In this study, a group of 4-substituted benzoyltaurinamide derivatives were designed, synthesized, and investigated for their anticancer activity against three cancer cell lines and one nontumorigenic cell line by MTT assay. Among the final compounds, methoxyphenyl derivatives 14, 15, 16 were found to be effective against all the tested cancerous cell lines with promising selectivity. The most active compounds were further evaluated to determine the molecular mechanism of their anticancer activity by using western blot assay and the Annexin V-FITC/PI test. Compound 14 (in SH-SY5Y and MDA-MB-231 cell lines) and 15 (in SH-SY5Y cell line) were found to induce intrinsic apoptotic pathway by upregulating BAX, caspase-3, and caspase-9, while downregulating Bcl-2 and Bcl-xL expression levels. According to mechanistic studies, compounds displayed their anticancer activity via three different mechanisms: a. caspase-dependent, b. caspase-independent, and c. caspase-dependent pathway that excluded caspase-9 activation. As a result, this study provides interesting data which can be used to design new taurine-based anticancer derivatives. Since cancer is one of the leading causes of death worldwide, extensive resources have been invested in the development of increasingly potent anticancer agents for clinical use. Despite the advances, issues related to drug toxicity and resistance during treatment persist, thereby promoting researchers to develop more efficient, safer therapeutic agents [1–3].Cancer, characterized by the abnormal proliferation of genetically altered cells, exhibits certain hallmarks, namely, sustained proliferative signaling, evasion of growth suppressors, replicant immortality, resistance to programmed cell death (apoptosis), induction of angiogenesis, and invasion of healthy tissue [4]. Since apoptosis is the programmed cell death process responsible for the controlled eradication of damaged cells, it plays a crucial role in regulating tissue homeostasis, balancing cell death and survival, and regulating cell proliferation [5]. Generally, receptor-mediated extrinsic and mitochondrial-dependent intrinsic pathways trigger the activation of caspase-3, thereby resulting in apoptosis. In the intrinsic pathway, proapoptotic  and antiapoptotic  proteins stabilize the outer membrane of the mitochondria and activate caspase-9 and caspase-3 [6]. Studies indicate that cancer cells evade apoptosis by deregulating this process, thereby resulting in increased drug resistance. Given this challenging situation, apoptotic proteins have become the prime target for anticancer research .Taurine (i.e. 2-aminoethanesulfonic acid) is the only endogenous amino acid that does not interfere with protein synthesis . It is renowned for its cytoprotective effects and is involved in diverse physiological processes, including membrane stabilization, osmoregulation, neuromodulation, regulation of calcium homeostasis, and antioxidation [9]. Additionally, multiple studies have reported radioprotective, antimutagenic, and chemo-preventive properties associated with this compound [10]. Taurine exhibits anticancer activity via the elevation of caspase-3 and caspase-9 expression levels and BAX/Bcl-2 ratios . It also induces apoptosis in human colon or breast cancer cell lines and has been reportedly used as a biomarker for breast and bladder cancer . Given its versatility, several studies have attempted to utilize taurine as a drug, and notably, the limited derivatization studies yielded effective anticonvulsant, antimicrobial, and anticancer compounds . The urea derivative tauromustine exhibits potent in vitro and in vivo antiproliferative activity against several cancer cell lines [17] while the sultam derivative taurolidine was shown to be a clinically useful anticancer agent that did not affect healthy cells  [18]. In addition to its usefulness as a versatile building block for anticancer agents, various taurine‑based amide derivatives have been employed as effective anticonvulsant and