Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.10.015

In a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline-2-sulfonamides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar concentration. In order to deepen the structure-activ

Full text of DOI:10.1016/j.bmc.2011.10.015

Bioorganic & Medicinal Chemistry 19 (2011) 7003–7007
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines
as selective carbonic anhydrase inhibitors
Rosaria Gitto a, , Francesca Maria Damiano a, , Laura De Luca a, Stefania Ferro a, Daniela Vullo b,
⇑
⇑
Claudiu T. Supuran b, Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-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 a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfon-
amides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar
concentration. In order to deepen the structure–activity relationships (SARs) and identify novel com-
pounds with improved activity, we synthesized a series of monomethoxy analogues of the previously
investigated dimethoxy derivatives. The evaluation of biological profile has been focused on in vitro
effects against several CA isoforms. The new monomethoxy derivatives showed higher hCA inhibitory
effects against several isoforms compared to the dimethoxy analogues. Particularly, some of these com-
pounds (e.g., 1b and 1h) showed low nanomolar KI values and excellent selectivity for hCA IX and hCA XIV
versus hCA I and II inhibition.
Received 26 July 2011
Revised 4 October 2011
Accepted 6 October 2011
Available online 12 October 2011
Keywords:
Isoquinolines
Microwave assisted organic synthesis
Carbonic anhydrase inhibitors
hCA IX
Ó 2011 Elsevier Ltd. All rights reserved.
hCA XIV
1. Introduction
O
NH2
S
The carbonic anhydrases (CAs, EC 4.2.1.1) are a family of zinc
metalloenzymes that catalyze the reversible hydration of CO2.
Since this reaction regulates a broad range of physiological func-
tions, the pharmacological modulation of CA activity could be
useful for the treatment of several human diseases.1 There are 16
human known CA (hCA) isoforms with different tissue distribution,
expression levels, and subcellular locations; some of these
isozymes (e.g., hCA II, IV, VA, VB, VII, IX, XII, XIII, and XIV) consti-
tute valid targets for the development of anticancer,2 antiglauco-
ma,3 antiobesity,4 or anticonvulsant drugs.5 However, the CA
diffuse localization in many tissues and organs limits their poten-
tial clinical applications. So the development of CA inhibitors (CAIs)
possessing high potency and selectivity against specific isoforms
represents an attractive strategy to obtain new active compounds
thus avoiding side effects and improving therapeutic safety.1,6
Most of known CAIs contain a specific moiety (sulfonamide, sulfa-
mate or sulfamide) able to coordinate the zinc ion of catalytic bind-
ing site7,8 (e.g., zonisamide, topiramate and its sulfamide analogue,
Fig. 1) inhibiting in this way the enzymatic activity. These inhibi-
tors also bear specific functional groups that interact with impor-
tant amino acid residues thus driving the selectivity against the
different isoforms as confirmed by X-ray crystallographic data of
O
S
O
X
O
O
NH2
O
O
N
O
O
O
Zonisamide
X = O, Topiramate
X = NH, Topiramate Sulfamide Analogue
Figure 1. Carbonic anhydrase inhibitors.
complexes between CAIs and isozymes available in the
literature.9–16
There is a significant interest in the development of selective
inhibitors targeting specific and druggable isoforms such as hCA IX
and hCA XIV.9,17 hCA IX is expressed in a limited number of normal
tissues, whereas it is overexpressed in many solid tumors and con-
sidered involved in critical processes connected with cancer pro-
gression.17 The expression of CA IX is strongly up-regulated by
hypoxia via the hypoxia inducible factor-1 (HIF-1) transcription fac-
tor.18,19 The overexpression of CA IX induces the pH imbalance of tu-
mor issue contributing significantly to the extracellular acidification
of solid tumor; thereby CA IX inhibitors could specifically bind hyp-
oxic tumor cells expressing this isoform, consequently they have
been proposed as antitumor agents.20 CA XIV is a transmembrane
⇑
Corresponding authors. Tel.: +39 0906766413; fax: +39 0906766402.
