A novel synthesis of fused uracils: Indenopyrimidopyridazines, pyrimidopyridazines, and pyrazolopyrimidines for antimicrobial and antitumor evalution

Article abstract of DOI:10.3390/molecules21121714

A variety of different compounds of fused uracils were prepared simply by the heating of 6-hydrazinyl-1-methyl-, 6-hydrazinyl-1-propyl-, or 6-hydrazinyl-1,3-dipropyluracil under reflux with ninhydrin, isatin, benzylidene malononitrile, benzylylidene ethyl cyanoacetate, benzil, and phenacyl bromide derivatives. The newly synthesized compounds were completely screened for antimicrobial and antitumor activity.

Full text of DOI:10.3390/molecules21121714

molecules
Article
A Novel Synthesis of Fused Uracils:
Indenopyrimidopyridazines, Pyrimidopyridazines,
and Pyrazolopyrimidines for Antimicrobial and
Antitumor Evalution
Samar El-Kalyoubi 1,2,* and Fatimah Agili 3
1
Department of Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City 11651,
Cairo, Egypt
2
Medical Chemistry Department, Faculty of Medicine (Female Section), Jazan University, Jazan 45142,
Saudi Arabia
3
Chemistry Department, Faculty of Science (Female Section), Jazan University, Jazan 82621, Saudi Arabia;
dr.fatmah2000@gmail.com
*
Correspondence: s.elkalyoubi@hotmail.com; Tel.: +20-109-535-9392; Fax: +966-173-217-540
Academic Editor: Artur Silva
Received: 17 October 2016; Accepted: 8 December 2016; Published: 14 December 2016
Abstract: A variety of different compounds of fused uracils were prepared simply by the heating of
6
-hydrazinyl-1-methyl-, 6-hydrazinyl-1-propyl-, or 6-hydrazinyl-1,3-dipropyluracil under reflux with
ninhydrin, isatin, benzylidene malononitrile, benzylylidene ethyl cyanoacetate, benzil, and phenacyl
bromide derivatives. The newly synthesized compounds were completely screened for antimicrobial
and antitumor activity.
Keywords: 6-chlorouracil; 6-hydrazinyluracils; ninhydrin; isatin indenopyrimidopyridazines;
pyrrolopyrimidines; pyrimidopyridazines
1
. Introduction
For the last several decades, fused pyrimidine derivatives have become a significant attraction in
the field of medicinal chemistry research. This is attributed to the fact that pyrimidine is the basic unit of
DNA and RNA structure. This fact explains the wide range of pharmacological activities of pyrimidine
derivatives. Pyrazolo[3,4-d]pyrimidine derivatives are a class of fused pyrimidines possessing
significant biological activities [
with antimicrobial [ ], antiviral [
and xanthine oxidase inhibitor activities [13,14].
Furthermore, pyrimidopyridazine derivatives have a significant interest owing to the fact that
they have a potent pharmacological effect as therapeutic agents [15 17]. They have monoamine oxidase
MAO) inhibitory effect and subsequent modification on the diazine ring results in different inhibitory
1,
2]. They act as purine analogs [3] and many of their derivatives act
2,
4,
5
1
,6
], antimetabolites [ ], anticancer [
7
8
,
9
], anti-inflammatory [10 12],
–
–
(
activities [18]. MAO inhibitory drugs play an important role in clinical management of depression,
as well as Alzheimer’s disease [19].
It has been established that cancer is spread worldwide and responsible for about 15% of
all deaths [20]. Many drugs with anticancer and antiviral activities have been developed [21],
such as zidovudine (AZT) [22], zalcitabine (DDC) [23], brivudine (BVDU) [24], and methotrexate
(
MTX) [25]. 4-Deazatoxaflavin (1,6-dimethyl-1,5,6,7-tetrahydropyrimido[4,5-c]pyridazine-5,7-dione)
binds to herring sperm DNA and inhibits growth of Pseudomonas 568 [21,26].
On account of these facts, a new series of substituted pyrimidopyridazines and pyrazolopyrimidines
have been synthesized starting from 6-hydrazinyluracil derivatives and their antimicrobial, as well as
antitumor activity has been evaluated and reported.
Molecules 2016, 21, 1714; doi:10.3390/molecules21121714
www.mdpi.com/journal/molecules

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2
. Results and Discussion
2
.1. Chemistry
Extending our work in the synthesis of non-nucleosidic compounds of fused uracils [27–29],
we tried to synthesize pyrimidopyridazines and pyrazolopyrimidines from 6-hydrazinyluracils.
The regioselective alkylation of 6-chlorouracil 1 [30] with methyl—and/or propyl iodide in dimethyl
sulfoxide (DMSO) in the presence of K CO as basic medium afforded about 60%–70% yield of
2
3
alkylated uracils 2a
hydrate afforded 6-hydrazinyluracils 3a
–10 min in the presence of AcOH resulted the desired compounds 4a–c
–
c
[
31
–
34]. The nucleophilic substitution of 6-chlorouracil 2a
34 35]. Heating of 3a under reflux with ninhydrin for
in good yield, as shown in
–c using hydrazine
–
c
[
,
–c
5
Scheme 1. The structure of which was confirmed on the basis of analytical and spectral data. Thus,
1
the H-NMR (DMSO-d ) spectrum of compounds 4a
,
b
showed a singlet around
4.36 ppm characteristic for
NCH of propyl group. Additionally, a characteristic signal of the phenyl group for compounds 4a
δ 12.24–12.23 ppm
6
exchangeable characteristic for NH, while in 4c a triplet splitting signal at
δ
-
–c
2
appears around
compounds 4a
for carbon atoms. While, refluxing of 3b
shown in Scheme 1. Compounds 5a
singlets at
for CH(5) of compound 5a and two singlets at
singlet at 5.74, characterized for CH(5) of compound 5b
δ
9.26–7.75 ppm and the characteristic signal disappeared at 5–6 ppm of CH (5) in
–
c
. 13C-NMR showed 14 signals for compound 4a and 16 signals for 4b characteristic
,
c
with isatin for 1–2 h in AcOH gave the open form 5a
,
b
as
1
,
b
were proved by the H-NMR spectrum, which showed three
δ
13.01, 11.35, 10.99 ppm characterized for three NH groups, a singlet at
δ 5.61 characterized
δ
13.03, 11.37 ppm characteristic for two NH groups, a
13
δ
.
C-NMR showed 15 signals for compound
5
a and 18 signals for 5b characteristic for carbon atoms.
O
O
R
HN
CH3I OR
CH3CH2CH2I
N
DMSO
O
N
R
Cl
O
N
H
Cl
1
1
1
2a. R = H, R = CH3-
1
2
b. R = H, R = propyl
1
2
c. R = R = propyl
NH NH .H O
2
2
2
O
R
N
O
N
R
NH NH2
1
ninhydrin
3a- c
isatin
O
O
R
R
O
N
NH
O
N
N
O
N
R
NH N
O
N
R
N
1
1
4a-c
1
1
a. R = H; R = CH3
5a. R = H, R = Propyl
1
b. R = H; R = Propyl
1
c. R = R = Propyl
1
5b. R = R = Propyl
Scheme 1. The reaction of 6-hydrazinyluracils with ninhydrin and isatin.
The mechanism formation of 4a–c is shown in Scheme 2.

