Synthesis and evaluation of pharmacological profile of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamides

Article abstract of DOI:10.1016/j.bmc.2009.03.066

In previous studies we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives displaying potent anticonvulsant effects in different animal models of epilepsy. With the aim to deepen the structure-activity relationships (SAR) fo

Full text of DOI:10.1016/j.bmc.2009.03.066

Bioorganic & Medicinal Chemistry 17 (2009) 3659–3664
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of pharmacological profile of
1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamides
a
a
a
b
b
Rosaria Gitto a, , Stefania Ferro , Stefano Agnello , Laura De Luca , Giovanbattista De Sarro , Emilio Russo ,
*
Daniela Vullo c, Claudiu T. Supuran c, Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Medicina Sperimentale e Clinica, Università Magna Gr
ꢀ
cia, Viale Europa Località Germaneto, 88100 Catanzaro, Italy
c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In previous studies we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline deriva-
tives displaying potent anticonvulsant effects in different animal models of epilepsy. With the aim to dee-
pen the structure–activity relationships (SAR) for this class of compounds and identify novel
anticonvulsant agents we synthesized a series of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-
sulfonamides. The new compounds incorporate the main features of the above-mentioned anticonvul-
sants and a sulfonamide function capable to inhibit the enzyme carbonic anhydrase (CA, EC 4.2.1.1),
which represents an attractive target in epilepsy. Pharmacological effects were evaluated in vivo against
audiogenic seizures in DBA/2 mice and in vitro against several CA isoforms. Some of the new molecules
showed anticonvulsant properties better than topiramate, but weak inhibitory activity and low selectiv-
ity in enzymatic assay.
Received 27 January 2009
Revised 26 March 2009
Accepted 29 March 2009
Available online 7 April 2009
Keywords:
Isoquinolines
Anticonvulsants
DBA/2 mice
Carbonic anhydrase inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
antagonists by interaction with glutamate ionotropic AMPA
receptor (AMPAR) complex.6–12 In particular, the 2-acetyl-1-(40-
Epilepsy affects ꢀ1% of the population worldwide; even though
most people become seizure-free with drug therapy there is a sig-
nificant group of patients (30%) who are resistant to the currently
available antiepileptic drugs (AEDs) and more effective therapies
are needed. The efficacy of anticonvulsant drugs may be connected
to different molecular targets to reduce the excitability of neurons
involved in seizures on-set. For anticonvulsant activity several
mechanisms can be considered and the different targets include
not only voltage-gated ion channels (Na+, Ca2+, K+, Clꢁ) or ligand-
gated ion channels (GABA, glutamate, glycine, etc.) but also neuro-
transmitter transporters (GAT1-4, EAAT1-5), and enzymes (GABA-
transaminase, carbonic anhydrase, specific protein kinases).1,2 The
marketed anticonvulsants predominantly target voltage-gated cat-
ion channels, the improvement of GABA-mediated inhibition, and
the blockade of excitatory glutamatergic neurotransmission.3,4
Nevertheless, the newer agents currently in preclinical or clinical
development are characterized by pioneering mechanisms of
action.5
chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (A)
and 1-(40-bromophenyl)-6,7-dimethoxy-2-(piperidin-1-yl-acet-
yl)-3,4-dihydroisoquinoline (B), the most active compounds of
the series (Fig. 1), showed stronger potency in in vivo and
in vitro tests when compared with other known AMPAR
antagonists.8,11
Starting from these promising results we synthesized a large
series of analogues exploring the effect of the modifications both
of the moiety at N-2 position and the substituent on the C-1 phenyl
group, as well as on the benzene fused ring. Structure-activity rela-
tionship (SAR) studies highlighted that the substituent at the N-2
MeO
MeO
MeO
MeO
N
Me
N
N
O
O
In previous papers, we planned the synthesis of N-substituted
1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines and identi-
fied some potent anticonvulsant agents acting as noncompetitive
Cl
Br
* Corresponding author. Tel./fax: +39 0906766413.
E-mail address: rgitto@pharma.unime.it (R. Gitto).
Figure 1. 1-Aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines as AMPAR antagonists.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.066

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R. Gitto et al. / Bioorg. Med. Chem. 17 (2009) 3659–3664
position of tetrahydroisoquinoline system plays the principal role
in the process of receptor recognition, as had been already sug-
gested by our computational studies.7,8
On the basis of these findings to identify new anticonvulsant
agents containing isoquinoline skeleton we herein report the syn-
thesis of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sul-
fonamides (1a–j, Fig. 2) in which we explored if the sulfonamide
function could be a feature useful to improve the anticonvulsant
efficacy.
O
NH2
S
O
O
O
O
NH2
S
O
O
O
N
O
O
O
zonisamide
topiramate
Figure 3. Chemical structure of CA inhibitors Zonisamide and Topiramate.