E-mail addresses: rgitto@unime.it (R. Gitto), fdamiano@unime.it (F.M. Damiano).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.015

7004
R. Gitto et al. / Bioorg. Med. Chem. 19 (2011) 7003–7007
MeO
R 2
MeO
MeO
MeO
MeO
O
O
N
N
NR 4
S
S
NH2
NH2
O
O
R 3
R 1
SO2NH2
III, R4 = H, SO2NH2
I, R1 = H, Br, Cl, CN, F, Me, NO2, NH2
2 = OMe
II, R3 = H, (cyclo)alkyl
R
1a-j R2 = H
Figure 2. Target compounds.
isozyme with the active site oriented extracellularly; it is highly
abundant in neurons and axons in the murine and human brain,
where it seems to play an important role in modulating excitatory
synaptic transmission.21
(TFA) to provide the desired isoquinolines 5a–h. Successively,
these intermediates reacted with a large excess of sulfamide
leading to the 1-aryl-6-methoxy-1,2,3,4-tetrahydroisoquinoline-
2-sulfonamides 1a–h as racemic mixture. Finally the aminophe-
nyl derivatives 1i–j were prepared by reduction of the corre-
sponding nitrophenyl analogues 1g–h. The structures of all
obtained compounds were supported by elemental analyses
and spectroscopic measurements.
Our previous studies indicated that some sulfamide-based het-
erocyclic compounds such as 1-aryl-6,7-dimethoxy-1,2,3,4-tetra-
hydroisoquinoline derivatives (I, Fig. 2) inhibited several CA
isoforms at micromolar concentration but showing poor selectiv-
ity.22 Two members of this series were also efficacious as anticon-
vulsants in audiogenic seizure test exhibiting more efficacy than
topiramate. Successively we extended our study to other 1,2,3,4-
tetrahydroisoquinoline-2-sulfonamides (II and III, Fig. 2)23,24 con-
taining some structural modifications in search of the enhancement
both of activity and selectivity. The results of this study were the
identification of potent and selective inhibitors towards hCA VII,
hCA IX and hCA XIV isoforms, making them as ideal candidates
for a potential treatment of several diseases in which these iso-
zymes are involved. In addition, some molecules displayed low
affinity for hCA II isoform that is considered responsible of un-
wanted side effects of CAIs currently in therapy. By means of dock-
ing experiments we suggested the main interactions that some
selective 1,2,3,4-tetrahydroisoquinoline-2-sulfonamides can en-
gage into catalytic pocket producing hCA IX and hCA XIV selectivity
over hCA II.23 Moreover, X-ray crystallographic studies of these
inhibitors in complex with wide-expressed hCA II isoform dis-
played the main interactions between its catalytic pocket and some
active 1,2,3,4-tetrahydroisoquinoline-2-sulfonamide derivatives.23
From these crystallographic data, it seems that the inhibitor
arrangement in catalytic pocket was also influenced by the meth-
oxy-groups and suitable substituents at C-1 position on isoquino-
line skeleton.23,25
The new synthesized 1-aryl-6-methoxy-1,2,3,4-tetrahydroiso-
quinoline-2-sulfonamides (1a–j) were subjected to evaluation of
their inhibition ability of the catalytic activity of hCA I, hCA II,
hCA IX and hCA XIV isozymes (Table 1). The data obtained were
compared with those of the previously reported dimethoxy ana-
logues Ia–j;22 topiramate and zonisamide were included as refer-
ence compounds in this study. The analysis of the screening
results displayed in Table 1 highlighted that the new synthesized
isoquinoline-N-sulfonamides 1a–j were generally micromolar
inhibitors (KI from 88 to 6003 nM values) of hCA I and hCA II iso-
forms, that are considered responsible of unwanted side-effects
characterizing non-selective inhibitors currently in therapy. On
the contrary, compounds 1a–j were potent inhibitors of hCA IX
and hCA XIV isoforms in the range of 5–78 nM and showed higher
activity than dimethoxy analogues Ia–j. Given that the unique dif-
ference between these two classes of molecules is the presence/ab-
sence of 7-methoxy group we can speculate that this substituent is
the main player influencing the interaction within catalytic site.
