Unravelling the potency of the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile scaffold with S-arylamide hybrids as PIM-1 kinase inhibitors: synthesis, biological activity and in silico studies

Abstract

PIM-1 is a type of serine/threonine kinase that plays a crucial role in controlling several vital processes, including proliferation and apoptosis. New synthetic S-amide tetrahydropyrimidinone derivatives were designed and synthesized as PIM-1 inhibitors with potential anticancer activity. Several biochemical assays were performed for anticancer assessment, including PIM-1 inhibitory assays, MTT, apoptosis and cell cycle, gene expression analysis, c-MYC analysis, and ATPase inhibitory assays. Compounds (8c, 8d, 8g, 8h, 8k, and 8l) exhibited strong in vitro broad antiproliferative activity against MCF-7, DU-145, and PC-3, with a relatively higher SI index suggesting minimal cytotoxicity to normal cells. Furthermore, these compounds induced mixed late apoptosis and necrosis with cell cycle arrest at the G2/M phase. Moreover, compounds 8b, 8f, 8g, 8k, and 8l showed potent inhibitory action against PIM-1 kinase, with corresponding IC₅₀ values of 660, 909, 373, 518, and 501 nM. In silico prediction studies of physiochemical properties, molecular dynamics, and induced fit docking studies were performed for these compounds to explain their potent biological activity. In conclusion, new pyrimidinone compounds (8c, 8d, 8g, 8h, 8k, and 8l) exhibit potential PIM-1 inhibitory activity and can be used as promising scaffolds for further optimization of new leads with selective PIM-inhibitors and anticancer activity.

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1 Introduction

Cancer is one of the top three causes of death in 177 of 183 countries.¹ In 2022, the global incidence of cancer was significant, with around 20 million new cases diagnosed and 9.7 million deaths.² Cancer is a serious societal, public health, and economic issue in the twenty-first century, accounting for about one in every six fatalities (16.8%) and one in every four deaths from noncommunicable diseases (22.8%) worldwide.¹

PIM (provirus integration site for Moloney leukaemia virus) kinases are oncoprotein-type serine/threonine kinases that regulate cell proliferation, survival, metabolism, cellular trafficking, and signalling.³ PIM kinases have three isoforms: PIM-1, PIM-2, and PIM-3; the first isoform has been found to be overexpressed in many haematological and solid malignancies.⁴ PIM-2 is overexpressed in myeloma, lymphoma, and leukaemia, while PIM-3 expression is higher in adenocarcinomas.⁵ PIM kinases play several roles in carcinogenesis, including multiple myeloma proliferation, anti-apoptosis, cell cycle control, and bone degradation.⁴ PIM kinases can block apoptosis and increase cell cycle progression in many cancer types, such as prostate cancer, and their overexpression correlates with grade and neoplastic transformation.⁴

Overexpression of PIM-1 in breast cancer is associated with a poor prognosis in HER-2 and hormone-negative tumors.⁶ PIM kinases phosphorylate various cellular substrates, such as myelocytomatosis (MYC), cell cycle modulators such as cyclin-dependent kinase inhibitor (CDKN) 1A and 1B, as well as cell division cycle (CDC) 25A and 25C, signalling intermediates such as neurogenic locus notch homolog protein 1 (Notch1), and apoptosis modulators (BCL-2-associated agonist).⁴ Myelocytomatosis oncogene (MYC) is an important transcription factor that works closely with PIM-1 and PIM-2 and affects cell proliferation, microRNA regulation, cell metabolism, and anti-apoptosis by activating or inhibiting signalling pathways.⁷ PIM kinases phosphorylate MYC at Thr-58, Ser-62, and Ser-329 on the N-terminal domain, boosting their stability and transcriptional activity and promoting the progression of cancer.⁷ PIM kinases are involved in several distinct signalling pathways linked to cancer cells, and their ATP binding pockets differ significantly from those of most other protein kinases, and they are catalytically active even when phosphorylated.⁸ Based on the aforementioned finding, targeting PIM kinases is an intriguing approach because knocking down PIM kinases causes non-fatal in vivo phenotypes, implying that clinical inhibition of PIM may have fewer adverse effects.⁹ Some small molecule inhibitors directly targeting the adenosine triphosphate (ATP)-binding domain of PIM proteins have been developed and have already been well-researched for getting into clinical trials. Structure–activity relationship studies revealed that potent PIM-1 inhibitors possess key structural features, including an imidazo[1,2-b]pyridazine scaffold as in SGI-1776 (I), a benzylidene-1,3-thiazolidine-2,4-dione scaffold as in AZD1208 (II) and SMI-4a (V), a 5-fluoropicolinamide moiety as in PIM447 (III), and an indolin-2-one nucleus as in CX-6258 (IV)^9–12 (Fig. 1). Various pharmacophoric nuclei of small-molecule inhibitors for PIM kinases have been generated and reported within the past few years. First of all, cyanopyridine and cyanopyridone cores (VII–XII) are frequently substituted with various aryl moieties at positions 4 and 6.^13–17 Furthermore, the literature showed that the pyridine ring is abundant in the skeletal backbones of several key anticancer medicines and PIM-1 inhibitors.^13,16 Second, the diaryl pyrimidone scaffold (XIII) has been reported as a potent PIM-1 kinase inhibitor.

Fig. 1 Some small molecule PIM kinase inhibitors in clinical and preclinical trials.

Third, the fused derivatives of benzofuropyrimidin-4-ones XIV, benzothienopyrimidin-4-ones XV, and pyridopyrimidin-4-ones XVI have been reported earlier and exhibited potent and selective PIM-1 kinase inhibitors^13,18 (Fig. 2).

Fig. 2 Some reported pyridine, pyridone and fused pyridine and pyrimidine as potent PIM-1kinase inhibitors.

However, there are no published data on 4-oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile as PIM-1 kinase inhibitors. Therefore, pyrimidines have been implicated in the structure scaffold in order to increase the PIM-1 kinase inhibitory activity of the previously published compounds (VII–XVII) due to many properties in common with pyridines, such as reduced resonance stabilization and difficulty in N-alkylation and N-oxidation.

Our rational design depends on the introduction of 4-oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile as a core structure in PIM kinase inhibitors, depending on the main reasons: firstly, the fragment merging approach between the 4-oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile scaffold resembling that of reported pyrimidone derivatives as PIM-1 inhibitors and amide functionalities to increase linker extension with different substitutions of electron-donating or withdrawing groups at the terminal phenyl ring in order to change the electronic configuration and possible conformations, establish a SAR for this study, and investigate their potential to create more hydrogen bonds in the PIM-1 kinase's hinge area. Second, the 6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile scaffold can be readily synthesized. Finally, this fragment has drug-like properties (MW = 229, tPSA = 64, clog P = 0.945, H-bond donor = 2, H-bond acceptor = 2). In addition, the carbonitrile group was also kept constant to ensure binding to Lys67, aiming to increase binding affinity and hence biological activity¹⁹ (Fig. 3).

Fig. 3 Rationale and design of targeted compounds as PIM-1 inhibitors.

In order to verify our hypothesis, we co-crystallized representative target molecules (8d, 8b, 8g, 8f, 8j, 8l, 8c, and 8a) to co-crystallized conformers of VII with PIM-1 kinase (PDB code: 2OBJ) using Cresset BMD Field Align. The idea is that molecules with similar field patterns will exhibit similar patterns of biological activity, which is the foundation for the significance of field alignment research. The suggested compounds' estimated molecular field similarity values (alignment score range: 0.72 to 0.65) showed a strong molecular field similarity with the co-crystallized VI within the active region of PIM-1. Given that VII and our compounds share the same electronic environment (Fig. 4), our compounds are expected to have better PIM-1 inhibitory activity.

Fig. 4 An investigation on the field alignment of specific tetrahydropyrimidine derivatives. [A] Correct alignment of VII (cyan) and compound 8d (green); score: 0.72. [B] Compound 8b (green) and VII (cyan) have proper field alignment; score: 0.72. [C] Compound 8g (green) and VII (cyan) are aligned; score: 0.71. [D] VII (cyan) and compound 8f field alignment (green); score: 0.708. [E] Compound 8j field alignment (green) and VII (cyan): score: 0.706. [F] Compounds 8l (green) and VII (cyan) have proper field alignment; score: 0.701. [G] Compounds 8c (green) and VII (cyan) have proper field alignment; score: 0.70. [H] Compound 8a (green) and VII (cyan) are aligned; score: 0.698. * Icosahedral field points stand for reference compounds, and spherical field points represent designed compounds. Fields can be computed thanks to field points, which are compressed field patterns. More possible points of interaction are indicated by larger field points. van der Waals surface field points are represented by yellow, hydrophobic field points by gold, negative field points by cyan, and positive field points by red.

The purpose of this work is to demonstrate the relationship between PIM-1 inhibition and in vitro anticancer activity against a wide screen of cancer cell lines and evaluate the biological activity of the most active compound(s) on the most sensitive cancer cell line(s). Computer-aided in silico studies such as ADME, molecular docking, and molecular dynamics (MD) studies will be performed for the synthesized potent PIM-1 inhibitors to illustrate their predicted pharmacokinetic properties and drug likeness, and forecast the compounds' binding mode and stability.

2 Results and discussion

2.1 Chemistry

Our convergent synthesis approach starts with the preparation of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile intermediates (4a–c) using the Biginelli reaction by the reaction of thiourea (1) with ethyl cyanoacetate (2) and appropriate aldehyde derivatives (3) in the presence of anhydrous K₂CO₃ and refluxing in absolute ethanol for 6–7 hours to obtain 4a–c after neutralization with acetic acid.²⁰ On the other hand, N-aryl-2-chloroacetamides (7a–e) were synthesized by the reaction of substituted anilines (5a–e) with 2-chloroacetylchloride (6) in dichloromethane and in the presence of triethylamine as a base to obtain 7a–e derivatives in good yields.^21,22 In Scheme 1, Williamson ether synthesis of thioethers was adopted to obtain targeted compounds (8a–n) through the interaction of key intermediates (4a–c) with N-aryl-2-chloroacetamides (7a–e) using anhydrous K₂CO₃/KI in DMF reflux for 10–12 hours to furnish the targeted compounds (8a–n) in high yields²³(Scheme 1).

Scheme 1 Reagents and conditions: (i) Abs. EtOH, K₂CO₃, reflux 10–12 h; (ii) neutralized with CH₃COOH; (iii) TEA, CH₂Cl₂, stirring for 3–4 h in an ice bath; (iv) K₂CO₃, KI, DMF, reflux 10–12 h.

The structures of the synthesized compounds (8a–n) were supported by spectral and microanalytical methods. ¹H NMR spectra showed the existence of the characteristic peak due to the S–CH₂ group at the 3.81–4.19 ppm range. ¹H NMR spectra showed one signal at around δ 10.19–11.14 ppm representing the D₂O exchangeable proton of the amide NH proton. Regarding compounds 8k, 8l, 8m, and 8n, additional aliphatic protons were shown as a singlet signal at δ 2.25–2.35 ppm of the extra methyl group. Compounds 8f–j showed a singlet signal at δ 3.76–3.81 ppm for the methoxy group proton.

¹³C NMR spectra of compounds 8a–n displayed three upfield signals assigned for the S–CH₂ group at 35.71–36.49 ppm, for OCH₃ at 55.01–55.92 ppm, and for the CH₃ group at 20.89–21.52 ppm. Concerning the carbamide group, it was characterized with a distinct signal of C=O at the 159.75–165.75 ppm range. Finally, the mass spectra of compounds 8a–e and 8i showed the presence of M⁺ and M⁺ + 2 peaks corresponding to chlorine isotopes.

2.2 Biological evaluation

2.2.1 In vitro PIM kinase enzymatic assay.
2.2.1.1 Initial screening at a single dose of 10 μM concentration. The PIM-1 kinase assays were performed at Thermo Fischer Scientific, USA. The Z'-LYTE biochemical assay was used to test the designed compounds' PIM-1 inhibitory activity using a fluorescence-based, coupled-enzyme method. It focused on the differences in the susceptibilities of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The emission ratio can be used to calculate the degree of FRET-peptide phosphorylation. When the FRET-peptide complex is phosphorylated (i.e., there is no kinase inhibition), the emission percentage will remain small, and when it is not phosphorylated (i.e., there is kinase inhibition), the emission percentage will remain large. All target compounds were tested against PIM-1 kinase in a single-point concentration assay at 10 μM. The new synthetic compounds showed either 56–89% or potent (90–100%) inhibitory activity against PIM-1 kinase (Table 1).