We selected this moiety taking in account that sulfonamide or
sulfamate functions are able to coordinate the metal ion of some
metalloenzymes13,14 such as carbonic anhydrase (CA) whose inhibi-
tion has been shown to play an important role in convulsion control
due to CO2 retention secondary to the inhibition of enzyme.15 In fact
the anticonvulsants zonisamide16 and topiramate17 (Fig. 3), cur-
rently in therapy, are CA inhibitors, although their mechanism of ac-
tion cannot be completely attributed to this enzyme inhibition.
Our hypothesis was that the introduction of the sulfonamide
function into tetrahydroisoquinoline scaffold could determine a
pharmacological additive effect. So the suitable synthetic approach
has been set up and the new derivatives obtained (1a–j) com-
pounds were evaluated as anticonvulsant agents against sound-in-
duced seizures in DBA/2 mice. Moreover their inhibitory activity
was assayed on several CA isoforms.
2. Results and discussion
2.1. Chemistry
Figure 4. The highest-ranked four-point hypothesis for noncompetitive AMPAR
antagonists derived using the Catalyst/HIPHOP program. Cyan: hydrophobic groups;
green: hydrogen bond acceptor feature (HBA) with a vector in the direction of the
putative hydrogen donor (HBA PP); orange: aromatic ring with proposed d-stacking
interaction shown by an arrow (aromatic ring PP). The alignment of 1j on the
pharmacophore is shown.
As depicted in Scheme 1, the synthesis of 1-aryl-6,7-dimethoxy-
3,4-dihydroisoquinoline-2(1H)-sulfonamides (1a–j) was carried
out through a multistep procedure with good yields. Following a
previously reported method11 and in Microwave Assisted Organic
Synthesis (MAOS) conditions,18 we prepared the 1-aryl-6,7-dime-
thoxy-1,2,3,4-tetrahydroisoquinolines (5a–h). By reaction of the
6,7-dimethoxyphenethylamine (2) with suitable aromatic alde-
hydes 3a–h we obtained the corresponding imine intermediates
4a–h, which without purification were treated with trifluoroacetic
acid (TFA) to provide the desired isoquinolines 5a–h. Successively,
the key intermediates 5a–h reacted with a large excess of sulfam-
ide leading to the 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-
2(1H)-sulfonamides (1a–h). In the first approach this step was per-
formed using a conventional method,19 then it was optimized by
application of MAOS, allowing a significantly reaction time reduc-
tion and a considerable enhancement of the yields. Finally the ami-
nophenyl derivatives 1i–j were prepared by reduction of the
corresponding nitrophenyl derivatives 1g–h. The structures of all
compounds obtained were supported by elemental analyses and
spectroscopic measurements.
ated against audiogenicseizures in DBA/2 mice, considered an excel-
lent animal model for generalized epilepsy and for the screening of
new anticonvulsant drugs.20 Table 1 reports the ED50 values mea-
sured after intraperitoneal (ip) administration of new synthesized
compounds; these values were compared with those of parent
unsubstituted 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquino-
lines (5a–j) and topiramateas reference compound. The obtained re-
sults suggest that the 1-(40-aminophenyl)-6,7-dimethoxy-3,4-
dihydroisoquinoline-2(1H)-sulfonamide (1j) is 2.5-fold more potent
than parent compound 5j; in addition, the 1-(40-chlorophenyl)-6,7-
dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamide (1c) is able
to protect in audiogenic seizure test at doses that are comparable to
those of unsubstituted analogue5c. Furthermore, 1c and 1j, the most
active compounds of the series, were more potent than topiramate.
Moreover, compound 1a displayed significant improvement of anti-
convulsant activity in tonic phase of the test. Nevertheless, com-
pounds 1c and 1j were less potent than our hits previously
synthesized (i.e., compounds A and B).8,11 The other sulfonamide
derivatives 1a–b and 1d–i are characterized by poor anticonvulsant
efficacy and lower activity than parent compounds 5a–b and 5d–i.
These findings highlighted that the introduction of the sulfonamide
moietyisnotconduciveto enhancetheanticonvulsanteffectsexcept
for compound 1j. Furthermore, we explored the modifications on C-
1 phenyl structural fragment. Pharmacological data showed that the
best substitution pattern involves the presence of a chlorine atom or
an amino group at 40-position of phenyl group for compounds 1c and
1j, respectively. These results suggest that the anticonvulsant prop-
ertiesof thisseriesof derivativesmightbe directlycorrelated neither
to the electronic nor to lipophilic properties of the 40-substituent on
the phenyl ring. Interestingly, the 4-aminophenyl or 4-chlorophenyl
substituents had been suggested, by our previous studies, as struc-
2.2. Anticonvulsant properties
Theanticonvulsanteffectsofthedesigned1-aryl-6,7-dimethoxy-
3,4-dihydroisoquinoline-2(1H)-sulfonamides (1a–j) were evalu-
MeO
O
N
MeO
S
NH2
O
R
1
Figure 2. Designed 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfona-
mides (1).

