Hybrid nucleobase–heterocycle–2-oxindole scaffolds as innovative cell cycle modulators with potential anticancer activity

Abstract

A series of hybrid molecules 6a–6d–13a–13d combining pyrazolo[3,4-d]pyrimidine or aminopurine frameworks with an oxindole moiety were designed as multitarget anticancer agents. Several compounds, especially 8b and 12a–12d, showed potent antiproliferative effects against three human cancer cell lines: A498 (kidney carcinoma), HepG-2 (hepatocellular carcinoma), and MDA-MB-231 (breast adenocarcinoma). Compounds 6b, 7b, 8b, and 12a–12c exhibited remarkable CDK6 inhibition (pIC₅₀ of up to 7.17), outperforming palbociclib, and VEGFR-2 inhibition comparable to sorafenib. These compounds also inhibited xanthine oxidase. Notably, 12a and 12c induced sub-G1 cell cycle arrest in HepG2 cells. Molecular modeling confirmed stable binding to CDK6 and VEGFR-2, while in silico ADMET profiling suggested favorable pharmacokinetics. These results support 8b and 12a–12c as strong leads for further multitarget cancer therapy development.

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Introduction

Cancer is a major global health and economic burden, causing nearly one in six deaths worldwide.^1,2 By 2050, more than 35 million new cases are projected due to population growth and aging.³ Current treatment generally involves surgery, radiation, and chemotherapy, which face challenges like drug resistance and severe side effects. There is a pressing need for new anticancer agents with greater efficacy and lower toxicity. A deeper understanding of cell cycle control, particularly key checkpoints (late G1, G2/M, and metaphase-to-anaphase transition), offers a promising strategy for developing more effective cancer therapies.⁴

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that regulate the cell cycle by forming heterodimeric complexes with cyclin subunits. Dysregulation of CDK or cyclin levels is frequently observed in cancers.^5,6 Among these, CDK4 and CDK6 are critical drivers of oncogenesis in specific tumors, making them promising targets for therapeutic development. Cyclin D-CDK4/6 complexes promote the transition from the G1 phase to the S phase by phosphorylating the retinoblastoma (RB) tumor suppressor protein, thereby facilitating cell cycle progression.^7–10 Inhibiting CDK4 and CDK6 is essential for targeting this critical cell cycle phase. Selective CDK4/6 inhibitors have shown effectiveness in treating various cancers, including melanoma, breast cancer, osteosarcoma, bladder cancer, lung cancer, and stomach cancer while minimizing toxicity in non-cancer cells. This therapeutic strategy holds promise for improving cancer treatment outcomes.^11–14 Various reported CDK4/6 inhibitors are based on various molecular scaffolds that enable specific binding to these kinases, thereby blocking their activity. Several “ciclib” scaffolds have been identified as core structural frameworks in the development of CDK4/6 inhibitors^15,16 are shown in Fig. 1. The pyrido[3,4-d]pyrimidin-4-one scaffold has been extensively explored for the development of experimental CDK4/6 inhibitors. Its core structure offers flexibility for optimizing its affinity towards CDK4/6, enabling the creation of selective inhibitors with varied pharmacokinetic properties. One of the first notable examples is palbociclib I,¹⁷ which became the first FDA-approved CDK4/6 inhibitor. Dalpiciclib V is another potent and orally bioavailable CDK4/6 inhibitor, which differs from palbociclib by featuring a piperidine ring in place of the piperazine ring.

Fig. 1 Representative scaffolds of some reported CDK4/6 inhibitors.

The pyrrolo[2,3-d] pyrimidine scaffold is represented by ribociclib II, the second FDA-approved oral CDK4/6 inhibitor. Birociclib VI is another selective CDK4/6 inhibitor containing this scaffold that has also gained FDA approval.^18,19

The 6-(pyrimidin-4-yl)-1H-benzo[d]imidazole scaffold is exemplified by abemaciclib III, an oral CDK4/6 inhibitor, approved by the FDA. BPI-16350 VII is a highly potent CDK4/6 inhibitor with structural similarity to abemaciclib.²⁰ Finally, the tricyclic lactam scaffold underlies trilaciclib IV, the first FDA-approved CDK4/6 inhibitor for chemotherapy-induced myelosuppression. Lerociclib VIII, a structural analog of trilaciclib, is equally potent and selective for CDK4/6.

Vascular endothelial growth factor receptors (VEGFRs) are crucial receptor protein tyrosine kinases involved in tumor-induced angiogenesis, which are essential for tumor growth, progression, and metastasis.²¹ Targeting the VEGFR-2 signaling pathway is a promising cancer therapy strategy, with FDA-approved VEGFR-2 inhibitors, like the “nibs” sorafenib, foretinib, regorafenib, and tivozanib.^22–24

Xanthine oxidase (XO) overexpression is linked to cancer due to its role in generating reactive oxygen species (ROS) and activating carcinogens. XO oxidizes hypoxanthine to xanthine and uric acid, producing ROS that promote tumor progression, inflammation, angiogenesis, and cell migration. XO inhibitors can suppress proinflammatory and proapoptotic proteins, potentially aiding cancer treatment. Elevated uric acid levels correlate with poorer survival rates in cancer patients, highlighting XO as a diagnostic and prognostic marker. Although XO inhibitors reduce oxidative stress and inflammation, their impact on cancer progression and metastasis remains largely unexplored.^25,26

Current studies focus on developing next-generation inhibitors targeting CDK4/6, VEGFR-2, and XO. These targets are critical in cancer progression, with CDK4/6 regulating the cell cycle, VEGFR-2 facilitating angiogenesis, and XO generating ROS.²⁷ By targeting all three, it may be possible to enhance anticancer efficacy by inhibiting cell proliferation and tumor blood supply. Specific chemical scaffolds, such as oxindoles, purines, pyrimidines, and pyrazolopyrimidines, are explored for their biological activities. Oxindoles exhibit anticancer properties by inhibiting key enzymes, with C3-substituted derivatives enhancing interactions for novel drug design. FDA-approved oxindole-based drugs, like sunitinib and nintedanib, treat renal cell carcinoma and adenocarcinoma, while indirubin shows CDK inhibition, and toceranib is used in veterinary oncology. Other oxindole inhibitors are orantinib and target receptor tyrosine kinases.^28,29 Additionally, thiazolidinone and thiazolylidene derivatives, featuring an –N=C–S– moiety, display diverse biological activities, with ponesimod gaining FDA approval for multiple sclerosis.^30,31 Purines and pyrimidines, which are fundamental to DNA and RNA, are crucial in drug discovery. Purine derivatives serve as anticancer antimetabolites, while pyrimidines exhibit diverse biological activities. Targeting nucleotide synthesis with analogs enhances chemotherapy.³² Pyrazolopyrimidines serve as bioisosteres of adenine, mimic ATP interactions, and function as CDK inhibitors. Allopurinol, a pyrazolopyrimidine, inhibits xanthine oxidase and exhibits cytotoxic effects. Its structural versatility and tautomeric forms enhance its therapeutic potential.^33,34 In this framework, structure-based drug design (SBDD) and molecular docking were performed to evaluate the binding affinities of the designed compounds within the active site of CDK6, a key enzyme in cancer progression. Their affinities were compared with palbociclib to validate the design strategy.

Design rational

A novel series of compounds incorporating pyrazolo[3,4-d] pyrimidine and aminopurine scaffolds, fused with an oxindole ring, was strategically designed to inhibit CDK6, VEGFR-2, and xanthine oxidase, which are critical enzymes involved in cancer progression. The structural design of these compounds focuses on enhancing both potency and multi-targeted therapeutic effectiveness and is summarized as follows (Fig. 2).

Fig. 2 Feature similarities of the palbociclib ligand and the newly designed compounds 10a–d–13a–d as potent CDK4/6 inhibitors.

(1) Scaffold modification

The pyrido[3,4-d] pyrimidine scaffold of palbociclib was replaced with pyrazolo[3,4-d] pyrimidine and aminopurine, combined with an oxindole ring. This adjustment enables noncovalent bonding with Val101 and Asp163 in the CDK6 active site.

(2) Oxindole substitutions

Variably substituted oxindole rings incorporating electron-donating (EDGs) and electron-withdrawing groups (EWGs) were designed to evaluate the effects of electronic modifications on biological activity.

(3) Thiosemicarbazide addition

A thiosemicarbazide motif was attached at the oxindole's C-3 position to boost the potency of the target compounds.

(4) Thiosemicarbazone cyclization

Cyclized forms, such as 2-hydrazinylidine thiazolidin-4-one and 2-hydrazinylidine-2,3-dihydrothiazole, were introduced at oxindole's C-3 position to enhance binding affinity and potency via alternative interactions.

These design elements maximize structural diversity and binding efficiency, paving the way for novel multi-targeted cancer therapies.

Results and discussion

Computational studies

Molecular docking of the design compounds onto the CDK4/6 inhibitor. In our study, we focused on developing new compounds that target the ATP binding site of the CDK6 enzyme. Our approach was inspired by the structural analysis of palbociclib, a known CDK6 inhibitor, which was studied in complex with the enzyme (PDB ID: 5L2I).³⁵ Fig. 3 shows how palbociclib binds to the ATP active site, serving as a foundation for designing improved CDK6 inhibitors.

Fig. 3 Schematic of the binding regions within the ATP active site of CDK6 in complex with palbociclib. The key interaction regions are (1) hinge (adenine) region—Val101, Leu152, Glu99, and Phe98 residues; (2) ribose pocket—Asp163 and Lys43 residues; (3) hydrophobic region—Phe98 and Leu152 residues; (4) solvent region—Asp163 and Lys43 residues; and (5) phosphate pocket—Lys43, Asp145, and Glu61 residues.

Docking studies for the newly designed compounds 6a–6d–13a–13d were conducted using MOE 2020.01 software, utilizing the three-dimensional structure of the CDK6 enzyme in complex with palbociclib, obtained from the Protein Data Bank (PDB).

To ensure the accuracy of the docking protocol, palbociclib was redocked into the CDK6 active site, yielding a root-mean-square deviation (RMSD) of 1.03 Å and a binding score of −8.23 kcal mol⁻¹. The docking procedure successfully reproduced all key interactions observed in the co-crystallized structure, confirming the reliability of the methodology. This validated approach was then employed to investigate the binding interactions of the newly designed compounds, allowing for a comparative analysis of palbociclib within the active site. The co-crystal structure of CDK6 with palbociclib revealed strong interactions within the ATP binding site, contributing to the compound's stability and selectivity. Palbociclib is anchored by three key hydrogen bonds: two with Val101 in the hinge region and one with Asp163 in the DFG (Asp–Phe–Gly) motif. Additionally, it establishes a favorable electrostatic interaction with a solvent-exposed ridge formed by Asp104 and Thr107. Further stabilization is provided by multiple polar interactions between the ribose and phosphate groups and residues Val27 and Ala162. A defining feature of palbociclib is its high specificity for CDK4/6, with minimal activity against other CDKs or non-CDKs. This selectivity is largely attributed to interactions with CDK4/6-specific residues, particularly His100 and Thr107 near the hinge region. Notably, His100 is exclusive to CDK4 and CDK6, further reinforcing its specificity. Additionally, kinase selectivity may be influenced by palbociclib's lipophilicity, as its cLog P value facilitates deeper accommodation within the ATP-binding cleft (Fig. S1, SI).

The designed compounds 6a–6d–13a–13d exhibited a common binding pattern, forming halogen bonds (XB) and/or hydrogen bonds (HB) with Val101, along with hydrogen bonding interactions with Asp163 residues in the hinge (adenine) region, which are critical for CDK6 inhibition. Additionally, these compounds engage in hydrophobic interactions with key gatekeeper residues, including Phe98, Val27, and Ile19. Beyond these core interactions, the new compounds also formed additional contacts with amino acids, such as Asp104, Asn150, Gln149, Gly20, Gly165, Leu96, Leu152, Leu166, Lys43, Lys147, Phe164, and Val77. These extended interactions, facilitated by the molecular design, contributed to the overall stability of the ligand–protein complex. Several compounds, specifically 8b, 9b, 10a–10c, 11b, 12a–12d and 13c–13d, demonstrated binding scores ranging from ΔG −8.29 to −9.00 kcal mol⁻¹, surpassing the binding score of palbociclib (−8.23 kcal mol⁻¹). In contrast, compounds 6a–6d and 7a-7d exhibited lower binding scores, ranging from −6.95 to −7.45 kcal mol⁻¹. However, these compounds still showed an affinity for the ATP binding site of CDK6 by interacting with Val101 and Asp163 and penetrating the ribose pocket through interactions with Leu152, Leu166, and Phe164, as exemplified by compounds 6b and 7b (Fig. 4). These findings support the synthesis of docked compounds and encourage further evaluation of their potential as CDK6 inhibitors.

Fig. 4 2D interaction of the designed compounds 6b and 7b.

Thiosemicarbazone groups 8a–8d and 9a–9d showed slightly better binding energy, ranging from −7.71 to −8.35 kcal mol⁻¹, due to their interaction with essential amino acids Val101 and His100, which matched the binding interaction with palbociclib. Additionally, they bind to extra amino acids, including Leu96, Gln149, Leu152, and Gly165, as depicted in Fig. 5. The higher ΔG of 8b was attributed to the strong electrostatic Cl⋯O attraction (d = 3.14 Å) contributed to CDK6 inhibition activity.

Fig. 5 2D interaction of the designed compounds 8b and 9b.

The thiazolidinone moiety of compounds 10a–10d and 11a–11d binds to the crucial amino acids Val101 and Glu99, plus the added interactions with Asn150, Asp 104, Leu96, Leu152, Gly20, Gly 165, and Tyr24. These compounds exhibit a higher binding energy than palbociclib. The sulphur atom within the thiazolylidene moiety in 12a–12d and 13a–13d binds in hydrogen bonding with the solvent region of the CDK6 enzyme's active site, particularly with Asp104. Meanwhile, the thiazolylidene moiety is positioned toward the ribose pocket, facilitating hydrophobic interactions with Gly20. These compounds not only bind to crucial amino acids but also interact with additional residues, like Leu96, Leu166, Phe164, Val77, Gln149, and Gly165. They exhibit the highest binding scores of ΔGs of 12a–12d and 13a–13d among the compounds studied, as shown in Fig. 6.

