An expeditious access to 5-pyrimidinol derivatives from cyclic methylglyoxal diadducts, formation of argpyrimidines under physiological conditions and discovery of new CFTR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.02.037

In the study of previously reported modulators of CFTR chloride channels that are cyclic methylglyoxal (MG) diadducts (CMGD) to aromatic α-aminoazaheterocycles, we optimized a new expeditious one pot route for preparing in water novel aromatic polycyclic azaheterocycles and described 5-pyrimidinols antioxidants through the formation of 2-oxoaldehyde diadducts to aromatic α-aminoazaheterocycles, amidines, guanidines and thiourea. In regard to the importance as biomarkers of diabetic complications of the 5-pyrimidinols "argpyrimidines" formed in proteins from MG and arginine residues, we demonstrated that argpyrimidines are slowly formed under physiological conditions from CMGD to arginine derivatives according to the synthesis route described. Among the 5-pyrimidinol derivatives prepared, two polycyclic derivatives appeared to inhibit strongly the activity of CFTR channels in wt-CHO cells.

Full text of DOI:10.1016/j.ejmech.2011.02.037

European Journal of Medicinal Chemistry 46 (2011) 1935e1941
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
An expeditious access to 5-pyrimidinol derivatives from cyclic methylglyoxal
diadducts, formation of argpyrimidines under physiological conditions and
discovery of new CFTR inhibitors
Brice-Loïc Renard a, Benjamin Boucherle a, Bruno Maurin a, Marie-Carmen Molina a, Caroline Norez b,
,
Frédéric Becq b, Jean-Luc Décout a
*
a University Joseph Fourier-Grenoble 1/CNRS, UMR 5063, Département de Pharmacochimie Moléculaire, ICMG FR 2607, Bât. E 470 rue de la Chimie,
BP 53 F-38041, Grenoble Cedex 9, France
b University of Poitiers/CNRS, UMR 6187, Institut de Physiologie et Biologie Cellulaires, F-86022 Poitiers, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In the study of previously reported modulators of CFTR chloride channels that are cyclic methylglyoxal
Received 10 November 2010
Received in revised form
14 February 2011
Accepted 16 February 2011
Available online 23 February 2011
(MG) diadducts (CMGD) to aromatic
route for preparing in water novel aromatic polycyclic azaheterocycles and described 5-pyrimidinols
antioxidants through the formation of 2-oxoaldehyde diadducts to aromatic -aminoazaheterocycles,
a-aminoazaheterocycles, we optimized a new expeditious one pot
a
amidines, guanidines and thiourea. In regard to the importance as biomarkers of diabetic complications
of the 5-pyrimidinols “argpyrimidines” formed in proteins from MG and arginine residues, we demon-
strated that argpyrimidines are slowly formed under physiological conditions from CMGD to arginine
derivatives according to the synthesis route described. Among the 5-pyrimidinol derivatives prepared,
two polycyclic derivatives appeared to inhibit strongly the activity of CFTR channels in wt-CHO cells.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Methylglyoxal
2-Oxoaldehydes
Argpyrimidine
Arginine
Antioxidants
5-Pyrimidinols
CFTR
1. Introduction
proteins like Cystic Fibrosis which is the most common lethal
autosomal recessive genetic disease in Caucasians [5], myotonia,
Previously, we have reported the remarkable stereoselective
diaddition of methylglyoxal (MG) to aromatic -aminoazahetero-
Bartler’s and Dent’s diseases and osteopetrosis, or due to loss of
regulation of CFTR activity (secretory diarrheas, cholera) [4].
In the study of the reactions of the CFTR modulators that we
have previously identified, here, we report on the remarkable
conversion in one pot in water of such CMGD to polycyclic 5-pyr-
imidinol derivatives. This conversion was extended to amidines and
guanidines derivatives and thiourea and, was related to the in vivo
formation of the fluorescent 5-pyrimidinols named argpyrimidines
(ArgPy) involved in diabete complications and formed from MG
and proteins carrying arginine residues. Among the 5-pyrimidinol
derivatives prepared, two polycyclic derivatives were found to be
strong inhibitors of the CFTR activity.
a
cycles, like, 2-aminopyridine (2AP), adenine, adenine nucleosides
and polyadenylic acid [1]. This reaction leads to cyclic methylglyoxal
diadducts (CMGD) which are highly functionnalized heterocycles
(for example CMGD to 2AP 1a and 1b, Fig. 1). More recently, we
showed that the CMGD to 20-deoxyadenosine and 1-amino-
isoquinoline (1IQ) 2a and 3a-b, respectively, are potent inhibitors of
wild-type Cystic Fibrosis Transmembrane conductance Regulator
(CFTR) chloride channels and CFTR affected by CF-related mutations
[2,3]. The 9-propyladenine CMGD 4a (Fig. 1) also were able to acti-
vate in vivo CFTR at micromolar concentrations [2,4].
Searching for potent and specific small molecules able to
modulate Clꢀ channels is crucial to understand their physiological
role in cell functions and also for the development of molecules of
therapeutic interest for diseases caused by mutations of these
2. Results and discussion
2.1. Chemistry
In the aim to identify the common pharmacophore present in
the prepared modulators of CFTR channels, it appeared interesting
* Corresponding author. Tel.: þ33 4 7663 5317; fax: þ33 4763 5298.
E-mail address: Jean-Luc.Decout@ujf-grenoble.fr (J.-L. Décout).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.037

1936
B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
CO2
CO2
OH
CO2
OH
OH
HN
HN
HN
N
N
N
N
OH
2a
OH
1a
OH
HO
N
N
1b
O
CO2
OH
CO2
OH
HN
OH
HN
N
N
OH
N
OH
3a-b
N
N
4a
CO2
S
OH
OH
NH
HN
Fig. 3. Kinetic profiles at 37 and 55 ꢁC of formation of ArgPy 10b from Na-acetyl-L-
arginine CMGD 11a,b (36 mg, 0.1 mmol, 1 mL of 100 mM aq. phosphate buffer, pH 7.4)
monitored by HPLC analysis.
