Synthesis of new series of pyrazolo[4,3-d]pyrimidin-7-ones and pyrido[2,3-d]pyrimidin-4-ones for their bacterial and cyclin-dependent kinases (CDKs) inhibitory activities

Article abstract of DOI:10.1007/s00044-010-9328-z

Two series of pyrazolo[4,3-d]pyrimidin-7-ones and pyrido[2,3-d]pyrimidin-4- ones were designed, synthesised, and evaluated for their antibacterial activities and CDKs inhibitory activities. The pyridazine derivative: 6-phenyl-5-phenylhydrazono-2,3,4,5-tetrahydropyridazine- 3,4-dione (3a) revealed activity against Staphylococcus aureus as Gram-positive bacteria while compound 2-(2- Ethoxyphenyl-5-Phenylpiperazinosulfonamido)-3H-pyrido [2,3-d]pyrimidin-4-one (13c) was showing moderate antifungal activity against Candida albicans.

Full text of DOI:10.1007/s00044-010-9328-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:408–420
DOI 10.1007/s00044-010-9328-z
ORIGINAL RESEARCH
Synthesis of new series of pyrazolo[4,3-d]pyrimidin-7-ones
and pyrido[2,3-d]pyrimidin-4-ones for their bacterial and
cyclin-dependent kinases (CDKs) inhibitory activities
•
Detlef Geffken Raafat Soliman Farid S. G. Soliman
•
•
•
Magdi M. Abdel-Khalek Doaa A. E. Issa
Received: 24 July 2009 / Accepted: 16 February 2010 / Published online: 12 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract Two series of pyrazolo[4,3-d]pyrimidin-7-ones
and pyrido[2,3-d]pyrimidin-4-ones were designed, syn-
thesised, and evaluated for their antibacterial activities
and CDKs inhibitory activities. The pyridazine derivative:
6-phenyl-5-phenylhydrazono-2,3,4,5-tetrahydropyridazine-
3,4-dione (3a) revealed activity against Staphylococcus
aureus as Gram-positive bacteria while compound 2-(2-
Ethoxyphenyl-5-Phenylpiperazinosulfonamido)-3H-pyrido
[2,3-d]pyrimidin-4-one (13c) was showing moderate anti-
fungal activity against Candida albicans.
revealed a large number of highly active CDK inhibitors
´
(Vesely et al., 1994). One of the first CDK specific
inhibitors, Olomoucine (II) was found to block selectively
CDK1, CDK2, and CDK5 kinases at micromolar concen-
´
trations (Vesely et al., 1994), molecular modifications lead
to the development of Roscovitine (I) (Havlicek et al.,
1997), with enhanced CDK1 selective inhibition and anti-
mitotic activity (Meijer et al., 1997). Successive studies
lead to the development of derivatives with enhanced
efficiency as Purvalanol (III), Olomoucine II (IV) (Fig. 1)
(Havlicek et al., 1997; Dreyer et al., 1998; Gray et al.,
1998; Haesselein and Jullian, 2002; Krystof et al., 2002).
In contrast to 2,6,9-trisubstituted purine inhibitors (I–
IV), substituted pyrazolo[4,3-d] pyrimidines, studied early
as cytokinin antagonists with antiproliferative effects on
plant cells (Hecht et al., 1971), showed potent human
CDK1 inhibition.
Keywords Pyrazole ꢀ Pyrazolo[4,3-d]pyrimidin-7-one ꢀ
Pyrido[2,3-d]pyrimidin-4-one ꢀ Hydrazono-pyridazine ꢀ
Antibacterial activity
Introduction
The published 7-(3-Chloroanilino)-3-isopropylpyrazolo
[4,3-d]pyrimidine and 7-(3-hydroxy-4-methoxybenzyl)-
amino-3-isopropylpyrazolo[4,3-d]pyrimidine (V) (Fig. 2)
showed inhibitory activity against CDK1/cyclin B with
IC50 values 0.9 and 1.1 lmol/dm3, respectively, and anti-
proliferative effect on myeloid leukemia cell line K-562
with IC50 values 18.4 and 26 lmol/dm3, respectively, using
Olomoucine as reference compound 7 and 163 lmol/dm3,
respectively (Moravcova et al., 2003). Conversely, pyr-
ido[2,3-d]pyrimidine derivatives showed antibacterial and
anticancer potency (Ravi Kanth et al., 2006; Dabiri et al.,
2007; Mohamed et al., 2007).
Cell division process is regulated by a diverse set of cel-
lular signals. CDKs play a role in regulating the cell cycle
through their activating agents and the endogenous CDK
inhibitors which are frequently mutated in human cancers,
making CDKs interesting targets for cancer chemotherapy.
Roscovitine (I) (purine inhibitor of CDKs) attracted
attention as potential anticancer drug lead (Moravec et al.,
2003). The discovery of 2,6,9-trisubstituted purines
D. Geffken
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
University of Hamburg, Hamburg, Germany
Our interest in modifying compounds V promoted us to
prepare a series of 3-(4-substituted phenyl)-1,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-ones (6a–d) which retains
the aromatic substitution at position 3 and a cyclic
amide keto-function at position 7. Synthesis of a bio-
isosteric series of 2-(2-ethoxyphenyl-5-sulfonamido)-3H-
R. Soliman ꢀ F. S. G. Soliman (&) ꢀ M. M. Abdel-Khalek ꢀ
D. A. E. Issa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Alexandria, Alexandria, Egypt
e-mail: solfarid@hotmail.com
123

Med Chem Res (2011) 20:408–420
409
Fig. 1 Selected purine
inhibitors of CDK1
HO
Cl
NH
N
N
N
N
N
NH
N
N
NH
N
NH
N
N
N
N
N
NH
N
N
OH
OH
NH
N
NH
NH
OH
OH
(I)
(II)
(III)
(IV)
R1
N
Fig. 2 Selected 3,7-
Results and discussion
disubstituted pyrazolo[4,3-
d]pyrimidines with recorded
inhibitory activity of CDK1/
cyclin B and antiproliferative
effect on myeloid leukemia cell
line K-562
NH
H
N
Chemistry
N
N
The target pyrazolo[4,3-d]pyrimidin-7-ones (6a–d) and
pyrido[2,3-d]pyrimidin-4-ones (13a–e) were synthesised
by the reactions depicted in Schemes 1 and 2.
(V)
The key intermediates 4-(4-substituted phenyl)-2,4-di-
oxobutanoic acid ethyl esters (1a–d) were synthesised by
condensing the appropriate acetophenone with diethyl
oxalate either in presence of sodium hydride (method I) as
previously reported by Roy and Batra (Roy and Batra,
2003), or using sodium ethoxide (method II) as formerly
described by Marvel and Dreger (1941), Yields from
method II were better than those from method I. Their
pyrido[2,3-d]pyrimidin-4-ones (13a–e) was also designed
as an interesting structural variant. The impact of these
modifications would lead to the improvement of the physi-
cochemical and pharmacodynamic properties of the syn-
thesised compounds. The CDKs inhibitory activity and in
vitro antimicrobial activity of the target compounds were
evaluated.
X
Scheme 1
O
O
X
X
O
O
phenyldiazonium salt
sodium acetate
N
N
N
NH
O
X
O
O
1a-d
O
O
O
O
O
2a-d
O
O
NH2NH2.H2O
O
HO
X
X
N
H
O
4c
H2 - Pd/C
N
N
N
O
5a
+
N
N
NH
O
N
O
H
3a-d
4a-d
SnCl2
X
X
N
NH2
Formamidine acetate
H
N
O
N
N
N
H
N
H
O
O
6a-d
5a-d
X-Key: a = H; b = F; c = Cl; d = Br
123

410
Med Chem Res (2011) 20:408–420
products; namely, 6-(4-Substituted phenyl)-5-phenylhyd-
razono-2,3,4,5-tetrahydropyridazin-3,4-diones (3a–d) and
5(3)-(4-substituted phenyl)-4-phenylazo-1H-pyrazole-3(5)-
carboxylic acid ethyl esters (4a–d) which could be sepa-
rated by fractional crystallisation.
