Hit identification and biological evaluation of anticancer pyrazolopyrimidines endowed with anti-inflammatory activity

Article abstract of DOI:10.1002/cmdc.201000165

(Chemical Equation Presented) Inhibiting COX: A small library of pyrazolopyriminines endowed with antiproliferative action was submitted to virtual screening against two COX isoforms. Three compounds were identified in silico as potentially selective COX-

Full text of DOI:10.1002/cmdc.201000165

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DOI: 10.1002/cmdc.201000165
Hit Identification and Biological Evaluation of Anticancer
Pyrazolopyrimidines Endowed with Anti-inflammatory Activity
Stefano Alcaro,*[a] Anna Artese,[a] Maurizio Botta,[b] Alessandra T. Zizzari,[b] Francisco Orallo,[c]
Francesco Ortuso,[a] Silvia Schenone,[d] Chiara Brullo,[d] and Matilde YꢀÇez[c]
This paper is dedicated to the memory of Prof. Francisco Orallo, who died unexpectedly during the preparation of this manuscript.
In recent years, several studies have proposed the cyclo-oxy-
genases (COXs) as therapeutic targets in the prevention of
cancer.[1] Some epidemiological investigations highlighted that
nonsteroidal anti-inflammatory drugs (NSAIDs) reduced the de-
velopment of several malignant diseases, including colorectal
cancer.[2,3,4] NSAIDs inhibit COXs and consequently decrease
the levels of prostaglandins (PG) that are significantly involved
in carcinogenesis.[5,6]
fied by indometacin, diclofenac, nimesulide, DuP697, SC-588
(shown).
In fact, COX (also known as prostaglandin G/H synthase) is a
heme-containing enzyme that catalyzes the conversion of
arachidonic acid (AA) into the hydroperoxide prostaglandin G2
(PGG2) and its subsequent reduction to prostaglandin H2
(PGH2). PGH2 is a precursor of several classes of eicosanoids,
which modulate central physiological functions, such as the in-
flammatory response, platelet aggregation, and the protection
of gastric mucosa.
Two main COX isoforms are known: the constitutive isoform
COX-1 and the inducible isoform COX-2. They show similar cat-
alytic activity and share 65% amino acid sequence homology.
COX-1 is involved in regulating vascular tone, and it is mainly
present in the gastrointestinal tract and platelets. Inhibition of
COX-1 by NSAIDs is believed to cause the adverse side effects
associated with these drugs. Conversely, the COX-2 isoform is
normally absent in healthy tissues, but it is rapidly induced in
response to inflammatory mediators, such as bacterial endo-
toxin, interleukin 1 and various growth factors.
COX-2 can be selectively inhibited despite the very similar
structures of the active sites of both proteins. A single muta-
tion of Ile in COX-1 with Val in the COX-2 binding site creates
an additional pocket that is hydrophobic in nature.[7]
The COX active site is a narrow cleft, approximately 8 ꢁ wide
and 25 ꢁ long. NSAIDs, with the exception of the acetylsalicilic
acid, show reversible and competitive COX inhibition. Several
scaffolds are able to exert selective COX inhibition, as exempli-
In tumor cells, high levels of PGE2 have been highlighted in
a number of experimental and clinical studies.[8] COX-2 has
been shown to be overexpressed in various cancer types, such
as head, neck, breast, skin, lung, bladder, tongue, colorectal,
stomach, and prostate tumors.[9] Evidence that COX-2 inhibi-
tion can prevent these types of cancer has been reported in
the literature.[10–13]
[a] Prof. S. Alcaro, Dr. A. Artese, Dr. F. Ortuso
Dipartimento di Scienze Farmacobiologiche
Universitꢀ degli Studi “Magna Græcia” di Catanzaro
Complesso Ninꢁ Barbieri, 88021 Roccelletta di Borgia, Catanzaro (Italy)
Fax: (+39)0961-391490
In a recent study, a dimethylamino pyrazolopyrimidine deriv-
ative (DPP) was identified as a potent inhibitor of PGE2 produc-
tion.[14] A further study confirmed the effect of DPP derivatives
in vivo using a 24 h zymosan-injected mouse air pouch
model.[15] Moreover, DPP was found to exert acute anti-inflam-