Molecules 2016, 21, 1714
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O
O
O
R
O
R
O
O
N
HO
N
-
2H O
2
O
H
N
R
NH N
HO
N
R
N
H
N
+
1
H
1
O
-
H
+
+
H
O
HO
H
O
R
O
R
O
N
-
H O
2
N
N
N
O
N
N
O
N
R
N
1
1
R
Scheme 2. The mechanism formation of 4a–c.
On the other hand, the reaction of 3b,c with even benzylidene malononitrile or benzylidene
ethyl cyanoacetate derivative via Michael addition reaction by heating under reflux for 6–8 h in
dimethylformamide (DMF) in the presence of triethylamine as basic medium furnished the same
products of pyrazolopyrimidines 6a–f, as shown in Scheme 3 by the elimination of malononitrile
and ethyl cyanoacetate moieties, respectively, as shown in Scheme 4. Compounds 6a
confirmed on the basis of analytical and spectral data. The H-NMR spectrum showed a characteristic
–
f
were
1
singlet at
δ
10.95, 11.19 ppm for NH(5) of compounds 6a and 6b, respectively, a singlet around
7.73–6.72 ppm
C-NMR for compounds 6b and 6e showed 12 and 17 signals characteristic for
δ
7.95–7.50 ppm for NH(1) and characteristic signals for the phenyl group around
δ
13
for compounds 6a–f.
carbon atoms respectively.
O
R
O
N
2
R
N
R
NH NH2
1
CN
O
2
R
1
3
3
3
a. R = H, R = CH
-
Br
3
1
b. R = H, R = propyl
EtOOC
1
c. R = R = propyl
OR
2
NC
R
Benzil
2
R
NC
2
R
O
O
R
O
N
R
O
N
R
O
N
N
N
O
N
R
N
N
H
N
R
1
N
N
1
N
R
1
6
a - f
8 a-c
1
1
2
1
2
R = H; R = Propyl; R = H, 4-Cl
7a. R = H, R = propyl
R = H; R = Propyl; R = H, 4 - OCH3, 4 -NO2
1
1
2
R = R = Propyl; R = H, 4-Br, 2-OH, 4-Cl
7b. R = R = propyl
Scheme 3. The reaction of 6-hydrazinyluracil with benzylidene malononitrile, benzylidene ethyl
cyanoacetate, benzil, and phenacyl bromides.

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CN
CN
NC
O
O
NC
R
N
R
O
H
H
+
N
2
- H
2
R
+
R
+
H
O
N
R
NH NH2
N
R
N
H
NH
H
1
1
-
H2
2
CN
R
NC
O
O
2
H
R
R
H
R
O
N
N
-
CH2 (CN)2
N
N
H
O
N
R
N
N
N
1
1
R
H
Scheme 4. The expected mechanism for the reaction of 6-hydrazinyluracil with malononitrile.
Heating of 3b
presence of triethylamine furnished 7a
hand, compound 7a was also obtained via heating of 3b with
,
c
under reflux conditions for 4–5 h with benzil in dimethylformamide in the
in moderate yield, as shown in (Scheme 3). On the other
-phenyl phenacyl bromide under
were confirmed on the basis of analytical and spectral
11.72 characteristic for NH(6) and signals of phenyl
,b around δ 7.26–7.10 ppm. C-NMR showed 16 signals characteristic for carbon atoms
,b
α
reflux conditions for 5 h. Compounds 7a
,
b
1
data. H-NMR spectra of 7a showed a singlet at
δ
13
groups for 7a
of compound 7a.
Finally, reaction of 3b with different phenacyl bromides such as phenacyl-, p-methoxyphenacyl-,
and p-nitrophenacyl bromide by heating under reflux for 4–6 h in dimethylformamide in the presence
of triethylamine afforded pyrimidopyridazines 8a
were identified on the basis of analytical and spectral data. The H-NMR spectra showed a singlet
–
c
in moderate yields (Scheme 3). Compounds 8a
–
c
1
around
around
δ
12.08–11.96 ppm characteristic for NH(6), a characteristic singlet aromatic proton at CH(4)
8.69–8.43 ppm of pyridazine ring, and signals of phenyl groups around 8.69–7.00 ppm.
δ
δ
13
C-NMR showed 14 signals for compound 8b characteristic for carbon atoms.
The expected mechanism for the reaction of 6-hydrazinyl uracil with benzylidene malononitrile
and/or benzylidene ethyl cyanoacetate (Scheme 4).
2
.2. Antimicrobial Screening
As shown in Table 1, the newly-synthesized compounds tested displayed variable in vitro
antibacterial and antifungal activities. From the screening results, it can be seen that compound 4b
showed the highest activity against Gram-positive bacteria Bacillus subtilis compared with the standard
drug, followed by compounds 4c
showed the highest activity against Gram-positive bacteria Streptococcus pneumonia in comparison to the
standard drug, followed by compounds 5a 4c 6b 6d 6c 4a, and 5b, respectively. On the other hand,
compound 4b showed the highest activity against Gram-negative bacteria Escherichia coli compared
with the standard drug, followed by compounds 4c 6b 5a 6d 4a, and 6c, respectively. However, the
order of activity against Pseudomona aeruginosa was 4b, followed by compounds 5a 4c 6b 6d 6c 4a
and 5b, respectively. Regarding the activity of the tested compounds against the tested filamentous
fungus Aspergillus fumigatus, the order of activity being 4b 4c 6b 5a 6d 4a 6c 6f, respectively.
, 5a, 6b, 6d, 4a, 5b, 6c and 6f, respectively. Similarly, compound 4b
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
Compound 6f showed a weak antimicrobial effect on Gram-positive bacteria Bacillus subtilis as well
as the tested filamentous fungus Aspergillus fumigatus. No antimicrobial activities were detected for
compounds 7a and 7b. None of the tested compounds exert any activity against the pathogenic yeast
species (Candida albicans) under these screening conditions.

Molecules 2016, 21, 1714
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Table 1. In vitro antimicrobial activity of the tested compounds by well diffusion agar assay expressed
as inhibition zone diameter (mm) in the form of mean ± SD *.
Gram-Positive Bacteria
Gram-Negative Bacteria
Fungi
Aspergillus
Tested Compounds
Bacillus
subtilis
Streptococcus
pneumoniae
Escherichia
coli
Pseudomonas
aeruginosa
Candida
albicans
fumigatus
4
a
20.6 ± 0.63
25.3 ± 1.2
23.6 ± 0.63
21.3 ± 0.72
18.1 ± 0.63
22.3 ± 1.5
17.3 ± 0.63
20.9 ± 1.5
15.2 ± 0.63
NA
18.3 ± 0.72
22.6 ± 0.72
21.1 ± 1.5
22.4 ± 0.63
17.3 ± 0.63
20.1 ± 0.58
19.2 ± 0.72
19.2 ± 1.2
NA
20.6 ± 1.2
28.3 ± 0.72
23.4 ± 1.2
21.3 ± 0.37
NA
22.4 ± 0.58
16.3 ± 0.46
21.3 ± 0.37
NA
15.2 ± 0.58
24.2 ± 0.58
19.2 ± 1.5
20.1 ± 0.63
17.3 ± 0.63
18.6 ± 1.2
17.3 ± 0.63
17.3 ± 0.63
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
18.3 ± 1.5
23.4 ± 1.5
21.5 ± 1.2
19.3 ± 0.63
NA
21.3 ± 1.2
17.3 ± 0.63
18.9 ± 1.2
13.6 ± 0.63
NA
4b
4
5
c
a
5
6
b
b
6
c
6d
6
7
f
a
NA
NA
NA
NA
NA
NA
7b
NA
NA
Tetracycline
Amphotericin B
28.7 ± 0.5
26.4 ± 0.7
30.2 ± 0.6
27.4 ± 0.8
-
-
-
-
25.4 ± 0.63
23.7 ± 0.72
*
NA: No activity under the screening conditions; -: Not tested.
The minimum inhibitory concentration of the six most active synthesized compounds were
detected, as shown in Table 2. It was shown that 4b showed the highest potential where its minimum
inhibitory concentration (MIC) was comparable with that of the standard compounds, whereas 4a
showed the lowest potential and a very high MICs in comparison to the standard.
Table 2. The MIC of the synthesized compounds.
4
a
4b
4c
5a
6d
Sample Tested Microorganisms
Standard
Minimum Inhibitory Concentration (µg/mL)
Fungi
Amphotericin B
Aspergillus fumigatus (RCMB 02568)
Candida albicans (RCMB 05036)
7.81
NA
1.95
NA
3.9
NA
3.9
NA
3.9
NA
1.95
0.98
Gram Positive Bacteria:
Streptococcuspneumonia (RCMB 010010)
Bacillis subtilis (RCMB 010067)
Ampicillin
1.95
7.81
3.9
1.95
0.98
3.9
0.98
3.9
1.95
3.9
3.9
0.49
Gram negative Bacteria:
Pseudomonas aeruginosa (RCMB 010043-5)
Escherichia coli (RCMB 010052-6)
Gentamicin
0.98
62.5
3.9
0.98
0.49
3.9
1.95
3.9
1.95
15.63
3.9
0.49
*
NA: No activity.
Anticancer Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in
comparison with the well-known anticancer standard drug 5-flourouracil under the same conditions
using colorimetric viability assay. Data generated were used to plot a dose response curve of which
the concentration of test compounds required to kill 50% of cell population (IC ) was determined.
50
The results revealed that all the tested compounds showed inhibitory activity to the tumor cell
lines in a concentration dependent manner. Cytotoxic activity was expressed as the mean IC50 of
three independent experiments. The results are represented in Table 3 and Figure 1a,b showed that
compound 4a was the most active against the breast carcinoma cell line (MCF-7), compared with the
reference drug with IC50 values of 3.6 and 4.1 µg/mL, respectively. Interestingly, compounds 4a, 4c,
and 8a exhibited potent antitumor activity against breast cancer, respectively, and were the most active
among their analogues. Moreover, the other compounds were less active.

