Neuroprotective efficacy of quinazoline type phosphodiesterase 7 inhibitors in cellular cultures and experimental stroke model

Article abstract of DOI:10.1016/j.ejmech.2011.10.040

A simple and efficient synthetic method for the preparation of quinazoline type phosphodiesterase 7 (PDE7) inhibitors, based on microwave irradiation, has been developed. The use of this methodology improved yields and reaction times, providing a scalable procedure. These compounds are pharmacologically interesting because of their in vivo efficacy both in spinal cord injury and Parkinson's disease models, as shown in previous studies from our group. Herein we describe for the first time that administration of one of the PDE7 inhibitors here optimized, 3-phenyl-2,4-dithioxo-1,2,3,4-tetrahydroquinazoline (compound 5), ameliorated brain damage and improved behavioral outcome in a permanent middle cerebral artery occlusion (pMCAO) stroke model. Furthermore, we demonstrate that these PDE7 inhibitors are potent anti-inflammatory as well as neuroprotective agents in primary cultures of neural cells. These results led us to propose PDE7 inhibitors as a new class of therapeutic agents for neuroprotection.

Full text of DOI:10.1016/j.ejmech.2011.10.040

European Journal of Medicinal Chemistry 47 (2012) 175e185
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Neuroprotective efficacy of quinazoline type phosphodiesterase 7 inhibitors
in cellular cultures and experimental stroke model
Miriam Redondo a, Juan G. Zarruk b, Placido Ceballos a, Daniel I. Pérez a, Concepción Pérez a,
Ana Perez-Castillo c, María A. Moro b, José Brea d, Cristina Val d, María I. Cadavid d, María I. Loza d,
,
Nuria E. Campillo a, Ana Martínez a, Carmen Gil a
a Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
*
b Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid, Avd. Complutense s/n, 28040 Madrid, Spain
c Instituto de Investigaciones Biomédicas (CSIC-UAM) and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Arturo Duperier,
4, 28029 Madrid, Spain
d Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur s/n, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and efficient synthetic method for the preparation of quinazoline type phosphodiesterase 7
(PDE7) inhibitors, based on microwave irradiation, has been developed. The use of this methodology
improved yields and reaction times, providing a scalable procedure. These compounds are pharmaco-
logically interesting because of their in vivo efficacy both in spinal cord injury and Parkinson’s disease
models, as shown in previous studies from our group. Herein we describe for the first time that
administration of one of the PDE7 inhibitors here optimized, 3-phenyl-2,4-dithioxo-1,2,3,4-
tetrahydroquinazoline (compound 5), ameliorated brain damage and improved behavioral outcome in
a permanent middle cerebral artery occlusion (pMCAO) stroke model. Furthermore, we demonstrate that
these PDE7 inhibitors are potent anti-inflammatory as well as neuroprotective agents in primary cultures
of neural cells. These results led us to propose PDE7 inhibitors as a new class of therapeutic agents for
neuroprotection.
Received 12 July 2011
Received in revised form
18 October 2011
Accepted 21 October 2011
Available online 4 November 2011
Keywords:
PDE7 inhibitors
Neuroprotection
Stroke
Quinazolines
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Recently, several reports have suggested that PDEs are new
targets for central nervous system (CNS) diseases [7,8]. The PDE
Phosphodiesterases (PDEs) selectively degrade cyclic purine
nucleotides (cAMP and cGMP) that serve as second messengers in
a number of cellular pathways. PDEs have proven to be druggable
targets, with compounds on the market or in late-stage clinical
development for a variety of diseases [1].
Theophylline was probably the first non-selective PDE inhibitor
to be used clinically for the treatment of asthma, acting as bron-
chodilator [2]. However, the known PDEs heterogeneity led to the
synthesis of highly selective inhibitors [3], which have demon-
strated efficacy in a variety of disorders such as cilostazol (PDE3
inhibitor) for intermittent claudication [4], roflumilast (PDE4
inhibitor) for chronic obstructive pulmonary disease [5], and sil-
denafil (PDE5 inhibitor) for male erectile dysfunction and pulmo-
nary hypertension [6].
family is comprised by 11 different subfamilies, being PDE1, PDE4,
PDE7 and PDE10 highly expressed in brain. Since it has been sug-
gested that cAMP pathways could be involved in neurodegenera-
tive diseases, interfering with both the inflammatory and
neurotransmitter cascades, selective inhibitors of these PDEs could
represent a novel approach to treat several CNS diseases [9,10].
PDE7 is a cAMP-specific enzyme not modulated by rolipram
(PDE4 inhibitor), expressed across a variety of brain structures
apart from its expression on T-cells [11]. PDE7 emerged as a new
therapeutic target, not only for a variety of immunological and
immunodeficiency conditions to alleviate chronic inflammation
[12], but also for several neurodegenerative disorders including
multiple sclerosis (MS) in which both autoimmune system and CNS
are implicated [13]. Very recently, the usefulness of this new drug
target has been studied in different animal models showing
preclinical positive data for the treatment of spinal cord injury [14]
and Parkinson’s disease (PD) [15].
Benzothiadiazine and benzothienothiadiazine derivatives,
developed in our group, constituted the first described heterocyclic
* Corresponding author. Tel.: þ34 91 5622900; fax: þ34 91 5644853.
E-mail address: cgil@iqm.csic.es (C. Gil).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.040

176
M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
family of compounds with PDE7 inhibitory properties [16,17].
Afterward, several diverse heterocyclic compounds such as spi-
roquinazolinones, sulfonamide and thiadiazole analogs have been
S
O
Ph
S
Ph
S
i
N
N
described as PDE7 inhibitors [13,18].
A ligand-based virtual
N
H
N
H
screening using the descriptors generated by CODES program [19],
pointed out the quinazoline derivatives as new PDE7 inhibitors
[20]. After a preliminary synthetic optimization, new and potent
quinazoline derivatives were obtained and the crystal structure of
one of them within the PDE7A1 catalytic domain was solved by X-
ray diffraction [21].
1
5
Scheme 1. Reagents: (i) Lawesson reagent, toluene, 120 ꢀC, 2 h, MW.
further development. The results are gathered in Table 2 demon-
strating that this methodology is valid also for more diverse
quinazolines.
We have recently demonstrated that PDE7 inhibitors belonging
to the quinazoline family enhance neuroprotection and diminish
neuroinflammation in well-characterized cellular and animal
models of PD and spinal cord injury [14,15]. Due to this fact, here we
propose the study of a possible neuroprotective activity of quina-
zoline type PDE7 inhibitors, both in primary cell cultures and in
a well-characterized in vivo model of stroke. With this aim, their
synthetic pathway has been optimized and new quinazoline
derivatives have been synthesized as continuation of our on-going
research on new quinazoline type PDE7 inhibitors.
The new designed quinazolines (6e25) were proposed based on
the fact that a major drawback of some of the previously prepared
quinazoline type PDE7 inhibitors was the lack of solubility. Thus,
some modifications were done to improve the solubility and also in
order to establish additional interactions in the PDE7 catalytic
domain based on the X-ray data reported previously [21,23].
First, a classical isosteric replacement of the thiocarbonyl at C-2
by carbonyl group was carried out. Following with the application
of isosteres the substitution of CH with N at whichever of the
aromatic rings should improve also the aqueous solubility [24].
Second, in order to improve solubility by disruption of mole-
cular planarity [25] we propose to increase the distance between
the quinazoline core and the aryl group at N-3, introducing 1 or 2
methylene groups.
Third, the introduction of a variety of substituents in the fused
ring was performed, to achieve an electronic enrichment of that
aromatic ring in order to improve the stacking interaction with the
conserved Phe416 which is a key feature in the binding mode of
quinazoline type PDE7 inhibitors with the catalytic domain of
PDE7A1 [21].
2. Chemistry
The synthesis of quinazoline type PDE7 inhibitors was previ-
ously described [20,21] by cyclocondensation of the corresponding
functionalized anthranillic acid derivatives with isothiocyanates,
subsequent thionation of the carbonyl group at 4-position and/or
methylation of sulfur at 2-position when required. The major
drawbacks of this procedure were the long reaction times and poor
yields. Due to their promising pharmacological profile, we were
interested in developing a more efficient synthetic pathway,
reducing the reaction times and improving the overall yields. In this
sense, we decided to prepare the PDE7 inhibitors 1e4 previously
described [20,21] by using microwave assisted organic synthesis
(MAOS) which has been successfully applied to obtain 2-
thioxoquinazolinones [22]. Noteworthy is the fact that the
protocol based on MAOS yielded the desired products faster than
the conventional heating (Table 1).
Table 2 summarizes the different compounds obtained with
these purposes, indicating the best reaction conditions found in
each case. In all cases the condensations were carried out under
microwave irradiation in shorter times and easier purification
procedures which confirms the convenience of this methodology
for the synthesis of this kind of compounds.
