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Case Studies in Chemical and Environmental Engineering

Dual Antioxidant and Cytotoxic Activities of Novel 1,2,3-Triazole-Decorated Unsymmetrical Monocarbonyl Curcumin Analogues

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Article Details
Authors
Bayu Ardiansah, Ahmad Farhan, Novita Sari Nurhasanah, Mochammad Arfin Fardiansyah Nasution, Noordini M. Salleh, Kenji Mizuguchi, Antonius Herry Cahyana, Lina Mardiana
Journal
Case Studies in Chemical and Environmental Engineering
DOI
10.1016/j.cscee.2024.101031
Table of Contents
Abstract
1. Introduction
2. Results And Discussion
2.1. Synthesis
2.2. Antioxidant Evaluation
2.3. Cytotoxic Evaluation
2.4. Molecular Docking Analysis
2.5. Physicochemical, ADME And Drug-Likeness Prediction Analysis
3. Experimental
3.1. Materials
3.2. Instrumentations
3.3. Synthesis Of Compounds 4 And 5a-5f
3.3.1. (1E,4E)-1-(3-Methoxy-4-(Prop-2-Yn-1-Yloxy)Phenyl)-5-(4methoxyphenyl)Penta-1,4-Dien-3-One (4)
3.3.2. General Procedure Of Azide-Alkyne Cycloaddition (Triazolation) To Synthesize Compounds 5a-5f
3.3.3. (1E,4E)-1-(4-((1-(4-Chlorophenyl)-1H-1,2,3-Triazol-4-Yl) Methoxy)-3-Methoxyphenyl)-5-(4-Methoxyphenyl)Penta-1,4-Dien-3-One (5a)
3.3.4. (1E,4E)-1-(4-((1-(4-Acetylphenyl)-1H-1,2,3-Triazol-4-Yl)Methoxy)3-Methoxyphenyl)-5-(4-Methoxyphenyl)Penta-1,4-Dien-3-One (5b)
3.3.5. (1E,4E)-1-(3-Methoxy-4-((1-(4-Nitrophenyl)-1H-1,2,3-Triazol-4-Yl) Methoxy)Phenyl)-5-(4-Methoxyphenyl)Penta-1,4-Dien-3-One (5c)
3.3.6. 4-(4-((2-Methoxy-4-((1E,4E)-5-(4-Methoxyphenyl)-3-Oxopenta1,4-Dien-1-Yl)Phenoxy)Methyl)-1H-1,2,3-Triazol-1-Yl)Benzoic Acid (5d)
3.3.7. Ethyl 4-(4-((2-Methoxy-4-((1E,4E)-5-(4-Methoxyphenyl)-3oxopenta-1,4-Dien-1-Yl)Phenoxy)Methyl)-1H-1,2,3-Triazol-1-Yl)Benzoate (5e)
3.3.8. 4-(4-((2-Methoxy-4-((1E,4E)-5-(4-Methoxyphenyl)-3-Oxopenta1,4-Dien-1-Yl)Phenoxy)Methyl)-1H-1,2,3-Triazol-1-Yl)Benzenesulfonamide (5f)
3.4. Procedure For Antioxidant Evaluation
3.5. Procedure For Cytotoxic Evaluation
3.6. Procedure For Molecular Docking
3.7. Procedure For Physicochemical, ADME, And Drug-Likeness Predictions Study
4. Conclusions
Acknowledgements
Appendix A. Supplementary Data
Abstract
Unsymmetrical monocarbonyl curcumin analogs containing 1,2,3-triazole scaffold (5a-5f) have been synthesized from vanillin. 5d, which contains a carboxylic group, exhibited the highest antioxidant and cytotoxic activities, with an IC50 of 0.56 mM against DPPH and an IC50 of 38.25 ± 4.79 μM against MCF-7 cells. Docking studies revealed that 5d has high binding affinity for the tubulin protein, with predicted binding value of − 10.4 kcal/ mol. Additionally, in silico ADME predictions indicated that 5d demonstrates high gastrointestinal absorption and moderate intrinsic clearance. These findings indicate that the designed compounds, particularly compound 5d, could be promising for developing a new drug candidate.
Case Report Dual antioxidant and cytotoxic activities of novel 1,2,3-triazole-decorated unsymmetrical monocarbonyl curcumin analogs Bayu Ardiansah a,*, Ahmad Farhan a, Novita Sari Nurhasanah a, Mochammad Arfin Fardiansyah Nasution b, Noordini M. Salleh c, Kenji Mizuguchi b, Antonius Herry Cahyana a, Lina Mardiana a a Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, 16424, Indonesia b Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan c Department of Chemistry, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia A R T I C L E I N F O Keywords: Curcumin analogs 1,2,3-triazole Hybrid compounds Cytotoxic Antioxidant A B S T R A C T Unsymmetrical monocarbonyl curcumin analogs containing 1,2,3-triazole scaffold (5a-5f) have been synthesized from vanillin. 5d, which contains a carboxylic group, exhibited the highest antioxidant and cytotoxic activities, with an IC50 of 0.56 mM against DPPH and an IC50 of 38.25 ± 4.79 μM against MCF-7 cells. Docking studies revealed that 5d has high binding affinity for the tubulin protein, with predicted binding value of − 10.4 kcal/ mol. Additionally, in silico ADME predictions indicated that 5d demonstrates high gastrointestinal absorption and moderate intrinsic clearance. These findings indicate that the designed compounds, particularly compound 5d, could be promising for developing a new drug candidate.
