Attachment of carbohydrates to methoxyaryl moieties leads to highly selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1016/j.bmc.2014.07.052

The transmembrane isoforms of carbonic anhydrase (hCA IX and XII) have been shown to be linked to carcinogenesis and their inhibition to arrest primary tumor and metastases growth. In this paper, we present a new class of C-glycosides incorporating the me

Full text of DOI:10.1016/j.bmc.2014.07.052

Accepted Manuscript
Attachment of carbohydrates to methoxyaryl moieties leads to highly selective
inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII
Leonardo E. Riafrecha, Oscar M. Rodríguez, Daniela Vullo, Claudiu T.
Supuran, Pedro A. Colinas
PII:
S0968-0896(14)00576-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.07.052
BMC 11747
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2014
22 July 2014
30 July 2014
Please cite this article as: Riafrecha, L.E., Rodríguez, O.M., Vullo, D., Supuran, C.T., Colinas, P.A., Attachment of
carbohydrates to methoxyaryl moieties leads to highly selective inhibitors of the cancer associated carbonic
anhydrase isoforms IX and XII, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.07.052
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Attachment of carbohydrates to methoxyaryl moieties leads to highly
selective inhibitors of the cancer associated carbonic anhydrase isoforms
IX and XII
Leonardo E. Riafrecha,a Oscar M. Rodríguez,a Daniela Vullo, b Claudiu T.
Supuran b,c * and Pedro A. Colinas a ∗
a
LADECOR, Departamento de Química, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, 47 y 115, 1900 La Plata, Argentina
b
Universitá degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3,I-50019 Sesto Fiorentino (Firenze), Italy
c
Università degli Studi di Firenze, NEUROFARBA Department, Section of
Pharmaceutical Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence),
Italy
Abstract
The transmembrane isoforms of carbonic anhydrase (hCA IX and XII) have
been shown to be linked to carcinogenesis and their inhibition to arrest primary
tumor and metastases growth. In this paper, we present a new class of C-
glycosides incorporating the methoxyaryl moiety, that was designed to
selectively target and inhibit the extracellular domains of the cancer-relevant CA
isozymes. The glycosides have been prepared by aldol reaction of glycosyl
ketones with the appropriate aromatic aldehydes. We also present the inhibition
profile of our new glycomimetics, against four isozymes of carbonic anhydrase
comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII
(tumor associated isozymes). In this study, per-O-acetylated glycoside 4, 6 and
deprotected compounds 7,9,10 and 12 were identified as potent and highly
selective inhibitors of hCA IX and XII. These results confirm that attaching
carbohydrate moieties to CA methoxyaryl pharmacophore improves and
enhances its inhibitory activity. These CA inhibitors have developmental
potential to selectively target cancer cells, leading to cell death.
∗Correspondence authors.Tel:+54-221-4243104;Fax:+54-221-4226947; Email: pcolinas@quimica.unlp.edu.ar (PAC); Tel:
+39-055-457 3005; Fax: +39-055-4573385; E-mail: claudiu.supuran@unifi.it (CTS).
1

1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are the most studied members of a
great family of metalloenzymes. CAs catalyze the reversible hydration of carbon
dioxide and they are found in multiple organism such as vertebrates, bacteria,
algae, etc.1 Five genetically distinct CA families are known to date, the α-, β-, γ-,
δ- and ζ-CAs. Mammals posses only α-CAs, while many pathogenic organisms
such as bacteria and fungi encoded β- CAs. These enzymes contain a zinc ion
(Zn2+) in their active site, which is coordinated by three histidine residues and a
water molecule/hydroxide ion (in the α-, γ- and δ-classes) or by two cysteine
and one histidine residues (in the β- and ζ-CA classes), with the fourth ligand
being a water molecule/hydroxide ion. Out of the 16 different CA isoforms
discovered so far in the α-class, human CA isoforms hCA I and II are cytosolic
enzymes that are widespread throughout the human body. Further, dimeric
transmembrane glycoproteins hCA IX and XII are also human associated CA
isoforms having extracellular active site and are marker for a broad spectrum of
hypoxic tumor types.2,3 The overexpression of these isoforms contributes to the
increased acidification of extracellular hypoxic environment (pH = 6.8) in
contrast to normal tissues (pH = 7.4) thus promoting tumor cell survival in an
acidic condition by decreasing uptake of weakly basic anticancer drugs.4 Thus
specifically targeting the tumor associated isoforms hCA IX and XII over the
main off target isoforms hCA I and II, which have a physiological relevance,
using specific inhibitors is considered to be a promising strategy in the cancer
therapy.
Carbonic anhydrase inhibitors (CAIs) are classified in three main classes:
• sulfonamides, sulfamates and sulfamides which bind in deprotonated
form, as anions, to the Zn(II) ion from the enzyme active site by replacing
the zinc-bound water/hydroxide ion and leading to a tetrahedral geometry
of Zn(II)2
• coumarins which exhibit a very different binding mode with no
interactions between the inhibitor molecule and the active site Zn(II) ion
observed, occluding the entrance to the active site cavity.5
2

