Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I, II, IX and XII

Article abstract of DOI:10.1016/j.bmc.2016.01.020

Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space.

Full text of DOI:10.1016/j.bmc.2016.01.020

Accepted Manuscript
Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activ-
ity against human carbonic anhydrase isoforms I, II, IX and XII
Mustafa Durgun, Hasan Turkmen, Mariangela Ceruso, Claudiu T. Supuran
PII:
S0968-0896(16)30019-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.020
BMC 12763
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
4 January 2016
8 January 2016
9 January 2016
Please cite this article as: Durgun, M., Turkmen, H., Ceruso, M., Supuran, C.T., Synthesis of 4-sulfamoylphenyl-
benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I, II, IX and XII,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.020
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Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against
human carbonic anhydrase isoforms I, II, IX and XII
Mustafa Durguna*, Hasan Turkmenb, Mariangela Cerusoc, Claudiu T. Supuran c,d
*
a Department of Chemistry, Faculty of Arts and Sciences, Harran University, 63190, Sanliurfa, Turkey
b Department of Medical Pharmacology, Faculty of Medicine, Harran University, 63190, Sanliurfa, Turkey
c Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della
Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
d Università degli Studi di Firenze, Neurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Via
U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
Abstract. Imine derivatives were obtained by condensation of sulfanilamide with substituted
aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride,
leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. These
sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC
4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII
(transmembrane, tumor-associated enzymes). We noted that the compounds incorporating
secondary amine moieties showed a better inhibitory activity against all CA isozymes
compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors
were detected, whereas the activity against hCA I was less potent. The secondary amines
incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for
designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the
facile preparation and possibility to explore a vast chemical space.
Keywords: carbonic anhydrase; sulfonamide; Schiff base; secondary amine; isoform
*Correspondingauthors. Tel./fax: +904143443000 (M.D.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: mustafadurgun@harran.edu.tr, mustafadurgun@yahoo.com (M.Durgun), claudiu.
supuran@unifi.it (C.T. Supuran).
1

1.Introduction
The carbonic anhydrase (CA, EC 4.2.1.1) family of metalloenzymes catalyzes the
-
reversible hydration of CO2 to HCO3 , and this regulatory reaction supports many
physiological processes associated with pH control, ion transport, fluid secretion and several
biosynthetic processes.1-3 The inhibition of CAs has been exploited clinically for many
decades, as most CA isoforms of the 15 known in humans are therapeutic targets with the
potential to be inhibited or activated.1-3 The physiologically dominant isoform is the red blood
cell CA II (present in many other tissues) and its inhibitors have widespread use in a variety
of applications as diuretics, antiglaucoma, and antiepileptic agents.1-4 For such reasons, the
design of novel classes of potent, possibly isoform-selective inhibitors targeting other human
(h) CA isoforms may lead to clinical applications for treating a multitude of diseases such as
edema, epilepsy, obesity, neuropathic pain and other neurological disorders, etc.2–4 More
recently, two CA isoforms, hCA IX and XII, have been shown to play an important role in
tumor progression5 and one CA IX/XII selective sulfonamide inhibitor is presently in Phase I
clinical trials for the management of metastatic solid tumors.5f
The importance of sulfonamides as pharmacological agents appeared when Domagk6
showed that sulfanilamide was the metabolite of the antibacterial drug Prontosil and possess
excellent bacteriostatic effects. Later, a great number of sulfanilamide derivatives started to be
employed in clinical medicine as antibacterials,7 whereas other sulfonamides have been and
are still used as pharmacological agents with a wide variety of biological actions.8-14 All of
them were designed considering sulfanilamide as lead molecule.8 Nowadays sulfonamides
have found widespread use in a variety of applications, among which as antibacterials,9
anticancer agents,5 diuretics,10 CA inhibitors (CAIs) with a variety of uses,11 hypoglycaemic
agents,12 anti-hyperthyroidism agents 13 and protease inhibitors.14
Schiff bases incorporating aromatic/heterocyclic sulfonamide moieties in their
molecules have been extensively investigated as CAIs.15-17 Some of these derivatives were the
first ones reported to have some selectivity for the inhibition of some human (h) CA isoforms,
such as hCA I, II or IV. The first Schiff bases obtained from aromatic sulfonamides and
aromatic/heterocyclic aldehydes showed CA IV-selective inhibition patterns, whereas they
inhibited less the off-target isoform CA II. 15-17
Schiff base compounds have also been extensively investigated due to their role as
chelating agents for main group and transition metal ions.18,19 In the present study, we extend
our previous research in the field of sulfonamide Schiff bases and their derivatives, and report
2

a new series of sulfanilamide-imine compounds as well as the corresponding secondary
amines obtained by their reduction. These sulfonamides were investigated as inhibitors of
four physiologically relevant enzymes, the cytosolic isoforms hCA I and II (drug targets for
antiglaucoma agents), as well as, the transmembrane, tumor-associated ones hCA IX and XII
(drug targets for antitumor agents, and respectively antiglaucoma/antitumor agents).
2. Results and Discussion
2.1. Chemistry
Schiff base derivatives (imine compounds) 1–11 were obtained by condensation of the
sulfanilamide (4-aminobenzenesulfonamide) with the corresponding aromatical dehydes in
methanol, with catalytic amounts of formic acid (Scheme 1) as reported earlier for many such
sulfonamides by our groups.15-17 The secondary amine sulfonamides 1i-11i were subsequently
prepared by reduction of the imine derivatives 1-11 with NaBH4 in methanol. The drug design
strategy for obtaining these secondary amines, which are not prone to hydrolysis is based on
the fact we observed that many Schiff bases of ariomatic sulfonamides, including
sulphanilamide, tend to hydrolyze to the starting materials by storage at room temperature for
several weeks.17 However, the reduced, secondary amines (suchas 1i-11i prepared here) are
stable and do not undergo any hydrolytic or decomposition process even after month or years
of storage.
All the synthesized compounds were fully characterized by analytical and spectral data. Some
imine compounds have been already synthesized and characterized and our results are in
complete agreement with those reported in the literature. The X-ray structure of one of the 4-
{[4-(dimethylamino)benzylidene]amino}benzenesulfonamide (11) has been published.18d In
the study, among the aromatic aldehyde derivatives used in the synthesis of the Schiff bases
were 2-hydroxybenzaldehyde (1), 3,5-dibromo-2-hydroxybenzaldehyde (2), benzaldehyde
(3), 2-hydroxy-3-methylbenzaldehyde (4), 4-methoxybenzaldehyde (5), 5-bromo-2-
hydroxybenzaldehyde (6), 4-methylbenzaldehyde (7), 5-chloro-2-hydroxybenzaldehyde (8),
4-(benzyloxy)benzaldehyde(9), 3,5-dichloro-2-hydroxybenzaldehyde (10), 4-(dimethylamino)
benzaldehyde(11).19 They were chosen in such a way as to incorporate both hydrophilic (OH,
dimethylamino) as well as hydrophobic (methyl, methoxy, phenethyloxy, halogens) moieties,
or combination of the two types of moieties in their molecules, in order to explore structure-
activity and a diverse chemical space.
3