Fig. 6 2D interaction of the designed compounds 12b and 13b.

The 5-chloro-substituent plays a significant role in binding to the CDK6 enzyme's active site. Among the designed compounds, variations at the 5-position include H, Cl, OCH₃, and F. Notably, compounds featuring 5-chloro substituents exhibit the most favorable binding interactions with the CDK6 enzyme's active site, particularly through interactions with essential residues Val101 and His100. Representative examples of compounds 6b, 6c and 6d contain similar structural substituents but differ solely in the 5-position, Fig. S2, SI.

To assess the selectivity of the newly designed compounds for CDK6 over CDK2, docking studies were conducted using the CDK2-sunitinib complex (PDB ID: 3TI1). The results showed that the tested compounds exhibited different binding modes compared to sunitinib, which interacts with CDK2 through hydrogen bonding with Glu81 and Leu83 in the ATP-binding site, along with hydrophobic interactions involving Phe80, Ile10, and Asp86.

The tested compounds displayed interaction energies ranging from ΔG = −7.07 to −8.23 kcal mol⁻¹, while the lead compounds had a slightly stronger interaction energy of ΔG = −8.65 kcal mol⁻¹. Notably, compounds 8b, 12a–12c, and 13b failed to establish crucial hydrogen bonds with Leu83 and lacked hydrophobic interactions with Phe80. Similarly, compounds 6b and 7b did not form key hydrogen bonds with Leu83 or interact hydrophobically with Phe80. These findings suggest that the designed compounds exhibit distinct binding preferences, reinforcing their selectivity for CDK6 over CDK2 (Fig. S3, SI).

Docking studies of the VEGFR-2 inhibitor. Docking studies were conducted on the most active compounds 6b, 7b, 8b, 12a–d, and 13b using the VEGFR-2 co-crystal structure with sorafenib (PDB ID: 3WZE)^36–39 and MOE 2020.01 software. The docking protocol was validated by redocking sorafenib into the VEGFR-2 active site, which yielded an RMSD of 1.02 Å and a binding score of −10.16 kcal mol⁻¹, successfully reproducing key interactions with critical binding site residues (Table 1). Sorafenib interacts with VEGFR-2 through five hydrogen bonds: two with Cys919, two with Glu885, and one with Asp1046. Its N-methyl picolinamide group fits into a pocket formed by Cys919, Phe918, Leu1035, Lys920, Glu917, Val848, and Leu889, while the terminal 3-trifluoromethyl-4-chlorophenyl group occupies a hydrophobic channel involving residues such as Phe1047, Cys1045, and Ile1044. The urea spacer plays a key role in binding, enhancing sorafenib's affinity for VEGFR-2 (Fig. 7). These findings highlight the potential of modifying linker structures to develop more potent VEGFR-2 inhibitors.

Table 1 Binding energy score of the docked most active cytotoxic compounds on VEGFR-2

Compound	Binding score ΔG (kcal mol^−1)	Amino acids involved in interaction
Sorafenib	−10.16	Asp1046, Glu885, Cys919, Phe1047, Leu840 and Leu1035
6b	−7.68	Asp1046, Glu885, Leu889, Cys1045 and Phe1047
7b	−7.33	Glu885, Cys1024, Leu889, Ile888, and His1026
8b	−9.13	Asp1046, Glu885, Cys919, Cys1045, Leu840 and Lys868
12a	−8.99	Asp1046, Glu885, Cys1045, Leu889, Ile888, Cys1024 and Arg1027
12b	−8.84	Asp1046, Glu885, Cys1045, Leu889 and Ile888
12c	−8.13	Asp1046, Glu885, Ile888, Cys1024, Ile1025 and Arg1027
13b	−7.89	Asp1046, Leu889, Ile888, Glu815, His816 and Lys868

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Fig. 7 2D and 3D interactions of co-crystallized sorafenib shown in green and red, adopting the Phe1047 “DFG-out” conformation.

Compound 8b exhibited a binding mode closely resembling that of sorafenib, with a binding score of −9.00 kcal mol⁻¹ and the formation of five hydrogen bonds. The pyrazolopyrimidine ring forms one hydrogen bond with Cys919, while the hydrazino groups establish three hydrogen bonds with Asp1046, Cys1045, and Glu885. Additionally, the sulfur atom of the thiosemicarbazide moiety forms a hydrogen bond with Lys868. The 1-ethyl pyrazolo pyrimidine scaffold fits into a pocket formed by Leu1035, Lys920, Cys919, Phe918, Glu917, Ala866, and Val848, while the terminal thiosemicarbazone tail occupies a hydrophobic channel created by Phe1046, Ile1044, Val916, Val914, and Leu889 (Fig. S4, SI). These key interactions likely contributed to the strong anticancer activity of compound 8b.

The predicted binding mode of compound 12a closely resembles that of sorafenib and 8b, with a binding score of −8.95 kcal mol⁻¹ and four hydrogen bonds. The pyrazolopyrimidine ring forms hydrogen bonds with Cys1024 and Ile888, while the ethylene linker interacts with Asp1046. Additionally, the NH group of the thiazolylidene forms a hydrogen bond with Glu885, and the sulfur atom establishes another with Cys1045. The 1-ethyl pyrazolopyrimidine and oxindole scaffolds fit within a hydrophobic pocket composed of Arg1027, Ile1025, Leu1019, Ile892, Leu889, and Glu815, while the terminal thiazolylidene tail is accommodated in a hydrophobic channel formed by His1026, Val916, Val899, Val898, and Lys898 (Fig. S5, SI).

Compound 12b displayed a binding score of −8.84 kcal mol⁻¹ and formed three hydrogen bonds. The ethylene linker interacts with Asp1046, while the NH group and sulfur atom of the thiazolylidene form hydrogen bonds with Glu885 and Cys1045, respectively. Its 1-ethyl pyrazolopyrimidine and oxindole scaffold occupy a hydrophobic pocket consisting of Arg1027, Cys1024, Ile1025, Leu1019, Ile892, Leu889, Lys868, and Glu815. Terminal thiazolylidene tail is positioned within a hydrophobic channel formed by Ile1044, His1026, Val916, Val899, and Val898 (Fig. S6, SI).

These docking results suggest that the linkers used in these compounds align with the urea linker in sorafenib, serving a similar function and enhancing affinity for the VEGFR-2 enzyme. The formation of hydrogen bonds with Cys919, Asp1046, and Glu889, along with key hydrophobic interactions, likely contributes to the strong binding of the heterocyclic pyrazolopyrimidine scaffold to VEGFR-2.

Molecular dynamics simulation

Compound 12b exhibited good stability within the CDK6 active site, maintaining an average RMSD of 2.5489 Å, which is comparable to palbociclib (2.4922 Å) (Fig. S7, SI). The interaction energies further supported this stability, with total electrostatic and van der Waals energies of −40.17 kcal mol⁻¹ for 12b and −36.94 kcal mol⁻¹ for palbociclib. Both compounds formed hydrogen bonds with key residues, contributing to their stable binding (Fig. S8 and S9, SI). MM-PBSA calculations revealed that 12b had a slightly better binding free energy (−9.3551 kcal mol⁻¹) than palbociclib (−9.0804 kcal mol⁻¹), aligning with its in vitro inhibitory activity (IC₅₀ = 0.123 μM). Protein stability assessments showed similar RMSD values (12b: 2.2934 Å, palbociclib: 2.2148 Å) (Fig. S10) and RMSF values (12b, 1.5675 Å; palbociclib, 1.6492 Å) profiles (Fig. S11), indicating the structural stability of the protein. Additional parameters, including the radius of gyration (12b: 19.42 Å, palbociclib: 19.47 Å) (Fig. S12 and S13) and solvent-accessible surface area (SASA ∼ 14 360 Ų) (Fig. S14), further confirmed the compactness and stability of the CDK6 complexes.

Overall, the 50 ns molecular dynamics simulation demonstrated that compound 12b exhibits strong stability and binding affinity, highlighting its potential as a potent CDK6 inhibitor (Fig. 8 and 9).

Fig. 8 Snapshots of palbociclib inside the active site of CDK6 over 50 ns long MDS (5, 10, 20, 30, 40 and 50 ns).

Fig. 9 Snapshots of compound 12b inside the active site of CDK6 over 50 ns long MDS (5, 10, 20, 30, 40 and 50 ns).

Chemistry

Target molecules 6a–d–13a–d were synthesized as outlined in Scheme 1, and their structures were confirmed through spectral analyses (Fig. S15–S30).

Scheme 1 Synthesis of the target compounds 6a–d–13a–d. (i) K₂CO₃, 1,2-dibromoethane, DMF, r.t., 24 h; (ii) K₂CO₃, DMF, 60 °C, 6–8 h; (iii) thiosemicarbazide, EtOH, reflux, 2–4 h; (iv) ethyl bromoacetate or monochloroacetic acid, sod. acetate, EtOH, reflux, 24 h; and (v) chloroacetone, sod. acetate, EtOH, reflux, 24 h.

It has been reported that the reaction of isatin with thiosemicarbazide is stereoselective, producing only the Z-diastereomer. The chemical shifts of the synthesized compounds 8a–8d–9a–9d are comparable with the reported ¹H NMR of Z-thiosemicarbazone^40,41 (Fig. S31–S46). These compounds exhibit highly deshielded hydrazine protons resonated at a far downfield shift around δ 12.22–12.09 ppm stemming from the strong intramolecular hydrogen bond formed with the lactam oxygen atom of oxindole (N⋯H⋯O d = 2.09 Å, and Φ = 130°) to stabilize the Z configuration,⁴² as depicted in Fig. 10.

Fig. 10 Representative example for the Z configuration of 9b.

The cyclization of the SCNH₂ moiety was performed using ethyl bromoacetate and a catalytic amount of anhydrous sodium acetate, yielding oxindole-allopurinol-2-hydrazonothiazolidin-4-one 10a–10d. Alternatively, cyclization in the presence of monochloroacetic acid resulted in oxindole-adenine-2-hydrazonothiazolidin-4-one 11a-11d.

Further modification of the SCNH₂ cyclization polarity was achieved using chloroacetone, leading to the formation of oxindole-allopurinol/adenine thiazol-2(3H)-ylidenes 12a–12d and 13a–13d. The structures and purity of the synthesized compounds were confirmed through spectral and elemental analyses (Fig. S47–S77).

As expected, the restricted rotation of the thiazolylidene ring and the imine group in 12a–12d and 13a–13d revealed E and Z diasteroisomers. The proton peaks of the E-diastereomer are significantly downfield shifted relative to those of the Z-diastereomer. For example, in the ¹H-NMR spectrum of compound 13a E/Z%, 38 : 62, the formation of the thiazolyidene ring is confirmed by the presence of two characteristic singlet peaks for SCH_(E) and SCH_(Z) at 6.51 and 6.36 ppm, respectively. Additionally, two characteristic doublet peaks were assigned to NCH_(E) and NCH_(Z) at 7.15 and 6.89 ppm, respectively. The chemical shift of the indole proton at C4H is particularly useful for differentiating between E- and Z-diastereomers. The C4H proton of the E-diastereomer resonates more downfield, appearing as a doublet around δ 8.26 ppm, while the C4H proton of the Z-diastereomer resonates relatively upfield, appearing as a doublet around δ 7.48 ppm. The same pattern was observed in ¹³C-NMR spectra, NCH_(E) and NCH_(Z), around δ 122.8 and 121.8 ppm, respectively, and was observed in C4_(E) and C4_(Z) around δ 130.0 and 129.5 ppm, respectively. The differentiation of E- and Z-diastereomers can be effectively achieved using 2D-NMR techniques. The ¹H–¹H NOESY experiment confirmed the presence of both diastereomers (E/Z) in isatin-thiazol-2(3H)ylidene, enabling the assignment of E and Z protons. As a representative example, compound 10b exhibited no NOE cross-peak between the NH and C4–H protons, indicating that these groups are spatially separated and oriented differently. Additionally, the presence of cross-peaks between the C7–H and N1–CH₂ protons of oxindole further supports the Z-configuration of compound 10b, which can be extrapolated to other derivatives, as illustrated in Fig. 11.

Fig. 11 ¹H–¹H-NOESY spectrum of compound 10b.

The purity of the synthesized compounds was confirmed by elemental analysis, with deviations within acceptable limits (±0.4%). Furthermore, HPLC chromatograms of representative compounds 6b, 8b, 12a, 12b, and 12c, using allopurinol as an internal standard, indicated high chemical purity, with all tested leads showing purity ≥95% and several exceeding 97% at their respective retention times. These findings confirm the successful synthesis and isolation of highly pure compounds suitable for subsequent biological studies and formulation efforts. Detailed chromatographic data are provided in the SI (Fig. S78–S82).

Biological evaluation

Antiproliferative activity assay

The antiproliferative activities of allopurinol-isatin and adenine-isatin derivatives 6a–6d and 7a–7d, along with thiosemicarbazone derivatives 8a–8d and 9a–9d, and cyclized products 2-hydrazinylidine thiazolidin-4-one 10a–10d and 11a–11d, as well as 2-hydrazinylidine-2,3-dihydrothiazole 12a–12d and 13a–13d, were evaluated against three cancer cell lines: A498 (kidney carcinoma), HepG-2 (hepatocellular carcinoma), and MDA-MB-231 (breast adenocarcinoma). Additionally, their effects on the normal murine embryonic fibroblast cell line BALB/3T3 (clone A31) were assessed using the sulforhodamine B (SRB) assay to determine antiproliferative activity compared to the reference drug sorafenib. The results shown in Tables 2, S1 and Fig. S83 clearly reveal that most of the synthesized compounds have pIC₅₀ in the range of 4.03–5.82, matching or higher than the reference sorafenib. Of these active compounds, 30 structures carry the allopurinol feature and nine carry the adenine. Among series 6a–6d and 7a–7d, the most active ligands are those with a higher lipophilic nature, carrying 5-chloroindoline-2-one fragment 6b (clog P = 0.77) and 7b (clog P = 1.54). The allopurinol compound 6b R₁ = Cl showed the most distinct effect against the three cancer cell lines pIC₅₀ (4.79, 4.82, and 4.88), and in the adenine, series 7b showed antiproliferative effects against HepG2 and MDA-MB-231 pIC₅₀ (5.22 and 4.71, respectively). Both most probably privileged the contribution of halogen bond formation that improved the binding affinity of ligands to the residues in the active site compared to 6d pIC₅₀ (4.21, 4.32, and 4.41) and 7d with promising activity against the three cancer cell lines pIC₅₀ (3.78, 4.76, and 4.08).

