Introduction

Tuberculosis (TB) is caused predominantly by Mycobacterium tuberculosis bacteria (MTB), an obligate aerobic bacillum that divides at an extremely slow rate. According to the World Health Organization (WHO), in 2011, an estimated 8.7 million people died from TB [1]. The multi-drug-resistant TB strains (MDR-TBs) exhibit resistance to the front-line drugs isoniazid (INH) and rifampicin (RIF), and extensively drug resistant TB strains (XDR-TBs) exhibit resistance to second-line drugs, including fluoroquinolones, capreomycin and kanamycin [2]. These reasons make a compelling case for the urgent need for new and effective antitubercular drugs. Nitrogen-containing heterocyclic compounds are widespread in nature, and their applications in biologically active pharmaceuticals, agrochemicals, and functional materials are becoming more and more important [3–6]. Furthermore, pyrazoles are usually the core fragment of many biologically active compounds, such as Celecoxib, Viagra, Pyrazofurine and so on [7–11]. Among a large variety of N-containing heterocyclic compounds, those containing an hydrazine moiety as a “fusion site” have received considerable attention because of their pharmacological properties and clinical applications [12]. Moreover, fused phthalazines were found to possess multiple biological activities, such as antimicrobial [13], anticonvulsant [14], antifungal [15], anticancer [16] and anti-inflammatory activities [17]. Nevertheless, the development of new synthetic methods for the efficient preparation of heterocycles containing a phthalazine ring fragment is an interesting challenge. Also, multi-component reactions (MCRs) were employed as a powerful tool to synthesize diverse and complex heterocyclic compounds due to their advantages of the intrinsic atom economy, simpler procedures, structural diversity, energy savings, and reduced waste [17–19].

Despite their importance from pharmacological and synthetic points of views, recently, several elegant multi-component strategies have emerged for the synthesis of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione by the cyclo condensation of phthalhydrazide, aldehydes, and malononitrile/ethyl cyanoacetate catalyzed by p-TSA [20], Et3N4 or [bmim]OH [21]. But, there was not even a single report in which 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-carbaldehyde was used. Thus, in view of the biological significance of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione, a modification on the 1-position of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione by 3-methyl-5-phenoxy-1-phenyl-1H-pyrazole was undertaken to check whether it brings significant changes in the bioactivities of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives. As a part of our current study in developing new antimicrobial agents via combination of two therapeutically active moieties [22–25], we report herein the preparation 1H-pyrazolo[1,2-b]phthalazine-5,10-dione 4a–p derivatives by an MCR approach. The structures of title derivatives were elucidated on the basis of FT-IR, 1H NMR, 13C NMR, mass spectra and elemental analysis. All these derivatives were screened for their in vitro antimicrobial activity against a representative panel of bacteria and fungi, antitubercular activity against M. tuberculosis H37Rv, antioxidant activity and cytotoxicity study against Artemia cysts.

In continuation of our interest on synthesizing biologically potent antimicrobials [22–25], we report herein a new series of 1H-Pyrazolo[1,2-b]phthalazine-5,10-dione derivatives (4a–p) via one-pot, three-component base-catalyzed cyclo condensation reaction of 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-carbaldehyde (1a–d), malononitrile or ethyl cyanoacetate (2a–b) and 2,3-dihydro-1,4-phthalazinedione (3a–b) in ethanol containing eco friendly base NaOH in good to excellent yields (Scheme 1). The required starting material, 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde was prepared by using the procedure in the literature [26]. 1-Aryl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde undergoes a nucleophilic substitution reaction with respective phenol at refluxing temperature for 4 h in the presence of a basic catalyst (K2CO3) in DMF which resulted in the required 3-methyl-5-aryloxy-1-aryl-1H-pyrazole-4-carbaldehyde [22, 23]. A possible mechanism for the reaction is outlined in Scheme 2. The reaction may occur via initial Knoevenagel condensation of 1a–d and 2a–b in the presence of NaOH base to give intermediate heterylidenenitrile which on subsequent Michael-type addition of the 2,3-dihydro-1,4-phthalazinedione 3a–b, followed by cyclization and tautomerization, affords the corresponding 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives 4a–p.

Scheme 1
scheme 1

Synthetic pathway for the compounds 4a–p

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Scheme 2
scheme 2

Possible mechanistic pathway for the synthesis of compounds 4a–p

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Experimental

All reactions were performed with commercially available reagents that were used without further purification. Organic solvents were purified by standard methods and stored over molecular sieves. All melting points were taken in open capillaries and are uncorrected. Thin-layer chromatography (TLC, on aluminum plates coated with silica gel 60 F254, 0.25 mm thickness, Merck) was used for monitoring the progress of all reactions, purity and homogeneity of the synthesized compounds. Elemental analysis (%C, H, N) was carried out using a Perkin-Elmer 2400 series-II elemental analyzer, and all compounds are within ±0.4 % of theory-specified values. The FTIR spectra were recorded using the potassium bromide disc on a Shimadzu FTIR 8401 spectrometer and only the characteristic peaks are reported in cm−1. 1H NMR and 13C NMR spectra were recorded in deuterated dimethylsilane (DMSO-d 6 ) on a Bruker Avance 400F (MHz) spectrometer using the solvent peak as the internal standard at 400 and 100 MHz, respectively. Chemical shifts are reported in parts per million (ppm). Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer. Ampicillin, ciprofloxacin, norfloxacin, chloramphenicol, griseofulvin, nystatin, isoniazid, rifampicin and l-ascorbic acid were commercial grade.

