.4 mmol) accord-
ing to the procedure used to synthesize 6a as brownish

Med Chem Res
white solid. Yield: 92%. 1H-NMR (400 MHz, DMSO-d6): δ
2.11–2.15 (m, 3H) 2.67 (t, J = 5.62 Hz, 1H) 2.77 (t, J =
5.62 Hz, 1H) 3.75 (t, J = 5.62 Hz, 1H) 3.81 (t, J = 5.62 Hz,
1H) 4.67 (s, 2H) 6.94–7.00 (m, 1H, Ar–H) 7.01–7.08 (m,
1H, Ar–H) 7.29–7.34 (m, 1H, Ar–H) 7.40 (d, J = 7.83 Hz,
1H, Ar–H) 10.85 (br. s., 1H, NH). 13C-NMR (400 MHz,
DMSO-d6): δC ppm 21.11, 22.43, 39.93, 44.57, 107.10,
107.74, 111.48, 117.95, 118.98, 121.32,127.03, 131.77,
136.57, 169.61.MS (EI+) m/z: 214 [M+].
(t, J = 7.46 Hz, 1H, Ar-H) 7.36 (d, J = 8.07Hz, 1H, Ar-H)
7.48 (d, J = 8.07 Hz, 1H, Ar-H). 13C-NMR (400 MHz,
CDCl3): δ 17.91, 22.14, 25.33, 25.36, 37.87, 40.70, 43.37,
107.54, 111.10, 117.72, 119.36, 121.58, 126.77, 130.65,
136.88, 174.27. MS (EI+) m/z: 254 [M+].
Cyclohexyl(3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)
methanone (6l)
Compound 6l (0.267 g) was synthesized from 5 (0.2 g, 1.16
mmol) and cyclohexanecarbonyl chloride (0.223 ml, 1.389
mmol) according to the procedure used to synthesize 6a as
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(4-
methoxyphenyl)methanone (6i)
1
brownish white solid. Yield: 81.7%. H-NMR (400 MHz,
Compound 6i (0.228 g) was synthesized from 5 (0.2 g, 1.16
mmol) and 4-methoxybenzoyl chloride (0.195 ml, 1.4
mmol) according to the procedure used to synthesize 6a as
golden yellow solid. Yield: 90.4%. 1H-NMR (400 MHz,
CDCl3): δ 2.86 (d, J = 2.20 Hz, 2H), 3.82 (d, J = 2.20 Hz,
2H) 3.88 (s, 3H, O-CH3) 4.97 (br. s., 2H) 7.05 (br. s., 2H,
Ar–H) 7.11–7.14 (m, 1H, Ar–H) 7.16 (d, J = 3.91 Hz, 1H,
Ar–H) 7.17–7.21 (m, 1H, Ar–H) 7.39 (t, J = 7.95 Hz, 2H,
Ar–H) 7.49 (d, J = 7.95 Hz, 1H, Ar–H) 8.52 (br. s., 1H,
NH). 13C-NMR (400 MHz, DMSO-d6): δ 22.16, 41.10,
46.06, 55.47, 111.13, 112.21, 114.36, 115.98, 117.87,
119.60, 121.86, 126.81, 129.49, 129.82, 129.97, 130.96,
137.27, 159.74, 170.47. MS (EI+) m/z: 306 [M+].
CDCl3): δ1.21–1.28 (m, 2H) 1.50–1.59 (m, 2 × 2H) 1.68
(br. s., 2H) 1.78 (br. s., 2H) 2.58 (t, J = 11.37 Hz, 1H) 2.80
(br. s., 2H) 3.75 (t, J = 5.50 Hz, 2H) 4.76 (s, 2H) 6.98–7.03
(m, 1H, Ar–H) 7.07 (t, J = 7.46 Hz, 1H, Ar–H) 7.25 (d, J =
7.83 Hz, 1H, Ar–H) 7.37 (d, J = 7.83 Hz, 1H, Ar–H). 13C-
NMR (400 MHz, CDCl3): δ 22.51, 25.42, 25.75, 25.92,
28.93, 29.72, 40.82, 41.04, 43.82, 107.44, 111.13, 117.72,
119.34, 121.54, 126.77, 130.87, 136.35, 175.86. MS (EI+)
m/z: 282 [M+].
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(furan-3-
yl)methanone (6m)
To a solution of compound 5 (0.120 g, 0.69 mmol) in DCM
(5 ml), furan-3-carboxylic acid (0.094 g, 0.84 mmol), HOBt
(0.113 g, 0.84 mmol), EDCI (0.2 g, 1.04 mmol) and Et3N
(0.48 ml, 3.44 mmol) were added and the mixture was
stirred overnight at room temperature. The mixture was then
partitioned between DCM and saturated NaHCO3 aqueous
solution and the organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated in vacuo. The crude
residue was further purified by flash chromatography using
silica gel and eluting by a mixture of DCM/methanol (98:2)
to afford the required amides (6m) as a yellowish white
1-(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)butan-1-
one (6J)
Compound 6J (0.272 g) was synthesized from 5 (0.224 g,
1.30 mmol) and butyryl chloride (0.162 ml, 1.559 mmol)
according to the procedure used to synthesize 6a as
brownish white solid. Yield: 84%. 1H-NMR (400 MHz,
CDCl3): δ 0.81–1.02 (m, 3H) 1.69 (d, J = 6.60 Hz, 2H) 2.40
(br. s., 2H) 2.76 (br. s., 2H) 3.71 (br. s., 2H)4.74 (br. s., 2H)
7.01 (t, J = 7.21 Hz, 1H, Ar–H) 7.07 (t, J = 7.21 Hz, 1H,
Ar–H) 7.24 (d, J = 7.82 Hz, 1H, Ar–H) 7.38 (d, J = 7.82
Hz, 1H, Ar–H) 8.79 (br. s., 1H, NH). 13C-NMR (400 MHz,
CDCl3): δ 14.04, 18.88, 22.10, 36.01, 40.17, 44.03, 107.45,
110.97, 117.73, 119.34, 121.56, 126.77, 130.72, 136.37,
172.68. MS (EI+) m/z: 242 [M+].
1
solid. Yield: 92%. H-NMR (400 MHz, DMSO-d6): δ 2.81
(br. s., 2H) 3.34 (s, 2H) 4.80 (br. s., 2H) 6.72–6.77 (m, 1H,
Ar–H) 6.98 (t, J = 7.34 Hz, 1H, Ar–H) 7.06 (t, J = 7.34,
Hz, 1H, Ar–H) 7.32 (d, J = 7.58 Hz, 1H, Ar–H) 7.41 (d, J
= 7.58 Hz, 1H, Ar–H) 7.80 (s, 1H, Ar–H) 8.14 (s, 1H,
Ar–H). 13C-NMR (400 MHz, DMSO-d6): δ 22.24, 40.15,
55.36, 110.78, 111.56, 118.02, 119.05, 121.35 126.98,
131.25, 136.49, 144.06, 164.05. MS (EI+) m/z: 266 [M+].
