Natural product hybrid and its superacid synthesized analogues: Dodoneine and its derivatives show selective inhibition of carbonic anhydrase isoforms I, III, XIII and XIV

Article abstract of DOI:10.1016/j.bmc.2013.04.041

The natural product dodoneine (a dihydropyranone phenolic compound), extracted from African mistletoe Agelanthus dodoneifolius, has been investigated as inhibitor of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. By using superacid chemistry

Full text of DOI:10.1016/j.bmc.2013.04.041

Bioorganic & Medicinal Chemistry 21 (2013) 3790–3794
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Natural product hybrid and its superacid synthesized analogues:
Dodoneine and its derivatives show selective inhibition of carbonic
anhydrase isoforms I, III, XIII and XIV
Hélène Carreyre a, Jean-Marie Coustard a, Grégoire Carré b, Clarisse Vandebrouck b, Jocelyn Bescond b,
Maurice Ouédraogo c, Jérôme Marrot d, Daniella Vullo e, Claudiu T. Supuran f, , Sébastien Thibaudeau a,
⇑
⇑
a Superacid Group in Organic Synthesis Team—Université de Poitiers, CNRS UMR 7285 IC2MP, 4, Avenue Michel Brunet, F-86022 Poitiers Cedex, France
b Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, FRE 3511, 1 rue Georges Bonnet, F-86022 Poitiers Cedex, France
c Université de Ouagadougou, 03 BP 7021, Ouagadougou 01, Burkina Faso
d Institut Lavoisier de Versailles, UMR CNRS 8180, Université de Versailles St-Quentin, 45 avenue des Etats Unis, 78035 Versailles Cedex, France
e Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
f Università degli Studi di Firenze, Neurofarba Department, Section of Pharmaceutical Sciences, Via Ugo Schiff 6, 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:
Received 22 February 2013
Revised 5 April 2013
Accepted 10 April 2013
Available online 23 April 2013
The natural product dodoneine (a dihydropyranone phenolic compound), extracted from African mistle-
toe Agelanthus dodoneifolius, has been investigated as inhibitor of several human carbonic anhydrase
(hCA, EC 4.2.1.1) isoforms. By using superacid chemistry, analogues of the lactone phenolic hybrid lead
compound have been synthesized and tested as CA inhibitors. Small chemical modifications of the basic
scaffold revealed strong changes in the selectivity profile against different CA isoforms. These new com-
pounds selectively inhibited isoforms CA I (KIs in the range of 0.13–0.76
l
M), III (KIs in the range of 5.13–
M), and can be
Keywords:
Dodoneine
Superacid
Metabolites
Carbonic anhydrase
Hybrid molecules
10.80 M), XIII (KIs in the range of 0.34–0.96 M) and XIV (KIs in the range of 2.44–7.24 l
l
l
considered as new leads, probably acting as non-zinc-binders, similar to other phenols/lactones investi-
gated earlier.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
cently discovered to act as good inhibitors.6 In the design of new
drugs, the selective inhibition of CAs became an essential parameter,
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in
all organisms. These metalloenzymes catalyse CO2 hydratation to
bicarbonate and protons.1 CO2, bicarbonate and protons are essen-
tial molecules/ions in many important physiological/pathologic
processes. The catalytic mechanism of CAs is understood in detail.
In all enzyme classes, a metal hydroxide species (L3–Zn2+–OHꢀ) of
the enzyme is the catalytically active species, acting as a strong
nucleophile (at neutral pH) on the CO2 molecule bound in a hydro-
especially considering the high number of isozymes in mammals
and their widespread distribution in the organism.4 Recently, a no-
vel strategy to attain good selectivity was developed: designing of
mechanism-based inhibitors that may interact in other part of the
enzyme than the classical zinc binding zone. Such a strategy led to
the discovery of new chemotypes that selectively inhibit some tar-
geted hCA isoforms. For instance coumarins, thiocoumarines7 and
more recently sulfocoumarins,8 located at the entrance of the en-
zyme active site, were found to act as selective inhibitors of several
isoforms, such as hCA IX isozyme, a validated anti-cancer target, and
some of these compounds are currently under clinical trials. Re-
cently, these and other groups discovered two new classes of inhib-
itors which do not bind to the metal ion within the CA active site.