antibacterial agents; however, there has been no study evaluating their anticancer activities so far  . To date, no extensive structure–activity relationship studies have been conducted to identify specific biological pathways that can accommodate taurine‑based derivatives [21]. This gap in the data prompted us to design and synthesize a group of novel 4-substituted benzoyltaurinamide derivatives and to determine their anticancer activity .In the structure–activity relationship studies conducted using 4-substituted benzoyltaurinamide derivatives, taurine’s sulfonic acid group was functionalized as a primary, secondary, or tertiary sulfonamide that was capable of demonstrating different ionization properties. The resulting compounds were categorized as phenyl, methoxyphenyl, sulfonamide, and morpholine derivatives based on the functional group located at the original sulfonamide position. Here, the amine moiety was functionalized as an amide with hydrogen-, chloro-, methoxy-, and methyl-substituted benzoic acids, while the sulfonamide groups remained untouched . This derivatization strategy allowed us to systematically study the impact of the sulfonamide and amide functional groups as well as the electronic and steric characteristics of the substituents on the aromatic ring of the benzoyltaurinamide derivatives on the compound’s anticancer activity. The resulting derivatives were evaluated against three cancer cell lines and one normal cell line. The most active compounds were subsequently employed to deduce the molecular mechanisms governing the anticancer activity of this class of compounds.(1-16)were produced via a 5-step reaction scheme (Scheme), and their structures were identified using spectroscopic  and elemental analytical methods. Briefly, taurine, phthalic anhydride, and potassium acetate were refluxed in an acetic acid solution to generate the 2-phthalimido-ethanesulfonic acid potassium salt,A, which was subsequently chlorinated to yieldBusing thionyl chloride via a previously reported procedure [22–26]. The phenyl and methoxyphenyl derivatives were prepared by treating 2-phtalimido-ethanesulfonyl chloride  in pyridine (5 mL) with aniline and 4-methoxyaniline (1 mmol) at 0–5 °C until the starting material was consumed, as evidenced by TLC analysis.N-phenyl-2-phtalimido-ethansulfonamide (C1) and 2--N-(4-methoxyphenyl)ethane-1-sulfonamide (C2) derivatives were achieved in yields of 12–60% after crystallization from acetic acid:water mixture (1:1) . The treatment ofBwith concentrated ammonium hydroxide solution produced 2-ethane-1-sulfonamide (C3) in 23% yield, whereas treatment with morpholine and triethylamine in dichloromethane solution yielded 2-(2-(morpholinosulfonyl)ethyl)isoindoline-1,3-dione (C4) [25]. In the last step, various benzoic acids were treated with 1,1’‑carbonyldiimidazole and diisopropylethylamine in dry acetonitrile, and aqueous solutions of the deprotected derivativesD1-D4were subsequently added to the respective reaction mixtures. The final compounds (1-16) were achieved in yields of 22–83%, as described in the experimental section. All compounds were characterized via 1H and 13C NMR, ESI–MS, ATR u1e7c, and u1e7d analysis. All spectral data obtained were consistent with the proposed structures .Herein, sixteen 4-substituted benzoyltaurinamide derivativesThe 1H NMR spectra of the derivatives exhibited phenyl protons between 6.92 and 7.89 ppm. The methylene protons of taurine and morpholine were observed between 3.20 and 3.72 ppm in the aliphatic field as expected. The secondary sulfonamide peaks for the phenyl and methoxyphenyl derivatives appeared between 9.50 and 10.43 ppm as broad singlets or singlets, whereas these peaks were not detected in the morpholine and sulfonamide derivatives. The sulfonamide derivatives exhibited specific signals for the protons of the primary sulfonamide group at 6.90–6.97 ppm. Finally, the proton signals at 8.46–8.88 ppm were verification of the formation of the amide bond. The carbonyl resonances of the