Synthesis and evaluation of 2-tosylamino and 2-tosyliminopyrimidine derivatives as inhibitors of some leukocyte functions

Article abstract of DOI:10.1016/S0223-5234(03)00013-8

We have studied the potential anti-inflammatory effects of 20 2-tosylamino and 2-tosyliminopyrimidine new derivatives in human neutrophils. We have evaluated their interference with some leukocyte functions and 5-lipoxygenase activity. All the compounds r

Full text of DOI:10.1016/S0223-5234(03)00013-8

European Journal of Medicinal Chemistry 38 (2003) 289ꢀ296
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and evaluation of 2-tosylamino and 2-tosyliminopyrimidine
derivatives as inhibitors of some leukocyte functions
Patricia Fernandez-Ferri a, Amalia Ubeda a,*, Isabel Guille´n a, Jamal Lasri b,
M. Eugenia Gonza´lez-Rosende c, Mohamed Akssira b,1, Jose´ Sepu´lveda-Arques b,*
a Departamento de Farmacolog´ıa, Universitat de Valencia, 46100 Burjassot, Spain
b Departamento de Qu´ımica Orga´nica, Universitat de Valencia, 46100 Burjassot, Spain
c Departamento de Qu´ımica, Bioqu´ımica y Biolog´ıa Molecular, Facultad de Ciencias Experimentales y de la Salud, Universidad Cardenal Herrera-CEU,
46113 Moncada, Valencia, Spain
Received 21 August 2002; received in revised form 8 December 2002; accepted 16 December 2002
Abstract
We have studied the potential anti-inflammatory effects of 20 2-tosylamino and 2-tosyliminopyrimidine new derivatives in human
neutrophils. We have evaluated their interference with some leukocyte functions and 5-lipoxygenase activity. All the compounds
reduced neutrophil degranulation process at concentrations in the mM range. Besides, compounds with a phenolic substitution
inhibited leukotriene B4 biosynthesis in neutrophils and decreased the cell-free 5-lipoxygenase activity. This study demonstrates that
2-tosylamino and 2-tosyliminopyrimidine derivatives can reduce the activation of neutrophil cells which may have relevance for the
modulation of the inflammatory response.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Leukocytes; Elastase; Lipoxygenase; 2-Tosyliminopyrimidines; 2-Tosylaminopyrimidines
1. Introduction
They can regulate leukocyte functions, smooth muscle
tone and vascular permeability [2].
Neutrophils play a crucial role in the inflammatory
response. Activated neutrophils migrate to ingest and
kill invading pathogens at the inflamed area. They
generate reactive oxygen species and secrete lytic pro-
teins during the oxidative burst which can mediate tissue
injury [1].
5-Lipoxygenase (5-LO) is the enzyme responsible for
the production of leukotrienes, such as LTB4 and
cysteinyl-leukotrienes. Leukotrienes are potent lipid
mediators of inflammation and immune response.
During a project directed toward the synthesis of new
anti-inflammatory compounds we reported in a previous
work that a serie of 2-tosyliminohexahydroimidazopyr-
imidines A exerted inhibition of leukocytes responses [3].
As an extension of this study we planned the prepara-
tion of 2-tosylamino and 2-tosyliminodihydropyrimi-
dines (B), that contain a similar functionality of
compounds A, but with a structure where the carbox-
amide group was moved to the para position of the
phenyl ring (Fig. 1).
The synthesis of the compounds B was attempted by
alkylation of 2-tosylaminopyrimidine, however in this
reaction in addition to derivatives from alkylation of the
pyrimidinic nitrogen, compounds 3, we also obtained
the derivatives 4, as a result of the exocyclic nitrogen
alkylation (Fig. 2). In this paper we describe the
synthesis of compounds 3 and 4 and the biological
evaluation on neutrophil degranulation and LTB4
biosynthesis.
Abbreviations: LTB4, leukotriene B4; 5-LO, 5-lipoxygenase; DMF,
dimethylformamide; DIPEA, diisopropylethylamine; Ts, tosyl; IC50
inhibitory concentration 50%.
,
* Corresponding authors.
E-mail addresses: ubedap@uv.es (A. Ubeda), jose.sepulveda@uv.es
(J. Sepu´lveda-Arques).
1
On leave from Universite´ Hassan II, Mohammedia, Maroc.
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00013-8

290
P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ296
/
Table 1
Inhibition of human neutrophil elastase release for compounds 3aꢀ
and 4aꢀ
/j
/j
Fig. 1. Comparison of functionality between tosyliminoihexahydro-
pyrimidines (A) and 2-tosylaminodihydropyrimidines (B).
Compound
R
Elastase
2. Chemistry
% Inhibition a IC50 (mM) b
Reaction of the 2-p-tosylaminopyrimidine 1 with the
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
H
H
60.29
61.09
53.79
45.59
48.69
62.09
CH2 92.09
CH2 70.39
/
2.2
2.6
2.6
3.4
2.7
2.3
2.1
1.9
1.9
2.5
3.2
3.5
3.3
3.2
2.2
2.8
2.6
3.5
4.3
2.6
5.0
ND
ND
ND
ND
ND
ND
appropriate p-bromomethylbenzamide (2aꢀj) in the
/
/
CH3 ꢀ
CH3 ꢀ
/
CH2
/
presence of Hunig’s base in DMF [4,5] provided the
¨
corresponding 2-tosylimino-1-substituted dihydropyri-
/CH2
/
C6H5 ꢀ
C6H5 ꢀ
4-HOꢀ
4-HOꢀ
4-FꢀC6H4 ꢀ
4-FꢀC6H4 ꢀ
2,4-Cl2 ꢀC6H3 ꢀ
2,4-Cl2 ꢀC6H3 ꢀ
/
CH2 ꢀ
/
CH2
/
midines (3aꢀ
The relative yields (3:4) in the alkylation reaction [6ꢀ
could be explained by steric factors due to the bulky
electrophiles as it has been reported in literature [10ꢀ12].
/
j) and 2-tosylaminopyrimidines (4aꢀ
/j).
/
CH2 ꢀ
/
CH2
/
/9]
/
C6H4 ꢀ
C6H4 ꢀ
CH2 ꢀ
CH2 ꢀ
/CH2 ꢀ
/
/
1.5 (1.0ꢀ
2.8 (1.6ꢀ
ND
ND
/
2.0)
/
/CH2 ꢀ
/
/
/5.0)
/
/
/
CH2
44.19
/
/
/
/
/
CH2
38.29
/
Compound 1 was obtained from the 2-aminopyrimidine
by treatment with p-toluenesulfonyl chloride in pyridine
/
/
CH2 ꢀ
/
CH2 46.19
CH2 37.59
40.49
53.99
81.99
80.69
38.89
29.99
40.59
23.89
83.99
/
ND
ND
4f
/
/
CH2 ꢀ
/
/
[4]. The required p-bromomethylbenzamides (2aꢀj) were
/
3g
4g
3h
4h
3i
C6H5
C6H5
/
ND
ND
prepared by amination of their corresponding p-bro-
momethylbenzoic acids [5].
