Synthesis,evaluation,and docking study of adamantyl-1,3,4-oxadiazol hybrid compounds as CaMKIIδ kinase inhibitor

Abstract

This study revealed a new inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), a crucial factor in cardiovascular disease and hypertension. The study focuses on the bioactivity compounds that combine adamantane/1,3,4-oxadiazole, potentially inhibiting CaMKIIδ. Various adamantyl-1,3,4-oxadiazole derivatives were synthesized and tested for their efficiency against CaMKIIδ kinase, with 6f being the most potent with an IC₅₀ value of 14.4 μM. Docking studies were carried out to determine the binding processes of these chemicals within the kinase’s active region. These discoveries are an important step toward the development of novel treatments for cardiovascular illnesses and hypertension, with the potential for more precise and efficient therapeutic interventions in the future.

Introduction

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), one of the Ca²⁺/calmodulin-dependent protein kinase family (comprising 3 subgroups: I, II, and IV), is a versatile serine/threonine kinase pivotal for modulating calcium signaling. It exerts significant influence over a multitude of cellular processes, encompassing excitation-contraction coupling, gene expression, and cell apoptosis. CaMKII phosphorylates a diverse array of substrates that participate in the regulation of calcium-induced adjustments in cellular function. The four distinct CaMKII isoforms (alpha, beta, gamma, and delta) are spread across different tissues: CaMKIIα, β, and γ predominantly inhabit the brain, while CaMKIIδ is primarily situated in the heart.^1,2 Within the cardiac context, CaMKIIδ is implicated in the development of heart failure, arrhythmias, and atrial fibrillation. Research has demonstrated heightened CaMKIIδ activity in individuals with heart failure and atrial fibrillation. Inhibiting CaMKIIδ has been observed to ameliorate cardiac function and reduce the occurrence of arrhythmias in animal models of heart failure.^3,4 Moreover, inhibition of CaMKIIδ has been shown to protect cardiac mitochondria from Ca²⁺ overload, loss of inner membrane potential, increased ROS, and reduce the incidence of arrhythmias in models of heart failure.^4–6

CaMKII’s activity is regulated by calmodulin (CaM), a calcium-sensing protein. When intracellular calcium levels surge, Ca²⁺ binds to CaM, initiating the activation of CaMKII. The sensitivity of CaMKII to calcium and CaM is subject to regulation through variable and self-associative domains.^4,7 The binding of CaM to CaMKIIδ promotes structural modifications in CaMKIIδ, alleviating autoinhibition and enabling complete enzymatic activity. ⁸

The potential of CaMKIIδ as a target for clinical intervention in cardiovascular disorders has been established through the emergence of clinical trials involving potent CaMKIIδ inhibitors, such as KN-93, KN-62, and staurosporine the natural CaMKIIδ inhibitor (see Figure 1).^4,8 However, therapeutic inhibition of CaMKIIδ is challenged by the diversity of CaMKII isoforms and splice variants and by physiological activities of CaMKII isoforms that contribute to learning and memory. ⁹

Figure 1.

Commercially available and natural used CaMKII inhibitor.

CaMKIIδ inhibitors encounter additional drawbacks encompass poor cellular permeability, a tendency for general toxicity that can impede their clinical applicability, a deficiency in specificity potentially resulting in unintended off-target effects and constraining their therapeutic promise. In addition, certain CaMKIIδ inhibitors may interfere with the interaction between CaMKIIδ and scaffolding proteins, potentially disrupting kinase signaling domains.^4,5,10–12

As part of our ongoing interest in the discovery and design of adamantyl-1,3,4-oxadiazole hybrids as bioactive compounds.^13–17 As potential CaMKIIδ kinase inhibitors, we had an interest in creating hybrid adducts of adamantane/1,3,4-oxadiazole. The synthesis of target compounds pharmacophore was obtained using a Cu-catalyzed Mannich reaction^13,14,18,19 (Scheme 1).

