A new convenient synthetic method and preliminary pharmacological characterization of triazinediones as prokineticin receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2014.05.030

A new efficient synthetic method to obtain prokineticin receptor antagonists based on the triazinedione scaffold is described. In this procedure the overall yield improves from 13% to about 54%, essentially for two factors: 1) N-(chlorocarbonyl) isocyanate is no more used, it represents the yield limiting step with an average yield not exceeding 30%. 2) The Mitsunobu reaction is not involved in the new synthetic scheme avoiding the use of time and solvent consuming column chromatography. All synthesized triazinediones were preliminary pharmacologically screened in vivo for their ability to reduce the Bv8-induced thermal hyperalgesia. In this assay all compounds displayed EC ₅₀ values in the picomolar-subpicomolar range, some triazinediones containing a 4-halogen substituted benzyl group in position 5 showed the best activity. The analogues containing a 4-fluorine atom (PC-7) and a 4-bromobenzyl group (PC-25) resulted 10 times more potent than the reference PC-1 that bears a 4-ethylbenzyl group. While the 4-trifluoromethylbenzyl substituted analog (PC-27) was 100 times more potent as compared to PC1.

Full text of DOI:10.1016/j.ejmech.2014.05.030

European Journal of Medicinal Chemistry 81 (2014) 334e340
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
A new convenient synthetic method and preliminary pharmacological
characterization of triazinediones as prokineticin receptor antagonists
,
a
b
Cenzo Congiu a, Valentina Onnis a , Alessandro Deplano , Severo Salvadori ,
*
Veronica Marconi c, Daniela Maftei c, Lucia Negri c, Roberta Lattanzi c,
**
Gianfranco Balboni a
,
a Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari,
I-09124 Cagliari, Italy
b Department of Chemical and Pharmaceutical Sciences, University of Ferrara, I-44100 Ferrara, Italy
c Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, I-00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new efficient synthetic method to obtain prokineticin receptor antagonists based on the triazinedione
scaffold is described. In this procedure the overall yield improves from 13% to about 54%, essentially for
two factors: 1) N-(chlorocarbonyl) isocyanate is no more used, it represents the yield limiting step with
an average yield not exceeding 30%. 2) The Mitsunobu reaction is not involved in the new synthetic
scheme avoiding the use of time and solvent consuming column chromatography. All synthesized tri-
azinediones were preliminary pharmacologically screened in vivo for their ability to reduce the Bv8-
induced thermal hyperalgesia. In this assay all compounds displayed EC50 values in the picomolar
esubpicomolar range, some triazinediones containing a 4-halogen substituted benzyl group in position 5
showed the best activity. The analogues containing a 4-fluorine atom (PC-7) and a 4-bromobenzyl group
(PC-25) resulted 10 times more potent than the reference PC-1 that bears a 4-ethylbenzyl group. While
the 4-trifluoromethylbenzyl substituted analog (PC-27) was 100 times more potent as compared to PC1.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 12 March 2014
Received in revised form
9 May 2014
Accepted 10 May 2014
Available online 14 May 2014
Keywords:
Triazinediones
Prokineticin antagonists
Analgesic activity
1. Introduction
system has been implicated in several pathological conditions,
including immunological response [13] and persistent pain [14].
The mammalian prokineticin 1 (PK1) and prokineticin 2 (PK2),
also known as Endocrine Gland-derived Vascular Endothelial
Growth Factor (EG-VEGF) and Bombina Variegata 8 (Bv8) are a pair
of bioactive peptides highly conserved across species [1]. PK1 and
PK2 make up a new family of chemokines which activate two G-
protein linked receptors (prokineticin receptor 1 and 2, PKR1 and
PKR2). Intensive research of the prokineticin system over the past
decade has revealed a dazzling array of physiological functions [2].
Since their initial identification, multiple physiological roles have
been discovered including: gastrointestinal motility [3,4], regula-
tion of circadian rhythms [5], angiogenesis [6,7], olfactory bulb
neurogenesis [8], neuroexcitation [9] inflammation [10,11], and
reproduction [12]. In addition, the disruption of prokineticin
Recent evidences implicated PK2 in a human disease in which
different point mutations in genes encoding PK2 or its receptor
(PKR2) lead to a type of Kallmann syndrome, a disease character-
ized by a deficiency in hypothalamic gonadotropin-releasing hor-
mones [15]. Furthermore PK2 is reported as an endangering
mediator of cerebral ischemia injury [16].
Considering the potential involvement of the prokineticin sys-
tem in different human disease, the availability of prokineticin re-
ceptor modulators represents an important task.
At the best of our knowledge, until now only two articles
describing the synthesis of triazinediones endowed with a prefer-
ential PKR1 antagonist activity have been reported [17,18]. Only one
unselective antagonist endowed with a morpholine scaffold is
published to date [16]. On the contrary, numerous patents were
filed about the synthesis and use of such antagonists endowed with
triazine and morpholine scaffolds. The two most recent patents
were filed in early 2012 [19,20]. Furthermore in the late 2013,
Takeda Pharmaceuticals filed a patent for new PKR antagonists
characterized by a sulfonylpiperidine scaffold [21].
* Corresponding author.
