Synthesis of 2,4-dihydroxyacetophenone derivatives as potent PDE-1 and -3 inhibitors: in vitro and in silico insights

Abstract

Aim: Synthesis of novel bis-Schiff bases having potent inhibitory activity against phosphodiesterase (PDE-1 and -3) enzymes, potentially offering therapeutic implications for various conditions. Methods: Bis-Schiff bases were synthesized by refluxing 2,4-dihydroxyacetophenone with hydrazine hydrate, followed by treatment of substituted aldehydes with the resulting hydrazone to obtain the product compounds. After structural confirmation, the compounds were screened for their in vitro PDE-1 and -3 inhibitory activities. Results: The prepared compounds exhibited noteworthy inhibitory efficacy against PDE-1 and -3 enzymes by comparing with suramin standard. To clarify the binding interactions between the drugs, PDE-1 and -3 active sites, molecular docking studies were carried out. Conclusion: The potent compounds discovered in this study may be good candidates for drug development.

GRAPHICAL ABSTRACT

Plain language summary

Summary points.

• Different bis-Schiff bases of 2,4-dihydroxyacetophenone (3–32) have been synthesized successfully.

• These derivatives were structurally elucidated through different spectroscopic techniques including HR-ESI-MS, ¹H- and ¹³C-NMR, and tested for their in vitro phosphodiesterase-1 and -3 inhibitory activities.

• Among the series, all the compounds attributed excellent to moderate inhibitory activity except seven compounds which were found inactive. However, 14 compounds were found the most potent inhibitors having IC₅₀ values of 0.05 ± 0.11 to 8.02 ± 1.03 μM, when compared with the standard suramin.

• In case of phosphodiesterase-3 inhibitory activity, 11 compounds displayed excellent inhibitory activity with IC₅₀ values from 0.012 ± 0.32 to 1.01 ± 0.22 μM better than the standard suramin (IC₅₀ = 1.05 ± 0.28 μM).

• All the synthesized derivatives showed excellent to less inhibitory activity in the range of IC₅₀ values from 1.23 ± 0.03 to 73.85 ± 0.61 μM, compared with acarbose IC₅₀ = 873.34 ± 1.67 μM.

• Structure–activity relationship study to know the contribution of various attached substituents.

• The docking investigations regarding the phosphodiesterase-1 and -3 binding sites were implemented for the most active bis-Schiff bases and Suramin in order to attain better comprehension with respect to the pattern in which binding mechanics occur between the new compounds and the phosphodiesterase-1 and -3 active sites, which illustrated a higher binding efficacy in appraisal with reference inhibitor and suramin.

• The absorption, distribution, metabolism and excretion molecular characteristics, estimation of toxicity and bioactivity scores were also assessed.

1. Background

Henry Hyde Salter's study from 1886 marks the beginning of the phosphodiesterase (PDE) tragedy. He was an asthmatic and noticed that when he drank a strong cup of coffee on an empty stomach, his breathing became easier [1]. This effect was linked to caffeine's bronchodilator qualities. Although its exact mechanism of action was not known at that time, caffeine has subsequently been demonstrated to function as a non-selective, albeit weak, PDE inhibitor [2]. 11 families of metallo phosphohydrolases known as phosphodiesterases (PDEs), hydrolyze the cyclic AMPs adenosine 3′5′ (cAMP) and guanosine 3′5′ (cGMP) to their inactive 5′ monophosphates [3–5]. Adenylate cyclase and guanylate cyclase, respectively, produce cyclic AMP and cyclic GMP, which transmit the effects of numerous hormones and cellular processes [6,7]. Proteins like protein kinase A, cyclic nucleotide gated ion channels and cAMP/cGMP stimulated guanyl triphosphatase (GTPase) exchange factors are among the intracellular regulatory proteins that cAMP and cGMP bind to. When cyclic nucleotide PDEs is inhibited, cells' levels of cAMP and cGMP rise [8,9]. Therefore, inhibiting PDE is an effective strategy to trigger a range of biological effects and can affect the activation of immune cells, inflammatory cells and smooth muscle contractile responses [10,11]. Each PDE family has a different level of selectivity for cAMP or cGMP and is distinguished by a special blend of enzymatic features and pharmacological inhibitory properties [12]. Each family has its own particular inhibitors, and some families may be tissue specific [13,14]. There are several expressed isoforms for each family. PDE-3 is an enzyme that exclusively hydrolyzes cAMP. PDE-3A and PDE-3B are its two isozymes [15,16]. While PDE-3B is prevalent in adipose tissue, PDE-3A is distributed in the heart, arterial and venous smooth muscle, bronchial and gastrointestinal smooth muscle, etc. PDE-3 activity is highly expressed in alveolar macrophages, endothelial cells, platelets and smooth muscle cells of the airways of the lung. PDE-1, PDE-2, PDE-3, PDE-4 and PDE-5 are PDE isozymes that are found in the trachea, with PDE-3 and PDE-4 serving as the primary cAMP hydrolyzing enzymes [17,18]. PDE-1 and PDE-3 play crucial roles in diverse biological processes, including bone softening and various types of cancers [19]. Asensio et al. reported elevated PDE-1 expression in rat brains and the brain cortex of Alzheimer's disease patients [20]. This finding suggests that PDE inhibitors could serve as innovative therapeutics for neurodegenerative diseases. Additionally, PDE-3 expression is linked to cancer cell metastasis, proposing it as a potential tumor marker [21–23]. To explore the potential of selective PDE inhibitors for novel therapeutic applications such as anti-cancer, anti-metastatic or anti-neurodegenerative drugs, the development of selective inhibitors is essential [24,25]. A rapid and straightforward screening method is required to investigate the inhibitory potential of a compound library, identify lead structures and optimize them as subtype-selective PDE inhibitors.

The diverse biological uses of Schiff bases as well as di-imines and azines, which are frequently created by combining aromatic or aliphatic aldehydes with aniline or aliphatic amines, have raised their worth during the past few decades [26,27]. Heteroatoms and azomethine or imine connection are responsible for this distinctive characteristic [28,29]. These compounds have the general formula R₁HC=N-N=CHR₂ which involves two double bonds among carbon and nitrogen (-HC=N-) [30,31]. They are frequently produced by reacting primary amines with aliphatic/aromatic aldehydes or ketones in an acidic environment [32,33]. R₁R₂=R₃R₄ are examples of asymmetric azines, depending on the type of R substituent [34–36]. Di-imines, among other compounds, have drawn more attention due to their variety of properties, such as their anti-tumor [37], anti-oxidant [38], anti-inflammatory [39], anti-cancer [40], anti-bacterial [41], anti-viral [42] and anti-convulsant properties [43]. Due to a number of biological activities of these compounds, our group has recently published a number of Schiff base and bis-Schiff base derivatives with promising results [44–49].

The present investigation is motivated by the diverse functions of PDE enzymes in controlling intracellular signaling pathways that are facilitated by cyclic nucleotides. Since PDE activity dysregulation has been linked to a number of clinical disorders, PDE inhibitors may be useful as therapeutics [50]. The goal of the synthesis and analysis of new bis-Schiff base derivatives targeting PDE-1 and PDE-3 enzymes is to aid in the creation of strong and specific inhibitors that may be used as treatments for ailments like neurological disorders, respiratory problems and cardiovascular diseases. Through the investigation of novel chemical entities with enhanced inhibitory action against PDE enzymes, the study seeks to provide innovative options for drug development by focusing on the core structure of 2,4-dihydroxyacetophenone, which is known for its pharmacological activities.

2. Experimental

2.1. General

In the current research work, the chemicals, solvents and reagents, all were purchased from Sigma Aldrich (MO, USA), TCI (Fukaya, Japan), Alfa Aesar (MA, USA) and Merck (NJ, USA) in analytical grade. Thin layer chromatography was performed on pre-coated silica gel plates and the chromatograms were seen under the UV light at 256 nm. Stuart SMP10 melting point apparatus was used to check the melting points of the synthetic product compounds. Structure elucidation of these derivatives was confirmed using nuclear magnetic resonance spectroscopic techniques which include ¹H- and ¹³C-NMR. The spectra were recorded on Avance Bruker NMR spectrophotometer on 600 MHz for ¹H- and 150 MHz for ¹³C-NMR. Similarly, the molar masses of these compounds were confirmed using high resolution electrospray ionization mass spectrometry (HR-ESI-MS). The splitting patterns for ¹H-NMR spectrum are: multiplet (m), triplet (t), doublet (d) and singlet (s), whereas the coupling constant (J) and chemical shift (δ) were taken in hertz and ppm, respectively.

