Benzenesulfonamides Incorporating Flexible Triazole Moieties Are Highly Effective Carbonic Anhydrase Inhibitors: Synthesis and Kinetic, Crystallographic, Computational, and Intraocular Pressure Lowering Investigations

Article abstract of DOI:10.1021/acs.jmedchem.6b01389

Herein we report the synthesis of two series of benzenesulfonamide containing compounds that incorporate the phenyl-1,2,3-triazole moieties. We explored the insertion of appropriate linkers, such as ether, thioether, and amino type, into the inner section

Full text of DOI:10.1021/acs.jmedchem.6b01389

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Article
Benzenesulfonamides incorporating flexible triazole moieties are highly
effective carbonic anhydrase inhibitors: synthesis, kinetic, crystallographic,
computational and intraocular pressure lowering investigations
Alessio Nocentini, Marta Ferraroni, Fabrizio Carta, Mariangela Ceruso,
Paola Gratteri, Cecilia Lanzi, Emanuela Masini, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01389 • Publication Date (Web): 14 Nov 2016
Downloaded from http://pubs.acs.org on November 16, 2016
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Benzenesulfonamides Incorporating Flexible Triazole Moieties are Highly Effective
Carbonic Anhydrase Inhibitors: Synthesis, Kinetic, Crystallographic, Computational and
Intraocular Pressure Lowering Investigations
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Alessio Nocentini,1,2 Marta Ferraroni,3 Fabrizio Carta,1 Mariangela Ceruso,3 Paola Gratteri,2
Cecilia Lanzi,4 Emanuela Masini4 and Claudiu T. Supuran1*
1
Università degli Studi di Firenze, Neurofarba Department., Sezione di Scienze Farmaceutiche, Via Ugo
Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
2 Università degli Studi di Firenze, Neurofarba Department., Sezione di Scienze Farmaceutiche, Laboratory
of Molecular Modeling Cheminformatics & QSAR, via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
3
Università degli Studi di Firenze, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino
(Florence), Italy.
4
Università degli Studi di Firenze, Neurofarba Department., Sezione di Farmacologia, Viale Pieraccini 6,
50139 Florence, Italy.
Abstract. Herein we report the synthesis of two series of benzenesulfonamide containing
compounds which incorporate the phenylꢀ1,2,3ꢀtriazole moieties. We explored the insertion of
appropriate linkers, such as ether, thioether and amino type, into the inner section of the molecules
with the intent to confer additional flexibility. All obtained compounds were screened in vitro as
inhibitors of the physiologically relevant human (h) isoforms of the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1). Many of them were low nanomolar/subnanomolar hCA II, IX and XII
inhibitors, whereas they did not potently inhibit hCA I. Computational and Xꢀray crystallographic
studies of the enzymeꢀinhibitor adducts helped us to rationalize the obtained results. Some of the
sulfonamides reported here showed significant intraocular pressure lowering activity in an animal
model of glaucoma.
Keywords: carbonic anhydrase; inhibitor; sulfonamide; glaucoma; click chemistry
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Introduction.
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Up to now, different approaches have been pursued with the intent to develop sulfonamideꢀlike
carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs)1ꢀ3 which possess better selectivity profiles
towards the different human (h) isoforms of the enzyme. Typically, the sulfonamide moiety
coordinates the Zn(II) ion present within the hCAs active sites and establishes two additional
hydrogen bonds with a residue nearby (Thr199). Such binding features are common among the
active site architectures of all the 15 human isozymes,1 which belong to the αꢀclass (seven distinct
genetic families have been identified so far, the αꢀ, βꢀ, γꢀ, δꢀ, ζꢀ. ηꢀ and θꢀCAs). These CA isoforms
differ by many molecular features, including the oligomeric organization (monoꢀ or homoꢀdimeric),
cellular localization, distribution in organs and tissues, expression levels, and kinetic
properties/affinities for the main classes of inhibitors.1,2
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In the last decades one of the research trends on hCAs, has tackled the design of isoformꢀ
selective sulfonamideꢀinhibitors by applying two principal methods: the ring and the tail
approaches.1,4,5 The first consists in modulating the ring (mainly its chemical nature) directly linked
to the sulfonamide group, whereas the latter resides in appending different tails to the
aromatic/heterocyclic ring bearing the zingꢀbindingꢀgroup (ZBG). This allows to modulate the
possible interactions the ligand establishes with the middle/outer parts of the active site cavity,
which are the most variable among the 15 isoforms mentioned above.1c
The many synthetic efforts greatly contributed in the CA field mainly by the introduction of
large sets of substitution patterns at the aromatic/heterocyclic ring.4,5 These allowed to explore
compounds possessing a variety of physicoꢀchemical properties as well as to carryꢀout detailed
structureꢀactivity relationship (SAR) studies.1 It should be stressed that, although the tail approach
has been extensively applied to aromatic sulfonamides and led to many novel potent CAIs, only a
rather limited number of such derivatives showed relevant isoformꢀselectivity inhibition profiles.4d
The role of the CAs in vertebrates, that is, the catalysis of the hydration reaction of carbon
dioxide to bicarbonate and a proton,1c,2a is pivotal for a variety of physiologicalꢀpathological
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processes,6ꢀ12 including respiration and transport of CO2/bicarbonate between metabolizing tissues
and lungs, pH andCO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic
reactions (such as gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification,
tumorigenicity, and many other physiological and pathological processes in humans.1ꢀ3 Indeed
atypical expression levels and/or activities of these enzymes were proved connected with several
human diseases, such as glaucoma (hCAs I, II, IVand XII) , oedema (hCA II, IV, XII and XIV),
osteoporosis (hCA IV and XIV), obesity (hCAs VA VB), cancer (hCAs IX and XII) and some
centralꢀnervous system affecting diseases (hCAs I, II and VII).1ꢀ3
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Primary sulfonamides such as sulfanilamide (SA), acetazolamide (AAZ), ethoxzolamide
(EZA) and dichlorphenamide (DCP) are clinically used for almost 70 years as antiglaucoma agents
for systematic administration (first generation CAIs; figure 1A), although they show a range of side
effects due to the lack of enzymatic as well as tissue selectivity. Consecutively they were also
approved for the treatment of oedema.1c,2a,7ꢀ10 The second generation antiglaucoma drugs, such as
dorzolamide (DZA) and brinzolamide (BRZ), act topically, thus leading to significant reduction of
the side effects (Figure 1B).
SO2NH2
SO2NH2
A)
SO2NH2
S
N
S
HN
O
SO2NH2
N
N
O
Cl
SO2NH2
NH2
Cl
AAZ
SA
EZA
DCP
HN
HN
B)
O
SO2NH2
SO2NH2
N
S
S
S
Me
S
O
O
O
O
DZA
BRZ
Figure 1: Molecular structures of the first (A) and second (B) generation antiglaucoma CAI drugs.
