New amides of 5-(4-chlorobenzoyl)aminoorotic acid: Their synthesis and biological activity

Article abstract of DOI:10.1002/ardp.200800034

The synthesis and in-vitro biological evaluation of the amide series 4 of 5-(4-chlorobenzoyl)aminoorotic acid 2 are presented. The biological properties of a few 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-N-substituted-4- pyrimidinecarboxamide derivatives 4 tested here were compared with those of the isosteric isothiazole derivative MR-2/94 (5-(4-chlorobenzoyl)amino-N-(4- chlorophenyl)-3-methyl-4-isothiazolecarboxamide), which possesses a strong immunosuppressive and anti-inflammatory activity [1, 2], It must be suggested that replacement of the isothiazole by a pyrimidine core ring system resulted in considerable lowering of the anti-inflammatory and immunotropic actions of the obtained amides. Physicochemical properties of 2-(4-chlorophenyl)-6,8-dihydroxy- 4H-pyrimido[5,4-d]-1,3-oxazin-4-on 3 are also briefly described.

Full text of DOI:10.1002/ardp.200800034

Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
A. Regiec et al.
677
Full Paper
New Amides of 5-(4-Chlorobenzoyl)aminoorotic Acid:
Their Synthesis and Biological Activity
1
2
1
3
´
Andrzej Regiec , Magdalena Spiewak , Ryszard Jasztold-Howorko , and Ryszard Mie˛dzybrodzki
1 Department of Organic Chemistry, Faculty of Pharmacy, Medical Academy of Wroclaw, Wroclaw, Poland
2 Department of Chemistry, Wroclaw University of Technology, Wroclaw, Poland
3 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
The synthesis and in-vitro biological evaluation of the amide series 4 of 5-(4-chlorobenzoyl)ami-
noorotic acid 2 are presented. The biological properties of a few 5-(4-chlorobenzoyl)amino-2,6-
dihydroxy-N-substituted-4-pyrimidinecarboxamide derivatives 4 tested here were compared with
those of the isosteric isothiazole derivative MR-2/94 (5-(4-chlorobenzoyl)amino-N-(4-chloro-
phenyl)-3-methyl-4-isothiazolecarboxamide), which possesses a strong immunosuppressive and
anti-inflammatory activity [1, 2], It must be suggested that replacement of the isothiazole by a
pyrimidine core ring system resulted in considerable lowering of the anti-inflammatory and
immunotropic actions of the obtained amides. Physicochemical properties of 2-(4-chlorophenyl)-
6,8-dihydroxy-4H-pyrimido[5,4-d]-1,3-oxazin-4-on 3 are also briefly described.
Keywords: 5-Aminoorotic acid / Immunotropic activity / Nitric oxide / Proinflammatory cytokines / Pyrimido[5,4-d]-4H-
1,3-oxazine /
Received: January 31, 2008; accepted: July 29, 2008
DOI 10.1002/ardp.200800034
Introduction
The different previously described derivatives of 5-amino-
3-methyl-4-isothiazolecarboxylic acid 5 (Fig: 1) displayed
remarkable antiviral, anti-inflammatory, cytostatic, anal-
gesic, and a recently discovered immunotropic activity
[1, 3–8]. Of this group of compounds, 5-benzoylamine-N-
(4-chlorophenyl)-3-methyl-4-isothiazolecarboxamide, i. e.
Denotivir (ITCL, Vratizolin) [9, 10] (see Fig. 1), an antiviral
drug with anti-inflammatory activity, has been put into
medical practice. Currently, it is mainly used against her-
pes virus infections [3, 11, 12]. It also has significant
immunotropic properties [10].
Based on the structure of Denotivir, a number of iso-
thiazole derivatives have been synthesized and biologi-
Correspondence: Andrzej Regiec, Department of Organic Chemistry,
Faculty of Pharmacy, Medical Academy of Wroclaw, ul. Grodzka 9, 50-
137 Wroclaw, Poland.
Figure 1. 5-amino-3-methyl-4-isothiazole carboxylic acid deriv-
atives and related compounds described previously.
E-mail: rykolf@mp.pl
Fax: +48 71 784-0341
cally tested recently [1, 4, 8]. Some new amides have
shown in-vitro and in-vivo immunosuppressive and anti-
inflammatory activities stronger than those of cyclospor-
Abbreviations: peripheral blood mononuclear cells (PBMCs); phytohe-
magglutinin (PHA)
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Scheme 1. Synthesis of compounds 3, 4, 10–12. (oxazinones 3, 12, amides 4a–e, ester 10 and
pyridinium complex salt 11).
ine A, leflunomide, and ibuprofen, for example the com- boxamide derivatives of 4-amino-1-methyl-5-imidazole-
pounds designated as MR-2/94 and MR-17/95W, i. e. 5-(4- carboxylic acid 6, despite the heteroaromatic core ring
chlorobenzoyl)amino-N-(4-chlorophenyl)-3-methyl-4-iso-
thiazolecarboxamide and 5-(4-chlorobenzoyl)amino-N-(4- lowering of toxicity while maintaining anti-inflamma-
trifluoromethylphenyl)-3-methyl-4-isothiazolecarboxa- tory and immunotropic properties. This strongly sug-
replacement. The results also indicated a considerable
mide, respectively [1, 2, 4, 7, 13] (see Fig. 1). It has been gests that the presence of the isothiazole ring system
shown that the amides of the 5-(4-chloro)benzoyl series of may not be a crucial factor for the anti-inflammatory and
derivatives of 5-amino-3-methyl-4-isothiazolecarboxylic immunotropic activity of N-(4-chorobenzoyl)amino-N-
acid 5 should be better anti-inflammatory agents than substituted heterocyclecarboxamides.
the non-substituted 5-benzoyl series [1]. In a previous
Because of the considerations described above and
study [13], the influence of exchanging the isothiazole data that some of the previously described and biologi-
for an imidazole core ring system on biological activity cally tested amides of 5-benzoylaminoorotic acid 7
was dealt with on the example of 4-amino-1-methyl-5-imi- (Fig. 1) [14–16] showed anti-inflammatory and analgesic
dazolecarboxylic acid 6 derivatives (Fig. 1). On the basis activity, the main aim of this study was to investigate the
of biological tests carried out previously for MR-2/94 influence of replacing the five-membered heterocycle-iso-
[1, 2, 4, 7] and presented in [13], it was suggested that the thiazole by a six-membered pyrimidine core ring on the
isothiazolecarboxamide derivatives are only slightly biological activity of heterocyclic carboxamides (5-(4-
more active than the structurally related imidazolecar- chlorobenzoyl)amino-2,6-dihydroxy-N-substituted-4-pyri-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
New Pyrimidine Derivatives and Their Biological Activity
679
Table 1. Physical constants and analytical data of compounds 2–4, and 10–12.
Compound
R
Formula M. wt. (g/mol)
Elemental analysis (calculated / found) (%)
C
H
N
2
–OH
–
C12H8N3O5Cl
309.67 (327.69a)
C12H6N3O4Cl
291.66
C18H12N4O4Cl2
419.24
43.99a)
44.06
49.42
49.03
51.57
51.36
3.08a)
3.24
2.07
2.13
2.89
3.11
12.82a)
12.61
14.41
14.28
13.36
13.75
3
4a
4b
4c
C19H12N4O4ClF3
452.78
50.40
50.10
2.67
2.46
12.37
12.41
C17H11N5O4Cl2
420.22
48.59
48.38
2.64
2.40
16.67
16.97
4d
4e
C18H19N4O5Cl
406.83
53.14
53.31
4.71
5.01
13.77
13.40
C22H23N4O4Cl
442.91
59.66
59.27
5.23
5.35
12.65
12.69
10
–O–CH2CH3
C14H12N3O5Cl
337.73
C17H11N4O4Cl
370.76
C12H4N3O2Cl3
328.56
49.79
49.86
55.07
54.76
43.87
43.79
3.58
3.80
2.99
2.67
1.23
1.41
12.44
12.24
15.11
15.15
12.79
12.90
11, 11a
12
–
–
a)
Data for monohydrate C12H8ClN3O5 N H2O.
midinecarboxamides 4) under consideration possessing amino-2,6-dioxo-1,2,3,6-tetrahydro-4-pyrimidinecarbox-
those substituents, which have been proven to be advan- ylic acid 1 [17], which had given the previously described
tageous in the 5-amino-3-methyl-4-isothiazolecarboxylic 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-4-pyrimidinecar-
acid 5 derivatives series.
boxylic acid 2 in reaction with 4-chlorobenzoyl chloride
in pyridine solution [14]. Compound 2 was transformed
into
a
semianhydride-type compound, 2-(4-chloro-
phenyl)-6,8-dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on
3, under the influence of dehydrating reagents such as
SOCl2 in solvents such as benzene, toluene, and carbon
tetrachloride. The single, very strong band of the stretch-
ing oscillations of the carbonyl group at 1770 cm– 1 and
the medium sharp band of the stretching oscillations of
Results and discussion
Chemistry
The new amides 4a–e (Table 1) were prepared using the
methods outlined in Scheme 1. The starting material
used in the synthesis was the 5-aminoorotic acid, i. e. 5-
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Table 2. IR (KBr tablets) data of the compounds 2–4, and 10–12.
