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. 2023 Aug 15;14(10):1981–1991. doi: 10.1039/d3md00300k

Design, synthesis, and anti-hepatocellular carcinoma of thiopyrimidine/chalcone hybrids as dual STAT3/STAT5 inhibitors†

Najla Altwaijry a, Rehab Sabour b, Mona H Ibrahim b, Omkulthom Al kamaly a, Omeima Abdullah c, Marwa F Harras b,✉
PMCID: PMC10583823  PMID: 37859719

Abstract

Among the promising therapeutic targets for treating cancer are the continuously active STAT proteins, which are important in the progression of many malignancies. Here, we detail the STAT3/5 inhibitory action and thiopyrimidine/chalcone hybrid design, production, and anti-hepatocellular carcinoma activity. The prepared hybrids were assessed for their cytotoxic effect on HepG2 and Huh7 liver cancer cells. The most active compounds 5e and 5h (IC50 range from 0.55 to 2.58 μM) were further evaluated against normal THLE cells to examine their safety profiles. The hybrids 5e and 5h were additionally tested for their potential to inhibit STAT3 and STAT5a. They showed dual inhibitory action, with a decrease in the level of STAT3 by 65 and 87 times, respectively, and a decrease in the level of STAT5 by 60 and 79.5 times, respectively, compared to the control. Additionally, western blot analysis of compound 5h revealed inhibition of STAT3 and STAT5 phosphorylation at Tyr705 and Tyr694, respectively, with only a slight decrease in the total expression of STAT3 and STAT5 proteins. And lastly, molecular docking research provided additional insight on the 5h binding mechanism in the STAT3 and STAT5 SH2 domains.

New thiopyrimidine/chalcone hybrids were synthesized for hepatocellular carcinoma treatment. Compound 5h was the most active one. Additionally, it displayed STAT3/STAT5 dual inhibitory action which was confirmed by western blot analysis.graphic file with name d3md00300k-ga.jpg

1. Introduction

Liver cancer is considered both a common and deadly disease. Despite major therapy advancements, the prognosis for people with hepatocellular carcinoma (HCC) remains poor.1,2 Death rates from many other prevalent malignancies are falling globally, while both male and female mortality rates from hepatocellular carcinoma are on the rise.3

The members of the signal transducer and activator of transcription (STAT) protein,4–6 perform a crucial role in tumor cells as one of the signaling intermediaries. They participate in transcription and immune response regulation and are located in the cytoplasm. The STAT proteins include seven different members. From them, STAT1, STAT3, and STAT5 are family members that can control cell cycle-related gene expression, maintaining cells, and angiogenesis.7–9

STATs are activated by phosphorylation, whereupon they dimerize and transfer into the nucleus, where they control gene expression. The over-activation of STATs has been linked to carcinogenesis.10 It has been shown in different studies that inhibiting the STAT3 or STAT5 signaling pathways causes tumor cells to self-destruct. Normal cells, on the other hand, were able to proliferate and survive despite low levels of STAT3 or STAT5 through a number of independent mechanisms.11,12

Thus, dual inhibition of both STAT3 and STAT5 or using chemicals targeting a number of STAT3 activators13 can lead to the creation of new, less hazardous anti-cancer medicines with the avoidance of drug resistance.9

From the potent STAT inhibitors (Fig. 1), S3I-201 (I) and its improved successors SF-1-066 (II) and S31-1757 (III)6,14 restricted STAT3 activity in breast and hepatocellular carcinoma cells.14 Stattic (IV) is another potent STAT3 inhibitor that can inhibit dimerization, activation and translocation of STAT3.15 Additionally, the STAT3 amount in the leukemia cell line (HL-60) was significantly reduced after treatment with chalcone V.16 Also, pyrazolopyrimidine/chalcone hybrid VI17 inhibited STAT3 expression, and showed promising anti-proliferative action. Compound VII, with a thiopyrimidine moiety, was reported by Xu et al. to have low micromolar IC50 values against STAT3.18 On the other hand, compounds VIII and SF-1-088 (IX) were described as selective inhibitors of STAT5b.19,20 Furthermore, Stafib-1 (X) and its optimized analogue, Stafib-2 (XI) were identified as selective STAT5b inhibitors.21

Fig. 1. Some reported STAT3 and/or STAT5 inhibitors and the designed target hybrids.

