6-Amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives as a new class of potent inhibitors of Interleukin-8-induced neutrophil chemotaxis

Article abstract of DOI:10.1016/j.bmc.2009.04.006

A series of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives was synthesized. The compounds demonstrated to be novel, potent and selective inhibitors of Interleukin-8-induced human neutrophil chemotaxis. A SAR study

Full text of DOI:10.1016/j.bmc.2009.04.006

Bioorganic & Medicinal Chemistry 17 (2009) 3580–3587
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
6-Amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl
derivatives as a new class of potent inhibitors of Interleukin-8-induced
neutrophil chemotaxis
a
a
a
b
b
Sara Cesarini a, , Andrea Spallarossa , Angelo Ranise , Olga Bruno , Nicoletta Arduino , Maria Bertolotto ,
*
Franco Dallegri b, Massimiliano Tognolini c, Thomas Gobbetti c, Elisabetta Barocelli c
a Dipartimento di Scienze Farmaceutiche, Universitá di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b Dipartimento di Medicina Interna, Universitá di Genova, Viale Benedetto XV, 6;16132 Genova, Italy
c Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universitá di Parma, Viale G.P. Usberti 27/A, 43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 December 2008
Revised 2 April 2009
Accepted 6 April 2009
Available online 11 April 2009
A series of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives
was synthesized. The compounds demonstrated to be novel, potent and selective inhibitors of Interleu-
kin-8-induced human neutrophil chemotaxis. A SAR study was performed by varying the carbonyl func-
tion at position 5 and the chain linked to the amino group at position 6 of the scaffold. All the compounds
of the series displayed inhibitory activity at nano- or picomolar concentrations against Interleukin-8-dri-
ven migration and no activity against fMLP- and C5a-induced chemotaxis. The binding tests of selected
compounds on CXCR1 and CXCR2 receptors were negative. The most potent derivative showed in vivo
efficacy in a mouse model of Zymosan-induced peritonitis.
Keywords:
Neutrophil
Chemotaxis
Interleukin-8
Inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
Peritonitis
1. Introduction
heterotrimeric Gi proteins are activated and, downstream from
them, a complex interrelated signalling network is activated: stim-
Neutrophils are key cellular elements of innate immune system,
providing protection from invading microorganisms, and play a
critical role in inflammatory response.1–3 Chemotaxis (i.e., directed
migration in a gradient of chemotactic stimuli, in contrast to ran-
dom cell migration, which is called chemokinesis) enables neutro-
phils to rapidly reach the site of infection and destroy the invading
pathogens.4,5 Chemotaxis is induced by chemoattractants that in-
clude chemokines such as Interleukin-8 (IL-8), bacterial products
ulation of phospholipase Cb results in hydrolysis of phosphatidyl-
inositol 4,5-bisphosphate, generating diacylglycerol which
activates protein kinase C isoforms, and inositol 1,4,5-triphosphate
which releases calcium from intracellular stores; activation of
phosphoinositide 3-kinase (PI3K) results in increased production
of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinosi-
tol 3,4-bisphosphate; activation of the small GTP-binding proteins
of the Rho family and of the mitogen-activated protein kinase
(MAPK) cascade, as well as activation of protein phosphatases
and tyrosine kinases.10–12
Compounds able to inhibit neutrophil chemotaxis are poten-
tially useful for the treatment of acute and chronic disorders asso-
ciated with excessive inflammatory responses and neutrophil-
mediated tissue damage, such as rheumatoid arthritis, psoriasis,
inflammatory bowel disease, asthma, chronic obstructive pulmon-
ary disease, acute respiratory distress syndrome, ulcerative colitis,
post-ischemia reperfusion injury and transplant rejection.13–19
Figure 1 illustrates examples of IL-8 induced neutrophil chemo-
taxis inhibitors under clinical trials: SB656933 and SCH527123,
CXCR2-selective and CXCR1/CXCR2 competitive antagonists,
respectively,20–22 and the CXCR1 non-competitive antagonist Rep-
arixin, which binds to an allosteric pocket in the transmembrane
such as the peptide N-formyl-L-methionyl-L-leucyl-L-phenylala-
nine (fMLP), the product of the complement cascade C5a anaphylo-
toxin, platelet activating factor (PAF), and products of arachidonic
acid metabolism such as leukotriene B4 (LTB4). The binding of
the chemoattractants to specific receptors located on the neutro-
phil membrane (e.g., CXCR1 and CXCR2 receptors for IL-8; FPR
and FPRL1 receptors6 for fMLP; C5aR and C5L2 receptors7 for
C5a; PAFR for PAF; BLT1 and BLT2 for LTB4)8 causes a series of
cytoplasmic events resulting in the actin cytoskeleton re-organisa-
tion that drives neutrophil motility.9 In particular, when chemotac-
tic factors bind to these seven-transmembrane-domain receptors,
* Corresponding author. Tel.: +39 010 353 8361; fax: +39 010 353 8358.
E-mail address: sara.cesarini@unige.it (S. Cesarini).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.006

S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
3581
Cl
(equiv) of phenylisothiocyanate in dry DMF in the presence of
2 equiv of sodium hydride. Compounds 6–9 were obtained in high
yields (83–89%). The successive methylation of 6–9, performed
with iodomethane in DMF at 65 °C in the presence of sodium bicar-
bonate as a base, afforded the corresponding thiomethyl-deriva-
tives 10–13 in very high yields (93–97%) and allowed to obtain a
good leaving group (–SMe) at the position 6 of the scaffold for
the following step of nucleophilic aromatic substitution. The meth-
ylation occurred at the sulfur atom at position 6, instead of at the
oxygen atom at position 4, because, although the enol–thione tau-
tomer is conformationally stabilized by a strong intramolecular
hydrogen bond24,27 (as depicted in Scheme 1), the soft electrophile
iodomethane prefers to react at the soft nucleophilic site, that is,
sulfur atom (cheto–thiol tautomer), as confirmed by 1H NMR
(singlet at d 2.30–2.31) and 13C NMR spectral data (thiomethyl
signal at d 18.14–18.57 and signal of the amidic carbonyl at posi-
tion 4 at d 156.45–156.54).
O
H
N
O
O
S
F
N
H
N
H
S
N
O
O
O
OH
Cl
NH
O
SB656933
Reparixin
O
N
O
N
N
H
H
O
OH
SCH527123
Figure 1. Chemical structures of SB656933, Reparixin and SCH527123.
region of CXCR1 and blocks the agonist-induced receptor signalling
in the intracellular compartment, not directly affecting the IL-8
binding affinity.22,23
The
displacement
of
the
thiomethyl group
with
dialkylaminoalkyl-, cycloaminoethyl-, phenylaminoethyl-, (di)
methoxyethyl- and (hetero)arylethyl-amines and 4-methylpipera-
zine led to compounds 1 and 14–29 (Table 1). Different reaction
times and temperatures and number of equivalents of amine have
been used depending on the different reactivity of the amines em-
ployed. The work-up simply required filtrations or extractions and
the final products were purified by crystallization. The yields ran-
ged from 47% to 95%. The structure of 1 and 14–29 was confirmed
by IR, 1H NMR and, for compounds 1, 16–18, 20, 22, 25, 26 and 29,
by 13C NMR spectral data. When tautomerism was possible (i.e., for
1 and 14–28, Fig. 3), only one tautomer was detected. Both the
enaminone form T1 and the enol–imine form T2 can be stabilized
by an intramolecular hydrogen bond (Fig. 3). However, the pres-
ence of a sharp band at 3140–3365 cmꢀ1 in the IR spectra (N–H
stretching) and of a signal at d 157.67–158.98 in the 13C NMR spec-
tra (amidic carbonyl at position 4) suggests that T1 is favoured. This
would be supported also by the presence of a broad singlet,
exchangeable with deuterium oxide, at d 5.94–8.00 in the 1H
NMR spectra recorded in DMSO-d6 or CDCl3. In this context, the
higher d value of this signal in the 1H NMR spectrum of 14 (d
11.92) might be due to the stronger N–Hꢁ ꢁ ꢁO hydrogen bond that
the ketone carbonyl is able to form (the ketone carbonyl is a better
hydrogen bond acceptor than the ester carbonyl).