Molecules 2016, 21, 1714
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(
a)
(b)
Figure 1.
5b 6a
drug 5-flourouracil; (
compounds 4a 4b 5a and 6b
drug 5-flourouracil.
(
,
a
) The dose response curve showing the in vitro inhibitory activity of the tested compounds
7a 7b 8a and 8c against breast carcinoma (MCF-7) cell line compared with the reference
) The dose response curve showing the in vitro inhibitory activity of the tested
against breast carcinoma (MCF-7) cell line compared with the reference
4
c,
,
,
,
b
,
,
–f
Table 3. The in vitro inhibitory activity of tested compounds against breast carcinoma cell line (MCF-7)
expressed as IC50 values (µg/mL) ± standard deviation from three replicates.
Tested Compounds
IC50 Values (µg/mL)
±Standard Deviation
4
a
3.6
47.6
4.6
0.4
2.8
0.3
>8
4
4
b
c
5
a
>200
95.1
106.7
42.2
160.4
189.9
49.8
34.8
104.5
68.1
20.4
86.1
4.1
5
6
b
a
2.6
2.5
1.9
5.8
7.6
2.4
3.2
4.9
1.7
0.8
1.7
0.6
6
6
b
c
6
6
d
e
6
f
7
a
7
8
b
a
8
c
5
-Flurouracil
3
. Experimental Section
3
.1. General
All melting points were determined with an Electrothermal Mel.-Temp. II (Registered trademark
of Barnstead, Barnstead, NH, USA) apparatus and were uncorrected. Element analyses were performed
at Regional Center for Mycology and Biotechnology at Al-Azhar University. The infrared (IR) spectra
were recorded using a potassium bromide disc technique on a Nikolet IR 200 FT IR spectrometer
(
Thermo Electron Scientific Instruments LLC, Madison, WI, USA) and carried out in Taif University,
Taif, KSA. Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A mass spectrometer
Shimadzu Corporation, Tokyo, Japan) at the Regional Center for Mycology and Biotechnology at
(
1
13
Al-Azhar University. H-NMR and C-NMR spectra were recorded in DMSO-d as a solvent using a
6
Varian Mercury spectrometer at 400 MHz and 125 MHz, respectively, Applied Nucleic Acid Research
Center, Zagazig University, Egypt. Chemical shifts (δ) are given in ppm and coupling constants

Molecules 2016, 21, 1714
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(J) are given in Hz. All reactions were monitored by TLC using pre-coated plastic sheet silica gel
(
0.25 mm, 20 × 20 cm, 60F254, E. Merck KGaA, Konstanz, Germany) and spots were visualized by
irradiation with UV light (254 nm). The used solvent system was chloroform:methanol (9:1) and ethyl
acetate:toluene (1:1).
6
6
6
-Chlorouracil (1) was prepared according to the reported method [30].
-Chloro-1-alkyl- and/or 1,3-Dialkyluracils 2a–c [31–34]
-Chloro-1-propyluracil (2b) and 6-chloro-1,3-dipropyluracil (2c): A solution of 6-chlorouracil (1) (40 mmol)
in dimethyl sulfoxide (25 mL) was heated gently until 6-chlorouracil dissolved, and then potassium
carbonate (20 mmol) was added with stirring. Propyl iodide (40 mmol) was added one time and the
mixture was stirred at room temperature for 6 h. Water (40 mL) was added, and cooled in an ice box
for several hours. The formed precipitate was collected by filtration, washed with water, dried in the
◦
oven at 80 C, and crystallized from methanol to give 3.8 g of a white crystalline precipitate (51% yield)
2
◦
b with m.p. = 165 C.
The mother liquor was evaporated in vacuo until dryness, then water (30 mL) was added,
followed by extraction with chloroform (40 mL × 3). The chloroformic layer was evaporated and the
◦
1
obtained colorless crystals was dried in desiccator to give 2.3g (25%) of 2c with m.p. = 58 C; H-NMR
DMSO-d6) ppm: 6.03 (s, 1H, CH-5), 4.26 (t, 2H, NCH ), 3.89 (t, 2H, NCH ), 1.52–1.62 (m, 4H, 2CH ),
(
δ
2
2
2
0
.84–0.88 (m, 6H, 2CH3).
6
-Hydrazinyl-1-methyl-, 1-Propyl- and/or 1,3-Dipropyluracils (3a–c) [34,35]
◦ ◦ ◦
3a: Yield 95%; m.p. 254 C, lit. [34] = 255 C; 3b: Yield 84%; m.p. 238–240 C; 3c: Yield 91%; m.p.
◦
1
20 C.
,4-Disubstituted-1H-indeno[2,1-c]pyrimido[5,4-e]pyridazine-1,3,7(2H,4H)-triones (4a–c)
A mixture of 6-hydrazinyl-1-substituted and/or -1,3-disubstituteduracils (3a–c) (1.9 mmol) and
2
ninhydrin (1.9 mmol) in acetic acid (5 mL) was heated under reflux for 5–10 min. The formed precipitate
after cooling was filtered, washed with ethanol and crystallized from DMF/ethanol (1:3).
◦
4
(
-Methyl-1H-indeno[2,1-c]pyrimido[5,4-e]pyridazine-1,3,7(2H,4H)-trione (4a): Yield: 48%; m.p. >300 C; IR
−
1
KBr) νmax (cm ): 3178 (NH), 3062 (CH arom), 2885 (CH aliph), 1687, 1631, 1597 (C=O), 1447 (C=C);
1
H-NMR (DMSO-d )
δ ppm: 12.24 (s, 1H, NH),9.20 (d, 1H, J = 7.6 Hz), 7.89–7.76 (m, 3H, arom), 3.66 (s,
= 188.2, 160.9, 153.5, 151.2, 149.8, 140.8, 137.5, 136.5, 134.8, 133.9,
31.4, 129.2, 109.9, 29.9; MS: m/z (%) = M , 280 (52), 251 (10), 182 (59), 155 (52), 154 (28), 153 (22),
6
3
1
H, CH3); 13C-NMR (DMSO-d6):
δ
+
1
38 (100), 126 (32), 111 (23), 99 (20), 76 (37). Anal. Calcd for C H N O : C, 60.00; H, 2.88; N, 19.99.
14 8 4 3
Found: C, 60.16; H, 2.85; N, 20.14.
◦
-Propyl-1H-indeno[2,1-c]pyrimido[5,4-e]pyridazine-1,3,7(2H,4H)-trione (4b): Yield: 51%; m.p. 281–283 C;
4
−
1
IR (KBr) νmax (cm ):3174 (NH), 3047 (CH arom), 2974, 2838 (CH aliph), 1720, 1692, 1555 (C=O), 1458
1
(
C=C); H-NMR (DMSO-d )
δ
ppm: 12.23 (s, 1H, NH, exchangeable), 9.23 (d, 1H, J = 7.6 Hz), 7.90–7.75
6
(
m, 3H, arom), 4.31 (t, 2H, J = 7.6 Hz, CH ), 1.77–1.71 (m, 2H, J = 7.6 Hz, CH ), 0.96 (t, 3H, 7.6 Hz,
2
2
CH3). 13C-NMR (DMSO-d6):
δ
=188.1, 160.8, 153.5, 151.1, 149.6, 141.0, 137.6, 136.5, 134.7, 133.9, 131.4,
+
1
29.2, 110.0, 44.0, 20.4, 11.1; MS: m/z (%) = M , 308 (100), 267 (94), 266 (80), 265 (36), 238 (50), 223 (77),
210 (32), 196 (48), 195 (34), 181 (49), 167 (36), 155 (33), 154 (37), 153 (15), 152 (24), 139 (49), 138 (39), 127
(
36), 126 (53), 125 (46), 112 (24), 99 (28). Anal. Calcd for C H N O : C, 62.33; H, 3.92; N, 18.17. Found:
16 12 4 3
C, 62.51; H, 3.95; N, 18.25.