Cyclobutyl(3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)
methanone (6k)
Compound 6k (0.272 g) was synthesized from 5 (0.22 g,
1.27 mmol) and cyclobutanecarbonyl chloride (0.176 ml,
1.542 mmol) according to the procedure used to synthesize
6a as an off-white solid. Yield: 92.2%. 1H-NMR (400 MHz,
CDCl3): δ1.99–2.11 (m, 2H) 2.20–2.31 (m, 2H) 2.38–2.54
(m, 2H) 2.84 (t, J = 5.14 Hz, 1H) 3.44 (br. s., 1H) 3.72 (br.
s., 2H) 4.83 (br. s., 2H) 7.12 (t, J = 7.46 Hz, 1H, Ar-H) 7.18
1-(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2-(1H-
indol-3-yl)ethanone (6n)
Compound 6n (0.145 g) was synthesized from 5 (0.120 g,
0.69 mmol) and indole-3-acetic acid (0.146 g, 0.836 mmol)
according to the procedure used to synthesize 6m as a white
1
solid. Yield: 63%. H-NMR (400 MHz, DMSO-d6): δ 2.59

Med Chem Res
(br. s., 1H) 3.86 (br. s., 1H) 3.92 (s, 1H) 4.70 (s, 1H)
6.90–7.12 (m, 5H, Ar-H) 7.25 −7.44 (m, 3H, Ar–H) 7.60
(d, J = 8.07 Hz, 1H, Ar–H) 10.84 (s, 1H, NH) 10.91 (br. s.,
1H, NH). 13C-NMR (400 MHz, DMSO-d6): δ 21.17, 31.51,
40.61, 44.38, 107.02, 107.71, 111.45, 111.81, 117.70,
117.92, 118.87, 118.92, 121.17, 121.52, 124.01, 126.96,
127.65, 131.39, 131.77, 136.60, 170.55. MS (EI+) m/z: 329
[M+].
2H) 3.89–3.92 (m, 2H) 4.73–4.80 (m, 2H) 6.97–7.03 (m,
1H, Ar–H) 7.05–7.10 (m, 1H, Ar–H) 7.25–7.30 (m, 2H,
Ar–H) 7.31–7.38 (m, 4H, Ar–H) 7.38–7.46 (m, 1H, Ar–H)
10.90 (s, 1H, NH). 13C-NMR (400 MHz, DMSO-d6): δ
21.95, 40.14, 40.56, 44.32, 107.02, 111.48, 117.95, 118.98,
121.24, 126.89, 126.95, 128.79, 128.83, 129.42, 129.45,
131.63, 136.36, 136.37, 170.18. MS (EI+)m/z: 290 [M+].
1-Methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-6-ol
(8)
(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)(thiophen-
3-yl)methanone (6o)
In a well oven dried round bottom flask and under inert
atmosphere, glacial acetic acid (2 ml) was added to a
solution of 3-(2-aminoethyl)-1H-indol-5-ol hydrochloride 7
(0.2 g) in 7 ml anhydrous ethanol. Acetaldehyde (0.2 ml)
was then added and the mixture was refluxed for 2 h,
making sure that the reaction was well-protected from air
and light. The mixture was then allowed to cool and ethanol
was evaporated completely to produce the salt, which was
then dissolved in water and adjusted to alkaline pH 9 using
aqueous ammonia. The mixture was then extracted using
ethyl acetate (3 × 30 ml) and washed with brine (1 × 30
ml). The organic phase was then dried over Na2SO4, fil-
tered, and concentrated in vacuo to yield 0.170 g (74%
yield) of the target compound 8 as a white powder, which
was used for the next step without further purification. MP:
Compound 6o (0.151 g) was synthesized from 5 (0.120 g,
0.69 mmol) and 3-thiophenecarboxylic acid (0.107 g, 0.834
mmol) according to the procedure used to synthesize 6m as
1
a yellow solid. Yield: 77%. H-NMR (400 MHz, DMSO-
d6): δ 2.77–2.88 (m, 2H) 3.79 (br. s., 1H) 3.99 (br. s., 1H)
4.83 (br. s., 2H) 6.99–7.05 (m, 1H) 7.09 (t, J = 7.58 Hz, 1H,
Ar–H) 7.30–7.39 (m, 2H, Ar–H) 7.45 (d, J = 7.58 Hz, 1H,
Ar–H) 7.69 (dd, J = 4.89, 1.71 Hz, 1H, Ar–H) 7.92 (d, J =
1.71 Hz, 1H, Ar–H) 10.96 (br. s., 1H, NH). 13C-NMR (400
MHz, DMSO-d6): δ 22.19, 41.02, 45.91, 107.20, 111.55,
118.04, 119.08, 121.38, 126.98, 127.24, 127.64, 131.18,
136.47, 137.00, 166.04. MS (EI+) m/z: 282 [M+]. HPLC
purity: method A: 99.0%, UV 275 nm, retention time 2.03
min, method B: 99.5%, UV 274 nm, retention time 3.88
min.
1
183–187 °C. H-NMR (400 MHz, DMSO-d6): δ 1.33 (d, J
= 6.60 Hz, 3H) 2.37–2.49 (m, 1H) 2.77–2.87 (m, 1H)
3.09–3.20 (m, 2H) 3.98 (q, J = 6.60 Hz, 1H) 6.52 (dd, J =
8.44, 2.32 Hz, 1H, Ar–H) 6.66 (d, J = 1.96 Hz, 1H, Ar–H)
7.05 (d, J = 8.44 Hz, 1H, Ar–H) 10.33 (s, 1H, NH). MS
(EI+) m/z: 202 [M+].
