Polynitrogen Bicyclic and Tricyclic Compounds as PDE4 Inhibitors

Abstract

We report here two new series of designed PDE4 inhibitors, the first one showing the quinoline scaffold recently investigated by us through a fragment-based drug design strategy, and the second consisting of pyrazolo [1′,5′:1,6]pyrimido[4,5-d]pyridazine derivatives. Both the new series were subjected to biological studies to assess their inhibitory effect on PDE4 enzymes, supported by molecular modelling experiments, to rationalize the different activities recorded in the in vitro tests. Interesting results were achieved for two compounds belonging to the tricyclic series, namely 10a and 10e, exhibiting IC₅₀ = 62 and 175.5 nM, respectively. These results could represent the starting point for further studies with the aim of developing new and effective PDE4 inhibitors for biomedical investigations.

1. Introduction

Phosphodiesterases (PDEs) are a family of enzymes responsible for the hydrolysis of the intracellular cyclic nucleotides adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP), causing their inactivation [1,2]. Currently, eleven PDE subfamilies (PDE1–PDE11) and at least twenty-one isoforms with numerous splice-site variants are known in humans, which are characterized by different functional roles, tissue distribution, and substrate specificity (cAMP/cGMP) [3]. The PDE4 subfamily, specific for cAMP, is encoded by four genes (A–D), translating into four isoforms: PDE4A–PDE4D [4,5]. These are found in several cell types and tissues, including airway, pulmonary and vascular smooth muscle, vascular endothelium, and brain [6]. Moreover, PDE4A, PDE4B, and PDE4D are additionally expressed in immunocompetent and inflammatory cells, as neutrophils, T-lymphocytes, macrophages, and eosinophils [7]. It is well known that the increase in intracellular cAMP concentrations by PDE4 inhibition affords a potent anti-inflammatory effect, useful for interfering with a broad range of diseases, including asthma, chronic obstructive pulmonary disease (COPD), Crohn’s disease, psoriasis, atopic dermatitis, and rheumatoid arthritis [8,9]. In recent years, other possible therapeutic applications have also emerged for PDE4 inhibitors, and relate to neurological disorders such as depression, Alzheimer’s disease, and anxiety, with some compounds currently in clinical trials for these pathologies [10,11,12].

Since 2010, four PDE4 inhibitors have been launched on the market (Figure 1). Firstly Roflumilast (Daxas^TM, AstraZeneca, Cambridge, UK) for COPD [13], then Apremilast (Celgene; 2014, Summit, NJ, USA) for psoriasis [14,15] and, lastly, Ibudilast (Medicinova; 2016, La Jolla, CA, USA) for Krabbe Disease [16] and Crisaborole (Pfizer; 2016, New York, NY, USA) for atopic dermatitis [17], were commercialized.

From the 1990s, we have been working in the field of PDE4 inhibitors, obtaining very interesting results in terms of developing several new hits endowed with high potency and selectivity [18,19]. Recent outcomes of this research [20] have focused on fragment-based drug design (FBDD) strategies [21] to identify new quinoline-based PDE4 inhibitors as an evolution of GSK-256066, one of the most potent PDE4 inhibitors synthesized to date (IC₅₀ = 8 pM), in clinical trials for COPD and asthma [22]. The structure–activity relationships (SARs) of this latest series have been finely assigned, and the most relevant results in terms of PDE inhibition are outlined in Figure 2, along with the most favorable groups and functions determining the activity [20].

To continue our investigation on the quinoline scaffold towards PDE4 inhibition, we have adopted compound A (5e in the original paper [20], i.e., the most active product of the series, IC₅₀ = 21.27 nM) (Figure 3) as the lead candidate, in order to plan the synthesis of new amide derivatives, by (1) introducing de novo an amide function in meta position of the aniline group (indicated as red circles) and (2) elaborating the primary amide at position 3 of the heterocyclic scaffold (indicated as blue circle) (Figure 3). The choice of inserting an additional amide group on the 3-methoxyaniline in position 4 results from the observation that a limited number of compounds developed earlier and bearing a secondary alkylaryl amide in this position possess an activity in the micromolar range [20], while the primary amide is completely inactive. With regard to the rationale behind modifying the primary amide at position 3 of the quinoline, we have previously developed compound B (9 in the original paper [20]), bearing the N-(3,5-dichloropyridin-4-yl)amide fragment of Roflumilast. This derivative, despite being less active than the lead compound A (5e), determines an appreciable level of PDE4 inhibition (i.e., 76%) at 1 µM, which prompted us to continue the chemical elaboration of this amide function.

In parallel, to expand our research on new PDE4 inhibitors and build up further evidence that the presence of amide functions in known PDE-targeting structures is a determinant for activity, we have additionally performed a biological screening of a new series of pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazines available within our in-house compound library. In particular, all the selected analogues possess a secondary or tertiary amide function at position C-1 and an ethyl group at N-3 of the tricyclic scaffold. This series is based on the results obtained through a wider investigation conducted earlier by us, on tricyclic structures to access effective PDE5 inhibitors [23], whereby SAR studies had clearly indicated that the N-3 substituent on the pyridazine is crucial for selectivity, enabling differentiation of the binding and inhibitory effect towards either PDE4 or PDE5 subfamilies. Specifically, the presence of a benzyl on N-3 leads to selective PDE5 inhibitors, whereas the ethyl group in the same position produces selective PDE4 inhibitors. Based on this evidence, we decided to test these novel tricyclic analogues, where the necessary structural requirements for the inhibitory effect on PDE4 (i.e., the ethyl group on N-3 of the heterocycle and an amide function in position C-1) are present (Figure 4).

2. Materials and Methods

2.1. Chemistry

Reagents and starting materials were obtained from commercial sources (Sigma Aldrich (Merck Spa), Via Monte Rosa, 93-20149 Milan, Italy; Fluorochem Limited, Unit 14, Graphite Way, Rossington Park, Hadfield, Derbyshire SK13 1QH, UK). Extracts were dried over Na₂SO₄, and the solvents were removed under reduced pressure. All melting points were determined on a Büchi apparatus (New Castle, DE, USA) and are uncorrected. All reactions were monitored by thin layer chromatography (TLC) using commercial plates pre-coated with Merck silica gel 60 F-254. Visualization was performed by UV fluorescence (λmax = 254 nm) or by staining with iodine or potassium permanganate. Chromatographic separations were performed on a silica gel column by gravity (Kieselgel 40, 0.063–0.200 mm; Merck, Rahway, NJ, USA) or flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. When reactions were performed in anhydrous conditions, the mixtures were maintained under a nitrogen atmosphere. Compounds were named following IUPAC rules, as applied by Beilstein-Institut AutoNom 2000 (4.01.305) or CA Index Name. The identity and purity of intermediates and final compounds were ascertained through TLC chromatography and NMR. ¹H NMR and ¹³C NMR spectra were recorded with Avance 400 or Bruker Avance 400 Neo (Billerica, MA, USA) instruments (Bruker Biospin Version 002 with SGU). Chemical shifts (δ) are reported in ppm to the nearest 0.01 ppm, using the solvent as an internal standard. Coupling constants (J values) are given in Hz and were calculated using ‘TopSpin 1.3′ software, rounded to the nearest 0.1 Hz. Data are reported as follows: chemical shift, multiplicity [exch, exchange; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; sept, septet; m, multiplet; or as a combination of these (e.g., dd, dt etc.)], integration, assignment and coupling constant (s).

High resolution mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan (San Jose, CA, USA) LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). Analyses were carried out in positive ion mode, monitoring protonated molecules, [M+H]⁺ species, and a proper dwell time acquisition was used to achieve 30,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental composition of compounds was evaluated on the basis of their measured accurate masses, accepting only results with an attribution error less than 2 ppm and a non-integer RDB (double bond/ring equivalents) value [24]. All new compounds possess a purity > 95%; microanalyses indicated by the symbols of the elements were performed with a Perkin-Elmer 260 (Waltham, MA, USA) elemental analyzer for C, H, and N, and they were within ± 0.4% of the theoretical values.

General procedure for 2a–g. A total of 0.98 mmol of commercial starting material 1 was dissolved in an excess of SOCl₂ (3 mL), and a catalytic amount of anhydrous DMF (0.15 mL) was added. The mixture was stirred at reflux for 4–5 h. After cooling, the excess of SOCl₂ was removed in vacuo, and the residual oil was dissolved in anhydrous tetrahydrofuran (2.5 mL) and added to a mixture previously prepared consisting of 1.18 mmol of suitable amine and 1.96 mmol of triethylamine in anhydrous tetrahydrofuran (5 mL) at 0 °C. The mixture was stirred for 2 h at 0 °C, and then at room temperature overnight. The mixture was filtered to remove the solid, and the filtrate was recovered. Evaporation of the solvent resulted in a residue that was purified by flash column chromatography using dichloromethane/methanol 9:1 (for 2a,2d) or 9.5:05 (for 2b,2c,2e,2f,2g) as eluents.

4-Chloro-8-methyl-N-(4-methylpiperazin-1-yl)quinoline-3-carboxamide, 2a. Yield = 44%; mp = 206–207 °C (EtOH); ¹H-NMR (400 MHz, CD₃OD-d₄) δ 2.33 (s, 3H, CH₃ piperazine), 2.60–2.70 (m, 4H, 2 × CH₂ piperazine), 2.79 (s, 3H, CH₃), 2.90–3.10 (m, 4H, 2 × CH₂ piperazine), 7.76 (t, 1H, Ar, J = 8.4 Hz), 7.76 (d, 1H, Ar, J = 7.2 Hz), 8.20 (d, 1H, Ar, J = 8.4 Hz), 8.81 (s, 1H, Ar). Anal. Calcd for C₁₆H₁₉ClN₄O: C, 60.28; H, 6.01; N, 17.57. Found C, 60.52; H, 6.03; N, 17.64.

4-Chloro-N-(2-hydroxyethyl)-8-methylquinoline-3-carboxamide, 2b. Yield = 98%; mp = 142–143 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 2.81 (s, 3H, CH₃), 3.73 (s, 2H, CH₂), 3.91 (s, 2H, CH₂), 6.78 (exch br s, 1H, NH), 7.58 (t, 1H, Ar, J = 8.4 Hz), 7.66 (d, 1H, Ar, J = 7.6 Hz), 8.15 (d, 1H, Ar, J = 7.6 Hz), 9.06 (s, 1H, Ar). Anal. Calcd for C₁₃H₁₃ClN₂O₂: C, 58.99; H, 4.95; N, 10.58. Found C, 58.75; H, 4.93; N, 10.54.

4-Chloro-8-methyl-N-propylquinoline-3-carboxamide, 2c. Yield = 97%; mp = 121–124 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.04 (t, 3H, NHCH₂CH₂CH₃, J = 6.8 Hz), 1.68–1.74 (m, 2H, NHCH₂CH₂CH₃), 2.81 (s, 3H, CH₃), 3.48–3.53 (m, 2H, NHCH₂CH₂CH₃), 6.28 (exch br s, 1H, NH), 7.58 (t, 1H, Ar, J = 7.6 Hz), 7.66 (d, 1H, Ar, J = 7.6 Hz), 8.16 (d, 1H, Ar, J = 8.0 Hz), 9.04 (s, 1H, Ar). Anal. Calcd for C₁₄H₁₅ClN₂O: C, 64.00; H, 5.75; N, 10.66. Found C, 64.25; H, 5.77; N, 10.70.

(4-Chloro-8-methylquinolin-3-yl)(4-methylpiperazin-1-yl)methanone, 2d. Yield = 98%; mp = 170–172 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 2.33 (s, 3H, CH₃ piperazine), 2.40–2.49 (m, 2H, CH₂ piperazine), 2.50–2.60 (m, 2H, CH₂ piperazine), 2.81 (s, 3H, CH₃), 3.25–3.35 (m, 2H, CH₂ piperazine), 3.85–3.95 (m, 2H, CH₂ piperazine), 7.58 (t, 1H, Ar, J = 7.2 Hz), 7.66 (d, 1H, Ar, J = 7.2 Hz), 8.13 (d, 1H, Ar, J = 8.4 Hz), 8.75 (s, 1H, Ar). Anal. Calcd for C₁₆H₁₈ClN₃O: C, 63.26; H, 5.97; N, 13.83. Found C, 63.51; H, 5.99; N, 13.88.

4-Chloro-N-heptyl-8-methylquinoline-3-carboxamide, 2e. Yield = 42%; mp = 95–97 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 0.86 (t, 3H, CH₂CH₃, J = 6.8 Hz), 1.27–1.36 (m, 8H, 4 × CH₂), 1.50–1.57 (m, 2H, CH₂), 2.74 (s, 3H, CH₃), 3.28 (t, 2H, NHCH₂, J = 6.8 Hz), 7.69 (t, 1H, Ar, J = 7.8 Hz), 7.77 (d, 1H, Ar, J = 6.8 Hz), 8.12 (d, 1H, Ar, J = 8.4 Hz), 8.67 (exch br t, 1H, NH, J = 5.4 Hz), 8.82 (s, 1H, Ar). Anal. Calcd for C₁₈H₂₃ClN₂O: C, 67.81; H, 7.27; N, 8.79. Found C, 67.54; H, 7.24; N, 8.75.

4-Chloro-8-methyl-N-undecylquinoline-3-carboxamide, 2f. Yield = 72%; mp = 101–102 °C (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 0.83 (t, 3H, -CH₂CH₃, J = 6.8 Hz), 1.20–1.40 (m, 16H, 8 × CH₂), 1.50–1.57 (m, 2H, CH₂), 2.74 (s, 3H, CH₃), 3.28 (t, 2H, -NHCH₂-, J = 6.8 Hz), 7.69 (t, 1H, Ar, J = 7.8 Hz), 7.77 (d, 1H, Ar, J = 7.2 Hz), 8.12 (d, 1H, Ar, J = 8.4 Hz), 8.67 (exch br t, 1H, NH, J = 5.4 Hz), 8.82 (s, 1H, Ar). Anal. Calcd for C₂₂H₃₁ClN₂O: C, 70.47; H, 8.33; N, 7.47. Found C, 70.18; H, 8.36; N, 7.49.

