Substituted quinolinones 15*. Preparation and enzymatic activity of some pyrazoloazines linked to the 4-hydroxy-1-methyl-quinolin-2(1H)-one moiety

Article abstract of DOI:10.1007/s10593-011-0806-0

The heterocyclization reaction of pyrazolinylquinolinone and its 4-chlorobenzylidene, 2-pyridylmethylene, pyridylaminomethylene, and 2-pyridylhydrazone derivatives with some active methylene nitriles and acrylonitriles, is described. These cyclization reactions afforded novel heterocyclic systems, such as pyrazolo[3,4-b]pyridines, pyrano[2,3-c]pyrazoles, and pyrazolo[4,3-c]pyridazines, linked to position 3 of 4-hydroxy-1- methylquinolin-2(1H)-one. The effect of the new products on the activity of α-amylase was examined. Some pyrano[2,3-c]pyrazoles revealed significant increase in α-D-glucose production by the enzyme.

Full text of DOI:10.1007/s10593-011-0806-0

Chemistry of Heterocyclic Compounds, Vol. 47, No. 5, August 2011 (Russian original Vol. 47, No. 5, May, 2011)
SUBSTITUTED QUINOLINONES
15*. PREPARATION AND ENZYMATIC
ACTIVITY OF SOME PYRAZOLOAZINES
LINKED TO THE 4-HYDROXY-1-METHYL-
QUINOLIN-2(1H)-ONE MOIETY
A. Khodairy1** and M. Abass2
The heterocyclization reaction of pyrazolinylquinolinone and its 4-chlorobenzylidene, 2-pyridylmethy-
lene, pyridylaminomethylene, and 2-pyridylhydrazone derivatives with some active methylene nitriles
and acrylonitriles, is described. These cyclization reactions afforded novel heterocyclic systems, such as
pyrazolo[3,4-b]pyridines, pyrano[2,3-c]pyrazoles, and pyrazolo[4,3-c]pyridazines, linked to position 3
of 4-hydroxy-1-methylquinolin-2(1H)-one. The effect of the new products on the activity of α-amylase
was examined. Some pyrano[2,3-c]pyrazoles revealed significant increase in α-D-glucose production by
the enzyme.
Keywords: pyrano[2,3-c]pyrazole, pyrazolinone, pyrazolo[4,3-c]pyridazine, pyrazolo[3,4-b]pyridine,
quinolinone, enzyme regulation.
3-Substituted 4-hydroxyquinolin-2(1H)-one derivatives represent one of the important classes of hetero-
cyclic compounds possessing a wide spectrum of medicinal activity. Biological screening of many prepared
and/or natural products of this heterocycle class has shown their antibacterial [2], antitumor [3], anti-HSV [4],
antineoplastic [5], anti-inflammatory [6], molluscicidal [7], and herbicidal activity [8]. On the other hand,
pyrazolinones have displayed fungicidal [9], antitumor [10], analgesic [11], antipyretic [12], and antineoplastic
activity [13]. They have also shown promise for developing congestive heart failure treatments [14].
Pyrazolinones with herbicidal activity, too, have been reported [15]. Fused pyrazoloazines, which are aza
isomers of indole, also display significant biological activity. Pyrazolo[3,4-b]pyridines are attractive compounds
due to their wide range of pharmaceutical applications. Some representatives of this class have displayed
important hypotensive, vasodilator, anti-inflammatory, analgesic, and antipyretic activity [16], others are
_______________
* For Communication 14, see [1].
** To whom correspondence should be addressed, e-mail: quinolinone@yahoo.com.
1 Department of Chemistry, Faculty of Science, Sohag University, Sohag 82524, Egypt.
2 Department of Chemistry, Faculty of Education, Ain Shams University, Cairo 11757, Egypt.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 736–749, May, 2011. Received
December 9, 2010, in revised form April 3, 2011.
0009-3122/11/4705-0611©2011 Springer Science+Business Media, Inc.
611

xanthine oxidase [17] or HIV reverse transcriptase inhibitors [18], antitumor agents [19, 20], or anxiolytics [21].