/
4-HOꢀ
4-HOꢀ
4-Fꢀ
4-Fꢀ
2,4ꢀCl2 ꢀ
2,4-Cl2 ꢀC6H3
/
C6H4
/
1.1 (0.6ꢀ
1.5 (1.0-2.2)
ND
/1.9)
/
C6H4
/
/
C6H4
C6H4
C6H3
/
4i
/
/
ND
3j
/
/
/
ND
3. Results and discussion
4j
/
/
ND
HIP-4
/
3.9 (2.0ꢀ5.5)
/
Inhibitory activity of pyrimidine derivatives was
evaluated in human neutrophils. As can be seen from
the data in Table 1, all the compounds at 10 mM
inhibited degranulation process assessed as elastase
release. None of the compounds, at 10 mM showed
cytotoxic effects on neutrophils as assayed by the
mitochondrial reduction of MTT (data not shown).
Comparing the pair of amino or imino derivatives, there
were not significant differences, which suggest the lack
of influence of this conversion. The unsubstituted
compounds (3a and 4a) showed moderate inhibition,
the addition of a phenyl or halogenated phenyl group
(3g, 4g, 3i, 4i, 3j and 4j) decreased the activity. However,
the presence of a phenolic substituent increased the
potency in phenyl and phenylethyl series of compounds,
HIP-4: 3-(4-fluorophenyl)-8-(5,5-dimethyl-1,3-dioxan-2-yl)-5-tosyli-
mino-2-oxo-1,2,3,5,6,8a-hexahydroimidazo[1,2-c]pyrimidine (Ref. [3]).
a
Compounds were assayed at 10 mM.
b
Concentration (mM) required for 50% inhibition of elastase release
with confidence limits in parentheses. Only compounds that reached
70% inhibition were assayed, ND: not determined.
being 3d, 4d, 3h and 4h the most active compounds, with
IC50 about 1 mM. The inhibition of the release of
proteolytic enzymes during neutrophil infiltration and
activation may limit tissue damage during the inflam-
matory response [1].
Data from active compounds on LTB4 release and 5-
LO activity are indicated in Table 2 (the rest of
Fig. 2.

P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ
/296
291
Table 2
Inhibition of human neutrophil LTB4 release and 5-LO activity for active compounds
Compound
LTB4 release % inhibition a
LTB4 release IC50 (mM) b
5-LO activity % Inhibition a
3d
4d
3e
4e
3f
49.59
51.59
NA
80.39
66.49
91.79
44.89
79.19
/
0.9
5.3 (3.6ꢀ
3.6 (2.0ꢀ
ND
1.2 (0.9ꢀ
1.4 (0.8ꢀ
0.5 (0.3ꢀ
5.9 (3.9ꢀ
0.4 (0.3ꢀ
/
8.9)
47.39
51.09
NA
65.29
61.29
67.19
46.99
67.59
/
5.4
/
3.4
/6.3)
/6.6
/
3.6
0.9
5.3
2.7
3.3
/
1.6)
2.2)
0.8)
8.9)
0.6)
/
7.8
1.2
3.6
4.6
2.6
/
/
/
4f
/
/
/
3h
4h
/
/
/
/
/
/
Reference inhibitor zileuton showed an IC50 of 0.23 (0.03ꢀ
/
0.9) mM for LTB4 release and for 5-LO activity showed an IC50 of 0.61 (0.5ꢀ
not active, ND: not determined.
12 experiments, IC50 in mM is given with confidence limits in parentheses.
/
0.8) mM.
a
Compounds were assayed at 5 mM. NAꢂ
/
b
Values are means of 6ꢀ
/
derivatives were not active at 10 mM). The presence of a
hydroxy substituent in the aromatic ring had a general
effect increasing the potency in both compounds 3 and
4, and in both series d and h derived, respectively, from
phenylethylamine and phenylamine. However, the pre-
sence of Cl as substituent had a more restricted effect
and only the compounds 3f, 4f, containing an ethylenic
chain presented activity. In the case of F as substituent
only the compound 4e behaved as a potent inhibitor.
This effect can be related to a direct inhibition of the 5-
LO activity, since these compounds exerted potent
inhibition in the cell free assay of 5-LO activity. LTB4
is a potent leukocyte chemotactic agent, thus the
modulation of LTB4 production contributes to reduce
leukocyte infiltration at the inflammatory site.
Neutrophils play a critical role in initiating and
maintaining inflammatory processes in the joint [13].
Besides, it has been reported that 5-LO inhibition may
be beneficial in some inflammatory and respiratory
diseases such as systemic lupus erythematous and
asthma [14,15].
Present study indicates that 2-tosyliminopyrimidine
derivatives exert in vitro inhibitory effects on human
neutrophil functions which may confer a modulatory
effect on inflammatory process.
(J values) are given in ppm and Hz, respectively. MS
and HRMS were obtained using a VG Autospec TRIO
1000 instrument. The ionization mode used in mass
spectra was electron impact (EI) or fast atom bombard-
1
ment (FAB). H- and 13C-NMR assignments have been
confirmed by homonuclear two dimensional correla-
tions and DEPT experiments. Elemental analyses were
recorded by the Servicio de Microana´lisis of the Uni-
versity of Valencia (SCSIE).
4.1.2. 2-(p-Toluenesulfonamido)pyrimidine (1)
p-Toluenesulfonyl chloride (30 mmol) in pyridine (6
mL) was slowly added to 2-aminopyrimidine (20 mmol)
dissolved in dry pyridine (8 mL) at 0 8C. The solution
was stirred for 2 h and allowed to reach room
temperature (r.t.). Water was added (100 mL) and the
solid was collected and recrystallized from chloroform.
Yield: 90%; m.p. 208ꢀ
/
210 8C (lit. [4] 204ꢀ206 8C).
/
4.1.3. General procedure for the synthesis of p-
bromomethylbenzamides (2aꢀj)
/
DMF (2 drops) was added to a stirred solution of p-
bromomethylbenzoic acid (1 g, 4.64 mmol) in dry
CH2Cl2 (8 mL) under argon and then cooled in an ice
bath at 0 8C. Oxalyl chloride (0.79 mL, 9.28 mmol) in
dry CH2Cl2 (1.1 mL) was added dropwise and the
reaction mixture was stirred at 0 8C for 3 h. The solvents
were removed in vacuo and then azeotroped with
4. Experimental
4.1. Chemistry
toluene (3ꢁ1.6 mL). The remaining oil was dissolved
/
in toluene (13.9 mL) and hexane (13.9 mL) and stirred at
r.t. The corresponding amine (3.86 mmol) and pyridine
(0.31 mL, 3.86 mmol) were added and the reaction
mixture was stirred overnight. The resulting solid was
filtered, and the solvents were removed in vacuo. The
4.1.1. General
All reagents were purchased from Aldrich and used
without purification unless stated otherwise. All experi-
ments were made under nitrogen atmosphere. Melting
points were determined with a Kofler hot-stage appara-
tus and are uncorrected. Flash column chromatography
solid was dissolved in EtOAcꢀH2O and the organic
/
layer washed with HCl 1 N, saturated NaHCO3 and
brine, and then dried over Na2SO4. The solvent was
removed in vacuo yielding a solid which was identified
was performed using silica gel (Merck 60, 70ꢀ230 mesh).