O
O
O
NH2
NH2
Cl
+
N
7
8
Reduction of (4a–d) afforded the respective 4-Amino-
5(3)-(4-substituted phenyl)-1H-pyrazole-3(5)-carboxylic
acid ethyl esters (5a–d). It was achieved using of stannous
chloride in hydrochloric acid, where the amines were
obtained in good yields. Alternatively catalytic hydrogena-
tion of 3(5)-(4-chlorophenyl)-4-phenylazo-1H-pyrazole-
5(3)-carboxylic acid ethyl ester (4c) in presence of palla-
dium over carbon resulted in the dehalogenated amine.
Compounds 5a–d were converted to the target 3-(4-
substituted phenyl)-1,6-dihydro-7H-pyrazolo[4,3-d]pyrim-
idin-7-ones (6a–d) in good yields upon reaction with
formamidine acetate in refluxing n-butanol. A remarkable
shift in the IR absorption band of the cyclic carbonyl of
the produced pyrazolopyrimidinones (6a–d) to about
1680 cm-1 was observed instead of about 1700 cm-1 in
case of the starting aminopyrazole esters (5a–d). The
assignment of the structure was based on the spectroscopic
data.
Acetonitrile / TEA
O
O
NH2
NaOH / Pyridine
NH
10
O
N
NH
O
N
N
O
9
SOCl2 / HSO3Cl
O
N
O
N
NH
O
NH
O
N
N
12a-e
O2S
O2S
Scheme 2 illustrates the applied synthetic sequences for
the achievement of the target novel 2-(2-ethoxyphenyl-5-
sulfonamido)-3H-pyrido[2,3-d]pyrimidin-4-ones (13a–e).
Thus reacting the freshly prepared acid chloride (7) with
2-aminonicotinamide (8) in presence of triethylamine in
acetonitrile, following previously described methods for
the synthesis of approximately similar compounds (Bell
et al., 1992; Piazza and Pamukcu, 2000), afforded the
desired carboxamide (9) in excellent yield. 2-aminonico-
tinamide (8) was previously prepared by many varying
yields methods (Taylor and Crovetti, 1954; Althuis et al.,
1979; Purcell et al., 1982). In this study we describe a new
method isolating pure compound in very good yield which
involves the activation of 2-aminonicotinic acid with 1,10-
carbodiimidazole (CDI) in dry THF, followed by injecting
ammonia gas.
R
Cl
11
13a-e
R-Key:
a = Piperidino; b = Morpholino; c = 4-Phenylpiperazino;
d = 4-Ethylpiperazino; e = Diethanolamino
Scheme 2
structures were established to exist in an enol form with
internal hydrogen bonding of H-chelate type as Z-isomer
(Zalesov et al., 2002). The resulting esters, although being
early published (Gardner et al., 1961; Walford et al., 1971;
Burch and Gray, 1972; van Herk et al., 2003; Braga et al.,
2004; Fadnavis and Radhika, 2004) yet the physical and
spectral data of some candidates were not satisfactory
integrated.
Refluxing 9 in pyridine and 2 N sodium hydroxide, as
earlier described by Piazza and Pamukcu (Piazza and
Pamukcu, 2000) for cyclizing their synthesised 2-prop-
oxybenzamido derivatives, followed by neutralisation with
2 N hydrochloric acid produced the 2-(2-ethoxyphenyl)-
3H-pyrido[2,3-d]pyrimidin-4-one (10) in feeble yield. The
1H-NMR and 13C-NMR spectra of the newly synthesised
carboxamide (9) and pyrido[2,3-d]pyrimidin-4-one (10)
coincide with their assigned structures. The X-ray crystal
structure of compound 10 is shown in Fig. 3.
Diazocoupling of (1a–d) with phenyldiazonium salt
in presence of sodium acetate yielded their respective
4-substituted phenyl-3-phenylazo-2,4-dioxobutanoic acid
ethyl esters (2a–d) in 70–80% yield. The chemical struc-
ture of the obtained esters would exist in different tauto-
meric forms (Mitchell, 1979) of which, one predominates
and separates. Thus the predominating structure of 2a is the
hydrazono tautomer showing a well-defined C=N signal at
d 171.15 ppm confirming the hydrazono functionality. By
contrast, the azo tautomer in the 4-halo substituted deriv-
atives (2b–d) is the predominating form.
The intermediate 2-(4-Chlorosulfonyl-2-ethoxyphenyl)-
3H-pyrido[2,3-d]pyrimidin-4-one (11) was synthesised
following Liu’s methodology (Liu, 2004) which involved
the treatment of 10 with a mixture of thionyl chloride and
Condensing the appropriate ester (2a–d) with hydrazine
hydrate in acetic acid at room temperature yielded two
123

Med Chem Res (2011) 20:408–420
411
comparison with that of some control drugs was pre-
liminary tested as shown in Table 1. The results revealed
that compounds 3a, 3d, 4b, and 4c showed activity against
Staphylococcus aureus as Gram-positive bacteria lower
than the control, ampicillin. Compound 3a was considered
the most potent with IZ 30 mm and equal MIC and MBC
62.5 lg/ml; however, it was less potent than ampicillin.
Compounds 3a, 5c showed antimicrobial activity against
Pseudomonas aeruginosa. Compound 3a was the most
active with IZ 24 mm and showed equal MIC and MBC
125 lg ml-1. As regards to the antimicrobial activity
against Candida albicans; the compounds were also tested
for their antifungal activity; compounds 6d and 13c were
active with IZ 28 and 25 mm, respectively. However, the
results indicated that compound 13c exhibited mild
activity when compared with clotrimazole. None of the
selected tested compounds possessed antimicrobial activ-
ity against Escherichia coli. The pyridazine derivative:
6-phenyl-5-phenylhydrazono-2,3,4,5-tetrahydropyridazine-
3,4-dione (3a) showed moderate activity against S. aureus
as Gram-positive bacteria while compound 2-(2-etho-
xyphenyl-5-phenylpiperazinosulfonamido)-3H-pyrido[2,3-d]
pyrimidin-4-one (13c) showed weak antifungal activity
against C. albicans.
Fig. 3 Perspective view of the X-ray crystal structure of compound
10
chlorosulfonic acid. Reacting 11 with the appropriate
amine (12a–e) in presence of triethylamine applying the
technique of Kim et al. (2001, 2004) afforded 2-(2-
ethoxyphenyl-5-sulfonamido)-3H-pyrido[2,3-d]pyrimidin-
4-ones (13a–e) in 70–80% yield.
Inhibition of CDk1/cyclin B, DYRK1A, CDK5/p25,
Antimicrobial evaluation
and GSK-3a/b kinases
The in vitro antimicrobial activity of twelve compounds;
Twenty-six compounds (2a–d, 3a–d, 4a–d, 5b–d, 6a–d, 9,
10, and 13a–e) were tested against four protein kinases;
2a, 2c, 3a, 3d, 4b, 4c, 5c, 5d, 6a, 6d, 13b, and 13c in
Table 1 The inhibition zones (IZ) in mm diameter, MIC and MBC in lg ml-1 of some selected compounds
Cpd no.