1. Introduction
Curcumin (Fig. 1A), a naturally occurring polyphenolic compound, has garnered significant attention in recent years due to its diverse pharmacological properties and therapeutic potential [1]. Derived from the rhizomes of the Curcuma longa plant, commonly known as turmeric, curcumin has a rich history in traditional medicine, particularly in Ayurveda and Traditional Chinese Medicine, where it has been utilized for centuries [2]. Its vibrant yellow color and culinary applications have made it a staple in various cuisines around the world [3]. The multifaceted nature of curcumin has propelled it into the spotlight of contemporary biomedical research [4]. Several investigations have confirmed that curcumin exhibits biological activities, including anti-inflammatory, antioxidant, anticancer, antibacterial, and antiviral properties [5–9]. However, despite its promising contribution, curcumin is not without limitations. One of the primary challenges in harnessing its full therapeutic potential lies in its poor bioavailability [10]. The low absorption, rapid metabolism, and limited stability of curcumin in vivo have raised concerns regarding its clinical utility. Researchers have been striving to overcome these hurdles through various approaches, including the development of novel curcumin analogs to find better molecules with improved bioactivity and bioavailability. In recent years, the synthesis and assessment of unsymmetrical analogs of curcumin have emerged as a captivating domain within the field of medicinal science [11]. These analogs as shown in Fig. 1B, which exhibit structural variations from the widely recognized polyphenolic compound curcumin, have generated significant intrigue due to their potential therapeutic utility. For instance, compound I was found to exhibit antitubercular activity [12]. On the other hand, molecular hybridization, a fundamental strategy in modern organic synthesis, represents an innovative approach that harmoniously amalgamates diverse molecular fragments to yield intricate and utilitarian compounds [13]. By capitalizing on the synergistic reactivity and structural attributes of distinct precursors, this pioneering methodology facilitates the creation of molecules tailored for specific functions and applications. In response to the mounting demand for novel organic compounds, spanning pharmaceuticals, agrochemicals, and advanced materials, the appeal of molecular hybridization has surged significantly in recent times [14]. In this regard, currently, unsymmetrical analogs of curcumin as hybrid molecules were synthesized by several research groups. Unsymmetrical curcumin analogs bearing piperazine moiety II was reported by Rahmawati et al. (2020) to have anti-inflammatory effects [15]. Meanwhile, the analog bearing phenylpyrazole functionality III was found to demonstrate anticancer activity, especially against MDA-MB-231 [16]. * Corresponding author. E-mail address: bayu.ardiansah@sci.ui.ac.id (B. Ardiansah). Contents lists available at ScienceDirect Case Studies in Chemical and Environmental Engineering journal homepage: www.sciencedirect.com/journal/case-studies-in-chemical- and-environmental-engineering https://doi.org/10.1016/j.cscee.2024.101031 Received 21 September 2024; Received in revised form 31 October 2024; Accepted 24 November 2024 Case Studies in Chemical and Environmental Engineering 11 (2025) 101031 Available online 26 November 2024 2666-0164/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). Moreover, dichloro-substituted curcumin analog clubbed 1,2,4-triazole ring IV showed antibacterial potency, as reported by Chen et al. (2020) [17]. While some hybrid compounds derived from curcumin analogs have been reported, synthesizing new structural derivatives of curcumin and evaluating their biological activities continue to be a compelling area of research. This is closely tied to ongoing efforts in identifying potential drug candidates. The 1,2,3-triazole, with its distinctive structural attributes, has evolved into a versatile and indispensable component of modern chemistry and drug discovery [18]. Its unique five-membered ring, composed of three nitrogen and two carbon atoms (Fig. 1C), imparts exceptional stability to this heterocyclic compound, rendering it an invaluable scaffold in the field of organic synthesis [19]. Notably, the simplicity of its synthesis through copper-catalyzed azide-alkyne cycloaddition (CuAAC) has propelled the 1,2,3-triazole to the forefront of chemical innovation [20]. The significance of 1,2,3-triazole extends beyond the laboratory, as it is an integral component in the structural framework of many commercially available drugs such as Rufinamide and Radezolid, underscoring its relevance in pharmaceuticals [21]. Furthermore, recent research has unveiled the remarkable biological activities associated with 1,2,3-triazole-containing compounds, such as antibacterial, anticancer, anti-inflammatory, and antioxidant effects [22–25]. Our motivation was to synthesize novel derivatives of unsymmetrical monocarbonyl curcumin analogs, specifically incorporating the 1,2,3- triazole ring system as shown in Fig. 1D, and to evaluate their potential as antioxidant and cytotoxic agents. This choice was driven by prior research underscoring the significance of the 1,2,3-triazole ring in medicinal chemistry and our interest in advancing curcumin analogs. Additionally, we performed an ADME analysis to assess their pharmacokinetic properties and conducted molecular docking studies to explore their interactions with the target enzyme, thereby complementing our in vitro investigations.
2. Results and discussion
2.1. Synthesis