Continuing of our research regarding quinazoline derivative
synthesis, we investigated the thionation reaction of this kind of
molecules by using also the MAOS technique. In this case,
The structures of all the compounds were determined by their
analytical and spectroscopic data (1H and 13C NMR), which are
described in the Experimental Section.
compound 5 (IC50 PDE7A ¼ 1.04
mM [21]) was successfully obtained
after reaction of 1 with Lawesson reagent in toluene, at 120 ꢀC after
2 h, in 50% yield. The improvement of the MAOS is reflected on
a significant reduction of reaction time, 2 h instead of 48 h in the
conventional synthesis (Scheme 1) [21].
3. Biological results and discussion
3.1. In vitro evaluation of PDE7 inhibition
These results encouraged us to apply this MAOS procedure to
the design of new quinazolines with major functionality with the
aim of obtaining a good lead with improved drug-like properties for
The new derivatives here synthetized (6e25) were tested for
their inhibitory potencies against the catalytic domain of PDE7A as
described in the Experimental Section (Table 3). Among them only
two of them (12 and 13) are potent PDE7 inhibitors in the micro-
molar range.
Table 1
These results led us to confirm a key stacking interaction
between the fused benzene ring and the enzyme. In this way we
can explain the activity of derivatives 12 and 13 which have
a chlorine atom attached. Electronic deficient rings have been
described as the preferred over the electron rich ones when
a stacking interaction is relevant in the molecular recognition in
biological systems [26]. On the other hand, the modifications here
proposed to increase solubility, such as the disruption of molecular
planarity and bioisosteric replacements, were incompatible with
optimal biological interaction. While these results are analyzed in
deep by different QSAR methods, the improvement of solubility for
this class of compounds is in progress, being performed by phar-
maceutical technology development.
MAOS of quinazoline type PDE7 inhibitors.
O
O
R3
NCS
MW, 120 ºC
40 min
OR1
N
R3
R2
R2
+
NH2
N
H
S
Comp.
R1
R2
R3
Solvent
Yield
IC50 (PDE7A)
1
2
3
4
Me
Me
Me
H
H
H
H
8-Me
H
2-Br
2,6-diF
2-Br
Toluene
70%
60%
60%
30%
5.5
1.9
5.5
1.7
m
m
m
m
M [20]
M [20]
M [20]
M [21]
DMSO/H2O
DMSO/H2O
Toluene

M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
177
Table 2
New cyclocondensation reactions for quinazoline synthesis.
O
O
R3
Z
R3
Z C N
MW
OR1
N
R2
R2
+
Y
Y
solvent
X
N
H
X
NH2
Comp.
R1
R2
X
Y
Z
R3
Solvent
MeCNa
Temp.
170 ꢀC
Time
Yield
6
Me
e
CH
CH
O
30 min
33%
7
8
9
Me
Me
Me
e
e
e
CH
N
CH
CH
CH
O
O
O
Dioxanea
MeCNa
170 ꢀC
170 ꢀC
160 ꢀC
30 min
30 min
45 min
84%
28%
46%
CH
1,2-DME
10
11
12
H
H
H
6-Br
e
CH
CH
CH
CH
N
O
O
S
MeCNa
MeCNa
145 ꢀC
170 ꢀC
120 ꢀC
45 min
60 min
40 min
17%
21%
63%
8-Cl
CH
DMSO/H2O
DMSO/H2O
13
H
8-Cl
CH
CH
S
120 ꢀC
40 min
18%
14
15
16
17
18
19
H
6-Me,8-Br
CH
CH
N
CH
CH
CH
CH
N
S
S
S
S
S
S
MeCN
150 ꢀC
175 ꢀC
150 ꢀC
160 ꢀC
150 ꢀC
170 ꢀC
45 min
30 min
30 min
30 min
30 min
45 min
55%
63%
5%
Me
Me
Me
H
6,7-diMeO
MeCNa
e
e
e
e
MeCNa
N
1,4-Dioxane
MeCNa
18%
35%
87%
CH
CH
Me
CH
MeCNa
(continued on next page)

178
M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
Table 2 (continued )
Comp.
R1
R2
X
Y
Z
R3
Solvent
Temp.
Time
Yield
20
21
22
23
24
Me
H
6,7-diMeO
CH
CH
CH
CH
CH
CH
N
S
S
S
S
S
MeCNa
180 ꢀC
140 ꢀC
150 ꢀC
170 ꢀC
175 ꢀC
25 min
30 min
10 min
45 min
30 min
59%
69%
62%
76%
20%
e
1,2-DME
1,2-DMEb
MeCN
Me
Me
Me
e
CH
CH
CH
e
6,7-diMeO
MeCNb
25
Me
6,7-diMeO
CH
CH
S
MeCN
170 ꢀC
45 min
59%
a
With 2% triethylamine.
With 2% DMAP.
b
3.2. Neuroprotective studies in vitro
Table 3
PDE7A inhibition of compounds 6e25.
Our next step was to explore whether the quinazoline type PDE7
inhibitors are neuroprotective in different cell based assays. Thus,
we used primary cultures of astrocytes, microglia and neurons
treated with lipopolysaccharide (LPS), a potent inflammatory agent.
The potential anti-inflammatory activity of the selected PDE7
inhibitors was first tested by evaluating the production of nitrites
from primary cultured glial cells (astrocytes and microglia).
Cultures were incubated with the indicated concentrations of the
compounds 1, 4, 5, 12 and 13 for 1 h, and then cells were cultured
for another 24 h with LPS. When primary astrocytes and microglial
cells were stimulated with LPS (Figs. 1 and 2) we observed
a significant induction of nitrite production in the culture medium
(6- and 20-fold, respectively), which was significantly diminished
by PDE7 inhibitors treatment.
Continuing with the neuroprotective capacity, we investigated
whether these compounds were also effective protecting neurons
from the injury induced by cell-free supernatant from LPS-
activated microglia. As shown in Fig. 3, treatment of neuronal
cultures with compounds 1, 4 and 5, also resulted in a significant
reduction in nitrite production.
Comp.
%Inhibition PDE7A @10
m
M
IC50 (PDE7A)
6
7
2.5
9.3
e
e
e
e
e
e
8
8.0
9
ꢁ7.2
ꢁ8.3
22.1
74.9
63.4
4.0
13.2
ꢁ3.1
12.8
9.3
10.0
19.0
2.7
21.2
13.2
12.7
17.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6.1
1.8
e
m
m
M
M
e
e
e
e
e
e
e
e
e
e
e
Fig. 1. Effect of PDE7 inhibitors on the inflammatory response of astrocyte cultures. Rat primary astrocyte cultures were treated for 24 h with LPS (10
mg/ml) in the absence or
presence of PDE7 inhibitors and the production of nitrite was evaluated by the Griess reaction. Values represent the means ꢂ SD from three different experiments. ***, p ꢃ 0.001.

M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
179
Fig. 2. Effect of PDE7 inhibitors on the inflammatory response of microglial cultures. Rat primary microglial cultures were treated for 24 h with LPS (10
m
g/ml) in the absence or
presence of PDE7 inhibitors and the production of nitrite was evaluated by the Griess reaction. Values represent the means ꢂ SD from three different experiments. ***, p ꢃ 0.001, **,
p ꢃ 0.01, *, p ꢃ 0.05.
Altogether these results indicate that PDE7 inhibitors, specially
those of quinazoline scaffold, are potent anti-inflammatory and
neuroprotective agents in primary neural cultures.
were determined. An assay validation was made comparing the
reported permeability values of commercial drugs with the
experimental data obtained employing this methodology. A good
correlation between experimental-described values was obtained
Pe (exp) ¼ 0.9541 (bibl) e 0.536 (R2 ¼ 0.987) (see Supplementary
data). From this equation and following the pattern established
in the literature for BBB permeation prediction [31] we could
3.3. Blood brain barrier permeation prediction
Drug’s penetration through the bloodebrain barrier (BBB) is one
of the major obstacles for the treatment of diseases in the central
nervous system (CNS). The BBB is a protective barrier which is
designed to keep the environment in the brain as stable as possible. It
prevents many dangerous substances from entering the brain [27].