During investigations on the pharmacological potential of 1,3-
diphenyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one derivatives,
compound 1 (Fig. 2) emerged as a potent inhibitor of human neu-
trophil chemotaxis induced by IL-8 (IC50 = 3.32 0.11 nM), not en-
dowed with cytotoxic activity like its isoster sulfur analogue I.24
The isosteric replacement of the sulfur atom with the NH group
is one of the strategies often successfully applied in medicinal
chemistry and that has led to the discovery of new drugs.25,26 In or-
der to perform a structure–activity relationship (SAR) study, a ser-
ies of analogues of 1 was synthesized (14–29, Table 1). Initially, the
(dimethylamino)ethylamino chain at position 6 was maintained
constant and group R1 was modified by substituting the methyl
with bulkier alkyl groups (ethyl 15, isopropyl 16) or by replacing
the ester function with a methylketone (14). Successively, the ef-
fects of the variations on the chain linked to the amino group at po-
sition 6 on the inhibitory activity were investigated. Thus, (i) the
dimethylamino substructure was replaced by the diethylamino
moiety (17 and 18), by cycloaliphatic amines (piperidine 20, mor-
pholine 21, N-methylpyrrolidine 22) and by aromatic moieties
bearing basic functions (2-pyridyl 23, aminophenyl 24) or acidic
functionalities (4-aminosulfonylphenyl 25 and 3-indolyl 26); (ii)
the ethyl spacer was lengthened to propyl (19); (iii) the (dimethyl-
amino)ethyl chain was replaced by the methoxy- (27) or dime-
thoxy- (28) -ethyl moiety; (iv) the (dimethylamino)ethylamino
substructure was incorporated in the 4-methylpiperazinyl ring
(29).
3. Biological results and discussion
Compounds 1 and 14–29 were tested to evaluate the inhibition
of IL-8- and fMLP-induced human neutrophil migration (the results
are expressed as IC50 values, Table 1) and the cytotoxicity against
neutrophils. The most potent IL-8-driven chemotaxis inhibitors
were assayed also for their ability to block the human neutrophil
migration induced by C5a anaphylotoxin. To shed some light on
the mechanism of action, selected compounds were submitted to
a binding assay on CXCR1 and CXCR2. The compound emerged
from the in vitro assays as the most potent chemotaxis inhibitor
was tested in vivo in a mouse model of peritonitis, using Dexa-
methasone as reference drug (Table 2).
No compounds showed cytotoxic activity against neutrophils,
the percentage of viable cells never being lower than 94%. All the
tested molecules blocked the IL-8-induced chemotaxis, while they
were inactive against fMLP-driven migration. All compounds dis-
played IC50 values (Table 1) in the nanomolar or even picomolar
concentration range (derivatives 15–18, 23 and 24). The most po-
tent analogues resulted to be the ethyl and isopropyl esters 15–
18 incorporating the dimethyl- or diethyl-aminoethyl chain and
they showed no inhibitory activity against C5a-induced
chemotaxis.
2. Chemistry
The synthesis of 6–9 (Scheme 1) was accomplished by a one-pot
procedure previously described by some of us for the synthesis of 6
and 8.24,27 The synthetic method requires the reaction of methyl
acetoacetate (2) or the suitable dialkyl malonate (3–5) with 3 equiv
O
O
X
4
5
3 N
O
6
N
2
S
N1
1: X = NH
I: X = S
Figure 2. Chemical structure of compounds 1 and I.

3582
S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
Table 1 (continued)
Table 1
Chemical structures and chemotaxis inhibition values of compounds 1 and 14–29a
Compound
R1
NR2R3
Inhibition of IL-8
-induced neutrophil
migration IC50 (nM)
O
N
O
N
R1
R2
NH
27
28
OCH2CH3
OCH3
1.36 0.47
31.22 1.24
14.8 3.25
S
N
O
O
R3
O
NH
N
Compound
R1
NR2R3
Inhibition of IL-
8-induced neutrophil
migration IC50 (nM)
29
CH3
N
NH
NH
NH
N
1
14
15
16
17
18
19
20
21
22
23
24
OCH3
3.32 0.11
6.86 0.76
0.10 0.05
0.18 0.07
0.02 0.01
0.21 0.10
3.89 0.69
85.44 8.20
171.63 8.71
1.61 0.12
0.49 0.14
0.61 0.13
a
Neutrophil chemotaxis was evaluated in Boyden Chamber Migration assay,
after stimulation with 10ꢀ9 M IL-8 or 10ꢀ8 M fMLP or 10ꢀ9 M C5a (C5a only for 15–
18), in the absence and in the presence of tested compounds. The net migration was
determined by subtracting spontaneous migration, that is, the distance travelled by
neutrophils in the absence of the stimulus, from the distance travelled by neutro-
phils towards the stimulus. The concentration of each compound giving 50% inhi-
bition (IC50) of net migration was obtained from nonlinear regression analysis of
concentration–response curves, and the results reported are the mean of at least
three experiments. No inhibitory activity was observed in fMLP- or C5a-induced
chemotaxis.
N
N
CH3
OCH2CH3
OCH(CH3)2
OCH2CH3
OCH(CH3)2
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
OCH2CH3
NH
NH
N
N
Regarding the substituent R1 of the carbonyl function at posi-
tion 5, the ethoxy group demonstrated to be more beneficial than
propoxy (15 vs 16; 17 vs 18) and much more favourable than
methoxy and methyl (15 vs 14 and 1).
Among the compounds bearing the aminoethylamino linker at
position 6, the diethylamino derivatives 17 and 18 showed very
high potency, similar to the corresponding dimethylamino ana-
logues 15 and 16, and resulted to be much more effective than
the cycloamino congeners (compare 20 and 21 with 17 and 15).
The lengthening of the ethyl spacer to propyl produced an approx-
imately 39-fold decrease in potency (compare 19 with 15).
In compound 20, the isosteric substitution of the methylene at
piperidine position 4 with an oxygen atom (21) caused the potency
halving, while the replacement of the six-membered ring with N-
methylpyrrolidine (five-membered ring with the basic and possi-
ble H-bond acceptor amino group shifted) afforded an approxi-
mately 53-fold increase in inhibitory activity (22 vs 20).
Moreover, the exchange of the cycloaliphatic amines with basic
but aromatic moieties, such as the 2-pyridyl ring (23) and the ani-
line substructure (24), led to picomolar inhibitors from five to six-
fold less potent than the corresponding dimethylamino analogue
15 (R1 = OEt).
