Triazine compounds as antagonists at Bv8-prokineticin receptors

Article abstract of DOI:10.1021/jm800854e

On the basis of a Janssen's patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N¹ and N ⁵ link a 4-methoxybenzy

Full text of DOI:10.1021/jm800854e

J. Med. Chem. 2008, 51, 7635–7639
7635
Brief Articles
Triazine Compounds as Antagonists at Bv8-Prokineticin Receptors
Gianfranco Balboni,*,†,| Ilaria Lazzari,† Claudio Trapella,† Lucia Negri,‡ Roberta Lattanzi,‡ Elisa Giannini,‡ Annalisa Nicotra,‡
Pietro Melchiorri,‡ Sergio Visentin,§ Chiara De Nuccio,§ and Severo Salvadori†
Department of Pharmaceutical Sciences, UniVersity of Ferrara, I-44100 Ferrara, Italy, Department of Physiology and Pharmacology “Vittorio
Erspamer”, UniVersity “La Sapienza”, I-00185 Rome, Italy, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita`, Rome,
Italy, Department of Toxicology, UniVersity of Cagliari, I-09124, Cagliari, Italy
ReceiVed July 11, 2008
On the basis of a Janssen’s patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential
non peptidic prokineticin receptor antagonists, containing the following substitutions: (N1 and N5 link a
4-methoxybenzyl and a 4-ethylbenzyl, respectively; C2 can link an amino-ethyl-guanidine (reference
compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were
assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-
induced intracellular Ca2+ mobilization.
Introduction
(PKR2), encoded within distinct chromosomes in both mouse
and human, share about 85% amino acid identity, with most
differences at the N-terminal. Their sequences are almost
identical in the transmembrane domains. Affinity of Bv8 and
PKs for their receptors is similar, with Bv8 showing a 10
times higher affinity for either receptor, while MIT-1 is a
PKR2 preferring ligand. PKRs have been reported to couple
either to Gi or to Gq/o proteins.4-6 Intensive research over
the past few years has shown that the biological activities of
Bv8/PK proteins range from angiogenesis and involvement
in reproduction and cancer, to neuronal survival and neuro-
genesis, hypothalamic hormone secretions, circadian rhythm
control, and modulation of complex behaviours such as
feeding and drinking. The high expression level of human
Bv8/PK2 in bone marrow, lymphoid organs, and leukocytes
suggested an involvement of these peptides in hematopoiesis
and in inflammatory and immunomodulatory processes.
Moreover, the dramatic reduction in the pain threshold
produced by Bv8 indicates that Bv8/PKs and their receptors
may act as mediators of inflammatory and neuropatic
pain.7-11 PKRs are present in DRG, in the outer layers of
the dorsal horns of the spinal cord, and in peripheral terminals
of nociceptor axons. Activation of nociceptor PKRs by Bv8
in rats and mice produces sensitization to thermal and
mechanical stimuli. A physiological role of Bv8/PKs as
peripheral and central pain modulators is supported by the
observation that mice lacking the PKRs or PK2 are less
sensitive to noxious stimuli than wild type mice. PKR1-null
mice also exhibited impaired development of hyperalgesia
after tissue injury. PK2 released by inflammatory cells can
bind and activate PKRs on the primary sensitive neurons,
contributing to inflammatory pain.11,12 Hence the PKRs are
potential targets for novel analgesic drugs that block the
nociceptive information before it reaches the brain. Identify-
ing of the structural determinants required for receptor
binding and hyperalgesic activity of Bv8-PKs is thus manda-
tory for the design of PKR antagonists. The highly conserved
amino terminal sequence AVITGA and the tryptophan (Trp)
residue in position 24 in all members of the Bv8/PK family
A small protein, named Bv8 to indicate its origin from
the skin secretion of Bombina Variegata and its molecular