2.2. Procedure for the synthesis of Bis-Schiff base derivatives of 2,4-dihydroxyacetophenone (2–32)

Bis-Schiff bases of 2,4-dihydroxyacetophenone (1) were synthesized in two step reactions. Initially, in a 100 ml round bottomed (RB) flask, 2,4-dihydroxyacetophenone were dissolved in 10 ml ethanol then excess of hydrazine hydrate was added to the reaction mixture and refluxed for 5–6 h with constant stirring. At the end of reaction, the mixture was transferred to a beaker consisting of cold water (distilled). The appeared precipitates were filtered, washed with water to remove unreacted hydrazine, dried under air and collected for further reactions. In the final step, a number of substituted aromatic and aliphatic aldehydes were refluxed with the desired hydrazone (2) in absolute ethanol and some drops of acetic acid to afford 30 different substituted bis-Schiff bases (3–32) in better yields (Figure 1). The consumption of reactants and formation of products were checked using the TLC technique in solvent system of n-hexane and ethyl acetate (7:3 = 30%). The mixtures were poured into a beaker containing distilled cold water. The precipitates appeared were washed through water after filtration and collected after drying overnight. These compounds were structurally deduced using modern spectroscopic techniques (¹H-, ¹³C-NMR and HR-ESI-MS) and finally tested for their in vitro phosphodiesterase inhibitory activity (PDE-1 and PDE-3).

Figure 1.

Synthesis of Bis-Schiff base derivatives of 2,4-dihydroxyacetophenone.

2.3. Spectral interpretation of the product compounds (3–32)

2.3.1. 4-(1-4-nitrobenzylidene)hydrazono)ethyl)benzene-1,3-diol (3)

Yellowish Amorphous Solid; Yield: 90%; M. P: 220–221°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.51 (s, 1H, -OH), 10.14 (s, IH, -OH), 8.72 (s, 1H, -CH=N-), 8.28 (d, J = 8.4 Hz, 2H, Ar-H), 8.08 (s, 1H, Ar-H), 7.54 (d, J = 9.0 Hz, 1H, Ar-H), 6.35 (d, J = 9.0 Hz, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 2.59 (s, 3H, -CH3). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 172.3, 162.5, 162.1, 157.2, 148.6, 140.1, 131.5, 129.3, 124.0, 111.0, 107.8, 102.9, 14.2. HR-EIMS (ESI⁺): 300.0856 [M+H]⁺ Calcd for C15H13N3O4: 299.2860.

2.3.2. 4-(1-((4-bromo-2-fluorobenzylidene)hydrazono)ethyl)benzene-1,3-diol (4)

Pale Yellow Amorphous Solid; Yield: 88%; M. P: 218–220°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.49 (s, 1H, -OH), 13.39 (s, 1H, -OH), 8.85 (s, 1H, -CH=N-), 8.64 (s, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.62–6.19 (m, 5H, Ar-H), 2.51 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.8, 166.9, 162.3, 161.8, 151.3, 131.4, 129.3, 128.3, 125.1, 121.1, 119.8, 111.3, 107.6, 102.9, 14.3. HR-EIMS (ESI⁺): 350.9855 [M+H]⁺ Calcd for C₁₅H₁₂BrFN₂O₂: 350.0066.

2.3.3. 4-(1-((2,4-dichlorobenzylidene)hydrazono)ethyl)benzene-1,3-diol (5)

Yellow Amorphous Solid; Yield: 89%; M. P: 232–234°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.54 (s, 1H, -OH), 10.13 (s, 1H, -OH), 8.73 (s, 1H, -CH=N-), 8.11 (d, J = 8.4 Hz, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.56–7.51 (m, 2H, Ar-H), 6.36 (d, J = 9.0 Hz, 1H, Ar-H), 6.25 (s, 1H, Ar-H), 2.57 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 172.1, 162.4, 153.7, 136.4, 135.0, 130.2, 129.5, 128.1, 111.0, 107.7, 102.9, 14.3. HR-EIMS (ESI⁺): 338.1111 [M+NH₄]⁺ Calcd for C₁₅H₁₂Cl₂N₂O₂: 321.0276.

2.3.4. 4-(1-((3-nitrobenzylidene)hydrazono)ethyl)benzene-1,3-diol (6)

Yellowish Amorphous Solid; Yield: 90%; M. P: 222–223°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.54 (s, 1H, -OH), 10.12 (s, 1H, -OH), 8.77 (s, 1H, -CH=N-), 8.64 (s, 1H, Ar-H), 8.31–7.77 (m, 3H, Ar-H), 7.56 (d, J = 8.4 Hz, 1H, Ar-H), 6.36–6.35 (m, 1H, Ar-H), 6.25 (d, J = 8.4 Hz, 1H, Ar-H), 6.25 (d, J = 8.4 Hz, 1H, Ar-H), 2.60 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.9, 162.4, 157.2, 148.2, 135.8, 134.2, 131.4, 130.5, 125.4, 122.5, 111.1, 107.7, 102.9, 14.2. HR-EIMS (ESI⁺): 300.1751 [M+H]⁺ C₁₅H₁₃N₃O₄: 299.2860.

2.3.5. 4-(1-((4-(diethylamino)benzylidene)hydrazono)ethyl)benzene-1,3-diol (7)

White Amorphous Solid; Yield: 80%; M. P: 187–189°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.96 (s, 1H, -OH), 9.93 (s, 1H, -OH), 8.38 (s, 1H, -CH=N-), 7.64 (d, J = 9.0 Hz, 2H, Ar-H), 7.47 (d, J = 9.0 Hz, 1H, Ar-H), 6.70 (d, J = 9.0 Hz, Ar-H), 6.33–6.22 (m, 2H, Ar-H), 3.39 (q, J = 6.6 Hz, 4H, -CH₂CH₃), 2.54 (s, 3H, -CH₃), 1.10 (t, J = 7.2 Hz, -CH₂CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 167.9, 162.0, 160.9, 159.1, 149.7, 130.6, 130.3, 120.4, 111.6, 111.0, 107.1, 102.9, 43.8, 13.7, 12.2. HR-EIMS (ESI⁺): 326.1755 [M+H]⁺ Calcd for C₁₉H₂₃N₃O₂: 326.1790.

2.3.6. 4-(1-((3-hydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (8)

Ash White Amorphous Solid; Yield: 72%; M. P: 190–192°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.66 (s, 1H, -OH), 10.07 (s, 1H, -OH), 9.65 (s, 1H, -OH), 8.49 (s, 1H, -CH=N-), 7.55 (d, J = 9.0 Hz, 1H, Ar-H), 7.27–7.24 (m, 1H, Ar-H), 6.87 (s, 1H, Ar-H), 6.35–6.23 (sm, 2H, Ar-H), 2.55 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.5, 167.0, 162.2, 161.6, 159.1, 157.7, 135.4, 131.1, 129.9, 119.9, 118.6, 114.0, 111.3, 107.6, 102.9, 14.3. HR-EIMS (ESI⁺): 270.3144 [M+H]⁺ Calcd for C₁₅H₁₄N₂O₃: 270.1004.

2.3.7. 4-(1-((2-hydroxy-3-methoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (9)

Off White Amorphous Solid; Yield: 78%; M. P: 231–232°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.45 (s, 1H, -OH), 10.82 (s, 1H, -OH), 10.10 (s, 1H, -OH), 8.84 (s, 1H, -CH=N-), 7.56 (d, J = 8.4 Hz, 1H, Ar-H), 7.28 (d, J = 7.2 Hz, 1H, Ar-H), 7.09 (d, J = 7.8 Hz, 1H, Ar-H), 6.89 (t, J = 7.8 Hz, 1H, Ar-H), 6.36–6.25 (m, 2H, Ar-H), 3.80 (s, 3H, -OCH₃), 2.51 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.1, 162.2, 161.8, 159.6, 148.3, 148.0, 131.4, 121.8, 119.3, 118.9, 115.0, 111.1, 107.6, 103.0, 55.9, 14.3. HR-EIMS (ESI⁺): 300.1769 [M+H]⁺ Calcd for C₁₆H₁₆N₂O₄: 300.1110.

2.3.8. 4-(1-(((2-hydroxynaphthalen-1-yl)methylene)hydrazono)ethyl)benzene-1,3-diol (10)

Yellowish Amorphous Solid; Yield: 73%; M. P: 260–261°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.31 (s, 1H, -OH), 12.88 (s, 1H, -OH), 10.11 (s, 1H, -OH), 9.71 (s, 1H, -CH=N-), 8.62 (d, J = 8.4 Hz, 1H, Ar-H), 7.97 (d, J = 9.0 Hz, 1H, Ar-H), 7.86 (d, J = 7.8 Hz, 1H, Ar-H), 7.58–7.54 (m, 2H, Ar-H), 7.39 (t, J = 7.8 Hz, 1H, Ar-H), 7.23 (d, J = 9.0 Hz, 1H, Ar-H), 6.38–6.28 (m, 2H, Ar-H), 2.53 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.2, 162.1, 161.8, 160.0, 158.8, 134.5, 132.1, 131.4, 128.9, 127.8, 123.8, 122.0, 118.7, 111.1, 109.0, 107.7, 103.1, 14.6. HR-EIMS (ESI⁺): 320.2963 [M+H]⁺ Calcd for C₁₉H₁₆N₂O₃: 320.1161.