final compounds were observed between 166.19 and 167.14 ppm in the 13C NMR spectra. All other aliphatic and aromatic carbons were observed in the expected regions at 34.65–51.76 ppm and 114.40–142.13 ppm, respectively. In the FT-IR spectra, N–H stretching, as well as SO2 symmetric and asymmetric stretching bands, provided confirmation of the products’ structures. All derivatives exhibited characteristic amide I and amide II peaks at 1606–1645 cm–1 and 1504–1548 cm–1, respectively. In the mass spectra (ESI-MS), the [M+1]+ ion peaks were observed for all titled compounds.The cytotoxicity of all derivatives was tested against one nontumorigenic cell line (i.e. MCF-10a) and three cancerous  cell lines using the colorimetric methyl tetrazolium test (MTT). The IC50 values of the tested compounds are summarized in Table. The MTT assay revealed that the cytotoxicity of the compounds varied depending on the moiety of the derivative and the type of cell lines tested (Table).The final compounds exhibited moderate to good cytotoxicity against PANC-1 cell lines, with IC50 values in the range of 1.2–120.3 µM. Compound 7, a morpholine derivative containing a 4-methoxyphenyl group on the amide functionality, was the most promising product obtained, with an IC50 of 1.2 µM. The other morpholine derivatives, namely, 6 (chloro‑), 5 (hydrogen‑), and 8 (methyl‑substituted) exhibited 8, 24, and 120 times lower activity than 7 (methoxy‑substituted), respectively, thereby proving that methyl substitution diminished the compound’s anticancer activity. The IC50 values of the morpholine derivatives (i.e. 5 and 6) were significantly lower than their sulfonamide (9-12) and phenyl (1-4) counterparts. Similarly, replacing the morpholine group in 7 with phenyl (3), methoxyphenyl (15), or hydrogen (11) moieties reduced the anticancer activity of the compound by 100-, 18-, and 80-fold, respectively. The methoxyphenyl derivatives (13-16) displayed good antiproliferative activity, with IC50 values in the range of 17.9–22.5 µM. From these results, it can be concluded that both morpholine and methoxyphenyl substitution at the sulfonamide group generally increased the compound’s antiproliferative activity against the PANC-1 cell line. In contrast, the ionization properties of the sulfonamide linker did not appear to have any impact on the compound’s activity.The derivatives also demonstrated well to moderate cytotoxicity against the brain cancer cell line (SH-SY5Y), with IC50 values in the range of 26.0–237.8 µM. The lowest IC50 value was obtained with the phenyl derivative (2). For the phenyl, methoxyphenyl, and sulfonamide derivatives, a chloro-substitution of the amide group produced more potent compounds when compared to the results obtained for their hydrogen‑, methoxy‑, and methyl‑substituted counterparts, with the only exceptions to this being the morpholine derivatives. Interestingly, replacing the phenyl, morpholine, or sulfonamide hydrogen with a methoxyphenyl ring lowered the IC50 values. These results indicated that the ionization properties of the sulfonamide group and chloro‑substitution on the benzamide functionality significantly affected the cytotoxicity of these derivatives against the SH-SY5Y cell line.The final compounds also exhibited excellent cytotoxicity against the nontumorigenic breast epithelial (MCF-10a) cell line, with IC50 values in the range of 70.7–217.8 µM. Compounds 1-13 displayed moderate IC50 activity (i.e. 77.1–140.4 µM), whereas 14, 15, and 16 (i.e. the methoxyphenyl derivatives) showed good activity against the breast cancer cell line (MDA-MB-231), with IC50 values of 19.9, 33.9, and 15.7 µM, respectively. We noted that all the tested cancerous cell lines were susceptible to the methoxyphenyl derivatives, indicating that the sulfonamide functionality significantly influenced cytotoxicity.Despite their effectiveness against cancerous cells and healthy cells that exhibit abnormally rapid proliferation, one persistent issue associated with conventional anticancer agents