Nowadays Xꢀray crystal structures are available for many CA complexes with inhibitors,1c,13
including the majority of the twelve catalytically active hCAs. Nevertheless the rationalisation of
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these data did not lead to the expected breakthrough in rational drug design of more selective
enzyme inhibitors, since too many factors influence the interaction between the rather large active
site of the enzyme and the various classes of inhibitors. It should be also stressed that most of the
reported coꢀcrystallized inhibitor complexes are with the model isozyme II, displaying a lack of
detailed knowledge about the interaction between inhibitors and the remaining human isoforms.
The copperꢀcatalyzed azide−alkyne cycloadditions (CuAAC), better known as “Click
Chemistry”, have acquired a prominent role in medicinal chemistry,5,14 thus becoming a versatile
and useful synthetic tool to generate 1,4ꢀdisubstitutedꢀ1,2,3ꢀtriazoles to be used as biologically
uncleavable linker between valuable molecular fragments and/or as moiety that can actively
contribute to additional enzymeꢀinhibitor interaction points. The 1,2,3ꢀtriazole ring is an amide
bioisoster endowed with a moderate dipole character, hydrogen bonding capability, rigidity,
stability in the in vivo environment and an aromatic character that may allow πꢀstacking interactions
with appropriate aminoacid residues within the enzymatic cavity sites.
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In the last years, click chemistry has often been used to obtain CAIs belonging to the sulfonamide
or coumarin classes. For example, aromatic sulfonamides incorporating glycosyl moieties as well as
aromaticꢀheteroaromatic or aliphatic groups by means of a triazole linker, were obtained and
showed to be potent inhibitors against physiologically/pathologically relevant isoforms such as hCA
I, II, IX, and XII.5 Noteworthy, Pala et al. recently reported two series of benzene and
tetrafluorobenzene sulfonamides bearing aliphatic or aromatic moieties through the Click Chemistry
approach. 5d
Herein we continue the exploration of this type of derivatives, including a molecular
flexibility element in the compounds structure by adopting two different synthetic strategies for the
benzene sulfonamide scaffold. The two series of triazole derivatives were investigated for the
inhibition of the physiologically dominant, cytosolic isoforms hCA I and II, as well as the
transmembrane, tumorꢀassociated ones hCA IX and XII. In addition, we examined the enzyme
inhibitory properties of such compounds at the molecular level by reporting the high resolution Xꢀ
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ray crystal structure for two derivatives bound to hCA II as well as molecular docking studies on
hCAII and IX. Finally, some of the new compounds were screened for their antiglaucoma activity
in an animal model of the disease.
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RESULTS AND DISCUSSION
Compound Design and Synthesis. Herein we report a deeper investigation based on the
benzenesulfonamideꢀcontaining 1,2,3ꢀtriazolyl moieties previously reported from Pala et al.5d
Compounds of the phenyltriazolylbenzenesulfonamide (PTB)ꢀtype, shown in figure 2, were
considered as analogs of SLCꢀ0111,4d,e since they incorporate the 1,2,3ꢀtriazolyl moiety as
bioisoster of the ureido group. Such derivatives were all characterized by the direct connections
between three main molecular fragments: the benzenesulfonamide head (α), the triazolyl spacer (β)
and the aromatic tailing portion (γ). PTBꢀbased structures possess a rather low conformational
freedom which is limited only to the torsion angles between the single portions (Figure 2).
Figure 2. General structures of the designed benzenesulfonamides in the present study.
Hence, considering these facts, the present work was conducted with the aim to investigate
whether an enhanced flexibility between these three molecular fragments will produce better
interactions within the CAs active sites, and thus will reinforce inhibitory properties, as well as the
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selectivity profiles for the different isoforms. The enhanced flexibility will reduce the molecular
tension and increase the degree of freedom, thus allowing the aromatic tail to better interact with the
most energetically favourable enzymatic subꢀpocket(s).13f
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We proceeded by means of introduction of a CH2X (X = O, S, NH) linker (φ) between β and γ
(derivatives 3ꢀ9) and α and β (derivatives 13ꢀ17). In comparison to the reference PTB, a larger set
of substituents on the aromatic tail has been explored, obtaining two novel series of
benzenesulfonamide not investigated as CAIs up to now.
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Scheme 1. General synthetic procedure for compounds 3ꢀ10, 13ꢀ17.
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Two different synthetic strategies were used for obtaining the aboveꢀmentioned derivatives,
and both involved the application of the Click chemistry approach (Scheme 1). For the first series,
the key intermediate 4ꢀazidobenzenesulfonamide 2 was obtained from sulfanilamide treated with
NaNO2 and NaN3 in acid aqueous media. Reaction of 2 with the propargyl amino, thioether or
ethers derivatives 18ꢀ24 afforded the first series of triazoles, 3ꢀ9, incorporating diverse aryl groups
at the triazoleꢀbenzenesulfonamide moiety by means of the linker CH2X. A double propargylation
which occurred at aniline NH2 suggested us to react the dipropargyl derivate 25 with key
intermediate 2 in order to obtain the novel double head, twinꢀtailed sulfonamide 10.
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For the second series, starting from 4ꢀhydroxyꢀbenzenesulfonamide, the propargyl ether key
intermediate 12 was obtained as an alkyne by using click chemistry. Reaction of 12 with freshly
prepared aromatic azides 26ꢀ30 afforded the second series of triazoles, 13ꢀ17, that on the contrary to
the preceding one, incorporated diverse arylꢀtriazole tails appended to the zincꢀbinding group
(ZBG) scaffold through a CH2O linker.
Carbonic Anhydrase Inhibition. We investigated the CA inhibitory activities of
compounds 3ꢀ10, and 13ꢀ17 by means of the stopped flow carbon dioxide hydrase assay,15 in
comparison to the lead compound PTB5d and acetazolamide (AAZ) as standard CAI, against four
physiologically significant isoforms, the cytosolic, hCA I and II, as well as the transmembrane,
tumorꢀassociated hCA IX and XII. The rational for the choice of these four isoforms is that hCA II
and XII (upregulated in the eyes of glaucomatous patients)7bꢀd, 10b are targets for antiglaucoma
drugs,1,4,7 whereas hCA IX (and XII) have been validated as targets for the treatment and prognosis
of hypoxic cancers.2b,12 Otherwise hCA I is one of the main offꢀtarget isoforms both for the
antiglaucoma or anticancer CAIs therapeutic application.1
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Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with sulfonamides 3ꢀ10, 13ꢀ
17 reported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow
CO2 hydrase assay.15
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KI (nM)
Compound
X
R
hCA I
hCA II
hCA IX
hCA XII
3
O
O
O
O
O
S
NH
ꢀ
H
m
m
349.9
406.8
351.1
512.6
123.0
195.7
7.9
1.0
2.1
1.1
5.4
1.1
1.0
6.0
4.9
1.1
10.2
1.1
101.6
3.2
4.3
7.1
6.0
5.7
4
ꢀCH3
1.5
2.3
5
ꢀOCH3
1.5
9.3
6
p
ꢀOCH3
4.3
14.3
17.2
2.4
7
Pyridylꢀ3ꢀyl
1.4
8
H
H
ꢀ
1.5
9
0.83
>10000
1.2
2.2
10
13
14
15
16
17
PTB
AAZ
>10000
565.6
278.1
949.8
>10000
92.8
12.1
2.6
ꢀ
H
ꢀ
m
ꢀOCH3
12.4
2.6
20.5
16.2
23.6
18.7
25.9
25.2
ꢀ
p
p
p
ꢀF
ꢀ
ꢀCF3
ꢀOH
15.7
1.0
ꢀ
342.0
250.2
43.9
12.0
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5ꢀ10 % of the reported
values).