Compound
m (cm– 1
)
2
3
3450 (m-NH, m-OH medium sharp band, intramolecular hydrogen bond), 3330 (m-NH, m-OH medium sharp band, intramo-
lecular hydrogen bond), 3220–3140 (m-OH, m-NH), 3500–2400 (m-COO-H), 1710–1660 (m-C=O), 1595, 1585 (m-C=N-, -C=C-
3600–3340, 3180, 3100 (m-OH, m-NH), 1770 (-C=O, very strong, a,b,d,c-unsaturated lactone carbonyl), 1720–1710, 1675
(m-CO-, strong, pyrimidinon ring carbonyls), 1615, 1585 (m-C=N-, -C=C-, strong)
)
4a
4b
4c
4d
3650–3400, 3280, 3200 (m-OH,m-NH),1730, 1660 (m-C=O), 1600, 1590 (m-C=N-, – C=C-
3600–3350, 3270, 3200 (m-OH,m-NH), 1730, 1670, 1650 (m-C=O), 1605, 1590 (m-C=N-, – C=C-
3650–3390, 3260, 3180 (m-OH,m-NH), 1727, 1700, 1680, 1660 (m-C=O), 1605–1590 (m-C=N-, – C=C-
3650–3100 (m-OH,m-NH, medium strong, very broad), 2950 (m-C-H, medium), 2865 (m-C-H, weak to medium), 1730 (m-C=O
very strong), 1700–1650 (m-C=O, very strong, broad), 1600, 1590 (m-C=N-, – C=C- medium)
)
)
)
,
4e
10
3650–3100 (m-OH,m-NH, strong, very broad), 2925 (m-C-H2-, strong), 2865 (m-C-H, strong), 2820 (m-C-H, medium), 1745 (m-C=O
very strong), 1700–1650 (m-C=O, very strong, broad), 1600, 1590 (m-C=N-, – C=C- medium)
3650–3400 (m-OH,m-NH , strong broad band), 3300 (m-OH,m-NH ,sharp and very strong), 3260, 3180(m-OH,m-NH), 2820 (m-CH3, -
,
CH2, weak), 1730 (m-C=O very strong), 1700 (m-C=O very strong), 1675 (m-C=O very strong), 1650 (m-C=O, medium), 1600 (m-C=N-
, – C=C-), 1190 (m-C-O-C=O, asymmetric, strong) 1050, 1010 (m-C-O-C symmetric, very strong)
+
11, 11a
3600–3350 (m-OH,m-NH weak), 3195 (m-OH,m-NH medium), 3060 (m=C-H medium), 2830, 2700-2300(m-HN =C-H weak to me-
+
dium, very broad), 1750, 1690 (m-C=O very strong), 1630–1590 (strong), 1530, 1485 (m-C=N -, – C=C- medium)
12
3080–3020 (m=C-H aromatic ring, weak), 1775 (m-C=O , strong, a,b,d,c-unsaturated lactone carbonyl), 1615, 1585 (m-C=N-,
-C=C-, very strong)
the ring imine bond at 1615 cm– 1 appeared in the IR
spectrum (see Table 2). Two medium sharp signals at
3450 and 3330 cm– 1 of the stretching oscillations of the
intramolecular hydrogen bonds (NH, OH) completely
vanished and the broad band in the range of 2400–
3300 cm– 1 of carboxyl group disappeared. The strong
stretching oscillation at 1660 cm– 1 of the carbonyl 4-
chlorobenzoylamino group at pyrimidine ring position 5
also vanished. However, the valence stretching oscilla-
tions of the pyrimidinone ring carbonyls in the 1720–
1675 cm– 1 range remained unchanged. Moreover, these
valence stretching oscillations of the pyrimidinone ring
carbonyls in the 1720–1675 cm– 1 range in IR spectrum
completely vanished after replacing of carbonyl oxygen
groups of the pyrimidinone ring with chlorine substitu-
ents (see compound 12 in Scheme 1, Table 2, and Experi-
mental, Section 4). After chlorination of oxazinonone 3,
the single, very strong band of the stretching oscillations
of the oxazinon carbonyl group at 1770 cm– 1 remain
practically unchanged except the vibration intensity
that slightly lowered. The chlorination also caused the
sharp band of the stretching vibrations of the imine
bond (-C=N-) and aromatic (-C=C-) with maximum at
1615 cm– 1 amplified itself (from medium to strongest
one in the spectrum) and broadened in the 1575–
1620 cm– 1 range. There is the lack of other bands of vibra-
tions between 1620–2800 cm– 1 and above 3080 cm-1 in
the IR spectrum of the chloroderivative 12 (Table 2). The
Figure 2. Substances 8, 9, and 10.
was created from 2 as a result of intramolecular dehydra-
tion of the imide form 2a under the influence of dehy-
drating reagents. The mechanism of intramolecular
dehydration of the imide form 2a of 5-(4-chlorobenzoyl)a-
mino acid 2 is probably similar to that initially described
for acyl antranilic acid cyclodehydration [18, 19] as well
as that for the corresponding 5-acylamino-4-isothiazole-
carboxylic acid derivatives [1, 8]. 2-(4-Chlorophenyl)-6,8-
dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on has been
obtained in [14], but the interpretation of its structure
there was incorrect. At that time, it was incorrectly inter-
preted and presented as 5-(4-chlorophenyl)-2,4-dihydoxy-
6-oxoazetino[3,2-d]pyrimidine 8 (lactam) [14] (Fig. 2). Its
6,8-dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on
3 has
been easily transformed into 6,8-dichloropyrimido[5,4-d]-
4H-1,3-oxazin-4-on 12 under phosphoryl oxychloride
(POCl3) influence. These spectral parameters support a
semianhydride structure of compound 3. Oxazinone 3
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Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
New Pyrimidine Derivatives and Their Biological Activity
681
Table 3. 1H-NMR and 13C-NMR data of the compounds 2–4, and 10–12.
Compound 1H-NMR, chemical shift: [ppm]
13C-NMR, chemical shift: [ppm]
2
(DMSO-d6, Bruker, 300.15 MHz): d = 7.57 (d, J = 8.6 Hz, (DMSO-d6, Bruker, 75.47 MHz): = 111.07 (quaternary carbon C5 of
2H, 3,5–H, 4-chlorobenzoyl), 7.91 (d, J = 8.6 Hz, 2H, pyrimidine ring), 128.48 (C3, C5 of 4- chlorobenzoyl ring), 129.57
2,6-H, 4-chlorobenzoyl), 9.65 (s, 1H, NH of 4-chloro- (C2, C6 of – chlorobenzoyl ring), 132.39 (quaternary carbon atom
benzamide group), 11.05 (s, 1H, NH or OH, sharp
C1 of 4- chlorobenzoyl ring), 136.57 (quaternary carbon atom C4
henol-lactamic), 11.62 (s, 1H, NH or OH, sharp phe- of 4- chlorobenzoyl ring), 139.20 (quaternary carbon C4 of pyrimi-
nol-lactamic) ppm.
dine ring), 149.60 ( carbonyl carbon atom C2 of pyrimidine ring),
161.75(carbonyl carbon atom C6 of pyrimidine ring), 161.84 (car-
bonyl carbon atom of 4-chlorobenzoyl group), 164.84 (carbonyl
carbon atom of carboxylic group) ppm.