Collecting structure–activity relationship data we mainly found
that the effect of the nature of R1 substituent on the C-1 phenyl
ring is rather variable. The unsubstituted derivative 1a was a very
potent hCA XIV inhibitor (KI value of 7.9 nM), whereas its inhibi-
tory efficacy towards hCA IX was significant lower than other ana-
logues bearing aryl substituted moiety. The presence of nitro group
at 40- or 30-position of C-1 phenyl substituent optimizes the inhib-
itory effects of 1-aryl-6-methoxy-1,2,3,4-tetrahydroisoquinoline-
2-sulfonamides (e.g., 1h and 1g) towards druggable hCA IX and
hCA XIV isoforms. Particularly, the 40-nitro derivative 1h behaves
as a very potent hCA IX inhibitor being about 15-fold more potent
than unsubstituted compound 1a. The 40-fluorine derivative 1e
displayed a potency very similar to that of the highest active
nitro-analogue 1g as hCA XIV inhibitor (6.8 vs 6.3 nM), even if
we observed that the introduction of halo-substituent generally
was detrimental for the affinity towards all studied CA isoforms.
The compounds incorporating hydrophilic R1 substituents (e.g.,
amino or cyano groups) possessed a similar potency of analogs
containing lipophilic ones (see Table 1). Overall, the majority of
KI values was clustered within a restricted range thus suggesting
a slight impact on SAR of the 1-aryl substituents; in fact sulfona-
mides 1g, 1e, 1i, 1f, 1a, 1b, 1j, and 1h were potent hCA XIV inhib-
itors showing KI values ranging from 6.3 to 9.5 nM. Notable, one of
the most active compound 1g displayed KI value of 6.3 nM against
hCA XIV, that is about 1000-fold lower than corresponding KI value
To obtain further SAR information about this class of compounds
we herein report the synthesis of novel 1-aryl-6-methoxy-1,2,3,4-
tetrahydroisoquinoline-2-sulfonamides (R2 = H, see Fig. 2) in which
we maintained the moiety able to coordinate the Zinc ion of metal-
loenzyme giving inhibitory effects,8 and we explored the effects of
7-methoxysubstituent deletion as well as the role of the various
substituents on arylgroup at C-1 position. The new synthesized
compounds were evaluated as CAIs towards various isozymes
(e.g., hCA I, hCA II, hCA IX, and hCA XIV).
2. Results and discussion
Scheme 1 describes the synthetic pathway for obtaining the
1-aryl-6-methoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfonamides
1a–j. First, following a previously reported method in Microwave
Assisted Organic Synthesis (MAOS) conditions,22 we prepared the
precursors 5a–h. In particular, by reaction of the 2-(30-methoxy-
phenyl)ethylamine (2) with suitable benzaldehydes 3a–h and in
solvent free condition, we obtained the corresponding imine
intermediates 4a–h, which were treated with trifluoroacetic acid

R. Gitto et al. / Bioorg. Med. Chem. 19 (2011) 7003–7007
7005
CHO
MeO
MeO
MeO
a
b
+
N
NH
NH2
R 1
2
3 a-h
4 a-h
R 1
5 a-h
R 1
c
R1
R1
MeO
a
f
4-CH3
H
b
c
N
O
g
4-Br
4-Cl
3-NO2
S
NH2
h
i
j
4-NO2
3-NH2
4-NH2
O
1 a-j
R 1
iv
4-CN
4-F
d
e
Scheme 1. Reagents and conditions: (a) MW: 10 min, 90 °C, 150 W; (b) TFA MW: 10 min, 90 °C, 150 W; (c) CH3CH(OCH3)2, NH2SO2NH2, two steps 20 min, 100 °C, 150 W; iv)
Zn/HCl conc.,
D, 2 h.
1b (KI value of 8.3 nM) showed excellent hCA XIV selectivity dis-
playing KI values more than 4000 against hCA I and hCA II.
In conclusion, this study allowed us to identify new CAIs con-
taining the isoquinoline scaffold, several of which proved to be very
efficient (KI <10 nM) inhibitors exhibiting good selectivity towards
the established antitumor target hCA IX and the potentially drugga-
ble one hCA XIV. Considering the structure–property relationships
we found that (a) the introduction of an additional 7-methoxy
group dramatically decreases the inhibitory efficacy; (b) the nature
of the substituent on 1-aryl moiety plays a key role in selectivity
over off-target hCA I and hCA II isozymes; (c) whereas this substitu-
ent seems to be not extremely relevant to control the potency of
inhibitory effects against hCA IX and hCA XIV. Finally, the most ac-
tive compounds were found to be more potent and selective inhib-
itors than well known CAIs such as topiramate and zonisamide.
Overall, these findings contribute to improve our knowledge on
SAR of the CAIs containing this sulfamide-based heterocyclic scaf-
fold which may allow interesting future developments for obtain-
ing isoform-selective inhibitors of these enzymes.