Scheme 1.

Compounds 6a–h synthetic pathways.

In silico profiling against many targets proposed our hybrid compounds block CaMKIIδ kinase, ²⁰ and were, therefore, assessed in vitro as CaMKIIδ kinase blockers. One of them showed micromolar IC₅₀s. It’s interesting to note that no prior research has been done using hybrid molecules of adamantyl-1,3,4-oxadiazole as CaMKIIδ kinase inhibitors.

Results and discussion

Chemistry

As shown in Scheme 1, our synthetic approach started from adamantane carboxylic acid (1) and proceeded to synthesize adamantyl hydrazide (3), as described earlier.^15,21 Then, after refluxing adamantyl hydrazide (3) in ethanol along with trimethylamine and carbon disulfide, compound (4) was produced with an excellent yield (85%).^22,23 After 12 h, the thiol of 4 was treated with sodium carbonate (Na₂CO₃) and propargyl bromide in acetonitrile to produce the thiopropargylated derivative (5) in 76% yield. ²⁴ Compound (5) served as the basis for the Mannich reaction, which produced a library of eight series compounds (6a–h) that are displayed in Table 1.

Table 1.

Structures of synthesized 6a–h compounds.

Compound	R1	Compound	R1
6a		6e
6b		6f
6c		6g
6d		6h

High-resolution mass spectrometry (HRMS), ¹H, ¹³C NMR, and IR spectroscopy were used to characterize the synthesize compounds (spectra of all compounds HRMS, IR, ¹H and ¹³C NMR, are available in SI file). The mass spectra were found to be in highly agreement with estimated values and provided the right molecular ion peaks. Aliphatic protons related to the adamantyl group in the region δ = 0.72–2.08 ppm and protons from aliphatic amines were detected in the ¹H NMR spectra. The SCH₂ protons showed as singlets at around δ = 4.00 ppm.

Resonances were observed for all aromatic rings at δ = 6.70–8.22 ppm. However, all of the produced compounds’ distinctive signals were seen in the ¹³C NMR spectra, which also included several aliphatic peaks for the aliphatic amine (region δ = 12.0–39.5 ppm) and adamantly (region δ = 41.0–58.0 ppm) moieties. In the aromatic area (region δ = 103.0–152.0 ppm), every aromatic moiety displayed accurate and distinct carbon signals. For majority of the produced compounds, the alkyne carbon atoms peaks (78.5–80.0 ppm) are easily identifiable. At approximately δ = 162.0 and 174.0 ppm, the two quaternary oxadiazole peaks were visible.

Evaluation of biological activities

Profiling against numerous pharmacophores revealed one of the molecules, namely, 6f, fits CaMKIIδ kinase pharmacophore, ²⁰ as in Figure 2. Clearly from the figure, the H-bond acceptor and donor features of the binding model map the phenol OH and the ionizable ammonium NH of the central piperazine within 6f, respectively, while a hydrophobic feature of the pharmacophore partly fits the phenol ring of 6f. The other hydrophobic feature is closely positioned to one of the linker backbone carbons within 6f, while the positive ionizable feature of the pharmacophore maps one of the oxadiazol ionizable nitrogens of 6f. To validate these observations, we assessed the inhibitory properties of the synthesized molecules against CaMKIIδ kinase. The bioactivity profiles of 6a–h are shown in Table 2. Obviously, 6f scored the best inhibitory percentage among other molecules encouraging us to follow its IC₅₀ value. The resulting dose/response curve is shown in Figure 3.

Figure 2.

6f was matched against the pharmacophore of CaMKIIδ kinase ²⁰ . Red spheres indicate positive ionizable features, blue spheres indicate hydrophobic characteristics, gray spheres are exclusion zones, and green vectored spheres represent hydrogen bond acceptor features, purple vectored spheres represent a hydrogen bond donor feature.

Table 2.