/
1H- and 13C-NMR spectra were recorded on a Bruker
AC-300 instrument in CDCl3, unless otherwise indi-
cated. Chemical shifts (d values) and coupling constants
1
as the corresponding benzamide (2). Spectroscopic H-
and 13C-NMR data are shown in Table 3.

292
P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ296
/
Table 3
Characteristic spectral data for compounds 2aꢀ
/
j
Product 13C-NMR (CDCl3 ꢀ
/TMS) d (ppm)
1H-NMR (CDCl3 ꢀ
TMS) d (ppm)
/
2a a
2b a
34.4 (CH2), 128.7 (CH), 129.9 (CH), 135.0 (C), 141.9 (C), 168.3 (CÄ
15.1 (CH3), 33.9 (CH2), 34.4 (CH2), 127.8 (CH), 129.5 (CH), 134.9 (C), 1.16 (t, Jꢂ
141.2 (C), 165.8 (CÄO) (d, Jꢂ 8, 2H), 7.83 (d, Jꢂ
32.7 (CH2), 36.0 (CH2), 41.5 (CH2), 127.0 (CH), 127.7 (CH), 129.4 2.86 (t, Jꢂ 6.8, 2H), 3.64 (c, Jꢂ
(CH), 129.9 (CH), 135.0 (C), 139.2 (CH), 141.1 (C), 141.5 (C), 162.1 1H, NH), 7.17 (m, 3H), 7.24 (d, Jꢂ
(C), 167.3 (CÄO) 7.60 (d, Jꢂ 8, 2H), 8.46 (d, Jꢂ 8, 1H)
33.9 (CH2), 34.7 (CH2), 41.2 (CH2) 122.0 (CH),127.8 (CH), 129.1 2.89 (t, 2H, Jꢂ 7), 3.66 (c, 2H, Jꢂ 7), 4.41 (s, 2H), 6.15 (bs, 1H,
(CH), 130.5 (CH), 132.5 (C), 134.7 (C), 135.5 (C), 146.8 (C), 154.1 (C), NH), 7.08 (d, Jꢂ 8.4, 2H), 7.21 (d, Jꢂ 8.4, 2H), 7.36 (d, Jꢂ 8.1,
166.1 (CÄO) 2H), 7.61 (d, Jꢂ 8, 2H)
33.7 (CH2), 35.2 (CH2), 41.7 (CH2), 116.0 (d, Jꢂ 2.83 (t, 2H, Jꢂ 7), 3.61 (c, 2H, Jꢂ
(CH), 130.5 (d, Jꢂ7.8, CH), 131.4 (CH), 134.9 (C), 141.1 (C), 144.9 8.6, 2H), 7.10 (dd, Jꢂ 8.6, Jꢂ 5, 2H), 7.35 (d, Jꢂ
(C), 162.1 (d, Jꢂ244.7, C), 167.3 (CÄO) Jꢂ 8, 1H)
32.7 (CH2), 33.2 (CH2), 40.0 (CH2), 127.7 (CH),129.2 (CH), 129.7 2.98 (t, Jꢂ
(CH), 129.8 (CH), 132.2 (CH), 133.5 (C), 134.7 (C), 135.1 (C), 135.5 6.17 ( bs, 1H, NH), 7.12 (bs, 2H), 7.32ꢀ
(C), 141.6 (C), 167.3 (CÄO) 8, 2H
33.8 (CH2), 120.6 (CH),124.1(CH), 128.4 (CH), 129.0 (CH), 129.6 4.75 (s, 2H), 7.20 (t, Jꢂ
(CH), 135.1 (C), 139.4 (C), 141.8 (C), 165.4 (CÄO) 2H), 7.80 (d, Jꢂ 8, 2H), 8.00 (d, Jꢂ
33.9 (CH2), 115.3 (CH), 122.5 (CH), 128.2 (CH), 129.5 (CH), 130.9 4.8 (s, 2H), 6.85 (d, Jꢂ 7, 2H), 7.40 (d, Jꢂ
(C), 135.3 (C), 141.8 (C), 154.1 (C), 164.8 (CÄO) 2H), 7.90 (d, Jꢂ 8, 2H), 9.3 (bs, 1H), 10.2 (s, 1H)
33.2 (CH2Br), 116.6 (d, Jꢂ22, CH), 124.5 (CH), 128.3 (CH), 129.2 4.43 (s, 2H), 7.0 (t, Jꢂ 8, 2H), 7.45 (d, Jꢂ 7, 2H), 7.6 (dd, Jꢂ
(CH), 135.0 (C), 135.8 (C), 141.4 (C), 157.0 (d, Jꢂ240, C), 165.4 (CÄ Jꢂ 5, 2H), 7.9 (d, Jꢂ 8, 2H)
O)
33.4 (CH2), 122.6 (CH),124.0 (C), 128.5 (CH), 129.5 (CH), 130.0 (C), 4.46 (s, 2H), 7.19 (s, 1H), 7.47 (d, Jꢂ
130.1 (CH),131.7 (CH), 134.0 (C), 142.6 (C), 145.0 (C), 165.0 (CÄO) 8.05 (d, Jꢂ 8, 1H), 8.46 (d, Jꢂ 8, 1H)
/
O) 4.73 (s, 2H), 7.51 (d, Jꢂ
7, 3H), 3.32 ( qd, Jꢂ
8, 2H), 8.50 (bs, 1H, NH)
6.8, 2H), 4.45 (s, 2H), 6.08 (bs,
7, 2H), 7.35 (d, Jꢂ 8, 2H),
/
7.9, 2H), 7.86 (d, Jꢂ
/
7.9, 2H)
/
/
7, Jꢂ5, 2H), 4.77 (s, 2H), 7.55
/
/
/
/
2c
2d
2e
2f
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
20.5, CH), 127.7
/
/
7), 4.41 (s, 2H), 6.93 (t, Jꢂ
/
/
/
/
/
8, 2H), 7.61 (d,
/
/
/
/
6.7, 2H), 3.63 (c, Jꢂ
/
6.7, 2H, CH2NH), 4.42 (s, 2H),
7.38 (m, 3H), 7.61 (d, Jꢂ
/
/
/
2g
/
7, 1H), 7.40 (t, Jꢂ
7, 2H), 10.5 (bs, 1H)
7, 2H), 7.60 (d, Jꢂ
/
7, 2H), 7.65 (d, Jꢂ
/
8,
7,
8,
/
/
/
2h a
2i a,b
/
/
/
/
/
/
/
/
/
/
/
/
/
2j
/
7, 2H), 7.81 (d, Jꢂ 7, 2H),
/
/
/
/
a
1H and 13C spectra recorded in DMSO-d6.