The synthesis of unsymmetrical monocarbonyl curcumin analog compounds with a 1,2,3-triazole scaffold commenced with a basecatalyzed aldol condensation reaction of vanillin (1) with acetone to produce dehydrozingerone (2) in 64 % yield. The second step was propargylation of compound 2 in DMF containing K2CO3 at room temperature to give propargylated dehydrozingerone (3). In order to create a monocarbonyl curcumin skeleton, an acid-catalyzed aldol condensation reaction was carried out between compound 3 and 4-methoxybenzaldehyde to afford compound 4. This compound was then used as the key intermediate in copper-mediated azide-alkyne [3+ 2] cycloaddition with various aryl azides to yield the final products (5a‒f) in medium to good yields (54–89 %) as shown in Scheme 1. The synthesized intermediates and final products were characterized by FTIR, HRMS, 1H and 13C NMR (see supplementary file). Compound 2 is confirmed by FTIR with Csp2‒H and C–C absorption at 3002 and 1583 cm− 1, respectively. The 1H NMR spectrum shows the presence of alkene protons of the α,β-unsaturated structure at 7.44 and 6.57 ppm (as a doublet) with trans configuration (J= 16.2 Hz). The next intermediate compound, propargylated dehydrozingerone (3), was identified by FTIR which showed characteristic peaks at 3258 and 2128 cm− 1, indicating the existence of Csp‒H and C ≡ C stretching vibrations, respectively. The methylene proton and alkyne terminal proton of the propargyl group was confirmed by 1H NMR at 4.81 ppm (as a doublet) and 2.54 ppm (as a triplet). Monocarbonyl curcumin analog compound (4) was also successfully confirmed. The carbonyl group conjugated with α,β-unsaturated was detected at 1645 cm− 1 by FTIR and 188.8 ppm by 13C NMR. For alkene protons in α,β-unsaturated group, the 1H NMR peaks was observed at around 7.72–7.66 and 6.99–6.92 ppm. Final confirmation with HRMS shows the experimental [M+H]+ value of 349.1427, which is close to the theoretical value 349.1427. Scheme 1. Synthetic route of curcumin analogs with 1,2,3-triazole scaffold. Based on the FTIR spectra, the most typical characteristics for all final products (5a‒f) are the absorption of conjugated C–O and C–C enone groups at 1641–1686 and 1598‒1601 cm− 1, respectively. The stretching vibrations of Csp2‒H was detected around 3162‒3014 while Csp3‒H at around 2976‒2837 cm− 1. For compound 5c, N–O vibration from the nitro group were detected at 1511 cm− 1, and then wide absorption was observed for compound 5d in the range 3500‒2600 cm− 1, which is identified the presence of a carboxylic group. Meanwhile, the sulfonamide group in compound 5f shows absorption at 3348‒3250 cm− 1 (‒NH2) and 1335 cm− 1 (‒SO2). The 1H NMR spectrum successfully confirmed the presence of olefin protons from the triazole at around 9.20–8.09 ppm as a singlet peak. For methylene protons between the phenoxy and triazole framework, singlet peak was observed in the range of 5.42‒5.31 ppm. The alkene proton of the enone group was confirmed for all compounds in the range 7.76‒6.90 ppm (as a doublet) with J coupling values around 15.9 Hz, which is indicating a trans alkene configuration. Compounds 5b and 5e have additional signals from protons bound to sp3 carbon. For compound 5b a singlet signal was observed at 2.65 ppm (3H), and for 5e signals were observed at 4.41 ppm (q, 2H) and 1.42 ppm (t, 3H). Meanwhile, for compound 5f, proton signal from the sulfonamide group was observed as a singlet at 7.57 ppm (s, 2H). Further characterization, 13C NMR confirmed the presence carbonyl of the enone group in all products at around 188.7–188.0 ppm. For example, the carbonyl group of compound 5awas observed at 188.7 ppm, while the aromatic quaternary carbon that binds the chlorine (4- Cl-Ph) was detected at a chemical shift of 134.7 ppm. In compounds 5b, 5d and 5e there are two carbonyl signals because they have an additional carbonyl group from the aryl azide. For example, carbonyl signals in compound 5d can be observed at 188 and 170.4 ppm, both signals were identified as signals from the carbonyl on the enone and carboxylate group, respectively. Finally, HRMS spectra confirmed the high precision of the experimental [M+H]+ values in all products compared with their theoretical values. For example, compound 5f shows an experimental [M+H]+ value of 547.1657 (theoretical value = 547.1651).
2.2. Antioxidant evaluation
Antioxidant substances have the ability to attach to free radicals, safeguarding materials from the process of oxidation [26]. The DPPH method, as established by Blois, was employed to assess the capacity of compounds to scavenge free radicals [27]. DPPH serves as a stable free radical that readily accepts an electron or hydrogen radical, transforming into a stable molecule. The capacity of antioxidants to reduce DPPH radicals was evaluated by measuring the reduction in absorbance at 517 nm. As antioxidants interact with these radicals, the DPPH radical’s absorbance decreases, leading to radical scavenging. This transformation is observable as a color change from deep purple DPPH solution to yellow and neutralize free radicals by partnering the odd DPPH electron with a hydrogen atom or by electron donation. Consequently, DPPH is commonly employed as a substrate for assessing antioxidant activity [28]. Although there are other techniques for determining antioxidant activity, the DPPH method is one of the most widely used, fastest, and most effective [29]. Based on the antioxidant activity assay with the DPPH method, all compounds (5a-f) at 1 mM showed antioxidant activity (Table 1). Compound 5d with a carboxylic group showed the highest antioxidant activity with inhibition of 80.27 ± 0.23 %. However, replacing the hydrogen atom in the carboxylate group with an ethyl group (compound 5e) reduces the antioxidant activity compared to compound 5d. The hydrogen atom in the carboxylic group is easier to donate and stabilizes the DPPH radical, so compound 5d has a higher antioxidant potential. The product compound with chloro (5a), acetyl (5b), and nitro (5c) substituents shows inhibitory activity of DPPH radicals of 73.35 ± 1.40, 71.94 ± 0.50, and 68.17 ± 1.10 %, respectively. This decrease in antioxidant activity is due to the presence of electron-withdrawing group substituents [30]. Chloro as a substituent has an electron-withdrawing inductive effect as well as an electron-donating resonance effect (EDG and EWG), resulting in increased inhibition compared to strong electron-withdrawing groups such as nitro. The nitro group is a strong electron-withdrawing group that can attract electron density from the radicals and reduce the stability of radicals. Meanwhile, compounds with sulfonamide (5f) substituents showed the weakest antioxidant activity compared to other compounds with inhibition of 60.22 % ± 0.50. Based on the evaluation, compound 5d with IC50 of 0.5613 mM was denoted as the most potent agent as antioxidant. This result provides a new alternative for the potential antioxidant agents from the class of curcumin analogs with 1,2,3-triazole scaffold.