** Corresponding author.
E-mail addresses: vonnis@unica.it (V. Onnis), gbalboni@unica.it (G. Balboni).
http://dx.doi.org/10.1016/j.ejmech.2014.05.030
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
335
In an attempt to identify one or more selective ligands for
prokineticin receptors, we started the synthesis of triazinediones
endowed with different substituted/unsubstituted benzyl groups at
positions 1 and 5. The substitution pattern of triazine scaffold was
selected taking into account the results of patent literature and the
availability of the synthetic precursors. Looking at published and
patented synthetic ways to triazinediones, we identified essentially
two different routes clearly reported by Ralbovsky et al. and our
group [18,17]. Unfortunately, each synthetic way is characterized by
limiting steps which significantly lowers the overall yield. In
Scheme 1 it is depicted the first of two synthetic methods published
by Ralbovsky et al. [18] and also partially adopted in our previous
paper [17].
The major drawback of this route is represented by the N-
acylation of isothiourea 24 to give the intermediate 25. This reac-
tion per se is not difficult, but often it is a source of side reactions
and it is quite easy to obtain considerable amounts of the disub-
stituted isothiourea. Indeed, in our hand this step never gave yields
higher than 50%. Furthermore, if the second benzyl group is
introduced by a Mitsunobu reaction, a column chromatography
purification is always necessary to obtain the intermediate 27 of
good purity.
The second route to triazinediones is summarized in Scheme 2.
In this synthetic way, the ring closure by N-(chlorocarbonyl) iso-
cyanate (step b) to give the intermediate 30 represents the yield
limiting step. The average yield of this reaction does not exceed
30%.
Taking into account these considerations, here we report a new
synthetic method that can give easy access to high yield and purity
new triazinediones as potential ligands for prokineticin receptors.
All triazinediones were screened in vivo in mice to evaluate their
ability to reduce the hyperalgesia induced by BV8, a selective
agonist for the prokineticin receptors expressed on the peripheral
endings of nociceptors.
A). Alternatively, cyclization and successive methylation steps can
be conveniently done one pot (Method B), avoiding the isolation
and purification of 33, with a further improvement of yield up to
95%. The 4-methoxybenzyl group was introduced as a chloride in
presence of potassium carbonate to give the intermediate 35.
Displacement of the thiomethyl group by ethylenediamine in
toluene at 90 ꢀC yielded 36, which was converted into the final
guanidine compound PC-25 with pyrazole-1-carboxamidine
hydrochloride.
This synthetic method is endowed with two important features:
high yield in each step, and the purification of intermediates never
needs the use of time and solvent consuming column chromatog-
raphy. In this way it is easy to obtain triazinediones in high amount
and high purity for furthermore in deep pharmacological in vitro
and in vivo studies.
2.2. Analgesic activity
All synthesized triazinediones were screened in vivo in mice
with a quick and simple test: by injecting them into one hind paw
of mice and evaluating their ability to reduce the hyperalgesia
induced by BV8, a selective agonist for the prokineticin receptors
expressed on the peripheral endings of nociceptors. The com-
pounds injected into the hind paw of mice up to doses of 120 pmol
never produced changes in the basal thermal sensitivity, measured
as the latency to paw withdrawal from 48 ꢀC water, indicating they
do not activate any receptor involved in nociceptor sensitization.
Hence we tested their antagonistic activity looking for effectiveness
in antagonizing the hyperalgesic effect of Bv8, a selective PKR-1 and
PKR-2 agonist which, binding these receptors, increases the noci-
ceptor sensitization, so reducing the paw withdrawal latency. As
already demonstrated [23] intraplantar (i.pl. e subcutaneous in-
jection in the paw) injection of 63 fmol of Bv8 induced more than
50% decrease in thermal threshold of the injected paw in 30 min.
We evaluated if and at which doses the new molecules were able to
reduce the Bv8-induced decrease in thermal threshold when
injected by intraplantar route 5 min before Bv8 injection. The
ability of different doses of each compound in antagonizing the
Bv8-induced hyperalgesia was evaluated. In Fig. 1 the dosee
response curves of all synthesized triazinediones are reported and
the corresponding EC50 values are showed in Table 1. Because of the
high number of compounds, the dose response curves are divided
in four panels. In each panel the doseeresponse curve of the
reference PC-1 is reported. In Panel A, the most active compounds
are shown while in panel B, C and D there are the compounds as/
less effective than PC-1.
2. Results and discussion
2.1. Chemistry
In Scheme 3 is depicted the new way we used for the synthesis
of PC-25 and all the other triazinediones reported in Table 1.
Coupling 4-bromobenzylurea 31, obtained upon reaction of 4-
bromobenzylamine hydrochloride with potassium cyanate [22],
with the commercially available ethoxycarbonyl isothiocyanate
gave the N-ethoxycarbonylthiourea 32 in 85% yield. Subsequently,
32 was cyclized under basic conditions to yield the triazine 33
(yield 93%), which was alkylated by methyl iodide under basic
conditions to provide the key intermediate 34 in 90% yield (method
In this in vivo assay all triazinediones showed picomolar EC50
values. The analogues PC-7 and PC-25 displayed about 0.3 pmolar
EC50 values and resulted 10 times more potent than the reference
PC-1, while PC-27 (EC50 value 0.033 pmol) was 100 times more
potent. The replacement of PC-1 4-ethylbenzyl group in position 5
with a 4-fluorine atom to give PC-7 or with a 4-bromobenzyl group
to give PC-25 highly improved analgesic activity. The presence in 4-
position of a chorine atom (PC18) produced antagonistic activity
comparable with that of PC1, while its substitution with an iodine
atom or a trifluoromethyl group led to reduction in activity. The
opposite effect is showed by analog PC27 bearing
trifluoromethylbenzyl group in position 5 and 4-methyl group in
position 1, that is the most potent. The presence of 4-
a 4-
a
methoxylbenzyl group in position 5 generally led to analogs that
were less effective than the reference PC1, except PC24, PC26, PC29
and PC31 that displayed antagonistic activity comparable with that
of PC1.