• polyamines6 and phenols,7 which bind by interacting with a water
molecule/hydroxide ion coordinated to Zn(II) through hydrogen bonding.
Methylation is one of the most common chemical modifications of the OH
moiety in phenol. Only very recently, methoxyphenyl derivatives have been
investigated as carbonic anhydrase inhibitors because it was considered that
they do not bear any moiety normally associated with CA inhibition in their
molecules.8 Unexpectedly, di- and tri-methoxybenzenes are rather similar or
better CAIs than phenol but they showed a flat inhibition profile. Docking studies
have been performed to explain the behavior of these compounds. It was found
that the binding of methoxy-substituted benzene within the enzyme active site is
done without interaction with the zinc ion, by means of different interactions with
amino acid residues and water molecules.8 The compounds were located
between the phenol-binding site and coumarin binding site, filling the middle of
the enzyme cavity, but this hypothesis was not yet confirmed by
crystallography. Thus this completely different binding mode offers the
possibility of design carbonic anhydrase inhibitors with a different inhibition
profile to the known inhibitors. However, up to now, only very few such
derivatives have been investigated so far.8
One of the most successful approaches for designing CAIs targeting all
isoforms known to date, was denominated ‘the tail approach’. The tail approach
originally consisted in attaching different tails to the scaffolds of sulfonamides to
modulate the physicochemical properties of these pharmacological agents. A
very good example of such ‘tails’ is constituted by sugars, which represent a
wide range of chemotypes, leading thus to a high number of new CAIs.9,10 The
stereochemical diversity across the carbohydrate tails provides the opportunity
for interrogation of subtle differences in active site topology of CA isozymes.
Glycosidic CAIs were explored previously by our group,11 Poulsen’s12 and
Winum´s13 groups and the presence of carbohydrate moieties in the molecules
of such compounds was associated with effective inhibition of physiologically
relevant isoforms, among which were also CA IX and XII. Recently our group
has applied the “sugar approach” to the preparation of C-glycosyl phenols,
where the carbohydrate moiety is tethered to a phenol CA pharmacophore
through a carbon chain.14 These compounds have been tested as inhibitors of
the M. tuberculosis β-CAs and have shown better inhibitory activity against
3

mtCAs than phenol. Thus, this confirms that attaching carbohydrate moieties to
phenol could improve its inhibitory activity. The antitubercular activity of the C-
glycosyl phenols was investigated, allowing us to identify the first mtCAs
inhibitor with antimycobacterial activity.15
Thus, in the search of non-sulfonamide CAIs belonging to different classes of
compounds, we report here the synthesis of a series of new C-glycosides
incorporating the methoxyaryl moiety, and their inhibitory activity against the off-
target hCA I and II, and tumor-associated hCA IX and XII.
OCH3
OCH3
OAc
OAc
OAc
O
O
O
OCH3
OCH3
AcO
AcO
AcO
AcO
AcO
AcO
OAc
OAc
OAc
O
O
O
2
3
1
OCH3
OCH3
OCH3
AcO
AcO
AcO
AcO
AcO
AcO
OAc
OAc
OAc
O
O
O
OCH3
OAc
OAc
OAc
O
O
O
6
5
4
OCH3
OCH3
OCH3
OH
OH
OH
O
O
O
OCH3
HO
HO
HO
HO
HO
HO
OH
OH
OH
O
O
O
8
9
7
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
O
O
O
OCH3
HO
HO
HO
OH
OH
OH
O
O
O
12
11
10
Figure 1. Peracetylated C-glycosides (1-6) and fully deprotected derivatives (7-12)
2. Results and discussion
2.1 Chemistry
A set of new C-cinnamoyl glycosides (Fig. 1) was synthesized as outlined in
Scheme 1. β-D-Glycosyl-propan-2-ones were prepared by Knoevenagel
condensation with 2,4-pentanedione in the presence of sodium carbonate or
sodium bicarbonate using water and THF as solvent.16 Crude mixtures
containing the C-glycosyl ketones were acetylated and then purified to afford
the peracetylated compounds in very good yields. C-glycosides have been
prepared by aldol condensation of β-C-glucosyl and β-C-galactosyl ketones with
different aldehydes incorporating the methoxyaryl moiety at room temperature
4

in the presence of pyrrolidine as catalyst.14,17 The crude mixtures could be
easily purified by flash column chromatography and/or crystallization to afford
the pure compounds 1-6 in good yields.
1
13
The H NMR, C NMR, 2D COSY and HSQC were in full agreement with their
structures (see Supplementary Information). The trans double bond in the
glycosides was established by the large coupling constant (J = 16 Hz) between
the two olefinic protons. The β-configuration of the C-glycosides was
established by the large coupling constant (J=9.7-11.4 Hz) observed between
H-1´ and H-2´.
The O-acetate protecting groups of the carbohydrate moiety were next easily
removed using triethylamine in methanol/water to afford the fully deprotected C-
glycosides 7–12 in very good yields (80-98%).
Table 1. Synthesis of C-glycosides
Aldehyde
Ketone
Reaction
time (h.)
78
Product
Yield
(%)
76
1
2
3
C-glucosyl
3-methoxybenzaldehyde
veratraldehyde
23
66
6-methoxy-2-naphtaldehyde
48
53
4
5
6
C-galactosyl
3-methoxybenzaldehyde
veratraldehyde
70
24
47
72
65
52
6-methoxy-2-naphtaldehyde
5

The inhibitory activities of C-glycosides 1–12 against cytosolic isoforms hCA I
and II as well as the membrane associated isoforms hCA IX and XII was
assayed by using stopped flow assay method18 and the results are presented in
Table 2.
A number of structure–activity relationships (SARs) were identified in this study
and are summarized as follows:
Off-Target CA Isozymes.
i) hCA I: Five of the peracetylated C-glycosides are micromolar inhibitors of the
hCA I; the exception is the C-glucosyl compound 3 which is a very poor hCA I
inhibitor with Ki >50 µM. A similar trend was found for the deprotected sugar
analogues 7-12, where also the deprotected derivative of compound 3
(glycoside 7) is a very poor inhibitor of hCA I. It is of great interest to relate this
behaviour of these compounds toward hCA I and it can be concluded that
combination of a glucosyl scaffold and the 6-methoxy-2-naphtyl moiety leads to
a steep decrease in the inhibitory potency of thesecompounds against hCA I.
ii) The C-glycosides showed a very interesting inhibition profile against hCA II. It
should be noted that all glycosyl derivatives of veratraldehyde 2,5,8 and 11
showed to be good inhibitors in the micromolar range. Compounds 1-3,5, 8 and
12 are good hCA II inhibitors in the micromolar range. However being a
ubiquitous, housekeeping isoform, this may not be a valuable property in
another context if compounds targeting other isoforms (hCA IX and hCA XII)
should also possess activity against hCA II. Thus it is interesting to note that
some of the novel compounds showed poor inhibition against hCA II while
retaining potent inhibition against hCA IX and hCA XII. C-galactosyl derivatives
4 and 6 and their deprotected analogues 10 and 12 were very poor inhibitors of
hCA II. Also C-glucosides 7 and 9 showed a highly reduced activity against this
isozyme.
6