4-Chloro-N-[2-(2-hydroxyethoxy)ethyl]-8-methylquinoline-3-carboxamide, 2g. Yield = 98%; yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ 1.09 (exch br t, 3H, OH, J = 2.8 Hz), 2.64 (s, 3H, CH₃), 3.48–3.65 (m, 8H, 2 × CH₂), 7.37 (t, 1H, Ar, J = 7.2 Hz), 7.47 (d, 1H, Ar, J = 6.4 Hz), 7.67 (exch br s, 1H, NH), 7.88 (d, 1H, Ar, J = 8.4 Hz), 8.78 (s, 1H, Ar). Anal. Calcd for C₁₅H₁₇ClN₂O₃: C, 58.35; H, 5.55; N, 9.07. Found C, 58.58; H, 5.57; N, 9.10.

General procedure for 3a–g. To a solution of intermediates 2a–g (0.53 mmol) in 2 mL of anhydrous acetonitrile, 0.58 mmol of 3-methoxyaniline was added. The mixture was stirred at reflux for 3–4 h. After cooling, the precipitate was recovered by vacuum filtration, and the crude product was purified by crystallization from ethanol (for 3b,3c,3e,3f,3g) or by flash column chromatography using dichloromethane/methanol 9:1 as eluent (for 3a and 3d).

4-[(3-Methoxyphenyl)amino]-8-methyl-N-(4-methylpiperazin-1-yl)quinoline-3-carboxamide, 3a. Yield = 56%; yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ 2.32 (s, 3H, CH₃ piperazine), 2.60–2.70 (m, 4H, 2 × CH₂ piperazine), 2.74 (s, 3H, CH₃), 2.90–3.05 (m, 4H, 2 × CH₂ piperazine), 3.69 (s, 3H, OCH₃), 6.47 (s, 2H, Ar), 6.57 (d, 1H, Ar, J = 6.0 Hz), 7.08–7.15 (m, 2H, Ar), 7.48 (s, 1H, Ar), 7.64 (d, 1H, Ar, J = 7.2 Hz), 7.98 (exch br s, 1H, NH), 8.90 (s, 1H, Ar), 10.65 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, CDCl₃) δ 16.69, 46.65, 54.70, 55.84, 59.47, 107.20, 110.98, 111.80, 116.53, 123.42, 125.56, 125.70, 130.08, 131.70, 137.13, 146.01, 151.87, 160.91, 164.08. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₃H₂₈N₅O₂ 406.2238; found 406.2243. Anal. Calcd for C₂₃H₂₇N₅O₂: C, 68.13; H, 6.71; N, 17.27. Found C, 68.40; H, 6.74; N, 17.33.

N-(2-Hydroxyethyl)-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide, 3b. Yield = 59%; mp = 180–183 °C (EtOH). ¹H-NMR (400 MHz, CDCl₃) δ 2.86 (s, 3H, CH₃), 3.72 (t, 2H, NHCH₂CH₂OH, J = 4.4 Hz), 3.80 (s, 3H, OCH₃), 3.99 (t, 2H, NHCH₂CH₂OH, J = 4.4 Hz), 6.78–6.83 (m, 2H, Ar), 6.92 (d, 1H, Ar, J = 8.4 Hz), 7.15 (t, 1H, Ar, J = 8.0 Hz), 7.34 (t, 1H, Ar, J = 8.0 Hz), 7.54 (d, 1H, Ar, J = 8.4 Hz), 7.60 (d, 1H, Ar, J = 6.8 Hz), 9.74 (s, 1H, Ar), 10.08 (exch br s, 1H, NH), 12.95 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, CDCl₃) δ 18.52, 43.61, 55.54, 61.07, 107.10, 109.88, 113.81, 116.60, 117.53, 125.32, 125.46, 129.60, 130.78, 135.07, 138.13, 140.71, 143.79, 156.87, 160.91, 167.03. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₀H₂₂N₃O₃ 352.1656; found 352.1650. Anal. Calcd for C₂₀H₂₁N₃O₃: C, 68.36; H, 6.02; N, 11.96. Found C, 68.63; H, 6.04; N, 11.91.

4-[(3-Methoxyphenyl)amino]-8-methyl-N-propylquinoline-3-carboxamide, 3c. Yield = 95%; mp = 161–164 °C (EtOH). ¹H-NMR (400 MHz, CDCl₃) δ 1.02 (t, 3H, NHCH₂CH₂CH₃, J = 7.2 Hz), 1.78 (sex, 2H, NHCH₂CH₂CH₃, J = 7.2 Hz), 2.90 (s, 3H, CH₃), 3.46 (q, 2H, NHCH₂CH₂CH₃, J = 6.8 Hz), 3.80 (s, 3H, OCH₃), 6.77–6.82 (m, 2H, Ar), 6.92 (d, 1H, Ar, J = 8.4 Hz), 7.15 (t, 1H, Ar, J = 8.4 Hz), 7.34 (t, 1H, Ar, J = 8.2 Hz), 7.54 (d, 1H, Ar, J = 8.8 Hz), 7.59 (d, 1H, Ar, J = 7.2 Hz), 9.28 (s, 1H, Ar), 9.73 (exch br s, 1H, NH), 13.02 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, CDCl₃) δ 11.64, 18.87, 22.47, 42.10, 55.52, 107.14, 109.85, 113.63, 116.48, 117.57, 125.19, 125.51, 130.10, 130.75, 135.03, 138.25, 140.77, 142.69, 156.79, 160.90, 166.67. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₁H₂₄N₃O₂ 350.1863; found 350.1857. Anal. Calcd for C₂₁H₂₃N₃O₂: C, 72.18; H, 6.63; N, 12.03. Found C, 72.47; H, 6.65; N, 12.08.

{4-[(3-Methoxyphenyl)amino]-8-methylquinolin-3-yl}(4-methylpiperazin-1-yl)methanone, 3d. Yield = 89%; yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ 2.28 (s, 3H, CH₃ piperazine), 2.75–2.85 (m, 4H, 2 × CH₂ piperazine), 2.81 (s, 3H, CH₃), 3.50–3.60 (m, 4H, 2 × CH₂ piperazine), 3.74 (s, 3H, OCH₃), 6.48 (s, 2H, Ar), 6.56 (d, 1H, Ar, J = 9.2 Hz), 7.13 (t, 1H, Ar, J = 8.0 Hz), 7.55 (d, 1H, Ar, J = 6.8 Hz), 7.67 (s, 1H, Ar), 7.74 (d, 1H, Ar, J = 8.0 Hz), 8.72 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 16.50, 46.75, 54.79, 55.85, 59.57, 107.20, 110.98, 111.80, 116.53, 123.42, 125.56, 125.70, 130.08, 131.70, 137.13, 142.70, 146.01, 152.87, 161.91, 164.08. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₃H₂₇N₄O₂ 391.2129; found 391.2132. Anal. Calcd for C₂₃H₂₆N₄O₂: C, 70.75; H, 6.71; N, 14.35. Found C, 70.47; H, 6.68; N, 14.29.

N-Heptyl-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide, 3e. Yield = 62%; mp = 198–200 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 0.88 (t, 3H, NH-CH₂-(CH₂)₅CH₃, J = 6.8 Hz), 1.10–1.30 (m, 10H, NH-CH₂-(CH₂)₅CH₃), 2.75 (s, 3H, CH₃), 2.80–2.90 (m, 2H, NH-CH₂-(CH₂)₅CH₃), 3.75 (s, 3H, OCH₃), 6.80–6.90 (m, 2H, Ar), 7.28 (t, 1H, Ar, J = 8.4 Hz), 7.61 (t, 1H, Ar, J = 7.6 Hz), 7.86 (d, 1H, Ar, J = 6.4 Hz), 8.45 (d, 1H, Ar, J = 8.0 Hz), 8.63 (s, 1H, Ar), 8.77 (s, 1H, Ar), 11.34 (exch br s, 1H, NH), 14.28 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 14.42, 18.53, 22.52, 26.99, 28.73, 28.87, 31.66, 55.62, 108.94, 112.15, 112.66, 115.85, 119.68, 123.20, 127.21, 130.10, 135.22, 141.12, 143.93, 153.35, 160.17, 164.34. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₅H₃₂N₃O₂ 406.2489; found 406.2485. Anal. Calcd for C₂₅H₃₁N₃O₂: C, 74.04; H, 7.71; N, 10.36. Found C, 74.34; H, 7.74; N, 10.40.

4-[(3-Methoxyphenyl)amino]-8-methyl-N-undecylquinoline-3-carboxamide, 3f. Yield = 54%; mp = 182–184 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 0.86 (t, 3H, NH-CH₂-(CH₂)₉CH₃, J = 6.8 Hz), 1.15–1.35 (m, 18H, NH-CH₂-(CH₂)₉CH₃), 2.74 (s, 3H, CH₃), 2.81–2.86 (m, 2H, NH-CH₂-(CH₂)₉CH₃), 3.74 (s, 3H, OCH₃), 6.80–6.90 (m, 3H, Ar), 7.28 (t, 1H, Ar, J = 8.4 Hz), 7.61 (t, 1H, Ar, J = 7.6 Hz), 7.87 (d, 1H, Ar, J = 7.2 Hz), 8.39 (s, 1H, Ar), 8.63 (s, 1H, Ar), 8.71 (exch br s, 1H, NH), 11.26 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 14.41, 18.40, 22.56, 27.02, 28.73, 29.17, 29.41, 29.48, 31.77, 55.62, 108.88, 112.58, 115.77, 119.69, 123.11, 127.21, 130.34, 135.17, 160.20, 164.39. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₉H₄₀N₃O₂ 462.3115; found 462.3111. Anal. Calcd for C₂₉H₃₉N₃O₂: C, 75.45; H, 8.52; N, 9.10. Found C, 75.75; H, 8.55; N, 9.13.

N-[2-(2-Hydroxyethoxy)ethyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide, 3g. Yield = 57%; mp = 92–95 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.75 (s, 3H, CH₃), 3.01 (q, 2H, CH₂, J = 6.0 Hz), 3.30 (t, 2H, CH₂, J = 6.0 Hz), 3.41 (t, 2H, CH₂, J = 6.0 Hz), 3.51 (t, 2H, CH₂, J = 6.0 Hz), 3.75 (s, 3H, OCH₃), 6.80–6.85 (m, 3H, Ar), 7.30 (t, 1H, Ar, J = 8.4 Hz), 7.62 (t, 1H, Ar, J = 7.8 Hz), 7.87 (d, 1H, Ar, J = 6.8 Hz), 8.46 (d, 1H, Ar, J = 8.4 Hz), 8.62 (s, 1H, Ar), 8.84 (exch br t, 1H, NH, J = 5.4 Hz), 11.27 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 18.48, 55.66, 60.60, 68.64, 72.57, 108.92, 112.03, 112.77, 115.88, 119.70, 123.13, 127.27, 130.10, 130.35, 135.26, 141.08, 144.04, 153.25, 160.16, 164.59. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₂H₂₆N₃O₄ 396.1918; found 396.1914. Anal. Calcd for C₂₂H₂₅N₃O₄: C, 66.82; H, 6.37; N, 10.63. Found C, 66.55; H, 6.34; N, 10.59.

General procedure for 5a–d. To a cooled (−5 °C) and stirred solution of intermediate 4 [20] (0.28 mmol) in anhydrous tetrahydrofuran (10 mL), Et₃N (0.99 mmol) was added. After 30 min, the mixture was allowed to warm up to 0 °C, and ethyl chloroformate (0.31 mmol) was added. After 1 h, a suitable commercially available alkyl amine (0.34 mmol) was added. The reaction was carried out at room temperature overnight. After evaporation of the solvent, ice-cold water (20–30 mL) was added, and the solid formed was recovered by vacuum filtration. The crude final compounds 5a–d were purified by column chromatography using dichloromethane/methanol 9:1 (for 5b,5c,5d) or cyclohexane/ethyl acetate 1:3 (for 5a) as eluents.

4-{[3-(Heptylcarbamoyl)-5-methoxyphenyl]amino}-8-methylquinoline-3-carboxamide, 5a. Yield = 24%; mp = 178–181 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 0.86 (t, 3H, NH(CH₂)₆CH₃, J = 6.4 Hz), 1.20–1.30 (m, 8H, 4 × CH₂), 1.45–1.50 (m, 2H, CH₂), 2.72 (s, 3H, CH₃), 3.18 (q, 2H, NHCH₂(CH₂)₅CH₃, J = 6.4 Hz), 3.72 (s, 3H, OCH₃), 6.62 (s, 1H, Ar), 6.98 (s, 1H, Ar), 7.04 (s, 1H, Ar), 7.28 (t, 1H, Ar, J = 7.6 Hz), 7.59 (d, 1H, Ar, J = 6.8 Hz), 7.67 (d, 1H, Ar, J = 8.4 Hz), 7.73 (exch br s, 1H, NH), 8.29 (exch br s, 2H, NH₂), 9.05 (s, 1H, Ar), 10.40 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 14.40, 18.58, 22.52, 26.88, 28.89, 29.49, 31.69, 55.78, 107.18, 108.13, 111.54, 114.53, 121.21, 123.67, 125.32, 131.18, 137.10, 137.24, 145.40, 148.69, 149.00, 160.27, 165.95, 170.07. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₆H₃₃N₄O₃ 449.2547; found 449.2549. Anal. Calcd for C₂₆H₃₂N₄O₃: C, 69.62; H, 7.19; N, 12.49. Found C, 69.34; H, 7.16; N, 12.44.

4-{[3-Methoxy-5-(undecylcarbamoyl)phenyl]amino}-8-methylquinoline-3-carboxamide, 5b. Yield = 14%; mp = 177–179 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 0.85 (t, 3H, NH(CH₂)₁₀CH₃, J = 6.8 Hz), 1.20–1.30 (m, 16H, 8 × CH₂), 1.43–1.49 (m, 2H, CH₂), 2.72 (s, 3H, CH₃), 3.18 (q, 2H, NHCH₂(CH₂)₉CH₃, J = 6.8 Hz), 3.71 (s, 3H, OCH₃), 6.61 (s, 1H, Ar), 6.97 (s, 1H, Ar), 7.03 (s, 1H, Ar), 7.27 (t, 1H, Ar, J = 7.6 Hz), 7.59 (d, 1H, Ar, J = 7.2 Hz), 7.65 (d, 1H, Ar, J = 8.4 Hz), 7.72 (exch br s, 1H, NH), 8.28 (exch br s, 2H, NH₂), 9.05 (s, 1H, Ar), 10.36 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 14.40, 18.58, 22.55, 26.91, 29.17, 29.22, 29.47, 31.75, 55.76, 107.09, 108.05, 111.47, 114.58, 121.24, 123.67, 125.27, 131.08, 137.09, 137.39, 145.47, 148.57, 148.87, 149.11, 160.26, 165.95, 170.13. ESI-HRMS (m/z) [M+H]⁺: calculated for C₃₀H₄₁N₄O₃ 505.3173; found 505.3165. Anal. Calcd for C₃₀H₄₀N₄O₃: C, 71.40; H, 7.99; N, 11.10. Found C, 71.68; H, 8.02; N, 11.14.