weight (8 kDa), is the first amphibian member of the Bv8-
Prokineticin family.1 Homologues of Bv8 are present in skin
secretion of other Bombina species, in the venom of the snake
black mamba (mamba intestinal toxin, MIT-1a), in lizards,
and in fishes. Striking characterization of these proteins are
their identical amino terminal sequence, AVITG, and the
presence of 10 cysteines with identical spacing that define a
five disulphide-bridged motif called a colipase fold.2 The high
degree of identity between amphibian Bv8 peptides, fish
peptides, and mamba MIT-1 (58%) suggested that similar
peptides could also be present in other species, including
mammals. In the mouse, rat, cattle, monkey, and man, cDNA
cloning identified orthologues of Bv8. The two mammalian
proteins were named prokineticin 1 (PK1, or EG-VEGF) that
is 80% homologous to MIT, and prokineticin 2 (PK2 or
mBv8) that is an orthologue of amphibian Bv8. The name
prokineticin refers to the ability of these peptides to contract
guinea pig ileum (GPI), a property shared with amphibian
Bv8.3 The two G-protein-coupled receptors for Bv8-PKs,
prokineticin receptor 1 (PKR1) and prokineticin receptor 2
* To whom correspondence should be addressed. Phone: (+39)-70-675-
8625. Fax: (+39)-70-675-8612. E-mail: gbalboni@unica.it; bbg@unife.it.
†
Department of Pharmaceutical Sciences, University of Ferrara.
Department of Toxicology, University of Cagliari.
Department of Physiology and Pharmacology “Vittorio Erspamer”,
|
‡
University “La Sapienza”.
§
Department of Cell Biology and Neuroscience, Istituto Superiore di
Sanita`.
a Abbreviations. In addition to the IUPAC-IUB Commission on Bio-
chemical Nomenclature (J. Biol. Chem. 1985, 260, 14-42), this paper uses
the following additional symbols and abbreviations: AcOEt, ethyl acetate;
Boc, tert-butyloxycarbonyl; CDCl3, deuterochloroform; DCM, dichlo-
romethane; DEAD, diethyl azodicarboxylate; DIPEA, N,N-diisopropylethy-
lamine; DMF, N,N-dimethylformamide; DMSO-d6, hexadeuteriodimethyl
sulfoxide; ESI, electrospray ionization; Et2O, diethyl ether; HPLC, high
performance liquid chromatography; MIT, mamba intestinal toxin; Pe,
petroleum ether; PKR1, prokineticin receptor 1; PKR2, prokineticin receptor
2; PPh3, triphenylphosphine; TEA, triethylamine; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TLC, thin-layer chromatography; Y, yield.
10.1021/jm800854e CCC: $40.75
2008 American Chemical Society
Published on Web 11/12/2008

7636 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Brief Articles
preventing tumoral development/progression. Recently, some
patents regarding the synthesis and pharmacological char-
acterization of non-peptide prokineticin antagonists were
deposited by Janssen Pharmaceutica and Merck.15-18 General
structures of patented compounds, summarized in Figure 1,
are as follows: 1,3,4-trisubstituted 1,2,3,6-tetrahydro-2,6-
dioxopyrimidines, 1,2,5-trisubstituted 1,4,5,6-tetrahydro-4,6-
dioxo-1,3,5-triazines, and N,4-disubstituted morpholine-2-
carboxamides, respectively. Considering the completely
different structures reported in Figure 1, we selected to study
the structure/activity relationship related to the triazine/
pirimidine scaffold on the basis of better pharmacological
data reported for its analogues.
Figure 1. General structures of patented non-peptide prokineticin
antagonists.
With the aim to ameliorate the synthetic way to triazine
compounds, among all patented 1,3,5-triazine prokineticin
antagonists, on the basis of its pharmacological data and its easy
synthetic feasibility, we focalized our attention on compound 1
[(2-(5-(4-ethylbenzyl)-1-(4-methoxybenzyl)-1,4,5,6-tetrahydro-
4,6-dioxo-1,3,5-triazin-2-ylamino)ethyl)guanidine] reported in
the patent WO2006104713 as compound 46 (Figure 2).