Scheme 1. Synthesis of the sulfonamide derivatives 1-11 and 1i-11i.
In general, FT-IR spectra of imine compounds (1–11) were characterized by the
presence of a strong band atabout 1620 cm-1 due to the stretching of the C=N bond, while it
was not observed in the spectra of amine compounds (1i–11i) were not observed this strong
1
band (C=N).On the other hand, in the H NMR spectra of 1–11 were observed a singlet at
1
about 8.50 ppm that attributed to azomethine (CH=N) chemical shift, while in the H NMR
spectrum of 1i–11i this peak (CH=N) was not observed. The derivatives 1i–11i showed a
peak at about 4.25 ppm due to the presence of -CH2- group. In the 13C-NMR spectrum, the
signals at about 163 ppm were assigned to azomethine group (CH=N) for 1–11. These signals
were not observed for 1i-11i (see experimental section for details).
2.2. CA inhibition
We have assayed the sulfonamides prepared here for the inhibition of four physiologically
relevant CAs, the cytosolic isoforms hCA I (offtarget isoform), hCA II (drug targets for
antiglaucoma agents but offtarget when other isoforms are considered for the inhibition,
suichas CA VA/VB, VII, IX and XII), as well as the transmembrane, tumor-associated ones
hCA IX and XII (drug targets for antitumor agents – hCA IX – and respectively
antiglaucoma/antitumor agents, hCA XII).
4

Table 1. Inhiibtion data of hCA I, II, IX and XII with sulfonamides 1-11 and 1i-11i reported
here, and acetazolamide AAZ as standard drug, by a stopped-flow CO2 hydrase assay.20
KI (nM)*
Compound
hCA I
250
hCA II
12
hCA IX
25
hCA XII
6.0
AAZ
1
428
258
952
10.3
38.5
11.1
11.6
11.2
10.4
9.9
2
>50000
>50000
463
251
360
3
289
454
4
264
465
5
>50000
458
464
883
6
248
257
7
>50000
>50000
>50000
442
469
23.7
47.9
36.0
35.6
41.5
8
404
9.4
9
>50000
290
9.7
10
11
7.5
421
328
8.8
1i
2i
48.7
42.3
20.0
24.8
21.9
42.5
17.9
30.7
37.9
20.9
21.5
8.1
2.1
1.7
2.2
1.8
3.4
1.9
2.6
3.5
2.3
1.9
2.9
2.3
2.2
2.3
2.2
2.6
2.1
2.4
2.5
2.2
2.4
3.0
31.9
19.8
131
3i
4i
5i
427
6i
213
7i
121
8i
21.0
404
9i
10i
11i
138
> 50000
*Errors were in the range of 5-10 % of the reported values (data not shown), from 3
different assays.
The following SAR can be observed from data of Table 1:
(i) The off-target slow isoform hCA I was poorly inhibited by most Schiff bases 1-11 (KI > 50
µM) except few derivatives (1, 4, 6, 10 and 11) which were weak inhibitors with inhibition
constants ranging between 421 and 463 nM. On the contrary, the secondary amines 1i-11i
showed a much better inhibitory action against this isoform, with KIs in the range of 17.9 –
48.7 nM (Table 1): Thus, the reduction of the imine functionality to the secondary amine
probably allows the inhibitors to adopt a favorable conformation when bound to the enzyme,
which cannot be adopted by the derivatives 1-11 which incorporate the double bond. This
effect has in fact observed earlier by this group17e for a series of sulfonamides incorporating a
longer linker between the two aromatic rings, of the type CH2CH2NHCH2 instead of NHCH2.
5

(ii) The dominant cytosolic isoform hCA II was weakly inhibited by the Schiff bases 1-11
(KIs in the range of 248 – 469 nM, except 9 which was not inhibited up to 50 µM) whereas
the secondary amines 1i-11i were highly effective CAIs, with KIs in the range of 1.7 – 8.1
nM. Thus all substitution patterns from these secondary amines lead to highly effective CAIs,
with difficulty to discuss a real SAR, since the variation of the inhibition constants is
minimal, as shown above.
Table 2. Selectivity ratios for inhibiting the tumor-associated isoforms hCA IX and XII over
the cytosolic ones hCA I and II, with AAZ and compounds 1-11 and 1i-11i.
Selectivity ratio
Compound
hCA II / hCA
XII
hCA I / hCA IX
hCA II / hCA IX hCA I / hCA XII
AAZ
1
10.00
0.45
0.48
0.27
41.67
41.57
2.00
25.04
6.52
2
>138.89
>110.13
1.00
0.70
>1298.70
>4504.50
39.91
3
0.64
26.04
22.76
41.43
23.85
47.37
42.98
>5154.64
38.67
37.27
2.700
0.066
0.086
0.017
0.004
0.016
0.016
0.124
0.009
0.017
0.00003
4
0.57
5
>56.63
1.78
0.53
>4464.29
44.04
6
0.96
7
>2109.70
>1043.84
>1388.89
12.42
19.79
8.43
>5050.51
>5319.15
>5154.64
58.93
8
9
>1388.89
8.15
10
11
1i
2i
3i
4i
5i
6i
7i
8i
9i
10i
11i
10.14
7.90
47.84
16.793
18.391
9.091
2.793
0.913
0.773
0.957
0.818
1.308
0.905
1.083
1.400
1.045
0.792
16.233
1.326
1.010
10.783
9.955
0.189
0.051
16.346
8.524
0.200
0.148
12.792
15.160
9.500
1.462
0.094
0.151
8.958
0.0004
(iii) The inhibition pattern of the trans-membrane isoform hCA IX with these compounds is
rather similar to what discussed above for hCA II: the imines 1-11 were poorer inhibitors
compared to the corresponding secondary amines 1i-11i. For the first subseries, derivatives 1-
5 showed KIs of 257-962 nM, being thus rather weak inhibitors, whereas 7-11 were better
hCA IX inhibitors, with KIs of 23.7 – 47.9 nM, the same range as the clinically used
6

