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Synthesis of pyrazolo-enaminones, bipyrazoles and pyrazolopyrimidines and evaluation of antioxidant and antimicrobial properties
⁎Corresponding author. mdpujol@ub.edu (Maria Dolors Pujol)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Abstract
New pyrazolo-enaminones have been synthesized in three steps from DHA in good yields. The pyrazolo-enaminone 3h shows antioxidant properties comparable to Vitamin C. Pyrazolopyridines were prepared by double condensation of the aminopyrazoles with keto-esters. The pyrazoles 3b, 3d, 3e and the pyrazolopyridine 4b showed activity against Gram+ and Gram− bacteria.
Abstract
A novel pyrazolo-enaminones, bipyrazoles and bipyrazolopyridines from 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)butane-1,3-dione and 4-methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6(3aH,7H)-dione have been synthesized by assisted heating with microwave radiation without any catalyst. The pyridine and pyrazole ring formation has been developed from easily accessible enamino keto esters by formylation followed by intramolecular cyclization. The general applicability for the synthesis of the important pyrazolo-enaminones, bipyrazoles and pyrazolo-pyridines heterocycles was attributed to simplicity of operation, synthesis without catalyst, energy efficiency (shorter reaction time under microwave irradiation), good yields, more environmentally friendly and more cost-effective procedure. The antioxidant activity of new heterocyclic compounds was evaluated by free radical scavenging by DPPH assay. Several of these compounds showed good activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria.
Keywords
Intramolecular cyclization
Pyrazolo-enaminones
Bipyrazolopyridines
Antioxidant activity
Antibacterial activity
1 Introduction
Pyrazolo(3,4-b)pyridines and related fused derivatives are considered important class of pharmaceutical agents (Zhang et al., 2010; El-Sayed, 2009; Misra et al., 2003; Nagender et al., 2014; El-Borai et al., 2012), which includes compounds that have antitumor properties (Stepanenko et al., 2011); antidepressant (Kunstmann et al., 1984), and cytotoxic activity (Manpadi et al., 2007). Furthermore, these heterocyclic systems are part of the chemical structure of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases (CDKs) inhibitors (Beutne et al., 2009), and others that have activity against Alzheimer's disease (AD) (Bhat et al., 2003). Likewise, the pyrazolo-enaminones, bipyrazoles and bipyrazolopyridines have a great importance in medicinal chemistry, possessing anti-inflammatory, antimicrobial and cytotoxic activities (Kumari and Venkatarao, 2020).
Conjugated nitrogenous systems in addition to therapeutic applications can also be used as semiconductors (Wang et al., 2021; Wu et al., 2012; Li et al., 2012; Gu et al., 2016). These derivatives also have been used to prevent various diseases induced by active oxygen and as trapping free radicals' agents. Due to the interest of this type of compounds, various synthetic methods have been developed that their authors have reported previously (Datta et al., 2011).
Recently, interest in these heterocyclic compounds has increased considerably due to their potential application in both organic and medicinal chemistry. Several syntheses of these derivatives have been carried out using different catalysts, which have proven to be extremely effective. Especially in the reaction of primary amines with β-dicarbonyl compounds to obtain pyrazolo-enaminones with high yields, iodine (Gogoi et al., 2005), copper nanoparticles (Figueiredo and Kascheres, 1997) and silica (SiO2) were used (Mohammadizadeh et al., 2009). The synthesis of pyrazolo[3,4-b]pyridine derivatives was carried out by a cyclocondensation reaction in one pot assisted by microwave irradiation or by conventional heating (Bennamane et al., 2008).
Badr et al. prepared a series of pyrazoles, bipyrazoles and polysubstituted pyranopyrazoles that were evaluated for their cytotoxic properties (Badr and Abd El Razik, 2018). Also, Kumar et al. reported the synthesis of a series of 1H-pyrazolo[3,4-b]pyridines from the corresponding β-ketoester in four steps (Kumar et al., 2016).
Preparation of a mixture of pyrazolo[3,4-b]pyridines and pyrazolo[4,3-c]pyridines from 3-acylpyridine-N-oxide of tosylhydrazones by reaction with electrophilic reagents in the presence of amines as base with high yields have been described (Lominac et al., 2012). The 1H-pyrazolo[3,4-b]pyridines were also prepared by condensation of amino-pyrazole and α,β-unsaturated ketones in ionic liquid without catalyst (Narsaiah et al., 2010). More recently, new compounds possessing skeletons characterized by a common pyrazole ring fused to a nitrogenous heterocycle entity to six membered were synthesized (Shekarrao et al., 2014). Recent works also describes the preparation of new pyrazoles (Tian et al., 2020) and chemical reactions involving the formation of enaminones (Guodong et al., 2020).
In previous work, we have developed a new method for the synthesis of pyrazolo[3,4-b]pyridine-3-ones and [1,5-a]pyrimidine-2(1H)-ones based on the reaction of 2-chloronicotinonitrile with some hydrazines at reflux of xylene or ethanol (Fadel et al., 2011). This work becomes the continuation of previous work of our research group, which studied the condensation of amino-pyrazole with α,β-unsaturated compounds. To develop a new and fast route with better performance by minimizing the reaction time of the condensation of the ethyl acetoacetate with 3-amino-1-phenyl-1H-pyrazol-5(4H)-one to synthesis of 4-methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6(3aH,7H)-dione (Akhramez et al., 2016). The main objective of this work consists in the efficient preparation of suitably substituted mono-pyrazoles, bipyrazoles and pyrazolo-pyrimidines that allow us to know the structural requirements that determine the antioxidant and antibacterial activities of these pyrazole derivatives (Scheme 1).
Pyrazole derivatives synthesized in this word.
2 Results and discussion
2.1 Chemistry
A general strategy was developed to obtain pyrazolo-enaminones from DHA. This procedure begins with condensation of commercially available DHA (dehydroacetic acid or 3-acetyl-4,6-dimethyl-2H-pyran-2-one) with phenyl-hydrazine to give the 1,3-dicarbonyl compound (2) in two steps, according to the procedure described in the literature with modifications (Bendaas et al., 1999). The condensation of the pyrazolo-diketone (2) with selected anilines leads to pyrazolo-enaminones 3 (Scheme 2).
Synthesis of pyrazolo-enaminone 3 from DHA.
The addition of the 3-bromoaniline to the diketo-N-phenyl-pyrazole (2) and heating at reflux of the solvent indicated in the Table 1 during the time indicated in h in the same Table 1 allows obtaining the pyrazolo-enaminone 3a with the indicated yields (Table 1, entries 1-11). a: 80 °C; b: room temperature. These reactions were carried out with 1 mmol of 2 and 1 mmol of 3-bromoaniline in 5 mL of the chosen solvent.
Entry
Temperature
Cat.
Solvent
Time (h)
Yield (%)
1
a
AcONa
EtOH
2
62
2
b
AcONa
EtOH
20
44
3
a
AcOH
EtOH
2
89
4
b
AcOH
EtOH
18
63
5
a
–
EtOH
4
81
6
a
AcOH
MeOH
2
77
7
b
AcOH
MeOH
20
58
8
a
–
Acetone
2
68
9
a
AcOH
CH2Cl2
2
70
10
a
–
CHCl3
2
68
11
a
–
Acetonitrile
2
65
The addition of AcONa as a base and using ethanol as solvent leads to moderate yields (Table 1, entries 1 and 2). While the addition of acetic acid (AcOH) in ethanol allows to increase the yields (entries 3 and 4). Also, good results will be achieved by maintaining acid medium in methanol (Table 1, entries 6 and 7) or dichloromethane (Table 1, entry 9). These results show that the presence of acid or base is not essential for this reaction (Table 1, entries 5, 8, 10 and 11). The studies carried out indicate that heating for short reaction times (2 h) lead to better results than long times stirring at room temperature (18-20 h) (Table 1, compare entries 1 and 2; 3 and 4; 6 and 7).