Peripheral nociceptors of mice contain both PKR1 and PKR2,
hence with this test we cannot evaluate selectivity for either
Scheme 1. Reagents and conditions: (a) 3 N NaOH; (b) R2-C6H4eCH2eNCO, H2O/
MeOH/THF, 0 ꢀC to rt; (c) methylchloroformate, NEt3, CH2Cl2, ꢁ10 ꢀC to rt; (d) NaOMe,
MeOH; (e) R1-C6H4eCH2eOH, PPh3, DEAD, THF (Mitsunobu reaction); or R1-C6H4e
CH2eCl, NaOMe, CH3CN, heat.

336
C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
Scheme 2. Reagents: (a) MeI, MeOH; (b) ClCONCO, DIEA, CH2Cl2; (c) R2-C6H4eCH2eOH, PPh3, DEAD, THF (Mitsunobu reaction).
Scheme 3. Reagents and conditions: (a) KOCN, H2O, 60 ꢀC, 30 min; (b) ethoxycarbonyl isothiocyanate, toluene reflux, 4 h; (c) cI) NaOMe, MeOH, 50 ꢀC, 0.5 h, cII) MeI, rt, 3 h; (d)
NaOMe, MeOH, 50 ꢀC, 0.5 h; (e) NaOMe, MeI, MeOH, rt, 3 h; (f) 4-MeOeC6H4eCH2eCl, K2CO3, DMF, rt, 48 h; (g) ethylenediamine, toluene, 90 ꢀC; (h) pyrazole-1-carboxamidine
hydrochloride, DIPEA, CH3CN.
receptor. Because thermal nociception is mainly mediated by PKR1
activation we might predict these compounds bind the PKR1
however we cannot exclude they bind also PKR2. To answer this
question next step of our research will be to evaluate the affinity
and selectivity for PKR1 and PKR2 by in vitro binding and in vivo test
in mice lacking the pkr1 or the pkr2 gene.
inflammatory and neuropathic pain [14], and of PC-7 for in vivo
studies in animal model of multiple sclerosis (manuscript in
preparation) and Parkinson disease [24].
4. Experimental section
4.1. Chemistry
3. Conclusions
4.1.1. General methods
We have reported a new improved synthetic method to tri-
azinediones as potential antagonists for prokineticin receptors with
an overall yield of about 54%, far better than 13% reported by us in a
previous paper [17]. The advantage of this new route derives
essentially by two factors: 1) the reaction of N-(chlorocarbonyl)
isocyanate to give the intermediate 30 is not used; in fact it rep-
resents the yield limiting step with an average yield not exceeding
30%. 2) The Mitsunobu reaction is not involved in the synthetic
scheme avoiding the use of time and solvent consuming column
chromatography.
Using the new synthetic method we easily synthesized more
than 20 triazinediones that were preliminary tested in vivo as po-
tential antagonists of the hyperalgesic effect induced by Bv8. All
triazinediones showed picomolar EC50 values and three com-
pounds (PC-7; PC-25 and PC-27) showed an efficacy better than the
reference PC-1.
All commercially available solvents and reagents were used
without further purification. 1H and 13C NMR spectra were recorded
on a Varian Inova 500 spectrometer. The chemical shifts (d) are
reported in part per million downfield from tetramethylsilane
(TMS), which was used as internal standard, and the spectra were
recorded in DMSO-d6. Infrared spectra were recorded on a Bruker
Vector 22 spectrometer in Nujol mull. The main bands are given in
cmꢁ1. Positive-ion electrospray ionization (ESI) mass spectra were
recorded on a double-focusing Finnigan MAT 95 instrument with
BE geometry. Melting points (mp) were determined on a Stuart
Scientific Melting point SMP1 apparatus and are uncorrected. All
products reported showed 1H and 13C NMR spectra in agreement
with the assigned structures. The purity of tested compounds was
determined by combustion elemental analyses conducted by the
Microanalytical Laboratory of the Chemistry Department of the
University of Ferrara with a Yanagimoto MT-5 CHN recorder
elemental analyzer. All tested compounds yielded data consistent
with a purity of at least 95% as compared with the theoretical
Finally, now we are able to provide large amounts of the refer-
ence PKR antagonist PC-1 for in vivo studies regarding the

C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
337
Table 1
values. Reaction courses and product mixtures were routinely
monitored by TLC on silica gel (precoated F254 Merck plates), and
compounds were visualized with aqueous KMnO4, ninhydrin (1%
ethanol, Merck) and chlorine spray reagents. Rf were determined in
one or more of the following solvent systems: (A) AcOEt/Pe (1:9, v/
v); (B) AcOEt/Pe/ammonia 30% in H2O (1:1:0.3, v/v/v); (C) AcOEt/Pe
(1:1, v/v); (D) AcOEt/Pe (1:2, v/v). Final crude compounds were
purified by preparative reversed phase HPLC [Waters Delta Prep LC
Structures of new synthesized triazinediones and their EC50 analgesic activity
values.