N
N
CO2
OH
5a-b
HO
6a-b
55 ꢁC of MG in excess with Na-Boc-arginine as a model reaction of
Fig. 1. Structures of the prepared cyclic methylglyoxal diadducts to
a-amino-
proteins (isolation after removal of the Boc group with HCl) [6].
azaheterocycles used in this study (one enantiomer drawn).
More recently, after reaction of MG with Na-acetyl-L-arginine at pH
to remove the carboxylic acid function and/or water for creating
a double bond in the heterocyclic ring formed from MG.
7.4 and 37 ꢁC during 14 days, the ArgPy 10b and also the CMGD 11
were isolated as major products (Fig. 2) [7].
The 2AP CMGD 1a-b were prepared in mixture as model
compounds in large scale with 63% yield (30 g of starting amine).
When heated under basic conditions, in aqueous NaOH, the mixture
of CMGD 1 led to 2AP and to the yellow fluorescent compound 7
which was isolated in low yield (Fig. 2). Interestingly, 7 results from
three reactions in a same pot: decarboxylation, dehydration and
oxidation. In order to optimize the formation of 7 and avoid the
formation of 2AP, the influence of NaOH concentration and temper-
ature onthecourseof thereactionwasstudied. Athigh concentration
(>0.5 M), the yield in 2AP decreased strongly. At temperature lower
than 70 ꢁC, the produced amount of 2AP increased. Finally, the best
result was obtained at 70 ꢁC in 1 M aqueous NaOH. Under these
conditions, 7 was obtained in high 81% yield.
The presence of the argpyrimidines also was detected in vivo by
immunochemistry in MG-modified proteins in the kidneys from
diabetic patients and in the rat brain suggesting that ArgPy may
contribute to the progression of not only long term diabetic
complications, such as nephropathy and atherosclerosis but also
the tissue injury caused by ischemia/reperfusion [7,8]. MG can be
formed in vivo by slow glucose degradation and is involved in the
development of diabetic complications and in mutagenesis [9e15].
It also has appeared to be a signal molecule in apoptosis [16] and
was used as an anti-cancer agent [17]. In diabetic complications,
MG produced from glucose reacts with nucleic acids and proteins to
form advanced glycation end products. The formation of ArgPy has
been explained in vitro through the condensation of two molecules
of MG to lead to the unstable 3-hydroxypentane-2,4-dione and
then reaction of this latter with Na-Boc-arginine [6].
We tried to prepare analogues of compound 7 from the CMGD 2a,
3a-b, 4a and 5a-b (5a-b were not described previously). The main
productsformedunderdifferentbasicconditionswerethearomatic
a
-
In regard to importance of ArgPys as biomarkers of pathologies,
their possible formation from CMGD to arginine derivatives was
investigated.
aminoazaheterocycle: 20-deoxyadenosine,1-aminoisoquinoline (1AI),
9-propyladenine, 2-aminobenzothiazole, respectively. However, the
1AI-CMGD 3 led to the phenolate 8 in low 10% yield (Fig. 2). A high 2 M
NaOH concentration inwater was alsorequired in order to increase the
yield. The ethylglyoxal diadducts (CEGD) to AP 6a-b (Fig. 1) were
prepared (30% yield) and converted under the same conditions to the
phenolate 9 in 55% yield (Fig. 2).
First, we studied the conversion of Na-Fmoc-L-arginine-CMGD to
ArgPy 10a under the conditions used for preparing the polycyclic
pyrimidinols 7 and 8 without isolation of the intermediate CMGD.
Interestingly, 10a was isolated in 22% after two short steps in one
pot: (i) formation of the adducts inwater in 40% aqueous MG at 80 ꢁC
(8 and 6 equiv. added in twice) and (ii) co-evaporation of MG/water
and then aromatisation by heating at 80 ꢁC in 3 M aqueous NaOH.
After this result, we studied the formation of ArgPys from CMGD
to arginine derivatives under the conditions previously used to
isolate ArgPys that are close to physiological conditions [6,7]. We
The structure of compounds 7e9 appeared to be related to the
structure of the fluorescent 5-pyrimidinol 10a named argpyr-
imidine (ArgPy, Fig. 2) isolated in the study of the reaction at 37 and
prepared [18] the Na-acetyl- -arginine CMGD 11 and studied its
L
O
O
O
N
behaviour in water at pH 7.4 (phosphate buffer) at 37 and 55 ꢁC
[6,7]. Under both conditions, the formation of ArgPy 10b was
detected by HPLC and the yields were measured after 7 and 14
N
N
N
N
N
N
9
7
8
CO2
OH
COO
CO2
OH
R
CO2H
AcHN
OH
OH
HN
HN
N
N
N
N
H
N
N
H
R
N
OH
H
OH
H
11
10a: R= NH3+; 10b: R= NHAc
12: R= CH3, 13: R= Bn
14: R= NH2, 15: R= NHC2H5
16: R= N(CH3)2, 17: R= SH
18a-b
Fig. 2. Structures of the prepared phenolates 7e9 compared to the structure of the
reported argpyrimidines 10a-b [6,7] and structures of the previously described CMGD
to Na-acetylarginine 11 [7].
Fig. 4. Structure of the prepared 5-pyrimidinols and benzamidine CMGD.

B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
1937
Table 1
Yields of preparation from methylglyoxal of the phenolates 7 and 8 and pyrimidinols 10a, 12e17 in water in two steps one pot (heating or microwave irradiation). Previously,
some of these compounds have been prepared from 2,4-dimethyl-5-acetyloxazole (*) or 3-acetoxypentan-2,4-dione (y), both prepared from commercially available 3-
chloropentan-2,4-dione (30 and 80% yields, respectively). The corresponding references and yields calculated from 3-chloropentan-2,4-dione are mentioned.
Reagent
2AP
1AI
Na-Fmoc-Arg
RC(NH)NH2, R]
CH3
Bn
NH2
NHAc
NHC2H5
N(CH3)2
SH
Product, yields (%, heating/microwave)
Previous synthesis: reference (), yield %
7 51/41
e
8 8/14
_
10a 22/19
[23]y 37
12 24/7
[19]* 18
13 48/44
_
14 7/3
[21]y 62
14 20/20
[21]y 62
15 27/40
_
16 25/27
[19]y 24
17 17/15
[27]y 38
days: at 37 ꢁC, 0.1 and 0.2%, respectively and, at 55 ꢁC, 4.3 and 6.4%,
respectively (Fig. 3). Clearly, ArgPys can be formed from arginine
CMGD under mild conditions close to physiological conditions.
