Synthesis of C-cinnamoyl glycosides and their inhibitory activity against mammalian carbonic anhydrases

Article abstract of DOI:10.1016/j.bmc.2012.09.002

A small series of C-cinnamoyl glycosides incorporating the phenol moiety has been prepared by reaction of glycosyl ketones with the appropriate benzaldehydes. Glycosides were tested for the inhibition of twelve mammalian isoforms of carbonic anhydrase. This is the first study in which α-CAs have been investigated for their interaction with C-glycosides, a novel carbohydrate scaffold in the design of carbonic anhydrase inhibitors. The C-cinnamoyl glycosides were generally effective CA inhibitors, with inhibition constants in the low micromolar range against CA I, II, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and ineffective inhibitors of CA III. These results confirm that attaching carbohydrate moieties to CA phenol pharmacophore improves its inhibitory activity.

Full text of DOI:10.1016/j.bmc.2012.09.002

Bioorganic & Medicinal Chemistry 21 (2013) 1489–1494
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of C-cinnamoyl glycosides and their inhibitory activity against
mammalian carbonic anhydrases
Leonardo E. Riafrecha a, Oscar M. Rodríguez a, Daniela Vullo b, Claudiu T. Supuran b, , 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 11 September 2012
A small series of C-cinnamoyl glycosides incorporating the phenol moiety has been prepared by reaction
of glycosyl ketones with the appropriate benzaldehydes. Glycosides were tested for the inhibition of
twelve mammalian isoforms of carbonic anhydrase. This is the first study in which
a-CAs have been
Keywords:
investigated for their interaction with C-glycosides, a novel carbohydrate scaffold in the design of car-
bonic anhydrase inhibitors. The C-cinnamoyl glycosides were generally effective CA inhibitors, with inhi-
bition constants in the low micromolar range against CA I, II, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and
ineffective inhibitors of CA III. These results confirm that attaching carbohydrate moieties to CA phenol
pharmacophore improves its inhibitory activity.
C-Glycosides
Carbonic anhydrase
Phenol
Enzyme inhibition
Ó 2012 Elsevier Ltd. All rights reserved.
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of zinc
metalloenzymes that catalyze the reversible hydration of carbon
dioxide to give bicarbonate and a proton.1 CAs constitute an excel-
in the active site mainly because of van der Waals contacts with
side chain residues of the hydrophobic side of the active site. Supu-
ran’s group has recently investigated the interaction of phenol and
lent example of convergent evolution, and in addition to
from mammals there are four distinct, unrelated families
a
-CAs
some derivatives with a- and b-carbonic anhydrases enzymes dis-
playing low micromolar/submicromolar inhibition.6,8 The different
mechanism of inhibition of phenols as compared to other inhibi-
tors and their inhibition profile, make this class of derivatives of
great interest to design novel CA inhibitors with selectivity and/
or specificity for some of the medicinal targets belonging to this
enzyme family.
(b-, c-, d- and n-CA families that encode these metalloenzymes in
organisms across the phylogenetic tree.2
Inhibition of carbonic anhydrases has pharmacologic applica-
tions in the field of antiglaucoma, anticonvulsant, and anticancer
agents but is emerging also for designing antifungal and antibacte-
rial agents with a novel mechanism of action. CA inhibitors (CAIs)
belong to four main classes: (i) sulfonamides and their isosteres
(sulfamates/sulfamides), which bind in deprotonated form, as an-
ions, to the Zn(II) ion from the enzyme active site in tetrahedral
geometry,3 (ii) coumarins which exhibited a completely unprece-
dent binding mode (in hydrolized form), with no interactions be-
tween the inhibitor molecule and the active site Zn(II) ion
observed,4 (iii) polyamines, which bind by anchoring to the water
molecule/hydroxide ion coordinated to Zn(II)5 and (iv) phenols
which bind rather similar but not identical to polyamines, by inter-
acting with a zinc-bound water molecule through hydrogen bond-
ing and with no direct interaction between the inhibitor and the
zinc.6 This binding mode was reported by Christianson and col-
leagues in a very elegant study of the X-ray structure for the adduct
of hCA II with phenol.7 Also they showed that the inhibitor bound
The use of carbohydrate scaffolds in the design of CA inhibitors
has proven to be a successful approach and now constitutes one of
the most attractive ways to develop new generations of effective