40 mm assembly column C18 (30 cm ꢂ 4 cm, 15
mm particle size)]
eluted at a flow rate of 20 mL/min with mobile phase solvent A (10%
CH3CN þ 0.1% TFA in H2O, v/v), and a linear gradient from 10% to
60% B (60% CH3CN þ 0.1% TFA in H2O, v/v) in 25 min. Analytical
HPLC analyses were performed on a Beckman System Gold (Beck-
man ultrasphere ODS column, 250 mm ꢂ 4.5 mm, 5
mm particle
Compound
R1
R2
EC50 (pmol)
[95% confidence intervals]
size). For analytical determinations and capacity factor (Kʹ) of then
products were used solutions A and B as reported above, pro-
grammed at a flow rate of 1 mL/min with a linear gradient from 0%
to 100% B in 25 min.
PC-1 (Reference) OMe
PC-7
PC-8
Et
F
5.8 [3.6e9.1]
0.31 [0.16e0.59]
63 [44e90]
4.4 [2.5e7.8]
0.053 [0.038e0.067]
4.0 [2.9e5.7]
51 [27e83]
4.3 [2.7e6.8]
0.36 [0.17e0.78]
4.5 [2.9e6.4]
0.033 [0.018e0.061]
5.1 [3.8e6.8]
4.5 [3.4e5.6]
36 [24e59]
OMe
H
OMe
Me
OMe
Cl
CF3
OMe
Br
PC-15
PC-17
PC-18
PC-23
PC-24
PC-25
PC-26
PC-27
PC-28
PC-29
PC-30
PC-31
PC-32
PC-33
PC-34
PC-35
PC-36
OMe
OMe
OMe
OMe
Br
4.1.2. 1-(4-Bromobenzyl)urea (31) was prepared according to the
procedure of Carmellino et al. [22]
Briefly, to a solution of 4-bromobenzylamine hydrochloride
(4.45 g, 20 mmol) in H2O (20 mL) potassium cyanate (1.62 g,
20 mmol) was added, and the mixture was heated at 60 ꢀC for
30 min. After cooling at room temperature, the precipitate was
filtered, rinsed with ice water and dried [25].
OMe
3,4-diCl OMe
Me CF3
3,4-diCl Me
Me
F
OMe
OMe
OMe
OMe
Cl
4.4 [3.1e5.9]
48 [33e70]
4.1.3. 1-((4-Bromobenzyl)aminocarbonyl)-3-(ethoxycarbonyl)
thiourea (32)
CF3
OMe
I
OMe
OMe
3,4-methylenedioxy 39 [29e53]
OMe
I
39 [27e52]
51 [32e80]
48 [35ee69]
A mixture of 1-(4-bromobenzyl)urea (31) (2.29 g, 10 mmol) and
ethoxycarbonyl isothiocyanate (12 mmol, 1.2 mL) in toluene
(15 mL) was refluxed for 4 h. The solvent was evaporated under
vacuum, and the crude product was collected and washed with i-
Pr2O to give a pale-yellow solid: yield 3.06 g (85%); Rf(D) 0.66; HPLC
Kʹ 3.24; mp 180e182 ꢀC; m/z 361 (MþH)þ; 1H NMR (DMSO-d6):
NO2
Fig. 1. Doseeresponse curves of all synthesized triazinediones.

338
C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
d
11.12 (s, 1H), 12.22 (s. 1H), 7.55e7.32 (m, 4H), 4.52 (d, J ¼ 6.0 Hz,
0.2 g (95%); Rf(D) 0.38; HPLC Kʹ 5.41; mp 180e182 ꢀC; m/z 461
2H), 4.26 (q, J ¼ 7.0 Hz, 2H), 1.35 (t, J ¼ 7.0 Hz, 3H); IR (nujol) 3320,
1746, 1731, 1654 cmꢁ1. Anal. Calcd. for C12H14BrN3O3S (360.23): C,
40.01; H, 3.92, N, 11.66. Found: C, 40.06; H, 3.91, N, 11.64.
(MþH)þ; 1H NMR (DMSO-d6):
d 7.53e6.88 (m, 8H), 4.98 (s, 2H),
4.87 (s, 2H), 3.73 (s, 3H), 3.28e3.25 (t, 2H, J ¼ 6.4 Hz), 2.53e2.50 (t,
2H, J ¼ 6.4 Hz); IR (nujol) 3337, 1702, 1570 cmꢁ1 Anal. Calcd. for
C20H22BrN5O3 (460.32): C, 52.18; H, 4.82, N, 15.21. Found: C, 52.12;
4.1.4. 3-(4-Bromobenzyl)-6-thioxo-1,3,5-triazine-2,4-dione (33)
To a solution of 1-((4-bromobenzyl)aminocarbonyl)-3-(ethox-
ycarbonyl)thiourea (32) (3.6 g, 10 mmol) in anhydrous MeOH
(5 mL) 5.4 M methanolic NaOMe (1.7 mL) was added at room
temperature. The reaction was heated at 50 ꢀC for 0.5 h. The solvent
was evaporated under vacuum, then H2O (10 mL) was added and
the solution was treated with acq. 10% hydrochloric acid to pH 5e6.