is the severe side effects. Given this, researchers have focused on developing therapeutic agents that exhibit selective cytotoxicity against cancerous cells. Thus, the safety profile of the titled compounds was determined by calculating their selectivity index (SI) values using the formula:SI = IC50 value calculated for the cancerous breast cell lines (MDA-MB-231) / IC50 value calculated for the normal breast cell line MCF-10a.Interestingly, compounds 13, 14, 15, and 16 exhibited high selectivity, with SI values of 3, 6, 4, and 9, respectively, for the cancerous when compared to the noncancerous breast cell lines.The substitution of the phenyl ring in the benzamide portion of the molecule with an electron‑withdrawing chloro-substituent improved the compound’s activity against the SH-SY5Y cell line only, whereas the substitution of phenyl ring with electron‑donating groups did not result in any significantly notable differences in activity against the PANC-1 and MDA-MB-231 cell lines. It was theorized that the derivatization of the sulfonamide part of the molecule was largely responsible for the compounds’ cytotoxicity. We noted that the phenyl and sulfonamide derivatives showed moderate activity against all tested cell lines. Replacing the primary sulfonamide with a secondary sulfonamide containing a phenyl ring did not significantly impact the compound’s cytotoxicity. The presence of heteroatoms increased the contact points in biologically active sites. Since morpholine and methoxyphenyl substitution were known to introduce heteroatoms into the taurine derivatives, their activity was expected to be higher than their counterparts. For our compounds, the morpholine‑substituted derivatives exhibited excellent cytotoxicity against the PANC-1 cell line only, whereas the 4-methoxyphenyl substituent enhanced cytotoxicity against all tested cell lines and was preferentially selective for the breast cancer cell line over the noncancerous breast cell line. This result indicated that the cytotoxicity and selectivity of taurine derivatives could be significantly improved by the incorporation of a substituted aromatic ring in the sulfonamide section of taurine.Cell death may occur via either necrosis or apoptosis. Necrosis causes inflammation of normal, healthy tissues, whereas apoptotic death is associated with minimal damage to the surrounding cells or tissue, a feature that is highly desired in new anticancer agents. In this study, the mechanism (i.e. via necrosis or apoptosis) triggered by the presence of the taurine derivatives was determined by subjecting compounds 7, 14, and 15 to Annexin V-FITC/PI via flow cytometry. Here, the rates of cell death for each experimental group were classified into three categories: “early apoptotic” in which a positive result was obtained for phosphatidylserine (PS), but a negative result was noted for PI, “late apoptotic” in which a positive result was obtained for both PS and PI, and “necrotic” in which a negative result was seen for PS, but a positive result was noted for PI [27]. We noted that the mitochondria-mediated intrinsic apoptotic pathway was controlled by proapoptotic (BAX) and antiapoptotic (Bcl-2 and Bcl-xL) Bcl-2 proteins. Stimulation of these proteins, therefore, resulted in changes in the mitochondrial membrane’s potential, which triggered the release of mitochondrial cytochrome c into the cytoplasm and activated both caspase-9 and caspase-3 proteins. This, in turn, initiated apoptosis. Both the intrinsic and extrinsic pathways converged on caspase-3 activation, whereas the initiator caspase-9 protein was activated only via the intrinsic pathway. Therefore, the activation of caspase-3 and caspase-9 was indicative of apoptosis via the intrinsic pathway [28].Emerging evidence suggested that alterations in the expression of antiapoptotic and proapoptotic Bcl-2 proteins were indicators for the presence of cancer cells. Moreover, these proteins were believed to be responsible for the development of resistance to apoptotic