sulfonamide AAZ (Table 1). It is obvious that rather small variations in the substitution
pattern at the aldehyde side of the imines (e.g., the pairs 3-7, differing by a methyl group; 1-8,
differing by a Cl atom) lead to important differences of the CA IX inhibitory pattern. The
secondary amines on the other hand were all highly similar in their inhibition behaviour
towards hCA IX , again with a very limited variation of the KIs, i.e., 2.1 – 3.9 nM (Table 1).
(iv) The most interesting inhibition profile was however observed for hCA XII. For this
isoform the Schiff bases 1-11 showed a similar behaviour of potent inhibitor, with KIs in the
range of 7.5 – 38.5 nM, whereas the secondary amines 1i-11i were generally weaker
inhibitors compared to the corresponding Schiff bases, with KIs in the range of 3.0 – 427 nM.
It is not possible to explain this very intriguing behaviour of the Schiff base/cognate
secondary amine, since no X-ray crystal structures for these types of CAIs were reported so
far.
(v) The selectivity ratios for inhibiting the tumor-associated isoforms hCA IX and XII over
the cytosolic ones hCA I and II, with AAZ and compounds 1-11 and 1i-11i are shown in
Table 2. It can be observed that some of the investigated derivatives do show some promising
levels of selectrive inhibition of the transmembrane over the cytosolic isoforms, among which
7-9, 1i. Thus, the biological activity of these sulfonamides constitute an interesting starting
point for exploring in more detail the secondary amines incorporating sulfonamide or other
zinc-binding group moieties, for designing isoform-selective metalloenzyme inhibitors.
3. Conclusion
In this study, Schiff base derivatives of sulfanilamide were prepared by condensation with
substituted benzaldehydes incorporating hydrophilic (OH, dimethylamino) as well as
hydrophobic (methyl, methoxy, phenethyloxy, halogens) moieties, or a combination of the
two types of moieties in their molecules. The secondary amine sulfonamides were thereafter
prepared by reduction reaction of the imine compounds with NaBH4. The obtained
sulfonamides were investigated as inhibitors of four CA isoforms, the cytosolic CA I and II,
as well as the transmembrane, tumor-associated CA IX and XII. It is interesting to note that
the compounds incorporating secondary amine moieties showed a better inhibitory activity
against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II
and IX inhibitors were detected, whereas the activity against hCA I and XII was less strong.
The secondary amines incorporating sulfonamide or other zinc-binding groups, poorly
investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting
7

opportunities in the field due to the facile preparation and possibility to explore a vast
chemical space.
4. Experimental
4.1.Chemistry
All the chemicals were obtained from commercial suppliers (Sigma-Aldrich, Merck) and used
without further purification. Elemental analysis was carried out on a LECO CHNS model 932
elemental analyser. FT-IR spectra were performed by using with a Perkin Elmer Spectrum
Two FT-IR spectrometer (400-4000 cm-1) with KBr pellets.NMR spectra were recorded at 25
oC in DMSO-d6 using TMS as an internal standard on an Agilent-VNMRS-400 spectrometer
1
operating at 400 MHz for H-NMR and 100 MHz for 13C-NMR.Melting points were
measured in open capillary tubes with an Electro thermal 9100 melting point apparatus and
were uncorrected. Mass Spectra results were recorded on a Shimadzu LCMS 8040 Model
spectrometer. TLC (on Merck silica gel 60 F254 sheets) was used to follow the course of the
reaction and assess product purity.
4.1.1. General procedure for the synthesis of imine derivatives 1-11:
The Schiff bases were synthesized according to the literature method with some
modifications. The benzaldehyde derivatives (1.0 mmol) in 30 mL of methanol were added
dropwise to the appropriate sulfanilamide (1.0 mmol) previously dissolved in 30 mL of
methanol. Catalytic amounts of formic acid were added and the reaction stirred for 3–4 h
under reflux. The solvent was thenevaporated. The obtained solid was washed with ice-cold
ethanol/methanol. Then, the obtained products were recrystallized from methanol (1-
9)/ethanol (10 and 11) and dried under vacuum to give the corresponding imino-derivatives
(1-11) (Scheme 1).
4.1.1.1. 4-[(2-hydroxybenzylidene)amino]benzenesulfonamide (1):
Yield: 85%; Color: Yellow; M.p: 212-213 ˚C (lit.: 212,9b 209-210,15 ˚C); Anal. Calcd. For
C13H12N2O3S (276.31) (%): C, 56.51; H, 4.38; N, 10.14; S, 11.60.Found (%): C, 56.42; H,
4.45; N, 10.26; S, 11.45; FT-IR (KBr pellets, cm-1): 3341, 3245 (NH2), 3100-3450 (O-H…N
broad), 3062 (Ar-H), 1616 (-CH=N-), 1311 (asymmetric), 1164 (symmetric) (S=O); 1H-NMR
8