Some pyrano[2,3-с]pyrazoles have demonstrated analgesic activity [22], as well as potential inhibitory activity
against human kinase Chk1 [23]. Pyrazolo[3,4-c]pyridazine derivatives are known to possess interesting
biological and pharmacological properties [24–26]. Some of them were identified in high-throughput screening
as potent inhibitors of cyclin-dependent kinases CDK1/cyclin-B [27]. In view of these important applications of
the three classes of pyrazoloazines it is thought that combination of both quinolinone and some pyrazoloazine
moieties in one-molecular frame may lead to a new biologically active series of heterocyclic compounds. In
continuation of our program dealing with the synthesis and study of new pyrazoles [28, 29] and substituted
quinolinones [30, 31], the present work focused on the synthesis of some fused pyrazoloazine derivatives
incorporating the 4-hydroxy-1-methylquinolin-2(1H)-one moiety as the substituent attached to pyrazolinone. An
important regulating effect of quinolinones on the activity of α-amylase in efficient conversion of starch to
D-glucose has been described previously [32, 33]. These results encouraged us to check the effect of the
described new compounds on the activity of this enzyme.
The Knoevenagel condensation reaction of two selected aldehydes, 4-chlorobenzaldehyde and pyridine-
2-carbaldehyde, with 4-hydroxy-1-methyl-3-(5-oxo-4,5-dihydro-1H-pyrazol-3-yl)quinolin-2(1H)-one (1) in the
presence of fused sodium acetate and glacial acetic acid is considered an efficient procedure to obtain 3-[4-ary-
lidene-5-oxo-4,5-dihydro-1H-pyrazol-3-yl]-4-hydroxy-1-methylquinolin-2(1H)-ones 2a and 2b [30, 31]. Michael
addition of pyrazolinone 1 to 3-aryl-2-cyanoacrylamides and/or thioacrylamides in the presence of piperidine as
a base catalyst furnished heterocyclized fused systems, pyrazolo[3,4-b]pyridines 3a–d, in 64–72% yields.
Scheme 1
RCHO,
AcOH,
AcONa anh.
H
H
N
N
N
N
O
R
O
Quin
2a, b
Quin
X
1
CN
NH2
Piperidine,
DMF
H2N
S
H
H
N
H
X
CN
N
N
N
N
H
N
CN
S
NH2
R
Quin
Quin
H
X
CN
Piperidine,
DMF
Piperidine,
DMF
R
CN
4
3a–d
Quin = 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl;
2a, 3a,b R = 4-chlorophenyl; 2b, 3c,d R = 2-pyridyl; 3 a,c X = O, b,d X = S
The structure of the products was deduced from their IR, 1H NMR, as well as mass spectra, and elemen-
1
tal analysis. The H NMR spectra of compounds 3a–d indicated the formation of a trans isomer. The two
protons at positions 4 and 5 of pyrazolo[3,4-b]pyridine appear as doublets with coupling constants
J = 11.7 ± 0.1 Hz, a characteristic coupling value for transoid protons. As an explanation, getting the trans iso-
mer is probably due to increased steric hindrance in its possible cis form.
Recently, Rahmati had used the multicomponent condensation reaction technique and obtained related
results; even though he had separated both isomers, the major product was the trans form [34]. Interestingly,
when we subjected compounds 2a,b to Michael addition with cyanoacetamide and cyanothioacetamide in the
presence of piperidine, the addition was accompanied by cyclization and we obtained the same products 3a–d in
76–84% yields. Similarly, the reaction of pyrazolinone 1 with 2-cyano-2-cyclohexylidenethioacetamide in the
presence of piperidine led to the spiro[cyclohexane-1,4'-pyrazolo[3,4-b]pyridine] derivative 4 (Scheme 1).
612

Scheme 2
H
N
O
N
O
H
H
O
Me
N
N
K2CO3/ DMF
N
N
O
OH
S
CN
CN
+
H
Quin
Quin
H
N
H
PhHN
H
Me
1
H
H
N
N
O
O
N
N
O
N
O
N
CN
_
– MeSH
H
CN
S
HN
NHPh
CN
O
N
O
Ph
Me
Me
Me
H
H
N
N
N
O
N
O
H
N
O
N
Quin
O
CN
HN
HN
N
CN
Ph
Ph
Me
5
The reaction of pyrazolinone 1 with [anilino(methylthio)methylidene]malononitrile in the presence of
dry potassium carbonate in DMF furnished compound 5. It is likely that a base-catalyzed nucleophilic addition
took place to give a tetrahedral adduct intermediate (Scheme 2). This intermediate loses methanethiol to give the
enamine intermediate which, in turn, undergoes intramolecular cyclization, followed by iminopyrane–pyridone
Dimroth-like rearrangement [35, 36] to give the pyrazolopyridine 5. The evolution of methanethiol during the
1
course of reaction was detectable, the H NMR spectrum of the product revealed four deuterium-exchangeable
protons, and elemental analysis showed the absence of sulfur.
