Potential Antileishmanial Activity of 4-N-Acylhydrazone Pyrazolo[3,4-d]pyridazin-7-ones: Synthesis, in vitro Biological Evaluations and Computational Studies

Abstract

In this work a series of 28 novel pyrazolo[3,4-d]pyridazin-7-ones were synthesized and tested against Leishmania amazonensis (strain WHOM/BR/75/JOSEFA) in promastigote and axenic amastigote forms. Five compounds were active against both cellular forms with IC₅₀ (inhibitory concentration growth of 50%) values of 20.2, 11.7, 16.2, 29.5 and 40.3 µM for promastigote and 17.4, 25.2, 3.84, 21.8 and 22.7 µM for axenic amastigote. All compounds were studied by the Lipinski rule, cytotoxicity both in silico and in vitro to fibroblast line (L929) and macrophages (J774A1), and the most active compound showed a selectivity index of 59.9.

Keywords:
pyrazolo[3,4-d]pyridazinone; N-acylhydrazone; Leishmania amazonensis

Introduction

Leishmaniasis is a major infection, a neglected disease caused by more than 20 species that belong to family Trypanosomatidae and genus Leishmania. This disease is distributed worldwide and can be found in subtropical and temperate regions in more than 88 countries, mainly affecting poor regions of developing countries. It is estimated that about 12-15 million people are already infected with leishmaniasis; 2 million new cases occur annually and around 350 million people live in risk areas. The parasites may be transferred to humans by bites from 30 species of sand fly and can cause three main clinical types of leishmaniasis: cutaneous, mucocutaneous and visceral.^1-7 The drugs used nowadays in the treatment of leishmaniasis have shown highly toxic effects and parasite resistance, demonstrating the necessity of development of novel antileishmanial compounds.^8-10

It is known that pyrazoles,^11-14 fused pyrazoles^15-17 and N-acylhydrazones^13,18 show antileishmanial activity. Recently, studies by our research group have shown the synthesis,¹⁹ and antileishmanial activity²⁰ of a series of 1,4,6-trisubstituted pyrazolo[3,4-d]pyridazin-7-one-N-acylhydrazone-(bi)thiophene hybrids. In continuation of our studies with pyrazolopyridazinone hybrids, a new series was synthesized using a phenyl substituent in the N-acylhydrazone moiety, which is a bioisostere of thiophene (Figure 1).

Figure 1
Pyrazolopyridazinones synthesized in previous work²⁰ and in this work.

Results and Discussion

The novel pyrazolo[3,4-d]pyridazin-7-ones 5-8 were obtained from the β-enamino diketone and hydrazines according to highly regioselective methodology reported by our research group,¹⁹ as shown in Scheme 1, and characterized by¹H and ¹³C nuclear magnetic resonance (NMR), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) data, high resolution mass spectrometry (HRMS), X-ray diffraction (XRD, CCDC 1576799) and melting point (mp) (see Supplementary Information for full details).

Scheme 1
General methodology for synthesis of new derivatives. (i) R¹ NHNH₂ (0.9 equiv.), EtOH; (ii) r² NHNH₂ (1.1 equiv.), AcOH (4.0 equiv.), EtOH; (iii) PhNHNH₂ (2.2 equiv.), AcOH (4.0 equiv.), EtOH; (iv) NH₂ NH₂ .H₂ O (20 equiv.), EtOH:MeCN (1:1); (v) aldehyde(R³) (1.0 equiv.), HCl_(cat.), DMSO.

All new compounds synthesized (3b, 4b, 5e-j, 6e-l, 7e-j and 8e-j) were evaluated against promastigote and axenic amastigote forms of L. amazonensis and the inhibitory concentration growth of 50% (IC₅₀) was determined for each compound. Results of IC₅₀ > 100 µM were considered as inactive. As observed for the compounds 3a,c,d and 4a,c,d,²⁰ the ester (3b) and hydrazide (4b) derivatives were also inactive against L. amazonensis, with values of IC₅₀ higher than 100 µM for both, although the formation of the phenyl-substituted pyrazolo[3,4-d]pyridazin-7-one-N-acylhydrazone 5-8 led to active compounds 6h, 6l, 7e, 8f, and 8i with IC₅₀ values of 20.2, 11.7, 16.2, 29.5 and 40.3 µM to promastigote forms and 17.4, 25.2, 3.84, 21.8 and 22.7 µM to axenic amastigote forms, respectively ( Table 1).

Table 1
In vitro antiproliferative activity in L. amazonensis and cytotoxicity in mammalian cells of new compounds synthesized

Compound	IC50(pro)a / µM	IC50(ama)b / µM	CC50(f)c / µM	CC50(m)d / µM	SIp-fe	SIp-mf	SIa-fg	SIa-mh
3b	> 100	> 100	-	> 100	-	-	-	-
4b	> 100	> 100	-	> 100	-	-	-	-
5e	> 100	> 100	-	> 100	-	-	-	-
5f	> 100	> 100	-	> 100	-	-	-	-
5g	> 100	> 100	-	> 100	-	-	-	-
5h	> 100	> 100	-	> 100	-	-	-	-
5i	> 100	> 100	-	> 100	-	-	-	-
5j	> 100	> 100	-	> 100	-	-	-	-
6e	> 100	> 100	-	> 100	-	-	-	-
6f	> 100	> 100	-	> 100	-	-	-	-
6g	> 100	> 100	-	> 100	-	-	-	-
6h	20.2 ± 0.2	17.4 ± 2.1	228.5 ± 9.1	234.0 ± 5.7	11.3	11.6	13.1	13.4
6i	> 100	> 100	-	> 100	-	-	-	-
6j	> 100	> 100	-	> 100	-	-	-	-
6k	> 100	> 100	-	> 100	-	-	-	-
6l	11.7 ± 1.1	25.2 ± 2.7	301.7 ± 7.4	298.5 ± 8.8	25.8	25.5	12.0	11.8
7e	16.2 ± 2.6	3.84 ± 2.1	145.54 ± 14.45	230.15 ± 10.8	9.0	14.2	37.9	59.9
7f	> 100	> 100	-	> 100	-	-	-	-
7g	> 100	> 100	-	> 100	-	-	-	-
7h	> 100	> 100	-	> 100	-	-	-	-
7i	> 100	> 100	-	> 100	-	-	-	-
7j	> 100	> 100	-	> 100	-	-	-	-
8e	> 100	> 100	-	> 100	-	-	-	-
8f	29.5 ± 3.0	21.8 ± 1.4	245.1 ± 7.8	370.8 ± 9.0	8.3	12.6	11.2	17.0
8g	> 100	> 100	-	> 100	-	-	-	-
8h	> 100	> 100	-	> 100	-	-	-	-
8i	40.3 ± 4.4	22.7 ± 0.9	273.2 ± 9.6	318.3 ± 3.6	6.8	7.9	12.0	14.0
8j	> 100	> 100	-	> 100	-	-	-	-

• ^a
  IC₅₀ (inhibitory concentration growth of 50%) values of promastigote form;
• ^b
  IC₅₀ values of axenic amastigote form;
• ^c
  cytotoxic concentration corresponding to 50% inhibition of fibroblast growth;
• ^d
  cytotoxic concentration corresponding to 50% inhibition of macrophage growth;
• ^e
  selectivity index (CC_50(f)/IC_50(pro));
• ^f
  selectivity index (CC_50(m)/IC_50(pro));
• ^g
  selectivity index (CC_50(f)/IC_50(ama));
• ^h
  selectivity index (CC_50(m)/IC_50(ama)). -: not determined

It is notorious that the N-acylhydrazone moiety in 4-position has a great influence on the activity;²⁰ however, the nature of the substituent in 1 and 6-positions of pyrazolo[3,4-d]pyridazinone also has an influence on the antileishmanial results for those substances (Figure 2), where the presence of a phenyl group in all positions (1, 4 and 6) resulted in substance 7e, which showed the best results of IC₅₀: 16.2 and 3.84 µM for promastigotes and amastigotes, respectively. The phenyl substituent can be found in 1-position in two more active compounds8f and 8i, where there is a 4-chlorophenyl substituent in 6-position; nevertheless, this combination led to less active compounds. Compounds 6h and 6l, which have not phenyl substituent in 1 or 6-position, were active with IC₅₀ of 20.2 and 11.7, and 17.4 and 25.2 µM for promastigotes and amastigotes, respectively. Although these results demonstrated that N-acylhydrazone moiety in 4-position and the nature of the substituent in 1 and 6-positions of pyrazolo[3,4-d]pyridazinone play an important role in antileishmanial activity, additional studies are necessary to investigate the action mechanism of these class of compounds in antileishmanial activity.