Optimized conditions for 3a (using acetic acid in ethanol and reflux for 2 h) have been applied to the condensation of diketo-N-phenyl-pyrazole (2) with different anilines to obtain pyrazolo-enaminones 3b-j in yields ranging from 68 to 89% (Scheme 3).
Synthesis of pyrazoloenaminones and bipyrazoles.
In general, the presence of electron donor or withdrawing substituents on aromatic nucleus of anilines does not affect the yield of these amination.
The synthesis of 3,4′-bipyrazol-5′-ols from 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one (dehydroacetic acid, DHA) and hetero-aryls hydrazines was previously reported in the literature (Parshad et al., 2014). In this work the treatment of 2 with hydrazines gave 3;4′bipyrazol-5′-ol heterocycles 4a-g in good yields (Scheme 3). The different electrophilicity of the two carbonyl groups of diketones compared to hydrazines explains the regioselectivity of this condensation reaction. The NH2– group of the hydrazine reacts with the less hindered carbonyl and the intermediate from this condensation undergoes another condensation, this time between the second hydrogen of the hydrazine and the benzyl carbonyl which, followed by dehydration, gives rise to the formation of the pyrazole derivative. Despite condensing the diketone with a monosubstituted hydrazine, only the presence of the indicated regioisomer 4 was detected. The experimental results proved that the structure of the [3,4′]-bipyrazoles is indicated in the Scheme 3. Then the reaction of pyrazolyl-1,3-butanedione 2 with an equimolar amount of 5-amino-2-phenyl-2,4-dihydro-3H-pyrazol-3-one 6, in refluxing ethanol leads exclusively to the 4-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridin-3(3αH)-one 5, with high yield (92%) (Scheme 4).
Preparation of pyrazolo-pyrimidine 5.
The condensation of the aminopyrazolone 6 with ethyl acetoacetate has been carried out under two different conditions (A and B) (Scheme 5).
Condition A: ethyl acetoacetate 5 eq, solvent free, 180 °C, 4 h. Condition B: ethyl acetoacetate 2 eq, toluene, 120 °C, overnight.
The treatment of 5-amino-2-phenyl-2,4-dihydro-3H-pyrazol-3-one 6 with 5 equivalents of ethyl acetoacetate without solvent and catalyst gives regioselectively 4-methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6(3aH,7H)-dione (7) in good yield (Condition A). While after several attempts to improve the yield of 8, this was obtained with 54% when treating 6 with acetoacetate in toluene at 120 °C overnight (Scheme 5, Condition B).
Previously, the pyrazolopyridine 8 was prepared with a complicated method in only 42% of yield (Akhramez et al., 2016).
The condensation reaction of aminopyrazolone 6 with ethyl acetoacetate to give 7 was carried out with three different methods and each method several conditions were tested to determine the reaction type that gives the best yield, so we brought in different solvents, different catalysts and changes of the amount of ethyl acetoacetate as described in the (Table 2). A: 180 °C, 4 h. B: 120 °C, overnight. MW: 100 °C. C: other conditions.
Conventional Heating
MW Irradiation
Sealed Tube
Entry
Cond. A or B
Solvent
ketoester ratio (eq)
Catalyst
Yield (%)
Time (h)
Yield (%)
Time (min)
Yield (%)
Time (h)
1
A
_
5
_
92
4
95
10
92
2
2
A
_
2
_
75
4
80
10
72
2
3
C
EtOH
1
_
63
6
66
15
60
3
4
C
EtOH
1.5
_
71
6
79
15
77
3
5
C
EtOH
1
APTS
72
6
80
15
66
3
6
C
EtOH
1
Acetic acid
76
6
83
15
67
3
7
C
EtOH
1.2
NaNO2/I2
43
8
44
15
39
4
8
C
EtOH
1
HCl
77
6
83
15
73
3
9
C
MeOH
1
_
64
6
73
15
59
3
10
C
MeOH
2
_
74
6
81
15
79
3
11
C
MeOH
1.5
APTS
75
6
81
15
77
3
12
B
Toluene
1
_
45
8
48
20
37
4
13
B
Toluene
1
APTS
51
8
54
20
41
4
The amino group of the pyrazolone undergoes nucleophilic addition to the ester and gives the intermediate keto-amide that was involved in an intramolecular cyclisation followed by dehydration giving the pyrazolo-pyridine 7. A plausible mechanism for the formation of 7 is detailed in the Scheme 6.
A plausible mechanism for the formation of 7 from the aminopyrazole 6.
The utilization of polar protic solvents decreased the yields (EtOH, Table 2 entries 3–8 and MeOH, entries 9–11), but lipophilic solvents such as toluene significantly lowered the yields, fact attributable to high lipophilicity that difficult the solubility of starting material (Table 2, entries 12–13).
Without solvent, catalyst free and starting from 5 equivalents of acetoacetate ester, they were the best conditions (Table 2, entry 1). The microwave irradiation assistance was used to improve the yields and to reduce the reaction time (Table 2, entries 1, 2, 5, 6, 7, 8 and 9).
Using the conditions B, a mixture of the pyrazolo-pyrimidines 7 and 8 was obtained. The results of the NMR spectra as well as the results of the X-ray diffraction allowed to confirm the proposed structure for compound 8 (to see ORTEP, Fig. 1) (Akhramez et al., 2016). It is important to highlight that the X-ray diffraction results confirm that the 8 isomer corresponds to the represented structure and not as a pyridine isomer as indicated in previous scientific works (Fadel et al., 2011).
ORTEP diagram of the pyrazolo-pyridine 8 (Lominac et al., 2012).
A retro-Claisen of the β-keto ester (ethyl acetoacetate) facilitates the acetylation of the aminopyrazole (6) followed by the condensation of this acetyl group with ethyl acetoacetate giving the corresponding unsaturated β-keto ester. Subsequently the intra-molecular condensation of the ketone function with the methylene group of the pyrazolone takes place yielding the pyrazolo-pyridine 8. A plausible mechanism for the formation of 8 from the aminopyrazole 6 is indicated in Scheme 7.
Proposed mechanism for the formation of 8.
2.2 Potential biological results
Many compounds containing pyrazole heterocycle are found to have potent antibacterial and antioxidant properties (Anush et al., 2018).
In this work and in accordance with the foreseen objectives, the antioxidant and antibacterial activity of selected compounds from each series was evaluated to know the effect of the skeleton of these heterocyclic structures on the activity according to validated procedures.
2.2.1 Antioxidant activity (Kedare and Singh, 2011)
The free radical scavenging potentials of synthesis compounds and ascorbic acid (Vitamin C) at different concentrations were evaluated by DPPH test. Antioxidants react with DPPH, which is a nitrogen-centered radical with a characteristic absorption at 517 nm and convert to 1,1-diphenyl-2-picryl hydrazine.
The degree of discoloration indicates the scavenging potentials of the antioxidant compounds as shown in Fig. 2, in which all representative pyrazole derivative compounds tested as well as ascorbic acid (Vitamin C) (positive control), have reduced the DPPH radical in dose dependent manner. Radical scavenging activity was expressed as the concentration (µg/mL) of compound that reduces DPPH (40 µg/mL) by 50% (IC50).
Antioxidant activity of pyrazole-derivatives evaluated by DPPH method.
All these newly synthesized compounds exhibited significantly antioxidant activity, with an IC50 ranged between 5.5 and 180 mg/mL (Table 3).