Table 1
Inhibition of hCA I, hCA II, hCA IX and hCA XIV isoforms by 1-aryl-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinolines-2-sulfonamides (Ia–j), 1-aryl-6-methoxy-1,2,3,4-tet-
rahydroisoquinoline-2-sulfonamides (1a–j), topiramate and zonisamide
MeO
O
N
R 2
S
NH2
O
R 1
R1
R2
KIa (nM)
hCA I
hCA II
hCA IX
hCA XIV
Iab
H
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
H
H
H
H
H
H
H
H
8980
25700
9440
4070
4430
3490
7690
330
3830
8070
6003
4790
101
995
5134
957
3514
5213
4815
4793
250
15700
10710
5300
4240
14980
10770
3820
600
9310
11300
422
4184
104
91
476
88
293
320
99.7
222
10
35
8440
8200
7540
4980
5590
6750
6450
480
8130
6780
78
60.7
21.8
7.0
31
7.5
3860
7020
8980
3040
6930
7050
5350
210
Ibb
40-Br
40-Cl
40-CN
40-F
Icb
Idb
Ieb
3. Experimental section
3.1. Chemistry
Ifb
40-Me
Igb
30-NO2
40-NO2
30-NH2
40-NH2
H
Ihb
Iib
6670
3070
7.9
Ijb
All starting materials and reagents commercially available
(Sigma–Aldrich Milan, Italy) were used without further purifica-
tion. Microwave-assisted reactions were carried out in a CEM
focused Microwave Synthesis System. Melting points were deter-
mined on a Buchi B-545 apparatus and are uncorrected. Elemental
analyses (C, H, N) were carried out on a Carlo Erba Model 1106 Ele-
mental Analyzer and the results are within 0.4% of the theoretical
values. Merck silica gel 60 F254 plates were used for analytical TLC.
Rf values were determined on TLC plates using a mixture of CH2Cl2/
CH3OH (96/4) as eluent. Flash Chromatography (FC) was carried
out on a Biotage SP1 EXP. 1H 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 cou-
pling constants (J) in Hz. All exchangeable protons were confirmed
by addition of D2O.
1a
1b
1c
1d
1e
1f
1g
1h
40-Br
8.3
40-Cl
53.9
19.8
6.8
7.7
6.3
9.5
7.1
8.6
1460
5250
40-CN
40-F
40-Me
30-NO2
40-NO2
30-NH2
40-NH2
16
5.0
18
12
58
5.1
1i
1j
H
Topiramateb
Zonisamideb
56
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.
b
Ref. 22
3.1.1. General procedure for the synthesis of 1-aryl-6-methoxy-
1,2,3,4-tetrahydroisoquinolines (5a–h)
of 5350 nM measured for analogue Ig. Furthermore, compounds
1a–j were also more selective against hCA IX and hCA XIV over
the off-target hCA II isoform. The most active hCA IX inhibitor 40-
nitro derivative 1h (KI value of 5.0 nM) had >1000-fold selectivity
over hCA I as well as >60-fold selectivity over hCA II. Compound
A mixture of 2-(30-methoxyphenyl)ethylamine (2) (2.0 mmol)
and suitable aldehyde 3a–h (2.4 mmol), was placed in a cylindrical
quartz tube (ø2 cm), in solvent free condition, then stirred and

7006
R. Gitto et al. / Bioorg. Med. Chem. 19 (2011) 7003–7007
irradiated in a microwave oven at 150 W for 10 min at 90 °C; after
cooling to room temperature trifluoroacetic acid (2.5 mL) was
added to crude intermediates 4a–h obtained in the previous step
and the mixture was irradiated at 150 W for 10 min at 90 °C. The
reaction was quenched by adding water and the mixture was basi-
fied (pH 8–9) with sodium hydroxide 2 N (10 mL). The crude prod-
uct was purified by crystallization with EtOH to afford compounds
5a–h as white powder. Compounds 5a–c, 5e, 5g–h were already
synthesized; by means of the application of microwave irradiation
as well as the employment of a different synthetic pathway these
compounds were re-synthesized thus reducing reaction times and
improving in same cases the yields; their analytical data are in
accordance with literature.26
NH2), 6.01 (1H, s, CH), 6.75–6.79 (2H, m, ArH), 6.88 (1H, d, ArH),
7.20 (2H, d, J = 8.5, ArH), 7.29 (2H, d, J = 8.5, ArH). Anal. (C, H, N).