Table 1 In vitro evaluation of PIM-1 inhibitory activity by compounds 8a–n at 10 μM concentration

Compd ID	R	R1	PIM-1 (% of inhibition)
8a	Cl	H	56%
8b	Cl	3-F	96%
8c	Cl	4-F	84%
8d	Cl	4-Cl	82%
8e	Cl	4-OCH3	72%
8f	OCH3	H	90%
8g	OCH3	3-F	100%
8h	OCH3	4-F	61%
8i	OCH3	4-Cl	77%
8j	OCH3	4-OCH3	87%
8k	CH3	H	96%
8l	CH3	3-F	97%
8m	CH3	4-Cl	72%
8n	CH3	4-OCH3	89%

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2.2.1.2 Measurement of potential enzyme inhibitory activity (IC₅₀). Promising candidates (8b, 8f, 8g, 8k, and 8l) demonstrating PIM-1 inhibition percentage above 90% at 10 μM concentration were selected for further assessment of their inhibitory and selectivity activity to PIM-1 kinase activity. TBB (4,5,6,7-tetrabromo-1H-benzotriazole) was used as a positive standard control in the experiment. The positive control should exert a well-known characteristic biological mechanism of action to validate experimental data and be used in comparative studies for the new compounds.²⁴ In our study, TBB was used as a positive control in the PIM-1 inhibitory assay since it is known and being tested in several studies as a PIM-1 inhibitory agent and even being used as a standard in the PIM-1 activity assay and chemically modified for the synthesis of new TBB analogues as PIM-1 inhibitors.^25–27 The assessment included dose-related enzymatic inhibition of PIM-1 at 5 different concentrations (1 nM, 10 nM, 100 nM, 1 μM, and 10 μM) to evaluate their IC₅₀ values.

TBB showed an IC₅₀ of 1120 nM (1.12 μM), while the five tested compounds displayed potent PIM-1 inhibitory activity compared to TBB, with IC₅₀ values in the nanomolar range (373–909 nM) (Table 2, Fig. S1†).

Table 2 IC₅₀ values against PIM-1 achieved by the most active derivatives

Compd ID	PIM-1 (% of inhibition)	IC50 (nM)
8b	96%	660
8f	90%	909
8g	100%	373
8k	96%	518
8l	97%	501
TBB	—	1120

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2.2.2 In vitro antiproliferative and cytotoxicity activity. The antiproliferative and cytotoxic properties of the synthetic compounds (8a–n) were examined in vitro against three different human cancer cell lines: MCF-7 (breast cancer), DU-145 (prostate cancer), and PC-3 (prostate cancer), in comparison with the normal human fibroblast cell line WI-38 for calculation of the safety index (SI). The MTT assay was employed to determine IC₅₀, with doxorubicin and quercetin being used as the standard drugs. The compounds were divided into three categories based on their IC₅₀ values (IC₅₀ < 10 μg mL⁻¹, IC₅₀ between 10 and 50 μg mL⁻¹, and IC₅₀ > 50 μg mL⁻¹).

Table S1† displays that doxorubicin has IC₅₀ values of 34.40 ± 1.1, 18.76 ± 1.9, and 59.47 ± 4.3 μg mL⁻¹ against MCF-7, DU-145, and PC-3, respectively. Also, quercetin showed comparable IC₅₀ values of 32.83 ± 4.7, 37.93 ± 5.1 and 37.83 ± 4.2 μg mL⁻¹ against MCF-7, DU-145, and PC-3, respectively. This may suggest that both doxorubicin and quercetin have moderate activity on the selected cancer cell lines and are matched with previously observed data in the literature.^28–33 Additionally, SI values for doxorubicin range from 0.55 to 1.73 and for quercetin, they range from 0.33 to 0.38, suggesting their lower safety index and potential adverse effect.^34–36

Regarding the use of a positive control in cell-based assays, it is well known that a positive control is used to generate a known, repeatable response of the key endpoint result being evaluated in the assay and hence, there are some important characteristics that should be available and considered when a positive control is chosen. The important characteristics of the positive control should be fit for purpose in the measurement system to provide the expected response. For example, a cell cytotoxicity assay would have a positive control material that is very well known to cause cell death, while an assay evaluating the potential of a chemical to cause inflammation would require a positive control material known to induce inflammation-like responses in different cell type systems.²⁴ However, the role of TBB in cell apoptosis, cell cycle arrest, and other related modulatory pathways has not yet been well settled in all biological systems.^24,25

Therefore, we used doxorubicin as a positive control in apoptosis and cell cycle assays and its key modulatory mediators at gene and protein levels since doxorubicin is well known for preventing relegation of topoisomerase-mediated DNA breaks, thus inhibiting replication, transcription, and cell proliferation, and inducing apoptosis and cycle arrest activities. Also, doxorubicin has the ability to modulate the key regulatory apoptotic and cell cycle arrest genes and proteins such as caspase-3,³⁷ cytochrome-C,³⁸ AKT1,³⁹ PI3K,⁴⁰ CDK-4,⁴¹c-Myc⁴² and cyclin-c.⁴³ Additionally, quercetin was used as a positive control with Pim-1 inhibitory activity as previously mentioned.^44,45

For MCF-7 cells, synthetic analogues 8c, 8g, 8k, 8l, 8h, and 8d showed potent antiproliferative activity on the selected cancer cell line with IC₅₀ values in the range of 2.221 ± 1.7–8.794 ± 6.8 μg mL⁻¹ and relatively higher SI values in the range of 4.68–33.08, suggesting their selectivity and safety margins. Compounds 8c (chlorophenyl with 4-fluorophenyl acetamide), 8g (methoxyphenyl with 3-fluorophenyl acetamide), 8l (methylphenyl with 3-fluorophenyl acetamide), and 8h (4-methoxyphenyl-4-fluorophenyl acetamide) showed the most potent antiproliferative activity. Among the variations of “small substituents” in the phenyl ring, fluorine (F),⁴⁶ chlorine (Cl)^47,48 methyl (CH₃),⁴⁹ and methoxy (OCH₃)⁵⁰ have been identified as key moieties in approved drugs and medicinal chemistry design.

The observed potent antiproliferative activity of the previous compounds may be due to the addition of fluorine at the third or fourth position of the phenyl acetamide linker, enhancing the anticancer activity, and this reason matched with previously observed data in the literature with structures of PIM-1 inhibitors in clinical trials as I, III, and V.¹² Fluorine-containing compounds are generally more stable due to the unique size and electronegativity of the C–F bond, which gives these molecules the ability to enhance their potency, selectivity, metabolic stability, and pharmacokinetic characteristics. Also, compounds 8d (4-chlorophenyl-4-chlorophenyl acetamide) and 8k (methylphenyl with phenyl acetamide) showed strong antiproliferative action resulting from the presence of electron-donating or electron-withdrawing groups, which may alter the electronic configuration, conjugation, and potential conformations inside the PIM-1 kinase active site with the goal of boosting binding affinity and, consequently, biological activity.⁵¹

Compounds 8f (4-methoxyphenyl-phenyl acetamide), 8b (4-chlorophenyl-3-fluorophenyl acetamide), 8n (4-methylphenyl-4-methoxyphenyl acetamide), 8m (4-methylphenyl-4-chlorophenyl acetamide), 8e with a 4-chlorophenyl-4-methoxyphenyl acetamide moiety, and 8i (4-methoxyphenyl-4-chlorophenyl acetamide) exhibited moderate activity with an IC₅₀ range of 10.68 ± 2.5–34.84 ± 4.3 μg mL⁻¹ and SI values in the range of 2.22–4.68, suggesting their moderate antiproliferative activity and potential side effects. The moderate antiproliferative activity of methoxy-containing compounds is due to the OCH₃ moiety: a methyl (CH₃) group and an oxygen (O) atom. The O atom is hydrophilic, and the CH₃ group is hydrophobic, and it would be expected that the O atom would offer a single pair of electrons that could function as a hydrogen bond acceptor (HBA) and change the molecule's electronic properties by acting as both an electron-withdrawing group (EWG) and an electron-donating group (EDG), which may stabilize the conformation of the molecules via intermolecular hydrogen bonding and, consequently, biological activity.⁵² Compound 8j with the (4-methoxyphenyl-4-methoxyphenyl acetamide) moiety is the only compound that showed the weakest antiproliferative activity (IC₅₀ = 55.86 ± 4.6 μg mL⁻¹) and a higher incidence of side effects (SI = 1.09) (Table S1,†Fig. 5).

Fig. 5 Dose–response curves for the determination of IC₅₀ values of synthetic analogs using the MTT assay: A) WI-38, B) MCF-7, C) DU-145, and D) PC-3. The percentage of cell viability was plotted against the logarithmic concentration of synthetic analogs. Data points represent the mean ± standard deviation from three independent experiments. E) Doxorubicin and quercetin in WI-38, MCF-7, DU-145 and PC-3, respectively (from left to right).

For DU-145 cells, synthetic analogues 8c (chlorophenyl with 4-fluorophenyl acetamide), 8f with 4-methoxyphenyl-phenyl acetamide substitution, 8k (methylphenyl with phenyl acetamide), 8g (methoxyphenyl with 3-fluorophenyl acetamide), 8l (methylphenyl with 3-fluorophenyl acetamide), 8b (4-chlorophenyl-3-fluorophenyl acetamide), 8n with 4-methylphenyl-4-methoxyphenyl acetamide substitution, 8e with a 4-chlorophenyl-4-methoxyphenyl acetamide moiety, 8i (4-methoxyphenyl-4-chlorophenyl acetamide) and 8j (4-methoxyphenyl-4-methoxyphenyl acetamide) showed potent antiproliferative activity on the selected cancer cell line with IC₅₀ values in the range of 2.217 ± 1.0–9.560 ± 6.8 μg mL⁻¹ and relatively higher SI values in the range of 8.08–34.71, suggesting their selectivity and safety margins. These potent antiproliferative activity results are due to the same reasons mentioned before, in addition to compounds with a methyl substituent enhancing the conformational properties of a given scaffold, increasing hydrophobic interactions, and modulating physicochemical properties like log P and aqueous solubility.⁵³

Compounds 8n with 4-methylphenyl-4-methoxyphenyl acetamide, 8h with 4-methoxyphenyl-4-fluorophenyl acetamide, 8d with 4-chlorophenyl-4-chlorophenyl acetamide, and 8m with 4-methylphenyl-4-chlorophenyl acetamide substitutions showed moderate activity with an IC₅₀ range of 25.16 ± 1.2–50.30 ± 3.6 μg mL⁻¹ and SI values in the range of 1.04–2.85, suggesting their moderate antiproliferative activity and higher incidence of side effects, Table S1.†

For PC-3 cells, synthetic analogues 8f, 8d, 8m, 8e, and 8a (4-chlorophenyl-3-fluorophenyl acetamide) showed potent antiproliferative activity on the selected cancer cell line with IC₅₀ values in the range of 3.745 ± 1.1–8.346 ± 1.6 μg mL⁻¹ and relatively higher SI values in the range of 7.66–20.54, suggesting their selectivity and safety margins. Compounds 8c, 8k, 8g, 8l, 8b, 8n, 8h, and 8j showed moderate activity with an IC₅₀ range of 14.10 ± 0.9–49.19 ± 3.7 μg mL⁻¹ and SI values in the range of 1.07–4.31, suggesting their moderate antiproliferative activity and higher incidence of side effects. Compound 8i is the only compound that showed the weakest antiproliferative activity (IC₅₀ = 74.09 ± 1.9 μg mL⁻¹) and a higher incidence of side effects (SI = 0.67), Table S1.†

PIM1 expression is significantly higher in breast cancer tissues than in healthy breast epithelium,^54–56 and it is overexpressed in TNBC and linked to tumor development, cell survival, and chemotherapy resistance, indicating that it may be a valid biomarker for TNBC.^57,58 PIM1 expression is influenced by a variety of cytokines, growth factors, and mitogens, and previous reports have suggested that PIM1 may be enhanced by estrogen and estrogen receptor alpha-binding areas [6]. Four estradiol-bound ER-α binding sites are located far upstream of the PIM1 promoter, and they may function as estrogen-regulated enhancers for PIM1.⁵⁹ Key proteins become phosphorylated as a result of estradiol-induced PIM1 overexpression, which inhibits the production of cell cycle inhibitors (CDKN1A and CDKN2B). This process makes breast cancer tumors more invasive, speeds up the cell cycle, and prevents apoptosis.

PIM1 kinase is found in higher concentrations in tissues from high-grade prostate intraepithelial neoplasia and castration-resistant prostate cancer than in normal prostatic tissue and benign prostatic hyperplasia,⁶⁰ and in human prostate cancer, PIM1 kinase and the Myc gene are frequently co-expressed, which greatly increases c-Myc-driven tumorigenesis in a way that depends on the kinase activity.⁶¹ PIM1 contributes to prostate cancer by phosphorylating the androgen receptor (AR) at serine 213 (S213), which is essential for the AR's function and influences the expression of its target gene. This specific phosphorylation event is linked to more aggressive tumors, castration-resistant forms of the disease, and changes in the AR's transcriptional activity.⁶²

In conclusion, all targeted compounds showed sensitivity to the selected cancer cell lines with a variable degree of activity for the newly synthesized analogues. MCF-7 and PC-3 cancer cell lines will be selected for further biological assessments based on some reasons. First, MCF-7 and PC-3 are examples of breast and prostate cancers, respectively, and both cancer types showed higher mortality rate incidence worldwide and poor diagnosis and recurrence.⁶³ Second, both breast and prostate cancers displayed notable drug resistance and recurrence, with limited options of drugs available for complete remission.⁶³ Ultimately, the two cancer cell lines are widely accessible and make excellent in vitro models for evaluating novel synthetic analogues that may have anticancer properties. Therefore, compounds 8g (methoxyphenyl with 3-fluorophenyl acetamide) (inhibition % = 100% on PIM-1), 8b (4-chlorophenyl-3-fluorophenyl acetamide) (inhibition % = 96% on PIM-1), 8c (chlorophenyl with 4-fluorophenyl acetamide) (inhibition % = 84% on PIM-1), 8j (4-methoxyphenyl-4-methoxyphenyl acetamide) (inhibition % = 87% on PIM-1), 8n (4-methylphenyl-4-methoxyphenyl acetamide) (inhibition % = 89% on PIM-1), 8f (4-methoxyphenyl-phenyl acetamide) (inhibition % = 90% on PIM-1) and 8m (4-methylphenyl-4-chlorophenyl acetamide) (inhibition % = 72% on PIM-1) exhibited performance similar to the standard treatment and will be used for further assessment on the selected cancer cell lines.