S. aureus (ATCC 6538)
E. Coli (NCTC 10418)
P. aeruginosa (ATCC 9027)
C. albicans (ATCC 10231)
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
2a
–
–
–
16
–
–
16
–
–
16
16
17.5
16.5
16
16
17
16
17
28
17
25
–
–
–
2c
–
–
–
16
–
–
17
–
–
–
–
3a
30
20
20
21
10
14
11
–
62.5
250
250
125
–
62.5
250
250
250
–
17
–
–
24
125
–
125
–
–
3d
16
–
–
17.5
17
–
–
–
4b
16
–
–
–
–
–
–
4c
16.5
17
–
–
16
–
–
–
–
5c
–
–
23
125
–
[500
–
–
5d
–
–
15.5
16
–
–
17
–
–
–
6a
–
–
–
–
17.5
17.5
15
–
–
–
–
6d
–
–
15.5
17
–
–
–
–
250
–
250
–
13b
10
12
40
–
–
–
–
–
–
–
13c
–
–
16.5
26
–
–
15.5
18
–
–
62.5
–
250
–
Ampicillin
Clotrimazol
3.9
–
15.62
–
7.81
–
31.25
–
250
–
500
–
–
–
28
7.81
15.62
123

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Med Chem Res (2011) 20:408–420
CDKl/cyclin B, DYRK1A, CDK5/p25, and GSK-3a/b.1
All tested compounds were considered inactive since they
displayed IC50 values above 10 lM.
filtrate was concentrated under vacuum and column chro-
matographed using n-hexane/ethyl acetate mixture (4:1), to
obtain the 2,4-diketo ester derivatives (1a–d).
Method II A mixture of the appropriate acetophenone
(30 mmole) and diethyl oxalate (6.58 g, 45 mmole) was
dropped slowly on the stirred cooled sodium ethoxide
solution [sodium metal (1.03 g, 45 mmol) in absolute
ethanol (40 ml)] (0–5°C) over a period of 1 h under
absolute anhydrous conditions. Stirring was continued for
further 2 h. The formed sodium salt was filtered, washed
with absolute ethanol, dried, and the product was dispersed
in ice-cold water and acidified with 2 N hydrochloric acid
till pH 2. The liberated ester was filtered and recrystallised
from the appropriate solvent. The yields %, melting points,
molecular formulas, and analysis are recorded in Table 2.
IR of compounds 1a–d (KBr, cm-1): 3448–3120,
2925–2920 (–CH=C–OH); 1733–1621 (C=O); 1588–1598,
1541–1509, 1479–1442 (C=C and Aromatics).
Experimental protocols
Melting points were determined in open glass capillaries on
a Mettler FP 62 apparatus and are uncorrected. The follow-
up of reactions and checking the homogeneity of com-
pounds were performed using Thin Layer Chromatography
(TLC) on DC-Mikrokarten Polygram SIL G/UV254
,
of thickness: 0.25 mm from the Macherey–Nagel Firm,
Duren. Column Chromatography was applied using Sili-
cagel, ICN Silica 100–200 active 60A, Silicagel 60 (par-
ticle size 0.015–0.040 mm), Merck. The drying agent used
for organic phases was dry magnesium sulfate. The IR
spectra were recorded in KBr disks or as film on NaCl plate
on a Shimadzu FT-IR 8300 infrared spectrophotometer.
The 1H-NMR (400 MHz) spectra were carried on a Bruker
AMX 400 spectrometer using tetramethylsilane (TMS) as
an internal standard. All samples were dissolved in DMSO-
d6 except when CDCl3 is stated. Chemical shifts are
expressed in d ppm. The type of signal is indicated by one
of the following letters: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. The coupling constant
J is measured in Hz. The 13C-NMR (100 MHz) spectra
were performed on Bruker AMX 400 using Trimethylsi-
lane (TMS) as internal standard. All samples were dis-
solved in DMSO-d6 except when CDCl3 is stated.
Chemical shifts are expressed in d ppm. The Mass Spectra
were run on HRFAB-Mass spectra: Mass spectrometer VG
70-250S and ESI- Mass spectra: Varian MS 1200L. Ele-
mental analyses were measured on Heraeus CHN-O-Rapid,
and the data were within 0.4% of the theoretical value.
MS of 1a m/z (relative abundance %): 222, 223 (M , M
?1) (11.3, 1.2%), 176 (2.1), 148 (10.0), 147 (100), 105
(25.0), 77 (16.2), 69 (40.0).
1H-NMR of compound 1b (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, O–CH2–CH3), 4.33 (q, 2H, J = 7.12 Hz,
O–CH2–CH3), 7.13 (s, 1H, CH), 7.39–8.20 (m, 4H, Ar–H).
13C-NMR of compound 1b (DMSO-d6, d ppm): 14.24
(O–CH2–CH3), 62.60 (O–CH2–CH3), 98.47 (=CH),
115.93–164.72 (Ar–C), 161.91 (=CHCOH), 167.23 (COO),
189.74 (C6H5–CO).
MS of 1b m/z (relative abundance %): 238, 239 (M ,
M ?1) (4.9, 0.7%), 210 (1.2), 166 (9.7), 165 (100), 138
(2.3), 123 (28.8), 95 (14.5), 69 (26.0).
1H-NMR of compound 1c (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, O–CH2–CH3), 4.32 (q, 2H, J = 7.12 Hz,
O–CH2–CH3), 7.12 (s, 1H, CH), 7.65 (d, 2H, J = 8.40 Hz,
Ar–H) 8.10 (d, 2H, J = 8.40 Hz, Ar–H).
13C-NMR of compound 1c (DMSO-d6, d ppm): 14.21
(O–CH2–CH3), 62.62 (O–CH2–CH3), 98.46 (=CH),
129.35-139.41 (Ar–C), 161.84 (=CHCOH), 169.28 (COO),
189.34 (C6H5–CO).
Synthetic methodology
Scheme 1
MS of 1c m/z (relative abundance %): 254, 256 (M , M
?2) (8.4, 2.3%), 183 (33.2), 181 (100), 141 (5.5), 139
(17.7), 111 (8.3).
4-(4-Substituted phenyl)-2,4-dioxobutanoic acid ethyl
esters (1a–d)
Method I To a solution of the appropriately 4-substituted
acetophenone (5 mmole) in anhydrous toluene (12 ml) was
added a suspension of sodium hydride in oil (60%; 0.4 g,
10 mmole) in portions, and the mixture was warmed to
45–50°C with stirring. At this temperature, a solution of
diethyl oxalate (1.09 g, 7.5 mmole) in anhydrous toluene
(3 ml) was added dropwise with stirring. The reaction
mixture was refluxed for further 1.5 h then filtered. The
1H-NMR of compound 1d (DMSO-d6, d ppm): 1.31 (t,
3H, J = 7.12 Hz, O–CH2–CH3), 4.32 (q, 2H, J = 6.95 Hz,
O–CH2–CH3), 7.10 (s, 1H, CH), 7.78 (d, 2H, J = 8.40 Hz,
Ar–H) 8.01 (d, 2H, J = 8.40 Hz, Ar–H).
13C-NMR of compound 1d (DMSO-d6, d ppm): 13.77
(O–CH2–CH3), 62.18 (O–CH2–CH3), 97.97 (=CH),
128.21–132.12 (Ar–C), 161.40 (=CHCOH), 168.95 (COO),
188.99 (C6H5–CO).
MS of 1d m/z (relative abundance %): 298, 300 (M -1,
M ?1) (9.0, 9.3%), 227 (97.7), 225 (100), 185 (15.8), 183
(16.6), 157 (7.0), 155 (7.3), 69 (34.1).
1
Testing was conducted by ‘‘Centre National de la Recherche
Scientifique (CNRS), Roscoff Cedex; France’’.
