Novel Triazole‐Linked Xanthine‐Steroid Hybrids: Molecular Docking and In Vitro Evaluation Against α‐Glucosidase and MCF‐7 Cells

ABSTRACT

Triazole hybrids have emerged as versatile scaffolds exhibiting diverse pharmacological profiles. In this study, four novel xanthine steroid hybrids linked via a 1,2,3‐triazole moiety (4–7) were efficiently synthesized through copper(I)‐catalyzed azide alkyne cycloaddition. Their bioactive potential was investigated using molecular docking and in vitro assays targeting both metabolic and apoptotic pathways. Docking simulations were performed against the anti‐apoptotic protein BCL‐2 (isoform 1), associated with human breast adenocarcinoma cells, and α‐glucosidase, a key enzyme involved in carbohydrate hydrolysis. Among the series, compound 4 displayed the highest binding affinity toward α‐glucosidase and significant affinity toward BCL‐2. In vitro assays confirmed that compound 4 exhibited the most potent dual effect, demonstrating α‐glucosidase inhibition with an IC₅₀ value of 123.9 µM (95% CI: 119–129 µM) and an antiproliferative effect on MCF‐7 cells, reaching a maximum viability reduction of 21% ± 3% at 250 µM. The dual activity is largely attributed to the 1,2,3‐triazole linker, which facilitates stacking and critical hydrogen‐bonding interactions with its biological targets.

Xanthine–steroid hybrids linked by 1,2,3‐triazole are synthesized and evaluated for dual bioactivity. Docking and in vitro assays reveal that compound 4 exhibits concurrent alfa‐glucosidase inhibition and antiproliferative effects on breast cancer cells. The triazole linker promotes key interactions, highlighting a promising scaffold for integrated metabolic and anticancer therapeutic development.

1. Introduction

Diabetes mellitus and cancer represent two of the most critical global health challenges due to their significant contributions to morbidity, mortality, and long‐term disability worldwide [1, 2]. According to the World Health Organization (WHO) and the international Agency for Research on Cancer (IARC), while cancer remains the second leading cause of death globally, with an estimated 20 million new cases and 9.7 million fatalities reported in 2022 [2]. Recent studies have revealed a close relationship between metabolic disorders and cancer progression, where chronic hyperglycemia, inflammation, and altered cellular metabolism contribute to tumor development and resistance to therapy [3, 4]. Consequently, the search for multifunctional agents capable of modulating both metabolic and oncogenic pathways has attracted considerable attention in medicinal chemistry [5]. Among therapeutic targets associated with diabetes, α‐glucosidase plays a key role in carbohydrate digestion by catalyzing the final step of polysaccharide hydrolysis into absorbable monosaccharides [1, 4]. Inhibition of this enzyme effectively reduces postprandial hyperglycemia and represents an established strategy for the management of type 2 diabetes mellitus. Likewise, in cancer therapy, the anti‐apoptotic protein BCL‐2 has emerged as an important molecular target due to its involvement in cell survival, tumor progression, and chemoresistance [5]. Therefore, multifunctional compounds capable of simultaneously modulating α‐glucosidase activity and apoptosis‐related pathways constitute an attractive strategy for the treatment of complex diseases such as diabetes and cancer [5, 6, 7]. Accordingly, the rational design of small molecules targeting both α‐glucosidase and the anti‐apoptotic protein BCL‐2 has emerged as a promising multitarget approach for addressing the interconnected metabolic and oncogenic processes underlying these disorders [5, 6, 8]. For instance, 3,4‐di‐O‐caffeoylquinic acid exhibited both α‐glucosidase inhibitory activity and apoptosis‐mediated cytotoxicity in lung adenocarcinoma cells [9]. Similarly, derivatives of 1‐deoxynojirimycin (DNJ) have provided valuable mechanistic insights as dual‐target inhibitors, acting against both α‐glucosidase and the human colon cancer cell line HCT‐116. These compounds exhibit half‐maximal inhibitory concentration (IC₅₀) values in the ranges of 2.76–40.96 µM and 1.37–57.59 µM, respectively [10]. Xanthine derivatives constitute a privileged class of heterocyclic compounds displaying a broad spectrum of biological activities, including anticancer, anti‐inflammatory, antioxidant, and antimicrobial effects [11]. Among them, theophylline and theobromine have attracted considerable attention due to their ability to regulate intracellular cAMP levels and interfere with signaling pathways associated with proliferation and apoptosis. Notably, theobromine has been reported to suppress glioblastoma cell proliferation through modulation of PDE4, Akt/mTOR, and NF‐κB pathways, whereas theophylline and related methylxanthines have demonstrated pro‐apoptotic effects accompanied by downregulation of anti‐apoptotic mediators such as BCL‐2, highlighting their potential as scaffolds for anticancer drug development [11, 12, 13].

1,2,3‐Triazoles constitute robust heterocyclic scaffolds widely present in biologically active compounds, including pharmaceuticals, agrochemicals, and biomolecules [14, 15]. Due to their structural stability, aromaticity, and favorable electronic properties, they are extensively used in medicinal chemistry as versatile linkers to connect pharmacophores within hybrid molecules. Their heteroatom‐rich structure enables strong interactions with biological targets, often enhancing binding affinity and solubility [16]. Notably, triazole‐containing compounds have been widely explored as α‐glucosidase inhibitors, frequently exhibiting in vitro and in silico activities comparable to acarbose [17]. For example, 1,2,4‐triazole hydrazone derivatives have demonstrated variable inhibitory potency, while 1,2,3‐triazole derivatives of hydrochlorothiazide displayed IC₅₀ values around 379 µM [18, 19]. In addition, triazole‐based compounds have exhibited selective cytotoxicity against various cancer cell lines, displaying pro‐apoptotic and antiproliferative effects. For instance, certain 1,2,3‐triazole derivatives showed significant activity in leukemia cell lines while exhibiting lower efficacy against MCF‐7 breast cancer cells [20]. Conversely, aminonaphthoquinone triazole conjugates synthesized via click chemistry demonstrated notable cytotoxicity against MCF‐7 cells, with mechanisms involving cell cycle arrest [21]. Triazole–steroid hybrids represent an emerging class of multifunctional molecules that combine two bioactive scaffolds within a single framework. Such conjugates have demonstrated promising anticancer activity; for example, celastrol triazole derivatives exhibited activity against A549 cells (IC₅₀ = 54.94 µM) [22], while diosgenin–triazole hybrids showed enhanced potency (IC₅₀ = 5.54 µM) [23]. Furthermore, steroid conjugation has been shown to improve selectivity and reduce toxicity, as observed with estradiol‐based hybrids [24]. These compounds are typically synthesized via copper(I)‐catalyzed azide alkyne cycloaddition (CuAAC), a highly efficient and regioselective “click chemistry” approach [25, 26, 27]. Therefore, the combination of methylxanthine, triazole, and steroid pharmacophores within a single molecular framework may provide synergistic biological effects through complementary mechanisms of action, potentially enhancing both enzyme inhibition and antiproliferative activity.

