Flavones and structurally related 4-chromenones inhibit carbonic anhydrases by a different mechanism of action compared to coumarins

Article abstract of DOI:10.1016/j.bmcl.2012.03.071

An inhibition study of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with flavones and aminoflavones, compounds possessing a rather similar scaffold with the coumarins, recently discovered inhibitors of this enzyme, is reported. The natural product

Full text of DOI:10.1016/j.bmcl.2012.03.071

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3063–3066
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Bioorganic & Medicinal Chemistry Letters
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Flavones and structurally related 4-chromenones inhibit carbonic
anhydrases by a different mechanism of action compared to coumarins
Gianfranco Balboni a, , Cenzo Congiu a, Valentina Onnis a, Alfonso Maresca b, Andrea Scozzafava b,
⇑
Jean-Yves Winum c, Annalisa Maietti d, Claudiu T. Supuran b,
⇑
a Dipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, Via Ospedale 72, I-09124 Cagliari, Italy
b Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, and CSGI, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
c Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue
de l’Ecole Normale, 34296 Montpellier Cedex, France
d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
An inhibition study of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with flavones and aminoflav-
ones, compounds possessing a rather similar scaffold with the coumarins, recently discovered inhibitors
of this enzyme, is reported. The natural product flavone and some of its hydroxylated derivatives did not
show time-dependent inhibition of the CAs, sign that they are not hydrolyzed within the enzyme active
site as the (thio)coumarins and lactones. These compounds were low micromolar inhibitors of hCA I, II, IX
Received 21 February 2012
Revised 16 March 2012
Accepted 19 March 2012
Available online 27 March 2012
and XII, with KIs in the range of 1.88–9.07 lM. A series of substituted 2-amino-3-phenyl-4H-chromen-4-
Keywords:
Carbonic anhydrase
Flavone
Coumarin
Inhibitor
ones, incorporating chloro- and methoxy substituents in various positions of the heterocycle, were then
prepared and assayed as hCA I and II inhibitors, showing activity in the micromolar range. Some of these
derivatives, as well as cis + trans resveratrol, were then assayed for the inhibition of all catalytically active
mammalian CA isoforms, hCA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and mCA XV (h = human,
m = murine enzyme). These derivatives inhibited these CAs in the submicromolar—low micromolar
range. Flavones, although not as active as the coumarins, may be considered as interesting leads for
the design of non-sulfonamide CA inhibitors.
Resveratrol
Ó 2012 Elsevier Ltd. All rights reserved.
Coumarins (2-chromenones) were recently discovered to act as
prodrug-type inhibitors1–3 of the metalloenzyme carbonic anhy-
drase (CA, EC 4.2.1.1).4 These compounds initially bind (probably
as benzopyrylium phenol tautomers) to the enzyme active site,
and undergo an in situ hydrolytic process of the lactone ring
through the esterase activity of the CAs. The 2-hydroxycinnamic
acids thus formed thereafter act as the real CA inhibitors
(CAIs).1–4 As substituted 2-hydroxycinnamic acids are rather bulky,
due to steric hindrance they cannot bind near the zinc ion from the
enzyme active site, where most other classes of inhibitors bind.4
Thus, the coumarin binding site is unique among al CAIs investi-
gated so far, being at the entrance of the cavity, far away from
the catalytically crucial Zn(II) ion, where only CA activators were
observed to bind earlier.5–9 As this is the active site region where
the amino acid residues are the most variable among the 16 mam-
malian CA isoforms known to date, many coumarins showed a high
degree of selectivity for inhibiting CA isoforms with pharmacologic
relevance for obtaining antiglaucoma (CA IV, CA XII),10 antiepilep-
tic (CA VII, CA XIV),11 antiobesity (CA VA, CA VB)12 or antitumor
(CA IX, CA XII)4,13 agents, over offtarget CA isoforms CA I and II.1–4
Furthermore, thiocoumarins,2 2-thioxo-coumarins,7 5- and 6-ring
membered lactones/thiolactones8 and coumarin/d-lactone oximes,9
possesssing scaffolds resembling the coumarin one, were recently
shown to act as CAIs with a similar mechanism of action as the
coumarins, that is, they are prodrugs undergoing active site hydro-
lysis through the CA esterase activity, with generation of the final
inhibitor, which does not interact with the Zn(II) ion from the
enzyme active site.