The addition of cyanoacetohydrazide to compound 2b in the presence of piperidine afforded a trihetero-
cyclic system, dipyrazolopyridine 6. A possible mechanism for the formation of compound 6 may involve a
cascade nucleophilic addition and cyclization with the removal of a water molecule (Scheme 3). The structure of
the dipyrazolopyridine 6 was established on the basis of its spectral and elemental analysis data.
4-((Pyridin-2-ylamino)methylidene)pyrazolin-5-one 7 [30], considered as a β-enaminone system, was
subjected to Michael addition reaction with some active methylene compounds – malononitrile, ethyl cyanoace-
tate, and cyanoacetohydrazide – in the presence of piperidine as a base catalyst. Treatment of the compound 7
with malononitrile afforded iminopyranopyrazole 8 (Scheme 4). Similarly, treatment of compound 7 with ethyl
cyanoacetate gave pyranopyrazolone 9. The most important feature of these last two reactions is that addition of
the active methylene nitrile at the β-enaminone is accompanied by elimination of 2-aminopyridine. This is follo-
wed by intramolecular cyclization via nucleophilic addition of the deprotonated pyrazole hydroxy group
to either the cyano group, in the case of malononitrile, or the ester group, in the case of ethyl cyanoacetate
(Scheme 4). The structure of both compounds 8 and 9 was established from their spectra and elemental analysis
data.
613

Scheme 3
H
H
N
O
H
N
N
N
N
N
NH2
NC
O
NH2
NH
Quin
O
Quin
H
H
Piperidine,
DMF
O
N
N
2b
H
H
OH
N
N
N
H
N
N
N
H
N
– H2O
Quin
H
NH
NH
NH
Quin
H
H
N
O
O
N
6
Scheme 4
H
CN
CN
N
N
O
NH
Quin
Piperidine,
DMF
CN
8
OEt
O
H
N
2-Aminopyridine,
CH(OEt)3,
(CH2OH)2
H
N
N
N
O
O
NC
Quin
O
1
Quin
NH
Piperidine,
DMF
CN
9
N
H
N
NH2
H
7
O
N
N
O
NC
Quin
N
Piperidine,
DMF
NH
HN
10
In an analogous way, the reaction of cyanoacetohydrazide with compound 7 is another cascade addition
followed by cyclization to give pyrazolo[4',3':5,6]pyrano[2,3-c]pyrazole 10. The IR spectrum of the product
showed that, unlike compounds 8 and 9, its structure does not include a nitrile group, and its proton NMR
spectrum has four deuterium-exchangeable protons. We suggest that the addition reaction may start with initial
intramolecular cyclization of cyanoacetohydrazide to give iminopyrazolinone. This active methylene-containing
614

ring adds to the enaminone, and the addition is followed by leaving of a 2-aminopyridine molecule and, finally,
cyclization with the loss of a water molecule (Scheme 5).
α-Arylazocarbonyl compounds are considered good precursors for preparation of their corresponding
pyridazine derivatives [37]. The behavior of the 4-(pyrid-2-ylhydrazono)pyrazolinone 11 [30], in the presence of
piperidine as a base catalyst, towards some nitriles containing an active methylene group was investigated. Thus,
treatment of compound 11 with malononitrile furnished pyrazolo[4,3-c]pyridazine-7-carbonitrile 12 (Scheme 6).