2.2.3 Apoptosis assay. Pyrimidine analogues are known for their anticancer properties, induction of apoptosis through diverse mechanisms such as the caspase-3-dependent pathway, kinase inhibition, cell cycle arrest, mitochondrial membrane potential reduction, and increased ROS production.^55,64 The apoptosis assay results are shown in Table S2† and Fig. 6, using annexin V/PI staining and flow cytometry to analyze cell viability and apoptosis. This was consistent with previous studies demonstrating doxorubicin's impact on PC-3 cells.⁶⁵

Fig. 6 Contour plots showing the distribution of viable (LL), early apoptotic (LR), late apoptotic (UL), and necrotic (UR) cells in PC-3 (top) and MCF-7 (bottom) cell lines after 24 hour treatment with doxorubicin, quercetin and synthetic analogs (8g, 8b, 8c, 8j, 8n, 8f, 8m) compared to the 0.1% DMSO control, assessed by AV/PI staining using flow cytometry.

In the PC-3 cell line, the negative untreated control cells showed a typical distribution of viable cells with 81.55 ± 7.4% with a minimal percentage of early, late, and necrotic cells. Quercetin and doxorubicin were used as positive controls. Doxorubicin was able to effectively reduce cellular PC-3 viability to 18.82% ± 1.1 (***p < 0.001) and induce both late apoptosis (54.70% ± 4.3%, ***p < 0.001) and necrosis (23.02% ± 1.7%, **p < 0.01) compared to the negative control (Table S2† and Fig. 6). For quercetin, it was able to effectively reduce cellular PC-3 viability to 15.46 ± 1.3% (***p < 0.001) and induce both early apoptosis (53.16 ± 1.1%, ***p < 0.001) and necrosis (31.05 ± 1.1%, ***p < 0.01) compared to the negative control. Some analogues, such as 8b (4-chlorophenyl-3-fluorophenyl acetamide) and 8c (chlorophenyl with 4-fluorophenyl acetamide), induced significant late apoptosis. This was matched with previous studies showing the apoptotic effect of these substituents on PC-3 cells, where compounds bearing 3-trifluoromethyl- and 4-chloro-3-trifluoromethylphenyl groups showed strong cytotoxic effects through activation of the mitochondrial apoptosis pathway in PC-3 cells.⁶⁶ The observed findings indicated that the presence of an electron-withdrawing group (EWG) compared to an electron-donating group (EDG) resulted in higher activity.^46,49 Compound 8g (58.19% ± 2.3) with significantly lower necrosis (9.17% ± 2.4) has an F atom in the C3 position on the 6-methoxyphenyl-pyrimidine scaffold and exhibited higher activity than compound 8j (18.66% ± 1.3) having a 4-OCH₃ phenyl acetamide group on the 6-methoxyphenyl-pyrimidine scaffold. Moreover, compound 8f (31.43% ± 2.1) with un-substituted phenyl acetamide on a 6-methoxyphenyl-pyrimidine scaffold induced significant late apoptosis. Also, 8n (4-methylphenyl-4-methoxyphenyl acetamide) showed a similar effect to doxorubicin in inducing significant late apoptosis of cells with a range of 18.66 ± 1.3–38.53 ± 2.4% and necrotic cells (14.49 ± 1.4–37.91 ± 2.6%), Table S2.†

In the MCF-7 cell line, the negative untreated control cells showed a typical distribution of viable cells with 88.79 ± 7.7% with a minimal percentage of early, late, and necrotic cells. Doxorubicin as a positive control was able to effectively reduce cellular viability to 17.08 ± 1.1 (***p < 0.001) and induced both late apoptosis (50.01 ± 5.1%, ***p < 0.001) and necrosis (29.12 ± 3.1%, **p < 0.01) compared to the negative control, Table S2.† Quercetin as an additional positive control reduced cellular viability to 20.72 ± 2.8% (***p < 0.001) and induced only early apoptosis (75.26 ± 5.4%, ***p < 0.001) compared to the negative control. This matched previous studies showing the effect of doxorubicin and quercetin on MCF-7 cells.⁶⁷ Interestingly, the synthetic analogues 8b, 8c, and 8j showed comparable effects to doxorubicin in inducing significant late apoptosis in the range of 43.85 ± 3.6–47.99 ± 3.8% and necrosis (12.87 ± 3.8–16.00 ± 4.1%). Compounds 8g, 8n, 8f, and 8m were able to induce significant late apoptosis only in the range of 31.97 ± 3.5–51.25 ± 4.3%. The observed apoptotic activity was due to the aforementioned reasons in PC-3 results.

All the synthetic compounds showed induction of late apoptosis, indicating advanced cell death with potential for adverse inflammatory responses due to the release of intracellular contents.⁶⁸

2.2.4 Cell cycle assay. The cell cycle analysis of PC-3 and MCF-7 cells treated with doxorubicin, quercetin and pyrimidine-based synthetic analogs (8g, 8b, 8c, 8j, 8b, 8f, and 8m) for 24 hours showed distinct effects on the cell cycle distribution across different phases, as summarized in Table S3† and Fig. 7.

Fig. 7 Cell cycle analysis of A) MCF-7 and B) PC-3 cancer cell lines after 24 hour treatment with doxorubicin, quercetin and synthetic analogs (8g, 8b, 8c, 8j, 8n, 8f, 8m). Histograms illustrate phases of cell cycle arrest compared to the 0.1% DMSO control.

In the PC-3 cell line, the control group demonstrated a typical cell cycle distribution, with the following percentages of cells in each phase: 8.20% ± 0.6 in the sub-G0–G1 phase, 44.94% ± 4.5 in the G0–G1 phase, 12.90% ± 1.3 in the S-phase, and 33.46% ± 1.3 in the G2/M phase. Doxorubicin only was able to induce sub-G0–G1 cell cycle arrest (27.33 ± 1.6%, ***p < 0.001), while quercetin did not show any significant change in the cell cycle phases. None of the synthetic pyrimidine analogs (8g, 8b, 8c, 8j, 8b, 8f, and 8m) demonstrated any significant cell cycle arrest in comparison with the negative control. The results suggest that apoptosis in PC-3 cells may occur independently of cell cycle arrest,⁶⁹ suggesting possible mechanisms of resistance. Moreover, these contradictions with apoptosis result from cell type specificity, as well as concentration and duration of exposure-related effects.^70,71 In contrast, the breast cancer cell line MCF-7 showed an increased percentage of apoptotic cells (including early, late, and necrotic cells) after treatment with synthetic compounds compared to the control. Doxorubicin and quercetin induced cell cycle arrest at the G2/M phase (32.25% ± 2.2% and 71.65 ± 6.7%, ***p < 0.001, respectively) and caused a significant reduction of cells in the G0–G1 phase (39.72 ± 2.8% and 8.79 ± 4.2%, respectively), aligning with its known mechanisms of inducing apoptosis through DNA damage.⁷²

Most synthetic pyrimidine derivatives (8g, 8b, 8c, 8j, 8b, 8f, and 8m) caused a significant shift in cell cycle distribution, particularly at the G2/M phase, which may be due to apoptosis induction of the tested compounds by cell-cycle arrest at G2/M that deteriorates the genetic material. Our results follow previous studies that reported that the PIM-1 inhibition would have apoptosis-inducing activity through cell cycle arrest at the G2/M phase transition.⁷² Additionally, it was reported by Bachmann et al.,⁷³ who proved a novel function for PIM-1 in the G2/M cell cycle process as a positive regulator.⁷⁴ It's interesting to note that fluorine has the inductive ability to increase the acidity and decrease the basicity of many compounds, improving the lipophilicity and membrane penetration of fluorinated compounds. The process of apoptosis is believed to be significantly influenced by lipophilicity.^66,75 That is, the complexes with the groups CF₃, SCF₃, and SF₅ showed higher lipophilicity than those with F or F₂, which in turn affected their cytotoxic properties.⁵¹ These characteristics enable adjustments to the compounds' permeability, solubility, and protein binding.⁴⁶

In our study, compound 8b (4-chlorophenyl-3-fluorophenyl acetamide) was one of the most potent compounds, arresting 84.04% ± 5.6 of cells at G2/M (p < 0.001) and reducing the G0–G1 phase to 5.72% ± 3.7, indicating that the cells were no longer dividing and were being arrested at the G2/M checkpoint. Compounds 8g (methoxyphenyl with 3-fluorophenyl acetamide) and 8c (chlorophenyl with 4-fluorophenyl acetamide) arrested 59.03% ± 4.9 of cells at the G2/M phase and increased sub-G0–G1 cells to 14.58% ± 2.4 (p < 0.05), suggesting DNA fragmentation and apoptosis.^46,51

Furthermore, in the aromatic ring, a methoxy or fluoro group at the meta position increases the G2/M cell cycle arrest. Similar effects on the G2/M cell cycle arrest have been observed for a methyl group in the aromatic ring.^53,67 Therefore, compound 8j (4-methoxyphenyl-4-methoxyphenyl acetamide) also demonstrated strong arrest at G2/M (79.03% ± 3.6 and 74.51% ± 7.5, respectively, p < 0.001), indicating strong anti-proliferative effects. Compounds 8n (4-methylphenyl-4-methoxyphenyl acetamide), 8f (4-methoxyphenyl-phenyl acetamide), and 8m (4-methylphenyl-4-chlorophenyl acetamide) significantly increased the percentage of sub-G0–G1 cells (23.19% ± 2.3, 31.50% ± 3.1, and 29.24% ± 2.8, respectively, p < 0.05) while arresting a substantial portion of cells at the G2/M phase (55.81% ± 6.1, 48.02% ± 5.2, and 48.96% ± 4.7, respectively, p < 0.001), which demonstrates that these analogs induce both sub-G0–G1 and G2/M phase arrest in MCF-7 breast cancer cells, indicating their ability to disrupt cell cycle progression and promote apoptosis.⁷⁶

2.2.5 ATPase inhibitory assay. The significant G2/M arrest caused by the pyrimidine analogs in MCF-7 cells may be attributed to their potential role as ATPase inhibitors. ATPase is critical for the energy-dependent processes of ABC transporters, which mediate drug resistance in cancer cells by effluxing chemotherapeutic agents.⁷⁷ Inhibition of ATPase could disrupt these transporters, leading to intracellular accumulation of drugs and subsequent DNA damage and cell cycle arrest at the G2/M phase.⁷⁸ The arrest in this phase could further trigger apoptosis, particularly in cancer cells that rely on ATP-dependent mechanisms for survival and proliferation.^79–81 The ATPase activity assay was performed to evaluate the impact of the new synthetic analogs on ATPase activity in MCF-7 breast cancer cells. ATPase enzymes, particularly those belonging to the ATP-binding cassette (ABC) transporter family, play a significant role in the development of multidrug resistance (MDR) by actively effluxing chemotherapeutic agents out of cancer cells, thereby reducing their intracellular concentrations and therapeutic efficacy.^82,83 Key members of the ABC transporters implicated in MDR include P-glycoprotein (P-gp or ABCB1), multidrug resistance-associated protein 1 (MRP1 or ABCC1), and breast cancer resistance protein (BCRP or ABCG2).^84,85 The simplest phosphoryl transfer reaction, the hydrolysis of the symmetrical PPi substrate to two molecules of inorganic phosphate (Pi), is catalyzed by highly active pyrophosphatases (PPases) found in most tissues.⁸²

In this study, the amount of inorganic phosphate (Pi) liberated from ATPase enzymes was measured after 24 hours of exposure to the synthetic analogs. The results were compared to a solvent control and to doxorubicin, a standard chemotherapeutic agent known to be affected by ATPase-mediated drug efflux.⁸⁶ Doxorubicin demonstrated a 54.6% reduction in ATPase activity compared to the control, as shown in Fig. 8.

Fig. 8 The total activity of ATPase activity after the exposure of MCF-7 cells to new synthetic analogs. MCF-7 cells were treated with the IC₅₀ concentration of compounds for 24 h, and the enzymatic activity was measured colorimetrically. The ATPase activity observed in the 0.1% DMSO control was normalized to 100%. The activity in the presence of synthetic analogs was then detected and compared to the control. The mean change in ATP-degradation activity ± SEM of three independent experiments (n = 3) (color is not required in print) is shown.