Cyclopentyl(3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-
yl)methanone (6p)
Compound 6p (0.145 g) was synthesized from 5 (0.120 g,
0.69 mmol) and cyclopentane carboxylic acid (0.091 ml,
0.838 mmol) according to the procedure used to synthesize
6m as white solid. Yield: 78%. 1H-NMR (400 MHz,
CDCl3): δ 1.63–1.73 (m, 4H) 1.77–1.90 (m, 2H) 1.92–1.99
(m, 2H) 2.90 (t, J = 5.62 Hz, 2H) 3.10 (t, J = 7.95 Hz, 1H)
3.89 (t, J = 5.62 Hz, 2H) 4.88 (s, 2H) 7.09–7.15 (m, 1H,
Ar–H) 7.18 (t, J = 7.46 Hz, 1H, Ar–H) 7.35 (d, J = 7.82 Hz,
1H, Ar–H) 7.49 (d, J = 7.82 Hz, 1H, Ar–H) 8.68 (br. s., 1H,
NH). 13C-NMR (400 MHz, CDCl3): δ 22.33, 26.11, 26.14,
30.14, 30.42, 40.95, 41.51, 43.90, 107.55, 110.89, 117.73,
119.35, 121.56, 126.81, 130.84, 136.34, 175.75. MS (EI+)
m/z: 268 [M+].
Tert-butyl 6-hydroxy-1-methyl-3,4-dihydro-1H-pyrido
[3,4-b]indole-2(9H)-carboxylate (9)
In a well oven dried round bottom flask and under argon,
compound 8 (0.170 g, 0.84 mmol) was dissolved in 5 ml
anhydrous THF before the addition of TEA (0.13 ml, 0.92
mmol) and di-tert-butyldicarbonate (0.21 g, 0.92 mmol).
The reaction was stirred protected from light for 3 h. The
solvent was then evaporated in vacuum and the residue was
purified by flash column chromatography using silica gel
and eluting by a mixture of DCM/methanol (98:2) to afford
0.240 g (95% yield) of the target compound 9 as a yellowish
white powder. MP: 110–115 °C. 1H-NMR (400 MHz,
CDCl3): δ 1.33 (d, 3H) 1.44 (s, 9H) 2.42–2.52 (t, 2H)
2.56–2.67 (t, 2H) 4.19 (q, 1H) 6.64 (d, J = 7.82 Hz, 1H,
Ar–H) 6.79–6.82 (m, 1H, Ar–H) 6.99 (br. s., 1H, Ar–H).
13C-NMR (400 MHz, CDCl3): δ 19.52, 27.44, 28.54, 80.02,
103.06, 111.38, 119.63, 127.64, 131.22, 149.63, 154.69,
172.12, 192.32, 212.60. MS (EI+) m/z: 302 [M+].
1-(3,4-Dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2-
phenylethanone (6q)
Compound 6q (0.175 g) was synthesized from 5 (0.150 g,
0.871 mmol) and phenylacetic acid (0.142 g, 1.043 mmol)
according to the procedure used to synthesize 6m as yellow
solid. Yield: 70%.1H-NMR (400 MHz, DMSO-d6): δ 2.64
(t, J = 5.14 Hz, 1H) 2.73 (t, J = 5.14 Hz, 1H) 3.85–3.89 (m,

Med Chem Res
Tert-butyl 6-(benzyloxy)-1-methylα,4-dihydro-1H-
pyrido[3,4-b]indole-2(9H)-carboxylate (10a)
Tert-butyl 6-((3-fluorobenzyl)oxy)-1-methyl-3,4-
dihydro-1H-pyrido[3,4-b]indole-2(9H)-carboxylate
(10d)
In a well oven dried round bottom flask and under argon,
Cs2CO3 (0.550 g, 1.69 mmol) was added to a solution of
compound 9 (0.170 g, 0.562 mmol) in anhydrous DMF
(5 ml) and benzyl chloride (0.064 ml, 0.562 mmol) and
the mixture was heated on stirrer at 95°C for 2 h. The
solution was partitioned between water and ethyl acetate
(3 × 30 ml), washed with brine (1 × 30 ml), and the organic
phase was evaporated in vacuo. The resultant residue was
then purified by flash column chromatography using silica
gel and eluting with hexane/ethyl acetate (85:15) to yield
compound 10a (0.090 g) as a colorless sticky oil. Yield:
Compound 10d (0.097 g) was synthesized from 9 (0.170 g,
0.562 mmol) and 1-(chloromethyl)-3-fluorobenzene (0.067 ml,
0.562 mmol) according to the procedure used to synthesize
1
10a as colorless sticky oil. Yield: 42.17%. H-NMR (400
MHz, CDCl3): δ 1.38 (dd, J = 17.36, 6.60 Hz, 3H) 1.52 (s,
9H) 2.69–2.77 (m, 2H) 3.16–3.35 (m, 2H) 4.32 –4.52 (m,
1H)5.12 (s, 2H) 6.69 (d, J = 9.41 Hz, 1H, Ar–H) 6.81 (t, J
= 8.93 Hz, 1H, Ar–H) 6.88 (t, J = 9.41 Hz, 1H, Ar–H) 7.03
(d, J = 2.20 Hz, 1H, Ar–H) 7.04–7.09 (m, 1H, Ar–H)7.25
(s, 1H, Ar–H) 7.32–7.40 (m, 1H, Ar–H). 13C-NMR (400
MHz, CDCl3): δ 19.53, 21.68, 28.43, 46.54, 64.52, 70.20,
79.97, 102.37, 114.18, 114.40, 114.49, 114.71 121.55,
122.16, 122.78, 129.99, 130.07, 140.26, 140.32, 153.21,
161.79, 164.46. MS (EI+) m/z: 410 [M+].
1
51.4%. H-NMR (400 MHz, CDCl3): δHppm 1.37 (d, 3H)
1.49 (s, 9H) 2.77 (m, 2H) 2.84 (m, 2H)4.32 −4.52 (m, 1H)
5.12 (s, 2H) 6.88–6.91 (m, 1H, Ar–H) 7.34–7.40 (m, 1H,
Ar–H) 7.50–7.57 (m, 1H, Ar–H) 7.55–7.61 (m, 1H, Ar–H)
7.66 (d, J = 7.58 Hz, 1H, Ar–H) 7.75 (s, 1H, Ar–H). MS
(EI+) m/z: 392 [M+].