1H spectra recorded in MeOD.
b
4.1.3.1. p-Bromomethylbenzamide (2a). Yield 62%;
m.p. 185ꢀ187 8C.
4.1.3.8. N-(4-hydroxyphenyl)-4-bromomethylbenzamide
(2h). Yield 55%; m.p. 207-210 8C; MS (C14H12BrNO2)
/
m/z 305 (88%), 307 (88%, Mꢃ2).
/
4.1.3.2. N-ethyl-p-bromomethylbenzamide (2b). Yield
55%; m.p. 110ꢀ112 8C.
4.1.3.9. N-(4-fluorophenyl)-4-bromomethylbenzamide
(2i). Yield 77%; m.p. 165-168 8C.
/
4.1.3.3. N-phenethyl-p-bromomethylbenzamide
Yield 60%; m.p. 115ꢀ117 8C.
(2c).
4.1.3.10. 1-(4-Bromomethylphenylcarboxamide)-2,4-
/
dichlorobenzene (2j). Yield 37%; m.p. 132ꢀ135 8C.
/
4.1.4. General procedure for preparation of compounds
3aꢀj and 4aꢀ
4.1.3.4. 1-[2-(4-
Bromomethylphenylcarboxamide)ethyl]-4-
/
/
j
Diisopropylethylamine, DIPEA (0.65 mL, 3.6 mmol)
was added dropwise to a stirred suspension of 2-(p-
toluenesulfonamido)pyrimidine 1 (0.75 g, 3 mmol) in
dry DMF (12 mL) under argon. After 40 min the
corresponding bromocarboxamide 2 (3.6 mmol) was
added. The reaction mixture was stirred at r.t. for 16 h
and then poured onto water (100 mL). The resulting
solid was collected, air dried and purified by flash
column chromatography to give the exocyclic nitrogen
hydroxybenzene (2d). Yield 40%; m.p. 170ꢀ173 8C.
/
4.1.3.5. 1-[2-(4-
Bromomethylphenylcarboxamide)ethyl]-4-fluorobenzene
(2e). Yield 63%; m.p. 101ꢀ114 8C.
/
4.1.3.6. 1-[2-(4-
Bromomethylphenylcarboxamide)ethyl]-2,4-
dichlorobenzene (2f). Yield 56%; m.p. 126ꢀ
(C16H14BrCl2NO) m/z 388 (100%), 390 (47%, Mꢃ
alkylated compounds 4aꢀ
/
j (hexane:EtOAc 1:1) and the
j (EtOAc:-
MeOH 9:1). Spectroscopic H- and 13C-NMR data for
compounds 3aꢀj and 4aꢀj are shown in Tables 4 and 5,
respectively.
/
129 8C; MS
2).
endocyclic nitrogen alkylated compounds 3aꢀ
/
/
1
/
/
4.1.3.7. N-phenyl-4-bromomethylbenzamide (2g). Yield
54%; m.p. 174ꢀ177 8C; MS (C14H12BrNO) m/z 289
(67%), 291 (66%, Mꢃ2).
/
/
4.1.4.1. 4-{[2-{[(4-Methylphenyl)sulfonyl]imino}-

Table 4
Characteristic Spectral Data for Compounds 3aꢀ
/j
Product 13C-NMR (CDCl3 ꢀ
/TMS) d (ppm)
1H-NMR (CDCl3 ꢀ
TMS) d (ppm)
/
3a a
21.2 (CH3), 55.8 (CH2), 108.8 (CH), 127.0 (CH), 128.0 (CH), 128.4 (CH), 128.9 (CH), 134.3 2.37 (s,3H), 5.35 (s,2H), 6.92(dd, Jꢂ
(C), 138.2 (C), 141.1 (C), 141.4 (C), 150.9 (CH), 154.2 (C), 164.7 (CH), 167.7 (CÄO) 2H), 7.50 (bs,1H,NH), 7.62 (d, Jꢂ 8.1,2H), 7.92 (d, Jꢂ
Jꢂ 6.4, Jꢂ 2.3,1H), 8.74 (dd, Jꢂ 4.3, Jꢂ 2.3,1H)
15.1 (CH3); 21.8 (CH3); 35.3 (CH2); 53.9 (CH2); 108.6 (CH); 127.8 (CH); 128.1 (CH); 129.1 1.1 (t, Jꢂ 7.1, 3H), 2.3 (s, 3H), 3.4 (qd, Jꢂ 7.1, Jꢂ
(CH); 129.2 (CH); 135.5 (C); 136.9 (C); 140.1 (C); 142.5 (C); 149.2 (CH); 154.9( C) 164.4 Jꢂ 4.3,1H); 7.05 (d, Jꢂ 8.1, 2H); 7.2 (d, Jꢂ 8.3, 2H); 7.6 (d, Jꢂ
(CH); 167.2 (CÄO) 2H); 8.03(dd, Jꢂ 6.4, Jꢂ 2.3,1H); 8.35 (dd, Jꢂ 4.3, Jꢂ 2.3,1H)
21.2 (CH3), 35.2 (CH2), 41.7(CH2), 55.8(CH2), 108.8 (CH), 120.6 (CH), 124.1(CH), 2.32 (s, 3H), 2.83 (t, Jꢂ 7, 2H), 3.62 (c, Jꢂ 7, 2H), 5.33(s,2H), 6.87 (dd, Jꢂ
127.1(CH), 128.2 (CH), 128.7 (CH), 129.0 (CH), 129.1(CH), 135.1(C), 138.6(C), 139.4(C), 7.10 (t, Jꢂ 7.5, 1H), 7.24 (d, Jꢂ 8.2, 2H), 7.34 (t, Jꢂ 7.5, 2H), 7.45 (d, Jꢂ
141.1(C), 141.4 (C), 150.9 (CH), 154.2 (C), 164.8 (CH), 165.5 (CÄO) Jꢂ 8.2, 2H), 7.80 (d, Jꢂ 7.5, 2H), 7.95(d, Jꢂ 8.1, 2H), 8.66 (dd, Jꢂ 6.6, Jꢂ
(dd, Jꢂ 4.3, Jꢂ 2.4,1H), 10.26 (s, 1H)
21.2 (CH3), 34.7 (CH2), 41.1 (CH2), 55.9 (CH2), 108.8 (CH), 122.0 (CH), 127.0 (CH), 127.7 d 2.32 (s, 3H), 2.83 (t, Jꢂ 7, 2H), 3.62 (c, Jꢂ
(CH), 128.8 (CH), 128.9 (CH), 130.0 (CH), 130.7 (C), 135.4 (C), 141.1 (C), 148.8 (C), 149.2 1H), 7.30 (d, Jꢂ 8.3, 2H), 7.40ꢀ7.62 (m, 6H), 7.80ꢀ
(CH), 154.2 (C), 164.7 (CH), 166.0 (CÄO) 1H), 8.66 (dd, Jꢂ 4.1, Jꢂ 2.4,1H)
