Synthesis, evaluation and absolute configuration assignment of novel dihydropyrimidin-2-ones as picomolar sodium iodide symporter inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.01.043

A small library of dihydropyrimidin-2-ones (DHPMs) was synthesized and evaluated for their potency to block iodide entrapment in rat thyroid cells. Synthesis was achieved using the multicomponent Biginelli reaction. Twelve compounds were tested for the inhibition of sodium iodide symporter (NIS) in a cell-based assay. One newly synthesized derivative exhibited a remarkably strong activity, with a half-maximum inhibitory concentration value (IC₅₀) of 65 pM. Three DHPMs were further resolved from racemates using chiral HPLC and absolute configurations were assigned using circular dichroism spectroscopy. Biological evaluation showed that most of the activity against NIS resides in one enantiomer. This study provides new insights for the development of anti-thyroid drugs, as well as for the synthesis of novel pharmacological tools designed to investigate iodide transport mechanisms at cellular and molecular levels.

Full text of DOI:10.1016/j.ejmech.2013.01.043

European Journal of Medicinal Chemistry 62 (2013) 722e727
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, evaluation and absolute configuration assignment
of novel dihydropyrimidin-2-ones as picomolar sodium iodide
symporter inhibitors
*
Pierre Lacotte, David-Alexandre Buisson, Yves Ambroise
CEA Saclay, Institut de Biologie et de Technologies de Saclay (iBiTecS), Service de Chimie Bioorganique et de Marquage (SCBM), 91191 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of dihydropyrimidin-2-ones (DHPMs) was synthesized and evaluated for their potency to
block iodide entrapment in rat thyroid cells. Synthesis was achieved using the multicomponent Biginelli
reaction. Twelve compounds were tested for the inhibition of sodium iodide symporter (NIS) in a cell-
based assay. One newly synthesized derivative exhibited a remarkably strong activity, with a half-
maximum inhibitory concentration value (IC50) of 65 pM. Three DHPMs were further resolved from
racemates using chiral HPLC and absolute configurations were assigned using circular dichroism spec-
troscopy. Biological evaluation showed that most of the activity against NIS resides in one enantiomer.
This study provides new insights for the development of anti-thyroid drugs, as well as for the synthesis
of novel pharmacological tools designed to investigate iodide transport mechanisms at cellular and
molecular levels.
Received 15 November 2012
Received in revised form
17 January 2013
Accepted 23 January 2013
Available online 9 February 2013
Keywords:
Absolute configuration
Dihydropyrimidinones
Membrane proteins
Sodium iodide symporter
Thyroid
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
pancreatic cancers, hepatoma, and melanoma [7]. In cases of acci-
dental exposure of the population to radioactive iodine species, NIS
Iodide translocation into thyroid cells is the first and rate-
limiting step in the biosynthesis of iodinated hormones T3 and T4
[1]. This process is mediated by the sodium iodide symporter (NIS),
a glycoprotein with 13 putative transmembrane domains mainly
expressed in the thyroid gland and also in other tissues including
salivary glands, gastric mucosa and mammary glands during lac-
tation [2]. NIS was cloned in 1996 [3]. Since then, extensive char-
acterization has unraveled the role of NIS in many thyroid as well as
non-thyroid diseases such as cancer (thyroid, breast), thyrotoxicosis
and congenital hypothyroidism [4]. Several studies conclude that
thyroid gland failure occurs in 5e7% of the population across dif-
ferent countries [5]. Furthermore, the ability of NIS-expressing cells
to efficiently take up iodide has provided a basis for extra-thyroid
cancer cell destruction by radioiodide after tumor-selective intro-
duction of exogenous NIS [6]. This strategy showed promising re-
sults with successful tumor growth inhibition and volume
reduction in models of many types of cancers including colon and
is directly responsible for human contamination, leading to an
increased risk of cancer and birth defects [8]. Solutions for body
decontamination are still awaited. At a cellular level, the post-
translational mechanisms of NIS regulation are still poorly under-
stood and small molecules modulating NIS function are promising
tools for investigation of the intracellular signaling pathways and
protein/protein interactions in which NIS is involved. Moreover, the
development of new drugs capable of disrupting NIS function for
the treatment of overactive thyroid may be relevant as it is known
that current anti-thyroid drugs such as methimazole, carbimazole
and propylthiouracil can cause severe adverse effects [9].
In 2008, a high-throughput screening campaign led to the dis-
covery of dihydropyrimidin-2-ones (DHPMs) as very potent iodide
uptake inhibitors [10]. Iodide transport blocker 9 (ITB9, compound
1 herein) was identified as the lead within the DHPM family, with
a half maximal inhibitory concentration (IC50) value of 89 nM
(Fig. 1) in rat thyroid-derived cells (FRTL5). Further analysis of the
effect of 1 on the iodide-induced current in NIS-expressing Xenopus
laevis oocytes showed that the inhibition was specific and imme-
diate [11]. Additional isotopic flux experiments showed that 1 can
trigger a rapid and total iodide discharge from preloaded hNIS-
HEK293 cells. Preliminary experiments run in our laboratory
showed that compound 1 has no impact on cell viability at con-
Abbreviations: CD, circular dichroism; DHPM, dihydropyrimidin-2-one; FRTL5,
Fischer rat thyroid 5; IC50, half maximal inhibitory concentration; ITB, iodide
transport blocker; MTT, methylthiazolyldiphenyl-tetrazolium bromide; NIS, sodium
iodide symporter; SAR, structureeactivity relationship.