4-{3-[(2-(2-Hydroxyethoxy)ethyl)carbamoyl]-5-(methoxyphenyl)amino}-8-methylquinoline-3-carboxamide, 5c. Yield = 16%; mp = 155–157 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.72 (s, 3H, CH₃), 3.30–3.40 (m, 4H, 2 × CH₂), 3.45–3.50 (m, 4H, 2 × CH₂), 3.72 (s, 3H, OCH₃), 4.58 (exch br s, 1H, OH), 6.65 (s, 1H, Ar), 7.00 (s, 1H, Ar), 7.05 (s, 1H, Ar), 7.31 (t, 1H, Ar, J = 7.6 Hz), 7.61 (d, 1H, Ar, J = 6.8 Hz), 7.73 (d, 1H, Ar, J = 8.4 Hz), 7.74 (exch br s, 1H, NH), 8.26 (exch br s, 1H, NH), 8.39 (exch br t, 1H, CONHCH₂, J = 5.4 Hz), 9.01 (s, 1H, Ar), 10.32 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 16.90, 40.65, 55.85, 61.03, 70.10, 107.20, 108.54, 111.70, 114.65, 121.86, 123.65, 125.40, 131.15, 137.01, 137.36, 145.50, 148.71, 149.15, 160.30, 166.25, 170.15. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₃H₂₇N₄O₅ 439.1976; found 439.1980. Anal. Calcd for C₂₃H₂₆N₄O₅: C, 63.00; H, 5.98; N, 12.78. Found C, 63.25; H, 6.00; N, 12.83.

4-{3-[(2-Hydroxyethyl)carbamoyl-5-methoxyphenyl]amino}-8-methylquinoline-3-carboxamide, 5d. Yield = 54%; mp = 233–234 °C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.72 (s, 3H, CH₃), 3.25–3.32 (m, 2H, CH₂), 3.45–3.50 (m, 2H, CH₂), 3.72 (s, 3H, OCH₃), 4.68 (exch br s, 1H, OH), 6.61 (s, 1H, Ar), 7.01 (s, 1H, Ar), 7.06 (s, 1H, Ar), 7.29 (t, 1H, Ar, J = 7.6 Hz), 7.60 (d, 1H, Ar, J = 6.8 Hz), 7.66 (d, 1H, Ar, J = 8.4 Hz), 7.72 (exch br s, 1H, NH), 8.25 (exch br s, 1H, NH), 8.32 (exch br t, 1H, CONHCH₂, J = 5.4 Hz), 9.04 (s, 1H, Ar), 10.35 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 18.58, 42.64, 55.79, 60.14, 107.17, 108.24, 111.67, 114.62, 121.26, 123.63, 125.38, 131.16, 136.93, 137.31, 145.46, 148.61, 149.05, 160.26, 166.20, 170.05. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₁H₂₃N₄O₄ 395.1714; found 395.1718. Anal. Calcd for C₂₁H₂₂N₄O₄: C, 63.95; H, 5.62; N, 14.20. Found C, 63.69; H, 5.60; N, 14.14.

General procedure for 7a-n. A mixture of compound 6 [19] (0.92 mmol), SOCl₂ (2–3 mL), and a catalytic amount of Et₃N was refluxed for 2 h. After cooling, the excess of SOCl₂ was removed in vacuo, the residue was dissolved in anhydrous THF (2–3 mL), and appropriate alkyl(aryl)amine was added (1.40–2.30 mmol). The mixture was stirred for 30–40 min at room temperature. After evaporation of the solvent, ice-cold water (20–30 mL) was added, and compounds 7a, 7h, 7j, 7k, and 7m were recovered by vacuum filtration, while compounds 7b-g, 7i, 7l, and 7n were recovered by extraction with CH₂Cl₂ (3 × 15 mL). The crude products were purified by crystallization with a suitable solvent or by flash column chromatography to obtain the desired compounds.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid diethylamide, 7a. Yield = 61%; mp = 98–101 °C (Cyclohexane); ¹H-NMR (400 MHz, CDCl₃) δ 1.40 (m, 6H, (CH₃CH₂)₂N), 1.45 (t, 3H, CH₃CH₂N, J = 6.8 Hz), 2.75 (s, 3H, C3-CH₃), 3.40 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.60 (q, 2H, CH₃CH₂)₂N, J = 6.8 Hz), 4.20 (q, 2H, CH₃CH₂N, J = 7.2 Hz). Anal. Calcd for C₁₃H₁₈N₄O₃: C, 56.10; H, 6.52; N, 20.13. Found C, 56.23; H, 6.53; N, 20.19.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid benzylmethylamide, 7b. Yield = 83%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.22 (t, 3H, CH₃CH₂, J = 6.8 Hz) 1.38 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.79 (s, 3H, C3-CH₃), 2.80 (s, 3H, C3-CH₃), 3.12 (s, 3H, NCH₃), 3.15 (s, 3H, NCH₃), 4.08 (q, 2H, CH₂CH₃, J = 7.2 Hz), 4.24 (q, 2H, CH₂CH₃, J = 7.2 Hz), 4.74 (s, 2H, NCH₂Ph), 4.80 (s, 2H, NCH₂Ph), 7.23–7.40 (m, 10H, Ar). Anal. Calcd for C₁₇H₁₈N₄O₃: C, 62.57; H, 5.56; N, 17.17. Found C, 62.43; H, 5.57; N, 17.22.

6-Ethyl-3-methyl-4-(morpholine-4-carbonyl)isoxazolo [3,4-d]pyridazin-7(6H)-one, 7c. Yield = 95%; mp = 135–136 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.35 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.78 (s, 3H, C3-CH₃), 3.80 (m, 4H, morpholine), 3.84 (m, 4H, morpholine), 4.20 (q, 2H, NCH₂CH₃, J = 7.0 Hz). Anal. Calcd for C₁₃H₁₆N₄O₄: C, 53.42; H, 5.52; N, 19.17. Found C, 53.54; H, 5.52; N, 19.12.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid methylpropylamide, 7d. Yield = 66%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.85 (t, 3H, CH₃CH₂CH₂N, J = 7.2 Hz), 0.99 (t, 3H, CH₃CH₂CH₂N, J = 7.2 Hz), 1.35 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.67–1.74 (m, 4H, CH₃CH₂CH₂N), 2.75 (s, 3H, C3-CH₃), 2.76 (s, 3H, C3-CH₃), 3.13 (s, 3H, NCH₃), 3.14 (s, 3H, NCH₃), 3.41 (t, 2H, NCH₂CH₂CH₃, J = 7.2 Hz), 3.54 (t, 2H, NCH₂CH₂CH₃, J = 7.2 Hz), 4.20 (m, 4H, NCH₂CH₃). Anal. Calcd for C₁₃H₁₈N₄O₃: C, 56.10; H, 6.52; N, 20.13. Found C, 56.23; H, 6.53; N, 20.19.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid butylmethyl amide, 7e. Yield = 41%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.88 (t, 3H, CH₃(CH₂)₃N, J = 6.8 Hz), 1.03 (t, 3H, CH₃(CH₂)₃N, J = 6.8 Hz), 1.25 (q, 2H, CH₃CH₂(CH₂)₂, J = 7.6 Hz), 1.38 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.43 (q, 2H, CH₃CH₂(CH₂)₂, J = 7.6 Hz), 1.68 (m, 4H, CH₃CH₂CH₂CH₂N), 2.76 (s, 3H, C3-CH₃), 2.78 (s, 3H, C3-CH₃), 3.14 (s, 3H, NCH₃), 3.15 (s, 3H, NCH₃), 3.45 (t, 2H, NCH₂(CH₂)₂CH₃, J = 7.6 Hz), 3.58 (t, 2H, NCH₂(CH₂)₂CH₃, J = 7.6 Hz), 4.20 (m, 4H, NCH₂CH₃). Anal. Calcd for C₁₄H₂₀N₄O₃: C, 57.52; H, 6.90; N, 19.17. Found C, 57.68; H, 6.91; N, 19.12.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid methylpentylamide, 7f. Yield = 82%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.87 (t, 3H, CH₃(CH₂)₄N, J = 6.8 Hz), 0.96 (t, 3H, CH₃(CH₂)₄N, J = 6.8 Hz), 1.18–1.36 (m, 8H, CH₃(CH₂)₂CH₂CH₂), 1.38 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.70 (m, 4H, CH₃(CH₂)₂CH₂CH₂N), 2.77 (s, 3H, C3-CH₃), 2.79 (s, 3H, C3-CH₃), 3.15 (s, 3H, NCH₃), 3.16 (s, 3H, NCH₃), 3.45 (t, 2H, NCH₂(CH₂)₃CH₃, J = 7.6 Hz), 3.58 (t, 2H, NCH₂(CH₂)₃CH₃, J = 7.6 Hz), 4.24 (m, 4H, CH₃CH₂N). Anal. Calcd for C₁₅H₂₂N₄O₃: C, 58.81; H, 7.24; N, 18.29. Found C, 58.67; H, 7.26; N, 18.24.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid ethylmethylamide, 7g. Yield = 66%; mp = 82–85 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.30 (t, 6H, CH₃CH₂NCO, J = 6.8 Hz), 1.40 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 2.79 (s, 3H, C3-CH₃), 2.80 (s, 3H, C3-CH₃), 3.15 (s, 6H, NCH₃), 3.50 (q, 2H, CH₃CH₂NCO, J = 6.8 Hz), 3.65 (q, 2H, CH₃CH₂NCO, J = 7.2 Hz), 4.24 (q, 4H, CH₃CH₂N, J = 6.8 Hz). Anal. Calcd for C₁₂H₁₆N₄O₃: C, 54.54; H, 6.10; N, 21.20. Found C, 54.43; H, 6.11; N, 21.15.

6-Ethyl-3-methyl-4-(4-phenylpiperazine-1-carbonyl)isoxazolo [3,4-d]pyridazin-7(6H)-one, 7h. Yield = 86%; mp = 166–168 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.39 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.83 (s, 3H, C3-CH₃), 3.31–3.37 (m, 4H, piperazine), 3.98 (m, 2H, piperazine), 4.06 (m, 2H, piperazine), 4.24 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.95–7.06 (m, 3H, Ar), 7.37 (m, 2H, Ar). Anal. Calcd for C₁₉H₂₁N₅O₃: C, 62.11; H, 5.76; N, 19.06. Found C, 62.29; H, 5.77; N, 19.02.

4-(4-Benzylpiperazine-1-carbonyl)-6-ethyl-3-methylisoxazolo [3,4-d]pyridazin-7(6H)-one, 7i. Yield = 77%; mp = 118–120 °C (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.25 (t, 3H, CH₃CH₂, J = 7.2 Hz ), 2.36 (m, 2H, piperazine), 2.54 (m, 2H, piperazine), 2.70 (s, 3H, C3-CH₃), 3.52 (s, 2H, NCH₂Ph), 3.57 (m, 2H, piperazine), 3.70 (m, 2H, piperazine), 4.06 (q, 2H, CH₃CH₂, J = 6.8 Hz), 7.26–7.34 (m, 5H, Ar). Anal. Calcd for C₂₀H₂₃N₅O₃: C, 62.98; H, 6.08; N, 18.36. Found C, 63.20; H, 6.09; N, 18.41.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid (3-methoxyphenyl)amide, 7j. Yield = 88%; mp = 178–180 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.48 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.19 (s, 3H, C3-CH₃), 3.88 (s, 3H, OCH₃), 4.34 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.77 (m, 1H, Ar), 7.18 (m, 1H, Ar), 7.33 (t, 1H, Ar, = 8.0 Hz), 7.39 (s, 1H, Ar), 8.88 (exch br s, 1H, NH). Anal. Calcd for C₁₆H₁₆N₄O₄: C, 58.53; H, 4.91; N, 17.06. Found C, 58.69; H, 4.90; N, 17.01.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]amide, 7k. Yield = 78%; mp = 142–144 °C (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.32 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.79 (t, 2H, NHCH₂CH₂, J = 6.8 Hz ), 2.95 (s, 3H, C3-CH₃), 3.48 (m, 2H, NHCH₂CH₂), 3.71 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 4.12 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.75–6.88 (m, 3H, Ar), 8.62 (exch br s, 1H, NH). Anal. Calcd for C₁₉H₂₂N₄O₅: C, 59.06; H, 5.74; N, 14.50. Found C, 59.22; H, 5.73; N, 14.54.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid (3-methoxybenzyl)methylamide, 7l Yield = 88%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.23 (t, 3H, CH₃CH₂, J = 6.8 Hz), 1.35 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.76 (s, 3H, C3-CH₃), 2.78 (s, 3H, C3-CH₃), 3.11 (s, 3H, NCH₃), 3.12 (s, 3H, NCH₃), 3.77 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 4.07 (q, 2H, CH₃CH₂, J = 6.8 Hz), 4.20 (q, 2H, CH₃CH₂, J = 6.8 Hz), 4.69 (s, 2H, NCH₂Ph), 4.74 (s, 2H, NCH₂Ph), 6.77–6.90 (m, 6H, Ar), 7.22–7.28 (m, 2H, Ar). Anal. Calcd for C₁₈H₂₀N₄O₄: C, 60.66; H, 5.66; N, 15.72. Found C, 60.82; H, 5.65; N, 15.78.

