Synthesis of 3,4-dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives and evaluation of the carbonic anhydrase I and II inhibition

Article abstract of DOI:10.3109/14756366.2014.983917

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with some 3,4-dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives, were investigated by using the esterase assay, with 4-nitrophenyl acetate (4-NPA) as substrate. Compounds 10-13 showed K_I values in the range of 112.7-441.5 μM for hCA I and of 3.5-10.76 μM against hCA II, respectively. These hydroxyl group containing compounds generally were competitive inhibitors. Some hydroxyl group containing compounds investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

Full text of DOI:10.3109/14756366.2014.983917

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2015 Informa UK Ltd. DOI: 10.3109/14756366.2014.983917
!
RESEARCH ARTICLE
Synthesis of 3,4-dihydroxypyrrolidine-2,5-dione and
3,5-dihydroxybenzoic acid derivatives and evaluation of
the carbonic anhydrase I and II inhibition
3
4
5
6
2
Mehmet Arslan1,2, Murat S¸entu¨rk , Ismail Fidan , Oktay Talaz , Deniz Ekinci , Sedat Cos¸gun , and
_
Claudiu T. Supuran7
1Engineering Faculty, Department of Polymer Engineering, Yalova University, Yalova, Turkey, 2Science and Art Faculty, Chemistry Department, Fatih
3
University, Istanbul, Turkey, Science and Art Faculty, Chemistry Department, Agri Ibrahim C¸ec¸en University, Agri, Turkey, Chemistry Department,
4
_
˘
˘
5
Gebze Institute of Technology, Kocaeli, Turkey, Department of Chemistry, Karamanoglu Mehmetbey University, Karaman, Turkey, Faculty of
6
˘
7
Agriculture, Department of Agricultural Biotechnology, Ondokuz Mayıs University, Samsun, Turkey, and Polo Scientifico, Laboratorio di Chimica
`
Bioinorganica, Universita degli Studi di Firenze, Sesto Fiorentino, Florence, Italy
Abstract
Keywords
The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II,
with some 3,4-dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives,
were investigated by using the esterase assay, with 4-nitrophenyl acetate (4-NPA) as substrate.
Compounds 10–13 showed KI values in the range of 112.7–441.5 mM for hCA I and of
3.5–10.76 mM against hCA II, respectively. These hydroxyl group containing compounds
generally were competitive inhibitors. Some hydroxyl group containing compounds
investigated here showed effective hCA II inhibitory effects, in the same range as the clinically
used sulfonamide acetazolamide, and might be used as leads for generating enzyme
inhibitors possibly targeting other CA isoforms which have not been yet assayed for their
interactions with such agents.
Benzoic acid, carbonic anhydrase, enzyme
inhibitor, hydroxyl, pyrrolidine
History
Received 9 September 2014
Revised 30 October 2014
Accepted 30 October 2014
Published online 6 March 2015
Introduction
anti-tumor activity. Recently, N-substituted maleimides and
5-ylidene pyrrol-2(5H)-ones have received growing attention6–8
,
Carbonic anhydrase (CA, EC 4.2.1.1) enzymes are involved in
important physiological and pathological functions, such as pH
and CO2 homeostasis, respiration and transport of CO2=HCO3ꢀ
between metabolizing tissues and the lungs, ion secretion in
different tissues/organs and biosynthetic reactions (e.g. gluconeo-
genesis, lipogenesis and ureagenesis)1. CA isoforms are found in a
variety of tissues where they participate in several important
biological processes such as acid–base balance, respiration,
carbon dioxide and ion transport, bone resorption, ureagenesis,
gluconeogenesis, lipogenesis and electrolyte secretion. Many CA
isozymes involved in these processes are important therapeutic
targets with the potential to be inhibited/activated for the
treatment of a range of disorders such as edema, glaucoma,
due to their biological characteristics and potentials for organic
synthesis as key compounds for synthesis of bio-active molecules,
which are Oteromycin, Talaroconvolutin A, Azaspirene, Fusarin,
Clausenamide, and so on9,10
.