3
(DMSO- d6, Bruker, 300.15 MHz): d = 7.65 (d, J = 8.7 Hz,(DMSO-d6, Bruker, 75.47 MHz): = 126.43 (quaternary carbon C5 of
2H, 3,5-H of 4-chlorophenyl ring), 8.07 (d, J = 8.7 Hz, pyrimidine ring), 128.22 (quaternary carbon atom C1 or C4 of 4-
2H, 2,6-H of 4-chlorophenyl ring), 11.80 (s, 1H, NH
or OH, phenollactamic), 11.83 (s, 1H, NH or OH,
sharp and high phenollactamic) ppm.
chlorophenyl ring), 128.80 (quaternary carbon atom C4 or C1 of 4-
chlorophenyl ring), 129.05 (C2, C6 of 4-chlorophenyl ring), 129.27
(C3, C5 of -chlorophenyl ring), 137.34 (quaternary carbon C4 of
pyrimidine ring), 149.32 (carbonyl carbon atom C2 of pyrimidine
ring), 153.36 (quaternary carbon atom C2 of oxazinon ring),
154.15 (carbonyl carbon atom C4 of oxazinon ring), 160.34 (car-
bonyl carbon atom C6 of pyrimidine ring) ppm.
4a
(DMSO- d6, Bruker, 300.13 MHz): d = 7.35(d, J = 8.7 Hz, (DMSO-d6, Bruker, 75.47 MHz): = 108.47 (quaternary carbon C5 of
2H, 3,5 -H 4-chlorophenyl), 7.51(d, J = 8.7 Hz, 2H, 3,5 - pyrimidine ring), 121.34 (C2, C6 of 4-chlorophenyl ring), 127.90
H 4-chlorobenzoyl), 7.55 (d, J = 8.7 Hz, 2H, 2,6-H 4-
chlorophenyl), 7.82 ( d, J = 8.4 Hz, 2H, 2,6-H 4-chlo-
robenzoyl), 8.53 (s, 1H, NH, carboxamide group at
(quaternary carbon atom C1 of 4-chlorobenzoyl ring), 128.42 (C2,
C6 of 4- chlorobenzoyl ring), 128.709 (C3,C5 of 4-chlorophenyl
ring), 129.49 (C3, C5 of 4-chlorobenzoyl ring), 132.27 (quaternary
pyrimidine position 4), 9.55 (s, 1H, -NH, amide group carbon atom C4 of 4- chlorophenyl ring), 136.52 (quaternary car-
at pyrimidine position 5), 10.74 (s, 1H, NH or OH, bon atom C1 of 4- chlorophenyl ring) 136.87 (quaternary carbon
phenol-lactamic of 2,6-dihydroxypyrimidine ring), atom C4 of 4- chlorobenzoyl ring), 144.36 (quaternary carbon C4
11.6 (s, 1H, NH or OH, phenol-lactamic of 2,6-dihy-
droxypyrimidine ring) ppm.
of pyrimidine ring), 150.34 ( carbonyl carbon atom C2 of pyrimi-
dine ring), 158.54 (carbonyl carbon atom C6 of pyrimidine ring),
161.85 (carbonyl carbon atom of 4-chlorobenzoyl group), 165.43
(carbonyl carbon atom of carboxamide group at 4th pyrimidine
ring position) ppm.
4b
(DMSO-d6, Bruker, 300.13 MHz): d = 7.50 (d, J = 7.8 Hz, (DMSO-d6, Bruker, 75.47 MHz): = 108.64 (quaternary carbon C5 of
2H, 3,5 -H 4-chlorobenzoyl), 7.66 (d, J = 8.4 Hz, 2H,
3,5 -H 4-trifluoromethylphenyl), 7.74 (d, J = 8.4 Hz,
2H, 2,6-H 4-trifluoromethylphenyl), 7.82 (d, J = 7.8
Hz, 2H, 2,6-H 4-chlorobenzoyl), 9.59 (s, 1H, -NH,
pyrimidine ring), 119.78 (C2, C6 of 4-chlorophenyl ring), 126.07,
126.12 (C3,C5 of 4-chlorophenyl ring), 128.41 (C2, C6 of 4- chloro-
benzoyl ring),129.49 (C3, C5 of 4-chlorobenzoyl ring), 132.16 (qua-
ternary carbon atom C4 of 4- chlorophenyl ring), 136.54 (quater-
amide group at pyrimidine position 5), 10.99 (s, 1H, nary carbon atom C4 of 4- chlorobenzoyl ring), 141.47 (quaternary
NH, carboxamide group at pyrimidine position 4), carbon atom C1 of 4-chlorophenyl ring), 143.61 (quaternary car-
11.60 (s, 1H, NH or OH, phenol-lactamic of 2,6-di-
hydroxypyrimidine ring), 11.66 (s, 1H, NH or OH,
bon C4 of pyrimidine ring), 149.97 (carbonyl carbon atom C2 of
pyrimidine ring), 158.74 (carbonyl carbon atom C6 of pyrimidine
phenol-lactamic of 2,6-dihydroxypyrimidine ring) ring), 161.70 (carbonyl carbon atom of 4-chlorobenzoyl group),
ppm.
165.41 (carbonyl carbon atom of carboxamide group at 4th pyrimi-
dine ring position) ppm.
4c
(DMSO- d6, Bruker, 300.13 MHz): d = 7.49 (d, J = 8.8 Hz,(DMSO-d6, Bruker, 75.47 MHz): = 108.85 (quaternary carbon C5 of
1H, H3' pyridyl), 7.53 (d, J = 8.2 Hz, 2H, 3,5-H 4-chlo- pyrimidine ring), 124.39 (ß-C of pyridine fragment), 128.42 (C2, C6
robenzoyl), 7.85 (d, J = 8.2 Hz, 2H, 2,6-H p-chloro-
benzoyl), 8.02 (dd, J1 = 8.7 Hz, J2 = 2.0 Hz, 1H, H4'
pyridyl), 8.56 (d, J = 2.0 Hz, 1H, H6' pyridyl), 9.62
of 4-chlorobenzoyl ring), 129.51 (C3, C5 of 4-chlorobenzoyl ring),
130.44 (-C of pyridinium fragment), 132.16, 134.29, 136.57, 140.88
(quaternary carbon atoms of pyridine and 4-chlorobenzoyl
(s, 1H, -NH, amide group at pyrimidine position 5), groups), 143.03 (carbonyl carbon atom C2 of pyrimidine ring),
11.03 (s, 1H, NH, carboxamide group at pyrimidine 144.64 (quaternary carbon C4 of pyrimidine ring), 149.89 (a-C of
position 4), 11.55 (s, 1H, NH or OH, phenol-lactamic pyridine fragment), 158.81 (carbonyl carbon atom C6 of pyrimi-
of 2,6-dihydroxypyrimidine ring), 11.64 (s, 1H, NH
or OH, phenol-lactamic of 2,6-dihydroxypyrimidine group), 165.32 (carbonyl carbon atom of carboxamide group at 4th
ring) ppm. pyrimidine ring position) ppm.
dine ring), 161.61 (carbonyl carbon atom of 4-chlorobenzoyl
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Table 3. Continued.