Figure 2
Influence of substituent in different positions.

The cytotoxicity of all compounds was investigated in mammalian fibroblast L929 cells and macrophages J774A1 through the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method.²¹ For the active compound the range of cytotoxicity was up to 1000 µM. The most active compound 7e was also the most selective against both forms of L. amazonensis, with selectivity index (SI) values of 37.9 and 59.9 to promastigote and amastigote forms, respectively. All the active compounds showed SI values higher than 10 for axenic amastigote forms, and all inactive compounds showed values of cytotoxic concentration corresponding to 50% inhibition (CC₅₀) > 100 µM.

In addition, all compounds were evaluated considering the Lipinski rules,²² aiming to determine the theoretical oral bioavailability using Molinspiration online software.²³ Some descriptors, such as nRot (number of rotatable bonds) and PSA (polar surface area) were included in the Lipinski rules, in which is desirable that there are 10 or fewer rotatable bonds and a polar surface area equal to or less than 140 Ų (Table 2). Five compounds, 5j, 6j, 7j, 8h and 8j showed one or two violations of Lipinski rules and may present problems in oral bioavailability; however, these did not show antileishmanial activity. All the active derivatives, 6h, 6l, 7e, 8f and 8i showed no violations of conditions, being able to participate in the drug development process ( Table 2).

Table 2
Physicochemical descriptors calculated for the set of pyrazolo[3,4-d]pyridazinone derivatives

Compound	Molecular weight / Dalton	nON	nOHNH	miLogP	nRot	PSA / Å2
5e	358.36	8	2	2.40	4	105.04
5f	374.36	9	3	1.93	4	125.27
5g	388.39	9	2	2.46	5	114.28
5h	401.43	9	2	2.51	6	108.28
5i	376.35	8	2	2.57	4	105.04
5j	406.36	11	2	2.36	5	150.87
6e	338.37	8	2	2.32	4	105.04
6f	354.37	9	3	1.84	4	125.27
6g	368.40	9	2	2.37	5	114.28
6h	381.44	9	2	2.42	5	140.28
6i	356.36	8	2	2.48	4	102.04
6j	383.37	11	2	2.27	5	150.87
6k	346.42	8	2	1.07	4	98.53
6l	428.53	8	2	2.77	5	98.53
7e	434.46	8	1	3.75	5	94.19
7f	450.46	9	2	3.27	5	114.41
7g	464.49	9	1	3.80	6	103.42
7h	477.53	9	1	3.45	6	97.82
7i	452.45	8	1	3.91	5	94.19
7j	479.46	11	1	3.71	6	140.01
8e	468.90	8	1	4.42	5	94.19
8f	484.90	9	2	3.95	5	114.41
8g	498.93	9	1	4.48	6	103.42
8h	511.97	9	1	4.53	6	97.42
8i	486.89	8	1	4.59	5	94.19
8j	513.90	11	2	4.38	6	140.31

• nRot: number of rotatable bonds; PSA: polar surface area.

The calculated electrostatic potential map (MEP) showed no important differences between the compounds studied. Considering compound 8f, regions of negative electrostatic potential near the nitrogen atom of the pyrazole ring, the oxygen atoms of the carbonyl moieties located on the lateral chain and the pyridazinone group were observed (Figure 3), and the positive electrostatic potentials were found in the lateral chain in all compounds studied.

Figure 3
3D visualization of the MEP from compound 8f, beyond the Van der Waals surface using Spartan for Window 08.²⁴ Color scheme: blue positive to red negative electrostatic potentials values (-40.000 to 67.000 kcal mol^-1).

For in silico toxicological studies, Osiris Property Explorer software²⁵ was used for calculating the fragment-based toxicity risks, and compared with hydrogen peroxide and meglumine antimoniate ( Table 3).

Table 3
Theoretical toxicity evaluation values of the active compounds pyrazolo[3,4-d]pyridazin-7-one derivatives compared to H₂O₂ and meglumine antimoniate

Compound	Toxicity risk
	Mutagenic	Tumorigenic	Irritant	Reproductive
3b	(+)	(+)	(+)	(+)
4b	(+++)	(+++)	(+)	(+)
5e	(+)	(+)	(+)	(+)
5f	(+)	(+)	(+)	(+)
5g	(+)	(+)	(+)	(+)
5h	(+)	(+)	(+)	(+)
5i	(+)	(+)	(+)	(+)
5j	(+)	(+)	(+++)	(+)
6e	(+)	(+)	(+)	(+)
6f	(+)	(+)	(+)	(+)
6g	(+)	(+)	(+)	(+)
6h	(+)	(+++)	(+)	(+)
6i	(+)	(+)	(+)	(+)
6j	(+)	(+)	(+)	(+)
6k	(+)	(+)	(+)	(+)
6l	(+)	(+)	(+)	(+)
7e	(+)	(+)	(+)	(+)
7f	(+)	(+)	(+)	(+)
7g	(+)	(+)	(+)	(+)
7h	(+)	(+)	(+)	(+)
7i	(+)	(+)	(+)	(+)
7j	(+)	(+)	(+++)	(+)
8e	(+)	(+)	(+)	(+)
8f	(+)	(+)	(+)	(+)
8g	(+)	(+)	(+)	(+)
8h	(+)	(+++)	(+)	(+)
8i	(+)	(+)	(+)	(+)
8j	(+)	(+)	(+)	(+)
Meglumine antimoniate	(++)	(+)	(+)	(+)
H2O2	(+++)	(+++)	(+++)	(+)

• The scale of toxicity risk ranges from low (+) to medium (++) and high (+++), calculated using Osiris software.²⁵

The theoretical toxicity evaluation of the tumorigenic profiles of pyrazolo[3,4-d]pyridazin-7-one derivatives 4b, 6h and 8h showed the same tumorigenic risk as H₂ O₂. The compound 4b has an hydrazide moiety without substitution, and 6h and 8h have a dimethylamino attached to the para-position of the benzene ring, which may increase the risk of these molecules to cause a tumorigenic effect. The compound 4b showed a high risk of mutagenic effect. Compounds 5j and 7j showed high risk of causing damage to the reproductive system. Other derivatives showed low risk of causing all toxic effects studied. This information is useful since the active compounds 6l, 7e, 8f, and 8i are able to be submitted to drug design development. All compounds showed lower risk of toxic effects when evaluated in comparison with the antileishmanial drug meglumine antimoniate, which showed a high risk of tumorigenic and mutagenic effects and effects on the reproductive system.

Conclusions

Our results suggest that our synthetic methodology to synthesize fused-ring pyrazolopyridazinone has a wide application, and those rings have potential antileishmanial activity, depending on the substituents that is present in 1, 4 and 6-positions, where the phenyl substituent led to higher-activity compounds in comparison to tert-butyl and 4-chlorophenyl substituents. All compounds synthesized showed low cytotoxicity with good selectivity index for promastigote and amastigote forms of L. amazonensis.