Tert-butyl 6-(cyclopropylmethoxy)-1-methyl-3,4-
dihydro-1H-pyrido[3,4-b]indole-2(9H)-carboxylate (10e)
Tert-butyl 6-(2-chlorobenzyloxy)-1-methyl-3,4-dihydro-
1H-pyrido[3,4-b]indole-2(9H)-carboxylate (10b)
Compound 10e (0.144 g) was synthesized from 9 (0.170 g,
0.562 mmol) and (Bromomethyl)cyclopropane (0.06 ml,
0.562 mmol) according to the procedure used to synthesize
10a as colorless sticky oil. Yield: 72%. 1H-NMR (400
MHz, CDCl3): δHppm 1.37 (d, 3H) 1.49 (s, 9H) 2.77 (m,
2H) 2.84 (m, 2H) 4.32 −4.52 (m, 1H) 5.12 (s, 2H)
6.88–6.91 (m, 1H, Ar–H) 7.75 (s, 1H, Ar–H) 7.17–7.23 (m,
1H, Ar–H) 7.32 (m, 1H, Ar–H)7.34–7.40 (m, 1H, Ar–H)
7.50–7.57 (m, 1H, Ar–H) 7.55–7.61 (m, 1H, Ar–H) 7.66 (d,
J = 7.58 Hz, 1H, Ar–H). MS (EI+) m/z: 356 [M+].
Compound 10b (0.113 g) was synthesized from 9 (0.170 g,
0.562 mmol) and 1-(chloromethyl)-3-chlorobenzene (0.064
ml, 0.562 mmol) according to the procedure used to syn-
1
thesize 10a as colorless sticky oil. Yield 38%. H-NMR
(400 MHz, CDCl3): δ 1.26–1.39 (m, 3H) 1.52 (s, 9H)
2.71–2.81 (m, 2H) 2.90 (m, 1H) 3.27 (d, J = 13.20 Hz, 1H)
4.31–4.50 (m, 1H) 5.23 (s, 2H) 6.35–6.44 (m, 1H, Ar–H)
6.99 (d, J = 8.56 Hz, 1H, Ar–H) 7.02–7.09 (m, 1H, Ar–H)
7.12 (br. s., 1H, Ar–H) 7.32 (td, J = 7.46, 1.71 Hz, 2H,
Ar–H) 7.41–7.46 (m, 1H, Ar–H) 7.66 (dd, J = 7.34, 1.71
Hz, 1H, Ar–H). MS (EI+) m/z: 426 [M+].
6-(Benzyloxy)-1-methyl-2,3,4,9-tetrahydro-1H-pyrido
[3,4-b]indole (11a)
In an oven dried round bottom flask and under argon at 0°C,
TFA (0.42 ml) was added to a solution of the corresponding
compound 10a (0.090 g) in 2 ml anhydrous DCM. The
mixture was stirred for 30 min and when the reaction was
finished, saturated Na2CO3 was added to the solution until
the pH turned alkaline. Then, the organic layer was sepa-
rated, washed with water, dried over Na2SO4, and evapo-
rated under vacuum to give 0.073 g of a yellow solid. Yield:
Tert-butyl 1-methyl-6-((3-(trifluoromethyl)benzyl)oxy)-
3,4-dihydro-1H-pyrido[3,4-b]indole-2(9H)-carboxylate
(10c)
Compound 10c (0.110 g) was synthesized from 9 (0.170 g,
0.562 mmol) and 1-(chloromethyl)-3-(trifluoromethyl) ben-
zene (0.087 ml, 0.562 mmol) according to the pro-
cedure used to synthesize 10a as colorless sticky oil. Yield:
1
87%. H-NMR (400 MHz, CDCl3): δ 1.43 (d, J = 6.60 Hz,
42.6%. 1H-NMR (400 MHz, CDCl3):
δ
1.32–1.47
3H) 2.72 (t, d, J = 8.62, 5.50 Hz, 2H) 3.09–3.24 (m, 2H)
4.14 (d, J = 6.60 Hz, 1H) 5.00–5.05 (s, 2H) 6.78 (dd, J =
8.80, 2.20 Hz, 1H, Ar–H) 6.85–6.91 (m, 1H, Ar–H) 6.94 (d,
J = 8.80 Hz, 1H, Ar–H) 6.99 (d, J = 2.20 Hz, 1H, Ar–H)
7.11–7.21 (m, 3H, Ar–H) 7.22–7.27 (m, 1H, Ar–H).
13C-NMR (400 MHz, CDCl3): δ 20.88, 22.25, 37.55, 46.48,
70.99, 102.13, 107.46, 110.34, 112.12, 125.87, 127.33,
(d, 3H) 1.52 (s, 9H) 2.69–2.80 (m, 2H) 2.89 (d, 2H)
4.32–4.52 (m, 1H) 5.18 (s, 2H) 6.84–6.93 (m, 1H, Ar–H)
7.05–7.12 (m, 1H, Ar–H) 7.37–7.44 (m, 1H, Ar–H)
7.50–7.57 (m, 1H, Ar–H) 7.57–7.63 (m, 1H, Ar–H) 7.68 (d,
J = 7.58 Hz, 1H, Ar–H) 7.78 (s, 1H, Ar–H). MS (EI+) m/z:
460 [M+].

Med Chem Res
127.53, 127.76, 128.51, 128.79, 132.38, 137.70, 137.75,
1H, Ar-H) 7.01 (t, J = 7.43 Hz, 1H, Ar–H)7.23 (d, J = 7.43
Hz, 1H, Ar–H) 7.55 (d, J = 7.07 Hz, 1H, Ar–H). 13C-NMR
(400 MHz, CDCl3): δ 20.90, 22.24, 44.87, 46.49, 68.09,
102.36, 107.89, 110.18, 112.21, 126.94, 127.29, 127.48,
128.96, 129.30, 131.76, 132.31, 135.02, 138.11, 153.26.
MS (EI+) m/z: 310 [M+]. HPLC purity: method A: 99.5%,
UV 254 nm, retention time 4.65 min, method B: 98.9%, UV
275 nm, retention time 2.15 min.