The precipitate was filtered off, washed with H2O (2 ꢂ 5 mL), air
dried, then crystallized with EtOH to give a white solid: yield 2.92þg
(93%); Rf(D) 0.49; HPLC Kʹ 7.24; mp 236e238 ꢀC; m/z 315 (MþH) ;
H, 4.80, N, 15.24.
4.1.8. (2-(5-(4-Bromobenzyl)-1-(4-methoxybenzyl)-1,4,5,6-
tetrahydro-4,6-dioxo-1,3,5-triazin-2-ylamino)ethyl)guanidine (11)
[PC-25]
To a solution of intermediate (36) (0.17 g, 0.37 mmol) in CH3CN
(10 mL) at room temperature, DIPEA (0.13 mL, 0.75 mmol) and
pirazole-1-carboxamidine hydrochloride (0.05 g, 0.37 mmol) were
added. The reaction mixture was stirred for 12 h at room temper-
ature. The white solid formed was collected by centrifugation and
washed twice with ethyl ether (2 ꢂ 10 mL). The crude final com-
pound was purified by reversed phase preparative HPLC. Yield
0.17 g (93%); Rf(D) 0.42; HPLC Kʹ 5.52; mp 215e217 ꢀC; m/z 503.37
1H NMR (DMSO-d6):
d 12.57 (s, 2H), 7.52e7.20 (m, 4H), 4.91 (s, 2H).
IR (Nujol) 3075, 1763, 1570 cmꢁ1. Anal. Calcd. for C10H8BrN3O2S
(314,16): C, 38.23; H, 2.57, N,13.38. Found: C, 38.20; H, 2.58, N,13.41.
(MþH)þ; 1H NMR (DMSO-d6):
d 7.52e6.89 (m, 8H), 4.98 (s, 2H),
4.1.5. 3-(4-Bromobenzyl)-6-(methylthio)-1,3,5-triazine-
2,4(1H,3H)-dione (34)
4.87 (s, 2H), 3.73 (s, 3H), 3.39e3.28 (m, 4H); 13C NMR (DMSO-d6):
158.55, 156.91, 154.04, 153.27, 150.90, 136.66, 131.08 (2 carbon
atoms), 129.77 (2 carbon atoms), 127.92 (2 carbon atoms), 126.99,
120.17, 113.87 (2 carbon atoms), 55.00, 44.28, 43.90, 36.98, 36.81; IR
(nujol) 3396, 3149, 1724, 1666, 1578 cm-1. Anal. Calcd. for
Method A) To a solution of 3-(4-bromobenzyl)-6-thioxo-1,3,5-
triazine-2,4-dione (33) (3.14 g, 10 mmol) and 5.4 M methanolic
NaOMe (1.7 mL) in anhydrous MeOH (5 mL) methyl iodide (1.9 mL,
30 mmol) was added at room temperature and the reaction
mixture was stirred for 3 h. The solvent was evaporated under
vacuum, then H2O (10 mL) was added. The precipitate was filtered
off, washed with H2O (2 ꢂ 5 mL), then crystallized with 2-PrOH to
give a pale-yellow solid: yield 2.95 g (90%); Rf(D) 0.28; HPLC Kʹ 3.56;
C25H26BrF6N7O7 (502.36) C, 50.21; H, 4.82, N, 19.52. Found: C,
50.26; H, 4.83, N, 19.55.
4.1.9. 1-((4-Fluorobenzyl)aminocarbonyl)-3-(ethoxycarbonyl)
thiourea (32-a)
mp 166e168 ꢀC; m/z 329 (MþH)þ; 1H NMR (DMSO-d6):
d
7.56e7.38
Commercially available 1-(4-fluorobenzyl)urea (31-a) [alterna-
tively, it can be prepared as reported for (31) starting from the
appropriate benzyl amine] was reacted with ethoxycarbonyl iso-
thiocyanate as reported for (32): yield 2.63 gþ(88%); Rf (D) 0.55;
HPLC Kʹ 3.18; mp 185e187 ꢀC; m/z 300 (MþH) ; 1H NMR (DMSO-
(m, 4H), 4.99 (s, 2H), 2.56 (s, 3H). IR (Nujol) 3421, 1756, 1737,
1644 cmꢁ1. Anal. Calcd. for C11H10BrN3O2S (328.19): C, 40.26; H,
3.07, N, 12.80. Found: C, 40.20; H, 3.05, N, 12.83.
Method B) To a solution of 1-((4-bromobenzyl)aminocarbonyl)-
3-(ethoxycarbonyl)thiourea (32) (3.6 g, 10 mmol) in anhydrous
MeOH (5 mL) 5.4 M methanolic NaOMe (1.7 mL) was added at room
temperature. The reaction was heated at 50 ꢀC for 0.5 h. After
cooling methyl iodide (0.8 mL, 12 mmol) was added at room tem-
perature and the reaction mixture was stirred for 3 h. The title
compound was isolated as described in method A: yield 3.11 g
(95%).
d6):
d 12.30 (s. 1H), 11.20 (s, 1H), 7.46 (m, 2H), 7.28 (m, 2H), 4.48 (d,
J ¼ 6.0 Hz, 2H), 4.26 (q, J ¼ 7.0 Hz, 2H), 1.35 (t, J ¼ 7.0 Hz, 3H); IR
(nujol) 3317, 1744, 1702 cmꢁ1. Anal. Calcd. for C12H14FN3O3S
(299.32): C, 48.15; H, 4.71, N, 14.04. Found: C, 48.20; H, 4.69, N,
14.08.
