Fragment merging approach for design, synthesis, and biological assessment of urea/acetyl hydrazide clubbed thienopyrimidine derivatives as GSK-3β inhibitors

New thienopyrimidine derivatives were designed and synthesized as GSK-3β inhibitors based on the structure of active binding site of GSK-3β enzyme. In this study, compounds 6b and 6a were found to be moderate GSK-3β inhibitors with IC50s 10.2 and 17.3 μM, respectively. Molecular docking study was carried out by docking the targeted compounds in the binding site of the GSK-3β enzyme using the MOE program. Moreover, ADME study was performed to predict certain pharmacokinetic properties. The results showed that all synthesized compounds may not be able to penetrate the blood brain barrier; so, the chances of CNS side effects are predicted to be low. CYP1D6 is predicted to be inhibited by compounds (5a, 5d, 6a, 9a and 9b), So drug-drug interactions are expected upon administration of these compounds. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13065-023-01026-w.


Introduction
Cancer is a genetic disease with two main characteristics: uncontrolled growth of the cells and tissue invasion/ metastasis of the tissues [1] Cell cycle control is dependent on the four stages (G1, S, G2, M).A variety of metabolic pathways have been identified as critical processes for the start of a certain cell cycle event [2].In these biochemical pathways, many protein kinases are active and employ regulated phosphorylation processes to relay biological information to downstream signaling pathways [3].Glycogen Synthase Kinase-3 (GSK-3) is a multitasking serine/threonine protein kinase because of its broad participation in different signaling pathways [4].GSK-3 is a cyclin-dependent kinase (CDK) proline-directed kinase, which also includes mitogen-activated protein kinases (MAPKs), cyclin-dependent kinases (CDKs), and CDK-like kinases (CLKs).GSK-3α and GSK-3β that encode 51 and 47 kDa proteins, respectively.Both isomers are found in cells and tissues, and their physiological activity is similar.The most common isoform is GSk3β which overexpressed in different human cancer, that why it considers as strong therapeutic target [5].It is a kinase that belongs to the phosphotransferase family.Originally thought to control glycogen synthase, it is now shown to phosphorylate a huge variety of substrates, hence regulating a large range of biological activities, involving Wnt and Hedgehog signaling, transcription, and the insulin system [6].Despite structural similarities between two isoforms, their roles are distinguished by phosphorylation at unique locations.For the activation of GSK 3α and GSK 3β, Tyr 279 and Tyr 216 are two sites situated at the T-loop of GSK 3 (activation domain) and are phosphorylated by upstream signaling molecules.Obviously, GSK 3α and GSK 3β are inhibited by sitespecific phosphorylation, which is closely regulated by a variety of processes.All of these pathways for GSK3α inactivation at Ser21 and GSK 3β at Ser9 have been linked to the phosphoinositide3 kinase (PI3K) dependent mechanism.Activation of PI3 kinase, protein kinase A (PKA), and protein kinase B (PKB) (also termed as Akt), protein kinase C (PKC) and p90Rsk contribute to GSK3 inactivation.Inactivation of GSK 3β is also implicated in glycogen production, protein synthesis, cell invasion and cell proliferation.Consequently, GSK 3β is regarded as a possible therapeutic target in cancer therapy.