Among the tested compounds, 8g (methoxyphenyl with 3-fluorophenyl acetamide), 8c (chlorophenyl with 4-fluorophenyl acetamide), and 8b (4-chlorophenyl-3-fluorophenyl acetamide) exhibited the highest reduction in ATPase activity, with percentages of 65.34%, 65.3%, and 55.7%, respectively. The inhibition is promoted by the presence of fluorine atoms in the previous analogues, which are potent and specific inhibitors of cytoplasmic PPases.⁸⁷ This significant inhibition suggests that these compounds may effectively inhibit ATPase activity, potentially overcoming MDR by decreasing drug efflux and enhancing intracellular drug accumulation.

Compound 8f (4-methoxyphenyl-phenyl acetamide) also showed considerable inhibition with a 57.3% reduction in ATPase activity, indicating its potential activity as an ATPase inhibitor.⁸⁸ Compound 8n (4-methylphenyl-4-methoxyphenyl acetamide) demonstrated moderate inhibitory effects, reducing ATPase activity by 43.55%, respectively. These findings indicate that it still possesses the ability to inhibit ATPase activity to a certain extent. Conversely, 8j (4-methoxyphenyl-4-methoxyphenyl acetamide) and 8m (4-methylphenyl-4-chlorophenyl acetamide) showed lower inhibitory effects on ATPase activity, with reductions of 23.5% and 33.4%, respectively. The modest inhibition by these compounds suggests that they may be less effective in overcoming MDR through ATPase inhibition.

2.2.6 Gene expression analysis.
2.2.6.1 Apoptosis-related genes in MCF-7 cells. The intrinsic apoptotic pathway is crucial for maintaining cellular homeostasis and suppressing tumors, and it is characterized by mitochondrial outer membrane permeabilization (MOMP). This process leads to the release of cytochrome-C (Cyt-C) and the activation of the apoptosome and caspases.⁸⁹ Disruption of this pathway in cancer cells often results in apoptosis resistance, facilitating tumor survival and progression.⁹⁰ Recent research has demonstrated that ATPase inhibitors significantly contribute to mitochondrial stress and cellular dysfunction by impairing ATP production.⁹¹ These inhibitors affect various cellular processes, including pH homeostasis and iron metabolism, which collectively enhance the activation of the intrinsic apoptotic pathway.⁸⁰ Consequently, ATPase inhibitors induce apoptosis primarily through this pathway, offering a promising approach to overcoming apoptotic resistance and improving cancer treatment outcomes.⁸¹

The intrinsic apoptotic pathway's efficacy was assessed by measuring the expression levels of caspase-3 and Cyt-C in MCF-7 cells treated with various synthetic analogs, and the results were compared to doxorubicin as shown in Fig. 9. Caspase-3 is recognized as an executioner caspase due to its pivotal role in the apoptotic process, where it facilitates the breakdown of essential cellular components, including DNA and cytoskeletal proteins. Research demonstrates that the activation of caspase-3 is frequently associated with cell cycle arrest, indicating a complex relationship between the mechanisms of apoptosis and cell cycle regulation in various types of cancer.^92–95 This enzyme specifically targets key regulators of the cell cycle, such as cyclin-dependent kinases (CDKs), resulting in irreversible arrest in cell cycle progression.⁹⁶ Additionally, studies have shown that the inhibition of ATPases can lead to reduced ATP levels, which in turn activate caspase-3.⁹⁷ A study on halogenated pyrrolo[2,3-d]pyrimidine derivatives, particularly those with chlorine and fluorine substitutions, has shown that these modifications significantly enhance their potency, selectivity, and therapeutic efficacy due to their role in upregulating pro-apoptotic proteins, including caspase-3.⁹⁸ Among the synthesized compounds, 8n (4-methylphenyl-4-methoxyphenyl acetamide) was particularly notable, with a 5.1-fold increase in caspase-3 expression, which is substantially higher than doxorubicin's 2.8-fold increase. Moreover, 8b (4-chlorophenyl-3-fluorophenyl acetamide) also demonstrated a significant effect, inducing a 4.4-fold increase in caspase-3 expression, surpassing the fold change seen with doxorubicin. In terms of Cyt-C expression, which reflects mitochondrial outer membrane permeabilization and subsequent apoptosome activation, compound 8b led to the highest fold change at 6.2, again showing superior performance compared to doxorubicin, which had a 3.2-fold increase, which may be attributed to the fluorine group that probably enhances the lipophilicity of the drug, increasing the drug cell penetration.⁵¹ Compound 8c (4-chlorophenyl-4-fluorophenyl acetamide) also proved to be highly effective, with a 4.3-fold increase in Cyt-C expression, outperforming doxorubicin. The results suggest that 8b and 8c are particularly effective in inducing apoptosis via the intrinsic pathway, as they exhibit a higher ability to activate both caspase-3 and Cyt-C compared to doxorubicin. Halogen bonding and methoxy substituents enhance the binding affinity of potential anticancer agents, increasing their efficacy.^50,51

Fig. 9 Analysis of key regulatory genes involved in intrinsic apoptosis following treatment of MCF-7 cells with IC₅₀ concentrations of new synthetic analogs for 24 hours. The quantification of target mRNA was normalized to the internal reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and compared to MCF-7 cells treated with the 0.1% DMSO vehicle for 24 hours. Relative gene expression was determined using the 2^−ΔΔCt method and is presented as the average of three independent experiments. Data are shown as mean ± SEM, n = 3 (color is not required in print).

2.2.6.2 Proliferation-related gene expression. The Akt1 and PI3K signaling pathways play a crucial role in regulating various cellular processes such as growth, survival, and metabolism.⁹² The PI3K (phosphoinositide 3-kinase) pathway initiates signaling through PI3K activation, which converts PIP2 (phosphatidylinositol-4,5-bisphosphate) into PIP3 (phosphatidylinositol-3,4,5-trisphosphate).⁹³ PIP3 is a secondary messenger that recruits and activates Akt (protein kinase B), specifically the Akt1 isoform, which then phosphorylates substrates involved in apoptosis, cell cycle progression, and survival.⁹⁹ In cancer research, the Akt1 and PI3K pathways have been extensively explored, particularly in MCF-7 breast cancer cells, due to their significant role in promoting tumor growth and therapy resistance.¹⁰⁰ These pathways contribute to cancer cell proliferation by preventing apoptosis and advancing the cell cycle, with dysregulation often leading to malignant cell growth.⁹²

Additionally, Akt1 and PI3K pathways interact with enzymes like ATPase, affecting cellular energy metabolism and further promoting cancer cell survival.¹⁰¹ This modulation of ATPase activity underscores the importance of these pathways in cancer energetics. The intrinsic apoptotic pathway, regulated by Akt1 and PI3K, is critical for programmed cell death, with Akt1 promoting survival by inhibiting pro-apoptotic factors and enhancing anti-apoptotic signals.^102,103 When disrupted, this balance can result in apoptosis resistance, a key factor in cancer progression. The Akt1–PI3K signaling axis is also linked to the c-Myc transcription factor, which influences cell cycle regulation.¹⁰⁴ Akt1 modulates c-Myc activity, which in turn regulates cellular growth and proliferation, making this interaction vital for understanding cancer development. Certain pyrimidine analogs have been found to target the Akt1 and PI3K pathways, suppressing cancer growth and inducing apoptosis.¹⁰⁴ In this study, several synthetic analogs were tested for their ability to inhibit the expression of Akt1 and PI3K, both of which are key regulators of cancer cell survival and proliferation. The results were compared to doxorubicin (Fig. 10).

Fig. 10 Analysis of AKT1 and PI3K gene expression following treatment of MCF-7 cells with IC₅₀ concentrations of new synthetic analogs and doxorubicin for 24 hours. The quantification of target mRNA was normalized to the internal reference gene GAPDH and compared to MCF-7 cells treated with the 0.1% DMSO vehicle control. Relative gene expression was determined using the 2^−ΔΔCt method, and the data are presented as the mean ± SEM from three independent experiments (color is not required in print).

Notably, 8n, 8j, and 8f demonstrated stronger inhibitory effects on the expression of one or both genes. For Akt1 expression, 8n (4-methylphenyl-4-methoxyphenyl acetamide) was the most effective, reducing levels to 0.1-fold—a significant decrease compared to doxorubicin's 0.2-fold reduction. This indicates that 8n could potentially be more effective at promoting apoptosis by inhibiting Akt1, a crucial protein in the PI3K/Akt signaling pathway (Fig. 10).

Regarding PI3K expression, compound 8j (4-methoxyphenyl-4-methoxyphenyl acetamide) showed the greatest suppression, lowering PI3K levels to 0.1-fold, compared to 0.5-fold for doxorubicin. PI3K activation is essential for survival pathways, and the ability of 8j to inhibit this gene more effectively highlights its therapeutic potential. The results of this study indicate that 8n, 8j, and 8f may be more effective than doxorubicin in suppressing the PI3K/Akt pathway, a key driver of cancer cell survival. The presence of methoxy groups on these pyrimidine derivatives appears to enhance their activity against this critical pathway. This is supported by research indicating that pyrimidine-5-carbonitrile derivatives can act as potential anticancer agents, particularly the trimethoxy derivative, which showed a multi-target inhibitor in the PI3K/Akt axis in breast cancer and leukemia models.¹⁰⁵ Although 8f (4-methoxyphenyl-phenyl acetamide) exhibited inhibition of PI3K that was not as strong as 8j, its moderate suppression of both genes suggests that it could effectively target multiple pathways, enhancing its therapeutic potential. Additionally, 8g and 8b were also effective, reducing PI3K expression to 0.2-fold. Compound 8g displayed balanced activity, with a 0.2-fold reduction in Akt1 and a 0.3-fold decrease in PI3K expression. Their enhanced ability to inhibit these pathways makes them promising candidates for cancer treatment, especially for overcoming drug resistance and improving therapeutic outcomes. Despite the lack of knowledge regarding intrinsic and acquired resistance to PI3K inhibitors, recent research on breast cancer has revealed that PIM protein kinase is another therapeutic target that makes cancer cells more susceptible to anticancer treatments that block this pathway.¹⁰⁶

2.2.6.3 Cell cycle-related genes in MCF-7 cells. The primary regulators of the cell cycle are cyclins and CDKs. CDKs are essential regulatory enzymes that control transcriptional processes and cell-cycle checkpoints in response to external and intracellular cues.¹⁰⁷ This leads to promoting cell proliferation by phosphorylating the target genes, such as the tumor suppressor protein retinoblastoma.¹⁰⁸ Dysregulation of CDKs is a common feature of cancer, making inhibition of individual members a promising target for cancer treatment.¹⁰⁹ There exist multiple mechanisms that govern the catalytic action of CDK4. The primary mechanism that governs its activation is its binding to cyclins, which exhibit a cyclical pattern of production and breakdown.¹¹⁰ A second step is also necessary for CDK4 activation, and that is CDK-activating kinase (CAK) phosphorylating the Thr160 residue of the CDK activation loop. By eliminating inhibitory phosphate groups from different tyrosine residues, Cdc25A phosphatase also contributes to CDK4 activation.¹¹⁰

In this study, CDK4 showed significant downregulation with several synthetic analogs. Fig. 11 shows that CDK4 gene expression was downregulated after treatment with compounds 8g, 8b, 8c, 8j, 8n, 8f, and 8m. However, compounds 8g, 8c, and 8n induced the most significant level of downregulation by 0.3% when compared to the doxorubicin group with a 0.2-fold change, while 8j showed the lowest downregulation with a 0.8-fold change. Interestingly, by downregulating CDK4, apoptosis is induced and MCF-7 cell viability is inhibited, resulting in cell cycle arrest.¹¹¹ Drugs that target CDK have revolutionized the treatment of metastatic breast cancer by regulating the cell cycle that induces apoptosis via blocking the cell cycle at G1/S and G2/M checkpoints through activation of caspases.¹¹² CDK4 inhibitors are now the new standard of therapy due to significant improvements in clinical outcomes.¹¹⁰

Fig. 11 Analysis of key regulatory genes for the cell cycle following treatment of MCF-7 cells with IC₅₀ concentrations of new synthetic analogs for 24 h. The quantification of target mRNA after retinoid treatment was relative to MCF-7 cells incubated with the 0.1% DMSO vehicle for 24 h and was normalized to the internal reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression was calculated by the 2^−ΔΔCt method and presented as an average of three independent experiments. The values are considered statistically significant compared to the solvent-treated control at **p < 0.01, ****p < 0.0001. Data are represented as mean ± SEM, n = 3 (color is not required in print).