Fig. 1

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However, some compounds are reported as dual STAT3/STAT5 inhibitors (Fig. 1), such as the 2-thiopyrimidine/chalcone hybrid XII22 and the sulfonyl compounds XIII and XIV.23,24

In the pyrimidine family, 2-thiopyrimidines are among the most useful compounds. Their numerous potential uses in medicinal chemistry attract the interest of researchers. Many thiopyrimidine derivatives were reported as potent anticancer agents against diverse types of cancer.25,26 As well, significant anticancer activity has been demonstrated in vitro for many synthetic chalcones.27–29

To address these issues, we used molecular hybridization to design and synthesize new 2-thiopyrimidine-chalcone derivatives with an acetamide group as a linker (Fig. 1). The prepared hybrids were assessed for their anti-proliferative ability on hepatocellular carcinoma HepG2 and Huh7 cells. The inhibitory actions of the most powerful bioactive compounds against STAT3 and STAT5 were evaluated in an effort to identify more effective anticancer treatments.

2. Discussion

2.1. Rational design

The formation of a dimer between two STAT monomers is a crucial stage in STAT activation, making it a promising target for blocking binding to DNA as well as transcription and other biological roles of STAT. A key step in the dimerization process is that one of the monomers SH2 domains binds to the pTyr (phosphorylated tyrosine) peptide of the second.30,31 Therefore, an effective strategy that targets the SH2 regions of STAT5 and STAT3 holds great promise. All of the STAT family members share remarkably similar backbone conformations in their SH2 domains,32 with minor variations between the SH2 binding regions of STAT3 and STAT5. This provides hope for the discovery of compounds that may target concurrently both SH2 regions.

Within the STAT3 SH2 domain, there are three highly variable regions: the hydrophobic pY-X (592–605), the polar pY+0 (591, 609–620), which is important for pTyr705 binding, and the pY+1 (626–639) binding hot spots.30,33 It was discovered that the unique STAT3 hydrophobic side pocket, pY-X, could be targeted by drugs. The SH2 domain of STAT5 has dual sub-regions, polar pY+0 (618–628) and hydrophobic region pY+1 (632–642).

Accordingly, new thiopyrimidine/chalcone hybrids were designed to be able to fit in both important binding sub-sites of STAT3 and STAT5 SH2 domains (Fig. 2); the amino-thiopyrimidine moiety is predicted to occupy the polar pY+0 pocket and form polar interactions with the critical residues in this pocket, while the chalcone moiety can form different non-polar interactions in the other hydrophobic binding sites pY+1 and pY-X.

Fig. 2. Rational design of the new thiopyrimidine/chalcone hybrids in the SH2 binding sites of STAT3 and STAT5.

Fig. 2

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2.2. Chemistry

Scheme 1 details the steps in the production of the desired compounds. Reacting p-aminoacetophenone 1 with the proper benzaldehyde derivative 2a–i, by stirring reagents in ethanol with a catalyst of NaOH produced the intermediate chalcones 3a–i.34 Aminochalcone compounds were N-acylated with chloroacetyl chloride in methylene chloride using K2CO3 for catalysis, resulting in the respective acylated derivatives 4a–i.35 The target compounds 5a–i were synthesized by heating 6-amino-2-thiouracil and the acylated chalcones 4a–i in dimethylformamide and K2CO3 at 60 °C.36,37 The 1H NMR spectra of hybrids 5a–i presented a common singlet at δ 4.68–6.17 ppm assigned to H5 of the pyrimidine ring. Besides, a single signal was found at δ 3.26–3.88 ppm related to the S–CH2– linker group. The 13C NMR spectra also revealed the linker signal in the range of 35.48–35.89 ppm and the carbon 5 of the pyrimidine ring signal at 89.10–92.23 ppm. As an illustration, the spectrum of the hybrid 5e1H NMR revealed singlet signals at δH 2.33, 3.88, and 5.70 ppm, which correspond to the methyl, linker S–CH2–, and pyrimidine H-5, respectively. Additionally, the doublets of the chalcone's unsaturated system at 6.81 and 7.52 ppm revealed the existence of a trans geometric isomer, which was verified by the coupling constant (J = 16 Hz). The 13C NMR spectrum revealed the methyl signal at δC 21.04 ppm, as well as signals at 35.79 and 90.41 ppm, corresponding to the linker S–CH2– and pyrimidine C5, respectively. In addition, three signals at δC 162.32, 164.19, and 187.58 ppm were detected, corresponding to three carbonyl groups.