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Med Chem Res (2011) 20:408–420
413
Table 2 4-(4-substituted phenyl)-2,4-dioxobutanoic acid ethyl esters (1a–d) and 4-Substituted phenyl-3-phenylazo-2,4-dioxobutanoic acid ethyl
esters (2a–d)
Cpd. no.
X
Yield %
*
M.P. °C (Cryst. solvent)
Mol. formula (Mol wt.)
Analysis %
Calcd.
**
Found
1a
1b
1c
1d
2a
H
F
59.5
66.7
57.6
50.3
80
76.2
79.2
70.5
73.8
35–37 (EtOH)
C12H12O4 (220.22)
48–49 (Benzene)
C12H11FO4 (238.22)
Cl
Br
H
62–64 (EtOH/H2O)
61–63 (EtOH/H2O)
118–19 (decomp. MeOH)
C12H11ClO4 (254.67)
C12H11BrO4 (299.12)
C
C
C
C
18H 16N2O4 (324.34)
18H15FN2O4 (342.33)
18H15ClN2O4 (358.78)
18H15BrN2O4 (403.23)
C
H
N
C
H
N
C
H
N
C
H
N
66.66
4.97
8.64
63.16
4.42
8.18
60.26
4.21
7.81
53.62
3.75
6.95
66.33
5.05
8.57
62.85
4.17
8.51
59.87
3.92
8.03
53.62
3.88
6.90
2b
2c
2d
F
73
71
80
123–24 (decomp. MeOH)
129–30 (decomp. MeOH)
135–36 (decomp. MeOH)
Cl
Br
* Method I Reported yields 90–95% (Roy and Batra, 2003)
** Method II
4-Substituted phenyl-3-phenylazo-2,4-dioxobutanoic acid
ethyl esters (2a–d)
MS of 2a m/z (relative abundance %): 324, 325 (M , M
?1) (67.3, 13.6%), 279 (3.9), 251 (35.2), 105 (100).
1H-NMR of compound 2b (DMSO-d6, d ppm): 1.06 (t,
3H, J = 6.99 Hz, CH3), 3.44 (q, 2H, J = 6.95 Hz, CH2),
7.30–8.22 (m, 10H, Ar–H and C3–H).
13C-NMR of compound 2b (DMSO-d6, d ppm): 18.45
(CH3), 55.92 (CH2), 92.10 (C3), 115.65–146.32 (Ar–C),
164.26 (COO), 183.77 (C6H5–CO), 203.54 (CO–COO).
MS of 2b m/z (relative abundance %): 342, 343 (M , M
?1) (27.9, 3.1%), 296 (4.2), 269 (20.4), 240 (3.5), 123 (100).
1H-NMR of compound 2c (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, CH3), 4.32 (q, 2H, J = 7.12 Hz, CH2),
7.11–8.11 (m, 10H, Ar–H and C3–H).
13C-NMR of compound 2c (DMSO-d6, d ppm): 14.22
(CH3), 62.61 (CH2), 98.46 (C3), 116.15–143.58 (Ar–C),
165.94(COO), 187.67 (C6H5–CO), 205.64 (CO–COO).
MS of 2c m/z (relative abundance %): 358, 360 (M , M
?2) (22.9, 2.0%), 285 (18.2), 139 (100), 111 (26.5).
1H-NMR of compound 2d (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.05 Hz, CH3), 4.32 (q, 2H, J = 7.03 Hz, CH2),
7.12-8.03 (m, 10H, Ar–H and C3–H).
To a cold stirred solution of aniline (0.465 g, 5 mmol) in
5 N hydrochloric acid (5 ml), a cold solution of sodium
nitrite (0.414 g, 6 mmol) in water (2 ml) was added
dropwise while keeping the temperature below 5°C. The
formed diazonium salt was allowed to stand for 1 h at 0°C
and then dripped at a very low rate onto a cold suspension
(0–5°C) of the appropriate diketoester (1a–d) (5 mmol)
and sodium acetate (0.82 g, 10 mmol) in ethanol/water
(1:1) (10 ml). The mixture was stirred at 0–5°C for 2 h. Ice
water (20 ml) was added and the formed yellow precipitate
was filtered, washed with water, and dried. The yields %,
melting points, molecular formulas, and analysis are
recorded in Table 1.
IR of compounds 2a (KBr, cm-1): 1743 (CO–COO);
1718 (C6H5–CO); 1686.13 (C=N); 1637.62 (COO);
1560.20, 1524.39 (C=C and Aromatics).
IR of compounds 2b–d (KBr, cm-1): 1743–1742 (CO–
COO); 1728–1717 (C6H5–CO); 1647–1644 (COO);
1586–1599, 1524–1528 (C=C and Aromatics).
1H-NMR of compound 2a (DMSO-d6, d ppm): 1.36 (t,
3H, J = 7.12 Hz, CH3), 4.43 (q, 2H, J = 7.12 Hz, CH2),
7.13–8.12 (m, 10H, Ar–H), 12.42, 13.84 (2 s, 1H, NH).
13C-NMR of compound 2a (DMSO-d6, d ppm): 14.53
(CH3), 62.15 (CH2), 116.15–142.29 (Ar–C), 165.63
(COO), 171.15 (C=N), 184.58 (C6H5–CO), 205.25 (CO–
COO).
13C-NMR of compound 2d (DMSO-d6, d ppm): 14.23
(CH3), 62.63 (CH2), 98.45 (C3), 116.35–143.41 (Ar–C),
165.41 (COO), 184.52 (C6H5–CO), 205.56 (CO–COO).
MS of 2d m/z (relative abundance %): 402, 404 (M -1,
M ?1) (8.2, 6.5%), 358 (4.5), 356 (5.6), 331 (10.8), 329
(11.1), 227 (19.2), 225 (20.1), 185 (96.2), 183 (100), 155
(26.5).
123

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Med Chem Res (2011) 20:408–420
Table 3 6-(4-Substituted phenyl)-5-phenylhydrazono-2,3,4,5-tetrahydropyridazine-3,4-diones (3a–d) and 3(5)-(4-Substituted phenyl)-4-phe-
nylazo-1H-pyrazole-5(3)-carboxylic acid ethyl esters (4a–d)
Cpd. no.
X
Yield %
M.P. °C (Cryst. solvent)
Mol. formula (Mol. wt.)
Analysis %
Calcd.
Found
3a
H
15.3
232–33 (decomp. DMF)
C16H12N4O2 (292.30)
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
65.75
4.14
65.79
4.48
19.17
61.93
3.57
18.84
61.74
3.65
3b
3c
3d
4a
4b
4c
4d
F
12.7
14.6
14.0
75.6
85.8
80.3
78.4
256–57 (decomp. HAc)
271–72 (decomp. HAc)
279–80 (decomp. HAc)
145–46 (Methanol)
155–56 (HAc)
C
C
C
C
C
C
C
16H11FN4O2 (310.29)
16H11ClN4O2 (326.74)
16H11BrN4O2 (371.19)
18H16N4O2 (320.35)
18H15FN4O2 (338.34)
18H15ClN4O2 (354.80)
18H15BrN4O2 (399.25)
18.06
58.82
3.39
17.80
58.51
3.51
Cl
Br
H
17.15
51.77
2.99
17.02
51.29
3.35
15.09
67.49
5.03
14.83
66.81
5.43
17.49
63.90
4.47
17.41
63.91
4.92
F
16.56
60.94
4.26
16.62
61.01
4.67
Cl
Br
150–51 (HAc)
15.79
54.15
3.79
15.58
53.88
4.03
157–59 (HAc)
14.03
13.78
6-(4-Substituted phenyl)-5-phenylhydrazono-2,3,4,5-tet-
rahydropyridazine-3,4-diones (3a–d) and 3(5)-(4-Substi-
tuted phenyl)-4-phenylazo-1H-pyrazole-5(3)-carboxylic acid
ethyl esters (4a–d)
13C-NMR of compound 3a (DMSO-d6, d ppm):
118.73–147.66 (Ar–H), 158.00 (C5=N), 167.38 (CO–NH),
195.75 (CO–CO–C=N).