Among all human cancer types, the locus encoding the transcription factor c-MYC is one of the most frequently amplified genes.¹¹³ This MYC gene family member is involved in a variety of biological processes, such as cell cycle regulation, protein synthesis, apoptosis, and cell adhesion, which regulate cell proliferation.¹¹⁴ Tumorigenesis and persistent tumor growth are linked to aberrant expression of c-MYC.¹¹⁵ Increased cellular proliferation is the outcome of coordinated changes in gene family expression levels caused by c-MYC overexpression.¹¹⁶ The control of the G1 phase transition in the cell cycle is the primary function of c-MYC.¹¹⁷

Here, c-MYC was downregulated in all analogs when pyrimidine derivatives were exposed to MCF-7 cells. Among the tested compounds, 8c (chlorophenyl with 4-fluorophenyl acetamide), 8b (chlorophenyl with 3-fluorophenyl acetamide), and 8m (4-methylphenyl-4-chlorophenyl acetamide) demonstrated potent effects, inducing a decrease in c-MYC expression compared to doxorubicin. Potential anticancer drugs' binding affinities are increased, and their efficacy is raised by halogen bonding, especially those with fluorine and chlorine replacement, which reduced the c-MYC level in a previous study.¹¹⁸ It is possible to explain the lower levels of c-MYC by the inhibitor's effects on protein stability and/or cell cycle progression.¹¹⁸ The widespread involvement of c-MYC in human tumors makes it a desirable target for therapy, and hence, blocking the metabolic pathways caused by c-MYC may result in a novel strategy for treating cancer.

As confirmation, we detected the expression of c-MYC protein in MCF-7 cells by the ELISA technique compared to the standard positive and negative groups. It is common for human malignancies to have dysregulated expression of the c-MYC protein, which is involved in energy-intensive processes including ribosome production and proliferation.¹¹⁹ It was demonstrated that MCF-7-treated cells had a substantially downregulated level of c-MYC protein expression with the compound 8n, which proved to be the most effective one with a 32.8 μg mL⁻¹ decrease in its expression, followed by 8g with 34.5 μg mL⁻¹ as compared to the positive (doxorubicin) and negative controls, as shown in Fig. 11 and 12. The least downregulated compounds were 8j and 8m with 77.4 μg mL⁻¹ and 66.4 μg mL⁻¹, respectively. Our study showed a consistent result with Shi et al., who reported that the plasma c-MYC gene level was correlated with the expression of c-MYC protein in breast cancer tissues.¹²⁰

Fig. 12 c-Myc protein determination in cell culture media of MCF-7 treated with new synthetic analogues compared to doxorubicin as a positive control. MCF-7 cells were treated with the IC₅₀ concentration of compounds for 24 h, and the protein concentration was measured colorimetrically using a standard curve of c-Myc protein. The c-Myc protein concentration was measured in comparison with the 0.1% DMSO control. The mean change in c-Myc protein concentration (ng mL⁻¹) ± SEM of three independent experiments (n = 3) is shown (color not required in print).

One highly conserved nuclear protein that enters the cell cycle is cyclin-c, which is represented by the CCNC gene.¹²¹ The dual function of cyclin-c is exemplified by its ability to both enhance and repress the development of cancer.¹²² Numerous investigations have reported on the function of cyclin-c in promoting cell division.¹²³ Cyclin-c has been demonstrated to collaborate with c-MYC and is thought to be involved in both the G1 and G2 stages of the cell cycle.¹²³ Through the regulation of Notch1 oncogene levels, cyclin-c functions as a tumor suppressor.¹²⁴ Regarding cyclin-c expression, the results were compared to the standard chemotherapy agent doxorubicin (Fig. 11). All compounds, especially 8m and 8b, showed downregulation of cyclin-c levels compared to doxorubicin. It was previously reported that compounds with chloride in their aromatic pyrimidine ring displayed significant enzymatic inhibitory activity, which was essential for the anticancer activity.¹²⁵ The halogenation of an aromatic ring stopped the G1 cell cycle, caused apoptosis, and inhibited cellular CDK8/cyclin-c as demonstrated through targeting p-STAT1S727.¹²⁶ The downregulation of cyclin-c likely contributes to indicating their ability to disrupt cell cycle progression and promote apoptosis.

Several studies showed that PIM-1 kinase acts cooperatively with c-Myc through the phosphorylation of a factor(s) that regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation. Therefore, assessment of key regulatory mediators such as caspase-3 and cytochrome-c was essential to ensure the link between PIM-1, c-Myc, and apoptosis stimulation.^7,127–129 Also, AKT1 and PI3K are two regulatory mediators whose overexpression contributes to carcinogenesis, proliferation, invasion, metastasis, and resistance to c-Myc-induced apoptosis in tumor cells.¹³⁰ Additionally, it was essential to gain a clearer understanding of the role of c-Myc in cellular proliferation; hence, we have performed gene expression analysis of the components that regulate cell cycle progression such as cyclin-c and CDK-4 levels.¹³¹ Therefore, all the selected markers are not only for standard analysis but also to correlate the effect of PIM-1 inhibition with key regulatory mediators of apoptosis and the cell cycle.

2.3 Structure–activity relationship (SAR)

The structure–activity link for the produced pyrimidinone derivatives' anticancer activity can be illustrated using the earlier findings. The pyrimidinone ring, a heterocyclic ring, was necessary for the anticancer action. SAR analysis among targeted compounds (8a–n) demonstrated that the p-Cl, p-OCH₃, and p-CH₃ substitutions on ring A improve the anticancer activity and selectivity. Furthermore, the anticancer activity was boosted by the fluorine substitution in ring B at the phenyl amide linker's third position. Because of the unique size and electronegativity of the C–F bond, molecules with a fluorine atom typically exhibit greater stability. The anticancer action is enhanced by the S-arylamide moiety's linker extension. Also, the presence of the amide linker lengthens the spacer between the pyrimidinone ring and the terminal phenyl ring (ring B) and may be a better fit for the PIM-1 active site (Fig. 13).

Fig. 13 Structure–activity relationship for design of the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile scaffold with S-arylamide linkers as potent PIM-1 inhibitors with high antiproliferative activity.

2.4 In silico investigation of predicting ADME parameters

To investigate the pharmacokinetic features of the synthesized compounds, a computer-aided ADME study was conducted using the SWISSADME tool. Blood brain barrier (BBB) and human intestinal absorption (HIA) plots were drawn using all the synthesized compounds (Fig. S2†). This type of study focuses on the chemical structure of the molecule and measures additional parameters like lipophilicity (log P), blood–brain barrier permeability (BBB), cytochrome P450 2D6 (CYP2D6), and polar surface area (TPSA). Every synthetic compound was located outside of the BBB ellipses in the BBB plot. Furthermore, the likelihood of central nervous system adverse effects is reduced since these targeted compounds may not be ready to cross the blood–brain barrier.

The HIA graphic indicates that four compounds had intestinal absorption predicted better since they fell outside the confidence ellipse (undefined), whereas the rest of the compounds fell inside the ellipse. The CYP2D6 value predicts the inhibitory and non-inhibitory aspects of the titled chemical structures of the cytochrome P450 2D6 enzyme. Most of the compounds are foreseen as non-inhibitors of CYP2D6; the side effects of liver dysfunction are not expected upon administration of these compounds. One facet of medication bioavailability is TPSA. Thus, it is considered that compounds that are actively absorbed and have a PSA greater than 140 have low bioavailability. With the exception of compound 8j, which has a PSA of 142.40 A⁰, all of the synthesized compounds have PSAs between 123.94 and 133.17. These values indicate good passive oral absorption. Table 3 lists the ADME study parameters.

Table 3 Computer aided ADMET screening of the synthesized compounds

Comp	HB^a donor	HB acceptor	Log P	TPSA^a (A^02)	Rotatable bonds	Lipinski violations	Veber violations	CYP2D6	CYP1A2
a HB: hydrogen bonding. TPSA: topological surface area.
8a	2	4	3.11	123.94	6	0	0	NO	YES
8b	2	5	3.32	123.94	6	0	0	NO	YES
8c	2	5	3.32	123.94	6	0	0	NO	YES
8d	2	4	3.63	123.94	6	0	0	NO	YES
8e	2	5	3.05	133.17	7	0	0	NO	YES
8f	2	5	2.58	133.17	7	0	0	NO	YES
8g	2	6	2.83	133.17	7	0	0	NO	YES
8h	3	6	2.89	133.17	7	0	0	NO	YES
8i	2	5	3.05	133.17	7	0	0	NO	YES
8j	2	6	2.62	142.40	8	0	1	NO	NO
8k	2	4	2.91	123.94	6	0	0	NO	YES
8l	2	5	3.15	123.94	6	0	0	NO	YES
8m	2	4	3.40	123.49	6	0	0	NO	YES
8n	2	5	2.89	133.17	7	0	0	NO	YES

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2.5 Molecular docking study

Human PIM-1 kinase is a two-lobed protein with 313 amino acids and a significant space separating its smaller N- and C-terminal lobes. A special hinge design using residues Glu121, Arg122, Pro123, Glu124, Pro125, and Val126 connects the two lobes. Two hydrogen bonds allow the hinge region of Ser/Thr kinases to connect with the adenine moiety of ATP. Between the two lobes and the hinge area is a hole where PIM-1 kinase's ATP-binding site is located.¹³² Based on how they bind, PIM-1 inhibitors can be broadly classified as either ATP-mimetic or non-ATP mimetic inhibitors. The first class showed excellent enzyme potency but limited or poor selectivity over other kinases. It interacts directly with the unique hinge region of the enzyme, in which Glu121 participates.^133,134 Second-class inhibitors connect with the part of the active site opposite the hinge region, which varies dramatically amongst kinases, while ATP or ATP mimetics engage with the ATP binding cleft (Lys67) in a different manner.^135,136 As a result, non-ATP mimetics have a tendency to be more potent and selective against the PIM-1 enzyme.

A number of inhibitors have been developed to combat drug-resistant PIM-1-associated illnesses. Most ATP-competitive inhibitors have been clinically tested for the treatment of multiple myeloma, acute myelogenous leukemia, and prostate cancer lymphomas; however, only a small number of them, such as SGI-1776 and AZD1208, have done so. Consequently, the new hope for inhibitor design that targets the non-hinge side of the ATP-binding pocket and provides structural variation among kinases is non-ATP mimetic inhibitors. Through the use of molecular docking, the ligand structure complex was studied in order to better understand the inhibitory mode of action for synthetic drugs. The goal of this work is to establish a relationship between the computational and experimental methods for examining the activity of pyrimidinone-S-amide derivatives. When the target compounds were docked, it became clear that the core scaffolds have taken on the lead compound's volumes and orientations at the hinge region. Validation of the docking algorithm was achieved by redocking VII into the active site of PIM-1 (2OBJ). This was found to retrieve the reported X-ray crystal structure binding mode of VII, with a root mean square difference (RMSD) between the top docking pose and original crystallographic geometry of 0.406 A° (Fig. 14).

Fig. 14 The alignment between the X-ray bioactive conformer of the lead compound (VII) (colored in green) and the docked pose of the same compound (orange) at the PIM-1 active site.

It was demonstrated that compound 8b forms the crucial critical contacts that are recognized for non-ATP mimetic inhibitors. As a result, a hydrogen bond was found between the NH group of the Lys 67 backbone and the NH and C=O of the pyrimidine core in PIM-1 kinase. Additionally, a second H-bond was created between the Glu89 residue and the C=O of the pyrimidine core. Additionally, a H-bonding network with the Phe187 residue was formed via a water bridge (HOH no. 413, 424, and 464). With the Asp186 residue, the carbonyl group of the 3-fluoro-phenyl acetamide molecule forms another hydrogen bond. Notably, Lys67 and Glu89 residues engage as “anchor residues,” and this association contributes more potently to the stability of the protein–ligand binding.¹³⁴ In general, our studies have pointed out the important functions that N–H–S interactions play in enzyme-binding pockets.¹³⁵ The design demonstrated that the substrate specificity and catalytic site efficiency were caused by a hydrogen bond between an S HB acceptor in compound 8f¹²⁶ and an N–H HB donor in the NH group of Lys 67, as well as a reaction with Phe187 through water bridge molecules.

Furthermore, a convincing binding of compound 8g to the ATP-binding region of PIM-1 suggests an ATP-competitive (ATP mimetic and non-mimetic) inhibitory mechanism. The oxygen methoxy group on the phenyl ring's notable interaction with the Lys67 side chain of PIM-1 and a hydrogen bond to a water molecule (HOH 413), which seemed to be an essential component of a wider H-bonding network in this area, was arguably the most important finding from this complex structure (Fig. 15 and 16). A second conserved water molecule (HOH 463) linked to Asp186 and Phe187, the catalytic residues of PIM-1 kinase, completed the apparent H-bonding network. Interestingly, the only potential interaction of compound 8g with the hinge region was at the Glu121 residue, where the complex structure formed a hydrogen connection between a nitrile group and the hydroxyl on the Glu121 main chain. Compound 8l demonstrated a crucial interaction with the Lys67 side chain of PIM-1, as well as a sulfur–hydrogen bond to two water molecules (HOH 464 and HOH 413) and a Glu89 residue that seemed to be a crucial component of another H-bond with the lysine residue and HOH 442 (Fig. 16). According to this SAR study, extending the amide linker in the hinge's pyrimidine core may offer more advantageous interactions with PIM-1 kinase. They indicated that the hydrogen bond donor–acceptor in the pyrimidine ring anchors at the sidechain of Asp186. An inhibitor with a C-shaped convex would point in the direction of the hinge. It would be located parallel to the hinge backbone and anchor at Asp128 and Phe187 through interactions mediated by water molecules. Along with being directed toward the hinge region, this amide linker extension binds at the Lys67 sidechain. We believe that the stable H-bonding network between two water molecules accounts for most of the inhibitor's efficacy.