Table 2. Inhibition of mammalian α-CA with the C-glycosides 1-12.a
C-glycoside
c Log Pb
Ki (nM)c
hCA II
Selectivity
II/IX
hCA I
5670
hCA IX
779
753
673
93
hCA XII
794
126
589
653
81
I/IX
7.3
I/XII
7.1
II/XII
0.53
4.3
1
2
0.12
-0.01
1.11
421
541
0.5
8360
11.1
>74.3
93.4
6.0
0.7
66.3
>85
3
>50000
8690
413
0.6
0.7
4
0.12
>50000
629
>538
0.81
>120
>81
13.3
57.7
83.1
10.9
89.2
>212
103.2
31.8
81.0
>76
7.8
5
-0.01
1.11
4670
773
417
615
840
462
3860
936
485
6
6650
>50000
>50000
438
80
15.9
12.1
12.1
>108
2.24
8.0
>625
>73
6.0
7
-0.82
-0.93
0.19
7440
682
73
8
6510
0.5
9
>50000
8670
>50000
>50000
552
236
84
>108
>13
>212
>595
2.3
10
11
12
-0.80
-0.93
0.19
7480
235
89
0.6
7210
>50000
14.9
>103
>562
a) All CAs are recombinant enzymes obtained in the authors' laboratory as reported earlier.5
b) Values calculated using ChemBioDraw Ultra 12.0.
c) Errors in the range of 5-10 % of the reported value, from 3 different determinations.
7

Cancer-Associated CA Isozymes.
iii) The tumor associated target isoform hCA IX was inhibited in the
submicromolar range by all the C-glycosides except compound 10, wich is a
micromolar inhibitor of this isozyme. The best inhibitor is compound 4 which is
also a very bad hCA II inhibitor.
iv) The second tumor-associated isoform, hCA XII, was the most inhibited
isoform by compounds 2, 5, 6 and 8-12. The least effective hCA XII inhibitors
were 1,3,4 and 7.
v) Selectivity for inhibiting the tumor-associated isoforms (hCA IX and XII) over
the widespread cytosolic forms (hCA I and II) is a key issue when designing
CAIs. As can be in Table 2 several compounds showed better activity profile
against hCA IX and XII over I and II which is highly desirable when only the
tumor-associated isoforms would be targeted. It was observed that several C-
glycoside which showed very good inhibition of isoform IX and XII were also
shown to be highly selective. The most effective hCA IX inhibitor 4 showed
excellent selectivity ratios over hCA I and II. Also the C-glycosides 6, 10, 12,
which are nanomolar inhibitors of hCA XII, showed excellent selectivity ratios.
It is interesting to note that all the peracetylated C-glucosides 1-3 were shown
to be no selective. Also C-glycosides incorporating the veratrole moiety (2, 5, 8
11) showed almost no selectivity and are not useful in the design of selective
inhibitors. Thus, the 3-methoxyphenyl and 6-methoxy-2-naphtyl moieties are
very useful in the design of inhibitors with high activity and selectivity.
Previously we have investigated the enzyme inhibition characteristics of a small
series of C-cinnamoyl glycosides incorporating the phenol moiety.14 The
inhibition profile found for those C-glycosyl compounds was flat and there was
only small variations in the Ki values (micromolar range) observed across all CA
isozymes investigated, leading to Ki selectivity ratios of ∼1.0 across isozymes,
that is, they were nonselective CA inhibitors. As can be seen in our present
report, a small structural change, replacement of a proton by a methyl group
lead to a high improvement in the activity and selectivity of the C-glycosides. It
could be explained in terms of the bind mode of methoxyaryl compounds,
described above.8 C-glycosides, where the carbohydrate moiety is tethered to
a phenol moiety, would bind by interacting with a water molecule/hydroxide ion
coordinated to Zn(II) through hydrogen bonding in the active site of carbonic
8

anhydrase. On the other hand when a methoxyaryl was attached to the
glycoside, the compounds would bind between the phenol-binding site and
coumarin binding site, filling the middle of the carbonic anhydrase cavity. Thus
the carbohydrate moiety should interact with amino acid residues found at the
entrance of the active site cavity. As these residues are generally different
between the various CA isozymes,19 this could lead to the selectivity found in
this work. Up to now, we were unable to crystallize a CA isozyme in complex
with one of the glycosides 1-12, thus it is not possible to confirm our hypothesis.
In the development of anti-cancer compounds that target selectively the
membrane bound isoform CA IX and CA XII versus the ubiquitous isoform CA
II, the design of membrane non-permeant inhibitors is crucial.10 The calculated
Log P (cLog P) parameter generally provides a good correlation with
experimental permeability data, and molecules with cLogP values between 1
and 3 typically have good passive membrane permeability properties while
those with cLogP values of <0 are more likely to have a low capacity for
penetrating cell membranes.20 As can be seen in Table 2, calculated log P
values for the C-glycosides show that several compounds fall within the range
indicative of molecules with poor membrane permeability, the exceptions are
naphtyl derivatives 3 and 6. Values of acetylated glycosides 1–6 are consistent
with the incorporated acetyl groups, decreasing the polarity of the resulting
carbohydrate moiety. It is expected that the
poor passive membrane
permeability of the C-glycosides would enhance the preferential inhibition of
CAs IX and XII over ubiquitous cytosolic hCA II.
3. Conclusion
A novel series of C-glycosides 1-12 containing the methoxyaryl scaffold have
been synthesized and investigated as inhibitors against four isozymes of
carbonic anhydrases comprising cytosolic, ubiquitous isozymes hCA I and II as
well as the transmembrane, tumor-associated isoforms hCA IX and XII which
are validated antitumor targets. In this study, per-O-acetylated glycoside 4, 6
and deprotected compounds 7, 9, 10 and 12 were identified as potent and
highly selective inhibitors of hCA IX and XII. These results confirm that
attaching carbohydrate moieties to CA methoxyaryl pharmacophore improves
9