Brain penetration is influenced by several processes, such as tissue
binding in the bloodstream and the brain, metabolism in the liver and
the brain and active influx and efflux. Most compounds enter the
brain by transcellular passive diffusion, which is driven by a
concentration gradient between the blood and the brain [28]. In early
drug discovery stages, evaluation of ADME (Absorption, Distribution,
Metabolism, Excretion) properties is of crucial importance to reduce
attrition in development process [29]. Parallel Artificial Membrane
Permeability Assay (PAMPA) is a high throughput technique
developed to predict passive permeability through biological
membranes. In order to explore the capacity of the quinazoline
derivatives to penetrate into the brain and select the best
compound for in vivo studies, we used the PAMPA-BBB method
described by Di et al. [30] which employed a brain lipid porcine
membrane. The in vitro permeabilities (Pe) of commercial drugs
through lipid membrane extract together with compounds 4 and 5
classify compounds as CNS
þ
when they present a per-
meability > 3.26 ꢄ 10ꢁ6 cm sꢁ1. Based on these results we can
consider that compounds 4 and 5 are able to cross the BBB by
passive permeation. Compound 1 was classified as CNS þ in
a previous experiment [15]. We selected compound 5 for in vivo
studies due to its better IC50 value on the target.
3.4. Neuroprotective studies in vivo
Finally, to evaluate the neuroprotective efficacy in vivo we used
a stroke model by permanent middle cerebral artery occlusion
(pMCAO) in mice. Our data demonstrate that compound 5 (5 mg/
kg; administered 15 min after pMCAO) is neuroprotective as shown
by a reduction in infarct volume (15.4 ꢂ 1.1 vs. 10.8 ꢂ 1.1% of total
hemisphere volume in pMCAO þ vehicle vs. pMCAO þ comp. 5,
respectively, n ¼ 8e10; p < 0.05) and neurological deficit (6.5 ꢂ 0.2
vs. 4.4 ꢂ 0.3 points in mNSS score in MCAO vs. MCAO þ comp. 5,
respectively, n ¼ 8e10; p < 0.05), determined 48 h after MCAO
(Fig. 4).
Fig. 3. Effect of PDE7 inhibitors on the inflammatory response of cultured neurons. Rat primary neuronal cultures were treated for 24 h with cell-free supernatant from LPS-
stimulated microglia in the absence or presence of PDE7 inhibitors and the production of nitrite was evaluated by the Griess reaction. Values represent the means ꢂ SD from
three different experiments. ***, p ꢃ 0.001. **, p ꢃ 0.01.

180
M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
Fig. 4. (A) Pharmacological treatment and infarct outcome evaluation protocol. Effect of compound 5 on infarct volume determined 48 h after pMCAO (B) and on the modified
neurological severity score (mNSS) 24 and 48 h after pMCAO (C). Data are expressed as mean value ꢂ SEM., n ¼ 9e10, *p < 0.05 vs. vehicle, treated group. Unpaired t-Student’s test.
4. Conclusion
Experiments were performed with temperature control mode in
sealed microwave process vials. The temperature was measured
with an IR sensor on the outside of the reaction vessel. Stirring was
provided by an in situ magnetic stirrer.
Based on the results obtained here, we can conclude that PDE7
inhibitors, and specifically those of quinazoline type, present neuro-
protective activity modulating nitrite production in neural cells and
they also have a role in stroke by reducing the infarct volume and the
associated motor deficits. These results emphasize the idea of
a potential use of PDE7 inhibitors as therapeutic agents in stroke.
Moreover we have provided enough data to show that MAOS is an
efficient synthetic pathway for obtaining this class of potential new
drugs.
5.1.1. 4-Oxo-3-phenyl-2-thioxo-1,2,3,4-tetrahydroquinazoline (1)
A mixture of methyl anthranilate (0.25 mL, 1.98 mmol) and
phenylisothiocyanate (0.20 mL, 1.98 mmol) in toluene (4 mL) was
heated under microwave irradiation (40 min, 120 ꢀC). After cooling
down the reaction in an ice/salt bath, a white precipitate was
formed. The solid was filtered, washed with diethylether and dried,
affording 1 as a pure product (yield 70%). Mp 300e302 ꢀC (Lit. [32]
305e306 ꢀC); 1H NMR (300 MHz, DMSO-d6)
7.95 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.78 (t, J ¼ 7.6 Hz, 1H, H-7,), 7.51e7.27
(m, 7H, H-6, H-8, Ph); 13C NMR (75 MHz, DMSO-d6)
(ppm): 176.0,
d (ppm): 13.05 (bs,1H),
5. Experimental section
d
5.1. Chemistry
159.7, 139.5, 139.2, 135.5, 128.9, 128.8, 128.0, 127.3, 124.3, 116.1,
115.6; ESI MS (m/z): 255 [M þ H]þ; purity >99% (by HPLC); Anal.
C14H10N2OS (C, H, N, O).
Substrates were purchased from commercial sources and used
without further purification. Melting points were determined with
a Mettler Toledo MP70 apparatus. Flash column chromatography
was carried out at medium pressure using silica gel (E. Merck, Grade
60, particle size 0.040e0.063 mm, 230e240 mesh ASTM) with the
indicated solvent as eluent. Compounds were detected with UV light
(254nm).1H NMRspectrawereobtainedonthe BrukerAVANCE-300
spectrometer working at 300 MHz or on a Varian INOVA 400 spec-
trometer working at 400 MHz. Typical spectral parameters: spectral
5.1.2. 3-(2-Bromophenyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydroquinazoline (2)
A mixture of methyl anthranilate (0.25 mL, 1.98 mmol) and 2-
bromophenylisothiocyianate (0.25 mL, 1.98 mmol) in DMSO/H2O
(1:1) (4 mL) was heated under microwave irradiation (40 min,
120 ꢀC). After cooling down the reaction in an ice/salt bath, a white
precipitate was formed. The solid was filtered and purified by
column chromatography on silica using hexane/ethyl acetate (9:1)
as eluent affording 2 as a pure product (yield 60%). Mp 252e254 ꢀC
width 10 ppm, pulse width 9
m
s (57ꢀ), data size 32 K 13C NMR
experiments were carried out on the Bruker AVANCE-300 spec-
trometer operating at 75 MHz or on a Varian INOVA 400 spec-
trometer working at 100 MHz. The acquisition parameters: spectral
(Lit [33] 256e258 ꢀC); 1H NMR (300 MHz, DMSO-d6)
d (ppm): 13.18
(bs, 1H), 7.98 (dd, J ¼ 7.8, 0.9 Hz, 1H, H-5), 7.81 (ddd, J ¼ 8.5, 8.4,
1.3 Hz,1H, H-7), 7.76 (dd, J ¼ 8.2, 0.6 Hz,1H, H-Ar), 7.51e7.45 (m, 3H,
H-Ar, H-8), 7.40e7.35 (m, 2H, H-6, H-Ar); 13C NMR (75 MHz, DMSO-
width 16 kHz, acquisition time 0.99 s, pulse width 9 m
s (57ꢀ), data
size 32 K. Chemical shifts are reported in values (ppm) relative to
internal Me4Si and J values are reported in Hz. HPLC analyses were
performed on Alliance Waters 2690 equipment, with a UV detector
photodiode array Waters 2996 with MS detector MicromassZQ
d6) d (ppm): 175.0,159.0,139.6,138.1,136.0,132.7,131.2,130.2,128.6,
127.5, 124.6, 122.3, 115.8, 115.7; ESI MS (m/z): 333/335 [M þ H]þ;
purity 98% (by HPLC); Anal. C14H9BrN2OS (C, H, N, O).
(Waters), using an XBridge C18, 3.5
m
m, 200 Å (2.1 nm ꢄ 100 mm)
column. The standard gradient consisted of a 10 min run from 5% to
100% of acetonitrile at a flowrateof 0.25 mL/min. Elemental analyses
were performed by the analytical department at CENQUIOR (CSIC),
and the results obtained werewithin ꢂ0.4% of the theoretical values.
The microwave assisted syntheses were carried out using a Biotage
Initiator eight single-mode cavity instrument from Biotage.
5.1.3. 3-(2,6-Difluorophenyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydroquinazoline (3)
A mixture of methyl anthranilate (0.30 mL, 2.27 mmol) and 2,6-
difluorophenylisothiocyanate (0.30 mL, 2.27 mmol) in DMSO/H2O
(1:1) (4 mL) was heated under microwave irradiation (40 min,

M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
181
120 ꢀC). After cooling down the reaction in an ice/salt bath, a white
precipitate was formed. The solid was filtered, washed with
diethylether and dried, affording 3 as a pure product (yield 60%);
Mp 283e284 ꢀC (Lit. [21] 262e263 ꢀC); 1H NMR (300 MHz, DMSO-
triethylamine (8 mg, 0.08 mmol) in 1,4-dioxane (4 mL) was heated
under microwave irradiation (45 min, 140 ꢀC). After cooling down
the reaction in an ice/salt bath, a pale yellow precipitate was formed.