O
O
H
Scheme 5
HN
H
H
HN
HN
H
H2N
N
NH
Base
H
H
H
N
N
N
N
N
N
O
O
O
O
O
_
O
Quin
NH
NH
Quin
Quin
+
NH
NH
H
NH
NH
– 2-H2NPy
H
HN
H
NH
HN
H
2-
Py
2-
Py
HN
HN
7
O
OH
H
H
H
H
H
O
O
O
N
N
N
H+
N
N
N
– H2O
NH
N
N
N
NH
NH
NH
Quin
Quin
Quin
NH
NH
10
H
CN
N
N
CN
CN
N
NH
Scheme 6
Quin
N
N
Piperidine,
DMF
12
H
N
OEt
O
i. NaOH
ii. 2-aminopyridine,
NaNO2/HCl,
0–5oC
CONH2
NH
H
N
N
N
NC
O
N
N
Quin
N
1
Quin
Piperidine,
DMF
N
NH
13
N
O
H
H
N
NH2
N
NH
N
O
N
11
NC
Quin
N
N
N
Piperidine,
DMF
14
615

The structure of compound 12 was elucidated through its spectra and elemental analysis. The IR spec-
trum showed the presence of an absorption due to the C≡N bond at 2214 cm–1, in addition to a sharp strong
vibrational band at 1644 cm–1 due to C=O of 2-quinolone, while the carbonyl of pyrazolinone was not detected.
1
The H NMR spectrum revealed the existence of three deuterium exchangeable protons at 8.31, 10.30, and
12.60 ppm due to NH and OH groups. Similarly, compound 11 was treated with cyanoacetamide to give the
corresponding pyrazolo[4,3-c]pyridazine-7-carboxamide 13 in 59% yield. The IR spectrum showed no evidence
for the presence of the nitrile function, indicating its involvement in the cyclization reaction, while characteristic
vibrations due to the amino group were observed. The 1H NMR spectrum contained a broad singlet at 6.05 ppm
due to the NH2 protons, which disappeared on addition of D2O.
A triheterocyclic system, dipyrazolo[3,4-c:3',4'-e]pyridazinone 14, was obtained by treating the hydra-
zone 11 with cyanoacetohydrazide under the above conditions. We suppose that iminopyrazolinone was initially
formed by intramolecular cyclization and this active methylene compound attacked the carbonyl carbon of
pyrazolinone with the removal of a water molecule. A subsequent cyclization took place accompanied by loss of
an ammonia molecule to give the dipyrazolopyridazinone 14 (Scheme 7).
The effect of the new compounds, 3a–d, 4–6, 8–10, and 12–14, on the activity of α-amylase in the hyd-
rolysis of starch was indicated by monitoring production of α-D-glucose. Screening of the new compounds
showed that pyranopyrazoles 8–10 are the most potent enzyme activator candidates, achieving 5–6-fold increase
in the enzyme activity, while pyrazolopyridazines 11–14 have moderate effects (Table). It is noted that all the
tested compounds activate α-amylase except for compounds 3c and 3d, which turned out to be inhibitors and
reduced the activity by about 30%. Both compounds possess a pyridyl group, which increases their basicity.
However, the importance of this factor in the regulation of this enzyme is not presently known.
Scheme 7
H
H
OH
N
O
N
N
N
H
O
O
O
N
N
O
NH
NH
_
H+
Quin
Quin
– H2O
NH
NH
NH
NH
H
Quin
N
N
H
H
NH
+
NH
N
NH
NH
Py
HN
2-
Py
NH
2-
Py
2-
11
O
O
H
O
H
H
N
N
N
N
NH
N
NH
N
NH
NH
NH
– NH3
N
Quin
NH
Quin
Quin
N
N
N
N N
NH
Py
NH2
Py
-2
2-
Py
2-
14
To conclude, this research work describes efficient and facile heterocyclization reactions with pyrazolin-
5-one and its 4-arylidene, 4-arylaminomethylene, and 4-arylazo derivatives. Active methylene nitriles and
acrylonitriles can be used in such cyclization reaction to obtain the heterocyclic systems, such as pyrazolo-
[3,4-b]pyridines, pyrano[2,3-c]pyrazoles, and pyrazolo[4,3-c]pyridazines, linked to position 3 of 4-hydroxy-
1-methylquinolin-2(1H)-one. Some quinolinylpyrano[2,3-c]pyrazoles revealed a significant effect on the activity
of α-amylase.