and enhances its inhibitory activity. Also the physicochemical properties of the
glycosides tested would enhance the preferential inhibition in vivo. Free
glycosides could be useful for chemotherapy if they are delivered through a
route of intravenous administration. For oral delivery peracetylated C-glycosides
may be used as ester prodrugs. Once in the body, the ester groups could be
readily hydrolyzed by ubiquitous esterases.21 As it has been evidenced that
isoforms IX and XII are overexpressed by tumours and are also potential targets
for diagnosis and treatment of cancer, discovery of these C-glycosyl selective
IX and XII inhibitors will be a promising step in the strategy for an effective
cancer therapy.
Acknowledgments
This work was financed in part by an EU grant (Metoxia) to CTS, and by UNLP
and CONICET (Argentina). P.A.C is member of the Scientific Research Career
of CONICET.
Experimental section
General
All starting materials and reagents, were purchased from commercial suppliers.
Reactions were monitored by TLC and TLC plates visualized with short wave
UV fluorescence (λ = 254 nm), sulfuric acid stain (5% H2SO4 in methanol).
Silica gel flash chromatography was performed using silica gel 60 Å (230–
1
400 mesh). All melting points are uncorrected. H and 13C NMR spectra were
recorded on a Varian Mercury 200 (200.055 and 50.309 MHz, respectively).
Chemical shifts were measured in ppm and coupling constants in Hz. High
resolution mass spectra were recorded using electrospray as the ionization
technique in positive ion or negative ion modes as stated. All MS analysis
samples were prepared as solutions in methanol.
General procedure 1: Synthesis of per-O-acetylated C-glycosides (1–6)
10

To a solution of per-O-acetylated β-C-glucopyranosyl or C-galactopyranosyl
ketone (1.0 equiv) in dry dichloromethane was added the desired aldehyde (1.0
equiv) and pyrrolidine (0.2 equiv). The reaction was stirred at room temperature
until the starting material was consumed as evidenced by TLC (see times in
Table 1). The reaction mixture was concentrated and the residue diluted in ethyl
acetate and washed with water (3×). The aqueous extracts were combined and
back extracted with ethyl acetate (1×). The organic extracts were combined,
dried over NaSO4, filtered and evaporated. The product was purified by column
chromatography (eluant 3:7 to 1:1 hexanes–EtOAc) to give compounds 1-6.
General procedure 2: Deprotection of per-O-acetylated C-glycosides (1-6
→ 7-12)
Deprotected compounds 7–12 were prepared by dissolving the corresponding
per-O-acetylated precursor 1–6 in methanol/triethylamine/water (8:2:1). The
reaction was kept at room temperature overnight and then concentrated. The
product was purified by column chromatography (eluant 5:1 EtOAc-MeOH) to
afforded pure material by 1H NMR and 13C NMR spectroscopy. Yields 80–98%.
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3-methoxyphenyl)but-3-
en-2-one (1)
The title compound 1 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
glucopyranosyl)-propan-2-one and 3-methoxybenzaldehyde according to
1
general procedure 1 to give a white solid. mp= H NMR (500 MHz, CDCl3) δ
7.53 (d, 1H, J = 16.2 Hz,H-4), 7.33 (t, 1H, J = 7.9 Hz,ArH), 7.16 (d, 1H, J = 7.6
Hz,ArH), 7.08 (m, 1H,ArH), 6.98 (dd, 1H, J = 8.2, 1.9 Hz,ArH), 6.74 (d, 1H, J =
16.2 Hz,H-3), 5.25 (t, 1H, J = 9.4 Hz,H-3’), 5.09 (t, 1H, J = 9.7 Hz,H-4’), 5.01 (t,
1H, J = 9.7 Hz,H-2’), 4.28 (dd, 1H, J = 12.4, 4.9 Hz,H-6´b), 4.14 (ddd, 1H, J =
10.0, 8.5, 3.2 Hz,H-1’), 4.04 (dd, 1H, J = 12.4, 2.2 Hz,H-6’a), 3.86 (s, 3H,CH3O),
3.74 (ddd, 1H, J = 10.1, 4.9, 2.2 Hz,H-5’), 3.04 (dd, 1H, J = 16.3, 8.5 Hz,H-1b),
2.70 (dd, 1H, J = 16.3, 3.2 Hz,H-1a), 2.05 (s, 3H,CH3COO), 2.04 (s,
13
3H,CH3COO), 2.03 (s, 6H,CH3COO). C NMR (126 MHz, CDCl3) δ 196.13 (C-
2), 170.62 (CH3COO), 170.22 (CH3COO), 169.98 (CH3COO), 169.55
(CH3COO), 159.97 (ArC), 143.70 (C-4), 135.57 (ArC), 130.01 (ArC), 126.49 (C-
3), 121.14 (ArC), 116.75 (ArC), 113.12 (ArC), 75.74 (C-5’), 74.19 (C-4’), 74.13
11