R. Gitto et al. / Bioorg. Med. Chem. 17 (2009) 3659–3664
3661
CHO
MeO
MeO
MeO
MeO
MeO
MeO
a
b
+
N
NH
NH2
R
2
3 a-h
4 a-h
5 a-h
R
R
c
R
H
4-Br
4-Cl
4-CN
R
4-CH3
3-NO2
MeO
MeO
1 a-j
a
f
b
c
N
O
g
h
i
S
NH2
4-NO2
3-NH2
4-NH2
O
iv
d
e
4-F
j
R
Scheme 1. Reagents and conditions: (a) MW: 5 min, 90 °C, 200 Psi, 150 W; (b) TFA MW: 5 min, 90 °C, 200 Psi, 150 W; (c) CH3CH(OCH3)2, NH2SO2NH2,
D
, 24–48 h or two steps
in the same conditions: 20 min, 100 °C, 200 Psi, 150 W; (iv) Zn/HCl concd,
D, 2 h.
Table 1
isoquinoline-2(1H)-sulfonamide (1j) demonstrated good alignment
with all the 3D pharmacophoric features requested for AMPA recep-
tor antagonism (Fig. 4). In particular the sulfonamide moiety gener-
ates the hydrogen bond acceptor (HBA) feature thus mimicking the
carbonyl function of active compounds A–B.
Anticonvulsant activity of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sul-
fonamides (1a–j), 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines (5a–j) and
reference compounds against audiogenic seizures in DBA/2 mice
MeO
2.3. Carbonic anhydrase inhibition
N
MeO
R'
To determine the enzymatic inhibitory activity we assayed the
new series of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-
2(1H)-sulfonamides (1a–j) on four important carbonic anhydrase
isoforms involved in several physiological and pathological pro-
cess, hCA I, hCA II, hCA IX and hCA XIV (Table 2). This preliminary
screening showed that all tested compounds have a moderate–
weak inhibitory activity against these isozymes, with inhibition
R
R
R0
ED50
l
mol/kga
Tonic phase
Clonic phase
5ab
H
4-Br
4-Cl
4-CN
4-F
4-Me
3-NO2
3-NH2
4-NO2
4-NH2
H
4-Br
4-Cl
4-CN
4-F
4-Me
3-NO2
4-NO2
3-NH2
4-NH2
H
H
H
H
H
H
H
H
H
H
44.8 (23.9–84.0)
30.6 (19.4–48.2)
20.1 (9.65–41.9)
>100
57.8(25.0–133)
48.6 (32.8–71.9)
19.3 (6.10–61.2)
40.4 (21.8–74.7)
101 (52.0–194)
46.6 (28.0–77.4)
>100
19.3 (9.05–41.3)
12.5 (6.90–22.6)
19.3(11.8–31.5)
48.6 (32.9–71.8)
26.4(15.5–44.9)
36.5 (28.2–47.3)
7.20 (2.45–21.2)
18.2 (7.76–41.8)
56.1 (26.9–117)
28.6 (14.4–56.8)
7.82 (6.66–9.19)
67.3 (44.6–102)
7.25 (4.54–11.6)
>100
constants (KI) in the range of 0.33–25.70 lM. The most interesting
5bb
compound was 6,7-dimethoxy-1-(40-nitrophenyl)-3,4-dihydroiso-
quinoline-2(1H)-sulfonamide (1h), which demonstrated KI value
lower than that of topiramate against hCA XIV; moreover, 1h and
topiramate inhibited hCA I with similar potency. Nevertheless,
the inhibitory effects of 1h were very similar against the different
tested isozymes, exhibiting poor selectivity when compared with
topiramate.
5cb
5d
5eb
5f
5gb
5ib
5hb
5jb
For the other tested compounds (1a–g and 1i–j), the data re-
1°
1b
1c
1d
1e
1f
1g
1h
SO2NH2
ported in Table 2 show that the nature of 40-substituent on the
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
>100
23.5 (14.4–38.5)
>100
>100
41.8 (19.7–88.9)
51.7 (36.9–104)
>100
97.1 (64.2–147)
>100
Table 2
Inhibition of hCA I, hCA II, hCA IX and hCA XIV isoforms by 1-aryl-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinolines (1a–j) and topiramate
>100
>100
>100
1i
1j
65.8 (41.7–104)
3.60 (1.45–8.97)
2.39 (1.30–4.40)
8.17 (4.03–16.6)
18.0 (10.2–31.6)
KI (l
M)a
17.7 (9.80–30.1)
4.18 (2.23–7.84)
12.7 (6.09–26.3)
35.7 (20.4–62.3)
Compound A
Compound B
Topiramate
hCA I
hCA II
hCA IX
hCA XIV
1a
1b
1c
1d
1e
1f
1g
1h
8.98
25.70
9.44
4.07
4.43
3.49
7.69
0.33
3.83
8.07
0.25
15.70
10.71
5.30
8.44
8.20
7.54
4.98
5.59
6.75
6.45
0.48
8.13
6.78
0.058
3.86
7.02
8.98
3.04
6.93
7.05
5.35
0.21
6.67
3.07
1.46
a
All data were calculated according to the method of Litchfield and Wilcoxon. At
least 32 animals were used to calculate each ED50
. 95% confidence limits are given
4.24
in parentheses.