Based on these considerations, this study focuses on the synthesis of novel triazole‐linked xanthine–steroid hybrids using CuAAC methodology. Their inhibitory potential against α‐glucosidase and BCL‐2 was evaluated through molecular docking studies, while their biological activity was assessed via in vitro assays, including α‐glucosidase inhibition and cytotoxicity against the MCF‐7 cell line. Specifically, estrone‐based compounds (4 and 5) and estradiol‐derived hybrids (6 and 7) were investigated to determine their dual therapeutic potential.

2. Results and Discussion

2.1. Chemistry

The synthetic route was designed following a convergent strategy that enabled the independent preparation of steroidal alkyne and azide‐functionalized methylxanthine building blocks prior to their coupling through click chemistry (Schemes 1 and 2). The steroidal precursor was obtained by O‐propargylation of the phenolic hydroxyl group of estrone. Accordingly, reaction of estrone with propargyl bromide in the presence of sodium hydride in anhydrous DMF afforded the corresponding propargyl ether 3 in 78% yield (Scheme 1).

SCHEME 1.

Synthesis of 3‐O‐Propargylestrone (3).

SCHEME 2.

Synthesis of triazole‐estrone and triazole‐estradiol conjugates.

Organic azides 1 and 2 were synthesized from the commercially available methylxanthines theophylline and theobromine through a two‐step sequence involving N‐alkylation and subsequent azidation. Alkylation with 1,3‐dibromopropane under basic anhydrous conditions generated the corresponding bromopropyl intermediates, which were used without isolation and directly converted into the desired azides by nucleophilic substitution with sodium azide (Scheme 2). Subsequent copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC) under Meldal conditions enabled the efficient assembly of the target steroidal hybrids. The reaction proceeded smoothly and with complete regioselectivity, affording the corresponding 1,4‐disubstituted triazoles. Thus, estrone‐derived hybrids 4 and 5 were obtained in 90% and 83% yields, respectively. Subsequent selective reduction of the C‐17 carbonyl group with NaBH₄ furnished the corresponding estradiol analogues 6 and 7 in 90% and 80% yields, respectively. The uniformly high yields obtained throughout the cycloaddition and reduction steps highlight the efficiency and robustness of the synthetic methodology. Overall, the CuAAC‐based approach proved to be a versatile platform for connecting steroidal and methylxanthine pharmacophores through a triazole linker, providing straightforward access to structurally diverse molecular hybrids for subsequent biological evaluation.

2.2. Spectroscopic Characterization of the Synthesized Compounds

The ¹H and ¹³C NMR spectral assignment for the steroidal fragments of compounds 4 to 7 was performed by comparison with the chemical shifts reported for estradiol [28] and estrone [29]. In turn, the assignment for azide 1 and 2 was done by comparison with the data reported for theophylline [30] and theobromine [31]. Additionally, the assignments for all compounds were compared with the spectra calculated by ACD/Lab showing good agreement (Table 1). The formation of the triazole fragment after the click reaction was confirmed by the presence of a signal around 145.0 ppm for C‐20 and 122.8 ppm for C‐21, and the two methyl groups corresponding to the theophylline and theobromine precursor. Also, the ¹H NMR spectra of azides 1 and 2, as well as triazol derivatives 4 to 7, show two singlets between 3.33–3.98 ppm for H‐30 and H‐31, two signals around 30.0 ppm in ¹³C corresponding to the methyl groups at C‐30 and C‐31 and the signal corresponding to C‐25 from 7.46 to 7.65 ppm in ¹H and 106.0 to 108.0 ppm in ¹³C. Finally, the reduced triazole hybrids 6 and 7 were unambiguously determined by the presence of a triple signal between 3.65–3.73 ppm corresponding to the H‐17 proton.

TABLE 1.

¹³C NMR chemical shifts for compounds 1‐7 in CDCl₃ at 126 MHz.

Carbons	Estradiol1	Estrone2	Theophylline3	Theobromine4	1	2	4	5	6	7
C‐1	127.05	127,0					126.5	126.4	126.5	126.4
C‐2	113.62	113.0					112.3	112.4	112.3	112.3
C‐3	155.96	156.0					156.2	156.3	156.1	156.2
C‐4	116.00	116.0					114.8	114.8	114.8	114.8
C‐5	138.47	138.0					138.0	137.8	138.3	138.1
C‐6	a	30.1					31.1	31.6	30.7	30.6
C‐7	28.18	27.4					26.5	26.5	27.3	27.2
C‐8	40.08	39.0					38.3	38.3	38.9	38.8
C‐9	44.96	45.0					44.0	44.0	44.0 a	44.0
C‐10	132.13	132.0					132.8	132.6	133.5	133.2
C‐11	27.30	26.4					25.9	25.9	26.4	26.3
C‐12	37.79	36.0					35.9	29.7	36.8	36.7
C‐13	44.09	48.0					48.0	48.0	46.7	48.3
C‐14	50.98	51.0					50.4	50.4	50.1	50.1
C‐15	23.84	22.3					21.6	21.6	23.2	23.1
C‐16	31.06	32.5					43.9	35.9	43.4	43.3
C‐17	81.89	219.0					220.9	221.0	81.9	81.9
C‐18	11.68	14.0					14.0	13.9	11.2	11.1
C‐19							62.0	62.1	62.1	62.1
C‐20							145.0	144.3	145.2	141.8 a
C‐21							122.9	122.8	122.9	122.8
C‐22					45.3	49.2	46.7	48.3	44.1 a	48.3
C‐23					28.1	27.6	28.1	28.9	28.1	28.9
C‐24					32.7	38.9	31.6	38.6	31.2	38.6
C‐25			140.2	138.0	141.7	141.6	142.0	141.8	142.0	141.8 a
C‐26			106.3	110.0	106.8	107.6	106.7	107.6	106.8	107.6
C‐27			154.3	156.0	155.2	155.2	155.2	155.2	155.3	155.2
C‐28			151.1	152.3	151.7	151.4	151.6	151.5	151.7	151.5
C‐29			147.9	143.0	149.4	148.9	149.3	148.9	149.5	149.0
C‐30			27.6	32.7	29.6 a	33.6	29.7 a	33.7	29.9 a	33.6
C‐31			29.1	28.4	29.9 a	29.7	29.9 a	29.8	30.0 a	29.8

^aCarbons may be interchanged.

