First synthesis and biological evaluation of indeno[2,1-e]pyrazolo[3,4-b] pyrazin-5-one and related derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.01.025

The synthesis of indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one was achieved by intramolecular Friedel-Crafts reaction of the acid chloride 3-methyl-1,6- diphenyl-1H-pyrazolo[3,4-b] pyrazine-5-carboxylic acid chloride (4) using AlCl₃ in boiling CS₂. Compound 4 proved to be a versatile compound for the synthesis of several Indenopyrazolopyrazinone derivatives. The antibacterial and antifungal activities of selected derivatives were evaluated.

Full text of DOI:10.1016/j.ejmech.2013.01.025

European Journal of Medicinal Chemistry 62 (2013) 478e485
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journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
First synthesis and biological evaluation of indeno[2,1-e]pyrazolo
[3,4-b]pyrazin-5-one and related derivatives
*
Talaat El-Emary , Hussein El-Kashef
Chemistry Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one was achieved by intramolecular FriedeleCrafts
reaction of the acid chloride 3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b] pyrazine-5-carboxylic acid chloride
(4) using AlCl3 in boiling CS2. Compound 4 proved to be a versatile compound for the synthesis of several
Indenopyrazolopyrazinone derivatives. The antibacterial and antifungal activities of selected derivatives
were evaluated.
Received 18 October 2012
Received in revised form
17 January 2013
Accepted 19 January 2013
Available online 26 January 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazolo[3,4-b]pyrazine
Indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one
Biological evaluation
1. Introduction
2. Results and discussions
Pyrazolopyrazines constitute an important class of heterocy-
clic compounds. They are used in the prevention and/or treat-
ment of a wide variety of conditions known to be related to
adenosine receptors such as depression, dementia, Parkinson’s
disease, anxiety, pain, cerebrovascular disease, and heart failure
[1e3].
A perusal of the literature revealed that there are rather limited
reports on the synthesis of pyrazolo[3,4-b]pyrazines in spite of
their importance as therapeutic agents. The latter compounds
were reported to play a role as bone metabolism improvers useful
for the treatment of osteoporosis, periodontosis, hypercalcemia,
Paget’s disease and rheumatoid arthritis [4a]. Some reports indi-
cated their use as blood platelets aggregation inhibitors, antiin-
flammatories [4b] and anticancer agents with low toxicity [5a,b].
As well as, it showed antiparasitic, antifungal and antibacterial
activities [6aec]. Other derivatives are also used as fluorescent
dyes [7a] or dispersed dyes for polyester fibers [7b]. Very recently,
a microwave-assisted synthesis of fused pyrazolo[3,4-b]pyrazines
by the reaction of ortho-aminonitrosopyrazoles and cyclic
2.1. Chemistry
In continuation to our interest in the synthesis of pyrazole-
based heterocycles [9e13], and in particular the synthesis pyr-
azolo[3,4-b]pyrazines of antiparasitic, antifungal and antibacterial
activities [6aec] we wish to report herein the first synthesis of
3-methyl-1-phenyl-1H-indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one
(5), the pyrazole isostere of indeno[1,2-b] quinoxalin-11-one (6),
and to evaluate the biological activities of this new tetrafused
heterocycle and its derivatives.
O
N
N
6
Indeno[1,2-b]quinoxalin-11-one
The synthesis of 5 was achieved by intramolecular Friedele
Crafts reaction of the acid chloride 4 using AlCl3 in boiling CS2
(Scheme 1). The synthesis of 4 was described earlier by us [6a]
starting from the easily accessible 5-amino-3-methyl-4-nitroso-1-
phenyl-1H-pyrazole (1) which was interacted with benzoyl aceto-
nitrile to give the pyrazolopyrazine carbonitrile 2. Alkaline hydro-
lysis of 2 gave the acid 3 followed by treatment of 3 with thionyl
chloride at reflux to give the acid chloride 4 as shown in Scheme 1.
b
-diketones was reported [8].
* Corresponding author. Tel.: þ20 882412264; fax: þ20 882342708.
E-mail address: emarytalaat@yahoo.com (T. El-Emary).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.025

T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
479
CN
COOH
N
O
O
N
N
CN
(ii)
(iii)
(i)
+
N
N
NH2
N
N
N
N
N
N
1
2
3
O
COCl
N
N
(iv)
N
N
N
N
N
N
5
4
Scheme 1. Synthesis of compounds 1e5. Reagent and conditions: i) pyridine, reflux 3 h.; ii) aq. NaOH (20%), reflux 6 h.; iii) SOCl2, reflux, 2 h.; iv) AlCl3/CS2, reflux, 6 h.
Several derivatives were obtained from the versatile compound
5. Thus, 5 was condensed with some active methylene compounds
such as malononitrile, acetylacetone, ethyl acetoacetate, ethyl
cyanoacetate, benzoyl acetonitrile, and diethyl malonate to give the
corresponding indenopyrazolopyrazin-5-ylidene derivatives 7e12
respectively (Scheme 2).
A series of anils 13aee were obtained when 5 was interacted
with the corresponding aromatic amines. The reaction of 5, in
boiling pyridine, with hydroxylamine hydrochloride, semicarbazide
hydrochloride and thiosemicarbazide gave the corresponding
oxime 14, semicarbazone 15 and thiosemicarbazone 16 respectively.
The hydrazones 17 and18 were obtained viathe reaction of 5 with
hydrazine hydrate and phenyl hydrazine respectively (Scheme 2).
It is of interest to note that when 7 was reacted with hydrazine
hydrate the product was identified as the hydrazone 17 and not the
expected diamino product 19 (Scheme 3).
When the hydrazone 17 was reacted with some aromatic alde-
hydes the corresponding arylidene derivatives 20aed were
obtained and when it was reacted with triethyl orthoformate it
gave the ethoxymethylene derivative 21 (Scheme 4).
The thiosemicarbazone 16 was allowed to reacted with some
a-
halo compounds such as diethyl bromomalonate, ethyl chloroacetate
Scheme 2. Synthesis of compounds 7e18. Reagent and conditions: i) CH2(CN)2, dry pyridine, rt.; ii) CH2(COCH3)2, dry pyridine, reflux 10 h.; iii) CH3COCH2COOC2H5, dry pyridine,
reflux 10 h.; iv) NCCH2COOC2H5, dry pyridine, reflux 10 h. (v) C6H5COCH2CN, dry pyridine, reflux 6 h.(vi) CH2(COOC2H5)2 dry pyridine, reflux 10 h.; vii) x-C6H5NH2, dry pyridine,
reflux 8 h.; viii) NH2OH$HCl, dry pyridine, reflux 3 h.

480
T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
N N
N
N
N
H2N
H2N
N
NH2
N
N
(i)
N
N
N
N
N
N
N
N
N
7
17
19
Scheme 3. Synthesis of compound 17. Reagent and conditions: i) NH2NH2$H2O, reflux 15 min.
and/or phenacyl bromide in boiling ethanol in the presence of
anhydrous sodium acetate, to give the corresponding thiazolidinyli-
dene derivatives 22e24 (Scheme 5).