Also the substitution of the dimethylamino group of compound
1 (R1 = OMe) with aromatic moieties bearing acidic functions (4-
aminosulfonylphenyl 25 and 3-indolyl 26) caused a drop in po-
tency, in particular in the case of the bulky indole ring (25 and
26 vs 1).
The replacement of the basic dimethylamino function with the
neutral and smaller methoxy group led to an approximately 14-
fold decrease in inhibitory activity (compare 27 with 15). The
introduction of another methoxy group was not beneficial (com-
pare 28 with 27 and 1).
NH
N
N
NH
NH
N
NH
N
O
NH
NH
N
N
NH
NH
N
H
The rigidification of the (dimethylamino)ethylamino chain of
compound 14 in a 4-methylpiperazinyl ring led to about the halv-
ing of the potency (29 vs 14). According to this result, the presence
of the H-bond donor function (NH) at position 6 does not seem to
be essential for inhibitory activity.
25
26
OCH3
OCH3
10.79 4.21
29.64 8.14
SO2NH2
NH
The selectivity of the series in inhibiting the human neutrophil
chemotaxis induced by IL-8 prompted us to investigate the possi-
ble ability of these molecules to interfere with the binding of IL-
8 to CXCR1 and CXCR2 receptors with a mechanism of competitive
N
H

S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
3583
H
O
O
O
O
S
O
O
S
O
O
Ph
S
Ph
S
Ph
S
N
R1
R2
N
R1
N
R1
(
a,b)
(c)
(d)
R
R1
O
N
N
N
N
Ph
R3
Ph
Ph
2:
3:
4:
5:
R1 = CH3; R = CH3
R1 = OCH3; R = CH3
R1 = OCH2CH3; R = CH2CH3
6
:
:
:
:
R1 = CH3
R1 = OCH3
R1 = OCH2CH3
R1 = OCH(CH3)3
1, 14-29
10
:
:
:
:
R1 = CH3
R1 = OCH3
R1 = OCH2CH3
R1 = OCH(CH3)2
7
8
9
11
12
13
R1 = OCH(CH3)2; R = CH(CH3)2
Scheme 1. Reagents and conditions: (a) NaH (2 equiv), Ph–N@C@S (3 equiv), dry DMF, 0 °C, 1 h, then rt, 20 h. (b) H3O+; (c) NaHCO3 (1.1 equiv), CH3I (1 equiv), DMF, 65 °C, 1 h;
(d) NHR2R3 (1–2 equiv), rt or 80 °C, 1.5–48 h. The structures of 1 and 14–29 are listed in Table 1.
H
with selectivity against fMLP- and C5a-induced chemotaxis. Deriv-
O
N
R1
O
N
O
ative 17 emerged as the most potent compound in vitro and
showed in vivo efficacy in a mouse model of peritonitis.
The investigation of the mechanism of action of the title com-
pounds is in progress. The understanding of the molecular target
will provide useful information to modify the structure of this
new class of inhibitors in order to improve their pharmacological
properties.
N
O
H
N
R1
S
N
S
N
R2
R2
enaminone tautomer T1
enol-imine tautomer T2
Figure 3. General structure of compounds 1 and 14–28 in the enaminone and enol–
imine forms.
5. Experimental protocols
5.1. Chemistry
Table 2
Effect of compound 17 on cell and granulocyte recruitment as well as on protein
5.1.1. General
concentration enhancement induced by Zymosan in peritoneal cavity of micea
All chemicals were purchased by Sigma–Aldrich Chemical Co.,
Alfa Aesar and Lancaster and used without further purification. Sol-
vents were reagent grade. DMF was dried on molecular sieves (5 Å
1/1600 inch pellets). Organic solutions were dried over anhydrous
sodium sulfate. Thin layer chromatography (TLC) system for rou-
tine monitoring the course of reactions and confirming the purity
of analytical samples employed aluminium-backed silica gel plates
(Merck DC-Alufolien Kieselgel 60 F254): petroleum ether/ethyl ace-
tate or CH2Cl2/methanol were used as developing solvents and
detection of spots was made by UV light and/or by iodine vapours.
The organic solutions were evaporated using a rotatory evaporator
operating at reduced pressure of about 10–20 Torr. Yields were not
optimized. Melting points were determined on a Fisher-Johns
apparatus and are uncorrected. IR spectra were recorded on a Per-
kin Elmer 398 spectrometer as KBr discs. 1H and 13C NMR spectra
were recorded on a Varian Gemini 200 instrument at 200 and
50.30 MHz, respectively, employing solutions in DMSO-d6 or
CDCl3. Chemical shifts were reported in ppm units relative to the
internal reference tetramethylsilane, and the splitting patterns
were described as follows: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet) and br s (broad singlet). The first order val-
ues reported for coupling constants J were given in hertz. Elemen-
tal analyses were performed by an EA1110 Elemental Analyser
(Fison-Instruments, Milan) and were within 0.4% of the theoreti-
cal values. The synthesis of compounds 5 and 7 was accomplished
according to the published procedure.24,27
Compound
No. peritoneal
cells (million/
cavity)
No. peritoneal
granulocytes
(million/cavity)
Peritoneal protein
concentration (
mL)
lg/
Saline
6.2 1.2
31.5 3.4
23.8 2.7
22.0 1.8
1.9 0.4
551 203
1961 160
1521 170
1404 144
Saline + Zymosan
17 + Zymosan
Dexamethasone +
Zymosan
27.1 3.5
17.7 1.6
18.5 1.6
**
**
**
P < 0.01 by Anova one-way followed by Bonferroni’s post-test compared to
saline + Zymosan. Data are expressed as mean SEM of 8–12 independent data.
a
Animals received compound 17 (50 mg/kg os) or Dexamethasone (3 mg/kg os)
1 h before intraperitoneal injection of Zymosan (1 mg/mouse). Peritoneal fluid was
collected and examined microscopically 4 h later.
antagonism. However, the binding tests of 17, 22, 23, 24 and 27 on
CXCR1 and CXCR2 receptors turned out to be negative (data not
shown), thus suggesting that these new chemotaxis inhibitors
might affect intracellular signal transduction activated by IL-8.
Interestingly, compound 17 demonstrated in vivo activity in a
mouse model of peritonitis (induced by the intraperitoneal admin-
istration of Zymosan). Table 2 shows the effects of 17 on cell and
granulocyte recruitment (neutrophils constitute the majority of
granulocytes, which include also eosinophils and basophils in
small percentage) as well as on protein concentration enhance-
ment induced by Zymosan in peritoneal cavity of mice. The treat-
ment with compound 17 caused
a significant decrease of
granulocytes present in the peritoneal cavity compared to negative
control. 17 displayed similar activity to Dexamethason used at a
17-fold lower dose.