Compound 1H-NMR, chemical shift: [ppm]
13C-NMR, chemical shift: [ppm]
4d
(DMSO-d6, Bruker, 500.13 MHz): d = 1.10-1.70 (m, 9H, DMSO-d6, Bruker, 75.47 MHz): = 26.53, 29.50, 30.76, 33.36 (methyl-
protons at position 2,3,4,5,6 of 4-hydroxycyclohexyl ene carbons of 4-hydroxycyclohexyl ring, equimolar cis and trans
ring), 4.35 (m, 1H, N-CHa, proton at position 1 of
4-hydroxycyclohexyl ring), 7.57 (d, J = 8.1 Hz, 2H,
3,5-H of 4-chlorobenzoyl), 7.89 (d, J = 8.1 Hz, 2H,
2,6-H of 4-chlorobenzoyl group), 8.16 (2s, 2 6 0.5H,
-NH of both cis and trans isomers, carboxamide
group at pyrimidine position 4), 9.40 (s, 1H, -NH,
amide group at pyrimidine position 5), 11.25 (s,
isomers), 47.607, 46.772, (-NH-C1 carbon of 4-hydroxycyclohexyl
ring), 64.281, 67.746 (HO-C4 carbon of 4-hydroxycyclohexyl ring),
107.70, 107.77 (quaternary carbon C5 of pyrimidine ring), 128.60
(C2, C6 of 4- chlorobenzoyl ring), 129.56 (C3, C5 of 4-chlorobenzoyl
ring), 132.17, 132.20 (quaternary carbon atom C1 of 4-chloroben-
zoyl ring), 136.77 (quaternary carbon atom C4 of 4- chlorobenzoyl
ring), 145.24, 145.41 (quaternary carbon C4 of pyrimidine ring),
1H, NH or OH, phenol-lactamic of 2,6-dihydroxypy- 150.214 (carbonyl carbon atom C2 of pyrimidine ring), 158.81,
rimidine ring), 11.46 (s, 1H, NH or OH, phenol-lacta- 158.91 (carbonyl carbon atom C6 of pyrimidine ring), 162.00,
mic of 2,6-dihydroxypyrimidine ring) ppm.
162.05 (carbonyl carbon atom of 4-chlorobenzoyl group), 165.77,
165.96 (carbonyl carbon atom of carboxamide group at 4th pyrimi-
dine ring position) ppm.
4e
(DMSO-d6, Bruker, 500.13 MHz): d = 1.47 (m, 12H,
DMSO-d6, Bruker, 75.47 MHz): = 28.61 (bridging carbon atoms of
protons of adamantyl ring), 1.92 (m, 3H, protons of adamantyl moiety), 35.77 (rear methylene carbons of adamantyl
adamantyl ring), 7.58 (s, 1H, -NH, carboxamide moiety), 40.52 (front methylene carbons of adamantyl moiety),
group at pyrimidine position 4), 7.60 (d, J = 8.5 Hz, 51.98 (quaternary carbon C1 of adamantyl moiety) 107.19 (quater-
2H, 3,5-H of 4-chlorobenzoyl), 7.92 (d, J = 8.5 Hz,
2H, 2,6-H of 4-chlorobenzoyl group), 9.48 (s, 1H,
nary carbon C5 of pyrimidine ring), 128.64 (C2, C6 of 4-chloroben-
zoyl ring), 129.52 (C3, C5 of 4-chlorobenzoyl ring), 132.15 (quater-
-NH, amide group at pyrimidine position 5), 11.30 nary carbon atom C1 of 4-chlorobenzoyl ring), 136.83 (quaternary
(s, 1H, NH or OH, phenol-lactamic of 2,6-dihydro- carbon atom C4 of 4- chlorobenzoyl ring), 145.84 (quaternary car-
xypyrimidine ring), 11.44 (s, 1H, NH or OH, phenol- bon C4 of pyrimidine ring), 150.24 ( carbonyl carbon atom C2 of
lactamic of 2,6-dihydroxypyrimidine ring) ppm.
pyrimidine ring), 158.62 (carbonyl carbon atom C6 of pyrimidine
ring), 162.18 (carbonyl carbon atom of 4-chlorobenzoyl group),
166.08 (carbonyl carbon atom of carboxamide group at 4th pyrimi-
dine ring position) ppm.
10
(DMSO-d6, Bruker, 300.15 MHz): d = 1.07 (t, J = 7.1 Hz, (DMSO-d6, Bruker, 75.47 MHz): = 13.67 (methyl carbon of ester
3H, methyl group protons of CH3CH2-), 4.15 (q, J =
7.1 Hz, 2H, methylen group protons of CH3CH2-),
group), 63.38 (methylen carbon of ester group), 111.64 (quaternary
carbon C5 of pyrimidine ring), 128.64 (C3, C5 of 4- chlorobenzoyl
7.58 (d, J = 8.5 Hz, 2H, 3,5 -H 4-chlorobenzoyl), 7.92 ring), 129.65 (C2, C6 of – chlorobenzoyl ring), 132.25 (quaternary
(d, J = 8.5 Hz, 2H, 2,6-H 4-chlorobenzoyl), 9.73 (s, carbon atom C1 of 4- chlorobenzoyl ring), 136.83 (quaternary car-
1H, -NH, high and sharp, 4-chlorobenzamide group bon atom C4 of 4- chlorobenzoyl ring), 137.53 (quaternary carbon
at pyrimidine position 5), 11.70 (s, 1H, NH or OH,
low and broad, phenol-lactamic of 2,6-dihydroxy-
pyrimidine ring) ppm.
C4 of pyrimidine ring), 149.64 ( carbonyl carbon atom C2 of pyri-
midine ring), 160.50 (carbonyl carbon atom C6 of pyrimidine
ring), 161.78 (carbonyl carbon atom of 4-chlorobenzoyl group),
165.02 (carbonyl carbon atom of ester group) ppm.
11, 11a
(DMSO- d6, Bruker, 300.15 MHz): d = 7.38 (dd, J1 = 5.7 (DMSO-d6, Bruker, 75.47 MHz): = 123.91 (ß-C of pyridinium frag-
Hz, J2 = 7.7 Hz, 2H, ß-H of pyridinium fragment),
7.64 (d, J = 8.6 Hz, 2H, 3,5-H 4-chlorophenyl), 7.78
ment), 126.48 (quaternary carbon C5 of pyrimidine ring), 128.26
(quaternary carbon atom C4 of 4-chlorophenyl ring), 128.85 (qua-
(m, J1 = 7.7 Hz, J2 = 1.8 Hz, 1H, -H of pyridinium frag- ternary carbon atom C1 of 4-chlorophenyl ring), 129.11 (C2, C6 of
ment), 8.06 (d, J = 8.6 Hz, 2H, 2,6-H 4-chlorophenyl), 4-chlorophenyl ring), 129.31 (C3, C5 of -chlorophenyl ring), 136.20
8.56 (d, J = 5.7 Hz, 2H, a-H of pyridinium fragment), (-C of pyridinium fragment), 137.40 (quaternary carbon C4 of pyri-
11.84 (s, 2H, NH or OH, sharp with broad base, phe- midine ring), 149.39 (carbonyl carbon atom C2 of pyrimidine
nol-lactamic of 2,6-dihydroxypyrimidine ring) ppm. ring), 149.51 (a-C of pyridinium fragment), 153.43 (quaternary car-
bon atom C2 of oxazinon ring), 154.21 (carbonyl carbon atom C4
of oxazinon ring), 160.42 (carbonyl carbon atom C6 of pyrimidine
ring) ppm.
12
(CDCl3, Bruker, 300.15 MHz): d = 7.54 (d, J = 8.7 Hz,
2H, 3,5-H of 4-chlorophenyl ring), 8.27 (d, J = 8.7 Hz,
2H, 2,6-H 4-chlorophenyl ring)
physicochemical properties have been very poorly ate amine at the carbon atom of the carbonyl group in
researched and documented to date. the oxazinone ring (Scheme 1). The reactions were con-
Pyrimidooxazinone 3 was used to obtain the diamides ducted mainly in anhydrous pyridine solutions. Pyridine,
4a–e as a result of a nucleophilic attack of the appropri- as in the case of 6-(4-chlorophenyl)-3-methylisothia-
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New Pyrimidine Derivatives and Their Biological Activity
683
Table 4. MS-ESI (Mass Spectrum-Electrospray Ionization) data of the compounds 3–4, 10–12.
Compound Calculated value of the
parent peak mass (a.m.u.)a)
m/z [Th]b) (%)c)
3
291.004685
(for the formula
C12H6ClN3O4)
(negative ionization, –Q1MS in methanol): 322.0(100) [M+MeOH–H]–- base peak, 292.0 (0.13)
[M+2–H]– – isotope peak, 290.0 (3) [M–H]– – quasi-molecular ion peak, 279.0 [M+MeOH, –CO2]–,
228.0 (5) [M–CO, –Cl]–
4a
418.023562
(for the formula
C18H12N4O4Cl2)
(negative ionization, –Q1MS in methanol): 419.9 (18) [M+4–H2]– 9 – isotope peak, 417.8(86)
[M+2–H2]– 9 – isotope peak, 416.1(100) [M–H2]– 9 – quasi-molecular ion and base peak.