2.3. Molecular Docking Studies

The binding energies obtained from the molecular docking studies are summarized in Table 2. Among the evaluated compounds, hybrid 4 exhibited the most favorable binding affinity toward α‐glucosidase and the second most favorable affinity toward BCL‐2 (isoform 1), suggesting a promising dual‐target profile. Docking analysis of compounds 4–7 against α‐glucosidase (Figure 1a) revealed that compound 4 established hydrogen‐bond interactions with residues ARG442 and ARG315, together with additional contacts involving SER311, PRO312, ASP307, SER304, and VAL308. The extensive interaction network observed within the catalytic site may contribute to the enhanced binding affinity predicted for this derivative. In the case of BCL‐2 (Figure 1b), compound 4 showed an interaction with residues GLN:190, THR:7, ASP:10, and TYR:9 via strong hydrogen bonds, while weaker hydrogen bond interactions were observed with residues ALA:4 and ARG:6. In both cases, compound 4 exhibits stronger binding affinity (more negative binding energies) compared to its respective positive control. Regarding α‐glucosidase, acarbose presented a binding energy of −8.2 kcal/mol, whereas compound 4 maintained an energy of −10.8 kcal/mol. For BCL‐2, doxorubicin (DOX) showed a binding energy of −7.6 kcal/mol compared to −8.3 kcal/mol for compound 4. These findings are consistent with the principle that lower binding energies and extensive interaction networks increase the likelihood of biological activity [32].

TABLE 2.

Molecular docking parameters and binding energies of compounds 4‐7 against α‐glucosidase and BCL‐2 targets.

Compounds	Bonding energy Kcal/mol
	BCL‐2	RMSD	α‐glucosidase	RMSD
Controls	−7.6	0.1194	−8,2	0.1222
4	−8.3	0.6753	−10.8	0.8888
5	−8.0	0.2183	−10.1	0.3185
6	−7.9	1.7938	−10.1	0.1219
7	−8.0	0.5556	−9.8	0.4074

FIGURE 1.

2D protein‐ligand interaction diagrams for compound 4. (a) In α‐glucosidase, the binding mode involves a cooperative network of conventional hydrogen bonds and alkyl‐type interactions. (b) In BCL‐2, the hybrid establishes predominantly conventional hydrogen interactions with the catalytic pocket residues.

2.4. Evaluation of the Inhibitory Activity of α‐Glucosidase

To evaluate the inhibitory effect of compounds 4‐7 on α‐glucosidase activity, assays were conducted using 500 µM of acarbose as a positive control and 250 µM of each synthesized hybrid. The results obtained demonstrated that compound 4 achieved a significant inhibition of 84.4% ± 2.9% inhibition. In contrast, compounds 5, 6 and 7 exhibited notably lower activities of 36.6% ± 1.4 %, 6.1% ± 1.6 %, and 9.7% ± 5.1% respectively (Table 3).

TABLE 3.

Percentage of α‐glucosidase inhibition by compounds 4‐7 (250 µM) and acarbose as a reference control (500 µM).

Compounds	Inhibition %
Acarbose (control)	49.7% ± 4.5%
4	84.4% ± 2.9%
5	36.6% ± 1.4%
6	6.1% ± 1.6%
7	9.7% ± 5.1%

Based on these preliminary findings, the half maximal inhibitory concentration (IC₅₀) for α‐glucosidase was determined for all compounds using concentration range from 15.6 to 250 µM. Compound 4 yielded a value of 123.9 µM (95% confidence interval [CI]; 119 – 129 µM) (Figure 2). The remaining compounds did not reach the threshold within the tested concentration range (Figures S13–S15). Higher concentrations were not evaluated due to solubility limits of the compounds and the requirement to maintain DMSO concentrations below 1%, a commonly recommended threshold to minimize interference with enzymatic activity, as applied in α‐glucosidase inhibition assays [33, 34].

FIGURE 2.

 Alpha glucosidase inhibitory activity and estimation of IC₅₀ value of compound 4 across a concentration range of 15.6–250 µM. Data are presented as mean ± SD from three independent experiments (n = 4 replicates per experiment). Statistical significance was assessed via two‐way ANOVA followed by Dunnett's multiple comparisons test against the control (*p < 0.0001).

The inhibition of α‐glucosidase activity has been widely explored using natural extracts; however, many of these require concentrations 10–20 times higher than Acarbose to achieve comparable efficacy [35], which limits their pharmacological viability. In our study, the results obtained by the compound 4 is comparable to certain steroidal derivatives. For instance, cholestane type derivatives isolated from Coffea canephora husk showed IC₅₀ and other hydroxycinnamic acid rich extracts have shown values ranging from 61.5 to 250 µM. These findings suggest that the steroidal scaffold represents a privileged structure for enzyme inhibition [36]. Its rigid hydrophobic core likely facilitates interactions within the hydrophobic pockets of the catalytic site or adjacent allosteric regions [37]. Consequently, future kinetic are warranted to further elucidate the precise inhibitory mechanisms of these hybrids.

On the other hand, the limited activity observed for compounds 5–7 can be rationalized by the specific spatial arrangement of hydroxyl groups, aromatic rings, and conjugated systems, which are essential for facilitating hydrogen bonding and π–π interactions with key residues in the enzyme´s active site [38]. The absence or unfavorable positioning of these functional groups likely reduces binding affinity, explaining the weak inhibition profiles observed for these compounds. Furthermore, subtle structural modifications have been shown to drastically enhance inhibitory potency, as evidenced by halogenated flavonoids and other derivatives that exhibit significantly improved values. For instance, the synthesis of brominated flavonoid derivatives has yielded compounds such as 8‐bromoquercetin, which demonstrates inhibitory activity up to thirty‐seven times greater than the standard drug acarbose [39].