616

TABLE 1. Effect of the New Compounds on the Activity of α-Amylase,
Indicated by the Amount of α-D-Glucose Produced
Compound
Glucose produced, μg/ml
Enhancement ratio (test/control)
3a
0.45
0.51
0.26
0.25
0.78
1.20
0.28
2.19
1.88
1.76
0.39
0.43
0.54
0.36
1.25
1.42
0.72
0.69
2.17
3.33
0.78
6.08
5.22
4.89
1.08
1.19
1.50

3b
3c
3d
4
5
6
8
9
10
12
13
14
Control (DMF)
EXPERIMENTAL
Melting points were determined in open capillary tubes on a digital Stuart SMP3 apparatus. IR spectra
were recorded on Perkin-Elmer FT-IR 1650 or Nicolet 710 FT-IR spectrometers using samples in KBr disks.
1H NMR spectra were recorded on a Varian Gemini-200 NMR spectrometer (200 MHz), using TMS as internal
reference. Electron-impact mass spectra were determined on a Carlo-Erba QMD-1000 instrument by direct inlet,
operating at 70 eV. Elemental analyses were performed on a Perkin Elmer CHN-2400 Analyzer. All reactions were
monitored by TLC on 0.2 mm silica gel F-254 (Merck) plates, using UV light (254 and 366 nm) for detection.
Compounds 1, 2a,b, 7, and 11 were synthesized according to the method described previously [30].
Quinolinylpyrazolopyridines 3a–d (General Method). A. A mixture of the pyrazolinone 1 (1.29 g,
5 mmol), the appropriate 3-aryl-2-cyanoacrylamide (5 mmol), and piperidine (0.5 ml) in DMF (25 ml) was
heated under reflux for 4 h. The reaction mixture was left to cool, and the solid precipitate was collected by
filtration, washed with cold methanol (20 ml), dried, and crystallized to give compounds 3ad, respectively.
B. To a solution of the arylmethylenepyrazolinones 2a (1.9 g, 5 mmol) or 2b (1.73 g, 5 mmol) in DMF
(30 ml), containing piperidine (0.2 ml), the appropriate active methylene compound (5 mmol) was added. The
reaction mixture was heated under reflux for 4 h. After cooling to room temperature, the obtained precipitate
was filtered and crystallized from an appropriate solvent to give the corresponding product 3ad.
trans-4-(4-Chlorophenyl)-3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-oxo-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (3a). Brown crystals from dioxane, yield 1.60 g (72%)
(procedure A), 1.78 g (80%) (procedure B); mp 209–211°C. IR spectrum, , cm1: 1652 (C=O), 1680 (C=O),
1
2204 (C≡N), 3100–3209 (N–H), 3407 (O–H). H NMR spectrum (CDCl3), δ, ppm (J, Hz): 3.63 (3H, s, CH3);
3.95 (1H, d, J = 11.7, H-5); 4.44 (1H, d, J = 11.6, H-4); 6.98–8.07 (8H, m, H Ar); 9.60 (1H, s, exch. with D2O,
NH); 10.34 (1H, s, exch. with D2O, NH); 12.62 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 445
[M]+ (50). Found, %: C 61.60; H 3.40; N 15.80. C23H16ClN5O3. Calculated, %: C 61.96; H 3.60; N 15.71.
trans-4-(4-Chlorophenyl)-3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-thioxo-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (3b). Yellow crystals from ethanol, yield 1.64 g (71%)
(procedure A), 1.93 g (84 %) (procedure B); mp 222–224°C. IR spectrum, , cm1: 1190 (C=S), 1650 (C=O),
2215 (C≡N), 3189–3317 (N–H), 3411 (O–H). 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 3.55 (3H, s, CH3);
3.87 (1H, d, J = 11.8, H-5); 4.35 (1H, d, J = 11.8, H-4); 6.92–8.04 (9H, m, H Ar + NH); 8.31 (1H, br. s, exch.
617

with D2O, NH); 12.06 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 461 [M]+ (28). Found, %:
C 59.60; H 3.20; N 15.00. C23H16ClN5O2S . Calculated, %: C 59.80; H 3.49; N 15.16.
trans-3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-oxo-4-pyrid-2-yl-4,5,6,7-tetrahydro-
1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (3c). Pale-yellow crystals from methanol, yield 1.34 g (65%)
(procedure A), 1.60 g (78%) (procedure B); mp 223–225°C. IR spectrum, , cm1: 1639 (C=O), 1682 (C=O),
2205 (C≡N), 3175–3209 (N–H), 3402 (O–H). 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 3.54 (3H, s, CH3);
3.96 (1H, d, J = 11.6, H-5); 4.28 (1H, d, J = 11.7, H-4); 7.02–8.10 (9H, m, H Ar + NH); 10.15 (1H, s, exch. with
D2O, NH); 11.10 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 412 [M]+ (16). Found, %: C 64.10;
H 3.60; N 20.20. C22H16N6O3. Calculated, %: C 64.07; H 3.91; N 20.38.