3. Conclusion
In conclusion, we have reported the synthesis novel heterocyclic
system; indeno[2,1-e]pyrazolo[3,4-b]pyrazine and its related de-
rivatives. Ten synthesized compounds have been screened for their
antibacterial and antifungal activities. The results obtained showed
that the compounds under investigation did not show significant
activity against the used bacterial strains. Meanwhile, 5 tested
compounds (16, 22, 23, 24, and 26b) showed weak inhibitory ac-
tivity against two fungal strains Trichophyton rubrum AUMC No.
1804 and A. flavus AUMC No. 3214.
On the other hand, the carbonyl group of 5 could be reduced
under WolffeKishner reduction conditions yielding the 3-methyl-
1-phenyl-1H,5H-indeno[2,1-e]pyrazolo[3,4-b]pyrazine (25). The
latter compound when reacted with some aromatic aldehydes in
refluxing ethanol in the presence of sodium ethoxide the corre-
sponding arylidene derivatives 26aec were formed, and the anils
27a,b were obtained when 25 was allowed to react with 4-nitroso-
N,N-diemethyl(or diethyl)aniline in refluxing ethanol in the pres-
ence of KOH (Scheme 6).
2.2. Biological results
4. Experimental
Ten selected indenopyrazolopyrazine derivatives (5, 10, 14, 16,
17, 22, 23, 24, 25, and 26b) were evaluated for their antibacterial
and antifungal activities.
4.1. Chemistry
All melting points were determined on a Kofler melting point
apparatus. IR spectra were recorded on a Pye Unicam SP3-100
spectrophotometer using KBr wafer technique. The 1H NMR
Thus, these compounds were screened against Staphylococcus
aureus, Bacillus cereus, Micrococcus luteus as a Gram positive bac-
teria and Escherichia coil, Pseudomonas aeruginosa, Serratia mar-
cescens as Gram negative bacteria. None of the tested compounds
showed significant activity against these bacterial strains (Table 1).
On the other hand, the compounds under testing (in a concen-
tration of 20 mg/ml) were screened for their antifungal activities
against fungal strains; (Candida albicans AUMC No. 418, Tricho-
phyton rubrum AUMC No. 1804, Aspergillus flavus AUMC No. 1276,
Fusarium oxysporum AUMC No. 5119, Scopulariopsis brevicaulis
AUMC No. 729, Geotrichum candidum AUMC No. 226 (Table 2). The
results indicated that compounds 16, 22, 23, 24 and 26b were active
against both Trichophyton rubrum AUMC No. 1804, A. flavus AUMC
No. 1276) [14]. The MIC of these compounds against the latter two
fungal strains was evaluated and the results were depicted in
Table 3. None of the results obtained was promising.
spectra were recorded on
a Bruker ARX 200 spectrometer
(200 MHz for 1H and 50 MHz for 13C) at the Faculty of Pharmacy,
University of Aix Marseille, France. 1H and 13C NMR chemical shifts
(d) were reported in parts per million (ppm) and were referenced
to the solvent peak; CDCl3 (7.26 ppm for 1H and 76.90 ppm for 13C)
and DMSO-d6 (2.50 ppm for 1H and 39.70 ppm for 13C). Multi-
plicities are represented by s (singlet), d (doublet), t (triplet), q
(quartet) and m (multiplet). Coupling constants (J) are reported in
Hertz (Hz). Mass spectra were taken on a Jeol JMS600 instrument
spectrometer at ionizing potential of 70 eV (EI) at Assiut University.
Elemental analyses were carried out using a PerkineElmer 240 C
Micro analyzer at Assiut University and they were found to be
within ꢀ0.4% of the theoretical values. Their results were found in
good agreement with the calculated values. The geometry around
X
O
N
H2N
N
N
N
N
N
N
N
ii
i
N
N
N
N
N
N
N
N
N
21
17
20a-d
a, X = H
b, X = 4-N(CH3)2
c, X = 4-NO2
d, X = 2-NO2
Scheme 4. Synthesis of compounds 20e21aed. Reagent and conditions: i) ArCHO, aq. NaOH, EtOH, reflux 3 h.; ii) CH(OEt)3 (5 ml), reflux 5 h.

T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
481
EtOOC
O
iii
i
16
S
N
NH
S
N
NH
O
N
N
S
N
NH
ii
N
N
N
N
N
N
N
N
N
N
24
22
N
N
N
23
Scheme 5. Synthesis of compounds 22e24. Reagent and conditions: i) BrCH(COOC2H5)2,fused CH3COONa, EtOH, reflux 10 h., i) C6H5COCH2Br, fused CH3COONa, EtOH, reflux 10 h.
the new double bond (E and/or Z) and the minimum energy of
each isomer was calculated and assigned according to ChemBio
3D11.0 (2008) program. Thus we depend on the minimum energy
calculation for the assignment of E-/and or in Z- isomers of the
compounds prepared.
was poured onto ice-water mixture. The precipitate thus formed
was collected by filtration, washed with water, dried and crystal-
lized from the proper solvent to afford 7e12.
4.1.2.1. 2-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-
5(1H)-ylidene) malononitrile (7). A mixture 5 (1.56 g, 0.005 mol)
and malononitrile (0.33 g, 0.005 mol) in pyridine (25 ml) was
stirred at room temperature for 15 min. The red precipitate formed
was filtered, dried and recrystallized fro DMF to give red needled,
4.1.1. 3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-5(1H)-
one (5)
A mixture of the acid chloride 4 [14] (1.74 g, 0.005 mol) and
anhydrous AlCl3 (2 g, 0.015 mol) in CS2 (10 ml) was heated under
reflux on a water bath for 6 h. After removal of the solvent under
reduced pressure diluted HCl was added to the residue and the
resulting mixture was stirred at 40 ꢁC for 1 h. The solid product
obtained was then collected, washed with water, dried and
recrystallized from ethanoledioxane (1:2) to give yellow crystals,
m.p. 314e316 ꢁC, yield quantitative. IR (KBr, cmꢂ1):
n
¼ 3050 (CH
arom.), 2910 (CH aliph.), 2210 (CN). 1H NMR, the product was
insoluble in most deuterated solvents. MS: 360.0 (100%), 361.1
(22.7%), 362.2 (3.97%), 344.8 (27.47.%), 318.9 (17.28%).
Anal. Calcd for C22H12N6 (360.37): C, 73.32; H, 3.36; N, 23.32.
Found: C, 73.23; H, 3.69; N, 23.63.
m.p. 227e229 ꢁC, yield 0.98 g (63%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH
arom.), 2910, 2850 (CH aliph.), 1715 (CO). 1H NMR (CDCl3):
d
¼ 2.55
4.1.2.2. 3-(Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-
5(1H)-ylidene)pentane-2,4-dione (8). A mixture of the oxo com-
pound 2 (0.624 g, 0.002 mol) and acetylacetone (0.2 g, 0.002 mol) in
dry pyridine (20 ml) was heated under reflux for 10 h. The cold
reaction mixture was poured onto iceewater and the solid product
formed was filtered, dried and crystallized from dioxaneewater
(3:2) to give fine yellowish-brown crystals, m.p. 281e283 ꢁC yield
(S, 3H, CH3), 7.15e8.18 (m, 9H, AreH). 13C NMR (CDCl3)
d ppm:
11.44,120.63 (2C),122.15,124.35,126.66, 129.17 (2C),132.32,134.80,
136.00, 136.20, 138.45, 140.34, 143.73, 144.32, 146.40, 158.41, 189.10.