5.1.2. Synthesis of alkyl 6-hydroxy-1,3-diphenyl-2,4-dithioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate 7 and 9
A 60% sodium hydride dispersion in mineral oil (12 g, 0.30 mol)
was added in portions to a stirred and ice-cooled solution of dim-
ethylmalonate (34.29 mL, 0.30 mol) (for 7) or diisopropylmalonate
(56.98 mL, 0.30 mol) (for 9) in dry DMF (300 mL). When hydrogen
evolution ceased, phenylisothiocyanate (107.48 mL, 0.90 mol) was
added, followed after 15 min by another amount of sodium hy-
dride dispersion (12 g, 0.30 mol). After stirring at 0 °C for 1 h, and
then at rt for 20 h, 1.0 L of an aqueous 0.6 M ammonium chloride
solution was added to the reaction mixture. After extraction with
4. Conclusions
A new class of IL-8-induced neutrophil chemotaxis inhibitors
was identified and a SAR study was performed by varying the car-
bonyl function at position 5 and the chain linked to the amino
group at position 6 of the scaffold. All the compounds of the series
displayed inhibitory activity at nano- or picomolar concentrations,

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S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
diethyl ether/petroleum ether (1:1) (5 ꢂ 400 mL), the aqueous
layer was cooled and acidified with a 6 M HCl solution (300 mL).
The precipitate was filtered, washed with water, dried under vac-
uum and purified by crystallization from CH2Cl2/acetone.
arom. H). 13C NMR (DMSO-d6) d: 18.42 (SCH3), 21.21 (2CH3),
69.61 (OCH), 116.46 (C-5), 128.09 (CH), 128.23 (2CH), 128.84
(CH), 129.00 (2CH), 129.11 (2CH), 129.46 (2CH), 139.56 (C),
141.22 (C), 153.18 (C-6), 156.54 (CON), 162.45 (COO), 178.65
(CS). Anal. Calcd for C21H20N2O3S2: C, 61.14; H, 4.89; N, 6.79; S,
15.54. Found: C, 60.93; H, 4.91; N, 6.87; S, 15.42.
5.1.2.1. Methyl
6-hydroxy-1,3-diphenyl-2,4-dithioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate 7. Mp 275 °C (dec); yield:
84%. IR (KBr) cmꢀ1: 1739, 1674, 1563. 1H NMR (CDCl3) d: 3.92 (s,
3H, CH3), 7.21–7.55 (m, 10H, arom. H), 9.05 (br s, 1H, OH). Anal.
Calcd for C18H14N2O3S2: C, 58.36; H, 3.81; N, 7.56; S, 17.31. Found:
C, 58.57; H, 3.89; N, 7.74; S, 17.04.
5.1.4. Synthesis of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,
3,4-tetrahydropyrimidine-5-carbonyl derivatives 1 and 14–29
A mixture of the 6-(methylthio)-1,3-diphenyl-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one derivative 10, 11, 12 or 13 (3 mmol)
and the suitable amine (3.6 mmol; 3 mmol for 25 and 26;
4.5 mmol for 28; 6 mmol for 16, 18 and 29) in DMF (10 mL) was
stirred at rt for 18 h (1.5 h for 22; 3 h for 14 and 23; 48 h for 29;
70 °C for 4 h for 16 and 18; 80 °C for 6 h for 25, 26 and 28). Then,
the reaction mixture was diluted with water (100 mL). For all com-
pounds with the exception of 16 and 18, the precipitate was fil-
tered, washed with water, dried under vacuum and purified by
crystallization from the suitable solvent or solvent mixture. For
16 and 18, the mixture was extracted with diethyl ether
(50 mL ꢂ 3) and the combined extracts were washed with water,
and dried over anhydrous Na2SO4. Evaporation of the solvent under
reduced pressure gave a residue which was purified by crystalliza-
tion from the suitable solvent or solvent mixture.
5.1.2.2. Isopropyl 6-hydroxy-1,3-diphenyl-2,4-dithioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate 9. Mp > 300 °C; yield:
83%. IR (KBr) cmꢀ1: 2980, 1696, 1675, 1558. 1H NMR (CDCl3) d:
1.36 (d, J = 6.3 Hz, 6H, 2CH3), 5.24 (m, 1H, OCH), 7.22–7.55 (m,
10H, arom. H), 8.85 (br s, 1H, OH). Anal. Calcd for C20H18N2O3S2:
C, 60.28; H, 4.55; N, 7.03; S, 16.09. Found: C, 60.24; H, 4.80; N,
7.16; S, 16.13.
5.1.3. Synthesis of 6-(methylthio)-1,3-diphenyl-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one derivatives 10–13
Iodomethane (6.224 mL, 0.10 mol) was added to the stirred
mixture of the suitable 6-hydroxy-1,3-diphenyl-2,4-dithioxo-
1,2,3,4-tetrahydropyrimidine derivative (6 for 10; 7 for 11; 8 for
12; 9 for 13, 0.10 mol) and sodium bicarbonate (9.24 g, 0.11 mol)
in DMF (150 mL). After stirring at 65 °C for 1 h, the reaction mix-
ture was diluted with water (200 mL). The precipitate was filtered,
washed with water, dried under vacuum and purified by crystalli-
zation from acetone or CH2Cl2/acetone.
5.1.4.1. Methyl 6-{[2-(dimethylamino)ethyl]amino}-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
1. Mp 219–221 °C; yield: 95% from acetone/methanol. IR (KBr)
cmꢀ1: 3359, 2956, 1704, 1681, 1610. 1H NMR (CDCl3) d: 1.94 (s,
6H, N(CH3)2), 2.19–2.30 (m, 2H, CH2N), 2.84–2.95 (m, 2H, NHCH2),
3.88 (s, 3H, OCH3), 6.85 (br s, 1H, NH, exchangeable), 7.22–7.61 (m,
10H, arom. H). 13C NMR (CDCl3) d: 41.79 (CH2N), 44.34 (N(CH3)2),
52.64 (NHCH2), 56.41 (OCH3), 88.42 (C-5), 128.43 (CH), 128.57
(2CH), 129.41 (2CH), 129.74 (2CH), 129.89 (CH), 130.35 (2CH),
138.42 (C), 140.07 (C), 153.65 (C-6), 158.86 (CON), 166.94 (COO),
178.86 (CS). Anal. Calcd for C22H24N4O3S: C, 62.24; H, 5.70; N,
13.20; S, 7.55. Found: C, 62.13; H, 5.69; N, 13.02; S, 7.46.
5.1.3.1. 5-Acetyl-6-(methylthio)-1,3-diphenyl-2-thioxo-2,3-di-
hydropyrimidin-4(1H)-one 10. Mp > 300 °C; yield: 95% from
acetone. IR (KBr) cmꢀ1: 1698, 1668, 1559. 1H NMR (DMSO-d6) d:
2.31 (s, 3H, SCH3), 2.65 (s, 3H, CH3), 7.18–7.55 (m, 10H, arom. H).
Anal. Calcd for C19H16N2O2S2: C, 61.93; H, 4.38; N, 7.60; S, 17.40.
Found: C, 61.63; H, 4.52; N, 7.99; S, 17.12.
5.1.3.2. Methyl
1,2,3,4-tetrahydropyrimidine-5-carboxylate
6-(methylthio)-4-oxo-1,3-diphenyl-2-thioxo-
11. Mp 247–
5.1.4.2.
5-Acetyl-6-{[2-(dimethylamino)ethyl]amino}-1,3-
14. Mp
diphenyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
249 °C; yield: 93% from CH2Cl2/acetone. IR (KBr) cmꢀ1: 1725,
1674, 1568. 1H NMR (DMSO-d6) d: 2.30 (s, 3H, SCH3), 3.80 (s, 3H,
OCH3), 7.23–7.53 (m, 10H, arom. H). 13C NMR (DMSO-d6) d :
18.14 (SCH3), 52.67 (OCH3), 114.97 (C-5), 128.13 (CH), 128.20
(2CH), 129.07 (2CH), 129.12 (2CH), 129.22 (CH), 129.49 (2CH),
139.52 (C), 141.04 (C), 154.49 (C-6), 156.45 (CON), 163.60 (COO),
178.56 (CS). Anal. Calcd for C19H16N2O3S2: C, 59.63; H, 4.19; N,
7.29; S, 16.68. Found: C, 59.36; H, 4.27; N, 7.29; S, 16.67.