1H-NMR of compound 3b (DMSO-d6,
d ppm):
7.30–7.70 (m, 9H, Ar–H), 12.60 (s, 1H, NHpyridazine), 14.21
To a solution of the appropriate ester (2a–d) (15 mmol)
in acetic acid (90 ml) was added hydrazine hydrate (80%)
(1.09 ml, 18 mmol) and the mixture was stirred for 4 h
at room temperature then allowed to stand overnight.
The resulting red precipitate was filtered and recrystallised
to obtain compounds (3a–d). The filtrate was diluted
with water (1:10) to yield an orange precipitate that was
filtered and recrystallised to obtain compounds (4a–d).
The yields %, melting points, molecular formulas, and
analysis of compounds 3a–d and 4a–d are recorded in
Table 3.
(s, 1H, NHhydrazono).
13C-NMR of compound 3b (DMSO-d6, d ppm):
114.57–146.79 (Ar–H), 158.08 (C5=N), 161.11 (C–F),
163.55 (CO–NH), 194.33 (CO–CO–C=N).
1H-NMR of compound 3c (DMSO-d6,
d ppm):
7.37–7.67 (m, 9H, Ar–H), 12.63 (s, 1H, NHpyridazine), 15.44
(s, 1H, NHhydrazono).
13C-NMR of compound 3c (DMSO-d6, d ppm):
118.76–146.56 (Ar–H), 158.05 (C5=N), 167.71 (CO–NH),
188.27 (CO–CO–C=N).
IR of compounds 3a–d (KBr, cm-1): 3178–3159 (NH);
1686–1684 (C=O); 1678–1675 (CO–NH); 1508–1389
(C=N and C=C).
1H-NMR of compound 3d (DMSO-d6,
d ppm):
7.36–7.69 (m, 9H, Ar–H), 12.60 (s, 1H, NHpyridazine), 14.49
(s, 1H, NHhydrazono).
1H-NMR of compound 3a (DMSO-d6,
d
ppm):
13C-NMR of compound 3d (DMSO-d6, d ppm):
118.93–146.84 (Ar–H), 158.57 (C5=N), 163.87 (CO–NH),
192.78 (CO–CO–C=N).
7.36–7.64 (m, 10H, Ar–H), 12.60 (s, 1H, NHpyridazine),
15.39 (s, 1H, NHhydrazono).
123

Med Chem Res (2011) 20:408–420
415
IR of compounds 4a–d (KBr, cm-1): 3277–3169 (NH);
1747–1734 (COO); 1699–1686 (C=N); 1560 (N=N);
1490–1458, 1261–1288 (C=N and C=C).
1H-NMR of compound 4a (DMSO-d6, d ppm): 1.22 (t,
3H, J = 7.13 Hz, CH3), 4.30 (q, 2H, J = 7.13 Hz, CH2),
7.15–7.98 (m, 10H, Ar–H), 14.19 (s, 1H, NH).
13C-NMR of compound 4a (DMSO-d6, d ppm): 14.41
(CH3), 61.34 (CH2), 122.44–152.43 (Ar–C), 163.97
(COO).
1H-NMR of compound 4b (DMSO-d6, d ppm): 1.23 (t,
3H, J = 7.12 Hz, CH3), 4.30 (q, 2H, J = 7.12 Hz, CH2),
7.43–8.04 (m, 9H, Ar–H), 14.18 (s, 1H, NH).
13C-NMR of compound 4b (DMSO-d6, d ppm): 13.90
(CH3), 60.88 (CH2), 116.13–152.00 (Ar–C), 159.69 (CF),
163.44 (COO).
gas. The mixture was reduced with hydrogen at 2 bar
pressure for 4 h with shaking. The reaction mixture was
filtered, the solvent was removed under vacuum, and the
crude product was recrystallised from methanol to afford
the compound 4-amino-3(5)-phenyl-1H-pyrazole-5(3)-car-
boxylic acid ethyl ester (5a), whose melting point and
spectral data coincided with those recorded for the com-
pound prepared as described above.
IR of compounds 5a–d (KBr, cm-1): 3396–3375 (NH);
1708–1693 (COO); 1609–1605, 1508–1450, 1417–1422,
1385–1382, 1299–1296 (C=N and C=C).
1H-NMR of compound 5a (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, CH3), 4.32 (q, 2H, J = 7.12 Hz, CH2),
4.96 (s, 2H, NH2), 7.29–7.73 (m, 5H, Ar–H), 13.17 (s, 1H,
NH).
1H-NMR of compound 4c (DMSO-d6, d ppm): 1.22 (t,
3H, J = 7.12 Hz, CH3), 4.30 (q, 2H, J = 7.12 Hz, CH2),
7.50–8.00 (m, 9H, Ar–H), 14.27 (s, 1H, NH).
13C-NMR of compound 4c (DMSO-d6, d ppm): 13.90
(CH3), 60.91 (CH2), 122.06–151.92 (Ar–C), 162.86
(COO).
1H-NMR of compound 4d (DMSO-d6, d ppm): 1.22 (t,
3H, J = 7.12 Hz, CH3), 4.30 (q, 2H, J = 7.12 Hz, CH2),
7.50–7.93 (m, 9H, Ar–H), 14.27 (s, 1H, NH).
13C-NMR of compound 4d (DMSO-d6, d ppm): 13.89
(CH3), 60.91 (CH2), 122.06–152.29 (Ar–C), 162.21
(COO).
1H-NMR of 5a (CDCl3, d ppm): 1.40 (t, 3H,
J = 7.13 Hz, CH3), 4.40 (q, 2H, J = 7.12 Hz, CH2), 5.81
(s, 3H, NH, NH2), 7.33–7.70 (m, 5H, Ar–H).
13C-NMR of compound 5a (DMSO-d6, d ppm): 14.25
(CH3), 59.73 (CH2), 125.52–155.49 (Ar–C), 182.26 (C=O).
MS of 5a m/z (relative abundance %): 231 (M ) (100%),
203 (5.0), 185 (14.7), 129 (37.4), 104 (35.9).
1H-NMR of compound 5b (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, CH3), 4.32 (q, 2H, J = 7.03 Hz, CH2),
4.94 (s, 2H, NH2), 7.27–7.76 (m, 4H, Ar–H), 13.21 (s, 1H,
NH).
13C-NMR of compound 5b (DMSO-d6, d ppm): 14.31
(CH3), 59.88 (CH2), 115.25–168.11 (Ar–C), 191.25 (C=O).
MS of 5b m/z (relative abundance %): 249, 250 (M ,
M ?1) (100, 6.2%), 24 (4.8), 203 (15.2), 175 (5.0), 147
(46.0), 146 (15.9), 122 (26.5), 121 (8.2).
1H-NMR of compound 5c (DMSO-d6, d ppm): 1.32 (t,
3H, J = 7.12 Hz, CH3), 4.32 (q, 2H, J = 7.03 Hz, CH2),
5.01 (s, 2H, NH2), 7.47–7.78 (m, 4H, Ar–H), 13.30 (s, 1H,
NH).
13C-NMR of compound 5c (DMSO-d6, d ppm): 14.25
(CH3), 59.88 (CH2), 127.36–152.50 (Ar–C), 191.60 (C=O).
MS of 5c m/z (relative abundance %): 265, 267 (M , M
?2) (100, 32.8%), 219 (33.2), 165 (27.5) 163 (72.03), 138
(52).
