Synthesis, antitumor evaluation and computational study of thiazolidinone derivatives of dehydroabietic acid-based B ring-fused-thiazole

<jats:title>Abstract</jats:title>         <jats:p>In an attempt to search for new natural product-based antitumor agents, a series of novel thiazolidinone derivatives of dehydroabietic acid-based B ring-fused-thiazole were designed and synthesized. The primary antitumor tests showed that compounds <jats:bold>5m </jats:bold>exhibited almost the best inhibitory activity against the tested cancer cells. The computational study suggested NOTCH1, IGF1R, TLR4, and KDR were the core targets of the title compounds, and the IC<jats:sub>50</jats:sub> of SCC9 and Cal27 is strong correlation with the binding ability of TLR4 and compounds.</jats:p>

section_content: Chemotherapy is still a main method for cancer treatment [1]. However, the tumor cells could resistant to some chemotherapeutic drugs intrinsically, and show acquired drug resistance after chemotherapy, which makes the tumor cells become insensitive to similar drugs later [2, 3]. For this reason, the development of the drug with novel structure and higher therapeutic efficiency is still an important mission for medicinal chemists. 

Natural products play an important role in drug development due to their chemical structure diversity, better biocompatibility and drug-like properties [4]. Dehydroabietic acid (DHA) is a natural tricyclic diterpenoid, isolated from pine resins. DHA and its derivatives were reported possessing a broad spectrum of biological activities [5]. Recent reports indicated that DHA and its derivatives exhibited antitumour activity with a variety of mechanisms, such as survivin inhibition [6], DNA binding [7], EGFR or MEK1 kinase binding [8, 9], PI3K/AKT/mTOR pathway signaling inhibition [10]. Therefore, using DHA as a promising starting material to synthesize leading compounds for the development of antitumor agents has received growing interest from medicinal chemists. 

On the other hand, thiazolidinone scaffolds are privileged structures in drug design, some of its derivatives made significant contribution in clinical treatment, such as Ponvory (antimultiple sclerosis and antipsoriasis), Ritalin (anticonvulsant) and Etozoline (antihypertensive) [11, 12]. In the development of antitumor agents, thiazolidinone scaffolds are also widely used as pharmacophore to form new candidate compounds, such as some synthetic indolethiazolidinones could trigger DNA damage and induce apoptosis of human tumor cells [13], some 2,3-disubstituted 1,3-thiazolidin-4-ones could prevent mitotic divisions of the human renal tumor cell though G1 cell cycle arrest [14], some 4-thiazolidinonephenylaminopyrimidine hybrids were found possessing certain inhibitory ability against K562 cells through possible Abl inhibition mechanisms [15], some hybrid molecules integrated Ciminalum and thiazolidinone moieties show significant anticancer activity against different tumor cells [16]. 

In addition, molecular-hybridization approaches is an effective strategy in drug development to integrate various pharmacophores to obtain novel molecules with synergistic effect [17]. In our previous study a series of (aryl) methyl-amine derivatives of DHA based B ring-fusedthiazole were synthesized and showed certain inhibitory ability against tongue squamous cancer cells SCC9. For the development of novel DHA based B ring-fused heterocyclic antitumor derivatives, thiazolidinone scaffolds was chosen as pharmacophore to link with DHA based B ring-fused-thiazole amine, which was synthesized before. The in vitro antitumor activities of the title compounds against the oral cavity cancer cells lines (SCC9, Cal27 and SAS) were evaluated and the action mechanisms were studied using a representative compound by the prediction of therapeutic targets, network pharmacology, and molecular docking.
section_name: Introduction

section_name: Results and discussion

section_content: The synthetic approaches adopted to afford the title compounds are outlined in Scheme 1. Compounds 1 were prepared according to the literatures [18, 19]. Compounds 2 were prepared according to our previous work [10], and further acylated with chloroacetyl chloride at room temperature to formed compound 3 in 80%-90% yield. The first thiazolidinone 4 was obtained by heterocyclization of compound 3 with potassium thiocyanate in the refluxing mixture of triethylamine and ethanol, through intramolecular cyclization and the Dimroth-like rearrangements [20]. The title compounds 5a-5n were synthesized in good yields by the condensation reaction of the thiazolidinone 4 with different excessive aryl aldehydes refluxed in a mixture of triethylamine and ethanol. In thin layer chromatography testing, compounds 5a-5n showed 2 close spot, whose color density can transform each other by changing the sample concentration. In the 1 H NMR spectra of thiazolidinone 4, two NH protons signals were found at 12. 25 and 9. 36 ppm, the H signals at benzene ring of DHA also show cleavages. It may be caused by the amino-imino tautomeric equilibrium [12]. 