2.3.9. 4-(1-((4-hydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (11)

Yellow Amorphous Solid; Yield: 75%; M. P: 188–189°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.85 (s, 1H, -OH), 10.10 (s, 1H, -OH), 10.06 (s, 1H, -OH), 8.77 (s, 1H, -CH=N-), 8.56 (s, 1H, Ar-H), 7.92–7.69 (m, 2H, Ar-H), 7.53 (d, J = 7.8 Hz, 2H, Ar-H), 6.95–6.86 (m, 2H, Ar-H), 2.59 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.2, 163.2, 162.0, 161.2, 160.4, 158.7, 131.0, 130.8, 128.4, 125.1, 115.8, 111.3, 107.2, 102.8, 13.8. HR-EIMS (ESI⁺): 270.3141 [M+H]⁺ Calcd for C₁₅H₁₄N₂O₃: 270.1004.

2.3.10. 4-(1-((2-methoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (12)

White Amorphous Solid; Yield: 80%; M. P: 188–189°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.77 (s, 1H, -OH), 10.09 (s, 1H, -OH), 8.79 (s, 1H, -CH=N-), 8.01 (d, J = 7.8 Hz, 1H, Ar-H), 7.55 (d, J = 7.2 Hz, Ar-H), 7.51 (t, J = 7.8 Hz, Ar-H), 7.14 (d, J = 7.2 Hz, 1H, Ar-H), 7.06 (t, J = 7.8 Hz, 1H, Ar-H), 6.39 (d, J = 7.2 Hz, 2H, Ar-H), 6.29 (s, 1H, Ar-H), 3.88 (s, 3H, -OCH₃), 2.60 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.4, 162.2, 161.8, 158.6, 153.8, 132.9, 131.0, 126.6, 121.8, 121.8, 120.7, 111.9, 111.1, 107.4, 102.8, 55.7, 14.0. HR-EIMS (ESI⁺): 284.0886 [M+H]⁺ Calcd for C₁₆H₁₆N₂O₃: 283.1161.

2.3.11. 4-(1-((4-methoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (13)

Orange Amorphous Solid; Yield: 82%; M. P: 180–182°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.82 (s, 1H, -OH), 10.07 (s, 1H, -OH), 8.56 (s, 1H, -CH=N-), 7.85 (d, J = 7.8 Hz, 2H, Ar-H), 7.54 (d, J = 7.2 Hz, 1H, Ar-H), 7.06 (d, J = 8.4 Hz, 2H, Ar-H), 6.40–6.30 (m, 2H, Ar-H), 3.83 (s, 3H, -OCH₃), 2.45 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.7, 162.1, 161.7, 161.3, 158.5, 130.8, 130.0, 126.6, 114.3, 111.3, 107.3, 102.8, 55.3, 14.3. HR-EIMS (ESI⁺): 285.1385 [M+H]⁺ Calcd for C₁₆H₁₆N₂O₃: 284.1161.

2.3.12. 4-(1-((3,4,5-trimethoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (14)

White Amorphous Solid; Yield: 88%; M. P: 170–171°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.76 (s, 1H, -OH), 10.11 (s, 1H, -OH), 8.65 (s, 1H, -CH=N-), 7.56 (d, J = 7.2 Hz, 1H, Ar-H), 7.23 (s, 2H, Ar-H), 6.40 (d, J = 7.2 Hz, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 3.85 (s, 6H, -OCH₃), 3.74 (s, 3H, -OCH₃), 2.62 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.3, 162.1, 161.3, 158.7, 153.1, 140.1, 131.0, 129.3, 111.2, 107.5, 105.5, 102.8, 60.1, 55.8, 14.0. HR-EIMS (ESI⁺): 346.1221 [M+H]⁺ Calcd for C₁₈H₂₀N₂O₅: 345.1372.

2.3.13. 4-(1-((thiophen-2-ylmethylene)hydrazono)ethyl)benzene-1,3-diol (15)

Yellow Amorphous Solid; Yield: 85%; M. P: 165–166°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.60 (s, 1H, -OH), 10.10 (s, 1H, -OH), 8.83 (s, 1H, -CH=N-), 7.79 (d, J = 7.8 Hz, Ar-H), 7.64 (d, J = 7.8 Hz, 1H, Ar-H), 7.54 (d, J = 7.2 Hz, 1H, Ar-H), 7.22 (t, J = 7.2 Hz, 1H, Ar-H), 6.41–6.8 (m, 2H, Ar-H), 2.45 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.1, 162.1, 161.5, 153.3, 138.8, 133.4, 131.0, 130.9, 128.2, 111.2, 107.5, 102.8, 14.3. HR-EIMS (ESI⁺): 260.0715 [M+H]⁺ Calcd for C₁₃H₁₂N₂O₂S: 260.0619.

2.3.14. 4-(1-((2-nitrobenzylidene)hydrazono)ethyl)benzene-1,3-diol (16)

Light Yellow Amorphous Solid; Yield: 88%; M. P: 181–182°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.58 (s, 1H, -OH), 10.20 (s, 1H, -OH), 8.85 (s, 1H, -CH=N-), 8.45–8.07 (m, 2H, Ar-H), 7.87 (t, J = 7.2 Hz, 1H, Ar-H), 7.84 (t, J = 7.2 Hz, 1H, Ar-H), 7.59 (d, J = 7.8 Hz, 1H, Ar-H), 6.41–6.30 (m, 2H, Ar-H), 2.58 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.2, 162.3, 162.0, 154.9, 148.7, 133.8, 131.6, 131.4, 129.5, 127.8, 124.5, 111.0, 107.6, 102.8, 14.2. HR-EIMS (ESI⁺): 300.0978 [M+H]⁺ Calcd for C₁₅H₁₃N₃O₄: 299.2860.

2.3.15. 4-(1-((3,4-dimethoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (17)

Yellow Amorphous Solid; Yield: 80%; M. P: 160–161°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.82 (s, 1H, -OH), 10.08 (s, 1H, -OH), 8.66 (s, 1H, -CH=N-), 8.54–7.54 (m, 3H, Ar-H), 7.06 (d, J = 7.8 Hz, 1H, Ar-H), 6.84–6.07 (m, 2H, Ar-H), 3.84 (s, 6H, -OCH₃), 2.52 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.5, 166.9, 162.2, 161.9, 161.6, 159.3, 136.7, 135.2, 133.5, 131.8, 130.5, 129.4, 128.7, 127.6, 126.8, 125.4, 124.8, 111.3, 107.5, 102.9, 14.4, 14.2. HR-EIMS (ESI⁺): 315.1461 [M+H]⁺ Calcd for C₁₇H₁₈N₂O₄: 314.1267.

2.3.16. 4-(1-((4-chlorobenzylidene)hydrazono)ethyl)benzene-1,3-diol (18)

Orange Amorphous Solid; Yield: 89%; M. P: 185–186°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.66 (s, 1H, -OH), 10.12 (s, 1H, -OH), 8.64 (s, 1H, -CH=N-), 7.92 (d, J = 7.2 Hz, 2H, Ar-H), 7.60 (d, J = 7.2 Hz, Ar-H), 6.40–6.29 (m, 3H, Ar-H), 2.52 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.0, 166.9, 162.2, 161.6, 157.8, 135.7, 132.9, 131.1, 129.9, 128.9, 111.2, 107.5, 102.8, 14.2. HR-EIMS (ESI⁺): 287.1468 [M+H]⁺ Calcd for C₁₅H₁₆ClN₂O₂: 286.0666.

2.3.17. 4-(1-((2-hydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (19)

Yellow Amorphous Solid; Yield: 70%; M. P: 203–204°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.77 (s, 1H, -OH), 10.41, (s, 1H, -OH), 9.25 (s, 1H, -OH), 9.21 (s, 1H, -CH=N-), 8.19 (m, 2H, Ar-H), 7.77 (t, J = 7.2 Hz, 1H, Ar-H), 7.71–7.59 (m, 2H, Ar-H), 6.71–6.35 (m, 2H, Ar-H), 2.69 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.5, 166.9, 162.0, 159.3, 133.5, 131.0, 130.4, 128.6, 127.6, 126.8, 125.3, 124.0, 111.3, 107.5, 102.9, 14.2. HR-EIMS (ESI⁺): 271.1252 [M+H]⁺ Calcd for C₁₅H₁₄N₂O₃: 270.1004.