Experimental

General information

Reagents were used as obtained from commercial suppliers without further purification. Solvents were dried and purified according to procedure.²⁶¹H NMR, ¹³C NMR, HSQC and HMBC experiments were run on VARIAN Mercury Plus apparatus operating at¹H 300.06 MHz and ¹³C 75.46 MHz, and Bruker Avance III HD apparatus operating at¹H 500.13 MHz and ¹³C 125.77 MHz. Chemical shifts are reported in ppm using tetramethylsilane (TMS) as the internal standard for CDCl₃ and dimethyl sulfoxide (DMSO-d₆). Melting points were determined on Micro-Química apparatus model MQAPF-301. The column chromatography used was silica gel 60, with 230-400 mesh (Merck). Electrospray ionization (ESI)(+)-MS and tandem ESI(+)-MS/MS were acquired using a hybrid high-resolution and high accuracy microTof (Q-TOF, quadrupole time-of-flight) mass spectrometer (Bruker®). For ESI(+)-MS, the energy for the collision induced dissociations (CDI) was optimized for each component. For data acquisition and processing, the Q-TOF-control data analysis software (Bruker Scientific) was used. The error was calculated for all compounds in ppm. Single crystal X-ray diffraction studies: X-ray intensity data measurements of compounds 8h (CCDC 1576799) were collected with a Bruker APEX II CCD area-detector diffractometer and graphite-monochromated MoKα radiation. The structure was solved by direct methods using SHELXS.²⁷ Subsequent Fourier-difference map analyses yielded the positions of the non-hydrogen atoms. Refinements were carried out by the SHELXS package.²⁷ All refinements were made by full matrix least squares on F² with anisotropic displacement parameters for all non-hydrogen atoms. Hydrogen atoms were included in the refinement in calculated positions, but the atoms (of hydrogens) that are performing special bond were located in the Fourier map. The ORTEP diagram were drawn with 50% probability displacement ellipsoids using ORTEP-3 for Windows.²⁸

Parasites and cell culture

Biological activity was determined in promastigotes and axenic amastigotes of L. amazonensis (strain WHOM/BR/75/JOSEFA). Promastigotes were cultured in Warren (brain heart infusion, hemin, and folic acid; pH 7.0) supplemented with 10% fetal calf serum (FCS) at 25 ºC. Axenic amastigotes were cultured in Schneider medium (pH 4.6) supplemented with 20% fetal bovine serum (FBS) at 32 ºC.

Cytotoxicity in mammalian cells was determined in a fibroblast line (L929) and macrophages (J774A1). Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM, pH 7.2) supplemented with 10% FBS at 37 ºC in a 5% CO₂ atmosphere. Macrophages were cultured in RPMI-1640 (pH 7.2) medium supplemented with 10% FBS at 37 ºC in a 5% CO₂ atmosphere.

Dilution of compounds

Stock solutions of the compounds were prepared in DMSO and then diluted in the respective medium. The groups (controls and treated) were tested with DMSO concentrations below 1%, with concentrations that do not affect viability of the protozoa, and mammalian cells.

Anti-proliferative assay

Promastigotes (1 × 10⁶ cells mL^-1) were cultured in 24-well plates in the presence and absence of different concentrations of compounds diluted in Warren medium supplemented with 10% FBS and incubated for 72 h. Axenic amastigotes (1 × 10⁶ cells mL^-1) were cultured in a 12-well plate in the presence and absence of different concentrations of compounds diluted in Schneider medium supplemented with 20% FBS and incubated for 72 h.

After incubation, the cell count was performed in a Neubauer chamber to determine the 50% inhibitory concentration of protozoa (IC₅₀).

Cytotoxicity assay in mammalian cells

A fibroblast (2.5 × 10⁵ cells mL^-1) suspension was prepared in DMEM medium supplemented with 10% FBS and added to 96-well plates. Then, the plates were incubated at 37 ºC in a CO₂ atmosphere for 24 h to obtain confluent cell growth. After incubation, cells were treated or not with different concentrations of compounds diluted in DMEM for 72 h. A macrophage (5 × 10⁵ cells mL^-1) suspension was prepared in RPMI-1640 medium supplemented with 10% FBS and added to 96-well plates. Then, the plates were incubated at 37 ºC in a CO₂ atmosphere for 24 h to obtain confluent cell growth. After incubation, cells were treated or not with different concentrations of compounds diluted in RPMI-1640 for 48 h.

After treatment, medium was removed and cells were incubated with MTT (2 mg mL^-1) for 4 h. Then, DMSO was added for solubilization of the formazan and analyzed with a reading microplate reader (BIO-TEK PowerWave XS spectrophotometer) at 392 nm. The percentage of viable cells was calculated in relation to the control to determine the cytotoxic concentration to 50% of the cells (CC₅₀).

Molecular modeling

Molecular modeling studies were performed on the pyrazolo[3,4-d]pyridazinone derivatives developed using the physicochemical properties of lipophilic, electronic and structural profiles in order to support the understanding of their antileishmanial mechanism of action and the potential toxicity effects.

The geometry of compounds was fully optimized by applying DFT B3LYP/6-31G* basis in gas phase²⁴ and it was used to calculate the stereoelectronic properties, E_HOMO (highest occupied molecular orbital energy), E_LUMO (lowest unoccupied molecular orbital energy), gap energy (E_LUMO - E_HOMO), and surface of electrostatic potential density (MEPs). The three-dimensional isosurfaces of the molecular MEPs at the van der Waals contact surface represented electrostatic potentials superimposed onto a surface of constant electron density (0.002 e au^-3). The color-coded isosurface values provide an indication of the overall molecular size and location of negative (red) or positive (blue) electrostatic potentials.

The compounds were also studied by applying the Lipinski's rule of five,²² through the use of online free web cheminformatics software Molinspiration,²³ where the following values were obtained: polar surface area (PSA), molecular weight, and number of acceptor groups and hydrogen bond donors (nON and nOHNH, respectively).

For the theoretical study of toxicity (through mutagenic effects, tumorigenic, irritant and impacts on their productive system) and the drug score and druglikeness profiles of pyrazolo[3,4-d]pyridazinone derivatives, the online web free Osiris Property Explorer (Actelion Pharmaceuticals Ltd, Allschwil, Switzerland, CH) computer program was used.

General synthetic procedure and spectra data

Diethyl 3-((dimethylamine)methylene)-2,4-dioxopentanedioate (1)

The β-enamino diketone substrate 1 was prepared according to the literature.¹⁹

5-(Ethoxycarbonyl)-4-[ethoxy(oxo)acetyl]-1-phenyl-1H-pyrazole (2a)

Pyrazole 2a was prepared according to the literature.¹⁹

1-(tert-Butyl)-5-(ethoxycarbonyl)-4-[ethoxy(oxo)acetyl]-1H-pyrazole (2b)

Pyrazole 2b was prepared according to the methodology described in the literature for the preparation of 2a¹⁹ with a little modification. A mixture of the compound 1 (0.271 g, 1 mmol) and tert-butylhydrazine hydrochloride (0.113 g, 0.9 mmol) in ethanol (2 mL) was stirred under reflux for 1 h. After, the solvent was evaporated under reduced pressure and the residue was washed with distilled water (25 mL), extracted with ethyl acetate (3 × 20 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated once again under reduced pressure and the obtained residue was purified on a silica gel chromatography column, utilizing a 95:5 mixture of hexane:ethyl acetate as the eluent, affording the product as a viscous light yellow oil: 79% yield (0.211 g);¹H NMR (300.06 MHz, CDCl₃) d 1.38 (t, 3H, J7.2 Hz, O-CH₂ -CH₃), 1.39 (t, 3H, J7.2 Hz, O-CH₂ -CH₃), 1.63 (s, 9H, t-Bu) 4.37 (q, 2H, J7.2 Hz, O-CH₂ -CH₃), 4.44 (q, 2H, J7.2 Hz, O-CH₂ -CH₃), 8.18 (s, 1H, H3); ¹³C NMR (75.46 MHz, CDCl₃) d 13.8, 14.1, 29.7, 62.7, 63.3, 63.5, 119.1, 138.3, 140.5, 161.4, 162.9, 177.0; HRMS (ESI(+)): calcd. for C₁₄ H₂₁ N₂ O₅⁺, [M + H]⁺: 297.1445, found 297.1469.