Our groups recently investigated the interaction of 12 mam-
malian CA isozymes with several types of phenolic compounds,
such as catechol, resorcinol, a series of phenols, a series of
phenolic acids and some other derivatives11–14. Here, we extend
these earlier investigations to series of phenolic compounds, some
of which are widely used as antioxidant food additives or as
drugs. Among the various natural or unnatural phenolic com-
pounds with antioxidant properties, these compounds are very
active in quenching reactive oxygen species13. They are reported
to possess anti-cancer, anti-carcinogenic, anti-mutagenic, anti-
bacterial, anti-viral or anti-inflammatory activities12,13. Phenol,
phenolic compounds and hydroxybenzoic acid derivatives are
widely used as prodrugs or drugs. Salicylic acid is known for its
ability to ease aches, pains, and reduce fevers. These medicinal
properties, particularly fever relief, have been known since
obesity, cancer, epilepsy and osteoporosis1–5
.
Pyrrolidine derivatives are widely used as prodrugs. Notably,
5-lactams such as pyrrol-2-one and its derivatives have illustrated
Address for correspondence: Murat S¸ entu¨rk, Science and Art Faculty,
_
Chemistry Department, Agri Ibrahim C¸ ec¸en University, 04100 Agri,
˘
Turkey. E-mail: senturkm36@gmail.com
˘
ancient times, and it was used as an anti-inflammatory drug12,13
.
Sedat Cos¸gun, Science and Art Faculty, Chemistry Department, Fatih
University, 34500 Istanbul, Turkey
In the present study, we have purified CA I and II (hCA I and
hCA II) from human erythrocytes and examined the in vitro
inhibition effects of above mentioned hydroxyl group containing
compounds on these enzymes, using the esterase activity of hCA I
and II, with 4-NPA as substrate.
`
Claudiu T. Supuran, Universita degli Studi di Firenze, Polo Scientifico,
Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy. E-mail: claudiu.supuran@
unifi.it

2
M. Arslan et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
Materials and methods
30 mmol, 3 eq.), (Benzotriazol-1-yloxy)tris(dimethylamino)phos-
phonium hexafluorophosphate (BOP) (4.42 g, 10 mmol, 1 eq.) and
benzylamine (1.07 g, 10 mmol, 1 eq.) were added. The mixture
Chemicals
The CNBr-activated Sepharose 4B, protein assay reagents, was stirred at room temperature for 6 h. The precipitate was
p-aminobenzene sulphonamide, L-tyrosine, 4-nitrophenylacetate filtered off and then washed with acetonitrile. The filtrate was
(4-NPA) and chemicals for electrophoresis were purchased from evaporated under vacuum, taken up with ethyl acetate then
Sigma-Aldrich Co. (St Louis, MO). All other chemicals were of washed with 20 ml of 2N HCl solution, 20 ml of saturated
analytical grade and obtained from either Sigma or Merck NaHCO3 solution and with 20 ml of a saturated solution of NaCl.
(Darmstadt, Germany). Reagents for syntheses of compounds 10– The product was purified by column chromatography on silica gel
13; L-(+)-tartaric acid, benzylamine, aniline, cyclohexylamine, eluting with mixture of ethyl acetate-hexane (2:1) to yield a white
3,5-dihydroxybenzoic acid and triethylamine were purchased solid (65%), m.p. 213–215 ꢁC. FT-IR (cmꢀ1): 3380, 3301, 3035,
1
from Aldrich and (benzotriazol-1-yloxy) tris (dimethylamino) 1655, 1536, 1154, 997. H-NMR (400 MHz, DMSO-d6): ꢀ 9.48
phosphonium hexafluorophosphate (BOP reagent) from Alfa (s, 2H, –OH), 8.83 (t, J ¼ 6.03 Hz, 1H, –NH), 7.33–7.19 (m, 5H,
Aesar (Ward Hill, MA). Other common chemicals and solvents ArH), 6.70(d, J ¼ 2.15 Hz, 2H, H2 and H6), 6.35 t, J ¼ 2.15 Hz,
are commercially available and were used after distillation or 1H, H4), 4.40(d, J ¼ 6.03 Hz, 2H, PhCH2). 13C-NMR (100 MHz,
treatment with drying agents. Progress of reactions was monitored DMSO-d6): ꢀ 166.5, 158.3, 139.9, 136.6, 128.2, 127.1, 126.6,
by thin layer chromatography (Merck, TLC Silica gel 60 F254) in a 105.4, 105.1, 42.5. ANAL. FOUND: C, 68.87; H, 5.64; N, 5.60. Calcd
suitable solvent system. Column chromatography was performed for C14H13NO3: C, 69.12; H, 5.39; N, 5.76.