159–161 °C; yield: 74% from acetone/methanol. IR (KBr) cmꢀ1
:
3140, 2972, 1682, 1621. 1H NMR (CDCl3) d: 2.14 (s, 6H, N(CH3)2),
2.21–2.33 (m, 2H, CH2N), 2.37–2.50 (m, 2H, NHCH2), 2.68 (s, 3H,
CCH3), 7.19–7.59 (m, 10H, arom. H), 11.92 (br s, 1H, NH, exchange-
able). Anal. Calcd for C22H24N4O2S: C, 64.68; H, 5.92; N, 13.71; S,
7.85. Found: C, 64.36; H, 5.95; N, 13.69; S, 7.84.
5.1.4.3. Ethyl
6-{[2-(dimethylamino)ethyl]amino}-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
15. Mp 195–196 °C; yield: 72% from CH2Cl2/diethyl ether. IR (KBr)
cmꢀ1: 3208, 2976, 1731, 1657, 1608. 1H NMR (CDCl3) d: 1.34 (t,
J = 7.1 Hz, 3H, CCH3), 1.89 (s, 6H, N(CH3)2), 2.12–2.42 (m, 2H,
CH2N), 2.77–3.00 (m, 2H, NHCH2), 4.16–4.45 (m, 2H, OCH2), 6.50
(br s, 1H, NH, exchangeable), 7.18–7.70 (m, 10H, arom. H). Anal.
Calcd for C23H26N4O3S: C, 62.99; H, 5.98; N, 12.78; S, 7.31. Found:
C, 62.76; H, 5.93; N, 12.76; S, 7.32.
5.1.3.3. Ethyl
1,2,3,4-tetrahydropyrimidine-5-carboxylate
6-(methylthio)-4-oxo-1,3-diphenyl-2-thioxo-
12. Mp 223–
225 °C; yield: 97% from CH2Cl2/acetone. IR (KBr) cmꢀ1: 2988,
1732, 1683, 1572. 1H NMR (DMSO-d6) d: 1.26 (t, J = 7.1 Hz, 3H,
CCH3), 2.31 (s, 3H, SCH3), 4.27 (q, J = 7.1 Hz, 2H, CH2), 7.24–7.50
(m, 10H, arom. H). 13C NMR (DMSO-d6) d: 13.68 (CCH3), 18.57
(SCH3), 61.70 (CH2), 115.66 (C-5), 128.10 (CH), 128.20 (2CH),
128.87 (CH), 129.03 (2CH), 129.11 (2CH), 129.46 (2CH), 139.53
(C), 141.12 (C), 153.86 (C-6), 156.50 (CON), 163.02 (COO), 178.59
(CS). Anal. Calcd for C20H18N2O3S2: C, 60.28; H, 4.55; N, 7.03; S,
16.09. Found: C, 60.08; H, 4.37; N, 7.08; S, 15.98.
5.1.4.4. Isopropyl 6-{[2-(dimethylamino)ethyl]amino}-4-oxo-
1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbox-
ylate 16. Mp 178–180 °C; yield: 64% from diethyl ether. IR (KBr)
cmꢀ1: 3356, 2952, 1690, 1605. 1H NMR (DMSO-d6) d: 1.23 (d,
J = 6.3 Hz, 6H, 2CH3), 1.85 (s, 6H, N(CH3)2), 2.19–2.23 (m, 2H,
CH2N), 2.85–2.98 (m, 2H, NHCH2), 4.99–5.03 (m, 1H, OCH), 6.00
(br s, 1H, NH, exchangeable), 7.17–7.59 (m, 10H, arom. H). 13C
NMR (DMSO-d6) d: 21.40 (2CH3), 40.81 (CH2N), 44.64 (N(CH3)2),
55.52 (NHCH2), 68.33 (OCH), 89.15 (C-5), 127.59 (CH), 128.73
5.1.3.4. Isopropyl 6-(methylthio)-4-oxo-1,3-diphenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate
13. Mp
225–
227 °C; yield: 94% from CH2Cl2/acetone. IR (KBr) cmꢀ1: 2980,
1720, 1677, 1588. 1H NMR (DMSO-d6) d: 1.27 (d, J = 6.3 Hz, 6H,
2CH3), 2.31 (s, 3H, SCH3), 5.10 (m, 2H, OCH), 7.24–7.52 (m, 10H,

S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
3585
(2CH), 129.00 (2CH), 129.34 (CH), 129.54 (2CH), 129.95 (2CH),
137.84 (C), 140.19 (C), 152.02 (C-6), 157.86 (CON), 164.76 (COO),
177.88 (CS). Anal. Calcd for C24H28N4O3S: C, 63.69; H, 6.24; N,
12.38; S, 7.08. Found: C, 63.71; H, 5.89; N, 12.59; S, 7.40.
cmꢀ1: 3288, 2963, 2815, 1697, 1670, 1589. 1H NMR (CDCl3) d:
1.35 (t, J = 7.1 Hz, 3H, CH3), 2.02–2.49 (m, 6H, 3 NCH2), 2.82–3.08
(m, 2H, NHCH2), 3.22–3.52 (m, 4H, 2 morph. OCH2), 4.18–4.55
(m, 2H OCH2), 6.82 (br s, 1H, NH, exchangeable), 7.25–7.68 (m,
10H, arom. H). Anal. Calcd for C25H28N4O4S: C, 62.48; H, 5.87; N,
11.66; S, 6.67. Found: C, 62.47; H, 5.87; N, 11.70; S, 6.70.
5.1.4.5. Ethyl 6-{[2-(diethylamino)ethyl]amino}-4-oxo-1,3-di-
phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
17. Mp 172–173 °C; yield: 79% from CH2Cl2/diethyl ether. IR
(KBr) cmꢀ1: 3298, 2969, 1733, 1661, 1611. 1H NMR (CDCl3) d:
0.70 (t, J = 6.8 Hz, 6H, N(CH2CH3)2), 1.35 (t, J = 7.2 Hz, 3H, CH3),
2.18 (q, J = 6.8 Hz, 4H, N(CH2CH3)2), 2.30–2.42 (m, 2H, CH2N),
2.93–3.05 (m, 2H, NHCH2), 4.32 (q, J = 7.2 Hz, 2H, OCH2), 6.29
(br s, 1H, NH, exchangeable), 7.15–7.57 (m, 10H, arom. H). 13C
NMR (CDCl3) d: 10.28 (N(CH2CH3)2), 13.45 (CH3), 40.28 (CH2N),
44.39 (N(CH2CH3)2), 49.50 (NHCH2), 61.10 (OCH2), 88.28 (C-5),
127.57 (CH), 127.74 (2CH), 128.59 (2CH), 128.82 (2CH), 129.20
(CH), 129.79 (2CH), 137.28 (C), 139.19 (C), 151.50 (C-6),
158.16 (CON), 165.70 (COO), 178.04 (CS). Anal. Calcd for
C25H30N4O3S: C, 64.35; H, 6.48; N, 12.01; S, 6.87. Found: C,
64.49; H, 6.53; N, 12.11; S, 6.54.