1,6-Disubstituted 4-(ethoxycarbonyl)-1H-pyrazolo [3,4-d]pyridazin-7-one (3a,b,d)

The pyrazolopyridazinone derivatives 3a,b,d were prepared according to the literature.¹⁹ A mixture of compound 2 (2a: 0.158 g; 2b: 0.148 g, 0.50 mmol), hydrazine (hydrazine hydrate, 80% in water: 0.022 g; 4-chlorophenylhydrazine: 0.078 g, 0.55 mmol) and acetic acid (2 mmol, 0.11 mL), was stirred under reflux in ethanol (5 mL) for 12 h. Then, the mixture was cooled to 0 ºC and the solid was filtered, washed with cold ethanol (10 mL) and dried under vacuum.

1-(tert-Butyl)-4-(ethoxycarbonyl)-6,7-dihydro-1H-pyrazolo [3,4-d]pyridazin-7-one (3b)

White solid; 87% yield (0.115 g); mp 177.3-178.9 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.36 (t, 3H, J7.1 Hz, O-CH₂ -CH₃), 1.76 (s, 9H, t-Bu), 4.39 (q, 2H, J7.1 Hz, O-CH₂ -CH₃), 8.28 (s, 1H, H3), 13.27 (bs, 1H, NH); ¹³C NMR (75.46 MHz, DMSO-d₆) d 14.0 (O-CH₂ -CH₃), 29.4 (C(CH₃)₃), 61.5 (O-CH₂ -CH₃), 62.4 (C(CH₃)₃), 120.7 (C3a), 131.3 (C7a), 132.3 (C3), 132.4 (C4), 153.4 (C7), 162.2 (COOEt); HRMS (ESI(+)): calcd. for C₁₂ H₁₇ N₄ O₃⁺, [M + H]⁺: 265.1295, found 265.1316.

4-(Ethoxycarbonyl)-1,6-diphenyl-1H-pyrazolo [3,4-d]pyridazin-7-one (3c)

The pyrazolopyridazinone derivative 3c was prepared according to the literature.²¹

1,6-Disubstituted 4-(hydrazinecarbonyl)-1H-pyrazolo [3,4-d]pyridazin-7-one (4a-d)

The hydrazides derivatives 4a-c were prepared according to the literature.²² A mixture of 3 (3a: 0.284 g; 3b: 0.264 g; 3c: 0.360 g; 3d: 0.394 g, 1.0 mmol) and hydrazine monohydrate (0.84 mL, 20 mmol) was stirred under reflux in ethanol:acetonitrile (1:1, 10 mL) for 24 h. Then, the mixture was cooled to room temperature and the solvent evaporated under vacuum.

1-(tert-Butyl)-4-hydrazinecarbonyl-6,7-dihydro-1H-pyrazolo [3,4-d]pyridazin-7-one (4b)

White solid; 95% yield (0.238 g); mp 291.4-292.7 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.75 (s, 9H, t-Bu), 4.58 (bs, 2H, CONHNH ₂), 8.30 (s, 1H, H3), 9.74 (bs, 1H, CONHNH₂), 13.03 (bs, 1H, NH); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.2 (C(CH₃)₃), 120.4 (C3a), 131.6 (C7a), 132.5 (C3), 135.4 (C4), 153.5 (C7), 161.4 (CONHNH₂); HRMS (ESI(+)): calcd. for C₁₀ H₁₅ N₆ O₂⁺, [M + H]⁺: 251.1251, found 251.1274.

1,6-Disubstituted 4-[(2E)-N'-(substituted)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (5, 6, 7, 8e-j, 6l,k)

The N-acylhydrazones derivatives 5e-j, 6e-l, 7 and 8e-j were prepared according to the literature.^21,22 A mixture of compound 4 (4a: 0.081 g; 4b: 0.075 g; 4c: 0.104 g; 4d: 0.114 g, 0.3 mmol), aldehyde (benzaldehyde: 0.032 g; 4-hydroxybenzaldehyde: 0.037 g; p-anisaldehyde: 0.041 g; 4-(dimethylamino)benzaldehyde: 0.045 g; 4-fluorobenzaldehyde: 0.037 g; 4-nitrobenzaldehyde: 0.045 g; 2-thiophenecarboxaldehyde: 0.034 g; 2,2'-bithiophene-5-carboxaldehyde: 0.058 g, 0.3 mmol) and HCl 37% (two drops) in DMSO (3 mL) was stirred at room temperature for 1 h. Then, cold distilled water (20 mL) was added to the mixture and the product was obtained as a white solid. The product was washed with cold water and dried under vacuum.

4-[(2E)-N'-(Benzylidene)hydrazinecarbonyl]-6,7-dihydro-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5e)

White solid; 95% yield (0.102 g); mp > 350 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.45-7.60 (m, 6H, PhA and PhB), 7.72-7.76 (m, 4H, PhA and PhB), 8.62 (bs, 1H, H8'), 8.66 (s, 1H, H3), 12.06 (bs, 1H, H6'), 13.33 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 119.9 (C3a), 125.5, 127.2, 128.6, 128.6, 128.9, 130.3 (PhA and PhB), 131.9 (C7a), 134.3 (PhB), 134.9 (C4), 136.5 (C3), 138.4 (PhA), 149.2 (C8'), 153.3 (C7), 158.7 (C5'); HRMS (ESI(+)): calcd. for C₁₉ H₁₅ N₆ O₂⁺, [M + H]⁺: 359.1251, found 359.1270.

6,7-Dihydro-4-[(2E)-N'-(4-hydroxybenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5f)

White solid; 94% yield (0.105 g); mp 328.4-330.6 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 6.86 (d, 2H, J8.6 Hz, 4-OHC₆H₄), 7.48-7.60 (m, 5H, 4-OHC₆H₄ and Ph), 7.72-7.75 (m, 2H, Ph), 8.49 (bs, 1H, H8'), 8.65 (bs, 1H, H3), 9.98 (bs, 1H, 4-OHC₆ H₄), 11.84 (bs, 1H, H6'), 13.29 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 115.8 (4-OHC₆ H₄), 120.0 (C3a), 125.2, 125.5, 128.5, 128.5, 129.0 (Ph and 4-OHC₆ H₄), 131.9 (C7a), 135.1 (C4), 136.6 (C3), 138.5 (Ph), 149.5 (C8'), 153.3 (C7), 158.4 (C5'), 159.6 (4-OHC₆ H₄); HRMS (ESI(+)): calcd. for C₁₉ H₁₅ N₆ O₃⁺, [M + H]⁺: 375.1200, found 375.1220.

6,7-Dihydro-4-[(2E)-N'-(4-methoxybenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5g)

White solid; 95% yield (0.111 g); mp > 350 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 3.82 (s, 3H, 4-OCH₃ C₆ H₄), 7.04 (d, 2H, J8.8 Hz, 4-OCH₃ C₆ H₄), 7.48-7.60 (m, 3H, Ph), 7.68 (d, 2H, J8.8 Hz, 4-OCH₃ C₆ H₄), 7.71-7.76 (m, 2H, Ph), 8.54 (bs, 1H, H8'), 8.65 (s, 1H, H3), 11.92 (bs, 1H, H6'), 13.30 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 55.3 (4-OCH₃ C₆ H₄), 114.4 (4-OCH₃C₆ H₄), 119.9 (C3a), 125.5 (Ph), 126.8 (4-OCH₃C₆ H₄), 128.5, 128.5 (Ph), 128.8 (4-OCH₃ C₆ H₄), 131.9 (C7a), 135.1 (C4), 136.5 (C3), 138.4 (Ph), 149.0 (C8'), 153.3 (C7), 158.5 (C5'), 161.0 (4-OCH₃C₆ H₄); HRMS (ESI(+)): calcd. for C₂₀ H₁₇ N₆ O₃⁺, [M + H]⁺: 389.1357, found 389.1374.