Rationale and design
The structural study of GSK-3β inhibitors were observed certain interactions between to the ATP binding site via hydrogen bonds with Asp133 or Val135 (two key binding residues) [13].Based on the urea moiety's substituent, we revealed two distinct mechanisms of binding.The first one, in the tetrahydropyridoisoindolone scaffold where the carbonyl group in one binding mode directs towards the catalytic Lys85, whereas the urea is parallel to the hinge area, forming a hydrogen bond contact between the urea and the backbone of Val135.In GSK3β, a second method of binding was more discovered.In the tetrahydropyridoisoindolone scaffold, the carboxyl group binds with the NH backbone of Val135 by a hydrogen link, and the catalytic Lys85 interacts with the urea via a hydrogen bond.If the ortho position contains nitrogen, the heterocyclic scaffold connected to the urea can form a further hydrogen bond with Lys85.The substituent connected to the saturated ring of the tetrahydropyridoisoindolone scaffold interacts with Thr138 through a hydrogen bond network or a van der Waals interaction, depending on the orientation of the residue side chain [14,15].According to the level of potency, activity of pyrrolopyrimidine (I), furopyrimidine (II), quinoline derivative (III) and tetrahydropyrido[1,2-a]isoindolone (IV) as GSK-3β inhibitors have IC 50 in micromolar or nanomolar range, ring replacement of pyrrolopyrimidine and furopyrimidine with thienopyrimidine core to explore additional interaction with the hinge region of kinase.
The goal of this work was to provide evidence to support the use of thienopyrimidine scaffold as a pharmacophoric constituent essential in the development of novel GSK-3β inhibitors [16].In this study thienopyrimidine scaffold was utilized to design novel GSK-3β kinase inhibitor depending on the main reasons: novelty of this fragment because it is not exploited yet in any clinically approved drug.Also, tetrahydrothienopyrimidine can be readily synthesized from commercially available materials.Based on the aforementioned findings, fragment merging approach and structure extension through linking the urea moiety or amide moiety with thienopyrimidine scaffold to explore if occupy a deep hydrophobic allosteric pocket.As a result, several substitution patterns on (R) on the terminal phenyl ring were studied to investigate the interaction with the terminal allosteric pocket (Fig. 2).
Several analytical and spectral approaches were used to understand the structural properties of the synthesized molecules.The 1 HNMR spectra for the targeted compounds ( 5a-d, 6a, b and 9a-c) were consistent with total number of protons.proton leads to a downfield shift in all the vinylic protons in the Z-isomer and an upfield shift in the vinylic protons in the E-isomer.Additionally, the spectra showed one signal of O-CH 2 showed as singlet around δ 5.19-4.70ppm as in compound (5a-d) confirming the E/Z configuration.Additional protons were shown as a three singlet peaks around δ 3.85, 3.84 and 3.78 ppm of methoxy groups in compound (5a), also, singlet peak around δ 2.97 ppm for two methyl groups of -N(CH 3 ) 2 as in compound (5d).Furthermore, the extra signal of aromatic protons are around δ 6.75-7.77ppm.
Regarding compound (6a) showed three signals around δ 10.62-9.34ppm representing the D 2 O exchangeable proton of 3NH group while two signals around 10.93-10.64ppm representing the D 2 O exchangeable proton of 2NH group in compound (6b).Additional aliphatic protons were shown in compounds (6a, b) as singlet peak around δ 4.73 and 4.82 ppm of the O-CH 2 group.Also, the extra signal of aromatic protons are around δ 7.18-8.10ppm.
On the other hand, 1 HNMR spectra of compounds (9a-c) revealed the appearance of the 2 characteristic signals of D 2 O exchangeable protons of NH groups around δ 11.23 and 8.72 ppm.Regarding compounds (9b) spectra, showed singlet peaks at δ 3.06-2.27ppm, respectively representing for the CH 3 groups.In addition to the signal of aromatic protons are around δ 6.15-7.40ppm.
In all synthesized compounds, protons were shown as a singlet peak at around δ 1.75-1.79ppm of four protons at C 6 , C 7 of cyclohexyl moiety, a singlet peak at around δ 2.74-2.78ppm for two protons at C 5 of cyclohexyl moiety, a singlet peak at δ around 2.80-2.88ppm for two protons at C 8 of cyclohexyl moiety.All spectra showed singlet peak of pyrimidine ring appear at around δ 8.34-8.29 ppm, The 13 CNMR spectra were consistent with number of carbons of the targeted compounds (6a, 6b, 9a, 9b), where shown as a singlet peak at around δ 22.18, 22.84, 25.00, 25.76 (cyclohexyl), 167.18 ppm (C=O) for (6a), two C=O signals at 164.26 and 166.64 ppm for compound (6b).Both compounds (6a, b) showed a signal around δ 48.03-46.93(OCH 2 ).IR data stretching signal revealed of N-H group around 3371-3433 cm −1 , aromatic C-H around 3000-3070 cm −1 , aliphatic C-H at 2908-2989 cm −1 , C=O or C=S around 1647-1708 cm −1 and C=N around 1600 cm-1 .Mass analysis was performed on compounds showed the presence of M + , M + + 1 and M + + 2 peaks with A 3:1 relative intensity ratio refers to chlorine isotopes as compounds (5b and 9c) while compound (5c) has relative intensity 1:1 corresponding to bromine isotopes.The founded molecular weights of the titled compounds were compared to their calculated molecular weights (Additional file 1: Sect."Introduction".)