Compound
IC50 µg/mL
Log P
Compound
IC50 µg/mL
Log P
2
80 ± 5.9
1.42
4b
100 ± 6.5
3.07
3a
18 ± 1.9
3.99
4d
180 ± 9.0
4.02
3b
34 ± 1.5
3.16
4e
40 ± 3.0
4.25
3c
21 ± 2.0
3.22
4f
80 ± 5.0
4.85
3d
40 ± 2.5
3.72
4g
40 ± 2.2
4.97
3e
20 ± 1.7
3.85
5
48 ± 4.0
3.48
3h
5.5 ± 0.3
2.77
7
21 ± 1.3
1.07
3j
90 ± 5.0
3.03
8
48 ± 2.0
1.83
4a
20 ± 1.4
4.06
Vitamin C
5 ± 0.7
−2.15
When analyzing the results, no correlation was found between antioxidant power and polarity (log P). Thus, diketone 2, which presents the lowest value of log P (1.42) of the compounds in the series of monopyrazoles, shows an antioxidant activity 16 times lower than that of 3h (Table 3, see 2 and 3h compounds). It should be noted that the aromatic hydroxyl group is essential for antioxidant activity but so is the position on the aromatic nucleus. The situation of the hydroxyl group in position para of the substituted aniline favors the antioxidant activity, as confirmed by the fact that compound 3h has an antioxidant activity four times higher than the pyrazoloenaminone 3e that has the hydroxyl group in position 3.
The compound 3h has been found to be the strongest of this series (IC50 = 5.5 μg/mL), it shows the best antioxidant activity and of the same order as vitamin C (ascorbic acid, IC50 = 5 μg/mL) used as a reference (standard antioxidant). This excellent free radical scavenging activity with the DPPH method can be attributed to the hydroxyl groups of pyrazolo-enaminone 3h.
The presence of the 4-hydroxyphenylamino group can be oxidized to the corresponding quinonimine, manifesting a high antioxidant power. The same functional phenol group was possessed by compound 3e, which show interesting activity (IC50 = 20 μg/mL), but less than 3h as we have indicated, a fact attributable to the position of the hydroxyl group which, being in the metha position, does not allow the formation of quinonimines. Compound 3j (IC50 = 90 μg/mL), which is equivalent to the methoxylated derivative of 3h, has an antioxidant activity 18 times less than 3h. This reduction in activity is attributed to the fact that the methoxyl group prevents the formation of the intermediate quinonimine responsible for the antioxidant power. The presence of halogens on the substituted aniline maintains the activity (see 3a and 3d). Among the bipyrazoles, it should be noted that 4a is the one that shows the highest antioxidant activity (IC50 = 20 μg/mL). Among the bipyrazoles, it should be noted that 4a is the one that shows the highest antioxidant activity. This activity seems to be related to the presence of nitro groups as substituents of the benzene ring. It is notable that 4a loses 50% of its activity when a phenyl group is introduced on the nitrogen atom (4e, (IC50 = 40 μg/mL). The bipyrazole derivative 4d (IC50 = 180 μg/mL), which has only one nitro group, has its antioxidant activity reduced by 4 times compared to 4e, which has two nitro groups in its structure.
Finally, pyrazolopyridines 7 and 8 also have antioxidant properties that in this case do correlate with the polarity of the structure, that is, compound 7 with the lowest log P is the one with the highest antioxidant power (Table 3).
These results reveal that these compounds can be inhibiting or preventing the consequences of the oxidative stress. The ability of some compounds to scavenge free radicals in DPPH test does not mean that these compounds will perform readily where complex mechanisms are operating such as those in physiological substrates.
For this reason, there is need to verify the antioxidant effect in scavenging specific species such as superoxide anion radical (O2• -), hydroxyl radical (OH) and hydrogen peroxide (H2O2). These oxygen species are often generated in organisms by diverse cellular processes, such as the electron transport chain in mitochondria, in microsomes and through enzymes like xanthine oxidase and NADPH oxidase or from exogenous factors. Studies are currently being carried out to determine the mechanism of action of the antioxidant effect of these compounds.
2.2.2 Antibacterial activity
Antibacterial properties of selected compounds were determined in vitro using a micro-dilution method (Balouiri et al., 2016) at different concentrations against both Gram-Negative (Escherichia coli ATCC 25922) (Table 4) and Gram-Positive (Staphylococcus aureus ATCC 25923) (Table 5) bacteria.
Compound
inhibition area (mm)
2 mg/mL
4 mg/mL
5 mg/mL
6 mg/mL
8 mg/mL
2
12 ± 0.2
13 ± 0.3
15 ± 0.1
15 ± 1.4
16 ± 0.6
3a
6 ± 0.1
7 ± 0.4
7 ± 0.2
7 ± 1.1
7 ± 0.7
3b
15 ± 0.4
14 ± 0.7
14 ± 0.5
17 ± 0.1
17 ± 0.1
3c
13 ± 0.1
9 ± 0.4
7 ± 0.4
7 ± 0.6
6 ± 0.2
3d
16 ± 0.3
14 ± 0.7
15 ± 07
17 ± 0.3
19 ± 0.8
3e
10 ± 0.2
27 ± 0.8
23 ± 2.1
16 ± 0.6
9 ± 0.4
3h
6 ± 0.1
9 ± 0.1
8 ± 0.9
8 ± 0.4
10 ± 0.5
3j
9 ± 0.1
18 ± 0.2
15 ± 1.1
12 ± 0.7
10 ± 0.9
4a
10 ± 0.4
8 ± 0.3
8 ± 0.1
6 ± 0.7
7 ± 0.1
4b
19 ± 0.4
18 ± 0.8
9 ± 0.7
11 ± 0.3
12 ± 1.2
4d
10 ± 0.3
11 ± 0.4
11 ± 0.3
13 ± 0.1
14 ± 0.6
4e
12 ± 0.5
8 ± 0.2
9 ± 0.1
9 ± 0.8
1 ± 0.5
4f
10 ± 0.8
5 ± 0.4
6 ± 0.6
8 ± 0.4
7 ± 0.4
4g
9 ± 0.2
10 ± 0.7
10 ± 0.7
12 ± 0.6
17 ± 0.5
5
15 ± 0.8
16 ± 0.6
17 ± 0.1
14 ± 0.8
16 ± 0.9
7
10 ± 0.3
11 ± 0.6
11 ± 0.3
10 ± 0.3
12 ± 0.7
8
7 ± 0.5
7 ± 0.5
8 ± 0.6
7 ± 0.4
8 ± 0.7
C
22 ± 0.9
–
–
–
–
CN
26 ± 0.7
–
–
–
–
TE
15 ± 0.1
–
–
–
–
AMC
7 ± 0.1
–
–
–
–
Compound
Inhibition area (mm)
2 mg/mL
4 mg/mL
5 mg/mL
6 mg/mL
8 mg/mL
2
9 ± 1.0
10 ± 1.2
12 ± 1.1
10 ± 1.0
10 ± 1.0
3a
12 ± 1.0
8 ± 0.98
8 ± 1.0
8 ± 1.0
7 ± 1.0
3b
12 ± 1.0
8 ± 1.0
7 ± 1.0
11 ± 1.0
11 ± 1.0
3c
13 ± 1.0
12 ± 1.0
8 ± 1.0
7 ± 1.0
10 ± 1.0
3d
16 ± 1.1
12 ± 1.0
12 ± 1.2
10 ± 0.8
11 ± 1.0
3e
19 ± 1.2
18 ± 1.0
17 ± 1.0
16 ± 1.1
18 ± 1.1
3h
11 ± 1.0
11 ± 1.0
10 ± 0.8
10 ± 1.0
10 ± 1.0
3j
14 ± 1.1
10 ± 1.0
10 ± 1.0
13 ± 1.0
13 ± 1.0
4a
15 ± 1.1
12 ± 1.0
16 ± 1.0
14 ± 0.9
12 ± 1.0
4b
14 ± 1.1
11 ± 0.9
12 ± 1.0
14 ± 1.1
16 ± 1.0
4d
9 ± 1.0
10 ± 1.0
10 ± 1.0
14 ± 1.0
12 ± 1.0
4e
9 ± 1.0
9 ± 0.96
13 ± 1.0
16 ± 1.1
12 ± 1.0
4f
11 ± 1.0
10 ± 1.0
13 ± 1.0
13 ± 0.8
15 ± 1.0
4g
7 ± 1.0
9 ± 1.0
8 ± 1.0
6 ± 1.0
5 ± 1.0
5
12 ± 1.0
9 ± 1.0
9 ± 1.0
10 ± 1.0
10 ± 1.0
7
12 ± 1.0
9 ± 1.0
11 ± 1.0
9 ± 1.0
8 ± 1.0
8
9 ± 1.0
13 ± 1.0
8 ± 1.0
8 ± 1.0
10 ± 1.0
C
23 ± 1.0
–
–
–
–
CN
15 ± 1.0
–
–
–
–
TE
21 ± 1.0
–
–
–
–
AMC
22 ± 1.0
–
–
–
–
The results are expressed by the halo diameter of inhibition (mm), which signifies the sensitivity of the strains to the molecules tested.