2-Substituted-3,6-dimethyl-5-pyrimidinols having the aromatic
ring of ArgPy have been recently synthesised for the study of their
radical-scavenging ability [19e22]. In the main preparation, the
commercially available 3-chloropentan-2,4-dione have been con-
verted to the intermediate 3-acetoxypentan-2,4-dione [19]. The
reaction of this dionewith different amidines and guanidines has led
to the corresponding 5-pyrimidinols. 3,6-Dialkyl-5-pyrimidinols
have appeared to transfer their phenolic hydrogen atom to peroxyl
radicals as quickly as equivalently substituted phenols, while their
reactivity toward alkyl radicals far exceeds that of the corresponding
phenols. 3,6-Dimethyl-5-pyrimidinol is very efficient to inhibit
enzyme-catalysed lipid peroxidation by ovine cyclooxygenase 1
(oCOX-1) and is non cytotoxic [22]. ArgPy also has been prepared
from 3-acetoxypentan-2,4-dione in 47% yield (37% from 3-chlor-
opentan-2,4-dione) and also has been reported to be an antioxidant
agent [23].
the pyrimidinols prepared, compounds 7 and 8 were found able to
inhibit strongly the CFTR channel activity in wt-CHO cells. Low IC50
values of 5 and 16 nM were measured for compounds 7 (GPinh-12)
and 8 (GPinh-3), respectively, whereas the known inhibitor gli-
benclamide showed a 15 mM IC50. The parent CMGD 1a-b have been
found inactive and 3a-b have shown a low 15 nM IC50 [3].
Interestingly, under physiological conditions, 7 and 8 are in the
zwitterionic form like the previously identified active adducts 2a,
3a-b and 4a (for 8, the pKa value of approximately 4 was deter-
mined by UV spectrophotometry, data not shown). This zwitter-
ionic character at physiological pH could be related to the inhibition
of CFTR channels observed. Molecular dockings are underway in
the search for a common targeted site using the recently reported
models of the CFTR 3D structure [24e26].
3. Conclusions
In the study of the synthesis of previously reported modulators
of CFTR chloride channels that are cyclic methylglyoxal diadducts
In regard to the observed conversion of CMGD and CEGD to 5-
pyrimidinols or azapolycyclic phenols, we investigated a possible
direct conversion of amidines, guanidines and thiourea to 5-pyr-
imidinols from MG without isolation of the intermediate MG-
adducts in two steps one pot in water: (i) formation at 80 ꢁC of the
CMGD in 40% aqueous MG (addition of 8 and then 6 equiv.) and (ii)
after co-evaporation of MG and water, aromatisation by heating
at 80 ꢁC in 3 M aqueous NaOH. The conversion was successful
with N,N-dimethylguanidine, N-ethylguanidine, N-acetylguanidine,
acetamidine, benzamidine and thiourea leading to the corre-
sponding 2-substituted-3,6-dimethyl-5-pyrimidinols 12e17 in low
to good yields (Fig. 4, Table 1). Like 10a-b, pyrimidinols 14e16
appeared to be fluorescent on TLC. The 2-aminopyrimidinol 14 was
obtained with a low 3.5% yield from guanidine (7% after partial
purification of the diadducts) and in 20% from N-acetylguanidine.
From benzamidine, the intermediate adducts 18a-b also were iso-
lated (88%) and then treated with 1 M aqueous NaOH to lead to the
pyrimidinol 13 (39%).
The procedure was strongly shortened upon microwave irradi-
ation according to the following procedure in one pot: (i) reaction
with MG (4 equiv.) in water at 80 ꢁC, 1 h and (ii) addition of aqueous
NaOH and reaction at 80 ꢁC, 1 h (Table 1).
Compounds 7 and 8 also were prepared using the developed
short procedures in one pot (heating and upon microcrowave)
(Table 1).
In comparison to the methods of synthesis of pyrimidinols
reported, the method described here appears to be competitive in
regard to its simplicity and rapidity (one step of purification) and to
the low cost of the commercially available reagents (commercial
aqueous MG solution) and solvent (water).
(CMGD) to aromatic a-aminoazaheterocycles, we optimized a new
one pot route for preparing in water novel aromatic polycyclic
azaheterocycles and described 5-pyrimidinols antioxidants.
The addition of 2-oxoaldehydes to
a-aminoazaheterocycles,
amidines, guanidines and thiourea is a general and versatile reac-
tion leading to cyclic diadducts which can be converted to 5-pyr-
imidinol derivatives. The involved reaction sequence constitutes
a new expeditious route for preparing such heterocycles that
allowed the preparation and identification of the new polycyclic
5-pyrimidinols 7 and 8 as strong inhibitors of wild-type CFTR
channels in wt-CHO cells.
In regard to the importance as biomarkers in diabetic compli-
cations of the 5-pyrimidinols “argpyrimidines” formed in proteins
from methylglyoxal and arginine residues, we demonstrated here
that argpyrimidines are slowly formed under physiological condi-
tions from CMGD to arginine derivatives according to the synthesis
route described. The observed great instability of the CMGD to
arginine derivatives and the corresponding easy formation of
ArgPys under physiological conditions show that the analysis and
quantification in vitro and in vivo of ArgPys and related CMGD have
to be performed carefully. The adenine-CMGD such as 2a which
could be formed in vivo in DNA during diabetic complications
appeared to be stable under physiological conditions.
4. Experimental protocols
4.1. Chemistry
All starting materials were commercially available research-
grade chemicals and used without further purification except
ethylglyoxal which was synthesised according to Gi et al. [28].