6,7-Dihydro-4-[(2E)-N'-(4-(dimethylamino)benzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5h)

Light yellow solid; 95% yield (0.114 g); mp 329.0-330.6 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 2.99 (s, 6H, 4-N(CH₃)₂ C₆ H₄), 6.77 (d, 2H, J9.1 Hz, 4-N(CH₃)₂ C₆H₄), 7.48-7.60 (m, 5H, Ph and 4-N(CH₃)₂ C₆H₄), 7.71-7.75 (m, 2H, Ph), 8.44 (bs, 1H, H8'), 8.65 (s, 1H, H3), 11.74 (bs, 1H, H6'), 13.27 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 39.8 (4-N(CH₃)₂ C₆ H₄), 111.8 (4-N(CH₃)₂ C₆ H₄), 120.0 (C3a), 121.5 (4-N(CH₃)₂C₆ H₄), 125.5, 128.5, 128.5 (Ph), 128.5 (4-N(CH₃)₂C₆ H₄), 131.9 (C7a), 135.3 (C4), 136.6 (C3), 138.5 (Ph), 149.9 (C8'), 151.6 (4-N(CH₃)₂C₆ H₄), 153.3 (C7), 158.2 (C5'); HRMS (ESI(+)): calcd. for C₂₁ H₂₀ N₇ O₂⁺, [M + H]⁺: 402.1673, found 402.1686.

6,7-Dihydro-4-[(2E)-N'-(4-fluorobenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5i)

White solid; 94% yield (0.106 g); mp > 350 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.32 (t, 2H,³ J_H-F 8.9 Hz, J8.9 Hz, 4-FC₆ H₄), 7.48-7.60 (m, 3H, Ph), 7.72-7.76 (m, 2H, Ph), 7.80 (dd, 2H,⁴ J_H-F 5.6 Hz, J8.8 Hz, 4-FC₆ H₄), 8.61 (bs, 1H, H8'), 8.66 (s, 1H, H3), 12.07 (bs, 1H, H6'), 13.33 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 116.0 (d,² J_C-F 22.0 Hz, 4-FC₆ H₄), 119.9 (C3a), 125.5, 128.6, 128.6 (Ph), 129.4 (d,³ J_C-F 8.7 Hz, 4-FC₆ H₄), 130.9 (d,⁴ J_C-F 2.8 Hz, 4-FC₆ H₄), 131.9 (C7a), 134.9 (C4), 136.5 (C3), 138.4 (Ph), 148.0 (C8'), 153.3 (C7), 158.7 (C5'), 163.2 (d,¹ J_C-F 248.0 Hz, 4-FC₆ H₄); HRMS (ESI(+)): calcd. for C₁₉ H₁₄ FN₆ O₂⁺, [M + H]⁺: 377.1157, found 377.1166.

6,7-Dihydro-4-[(2E)-N'-(4-nitrobenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (5j)

Light yellow solid; 94% yield (0.114 g); mp > 350 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.48-7.60 (m, 3H, Ph), 7.71-7.74 (m, 2H, Ph), 7.99 (d, 2H, J9.0 Hz, 4-NO₂ C₆ H₄), 8.32 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.66 (s, 1H, H3), 8.71 (bs, 1H, H8'), 12.38 (bs, 1H, H6'), 13.33 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 119.9 (C3a), 124.2 (4-NO₂ C₆ H₄), 125.6 (Ph), 128.2 (4-NO₂ C₆ H₄), 128.7, 128.7 (Ph), 131.9 (C7a), 134.7 (C4), 136.5 (C3), 138.5 (Ph), 140.6 (4-NO₂ C₆ H₄), 146.8 (C8'), 148.0 (4-NO₂ C₆ H₄), 153.4 (C7), 159.1 (C5'); HRMS (ESI(+)): calcd. for C₁₉ H₁₄ N₇ O₄⁺, [M + H]⁺: 404.1102, found 404.1093.

4-[(2E)-N'-(Benzylidene)hydrazinecarbonyl]-1-(tert-butyl)-6,7-dihydro-1H-pyrazolo[3,4-d]pyridazin-7-one (6e)

White solid; 95% yield (0.096 g); mp 304.8-306.9 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.79 (s, 9H, t-Bu), 7.43-7.52 (m, 3H, Ph), 7.71-7.75 (m, 2H, Ph), 8.39 (s, 1H, H3), 8.59 (bs, 1H, H8'), 12.00 (bs, 1H, H6'), 13.24 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.4 (C(CH₃)₃), 120.4 (C3a), 127.1, 128.9, 130.2 (Ph), 131.7 (C7a), 132.6 (C3), 134.3 (Ph), 135.1 (C4), 149.0 (C8'), 153.7 (C7), 158.8 (C5'); HRMS (ESI(+)): calcd. for C₁₇ H₁₉ N₆ O₂⁺, [M + H]⁺: 339.1564, found 339.1568.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(4-hydroxybenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (6f)

Light yellow solid; 95% yield (0.101 g); mp 310.4-312.8 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.78 (s, 9H, t-Bu), 6.85 (d, 2H, J8.7 Hz, 4-OHC₆H₄), 7.56 (d, 2H, J8.7 Hz, 4-OHC₆H₄), 8.37 (s, 1H, H3), 8.46 (bs, 1H, H8'), 9.96 (bs, 1H, 4-OHC₆ H₄), 11.78 (bs, 1H, H6'), 13.19 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.3 (C(CH₃)₃), 115.8 (4-OHC₆ H₄), 120.5 (C3a), 125.2, 128.9 (4-OHC₆ H₄), 131.7 (C7a), 132.6 (C3), 135.3 (C4), 149.3 (C8'), 153.6 (C7), 158.5 (C5'), 159.6 (4-OHC₆ H₄); HRMS (ESI(+)): calcd. for C₁₇ H₁₉ N₆ O₃⁺, [M + H]⁺: 355.1513, found 355.1515.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(4-methoxybenzylidene)hydrazinecarbonyl]-1H-pyrazolo [3,4-d]pyridazin-7-one (6g)

White solid; 95% yield (0.105 g); mp 271.5-272.0 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.79 (s, 9H, t-Bu), 3.82 (s, 3H, 4-OCH₃ C₆ H₄), 7.04 (d, 2H, J8.8 Hz, 4-OCH₃ C₆ H₄), 7.67 (d, 2H, J8.9 Hz, 4-OCH₃ C₆H₄), 8.38 (s, 1H, H3), 8.52 (bs, 1H, H8'), 11.86 (bs, 1H, H6'), 13.21 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 55.3 (4-OCH₃ C₆ H₄), 62.3 (C(CH₃)₃), 114.4 (4-OCH₃ C₆ H₄), 120.5 (C3a), 126.8, 128.8 (4-OCH₃C₆ H₄), 131.7 (C7a), 132.6 (C3), 135.2 (C4), 148.9 (C8'), 153.7 (C7), 158.6 (C5'), 161.0 (4-OCH₃C₆ H₄); HRMS (ESI(+)): calcd. for C₁₈ H₂₁ N₆ O₃⁺, [M + H]⁺: 369.1670, found 369.1674.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(4-(dimethylamino)benzylidene)hydrazinecarbonyl]-1H-pyrazolo [3,4-d]pyridazin-7-one (6h)