trans-3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-4-pyrid-2-yl-6-thioxo-4,5,6,7-tetra-
hydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (3d). Pale-yellow crystals from ethanol, yield 1.37 g (64%)
(procedure A), 1.63 g (76%) (procedure B); mp >300°C. IR spectrum, , cm1: 1178 (C=S), 1639 (C=O), 2209
1
(C≡N), 3177–3209 (N–H), 3405 (O–H). H NMR spectrum (CDCl3), δ, ppm (J, Hz): 3.63 (3H, s, CH3); 3.96
(1H, d, J = 11.7, H-5); 4.40 (1H, d, J = 11.7, H-4); 7.00–8.08 (9H, m, H Ar + NH); 10.31 (1H, br. s, exch. with
D2O, NH); 11.38 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 428 [M]+ (15). Found, %: C 61.40;
H 3.90; N 19.50. C22H16N6O2S. Calculated, %: C 61.67; H 3.76; N 19.61.
3'-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6'-thioxo-1',5',6',7'-tetrahydrospiro[cyclo-
hexane-1,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (4). A mixture of the pyrazolinone 1 (1.29 g, 5 mmol),
2-cyano-2-cyclohexylidenethioacetamide (0.9 g, 5 mmol), and piperidine (0.2 ml) in DMF (20 ml) was heated
under reflux for 4 h. The reaction mixture was left overnight, and the solid precipitate was collected by filtration,
washed with cold methanol (20 ml), dried, and recrystallized from DMF to give white crystals. Yield 1.70 g
(81%); mp 202–204°C. IR spectrum, , cm1: 1176 (C=S), 1650 (C=O), 2206 (C≡N), 3150–3200 (N–H), 3422
1
(O–H). H NMR spectrum (CDCl3), δ, ppm: 1.32–1.78 (6H, m, 3 CH2); 2.25–2.49 (4H, m, 2 CH2); 3.50 (3H, s,
CH3); 3.94 (1H, s, H-5'); 6.50–7.90 (5H, m, H Ar + NH); 10.20 (1H, br. s, exch. with D2O, NH); 11.53 (1H, s,
exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 419 [M]+ (53). Found, %: C 62.60; H 4.90; N 16.40.
C22H21N5O2S. Calculated, %: C 62.99; H 5.05; N 16.69.
4-Anilino-3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-oxo-6,7-dihydro-1H-pyrazolo-
[3,4-b]pyridine-5-carbonitrile (5). A mixture of the pyrazolinone 1 (1.29 g, 5 mmol), [anilino(methylthio)methy-
lidene]malononitrile (1.08 g, 5 mmol), and anhydrous potassium carbonate (2.76 g) in DMF (25 ml) was heated
under reflux for 6 h. The reaction mixture was filtered while hot. The filtrate was treated with diluted hydrochloric
acid (5 ml, 1M). The precipitated material obtained was collected by filtration, washed with cold methanol (10 ml),
dried, and crystallized from DMF to give dark-brown crystals. Yield 1.36 g (64%); mp 294–295°C. IR spectrum,
, cm1: 1650 (C=O), 1682 (C=O), 2202 (C≡N), 3175–3209 (N–H), 3420 (O–H). 1H NMR spectrum (DMSO-d6),
δ, ppm: 3.68 (3H, s, CH3); 6.95–7.80 (8H, m, H Ar); 7.90 (1H, s, exch. with D2O, NH); 8.08 (1H, d, J = 8.0, H Ar);
9.10, 9.35 (2H, br. s, exch. with D2O, 2NH); 12.03 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 424
[M]+ (17). Found, %: C 65.00; H 3.50; N 19.60. C23H16N6O3 Calculated, %: C 65.09; H 3.80; N 19.80.