The results obtained were compared with those obtained from commercial antibiotics (C (Chloramphenicol), CN (Gentamicin), TE (Tetracycline), AMC (Amoxicillin-Clavulanic acid)).
All the compounds studied possessed some antibacterial activity against E. coli (Table 4). Growth of E. coli was significantly inhibited in the presence of 4b, the most active compound. The compounds 3d, 5 and 3b also show interesting antibacterial activity superior to amoxicillin. The antibacterial activity is related to polarity / lipophilicity, the most active compounds are neither the most polar nor the most lipophilic (4b (log P = 3.07); 3d (log P = 3.72); 5 (log P = 3.48); 3b (log P = 3.16). Regarding the structure, bipyrazoles like 4b show greater activity against Gram-negative bacteria (E. coli) than monopyrazole derivatives such as 3d and 3b. With regard to the substituents on the aromatic ring, the presence of electron donating (4c, 4f) or electron-withdrawing substituents (4a, 4d, 4e) leads to a decrease in antibacterial activity. While in the series of enanimones, the presence of chlorine as a substituent in the 4-position of aniline (3d) presents activity of the same order as that derived with aniline without substituents (3b). However, the presence of hydroxyl groups (3h, 3e) leads to a decrease in activity against E. coli.
From these results, compounds 3e, 3d, 3j and 4a were more active than other compounds against Staphylococcus aureus (Table 5). To these must be added 4b, which has an important antibacterial activity against both Gram (+) (Table 5) and Gram (−) (Table 4). The presence of 4-chlorophenyl and 3-hydroxy groups as well as 4-nitrophenyl enhances the antibacterial activity of pyrazolo-enaminones, and 4-nitrophenyl enhances the activity of bipyrazole compounds. With regard to enaminones, the presence of a hydroxyl substituent in the metha position of the aniline (3e) is more favorable than in the para position (3h). While in the case of halogens, the substitution in para (3d) is more appropriate than in metha position (3a). Furthermore, bipyrazolopyridines (5) and bipyrazoles (7) have often exhibited good activity. From the results of the antioxidant activity, compound 3h appears to be the most promising, while 3e shows the best inhibitory effect against S. aureus and bipyrazole 4b satisfactorily inhibit the growth of E. coli. Due to the serious problems of resistance to the antibacterials currently used for the treatment of infections, it is of interest and the search for new antimicrobial agents is vital.
3 Conclusion
A series of heterocyclic enaminones (3a-j) has been synthesized with good yields under classical conditions. This preparation was based on a condensation of diketo-N-phenyl-pyrazole with different substituted anilines in the presence of catalytic amount of acid. Subsequently, bipyrazole structures (4a-g) were obtained by condensing the diketo-N-phenyl-pyrazole (2) with different phenyl-hydrazines. Furthermore, it was carried out, a new synthesis of bi-pyrazolopyridine (5) by a fast one-step condensation of diketo-N-phenyl-pyrazole (2) with 3-amino-1-phenyl-1H-pyrazol-5(4H)-one (6). Several of this nitrogenated compounds have shown an interesting antibacterial activity against E. coli (3b, 3d, 3e and 4d) or S. aureus (3e and 4a). In addition, some compounds have presented good antioxidant results, especially the enaminone 3h with results of the same order as vitamin C. Finally, it should be noted that the not inconsiderable activity of compounds 3h (antioxidant), 3b (antibacterial activity against E. coli), and 3e (antibacterial activity against S. aureus), turns them into leads that, through new modifications, can optimize both their activity and their pharmacokinetics.
4 Experimental section
4.1 Chemistry
4.1.1 General
Microwave-assisted reactions were carried out in a CEM Discover LabMate Microwave synthesis instrument in a glass tube (10 mL) sealed with a silicon septum under magnetic stirring and an IR sensor measured the temperature of vessel surface. Fixed temperature of 100 °C with a variable MW power (maximum 300 W) was used. The reactions were monitored by thin-layer chromatography (TLC) analysis using silica gel (60 F254, Merck) plates. Compounds were visualized by UV irradiation.
1H and 13C NMR spectra were recorded with a Bruker spectrometer at 300 MHz (13C, 75.5 MHz). Chemical shifts are given in parts per million (ppm) from tetramethylsilane (TMS) as internal standard in CDCl3, and the residual peak of DMSO in DMSO‑d6. Melting points (mp [°C]) were taken on samples in open capillary tubes and are not corrected. The following abbreviations are used for the 1H NMR spectra multiplicities: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet. Coupling constants (J) are reported in Hertz [Hz]. Elemental analyzes have been performed on a LECO TruSpec CHN Elemental Analyzer. ESI Mass Spectrums have been carried out with Agilent LC/MSD/ToF mass spectrophotometer (CCITUB, Chemical Faculty, University of Barcelona).
Compounds 1, 2, 4d, 4e and 4g were reported previously by Singh, S. P. Naithani, R.; Aggarwal, R.; Prakash, O. C-C Bond Cleavage Studies in Bipyrazoles: A Convenient Synthesis of Pyrazolo-5-ols Synth. Commun. 2005, 35, 611–619.
Compound 3b was previously described by Bendaas, A.; Hamdi, M.; Sellier, N. Synthesis of bipyrazoles and pyrazoloisoxazoles from 3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one. J. Heterocycl. Chem. 1999, 35, 611–619.
Compound 3j was previously reported by Asiri, A. M.; Al-Youbi, O. A.; Faidallah, H. M.; Weng, S.; Tiekinkc, R. T. (2Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-(4-methoxyanilino)-but-2-en-1-one. Acta Cryst., Section E: Structure Reports Online, 2011, 67, o2353.
Compound 3g was previously reported by Asiri, Abdullah M. Acta Cryst., Section E: Structure Reports Online, 2012, 68, o764.
Compound 3d was previously reported by Asiri, Abdullah M. Acta Cryst., Section E: Structure Reports Online, 2011, 67, o2157.