matory, analgesic and antiangiogenic effects that may be asso-
ciated with COX-2 inhibition. DPP derivatives showed inhibito-
ry activity comparable to that of the reference compound,
NS398, a potent selective COX-2 inhibitor.[15]
E-mail: alcaro@unicz.it
[b] Prof. M. Botta, Dr. A. T. Zizzari
Dipartimento Farmaco Chimico Tecnologico, Universitꢀ degli Studi di Siena
Via Alcide de Gasperi 2, 53100 Siena (Italy)
[c] Prof. F. Orallo, Dr. M. YꢂÇez
Departamento de Farmacologꢃa and Instituto de Farmacia Industrial
Universidad de Santiago de Compostela, Campus Universitario Sur
15782 Santiago de Compostela (Spain)
In studies previously reported by us, novel 4-amino-substi-
tuted pyrazolopyrimidines were shown to reduce epidermal
growth factor (EGF)-stimulated Src activation, causing a de-
crease in proliferation of A431 epidermoid tumor cells and
8701-BC breast cancer cells.[16–18] These derivatives, by reducing
[d] Prof. S. Schenone, Dr. C. Brullo
Dipartimento di Scienze Farmaceutiche Universitꢀ degli Studi di Genova
Viale Benedetto XV 3, 16132 Genova (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000165.
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ChemMedChem 2010, 5, 1242 – 1246

the production of the vascular endothelial growth factor
(VEGF) released by tumor cells, also showed a highly selective
antiangiogenic effect.[19] Moreover, some of them showed a
significant antiproliferative effect on osteosarcoma cells and
are active in a xenograft mouse model of this tumor.[20]
the 2-chloro-2-phenylethyl substituent of the enantiomers of 1
docked in the same regions as the pyrazole and p-bromophen-
yl moieties of SC-588, respectively. Moreover, in both cases the
propylamino group occupied the same trifluoromethyl recog-
nition region of SC-588 (Figure 1). Inversion of the asymmetric
carbon only seemed to affect the relative position of the pyra-
zolopyrimidine scaffold within the binding pocket, maintaining
the two extreme phenyl and propyl moieties in the same loca-
tions.
On the basis of these reports, and considering the great in-
terest in antitumor agents acting with a dual mechanism (i.e.,
tyrosine kinase inhibition/anti-inflammatory action), we decid-
ed to investigate the potential anti-inflammatory activity of
our pyrazolopyrimidines. With this aim, a three-dimensional
chemical library containing 423 pyrazolo- and four pyrrolopyri-
midines, variously substituted, has been designed and
screened in silico against structural models of both COX-1 and
COX-2. The compounds in our library possess a wide range of
molecular weights (277.32–529.49), and 402 derivatives contain
at least one asymmetric carbon atom. Enantiomers and diaste-
reoisomers have been taken into account considering all possi-
ble configurations of their asymmetric centers. In total, 427
compounds were modeled.
All of the compounds were evaluated using Instant JChem
(v. 1.0)[21] to predict their Log P; values ranged from 1.04 to
7.54. As reported previously,[22] the virtual screening tool
Glide[23] was used to predict the binding of our compounds to
COX-1 and COX-2 obtained from Protein Data Bank (PDB).[24]
The crystal structures used for COX-1 and COX-2 were PBD ID:
1Q4G[25] and 1PXX,[26] respectively. The computed ligand–
enzyme interaction energies were considered for ranking the
theoretical affinity of our molecules with respect to both tar-
gets. The highest ranked COX-2 compounds, reporting no in-
teraction with the COX-1 enzymatic cleft, were selected for ex-
perimental evaluation (Table 1).
Table 1. Structures of compounds 1–3 predicted to be the most selective
COX-2 inhibitors together with their enzymatic (Ki) and cell data (IC50).
Figure 1. The best predicted docking poses of a) (R)-1 and b) (S)-1 (white
carbon sticks) superimposed on that of the reference COX inhibitor SC588
(gray carbon sticks), modeled in the COX-2 binding site (PDB ID: 1PXX). The
binding site is shown as a surface.