2.3.18. 4-(1-((naphthalen-1-ylmethylene)hydrazono)ethyl)benzene-1,3-diol (20)

White Amorphous Solid; Yield: 72%; M. P: 179°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.50 (s, 1H, -OH), 10.20 (s, 1H, -OH), 8.87 (s, 1H, -CH=N-), 7.73 (dd, J = 7.2 Hz, J = 4.4 Hz, 1H, Ar-H), 7.60 (d, J = 7.8 Hz, 1H. Ar-H), 7.40 (t, J = 7.2 Hz, 1H, Ar-H), 6.99–6.95 (m, 3H, Ar-H), 6.41–6.30 (m, 2H, Ar-H), 2.55 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.0, 166.9, 162.1, 162.0, 161.7, 161.6, 159.5, 158.3, 133.1, 132.9, 131.2, 130.6, 119.5, 118.6, 116.5, 111.2, 111.0, 107.5, 102.9, 14.2. HR-EIMS (ESI⁺): 326.1919 [M+Na]⁺ Calcd for C₁₉H₁₆N₂O₂: 303.1212.

2.3.19. 4-(1-(octylidenehydrazono)ethyl)benzene-1,3-diol (21)

Brownish Gel; Yield: 60%; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.53 (s, 1H, -OH), 12.45 (s, 1H, -OH), 8.30 (s, 1H, -CH=N-), 2.55 (s, 3H, -CH₃), 2.31 (q, J = 7.2 Hz, 2H), 1.45–1.30 (m, 8H), 0.90 (t, J = 7.2 Hz, 3H). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 168.0, 163.7, 162.5, 162.2, 129.8, 108.6, 103.7, 31.8, 29.4, 29.0, 26.6, 26.0, 22.7, 14.1. HR-EIMS (ESI⁺): 277.0121 [M+H]⁺ Calcd for C₁₆H₂₄N₂O₂: 276.1838.

2.3.20. 4-(1-((4-methylbenzylidene)hydrazono)ethyl)benzene-1,3-diol (22)

Off White Amorphous Solid; Yield: 76%; M. P: 158–160°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.77 (s, 1H, -OH), 10.10 (s, 1H, -OH), 8.58 (s, 1H, -CH=N-), 7.79 (d, J = 6.6 Hz, 2H, Ar-H), 7.55 (d, J = 8.4 Hz, 1H, Ar-H), 7.31 (d, J = 6.6 Hz, 2H, Ar-H), 6.42–6.30 (m, 2H, Ar-H), 2.60 (s, 3H, -CH₃), 2.36 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 168.0, 162.5, 162.2, 149.4, 140.7, 132.7, 129.8, 129.1, 111.4, 108.6, 21.3, 14.3. HR-EIMS (ESI⁺): 269.1269 [M+H]⁺ Calcd for C₁₆H₁₆N₂O₂: 268.1212.

2.3.21. 4-(1-(((5-methylfuran-2-yl)methylene)hydrazono)ethyl)benzene-1,3-diol (23)

White Amorphous Solid; Yield: 68%; M. P: 149–151°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.67 (s, 1H, -OH), 10.08 (s, 1H, -OH), 8.37 (s, 1H, -CH=N-), 7.53 (d, J = 7.2 Hz, 1H, Ar-H), 7.01 (d, J = 7.2 Hz, 1H, Ar-H), 6.78–6.27 (m, 2H, Ar-H), 2.55 (s, 3H, -CH₃), 2.39 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.7, 166.9, 162.0, 161.5, 161.3, 156.3, 147.8, 131.0, 130.0, 119.3, 111.3, 111.2, 109.2, 108.0, 107.5, 107.3, 102.8, 14.2, 13.6. HR-EIMS (ESI⁺): 258.2427 [M+H]⁺ Calcd for C₁₄H₁₄N₂O₃: 258.1004.

2.3.22. 4-(1-((2-chlorobenzylidene)hydrazono)ethyl)benzene-1,3-diol (24)

Yellow Amorphous Solid; Yield: 85%; M. P: 166–167°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.59 (s, 1H, -OH), 13.54 (s, 1H, -OH), 8.81 (s, 1H, -CH=N-), 8.16 (d, J = 6.6 Hz, Ar-H), 8.13–7.45 (m, 4H, Ar-H), 6.41–6.30 (m, 2H, Ar-H), 2.62 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.3, 162.0, 161.5, 157.5, 135.9, 133.6, 131.0, 130.7, 127.8, 127.2, 111.2, 107.5, 102.9, 14.2. HR-EIMS (ESI⁺): 290.2668 [M+H]⁺ Calcd for C₁₅H₁₆ClN₂O₂: 289.0666.

2.3.23. 4-(1-((furan-2-ylmethylene)hydrazono)ethyl)benzene-1,3-diol (25)

White Amorphous Solid; Yield: 80%; M. P: 145–146°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.61 (s, 1H, -OH), 10.54 (s, 1H, -OH), 8.48 (s, 1H, -CH=N-), 7.95 (s, 1H, Ar-H), 7.55 (d, J = 7.2 Hz, Ar-H), 7.12 (d, J = 7.2 Hz, 1H, Ar-H), 6.78–6.28 (m, 3H, Ar-H), 2.59 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.4, 162.1, 161.5, 149.3, 147.8, 146.5, 131.0, 117.3, 112.5, 111.2, 107.4, 102.8, 14.2. HR-EIMS (ESI⁺): 244.1137 [M+H]⁺ Calcd for C₁₃H₁₂N₂O₃: 244.0848.

2.3.24. 4-(1-((3-chlorobenzylidene)hydrazono)ethyl)benzene-1,3-diol (26)

Light Orange Amorphous Solid; Yield: 78%; M. P: 178–180°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.63 (s, 1H, -OH), 10.16 (s, 1H, -OH), 8.63 (s, 1H, -CH=N-), 7.93 (s, 1H, Ar-H), 7.85 (d, J = 6.6 Hz, 1H, Ar-H), 7.60–7.51 (m, 3H, Ar-H), 6.42–6.30 (m, 2H, Ar-H), 2.61 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.3, 162.0, 161.5, 157.5, 135.9, 133.6, 131.0, 130.7, 127.8, 127.2, 111.2, 107.5, 102.9, 14.2. HR-EIMS (ESI⁺): 312.1599 [M+Na]⁺ Calcd for C₁₅H₁₆ClN₂O₂: 289.0666.

2.3.25. 4-(1-((3-ethoxy-4-hydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (27)

Yellow Amorphous Solid; Yield: 86%; M. P: 199–201°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.87 (s, 1H, -OH), 9.91 (s, 1H, -OH), 8.48 (s, 1H, -CH=N-), 7.59 (d, J = 6.6 Hz, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.32 (d, J = 6.6 Hz, 1H, Ar-H), 6.92 (d, J = 7.2 Hz, 1H, Ar-H), 6.42–6.30 (m, 3H, Ar-H), 4.11 (q, J = 6.6 Hz, 2H, -OCH₂CH₃), 2.60 (s, 3H, -CH₃), 1.39 (t, J = 6.6 Hz, 3H, -OCH₂CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.2, 162.0, 161.5, 158.9, 150.2, 147.0, 131.1, 125.4, 123.2, 115.5, 112.0, 111.2, 107.2, 102.8, 63.8, 14.6, 13.8. HR-EIMS (ESI⁺): 314.1106 [M+H]⁺ Calcd for C₁₇H₁₈N₂O₄: 314.1067.

2.3.26. 4-(1-((3-methoxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (28)

Yellow Amorphous Solid; Yield: 76%; M. P: 142–144°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.81 (s, 1H, -OH), 10.15 (s, 1H, -OH), 8.60 (s, 1H, -CH=N-), 8.05 (s, 1H, Ar-H), 7.85 (d, J = 7.2 Hz, Ar-H), 7.54 (d, J = 6.6 Hz, 2H, Ar-H), 7.07 (d, J = 7.2 Hz, 1H, Ar-H), 6.41–6.28 (m, 1H, Ar-H), 3.83 (s, 3H, -OCH₃), 2.59 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 169.7, 162.1, 162.0, 161.7, 158.5, 131.0, 130.1, 126.5, 114.3, 111.2, 107.3, 102.8, 55.3, 14.2. HR-EIMS (ESI⁺): 284.3313 [M+H]⁺ Calcd for C₁₆H₁₆N₂O₃: 284.1161.

2.3.27. 4-(1-((3-bromobenzylidene)hydrazono)ethyl)benzene-1,3-diol (29)

Yellow Amorphous Solid; Yield: 88%; M. P: 189–191°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.63 (s, 1H, -OH), 10.15 (s, 1H, -OH), 8.60 (s, 1H, -CH=N-), 8.05 (s, 1H, Ar-H), 7.88 (d, J = 6.0 Hz, 1H, Ar-H), 7.69 (d, J = 6.0 Hz, 1H, Ar-H), 7.67 (d, J = 7.2 Hz, 1H, Ar-H), 7.56 (d, J = 7.2 Hz, 1H, Ar-H), 7.45 (t, J = 6.0 Hz, 1H, Ar-H), 6.29–6.20 (m, 1H, Ar-H), 2.60 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 171.3, 162.3, 161.5, 157.4, 136.4, 131.3, 130.9, 130.7, 130.4, 127.2, 122.1, 111.0, 107.5, 102.9, 14.1. HR-EIMS (ESI⁺): 334.1155 [M+H]⁺ Calcd for C₁₅H₁₃BrN₂O₂: 333.0160.