4.1.2 General procedure for the synthesis of (E)-4-hydroxy-6-methyl-3-(1-(2-phenylhydrazono)ethyl)-2H-pyran-2-one (1)
Phenylhydrazine (17.8 mmol, 1.75 mL) was added to a solution of dehydroacetic acid (17.9 mmol, 3.02 g) in hot ethanol (20 mL). After heating for 5 min under reflux, the yellow precipitate formed was collected by filtration and the solid obtained is recrystallized from ethanol to give (1) as yellow crystalline product, mp 205–207 °C; 95% yield (4.370 g, 0.017 mol). 1H NMR (300 MHz, CDCl3): δ(ppm) 16.1 (s, 1H, OH), 7.77 (d, J = 7.5, 2H) 7.19 (t, J = 8.1, 2H) 7.03 (t, J = 8.4, 1H), 6.43(s, 1H, NH), 5.81 (s, 1H, H), 2.53 (s, 3H, CH3), 2.10 (s, 3H, CH3). 13C-RMN (75.5 MHz, CDCl3): δ(ppm)181.0; 169.5; 163.7; 163.6; 145.0; 130.1; 122.5; 113.8; 105.8; 96.6; 20.3; 16.4.
4.1.3 Synthesis of 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)butane-1,3-dione (2)
A solution of N-2-phenylhydrazone (1) (2.6 g, 10.2 mmol) in glacial acetic acid (20 mL) is refluxed for 2 h, and then the crude reaction was extracted with CH2Cl2/water. After evaporation of the solvent, the crude product (2.0 g, 7.740 mmol) was recrystallized from acetonitrile to give 2 as a yellow solid, mp 89–91 °C; 75% yield (1.970 g, 7.630 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 14.70 (br, 1H, OH), 11.33 (s, 1H, OH), 8.11 (d, J = 7.5, 2H, Ar), 7.12 (t, J = 8.1, 2H, Ar), 6.90 (t, J = 8.1, 1H, Ar), 5.66 (s,1H), 2.47 (s, 3H, CH3), 1.79 (s, 3H, CH3); 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 188.6; 171.2; 163.5; 144.5; 139.9; 128.4 (x 2); 123.4; 117.6 (x 2); 95.2; 58.6; 23.0; 15.6.
4.1.4 General procedure for the synthesis of pyrazolo-enaminones 3a-j
To a solution of 1-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)butane-1,3-dione (2) (258 mg, 1 mmol) in absolute ethanol (5 mL), a suitable amine (1 mmol) was added with a few drops of acetic acid. The crude reaction is refluxed under magnetic stirring. Once the reaction was judged complete, the expected product was isolated by chromatography on silica gel (eluent: hexane/ethyl acetate: 7/3).
(Z)-3-((3-Bromophenyl)amino)-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-2-en-1-one 3a: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 176–178 °C; 89% yield (0.370 g, 0.897 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.98 (s, OH); 7.94 (s, 1H, NH); 7.91–6.56 (m, 9H, ArH); 5.74 (s, 1H, CH); 2.41 (s, 3H, CH3); 2.01 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 191.5; 160.7; 149.3; 145.8; 136.4; 131.7; 129.9; 129.1 (x 2); 126.9; 126.1; 122.0 (x 2); 121.1; 112.8; 103.5; 97.4; 24.2; 14.1. MS m/z (%): M+= 412.09 (100%). Anal. Calcd. for C20H18BrN3O2: C, 58.38; H, 4.40; N, 10.20. Found: C, 58.30; H, 4.31; N, 10.17.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-(phenylamino)but-2-en-1-one 3b: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 170–172 °C; 82% yield (0.270 g, 0.809 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.97 (s, OH); 7.68–7.40 (m, 5H, ArH); 7,39 (s, 1H, NH); 7,23–6,74 (m, 5H, ArH); 5,66 (s, 1H, CH); 2,70 (s, 3H, CH3); 2,25 (s, 3H, CH3). 13C-RMN (75.5 MHz, DMSO‑d6): δ(ppm) 192.8; 159.7; 149.1); 138.1; 134.5; 130.6 (x 2); 129.7 (x 2); 128.9 (2 x); 128.4; 126.1; 122.8 (x 2); 122.4; 102.4; 98.9; 22.7; 14.0. MS m/z (%): M+= 343.01 (100%). Anal. Calcd. for C20H19N3O2: C, 72.05; H, 5.74; N, 12.60. Found: C, 71.93; H, 5.61; N, 12.59.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-((4-methoxy-2-nitrophenyl)amino)but-2-en-1-one 3c: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a pink solid. mp 178–180 °C; 79% yield (0.322 g, 0.788 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.98 (s, OH); 7.65–7.42 (m, 5H, ArH); 7.35 (s, 1H, ArH); 7.24 (s, 1H, NH); 7.13 (d, J = 9.3, 1H, ArH); 6.96 (d, J = 9.3, 1H, ArH); 5.79 (s, 1H, CH); 3.70 (s, 3H, OCH3); 2.65 (s, 3H, CH3); 2.29 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 191.3; 160.2; 155.6; 149.5; 138.5; 137.2; 132.5; 130.3 (x 2); 128.4 (x 2); 126.5); 122.8 (x 2); 121.4; 118.9; 105.4; 101.3; 99.7; 55.9; 23.8; 14.5. MS m/z (%): M+= 409.11 (100%). Anal. Calcd. for C21H20N4O5: C, 61.74; H, 4.90; N, 13.72. Found: C, 61.63; H, 4.81; N, 13.58.
(Z)-3-((4-Chlorophenyl)amino)-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-2-en-1-one 3d: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 191–193 °C; 85% yield (0.312 g, 0.848 mmol). 1H NMR (400 MHz, CDCl3): δ(ppm) 11.96 (s, OH); 7.68 (d, J = 8 Hz, 2H, ArH); 7.49 (t, J = 8 Hz, 2H, ArH); 7.36 (d, J = 8 Hz, 2H, ArH); 7.24–7.26 (m, 1H, Ar); 7.12 (d, J = 8 Hz, 2H, ArH); 5.78 (s, 1H, CH); 2.50 (s, 3H, CH3); 2.25 (s, 3H, CH3). 13C NMR (100.6 MHz, CDCl3): δ(ppm) 185.2; 160.1; 146.2; 138.1; 136.1; 135.4; 135.1; 132.6; 129.8 (x 2); 128.9 (x 2); 126.8 (x 2); 122.2 (x 2); 101.5; 97.7; 22.6; 15.2. MS m/z (%): M+= 368.02 (100%). Anal. Calcd. for C20H18ClN3O2: C, 65.37; H, 4.90; N, 11.44. Found: C, 65.28; H, 4.78; N, 11.29.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-((3-hydroxyphenyl)amino)but-2-en-1-one 3e: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as an orange solid. mp 228–230 °C; 78% yield (0.272 g, 0.778 mmol). 1H NMR (400 MHz, CDCl3): δ(ppm) 11.92 (s, OH); 7.88 ((d, J = 8 Hz, 2H, ArH); 7.45 (t, J = 8 Hz, 2H, ArH); 7.27 (t, J = 8 Hz, 1H, ArH); 6.65–6.72 (m, 3H, ArH); 5.70 (s, 1H, CH); 2.51 (s, 1H, CH3); 2.18 (s, 1H, CH3). 13C NMR (100.6 MHz, CDCl3): δ(ppm) 187.5; 157.2; 149.0; 140.3; 130.9; 128.9 (x 2); 126.5; 122.9 (x 2); 115.6; 109.9; 100.0; 99.7; 98.2; 20.8; 15.6. Anal. Calcd. for C20H19N3O3: C, 68.75; H, 5.48; N, 12.03. Found: C, 68.30; H, 5.31; N, 12.17.