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The following SAR can be unveiled from the inhibition data shown in Table 1:
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i)
The cytosolic isoforms hCA I was moderately inhibited by most of the sulfonamides of the
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first series with inhibition constants (KIs) ranging between 123.0 and 512.6 nM and comparable to
the lead PTB (KI 342.0 nM) and standard AAZ (KI 250 nM). The only exceptions were derivative
9, which was an effective hCA I inhibitor (KI of 7.9 nM), and the double head derivative 10 which
resulted inactive (KI > 10000 nM). We may hypothesize that the presence of hydrophilic moieties in
these molecular structures is well tolerated within the hCA I active site. Indeed the substitution of
the phenyl with a more hydrophilic pyridyl ring (7) or the substitution of the oxymethylene linker
with a strongly hydrophilic aminomethylene one (9) increased the inhibitory activity up to 123.0
and 7.91 nM respectively.
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The sulfonamides belonging to the second series showed a diminished hCA I inhibitory
action. Indeed only derivate 17 which incorporates a hydroxy group to the phenyl ring acted as a
strong hCA I inhibitor, with KI of 92.8 nM, whereas the remaining compounds possessed inhibition
constants ranging between 278.1 and 949.8 nM, whereas derivative 16 did not inhibit hCA I up to
10000 nM.
ii) The physiologically dominant isoform hCA II was very potently inhibited by all the investigated
compounds (KI values ranging between 0.83 and 15.7 nM, Table 1), with the exception of the
double head derivative 10 which did not inhibit hCA II up to 10000 nM. The definition of a proper
SAR for hCA II is not feasible, considering that the remaining derivatives showed rather similar
affinities. However it could be stressed that the presence of a hydrophilic aminomethylene linker in
the first series of derivatives led to the strongest inhibitor reported here (9) with its subnanomolar
activity of 0.83 nM for this isoform, whereas the presence of a para substituent on the phenyl ring,
represented herein by the methoxy group of 6, reduced the inhibitory potency by four times.
Within the second series of derivatives, the incorporation of a methoxy or trifluoromethyl group in
meta or para positions to the sulfamoyl ZBG, respectively, led to a ten fold decrease of the
inhibitory activity. Generally all the investigated compounds (except derivative 10) were better
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inhibitors compared to the lead PTB or the clinically used AAZ (KI values of 43.9 and 12.1 nM),
thus proving that compounds endowed with enhanced flexibility between their molecular fragments
act as more effective hCA II inhibitors compared to the rigid compound PTB.
9
iii) All the reported derivatives acted as very efficient inhibitors for the tumorꢀassociated isoform
hCA IX. The general inhibitory tendency was slightly less compact compared to the behaviour
observed against hCA II, with KIs ranging between 2.1 and 23.6 nM, but the data in Table 1
allowed us to draw a very clearꢀcut SAR: for both reported series, the derivates bearing an
unsubstituted phenyl ring are the most efficient inhibitors regardless on the nature of the XCH2
linker. Indeed derivatives 3, 8, 9 and 13 were low nanomolar hCA IX inhibitors, with KIs of 2.1,
2.4, 2.2 and 2.6 nM respectively. Only compound 4 represented the exception to this general rule
with a KI of 2.34 nM, although it has a methyl substituent at position 3 of the aromatic tail.
Conversely, for the first series of derivatives, the insertion of a methoxy group on the ring or its
substitution with a pyridyl moiety decreased the inhibitory potency to the range of 9.3ꢀ17.3 nM.
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Within the second series f CAIs, the incorporation of pꢀFꢀ, p ꢀOHꢀ, mꢀMeOꢀ and p ꢀCF3ꢀ moiety on
the tail drove to KIs ranging between 16.2 and 23.6 nM. It shoud be also highlighted the potent
inhibitory activity of the double head derivate 10 against this isoform with a K of 12.1 nM, in
I
comparison to its ineffective activity on the cytosolic isoforms hCA I and hCA II, making this
compound selective for the tumorꢀassociated over the cytosolic isoforms.
All the reported derivatives were more potent inhibitors compared to the lead PTB or the clinically
used AAZ (KI values of 25.9 and 25.0 nM), especially the unsubstituted compounds 3, 8 ,9 and 13
which showed a more than ten times better inhibitory activity, confirming the importance of the
flexible features to better interact with the CA IX active site.
iv) The reported derivatives showed a very efficient inhibitory activity also against the other tumorꢀ
associated isoform hCA XII (which is also involved in glaucoma). However in this case the outline
of a straightforward SAR, as for CA IX, was not observed. Indeed within the first series,
compounds 3 and 9, unsubstituted on the external phenyl ring and compounds 5 and 6,
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incorporating a methoxy group respectively at the meta and para positions, acted as low nanomolar
inhibitors with KIs ranging between 1.0 and 1.1 nM. On the contrary, derivative 8, bearing a ꢀCH2Sꢀ
linker, showed in this case a fiveꢀfold reduction in the inhibitory activity. The insertion of a methyl
group at the meta position (4) or the substitution of the phenyl ring with a pyridyl one (7) reduced
the activity by five and six times respectively.
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Conversely, for the second group of compounds, the unsubstituted derivative 13 acted as the most
potent inhibitor with a KI of 1.1 nM, whereas the insertion of substituents on the phenyl ring
generally reduced the inhibitory efficiency. In particular derivative 17, bearing a meta methoxy
group showed a KI increased by a hundred times compared to its unsubstituted counterpart.
Differently from the previously discussed isoforms, not all the new sulfonamides inhibited CA XII
better than the lead PTB or the clinically used AAZ (KI values of 6.0 and 5.7 nM). These data
suggested that the enhancement of the molecular flexibility did not generally increase the
interaction with the CA XII active site, but depending on the nature and position of the substituents
on the external phenyl ring, a more intricate interaction was probably in act between the enyme and
the inhibitor.
Finally it should be stressed the potent inhibitory activity of the double head derivate 10 against this
isoform too, with a KI of 10.2 nM. Although its inhibitory potency was slightly reduced in
comparison to lead PTB or AAZ, this data was surprising and extremely interesting when merged
with the KI of compound 10 against CA I, II and IX. In fact 10 acted as a strong and selective
inhibitor of the tumorꢀassociated isoform hCA IX and XII over the cytosolic isoforms hCA I and II.