Molecules 2016, 21, 1714
8 of 14
◦
2
,4-Dipropyl-1H-indeno[2,1-c]pyrimido[5,4-e]pyridazine-1,3,7(2H,4H)-trione (4c): Yield: 71%, m.p. 260–262 C;
−
1
IR (KBr) νmax (cm ): 3050 (CH arom), 2961, 2873 (CH aliph), 1710, 1667, 1566 (C=O), 1432 (C=C);
1
H-NMR (DMSO-d6)
δ ppm: 9.26 (d, 1H, J = 7.6 Hz, arom), 8.11–7.61 (m, 3H, arom), 4.37 (t, 2H,
J = 6.8 Hz, CH ), 3.95 (t, 2H, J = 6.8 Hz, CH ), 1.76–1.73 (m, 2H, CH ), 1.66–1.65 (m, 2H, CH ), 0.96 (t,
2
2
2
2
+
3
H, J = 6.8 Hz, CH ), 0.93 (t, 3H, J = 6.8 Hz, CH ); MS: m/z (%) = M , 350 (62), 323 (33), 290 (27), 268
3 3
(28), 262 (50), 240 (20), 238 (16), 236 (42), 223 (26), 209 (17), 196 (15), 195 (19), 192 (47), 180 (63), 177 (41),
169 (64), 154 (21), 140 (22), 138 (83), 123 (46), 112 (45), 99 (17), 180 (63), 98 (45), 97 (47), 94 (53), 74 (81),
7
3 (100); Anal. Calcd for C H N O : C, 65.13; H, 5.18; N, 15.99. Found: C, 65.45; H, 5.24; N, 16.17.
19 18 4 3
6
-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)-1-propyl- and/or 1,3-Dipropylpyrimidine-2,4(1H,3H)-diones 5a,b
A mixture of 6-hydrazinyl-1-propyl- and/or 1,3-dipropyluracils (3b ) (1.6 mmol) and isatin
1.6 mmol) in acetic acid (5 mL) was heated under reflux for 1–2 h. The formed precipitate after cooling
was filtered, washed with ethanol, and crystallized from DMF/ethanol (1:3).
,c
(
6
>
-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)-1-propylpyrimidine-2,4(1H,3H)-dione (5a): Yield: 53%; m.p.
◦
−1
300 C; IR (KBr) νmax (cm ): 3195 (br., NH), 3095 (CH arom), 2956, 2815 (CH aliph), 1702, 1594, 1515
1
(
C=O), 1458 (C=C); H-NMR (DMSO-d )
δ
ppm: 13.01 (s, 1H, NH), 11.35 (s, 1H, NH), 10.99 (s,1H, NH),
6
7
.63 (d, 1H, J = 7.6 Hz, arom), 7.39–7.36 (m, 1H, arom), 7.12–7.09 (m, 1H, arom), 6.97 (d, 1H, J = 7.6 Hz,
arom), 5.61 (s, 1H, CH-5), 3.81 (t, 2H, J = 7.6 Hz, CH ), 1.69–1.64 (m, 2H, J = 7.6 Hz, CH ), 0.94 (t, 3H,
2
2
J = 7.6 Hz, CH3); 13C-NMR (DMSO-d6):
1
δ
= 163.5, 162.3, 162.3, 151.0, 141.9, 136.1, 131.4, 122.7, 120.7,
19.5, 111.3, 78.5, 42.8, 20.9, 10.7; MS: m/z (%) = M , 313 (21), 285 (34), 253 (30), 243 (16), 226 (11), 213
+
(12), 200 (14), 158 (10), 147 (14), 145 (19), 132 (13), 118 (39), 117 (35), 104 (34), 103 (22), 101 (19), 90 (32),
7
4
7 (47), 76 (29), 68 (100); Anal. Calcd for C H N O : C, 57.50; H, 4.83; N, 22.35. Found: C, 57.78; H,
15 15 5 3
.90; N, 22.52.
6
2
-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)-1,3-dipropylpyrimidine-2,4(1H,3H)-dione (5b): Yield: 65%, m.p.
◦
−1
83–285 C; IR (KBr) νmax (cm ):3137 (br., NH), 3084 (CH arom), 2966, 2877 (CH aliph), 1691, 1600,
1
1
542 (C=O), 1458 (C=C); H-NMR (DMSO-d )
δ
ppm: 13.03 (s, 1H, NH), 11.37 (s, 1H, NH), 7.95–7.87
6
(m, 1H, arom), 7.65 (d, 1H, J = 6.8 Hz, arom), 7.40–7.36 (m, 1H, arom), 6.98 (d, 1H, J = 6.8 Hz, arom),
5
.74 (s, 1H, CH-5), 4.25 (t, 2H, J = 7.4 Hz, NCH ), 3.87 (t, 2H, J = 7.4 Hz, NCH ), 1.76–1.69 (m, 2H,
2
2
13
J = 7.4 Hz, CH ), 1.54–1.53 (m, 2H, J = 7.4 Hz, CH ), 0.96–0.89 (m, 6H, 2CH ); C-NMR (DMSO-d6):
2
2
3
δ = 163.5, 161.2, 158.0, 149.6, 141.9, 136.2, 131.4, 122.7, 120.7, 119.4, 111.3, 78.1, 43.2, 41.8, 20.1, 19.6, 11.1,
+
1
0.7; MS: m/z (%) = M , 355 (86), 338 (20), 327 (47), 313 (24), 285 (34), 243 (59), 227 (20), 226 (12), 213
(
27), 200 (52), 187 (36), 186 (54), 172 (12), 166 (41), 161 (100), 158 (32), 153 (31), 148 (50), 147 (42), 146 (17),
1
45 (69), 125 (17), 118 (42), 117 (44), 111 (66); Anal. Calcd for C H N O : C, 60.83; H, 5.96; N, 19.71.
18 21 5 3
Found: C, 61.07; H, 6.03; N, 19.94.
3
-Substituted-7-propyl- and/or 5,7-Dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-diones 6a–f
Method A: A mixture of 6-hydrazinyl-1-propyl- and/or 1,3-dipropyluracils (3b ) (1.7 mmol)
,c
and appropriate benzylidene malononitriles (1.7 mmol) in DMF (5 mL) in the presence of TEA (1mL)
was heated under reflux for 6–8 h. The reaction mixture was evaporated under reduced pressure.
The residue was treated with ethanol (10 mL), the formed precipitate was filtered, washed with ethanol,
and crystallized from DMF/ethanol (2:1) to afford 6a–f.
Method B: A mixture of 6-hydrazinyl-1,3-dipropyluracil (3c) (1.7 mmol) and 4-chlorobenzylidene
ethyl cyanoacetate (1.7 mmol) in DMF (5 mL) in the presence of TEA (1 mL) was heated under reflux
for 8 h. The reaction mixture was evaporated under reduced pressure. The residue was treated with
ethanol (10 mL), the formed precipitate was filtered, washed with ethanol, and crystallized from
DMF/ethanol (2:1) to afford 6f.