5.1.4.10. Ethyl 6-{[2-(1-methylpyrrolidin-2-yl)ethyl]amino}-4-
oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate 22. Mp 155–157 °C; yield: 84% from acetone/ethanol. IR
(KBr) cmꢀ1: 3150, 2955, 1727, 1660, 1586. 1H NMR (CDCl3) d: 1.38
(t, J = 7.1 Hz, 3H, CCH3), 1.45–1.80 (m, 6H, 3 CCH2C), 1.87 (s, 3H,
NCH3), 2.10–2.25 (m, 1H, NCH), 2.44–2.62 (m, 2H, CH2N), 2.95–
3.25 (m, 2H, NHCH2), 4.34 (q, J = 7.1 Hz, 2H, OCH2), 7.18–7.63 (m,
10H, arom. H), 8.00 (br s, 1H, NH, exchangeable). 13C NMR
(DMSO-d6) d: 13.83 (CH3), 22.31 (CCH2C), 27.54 (CCH2C), 28.74
(CCH2C), 41.93 (NCH3), 42.02 (NCH), 55.93 (NCH2), 60.75 (NHCH2),
63.87 (OCH2), 89.28 (C-5), 127.54 (CH), 128.72 (2CH), 129.40
(2CH), 129.52 (2CH), 129.81 (CH), 130.00 (2CH), 138.38 (C),
140.28 (C), 151.92 (C-6), 158.06 (CON), 165.64 (COO), 178.21
(CS). Anal. Calcd for C26H30N4O3S: C, 65.25; H, 6.32; N, 11.71; S,
6.70. Found: C, 65.12; H, 6.41; N, 11.69; S, 6.67.
5.1.4.6. Isopropyl 6-{[2-(diethylamino)ethyl]amino}-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
18. Mp 185–187 °C; yield: 48% from CH2Cl2/methanol. IR (KBr)
cmꢀ1: 3237, 2971, 1707, 1672, 1604. 1H NMR (DMSO-d6) d: 0.66
(t, J = 7.1 Hz, 6H, 2CH3), 1.23 (d, J = 6.2 Hz, 6H, CH(CH3)2), 2.15 (q,
J = 7.1 Hz, 4H, N(CH2CH3)2), 2.33–2.38 (m, 2H, CH2N), 2.85–2.95
(m, 2H, NHCH2), 4.94–5.06 (m, 1H, OCH), 5.94 (br s, 1H, NH,
exchangeable), 7.16–7.20 (m, 2H, arom. H), 7.30–7.59 (m, 8H,
arom. H). 13C NMR (DMSO-d6) d: 10.87 (2CH3), 21.37 (CH(CH3)2),
40.45 (CH2N), 44.67 (N(CH2CH3)2), 49.46 (NHCH2), 68.46 (OCH),
89.17 (C-5), 127.61 (CH), 128.63 (CH), 128.75 (2CH), 129.51
(2CH), 129.56 (2CH), 130.14 (2CH), 137.74 (C), 140.18 (C), 151.13
(C-6), 157.98 (CON), 164.79 (COO), 177.93 (CS). Anal. Calcd for
C26H32N4O3S: C, 66.17; H, 6.89; N, 11.71; S, 7.15. Found: C,
65.97; H, 6.71; N, 11.66; S, 6.97.
5.1.4.11. Ethyl
4-oxo-1,3-diphenyl-6-[(2-pyridin-2-ylethyl)-
amino]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
23. Mp 213–215 °C; yield: 83% from acetone/ethanol. IR (KBr)
cmꢀ1: 3310, 2991, 1702, 1673, 1593. 1H NMR (CDCl3) d: 1.37 (t,
J = 7.1 Hz, 3H, CH3), 2.68–2.97 (m, 2H, 3 CH2Py), 3.15–3.48 (m,
2H, NHCH2), 4.37 (q, J = 7.1 Hz, 2H, OCH2), 6.70 (br s, 1H, NH,
exchangeable), 6.92–7.67 (m, 13H, arom. H), 7.93–8.09 (m, 1H,
pyrid. H). Anal. Calcd for C26H24N4O3S: C, 66.08; H, 5.12; N,
11.86; S, 6.79. Found: C, 65.96; H, 5.30; N, 11.87; S, 6.86.
5.1.4.12. Ethyl 6-[(2-anilinoethyl)amino]-4-oxo-1,3-diphenyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 24. Mp
180–182 °C; yield: 88% from acetone/ethanol. IR (KBr) cmꢀ1
:
3318, 2933, 1692, 1602. 1H NMR (CDCl3) d: 1.35 (t, J = 7.1 Hz, 3H,
CH3), 2.76–3.20 (m, 4H, 2CH2), 4.37 (q, J = 7.1 Hz, 2H, OCH2),
6.46–7.95 (m, 17H, 15 arom. H + 2NH). Anal. Calcd for
C27H26N4O3S: C, 66.65; H, 5.39; N, 11.51; S, 6.59. Found: C,
66.69; H, 5.48; N, 11.59; S, 6.69.
5.1.4.7. Ethyl 6-{[3-(dimethylamino)propyl]amino}-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
19. Mp 176–178 °C; yield: 47% from acetone/ethanol. IR (KBr)
cmꢀ1: 3134, 2954, 2818, 1721, 1658, 1589. 1H NMR (CDCl3) d:
1.37 (t, J = 7.1 Hz, 3H, CCH3), 1.42–1.66 (m, 2H, CCH2C), 1.77 (s,
6H, N(CH3)2), 2.05–2.35 (m, 2H, CH2N), 2.95–3.25 (m, 2H, NHCH2),
4.16–4.53 (m, 2H, OCH2), 7.10–7.67 (m, 10H, arom. H), 7.86 (br s,
1H, NH, exchangeable). Anal. Calcd for C24H28N4O3S: C, 63.69; H,
6.24; N, 12.38; S, 7.08. Found: C, 63.87; H, 6.38; N, 12.46; S, 7.10.
5.1.4.13. Methyl 6-({2-[aminosulfonyl)phenyl]ethyl}amino)-4-
oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate 25. Mp 287–289 °C; yield: 82% from acetone/methanol.
IR (KBr) cmꢀ1: 3342, 3193, 1722, 1657, 1606. 1H NMR (DMSO-d6)
d: 2.72–2.79 (m, 2H, CH2Ph), 3.02–3.15 (m, 2H, NHCH2), 3.72 (s,
3H, OCH3), 6.15 (br s, 1H, NH, exchangeable), 7.15–7.55 (m, 12H,
arom. H), 7.71 (d, J = 8.3 Hz, 2H, arom. H). 13C NMR (DMSO-d6) d:
34.03 (CH2Ph), 45.29 (NHCH2), 51.97 (OCH2), 88.35 (C-5), 125.79
(2CH), 127.63 (CH), 128.62 (2CH), 128.77 (2CH), 128.83 (2CH),
129.59 (CH), 129.71 (2CH), 129.93 (2CH), 137.78 (C), 140.21 (C),
141.75 (C), 142.33 (C), 152.85 (C-6), 157.67 (CON), 166.31 (COO),
178.25 (CS). Anal. Calcd for C26H24N4O5S2: C, 58.19; H, 4.51; N,
10.44; S, 11.95. Found: C, 57.99; H, 4.59; N, 10.23; S, 11.86.