Phenol derivatives9 and lactones10 were found to be low micromolar
CAIs. Phenol was found anchored to the Zn(II)-coordinated water
molecule, binding through two hydrogen bonds to this molecule
and the OH of Thr199,11 and can be considered as a non-zinc binding
inhibitor. In the same time, our group recently isolated and charac-
terized a new dihydropyranone phenolic compound named dodone-
ine, 1, from an African mistletoe Agelanthus dodoneifolius (Fig. 1).12
phobic pocket nearby.2 So far, 16 different
a-CA isoforms were iso-
lated and characterized in mammals, where they play important
physiological roles.3 Many mammalian CAs are well established
therapeutic targets, with the potential to be inhibited to treat a wide
range of disorders, including glaucoma, epilepsy, obesity, hypergly-
cemia and more recently cancer.4 In the quest of CA inhibitors (CAIs),
beside the classical primary sulfonamides,5 new families were re-
⇑
Corresponding authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.);
tel.: +33 549454588; fax: +33 549453501 (S.T.).
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), sebastien.thibau-
deau@univ-poitiers.fr (S. Thibaudeau).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.041

H. Carreyre et al. / Bioorg. Med. Chem. 21 (2013) 3790–3794
3791
lytic activity, this compound inhibits isoforms differing strongly
for subcellular localization (hCA I, III and XIII are cytosolic, hCA
IV is localized in plasma membrane and hCA XIV is a membrane
bounded isozyme). This inhibitory profile let us envisage a new
mode of action, results which deserve to test other analogues.
However, since the discovery of dodoneine, even if various groups
elegantly performed its synthesis,14 the difficulty to access easily
and efficiently to a variety of analogues, probably discouraged
ambitious projects in this direction.
The possibility to selectively access to novel compounds starting
from usually non reactive substrates, via superacid-catalyzed reac-
tions15 (e.g., CH activation,16 fluorination,17 hydroxylation,18 Fri-
edel–Crafts and other reactions)19 has been largely documented.
On account of the exceedingly nature of such catalysts, under HF/
SbF5 superacid conditions, natural products react in their (poly)pro-
tonated forms (one could consider the substrate being protected by
protonation). As a consequence, novel modifications, than are ob-
served with conventional media, can be performed directly to the
elaborated products, without suffering from long synthetic routes,
non compatible with rare natural products modifications. This strat-
egy has been previously applied to indole, Cinchona and Vinca alka-
loids.20 The commercialization of the difluorinated anticancer agent
vinflunine (JavlorÒ)21 synthesized in superacid HF/SbF5 additionally
demonstrates that this technology is already operational at an
industrial scale. This chemistry was applied to the synthesis of
new dodoneine analogues 2 and 3 (Scheme 1).
O
OH
O
dodoneine 1
HO
Figure 1.
Considering the fact that this compound incorporates both the
phenol and lactone chemotypes known to act as CAIs, dodoneine
may be considered as an interesting natural product hybrid which
might possess CA inhibitory action.13 A question arises as to whether
combining these two moieties in the natural product hybrid mole-
cule dodoneine, could be of interest to access to novel selective CAIs.
Here we investigated this possibility, and report the evaluation of
dodoneine and its superacid synthesized analogues as CAIs. Among
the tested compounds, new potent hCA I, hCA III, mCA XIII and hCA
XIV (h = human, m = murine isoforms) selective inhibitors have
been identified.
2. Results and discussion
2.1. Chemistry
Initially, dodoneine 1 was tested against all the catalytically ac-
Starting from dodoneine 1 after superelectrophilic activation22
in the presence of H2O2,23 the hydroxylated analogue 2 was syn-
thesized. Interestingly, this compound was also found in very small
quantity in the plant methanolic extract,24 confirming that super-
acid electrophilic hydroxylation can be considered as a new direct
tool to synthesize hydroxylated metabolites of natural products.