4b
452.049925
(for the formula
C19H12N4O4ClF3)
(negative ionization, –Q1MS in methanol): 452.1 (38) [M+2–H2]– 9 – isotope peak, 451.2(36)
[M+1–H2]– 9 – isotope peak, 450.1(100) [M–H2]– 9 – quasi-molecular ion and base peak.
4c
419.018811
(for the formula
C17H11N5O4Cl2)
(negative ionization, –Q1MS in methanol): 421.8 (15) [M+4–H]– – isotope peak, 419.8 (73)
[M+2–H]– – isotope peak, 417.8 (100) [M–H]– – quasi-molecular ion and base peak.
4d
4e
406.104402
(for the formula
C18H19N4O5Cl)
(negative ionization, –Q1MS in methanol): 408.1 (2) [M+4–H]– – isotope peak, 407.1 (31) [M+3–
H]– – isotope peak, 406.1 (12) [M+1–H]– – isotope peak, 405.1 (100) [M–H]– – quasi-molecular
ion and base peak
442.140788
(for the formula
C22H23N4O4Cl)
(negative ionization, –Q1MS in methanol): 444.1 (3) [M+4–H]– – isotope peak, 443.1 (36) [M+3–
H]– – isotope peak, 442.1 (15.5) [M+1–H]– – isotope peak, 441.1 (100) [M–H]– – quasi-molecular
ion and base peak.
10
337.046551
(for the formula
C14H12ClN3O5)
(negative ionization, –Q1MS in methanol): 339(1.5) [M+3–H]– isotope peak, 338(27) [M+2–H]–
isotope peak, 337(13) [M+1–H]– isotope peak, 336(100) [M–H]– – quasi-molecular ion and base
peak
11, 11a
370.046886
(for the formula
C17H11N4O4Cl)
(positive ionization, +Q1MS in methanol): 370.0(3) [M+2–H2]+ 9 – isotope peak, 368.0 (21)
[M–H2]+ 9 – quasi-molecular ion peak, 346 (100)- base peak
(negative ionization, –Q1MS in methanol): 324.0(21) [M+2+MeOH–H–C5H5N (pyridine moi-
ety)]–, 322.0(100) [M+MeOH–H–C5H5N (pyridine moiety)]– , 292.0 (0.13) [M+2–H]– – isotope peak,
290.0 (3) [M–H–C5H5N (pyridine moiety)]– , 279.0 [M+MeOH,–CO2]–, 228.0 (5) [M–CO, –Cl]–
12
326.936907
(for the formula
C12H4Cl3N3O2)
(negative ionization, -Q1MS in methanol/water mixture): = 362(7) [M+4+MeOH–H]– – isotope
peak, 359.9 (46) [M+2+MeOH–H]– – isotope peak, 358(44) [M+MeOH–H]–, 348(12) [M+4+H2O–H]–
isotope peak, 345.9 (100) [M+2+H2O–H]– isotope and base peak, 344 (90) [M+H2O–H]–, 326 (2)
[M–H]– – quasi-molecular ion peak
a a.m.u. is atom mass unit., b mass ion (m) to ion charge (z) ratio expressed in Th (Thomson) unit, c value in parenthesis means the
relative intensities of peaks expressed in percents.
zolo[5,4-d]-4H-1,3-oxazin-4-one 9 [1], proved to be a much ing but anhydrous ethanol caused the creation of ethyl
better solvent for this type of nucleophilic reaction ester of 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-4-pyri-
because of its catalytic properties [20], particularly for midinecarboxylic acid 10 (Scheme 1) with a very high
the much less reactive aromatic amines such as 4-chlor- yield (see Experimental, Section 4). This is in contrary to
oaniline, 4-trifluoromethylaniline, and 5-amino-2-chloro- the earlier described 6-(4-chlorophenyl)-3-methylisothia-
pyridine. Application of pyridine significantly increased zolo[5,4-d]-4H-1,3-oxazin-4-one 9, in which case even long
the reaction yield, and in the case of weak aromatic heating of isothiazolooxazinone 9 with anhydrous etha-
amines such as 4-trifluoromethylaniline and 5-amino-2- nol did not cause any transformation of substrate 9 [1].
chloropyridine, it made the reaction occur at all. Etha- This is probably connected with the different electron-
nol, used previously as a solvent for reactions with density set of the compared oxazines 3 and 9 on oxazinon
amines, appeared useless in these cases because the previ- carbonyl carbon. Pyrimidooxazine 3 is also more reactive
ously reported and described ethyl ester of 5-(4-chloro- because there is no steric hindrance close to the carbonyl
benzoyl)amino-2,6-dihydroxy-4-pyrimidinecarboxylic
group under consideration, in contrast to isothiazolooxa-
acid 10 (Fig. 2) was also created as a side product [14]. zine [1], where the methyl group could repulse approach-
Long heating (10 h) of pyrimidooxazinone 3 with noth- ing nucleophilic agents. This huge difference of the reac-
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684
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
Table 5. Lymphocytes proliferation inhibition by tested amides
4a–e and reference MR-2/94.
tivity and chemical durability of both these oxazines 3
and 9 is also supported by electrospray MS spectra (see
Table 4) recorded in methanolic (or methanol/water mix-
ture) solution conditions. The quasi-molecular parent
peak was observed with 100% relative (base peak) inten-
sity in case of isothiazolooxazine 9 [1], while in case of
pyrimidooxazines 3 and 12 no quasi-molecular peak was
observed or this peak had very, very low intensity (2–3%)
(see Table 4). It is also interesting that the phenolo-lacta-
mic protons of the pyrimidine ring of 2-(4-chlorophenyl)-
Compound
Inhibition of PMBCs proliferation IC50 (lM)a)
Non-stimulated Stimulated
with PHAb)
Stimulated
with ConAc)
4a
4b
4c
4d
n.e.d)
79.1 l 5.2
57.6 l 8.5
n.e.d)
408.8 l 150.8
88.1 l 11.3
n.e.d)
n.e.d)
n.e.d)
n.e.d)
n.e.d)
n.e.d)
4e
MR-2/94
139.8 l 65.4
22.7 l 1.8
49.1 l 10.6
1.6 l 0.4
50.8 l 15.4
0.6 l 0.2
6,8-dihydroxy-4H-pyrimido[5,4-d]-1,3-oxazin-4-on
3 are
acidic enough to give a pyridinium complex salt-type
compound 11 (this could be a charge-transfer type com-
pound, donor-acceptor complex) (Scheme 1). The com-
pound 11 forms yellow needle crystals (see Experimental,
Section 4) which are stable enough to separate but which
are sensitive to water and water vapor, which cause
hydrolyzation after some time, and then decompose to
the starting 2-(4-chlorophenyl)-6,8-dihydroxy-4H-pyri-
mido[5,4-d]-1,3-oxazin-4-on 3 and 5-(4-chlorobenzoyl)a-
mino-2,6-dihydroxy-4-pyrimidinecarboxylic acid 2. The
structure of 11 can also be explained as a betaine-type
compound 11a (Scheme 1). In the case of isothiazolooxa-
zinon, the betaine intermediate was too reactive and has
not been liberated [1]. More results of research on the
reactivity of the 2-(4-chlorophenyl)-6,8-dihydroxy-4H-pyri-
mido[5,4-d]-1,3-oxazin-4-on 3 will be presented in subse-
quent papers.
a)
50% inhibition concentration, l SE.
PHA – phytohemagglutinin.
ConA – concanavalin A.
b)
c)
d)
Value very high or difficult to estimate (not estimated).
Influence of the tested diamides 4a-e on inhibition of
proinflammatory cytokine production by PBMCs
The activity of the new compounds 4a–e were tested at
concentrations of 0.3, 1, 3, 10, 30, and 100 lM. Their IC50
values (inhibition concentrations, the concentrations
that cause 50% inhibition of production) were calculated
on the basis of their biological response. The calculated
IC50 values of compounds 4, which were higher than
1000 lM, were acknowledged to be inactive. Only one of
the tested compounds, i. e. 4e with an adamantyl moiety,
inhibited TNF-a (IC50 = 14.0 l 7.7 lM) and IL-6 (IC50
=
11.1 l 3.0 lM) PBMC production at the levels of the refer-
ence isothiazole compound MR-2/94 (31.4 l 5.7 lM and
33.7 l 4.9 lM, respectively [13]) and the reference drug
leflunomide (50.0 l 5.1 lM and 20.3 l 7.3 lM, respec-
tively [13]). Weak TNF-a-inhibiting activity was shown by
amide 4a with a N-4-chlorophenyl fragment (about 30%
inhibition in the 1–100 lM concentration range) and
very weak activity was shown by the N-(4-hydroxycyclo-
hexyl) derivative 4d (20% inhibition in the concentration
range of 30–100 lM), but both amides 4a and 4d were
inactive as IL-6 production inhibitors (IC50 A 1000 lM).