Table 2 Antiproliferative activity of the synthesized compounds against A-498, HepG2, and MDA-MB-231 tumor cell lines using SRB assay with sorafenib as a positive control

Entry	R1	pIC50^b			Entry	pIC50^b
		A-498	HepG2	MDA-MB-231		A-498	HepG2	MDA-MB-231
a n.a. – not active.b pIC50 = −log IC50 (molar concentration).
6a	H	4.23	3.82	4.04	7a	n.a.^a	n.a.	3.68
6b	Cl	4.79	4.82	4.88	7b	4.28	5.22	4.71
6c	OCH3	4.52	4.03	4.32	7c	3.74	4.11	3.87
6d	F	4.41	4.21	4.32	7d	3.78	4.76	4.08
8a	H	n.a.	n.a.	n.a.	9a	n.a.	n.a.	n.a.
8b	Cl	4.85	4.88	4.27	9b	n.a.	n.a.	n.a.
8c	OCH3	n.a.	n.a.	n.a.	9c	n.a.	4.53	4.14
8d	F	3.98	4.60	n.a.	9d	n.a.	4.25	n.a.
10a	H	n.a.	n.a.	n.a.	11a	n.a.	n.a.	n.a.
10b	Cl	4.03	5.27	4.29	11b	n.a.	n.a.	n.a.
10c	OCH3	n.a.	n.a.	n.a.	11c	n.a.	n.a.	n.a.
10d	F	n.a.	n.a.	n.a.	11d	3.72	n.a.	3.72
12a	H	5.12	5.82	5.35	13a	3.72	n.a.	n.a.
12b	Cl	5.29	5.76	5.41	13b	n.a.	n.a.	n.a.
12c	OCH3	4.26	5.25	4.81	13c	3.98	3.84	3.75
12d	F	4.51	5.21	4.93	13d	3.85	3.72	n.a.
Sorafenib		4.20	4.35	4.09

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However, the other substituents H and OMe in 6a, 6c, 7a and 7c displayed relatively lower potency against the three cell lines.

The attachment of the thiosemicarbazide fragment to the ligands 6a–6d and 7a–7d demoted the scope and potency of the produced thiosemicarbazones against the three cancer cell lines. Nonetheless, the thiosemicarbazones 8b, 8d, and 9c showed potent antiproliferative activity pIC₅₀ (4.53–4.88) against the A-498 and HepG2 cell lines. Further modifications of 8a–8d and 9a–9d were conducted via two cyclization reactions of the SCNH₂ moiety. The first cyclized moiety was 2-hydrazinylidenethiazolidin-4-ones 10a–10d and 11a–11d. Incorporation of the lactam scaffold resulted in a reduction of one hydrogen bond donor (HBD) and an increase of two hydrogen bond acceptors (HBAs) per molecule relative to precursors 8a–8d and 9a–9d. The conversion of the –SCNH₂ functionality into a lactam framework led to a restricted antiproliferative profile and diminished cytotoxic potency when compared with the corresponding thiosemicarbazone analogues. An exception was 10b, which displayed high potency (pIC₅₀ = 5.27) against the HepG-2 cell line, and moderate potency against A-498 and MDA-MB-231 (pIC₅₀ = 4.03 and 4.29, respectively). The other compounds in this series exhibited lower activity. This observed inconsistency is most probably attributed to the spatial orientation of the majority of the 2-iminothiazolidin-4-one moiety within the active site of the targeted cell lines, which favors the two effectors, allopurinol and 5-chloroxindole nuclei. To improve the activities of 10a–10d and 11a–11d, we intended to attenuate the polarity and to avoid the HBA motif provided by the O in the lactam fragment. This goal was fulfilled by the cyclization of the terminal SCNH₂ fragment following the procedure outlined in Scheme 1 to yield a series of allopurinol/adenine-oxindole 2-hydrazinylideno thiazole 12a–12d and 13a–13d. Fortunately, the antiproliferative potency and scope of action of the allopurinol series shown by 12a–12d have surpassed the pIC₅₀ of the reference drug sorafenib. The activities of 12a and 12b against A-498 are 2-fold, against HepG2 are 8-fold, and against MDA-MB-231 are 5-fold those of sorafenib, which exhibits low cytotoxicity towards BALB/3T3 cells, indicating safety for normal cells. The activities of 12c and 12d against A-498 are 8-fold, against HepG2 are 30-fold, and against MDA-MB-231 are 20-fold those of sorafenib. However, adenine series 13a–13d showed marginal activity against any one of the cell lines. The observed strong anticancer activity of compounds 12a–12d can be attributed to the robust interactions of the motifs attached to the terminals of the dimethylene linker, namely the pyrazolo pyrimidinone, on one hand, and the fragments attached to the oxindole nucleus, on the other hand, showing multiple binding forces in the active site of the CDK6 enzyme. This interaction is further supported by their high binding scores, confirming their strong affinity for the target.

The selectivity indices (SI) were calculated for the most active compounds using the ratio SI = (IC₅₀ μM against normal cell line BALB/3T3)/(IC₅₀ μM against respective cancerous cell line) to evaluate the toxicity of tested compounds against normal cells and to predict their therapeutic potential. A favorable SI > 1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells. The most active compounds 6b, 7b, 8b and 12a–12d displayed an SI median of 2.8 against A498; against HepG2, 6a, 7b, and 8b showed an SI median of 2.03; and against MDA-MB-231 compounds, 6b, 7b and 12b showed an SI median of 2.49 (Table 3). The most active compounds with pIC₅₀ ≥ 4.5 showed SI values higher than the reference, which entitled them to further development as therapeutic candidates, especially for targeting the HepG2 cell line.

Table 3 SI of the most active compounds 6b, 7b, 8b, 12a–d and sorafenib

Entry	Selectivity index (SI)
Cell lines	A498	HepG2	MDA-MB-231
6b	2.03	2.15	2.49
7b	1.37	11.89	3.64
8b	2.45	2.60	0.63
12a	0.59	2.95	1.00
12b	0.79	2.80	1.25
12c	0.29	9.92	0.99
12d	0.35	1.78	0.91
Sorafenib	0.014	0.02	0.011

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Cell cycle analysis

Cell cycle distribution for compounds 6b, 7b, 12a, and 12c was assessed at two concentrations using the MV4-11 cell line, corresponding to their anti-proliferative IC₅₀ values (5.85, 11.77, 0.29, and 0.34 μM), with comparisons to a control group. These compounds, which exhibited potent cytotoxicity, were chosen for further investigation of their effects on cell cycle progression. The results, presented in Table 4 and Fig. S84, reveal that compounds 12a and 12c notably reduced the percentage of cells in the G2/M phase (6.64% and 10.59%, respectively) compared to the control group (20.98%). Additionally, the percentage of cells in the subG1 phase increased by approximately 4- and 3-fold, relative to the control, indicating that these compounds induced cell arrest at the subG1 phase. At higher concentrations (1 μg mL⁻¹), compounds 12a and 12c caused an increase in cell death, as evidenced by a higher proportion of cells in the subG1 phase, reflecting an enhanced cytotoxic effect. In contrast, compound 7b at 10 μg mL⁻¹ caused a statistically significant (p < 0.05) increase in the S phase by 1.49-fold more than the control and a decrease in the G2/M phase but did not induce cell death, as indicated by the subG1 percentage (6.15 ± 2.74%). The subG1 checkpoint prevents the replication of damaged DNA, allowing repair and inhibiting the proliferation of damaged cells. Arrest at this phase indicates severe DNA damage, potentially triggering apoptosis. The sub G1 phase arrest induced by compounds 12a and 12c highlights their selectivity for CDK6, a key regulator of the G1 phase transition. Palbociclib was reported to induce a cell cycle arrest at the G1 phase in the MV4-11 cancer cell line.⁴³

Table 4 Cell cycle analysis results presented as the means of cell percentage [%] ± SD

Entry	Cell percentage [%] ± SD
	SubG1	G0/G1	S	G2/M
Ctrl	7.12 ± 0.569	45.40 ± 3.051	24.54 ± 2.475	20.98 ± 0.614
6b, 1 μg mL^−1	3.48 ± 1.178	50.37 ± 4.619	27.15 ± 3.651	17.67 ± 1.285
7b, 10 μg mL^−1	6.15 ± 2.739	44.36 ± 4.193	36.66 ± 4.326	13.32 ± 2.763
12a, 1 μg mL^−1	31.28 ± 10.88	35.43 ± 5.182	24.34 ± 8.875	6.64 ± 2.959
12c, 1 μg mL^−1	21.16 ± 6.283	38.41 ± 4.391	29.22 ± 4.063	10.59 ± 1.830

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Enzymatic caspase-3/7 activity assay

Apoptosis is essential for controlling tumor growth and treatment efficacy. Many cancer therapies target this process by activating apoptotic pathways. A central mediator of apoptosis is caspase-3, a protein produced by the CASP3 gene, which plays a pivotal role in executing programmed cell death.⁴⁴ In this study, compounds 6b, 7b, 12a, and 12c were tested. Based on the test results, hepatic cancer lines (HepG2) were selected as the targeted model due to their high sensitivity to the synthesized derivatives, as well as the presence of relevant caspases, such as caspase-3/7, and their expression of multiple metabolizing enzymes.⁴⁵ Evaluating the activity of the released caspase-3 is considered the most accurate method to examine the sound effects of the most active compounds 6b, 7b, 12a and 12c on the apoptotic role of caspase 3. The results presented in Table 5 and Fig. S85 reveal the capability of compound 7b at a 5 μg mL⁻¹ dose to elevate the activity of caspase-3/7 more than that of the control. Additionally, 6b, 12a, and 12c did not increase caspase 3/7 activity, compared to the control. Among these compounds, 7b, containing an adenine nucleus, induced apoptosis through the contribution of caspase-3/7 activity slightly more than those containing allopurinol. The results suggest that caspase activation is not a primary mechanism of action for most of the tested compounds.

Table 5 Relative caspase-3/7 activity compared with the control against HepG2 cells

Entry	Relative caspase-3/7 activity ± SD
Ctrl	1
6b, 5 μg mL^−1	0.982 ± 0.128
7b, 5 μg mL^−1	1.296 ± 0.190
12a, 5 μg mL^−1	0.840 ± 0.200
12c, 5 μg mL^−1	0.927 ± 0.185

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In vitro CDK6 inhibitory assay

The most active towards cell proliferation compounds 6b, 7b, 8b and 12a–12c were screened owing to their potential inhibition of CDK6 enzyme using palbociclib as the reference drug, as shown in Table 6 and Fig. S86. Fortunately, all compounds proved to be potent inhibitors with pIC₅₀ ranging from 5.82 ± 0.059 to 7.17 ± 0.003. Notably, compound 12a exhibited pIC₅₀, matching the reference palbociclib (pIC₅₀ 6.79 ± 0.006), while compounds 8b, 12b and 12c showed a higher pIC₅₀ range of 6.91 ± 0.048 to 7.17 ± 0.003. These results position these compounds as strong candidates for further exploration and development as potential therapeutic CDKI leads. The lowest CDK6 inhibition effect of compound 7b has a pIC₅₀ value of 5.82. In the studied series, the most active compounds 12a–12d bind with the assigned fingerprint amino acids of the targeted active site. Furthermore, they bind with His100, which denotes selectivity for the CDK6 inhibitor. Overall, the ligand–protein binding pattern of 12a–12d with some extra amino acids out of the fingerprint residues kept within the ATP pocket enhances the binding affinity to the target site, possibly enhancing their inhibitory efficacy.

Table 6 In vitro inhibitory activities of 6b, 7b, 8b, 12a–c, and 13b against CDK6, VEGFR-2 and xanthine oxidase enzymes compared with palbociclib, sorafenib and allopurinol reference compounds, respectively

Entry	pIC50 CDK6 ± SD	pIC50 VEGFR-2 ± SD	pIC50 XO ± SD
6a	6.22 ± 0.023	6.51 ± 0.012	4.36 ± 0.55
7b	5.82 ± 0.059	6.29 ± 0.02	4.18 ± 0.82
8b	7.17 ± 0.003	7.15 ± 0.003	4.66 ± 0.33
12a	6.81 ± 0.006	7.27 ± 0.002	4.94 ± 0.16
12b	6.91 ± 0.048	6.92 ± 0.006	4.51 ± 0.62
12c	6.93 ± 0.005	6.84 ± 0.009	4.83 ± 0.23
13b	6.09 ± 0.032	6.49 ± 0.012	4.41 ± 0.49
Palbociclib	6.79 ± 0.006	—	—
Sorafenib	—	7.25 ± 0.002	—
Allopurinol	—	—	4.57 ± 0.13

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In vitro VEGFR-2 inhibitory assay

The most potent antitumor derivatives 6b, 7b, 8b and 12a–12c were tested for VEGFR-2 kinase inhibition using sorafenib as the reference compound. The results of pIC₅₀ values are presented in Table 6 and Fig. S86. Compounds 8b and 12a showed the most potent inhibition of VEGFR-2, achieving pIC₅₀ values of 7.15 and 7.27, respectively, comparable to the standard inhibitor, sorafenib (pIC₅₀ = 7.25).