2.5. Cytotoxicity of Compounds 4–7 Against MCF‐7 Breast Cancer Cells

In the cytotoxicity assays, the maximum evaluable concentration was limited to 250 M due to the solubility constraints of the compounds in DMSO. To account for potential vehicle induced toxicity, cell viability percentages were strictly normalized against their respective DMSO controls (1% for 250 M and 0.5% for 125 M). Based on these parameters, compound 4 was the only derivative to exhibit a concentration dependent cytotoxic profile. Statistical analysis via two‐way ANOVA followed by Dunnett's multiple comparisons test revealed that compound 4 demonstrated a statistically significant difference compared to the control starting at 15.6 M (*p < 0.05) within the responding replicates. The onset of a prominent biological effect was consolidated at 62.5 M (**p < 0.0001), reaching a maximum average viability reduction of 21% ± 3% at 250 M (Figure 3). Notably, this average profile reflects distinct inter‐assay variability; specifically, one of the three independent biological trials‐maintained baseline viability across all concentrations, thereby flattening the overall mean inhibition. According to the ISO 10993‐5:2009 standard, a substance is classified as cytotoxic only if it induces a reduction in cell viability equal to or greater than 30% [40]. Consequently, all four evaluated hybrids (4–7) are formally classified as noncytotoxic under the tested conditions, as the maximum average inhibition remained safely below the established regulatory threshold. The atypical absorbance fluctuations and high standard deviations observed for compounds 5, 6, and 7 at intermediate and high concentrations likely represent experimental variation or compound interference rather than a functional biological response. While all derivatives are noncytotoxic by official criteria, compound 4 stands out as the only derivative to display a discernible antiproliferative trend. These results align with previous studies on triazole‐containing estrone and estradiol analogs, which often require specific structural modifications or higher concentrations to elicit potent apoptotic responses in MCF‐7 cells [41]. Further trials would be required to fully establish the consistency of this trend, given the observed inter assay discrepancy.

FIGURE 3.

Cell viability of MCF‐7 cells treated with compounds 4–7 (7.8–250 µM) for 48 h. Panels show the effects of (a) compound 4, (b) compound 5, (c) compound 6, and (d) compound 7. Bars represent the mean ± SD of three independent biological experiments (n = 3 technical replicates), illustrating inter assay variability. Viability was normalized to control (100%). Statistical significance was determined by two‐way ANOVA followed by Dunnett's post‐hoc test against the control (* p < 0.05, p < 0.01, *** p < 0.001, **** p < 0.0001).

The concentrations tested in this study align with reports where various steroidal derivatives with heterocyclic substitutions did not exhibit significant antiproliferative activity at concentrations below 50 µM, often showing values exceeding 100 µM [42]. Similar trends have been observed when evaluating sarsapogenin derivatives with heterocycles, which lacked antiproliferative activity below 100 µM [43]. However, estradiol derived compounds have been reported to be highly successful against skin cancer (B16‐F10) [44]. In contrast, estradiol derived compounds have been successfully employed as antitumor agents in various cancer models [24]. Consequently, the results obtained suggest that these hybrids could serve as valuable scaffolds for further optimization. Previous studies have highlighted the use of estradiol derivatives as auxiliary agents due to their high affinity for MCF‐7 cells, which has led to combinations with other potent anticancer drugs to enhance cytotoxic effect [41]. While a positive reference drug was not experimentally evaluated in this initial screening (representing a limitation) these results establish the necessary groundwork for future side‐by‐side evaluations against standard reference drugs.

Finally, our findings identify compound 4 as a novel agent with a dual effect: the inhibition of α‐glucosidase activity and a discernible antiproliferative effect on MCF‐7 cells, correlating with the molecular docking predictions. The inhibitory activity of compound 4 is attributed to its hybrid structure, which effectively balances hydrophobicity and hydrogen bonding capacity. The steroidal core likely promotes strong hydrophobic interactions within the α‐glucosidase enzyme, while the carbonyl and hydroxyl groups enable hydrogen bonding with key catalytic residues [45]. For BCL‐2, the rigid scaffold of compound 4 may facilitate binding within the hydrophobic BH3 groove, where polar groups reinforce ligand anchoring and potentially disrupt anti‐apoptotic function [46]. Similar dual‐target trends have been reported for natural antioxidants such as 3,4‐di‐O‐caffeoylquinic acid, which demonstrates both α‐glucosidase inhibition and the induction of apoptosis in adenocarcinoma cell lines [9]. Furthermore, derivatives of 1‐deoxynojirimycin (DNJ) have provided valuable mechanistic insights as dual‐target inhibitors, acting against both α‐glucosidase and the human colon cancer cell line HCT‐116. These compounds exhibit IC₅₀ values in the ranges of 2.76–40.96 µM and 1.37–57.59 µM, respectively. DNJ derivatives have provided mechanistic insights into this interplay; specifically, a tegafur containing derivative induces apoptosis in HCT‐116 cells through mitochondrial dysfunction and oxidative stress, accompanied by downregulation of BCL‐2 and upregulation of Bax expression [10]. Further studies are necessary to fully elucidate the inhibitory mechanisms of compound 4 and to enhance its dual efficacy.

3. Materials and Methods

3.1. Chemistry

Commercially available reagents were purchased from Sigma Aldrich and used without previous purification. Theophylline and theobromine were commercially available. The solvents were commercially available and were distilled under argon atmosphere from sodium and benzophenone (THF) or calcium hydride (CH₂Cl₂, DMF). Oxidation reactions were carried out in a sealed tube and heated at 140°C using an oil bath under air atmosphere and solvents that were not anhydrous. The temperature was regulated using a digital contact thermometer. Unless otherwise indicated, reactions sensitive to air or moisture were carried out under argon atmosphere using oven dried glassware and freshly distilled dried solvents. Reactions were monitored by thin‐layer chromatography (TLC). Purifications of products were performed by column chromatography using silica gel (230‐400 mesh). Melting points are not corrected and were obtained with a FisherScientific 12144 melting point apparatus. IR spectra were recorded on an FT‐IR Bruker Tensor 27 spectrophotometer, by means of attenuated total reflection (ATR) technique, and all data is expressed as cm⁻¹. NMR spectra were acquired with a Bruker 500 MHz spectrometer and reported as δ values (ppm). High resolution mass spectra (HRMS) were recorded on AccuTOFTM Mass Spectrometer and acquired in electrospray ionization (ESI) mode using a TOF or FAB mode. Low resolution mass spectra were recorded on Agilent Technologies 6120 quadrupole LC/MS. Optical rotations were measured using a Perkin Elmer polarimeter.