(Z)-3-((4-Hydroxy-2-methylphenyl)amino)-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-2-en-1-one 3f: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 235–237 °C; 68% yield (0.246 g, 0.677 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.96 (s, OH); 7.59–7.45 (m, 5H, ArH); 7.22 (s, 1H, NH); 6.59 (s, 1H, ArH); 6.43 (d, J = 9.3, 1H, ArH); 6.38 (d, J = 9.3, 1H, ArH); 5.72 (s, 1H, CH); 2.58 (s, 3H, CH3); 2.36 (s, 3H, CH3); 2.24 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 189.2; 160.3; 153.6; 149.3; 138.5; 132.8; 130.3 (x 2); 128.2; 124.5 (x 2); 121.0; 114.7; 105.1; 88.7; 20.8; 19.1; 13.7. Anal. Calcd. for C21H21N3O3: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.33; H, 5.93; N, 11.58.
(Z)-3-((4-Fluorophenyl)amino)-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-2-en-1-one 3g: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 180–182 °C; 83% yield (0.291 g, 0.828 mmol). 1H NMR (400 MHz, CDCl3) δ(ppm) 11.98 (s, OH); 7.78 (d, J = 8 Hz, 2H, ArH); 7.45 (t, J = 8 Hz, 2H, ArH); 7,35 (t, J = 8 Hz, 2H, ArH); 7.22 (d, J = 7.8, 2H, ArH); 7.12 (d, J = 7.8, 2H, ArH); 5.45 (s, 1H, CH); 2.54 (s, 3H, CH3); 2.16 (s, 3H, CH3). 13C NMR (100.6 MHz, CDCl3)): δ(ppm) 185.1; 162.2; 161.0; 160.2; 146.3; 138.3; 137.5; 133.9; 130,1 (x 2); 129.1; 127.4 (x 2); 125.9 (x 2); 121.3; 120.2 (x 2); 115.7; 104.6; 96.9; 20.2; 15.3. MS m/z (%): M+= 352.08 (100%). Anal. Calcd. for C20H18FN3O2: C, 68.35; H, 5.16; N, 11.96. Found: C, 68.30; H, 5.02; N, 11.80.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-((4-hydroxyphenyl)amino)but-2-en-1-one 3 h: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 226–228 °C; 81% yield (0.283 g, 0.809 mmol). 1H NMR (400 MHz, CDCl3): δ(ppm) 11.55 (s, OH); 7.87 (d, J = 8 Hz, 2H, ArH); 7.48 (t, J = 8 Hz, 2H, ArH); 7.27 (t, J = 8 Hz, 2H, ArH); 7.12 (d, J = 8 Hz, 2H, ArH); 6.58 (d, J = 8 Hz, 2H, ArH); 5.38 (s, 1H, CH); 2.46 (s, 3H, CH3); 2.11 (s, 3H, CH3). 13C NMR (100.6 MHz, CDCl3): δ(ppm) 185.7; 170.0; 160.2; 151.9; 150.4; 138.5; 136.4; 129.2 (x 2); 125.1; 122.8 (x 2); 116.2; 114.0; 101.5; 98.6; 21.5; 14.1. Anal. Calcd. for C20H19N3O3: C, 68.75; H, 5.48; N, 12.03. Found: C, 68.99; H, 5.42; N, 11.98.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-(thiazol-2-ylamino)but-2-en-1-one 3i: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a purple solid. mp 173–175 °C; Yield 84%; NMR (300 MHz, DMSO‑d6): δ(ppm) 8.55 (d, J = 5.1 Hz, 1H, CH-N); 8.03 (dd, J1 = 8.7 Hz, J2 = 1.2 Hz, 2H, CH-Ar(ortho); 7.97 (d, J = 4.8 Hz, 1H, CH-S); 7.42 (s, 1H, CH); 7.32 (dt, J1 = 8.4 Hz, J2 = 1.2 Hz, 2H, CH-Ar(meta)); 7.03 (dt, J1 = 7.5 Hz, J2 = 1.2 Hz, 1H, CH-Ar(para)); 2.58 (s, 3H, CH3); 2.31 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 164.0; 163.8; 162.6; 149.7; 147.1; 140.7; 129.9; 128.8 (x 2); 123.2; 118.4 (x 2); 116.1; 112.7; 94.2; 24.4; 16.1. Anal. Cald.C17H16N4O2S: C, 59.98; H, 4.74; N, 16.46. Found: C, 60.04; H, 4.88; N, 16.61.
(Z)-1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-((4-methoxyphenyl)amino)but-2-en-1-one 3j: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 222–224 °C; 78% yield (0.283 g, 0.778 mmol). 1H NMR (400 MHz, CDCl3): δ(ppm) 11.62 (s, OH); 7.80 (d, J = 8 Hz, 2H, ArH); 7.48 7.24 (t, J = 8 Hz, 3H, ArH); 7.22 (d, J = 8 Hz, 2H, ArH); 7.12 (d, J = 8 Hz, 2H, ArH); 5.46 (s, 1H, CH); 3.73 (s, 3H, OCH3); 2.49 (s, 3H, CH3); 2.17 (s, 1H, CH3); 2.05 (H2O). 13C NMR (100.6 MHz, CDCl3): δ(ppm) 187.2; 159.1; 155.6; 149.5; 137.2; 132.5; 129.3 (x 2); 127.3 (x 2); 126.3; 122.8 (x 2); 121.4; 117.5 (x 2); 102.2; 99.7; 53.8; 22.6; 17.0. Anal. Calcd. for C21H21N3O3: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.49; H, 5.79; N, 11.48.
4.1.5 General procedure for the synthesis of 3,4′-bipyrazol-5′-oles 4a-g
A mixture of pyrazolylbutane-1,3-dione (2) (258 mg, 1 mmol) and an aromatic hydrazine (1 mmol) in absolute ethanol (5 mL) was refluxed for 2 h. The precipitate obtained from the hot solution is collected by filtration and washed several times with diethyl ether and recrystallized from ethanol.
2-(2,4-Dinitrophenyl)-3′,5-dimethyl-1′H,2H-[3,4′-bipyrazol]-5′-ol 4a: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a yellow solid. mp 262–264 °C; 89% yield (0.307 g, 0.89 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 12.01 (s, 1H, exchangeable OH); 8.66 (s, 1H, ArH); 8.21 (d, J = 7.4, 1H, ArH); 8.16 (s, 1H, NH); 7.84 (d, J = 7.3, 1H, ArH); 6.14 (s, 1H, CH); 2.31 (s, 3H, CH3); 1.81 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 161.2; 150.1; 147.8; 144.7; 142.3; 140.1; 137.8; 128.3; 124.9; 119.6; 111.2; 106.3; 13.6; 12.1. MS m/z (%): M+= 345.12 (100%). Anal. Calcd. for C14H12N6O5: C, 48.84; H, 3.51; N, 24.41. Found: C, 48.67; H, 3.42; N, 24.29.
3′,5-Dimethyl-2-phenyl-1′H,2H-[3,4′-bipyrazol]-5′-ol 4b: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as an yellow solid. mp 253–255 °C; 87% yield (0.221 g, 0.869 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.91 (s, 1H, exchangeable OH); 8.10 (s, 1H, NH); 7.69 (d, J = 8,2, 2H, ArH); 7.41 (t, J = 8.4, 2H, ArH); 7.25 (t, J = 8.4, 1H, ArH); 6.09 (s, 1H, CH); 2.29 (s, 3H, CH3); 1.84 (s, 3H, CH3). 13C NMR (δ-ppm): 159.8; 152.8; 142.7; 140.0; 138.8; 129.7 (x 2); 127.2; 123.8 (x 2); 106.7; 104.1; 13.1; 12.7. MS m/z (%): M+= 255.10 (100%). Anal. Calcd. for C14H14N4O: C, 66.13; H, 5.55; N, 22.03. Found: C, 65.98; H, 5.44; N, 21.93.