Anal. Calcd. for C19H12N4O (312. 32) C, 73.07; H, 3.87; N, 17.94.
Found: C, 73.23; H, 3.89; N, 17.73.
4.1.2. General procedure for the reaction of 5 with active methylene
compounds. Formation of compounds 7e12
An equimolar mixture (0.002 mol) of compound 5 and the active
methylene compound in dry pyridine (20 ml) was heated under
reflux for 6 h. The reaction mixture was allowed to cool and then it
(38%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH arom.) 2910 (CH aliph.), 1720
(CO). 1H NMR (CDCl3):
d
ppm ¼ 2.65 (s, 6H, 2 COCH3), 2.85( s, 3H,
CH3), 7.22e8.53 (m, 9H, AreH).
Anal. Calcd. for C24H18N4O2 (394.43): C, 73.08; H, 4.60; N, 14.20.
Found: C, 73.23; H, 4.89; N, 14.33.
5
R
N
X
R
(i)
(ii) for 26a
(iii)for 26b
(iv) for 26c
N
(vi) for 27a
(vii) for 27b
N
(v) for 26d
N
N
N
N
N
N
N
N
N
N
25
N
27a,b
26a-d
a, R = CH3
b, R = C2H5
a, X = H
b, X = 2-Cl
c, X = 4-NO2
d, X = 4-N(CH3)2
Scheme 6. Synthesis of compounds 25e27a,b. Reagent and conditions: i) KOH, NH2NH2$H2O, HO(CH2)2OH, 150 ꢁC, 14 h.; ii) 4-(R)2NC6H4NO, C2H5ONa, EtOH, reflux 2 h.

482
T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
Table 1
CH2CH3), 2.68 (s, 3H, CH3), 4.52e4.63 (q, 2H, CH2CH3), 7.36e8.55 (m,
9H, AreH).13C NMR (CDCl3)
ppm: 11.24,13.91, 63.34,101.20,115.03,
120.61, 120.70(2C), 122.68, 125.46, 126.56, 129.24(2C), 132.00,
133.28, 136.18, 137.49, 138.71, 143.81, 144.88, 145.27, 153.51, 161.64.
Anal. Calcd. for C24H17N5O2 (407.42): C, 70.75; H, 4.21; N, 17.19.
Found: C, 70.43; H, 4.11; N, 17.13.
Antibacterial activity of tested compounds at 20 mg concentration, (inhibition zone
in mm).
d
Compound no.
Bacterial strains
5
10 14 16 17 22 23 24 25 26b Control
Staphylococcus aureus
(þve) AUMC No. B-54
Bacillus cereus
8
0
8
0
8
8
0
0
8
0
0
8
0
0
8
0
0
0
0
0
8
0
8
0
0
0
8
0
8
8
0
0
10 10
8
0
0
0
0
0
10 18
0
8
8
0
0
0
8
0
0
0
0
8
8
0
0
22
18
18
20
20
4.1.2.5. (Z)
2-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyr-
(þve) AUMC NoB-52
azin-5(1H)-ylidene)-3-oxo-3-phenylpropanenitrile (11). A mixture
of the oxo compound 2 (0.62 g, 0.002 mol) and benzoyl acetonitrile
(0.29 g, 0.002 mol) in dry pyridine (20 ml) was heated under reflux
for 6 h. The cold reaction mixture was then poured onto iceewater.
The precipitate that formed was collected by filtration washed with
water, dried and crystallized from DMF-H2O (1:1) to give small
brownish-yellow needles, m.p. 294e296 ꢁC, yield 0.51 g (58%). IR
Escherichia coli
10
0
(ꢂve) AUMC No. B-53
Pseudomonas aeruginosa
(ꢂve) AUMC No. B-73g
Serratia marcescens
(ꢂve) AUMC No. B-55
Micrococcus luteus
0
0
(þve) AUMC NoB-112
(KBr, cmꢂ1):
(CO). 1H NMR (CDCl3):
14H, AreH). 13C NMR (CDCl3)
n
¼ 3050 (CH arom.) 2900 (CH aliph.), 2200 (CN), 1681
ppm ¼ 2.73 (s, 3H, CH3), 7.32e8.28 (m,
ppm: 10.86, 107.59, 115.50,
The amount added in each pore is 50
m
L.
d
p.i. ¼ Partial Inhibition.
d
AUMC ¼ Assiut University Mycological Center.
Control ¼ Chloramphenicol as antibacterial standard.
120.62(2C), 122.73(2C), 125.44(2C), 126.46(2C), 128.90 (2C),
129.20(2C), 129.45(2C), 131.99, 133.11, 134.28, 136.08, 136.29,
138.69, 145.04, 145.21, 145.82, 153.37, 188.06. MS: 439.19 (0.6%),
439.97 (0.4%), 440.99 (0.2%), 411.86 (30.1%), 400.20 (38.9%).
Anal. Calcd. for C28H17N5O (439.47): C, 76.52; H, 3.90; N, 15.94.
Found: C, 76.23; H, 3.89; N, 15.73.
4.1.2.3. (Z) Ethyl 2-(3-methyl-1-phenyl-1H,5H-indeno[2,1-e]pyrazolo
[3,4-b]pyrazin-5(1H)-ylidene)oxobutanoate (9). A mixture of the
oxo compound 2 (0.624 g, 0.001 mol) and ethyl acetoacetate
(0.26 g, 0.001 mol) in dry pyridine (20 ml) was heated under reflux
for 10 h. The cold reaction mixture was poured onto iceewater
and the solid product formed was filtered, dried and crystallized
from dioxaneewater (5:2) to give fine yellowish-brown crystals,
4.1.2.6. Diethyl
2-(3-methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]
pyrazin-5(1H)-ylidene) malonate (12). A mixture of the oxo com-
pound 2 (0.624 g, 0.002 mol) and diethyl malonate (0.32 g,
0.002 mol) in dry pyridine (20 ml) was heated under reflux for 10 h.
The cold reaction mixture was poured onto iceewater and the solid
product formed was filtered, dried and crystallized from dioxanee
water (5:2) to give fine yellowish-brown crystals, m.p. 231e233 ꢁC
m.p. 234e236 ꢁC, yield 0.4 g (49%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH
arom.), 2950, 2920 (CH. aliph.), 1728(CO). 1H NMR (CDCl3):
d
ppm ¼ 1.32e1.52 (t, 3H, CH2CH3), 2.62 (s, 3H, COCH3), 2.92 (s, 3H,
CH3), 4.25e4.50 (q, 2H, CH2CH3), 7.35e8.52 (m, 9H, Ar).