(DMSO-d6, TMS, 400 MHz, δ ppm): 12.62 (1H, s, Ar-OH), 8.99 (1H, s, -CH=N-), 7.39 (2H,
s, -SO2NH2), 7.87-7.89 (2H, d, J= 8.8, Ar-H), 7.54-7.57 (2H, d, J= 8.8, Ar-H), 7.69-7.71 (1H,
d, J= 9.6, Ar-H), 7.47-7.43 (1H, t, J= 17.6, Ar-H), 6.98-7.02 (2H, m, Ar-H);13C-NMR
(DMSO-d6, TMS, 100 MHz, δ ppm): 166.44 (-C=N-), 160.68, 151.67, 142.43, 134.36,
132.99, 127.52, 122.26, 119.81, 117.15, 112.48 (Aromatic).
4.1.1.2. 4-[(3,5-dibromo-2-hydroxybenzylidene)amino]benzenesulfonamide (2):
Yield: 85%; Color: Bright red; M.p.: 246-248 ˚C; Anal. Calcd. For C13H10Br2N2O3S (434.10)
(%): C, 35.97; H, 2.32; N, 6.45; S, 7.39.Found (%): C, 35.77; H, 2.25; N, 6.55; S, 7.30.FT-IR
(KBr pellets, cm-1): 3313, 3237 (NH2), 3130-3550 (O-H…N broad), 3051 (Ar-H), 1621 (-
C=N-), 1325 (asymmetric), 1161 (symmetric) (S=O); 1H-NMR (DMSO-d6, TMS, 400 MHz, δ
ppm): 14.12 (1H, s, -Ar-OH), 9.03 (1H, s, -CH=N-), 7.43 (2H, s, -SO2NH2), 7.99-7.98 (1H, d,
J= 2.4, Ar-H), 7.91 (1H, s, Ar-H), 7.92-7.93 (2H, d, J= 6, Ar-C-H), 7.62-7.65 (2H, d, J= 8.8,
Ar-C-H).13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm):164.65 (-C=N-), 157.43, 149.59,
143.29, 138.43, 135.03, 127.61, 122.53, 121.35, 112.00, 110.23 (Aromatic).
4.1.1.3. 4-(benzylideneamino)benzenesulfonamide (3):
Yield: 80%; Color: White; M.p.: 189 ˚C (lit.: 188-190,19a190 9b˚C); Anal. Calcd. For
C13H12N2O2S (260.31) (%): C, 59.98; H, 4.65; N, 10.76; S, 12.32. Found (%): C, 59.78; H,
4.73; N, 10.93; S, 12.21; FT-IR (KBr pellets, cm-1): 3295, 3165 (NH2), 3002 (Ar-C-H), 1622
(-C=N-), 1334 (asymmetric), 1151 (symmetric) (S=O); 1H-NMR (DMSO-d6, TMS, 400 MHz,
δ ppm): 8.55 (1H, s, -CH=N-), 7.33 (2H, s, -SO2NH2), 7.92-7.90 (2H, d, J= 8.8, Ar-H), 7.82-
13
7.84 (2H, d, J= 8.4, Ar-H)), 7.34-7.36 (3H, m, Ar-H), 7.16-7.18 (2H, d, J= 8.8, Ar-H); C-
NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 162.26 (-C=N-), 155.14, 141.25, 137.03, 131.31,
128.94, 128.28, 127.37, 121.64 (Aromatic). MS (m/z): 260.06219a.
4.1.1.4. 4-[(2-hydroxy-3-methylbenzylidene)amino]benzenesulfonamide (4):
Yield: 85%; Color: Yellow; M.p.: 168-170 ˚C; Anal. Calcd. For C14H14N2O3S(290.34) (%):
C, 57.92; H, 4.86; N, 9.65; S, 11.04. Found (%): C, 57.78; H, 4.75; N, 9.76; S, 11.10; FT-IR
(KBr pellets, cm-1):3356, 3259 (NH2), 3160-3450 (O-H…N broad), 3061 (Ar-H), 2932 (Ar-
CH3), 1614 (-C=N-), 1318 (asymmetric), 1159 (symmetric) (S=O).1H-NMR (DMSO-d6,
TMS, 400 MHz, δ ppm): 13.244 (1H, s, Ar-OH), 8.983 (1H, s, -CH=N-), 7.39 (2H, s,
SO2NH2), 7.89-7.87 (2H, d, J= 8.8, Ar-H), 7.59-7.56 (2H, d, J= 8.8, Ar-H), 7.49-7.47 (1H, d,
J= 8, Ar-H), 7.34-7.32 (1H, d, J= 7.2, Ar-H), 6.92-6.88 (1H, t, J= 14.8, Ar-H), 2.21 (3H, s,
9

Ar-CH3);13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 166.50 (-C=N-), 159.24, 151.01,
142.54, 135.15, 131.45, 127.54, 125.67, 122.30, 119.29, 118.62 (Aromatic), 15.64 (Ar-
CH3).LC-MS/MS, MS (m/z): 291.05 [M+H].
4.1.1.5. 4-[(4-methoxybenzylidene)amino]benzenesulfonamide (5):
Yield: 80%; Color: White; M.p.: 197-198 ˚C, (lit:195-19719a, 199-20119b, 2059b ˚C); Anal.
Calcd. for C14H14N2O3S (290.34) (%): C, 57.92; H, 4.86; N, 9.65; S, 11.04.Found (%): C,
57.78; H, 4.78; N, 9.73; S, 11.12; FT-IR (KBr pellets, cm-1): 3277, 3146 (NH2), 3068, 3025
(Ar-H), 2960-2840 (Al-H), 1610 (-C=N-), 1332 (asymmetric), 1151 (symmetric) (S=O), 1269
(Ar-O-C);1H-NMR (DMSO-d6, TMS, 400 MHz, δ ppm): 8.53 (1H, s, -CH=N-), 7.32 (2H, s, -
SO2NH2), 7.90-7.88 (2H, d, J= 8.8, Ar-H), 7.35-7.32 (2H, d, J= 8.8, Ar-H), 7.83-7.81 (2H, d,
J= 8.8, Ar-H), 7.08-7.06 (2H, J= 8.8, Ar-H), 3.83 (3H, s, -O-CH3);13C-NMR (DMSO-d6,
TMS, 100 MHz, δ ppm): 162.75 (-C=N-), 162.27, 155.14, 141.21, 131.30, 128.98, 127.36,
121.62, 114.81 (Aromatic), 55.92 (-O-CH3); MS (m/z): 290.073.
4.1.1.6. 4-[(5-bromo-2-hydroxybenzylidene)amino]benzenesulfonamide (6):
Yield: 85%; Color: Orange; M.p.: 217-219 ˚C (lit.19: 212-214°C); Anal. Calcd. For
C13H11BrN2O3S (355.21) (%):C, 43.96; H, 3.12; N, 7.89; S, 9.03. Found (%): C, 43.90; H,
3.07; N, 7.98; S, 8.96; FT-IR (KBr pellets, cm-1): 3360, 3270 (NH2), 3150-3450 (O-H…N
broad), 3094, 3068 (Ar-C-H), 1616 (-C=N-), 1336 (asymmetric), 1154 (symmetric) (S=O);
1H-NMR (DMSO-d6, TMS, 400 MHz, δ ppm): 12.50 (1H, s, Ar-OH), 8.94 (1H, s, -CH=N-),
7.40 (2H, s, -SO2NH2), 7.92-7,91 (1H, d, J= 2.8, Ar-H), 7.90-7.88 (2H, d, J= 8.4, Ar-H),
7.60-7.57 (1H, m, Ar-H), 7.547-7.525 (2H, d, J= 8.8, Ar-H), 6.99-6.97 (1H, d, J= 8.8, Ar-
H);13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 163.54 (-C=N-), 159.64, 151.66, 142.66,
136.51, 134.13, 127.54, 122.27, 121.82, 119.63, 110.61 (Aromatic); MS (ESI) [M]+ =
354.519c
.
4.1.1.7. 4-[(4-methylbenzylidene)amino]benzenesulfonamide (7):
Yield: 85%; Color: White; M.p: 196-198 ˚C(lit.19:198-200 ˚C); Anal. Calcd. for C14H14N2O2S
(274.34) (%): C, 61.29; H, 5.14; N, 10.21; S, 11.69. Found (%): C, 61.21; H, 5.06; N, 10.27;
S, 11.63; FT-IR (KBr pellets, cm-1): 3286, 3186 (NH2), 3085, 3043 (Ar-H), 1622 (-C=N-),
1334 (asymmetric), 1154 (symmetric) (S=O);1H-NMR (DMSO-d6, TMS, 400 MHz, δ ppm):
8.57 (1H, s, -CH=N-), 7.33 (2H, s, -SO2NH2), 7.84-7.84 (2H, d, J= 2.8, Ar-H), 7.82-7.82 (2H,
d, J= 3.2, Ar-H), 7.37-7.36 (2H, d, J= 2, Ar-H), 7.35-7.34(2H, J= 2, Ar-H), 2.37 (3H, s, Ar-
10