The identities of the title compounds were confirmed using physicochemical analytical methods. 1 H NMR spectra revealed that the protons of NH which belong to the imino-amino tautomers exhibited 2 single signals in the 12. 32-12. 18 and 10. 40-9. 24 ppm range. Due to this tautomer, some H signals were showed paired cleavages with varying degrees, especially the H on the conjugate plane. This The singlets at 8. 00-7. 67 ppm were assigned to the protons of the olefinic bond. The aromatic protons of the dehydroabietic acid scaffold showed as signals at 7. 81-7. 63 and 7. 24-7. 14. The methyl ester group protons showed a signal in the 3. 70-3. 64 ppm range. The 13 C NMR spectra signals of all the target compounds were match with the number of the carbon type. The high-resolution mass spectra confirmed the expected molecular weights of the final products.
section_name: Chemistry

section_content: The synthesized title compounds 5a-5n were evaluated for their in vitro antitumor activities by CCK-8 assay against the 3 oral cavity cancer cells lines (SCC9, Cal27 and SAS), which were belong to tongue squamous cell carcinoma. The results are summarized in Table 1. 

It was found that the synthesized compounds showed effective antitumor activities against SCC9 and Cal27, most of them exhibited better inhibitory ability than thiazolidinone 4 with IC 50 of 15. 3 ± 0. 8 and 20. 1 ± 2. 5 µM, respectively, which almost showed as the best compound against SAS cells line with IC 50 of 6. 2 ± 0. 3 µM. It is evident that the arylidene groups integrated are important contributors to their antitumor activities against SCC9 and Cal27, but paly as opposite role against SAS cell line. The substituents on the benzene ring showed different contribution to the antitumor activities, such as compounds 5a (R = 2Br-Ph), 5a (R = 2Br-Ph), 5d (R = 2Me-Ph), 5f (R = 4Br-Ph), and 5 h (R = 4F-Ph) showed better inhibitory ability than the non-substituted compound 5n against SCC9 and Cal27, while compounds 5e (R = 2MeO-Ph) and 5i (R = 4Me-Ph) showed lower inhibitory ability than 5n. Moreover, the substituents on the benzene ring almost caused activity decrease against SAS. The compounds with nitrogenous heterocyclic arylidene groups, 5 m (R = 1H-pyrrol-2-), 5 k (R = pyridin-3-), and 5 l (R = 6-Cl-pyridin-3-) showed the lowest half-inhibitory concentration (IC 50 ) in SCC9 and Cal27 assays. It suggested that the lone electron pair on the aryl groups might act as important role to interact with treatment targets. The trend of antitumor activities of the compounds with different substituents are showed in Fig. 1. 

Compound 5 m was chosen as representative for the further computational action mechanism study due to its excellent IC 50 of 0. 2 ± 0. 1, 0. 2 ± 0. 1 and 7. 3 ± 0. 4 µM, against SCC9, Cal27, and SAS, respectively.
section_name: Antitumor activity

section_name: Scheme 1 Synthetic route of thiazolidinone derivatives of dehydroabietic acid-based B ring-fused-thiazole 5a-5n

section_name: Computational study

section_content: About 240-252 targets in human cells of each compound were obtained by PharmMapper Server, and 145 common targets were found in each group of 5a-5n targets. A total of 660 targets associated with the 3 oral cavity cancer cells lines, were collected by OMIM database and Genecards database after eliminating duplicates. Finally, 16-19 potential therapeutic targets of each compound were obtained from the intersection of compound targets and the cancer cell lines associated targets. The results are summarized in Supplementary Table S1-S3.
section_name: Prediction of 5a-5n therapeutic targets in the 3 cancer cell lines

section_content: A total of 16 potential therapeutic targets of the representative compound 5 m was imported into the STRING database to obtain the data of PPI, summarized in Supplementary Table S4, and the visualized PPI network, shown in Fig. 2, the nodes in the network represent the potential targets, and the edges represent the interaction between the targets. The network was comprised of 16 nodes and 16 edges, and the average node degree was 2. 0. The targets with the degree greater than 1. 9 times median and could found in the 145 common targets of compounds 5a-5n, NOTCH1, IGF1R, TLR4, and KDR, were selected as the core targets. 