Bromination of dodoneine 1 by using HF/SbF5/NBS procedure25
led to the formation of dibrominated analogue 3.26
tive mammalian CA isoforms, hCA I–XIV (Table 1).
Dodoneine showed inhibition in the range of 5.5–10.4 lM
against isoforms I, III, IV, XIII and XIV and did not inhibit the other
isoforms. To the best of our knowledge, in terms of selectivity, the
inhibition profile of this compound was really original and let us
envisages that dodoneine 1 could be considered as a new lead com-
pound in the design of selective CAIs. Beside its micromolar level of
inhibition toward isoforms known for their low to very low cata-
Table 1
hCA inhibition data of dodoneine and analogues by a stopped flow CO2 hydrase assay33
Compound
Kia
(lm)
hCA I
hCA II
hCA III
10.35
hCA IV
hCA Va
hCA Vb
hCA VI
hCA VII
hCA IX
hCA XII
mCA XIII
hCA XIV
b
b
b
b
b
b
b
1
2
3
4
5
6
5.48
0.38
0.76
—
—
9.61
4.12
13.7
—
—
—
—
—
—
—
—
—
—
—
—
9.27
8.13
7.89
0.91
0.96
0.34
9.34
7.24
12.5
b
b
b
b
—
—
21.6
8.55
13.7
6.32
32.6
15.7
24.5
10.8
b
b
b
21.8
b
b
b
b
b
b
b
b
b
b
—
—
10.80
—
—
—
—
—
—
b
b
0.13
36.9
—
5.36
7.13
1.36
—
24.9
3.57
1.48
2.44
b
b
b
b
b
b
b
b
b
b
—
—
5.13
—
—
—
—
—
—
—
—
a
Errors in the range of 5% of the reported data from three different assays.
Not active >100.
b
O
O
O
O
O
OH
OH
OH
O
HF/SbF5/NBS
Br
HF/SbF5/H2O2
10 %
HO
28 %
HO
HO
Br
OH
dodoneine 1
2
3
K2CO3
98 %
O
DAST
90 %
O
O
O
H
O
F
O
H
HF/SbF5/H2O2
O
H
O
H
10 %
HO
H
H
HO
HO
OH
5
4
6
Scheme 1. Superacid chemistry on dodonein 1 leading to derivatives 2–6.

3792
H. Carreyre et al. / Bioorg. Med. Chem. 21 (2013) 3790–3794
2.2. CA inhibition
selective hCA III and XIII inhibitors were discovered. These new hy-
brid molecules become targets of choice for further therapeutic
evaluations.
Dodonein 1, and its analogues 2–6 were evaluated as hCA inhib-
itors and tested against the hCA I–hCA XIV isoforms (Table 1).
Interestingly the modification of the aromatic ring substitution
strongly modifies the inhibition profile of the compound. Hydrox-
ylated analogue 2 inhibits hCA I at nanomolar level and hCA IV, V,
IX, XII, XIII and XIV at low micromolar level (Table 1). However
compound 2 was found to be inactive toward hCA II, III, VI and
VII. The substitution of the aromatic ring with halogen atoms dra-
matically changes inhibition properties. Against isozymes hCA III,
VI and VII, compound 3 was inactive, and the other isoforms were
inhibited at micromolar level by this dibrominated analogue. These
results confirmed that these compounds could interact differently
within the active sites of the enzyme. Back to the dodoneine ex-
tract study, we identified a bicyclic analogue of dodoneine (com-
pound 4) present in low quantity in the methanolic extract. To
test it, the compound was synthesized from dodoneine. Under ba-
sic conditions, 1 underwent an easy intramolecular cyclization by
conjugate addition of the hydroxyl group to the double bond, to af-
ford the thermodynamically stable bicyclic lactone 4, metabolite of
dodoneine.12,27 Compound 4 was found to be the first highly selec-
4. Experimental part
4.1. Chemistry
4.1.1. General procedure
The authors draw the reader’s attention to the dangerous fea-
tures of superacidic chemistry. Handling of hydrogen fluoride
and antimony pentafluoride must be done by experienced chem-
ists with all the necessary safety arrangements in place.