and selective inhibitors.9 Attaching a carbohydrate moiety should
induce the desired physicochemical properties such as water
solubility, low permeability, etc. Also the stereochemical diver-
sity across the carbohydrate tails provides the opportunity for
interrogation of subtle differences in active site topology of CA
isozymes. A particular problem for the application of carbohy-
drates as drugs is the lability of the glycosidic bond. To circumvent
metabolic problems in the organism, stable mimetics (glycomi-
metics) have to be prepared.10 In recent years a variety of replace-
ment for the glycosidic linkage have raised considerable interest as
stable carbohydrate mimetics due to their enormous stability to
enzymatic degradation and the resulting possibilities as enzyme
inhibitors. The most common modifications with respect to the
linkage are N-glycosides, C-glycosides and S-glycosides.11 On the
last years one of our groups has developed several synthesis of
N-glycosyl sulfonamides by sulfonamidoglycosylation of carbohy-
drate derivatives.12 Recently we reported the synthesis of a series
⇑
Corresponding authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.);
tel.: +54 221 4243104; fax: +54 221 4226947 (P.A.C.).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), pcolinas@quimi
ca.unlp.edu.ar (P.A. Colinas).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.002

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L. E. Riafrecha et al. / Bioorg. Med. Chem. 21 (2013) 1489–1494
of
a
-
D
-hex-2-enopyranosyl sulfonamides, which was evaluated
reaction gave the C-cinnamoyl glycosides in good yields with 3-,
and 4-hydroxybenzaldehydes. When salicylaldehyde was used as
starting material, very complex mixtures were found and no prod-
uct could be isolated. This may be due to the presence of hydroxyl
group at the o-position to the aldehyde group, which induces the
steric hindrance. On the other hand, the reaction of 3,4-dihydroxy-
benzaldehyde with C-glucosyl or C-galactosyl ketones afforded a
dark red tar and no reactives could be recovered from the reaction
mixtures. No reaction was found when triethylamine was used as
catalyst.
The reaction mixtures were easily purified by flash chromatog-
raphy and/or crystallization to afford the pure C-cinnamoyl glyco-
sides 1–4. The 1H NMR, 13C NMR, 2D COSY and HSQC were in full
agreement with their structures. The trans double bond in the gly-
cosides was established by the large coupling constant (J = 16 Hz)
between the two olefinic protons.
against human hepatocellular liver carcinoma (HepG2) and human
lung adenocarcinoma (A549) cell lines and showed antiprolifera-
tive activity in the micromolar range.13 Very recently we have pre-
pared novel N-b-glycosyl sulfamides and shown that they
selectively target cancer-associated CAs (IX and XII) with Kis in
the low nanomolar range.14
Now our approach is based on the synthesis of C-cinnamoyl gly-
cosides, where the carbohydrate moiety is tethered to a phenol CA
pharmacophore through a carbon chain. The replacement of the
naturally occurring O-glycoside bond by C-glycoside bond is an ap-
proach practiced in the synthesis of carbohydrate-containing com-
pounds for the downstream biological applications.12 This isosteric
replacement seeks to enhance stability of the small molecule gly-
coside toward enzymatic hydrolysis of the glycosidic bond while
retaining vital molecular recognition interactions with the biolog-
ical target.15 C-cinnamoyl glycosides derivated from b-C-glycosyl
The O-acetate protecting groups of the carbohydrate moiety
were next removed using triethylamine in methanol/water to af-
ford the fully deprotected C-glycosides 5–8 in very good yields.
The inhibitory activity of C-glycosides 1–8 and phenol against
ketones were shown to possess a
-glucosidase inhibition activity16
and to be effective as antimycobacterial agents.17
Thus, in the search of non-sulfonamide CAIs belonging to differ-
ent classes of compounds, we report here the synthesis of a series
of new C-cinnamoyl glycosides incorporating the phenol moiety,
and their inhibitory activity against the 12 catalytically active
mammalian CA isozymes.
all the catalytically active mammalian
sented in Table 2.
a-CA isozymes are pre-
In contrast to standard CA inhibitors, the inhibition profile with
a-CAs for the peracetylated C-glycosyl compounds 1–4 is flat and
there is small variations in the Ki values observed across all CA iso-
zymes investigated, leading to Ki selectivity ratios of ꢀ1.0 across
isozymes, that is, they are nonselective CA inhibitors. On the other
hand deprotected glycosides 5–8 showed a more complicated inhi-
bition profile.