3′,5-Dimethyl-2-(p-tolyl)-1′H,2H-[3,4′-bipyrazol]-5′-ol 4c: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as an orange solid. mp 267–269 °C; 79% yield (0.345 g, 0.919 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 12.20 (s, 1H, exchangeable OH); 8.23 (s, 1H, NH); 7.61 (d, J = 7.1, 2H, ArH); 7.45 (d, J = 7.1, 2H, ArH); 6.39 (s, 1H, CH); 2.39 (s, 3H, CH3); 2.32 (s, 3H, CH3); 1.84 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm)157.9; 148.3; 141.4; 138.7; 137.8; 137.1; 129.0; 124.6; 109.2; 105.0; 21.4; 14.1; 13.6. MS m/z (%): M+= 269.22 (100%). Anal. Calcd. for C15H16N4O: C, 67.15; H, 6.01; N, 20.88. Found: C, 66.95; H, 5.84; N, 20.83.
3′,5-Dimethyl-2-(4-nitrophenyl)-1′H,2H-[3,4′-bipyrazol]-5′-ol 4d: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as an orange solid; mp 239–241 °C; 92% yield (0.345 g, 0.919 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 11.99 (s, 1H, exchangeable OH); 8.24 (d, J = 9.3, 2H, ArH); 7.65 (m, 3H, ArH); 7.41 (t, J = 7.5, 2H, ArH); 7.24 (t, J = 7.2, 1H, ArH); 6.38 (s, 1H, CH); 2.28 (s, 3H, CH3); 1.93 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 150.5; 149.2; 147.3; 145.9; 145.2; 138.3; 135.5; 129.3 (x 4); 125.9; 125.0; 123.2 (x 2); 120.9 (x 2); 111.4; 99.1; 13.9; 12.9. MS m/z (%): M+= 376.10 (100%). Anal. Calcd. for C20H17N5O3: C, 63.99; H, 4.56; N, 18.66. Found: C, 66.80; H, 4.44; N, 18.53.
2-(2,4-Dinitrophenyl)-3′,5-dimethyl-1′-phenyl-1′H,2H-[3,4′-bipyrazol]-5′-ol 4e: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as an orange solid; mp 230–232 °C; 87% yield (0.365 g, 0.868 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 12.10 (s, 1H, exchangeable OH); 9.31 (s, 1H, ArH); 8.80 (d, J = 9.6, 1H, ArH); 8.29 (d, J = 9.6, 1H, ArH); 7.62–7.41 (m, 5H, ArH); 6.68 (s, 1H, CH); 2.44 (s, 3H, CH3); 1.89 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm)150.7; 148.4; 146.2; 145.6; 143.1; 139.9; 137.9; 136.8; 129.4 (x 2); 127.7; 126.1; 124.5; 121.0 (x 2); 120.2; 108.7; 97.1; 14.4; 13.6. MS m/z (%): M+= 350.26 (100%). Anal. Calcd. for C20H16N6O5: C, 57.14; H, 3.84; N, 19.99. Found: C, 57.60; H, 3.37; N, 19.93.
3′,5-Dimethyl-1′-phenyl-2-(p-tolyl)-1′H,2H-[3,4′-bipyrazol]-5′-ol 4f: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a brown solid. mp 235–237 °C; 83% yield (0.285 g, 0.827 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 12.21 (s, 1H, exchangeable OH); 7.68 (d, J = 8.7, 2H, ArH); 7.49 (t, J = 8.4, 2H, ArH); 7.44 (d, J = 8.2, 2H, ArH); 7.35 (d, J = 8.1, 2H, ArH); 7.24 (t, J = 7.2, 1H, ArH); 6.24 (s, 1H, CH); 2.31 (s, 3H, CH3); 2.26 (s, 3H, CH3); 1.74 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 152,3; 149.7; 148.4; 138.4; 136.3; 134.5; 130.4 (x 2); 129.7 (x 2); 129.2 (x 2); 126.3; 125.7; 122.6 (x 2); 109.6; 95.4; 20.9; 13.9; 12.9. MS m/z (%): M+= 345.04 (100%). Anal. Calcd. for C21H20N4O: C, 73.23; H, 5.85; N, 16.27. Found: C, 72.99; H, 5.70; N, 16.13.
3′,5-Dimethyl-1′,2-diphenyl-1′H,2H-[3,4′-bipyrazol]-5′-ol 4 g: Starting from 1 mmol (258 mg) of 2 and following the general procedure indicated above, the desired compound was obtained as a white solid. mp 228–230 °C; 84% yield (0.277 g, 0.838 mmol). 1H NMR (300 MHz, DMSO‑d6): δ(ppm) 12.07 (s, 1H, exchangeable OH); 7.66–7.44 (m, 10H, ArH); 6.33 (s, 1H, CH); 2.29 (s, 3H, CH3); 1.94 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ(ppm) 165.4; 160.2; 152.7; 150.0; 146.0; 141.6; 140.6; 128.9 (x 2); 128.8 (x 2); 123.3; 122.8; 118,3 (x 2); 102.6; 101.3; 91.9; 14.2; 13.1. MS m/z (%): M+= 330.15 (100%). Anal. Calcd. for C20H18N4O: C, 72.71; H, 5.49; N, 16.96. Found: C, 72.05; H, 5.30; N, 16.86.
4.1.6 4-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-methyl-2-phenyl-2,3a-dihydro-3H-pyazolo[3,4-b]pyridine-3-one (5)
A mixture of 1-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)butane-1,3-dione 2 (258 mg, 1 mmol) and 3-amino-1-phenyl-1H-pyrazol-5(4H)-one (1 mmol) in ethanol (4 mL) was refluxed for 4 h. The precipitate, obtained from the hot solution, was collected and washed several times with ethanol, the pure product was recrystallized from ethanol. Orange solid; mp 182–184 °C; 92% yield (0.365 g, 0.918 mmol). 1H NMR (300 MHz, DMSO‑d6): δ (ppm) 14.08 (s, 2H, OH); 8.19–7.03 (m, 10H, ArH); 6.11 (s, 1H, CH); 2.36 (s, 3H, CH3); 2.06 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO‑d6): δ (ppm) 165.3; 160.2; 152.7; 150.0; 146.3; 146.0; 141.5; 140.6; 128.9 (x 2); 128.7 (x 2); 123.3; 122.8; 118.3 (x 4); 102.6; 91.8; 31.1; 17.9; 17.5. MS m/z (%): M+= 398.10 (100%). Anal. Calcd. for C23H19N5O2: C, 69.50; H, 4.82; N, 17.62. Found: C, 69.39; H, 4.69; N, 17.57.
4.1.7 4-Methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6(3aH,7H)-dione (7)
4.1.7.1 Conventional heating
In a 100 mL flask was dissolved (0.875 g, 5 mmol) of 3-amino-1-phenyl-1H-pyrazol-5(4H)–one 6 in a suitable volume of ethyl acetoacetate (3.19 mL, 25 mmol). The reaction mixture was heated at 180 °C for 4 h, after the reaction is judged complete by TLC plate, filtered immediately and washed with cold diethyl ether, to give a gray precipitate of compound 7.
4.1.7.2 Microwave irradiation
A mixture of (5 mmol; 0,875 g) of 3-amino-1-phenyl-1H-pyrazol-5(4H)–one (6) in a suitable volume of ethyl acetoacetate (3.19 mL, 25 mmol) was introduced to microwave oven at 100 °C. After the completion of the reaction, the solid product was collected by filtration and crystallized from diethyl ether to give the corresponding compound 7.