Reactions performed in superacid were carried out in a sealed
TeflonÒ flask with a magnetic stirrer. No further precautions have
to be taken to prevent mixture from moisture (test reaction
worked out in anhydrous conditions leads to the same results as
expected).
Yields refer to isolated pure products.
1H, 13C and 19F NMR were recorded on a 400 MHz Bruker Ad-
vance DPX spectrometer using CDCl3, or CD3OD as solvent. COSY
1H–1H and 1H–13C experiments were used to confirm the NMR
peaks assignments.
tive hCA III (Ki = 10.8 lM) and mCA XIII inhibitor (Ki = 910 nM) re-
ported to date.
All separations were done on preparative TLC plate.
High Resolution Mass Spectrometry (HRMS) spectra were per-
formed at the Centre Régional de Mesures Physiques de l’Ouest
of the University of Rennes 1, France or at the Institut de Chimie
Organique et Analytique of the University of Orleans, France.
Among the 15 human isoforms, hCA III is by far, the least under-
stood and investigated.28 However, the recently found physiologic/
pathologic functions of hCA III make this enzyme a target of choice
in the quest of new drugs.29 To evaluate whether dodoneine could
be considered as a prodrug of 4, the cyclisation process in physio-
logical conditions was evaluated. After 12 h half of dodoneine was
converted to its bicyclic analogue 4.24 However, the different inhi-
bition profile of both compounds does not confirm this hypothesis.
To pursue this investigation we envisaged the synthesis of a
dodoneine analogue, which could not metabolizes to the bicyclic
analogue. Carbon–fluorine bond being really strong and fluorine
atom being classically used to stabilize compound against meta-
bolic modification,30 fluorinated analogue 5 was synthesized by
fluorodehydroxylation reaction in the presence of DAST, and eval-
uated as carbonic anhydrase inhibitor (Table 1).31 As mentioned
above, again, the different inhibitory profile of the fluorinated ana-
logue 5 inhibiting at low micromolar level all hCA isoforms, despite
hCA III and VI, seems to refute the hypothesis that dodoneine 1
could be considered as a prodrug of the bicyclic highly selective
inhibitor 4. However, the modification of the alcohol function of
the aliphatic chain strongly modifies the the inhibitor properties
of these compounds, a result which emphasizes the importance
of the aliphatic core structure on the inhibition profile. Considering
the previously reported vasodilatator effect of dodoneine12 and its
CA inhibitory profile, further investigations could be pursue to
evaluate possible correlations.32 To confirm that the phenolic bicy-
clic lactone is the essential pharmacophore to attain the CA III and
CA XIII inhibition selectivity, an hydroxylated analogue of 4 was
synthesized and evaluated. Compound 6 also inhibited selectively
4.1.2. Preparation of HF/SbF5 mixture
After condensation of the desired quantity of hydrogen fluoride
in a TeflonÒ reactor at ꢀ30 °C, antimony pentafluoride was slowly
added to the reactor at the same temperature and the reactor was
then sealed and maintained at reaction temperature. In these con-
ditions, the SbF5 molar percentage was determined accordingly:
for example, 1 mL of SbF5 (13.8 ꢁ 10ꢀ3 mol) was added to 3 mL
of anhydrous liquid HF (0.15 mol) to give a HF/SbF5 solution with
a SbF5 molar percentage of 8.4 mol %.
4.1.3. Synthesis of compounds 2 and 6
Optimized procedure in superacidic media: to a mixture of HF/
SbF5 (4 mL, SbF5 mol % = 8.4) maintained at ꢀ20 °C, was added sub-
strate and H2O2 by dropwise addition of 0.6 mmol. The mixture
was magnetically stirred at the same temperature during 30 min.