Inhibitory profiles of synthesized compounds against CaMKIIδ.

Compound	% Inhibition at 10 μM a	IC50 (μM)
6a	12	60.9
6b	10	63.4
6c	14	83.4
6d	11	76.4
6e	21	74.3
6f	34	14.4
6g	20	52.3
6h	25	59.7
Staurosporine b	0.289 nM

a

Average of duplicate measurements ± standard deviation.

b

Standard inhibitor.

Figure 3.

Dose-response curves of compound 6f against CaMKIIδ kinase with IC₅₀ = 14.4 μM.

Docking study

In order to gain a mechanistic understanding of the bioactivity of 6f, we chose to dock 6f into the CaMKIIδ kinase binding site. Using CDOCKER, the docking investigation was completed.^25,26

However, prior to docking 6f, the docking parameters had to be validated by comparing the docked pose of the same ligand with the crystallographic pose of the co-crystallized inhibitor within CaMKIIδ kinase (PDB code: 5VLO). Fortunately, with a root mean square difference of 0.27 Å, the docking settings nearly matched the crystallographic position (Figure 4), which gave rise to the decision to dock 6f with the same parameters.

Figure 4.

Docked and crystallographic positions of a ligand inside CAMKIIδ (PDB code: 5VLO) (a) The bound ligand in its docked position. The hinge region’s DFG amino acids are represented by green residues. (b) A comparison of the complexed ligand’s crystallographic structure within the CAMKIIδ binding pocket (green) and its docked pose (red).

Figure 5 shows docked 6f. Clearly from Figure 5, the ionizable sp² N-atom of the oxadiazol ring of docked 6f is positioned close to the carboxylic acid moiety of Glu97 within CaMKIIδ kinase binding pocket suggesting mutual electrostatic attraction. This interaction agrees with mapping the same group against negative ionizable feature in the CaMKIIδ pharmacophore in Figure 2. Hydrogen bonding and electrostatically-reinforced hydrogen bonding interactions can be seen tethering the o-phenol OH and the central piperazine ammonium of 6f with the backbone NH of Ala156 and the carboxylic acid side chain of Asp157 (which belongs to the hinge DFG amino acids ²⁷ ), respectively. These two interactions correspond to mapping the same chemical groups of the ligand (i.e. the phenolic hydroxyl and piperazine ammonium) against the central hydrogen bond donor and acceptor features within the CaMKIIδ pharmacophore (Figure 2). The phenolic benzene ring of 6f is docked at close proximity to the side chains of hydrophobic amino acids Ala156, Phe90, Ala41, Val74, and Val28 suggesting mutual hydrophobic interactions. These interactions correspond to fitting the phenolic aromatic ring against one of the hydrophobic features in the CaMKIIδ pharmacophore (Figure 2). Finally, the ligand’s carbon atom separating the piperazine ammonium and the linker alkyne is positioned close to the α-carbon of Asn141 in the docked pose suggesting certain hydrophobic attraction, which probably corresponds to the less-than-optimal mapping of the same atom by the second hydrophobic feature in the CaMKIIδ pharmacophore (Figure 2).

Figure 5.

Docked pose of active compounds 6f. Green represents the DFG sequence, which is Gly159-Asp157-Phe158. Dotted lines linking interacting functional groups and moieties represent binding interactions. Orange lines show interactions involving electrostatic attraction and charge transfer, light pink lines show hydrophobic interactions, and green lines represent hydrogen bonds.

Conclusion

Due to CaMKIIδ kinase plays a crucial role in mitosis and cell cycle regulation, targeting it in anticancer research shows potential. This study focuses on using the copper-catalyzed Mannich reaction to generate hybrid adamantine/1,3,4-oxadiazole compounds as potential CaMKIIδ kinase inhibitors. Compound 6f was found to be a potent inhibitor of CaMKIIδ kinase using pharmacophore profiling and inhibitory assessments. The bioactivities of these compounds were validated by molecular docking, and in vitro bioassay testing demonstrated that compound 6f had an anti-CaMKIIδ kinase IC₅₀ value of 14.4 μM. These results give hope for more efficient and focused therapies in the future and support the ongoing efforts to create innovative cancer medicines.