stimuli, chemotherapy, and radiation that have recently been noted in cancer cells. Interestingly, a group of Bcl-2 protein inhibitors were reported as potential anticancer agents and has been adopted for clinical use. Therefore, the inhibition of one or more of these proteins has become an attractive anticancer target for researchers tackling apoptosis resistance in tumor cells [29].In light of these findings, the extent of protein expression, namely, BAX, Bcl-2, Bcl-xL, cleaved caspase-3, and caspase-9, in the apoptotic pathway was examined to determine the mechanism through which cellular apoptosis was induced. Compounds 2, 6, 7, 10, 14, and 15 were chosen for further evaluation, and the PANC-1, SH-SY5Y, and MDA-MB-231 cell lines were treated with the determined cytotoxic concentrations of the selected compounds for 24 h and 48 h .Here, Western Blot analysis revealed that compound 14 (i.e. the chloro‑substituted methoxyphenyl derivative) was the only molecule that increased BAX, caspase-3, and caspase-9 expression levels, whereas there was a decrease in the expression of Bcl-2 and Bcl-xL in the PANC-1 cell line after 24 and 48 h of exposure . Replacing the chloro group (i.e. the methoxy derivative 15) resulted in the opposite effect on the expression of Bcl-xL after 24 h, and caspase‑3 and caspase‑9 after 48 h. Compound 7 (i.e. the methoxy‑substituted morpholine derivative) was the most cytotoxic molecule tested, with an IC50 value of 1.2 µM against the PANC-1 cell line; we noted that 7 effectively upregulated the expression of BAX, caspase-3, and caspase-9. Conversely, compound 7 did not show any effect on the expression of Bcl-2 and seemed to slightly decreased Bcl-xL levels after only 24 h. Thus, it would seem that substituting the methoxyphenyl moiety (i.e. 15) with a morpholine group retarded Bcl-2 expression while potentiating the impact on the caspase‑3 and caspase‑9 levels. Interestingly, 2 (i.e. the chloro‑substituted phenyl derivative) possessed a high IC50 value of 86.64 µM, and significantly upregulated the expression of BAX after 24 h and Bcl-2 after 48 h when compared to chloro‑substituted compounds such as 6, 10, and 14 . Except for the methoxy‑substituted methoxyphenyl 15, all compounds triggered a notable increase in the expression of caspase-9. In contrast, only 7 (i.e. the methoxy‑substituted morpholine derivative), 10 (i.e. the chloro‑substituted sulfonamide derivative), and 14 (i.e. the chloro‑substituted methoxyphenyl derivative) effectively increased the expression of caspase-3 after 24 and 48 h. Except for 6, all compounds containing the chloro‑substituted morpholine derivative increased caspase‑3 expression better than the reference drug cisplatin after 24 h. The effects of 2 and 6 on the expression of caspase‑9 levels, 7 on the BAX levels, 15 on the Bcl-2 levels, and compounds 2, 10, and 15 on the expression of Bcl‑xL were found to be better than the expression observed with the reference drug cisplatin after 48 h. Annexin V-FITC/PI experiments have revealed that treatment of the cell lines with 7, 14, and 15 at their cytotoxic concentrations for 48 h resulted in no significant changes in the apoptotic cell ratios . Despite the impact exerted by these molecules on apoptotic proteins, Annexin V-FITC/PI results showed that there might be another mechanism at play that may control the cytotoxicity of these compounds. Further study on this topic is required.Treating the MDA-MB-231 cell lines with compounds 2, 6, 7, 10, 14, and 15, which had IC50 values of 99.7, 79.1, 136.5, 77, 19.9, and 33.9 µM, respectively, significantly increased BAX protein levels after only 24 h, whereas the expression of Bcl-xL decreased by 2.08- to 4.55-fold after 24 h and by 2- to 5-fold after 48 h when compared to the control samples . Interestingly, only 7 (i.e. the methoxy‑substituted morpholine derivative) and 15 (i.e. the methoxy‑substituted methoxyphenyl derivative) decreased the expression of Bcl-2 by 2- to 3-fold when compared to the control sample after 