The N-(6-chloro-3-pyridyl) amide derivative 4c inhibited
the production of IL-6 slightly at a level of 20% in the con-
centration range of 30-100 lM, but did not inhibit the
production of TNF-a at all (IC50>1000 lM). It is intriguing
that amide 4b with the N-4-trifluoromethylphenyl sub-
stituent did not inhibit the production of either pro-
inflammatory cytokine (IC50 A 1000 lM). This is in con-
trast to the structurally similar isothiazole analogue MR-
17/95W (see Fig. 1), which inhibits pro-inflammatory
The structures of the compounds 2–4, 11, and 12 have
been proven by IR (Table 2), 1H-NMR and 13C-NMR
(Table 3), and MS (Table 4) spectroscopic techniques and
elemental analysis (see Table 1).
Pharmacology
The biological activities of 5-(4-chlorobenzoyl)amino-2,6-
dihydroxy-4-pyrimidinecarboxamides 2 were tested in
preliminary biological studies in vitro for their influence
on the secretion of two pro-inflammatory cytokines,
tumor necrosis factor a (TNF-a), and interleukin-6 (IL-6),
by human peripheral blood mononuclear cells (PBMCs),
the production of nitric oxide by rat peritoneal macro-
phages, erythrocyte membrane stabilization, and lym-
phocyte proliferation.
MR-2/94, i. e. 5-(4-chlorobenzoyl)amino-N-(4-chloro-
phenyl)-3-methyl-4-isothiazolecarboxamide, was chosen
as the reference isothiazole for comparison because this
compoundprovedtobeoneofthemostactiveisothiazoles
(in immunosuppressive and anti-inflammatory tests) and
is structurally similar to the tested 4-pyrimidinecarboxa-
mides 2 [1, 2], and leflunomide, a drug that is known for
its immunosuppressive, and anti-inflammatory activity,
was alsoused as areference inthese tests [21].
cytokine production exceptionally strongly (TNF-a (IC50
=
6.5 l 1.2 lM) and IL-6 (IC50 = 8.2 l 0.9 lM) [7]) and is one of
the most active isothiazole derivatives [2, 4] It is interest-
ing that, as in the case of leflunomide (CC50, i. e. 50% cyto-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
New Pyrimidine Derivatives and Their Biological Activity
685
toxic concentration A 1000 lM) [13], no toxic effect of any with that of the reference isothiazole MR-2/94. The result-
of the new amides 4a–e (CC50 A 1000 lM) was observed ing IC50s are collected in Table 5. Inhibition of lympho-
on PBMCs stimulated with LPS. The CC50s of the reference cyte production by unstimulated PMBCs at the level of
isothiazole MR-2/94 and the analogue MR-17/95W esti- the reference isothiazole MR-2/94 (IC50 = 22.7 l 1.8 lM)
mated previously for PMBCs were 454 l 335 lM [13] and was shown only by amide 4e with the adamantyl sub-
1473 l 596 lM [7], respectively.
stituent (IC50 = 139.8 l 65.4 lM). Compound 4b (N-4-tri-
fluoromethylphenyl amide) inhibited proliferation by
25% at concentrations of 10 and 30 lM. Derivative 4c
with the N-(6-chloro-3-pyridyl) substituent inhibited pro-
Influence of the tested amides 4a–e on the NO production
by macrophages
Inhibition of nitric oxide (NO) secretion by LPS-stimu- liferation by 28% at a concentration of 10 lM, but con-
lated macrophages was estimated indirectly by measur- centrations above 30 lM slightly stimulated prolifera-
ing the concentration of the nitrite ion, which is a stable tion. The inhibitory effects of the compounds N-4-chloro-
metabolite of NO and has been previously used as a phenyl amide 4a, N-4-trifluoromethylphenyl amide 4b,
marker of NO production in different fluids [22]. None of and N-adamantyl amide 4e on the proliferation of PBMCs
the tested diamides 4a–e had the capability of specifi- stimulated with PHA were comparable, but much weaker
cally inhibiting NO secretion at the tested concentrations than that of the reference MR-2/94 (IC50 = 1.6 l 0.4 lM).
(0.3, 1, 3, 10, 30, and 100 lM). Compound 4e showed The N-(4-hydroxycyclohexyl) derivative 4d inhibited PHA-
nitric oxide secretion inhibition at a concentration (IC50 stimulated PBMCs by 33% at a concentration of 1 lM, but
= 101.9 l 6.0 lM), at which a remarkable toxic effect (CC50 30 and 100 lM increased the number of cells. The N-(6-
= 33.7 l 5.6 lM) appeared. There was no NO production chloro-3-pyridyl) derivative 4c did not affect the prolifera-
inhibitory activity beyond the toxic concentrations of tion PBMCs up to a concentration of 3 lM, but at higher
compounds 4 against macrophages. The corresponding concentrations compound 4c stimulated it. In case of the
values for the isothiazole reference MR-2/94 are: IC50
=
influence of the tested compounds on the lymphocyte
15.4 l 3.0 lM and CC50 = 35.2 l 3.3 lM [13], for isothiazole proliferation of PBMCs stimulated with concanavalin A
MR-17/95W IC50 = 7.8 l 0.2 lM and CC50 = 17.2 l 1.6 lM [7], (ConA), comparable inhibitory activities were revealed by
and for leflunomide IC50 = 280.3 l 108.4 lM, CC50
125.8 l 31.2 lM [13].
=
the adamantyl derivative 4e and the N-4-trifluoromethyl-
phenyl amide 4b. The N-4-chlorophenyl amide 4a dis-
played a weaker activity than both 4e and 4b, but all
these compounds, 4a, b, and e, showed significantly less
Membrane stabilization
Stabilization of the erythrocyte membrane is a character- activity of this type than the reference MR-2/94 (IC50
=
istic of some non-steroidal anti-inflammatory agents 0.6 l 0.2 lM). However, amides 4c and 4d inhibited prolif-
(NSAIDs) [23]. The activities of the new compounds 4a–e eration in the concentration range of 0.3 to 3 lM by
were tested at the concentrations of 0.001, 0.01, 0.1, 1, about 20%. But at higher concentrations, amide 4c
and 10 lg/mL. None of the tested pyrimidinecarboxa- slightly stimulated proliferation and derivative 4d did
mide derivatives 4a–e rendered a stabilizing effect on not influence the proliferation of PBMCs stimulated with
the erythrocyte membrane. The MR-2/94 reference concanavalin A (ConA). Such a profile of this type of activ-
showed strong membrane stabilization activity at con- ity could suggest immunomodulating properties of dia-
centrations of 1 and 10 lg/mL, with an estimated EC50 mides 4c and 4d.
(50% effective concentration, i. e. the concentration at
which relative hemolysis equals half of the control value)
of 8.1 l 0.98 lM.