The docking results revealed that the ethylene linker between the two heteroaromatic rings shared the same groove as the urea linker in sorafenib, contributing to a greater affinity for the VEGFR-2 enzyme. The formation of hydrogen bonds with Cys919, Asp1046, and Glu889, along with hydrophobic interactions, likely enhances the affinity of the heterocyclic pyrazolopyrimidine scaffold for the VEGFR-2 enzyme. In addition to the strong correlation between binding energies and pIC₅₀ values, as described in eqn (1), Fig. 12 indicates that ligand binding is primarily driven by interactions with the protein. This relationship is expressed as follows:

Binding energy = −1.8467 × pIC₅₀ + 4.2391. (1)

N = 7, r² = 0.91, p < 0.05

Fig. 12 Correlation between the calculated binding energy score ΔG and pIC₅₀ for compounds 6b, 7b, 8b, 12a–c and 13b.

Structure–activity relationship (SAR). Although a full SAR study is premature at this stage, initial observations reveal that the substituent pattern defined in Scheme 1 plays a pivotal role in modulating inhibitory activity. Compounds 6b, 7b, 8b, 10b, and 12b, all of which carry a 5-chloro substituent, demonstrate significantly enhanced potency. This enhancement may be explained by chlorine's capacity to participate in specific non-covalent interactions, including hydrogen and halogen bonding, which contribute to tighter receptor binding. Structural modeling indicates that the chlorine atom in these compounds forms halogen bonds with the carbonyl oxygen of Val101 (3.01–3.26 Å), supported by bond angles (C–Cl⋯O) in the range of 170–174.9° and favorable Cl⋯O–C angles (111–121°). These features enable stable and directed interactions within the CDK6 active site. In compound 12b, for example, the 5-chloro group facilitates binding to both His100 and Val101 (Fig. 13), further enhancing receptor affinity. Moreover, compounds 12a–12d, bearing a fused pyrazolopyrimidine and thiazole framework, exhibit an extended binding mode within the CDK6 pocket, which may account for their potent inhibitory effects.

Fig. 13 Representative example of a halogen bond in compound 12b with His100 and vicinal Val101 in the CDK6 active site.

Additionally, the predicted binding modes of compounds 8b and 12b within the CDK6 template structures were well-aligned, as shown in SI Fig. S87. These compounds demonstrated strong affinity for the ATP-binding site of CDK6, particularly interacting with Val101 and Asp163, with alignment scores of −126.70 and −116.67 kcal mol⁻¹, respectively.

Evaluation of xanthine oxidase inhibitory activity

The results of the in vitro XO inhibitory activity assay, as presented in Table 6 and Fig. S86, demonstrated that screened compounds 6b, 7b, 8b and 12a–c exhibited significant xanthine oxidase inhibitory activity with pIC₅₀ values ranging from 4.18 ± 0.82 to 4.94 ± 0.16. Among this series, the most active compounds 12a and 12c against XO have pIC₅₀ 4.94 and 4.83 distinct from allopurinol pIC₅₀ 4.57, respectively.

Kinetics of xanthine oxidase (XO) inhibition. Enzyme inhibition can generally be categorized as competitive, uncompetitive, noncompetitive, or mixed.^46,47 The Lineweaver–Burk plot (Fig. S88) for compound 12a displays intersecting lines to the left of the y-axis and above the X-axis, which is characteristic of mixed-type inhibition. This indicates that the inhibitor can bind both to the free enzyme and to the enzyme–substrate complex although with varying affinities.^48,49 Simultaneous increase in K_m and decrease in Vmax shown by 12a and the reference allopurinol support the suggested mixed XOI mechanism. These conclusions are supported by strong correlation coefficients of 0.996 for 12a and 0.958 for allopurinol (Table 7).

Table 7 Kinetic parameters of allopurinol and 12a against xanthine oxidase in the enzyme kinetic study

Conc. (μM)	12a		Allopurinol
	Km	Vmax	Km	Vmax
XO	40	2.25	40	2.21
0.1	75	1.66	50	1.51
1	112	1.38	64	1.33
10	123	0.98	51	0.55

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2D-QSAR study

A 2D-Quantitative Structure–Activity Relationship (2D-QSAR) model was developed to predict the root for the perspective design of new ligands against HepG2 hepatocellular carcinoma. The synthesized compounds were divided into a training set of 16 compounds for model development and a test set of 3 derivatives reserved for external validation. Molecular descriptors were calculated using the MOE (Molecular Operating Environment) software and categorized into seven clusters: physical properties, subdivided surface areas, atom and bond counts, connectivity-based descriptors, partial charge descriptors, pharmacophore feature descriptors, and distance matrix descriptors.^50–52

To identify the most significant structural predictors of activity, Stepwise Linear Regression Analysis (SLRA) was employed.⁵³ The QSAR model was developed using the Partial Least Squares (PLS) method with a dataset of 16 compounds. In accordance with QSAR modeling guidelines, the number of descriptors was limited to a maximum of three, ensuring a descriptor-to-compound ratio of approximately 1 : 5 to maintain model robustness and prevent overfitting.

The best QSAR model was selected based on its performance, which was evaluated using the highest correlation coefficient (R² = 0.90) and the lowest root mean square error (RMSE = 0.203), as shown in Fig. S89 and Table S2. The final model was a linear equation derived from using the Partial Least Squares (PLS) method and was based on three key molecular descriptors, as demonstrated in eqn (2):

pIC₅₀ = 6.42560 + 0.10829 × E_vdW − 0.00705 × TPSA + 0.29934 × log P(o/w). (2)

n = 16, RMSE = 0.20301, r² = 0.90112

The best-fit QSAR model revealed that the antiproliferative activity of the synthesized derivatives was influenced by three key molecular descriptors. van der Waals energy (E_vdW) exhibited a positive correlation with anticancer activity, as it plays a vital role in the formation of protein–ligand complexes. Similarly, lipophilicity (log P(o/w)) is crucial for determining a compound's ability to penetrate hydrophobic barriers, such as cell membranes, to reach its site of action. However, the polar surface area (TPSA) showed an inverse correlation with the antiproliferative activity of the synthesized derivatives. TPSA reflects the ability of a ligand to form polar interactions, particularly hydrogen bonds, with specific biological targets. The Z-score method was used to identify outliers in the QSAR model, as presented in Table S2. In our study, none of the compounds had a Z-score above 2.5, indicating that all the compounds were consistent with the model predictions. This finding underscores the robustness and accuracy of the QSAR model. To validate the model, an independent test set comprising five compounds (7b, 9c, and 12c) was used for external validation. Additionally, two approved drugs, sunitinib and cisplatin, were included to strengthen the correlation and assess the model's predictive accuracy. The model's predictive performance was assessed by plotting the experimental pIC₅₀ values against the predicted pIC₅₀ values for these compounds. The model demonstrated a high predictive accuracy, with a correlation coefficient of R² = 0.93, as shown in Fig. S90. This strong correlation between the predicted and experimental values confirmed the model's robustness and reliability.

For the future development of novel compounds in this series, it is recommended to prioritize structural modifications that enhance E_vdW and log P(o/w). This strategy is expected to improve the binding affinity of the compounds to biological targets, increase membrane permeability, and strengthen interactions.

In vitro chemical stability of compounds 6b, 8b, and 12a–12c

To assess the chemical stability of the most active compounds 6b, 8b, and 12a–12c under simulated physiological conditions, each compound was incubated in phosphate buffer (pH 7.4) at 37 °C, and UV-Vis spectra were recorded over the 190–800 nm range at regular intervals up to 24 hours. The spectra remained consistent throughout the study, with no significant changes in absorbance intensity or the appearance of new peaks, indicating no degradation. These results support the chemical integrity of the compounds in buffered aqueous solutions, confirming their suitability for further biological studies. The full data are given in the SI, Fig. S91.

Determination of global reactivity descriptors

Density Functional Theory (DFT) calculations were conducted at the B3LYP level using the 6-31G basis set to investigate the structural characteristics of compounds 6b, 7b, 8b and 12a–12c, which exhibit the highest activity on the target enzyme CDK6. These calculations provided key insights into the molecular electronic structure through global reactivity descriptors. The descriptors include the energies of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), as well as derived reactivity parameters, such as ionization potential (I), electron affinity (A), electronegativity (χ), chemical hardness (η), softness (s), chemical potential (μ), and electrophilicity (ω) (Table S3). These descriptors, particularly HOMO and LUMO energies, are critical for predicting the charge transfer, chemical reactivity, bioactivity, and stability of the compounds.^54–57 A higher HOMO energy suggests a stronger electron-donating ability and, therefore, greater chemical reactivity. In contrast, a lower LUMO energy indicates a higher electron-accepting capacity, also pointing to enhanced reactivity. In ligands 6b, 8b and 12a–12c, HOMO electron density is primarily localized over the ethylene link and extended in the direction of the 5-Cl-oxindole and the thiazolylidene ring, indicating a propensity for electron donation during interactions with the CDK6 enzyme. The LUMO electron density, however, is distributed over the oxindole ring, as well as the sulfur and nitrogen atoms in the thiazolylidene ring, facilitating electron delocalization and influencing interactions with target molecules. Fig. 14 illustrates the orbital isosurface of the frontier molecular orbitals (HOMO and LUMO) of compounds 6b, 8b and 12b, with positive phases depicted in green and negative phases in red. The energy gap (ΔE = LUMO − HOMO) serves as a key indicator of both chemical and biological activity; smaller energy gaps indicate higher reactivity and lower excitation energy requirements. In the studied series, the energy gap follows the trend: thiazolylidene derivatives 12a–12c < thiosemicarbazone 8b < unsubstituted compounds 6b and 7b. Among the 5-substituted oxindole derivatives, ΔE: 5-MeO < 5-Cl < 5-F < 5-H, signifying the increased reactivity of 5-substituted oxindole derivatives relative to their unsubstituted counterparts. Electrophilic and nucleophilic indices determine a molecule's ability to exchange electron density during reactions. The order of electron affinity, electronegativity, and chemical potential follows unsubstituted 6b, 7b > thiosemicarbazone 8b > thiazolylidene derivatives 12a–12c, indicating that thiazolylidene derivatives have the highest electron transfer capacity.

Fig. 14 Representative frontier molecular orbitals of compounds 6b, 8b and 12b.

Molecular polarizability is linked to softness and hardness: “soft” molecules with lower hardness and higher softness are more polarized. Among the synthesized compounds, thiazolylidene derivatives 12a–12c, with small energy gaps (ΔE = 2.558–2.800 eV), are classified as soft molecules, making them highly polarizable and biologically active. The electrophilicity index is essential for assessing a drug candidate's binding affinity to biological targets. Higher electrophilicity strengthens covalent interactions with nucleophilic residues in enzymes or receptor proteins, enhancing binding strength and specificity. Additionally, electron-withdrawing groups boost electrophilicity by stabilizing the LUMO, reducing the HOMO–LUMO energy gap, and increasing reactivity toward nucleophilic attack.

In conclusion, compounds 12b and 12c, which demonstrated the highest CDK6 inhibitory activity, exhibited a small energy gap and lower electrophilicity. These calculated descriptors provide valuable insights into drug design strategies, particularly in the optimization studies of these compounds.

Prediction of physicochemical properties of the most active compounds

The drug-likeness and molecular properties of the most active compounds, along with the reference drugs palbociclib and sorafenib, were evaluated using Molinspiration cheminformatics software⁵⁸ (Table S4). These parameters are essential for predicting the pharmacokinetics of the compounds, including their transport properties, reactivity, and metabolic stability within biological systems. All the targeted compounds, except for 10b and 12c, fully complied with Lipinski's Rule of Five. The number of rotatable bonds in the synthesized compounds was fewer than 10, indicating compliance with Veber's criteria⁵⁹ for molecular flexibility, suggesting that these compounds are likely to exhibit good oral bioavailability. Additionally, the TPSA values of the synthesized compounds ranged from 92.35 to 139.41 Ų, further supporting their potential for oral bioavailability. Substituents at the C-5 position of the oxindole ring (–F, –Cl, and –OCH₃) increased the log P values and enhanced hydrophobicity and, consequently, the inhibitory potency against CDK6 through hydrophobic and van der Waals interactions. The most active compounds (6b, 8b, and 12a–12d) adhered to both Lipinski's and Veber's rules, highlighting their strong potential to inhibit the activities of CDK6, VEGFR-2, and xanthine oxidase.

Conclusion

This study presents a new class of oxindole derivatives derived from allopurinol and adenine, with promising multi-target anticancer potential. Compounds 6b, 7b, 8b, and 12a–d displayed potent cytotoxic effects, particularly toward HepG2 cells. Notably, allopurinol-derived hybrids (12a–d) showed enhanced activity over adenine-based analogs, attributable to chlorine-induced electronic effects. Mechanistic assays demonstrated pro-apoptotic activity (12a and 12c), S-phase arrest (7b), and caspase activation. Enzymatic profiling and molecular modeling confirmed potent inhibition of CDK6, VEGFR-2, and xanthine oxidase, with key halogen/hydrogen bonding interactions at the CDK6 hinge region. The superior bioactivity of 12a–c correlated with its soft electronic character. These findings position compounds 8b, 12a, and 12b as compelling candidates for further development in anticancer therapeutics.

Experimental

Molecular docking study

The molecular docking simulation studies were performed on a Dell Precision T3600 workstation with Intel Xeon® CPU-1650.0 @ 3.20 GHz with a Windows 10 operating system using Molecular Operating Environment software (MOE 2020.01). The X-ray crystal structure of CDK6 co-crystalized with palbociclib (PDB ID: 5L2I) and Sorafenib with VEGFR-2 (PDB code 3WZE) was obtained from the protein data bank. Since the crystal structure contains a ligand molecule, the program automatically identifies the binding site, and we dock the tested ligands on it. Docking of the conformation database of the target ligands was done using the MOE-DOCK software wizard. To compare the conformers, London dG was used as a scoring function; lower values indicate more favorable poses. All the key interactions accomplished by the co-crystalized ligand with the key amino acids in the binding site are reproducible using the following docking setup.