Experimental

Chemicals and instruments

All compounds were purchased commercially from Across Organics and Sigma-Aldrich Co. and were utilized exactly as supplied, without further purification. The study’s solvents were procured from Scharlau, Fluka, and Aldrich. Thin layer chromatography (TLC) was used to monitor all reactions. Merk aluminum plates pre-coated with silica gel PF254; 20 × 20 × 0.25 mm; these plates were seen under a UV light to identify the reactions. Using TMS as an internal standard, ¹H NMR spectra were captured using a Bruker Avance III-500 MHz spectrometer. ¹³C NMR spectra were collected at 125 MHz on the same equipment. Chemical shifts were expressed as ppm-values for δ-. Spectra were obtained in CDCl₃. In addition, 2D (COZY, HMQC, and HMBC) and DEPT spectra were acquired. On a Bruker APEX-IV (7 Tesla) instrument, HR-MS were obtained using the electrospray ion trap (ESI) technique. Using a Thermo-Nicolet Nexus 670 FTIR device, FTIR spectra were captured.

Synthesis and characterization

Methyl adamantane-1-carboxylate (2)

Following the literature procedure,^14,15 compound 2 was synthesized to produce 4.76 gm with yield 95% (m.p. 38–40 °C)

1-adamantanecarbohydrazide (3)

Following the literature procedure,^14,15 compound 3 was synthesized to obtain 4.35 gm with yield 92% (m.p. = 149–151 °C).

5-(1-adamantyl)-1,3,4-oxadiazole-2-thiol (4)

Following the literature procedure, compound 4 was synthesized to obtain a pure compound (m.p. = 190–191 °C).^13,23

2-(1-adamantyl)-5-(prop-2-yn-1-ylthio)-1,3,4-oxadiazole (5)

Na₂CO₃ (44.80 mmol) was added to a stirred solution of 4 (14.93 mmol) in ACN (50 mL). After stirring the mixture for 20 min at room temperature, the reaction mixture was heated to 50 °C. After that, the reaction was supplemented with a 22.40 mmol, 80% toluene solution of 3-bromopropyne, and the mixture was stirred for 12 h at 50 °C. Filtration was used to remove Na₂CO₃ from the reaction mixture, and the solvent was then evaporated. Then 30 mL of water was added to the mixture and extracted with ethyl acetate (EtOAc) (3 × 20 mL). After washing the organic layer, drying it on MgSO₄, and vacuum-evaporating it, pure compound (5) (12.23 mmol) was produced as a light brown solid.^{13,23 1}H NMR (500 MHz, CDCl₃): δ 1.76 (s, 6H, Adamantyl), 2.03 (s, 6H, Adamantyl), 2.08 (s, 3H, Adamantane), 2.30 (s, 1H, alkyne), 3.96 (s, 2H, CH₂). ¹³C NMR (125 MHz, CDCl₃): δ 21.0, 27.7, 34.5, 36.2, 39.8, 72.8, 77.5, 161.9, 174.2. HRMS (ESI) m/z = calc. for C₁₅H₁₉N₂OS, [M+H]: 275.11205, found 274.11110.