4.1.10. 3-(4-Fluorobenzyl)-6-thioxo-1,3,5-triazine-2,4-dione (33-a)
1-((4-fluorobenzyl)aminocarbonyl)-3-(ethoxycarbonyl)thio-
urea (32-a) was reacted with NaOMe as reported for (33): yield
2.33 g (92%); Rf(D) 0.45; HPLC K0 6.84; mp 230e232 ꢀC; m/z 254
4.1.6. 3-(4-Bromobenzyl)-1-(4-methoxybenzyl)-6-(methylthio)-
1,3,5-triazine-2,4(1H,3H)-dione (35)
To
a
solution of 3-(4-bromobenzyl)-6-(methylthio)-1,3,5-
(MþH)þ; 1H NMR (DMSO-d6):
d 12.84 (s, 2H), 7.48 (m, 2H), 7.26 (m,
triazine-2,4(1H,3H)-dione (34) (3.28 g, 10 mmol) in anhydrous
DMF (5 mL) anhydrous potassium carbonate (2.76 g, 20 mmol) and
4-methoxybenzyl chloride (1.88 g, 12 mmol) were added. The
mixture was stirred for 48 h at room temperature monitoring the
reaction via HPLC. H2O (20 mL) was added and the precipitate was
filtered off, washed with H2O (2 ꢂ 5 mL) and crystallized with
MeCN to give a white solid: yield 3.63 g (81%); Rf(D) 0.53; HPLC Kʹ
6.81; mp 171e173 ꢀC; m/z 449 (MþH)þ; 1H NMR (DMSO-d6):
2H), 4.91 (s, 2H); IR (nujol) 3085, 1765, 1691 cmꢁ1. Anal. Calcd. for
C10H8FN3O2S (235.25): C, 47.43; H, 3.18, N, 16.59. Found: C, 47.47; H,
3.19, N, 16.54.
4.1.11. 3-(4-Fluorobenzyl)-6-(methylthio)-1,3,5-triazine-
2,4(1H,3H)-dione (34-a)
3-(4-Fluorobenzyl)-6-thioxo-1,3,5-triazine-2,4-dione
(33-a)
was reacted with methyl iodide as reported for (34): yield 2.38þg
d
7.66e7.07 (m, 8H), 5.00 (s, 2H), 4.87 (s, 2H), 3.86 (s, 3H), 2.58 (s,
(89%); Rf(D) 0.31; HPLC Kʹ 3.42; mp 171e173 ꢀC; m/z 268 (MþH) ;
3H). IR (nujol) 1726,1670,1610 cmꢁ1. Anal. Calcd. for C19H18BrN3O3S
(448.33): C, 50.90; H, 4.05, N, 9.37. Found: C, 50.95; H, 4.06, N, 9.34.
1H NMR (DMSO-d6):
d 12.85 (brs, 1H), 7.26 (m, 2H), 7.46 (m, 2H),
4.97 (s, 2H), 2.57 (s, 3H); IR (nujol) 3136, 1733, 1658 cmꢁ1. Anal.
Calcd. for C11H10FN3O2S (267.28): C, 49.43; H, 3.77, N, 15.72. Found:
C, 49.50; H, 3.75, N, 15.70.
4.1.7. 6-(2-Aminoethylamino)-3-(4-bromobenzyl)-1-(4-
methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (36)
To a solution of intermediate (35) (0.2 g, 0.45 mmol) in toluene
(10 mL) at room temperature, ethylenediamine (0.16 mL,
2.41 mmol) was added. The reaction mixture was refluxed for 12 h.
After solvent evaporation, the residue was dissolved in EtOAc and
washed twice with H2O (2 ꢂ 10 mL). The organic layer was dried
(Na2SO4) and evaporated to afford (36) as a pale-yellow oil: yield
4.1.12. 3-(4-Fluorobenzyl)-1-(4-methoxybenzyl)-6-(methylthio)-
1,3,5-triazine-2,4(1H,3H)-dione (35-a)
3-(4-Fluorobenzyl)-6-(methylthio)-1,3,5-triazine-2,4(1H,3H)-
dione (34-a) was reacted with 4-methoxybenzyl chloride as re-
ported for (35): yield 3.21 g (83%); Rf(D) 0.49; HPLC Kʹ 6.71; mp
175e177 ꢀC; m/z 388 (MþH)þ; 1H NMR (DMSO-d6):
d 7.49 (m, 2H),

C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
339
7.37 (d, J ¼ 8.4 Hz, 2H), 7.27 (m, 2H), 7.02 (d, J ¼ 8.4 Hz, 2H), 5.15 (s,
4.1.18. 3-(4-(Trifluoromethyl)benzyl)-1-(4-methoxybenzyl)-6-
(methylthio)-1,3,5-triazine-2,4(1H,3H)-dione (35-b)
2H), 5.05 (s, 2H), 3.85 (s, 3H), 2.59 (s, 3H); IR (nujol) 1721, 1679,
1562 cmꢁ1. Anal. Calcd. for C19H18FN3O3S (387.43): C, 58.90; H, 4.68,
N, 10.85. Found: C, 58.85; H, 4.70, N, 10.81.