3-[(6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carbonyl)amino]benzoic acid ethyl ester, 7m. Yield = 84%; mp = 156–157 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.47 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 1.50 (t, 3H, CH₃CH₂O, J = 7.2 Hz), 3.20 (s, 3H, C3-CH₃), 4.36 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 4.44 (q, 2H, CH₃CH₂O, J = 7.2 Hz), 7.52 (t, 1H, Ar, J = 8 Hz), 7.89 (d, 1H, Ar, J = 7.6 Hz), 8.13 (d, 2H, Ar, J = 8 Hz), 8.15 (s, 1H, Ar), 9.02 (exch br s, 1H, NH). Anal. Calcd for C₁₈H₁₈N₄O₅: C, 58.37; H, 4.90; N, 15.13. Found C, 58.21; H, 4.90; N, 15.17.

6-Ethyl-3-methyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid benzylamide, 7n. Yield = 75%; mp = 140–142 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.46 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.80 (s, 3H, C3-CH₃), 4.28 (q, 2H, CH₃CH₂, J = 6.8 Hz), 4.63 (m, 2H, NHCH₂Ph), 7.36–7.52 (m, 5H, Ar), 8.87 (exch br s, 1H, NH). Anal. Calcd for C₁₆H₁₆N₄O₃: C, 61.53; H, 5.16; N, 17.94. Found C, 61.41; H, 5.15; N, 17.97.

General Procedure for 8a, 8c–o. A suspension of 7a–n (0.72 mmol) in N,N-dimethylformammide dimethyl acetal (7.53–15.06 mmol) was stirred at 100 °C for 30–60 min. The mixture was cooled, and the compounds 8a, 8h–i, 8k–l and 8n–o were recovered by vacuum filtration. For compounds 8c–g, 8j, and 8m, after cooling, the mixture was diluted with cold water (10 mL), and the suspension was extracted with CH₂Cl₂ (3 × 15 mL). Evaporation of the solvent afforded the desired compounds. Compound 8j was purified by flash column chromatography using CH₂Cl₂/CH₃OH 9.5:0.5 as eluent.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid diethylamide, 8a. Yield = 75%; mp = 178–180 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.25 (t, 3H, (CH₃CH₂)₂N, J = 6.8 Hz), 1.32 (t, 3H, (CH₃CH₂)₂N, J = 7.2 Hz), 1.37 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 3.00 (s, 6H, N(CH₃)₂), 3.35 (q, 2H, (CH₃CH₂)₂N, J = 6.8 Hz), 3.60 (q, 2H, (CH₃CH₂)₂N, J = 6.8 Hz), 4.20 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 5.25 (m, 1H, CH=CHN), 7.58 (m, 1H, CH=CHN). Anal. Calcd for C₁₆H₂₃N₅O₃: C, 57.64; H, 6.95; N, 21.01. Found C, 57.96; H, 4.94; N, 21.07.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid benzylmethylamide, 8c. Yield = 94%; mp = 133–135 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.24 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.37 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.06 (s, 12H, N(CH₃)₂, 3.08 (s, 6H, NCH₃), 4.08 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.23 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.63 (s, 2H, NCH₂Ph), 4.79 (s, 2H, NCH₂Ph), 5.38 (m, 2H, CH=CHN), 7.29–7.40 (m, 10H, Ar), 7.59 (m, 2H, CH=CHN). Anal. Calcd for C₂₀H₂₃N₅O₃: C, 62.98; H, 6.08; N, 18.36. Found C, 62.83; H, 6.07; N, 18.31.

3-(2-Dimethylaminovinyl)-6-ethyl-4-(morpholine-4-carbonyl)isoxazolo [3,4-d]pyridazin-7(6H)-one, 8d. Yield = 84%; mp = 168–169 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.34 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.10 (s, 6H, N(CH₃)₂), 3.60 (m, 2H, morpholine), 3.70 (m, 2H, morpholine), 3.82 (m, 4H, morpholine), 4.17 (q, 2H, CH₃CH₂, J = 7.2 Hz), 5.30 (d, 1H, CH=CHN, J = 13.2 Hz), 7.58 (d, 1H, CH=CHN, J = 12.8 Hz). Anal. Calcd for C₁₆H₂₁N₅O₄: C, 55.32; H, 6.09; N, 20.16. Found C, 55.51; H, 6.08; N, 20.19.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid methylpropylamide, 8e. Yield = 92%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.80 (t, 3H, CH₃(CH₂)₂N, J = 6.8 Hz), 1.00 (t, 3H, CH₃(CH₂)₂N, J = 6.8 Hz), 1.35 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.60–1.75 (m, 4H, CH₃CH₂CH₂N), 3.05 (s, 6H, NCH₃), 3.10 (s, 12H, N(CH₃)₂), 3.28 (t, 2H, NCH₂CH₂CH₃, J = 7.2 Hz), 3.51 (t, 2H, NCH₂CH₂CH₃, J = 7.2 Hz), 4.15 (m, 4H, CH₃CH₂N), 5.21 (d, 1H, CH=CHN, J = 12.8 Hz), 5.27 (d, 1H, CH=CHN, J = 12.8 Hz), 7.55 (d, 2H, CH=CHN). Anal. Calcd for C₁₆H₂₃N₅O₃: C, 57.64; H, 6.95; N, 21.01. Found C, 57.50; H, 6.96; N, 21.06.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid butylmethylamide, 8f. Yield = 91%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.85 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 0.98 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 1.18–1.32 (m, 2H, CH₃CH₂(CH₂)₂N), 1.35 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.36–1.45 (m, 2H, CH₃CH₂(CH₂)₂N), 1.59–1.71 (m, 4H, CH₃CH₂CH₂CH₂N), 3.00 (s, 6H, NCH₃), 3.15 (s, 12H, N(CH₃)₂), 3.31 (t, 2H, NCH₂(CH₂)₂CH₃), 3.57 (t, 2H, NCH₂(CH₂)₂CH₃), 4.20 (m, 4H, CH₃CH₂N), 5.22 (d, 1H, CH=CHN, J = 13.2 Hz), 5.28 (d, 1H, CH=CHN, J = 13.2 Hz), 7.55 (d, 2H, CH=CHN, J = 12.4 Hz). Anal. Calcd for C₁₇H₂₅N₅O₃: C, 58.77; H, 7.25; N, 20.16. Found C, 58.90; H, 7.23; N, 20.23.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid methylpentylamide, 8g. Yield = 96%; oil; ¹H-NMR (400 MHz, DMSO-d₆) δ 0.75 (t, 3H, CH₃(CH₂)₄N, J = 6.8 Hz), 0.89 (t, 3H, CH₃(CH₂)₄N, J = 6.8 Hz), 1.00–1.20 (m, 8H, CH₃(CH₂)₂CH₂CH₂N), 1.30 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.50–1.65 (m, 4H, CH₃(CH₂)₂CH₂CH₂N), 2.95 (s, 6H, N(CH₃)₂), 3.02 (s, 6H, N(CH₃)₂), 3.18 (s, 6H, NCH₃), 3.30 (m, 2H, CH₃(CH₂)₃CH₂N), 3.45 (m, 2H, CH₃(CH₂)₃CH₂N), 4.05 (m, 4H, CH₂CH₃), 5.05 (m, 2H, CH=CHN), 7.81 (m, 2H, CH=CHN). Anal. Calcd for C₁₈H₂₇N₅O₃: C, 59.81; H, 7.53; N, 19.38. Found C, 59.65; H, 7.54; N, 19.33.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid ethylmethylamide, 8h. Yield = 67%; mp = 115–118 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.23–1.30 (m, 6H, CH₃CH₂NCO), 1.38 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 3.05 (s, 12H, N(CH₃)₂), 3.15 (s, 6H, NCH₃), 3.41 (q, 2H, CH₃CH₂NCO, J = 6.8 Hz), 3.63 (q, 2H, CH₃CH₂NCO, J = 6.8 Hz), 4.22 (m, 4H, CH₃CH₂N), 5.30 (m, 2H, CH=CHN), 7.57 (m, 2H, CH=CHN). Anal. Calcd for C₁₅H₂₁N₅O₃: C, 56.41; H, 6.63; N, 21.93. Found C, 56.56; H, 6.62; N, 21.97.

3-(2-Dimethylaminovinyl)-6-ethyl-4-(4-phenylpiperazine-1-carbonyl)-6H-isoxazolo [3,4-d]pyridazin-7-one, 8i. Yield = 77%; mp = 189–191 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.37 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.90 (s, 3H, (CH₃)₂N), 2.97 (s, 3H, (CH₃)₂N), 3.08–3.33 (m, 4H, piparazine), 3.78 (m, 2H, piperazine), 4.05 (m, 2H, piperazine), 4.20 (q, 2H, CH₃CH₂, J = 6.8 Hz), 5.31 (d, 1H, CH=CHN, J = 13.2), 7.00 (m, 3H, Ar), 7.43 (m, 2H, Ar), 7.60 (d, 1H, CH=CHN, J = 13.2 Hz). Anal. Calcd for C₂₂H₂₆N₆O₃: C, 62.54; H, 6.20; N, 19.89. Found C, 62.70; H, 6.21; N, 19.83.

4-(4-Benzylpiperazine-1-carbonyl)-3-(2-dimethylaminovinyl)ethyl-6H-isoxazolo [3,4-d]pyridazin-7(6H)-one, 8j. Yield = 49%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.34 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.47–2.57 (m, 4H, piparazine), 2.90–3.10 (m, 6H, (CH₃)₂N), 3.60 (m, 4H, piperazine), 3.85 (s, 2H, CH₂Ph), 4.18 (q, 2H, CH₃CH₂, J = 7.2 Hz), 5.28 (d, 1H, CH=CHN, J = 12.8 Hz), 7.45 (m, 5H, Ar), 7.60 (d, 1H, CH=CHN, J = 13.2 Hz). Anal. Calcd for C₂₃H₂₈N₆O₃: C, 63.29; H, 6.47; N, 19.25. Found C, 63.08; H, 6.46; N, 19.30.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid (3-methoxyphenyl)amide, 8k. Yield = 66%; mp = 161–163 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.45 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.12 (s, 6H, (CH₃)₂N), 3.87 (s, 3H, OCH₃), 4.28 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.75 (d, 1H, CH=CHN, J = 16 Hz), 7.19 (m, 1H, Ar), 7.33 (m, 2H, Ar), 7.42 (s, 1H, Ar), 7.60 (m, 1H, CH=CHN), 9.05 (exch br s, 1H, NH). Anal. Calcd for C₁₉H₂₁N₅O₄: C, 59.52; H, 5.52; N, 18.27. Found C, 59.68; H, 5.50; N, 18.31.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]amide, 8l. Yield = 69%; mp = 165–167 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.33 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.89 (t, 2H, NHCH₂CH₂, J = 7.2 Hz), 3.10 (s, 6H, (CH₃)₂N), 3.67 (m, 2H, NHCH₂CH₂), 3.87 (s, 6H, 2 OCH₃), 4.18 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.78–6.85 (m, 3H: 1H, CH=CHN; 2H, Ar), 7.28 (s, 1H, Ar), 7.63 (m, 1H, CH=CHN), 9.10 (exch br s, 1H, NH). Anal. Calcd for C₂₂H₂₇N₅O₅: C, 59.85; H, 6.16; N, 15.86. Found C, 59.97; H, 6.15; N, 15.80.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydro-isoxazolo [3,4-d]pyridazine-4-carboxylic acid (3-methoxybenzyl)methylamide, 8m. Yield = 72%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.26 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.39 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.90 (s, 3H, NCH₃), 2.98 (s, 3H, NCH₃), 3.06 (s, 6H, N(CH₃)₂), 3.08 (s, 6H, N(CH₃)₂), 3.81 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 4.10 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.21(q, 2H, CH₃CH₂, J = 7.2 Hz), 4.60 (s, 2H, NCH₂), 4.76 (s, 2H, NCH₂), 5.40 (m, 2H, CH=CHN), 6.81–6.91 (m, 6H, Ar), 7.24 (m, 2H, Ar), 7.58 (m, 2H, CH=CHN). Anal. Calcd for C₂₁H₂₅N₅O₄: C, 61.30; H, 6.12; N, 17.02. Found C, 61.49; H, 6.13; N, 17.07.

3-{[3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carbonyl]amino}benzoic acid ethyl ester, 8n. Yield = 80%; mp = 192–193 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.40–1.50 (m, 6H: 3H, CH₃CH₂N; 3H, CH₃CH₂O), 3.13 (s, 6H, N(CH₃)₂), 4.28 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 4.45 (q, 2H, CH₃CH₂O, J = 7.2 Hz), 6.22 (m, 1H, CH=CHN), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.67 (m, 1H, CH=CHN), 7.88 (d, 1H, Ar, J = 7.6 Hz), 8.10 (m, 1H, Ar), 8.20 (s, 1H, Ar), 9.15 (exch br s, 1H, NH). Anal. Calcd for C₂₁H₂₃N₅O₅: C, 59.29; H, 5.45; N, 16.46. Found C, 59.40; H, 5.44; N, 16.51.

3-(2-Dimethylaminovinyl)-6-ethyl-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid benzylamide, 8o.Yield = 63%; mp = 158–160 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.43 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.10 (s, 6H, N(CH₃)₂), 4.31 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.65 (m, 2H, NHCH₂Ph), 7.28–7.40 (m, 5H, Ar), 8.28 (exch br s, 1H, NH). Anal. Calcd for C₁₉H₂₁N₅O₃: C, 62.11; H, 5.76; N, 19.06. Found C, 62.28; H, 5.75; N, 19.11.

6-Ethyl-3-(2-furan-3-yl-vinyl)-7-oxo-6,7-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid diethylamide, 8b. To a suspension of 7a (0.36 mmol) and 3-furylaldehyde (0.54 mmol) in anhydrous methanol (1 mL), CH₃ONa (0.9 mmol) was added. The mixture was refluxed for 10 min. After cooling, the suspension was concentrated under vacuum, and the solid was recovered by vacuum filtration and purified by crystallization from ethanol. Yield = 46%; mp = 195–197 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.28–1.46 (m, 9H, 3H, CH₃CH₂N, 6H, (CH₃CH₂)₂N), 3.44 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.64 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 4.28 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.65 (s, 1H, furane), 7.02 (d, 1H, CH=CH, J = 16 Hz), 7.50 (m, 1H, furane), 7.61 (d, 1H, CH=CH, J = 16 Hz), 7.70 (m, 1H, furane). Anal. Calcd for C₁₈H₂₀N₄O₄: C, 60.66; H, 5.66; N, 15.72. Found C, 60.52; H, 5.65; N, 15.76.