(C-3’), 71.70 (C-1’), 68.50 (C-2’), 62.04 (C-6’), 55.33 (CH3O), 42.55 (C-1), 20.72
(CH3CO), 20.66 (CH3CO), 20.64 (CH3CO), 20.62 (CH3CO). HRMS m/z: calcd
for C25H30O11, 506.1788; found, 506.1776
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3,4–dimethoxy-phenyl)
but-3-en-2-one (2)
The title compound 2 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
glucopyranosyl)-propan-2-one and veratraldehyde according to general
procedure 1 to give a white solid. mp=135-136°C. 1H NMR (500 MHz, CDCl3) δ
7.51 (d, 1H, J = 16.1 Hz,H-4), 7.16 (d, 1H, J = 8.3 Hz,ArH), 7.09 (s, 1H,ArH),
6.90 (d, 1H, J = 8.3 Hz,ArH), 6.64 (d, 1H, J = 16.1 Hz,H-3), 5.25 (t, 1H, J = 9.4
Hz,H-3’), 5.09 (t,1H, J = 9.7 Hz,H-4’), 5.00 (t, 1H, J = 9.7 Hz,H-2’), 4.28 (dd, 1H,
J = 12.4, 4.9 Hz,H-1’), 4.14 (dd, 1H, J = 18.4, 3.2 Hz,H-6’b), 4.04 (dd, 1H, J =
12.3, 1.8 Hz,H-6’a), 3.94 (s, 6H,CH3O), 3.74 (ddd, 1H, J = 10.0, 4.8, 2.0 Hz,H-
5’), 3.03 (dd, 1H, J = 16.2, 8.4 Hz,H-1b), 2.68 (dd, 1H, J = 16.2, 3.2 Hz,H-1a),
2.04 (s, 1H,CH3COO), 2.04 (s, 1H,CH3COO), 2.02 (s, 1H,CH3COO), 2.02 (s,
1H,CH3COO). 13C NMR (126 MHz, CDCl3) δ 196.00 (C-2), 170.63 (CH3COO),
170.23 (CH3COO), 170.03 (CH3COO), 169.57 (CH3COO), 151.60 (ArC), 149.30
(ArC), 143.85 (C-4), 127.11 (ArC), 124.28 (C-3), 123.33 (ArC), 111.08 (ArC),
109.70 (ArC), 75.71 (C-5’), 74.21 (C-1’, C-3’),71.72 (C-4’), 68.52 (C-2’), 62.04
(C-6’), 56.01 (CH3O), 55.92 (CH3O), 42.39 (C-1), 20.75 (CH3CO), 20.68
(CH3CO), 20.65 (CH3CO), 20.63 (CH3CO). HRMS m/z: calcd for C26H32O12,
536.1894; found, 536.1898.
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(6-methoxy-2-naphthyl)
but-3-en-2-one (3)
The title compound 3 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
glucopyranosyl)-propan-2-one and 6-methoxy-2-naphtaldehyde according to
general procedure 1 to give a yellow solid. mp=178-179°C. 1H NMR (500 MHz,
CDCl3) δ 7.93 (d, 1H, J = 8.8 Hz, ArH), 7.74 (m, 3H, ArH), 7.66 (dd, 1H, J = 8.6,
1.5 Hz, ArH), 7.20 (dd, 1H, J = 9.0, 2.5 Hz, ArH), 7.16 (d, 1H, J = 2.4 Hz,ArH),
6.83 (d, 1H, J = 16.1 Hz,H-3), 5.26 (t, 1H, J = 9.4 Hz,H-3’), 5.11 (t, 1H, J = 9.7
Hz,H-4’), 5.03 (t, 1H, J = 9.7 Hz,H-2’), 4.29 (dd, 1H, J = 12.4, 4.9 Hz,H-1’), 4.17
12

(dd, 1H, J = 18.3, 3.3 Hz,H-6’b), 4.05 (dd,1H, J = 12.4, 2.1 Hz,H-6’a), 3.96 (s,
3H,CH3O), 3.75 (ddd, 1H, J = 10.0, 4.8, 2.1 Hz,H-5’), 3.07 (dd, 1H, J = 16.2, 8.4
Hz,H-1b), 2.74 (dd, 1H, J = 16.2, 3.3 Hz,H-1a), 2.05 (s, 3H,CH3COO), 2.05 (s,
3H,CH3COO), 2.04 (s, 3H,CH3COO), 2.02 (s, 3H,CH3COO). 13C NMR (126
MHz, CDCl3) δ 196.06 (C-2), 170.64 (CH3COO), 170.24 (CH3COO), 170.00
(CH3COO), 169.57 (CH3COO), 159.08 (ArC), 144.12 (C-4), 136.00 (ArC),
130.61 (ArC), 130.23 (ArC), 129.56 (ArC), 128.67 (ArC), 127.64 (ArC), 125.30
(ArC), 124.16 (C-3), 119.62 (ArC), 106.02 (ArC), 75.75 (ArC), 74.24 (C-3’, C-
1´), 71.76 (C-4’), 68.54 (C-2’), 62.07 (C-6’), 55.41 (CH3O), 42.62 (C-1), 20.74
(CH3CO), 20.68 (CH3CO), 20.65 (CH3CO), 20.63 (CH3CO). HRMS m/z: calcd
for C29H32O11, 556.1945; found, 556.1968.
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-galactopyranosyl)-4-(3-methoxyphenyl)but-
3-en-2-one (4)
The title compound 4 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
galactopyranosyl)-propan-2-one and 3-methoxybenzaldehyde according to
1
general procedure 1 to give a white solid. mp=111-112°C. H NMR (500 MHz,
CDCl3) δ 7.54 (d, 1H ,J = 16.2 Hz,H-4), 7.33 (t, 1H, J = 7.9 Hz,ArH), 7.16 (d,
1H, J = 7.6 Hz,ArH), 7.08 (s, 1H,ArH), 6.98 (dd, 1H, J = 8.2, 2.3 Hz,ArH), 6.75
(d, 1H, J = 16.2 Hz,H-3), 5.46 (d, 1H, J = 3.2 Hz,H-3’), 5.20 (t, 1H, J = 9.9 Hz,H-
4’), 5.07 (m, 1H,H-2’), 4.12 (dd, 1H, J = 11.4, 4.1 Hz,H-1’), 4.07 (m, 2H,H-6’a,H-
6’b), 3.96 (m, 1H,H-5’), 3.86 (s, 3H,CH3O), 3.09 (dd, 1H, J = 16.1, 8.4 Hz,H-1b),
2.71 (dd, 1H, J = 16.1, 3.2 Hz,H-1a), 2.17 (s, 3H,CH3COO), 2.05 (s,
3H,CH3COO), 2.00 (s, 3H,CH3COO), 1.98 (s, 3H,CH3COO). 13C NMR (126
MHz, CDCl3) δ 196.45 (C-2), 170.45 (CH3COO), 170.26 (CH3COO), 170.24
(CH3COO), 170.10 (CH3COO), 159.94 (ArC), 143.66 (C-4), 135.58 (ArC),
130.01 (ArC), 126.52 (C-3), 121.15 (ArC), 116.70 (ArC), 113.15 (ArC), 74.66
(C-5’), 74.23 (C-1’), 71.99 (C-4’), 69.12 (C-3’), 67.72 (C-2’), 61.40 (C-6’), 55.34
(CH3O), 42.75 (C-1), 20.83 (CH3CO),20.71 (CH3CO),20.62 (CH3CO),20.61
(CH3CO). HRMS m/z: calcd for C25H30O11, 506.1788; found, 506.1794
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-galactopyranosyl)-4-(3,4-dimethoxyphenyl)
but-3-en-2-one (5)
13