The solid was filtered, washed with diethyletherand dried, affording
7 as awhite solid (yield 84%). Mp 216.7 ꢀC (Lit. [34] 213.8 ꢀC); 1H NMR
d6)
d
(ppm): 13.47 (bs, 1H), 8.00 (dd, J ¼ 7.9, 0.9 Hz, 1H, H-5), 7.85
(ddd, J ¼ 8.4, 7.1, 0.9 Hz, 1H, H-7), 7.66e7.56 (m, 1H, H-Ar), 7.48 (d,
(300 MHz, DMSO-d6)
d
(ppm): 11.48 (s, 1H, NH), 7.92 (dd, J ¼ 7.2,
J ¼ 8.4 Hz, 1H, H-8), 7.41 (dd, J ¼ 7.9, 7.1 Hz, 1H, H-6), 7.35e7.29 (m,
0.9 Hz, 1H, H-5), 7.65 (dt, J ¼ 7.5, 1.6 Hz, 1H, H-7), 7.27 (d, J ¼ 8.7 Hz,
2H, H-30), 7.17 (t, J ¼ 7.8 Hz,1H, H-6), 6.85 (d, J ¼ 7.8 Hz,1H, H-8), 6.83
(d, J ¼ 8.7 Hz, 2nH- H-40), 5.00 (s, 2H, CH2), 3.69 (s, 3H, MeO); 13C
2H); 13C NMR (75 MHz, DMSO-d6)
d (ppm): 174.7, 158.7, 157.9, 139.7,
136.7, 131.5, 127.6, 125.2, 116.2, 115.2, 114.7, 112.3; ESI MS (m/z): 271
[M ꢁ F]þ; purity >99% (by HPLC); Anal. C14H8F2N2OS (C, H, N, O).
NMR (75 MHz, DMSO-d6)
d (ppm): 162.1, 158.5, 154.1, 141.2, 137.5,
132.8, 128.9, 126.0, 124.6, 117.8, 116.5, 114.7, 55,7, 43.9; ESI MS (m/z):
5.1.4. 3-(2-Bromophenyl)-8-methyl-4-oxo-2-thioxo-1,2,3,4-
tetrahydroquinazoline (4)
283 [M þ H]þ, purity 97% (by HPLC); Anal. C16H14N2O3 (C, H, N, O).
A
mixture of 2-amino-3-methylbenzoic acid (100 mg,
5.1.8. 3-(4-Methoxybenzyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido
[2,3-d]pyrimidine (8)
0.66 mmol) and 2-bromophenylisothiocyanate (0.089 mL,
0.66 mmol) in toluene (4 mL) was heated under microwave irra-
diation (40 min, 120 ꢀC). After cooling down the reaction in an ice/
salt bath, a white precipitate was formed. The solid was filtered and
purified by column chromatography on silica using hexane/ethyl
acetate (9:1) as eluent affording 4 as a white solid (yield 30%). Mp
209e210 ꢀC (Lit. [21] 209e211 ꢀC); 1H NMR (300 MHz, DMSO-d6)
A mixture of methyl 2-aminonicotinate (150 mg, 0.98 mmol), 1-
isocyanatomethyl-4-methoxybenzene (160 mg, 0.98 mmol) and
triethylamine (10 mg, 0.10 mmol) in acetonitrile (3 mL) was heated
under microwave irradiation (30 min, 170 ꢀC). After cooling down
the reaction in an ice/salt bath, a pale brown precipitate was
formed. The solid was filtered, washed with diethylether and dried,
affording 8 as a yellowish solid (yield 28%). Mp 251e253 ꢀC; 1H
d
(ppm): 11.98 (bs, 1H); 7.86 (d, J ¼ 7.9 Hz, 1H, H-5), 7.76 (d,
J ¼ 8.0 Hz, 1H, H-Ar), 7.65 (d, J ¼ 7.5 Hz, 1H, H-7), 7.56e7.46 (m, 2H,
H-Ar), 7.42e7.35 (m, 1H, H-Ar), 7.29 (dd, J ¼ 7.9, 7.5 Hz, 1H, H-6),
NMR (300 MHz, DMSO-d6)
J ¼ 6.7 Hz,1H), 8.31 (d, J ¼ 7.0 Hz,1H), 7.30 (m,1H), 7.17 (m,1H), 6.84
(m, 1H), 4.99 (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6)
(ppm):
d (ppm): 12.01 (s, 1H, NH), 8.66 (d,
2.53 (s, 3H, Me); 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 175.4, 159.0,
d
138.3, 138.0, 137.2, 132.7, 131.1, 130.2, 128.6, 125.3, 124.7, 124.4,
122.2, 116.0, 17.4; ESI MS (m/z): 267 [M ꢁ Br]; purity >99% (by
HPLC); Anal. C15H11BrN2OS (C, H, N, O).
160.4, 158.6, 152.2, 152.1, 148.4, 137.6, 130.9, 128.7, 121.1, 114.7, 108.2,
55,8, 46.1; ESI MS (m/z): 284 [M þ H]þ, purity 95% (by HPLC); Anal.
C15H13N3O3 (C, H, N, O).
5.1.5. 3-Phenyl-2,4-dithioxo-1,2,3,4-tetrahydroquinazoline (5)
A mixture of compound 1 (50 mg, 0.196 mmol) and Lawesson’s
reagent (120 mg, 0.294 mmol) in toluene (4 mL) was heated under
microwave irradiation (2 h, 120 ꢀC). After cooling down the reaction
in an ice/salt bath, an orange precipitate was formed. The solid was
filtered and purified by column chromatography on silica using
hexane/ethyl acetate (12:1) as eluent affording 5 as a pure product
(yield 50%). Mp 253e256 ꢀC (Lit. [21]. 252e254 ꢀC); 1H NMR
5.1.9. 3-(4-Methoxyphenethyl)-2,4-dioxo-1,2,3,4-
tetrahydroquinazoline (9)
A mixture of methyl anthranilate (292 mg, 1.93 mmol) and 1-(2-
isocyanatoethyl)-4-methoxybenzene (341 mg, 1.93 mmol) in 1,2-
dimethoxyethane (3 mL) was heated under microwave irradiation
(45 min, 160 ꢀC). After cooling down the reaction in an ice/salt bath,
a pale brown precipitate was formed. The solid was filtered, washed
with diethylether and dried, affording 9 as a white solid (yield 46%).
Mp 223.8 ꢀC (Lit. [35] 76e78 ꢀC); 1H NMR (300 MHz, DMSO-d6)
(300 MHz, DMSO-d6)
d
(ppm): 13.48 (bs,1H), 8.32 (dd, J ¼ 8.2,1.0 Hz,
1H, H-5), 7.78 (ddd, J ¼ 8.3, 7.0,1.0 Hz,1H, H-7), 7.50e7.42 (m, 3H, H-
d
(ppm): 11.46 (s, 1H, NH), 7.94 (d, J ¼ 7.1 Hz, 1H), 7.65 (t, J ¼ 7.2 Hz,
Ar, H-6), 7.39e7.32 (m, 2H, H-Ar, H-8), 7.21 (dd, J ¼8.3,1.1 Hz, 2H); 13C
1H), 7.20e7.08 (m, 4H), 6.83 (m, 2H), 4.03 (m, 2H, N-CH2), 3.82 (s,
NMR (75 MHz, DMSO-d6)
d (ppm): 189.8, 172.8, 144.2, 135.8, 135.2,
3H, MeO), 2.79 (m, 2H, CH2-Ar); 13C NMR (75 MHz, DMSO-d6)
131.7, 129.2, 128.5, 127.9, 125.2, 123.7, 115.9; ESI MS (m/z): 271
d (ppm): 161.1, 158.1, 150.3, 139.7, 135.2, 130.8, 129.9, 127.6, 122.7,
[M þ H]þ; purity 98% (by HPLC); Anal. C14H10N2S2 (C, H, N, O).
115.4, 114.1, 114.0, 55.3, 41.8, 32.8; ESI MS (m/z): 297 [M þ H]þ,
purity 98% (by HPLC); Anal. C17H16N2O3 (C, H, N, O).