2.3. Cytotoxic evaluation
Cancer arises from the unchecked proliferation of cells and represents a complex ailment with various contributing factors, resulting in millions of deaths annually on a global scale. Particularly, breast cancer remains prevalent as one of the leading cancers affecting women. Thus, exploration of new drug candidates through synthetic organic approach is urgently required. The MTT assay is a method to assess cytotoxic activity of novel drug candidates, specifically through the inhibition of cancer cell proliferation by molecules (anticancer activity). This method involves the reduction of a tetrazolium salt called MTT (yellow) to form formazan crystals (purple) and the oxidation of NADH to NAD+ [31]. The reduction of MTT salts to formazan products is catalyzed by the mitochondrial reductase enzyme. The more NADH is oxidized to NAD+, the more cells are still alive and the more purple formazan crystals are formed [32]. The cytotoxicity of a compound is generally expressed by the IC50 value which shows the concentration of a substance that can inhibit half of the biological activity of living cells [33]. The lower IC50 value, the more effective the cytotoxic activity. The IC50 value is determined by analyzing the linear regression curve between the probit values of response (inhibition) and the logarithm of the concentration. The results of cytotoxic evaluation against breast cancer cells (MCF7) using MTT assay are shown in Table 2. Compound 5dwith carboxylic group showed the highest cytotoxic activity with an IC50 value of 38.25 ± 4.79 μM. Substitution of the hydrogen atom with an ethyl group in compound 5e (IC50 > 200 μM) causes the decrease cytotoxic activity. Similar to compound 5e, the product with chloro (5a), acetyl (5b), and nitro (5c) substituents did not show cytotoxic activity against MCF-7 cells, with IC50 values of >200 μM. Meanwhile, compounds with sulfonamide substituents (5f) showed moderate cytotoxic activity with an IC50 value of 75.28 ± 7.48 μM. Based on the classification in anticancer strength by Kuete & Efferth [34], compound 5d has strong cytotoxicity and 5f is moderate. Comparison of cytotoxic activity by our lead compound to normal curcumin as well as previously synthesized compounds in the literatures was listed in Table 3 [35–38]. Although our compound, 5d, showed slightly lower activity compared to literature compounds, this compound showed higher activity than its parent compound, curcumin. Therefore, this synthetic pathway can open the windows and provide an alternative strategy for exploring new anticancer agents with moderate to strong activity against MCF-7 cell lines. Various studies indicate that the apoptosis induced by anticancer drugs is an important mechanism in cancer therapy [39]. Apoptosis describes the cell death that characterized by cell wall deformation, cell shrinkage, and reduction in number of viable cells [40]. Fig. 2 shows the results of microscope observations in cell morphology changes when treated with the active compound 5d. It was observed the effect on cell shrinkage and reduction in number of viable cells compared to the control which only contained MCF-7 cells without samples. The higher concentration of sample, the more shrinkage and reduction in cell number is observed. Microscope observation at 50 μ M showed that more than 50 % of cells were reduced, this is in accordance with the IC50 result of 5d that lower than 50 μ M. Moreover, the structure-activity relationship was pointed in Fig. 3.
2.4. Molecular docking analysis
Herein, molecular docking studies were conducted to investigate the potential interactions between the synthesized monocarbonyl curcumin analog compounds and tubulin, as well as to determine their binding conformations within tubulin’s binding sites. Prior to this study, various protein such as tubulin [41–43], estrogen receptor alpha (ERɑ) [44,45], human epidermal growth factor receptor 2 (HER2) [46,47], and B-cell lymphoma 2 (Bcl-2) [48,49], expressed in MCF-7 cells, were extensively studied to determine the specific binding interactions of the investigated ligand, which shows cytotoxic activity against MCF-7 cells. Among these, inhibiting tubulin was found to disrupt the dynamic equilibrium of microtubules, crucial for cell division, leading to mitotic arrest, cell death, and effectively halting cancer cell proliferation [50]. Tubulin consists of two subunits, α-tubulin and β-tubulin, which form heterodimers, and it needs to function as heterodimers to work properly [51]. Therefore, the docking studies in this research were performed using the tubulin heterodimers by selecting the binding pocket around colchicine’s binding site, which also includes the GTP binding site (Fig. 4). As listed in Table 4, docking studies show that compound 5d possesses the highest binding affinity for tubulin, with a predicted binding score of − 10.4 kcal/mol, followed by compounds 5a and 5f, both of which have predicted binding score of − 10.0 kcal/mol. The trends in binding affinities of the synthesized monocarbonyl curcumin analog compounds are consistent with the results from in vitro studies, except for compound 5a. The binding interaction analysis of the six compounds within the tubulin binding pocket, as depicted in Fig. 5, reveals different binding configurations among the compounds. Although the synthesized compounds share the same basic structure, from curcumin to the triazole group, the variations in the –R groups significantly affect the binding configurations of the ligands within the tubulin binding pocket. Compound 5d, which exhibits the highest binding affinity and cytotoxic activity in vitro, forms four hydrogen bonds (H-bonds) within the tubulin binding pocket. These H-bonds are with Asn α101, Asn α206, Cys β241, and Lys β254, making it the most H-bond interaction-rich among all synthesized compounds. Furthermore, the –COO– group in the –R of compound 5d significantly contributes to interactions within the tubulin binding pocket by forming a H-bond with the backbone (-NH) of Cys β241. In contrast, the role of the –R group in other synthesized compounds is almost non-existent, except for compound 5a, where the -Cl group forms a H-bond with the C–O group of the side chain of Asn α228. These observations explain the high binding affinity of compound 5d for the tubulin protein and may account for its significant cytotoxic activity against MCF-7 cells.