The reaction mixture was then hydrolyzed in ice and neutralized
with Na2CO3 until pH 6.5, extracted with dichloromethane (ꢁ2)
and then with ethyl acetate (ꢁ2). The combined organic phases
were dried (MgSO4) and concentrated in vacuo. Products were then
purified by preparative TLC plate.
4.1.3.1. Compound 2: (R)-6-[(S)-2-hydroxy-4-(3,4-dihydroxy-
phenyl)butyl]-5,6-dihydropyran-2-one.
This
compound
was obtained from dodoneine 1 (100 mg, 0.38 mmol) following
the optimized procedure. The reaction crude was purified with
CH2Cl2/EtOH: 95/5 as eluent. Compound 2 (29 mg) was eluted
(R = 28%). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.55–1.70 (m, 2H,
H-9), 1.70–1.90 (m, 2H, H-7), 2.20–2.30 (ddd, 1H, J = 18.6 Hz,
J = 11.6 Hz, J = 2.6 Hz, H-5), 2.35 (m, 1H, H-5), 2.45–2.60 (m, 2H,
H-10), 3.60 (m, 1H, H-8), 4.5 (m, 1H, H-6), 5.83 (m, 1H, H-3),
6.11 (dd, 1H, J = 8.1 Hz, J = 2.5 Hz, H-60), 6.16 (d, 1H, J = 2.4 Hz,
1H, H-30), 6.77 (d, 1H, J = 8.1 Hz, H-50), 6.93 (m, 1H, H-4). 13C
NMR (100 MHz, CD3OD, ppm) d: 26.5 (CH2, C-10), 30.1 (CH2, C-
5), 39.2 (CH2, C-9), 43.0 (CH2, C-7), 68.3 (CH, C-8), 77.8 (CH, C-6),
103.5 (CH, C-30), 107.5 (CH, C-60), 120.6 (C, C-10), 121.3 (@CH,
C-3), 131.5 (CH, C-50), 148.4 (@CH, C-4), 157.0 (C, C-40), 157.5 (C,
hCA III (Ki = 5.1 lM) and mCA XIII (Ki = 340 nM) confirming the
high potential of this new chemotype.
3. Conclusion
In conclusion, several natural product hybrids have been syn-
thesized by using direct superacid catalyzed reactions on natural
product dodoneine extracted from Agelanthus dodoneifolius, and
evaluated as new CA inhibitors. The interesting inhibitory profile
diversity of these new compounds make them important leads in
the quest of new non-zinc-binding selective inhibitors, probably
interfering differently in the enzymes active sites. Two new highly

H. Carreyre et al. / Bioorg. Med. Chem. 21 (2013) 3790–3794
3793
C-20), 167.1 (CO, C-2). HRMS (ESI, MeOH): Calcd for C15H18O5
[M+Na]+: 301.1052, found 301.1053.
(396 MHz, CDCl3, ppm) d: ꢀ184.5. HRMS (ESI, MeOH): Calcd for
C15H17O3F [M+Na]+: 287.10594, found 287.1058.
4.1.3.2. Compound 6: (1S,5R,7S)-7-(2,4-dihydroxyphenethyl)-
4.1.6. Conversion of dodoneine to compound 4
2,6-dioxabicyclo[3.3.1]nonan-3-one.