Reactions were monitored by analytical TLC on silica gel (Alugram
Sil G/UV254) from MachereyeNagel with fluorescent indicator
UV254. Melting points were determined in open glass capillaries
using a Büchi 510 apparatus and are reported uncorrected. LRMS
were achieved with a ZQ Waters for the ESI. HRMS and elemental
2.2. Biological activity
Previously, we have used a simple and robust robotic HTS assay
to screen small molecules able to modulate CFTR Clꢀ channel
activity through measurements of iodide (125I) efflux [2e4]. Among

1938
B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
analysis were obtained from the Mass Spectrometry Service,
CRMPO, at the University of Rennes I, France, using a Micro-Tof-Q-II
Micromass Zabspec-Tof spectrometer for ESI and a Varian Mat311
spectrometer for EI technique, and a Microanalyseur Flash EA1112
CHNS/O Thermo Electron.
13C (100 MHz, D2O)
d (ppm): 176.1 (COOH), 149.5 (CIV), 141.8 (CHAr),
133.0 (CHAr), 114.3 (CHAr), 112.9 (CHAr), 91.0 (CIV), 68.7 (CH), 65.9
(CIV), 30.1 (CH2), 29.1 (CH2), 7.3 (CH3), 6.4 (CH3); HRMS (ESI) calc. for
C13H18N2O4: [M þ H]þ 267.1345, found 267.1345.
1H and 13C NMR spectra were recorded on a Bruker Avance 400
at 400 MHz and 100 MHz, respectively, using the residual solvent
signal as internal standard. Chemical shifts are reported in ppm
(parts per million) relative to the residual signal of the solvent, and
the signals are described as singlet (s), broad singlet (bs), doublet
(d), triplet (t), doublet of doublet (dd), quartet (q), multiplet (m);
coupling constants are reported in Hertz (Hz). Columns chroma-
tography was performed on silica gel (MN Kieselgel 60, 0.063e0.2
mm/70e230 mesh, MachereyeNagel) or on C18 or C8 reversed
phase (MachereyeNagel polygoprep 60-50C18).
4.1.1.3. Na-Acetyl-
solution of MG (40%) (10 mL, 66.6 mmol), Na-Acetyl-
L
-arginine CMGD 11a,b. To a commercial aqueous
-arginine
L
(2.5 g, 11.56 mmol) was added under Argon and the mixture was
heated at 70 ꢁC for 16 h. After two co-evaporation with water and
MeOH, the solid was crystallised in THF then in MeOH to afford
11a,b [7] (1.30 g, 3.61 mmol, 31%), mp: 175 ꢁC (dec.). LRMS (ESI)
calc. for C14H25N4O7: [M þ H]þ 361.5, found 361.1.
4.1.1.4. Benzamidine CMGD 18a,b (70:30). To the aqueous solution
of MG (40%) (11.8 mL, 76.8 mmol), benzamidine chlorhydrate (1.5 g,
9.6 mmol), water (2 mL) and ethanol (2 mL) were added. Argon was
flushed through the solution and the mixture was heated at 50 ꢁC
for 24 h. After evaporation, the residue was chromatographed on
Sep-PakÒ C18 cartridges (10 g) eluting with H2O and H2O-MeOH
(9:1). Crystallization from propan-2-ol afforded 18a,b (2.23 g,
8.45 mmol, 88%) as a white solid. The adducts 18a were isolated as
a white solid (651 mg, 2.23 mmol) after chromatography on silica
gel in DCM-MeOH (9:1 then 8:2), mp: 177 ꢁC (dec.). Found C 52.42,
H 6.37, N 8.39. C13H16N2O4, HCl,1/2C3H7OH requires C 52.65, H 6.40,
The synthesis and characterization of compounds 1, 2 and 3
have been previously described [1]. Only one enantiomer of the
racemic mixture of compounds 1, 6, 18 is drawn here. When indi-
cated, the ratio of diadducts a and b are measured by NMR.
Microwave reactions were conducted using a CEM Discover
Synthesis Unit (CEM Corp., Matthews, NC) with PC control, equip-
ped with a continuous focused microwave power delivery system
which operates at 2.45 GHz with selectable power output from 0 to
300 W. The reaction was performed in glass vessels (capacity
10 mL) sealed with a septum. The temperature of the reaction
mixture was monitored using a calibrated infrared temperature
control mounted under the reaction vessel. All experiments were
conducted under magnetic stirring (rotating magnetic plate located
under the microwave cavity and using Teflon-coated magnetic stir
bar in the vessel). The solutions were irradiated in a sealed tube at
80 ꢁC for 1 h (ramp time 1 min, Tmax ¼ 80 ꢁC, Power max ¼ 150 W).
The kinetic study was performed with an Agilent 1100 series
HPLC machine using a diode array detector and a C18 reversed phase
N 8.47; 1H (400 MHz, D2O)
7.57e7.53 (2H, m, CHAr), 4.21 (1H, s, CH), 1.65 (3H, s, CH3), 1.64 (3H,
s, CH3); 13C (100 MHz, D2O)
(ppm): 176.6 (COOH), 161.9 (CIV), 135.6
d (ppm): 7.70e7.66 (3H, m, CHAr),
d
(CIV), 130.9 (CHAr), 129.4 (CHAr), 128.8 (CHAr), 82.0 (CIV), 70.8 (CH),
62.9 (CIV), 24.8 (CH3), 22.3 (CH3); HRMS (ESI) calc. for C13H16N2O4:
[M þ H]þ 265.1188, found 265.1172.
4.1.2. Preparation of phenolates 7, 8, 9 and pyrimidinols 10a, 13
after isolation of the diadducts
column (Nucleodur C18 ISIS, MachereyeNagel, 5 mm particle size,
4.1.2.1. Phenolate 7. A solution of 1a,b (200 mg, 0.8 mmol) in 1 M
aqueous NaOH (25 mL) was heated at 70 ꢁC for 5 h. After cooling at
rt, the solution was neutralized by addition of 1 M aqueous HCl.
After evaporation, the residue was chromatographed on Sep-PakÒ
C18 cartridges (10 g) eluting with H2O and H2O-MeOH (95:5) to give
7 (119 mg, 0.68 mmol, 81%) as a yellow solid, mp: 140 ꢁC (dec.). 1H
250 mm ꢂ 4.6 mm), with a mobile phase composed of A ¼water and
B ¼ methanol containing 0.5% TFAwith a gradient 90:10 to 0:100 A:B
over 30 min, 0.7 mL/min, 50 mL injection, detection at 340 nm.