Synthesis of compounds (6a–h)

4-((5-((3r,5r,7r)-adamantan-1-yl)-1,3,4-oxadiazol-2-yl)thio)-N, N-2-yn-1-amine (6a–h)

CuI (25 mg) and aqueous formaldehyde (35%, 1 mL) in 2 mL dimethyl sulfoxide (DMSO) were combined with an alkyne 5 (0.60 mmol) and the suitable amine (0.72 mmol) in a stirred solution. For 3 h, the reaction mixture was stirred at room temperature. Following the addition of 8 mL of water, the mixture was extracted using 2 × 25 mL of EtOAc. 6a–h was the result of purifying the residue using column chromatography and an EtOAc-MeOH (10:1, v/v) eluent.^13,23

4-(4-((5-((3r,5r,7r)-adamantan-1-yl)-1,3,4-oxadiazol-2-yl)thio)but-2-yn-1-yl)morpholine (6a)

Pale brown oily product (0.200 g, 69%). IR (cm^–1): 3417, 2910, 2852, 2818, 2763, 2644, 2250, 1568, 1485, 1240, 1176, 1116, 1053, 756. ¹H NMR (500 MHz, CDCl₃): δ 1.66 (br s, 6H, Adamantyl), 1.90 (s, 6H, Adamantane), 1.96 (br s, 3H, Adamantyl), 2.39 (s, 4H, –NCH₂CH₂O), 3.17 (s, 2H, –NCH₂), 3.56 (s, 4H, –NCH₂CH₂O), 3.90 (s, 2H, –SCH₂). ¹³C NMR (125 MHz, CDCl₃): δ 12.2, 21.6, 27.6, 34.5, 36.2, 39.8, 40.8, 47.2, 78.8, 78.9, 162.1, 174.1. HRMS (ESI) m/z = calc. for C₂₀H₂₈N₃O₂S, [M+H]: 274.18966 found 274.18898.

2-((3r,5r,7r)-adamantan-1-yl)-5-((4-thiomorpholinobut-2-yn-1-yl)thio)-1,3,4-oxadiazole ( 6b )

Pale brown oily product (0.199 g, 65%). IR (cm^–1): 3450, 2908, 2852, 1485, 1454, 1417, 1242, 1174, 1053, 754. ¹H NMR (500 MHz, CDCl₃): δ 1.76 (m, 6H, Adamantyl), 2.02 (s, 6H, Adamantane), 2.08 (br s, 3H, Adamantyl), 2.68 (pst, 4H, –NCH₂CH₂S), 2.75 (pst, 4H, –NCH₂CH₂S), 3.28 (s, 2H, –NCH₂), 4.00 (s, 2H, –SCH₂). ¹³C NMR (125 MHz, CDCl₃): δ 21.5, 27.3, 27.9, 34.5, 36.2, 39.8, 48.3, 53.8, 79.0, 79.5, 162.1, 174.2. HRMS (ESI) m/z = calc. for C₂₀H₂₈N₃OS₂, [M+H]: 390.16684 found 390.16753.

2-((3r,5r,7r)-adamantan-1-yl)-5-((4-(4-benzylpiperidin-1-yl)but-2-yn-1-yl)thio)-1,3,4-oxadiazole (6c)

Pale brown oily product (0.273 g, 70%). IR (cm^–1): 2910, 2850, 1568, 1485, 1174, 1053, 750. ¹H NMR (500 MHz, CDCl₃): δ 1.27 (pst, 2H, –NCH₂CH₂CH), 1.60 (d, 2H, J= 12.0 Hz, –NCH₂CH₂CH), 1.72 (m, 6H, Adamantyl), 1.98 (s, 6H, Adamantane), 2.03 (br s, 3H, Adamantyl), 2.07 (2H, –NCH₂CH₂CH), 2.48 (d, 2H, J= 7.0 Hz, −CH₂Ph), 2.78 (d, 2H, J= 11.0 Hz, –NCH₂CH₂CH), 3.22 (s, 2H, –NCH₂), 3.95 (s, 2H, –SCH₂), 7.08–7.21 ( pst, 5H-Ar). ¹³C NMR (125 MHz, CDCl₃): δ 21.6, 27.6, 31.9, 34.5, 36.2, 37.2, 39.8, 43.0, 46.3, 52.5, 78.5, 80.1, 125.8, 128.1, 129.1, 141.4, 162.1, 174.0. HRMS (ESI) m/z = calc. for C₂₈H₃₆N₃OS, [M+H]: 462.25737 found 462.25756.