Figure 2. Structure of patented reference 1.
Table 1. Effect of Triazine Compounds 1-3 on 1 nM Bv8 Induced
Intracellular Ca2+ Mobilization
treatment
PKR1% active cells
PKR2 % active cells
BV8 1 nM
93 ( 8.6
52 ( 4.8
4 ( 0.7
90 ( 9.3
0.3 ( 0.7
91 ( 12
95 ( 7.8
48 ( 3.7
6 ( 0.9
89 ( 9.2
2 ( 1
+(1) 100 nM
+(1) 300 nM
+(2) 10 µM
+(2) 1 mM
+(3) 100 µM
Chemistry
A useful amelioration in the new synthetic way for the
synthesis of compound 1 (Scheme 1) regards the preparation
of the intermediate 1-(4-methoxybenzyl)-2-methylisothiourea.
In the patented scheme (Supporting Information, Scheme 2), it
was prepared by alkylation of 1-(4-methoxybenzyl)thiourea with
methyl iodide. This reaction per se is not difficult, but the
N-alkylation of thiourea often is a source of side reactions and
it is quite easy to obtain considerable amounts of the disubsti-
tuted thiourea with an important yield decrease. To avoid this
drawback, it is convenient to start the synthesis from the
commercially available (Sigma-Aldrich) 1,3-bis(tert-butoxycar-
bonyl)-2-methyl-2-thiopseudourea that, under Mitsunobu condi-
tions, is treated with 4-methoxybenzyl alcohol to yield the N,N′-
Boc diprotected 1-(4-methoxybenzyl)-2-methylisothiourea (4),
which upon TFA treatment gives the Boc deprotected (5) in
good yield. The reaction of (5) with the commercially available
N-(chlorocarbonyl) isocyanate provides the ring closure to the
triazine intermediate (6) in 31% yield, in accord with the
corresponding patented reaction. A second Mitsunobu reaction
92 ( 8.8
are required for biological activity: deletions and substitutions
in these conserved residues produces antagonist molecules.13,14
The N-terminal deletion of alanine and valine, in Bv8
molecule (dAV-Bv8), yields an analogue lacking any biologi-
cal activity but still able to bind the receptors acting as PKR-
antagonist in vitro and in vivo.14 In rats and mice, dAV-Bv8
dose-dependently reduced and abolished Bv8-induced hype-
ralgesia.14 Owing to the involvement of the prokineticin
system in various biological and pathological functions, the
availability of effective antagonists of the PKRs may be
useful in the treatment and prevention of various mammalian
disease states, for example, visceral pain that is associated
with irritable bowel syndrome (IBS) and inflammatory bowel
disease (IBD). Furthermore, PK receptor antagonists could
be useful in treating cancer-specific angiogenesis, hence in
Figure 3. Effect of 1 on 1 nM Bv8-triggered cytoplasmic Ca2+ signals.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7637
Scheme 1. Synthesis of the Triazine Compounds (1-3)a
a Reagents: (a) THF, DEAD, PPh3, room temp. Y ) 78%; (b) TFA, room temp. Y ) 91%; c) DCM, Cl(CdO)-NdCdO, DIPEA, Y ) 31%; (d)
THF, DEAD, PPh3 4-ethylbenzyl alcohol, room temp. Y ) 80%; (e) toluene, NH2CH2CH2NH2, reflux Y ) 95%; (f) 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea, THF/H2O, 50 °C, Y ) 77%; (g) TFA, room temp. Y ) 95%; (h) 4,5-dihydro-2-(methylthio)-1H-imidazole, THF/H2O, 50
°C, Y ) 65%.
inserts the second benzyl group providing the intermediate (7),
which upon treatment with ethylenediamine yields the com-
pound 2. Finally, (2) can be converted to the reference (1) by
the reaction with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thi-
opseudourea and subsequent TFA deprotection or to compound
3 by the reaction with 4,5-dihydro-2-(methylthio)-1H-imidazole.