4.1.1. Synthesis of cyclic methylglyoxal diadducts (CMGD)
4.1.1.1. Synthesis in large scale of the 2-aminopyridine methylglyoxal
diadducts (2AP CMGD) 1a,b (60:40, respectively). To the commer-
cially available concentrated aqueous solution of MG (40%)
(380 mL, 1.9 mol), 2-aminopyridine (30.0 g, 318.8 mmol) was
added. Argon was flushed through the solution and the mixture
was heated at 60 ꢁC for 20 h. After evaporation under reduced
pressure, the residue was dissolved in methanol and precipitated in
tetrahydrofuran (300 mL). After filtration, the solid was washed
with diethyl ether to give the adducts 1a,b [1] (47.9 g, 201.1 mmol,
63%) as a white solid.
(400 MHz, D2O)
CHAr), 8.26e8.23 (1H, m, CHAr), 8.00e7.96 (1H, m, CHAr), 2.89 (3H, s,
CH3), 2.81 (3H, s, CH3); 13C (100 MHz, D2O)
(ppm): 164.6 (CIV),
145.8 (CIV), 143.9 (CIV), 137.5 (CHAr), 136.8 (CIV), 131.0 (CHAr), 126.7
(CHAr), 123.3 (CHAr), 20.7 (CH3), 12.0 (CH3); HRMS (EI) calc. for
C10H10N2O: [M]þ 174.0793, found 174.0780.
d (ppm): 8.92 (1H, m, CHAr), 8.36e8.32 (1H, m,
d
4.1.2.2. Phenolate 8. A solution of 3a,b [3] (200 mg, 0.63 mmol) in
2 M aqueous NaOH (3 mL) was heated at 70 ꢁC for 3 h. After cooling
at rt, the solution was neutralized by addition of aqueous H3PO4
(30%) and then, extracted with DCM. After evaporation, the residue
was chromatographed on silica gel in DCM-MeOH (95:5) to give 8
(14 mg, 0.06 mmol, 10%) as a yellow solid, mp: 187 ꢁC (dec.). Found
C 69.84, H 5.81, N 11.37. C14H12N2O, 1/2H2O requires C 69.86, H 6.06,
4.1.1.2. 2-Aminopyridine ethylglyoxal adducts (2AP CEGD) 6a,b
(75:25). To a solution of 2-aminopyridine (1.0 g, 10.6 mmol) in
water (10 mL), ethylglyoxal (4.1 g, 47.6 mmol) was added. Argon
was flushed through the solution and the mixture was heated at
60 ꢁC for 10 h. After evaporation, the residue was chromatographed
on Sep-PakÒ C18 cartridges (10 g) eluting with H2O and then
H2O-MeOH (95:5) to give 6a,b (850 mg, 3.2 mmol, 30%).
N 11.24; 1H (400 MHz, CD3OD)
d (ppm): 8.74e8.71 (1H, m, CHAr),
8.14 (1H, d, J 7.6, CHAr), 7.80e7.78 (1H, m, CHAr), 7.73e7.66 (3H, m,
CHAr), 2.67 (3H, s, CH3), 2.63 (3H, s, CH3), 13C (100 MHz, CD3OD)
d
(ppm): 166.3 (CIV), 160.0 (CIV), 136.6 (CIV), 136.4 (CIV), 132.1 (CHAr),
The adducts 6a were isolated as a white solid (600 mg,
2.3 mmol, purification yield 94%) after chromatography on Sep-
PakÒ C18 cartridges (10 g) eluting with H2O and H2O-MeOH (95:5),
mp: 152e154 ꢁC. Found C 56.88, H 6.67, 10.46. C13H18N2O4, 1/2H2O
131.1 (CHAr), 130.8 (CIV), 128.0 (CHAr), 128.0 (CIV), 126.4 (CHAr), 125.4
(CHAr), 122.6 (CHAr), 22.0 (CH3), 12.3 (CH3); HRMS (EI) calc. for
C14H12N2O: [M]þ 224.0950, found 224.0947.
requires C 56.72, H 6.96, N 10.18; 1H (400 MHz, D2O)
d
(ppm): 8.16
4.1.2.3. Phenolate 9. A solution of 6a,b (50 mg, 0.19 mmol) in 1 M
aqueous NaOH (20 mL) was heated at 60 ꢁC for 6 h. After cooling at
rt, the solution was neutralized by addition of 1 M aqueous HCl and,
then, extracted with DCM. After evaporation, the residue was
(1H, d, J 7.2, CHAr), 7.75 (1H, t, J 8.4, CHAr), 7.05 (1H, d, J 8.4, CHAr),
6.89 (1H, t, J 7.2, CHAr), 4.55 (1H, s, CH), 2.02e1.93 (1H, m, CH2),
1.87e1.78 (3H, m, CH2), 0.97 (3H, t, J 7.2, CH3), 0.91 (3H, t, J 7.2, CH3);

B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
1939
chromatographed on a C18 reversed phase eluting with H2O and
HCl and, then, extracted with AcOEt. After evaporation, the residue
was chromatographed on silica gel eluting with AcOEt and AcOEt-
MeOH (9:1) to give 8 (114 mg, 0.51 mmol, 14%) as a yellow solid
(characteristics described in Section 4.1.2.2.).
H2O-MeOH (9:1) to afford 9 (21 mg, 0.10 mmol, 55%) as a yellow
hygroscopic solid. 1H (400 MHz, CD3OD)
d (ppm): 8.68 (1H, d, J 6.8,
CHAr), 8.05 (1H, d, J 8.4, CHAr), 7.82e7.78 (1H, m, CHAr), 7.68e7.65
(1H, m, CHAr), 3.39e3.34 (2H, m, CH2), 3.11e3.06 (2H, m, CH2),
1.40e1.33 (3H, m, CH3), 1.31e1.27 (3H, m, CH3); 13C (100 MHz,
4.1.3.3. ArgPy 10a. Thermic procedure: Fmoc-Arg-OH (1.455 g,
3.67 mmol). After cooling at rt, the pH was adjusted to 7.5 by
addition of 3 M aqueous HCl. After evaporation, the residue was
chromatographed on silica gel eluting with AcOEt-MeOH (1:2) and
on a C18 reversed phase eluting with H2O and H2O-MeOH (9:1) to
afford 10a (205 mg, 0.81 mmol, 22%) as a white solid.