* Corresponding author. Tel.: þ33 1 69 08 24 70; fax: þ33 1 69 08 79 91.
centrations up to 200 mM. Altogether, these results show that the
E-mail address: yves.ambroise@cea.fr (Y. Ambroise).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.043

P. Lacotte et al. / European Journal of Medicinal Chemistry 62 (2013) 722e727
723
2. Chemistry
The 3,4-dihydropyrimidin-2(1H)-ones reported herein were
prepared using the three-component ring-forming Biginelli reac-
tion as depicted in Fig. 2A [13]. This reaction involves the con-
densation of an aldehyde, a b-keto ester and a urea derivative, and
is catalyzed with acid. First, we assayed several combinations of
Brønsted and Lewis catalysts, solvents and temperature in a model
reaction using dimethylurea in order to optimize the conversion
rates. The optimal conditions were obtained using 0.1 eq zinc tri-
flate in refluxing acetonitrile. Using these conditions, we combined
4 acetoacetates and 3 urea derivatives while keeping the 2-
furaldehyde unchanged to produce a set of 12 individual DHPMs
(Fig. 2B). Biginelli condensation using monomethyl urea provided
exclusively the N1 methylated DHPM (7, 10, 13, 16). This was veri-
fied by 1H and 13C NMR, and was concordant with previous ob-
servations [14]. The 12 DHPMs were obtained after flash
chromatography on silica gel with satisfactory yields (30e73%) and
excellent chemical purities (>98%). Acetoacetates 2e5 were pre-
pared from 2,2,6-trimethyl-4H-1,3-dioxin-4-one and diverse R5OH
in the presence of potassium acetate (Fig. 2A) using a slightly
modified version of the procedure of Menéndez et al. [15].
Fig. 1. Structure of ITB9 (compound 1) and summary of a SAR study from previous
work [12].
dihydropyrimidin-2-one core is a promising chemical platform for
development of new anti-thyroid drugs.
We recently reported the results of an extensive SAR study of
compound 1 using single-point modifications at five key positions
on the pyrimidinone ring [12]. The synthesis and evaluation of 115
derivatives provided valuable data and identified a novel potent
and non-toxic compound, exhibiting an IC50 value of 3.2 nM in
FRTL5 (compound 6, Fig. 2). In this work, it was shown that the
furan-2-yl group at C4, methyl and H on N1 and N3, and methox-
ybenzyl ester groups at C5 were optimal for potency (Fig. 1). With
these data in hand, we decided to synthesize and evaluate a second
generation of compounds by combining these optimal modifica-
tions. A small library of DHPMs was generated and its evaluation
led to a spectacular IC50 value improvement from 3.2 nM to 65 pM.
3. Biological evaluation
We evaluated the potency of each DHPM (6e17) by measuring
their effect on iodide entrapment in the rat thyroid-derived cell
lines FRTL5 [16]. The half maximal inhibitory concentration (IC50
)
values were measured in at least two independent experiments
Fig. 2. A: Synthesis of target 3,4-dihydropyrimidinones by the Biginelli reaction. Reagents and conditions: (a) AcOK, microwave irradiation, 120 ꢀC, 30 min, 70e91%; (b) Zn(OTf)2,
MeCN, reflux, 2 h, 30e73%. B: Structure of the 12 DHPMs.

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P. Lacotte et al. / European Journal of Medicinal Chemistry 62 (2013) 722e727
Table 1
This compound is the most potent NIS inhibitor reported to date. On
the urea motif, potency improved in the order N,N-dimethyl > non-
methyl > N-monomethyl and the ranking of activities for the ester
IC50 values of the 12 DHPMs (6e17) for the inhibition
of iodide entrapment in FRTL5 cells.a
Compound
IC50 (nM)
89b
3.2
90
1.6
position
was
3-methoxybenzyl
>
piperonyl
>
4-
1
6
7
8
methoxybenzyl > 3,4-dimethoxybenzyl. A cell-based MTT assay
was performed and none of the compounds tested herein was
shown to be toxic to FRTL5 cell lines at 1
mM concentration. In
addition, Lipinski’s rule of five is respected for DHPMs 6e17. Mo-
lecular weights are in the range 342e386 g/mol and ClogP are in
the range 2.5e3.7 [18]. Taken together, among the analogs tested
herein, compounds 6, 8, 9, 11, 15 and 17 are the most promising
leads for further investigations in animal models.