2-((3r,5r,7r)-adamantan-1-yl)-5-((4-(4-(3-methoxyphenyl)piperazin-1-yl)but-2-yn-1-yl)thio)-1,3,4-oxadiazole (6d)

Pale brown oily product (0.281 g, 72%). IR (cm^–1): 3377, 2852, 1778, 1668, 1485, 1298, 1236, 1215, 1172, 1043, 768. ¹H NMR (500 MHz, CDCl₃): δ 1.73 (br s, 6H, Adamantane), 2.00 (s, 6H, Adamantane), 2.05 (br s, 3H, Adamantyl), 2.45 (br s, 2H, –NCH₂CH₂N), 2.59 (br s, 2H, –NCH₂CHN), 2.76 (br s, 2H, –NCH₂CH₂N), 3.31 (s, 2H, –NCH₂), 3.75 (s, 3H, -OCH₃), 3.82(br s, 2H, –NCHCH₂N), 4.00 (s, 2H, –SCH₂), 6.39 (d, 1H, J= 8.0 Hz, Ar), 6.43 (s, 1H, Ar), 6.51 (d, 1H, J= 8.0 Hz, Ar), 7.12 (t, 1H, J= 8.0 Hz, Ar). ¹³C NMR (125 MHz, CDCl₃): δ 21.6, 27.7, 34.5, 36.2, 39.8, 47.3, 51.1, 52.2, 55.1, 57.8, 78.9, 79.7, 103.8, 104.6, 110.1, 129.7, 151.5, 160.6, 162.1, 162.1, 174.1. HRMS (ESI) m/z = calc. for C₂₇H₃₅N₄O₂S, [M+H]: 479.24753 found 479.24878.

2-((3r,5r,7r)-adamantan-1-yl)-5-((4-(4-(3-chlorophenyl)piperazin-1-yl)but-2-yn-1-yl)thio)-1,3,4-oxadiazole (6e)

Pale brown oily product (0.276 g, 68%). IR (cm^–1): 2910, 2852, 1568, 1485,1236, 1174, 945, 769. ¹H NMR (500 MHz, CDCl₃): δ 1.70 (br s, 6H, Adamantyl), 1.97 (s, 6H, Adamantane), 2.01 (br s, 3H, Adamantyl), 2.60 (br s, 4H, –NCH₂CH₂N), 3.14 (br s, 4H, –NCH₂CH₂N), 3.30 (s, 2H, –NCH₂), 3.97 (s, 2H, –SCH₂) 6.73, 6.81, 7.10 (H, Ar). ¹³C NMR (125 MHz, CDCl₃): δ 21.5, 27.6, 34.5, 36.2, 39.8, 47.1, 48.5, 51.7, 79.1, 79.4, 119.2, 115.7, 119.4, 130.0, 134.9, 152.2, 162.0, 174.1. HRMS (ESI) m/z = calc. for C₂₆H₃₂ClN₄OS, [M+H]: 483.19800 found 483.19637.

2-(4-(4-((5-((3r,5r,7r)-adamantan-1-yl)-1,3,4-oxadiazol-2-yl)thio)but-2-yn-1-yl)piperazin-1-yl)phenol (6f)

Pale brown oily product (0.288 g, 71%). IR (cm^–1): 3304, 2914, 2850, 1623, 1568, 1485,1247, 1174, 1053, 927, 763. ¹H NMR (500 MHz, CDCl₃): δ 1.73 (br s, 6H, Adamantyl), 2.01 (s, 6H, Adamantane), 2.05 (br s, 3H, Adamantyl), 2.68 (br s, 4H, –NCH₂CH₂N), 3.05 (br s, 4H, –NCH₂CH₂N), 3.33 (s, 2H, –NCH₂), 3.99 (s, 2H, –SCH₂), 7.08, 7.12, 7.19, 7.21 (4H, Ar). ¹³C NMR (125 MHz, CDCl₃): δ 21.5, 27.6, 34.5, 36.2, 39.7, 47.0, 50.5, 51.9, 79.0, 79.3, 114.7, 118.7, 123.3, 127.6, 139.7, 139.7, 155.9, 163.3, 174.3. HRMS (ESI) m/z = calc. for C₂₆H₃₃N₄O₂S, [M+H]: 465.23188 found 465.23049.