Anal. Calcd. for C25H20N4O3 (424. 45): C, 70.74; H, 4.75; N, 13.20.
Found: C, 70.53; H, 4.89; N, 13.33.
yield (38%). IR (KBr, cmꢂ1):
1720 (CO). 1H NMR (CDCl3):
(s, 3H, CH3), 4.15e4.35 (q, 4H, CH2CH3), 6.56e8.32 (m, 9H, Ar-H).
Anal. Calcd. for C26H22N4O4 (454.48): C, 68.71; H, 4.88; N, 12.33.
Found: C, 68.63; H, 4.89; N, 13.23.
n
¼ 3500 (CH arom.) 2910 (CH aliph.),
d
ppm ¼ 1.11e1.28 (t, 6H,CH2CH3), 2.53
4.1.2.4. (E) Ethyl 2-cyano-2(3-methyl-1-phenylindeno[2,1-e]pyrazolo
[3,4-b]pyrazin-5(1H)-ylidene)acetate (10). A mixture of 5 (0.624 g,
0.002 mol) and ethyl cyanoacetate (0.226 g, 0.001 mol) in dry pyr-
idine (10 ml) was heated under reflux for 10 h. The cold reaction
mixture was poured onto iceewater and the solid product formed
was filtered, dried and crystallized from dioxaneewater (5:2) to give
brownish-yellow crystals, m.p. 286e288 ꢁC, yield 0.47 g (58%). IR
4.1.3. Generalprocedure for the reaction of compound 5 with aromatic
amines. Formation of 13aee
An equimolar mixture of 5 (0.002 mol) and appropriate aromatic
amine namely (aniline, 2-chloroaniline, 2-phenelenediamine, 2-
nitroaniline and 4-nitroaniline) in dry pyridine (20 ml) was heat-
ed under reflux for 8 h. After cooling to room temperature the re-
action mixture was then poured onto iceewater mixture. The
precipitate thus formed was filtered, washed with water, dried and
crystallized from the appropriate solvent.
(KBr, cmꢂ1):
n
¼ 3500 (CH arom.) 2910 (CH aliph.) 2200 (CN) 1700
(CO). MS, 407 (58%). 1H NMR (CDCl3):
d
ppm ¼ 1.43e1.55 (t, 3H,
Table 2
Antifungal sensitivity (inhibition zone in mm) of the tested compounds at 20 mg
concentration.
4.1.3.1. (E)
N-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyr-
Compound no.
Fungal strains
5 10 14 16
17 22 23 24 25 26b Clotrimazole
azin-5(1H)-ylidene)aniline (13a). Yellowish-orange needles from
dioxaneewater (1:1), m.p. 229e231 ꢁC, yield 0.35 g (46%). IR (KBr,
cmꢂ1):
d
n
¼ 3050 (CH arom.) 2910 (CH aliph.). 1H NMR (CDCl3):
Candida albicans
AUMC No. 418
0 10 0
0
0
0
0
0
0
0
0
0 0
0
24
ppm ¼ 2.82 (s, 3H, CH3), 6.70e8.37 (m, 14H, AreH). 13C NMR
Trichophyton rubrum
AUMC No. 1804
Aspergillus flavus
AUMC No. 1276
Fusarium oxysporum
AUMC No. 5119
Scopulariopsis brevicaulis 0 0 0
AUMC No. 729
Geotrichum candidum
AUMC No. 226
0
0
0
0 0 30
0 0 16
30 26 30 0 26 36
(CDCl3) d ppm: 11.75,118.09 (2C),120.66(2C),122.43,124.62,126.32,
126.92, 129.20(2C), 129.36(2C), 131.15, 132.86, 133.32, 134.42,
138.95, 139.51, 144.39, 145.55, 147.11, 151.35, 154.87, 158.53.
Anal. Calcd. for C25H17N5 (387.44): C, 77.50; H, 4.42; N, 18.08.
Found: C, 77.23; H, 4.23; N, 18.23.
13 12 16 0
9
0
0
0
44
20
28
26
0 0
0
0
0
0
0
0
0 0
0 0
4.1.3.2. (E) 2-Chloro-N-(3-methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-
b]pyrazin-5(1H)-ylidene)aniline (13b). Crystallised from dioxanee
water (1:1) to give brownish-yellow crystals, m.p. 164e166 ꢁC,
0
0 0 17 pi. 0 10 10 10 0
The amount added in each pore is 50 mL.
yield 0.55 g (41%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH arom.) 2910 (CH
p.i. ¼ Partial Inhibition.
aliph.). 1H NMR (CDCl3):
d
ppm ¼ 2.65 (s, 3H, CH3), 7.35e8.35 (m,
AUMC ¼ Assiut University Mycological Center.
Clotrimazole (as antifungal reference drug).
13H, AreH).

T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
483
Table 3
4.1.4.2. (Z)
2-(3-Methyl-1-phenyindeno[2,1-e]pyrazolo[3,4-b]pyr-
Antifungal activity of 16, 22, 23, 24 and 26b.
azin-5(1H)-ylidene) hydrazinecarboxamide (15). Yellow crystals
from dioxane, m.p. 267e269 ꢁC, yield 0.45 g (61%). IR (KBr, cmꢂ1):
Sample no.
Trichophyton rubrum
AUMC no. 1804
Aspergillus flavus AUMC
no. 3214
n
¼ 3400e3200 (NH2 þ NH), 3050 (CH arom.), 2900 (CH aliph),
1650 (CO), 1H NMR (CDCl3):
ppm ¼ 2.72 (s, 3H, CH3), 3.73 (s, 1H,
NH), 6.45 (s, 2H, NH2) 7.25e8.35 (m, 9H, AreH) Anal. Calcd. for
20H15N7O (369.38): C, 65.03; H, 4.09; N, 26.54. Found: C, 65.32; H,
d
Inhibition
MIC
Inhibition
MIC
zone (mm)
(mg/mL)
zone (mm)
(mg/mL)
C
16
22
24
23
14
16
14
12
10
35
0.3
0.3
0.3
0.6
0.6
0.08
14
10
12
10
9
2.5
2.5
2.5
5
3.94; N, 26.23.
4.1.4.3. (Z)
2-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyr-
26b
20
Clotrimazolea
15
0.15
azin-5(1H)-ylidene) hydrazinecarbothioamide (16). Yellow crystals
from DMFeH2O (3:1), m.p. 264e266 ꢁC, yield 0.45 g (61%). IR (KBr,
a
Clotrimazole was used as standard reference drug.
cmꢂ1):
n
¼ 3400e3200 (NH2 þ NH), 3050 CH (arom.), 2900 CH.
aliph). 1H NMR (CDCl3):
ppm ¼ 2.73 (s, 3H, CH3), 3.70 (s, 1H, NH),
6.49 (s, 2H, NH2) 7.26e8.30 (m, 9H, AreH). 13C NMR (CDCl3)
ppm:
d
Anal. Calcd. for C25H16ClN5(421.88): C, 71.17; H, 3.82; N, 16.60.