21.3 (CH3), 34.6 (CH2), 41.3 (CH2), 55.8 (CH2), 108.1 (CH), 115.1 (d, Jꢂ 21, CH), 127.0 2.25 (s, 3H), 2.77 (t, Jꢂ 7, 2H), 3.50 (c, Jꢂ
(CH), 127.6 (CH), 128.5 (CH), 128.7 (CH), 130.0 (d, Jꢂ 7.9, CH), 134.6 (C), 134.7 (C), 136.6 1H), 6.82 (t, Jꢂ 8.6, 2H), 7.08 (d, Jꢂ
(C), 139.6 (C), 142.1 (C), 148.7 (CH), 154.4 (C), 161.3 (d, Jꢂ 244, C), 163.9 (CH), 166.8 (CÄ 8.1, 2H), 7.67 (d, Jꢂ 8.2, 2H), 7.98 (dd, Jꢂ
O)
22.0 (CH3), 33.1 (CH2), 39.9 (CH2), 56.3 (CH2), 108.6 (CH), 127.5 (CH), 128.1 (CH), 129.0 2.28 (s, 3H), 2.95 (t, Jꢂ
(CH), 129.2 (CH), 129.4 (CH), 129.6 (CH), 132.2 (CH), 133.1 (C), 135.1 (C), 135.8 (C), 137.1 1H), 7.06ꢀ7.25 (m, 8H), 7.59ꢀ
(C), 140.1 (C), 142.5 (C), 149.1 (CH), 154.9 (C), 164.4 (CH), 167.4 (CÄO) Jꢂ 2.2, 1H)
21.2 (CH3), 55.8 (CH2), 108.8 (CH), 120.7 (CH), 124.1 (CH), 127.0 (CH), 128.2 (CH), 128.6 2.32 (s, 3H), 5.34 (s, 2H,), 6.88 (dd, Jꢂ
(CH), 128.9 (CH), 135.1 (C), 138.5 (C), 139.4 (C), 141.1 (C), 141.4 (C); 150.9 (C), 154.2 (CH), 8.2, 2H), 7.36 (t, Jꢂ 7.5, 2H), 7.48 (d, Jꢂ
164.8 (CH), 165.5 (CÄO) 2H), 7.94 (d, Jꢂ 8.1, 2H), 8.65 (dd, Jꢂ 6.6, Jꢂ
(s, 1H)
20.9 (CH3), 55.5 (CH2), 108.5 (CH), 115.0 (CH), 122.2 (CH), 126.7(CH), 127.7 (CH), 128.2 2.32 (s, 3H), 5.32 (s, 2H), 6.89 (dd, Jꢂ
(CH), 128.6 (CH), 130.6 (C), 135.2 (C), 140.8 (C), 141.0 (C), 150.5 (CH), 153.8 (C), 164.4 6H), 7.84ꢀ7.99 (m, 4H), 8.64 (dd, Jꢂ 6.6, Jꢂ
(CH), 165.3 (CÄO) 1H), 10.0 (s, 1H)
20.9 (CH3), 55.5 (CH2), 108.5 (CH), 115.2 (d, Jꢂ 22, CH), 122.2 (CH), 126.7 (CH), 127.9 2.32 (s, 3H), 5.34 (s, 2H), 6.88 (dd, Jꢂ
(CH), 128.3 (CH), 128.6 (CH), 134.6 (C), 135.4 (C), 138.3 (C), 140.8 (C), 141.1 (C), 150.6 2H), 7.47 (d, Jꢂ 8.3, 2H), 7.60 (d, Jꢂ
(CH), 153.9 (C), 160.5 (d, Jꢂ 240, C), 164.5 (CH), 165.1 (CÄO) 2H), 8.64 (dd, Jꢂ 6.4, Jꢂ 2.2, 1H), 8.71 (dd, Jꢂ
21.8 (CH3), 56.4 (CH2), 108.5 (CH), 123.4 (CH), 124.9 (C), 127.6 (CH), 127.9 (CH), 128.3 2.31 (s, 3H); 5.20 (s, 2H); 6.48 (dd, Jꢂ 6.6, Jꢂ 4.1, 1H), 7.15 (d, Jꢂ
(CH), 129.2 (CH), 129.8 (CH), 130.1 (C), 130.6 (CH), 133.6 (C), 134.7 (C), 138.3 (C), 140.3 Jꢂ 2.46, 1H), 7.36 (d, Jꢂ 2.46, 1H), 7.38 (d, Jꢂ 8.46, 2H), 7.78 (m, 4H), 7.94 (dd, Jꢂ
(C), 142.5 (C), 149.2 (CH), 154.8 (C), 164.5 (CH), 165.0 (CÄO) Jꢂ 2, 1H), 8.30 (bs, 1H, NH), 8.40 (d, Jꢂ 9, 1H), 8.52 (dd, Jꢂ 4.1, Jꢂ 2, 1H)
/
6.4, Jꢂ
/
4.3,1H), 7.27(d, Jꢂ
/
8.1, 2H), 7.45 (d, Jꢂ 8.3,
/
/
/
/
8.3,2H), 8.01 (s, 1H, NH), 8.68 (dd,
/
/
/
/
3b
/
/
/
5.7, 2H); 5.1 (s, 2H), 6.45 (dd, Jꢂ
/
6.4,
/
/
/
/
8.1, 2H); 7.7(d, Jꢂ 8.3,
/
/
/
/
/
/
3c
/
/
/
6.6, Jꢂ
8.1, 2H), 7.62 (d,
2.4, 1H), 8.71
/
4.3,1H),
/
/
/
/
/
/
/
/
/
/
/
/
3d a
3e
/
/
7, 2H), 5.30 (s, 2H), 6.80 (dd, Jꢂ
/
6.6, Jꢂ
/
4.1,
2.4,
/
/
/7.89 (m, 4H), 8.64 (dd, Jꢂ
/
6.6, Jꢂ
/
/
/
/
/
/
/
7, 2H), 5.09 (s, 2H), 6.42 (dd, Jꢂ
/
6.4, Jꢂ
/
4.3,
/
/
/
8.1, 2H), 7.10 (m, 4H), 7.25 (bs, 1H, NH), 7.56 (d, Jꢂ
/
/
/
/
/
6.4, Jꢂ 2.2, 1H), 8.33 (dd, Jꢂ 4.3, Jꢂ 2.2, 1H)
/
/
/
3f
/
6.8, 2H), 3.58 (c, Jꢂ
/
6.8, 2H), 5.12 (s, 2H), 6.46 (dd, Jꢂ
/
6.6, Jꢂ
/
4.1,
4.1,
/
/
7.73 (m, 3H), 7.90 (dd, Jꢂ
/
6.6, Jꢂ 2.2, 1H), 8.40 (dd, Jꢂ
/
/
/
/
3g a
/
6.6, Jꢂ
8.1, 2H), 7.63 (d, Jꢂ
2.4, 1H), 8.7 (dd, Jꢂ
/
4.3, 1H), 7.10 (t, Jꢂ
8.2, 2H), 7.78 (d, Jꢂ
4.3, Jꢂ 2.4, 1H), 10.25
/
7.5, 1H), 7.24 (d, Jꢂ
/
/
/
/
/
7.5,
/
/
/
/
/
/
3h a
3i a
3j
/
6.6,Jꢂ
/
4.1, 1H), 7.24 (d, Jꢂ
/
8, 2H), 7.44ꢀ/7.63 (m,
/
/
/
2.4, 1H), 8.66 (dd, Jꢂ
/
4.1, Jꢂ
/
2.4, 1H), 9.3 (s,
/
/
/
6.4, Jꢂ
8.2, 2H), 7.79 (dd, Jꢂ
4.3, Jꢂ 2.2, 1H), 10.35 (s, 1H, NH)
8, 2H), 7.23 (dd, Jꢂ
6.6,
/
4.3, 1H), 7.20 (t, Jꢂ
/
9.1, 2H), 7.23 (d, Jꢂ
/
8.2,
/
/
/
9.1, Jꢂ
/
5, 2H), 7.93 (d, Jꢂ
/
8.3,
/
/
/
/
/
/
/
/
/
/
9,
/
/
/
/
/
/
/
/
/
a
1H and 13C spectra recorded in DMSO-d6.