24 and 48 h. Conversely, only 6 (i.e. the chloro‑substituted morpholine derivative), 10 (i.e. the chloro‑substituted sulfonamide derivative), and 14 (i.e. the chloro‑substituted methoxyphenyl derivative) slightly increased caspase-9 activity by 1.06-, 1.12-, and 1.18-fold relative to the control after only 48 h. Except for 7, all compounds were shown to improve caspase-3 expression levels by 3.96- to 8.2-fold relative to the control group. Interestingly, while 7 and 10 exhibited almost the same cytotoxicity  against the MDA-MB-231 cell line, their influence on the caspase‑3 levels differed by almost 12-fold. The effects exerted by 7, 10, and 15 on BAX expression and by compound 15 on Bcl-xL expression were statistically higher than the results associated with the reference drug. Annexin V-FITC/PI results have revealed that 14 and 15 slightly increased the percent of the positive apoptotic cells from 1.2% to 1.6% and 1.4%, respectively, even though compound 7 exhibited no changes . Given these results, it was theorized that 7 exerted its influence via a caspase-independent mechanism, as evidenced by the lack of change associated with the expression of caspase‑3 in the presence of this compound. Interestingly, replacing the chloro group (i.e. 14) with a methoxy group (i.e. 15) resulted in a change in the anticancer mechanistic pathway of the methoxyphenyl derivatives, as 14 appeared to follow the intrinsic apoptotic pathway; on the other hand, 15 did not exert any effect on the caspase-9 expression levels but increased the expression of caspase-3. Therefore, it was proposed that 15 induced apoptosis via a different caspase-dependent pathway [30].As noted, compounds 2, 6, 7, 10, 14, and 15 exerted their influence by increasing BAX (1.740- to 7.5-fold), caspase-3 (1.259- to 3.191-fold relative to the control), caspase-9 (1.214- to 2.068-fold relative to the control), while decreasing Bcl-2  and Bcl-xL (1.288- to 3.125-fold relative to the control) expression levels in the SH-SY5Y cell line . Interestingly, while 6 and 7 were the least cytotoxic compounds in the SH-SY5Y cell line with IC50 values of 80.62 and 134 µM, respectively, there were uncanny similarities in their activity values and protein expression levels when compared to the other compounds. Compounds 6 (i.e. the chloro‑substituted morpholine derivative), 7 (i.e. the methoxy‑substituted morpholine derivative), and 10 (i.e. the chloro‑substituted sulfonamide derivative) influenced the expression of BAX. Compounds 2 (i.e. the chloro‑substituted phenyl derivative), 7, 10, and 14 influenced the caspase-3 expression levels, whereas the expression of caspase‑9 was affected by the presence of 7 and 10. In these cases, the expression levels of the respective proteins were better when compared to the standard drug cisplatin. Compound 7 gave no positive results when subjected to Annexin V-FITC/PI experiments, whereas apoptotic cell ratios were slightly increased from 3.2% to 3.8% and 3.9%, respectively, after the application of 14 and 15 . According to these results, 14 and 15 acted through the intrinsic apoptotic pathway, whereas the cytotoxicity of 7 was supported by other cell death mechanisms.In this study, sixteen new 4-substituted benzoyltaurinamide derivatives were synthesized, and their anticancer activity was investigated using one normal (i.e. MCF-10a) and three cancerous cell lines  via MTT assay. The mechanism through which this group of compounds exerted their cytotoxic activity was determined using the most active compounds. Here, the expression levels of BAX, Bcl-2, and Bcl-xL, as well as cleaved caspase-3 and caspase-9, were examined via Western Blot assay, and apoptosis was determined using the Annexin V-FITC/PI test in combination with flow cytometry.