1H-NMR of compound 5d (DMSO-d6, d ppm): 1.33 (t,
3H, J = 7.12 Hz, CH3), 4.32 (q, 2H, J = 7.04 Hz, CH2),
7.64–7.69 (m, 7H, Ar–H, NH, NH2).
13C-NMR of compound 5d (DMSO-d6, d ppm): 14.24
(CH3), 59.81 (CH2), 119.66–154.24 (Ar–C), 193.68 (C=O).
MS of 5d m/z (relative abundance %): 309, 311 (M -1,
M ?1) (100, 97.6%), 265 (15.65), 263 (14.8), 209 (35.0),
207 (33.0), 148 (25.2), 182 (19.4).
MS of 4d m/z (relative abundance %): 398, 400 (M -1,
M ) (73.5, 82.2%), 323 (100), 321 (94.7), 251 (22.5), 249
(29.4).
4-amino-3(5)-(4-substituted phenyl)-1H-pyrazole-5(3)-
carboxylic acid ethyl esters (5a–d)
To a cold solution of stannous chloride (1.8 g, 8 mmol)
in hydrochloric acid (10 ml) was added dropwise a sus-
pension of the appropriate phenylazopyrazole (4a–d) (2
mmol) in ethanol (15 ml) over a period of 15 min. The
reaction mixture was kept in ice chest for an overnight. The
solvent was then removed under high vacuum at ambient
temperature. The crude residue left was mixed with
anhydrous sodium carbonate (2.12 g, 20 mmol), trans-
ferred to a continuous extraction apparatus and extracted
with ethanol. The ethanolic extract of the amine was con-
centrated under vacuum, and the resulting crude amine was
recrystallised from the appropriate solvent. The yields %,
melting points, molecular formulas, and analysis are
recorded in Table 4.
Catalytic hydrogenation of 3(5)-(4-chlorophenyl)-4-
phenylazo-1H-pyrazole-5(3)-carboxylic acid ethyl esters
(4c)
3-(4-Substituted phenyl)-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-ones (6a–d)
In a hydrogenation vessel, 4-chlorophenylazopyrazole
derivative (4c) (1.77 g, 5 mmole) was introduced followed
by methanol (100 ml) and Pd/C (100 mg). The vessel was
flushed twice with nitrogen gas then twice with hydrogen
To a solution of formamidine acetate (0.21 g, 2 mmol)
in n-butanol (10 ml) was added the appropriate pyrazole
amine (5a–d) (1 mmol). The reaction mixture was refluxed
123

416
Med Chem Res (2011) 20:408–420
Table 4 4-Amino-3(5)-(4-substituted phenyl)-1H-pyrazole-5(3)-carboxylic acid ethyl esters (5a–d) and 3-(4-Substituted phenyl)-1,6-dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-ones (6a–d)
Cpd. no.
X
Yield %
M.P. °Ca (Cryst. solvent)
Mol. formula (Mol. wt.)
Analysis %
Calcd.
Found
5a
H
64.3b
(MeOH)
C12H13N3O2 (231.25)
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
62.33
5.67
62.57
5.43
18.17
57.83
4.85
17.96
57.45
4.59
5b
5c
5d
6a
6b
6c
6d
a
F
70.6
76.8
80.2
60.5
62.4
77.2
70.7
(MeOH)
(EtOH)
C
C
C
C
C
C
C
12H12FN3O (249.24)
12H12ClN3O2 (265.70)
12H12BrN3O2 (310.15)
11H8N4O (212.21)
16.86
54.25
4.55
16.83
54.50
4.90
Cl
Br
H
15.81
46.47
3.90
15.60
46.69
4.38
(MeOH)
(MeOH)
(MeOH)
(MeOH)
(EtOH)
13.55
62.33
5.67
13.28
62.57
5.43
18.17
57.83
4.85
17.96
57.45
4.59
F
11H7FN4O (230.20)
11H7ClN4O (246.66)
11H7BrN4O (291.11)
16.86
54.25
4.55
16.83
54.50
4.90
Cl
Br
15.81
46.47
3.90
15.60
46.69
4.38
13.55
13.28
All melting points were above 350°C
Yield from catalytic hydrogenation 74.7%
b
for 8 h, left to cool, and the obtained product was filtered
and recrystallised from the appropriate solvent. The yields
%, melting points, molecular formulas, and analysis are
recorded in Table 4.
MS of 6b m/z (relative abundance %): 230, 231 (M ,
M ?1) (100, 6.8%), 202 (7.1), 174 (5.4), 121 (20.0).
1H-NMR of compound 6c (DMSO-d6, d ppm): 7.56 (d,
2H, J = 8.65 Hz, Ar–H), 8.01 (d, 1H, J = 3.31 Hz,
CHpyrimidine), 8.84 (d, 2H, J = 8.64 Hz, Ar–H), 12.42 (s,
1H, NHpyrazole), 14.34 (s, 1H, NHpyrimidine).
IR of compounds 6a–d (KBr, cm-1): 3219–3159,
2982–2902 (NH); 1694–1685 (C=O); 1686–1671 (C=N);
1601–1580, 1458–1386 (C=C and Aromatics).
13C-NMR of compound 6c (DMSO-d6, d ppm):
114.45–152.93 (Ar–C), 163.60 (C=O).
1H-NMR of compound 6a (DMSO-d6,
d ppm):
7.36–8.32 (m, 6H, Ar–H and CHpyrimidine), 12.41 (d, 1H,
MS of 6c m/z (relative abundance %): 246, 248 (M ,
M ?2) (100, 32.5%), 218 (1.6), 191 (1.4), 138 (12.2), 137
(7.8), 102 (4.8).
1H-NMR of compound 6d (DMSO-d6, d ppm): 7.70 (d,
2H, J = 8.65 Hz, Ar–H), 8.02 (d, 1H, J = 3.31 Hz,
CHpyrimidine), 8.28 (d, 2H, J = 8.64 Hz, Ar–H), 12.45 (s,
1H, NHpyrazole), 14.37 (s, 1H, NHpyrimidine).
NHpyrazole), 14.27 (s, 1H, NHpyrimidine).
13C-NMR of compound 6a (DMSO-d6, d ppm):
126.02–156.22 (Ar–C), 162.55 (C=O).
MS of 6a m/z (relative abundance %): 212, 213 (M , M
?1) (100, 18.4%), 184 (5.0), 157 (4.2), 104 (16.8).
1H-NMR of compound 6b (DMSO-d6, d ppm): 7.31-
8.37 (m, 5H, Ar–H and CHpyrimidine), 12.41 (s, 1H,
NHpyrazole), 14.27 (s, 1H, NHpyrimidine).
13C-NMR of compound 6d (DMSO-d6, d ppm):
121.10–152.97 (Ar–C), 158.91 (C=O).
13C-NMR of compound 6b (DMSO-d6, d ppm):
115.64–160.63 (Ar–C), 163.07 (C=O).
MS of 6d m/z (relative abundance %): 290, 291 (M -1,
M ) (100, 98%).
123

Med Chem Res (2011) 20:408–420
417
Scheme 2
2-(2-ethoxyphenyl)-3H-pyrido[2,3-d]pyrimidin-4-one
(10)
2-Aminonicotinamide (8)
A stirred mixture of 2-(2-ethoxybenzamido)pyridine-3-
carboxamide (9) (0.74 g, 2.6 mmol), pyridine (0.8 ml) and
2 N sodium hydroxide (20 ml) was heated under reflux for
30 min then cooled. The reaction mixture was neutralised
with 2 N hydrochloric acid to precipitate the crude product
which was recrystallised from methanol/ether (Table 5).
IR (KBr, cm-1): 3299 (NH), 1686 (C=O), 1586, 1560,
1458 (C=N and aromatics).