CD3OD)
d (ppm): 170.8 (CIV), 156.9 (CIV), 138.6 (CIV), 136.2 (CIV),
128.9 (CHAr), 127.4 (CHAr), 127.1 (CHAr), 121.4 (CHAr), 26.3 (CH2), 17.2
(CH2), 9.3 (CH3), 8.0 (CH3); HRMS (ESI) calc. for C12H15N2O:
[M þ H]þ 203.1184, found 203.1182.
4.1.2.4. ArgPy 10b. 2 mL of 3 M aqueous NaOH was added to 50 mg
of 11a,b (0.14 mmol) and the mixture was stirred 24 h at 70 ꢁC. After
cooling, the pH was adjusted to 7.5 by addition of 3 M aqueous HCl.
After evaporation, the residue was chromatographed on silica gel
eluting with AcOEt-MeOH (1:2) and on a C18 reversed phase eluting
with H2O and H2O-MeOH (9:1) to afford 10b (25 mg, 0.08 mmol,
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 7 by addition of 3 M aqueous NaOH, and then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt-MeOH (1:2) and fol-
lowed by a purification on a C18 reversed phase eluting with H2O
and H2O-MeOH (9:1) to afford 10a (182 mg, 0.70 mmol, 19%) as
a white solid, mp: 213 ꢁC (dec.) (207 ꢁC, dec., [23]). 1H (400 MHz,
61%) as a white solid, mp: 187 ꢁC (dec.). 1H (400 MHz, D2O)
d (ppm):
4.10 (1 H, q, CH), 3.23 (2 H, t, J 6.4, N-CH2), 2.24 (6 H, s, CH3), 1.95
D2O)
d (ppm): 3.72 (2 H, t, J 6, CH), 3.26 (2 H, t, J 9.6, NeCH2), 2.23
(3 H, s, CH3), 1.81 (1 H, m, CH2), 1.62 (3 H, m, CHþCH2); 13C
(6 H, s, CH3), 1.86 (2 H, m, CH2), 1.62 (2 H, m, CH2); 13C (100 MHz,
(ppm): 180.8 (COOꢀ), 175.1 (CO), 159.5 (2ꢂCIV-
D2O)
d
(ppm): 176.2 (COOꢀ), 159.6 (2 ꢂ CIV-CH3) 158.1 (CIV-OH),
(100 MHz, D2O)
d
CH3) 157.9 (CIV-OH), 139.9 (CIV-NH), 56.7 (CH), 42.7 (HNeCH2), 30.6
(CH2), 26.7 (CH2), 23.4 (CH3), 19.2 (2ꢂCH3); LRMS (ESI) calc. for
C13H20N4O4: [M ꢀ H]ꢀ 295.3, found 294.9.
140.1 (CIV-NH), 56.1 (CH), 42.4 (HNeCH2), 29.4 (CH2), 26.0 (CH2),
19.3 (2ꢂCH3); HRMS (ESI) calc. for C11H17N4O3: [M ꢀ H]ꢀ 253.1301,
found 253.1306.
4.1.2.5. 2-Phenyl-5-pyrimidinol 13. A solution of 18a,b (100 mg,
0.30 mmol) in 1 M aqueous NaOH (20 mL) was heated to 50 ꢁC for
3 h. After cooling at rt, the solution was neutralized by addition of
1 M aqueous HCl and then, extracted with DCM. After evaporation
under reduced pressure, the residue was chromatographed on
silica gel eluting with DCM and DCM-MeOH (95:5) to give 13
(24 mg, 0.12 mmol, 39%) as a white solid, mp: 142 ꢁC (dec.). Found:
C, 71.77; H, 5.93; N, 13.86. Calc. for C12H12N2O: C, 71.98; H, 6.04; N,
4.1.3.4. 2,4,6-Trimethyl-5-pyrimidinol 12. Thermic procedure: Acet-
amidine hydrochloride (347 mg, 3.67 mmol). After cooling at rt, the
pH was adjusted to 6.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chromato-
graphed on silica gel eluting with AcOEt and AcOEt-MeOH (9:1) to
give 12 (120 mg, 0.87 mmol, 24%) as a white solid.
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 6.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt and AcOEt-MeOH (9:1)
to give 12 (38 mg, 0.28 mmol, 7.5%) as a white solid, mp:
13.99; 1H (400 MHz, CDCl3)
(3H, m, CHAr), 2.47 (6H, s, CH3); 13C (100 MHz, CDCl3)
d
(ppm): 8.24 (2H, m, CHAr), 7.42e7.38
(ppm): 156.7
d
(CIV), 153.0 (CIV), 146.0 (CIV), 138.0 (CIV), 129.7 (CHAr), 128.6 (CHAr),
127.8 (CHAr), 18.8 (CH3); HRMS (EI) calc. for C12H12N2O: [M]þ
200.0950, found 200.0947.
150e152 ꢁC (152e154 ꢁC, [29]). 1H (400 MHz, CD3OD)
d
(ppm): 2.39
(ppm):
(3 H, s, CH3), 2.29 (6 H, s, 2 ꢂ CH3); 13C (100 MHz, CD3OD)
d
158.5 (CIV-OH), 155.2 (2ꢂCIV-CH3), 147.4 (CIV-CH3), 24.1 (CH3), 18.5
(2 ꢂ CH3); HRMS (ESI) calc. for C7H11N2O: [M þ H]þ 139.0871, found
139.0873.
4.1.3. Expeditious synthesis of 5-pyrimidinols and phenolates
without isolation of the intermediate diadducts
General procedure for preparation of phenolates and pyr-
imidinols upon conventional heating: To a commercial aqueous
solution of MG (40%) (4.41 mL, 29.4 mmol), 3.67 mmol of reactant
were added and the mixture was heated at 80 ꢁC for 6 h, Then 6
equiv of MG (40%) (3.30 mL, 22.02 mmol) was added and the
mixture stirred at 80 ꢁC for 6 h. After two co-evaporation with
water, the residue was dissolved in 3 M aqueous NaOH (15 mL) and
the solution was heated at 80 ꢁC for 8 h.