The following should be noted regarding the inhibition of the
mammalian a-CA isozime with C-glycosides 1–8:
1. Chemistry
A set of new C-cinnamoyl glycosides (Fig. 1) was synthesized as
outlined in Scheme 1. 1-(b-D-Glucopyranosyl)-propan-2-one was
prepared by Knoevenagel condensation with 2,4-pentanedione in
the presence of sodium carbonate using water as solvent.18 We
found that the reaction of D-galactose in these conditions resulted
(i) All investigated C-cinnamoyl glycosides were efficient,
in a mixture of several compounds that were difficult to separate
due to their similar polarity. Thus b-C-galactosyl ketone was syn-
thesized by the same reaction but using sodium bicarbonate fol-
lowing an earlier protocol.19 Crude mixtures containing the
C-glycosyl ketones were acetylated and then purified to afford
the peracetylated compounds in good yields. 1-(2,3,4,6-Tetra-O-
micromolar hCA I and hCA II inhibitors with inhibition con-
stants in the range of 3.6–9.3
(CA II).
lM (CA I) and of 3.1–8.8 lM
(ii) Isozyme hCA III was not inhibited by C-cinnamoyl glyco-
sides with only compound 8 behaving as an effective inhib-
itor (Ki of 8.4 lM.) Glycosides, similarly to other CA
acetyl-b-
the Knoevenagel condensation of
C-mannosyl ketone could be rationalized considering that
nose could be obtained by alkaline epimerization of -glucose.
D
-mannosyl)-propan-2-one was found as by-product in
-glucose. The formation of the
-man-
inhibitors, may have steric clashes with Phe198, a bulky
amino acid residue place in the middle of the CA II active
site, explaining their inefficient inhibitory activity.20
D
D
D
(iii) Peracetylated C-glycosides 1–4 act as good inhibitors of the
mitochondrial isoforms hCA VA and VB. A quite similar
inhibition was observed with the deprotected compounds
C-cinnamoyl glycosides have been prepared by aldol condensa-
tion of b-C-glucosyl and b-C-galactosyl ketones with different aro-
matic aldehydes incorporating the phenol moiety at room
temperature in the presence of pyrrolidine as catalyst.16,17 We
found that reaction times and the yields are highly dependent on
the position of the hydroxyl group in the aromatic ring. Thus the
5–8, which inhibited hCA VA with Kis of 4.4–8.0 lM. On
the other hand C-glycosides 5–8 behave as ineffective
inhibitors of hCA VB. One should note the important differ-
ences in the pattern of inhibition of the two mitochondrial
OH
OH
AcO
AcO
AcO
OAc
OAc
OAc
OAc
O
O
O
O
OH
OH
OH
AcO
AcO
AcO
AcO
AcO
OAc
OAc
OAc
OAc
O
O
O
O
2
1
4
3
OH
OH
HO
HO
HO
OH
OH
OH
OH
O
O
O
O
OH
HO
HO
HO
HO
HO
OH
OH
OH
OH
O
O
O
O
8
5
7
6
Figure 1. Per-O-acetyl C-glycosides (1–4) and fully deprotected derivatives.

L. E. Riafrecha et al. / Bioorg. Med. Chem. 21 (2013) 1489–1494
1491
O
O
O
O
O
O
O
O
(HO)n
(HO)n
(AcO)n
OH
py
HCO3Na
O
O
OH
OHC
pyrrolidine
O
O
CH3OH/Et3N/H2O
(HO)n
(AcO)n
OH
OH
O
O
1-4
5-8
Scheme 1. Preparation of C-glycosides.
(vi) The two tumor-associated isoforms hCA IX and XII showed
a similar inhibition profile with compounds 1–8. Thus,
these derivatives inhibited hCA IX with inhibition constants
Table 1
Synthesis of C-cinnamoyl glycosides
Ketone
Benzaldehyde
Reaction time (hs.)
Product
Yield (%)
in the range of 2.9–9.2
lM, and hCA XII with Kis of 3.9–
8.7 M. In the development of anti-cancer compounds that
l
C-Glucosyl
2-Hydroxy
3-Hydroxy
28
—
1
—
1.5
24a
48
68
n.r.
44
—
target selectively the membrane bound isoform hCA IX
(and hCA) versus the ubiquitous isoform hCA II, the design
of membrane non-permeant inhibitors is crucial. In recent
years, several parameters have been introduced for mem-
brane permeability prediction.21 Topological polar surface
area (TPSA) is now been recognized as a good indicator of
drug absorbance in the intestines, Caco-2 monolayers pen-
etration, and blood–brain barrier crossing.22 Molecules
with a TPSA greater than 140 Å2 are likely to have a low
capacity for penetrating cell membranes. We had calcu-
lated the lipophilicity and topological polar surface area
for the S-glycosyl sulfonamides showing that all com-
pounds fall within the range indicative of molecules with
poor membrane permeability.23 Though C-glycosides
showed no selectivity for the cancer associated CAs, their
pysicochemical properties would lead to preferential inhi-
bition of the transmembrane CA IX over cytosolic CA II.