Found: C, 71.23; H, 3.89; N, 16.73.
d
11.27, 120.60, 120.80, 122.43, 122.45, 126.51, 129.20, 129.23, 131.06,
131.68, 132.39, 134.88, 135.67, 137.69, 144.62, 144.49, 153.83, 158.54,
179.88.
Anal. Calcd. for C20H15N7S (385.44): C, 62.32; H, 3.92; N, 25.44.
Found: C, 62.23; H, 3.89; N, 25.73.
4.1.3.3. (E) N1-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyr-
azin-5(1H)-ylidene) benzene-1,2- diamine (13c). Crystallised from
dioxaneewater (1:1) to give brownish-red needles, m.p. 246e
248 ꢁC, yield 0.42 g (53%). IR (KBr, cmꢂ1):
3050 (CH arom.) 2910 (CH aliph.).
n
¼ 3400e3300 (NH2)
4.1.5. (E) 5-Hydrazono-3-methyl-1-phenyl-1,5-dihydroindeno[2,1-
e]pyrazolo[3,4-b]pyrazine (17)
1H NMR (CDCl3):
d
ppm ¼ 2.73 (s, 3H, CH3), 5.88 (s, 2H, NH2),
7.26e8.37 (m, 13H, AreH). MS: 401.62 (85.5%), 402.08 (27.6%),
403.72 (19.3), 312.3 (100%).
Anal. Calcd. for C25H18N6 (402.45): C, 74.61; H, 4.51; N, 20.88.
Found: C, 74.53; H, 4.89; N, 20.73.
4.1.5.1. Method A. A mixture of compound 5 (1.56 g, 0.005 mol) and
excess hydrazine hydrate (80%, 5 ml) in ethanol (20 ml) was heated
under reflux for half an hour. The solid product formed was filtered,
washed with water, dried and recrystallized from dioxaneeethanol
(1:1) to give small yellow needles, m.p. 236e288 ꢁC, yield 1.1 g
4.1.3.4. (E) N-(3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-
5(1H)-ylidene)-2-nitroaniline (13d). Crystallised from dioxanee
water (3:1) to give brownish-red needles, m.p. 261e263 ꢁC, yield
(66%). IR (KBr, cmꢂ1):
(CH aliph.), 1H NMR (CDCl3):
9H, AreH), 9.85 (NH2). 13C NMR (CDCl3)
n
¼ 3328e3175 (NH2), 3050 (CH arom.) 2900
ppm ¼ 2.65 (s, 3H, CH3), 7.25e8.7 (m,
ppm: 11.40, 119.90,
d
d
0.42 g (53%). IR (KBr, cmꢂ1):
NMR (CDCl3):
n
¼ 3040 (CH arom.) 2920 (CH aliph.).1H
120.59(4C), 122.01, 126.07, 128.16, 129.16(4C), 131.31, 133.26, 134.56,
139.17, 139.83, 142.66, 143.63, 143.98, 152.50. MS: 326.1 (100%),
327.1 (27.9%), 328.1 (3.69%), 297.9 (38.3%), 282.8 (17.6%).
Anal. Calcd. for C19H14N6 (326.35): C, 69.92; H, 4.32; N, 25.75.
Found: C, 69.73; H, 4.59; N, 25.73.
d
ppm ¼ 2.73 (s, 3H, CH3), 7.35e8.35 (m, 13H, AreH).
Anal. Calcd. for C25H16N6O2 (432.43): C, 69.44; H, 3.73; N,19.43.
Found: C, 69.23; H, 3.82; N, 19.73.
4.1.3.5. (E) N-(3-methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-
5(1H)-ylidene)-4-nitroaniline. (13e). Reddish-brown crystals from
DMFeH2O (2:1), m.p. 258e260 ꢁC, yield 0.46 g (53%). IR (KBr, cmꢂ1):
4.1.5.2. Method B. A mixture of 7 (0.36 g, 0.001 mol) and excess
hydrazine hydrate (2 ml, 80%) was heated under reflux for 15 min.
The heavy yellowish precipitate filtered, washed with water, dried
and recrystallized from dioxaneeethanol (1:1) to give small yellow
needles, m.p., mixed m.p. (238 ꢁC) and IR spectrum were found to
be identical with those of an authentic sample (method A), yield
0.24 g (68%).
n
¼ 3050 (CH arom.), 2970 (CH aliph.). 1H NMR (DMSO-d6):
ppm ¼ 2.70 (s, 3H, CH3), 7.32e8.35 (m, 13H, AreH).
d
Anal. Calcd. for C26H19N6O2 (447.47): C, 69.79; H, 4.28; N, 18.78.
Found C, 69.58; H, 4.32; N, 18.89.
4.1.4. Reaction of compound 5 with hydroxylamine hydrochloride,
semicarbazide hydrochloride and thiosemicarbazide. Formation of
compounds 14e16
4.1.6. (Z) 3-Methyl-1-phenyl-5-(2-phenylhydrazono)1,5-dihydroin-
deno[2,1-e]pyrazolo[3,4-b] pyrazine (18)
An equimolar mixture of compound 5(0.002 mol) and hydrox-
ylamine hydrochloride, semicarbazide hydrochloride or thio-
semicarbazide in dry pyridine (15 ml) was heated under reflux for
3 h. After cooling, the reaction mixture was then poured onto icee
water mixture. The solid precipitate thus formed was then filtered,
washed with water, dried and crystallized the proper solvent to
give compounds 14e16.
A mixture of 5 (0.312 g, 0.001 mol) and excess phenyl hydrazine
(2 ml) was heated for 15 min (no solvent at reflux). The orange
precipitate was filtered washed with water, dried and crystallized
from dioxane to give orange needles, m.p. 294e296 ꢁC, yield 0.27 g
(68%). IR (KBr, cmꢂ1):
n
¼ 3220 (NH) 3050 (CH arom.) 2910 (CH
aliph.). 1H NMR (CF3COOD):
d
ppm ¼ 2.95 (s, 3H, CH3), 7.75 (m, 14H,
AreH).
Anal. Calcd. for C25H18N6 (402.45): C, 74.61; H, 4.51; N, 20.88.
Found: C, 74.23; H, 4.82; N, 20.73.
4.1.4.1. (Z) 3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]pyrazin-
5(1H)-one-oxime (14). Yellow needles from ethanoledioxane (1:1),
m.p. 304e306 ꢁC, yield 0.95 g (58%). IR (KBr, cmꢂ1):
n
¼ 3200 (OH),
4.1.7. General procedure for the reaction of the hydrazono derivative
17 with some aromatic aldehydes. Formation of compounds 20ae
d25 ml
To an equimolar mixture of 17 (0.02 mol) and the appropriate
aldehyde in ethanol (), aqueous sodium hydroxide solution (5 ml,
20%) was added and the mixture was allowed to reflux for 3hr. After
cooling, the obtained precipitate was filtered, washed with water,
dried and crystallized from the suitable solvent.
3050 (CH arom.) 2900 (CH aliph.). 1H NMR (DMSO-d6):
d
ppm ¼ 2.93 (s, 3H, CH3), 7.73 (m, 9H, AreH), 9.9 (s, 1H, OH). 13
C
NMR (CDCl3)
d
ppm: 11.33, 120.12(2C), 121.92, 126.20, 128.63,
129.48(4C), 132.21, 132.35, 133.07, 135.54, 138.83, 143.39, 144.06,
146.87, 152.11.