294
P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ296
/
Table 5
Characteristic spectral data for compounds 4aꢀ
/
j
Product 13C-NMR (CDCl3 ꢀ
/TMS) d (ppm)
1H-NMR (CDCl3 ꢀ
TMS) d (ppm)
/
4a a
4b
21.9 (CH3), 50.0 (CH2), 117.2 (CH), 127.3 (CH), 128.4 (CH), 129.1 d 2.36 (s, 3H), 5.46(s, 2H), 7.1(t, Jꢂ
(CH), 130.1 (CH), 133.9 (C), 138.0 (C), 142.4 (C), 144.7 (C), 157.7 7.40 (d, Jꢂ 7.9, 2H), 7.83 (d, Jꢂ 8.2, 2H), 7.89 (d, Jꢂ
(C), 158.4 (CH), 168.5 (CÄO) 8.54 (d, Jꢂ 4.8, 2H)
15.2 (CH3), 21.9 (CH3), 35.2 (CH2), 49.9 (CH2), 116.0 (CH), 127.4 1.13 (t, Jꢂ 7.2, 3H), 2.28 (s, 3H), 3.34 (qd, Jꢂ
(CH), 128.2 (CH),129.2 (CH), 129.3 (CH), 134.1 (C), 137.6 (C), 141.7 5.45 (s, 2H), 6.40 (bs, 1H), 6.75 (t, Jꢂ 4.8, 1H), 7.12 (d, Jꢂ
(C), 144.4(C), 157.9 (C), 158.5 (CH), 167.6 (CÄO) 7.34 (d, Jꢂ 8.4, 2H), 7.61 (d, Jꢂ 8, 2H), 7.74 (d, Jꢂ 8.4, 2H), 8.30
(d, Jꢂ 4.8, 2H)
22.0 (CH3), 36.0 (CH2), 41.3 (CH2), 50.0 (CH2), 116.1 (CH), 126.5 2.32 (s, 3H), 2.86 (t, Jꢂ
(CH), 127.4 (CH), 127.6 (CH), 128.3 (CH), 129.3 (CH), 129.4 (CH), 6.81 (t, Jꢂ 4.71, 1H), 7.1 (t, Jꢂ
129.5 (CH), 134.0 (C), 137.6 (C), 139.4 (C), 142.0 (C), 144.4 (C), (t, Jꢂ 7.5, 2H), 7.46 (d, Jꢂ 8.1, 2H), 7.58 (d, Jꢂ
157.9 (CH), 158.5 (C), 167.9 (CÄO) Jꢂ 8.4, 2H), 7.80 (d, Jꢂ 8.4, 2H), 8.35 (d, Jꢂ 4.71, 2H)
21.2 (CH3), 34.7 (CH2), 41.1 (CH2), 55.9 (CH2), 108.8 (CH), 122.0 2.32 (s, 3H), 2.88 (t, Jꢂ 7, 2H), 3.64 (c, Jꢂ 7, 2H), 5.45 (s, 2H),
(CH), 127.0 (CH), 127.7 (CH), 128.8 (CH), 128.9 (CH), 130.0 (CH), 6.16 (bs, 1H), 6.80 (t, Jꢂ 4.8, 1H), 7.09ꢀ7.16 (m, 4H), 7.40 (d, Jꢂ
130.7 (C), 135.4 (C), 141.1 (Car), 141.4 (C), 148.8 (C), 148.8 (C), 149.2 8.1, 2H), 7.58 (d, Jꢂ 8.1, 2H), 7.81 (d, Jꢂ 8.3, 2H), 8.1 (d, Jꢂ 8.3,
(CH), 154.2 (C), 164.7 (CH), 166.0 (CÄO) 2H), 8.35 (d, Jꢂ 4.8, 2H)
21.9 (CH3), 35.3 (CH2), 41.6 (CH2), 49.9 (CH2), 115.9 (d, Jꢂ 2.30 (s, 3H), 2.80 (t, Jꢂ 7, 2H), 3.56 (c, Jꢂ
CH), 116.0 (CH), 127.3 (CH), 128.2 (CH), 129.2 (CH), 129.3 (CH), 6.23 (bs, 1H), 6.77 (t, Jꢂ 4.7, 1H), 6.90 (t, Jꢂ
130.6 (d, Jꢂ 7.9, CH), 133.8 (C), 134.9 (C), 137.6 (C), 142.0 (C), Jꢂ 8.6, Jꢂ 5.4, 2H), 7.14 (d, Jꢂ 8.1, 2H), 7.37 (d, Jꢂ
144.3 (C), 157.9 (CH), 158.5 (C), 161.0 (d, Jꢂ 240, C), 167.6 (CÄO) 7.55 (d, Jꢂ 8.3, 2H), 7.76 (d, Jꢂ 8.3, 2H), 8.32 (d, Jꢂ
22.0 (CH3), 33.2 (CH2), 39.9 (CH2), 49.9 (CH2), 116.1 (CH), 127.4 2.29 (s, 3H), 2.92 (t, Jꢂ 6.7, 2H), 3.56 (c, Jꢂ
(CH), 127.6 (CH), 128.1 (CH), 129.2 (CH), 129.4 (CH), 129.7 (CH), 6.47 (t, Jꢂ 5.6, 1H, NH), 6.75 (t, Jꢂ 4.8, 1H), 7.06 (m, 2H), 7.13
132.2 (CH), 133.3 (C), 133.7 (C), 135.1 (C), 135.7 (C), 137.5 (C), (d, Jꢂ 8.3, 2H), 7.25 (s, 1H), 7.35 (d, Jꢂ 8.4, 2H), 7.56 (d, Jꢂ 8.3,
142.0 (C), 144.4 (C), 157.9 (CH), 158.4 (C), 167.8 (CÄO) 2H), 7.76 (d, Jꢂ 8.4, 2H), 8.30 (d, Jꢂ 4.8, 2H)
22.0 (CH3), 50.0 (CH2), 116.0 (CH), 120.5 (CH), 124.8 (CH), 127.5 2.32 (s, 3H), 5.48 (s, 2H), 6.80 (t, Jꢂ 4.71, 1H), 7.09 (t, Jꢂ