150.06. MS (EI+) m/z: 292 [M+].
6-((2-Chlorobenzyl)oxy)-1-methyl-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole (11b)
Compound 11b was prepared from 10b following the same
procedure used for the synthesis of 11a to give 0.040 g of
compound 11a as a white solid. Yield: 58.8%. MP:
1
149–155 °C. H-NMR (400 MHz, CDCl3): δ 1.22 (d, J =
6-(Cyclopropylmethoxy)-1-methyl-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole (11e)
6.60 Hz, 3H) 2.60–2.71 (m, 2H) 3.01–3.19 (m, 2H) 4.04 (q,
J = 6.60 Hz, 1H) 5.04–5.18 (m, 2H) 6.21 (d, J = 7.58 Hz,
1H, Ar–H) 6.75 (d, J = 8.80 Hz, 1H, Ar–H) 6.85 (d, J =
8.80 Hz, 1H, Ar–H) 6.90 (t, J = 7.46 Hz, 1H, Ar–H) 7.04 (t,
J = 7.46 Hz, 1H, Ar–H)7.26 (d, J = 7.46 Hz, 1H, Ar–H)
7.51 (d, J = 7.09 Hz, 1H, Ar–H). 13C-NMR (400 MHz,
CDCl3): δ 20.90, 22.24, 44.87, 46.49, 68.09, 102.36,
107.89, 110.18, 112.21, 126.94, 127.29, 127.48, 128.96,
129.30, 131.76, 132.31, 135.02, 138.11, 153.26. MS
(EI+) m/z: 356 [M+]. HPLC purity: method A: 99.2%, UV
276 nm, retention time 4.89 min, method B: 95.1%, UV
275 nm, retention time 3.49 min.
Compound 11e was prepared from 10e following the same
procedure used for the synthesis of 11a to give 0.063 g of a
colorless sticky oil. Yield: 61%. 1H-NMR (400 MHz,
CDCl3): δ 0.16–0.25 (m, 2H) 0.45–0.53 (m, 2H) 1.08–1.14
(m, 1H) 1.22–1.32 (m, 3H) 1.43 (s, 1H, NH) 2.80- 2.90 (m,
2H) 3.11–3.22 (m, 2H) 3.67 (d, J = 7.09 Hz, 2H) 3.97–4.04
(m, 1H) 6.57–6.69 (m, 1H, Ar–H) 6.70–6.81 (m, 1H, Ar–H)
7.01–7.06 (m, 1H, Ar–H). 13C-NMR (400 MHz, CDCl3):
δ 3.16, 3.19, 10.54, 41.95, 48.51, 83.18, 101.79, 107.29,
111.65, 112.33, 127.38, 131.04, 153.39. MS (EI+) m/z: 256
[M+]. HPLC purity: method A: 98.8%, UV 275 nm,
retention time 1.90 min, method B: 96.0%, UV 276 nm,
retention time 2.70 min.
1-Methyl-6-((3-(trifluoromethyl)benzyl)oxy)-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole (11c)
Compound 11b was prepared from 10b following the same
procedure used for the synthesis of 11a to give a colorless
sticky oil. Yield: 87.2%. H-NMR (400 MHz, CDCl3): δ
Biological evaluation
1
All the synthesized 22 tetrahydro-β-carboline compounds
were tested for their in vitro inhibitory activity against
PDE4B enzyme by detecting the reaction product, AMP, by
Transcreener Fluorescence Polarization assay. Compounds
were tested in a 5-dose IC50 assay with 10-fold serial
dilution starting at 100 μM and the fold decrease of the
cAMP level caused by the test compounds over DMSO
control was determined. Three types of controls were tested
to make sure the assay is consistent from batch to batch.
The DMSO only reactions are used as full activity control.
The EDTA only reactions are used as the full no activity
control. In addition, one standard control compound’s 10-
dose IC50 assay for every target in every assay was added,
which in our case, was the non-selective PDE inhibitor,
isobutyl methyl xanthine (IBMX). Control Compound
IBMX was tested in a 10-dose IC50 with 3-fold serial
dilution starting at 100 uM.
1.44–1.49 (d, 3H) 2.82–2.90 (m, 1H) 2.96 (d, J = 7.83 Hz,
1H) 3.35 (dd, J = 7.83, 4.40 Hz, 2H) 4.43 (d, J = 7.83 Hz,
1H) 5.17 (s, 2H) 6.98 (s, 1H, Ar–H) 7.00 (s, 1H, Ar–H)
7.08 (d, J = 2.45 Hz, 1H, Ar-H) 7.53 (dd, J = 7.82, 4.77 Hz,
1H, Ar-H) 7.60 (d, J = 7.82 Hz, 1H, Ar-H) 7.68 (d, J =
7.82 Hz, 1H, Ar–H) 7.77 (s, 1H, Ar–H). 13C-NMR (400
MHz, CDCl3): δ 20.90, 22.14, 38.80, 46.38, 46.56, 70.24,
102.49, 107.94, 110.25, 112.23, 122.66, 122.82, 124.33,
124.58, 125.32, 127.65, 129.00, 129.48, 130.71, 132.35,
138.25, 138.72, 153.22. MS (EI+) m/z: 360 [M+]. HPLC
purity: method A: 96.0%, UV 275 nm, retention time 4.23
min, method B: 99.0%, UV 276 nm, retention time 9.31
min.
6-((3-Fluorobenzyl)oxy)-1-methyl-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole (11d)
Compounds were sent in powder form at room tem-
perature, 2 mg each, and the exact molecular weight and
exact weight of each compound were given to the service
providers, who re-suspend them in DMSO. The reaction
was carried out at room temperature using 1 μM AMP
(substrate). The buffer used for the assay consisted of 10
mM Tris, pH 7.5, 5 mM MgCl2, 0.01% Brij 35, 1 mM DTT
(Dithiothreitol) and 1% DMSO. The procedure involved
Compound 11c was prepared from 10c following the same
procedure used for the synthesis of 11a to give 0.057 g of a
colorless sticky oil. Yield: 78.1%. 1H-NMR (400 MHz,
CDCl3): δ 1.23 (d, J = 6.58 Hz, 3H) 2.66–2.74 (m, 2H)
3.08–3.14 (m, 2H) 4.05 (q, J = 6.58 Hz, 1H) 5.08 (s., 2H)
6.20 (d, J = 7.55 Hz, 1H, Ar–H) 6.73 (d, J = 8.78 Hz, 1H,
Ar–H) 6.82 (d, J = 8.78 Hz, 1H, Ar–H) 6.88 (t, J = 7.43 Hz,

Med Chem Res
adding the compounds in DMSO to enzyme by using
Acoustic Technology followed by the addition of the sub-
strate. The mixture was then incubated for 1 h. The reaction
was halted by the addition of Stop/detection mixture and the
FP (fluorescence polarization) was measured after 90 min
incubation at room temperature and the polarization was
calculated in mP (millipolarization) units.
active site metal ion. In addition, the rigid chemical struc-
ture of its β-carboline scaffold has also contributed to its
high binding affinity since it loses less entropy upon protein
binding compared to other PDE ligands.