Molecular modeling. In order to gain a better understanding on the binding modes of the
synthesized compounds, docking studies were carried out for the inhibitors 3ꢀ9 and 13ꢀ17 with hCA
II (PDB ID: 4Q6E) and hCA IX (PDB ID: 3IAI)13a. In hCA II, all compounds were predicted to
orient the aromatic sulfonamide moiety deeply into the catalytic cleft of the active site forming a
hydrogen bond with the hydroxyl group of T199 side chain, while the NHꢀ coordinates the zinc ion
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and the phenyl ring is neighboured by several hydrophobic residues (V121, H94 and L198). The
1,2,3ꢀtriazole ring, linked to the position 4 of the benzosulfonamide directly (first series:
compounds 3ꢀ9), or through an oxymethylene linker (second series: compounds 13ꢀ17), mostly
forms edgeꢀtoꢀface πꢀ π interactions with the phenyl group of the F131side chain. The flexible
hydrophobic tail of the compounds is oriented towards the lipophilic half of the CA active site, in
the region delimited by residues F131, G132, V135, Q136, P202 and L204 (Figure 3).
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A)
B)
Figure 3. Representation of the binding orientation of compounds A) 3ꢀ9 and B) 13ꢀ17 in hCA II
(PDB code 4Q6E). The hydrophobic half of the active site cleft is shown as a red surface, while the
hydrophilic half as a blue one.
An experimental validation of the docking procedure was achieved by the Xꢀray crystallographic
structures of the adducts formed by hCA II with compounds 3 and 13 (at 1.0 Å atomic resolution).
Indeed the superposition of the in silico and the experimental poses highlight a very good matching
(Figure 4).
A)
B)
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Figure 4. Docking (grey) vs. Xꢀray (green) orientation in hCAII active site for compounds 3 (A)
and 13 (B).
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9
As mentioned above, docking studies were also performed in the hCA IX active site (PDB ID:
3IAI).13a Compounds 3ꢀ9 oriented themselves in quite similar poses as observed for hCA II. The
triazole ring established hydrophobic interactions with V131, whereas the tail aryl groups extended
in a partly exposed pocket formed by hydrophobic residues. The different orientation of the
aromatic tails could be correlated to the mutation of F131 replaced by V131 in hCA IX active site.
Conversely compounds 13ꢀ17, which bear the aryltriazole moieties linked to benzenesulfonamide
through an oxymethylene linker, extend their tails towards the hydrophilic region delimited by
residues N62, H64, Q67 and Q92, where the triazole forms polar interactions with Q67 and Q92.
The different orientation assumed by such moieties within hCA IX active site is likely due to the
F131V CAII/IX mutation. In absence of the πꢀ π interaction driving force the aromatic tails were
not driven near the hydrophobic side anymore, but instead found good interaction points on the
hydrophilic region (Figure 5B).
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The different set of interactions established by the aromatic tails of the two series of compounds
within hCA II and hCA IX active sites could explain the general reduced inhibitory activity against
the tumorꢀassociated isoforms compared to the cytosolic one.
A)
B)
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Figure 5. Representation of the binding orientation of compounds A) 3ꢀ8 and B) 13ꢀ17 in hCAIX
(PDB code 3IAI). The hydrophobic half of the active site cleft is shown as a red surface, while the
hydrophilic half as a blue one.
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Xꢀray Crystallography. The Fo ꢀFc electron density maps (Figure 6) show a well defined
density for all the atoms of the inhibitors 3 and 13 bound within the hCA II active site, which are
coordinated to the Zn(II) ion by means of the sulfonamide moiety. The sulfonamide nitrogen
coordinated to the zinc ion also makes a strong Hꢀbond with the OH of Thr199. These interactions
are common to all CA ꢀ sulfonamide adducts reported to date.1,13 The scaffold of the inhibitors
occupied the entire available space inside the cavity and were oriented toward the hydrophobic part
of the active site cleft, establishing strong van der Waals interactions. In particular the phenyl ring
attached to the sulfonamide group made hydrophobic interactions with residues V121 and L198.
The 1,2,3ꢀtriazole ring forms hydrogen bonds with water molecules and in the inhibitor 13 an edgeꢀ
toꢀface πꢀ π stacking interaction with the phenyl group of the F131 side chain. Furthermore the
terminal phenyl ring of inhibitors 3 and 13 forms strong hydrophobic interactions with V135 and
F131.
A)
B)
Figure 6. Active site region in the hCA IIꢀ3 (A) and 13 (B) complexes. The omit FoꢀFc electron
density maps (contoured at 2.0 σ) for the two inhibitors are also shown.
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Table 2.
Summary of Data Collection and Atomic Model Refinement Statistics.a
8
9
HCAII + 3
HCAII + 13
10
11
12
13
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17
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PDB ID
5LJQ
5LJT
Wavelength (Å)
Space Group
0.976
P21
0.976
P21
Unit cell (a,b,c,β) (Å, °)
42.37, 41.44, 72.22, 104.45 42.36, 41.40, 72.17 104.41
Limiting resolution (Å)
Unique reflections
Rsym (%)
40.0ꢀ1.05 (1.11ꢀ1.05)
109788
41.0ꢀ1.00 (1.06ꢀ1.00)
121936
6.1 (57.9)
6.0 (67.2)
Redundancy
4.75 (3.77)
3.5 (2.6)
Completeness overall (%)
<I/(I)>
97.1 (86.1)
93.7 (75.6)
10.22 (1.30)
12.61 (2.07)
Refinement statistics
Resolution range (Å)
Unique reflections, working/free
Rfactor (%)
30.0ꢀ1.05
104237/5466
13.35
30.0ꢀ 1.00
115805/6012
13.82
Rfree(%)
14.85
15.72
No. of protein atoms
No. of water molecules
No. of heterogen atoms
r.m.s.d. bonds(Å)
r.m.s.d. angles (°)
Average B factor (Å2)
All atoms
4176
4328
418
439
52
66
0.0046
1.176
0.0047
1.189
12.48
14.33
26.55
11.84
10.81
26.07
inhibitor
solvent
a
Values in parentheses are for the highest resolution shell.
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IOP Lowering Activity. We have investigated the intraocular pressure (IOP) lowering
properties of some of the herein reported derivatives, more precisely 3, 13 (for which the Xꢀray
structures of the adducts with hCA II was reported) and 9 (which showed a unique subnanomolar
inhibitory activity against hCA II) in an animal model of glaucoma.16 The three compounds showed
a sufficient water solubility to be formulated as 1% eye drops at the neutral pH value (dorzolamide,
DRZ, the clinically used drug is a hydrochloride salt with a pH of the eye drops of 5.5 which
produces eye irritation and stinging as side effects).7bꢀd, 10b The drugs were administered to rabbits
with high IOP, induced by the injection of 0.1 mL of hypertonic saline solution (5% in distilled
water) into the vitreous of both eyes (Figure 6). DZA hydrochloride was used as a standard drug,
whereas the control experiments were done by using the vehicle (hydroxypropylcellulose at 0.05%).
The selected derivatives were very potent inhibitors of isoforms hCA II (responsible for aqueous
humor secretion; KI of 0.83ꢀ1.2 nM, Table 1) and hCA XII (isoform that is overexpressed in the
eyes of glaucomatous patients;17 KI of 1.1 nM, Table 1).