The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis were performed using the 16 potential targets of compound 5 m. 87 GO-terms of biological process (BP), 1 GO-term of molecular function (MF) and 5 pathways of KEGG pathways were enriched significantly, summarized in Supplementary Table S5 -S7. Besides the top 5 GO-terms of BP with the lowest false, the terms of MF and KEGG were shown in Table 2. These results showed that compound 5 m inhibited the cancer cells tested might mainly though participating in the cancer related pathways and regulating the biological process of cells death, apoptosis, proliferation, and so on. Fig. 1 The trend of antitumor activities of the compounds with different substituents. The compounds with inhibitory ability lower than compound 4 were marked as green
section_name: Analysis of the core targets and network pharmacology of 5 m against the cancer cells tested

section_content: In an attempt to study the possible interaction mode between synthesized compounds 5a-5n and the core targets predicted, NOTCH1, IGF1R, TLR4, and KDR (PBD ID: 2f8x, 1igr, 3fxi, and 1y6a, respectively), molecular docking was conducted using AutoDock Tools. Each docking result contained 50 conformations predicted, and the conformation with the lowest binding energy was chosen as representative to analysis. The correlation between the antitumor activity of compounds and the binding ability to the targets were investigated by Spearman's rank correlation coefficient analysis through compare the IC 50 with the binding energies. The results were shown in Table 3. 

The docking results showed the lowest binding energy of compounds to NOTCH1, IGF1R, TLR4 and KDR were -6. 67 to -8. 15, -5. 94 to -7. 04, -7. 04 to -8. 24, and -7. 71 to -8. 19 kcal/mol, respectively. These result indicated that the compounds might possess certain binding ability to the 4 targets, which were found overexpression in caner cells. 

The binding mode prediction between compound 5 m and NOTCH1 were performed at the binding pocket of MAML, the co-activator protein of NOTCH1 [21], and shown in Fig. 2. Compound 5 m possibly binds to the pocket through 10 non-bonding interactions with residues of Glu2072, Tyr381, Pro366, Val2039, Val2042, Asp2043, Asn2041, Ser2074, Glu2076, Gly2073 and 2 hydrogen bonds interacted residues of Arg382 and Asn2040. (Fig. 3a, b ). 

The binding mode prediction between compound 5 m and IGF1R were performed at the ligand binding site of L1 domain of IGF1R [22], and shown in Fig. 3. Compound 5 m possibly binds to the binding site through 13 non-bonding interactions with residues of Gly243, Tyr224, Arg222, His223, Thr107, Leu131, Asp132, Gly109, Ala133, Arg108, Ile106, Trp79 and Arg77 (Fig. 4a, b ). 

The binding mode prediction between compound 5 m and TLR4 were performed at the ligand binding pocket of the adaptor protein of TLR4 [23], and shown in Fig. 4. Compound 5 m were surrounded by the hydrophobic residues, Ile32, Ile153, Phe151, Ile52, Phe76, Phe104, Ile94, Ile117, Leu61, Phe119 and Phe121 (Fig. 5a, b ). 

The binding mode of compound 5 m docked within KDR were performed at the inhibitor-binding pocket [24], and shown in Fig. 5. The binding between compound 5 m and KDR possibly formed through 15 non-bonding interactions with residues Lys866, Cys1043, Phe1045, Gly839, Arg1030, Cys917, Phe916, Gly920, Asn921, Leu838, Phe919, Leu1033, Val914, Glu883 and Val846 (Fig. 6a, b ). 