Compd
R
Ki [mm]
IC50[a] [mm]
Src
Abl
A431
8701-BC
Compound 2, the previously reported anticancer agent,[16–20]
and the novel parent compound 3, were also well-ranked, with
larger differences in the predicted binding affinities of the
enantiomers. In particular, compound 3 appeared as interest-
ing as 1 due to their similar binding modes. In fact, both enan-
tiomers of compound 1 and the (S)-enantiomer of 3 were pre-
dicted to bind with the propylamino substituent oriented simi-
larly to the trifluoromethyl moiety of SC-588, and the R group
in the same region as the phenylsulfonamide group of SC-588
(data not shown). Since we were mainly interested in the hit
identification of dual action compounds, we selected 1–3 for
biological evaluation.
1
2
3
N(CH3)2
S-C2H5
S-iC3H7
4.10Æ0.5
0.70Æ0.1
0.52Æ0.1
0.32Æ0.04
0.41Æ0.03
0.65Æ0.2
20.0Æ0.9
79.1Æ1.8
13.2Æ0.7
12.4Æ0.6
29.5Æ0.9
18.1Æ0.9
[a] A431 epidermoid tumor cells and 8701-BC breast cancer cells. Ki
values are the means SD of three independent experiments. IC50
Æ
values are the means Æ SEM of four independent experiments.
The binding of the selected compounds to the COX-2 active
site was compared by superimposition using the co-crystal
structure of the known, selective inhibitor, SC-588 bound to
COX-2 (PDB ID: 6COX).[27] The docking experiments suggested
to consider especially 1, since both its enantiomers were able
to fit into the COX-2 binding clefts with comparable theoretical
affinities. In both cases, the pyrazolopyrimidine scaffold and
Moreover, among our ligands, only the enantiomers of deriv-
ative 1 were predicted to establish a hydrogen bond with the
enzyme through the Ser530 residue of COX-2. This may ex-
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the eluant. The enantiomeric
excess was then determined by
analytical Chiracel OD column
using an UV detector. The abso-
lute stereochemistry of the
chiral center on the side chain
of compounds 1, 2 and 3 was
established via CD spectra com-
parison with those of com-
pound
4 of known absolute
configuration, obtained through
X-ray crystal structure,[31] and
confirmed with that of a very
similar compound 5 reported by
Da Settimo and co-workers.[32]
The structure of compound 5
and CD spectra analyses are re-
ported in the Supporting Infor-
mation. Stereogenic assignment
demonstrated that the (À) and
(+) isomers of compounds 1–3
correspond to the R and S abso-
lute configurations, respectively.
Figure 2. Predicted recognition of the COX-2 active site by (S)-1. The ligand (white carbon sticks) is shown inter-
acting with key residues in the active site (gray carbon sticks); the heme cofactor is shown in slate spacefill
model, and the rest of the enzyme is in transparent gray cartoon. The dotted line indicates a hydrogen bond.
plain the better affinity of compound 1 for COX-2 compared
with the other studied compounds. Figure 2 shows the pre-
dicted, most stable binding mode of the (S)-1–COX-2 complex.
The synthesis of compounds 1 and 2 was previously report-
ed.[28,29] Compound 3 was prepared as shown in Scheme 1,
starting from intermediate 6.[30] Synthetic details regarding the
preparation of compound 3 are reported in the Supporting In-
formation.
The biological tests were carried out using a chromogenic
assay based on the oxidation of N,N,N’,N’-tetramethyl-p-phenyl-
enediamine (TMPD) during the reduction of PGG2 to PGH2. The
enzyme activity was measured by estimation of the initial reac-
tion rate as followed by the increase in absorbency at 600 nm.
Control experiments using H2O2 (0.4 mm) as the substrate
showed that inhibitors 1–3 did not affect the peroxidase activi-
ty of COX under the assay conditions. The reference COX in-
hibitors were preincubated at 378C for 5 min before initiation
of the enzymatic reaction by the addition of 1 mm arachidonic
acid. Our compounds were submitted to the same analysis
using pure enantiomers after stereogenic assignment (Support-
ing Information). The results are reported in Table 2.