9
0.57
75
0.065
400
1000
65
4.0
73
0.85
10
11
12
13
14
15
16
17
4. Assignment of the bioactive absolute configuration
a
IC50 values were averaged from at least two in-
dependent experiments. NaClO4 and 1 were used as
controls. A two-fold standard deviation was deemed
acceptable. DHPMs were tested as racemic mixtures.
The resolution of 1, 6 and 17 into their respective enantiomers
was performed in order to evaluate the impact of stereochemistry
on activity. The three compounds were applied to chiral HPLC using
analytical Chiralcel OD or Chiralpak AD columns (250 ꢁ 4.6 mm).
Each pair of enantiomers was obtained with excellent stereo-
chemical purities (>99%). The absolute configuration of each
enantiomer was assigned by circular dichroism (CD) spectroscopy.
Based on the comparison of experimental CD spectra (Fig. 3) with
reference CD spectra of DHPMs with known absolute configuration
[19], the enantiomers showing a negative Cotton effect around
280e300 nm were identified as (R)-1, (S)-6 and (S)-17. Note-
worthily, (R)-1, (S)-6 and (S)-17 have the same stereochemical
orientation at C4 [20]. Biological evaluation of each pair of enan-
tiomers showed that the activity is concentrated in one enantiomer
whereas the other does not contribute significantly to the observed
activity (Table 2). Therefore, it can be concluded that the mecha-
nism of action of 1, 6 and 17 for the inhibition of NIS is strongly
stereoselective. It probably involves a highly specific interaction
between the DHPM and the chiral environment of the recognition
site. However, it must be kept in mind that pharmacodynamic
processes in cellulo may, at least partially, account for the observed
difference in activity between two enantiomers. It can also be noted
that Pfeiffer’s rule is not violated [21], as the eudismic ratios of the
two most active compounds (14,000 and 900 for 6 and 17, resp.) are
higher than that of the less active derivative (21 for 1) [22].
b
From Ref. [12].
using the non-radioactive arsenic/cerium titration method devel-
oped earlier by our group [17]. Compound 1 was used as the ref-
erence compound (IC50 ¼ 89 nM) and sodium perchlorate as an
assay control (IC50 ¼ 0.1
mM).
Table 1 reports the IC50 values for the 12 tested DHPMs. As
reported earlier, replacing the C4-phenyl ring of 1 by a furan-2-yl
group improved potency, as shown by the IC50 value of 3.2 nM
for compound 6 [12]. When the 4-methoxybenzyl ester group was
replaced by a 3-methoxybenzyl ester, the resulting compound 9
showed a great improvement in activity with an IC50 value of
0.57 nM. A significant loss of activity for the 3,4-dimethoxy deriv-
ative 12 was observed (IC50 ¼ 0.4
mM). However, the potency was
almost completely recovered with the piperonyl group (15,
IC50 ¼ 4.0 nM). The N1 methylation had a negative impact on ac-
tivity as judged by the IC50 values of 7, 10, 13 and 16, which ranged
from 73 nM to 1 mM. Very interestingly, the N1,N3-dimethylated
compounds (8, 11, 14, 17) exhibited much stronger potencies
(IC50 ¼ 65 pMe65 nM) than their non-methylated counterparts.
This observation was especially true for the 3-methoxybenzyl ester
derivative (11), which exhibited a remarkable IC50 value of 65 pM.
Fig. 3. CD spectra and structure of (R)- and (S)-1, (R)- and (S)-6, (R)- and (S)-17.

P. Lacotte et al. / European Journal of Medicinal Chemistry 62 (2013) 722e727
725
20 min at 130 ꢀC and the resulting mixture was chromatographed
on silica gel (cHex/EtOAc 100/0e70/30).
Table 2
IC50 values of separated enantiomers of 1, 6 and 17 in
the inhibition of iodide entrapment in FRTL5 cells.a
Compound
IC50 (nM)
6.2.1. 4-Methoxybenzyl 3-oxobutanoate (2)
(R)-1
(S)-1
(R)-6
(S)-6
(R)-17
(S)-17
70
Compound 2 was prepared using Method A and isolated as
a yellow liquid (73%). 1H NMR (400 MHz, CDCl3)
d 2.16 (s, 3H), 3.64
1500
14,000
1.0
630
0.70
(s, 2H), 3.75 (s, 3H), 5.06 (s, 2H), 6.93 (d, J ¼ 8.0 Hz, 2H), 7.31 (d,
J ¼ 8.0 Hz, 2H). 13C NMR (100 MHz, CDCl3)
d 30.5, 50.0, 55.5, 66.3,
114.2, 128.1, 130.5, 159.6, 167.7, 202.2.
a
IC50 values were averaged from at least two in-
dependent experiments. Each compound had a ste-
reochemical purity >99% (chiral HPLC).