Conclusion
Influence of the tested amides 4a-e on lymphocyte
proliferation
All the new tested amides 4, apart from 4e, showed very
low or no activity in the biological in-vitro tests presented
The new derivatives 4a–e were tested in vitro for their in this study. These results thus indicate that the distin-
influence on the proliferation of human peripheral guishing activity of derivative 4e in these tests is rather
blood mononuclear cells (PBMCs), both unstimulated connected with the presence of the adamantyl moiety in
and stimulated with phytohemagglutinin (PHA) and con- the molecular structure of 4e and the pyrimidine core
canavalin A (ConA), at the concentrations of 0.3, 1, 3, 10, fragment is an unnecessary ballast in this case. It is
30, and 100 lM. The IC50s (concentrations that caused known that 1-aminoadamantane (amantadine) has anti-
50% proliferation inhibition) of amides 4a–e were calcu- parkinsonian activity and is used in Parkinson's disease
lated on the basis of biological response and compared therapy [24]. In addition, amantadine is an effective anti-
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686
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
liberate 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-4-pyrimidine-
carboxylic acid. The mixture was stirred for 30 minutes, free
acid was filtered off (92%), and the crude solid was washed with
distilled water and was solvated in sodium hydroxide solution,
filtered off, and then the free acid 5 was precipitated from basic
solution with 10% water solution of HCl, then the precipitate
was dried, and a sample for elemental and spectral analysis was
crystallized from DMF (dimethylformamide). M.p. = 3308C (dec.),
(m.p. reported in [14] = 327–3308C (dec.)).
viral agent [25]. Its derivatives also show a broad scope of
activity, namely the antiviral [25], anti-inflammatory
[26], immunosuppressive [27], and antimicrobial [28]
activities already mentioned. In summary, on the basis of
the biological tests conducted, exchanging the isothia-
zole for a pyrimidine core ring caused a decrease in the
anti-inflammatory and immunotropic profile of biologi-
cal activity.
This study was supported by the Medical Academy of Wroclaw
(internal project no. 1526). We would also like to thank our
technicians, Ms. Barbara Bubak and Ms. Boguslawa Biadun,
for their skilful assistance.
2-(4-Chlorophenyl)-6,8-dihydroxypyrimido[5,4-d]-4H-1,3-
oxazin-4-on 3
A mixture of 5.0 g (0.016 mol) of 5-(4-chlorobenzoyl)amino-2,6-
dihydroxy-4-pyrimidinecarboxylic acid and 100 mL of toluene
was heated to 808C, then, ten drops of DMF (dimethylforma-
mide) were added and 10 mL thionyl chloride (SOCl2) was
dropped in. The whole mixture was stirred and heated at 808C
for 8 h. After cooling, the crude solid was filtered off and dried,
giving 4.65 g (100%) of crude compound. A sample for elemental
and spectral analysis was crystallized from a toluene / DMF
(dimethylformamide) 9 : 1 mixture. M.p. = 330–3408C (dec.).
The authors have declared no conflict of interest.
Experimental
Chemistry
The melting points of all the new compounds were measured on
a Bꢀchi 510 apparatus (Bꢀchi Laboratoriums-Technik AG, Flawil,
Switzerland) and were uncorrected. Elemental analyses were
conducted by the Microlaboratory of the Department of Phar-
macy, Medical Academy of Wroclaw with Carlo Erba series NA
1500 elemental analyzer (Carlo Erba, Milan, Italy). IR spectra:
Pye-Unicam SP-1000 apparatus (Pye Unicam Ltd. Cambridge,
England), KBr tablets. 1H-NMR spectra (300.14 MHz and
500.13 MHz), 13C-NMR spectra (broadband full decoupling
method: 75.47 MHz), 5 mm tubes, concentration 20–30 mg of
compound in 0.6 mL of solvent (L 0.1 M solution), TMS (tetrame-
thylsilane) as internal reference: Bruker Fourier spectrometer
(Bruker, Rheinstetten, Germany), MS, Electrospray Ionization
(ESI): mass spectrometer micrOTOF-Q Bruker Daltonics. Thin-
layer chromatography method (TLC) was applied to monitor the
reaction course as well as to confirm the purity of the synthe-
sized compounds. Polygram SIL G/UV254 plates (Macherey-Nagel,
Dꢀren, Germany) for TLC were used, eluting medium was
chloroform / methanol (9 : 1), detection of the compounds on
the chromatograms was done with UV light and / or by treat-
ment with iodine vapors.
General methods for amides 4 synthesis on the example
of 5-(4-chlorobenzoyl)amino-N-(4-chlorophenyl)-2,6-
dihydroxy-4-pyrimidinecarboxamide 4a
870 mg (6.82 mmol) of 4-chloroaniline was solved in 15 mL of
anhydrous pyridine, then the solution was heated to boiling and
1.0 g (3.43 mmol) of 2-(4-chlorophenyl)-6,8-dihydroxypyri-
mido[5,4-d]-4H-1,3-oxazin-4-on 3 was added. The whole mixture
was refluxed and stirred for 10 h, then cooled, and the yellow
needle crystals of the pyridinium complex salt of 2-(4-chloro-
phenyl)-6,8-dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on 11 was
filtered out (in case it formed). The filtration was evaporated to
dryness in vacuo. 200 mL of a 10% water solution of sulphuric
acid was added to the crude residue, the mixture was stirred for
30 minutes to remove excess amine that did not react and
remaining residue of the pyridine solvent. Then, the crude com-
pound was filtered off and washed with distilled water to neu-
tral pH. 1.32 g (92%) of the crude solid was obtained that was
crystallized from methanol to give 880 mg (61%) of pure com-
pound 4a. M.p. = 248–2508C.
5-(4-Chlorobenzoyl)amino-2,6-dihydroxy-N-(4-
Synthesis and physicochemical properties of compounds
5-(4-Chlorobenzoyl)amino-2,6-dihydroxy-4-
pyrimidinecarboxylic acid 5
5-(4-Chlorobenzoyl)amino-2,6-dihydroxy-4-pyrimidinecarboxylic
acid was obtained with the method described in [14].
trifluoromethylphenyl)-4-pyrimidinecarboxamide 4b
The crude solid (yield = 34%) was crystallized from methanol to
give pure 4b with a yield of 17%. M.p. = 297–2988C. Pyridinium
complex salt of 2-(4-chlorophenyl)-6,8-dihydroxy-4H-pyri-
mido[5,4-d]-4H-1,3-oxazin-4-on 11 was also formed (39%).
Modification of the procedure described in [14]
3.5 g (0.0205 mol) of 5-aminoorotic acid (5-amino-2,6-dihydroxy-
4-pyrimidinecarboxylic acid) was suspended (partly solved) in
50 mL of anhydrous pyridine and was continuously stirred. The
mixture was cooled to 5–108C, then, while cooling was sus-
tained, 3.9 mL (0.030 mol) of 4-chlorobenzoyl chloride were
dropped in. Afterwards, the mixture was heated for 6 h at about
808C. After cooling, the precipitate was filtered off, pressed, and
the crude solid was suspended in a 10% water solution of HCl to
5-(4-Chlorobenzoyl)amino-N-(6-chloro-3-pyridyl)-2,6-
dihydroxy- 4-pyrimidinecarboxamide 4c
The crude solid (yield = 56%) was crystallized from a methanol /
water mixture of in a ratio of 2.3 / 1 to give pure 4b with a yield
of 26%. M.p. = 302.5–3038C (dec.). Pyridinium complex salt of 2-
(4-chlorophenyl)-6,8-dihydroxy-4H-pyrimido[5,4-d]-4H-1,3-oxa-
zin-4-on 11 was also created (19%).
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Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
New Pyrimidine Derivatives and Their Biological Activity
687
(5% CO2 in air, 378C) with compounds at the tested concentra-
tions (0.3, 1, 3, 10, 30, 100 lM) in 2–3 replicates on 96-well plates
(Costar, Vitaris AG, Baar, Switzerland) and simultaneously
stimulated with 1 lg/mL of E. coli O1 lipopolysaccharide. After
18 h, the plates were centrifuged and the supernatants for cyto-
kine determination were collected and kept at –708C until
assay. Cell viability was determined immediately as described
below.
5-(4-Chlorobenzoyl)amino-2,6-dihydroxy-N-(4-
hydroxycyclohexyl)-4-pyrimidinecarboxamide 4d (as
equimolar mixture of isomer cis and trans)
The crude product (87%) was crystallized from methanol. M.p. =
248–2528C (dec.).
5-(4-Chlorobenzoyl)amino-N-(1-adamantyl)-2,6-
dihydroxy-4-pyrimidinecarboxamide 4e
The crude solid (95%) was crystallized from methanol. M.p. =
239–2418C (dec.).
Measurement of tumor necrosis factor a activity
Tumor necrosis factor a (TNF-a) activity in the supernatants was
determined according to Espevik and Nissen–Meyer [30]. Briefly,
WEHI-164.13 cells were cultured in 96-well plates (Nunc, Ros-
kilde, Denmark) at a density of 26104 cells/well with serial dilu-
tions of the tested supernatants. Cell proliferation was evaluated
after 20 h of culture using the 3-(4,5-dimethyl-2-tiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT, Sigma) colorimetric
method. Absorbance was measured at 550 nm (reference wave-
length: 630 nm) with a colorimetric microplate reader (Dynex
Opsys MR, Dynex Technologies GmbH, Berlin, Germany) [31].