Scheme 1. Synthesis of target thiopyrimidine/chalcone derivatives 5a–i.

Scheme 1

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2.3. Biological activity

2.3.1. The in vitro anti-hepatocellular carcinoma study

The anti-hepatocellular carcinoma activity of the prepared thiopyrimidine–chalcone hybrids against HepG2 and Huh7 liver cancer cells was evaluated. The results, presented as IC50 values, are listed in Table 1. Generally, all hybrids displayed potent to moderate anti-tumor activity on the studied hepatocellular carcinoma cells. Compound 5h with 3,4,5-trimethoxy substituted chalcone was the most potent one against both cell lines. It showed 3.7 and 4.2 times the anticancer potency of doxorubicin and static, respectively on HepG2 and 10.4 and 14.3 times their cytotoxicity against the Huh7 cell line, respectively. Also, compounds 5b, 5e and 5g were more cytotoxic than reference in the two cell lines used. They revealed IC50 values of 2.58–5.08 μM against HepG2 and 1.58–2.44 μM against Huh7, better than the used references doxorubicin and static. Additionally, the tolyl derivative 5c displayed superior activity against HepG2 (IC50 = 4.94 μM) and potent activity against Huh7 (IC50 = 8.32 μM). Moreover, significant anti-hepatocellular carcinoma effects were detected by compounds 5a and 5f (IC50 = 11.20 and 8.14 μM, respectively, against HepG2 and 7.27 and 6.70 μM, respectively, against Huh7), while hybrids 5d and 5i displayed moderate activity on the cells tested (IC50 = 22.55–31.14 μM).

The anti-hepatocellular carcinoma activity of compounds 5a–i and doxorubicin against HepG2 and Huh7.
Compound IC50a (μM)
HepG2 Huh7
5a 11.20 ± 1.92 7.27 ± 0.39
5b 3.57 ± 3.08 2.17 ± 0.83
5c 4.94 ± 1.64 8.32 ± 0.29
5d 22.55 ± 3.24 25.01 ± 3.11
5e 2.58 ± 2.9 2.44 ± 0.81
5f 8.14 ± 0.06 6.70 ± 0.73
5g 5.08 ± 0.65 1.58 ± 1.15
5h 1.95 ± 2.41 0.55 ± 0.52
5i 31.14 ± 1.76 23.08 ± 1.1
Doxorubicin 7.34 ± 0.09 5.70 ± 0.21
Stattic 8.35 ± 0.30 7.89 ± 0.25
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a

The IC50 values displayed here are the mean of three distinct studies ± S.D.

2.3.2. Cytotoxic impacts on normal cell line

The hybrids 5e and 5h, with the best anti-liver cancer activity, were examined against normal hepatic THLE2 cells to assess the safety and selectivity on the normal cells. High IC50 values of 49.51 and 63.20 μM for 5e and 5h, respectively, indicated preferential cytotoxic effects on carcinoma cells and less toxicity on normal cells for the evaluated hybrids. As shown in Table 2, Compound 5h had the highest selectivity index (SI = 114.9) for the Huh7 cell line and SI = 32.41 for the HepG2 cell line.

The cytotoxic effect of 5e and 5h on normal cells.
Compound IC50 (μM) Selectivity indexa (SI)
THLE2 HepG2 Huh7
5e 49.51 ± 0.14 19.18 20.29
5h 63.20 ± 0.09 32.41 114.90
Stattic >100 — —
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a

SI = THLE IC50/cancer cells IC50.

2.3.3. STAT3 and STAT5 inhibitory activity of 5e and 5h

The most effective hybrids in the cytotoxic screening against hepatocellular cancer cell lines were evaluated as inhibitors for STAT3 and STAT5 enzymes using STAT3/STAT5 ELISA kits. Table 3 and Fig. 3 show the results. Both STAT3 and STAT5 were shown to be inhibited by the 5e and 5h hybrids at a concentration of 10 μM. However, compound 5h was a more potent inhibitor than compound 5e as it decreased the level of STAT3 by 4.5 times and STAT5 by 4.8 times relative to the control cells, while the hybrid 5e reduced the amount of STAT3 by 2.8 times and STAT5 by 2.5 times compared to the control cells. The STAT3 inhibitor, stattic, showed inhibitory activity close to the hybrids 5e and 5h against STAT3. On the other hand, 5e and 5h displayed more potent inhibitory activity against STAT5 than static indicating their dual STAT3 and STAT5 inhibitory potential.