6.2.2. 3-Methoxybenzyl 3-oxobutanoate (3)
Compound 3 was prepared using Method A and isolated as
a yellow liquid (87%). 1H NMR (400 MHz, CDCl3)
2.24 (s, 3H), 3.49
(s, 2H), 3.80 (s, 3H), 5.14 (s, 2H), 6.84e6.92 (m, 3H), 6.90 (m, 1H). 13C
NMR (100 MHz, CDCl3) 30.4, 50.2, 55.5, 67.2, 113.9, 114.2, 120.6,
d
5. Conclusion
d
In summary, a series of dihydropyrimidin-2-ones (6e17) was
synthesized and evaluated as NIS inhibitors in a cellular assay.
Among the derivatives, compounds 6, 8, 9,11,15 and 17 are the most
potent and 11 exhibited a remarkable IC50 value of 65 pM. The in-
hibition of NIS function by DHPMs was shown to be stereoselective
and the absolute configuration of the most active enantiomers was
assigned. This study provides important SAR information that will
be valuable for the development of compounds with in vivo efficacy
in the fields of thyroidology and radioprotection.
129.9, 137.0, 160.0, 167.1, 200.5.
6.2.3. 3,4-Dimethoxybenzyl 3-oxobutanoate (4)
Compound 4 was prepared using Method A and isolated as
a yellow liquid (70%). 1H NMR (400 MHz, CDCl3)
d 2.24 (s, 3H), 3.48
(s, 2H), 3.76 (s, 3H), 3.77 (s, 3H), 5.19 (s, 2H), 6.78e6.83 (m, 2H), 6.90
(d, J ¼ 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 30.3, 50.3, 56.0, 56.2,
62.8, 111.8, 114.4, 115.9, 124.7, 151.9, 153.7, 167.2, 200.7.
6.2.4. 3,4-(Methylenedioxy)benzyl 3-oxobutanoate (5)
6. Experimental section
Compound 5 was prepared using Method A and isolated as
a yellow liquid (91%). 1H NMR (400 MHz, CDCl3)
d
2.24 (s, 3H), 3.48
(s, 2H), 5.07 (s, 2H), 5.97 (s, 2H), 6.77e6.85 (m, 3H). 13C NMR
(100 MHz, CDCl3) 30.5, 50.0, 66.4, 101.5, 108.5, 109.3, 122.6, 129.9,
6.1. General methods
d
Reagents and solvents were from SigmaeAldrich without fur-
ther purification. Microwave-assisted reactions were run on a Dis-
cover SP system (CEM) equipped with an explorer module. Flash
chromatography was performed on a CombiFlash Rf system (Tele-
dyne Isco) using normal phase Redisep (Teledyne Isco) or SNAP
(Biotage) cartridges. The HPLCeMS analysis was performed on
a system equipped with a binary gradient solvent delivery system
(LC-20AB, Shimadzu), a SIL-20A autosampler (Shimadzu) and
a photodiode array detector (SPD-20A, Shimadzu, 200e400 nm).
This system was coupled to an electrospray ionization
Micromass-ZQ spectrometer (Waters) operating in both positive
147.6, 147.7, 167.6, 202.0.
6.3. General procedure (Method B) for the parallel synthesis of
DHPMs 6e17 by the Biginelli reaction
Acetoacetate 2e5 (0.50 mmol), aldehyde (0.60 mmol), urea
(0.75 mmol) and Zn(OTf)2 (10 mol%) were dissolved in acetonitrile
and refluxed for 2e16 h. The reaction mixture was then allowed to
cool to room temperature. The solvent was evaporated under
reduced pressure and the residue was chromatographed on silica
gel (cHex/EtOAc 100/0e50/50).
and negative mode. Each compound (8e15
mg) was applied to
a 250 ꢁ 4.6 mm (5
m
m) Zorbax SB-C18 (Agilent) equilibrated with
6.3.1. 4-Methoxybenzyl 4-(furan-2-yl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (6)
acetonitrile/water ¼ 30/70 (1 mL/min). Samples were eluted by
increasing acetonitrile to 45% (10 min), then 85% (25e30 min). 1H
and 13C NMR spectra were recorded on a Bruker Avance DPX 400
spectrometer operating at 400 MHz (1H) and 100 MHz (13C). The
Compound 6 was prepared using Method B and isolated as an
ocher powder (40%). mp 158e159 ꢀC. TLC Rf ¼ 0.26 (cHex/EtOAc 1/
1). 1H NMR (400 MHz, DMSO-d6)
d 2.23 (s, 3H), 3.74 (s, 3H), 4.99 (s,
chemical shifts (d) were expressed in ppm. Melting points (B-540,
2H), 5.20 (d, J ¼ 3.2 Hz, 1H), 6.04 (d, J ¼ 2.8 Hz, 1H), 6.35 (dd, J ¼ 2.0,
Büchi) are uncorrected. High-resolution mass spectra (HRMS) were
performed on the imagif platform (CNRS e Gif sur Yvette, France),
and recorded on a ESI/TOF LCP premier XE mass spectrometer
(Waters) using flow injection analysis mode. Circular dichroism
spectra were recorded on a Jasco-815 (Jasco) equipped with
a Peltier type thermostating accessory (CDF-426S, Jasco). Mea-
surements were carried out at 20 ꢀC using a 1-mm quartz cell in
3.2 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 2H), 7.20 (d, J ¼ 8.4 Hz, 2H), 7.55 (s,
1H), 7.76 (s, 1H), 9.29 (s, 1H). 13C NMR (100 MHz, DMSO-d6)
d 18.2,
48.1, 55.5, 65.2, 96.9, 105.8, 110.8, 114.2, 128.9, 129.9, 142.6, 150.4,
152.8,156.3,159.4,165.5. HPLC tR ¼ 13.1 min. MS m/z 343 ([M þ H]þ).