Flavones, 4-chromenones, are isomers of coumarins (2-chrome-
nones), but this class of derivatives was scarcely investigated as
CAIs. Only one such paper reported the inhibition of four mamma-
lian CAs, i.e., hCA I, II, bCA III and hCA IV (h = human, b = bovine iso-
form) with several natural product flavones, such as quercetin,
catechin, apigenin, luteolin and morin.14 In thats paper, an esterase
method to assay CA inhibition has been used, with 4-nitrophenyl
acetate as substrate.14 As many CAs are weak esterases, it is not
surprising that only micromolar inhibition has been observed with
these naturl product flavones in the study by Ekinci et al.14
In the present study we investigated in more detail the inhibi-
tion mechanism of CAs with flavones on one hand, and on the other
one we report the synthesis of a series of 2-amino-4-chromenones,
⇑
Corresponding authors. Tel.: +39 070 675 8625; fax: +39 070 675 8612 (G.B.);
tel.: +39 055 457 3005; fax: +39 055 457 3385 (C.T.S.).
E-mail addresses: gbalboni@unica.it (G. Balboni), claudiu.supuran@unifi.it
(C.T. Supuran).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.071

3064
G. Balboni et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3063–3066
Table 1
Substituted methyl salicylates 1 and benzylcyanides 2 were the
starting materials for obtaining the 2-aminoflavones 3–18,21 by
CA inhibition data of isoforms hCA I, II, IX and XII with flavones A–D, by a stopped-
flow CO2 hydrase assays15
a
classical cyclization reaction leading to this ring system
(Scheme 1).22 In an analogous manner were then obtained the
iminoflavone derivatives 20 and 21, by using ortho-amino methyl
benzoates instead of salicylates (Scheme 2). Coumarin 24 was on
the other hand obtained by condensation of 5-chloro-salicyclic alde-
hyde with 4-methoxybenzyl cyanide, as shown in Scheme 3. The
moieties present in compounds 3–24 as substituents of the 3-phe-
nyl ring and of the 4-chromenone benzo-moiety were of the
halogeno (chlorine) and methoxy type, in order to explore struc-
ture–activity relationship (SAR) for such substitution patterns.
Compounds 3–24 were initially assayed for the inhibition of the
major cytosolic isoforms hCA I and II (Table 2). It may be observed
that both isoforms were inhibited with IC50-s in the micromolar
O
O
O
O
O
O
O
OH
OH
Ph
O
Ph
HO
Ph
HO
Ph
A
B
C
D
Compd
KIa
(lM)
hCA I
hCA II
hCA IX
hCA XII
15 min 12 h 15 min 12 h 15 min 12 h 15 min 12 h
A
B
C
D
1.89
5.21
3.37
5.49
1.88 8.71
5.10 9.30
3.43 2.24
5.42 4.38
8.80 4.13
9.20 5.54
2.32 4.92
4.31 2.65
4.22 7.22
5.63 6.59
4.85 8.40
2.71 8.96
7.33
6.65
8.51
9.07
range, i.e., IC50 of 4.61–95.7
lM against hCA I, and of 3.62–
36.7 M against hCA II. The SAR for flavones/coumarins 3–24 is in
l
fact rather straightforward. Thus, for hCA I, three compounds were
weakly active, that is, 4, 14 and 16, possessing IC50-s in the range
Enzyme and inhibitor were incubated for 15 min (standard assay and for 12 h,
respectively).15
Errors in the range of 10% of the reported values, from three different assays
(data not shown).