(vii) C-glycosides 1–7 were good inhibitors of mCA XIII, another
4-Hydroxy
3,4-Dihydroxy
2
—
24
C-Galactosyl
2-Hydroxy
3-Hydroxy
28
4
—
3
—
62
n.r.
40
—
24a
48
24
4-Hydroxy
3,4-Dihydroxy
4
—
a
Reaction performed using triethylamine as catalyst.
enzymes (hCA VA and hCA VB) by the deprotected C-glyco-
sides, in contrast with sulfonamides which are strong
inhibitors for both of them.
(iv) C-cinnamoyl glycosides 1–4 are good inhibitors of the
secreted isozyme hCA VI (Kis of 6.2–8.8 lM). On the other
hand only deprotected glycoside 7 showed a high affinity
for this isozyme.
cytosolic isoform (Kis in the range 4.9–8.8 lM) whereas the
(v) Isoform hCA VII was also inhibitied by compounds 1–8
remaining derivative behaved as an ineffective inhibitor.
(viii) The last human isoform hCA XIV (transmembrane one,
similarly to hCA IX and XII, but not tumor associated)
with Kis in the range 4.9–9.5
less variation of the inhibitory power.
lM, showing a flat SAR with
Table 2
Inhibition of mammalian
a
-CA with the C-cinnamoyl glycosydes 1–8 and phenol
Isozymea
Ki (l
M)b
1
2
3
4
5
6
7
8
Phenol
hCA I
hCA II
8.5
7.0
>100
5.6
9.8
6.0
8.8
9.5
5.2
6.7
5.1
5.9
5.7
3.9
>100
4.9
8.4
4.0
6.2
6.3
5.9
6.2
4.9
5.6
5.1
7.1
>100
7.8
9.5
6.9
7.7
7.1
3.3
3.9
7.2
4.6
9.3
5.5
>100
6.7
9.3
5.2
7.9
5.8
2.9
4.2
6.7
2.3
6.8
7.8
>100
8.3
3.7
8.8
>100
7.1
4.4
>100
>100
8.5
8.2
8.7
3.6
3.1
>100
9.2
3.4
>100
8.1
9.0
9.2
8.4
5.5
6.8
8.4
8.4
10.2
5.5
2.7
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IX
hCA XII
hCA XIII
hCA XIV
9.5
7.4
8.0
218
543
208
710
8.8
9.2
697
11.5
>100
>100
4.9
4.5
7.4
>100
>100
9.3
8.2
6.8
8.8
4.3
8.6
7.1
8.6
>100
>100
>100
a
All CAs are recombinant enzymes obtained in the authors’ laboratory as reported earlier.4
Errors in the range of 5–10% of the reported value, from 3 different determinations.
b

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L. E. Riafrecha et al. / Bioorg. Med. Chem. 21 (2013) 1489–1494
was generally well inhibited by the C-cinnamoyl glycosides
1–6, which showed Kis in the range of 2.3–7.1 M. Depro-
tected galactosyl derivatives 7–8 were not all inhibitory.
The organic extracts were combined, dried over NaSO4, filtered
and evaporated. The product was purified by column chromatogra-
phy (eluant 4:6 to 1:1 hexanes–EtOAc) to give compounds 1–4.
l
Neither the stereochemistry presented by the differing carbo-
hydrate moiety nor the nature of the carbohydrate hydroxyl
groups, either as the C-glycosides (5–8) or less polar and bulkier
acetylated sugar (1–4) impacted to alter enzyme inhibition char-
acteristics (with the exception of isozymes hCA VB and hCA VI).
Also there is no influence of the position of hydroxyl in the aro-
matic ring on the inhibition profile. However per-O-acetyl and
deprotected C-glycosides showed better inhibitory activity
3.3. General procedure 2: Deprotection of per-O-acetylated C-
glycosides (1–4 ? 5–8)
Deprotected compounds 5–8 were prepared by dissolving the
corresponding per-O-acetylated precursor 1–4 in methanol/trieth-
ylamine/water (8:2:1). The reaction was kept at room temperature
overnight and then concentrated. The product was purified by col-
umn chromatography (eluant 5:1 EtOAc–MeOH) to afforded pure
material by 1H NMR and 13C NMR spectroscopy. Yields 75–95%.
against almost all
a-CA than phenol. Thus, this confirms that
attaching carbohydrate moieties to CA phenol pharmacophore
could improve its inhibitory activity.