General Procedure for 9a–o. A mixture of 8a–o (0.47 mmol) and hydrazine hydrate (7.5–16 mmol) in 2–3 mL of ethanol was stirred at room temperature for 1–3 h (compounds 9b–f). For compounds 9a and 9g–o the reaction was carried out at 60 °C for 1–8 h. After cooling, the mixture was concentrated in vacuo, and compounds 9a, 9c, 9f–h and 9l were isolated by filtration. For compounds 9b and 9d-e, after concentration, cold water was added, and the suspension was extracted with CH₂Cl₂. Evaporation of the solvent afforded the desired final compounds, which were purified by crystallization from ethanol (for 9b and 9e–f) or by flash column chromatography using CH₂Cl₂/CH₃OH 9:1 as eluent (for 9d). Finally, for compounds 9i–k and 9m–o, the solvent was removed under reduced pressure and the residue was directly purified by flash column chromatography using as eluent CH₂Cl₂/CH₃OH 9.5:0.5 (for 9i–j and 9m), cyclohexane/ethyl acetate 1:3 (for 9k) or CH₂Cl₂/CH₃OH 9:1 (for 9n–o) as eluents.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid diethylamide, 9a. Yield = 71%; mp = 200–201 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.00 (t, 3H, (CH₃CH₂)₂N, J = 7.2 Hz), 1.20 (t, 3H, (CH₃CH₂)₂N, J = 7.2 Hz), 1.42 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 3.15 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.55 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 4.25 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.60 (d, 1H, pyrazole, J = 6.8 Hz), 7.60 (d, 1H, pyrazole, J = 6.8 Hz). Anal. Calcd for C₁₄H₂₀N₆O₂: C, 55.25; H, 6.62; N, 27.61. Found C, 55.37; H, 6.61; N, 27.70.

5-Amino-1-ethyl-4-(5-furan-3-yl-2H-pyrazol-3-yl)-6-oxo-1,6-dihydropyridazine-3-carboxylic acid diethylamide, 9b. Yield = 56%; mp = 185 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.09 (t, 3H, (CH₃CH₂)₂N, J = 7.2 Hz), 1.23 (t, 3H, (CH₃CH₂)₂N, J = 7.2 Hz), 1.41 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 3.19 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.56 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 4.25 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.63 (s, 1H, pyrazole), 6.67 (s, 1H, furane), 7.49 (s, 1H, furane), 7.81 (s, 1H, furane). Anal. Calcd for C₁₈H₂₂N₆O₃: C, 58.37; H, 5.99; N, 22.69. Found C, 58.53; H, 5.98; N, 22.62.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid benzyl-methylamide, 9c. Yield = 60%; mp = 191–193 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.31 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.43 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.78 (s, 3H, NCH₃), 2.99 (s, 3H, NCH₃), 4.21 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.27 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.34 (s, 2H, CH₂Ph), 4.72 (s, 2H, CH₂Ph), 6.55 (m, 1H, pyrazole), 6.65 (m, 1H, pyrazole), 7.10 (exch br s, 4H, NH₂), 7.30–7.45 (m, 10H, Ar), 7.58 (m, 1H, pyrazole), 7.69 (m, 1H, pyrazole). Anal. Calcd for C₁₈H₂₀N₆O₂: C, 61.35; H, 5.72; N, 23.85. Found C, 61.15; H, 5.71; N, 23.91.

4-Amino-2-ethyl-6-(morpholine-4-carbonyl)-5-(2H-pyrazol-3-yl)pyridazin-3(2H)-one, 9d. Yield = 92%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.41 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.28 (m, 2H, morpholine), 3.44 (m, 2H, morpholine), 3.71 (m, 2H, morpholine), 3.77 (m, 2H, morpholine), 4.26 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.63 (m, 1H, pyrazole), 7.69 (m, 1H, pyrazole). Anal. Calcd for C₁₄H₁₈N₆O₃: C, 52.82; H, 5.70; N, 26.40. Found C, 52.70; H, 5.70; N, 26.47.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid methyl-propylamide, 9e. Yield = 47%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 0.76 (t, 1.5H, CH₃(CH₂)₂N), 0.94 (t, 3H, CH₃(CH₂)₂N), 1.30–1.68 (m, 5H, 3H CH₃CH₂N and 2H CH₃CH₂CH₂N), 2.80 (s, 3H, NCH₃), 3.08 (s, 1.5H, NCH₃), 2.92 (m, 1H, NCH₂CH₂CH₃), 3.50 (m, 1H, NCH₂CH₂CH₃), 4.26 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.58 (m, 1H, pyrazole), 7.64 (m, 1H, pyrazole). Anal. Calcd for C₁₄H₂₀N₆O₂: C, 55.25; H, 6.62; N, 27.61. Found C, 55.10; H, 6.62; N, 27.69.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydro-pyridazine-3-carboxylic acid butyl-methylamide, 9f. Yield = 73%; mp = 197–198 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 0.83 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 0.97 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 1.38 (m, 4H, CH₃CH₂(CH₂)₂), 1.42 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.60 (m, 4H, CH₃CH₂CH₂CH₂N), 2.80 (s, 3H, NCH₃), 3.10 (s, 3H, NCH₃), 3.22 (t, 2H, NCH₂(CH₂)₂CH₃, J = 6.8 Hz), 3.50 (t, 2H, NCH₂(CH₂)₂CH₃, J = 6.8 Hz), 4.26 (q, 4H, CH₃CH₂N, J = 7.2 Hz), 6.58 (m, 1H, pyrazole), 6.31 (m, 1H, pyrazole), 7.68 (m, 1H, pyrazole), 7.71 (m, 1H, pyrazole). Anal. Calcd for C₁₅H₂₂N₆O₂: C, 56.59; H, 6.96; N, 26.40. Found C, 56.74; H, 6.95; N, 26.54.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid methylpentylamide, 9g. Yield = 33%; mp = 198–199 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 0.82 (t, 3H, CH₃(CH₂)₄N, J = 7.2 Hz), 0.95 (t, 3H, CH₃(CH₂)₄N, J = 7.2 Hz), 1.10–1.38 (m, 8H, CH₃(CH₂)₂CH₂CH₂N), 1.42 (t, 6H, CH₃CH₂N, J = 6.8 Hz), 1.54 (m, 4H, CH₃(CH₂)₂CH₂CH₂N), 2.92 (s, 3H, NCH₃), 3.12 (s, 3H, NCH₃), 3.18 (t, 2H, CH₃(CH₂)₃CH₂N, J = 7.2 Hz) 3.45 (t, 2H, CH₃(CH₂)₃CH₂N, J = 7.2 Hz), 4.25 (q, 4H, CH₃CH₂N, J = 6.8 Hz), 6.60 (m, 1H, pyrazole), 6.85 (m, 1H, pyrazole), 7.70 (m, 2H, pyrazole), 8.21 (exch br s, 2H, NH). Calcd for C₁₆H₂₄N₆O₂: C, 57.81; H, 7.28; N, 25.28. Found C, 57.95; H, 7.28; N, 25.34.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid ethylmethylamide, 9h. Yield = 55%; mp = 178–180 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.06 (t, 3H, CH₃CH₂NCO, J = 7.2 Hz), 1.22 (t, 3H, CH₃CH₂NCO, J = 7.2 Hz), 1.42 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 2.81 (s, 3H, NCH₃), 3.10 (s, 3H, NCH₃), 3.21 (q, 2H, CH₃CH₂NCO, J = 7.2 Hz), 3.60 (q, 2H, CH₃CH₂NCO, J = 7.2 Hz), 4.27 (q, 4H, CH₃CH₂N, J = 7.2 Hz), 6.60 (m, 2H, pyrazole), 6.9–7.1 (exch br s, 4H, NH₂), 7.60 (m, 2H, pyrazole). Anal. Calcd for C₁₃H₁₈N₆O₂: C, 53.78; H, 6.25; N, 28.95. Found C, 53.65; H, 6.24; N, 29.05.

4-Amino-2-ethyl-6-(4-phenylpiperazine-1-carbonyl)-5-(2H-pyrazol-3-yl)pyridazin-3(2H)-one, 9i. Yield = 21%; mp = 224–226 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.40 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.94 (m, 2H, piparazine), 3.24 (m, 2H, piperazine), 3.45 (m, 2H, piperazine), 3.97 (m, 2H, piperazine), 4.26 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.60 (m, 1H, pyrazole), 6.94 (m, 3H, Ar), 7.29 (m, 2H, Ar), 7.57 (m, 1H, pyrazole), 11.21 (exch br s, 1H, NH). Anal. Calcd for C₂₀H₂₃N₇O₂: C, 61.05; H, 5.89; N, 24.92. Found C, 61.22; H, 5.88; N, 24.98.

4-Amino-6-(4-benzyl-piperazine-1-carbonyl)-2-ethyl-5-(2H-pyrazol-3-yl)-2H-pyridazin-3-one, 9j. Yield = 53%; mp = 118–120 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.39 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.28 (m, 2H, piparazine), 2.55 (m, 2H, piperazine), 3.31 (m, 2H, piperazine), 3.55 (s, 2H, NCH₂Ph), 3.83 (m, 2H, piperazine), 4.24 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.53 (d, 1H, pyrazole, J = 2.4 Hz), 7.28–7.35 (m, 5H, Ar), 7.53 (d, 1H, pyrazole, J = 2.4 Hz). Anal. Calcd for C₂₁H₂₅N₇O₂: C, 61.90; H, 6.18; N, 24.06. Found C, 61.73; H, 6.20; N, 24.13.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid (3-methoxyphenyl)amide, 9k. Yield = 15%; mp = 250–251 °C (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.32 (t, 3H, CH₃CH₂, J = 6.8 Hz), 3.74 (s, 3H, OCH₃), 4.13 (q, 2H, CH₃CH₂, J = 6.8 Hz), 6.31 (d, 1H, pyrazole, J = 1.6 Hz), 6.70 (m, 1H, Ar), 7.21–7.28 (m, 2H, Ar), 7.36 (s, 1H, Ar), 7.81 (d, 1H, pyrazole, J = 1.6 Hz), 10.66 (exch br s, 1H, NH), 13.21 (exch br s, 2H, NH₂). Anal. Calcd for C₁₇H₁₈N₆O₃: C, 57.62; H, 5.12; N, 23.72. Found C, 57.46; H, 5.11; N, 23.78.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]amide, 9l. Yield = 38%; mp = 177–179 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.38 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.83 (t, 2H, NHCH₂CH₂, J = 6.4 Hz), 3.59 (m, 2H, NHCH₂CH₂), 3.87 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 4.19 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.41 (m, 1H, pyrazole), 6.69–6.77 (m, 3H, Ar), 6.96 (exch br s, 1H, NH), 7.72 (m, 1H, pyrazole). Anal. Calcd for C₂₀H₂₄N₆O₄: C, 58.24; H, 5.87; N, 20.38. Found C, 58.07; H, 5.86; N, 20.32.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid (3-methoxybenzyl)methylamide, 9m. Yield = 26%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.31 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.40 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.79 (s, 6H, NCH₃), 2.99 (s, 6H, NCH₃), 3.76 (s, 6H, OCH₃), 3.81 (s, 6H, OCH₃), 4.19–4.30 (m, 4H, CH₃CH₂), 4.69 (s, 4H, NCH₂), 6.47 (m, 1H, pyrazole), 6.57 (m, 1H, pyrazole), 6.75–6.90 (m, 6H, Ar), 7.19–7.28 (m, 2H, Ar), 7.55 (m, 1H, pyrazole), 7.65 (m, 1H, pyrazole). Anal. Calcd for C₁₉H₂₂N₆O₃: C, C, 59.67; H, 5.80; N, 21.98. Found C, 59.55; H, 5.81; N, 21.92.

3-{[5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carbonyl]amino}benzoic acid ethyl ester, 9n. Yield = 16%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.33 (m, 6H: 3H CH₃CH₂N and 3H CH₃CH₂O), 4.20 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 4.43 (q, 2H, CH₃CH₂O, J = 7.2 Hz), 6.30 (m, 1H, pyrazole), 7.51 (t, 1H, Ar, J = 8.0 Hz), 7.71 (d, 1H, Ar, J = 7.2 Hz), 7.72 (m, 1H, pyrazole), 7.90 (d, 1H, Ar, J = 7.2 Hz), 8.34 (s, 1H, Ar), 10.90 (exch br s, 2H, NH₂), 13.22 (exch br s, 1H, NH). Anal. Calcd for C₁₉H₂₀N₆O₄: C, 57.57; H, 5.09; N, 21.20. Found C, 57.44; H, 5.09; N, 21.24.

5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carboxylic acid benzylamide, 9o. Yield = 37%; mp = 195–197 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.45 (t, 3H, CH₃CH_2, J = 7.2 Hz), 4.34 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.65 (m, 2H, NHCH₂Ph), 6.34 (m, 1H, pyrazole), 7.41–7.60 (m, 5H, Ar), 7.72 (m, 1H, pyrazole), 8.86 (exch br s, 1H, NH), 10.87 (exch br s, 2H, NH₂), 13.02 (exch br s, 1H, NH). Anal. Calcd for C₁₇H₁₈N₆O₂: C, 60.34; H, 5.36; N, 24.84. Found C, 60.21; H, 5.35; N, 24.91.

3-{[5-Amino-1-ethyl-6-oxo-4-(2H-pyrazol-3-yl)-1,6-dihydropyridazine-3-carbonyl]-amino}-benzoic acid, 9p. To a suspension of 9n (0.1 mmol) in 2 mL of ethanol, 0.5 mL of 2N NaOH was added, and the mixture was stirred at room temperature for 15 min. Then, the mixture was concentrated in vacuo, diluted with cold water (5–10 mL), and acidified with 2N HCl. Finally, the suspension was extracted with CH₂Cl₂ (3 × 10 mL), and the evaporation of the solvent afforded the desired compound 9p. Yield = 66%; oil; ¹H-NMR (400 MHz, DMSO-d₆) δ 1.33 (m, 3H, CH₃CH₂, J = 7.2 Hz), 4.15 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.32 (m, 1H, pyrazole), 7.48–7.88 (m, 6H: 3H, Ar; 1H, pyrazole; exch br, 2H, NH₂), 8.32 (m, 1H, Ar), 10.90 (exch br s, 1H, NH), 13.22 (exch br s, 1H, NH). Anal. Calcd for C₁₇H₁₆N₆O₄: C, 55.43; H, 4.38; N, 22.82. Found C, 55.56; H, 4.39; N, 22.88.