Synthesis of compounds 4a–p

An appropriate mixture of 1H-pyrazole-4-carbaldehyde (1a–d; 5 mmol), malononitrile or ethylcyanoacetate (2a–b; 5 mmol), and 2,3-dihydro-1,4-phthalazinedione (3a–b, 5 mmol) in ethanol (10 mL) containing NaOH (5 mmol, 10 mL) was heated under reflux for 3.5–4 h. On completion of reaction, monitored by TLC, the separated solid was filtered and washed well with ethanol to obtain the pure solid samples 4a–p.

3-Amino-1-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4a)

Yield: 88 %; mp 240–242 °C; IR (KBr, ν max, cm−1): 3395 and 3180 (asym. and sym. stretching of –NH2), 2205 (C≡N stretching), 1695 (C=O stretching), 1680 (C=O stretching), 1210 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.33 (s, 3H, CH3), δ 6.55 (s, 1H, C1H), 7.54–8.41 (m, 13H, Ar–H), 8.78 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.02, (Ar–CH3), 59.32 (C1), 61.32 (C2), 116.05, 124.35, 127.33, 127.90, 128.13, 128.32, 128.90, 129.40, 131.89, 132.20, 134.51, 135.41, 147.20, 151.85, 154.30, 157.87, 158.09 (Ar–C), 156.88 (C=O), 158.21 (C=O); Anal. Calcd for C28H19FN6O3 (506.49 g/mol): C, 66.40; H, 3.78; N, 16.59 (%); Found: C, 66.22; H, 3.62; N, 16.87 (%). MS: 506 [M + H]+.

3-Amino-1-(5-(4-cyanophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4b)

Yield: 85 %; mp 257–259 °C; IR (KBr, ν max, cm−1): 3375 and 3170 (asym. and sym. stretching of –NH2), 2190 (C≡N stretching), 1680 (C=O stretching), 1665 (C=O stretching), 1195 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.54 (s, 3H, CH3), 6.50 (s, 1H, C1H), 7.59–8.28 (m, 13H, Ar–H), 8.70 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.05 (Ar–CH3), 59.90 (C1), 62.07 (C2), 116.44, 117.14, 125.05, 127.17, 127.78, 128.84, 129.35, 130.13, 131.45, 132.37, 133.95, 134.56, 135.38, 137.92, 142.14, 151.71, 154.79, 156.15 (Ar–C), 157.18 (C=O), 158.25 (C=O); Anal. Calcd for C29H19N7O3 (513.51 g/mol): C, 67.83; H, 3.73; N, 19.09 (%); Found: C, 68.05; H, 3.89; N, 19.15 (%). MS: 513 [M + H]+.

3-Amino-1-(5-(4-fluorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4c)

Yield: 87 %; mp 231–233 °C; IR (KBr, ν max, cm−1): 3395 and 3180 (asym. and sym. stretching of –NH2), 2205 (C≡N stretching), 1685 (C=O stretching), 1665 (C=O stretching), 1200 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.10, 2.32 (s, 6H, 2 × CH3), 6.53 (s, 1H, C1H), 7.35–8.34 (m, 12H, Ar–H), 8.69 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.06, 20.80 (Ar–CH3), 60.18 (C1), 62.50 (C2), 117.17, 124.80, 127.12, 127.40, 127.80, 128.50, 128.90, 131.16, 132.15, 133.70, 135.16, 137.17, 145.80, 151.01, 154.96, 157.13, 158.01 (Ar–C), 157.34 (C=O), 158.43 (C=O); Anal. Calcd for C29H21FN6O3 (520.51 g/mol): C, 66.92; H, 4.07; N, 16.15 (%); Found: C, 66.73; H, 3.90; N, 16.37 (%). MS: 520 [M + H]+.

3-Amino-1-(5-(4-cyanophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4d)

Yield: 86 %; mp 250–252 °C; IR (KBr, ν max, cm−1): 3380 and 3185 (asym. and sym. stretching of –NH2), 2190 (C≡N stretching), 1685 (C=O stretching), 1660 (C=O stretching), 1210 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.99, 2.39 (s, 6H, 2 × CH3), 7.82–8.35 (m, 12H, Ar–H), 8.76 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.00, 20.75 (Ar–CH3), 59.23 (C1), 61.80 (C2), 116.90, 117.16, 124.60, 126.80, 127.18, 127.90, 128.17, 129.44, 131.40, 132.50, 133.31, 133.60, 134.50, 135.19, 147.17, 151.91, 154.24, 157.25 (Ar–C), 157.00 (C=O), 158.30 (C=O); Anal. Calcd for C30H21N7O3 (527.53 g/mol): C, 68.30; H, 4.01; N, 18.59 (%); Found: C, 67.98; H, 4.18; N, 18.80 (%). MS: 527 [M + H]+.