Bv8 induced transient increases in [Ca2+]i in a concentration-
dependent number of cells.14 As shown in Figure 3, PKR1
expressing cells were challenged with 1 nM Bv8 in the absence
(upper panels) or presence (lower panels) of 100 nM (1).
In the left panels is reported the time-course of the intracel-
lular Ca2+ concentration (expressed as ratio between the
emissions at 340 and 380 nm). In the right panels are reported
the pseudocolor images of the emission intensity ratios recorded
just before (240 s) and during (320 s) Bv8 challenge. Compound
1 dose dependently antagonized the 1 nM Bv8-induced intra-
cellular Ca2+ mobilization ([Ca2+]i) in PKR1- and PKR2-
transfected CHO cells (IC50 ) 100 nM; IC100 ) 300 nM). 2
and 3 slightly reduced Bv8-induced [Ca2+]i at 10 and 100 µM.
Compound 2 completely blocked Bv8-induced intracellular Ca2+
mobilization at 1 mM concentration (Table 1).
Results and Discussion
Binding of Triazine Compounds to Prokineticin Receptors.
The affinity of the new compounds for the prokineticin receptors
was expressed as inhibition constant (Ki) of the binding of 125I-
MIT on PKR1 and PKR2 as detailed in the Supporting
Information. The results from the binding assay indicated that
the reference (1) is a PKR1 preferring ligand with an apparent
affinity about 70-fold higher for PKR1 (Ki ) 22 nM) than for
PKR2 (Ki ) 1610 nM). PKR1 affinity of 2 (Ki ) 440 nM) and
3 (Ki ) 4719 nM) was 20- and 200-fold lower than that of (1).
PKR2 affinity of (2) and (3) is negligible, on the order of µM
(Figures 4-5, Supporting Information).
Conclusion
Here we improved the synthesis and confirmed the activity
of a non-peptidic prokineticin antagonist characterized by a
structure much more simple than the corresponding cysteine-
rich small proteins endowed with the same activity. Making
a comparison between the two synthetic ways to the reference
1, our method, reported in Scheme 1, provides an overall
yield of the final compound of about 13% vs an overall yield
of about 3% calculated in accord with the synthesis reported
in the patent (Scheme 2, Supporting Information). The same
synthetic method we applied to (1), can be extended to a
The competitive displacement of 125I-MIT from PKR1 and
PKR2 by graded concentration of compounds 2 and 3 indicated
that the lack of the guanidine function reduced the affinity for
both receptors.
Effect of Triazine Compounds on Bv8 Triggered [Ca2+]i.
Fura-2 loaded CHO cells stably transfected with the PKR1 or
PKR214 were utilized to evaluate the antagonistic effect of
compounds 1-3 on Bv8-triggered cytoplasmatic Ca2+ signals.

7638 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Brief Articles
ethylenediamine (0.38 mL, 5.7 mmol) was added. The reaction
mixture was refluxed for 18 h. After solvent evaporation, the residue
was dissolved in EtOAc and washed twice with water (2 × 10
mL). The organic layer was dried (Na2SO4) and evaporated to afford
the final compound 2 as a pale-yellow oil: yield 0.37 g (95%); Rf(B)
0.92; HPLC K′ 5.22; mp oil; m/z 410 (M + H)+. 1H NMR (DMSO-
d6): δ 7.21-7.10 (m, 6H), 6.92-6.88 (d, 2H, J ) 8 Hz), 5.02-4.98
(d, 2H, J ) 8.8 Hz), 4.86 (s, 2H), 4.08-3.98 (q, 2H, J ) 7 Hz)
3.73 (s, 3H), 3.28-3.25 (t, 2H, J ) 6.4), 2.53-2.50 (t, 2H, J )
6.4 Hz), 1.14-1.10 (t, 3H, J ) 7 Hz). Anal. C26H29F6N5O7: C; H;
N.
wide range of triazine analogues deriving for example by
the insertion of different benzyl groups. Interestingly, (1)
showed comparable affinity but higher selectivity for PKR1
than the nonselective prokineticin receptor antagonist dAV-
Bv8 obtained from Bv8 by N-terminal deletion of alanine
and valine residues.14 Preliminary in vivo studies (data not
shown) indicate that (1) and (2) both behave as antihyper-
algesic and anti-inflammatory drugs.