General procedure for 10a, 10c–d, 10f–i and 10p–q. A mixture of 4-amino-5-pyrazoyl derivatives 9a–g and 9o–p (0.23 mmol), acetic anhydride (10–21 mmol), and a catalytic amount of concentrated sulfuric acid was stirred at room temperature for 15–20 min. The mixture was diluted with cold water (10 mL) and neutralized with 10% KHCO₃. Compound 10p was filtered off and recrystallized from ethanol, while for compounds 10a, 10c–d, 10f–i and 10q the mixture was extracted with CH₂Cl₂ (3 × 15 mL), and the solvent was evaporated under vacuum. Final compounds were purified by flash column chromatography using CH₂Cl₂/CH₃OH as eluent in different ratios.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N,N-diethylcarboxamide), 10a. Yield = 59%; mp = 221–222 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.20 (t, 3H, CH₃CH₂N, J = 6.8 Hz), 1.45 (m, 6H, 3H (CH₃CH₂)₂N), 3.10 (s, 3H, C6-CH₃), 3.25 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.70 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 4.40 (q, 2H, CH₃CH₂N, J = 6.8 Hz), 6.99 (m, 1H, Ar), 8.20 (m, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.21, 12.84, 18.67, 36.48, 43.53, 96.92, 119.91, 133.81, 141.75, 146.39, 152.94, 157.24, 158.79, 167.75. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₆H₂₁N₆O₂ 329.1721; found 339.1723. Anal. Calcd for C₁₆H₂₀N₆O₂: C, 58.52; H, 6.14; N, 25.59. Found C, 58.68; H, 6.13; N, 25.53.

3-Ethyl-9-furyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N,N-diethylcarboxamide), 10c. Yield = 24%; mp = 238–241 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.20 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 1.44–1.50 (m, 6H, 3H (CH₃CH₂)₂N), 3.16 (s, 3H, C6-CH₃), 3.31 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.75 (q, 2H, (CH₃CH₂)₂N, J = 6.8 Hz), 4.39 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 6.91 (s, 1H, furane), 7.05 (m, 1H, furane), 7.57 (m, 1H, furane), 8.02 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.22, 12.79, 18.60, 36.53, 43.52, 101.68, 108.82, 119.90, 129.32, 131.04, 138.50, 141.27, 144.12, 146.42, 153.01, 157.28, 158.82, 167.78. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₀H₂₃N₆O₃ 395.1826; found 395.1832. Anal. Calcd for C₂₀H₂₂N₆O₃: 60.90; H, 5.62; N, 21.31. Found C, 61.07; H, 5.61; N, 21.38.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-benzyl-N-methylcarboxamide), 10d. Yield = 96%; mp = 153–156 °C dec. (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.22 (t, 3H, CH₃CH₂, J = 6.8 Hz), 1.45 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.89 (s, 3H, NCH₃), 3.02 (s, 6H, C6-CH₃), 3.10 (s, 3H, NCH₃), 4.15 (q, 2H, CH₃CH₂, J = 6.8 Hz), 4.23 (q, 2H, CH₃CH₂, J = 6.8 Hz), 4.49 (s, 2H, CH₂Ph), 4.82 (s, 2H, CH₂Ph), 6.74 (m, 1H, Ar), 6.89 (m, 1H, Ar), 7.22–7.46 (m, 10H, Ar), 8.36 (m, 1H, Ar), 8.45 (m, 1H, Ar). ¹³C-NMR (100 MHz, DMSO-d₆) δ 12.21, 18.63, 35.61, 36.53, 54.53, 96.64, 119.91, 127.05, 127.91, 128.53, 134.01, 136.42, 142.01, 146.43. 153.03, 157.23, 159.42, 167.72. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₀H₂₁N₆O₂ 377.1721; found 377.1715. Anal. Calcd for C₂₀H₂₀N₆O₂: C, 63.82; H, 5.36; N, 22.33. Found C, 63.65; H, 5.35; N, 22.39.

3-Ethyl-6-methyl-1-(morpholin-4-ylcarbonyl)pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one, 10f. Yield = 73%; mp = 278–280 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.46 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.18 (s, 3H, C6-CH₃), 3.48 (m, 2H, morpholine), 3.67 (m, 2H, morpholine), 3.90 (m, 2H, morpholine), 3.99 (m, 2H, morpholine), 4.39 (q, 2H, CH₃CH₂, J = 7.2 Hz), 7.03 (d, 1H, Ar, J = 2.0 Hz), 8.26 (d, 1H, Ar, J = 2.0 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ 12.21, 18.57, 36.51, 47.96, 66.21, 96.71, 119.92, 133.81, 141.76, 146.43, 153.01, 157.18, 158.81, 167.67. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₆H₁₉N₆O₃ 343.1513; found 343.1516. Anal. Calcd for C₁₆H₁₈N₆O₃: C, 56.13; H, 5.30; N, 24.55. Found C, 56.29; H, 5.31; N, 24.50.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-ethyl-N-methylcarboxamide), 10g. Yield = 39%; mp = 182–185 °C (Cyclohexane); ¹H-NMR (400 MHz, CDCl₃) δ 0.78 (t, 3H, CH₃(CH₂)₂N, J = 7.2 Hz), 1.08 (t, 3H, CH₃(CH₂)₂N, J = 7.2 Hz), 1.44 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.60 (m, 2H, CH₃CH₂CH₂N), 1.81 (m, 2H, CH₃CH₂CH₂N), 2.97 (s, 3H, C6-CH₃), 3.15 (s, 3H, C6-CH₃), 3.22 (t, 2H, NCH₂CH₂CH₃, J = 7.6 Hz), 3.25 (s, 6H, NCH₃), 3.65 (t, 2H, NCH₂CH₂CH₃, J = 7.6 Hz), 4.34–4.40 (q, 4H, CH₂CH₃, J = 7.2 Hz), 6.92 (d, 1H, Ar, J = 2.0 Hz), 7.01 (d, 1H, Ar, J = 2.0 Hz), 8.21 (d, 2H, Ar, J = 2 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ 11.54. 12.20, 18.62, 20.61, 36.06, 36.59, 54.06, 96.61, 120.01, 133.85, 141.72, 146.43, 153.02, 157.21, 159.10, 167.72. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₆H₂₁N₆O₂ 329.1721; found 339.1726. Anal. Calcd for C₁₆H₂₀N₆O₂: C, 58.52; H, 6.14; N, 25.59. Found C, 58.67; H, 6.13; N, 25.52.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-butyl-N-methylcarboxamide), 10h. Yield = 58%; mp = 199–200 °C (Cyclohexane); ¹H-NMR (400 MHz, CDCl₃) δ 0.81 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 1.06 (t, 3H, CH₃(CH₂)₃N, J = 7.2 Hz), 1.21 (m, 2H, CH₃CH₂CH₂CH₂N) 1.44 (t, 6H, CH₃CH₂N, J = 7.6 Hz), 1.55–1.80 (m, 8H, CH₃(CH₂)₂CH₂N), 2.98 (s, 3H, C6-CH₃), 3.12 (s, 3H, C6-CH₃), 3.23 (t, 2H, NCH₂(CH₂)₂CH₃, J = 7.2 Hz), 3.28 (s, 6H, NCH₃), 3.70 (t, 2H, NCH₂(CH₂)₂CH₃, J = 7.2 Hz), 4.40 (q, 4H, CH₃CH₂N, J = 7.6 Hz), 6.93 (d, 1H, Ar, J = 2.0 Hz), 7.01 (d, 1H, Ar, J = 2.0 Hz), 8.25 (d, 2H, Ar, J = 2.0 Hz). ¹³C-NMR (100 MHz, CDCl₃) δ 12.23, 13.80, 18.67, 20.03, 30.01, 36.05, 36.57, 51.54, 96.68, 120.02, 133.81, 141.74, 146.43, 153.00, 157.21, 159.18, 167.71. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₇H₂₃N₆O₂ 343.1877; found 343.1878. Anal. Calcd for C₁₇H₂₂N₆O₂: C, 59.63; H, 6.48; N, 24.54. Found C, 59.79; H, 6.47; N, 24.62.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-methy-N-pentylcarboxamide), 10i. Yield = 56%; mp = 177–178 °C (Cyclohexane); ¹H-NMR (400 MHz, CDCl₃) δ 0.78 (t, 3H, CH₃(CH₂)₄N, J = 7.2 Hz), 1.18 (t, 3H, CH₃(CH₂)₄N, J = 7.2 Hz), 1.10–1.27 (m, 8H, CH₃(CH₂)₂CH₂CH₂N), 1.46 (t, 6H, CH₃CH₂N, J = 7.2 Hz), 1.80 (m, 4H, CH₃(CH₂)₂CH₂CH₂N), 2.98 (s, 3H, C6-CH₃), 3.20 (s, 3H, C6-CH₃), 3.26 (t, 2H, CH₃(CH₂)₃CH₂N, J = 7.6 Hz), 3.30 (s, 6H, NCH₃), 3.68 (t, 2H, CH₃(CH₂)₃CH₂N, J = 7.6 Hz), 4.40 (q, 4H, CH₃CH₂N, J = 7.2 Hz), 6.94 (d, 1H, Ar, J = 2.0 Hz), 7.05 (d, 1H, Ar, J = 2.0 Hz), 8.22 (m, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.23, 14.10, 18.71, 22.15, 22.49, 28.72, 36.07, 36.89, 51.83, 96.64, 119.89, 188.82, 141.72, 146.43, 153.02, 157.21, 158.98, 167.82. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₈H₂₅N₆O₂ 357.2034; found 357.2023. Anal. Calcd for C₁₈H₂₄N₆O₂: C, 60.66; H, 6.79; N, 23.58. Found C, 60.49; H, 6.78; N, 23.52.

3-{[(3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-1-yl) carbonyl]amino}benzoic acid, 10p. Yield = 50%; mp = 290–295 °C dec. (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.36 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.00 (s, 3H, C6-CH₃), 4.26 (q, 2H, CH₃CH₂, J = 7.2 Hz), 7.03 (m, 1H, Ar), 7.56 (m, 1H, Ar), 7.77 (m, 1H, Ar), 8.03 (m, 1H, Ar), 8.42 (m, 1H, Ar), 11.10 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 12.19, 18.62, 36.49, 96.70, 119.89, 121.08, 126.01, 127.02, 128.31, 129.04, 133.79, 135.81, 141.68, 146.38, 153.02, 157.18, 160.88, 166.29, 167.72. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₉H₁₇N₆O₄ 393.1306; found 393.1310. Anal. Calcd for C₁₉H₁₆N₆O₄: C, 58.16; H, 4.11; N, 21.42. Found C, 58.30; H, 4.10; N, 21.48.

3-Ethyl-6-methyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-benzyl)carboxamide, 10q. Yield = 38%; mp = 212–214 °C (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.37 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.00 (s, 3H, C6-CH₃), 4.24 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.57 (exch br d, 2H, NHCH₂Ph, J = 6.0 Hz), 6.97 (d, 1H, Ar, J = 2.0 Hz), 7.29–7.42 (m, 5H, Ar), 8.36 (d, 1H, Ar, J = 2.0 Hz), 9.43 (exch br t, 1H, NH, J = 6.0 Hz). ¹³C-NMR (100 MHz, DMSO-d₆) δ 12.23, 18.61, 36.49, 43.58, 96.58, 120.03, 126.72, 127.02, 128.54, 133.83, 138.05, 141.67, 146.42, 152.89, 157.18, 164.02, 167.69. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₉H₁₉N₆O₂ 363.1564; found 363.1566. Anal. Calcd for C₁₉H₁₈N₆O₂: C, 62.97; H, 5.01; N, 23.19. Found C, 62.83; H, 5.02; N, 23.13.

General procedure for 10b, 10e, and 10j-o. A mixture of compounds 9a, 9c, 9h-m (0.24 mmol), triethylorthoformate (12–20 mmol), and a catalytic amount of concentrated sulfuric acid in anhydrous DMF (0.5–1 mL) was stirred at room temperature for 10–30 min. Cold water was added, and the mixture was neutralized with NaHCO₃ and extracted with CH₂Cl₂. Evaporation of the solvent afforded the desired final compounds, which were purified by crystallization with ethanol.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N,N-diethylcarboxamide), 10b. Yield = 48%; mp = 213–15 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.20 (t, 3H, CH₃CH₂N, J = 7.2 Hz), 1.41–1.49 (m, 6H, 3H (CH₃CH₂)₂N), 3.33 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 3.72 (q, 2H, (CH₃CH₂)₂N, J = 7.2 Hz), 4.40 (q, 2H, CH₃CH₂N, J = 7.2 Hz), 7.00 (d, 1H, Ar, J = 1.5 Hz), 8.27 (s, 1H, Ar, J = 1.5 Hz), 9.45 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.23, 12.85, 36.52, 43.58, 96.61, 122.88, 133.84, 141.74, 142.22, 146.46, 152.86, 157.05, 158.76. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₅H₁₉N₆O₂ 315.1564; found 315.1570. Calcd for C₁₅H₁₈N₆O₂: C, 57.31; H, 5.77; N, 26.74. Found C, 57.44; H, 5.76; N, 26.67.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-benzyl-N-methylcarboxamide), 10e. Yield = 98%; mp = 164–166 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.35 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.48 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.99 (s, 3H, NCH₃), 3.25 (s, 3H, NCH₃), 4.32 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.42 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.55 (s, 2H, CH₂Ph), 4.90 (s, 2H, CH₂Ph), 6.87 (m, 1H, Ar), 7.00 (m, 1H, Ar), 7.20–7.31 (m, 6H, Ar), 7.42–7.50 (m, 4H, Ar), 8.19 (d, 1H, Ar, J = 2.4), 8.27 (d, 1H, Ar, J = 2.4), 9.43 (s, 2H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 11.98, 35.61, 36.53, 54.49, 96.60, 123.01, 127.10, 127.91, 128.62, 133.82, 136.43, 141.71, 142.26, 146.43, 152.98, 156.99, 159.44. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₉H₁₉N₆O₂ 363.1564; found 363.1561. Anal. Calcd for C₁₉H₁₈N₆O₂: C, 62.97; H, 5.01; N, 23.19. Found C, 62.79; H, 5.00; N, 23.25.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-(N-ethyl-N-methylcarboxamide, 10j. Yield = 28%; mp = 213–214 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.20 (t, 3H, CH₃CH₂NCO, J = 6.8 Hz), 1.40 (t, 3H, CH₃CH₂NCO, J = 6.8 Hz), 1.47 (t, 6H, CH₃CH₂N, J = 6.8 Hz), 3.09 (s, 3H, NCH₃), 3.27 (s, 3H, NCH₃), 3.34 (q, 2H, CH₃CH₂NCO, J = 6.8 Hz), 3.76 (q, 2H, CH₃CH₂NCO, J = 6.8 Hz), 4.39 (q, 4H, CH₃CH₂N, J = 6.8 Hz), 6.95 (d, 1H, Ar, J = 2.0 Hz), 7.01 (d, 1H, Ar, J = 2.0 Hz), 8.26 (d, 2H, Ar, J = 2.0 Hz), 9.45 (s, 2H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.21, 12.49, 35.71, 36.54, 46.03, 96.62, 123.01, 133.80, 141.72, 142.23, 146.44, 152.88, 156.91, 159.21. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₄H₁₇N₆O₂ 301.1408; found 301.1411. Anal. Calcd for C₁₄H₁₆N₆O₂: C, 55.99; H, 5.37; N, 27.98. Found C, 55.84; H, 5.36; N, 28.03.