Among the known COX inhibitors, DuP697 resulted the
most potent and selective against COX-2 with inhibitory activi-
ty in the nanomolar range. As reported in Table 2, our com-
pounds exhibited significant COX-2 selectivity, with inhibitory
activity in the micromolar range. In particular, both enantio-
mers of 1 were more potent than the reference inhibitors indo-
metacin and diclofenac. It is worth noting that the inhibitory
activities for these two reference compounds were recalculated
by us since no convergence to similar IC50 values were found
in the literature. Compound 2 showed inhibitory activity com-
Scheme 1. Synthesis of compound 3. Reagents and conditions: a) (CH3)2CHBr,
K2CO3, DMF, RT, 8 h, 57% yield; b) POCl3/DMF, CHCl3, reflux, 8 h, 74% yield;
c) n-propylamine, anhyd toluene, RT, 48 h, 97% yield.
A direct method for the enantiomeric separation was used
to obtain sizeable amounts of the individual enantiomers of
compounds 1–3. Separation was performed on a semiprepara-
tive Chiralpak AS column, using n-hexane and 2-propanol as
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Table 2. IC50 values and COX-1 selectivity ratio obtained using the pure
enantiomers of compounds 1–3 with respect to reference compounds.
Keywords: COX · dual activity · enantiomeric separation ·
pyrazolopyrimidines · virtual screening
Compd
IC50 values[a] [mm]
Selectivity
ratio[b]
COX-1[d]
COX-2
Indometacin[c]
Diclofenac
Nimesulide
DuP697[c]
(R)-(À)-1
12.16Æ1.16
35.20Æ1.41
23.62Æ1.97
231.40Æ19.84
0.12632Æ0.00741
21.60Æ1.18
20.35Æ1.87
36.73Æ3.16
30.85Æ2.79
65.26Æ2.41
66.48Æ5.30
2.9
1.3
[1] M. J. Thun, M. M. Namboodiri, C. W. Heath Jr. , N. Engl. J. Med. 1991,
325, 1593–1596.
18.23Æ1.73
<0.46[e]
0.0056
<0.22[d]
<0.20[e]
<0.37[e]
<0.31[e]
<0.65[e]
<0.66[e]
[2] D. M. Schreinemachers, R. B. Everson, Epidemiology 1994, 5, 138–146.
[3] D. C. Farrow, T. L. Vaughan, P. D. Hansten, J. L. Stanford, H. A. Risch,
M. D. Gammon, W. H. Chow, R. Dubrow, H. Ahsan, S. T. Mayne, J. B.
Schoenberg, A. B. West, H. Rotterdam, J. F. Fraumeni Jr. , W. J. Blot,
Cancer Epidemiol., Biomarkers Prev. 1998, 7, 97–102.
[4] M. J. Langman, K. K. Cheng, E. A. Gilman, R. J. Lancashire, BMJ 2000,
320, 1642–1646.
[5] P. Ben-Av, L. J. Crofford, R. L. Wilder, T. Hla, FEBS Lett. 1995, 372, 83–87.
[6] J. L. Masferrer, K. M. Leahy, A. T. Koki, B. S. Zweifel, S. L. Settle, B. M.
Woerner, D. A. Edwards, A. G. Flickinger, R. J. Moore, K. Seibert, Cancer
Res. 2000, 60, 1306–1311.
[7] R. M. Garavito, M. G. Malkowski, D. L. DeWitt, Prostaglandins Other Lipid
Mediators 2002, 68–69, 129–152.
[8] A. Raz, Biochem Pharm. 2002, 63, 343–347.
[9] A. J. Dannenberg, N. K. Altorki, J. O. Boyle, C. Dang, L. R. Howe, B. B.
Weksler, K. Subbaramaiah, Lancet Oncol. 2001, 2, 544–551.
[10] R. E. Harris, J. Beebe-Donk, G. A. Alshafie, BMC Cancer 2008, 8, 237.
[11] Y. Dai, W. H. Wang, World J. Gastroenterol. 2006, 12, 2884–2889.
[12] L. Gallicchio, K. Visvanathan, A. Burke, S. C. Hoffman, K. J. Helzlsouer, Int.