CH3);13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 162.93 (-C=N-), 154.96, 142.60,141.46,
133.56, 129.96, 129.44, 127.36,121.65 (Aromatic), 21.67(Ar-CH3); MS (m/z): 274.07719a
.
4.1.1.8. 4-[(5-chloro-2-hydroxybenzylidene)amino]benzenesulfonamide (8):
Yield: 85%; Color: Bright orange; M.p: 199-201 ˚C (lit. 19d: 196-198˚C); Anal. Calcd. For
C13H11ClN2O3S (310.76) (%): C, 50.24; H, 3.57; N, 9.01; S, 10.32.Found (%):C, 50.16; H,
3.53; N, 9.13; S, 10.23; FT-IR (KBr pellets, cm-1): 3359, 3270 (NH2), 3150-3550 (O-H…N
1
broad), 3107, 3077 (Ar-H), 1618 (-C=N-), 1336 (asymmetric), 1154 (symmetric) (S=O); H-
NMR (DMSO-d6, TMS, 400 MHz, δ ppm): 12.48 (1H, s, Ar-OH), 8.95 (1H, s, -CH=N-), 7.40
(2H, s, -SO2NH2), 7.90-7.88 (2H, d, J= 8.8, Ar-H), 7.792-7.785 (1H, d, J= 2.8, Ar-H), 7.55-
7.53 (2H, d, J= 8.8, Ar-H), 7.49-7.46 (1H, m, Ar-H), 7.04-7.02 (1H, d, J= 8.8, Ar-H); 13C-
NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 163.61 (-C=N-), 159.24, 151.67, 142.66, 133.75,
131.15, 127.54, 122.27, 123.25, 121.21, 119.22 (Aromatic). MS (ESI) [M]+ = 31019d
.
4.1.1.9. 4-{[4-(benzyloxy)benzylidene]amino}benzenesulfonamide (9):
Yield: 75%; Color: white; M.p: 217-219 ˚C; Anal. Calcd. For C20H18N2O3S (366.43) (%): C,
65.55; H, 4.95; N, 7.64; S, 8.75. Found (%): C, 65.47; H, 5.01; N, 7.77; S, 8.69; FT-IR
(KBrpellets, cm-1): 3337, 3253 (NH2), 3068, 3025 (Ar-H), 2950-2850 (Al-H), 1627 (-C=N-),
1293 (asymmetric), 1156 (symmetric) (S=O), 1247 (-O-C-); 1H-NMR (DMSO-d6, TMS, 400
MHz, δ ppm): 8.59 (1H, s, -CH=N-), 7.33 (2H, s, -SO2NH2), 7.92-7.90 (2H, d, J= 8.8, Ar-H),
7.84-7.82 (2H, d, J= 8.4, Ar-H), 7.49-7.39 (5H, m, Ar-H), 7.34-7.36 (2H, d, J= 8.4, Ar-H),
7.18-7.16 (2H, J= 8.8, Ar-H), 5.21 (2H, s, Ar-O-CH2-Ar);13C-NMR (DMSO-d6, TMS, 100
MHz, δ ppm): 162.26 (-C=N-), 161.84, 155.13, 141.25, 137.03, 129.17, 128.45, 131.31,
127.37, 128.94, 128.28, 121.64, 115.94 (Aromatic), 69.93 (Ar-CH2-O-Ar).LC-MS/MS (m/z):
367.05 [M+H].
4.1.1.10. 4-[(3,5-dichloro-2-hydroxybenzylidene)amino]benzenesulfonamide (10):
Yield: 85%; Color: Bright red; M.p.: 242-244 ˚C; Anal. Calcd. for C13H10Cl2N2O3S (345.20)
(%): C, 45.23; H, 2.92; N, 8.12; S, 9.29, Found (%): C, 45.19; H, 2.80; N, 8.25; S, 9.20;FT-IR
(KBr pellets, cm-1): 3344, 3245 (NH2), 3120-3600 (O-H…N broad), 3063 (Ar-C-H), 1619 (-
C=N-), 1324 (asymmetric), 1159 (symmetric) (S=O); 1H-NMR (DMSO-d6, TMS, 400 MHz, δ
ppm): 13.99 (1H, s, -Ar-OH), 9.54 (1H, s, -CH=N-), 7.43 (2H, s, -SO2NH2), 7.93-7.91 (2H, d,
J=8.8, Ar-H), 7.80-7.79 (1H, d, J=2.8, Ar-H), 7.772-7.765 (1H, d, J= 2.8, Ar-H), 7.65-7.62
(2H, d, J= 8.8, Ar-H); 13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 164.99 (-C=N-),
11