4.1.7.3 Sealed tube
The mixture of (0.875 g, 5 mmol) of 3-amino-1-phenyl-1H-pyrazol-5(4H)–one (6) in a suitable volume of ethyl acetoacetate (3.19 mL, 25 mmol) was placed in a sealed tube in a sand bath (180–185 °C). After the reaction was complete, the reaction mixture was cooled and filtered. The precipitate obtained was washed thoroughly with diethyl ether to give the pure compound 7.
Gray powder; mp 276–278 °C; 92% yield (1.11 g, 4.601 mmol); Rf = 0.22 (Ethyl Acetate–Hexane, 2:3). 1HNMR (300 MHz, DMSO‑d6): δ(ppm) 2.41 (s, 3H, CH3); 6.02 (s, 1H, H); 7.20 (t, J = 7.4 Hz, 1H, Ar-H); 7.45 (t, J = 8.0 Hz, 2H, Ar-H); 7.74 (d, J = 7.7 Hz, 2H, Ar-H); 11.57 (s, 1H, NH); 13C NMR (75.5 MHz, DMSO‑d6): δ 17.6; 108.9; 119.6 (x 2); 124.9; 129.3 (x 2); 138.2; 149.9; 155.0; 159.8; 166.6. MS m/z (%): M+= 242.06 (100%). Anal. Calcd. for C13H11N3O: C, 64.72; H, 4.60; N, 17.42. Found: C, 64.59; H, 4.49; N, 17.37.
4.1.8 Ethyl 4,6-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (8)
A solution of 3-amino-1-phenyl-1H-pyrazol-5(4H)-one (6) (350 mg, 2 mmol) and ethyl acetoacetate (3.19 mL, 25 mmol) was refluxed in a sand bath (120 °C) under stirring overnight. The orange crystals formed (compound 8) were collected by filtration and washed with diethyl ether.
Orange crystal; mp 260–262 °C; 54% yield (0.336 g, 1.079 mmol). 1H NMR (300 MHz, CDCl3): δ(ppm) 1.39 (t, J = 5.9 Hz, 3H), 2.28 (s, 3H, CH3), 2.77 (s, 3H, CH3), 4.40 (q, J = 5.9 Hz, 2H, CH2), 7.21 (t, J = 7.4 Hz, 1H, Ar-H), 7.40 (t, J = 7.5 Hz, 2H, t, Ar-H), 7.85 (d, J = 7.6 Hz, 2H, Ar-H); 13C NMR (75.5 MHz, CDCl3): δ(ppm) 14.6; 22.2; 61.7; 109.0; 122.3 (x 2); 125.7; 129.1 (x 2); 137.4; 150.9; 153.7; 157.3; 159.2; 167.3. MS m/z (%): M+= 312.09 (100%). Anal. Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: C, 65.29; H, 5.34; N, 13.35.
4.2 Antioxidant activity determination
The antioxidant activity of the synthesized compounds was measured following the Kedare procedure (Kedare and Singh, 2011). The antioxidant activity of the synthesized compounds was measured using more stable free radicals. DPPH (2,2-diphenyl-1-picrylhydrazyl free radical) is a very stable free radical in the crystalline state and in solution, with a violet coloration. The antioxidant activity of this method is the ability of antioxidants to act as scavengers of free radicals. They act by transferring a hydrogen atom, which leads to the disappearance of the DPPH during the reaction and to a change of color in the initial solution. The progress of the reaction was followed by the spectrophotometric method at 517 nm.
The standard solution of vitamin C (positive control) was 1 mg / 100 μL of methanol. A solution of DPPH (1 mg / 25 mL of methanol) was prepared as a control. In 96 well plates, the products were solubilized in a suitable volume of DMSO to have a concentration of 20 mg / mL. In 96 well plates, 16 μL of the solution of each product and 16 μL of vitamin C (10 mg / mL) were added to methanol to have a final volume of 200 μL. Then 100 μL was transferred to the next well containing 100 μL of methanol (1/2 dilution).
The same procedure was repeated to perform the cascade 1/4, 1/8, 1/16, 1/32, 1/64 and 1/128 dilutions. After which, 150 μL of DPPH (1 mg / 25 mL methanol) were added to the wells containing 100 μL of the mixture and then incubated for 30 min in the dark at 37 °C.
Absorbance was measured by a spectrophotometer at 517 nm. The white test is a mixture of methanol and the DPPH reagent under the same experimental conditions. The results are expressed as the percentage of anti-radical activity. The value of the IC50 determined from the graphical plot of scavenging activity according to the different concentrations of each product, was defined as the concentration of the antioxidant necessary to reduce the initial concentration of DPPH to 50%. The results obtained for the compounds evaluated were compiled in Table 3.
4.3 Antibacterial activity determination
The antibacterial activity of the synthesized molecules 2, 3a, 3b, 3j, 3d, 3e, 3h, 4a, 4b, 4d, 4e, 4f, 4g, 5, 6a and 6b was tested on two Gram+ bacterial strains (Staphylococcus aureus ATCC 25923) and Gram-negative (Escherichia coli ATCC 25922) following the applied disc diffusion method (Balouiri et al., 2016). This method is based on the migratory power of molecules on a solid medium inside a Petri dish. It allows us to demonstrate the antibacterial effect of the molecules to be tested, as well as the determination of the resistance or sensitivity of these bacteria to the molecules.
-
Preparation of bacterial suspensions
For each test bacteria, an inoculum was produced from a culture of 24 h at 37 °C, suspended in liquid Luria-Bertani medium (10 g of peptone; 5 g of yeast extract; 10 g of NaCl) sterile so as to obtain an optical density between 0.07 and 0.08 (approximately 108 mL−1 CFU). Antibacterial tests are carried out using young cultures in the exponential growth phase.
Preparation of discs
The disks were obtained from Wattman paper, with a diameter of 6 mm by the punch. They were autoclaved and then stored at room temperature.
Seeding
A volume of 15 mL of Muller Hinton's agar (Infusion of beef: 300 mL; casein peptone: 17.5 g; corn starch: 1.5 g; agar: 17.0 g and pH = 7.4.) was poured in Petri dishes. After solidification of the culture medium, 0. 2 mL of the bacterial suspension to be tested (108 CFU. mL−1) was spread on the surface, then the excess was removed.
Deposit of discs
Under aseptic conditions and using sterile forceps, the Wattman paper discs are placed on the Muller Hinton culture medium beforehand, seeded at the surface with the target microorganisms, the concentration of which is 108 CFU. mL−1 then soaked with 10 μL of each molecule to be studied. Previously inoculated with the chosen microorganism, the dishes are kept at 4 °C for 1 h so that the molecule can diffuse and subsequently incubated in the oven at 37 °C for 24 h. The study is done by measuring the diameter of the zone of inhibition around the disc using a caliper or a ruler graduated in millimeters (mm). The results obtained for the compounds evaluated have been detailed in Tables 4 and 5.
4.3.1 Partition coefficient (log P)
It has been determined theoretically by the program ChemDraw Professional 20. ChemDraw: PerkinElmer Inc. Addlink Software Científic. Creative Commons (CC), CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/deed.es). https://www.addlink.es/productos/chembiodraw
Acknowledgements
The financial support from University Sultan Moulay Slimane is gratefully acknowledged. We also thank to CNRST scientific center (Rabat, Morocco) for the NMR results and the Serveis Cientifico-Tècnics (CCIT) of the University of Barcelona (Spain) for the NMR, masses spectra and analytical tests.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103527.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary data 1
Supplementary data 1