2.5. Physicochemical, ADME and drug-likeness prediction analysis
In this research, the physicochemical properties, pharmacokinetics (absorption, distribution, metabolism, and excretion - ADME), and druglikeness predictions for the final products (5a-f) were computed using SwissADME and DruMAP, with the results presented in Table 5. The physicochemical analysis was based on Lipinski’s Rule of Five (RO5), which includes criteria such as molecular weight (MW ≤ 500), number of rotatable bonds (NROTB ≤10), hydrogen-bond acceptors (HBA ≤10), hydrogen-bond donors (HBD ≤5), and partition coefficient (MlogP ≤4.15) [52]. A compound is considered orally available if it violates none or only one of Lipinski’s RO5 criteria. According to the results, the MW of the compounds ranged from 501.96 to 546.59 g/mol, and the computed LogP values ranged from 1.66 to 3.51. Only compound 5awas predicted to be an orally active substance, as it violated only one criterion of Lipinski’s RO5 by having an MW slightly above the desirable threshold. The other five compounds violated two criteria: they had both a higher MW than the threshold and a higher number of rotatable bonds, making them not predicted to be orally bioavailable according to the Lipinski’s RO5. Furthermore, we analyzed additional physicochemical parameters, such as topological polar surface area (TPSA; ≤130 Å2) and water solubility (LogS; − 3.5 to − 6.5). The results indicate that all synthesized ligands are predicted to have low-to-moderate water solubility, as shown by their LogS values, which range from − 6.30 (5a) to − 5.18 (5f). Compounds 5a and 5e, with the lowest LogS values, are categorized as "Poor" in solubility, despite having TPSA values below 130 Å2 and LogP values less than 4.15. This suggests that the ligand’s solubility is consistent with their LogS values, and that while TPSA and LogP are important, the solubility as indicated by LogS plays a crucial role in determining bioavailability [53]. In addition, the ADME predictions for the synthesized monocarbonyl curcumin analog compounds were carried out in this study. First, gastrointestinal (GI) absorption and blood–brain barrier (BBB) permeability were predicted due to its crucial role in optimal pharmacokinetics, as the GI tract is essential for oral drug administration and plays a significant role in the oral drug release mechanism [54,55] and BBB acts as a natural protective membrane that protects central nervous system (CNS) from toxins and pathogens in blood [56]. According to SwissADME predictions, all compounds were predicted not to exhibit any BBB permeability, primarily due to their high MW and relatively high TPSA, which hinders their ability to cross the BBB [57]. In contrast, all compounds exhibited high GI absorption, except for compounds 5c and 5f. Their reduced GI absorption is due to their high TPSA, which exceeds 120 Å2 and is likely attributed to the presence of strong electron-withdrawing groups, such as –NO₂ and –SO₂NH₂, respectively. A high TPSA correlates with low GI absorption [58], as these groups increase the hydrophilicity of the compounds, ultimately reducing their GI absorption. In addition, ADME predictions from DruMAP indicated that all ligands have a good retention profile, demonstrated by their low P-gp net efflux ratio. However, their low fraction unbound in plasma suggests lower bioavailability, low drug efficiency and slower clearance, contributing to longer half-lives in the body [59,60]. Furthermore, CYP3A4 appears to be the primary CYP450 enzyme responsible for metabolizing all compounds, with CYP1A2 also predicted to play a role in the metabolism of compounds 5a and 5c. This result may correspond to the structural similarity of these ligands to curcumin, which has been previously reported to be efficiently metabolized by CYP3A4 [61]. All ligands, except for compound 5d, share similar properties, including a high fraction of dose absorbed and high permeability coefficients. However, compound 5d has a lower fraction of dose absorbed and reduced permeability, likely due to the presence of a –COOH group. This group decreases membrane permeability of the compound because it exists in a deprotonated state (-COO⁻) at physiological pH, leading to lower absorption and poorer permeability [62]. To address this issue, a protecting group could be introduced to the –COOH group, such as by creating a prodrug in the form of a carboxylate-derived ester or lactone, which would enhance absorption and permeability [63]. After absorption, the prodrug would be metabolically transformed back to –COOH inside the body [63]. This is a common strategy for drugs facing similar challenges with the –COOH functional group, as seen in statin-based drugs for HMG-CoA reductase inhibition [64] or benazepril as an ACE inhibitor [65]. The use of a methyl or ethyl ester as a protecting group for –COOH is further supported by the DruMAP results for compound 5e, which indicate that its -COOEt moiety exhibits unstable intrinsic clearance. This instability is attributed to its rapid metabolism and susceptibility to enzymatic degradation, likely due to minimal steric hindrance [66]. In contrast, the other compounds show moderate intrinsic clearance, which further emphasizes that the ester moiety contributes to the intrinsic clearance instability observed in compound 5e.
3. Experimental
3.1. Materials
Organic azides have the potential to be explosive and dangerous. Even though there haven’t been any significant mishaps in our research, every manipulation needs to be done carefully behind a safety screen in a hood to prevent an explosion. To transfer the sodium azide, a plastic spatula is needed. Smith’s ratio indicates that azido compounds with (C + O)/N < 3 require special consideration. The progress of the reaction was monitored using silica gel thin layer chromatography (TLC, Merck TLC Silica Gel 60 F254 plates) and a UV lamp. Column chromatography was performed using Merck silica gel 60. All reagents were supplied by Merck and Sigma-Aldrich. Anhydrous solvents such as ethanol were purchased from Merck and Supelco Inc. distributor in Jakarta, Indonesia. Distilled water was utilized as the reaction solvent, in the quenching reactions, and during the separation processes.
3.2. Instrumentations
The 1H and 13C nuclear magnetic resonance (NMR) spectra were collected at the Integrated Laboratory and Research Center (ILRC) of Universitas Indonesia using a Bruker Avance Neo 500 MHz spectrometer. Chemical shifts are expressed in parts per million (ppm) in deuterated chloroform (CDCl3), and the solvent peak (1H: 7.26, 13C: 77.00 ppm) was used for the calibration. The spectra of samples that are poorly soluble in deuterated chloroform were assessed using dimethylsulfoxide (DMSO‑d6, 1H: 2.50, 13C: 39.52 ppm). The abbreviations s (singlet), d (doublet), t (triplet), q (quartet), quint. (quintet), and m (multiplet) are used. Electrothermal IA9100 was used to measure melting points. For the determination of measure infrared spectra, a Shimadzu IRPrestige-21 FTIR spectrometer was employed. At the Indonesia National Police Forensic Laboratory Centre, High Resolution Mass Spectra (HRMS) were acquired using a Xevo G2-XS QTof (Waters, USA) in detection mode with ESI-TOF.