Anal. Calcd. for C19H13N5O (327.34) C, 69.71; H, 4.00; N, 21.39.
Found: C, 69.83; H, 3.89; N, 21.53.

484
T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
4.1.7.1. (Z) 5-(E)(Benzylidenehydrazono)-3-methyl-1-phenyl-1,5-
dihydroindeno[2,1-e] pyrazolo[3,4-b]pyrazine (20a). As reddish-
brown fine needles, from dioxaneewater (3:1), m.p. 241e243 ꢁC,
3.75 (s, 1H, CH), 4.15e4.30 (q, 2H, CH2CH3), 7.35e8.35 (m, 9H,
AreH).
Anal. Calcd. for C25H19N7O3S (497.53): C, 60.35; H, 3.85; N, 19.71.
Found: C, 60.23; H, 3.89; N, 19.73.
yield 0.46 g (56%). IR (KBr, cmꢂ1):
n
¼ 3050, 3020 (CH, arom.),
2960, 2920(CH, aliph.). 1H NMR (CDCl3): 2.72 (s, 3H, CH3), 7.25e
8.35 (m, 14H, AreH), 8.99 (s, 1H, CH).
4.1.8.2. (Z)
2-(E)((3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]
Anal. Calcd. for C26H18N6 (414.46): C, 75.35; H, 4.38; N, 20.28.
Found: C, 75.23; H, 4.59; N, 20.53.
pyrazin-5(1H)-ylidene) hydrazono)thiazolidine-4-one (23). Bro-
wnish-orange crystals from DMF-water (3:1), m.p. 238e240 ꢁC,
yield 0.54 g (47%). IR (KBr, cmꢂ1):
n
¼ 3335 (NH), 3050 (CH,
4.1.7.2. N,N-Dimethyl-4-(E)-(Z)((3-methyl-1-phenylindeno[2,1-e]
pyrazolo[3,4-b]pyrazin-5 (1H)-ylidene)hydrazinyl)aniline (20b).
Golden needles from DMFeH2O (2:1), m.p. 173e175 ꢁC, yield 0.48 g
arom.), 2950, 2920, 2890 (CH, aliph.). 1H NMR (CDCl3): 2.76 (s, 3H,
CH3), 4.15 (s, 2H, CH2), 7.35e8.35 (m, 9H, AreH).
Anal. Calcd. for C22H15N7OS (425.47): C, 62.10; H, 3.55; N, 23.04.
Found: C, 62.43; H, 3.65; N, 23.13.
(53%). IR (KBr, cmꢂ1):
n
¼ 3070, 3020 (CH. arom). 2970, 2930 (CH.
aliph.) 1H NMR (CDCl3) 2.65 (s, 3H, CH3), 2.95 (s, 6H, 2CH3), 6.65e
8.50 (m, 13H, AreH), 8.9 (s, 1H, CH).
4.1.8.3. (Z)
2-(E)((3-Methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-b]
Anal. Calcd. for C28H23N7 (457.53): C, 73.50; H, 5.07; N, 21.43.
Found: C, 73.23; H, 5.21; N, 21.73.
pyrazin-5(1H)-ylidene) hydrazono)-4-phenyl-2,3-dihydrothiazole
(24). Yellowish-brown crystals, from DMFewater (3:1), yield 0.46
(47%), m.p. 282e284 ꢁC, IR (KBr, cmꢂ1):
n
¼ 3300 (NH), 3050 (CH,
arom.), 2920 (CH, aliph.). 1H NMR: (CDCl3), 2.84 (s, 3H, CH3), 7.03 (s,
1H, CH), 7.33e8.35 (m, 14H, AreH). 13C NMR (CDCl3)
ppm: 11.66,
4.1.7.3. (Z) 3-(E)-Methyl-5-((4-nitrobenzylidene)hydrazono)-1-
phenyl-1,5-dihydroindeno [2,1-e] pyrazolo[3,4-b]pyrazine (20c).
Brownish-red crystals from DMF-H2O (4:1), yield 0.53 g (58%), m.p.
d
105.02, 120.69(3C), 121.03, 122.23, 125.99(3C), 127.92, 128.67(3C),
129.18(3C), 129.66, 131.56, 134.56, 138.34, 138.93, 142.98, 143.30,
144.12, 152.16, 153.38, 167.39.
Anal. Calcd. for C28H19N7S (485.56): C, 69.26; H, 3.94; N, 20.19.
Found: C, 69.23; H, 3.89; N, 20.73.
244e246 ꢁC. IR (KBr, cmꢂ1):
n
¼ 3020 (CH, arom.), 2950, 2920(CH,
aliph.). 1H NMR (DMSO-d6) 2.75 (s, 3H, CH3), 7.45e8.30 (m, 13H,
AreH), 8.85 (s, 1H, CH).
Anal. Calcd. for C26H17N7O2 (459.46): C, 67.97; H, 3.73; N, 21.34.
Found: C, 67.73; H, 3.81; N, 21.53.
4.1.8.4. 3-Methyl-1-phenyl-1,5-dihydroindeno[2,1-e]pyrazolo[3,4-b]
pyrazine (25). A mixture of compound 5 (1.56 g, 0.005 mol), KOH
(0.75 g, 0.014 mol) and hydrazine hydrate (2 ml) in ethylene
glycol (30 ml) was heated under reflux in an oil bath at 150 ꢁC for
14 h. After cooling, the reaction mixture was poured onto water
and acidified with HCl. The solid product was filtered off, washed
with water, dried and crystallized from dioxaneewater (1:1) as
buff crystals, m.p. 175e177 ꢁC, yield 0.6 g (52%). IR (KBr, cmꢂ1):
4.1.7.4. (Z)-3-(E)(Methyl-5-(2-nitrobenzylidene)hydrazono)-1-
phenyl-1,5-dihydroindeno[2,1-e]pyrazolo[3,4-b]pyrazine
(20d).
Brownish-red crystals from DMFeH2O (2:1), mp 236e238 ꢁC,
yield 0.47 g (51%) IR (KBr, cmꢂ1):
n
¼ 3050 (CH arom.), 2920 (CH
aliph.), 1H NMR (DMSO-d6): 2.75 (s, 3H, CH3), 7.40e8.50 (m, 13H,
AreH), 8.65 (s, 1H, CH).
Anal. Calcd. for C26H17N7O2 (459.46): C, 67.97; H, 3.73; N, 21.34.