Ethyl 3-amino-1-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4e)

Yield: 84 %; mp 219–221 °C; IR (KBr, ν max, cm−1): 3395 and 3300 (asym. and sym. stretching of –NH2), 1700 (ester C=O stretching), 1670 (C=O stretching), 1650 (C=O stretching), 1195 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.07 (t, 3H, CH3), 2.48 (s, 3H, CH3), 3.99 (q, 2H, OCH2), 6.56 (s, 1H, C1H), 7.61–8.40 (m, 13H, Ar–H), 8.82 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.32 (CH3), 13.08, (Ar–CH3), 58.98 (C1), 63.55 (OCH2), 82.07 (C2), 124.18, 126.85, 127.11, 127.24, 127.40, 127.75, 129.54, 131.40, 132.44, 134.08, 135.96, 146.50, 151.10, 154.90, 157.33, 158.23 (Ar–C), 156.37 (C=O), 158.55 (C=O), 164.35 (COOEt); Anal. Calcd for C30H24FN5O5 (553.54 g/mol): C, 65.09; H, 4.37; N, 12.65 (%); Found: C, 65.23; H, 4.08; N, 12.39 (%). MS: 553 [M + H]+.

Ethyl 3-amino-1-(5-(4-cyanophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4f)

Yield: 82 %; mp 260–262 °C; IR (KBr, ν max, cm−1): 3425 and 3335 (asym. and sym. stretching of –NH2), 2210 (C≡N stretching), 1710 (ester C=O stretching), 1675 (C=O stretching), 1650 (C=O stretching), 1190 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.09 (t, 3H, CH3), 2.38 (s, 3H, CH3), 3.95 (q, 2H, OCH2), 6.55 (s, 1H, C1H), 7.64–8.29 (m, 13H, Ar–H), 8.73 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.28 (CH3), 13.04, (Ar–CH3), 60.03 (C1), 63.70 (OCH2), 81.90 (C2), 117.29, 124.80, 127.37, 127.50, 127.78, 128.93, 129.06, 130.17, 131.06, 133.72, 134.67, 135.19, 136.94, 145.22, 151.12, 153.58, 155.19 (Ar–C), 156.88 (C=O), 158.07 (C=O), 164.56 (COOEt); Anal. Calcd for C31H24N6O5 (560.56 g/mol): C, 66.42; H, 4.32; N, 14.99 (%); Found: C, 66.65; H, 4.18; N, 14.71 (%). MS: 560 [M + H]+.

Ethyl 3-amino-1-(5-(4-fluorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4g)

Yield: 85 %; mp 227–229 °C; IR (KBr, ν max, cm−1): 3465 and 3345 (asym. and sym. stretching of –NH2), 1695 (ester C=O stretching), 1665 (C=O stretching), 1635 (C=O stretching), 1205 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.02 (t, 3H, CH3), 2.09, 2.36 (s, 6H, 2 × CH3), 4.01 (q, 2H, OCH2), 6.44 (s, 1H, C1H), 7.30–8.37 (m, 12H, Ar–H), 8.46 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.30 (CH3), 13.10, 21.00 (Ar–CH3), 59.88 (C1), 63.71 (OCH2), 82.50 (C2), 123.71, 127.24, 127.57, 127.62, 128.93, 129.10, 129.36, 130.19, 132.40, 134.28, 135.20, 144.70, 149.16, 150.80, 155.56, 158.20 (Ar–C), 157.14 (C=O), 158.17 (C=O), 164.17 (COOEt); Anal. Calcd for C31H26FN5O5 (567.57 g/mol): C, 65.60; H, 4.62; N, 12.34 (%); Found: C, 65.78; H, 4.37; N, 12.21 (%). MS: 567 [M + H]+.

Ethyl 3-amino-1-(5-(4-cyanophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4h)

Yield: 89 %; mp 270–272 °C; IR (KBr, ν max, cm−1): 3450 and 3340 (asym. and sym. stretching of –NH2), 2205 (C≡N stretching), 1710 (ester C=O stretching), 1670 (C=O stretching), 1655 (C=O stretching), 1210 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): 0.99 (t, 3H, CH3), 1.95, 2.25 (s, 6H, 2 × CH3), 3.99 (q, 2H, OCH2), 6.57 (s, 1H, C1H), 7.80–8.35 (m, 12H, Ar–H), 8.75 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.50 (CH3), 13.07, 20.25 (Ar–CH3), 59.88 (C1), 63.22 (OCH2), 82.45 (C2), 117.23, 124.16, 124.84, 127.11, 127.55, 128.15, 128.53, 130.18, 132.40, 132.81, 133.17, 133.99, 135.28, 146.80, 151.30, 153.37 155.12 (Ar–C), 157.23 (C=O), 158.67 (C=O), 164.22 (COOEt); Anal. Calcd for C32H26N6O5 (574.59 g/mol): C, 66.89; H, 4.56; N, 14.63 (%); Found: C, 67.16; H, 4.82; N, 14.77 (%). MS: 574 [M + H]+.