a
of 46.2–95.7
of magnitude more effective as hCA I inhibitors, with IC50-s in the
range of 4.61–9.85 M (Table 1). Small structural changes in these
lM. The remaining derivatives were around one order
l
compounds led to important differences of inhibitory power. For
example compounds 6 and 7, regiomers differing only by the posi-
tion of the chlorine atom in position 6 or 7 of the heterocyclic ring,
differed by a factor of 5.1 in inhibiting this isoform. Compounds 4
and 20 differ only by the substitution of the endocyclic O atom by
an NH group, and again their activity against hCA I differed by a fac-
tor of 4.75. It is interesting to note that coumarin 24 was also weakly
active (low micromolar inhibitor) against these two isoforms, pos-
sessing the same activity as most of the flavones investigated here.
In fact, we showed earlier1b,2,3 that a bulky substituent in position 3
of the coumarin ring, interferes with the hydrolysis of the lactone
moiety from the coumarin prodrug inhibitor. As a consequence, 3-
substituted coumarins (possessing bulky, aromatic 3-substituents)
do not show time-dependent CA inhibition (as other coumarins
do)1b,2,3 but inhibit these enzymes similarly to the phenols, that is,
probably a benzopyrylium phenol is the real CAI. This observation1b
has been reconfirmed here by the inhibition data of compound 24.
hCA II was also effectively inhibited by compounds 3, 5–13, 15,
a chemotype not investigated earlier for CA inhibition, and the en-
zyme inhibitory properties of such compounds against a rather
large range of CA isoforms.
Initially, we investigated whether the simple, natural product
flavone A, 3-hydroxyflavone B, 7-hydroxy-flavone C and 5,7-
dihydroxyflavone D, act as CAIs against the physiologically rele-
vant isoforms hCA I, II (cytosolic), IX and XII (transmembrane,
tumor-associated enzymes)—Table 1.15 As seen from data of Table
1, both the standard 15 min incubation between enzyme and
inhibitors has been used,15 as well as the 12 h incubation period,
which may afford for an eventual in situ hydrolytic process of
the chromenone, as for the coumarins, or as for carbonyl com-
pounds, which have been reported by Pocker’s group to be sub-
strates and subsequently inhibitors for CA II.16,17 Indeed, both
aliphatic and aromatic aldehydes, as well as a-ketoesters were re-
ported to be hydrated by CA II, with formation of aldehyde/ketone
diols which subsequently inhibited the enzyme activity.16,17
Data of Table 1 show that the flavone A and its hydroxylated
derivatives B–D act as low micromolar inhibitors against isoforms
hCA I, II, IX and XII, and that the incubation period (of 15 min–12 h)
between enzyme and inhibitor has no consequences on the inhib-
itory power of these compounds, which showed KIs in the range of
17–24, with IC50-s in the range of 3.62–9.43
pounds, among which 4, 14, and 16, showed weaker inhibition,
with IC50-s in the range of 13.7–36.7 M (Table 2). SAR was more
lM. Several com-
l
or less similar with what discussed above for hCA I inhibition, with
the nature and position of the substituents on the heterocyclic ring
being the main factors influencing enzyme inhibitory activity.