(CH), 128.3 (CH), 129.2 (CH), 129.3 (CH), 129.4 (CH), 134.2 (C), 7.16 (d, Jꢂ 8.1, 2H), 7.29 (t, Jꢂ 7.5, 2H), 7.45 (d, Jꢂ 8.1, 2H),
137.6 (C), 138.4 (C), 142.5 (C), 144.4 (C), 157.9 (CH), 158.5 (C), 7.58 (d, Jꢂ 7.5, 2H), 7.72 (d, Jꢂ 8.4, 2H), 7.79 (d, Jꢂ 8.4, 2H),
165.9 (CÄO) 8.35 (d, Jꢂ 4.71, 2H)
21.1 (CH3), 50.0 (CH2), 115.1 (CH), 116.5 (CH), 122.4 (CH), 126.7 2.36 (s, 3H), 5.48 (s, 2H), 6.74 (d, Jꢂ
/
4.8,1H), 7.36 (d, Jꢂ
/
8.2, 2H),
/
/
/
7.9, 2H),
/
/
/
/
7.2, Jꢂ
/
5.8, 2H),
8, 2H),
/
/
/
/
/
/
/
4c
4d
4e
4f
/
7, 2H), 3.61 (c, Jꢂ
7.5, 1H), 7.16 (d, Jꢂ
7.5, 2H), 7.72 (d,
/
7, 2H), 5.48 (s, 2H),
/
/
/
8.1, 2H), 7.3
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
21,
/
/
7, 2H), 5.43 (s, 2H),
8.6, 2H), 7.08 (dd,
/
/
/
/
/
/
/
8.1, 2H),
4.7, 2H)
/
/
/
/
/
/
/
6.7, 2H), 5.42 (s, 2H),
/
/
/
/
/
/
/
/
4g
4h a
4i
/
/
7.5, 1H),
/
/
/
/
/
/
/
/
/
9, 2H), 7.10 (t, Jꢂ
8.3, 2H), 7.51 (d, Jꢂ 9, 2H), 7.87
8.1, 2H), 8.53 (d, Jꢂ 4.8, 2H), 10.0 (s,
/
4.8, 1H),
(CH), 127.7 (CH), 128.3 (CH), 129.4 (CH), 130.8 (C), 134.0 (C),
7.38 (d, Jꢂ
(d, Jꢂ 8.1, 2H), 7.91 (d, Jꢂ
1H)
2.32 (s, 3H), 5.47 (s, 2H), 6.80 (t, Jꢂ
(CH), 127.5 (CH), 128.3 (CH), 129.2 (CH), 129.4 (CH), 133.9 (C), 7.17 (d, Jꢂ 8.2, 2H), 7.45 (d, Jꢂ 8.4, 2H), 7.52 (dd, Jꢂ
134.3 (C), 137.6 (C), 142.6 (C), 144.4 (C), 157.9 (CH), 158.4 (C), 2H), 7.71 (d, Jꢂ 8.2, 2H), 7.80 (d, Jꢂ 8.4, 2H), 8.35 (d, Jꢂ
161.0 (d, Jꢂ 240, C), 165.9 (CÄO) 2H)
22.0 (CH3), 50.0 (CH2), 116.1 (CH), 122.6 (CH), 124.0 (C), 127.6 2.32 (s, 3H), 5.50 (s, 2H), 6.81 (t, Jꢂ
(CH), 128.4 (CH), 128.5 (CH), 129.1 (CH), 129.3 (CH), 129.4 (CH), 7.24 (dd, Jꢂ 8.8, Jꢂ 2.43, 1H), 7.35 (d, Jꢂ
129.5 (C), 133.4 (C), 133.9 (C), 137.6 (C), 143.2 (C), 144.4 (C), 158.0 8.2, 2H), 7.79 (m, 4H), 8.29 (bs, 1H, NH), 8.36 (d, Jꢂ
(CH), 158.5 (C), 165.4 (CÄO) (d, Jꢂ 8.8, 1H)
/
8.3, 2H), 7.44 (d, Jꢂ
/
/
137.3 (C), 141.8 (C), 144.0 (C), 153.8 (C), 157.6 (C), 158.2 (CH),
/
/
/
164.9 (CÄ
/
O)
22.0 (CH3), 50.0 (CH2), 116.0 (CH), 116.1 (d, Jꢂ
/
22, CH), 122.5
/
4.9, 1H), 6.97 (t, Jꢂ
9, Jꢂ
4.9,
/
9, 2H),
/
/
/
/
4.7,
/
/
/
/
/
4j
/
4.7, 1H), 7.19 (d, Jꢂ
2.4, 1H), 7.51 (d, Jꢂ
4.7, 2H), 8.46
/
9, 2H),
/
/
/
/
/
/
/
a
1H and 13C spectra recorded in DMSO-d6.
pyrimidin-1-(2H)-yl]methyl]benzamide (3a). Yield
53%; m.p. 232ꢀ234 8C.
4.1.4.5. 4-{[2-{[(4-
Methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-
yl]methyl]-N-(2-phenylethyl)benzamide (3c). Yield
/
47%; oil.
4.1.4.2. 4-{[[4-Methylphenylsufonyl)](pyrimidin-2-
yl)amino]methyl}benzamide (4a). Yield 35%; m.p. 194ꢀ
/
196 8C.
4.1.4.6. 4-{[[4-Methylphenylsufonyl)](pyrimidin-2-
yl)amino]methyl}-N-(2-phenylethyl)benzamide
Yield 30%; m.p. 156ꢀ158 8C.
(4c).
/
4.1.4.3. N-ethyl-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-
yl]methyl]benzamide (3b). Yield 54%; m.p. 230ꢀ
233 8C.