Ethyl 5-(4-chlorobenzoyl)amino-2,6-dihydroxy-4-
pyrimidinecarboxylate 10 [14]
200 mg (685.8 lmol) of 2-(4-chlorophenyl)-6,8-dihydroxypyri-
mido[5,4-d]-4H-1,3-oxazin-4-on 3 was heated with 20 mL of anhy-
drous ethanol with molecular sieves for 10 h. After the mixture
had cooled, the small white needle crystals were filtered off and
dried, giving 217 mg (94%) of pure ester 10. M.p. = 2718C (dec.)
(m.p. reported in [14] = 264–2678C).
Measurement of interleukin-6 concentration
Pyridinium complex salt of 2-(4-chlorophenyl)-6,8-
Interleukin-6 (IL-6) concentration in the supernatants was deter-
mined by enzyme-linked immunosorbent assay according to [32]
and the Cytokine ELISA Protocol (May 29, 1998, 2nd Edition, Phar-
mingen). Shortly, 100 lL per well of the ten times diluted super-
natants or serial dilutions of recombinant human IL-6 (Pharmin-
gen, BD Bioscience, USA) were incubated in duplicate for 18 h at
48C on 96-well plates (Nunc-Immuno Plates, MaxiSorp Surface,
Nunc, Denmark) previously covered with rat anti-human IL-6
antibody (1 lg/mL, Pharmingen). After washings, biotinylated
rat anti-human IL-6 antibody (5 lg/mL, Pharmingen) was incu-
bated on the plate for 60 min at room temperature. Then avi-
dine conjugated with horse radish peroxidase (1 : 4000, Dako)
was added for 30 min at room temperature. The colorimetric
reaction was elicited by addition of ABTS substrate solution
(0.03% 2,29-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) in
0.1 M citric acid, pH = 4.35, with addition of 0.03% hydrogen per-
oxide). Absorbance was measured at 405 nm (reference wave-
length: 630 nm) after 30 min of incubation at 208C. The IL-6 con-
centration was calculated from an IL-6 standard curve.
dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on 11 (11a)
Yellow needle crystals, m.p. A2858C (dec.).
2-(4-Chlorophenyl)-6,8-dichloropyrimido[5,4-d]-4H-1,3-
oxazin-4-on 12 [16]
The mixture of 250 mg (857.2 lmol) of 2-(4-chlorophenyl)-6,8-
dihydroxypyrimido[5,4-d]-4H-1,3-oxazin-4-on 3, 10 mL of phos-
phoryl chloride (POCl3) and a few drops of N,N,N-triethylamine
was heated in reflux for 5 h. Afterwards POCl3 was evaporated.
The obtained crude solid was treated with boiling benzene and
then, the benzene solution was filtered off from solid insoluble
impurities. The filtrate was evaporated in vacuo to give a dry
solid that was crystallized from cyclohexane to give 153 mg
(54%) of pure compound 12. M.p. = 223–2258C. 2-(4-Chloro-
phenyl)-6,8-dichloropyrimido[5,4-d]-4H-1,3-oxazin-4-on 12 cre-
ates very durable yellow complex with N,N-dimethylaniline,
m.p. = 235–2378C.
Pharmacology
Dilution of the compounds for in-vitro tests
Nitrite measurement
Macrophages elicited with thioglycolate according to [33] were
derived from the peritoneal cavity of Wistar rats and suspended
after washing in culture medium (RPMI 1640 without phenol
red, supplemented with 10% fetal calf serum, 2 mM L-glutamine,
100 U/mL penicillin, and 100 mg/mL streptomycin). Then, they
were incubated (1.26106 cells per well, 5% CO2 in air, 378C) for
24 h with the tested compounds at the specified concentrations
(0.3, 1, 3, 10, 30, 100 lM) in duplicate in 96-well plates and simul-
taneously stimulated with 10 lg/mL of E. coli O1 LPS. Then, the
plates were centrifuged and supernatants for cytokine determi-
nation were collected. The cells were detached for viability
assessment as described below immediately after centrifuga-
tion.
For the in-vitro tests, the compounds were dissolved initially in
dimethyl sulphoxide (DMSO, ICN) and then in culture medium,
i. e. RPMI 1640 supplemented with 25 mM HEPES buffer, 10%
fetal calf serum (Life Technologies, St. Paul, MN, USA), 2 mM L-
glutamine (Sigma, Poznan, Poland), 100 U/mL penicillin, and
100 mg/mL streptomycin (both from Polfa Tarchomin; Warsaw,
Poland) supplemented with DMSO to maintain a constant con-
centration. The final concentration of the compounds ranged
from 0.001 to 100 lM and the DMSO dilution was 1 : 1000. At
this dilution, DMSO in culture medium was used as a control.
Cell isolation and stimulation for cytokine production
Human peripheral blood mononuclear cells (PBMCs) were iso-
lated from the buffy coats (enriched with 2500 UI/mL heparin) of
volunteer blood donors according to Bøyum using Lymphoprep
(Nycodenz, Aachen, Germany) and resuspended (106 cells/mL) in
culture medium [29]. The cells (26105 per well) were incubated
For an indirect determination of NO secretion by the stimu-
lated macrophages, 100 lL of the supernatant was incubated at
room temperature with 100 lL of Griess reagent (1% sulfanila-
mide / 0.1% naphthylenediamine dihydrochloride, both from
Sigma, in 2.5% H3PO4) [34]. After 10 min, absorbance at 550 nm
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688
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 677–689
´
was measured. The concentration of nitrite ion was calculated
from a NaNO2 standard curve.
[3] Z. Machon, Drugs Future 1988, 13, 426–428 and the refer-
ences cited therein.
[4] A. Regiec, Ph. D. Dissertation (in Polish), Faculty of Phar-
macy, Wroclaw Medical University, 1998 and unpublished
data.
Membrane stabilization
Stabilization of erythrocyte membranes was determined by
hemoglobin release according to Seeman and Weinstein [35]. A
suspension of rabbit erythrocytes was used (46106 cells/mL in
PBS without Ca2+ and Mg2+, pH = 7.0). The compounds were dis-
solved in DMSO and further diluted with hypotonic phosphate
buffer to a concentration ranging from 0.001 to 10 lg/mL.
Five mL of dissolved compound was incubated with 0.1 mL of
the erythrocyte suspension for 5 min at room temperature and
then the absorbance of the supernatant at 543 nm (correspond-
ing to the release of hemoglobin) was measured. Inhibition of
hemoglobin release by more than 20% compared with controls
(control hemolysis was set at around 50%) was considered a posi-
tive effect.
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[5] U. Lipnicka, A. Regiec, Z. Machon, Polish Patent P172969
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[6] U. Lipnicka, A. Regiec, Z. Machon, Polish Patent P172967
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of Immunology and Experimental Therapy, Polish Academy
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[10] Z. Machon, Z. Wieczorek, M. Zimecki, Pol. J. Pharmacol.
Lymphocyte proliferation assay
2001, 53, 377–383.
PBMCs were isolated according to Bøyum as described earlier
[29]. One hundred five cells per well were not stimulated or
stimulated with 10 lg/mL PHA or ConA (in 2–3 replicates) and
incubated with the tested compounds at concentrations of 0.3,
1, 3, 10, 30, or 100 lM in culture medium in 96-well plates. Cell
proliferation was evaluated after 72 h using the 3-(4,5-dimethyl-
2-tiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma)
colorimetric method as described earlier [29]. Control cells were
incubated with DMSO at the corresponding concentrations.
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Cytotoxic activity
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The cytotoxic activities of the compounds on PBMCs and macro-
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diphenyl-2H-tetrazolium bromide (MTT; Sigma) colorimetric
method as described earlier in methods. Control cells were incu-
bated with DMSO at the appropriate concentration. In the prolif-
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from two wells for each compound dilution was assessed with
an acridine orange / ethidium bromide mixture solution with a
fluorescence microscope (viable cells appear green, dead cells
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