3.3. Synthesis of compounds 4 and 5a-5f
The synthetic route for curcumin analogs with a 1,2,3-triazole scaffold (5a-5f) is illustrated in Scheme 1. The intermediate compounds, including aryl azides such as 1-azido-4 chlorobenzene, 1-(4-azidophenyl)ethan-1-one, 1-azido-4-nitrobenzene, and ethyl 4 azidobenzoate, were synthesized following methods reported in the literature with small modifications [67–71]. This was followed by the preparation of 4-azidobenzoic acid [72] and 4-azidobenzenesulfonamide [73]. The preparation of compounds 2 and 3 was also based on literature procedures [70]. Detailed experimental procedures and spectroscopic data of the intermediate compounds were given in Supplementary Material. Meanwhile, the synthesis procedures for compounds 4 and 5a-5f are described as follows:
3.3.1. (1E,4E)-1-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)-5-(4methoxyphenyl)penta-1,4-dien-3-one (4)
In a 10-mL round-bottom flask containing 3 (461 mg, 2.0 mmol) and 4-methoxybenzaldehyde (0.24 mL, 2.0 mmol) in ethanol (3.5 mL), concentrated hydrochloric acid (3 mL) was added dropwise. The mixture was stirred at room temperature for 67 h and then at 50 ◦C for 13 h. Afterward, the mixture cooled to room temperature, and then poured into ice-cold water. The solid obtained was purified by silica gel column chromatography (hexane/ethyl acetate = 6/1 to 1/1) to produce compound 4 (226 mg, 32 % yield) as a yellow solid. Rf value 0.21 (hexane/ethyl acetate = 2/1); m.p. 109–111 ◦C; IR (KBr, disc) νmax 3293, 3008, 2940, 2839, 2132, 1645, 1601, 1583, 1511, 1424, 1267, 1140, 1016, 977 cm− 1; 1H NMR (500 MHz, CDCl3) δ 7.72–7.66 (m, 2H), 7.58 (d, 2H, J = 8.7 Hz), 7.20 (dd, 1H, J = 8.4, 1.8 Hz), 7.15 (d, 1H, J = 1.7 Hz), 7.05 (d, 1H, J= 8.3 Hz), 6.99–6.92 (m, 4H), 4.82 (d, 2H, J= 2.3 Hz), 3.94 (s, 3H), 3.85 (s, 3H), 2.55 (t, 1H, J = 2.3 Hz); 13C NMR (126 MHz, CDCl3) δ 188.8, 161.6, 149.6, 148.8, 142.9, 142.7, 130.1, 127.5, 127.3, 124.3, 123.1, 122.5, 114.4, 110.33, 110.30, 77.9, 76.3, 56.5, 55.9, 55.4; HRMS (ESI-TOF) calcd for C22H21O4 [M+H]+ 349.1440, found 349.1427.
3.3.2. General procedure of azide-alkyne cycloaddition (triazolation) to synthesize compounds 5a-5f
In a 10-mL round-bottom flask containing 4 (87 mg, 0.25 mmol), aryl azide (0.30 mmol), copper(II) sulfate pentahydrate (25 mg, 0.10 mmol), and ascorbic acid (35 mg, 0.20 mmol) in DMF/water (1.5 mL/1.5 mL) was stirred at room temperature. The mixture then was poured into icecold water. The resulting solid was filtered, washed with water, and dried overnight to obtain the final product. Compounds 5a-5f were synthesized according to the general condition unless stated otherwise.
3.3.3. (1E,4E)-1-(4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl) methoxy)-3-methoxyphenyl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one (5a)
The reaction of 4 with 1-azido-4-chlorobenzene (40 μL, 0.30 mmol) for 24 h following general procedure above yielded compound 5a (105 mg, 83 % yield) as a brown solid. Rf value 0.65 (hexane/ethyl acetate = 1/1); m.p. 154–156 ◦C; IR (KBr, disc) νmax 3078, 2933, 2838, 1664, 1645, 1599, 1511, 1423, 1338, 1252, 1175, 1137, 1096 cm− 1; 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.72–7.68 (m, 4H), 7.57 (d, 2H, J= 6.8 Hz), 7.49 (d, 2H, J = 6.8 Hz), 7.18–7.12 (m, 3H), 6.98–6.92 (m, 4H), 5.40 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 188.7, 161.5, 149.50, 149.47, 142.9, 142.6, 135.3, 134.7, 130.1, 129.9, 128.7, 127.4, 124.2, 123.0, 122.7, 121.7, 121.2, 114.3, 113.3, 110.3, 62.7, 55.9, 55.4; HRMS (ESI-TOF) calcd for C28H25ClN3O4 [M+H]+ 502.1534, found 502.1503.
3.3.4. (1E,4E)-1-(4-((1-(4-acetylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)3-methoxyphenyl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one (5b)
The reaction of 4 with 1-(4-azidophenyl)ethan-1-one (48 mg, 0.30 mmol) for 47 h following general procedure above yielded compound 5b (110 mg, 86 % yield) as a yellow solid. Rf value 0.29 (hexane/ethyl acetate = 1/1); m.p. 149–151 ◦C; IR (KBr, disc) νmax 2925, 1686, 1616, 1601, 1511, 1253, 1174, 1098 cm− 1; 1H NMR (500 MHz, CDCl3) δ 8.20 (s, 1H), 8.12 (d, 2H, J = 8.6 Hz), 7.87 (d, 2H, J = 8.6 Hz), 7.70 (d, 1H, J = 15.9 Hz), 7.66 (d, 1H, J = 15.9 Hz), 7.57 (d, 2H, J = 8.7 Hz), 7.19 (d, 1H, J = 8.3 Hz), 7.15 (s, 1H), 7.11 (d, 1H, J = 8.3 Hz), 6.98–6.91 (m, 4H), 5.42 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 2.65 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 196.6, 188.7, 161.5, 149.48, 149.47, 142.9, 142.6, 139.8, 136.9, 130.1, 128.7, 127.4, 124.2, 123.0, 122.7, 121.1, 120.1, 114.4, 113.3, 110.3, 62.7, 55.9, 55.4, 26.7; HRMS (ESI-TOF) calcd for C30H28N3O5 [M+H]+ 510.2029, found 510.2004.
3.3.5. (1E,4E)-1-(3-methoxy-4-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one (5c)
The reaction of 4 with 1-azido-4-nitrobenzene (49 mg, 0.30 mmol) for 31 h following general procedure above yielded compound 5c (90 mg, 70 % yield) as an orange solid. Rf value 0.23 (hexane/ethyl acetate = 1/1); m.p. 191–193 ◦C; IR (KBr, disc) νmax 3099, 3014, 2957, 1643, 1598, 1511, 1340, 1270, 1173, 1030, 854 cm− 1; 1H NMR (500 MHz, DMSO‑d6) δ 9.20 (s, 1H), 8.47 (d, 2H, J = 8.9 Hz), 8.26 (d, 2H, J = 8.9 Hz), 7.76–7.74 (m, 3H), 7.71 (d, 1H, J = 7.2 Hz), 7.44 (s, 1H), 7.35 (d, 1H, J= 8.5 Hz), 7.28 (d, 1H, J= 8.4 Hz), 7.26–7.20 (m, 2H), 7.02 (d, 2H, J = 8.5 Hz), 5.32 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H); 13C NMR (126 MHz, DMSO‑d6) δ 188.2, 161.3, 149.5, 149.2, 146.8, 144.2, 142.6, 142.3, 140.8, 130.4, 128.3, 127.4, 125.7, 124.2, 123.8, 123.5, 123.0, 120.8, 114.5, 113.3, 110.8, 61.4, 55.6, 55.4; HRMS (ESI-TOF) calcd for C28H25N4O6 [M+H]+ 513.1774, found 513.1775.