14.98
10.77
3.82
0.60
9.31
b
From Ref. 21.
1i
1j
11.30
0.010
tural requirements able to positively influence the anticonvulsant
activity of AMPA receptor antagonists.4 With the aim to corroborate
this hypothesis we performed the overlapping of the most active
derivative on our previously developed 3D-pharmacophore model.
The fitting of the 1-(40-aminophenyl)-6,7-dimethoxy-3,4-dihydro-
Topiramate
a
Errors in the range of 10% of the reported value, from three different assays.
Recombinant full length hCA I, II and XIV and catalytic domain of hCA IX were
used.24–27

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R. Gitto et al. / Bioorg. Med. Chem. 17 (2009) 3659–3664
phenyl ring does not significantly influence inhibitory effects
against hCA I, hCA II, hCA IX and hCA XIV isoforms. Considering
these results we could speculate that this molecular frame is not
involved in the binding recognition process with the active site.
Furthermore, with the aim to confirm that the introduction of sul-
fonamide moiety determines CA inhibitory effects, we also evalu-
ated the activity of some selected free amines (e.g., 5c and 5j)
and corresponding N-acetyl derivatives on hCA I, hCA II, hCA IX
and hCA XIV isoforms. These compounds were not able to produce
C18H18N2O2: C, 73.45; H, 6.16; N, 9.52. Found: C, 73.68; H, 6.33;
N, 9.24.
4.1.1.2. 6,7-Dimethoxy-1-(40-methylphenyl)-1,2,3,4-tetrahydro-
isoquinoline (5f). Yield 72%. Mp 140–142 °C. 1H NMR (CDCl3, d):
2.34 (3H, s, CH3), 2.73–3.26 (4H, m, CH2CH2), 3.64 (3H, s, OCH3),
3.87 (3H, s, OCH3), 5.03 (1H, s, CH), 6.26 (1H, s, ArH), 6.62 (1H, s,
ArH) 7.13 (4H, s, ArH). Anal. Calcd for C18H21NO2: C, 76.30; H,
7.47; N, 4.94. Found: C, 76.52; H, 7.15; N, 5.13 .
any inhibitory effects at higher tested doses (Ki > 200 lM).
4.1.2. General procedure for the synthesis of 1-aryl-6,7-
dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamides (1a–h)
A mixture of the appropriate 1-aryl-6,7-dimethoxy-1,2,3,4-tet-
rahydroisoquinoline (5a–h) (1.0 mmol) and sulfamide (6 mmol,
576 mg) in dimethoxyethane (2 mL) was placed in a cylindrical
quartz tube (Ø 2 cm), then stirred and irradiated in a microwave
oven at 150 W for two steps of 20 min at 90 °C. The reaction was
quenched by addition of water (5 mL) and extracted with ethyl
acetate (3 ꢂ 5 mL). The organic layer was washed with an aqueous
saturated solution of NaHCO3 (2 ꢂ 5 mL), dried over Na2SO4 and
concentrated until dryness under reduced pressure. The residue
crystallized from diethyl ether to give the desired compounds
1a–h as pure products.
3. Conclusions
An innovative approach for the preparation of sulfonamide
derivatives has been set up and used to obtain a new series of N-
substituted 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinolines (1a–
j). Among the new synthesized compounds, derivatives 1c and 1j
proved to be more potent than topiramate against audiogenic sei-
zures in DBA/2 mice. The evaluation of their inhibitory effects on
several CA isozymes demonstrated that anticonvulsant properties
of 1c and 1j are not related to inhibition of carbonic anhydrase.
On the contrary, compound 1h provided good CA inhibitory activ-
ity, even if it lacked in vivo properties. Collecting the in vivo and
in vitro results we can summarize that the introduction of sulfon-
amide function on isoquinoline skeleton provides potent anticon-
vulsant agents, but their activity does not appear correlated only
to CA inhibition.