STAT3 and STAT5a inhibitory activity of 5e, 5h, and stattic.
Compound STAT3 (ng mL−1) STAT5 (ng mL−1)
5e 5.09 ± 0.21 4.44 ± 0.28
5h 3.19 ± 0.07 2.28 ± 0.06
Stattic 4.90 ± 0.09 9.43 ± 0.08
Control 14.53 ± 0.4 11.15 ± 0.91
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Fig. 3. Effects of compounds 5e and 5h and stattic on the levels of phosphorylated STAT3 and STAT5.

Fig. 3

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2.3.4. Western blot analysis

On the basis of our findings of inhibitory activities on STAT3 and STAT5 transcription, cytotoxicity, and selectivity, we evaluated the inhibitory effect of compound 5h (at its IC50 concentration) on phosphorylation in both STAT3 and STAT5 of HepG2 cells using Western blotting. Phosphorylation of STAT3 at Tyr705 and STAT5 at Tyr694 can result in their dimerization, nuclear translocation, binding to DNA, and subsequent transcription.37–40 Thus, the degree of STAT3 phosphorylation at Tyr705 and STAT5 phosphorylation at Tyr694 was measured. As demonstrated in Fig. 4, 5h inhibited STAT3 and STAT5 phosphorylation at Tyr705 and Tyr694, respectively, after incubation for 24 hours, with only a little decrease in the total STAT3 and STAT5 protein expression. It was evident from these findings that the decline in Tyr705-phosphorylated STAT3 and Tyr694-phosphorylated STAT5 was not due to the institutional decrease in total STAT proteins. Further study of compound 5h on STAT1 revealed a negligible effect on the level of STAT1 and a little decrease in phosphorylation on Tyr701, indicating that this compound has a degree of selectivity on STAT3/5 over STAT1.

Fig. 4. (A) Western blot analysis of the compound 5h effect on total and phosphorylated STAT3, STAT5, and STAT1 in the HepG2 cells. (B) Expression levels of different STATs in control cells and after treatment with effect of compound 5h.

Fig. 4

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2.4. Molecular docking study

Docking investigation was done using the STAT3/STAT5 homodimer crystal structures to gain insight on the process of binding of compound 5h with the SH2 domains of STAT3 having PDB code: 1BG1 (ref. 41) and STAT5 having PDB code: 1Y1U.42

For comparison, a docking analysis of the STAT3 inhibitor S3I-201 (I) was conducted. The salicylic acid moiety formed three hydrogen bonds in the polar pY+0 binding site of STAT3; to Lys591, Arg-609, and Ser611, while the NH group formed an H-bond with Ser636 (see ESI†).

Molecular docking of compound 5h to STAT3 (Fig. 5) revealed the following interactions: the aminopyrimidine motif formed 3 H-bonds with Arg609, Ser611, and Glu612 in the pY+0 pocket, in addition to an H-bond with Val639 in the pY+1 site. The sulfur atom was also bonded to the pY+0 pocket through a sulfur bond with Ser613. The important amino acid Lys591 formed a network of non-polar interactions with all the aryl groups of compound 5h. Moreover, the chalcone carbonyl group formed an H-bond with Ser636 in the pY+1 site. The trimethoxyphenyl moiety of the compound was oriented in the unique pY-X binding pocket of STAT3, forming non-polar interactions in addition to hydrogen bonding with Arg595.

Fig. 5. Binding poses of 5h inside STAT3 active site.

Fig. 5

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Concerning docking into the SH2 domain of STAT5, the potent STAT5 inhibitor SF-1-088 (VIII) formed hydrogen bonds with Lys600 and Arg618 in the pY+0 region in addition to non-polar interactions with the residues in the pY+1 subunit (see ESI†), which was consistent with the reported binding mode.20 As well, the aminothiopyrimindine group of the hybrid 5h contributed to the fitting to the SH2 domain of STAT5 through binding to the essential pY+0 residues; it formed 2 H-bonds with Asp621 and Ser622 together with Pi–alkyl interaction with Lys600. However, the trimethoxyphenyl group formed both non polar interactions (with Trp641 and Asn642) and polar interactions (with Pro636 and Asp637) in the pY+1 binding pocket (Fig. 6).