2.3.28. 4-(1-((4-(dimethylamino)benzylidene)hydrazono)ethyl)benzene-1,3-diol (30)

Yellow Amorphous Solid; Yield: 90%; M. P: 215–217°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.98 (s, 1H, -OH), 10.01 (s, 1H, -OH), 8.44 (s, 1H, -CH=N-), 7.70 (d, J = 7.2 Hz, 2H, Ar-H), 7.51 (d, J = 6.6 Hz, 1H, Ar-H), 6.77 (d, J = 6.6 Hz, 2H, Ar-H), 6.41–6.27 (m, 2H, Ar-H), 3.03–2.58 (m, 13H). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 168.1, 162.0, 160.9, 159.0, 152.2, 131.08, 130.5, 129.9, 121.1, 111.6, 111.5, 107.1, 102.8, 13.7. HR-EIMS (ESI⁺): 298.1110 [M+H]⁺ Calcd for C₁₇H₁₉N₃O₂: 297.1477.

2.3.29. 4-(1-((3,5-dibromo-4-hydroxybenzylidene)hydrazono)ethyl)benzene-1,3-diol (31)

Ash Whita Amorphous Solid; Yield: 89%; M. P: 258–260°C; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 13.66 (s, 1H, -OH), 13.58 (s, 1H, -OH), 10.13 (s, 1H, -OH), 8.49 (s, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 7.54 (d, J = 6.6 Hz, 1H, Ar-H), 6.40–6.17 (m, 1H, Ar-H), 2.58 (s, 3H, -CH₃). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 170.7, 162.2, 161.5, 156.1, 153.1, 131.9, 131.1, 128.4, 112.0, 11.1, 107.4, 102.8, 14.2. HR-EIMS (ESI⁺): 425.0662 [M+H]⁺ Calcd for C₁₅H₁₂Br₂N₂O₃: 425.9215.

2.3.30. 4-(1-(hexylidenehydrazono)ethyl)benzene-1,3-diol (32)

Brownish Gel; Yield: 60%; ¹H-NMR (600 MHz, DMSO-d₆, δ, ppm): 12.60 (s, 1H, -OH), 10.10 (s, 1H, -OH), 7.94 (s, 1H, Ar-H), 7.45–7.30 (m, 2H, Ar-H), 2.58 (s, 3H, -CH₃), 2.35–0.78 (m, 11H). ¹³C-NMR (150 MHz, DMSO-d₆, δ, ppm): 168.0, 163.7, 162.5, 162.2, 129.4, 111.4, 108.6, 103.7, 32.5, 28.6, 26.2, 23.7, 14.8, 14.2. HR-EIMS (ESI⁺): 248.2101 [M+H]⁺ Calcd for C₁₄H₂₀N₂O₂: 248.1525.

2.4. Enzyme inhibition assay

In this assay, the activity against PDE-1 and -3 was assayed by employing the reported method with some modifications, such as, 33 mM tris-HCl was used as buffer with pH 8.8 to which 30 mM Mg–acetate was added as a cofactor with 0.000742 mM of enzyme PDE-1 as the final concentration, using 96-well flat bottom plate and 0.33 mM bis (p-nitrophenyl) phosphonate as a substrate. EDTA was used as the positive control, having IC₅₀ = 277.69 ± 2.52 μM. The test compounds were incubated with the enzyme for 30 min, the product formation was monitored by the addition of the substrate after incubation period at 37°C for 30 min with 1 min intervals. M2 and spectra max 384 (from Molecular Devices, CA, USA) were used at the particular wavelength of 410 nm. All the reactions were performed in triplicate, and the initial rates were measured as the rates of changes in the Vmax/min (absorbance/min) and used in subsequent calculations.

2.5. Molecular docking

GOLD was implemented to conduct a docking investigation for the target molecules (version 5.2) alongside its employment in the discovery studio [51,52]. After attaining the crystallized PDE-1 and PDE-3, the H₂O and inhibitors were removed, and the H atoms were added. The examined ligands were redocked into the vacant active site after the standard inhibitor was removed from it. The ChemPLP scoring function was generated for measuring the binding affinity, and the charges were assigned with the CHARMM force field [53,54]. The structure with the lowest root-mean-square deviation (RMSD) score was utilized to generate different ligand poses. For calculating the binding affinity, the ChemPLP scoring function was integrated, and the CHARMM force field was used to assign charges [55].

2.6. Docking simulation

Molecular Dynamics Simulation and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSAMD) [56] simulation and MM/PBSA were performed by GROMACS [57–59] as mentioned in (Supplementary Data).

3. Results & discussion

3.1. Chemistry

Thirty bis-Schiff base derivatives of 2,4-dihydroxyacetophenone (1) have been effectively synthesized through two-step reactions as part of our being conducted search for medicinally powerful PDE inhibitors. Primarily, 2,4-dihydroxyacetophenone (1) was refluxed with excess of hydrazine hydrate in ethanol solvent for 4–5 h with constant stirring to obtain the desired hydrazone (2), which was finally treated with different substituted aromatic aldehydes in absolute ethanol containing catalytic amount of acetic acid for 3–4 h to afford bis-Schiff bases in outstanding yields (3–32) (Figure 1). The formation of these derivatives was monitored by thin layer chromatographic (TLC) technique in the solvent system of n-hexane and ethyl acetate (7:3). At the end of reaction, the mixtures were cooled to room temperature and decanted to a beaker containing chilled distilled water. The appeared precipitates were washed with water after filtration and kept under the air for drying. After drying, the product compounds were collected and recrystallized with pure ethanol where needed and structurally elucidated via modern spectroscopic techniques including ¹H-, ¹³C-NMR and HR-ESI-MS. These bis-Schiff base derivatives were subjected to in vitro phosphodiesterase inhibitory activity (Supplementary Table S1).

Analyzing the synthetic derivatives, ¹H-NMR spectra revealed evidence for several methine protons, methyl and methylene. Signals resonated in the down field region of the spectrum at δ 13.98 to 9.91 ppm were due to the presence of two hydroxyl groups attached to the benzene ring. Similarly, signals were seen in the region δ 8.87–8.30 ppm were due to the presence of azomethine protons, which confirmed the formation of the product compounds. Besides, signals of the aromatic protons appeared in the region δ 8.28–6.19 ppm, while in the up field region of the spectrum, signals resonated at δ 2.60–2.49 ppm were due to the methyl group. The ¹³C-NMR (broad band and DEPT) spectra of the compounds showed signals for quaternary carbons, methine, methylene and methyl. In the down field region of the spectrum, signals appeared at δ 169.2–168.1 ppm were due to the presence of azomethine carbon atoms. Furthermore, signals observed in the region δ 162.5–160.0 ppm were due to the carbon atoms to which the two hydroxyl groups are attached, while in the up field region of the spectrum, signals appeared at δ 15.9–13.1 ppm were because of the presence of methyl group. On the other hand, molar masses of the synthetic derivatives have been confirmed by means of HR-ESI-MS spectra showing there molecular ion peaks. The detailed spectral interpretation of compounds is presented in experimental section.

3.2. In vitro phosphodiesterase inhibitory activity

The synthesized bis-Schiff base derivatives (3–32) of 2,4-dihydroxyacetophenone (1) were screened for their in vitro phosphodiesterase-1 and phosphodiesterase-3 inhibitory activity. Among the series, all the compounds attributed excellent to moderate inhibitory activity except seven compounds (8, 9, 10, 11, 12, 19 and 21) which were found inactive. Similarly, 14 compounds 30 (IC₅₀ = 0.05 ± 0.11 μM), 31 (IC₅₀ = 0.5 ± 0.41 μM), 29 (IC₅₀ = 1.01 ± 0.14 μM), 20 (IC₅₀ = 1.30 ± 1.00 μM), 3 (IC₅₀ = 2.31 ± 0.87 μM), 22 (IC₅₀ = 2.36 ± 0.51 μM), 23 (IC₅₀ = 3.27 ± 0.32 μM), 18 (IC₅₀ = 3.65 ± 0.36 μM), 17 (IC₅₀ = 4.05 ± 0.62 μM), 24 (IC₅₀ = 4.36 ± 0.89 μM), 5 (IC₅₀ = 5.89 ± 2.36 μM), 4 (IC₅₀ = 6.28 ± 1.01 μM), 13 (IC₅₀ = 7.89 ± 0.58 μM) and 25 (IC₅₀ = 8.02 ± 1.03 μM) displayed excellent inhibitory potential when compared with the standard suramin (IC₅₀ = 8.02 ± 0.12 μM) [60,61]. Moreover, four compounds 14, 32, 16 and 15 showed significant inhibitory activity with IC₅₀ values of 10.65 ± 0.14, 11.62 ± 0.47, 14.80 ± 0.29 and 17.41 ± 1.05 μM respectively, while the remaining five derivatives (26, 6, 7, 27 and 28) displayed good to moderate inhibition in the range of IC₅₀ values from 28.12 ± 0.51 to 55.32 ± 0.31 μM against phosphodiesterase-1 inhibitory activity (Supplementary Table S1).