Yellow solid; 96% yield (0.110 g); mp 270.4-272.4 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.78 (s, 9H, t-Bu), 2.99 (s, 6H, 4-N(CH₃)₂ C₆ H₄), 6.77 (d, 2H, J8.9 Hz, 4-N(CH₃)₂ C₆H₄), 7.54 (d, 2H, J8.9 Hz, 4-N(CH₃)₂ C₆H₄), 8.37 (s, 1H, H3), 8.41 (bs, 1H, H8'), 11.67 (bs, 1H, H6'), 13.18 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 39.8 (4-N(CH₃)₂ C₆ H₄), 62.3 (C(CH₃)₃), 111.8 (4-N(CH₃)₂C₆ H₄), 120.5 (C3a), 121.5, 128.5 (4-N(CH₃)₂C₆ H₄), 131.7 (C7a), 132.6 (C3), 135.4 (C4), 149.8 (C8'), 151.6 (4-N(CH₃)₂C₆ H₄), 153.6 (C7), 158.3 (C5'); HRMS (ESI(+)): calcd. for C₁₉ H₂₄ N₇ O₂⁺, [M + H]⁺: 382.1986, found 382.1998.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(4-fluorobenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (6i)

White solid; 94% yield (0.100 g); mp 314.4-315.4 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.79 (s, 9H, t-Bu), 7.32 (t, 2H,³ J_H-F 8.9 Hz, J8.9 Hz, 4-FC₆ H₄), 7.79 (dd, 2H,⁴ J_H-F 5.7 Hz, J8.7 Hz, 4-FC₆ H₄), 8.39 (s, 1H, H3), 8.59 (bs, 1H, H8'), 12.01 (bs, 1H, H6'), 13.24 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.3 (C(CH₃)₃), 116.0 (d,² J_C-F 22.0 Hz, 4-FC₆ H₄), 120.4 (C3a), 129.3 (d,³ J_C-F 8.6 Hz, 4-FC₆ H₄), 130.9 (d,⁴ J_C-F 3.1 Hz, 4-FC₆ H₄), 131.7 (C7a), 132.5 (C3), 135.1 (C4), 147.9 (C8'), 153.7 (C7), 158.8 (C5'), 163.2 (d,¹ J_C-F 247.8 Hz, 4-FC₆ H₄); HRMS (ESI(+)): calcd. for C₁₇ H₁₈ FN₆ O₂⁺, [M + H]⁺: 357.1470, found 357.1473.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(4-nitrobenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (6j)

Light yellow solid; 94% yield (0.108 g); mp 317.4-319.5 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 1.79 (s, 9H, t-Bu), 7.98 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.31 (d, 2H, J8.8 Hz, 4-NO₂ C₆ H₄), 8.39 (s, 1H, H3), 8.70 (bs, 1H, H8'), 12.30 (bs, 1H, H6'), 13.28 (bs, 1H, H6); ¹³C NMR (75.46 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.4 (C(CH₃)₃), 120.4 (C3a), 124.1, 128.0 (4-NO₂ C₆ H₄), 131.7 (C7a), 132.5 (C3), 134.8 (C4), 140.6 (4-NO₂ C₆ H₄), 146.5 (C8'), 147.9 (4-NO₂ C₆ H₄), 153.7 (C7), 159.1 (C5'); HRMS (ESI(+)): calcd. for C₁₇ H₁₈ N₇ O₄⁺, [M + H]⁺: 384.1415, found 384.1385.

1-(tert-Butyl)-6,7-dihydro-4-[(2E)-N'-(2-thienylmethylene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (6k)

White solid; 95% yield (0.098 g); mp 319.2-322.1 ºC;¹H NMR (500.13 MHz, DMSO-d₆) d 1.77 (s, 9H, t-Bu), 7.15 (dd, 1H, J5.1, 3.6 Hz, 2-thienyl), 7.44 (dd, 1H, J3.7, 1.1 Hz, 2-thienyl), 7.69 (dd, 1H, J5.0 Hz, 2-thienyl), 8.36 (s, 1H, H3), 8.76 (bs, 1H, H8'), 11.99 (bs, 1H, H6'), 13.21 (bs, 1H, H6); ¹³C NMR (125.77 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.4 (C(CH₃)₃), 120.4 (C3a), 127.9, 129.2, 131.1 (2-thienyl), 131.7 (C7a), 132.6 (C3), 135.1 (C4), 139.1 (2-thienyl), 144.0 (C8'), 153.7 (C7), 158.7 (C5'); HRMS (ESI(+)): calcd. for C₁₅ H₁₇ N₆ O₂ S⁺, [M + H]⁺: 345.1128, found 345.1130.

4-[(2E)-N'-(2,2'-Bithienyl-5-methylene)hydrazinecarbonyl]-1-(tert-butyl)-6,7-dihydro-1H-pyrazolo[3,4-d]pyridazin-7-one (6l)

Yellow solid; 96% yield (0.123 g); mp 223.2-225.3 ºC;¹H NMR (500.13 MHz, DMSO-d₆) d 1.77 (s, 9H, t-Bu), 7.13 (dd, 1H, J5.1, 3.6 Hz, 2,2'-bithienyl), 7.32 (d, 1H, J3.8 Hz, 2,2'-bithienyl), 7.40 (d, 1H, J3.9 Hz, 2,2'-bithienyl), 7.44 (dd, 1H, J3.7, 1.2 Hz, 2,2'-bithienyl), 7.59 (dd, 1H, J5.1, 1.2 Hz, 2,2'-bithienyl), 8.37 (s, 1H, H3), 8.71 (bs, 1H, H8'), 12.05 (bs, 1H, H6'), 13.21 (bs, 1H, H6); ¹³C NMR (125.77 MHz, DMSO-d₆) d 29.5 (C(CH₃)₃), 62.4 (C(CH₃)₃), 120.4 (C3a), 124.3, 125.2, 126.6, 128.6 (2,2'-bithienyl), 131.7 (C7a), 132.2 (2,2'-bithienyl), 132.6 (C3), 135.1 (C4), 136.1, 137.8, 138.9 (2,2'-bithienyl), 143.6 (C8'), 153.7 (C7), 158.6 (C5'); HRMS (ESI(+)): calcd. for C₁₉ H₁₉ N₆ O₂ S₂⁺, [M + H]⁺: 427.1005, found 427.1008.

4-[(2E)-N'-(Benzylidene)hydrazinecarbonyl]-1,6-diphenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (7e)

Light yellow solid; 94% yield (0.122 g); mp 259.3-260.6 ºC;¹H NMR (300.06 MHz, CDCl₃) d 7.36-7.58 (m, 1H, PhA, B and C), 7.66-7.71 (m, 2H, PhA), 7.75-7.79 (m, 2H, PhC), 8.23 (bs, 1H, H8'), 8.87 (s, 1H, H3), 10.18 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, CDCl₃) d 120.0 (C3a), 125.7, 126.5, 127.9, 128.7, 128.9, 128.9, 129.1, 129.1, 130.9, 133.4 (PhA, B and C), 132.1 (C7a), 134.4 (C4), 137.5 (C3), 138.5, 140.8 (PhA and B), 149.4 (C8'), 152.6 (C7), 157.9 (C5'); HRMS (ESI(+)): calcd. for C₂₅ H₁₉ N₆ O₂⁺, [M + H]⁺: 435.1564, found 435.1576.

1,6-Diphenyl-4-[(2E)-N'-(4-hydroxybenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (7f)

Light yellow solid; 95% yield (0.128 g); mp > 350 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 6.85 (d, 2H, J8.6 Hz, 4-OHC₆H₄), 7.43-7.58 (m, 8H, 4-OHC₆H₄, PhA and B), 7.68-7.75 (m, 4H, PhA and B), 8.47 (bs, 1H, H8'), 8.69 (s, 1H, H3), 9.98 (bs, 1H, 4-OHC₆ H₄), 11.72 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 115.8 (4-OHC₆ H₄), 119.6 (C3a), 125.1 (4-OHC₆ H₄), 125.7, 126.8, 128.2, 128.5, 128.6, 128.7 (PhA and B), 129.0 (4-OHC₆ H₄), 132.3 (C7a), 134.6 (C4), 136.5 (C3), 138.4, 140.9 (PhA and B), 149.9 (C8'), 152.0 (C7), 158.1 (C5'), 159.7 (4-OHC₆ H₄); HRMS (ESI(+)): calcd. for C₂₅ H₁₉ N₆ O₃⁺, [M + H]⁺: 451.1513, found 451.1514.