Two of the most active compounds against hCA II, that is, com-
pounds 7 and 10, as well as another important natural product
phenol, resveratrol (as a mixture of 50% cis + 50% trans isomers,
RVT) were investigated in detail for the inhibition of all catalyti-
cally active human isoforms, that is, hCA I, II, III, IV, VA, VB, VI,
VII, IX, XII, XIII, XIV and mCA XV (m = murine isoform, as primates,
including humans, do not express CA XV).23 As seen from data of
Table 3, where the inhibition constants (KI) of these compounds
are presented, the three derivatives were low micromolar or sub-
micromolar CAIs. Amino-flavone 7 showed effective inhibition of
1.88–8.80
2.24–8.51
l
l
M against hCA I, of 5.10–9.30
M against hCA IX and of 2.65–9.07
l
M against hCA II, of
l
M against hCA
XII, respectively (Table 1). This is a clear sign that the flavones
A–D do not undergo any hydrolytic process catalyzed by the CAs,
and that they inhibit the enzyme in a similar manner with the phe-
nols.18–20 Simple phenol, PhOH, as well as many of its substituted
derivatives, are known to effectively inhibit all CA isoforms, usually
in the micromolar range.18–20 Furthermore, the adduct of hCA II
with PhOH has been characterized by means of X-ray crystallogra-
phy by Christianson’s group,18 who showed this compound to pos-
sess a particular inhibition mechanism compared to other classes
of CAIs. PhOH was found anchored by means of its OH moiety to
the zinc-coordinated water molecule/hydroxide ion, whereas a
second hydrogen bond between its OH moiety and the OH of
Thr199, further stabilized the adduct.18
Considering the interesting CA inhibitory data obtained with the
simple natural product flavones A–D, and using the A scaffold as
lead molecule, we report the synthesis of a series of aminoflavones
and structurally related derivatives, of types 3–23 as well as a cou-
marin (compound 24) structurally related to these scaffolds
(Schemes 1–3) and their CA inhibitory properties (Tables 2 and 3).
hCA I and II (KIs of 0.91–1.25
l
M) and was slightly less effective
CN
O
O
R1
OMe
R
R1
R
+
OH
O
NH2
1
3 -18
2
Scheme 1. Reagents and conditions: THF, NaH, 60 °C, 24 h, then 2 N HCl.

G. Balboni et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3063–3066
3065
Table 3
O
O
CA I-XV inhibition data (KI, l
M)a with chromenones 7 and 10, and cis+trans-RVT
OMe
NH2
R
R
HO
OH
N
H
NH2
HO
20 - 21
19
OH
Scheme 2. Reagents and conditions: benzyl cyanide, THF, NaH, 60 °C, 24 h.
OH
OH
trans-RVT
cis-RVT
OMe
CN
7
10
c+t-RVT
Cl
Cl
O
hCA I
hCA II
1.25
0.91
5.90
3.91
2.27
2.48
8.58
7.95
3.40
9.31
9.67
3.28
3.75
2.26
2.01
4.58
3.60
2.58
2.82
8.95
6.75
2.41
6.49
7.85
2.83
1.65
2.18
1.75
6.75
4.01
2.86
3.24
8.63
8.98
9.53
8.13
8.25
1.95
2.57
+
OH
O
O
OMe
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IX
hCA XII
hCA XIII
hCA XIV
mCA XV
24
22
23
Scheme 3. Reagents and conditions: MeOH, 50% aq NaOH, rt, 24 h, then N HCl.
Table 2
Inhibitory activity (IC50, l
M)a of chromenone derivatives and cis+trans-Resveratrol
(c+t-RVT) against hCA I and II
a
Mean from 3 different assays, by a stopped-flow CO2 hydrase assay.15
O
X
R1
R
NH2
hydroxylated derivatives did not show time-dependent inhibition
of the CAs, sign that they are not hydrolyzed within the enzyme ac-
tive site as the coumarins and lactones. These compounds were
low micromolar inhibitors of hCA I, II, IX and XII, with KIs in the
3
21
Compd
R
R1
X
hCA I
hCA II
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
20
21
24
H
6-Cl
7-Cl
H
6-Cl
7-Cl
H
6-Cl
7-Cl
H
6-Cl
7-Cl
7-MeO
7-MeO
7-MeO
7-MeO
6-Cl
H
H
H
4-Cl
4-Cl
4-Cl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
4.87
46.2
9.07
7.66
4.61
8.12
8.06
9.56
7.10
8.37
7.68
54.1
9.85
95.7
5.85
8.30
9.72
8.51
4.81
9.43
36.2
6.79
8.66
3.62
8.25
6.51
3.66
8.07
8.28
7.59
13.7
7.87
36.7
8.74
8.35
9.02
6.44
9.33
range of 1.88–9.07 lM. A series of substituted 2-amino-3-phenyl-
4H-chromen-4-ones, incorporating chloro- and methoxy substitu-
ents in various positions of the heterocycle, were then prepared
and assayed as hCA I and II inhibitors, showing activity in the
micromolar range. Some of these derivatives, as well as cis+trans
resveratrol, were then assayed for the inhibition of all catalytically
active mammalian CA isoforms, hCA I, II, III, IV, VA, VB, VI, VII, IX,
XII, XIII, XIV and mCA XV. These derivatives inhibited these en-
zymes in the submicromolar—low micromolar range. Flavones,
although not as active as the coumarins, may be considered as
interesting leads for the design of non-sulfonamide CAIs.