In case of phosphodiesterase-3 inhibitory activity, 11 compounds 25 (IC₅₀ = 0.012 ± 0.32 μM), 27 (IC₅₀ = 0.047 ± 0.35 μM), 26 (IC₅₀ = 0.11 ± 0.12 μM), 11 (IC₅₀ = 0.27 ± 0.32 μM), 24 (IC₅₀ = 0.32 ± 0.21 μM), 14 (IC₅₀ = 0.32 ± 0.89 μM), 7 (IC₅₀ = 0.57 ± 0.65 μM), 19 (IC₅₀ = 0.58 ± 0.62 μM), 3 (IC₅₀ = 0.87 ± 0.36 μM), 16 (IC₅₀ = 1.00 ± 0.57 μM) and 32 (IC₅₀ = 1.01 ± 0.22 μM) among the series were found potent inhibitors of PDE-3 enzyme better than the standard suramin (IC₅₀ = 1.05 ± 0.28 μM). Similar to this, 13 derivatives 17, 28, 15, 31, 20, 30, 18, 8, 29, 21, 9, 13 and 10 showed significant to less activity in the range of IC₅₀ values from 2.01 ± 1.35 to 15.21 ± 0.15 μM, while the remaining six bis-Schiff bases (4, 5, 6, 12, 22 and 23) were found inactive towards the PDE-3 enzyme.

3.2. Structure–activity relationship study

3.2.1. Phosphodiesterase-1

Compounds 30 (IC₅₀ = 0.05 ± 0.11 μM), 31 (IC₅₀ = 0.5 ± 0.41 μM), 29 (IC₅₀ = 1.01 ± 0.14 μM), 20 (IC₅₀ = 1.30 ± 1.00 μM), 3 (IC₅₀ = 2.31 ± 0.87 μM), 22 (IC₅₀ = 2.36 ± 0.51 μM), 23 (IC₅₀ = 3.27 ± 0.32 μM), 18 (IC₅₀ = 3.65 ± 0.36 μM), 17 (IC₅₀ = 4.05 ± 0.62 μM), 24 (IC₅₀ = 4.36 ± 0.89 μM), 5 (IC₅₀ = 5.89 ± 2.36 μM), 4 (IC₅₀ = 6.28 ± 1.01 μM), 13 (IC₅₀ = 7.89 ± 0.58 μM) and 25 (IC₅₀ = 8.02 ± 1.03 μM) were the most promising inhibitors of phosphodiesterase-1 enzyme. The promising result of compound 30 could be due to the attachment of dimethyl amino substituent at para position of the benzene ring compared with the activity of compound 7 (IC₅₀ = 39.07 ± 0.89 μM) having less activity it could be due to the attachment of diethyl amino group at para position of the benzene ring. Similar to this, by comparing compound 3 (IC₅₀ = 2.31 ± 0.87 μM) with 16 (IC₅₀ = 14.80 ± 0.29 μM) and 6 (IC₅₀ = 32.20 ± 1.03 μM), the highest activity of compound 3 could be due to the attachment of electron withdrawing nitro substituent attached to the benzene ring at para position (Figure 2). A decrease occurs in the activities of compound 16 and 6 may be due to the change in position of nitro group from para to ortho and meta respectively. Furthermore, comparing compound 5 (IC₅₀ = 5.89 ± 2.36 μM), with 4 (IC₅₀ = 6.28 ± 1.01 μM), both derivatives showed approximately same activity, but a little bit good activity of compound 5 might be due to the attachment of chlorine atoms at para position of the benzene ring. The change of substituents from chlorine (2,4-dichloro) to bromine and fluorine is responsible for the slight decrease in the inhibitory activity of compound 4. On the other hand, comparing compounds 12 (inactive), 13 (IC₅₀ = 7.89 ± 0.58 μM) and 28 (IC₅₀ = 55.32 ± 0.31 μM), among these derivatives, compound 13 showed excellent activity having a methoxy group at para position of the benzene ring. By changing the same methoxy group to meta position can cause a great decrease in the activity of compound 28, while compound 12 having methoxy substituent at ortho position did not show any activity. Similar to this comparing compound 20 (IC₅₀ = 1.30 ± 1.00 μM) with 10 which was found inactive it may be due to the attachment of hydroxyl group at ortho position of the naphthalene moiety, by removing the hydroxyl substituent compound 20 showed highest inhibitory activity among the series. When we compare compounds 8 with 11 and 19 these derivatives were found inactive towards PDE-1 enzyme and could be due to the attachment of hydroxyl group at ortho, meta and para position of the benzene ring. In case of tri substituted compounds 14 (IC₅₀ = 10.65 ± 0.14 μM), compared with 31 (IC₅₀ = 0.5 ± 0.41 μM), the most potent activity of compound 31 can be due to the presence of hydroxyl group at para position and bromine groups attached at meta position of the benzene ring, while replacing these three groups with electron donating methoxy substituents are responsible for the decrease in the activity of compound 14.

Figure 2.

Most active inhibitors of phosphodiesterase-1 enzyme.

3.2.2. Phosphodiesterase-3

11 compounds 25 (IC₅₀ = 0.012 ± 0.32 μM), 27 (IC₅₀ = 0.047 ± 0.35 μM), 26 (IC₅₀ = 0.11 ± 0.12 μM), 11 (IC₅₀ = 0.27 ± 0.32 μM), 24 (IC₅₀ = 0.32 ± 0.21 μM), 14 (IC₅₀ = 0.32 ± 0.89 μM), 7 (IC₅₀ = 0.57 ± 0.65 μM), 19 (IC₅₀ = 0.58 ± 0.62 μM), 3 (IC₅₀ = 0.87 ± 0.36 μM), 16 (IC₅₀ = 1.00 ± 0.57 μM) and 32 (IC₅₀ = 1.01 ± 0.22 μM) among the series were found potent inhibitors of PDE-3 enzyme better than the standard suramin (IC₅₀ = 1.05 ± 0.28 μM). The highest activity of compound 25 could be due to the furan moiety. Similarly, comparing compounds 3 (IC₅₀ = 0.87 ± 0.36 μM), with 6 (inactive), and 16 (IC₅₀ = 1.00 ± 0.57 μM), the excellent inhibitory activity of compound 3 might be due to the attachment of nitro group at para position, by changing the position from para to ortho can slightly decrease the activity of compound 16, while the nitro group attached at meta position of the benzene ring in compound 6 did not show any activity. Additionally, by comparison of compound 26 (IC₅₀ = 0.11 ± 0.12 μM), with 24 (IC₅₀ = 0.32 ± 0.21 μM), and 18 (IC₅₀ = 2.01 ± 1.35 μM), the highest activity of compound 26 might be due to the presence of chlorine group attached to the benzene ring at meta position, a little bit decrease occurs in the activity of compound 24 could be due to the change in position of chlorine group to ortho position while a great decrease occurs in the activity of compound 18 may be due to the change in position of chlorine group to para position of the benzene ring (Figure 3). When we compare compound 11 (IC₅₀ = 0.27 ± 0.32 μM), with 19 (IC₅₀ = 0.58 ± 0.62 μM), and 8 (IC₅₀ = 5.97 ± 0.14 μM), these three compounds have same substituents attached at different position of the benzene ring. Compound 11 showed the best inhibitory activity may be due to the presence of hydroxyl group at para position instead of ortho and meta in compound 19 and 8, respectively, showing less activity than compound 11. In case of tri substituted compounds, 14 (IC₅₀ = 0.32 ± 0.89 μM), are the most promising inhibitor and could be due to the existence of methoxy groups at meta and para position, replacing these three groups to bromine and hydroxyl may lower the activity of compound 31 (IC₅₀ = 3.01 ± 1.02 μM). On the other hand, compound 7 showing excellent inhibition with IC₅₀ of 0.57 ± 0.65 μM might be due to the ethyl amino group at para position of the benzene ring. A decrease occurs in the activity of compound 31, which could be possibly due to the change of substituent ethyl amino to methyl amino at the same (para) position of the benzene ring. Furthermore, in case of straight chain substituents, compound 32 (IC₅₀ = 1.01 ± 0.22 μM) showed highest activity than compound 21 (IC₅₀ = 7.26 ± 0.28 μM), which could be due to the pentane moiety instead of heptane chain in compound 21 showing less activity.