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time/min
0
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120
240
1.0
0.5
0.0
Vehicle
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
-3.5
DRZ
13
3
9
Figure 7. Drop of intraocular pressure (ꢁIOP, mmHg) versus time (min) in hypertonic salineꢀ
induced ocular hypertension in rabbits, treated with 50 ꢂL of 1 % solution of compounds 3, 9 and
13 and DRZ as the standard drug and vehicle. Errors were within 10−15% of the reported IOP
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values (from three different measurements for each of the four animals in the study group) and were
statistically significant (p = 0.045 by the Student’s t test).
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9
As seen from the data of Figure 7, all three compounds proved to be more effective than DRZ, even
if at different times postꢀadministration. Indeed, a comparable behaviour to DRZ, which caused an
IOP drop of 1.5 mm Hg at 60 min postꢀadministration, emerged only for derivative 13, that effected
an IOP drop increased to 2.5 mm Hg after the same time. Another analogy between 13 and DRZ
was the IOP rise in shortꢀterm, so that after 4 h no IOP decrease was seen. Conversely for
derivatives 3 and 9, the peak of IOP drop, that reached 3.0 and 2.0 mm Hg respectively, was at 2 h
postꢀadministration, whereas their IOP lowering activity after 1h was reduced in comparison to
DRZ. In addition, while for compound 9 after 4 h no IOP decrease was seen, 3 showed the ability to
protract its IOP lowering action up to the same time point.
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It should be mentioned that the animal model employed here is of normotensive rabbits, and this is
why the absolute IOP drops are not very high, but the advantage of this model is that the
measurements can be done rapidly and are highly reproducible.
Conclusions. We report two new series of benzenesulfonamides incorporating phenylꢀ1,2,3ꢀ
triazole moieties, which have been prepared by means of click chemistry. The sulfonamides,
designed in order to be endowed with an enhanced flexibility between their molecular fragments in
comparison to their lead PTB, has been screened for the inhibition of four physiologically relevant
CA isoforms: hCA I, II, IX, and XII. Hence, interesting SAR have been extrapolated and afterwards
the Xꢀray crystal structures of two of them bound to hCA II shed light on their inhibitory behaviour
at the molecular level. The crystallographic results supported a deeper molecular modeling analysis
concerning all the reported derivatives and both hCA II and hCA IX. Three of these compounds
also showed highly effective in vivo antiglaucoma activity in an animal model of the disease being
more effective compared to the clinically used drug dorzolamide, even if at different time postꢀ
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administration. The attempt to functionalize the undesired dipropargylaniline, obtained during the
synthetic pathway, with benzenesulfonamide moieties has been rewarded with the most surprising
and excellent inhibitory profile, that belongs to compound 10. Indeed this double head sulfonamide
act as the unique totally selective inhibitors within the two series. Preliminary hypothesis about
such a selectivity have to be underpinned with crystallographic studies (currently ongoing), but it is
clear that this innovative substitution pattern for the benzensulfonamideꢀlike inhibitors is worth to
be deeper investigated with additional similar derivatives.
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Experimental protocols
Chemistry. Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar
and TCI. All reactions involving airꢀ or moistureꢀsensitive compounds were performed under a
nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear
13
magnetic resonance (1HꢀNMR, CꢀNMR) spectra were recorded using a Bruker Advance III 400
MHz spectrometer in DMSOꢀd6. Chemical shifts are reported in parts per million (ppm) and the
coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s,
singlet; d, doublet; sept, septet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double
of doubles, appt, aparent triplet, appq, aparent quartet. The assignment of exchangeable protons
(OH and NH) was confirmed by the addition of D2O. Analytical thinꢀlayer chromatography (TLC)
was carried out on Merck silica gel Fꢀ254 plates. Flash chromatography purifications were
performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the stationary phase and ethyl
acetate/nꢀhexane were used as eluents. Melting points (mp) were measured in open capillary tubes
with a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected. HPLC was performed by using
a Waters 2690 separation module coupled with a photodiode array detector (PDA Waters 996) and
as column a NovaꢀPak C18 4 ꢂm 3.9 mm × 150 mm (Waters), silicaꢀbased reverse phase column.
Sample was dissolved in acetonitrile 10%, and an injection volume of 45 ꢂL was used. The mobile
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phase, at a flow rate of 1 mL/min, was a gradient of water + trifluoroacetic acid (TFA) 0.1% (A)
and acetonitrile + TFA 0.1% (B), with steps as follows: (A%:B%), 0−10 min 90:10, 10−25 min
gradient to 60:40, 26:28 min isocratic 20:80, 29−35 min isocratic 90:10. TFA 0.1% in water as well
in acetonitrile was used as counterion. All compounds reported here were >96% HPLC pure. The
solvents used in MS measures were acetone, acetonitrile (Chromasolv grade), purchased from
SigmaꢀAldrich (Milan ꢀ Italy), and mQ water 18 Mꢃ, obtained from Millipore's Simplicity system
(MilanꢀItaly). The mass spectra were obtained using a Varian 1200L triple quadrupole system (Palo
Alto, CA, USA) equipped by Electrospray Source (ESI) operating in both positive and negative
ions. Stock solutions of analytes were prepared in acetone at 1.0 mg mLꢀ1 and stored at 4°C.
Working solutions of each analyte were freshly prepared by diluting stock solutions in a mixture of
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mQ H2O/ACN 1/1 (v/v) up to a concentration of 1.0
acquired by introducing, via syringe pump at 10
µ
g mLꢀ1 The mass spectra of each analyte were
L minꢀ1, of the its working solution. Rawꢀdata
µ
were collected an processed by Varian Workstation Vers. 6.8 software.
General synthetic procedure of compounds 3ꢀ10, 13ꢀ17.5d
The appropriate alkyne 12, 18ꢀ24 (1.0 eq) was added to a suspension of aryl azide 2, 26ꢀ30 (1.1 eq)
in H2O/tꢀBuOH 1/1 (4 ml) at r.t., followed by copper (0) nanosized (0.1 eq) and TEA HCl or
TMACl (1.0 eq). The suspension was stirred at r.t. or 60°C until starting materials were consumed
(TLC monitoring), then quenched with H2O (20 ml) and the formed precipitate was filteredꢀoff and
washed with H2O. The residue was purified by silica gel column chromatography eluting with the
appropriate mixture of ethyl acetate in nꢀhexane, or alternatively was dissolved in a minimal
amount of acetone. Thus the obtained solution was filtered through Celite 521® and concentrated
under vacuo to give a residue that was triturated with Et2O to afford the titled compounds 3ꢀ10, 13ꢀ
17.
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4-(4-Phenoxymethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide (3).
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8
Compound 3 was obtained according the general procedure earlier reported using 18 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 27h at r.t. to give the titled compound 3 as a light yellow solid.
72% yield; m.p. 200ꢀ202°C; silica gel TLC Rf 0.22 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.29 (s, 2H, CH2), 7.01 (t, J = 7.2, 1H), 7.12 (d, J = 7.6, 2H), 7.37 (t, J = 7.6, 2H), 7.57
(s, 2H, exchange with D2O, SO2NH2), 8.07 (d, J = 8.8, 2H), 8.19 (d, J = 8.8, 2H), 9.10 (s, 1H); δC
(100 MHz, DMSOꢀd6): 61.7, 115.6, 121.3, 121.9, 124.0, 128.4, 130.5, 139.5, 144.8, 145.3, 158.9;
m/z (ESI positive) 331.0 [M+H]+
9
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4-(4-m-Tolyloxymethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide (4).