5.1.6. 3-(4-Bromo-2-fluorophenyl)-2,4-dioxo-1,2,3,4-
tetrahydroquinazoline (6)
5.1.10. 6-Bromo-3-(4-methoxyphenethyl)-2,4-dioxo-1,2,3,4-
tetrahydroquinazoline (10)
A mixture of methyl anthranilate (117 mg, 0.77 mmol), 4-
bromo-2-fluoro-1-isocyanatobenzene (200 mg, 0.93 mmol) and
triethylamine (8 mg, 0.08 mmol) in acetonitrile (3 mL) was heated
under microwave irradiation (30 min, 170 ꢀC). After cooling down
the reaction in an ice/salt bath, a pale yellow precipitate was
formed. The solid was filtered, washed with diethylether and dried,
affording 6 as a pure product (yield 33%). Mp 263e264 ꢀC; 1H NMR
A mixture of 2-amino-5-bromobenzoic acid (200 mg, 0.93 mmol),
1-(2-isocyanatoethyl)-4-methoxybenzene (213 mg, 1.20 mmol) and
triethylamine (9 mg, 0.09 mmol) in acetonitrile (3 mL) was heated
under microwave irradiation (45 min,145 ꢀC). After cooling down the
reaction in an ice/salt bath, a pale yellow precipitate was formed. The
solid was filtered, washed with diethyletherand dried, affording 10 as
a pure product (yield 17%). Mp 180e181 ꢀC; 1H NMR (400 MHz,
(400 MHz, DMSO-d6)
d
(ppm): 11.76 (s, 1H), 7.95 (dd, J ¼ 8.1, 1.2 Hz,
1H), 7.78 (dd, J ¼ 9.5, 2.1 Hz, 1H), 7.73 (ddd, J ¼ 8.2, 7.3, 1.5 Hz, 1H),
DMSO-d6)
MeO), 3.15 (m, 2H, N-CH2-CH2), 2.58 (m, 2H, CH2-CH2-Ar); 13C NMR
(100 MHz, DMSO-d6) (ppm): 160.7, 157.8, 157.5, 149.7, 138.6, 137.5,
d (ppm):7.12 (m, 2H), 7.08 (m, 2H), 6.84(m, 3H), 3.70 (s, 3H,
7.57 (ddd, J ¼ 8.5, 2.1, 0.8 Hz, 1H), 7.50 (t, J ¼ 8.1 Hz, 1H), 7.26 (m,
2H); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 161.4, 158.9, 156.4,
d
149.2, 139.8, 135.7, 133.0, 128.0, 127.6, 122.9, 119.6, 119.4, 115.5,
113.6; ESI MS (m/z): 335/337 [M þ H]þ, purity >99% (by HPLC);
Anal. C14H8BrFN2O2 (C, H, N, O).
131.6, 129.5 (2xCH), 129.2, 117.6, 113.8, 113.7 (2xCH), 54.9, 41.1, 35.2;
ESI MS (m/z): 375/377 [M þ H]þ, purity >99% (by HPLC); Anal.
C17H15BrN2O3 (C, H, N, O).
5.1.7. 3-(4-Methoxybenzyl)-2,4-dioxo-1,2,3,4-
tetrahydroquinazoline (7)
5.1.11. 3-(4-Methoxyphenethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido
[3,4-d]pyrimidine (11)
A mixture of methyl anthranilate (116 mg, 0.77 mmol), 1-
isocyanatomethyl)-4-methoxybenzene (125 mg, 0.77 mmol) and
A mixture of 3-aminoisonicotinic acid (200 mg, 0.93 mmol), 1-
(2-isocyanatoethyl)-4-methoxybenzene (213 mg, 1.20 mmol) and

182
M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
triethylamine (8 mg, 0.08 mmol) in acetonitrile (3 mL) was heated
under microwave irradiation (45 min, 145 ꢀC). After cooling down
the reaction in an ice/salt bath, a white precipitate was formed. The
solid was filtered, washed with diethylether and dried, affording 11
as a pure product (yield 21%). Mp 238 ꢀC; 1H NMR (300 MHz,
triethylamine (9 mg, 0.09 mmol) in acetonitrile (3 mL) was heated
under microwave irradiation (30 min, 175 ꢀC). After cooling down
the reaction in an ice/salt bath, a pale orange precipitate was
formed. The solid was filtered, washed with diethylether and dried,
affording 15 as a white solid (yield 63%). Mp 316 ꢀC; 1H NMR
DMSO-d6)
d
(ppm): 8.58 (s, 1H), 8.39 (d, J ¼ 7.1 Hz, 1H), 7.78 (d,
(300 MHz, DMSO-d6)
d
(ppm): 7.45 (m, 3H), 7.26 (m, 3H), 7.02 (s,
(ppm):
J ¼ 7.1 Hz, 1H), 7.13 (d, J ¼ 8.9 Hz, 2H), 6.81 (d, J ¼ 8.9 Hz, 2H), 4.04
1H), 3.90 (s, 3H), 3.82 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d
(m, 2H, N-CH2-CH2), 3.75 (s, 3H, MeO), 2.81 (m, 2H, CH2-Ar); 13C
174.8, 159.3, 155.3, 146.5, 139.4, 135.4, 129.0, 128.9, 128.1, 108.5,
106.9, 97.8, 56.0, 55.8; ESI MS (m/z): 315 [M þ H]þ, purity 98% (by
HPLC); Anal. C16H14N2O3S (C, H, N, O).
NMR (125 MHz, DMSO-d6) d (ppm): 161.5, 158.3, 150.2, 143.2, 138.9,
135.1, 132.0, 130.0, 119.8, 119.5, 114.3, 55.4, 42.2, 41.6; ESI MS (m/z):
298 [M þ H]þ, purity >99% (by HPLC); Anal. C16H15N3O3 (C, H, N, O).
5.1.16. 4-Oxo-3-phenyl-2-thioxo-1,2,3,4-tetrahydropyrido[2,3-d]
pyrimidine (16)
5.1.12. 8-Chloro-3-(2,6-difluorophenyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydroquinazoline (12)
A mixture of methyl 2-aminonicotinate (200 mg, 1.27 mmol)
and phenylisothiocyanate (0.199 mL, 1.34 mmol) with triethyl-
amine (0.035 mL, 0.24 mmol) in acetonitrile (4 mL) was heated
under microwave irradiation (30 min, 150 ꢀC). After cooling down
the reaction in an ice/salt bath, a white precipitate was formed. The
solid was filtered affording 16 as a pure product (yield 5%). Mp
301e302 ꢀC (Lit. [36] 275e276 ꢀC); 1H NMR (300 MHz, DMSO-d6)
A mixture of 2-amino-3-chlorobenzoic acid (140 mg, 0.818 mmol)
and 2,6-difluorophenylisothiocyanate (0.105 mL, 0.818 mmol) in
DMSO/H2O (1:1) (4 mL) was heated under microwave irradiation
(40 min, 120 ꢀC). After cooling down the reaction in an ice/salt bath,
a yellow precipitate was formed. The solid was filteredand purifiedby
column chromatography on silica using hexane/ethyl acetate (9:1) as
eluent affording 12 as a pure product (yield 63%). Mp 206e208 ꢀC; 1H
d
(ppm): 13.50 (s, 1H); 8.76 (d, J ¼ 4.6 Hz, 1H, H-7); 8.34 (d,
NMR (300 MHz, DMSO-d6)
d
(ppm): 12.37 (bs, 1H), 7.97 (t, J ¼ 1.2 Hz,
J ¼ 7.7 Hz,1H, H-5); 7.50e729 (m, 6H, H-Ar, H-6); 13C NMR (75 MHz,
1H, H-7), 7.99 (t, J ¼ 1.5 Hz, 1H, H-5), 7.66e7.56 (m, 1H, H-6), 7.40 (t,
DMSO-d6) d (ppm): 177.8 (C-2); 160.4 (C-4); 155.6 (C-6); 154.3 (C-
J ¼ 7.8 Hz, 1H, H-Ar), 7.33 (tt, J ¼ 8.2, 2.5 Hz, 2H, H-Ar); 13C NMR
7); 151.0 (C-8a); 139.5 (C-Ar); 137.3 (C-Ar); 129.3 (C-Ar); 128.5 (C-
Ar); 120.7 (C-4a); 112.3 (C-5); ESI MS (m/z): 256 [M þ H]þ;
purity > 99% (by HPLC). Anal. C13H9N3OS (C, H, N, O).
(75 MHz, DMSO-d6)
d (ppm): 176.0, 160.2, 158.7, 137.6, 137.4, 132.5,
127.8,126.4,120.0,117.6,113.3; ESI MS (m/z): 325 [Mþ H]þ; purity98%
(by HPLC); Anal. C14H7OSN2ClF2 (C, H, N, O).
5.1.17. 3-(3-Bromophenyl)-4-oxo-2-thioxo-1,2,3,4-
5.1.13. 3-(2-Bromophenyl)-8-chloro-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrido[2,3-d]pyrimidine (17)
tetrahydroquinazoline (13)
A mixture of methyl 2-aminonicotinate (150 mg, 0.98 mmol)
and 1-bromo-3-isothiocyanatobenzene (210 mg, 0.98 mmol) in 1,4-
dioxane (3 mL) was heated under microwave irradiation (30 min,
160 ꢀC). After cooling down the reaction in an ice/salt bath, a yellow
precipitate was formed. The solid was filtered, washed with
diethylether and dried, affording 17 as a pure product (yield 18%).