The correlation study result showed that the IC 50 against SCC9 and Cal27 exhibited moderate positive correlation with the lowest binding energies of IGF1R at significant level of 0. 05, and exhibited positive strong correlation with the lowest binding energies of TLR4 at significant level of 0. 01. However, in SAS assay, the IC 50 showed moderate negative correlation with the lowest binding energies to IGF1R and KDR at significant level of 0. 05. These results indicated that the binding ability of compounds to TLR4 might be an important factor effected the antitumor activity against SCC9 and Cal27.
section_name: Molecular docking study of the core targets

section_content: In summary, 15 new thiazolidinone derivatives of dehydroabietic acid-based B ring-fused-thiazole were strong correlation between the IC 50 and the lowest binding energies. These results indicated that the synthetic strategy in this study is feasible. The compound 5 m might serve as a leading compound for potent antitumor agents and the TLR4 inhibitor.
section_name: Conclusions

section_name: Materials and methods

section_content: The melting points (m. p. ) were determined on an X-5 A digital melting point apparatus (Gongyi Kerui Instruments, Gongyi, China). NMR spectra were recorded in CDCl 3 on a Bruker AVANCE III HD500 spectrometer (Bruker, Switzerland) and chemical shifts were expressed in ppm (δ) downfield relative to TMS as an internal standard; NMR abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. MS spectra were obtained by means of the electrospray ionization (ESI) on a high-resolution mass spectrometer Q Exactive HR-MS (Thermo Fisher, USA). Reaction products were isolated by using column chromatography on silica gel column (200-300 mesh). All chemicals and reagents were purchased from commercial suppliers and used as-received. Compounds 1 and 2 were prepared according to the literatures. Chloracetyl chloride (0. 97 g, 8. 6 mmol) was added dropwise to a solution of compound 2 (3. 0 g, 7. 8 mmol) in CH 2 Cl 2 (20. 0 mL) at room temperature, and then continuously stirred for 15 min. The mixture was washed with excess aqueous Na 2 CO 3 solution and subsequently with deionized water. After desiccation, the residue was chromatographed over silica gel (petroleum ether-ethylacetate, 1:5) to afford compound 3. Yield 75%. 1 A solution of compound 3 (3. 0 g, 6. 5 mmol), potassium thiocyanate (0. 95 g, 9. 8 mmol), and triethylamine (1. 0 mL) in ethanol (10. 0 mL) was heated to 70 °C for 5 h. Then, the solvent was removed in vacuo.
section_name: Chemistry

section_name: Preparation of (7bS,11R

section_content: Compound 4 (0. 1 g, 0. 2 mmol) and corresponding aldehydes (0. 3 mmol) were dissolved in a mixture of triethylamine (1. 0 mL) and ethanol (1. 0 mL). The resulting solution was heated to 80 °C under stirring for 8 h. Then, the mixture was purified by column chromatography on silica gel (CH 2 Cl 2 -MeOH, 130:1) to afford target compounds 5a-5n. Cell proliferation was measured with the Cell Counting Kit-8 (CCK-8) assay kit (UElandy, Su Zhou, China). Cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 5 × 10 3 cells/well, and cultured at 37 • C in a humidified incubator (5% CO 2 ) for 24 h, followed by exposure to various concentrations of compounds for 48 h. Subsequently 10 μL of CCK-8 was added to each well, the cells were then incubated for an additional 1 h at 37 °C. Cell growth inhibition was determined by measuring the optical density value (OD) at λ = 450 nm using a microplate reader. Three independent experiments were performed. Cell growth inhibition was calculated according to the following equation:
section_name: Synthesis of thiazolidinone derivatives of dehydroabietic acid-based B ring-fused-thiazole 5a-5n

section_content: The IC 50 were obtained from liner regression analysis of the concentration-response curves plotted for each tested compound.
section_name: (2E,7bS,11R)-methyl 2-((Z)-5-(2-bromobenzylidene)-

section_content: The structure of 5a-5n was imported into the database of PharmMapper Server (http:// www. lilab-ecust. cn/ pharm mapper/) for prediction [25]. The results, whose scores of Norm Fit were greater than zero, were chosen as the predicted targets. Their information, including the gene names and gene ID, were further extracted using UniProtKB (http:// www. unipr ot. org) [26]. 

"Tongue squamous cell carcinoma" were used as keywords to retrieve the 3 cancer cell lines associated targets in Online Mendelian Inheritance in Man database (OMIM, https:// omim. org) and Genecards database (https:// www. genec ards. org) [27]. 