4-MeO
4-MeO
4-MeO
3,4-(MeO)2
3,4-(MeO)2
3,4-(MeO)2
H
4-Cl
4-MeO
3,4-(MeO)2
H
Acknowledgments
7-Cl
See Scheme 3
H
Dr. Alessio Innocenti participated in the early phases of this pro-
ject with testing some flavones as CA inhibitors. This research was
financed by an FP7 EU project (Metoxia, to AS and CTS)
Mean from 3 different assays, by a stopped-flow CO2 hydrase assay.15
a
as inhibitor of hCA IV, VA, VB, hCA IX, hCA XIV and mCA XV (KIs of
2.27–3.75 M). hCA III was inhibited with a KI of 5.90 M, whereas
the remaining isoforms (hCA VI, VII, XII and XIII) were the least
inhibited ones, with KIs of 7.95–9.67 M. Compound 10, in which
one of the chlorine atoms of 7 was substituted by a methoxy group,
also showed interesting inhibitory properties against these iso-
forms. Thus, hCA I, II, VA, VB, IX, XIV and mCA XV were inhibited
Supplementary data
l
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.071.
l
References and notes
with KIs in the range of 1.65–2.83
showed KIs in the range of 3.60–4.58
and XIII had KIs in the range of 6.49–8.95
The mixture of 50% cis + 50% trans isomers of RVT also inhibited
all CAs in the low micromolar range, with KIs of 1.75–9.53 M.
The best inhibited isoform was hCA II and the least inhibited one
hCA IX.
In conclusion, we present here a detailed ihibition study of sev-
eral CA isoforms with flavones and aminoflavones, compounds
possessing a rather similar scaffold with the coumarins, recently
discovered CAIs. The natural product flavone and some of its
l
l
M (Table 3). hCA III and IV
M, whereas hCA VI, VII, XII
M with this compound.
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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 the assay buffer. Inhibitor and
enzyme solutions were preincubated together for 15 min–12 h at room
temperature (15 min) or 4 °C (all other incubation times) prior to assay, in
order to allow for the formation of the E–I complex or for the eventual active
site mediated hydrolysis of the inhibitor. Data reported in Table 1 show the
inhibition after 12 h incubation, which led to the eventual completion of the
in situ hydrolysis of the heterocyclic ring, as reported earlier for couamrins.1b,2
The inhibition constants were obtained by non-linear least-squares methods
using PRISM 3, as reported earlier,1b,2 and represent the mean from at least
three different determinations. CA isofoms were recombinant ones obtained in
house as reported earlier.1,2
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2008, 16, 9101; (b) Sßentürk, M.; Gülçin, I.; Dasßtan, A.; Kufrevioglu, O. I.;
Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 3207; Innocenti, A.; Öztürk Sarıkaya,
S. B.; Gülçin, I.; Supuran, C. T. Bioorg. Med. Chem. 2010, 18, 2159.
21. General procedure for 3-aryl-4H-chromen-4-ones 3–18. To a suspension of 80%
sodium hydride (40 mmol) in THF (30 mL) was added a methyl salicylate 1
(11 mmol) and a benzyl cyanide 2 (10 mmol). The mixture was stirred at 60 °C
for 24 h. After cooling, 2 N hydrochloric acid (20 mL) was added, and the
formed precipitate filtered off, air dried, and crystallized with EtOH.