1,6-Diphenyl-4-[(2E)-N'-(4-methoxybenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (7g)

White solid; 96% yield (0.134 g); mp 247.3-248.6 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 3.81 (s, 3H, 4-OCH₃ C₆ H₄), 7.04 (d, 2H, J8.8 Hz, 4-OCH₃ C₆ H₄), 7.44-7.58 (m, 6H, PhA and B), 7.65-7.76 (m, 6H, 4-OCH₃ C₆H₄, PhA and B), 8.52 (bs, 1H, H8'), 8.69 (s, 1H, H3), 11.79 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 55.3 (4-OCH₃ C₆ H₄), 114.4 (4-OCH₃C₆ H₄), 119.6 (C3a), 125.7, 126.8, 128.2, 128.5, 128.6, 128.7 (PhA and B), 126.7, 128.9 (4-OCH₃C₆ H₄), 132.3 (C7a), 134.5 (C4), 136.5 (C3), 138.4, 140.9 (PhA and B), 149.5 (C8'), 152.0 (C7), 158.2 (C5'), 161.0 (4-OCH₃C₆ H₄); HRMS (ESI(+)): calcd. for C₂₆ H₂₁ N₆ O₃⁺, [M + H]⁺: 465.1670, found 465.1678.

4-[(2E)-N'-(4-(Dimethylamino)benzylidene)hydrazinecarbonyl]-1,6-diphenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (7h)

Yellow solid; 96% yield (0.137 g); mp 267.5-268.9 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 2.98 (s, 6H, 4-N(CH₃)₂ C₆ H₄), 6.76 (d, 2H, J9.0 Hz, 4-N(CH₃)₂ C₆H₄), 7.43-7.58 (m, 8H, 4-N(CH₃)₂ C₆H₄, PhA and B), 7.68-7.75 (m, 4H, PhA and B), 8.41 (bs, 1H, H8'), 8.68 (s, 1H, H3), 11.62 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 39.5 (4-N(CH₃)₂ C₆ H₄), 111.8 (4-N(CH₃)₂ C₆ H₄), 119.7 (C3a), 121.3 (4-N(CH₃)₂C₆ H₄), 125.7, 126.8, 128.2, 128.5, 128.5, 128.6 (PhA and B), 128.6 (4-N(CH₃)₂C₆ H₄), 132.2 (C7a), 134.8 (C4), 136.5 (C3), 138.4, 140.9 (PhA and B), 150.4 (C8'), 151.7 (4-N(CH₃)₂ C₆ H₄), 152.0 (C7), 157.9 (C5'); HRMS (ESI(+)): calcd. for C₂₇ H₂₄ N₇ O₂⁺, [M + H]⁺: 478.1986, found 478.1985.

1,6-Diphenyl-4-[(2E)-N'-(4-fluorobenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (7i)

White solid; 95% yield (0.129 g); mp 251.2-252.2 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.31 (t, 2H,³ J_H-F 8.9 Hz, J8.9 Hz, 4-FC₆ H₄), 7.43-7.58 (m, 6H, PhA and B), 7.68-7.75 (m, 4H, PhA and B), 7.79 (dd, 2H,⁴ J_H-F 5.6 Hz, J8.9 Hz, 4-FC₆ H₄), 8.58 (bs, 1H, H8'), 8.69 (s, 1H, H3), 11.93 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 116.0 (d,² J_C-F 22.0 Hz, 4-FC₆ H₄), 119.6 (C3a), 125.7, 126.8, 128.3, 128.5, 128.6, 128.7 (PhA and B), 129.4 (d,³ J_C-F 8.7 Hz, 4-FC₆ H₄), 130.8 (d,⁴ J_C-F 2.9 Hz, 4-FC₆ H₄), 132.2 (C7a), 134.3 (C4), 136.5 (C3), 138.4, 140.9 (PhA and B), 148.5 (C8'), 152.0 (C7), 158.4 (C5'), 163.3 (d,¹ J_C-F 248.2 Hz, 4-FC₆ H₄); HRMS (ESI(+)): calcd. for C₂₅ H₁₈ FN₆ O₂⁺, [M + H]⁺: 453.147, found 453.1484.

1,6-Diphenyl-4-[(2E)-N'-(4-nitrobenzylidene)hydrazinecarbonyl]-1H-pyrazolo[3,4-d]pyridazin-7-one (7j)

White solid; 94% yield (0.135 g); mp 277.6-278.2 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.45-7.58 (m, 6H, PhA and B), 7.68-7.76 (m, 4H, PhA and B), 7.98 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.32 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.70 (bs, 1H, H8'), 8.71 (s, 1H, H3), 12.22 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 119.6 (C3a), 124.1 (4-NO₂ C₆ H₄), 125.7, 126.9, 128.3, 128.5, 128.6, 128.7 (PhA and B), 128.1 (4-NO₂ C₆ H₄), 132.3 (C7a), 134.1 (C4), 136.4 (C3), 138.4, 140.9 (PhA and B), 140.4 (4-NO₂ C₆ H₄), 147.1 (C8'), 148.0 (4-NO₂ C₆ H₄), 152.0 (C7), 158.8 (C5'); HRMS (ESI(+)): calcd. for C₂₅ H₁₈ N₇ O₄⁺, [M + H]⁺: 480.1415, found 480.1422.

4-[(2E)-N'-(Benzylidene)hydrazinecarbonyl]-6-(4-chlorophenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (8e)

White solid; 96% yield (0.135 g); mp 255.7-257.5 ºC;¹H NMR (500.13 MHz, CDCl₃) d 7.37-7.42 (m, 3H, PhB), 7.44-7.51 (m, 5H, PhA and 4-ClC₆ H₄), 7.54 (d, 2H, J8.8 Hz, 4-ClC₆ H₄), 7.66-7.68 (m, 2H, PhA), 7.75-7.76 (m, 2H, PhB), 8.26 (bs, 1H, H8'), 8.85 (s, 1H, H3), 10.17 (bs, 1H, H6'); ¹³C NMR (125.77 MHz, CDCl₃) d 120.0 (C3a), 125.7, 127.8 (PhA and 4-ClC₆ H₄), 127.9 (PhB), 128.8, 128.9, 129.2 (PhA and 4-ClC₆ H₄), 129.2, 131.0 (PhB), 132.0 (C7a), 133.4 (PhB), 134.7 (4-ClC₆ H₄), 134.8 (C4), 137.6 (C3), 138.5, 139.3 (PhA and 4-ClC₆ H₄), 149.6 (C8'), 152.5 (C7), 157.8 (C5'); HRMS (ESI(+)): calcd. for C₂₅ H₁₈ ClN₆ O₂⁺, [M + H]⁺: 469.1174, found 469.1169.