3.4. (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)-4-(3-
hydroxyphenyl)but-3-en-2-one (1)
2. Conclusions
The title compound 1 was prepared from 1-(20,30,40,60-tetra-O-
acetyl-b- -glucopyranosyl)-propan-2-one and 3-hydroxybenzalde-
hyde according to general procedure 1 to give a white solid.
Mp = 152–153 °C 1H NMR (200 MHz, CDCl3)
7.50 (d, 1H,
D
We have investigated the enzyme inhibition characteristics of a
small series of C-cinnamoyl glycosides incorporating the phenol
moiety, against mammalian CAs. This is the first study in which
d
J = 16.2 Hz, H-4), 7.27 (dd, 1H, J = 9.2, 6.5 Hz, ArH), 7.07 (t,
J = 4.4 Hz, 2H, ArH), 6.91 (m, 1H, ArH), 6.71 (d,1H, J = 16.2 Hz, H-
3), 6.69 (s, 1H, OH), 5.22 (d, 1H, J = 9.2 Hz, H-30), 5.11 (d, 1H,
J = 9.8 Hz, H-40), 4.96 (d, 1H, J = 9.3 Hz, H-20), 4.27 (dd, 1H,
J = 12.4, 4.7 Hz, H-60a), 4.11 (ddd, 1H, J = 9.7, 8.6, 2.8 Hz, H-10),
4.05 (dd, 1H, J = 12.4, 2.2 Hz, H-60b), 3.73 (ddd, 1H, J = 9.7, 4.7,
2.2 Hz, H-50), 3.02 (dd, 1H, J = 16.3, 8.6 Hz, H-1a), 2.68 (dd, 1H,
J = 16.3, 3.1 Hz, H-1b), 2.04 (s, 3H, CH3COO), 2.03 (s, 3H, CH3COO),
2.02 (s, 3H, CH3COO), 2.01 (s, 3H, CH3COO). 13C NMR (50 MHz,
a
-CAs have been investigated for their interaction with
C-glycosides, a novel carbohydrate scaffold in the design of carbonic
anhydrase inhibitors. The C-cinnamoyl glycosides were generally
effective CA inhibitors, with inhibition constants in the low micro-
molar range against CA I, II, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and
ineffective inhibitors of CA III. These results confirm that attaching
carbohydrate moieties to CA phenol pharmacophore improves its
inhibitory activity.
The physicochemical properties of the glycosides tested would
enhance the preferential inhibition of transmembrane isozymes
in vivo. Free C-cinnamoyl glycosydes could be useful for chemo-
therapy if they are delivered through a route of intravenous admin-
istration. For oral delivery peracetylated glycosides may be used as
ester prodrugs. Once in the body, the ester groups could be readily
hydrolyzed by ubiquitous esterases.24
CDCl3)
d 196.99 (C-2’), 171.27 (CH3COO), 170.63 (CH3COO),
170.37 (CH3COO), 169.95 (CH3COO), 156.83 (ArC), 144.22 (C-4),
135.79 (ArC), 130.44 (ArC), 126.46 (C-3), 121.29 (ArC), 118.46
(ArC), 114.86 (ArC), 75.84 (C-50), 74.40 (C-30), 74.30 (C-40), 71.84
(C-10), 68.67 (C-20), 62.29 (C-60), 42.74 (C-1), 20.96 (CH3COO),
20.93 (CH3COO), 20.878 (CH3COO), 20.878 (CH3COO). HRMS m/z:
calcd for C24H28O11, 492.1632; found, 492.1630
3. Experimental section
3.1. General
3.5. (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)-4-(4-
hydroxyphenyl)but-3-en-2-one (2)
The title compound 2 was prepared from 1-(20,30,40,60-tetra-O-
acetyl-b- -glucopyranosyl)-propan-2-one and 4-hydroxybenzalde-
hyde according to general procedure 1 to give a white solid.
Mp = 176–177 °C 1H NMR (200 MHz, CDCl3)
7.50 (d, 1H,
All starting materials and reagents, were purchased from com-
mercial suppliers. Reactions were monitored by TLC and TLC plates
visualized with short wave UV fluorescence (k = 254 nm), sulfuric
acid stain (5% H2SO4 in methanol). Silica gel flash chromatography
was performed using silica gel 60 Å (230–400 mesh). All melting
points are uncorrected. 1H 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.