Biological evaluation GSK-3β serine/threonine kinase inhibitory activity in vitro
Preliminary screening at 100 µM )single dose Concentration) The GSK-3β tyrosine kinase tests were carried out at Thermo Fischer Scientific USA (www.therm ofisc her.com/ selec tscre en).The investigation was carried out to evaluate the GSK-3β inhibitory activity of the synthesized compounds.During the main reaction of the kinase, a single residue of serine, threonine, or tyrosine in a produced FRET-peptide receives gamma-phosphate of ATP through the biological test Z'-LYTE.In the secondary process, Non-phosphorylated FRET-peptides are identified and degraded by a site-specific protease.The phosphorylation of FRET-peptides inhibits the development reagent from cleaving them.FRET between the donor (coumarin) and acceptor (fluorescein) fluorophores on the FRET-peptide is disrupted by cleavage, while FRET is maintained by uncleaved, phosphorylated FRET-peptides.The percentage of enzymatic activity that the tested substances inhibited GSK-3β kinase was compared to a 100 µM as refer-Scheme 2 Reagent and conditions: (i) S 18 , TEA, EtOH, RT, overnight (ii) HCONH 2 , reflux, 5 h (iii) PhSCN/ PhCNO derivatives, CH 3 CN, reflux, 24-48 h ence concentration of the kinase inhibitor (Staurosporine with IC 50 = 10 nM).As shown in Table 1, the compounds demonstrated moderate to outstanding inhibitory efficacy against GSK-3β kinases (Supplementary Material).
SAR study among the assessed thienopyrimidine derivatives with respect to the linker extension; it was found that the order of inhibitory activity seemed to be acetyl hydrazine-1-carbothioamide series (6a-b) more active than acetyl hydrazide series (5a-d).Unfortunately, other compounds showed poor inhibition on GSk-3β kinase.Nevertheless, the investigated thienopyrimidine derivatives (9b-c) with urea moiety either electron-donating or electron-withdrawing groups on terminal rings can be tolerated (Fig. 3).
Antiproliferative efficacy in vitro against NCI 60-cell line The National Cancer Institute "NCI", NIH, Bethesda, Maryland, USA (www.dtp.nci.nih.gov) chose six of the final compounds for the Developmental Therapeutic Program (DTP), codes (5a, 5b, 5c, 5d, 6a, 6b).The NCI screening service will favor compounds with drug-like modes of action based on computer-aided design.Selection The criterion for screening is the possibility of the submitted compounds to provide diversity to the NCI small molecule chemical collection.This Table 1 Percent inhibition against GSK-3β enzyme formed by the urea and amide-depend on analogues ( 5a-d, 6a-b and 9a-c)  2.

Results of in vitro NCI 60 cell panel assay
On the entire NCI 60 cell panel, an initial in vitro one dose anticancer investigation was performed.The results for each compound were presented as a mean graph of the percentage growth of the treated cells compared to the untreated control cells.The mean graph of 5d and 6a results of the NCI 60 cell line screening program are shown in (Additional file 1: Section 2).
Only acyl hydrazide derivatives (6b) and (6a) showed moderate inhibitory activity on GSK-3β kinase with IC 50 s of 10.2 and 17.3 uM, respectively, these compounds may be a gloss of hope to develop more selective GSK-3β inhibitors in the future perspective.