6-(4-Chlorophenyl)-4-[(2E)-N'-(4-hydroxybenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (8f)

Yellow solid; 94% yield (0.137 g); mp > 350 ºC;¹H NMR (500.13 MHz, DMSO-d₆) d 6.85 (d, 2H, J8.6 Hz, 4-OHC₆H₄), 7.49-7.58 (m, 5H, 4-OHC₆H₄ and Ph), 7.61 (d, 2H, J8.7 Hz, 4-ClC₆ H₄), 7.73-7.74 (m, 2H, Ph), 7.77 (d, 2H, J8.7 Hz, 4-ClC₆ H₄), 8.46 (bs, 1H, H8'), 8.69 (s, 1H, H3), 9.98 (bs, 1H, 4-OHC₆ H₄), 11.73 (bs, 1H, H6'); ¹³C NMR (125.77 MHz, DMSO-d₆) d 115.8 (4-OHC₆ H₄), 119.6 (C3a), 125.1 (4-OHC₆ H₄), 125.6, 128.5, 128.5, 128.6, 128.7 (Ph and 4-ClC₆ H₄), 129.0 (4-OHC₆ H₄), 132.1 (C7a), 132.6 (4-ClC₆ H₄), 134.8 (C4), 136.5 (C3), 138.4, 139.7 (Ph and 4-ClC₆ H₄), 149.9 (C8'), 151.9 (C7), 158.0 (C5'), 159.7 (4-OHC₆ H₄); HRMS (ESI(+)): calcd. for C₂₅ H₁₈ ClN₆ O₃⁺, [M + H]⁺: 485.1123, found 485.1119.

6-(4-Chlorophenyl)-4-[(2E)-N'-(4-methoxybenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (8g)

White solid; 94% yield (0.140 g); mp 154.6-155.8 ºC;¹H NMR (300.06 MHz, CDCl₃) d 3.83 (s, 3H, 4-OCH₃ C₆ H₄), 6.91 (d, 2H, J8.8 Hz, 4-OCH₃ C₆ H₄), 7.44-7.57 (m, 7H, Ph and 4-ClC₆ H₄), 7.65-7.73 (m, 4H, 4-OCH₃ C₆ H₄ and Ph), 8.18 (bs, 1H, H8'), 8.86 (s, 1H, H3), 10.06 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, CDCl₃) d 55.5 (4-OCH₃ C₆ H₄), 114.4 (4-OCH₃C₆ H₄), 120.0 (C3a), 125.7, 127.8, 128.8, 129.1, 129.2 (Ph and 4-ClC₆ H₄), 126.0, 129.6 (4-OCH₃C₆ H₄), 131.9 (C7a), 134.6 (4-ClC₆ H₄), 134.9 (C4), 137.6 (C3), 138.5, 139.3 (Ph and 4-ClC₆ H₄), 149.4 (C8'), 152.5 (C7), 157.6 (C5'), 162.0 (4-OCH₃C₆ H₄); HRMS (ESI(+)): calcd. for C₂₆ H₂₀ ClN₆ O₃⁺, [M + H]⁺: 499.1280, found 499.1280.

6-(4-Chlorophenyl)-4-[(2E)-N'-(4-(dimethylamino)benzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo [3,4-d]pyridazin-7-one (8h)

Yellow solid; 96% yield (0.147 g); mp 245.2-246.7 ºC;¹H NMR (500.13 MHz, CDCl₃) d 2.99 (s, 6H, 4-N(CH₃)₂ C₆ H₄), 6.63 (d, 2H, J9.0 Hz, 4-N(CH₃)₂ C₆H₄), 7.43-7.50 (m, 5H, 4-ClC₆ H₄ and Ph), 7.55 (d, 2H, J8.8 Hz, 4-ClC₆ H₄), 7.59 (d, 2H, J8.8 Hz, 4-N(CH₃)₂ C₆H₄), 7.66-7.68 (m, 2H, Ph), 8.08 (bs, 1H, H8'), 8.85 (s, 1H, H3), 9.99 (bs, 1H, H6'); ¹³C NMR (125.77 MHz, CDCl₃) d 40.2 (4-N(CH₃)₂ C₆ H₄), 111.7 (4-N(CH₃)₂ C₆ H₄), 120.1 (C3a), 120.8 (4-N(CH₃)₂C₆ H₄), 125.7, 127.8, 128.7, 129.0, 129.2 (Ph and 4-ClC₆ H₄), 129.5 (4-N(CH₃)₂C₆ H₄), 132.0 (C7a), 134.5 (4-ClC₆ H₄), 135.2 (C4), 137.7 (C3), 138.6, 139.4 (Ph and 4-ClC₆ H₄), 150.3 (C8'), 152.2 (4-N(CH₃)₂C₆ H₄), 152.5 (C7), 157.3 (C5'); HRMS (ESI(+)): calcd. for C₂₇ H₂₃ ClN₇ O₂⁺, [M + H]⁺: 512.1596, found 512.1593.

6-(4-Chlorophenyl)-4-[(2E)-N'-(4-fluorobenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (8i)

White solid; 95% yield (0.139 g); mp 279.9-280.2 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.32 (t, 2H,³ J_H-F 8.9 Hz, J8.9 Hz, 4-FC₆ H₄), 7.50-7.63 (m, 5H, Ph and 4-ClC₆ H₄), 7.72-7.82 (m, 6H, Ph, 4-ClC₆ H₄ and 4-FC₆ H₄), 8.58 (bs, 1H, H8'), 8.69 (s, 1H, H3), 11.94 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 116.0 (d,² J_C-F 22.0 Hz, 4-FC₆ H₄), 119.6 (C3a), 125.7, 128.5, 128.5, 128.6, 128.7 (Ph and 4-ClC₆ H₄), 129.4 (d,³ J_C-F 8.8 Hz, 4-FC₆ H₄), 130.7 (d,⁴ J_C-F 3.1 Hz, 4-FC₆ H₄), 132.2 (C7a), 134.5 (C4), 136.5 (C3), 132.7, 138.4, 139.7 (Ph and 4-ClC₆ H₄), 148.5 (C8'), 152.0 (C7), 158.3 (C5'), 163.3 (d,¹ J_C-F 248.1 Hz, 4-FC₆ H₄); HRMS (ESI(+)): calcd. for C₂₅ H₁₇ ClFN₆ O₂⁺, [M + H]⁺: 487.1080, found 487.1082.

6-(4-Chlorophenyl)-4-[(2E)-N'-(4-nitrobenzylidene)hydrazinecarbonyl]-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (8j)

Light yellow solid; 96% yield (0.148 g); mp 303.5-304.3 ºC;¹H NMR (300.06 MHz, DMSO-d₆) d 7.48-7.58 (m, 3H, Ph), 7.62 (d, 2H, J8.8 Hz, 4-ClC₆ H₄), 7.71-7.79 (m, 4H, Ph and 4-ClC₆ H₄), 7.98 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.31 (d, 2H, J8.9 Hz, 4-NO₂ C₆ H₄), 8.69 (bs, 2H, H3 and H8'), 12.21 (bs, 1H, H6'); ¹³C NMR (75.46 MHz, DMSO-d₆) d 119.5 (C3a), 124.2 (4-NO₂ C₆ H₄), 125.7, 128.5, 128.6, 128.7, 128.7 (Ph and 4-ClC₆ H₄), 128.2 (4-NO₂ C₆ H₄), 132.2 (C7a), 134.3 (C4), 136.5 (C3), 132.7, 138.3, 139.7 (Ph and 4-ClC₆ H₄), 140.4 (4-NO₂ C₆ H₄), 147.2 (C8'), 148.0 (4-NO₂ C₆ H₄), 152.0 (C7), 158.7 (C5'); HRMS (ESI(+)): calcd. for C₂₅ H₁₇ ClN₇ O₄⁺, [M + H]⁺: 514.1025, found 514.1017.

Acknowledgments

The authors are grateful for the financial support from CNPq, Brazil (Universal Process No. 405107/2013-7 and 454920/2014-8) and Fundação Araucária (Brazil). Fellowships from CAPES (Brazil) are also acknowledged.

Supplementary Information

Crystallographic data for compound 8h in this work were deposited in the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1576799. Copies of the data can be obtained, free of charge, via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: [phone]. E-mail: [e-mail].

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

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Publication Dates

• Publication in this collection
  Dec 2018

History

• Received
  14 May 2018
• Accepted
  12 July 2018