Fig. 15 The molecular structure of PIM-1 kinase (ID PDB: 2OBJ), the basic structure of PIM-1 protein. The N-terminal leaf β-sheet is indicated by an arrow, and the C-terminal leaf α-helix is represented by a spiral.

Fig. 16 Interactions between the inhibitors and the typical interactive residues in the ATP-binding site of PIM-1 (PDB: 2OBJ). [A] Redocking of the lead compound (VII) in the PIM-1 kinase active site (PDB: 2OBJ). [B] Binding mode of 8b (purple) in the PIM-1 active site. [C] Compound 8f (purple) interaction in the active site of PIM-1 kinase. [D] Binding interaction of 8g (purple) in the PIM-1 active site. [E] Compound 8k (purple) and essential interaction in PIM-1 kinase. [F] Compound 8l (purple) interaction in the PIM-1 kinase active site. Cyan or green ellipses denote protein residues. Water molecules mediating protein–ligand interactions are shown as red circles.

This study clarifies the signaling pathways that regulate PIM kinases as a means of successfully treating human cancer. Genes involved in cancer cell survival and cell cycle progression are transcriptionally activated by all PIM kinases. A variety of pro-tumorigenic signaling molecules, such as MYC,⁷ CDKs (CDC25)³ and the PI3K/Akt¹⁰⁵ pathway have been identified as the downstream targets of PIM kinases. The release of cyt-c is crucial for caspase-3 activation.¹³⁶ On the other hand, PIM kinases promote Bcl-2 release.¹³⁶ By forming a heterodimer with BAX, the overexpressed Bcl-2 blocks the delivery of cyt-c and closes the mitochondrial permeability transition pore, preventing caspase-3 activation.¹³⁷ Apoptosis was further encouraged by the targeted compounds' increased expression of cleaved caspase-3.¹³⁸ According to the results, PIM1 inhibitors exhibited their enzymatic activity through significant apoptosis induction; compounds 8b, 8f, 8g, 8k, and 8l drastically hindered cell migration and proliferation. This made them appealing lead compounds for the development of chemotherapeutics to treat cancer (Table 4).

Table 4 Binding score of some targeted compounds within the active site of the PIM-1 enzyme

Compound	Binding score (kcal mol^−1)	Compound	Binding score (kcal mol^−1)
Lead (VII)	−8.1	8g	−8.3
8b	−9.1	8k	−8.5
8f	−8.0	8l	−8.6

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2.6 Molecular dynamics study

2.6.1 RMSD and RMSF analysis. The current study conducted extensive in silico investigations using molecular dynamics (MD) simulations. MD simulation provides important data and parameters for studying the dynamics of biological systems, such as accurately estimating the binding strength of a docked ligand–target complex. As a result, the anticipated binding coordinates obtained from PIM-1 docking with 8b, 8g, and the co-crystallized ligand (VII) were used for MD simulation. To assess the influence of each ligand on PIM-1 enzyme stability, the latter was subjected to MDS in its Apo form. As shown in Fig. 17A, the inhibitory compounds (8b and 8g) were able to stabilize the PIM-1 enzyme, as evidenced by similar or lower RMSD values compared to Apo PIM-1. The RMSD value of the PIM–8g complex was 0.115 Å, similar to that of the PIM-co-crystallized ligand (0.118 Å), while the Apo PIM achieved 0.123 Å. Meanwhile, that of the PIM–8b complex was 0.127 Å, similar to that of the apoprotein.

Fig. 17 A: RMSD analysis for 100 ps of MDS, Apo protein PIM-1 (black), PIM-1–8b (blue), PIM-1–8g (orange) and PIM-1-co-crystallized ligand (VII) (green); B: RMSF analysis for 100 ps of MDS, Apo protein PIM-1 (black), PIM-1–8b, PIM-1–8g, and PIM-1-co-crystallized ligand; C: time evolution of the radius of gyration (R_g) value for the co-crystallized ligand, 8g and 8b; D: solvent accessible surface area (SASA) plot for the co-crystallized ligand, 8g and 8b.

The capacity of compounds 8b and 8g to limit the dynamic character of PIM-1 by creating stable complexes, as demonstrated by lower RMSD values, provides clear evidence of its inhibitory activity on PIM-1 (Fig. 17A). To supplement the RMSD calculation results, the RMSF value for each residue was calculated. As expected, the RMSF number matched the conclusions reached from the RMSD calculations. The average RMSF of the Apo PIM-1 residues was 0.056 Å, while the average RMSF values for the PIM-1 residues in association with 8b, 8g, and the co-crystallized ligand (VII) were 0.059, 0.056 and 0.060 Å, respectively (Fig. 17B). The suggested binding modes of 8b and 8g with the PIM-1 active site were thus confirmed by the RMSD and RMSF values, which suggested that the inhibitory activity of these molecule was due to their capacity to form stable complexes with PIM-1 kinase. The protein's compactness during the simulation is gauged by the radius of gyration (R_g).

Compounds 8b and 8g and the co-crystallized ligand (VII) had R_g values of 1.952, 1.946 and 1.950 Å (mean), respectively, as seen in Fig. 17C. Since R_g remains constant for every protein during the simulation, no significant conformational changes take place.

We applied the solvent-accessible surface area (SASA) to find the behavior of the hydrophilic and hydrophobic residues of VII, 8b, and 8g. The results revealed that the amino acid residues of the 8b and 8g structures have similar SASA values as compared to the VII structure, and they maintained the solvent accessibility during a simulation time of 100 ps (Fig. 17D).

To find out how hydrogen bonds contributed to the total binding interactions, a hydrogen bonding study between each compound and the protein was conducted. The number of H-bonds created over the simulation period is shown in Fig. 18. The reference compound (VII) shows a lower number of formed H-bonds. In contrast, compounds 8b and 8g have the greatest amount of H-bonds to the protein, indicating a strong binding and a high docking score, and also reflecting their high enzymatic activity in the biological evaluation (Fig. 18).

Fig. 18 Number of intermolecular hydrogen bonds between targeted compounds (8b, 8g) and the PIM-1 enzyme.

3 Experimental

3.1 Chemistry

Melting points were ascertained using a Stuart digital device. The infrared spectra were recorded using a Shimadzu FT-IR 8400S infrared spectrophotometer. Using a Bruker spectrophotometer, ¹H NMR and ¹³C NMR spectra were recorded at different frequencies (100 MHz for ¹³C NMR and 400 MHz for ¹H NMR) in deuterated dimethyl sulfoxide (DMSO) at the Beni Suef University Faculty of Pharmacy. At El-Fayoum and Al-Azhar Universities in Egypt, mass spectra were obtained.

3.1.1 Synthesis of targeted compounds (4a–c).
General procedure. Thiourea (1.5 g, 20 mmol), ethyl cyanoacetate (2.6 g, 2.1 ml, 20 mmol), suitable aldehyde (20 mmol), and K₂CO₃ (2.8 g, 20 mmol) were combined in 50 ml of 95% EtOH and refluxed for 12 hours before being cooled. After filtering, the precipitate was cleaned with ethanol. The desired thione was then extracted by dissolving the product in hot H₂O, filtering it while still hot, and neutralizing it with a small amount of HOAc. From DMF/H₂O, it was filtered, rinsed with water, dried, and crystallized.^139–141

3.1.2 Synthesis of targeted intermediates (7a–e).
General procedure. Chloroacetyl chloride (1.1 equiv.) was added at 0 °C to an amine (1.0 equiv.) solution in DCM, and the reaction mixture was agitated for 4 hours at room temperature. Next, TEA (1.1 equiv.) was added. By recrystallizing the crude product in ethanol, the excess solvent DCM was eliminated in vacuo.^142,143