156.15, 149.77, 143.29, 133.18, 131.36, 127.61, 122.85, 122.51, 122.21, 121.00 (Aromatic);
LC-MS/MS (m/z): 342.95 [M-H].
4.1.1.11. 4-{[4-(dimethylamino)benzylidene]amino}benzenesulfonamide (11):
Yield: 85%; Color: Lightyellow; M.p: 230-232 ˚C (lit: 226-22715 , 212-21419a, 222-22519e ˚C);
Anal. Calcd. for C15H17N3O2S (303.38) (%): C, 59.38; H, 5.65; N, 13.85; S, 10.57.Found
(%):C, 59.31; H, 5.60; N, 13.96; S, 10.48. FT-IR (KBr pellet, cm-1): 3321, 3282 (NH2), 3086,
3047 (Ar-C-H), 2950-2800 (-CH3), 1605 (-C=N-), 1331 (asymmetric), 1149 (symmetric)
1
(S=O), 1231 (-N-C). H-NMR (DMSO-d6, TMS, 400 MHz, δ ppm): 8.41 (1H, s, -CH=N-),
7.27 (2H, s, -SO2NH2), 7.79-7.77 (2H, d, J= 8.8, Ar-H), 7.75-7.73 (2H, d, J= 9.2, Ar-H), 7.30-
7.28 (2H, d, J= 8.8, Ar-H), 6.79-6.77 (2H, J= 8.8, Ar-H), 3.01 (6H, s, N-(CH3)2). 13C-NMR
(DMSO-d6, TMS, 100 MHz, δ ppm): 162.15 (-C=N-), 155.73, 153.14, 140.54, 131.14,
127.32, 123.78, 121.53, 111.87 (Aromatic), 40.14 (-N-(CH3)2). MS (m/z): 303.104119a.
4.1.2. General procedure for the synthesis of amine derivatives 1i-11i:
To a stirred suspension of the imino-compound (1-11) (1.0 mmol) in methanol (30 mL) at 0
oC was added solid sodium borohydride (NaBH4) (6.0 mmol) in small portions, over 1h. The
mixture is left under stirring for 24 more hours at room temperature. After the reduction was
complete, half of the solvent in the reaction mixture was evaporated and the remaining
mixture was poured on ice. The resulting fine white precipitate was filtered, washed with cold
water and recrystallized from absolute methanol (1i-4i), acetonitrile (5i-7i), acetone (8i, 9i),
chloroform (10i) or dichloromethane (11i) and dried under vacuum to give the corresponding
amino-derivative (1i-11i) in high yields.
4.1.2.1.4-[(2-hydroxybenzyl)amino]benzenesulfonamide (1i):
Yield: 65%; Color: white; M.p.: 185-187 ˚C; Anal. Calcd. For C13H14N2O3S (278.33) (%): C,
56.10; H, 5.07; N, 10.06; S, 11.52.Found (%): C, 56.25; H, 4.98; N, 10.20; S, 11.45;FT-IR
(KBr pellet, cm-1): 3337 (NH, NH2),3253 (OH), 3077 (Ar-C-H), 2853, 2942 (Al-H), 1616 (-
1
C=N-)(disappeared),1295 (asymmetric), 1154 (symmetric) (S=O); H-NMR (DMSO-d6,
TMS, 400 MHz, δ ppm): 9.54 (1H, s, -Ar-OH), 6.86 (2H, s, -SO2NH2), 7.44-7.47 (2H, d, J=
8.8, Ar-H), 6.58-6.60 (2H, d, J= 8.8, Ar-H), 6.76 (1H, s, -Ar-NH), 7.11-7.09 (1H, d, J= 8.8,
Ar-H), 6.69-6.73 (1H, t, J= 16, Ar-H), 7.01-7.05 (1H, t, J= 15.2, Ar-H), 6.79-6.82 (1H, d, J=
9.2, Ar-H), 4,23-421 (2H, d, Ar-CH2);13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 155.44,
12

151.86, 130.41, 128.46, 128.14, 127.72, 111.30, 119.23, 125.26, 115.36 (Aromatic), 41.28
(Ar-CH2-N-).
4.1.2.2. 4-[(3,5-dibromo-2-hydroxybenzyl)amino]benzenesulfonamide (2i):
Yield: 55%; Color: white; M.p.: 163 ˚C; Anal. Calcd. For C13H12Br2N2O3S (436.12) (%): C,
35.80; H, 2.77; N, 6.42; S, 7.35.Found (%): C, 35.72; H, 2.55; N, 6.55; S, 7.28; FT-IR (KBr
pellet, cm-1): 3438, 3353(NH, NH2), 3266 (O-H), 3116, 3085 (Ar-H), 2830-2965 (Al-H),
1
1621 (-C=N-)(disappeared),1305 (asymmetric), 1138 (symmetric) (S=O); H-NMR (DMSO-
d6, TMS, 400 MHz, δ ppm): 9.65 (1H, s, Ar-OH), 6.92 (2H, s, -SO2NH2), 6.90 (1H, broad, N-
H), 7.614-7.608 (1H, d, J= 2.4, Ar-H), 7.254-7.248 (1H, d, J= 2.4, Ar-H), 7.50-7.52 (2H, d,
J= 8.8, Ar-H), 6.59-6.61 (2H, d, J= 8.8, Ar-H), 4.32 (2H, s, -Ar-CH2); 13C-NMR (DMSO-d6,
TMS, 100 MHz, δ ppm): 151.42, 151.26, 133.13, 129.98, 131.32, 131.20, 127.89, 112.85,
111.77, 111.52 (Aromatic), 41.82 (Ar-CH2-N).LC-MS/MS MS (m/z): 434.85 [M-H].
4.1.2.3. 4-(benzylamino)benzenesulfonamide (3i):
Yield: 80%; Color: white; M.p.: 174-176 ˚C; Anal. Calcd. For C13H12N2O2S (262.33) (%): C,
59.52; H, 5.38; N, 10. 68; S, 12.22.Found (%):C, 59.65; H, 5.27; N, 10.90; S, 12.11; FT-IR
(KBr pellet, cm-1):3395, 3343, 3264 (NH, NH2), 3106, 3029 (Ar-H), 2868-2954 (Al-H),1622
(-C=N-)(disappeared),1294 (asymmetric), 1147 (symmetric) (S=O);1H-NMR (DMSO-d6,
TMS, 400 MHz, δ ppm): 7.48-7.46 (2H, d, J= 8.8, Ar-H), 7.31-7.320 (4H, m, Ar-H), 7.25
(1H, broad, Ar-NH-C); 6.96-6.99 (1H, t, Ar-H), 6.883 (2H, s, -SO2NH2), 6.60-6.63 (2H, d, J=
9.2, Ar-H), 4.33-4.31 (2H, d, J= 6, Ar-CH2-N );13C-NMR (DMSO-d6, TMS, 100 MHz, δ
ppm): 151.66, 139.82, 130.72, 128.81, 127.74, 127.53, 127.23, 111.54(Aromatic), 46.29 (Ar-
CH2-N); LC-MS/MS (m/z): 263.10 [M+H].
4.1.2.4. 4-[(2-hydroxy-3-methylbenzyl)amino]benzenesulfonamide (4i):
Yield: 75%; Color: white; M.p.: 177 ˚C; Anal. Calcd. For C14H16N2O3S (292.35) (%): C,
57.52; H, 5.52; N, 9.58; S, 10.97. Found (%): C, 57.45; H, 5.55; N, 9.66; S, 11.10; FT-IR
(KBr pellet, cm-1): 3358, 3257(NH, NH2), 3123(OH), 3034 (Ar-H), 2861-2953 (Al-H), 1614
1
(-C=N-)(disappeared),1327 (asymmetric), 1156 (symmetric) (S=O); H-NMR (DMSO-d6,
TMS, 400 MHz, δ ppm): 8.43(1H, broad, Ar-OH), 6.75 (1H, broad,-N-H-), 6.86 (2H, s, -
SO2NH2), 7.47-7.44 (2H, d, J= 8.8, Ar-H), 6.59-6.57 (2H, d, J= 8.8, Ar-H), 6.96-6.94 (2H, d,
J= 7.6, Ar-H), 6.684-6.647 (1H, t, J= 14.8, Ar-H), 4.27-4.260 (2H, d, J= 5.2, Ar-CH2-N-),
2.17 (3H, s, Ar-CH3); 13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm):153.15, 151.85, 130.48,
13