The title compound 5 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
galactopyranosyl)-propan-2-one and veratraldehyde according to general
procedure 1 to give a yellow solid. mp=89.5-90°C.1H NMR (500 MHz, CDCl3) δ
7.53 (d, 1H, J = 16.1 Hz,H-4), 7.16 (d, 1H ,J = 8.3 Hz,ArH), 7.10 (s, 1H, J = 7.9
Hz,ArH), 6.90 (d, 1H, J = 8.3 Hz,ArH), 6.65 (d, 1H, J = 16.1 Hz,H-3), 5.46 (d,
1H, J = 3.0 Hz,H-3’), 5.21 (t, 1H, J = 9.9 Hz,H-4’), 5.09 (dd, 1H ,J = 10.1, 3.4
Hz,H-2’), 4.13 (dd, 1H, J = 10.2, 4.7 Hz,H-1’), 4.11 (m, 2H,H-6’), 4.04 (dd, 1H ,J
= 11.2, 6.6 Hz,H-5’), 3.94 (d, 6H, J = 1.1 Hz,CH3O), 3.08 (dd, 1H, J = 16.1, 8.4
Hz,H-1b), 2.69 (dd, 1H, J = 16.0, 3.2 Hz,H-1a), 2.18 (s, 3H,CH3COO), 2.05 (s,
3H,CH3COO), 2.00 (s, 3H,CH3COO), 1.98 (s, 3H,CH3COO). 13C NMR (126
MHz, CDCl3) δ 196.27 (C-2), 170.42 (CH3COO), 170.26 (CH3COO), 170.23
(CH3COO), 170.08 (CH3COO), 151.59 (ArC), 149.29 (ArC), 143.80 (C-4),
127.14 (ArC), 124.34 (C-3), 123.33 (ArC), 111.09 (ArC), 109.71 (ArC), 74.76
(C-5’), 74.22 (C-1’), 72.00 (C-3’), 69.16 (C-4’), 67.72 (C-2’), 61.35 (C-6’), 56.01
(CH3O), 55.92 (CH3O), 42.59 (C-1), 20.85 (CH3CO), 20.71 (CH3CO), 20.62
(CH3CO). HRMS m/z: calcd for C26H32O12, 536.1894; found, 536.1878.
(E)-1-(2’,3’,4’,6’-Tetra-O-acetyl-β-D-galactopyranosyl)-4-(6-methoxy-2-naphthyl)
but-3-en-2-one (6)
The title compound 6 was prepared from 1-(2´,3´,4´,6´-tetra-O-acetyl-β-D-
galactopyranosyl)-propan-2-one and 6-methoxy-2-naphtaldehyde according to
1
general procedure 1 to give a yellow solid. mp=114-115°C. H NMR (500 MHz,
CDCl3) δ 7.92 (d, 1H, J = 8.2 Hz,ArH), 7.73 (m, 3H,H-4,ArH), 7.66 (dd,1H, J =
8.6, 1.2 Hz,ArH), 7.19 (dd, 1H, J = 8.9, 2.4 Hz,ArH), 7.15 (d, 1H, J = 2.1
Hz,ArH), 6.84 (d, 1H, J = 16.1 Hz,H-3), 5.47 (d, 1H, J = 3.2 Hz,H-4’), 5.23 (t,1H,
J = 9.9 Hz,H-2’), 5.10 (dd, 1H, J = 10.1, 3.4 Hz,H-3’), 4.13(m, 2H,H-5’,H-1’),
4.06 (dd, 1H, J = 11.2, 6.4 Hz,H-6’b), 3.96(m, 1H,H-6’a), 3.95 (s, 3H,CH3O),
3.12(m, 1H,H-1b), 2.75 (m, 1H,H-1a), 2.18 (s, 3H,CH3COO), 2.05 (s,
3H,CH3COO), 2.00 (s, 3H,CH3COO), 1.97 (s, 3H,CH3COO). 13C NMR (126
MHz, CDCl3) δ 196.36 (C-2), 170.42 (CH3COO), 170.26 (CH3COO), 170.23
(CH3COO), 170.09 (CH3COO), 159.06 (ArC), 144.05 (C-4), 135.98 (ArC),
130.60 (ArC), 130.22 (ArC), 129.58 (ArC), 128.66 (ArC), 127.63 (ArC), 125.34
(ArC), 124.16 (C-3), 119.61 (ArC), 106.00 (ArC), 74.71 (C-5’), 74.25 (C-1’),
72.11 (C-3’), 69.19 (C-4’), 67.75 (C-2’), 61.42 (C-6’), 55.41 (CH3O), 42.84 (C-1),
14