J. Cancer 2007, 121, 211–215.
[13] S. Jain, G. Chakraborty, R. Raja, S. Kale, G. C. Kundu, Cancer Res. 2008,
68, 7750–7759.
[14] J. M. Quintela, C. Peinador, L. Gonzꢃlez, I. Devesa, M. L. Ferrꢃndiz, M. J.
Alcaraz, R. Riguera, Bioorg. Med. Chem. 2003, 11, 863–868.
[15] I. Devesa, M. J. Alcaraz, R. Riguera, M. L. Ferrꢃndiz, Eur. J. Pharmacol.
2004, 488, 225–230.
[16] F. Carraro, A. Pucci, A. Naldini, S. Schenone, O. Bruno, A. Ranise, F. Bon-
davalli, C. Brullo, P. Fossa, G. Menozzi, L. Mosti, F. Manetti, M. Botta, J.
Med. Chem. 2004, 47, 1595–1598.
[17] S. Schenone, O. Bruno, A. Ranise, F. Bondavalli, C. Brullo, P. Fossa, L.
Mosti, G. Menozzi, F. Carraro, A. Naldini, C. Bernini, F. Manetti, M. Botta,
Bioorg. Med. Chem. Lett. 2004, 14, 2511–2517.
[18] S. Schenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P.
Fossa, S. Donnini, A. Santoro, M. Ziche, F. Manetti, M. Botta, Eur. J. Med.
Chem. 2004, 39, 939–946.
[19] S. Donnini, M. Monti, C. Castagnini, R. Solito, M. Botta, S. Schenone, A.
Giachetti, M. Ziche, Int. J. Cancer 2007, 120, 995–1004.
[20] F. Manetti, A. Santucci, G. A. Locatelli, G. Maga, A. Sreafico, T. Serchi, M.
Orlandini, G. Bernardini, N. P. Caradonna, A. Spallarossa, C. Brullo, S.
Schenone, O. Bruno, A. Ranise, F. Bondavalli, O. Hoffmann, M. Bologna,
A. Angelucci, M. Botta, J. Med. Chem. 2007, 50, 5579–5588.
[21] a) Instant JChem, version 1.0 (ChemAxon Ltd); Full product details and
a user’s guide can be found here: http://www.chemaxon.com/prod-
ucts/instant-jchem/; b) V. N. Viswanadhan, A. K. Ghose, G. R. Revankar,
R. K. Robins, J. Chem. Inf. Comput. Sci. 1989, 29, 163–172; c) G. Klop-
man, J.-Y. Li, S. Wang, M. Dimayuga, J. Chem. Inf. Comput. Sci. 1994, 34,
752–781; d) F. Csizmadia, A. Tsantili-Kakoulidou, I. Panderi, F. Darvas, J.
Pharm. Sci. 1997, 86, 865–871; e) G. Bouchard, P. A. Carrupt, B. Testa, V.
Gobry, H. H. Girault, Pharm. Res. 2001, 18, 702–708.
***
22.61Æ1.56
**
**
**
**
**
**
(S)-(+)-1
(R)-(À)-2
(S)-(+)-2
(R)-(À)-3
(S)-(+)-3
[a] All IC50 values are the mean ÆSEM (n=5). [b] COX-1 ratio=[IC50 (COX-
2)]/[IC50 (COX-1)]. [c] Level of statistical significance: P<0.01 (for indome-
tacin) and P<0.05 (for DuP697) versus the corresponding IC50 values ob-
tained against COX-2, as determined by ANOVA/Dunnett’s test. [d] , in-
**
active at 100 mm (highest concentration tested);
(highest concentration tested). [e] Values obtained under the assumption
that the corresponding IC50 against COX-1 or COX-2 is the highest con-
centration tested.
, inactive at 500 mm
***
parable to indometacin, but with improved COX-2 selectivity.
Despite the binding modes predicted by the docking experi-
ments, both enantiomers of compound 3 showed moderate
COX-2 inhibition and selectivity.