3-Amino-1-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4i)

Yield: 80 %; mp 235–237 °C; IR (KBr, ν max, cm−1): 3400 and 3250 (asym. and sym. stretching of –NH2), 2205 (C≡N stretching), 1685 (C=O stretching), 1670 (C=O stretching), 1195 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.44 (s, 3H, CH3), δ 6.50 (s, 1H, C1H), 7.61–8.58 (m, 12H, Ar–H), 8.90 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.06, (Ar–CH3), 58.90 (C1), 61.02 (C2), 116.30, 125.75, 126.80, 127.45, 127.98, 128.19, 129.40, 129.60, 130.42, 131.14, 132.16, 134.17, 134.80, 146.40, 147.70, 151.93, 152.24, 154.70, 158.23 (Ar–C), 157.65 (C=O), 158.40 (C=O); Anal. Calcd for C28H18FN7O5 (551.48 g/mol): C, 60.98; H, 3.29; N, 17.78 (%); Found: C, 61.19; H, 2.96; N, 17.93 (%). MS: 551 [M + H]+.

3-Amino-1-(5-(4-cyanophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2- b]phthalazine-2-carbonitrile (4j)

Yield: 81 %; mp 246–248 °C; IR (KBr, ν max, cm−1): 3385 and 3185 (asym. and sym. stretching of –NH2), 2200 (C≡N stretching), 1680 (C=O stretching), 1660 (C=O stretching), 1185 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.31 (s, 3H, CH3), 6.53 (s, 1H, C1H), 7.55–8.60 (m, 12H, Ar–H), 8.80 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.01, (Ar–CH3), 59.67 (C1), 61.49 (C2), 116.18, 117.17, 124.95, 125.64, 126.30, 127.81, 128.55, 128.80, 129.30, 131.74, 132.18, 133.16, 134.11, 134.70, 135.85, 144.14, 148.23, 151.40, 152.56, 156.81 (Ar–C), 157.46 (C=O), 158.38 (C=O); Anal. Calcd for C29H18N8O5 (558.50 g/mol): C, 62.36; H, 3.25; N, 20.06(%); Found: C, 62.47; H, 3.46; N, 19.87 (%). MS: 558 [M + H]+.

3-Amino-1-(5-(4-fluorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4k)

Yield: 76 %; mp 254–256 °C; IR (KBr, ν max, cm−1): 3385 and 3200 (asym. and sym. stretching of –NH2), 2210 (C≡N stretching), 1685 (C=O stretching), 1670 (C=O stretching), 1205 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.17, 2.39 (s, 6H, 2 × CH3), 6.60 (s, 1H, C1H), 7.25–8.60 (m, 11H, Ar–H), 8.70 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.15, 20.50 (Ar–CH3), 60.00 (C1), 61.90 (C2), 116.33, 124.17, 127.21, 127.84, 128.14, 128.70, 129.20, 130.05, 131.30, 133.08, 134.15, 135.50, 137.18, 143.40, 149.80, 150.16, 151.96, 154.30, 157.90 (Ar–C), 158.80 (C=O), 158.95 (C=O); Anal. Calcd for C29H20FN7O5 (565.51 g/mol): C, 61.59; H, 3.56; N, 17.34 (%); Found: C, 61.81; H, 3.29; N, 17.45 (%). MS: 565 [M + H]+.

3-Amino-1-(5-(4-cyanophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carbonitrile (4l)

Yield: 78 %; mp 222–224 °C; IR (KBr, ν max, cm−1): 3390 and 3180 (asym. and sym. stretching of –NH2), 2205 (C≡N stretching), 1690 (C=O stretching), 1675 (C=O stretching), 1190 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 2.15, 2.43 (s, 6H, 2 × CH3), δ 6.54 (s, 1H, C1H), 7.82–8.65 (m, 11H, Ar–H), 8.72 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 13.11, 20.65 (Ar–CH3), 60.12 (C1), 61.60 (C2), 116.61, 117.14, 124.20, 127.27, 127.80, 128.65, 128.84, 129.17, 130.20, 131.68, 132.80, 133.23, 133.92, 134.80, 135.30, 144.18, 147.40, 151.85, 152.04, 154.55 (Ar–C), 156.98 (C=O), 158.53 (C=O); Anal. Calcd for C30H20N8O5 (572.53 g/mol): C, 62.93; H, 3.52; N, 19.57 (%); Found: C, 63.11; H, 3.28; N, 19.86 (%). MS: 572 [M + H]+.