129.64, 127.72, 126.04, 125.03, 119.71, 111.37 (Aromatic), 41.95 (Ar-CH2-N), 16.96 (Ar-
CH3); LC-MS/MS (m/z): 291.10 [M-H].
4.1.2.5. 4-[(4-methoxybenzyl)amino]benzenesulfonamide (5i):
Yield: 70%; Color: white; M.p: 181-183 ˚C ; Anal. Calcd. for C14H16N2O3S (292.35) (%): C,
57.52; H, 5.52; N, 9.58; S, 10.97.Found (%): C, 57.48; H, 5.58; N, 9.73; S, 11.05. FT-IR (KBr
pellet, cm-1): 3388, 3333, 3258 (NH, NH2), 3074, 3035 (Ar-H), 2955-2835 (Al-H), 1610 (-
1
C=N-)(disappeared), 1305 (asymmetric), 1161(symmetric(S=O), 1247 (Ar-O-C). H-NMR
(DMSO-d6, TMS, 400 MHz, δ ppm): 6.86 (2H, s, -SO2NH2), 6.89 (1H, s, broad, N-H), 7.46-
7.44 (2H, d, J= 8.8, Ar-H), 7.25-7.22 (2H, d, J= 8.8, Ar-H), 6.88-6.86 (2H, d, J= 9.2, Ar-H),
13
6.61-6.59(2H, J= 8.8, Ar-H), 4.24-4.22 (2H, d, Ar-CH2-N), 3.698 (3H, s, -O-CH3). C-NMR
(DMSO-d6, TMS, 100 MHz, δ ppm): 158.64, 151.65, 131.54, 130.59, 128.80, 127.69, 114.22,
111.53(Aromatic), 55.48 (-O-CH3), 45.75 (Ar-CH2-N). LC-MS/MS (m/z): 291.10 [M+H].
4.1.2.6. 4-[(5-bromo-2-hydroxybenzyl)amino]benzenesulfonamide (6i):
Yield: 60%; Color: white; M.p: 213-215 ˚C; Anal. Calcd. For C13H13BrN2O3S (357.22)
(%):C, 43.71; H, 3.67; N, 7.84; S, 8.98.Found (%):C, 43.61; H, 3.59; N, 7.94; S, 8.92; FT-IR
(KBr pellet, cm-1): 3370, 3278 (NH, NH2), 3235 (OH), 3073, 3047 (Ar-C-H), 2820-2965 (Al-
H), 1616 (-C=N-)(disappeared),1342 (asymmetric), 1142 (symmetric) (S=O);1H-NMR
(DMSO-d6, TMS, 400 MHz, δ ppm):9.94 (1H, s, -Ar-OH), 6.88 (2H, s, -SO2NH2), 6.82 (1H,
broad, N-H), 7.49-7.46 (2H, d, J= 8.8, Ar-H), 7.21-7.18 (2H, m, J= 2.4, Ar-H), 6.78-6.76
(1H, d, J= 8.4, Ar-H), 6.60-6.58 (2H, d, J= 8.8, Ar-H)), 4.20-4.22 (2H, d, J= 6, -Ar-CH2);
13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 154.82, 151.53, 130.82, 130.65, 130.54,
128.36, 127.83,117.50, 111.36, 110.49 (Aromatic), 40.85 (Ar-CH2-N).LC-MS/MS (m/z):
357.00 [M-H].
4.1.2.7. 4-[(4-methylbenzyl)amino]benzenesulfonamide (7i):
Yield: 70%; Color: white; M.p: 194-196 ˚C; Anal. Calcd. for C14H16N2O2S (276.35) (%): C,
60.85; H, 5.84; N, 10.14; S, 11.60.Found (%): C, 60.79; H, 5.88; N, 10.23; S, 11.54. FT-IR
(KBr pellet, cm-1): 3387, 3329, 3257 (NH, NH2), 3096, 3028 (Ar-C-H), 2850-2950 (Al-C-H),
1
1622 (-C=N-) disappeared, 1301 (asymmetric), 1156 (symmetric) (S=O). H-NMR (DMSO-
d6, TMS, 400 MHz, δ ppm): 6.86(2H, s, -SO2NH2), 6.93 (1H, s, N-H), 7.46-7.43 (2H, d, J=
8.8, Ar-C-H (C2, C6)), 7.21-7.19 (2H, d, J= 8, Ar-C-H (C’2, C’6)), 7.12-7.10 (2H, J= 7.6, Ar-
14