Found: C, 67.73; H, 3.89; N, 21.73.
n
¼ 3010 (CH arom) 2910 (CH aliph.). 1H NMR: (CDCl3): 2.76 (s,
3H, CH3), 4.07 (s, 2H, CH2), 7.32e8.39 (m, 9H, AreH). 13C
4.1.7.5. (1Z,N’Z) Ethyl 2-((3-methyl-1-phenylindeno[2,1-e]pyrazolo
[3,4-b]pyrazin-5(1H)-ylidene) hydrazono)acetate (21). A mixture of
the hydrazono derivative 14 (0.02 mol) and triethyl orthoformate
(5 ml) was heated under reflux for 4 h. The reaction mixture was
evaporated under reduced pressure and the residue was triturated
with cold ethanol, filtered, dried and crystallized from ethanol to
give yellow crystals, yield 0.4 g (52%) m.p. 161e163 ꢁC. IR (KBr,
NMR (CDCl3) d ppm: 11.40, 35.41, 120.35(2C), 122.46, 125.56,
125.71, 127.66, 127.80, 129.10(2C), 130.61, 132.41, 138.07, 139.44,
143.03, 144.93, 153.18, 154.82. MS: 298.01 (100%), 298.36 (0.4%),
298.76 (0.2%).
Anal. Calcd. For C19H14N4 (298.34): C, 76.49; H, 4.73; N, 18.78.
Found: C, 76.23; H, 4.82; N, 18.73.
cmꢂ1):
n
¼ 3020 (CH, arom.), 2960, 2920 (CH, aliph.). 1H NMR
4.1.9. General procedure for the reaction of the indeno derivative 25
with some aromatic aldehydes. Formation of 26aec
(CDCl3): 1.13e1.35 (t, 3H, CH2CH3), 2.75(s, 3H, CH3), 4.15e4.40 (q,
4H, CH2CH3), 7.35e8.75 (m, 10H, AreH þ CH).
An equimolar mixture of the indeno derivative 25 (0.002 mol),
the appropriate aromatic aldehyde and sodium ethoxide (0.002 mol)
in absolute ethanol (25 ml) was heated under reflux for 2 h. The
reaction mixture was poured onto water, neutralized with diluted.
HCl. The solid precipitate formed was filtered off, washed with wa-
ter, dried and recrystallized from proper solvent.
Anal. Calcd. for C22H18N6O (382.42): C, 69.10; H, 4.74; N, 21.98.
Found: C, 69.23; H, 4.89; N, 21.73.
4.1.8. General procedure for the reaction of the thiosemicarbazone
16 with some
An equimolar (0.002 mol) mixture of the thiosemicarbazone 16
the respective -halocarbonyl compound (diethyl bromomalonate,
a-halocarbonyl compounds. Formation of 22e24
a
4.1.9.1. (E) 5-Benzyledine-3-methyl-1-phenyl-1,5-dihydrindeno[2,1-
e]pyrazolo[3,4-b] pyrazine (26a). Small Orange-red flakes from
dioxaneeH2O (2:1), m.p. 231e233 ꢁC, yield 0.42 g, (54%). IR (KBr,
ethyl chloroacetate and/or phenacyl bromide), and fused sodium
acetate (0.005 mol) in absolute ethanol (25 ml) was refluxed for
10 h. After cooling the reaction mixture was poured onto cold
water, and the precipitate formed was filtered off, washed with
water, dried and crystallized from the proper solvent.
cmꢂ1):
n
¼ 3010 (CH arom.) 2950 (CH aliph.). 1H NMR (CDCl3): 2.85
(s, 3H, CH3), 7.35e8.30 (m, 14H, AreH), 8.9 (s, 1H, CH), MS: 385
(100), 386 (75%), 387 (18%), 344.1 (7.1%), 308.9 (0.4%).
Anal. Calcd. for C26H18N4 (386.45): C, 80.81; H, 4.69; N, 14.50.
Found: C, 80.68; H, 4.89; N, 14.73.
4.1.8.1. (Z) Ethyl-2-(E)((3-methyl-1-phenylindeno[2,1-e]pyrazolo[3,4-
b]pyrazin-5(1H)-ylidene) hydrazono)-4-oxothiazolidine-5-
carboxylate (22). As reddish-brown crystals, from dioxaneewa-
ter (3:1), m.p. 244e246 ꢁC, yield 0.42 (42%). IR (KBr, cmꢂ1):
4.1.9.2. (E) 5-(2-Chlorobenzyledine)-3-methyl-1-phenyl-1,5-
dihydroindeno[2,1-e]pyrazolo[3,4-b]pyrazine (26b). Reddish brown
needles, m.p. 247e249 ꢁC from dioxaneewater (3:1), yield 0.46 g
n
¼ 3270 (NH), 3050 (CH, arom.), 2950, 2920, 2890 (CH, aliph.).
1H NMR (CDCl3), 1.10e1.25 (t, 3H, CH2CH3), 2.75 (s, 3H, CH3),
(53%), IR (KBr, cmꢂ1):
n
¼ 3020 (CH arom) 2950C (CH aliph.). 1H

T. El-Emary, H. El-Kashef / European Journal of Medicinal Chemistry 62 (2013) 478e485
485
NMR (CDCl3): 2.70 (s, 3H, CH3), 7.25e8.35 (m, 13H, AreH), 8.95 (s,
1H, CH).
Anal. Calcd. for C26H17ClN4 (420.89): C, 74.19; H, 4.07; N, 13.31.
Found: C, 74.23; H, 4.19; N, 13.23.
animal diseases (Trichophyton rubrum, Candida albicans, Geo-
trichum candidum, S. brevicaulis, A. flavus), plant diseases (F. oxy-
sporum) or frequently reported from contaminated soil, water and
food substances (Escherichia coli, B. cereus, Pseudomonas aeruginosa,
S. marcescens, S. aureus, M. luteus).
4.1.9.3. (E) 3-Methyl-5-(4-nitrobenzylidene)-1-phenyl-1,5-dihydroin-
deno[2,1-e]pyrazolo[3,4-b]pyrazine (26c). Reddish-brown needles,
m.p. 287e289 ꢁC from DMFewater (3:1), yield 0.46 g (53%), IR (KBr,
To prepare inocula for bioassay, bacterial strains were individ-
ually cultured for 48 h in 100 ml conical flasks containing 30 ml
nutrient broth medium. Fungi were grown for 7 days in 100 ml
conicals containing 30 ml Sabouraud’s dextrose broth.
Bioassay was done in 10 cm sterile plastic Petri plates in which
microbial suspension (1 ml/plate) and 15 ml of appropriate agar
medium (15 ml/plate) were poured. Nutrient agar and Sabouraud’s
dextrose agar were respectively used for bacteria and fungi. After
solidification of the media, 5 mm diameter cavities were cut in the
solidified agar (4 cavities/plate) using sterile cork borer. The tested
compounds were dissolved in dimethyl sulfoxide (DMSO) at 2%w/v
cmꢂ1):
n
¼ 3020 (CH arom.) 2950C (CH aliph.). 1H NMR (CDCl3): 2.65
(s, 3H, CH3), 7.35e8.45 (m, 13H, AreH).
Anal. Calcd. for C26H17N5O2 (431.45): C, 72.38; H, 3.97; N, 16.23.
Found: C, 72.23; H, 3.89; N, 16.43.