Ethyl 3-amino-1-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4m)

Yield: 74 %; mp 264–266 °C; IR (KBr, ν max, cm−1): 3455 and 3330 (asym. and sym. stretching of –NH2), 1715 (ester C=O stretching), 1675 (C=O stretching), 1660 (C=O stretching), 1210 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.05 (t, 3H, CH3), 2.36 (s, 3H, CH3), 3.96 (q, 2H, OCH2), 6.48 (s, 1H, C1H), 7.47–8.55 (m, 12H, Ar–H), 8.80 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.22 (CH3), 13.03, (Ar–CH3), 59.94 (C1), 63.52 (OCH2), 82.15 (C2), 125.07, 126.84, 127.11, 127.43, 127.84, 128.53, 129.15, 129.44, 129.90, 131.20, 134.18, 135.82, 145.75, 151.26, 152.43, 154.82, 157.33, 158.40 (Ar–C), 156.50 (C=O), 158.72 (C=O), 164.19 (COOEt); Anal. Calcd for C30H24FN6O7 (598.54 g/mol): C, 60.20; H, 3.87; N, 14.04 (%); Found: C, 59.97; H, 4.06; N, 13.83 (%). MS: 598 [M + H]+.

Ethyl 3-amino-1-(5-(4-cyanophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4n)

Yield: 72 %; mp 235–237 °C; IR (KBr, ν max, cm−1): 3420 and 3300 (asym. and sym. stretching of –NH2), 2215 (C≡N stretching), 1700 (ester C=O stretching), 1680 (C=O stretching), 1665 (C=O stretching), 1195 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 0.99 (t, 3H, CH3), 2.51 (s, 3H, CH3), 3.99 (q, 2H, OCH2), 6.57 (s, 1H, C1H), 7.68–8.69 (m, 12H, Ar–H), 8.80 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.62 (CH3), 13.14, (Ar–CH3), 59.74 (C1), 63.00 (OCH2), 82.16 (C2), 117.34, 124.24, 125.72, 127.11, 127.64, 128.75, 129.20, 129.78, 131.25, 131.82, 133.55, 134.02, 135.23, 136.42, 144.91, 145.61, 151.20, 151.35, 154.72 (Ar–C), 156.17 (C=O), 158.03 (C=O), 164.35 (COOEt); Anal. Calcd for C31H23N7O7 (605.56 g/mol): C, 61.49; H, 3.83; N, 16.19 (%); Found: C, 61.68; H, 3.72; N, 15.91 (%). MS: 605 [M + H]+.

Ethyl 3-amino-1-(5-(4-fluorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4o)

Yield: 70 %; mp 249–251 °C; IR (KBr, ν max, cm−1): 3470 and 3345 (asym. and sym. stretching of –NH2), 1705 (ester C=O stretching), 1685 (C=O stretching), 1650 (C=O stretching), 1200 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): δ 1.03 (t, 3H, CH3), 1.97, 2.39 (s, 6H, 2 × CH3), 3.93 (q, 2H, OCH2), 6.53 (s, 1H, C1H), 7.30–8.59 (m, 11H, Ar–H), 8.60 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.50 (CH3), 13.08, 20.20 (Ar–CH3), 59.98 (C1), 63.55 (OCH2), 82.55 (C2), 123.20, 127.32, 127.68, 128.12, 128.67, 129.03, 129.60, 132.27, 133.34, 134.85, 135.17, 136.79, 143.16, 149.70, 151.11, 152.30, 156.22, 158.42 (Ar–C), 156.23 (C=O), 158.39 (C=O), 164.60 (COOEt); Anal. Calcd for C31H25FN6O7 (612.56 g/mol): C, 60.78; H, 4.11; N, 13.72 (%); Found: C, 60.99; H, 3.89; N, 14.03 (%). MS: 612 [M + H]+.

Ethyl 3-amino-1-(5-(4-cyanophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-7-nitro-5,10-dioxo-5,10-dihydro-1H-pyrazolo[1,2-b]phthalazine-2-carboxylate (4p)

Yield: 72 %; mp 238–240 °C; IR (KBr, ν max, cm−1): 3395 and 3285 (asym. and sym. stretching of –NH2), 2210 (C≡N stretching), 1705 (ester C=O stretching), 1685 (C=O stretching), 1670 (C=O stretching), 1205 (C–O–C ether stretching). 1H NMR (400 MHz, DMSO-d 6 ): 0.96 (t, 3H, CH3), 1.94, 2.33 (s, 6H, 2 × CH3), 3.99 (q, 2H, OCH2), 6.50 (s, 1H, C1H), 7.78–8.61 (m, 11H, Ar–H), 8.70 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d 6 ) δ: 14.45 (CH3), 13.07, 20.75 (Ar–CH3), 60.17 (C1), 63.34 (OCH2), 82.60 (C2), 117.15, 124.33, 127.17, 127.70, 128.35, 128.84, 129.20, 129.65, 130.20, 131.24, 133.12, 133.60, 134.17, 135.40, 145.65, 146.17, 151.20, 152.63, 155.18 (Ar–C), 155.93 (C=O), 158.18 (C=O), 164.38 (COOEt); Anal. Calcd for C32H25N7O7 (619.58 g/mol): C, 62.03; H, 4.07; N, 15.82 (%); Found: C, 62.19; H, 4.23; N, 15.65 (%). MS: 619 [M + H]+.