A
mixture of 2-amino-3-chlorobenzoic acid (300 mg,
1.66 mmol) and 2-bromophenylisothiocyanate (0.341 mL,
2.49 mmol) in DMSO/H2O (1:1) (4 mL) was heated under micro-
wave irradiation (40 min, 120 ꢀC). After cooling down the reaction
in an ice/salt bath, a white precipitate was formed. The solid was
filtered and purified by column chromatography on silica using
hexane/ethyl acetate (9:1) as eluent affording 13 as a white solid
Mp 292 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 13.51 (bs, 1H, NH), 8.74
(dd, J ¼ 4.7, 1.8 Hz, 1H, H-7), 8.31 (dd, J ¼ 7.8, 1.8 Hz, 1H, H-5), 7.60
(m, 1H, H-60), 7.56 (bt, 1H, H-20), 7.44 (t, J ¼ 8.0 Hz, 1H, H-50), 7.37
(dd, J ¼ 7.8, 4.8 Hz, 1H, H-6), 7.32 (m, 1H, H-40). 13C NMR (125 MHz,
(yield 18%). Mp 192e193 ꢀC; 1H NMR (300 MHz, CDCl3)
d (ppm):
9.50 (s, 1H), 8.06 (dd, J ¼ 7.9, 0.8 Hz, 1H, H-5), 7.74 (d, J ¼ 1.4 Hz, 1H,
H-7), 7.71 (d, J ¼ 1.4 Hz, 1H, H-Ar), 7.46 (td, J ¼ 7.6, 1.4 Hz, 1H, H-Ar),
7.35 (dd, J ¼ 7.6, 1.7 Hz, 1H, H-Ar), 7.31 (d, J ¼ 1.6 Hz, 1H, H-6), 7.29
DMSO-d6)
d (ppm): 177.2, 159.9, 155.2, 150.7, 140.6, 136.8, 131.8,
131.1, 130.7, 128.4, 121.0, 120.4, 112.1; ESI MS (m/z): 334/336
(m, 1H, H-Ar); 13C NMR (75 MHz, CDCl3)
d (ppm): 175.9, 158.8,
[M þ H]þ, purity >99% (by HPLC); Anal. C13H8BrN3OS (C, H, N, O).
138.0, 136.1, 135.8, 134.1, 131.1, 130.6, 129.2, 128.2, 125.4, 123.0,
119.21, 117.76; ESI MS (m/z): 369 [M þ H]þ; purity >99% (by HPLC).
Anal. C14H8OSN2ClBr (C, H, N, O).
5.1.18. 4-Oxo-3-phenyl-2-thioxo-1,2,3,4-tetrahydropyrido[3,4-d]
pyrimidine (18)
A mixture of 3-aminoisonicotinic acid (150 mg, 1.03 mmol) and
phenylisotiocianate (0.161 mL, 1.34 mmol) with triethylamine
(0.143 mL, 0.10 mmol) in acetonitrile (4 mL) was heated under
microwave irradiation (30 min, 150 ꢀC). After cooling down the
reaction in an ice/salt bath, a white precipitate was formed. The
solid was filtered and purified by column chromatography on silica
using hexane/ethyl acetate (6:1) as eluent affording 18 as a pure
product (yield 35%). Mp 280e281 ꢀC; 1H NMR (300 MHz, DMSO-d6)
5.1.14. 8-Bromo-6-methyl-4-oxo-3-phenyl-2-thioxo-1,2,3,4-
tetrahydroquinazoline (14)
A mixture of 2-amino-3-bromo-5-methylbenzoic acid (200 mg,
0.87 mmol) and isothiocyanatobenzene (152 mg, 1.13 mmol) in
acetonitrile (3 mL) was heated under microwave irradiation
(45 min, 150 ꢀC). After cooling down the reaction in an ice/salt bath,
a white precipitate was formed. The solid was filtered, washed with
diethylether and dried, affording 14 as a pure product (yield 55%).
d
(ppm): 13.22 (s, 1H), 8.82 (s, 1H, H-6), 8.52 (d, J ¼ 5.1 Hz, 1H, H-8),
Mp 249 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 10.89 (s, 1H),
7.81 (d, J ¼ 5.1 Hz, 1H, H-5), 7.41 (m, 5H, H-Ar); 13C NMR (75 MHz,
7.94 (d, J ¼ 1.1 Hz, 1H), 7.78 (s, 1H), 7.48 (t, J ¼ 7.5 Hz, 2H), 7.40 (t,
DMSO-d6) d (ppm): 177.2, 159.4, 144.5, 139.3, 135.2, 129.3, 129.2,
J ¼ 7.3 Hz, 1H), 7.27 (d, J ¼ 7.4 Hz, 2H), 2.37 (s, 3H); 13C NMR
128.6, 122.1, 119.7; ESI MS (m/z): 256 [M þ H]þ; purity 98% (by
(100 MHz, DMSO-d6)
d
(ppm): 176.7, 159.6, 140.1, 139.9, 136.2, 135.7,
HPLC). Anal. C13H9N3OS (C, H, N, O).
129.7, 129.4, 128.9, 127.7, 118.5, 108.1, 20.6; ESI MS (m/z): 347/349
[M þ H]þ, purity >99% (by HPLC); Anal. C15H11BrN2OS (C, H, N, O).
5.1.19. 4-Oxo-3-(pyridin-3-yl)-2-thioxo-1,2,3,4-
tetrahydroquinazoline (19)
5.1.15. 6,7-Dimethoxy-4-oxo-3-phenyl-2-thioxo-1,2,3,4-
tetrahydroquinazoline (15)
A mixture of methyl anthranilate (117 mg, 0.77 mmol), 3-
isothiocyanatopyridine (137 mg, 1.00 mmol) and triethylamine
A mixture of methyl 2-amino-4,5-dimethoxybenzoate (200 mg,
0.95 mmol), isothiocyanatobenzene (179 mg, 1.32 mmol) and
(21
mL, 0.15 mmol) in acetonitrile (3 mL) was heated under
microwave irradiation (45 min, 175 ꢀC). After cooling down the

M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
183
reaction in an ice/salt bath, an orange precipitate was formed. The
solid was filtered, washed with diethylether and dried, affording 19
as a pale brown solid (yield 87%). Mp does not melt at 350 ꢀC; 1H
acetonitrile (3 mL) was heated under microwave irradiation
(45 min, 170 ꢀC). After cooling down the reaction with an ice/salt
bath, a white, precipitate was formed. The solid was filtered and
washed with diethylether and dried, affording 24 as a white, silky
NMR (300 MHz, DMSO-d6) d (ppm): 8.57 (m, 1H), 8.48 (bs, 1H), 7.95
(d, J ¼ 6.9 Hz, 1H), 7.78 (m, 2H), 7.53 (dt, J ¼ 7.8, 1.5 Hz, 1H), 7.44 (bd,
solid (yield 76%). Mp 199 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.85 (bs,
1H), 7.34 (bt, J ¼ 8.1 Hz,1H); 13C NMR (125 MHz, DMSO-d6)
d
(ppm):
1H, NH), 8.21 (d, J ¼ 6.8 Hz, 1H, H-Ar), 7.64 (t, J ¼ 6.7 Hz, 1H, H-Ar),
7.28 (m, 1H, H-Ar), 7.20 (d, J ¼ 7.0 Hz, 1H, H-Ar), 4.79 (m, 2HN-CH2-
CH2-morpholine), 3.91 (bs, 4H), 2.96 (m, 2H), 2.85 (m, 4H); 13C NMR
176.5, 160.4, 150.3, 149.3, 140.0, 137.4, 136.6, 136.2, 127.9, 124.9,
124.4, 116.6, 116.3; ESI MS (m/z): 256 [M þ H]þ, purity > 99% (by
HPLC); Anal. C13H9N3OS (C, H, N, O).
(75 MHz, CDCl3): d 176.0,160.2, 138.9, 135.8, 128.8, 125.4, 116.5, 67.2,
55.3, 54.1, 43.4; m/z (ES) 292 (M þ H)þ; purity 98% (by HPLC); Anal.
5.1.20. 6,7-Dimethoxy-4-oxo-3-(pyridin-3-yl)-2-thioxo-1,2,3,4-
C14H17N3O2S (C, H, N, O).
tetrahydroquinazoline (20)
A
mixture of methyl 2-amino-4,5-dimethoxybenzoate
5.1.24. 6,7-Dimethoxy-4-oxo-3-(2-(piperidin-1-yl)ethyl)-2-thioxo-
1,2,3,4-tetrahydroquinazoline (24)
(200 mg, 0.94 mmol), 3-isothiocyanatopyridine (152 mg,
1.13 mmol) and triethylamine (9 mg, 0.09 mmol) in acetonitrile
(3 mL) was heated under microwave irradiation (25 min, 180 ꢀC).
After cooling down the reaction in an ice/salt bath, a yellow
precipitate was formed. The solid was filtered, washed with
diethylether and dried, affording 20 as a pure product (yield 59%).