3.1.1. General Methodology for the Synthesis of Alkyl Azido Xanthines

To a solution of theophylline or theobromine (1.00 g, 5.55 mmol) in DMF (8 mL) at 0°C was added NaH (60% dispersion in mineral oil; 0.26 g, 11.1 mmol) and 1,3‐dibromopropane (0.56 mL, 5.55 mmol) and allowed to stand for 10 min. After this time, the reaction mixture was stirred for 48 h at room temperature, then cooled to 0°C and H₂O (5 mL) was added dropwise. The organic phase was extracted with EtOAc (3 × 10 mL), the combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The crude product was used in the next reaction without further purification. To a solution of the crude reaction mixture in DMF (10 mL) was added NaN₃ (0.209 g, 4.98 mmol) and the reaction was stirred for 6 h at room temperature. The solvent was removed under reduced pressure and the product purified by column chromatography over silica gel with mixtures of hexane/EtOAc as eluent.

3.1.1.1. 7‐(3‐Azidopropyl)‐1,3‐dimethyl‐3,7‐dihydro‐1H‐purine‐2,6‐dione (1)

Eluent (hexane/EtOAc, 1:1). Compound 1 isolated: 0.57 g (65%) obtained as a yellow solid, m.p = 89°C–91°C. ¹H NMR (500 MHz, CDCl₃) δ: 7.64 (s, 1H, H‐25), 4.48 (t, J = 6.4 Hz, 2H, H‐24), 3.60 (s, 3H, H‐31), 3.41 (s, 3H, H‐30), 3.33 (t, J = 6.0 Hz, 2H, H‐22), 2.46 (tt, J = 6.4, 6.0 Hz, 2H, H‐23). ¹³C NMR (126 MHz, CDCl ₃) δ: 155.2 (C‐27), 151.7 (C‐28), 149.4 (C‐29), 141.7 (C‐25), 106.8 (C‐26), 45.3 (C‐22), 32.7 (C‐24), 29.9 (C‐31), 29.6 (C‐30), 28.1 (C‐23). FT‐IR/ATR v_max (cm⁻¹) 3111, 2101, 1653, 1542, 1402, 1364, 1220, 1183,1025, 761. MS Calculated for C₁₀H₁₄N₇O₂ [M + H]⁺ = 264.1, found = 264.1.

3.1.1.2. 1‐(3‐Azidopropyl)‐3,7‐dimethyl‐3,7‐dihydro‐1H‐purine‐2,6‐dione (2)

Eluent (hexane/EtOAc, 1:2). Compound 2 isolated: 0.52 g (60%) obtained as a white solid, m.p = 77°C–79°C. ¹ H NMR (500 MHz, CDCl₃) δ: 7.46 (s, 1H, H‐25), 4.04 (t, J = 6.9 Hz, 2H, H‐24), 3.93 (s, 3H, H‐30), 3.51 (s, 3H, H‐31), 3.32 (t, J = 6.8 Hz, 2H, H‐22), 1.90 (q, J = 6.9 Hz, 2H, H‐23). ¹³ C NMR (126 MHz, CDCl₃) δ: 155.2 (C‐27), 151.4 (C‐28), 148.9 (C‐29), 141.6 (C‐25), 107.6 (C‐26), 49.4 (C‐22), 38.9 (C‐24), 33.6 (C‐30), 29.7 (C‐31), 27.6 (C‐23). FT‐IR/ATR v_max (cm⁻¹) 3120, 2093, 1370, 16461, 1459, 1364, 1182, 1074, 1025, 763. MS Calculated for C₁₀H₁₄N₇O₂ [M + H]⁺ = 264.1, found = 264.2.

3.1.1.3. 3‐O‐Propargylestrone (3)

To a solution of estrone (0.100 g, 0.3699 mmol) in THF (10 mL) was added NaH (60% dispersion in mineral oil; 0.03 g, 0.6796 mmol) and propargyl bromide (solution 80 wt. % in toluene; 0.06 mL (0.6796 mmol) at 0°C. After 10 min, the reaction mixture was stirred for 12 h at 60°C on an oil bath. Then, the reaction mixture was cooled to room temperature and H₂O (5 mL) was added dropwise. The organic phase was extracted with EtOAc (3 × 10 mL), the combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product was purified by chromatography on silica gel (hexane/EtOAc, 10:1) to give 0.089 g (78%) as a white solid, m.p. = 130°C–133°C, (Lit [47]:132°C–134°C). The physical properties and the 1H and 13C NMR spectra of the product agree with data previously reported [47] Scheme 1.

3.1.2. General Procedure for Synthesis of Triazole‐Estrone Conjugates 4 and 5

A mixture of the CuI catalyst (10 mg) and EtOH/H₂O (3:1 v/v, 2 mL) was placed in a round‐bottom flask equipped with a magnetic stirrer. Propargyl estrone 3 (1.00 mmol), azido xanthine 1 or 2 (1.2 mmol), and sodium ascorbate (0.01 mmol) were then added sequentially. The reaction mixture was stirred under conventional thermal heating at 100°C until complete consumption of the starting materials, as monitored by TLC (typically 1–3 h). After completion, the reaction mixture was diluted with EtOAc and filtered through a short pad of silica gel to remove the catalyst. The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the desired triazole derivatives 4 and 5.