3.3.6. 4-(4-((2-methoxy-4-((1E,4E)-5-(4-methoxyphenyl)-3-oxopenta1,4-dien-1-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (5d)
The reaction of 4 with 4-azidobenzoic acid (49 mg, 0.30 mmol) for 30 h following general procedure above, followed by purification using silica gel column chromatography (n-hexane/ethyl acetate= 3/1 to 1/3) yielded compound 5d (114 mg, 89 % yield) as a yellow solid. Rf value 0.18 (n-hexane/ethyl acetate = 1/1 + a drop of glacial acetic acid); m.p. 151–154 ◦C; IR (KBr, disc) νmax 3526, 3150, 3078, 2939, 2838, 1713, 1641, 1600, 1511, 1422, 1255, 1173 cm− 1; 1H NMR (500 MHz, DMSO‑d6) δ 9.02 (s, 1H), 8.32 (s, 2H), 7.73–7.68 (m, 4H), 7.55–7.18 (m, 6H), 7.00 (d, 2H, J = 7.7 Hz), 6.93 (m, 1H), 5.24 (s, 2H), 3.80 (s, 6H); 13C NMR (126 MHz, DMSO‑d6) δ 188.1, 170.4, 161.1, 149.4, 149.1, 143.7, 142.0, 139.3, 130.3, 128.1, 127.3, 124.1, 123.3, 122.9, 114.4, 113.1, 110.6, 61.2, 55.5, 55.3; HRMS (ESI-TOF) calcd for C29H26N3O6 [M+H]+ 512.1822, found 512.1835.
3.3.7. Ethyl 4-(4-((2-methoxy-4-((1E,4E)-5-(4-methoxyphenyl)-3oxopenta-1,4-dien-1-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoate (5e)
The reaction of 4with ethyl 4-azidobenzoate (57 mg, 0.30 mmol) for 48 h following general procedure above yielded compound 5e (94 mg, 70 % yield) as a yellow solid. Rf value 0.47 (n-hexane/ethyl acetate= 1/ 1); m.p. 148 − 150 ◦C; IR (KBr, disc) νmax 3151, 3065, 2976, 2837, 1705, 1646, 1600, 1510, 1264, 1172 cm− 1; 1H NMR (500 MHz, CDCl3) δ 8.20 (m, 3H), 7.83 (d, 2H, J= 8.5 Hz), 7.70 (d, 1H, J = 15.9 Hz), 7.66 (d, 1H, J = 16.0 Hz), 7.57 (d, 2H, J = 8.6 Hz), 7.19 (d, 1H, J = 8.1 Hz), 7.15 (s, 1H), 7.11 (d, 1H, J= 8.2 Hz), 6.95 (m, 4H), 5.42 (s, 2H), 4.41 (q, 2H, J= 7.1 Hz), 3.94 (s, 3H), 3.85 (s, 3H), 1.42 (t, 3H, J= 7.1 Hz); 13C NMR (126 MHz, CDCl3) δ 188.7, 165.3, 161.5, 149.5, 142.9, 142.6, 139.8, 131.3, 130.7, 130.1, 128.7, 127.4, 124.2, 123.0, 122.7, 121.1, 119.9, 114.4, 113.3, 110.3, 62.7, 61.5, 55.9, 55.4, 14.3; HRMS (ESI-TOF) calcd for C31H30N3O6 [M+H]+ 540.2135, found 540.2150.
3.3.8. 4-(4-((2-methoxy-4-((1E,4E)-5-(4-methoxyphenyl)-3-oxopenta1,4-dien-1-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzenesulfonamide (5f)
The reaction of 4 with 4-azidobenzenesulfonamide (59 mg, 0.30 mmol) for 48 h following general procedure above, followed by purification using silica gel column chromatography (n-hexane/ethyl acetate = 1/1 to 1/5) yielded compound 5f (73 mg, 54 % yield) as a yellow solid. Rf value 0.20 (n-hexane/ethyl acetate = 1/2); m.p. 201 − 203 ◦C; IR (KBr, disc) νmax 3348, 3250, 3162, 3100, 2923, 2852, 1649, 1599, 1509, 1335, 1253, 1157 cm− 1; 1H NMR (500 MHz, DMSO‑d6) δ 9.10 (s, 1H), 8.16 (d, 2H, J = 8.5 Hz), 8.03 (d, 2H, J = 8.6 Hz), 7.76–7.70 (m, 4H), 7.57 (s, 2H), 7.44 (s, 1H), 7.35 (d, 1H, J = 7.3 Hz), 7.30–7.21 (m, 3H), 7.02 (d, 2H, J= 8.6 Hz), 5.31 (s, 2H), 3.84 (s, 3H), 3.82 (s, 3H); 13C NMR (126 MHz, DMSO‑d6) δ 188.6, 161.7, 150.0, 149.6, 144.4, 144.3, 143.0, 142.7, 139.0, 130.8, 128.7, 128.0, 127.8, 124.6, 124.0, 123.9, 123.5, 120.9, 114.9, 113.6, 111.2, 61.8, 56.0, 55.8; HRMS (ESI-TOF) calcd for C28H27N4O6S [M+H]+ 547.1651, found 547.1657.