Molecular dynamics simulation

The initial CDK6 complexes with palbociclib and compound 12b were obtained via molecular docking. Molecular dynamics simulations (MDS) were conducted using NAMD 3.0 with the CHARMM27 force field.⁶⁰ Hydrogen atoms were added via the psfgen plugin in VMD 1.9. The systems were solvated in TIP3P water with 0.15 M Na⁺ and Cl⁻ ions, energy minimized, heated to 300 K, and equilibrated for 1 ns. A 50 ns MDS was performed,⁶¹ recording trajectory data every 0.1 ns to analyze RMSD, RMSF, hydrogen bonds, radius of gyration, and SASA. Ligand parameters were determined using CHARMM-GUI. Binding free energy was estimated via MM-PBSA using the fastDRH webserver with AmberTools 21, calculated as follows: ΔG_binding = G_complex − (G_receptor + G_inhibitor). Each term involves calculating energy components, such as van der Waals, electrostatic, molecular mechanics, internal, and polar solvation energy.

Chemistry

Materials and methods. All the chemicals used for the synthesis of the target compounds were commercially available reagent grade and used without further purification, except the solvents, which were purified before use. N,N-Dimethylformamide (DMF) was purchased from Biochem and dried over molecular sieves (4 Å) prior to use. Isatin 98%, 5-chloroisatin 98%, and adenine 98% were obtained from Alfa Aesar Co., Germany. 5-Methoxyisatin 97% and 5-fluoroisatin 98% were obtained from Acros Organic Co., United Kingdom. Chemical reactions were monitored on thin layer chromatography (TLC) using precoated silica gel plates, 60 G F254, obtained from Merck, Darmstadt (Germany).

Silica gel (60–120 mesh) was used for column chromatography, and the developing systems used were: system (A): dichloromethane/methanol (9.5 : 0.5 v/v), system (B): chloroform/ethanol (9.7 : 0.3 v/v) and system (C): chloroform/ethyl acetate (2 : 1 v/v).

Melting points were measured using a Stuart® SMP10 melting point apparatus (Stuart Scientific, England) and were uncorrected. Infrared spectra (IR) were recorded using a Nicolet® iS10 FT-IR Spectrometer as KBr pellets at the Faculty of Science-Assiut University, Assiut, Egypt. The data are reported as the frequency of absorption (cm⁻¹). The NMR spectra were recorded using an AVANCE-III High Performance FT-NMR spectrometer, Brucker-Biospin International AG, Switzerland (400 MHz for ¹H and 100 MHz for ¹³C) at the Faculty of Science-Zagazig University, Zagazig/Egypt. Chemical shifts are reported in parts per million (ppm, δ scale) using TMS as referenced relative to residual solvent DMSO-d6 (δ ¹H = 2.50, ¹³C = 39.5 ppm). Coupling constants (J) for ¹H were reported in Hz, and spin multiplicities are represented by the following signals: singlet (s), doublet (d), triplet (t), doublet of doublet (dd) and multiplet (m). All raw fid files were processed, and the resulting spectra were analyzed using the program MestReNOVA 14.2 from Mestrelab Research S.L. Mass spectra (MS) were obtained using the inlet part to mass analyser in ThermoFisher scientific DPC-Direct Probe Controller model ISQ7000 single quadruple mass spectrometer at the Faculty of Science, Assiut University, Assiut, Egypt. Values are reported as the ratio of mass to charge in Daltons. The purity of the most active compounds 6b, 8b, 12a, 12b and 12c is assessed by HPLC analysis at the Institute for Drug Development and Innovation Research, Assiut University, Assiut, Egypt.

General method for synthesis of 2-bromoethyl-substituted pyrazolopyrimidine and purine derivatives 3, 4

To a stirred suspension of allopurinol 1 or adenine 2 (1 mmol) and potassium carbonate (0.32 g, 2.3 mmol) in DMF (20 mL), 1,2-dibromoethane (0.75 g, 4 mmol) was added in a single portion. The mixture was stirred at room temperature for 24 h, after which the starting material was no longer detectable by TLC MeOH : DCM 1 : 9 and a new spot was detected. Then, the mixture was poured into crushed ice and left for 1 h. The precipitate was filtered, washed with cold water and dried at 80 °C.

1-(2-Bromoethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 3. Yield 2.0 g (82%), a pale-orange solid, mp 261–264 °C, recrystallized from ethanol. FT IR: 3435, 3083, 2969, 1694, 1582, 782, 695. ¹H NMR δ: 8.44–8.12 (m, 3H), 4.70–4.66 (t, J = 4 Hz, 2H), 4.37–4.28 (t, J = 4 Hz, 1H), 3.93–3.91 (t, J = 4 Hz, 1H). Mass spectrum = 242.02 (M. wt: 243.06).

9-(2-Bromoethyl)-9H-purin-6-amine 4. Yield 1.75 g (72%), an orange solid, mp: 295–298 °C (297–298 lit.⁶²), washed with water. IR: 3362, 3323, 3150, 1652, 1599, 1385, 1325, 1309, 1246, 1074, 796, 708, 647. ¹H NMR δ: 8.19 (s, 1H), 8.15 (s, 1H), 7.27 (s, 2H), 4.57 (t, J = 5.97, 2H), 3.95 (t, J = 5.97, 2H).

General method for the synthesis of 1,5-disubstitutedindole-2,3-dione 6a–d and 7a–d

To a stirred solution of 5-(un) substituted-1H-indole-2,3-dione 5a–d (4 mmol) and anhydrous potassium carbonate (0.83 g, 6 mmol) in dry DMF (5 mL), compounds 3 and 4 (4 mmol) were added. The reaction mixture was stirred at 60 °C for 6–8 h. The reaction mixture was then cooled to room temperature, poured into crushed ice and left for 1 h. The products were separated by filtration, dried and purified by column chromatography on a silica gel column using developing systems (A) as the mobile phase.

1-[2-(1,5-Dihydro-4-oxopyrazolo[3,4-d]pyrimidin-1-yl) ethyl] indole-2, 3-dione 6a. Yield 1.35 (88%), orange powder, reaction time 6 h, mp: 223–225 °C. IR: 3435, 3084, 2952, 1745, 1694, 1608, 1582, 1473. ¹H NMR δ: 8.33–8.00 (m, 3H), 7.55 (dd, J = 13.1, 6.9 Hz, 2H), 7.09 (dd, J = 14.9, 7.0 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 4.55 (s, 2H), 4.19–4.08 (m, 2H). ¹³C NMR δ: 183.0, 158.3, 156.5, 151.8, 150.4, 150.1, 138.1, 135.0, 128.1, 124.5, 123.2, 110.3, 105.0, 44.3, 43.3. Elemental analyses found: C%, 58.43; H%, 3.77; N%, 22.91. Calc. for C₁₅H₁₁N₅O₃: C%, 58.25; H%, 3.58; O%, 22.64.

5-Chloro-1-[2-(1,5-dihydro-4-oxopyrazolo[3,4-d]pyrimidin-1-yl)ethyl]indole-2, 3-dione 6b. Yield 1.2 (70%), pale orange powder, reaction time 6.5 h, mp: 214–215 °C. IR: 3467, 3084, 2953, 1745, 1694, 1610, 1584, 1473. ¹H NMR δ: 8.11–7.97 (m, 3H), 7.60 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 2.3 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.60–4.53 (m, 2H), 4.34–4.23 (m, 2H). ¹³C NMR δ: 181.9, 157.8, 156.5, 151.6, 150.7, 148.6, 137.1, 136.0, 127.4, 124.0, 118.4, 111.9, 105.0, 44.3, 42.8. Elemental analyses found: C%, 52.67; H%, 3.05; N%, 20.63. Calc. for C₁₅H₁₀ClN₅O₃: C%, 52.41; H%, 2.93; N%, 20.37.

1-[2-(1,5-Dihydro-4-oxopyrazolo[3,4-d]pyrimidin-1-yl)ethyl]5-methoxyindole-2,3-dione 6c. Yield 1.2 g (71%), reddish orange powder, reaction time 7 h, mp: 226–228 °C. IR: 3458, 3084, 2945, 1736, 1697, 1621, 1598, 1490. ¹H NMR δ: 8.10–7.96 (m, 3H), 7.11 (d, 1H), 7.09 (d, J = 18.6 Hz, 2H), 6.79 (s, 1H), 4.64–4.27 (m, 2H), 4.27–4.03 (s, 1H), 4.03–3.90 (m, 1H), 3.73 (s, 3H). ¹³C NMR δ: 183.2, 158.1, 156.5, 155.6, 151.6, 150.8, 144.2, 135.0, 124.0, 117.7, 111.1, 109.2, 105.0, 55.8, 44.3, 43.2. Elemental analyses found: C%, 56.82; H%, 4.03; N%, 20.89. Calc. for C₁₆H₁₃N₅O₄: C, % 56.64; H%, 3.86; N%, 20.64.

5-Fluoro-1-[(2-(1,5-dihydro-4-oxopyrazolo[3,4-d]pyrimidin-1-yl)ethyl)]indole-2,3-dione 6d. Yield 1 g (63%), pale orange powder, reaction time 6.5 h, mp 219–220 °C, IR: 3450, 3056, 2952, 1741, 1697, 1624, 1583,1486. ¹H NMR δ: 8.35–8.01 (m, 2H),7.46 (s, 2H), 6.93 (s, 1H), 4.54 (s, 2H), 4.22–4.09 (m, 2H). ¹³C NMR δ: 182.8, 160.1, 158.5, 157.6, 156.9, 152, 147.1, 136.9, 135.4, 128.5, 124.5, 112.0, 105.4, 44.7, 43.9. Elemental analyses found: C%, 55.31; H%, 3.20; N%, 21.67. Calc. for C₁₅H₁₀FN₅O₃: C%, 55.05; H%, 3.08; N%, 21.40.

1-[2-(6-Amino-9H-purin-9-yl)ethyl]-2,3-dihydro-1H-indole-2,3-dione 7a. Yield 1.4 g (91%), orange powder, reaction time 8 h, mp: 244–246 °C (decomp.), (lit. 242 °C).⁶² IR: 3441, 3317, 3157, 3103, 2940, 1740, 1651, 1610, 1596, 1473. ¹H NMR δ: 8.16 (s, 1H), 7.95 (s, 1H), 7.65–7.38 (m, 2H), 7.16 (s, 2H), 7.04 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 7.9 Hz, 1H), 4.44 (t, J = 5.3 Hz, 2H), 4.11 (t, J = 5.3 Hz, 2H). ¹³C NMR δ: 183.3, 158.7, 156.2, 152.7, 150.7, 150.1, 141.3, 138.2, 124.7, 123.5,119, 117.8, 110.3, 41.1, 40.3. Mass spectrum: m/z 308.26 [M]⁺; M. wt: 308.29. Elemental analyses found: C%, 58.67; H%, 4.08; N%, 27.49. Calc. for C₁₅H₁₂N₆O₂: C%, 58.44; H%, 3.92; N%, 27.26.

1-[2-(6-Amino-9H-purin-9-yl)ethyl]-5-chloro-2,3-dihydro-1H-indole-2,3-dione 7b. Yield 1.5 g (87.5%), pale orange powder, reaction time 7.5 h, mp: 259–261 °C. IR: 3441, 3320, 3174, 3031, 1740, 1654, 1605, 1577, 1477. ¹H NMR δ: 8.15 (s, 1H), 7.93 (s, 1H), 7.62–7.48 (m, 2H), 7.16 (s, 2H), 6.93 (d, J = 8.3 Hz, 1H), 4.41 (s, 2H), 4.11 (d, J = 4.7 Hz, 1H). ¹³C NMR δ: 181.9, 159.2, 158, 155.8, 152.4, 148.8, 140.9, 136.7, 127.2, 123.7, 118.6, 113.8, 111.6, 42.7, 40.6. Elemental analyses found: C%, 52.73; H%, 3.42; N%, 24.68. Calc. for C₁₅H₁₁ClN₆O₂: C, 52.56; H, 3.23; N, 24.52.

1-[2-(6-Amino-9H-purin-9-yl)ethyl]-2,3-dihydro-5-methoxy-1H-indole-2,3-dione 7c. Yield 1.2 g (71%), reddish orange powder, reaction time 6 h, mp: 260–264 °C. IR: 3365, 3308, 3143, 2963, 1740, 1667, 1600, 1576, 1492. ¹H NMR δ: 8.14 (s, 1H), 7.96 (s, 1H), 7.15 (s, 2H), 7.05 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 7.3 Hz, 1H) 4.41 (s, 2H), 4.07 (s, 2H), 3.72 (s, 3H). ¹³C NMR δ: 179.1, 161.3, 156.2, 152.7, 150.1, 141.3, 136.7, 131.5, 120.7, 119, 117, 110.4, 106.6, 56.0, 41.3, 40.5. Elemental analyses found: C%, 56.88; H%, 4.35; N%, 24.99. Calc. for C₁₆H₁₄N₆O₃: C%, 56.80; H%, 4.17; N%, 24.84.

1-[2-(6-Amino-9H-purin-9-yl)ethyl]-2,3-dihydro-5-fluoro-1H-indole-2,3-dione 7d. Yield 1.45 g (92%), pale orange powder, reaction time 6.5 h, mp: 230–233 °C. IR: 3368, 3325, 3159, 3103, 1739, 1652, 1603, 1577, 1485. ¹H NMR δ: 8.15 (s, 1H), 7.93 (s, 1H), 7.36 (d, J = 7.0 Hz, 2H), 7.16 (s, 2H), 6.93 (d, J = 5.5 Hz, 1H), 4.44 (t, J = 5.3, 2H), 4.10 (t, J = 5.3, 2H). ¹³C NMR δ: 182.9, 159.9, 158.7, 157.5, 156.2, 152.8, 150.1, 146.9, 141.4, 124.3, 124.1, 118.9, 111.6, 43.0, 41.1. Elemental analyses found: C%, 55.41; H%, 3.57; N%, 26.04. Calc. for C₁₅H₁₁FN₆O₂: C%, 55.22; H%, 3.40; N%, 25.76.