on Merck Silica Gel 60 (70–230 mesh). Melting points were
determined with a Reichert thermovar micro melting point Synthesis of 3,5-dihydroxy-N-(cyclohexyl)benzamide (13)
apparatus and are uncorrected. Nuclear magnetic resonance
In a flask, 3,5-dihydroxybenzoic acid (1.54 g, 10 mmol, 1 eq.) was
(NMR) spectra were taken on a Bruker Ultra Shield Plus 400
dissolved in 20 ml of acetonitrile, and triethylamine (4.18 ml,
operating at 400 MHz for 1H- and 100 MHz for 13C-nuclei. NMR
30 mmol, 3 eq.), (benzotriazol-1-yloxy)tris(dimethylamino)phos-
spectra were recorded in deutorated dimethyl sulfoxide (DMSO-
phonium hexafluorophosphate (BOP) (4.42 g, 10 mmol, 1 eq.)
d6) solvent using TMS as internal standard and chemical shifts
and cyclohexylamine (0.99 g, 10 mmol, 1 eq.) were added.
referred to ꢀ were expressed in parts per million, ppm. FT-IR
The mixture was stirred at room temperature for 6 h and then
spectra were measured with a Perkin–Elmer BXII ATR spec-
the precipitate was filtered off by washing with acetonitrile. The
trometer. Elemental analyses were carried out by a Carlo Erba
filtrate was evaporated under vacuum and taken up with ethyl
1106 instrument.
acetate. Successive washings with 20 ml of a solution of 2N HCl,
20 ml of saturated NaHCO3 solution and 20 ml of a saturated
solution NaCl provided the crude. The product was purified by
Synthesis of (3R, 4R)-1-benzyl-3,4-dihydroxy-2,5-dioxopyrroli-
dine (10)
chromatography on silica (eluent: EtOAc/hexane: 2/1) to yield a
white solid (69%). Melting point 203–205 ꢁC. FT-IR (cmꢀ1):
Benzylamine (5.35 g, 0.05 mol) and L-(+)-tartaric acid (7.5 g,
3382, 3281, 1636, 1553, 1149, 858. 1H-NMR (400 MHz, DMSO-
0.05 mol) were refluxed in xylene overnight with a Dean-Stark
apparatus. The reaction mixture was cooled to ambient temperature
and resulting crystalline product was filtered off. After successive
d6): ꢀ 9.39 (s, 2H, –OH), 7.97 (t, J ¼ 8.02 Hz, 1H, –NH), 6.64
(d, J ¼ 2.14 Hz, 2H, H2 and H6), 6.32(t, J ¼ 2.10 Hz, 1H, H4),
1.76–1.04 (m, 11H, aliphatic hydrogens). 13C-NMR (100 MHz,
washing with portions of hexane, crude product was recrystallized
from ethanol to give product as pale yellow solid, 83%, m.p. 198–
DMSO-d6): ꢀ 165.6, 158.1, 137.1, 105.4, 104.8, 48.1, 32.3, 25.2,
200 ꢁC (200–201 ꢁC)15. FT-IR (cmꢀ1): 3189 br, 2885, 1710, 1452,
24.9. Anal. found: C, 66.81; H, 7.45; N, 5.87. Calcd for
C13H17NO3: C, 66.36; H, 7.28; N, 5.95.