4.1.2.1. 6,7-Dimethoxy-1-phenyl-3,4-dihydroisoquinoline-2(1H)-
sulfonamide (1a). Yield 74%. Mp 186–188 °C. 1H NMR (CDCl3, d):
2.69–2.77 (1H, m, CH2CH2), 3.15–3.29 (2H, m, CH2CH2), 3.73 (3H, s,
CH3O), 3.79–3.85 (1H, m, CH2), 3.90 (3H, s, CH3O), 4.06 (2H, br s,
NH2), 6.00 (1H, s, CH), 6.41 (1H, s, ArH), 6.68 (1H, s, ArH), 7.27–
7.36 (5H, m, ArH). 13C NMR (CDCl3, d): 26.4, 38.6, 55.9, 59.4,
111.8, 111.9, 125.8, 127.8, 128.9, 129.0, 140.2, 148.2, 148.3. Anal.
Calcd for C17H20N2O4S: C, 58.60; H, 5.79; N, 8.04. Found: C,
58.86; H, 5.62; N, 8.21.
4. Experimental
4.1. Chemistry
Microwave-assisted reactions were carried out in a CEM fo-
cused Microwave Synthesis System. Melting points were deter-
4.1.2.2. 1-(40-Bromophenyl)-6,7-dimethoxy-3,4-dihydroisoquino-
line-2(1H)-sulfonamide (1b). Yield 86%. Mp 158–160 °C. 1H NMR
(CDCl3, d): 2.69–2.74 (1H, m, CH2CH2), 3.13–3.21 (2H, m, CH2CH2),
3.75 (3H, s, CH3O), 3.80–3.86 (1H, m, CH2CH2), 3.89 (3H, s, CH3O),
4.16 (2H, br s, NH2), 5.96 (1H, s, CH), 6.39 (1H, s, ArH), 6.68 (1H, s,
ArH), 7.15 (2H, d, J = 8.5, ArH), 7.45 (2H, d, J = 8.5, ArH). 13C NMR
(CDCl3, d): 26.8, 39.1, 55.9, 58.7, 110.6, 111.3, 122.2, 125.0, 125.8,
130.7, 131.7, 140.1, 147.8, 148.5. Anal. (C, H, N). Anal. Calcd for
C17H19BrN2O4S: C, 47.78; H, 4.48; N, 6.56. Found: C, 47.56; H,
4.78; N, 6.86.
mined on
a Stuart SMP10 apparatus and are uncorrected.
Elemental analyses (C, H, N) were carried out on a Carlo Erba Model
1106 Elemental Analyzer and the results are within 0.4% of the
theoretical values. Merck Silica Gel 60 F254 plates were used for
analytical TLC. 1H NMR and 13C NMR spectra were measured in
CDCl3 (TMS as internal standard) or DMSO-d6 with a Varian Gemini
300 spectrometer; chemical shifts are expressed in d (ppm) and
coupling constants (J) in hertz. All exchangeable protons were
confirmed by addition of D2O.
4.1.2.3. 1-(40-Chlorophenyl)-6,7-dimethoxy-3,4-dihydroiso-
quinoline-2(1H)-sulfonamide (1c). Yield 51%. Mp 178–180 °C.
1H NMR (CDCl3, d): 2.69–2.74 (1H, m, CH2CH2), 3.13–3.21 (2H, m,
CH2CH2), 3.75 (3H, s, CH3O), 3.79–3.85 (1H, m, CH2CH2), 3.90
(3H, s, CH3O), 4.16 (2H, br s, NH2), 5.97 (1H, s, CH), 6.39 (1H, s,
ArH), 6.68 (1H, s, ArH), 7.21 (2H, d, J = 8.5, ArH), 7.30 (2H, d,
J = 8.5, ArH). 13C NMR (CDCl3, d): 27.1, 40.0, 56.1, 58.7, 110.8,
111.5, 125.0, 125.8, 129.3, 129.8, 131.7, 140.8, 147.7, 148.6. Anal.
Calcd for C17H19ClN2O4S: C, 53.33; H, 5.00; N, 7.32. Found: C,
53.68; H, 5.23; N, 7.43.