Mp does not melt at 350 ꢀC; 1H NMR (300 MHz, DMSO-d6)
A mixture of methyl 2-amino-4,5-dimethoxybenzoate (200 mg,
0.95 mmol), 1-(2-isothiocyanatoethyl)piperidine (224 mg,
1.32 mmol) and a catalytic amount of DMAP in acetonitrile (3 mL)
was heated under microwave irradiation (30 min, 175 ꢀC). After
cooling down the reaction with an ice/salt bath, a pale yellow
precipitate was formed. The solid was filtered and washed with
diethylether and dried, affording 24 as a white solid (yield 20%). Mp
d
(ppm): 12.91 (s, 1H, NH), 8.58 (dd, J ¼ 6.8, 1.1 Hz, 1H, H-40), 8.44
(d, J ¼ 1.8 Hz, 1H, H-20), 7.76 (dt, J ¼ 11.4, 2.1 Hz, 1H, H-60), 7.52 (dd,
J ¼ 11.4, 7.2 Hz, 1H, H-50), 7.27 (s, 1H, H-5), 6.99 (s, 1H, H-8), 3.87 (s,
210 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.25 (1H, s, H-5), 6.88 (1H,
s, H-8), 4.48 (2H, m, N-CH2), 3.81 (3H, s, MeO), 3.80 (3H, s, MeO),
3.32 (1H, bs, NH), 2.53 (2H, m, CH2-N), 2.33 (4H, bs, 2 ꢄ CH2), 1.45
(4H, bs, 2 ꢄ CH2), 1.34 (2H, bs, CH2); 13C NMR (100 MHz, DMSO-d6):
3H, MeO), 3.84; 13C NMR (75 MHz, DMSO-d6)
d (ppm): 175.2, 159.7,
155.8, 150.2, 149.1, 147.0, 137.3, 136.6, 135.9, 124.3, 108.8, 107.3,
98.3, 56.4, 56.2; ESI MS (m/z): 316 [M þ H]þ, purity > 99% (by
HPLC); Anal. C15H13N3O3S (C, H, N, O).
d
173.8, 158.7, 155.2, 146.7, 134.9, 107.9, 106.7, 97.7, 55.9, 55.7, 54.5,
54.2, 43.0, 25.6, 23.9; m/z (ES) 350 (M þ H)þ; purity > 99% (by
HPLC). Anal. (C13H9N3OS) (C, H, N, O).
5.1.21. 4-Oxo-3-(pyridin-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrido
[3,4-d]pyrimidine (21)
5.1.25. 6,7-Dimethoxy-3-(2-morpholinoethyl)-4-oxo-2-thioxo-
1,2,3,4-tetrahydroquinazoline (25)
A mixture of 3-aminoisonicotinic acid (100 mg, 0.72 mmol) and
3-isothiocyanatopyridine (128 mg, 0.94 mmol) in 1,2-
dimethoxyethane (3 mL) was heated under microwave irradiation
(30 min, 150 ꢀC). After cooling down the reaction in an ice/salt bath,
a yellow precipitate was formed. The solid was filtered, washed with
diethylether and dried, affording 21 as a pure product (yield 69%).
A mixture of methyl 2-amino-4,5-dimethoxybenzoate (115 mg,
0.55 mmol) and 4-(2-isothiocyanatoethyl)morpholine (113 mg,
065 mmol) in acetonitrile (3 mL) was heated under microwave
irradiation (45 min, 170 ꢀC). After cooling down the reaction with
an ice/salt bath, a white precipitate was formed. The solid was
filtered and washed with diethylether and dried, affording 25 as
a white solid (yield 59%). Mp 227 ꢀC; 1H NMR (300 MHz, CDCl3):
Mp 341 ꢀC; 1H NMR (300 MHz, DMSO-d6)
d (ppm): 13.37 (bs,1H, NH),
8.83 (s,1H, H-8), 8.59 (dd, J ¼ 4.5,1.2 Hz,1H, H-40), 8.52 (d, J ¼ 5.1 Hz,
1H, H-6), 8.48 (d, J ¼ 2.4 Hz,1H, H-20), 7.82 (d, J ¼ 5.1 Hz,1H, H-5), 7.77
(dt, J ¼ 8.1, 1.8 Hz, 1H, H-60), 7.54 (dd, J ¼ 7.8, 4.8 Hz, 1H, H-50); 13C
d
7.52 (1H, s, H-Ar), 6.78 (1H, s, H-Ar), 4.83 (m, 2H, N-CH2eCH2-
morpholine), 3.93 (s, 6H, 2 ꢄ MeO), 3.86 (m, 4H, 2 ꢄ CH2), 2.91 (m,
4H, 2 ꢄ CH2), 2.85 (m, 2H, NeCH2eCH2-morpholine); 13C NMR
NMR (75 MHz, DMSO-d6)
d (ppm): 176.7, 159.1, 149.5, 149.0, 144.3,
139.0, 136.7, 135.7, 134.7, 123.9, 121.6, 119.2; ESI MS (m/z): 257
(75 MHz, CDCl3): d 174.8, 159.9, 156.2, 147.9, 135.0, 109.3, 107.9, 97.2,
[M þ H]þ, purity >99% (by HPLC); Anal. C12H8N4OS (C, H, N, O).
67.0, 56.9, 56.7, 55.5, 54.7, 43.7; m/z (ES) 352 (M þ H)þ; purity 97%
(by HPLC); Anal. (C16H21N3O4S) (C, H, N, O).
5.1.22. 4-Oxo-3-(2-(piperidin-1-yl)ethyl)-2-thioxo-1,2,3,4-
tetrahydroquinazoline (22)
A mixture of methyl anthranilate (50 mg, 0.33 mmol), 1-(2-
isothiocyanatoethyl)piperidine (79 mg, 0.46 mmol) and a catalytic
amount of DMAP in 1,2-dimethoxyethane (3 mL) was heated under
microwave irradiation (50 min, 160 ꢀC). After cooling down the
reaction in an ice/salt bath, a yellow precipitate was formed. The
solid was filtered, washed with diethylether and dried, affording 22
as a pure product (yield 62%). Mp 199 ꢀC; 1H NMR (300 MHz, DMSO-
5.2. Biological assays
5.2.1. Phosphodiesterase inhibition assay
The methodology used for measuring PDE7A1 activity was
based in a Scintillation Proximity Assay (SPA) from Perkin Elmer
(TRKQ7090). The activity of PDE7A1 is measured by co-incubating
the enzyme with [3H]cAMP and the hydrolysis of the nucleotide
is quantified by radioactivity measurement after binding of [3H]
AMP to scintillation binding bead.
d6)
d
(ppm): 7.95 (dd, J ¼ 7.8, 0.9 Hz, 1H), 7.74 (dt, J ¼ 8.7, 1.5 Hz, 1H),
7.36 (d, J ¼ 8.1 Hz, 1H), 7.30 (dt, J ¼ 7.1, 0.9 Hz, 1H), 4.51 (t, J ¼ 7.2 Hz,
2H, N-CH2eCH2-piperidine), 2.58 (t, J ¼ 7.5 Hz, 2H, NeCH2eCH2-
piperidine), 2.45 (bs, 4H, 2 ꢄ CH2), 1.47 (m, 4H, 2 ꢄ CH2), 1.35 (m,
0.02 units of PDE7A1 (Calbiochem # 524751) were incubated in
a 96-well flexiplate with 5 nCi of [3H]cAMP and inhibitors in 100
ml
CH2); 13C NMR (75 MHz, DMSO-d6)
d
(ppm): 175.0, 170.3, 159.2,
of assay buffer (contained in the kit) for 20 min at 30 ꢀC. After the
incubation time 1 mg of scintillation beads were added to each well
and plate was shaken for 1 h at room temperature. Finally, beads
were settled for 30 min and radioactivity was detected in
a Microbeta Trilux reader.
139.0, 135.4, 127.2, 124.4, 115.5, 54.8, 42.9, 25.4, 23.8; ESI MS (m/z):
290 [M þ H]þ, purity > 99% (by HPLC); Anal. C15H19N3OS (C, H, N, O).
5.1.23. 3-(2-Morpholinoethyl)-4-oxo-2-thioxo-1,2,3,4-
tetrahydroquinazoline (23)
A mixture of methyl anthranilate (83 mg, 0.55 mmol) and 4-(2-
isothiocyanatoethyl)morpholine (113 mg, 0.65 mmol) in
IC50 values were calculated by non-linear regression fitting using
GrpahPad Prism. Data (radioactivity vs log concentra-
tion) was fitted to
a
sigmoidal doseeresponse equation:

184
M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
Y ¼ Bottom þ (Top-Bottom)/(1 þ 10^((logIC50-X)*n)), where Bottom
and Top were the minimum and maximal inhibition for PDE,
respectively, IC50 was the concentration of compound that inhibited
the PDE activity in a 50% and n was the slope of the concentration-
response curve.