This compound was
4.1.6.1. Compound 4: (1S,5R,7S)-7-(4-hydroxyphenethyl)-2,6-
obtained from bicyclic lactone 4 (100 mg, 0.38 mmol) following
the optimized procedure. The reaction crude was purified with
CH2Cl2/EtOH: 95/5 as eluent. Compound 6 (10 mg) was eluted
(R = 10%). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.51 (m, 1H, H-8),
1.61 (m, 2H, H-10), 1.83 (m, 1H, H-8), 1.88 (m, 2H, H-9), 2.40–
2.55 (m, 2H, H-11), 2.65 (d, 1H, J = 19.3 Hz, H-4), 2.77 (dd, 1H,
J = 19.3 Hz, J = 5.3 Hz, H-4), 3.56 (m, 1H, H-7), 4.22 (m, 1H, H-5),
4.85 (m, 1H, H-1), 6.11 (dd, 1H, J = 8.1 Hz, J = 2.5 Hz, H-60), 6.15
(d, 1H, J = 2.4 Hz, 1H, H-20), 6.73 (d, 1H, J = 8.1 Hz, H-50). 13C NMR
(100 MHz, CD3OD, ppm) d: 26.2 (CH2, C-11), 30.4 (CH2, C-9), 37.0
(CH2, C-4), 37.3 (CH2, C-10), 38.1 (CH2, C-8), 66.1 (CH, C-7), 67.3
(CH, C-5), 75.3 (CH, C-1), 103.5 (CH, C-30), 107.3 (CH, C-60), 120.3
(C, C-10), 131.6 (CH, C-50), 157.0 (C, C-40), 157.5 (C, C-20), 173.1
(CO, C-3). HRMS (ESI, MeOH): Calcd for C15H18O5 [M+Na]+:
301.1052, found 301.1053.
dioxabicyclo[3.3.1]nonan-3-one.
To a mixture of dodoneine
1 (10 mg, 0.04 mmol) in methanol (1.5 mL) was added Na2CO3
(5.3 mg, 0.05 mmol). The mixture was magnetically stirred at room
temperature during 2 h. After evaporation of methanol, the crude
was solubilized in dichloromethane and filtered. The organic phase
was dried (MgSO4) and concentrated in vacuo. Compound 4
(9.5 mg) was obtained without further purification (R = 95%). 1H
NMR (CDCl3, 400 MHz, ppm) d: 1.65 (m, 1H, H-8), 1.73 (m, 2H,
H-10), 1.95 (m, 1H, H-8), 2.0 (m, 2H, H-9), 2.65 (m, 2H, H-11),
2.72 (d, 1H, J = 18.9 Hz, H-4), 2.9 (dd, 1H, J = 19.2 Hz, J = 5.3 Hz,
H-4), 3.66 (m, 1H, H-7), 4.34 (m, 1H, H-5), 4.9 (m, 1H, H-1), 6.71
(d, J = 8.5 Hz, 2H, H-30 and H-50), 7.00 (d, J = 8.4 Hz, 2H, H-20 and
H-60). 13C NMR (100 MHz, CDCl3, ppm) d: 30.4 (CH2, C-9), 31.4
(CH2, C-11), 37.1 (CH2, C-4), 38.0 (CH2, C-8), 39.0 (CH2, C-10),
65.7 (CH, C-7), 67.3 (CH, C-5), 75.2 (CH, C-1), 116.2 (CH, C-30 and
C-50), 130.4 (CH, C-20 and C-60), 133.8 (C, C-10), 156.5 (C, C-40),
173 (C@O, C-3). HRMS (ESI, MeOH): Calcd for C15H18O4 [M+Na]+:
285.11028, found 285.1093.
4.1.4. Synthesis of compound 3
4.1.4.1. Compound 3: (R)-6-[(S)-2-hydroxy-4-(2,5-dibromo-4-
hydroxyphenyl)butyl]-5,6-dihydropyran-2-one.