D
d
J = 16.1 Hz,H-4), 7.43 (d, 2H, J = 8.6 Hz, ArH), 6.97 (s, 1H, OH),
6.87 (d, 2H, J = 8.6 Hz, ArH), 6.59 (d,1H, J = 16.1 Hz, H-3), 5.24 (t,
1H, J = 9.2 Hz, H-30), 5.10 (d, 1H, J = 9.8 Hz, H-40), 5.00 (dd, 1H,
J = 13.6, 5.5 Hz, H-20), 4.27 (dd, 1H, J = 12.4, 4.8 Hz, H-60a), 4.13
(ddd, 1H, J = 11.1, 9.1, 3.9 Hz, H-10), 4.03 (dd, 1H, J = 12.4, 2.1 Hz,
H-60b), 3.72 (ddd, 1H, J = 9.8, 4.8, 2.1 Hz, H-50), 3.01 (dd, 1H,
J = 16.3, 8.3 Hz, H-1a), 2.67 (dd, 1H, J = 16.3, 3.2 Hz, H-1b), 2.03
(s, 3H, CH3COO), 2.02 (s, 3H, CH3COO), 2.01 (s, 3H, CH3COO), 2.01
(s, 3H, CH3COO). 13C NMR (50 MHz, CDCl3) d 196.84 (C-2), 171.16
(CH3COO), 170.59 (CH3COO), 170.41 (CH3COO), 169.94 (CH3COO),
159.18 (ArC), 144.42 (C-4), 130.75 (ArC), 126.67 (C-3), 123.84
(ArC), 116.34 (ArC), 75.89 (C-50), 74.45 (C-30),74.37 (C-40), 71.98
(C-10), 68.74 (C-20), 62.31 (C-60), 42.57 (C-1), 20.95 (CH3COO),
20.95 (CH3COO), 20.85 (CH3COO), 20.85 (CH3COO). HRMS m/z:
calcd for C24H28O11, 492.1632; found, 492.1651
3.2. General procedure 1: Synthesis of per-O-acetylated C-
cinnamoyl glycosides (1–4)
To
a solution of per-O-acetylated b-C-glucopyranosyl or
C-galactopyranosyl ketone (1.0 equiv) in dry dichloromethane was
added the desired benzaldehyde (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 di-
luted in ethyl acetate and washed with water (3ꢁ). The aqueous ex-
tracts were combined and back extracted with ethyl acetate (1ꢁ).
3.6. (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-galactopyranosyl)-4-(3-
hydroxyphenyl)but-3-en-2-one (3)
The title compound 3 was prepared from 1-(20,30,40,60-tetra-O-
acetyl-b- -galactopyranosyl)-propan-2-one and 3-hydroxybenzal-
D

L. E. Riafrecha et al. / Bioorg. Med. Chem. 21 (2013) 1489–1494
1493
dehyde according to general procedure 1 to give a sticky white so-
lid. 1H NMR (200 MHz, CDCl3) d 7.51 (d, 1H, J = 16.1 Hz, H-4), 7.26
(t, 1H, J = 7.9 Hz, ArH), 7.08 (d, 2H, J = 6.8 Hz, ArH), 6.92 (d, 1H,
J = 8.9 Hz, ArH), 6.72 (d, 1H, J = 16.1 Hz, H-3), 5.45 (dd, 1H, J = 3.2,
0.9 Hz, H-40), 5.20 (m, 1H, H-2), 5.08 (dd, 1H, J = 10.0, 3.3 Hz,
H-30), 4.04 (m, 4H, H-10, H-50, 2 ꢁ H-60), 3.06 (dd, J = 16.1, 8.5 Hz,
1H, H-1a), 2.69 (dd, J = 16.1, 3.2 Hz, 1H, H-1b), 2.16 (s, 3H,
CH3COO), 2.04 (s, 3H, CH3COO) 1.99 (s, 3H, CH3COO), 1.96 (s, 3H,
69.81 (C-40), 60.83 (C-60), 42.36 (C-1). HRMS (m/z): [M+Na]+ calcu-
lated for C16H20O7Na 347.1107, found 347.1112.
3.10. (E)-1-b-D-galactopyranosyl-4-(3-hydroxyphenyl)but-3-en-
2-one (7)
The title compound 7 was prepared from compound 3 accord-
ing to general procedure to give pale yellow solid.
Mp = 126.5–127 °C 1H NMR (200 MHz, D2O)
7.53 (d, 1H,
2
a
CH3COO). 13C NMR (50 MHz, CDCl3)
d 197.19 (C-2), 170.99
d
(CH3COO), 170.61 (CH3COO), 170.57 (CH3COO), 170.44 (CH3COO),
156.88 (ArC), 144.12 (C-4), 135.85 (ArC), 130.39 (ArC), 126.49 (C-
3), 121.23 (ArC), 118.42 (ArC), 114.86 (ArC), 74.87 (C-50), 74.43
(C-10), 72.24 (C-30), 69.33 (C-20), 67.99 (C-40), 61.71 (C-60), 42.98
(C-1), 21.03 (CH3COO), 20.89 (CH3COO), 20.82 (CH3COO), 20.82
(CH3COO). HRMS m/z: calcd for C24H28O11, 492.1632; found,
492.1630.