4.1.1. General procedure for the synthesis of 1-aryl-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinolines (5a–h)
A
mixture of 2-(30,40-dimethoxyphenyl)ethylamine (2)
(1.0 mmol, 181.24 mg) and suitable aldehyde derivative
(1.2 mmol) 3a–h was placed in a cylindrical quartz tube (Ø
2 cm), then stirred and irradiated in a microwave oven at 280 W
for 5 min at 90 °C; after cooling to room temperature trifluoroace-
tic acid (2 mL) was added to crude intermediates 4a–h obtained in
the previous step and the mixture was irradiated at 280 W for
5 min at 90 °C. The reaction was quenched by adding water, and
the mixture was basified (pH 8–9) with sodium hydroxide to give
the isoquinoline derivatives as a solid. The crude product was col-
lected by filtration and purified by crystallization with EtOH to af-
ford compounds 5a–h. Analytical data for compounds 5a–c, 5e,
5g–h are in accordance with literature.21
4.1.2.4. 1-(40-Cyanophenyl)-6,7-dimethoxy-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1d). Yield 98%. Mp 195–197 °C. 1H
NMR (CDCl3, d): 2.68–2.78 (1H, m, CH2CH2), 3.14–3.17 (2H, m,
CH2CH2), 3.76 (3H, s, CH3O), 3.83–3.88 (1H, m, CH2CH2), 3.90
(3H, s, CH3O), 4.30 (2H, br s, NH2), 6.02 (1H, s, CH), 6.41 (1H, s,
ArH), 6.70 (1H, s, ArH), 7.39 (2H, d, J = 8.1, ArH), 7.62 (2H, d,
J = 8.1, ArH). 13C NMR (CDCl3, d): 26.8, 40.0, 56.4, 59.2, 110.9,
111.9, 112.3, 118.9, 124.5, 126.4, 129.9, 132.7, 146.9, 148.3,
149.2. Anal. Calcd for C18H19N3O4S: C, 57.90; H, 5.13; N, 11.25.
Found: C, 57.75; H, 5.33; N, 11.43.
4.1.1.1. 1-(40-Cyanophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydro-
isoquinoline (5d). Yield 46%. Mp 217–219 °C. 1H NMR (CDCl3,
d): 2.85–3.18 (4H, m, CH2CH2), 3.63 (3H, s, OCH3), 3.90 (3H, s,
OCH3), 5.42 (1H, s, CH), 6.07 (1H, s, ArH), 6.67 (1H, s, ArH), 7.44
(2H, d, J = 8.24, ArH), 7.68 (2H, d, J = 8.24, ArH). Anal. Calcd for

R. Gitto et al. / Bioorg. Med. Chem. 17 (2009) 3659–3664
3663
4.1.2.5. 6,7-Dimethoxy-1-(40-fluorophenyl)-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1e). Yield 70%. Mp 116–118 °C. 1H
NMR (CDCl3, d): 2.73–2.78 (1H, m, CH2CH2), 3.18–3.30 (2H, m,
CH2CH2), 3.79 (3H, s, CH3O), 3.84–3.90 (1H, m, CH2CH2), 3.94
(3H, s, CH3O), 4.21 (2H, br s, NH2), 6.03 (1H, s, CH), 6.44 (1H, s,
ArH), 6.72 (1H, s, ArH), 7.02–7.08 (2H, m, ArH), 7.27–7.31 (2H, m,
ArH). 13C NMR (CDCl3, d): 27.3, 39.0, 56.2, 57.8, 110.7, 111.2,
116.0, 125.7, 129.0, 129.8, 140.0, 147.7, 148.6, 158.0. Anal. Calcd
for C17H19FN2O4S: C, 55.73; H, 5.23; N, 7.65. Found: C, 55.64; H,
5.48; N, 7.56.
Anal. Calcd for C17H21N3O4S: C, 56.18; H, 5.82; N, 11.56. Found: C,
56.32; H, 5.71; N, 11.75.
4.1.3.2. 1-(40-Aminophenyl)-6,7-dimethoxy-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1j). Yield 77%. Mp 197–199 °C. 1H
NMR (CDCl3, d): 2.72–2.77 (1H, m, CH2CH2), 3.09–3.30 (2H, m,
CH2CH2), 3.68–3.81 (4H, m, CH3O and CH2CH2), 3.88 (3H, s,
CH3O), 3.96 (2H, br s, NH2), 5.90 (1H, s, CH), 6.38 (1H, s, ArH),
7.62 (2H, d, J = 8.5, ArH), 6.65 (1H, s, ArH), 7.07 (2H, d, J = 8.5,
ArH). 13C NMR (CDCl3, d): 27.6, 39.8, 56.2, 57.4, 110.7, 111.2,
116.8, 125.7, 127.3, 129.8, 132.7, 145.9, 147.3, 148.6. Anal. Calcd
for C17H21N3O4S: C, 56.18; H, 5.82; N, 11.56. Found: C, 56.31; H,
5.56; N, 11.72.
4.1.2.6. 6,7-Dimethoxy1-(40-methylphenyl)-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1f). Yield 56%. Mp 162–164 °C. 1H
NMR (CDCl3, d): 2.33 (3H, s, CH3), 2.71–2.77 (1H, m, CH2CH2),
3.14–3.25 (2H, m, CH2CH2), 3.72 (3H, s, CH3O), 3.76–3.82 (1H, m,
CH2CH2), 3.89 (3H, s, CH3O), 4.00 (2H, br s, NH2), 5.97 (1H, s, CH),
6.39 (1H, s, ArH), 6.67 (1H, s, ArH), 7.12–7.19 (4H, m, ArH). 13C
NMR (CDCl3, d): 21.1, 27.2, 38.8, 55.9, 59.2, 110.7, 111.1, 125.7,
126.0, 128.9, 129.2, 137.8, 138.0, 147.9, 148.2. Anal. Calcd for
C18H22N2O4S: C, 59.65; H, 6.12; N, 7.73. Found: C, 59.34; H, 6.38;
N, 7.45.