Compound 4 was obtained according the general procedure earlier reported using 19 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH /H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 24h at r.t. to give the titled compound 4 as a yellow solid.
68% yield; m.p. 176ꢀ178°C; silica gel TLC Rf 0.13 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 2.33 (s, 3H, CH3), 5.27 (s, 2H, CH2), 6.83 (d, J = 7.6, 1H), 6.91 (d, J = 7.6, 1H), 6.94
(s, 1H), 7.23 (t, J = 7.6, 1H), 7.57 (s, 2H, exchange with D2O, SO2NH2), 8.07 (d, J = 8.8, 2H), 8.19
(d, J = 8.8, 2H), 9.09 (s, 1H); δC (100 MHz, DMSOꢀd6): 22.1, 61.8, 112.6, 116.3, 121.3, 122.7,
124.0, 128.5, 130.3, 139.5, 140.0, 144.8, 145.4, 158.9; m/z (ESI positive) 345.0 [M+H]+
4-[4-(3-Methoxy-phenoxymethyl)-[1,2,3]triazol-1-yl]-benzenesulfonamide (5).
Compound 5 was obtained according the general procedure earlier reported using 20 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 17h at r.t. to give the titled compound 5 as a yellow solid.
70% yield; m.p. 159ꢀ161°C; silica gel TLC Rf 0.14 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 3.78 (s, 3H, CH3), 5.28 (s, 2H, CH2), 6.60 (dd, J = 2.0, 8.0, 1H), 6.68 (s, 1H), 6.70 (dd,
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J = 2.0, 8.0, 1H), 7.25 (t, J = 8.0, 1H), 7.59 (s, 2H, exchange with D2O, SO2NH2), 8.07 (d, J = 8.8,
2H), 8.19 (d, J = 8.8, 2H), 9.11 (s, 1H); δC (100 MHz, DMSOꢀd6): 56.1, 61.9, 102.0, 107.7, 107.8,
121.3, 124.0, 128.5, 131.0, 139.5, 144.9, 145.2, 160.1, 161.5; m/z (ESI positive) 361.0 [M+H]+
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4-[4-(4-Methoxy-phenoxymethyl)-[1,2,3]triazol-1-yl]-benzenesulfonamide (6).
Compound 6 was obtained according the general procedure earlier reported using 21 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 24h at r.t. to give the titled compound 6 as a yellow solid.
68% yield; m.p. 209ꢀ210°C; silica gel TLC Rf 0.18 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 3.74 (s, 3H,CH3), 5.22 (s, 2H,CH2), 6.92 (d, J = 8.8, 2H), 7.05 (d, J = 8.8, 2H), 7.58 (s,
2H, exchange with D2O, SO2NH2), 8.06 (d, J = 8.0, 2H), 8.19 (d, J = 8.0, 2H), 9.09 (s, 1H); δC
(100 MHz, DMSOꢀd6): 56.3, 62.4, 115.6, 116.7, 121.3, 123.9, 128.5, 139.5, 144.8, 145.5, 152.9,
154.6; m/z (ESI positive) 361.0 [M+H]+
4-(4-((Pyridin-3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (7).
Compound 7 was obtained according the general procedure earlier reported using 22 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 48h at r.t. to give the titled compound 7 as a yellow solid.
51% yield; m.p. 277ꢀ280d; silica gel TLC Rf 0.09 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.29 (s, 2H, CH2), 6.31 (t, J = 6.4, 1H), 6.45 (d, J = 9.4, 1H), 7.48 (t, J = 9.4, 1H), 7.55
(s, 2H, exchange with D2O, SO2NH2), 7.90 (d, J = 6.4, 1H), 8.04 (d, J = 8.8, 2H), 8.17 (d, J = 8.8,
2H), 8.89 (s, 1H); δC (100 MHz, DMSOꢀd6): 44.1, 106.5, 120.6, 121.2, 123.2, 128.4, 139.4, 140.1,
141.2, 144.8, 152.8, 162.1; m/z (ESI negative) 330.0 [M−H]ꢀ
4-(4-Phenylsulfanylmethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide (8).
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Compound 8 was obtained according the general procedure earlier reported using 23 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 24h at r.t. to give the titled compound 8 as a yellow solid.
63% yield; m.p. 202ꢀ204°C; silica gel TLC Rf 0.62 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 4.43 (s, 2H, CH2), 7.25 (t, J = 7.2, 1H), 7.37 (t, J = 7.6, 2H), 7.45 (d, J = 7.6, 2H), 7.53
(s, 2H, s, 2H, exchange with D2O, SO2NH2), 8.04 (d, J = 8.4, 2H), 8.12 (d, J = 8.4, 2H), 8.82 (s,
1H); δC (100 MHz, DMSOꢀd6): 28.1, 121.1, 122.6, 127.0, 128.4, 129.8, 130.0, 136.4, 139.4, 144.7,
146.2; m/z (ESI positive) 347.0 [M+H]+
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4-(4-Phenylaminomethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide (9).
Compound 9 was obtained according the general procedure earlier reported using 24 (1.0 eq), 2
(0.12 g, 1.1 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 20h at r.t. to give the titled compound 9 as a yellow solid.
78% yield; m.p. 221ꢀ223°C; silica gel TLC Rf 0.57 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 4.42 (d, J = 5.6, 2H, CH2), 6.20 (t, J = 5.6, 1H, exchange with D2O, NH) ,6.59 (t, J =
7.2, 1H), 6.71 (d, J = 8.0, 2H), 7.12 (t, J = 8.0, 2H), 7.57 (s, 2H, exchange with D2O, SO2NH2),
8.04 (d, J = 8.4, 2H), 8.16 (d, J = 8.4, 2H), 8.85 (s, 1H); δC (100 MHz, DMSOꢀd6): 39.5, 113.3,
117.1, 121.0, 122.2, 128.5, 129.8, 139.6, 144.6, 148.3, 149.2; m/z (ESI positive) 330.0 [M+H]+
N,N-Di-(1-(4-sulfamoylphenyl)-[1,2,3]triazol-4-yl-methyl)-aniline (10).
Compound 10 was obtained according the general procedure earlier reported using 25 (1.0 eq), 2
(0.12 g, 2.2 eq) in tꢀBuOH/H2O 1/1 (4 ml), TEA HCl (2.0 eq) and copper nanosize (0.2 eq). The
reaction mixture was stirred for 72h at r.t. to give the titled compound 10 as a yellow solid.
65% yield; m.p. 253ꢀ254°C; silica gel TLC Rf 0.14 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 4.83 (s, 4H, 2 x CH2) ,6.59 (t, J = 7.6, 1H), 6.96 (d, J = 7.8, 2H), 7.16 (t, J = 7.8, 2H),
7.52 (s, 4H, exchange with D2O, 2 x SO2NH2), 8.05 (d, J = 8.4, 4H), 8.17 (d, J = 8.4, 4H), 8.94 (s,
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2H); δC (100 MHz, DMSOꢀd6): 46.6, 113.9, 117.8, 121.1, 122.5, 128.4, 130.0, 139.6, 144.7, 147.2,
148.6; m/z (ESI negative) 564.0 [M−H]ꢀ
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4-(1-Phenyl-1H-[1,2,3]triazol-4-ylmethoxy)-benzenesulfonamide (13).