3.1.2.1. Theophylline–Triazole–Estrone (4)

Eluent (hexane/EtOAc, 1:2). Compound 4 isolated: 0.198 g (90%) as a yellow solid, m.p = 130°C–132°C. ${[\alpha ]}_{\mathrm{D}}^{20}$ = +51.5 (c = 1.00, CHCl₃). ¹H NMR (500 MHz, CDCl₃) δ: 7.62 (s, 1H, H‐21), 7.60 (s, 1H, H‐25), 7.13 (d, J = 8.6 Hz, 1H, H‐1), 6.72 (dd, J = 8.6, 2.7 Hz, 1H, H‐2), 6.66 (d, J = 2.7 Hz, 1H, H‐4), 5.12 (s, 2H, H‐19), 4.30 (t, J = 6.5 Hz, 2H, H‐24), 4.26 (t, J = 6.5 Hz, 2H, H‐22), 3.52(s, 3H, H‐31), 3.34 (s, 3H, H‐30), 2.81 (m, 2H, H‐23), 0.83 (s, 3H, H‐18).¹³C NMR (126 MHz, CDCl₃) δ 220.9 (C‐17), 156.2 (C‐3), 155.2 (C‐27), 151.6 (C‐28), 149.3 (C‐29), 145.0 (C‐20), 142.0 (C‐25), 138.0 (C‐5), 132.8 (C‐10), 126.5 (C‐1), 122.9 (C‐21), 114.8 (C‐4), 112.3 (C‐2), 106.7 (C‐26), 62.0 (C‐19), 50.4 (C‐14), 48.0 (C‐13), 46.7 (C‐22), 44.0 (C‐9), 43.9(C‐16), 38.3 (C‐8), 35.9 (C‐12), 31.6 (C‐24), 31.1 (C‐6), 29.9 (C‐30), 29.7 (C‐31), 28.1 (C‐23), 26.5 (C‐7), 25.9 (C‐11), 21.6 (C‐15) and 14.0 (C‐18). FT‐IR/ATR v_max(cm⁻¹): 2921, 2851,1702, 1655, 1459, 1246. HRMS (ESI‐ TOF) Calculated for C₃₁H₃₈N₇O₂ [M + H]⁺ = 572.2985, found = 572.2936.

3.1.2.2. Theobromine–Triazole–Estrone (5)

Eluent (hexane/EtOAc, 1:2). Compound 5: isolated 0.160 g (83%) of as a yellow solid, m.p. = 92°C–93°C. ${[\alpha ]}_{\mathrm{D}}^{20}$ = +38.0 (c = 1.00, CHCl₃) ¹H NMR (500 MHz, CDCl₃) δ 7.79 (s, 1H, H‐21), 7.54 (s, 1H, H‐25), 7.20 (d, J = 8.6 Hz, 1H, H‐1), 6.80 (dd, J = 8.6, 2.8 Hz, 1H, H‐2), 6.73 (d, J = 2.8 Hz, 1H, H‐4), 5.17 (s, 2H, H‐19), 4.44 (t, J = 7.1 Hz, 2H, H‐22), 4.13 (t, J = 6.5 Hz, 2H, H‐24), 3.89 (m, 3H, H‐30), 3.58 (s, 3H, H‐31), 2.89 (m, 2H, H‐23), 0.91 (s, 3H, H‐18). ¹³C NMR (126 MHz, CDCl₃) δ 221.0 (C‐17), 156.3 (C‐3), 155.2 (C‐27), 151.5 (C‐28), 148.9 (C‐29), 144.3 (C‐20), 141.8 (C‐25), 137.8 (C‐5), 132.6 (C‐10), 126.4 (C‐1), 122.8 (C‐21), 114.8 (C‐4), 112.4 (C‐2), 107.6 (C‐26), 62.1(C‐19), 50.4 (C‐14), 48.3 (C‐22), 48.0 (C‐13), 44.0 (C‐9), 38.6 (C‐24), 38.3 (C‐8), 35.9 (C‐16), 33.7 (C‐30), 31.6 (C‐6), 29.8 (C‐31), 29.7 (C‐12), 28.9 (C‐23), 26.5 (C‐7), 25.9 (C‐11), 21.6 (C‐15) and 13.9 (C‐18). FT‐IR/ATR v_max (cm⁻¹): 2911, 2855,1711, 1655, 1459, 1240. HRMS (ESI‐TOF). Calculated for C₃₁H₃₈N₇O₂ [M + H] ⁺ = 572.2985, found = 572.2938.

3.1.3. General Procedure for Synthesis of Triazole‐Estradiol Conjugates 6 and 7

To a solution of the triazole estrone 4 or 5 (0.156 g, 0.273 mmol) in EtOH (10 mL) was added NaBH₄ (0.021 g, 0.5457 mmol) and H₂O (0.5 mL) at 0°C. After 1.5 h, the reaction mixture was cooled to room temperature, and the organic phase was extracted with EtOAc (3 × 10 mL). The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure.

3.1.3.1. Theophylline–Triazole–Estradiol (6)

Eluent (hexane/EtOAc, 1:1). Compound 6 isolated: 0.178 g (90%) as a white solid, m.p. = 184°C–185°C. ${[\alpha ]}_{\mathrm{D}}^{20}$ = +12.0 (c 1.00, CHCl₃). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (s, 1H, H‐21), 7.59 (H‐25), 7.13 (d, J = 8.6 Hz, 1H, H‐1), 6.70 (dd, J = 8.7, 2.7 Hz, 1H, H‐2), 6.63 (d, J = 2.7 Hz, 1H, H‐4) 5.11 (s, 2H, H‐19), 4.29 (t, J = 6.4 Hz, 2H, H‐24), 4.26 (t, J = 6.7 Hz, 2H, H‐22), 3.65 (t, J = 8.5 Hz, 1H, H‐17), 3.52 (s, 3H, H‐31), 3.34 (s, 2H, H‐30), 2.77 (m, 2H, H‐23), 0.70 (s, 3H, H‐18). ¹³C NMR (126 MHz, CDCl₃) δ 156.1 (C‐3), 155.3 (C‐27), 151.7 (C‐28), 149.5 (C‐29), 145.2 (C‐20), 142.0 (C‐25), 138.3 (C‐5), 133.5 (C‐10), 126.5 (C‐1), 122.9 (C‐21), 114.8 (C‐4), 112.3 (C‐2), 106.8 (C‐26), 81.9 (C‐17), 62.1 (C‐19), 50.1 (C‐14), 46.7 (C‐13), 44.1 (C‐22*), 44.0 (C‐9*), 43.4 (C‐16), 38.9 (C‐8), 36.8 (C‐12), 31.2 (C‐24), 30.7 (C‐6), 30.0 (C‐31), 29.9 (C‐30), 28.1 (C‐23), 27.3 (C‐7), 26.4 (C‐11), 23.2 (C‐15) and 11.2 (C‐18). FT‐IR/ATR v_max (cm⁻¹): 3210, 3113, 1698, 1652, 1545, 1222. HRMS (ESI‐TOF) Calculated for C₃₁H₄₀ N₇ O₄ [M + H] ⁺ = 574.3142, found = 574.3134.