1390, 1155, 1099, 1003. 1H-NMR (400 MHz, DMSO-d6): ꢀ 7.35–
7.23 (m, 5H, ArH), 6.30 (d, J ¼ 5.24 Hz, 2H, –OH), 4.55 (d,
J ¼ 7.22 Hz, 2H, –CH, H3 and H4), 4.38 (d, J ¼ 4.62 Hz, 2H,
CA purification assay
–PhCH2). 13C NMR (100 MHz, DMSO-d6): ꢀ 174.6, 135.9, 128.5,
127.5, 127.5, 74.5, 41.1. Anal. found: C, 59.51; H, 5.07; N, 6.27.
Purification of two human CA isozymes (hCA I and hCA II)
were previously described with a simple one-step method by a
Calcd for C11H11NO4: C, 59.73; H, 5.01; N, 6.33.
Sepharose-4Baniline-sulfanilamide affinity column chromatog-
17,18
Synthesis of (3R, 4R)-1-phenyl-3,4-dihydroxy-2,5-dioxopyrroli-
dine (11)
raphy
.
CA activity assay and kinetic studies
Freshly distilled aniline (4.65 g, 0.05 mol) and L-(+)-tartaric acid
(7.5 g, 0.05 mol) were refluxed in xylene with a Dean-Stark
apparatus. After overnight reaction, the resulting crystalline
product was filtered off and washed several times with hexane.
Recrystallization from ethanol provided white crystalline product
with 87% yield, m.p. 248–250 ꢁC (249–250 ꢁC).16 FT-IR (cmꢀ1):
3338 br, 3064, 1710, 1497, 1393, 1182, 1103, 999. 1H-NMR
(400 MHz, DMSO-d6): ꢀ 7.51–7.39 (m, 3H, ArH), 7.32–7.30 (m,
2H, ArH), 6.39 (dd, J ¼ 4.64 Hz and J ¼ 1.78 Hz, 2H, –OH), 4.57
(dd, J ¼ 4.69 Hz and J ¼ 1.65 Hz, 2H, –CH, H3 and H4).
13C-NMR (100 MHz, DMSO-d6): ꢀ 174.0, 132.0, 128.9, 128.3,
126.9, 74.4. Anal. found: C, 58.11; H, 5.51; N, 6.54. Calcd for
C10H9NO4: C, 57.97; H, 4.38; N, 6.76.
CA activity was assayed by following the change in absorbance at
348 nm of 4-NPA to 4-nitrophenolate ion over a period of 3 min at
25 ꢁC using a spectrophotometer (Shimadzu UV–VIS, Kyoto,
Japan) according to the method described by Verpoorte et al.19
The inhibitory effects of compounds 1–13 and AZA were
examined. All compounds were tested in triplicate at each
concentration used. Control cuvette activity in the absence of
inhibitor was taken as 100%. For each inhibitor an Activity
%ꢀ[Inhibitor] graph was drawn. To determine KI values, three
different inhibitor concentrations were tested; In these experi-
ments, 4-NPA was used as substrate at five different concentra-
tions (0.15–0.75 mM). The Lineweaver–Burk curves were
drawn20. Regression analysis graphs were drawn for IC50 using
inhibition % values by a statistical package (SPSS for windows,
version 10.0; SPSS Inc., Chicago, IL) on a computer (Student’s t-
test; n: 3).
Synthesis of 3,5-dihydroxy-N-(benzyl)benzamide (12)
3,5-dihydroxybenzoic acid (1.54 g, 10 mmol, 1 eq.) was dissolved
in 20 ml of acetonitrile. To solution, triethylamine (4.18 ml,

DOI: 10.3109/14756366.2014.983917
3,4-Dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives
3
O
HO
HO
HO
COOH
COOH
a
+
R-NH2
N
R
HO
O
10
11
R: phenyl
R: benzyl
OH
OH
b
+
R-NH2
H
N
OH
HO
HO
R
O
O
6
12 R: phenyl
13 R: cyclohexyl
Scheme 1. Reagents and conditions. (a) xylene reflux; (b) acetonitrile, rt, TEA, BOP reagent.
Table 1. hCA I and II inhibition data some compounds, by an esterase
assay with 4-NPA as substrate19
Results and discussion
.