1H-NMR (DMSO-d6, d ppm): 1.35 (t, 3H, J = 6.99 Hz,
CH3), 4.17 (q, 2H, J = 6.95 Hz, CH2), 7.10–8.98 (m, 7H,
Ar–H), 12.34 (s, 1H, NH).
13C-NMR (DMSO-d6, d ppm): 14.36 (CH3), 64.04
(CH2), 112.69–156.64 (Ar–C), 169.80 (C=O).
To a stirred suspension of 2-aminonicotinic acid (2.76 g,
20 mmol) in dry THF (50 ml) was added portion wise 1,10-
carbonyldiimidazole (CDI) (3.65 g, 22 mmol). The reac-
tion mixture was kept on stirring for 12 h until complete
dissolution, which was indicated by the disappearance of
the acid carbonyl band at 1717 cm-1 in IR monitoring and
the appearance of the imidazocarbonyl at 1680 cm-1. The
reaction mixture was injected with ammonia gas (5 g) and
left under stirring for 8–10 h until the complete disap-
pearance of the 1680 cm-1 peak and the appearance of a
new peak at 1665 cm-1 in IR. The solvent was removed
under reduced pressure, and the residue was treated with
cold water. The crude product was filtered and recrystal-
lised from methanol. The yield was 2.13 g (77.7%).2 m.p.
198–99°C.
MS (relative abundance %): 267, 268 (M , M ?1)
(23.4, 2.9%), 253 (14.2), 252 (100), 223 (14.6), 120 (77.8),
93 (20.1).
1H-NMR (DMSO-d6, d ppm): 6.56 (dd, 1H, J = 7.63,
4.83 Hz, C5–H), 7.18 (s, 2H, NH2), 7.31 (s, 1H, C4–H),
7.93 (dd, 2H, J = 7.88, 1.78 Hz, CO–NH2), 8.07 (dd, 1H,
J = 4.58, 1.53 Hz, C6–H).
2-(4-chlorosulfonyl-2-ethoxyphenyl)-3H-pyrido[2,3-
d]pyrimidin-4-one (11)
In a 100-ml three-neck flask, 2-(2-ethoxyphenyl)-3H-
pyrido[2,3-d]pyrimidin-4-one (10) (5.3 g, 20 mmol) was
added portion wise to a stirred ice cooled mixture of
thionyl chloride (2.38 g, 20 mmol) and chlorosulfonic acid
(9.51 g, 82 mmol) keeping the temperature below 25°C.
The mixture was stirred at room temperature for 18 h,
poured on ice water while stirring, where a white deposit
appeared. Stirring was continued for another hour and the
product was filtered, washed with water, and dried in
vacuum. The crude product was used directly in the next
step.
MS (relative abundance %): 137, 138 (M , M ?1)
(100, 6.0%), 121 (15.4), 120 (22.1), 93 (51.2), 67 (19.3).
2-(2-ethoxybenzamido)pyridine-3-carboxamide (9)
A mixture of 2-ethoxybenzoic acid (0.88 g, 5.3 mmol)
and thionyl chloride (3.15 g, 26.5 mmol) was refluxed for
1 h. Excess thionyl chloride was removed under reduced
pressure by the aid of dry benzene (3 9 10 ml). The
formed acid chloride (7) was dissolved in acetonitrile
(7.5 ml) and added dropwise over 5 min to a 0°C cooled
stirred solution of 2-aminonicotinamide (8) (0.69 g,
5 mmol) and triethylamine (0.51 g, 5 mmol) in acetonitrile
(7.5 ml). The reaction mixture was stirred at ambient
temperature for further 1.5 h, allowed to stand over night,
and then evaporated to dryness under reduced pressure.
The residue was washed with water to afford a solid
product which was recrystallised from methanol (Table 5).
IR (KBr, cm-1): 3376–3206 (NH), 1676 (CO–NH),
1648 (CO–NH2), 1623, 1599, 1446, 1313 (C=N and
aromatics).
2-(2-ethoxyphenyl-5-sulfonamido)-3H-pyrido[2,3-
d]pyrimidin-4-ones (13a–e)
To a solution of the appropriate amine (12a–e) (1 mmol)
and triethylamine (0.138 g, 1 mmol) in methanol (7 ml),
2-(4-chlorosulfonyl-2-ethoxyphenyl)-3H-pyrido[2,3-d]pyr-
imidin-4-one (11) (0.384 g, 1.05 mmol) was added to the
solution. The mixture was stirred at room temperature for
2 h. The resulting solid was filtered, washed with ethyl
acetate (2 9 5 ml) to obtain the crude solid product, which
was recrystallised from the appropriate solvent. The yields
%, melting points, molecular formulas, and analysis are
recorded in Table 5.
IR of compound (13a–e) (KBr, cm-1): 3373–3302,
2938–2810 (NH (13a–e) and OH 13e), 1709–1688 (C=O),
1585–1579, 1561–1559, 1458–1420 (C=N, C=C and aro-
matics), 1349–1334, 1169–1149 (SO2–N).
1H-NMR (DMSO-d6, d ppm): 1.40 (t, 3H, J = 6.99 Hz,
CH3), 4.24 (q, 2H, J = 6.95 Hz, CH2), 7.05–8.48 (m, 9H,
Ar–H and NH2), 11.55 (s, 1H, NH).
13C-NMR (DMSO-d6, d ppm): 14.11 (CH3), 64.19
(CH2), 112.88–156.09 (Ar–C), 162.76 (CO–NH2), 168.52
(CO–NH).
1H-NMR of compound 13a (DMSO-d6, d ppm):
1.35–1.38 (m, 5H, CH2 C4-piperidino and O–CH2–CH3),
1.50–1.64 (m, 4H, CH2 C3-piperidino), 2.92 (t, 4H,
J = 5.17 Hz, CH2 C2-piperidino), 4.26 (q, 2H,
J = 6.94 Hz, O–CH2–CH3), 7.41–9.00 (m, 6H, Ar–H),
12.51 (s, 1H, NH).
MS (relative abundance %): 285 (M ) (25%), 252
(40.2), 240 (100), 223 (9.8), 149 (40.3), 121 (61.3), 120
(26.9).
2
Reported yields from different methods 66, 55, 45%, respectively
(Taylor and Crovetti, 1954; Althuis et al., 1979; Purcell et al., 1982).
123

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Med Chem Res (2011) 20:408–420
Table 5 2-(2-Ethoxybenzamido)pyridine-3-carboxamide (9), 2-(2-Ethoxyphenyl)-3H-pyrido[2,3-d] pyrimidin-4-one (10) and 2-(2-Ethox-
yphenyl-5-sulfonamido)-3H-pyrido[2,3-d]pyrimidin-4-ones (13a–e)
Cpd. no.
R
Yield %
M.P.°C (Cryst. solvent)
Mol. formula (Mol. wt.)
Analysis %
Calcd.
Found
9
–
96.80
180–81 (MeOH)
C15H15N3O3 (285.30)
C
H
N
C
H
N
C
H
N
S
63.15
5.30
63.10
5.76
14.73
67.41
4.90
14.65
67.34
5.04
10
–
36.00
79.80
176–77 (MeOH/Et2O)
238–39 (DMF/(H2O)
C15H13N3O2 (276.29)
15.72
53.52
5.82
15.69
53.33
5.71
13a
Piperidino
C20H22N4O4S.2H2O (450.51)
12.44
7.12
12.22
7.30
13b
13c
13d
13e
Morpholino
80.60
89.10
82.30
70.20
251–52 (DMF/(H2O)
C19H20N4O5S (416.45)
C
H
N
S
54.80
4.84
54.67
4.88
13.45
7.70
13.11
7.64
4-Phenylpiperazino
4-Ethylpiperazino
Diethanolamino
243–44 (decomp.) (DMF/MeOH)
228–29(decomp.) (DMF)
236–37 (MeOH)
C
C
C
25H25N5O4S (491.56)
21H25N5O4S (443.52)
19H22N4O6S (434.47)
C
H
N
S
61.09
5.13
61.06
5.25
14.25
6.52
14.23
6.34
C
H
N
S
56.87
5.68
56.49
5.82
15.79
7.23
15.38
7.06
C
H
N
S
52.53
5.10
52.41
5.28
12.90
7.38
12.72
7.21
13C-NMR of compound 13a (DMSO-d6, d ppm): 14.62
(O–CH2–CH3), 23.27 (C4-piperidino), 25.01 (C3-piperidi-
no), 46.99 (C2-piperidino), 65.39 (O–CH2–CH3),
116.85–160.20 (Ar–C), 162.16 (C=O).