Anal. Calcd for C16H17ClN2O3S: C, 54.47; H, 4.86; N, 7.94. Found:
C, 54.10; H, 4.46; N, 7.54.
3.1.2.4. (R,S)-1-(40-Cyanophenyl)-6-methoxy–1,2,3,4-tetrahydro-
isoquinoline-2-sulfonamide (1d).
Yield 60%. Mp 158–
162 °C. Rf = 0.35. 1H NMR (DMSO-d6) d: 2.66–2.71 (1H, m, CH2CH2),
3.14–3.31 (2H, m, CH2CH2), 3.52–3.58 (1H, m, CH2CH2), 3.82 (3H, s,
OCH3), 5.88 (1H, s, CH), 6.75–6.80 (2H, m, ArH), 6.94 (2H, br s,
NH2), 6.97–7.00 (1H, m, ArH), 7.25 (2H, d, J = 7.4, ArH), 7.78 (2H,
d, J = 7.4, ArH). Anal. Calcd for C17H17N3O3S: C, 59.46; H, 4.99; N,
12.24. Found: C, 59.16; H, 5.39; N, 12.64.
3.1.1.1. (R,S)-1-(40-Cyanophenyl)-6-methoxy-1,2,3,4-tetrahydro-
isoquinoline (5d).
3.1.2.5.
droisoquinoline-2-sulfonamide (1e).
(R,S)-1-(40-Fluorophenyl)-6-methoxy-1,2,3,4-tetrahy-
Yield 30%. Mp 46–
Yield 40%. Mp 211–215 °C. Rf = 0.30. 1H
NMR (DMSO-d6) d: 2.66–3.06 (4H, m, CH2CH2), 3.63 (3H, s,
OCH3), 4.96 (1H, s, CH), 6.50 (1H, d, J = 8.5, ArH), 6.56–6.60 (1H,
dd, J = 3.2, J = 7.5, ArH), 6.68 (1H, d, J = 3.2, ArH), 7.29 (2H, d,
J = 7.5, ArH), 7.78 (2H, d, J = 7.5, ArH), 7.91 (br s, 1H, NH). Anal.
Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60. Found: C, 76,85;
H, 6.50; N, 10.26.
50 °C. Rf = 0.81. 1H NMR (CDCl3) d: 2.74–2.80 (1H, m, CH2CH2),
3.12–3.29 (2H, m, CH2CH2), 3.81–3.83 (3H, m, OCH3 and 1H,
CH2CH2), 4.12 (2H, br s, NH2), 6.01 (1H, s, CH), 6.73–6.77 (2H, m,
ArH), 6.87–6.89 (1H, m, ArH), 6.97–7.23 (4H, m, ArH). Anal. (C, H,
N). Anal. Calcd for C16H17FN2O3S: C, 57.13; H, 5.09; N, 8.33. Found:
C, 57.53; H, 5.49; N, 8.73.
3.1.1.2.
droisoquinoline (5f).
(R,S)-6-Methoxy-1-(40-methylphenyl)-1,2,3,4-tetrahy-
3.1.2.6.
droisoquinoline-2-sulfonamide
(R,S)-6-Methoxy-1-(40-methylphenyl)-1,2,3,4-tetrahy-
(1f). Yield 45%. Mp
Yield 45%. Mp 105–108 °C. Rf = 0.06.
1H NMR (CDCl3) d: 2.45 (3H, s, Ch3), 2.95–3.02 (1H, m, CH2CH2),
3.19–322 (2H, m, CH2CH2), 3.30–3.38 (1H, m, CH2CH2), 3.90 (3H,
s, OCH3), 5.47 (1H, s, CH), 6.73–6.82 (3H, m, ArH), 7.25–7.38 (4H,
m, ArH). Anal. Calcd for C17H19NO: C, 80.60; H, 7.56; N, 5.53.
Found: C, 81.02; H, 7.96; N, 5.13.