Biological assay

In vitro evaluation of antimicrobial activity

The minimum inhibitory concentrations (MICs) of synthesized compounds were carried out by a broth micro dilution method [27]. DMSO was used as the diluents to obtain the desired concentration of compounds to test upon standard bacterial strains. Serial dilutions were prepared in primary and secondary screening. The control tube containing no antibiotic was immediately sub cultured (before inoculation) by spreading a loopful evenly over a quarter plate of medium suitable for the growth of the test organism and put for incubation at 37 °C overnight. The tubes were then incubated overnight. The MIC of the control organism was checked against the accuracy of the compound concentrations. The MIC was defined as the lowest concentration of the antibiotic or test sample allowing no visible growth. All the tubes not showing visible growth (in the same manner as the control tube described above) were sub cultured and incubated overnight at 37 °C. The amount of growth of the control tube before incubation (which represents the original inoculum) was compared. Subcultures might show: a similar number of coloniesn indicating bacteriostatic condition; a reduced number of colonies, indicating a partial or slow bactericidal activity and no growth if the whole inoculum has been killed. The test must include a second set of the same dilutions inoculated with an organism of known sensitivity. Each compound was diluted, obtaining a 2000 µg/mL concentration as a stock solution. In primary screening, 500, 250 and 200 µg/mL concentrations of the synthesized compounds were taken. The active synthesized compounds found in this primary screening were further tested in a second set of dilutions against all microorganisms. The compounds found active in primary screening were similarly diluted to obtain 100, 62.5, 50 and 25 µg/mL concentrations. The highest dilution showing at least 99 % inhibition is taken as the MIC.

In vitro evaluation of antituberculosis activity

A primary screen was conducted at 250 µg/mL against M. tuberculosis H37Rv by a Lowenstein-Jensen (LJ) MIC method [28] where primary 250 µg/mL dilutions of each test compound were added to liquid Lowenstein-Jensen medium and then the media were sterilized by an inspissations method. A culture of M. tuberculosis H37Rv growing on Lowenstein-Jensen medium was harvested in 0.85 % saline in bijou bottles. DMSO was used as a vehicle to obtain the desired concentration. The tubes were then incubated at 37 °C for 24 h followed by streaking of M. tuberculosis H37Rv (5 × 104 bacilli per tube). The tubes were then incubated at 37 °C. Growth of bacilli was seen after 12, 22, and, finally, 28 days of incubation. Tubes having the compounds were compared with control tubes where medium alone was incubated with M. tuberculosis H37Rv. The concentration at which complete inhibition of colonies occurred was taken as the active concentration of the test compound. The standard strain M. tuberculosis H37Rv was tested with known drugs Isoniazid and Rifampicin. The screening results are summarized as % inhibition relative to standard drugs Isoniazid and Rifampicin.

Brine shrimp lethality bioassay for evaluation of cytotoxicity

A brine shrimp lethality bioassay technique was applied for determining the general toxicity of the compounds. The in vitro lethality test has been carried out using brine shrimp eggs (Artemia cysts). Brine shrimp eggs were hatched in a shallow rectangular plastic dish (22 × 32 cm) filled with artificial seawater prepared with a commercial salt mixture and double-distilled water. An unequal partition was made in the plastic dish with the help of a perforated device. Approximately 50 mg of eggs was sprinkled into the large compartment, which was darkened while the minor compartment was opened to ordinary light. After 2 days, nauplii were collected in a pipette from the lighter side. A stock solution of the test complex was prepared in DMSO. From this stock solution, solutions were transferred to the vials to make final concentrations of 5, 10, 20, 30, 40, 50 mg/mL (dilutions were used in triplicate for each test sample, and the LC50 is the mean of three values) and three vials were kept as controls having DMSO only. After 2 days, when the nauplii were ready, 1 mL of seawater and 10 nauplii were added to each vial and the volume was adjusted with seawater to 2.5 mL per vial [29]. After 24 h, each vial was observed using a magnifying glass and the number of survivors in each vial was counted and noted. Data were analysed by a simple logic method to determine the LC50 values, in which log× of the dose concentration of samples was plotted against the percent mortality of nauplii [30].

In vitro evaluation of antioxidant activity

The ferric reducing antioxidant power (FRAP) assay was employed to measure the total antioxidant capacity of the compounds, converting ferric tripyridyl triazine [Fe(III)-TPTZ] complex into a blue ferrous tripyridyl triazine [Fe(II)-TPTZ] complex at a low pH, measurable at 593 nm [31].

Reagents: (1) Buffer solution: 0.187 gm sodium acetate and 1.6 mL acetic acid dissolved in double-distilled water to make 100 mL. (2) TPTZ: 0.155 gm TPTZ was dissolved in 100 mL of 40-mM HCl. (3) FeCl3 solution: 0.324 gm FeCl3 was dissolved in 100 mL of distilled water. (4) Standard ascorbic acid: 0.176 gm of standard ascorbic acid was dissolved in 100 mL of distilled water.