Several High-resolution crystal structures of the catalytic
domains of various phosphodiesterases have been published
(Huai et al. 2003; D’Ursi et al. 2016). The active site of
PDE4 can be subdivided into three pockets: (1) a metal
binding pocket (M), which contains two metal ions, Zn2+
and Mg2+; (2) a solvent-filled pocket (S), which consists of
hydrophilic amino acids and is filled with water molecules;
and (3) a pocket containing the invariant glutamine residue
and the hydrophobic clamp (Q) which constitutes an
essential binding site that anchors the ligand within the
active site of the enzyme. All known inhibitors co-
crystallized with PDE4B primarily utilize this pocket for
intermolecular interactions. The Q-pocket can be sub-
divided into two narrow but deep hydrophobic pockets (Q1
and Q2). The Q1 pocket is small and points away from the
S-pocket, whereas the Q2 pocket is larger and adjacent to
the S-pocket (Card et al. 2004; Fox et al. 2014). The
selectivity of tadalafil for PDE5 over PDE4 is attributed to
its bulky methylenedioxyphenyl group (Fig. 2). This sub-
stituent does not fit into the smaller Q2 pocket of PDE4 and
Docking
Compounds were built and energy-minimized using in
Sybyl X.1 with 2000 steps of the conjugate gradient algo-
rithm using the MMFF force field to a gradient of 0.001
kcal/Å (Cheng et al. 2000). CCDC GOLD 4.12 software was
used for docking of the minimized compounds into the
active site of the X-ray crystal structures of PDE4 (PDB ID:
3D3P) (Jones et al. 1997). This software uses a genetic
algorithm to determine potential ligand binding modes by
changing dihedrals of ligand rotatable bonds, ligand ring
geometries, and dihedrals of protein OH and NH2 groups
(Helal et al. 2013). The binding site was defined to include
all amino acid residues within 10 Å of the side chain oxygen
of Gln443 with H bond constraint to its side chain carbonyl.
Docking was carried out using the standard mode settings
and GoldScore,
a molecular mechanics-like function
(Annamala et al. 2007). A total of 10 genetic algorithm runs
were performed for each ligand. A value of 1.5 Å root-mean-
square deviation was set as the early termination criteria.
Results and discussion
Compounds design
Compounds possessing the tetrahydro-β-carboline scaffold
have been isolated from several plants and marine organ-
isms, and were reported to exhibit pronounced biological
activity, such as antitumor, antimicrobial, antiparasitic, and
antiviral activity (Kulick et al. 2014). In addition, tadalafil
(Cialis®), a potent inhibitor of PDE5 approved for the
treatment of erectile dysfunction, possesses a tetrahydro-β-
carboline ring system and has been used safely for more
than 10 years (Daugan et al. 2003). Therefore, we assumed
that the tetrahydro-β-carboline scaffold could represent a
suitable starting point for the development of novel PDE4
inhibitors with improved pharmacokinetic properties. The
rationale behind the design of PDE4 inhibitors by structural
modifications imposed on this nucleus depends on the
structure and properties of the PDE5 inhibitor “tadalafil.”
This compound is one of the most potent and long acting
PDE5 inhibitors. The bulky nature of tadalafil allows it to
make many hydrophobic interactions contributing to its
high potency without having any polar contacts with the
Fig. 2 Upper: Binding mode of tadalafil in PDE5 (PDB: 1XOZ);
Lower: Binding mode of piclamilast in PDE4 (PDB: 1XM4) The Q2
pocket is much larger in case of PDE5

Med Chem Res
may cause steric clashes with Met411 (Fig. S1, Supple-
mentary data) (Daugan et al. 2003). Interestingly, on the
other hand, we noticed that many PDE4 inhibitors are
characterized by the presence of the smaller cyclopentyloxy
or cyclopropylmethoxy group that fits into the aforemen-
tioned smaller Q2 pocket of PDE4. Examples include
rolipram, cilomilast, roflumilast, and piclamilast (Fig. 1)
(Mata et al. 2012). Our idea depends on combining the rigid
tetrahydro-β-carboline scaffold of tadalafil with relatively
smaller substituents like cyclopropyl or cyclopentyl groups
that could fit perfectly into the Q2 pocket of PDE4. This
arrangement allowed us to exploit the unique characteristics
of both tadalafil scaffold and piclamilast to get the best
shape complementarity with the PDE4 enzyme. We have
designed two series of 1,2,3,4-tetrahydro-β-carboline deri-
vatives, the first is substituted on the basic nitrogen and the
second possesses alkoxy substituent on position 6 of the β-
carboline ring. (Fig. 1). In both cases, we envisioned an H
bond between the aromatic NH of the compounds and the
critical Gln443 of PDE4 with the alkyl/aryl substituent fit-
ting into the selectivity pocket, Q2 (discussed later).
chlorides in presence of DIPEA to yield 6a-l (Table 1), or
through coupling of the tetrahydro-β-carboline 5 with the
proper carboxylic acid using 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide (EDCI) to give 6m-q (Table 1). Both
methods afforded the desired derivatives with good yields
(Elshihawy et al. 2014).
Another series of tetrahydro-β-carboline derivatives with
the substitution carried out on the 6- position was synthe-
sized. 5-hydroxytryptamine (serotonin) was used, in which
the phenolic hydroxy group in the 6 position can be
exploited for nucleophilic substitution. Due to its unduly
high sensitivity to light and moisture, we decided to start
from commercially available serotonin (Corona-Avendano
et al. 2005). These properties of serotonin also necessitated
performing all the reactions under strictly dry conditions.
Initial step in the synthesis involved Pictet-Spengler cycli-
zation of serotonin 7 (scheme 2). The polarity, and hence
the solubility, of serotonin in organic solvents was another
obstacle against performing cyclization under the standard
procedure. Different conditions were used to perform such
step in which the aldehyde was added to a solution of
serotonin in either glacial acetic acid, TFA/DCM, or glacial
acetic acid/ethanol. The latter gave the best result in the
shortest time as determined by thin layer chromatography.