4.2. Pharmacology
4.2.1. Testing of anticonvulsant activity against audiogenic
seizures in DBA/2 mice
All experiments were performed with DBA/2 mice which are
genetically susceptible to sound-induced seizures.20 DBA/2 mice
(8–12 g; 22–25-days-old) were purchased from Harlan Italy (Core-
zzano, Italy). Groups of 10 mice of either sex were exposed to audi-
tory stimulation 30 min following administration of vehicle or
each dose of drugs studied. The compounds were given intraperi-
toneally (i.p.) (0.1 mL/10 g of body weight of the mouse) as a
freshly-prepared solution in 50% dimethylsulfoxide (DMSO) and
50% sterile saline (0.9% NaCl). Individual mice were placed under
a hemispheric perspex dome (diameter 58 cm), and 60 s were al-
lowed for habituation and assessment of locomotor activity. Audi-
tory stimulation (12–16 kHz, 109 dB) was applied for 60 s or until
tonic extension occurred, and induced a sequential seizure re-
sponse in control DBA/2 mice, consisting of an early wild running
phase, followed by generalized myoclonus and tonic flexion and
extension sometimes followed by respiratory arrest. The control
and drug-treated mice were scored for latency to and incidence
of the different phases of the seizures. The experimental protocol
and all the procedures involving animals and their care were con-
ducted in conformity with the institutional guidelines and the
European Council Directive of laws and policies.
4.1.2.7. 6,7-Dimethoxy-1-(30-nitrophenyl)-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1g). Yield 90%. Mp 173–175 °C. 1H
NMR (CDCl3, d): 2.68–2.76 (1H, m, CH2CH2), 3.14–3.25 (2H, m,
CH2CH2), 3.76 (3H, s, CH3O), 3.81–3.88 (1H, m, CH2CH2), 3.91
(3H, s, CH3O), 4.38 (2H, bs, NH2), 6.08 (1H, s, CH), 6.44 (1H, s,
ArH), 6.72 (1H, s, ArH), 7.52 (1H, t, J = 8.1, H-50), 7.71 (1H, d,
J = 8.1, H-60), 8.03 (1H, s, H-20), 8.16 (1H, d, J = 8.1, H-40). 13C NMR
(DMSO-d6, d): 25.9, 38.7, 55.4, 55.6, 57.4, 111.5, 111.8, 122.3,
122.7, 125.1, 126.8 129.7, 134.8, 145.4, 147.1, 147.6, 148.0. Anal.
Calcd for C17H19N3O6S: C, 51.90; H, 4.87; N, 10.68. Found: C,
51.72; H, 4.62; N, 10.85.
4.1.2.8. 6,7-Dimethoxy-1-(40-nitrophenyl)-3,4-dihydroisoquino-
line-2(1H)-sulfonamide (1h). Yield 97%. Mp 194–196 °C. 1H
NMR (CDCl3, d): 2.68–2.78 (1H, m, CH2CH2), 3.10–3.25 (2H, m,
CH2CH2), 3.77 (3H, s, CH3O), 3.84–3.87 (1H, m, CH2CH2), 3.91
(3H, s, CH3O), 4.33 (2H, br s, NH2), 6.06 (1H, s, CH), 6.42 (1H, s,
ArH), 6.71 (1H, s, ArH), 7.45 (2H, d, J = 8.5, ArH), 8.17 (2H, d,
J = 8.5, ArH). 13C NMR (CDCl3, d): 29.1, 39.8, 55.83, 59.8, 110.5,
111.6, 123.6, 127.7, 128.2, 129.8, 145.4, 146.3, 147.9, 148.6. Anal.
Calcd for C17H19N3O6S: C, 51.90; H, 4.87; N, 10.68. Found: C,
52.04; H, 4.71; N, 10.55.