Molecules 2016, 21, 1714
9 of 14
3
>
-Phenyl-7-propyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6a): method A: Yield: 72%; m.p.
◦
−1
300 C; IR (KBr) νmax (cm ): 3170 (br., NH), 3058 (CH arom), 2965 (CH aliph), 1679, 1595 (C=O),
1
1452 (C=C); H-NMR (DMSO-d )
δ
ppm: 10.95 (s, 1H, NH), 7.95 (s, 1H, NH), 7.41–7.25 (m, 5H, arom),
6
3
.84 (t, 2H, J = 7.2 Hz, NCH ), 1.71–1.69 (m, 2H, J = 7.2 Hz, CH ), 0.99 (t, 3H, J = 7.2 Hz, CH ); MS:
2
2
3
+
m/z (%) = M , 270 (4), 231 (8), 184 (25), 176 (10), 165 (19), 139 (10), 130 (11), 111 (23), 109 (14), 107 (11),
8 (17), 96 (16), 95 (17), 83 (30), 81 (23), 71 (30), 69 (99), 67 (26), 55 (100), 44 (20), 43 (86), 41 (75); Anal.
Calcd for C H N O : C, 62.21; H, 5.22; N, 20.73. Found: C, 62.48; H, 5.24; N, 21.04.
9
14
14
4
2
3
-(4-Chlorophenyl)-7-propyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6b): Method A: Yield: 69%;
◦
−1
m.p. >300 C; IR (KBr) νmax (cm ): 3215 (br., NH), 3050(CH arom), 2965, 2869 (CH aliph), 1684, 1614
1
(
(
C=O), 1435 (C=C), 810 (p-substituted); H-NMR (DMSO-d )
d, 2H, J = 8.6 Hz, arom), 7.29 (d, 2H, J = 8.6 Hz, arom), 4.05 (t, 2H, J = 7.4 Hz, NCH ), 1.65–1.63 (m, 2H,
δ
: 11.19 (s, 1H, NH), 7.82 (s, 1H, NH), 7.47
6
2
13
J = 7.4 Hz, CH ), 0.91 (t, 3H, J = 7.4 Hz, CH ); C-NMR (DMSO-d6)
δ
ppm: 160.4, 154.6, 150.3, 135.9,
2
3
+
1
33.0, 129.4, 128.7, 115.3, 99.2, 42.7, 20.6, 11.1; MS: m/z (%) = 306 (M+2, 9), M , 304 (25), 271 (30), 265
(42), 211 (20), 176 (27), 145 (21), 138 (21), 131 (38), 125 (34), 116 (34), 114 (23), 110 (100), 87 (64), 84 (86),
82 (34), 43 (84), 42 (48); Anal. Calcd for C H ClN O : C, 55.18; H, 4.30; N, 18.39. Found: C, 55.37; H,
14 13 4 2
4
.36; N, 18.57.
3
>
-Phenyl-5,7-dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6c): Method A: Yield: 77%; m.p.
◦
−1
300 C; IR (KBr) νmax (cm ): 3195 (br., NH), 3040 (CH arom), 2964 (CH aliph), 1605, 1598 (C=O),
1
1449 (C=C); H-NMR (DMSO-d )
δ
ppm: 7.95 (s, 1H, NH), 7.56–7.15 (m, 5H, arom), 3.89 (t, 2H, NCH2),
6
3
.84 (t, 2H, J = 8.8 Hz, NCH ), 1.70–1.68 (m, 2H, CH ), 1.60–1.58 (m, 2H, CH ), 0.91–0.89 (m, 6H, 2CH );
2
2
2
3
+
MS: m/z (%) = M , 312 (21), 282(18), 255 (20), 247 (14), 194 (31), 180 (16), 163 (46), 126 (17), 125 (60),
21 (33), 105 (22), 97 (77), 83 (42), 81 (19), 80 (38), 69 (58), 57 (31), 56 (100), 43 (71); Anal. Calcd for
C H N O : C, 65.37; H, 6.45; N, 17.94. Found: C, 65.48; H, 6.53; N, 18.09.
1
17
20
4
2
3
-(4-Bromophenyl)-5,7-dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6d): Method A: Yield: 78%;
◦
−1
m.p. >300 C; IR (KBr) νmax (cm ): 3135 (NH), 3023 (CH arom), 2966, 2837 (CH aliph), 1678, 1676
1
(C=O), 1496 (C=C); H-NMR (DMSO-d )
δ
ppm: 7.84 (s, 1H, NH, exchangeable), 7.73–7.21 (m, 4H, arom),
6
+
4.0–3.64 (m, 4H, 2NCH ), 1.68–1.61 (m, 4H, 2CH ), 0.91–0.80 (m, 6H, 2CH ), MS: m/z (%) = M + 2, 393
2
2
3
+
(
(
1), M , 391 (3), 355 (10), 327 (19), 283 (13), 262 (17), 220 (17), 207 (14), 160 (15), 159 (13), 157 (16), 141
13), 129 (12), 119 (16), 115 (12), 109 (34), 97 (46), 95 (34), 87 (17), 85 (24), 84 (70), 81 (100), 71 (33); Anal.
Calcd for C H BrN O : C, 52.19; H, 4.89; N, 14.32. Found: C, 52.53; H, 4.91; N, 14.39.
17
19
4
2
3
-(2-Hydroxyphenyl)-5,7-dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6e): Method A: Yield: 72%;
◦
−1
m.p. >300 C; IR (KBr) νmax (cm ): 3435 (OH), 3180 (NH), 3055 (CH arom), 2965 (CH aliph), 1605, 1545
1
(C=O), 1485 (C=C), 750 (o-substituted phenyl); H-NMR (DMSO-d )
δ
ppm: 9.41 (s, 1H, OH), 7.59–6.72
6
(
m, 5H, 1NH & 4H arom), 3.80–3.66 (m, 4H, 2NCH ), 1.67–1.65 (m, 2H, CH ), 1.52–1.50 (m, 2H, CH ), 1.16
2 2 2
13
(t, 3H, J = 7.6 Hz, CH ), 0.91 (t, 3H, J = 7.6 Hz, CH ); C-NMR (DMSO-d6): δ = 162.5, 158.0, 152.0, 151.9,
3
3
+
1
34.4, 125.3, 124.9, 118.6, 116.4, 115.9, 97.3, 43.1, 42.8, 20.2, 20.0, 11.1, 11.0; MS: m/z (%) = M , 328 (57), 296
(
23), 278 (48), 266 (15), 223 (14), 179 (14), 165 (15), 140 (10), 136 (14), 129 (16), 127 (17), 125 (20), 116 (31), 115
25), 113 (20), 111 (31), 109 (23), 107 (27), 97 (33), 81 (19), 77 (40), 69 (77), 67 (48), 59 (20), 56 (94), 43 (100);
Anal. Calcd for C H N O : C, 62.18; H, 6.14; N, 17.06. Found: C, 62.44; H, 6.21; N, 17.23.
(
17
20
4
3
3
-(4-Chlorophenyl)-5,7-dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (6f): Method A: Yield: 69%,
◦
−1
method B: Yield: 61%; m.p. >300 C; IR (KBr) νmax (cm ): 3187 (NH), 3053 (CH arom), 2965, 2870
1
(
CH aliph), 1685, 1613 (C=O), 1497 (C=C), 814 (p-substituted); H-NMR (DMSO-d )
δ
ppm: 7.95 (s, 1H,
6
NH), 7.52 (d, 2H, J = 8.8 Hz, arom), 7.32 (d, 2H, J = 8.8 Hz, arom), 4.99–3.81 (m, 4H, 2NCH ), 1.72–1.55
2
+
+
(
m, 4H, 2CH ), 0.88–0.86 (m, 6H, 2CH ); MS: m/z (%) = M + 2, 348 (3), M , 346 (8), 341(21), 314(25),
2 3
311 (16), 280 (29), 269 (17), 266 (23), 247 (22), 245 (17), 238 (25), 226 (20), 207 (18), 206 (19), 203 (30), 184
(22), 154 (33), 146 (33), 145 (40), 127 (56), 125 (26), 123 (25), 119 (43), 89 (82), 82 (100), 73 (51), 67 (63),
6
6 (45), 40 (87); Anal. Calcd for C H ClN O : C, 58.87; H, 5.52; N, 16.15. Found: C, 59.05; H, 5.61;
17 19 4 2
N, 16.23.