Fig. 6. Binding pose of 5h inside STAT5 active site.

Fig. 6

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3. Conclusion

The novel 2-thiopyrimidine/chalcone hybrids 5a–i were developed to act as anticancer drugs. Several spectroscopic methods were used for their identification. The cytotoxic effects were tested using two various hepatocellular carcinoma cell lines: HepG2 and Huh7. The anti-cancer outcome of the new hybrids revealed moderate to high results. Compounds 5e and 5h presented significant anti-proliferative effects relative to the reference drug. The potential of hybrids 5e and 5h to inhibit STAT3 and STAT5a was also investigated. The levels of STAT3 and STAT5 were inhibited by 60–87 folds, demonstrating a dual inhibitory effect. In addition, compound 5h inhibited STAT3 and STAT5 phosphorylation at Tyr705 and Tyr694, respectively, with only a minor reduction in overall expression of STAT3 and STAT5 proteins, as shown by western blotting. Furthermore, a molecular docking study elucidated further details of the process by which 5h binds to the STAT3 and STAT5 SH2 domains.

4. Experimental

4.1. Chemistry

See ESI† for the equipment used and materials.

The chalcones 3a–i43,44 and the acylated chalcones 4a–h43 are synthesized as reported and their structures were validated by reported melting points.

2-Chloro-N-(4-{3-[4-(dimethylamino)phenyl]acryloyl}phenyl)acetamide (4i)

Melting point: 189–190 °C; yield 87%; IR (KBr, cm−1): 3315 (NH), 3055 (aroma. CH), 2928 (alipha. CH), 1660 (2C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C); 1H NMR (DMSO-d6) δ (ppm): 3.07 (s, 3H, N–CH3), 3.08 (s, 3H, N–CH3), 4.34 (s, 2H, CH2), 6.69 (d, 2H, phenyl-H, J = 8 Hz), 7.55 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.63 (d, 2H, phenyl-H, J = 8 Hz), 7.83 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.90 (d, 2H, phenyl-H, J = 8 Hz), 7.95 (d, 2H, phenyl-H, J = 8 Hz), 10.06 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 41.21, 44.20, 111.49, 121.44, 123.91, 125.04, 129.74, 130.07, 131.98, 153.17, 154.64, 157.09, 161.83, 190.26; MS, m/z: 344 [M + 2]+, 342 [M]+; C19H19ClN2O2 (342.82) calculated analysis (found analysis): C, 66.57 (66.56); H, 5.59 (5.56); N, 8.17 (8.20).

General procedure for the synthesis of 2-[(4-amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[aryl]acryloyl}phenyl)acetamides 5a–i

A mixture of acylated chalcones 4a–i (0.01 mol) and 6-aminothiouracil (0.01 mol) in 20 mL DMF and K2CO3 (0.01 mol) was heated for 5–6 hours at 60 degrees Celsius. The completed reaction was quenched with water, neutralized with diluted HCl, and filtered. The formed precipitate was ethanol crystallized to yield the target compounds 5a–i.

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[4-chlorophenyl]acryloyl}phenyl)acetamide (5a)

Melting point: 287–289 °C; yield 77%; IR (KBr, cm−1): 3355, 3221, 3137, (NH2 + 2NH), 3054 (aroma. CH), 2928 (alipha. CH), 1628 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1594 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.70 (s, 2H, CH2–S), 6.14 (s, 1H, pyrimidine-H), 6.64 (s, 2H, NH2; exchangeable with D2O), 7.16–7.27 (m, 4H, phenyl-H), 7.55 (d, 2H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.66 (d, 2H, phenyl-H), 7.80 (d, 2H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 8.16 (s, 1H, NH; exchangeable with D2O), 10.56 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.77, 89.10, 112.79, 125.30, 127.45, 129.34, 129.93, 131.42, 132.76, 132.80, 134.02, 136.09, 155.10, 157.13, 162.35, 163.02, 189.50; MS, m/z: 442 [M + 2]+, 440 [M+]; C21H17ClN4O3S (440.90) calculated analysis (found analysis): C, 57.21 (57.20); H, 3.89 (3.98); N, 12.71 (12.73).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[2-chlorophenyl]acryloyl}phenyl)acetamide (5b)