HRMS-ESI-TOF m/z calculated 341.1137, found 341.1141 ([M ꢂ H]ꢂ).
6.3.2. 4-Methoxybenzyl 4-(furan-2-yl)-1,6-dimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (7)
a volume of 300e350
mL. Compounds (200
mg) were dissolved in
MeOH (500 L)/DMSO (26e29
bandwidth, 1.0 nm; data pitch, 0.2 nm; speed, 50 nm/min; accu-
mulation, 2; wavelengths, 370e220 nm.
m
m
L). The instrument settings were:
Compound 7 was prepared using Method B and isolated as
a yellow solid (49%), mp 112e113 ꢀC. TLC Rf ¼ 0.28 (cHex/AcOEt 1/
1). 1H NMR (400 MHz, CDCl3)
d 2.49 (s, 3H), 3.15 (s, 3H), 3.76 (s, 3H),
5.03 (s, 1H), 5.04 (s, 1H), 5.41 (d, J ¼ 3.6 Hz, 1H), 5.97 (d, J ¼ 3.2 Hz,
1H), 6.15e6.19 (m, 1H), 6.32 (d, J ¼ 3.2 Hz, 1H), 6.82 (d, J ¼ 8.8 Hz,
2H), 7.16 (d, J ¼ 8.8 Hz, 2H), 7.24 (d, J ¼ 0.8 Hz, 1H). 13C NMR
6.2. General procedure (Method A) for the synthesis of intermediate
acetoacetates 2e5
(100 MHz, CDCl3)
d 16.6, 30.4, 47.6, 55.3, 65.9, 101.3, 105.8, 110.2,
2,2,6-trimethyl-4H-1,3-dioxin-4-one (1.0 mL, 7.7 mmol) and
alcohol (5.9 mmol) were mixed with potassium acetate (241 mg,
2.9 mmol) in a microwave vial. The mixture was microwaved for
113.9, 128.3, 129.8, 142.3, 151.4, 154.5, 155.0, 159.5, 165.6. HPLC
tR ¼ 16.1 min. MS m/z 357 ([M þ H]þ). HRMS-ESI-TOF m/z calculated
357.1450, found 357.1464 ([M þ H]þ).

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P. Lacotte et al. / European Journal of Medicinal Chemistry 62 (2013) 722e727
6.3.3. 4-Methoxybenzyl 4-(furan-2-yl)-1,3,6-trimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (8)
6.3.8. 3,4-Dimethoxybenzyl 4-(furan-2-yl)-1,6-dimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (13)
Compound 8 was prepared using Method B and isolated as
Compound 13 was prepared using Method B and isolated as
a yellow oil (46%). TLC Rf ¼ 0.28 (cHex/EtOAc 1/1). 1H NMR
a yellow solid (48%), mp 155e156 ꢀC. TLC Rf ¼ 0.28 (cHex/AcOEt 1/
(400 MHz, CDCl3)
d
2.48 (s, 3H), 2.96 (s, 3H), 3.20 (s, 3H), 3.78 (s,
1). 1H NMR (400 MHz, CDCl3)
d 2.51 (s, 3H), 3.18 (s, 3H), 3.69 (s, 3H),
3H), 5.02 (d, J ¼ 12.4 Hz, 1H), 5.08 (d, J ¼ 12.0 Hz, 1H), 5.28 (s, 1H),
6.00 (d, J ¼ 2.8 Hz, 1H), 6.22e6.23 (m, 1H), 6.84 (d, J ¼ 8.8 Hz, 2H),
7.18 (d, J ¼ 8.8 Hz, 2H), 7.26 (s, J ¼ 0.8 Hz, 1H). 13C NMR (100 MHz,
3.72 (s, 3H), 5.12 (d, J ¼ 12.8 Hz,1H), 5.17 (d, J ¼ 13.2 Hz, 1H), 5.45 (d,
J ¼ 3.2 Hz, 1H), 6.00e6.02 (m, 2H), 6.19-6.20 (m, 1H), 6.74e6.79 (m,
3H), 7.25 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 16.6, 30.5, 47.7, 55.9,
CDCl3)
d
16.7, 31.3, 34.9, 54.5, 55.4, 66.0, 100.5, 107.0, 110.2, 114.0,
56.0, 61.6, 101.5, 105.9, 110.3, 111.5, 113.7, 115.3, 125.6, 142.4, 151.4,
151.6, 153.5, 154.5, 154.9, 165.6. HPLC tR ¼ 16.6 min. MS m/z 387
([M þ H]þ). HRMS-ESI-TOF m/z calculated 387.1556, found 387.1554
([M þ H]þ).