3.1.3.2. Theobromine–Triazole–Estradiol (7)

Eluent (hexane/EtOAc, 1:1). Compound 7 isolated: 0.125 g (80%) as a white solid, m.p. = 187°C–188°C. ${[\alpha ]}_{\mathrm{D}}^{20}$ = −41.7 (c 1.00, CHCl₃) ¹H NMR (500 MHz, CDCl₃) δ 7.78 (s, 1H, H‐21), 7.52 (s, 1H, H‐25), 7.20 (d, J = 8.6 Hz, 1H, H‐1), 6.78 (dd, J = 8.6, 2.8 Hz, 1H, H‐2), 6.71 (d, J = 2.8 Hz, 1H, H‐4), 5.16 (s, 2H, H‐19), 4.44 (t, J = 7.2 Hz, 2H, H‐22), 4.13 (t, J = 6.6 Hz, 2H, H‐24), 3.98 (s, 3H, H‐30), 3.73 (t, J = 8.5 Hz, 1H, H‐17), 3.57 (s, 3H, H‐31), 2,84 (m, 2H, H‐23), 0.78 (s, 3H H‐18,). ¹³C NMR (126 MHz, CDCl₃) δ 156.2 (C‐3), 155.2 (C‐27), 151.5 (C‐28), 149.0 (C‐29), 141.8 (C‐20 and C‐25), 138.1 (C‐5), 133.2 (C‐10), 126.39 (C‐1), 122.8 (C‐21), 114.8 (C‐4), 112.3 (C‐2), 107.6 (C‐26), 81.9 (C‐17), 62.1 (C‐19), 50.1 (C‐14), 48.3 (C‐13 and C‐22), 44.0 (C‐9), 43.3 (C‐16), 38.8 (C‐8), 38.6 (C‐24), 36.7 (C‐12), 33.6 (C‐30), 30.6 (C‐6), 29.8 (C‐31), 28.9 (C‐23), 27.2 (C‐7), 26.3 (C‐11), 23.1 (C‐15) and 11.1 (C‐18). FT‐IR/ATR v_max (cm⁻¹): 3200, 3120, 1695, 1642, 1535, 1215 HRMS (ESI‐TOF) Calculated for C₃₁H₄₀ N₇O₄ [M + H] ⁺ = 574.3142, found = 574.3145.

3.2. Molecular Docking

The computational binding assays and molecular docking parameters were adjusted following structural refinement methodologies previously documented in literature [48]. The anti‐apoptotic protein BCL‐2 (isoform 1), associated with human breast adenocarcinoma MCF‐7 cells (ATCC HTB‐22), was retrieved from the Protein Data Bank using PDB ID 1G5M, while PDB ID 3A4A was utilized for α‐glucosidase. Protein integrity analysis, visualization, and structure preparation were performed using Discovery Studio (BIOVIA, RRID:SCR_015651). Ligand geometries were sketched using ChemSketch and processed in FreeViewer (ACD/Labs, RRID:SCR_019272), followed by conversion to .pdb format in Discovery Studio. Compounds 4, 5, 6, and 7 were energy minimized to their lowest conformational energy state using Vega ZZ, applying 3000 conjugate gradient steps. Molecular docking analyses were conducted using AutoDock Vina (RRID:SCR_011958) via the MGLTools graphical interface, with an exhaustiveness value of 50. For BCL‐2, the grid box dimensions were set to 22 × 22 × 22 Å, centered at coordinates 4.670, 2.100, 5.080 [49]. For α‐glucosidase, a grid box of 26 × 26 × 26 Å was used, centered at 21.276, −0.752, 18.634. A grid spacing of 0.375 Å was applied to both targets. Residue ligand interaction analyses were carried out using Discovery Studio, focusing on hydrogen bonding interactions between the ligands and amino acid residues [37]. Acarbose and DOX were employed as positive controls for α‐glucosidase and BCL‐2 respectively.

The docking protocol was validated to ensure its reliability and predictive accuracy. For the α‐glucosidase (3A4A), a self‐docking (re‐docking) procedure was executed by comparing the docked native ligand against its co‐crystallized conformation [50]. A Root‐Mean‐Square Deviation (RMSD) threshold of ≤ 2.0 Å was established as the criterion for a valid docking setup. The re‐docking successfully reproduced the experimental crystal structure with an excellent RMSD value of 0.7777 Å (Figure 4). Since the BCL‐2 (1G5M) lacks a co‐crystallized ligand, validation was performed via cross‐docking with established reference inhibitors, ensuring that predicted complexes consistently replicated the key catalytic interactions and spatial orientations reported in the literature [49].

FIGURE 4.

Structural overlay comparing the co‐crystallized (red) and re‐docked (green) conformations of glucose within the active site of α‐glucosidase (PDB ID: 3A4A). The re‐docking procedure accurately reproduced the experimental position with an RMSD of 0.7777 Å, which is well within the standard validation threshold of 2.0 Å.

3.3. Sample Preparation

Four compounds (4, 5, 6, and 7) were prepared at a stock concentration of 27 mM in DMSO. These stocks were filtered through a 0.2 µm pore size filter. Serial dilutions were then prepared in complete culture medium at 500, 250, 125, 62.5, 31.3, and 15.6 µM with DMSO less than 0.1% at concentrations below 125 µM. For 500 and 250 µM, a control was taken with its respective DMSO concentration [51].