The rationale of investigating phenols as CA inhibitors (CAIs) is
due to the fact that the simple phenol (PhOH) has been shown
to be the only competitive inhibitor with CO2 as substrate for the
main isoform of CA, i.e. human CA II (hCA II)14. The X-ray
crystal structure for the adduct of hCA II with phenol14. The
phenyl moiety of phenol was found to lay in the hydrophobic part
of the hCA II active site, where CO2, the physiologic substrate
of the CAs, binds in the precatalytic complex, explaining thus
the behavior of phenol as a unique CO2 competitive inhibitor.
Only recently, our groups investigated the interactions of some
simple phenols, salicylic acid derivatives, some antioxidant
phenolic compounds, some natural product polyphenols and
KI (mM)*
Compound
hCA I hCA II
Phenol (1)y
10.2
4003
795
5.5
9.91
7.7
Catechol (2)y
Resorcinol (3)y
Hydroquinone (4)y
Pyrogallol (5)y
10.7
7.41
0.55
37.3
14.6
36.2
0.09
0.54
0.51
23.1
0.51
0.37
8.0
3,5-Dihydroxybenzoic acid (6)y
1-Tosyl-1H-pyrrole-2(5H)-one (7)z
5-Hydroxy-1-tosyl-1H-pyrrole-2(5H)-one (8)z
Acetazolamide (9)
phenolic acids with all mammalian isozymes, CA I–XV11–14
,
(3R, 4R)-1-Benzyl-3,4-dihydroxy-2,5-dioxopyrrolidine (10) 133
(3R, 4R)-1-Phenyl-3,4-dihydroxy-2,5-dioxopyrrolidine (11) 112.7
3.5
evidencing some low micromolar/submicromolar inhibitors as
well as the possibility to design isozyme selective CAIs. Indeed,
the inhibition profile of various isozymes with this class of
agents is very variable, with inhibition constants ranging from
the millimolar to the submicromolar range for many simple
3,5-Dihydroxy-N-(benzyl)benzamide (12)
3,5-Dihydroxy-N-(cyclohexyl)benzamide (13)
441.5 10.76
392.2 9.48
*Mean from at least three determinations. Errors in the range of 3–8% of
the reported value (data not shown).
yFrom Ref 24.
phenols11–13
.
zFrom Ref 25.
(3R, 4R)-3,4-dihydroxy-2,5-dioxopyrrolidine derivatives (10
and 11) were synthesized15,16 by the reaction of L-(+)-tartaric
acid with primary amines. Benzamide structures (12 and 13) were competitive inhibitors with 4-NPA as substrate, i.e. they bind in
established (Scheme 1) by coupling 3,5-dihydroxybenzoic acid different regions of the active site cavity as compared to the
with primary amines using BOP (benzotriazol-1-yloxy-tris substrate. However, the binding site of 4-NPA itself is unknown,
(dimethylamino)-phosphoniumhexafluorophosphate) as coupling but it is presumed to be in the same region as that of CO2, the
reagent. Hydroxyl groups of benzoic acid were not protected since physiological substrate of this enzyme11–14
.
(ii) A better inhibitory activity has been observed with
compounds 10–13 investigated here for the inhibition of the
BOP coupling is safely mediated in the presence of unprotected
alkyl and phenolic hydroxyl groups in many examples21–23
.