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Compound 13 was obtained according the general procedure earlier reported using 12 (0.05g, 1.0
eq), 26 (1.1 eq) in tꢀBuOH/H2O 1/1 (3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred at 60°C for 2.5h and the obtained residue was purified by silica gel
column chromatography eluting with 50% ethyl acetate in nꢀhexane to afford 13 as a white solid.
70% yield; m.p. 200ꢀ202°C; silica gel TLC Rf 0.24 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.38 (s, 2H, CH2), 7.26 (s, 2H, exchange with D2O, SO2NH2) 7.28 (m, 2H, overlap with
signal at 7.26), 7.55 (t, J = 7.6, 1H), 7.65 (t, J = 7.6, 2H), 7.81 (d, J = 8.4, 2H), 7.95 (d, J = 7.6,
2H), 9.03 (s, 1H); δC (100 MHz, DMSOꢀd6): 62.3, 115.7, 121.1, 124.0, 128.6, 129.8, 130.9, 137.5,
137.6, 144.3, 161.2; m/z (ESI positive) 331.0 [M+H]+
4-[1-(3-Methoxy-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-benzenesulfonamide (14).
Compound 14 was obtained according the general procedure earlier reported using 12 (0.05g, 1.0
eq), 27 (1.1 eq) in tꢀBuOH/H2O 1/1 (3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred at 60°C for 3h and the obtained residue was purified by silica gel
column chromatography eluting with 50% ethyl acetate in nꢀhexane to afford 14 as a white solid.
72% yield; m.p. 184ꢀ186°C; silica gel TLC Rf 0.21 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 3.90 (s, 3H, CH3), 5.37 (s, 2H, CH2), 7.11 (d, J = 6.8, 1H), 7.27 (s, 2H, exchange with
D2O, SO2NH2), 7.28 (m, 2H, overlap with signal at 7.27), 7.54 (m, 3H), 7.82 (d, J = 8.8, 2H), 9.05
(s, 1H); δC (100 MHz, DMSOꢀd6): 56.6, 62.3, 106.7, 113.1, 115.5, 115.7, 124.1, 128.6, 131.8,
137.6, 138.5, 144.2, 161.1, 161.2; m/z (ESI positive) 361.0 [M+H]+
4-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-benzenesulfonamide (15).
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Compound 15 was obtained according the general procedure earlier reported using 12 (0.05g, 1.0
eq), 28 (1.1 eq) in tꢀBuOH/H2O 1/1 (3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred at 60°C for 2h and the obtained residue was purified by silica gel
column chromatography eluting with 50% ethyl acetate in nꢀhexane to afford 15 as a white solid.
70% yield; m.p. 211ꢀ213°C; silica gel TLC Rf 0.29 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.37 (s, 2H, CH2), 7.27 (s, 2H, exchange with D2O, SO2NH2), 7.28 (m, 2H, overlap
with signal at 7.27), 7.51 (at, J = 8.4, 2H), 7.81 (d, J = 8.4, 2H), 8.00 (m, 2H), 9.00 (s, 1H); δF (376
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MHz, DMSOꢀd6): ꢀ112.97 (s, 1F); δC (100 MHz, DMSOꢀd6): 62.2, 115.8, 117.8 (d, J2 = 23.1),
CF
123.6 (d, J3 = 8.8), 124.4, 128.7, 134.1, 137.6, 144.3, 161.2, 162.7 (d, J1 = 244.5); m/z (ESI
CF
CF
positive) 349.0 [M+H]+
4-[1-(4-Trifluoromethyl-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-benzenesulfonamide (16).
Compound 16 was obtained according the general procedure earlier reported using 12 (0.05g, 1.0
eq), 29 (1.1 eq) in tꢀBuOH/H2O 1/1 (3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq).The
reaction mixture was stirred at 60°C for 1.5h and the obtained residue was purified by silica gel
column chromatography eluting with 50% ethyl acetate in nꢀhexane to afford 16 as a white solid.
69% yield; m.p. 198ꢀ199 °C; silica gel TLC Rf 0.16 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.36(s, 2H, CH2), 7.23 (s, 2H, exchange with D2O, SO2NH2), 7.24 (m, 2H, overlap with
signal at 7.23), 7.78 (d, J = 8.8, 2H), 8.00 (d, J = 8.6, 2H), 8.18 (d, J = 8.6, 2H), 9.14 (s, 1H); δF
(376 MHz, DMSOꢀd6): ꢀ61.03 (s, 3F); δC (100 MHz, DMSOꢀd6): 62.2, 115.8, 121.6, 124.3, 124.7
(d, J1CF = 270.6), 128.2 (q, J3CF = 3.8), 128.6, 129.8 (q, J2CF = 32.2), 137.6, 140.3, 144.7, 161.2; m/z
(ESI positive) 399.0 [M+H]+
4-[1-(4-Hydroxy-phenyl)-1H-[1,2,3]triazol-4-ylmethoxy]-benzenesulfonamide (17).
Compound 17 was obtained according the general procedure earlier reported using 12 (0.05g, 1.0
eq), 30 (1.1 eq) in tꢀBuOH/H2O 1/1 (3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq). The
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reaction mixture was stirred at 60°C for 1h and the obtained residue was purified by silica gel
column chromatography eluting with 60% ethyl acetate in nꢀhexane to afford 17 as a white solid.
49% yield; m.p. 229ꢀ231°C; silica gel TLC Rf 0.09 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 5.34 (s, 2H, CH2), 6.97 (d, J = 8.8, 2H), 7.26 (m, 2H, overlap with signal at 7.27), 7.27
(s, 2H, exchange with D2O, SO2NH2), 7.71 (d, J = 8.8, 2H), 7.81 (d, J = 8.8, 2H), 8.84 (s, 1H),
10.02 (s, 1H, exchange with D2O, OH); δC (100 MHz, DMSOꢀd6): 62.3, 115.8, 117.0, 123.0, 123.9,
128.6, 129.6, 137.6, 143.9, 158.8, 161.3; m/z (ESI positive) 347.0 [M+H]+
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General synthetic procedure of alkynes 12, 18ꢀ24.18
Propargyl bromide (1.2 eq) was added to a suspension of the proper phenol, thiophenol or aniline
(0.5 g, 1.0 eq) and K2CO3 (2.0 eq) in dry DMF (4 ml) under a nitrogen atmosphere and that was
stirred at r.t. until starting material was consumed (TLC monitoring). The reaction mixture was
quenched with H2O (20 ml) and extracted with Et2O or EtOAc (25 ml). The organic layer was
washed with brine (4x15ml), dried over Na2SO4 filteredꢀoff and concentrated under vacuo to give
the titled compounds 12, 18ꢀ24.
Prop-2-ynyloxy-benzene (18).
Compound 18 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 2h to give the titled compound 18.