Melting point: 280–282 °C; yield 62%; IR (KBr, cm−1): 3325, 3228, 3139, (NH2 + 2NH), 3055 (aroma. CH), 2934 (alipha. CH), 1629 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1582 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.72 (s, 2H, CH2–S), 6.17 (s, 1H, pyrimidine-H), 6.42 (s, 2H, NH2; exchangeable with D2O), 6.61 (d, 2H, phenyl-H, J = 8 Hz), 7.42–7.44 (m, 4H, 3phenyl-H + CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH) 7.53 (t, 1H, phenyl-H), 7.91–7.94 (m, 3H, 2phenyl-H + CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 7.95 (s, 1H, NH; exchangeable with D2O), 8.15 (t, 1H, phenyl-H), 12.11 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.78, 89.11, 112.78, 125.02, 125.27, 127.61, 128.34, 129.93, 131.31, 131.36, 132.80, 133.97, 136.07, 154.10, 157.31, 162.30, 163.00, 185.48; MS, m/z: 442 [M + 2]+, 440 [M+]; C21H17ClN4O3S (440.90) calculated analysis (found analysis): C, 57.21 (57.30); H, 3.89 (3.90); N, 12.71 (12.72).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[2,4-dichlorophenyl]acryloyl}phenyl)acetamide (5c)

Melting point: 290–291 °C; yield 68%; IR (KBr, cm−1): 3348, 3225, 3180, (NH2 + 2NH), 3054 (aroma. CH), 2927 (alipha. CH), 1625 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1595 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.73 (s, 2H, CH2–S), 6.05 (s, 1H, pyrimidine-H), 6.32 (s, 2H, NH2; exchangeable with D2O), 6.55 (d, 2H, phenyl-H, J = 8 Hz), 7.31–8.21 (m, 7H, 5 phenyl-H + 2CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 8.34 (s, 1H, NH; exchangeable with D2O), 10.51 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.80, 89.12, 112.66, 118.57, 124.30, 127.29, 128.10, 128.51, 129.37, 130.33, 130.92, 131.35, 131.95, 133.87, 138.10, 513.56, 158.46, 162.33, 195.51; MS, m/z: 479 [M + 2]+, 477 [M + 2]+, 475 [M+]; C21H16Cl2N4O3S (475.34) calculated analysis (found analysis): C, 53.06 (53.06); H, 3.39 (3.38); N, 11.79 (11.81).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[4-fluorophenyl]acryloyl}phenyl)acetamide (5d)

Melting point: 275–276 °C; yield 80%; IR (KBr, cm−1): 3341, 3225, 3127, (NH2 + 2NH), 3037 (aroma. CH), 2925 (alipha. CH), 1622 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.88 (s, 2H, CH2–S), 5.93 (s, 1H, pyrimidine-H), 6.25 (s, 2H, NH2; exchangeable with D2O), 6.54 (d, 2H, phenyl-H, J = 8 Hz), 6.71 (d, 2H, phenyl-H, J = 8 Hz), 6.95 (d, 2H, phenyl-H, J = 8 Hz), 7.40 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.67 (d, 2H, phenyl-H, J = 8 Hz), 7.94 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 8.34 (s, 1H, NH; exchangeable with D2O), 10.51 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.78, 89.15, 111.06, 112.50, 123.30, 124.65, 125.24, 130.45, 131.59, 132.10, 137.11, 143.39, 150.90, 157.18, 162.31, 163.56, 190.00; MS, m/z: 424 [M+]; C21H17FN4O3S (424.45) calculated analysis (found analysis): C, 59.43 (59.39); H, 4.04 (3.99); N, 13.20 (13.23).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[p-tolyl]acryloyl}phenyl) acetamide (5e)

Melting point: 278–279 °C; yield 73%; IR (KBr, cm−1): 3350, 3222, 3145, (NH2 + 2NH), 3037 (aroma. CH), 2928 (alipha. CH), 1625 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1600 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 2.33 (s, 3H, CH3), 3.88 (s, 2H, CH2–S), 5.70 (s, 1H, pyrimidine-H), 6.14 (s, 2H, NH2; exchangeable with D2O), 6.60 (d, 2H, phenyl-H, J = 8 Hz), 6.81 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.52 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.70 (d, 2H, phenyl-H, J = 8 Hz), 7.76 (d, 2H, phenyl-H, J = 8 Hz), 7.90 (d, 2H, phenyl-H, J = 8 Hz), 7.94 (s, 1H, NH; exchangeable with D2O), 12.03 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 21.04, 35.79, 90.41, 112.73, 120.79, 125.41, 127.65, 128.03, 129.47, 131.83, 132.43, 134.11, 139.88, 152.70, 157.41, 162.32, 164.19, 187.58; MS, m/z: 420 [M+]; C22H20N4O3S (420.49) calculated analysis (found analysis): C, 62.84 (62.80); H, 4.79 (4.82); N, 13.32 (13.30).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[4-methoxyphenyl]acryloyl}phenyl)acetamide (5f)