J = 16.3 Hz, H-4), 7.29 (t, 1H, J = 7.8 Hz, ArH), 7.14 (d, 1H,
J = 7.8 Hz, ArH), 7.04 (d, 1H, J = 1.6 Hz, ArH), 6.93 (dd, 1H, J = 7.6,
2.1 Hz, ArH), 6.75 (d, 1H, J = 16.3 Hz, H-3), 3.95 (d, 1H, J = 3.2 Hz,
H-20), 3.66 (m, 6H, H-10, H-30, H-40, H-50, 2 ꢁ H-60), 3.18 (dd, 1H,
J = 16.2, 3.1 Hz, H-1a), 2.94 (dd, 1H, J = 16.3, 8.7 Hz, H-1b). 13C
NMR (50 MHz, D2O) d 202.80 (C-2), 156.22 (ArC), 145.28 (C-4),
135.97 (ArC), 130.70 (ArC), 126.32 (C-3), 121.51 (ArC), 118.51
(ArC), 115.05 (ArC), 78.84 (C-50), 76.46 (C-10), 74.27 (C-40), 70.98
(C-30), 69.48 (C-20), 61.49 (C-60), 42.89(C-1). HRMS (m/z):
[M+Na]+ calculated for C16H20O7Na 347.1107, found 347.1128.
3.7. (E)-1-(20,30,40,60-tetra-O-acetyl-b-
D-galactopyranosyl)-4-(4-
hydroxyphenyl)but-3-en-2-one (4)
The title compound 4 was prepared from 1-(20,30,40,60-tetra-O-
acetyl-b- -glucopyranosyl)-propan-2-one and 4-hydroxybenzalde-
hyde according to general procedure 1 to give a pale yellow solid.
mp = 119-120 °C 1H NMR (200 MHz, CDCl3)
7.51 (d, 1H,
3.11. (E)-1-b-D-galactopyranosyl)-4-(4-hydroxyphenyl)but-3-en-
2-one (8)
D
d
The title compound 8 was prepared from compound 4 accord-
ing to general procedure 2 to give a pale yellow solid. Mp = 162–
163 °C
J = 16.2 Hz, H-4), 7.43 (d, 2H, J = 8.6 Hz, ArH), 6.88 (d, 2H, J = 8.6 Hz,
ArH), 6.61 (d, 1H, J = 16.1 Hz, H-3), 5.45 (d, 1H, J = 2.5 Hz, H-40),
5.20 (m, 1H, H-20), 5.07 (dd, 1H, J = 10.0, 3.2 Hz, H-30), 4.06 (m, 4H,
H-10, H-50, 2 ꢁ H-60), 3.06 (dd, 1H, J = 16.1, 8.4 Hz, H-1a), 2.68 (dd,
1H, J = 16.1, 3.3 Hz, H-1b), 2.16 (s, 3H, CH3COO), 2.04 (s, 3H,
CH3COO), 1.99 (s, 3H, CH3COO), 1.97 (s, 3H, CH3COO). 13C NMR
(50 MHz, CDCl3) d 197.17 (C-2), 170.96 (CH3COO), 170.67 (CH3COO),
170.49 (CH3COO), 159.40 (ArC), 144.48 (C-4), 130.75 (ArC), 123.79
(C-3), 116.35 (ArC), 74.91 (C-50), 74.40 (C-10), 72.26 (C-30), 69.40
(C-20), 67.99 (C-40), 61.69 (C-60), 42.75 (C-1), 21.06 (CH3COO),
20.92 (2 ꢁ CH3COO), 20.84 (CH3COO). HRMS m/z: calcd for
1H NMR (200 MHz, D2O) d 7.51 (dd, 3H, J = 14.5, 12.5 Hz, H-4,
ArH), 6.86 (d, 2H, J = 8.6 Hz, ArH), 6.64 (d,1H, J = 16.1 Hz, H-3),
3.95 (d, 1H, J = 3.2 Hz, H-20), 3.64 (m, 6H, H-10, H-30, H-40, H-50,
2 ꢁ H-60), 3.14 (dd, 1H, J = 13.3, 6.6 Hz, H-1a), 2.90 (dd, 1H,
J = 16.1, 8.8 Hz, H-1b). 13C NMR (50 MHz, D2O) d 202.84 (C-2),
159.01 (ArC), 146.02 (C-4), 131.27 (ArC), 126.63 (C-3), 116.31
(ArC), 78.84 (C-50), 76.65 (C-10), 74.28 (C-30), 71.02 (C-20), 69.49
(C-40), 61.50 (C-60), 42.70 (C-1). HRMS (m/z): [M+Na]+ calculated
for C16H20O7Na 347.1107, found 347.1115.