However, unfortunately, the high polarity of the resultant
compound complicated its separation and caused a con-
siderable loss during the work up (isolated yield: 20–25%).
Therefore, we decided to use a more lipophilic aldehyde
such as acetaldehyde to overcome this problem. To check
the effect of adding a methyl group at carbon number 1 in
our scaffold, it was docked into the crystal structure of
PDE4. Actually, the obtained scores were encouraging and
indicated that the methyl group, in either configuration α or
β, would not negatively affect the binding of this molecule
to the active site. The next step after cyclization was Boc
protection of the basic nitrogen. That was mandatory prior
to carrying out the nucleophilic substitution on the phenolic
OH group using alkyl or aryl halides in the presence of
cesium carbonate. Finally, removal of the protecting group
using TFA gave the corresponding compounds 11a–e in a
reasonable yield (50–70%).
Chemistry
The synthesis of our first series of 1,2,3,4-tetrahydro-β-
carboline derivatives is illustrated in Scheme 1. Preparation
of the target compounds started with Vilsmeier formylation
of indole using formaldehyde and phosphorous oxychloride
(Huang et al. 2012). Henry reaction, which is an Aldol-type
reaction, was performed on the obtained aldehyde with
nitromethane to obtain the nitro olefin 3 (Vijaya et al. 2016).
The resultant nitro olefin was then reduced using lithium
aluminum hydride (LiAlH4) to afford tryptamine 4. Pictet-
Spengler cyclization is one of the most powerful methods to
prepare tetrahydro-β-carboline ring system. It involves the
reaction of the synthesized tryptamine 4 with formaldehyde
in the presence of acidic catalyst to obtain the correspond-
ing tetrahydro-β-carboline 5 (Overvoorde et al. 2015). The
latter is the core scaffold of our scheme. At this stage, two
synthetic pathways were pursued to synthesize the required
amides; either through treatment of 5 with different acyl
Scheme 1 Reagents and
conditions: a POCl3, DMF,
0 °C; b NH4OAc, MeNO2, 1 h,
reflux; c (i) LiAlH4, THF, 0 °C;
36 h, rt; (ii) Rochelle salt, H2O,
Et2O, 24 h, rt; d AcOH, HCHO,
reflux, 3 h; e RCOCl, DCM,
0 °C, 3 h; or RCOOH, HOBt,
EDCI, Et3N, DCM, rt, 24 h

Med Chem Res
Table 1 In vitro PDE4B inhibitory activity in Transcreener Fluorescence Polarization assay*
*
Empty cells indicate no inhibition or compound activity that could not be fit to an IC50 curve
*
IC50 value higher than 100 μM is estimated based on the best curve fitting available
Shaded column indicates derivatives belonging to series 2
Biological evaluation
et al. 2010). Compounds were tested in a 10-fold serial
dilution starting at 100 μM and the fold increase of the
cAMP level caused by the test compounds over DMSO
control was determined. In the first series, the assay indi-
cated that three compounds, 6c, 6d, and 6o demonstrated
All the synthesized final tetrahydro-β-carboline derivatives
were tested for their in vitro inhibitory activity against
PDE4B isoenzyme by detecting the reaction product, AMP,
in transcreener fluorescence polarization assay (Staeben
moderate inhibitory activity against PDE4 enzyme (IC50
=

Med Chem Res
Scheme 2 Reagents and
conditions: a CH3CHO, AcOH,
EtOH, reflux, 2 h; b (BOC)2O,
Et3N, THF, rt, 3 h; c RCl,
Cs2CO3, DMF, 95 °C, 2 h; d
TFA, DCM, 0 °C
2.11, 35.16, and 83.7 μM, respectively), which in case of 6–
c was superior to the reference IBMX (IC50 = 21.45 μM). It
is clearly shown that the 3, 4-dimethoxy in 6-c is better than
the other substituents on the benzoyl moiety. This activity
was reduced when the two methoxy groups were removed
in 6–d (IC50 = 35.16 μM). Interestingly, the activity was
greatly deteriorated in case of derivatives mono-substituted
with methoxy group (6i, IC50 > 100 μM). Also, the activity
vanished when replacing the benzoyl with phenylacetyl
group in 6q. This indicates an optimal length for the linker
separating the terminal phenyl group from the tetrahydro-β-
carboline scaffold. This is also applicable for the inactive
compound 6n which possesses a bulkier indole ring.
Unfortunately, changing the substituents on the benzoyl
group to 3-CF3, 3-F, 3-NO2 and 4-F, as in 6a, 6b, 6e, and
6 g, respectively, led to inactive compounds. Investigation
of the compounds bearing the heterocyclic rings furan and
thiophene, 6m and 6o, respectively, reveals a 4-fold loss of
PDE4 inhibitory activity of 6o (IC50 = 83.7 μM) compared
to 6c, while the furan derivative 6m was inactive. The
higher activity of the thiophene derivative may be attributed
to the stronger aromatic stabilization in the thiophene ring
which is a better isostere to benzene. The remaining com-
pounds with alkyl and cycloalkyl substituents exhibited no
inhibition of PDE4 except the cyclopentyl derivative 6p.
In the second series, substitution was carried out on the
opposite side of the tetrahydro-β-carboline scaffold. We
assumed that these compounds would be flipped inside the
active site so that the substituents on position 6 could form
hydrophobic interactions with the residues of the Q2 pocket.
To our delight, this series exhibited more promising results
than the 2-substituted tetrahydro-β-carbolines. Four out of five
compounds showed decent inhibitory activities against
PDE4B isoenzyme which reflects the improved binding affi-
nity of these compounds compared to the previous series. The
benzyloxy derivative 11a showed inhibitory activity similar to
the benzoyl derivative 6d of the previous series with IC50 of
43.3 μM. Introduction of a chlorine atom at the ortho position
of the benzyl group caused more than 3-fold increase in
activity (11b, IC50 = 13.3 μM). Fortunately, the activity did
not drop significantly when the substituent was shifted to the
meta position on the benzyl ring and changed to the bulkier
CF3 group in 11c (IC50 = 14.6 μM). Nevertheless, the activity
was completely lost when this substituent was replaced by the
smaller fluorine atom in 11d. The peculiar behavior of this
derivative might be attributed to formation inter- or intramo-
lecular H bonding, with the fluorine atom, forcing a con-
formation that is not suitable for enzyme interaction. As
expected, changing the entire benzyloxy group to the smaller
cyclopropylmethoxy, a fragment of the potent PDE4 inhibitor
roflumilast, led to the most active compound 11e (IC50 = 1.85
μM). It is worth noting that the two most active compounds in
this study, 6c and 11e, as well as the thiophene derivative 6o
were tested for PDE5A activity and none of them showed any
detectable inhibition at 10 µM. This indicates successful tar-
geting of PDE4 and selectivity over PDE5.