5.1.4.8. Ethyl
4-oxo-1,3-diphenyl-6-[(2-piperidin-1-ylethyl)-
amino]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
20. Mp 170–171 °C; yield: 86% from acetone/ethanol. IR (KBr)
cmꢀ1: 3243, 2938, 2816, 1683, 1592. 1H NMR (CDCl3) d: 1.13–
1.32 (m, 6H, CCH2CH2CH2C), 1.38 (t, J = 7.1 Hz, 3H, CH3), 2.03–
2.17 (m, 4H, 2 piper. NCH2), 2.27 (t, J = 5.9 Hz, 2H, CH2N), 2.97–
3.09 (m, 2H, NHCH2), 4.35 (q, J = 7.1 Hz, 2H, OCH2), 6.23 (br s, 1H,
NH, exchangeable), 7.18–7.67 (m, 10H, arom. H). 13C NMR (CDCl3)
d: 14.35 (CH3), 24.11 (NCH2CH2CH2), 25.52 (2 NCH2CH2CH2), 41.01
(CH2N), 53.51 (2 NCH2CH2CH2), 55.98 (NHCH2), 61.93 (OCH2),
89.08 (C-5), 128.43 (CH), 128.60 (2CH), 129.46 (2CH), 129.66
(2CH), 130.28 (CH), 130.68 (2CH), 138.04 (C), 140.06 (C), 152.35
(C-6), 158.98 (CON), 166.17 (COO), 178.86 (CS). Anal. Calcd for
C26H30N4O3S: C, 65.25; H, 6.32; N, 11.71; S, 6.70. Found: C,
65.13; H, 6.51; N, 11.84; S, 6.78.
5.1.4.14. Methyl 6-{[2-(1H-indol-3-yl)ethyl]amino}-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
26. Mp 231–233 °C; yield: 69% from acetone. IR (KBr) cmꢀ1: 3332,
2947, 1679, 1645, 1618. 1H NMR (DMSO-d6) d: 2.72–2.79 (m, 2H,
CCH2C), 3.05–3.20 (m, 2H, NHCH2), 3.71 (s, 3H, OCH3), 5.91 (br s,
1H, NH, exchangeable), 6.78 (s, 1H, indole H-2), 6.80–7.45 (m,
14H, arom. H), 10.82 (br s, 1H, indole NH, exchangeable). 13C
NMR (DMSO-d6) d: 24.59 (CCH2C), 44.68 (NHCH2), 51.88 (OCH3),
88.14 (C-5), 109.31 (CH), 111.41 (CH), 118.06 (CH), 118.28 (CH),
121.11 (CH), 123.06 (CH), 126.41 (C), 127.60 (C), 128.64 (2CH),
5.1.4.9. Ethyl
6-[(2-morpholin-4-ylethyl)amino]-4-oxo-1,3-
diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
21. Mp 194–195 °C; yield: 49% from acetone/ethanol. IR (KBr)

3586
S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
128.74 (2CH), 129.56 (CH), 129.64 (2CH), 129.86 (2CH), 136.30 (C),
137.74 (C), 140.23 (C), 152.80 (C-6), 157.67 (CON), 166.18 (COO),
178.16 (CS). Anal. Calcd for C28H24N4O3S: C, 67.72; H, 4.87; N,
11.28; S, 6.46. Found: C, 67.35; H, 5.12; N, 10.98; S, 6.40.
staining solution (2 mg/mL fluorescein diacetate, 4 lg/mL ethi-
dium bromide) in HBSS and incubated for 10 min at room temper-
ature. Thereafter, a drop of cell suspension was placed on a slide,
sealed with a coverslip, and analysed under UV light in a dark field
illumination. Neutrophils with intact membrane (i.e., viable cells)
appeared as green fluorescent cells, whereas neutrophils with
damage and ethidium bromide-permeable membrane (i.e., necro-
tic cells) displayed a fluorescent red nucleus.
5.1.4.15. Ethyl 6-[(2-methoxyethyl)amino]-4-oxo-1,3-diphenyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 27. Mp
201–203 °C; yield: 81% from acetone/ethanol. IR (KBr) cmꢀ1: 3365,
2985, 1733, 1661, 1601. 1H NMR (DMSO-d6) d: 1.22 (t, J = 7.1 Hz,
3H, CH3), 2.83–3.35 (m, 4H, 2CH2), 3.07 (s, 3H, COOCH3), 4.23 (q,
J = 7.1 Hz, 2H, OCH2), 6.38 (br s, 1H, NH, exchangeable), 7.05–7.68
(m, 10H, arom. H). Anal. Calcd for C22H23N3O4S: C, 62.10; H, 5.45;
N, 9.88; S, 7.54. Found: C, 62.12; H, 5.64; N, 9.88; S, 7.71.
5.2.4. Boyden chamber migration assay
Neutrophil locomotion was studied by means of the leading
front method, as previously described by Corcione et al.31 Neutro-
phils were preincubated in the absence or presence of appropriate
doses of compounds 1 and 14–29 for 15 min at room temperature.
Tests were conducted in duplicate, using blind well chambers
5.1.4.16. Methyl 6-[(2,2-dimethoxyethyl)amino]-4-oxo-1,3-di-
phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
28. Mp 177–179 °C; yield: 86% from CH2Cl2/diethyl ether. IR (KBr)
cmꢀ1: 1696, 1650, 1621. 1H NMR (CDCl3) d: 2.68–2.90 (m, 2H, CH2),
3.24 (s, 6H, 2CH3), 3.88 (s, 3H, COOCH3), 4.20–4.42 (m, 1H, OCH),
7.25–7.75 (m, 10H, arom. H), 7.96 (br s, 1H, NH, exchangeable).
Anal. Calcd for C22H23N3O5S: C, 59.85; H, 5.25; N, 9.52; S, 7.26.
Found: C, 59.68; H, 5.35; N, 9.38; S, 7.18.
(Neuro-Probe, Gathersburg, MD) with a 3 lm pore size cellulose
ester filter (Millipore, Milan, Italy) separating the upper from the
lower compartment of the chambers. Then, cells (4 ꢂ 105) in the
absence or presence of various amounts of tested compounds were
placed in the upper compartment of the chambers. Chemoattrac-
tants (10ꢀ9 M IL-8 or 10ꢀ8 M fMLP or 10ꢀ9 M C5a) were placed in
the lower compartment of the chambers. Experiments were also
carried out without chemoattractants in the lower compartment
(spontaneous migration). After incubation at 37 °C for 45 min,
the filters were removed, fixed in ethanol, stained with Harris
hematoxylin, dehydrated, cleared with xylene, and mounted in Eu-
kitt (Kindler, GmbH). Then, the distance (mm) travelled by the
leading front of cells was measured ꢂ 400 magnification. Five ran-
domly chosen fields were read for each filter. The net migration
was determined by subtracting spontaneous migration (i.e. the dis-
tance travelled by neutrophils in the absence of the stimulus) from
the distance travelled by neutrophils toward the stimulus. The con-
centration of each compound giving 50% inhibition (IC50) of net
migration was obtained from nonlinear regression analysis with
SPSS for Windows version 6.0, Wacker Drive, Chicago, IL.
5.1.4.17.