4-(4-(4-((5-((3r,5r,7r)-adamantan-1-yl)-1,3,4-oxadiazol-2-yl)thio)but-2-yn-1-yl)piperazin-1-yl)phenol (6g)

Pale brown oily product (0.350 g, 79%). IR (cm^–1): 3242, 2916, 2852, 2825, 1588, 1490,1217, 827, 759. ¹H NMR (500 MHz, CDCl₃): δ 1.74 (br s, 6H, Adamantyl), 2.01 (s, 6H, Adamantane), 2.05 (br s, 3H, Adamantyl), 2.86 (br s, 4H, –NCH₂CH₂N), 3.05 (br s, 4H, –NCH₂CH₂N), 3.33 (s, 2H, –NCH₂), 3.98 (s, 2H, –SCH₂), 6.76, 6.78 (pst, 4H, Ar). ¹³C NMR (125 MHz, CDCl₃): δ 21.5, 27.6, 34.5, 36.2, 39.7, 47.0, 50.6, 52.0, 79.1, 79.4, 116.1, 118.7, 144.7, 150.9, 162.1, 174.3. HRMS (ESI) m/z = calc. for C₂₆H₃₃N₄O₂S, [M+H]: 465.23188 found 465.23111.

2-((3r,5r,7r)-adamantan-1-yl)-5-((4-(4-(3-methoxyphenyl)-2-methylpiperazin-1-yl)but-2-yn-1-yl)thio)-1,3,4-oxadiazole (6h)

Pale brown oily product (0.290 g, 73%). IR (cm^–1): 3377, 2852, 1778, 1668, 1485, 1298, 1236, 1215, 1172, 1043, 768. ¹H NMR (500 MHz, CDCl₃): δ 1.04 (d, 3H, J= 6.4 Hz, CH₃), 1.74 (br s, 6H, Adamantyl), 2.01 (s, 6H, Adamantane), 2.05 (br s, 3H, Adamantyl), 2.45 (br s, 2H, –NCH₂CH₂N), 2.69 (br s, 2H, –NCH₂CHN), 2.76 (br s, 2H, –NCH₂CH₂N), 3.31 (s, 2H, –NCH₂), 3.75 (s, 3H, -OCH₃), 3.80 (br s, 2H, –NCHCH₂N), 4.00 (s, 2H, –SCH₂), 6.38 (d, 1H, J= 8.1 Hz, Ar), 6.43 (s, 1H, Ar), 6.51 (d, 1H, J= 8.1 Hz, Ar), 7.13 (t, 1H, J= 8.1 Hz, Ar). ¹³C NMR (125 MHz, CDCl₃): δ 13.5, 21.6, 27.7, 34.4, 36.2, 39.8, 47.3, 51.1, 52.2, 55.1, 57.8, 78.9, 79.7, 103.8, 104.6, 110.1, 129.7, 151.5, 160.6, 162.1, 174.1. HRMS (ESI) m/z = calc. for C₂₈H₃₇N₄O₂S, [M+H]: 493.26318 found 493.26273.