Computational study
All the prepared compounds were docked into the active site
of the PDE4B crystal structure complexed with piclamilast,
PDB ID: 3D3P, using GOLD suite 4.1. The binding site was
defined as 10 Å around Gln443 and all the parameters were
set as default (Verdonk et al. 2003; Helal et al. 2014). The
only constraint used was the H bond with the critical Gln443
which is the anchor point for all the crystallized PDE4
ligands. Although we did not specify any H bonding atom in
the ligands as a docking constraint, all of them showed the
expected H bonds with Gln433 using their aromatic NH in a
similar manner to the binding mode of tadalafil in PDE5. In
addition, the tertahydro-β-carboline scaffold was docked into
the aforementioned hydrophobic (P) clamp, which is com-
posed of two highly conserved hydrophobic residues forming
the two jaws of the clamp; Phe446 and, on the upper side,
Ile410. In the first series, compounds 6a–q, the diverse
hydrophobic substituents on position 2 showed contacts with
residues of the hydrophobic Q2 pocket. On the other hand,
and as expected, the scaffold of the second series, com-
pounds 11a–e, was inverted to bring the substituents on
position 6 close to the same pocket, again without using any
distance constraints. The proposed binding mode of two of
the most active compounds, one from each series, is shown
in Fig. 3. The dimethoxybenzoyl derivative 6c (Fig. 3a) fits
into the
P
clamp as mentioned above with the

Med Chem Res
inhibition while the unsubstituted benzyloxy derivative 11a,
suffered from a 3-fold drop in activity.
To gain further insights into the binding mode of both
series of compounds, we performed a 20 ns molecular
dynamics (MD) simulation using the docking-generated
complexes of PDE4 with compounds 6c and 11e. We used
the system builder tool within the Desmond software
(Schrodinger Inc., Portland, OR, USA) to insert each com-
plex into a pre-equilibrated TIP3P water orthorhombic box.
MD simulations were carried out using the Desmond appli-
cation in Maestro, employing the OPLS-AA force field
parameters and following the procedure described in our
recent publication (Helal et al. 2013). A constant-volume MD
simulation was performed for the first 100 ps during which
the temperature of the system was raised from 0 → 310 °K.
The system was then equilibrated in an NPT ensemble for 1
ns and then was simulated for 20 ns with a target pressure of
1.0132 bar. The docked poses of 6c and 11e were monitored
during the whole MD simulation and the stability of the
critical H bonds with Gln443 was examined. To our delight,
the proposed H bond between the β-carboline aromatic NH
and the key Gln443 remained stable in both complexes with
a more significant fluctuation in case of 6c (Fig. S3, Sup-
plementary data). Moreover, it was noticed that the tetra-
hydro β-carboline scaffold of both compounds maintained its
GOLD-generated binding mode in the hydrophobic clamp of
PDE4. These MD simulation results give more emphasis on
the proposed binding and activity determinants of this novel
series of PDE4 inhibitors.
Fig. 3 Proposed binding mode of 6c a, and 11e b in the crystal
structure of PDE4 (PDE ID: 3D3P). Active site residues are shown as
grey sticks. Docked compounds are displayed as green or cyan sticks.
Only residues having significant interaction with the ligand are shown
Conclusions
It is worth noting that the primary goal of this study was
not the optimization of a potent final drug candidate, but
the discovery of an initial lead compound based on a
scaffold with well-studied pharmacokinetics. Therefore,
more work is certainly needed in order to optimize the
steric and electronic characteristics of the substituents tar-
geting the Q2 pocket which is a crucial point for the
activity. This is the first report of tetrahydro-β-carboline
as a chemical scaffold for PDE4 inhibitors. Further studies
are being performed in our laboratory to explore the effect
of substitution on other positions of this nucleus. More-
over, smaller polar substituents could be introduced in the
proper position and orientation to target the metal pocket
and add to the ligands affinity. We hope that the proposed
modifications could finally contribute to the development
of a novel PDE4 inhibitor with improved safety and
pharmacokinetics.
dimethoxyphenyl moiety sandwiched between Met431 and
Phe414 in Q2 pocket. The superior activity of this com-
pound, compared to the other derivatives, may be attributed
to the suitable size and hydrophobicity required to make
interactions with the P clamp as well as Q2 hydrophobic
residues. It is worth noting that we could not get these
dimethoxy groups to interact with Gln433, similar to the
previously reported PDE4 inhibitors (Fig. 1), even when
using constraints to force this binding orientation. In the
second series, compound 11e, the cyclopropylmethoxy
derivative, was found to be inverted in the active site
allowing the cyclopropyl group to fit nicely into the Q2
pocket, also making contacts with Met431 and Phe414 (Fig.
3b). It was noticed that a combination of optimum hydro-
phobicity and size is required in the substituent to perfectly
fill this pocket. For instance, the substituted benzyloxy
derivatives, 11b and 11c, showed comparable enzyme
Acknowledgements This study was entirely funded by the Science
and Technology Development Fund (STDF), Egypt, Grant #4711,

Med Chem Res
awarded to the Principal Investigator Dr. Mohamed A. Helal.
The authors would like to thank Dr. Khaled M. Darwish, Medicinal
Chemistry Department, Faculty of Pharmacy, Suez Canal University,
for his insights in the design of the target compounds and during the
synthetic steps.
Helal MA, Darwish KM, Hammad MA (2014) Homology modeling
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Compliance with ethical standards
Huai Q, Colicelli J, Ke H (2003) The crystal structure of AMP-bound
PDE4 suggests a mechanism for phosphodiesterase catalysis.
Biochemistry 42(45):13220–13226
Conflict of interest The authors declare that they have no competing
interests.
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