20.84 (CH3CO), 20.72 (CH3CO), 20.63 (CH3CO), 20.62 (CH3CO). HRMS m/z:
calcd for C29H32O11, 556.1945; found, 556.1938.
1-(β-D-glucopyranosyl)-4-(3-methoxyphenyl)but-3-en-2-one (7)
The title compound 7 was prepared from compound 1 according to general
procedure 2 to give a white solid. mp=116-117°C
1H NMR (500 MHz, DMSO) δ 7.57 (d, 1H, J = 16.2 Hz,H-4), 7.35 (m, 1H,ArH),
7.29 (m, 2H, ArH), 7.00 (ddd, 1H, J = 8.2, 2.4, 1.1 Hz,ArH), 6.97 (m, 1H,H-3),
5.08 (d, 1H, J = 5.7 Hz,OH), 4.95 (t, 1H ,J = 5.7 Hz,OH), 4.88 (d, 1H, J = 4.6
Hz,OH), 4.37 (t, 1H, J = 5.7 Hz,OH), 3.80 (s, 3H,CH3O), 3.65 (dd, 1H, J = 9.2,
2.6 Hz,H-4), 3.61 (dd, 1H, J = 11.2, 4.0 Hz,H-6b), 3.41 (bs, 1H,H-6’a), 3.18 (dd,
1H, J = 8.3, 3.8 Hz,H-1’), 3.09 (m, 2H,H-2’,H-5’), 2.97 (m, 2H,H-1b,H-3’), 2.82
(dd,1H, J = 16.1, 8.8 Hz,H-1a). 13C NMR (126 MHz, DMSO) δ 198.60 (C-2),
160.05 (ArC), 142.49 (C-4), 136.41 (ArC), 130.42 (ArC), 127.57 (C-3), 121.52
(ArC), 116.92 (ArC), 113.53 (ArC), 81.15 (C-5’), 78.57 (C-1’), 76.26 (C-4’),
74.04 (C-3’), 70.74 (C-2’), 61.59 (C-6’), 55.68 (CH3O), 43.88 (C-1). HRMS m/z:
calcd for C17H22O7, 338.1366; found, 338.1378.
1-(β-D-glucopyranosyl)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (8)
The title compound 8 was prepared from compound 2 according to general
1
procedure 2 to give a yellow solid. mp=141-142°C. H NMR (500 MHz, DMSO)
δ 7.54 (d, 1H, J = 16.1 Hz,H-4), 7.34 (d, 1H, J = 1.6 Hz,ArH), 7.27 (dd, 1H ,J =
8.3, 1.7 Hz,ArH), 7.01 (d, 1H, J = 8.4 Hz,ArH), 6.87 (m, 1H,H-3), 5.07 (d, 1H ,J
= 4.9 Hz,OH), 4.95 (s, 1H,OH), 4.88 (s, 1H,OH), 4.36 (t, 1H ,J = 5.7 Hz,OH),
3.82 (s, 3H,CH3O), 3.81 (s, 3H,CH3O), 3.62(m, 2H,H-6’b,H-4’), 3.39 (m, 1H,H-
6’a), 3.18 (t, 1H, J = 8.1 Hz,H-1’), 3.08 (m, 2H,H-2’,H-5’), 2.95(m, 2H,H-1b,H-3’),
2.79 (dd, 1H, J = 16.0, 8.8 Hz,H-1a).13C NMR (126 MHz, DMSO) δ 198.35 (C-
2), 151.43 (ArC), 149.41 (ArC), 142.92 (C-4), 127.71 (ArC), 125.20 (C-3),
123.62 (ArC), 112.03 (ArC), 110.84 (ArC), 81.14 (C-5’), 78.59 (C-1’), 76.34 (C-
4’), 74.06 (C-3’), 70.76 (C-2’), 61.60 (C-6’), 56.03 (2xCH3O), 43.68 (C-1). HRMS
m/z: calcd for C18H24O8, 368.1471; found, 368.1482.
15

1-(β-D-glucopyranosyl)-4-(6-methoxy-2-naphthyl)but-3-en-2-one (9)
The title compound 9 was prepared from compound 3 according to general
procedure 2 to give a white solid. mp=130-131°C. 1H NMR (500 MHz, DMSO)
δ 8.16 (s, 1H,ArH), 7.87 (m, 3H,H-4,ArH), 7.72 (d, 1H, J = 16.1 Hz,ArH), 7.37
(d, 1H ,J = 2.3 Hz,ArH), 7.22 (dd, 1H, J = 9.0, 2.4 Hz,ArH), 7.04 (d, 1H, J = 16.2
Hz,H-3), 5.09 (d, 1H, J = 5.7 Hz,OH), 4.96 (d, 1H, J = 3.9 Hz,OH), 4.89 (d, 1H, J
= 4.0 Hz,OH), 4.39 (t, 1H, J = 5.6 Hz,OH), 3.90 (s, 3H,CH3O), 3.67 (dd, 1H, J =
9.1, 2.3 Hz,H-4’), 3.63 (m, 1H,H-6’b), 3.39 (bs, 1H, H-6’a), 3.20 (dd, 1H, J = 8.1,
5.2 Hz,H-1’), 3.10(m, 2H,H-2’,H-5’), 3.00 (dd, 2H, J = 12.7, 3.5 Hz,H-1b,H-3’),
2.83 (dd, 1H, J = 16.0, 8.8 Hz,H-1a).13C NMR (126 MHz, DMSO) δ 198.42 (C-
2), 158.90 (ArC), 142.79 (C-4), 135.90 (ArC), 130.58 (ArC), 130.51 (ArC),
130.30 (ArC), 128.72 (ArC), 127.88 (ArC), 126.44 (ArC), 125.13 (C-3), 119.70
(ArC), 106.71 (ArC), 81.19 (C-5’), 78.60 (C-1’), 76.36 (C-4’), 74.08 (C-3’), 70.77
(C-2’), 61.61 (C-6’), 55.80 (CH3O), 43.97 (C-1). HRMS m/z: calcd for C21H24O7,
388.1522; found, 388.1531.
1-(β-D-galactopyranosyl)-4-(3-methoxyphenyl)but-3-en-2-one (10)
The title compound 10 was prepared from compound 4 according to general
procedure 2 to give a white solid. mp=91-92 °C. 1H NMR (500 MHz, DMSO) δ
7.56 (d, 1H, J = 16.2 Hz,H-4), 7.35 (t, 1H, J = 8.1 Hz,ArH), 7.29 (m, 2H,ArH),
6.99 (m, 2H,ArH,H-3), 4.89 (dd, 1H, J = 16.2, 4.6 Hz,OH), 4.70 (s, 1H,OH), 4.50
(t, 1H, J = 5.5 Hz,OH), 4.33 (dd, 1H, J = 11.9, 4.6 Hz,OH), 3.80 (s, 3H,CH3O),
3.74 (dd, 1H, J = 21.3, 3.0 Hz,H-2’), 3.59 (td, 1H, J = 8.8, 2.5 Hz,H-1’), 3.48 (m,
1H,H-6’b), 3.38 (bs, 1H, H-6’a), 3.34 (m, 3H,H-3’,H-4’,H-5’), 2.96 (dd, 1H, J =
15.9, 2.5 Hz,H-1b), 2.83 (dd, 1H, J = 15.9, 8.9 Hz,H-1a).
13C NMR (126 MHz, DMSO) δ 198.82 (C-2), 160.05 (ArC), 142.42 (C-4), 136.42
(ArC), 130.42 (ArC), 127.59 (C-3), 121.50 (ArC), 116.91 (ArC), 113.51 (ArC),
79.23 (C-5’), 76.89 (C-1’), 75.09 (C-4’), 70.94 (C-3’), 69.07 (C-2’), 60.87 (C-6’),
55.68 (CH3O), 43.96 (C-1). HRMS m/z: calcd for C17H22O7, 338.1366; found,
338.1372.
1-(β-D-galactopyranosyl)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (11)
16