4.1.9.4. (E) N,N-Dimethyl-4-((3smethyl-1-phenylindeno[2,1-e]pyrazolo
[3,4-b]pyrazin-5(1H)-ylidene)methyl)aniline (26d). Orange needles,
m.p. 241e243 ꢁC from DioxaneeH2O 3:1, yield 0.44 g (51%). IR
(KBr, cmꢂ1):
n
¼ 3020 (CH arom.) 2900 (CH aliph.). 1H NMR (CDCl3):
(¼20 mg/ml), pipetted and poured in the cavities (20
mL/cavity).
2.75 (s, 3H, CH3), 2.97 (s, 6H, 2CH3), 6.60e8.50 (m,13 H, AreH), 9.0 (s,
1H, CH).
Cultures were then incubated at 28 ꢁC for 48 h in case of bacteria
and up to 7 days in case of fungi. Results were read as the diameter
(in mm) of inhibition zone around cavities [14].
Anal. Calcd. for C28H23N5 (429.52): C, 78.30; H, 5.40; N, 16.31.
Found: C, 78.23; H, 5.69; N, 16.13.
To determine the minimum inhibitory concentrations (MICs),
several concentrations in DMSO, of the compounds under testing
that gave positive results, have been prepared in descending
manner down to a concentration of 0.02 mg/ml. The solutions of
different compounds were similarly assayed as mentioned before
and the least concentration (below which no activity was observed)
was recorded as the MIC.
4.1.10. General procedure for the reaction of 25 with 4-nitroso-N,N-
dimethyl(and/or diethyl)aniline. Formation of compounds 27a,b
To an equimolar mixture of 25 (0.002 mol) and 4-nitroso-N,N-
dimethyl (and/or N,N-diethyl) aniline in ethanol (25 ml), 5 ml of an
aqueous KOH (20%) was added and the mixture was heated under
reflux for 2 h. The solid product formed after cooling was filtered
off, washed with water, dried and crystallized from ethanol.
References
[1] A. Akahane, S. Kuroda, H. Itani, S. Tabuchi, Y. Sato, N. Matsuoka, M. Tada, H.
Matsuoka, T. Oku, A. Tanaka, WO 2001040230, 2001, Chem. Abstr. 135 (2001)
33489a.
[2] N. Matsuoka, A. Moriguchi, M. Tada, T. Mihara, WO 2000069464, Chem. Abstr.
134 (2000) 533m.
4.1.10.1. (E)
N1,N1-Dimethyl-N4-(3-methyl-1-phenylindeno[2,1-e]
pyrazolo[3,4-b]pyrazin-5(1H)-ylidene) benzene-1,4-diamine (27a).
Violet needles, crystallized from dioxaneewater (1:1), m.p. 241e
243 ꢁC, yield 0.56 (54%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH arom.)
2950, 2910 (CH aliph.). 1H NMR CDCl3: 2.25 (s, 3H, CH3), 2.95 (s, 6H,
2CH3), 6.55e8.35 (m, 13H, AreH).
[3] A. Akahane, A. Tanaka, WO 2002100864, 2002, Chem. Abstr. 138 (2003)
24732a.
[4] [a] K. Imaizumi, T. Sado, JP 06080570, 1994, Chem. Abstr. 121 (1994) 91797;
[b] T. Sado, A. Inoue, Jpn. Kokai Tokkyo Koho JP 02 101078, 90 101781, Cl.
C07D487/04, 12 APr. 1990, Chem. Abstr. 113 (1990) 78422k.
[5] [a] S. Suzuki, A. Inoue, Akira, Jpn. Kokai Tokkyo Koho JP 02 172 988, 90 172
9881 Cl. C07D487/04, 4 Jul. 1990, Chem. Abstr. 113 (1990) 218276t;
[b] S. Suzuki, Jpn. Kokai Tokkyo Koho, 1990, Chem. Abstr. 114 (1991)
178382m.
Anal. Calcd. for C27H22N6 (430.50): C, 75.33; H, 5.15; N, 19.52.
Found: C, 75.23; H, 5.25; N, 19.73.
4.1.10.2. (E) N1,N1-Diethyl-N4-(3-methyl-1-phenylindeno[2,1-e]pyr-
azolo[3,4-b]pyrazin-5(1H)-ylidene) benzene-1,4-diamine (27b).
Brownish-violet needles, crystallized from dioxaneewater (1:1),
[6] [a] T.I. El-Emary, A.M. Kamal El-Dean, H.S. El-Kashef, Il Farmaco 53 (1998)
383e388;
m.p. 197e199 ꢁC, yield 0.47 g (52%). IR (KBr, cmꢂ1):
n
¼ 3050 (CH
[b] H.S. El-Kashef, T.I. El-Emary, M. Gasquet, P. Timone David, J. Maldonado,
P. Vanelle, Pharmazie 55 (2000) 572e576;
[c] T.I. El-Emary, J. Chin. Chem. Soc. 53 (2006) 391e401.
[7] [a] J. Hofmann, D. Sicker, G. Mann, Ger. (East) DD 276688, Cl. CO7 D487/04, 7
Mar. 1990, Chem. Abstr. 113 23 (1990) 1409h; [b] D.W. Rangnekar,
S.V. Dhamnaskar, Dyes Pigm. 13 (4) (1990) 241e250.
arom.) 2950 (CH aliph.). 1H NMR (CDCl3): 1.15e1.28 (t, 6H,
2CH2CH3), 2.75 (s, 3H, CH3), 3.35e3.50 (q, 4H, 2CH2CH3), 7.52 (m,
13H, AreH).
Anal. Calcd. for C29H26N6 (458.56): C, 75.69; H, 5.71; N, 18.33.
Found: C, 75.73; H, 5.89; N, 18.23.
[8] J. Quiroga, N.E. Sánchez, P. Acosta, B. Insuasty, A. Rodrigo, Tetrahedron Lett. 53
(2012) 3181e3187.
[9] P.G. Baraldi, F. Fruttarolo, M.A. Tabrizi, D. Preti, R. Romagnoli, H. El-Kashef,
A. Moorman, K. Varani, S. Gessi, S. Merighi, P.A. Borea, J. Med. Chem. 46 (2003)
1229e1241.
4.2. Biology
[10] A. Farghaly, H. El-Kashef, Monatsh. Chem. 136 (2) (2005) 217e227.
[11] A. Farghaly, Hussein El-Kashef, Monatsh. Chem. 137 (9) (2006) 1195e1202.
[12] P.G. Baraldi, F. Fruttarolo, M.A. Tabrizi, R. Romagnoli, D. Petri, E. Ongini, H. El-
Kashef, M.D. Carrion, P.A. Borea, J. Heterocycl. Chem. 44 (2007) 355e361.
[13] A. Farghaly, H. El-Kashef, J. Heterocycl. Chem. 48 (2011) 678e683.
[14] K.J. Kwon-Chung, J.W. Bennett, Med. Mycol. Lea and Febiger, Philadelphia,
1992, pp. 81e102.
The antimicrobial activity of 10 selected compounds was eval-
uated against 6 bacterial and 6 fungal strains. All microbial strains
were kindly provided by the Assiut University Mycological Centre
(AUMC). These strains are common contaminants of the environ-
ment in Egypt and some of which are involved in human and