74% yield; silica gel TLC Rf 0.68 (EtOAc/nꢀhexane 20 % v/v); δH (400 MHz, CDCl3): 2.58 (t, J =
2.4, 1H), 4.76 (d, J = 2.4, 2H), 7.06 (m, 3H), 7.37 (t, J = 8.8, 2H); δC (100 MHz, CDCl3): 56.1,
75.8, 79.0, 115.3, 121.9, 129.8, 157.9.
Experimental in agreement with reported data.18
1-Methyl-3-prop-2-ynyloxy-benzene (19).
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Compound 19 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 2h to give the titled compound 19.
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81% yield; silica gel TLC Rf 0.48 (EtOAc/nꢀhexane 20 % v/v); δH (400 MHz, DMSOꢀd6): 2.45 (s,
3H, CH3), 2.52 (t, J = 2.4, 1H), 4.69 (d, J = 2.4, 2H), 6.81 (m, 3H), 7.20 (t, J = 7.2, 1H); δC (100
MHz, CDCl3): 21.9, 56.0, 75.7, 79.1, 112.0, 116.1, 122.8, 129.5, 139.9, 157.9.
Experimental in agreement with reported data.19
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1-Methoxy-3-prop-2-ynyloxy-benzene (20).
Compound 20 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 2h to give the titled compound 20.
78% yield; silica gel TLC Rf 0.78 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz, CDCl3): 2.53 (t, J =
2.4, 1H), 3.80 (s, 3H, CH3), 4.68 (d, J = 2.4, 2H), 6.57 (m, 3H), 7.20 (t, J = 7.4, 1H); δC (100 MHz,
CDCl3): 55.6, 56.2, 75.8, 78.9, 101.9, 107.2, 107.6, 103.2, 159.2, 161.2.
Experimental in agreement with reported data.19
1-Methoxy-4-prop-2-ynyloxy-benzene (21).
Compound 21 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 2h to give the titled compound 21.
71% yield; silica gel TLC Rf 0.65 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz, CDCl3): 2.51 (t, J =
2.4, 1H), 3.78 (s, 3H, CH3), 4.64 (d, J = 2.4, 2H), 6.85 (d, J = 9.2, 2H), 6.93 (d, J = 9.2, 2H); δC
(100 MHz, CDCl3): 56.0, 56.9, 75.6, 79.2, 114.9, 116.5, 152.0, 154.8.
Experimental in agreement with reported data.19
2-Prop-2-ynyloxy-pyridine (22).
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Compound 22 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 3h to give to an oil that was purified by silica gel column chromatography
eluting with 5% MeOH in DCM to afford 22.
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50% yield; silica gel TLC Rf 0.49 (MeOH/CH2Cl2 10 % v/v); δH (400 MHz, CDCl3): 2.49 (t, J =
2.4, 1H), 4.76 (d, J = 2.4, 2H), 6.25 (t, J = 6.8, 1H), 6.61 (d, J = 9.2, 1H), 7.35 (m, 1H), 7.64 (d, J
= 6.8, 1H); δC (100 MHz, CDCl3): 31.3, 41.2, 73.5, 79.0, 116.6, 121.0, 129.6, 147.5.
Experimental in agreement with reported data.20
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Prop-2-ynylsulfanyl-benzene (23).
Compound 23 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at r.t. for 2h to give the titled compound 23.
96% yield; silica gel TLC Rf 0.61 (EtOAc/n-hexane 10 % v/v); δH (400 MHz, CDCl3): 2.30 (t, J =
2.4, 1H), 3.67 (d, J = 2.4, 2H), 7.32 (m, 1H), 7.39 (t, J = 7.6, 2H), 7.52 (m, 2H); δC (100 MHz,
CDCl3): 22.9, 71.9, 80.2, 127.3, 129.3, 129.4, 130.4.
Experimental in agreement with reported data.21
Phenyl-prop-2-ynyl-amine (24) and phenyl-di-prop-2-ynyl-amine (25).
Compound 24 and compound 25 were obtained according the general procedure earlier reported.
The reaction mixture was stirred at r.t. for 4h and the obtained oil was purified by silica gel column
chromatography eluting with EtOAc/petroleum ether 1/20 to afford the titled compounds 24 and 25
as two yellow liquids.
24: 35% yield; silica gel TLC Rf 0.76 (EtOAc/nꢀhexane 20 % v/v); δH (400 MHz, DMSOꢀd6): 2.24
(t, J = 2.4, 1H), 3.96 (d, J = 2.4, 2H), 6.78 (d, J = 7.4, 2H), 6.85 (t, J =7.4, 1H), 7.31 (t, J =7.4,
2H); δC (100 MHz, CDCl3): 34.4, 72.1, 80.8, 114.5, 119.7, 129.6, 146.3.
Experimental in agreement with reported data.22a
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25: 19% yield; silica gel TLC Rf 0.88 (EtOAc/nꢀhexane 20 % v/v); δH (400 MHz, DMSOꢀd6): 2.27
(t, J = 2.4, 2H), 4.14 (d, J = 2.4, 4H), 6.93 (t, J = 7.6, 1H), 7.02 (d, J =7.6, 2H), 7.24 (t, J = 7.6,
2H); δC (100 MHz, CDCl3): 41.1, 73.4, 79.1, 116.5, 120.8, 129.6, 147.6.
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Experimental in agreement with reported data.22b
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4-Prop-2-ynyloxy-benzenesulfonamide (12).
Compound 12 was obtained according the general procedure earlier reported. The reaction mixture
was stirred at 60°C for 5h and then was quenched with H2O (20 ml). The obtained suspension was
stirred overnight at r.t.. The formed precipitate was filteredꢀoff and purified by silica gel column
chromatography eluting with 50 % EtOAc in nꢀhexane to afford the title compound 12.
71% yield; m.p. 131ꢀ132°C; silica gel TLC Rf 0.47 (EtOAc/nꢀhexane 50 % v/v); δH (400 MHz,
DMSOꢀd6): 3.66 (t, J = 2.4, 1H), 4.94 (d, J = 2.4, 2H), 7.17 (d, J= 8.8, 2H), 7.26 (s, 2H, exchange
with D2O, SO2NH2), 7.80 (d, J = 8.8, 2H); δC (100 MHz, DMSOꢀd6): 56.7, 79.6, 79.7, 115.9, 128.6,
137.8, 160.4.
Experimental in agreement with reported data.23
General synthetic procedure of phenylazides 2, 26ꢀ30.24
The proper aniline (0.5g, 1.0eq) was dissolved in a 4M HCl aqueous solution (5 ml) at 0°C. NaNO2
(1.2 eq) was slowly added and the resulting solution was stirred at the same temperature for 0.5h.
Then NaN3 (1.5 eq) was added portionꢀwise and the mixture was stirred at r.t. for 0.5h. Reaction
mixture was filteredꢀoff or extracted with Et2O (2 x 15 ml) and the combined organic layers were
dried over Na2SO4, filteredꢀoff and the solvent evaporated in vacuo to afford the corresponding
phenylazide which was used without further purification.
4-Azido-benzenesulfonamide (2).
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