General procedure for preparation of phenolates and pyr-
imidinols upon microwave irradiation: To a commercial aqueous
solution of MG (40%) (2.21 mL, 14.7 mmol) in a sealed tube,
3.67 mmol of reactant were added and the mixture was heated at
80 ꢁC for 1 h upon microwave irradiation. Then 3 M aqueous NaOH
(10 mL) was added and the solution was heated at 80 ꢁC for 1 h
upon microwave irradiation.
4.1.3.5. 2-Phenyl-5-pyrimidinol 13. Thermic procedure: To the
concentrated aqueous solution of MG (40%) (4.1 mL, 25.6 mmol),
water (2 mL) and benzamidine chlorhydrate (500 mg, 3.2 mmol)
were added. Argon was flushed through the solution and the
mixture was heated at 50 ꢁC for 24 h. After two co-evaporation with
water, the residue was chromatographed on C8 reversed phase
eluting with water. The fractions containing 18a,b were evaporated
to dryness. A solution of 18a,b in 1 M aqueous NaOH (20 mL) was
heated at 50 ꢁC for 5 h. After cooling at rt, the solution was
neutralized by addition of 1 M aqueous HCl. After evaporation, the
residue was chromatographed on silica gel eluting with ethyl
acetate to lead to 12 (300 mg, 1.5 mmol, 48%) as a white solid.
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 7 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt-cHx (7:3) and AcOEt to
give 13 (322 mg, 1.62 mmol, 44%) as a white solid (characteristics
described in Section 4.1.2.5.).
4.1.3.1. Phenolate 7. Microwave procedure: After reaction and
cooling at rt, the pH was adjusted to 7 by addition of 3 M aqueous
HCl. After evaporation, the residue was chromatographed on a C18
reversed phase eluting with H2O and H2O-MeOH (9:1) to give 7
(260 mg, 1.49 mmol, 41%) as a yellow solid (characteristics
described in Section 4.1.2.1.).
4.1.3.6. 2-Amino-4,6-dimethyl-5-pyrimidinol 14 from N-acetylgua-
nidine. Thermic procedure: N-acetylguanidine (371 mg, 3.67 mmol).
After cooling at rt, the pH was adjusted to 7.5 by addition of 3 M
aqueous HCl and, then, extracted with AcOEt. After evaporation, the
4.1.3.2. Phenolate 8. Microwave procedure: After reaction and
cooling at rt, the pH was adjusted to 7 by addition of 3 M aqueous

1940
B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
residue was chromatographed on silica gel eluting with AcOEt and
AcOEt-MeOH (9:1) to give 14 (105 mg, 0.75 mmol, 20%) as a beige
solid.
kept around 6. After cooling at rt, the pH was adjusted to 7.5 by addition of
3 M aqueous HCl and, then, extracted with AcOEt. After evaporation, the
residue was chromatographed on silica gel eluting with AcOEt-cHx (7:3)
and AcOEt to give 16 (155 mg, 0.93 mmol, 25%) as a beige solid.
Microwave procedure:After reaction andcooling at rt, thepH was
adjusted to 7.5 by addition of 3 M aqueous HCl and, then, extracted
with AcOEt. After evaporation, the residue was chromatographed on
silica gel eluting with AcOEt and AcOEt-MeOH (9:1) to give 14
(100 mg, 0.72 mmol, 20%) as a beige solid, mp: 208 ꢁC (dec.). 1H
(400 MHz, CD3OD): 2.17 (6 H, s, CH3); 13C (100 MHz, CD3OD): 157.9
(2 ꢂ CIV-CH3), 156.3 (CIV-OH), 139.4 (CIV-NH2), 17.6 (2 ꢂ CH3); HRMS
(ESI) calc. for C6H9N3ONa: [M þ Na]þ 162.0643, found 162.0644.
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 7.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt-cHx (7:3) and AcOEt to
give 16 (166 mg, 1 mmol, 27%) as a beige solid, mp: 146e148 ꢁC
(149e151 ꢁC, [19]). 1H (400 MHz, CD3OD)
d (ppm): 3.20 (6 H, s,
2 ꢂ CH3), 2.41 (6 H, s, 2 ꢂ CH3); 13C (100 MHz, CD3OD)
d (ppm):
158.7 (CIV-OH), 157.2 (2ꢂCIV-CH3), 140.0 (CIV-N(CH3)2), 38.2
(2 ꢂ CH3), 19.1 (2 ꢂ CH3); HRMS (ESI) calc. for C8H14N3O: [M þ H]þ
168.1137, found 168.1137.
4.1.3.7. 2-Amino-4,6-dimethyl-5-pyrimidinol 14 from guanidine
hydrochloride. Thermic procedure: Guanidine hydrochloride (350 mg,
3.67 mmol). After cooling at rt, the pH was adjusted to 7.5 by addition
of 3 M aqueous HCl and, then, extracted with AcOEt. After evapora-
tion, the residue was chromatographed on silica gel eluting with
AcOEt and AcOEt-MeOH (9:1) to give 14 (17 mg, 0.12 mmol, 3.5%) as
a beige solid.
Thermic procedure with a short partial purification by chro-
matography on C18: After two co-evaporation with water, the
residue was chromatographed on a C18 reversed phase eluting with
H2O and H2O-MeOH (9:1). The fractions containing the adducts
were evapored and then dissolve in 3 M aqueous NaOH (17 mL) and
the solution was heated at 70 ꢁC for 14 h. After cooling at rt, the pH
was adjusted to 7.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt and AcOEt-MeOH (9:1)
to give 14 (36 mg, 0.26 mmol, 7%) as a beige solid.
4.1.3.10. 2-Thio-4,6-dimethyl-5-pyrimidinol 17. Thermic procedure:
After cooling at rt, the pH was adjusted to 6.5 by addition of 3 M
aqueous HCl and, then, extracted with AcOEt. After evaporation,
the residue was chromatographed on silica gel eluting with AcOEt-
cHx (7:3) and AcOEt to give 17 (95 mg, 0.61 mmol, 17%) as a yellow
solid.