Fe(II)-TPTZ(2,4,6-tripyridyl-s-triazine) reagent was prepared by mixing a 10.0 mL of TPTZ solution, 10 mL of FeCl36H2O solution and 100 mL of acetate buffer at pH 3.6. A mixture of 200.0 mL of sample solution and 3 mL of Fe(II)-TPTZ reagent was incubated at 37 °C for 15 min. The absorbance of colour complex Fe(II)-TPTZ was measured at 593 nm using ascorbic acid as the standard. The results were expressed as ascorbic equivalent (mmol/100 gm compound). Ascorbic acid taken = 1.99 × 10−4 mm. Sample taken = 0.04 mg. The FRAP can be calculated using the following equation:

$$ {\text{FRAP}}\,{\text{value }}\left( {{\text{mm}}\,{\text{A}}.{\text{A}}./100\,{\text{gm}}\,{\text{sample}}} \right) = \frac{{\Delta {\text{OD}}593\,{\text{nm}}\,{\text{of}}\,{\text{test}}\,{\text{sample}} \, \times \,{\text{standard}} \,\left( {\text{mm}} \right) \, \times \,105}}{{\Delta {\text{OD}}593\,{\text{nm }}\,{\text{of}}\,{\text{standard}}\, \times \,{\text{sample }}\,\left( {\text{mg}} \right)}} $$

Results and discussion

Characterization of compounds 4a–p

In the IR spectra, some significant stretching bands due to NH2, C=O, C≡N and C–O–C are observed about 3470–3180, 1715–1635, 2205–2190 and 1210–1185 cm−1 , respectively. The 1H NMR spectrum of compounds 4a–p indicated the presence of one singlet in the range δ 6.44–6.60 ppm of a –CH proton of a C1H-pyr ring, and the disappearance of a singlet from δ 9.57–9.63 ppm of –CHO, clearly confirming the cyclization of the Knoevenagel intermediate. Moreover, multiplets in the range δ 7.46–8.29 ppm appeared for aromatic protons and all –NH2 protons appear in the range 8.46–8.80 ppm. In the 13C NMR spectral data of the title compounds 4a–p, the most characteristic signal around δ 58.90–60.18 ppm (C1-pyr) indicated the formation of a pyrazolo[1,2-b]phthalazine ring. The signal at around δ 61.02–62.50 ppm (C2-pyr) is assigned to carbon attached to carbonitrile, and δ 81.90–82.55 ppm (C2-pyr) is assigned to carbon attached to an ester group. Also, δ 164.17–164.60 ppm is assigned to a carbonyl carbon of ester (O–C=O), while signals around δ 116.05–160.20 ppm are attributed to all the aromatic carbons of compounds 4a–p. The obtained elemental analysis values are in consonance with theoretical data. Mass spectra of title compounds showed expected molecular ion peak M+ corresponding with proposed molecular mass.

Antimicrobial activity

Upon examination of bioactivity data of compounds 4a–p (Table 1), it was noticed that almost all the compounds were equipotent or more potent compared to the standard drug ampicillin, and a few compounds were equipotent or more potent to norfloxacin. Against Gram positive bacteria, B. subtilis compounds 4a, 4d, 4i, 4m and 4o (MIC = 100 µg/mL) displayed efficacy, while compounds 4e, 4g and 4l (MIC = 200 µg/mL) were far better than ampicillin (MIC = 250 µg/mL). For inhibiting C. tetani, compounds 4f, 4g, 4h, 4l and 4p displayed efficacy (MIC = 100 µg/mL), while compounds 4a, 4c, 4e, 4k, 4m and 4o displayed (MIC = 200 µg/mL) activity much higher than that of ampicillin (MIC = 250 µg/mL). Against S. pneumonia, compounds 4a, 4e, 4g, 4i and 4o (MIC = 100 µg/mL) were found to be equipotent to ampicillin while none of the compounds were found to be more potent than that of ampicillin. Also, compounds 4f, 4g, 4h, 4l and 4p (MIC = 100 µg/mL) showed activity comparable to ciprofloxacine (MIC = 100 μg/mL) towards C. tetani. Compounds 4a, 4d, 4i and 4o (MIC = 100 µg/mL) were found to have activity comparable to norfloxacin (MIC = 100 µg/mL) towards B. subtilis.

Table 1 In vitro antimicrobial activity of pyrazolo[1,2-b]phthalazine 4a–p
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Against Gram negative bacteria E. coli, compound 4o (MIC = 50 µg/mL), 4h and 4k (MIC = 62.5 µg/mL) were found to be more potent whereas 4c, 4g and 4m (MIC = 100 μg/mL) showed comparable activity to ampicillin (MIC = 100 μg/mL). Moreover, compound 4g (MIC = 50 µg/mL) is found to possess pronounced activity against S. typhi compared to chlormphenicol (MIC = 50 µg/mL). Compounds 4e and 4h (MIC = 100 μg/mL) showed activity comparable to ampicillin (MIC = 100 μg/mL) towards S. typhi. Against V. cholera, compound 4o (MIC = 50 µg/mL) was found to bear excellent activity upon comparison with ampicillin (MIC = 100 µg/mL), and was equipotent with chlormphenicol (MIC = 50 µg/mL).