C-H (C’3, C’5)), 6.60-6.58 (2H, d, J= 8.8, Ar-C-H (C3, C5)), 4.27-4.25 (2H, d, J= 4.8, Ar-
CH2-N), 2.24 (3H, s, Ar-CH3). 13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 151.66,
136.69, 136.24, 130.62, 129.36, 127.69, 127.49, 111.53(Aromatic), 46.05 (Ar-CH2-N), 21.10
(Ar-CH3). LC-MS/MS (m/z): 277.10 [M+H].
4.1.2.8. 4-[(5-chloro-2-hydroxybenzyl)amino]benzenesulfonamide (8i):
Yield: 60%; Color: white; M.p: 195-197 ˚C; Anal. Calcd. For C13H13ClN2O3S (312.77) (%):
C, 49.92; H, 4.19; N, 8.96; S, 10.25. Found (%):C, 50.02; H, 4.11; N, 9.02; S, 10.17; FT-IR
(KBr pellet, cm-1): 3368, 3337(NH, NH2), 3262 (OH), 3077, 3051 (Ar-C-H), 2850-2965 (Al-
H), 1618 (-C=N-) disappeared,1323 (asymmetric), 1138 (symmetric) (S=O);1H-NMR
(DMSO-d6, TMS, 400 MHz, δ ppm): 9.93 (1H, s, -Ar-OH), 6.89 (2H, s, -SO2NH2), 6.86 (1H,
broad, N-H), 7.50-7.48 (2H, d, J= 8.8, Ar-H), 7.10-7.07 (2H, m, J= 2.4, Ar-H), 6.84-6.82
(1H, d, J= 8.8, Ar-H), 6.620-6.59 (2H, d, J= 8.8, Ar-H), 4.23-4.22 (2H, d, J= 6, -Ar-CH2-N);
13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 154.38, 151.56, 130.83, 127.84, 127.76,
127.70, 122.85, 116.95, 111.38(Aromatic), 40.92 (Ar-CH2-N); LC-MS/MS (m/z): 311.00 [M-
H].
4.1.2.9. 4-{[4-(benzyloxy)benzyl]amino}benzenesulfonamide (9i):
Yield: 70%; Color: white; M.p: 183-185 ˚C; Anal. Calcd. For C20H20N2O3S (368.45) (%): C,
65.20; H, 5.47; N, 7.60; S, 8.70.Found (%):C, 65.11; H, 5.39; N, 7.67; S, 8.63; FT-IR (KBr
pellet, cm-1): 3339, 3253 (NH, NH2), 3060, 3032 (Ar-H), 2950-2850 (Al-H), 1627(-C=N-)
disappeared,1323 (asymmetric), 1148 (symmetric) (S=O), 1245 (Ar-O-C); 1H-NMR (DMSO-
d6, TMS, 400 MHz, δ ppm): 7.36 (2H, s, -SO2NH2), 7.48-7.45 (2H, d, J= 8.8, Ar-H), 7.41-
7.31 (5H, m, Ar-H), 7.26-7.24 (2H, d, J= 8.8, Ar-H), 6.97-6.95 ( 2H, d, J= 8.8, Ar-H), 6.62-
6.60 (2H, J= 9.2, Ar-H), 6.91 (1H, broad, -NH-Ar), 5.07 (2H, s, Ar-O-CH2-Ar), 4.25-4.24
(2H, d, J= 5.6, Ar-CH2-N-).13C-NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 157.74, 151.68,
137.62, 131.83, 130.62, 128.23, 128.86, 128.09,127.83, 127.71, 115.14, 111.55 (Aromatic),
69.61(Ar-CH2-O-Ar), 45.76 (Ar-CH2-N-).LC-MS/MS (m/z): 369.05 [M+H].
4.1.2.10. 4-[(3,5-dichloro-2-hydroxybenzyl)amino]benzenesulfonamide (10i):
Yield: 85%; Color: white; M.p: 174-176 ˚C; Anal. Calcd. For C13H12Cl2N2O3S (347.22) (%):
C, 44.97; H, 3.48; N, 8.07; S, 9.23.Found (%): C, 44.95; H, 3.43; N, 8.13; S, 9.19; FT-IR
(KBr pellet, cm-1): 3352, 3263 (NH, NH2), 3245 (OH), 3120, 3092 (Ar-H), 2850-2950 (Al-H),
1
1619(-C=N-) disappeared,1325 (asymmetric), 1156 (symmetric) (S=O); H-NMR (DMSO-
15

d6, TMS, 400 MHz, δ ppm): 9.77 (2H, broad, Ar-OH, N-H), 6.89 (2H, s, -SO2NH2), 7.50-7.48
(2H, d, J= 8.8, Ar-H), 7.37-7.36 (1H, d, J= 2.8, Ar-H), 7.083-7.077 (1H, d, J= 2.4, Ar-H),
7.60-7.58 (2H, d, J= 8.8, Ar-H), 4,30 (2H, s, -Ar-CH2); 13C-NMR (DMSO-d6, TMS, 100
MHz, δ ppm): 151.29, 150.14, 131.14, 130.83, 127.87, 127.60, 126.55, 123.61, 122.17,
111.48 (Aromatic), 41.61 (Ar-CH2-N).LC-MS/MS (m/z): 344.95 [M-H].
4.1.2.11. 4-{[4-(dimethylamino)benzyl]amino}benzenesulfonamide (11i):
Yield: 70%; Color: whitish; M.p: 181-183 ˚C; Anal. Calcd. For C14H16N3O2S (305.40) (%):
C, 58.99; H, 6.27; N, 13.76; S, 10.50.Found (%): C, 58.90; H, 6.21; N, 13.88; S, 10.47. FT-IR
(KBr pellet, cm-1): 3384, 3356, 3252 (NH, NH2), 3107, 3073 (Ar-H), 2950-2800 (Al-H), 1605
1
(-C=N-) disappeared,1308 (asymmetric), 1152(symmetric) (S=O), 1239 (Ar-N-C). H-NMR
(DMSO-d6, TMS, 400 MHz, δ ppm): 6.87 (2H, s, -SO2NH2), 5.90 (1H, s, N-H), 7.46-7.44
(2H, d, J= 8.8, Ar-H), 7.16-7.14 (2H, d, J= 8.4, Ar-H), 6.69-6.67 (2H, d, J= 8.8, Ar-H), 6.62-
6.60 (2H, J= 8.8, Ar-H), 4.19-4.17 (2H, d, J= 6, Ar-CH2-N), 2.84 (6H, s, N-(CH3)2). 13C-
NMR (DMSO-d6, TMS, 100 MHz, δ ppm): 151.78, 150.03, 130.39, 128.99, 128.49, 127.66,
112.93, 111.50 (Aromatic), 45.95 (Ar-CH2-N), 40.75 (-N-(CH3)2); LC-MS/MS (m/z): 306.10
[M+H].
4.2. Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA-
catalyzed CO2 hydration activity.20 Phenol red (at a concentration of 0.2 mM) has been used
as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes buffer (pH
7.5) and 20 mM NaClO4 for maintaining constant ionic strength, following the initial rates of
the CA-catalyzed CO2 hydration reaction for a period of 10-100 s, at 20 °C. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction
have been used to determine the initial velocity. The uncatalyzed rates were measured in the
same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1
mM) were prepared in distilled-deionized water and dilutions down to 0.01 nM were made
thereafter with the assay buffer. 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 obtained by non-linear least-squares methods using
PRISM 3 and the Cheng-Prusoff equation as reported earlier,21 and represent the mean from
16

at least three different determinations. All CA isoforms were recombinant ones obtained in
huse as reported earlie,22-24 and their concentrations in the assay system were: 11.4 nM for
hCA I, 9.0 nM for hCA II; 7.8 nM for hCA IX and 14.7 nM for hCA XII, respectively.
Acknowledgments. This study supported by Harran University Scientific Research Project
Coordinator (HUBAK Project nos: 12040, 12059 and 12061) and by an European Union 7th
FP project (Dynano, to CTS).
17

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21

Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against
human carbonic anhydrase isoforms I, II, IX and XII
Mustafa Durgun*, Hasan Turkmen, Mariangela Ceruso, Claudiu T. Supuran *
*Correspondingauthors. Tel./fax: +904143443000 (M.D.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: mustafadurgun@harran.edu.tr, mustafadurgun@yahoo.com (M.Durgun), claudiu.
supuran@unifi.it (C.T. Supuran).
22