General method of synthesis of oxindole-3-thio-semicarbazone derivatives 8a–d–9a–d

The appropriate isatin derivative 6a–d, 7a–d (1 mmol) and thiosemicarbazide (0.09 g, 1 mmol) were dissolved in warm ethanol (20 mL). The reaction mixture was acidified by the addition of few drops of glacial acetic acid and refluxed for 2–4 h. The mixture was cooled to room temperature, and the separated precipitate was filtrated, washed with ethanol, and dried. The product was purified by recrystallization from ethanol.

(2Z)-2-{2-Oxo-1-[2-(4-oxo-1,5-dihydrpyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 8a. Yield 0.28 g (75%), yellow powder, reaction time 2 h, mp 245–248 °C. IR: 3420, 3260, 3163, 3083, 2951, 1694, 1612, 1358, 782. ¹H NMR δ: 12.22 (s, 1H), 9.09 (s, 1H), 8.72 (s, 1H), 8.26–7.96 (m, 2H), 7.67–7.62 (t, J = 8 Hz, 1H), 7.30–7.26 (m, J = 8 Hz, 1H), 7.08 (d, J = 6.8 Hz, 1H), 6.79 (dd, J = 8, 7.5 Hz, 1H), 4.65 (s, 2H), 4.30–4.07 (m, 2H). ¹³C NMR δ: 179.4, 162.8, 161.2, 156.7, 152.1, 151.1, 142.7, 135.4, 131.4, 123.1, 121.1, 119.7, 109.8, 105.7, 44.7, 43. Elemental analyses, found: C%, 50.49; H%, 3.80; N%, 29.47; S%, 8.46. Calc. for C₁₆H₁₄N₈O₂S: C%, 50.25; H%, 3.69; N%, 29.30; S%, 8.39.

(2Z)-2-{5-Chloro-2-oxo-1-[2-(4-oxo-1,5-dihydropyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 8b. Yield 0.3 g (70%), yellow powder, reaction time 2.5 h, mp: 250–252 °C. IR: 3431, 3265, 3172, 3056, 2928, 1694, 1597, 1348, 782. ¹H NMR δ: 12.07 (s, 1H), 9.18 (s, 1H), 8.85 (s, 1H), 8.27–7.98 (m, 3H), 7.77 (d, J = 16.5 Hz, 1H), 7.36 (s, 1H), 6.84 (d, J = 14.8 Hz, 1H), 4.58 (s, 2H), 4.29–4.14 (m, 2H). ¹³C NMR δ: 178.7, 160.5, 156.4, 151.5, 150.6, 140.9, 135.0, 130.1, 129.3, 127.1, 121.0, 120.3, 110.7, 105.0, 45.3, 44.5. Elemental analyses found: C%, 46.32; H%, 3.40; N%, 27.15; S%, 7.78. Calc. for C₁₆H₁₃ClN₈O₂S: C%, 46.10; H%, 3.14; N%, 26.88; S%, 7.69.

(2Z)-2-{5-Methoxy-2-oxo-1-[2-(4-oxo-1,5-dihydropyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 8c. Yield 0.25 g 68.5%, pale orange powder, reaction time 4 h, mp: 252–253 °C. IR: 3429, 3265–3168, 3056, 2943, 1693, 1596, 1357, 782. ¹H NMR δ: 12.16 (s, 1H), 9.09 (s, 1H), 8.75 (s, 1H), 8.21–7.99 (m, 3H), 7.37 (d, J = 14.4 Hz, 1H), 7.04–6.73 (m, 2H), 4.53 (s, 2H), 4.29–4.14 (m, 2H), 3.73 (s, 3H). ¹³C NMR δ: 179.2, 160.8, 156.4, 155.6, 151.6, 150.6, 136.1, 134.9, 130.9, 120.1, 116.8, 110.1, 106.3, 105.0, 55.6, 44, 43.7. Elemental analyses found: C%, 49.78; H%, 4.05; N%, 27.30; S%, 7.91. Calc. for C₁₇H₁₆N₈O₃S: C%, 49.51; H%, 3.91; N%, 27.17; S%, 7.77.

(2Z)-2-{5-Fluoro-2-oxo-1-[2-(4-oxo-1,5-dihydropyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 8d. Yield 0.3 g (82%), yellow powder, reaction time 2.5 h, mp 230–233 °C. IR: 3430, 3268, 3168, 3056, 2952, 1694, 1583, 1352, 782. ¹H NMR δ: 12.09 (s, 1H), 9.18 (s, 1H), 8.83 (s, 1H), 8.28–8 (m, 3H), 7.53 (s, 1H), 7.16 (s, 1H), 6.84 (d, J = 17.7 Hz, 1H), 4.55 (s, 2H), 4.35–4.13 (m, 2H). ¹³C NMR δ: 180.1, 161.2, 157.7, 156.9, 152.1, 151.1, 139.0, 135.6, 130.4, 121.3, 117.6, 110.9, 108.2, 105.0, 44.9, 43.2. Elemental analyses found: C%, 48.23; H%, 3.45; N%, 28.17; S%, 8.14. Calc. for C₁₆H₁₃FN₈O₂S: C%, 48.00; H%, 3.27; N%, 27.99; S%, 8.01.

(2Z)-2-{2-Oxo-1-[2-(6-aminopurine-9-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 9a. Yield 0.35 g (94%), bright yellow powder, reaction time 2.5 h, mp: 276–277 °C, IR: 3373, 3312, 3265, 3170, 2931, 1677, 1600, 1304, 1172. ¹H NMR δ: 12.26 (s, 1H), 9.10 (s, 1H), 8.74 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.16 (s, 2H), 7.06 (t, J = 7.4 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 4.46 (t, J = 5.5 Hz, 2H), 4.18 (t, J = 5.5 Hz, 2H). ¹³C NMR δ: 179.1, 161.4, 156.2, 152.8, 150.1, 143.0, 141.2, 131.4, 131.3, 123.2, 121.1, 119.8, 119.1, 109.5, 41.3, 40.3. Elemental analyses found: C%, 50.52; H%, 4.17; N%, 33.19; S%, 8.53. Calc. for C₁₆H₁₅N₉OS: C%, 50.38; H%, 3.96; N%, 33.05; S%, 8.41.

(2Z)-2-{5-Chloro-2-oxo-1-[2-(6-aminopurine-9-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 9b. Yield 0.4 g (94%), yellow powder, reaction time 3.5 h, mp 281–283 °C. IR: 3450, 3248, 3289, 3248, 3110, 2951, 1697, 1610, 1303, 1170. ¹H NMR δ: 12.07 (s, 1H), 9.15 (s, 1H), 8.84 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.29 (dd, J = 8.5, 2.2 Hz, 1H), 7.14 (s, 2H), 6.84 (d, J = 8.5 Hz, 1H), 4.44 (t, J = 5.5 Hz, 2H), 4.17 (t, J = 5.5 Hz). ¹³C NMR δ: 178.7, 160.6, 155.8, 152.3, 149.6, 141.2, 140.8, 140.4, 129.9, 126.9, 121.1, 120.3, 118.5, 110.6, 42.6, 40.8. Elemental analyses found: C%, 46.34; H%, 3.48; N%, 30.17; S%, 7.84. Calc. for C₁₆H₁₄ClN₉OS: C%, 46.21; H%, 3.39; N%, 30.31; S%, 7.71.

(2Z)-2-{5-Methoxy-2-oxo-1-[2-(6-aminopurine-9-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene} hydrazine-1-carbothioamide 9c. Yield 0.3 g (82%), yellowish orange powder, and reaction time 4 h, mp 244–245 °C. IR: 3446, 3294, 3244, 3142, 2927, 1694, 1603, 1304, 1164. ¹H NMR δ: 12.19 (s, 1H), 9.08 (s, 1H), 8.74 (s, 1H), 8.06 (s, 1H), 8.01 (s, 1H), 7.29 (s, 1H), 7.13 (s, 2H), 6.80–6.74 (m, 2H), 4.43 (s, 2H), 4.13 (s, 2H), 3.72 (s, 3H). ¹³C NMR δ: 178.7, 160.9, 155.8, 155.6, 152.3, 149.7, 140.8, 136.3, 131.1, 120.16, 118.6, 116.6, 109.9, 106.2, 55.6, 42.6, 40.8. Elemental analyses found: C%, 49.89; H%, 4.30; N%, 30.82; S%, 7.90. Calc. for C₁₇H₁₇N₉O₂S: C%, 49.63; H%, 4.16; N%, 30.64; S%, 7.79.

(2Z)-2-{5-Fluoro-2-oxo-1-[2-(6-aminopurine-9-yl)ethyl]-1,2-dihydro-3H-indol-3-ylidene}hydrazine-1-carbothioamide 9d. Yield 0.35 g (95%), yellow powder, reaction time 3 h, mp 279–281 °C. IR: 3420, 3362, 3321, 3150, 2951, 1652, 1599, 1309, 1174; ¹H NMR δ: 12.12 (s, 1H), 9.15 (s, 1H), 8.78 (s, 1H,), 8.08 (s, 1H), 7.97 (s, 1H), 7.45 (d, J = 5.3 Hz, 1H), 7.14 (s, 2H), 7.08 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 4.44 (s, 2H), 4.16 (s, 2H). ¹³C NMR δ: 178.8, 161.0, 157.3, 155.8, 152.4, 149.7, 140.9, 138.9, 130.4, 120.8, 118.5, 116.9, 110.3, 107.9, 40.9, 40.1. Elemental analyses found: C%, 48.27; H%, 3.49; N%, 31.75; S%, 8.11. Calc. for C₁₆H₁₄FN₉OS: C%, 48.11; H%, 3.53; N%, 31.56; S%, 8.03.

Synthesis of 1,5-disubstituted-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 10a–d and 11a–d

A mixture of appropriate 2-substituted hydrazine-1-carbothioamides series 8a–d, 9a–d (1 mmol), ethyl bromoacetate for 8a–d or monochloroacetic acid for 9a–d (0.17 g, 1 mmol) and anhydrous sodium acetate (0.16 g, 2 mmol) in ethanol (50 ml) were refluxed for 24 h, the reaction mixture was cooled, poured into crushed ice. The formed precipitate was filtered, dried, and purified by silica gel column using the developing system (B) for compounds 10a–d and (C) for 11a–d.

1-[2-(4-Oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl) ethyl]-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 10a. Yield 0.3 g (68%), orange powder, mp: 265–267 °C, IR: 3435, 3050, 2954, 1744, 1697, 1597, 1351, 783. ¹H NMR δ: 12.23 (s, 1H), 8.24–7.97 (m, 3H), 7.49 (dd, J = 13.6, 7.3 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.00 (d, J = 6.6 Hz, 2H), 6.87–6.61 (m, 1H), 4.55 (s, 2H), 4.19 (d, J = 7.8 Hz, 3H) 3.97 (s, 2H). ¹³C NMR δ: 171.8, 166.4, 156.9, 156.3,151.5, 150.4, 145.9, 134.8, 132.3, 130, 122.2, 121.2, 119.5, 108.7, 104.9, 48, 44.6, 32.2. Mass spectrum: m/z [M]⁺ = 422.42; found 422.21. Elemental analyses found: C%, 51.42; H%, 3.52; N%, 26.80; S%, 7.71. Calc. for C₁₆H₁₃FN₈O₂S: C%, 51.18; H%, 3.34; N%, 26.53; S%, 7.59.

5-Chloro-1-[2-(4-Oxo-1,5-dihydro-4H pyrazolo[3,4-d]pyrimidine-1-yl) ethyl]-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 10b. Yield 0.27 g (61.5%), reddish orange powder, mp: 288–290 °C dec., IR: 3435, 3084, 2952, 1694 (2C=O), 1582, 1374, 783. ¹H NMR δ: 12.06 (s, 1H), 8.26–7.97 (m, 3H), 7.74–7.34 (m, 2H), 6.80–6.70 (m, 1H), 4.53 (s, 2H), 4.21–4.07 (m, 2H), 3.94 (s, 2H). ¹³C NMR δ: 171.9, 166.7, 163.0, 158.4, 157.8, 156.6, 151.8, 150.75, 149.6, 145.0, 135.1, 130.2, 126.4, 106.0, 104.9, 45.4, 44.6, 31.2. Elemental analyses found: C%, 47.59; H%, 3.05; N%, 24.71; S%, 7.14. Calc. for C₁₈H₁₃ClN₈O₃S: C%, 47.32; H%, 2.87; N%, 24.53; S%, 7.02.

5-Methoxy-1-[2-(4-oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl)ethyl]-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 10c. Yield 0.25 g (57%), orange powder, mp 290–291 °C dec., IR: 3446, br., 3084, 2952, 1696, 1596, 1352, 783. ¹H NMR δ: 12.58 (s, 1H), 8.50–7.97 (m, 3H), 7.00–6.63 (m, 3H), 466–4.50 (m, 2H), 4.19 (s, 2H), 3.94 (s, 2H), 3.74 (s, 3H). ¹³C NMR δ: 171.8, 166.7, 158.4, 157.8, 157.1, 156.4, 155.1, 151.5, 150.5, 135.0, 130.1, 118.0, 109.3, 106.0, 105.0, 55.6, 44.5, 43.7, 32.3. Elemental analyses found: C%, 50.67; H%, 3.62; N%, 24.85; S%, 7.18. Calc. for C₁₉H₁₆N₈O₄S: C%, 50.44; H%, 3.56; N%, 24.77; S%, 7.09.