3.1.3 Synthesis of targeted compounds (8a–n). Compounds 4a–c (0.79 mmol), appropriate intermediates (7a–e) (0.79 mmol), potassium carbonate (1.18 mmol), and the catalytic amount of KI were all weighed and then added in turn to a 25 mL round-bottom flask along with the appropriate amount of DMF. The reaction was heated to 90 °C and agitated for 10 to 12 hours while TLC was used to gauge its completion. Following the completion of the reaction, the system was allowed to cool to room temperature before the appropriate volume of water was added. To obtain the required compounds (8a–n), the resultant solid was vacuum-filtered, dried, and then recrystallized from ethanol.
3.1.3.1 2-((4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-phenylacetamide (8a). The titled compound was separated as buff crystals (yield 70%); m.p. 230–232 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.34 (s, 1H, NH D₂O exchangeable), 7.88 (d, J = 8.8 Hz, 2H, ArH), 7.57 (d, J = 8.0 Hz, 2H, ArH), 7.36 (d, J = 8.8 Hz, 2H, ArH), 7.33 (t, J = 8.0 Hz, 2H, ArH), 7.08 (t, J = 7.2 Hz, 1H, ArH), 4.17 (s, 2H, –C̲H̲_2̲–S); ¹³C NMR (101 MHz, DMSO-d₆)δ 166.69, 166.33, 165.72, 161.71, 139.39, 136.92, 134.41, 131.07, 129.28, 128.90, 123.91, 119.41, 116.24, 93.62, 36.10 (CH₂); FT-IR (max, cm⁻¹): 3325,3201 (2NH), 3059 (CH aromatic), 2935 (CH aliphatic), 2191 (C≡N), 1678 (C=O amide); EI-MS: (M_wt.: 396.85): m/z (% rel. int.), 396.27 (M⁺, 27.65%), 398.19 (M⁺ + 2, 20.71%) 75.07 (100%); anal. calcd. for C₁₉H₁₃N₄O₂S Cl: C, 57.51; H, 3.30; N, 14.12; S, 8.08; found: C, 57.75; H, 3.41; N, 14.39; S, 8.20.
3.1.3.2 2-((4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(3-fluorophenyl)acetamide (8b). The titled compound was separated as buff crystals (yield 63%); m.p. 240–242 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.53 (s, 1H, NH D₂O exchangeable), 7.87 (d, J = 8.0 Hz, 2H, ArH), 7.52 (d, J = 11.0 Hz, 1H, ArH), 7.37 (d, J = 8.0 Hz, 2H, ArH), 7.35 (t, J = 8.0 Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 1H, ArH), 6.90 (t, J = 8.0 Hz, 1H, ArH), 4.19 (s, 2H, –C̲H̲_2̲–S); ¹³C NMR (101 MHz, DMSO-d₆)δ 166.42, 166.36, 166.11, 163.82, 161.42, 141.07, 140.96, 136.97, 134.34, 131.04, 130.97, 130.88, 128.89, 116.10, 115.23, 110.49, 110.28, 106.37, 106.10, 93.80, 36.10 (CH₂); FT-IR (max, cm⁻¹): 3286,3147 (2NH), 3086 (CH aromatic), 2985 (CH aliphatic), 2214 (C≡N), 1658 (C=O amide); EI-MS: (M_wt.: 414.84): m/z (% rel. int.), 414.66 (M⁺, 26.22%), 416.39 (M⁺ + 2, 19.86%) 86.32 (100%); anal. calcd. for C₁₉H₁₂N₄O₂S ClF: C, 55.01; H, 2.92; N, 13.51; S, 7.73; found: C, 55.28; H, 3.06; N, 13.76; S, 7.68.
3.1.3.3 2-((4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(4-fluorophenyl)acetamide (8c). The titled compound was separated as buff crystals (yield 60%); m.p. 180–182 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 11.04 (s, 1H, NH D₂O exchangeable), 7.80 (d, J = 8.0 Hz, 2H, ArH), 7.56 (d, J = 11.0 Hz, 1H, ArH), 7.47 (d, J = 8.0 Hz, 2H, ArH), 7.33 (d, J = 7.2 Hz, 1H, ArH), 7.29 (t, J = 8.4 Hz, 1H, ArH), 6.87 (t, J = 8.4 Hz, 1H, ArH), 3.85 (s, 2H, –C̲H̲_2̲–S); ¹³C NMR (101 MHz, DMSO-d₆)δ 172.08, 170.35, 168.44, 166.45, 161.45, 141.32, 141.21, 136.56, 135.16, 130.84, 130.49, 128.66, 119.90, 115.11, 110.19, 109.88, 106.21, 105.95, 89.95, 35.71 (CH₂); FT-IR (max, cm⁻¹): 3282,3147 (2NH), 3086 (CH aromatic), 2989 (CH aliphatic), 2214 (C≡N), 1658 (C=O amide); EI-MS: (M_wt.: 414.84): m/z (% rel. int.) 414.54 (M⁺, 100.00%), 416.73 (M⁺ + 2, 25.16%); anal. calcd. for C₁₉H₁₂N₄O₂S ClF: C, 55.01; H, 2.92; N, 13.51; S, 7.73; found: C, 55.27; H, 3.08; N, 13.69; S, 7.79.
3.1.3.4 N-(4-Chlorophenyl)-2-((4-(4-chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetamide (8d). The titled compound was separated as buff crystals (yield 69%); m.p. 200–202 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.30 (s, 1H, NH D₂O exchangeable), 7.88 (d, J = 7.6 Hz, 2H, ArH), 7.42 (t, J = 7.6 Hz, 4H, ArH), 7.12 (d, J = 5.2 Hz, 2H, ArH), 4.12 (s, 2H, –C̲H̲_2̲–S); FT-IR (max, cm⁻¹): 3448, 3394 (2NH), 3066 (CH aromatic), 2927 (CH aliphatic), 2220 (C≡N), 1651 (C=O amide); EI-MS: (M_wt.: 431.29): m/z (% rel. int.) 431.56 (M⁺, 47.85%), 433.73(M⁺ + 2, 34.82%), 435.01(M⁺ + 4, 16.83%), 178.73 (100%); anal. calcd. for C₁₉H₁₂N₄O₂S Cl₂: C, 52.91; H, 2.80; N, 12.99; S, 7.42; found: C, 53.14; H, 2.97; N, 13.26; S, 7.59.¹⁴³
3.1.3.5 2-((4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(4-methoxyphenyl)acetamide (8e). The titled compound was separated as brown crystals (yield 68%); m.p. 200–202 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.59 (s, 1H, NH D₂O exchangeable), 7.80 (d, J = 8.4 Hz, 2H, ArH), 7.84 (dd, J = 3.6/9.2 Hz, 4H, ArH), 6.87 (d, J = 8.8 Hz, 2H, ArH), 3.81 (s, 2H, –C̲H̲_2̲–S), 3.72 (s, 3H, –OC̲H̲_3̲); FT-IR (max, cm⁻¹): 3263, 3132 (2NH), 3070 (CH aromatic), 2958 (CH aliphatic), 2218 (C≡N), 1654 (C=O amide); HR-MS: (M_wt.: 426.88): m/z (% rel. int.), 427.06326 (M⁺ + H), 429.0607 (M⁺ + 2 + H); anal. calcd. for C₂₀H₁₅N₄O₃SCl: C, 56.27; H, 3.54; N, 13.13; S, 7.51; found: C, 56.43; H, 3.65; N, 13.40; S, 7.70.
3.1.3.6 2-((5-Cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-phenylacetamide (8f). The titled compound was separated as brown crystals (yield 73%); m.p. 317–319 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.37 (s, 1H, NH D₂O exchangeable), 7.92 (d, J = 8.8 Hz, 2H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 7.33 (t, J = 8.0 Hz, 2H, ArH),7.08 (t, J = 7.6 Hz, 1H, ArH), 6.82 (d, J = 8.8 Hz, 2H, ArH), 4.18 (s, 2H, –C̲H̲_2̲–S), 3.78 (s, 3H, –OC̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 166.67, 165.74, 165.70, 162.57, 161.82, 139.43, 131.36, 129.30, 127.57, 123.93, 119.44, 116.78, 114.20, 91.84, 55.91 (O–C̲H̲_3̲), 36.01 (CH₂); FT-IR (max, cm⁻¹): 3471,3251 (2NH), 3059 (CH aromatic), 2947 (CH aliphatic), 2233 (C≡N), 1654 (C=O amide); HR-MS: (M_wt.: 392.43): m/z (% rel. int.), 393.10178 (M⁺ + H); anal. calcd. for C₂₀H₁₆N₄O₃S: C, 61.21; H, 4.11; N, 14.28; S, 8.17; found: C, 60.98; H, 4.27; N, 14.54; S, 8.09.¹⁴⁴
3.1.3.7 2-((5-Cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(3-fluorophenyl)acetamide (8g). The titled compound was separated as buff crystals (yield 68%); m.p. 248–250 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.63 (s, 1H, NH D₂O exchangeable), 10.33 (s, 1H, NH D₂O exchangeable), 7.88 (d, J = 8.4 Hz, 1H, ArH), 7.55 (t, J = 10.0 Hz, 1H, ArH), 7.35 (m, 2H, ArH),6.89 (d, J = 8.4 Hz, 1H, ArH), 6.83 (d, J = 8.8 Hz, 1H, ArH), 4.14 (s, 1H, –C̲H̲_2̲–S), 3.78 (s, 3H, –OC̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 167.88, 161.66, 135.85, 131.29, 121.48, 121.41, 115.88, 115.66, 114.23, 106.23, 103.78, 55.92 (O–C̲H̲_3̲), 36.49 (CH₂); FT-IR (max, cm⁻¹): 3267,3221 (2NH), 3066 (CH aromatic), 2989 (CH aliphatic), 2222 (C≡N), 1662 (C=O amide); HR-MS: (M_wt.: 410.42): m/z (% rel. int.), 411.09249 (M⁺ + H); anal. calcd. for C₂₀H₁₅N₄O₃SF: C, 58.53; H, 3.68; N, 13.65; S, 7.81; found: C, 58.80; H, 3.72; N, 13.89; S, 7.96.
3.1.3.8 2-((5-Cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(4-fluorophenyl)acetamide (8h). The titled compound was separated as buff crystals (yield 62%); m.p. 230–232 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.51 (s, 1H, NH D₂O exchangeable), 7.90 (d, J = 8.8 Hz, 2H, ArH), 7.60 (t, J = 8.4 Hz, 2H, ArH), 7.16 (t, J = 8.8 Hz, 2H, ArH), 6.88 (d, J = 8.4 Hz, 2H, ArH), 4.12 (s, 2H, –C̲H̲_2̲–S), 3.80 (s, 3H, –OC̲H̲_3̲); FT-IR (max, cm⁻¹): 3454, 3282 (2NH), 3084 (CH aromatic), 2993 (CH aliphatic), 2220 (C≡N), 1637 (C=O amide); EI-MS: (M_wt.: 410.42): m/z (% rel. int.), 410.57 (M⁺, 40.00%), 41.13 (100%); anal. calcd. for C₂₀H₁₅N₄O₃SF: C, 58.53; H, 3.68; N, 13.65; S, 7.81; found: C, 58.74; H, 3.79; N, 13.76; S, 7.91.
3.1.3.9 N-(4-Chlorophenyl)-2-((5-cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)acetamide (8i). The titled compound was separated as buff crystals (yield 65%); m.p. 268–270 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.64 (s, 1H, NH D₂O exchangeable), 10.05 (s, 1H, NH D₂O exchangeable), 7.83 (s, 1H, ArH), 7.47 (m, 3H, ArH), 7.10 (m, 3H, ArH), 6.94 (s,1H, ArH), 3.85 (s, 1H, –C̲H̲_2̲–S), 3.81 (s, 3H, –OC̲H̲_3̲); FT-IR (max, cm⁻¹): 3454,3282 (2NH), 3084 (CH aromatic), 2993 (CH aliphatic), 2220 (C≡N), 1637 (C=O amide); EI-MS: (M_wt.: 426.88): m/z (% rel. int.), 426.69 (M⁺, 19.33%), 428.41(M⁺ + 2, 10.83%), 263.76 (100%); anal. calcd. for C₂₀H₁₅N₄O₃SCl: C, 56.27; H, 3.54; N, 13.13; S, 7.51; found: C, 56.44; H, 3.68; N, 13.41; S, 7.63.¹⁴⁵
3.1.3.10 2-((5-Cyano-4-(4-methoxyphenyl)-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(4-methoxyphenyl)acetamide (8j). The titled compound was separated as buff crystals (yield 61%); m.p. 292–294 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.25 (s, 1H, NH D₂O exchangeable), 7.92 (d, J = 8.4 Hz, 2H, ArH), 7.48 (d, J = 9.2 Hz, 2H, ArH), 6.88 (t, J = 8.0 Hz, 4H, ArH), 4.12 (s, 2H, –C̲H̲_2̲–S), 3.79 (s, 3H, –OC̲H̲_3̲), 3.72 (s, 3H, –OC̲H̲_3̲); FT-IR (max, cm⁻¹): 3243,3167 (2NH),3089 (CH aromatic), 2935 (CH aliphatic), 2222 (C≡N), 1654 (C=O amide); EI-MS: (M_wt.: 422.46): m/z (% rel. int.), 422.39 (M⁺, 11.7793%), 318.33 (100%); anal. calcd. for C₂₁H₁₈N₄O₄S: C, 59.71; H, 4.29; N, 13.26; S, 7.59; found: C, 59.98; H, 4.35; N, 13.53; S, 7.64.¹⁴⁴
3.1.3.11 2-((5-Cyano-6-oxo-4-(p-tolyl)-1,6-dihydropyrimidin-2-yl)thio)-N-phenylacetamide (8k). The titled compound was separated as buff crystals (yield 78%); m.p. 264–266 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.32 (s, 1H, NH D₂O exchangeable), 7.78 (d, J = 8.0 Hz, 2H, ArH), 7.56 (d, J = 7.6 Hz, 2H, ArH), 7.33 (t, J = 8.0 Hz, 2H, ArH),7.14 (d, J = 8.0 Hz, 1H, ArH), 7.08 (t, J = 7.2 Hz, 1H, ArH), 4.18 (s, 2H, –C̲H̲_2̲–S), 2.33 (s, 3H, –C̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 165.98, 165.92, 165.65, 161.75, 142.49, 139.33, 132.68, 129.48, 129.28, 123.97, 119.52, 116.42, 92.93, 35.93 (CH₂–S), 21.30 (C̲H̲_3̲); FT-IR (max, cm⁻¹): 3229,3197 (2NH), 3066 (CH aromatic), 2935 (CH aliphatic), 2191 (C≡N), 1674 (C=O amide); EI-MS: (M_wt.: 376.33): m/z (% rel. int.), 376.33 (M⁺, 20.92%), 298.28 (100%); anal. calcd. for C₂₀H₁₆N₄O₄S: C, 63.81; H, 4.28; N, 14.88; S, 8.52; found: C, 63.92; H, 4.45; N, 15.07; S, 8.68.
3.1.3.12 2-((5-Cyano-6-oxo-4-(p-tolyl)-1,6-dihydropyrimidin-2-yl)thio)-N-(3-fluorophenyl)acetamide (8l). The titled compound was separated as buff crystals (yield 67%); m.p. 200–202 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 11.14 (s, 1H, NH D₂O exchangeable), 7.69 (d, J = 8.0 Hz, 2H, ArH), 7.53 (d, J = 11.6 Hz, 1H, ArH), 7.32 (q, J = 8.0 Hz, 1H, ArH), 7.26 (d, J = 8.8 Hz, 1H, ArH), 7.23 (d, J = 8.0 Hz, 2H, ArH),6.86 (t, J = 9.2 Hz, 1H, ArH), 3.83 (s, 2H, –C̲H̲_2̲–S), 2.35 (s, 3H, –C̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 171.18, 170.49, 168.58, 167.64, 161.45, 161.75, 140.17, 134.98, 130.93, 130.83, 129.18, 128.61, 120.19, 115.11, 110.16, 109.95, 106.18, 105.92, 89.71, 35.68 (CH₂–S), 21.39 (C̲H̲_3̲); FT-IR (max, cm⁻¹): 3305, 3221 (2NH), 3070 (CH aromatic), 2981 (CH aliphatic), 2214 (C≡N), 1666 (C=O amide); EI-MS: (M_wt.: 394.42): m/z (% rel. int.), 394.30 (M⁺, 7.51%), 272.26 (100%); anal. calcd. for C₂₀H₁₅N₄O₂SF: C, 60.90; H, 3.83; N, 14.21; S, 8.13; found: C, 61.18; H, 3.92; N, 14.36; S, 8.27.
3.1.3.13 N-(4-Chlorophenyl)-2-((5-cyano-6-oxo-4-(p-tolyl)-1,6-dihydropyrimidin-2-yl)thio)acetamide (8m). The titled compound was separated as buff crystals (yield 77%); m.p. 245–247 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.28 (s, 1H, NH D₂O exchangeable), 7.87 (d, J = 8.4 Hz, 2H, ArH), 7.43 (d, J = 8.4 Hz, 2H, ArH), 7.39 (d, J = 8.0 Hz, 2H, ArH), 7.11 (d, J = 8.0 Hz, 2H, ArH), 4.13 (s, 2H, –C̲H̲_2̲–S), 2.25 (s, 3H, –C̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 167.49, 166.09, 165.44, 161.75, 142.54, 136.79, 132.95, 132.70, 129.62, 129.62, 129.46, 129.28, 119.58, 116.50, 92.83, 35.95 (CH₂–S), 20.89 (C̲H̲_3̲); FT-IR (max, cm⁻¹): 3448,3288 (2NH), 3078 (CH aromatic), 2936 (CH aliphatic), 2218 (C≡N), 1639 (C=O amide); EI-MS: (M_wt.: 410.88): m/z (% rel. int.), 410.82 (M⁺, 52.61%), 412.60 (M⁺ + 2, 24.58%),97.20 (100%); anal. calcd. for C₂₀H₁₅N₄O₂SCl: C, 58.47; H, 3.68; N, 13.64; S, 7.80; found: C, 58.75; H, 3.80; N, 13.91; S, 7.69.
3.1.3.14 2-((5-Cyano-6-oxo-4-(p-tolyl)-1,6-dihydropyrimidin-2-yl)thio)-N-(4-methoxyphenyl)acetamide (8n). The titled compound was separated as buff crystals (yield 79%); m.p. 239–241 °C; ¹H NMR (400 MHz, DMSO-d₆)δ 10.19 (s, 1H, NH D₂O exchangeable), 7.78 (d, J = 8.0 Hz, 2H, ArH), 7.44 (d, J = 9.2 Hz, 2H, ArH), 7.16 (d, J = 8.4 Hz, 2H, ArH),6.87 (d, J = 8.8 Hz, 2H, ArH), 4.10 (s, 2H, –C̲H̲_2̲–S), 3.70 (s, 3H, –OC̲H̲_3̲), 2.32 (s, 3H, –C̲H̲_3̲); ¹³C NMR (101 MHz, DMSO-d₆)δ 167.46, 165.97, 165.06, 155.83, 142.56, 132.52, 129.47, 129.32, 129.26, 121.08, 116.42, 114.38, 92.89, 55.70 (OCH₃), 35.94 (CH₂–S), 21.52 (C̲H̲_3̲); FT-IR (max, cm⁻¹): 3447,3282 (2NH), 3075 (CH aromatic), 2931 (CH aliphatic), 2218 (C≡N), 1649 (C=O amide); EI-MS: (M_wt.: 406.46): m/z (% rel. int.), 406.36 (M⁺, 16.22%), 272.26 (100%); anal. calcd. for C₂₁H₁₈N₄O₃S: C, 62.06; H, 4.46; N, 13.78; S, 7.89; found: C, 61.92; H, 4.73; N, 14.06; S, 8.01.