The intersection of 5a-5n respective targets and the 3 cancer cell lines associated targets were chosen as the treatment targets of 5a-5n against tumor call tested.
section_name: Prediction of 5a-5n therapeutic targets in the 3 cancer cell lines

section_content: The potential targets gene list was imported into the STRING database (https:// string-db. org), with the species as "Homo sapiens" and a confidence score greater than 0. 4, to obtain the data of PPI and the visualized PPI network [28]. The targets whose degrees were greater than 1. 9 times median, and can find in all the treatment targets were selected as the core targets. 

The biological functions of the target genes were assessed through GO and KEGG pathways enrichment analysis. The predicted results were also obtained for the STRING database.
section_name: Analysis of the core targets and network pharmacology of 5 m against the cancer cells tested

section_content: All docking procedures were performed using AutoDock 4. 2. 6 software (ADT) according to the reported paper [29].
section_name: Molecular docking study of the core targets

section_content: OD of control -OD of treatment OD of control -OD of blank × 100%

All the proteins in the PDB files were cleaned by removing small molecules, ions, and original ligand in the crystal of the proteins. Then, polar hydrogen atoms were added, Gasteiger charges were computed, and the atoms were set as "Assign AD4 type" in ADT. The compounds for docking program were drawn using ChemDraw 8. 0 and automatically set the torsional bonds by the AUTOTORS module in ADT. The docked sites of the protein were set in the place of the original ligand or according the literature [21]. The binding energy between the docked compound and the protein was calculated using the AutoGrid program with a grid spacing of 0. 375 Å by the Lamarckian genetic algorithm as a searching method. The interaction between compounds and the amino acid residues of the proteins was found by LigPlot + 2. 1 and visualized by PyMOL 2. 4. 0a0 software. 

The correlation between the antitumor activity and the binding energies were analysed using spearman rank correlation coefficient. The strength of the correlation were guided as very weak, weak, moderate, strong and very strong by the correlation coefficient in absolute value in the intervals of [0, 0. 2), [0. 2, 0. 4), [0. 4, 0. 6), [0. 6, 0. 8), and [0. 8, 1. 0], respectively [30].
section_name: Growth inhibition =

section_content: Acknowledgements This research was supported by Guangxi Natural Science Foundationunder under (Grant Nos. 2019GXNSFAA245057 and 2018GXNSFAA138023 ).

section_content: 4 -7. 37 -6. 25 -7. 04 -7. 87 5a ( R = 2Br-Ph) -7. 32 -6. 84 -8. 1 -8. 06 5b ( R = 2Cl-Ph) -7. 28 -6. 2 -8. 1 -7. 77 5c (R = 2F-P-h) -6. 95 -5. 94 -8. 1 -7. 80 5d ( R = 2Me--Ph) -6. 86 -6. 81 -7. 93 -7. 98 5e ( R = 2MeO-Ph) -6. 17 -5. 9 -7. 69 -8. 05 5f ( R = 4Br-Ph) -7. 76 -6. 48 -7. 78 -7. 92 5 g (R = 4Cl-Ph) -7. 17 -6. 25 -7. 29 -7. 84 5 h (R = 4F-Ph) -7. 15 -6. 64 -7. 38 -8. 12 5i ( R = 4Me-Ph) -7. 66 -6. 0 -7. 67 -7. 71 5j ( R = 4MeO-Ph) -7. 31 -6. 17 -6. 96 -7. 94 5 k (R = pyridin-3-) -7. 90 -7. 04 -8. 03 -8. 08 5 l (R = 6-Cl-pyridin-3-) -7. 35 -6. 37 -8. 24 -8. 19 5 m (R = 1H-pyrrol-2-) -7. 35 -6. 18 -7. 84 -7. 54 5n ( R = H-Ph) -7. 27 -6. 03 -7. 77 -7. 97

section_name: Supplementary Information

section_content: The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11030-023-10626-6. 

Author contributions Nai-Yuan Chen wrote the main manuscript text, and Cui-Ping Li provided the data of antitumor activity, and Hong-Fei Huang provided the data of the high-resolution mass spectra.
section_name: Supplementary Information

section_content: Competing interests The authors declare no competing interests.
section_name: Declarations

section_content: The authors declare that they have no confict of interest.
section_name: Conflict of interest

section_content: Nai-Yuan Chen
section_name: Authors and Affiliations