Molecules 2016, 21, 1714
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3
,4-Diphenyl-8-propyl- and/or 6,8-Dipropylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-diones (7a,b)
Method A: A mixture of 6-hydrazinyl-1-propyl and/or 1,3-dipropyluracils (3b ) (1.6 mmol) and
,
c
benzil (1.6 mmol) in DMF (5 mL) in the presence of TEA (1 mL) was heated under reflux for 4–5 h.
The reaction mixture was evaporated under reduced pressure. The residue was treated with ethanol
(
10 mL), the formed precipitate was filtered, washed with ethanol, and crystallized from DMF/ethanol
(2:1) to afford compounds 7a,b.
Method B: A mixture of 6-hydrazinyl-1-propyluracil (3b) (1.6 mmol) and α-phenylphenacyl
bromide (1.6 mmol) in DMF (5 mL) in the presence of TEA (1 mL) was heated under reflux for 5 h.
The reaction mixture was evaporated under reduced pressure. The residue was treated with ethanol
(
10 mL), the formed precipitate was filtered, washed with ethanol, and crystallized from DMF/ethanol
(2:1) to afford 7a.
3
,4-Diphenyl-8-propylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione (7a): Yield: method A: 76%, method B:
◦
−1
68%; m.p. 226–228 C; IR (KBr) νmax (cm ): 3159 (NH), 3003 (CH arom), 2966, 2835 (CH aliph), 1674,
1
1538 (C=O), 1496 (C=C); H-NMR (DMSO-d )
δ
ppm: 11.72 (s, 1H, NH), 7.25–7.10 (m, 10H, arom),
6
13
4
.34 (t, 2H, J = 7.6 Hz, CH ), 1.82–1.77 (m, 2H, J = 7.6 Hz, CH ),0.99 (t, 3H, J = 7.6 Hz, CH ); C-NMR
2
2
3
(
2
DMSO-d6):
δ
= 159.8, 157.5, 151.7, 149.8, 139.3, 136.5, 134.6, 129.6, 128.9, 128.0, 127.6, 127.3, 111.7, 43.1,
+
0.5, 11.1; MS: m/z (%) = M , 358 (16), 317 (10), 316 (46), 315 (100), 255 (10), 189 (9), 171 (9), 128 (4), 77
(
5); Anal. Calcd for C H N O : C, 70.38; H, 5.06; N, 15.63. Found: C, 70.49; H, 5.10; N, 15.84.
21 18 4 2
3
,4-Diphenyl-6,8-dipropylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione (7b): Yield: method A: 64%; m.p.
◦
−1
220–222 C; IR (KBr) νmax (cm ): 3056 (CH arom), 2963, 2873 (CH aliph), 1718, 1670 (C=O), 1495
1
(C=C); H-NMR (DMSO-d )
δ
ppm: 7.26–7.10 (m, 10H, arom), 4.42 (t, 2H, J = 7.4 Hz, NCH ), 3.75 (t,
6
2
2
3
H, J = 7.4 Hz, NCH ), 1.85–1.79 (m, 2H, J = 7.4 Hz, CH ), 1.53–1.47 (m, 2H, J = 7.4 Hz, CH ), 1.00 (t,
2 2 2
+
H, J = 7.4 Hz, CH ), 0.83 (t, 3H, J = 7.4 Hz, CH ); MS: m/z (%) = M , 400 (6), 383 (10), 369 (7), 267
3
3
(
(
22), 223 (17), 135 (12), 133 (26), 127 (11), 125 (11), 112 (20), 110 (17), 101 (18), 95 (26), 90 (23), 86 (24), 83
25), 80 (24), 76 (41), 72 (31), 70 (31), 69 (29), 59 (43), 55 (42), 44 (100), 42 (81), 40 (62); Anal. Calcd. for
C H N O : C, 71.98; H, 6.04; N, 13.93. Found: C, 72.21; H, 6.12; N, 14.12.
24
24
4
2
3
-Substituted-8-propylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-diones 8a–c
A mixture of 6-hydrazinyl-1-propyluracil (3b) (1.6 mmol) and appropriate phenacyl bromides
(
1.6 mmol) in DMF (5 mL) in the presence of TEA (1 mL) was heated under reflux for 4–6 h. The reaction
mixture was evaporated under reduced pressure. The residue was treated with ethanol (10 mL), the
formed precipitate was filtered, washed with ethanol, and crystallized from DMF/ethanol (2:1) to
afford 8a–c.
◦
3
-Phenyl-8-propylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione (8a): Yield: 56%; m.p. 262–264 C; IR (KBr)
−
1
νmax (cm ): 3178 (NH), 3039 (CH arom), 2965, 2866 (CH aliph), 1668, 1592 (C=O), 1496 (C=C);
H-NMR (DMSO-d6) δ ppm: 11.99 (s, 1H, NH), 8.50 (s,1H, arom), 8.22–8.19 (m, 2H, arom), 7.57–7.46
1
(
m, 3H, arom), 4.29 (t, 2H, J = 7.4 Hz, NCH ), 1.79–1.73 (m, 2H, J = 7.4 Hz, CH ), 0.97 (t, 3H, J = 7.4 Hz,
2
2
+
CH ); MS: m/z (%) = M , 282 (13), 254 (9), 241 (23), 240 (89), 239 (19), 197 (36), 77 (14), 44 (30), 40 (100);
3
Anal. Calcd. for C H N O : C, 63.82; H, 5.00; N, 19.85. Found: C, 63.97; H, 5.08; N, 20.02.
15
14
4
2
◦
-(4-Methoxyphenyl)-8-propylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione (8b): Yield; 61%; m.p. 219–221 C;
3
−
1
IR (KBr) νmax (cm ): 3162 (NH), 3039 (CH arom), 2967, 2826 (CH aliph), 1670, 1600 (C=O), 1449
1
(
C=C), 838 (p-substituted); H-NMR (DMSO-d )
δ
ppm: 11.96 (s, 1H, NH), 8.43 (s, 1H, arom), 8.17 (d,
6
1H, J = 7.0 Hz, arom), 7.46 (d, 1H, J = 8.4 Hz, arom), 7.10 (d, 1H, J = 7.0 Hz, arom), 7.01 (d, 1H, J = 8.4
Hz, arom), 4.28 (t, 2H, J = 7.4 Hz, NCH ), 3.84 (s, 3H, CH ), 1.78–1.72 (m, 2H, J = 7.4 Hz, CH ), 0.96 (t,
2
3
2
3
H, J = 7.4 Hz, CH3); 13C-NMR (DMSO-d6):
δ
= 160.8, 160.1, 160.0, 150.9, 149.8, 131.1, 127.4, 126.3,
+
1
20.7, 114.5, 55.3, 42.8, 20.5, 11.1; MS: m/z (%) = M , 312 (19), 271 (12), 270 (50), 269 (100), 255 (21), 239
(
24), 135 (20), 40 (23); Anal. Calcd for C H N O : C, 61.53; H, 5.16; N, 17.94. Found: C, 61.71; H, 5.23;
16 16 4 3
N, 18.13.