To a mix-
ture of HF/SbF5 (4 mL, SbF5 mol % = 3.8) maintained at ꢀ20 °C,
was added N-bromosuccinimide (1.2 equiv) and dodoneine 1
(50 mg, 0.19 mmol). The mixture was magnetically stirred at the
same temperature during 30 min. The reaction mixture was then
hydrolyzed in ice and neutralized with Na2CO3 until pH 6–7, ex-
tracted with dichloromethane (ꢁ2) and then with ethyl acetate
(ꢁ2). The combined organic phases were dried (MgSO4) and con-
centrated in vacuo. The reaction crude was purified with prepara-
tive TLC plate and CH2Cl2/EtOH: 95/5 as eluent. Compound 3
(8 mg) was eluted (R = 10%). 1H NMR (CDCl3, 400 MHz, ppm) d:
1.75 (m, 2H, H-9), 1.85 (m, 2H, H-7), 2.01 (m, 1H, H-7), 2.40 (m,
2H, H-10), 2.75 (m, 1H, H-5), 2.85 (m, 1H, H-5), 3.90 (m, 1H, H-
8), 4.68 (m, 1H, H-6), 5.65 (br s, 1H, OH), 6.03 (m, 1H, H-3), 6.90
(m, 1H, H-4), 7.21 (s, 1H, H-30), 7.35 (s, 1H, H-60). 13C NMR
(100 MHz, CDCl3, ppm) d: 29.6 (CH2, C-10), 31.0 (CH2, C-5), 37.7
(CH2, C-9), 42.0 (CH2, C-7), 68.6 (CH, C-8), 77.2 (CH, C-6), 109.2
(C, C-50), 120.1 (CH, C-30), 121.3 (@CH, C-4), 123.7 (C, C-20), 132.7
(CH, C-60), 134.6 (C, C-10), 145.1 (@CH, C-3), 151.2 (C, C-40), 163.8
(CO, C-2). HRMS (ESI, MeOH): Calcd for C15H17Br2O4 [M+NH3]+:
435.97507, found 435.97536.
4.2. CA inhibition assay
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.33 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining con-
stant the ionic strength), following the initial rates of the CA-cata-
lyzed 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 inhib-
itor 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 to-
tal observed rates. Stock solutions of inhibitor (0.1 mM) were pre-
pared in distilled–deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min–6 h at room tem-
perature (15 min) or 4 °C (6 h) 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, as
reported earlier,34 and represent the mean from at least three dif-
ferent determinations. All CA isofoms were recombinant ones ob-
tained in-house as reported earlier.35,36
4.1.5. Synthesis of compound 5
4.1.5.1. Compound 5: (R)-6-[(R)-2-fluoro-4-(4-hydroxyphenyl)
butyl)-5,6-dihydropyran-2-one.
To a mixture of CH2Cl2
(2 mL) and dodoneine (50 mg, 0.19 mmol) maintained at
1
Acknowledgments
ꢀ40 °C under nitrogen, was added DAST (0.15 mL, 1.14 mmol).
The mixture allowed to reach room temperature after addition,
was magnetically stirred during 5 h. The reaction crude was di-
rectly purified with preparative TLC plate and CH2Cl2/EtOH: 95/5
as eluent. Compound 5 (45 mg) was eluted (R = 90%). 1H NMR
(CDCl3, 400 MHz, ppm) d: 1.75 (m, 2H, H-9), 1.85 (m, 2H, H-7),
2.37 (m, 2H, H-10), 2.70 (m, 2H, H-5), 4.65 (m, 1H, H-6), 4.75 (br
s, 1H, OH), 4.85 (dm, 1H, J = 48.0 Hz, H-8), 6.03 (ddd, 1H,
J = 9.7 Hz, J = 2.5 Hz, J = 1.2 Hz, H-3), 6.76 (d, 2H, J = 6.0 Hz, H-30
and H-50), 6.89 (ddd, 1H, J = 9.7 Hz, J = 5.7 Hz, J = 2.7 Hz, H-4),
7.05 (d, 2H, J = 6.0 Hz, H-20 and H-60). 13C NMR (100 MHz, CDCl3,
ppm) d: 29.8 (d, J = 6 Hz, CH2, C-5), 30.3 (d, J = 17 Hz, CH2, C-10),
37.7 (d, J = 21 Hz, CH2, C-9), 41.1 (d, J = 20 Hz, CH2, C-7), 74.4
(CH, C-6), 89.3 (d, J = 167 Hz, CFH, C-8), 115.4 (CH, C-20 and C-60),
121.5 (@CH, C-4), 129.5 (CH, C-30 and C-50), 133.23 (C, C-10),
144.9 (@CH, C-3), 153.9 (C, C-40), 164.0 (CO, C-3). 19F{1H} NMR
This work was financed in part by two FP7 EU Grant (METOXIA
and DYNANO) to C.T.S. and from the CNRS, the University of Poi-
tiers. We also would like to thank Dr. Alfonso Maresca for technical
assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.041.
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