111–115 °C. Rf = 0.83. 1H NMR (DMSO-d6) d: 2.25 (3H, s, CH3),
2.62–2.67 (1H, m, CH2CH2), 3.03–3.14 (2H, m, CH2CH2), 3.54–
3.58 (1H, m, CH2CH2), 3.74 (3H, s, OCH3), 5.81 (1H, s, CH), 6.73–
6.78 (2H, m, ArH), 6.86 (2H, br s, NH2), 6.91–6.94 (1H, m, ArH),
7.03 (2H, d, J = 7.9, ArH), 7.09 (2H, d, J = 7.9, ArH). Anal. (C, H, N).
Anal. Calcd for C17H20N2O3S: C, 61.42; H, 6.06; N, 8.43. Found: C,
61.03; H, 5.66; N, 8.03.
3.1.2. General procedure for the synthesis of 1-aryl-6-methoxy-
1,2,3,4-tetrahydroisoquinoline-2-sulfonamides (1a–h)
A solution of the appropriate 1-aryl-6-methoxy-1,2,3,4-tetrahy-
droisoquinoline 5a–h (1.0 mmol) and sulfamide (6 mmol) in dime-
thoxyethane (2 mL) was placed in a cylindrical quartz tube (ø2 cm),
then stirred and irradiated in a microwave oven at 150 W for two
steps of 20 min at 100 °C. The reaction was quenched by addition
of water (5 mL) and extracted with ethyl acetate (3 ꢀ 5 mL). The or-
ganic layer was washed with an aqueous saturated solution of NaH-
CO3 (2 ꢀ 5 mL), dried over Na2SO4 and concentrated until dryness
under reduced pressure. From the obtained residue compound 1d
and 1g were crystallized using diethyl ether to give white powders.
The other compounds were purified by flash chromatography using
a mixture of CH2Cl2/CH3OH (96/4) as eluent.
3.1.2.7. (R,S)-6-Methoxy-1-(30-nitrophenyl)-1,2,3,4-tetrahydro-
isoquinoline-2-sulfonamide (1g). Yield 60%. Mp 140–144 °C.
Rf = 0.71. 1H NMR (DMSO-d6) d: 2.70–2.77 (1H, m, CH2CH2),
3.06–3.27 (2H, m, CH2CH2), 3.51–3.57 (1H, m, CH2CH2), 3.76 (3H, s,
OCH3), 5.99 (1H, s, CH), 6.78–6.84 (2H, m, ArH), 7.01 (2H, br s,
NH2), 7.05–7.08 (1H, m, ArH), 7.59–7.68 (2H, m, ArH), 8.01–8.14
(2H, m, ArH). Anal. (C, H, N). Anal. Calcd for C16H17N3O5S: C, 52.88;
H, 4.72; N, 11.56. Found: C, 52.55; H, 4.38; N, 11.16.
3.1.2.8. (R,S)-6-Methoxy-1-(40-nitrophenyl)-1,2,3,4-tetrahydro-
isoquinoline-2-sulfonamide (1h).
Yield 40%. Mp 55–59 °C.
Rf = 0.77. 1H NMR (CDCl3) d: 2.73–3.79 (4H, m, CH2CH2), 3.81
(3H, s, OCH3), 4.42 (2H, br s, NH2), 6.06 (1H, s, CH), 6.76–6.93
(3H, m, ArH), 7.43 (2H, d, J = 8.8, ArH), 8.13 (2H, d, J = 8.8, ArH).
Anal. (C, H, N). Anal. Calcd for C16H17N3O5S: C, 52.88; H, 4.72; N,
11.56. Found: C, 52.48; H, 4.30; N, 11.18.
3.1.2.1. (R,S)-6-Methoxy-1-phenyl-1,2,3,4-tetrahydroisoquino-
line-2-sulfonamide (1a). Yield 55%. Mp 124–128 °C. Rf = 0.80.
1H NMR (CDCl3) d :2.75–3.78 (4H, m, CH2CH2), 3.81 (3H, s, OCH3),
4.11 (2H, br s, NH2), 6.02 (1H, s, CH), 6.73 (2H, m, ArH), 6.88 (1H, m,
ArH), 7.27–7.35 (5H, m, ArH). Anal. Calcd for C16H18N2O3S: C,
60.36; H, 5.70; N, 8.80. Found: C, 59.96; H, 6.12; N, 9.21.
3.1.3. General procedure for the synthesis of 1-(30-amino-
phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-2-
sulfonamide (1i) and 1-(40-aminophenyl)-6-methoxy-1,2,3,
4-tetrahydroisoquinoline-2-sulfonamide (1j)
3.1.2.2.
droisoquinoline-2-sulfonamide (1b).