Molecules 2016, 21, 1714
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◦
3
-(4-Nitrophenyl)-8-propylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-dione (8c): Yield: 54%; m.p. 190–192 C;
−
1
IR (KBr) νmax (cm ): 3176 (NH), 3063 (CH arom), 2965, 2873 (CH aliph), 1684, 1595 (C=O), 1449 (C=C),
ppm: 12.08 (s, 1H, NH), 8.69 (s, 1H, arom),
.52 (d, 2H, J = 8.8 Hz, arom), 8.38 (d, 2H, J = 8.8 Hz, arom), 4.31 (t, 2H, J = 7.4 Hz,NCH ), 1.77–1.74 (m,
1
1
8
2
510, 1332 (NO ), 851 (p-substituted); H-NMR (DMSO-d )
δ
2
6
2
+
H, J = 7.4 Hz, CH ), 0.97 (t, 3H, J = 7.4 Hz, CH ); MS: m/z (%) = M , 327 (13), 286 (44), 285 (100), 243
2
3
(
14), 242 (90), 40 (24); Anal. Calcd for C H N O : C, 55.05; H, 4.00; N, 21.40. Found: C, 55.12; H, 4.09;
15 13 5 4
N, 21.57.
3
.2. Biological Evaluation
3
.2.1. Antimicrobial Bioassay by Using the Agar Diffusion Cylinder Method [36]
All microbial strains were provided from the culture collection of the Regional Center for
Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.
The newly-synthesized target compounds were tested in vitro against different types of
bacteria, Streptococcus pneumoniae and Bacillus subtilis as examples of Gram-positive bacteria, and
Pseudomonas aeruginosa and Escherichia coli as examples of Gram-negative bacteria. Fungi, as well as
bacteria, were used for testing the antifungal activity of the synthesized compounds. Aspergillus fumigates
and Candida albicans were used as example of fungi and yeast, respectively. The stock solution of
concentrations (1 mg/mL) of the synthesized compounds were used. The plates were incubated at
◦
3
7 C for 24 h for bacteria and yeast, and for 48–72 h for fungi. Tetracycline was used as the standard
antibacterial drug while amphotericin B was used as the standard antifungal drug. The diameters of
the inhibition zones (mm) were measured and used as criterion for the antimicrobial activity.
3
.2.2. Determination of the Minimum Inhibitory Concentration (MIC)
Serial dilutions of the promising compounds were subjected to MIC determination. The different
concentrations of each compound were tested with the modified agar diffusion cylinder method as
was described before.
3
.2.3. Evaluation of the Antitumor Activity Using Viability Assay
All human anticancer cell lines were obtained from the American Type Culture Collection.
The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum
◦
and 50
µ
g/mL gentamycin. The cells were maintained at 37 C in a humidified atmosphere with
5
% CO and were subcultured two to three times a week. For antitumor assays, the tumor cell lines
2
4
®
were suspended in medium at concentrations of 5
plates, then incubated for 24 h. The tested compounds were then added into 96-well plates (three
replicates) to achieve eight concentrations for each compound. Six vehicle controls with media or
×
10 cell/well in Corning 96-well tissue culture
0.5% DMSO were run for each 96-well plate as a control. After incubating for 24 h, the numbers of
viable cells were determined by staining the cells with crystal violet [37 38], followed by cell lysing
,
using 33% glacial acetic acid and read the absorbance at 590 nm using microplate reader (Sunrise,
TECAN, Inc., Morrisville, NC, USA) after well mixing. The percentage of viability was calculated as
[
1
−
(ODt/ODc)]
×
100%, where ODt is the mean optical density of wells treated with the tested sample
and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug
concentration is plotted to obtain the survival curve of each tumor cell line after treatment with the
specified compound. The 50% inhibitory concentration (IC ), the concentration required to cause
50
toxic effects in 50% of intact cells, was estimated from graphic plots [37].

Molecules 2016, 21, 1714
12 of 14
4
. Conclusions
The newly synthesized compounds of indeno[2,1-c]pyrimido[5,4-e]pyridazines, oxoindolinylidene
hydrazinyl pyrimidines, pyrazolo[3,4-d]pyrimidines and pyrimido[4,5-c]pyridazines were prepared by
a simple method. The novel compounds were screened for both antimicrobial and anticancer activities.
Compound 4b showed a very high MICs in comparison to the standard drug tetracycline. Compounds
4a, 4c and 8a exhibited potent antitumor activity against breast cancer in comparison to the standard
drug 5-flourouracil.
Acknowledgments: The Author wishes to thanks Mahmoud Elaasser to carry out the biological activity of this
work at Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.
Author Contributions: Samar El-Kalyoubi formulated the research idea, conceived and prepared the manuscript;
Samar El-Kalyoubi and Fatimah Agili performed the experiments and analyzed the data; Samar El-Kalyoubi
wrote the paper. The authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Rashad, A.E.; Shamroukh, A.H.; Abdel-Megeid, R.E.; Ali, S.H. Synthesis and isomerization of some novel
pyrazolopyrimidine and pyrazolotriazolopyrimidine derivatives. Molecules 2014, 19, 5459–5469. [CrossRef]
[PubMed]
2
3
4
.
.
.
Shamroukh, A.H.; Rashad, A.E.; Ali, H.S.; Abdel-Megeid, F.M.E. Synthesis, reactions and antimicrobial
evaluation of β-enaminonitriles of pyrazole. J. Heterocycl. Chem. 2013, 50, 758–765. [CrossRef]
Chauhan, M.; Kumar, R. Medicinal attributes of pyrazolo[3,4-d]pyrimidines: A review. Bioorg. Med. Chem.
2
013, 21, 5657–5668. [CrossRef] [PubMed]
Eweas, A.F.; Swelam, S.A.; Fathalla, O.A.; Fawzy, N.M.; Abdel-Moez, S.I. Synthesis, anti-microbial evaluation,
and molecular modeling of new pyrazolo[3,4-d]pyrimidine derivatives. Med. Chem. Res. 2012, 21, 3848–3857.
[CrossRef]
5
.
Aggarwal, R.; Sumran, G.; Garg, N.; Aggarwal, A.A. Regioselective synthesis of some new
pyrazol-1 -ylpyrazolo[1,5-a]pyrimidines in aqueous medium and their evaluation as antimicrobial agents.
0
Eur. J. Med. Chem. 2011, 46, 3038–3046. [CrossRef] [PubMed]
6
.
.
Gudmundsson, K.S.; Johns, B.A.; Weatherhead, J. Pyrazolopyrimidine and pyrazolo-triazines with potent
activity against herpesviruses. Bioorg. Med. Chem. Lett. 2009, 19, 5689–5692. [CrossRef] [PubMed]
Rajendran, A.; Raghupathy, D.; Priyadarshini, M. Green Synthesis of Biologically Active Pyrazolopyrimidine
Derivatives Using an Ionic liquid 2-Methyl-3-butylimidazolium chloride. Int. J. ChemTech Res. 2011, 3,
7
2
93–297.
8
9
1
.
.
Ghorab, M.M.; Ragab, F.A.; Alqasoumi, S.I.; Alafeefy, A.M.; Aboulmagd, S.A. Synthesis of some new
pyrazolo[3,4-d]pyrimidine derivatives of expected anticancer and radioprotective activity. Eur. J. Med. Chem.
2
010, 45, 171–178. [CrossRef] [PubMed]
Abd El Razik, H.A.; Abdel Wahab, A.E. Synthesis and biological evaluation of some novel fused
pyrazolopyrimidines as potential anticancer and antimicrobial agents. Arch. Pharm. 2011, 344, 184–196.
[
CrossRef] [PubMed]
0. Yewale, S.B.; Ganorkar, S.B.; Baheti, K.G.; Shelke, R.U. Novel 3-substituted-1-aryl-5-phenyl-6-anilinopyrazolo
3,4-d]pyrimidin-4-ones: Docking, synthesis and pharmacological evaluation as a potential anti-inflammatory
[
agents. Bioorg. Med. Chem. Lett. 2012, 22, 6616–6620. [CrossRef] [PubMed]
1
1. El-Tombany, A.A. Synthesis, anti-Inflammatory, and ulcerogenicity studies of novel substituted and fused
pyrazolo[3,4-d]pyrimidin-4-ones. Sci. Pharm. 2013, 81, 393–422. [CrossRef] [PubMed]
1
2. Alcaro, S.; Artese, A.; Botta, M.; Zizzari, A.T.; Orallo, F.; Ortuso, F.; Schenone, S.; Brullo, C.;
Yáñez, M. Hit identification and biological evaluation of anticancer pyrazolopyrimidines endowed with
anti-inflammatory activity. ChemMedChem 2010, 5, 1242–1246. [CrossRef] [PubMed]
1
3. Gupta, S.; Rodrigues, L.M.; Esteves, A.P.; Oliveira-Campos, A.M.F.; Jose Nascimento, M.S.; Nazareth, N.;
Cidade, H.; Neves, M.P.; Fernandes, E.; Pinto, M.; et al. Synthesis of N-aryl-5-amino-4-cyanopyrazole
derivatives as potent xanthine oxidase inhibitors. Eur. J. Med. Chem. 2008, 43, 771–780. [CrossRef] [PubMed]