4.2.2. Statistical analysis
Statistical comparisons between groups of control and drug-
treated animals were made using Fisher’s exact probability test
(incidence of the seizure phases). The ED50 values of each phase
of audiogenic seizures was determined for each dose of compound
administered, and dose–response curves were fitted using a com-
puter program by Litchfield and Wilcoxon’s method.22
4.1.3. General procedure for the synthesis of 6,7-dimethoxy-1-
(30-aminophenyl)-3,4-dihydroisoquinoline-2(1H)-sulfonamide
(1i) and 6,7-dimethoxy-1-(40-aminophenyl)-3,4-
dihydroisoquinoline-2(1H)-sulfonamide (1j)
4.3. CA inhibition assay
A solution of appropriate 6,7-dimethoxy-1-(40-aminophenyl)-
3,4-dihydroisoquinoline-2(1H)-sulfonamide (0.6 mmol) in 3 mL of
HCl and 4 mL of EtOH was stirred vigorously and zinc dust
(20 mmol) was added in several portions at room temperature.
The reaction mixture was heated in a water bath for 1 h, cooled,
made alkaline with a solution of NaOH 2 N, and then extracted
with ethyl acetate. The organic phase washed with water, dried
over Na2SO4 and evaporated. The residue was crystallized from
ethanol to give the desired products (1i–1j).
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.23 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 10–20 mM
Hepes (pH 7.5) or Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or
20 mM NaClO4 (for maintaining constant the ionic strength), fol-
lowing the initial rates of the CA-catalyzed CO2 hydration reaction
for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and inhibi-
tion constants. For each inhibitor at least six traces of the initial 5–
10% of the reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the same man-
ner and subtracted from the total observed rates. Stock solutions of
inhibitor (10 mM) were prepared in distilled–deionized water and
dilutions up to 0.01 nM were done thereafter with distilled–deion-
ized water. Inhibitor and enzyme solutions were preincubated to-
4.1.3.1. 1-(30-Aminophenyl)-6,7-dimethoxy-3,4-dihydroisoquin-
oline-2(1H)-sulfonamide (1i). Yield 35%. Mp 166–168 °C. 1H
NMR (CDCl3, d): 2.72–2.77 (1H, m, CH2CH2), 3.13–3.34 (2H, m,
CH2CH2), 3.69–3.84 (4H, m, CH3O and CH2CH2), 3.89 (3H, s,
CH3O), 4.03 (2H, br s, NH2), 5.89 (1H, s, CH), 6.42–7.13 (8H, m,
ArH). 13C NMR (CDCl3, d): 29.3, 39.5, 55.8, 55.9, 57.4, 111.0, 111.3,
114.2, 115.5, 119.3, 127.5, 129.2, 129.8, 146.0, 146.5, 147.6, 148.0.

3664
R. Gitto et al. / Bioorg. Med. Chem. 17 (2009) 3659–3664
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gether for 15 min at room temperature prior to assay, in order to
allow for the formation of the E-I complex. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3,
as reported earlier, and represent the mean from at least three dif-
ferent determinations. CA isoforms were recombinant ones ob-
tained as reported earlier by this group.24–27
4.4. Computational studies
The structure of compound 1j was generated using 2D/3D edi-
tor sketcher in CATALYST 4.10.28 software package and minimized
to their closest local energy minimum using a molecular mechan-
ics approach. Regarding the chirality of the asymmetric carbon, as
no experimental data on the biologically relevant conformations of
this molecule is available, it was arbitrarily decided to assign
‘undefined’ chirality.
To build conformational models of up to 250 conformers for
each molecule, the ‘best conformer generation’ option and a
10 kcal/mol energy cutoff were chosen.
15. Thiry, A.; Dogne, J. M.; Supuran, C. T.; Masereel, B. Curr. Top. Med. Chem. 2007, 7,
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Chem. 2008, 23, 271.
18. Gitto, R.; Ficarra, R.; Stancanelli, R.; Guardo, M.; De Luca, L.; Barreca, M. L.;
Pagano, B.; Rotondo, A.; Bruno, G.; Russo, E.; De Sarro, G.; Chimirri, A. Bioorg.
Med. Chem. 2007, 15, 5417.
19. McManus, J. M.; McFarland, J. W.; Gerber, C. F.; Mcamore, W. M.; Laubach, G. D.
J. Org. Chem. 1965, 8, 766.
Acknowledgments
20. Chapman, A. G.; Croucher, M. J.; Meldrum, B. S. Arzneimittelforschung 1984, 34,
1261.
21. Gitto, R.; Caruso, R.; Orlando, V.; Quartarone, S.; Barreca, M. L.; Ferreri, G.;
Russo, E.; De Sarro, G.; Chimirri, A. Farmaco 2004, 59, 7.
22. Litchfield, J. T., Jr. J. Pharmacol. Exp. Ther. 1949, 97, 399.
23. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
24. Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. J. Med. Chem. 2005, 48, 7860.
25. Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3828.
26. Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J.
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Financial support for this research by Fondo di Ateneo per la
Ricerca (PRA 2005—Università di Messina) and MiUR (FIRB_RB-
NE03YA3L_007) is gratefully acknowledged. The work was also
partially supported by grants from the EU projects DeZnIT and
Metoxia (to C.T.S.).
References and notes
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