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 6.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt-cHx (7:3) and AcOEt to
give 17 (84 mg, 0.54 mmol, 15%) as a yellow solid, mp: 186 ꢁC (dec.).
1H (400 MHz, DMSO d6)
DMSO d6)
d
(ppm): 2.30 (6 H, s, CH3); 13C (100 MHz,
d
(ppm): 155.8 (CIV-OH), 155.0 (2 ꢂ CIV-CH3), 146.0 (CIV-
SH), 19.0 (2 ꢂ CH3); HRMS (ESI) calc. for C6H9N2OS: [M þ H]þ
157.0436, found 157.0437; LRMS (ESI) calc. for C12H15N4O2S2:
[2M þ H]þ 311.5, found 311.1.
Microwave procedure: After reaction and cooling at rt, the pH
was adjusted to 7.5 by addition of 3 M aqueous HCl and, then,
extracted with AcOEt. After evaporation, the residue was chroma-
tographed on silica gel eluting with AcOEt and AcOEt-MeOH (9:1)
to give 14 (15 mg, 0.10 mmol, 3%) as a beige solid.
4.2. Biological activity: inhibition of CFTR channels in wt-CHO cells,
iodide efflux assay
4.1.3.8. 2-Ethylamino-4,6-dimethyl-5-pyrimidinol 15. Thermic proce-
dure: N-ethylguanidine hydrosulfate (500 mg, 3.67 mmol). After
cooling at rt, the pH was adjusted to 7.5 by addition of 3 M aqueous HCl
and, then, extracted with AcOEt. After evaporation, the residue was
chromatographed on silica gel eluting with AcOEt-cHx (7:3) and AcOEt
to give 15 (165 mg, 0.99 mmol, 27%) as a white solid.
Screening of small molecules and concentration response
curves were determined by measuring the rate of iodide (125I)
efflux with a high-capacity robotic system (MultiProbe II EXT;
PerkinElmer Life and Analytical Sciences, Courtaboeuf, France)
adapted to the determination of iodide efflux as described
previously [30e32]. Our protocol of screening is as follows. CHO
cells overexpressing wild-type CFTR (wt-CFTR) were cultured in
multiwell plates and incubated at 37 ꢁC in Krebs’ solution con-
Microwave procedure: After cooling at rt, the pH was adjusted
to 7.5 by addition of 3 M aqueous HCl and, then, extracted with
AcOEt. After evaporation, the residue was chromatographed on
silica gel eluting with AcOEt-cHx (7:3) and AcOEt to give 15
(240 mg, 1.42 mmol, 40%) as a white solid, mp: 122e123 ꢁC. Found:
C, 57.55; H, 7.73; N, 23.73. Calc. for C8H13N3O: C, 57.47; H, 7.84; N,
taining 1
mM KI and 1 m
Ci of Na125I/mL (PerkinElmer Life and
Analytical Sciences, Boston, MA) during 30 min to permit 125I to
reach equilibrium. The first three aliquots were used to establish
a stable baseline in cold Krebs’ buffer (from t0 to t2). A medium
containing the appropriate drug was then used for the remaining
aliquots from t3 to t8. Residual radioactivity was extracted at the
end of the experiment with a mixture of 0.1 N NaOH and 0.1%
SDS and determined using a gamma counter (Cobra II; Perki-
nElmer Life and Analytical Sciences). The fraction of initial
intracellular 125I lost during each time point was determined, and
time-dependent rates (k peak rate, per minute) of 125I efflux were
25.13%; 1H (400 MHz, CD3OD)
d (ppm): 3.35 (2 H, q, J 7.2, CH2), 2.30
(6 H, s, 2 ꢂ CH3), 1.19 (3 H, t, J 7.2, CH3); 13C (100 MHz, CD3OD)
d
(ppm): 158.1 (CIV-OH), 157.7 (2ꢂCIV-CH3), 140.6 (CIV-NH), 37.6
(2ꢂCH3), 18.9 (CH2), 15.5 (CH3); HRMS (ESI) calc. for C8H14N3O:
[M þ H]þ 168.1137, found 168.1137.
Comparison thermic procedure/microwave irradiation under
the same other conditions: To a commercial aqueous solution of
MG (40%) (2.21 mL, 14.7 mmol), N-ethylguanidine hydrosulfate
(500 mg, 3.67 mmol) was added and the mixture was heated at
80 ꢁC for 1 h. Then 3 M aqueous NaOH (17 mL) was added and the
solution was heated at 80 ꢁC for 1 h. After cooling at rt, the pH was
adjusted to 7.5 by addition of 3 M aqueous HCl and, then, extracted
with AcOEt. After evaporation, the residue was chromatographed
on silica gel eluting with AcOEt-cHx (7:3) and AcOEt to give 15
(90 mg, 0.54 mmol, 15%) as a white solid.
calculated from the following equation:
k
¼
ln(125It1/125It2)/
(t1 ꢀ t2), where 125It is the intracellular 125I at time t, and t1 and
t2 are successive time points [31]. Relative rates were calculated
as kpeak ꢀ kbasal (per minute), i.e., the maximal value for the time-
dependent rate (kpeak, per minute) excluding the third point used
to establish the baseline (kbasal, per minute). Concentration-
dependent activation curves were constructed as a percentage of
maximal activation (set at 100%) transformed from the calculated
relative rates. CFTR-dependent iodide efflux was stimulated by
forskolin. Other details have been described elsewhere [30,32].
4.1.3.9. 2-Dimethylamino-4,6-dimethyl-5-pyrimidinol 16. Thermic proce-
dure: N-dimethylguanidine hydrosulfate (503 mg, 3.67 mmol) with pH

B.-L. Renard et al. / European Journal of Medicinal Chemistry 46 (2011) 1935e1941
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Acknowledgments
This work was supported by the French National Research
Agency » Emergence de projets de biotechnologie à fort potentiel
de valorisation » and by the French Association “Vaincre la Muco-
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