3-Ethyl-1-[(4-phenylpiperazin-1-yl)carbonyl]pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one, 10k. Yield = 57%; mp = 244–246 °C dec. (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.48 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.20 (m, 2H, piparazine), 3.43 (m, 2H, piperazine), 3.70 (m, 2H, piperazine), 4.18 (m, 2H, piperazine), 4.41 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.90–7.10 (m, 4H, Ar), 7.30–7.40 (m, 2H, Ar), 8.30 (m, 1H, Ar), 9.47 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.15, 36.45, 49.51, 53.34, 96.62, 114.32, 122.01, 122.91, 129.62, 133.78, 141.68, 142.23, 146.38, 149.64, 153.04, 157.15, 158.82. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₁H₂₂N₇O₂ 404.1829; found 404.1821. Anal. Calcd for C₂₁H₂₁N₇O₂: C, 62.52; H, 5.25; N, 24.30. Found C, 62.73; H, 5.26; N, 24.35.

3-Ethyl-1-[(4-benzylpiperazin-1-yl)carbonyl]pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one, 10l. Yield = 18%; mp = 178–180 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.45 (t, 3H, CH₃CH₂, J = 6.8 Hz), 2.44 (m, 2H, piparazine), 2.69 (m, 2H, piperazine), 3.49–3.60 (m, 4H, 2H piperazine; 2H NCH₂Ph), 3.99 (m, 2H, piperazine), 4.38 (q, 2H, CH₃CH₂, J = 6.8 Hz), 7.00 (d, 1H, Ar, J = 2.0 Hz), 7.20–7.50 (m, 5H, Ar), 8.30 (d, 1H, Ar, J = 2.0 Hz), 9.45 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.24, 36.51, 50.03, 54.56, 64.38, 96.78, 122.91, 127.26, 128.43, 128.90, 133.78, 138.64, 141.72, 142.24, 146.38, 153.02, 157.12, 158.91. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₂H₂₄N₇O₂ 418.1986; found 418.1979. Anal. Calcd for C₂₂H₂₃N₇O₂: C, 63.30; H, 5.55; N, 23.49. Found C, 63.43; H, 5.54; N, 23.43.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1′,5’:1,6]pyrimido [4,5-d]pyridazine-1-N-(3-methoxyphenylcarboxamide), 10m. Yield = 98%; mp = 240–243 °C dec. (EtOH); ¹H-NMR (400 MHz, DMSO-d₆) δ 1.36 (t, 3H, CH₃CH₂, J = 7.2 Hz), 3.76 (s, 3H, OCH₃), 4.25 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.80 (d, 1H, Ar, J = 6.4 Hz), 6.99 (s, 1H, Ar), 7.30–7.40 (m, 2H, Ar), 8.40 (d, 1H, Ar, J = 1.6 Hz), 9.75 (s, 1H, Ar), 10.93 (exch br s, 1H, NH). ¹³C-NMR (100 MHz, DMSO-d₆) δ 12.18, 36.52, 55.82, 96.58, 110.21, 114.02, 116.38. 123.01, 129.78, 133.83, 137.03, 141.69, 142.18, 146.45, 153.02, 157.12, 161.03, 160.90. ESI-HRMS (m/z) [M+H]⁺: calculated for C₁₈H₁₇N₆O₃ 365.1357; found 365.1353. Anal. Calcd for C₁₈H₁₆N₆O₃: C, 59.34; H, 4.43; N, 23.07. Found C, 59.50; H, 4.42; N, 23.01.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1’,5’:1,6]pyrimido [4,5-d]pyridazine-1-N-[2-(3,4-dimethoxyphenyl)ethylcarboxamide], 10n. Yield = 80%; mp = 159–161 °C (EtOH); ¹H-NMR (400 MHz, CDCl₃) δ 1.44 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.96 (t, 2H, NHCH₂CH₂, J = 6.8 Hz), 3.80 (m, 2H, NHCH₂CH₂), 3.88 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.34 (q, 2H, CH₃CH₂, J = 7.2 Hz), 6.82 (s, 3H, Ar), 6.92 (exch br s, 1H, NH), 7.54 (d, 1H, Ar, J = 2.0 Hz), 8.25 (s, 1H, Ar, J = 2.0 Hz), 9.42 (s, 1H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.28, 35.41, 36.57, 40.61, 56.10, 96.61, 112.32, 112.87, 122.10, 123.01, 130.81, 133.82, 141.71, 142.23, 146.40, 147.12, 149.68, 153.02, 157.12, 163.63. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₁H₂₃N₆O₄ 423.1775; found 423.1779. Anal. Calcd for C₂₁H₂₂N₆O₄: C, 59.71; H, 5.25; N, 19.89. Found C, 59.85; H, 5.26; N, 59.67.

3-Ethyl-4-oxo-3,4-dihydropyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazine-1-[N-(3-methoxybenzyl)-N-methyl-carboxamide], 10o. Yield = 98%; oil; ¹H-NMR (400 MHz, CDCl₃) δ 1.38 (t, 3H, CH₃CH₂, J = 7.2 Hz), 1.47 (t, 3H, CH₃CH₂, J = 7.2 Hz), 2.99 (s, 3H, NCH₃), 3.25 (s, 3H, NCH₃), 3.71 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.32 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.40 (q, 2H, CH₃CH₂, J = 7.2 Hz), 4.43 (s, 2H, NCH₂Ar), 4.87 (s, 2H, NCH₂Ar), 6.73–7.10 (m, 8H, Ar), 7.15 (t, 1H, Ar, J = 8.0 Hz), 7.38 (t, 1H, Ar, J = 8.0 Hz), 8.20 (d, 1H, Ar, J = 2.0 Hz), 8.28 (d, 1H, Ar, J = 2.0 Hz), 9.44 (s, 2H, Ar). ¹³C-NMR (100 MHz, CDCl₃) δ 12.18, 35.59, 36.48, 54.78, 55.78, 96.57, 112.11, 112.89, 120.42, 123.02, 129.47, 134.02, 137.36, 141.80, 142.29, 164.38, 153.02, 157.10, 159.11, 160.89. ESI-HRMS (m/z) [M+H]⁺: calculated for C₂₀H₂₁N₆O₃ 393.1670; found 393.1663. Anal. Calcd for C₂₀H₂₀N₆O₃: C, 61.21; H, 5.14; N, 21.42. Found C, 61.33; H, 5.13; N, 21.36.

2.2. Molecular Docking and Complex Minimization

The enzyme 3GWT (resolution 1.75 Å) was chosen for our docking, and all the 3D structures of the molecules were designed by DS ViewerPro 6.0 [25].

For molecular docking, we used the AUTODOCK suite [26]. Autodock furnished 100 poses of the ligand–enzyme complex, which were grouped into clusters within 2 rmsd; the clusters at the lowest energy and covering at least 80% of all poses were considered. For the potential energy minimization of the ligand–enzyme complexes, GROMACS version 2023 [27] was used. From the minimized complexes, the lengths of hydrogen bond interactions between ligand, amino acid residues, and water of the catalytic site, with a cutoff of 3.5 Å, were evaluated. In particular, the hydrogen bond is collected if the distance between hydrogen and hydrogen acceptor (A) is lower than 3.5 Å, with included angles between 120° and 170° (ligand-D-H…A-enzyme and ligand-A…H-D-enzyme).

2.3. PDE4 Inhibition Assay

2.3.1. PDE4 Enzyme Preparation

The U937 human monocytic cell line was used as a source of PDE4 enzyme. Cells were cultured, harvested, and supernatant fraction prepared essentially as described in Torphy T. et al. [28]. PDE4 activity was determined in cell supernatants by assaying cAMP disappearance from the incubation mixtures. A volume of 50 mL of cell supernatant was incubated at 30 °C for 30 min in a final volume of 200 mL in the presence of 1.6 mM cAMP with or without the test compound (50 mL). Reactions were stopped by heat inactivation (2.5 min at 100 °C), and residual cAMP was measured using an electrochemiluminescence (ECL)-based immunoassay (Bioveris Corporation, Gaithersburg, MD, USA).

2.3.2. cAMP Hydrolysis Assay: PDE4 Inhibitory Activity Determination and IC₅₀ Calculation

PDE4 activity was calculated as nmol of cAMP disappeared/min mg prot. The percentage of inhibition of PDE4 activity was calculated, assuming cAMP disappearance in the absence of inhibitors as 100% and cAMP disappearance in heat-inactivated samples as 0%. IC_50s were determined by assaying PDE4 activity in the presence of different concentrations of test compound (concentration range: 10⁻¹² M–10⁻⁶ M) and calculated with non-linear regression (sigmoidal dose response) by GraphPad Version 4.03.

3. Results and Discussion

3.1. Chemistry

All the final compounds 3a–g, 5a–d (quinoline-based, Scheme 1 and Scheme 2), and 10a-p (pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-ones, Scheme 3 and

Table 1. R residues for compounds 7a–n.

Compd. 7	R1	Compd. 7	R1
a	--N(CH2CH3)2	h
b	--N(CH3)CH2Ph	i
c		j
d	--N(CH3)C3H7	k
e	--N(CH3)C4H9	l
f	--N(CH3)C5H11	m
g	--N(CH3)C2H5	n	--NHCH2Ph

,

Table 2. R residues for compounds 8a–o and 9a–o.

Compd. 8–9	R1	R9
a	--N(CH2CH3)2	--N(CH3)2 or H (for 9)
b	--N(CH2CH3)2	3-furyl
c	--N(CH3)CH2Ph	--N(CH3)2 or H (for 9)
d		--N(CH3)2 or H (for 9)
e	--N(CH3)C3H7	--N(CH3)2 or H (for 9)
f	--N(CH3)C4H9	--N(CH3)2 or H (for 9)
g	--N(CH3)C5H11	--N(CH3)2 or H (for 9)
h	--N(CH3)C2H5	--N(CH3)2 or H (for 9)
i		--N(CH3)2 or H (for 9)
j		--N(CH3)2 or H (for 9)
k		--N(CH3)2 or H (for 9)
l		--N(CH3)2 or H (for 9)
m		--N(CH3)2 or H (for 9)
n		--N(CH3)2 or H (for 9)
o	--NHCH2Ph	--N(CH3)2 or H (for 9)

and

Table 3. R residues for compounds 10a–q.

Compd. 10	R1	R9	R6
a	--N(CH2CH3)2	H	CH3
b	--N(CH2CH3)2	H	H
c	--N(CH2CH3)2	3-furyl	CH3
d	--N(CH3)CH2Ph	H	CH3
e	--N(CH3)CH2Ph	H	H
f		H	CH3
g	--N(CH3)C3H7	H	CH3
h	--N(CH3)C4H9	H	CH3
i	--N(CH3)C5H11	H	CH3
j	--N(CH3)C2H5	H	H
k		H	H
l		H	H
m		H	H
n		H	H
o		H	H
p		H	CH3
q	--NHCH2Ph	H	CH3

) were synthesized following an established synthetic procedure previously described by us and the chemical structures were assigned on the basis of spectral data which are in agreement with those of already published analogues [20,29,30]. In Scheme 1, the synthetic pathway affording the final compounds 3a–g is reported, which are functionalized with various amides in position 3. The treatment of commercially available intermediate 1 with thionyl chloride and the subsequent addition of the suitable amines, in the presence of triethylamine in anhydrous tetrahydrofuran, yielded derivatives 2a–g. The displacement of the chlorine at position 4 of the quinolines 2a–g, to obtain the final compounds 3a–g, was carried out with 4-methoxyaniline in solution at reflux. Scheme 2 shows the introduction of an additional amide group on the phenyl at position 4 of the 3-carboxyamidequinoline nucleus. To this end, the intermediate 4 [20] was treated with suitable alkyl amines in anhydrous tetrahydrofuran, triethylamine, and ethyl chloroformate to afford the target amides 5a–d. Scheme 3 depicts the synthetic route to obtain the final pyrazolo [1′,5′:1,6]pyrimido [4,5-d] pyridazin-4(3H)-ones of type 10. The reaction of 6-ethyl-3-methyl-7-oxo-dihydroisoxazolo [3,4-d]pyridazine-4-carboxylic acid 6 [19] with SOCl₂, Et₃N and appropriate alkyl or arylamine in anhydrous THF, furnished the 4-carboxyamides 7a-n which were transformed in the vinyl derivatives 8a–o with N,N-dimethylformammide dimethyl acetal (compounds 8a and 8c–o) or with 3-furylaldehyde and CH₃ONa in anhydrous CH₃OH (8b). The treatment of 8a–o with hydrazine hydrate in ethanol afforded the 4-amino-5-pyrazolyl derivatives (9a–o) in good yields. These latter were lastly cyclized to pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-ones as follows: acetic anhydride and a catalytic amount of concentrated H₂SO₄ was used at room temperature to obtain the 6-methylderivatives 10a, 10c,d, 10f–i, 10p,q, while the 6-unsubstituted 10b, 10c and 10j–o were obtained by treatment with triethylortoformate in anhydrous DMF in the presence of a catalytic amount of concentrated H₂SO₄ at room temperature.