PBS pH 7.4 buffer, filtered and then added to the donor 96-well
plate. Then the donor plate was carefully put on the acceptor
plate to form a “sandwich”, which was left undisturbed for 2h and
30 min at 25 ꢀC. During this time the compounds diffused from the
donor plate through the brain lipid membrane into the acceptor
plate. After incubation, the donor plate was removed. UV plate
reader determined the concentration of compounds and commer-
cial drugs in the acceptor and the donor wells. Every sample was
analyzed at three to five wavelengths, in 3 wells and in two inde-
pendent runs. Results are given as the mean [standard deviation
(SD)] and the average of the two runs is reported. 10 quality control
compounds (previously mentioned) of known BBB permeability
were included in each experiment to validate the analysis set.
5.2.2. Primary cell cultures
Primary cortical neuronal cultures were prepared from the
cerebral cortex of E18 rats, as previously reported by Luna-Medina
et al [37]. Briefly, after dissecting the cerebral cortex, cells were
plated on poly-lysine (20 mg/ml; Sigma) and gelatin (250 mg/ml;
Sigma)-coated plates or cover-slips and the cultures were main-
tained in Neurobasal medium with B-27 supplements. All experi-
ments were carried out with 7-day-old cultures.
Astrocytes were prepared from neonatal (P2) rat cerebral
cortex, as previously described by Luna-Medina et al [37]. Briefly,
after removal of the meninges the cerebral cortex was dissected,
dissociated, and incubated with 0.25% trypsin/EDTA at 37 ꢀC for
1 h. After centrifugation, the pellet was washed 3 times with HBSS
(Gibco) and the cells were plated on non-coated flasks and
maintained in HAMS/DMEM (1:1) medium containing 10% FBS.
After 15 days the flasks were agitated on an orbital shaker for 12 h
at 250 rpm at 37 ꢀC, and the non-adherent oligodendrocytes and
microglial cells were removed. After 7 days the flasks were
agitated again to remove any remaining microglia cells and then
trypsinized and expanded at a 1:5 ratio in complete medium.
Primary microglial cells were prepared from 15-day-old mixed
glial cultures, as described above. Fifteen-day-old mixed glial
cultures were agitated on an orbital shaker for 12 h at 250 rpm at
37 ꢀC. The supernatant was collected, centrifuged, and the cellular
pellet containing the microglial cells resuspended in HAMS/DMEM
(1:1) containing 10% FBS and seeded on uncoated plates. Cells
were allowed to adhere for 2 h and the medium was removed to
eliminate non-adherent oligodendrocytes and new fresh medium
containing 10 ng/ml of GM-CSF was added. The purity of microglia
obtained by this procedure was >98% as determined by immu-
nofluorescence with the OX42 antibody.
5.2.4. Induction of permanent focal ischemia
Adult male Swiss mice (8e10 weeks of age) were used. All
animals were kept in a room with controlled temperature, a 12 h
dark/light cycle and fed with standard food and water ad libitum.
All experimental protocols adhered to the guidelines of the Animal
Welfare Committee of the Universidad Complutense (EU directives
86/609/CEE and 2003/65/CE).
Surgery leading to focal cerebral ischemia was conducted
under anesthesia with isoflurane in a mix of O2 and N2O (0.3/0.7 L/
min). During surgery, body temperature was maintained at
37.0 ꢂ 0.5 ꢀC using a servo-controlled rectal probeeheating pad.
The surgical procedure was a variant of that described by Chen
et al. [38] and Liu et al. [39]. A small craniotomy was made over
the trunk of the left middle cerebral artery and above the rhinal
fissure. The permanent middle cerebral artery (MCA) occlusion
(pMCAO) was done by ligature of the trunk just before its bifur-
cation between the frontal and parietal branches with a 9-
0 suture. Complete interruption of blood flow was confirmed
under an operating microscope. Additionally, the left common
carotid artery was then occluded. Mice in which the MCA was
exposed but not occluded served as sham-operated controls
(sham). After surgery, individual animals were returned to their
cages with free access to water and food.
Physiological parameters (rectal temperature, mean arterial
pressure, blood glucose levels) were not significantly different
between all studied groups (data not shown). No spontaneous
mortality was found after MCAO with this model, and this was
unaffected by the different experimental treatments.
5.2.3. CNS penetration: In vitro parallel artificial membrane
permeability assay (PAMPA)-Blood brain barrier (BBB)
Prediction of the brain penetration was evaluated using
a parallel artificial membrane permeability assay (PAMPA) [30]. Ten
commercial drugs, phosphate buffer saline solution at pH 7.4 (PBS),
Ethanol and dodecane were purchased from Sigma, Acros organics,
Merck, Aldrich and Fluka. The porcine polar brain lipid (PBL)
(catalog no. 141101) was from Avanti Polar Lipids. The donor plate
was a 96-well filtrate plate (MultiscreenÒ IP Sterile Plate PDVF
5.2.5. Infarct outcome determination
For infarct volume determination, brains were removed 48 h
after pMCAO, and cut into eight coronal brain slices of 1-mm (Brain
Matrix, WPI, UK), which were stained in 2% TTC (2,3,5-triphenyl-
tetrazolium chloride, Merck, Madrid, Spain) in 0.2 mol/L phosphate
buffer. Infarct size was determined as follows: infarct volumes were
measured by sampling stained sections with a digital camera
(Nikon Coolpix 990, Nikon Corporation, Tokyo, Japan), and the
image of each section was analyzed using ImageJ 1.44l (NIH,
Bethesda, MD, USA). The digitalized image was displayed on a video
monitor. With the observer masked to the experimental conditions,
the ratio between the volume of the spared cortex in the damaged
hemisphere (LN) and that in the whole neocortex of the contrala-
teral hemisphere (R) was calculated, and used to detect differences
in the amount of cortex that was damaged by the infarct in each
animal. To calculate the percent of hemisphere infarcted volume
(HIV%) we used the formula: HIV% ¼ [1ꢁ(LN/R)] ꢄ 100.
membrane, pore size is 0.45 mM, catalog no. MAIPS4510) and the
acceptor plate was an indented 96-well plate (MultiscreenÒ,
catalog no. MAMCS9610) both from Millipore. Filter PDVF
membrane units (diameter 30 mm, pore size 0.45 mm) from Symta
were used to filtered the samples. A 96-well plate UV reader
(Thermoscientific, Multiskan spectrum) was used for the UV
measurements. Test compounds [(3e5 mg of Caffeine, Enoxacine,
Hydrocortisone, Desipramine, Ofloxacine, Piroxicam, Testosterone),
(12 mg of Promazine) and 25 mg of Verapamile and Atenolol] were
dissolved in EtOH (1000
m
L).100
m
L of this compound stock solution
L of PBS pH 7.4 buffer
was taken and 1400 L of EtOH and 3500
m
m
were added to reach 30% of EtOH concentration in the experiment.
These solutions were filtered. The acceptor 96-well microplate was
filled with 180
coated with 4
m
m
L of PBS/EtOH (70/30). The donor 96-well plate was
To determine neurological impairment, the modified Neuro-
logical Severity Score (mNSS) was applied to every animal 24 and
48 h after pMCAO as previously described [40]. This neurological
score evaluates motor symptoms (hemiparesis and gait), balance
and reflexes.
L of porcine brain lipid in dodecane (20 mg mLꢁ1
)
and after 5 min, 180
mL of each compound solution was added.
1e2 mg of every compound to be determined their ability to pass
the brain barrier were dissolved in 1500 L of EtOH and 3500 L of
m
m

M. Redondo et al. / European Journal of Medicinal Chemistry 47 (2012) 175e185
185
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inhibitors as new drugs for neurological and inflammatory disorders, Expert
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Acknowledgments
[19] M. Stud, CODESÒ v1.0 (revision 3).
The authors gratefully acknowledge the financial support of
Ministry of Science and Innovation (MICINN), projects nos.
SAF2007-62811, SAF2009-13015-C02-01, SAF2009-08145 and
SAF2010-16365; Instituto de Salud Carlos III (ISCiii), project no.
RD07/0060/0015 and RD06/0026/0005 (RETICS program) and
CIBERNED; Fundación Española para la Ciencia y la Tecnología
(FECYT), project no. FCT-09-INC-0367 and Consejo Superior de
Investigaciones Científicas (CSIC), project no. PIE 200780I012. M. R.
and D. I. P. acknowledge pre- and post-doctoral fellowship from the
CSIC (JAE program) respectively. J. G. Z. was supported by the Pro-
gramme Alban, scholarship No. E07D400805CO. BRAINco Bio-
pharma is acknowledged.
[20] A. Castro, M.J. Jerez, C. Gil, F. Calderon, T. Domenech, A. Nueda, A. Martinez,
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