(R,S)-1-(40-Bromophenyl)-6-methoxy-1,2,3,4-tetrahy-
Yield 40%. Mp 110–
A solution of appropriate nitro-derivatives (1g or 1h, 0.6 mmol)
in 3 mL of HCl and 4 mL of EtOH was vigorously stirred 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 (10 mL), and then ex-
tracted with ethyl acetate (3 ꢀ 5 mL). The organic phase washed
with water, dried over Na2SO4 and evaporated. The residue was
crystallized from ethanol to give the desired products 1i and 1j.
114 °C. Rf = 0.81. 1H NMR (DMSO-d6) d: 2.64–2.69 (1H, m, CH2CH2),
3.02–3.17 (2H, m, CH2CH2), 3.52–3.57 (1H, m, CH2CH2), 3.75 (3H, s,
OCH3), 5.83 (1H, s, CH), 6.75–6.79 (2H, m, ArH), 6.92 (2H, br s,
NH2), 6.96–6.99 (1H, d, ArH), 7.13 (2H, d, J = 8.2, ArH), 7.48 (2H,
d, J = 8.2, ArH). Anal. (C, H, N). Anal. Calcd for C16H17BrN2O3S: C,
48.37; H, 4.31; N, 7.05. Found: C, 47.97; H, 3.91; N, 7.45.
3.1.2.3.
(R,S)-1-(40-Chlorophenyl)-6-methoxy-1,2,3,4-tetrahy-
droisoquinoline-2-sulfonamide (1c).
Yield 60%. Mp 100–
3.1.3.1.
droisoquinoline-2-sulfonamide (1i).
120 °C. Rf = 0.49. 1H NMR (DMSO-d6) d: 2.62–2.87 (1H, m, CH2CH2),
(R,S)-1-(30-Aminophenyl)-6-methoxy-1,2,3,4-tetrahy-
Yield 42%. Mp 116–
104 °C. Rf = 0.82. 1H NMR (CDCl3) d: 2.64–2.69 (1H, m, CH2CH2),
3.23–3.80 (3H, m, CH2CH2), 3.82 (3H, s, OCH3), 4.12 (2H, br s,

R. Gitto et al. / Bioorg. Med. Chem. 19 (2011) 7003–7007
7007
3.12–3.29 (2H, m, CH2CH2), 3.54–3.68 (1H, m, CH2CH2), 3.73 (3H, s,
References and notes
OCH3), 5.84 (1H, s, CH), 6.73–6.76 (2H, m, ArH), 6.77 (2H, br s,
NH2), 6.90–6.96 (1H, m, ArH), 7.07–7.20 (6H, m, ArH). Anal. Calcd
for C16H19N3O3S: C, 57.64; H, 5.74; N, 12.60. Found: C, 58.04; H,
6.14; N, 12.26.
1. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
2. Thiry, A.; Supuran, C. T.; Masereel, B.; Dogne, J. M. J. Med. Chem. 2008, 51, 3051.
3. Mincione, F.; Scozzafava, A.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 649.
4. De Simone, G.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 879.
5. Thiry, A.; Dogne, J. M.; Supuran, C. T.; Masereel, B. Curr. Top. Med. Chem. 2007, 7,
855.
3.1.3.2.
droisoquinoline-2-sulfonamide
(R,S)-1-(40-Aminophenyl)-6-methoxy-1,2,3,4-tetrahy-
(1j). Yield 70%. Mp
6. Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467.
7. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146.
8. Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C. T. Med. Res. Rev. 2006,
26, 767.
9. Supuran, C. T.; Di Fiore, A.; Alterio, V.; Monti, S. M.; De Simone, G. Curr. Pharm.
Des. 2010, 16, 3246.
10. Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal, M.; Robbins, A. H.;
McKenna, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2011, 54, 3977.
11. Di Fiore, A.; Truppo, E.; Supuran, C. T.; Alterio, V.; Dathan, N.; Bootorabi, F.;
Parkkila, S.; Monti, S. M.; De Simone, G. Bioorg. Med. Chem. Lett. 2010, 20, 5023.
12. Temperini, C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2009, 52,
322.