We report here the first study on the inhibitory effects of rapid cytosolic isozyme hCA II (Table 1). Structure–activity
compounds 10–13 on the esterase activity of hCA I and II. The relationship (SAR) is thus quite sharp for this small series of
previous reports by Innocenti et al.11 investigated other phenol hydroxylic compounds: the 2,5-dioxopyrrolidine containing com-
derivatives (including catechol and resorcinol) by using a stopped pounds 10 and 11 are ineffective leads, with two 3, 5 dihidroxy
flow, CO2 hydration assay for monitoring CA inhibition. Data of moieties is already a submicromolar hCA II inhibitor. This trend
Table 1 show the following regarding inhibition of hCA I and II is maintained when different groups are present in the meta
with compounds 10–13, by an esterase assay19, with 4-NPA as position to the phenol OH moiety, such as in resorcinol. The best
substrate:
hCA II inhibitor in this series of derivatives were compounds 10
(i) Against the slow cytosolic isozyme hCA I, compounds and 11, which with a KI of 8.0–3.5 mM. It must be stressed that
10–13 behave as good inhibitors (Figure 1), with KI values in the KIs measured with the esterase method are always in the
range of 112.7–441.5 mM similarly to the structurally related micromolar range because hCA I and II are weak esterases.24–27
compounds 1–8 and acetazolamide (AZA) (KIs of 0.55–
In a recent study, it was reported that catechol and resorcinol 26
4003 mM). It is interesting to note that the compounds 10 and a simple compound lack of the sulfonamide, sulfamate, or related
11 were much better hCA I inhibitors as compared to the functional groups which are typically found in all known CAIs –
corresponding compounds 12 and 13 from which they were act as a CAI inhibitor, and could represent the starting point for a
prepared. Kinetic investigations (Lineweaver–Burk plots, data not new class of inhibitors that may have advantages for patients with
shown) indicate that similarly to sulfonamides and inorganic sulfonamide allergies24–30. The sulfonamide zinc-binding group is
anions3–5,17–20, all the investigated natural compounds act as thus superior to the thiol one (from the thioxolone hydrolysis

4
M. Arslan et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
OH
OH
HO
HO
HO
OH
HO
HO
HO
HO
1
2
3
4
5
OH
O
O
O
S
H
S
O
S
N
N
SO NH
2 2
O
HO
N
O
N
N
OH
O
OH
OH
O
6
7
8
9
OH
O
N
O
HO
HO
HO
H
H
N
N
N
HO
HO
HO
O
O
O
O
10
11
12
13
Figure 1. Structure of tested compounds.
5. Cavdar H, Ekinci D, Talaz O, et al. a-Carbonic anhydrases are
sulfatases with cyclic diol monosulfate esters. J Enzyme Inhib Med
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5-hydroxy-4-(alkylamino)-5-phenyl-1-(arylsulfonyl)-1,5-dihydro-
2H-pyrrol-2-ones via multicomponent reaction. Helv Chim Acta
2007;90:2414–20.
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din-2-one ring systems. J Org Chem 2004;69:8952–5.
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11. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors: Interactions of phenols with the 12 catalytic-
ally active mammalian isoforms (CA I-XIV). Bioorg Med Chem
Lett 2008;18:1583–7.
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product) for generating CAIs with a varied and sometimes
isozyme-selective inhibition profile against the mammalian
enzymes. However, it is critically important to explore further
classes of potent CAIs in order to detect compounds with a
different inhibition profile as compared to the sulfonamides and
their bioisosteres and to find novel applications for the inhibitors
of these widespread enzymes.
Conclusion
Compounds 10–13 used in this study affect the activity of CA
isozymes due to the presence of the different functional groups
(OH, phenyl, benzyl, and cyclohexyl) present in their aromatic
scaffold. Our findings here indicate thus another class of possible
CAIs of interest, in addition to the well-known sulfonamides/
sulfamates/sulfamides, the phenols/biphenyl diphenols bearing
bulky ortho moieties in their molecules. Indeed, some hyroxylic
compounds investigated here showed effective hCA I and II
inhibitory activity, in the low micromolar range, by the esterase
method which usually gives KI-s an order of magnitude higher as
compared to the CO2 hydrase assay30–45. These findings point out
that substituted hydroxylic compounds may be used as leads for
generating potent CAIs eventually targeting other isoforms which
have not been assayed yet for their interactions with such agents.
13. Sentu¨rk M, Gu¨lc¸in I, Dastan A, et al. Carbonic anhydrase inhibitors.
Inhibition of human erythrocyte isozymes I and II with a series of
antioxidant phenols. Bioorg Med Chem 2009;17:3207–11.
14. Supuran CT. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett
2010;20:3467–74.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the article.
15. Gonsalves AMAR, Serra MES, Murtinho D, et al. Pyrrolidine-based
amino alcohols: novel ligands for the enentioselective alkylation of
benzaldehyde. J Molec Cat A: Chem 2003;195:1–9.
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