128.5, 129.9, 142.5, 151.5, 153.3, 154.1, 159.6, 165.6. HPLC
tR ¼ 19.1 min. MS m/z 371 ([M þ H]þ). HRMS-ESI-TOF m/z calculated
371.1607, found 371.1592 ([M þ H]þ).
6.3.4. 3-Methoxybenzyl 4-(furan-2-yl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (9)
6.3.9. 3,4-Dimethoxybenzyl 4-(furan-2-yl)-1,3,6-trimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (14)
Compound 9 was prepared using Method B and isolated as an
Compound 14 was prepared using Method B and isolated as
orange solid (70%). mp 165e166 ꢀC. TLC Rf ¼ 0.35 (cHex/EtOAc 4/6).
a white solid (44%), mp 122e124 ꢀC. TLC Rf ¼ 0.28 (cHex/AcOEt 6/4).
1H NMR (400 MHz, CDCl3)
d
2.38 (s, 3H), 3.78 (s, 3H), 5.07 (d,
1H NMR (400 MHz, CDCl3)
d 2.50 (s, 3H), 2.98 (s, 3H), 3.21 (s, 3H),
J ¼ 12.8 Hz, 1H), 5.13 (d, J ¼ 12.8 Hz, 1H), 5.52 (m, 2H), 6.09 (d,
3.71 (s, 3H), 3.74 (s, 3H), 5.11 (d, J ¼ 13.2 Hz, 1H), 5.19 (d, J ¼ 12.8 Hz,
1H), 5.32 (s, 1H), 6.04 (d, J ¼ 3.2 Hz, 1H), 6.21e6.22 (m, 1H), 6.77e
6.78 (m, 3H), 7.26 (s, J ¼ 0.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
J ¼ 3.2 Hz, 1H), 6.25e6.27 (m, 1H), 6.78e6.85 (s, 3H), 7.21e7.32 (m,
3H). 13C NMR (100 MHz, CDCl3)
d 19.1, 49.1, 55.4, 66.1, 89.1, 106.4,
110.5, 113.5, 113.7, 120.3, 129.8, 137.8, 142.7, 154.7, 157.1, 159.9, 171.8.
HPLC tR ¼ 13.4 min. MS m/z 343 ([M þ H]þ). HRMS-ESI-TOF m/z
calculated 341.1137, found 341.1140 ([M ꢂ H]ꢂ).
d 16.7, 30.4, 34.9, 54.5, 55.9, 56.1, 61.6, 100.7, 107.0, 110.2, 111.6, 113.7,
115.4, 125.8, 142.5, 151.5, 151.7, 153.3, 153.6, 154.2, 165.6. HPLC
tR ¼ 19.5 min. MS m/z 401 ([M þ H]þ). HRMS-ESI-TOF m/z calculated
401.1713, found 401.1719 ([M þ H]þ).
6.3.5. 3-Methoxybenzyl 4-(furan-2-yl)-1,6-dimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (10)
6.3.10. Benzo[d][1,3]dioxol-5-ylmethyl 4-(furan-2-yl)-6-methyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15)
Compound 10 was prepared using Method B and isolated as
a yellow solid (51%), mp 155e156 ꢀC. TLC Rf ¼ 0.27 (cHex/AcOEt 1/
Compound 15 was prepared using Method B and isolated as
1). 1H NMR (400 MHz, CDCl3)
d 2.51 (s, 3H), 3.18 (s, 3H), 3.74 (s,
a white solid (45%), mp 169e171 ꢀC. TLC Rf ¼ 0.33 (cHex/EtOAc 4/6).
3H), 5.06 (d, J ¼ 8.4 Hz, 1H), 5.12 (d, J ¼ 8.4 Hz, 1H), 5.45 (d,
J ¼ 2.8 Hz, 1H), 6.01e6.02 (d, J ¼ 2.8 Hz, 1H), 6.07 (d, J ¼ 2.8 Hz,
1H), 6.20e6.22 (m, 1H), 6.77e6.82 (m, 3H), 7.19e7.23 (m, 1H), 7.26
1H NMR (400 MHz, DMSO-d6)
d
2.24 (s, 3H), 4.95 (d, J ¼ 12.0 Hz,
1H), 4.99 (d, J ¼ 12.4 Hz, 1H), 5.21 (d, J ¼ 3.2 Hz, 1H), 6.00 (s, 2H),
6.05 (d, J ¼ 3.2 Hz, 1H), 6.34e6.35 (m, 1H), 6.76 (d, J ¼ 8.0 Hz, 1H),
6.81 (s, 1H), 6.86 (d, J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 0.8 Hz, 1H), 7.77 (s,
(s, 1H). 13C NMR (100 MHz, CDCl3)
d 16.7, 30.5, 47.6, 55.3, 66.0,
101.2, 105.9, 110.3, 113.4, 113.7, 120.2, 129.6, 137.8, 142.4, 151.8,
1H), 9.30 (s, 1H). 13C NMR (100 MHz, DMSO-d6)
d 18.2, 48.1, 65.3,
154.5, 154.9, 159.8, 165.5. HPLC tR ¼ 16.3 min. MS m/z 357
96.8, 101.4, 105.8, 108.5, 108.8, 110.8, 122.0, 130.7, 142.6, 147.3, 147.7,
([M
þ
H]þ). HRMS-ESI-TOF m/z calculated 357.1450, found
150.5, 152.7, 156.3, 165.2. HPLC tR ¼ 12.7 min. MS m/z 357
357.1459 ([M þ H]þ).