Melting point: 294–296 °C; yield 80%; IR (KBr, cm−1): 3387, 3212, 3135, (NH2 + 2NH), 3055 (aroma. CH), 2927 (alipha. CH), 1625 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.69 (s, 2H, CH2–S), 3.80 (s, 3H, OCH3), 5.29 (s, 1H, pyrimidine-H), 6.10 (s, 2H, NH2; exchangeable with D2O), 6.60 (d, 2H, phenyl-H, J = 8 Hz), 6.97 (d, 2H, phenyl-H, J = 8 Hz), 7.56 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.70 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.77 (d, 2H, phenyl-H, J = 8 Hz), 7.89 (d, 2H, phenyl-H, J = 8 Hz), 7.94 (s, 1H, NH; exchangeable with D2O), 12.03 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.89, 55.40, 90.61, 112.83, 114.40, 119.97, 125.63, 127.84, 129.71, 130.78, 131.06, 141.47, 153.46, 157.19, 162.46, 163.12, 186.05; MS, m/z: 436 [M+]; C22H20N4O4S (436.49) calculated analysis (found analysis): C, 60.54 (60.52); H, 4.62 (4.68); N, 12.84 (12.82).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[3,5-dimethoxyphenyl]acryloyl}phenyl)acetamide (5g)

Melting point: 298–299 °C; yield 79%; IR (KBr, cm−1): 3352, 3248, 3145, (NH2 + 2NH), 3061 (aroma. CH), 2952 (alipha. CH), 16 228 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1595 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.67 (s, 2H, CH2–S), 3.79 (s, 6H, 2OCH3), 4.68 (s, 1H, pyrimidine-H), 6.11 (s, 2H, NH2; exchangeable with D2O), 6.55 (d, 2H, phenyl-H, J = 8 Hz), 6.62 (d, 2H, phenyl-H, J = 8 Hz), 7.00 (d, 2H, phenyl-H, J = 3 Hz), 7.49 (d, 1H, phenyl-H, J = 3 Hz), 7.68 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.80 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.96 (s, 1H, NH; exchangeable with D2O), 12.16 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.78, 55.67, 55.73, 92.23, 11.04, 112.35, 117.31, 122.45, 124.06, 125.52, 130.29, 135.73, 143.50, 152.40, 153.27, 153.69, 162.31, 186.05; MS, m/z: 466 [M+]; C23H22N4O5S (466.51) calculated analysis (found analysis): C, 59.22 (59.30); H, 4.75 (4.73); N, 12.01 (12.03).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[3,4,5-trimethoxyphenyl]acryloyl}phenyl)acetamide (5h)

Melting point: 295–297 °C; yield 81%; IR (KBr, cm−1): 3355, 3327, 3168, (NH2 + 2NH), 3045 (aroma. CH), 2924 (alipha. CH), 1628 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C);1H NMR (DMSO-d6) δ (ppm): 3.61 (s, 2H, CH2–S), 3.70 (s, 3H, OCH3), 3.85 (s, 6H, 2 OCH3), 5.56 (s, 1H, pyrimidine-H), 6.13 (s, 2H, NH2; exchangeable with D2O), 6.61 (d, 2H, phenyl-H, J = 8 Hz), 7.16 (s, 2H, phenyl-H), 7.54 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.79 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.93 (d, 2H, phenyl-H, J = 8 Hz), 8.17 (s, 1H, NH; exchangeable with D2O), 10.54 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.48, 56.10, 60.11, 89.61, 106.10, 106.47, 112.68, 118.50, 121.59, 125.40, 129.90, 130.31, 131.13, 132.54, 139.20, 141.88, 151.93, 153.08, 153.84, 185.83; MS, m/z: 496 [M+]; C24H24N4O6S (496.54) calculated analysis (found analysis): C, 58.05 (58.10); H, 4.87 (4.88); N, 11.28 (11.32).