3-(4-(trifluoromethyl)benzyl)-6-(methylthio)-1,3,5-triazine-
2,4(1H,3H)-dione (34-b) was reacted with 4-methylbenzyl chloride
as reported for (35): yield 3.50 gþ(80%); Rf(D) 0.45; HPLC Kʹ 6.56; mp
178e180 ꢀC; m/z 422,44 (MþH) ; 1H NMR (DMSO-d6):
d 7.69e7.11
4.1.13. 6-(2-Aminoethylamino)-3-(4-fluorobenzyl)-1-(4-
methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (36-a)
(m, 8H), 5.02 (s, 2H), 4.99 (s, 2H), 2.58 (s, 3H), 2.27 (s, 3H). 7.49 (m,
4H), 7.28 (m, 4H), 7.17 (s, 2H), 5.05 (s, 2H), 3.86 (s, 3H), 2.58 (s, 3H);
IR (nujol) 1728, 1688, 1519 cmꢁ1. Anal. Calcd. for C20H18F3N3O2S
(437.44): C, 54.91; H, 4.15, N, 9.61. Found: C, 54.86; H, 4.13, N, 9.58.
The intermediate (35-a) was treated with ethylenediamine as
reported for (36): yield 017þg (93%); Rf(D) 0.35; HPLC Kʹ 5.28; mp
d 7.34e6.87 (m,
184e186 ꢀC; m/z 400 (MþH) ; 1H NMR (DMSO-d6):
8H), 4.96 (s, 2H), 4.86 (s, 2H), 3.73 (s, 3H), 3.28e3.25 (t, 2H,
J ¼ 6.4 Hz), 2.53e2.50 (t, 2H, J ¼ 6.4 Hz); IR (nujol) 3331, 1720,
1561 cmꢁ1. Anal. Calcd. for C20H22FN5O3 (399.42): C, 60.14; H, 5.55,
N, 17.53. Found: C, 60.10; H, 5.57, N, 17.50.
4.1.19. 6-(2-Aminoethylamino)-3-(4-(trifluoromethyl)benzyl)-1-(4-
methylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (36-b)
The intermediate (35-b) was treated with ethylenediamine as
reported for (36): yield 0.19 g (92%); Rf(D) 0.34; HPLC Kʹ 5.19; mp
187e189 ꢀC; m/z 434,43 (MþH)þ; 1H NMR (DMSO-d6):
d 7.69e7.11
(m, 8H), 5.02 (s, 2H), 4.99 (s, 2H), 3.28e3.25 (t, 2H, J ¼ 6.4 Hz), 2.53e
4.1.14. (2-(5-(4-Fluorobenzyl)-1-(4-methoxybenzyl)-1,4,5,6-
tetrahydro-4,6-dioxo-1,3,5-triazin-2-ylamino)ethyl)guanidine (3)
[PC-7]
2.50 (t, 2H, J ¼ 6.4 Hz). 2.27 (s, 3H); IR (nujol) 3298,1697,1588 cmꢁ1
.
Anal. Calcd. for C21H22F3N5O2 (433.43): C, 58.19; H, 5.12, N, 16.16.
Found: C, 58.16; H, 5.13, N, 16.18.
The intermediate (36-a) was treated with pyrazole-1-
carboxamidine hydrochloride as reported for (11): yield 0.15 g
(92%); Rf(D) 0.43; HPLC Kʹ 6.05; mp 221e223 ꢀC; m/z 442.47
4.1.20. (2-(5-(4-(Trifluoromethyl)benzyl)-1-(4-methylbenzyl)-
1,4,5,6-tetrahydro-4,6-dioxo-1,3,5-triazin-2-ylamino)ethyl)
guanidine (13) [PC-27]
The intermediate (36-b) was treated with pyrazole-1-
carboxamidine hydrochloride as reported for (11): yield 0.17 g
(92%); Rf(D) 0.52; HPLC K0 6.51; mp 225e227 ꢀC; m/z 476,47
(MþH)þ; 1H NMR (DMSO-d6):
d 8.28 (br, 1H), 7.81 (s, 1H), 7.39 (m,
2H), 7.29 (d, J ¼ 8.4 Hz, 2H), 7.17 (m, 2H), 6.94 (d, J ¼ 8.6 Hz, 2H), 5.11
(s, 2H), 4.93 (s, 2H), 3.77 (s, 3H), 3.40 (m, 4H); 13C NMR (DMSO-d6):
163.01, 160.61, 159.10, 157.49, 154.60, 153.86, 151.48, 134.01, 130.26
(2 carbon atoms), 128.50 (2 carbon atoms), 115.39 (2 carbon atoms),
114.41 (2 carbon atoms), 55.56, 44.82, 44.31, 36.98, 36.81; IR (nujol)
3405, 3157, 1719, 1661, 1572 cmꢁ1 Anal. Calcd. for C25H26F7N7O7
(441.46): C, 57.13; H, 5.48, N, 22.21. Found: C, 57.10; H, 5.47, N, 22.25.