Figure 3.

Most active derivatives against phosphodiesterase-3 enzyme.

3.3. Molecular docking analysis

The docking analysis was performed to explicate the potency of these desirable molecules in vitro against the phosphodiesterase PDE-1 and PDE-3, respectively, through their potential interaction mechanisms with their crystal frameworks (Protein Data Bank ID: 4NPW [62] and 1SO2 [63]). The docking investigation was implemented through Glide's module. The preliminary inhibitors 4-aminoquinazoline, dihydropyridazine acid and Suramin (control inhibitor in vitro used) were redocked into the examined crystal frameworks to verify the docking methodology. Furthermore, the efficacious performance of the targeted molecules was authenticated via the low values of RMSD (0.209 and 1.9 Å) for PDE-1 and (1.3 and 2.40 Å) for PDE-3, respectively, which was acquired through the root mean square deviation between the native and redocked poses of the co-crystallized inhibitor. The binding free energies ΔG were listed in (Supplementary Table S2).

The initial inhibitors have been adequately installed into their binding sites in order to attain their crystal configurations. The lowest score poses and RMSD revealed increased stability in the binding pocket. The data was utilized to rank the docked poses and to select the most capable docked conformation of each compound. The quinazoline core is spun counterclockwise in the original 4-aminoquinazoline inhibitor of PDE-1 to take an H-bond from the side chain nitrogens of Glu421, Thr334 and His373 (Supplementary Figure S1). This rotation places the quinazoline in a position to chelate the catalytic Zn metal that coordinates with Asp264 and Asp370 [64]. While in PDE-3 dihydropyridazine caped (His737, Glu851 and Gln988), besides, magnesium ion in PDE-1 and PDE-3 is the most common ion to produce maximal activity of PDE families [63] (Supplementary Figure S2). The molecule docked prolifically into active sites in a similar mechanism to the original inhibitors. Then redocked 3–32 compounds and suramin and compared the results to the reference inhibitors (dihydropyridazine and aminoquinazoline), and obtained a RMSD in the range of 1.00 to 1.9 Å for PDE-1, and 1.02 to 7.92 Å (Supplementary Table S2). All synthesized compounds showed lower binding efficiency than reference inhibitors and suramin in both PDE proteins. Using the fingerprint interaction between ligand and protein (PLIF), binding effectiveness was assessed. The ‘Oples3e’ molecular-mechanics force field created the poses then picked the pose with the lowest ‘G’ and ‘RMSD’ to evaluate the binding affinities of 3–32 and suramin molecules. The glide ΔG score, which calculates the free energy of binding between the ligand and the receptor protein, was used to assess the binding mechanism and stability of the docked bis-Schiff bases 3–32 and suramin compounds. The bis-Schiff bases poses with the lowest score and RMSD is the more stable in the binding pocket. The interactions between the bis-Schiff bases and residues of active sites for PDE-1 and 3 were mainly, metal ion, polar bonds, hydrogen bonding, π–π and π–H interactions, which contributed to a strong alignment with the enzyme backbone. Further validation of the docking protocol the co-crystallized docked poses and reference inhibitors were superimposed in the binding site. The superimposed conformation occupied the same binding pocket (Figures 4 & 5).

Figure 4.

3D docking poses of nine active bis-Schiff bases and overlay there with reference inhibitors inside phosphodiesterase-1 active site.

Figure 5.

3D docking poses of nine most active bis-Schiff's bases and overlay there with reference inhibitors inside phosphodiesterase-3 active site.

The most promising inhibitors of phosphodiesterase-1 enzyme showed binding efficiency including compounds 30 (ΔG = -6.65 kcal/mol), 31 (ΔG = -7.20 kcal/mol), 29 (ΔG = -6.08 kcal/mol), 20 (ΔG = -6.92 kcal/mol), 3 (ΔG = -6.49 kcal/mol), 22 (ΔG = -6.60 kcal/mol), 23 (ΔG = -6.44 kcal/mol), 18 (ΔG = -6.28 kcal/mol), 17 (ΔG = -6.95 kcal/mol), 24 (ΔG = -6.43 kcal/mol), 5 (ΔG = -6.95 kcal/mol), 4 (ΔG = -5.70 kcal/mol), 13 (ΔG = -6.84 kcal/mol) and 25 (ΔG = -5.25 kcal/mol) were the most significant inhibitors of phosphodiesterase-1 enzyme.

The most active PDE-1 inhibitors in first series 30 and 31 were interacted with catalytic Zn603 ion which chelated with important amino acids Asp264 and Asp370, and at same time, the most active compound 30 forms; H-interaction with Thr334, π–π interaction with Phe392 and coordinated with important Mg metal ion (Figure 4). In the second series which arranged 20 (IC₅₀ = 1.30 ± 1.00 μM) >3 (IC₅₀ = 2.31 ± 0.87 μM) >22 (IC₅₀ = 2.36 ± 0.51 μM) and has higher PDE-1 inhibition than suramin, these series comforted in binding pocket by the same way through chelating with Zn and Mg metal ions that bonded with Asp370 and Asp264. Compounds 20 and 3 which are the most active compounds in this series forms extra H-bond by Thr334 with 20 and His373 with 3. Similarly, in third series in activity which arranged 23 > 18 > 17, they chelate with Zn and Mg to coordinate with same manner with Asp370 and Asp264. But compounds 23 and 17 connected with His227 and Thr334 (Figure 4).

In PDE-3, the binding efficiency for most active compounds compared with suramin; 25 (ΔG = -5.61 kcal/mol.), 27 (ΔG = -7.44 kcal/mol.), 26 (ΔG = -5.75 kcal/mol.), 11 (ΔG = -6.82 kcal/mol.), 24 (ΔG = -6.14 kcal/mol.), 14 (ΔG = -7.78 kcal/mol.), 7 (ΔG = -7.63 kcal/mol.), 19 (ΔG = -6.59 kcal/mol.), 3 (ΔG = -7.71 kcal/mol.), 16 (ΔG = -6.51 kcal/mol.) and 32 (ΔG = -5.79 kcal/mol.).

The most active PDE-3 inhibitors in first series 25 and 26 were localized in binding pocket in the same manner to interact by strong H-bond with Glu851 and H–π with Ilu955, but compound 27 coordinate with Zn and Mg metal ions which chelated with important amino acid Asp264 and form H–π interaction with Ilu955 (Figure 6). At same time, compounds 7 and 19 in second active series which arranged (7 > 19 > 3) were comforted in binding pocket by the same way through formation H–π with Ilu955, while most active compound 7 in this series formed extra three interactions; π–π with His737, H–π with His948 and π–π contact with Gln988, Ile955 and Asp937 and π–π contact with Phe991.

Figure 6.

The values for the Gibbs free energy of ligand–phosphodiesterase-1 and -phosphodiesterase-3 protein.

MM-PBSA: Molecular mechanics-Poisson-Boltzmann surface area.

Finally, two members 11 and 14 in third series formed only one π–π contact with Ile955. Only compound 24 in this series occupied binding pocket by formation four interactions, H–π, His948, π–π with Phe991, H-bond with Ile955 and Asp937.

3.4. Molecular dynamic simulations for the active compounds

To validate and get a clear understanding, ligand–protein interactions which resulted from the molecular docking results, molecular dynamic (MD) simulation were performed at nano scale (ns), using the Molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. That was used to calculate the binding free energy changes between most active bis-Schiff bases against PED-1 and PDE-3 (30, 31 and 29) and (25, 27 and 26) with the protein complexes. The MD simulations for ligand–protein interactions are carried out over a time period of 150 ns. The interactions between ligands and proteins are analyzed every 2 ns. Figure 6 illustrating the variations in binding strength between investigated bis-Schiff base molecules and the PDE-1 and PDE-3 complexes. The binding free energy changes of the most active ligands for PDE-1 and PDE-3. Negative values for MM-PBSA free energies indicate stronger binding interactions. The small divergence for MM-PBSA for (30, 31 and 29) and complexed with PDE-1 and (25, 27 and 26) complexed with PDE-3 over the entire time-scale indicated a robust contact between ligand and amino acid backbone, indicating a firm H-bonding pattern. The steady state appeared at 20 ns for 30, 31 and 29 complexes with PDE-1, and after 18 ns for 25, 27 and 26 with PDE-3. The low variation degree for MM-PBSA profiles along the simulation trajectories, which demonstrated their stability nature. These profiles did not vary significantly over time. This suggests that the complexes are stable and do not change their structure significantly over 150 ns, and able to bind to the protein complexes in a similar way (Figure 6).

3.5. In silico absorption, distribution, metabolism, excretion & toxicity toxicological profile

The oral bioavailability for bis-Schiff bases 3, 7, 11, 14, 17-20, 22, 24-27, 29, 30 and 31 which are most active compounds against PDE-1 and PDE-3 and suramin as standard inhibitor, were calculated and introduced in radar (Supplementary Figure S3). The ADME prediction showed that the bis-Schiff bases possess several desirable ADME properties.