In conclusion, we have demonstrated that antiproliferative
pyrazolopyrimidines can exert a dual activity, with anti-inflam-
matory effects comparable to known COX inhibitors. Even
through the anti-inflammatory potency of these compounds
was not as high as the known COX-2-selective inhibitor DuP
697, compound 1 revealed interesting COX-2 activity and se-
lectivity compared with the other three reference drugs. This
aspect, together with the sub-micromolar activity of all three
compounds against cytoplasmic tyrosine kinases Src and Abl,
highlights the potential of this class of molecules as dual anti-
inflammatory/tyrosine kinase inhibitors that could represent a
therapeutic opportunity for the prevention and treatment of
cancer.
Experimental Section
The Supporting Information contains full experimental details for
the docking simulations, synthesis, enantiomeric separation and
biological evaluation.
[22] A. Procopio, S. Alcaro, M. Nardi, M. Oliverio, F. Ortuso, P. Sacchetta, D.
Pieragostino, G. Sindona, J. Agric. Food Chem. 2009, 57, 11161–11167.
[23] a) Glide: A complete solution for ligand–receptor binding (Schrçdinger,
LLC); Full product details and a list of publications can be found here:
http://www.schrodinger.com/products/14/5/ (Last accessed: June 02,
2010); b) M. D. Eldridge, C. W. Murray, T. R. Auton, G. V. Paolini, R. P. Mee,
J. Comput.-Aided Mol. Des. 1997, 11, 425–445.
[24] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig,
I. N. Shindyalov, P. E. Bourne, Nucl. Acids Res. 2000, 28, 235–242.
[25] K. Gupta, B. S. Selinsky, C. J. Kaub, A. K. Katz, P. J. Loll, J. Mol. Biol. 2004,
335, 503–518.
Acknowledgements
This research was supported by grants from the Ministero dell’Is-
truzione, dell’Universitꢀ
e della Ricerca (MIUR), Italy (PRIN
2007 JERJPC), the Ministerio de Sanidad y Consumo, Spain (FISS
PI061537), and the Consellerꢃa de Innovaciꢄn e Industria de la
Xunta de Galicia, Spain (INCITE07PXI203039ES, INCI-
TE08E1R203054ES and 08CSA019203PR). Dr. Matilde YꢂÇez is a
researcher of the Angeles AlvariÇo program (Xunta de Galicia,
Spain).
[26] S. W. Rowlinson, J. R. Kiefer, J. J. Prusakiewicz, J. L. Pawlitz, K. R. Kozak,
A. S. Kalgutkar, W. C. Stallings, R. G. Kurumbail, L. J. Marnett, J. Biol.
Chem. 2003, 278, 45763–45769.
ChemMedChem 2010, 5, 1242 – 1246
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1245

MED
[27] R. G. Kurumbail, A. M. Stevens, J. K. Gierse, J. J. McDonald, R. A. Stege-
man, J. Y. Pak, D. Gildehaus, J. M. Miyashiro, T. D. Penning, K. Seibert,
P. C. Isakson, W. C. Stallings, Nature 1996, 384, 644–648.
[28] S. Schenone, C. Brullo, O. Bruno, F. Bondavalli, L. Mosti, G. Maga, E. Cre-
span, F. Carraro, F. Manetti, C. Tintori, M. Botta, Eur. J. Med. Chem. 2008,
43, 2665–2676.
[30] S. Schenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P.
Fossa, F. Manetti, L. Morbidelli, L. Trincavelli, C. Martini, A. Lucacchini,
Eur. J. Med. Chem. 2004, 39, 153–160.
[31] M. Botta, unpublished results.
[32] F. Da Settimo, G. Primofiore, C. La Motta, S. Taliani, F. Simorini, A. M.
Marini, L. Mugnaini, A. Lavecchia, E. Novellino, D. Tuscano, C. Martini, J.
Med. Chem. 2005, 48, 5162–5174.
[29] F. Carraro, F. Naldini, A. Pucci, G. A. Locatelli, G. Maga, S. Schenone, O.
Bruno, A. Ranise, F. Bondavalli, C. Brullo, P. Fossa, G. Menozzi, L. Mosti,
M. Modugno, C. Tintori, F. Manetti, M. Botta, J. Med. Chem. 2006, 49,
1549–1561.
Received: April 18, 2010
Revised: June 1, 2010
Published online on June 16, 2010
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ChemMedChem 2010, 5, 1242 – 1246