In vitro bioassay against CaMKIIδ kinase

The Invitrogen Z’-LYTE Kinase Assay Kit was utilized to evaluate the effectiveness of the generated compounds. The bioassay was carried out using 500 μM of ATP and CaMKIIδ kinase at an ideal concentration range of 2.7–4.7 nM. Molecules were first made into stock solutions with a concentration of 10 millimolar (mM) in DMSO. After that, these stock solutions were gradually diluted in a buffer solution to produce final molecular concentrations that ranged from 0.01 micromolar (µM) to 100 µM. In the final kinase reaction (10 μL), the DMSO content did not go above 1%. Using GraphPad Prism 7.04, nonlinear regression analysis of the log (concentration) versus inhibition % data was performed to estimate the inhibition percentage and IC50 value.

Docking study

Compounds 6a–h were subjected to molecular docking analysis within the binding pocket of CaMKIIδ kinase (PDB code: 5VLO, resolution = 2.06 Ǻ) in conjunction with the highly effective ligand 9EJ (IC₅₀ = 0.2 µM). ²⁷ The Discovery Studio 4.5 program (BIOVIA, USA) was used to carry out the docking studies. Using templates made especially for hydrogen atoms in Discovery Studio 4.5, hydrogen atoms were added to the protein structure. In later docking studies, the protein structure was used without energy minimization. Using a stiff receptor, CDOCKER combines simulated annealing and molecular dynamics techniques to execute lignad docking. ²⁵ The following steps make up the CDOCKER protocol: (1) High-temperature molecular dynamics simulations are used to construct ligand conformations; these simulations are started with different random seeds. (2) The ligand center is moved to a precise location within the receptor’s active site, and it is then randomly rotated to create the conformations. (3) After assessing the energy, the orientation is maintained but marginally reduced if it drops below a set threshold. This process continues until the maximum number of unfavorable orientations has been attempted or the target amount of low-energy orientations has been reached. (4) A simulated annealing molecular dynamics process is applied to each orientation, in which the temperature is first raised to a predetermined high temperature and then lowered to a desired temperature. (5) A hard receptor uses a potential that hasn’t been relaxed to carry out the ligand’s final shrinkage. Using the CHARMm force field to estimate the ligand strain, together with the interaction energy, the energy of each final position is calculated. Furthermore, the calculation of the interaction energy alone is also done. Only the postures with the highest scores (more negative, suggesting a larger possibility of binding) are preserved after the stances are ordered according on their CHARMm energy. Interaction energy calculations use a to increase efficiency and decrease computation time, interaction energy calculations utilize a non-bond energy grid rather than full potential energy terms. ²⁷

The current project has implemented the following CDOCKER parameters: A binding site sphere with a radius of 9.0 Å, centered around the co-crystallized ligand (PDB code: 5VLO), was incorporated into the crystallographic structures of CaMKIIδ kinase. The initial conformations of the ligands were subjected to energy minimization and subsequently heated to a temperature of 1000 K using 1000 molecular dynamics steps. For every ligand, this procedure was carried out 10 times, producing 10 randomly generated initial conformations. To modify its energy, each arbitrary conformer rotated 10 times inside the binding pocket. After determining the Van der Waals energies of the resultant conformers/poses, any energies that were 300 kcal/mol or higher were disregarded. A simulated annealing process including 2000 heating steps up to a target temperature of 700 K and 5000 cooling steps down to a target temperature of 300 K was applied to the conformers/poses that survived. Energy minimization was applied to the docked postures until a final minimization gradient tolerance of 0 kcal/mol/Å was reached.

Supplemental Material

sj-doc-1-chl-10.1177_17475198241262467 – Supplemental material for Synthesis, evaluation, and docking study of adamantyl-1,3,4-oxadiazol hybrid compounds as CaMKIIδ kinase inhibitor

Supplemental material, sj-doc-1-chl-10.1177_17475198241262467 for Synthesis, evaluation, and docking study of adamantyl-1,3,4-oxadiazol hybrid compounds as CaMKIIδ kinase inhibitor by Mohammed M Al-Mahadeen, Areej M Jaber, Raed A Al-Qawasmeh and Mutasem O Taha in Journal of Chemical Research