Experimental Section
Chemistry. 1,3-Bis-(tert-butoxycarbonyl)-1-(4-methoxybenzyl)-
2-methyl-3-thiopseudourea (4). To a solution of 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea (2.0 g, 6.90 mmol),
4-methoxybenzyl alcohol (0.94 mL, 7.59 mmol) and triphenylphos-
phyne (1.99 g, 7.59 mmol), at 0 °C in anhydrous THF, was added
a solution of diethyl azodicarboxylate (1.4 mL, 7.59 mmol)
dissolved in anhydrous THF. After 10 min, the reaction was warmed
at room temperature and stirred overnight. The solvent was removed
under vacuum, and the crude intermediate was purified by flash
chromatography (EtOAc/Pe, 1:9, v/v): yield 2.21 g (78%); Rf(A)
0.60; HPLC K′ 9.54; mp oil; m/z 412 (M + H)+. 1H NMR (CDCl3):
δ 7.27-7.25 (d, 2H, J ) 8.4 Hz), 6.84-6.82 (d, 2H, J ) 8.4 Hz),
4.69 (s, 2H), 3.77 (s, 3H), 2.24 (s, 3H), 1.50 (s, 9H), 1.40 (s, 9H).
13C NMR (CDCl3): 163.28, 158.99, 158.04, 152.00, 129.53, 129.35
(2 carbon atoms), 113.69 (2 carbon atoms), 82.59, 81.74, 55.21,
51.82, 28.01 (6 carbon atoms), 15.57.
(2-(5-(4-Ethylbenzyl)-1-(4-methoxybenzyl)-1,4,5,6-tetrahydro-
4.6-dioxo-1,3,5-triazin-2-ylamino)ethyl)-N,N′-di-tert-butyloxycarbo-
nyl-guanidine (N,N′-di-Boc-1). To a solution of 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea (0.15 g, 0.51 mmol)
in distilled THF (5 mL) and H2O (50 µL) (2) (0.2 g, 0.49 mmol)
was added at room temperature. The reaction was heated at 50 °C
for 3 h. The solvent was evaporated under vacuum and the crude
intermediate was precipitated from Et2O/Pe (1:9, v/v) to give a white
solid: yield 0.25 g (77%); Rf(D) 0.62; HPLC K′ 6.45; mp 132-134
1
°C; m/z 653 (M + H)+. H NMR (CDCl3): 7.41-7.14 (m, 6H),
6.95 (bs, 1H), 6.87-6.85 (d, 1H, J ) 8.8), 6.75-6.73 (d, 1H, J )
8.8), 6.43 (bs, 1H), 5.07 (s, 2H), 5.00 (s, 2H), 3.79 (s, 3H),
3.46-3.27 (m, 4H), 2.62-2.58 (q, 2H, J ) 7.6), 1.52 (s, 9H), 1.42
(s, 9H), 1.22-1.18 (t, 3H, J ) 7.6).
(2-(5-(4-Ethylbenzyl)-1-(4-methoxybenzyl)-1,4,5,6-tetrahydro-
4.6-dioxo-1,3,5-triazin-2-ylamino)ethyl)-guanidine (reference 1).
N,N′-di-Boc-1 (0.21 g, 0.32 mmol) was treated with TFA (2 mL)
for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) were added to
the solution until the product precipitated: yield 0.21 g (95%); Rf(D)
1-(4-Methoxy-benzyl)-2-methyl-3-thiopseudourea (5). Intermedi-
ate (4) (1.51 g, 3.68 mmol) was treated with TFA (10 mL) for 2 h.
at room temperature. TFA was removed under vacuum, and the
deprotected intermediate was precipitated from Et2O: yield 1.08 g
(91%); Rf(B) 0.32; HPLC K′ 6.51; mp >250 °C; m/z 211 (M +
H)+.