13C-NMR of compound 13c (DMSO-d6, d ppm): 14.59
(O–CH2–CH3), 46.23 (C2-piperazino), 48.29 (C3-piperazi-
no), 65.43 (O–CH2–CH3), 116.25-160.10 (Ar–C), 162.09
(C=O).
MS of compound 13a (relative abundance %): 414 (M )
(3.0%), 399 (2.7), 268 (16.6), 267 (100), 223 (7.2).
1H-NMR of compound 13b (DMSO-d6, d ppm): 1.36 (t,
3H, (J = 6.99 Hz, O–CH2–CH3), 2.90 (t, 4H, J = 4.45 Hz,
CH2 C2-morpholino), 3.65 (t, 4H, J = 4.58 Hz, CH2 C3-
morpholino), 4.26 (q, 2H, J = 6.87 Hz, O–CH2–CH3),
7.44–9.00 (m, 6H, Ar–H), 12.56 (s, 1H, NH).
13C-NMR of compound 13b (DMSO-d6, d ppm): 14.13
(CH3), 45.82 (C2-morpholino), 64.97 (CH2–CH3), 65.14
(C3-morpholino), 116.39–160.00 (Ar–C), 161.72 (C=O).
1H-NMR of compound 13c (DMSO-d6, d ppm): 1.35 (t,
3H, J = 6.99 Hz, O–CH2–CH3), 3.05 (t, 4H, J = 4.71 Hz,
CH2 C2-piperazino), 3.23 (t, 4H, J = 4.71 Hz, CH2 C3-
piperazino), 4.26 (q, 2H, J = 6.87 Hz, O–CH2–CH3),
6.78–9.00 (m, 11H, Ar–H), 12.54 (s, 1H, NH).
MS of compound 13c (relative abundance %): 387
(5.4%), 359 (26.0), 267 (18.8), 161(100), 160 (53.3), 120
(21.8).
1H-NMR of compound 13d (DMSO-d6, d ppm): 0.93 (t,
3H, J = 7.25 Hz, CH3-ethylpiperazino), 1.36 (t, 3H,
J = 6.99 Hz, O–CH2–CH3), 2.30 (q, 2H, J = 7.03 Hz,
CH2-ethylpiperazino), 2.43 (m, 4H, CH2 C2-piperazino),
2.90 (m, 4H, CH2 C3-piperazino), 4.26 (q, 2H,
J = 7.04 Hz, O–CH2–CH3), 7.42–9.00 (m, 6H, Ar–H),
12.54 (s, 1H, NH).
13C-NMR of compound 13d (DMSO-d6, d ppm): 12.66
(CH3-ethylpiperazino), 14.71 (O–CH2–CH3), 46.35 (C2-
piperazino), 51.40 (CH2-ethylpiperazino), 51.54 (C3-pipe-
razino), 65.41 (O–CH2–CH3), 113.79–160.33 (Ar–C),
165.91 (C=O).
123

Med Chem Res (2011) 20:408–420
419
MS of compound 13d (relative abundance %): 443
(22%), 359 (47.5), 267 (15.3), 114 (11.3), 113 (100).
1H-NMR of compound 13e (DMSO-d6, d ppm): 1.36 (t,
3H, J = 6.99 Hz, O–CH2–CH3), 3.18 (t, 4H, J = 6.36 Hz,
N–CH2–CH2–OH), 3.55 (q, 4H, J = 5.76 Hz, N–CH2–
CH2–OH), 4.24 (q, 2H, J = 6.95 Hz, O–CH2–CH3), 4.86
(t, 2H, J = 5.21 Hz, OH), 7.38–9.00 (m, 6H, Ar–H), 12.52
(s, 1H, NH).
study it has been revealed that the pyridazine derivative:
6-phenyl-5-phenylhydrazono-2,3,4,5-tetrahydropyridazine-
3,4-dione (3a) showed activity against S. aureus as Gram-
positive bacteria while compound 2-(2-ethoxyphenyl-5-
phenylpiperazinosulfonamido)-3H-pyrido[2,3-d]pyrimidin-4-
one (13c) was showing moderate antimycotic activity against
C. albicans.
13C-NMR of compound 13e (DMSO-d6, d ppm): 14.63
(O–CH2–CH3), 51.57 (N–CH2–CH2–OH), 60.32 (N–CH2–
CH2–OH), 65.34 (O–CH2–CH3), 113.83–159.96 (Ar–C),
162.18 (C=O).
Inhibition of CDk1/cyclin B, DYRK1A, CDK5/p25,
and GSK-3a/b kinases
All assays were run in the presence of 15 lM [c-33P]-ATP
and appropriate protein substrates (histone H1 for CDKs,
GS-l peptide for GSK-3). Kinases activities were assayed
in the appropriate buffer at 30°C. Blank values were sub-
tracted and activities were calculated as molar amounts of
phosphate incorporated in the protein substrate after
10 min incubation. The activities were expressed in per-
centage of the maximal activity, i.e., in the absence of
inhibitors. Controls were performed with appropriate
dilutions of dimethylsulfoxide, and IC50 (50% inhibitory
concentration) values were determined from dose–response
curves. A compound is considered inhibitor if its IC50 is
below 10 lM. All tested compounds were considered
inactive since they displayed IC50 values above 10 lM.
Biological evaluation
In vitro antimicrobial activity
The tested compounds were evaluated using agar cup dif-
fusion technique (Jain and Kar, 1971) using a 2 mg ml-1
solution in DMF. The test organisms were S. aureus
(ATCC 6538) as a representative of Gram-positive bacte-
ria, both Escherichia coli (NCTC 10418) and P. aerugin-
osa (ATCC 9027) as representatives of Gram-negative
bacteria. The compounds were also evaluated for their in
vitro antifungal activity against C. albicans (ATCC
10231). A control using DMF without the test compounds
was included for each organism. Compounds which
showed growth inhibition zone diameters \18 mm were
considered to have nonsignificant activity, while com-
pounds showing inhibition zones C18 mm were considered
to be active and were further evaluated for their minimum
inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) values, using the two-fold serial
dilution technique (Scott 1989). Ampicillin was used as the
standard antibacterial agent while Clotrimazole was used
as the antifungal reference.
Acknowledgments Sincere gratefulness is granted to the ‘‘Deut-
scher Akademischer Austausch Dienst’’ (DAAD, German Academic
Exchange Service) that offered the financial support to fulfill this
study in The Pharmaceutical Chemistry Department, Institute of
Pharmacy, Hamburg University, Hamburg. Profound gratitude to
‘‘Centre de la Recherche Scientifique (CNRS), Roscoff Cedex,
France,’’ for conducting Cyclin-dependent kinases (CDKs) Inhibition
assays. Extendable thanks are due to the Department of Microbiology,
Faculty of Pharmacy, University of Alexandria for conducting the
antimicrobial screening of the selected synthesised compounds.
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