(1-4)and sulfonamide(9-12)derivatives prepared in this study, only the chloro‑bearing compounds 2 and 10 exhibited good cytotoxicity with IC50 values of 29.0 and 38.6 µM, respectively. These compounds were preferentially selective against SH-SYH cells when compared to the other cell lines. All morpholine derivatives (5-8), except 8, exhibited low IC50 values  against the PANC-1 cell line only. In contrast, the methoxyphenyl derivatives (13-16) were effective against all tested cell lines, with low cytotoxic doses between 21.4 and 47.5 µM relative to their counterparts. Moreover, these compounds exhibited preferential selectivity toward cancerous breast cells than noncancerous cells, as evidenced by SI values between 3 and 9. Western Blot analysis revealed that all compounds, except for 7, induced the intrinsic apoptotic pathway by upregulating BAX, caspase-3, and caspase-9, while downregulating Bcl-2 and Bcl-xL expression levels in PANC-1 cell line either after 24 or 48 h. The same trend was noted in the SH-SY5Y cell line for all compounds after 24 and 48 h. For the MDA-MB-231 cell line, only 14 seemed to alter the intrinsic pathway-related expression of protein at the 24 or 48 h time mark. Annexin V-FITC/PI test results revealed that 7, 14, and 15 followed different cell death pathways in the MDA-MB‑231 cell line. These mechanisms can be categorized as: i) a caspase-dependent apoptotic pathway that includes the upregulation of BAX, caspase-3, and caspase-9 along with the downregulation of Bcl-2 and Bcl-xL expression, ii) a caspase-independent mechanism, and iii) a caspase-dependent apoptotic pathway that excludes caspase-9 activation. Even though the Annexin V-FITC/PI test results were negative for the PANC-1 cell lines for compounds 7, 14, and 15, the intrinsic pathway-related expression of the protein was altered as expected. Therefore, the anticancer effects of these compounds seem to support evidence of other cell death mechanisms. The same mechanism was detected for 7 in the SH-SYH cell lines, even though this cell line was subjected to the intrinsic pathway in the presence of compounds 14 and 15. Compounds 14 and 15 deserve further in vivo studies as they comprise an underexplored taurine structure compared to the classical anticancer drugs which already have resistance problems. As a result, this study has shown that modifying both the sulfonic acid and amine groups of taurine influences the selectivity, cytotoxicity, and the mechanism of cell death, thereby offering valuable insight into the design of new taurine-based anticancer derivatives.Of the phenyld6as a solvent. Chemical shifts were reported in parts per million (ppm), and the coupling constants (J) were expressed in hertz (Hz). Splitting patterns were designated as follows:s, singlet;d, doublet;t, triplet;m, multiplet;brs, broad singlet. Infrared spectra were run on a Perkin Elmer Spectrum 100 FT-IR equipped with a Universal ATR Sampling Accessory, and the frequencies were expressed in cm–1. Analytical thin-layer chromatography (TLC) was conducted on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck silica gel 60 (230–400 mesh ASTM) as the stationary phase. Silica gel was packed in with 2 cm diameter glass column to a 6 cm height bed with a solvent mixture consisting of ethyl acetate and hexane. Sample was loaded onto the column and eluted with a mobile phase of the aforementioned mixture. Melting points were taken with a Stuart® (SMP30) melting point apparatus in open capillary tubes and were uncorrected. The solvents used in MS measurements were ethanol, acetonitrile, and water (LC-MS grade), purchased from Sigma-Aldrich (Germany). The ESI-MS (electrospray ionization-mass spectrophotometry) spectra were obtained using a Thermo Scientific DSQ II  equipped with an electrospray source (ESI) operating in both positive and negative ions. Raw data were collected and processed by XcaliburTM 2.0 software. Elemental analyses  were performed using a Leco TruSpec CHNS Microanalyzer , and results were within ±0.4% of calculated values.Phthalic anhydride, potassium acetate, aniline, 4-methylbenzoic acid, and 4-chloro benzoic acid were purchased from Merck (Germany). All the other reagents, reactants, and anhydrous solvents were purchased from Sigma-Aldrich (Germany), Carlo Erba (France), and Interlab (Germany). All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes to transfer solutions. 