Molecular docking
In order to better understand the binding modalities and directions of active compounds into binding site of ATP for GSK-3β kinase enzyme, docking procedure is carried out using MOE software version 2019, and one of the ten retrievable docking configurations was picked.GSK-3β crystal structure in interaction with lead compound (IV) was obtained (PDB code: 1J1B).
The binding modes of GSK-3β inhibitors showed that they interact with the ATP binding site via hydrogen bonding with Asp133 or Val135 which represent two out of the three interactions in all inhibitors namely: two hydrogen bond donors interaction with the backbone carbonyls of Val135 and Asp133, and one hydrogen bond acceptor interacts with Val 135's -NH.The ligands also interact hydrophobically with Ile62, Phe67, Leu188, and Cys199 side chains, as well as polarly with Thr138, Glu185, and Asp200.

Validation of docking protocol
Redocking phosphoaminophosphonic acid-adenylate ester (ANP) was used to validate the docking algorithm into the active site of GSK-3β.With a root mean square difference (RMSD) of 1.83 Å between the top docking pose and the original crystallographic ligand, this was found to be effective in retrieving the previously described X-ray crystal structure binding site of ligand (Fig. 4).O 40-50% inhibition of growth, O 50-60% inhibition of growth, O 60-70% inhibition of growth, O 70-80% inhibition of growth, O 80-100% inhibition of growth Fig. 4 The aligning of the lead compound's X-ray active conformer (coloured in cyan) with the redocked posture (purple) at the GSK-3β binding site

Binding mode of the targeted compounds with GSK-3β binding site (PDB ID: 1J1B)
See Fig. 5.

In silico predictive ADMET study
The pharmacodynamic properties of the synthesized compounds were studied in silico for the prediction of their possible transport to the site of action.Also, due to the importance of their molecular characteristics for human pharmacokinetics, molecular properties of new drugs were examined throughout development.
For example, Lipinski's rule of five determines if a molecule of interest possesses chemical and physical qualities that would make it likely to be active orally.Oral bioavailability is likely to happen if at least three of the following requirements were met: There are a maximum of 5 H-bond donors, a maximum molecular weight of 500 Da, a maximum log P of 5, a maximum number of violations of 1 and a maximum number of H-bond acceptors of 10.The quantity of rotatable bonds, polar surface area, and drug oral bioavailability are all strongly correlated.According to the Veber rule, oral bioavailability for compounds with less than 10 rotatable bonds and a polar surface area (PSA) > 140 Å 2 is acceptable.Additionally, ADME was determined using the SWISSADME program.Additionally, the SWISSADME made a prediction regarding the potential pharmacokinetic effects on a number of cytochromes P450 enzymes (CYP450), including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, as well as the likelihood that these enzymes will serve as substrates (inducers) of Permeability glycoprotein (P-gp) (Table 3).Boiled-egg plot chart ADMET-Graphic, a 2D plot created utilizing determined by Swiss ADME, is used to display the findings of the ADMET study.All the substances were plotted outside the in yellow circle that Blood Brain Barrier (BBB) and Human Intestinal Absorption (HIA) graphs some compounds are in white circle and 3 compound are outside (Fig. 6).
Pharmacokinetic analyses of compounds (5a-d, 6a, b and 9a-c) on cytochrome P450 enzymes and P-glycoprotein revealed that CYP3A4 is predicted to be inhibited by all compounds, and all compounds except compound (6b) are predicted to be inhibitors of CYP2C9 & CYP2C19, while CYP1D6 is predicted to be inhibited by compounds (5a, 5d, 6a, 9a and 9b) and all compounds, with the exception of those (5a, 5d and 6b), are inhibitors of CYP1A2.Regarding P-gp, all substances are impermeable to glycoprotein except for (9a-9b).Additionally, the blood-brain barrier (BBB) is predicted to not be pass by all of the compound so it is expected to have no side effect on brain.