3.2 Biological evaluation

3.2.1 Chemical reagents and cell culture. DMSO (Sigma-Aldrich, St. Louis, MO, USA) was used to create stock solutions of the synthesized compounds up to a final concentration of 10 mM. The stock solutions were divided into aliquots and stored at −20 °C. Doxorubicin and quercetin (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. Piochem (Giza, Egypt) provided ammonium molybdate, ATP, polyvinyl alcohol, and malachite green. The materials were maintained according to the manufacturer's instructions.

All cancer cell lines as well as normal cells were acquired from the tissue culture unit's cell culture bank at the Holding Company for the Production of Vaccines, Sera, and Drugs (VACSERA), situated in Giza, Egypt. According to previous protocols, the cell lines were examined for mycoplasma contamination while being maintained at the Helwan Structural Biology Research (HSBR) Center of Scientific Excellence.

3.2.2 In vitro PIM-1 kinase enzyme inhibition. The test compounds were screened in 1% DMSO in 96-wells, and 3-fold serial dilutions of compounds were performed. A 2× PIM1/Ser/Thr07 mixture was prepared in 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl₂, and 1 mM EGTA. The final 10 μL kinase reaction consists of 0.3–1.19 ng PIM1 and 2 μM Ser/Thr 07 in 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl₂, and 1 mM EGTA. After the 1 hour kinase reaction incubation, 5 μL of a 1 : 45 000 dilution of Development Reagent A was added. The developed FRET signal was measured using a fluorescence plate reader at 400 nm and data were analyzed using the following equation:

3.2.3 In vitro antiproliferative activity. The standard positive controls (doxorubicin and quercetin) as well as all synthetic compounds (8g, 8b, 8c, 8j, 8n, 8f, and 8m) were assessed as well. The antiproliferation and cytotoxicity were assessed using the colorimetric assay for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Serva) according to a previously used protocol. The absorbance of solubilized violet formazan crystals was measured at 570 nm using a Biotek 800 TS microplate reader. The IC₅₀ was found to be the concentration of the substance that caused 50% inhibition of cell growth. The normal human fibroblast cells, WI-38, were used for the cytotoxicity assay. A SI value greater than 1 indicates a medication that is safer and more effective against cancer than normal tissues.¹⁴⁶
3.2.3.1 Apoptosis assay using annexin V (AV)/propidium iodide (PI). The standard HSBR approach was used while performing the apoptosis experiment.^147,148 After seeding 1 × 10⁶ cancer cells onto 6-well plates and letting them adhere for the whole night, the cells were treated for an additional 24 h using IC₅₀ doses of either doxorubicin, quercetin, or the synthetic analogs (8g, 8b, 8c, 8j, 8n, 8f, and 8m). The cells were rinsed with 1 mL of binding buffer and then suspended in PBS (phosphate-buffered saline; Lonza) once more. 100 μL of cell suspension was treated with 1 μL of FITC-labeled annexin-V and 5 μL of PI for 15 minutes at 4 °C in the dark. The apoptotic cells in each sample were then examined using a Cytoflex flow cytometer (Beckman Coulter, USA). Version 2.4.0.28 of the CytExpert program was utilized.
3.2.3.2 Cell cycle assay using propidium iodide (PI) staining. The standard HSBR protocol was used while performing the cell cycle experiment.^147,148 Briefly, 6-well plates were seeded with 1 × 10⁶ cancer cells, and the cells were left to adhere for the whole night. The cells were then exposed to IC₅₀ doses of either synthetic analogs (8g, 8b, 8c, 8j, 8n, 8f, and 8m) or doxorubicin and quercetin as a positive control for an additional 24 h. The cells were concentrated at 1500g and suspended in 50 g ml⁻¹ propidium iodide (PI) staining solution and 20 g ml⁻¹ RNase A in order to detect cells with a sub-G1 DNA content. CytExpert software (version 2.4.0.28) was used to analyze the data from the Cytoflex flow cytometer (Beckman Coulter, USA).

The gating approach used to eliminate doublets is based on plotting the population against a 2D contour map in four quadrants: Q1 (LL), Q2 (LR), Q3 (UL), and Q4 (UR). This map illustrates the distribution of the annexin-FITC-H core cell population vs. PI PE-H.

3.2.3.3 Assessment of ATPase activity. Following the instructions in our previous approach, colorimetric techniques were employed to measure the total activity of ATPase and calcium-independent ATPase activity.¹⁴⁹ In short, cancer cells were exposed to doxorubicin as a positive control for the whole day, as well as the IC₅₀ of synthetic analogues (8g, 8b, 8c, 8j, 8n, 8f, and 8m). The release of inorganic phosphate (Pi) following the hydrolysis of ATP by ATPase enzymes was complexed into a colorful result in a single step by adding a mixed reagent consisting of polyvinyl alcohol, ammonium molybdate, and malachite green (2 : 1 : 1). The intensity of absorbance at 630 nm, which was directly proportional to the quantity of liberated Pi, was measured using a BioTek 800 TS absorbance plate reader (USA). To exclude any color resulting from the non-enzymatic hydrolysis of ATP, negative control reactions were created using just ATP in the reaction buffer. After that, the absorbance intensity was deducted from the sample and standard values.¹⁵⁰
3.2.3.4 Gene expression analysis. Genes linked to apoptosis (caspase-3 and cytochrome-C), proliferation (AKT1 and PI3K), and the cell cycle (CDK4, c-myc, and cyclin-c) were examined after the cancer cell line was treated with doxorubicin and new synthetic analogues (8g, 8b, 8c, 8j, 8n, 8f, and 8m) for 24 h using their IC₅₀ dose. As previously reported, the gene expression study was conducted using real-time quantitative PCR (RT-qPCR).¹⁵¹ A Favor-PrepTM blood/cultured cell total RNA purification micro kit (Favorgen Biotech Corp., Ping-Tung, Taiwan) was used to extract total RNA from the cells after they had been removed during incubation. A Revert Aid First-Strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) was then used to reverse transcribe the purified RNA into the first-strand cDNA. A HERAPLUS SYBR® Green qPCR kit (Willowfort, Nottingham, UK) was used for all qPCR reactions. Table 5 contains a list of every primer sequence. Using GAPDH as a reference gene, the 2^−ΔΔCT technique was used to perform differential gene expression.

Table 5 Primer sequence of key modulatory genes used for gene expression analysis

Gene	Forward primer sequence	Reverse primer sequence
Caspase-3	5′-ACATGGAAGCGAATCAATGGACTC-3′	5′-AAGGACTCAAATTCTGTTGCCACC-3′
Cyto-c	5′-GAGGCAAGCATAAGACTGGA-3′	5′-TACTCCATCAGGGTATCCTC-3′
AKT1	5′-TCTATGGCGCTGAGATTGTG-3′	5′-CTTAATGTGCCCGTCCTTGT-3′
CDK4	5′-CTGGTGTTTGAGCATGTAGACC-3′	5′-AAACTGGCGCATCAGATCCTT-3′
C-myc	5′-AAACACAAACTTGAACAGCTAC-3′	5′-ATTTGAGGCAGTTTACATTATGG-3′
Cyclin-c	5′-GGACATGGGCCAAGAAGACA-3′	5′-CTTTTCTGCTAGGCTGGGCT-3′
GAPDH	5′-CTGACTTCAACAGCGACACC-3′	5′-TAGCCAAATTCGTTGTCATACC-3′

---

3.2.3.5 ELISA assay of c-Myc inhibitory activity. To measure c-Myc in cell lysates, cancer cells were exposed to the doxorubicin IC₅₀ as a positive control or synthetic drugs (8g, 8b, 8c, 8j, 8n, 8f, and 8m) for 24 h. Cell pellets were lysed in RIPA buffer, and the entire lysate that resulted was collected. The total protein was estimated using a commercially available ELISA kit (NBP2-75725) in accordance with the manufacturer's instructions. ELISA plates were coated with the capture antibody, and the coating was blocked for an hour using 5% BSA in PBS-Tween solution. The samples were allowed to incubate for two hours before a secondary HRP-detection antibody was added and incubated for an additional two hours.

3.3 In silico ADMET study prediction

The ADME study was performed using the SWISSADME server.

3.4 Molecular docking study

The docking study was performed using AutoDockVina 1.5.7. The three-dimensional (3D) structure of the human PIM-1 (PDB: 2OBJ) protein binding site was obtained from the Protein Data Bank (https://www.rcsb.org). The ideal docking posture is expected to be the most stable conformation of every molecule attached to the protein active site. Docking energies were used to select the docked model, and the target–ligand interactions were displayed using the Pymol Molecular Graphics System Version 3.0.4.

3.5 Molecular dynamics study

Molecular dynamics simulation was performed using GROMACS.

4 Conclusion

To summarize, we report on a new class of S-aryl amide derivatives of pyrimidinones for PIM-1 kinase inhibition. We designed and synthesized several compounds' derivatives, including 8b, 8f, 8g, 8k, and 8l that showed promising antiproliferative activity against a variety of cancer cell lines, including MCF-7, DU-145, and PC-3, with a relatively higher safety index suggesting their efficacy and minimal cytotoxicity to normal cells. All compounds induced cellular late apoptosis with necrosis in both PC-3 and MCF-7 cells, confirmed by downregulation of caspase-3 and cytochrome-c key apoptotic genes as well as AKT1 and PI3K as key proliferating genes. Also, these synthetic compounds showed promising cell cycle arrest of MCF-7 at the G2/M phase, verified by the downregulation of CDK4, c-Myc, and cyclin-c key regulatory genes of cell cycle progression. Moreover, the compounds showed a promising ATPase inhibitory effect that leads to intracellular accumulation of compounds and subsequent DNA damage and cell cycle arrest at the G2/M phase. PIM-1 was suggested as a molecular target, and compounds 8b, 8f, 8g, 8k, and 8l showed significant potent PIM-1 inhibitory activity with good binding interaction using structure-based molecular docking design. Furthermore, we found via the SAR that the G-loop of PIM-1 was flexible, creating a pocket that could potentially form a hydrogen bond with the DFG motif. The current design approach makes use of a variety of interactions, such as polar, hydrophobic, and water-mediated interactions. However, it seems that the inhibitor's core structure (pyrimidinone) forming a hydrogen bond with Lys67 is the best binding mode for selectivity. Potency can be further increased by additional direct water-mediated hydrogen bonding with Asp128, Glu171, and Asp 186. The future perspectives for the current study include compounds of similar framework structures, such as TBB as a positive control reference in different biochemical assays, to determine its role in different pathways such as apoptosis and the cell cycle. The structural stability of 8b and 8g protein complexes was assessed through molecular dynamics simulation. The results showed that there were no considerable changes in their structural stability in comparison with the lead compound (VII). However, both 8b and 8g reveal more hydrogen bond interaction and thermodynamic stability during the simulation time. This will help to open the gate for our research group for further future investigations on the most promising candidates using other detailed mechanisms, including western blotting. The current new synthetic S-aryl amide derivatives of pyrimidinone analogues can be promising scaffolds for further optimization and selectivity to PIM protein with potential anticancer activity.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Soha R. Abd El Hadi: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft, visualization, supervision. Manar A. Eldinary: methodology, formal analysis, investigation, resources, visualization. Amna Ghith: methodology, investigation, resources, visualization. Hesham Haffez: methodology, formal analysis, investigation, data curation, writing – review & editing. Aya Salman: methodology, investigation, data curation, writing – original draft, visualization. Ghadir A. Sayed: methodology, investigation, data curation, writing – original draft, visualization.

Conflicts of interest

The authors declare that they have no conflict that could have appeared to influence the work reported in this paper.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5md00021a

‡ These authors contributed equally to this work.

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