175–179 °C. Rf = 0.50. 1H NMR (DMSO-d6) d: 2.59–2.63 (1H, m,
CH2CH2), 3.02–3.08 (2H, m, CH2CH2), 3.54 (1H, m, CH2CH2), 3.72
(3H, s, OCH3), 4.98 (2H, br s, NH2), 5.69 (1H, s, CH), 6.42–6.87 (9H,
m, ArH). Anal. Calcd for C16H19N3O3S: C, 57.64; H, 5.74; N, 12.60.
Found: C, 58.04; H, 6.14; N, 12.36.
3.2. CA inhibition assay
13. Di Fiore, A.; Monti, S. M.; Hilvo, M.; Parkkila, S.; Romano, V.; Scaloni, A.;
Pedone, C.; Scozzafava, A.; Supuran, C. T.; De Simone, G. Proteins 2009, 74, 164.
14. Srivastava, D. K.; Jude, K. M.; Banerjee, A. L.; Haldar, M.; Manokaran, S.; Kooren,
J.; Mallik, S.; Christianson, D. W. J. Am. Chem. Soc. 2007, 129, 5528.
15. Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B. S.; Robbins, A. H.;
Scozzafava, A.; McKenna, R.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 3105.
16. De Simone, G.; Supuran, C. T. Biochim. Biophys. Acta 2010, 1804, 404.
17. Guler, O. O.; De Simone, G.; Supuran, C. T. Curr. Med. Chem. 2010, 17, 1516.
18. Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S.; Ostlund, C.; Ahmadi, A.; Kyle, A.;
Auf dem Keller, U.; Leung, S.; Huntsman, D.; Clarke, B.; Sutherland, B. W.;
Waterhouse, D.; Bally, M.; Roskelley, C.; Overall, C. M.; Minchinton, A.;
Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.;
Dedhar, S. Cancer Res. 2011, 71, 3364.
19. Chiche, J.; Ilc, K.; Laferriere, J.; Trottier, E.; Dayan, F.; Mazure, N. M.; Brahimi-
Horn, M. C.; Pouyssegur, J. Cancer Res. 2009, 69, 358.
20. Ahlskog, J. K.; Dumelin, C. E.; Trussel, S.; Marlind, J.; Neri, D. Bioorg. Med. Chem.
Lett. 2009, 19, 4851.
21. Hilvo, M.; Supuran, C. T.; Parkkila, S. Curr. Top. Med. Chem. 2007, 7, 893.
22. Gitto, R.; Ferro, S.; Agnello, S.; De Luca, L.; De Sarro, G.; Russo, E.; Vullo, D.;
Supuran, C. T.; Chimirri, A. Bioorg. Med. Chem. 2009, 17, 3659.
23. Gitto, R.; Agnello, S.; Ferro, S.; De Luca, L.; Vullo, D.; Brynda, J.; Mader, P.;
Supuran, C. T.; Chimirri, A. J. Med. Chem. 2010, 53, 2401.
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity. Phenol
red (at a concentration of 0.2 mM) has been used as indicator, work-
ing 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), following
the initial rates of the CA-catalyzed CO2 hydration reaction for a per-
iod of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and inhibition con-
stants. 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 manner and sub-
tracted 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-deionized water.
Inhibitor and enzyme solutions were preincubated together 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 ob-
tained by non-linear least-squares methods using PRISM 3, as re-
ported earlier, and represent the mean from at least three
different determinations. CA isoforms were recombinant ones ob-
tained as reported earlier by this group.27–30
24. Gitto, R.; Agnello, S.; Ferro, S.; Vullo, D.; Supuran, C. T.; Chimirri, A.
ChemMedChem 2010, 5, 823.
25. Mader, P.; Brynda, J.; Gitto, R.; Agnello, S.; Pachl, P.; Supuran, C. T.; Chimirri, A.;
Rezacova, P. J. Med. Chem. 2011, 54, 2522.
26. De Luca, L.; Gitto, R.; Barreca, M. L.; Caruso, R.; Quartarone, S.; Citraro, R.; De
Sarro, G.; Chimirri, A. Arch. Pharm. (Weinheim) 2006, 339, 388.
27. Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3828.
28. Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. J. Med. Chem. 2005, 48, 7860.
Acknowledgments
29. Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2007, 50, 381.
30. Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.; Onishi, S.; Takeuchi, H.;
Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 2117.
Financial support for this research by MiUR (Prin2008, grant
number No 20085HR5JK_002) is gratefully acknowledged. Research
from CTS lab was financed by a 7th FP grant (METOXIA project).