Molecules 2016, 21, 1714
13 of 14
1
4. Ishibuchi, S.; Morimoto, H.; Oe, T.; Ikebe, T.; Inoue, H.; Fukunari, A.; Kamezawa, M.; Yamada, I.;
Naka, Y. Synthesis and structure—Activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors.
Bioorg. Med. Chem. Lett. 2001, 11, 879–882. [CrossRef]
1
5. Rimaz, M.; Pourhossein, P.; Khalili, B. Regiospecific one-pot, combinatorial synthesis of new substituted
pyrimido[4,5-c]pyridazines as potential monoamine oxidase inhibitors. Turk. J. Chem. 2015, 39, 244–254.
6. Khalafy, J.; Rimaz, M.; Panahi, L.; Rabiei, H. A Regiospecific One-Pot, Three Component Synthesis of
1
4-Aryl-6,8-dimethylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-diones as New Potential Monoamine Oxidase
Inhibitors. Bull. Korean Chem. Soc. 2011, 32, 2428–2432. [CrossRef]
1
1
7. Hamilton, G.A. Progress in Bioorganic Chemistry; Kaiser, E.T., Kezdy, F.J., Eds.; Wiley: New York, NY, USA,
1
971; Volume 1, p. 83.
8. Altomare, C.; Cellamare, S.; Summo, L.; Catto, M.; Carotti, A.; Thull, U.; Carrupt, P.; Testa, B.;
Stoeckli-Evans, H. Inhibition of monoamine oxidase-B by condensed pyridazines and pyrimidines: Effects
of lipophilicity and structure-activity Relationships. J. Med. Chem. 1998, 41, 3812–3820. [CrossRef] [PubMed]
9. Patil, P.O.; Bari, S.B.; Firke, S.D.; Deshmukh, P.K.; Donda, S.T.; Patil, D.A. A comprehensive review on
synthesis and designing aspects of coumarin derivatives as monoamine oxidase inhibitors for depression
and Alzheimer’s disease. Bioorg. Med. Chem. 2013, 21, 2434–2450. [CrossRef] [PubMed]
1
2
0. Al-Abdullah, E.S. Synthesis and anticancer activity of some novel tetralin-6-yl-pyrazoline, 2-thioxopyrimidine,
2-Oxopyridine, 2-thioxo-pyridine and 2-iminopyridine derivatives. Molecules 2011, 16, 3410–3419. [CrossRef]
[PubMed]
2
1. Prajapati, D.; Baruah, P.; Gogoi, B.J.; Sandhu, J.S. One-pot synthesis of novel 1H-pyrimido[4,5-c][1,2]
diazepines and pyrazolo[3,4-d]pyrimidines. Beilstein J. Org. Chem. 2006, 2, 1–5.
2
2. Ribone, S.R.; Schenfeld, E.M.; Madrid, M.; Pierini, A.B.; Quevedo, M.A. Evaluation and synthesis of AZT
prodrugs with optimized chemical stabilities: Experimental and theoretical analyses. New J. Chem. 2016, 40,
2
383–2392. [CrossRef]
2
2
2
3. Huang, S.N.; Murai, J.; Rosa, I.D.; Dexheimer, T.S.; Naumova, A.; Gmeiner, W.H.; Pommier, Y. TDP1 repairs
nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside
analogs. Nucleic Acids Res. 2013, 41, 7793–7803. [CrossRef] [PubMed]
4. Zhang, N.; Zhao, L.; Ma, S.; Gu, M.; Zheng, X. Potent Anticancer Effects of Lentivirus Encoding a Drosophila
Melanogaster Deoxyribonucleoside Kinase Mutant Combined with Brivudine. Asian Pac. J. Cancer Prev.
2
012, 13, 2121–2127. [CrossRef] [PubMed]
5. Wang, R.; Cui, H.; Wang, J.; Li, N.; Zhao, Q.; Zhou, Y.; Lv, Z.; Zhong, W. Enhancing the antitumor effect of
methotrexate in vitro and in vivo by a novel targeted single-walled carbon nanohorn-based drug delivery
system. RSC Adv. 2016, 6, 47272–47280. [CrossRef]
2
2
2
6. Billings, B.K.; Wagner, J.A.; Cook, P.D.; Castle, R.N. The synthesis of pyrimido[4,5-c]pyridazines and
pyrido[2,3-d]pyrimidines related to toxoflavine and fervenulin. J. Het. Chem. 1975, 12, 1221–1224. [CrossRef]
7. El-kalyoubi, S.; Agili, F.; Youssif, S. Novel 2-thioxanthine and dipyrimidopyridine derivatives: Synthesis and
antimicrobial activity. Molecules 2015, 20, 19263–19276. [CrossRef] [PubMed]
8. Mousa, B.A.; Bayoumi, A.H.; Korraa, M.M.; Assy, M.G.; El-Kalyoubi, S.A. A Novel One-Pot and Efficient
Procedure for Synthesis of New Fused Uracil Derivatives for DNA Binding. Int. J. Org. Chem. 2015, 5, 37–47.
[CrossRef]
2
3
3
3
3
9. Mousa, B.A.; Bayoumi, A.H.; Korraa, M.M.; Assy, M.G.; El-Kalyoubi, S.A. Synthesis, DNA Binding Studies
Of New Pyrimidothiazepine And Pyrimidobenzothiazepine Derivatives. Afinidad 2012, 69, 144–149.
0. Cresswell, R.M.; Wood, H.C.S. The biosynthesis of pteridines. Part I. The synthesis of riboflavin. J. Chem. Soc.
1
960, 4768–4775. [CrossRef]
1. Youssif, S.; El-Kafrawy, A.; Bayoumy, B.; El-Bahaie, S. A novel synthesis of 3,9-dialkyl and
-aryl-3,9-dimethylxanthines. Bull. Korean Chem. Soc. 2002, 23, 374–380.
8
2. Youssef, S.; Pfleiderer, W. Purines XIV. Reactivity of 8-bromo-3,9-dimethylxanthine towards some
nucleophilic reagent. J. Heterocycl. Chem. 1998, 35, 949–954. [CrossRef]
3. Dudkin, S.; Iaroshenko, V.O.; Sosnovskikh, V.Y.; Tolmachev, A.A.; Villingera, A.; Langer, P. Synthesis and
reactivity of 5-polyfluoroalkyl-5-deazaalloxazines. Org. Biomol. Chem. 2013, 11, 5351–5361. [CrossRef]
[PubMed]
3
4. Furukawa, Y.; Maki, Y. 3-Aminopyrazolo[3,4-d]pyrimidine Derivatives and Production Thereof. U.S. Patent
Number 4,603,203, 29 July 1986.

Molecules 2016, 21, 1714
14 of 14
3
3
5. Youssif, S.; Assy, M. Fervenulin, 4-deazafervenulin and 5-deazaalloxazine analogues. J. Chem. Res. 1996, 442, 2546.
6. Ibrahim, H.S.; Eldehna, W.M.; Abdel-Aziz, H.A.; Elaasser, M.M.; Abdel-Aziz, M.M. Improvement of
antibacterial activity of some sulfa drugs through linkage to certain phthalazin-1(2H)-one scaffolds. Eur. J.
Med. Chem. 2014, 85, 480–486. [CrossRef] [PubMed]
3
7. Gomha, S.M.; Riyadh, S.M.; Mahmmoud, E.A.; Elaasser, M.M. Synthesis and anticancer activities of thiazoles,
1
,3-thiazines, and thiazolidine using chitosan-grafted-poly(vinylpyridine) as basic catalyst. Heterocycles 2015
,
9
1, 1227–1243.
3
8. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
Sample Availability: Samples of the compounds are available upon request.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).