Conclusion
This work involved the design and synthesis of new GSK-3β inhibitors containing urea /acetyl hydrazide coupled thienopyrimidine inhibitors.The synthesized compounds were examined for their in vitro GSK-3β inhibitory activities.Compounds 6a, 6b, 9a, 9b and 9c were found to be moderately active as GSK-3β inhibitors with IC 50 range (10.2-41.8µM).In addition, six of the final compounds (5a, 5b, 5c, 5d, 6a and 6b).The National Cancer Institute "NCI" chose them for a single dose screening programmed at 10 μM in the whole NCI 60 cell panel.The thienopyrimidine-based derivative Table 3 Predicted AMDE and pharmacological parameters for targeted compounds ( 5a-d, 6a-b, 9a-c (5d and 6a) showed good anti-proliferative activity against specific cell lines as renal cancer cell line.
Finally, molecular docking study was performed to predict how these compounds bind to the GSK-3β active site.Compounds (6a, 6b, 9b) showed similar orientation and binding interactions with a larger likelihood to enter the GSK-3β pocket.The study of computer aided ADMET was also carried out, using Swiss ADME to investigate the pharmacokinetic properties of the tested compounds.

Chemistry
Loba chemie and alfa-aesar organics provided the starting ingredients and reagents, which were employed without further purification.Fisher Scientific or Sigma-Aldrich solvents were obtained and used without further purification.Drying of the solvent were done using molecular Sieves.TLC was used to monitor chemical reactions using silica gel 60 F254 packed on aluminium sheets purchased from Merck and viewed under ultraviolet light (λ = 254 nm).Silica (240-70 mm) was used for column chromatography.

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate (1)
To a stirred solution of cyclohexanone (5.2 g, 53 mmol) in ethanol (15 mL), were added ethyl cyanoacetate (6.37 g, 57 mmol), sulfur powder (1.60 g, 50 mmol) and piperidine (1.3 g, 15 mmol) and the resulting reaction mixture was stirred at either 50 °C overnight in water bath.The progress of reaction was monitored by TLC, after completion of disappearance of starting materials, cooled the reaction mixture to ambient temperature and the resulting solids were filtered and dried under vacuum.The obtained crude solid was further purified by recrystallization by ethanol to afford desired compound-1 (9.76 g, yield: 80.1%) as a yellow crystal [24].
Fig. 6 The newly synthesized compounds' Blood Brain Barrier (BBB) plot and human intestinal absorption (HIA)

Synthesis of targeted compounds (6a, b)
A mixture of (4) (0.17 g, 0.63 mmol) with phenyl isothiocyanate (0.17 g, 1.26 mmol) were dissolved in 15 ml dry toluene under reflux for 48 h and follow up the reaction with TLC till end the reaction, then cool to room temperature.A solid product was precipitated which was filtrated, dried to give compound 6a (yield 64%) as white powder (Spectral data in supplementary material).A mixture of compound (4) (0.3 g, 1.077 mmol) with 4-nitrobenzoyl chloride (0.2 g 1.077 mmol) was dissolved in 15 ml dry toluene and add drops of TEA in ice bath, stirring for 3 h and follow up the reaction with TLC till end the reaction.A solid product was precipitated during stirring, which was filtered off, washed well, dried and then recrystallized from ethanol to afford 6b (yield 64%) as yellow powder (Spectral data in supplementary material).

Biological assessment In vitro GSK-3β inhibitory assays
The assay was run to gauge the designed compounds' ability to inhibit GSK-3.The biochemical test Z'-LYTE A synthesized FRET-peptide receives the gamma-phosphate of ATP produced by the kinase during the main reaction at a single tyrosine, serine, or threonine residue.Non-phosphorylated FRET-peptides are found and degraded by a site-specific protease in the secondary reaction.Development Reagent cleavage is prevented by FRET-peptide phosphorylation.While FRET is maintained by uncleaved, phosphorylated FRET-peptides, FRET between the donor (coumarin) and acceptor (fluorescein) fluorophores on the FRET-peptide is broken by cleavage.The radiometric approach, which quantifies reaction progress, determines the ratio (the Emission Ratio) of donor emission to acceptor emission following excitation of the donor fluorophore at 400 nm.By calculating how much ATP is still in solution after a kinase reaction, it quantifies kinase activity.The quantity of ATP present and the degree of kinase activity are both negatively connected with the luminous signal from the experiment.