5-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-4-(pyrid-2-yl)-1,4,7,8-tetrahydrodipyrazolo-
[3,4-b:4',3'-e]pyridin-3(2H)-one (6). A mixture of compound 2b (1.73 g, 5 mmol), cyanoacetohydrazide (0.5 g,
5 mmol), and piperidine (0.5 ml) in DMF (25 ml) was heated under reflux for 6 h. The reaction mixture was left
overnight, and the solid precipitate was collected by filtration, washed with cold methanol (20 ml), dried, and
crystallized from dioxane to give light-brown crystals. Yield 1.39 g (65%); mp 294–296°C. IR spectrum, ,
1
cm1: 1639 (C=O), 1683 (C=O), 3151–3225 (N–H), 3300 (O–H). H NMR spectrum (DMSO-d6), δ, ppm
(J, Hz): 3.62 (3H, s, CH3); 5.04 (1H, s, H-5); 7.06–7.90 (9H, m, H Ar + 2 NH); 8.09 (1H, d, J = 7.8, H Ar);
10.30–10.70 (2H, br. s, exch. with D2O, 2NH); 12.45 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %):
427 [M]+ (11). Found, %: C 61.60; H 3.80; N 22.70. C22H17N7O3. Calculated, %: C 61.82; H 4.01; N 22.94.
Reaction of Active Methylene Compounds with Enaminone 7 (General Method). To a solution of
the enaminone 7 (1.80 g, 5 mmol) in DMF (20 ml) containing piperidine (0.2 ml), the appropriate active
methylene compound (5 mmol) was added. The reaction mixture was heated under reflux for 4 h. After cooling
618

at room temperature and dilution with cold water (20 ml), the precipitate was filtered and crystallized from an
apropriate solvent to give the pyranopyrazoles 8, 9, and 10, respectively.
3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-imino-1,6-dihydropyrano[2,3-c]pyrazo-
le-5-carbonitrile (8). Yellow crystals from methanol, yield 0.92 g (55%); mp 213–215°C. IR spectrum, , cm1:
1
1608 (C=N), 1639 (C=O), 2205 (C≡N), 3175–3209 (N–H), 3402 (O–H). H NMR spectrum (DMSO-d6), δ,
ppm: 3.48 (3H, s, CH3); 6.70 (1H, br. s, exch. with D2O, NH); 7.28–8.05 (5H, m, H Ar); 9.50 (1H, br. s, exch.
with D2O, NH); 11.36 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 333 [M]+ (35). Found, %:
C 61.10; H 3.40; N 20.80. C17H11N5O3 Calculated, %: C 61.26; H 3.33; N 21.01.
3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-oxo-1,6-dihydropyrano-[2,3-c]pyrazole-
5-carbonitrile (9). Amber-yellow crystals from ethanol, yield 1.33 g (80%); mp 272–274°C. IR spectrum, ,
cm1: 1638 (C=O), 1712 (C=O), 2200 (C≡N), 3155 (N–H), 3408 (O–H). 1H NMR spectrum (DMSO-d6), δ, ppm
(J, Hz): 3.68 (3H, s, CH3); 7.32–8.12 (5H, m, H Ar); 9.90 (1H, s, exch. with D2O, NH); 11.54 (1H, s, exch. with
D2O, OH). Mass spectrum, m/z (Irel, %): 334 [M]+ (13). Found, %: C 61.30; H 3.33; N 16.50. C17H10N4O4.
Calculated, %: C 61.08; H 3.02; N 16.76.
4-Hydroxy-3-(5-imino-5,6-dihydro-1H-pyrazolo[4',3':5,6]pyrano[2,3-c]pyrazol-3-yl)-1-methylqui-
nolin-2(1H)-one (10) was crystallized from acetic acid and appears as brown crystals, yield 1.12 g (64%);
1
mp 250–252oC. IR spectrum, , cm1: 1602 (C=N), 1639 (C=O), 3100–3300 (N–H), 3426 (O–H). H NMR
spectrum (DMSO-d6), δ, ppm: 3.48 (3H, s, CH3); 6.80–7.96 (5H, m, H Ar); 8.40 (1H, s, exch. with D2O, NH);
9.52–9.78 (2H, br. s, exch. with D2O, 2 NH); 11.30 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %):
348 [M]+ (26). Found, %: C 58.50; H 3.30; N 24.00. C17H12N6O3. Calculated, %: C 58.62; H 3.47; N 24.13.