2-Amino-3-phenyl-4H-chromen-4-one (3). Yield 46%, mp 226–228 °C. 1H NMR: d
7.24 (s, 2H), 7.44–7.52 (m, 7H), 7.77–8.08 (m, 2H); IR (Nujol) 3484, 3230, 3133,
1636, 1604 cmÀ1. Anal. Calcd for C15H11NO2: C, 75.94; H, 4.67; N, 5.90. Found:
C, 75.90; H, 4.66; N, 5.87.
13. (a) McDonald, P. C.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Oncotarget 2012, 3,
´
ˇ
84; (b) Švastová, E.; Hulıková, A.; Rafajová, M.; Zat’ovicová, M.; Gibadulinová,
A.; Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.; Pastorek, J. FEBS Lett. 2004,
577, 439; (c) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.;
Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin, P.; Wouters,
B. G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.;
Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. J. Enzyme Inhib. Med.
Chem. 2009, 24, 1; (d) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.;
Harris, A. L.; Vaughan-Jones, R. D. J. Biol. Chem. 2008, 283, 20473; (e) Abbate, F.;
Winum, J.-Y.; Potter, B. V. L.; Casini, A.; Montero, J.-L.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2004, 14, 231.
General Procedure for 2-Amino-arylquinolin-4(1H)-ones 20, 21 To a suspension
of 80% sodium hydride (30 mmol) in THF (30 mL) was added benzyl cyanide
(1.30 g, 11 mmol) and methyl 4- or 5-chloroanthranilate 19 (1,86 g, 10 mmol).
The mixture was stirred at 60 °C for 24 h. The MeOH was then removed by
rotary evaporation. Water (30 mL) was added, and the formed precipitate was
filtered off, air dried, and crystallized with EtOH.
14. (a) Ekinci, D.; Karagoz, L.; Ekinci, D.; Senturk, M.; Supuran, C.T. J. Enzyme Inhib.
Med. Chem. 27, in press (doi: 10.3109/14756366.2011.643303).; (b) Sahin, H.;
Aliyazicioglu, R.; Yildiz, O.; Kolayli, S.; Innocenti, A.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2011, 26, 440; (c) Kolayli, S.; Karahalil, F.; Sahin, H.; Dincer,
B.; Supuran, C. T. J. Enzyme Inhib Med. Chem. 2011, 26, 895.
15. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO2 hydration
activity. 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 NaClO4 (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
Amino-6-chloro-3-phenylquinolin-4(1H)-one (20). Yield 78%, mp 246–248 °C. 1
H
NMR: d 5.84 (s, 2H), 7.41–8.03 (m, 8H), 11.30 (s, 1H); IR (Nujol) 3493, 3263,
1640, 1628 cmÀ1. Anal. Calcd for C15H11ClN2O: C, 66.55; H, 4.10; N, 10.35.
Found: C, 66.49; H, 4.12; N, 10.33.
22. Quezada, E.; Delogu, G.; Picciau, C.; Santana, L.; Podda, G.; Borges, F.; García-
Morales, V.; Viña, D.; Orallo, F. Molecules 2010, 15, 270.
23. Hilvo, M.; Tolvanen, M.; Clark, A.; Shen, B.; Shah, G. N.; Waheed, A.; Halmi, P.;
Hänninen, M.; Hämäläinen, J. M.; Vihinen, M.; Sly, W. S.; Parkkila, S. Biochem. J.
2005, 392, 83.