Assay for anti-proliferative action against a range of NCI 60-cell lines in vitro
The NCI cancer screening procedure includes a comparison of all substances to the sixty NCI cell line panel representing leukemia, NSCLC, cell lines from breast cancer, prostate cancer, melanoma, CNS cancer, renal cancer, ovarian cancer, and colon cancer at a single point of 10 uM.The results of the single-dose screen can be presented using a mean graph.

Protein preparation
The protein data bank was utilized to get the structure of the GSK3-β proteins, which has (PDB ID: 1JIB).Partial charges were calculated and the enzymes were protonated.In the simulations, water molecules surrounding the cocrystallized ligands were deleted.The defined and isolated binding pocket was determined.

Ligand preparation
The following steps are taken by test ligand structures to prepare them for docking: The target molecules were created using ChemBioDraw Ultra 14.0, which was then repeated to MOE. (2) In three dimensions, the chemicals were protonated.(3) The constructions were built to use as little energy as possible The Merk Molecular Force Field produces a gradient of 0.5 (MMff94x).( 4) Each molecule's force field partial charges were calculated.(5) Each molecule was subjected to a stochastic conformational analysis using default parameters, and the results were saved in a separate conformational database.(6) The most stable conformers of each molecule were recorded in a separate database in order for each molecule to dock onto the active site of the androgen receptor.

In silico ADMET study
Measuring drug probability and pharmacokinetics properties is critical in the development of novel medications.The Swiss Institute of Bioinformatics' SWISSADME server, a free web service, our target compounds' physicochemical characteristics were computed using ' ADME characteristics, pharmacokinetic qualities, and druglike nature may all be predicted.
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Fig. 2
Fig. 2 Schematic representation of targeted compounds screen employs 60 different Leukemia, melanoma, and additional tumor cell lines of the human from brain, kidney, lung, ovary, colon, breast, and prostate cancers.All compounds were picked based on their NCI codes.NSC: D-820696/1, NSC: D-820697/1, NSC: D-820698/1, NSC: D-820699/1, NSC: D-820700/1, NSC: D-820701/1.The initial 10 µM one dose percent inhibition assay was performed on the entire NCI 60 cell panel to examine the different chemotypes of this work.The results are presented as a percentage of cell growth on each of the 60 NCI cell line panels for each of the investigated substances in Table

Fig. 5
Fig. 5 Targeted compounds bind with GSK-3β binding site (PDB ID: 1J1B).Compound 6a in ATP binding site of GSK-3β showing four hydrogen bond interactions with Val135, Arg141, Ille62, Cys 199 residues, and hydrophobic interaction with Gln185 while compound 6b identified a critical hydrogen bond and its distances from the GSK-3β binding site; makes hydrogen bond interaction with Val135, Asp133, Lys85, Lys183 residues, and hydrophobic interaction with Val70 and Docking pose of compound 9b show the same critical interactions with the GSK-3β binding site; hydrogen bond interaction with Val135 residue and hydrophobic interaction with Val70.(Dotted blue lines are represent for hydrogen bonds and pale green bold line for hydrophobic interaction) Stuart Scientific gear was used to determine melting points and were uncorrected.NMR spectra were acquired on a Bruker at 400 MHz for 1 HNMR and at 100 MHz for 13 CNMR in ppm scale.Chemical shifts (δH) were provided relative to DMSO-d6.All coupling constant (J) values were given in Hz.The acronyms are as follows: s: singlet; d: doublet; and m: multiplet.IR spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer.An EI-MS LC/Ms/Ms mass spectrometer API 200 (AB Sciex Instrument) was utilized to get EI-MS spectra.Also, elemental analyses were also performed.

Table 2
Cell growth inhibition % of NCI 60 cancer cell lines displayed by studied final compounds (5a, 5b