5-Acetyl-6-(4-methylpiperazin-1-yl)-1,3-diphenyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one 29. Mp 243–245 °C;
yield: 74% from acetone/methanol. IR (KBr) cmꢀ1: 2968, 1678,
1580. 1H NMR (CDCl3) d: 1.68–1.83 (m, 4H, 2CH2N), 2.06 (s, 3H,
CH3), 2.63 (s, 3H, CCH3), 2.93–3.02 (m, 2H, 2CH2N), 7.18–7.36 (m,
4H, arom. H), 7.39–7.58 (m, 6H, arom. H). 13C NMR (CDCl3) d:
32.36 (CH3), 46.12 (NCH3), 51.39 (2CH2N), 53.23 (2CH2N), 110.32
(C-5), 128.22 (2CH), 128.72 (CH), 128.91 (CH), 129.15 (2CH),
129.74 (2CH), 130.40 (2CH), 139.91 (C), 141.51 (C), 160.02 (C-6),
160.47 (CON), 180.32 (CS), 198.07 (CO). Anal. Calcd for
C23H24N4O2S: C, 65.69; H, 5.75; N, 13.32; S, 7.62. Found: C,
65.74; H, 5.78; N, 13.38; S, 7.35.
5.2. Biology
5.2.5. Induction of peritonitis, cell count, cell composition and
determination of protein concentration in the peritoneal
exudate
5.2.1. General
Hank’s balanced salt solution without phenol red (HBSS, ICN,
Biomed, Milan, Italy) mixed with Dulbecco’s phosphate-buffered
saline (PBS, ICN Biomed) (HBSS:PBS = 3:1) containing 1 mg/mL bo-
vine serum albumin (BSA, Sigma, Milan, Italy) was used as incuba-
tion medium. IL-8, fMLP, C5a, ethidium bromide, and fluorescein
diacetate were from Sigma Chemical, St. Louis, MO.
The experiments were performed using male Swiss mice (25–
30 g) fasted 16 h before the experiment and with free access to
water. Mice were randomly assigned to groups of 8–12 animals or-
ally treated with vehicle or the compound under examination (17)
(50 mg/kg os) 1 h before the induction of peritonitis. Dexametha-
sone (3 mg/kg os) was used as reference drug. Peritonitis was in-
duced following a modification of Thurmond’s method.32 Briefly,
5 mg/mL Zymosan or phosphate-buffered saline (PBS) was injected
into the peritoneal space of mice (final volume 0.2 mL). After 4 h,
the animals were euthanized, the peritoneal cavities were washed
with 3 mL of PBS containing 3 mM EDTA and the volume was col-
lected. Total leukocyte counts were performed by an observer una-
ware of the treatment using a Neubauer chamber and an optical
microscope after diluting the samples of the peritoneal fluid with
Türk solution (1:20). Differential cell counts were performed using
a light microscope. Chromatic characteristics and the shape of the
nucleus relative to the cytoplasm were used to differentiate leuko-
cyte subpopulations. The total cell number/cavity and the granulo-
cyte number/cavity were measured.
5.2.2. Neutrophilic polymorphonuclear leukocyte preparation
Heparinized venous blood (10 U/mL heparin) was obtained
from healthy volunteers (20–37 years old) after informed consent.
Neutrophilic polymorphonuclear leukocytes (neutrophils) were
prepared by dextran sedimentation, followed by centrifugation
(400g, 30 min) on a Ficoll-Hypaque density gradient, as previously
described.28 Contaminating erythrocytes were removed by hypo-
tonic lysis. Neutrophils resuspended in incubation medium were
>97% pure, as determined by morphologic analysis of Giemsa-
stained cytopreparation.
5.2.3. Assessment of neutrophil viability
Neutrophils (2 ꢂ 106/mL) were incubated for 2 h in tissue cul-
ture tubes (17 ꢂ 100 mm, Falcon, Becton Dickinson) in incubation
medium at 37 °C in a CO2 atmosphere (0.5 mL final volume), with
appropriate doses of compounds 1 and 14–29. Cell viability, mea-
sured as integrity of membrane, was assessed by an ethidium bro-
mide–fluorescein diacetate test according to Dankberg,29 as
previously described by Ottonello et al.30 Briefly, cells (4 ꢂ 104/
100 mL) harvested from culture tubes were mixed with 50 mL of
Protein content (lg/mL) was spectrophotometrically deter-
mined applying the bicinchonate method with a commercial kit
(Pierce, BCA protein assay kit).
Data are expressed as mean SEM. Statistical analysis was per-
formed adopting ANOVA one way test followed by Bonferroni’s
**
post test; P < 0.01 indicated significant differences compared to
negative control (Graph pad PRISM 5.0, San Diego, CA, USA). Exper-

S. Cesarini et al. / Bioorg. Med. Chem. 17 (2009) 3580–3587
3587
16. Luster, A. D.; Alon, R.; Von Andrian, U. H. Nat. Immunol. 2005, 6, 1182.
17. Murdoch, C.; Finn, A. Blood 2000, 95, 3032.
18. Harada, A.; Mukaida, N.; Matsushima, K. Mol. Med. Today 1996, 2, 482.
iments were carried out in accordance with Italian law (DL 116/92)
and approved by the Ministry of Health.
19. Fujishima, S.; Aikawa, N. Intens. Care Med. 1995, 21, 277.
20. Dwyer, M. P.; Yu, Y.; Chao, J.; Aki, C.; Chao, J.; Biju, P.; Girijavallabhan, V.;
Rindgen, D.; Bond, R.; Mayer-Ezel, R.; Jakway, J.; Hipkin, R. W.; Fossetta, J.;
Gonsiorek, W.; Bian, H.; Fan, X.; Terminelli, C.; Fine, J.; Lundell, D.; Merritt, J. R.;
Rokosz, L.; Kaiser, B.; Li, G.; Wang, W.; Stauffer, T.; Ozgur, L.; Baldwin, J.;
Taveras, A. G. J. Med. Chem. 2006, 49, 7603.
21. Jin, Q.; Nie, H.; McCleland, B. W.; Widdowson, K. L.; Palovich, M. R.; Elliott,
J. D.; Goodman, R. M.; Burman, M.; Sarau, H. M.; Ward, K. W.; Nord, M.;
Orr, B. M.; Gorycki, P. D.; Busch-Petersen, J. Bioorg. Med. Chem. Lett. 2004,
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S.; Beccari, A. S.; Bizzarri, C.; Cavicchia, M. R.; Locati, M.; Galliera, E.; Di
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Acknowledgements
This work was supported by MURST (Cofinanziamento Nazio-
nale), CNR (Rome) and Ministero Italiano della Salute - Istituto
Superiore di Sanità (Grant No. 40D.46). Fondazione Carige is grate-
fully acknowledged for financially supporting S.C. The authors
thank Mr. O. Gagliardo for the microanalyses, Mr F. Tuberoni for
the IR spectra, Professor F. Lucchesini, Dr. M. Anzaldi, Dr. R. Raggio
and Dr. C. Rossi for the NMR spectra.
23. Allegretti, M.; Bestini, R.; Cesta, M. C.; Bizzarri, C.; Di Bitondo, R.; Di Cioccio, V.;
Galliera, E.; Berdini, V.; Topai, A.; Zampella, G.; Russo, V.; Di Bello, N.; Nano, G.;
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