5-Fluoro-1-[2-(4-oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl)ethyl]-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 10d. Yield 0.3 g (68%), orange powder, mp: 256–257 °C dec., IR: 3467, 3084, 2952, 1698. ¹H NMR δ: 12.75 (s, 1H), 8.50–7.98 (m, 3H), 7.74–6.62 (m, 3H), 4.51 (s, 2H), 4.21–3.96 (m, 4H). ¹³C NMR δ: 171.8, 166.7, 157.0, 156.4, 151.5, 150.5, 147.4, 145.5, 134.9, 128.0, 116.6, 114.9, 110.2, 105.0, 44.5, 43.6, 32.6. Elemental analyses found: C%, 49.31; H%, 3.17; N%, 25.30; S%, 7.42. Calc. for C₁₈H₁₃FN₈O₃S: C%, 49.09; H%, 2.98; N%, 25.44; S%, 7.28.

1-[2-(6-Amino purine-9-yl)ethyl]-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 11a. Yield 0.25 g (65%), dark yellow powder, mp: 302 °C dec., IR: 3366, 3188, 3067, 2983, 1698, 1613, 1541, 1337. ¹H NMR δ: 12.53 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.47 (d, J = 7.4 Hz, 1H), 7.28 (t, J = 8 Hz, 1H), 7.20 (s, 2H), 7.01 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 4.42 (t, J = 4 Hz, 2H), 4.12 (t, J = 4 Hz, 2H), 3.95 (s, 2H). ¹³C NMR δ: 172.1, 163.5, 155.5, 155.2, 152.3, 151.9, 140.9, 134.9, 132.3, 131.8, 129.4, 128.2, 122.3, 118.5, 108.5, 42.2, 41.8, 33.3. Elemental analyses found: C%, 51.47; H%, 3.65; N%, 30.14; S%, 7.69. Calc. for C₁₈H₁₅N₉O₂S: C%, 51.30; H%, 29.91; S%, 7.61.

1-[2-(6-Amino purine-9-yl) ethyl]-5-chloro-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 11b. Yield 0.28 g (64%), yellowish green powder, mp: 285 °C dec (decomp.), IR: 3429, 3232, 3079, 2927, 1705, 1622, 1540, 1334. ¹H NMR δ: 12.64 (br. s, 1H), 8.08 (s, 1H), 7.97 (s, 1H), 7.40 (d, J = 1.3 Hz, 1H), 7.33 (dd, J = 4.6, 2.4 Hz, 1H), 7.15 (s, 2H), 6.85 (t, J = 8 Hz, 1H), 4.42 (d, J = 4.7 Hz, 2H), 4.12 (t, J = 5.2 Hz, 2H), 4.03 (s, 2H). ¹³C NMR δ: 174.5, 163.7, 156.1, 153, 152.6, 150.1, 146.1, 143.2, 141.3, 131.8, 128, 126.7, 119.0, 118.1, 110.4, 41.9, 41.2, 34.2. Elemental analyses found: C%, 47.60; H%, 3.24; N%, 27.88; S%, 7.12. Calc. for C₁₈H₁₄ClN₉O₂S: C%, 47.42; H%, 3.10; N%, 27.65; S%, 7.03.

1-[2-(6-Amino purine-9-yl) ethyl]-5-methoxy-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 11c. Yield 0.25 g (57%), dark orange powder, mp: 290 °C, IR: 3362, 3145, 3070, 2928, 1701, 1600, 1541, 1337. ¹H NMR δ. 12.58 (s, 1H), 8.50–7.97 (m, 3H), 7.00–6.63 (m, 3H), 4.43 (s, 2H), 4.26 (s, 2H), 4.13 (s, 2H), 3.74 (s, 3H). ¹³C NMR δ: 71.8, 166.7, 156.4, 155.1, 151.5, 150.5, 146.3, 144.1, 135.0, 130.1, 125.3, 120.6, 119.2, 118.0, 109.3, 55.6, 44.5, 43.7, 33.1. Elemental analyses found: C%, 50.74; H%, 3.97; N%, 27.78; S%, 7.21. Calc. for C₁₉H₁₇N₉O₃S: C%, 50.55; H%, 3.80; N%, 27.92; S%, 7.10.

1-[2-(6-Amino purine-9-yl) ethyl]-5-fluoro-(3Z)-3-[(2E/Z)-(4-oxo-1,3-thiazolidin-2-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 11d. Yield 0.3 g (68%), dark yellow powder, mp: 298–299 °C, IR: 3378, 3336, 3072, 2926, 1705, 1631, 1543, 1336. ¹H NMR δ: 12.66 (s, 1H), 8.06 (s, 1H), 7.97 (s, 1H), 7.22 (d, J = 6.2 Hz, 1H,), 7.13 (s, 3H), 6.84 (d, J = 3.3 Hz, 1H), 4.40 (t, J = 4 Hz, 2H), 4.11 (t, J = 4 Hz, 2H), 3.96 (s, 2H). ¹³C NMR δ: 174.6, 165.8, 157.1, 156.6, 155.5, 152.0, 149.7, 140.8, 140.3, 139.4, 121.3, 118.5, 118.1, 117.7, 109.8, 41.7, 40.8, 33. 2. Elemental analyses found: C%, 49.08; H%, 3.40; N%, 28.92; S%, 7.45. Calc. for C₁₈H₁₄FN₉O₂S: C%, 3.21; N%, 28.69; S%, 7.30.

General method for the synthesis of 1,5-disubstituted-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene) hydrazinylidene]-1,3-dihydro-2H-indol-2-one 12a–d and 13a–d

A mixture of appropriate isatin-3-thiosemicarbazones 8a–d, 9a–d (1 mmol), chloroacetone (0.09 g, 1 mmol) and anhydrous sodium acetate (0.16 g, 2 mmol) in ethanol (50 mL) was refluxed for 24 h. The reaction mixture was cooled and poured into crushed ice. The precipitate was filtered and dried to give yellow to orange products. The crude powder was purified by column chromatography on a silica gel column using developing systems (A) as the mobile phase.

1-[2-(4-Oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl)ethyl]-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 12a. Yield 0.24 g (59%), reddish brown powder, mp: 223–225 °C, IR: 3435, 3084, 2961, 1694, 1583, 1351, 783. ¹H NMR δ: (E/Z%, 50 : 50): 12.92 (s, 1H, NH_(E)), 12.44 (s, 1H, NH_(Z)), 8.70–8.45 (m, 1H_(E)), 8.33–8.15 (m, 1H_(Z)), 8.02–7.96 (m, 3H), 7.75 (m, 1H_(E)), 7.55–7.36 (m, 1H_(Z)), 7.26 (dd, J = 12.8, 7.5 Hz, 1H), 7.17 (dd, J = 13.4, 6.3 Hz, 1H_(Z)), 7.06 (dd, J = 7.1, 3.6 Hz, 1H_(E)), 7.04–6.91 (m, 1H_(Z)), 6.84–6.75 (m, 1H_(E)), 6.68–6.57 (m, 1H_(Z)), 6.49 (s, 1H_(E)), 6.33 (s, 1H_(Z)), 4.53 (d, J = 4.7 Hz, 2H), 4.36–3.97 (m, 2H). ¹³C NMR δ: 178.5_(E), 178.1_(Z), 164.7_(E), 164.6_(Z), 156.8, 152.1_(E), 151.9_(Z), 150.9_(E), 150.5_(Z), 141.9, 136.1, 135.3_(E), 135.1_(Z),130.3_(E), 130.1_(Z), 129.8, 126.5_(E), 126.3_(Z), 122.2_(E), 121.9_(Z), 118.5_(E), 117.8_(Z), 108.3, 105.9_(E), 105.3_(Z), 102.1_(E), 101.5_(Z), 48.3, 44.9. Elemental analyses found: C%, 53.40; H%, 3.59; N%, 27.41; S%, 8.01. Calc. for C₁₈H₁₄N₈O₂S: C%, 53.19; H%, 3.47; N%, 27.57; S%, 7.89.

5-Chloro-1-[2-(4-oxo-1,5-dihydro-4H-pyrazolo[3,4-d] pyrimidine-1-yl)ethyl]-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 12b. Yield 0.29 g (68%), yellowish brown powder, mp 234–237 °C dec., IR: 3444, 3084, 2925, 1694, 1583, 1351, 783. ¹H NMR δ E/Z %, (40 : 60): 12.69 (s, 1H_(E)), 12.06 (s, 1H_(Z)), 8.58–8.38 (m, 1H_(E)), 8.39–7.73 (m, 3H), 7.84–7.50 (m, 1H_(Z)), 7.26 (d, J = 6.3 Hz, 1H), 7.18 (d, J = 6.4 Hz, 1H_(Z)), 7.05 (s, 1H), 6.59 (d, J = 8.8 Hz, 1H), 6.40 (s, 1H) 4.50 (s, 2H), 4.39–3.96 (m, 2H). ¹³C NMR δ: 178.7_(E), 178.4_(Z), 164.1_(E), 163.9_(Z), 156.3, 151.6, 150.5_(E),150.3_(Z), 140.1, 135.7, 134.8, 128.4_(E), 128.2_(Z), 125.8, 125.5_(E), 125.4_(Z), 125.1_(E), 124.9_(Z), 118.7_(E), 118.4_(Z), 108.8, 105.0, 101.9_(E), 101.4_(Z), 45.0, 43.2. Elemental analyses found: C%, 48.79; H%, 3.13; N%, 25.68; S%, 7.41. Calc. for C₁₈H₁₃ClN₈O₂S: C%, 49.04; H%, 2.97; N%, 25.42; S%, 7.27.

5-Methoxy-1-[2-(4-oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl) ethyl]-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 12c. Yield 0.26 g (61%), reddish brown powder, mp: 226–228 °C dec., IR: 3436, br (NH), 3084 (CH aromatic), 2945, 1694, 1542, 1356, 782. ¹H NMR δ: (E/Z%, 30 : 70): 12.97 (s, 1H_(E)), 12.41 (s, 1H, NH_(Z)), 8.50–8.30 (m, 1H_(E)), 8.26–7.91 (m, 3H), 7.83–7.55 (m, 1H_(Z)), 7.43 (d, J = 12.6 Hz, 1H _(E)), 7.02 (d, J = 13.1 Hz, 1H_(Z)), 6.83 (s, 1H_(E+Z)), 6.74 (s, 1H,), 6.56 (s, 1H_(E)), 6.34 (s, 1H_(Z)), 4.52 (s, 2H),4.05–4.18 (m, 2H), 3.75 (s, 3H). ¹³C NMR δ: 178.4_(E), 177.6_(Z), 164.9_(E), 164.7_(Z), 157.5_(E), 156.9_(Z), 155.7, 155.4, 155.2, 152.4, 152.1, 151.1_(E), 151.0_(Z), 136.0, 135.4, 118.8, 114.4, 113.5, 105.6, 101.9_(E), 101.6_(Z), 56.1, 45.3, 43.7. Elemental analyses found: C%, 52.43; H%, 3.81; N%, 25.89; S%, 7.52. Calc. for C₁₉H₁₆N₈O₃S: C%, 52.29; H%, 3.70; N%, 25.67; S%, 7.35.

5-Fluoro-1-[2-(4-oxo-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-1-yl) ethyl]-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 12d. Yield 0.22 g (52%), yellowish brown powder, mp 242–246 °C dec., IR: 3435, 3107, 2927, 1697, 1541, 1355, 782. ¹H NMR δ (E/Z %, 35 : 65): 12.59 (s, 1H), 8.37–8.16 (m, 1H_(E)), 8.14–7.89 (m, 4H_(Z)), 7.77 (dd, J = 17.5, 8.4 Hz, 1H), 7.14–6.90 (m, 1H), 6.62 (dd, J = 8.3, 4.0 Hz, 1H), 6.55 (s, 1H_(E)), 6.40 (d, J = 3.1 Hz, 1H_(Z)), 4.51 (d, J = 4.6 Hz, 2H), 4.39–3.89 (m, 2H). ¹³C NMR δ: 178.7 (C=O_(E)), 177.6 (C=O_(Z)), 164.7 (thiazol C=N_(E)), 164.5_(Z), 157.2_(E), 156.9_(Z), 155.9, 154.8, 152, 151_(E), 150.8_(Z), 140.4, 138.7, 138.2, 136.9, 136.2, 135.2, 108.6, 105.4, 102.2_(E), 101.9_(Z), 45.4, 45.0. Elemental analyses found: C%, 51.20; H%, 3.23; N%, 26.61; S%, 7.68. Calc. for C₁₈H₁₃FN₈O₂S: C%, 50.94; H%, 3.09; N%, 26.40; S%, 7.56.

1-[2-(6-Amionpurine-9-yl)ethyl]-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 13a. Yield 0.23 g (62%), brown powder, mp 249–250 °C dec., IR: 3338, 3181, 3104, 2924, 1682, 1603, 1544, 1329. ¹H NMR δ: (E/Z %, 38 : 62): 12.93 (s, 1H_(E)), 12.44 (s, 1H_(Z)), 8.26 (d, J = 7.4 Hz, 2H_(E)), 8.09 (s, 1H), 8.02 (s, 1H), 7.48 (d, J = 7.3 Hz, 1H_(Z)), 7.24 (t, J = 7.2 Hz, 1H_(E)), 7.15–7.07 (m, 3H), 7.05 (t, J = 7.2 Hz, 1H_(Z)), 6.96 (t, J = 7.5 Hz, 1H_(E)), 6.90 (d, J = 7.9 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.51 (s, 1H_(E)), 6.36 (s, 1H_(Z)), 4.44 (dd, J = 13.3, 8.0 Hz, 2H), 4.19 (dd, J = 14.8, 9.5 Hz, 2H). ¹³C NMR δ: 177.9, 164.3, 155.7, 152.3, 149.8, 141.8, 141.4_(E), 141.0_(Z), 137.2, 135.8, 130.0_(E), 129.5_(Z), 126.1, 122.9_(E), 121.9_(Z), 119.2_(E), 118.7_(Z), 117.6, 109.2_(E) 107.7_(Z), 101,4, 41.3, 41.1. Elemental analyses found: C%, 53.57; H%, 3.65; N%, 31.30; S%, 7.85. Calc. for C₁₈H₁₅N₉OS: C%, 53.32; H%, 3.73; N%, 31.09; S%, 7.91.