The title compound 11 was prepared from compound 5 according to general
procedure 2 to give a yellow solid. mp=89-89.5°C. 1H NMR (500 MHz, DMSO)
δ 7.53 (d, 1H, J = 16.1 Hz,H-4), 7.34 (d, 1H, J = 1.7 Hz,ArH), 7.27 (dd, 1H,J =
8.3, 1.7 Hz,ArH), 7.01 (d, 1H, J = 8.4 Hz,ArH), 6.87 (d, 1H, J = 16.2 Hz,H-3),
4.89 (d, 1H, J = 4.0 Hz,OH), 4.69 (s, 1H,OH), 4.49 (t, 1H, J = 5.6 Hz,OH), 4.33
(d, 1H, J = 4.5 Hz,OH), 3.82 (s, 3H, J = 3.4 Hz,CH3O), 3.81 (s, 3H,CH3O), 3.71
(d, 1H ,J = 2.7 Hz,H-2’), 3.57 (td,1H, J = 8.8, 2.5 Hz,H-1’), 3.47 (m, 1H,H-6’b),
3.36 (bs, 1H,H-6’a), 3.30 (s, 3H, J = 6.3 Hz,H-3’,H-4’H-5’ 2.93 (dd, 1H ,J = 15.8,
2.4 Hz,H-1b), 2.80 (dd, 1H, J = 15.9, 9.0 Hz,H-1a). 13C NMR (126 MHz, DMSO)
δ 198.59 (C-2), 151.42 (ArC), 149.42 (ArC), 142.86 (C-4), 127.71 (ArC), 125.24
(C-3), 123.60 (ArC), 112.04 (ArC), 110.82 (ArC), 79.21 (C-5’), 76.98 (C-1’),
75.11 (C-4’), 70.95 (C-3’), 69.06 (C-2’), 60.87 (C-6’), 56.03 (2 x CH3O), 43.75
(C-1). HRMS m/z: calcd for C18H24O8, 368.1471; found, 368.1484.
1-(β-D-galactopyranosyl)-4-(6-methoxy-2-naphthyl)but-3-en-2-one (12)
The title compound 12 was prepared from compound 6 according to general
procedure 2 to give a yellow solid. mp= 120-121°C. 1H NMR (500 MHz,
DMSO) δ 8.16 (s, 1H,ArH), 7.87 (m, 3H,H-4,ArH), 7.72 (dd, 1H, J = 15.5, 7.5
Hz,ArH), 7.38 (d, 1H, J = 2.3 Hz,ArH), 7.22 (dd, 1H, J = 8.9, 2.4 Hz,ArH), 7.03
(d, 1H, J = 16.2 Hz,H-3), 4.92 (d, 1H, J = 2.8 Hz,OH), 4.71 (s, 1H,OH), 4.51 (t,
1H, J = 5.5 Hz,OH), 4.35 (d, 1H, J = 4.4 Hz,OH), 3.90 (s, 3H, J = 4.3 Hz,CH3O),
3.72 (s, 1H,H-2’), 3.61 (dd, 1H, J = 8.8, 6.5 Hz,H-1’), 3.48 (m, 1H,H-6’b), 3.36
(bs, 1H, H-6’a), 3.33 (d, 3H, J = 6.0 Hz,H-3’,H-4’,H-5’), 2.99 (dd, 1H, J = 15.8,
2.3 Hz,H-1b), 2.84 (dd, 1H, J = 15.8, 9.0 Hz,H-1a). 13C NMR (126 MHz, DMSO)
δ 198.64 (C-2), 158.90 (ArC), 142.73 (C-4), 135.89 (ArC), 130.57 (ArC), 130.49
(ArC), 130.30 (ArC), 128.72 (ArC), 127.88 (ArC), 126.46 (ArC), 125.11 (C-3),
119.70 (ArC), 106.71 (ArC), 79.26 (C-5’), 77.00 (C-1’), 75.11 (C-4’), 70.98 (C-
3’), 69.09 (C-2’), 60.88 (C-6’), 55.80 (CH3O), 44.05 (C-1). HRMS m/z: calcd for
C21H24O7, 388.1522; found, 388.1511.
17

CA Inhibiton studies
An Applied Photophysics stopped-flow instrument has been used for assaying
the CA catalysed CO2 hydration activity as reported by Khalifah.18 Phenol red
(at a concentration of 0.02 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20
mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determination of the
kinetic parameters and inhibition constants. For each inhibitor at least six traces
of the initial 5-10% of the reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM)
were prepared in distilled-deionized water and dilutions up to 0.01 nM were
done thereafter with distilled-deionized water. Inhibitor and enzyme solutions
were preincubated together for 15 min at room temperature prior to assay, in
order to allow for the formation of the E-I complex. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3, and the Cheng-
Prussoff equation (Cheng, Y.; Prusoff, W.H. Biochem. Pharmacol. 1973, 22,
3099) as reported earlier and represent the mean from at least three different
determinations.
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21

Attachment of carbohydrates to methoxyaryl moieties leads to highly selective inhibitors
of the cancer associated carbonic anhydrase isoforms IX and XII
Leonardo E. Riafrecha, Oscar M. Rodríguez, Daniela Vullo, Claudiu T. Supuran* and Pedro A.
Colinas*
22