1
0.46; HPLC K′ 5.34; mp >250 °C; m/z 453 (M + H)+. H NMR
(CDCl3): δ 8.24 (bs, 1H), 7.78 (bs, 1H), 7.26-7.10 (m, 6H),
6.92-6.87 (d, 2H, J ) 8.8 Hz) 5.06 (s, 2H), 4.86 (s, 2H), 3.72 (s,
3H), 3.51 (q, 2H, J ) 7 Hz), 2.57-2.50 (m, 4H), 1.78-1.10 (t,
3H, J ) 7 Hz). 13C NMR (CDCl3): 163.03, 158.55, 157.04, 153.89,
153.35, 150.99, 142.58, 134.51, 128.21 (2 carbon atoms), 127.58
(2 carbon atoms), 127.20 (2 carbon atoms), 113.83 (2 carbon atoms),
55.02, 44.16, 40.65, 38.57, 27.74, 24.37, 15.63. Anal.
C27H31F6N7O7: C; H; N.
1-(4-Methoxybenzyl)-6-(methylthio)-1,3,5-triazine-2,4(1H,3H)-
dione (6). To a solution of (5) (0.5 g, 1.54 mmol) in dichloromethane
(10 mL) at 0 °C, diisopropylethylamine (0.79 mL, 4.63 mmol) was
added. At the same temperature, N-chlorocarbonyl isocianate (0.12
mL, 1.54 mmol), dissolved in dichloromethane (3 mL), was added
dropwise. The reaction mixture was allowed to stir while slowly
warming to room temperature (1 h) and was then stirred for an
additional 24 h. The solvent was evaporated, and the residue was
partitioned between EtOAc and H2O. The EtOAc layer was washed
with brine and dried over Na2SO4. The solution was filtered, the
solvent evaporated, and the residual oil was precipitated from
methanol: yield 0.13 g (31%); Rf(C) 0.25; HPLC K′ 5.33; mp
6-(2-(4,5-Dihydro-1H-imidazol-2-ylamino)ethylamino)-3-(4-eth-
ylbenzyl)-1-(4-methoxybenzyl)-1,3,5-triazine2,4(1H,3H)-dione (3).
To a solution of 4,5-dihydro-2-(methylthio)-1H-imidazole, (0.03
g, 0.25 mmol) in distilled THF (5 mL) and H2O (50 µL) (2) (0.1
g, 0.24 mmol) was added at room temperature. The reaction was
heated at 50 °C for 3 h. The solvent was evaporated under vacuum,
and the crude intermediate was precipitated from Et2O/Pe (1:9, v/v)
to give a pale-yellow solid: yield 0.11 g (65%); Rf(D) 0.53; HPLC
K′ 6.08; mp 220-222 °C; m/z 479 (M + H)+. 1H NMR (CDCl3):
7.41-7.14 (m, 6H), 6.95 (bs, 1H), 6.87-6.85 (d, 1H, J ) 8.8),
6.75-6.73 (d, 1H, J ) 8.8), 6.43 (bs, 1H), 5.07 (s, 2H), 5.00 (s,
2H), 3.83 (s, 4H) 3.79 (s, 3H), 3.46-3.27 (m, 4H), 2.62-2.58 (q,
2H, J ) 7.6), 1.22-1.18 (t, 3H, J ) 7.6). 13C NMR (CDCl3):
163.03, 158.55, 157.04, 153.89, 153.35, 150.99, 142.58, 134.51,
128.21 (2 carbon atoms), 127.58 (2 carbon atoms), 127.20 (2 carbon
atoms), 113.83 (2 carbon atoms), 55.02, 51.3, 44.16, 40.65, 38.57,
33.6, 27.74, 24.37, 15.63. Anal. C29H33F6N7O7: C; H; N.