/
4.1.4.7. N-[2-(4-hydroxyphenyl)ethyl)-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-
yl]methyl]benzamide (3d). Yield 56%; m.p. 205ꢀ
/
208 8C; HRMS (FAB) Calc. for C27H27N4O4S (Mꢃ1)
503.1102, Found 503.1103.
/
4.1.4.4. N-ethyl-4-{[[4-
methylphenylsufonyl)](pyrimidin-2-
yl)amino]methyl}benzamide (4b). Yield 32%; m.p. 195ꢀ
/
197 8C.
4.1.4.8. N-[2-(4-hydroxyphenyl)ethyl]-4-{[[4-

P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ
/296
295
methylphenylsufonyl)](pyrimidin-2-
4.1.4.19. N-(2,4-dichlorophenyl)-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-
yl)amino]methyl}benzamide (4d). Yield 18%; m.p. 155ꢀ
/
158 8C.
yl]methyl]benzamide (3j). Yield 46%; m.p. 185ꢀ
/
187 8C;
HRMS (FAB) Calcd for C25H21Cl2N4O3S [Mꢃꢃ
1]
527.0711, Found 527.0703.
/
4.1.4.9. N-[2-(4-fluorophenyl)ethyl]-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1(2H)-
yl]methyl}benzamide (3e). Yield 46%; oil.
4.1.4.20. N-(2,4-dichlorophenyl)-4-{[[4-
methylphenylsufonyl)](pyrimidin-2-yl)amino]
methyl}benzamide (4j). Yield 31%; m.p. 190-192 8C;
4.1.4.10. N-[2-(4-fluorophenyl)ethyl]-4-{[[(4-
methylphenyl)sulfonyl](pyrimidin-2-
HRMS (FAB) calcd for C25Cl2H21N4O3S [Mꢃꢃ
/1]
527.0711, Found 527.0718.
4.2. Biopharmacological methods
4.2.1. Materials
yl]amino]methyl}benzamide (4e). Yield 31%; m.p. 158ꢀ
160 8C; HRMS (EI) Calc. for C27H25FN4O3S 504.1631,
Found 504.1637.
/
4.1.4.11. N-[2-(2,4-dichlorophenyl)ethyl]-4-{[2-{[(4-
[5,6,8,9,11,12,14,15(n)-3H]leukotriene B4 was pur-
chased from Amersham Iberica, (Madrid, Spain). The
rest of reagents were from Sigma Chemical Co. (St.
Louis, MO).
methylphenyl)sulfonyl]imino}
methyl}benzamide (3f). Yield 53%; oil; HRMS (FAB)
Calc. for C27H25Cl2N4O3S [Mꢃꢃ
1] 555.1024, Found
555.0999.
pyrimidin-1(2H)-yl]-
/
4.1.4.12. N-[2-(2,4-dichlorophenyl)ethyl]-4-{[[(4-
methylphenyl)sulfonyl](pyrimidin-2-yl)
4.2.2. Isolation of human neutrophils
Venous blood was obtained, with informed consent,
from healthy volunteers. Leukocytes were obtained and
purified as described previously [16]. Viability was
greater than 95% by the trypan blue exclusion test.
The mitochondrial dependent reduction of 3-(4,5-di-
amino]methyl]}benzamide (4f). Yield 33%, m.p. 137ꢀ
139 8C. HRMS (EI) Calc. for C27H24Cl2N4-
O3S554.0946, Found 554.0961.
/
4.1.4.13. 4-{[2-{[(4-
Methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-
yl]methyl]-N-phenylbenzamide (3g). Yield 54%; m.p.
methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) to formazan [17], was used to assess the possible
cytotoxic effect of test compounds on human neutro-
phils
234ꢀ
/
237 8C; HRMS (FAB) Calc. for C25H23N4O3S
(Mꢃ
/
1) 459.1491, Found 459.1506.
4.2.3. Elastase release by human neutrophils
A suspension of neutrophils (2.5ꢁ
106 cells mlꢄ1) in
/
4.1.4.14. 4-{[[4-Methylphenylsufonyl)](pyrimidin-2-
yl)amino]methyl}-N-phenylbenzamide (4g). Yield 33%;
phosphate buffer saline containing 1.26 mM Ca2ꢃ and
0.9 mM Mg2ꢃ (pH 7.4) were preincubated with test
compounds or vehicle for 5 min and then stimulated
m.p. 195ꢀ197 8C.
/
4.1.4.15. N-(4-Hydroxyphenyl)-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1-(2H)-yl]-
with cytochalasin B (10 mM) and N-formyl-
-leucyl- -phenylalanine (fMLP, 10 nM) for 10 min at
37 8C. Elastase activity was determined in supernatants,
using N-ter-butoxy-carbonyl- -alanine p-nitrophenyl
L-methionyl-
L
L
methyl]benzamide (3h). Yield 35%; m.p. 150ꢀ
/
152 8C.
L
4.1.4.16. N-(4-hydroxyphenyl)-4-{[[4-
methylphenylsufonyl)](pyrimidin-2-yl)amino]
methyl}benzamide (4h). Yield 26%; m.p. 135ꢀ
ester (200 mM) as substrate and p-nitrophenol release
was measured. Possible direct inhibitory effects on
elastase activity were also assessed [18].
/
137 8C.
4.1.4.17. N-(4-fluorophenyl)-4-{[2-{[(4-
methylphenyl)sulfonyl]imino}pyrimidin-1(2H)-
4.2.4. Synthesis and release of LTB4 by human
neutrophils
A suspension of human neutrophils (5ꢁ
106 cells
/
yl]methyl}benzamide (3i). Yield 44%; m.p. 160ꢀ
/
162 8C;
HRMS (EI) Calc. for C25H23FN4O3S [Mꢃꢃ
478.1475, Found 478.1473.
/
2]
mlꢄ1) was preincubated with test compounds or vehicle
for 5 min and then stimulated with calcium ionophore
A23187 (1 mM) for 10 min at 37 8C. Leukotriene B4
levels in supernatants were measured by radioimmu-
4.1.4.18. N-(4-fluorophenyl)-4-{[[(4-
methylphenyl)sulfonyl](pyrimidin-2-yl)amino]
noassay [19]. High-speed (100,000ꢁg) supernatants
/
methyl}benzamide (4i). Yield 39%; m.p. 208ꢀ
HRMS (EI) Calcd for C25H21FN4O3S 476.1318, Found
476.1321.
/
210 8C;
from sonicated human neutrophils were obtained and
incubated under appropiate conditions with 10 mM
arachidonic acid to assess 5-lipoxygenase activity [20].

296
P. Fernandez-Ferri et al. / European Journal of Medicinal Chemistry 38 (2003) 289ꢀ296
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The results are presented as mean9S.E.M.; n repre-
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Acknowledgements
This work was supported by grant 1FD97-0373-C02-
01; CICYT-FEDER. P. Fernandez-Ferri thanks Gen-
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