Against fungal pathogen C. albicans, compounds 4g and 4o were found to possess excellent activity (MIC = 100 µg/mL), while compounds 4a, 4h and 4k (MIC = 250 µg/mL) were found to be more potent than that of the standard drug griseofulvin (MIC = 500 µg/mL). None of the compounds were found to be active against fungal pathogen A. fumigates.

Antituberculosis activity

The encouraging results from the antimicrobial studies prompted us to proceed to the preliminary screening of the title compounds for their in vitro antituberculosis activity against M. tuberculosis H37Rv bacteria (Table 2). Of the compounds screened for antituberculosis activity, compound 4g (MIC = 25 mg/mL) was found to possess the highest potency against M. tuberculosis with 98 % inhibition as compared to rifampicin (MIC = 40 mg/mL). Compounds 4k (MIC = 62.5 mg/mL) and 4o (MIC = 50 mg/mL) exhibited inhibition of 95 % and 96 %, respectively. Also, compounds 4c and 4h (MIC = 100 mg/mL) displayed moderate inhibition of 92 and 91 %, respectively (Table 3).

Table 2 In vitro antituberculosis activity (% inhibition) of pyrazolo[1,2-b]phthalazine 4a–p against M. tuberculosis H37Rv (at concentration 250 µg/mL)
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Table 3 In vitro antituberculosis activity of title compounds exhibiting higher % inhibition against M. tuberculosis H37Rv (MICs, mg/mL)
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It is interesting to note that substitutions at R1, R2 and R3 positions make a wide impact on antituberculosis activity rather than substitutions at the R4 position. Out of five active compounds, four compounds having R1 = CH3 and R2 = F effectively inhibited the growth of M. tuberculosis (i.e., 4c, 4g, 4k and 4o except 4h). Also, at the R3 position, the ester group has more impact than the cyanide group. So, there is a combination effect of CH3, F and COOEt groups to improve the tuberculosis activity. Compound 4g (R1 = CH3, R2 = F, R3 = COOEt) emerged out as the most potent member of the series and opens up a new door to optimize this series for a new class of antitubercular agents. From the antitubercular activity results, it is worth mentioning that the presence of lipophilic groups at the R3 position improve the lipophilicity of the whole molecule. As a result, the molecule may be expected to more easily penetrate the bacterial cell line.

Cytotoxicity

The LC50 values obtained for the five compounds exhibiting the highest % inhibition are shown in Table 4. As can be seen, for the five compounds, there was no significant toxicity observed for compounds 4g, 4h and 4o after a 24-h incubation. Among the compounds tested, compounds 4c and 4k showed greater toxicity.

Table 4 Cytotoxicity of title compounds exhibiting higher MICs against M. tuberculosis H37Rv (LC50, mg/mL)
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Antioxidant activity

Examination of the data (Table 5) revealed that compounds 4g and 4h showed relatively high antioxidant power while compounds 4c, 4d, 4o and 4p were found to have better ferric reducing power. Compounds 4k and 4l displayed promising antioxidant potency. From the ferric reducing power results, it can be stated that compounds carrying an electron donating CH3 group at the R1 position and an ester group at the R3 position exhibited excellent ferric reducing power, but when the electron-withdrawing group NO2 entered the R4 position, a decrease in the antioxidant activity occurred. In addition, it should be noted that the compounds with R1 = CH3 and R2 = F give better results than cyanide substitution (R2 = CN). It may be due to the combination effect of R1 = CH3, R2 = F or CN and the ester group at the R3 position.

Table 5 In vitro antioxidant activity of compounds 4a–p derivatives
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In conclusion, we have demonstrated the use of NaOH as an efficient green reaction medium for the synthesis of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives (i.e., 4a–p) bearing 5-aryloxypyrazole for probing antimicrobial, antituberculosis and antioxidant activity. Compounds 4e, 4g, 4h, 4k and 4o exhibited excellent antimicrobial inhibition, while compounds 4c, 4d, 4g and 4 h showed the highest ferric reducing power. Compounds 4g and 4o emerged as the promising antimicrobial members with better antitubercular activity and lower toxicity. Consequently, such type of compounds represent a fertile matrix for further development of more biologically potent agents that deserve further investigation and derivatization in order to discover the scope and limitation of their biological activities.

Conclusion

A novel series of pyrazolo[1,2-b]phthalazine derivatives (4a–p) have been successfully synthesized and characterized. The antimicrobial activity results showed that compounds 4e, 4g, 4h, 4k and 4o exhibited excellent antimicrobial activity compared with first line drugs. In vitro antituberculosis activity was evaluated against M. tuberculosis H37Rv and compounds 4g and 4o emerged as the promising antimicrobial members with better antituberculosis activity. Compounds 4c, 4d, 4g and 4h showed the highest antioxidant potency.