2-[(4-Amino-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-{3-[4-(dimethylamino)phenyl]acryloyl}phenyl)acetamide (5i)

Melting point: >300 °C; yield 80%; IR (KBr, cm−1): 3327, 3230, 3168, (NH2 + 2NH), 3055 (aroma. CH), 2925 (alipha. CH), 1625 (broad, 3C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 1598 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 C); 1H NMR (DMSO-d6) δ (ppm): 3.03 (s, 3H, N–CH3), 3.07 (s, 3H, N–CH3), 3.26 (s, 2H, CH2–S), 4.87 (s, 1H, pyrimidine-H), 5.39 (s, 2H, NH2; exchangeable with D2O), 6.20 (d, 2H, phenyl-H, J = 8 Hz), 6.81 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.25 (d, 1H, CH Created by potrace 1.16, written by Peter Selinger 2001-2019 CH, J = 16 Hz), 7.60 (d, 2H, phenyl-H, J = 8 Hz), 7.85 (d, 2H, phenyl-H, J = 8 Hz), 7.90 (s, 1H, NH; exchangeable with D2O), 8.23 (d, 2H, phenyl-H, J = 8 Hz), 10.53 (s, 1H, NH; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 35.81, 45.60, 89.60, 107.52, 114.19, 114.81, 116.17, 118.56, 121.94, 128.46, 130.96, 131.13, 134.67, 152.66, 158.87, 161.73, 163.80, 175.37; MS, m/z: 450 [M + 1]+; C23H23N5O3S (449.53) calculated analysis (found analysis): C, 61.45 (61.36); H, 5.16 (5.18); N, 15.58 (15.60).

4.2. Biological activity

4.2.1. In vitro anti-hepatocellular carcinoma activity

In vitro MTT cytotoxicity assay was carried out following the methods described (see ESI†).

4.2.2. STAT3 and STAT5 inhibitory activity

The STAT3 and STAT5 inhibitory activity of compounds 5e and 5h was examined using STAT3 (pY705) ELISA Kit (catalog number# ab126458) and STAT5A/B (pY694/699) ELISA kits (catalog number# ab176656) according to the kits protocol. The tests detect the endogenous levels of STATs in cellular lysates only when phosphorylated.

4.2.3. Western blot analysis

Proteins under investigation were isolated first via SDS-PAGE, electrophoresed, then removed to a HybondTM nylon film then incubated in Blocking Solution for 60 min at room temperature. In the assay, β-actin was used as a reference protein. Anti-STAT3 antibody [EPR787Y], anti-STAT3 (phospho/Y705) antibody [EPR23968/52], anti-STAT5 antibody [9F7], anti-STAT5 (phospho Y694) antibody [E208], anti-STAT1 antibody [EPR21057-141], or anti-STAT1 (phospho Y701) antibody [EPR3147], was added, and the membrane was incubated with the mixture for one night at 4 degrees Celsius. Then, the membrane was washed 5 times with blotting buffer. A diluted HRP-coupled secondary antibody was applied to the membrane in antibody solution for 60 min. It's between 0.1 and 0.5 micrograms per milliliter. The optimal signal strength and low background can be achieved by adjusting the antibody concentration between 0.5 and 2.0 micrograms per mL. Eventually, after an hour and 5+ changes of blotting buffer, the membrane was washed. Totallab analysis software, https://www.totallab.com, was used to analyze data recorded with the Gel documentation system (Geldoc-it, UVP, England) (Ver.1.0.1).

4.3. Molecular docking

Here, MOE 2014.09 was utilized for the docking operation. The STAT3 and STAT5 crystal structures were aligned with the under consideration hybrid 5h (PDB: 1BG1 and 1Y1U, respectively). For molecular docking, the “Docking” module of MOE was applied. The docking results of the investigated molecules were displayed by the Biovia discovery-studio 2020 visualizer, which produced approximated interactions.

Conflicts of interest

The authors report no conflicts of interest.

Supplementary Material

MD-014-D3MD00300K-s001

Acknowledgments

The authors extend their appreciation to Princess Nourah Bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R89), Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia for funding this work.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00300k

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MD-014-D3MD00300K-s001
MD-014-D3MD00300K-s001.pdf (2.5MB, pdf)

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