([M
þ
H]þ). HRMS-ESI-TOF m/z calculated 355.0930 found
355.0930 ([M ꢂ H]ꢂ).
6.3.6. 3-Methoxybenzyl 4-(furan-2-yl)-1,3,6-trimethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (11)
6.3.11. Benzo[d][1,3]dioxol-5-ylmethyl 4-(furan-2-yl)-1,6-dimethyl-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (16)
Compound 11 was prepared using Method B and isolated as
a yellow oil (43%). TLC Rf ¼ 0.31 (cHex/EtOAc 6/4). 1H NMR
Compound 16 was prepared using Method B and isolated as
(400 MHz, CDCl3)
d 2.50 (s, 3H), 2.97 (s, 3H), 3.21 (s, 3H), 3.75 (s,
a yellow solid (30%), mp 141e143 ꢀC. TLC Rf ¼ 0.42 (cHex/AcOEt 4/
3H), 5.05 (d, J ¼ 12.4 Hz, 1H), 5.13 (d, J ¼ 12.8 Hz, 1H), 5.32 (s, 1H),
6.03 (d, J ¼ 3.2 Hz, 1H), 6.21e6.23 (m, 1H), 6.79e6.82 (m, 3H), 7.20e
7.24 (m, 1H), 7.27 (s, J ¼ 0.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
6). 1H NMR (400 MHz, DMSO-d6)
d 2.49 (s, 3H), 3.10 (s, 3H), 5.00 (s,
2H), 5.21 (d, J ¼ 4.0 Hz, 1H), 6.01 (s, 2H), 6.05 (d, J ¼ 3.2 Hz, 1H),
6.34e6.35 (m, 1H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.82 (s, 1H), 6.87 (d,
J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 0.8 Hz, 1H), 7.96 (d, J ¼ 4.0 Hz, 1H). 13C
d
16.7, 31.3, 34.9, 54.4, 55.3, 66.0, 100.3, 107.0, 110.2, 113.4, 113.7,
120.2, 129.7, 137.9, 142.5, 151.8, 153.3, 154.1, 159.9, 165.4. HPLC
tR ¼ 19.3 min. MS m/z 371 ([M þ H]þ). HRMS-ESI-TOF m/z calculated
371.1607, found 371.1595 ([M þ H]þ).
NMR (100 MHz, DMSO-d6) d 16.5, 30.3, 46.9, 65.6, 100.2, 101.5,
105.9, 108.5, 108.9, 110.8, 122.1, 130.5, 142.7, 147.4, 147.7, 152.7, 153.6,
155.9, 165.4. HPLC tR ¼ 15.7 min. MS m/z 371 ([M þ H]þ). HRMS-ESI-
TOF m/z calculated 371.1243, found 371.1230 ([M þ H]þ).
6.3.7. 3,4-Dimethoxybenzyl 4-(furan-2-yl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (12)
6.3.12. Benzo[d][1,3]dioxol-5-ylmethyl 4-(furan-2-yl)-1,3,6-
trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (17)
Compound 17 was prepared using Method B and isolated as
a brown oil (70%). TLC Rf ¼ 0.28 (cHex/EtOAc 1/1). 1H NMR
Compound 12 was prepared using Method B and isolated as
a yellow solid (73%), mp 72e75 ꢀC. TLC Rf ¼ 0.22 (cHex/AcOEt 1/1).