3.2. Biological Evaluation

All the new compounds were evaluated for PDE4 inhibitory activity, and the biological results, expressed as inhibition (%) at 1 µM or IC_50, are reported in

Table 4. PDE4 inhibitory activity of quinoline-based compounds 3a–g and 5a–d.

Comp.	R3	R4	IC50 (µM) a or % Inhib (1 µM)
3a		H	46%
3b	---NH(CH2)2OH	H	64%
3c	--NH(CH2)2CH3	H	48%
3d		H	43%
3e	--NH(CH2)6CH3	H	11%
3f	--NH(CH2)10CH3	H	NA b
3g	--NH(CH2)2-O-(CH2)2OH	H	25%
5a	NH2	--CONH(CH2)6CH3	4.92 ± 0.71
5b	NH2	--CONH(CH2)10CH3	42%
5c	NH2	--CONH(CH2)2-O-(CH2)2OH	4.18 ± 0.98
5d	NH2	--CONH(CH2)2OH	3.54 ± 0.56
Roflumilast			0.00084

^a All compounds are tested in triplicate, and the IC₅₀ values are presented as the mean ± SD of three independent experiments. ^b NA = no inhibitory activity was found at 1 μM.

and

Table 5. PDE4 inhibitory activity of pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-ones 10a–q.

Compd. 10	R1	R6	R9	IC50 (nM) a or % Inhib. (1 µM)
a	--N(CH2CH3)2	CH3	H	62 ± 5.3
b	--N(CH2CH3)2	H	H	NA b
c	--N(CH2CH3)2	CH3	3-furyl	48%
d	--N(CH3)CH2Ph	CH3	H	26%
e	--N(CH3)CH2Ph	H	H	175.5 ± 15.8
f		CH3	H	17%
g	--N(CH3)C3H7	CH3	H	NA b
h	--N(CH3)C4H9	CH3	H	NA b
i	--N(CH3)C5H11	CH3	H	NA b
j	--N(CH3)C2H5	H	H	NA b
k		H	H	6%
l		H	H	NA b
m		H	H	NA b
n		H	H	NA b
o		H	H	NA b
p		CH3	H	5%
q	--NHCH2Ph	CH3	H	4%
Roflumilast				0.84

^a All compounds are tested in triplicate, and the IC₅₀ values are presented as the mean ± SD of three independent experiments. ^b NA = no inhibitory activity was found at 1 μM.

, together with the reference drug Roflumilast [13].

Based on the data obtained for the quinoline-based compounds (3a–g and 5a–d) (Table 4), we have carried out an evaluation of SARs to rationalise their inhibitory effects on PDE4, which appear to be influenced by the nature of the substituents at both R₃ and R₄ positions. For the compounds 3a–g (with hydrogen in position R₄, see general structure in Table 4), which feature various groups in R₃, the inhibition is overall low. 3a (R₃ = 1-amino-4-methylpiperazine) determines a 46% inhibition at 1 μM (baseline), while for the primary alcohol 3b (R₃ = NH(CH₂)₂OH), the activity increases to 64%, suggesting the introduction of a polar hydroxyethylamino group in this position enhances PDE4 binding. Derivatives 3c (R₃ = NH(CH₂)₂CH₃) and 3d (R₃ = N,N’-dimethylpiperazine) have an effect in line with 3a, confirming that the piperazine ring in this position lowers the activity in comparison to 3b, as well as that replacing hydroxyl alcohol with a hydrophobic methyl decreases the inhibitory effect. Clearly, the presence of long hydrophobic chains is detrimental in this series, as demonstrated by compounds 3e (R₃ = NH(CH₂)₆CH₃) and 3f (R₃ = NH(CH₂)₁₀CH₃), which demonstrate a sharp drop (11%) or complete lack of inhibition. A low response has been also recorded for 3g (R₃ = NH(CH₂)₂-O-(CH₂)₂OH) suggesting that increasing the length (or steric hindrance) of the substituent in R₃ (although polar and flexible chains, like the hydroxyethoxyethyl) is detrimental for the activity (i.e., only 25% inhibition), which in this series results instead maximized with a smaller, shorter and polar hydroxyethyl group (3b). Differently, for the series 5a–d (with a fixed NH₂ in position R₃, i.e., primary amides) featuring substitutions on the methoxy-aniline ring, we have recorded an overall moderate potency for 5a, 5c and 5d (R₄ = CONH(CH₂)₆CH₃, CONH(CH₂)₂-O-(CH₂)₂OH, or NH(CH₂)₂OH) with IC₅₀ values in the low micromolar range (IC₅₀ = 3.54–4.92 µM). In contrast, 5b (R₄ = CONH(CH₂)₁₀CH₃) demonstrated a low activity (42% inhibition), somewhat in line with the result obtained with the long chain-based analogues of the first series (i.e., 3e–g). In general, these data suggest that long and/or aliphatic chains reduce activity, either in R₃ or R₄, while short-chain polar terminal groups in both positions produce better inhibitory effects (see IC₅₀ for 3b and 5d). As a result, 5d demonstrates the most potent compound (IC₅₀ = 3.54 µM) within these new products, supporting the evidence that the presence of NH(CH₂)₂OH in R₄ is highly beneficial for activity on PDE4.

For the series of pyrazolo [1′,5′:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one derivatives (10a–q), our structure–activity relationships highlights that the structural variations at R₁ and R₆ also influence the inhibitory activity toward PDE4 significantly, as expressed by the IC₅₀ values (nM) or % inhibition at 1 µM (Table 5). Preliminarily, it is worth noting that in position R₉, the hydrogen is present throughout the series, except in the case of 10c (i.e., introduction of a 3-furyl group at R₉). This latter possesses a moderate inhibition (48%), substantially lower than 10a (i.e., the correspondent term, only differing for the presence of H at R₉, IC₅₀ = 62 nM), indicating that the introduction of the furyl heterocycle (and, perhaps, also other bulky, or aromatic groups) in R₉ may interfere with the binding. Therefore, the H has been kept constant in R₉ for the design of this library of tricyclic analogues, in order not to hinder optimal interactions with PDE4, thereby favoring activity. With regard to the influence recorded for chemical variations explored at R₁ (i.e., alkyl, (alkyl)aryl or piperazinyl groups) and R₆ substituents (i.e., H or CH₃) we have found that 10a (R₁ = N(CH₂CH₃)₂, R₆ = CH₃) has the most potent inhibitory effect (IC₅₀ = 62 nM) within the series. In contrast, for all the other analogs, the activity is generally reduced drastically, varying from moderate (10c, 10d) to very low (10f, 10k, 10p, 10q), or completely absent (10b, 10g–j, 10l–o). These data suggest that the introduction of residues too bulky (e.g., see alkyl chain length in 10g–l or (alkyl)aryl groups in 10m–q), or basic (piperazine in 10k,l), on the nitrogen of the amide group at R₁ is detrimental for the activity of this class of compounds. In addition to N,N-diethyl group, which clearly results the most valid choice in terms of substituent at R1 and present in the most potent compound 10a, the only substituent tolerated at R₁ is the N-methyl-N-benzylic group (see, e.g., 10e, IC₅₀ = 175.5 nM), perhaps due to favorable π–π interactions or hydrophobic effects in the PDE4 binding site, but only when R₆ = H, being the corresponding 6-methyl derivative 10d a very weak PDE4 inhibitor (26% inhibition at 1 µM). Still more remarkable is the complete inactivity of 10b, which is the 6-norderivative of 10a, the most potent product in this series (IC₅₀ = 62 nM). This result highlights that the substituent at position 6 may also play a relevant role in the binding, although the activity data of the 10d/10e pair are in contrast with the data recorded for 10a/10b pair discussed earlier, where the most potent 10a shows a methyl group at R₆, while the inactive 10b is 6-unsubstituted.

3.3. Molecular Modelling

To further rationalise the biological results with regard to the presence of a methyl group in position 6 of the scaffold, a molecular modelling study on the two pairs of compounds with a tricyclic structure (Figure 5), namely 10a/10b (IC₅₀= 62 nM and inactive, respectively) and 10d/10e (inactive and IC₅₀ = 175.5 nM, respectively) has been performed.

The 3D structures of the molecules were built with the ‘DS ViewerPro 6.0′ [25] and the lengths of the hydrogen bonds between the compounds and amino acids of the catalytic domain of human phosphodiesterase 4B2B (PDB-ID 3GWT, resolution 1.75 Å) [31] were searched and measured. For each compound, the best docking position (best binding energy) into the catalytic site was calculated with Autodock [26]. The ligand–enzyme complex structure was solvated and optimized through a complete minimization of the potential energy with GROMACS (version 2023) [27]. The lengths of the hydrogen bonds between the ligand and amino acids or with water that acts as a ‘bridge’ between ligands and residues are reported in

Table 6. Hydrogen bond interactions for compounds 10a,b,d,e.

Comp.	IC50 (nM)	Atom Ligand a	Atom/Residue b	HB length (Å) c
10a	62	O14	HE22/GLN 443	2.36
		N16	HE22/GLN 443	1.93
10b	NA	O7	HE22/GLN 443	2.02
10d	NA	-	-	-
10e	175.5	O11	HE22/GLN 443	1.98
		N20	HW2/SOL 508 *—TYR 233	2.18
		O18	HW2/SOL 528 *—ASP 392	1.91

^a atom numbered by DS ViewerPro 6.0; ^b residue atom attributed by PDB; * water molecules; ^c length of hydrogen bond (HB), Å; NA: not active

, while Figure 6, Figure 7, Figure 8 and Figure 9 show a visual representation of the pose of the molecules in the catalytic site and hydrogen bond interactions, respectively.

Starting the discussion from the pair 10a and 10b, bearing at 1-position a diethyl carboxamide group and at 6-position a methyl or a hydrogen respectively (see Figure 6), we can notice a different ‘mode of accommodation’ in the binding site (Figure 6, 10a green, 10b red) which could potentially help to explain the very different activity recorded for 10a and 10b. Specifically, compound 10a can engage a three-point hydrogen bond between the carbonyl oxygen (O14, Table 6) of the pyridazinone ring, the pyrimidine-N5 (N16, Table 6), and the hydrogen of the side chain amino group of Glu443. In contrast, compound 10b can form only one hydrogen bond interaction between the oxygen of the carbonyl (O7, Table 6) in the acetamido chain and the amino group of the side chain of the same Gln443.

In the same way, compounds 10d and 10e, showing the methyl(benzyl)acetamido group at position 1 and methyl or hydrogen at the 6-position, respectively (see Figure 8), exhibit a different orientation of the scaffold in the catalytic site (Figure 8, 10d red, 10e green).

Even in this case, the opposite accommodations are responsible for engaging hydrogen bond interactions. Compound 10e interacts with the Gln433 by a strong hydrogen bond (length 1.98 Å) through the oxygen atom (O11, Table 6) of the carbonyl group of the piridazinone ring. Moreover, the molecule is firmly anchored to a catalytic site by two other hydrogen bonds involving the carbonyl oxygen (O18, Table 6) of the acetamido chain and pyrimidine-N5 (N20, Table 6), water molecules (SOL508 and SOL528) bridging with Tyr233 and Asp392. Compound 10d shows no interactions, which could explain the lack of activity (Figure 9).

Interesting information arises from the evaluation of the whole volume occupied by the molecules (Figure 10), which adds further explanation for the difference in the inhibitory activity between the pairs 10a/10e (active terms, mesh surface) and 10b/10d (inactive terms, transparent surface).

Nonetheless, molecular modelling results do not enable us to conclusively clarify the role of the methyl at position 6, which seems to be a discriminating factor for biological activity, although in opposite fashion for the two pairs of compounds. That is, in the pair 10a/10b, the more potent analogue (10a) bears a methyl at the 6-position. In contrast, in the pair 10d/10e, it is the inactive derivative (10d) that has a methyl group in the same position.

On the other hand, the modelling studies enabled to draw other considerations which can be also relevant to explain the difference in the activity of the two pairs of compounds: (i) the inactive 10d does not possess hydrogen bond interactions within the catalytic site of the enzyme; (ii) the active compounds 10a and 10e have strong hydrogen bond interactions (HB length 1.93–2.36 Å) with the key amino acid Glu443, which could support the high potency; (iii) interestingly, compound 10e also engages two additional hydrogen bonds with two molecules of water (SOL508 and SOL528), bridging the residues Tyr233 and Asp392; (iv) the total volume (surface volume) occupied by the inactive compounds is bigger than that occupied by the active compounds.

4. Conclusions

In this paper, we have reported the development of a new series of heterocyclic small-molecule PDE4 inhibitors as further elaboration of quinoline-based analogues previously developed by us, along with a new class of derivatives obtained through structural functionalization of the pyrazolo [1’,5’:1,6]pyrimido [4,5-d]pyridazin-4(3H)-one scaffold. Both the new series were subjected to biological studies to assess their inhibitory effect on PDE4 enzymes, supported by molecular modelling experiments, in order to gain further insight into the different activities recorded in the in vitro tests. In summary, in quinoline-based series (i.e., compounds of type 3 and 5), when R3 is NH₂, the best activity is achieved with polar, short-chain substituents at R4, while the presence of hydroxyl groups suggests enhancing the binding, perhaps through the presence of hydrogen bonding. In agreement with this, short, polar, and flexible side chains (particularly hydroxyethyl or hydroxyethoxyethyl) at either R3 or R4 enhance PDE4 inhibitory activity, as opposed to long hydrophobic chains at either position, which reduce the activity and, possibly, the binding affinity.

For the tricyclic series 10a–q the best activity is achieved with the hydrogen at R9 and a benzylamide residue, as well as small and linear alkyl groups (i.e., diethylamide), at R1. Differently, bulky groups, or long and flexible alkyl chains, at R1 reduce activity. Moreover, the presence of both a methyl group and a hydrogen at R6 results in a suitable. Overall, compounds 5d (quinoline series) and 10a (tricyclic series) showed the best inhibitory activities within the two libraries (IC₅₀ = 3.54 µM and 62 nM, respectively), making them promising leads for further molecular optimization to access new and effective PDE4 inhibitors for biomedical investigations.