Reaction of Active Methylene Compounds with Hydrazonopyrazolinone 11 (General Method). To
a solution of the hydrazone 11 (1.81 g, 5 mmol) in DMF (20 ml) containing piperidine (0.2 ml), the appropriate
active methylene compound (5 mmol) was added. The reaction mixture was heated under reflux for 4 h. After
cooling at room temperature, the precipitate that formed was filtered and crystallized from an appropriate
solvent to give the pyrazolopyridazines 12, 13, and 14, respectively.
3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-imino-5-pyrid-2-yl-5,6-dihydro-1H-pyr-
azolo[4,3-c]pyridazine-7-carbonitrile (12). White crystals from dioxane, yield 1.27 g (62%); mp 253–254°C.
IR spectrum, , cm1: 1644 (C=O), 2214 (C≡N), 3156–3204 (N–H), 3400 (O–H). 1H NMR spectrum (DMSO-d6),
δ, ppm: 3.63 (3H, s, CH3); 7.24–8.15 (8H, m, H Ar); 8.31 (1H, s, exch. with D2O, NH); 10.30 (1H, br. s, exch.
with D2O, NH); 12.60 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel, %): 410 [M]+ (33). Found, %:
C 61.30; H 3.70; N 27.50. C21H14N8O2. Calculated, %: C 61.46; H 3.44; N 27.30.
3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-6-imino-5-pyrid-2-yl-5,6-dihydro-1H-pyr-
azolo[4,3-c]pyridazine-7-carboxamide (13). Pale-yellow crystals from DMF, yield 1.26 g (59%); mp 272–
1
274°C. IR spectrum, , cm1: 1640 (C=O), 1677 (C=O), 3156–3221 (N–H), 3340–3430 (O–H, NH2). H NMR
spectrum (DMSO-d6), δ, ppm: 3.62 (3H, s, CH3); 6.05 (2H, br. s, exch. with D2O, NH2); 7.22–8.18 (8H, m,
H Ar); 8.22 (1H, s, exch. with D2O, NH); 9.32 (1H, s, exch. with D2O, NH); 12.60 (1H, s, exch. with D2O, OH).
Mass spectrum), m/z (Irel, %): 428 [M]+ (64). Found, %: C 58.60; H 3.60; N 26.00. C21H16N8O3. Calculated, %:
C 58.88; H 3.76; N 26.16.
3-(4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-5-pyrid-2-yl-5,7-di-hydrodipyrazolo[3,4-c:
3',4'-e]pyridazin-8(1H)-one (14). Greenish-brown crystals from dioxane, yield 1.40 g (66%); mp >300°C. IR
1
spectrum, , cm1: 1639 (C=O), 1680 (C=O), 3170–3209 (N–H), 3402 (O–H). H NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 3.56 (3H, s, CH3); 7.24–8.08 (7H, m, H Ar); 8.18 (1H, d, J = 7.8, Ar); 9.24 (1H, br. s, exch. with
D2O, NH); 11.30 (1H, br. s, exch. with D2O, NH); 12.60 (1H, s, exch. with D2O, OH). Mass spectrum, m/z (Irel,
%): 426 [M]+ (51). Found, %: C 58.90; H 3.20; N 26.00. C21H14N8O3. Calculated, %: C 59.15; H 3.31; N 26.28.
Examination of the Effect on α-Amylase. The procedure for determination of the effect of the tested
compounds on α-amylase activity was based on measuring the amount of α-D-glucose (μg/ml) that liberated
from enzymatic hydrolysis of starch using the standard enzymatic glucose oxidase method [38, 39]. The tests
were carried out on α-amylase enzyme produced by Thermomyces lanuginosus, a thermophilic fungus which is
619

grown on starch-containing substances. The tested samples were added as a DMF solution (0.1 ml, 100 μg/ml)
to an assay mixture consisting of enzyme solution (0.5 ml) and citrate phosphate buffer (4.5 ml, pH 5.0),
containing 1% starch. Then it was incubated at 40°C for 30 min, and the released α-D-glucose was determined
on a Spekol-k colorimeter at λ = 505 nm. Tests were carried out in triplicates, and the mean reading was
recorded. A blank test was prepared using distilled water instead of the enzyme, and it was used for zero-setting
of the colorimeter. A control test was prepared using DMF without the addition of the candidate compounds.
The authors thank Prof. Dr. Usama F. Ali, Department of Biology, Faculty of Education, Ain Shams
University, Cairo, Egypt, for evaluation of the enzymatic activity.
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