1H NMR (400 MHz, CDCl3)
d 2.31 (s, 3H), 3.68 (s, 3H), 3.70 (s, 3H),
5.10 (d, J ¼ 12.8 Hz, 1H), 5.15 (d, J ¼ 13.2 Hz, 1H), 5.48 (d, J ¼ 2.8 Hz,
1H), 6.04 (d, J ¼ 3.2 Hz, 1H), 6.17e6.19 (m, 1H), 6.52 (s, 1H), 6.72e
6.75 (m, 3H), 7.24 (s, 1H), 8.94 (s, 1H). 13C NMR (100 MHz, CDCl3)
(400 MHz, CDCl3) d 2.51 (s, 3H), 2.99 (s, 3H), 3.23 (s, 3H), 5.00 (d,
J ¼ 12.0 Hz, 1H), 5.08 (d, J ¼ 12.4 Hz, 1H), 5.32 (s, 1H), 5.94 (s, 2H),
6.05 (d, J ¼ 3.2 Hz,1H), 6.25e6.26 (m,1H), 6.74e6.75 (m, 3H), 7.30 (d,
d
18.5, 48.8, 55.8, 56.0, 61.4, 98.3, 106.1, 110.3, 111.5, 113.6, 115.3,
J ¼ 0.8 Hz,1H).13C NMR (100 MHz, CDCl3)
d 16.5, 31.1, 34.7, 54.2, 65.9,
125.6, 142.4, 148.7, 151.6, 153.5, 154.7, 154.9, 165.4. HPLC
tR ¼ 13.8 min. MS m/z 373 ([M þ H]þ). HRMS-ESI-TOF m/z calcu-
lated 373.1400, found 373.1398 ([M þ H]þ).
100.2, 101.1, 106.8, 108.1, 108.7, 110.1, 121.8, 130.0, 142.3, 147.4, 147.7,
151.5, 153.1, 153.9, 165.3. HPLC tR ¼ 18.5min. MS m/z 385 ([M þ H]þ).
HRMS-ESI-TOF m/z calculated 385.1400, found 385.1386 ([M þ H]þ).

P. Lacotte et al. / European Journal of Medicinal Chemistry 62 (2013) 722e727
727
6.4. Chiral resolution
Acknowledgments
6.4.1. Chiral separation of 1
We thank Celine Puente (CEA-iBiTecS, Saclay, France) for per-
forming the LC-MS analysis and Jean-Christophe Cintrat for review-
ingthemanuscript. ThisworkwassupportedbyCEA-iBiTecS(France),
Université Paris-Sud (France) and the Laboratory of Excellence in
Research on Medication and Innovative Therapeutics (LERMIT).
Compound 1 (8 batches of 0.48 mg each) was applied to
a 250 ꢁ 4.6 mm Chiralcel OD column (DAICEL) equilibrated with
nHex/EtOH ¼ 92/8. (S)-1 (1.70 mg, tR ¼ 23.9 min) and (R)-1
(1.85 mg, tR ¼ 28.3 min) were isolated and further HPLC analysis of
separated enantiomers showed >99% stereochemical purity. (S)-1
and (R)-1 did not racemize after 5 months at ꢂ20 ꢀC in 20 mM
DMSO.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.043.
6.4.2. Chiral separation of 6
Compound 6 (10 batches of 0.36 mg each) was applied to
a 250 ꢁ 4.6 mm Chiralcel OD column (DAICEL) equilibrated with
nHex/EtOH ¼ 90/10. (S)-6 (1.64 mg, tR ¼ 27.6 min) and (R)-6
(1.65 mg, tR ¼ 30.4 min) were isolated and further HPLC analysis of
separated enantiomers showed >99% stereochemical purity. (S)-6
and (R)-6 did not racemize after 4 months at ꢂ20 ꢀC in 20 mM
DMSO.
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6.5. Biological evaluation of the synthesized compounds
The biological activity of each compound was determined in
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described elsewhere [17]. Compound potency was expressed as
IC50, the concentration of compound necessary to achieve 50% in-
hibition of iodide uptake. Briefly, to FRTL5 cells at 70e90% con-
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fluence was added the compound (200
1.2 nM, 60 pM, 30 pM, and 0.15 pM), followed by NaI (10
mM, 10
mM, 0.5
m
M, 25 nM,
mM). After
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1-h incubation at 20 ꢃ 1 ꢀC, supernatant was removed and the cells
were immediately assayed for iodide content using the modified
As/Ce SandelleKolthoff reaction. Stock solutions of DHPMs (1, 6e
17) were in DMSO (20 mM) and NaClO4 was in water at (20 mM).
Compounds were tested in columns 4e11 of 96-well polystyrene
microplates (Costar 9017, VWR). NaClO4 and 1 were tested in each
microplate (in columns 2 and 3) as assay controls. Columns 1 and 12
were for iodide standards. For IC50 determination, experimental
data were fitted by non-linear regression to the four-parameter
sigmoidal Hill equation using an “in-house” application devel-
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6.6. Cell viability
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[20] (R)-1, (S)-6 and (S)-17 are homochirals. There is an inversion of Cahne
IngoldePrelog priority (CIP) rules between 6/17 and 1 due to the replacement
of the phenyl ring by a furan-2-yl.
Cell viability was tested according to an MTT-based assay [23].
Briefly, to FRTL5 cells at w50% confluence was added the com-
pound (1 mM). Cell viability was determined at the 24-h end point
before the addition of MTT (1.2 mg/mL). Absorbance at 570 nm
was determined after 3-h incubation at 37 ꢀC using a 96-well
plate reader (Spectramax plus 384, Molecular Devices). Ouabain
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mer is the enantiomer presenting the highest activity. Distomer is the
enantiomer presenting the lowest activity.
(2
m
Me1 mM).
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