The physicochemical properties and drug-likeness of form most active bis-Schiff bases 3, 7, 11, 14, 17–20, 22, 24–27, 29, 30 and 31 was calculated using SwissADME and compared with stander inhibitor suramin [65]. The physicochemical properties, which include molecular weight, solubility, molar refractivity, topological polar surface area and hydrogen bonding capacities, are used to select novel drugs and formulations. As a result, these properties bis-Schiff bases 3, 7, 11, 14, 17–20, 22, 24–27, 29, 30, 31 and suramin (Table-Supplementary Table S3) are crucial for identifying new drug candidates and should be in accordance with the various rule-based filters. Based on the findings, we discovered that most active bis-Schiff bases complies with the five drug similarity guidelines (Lipinski, Ghose, Veber, Egan and Muegge rules) and are approved as a unique drug candidate [30].

The analysis of the drug-likeness model score is based on the positive and negative values of the target compounds; in contrast, if the drug-likeness score is negative, the compound is not a drug. In accordance with this rule, we discovered that the compounds had the highest ratings for drug-likeness with positive value, therefore deemed to be drug like pharmaceuticals (Supplementary Table S3). Before entering the systemic circulation, the new drug must pass through intestinal cell membranes. We used the human colon adenocarcinoma cell lines (Caco-2) to assess drug permeability. As a result, Caco-2 cell permeability has been a crucial indicator for the new medication. When a target compounds projected value is >-5.15 log.cm.s^-1, it is deemed to fit proper Caco-2 permeability. Based on this rule, we found that the bis-Schiff bases have a fit proper Caco-2 permeability within the range of -4.8 to -5.0 log cm.s^-1 in compared with suramin unacceptable value -6.6 log.cm.s^-1.

The toxic properties of bis-Schiff bases 3, 7, 11, 14, 17–20, 22, 24–27, 29, 30, 31 and suramin were calculated using ADMET lab and all the properties were summed in (Supplementary Table S3). Human hepatotoxicity (H-HT) is essential to risk inspection for new drugs. The new target bis-Schiff bases and suramin are non-toxic for the liver, H-HT negative, with a value of 0 to 0.3, which toxic for the liver, H-HT positive, with two categories; the first is moderate toxicity with a predicted value of 0.3 to 0.7 and the second is strong toxicity value from 0.7 to 1.0. As a result, all investigated bis-Schiff bases and suramin as significant carcinogens are classified as moderate-to-excellent carcinogen values range (0.83 to 0.95) at the same time. Target bis-Schiff bases substances are classified as non-mutagenic when their anticipated values range from 0.0 to 0.3 (gray color), medium mutagenic when their predicted values range from 0.3 to 0.7 and strong-mutagenic as predicted in suramin when their values range from 0.7 to 1 (aqua color). The bio-availability radar for examined bis-Schiff and suramin were represented in Figure 7A. The consensus log Po/w, the classical descriptor for liphophilicity, showed that all the tested compounds are lipophilic because their values are above 0. Only suramin showed lowest bioavailability, with lowest water solubility, polarity behaviors (Figure 7A). Compounds 7, 18 and 21 showed highest bioavailability, which their radar has optimal range as red color representation. Moreover, these examined bis-Schiff and suramin reached a bioavailability score in value range of 0.11 to 0.55 which can be categorized as drug-like, and has no undesirable functional groups which could lead to carcinogenic, mutagenic and hepatotoxic effects [66].

Figure 7.

Absorption, distribution, metabolism, excretion and toxicity properties of the investigated compounds. (A) The optimal range as red color for; lipophilicity: -0.7 ≤ XLOGP3 ≤ +5.0, size: 150 ≤ MW ≤ 500 g/mol, polarity: TPSA ≤ 140 Å2, solubility: log S ≤ 6, saturation fraction of carbons in the hybridization sp3 ≥ 0.25 and flexibility: rotatable bonds ≤9. (B) BOILED-Egg plot, white and yellow color represented highly probable for HIA (GI) absorption and BBB permeation, respectively, gray outside for molecules are low absorption and not brain penetration, blue and red points represented as P-gp substrate (PGP+) and P-gp non-substrate (PGP-), respectively.

BBB: Blood–brain barrier; HIA: Human intestinal absorption; GI: Gastrointestinal; MW: Molecular weight; TPSA: Topological polar surface area.

Brain or intestinal estimated permeability approach (BOILED-Egg) was presented as an effective prediction model based on small molecule liphophilicity and polarity calculations in order to attain this objective. The BOILED-Egg model offers a rapid, simple, easily reproducible and statistically unmatched method for predicting the excellent gastrointestinal absorption and brain permeability of tiny compounds that may be used in drug development and discovery [66]. The white part of the egg (the yolk) seems to be the physicochemical zone of substances that are likely to be absorbed by the gastrointestinal tract. The blood–brain barrier (BBB), shown in yellow, is a physical and chemical zone where chemicals that are likely to get into the brain (BBB permeation) are kept. Bis-Schiff bases 3, 11, 14, 22, 25, 26, 27, 29, 30 and 31 have High GI absorption and high permeability against BBB. None of these bis-Schiff bases compounds and suramin can be transported out of the cells by P-glycoprotein, which is a protein that pumps foreign substances out of cells. This implies that the compounds can stay longer in the cells and have more effects on the receptors [67]. Nowadays, the metabolism prediction model had concentrated on the interaction between the target compounds with cytochromes P450 mono oxygenase enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4) which catalyze the phase 1 metabolism of pharmaceuticals and concentrated in the liver. The inhibition of cytochromes enzymes is one of the principal mechanisms which cause pharmacokinetic drug-drug interactions [68]. Supplementary Table S2 demonstrated that bis-Schiff bases 3, 7, 11, 14, 17–20, 22, 24–27, 29 and 30 were predicted as inhibitors of their enzymes and CYP2D6 which represented as blue color. The skin permeability coefficient (Kp) is linearly correlated with molecular size and liphophilicity and the more negative the log Kp (cm/s), the less skin permeation [69]. Thus, Suramin has the lowest skin penetration (-13.12 cm/s) which allows its application through skin massage (Supplementary Table S3). The low synthetic accessibility score (2.40 to 2.73) of investigated bis-Schiff bases compared higher value for 6.41 for suramin, which is crucial in the process of choosing the most promising virtually tested molecules that can be produced and used in biological experiments (Supplementary Table S3).

4. Conclusion

Thirty novel bis-Schiff base derivatives (3–32) based on 2,4-dihydroxyacetophenone (1) were synthesized, characterized and tested for their in vitro PDE-1 and -3 inhibitory activity. In the series, 14 derivatives (30, 31, 29, 20, 3, 22, 23, 18, 17, 24, 5, 4, 13 and 25) showed excellent PDE-1 inhibitory activity having IC₅₀ values from 0.05 ± 0.11 to 8.02 ± 1.03 μM better than the standard suramin (IC₅₀ = 8.02 ± 0.12 μM). Moreover, nine derivatives presented significant to moderate activity, while the remaining seven compounds (8–12, 19 and 21) were found inactive. In case of PDE-3 activity, 11 compounds 25, 27, 26, 11, 24, 14, 7, 19, 3, 16 and 32 among the series were found potent inhibitors better than standard suramin (IC₅₀ = 1.05 ± 0.28 μM). Furthermore, 13 derivatives showed significant to less activity while the remaining six product compounds were inactive. At last, docking investigations regarding the PDE-1 and PDE-3 binding sites were implemented for the most targeted bis-Schiff bases and suramin in order to attain better comprehension with respect to the pattern in which binding mechanics occur between the bis-Schiff bases molecules and the PDE-1 and PDE-3 active sites, which illustrated a higher binding efficacy in appraisal with reference inhibitor and suramin. Additionally, the absorption, distribution, metabolism and excretion, molecular characteristics, estimation of toxicity and bioactivity scores were assessed.

Funding Statement

Authors would like to extent their appreciation for funding this research from the Researchers Supporting Project Number (RSPD2024R1100), King Saud University, Riyadh, Saudi Arabia.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2342707

Author contributions

A Alam, S Gul and Zainab synthesized the compounds, while M Khan and SA Ali Shah screened the compounds against the activities. A Alam and Zainab, performed structural elucidation and wrote the original draft of the manuscript. AA Elhenawy and A Latif performed molecular docking study of the synthesized compounds. M Ahmad, M Ali, supervised the project, assisted in the writing, the reviewing, and the editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Financial disclosure

Authors would like to extent their appreciation for funding this research from the Researchers Supporting Project Number (RSPD2024R1100), King Saud University, Riyadh, Saudi Arabia. The authors have no other financial interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Data availability statement

The spectroscopic data presented in this study are available in the supporting information.