(MþH)þ; 1H NMR (DMSO-d6):
d 7.69e7.11 (m, 8H), 5.02 (s, 2H), 4.99
(s, 2H), 3.40e3.28 (m, 4H). 2.27 (s, 3H); 13C NMR (DMSO-d6):
156.95, 154.17, 153.30, 150.95, 142.02, 136.52, 132.07 (2 carbon
atoms), 129.02 (2 carbon atoms), 128.03 (2 carbon atoms), 126.24 (2
carbon atoms), 125.14 (2 carbon atoms), 125.10, 44.61, 44.15, 36.98,
36.81, 20.53; IR (nujol) 3397, 3166, 1721, 1660, 1579 cmꢁ1. Anal.
Calcd. for C26H26F9N7O6 (475.45): C, 55.57; H, 5.09, N, 20.62. Found:
C, 55.63; H, 5.11, N, 20.68.
4.1.15. 1-((4-(Trifluoromethyl)benzyl)aminocarbonyl)-3-
(ethoxycarbonyl)thiourea (32-b)
Commercially available 1-(4-(trifluoromethyl)benzyl)urea (31-
b) (alternatively, it can be prepared as reported for (31) starting
from the appropriate benzyl amine) [26] was reacted with ethox-
ycarbonyl isothiocyanate as reported for (32): yield 2.8þ6 g (82%);
Rf(D) 0.51; HPLC Kʹ 3.11; mp 189e191 ꢀC; m/z 350 (MþH) ; 1H NMR
4.2. Biological activity
4.2.1. Animals
(DMSO-d6):
d 12.27 (s. 1H), 11.19 (s, 1H), 7.55e7.32 (m, 4H), 4.49 (d,
Male C57Bl/6 mice weighing 25e30 g were used for behaviorals
experiments. Mice were housed in plastic cages (5 for each group)
and maintained under 12:12 lightedark cycle at 21 ꢃ 1 ꢀC and
50 ꢃ 5% humidity with food and water ad libitum.
J ¼ 6.0 Hz, 2H), 4.28 (q, J ¼ 7.0 Hz, 2H), 1.33 (t, J ¼ 7.0 Hz, 3H); IR
(nujol) 3303, 3177, 1734, 1683 cmꢁ1. Anal. Calcd. for C13H14F3N3O3S
(349.33): C, 44.70; H, 4.04, N, 12.03. Found: C, 44.64; H, 4.05, N,
12.06.
All animal experiments were conducted under protocols
approved by the Animal Care and Use Committee of the Italian
Ministry of Health. Animal care was in compliance with the IASP
and European Community (E.C.L.358/118/12/86) guidelines on the
use and protection of animals in experimental research. All efforts
were made to minimize animal suffering and to reduce the number
of animal used.
4.1.16. 3-(4-(Trifluoromethyl)benzyl)-6-thioxo-1,3,5-triazine-2,4-
dione (33-b)
1-((4-(trifluoromethyl)benzyl)aminocarbonyl)-3-(ethox-
ycarbonyl)thiourea (32-b) was reacted with NaOMe as reported for
(33): yield 2.76 g (91%); Rf(D) 0.40; HPLC Kʹ 6.66; mp 233e235 ꢀC;
m/z 304 (MþH)þ; 1H NMR (DMSO-d6):
d 12.70 (s, 2H), 7.70e7.54 (m,
4.2.2. Measurement of nociceptive threshold
The nociceptive threshold to thermal stimuli was evaluated by
the PaweImmersion test.
4H), 4.90 (s, 2H); IR (nujol) 3073, 1766, 1691, 1571 cmꢁ1. Anal. Calcd.
for C11H8F3N3O2S (303.26): C, 43.57; H, 2.66, N, 13.86. Found: C,
43.62; H, 2.65, N, 13.70.
This test was performed by dipping one mouse hind-paw into
hot water (48 ꢀC) and measuring the latencies to paw withdrawal.
For measurement of the nociceptive threshold, mice were trained
in paw withdrawal test during the week preceding the experiment.
This adaptation protocol reduced variability in threshold mea-
surements, giving a more stable baseline and making drug-induced
changes easier to detect. On the day of the experiment, nociceptive
threshold was measured for 2 h at 30 min intervals before drug
injection. The mean of the last three of these threshold measure-
ments were taken as baseline nociceptive threshold (NTB). Noci-
ceptive threshold was then determined three times at 15, 30, 60, 90,
4.1.17. 3-(4-(Trifluoromethyl)benzyl)-6-(methylthio)-1,3,5-triazine-
2,4(1H,3H)-dione (34-b)
3-(4-(trifluoromethyl)benzyl)-6-thioxo-1,3,5-triazine-2,4-dione
(33-b) was reacted with methyl iodide as reported for (34): yield
2.85 g (90%); Rf(D) 0.28; HPLC Kʹ 3.39; mp 175e177 ꢀC; m/z 318.29
(MþH)þ; 1H NMR (DMSO-d6):
d
12.80 (br s, 1H), 7.65 (d, J ¼ 7.5 Hz,
2H), 7.49 (d, J ¼ 7.5 Hz, 2H), 4.95 (s, 2H), 2.49 (s, 3H); IR (nujol) 1757,
1644 cmꢁ1. Anal. Calcd. for C12H10F3N3O2S (317.29): C, 45.43; H,
3.18, N, 13.24. Found: C, 45.49; H, 3.17, N, 13.20.

340
C. Congiu et al. / European Journal of Medicinal Chemistry 81 (2014) 334e340
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This study was supported in part by the Italian Ministero del-
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20105YY2HL_002), the Research Funds of the University of Cagliari
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