1-[2-(6-Amionpurine-9-yl) ethyl]-5-chloro-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 13b. Yield 0.25 g (59%), brownish orange powder, mp 214–215 °C dec., IR: 3365, 3152, 3076, 2926, 1672, 1601, 1545, 1325. ¹H NMR δ: 12.70 (s, 1H), 8.28 (s, 1H), 8.05 (s, 1H), 8.01 (s, 1H), 7.79 (s, 1H), 7.15 (d, J = 9.3 Hz, 3H), 6.77 (d, J = 8.3 Hz, 1H), 6.44 (s, 1H), 4.41 (s, 2H), 4.15 (s, 2H). ¹³C NMR δ: 178.4, 163.9, 155.7, 152.6, 149.7, 140.8, 140.5, 140.2, 135.5, 128.2, 125.6, 125.0, 118.7, 118.6, 108.6, 102.0, 42.6, 41. Elemental analyses, found: C%, 49.42; H%, 3.47; N%, 28.90; S%, 7.41. Calc. for C₁₈H₁₄ClN₉OS: C%, 49.15; H%, 3.21; N%, 28.66; S%, 7.29.

1-[2-(6-Amionpurine-9-yl) ethyl]-5-methoxy-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 13c. Yield 0.27 g (64%), dark orange powder, mp: 265–267 °C dec., IR: 3338, 3176, 3108, 2925, 1675, 1598, 1544, 1325. ¹H NMR δ: 12.98 (s, 1H_(E)), 12.41 (s, 1H_(Z)), 8.08 (s, 1H), 8.05 (s, 1H_(E)), 8.01 (s, 1H), 7.94 (s, 1H_(Z)), 7.15 (s, 2H), 7.00 (s, 1H), 6.83 (s, 1H), 6.73–6.70 (m, 1H), 6.68 (s, 1H), 4.42 (d, J = 16 Hz, 2H), 4.14 (d, J = 20 Hz, 2H), 3.74 (s, 3H). ¹³C NMR δ: 177.6, 164.2, 155.7, 154.8, 152.2, 149.6, 140.8, 137.4, 135.5, 135.1, 118.6, 118.2, 113.8, 113.0, 107.9, 101.2, 55.6, 41.1, 40.9. Elemental analyses found: C%, 52.62; H%, 4.18; N%, 29.07; S%, 7.43. Calc. for C₁₉H₁₇N₉O₂S: C%, 52.40; H%, 3.93; N%, 28.95; S%, 7.36.

1-[2-(6-Amionpurine-9-yl) ethyl]-5-fluoro-(3Z)-3-[(2E/Z)-(1,3-thiazol-2(3H)-ylidene)hydrazinylidene]-1,3-dihydro-2H-indol-2-one 13d. Yield 0.26 g (61%), brownish orange powder, mp: 232 °C dec., IR: 3362, 3152, 3067, 2926, 1680, 1599, 1543, 1324. ¹H NMR δ: 12.59 (s, 1H), 8.05 (s, 2H), 8.01 (s, 1H), 7.14 (s, 2H), 6.98 (s, 1H), 6.77 (s, 1H), 6.42 (s, 1H,), 4.41 (s, 2H), 4.15 (s, 2H). ¹³C NMR δ: 178.2, 164.2, 155.7, 152.2, 149.7,140.8, 137.8, 136.3, 135.8, 118.6, 115, 114.8, 112.8, 112.5, 108.3, 101.8, 41.0, 40.8. Elemental analyses found: C%, 51.27; H%, 3.45; N%, 29.64; S%, 7.49. Calc. for C₁₈H₁₄FN₉OS: C%, 51.06; H%, 3.33; N%, 29.77; S%, 7.57.

HPLC purity assessment of compounds 6b, 8b, 12a, 12b, and 12c

The purity of the most active compounds (6b, 8b, 12a, 12b, and 12c) was evaluated using high-performance liquid chromatography (HPLC). Analyses were performed using a reverse-phase C18 column (250 mm × 4.6 mm, 5 μm) at ambient temperature. The mobile phase consisted of phosphate buffer (pH 6), adjusted with 0.1 N NaOH and acetonitrile in a 40 : 60 (v/v) ratio, filtered and degassed before use. The flow rate was set to 1.0 mL min⁻¹, with UV detection at 254 nm. Allopurinol was used as an internal standard in all samples. Purity was quantified by calculating the ratio of the analyte peak area to that of the internal standard. Full chromatographic data are provided in the SI.

Biological investigation

Cell lines. All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) and are maintained at the Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (Wroclaw, Poland). Enzyme assay studies were conducted at the confirmatory diagnostic unit in VACSERA, Egypt.

Culture media. The HepG2 cell line was cultured in Eagle's medium (IIET PAS, Wroclaw, Poland) containing 10% fetal bovine serum (HyClone, Life Technologies Poland) and 2 mM L-glutamine (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The MV-4-11 cell line was cultured in RPMI 1640 with a stable glutamine medium (Biowest, Nuaillé, France) containing 10% fetal bovine serum and 1.0 mM sodium pyruvate (both Sigma-Aldrich Chemie GmbH, Steinheim, Germany). All culture media were supplemented with 100 U mL⁻¹ penicillin (Polfa Tarchomin S.A., Warsaw, Poland) and 100 μg mL⁻¹ streptomycin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The cells were grown at 37 °C in a humid atmosphere saturated with 5% CO₂.

Compounds. 7 compounds were tested. Cisplatin (CDDP, Cisplatin 1 mg mL⁻¹ Concentrate for Solution for Infusion, Accord Healthcare Poland), the active cytostatic drug, was used as a control. Sunitinib and sorafenib (both from Sigma-Aldrich, Merck, Darmstadt, Germany) were used as positive controls for the compounds. The samples of the compounds were prepared freshly before being added to the cells. Prior to usage, the compounds were weighed and dissolved in DMSO to a concentration of 10 mg mL⁻¹ and subsequently diluted in culture medium to reach the required concentration depending on the type of test. Camptothecin (Pol-Aura, Poland) and doxorubicin (Doxorubicin Accord 2 mg mL⁻¹, Accord Healthcare Poland) were used as controls for the caspase-3/7 activity assay. Sunitinib, sorafenib and camptothecin were dissolved in DMSO. All cytostatics were diluted in culture medium to reach the required concentrations depending on the type of test.

Antiproliferative assay

The antiproliferative activity of the tested analogs was evaluated using the SRB assay for adherent cells and the MTT assay for suspension (leukemic) cells. The A-498 cells were seeded at 1500 cells per well, HepG2, BALB/3T3 clone A31 and MV4-11 at 2000 cells per well, MDA-MB-231 at 2500 cells per well in 384-well plates (Greiner Bio-One, Kremsmünster, Austria). After 24 hours, cells were treated with tested compounds (0.1–100 μg mL⁻¹) and incubated for 72 hours at 37 °C with 5% CO₂. For the SRB assay, the cells were fixed with cold 50% (w/v) trichloroacetic acid (Avantor Performance Materials, Gliwice, Poland) and stained with a 0.4% (w/v) solution of sulforhodamine B (Sigma-Aldrich, Germany). Protein-bound dye was extracted from the stained cells with a 10 mM TRIS base (Avantor Performance Materials, Gliwice, Poland) solution, and absorbance was measured at 540 nm using a Biotek Hybrid H4 reader (BioTek Instruments, Inc., Winooski, Vermont, USA). For the MTT assay, MTT solution was added. After 4 h of incubation, formazan crystals were dissolved in DMF solution, and absorbance was recorded at a wavelength of 570 nm. Sorafenib served as a reference, and IC₅₀ values were determined using Cheburator 0.4 software. Each compound was tested in triplicate, and the experiments were repeated at least three times.

Cell cycle analysis

Cells (1 × 10⁶) were washed twice with cold PBS and then fixed in 70% ethanol at −20 °C for 24 hours. Following PBS washing, the cells were treated with RNAse (8 mg mL⁻¹, Thermo Scientific, Waltham, MA, USA) at 37 °C for 1 hour. Next, the cells were stained with propidium iodide (0.5 mg mL⁻¹, Sigma-Aldrich Chemie GmbH, Germany) at 4 °C for 30 minutes, and the cellular DNA content was analyzed using BD LSR Fortessa II (Becton Dickinson, San Jose, CA, USA). The percentage of stained cells was calculated using ModFit LT version 3.2 (Verity Software House, USA) and Flowing Software version 2.5.1 (freeware developed by Perttu Terho, Turku Centre for Biotechnology, Finland).

Enzymatic caspase-3/7 activity assay

An enzymatic caspase-3/7 activity assay was conducted using the HepG2 (hepatocellular carcinoma) cell line to evaluate the four test compounds. Cisplatin served as a cytostatic control. Camptothecin and doxorubicin were used as positive controls for the caspase-3/7 activity assay. All test compounds were dissolved in DMSO, diluted in culture medium, and applied to cells seeded in 24-well plates at a density of 1.0 × 10⁵ cells per mL. After a 72 hour treatment, the cells were lysed, and caspase-3/7 activity was measured using Ac-DEVD-ACC as a fluorogenic substrate. Fluorescence was recorded for 2 hours at 37 °C. In parallel, an SRB antiproliferative assay was performed to assess cell viability. After 72 hours of compound exposure, the cells were fixed with trichloroacetic acid and stained with sulforhodamine B, and absorbance was measured at 540 nm. Both assays were conducted in duplicate, and the experiments were repeated three times.

In vitro CDK6 inhibitory assay

CDK6 inhibitory activity was evaluated using the ADP-Glo™ Kinase Assay CDK6 kit (BPS Bioscience, San Diego, CA, USA) following the provided protocol. This assay utilizes the Kinase-Glo® MAX reagent for detection and is formatted in a 96-well plate with sufficient quantities of purified recombinant CDK6/Cyclin D3 enzyme and CDK substrate peptide. The reaction was initiated by adding 20 μL of diluted CDK6/Cyclin D3 enzyme to each well, which contained varying concentrations of test compounds and a test inhibitor control. The plate was then incubated at 30 °C for 45 minutes. After incubation, 50 μL of Kinase-Glo Max reagent was added to each well, and the plate was covered with aluminum foil and incubated at room temperature for an additional 15 minutes. Luminescence was measured using a Tecan Spark reader with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Blank values were subtracted from all measurements to ensure accuracy.

In vitro VEGFR-2 inhibitory activity assay

VEGFR-2 kinase inhibition was evaluated using the VEGFR-2 Kinase Assay Kit (San Diego, CA, USA) following the manufacturer's protocol (https://bpsbioscience.com/vegfr2-kdrkinase-assay-kit-40325). This 96-well format assay utilized Kinase-Glo Max for detection. A master mixture containing kinase buffer, ATP, PTK substrate, and water was prepared and added to each well. Test inhibitor wells received 5 μL of inhibitor solution, while control and blank wells received a solution without the inhibitor. VEGFR-2 enzyme was diluted to 1 ng μL⁻¹ and added to test and positive control wells, followed by incubation at 30 °C for 45 minutes. Afterward, Kinase-Glo Max reagent was added, and luminescence was measured using a microplate reader. IC₅₀ values were determined using non-linear regression analysis, with results presented as mean IC₅₀ (nM) ± SD from at least two independent experiments performed in triplicate.

Xanthine oxidase (XO) inhibition assay

The xanthine oxidase (XO) inhibitory activity of compounds 6b, 7b, 8b, 12a–12c, and 13b was evaluated by measuring uric acid production at 292 nm using a spectrophotometric assay. Allopurinol served as a positive control. The reaction mixture comprised 1.5 mL of 50 mM potassium phosphate buffer (pH 7.4), 1 mL of the test compound at varying concentrations, and 0.5 mL of freshly prepared XO enzyme solution. After a 15 minute pre-incubation to allow enzyme–compound complex formation, 1 mL of 0.10 mM xanthine solution was added, followed by 30 minute incubation. The reaction was terminated with 1 mL of 1 M HCl. Control experiments were performed using phosphate buffer instead of the xanthine substrate. All measurements were conducted in triplicate, and the results were reported as IC₅₀ values ± standard deviation (SD). Kinetic parameters, including the Michaelis–Menten constant (K_m) and maximum velocity (V_max), were determined using either non-linear interpolation of the Michaelis–Menten equation or Lineweaver–Burk plot analysis.

In vitro chemical stability assessment

To investigate chemical stability under simulated physiological conditions, compounds 6b, 8b, and 12a–12c were prepared at 30 μM in phosphate-buffered saline (PBS, pH 7.4) and incubated at 37 ± 0.5 °C. Samples were collected at predetermined intervals (0, 0.5, 1, 2, 4, 6, 8, 10, and 24 h) and analyzed by UV-Vis spectrophotometry across the 190–800 nm range using a 1 cm quartz cuvette. The instrument was blanked with PBS prior to each measurement. Particular attention was given to identifying shifts in the absorption maxima (λ_max), variations in absorbance, or new spectral features indicating chemical degradation.

Author contributions

All authors contributed significantly to the work. Gehan Ahmed Abdel Hafez: conducted the synthesis, biological evaluation, data curation, and prepared the original draft of the manuscript. Dagmara Kłopotowska, Beata Filip Psurska, Joanna Wietrzyk: performed biological evaluation and contributed to the experimental work. Ahmed S. Aboraia: carried out data analysis, molecular docking studies, figure preparation, and manuscript editing. Tarek Aboul Fadl: conceptualization, methodology design, supervision, project administration, and critical revision of the manuscript. Adel F. Youssef: conceptualization, supervision, project administration, data analysis, and critical manuscript revision. All authors reviewed and approved the final version of the manuscript.

Conflicts of interest

The authors declare that they have no conflicts of interest regarding the publication of this article.

Data availability

The authors declare that all the data generated or analyzed during this study are included in this manuscript and its SI. See DOI: https://doi.org/10.1039/d5ra04997k.

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