3.4. Biologic Maintenance

MCF‐7 cancer cells (ATCC HTB‐22, RRID:CVCL_0031) were used, which were routinely cultured in complete DMEM‐F12 medium (Sigma Aldrich) supplemented with antibiotic‐antifungal (penicillin‐streptomycin‐amphotericin; Gibco) and 10% fetal bovine serum (Gibco, Cat. 16000044). The cells were cultured in 75 cm² T‐flasks at 37°C and 5% CO₂ in a humidified incubator. For the assays, when the cells reached 90% confluence, they were washed with PBS (1X, Gibco), and 0.05% trypsin‐EDTA (Gibco) was added. After 5 min, medium was added to recover the supernatant with the cells. The supernatant was then centrifuged, and the cells were resuspended in culture medium for counting. Counting was performed using the trypan blue technique in a Neubauer counting chamber [52].

3.5. Cell viability of MCF‐7 Cancer Cells Measurement Using an MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) Assay

The initial in vitro screening assays were conducted without a positive reference pharmacological control, as this exploratory study focused on the preliminary evaluation of the novel hybrid scaffolds. To ensure methodological rigor and reproducibility, cell viability assays on the MCF‐7 breast cancer line were executed following standardized metabolic protocols, with results strictly normalized vehicle treated controls to establish a consistent experimental baseline [53]. This approach aligns with established frameworks for the early‐stage assessment of novel triazole based medicinal conjugates, where identifying relative biological trends across a synthesized series is prioritized.

The cells were seeded in 96‐well plates at a density of 10,000 cells/well in 100 µL of complete culture medium and incubated under standard conditions for 24 h to allow adhesion. Subsequently, 100 µL of medium containing the compounds (4‐7) were added to obtain final concentrations of 250, 125, 62.5, 31.3, 15.6, and 7.8 µM, and incubated for 48 h. Controls included cells with culture medium only and cells treated with the vehicle (DMSO) in percentages equivalent to those used in the samples. At the end of the treatment, 20 µL of an MTT solution (Sigma Aldrich, 5 mg/mL in PBS) were added to each well and incubated for 4 h. After this time, the supernatant was removed, and the formazan crystals were dissolved by adding 50 µL of DMSO. The absorbance was measured at 570 nm in a microplate reader. The percentage of viability was calculated by normalizing the data with respect to the absorbance of the controls with the corresponding percentage of DMSO. Cell viability was determined by MTT assay after 48 h of treatment at concentrations ranging from 7.8 to 250 uM. Data are expressed as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA followed by Dunnett's multiple comparisons test; *p < 0.0001 compared to the untreated control.

3.6. Evaluation of Inhibitory Activity in α‐glucosidase

The inhibitory capacity of compounds 4‐7 against the enzyme α‐glucosidase was evaluated following the protocols described by Cong et al. [33] and [54]. A maximum concentration of 250 µM was utilized for the test compounds, while Acarbose was employed as a positive control at 500 µM. The α‐glucosidase enzyme (Sigma Aldrich) was inoculated with the compounds of interest together with a positive control (Acarbose) in 96‐well plates for 5 min. Subsequently, the substrate p‐nitrophenylglucopyranose (PNPG; Sigma Aldrich) was added to reach a final reaction volume of 80 µL. Each well contained 20 µL 2 M phosphate buffer, 20 µL α‐glucosidase (0.2 U/mL), 20 µL inhibitor, and 20 µL of PNPG and incubated for 15 min. The same volume of 2 M sodium carbonate was then added and immediately read in the plate reader (Thermo Scientific) at 405 nm. To determine the half maximal inhibitory concentration (IC₅₀) for all compounds, a concentration range of 15.6, 31.7, 63.5, 125, 200 and 250 µM was evaluated. All assays were performed in at least triplicate to ensure reliability and reproducibility.

3.7. Statistics

Data are expressed as the mean ± standard deviation (SD) from at least three independent biological replicates (n = 4 for α‐glucosidase assay and n = 3 for cell viability). Statistical analyses were performed using GraphPad Prism 8 software. To evaluate the significance of the observed effects, a two‐way ANOVA was conducted, considering concentration and compound type as factors, followed by Dunnett's post‐hoc multiple comparisons test to determine differences against the untreated control (*p < 0.0001). The half maximal inhibitory concentration (IC₅₀) and its corresponding 95% confidence interval (CI) were determined through nonlinear regression analysis using curve fit model. This approach ensures a robust estimation of a potency by accounting for the minimum and maximum plateaus of the dose response relationship.

4. Conclusion

Among the synthesized estrone and estradiol derivatives, compound 4 exhibited the most promising dual biological profile, with an IC50 value of 123.9 µM against α‐glucosidase (95% CI: 119–129 µM). In addition, compound 4 reduced MCF‐7 cell viability by 21% ± 3% at 250 µM. Although the antiproliferative effect was moderate, these findings demonstrate measurable dual activity toward both α‐glucosidase inhibition and breast cancer cell growth suppression. Molecular docking studies indicated that structural modifications of the estrone and estradiol scaffolds in compounds 4–7 enhanced interactions within the BCL‐2 hydrophobic groove and the α‐glucosidase catalytic site. These results provide valuable insights into the structure‐activity relationships governing the biological behavior of triazole based xanthine steroid hybrids. Collectively, this work establishes a foundation for the rational optimization of dual‐target compounds with improved affinity toward BCL‐2 and enhanced α‐glucosidase inhibitory potency, both of which warrant further investigation.

Author Contributions

Synthesis and characterization: Elsie Ramírez‐Domínguez, Alma Sánchez‐Eleuterio, Delfino Chamorro, Guillermo E. Negrón, Rosa L. Santillan. Investigation and methodology: Elsie Ramírez‐Domínguez, Alma Sánchez‐Eleuterio, Delfino Chamorro, Guillermo E. Negrón, Alejandro Zepeda, Diego Martinez, Leydi Carrillo‐Cocom, Susana Rincón, Rosa L. Santillan, Leticia Lomas. Conceptualization and writing – original draft preparation: Elsie Ramírez‐Domínguez, Alma Sánchez‐Eleuterio, Delfino Chamorro, Guillermo E. Negrón, Alejandro Zepeda, Diego Martinez, Leydi Carrillo‐Cocom, Susana Rincón, Rosa L. Santillan. Funding acquisition and writing – review and editing: Alma Sánchez‐Eleuterio, Guillermo E. Negrón, Alejandro Zepeda, Diego Martinez, Leydi Carrillo‐Cocom, Susana Rincón, Rosa L. Santillan. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting information for this article is available on the WWW under https://doi.org/10.1002/cbdv.71531.

Supporting Information File 1: cbdv71531‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.