1
210-212 °C; m/z 280 (M + H)+. H NMR (DMSO-d6): δ 11.60
(bs, 1H), 7.24-7.22 (d, 2H, J ) 7.6 Hz), 6.91-6.89 (d, 2H, J )
7.6 Hz), 4.97 (s, 2H), 3.73 (s, 3H), 2.45 (s, 3H). 13C NMR (CDCl3):
171.08, 158.62, 152.15, 149.98, 128.34 (2 carbon atoms), 126.86,
113.87 (2 carbon atoms), 55.00, 46.13, 14.68.
3-(4-Ethylbenzyl)-1-(4-methoxybenzyl)-6-(methylthio)-1,3,5-tri-
azine-2,4(1H,3H)-dione (7). To a solution of intermediate (6) (0.55
g, 1.97 mmol), triphenylphosphine (0.57 g, 2.17 mmol) and
4-ethylbenzyl alcohol (0.29 mL, 2.17 mmol), at 0 °C in anhydrous
THF, a solution of diethyl azodicarboxylate (0.4 mL, 2.17 mmol)
in anhydrous THF (3 mL) was added dropwise. The reaction
mixture was stirred overnight at room temperature, the solvent was
evaporated in vacuo, dissolved in EtOAc, and washed twice with
water (20 mL each). After solvent evaporation, the residue was
purified by flash chromatography (EtOAc/Pe, 1:2, v/v) to give a
colorless oil: yield 0.62 g (80%); Rf(D) 0.35 HPLC K′ 6.23; mp
Pharmacology. Receptor Binding Assay. Affinity of com-
pounds 1-3 for prokineticin receptors was assayed on membrane
preparation from PKR1- or PKR2-transfected CHO cells.14 The
prokineticin binding sites were labeled with 125I-MIT (Kd ) 4 pM
for PKR1; Kd ) 1.24 pM for PKR2, PerkinElmer, Membrane Target
Systems). The inhibition constant (Ki) of the three compounds was
calculated from competitive binding curves with the PRISM
software (GraphPad Software, San Diego, CA)
1
oil; m/z 398 (M + H)+. H NMR (DMSO-d6): δ 7.27-7.16 (m,
6H), 6.92-6.88 (d, 2H, J ) 8 Hz), 5.02 (s, 2H), 4.91 (s, 2H),
4.05-4.01 (q, 2H, J ) 7 Hz) 3.73 (s, 3H), 3.43 (s, 3H), 1.10-1.04
(t, 3H, J ) 7 Hz). 13C NMR (CDCl3): 170.06, 158.77, 151.58,
150.35, 142.88, 133.78 128.52 (2 carbon atoms), 127.73 (2 carbon
atoms), 126.72 (2 carbon atoms), 113.87 (2 carbon atoms), 63.29,
55.10, 47.56, 44.72, 27.85, 15.69, 14.75.
Intracellular Ca2+ Imaging. PKR1- or PKR2-transfected CHO
cells, were loaded for 50 min at room temperature with 2 µM-
Fura-2-AM in a balanced saline solution. Bv8 1 nM induced
increases in [Ca 2+]I in 10-40 s in about 95% of cells. Compounds
were added and incubated for 4 min, and then 1 nM Bv8 was added
and the fluorescent signal was evaluated for 2 min. The IC50 is
6-(2-Aminoethylamino)-3-(4-ethylbenzyl)-1-(4-methoxybenzyl)-
1,3,5-triazine-2,4(1H,3H)-dione (2). To a solution of intermediate
(7) (0.38 g, 0.95 mmol) in toluene (10 mL) at room temperature,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7639
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defined as the amount of a given compound required to inhibit 50%
of the maximum signal that is generated by 1 nM Bv8.
Acknowledgment. This study was supported in part by the
University of Cagliari (G.B.), and the University of Ferrara
(S.S.). Pharmacological tests were supported by grants of the
University of Rome “La Sapienza” (L.N.).
Supporting Information Available: Chemistry general methods,
patented synthetic scheme of Reference 1, receptor binding assay,
figures 1 and 2 related to prokineticin receptors binding, elemental
analysis and MS data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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