3.4. Procedure for antioxidant evaluation
Antioxidant evaluation using the DPPH method was performed according to our previous work [70]. A total of 1 mL of DPPH solution (4 mg/100 mL ethanol) was added to 2 mL of test solution (0.5 mg/mL ethanol) and mixed thoroughly. The mixture was then incubated for 30 minutes under dark conditions. Changes or fading in the purple color intensity of the DPPH solution were observed, and the absorbance of the solution was measured at 517 nm by a UV–Vis spectrophotometer. In this experiment, ascorbic acid was used as a positive control. Absorbance measurements for each test solution were conducted in triplicate. The obtained absorbance data were compared with the DPPH blank to determine the percentage of inhibition for each test compound.
3.5. Procedure for cytotoxic evaluation
Cytotoxic evaluation against MCF-7 cell lines with MTT assay was performed according to previous report [74]. MCF-7 cells (ATCC HTB 22) were grown at a concentration of 5000 cells in 100 μL of growth medium (RPMI1640 supplemented with 20 % Fetal Bovine Serum/FBS and 1 % penicillin antibiotic). After the cells had 50 % confluence in the growth medium (incubated for 24 h at 37 ◦C), sample solutions were added with various concentrations (200, 100, 50, 25, 12.5 μM) and incubated for 48 h at 37 ◦C. The MTT assay was carried out by adding 10 μL of MTT (5 mg/mL) per well and incubated for 4 h at 37 ◦C. The formed formazan crystals dissolved in ethanol, and then absorbance was measured with an ELISA reader (Enzyme-linked immunosorbent assay) at a wavelength of 595 nm.
3.6. Procedure for molecular docking
The docking studies were designed based on a modification of our previous methodology [75,76] and were carried out by using MOE 2022.02 software [77,78]. The x-ray crystallography structures of tubulin (PDB ID: 4O2B [79]) was retrieved from the PDB website [80]. Since the PDB file consisted of multiple chains, we first deleted the unused chains and kept only chain A (α-tubulin) and chain B (β-tubulin) in the system. Then, the pre-docking preparation was performed by adding missing hydrogen atoms, deleting unnecessary solvents and ions, and capping the N- and C-termini using ACE and NME, respectively. The molecular structure of the ligand was then prepared using ChemDraw 23.1.1 software and converted into an.mdb file within MOE 2022.02. The drawn ligands were optimized using at a pH of 7.4 and minimized using the MMFF94x force field, with an RMS gradient of 0.001 kcal/mol⋅Å2. The docking studies were subsequently performed in the colchicine binding site, generating 100 binding conformations. The 10 top-ranked poses, based on the calculated binding affinities (ΔGbinding, shown in Kcal/mol), were selected. Binding interactions from the docking results were further analyzed using ‘Ligand Interaction’ feature in MOE 2022.02 and visualized with ChimeraX v1.8.0 software [81]. In the end, the best docking pose of each ligand for each protein was selected.
3.7. Procedure for physicochemical, ADME, and drug-likeness predictions study
The physicochemical properties, ADME and drug-likeness predictions of the synthesized compounds were computed using SwissADME software (https://www.swissadme.ch) provided by the Swiss Institute of Bioinformatics (SIB) [82], and the DruMAP web server (https://drumap.nibiohn.go.jp/) [83]. SwissADME primarily focuses on physicochemical properties, drug-likeness predictions, and some ADME predictions, such as gastrointestinal absorption and blood-brain barrier (BBB) permeability, while the remaining ADME predictions are performed using DruMAP.
4. Conclusions
The synthesis of unsymmetrical monocarbonyl curcumin analogs with 1,2,3-triazole scaffold was successfully carried out from vanillin precursor. Compound 5a − 5f were obtained as final products subjected to in vitro antioxidant and cytotoxic activity assay. Compound with carboxylic group (5d) exhibited good radical scavenging performance against DPPH radical. For cytotoxic evaluation, compounds 5d and 5f were denoted as the most active agents against MFC-7 cell lines. The docking studies reveal that both compounds 5d and 5f exhibit high binding affinity for tubulin, one of the key proteins in MCF-7 cells responsible for cancer cell proliferation, with predicted binding score of − 10.4 kcal/mol for compound 5d and − 10.0 kcal/mol for compound 5f, respectively. Furthermore, computational analysis of physicochemical properties and ADME prediction using SwissADME and DruMAP shows that all synthesized compounds have moderate oral bioavailability and decent ADME properties. Specifically, compound 5d exhibits high GI absorption and moderate intrinsic clearance, despite its low fraction of dose absorbed and reduced permeability due to the –COOH group. Conversely, compound 5f demonstrates a high fraction of dose absorbed and good permeability, although its elevated TPSA results in decreased bioavailability and low GI absorption. These results suggested that by synthesizing some unsymmetrical monocarbonyl curcumin analogs linked 1,2,3-triazole scaffold (especially 5d) would prove to be useful as molecules for the creation of new antioxidant and anticancer drugs. CRediT authorship contribution statement Bayu Ardiansah: Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Conceptualization. Ahmad Farhan: Writing – original draft, Software, Investigation, Formal analysis, Data curation. Novita Sari Nurhasanah: Software, Investigation, Formal analysis, Data curation. Mochammad Arfin Fardiansyah Nasution: Writing – original draft, Software, Investigation, Formal analysis, Data curation. Noordini M. Salleh: Writing – review & editing, Validation, Supervision. Kenji Mizuguchi: Writing – review & editing, Validation, Supervision. Antonius Herry Cahyana: Writing – review & editing, Supervision, Resources. Lina Mardiana: Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research was fully supported by the Directorate of Research and Development (Direktorat Riset dan Pengembangan), Universitas Indonesia through Hibah Publikasi International Terindeks (PUTI Q1 Research Grant) with contract number No. NKB-462/UN2.RST/ HKP.05.00/2023. The authors would like to thank Ms. Pratiwi Puji Lestari, M.Si. (Integrated Laboratory and Research Center, Universitas Indonesia) for recording proton and carbon NMR spectra, Mr. Azhar Darlan (Forensic Laboratory Center, POLRI) for measuring HRMS spectra, and Dr. Reiko Watanabe (Institute for Protein Research, Osaka University) for her contributions to engaging discussions and for providing valuable comments regarding DruMAP.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.cscee.2024.101031. Data availability Detailed procedures and spectral data are available in the Supplementary Material.
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