C24H28O11, 492.1632; found, 492.1626.
4. CA Inhibiton studies
3.8. (E)-1-b-D-glucopyranosyl-4-(3-hydroxyphenyl)but-3-en-2-
one (5)
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity as re-
ported by Khalifah.25 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 inhibi-
tion constants. For each inhibitor at least six traces of the initial
5–10% of the reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the same man-
ner and subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mM) were prepared in distilled–deionized water and
dilutions up to 0.01 nM were done thereafter with distilled–deion-
ized water. Inhibitor and enzyme solutions were preincubated to-
gether for 15 min at room temperature prior to assay, in order to
allow for the formation of the E-I complex. The inhibition constants
were obtained by non-linear least-squares methods using PRISM 3,
The title compound 5 was prepared from compound 1 accord-
ing to general procedure 2 to give a white solid. Mp = 172–173 °C
1H NMR (200 MHz, D2O) d 7.51 (d, 1H, J = 16.2 Hz,H-4), 7.28 (t,
1H, J = 7.8 Hz,ArH), 7.12 (d, 1H, J = 7.8 Hz,ArH), 7.03 (d, 1H,
J = 2.0 Hz,ArH), 6.92 (dd, 1H, J = 8.1, 1.6 Hz,ArH), 6.73 (d, 1H,
J = 16.2 Hz,H-3), 3.72 (m, 3H, H-50, 2 ꢁ H-60), 3.35 (m, 4H, H-10,H-
20,H-30,H-40), 3.07 (dd, 1H, J = 16.2, 5.4 Hz,H-1a), 2.89 (dd, 1H,
J = 16.2, 8.8 Hz,H-1b). 13C NMR (50 MHz, D2O) d 202.65 (C-2),
156.23 (ArC), 145.30 (C-4), 135.93 (ArC),130.70 (ArC),126.31 (C-
3), 121.51 (ArC), 118.54 (ArC), 115.06 (ArC), 79.85 (C-50), 77.67
(C-30), 76.0 2 (C-40), 73.58 (C-10), 70.09 (C-20), 61.16 (C-60), 42.74
(C-1). HRMS (m/z): [M+Na]+ calculated for C16H20O7Na 347.1107,
found 347.1126.
3.9. (E)-1-b-D-glucopyranosyl)-4-(4-hydroxyphenyl)but-3-en-2-
one (6)
The title compound 6 was prepared from compound 2 accord-
ing to general procedure 2 to give a white solid. Mp = 123–
124 °C. 1H NMR (200 MHz, D2O) d 7.64 (d, 1H, J = 16.2 Hz, H-4),
7.56 (d, 2 H, J = 8.5 Hz, ArH), 6.90 (d, 2 H, J = 8.5 Hz, ArH), 6.73 (d,
1H, J = 16.2 Hz, H-3), 3.68 (m, 3H, H-50, 2 ꢁ H-60), 3.27 (m, 4H,
H-10, H-20, H-30, H-40), 3.06 (dd, 1H, J = 16.2, 5.3 Hz, H-1a), 2.87
(dd, 1H, J = 16.2, 8.7 Hz, H-1b). 13C NMR (50 MHz, D2O) d 202.80
(C-2), 156.23 (ArC), 146.28 (C-4), 131.13 (ArC), 126.44 (C-3),
116.13 (ArC), 79.68 (C-50), 77.40 (C-10), 76.05 (C-30), 73.15 (C-20),
26
and the Cheng-Prussoff equation as reported earlier and represent
the mean from at least three different determinations.
Acknowledgments
This work was financed in part by an EU grant (Metoxia) to
C.T.S., and by UNLP and CONICET (Argentina). P.A.C. is member of
the Scientific Research Career of CONICET.

1494
L. E. Riafrecha et al. / Bioorg. Med. Chem. 21 (2013) 1489–1494
8. Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo, D.;
Supplementary data
Innocenti, A.; Supuran, C. T.; Poulsen, S.-A. J. Med. Chem. 2011, 54, 1682.
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12. Colinas, P. A. Curr. Org. Chem. 2012, 16, 1670.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.002.
13. Crespo, R.; de Bravo, M. G.; Colinas, P. A.; Bravo, R. D. Bioorg. Med. Chem. Lett.
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