1H NMR spectra were recorded on a Varian AS 400 Mercury Plus NMR spectrometer using DMSO-N-phenylethane-1-sulfonamide.HCl (D1) and 2-(morpholinosulfonyl) ethan-1-amine.HCl (D2) were prepared according to the procedure that was described before [25].2-Amino-C3) and 2--N-(4-methoxyphenyl)ethane-1-sulfonamide (C4) were synthesized as previously reported [26]. To a 2.8 mmol solution of compoundC3orC4in 10 mL ethanol, hydrazine hydrate solution (7.0 mmol) was added and refluxed. After the consumption of the starting compounds, concentrated HCl (7.5 mL) was added and stirred for 1 h at room temperature. The mixture was filtered, and the solvent evaporated under vacuum. The crude product was triturated with dichloromethane/diethyl ether to produce 2-aminoethane-1-sulfonamide.HCl (D3) and triturated with ethanol/petroleum ether to produce 2-amino-N-(4-methoxyphenyl)ethane-1-sulfonamide.HCl (D4) as a white solid.The starting compounds 2-ethane-1-sulfonamide ethyl)benzamide (1-4), 4-substituted-N-(2-(morpholinosulfonyl)ethyl)benzamide derivatives (5-8), 4-substituted-N-(2-sulfamoylethyl)benzamide (9-12), and 4-substituted-N-(2-(N-(4-methoxyphenyl)sulfamoyl)ethyl)benzamide derivatives (13-16) as white solid . Compound 9 was reported previously for its anticonvulsant activity . For compounds 1-13 registered CAS numbers have been assigned previously. However, their synthesis procedures, chemical properties, and structural characterization are not available in the literature.Various benzoic acids (1.0 mmol), 1-1’-carbonyldiimidazole , and diisopropylethylamine  were dissolved in dry acetonitrile  stirred for ½ h at room temperature. Hydrolyzed derivatives (1.0 mmol)N-phenylethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of benzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:1) and crystallized from isopropanol to afford the title compound 1 as a white solid ;Rf= 0.36 (ethyl acetate:hexane 1:1); m.p. 123.7–124.7 °C; 1H NMR  δ: 3.38–3.45 , 3.66–3.71 , 7.12–7.16 , 7.25–7.28 , 7.33–7.37 , 7.47–7.50 , 7.54–7.58 , 7.80–7.83 , 8.59–8.61 , 9.88 . 13C NMR  δ: 35.1 (Aliph-C), 50.7 (Aliph-C), 120.9 (2 × Ar-C), 124.9 (Ar-C), 128.0 (2 × Ar-C), 129.2 (2 × Ar-C), 130.2 (2 × Ar-C), 132.2 (Ar-C), 134.8 (Ar-C), 138.9 (Ar-C), 167.3 (C=O). FT-IR υmaks (cm–1): 3411 (N-H stretching), 1645 (amide I band), 1538 (amide II band), 1312 (SO2 asymmetric stretching), 1142 (SO2 symmetric stretching) cm–1. Exact mass: 304.09, MS (EI):m/z(%); 305 , 105 (100). Elemental analysis calculated (%) for C15H16N2O3S: C, 59.19; H, 5.30; N, 9.20; S, 10.54. Found: C, 58.91; H, 5.14; N, 9.09; S, 10.75.A solution of 2-amino-N-phenylethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-chlorobenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:1) and crystallized from isopropanol:water to afford the title compound 2 as a white solid ; Rf = 0.39 (ethyl acetate:hexane 1:1); m.p. 143.8–144.8 °C; 1H NMR  δ: 3.38–3.40 , 3.64–3.69 , 7.14 , 7.26 , 7.33–7.37 , 7.57 , 7.83 , 8.67-8.70 , 9.86 . 13C NMR  δ: 35.2 (Aliph-C), 50.5 (Aliph-C), 120.8 (2 × Ar-C), 124.9 (Ar-C), 129.3 (2 × Ar-C), 129.9 (2 × Ar-C), 130.2 (2 × Ar-C), 133.6 (Ar-C), 137.1 (Ar-C), 138.9 (Ar-C), 166.2 (C=O). FT-IR υmaks (cm–1): 3391 (N-H stretching), 1645 (amide I band), 1541 (amide II band), 1311 (SO2 asymmetric stretching), 1142 (SO2 symmetric stretching) cm–1. Exact mass: 338.05, MS (EI): m/z (%); 339 , 41 (100). Elemental analysis calculated (%) for; C15H15ClN2O3S: C, 53.18; H, 4.46; N, 8.27; S, 9.46. Found: C, 53.15; H, 4.66; N, 8.32; S, 9.23.A solution of 2-amino-N-phenylethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methoxybenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 ml) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (2:1) to afford the title compound 3 as a white solid ; Rf = 0.33 (ethyl acetate:hexane 2:1); m.p. 121.3–122.3 °C; 1H NMR  δ: 3.35–3.38 , 3.63–3.68 , 3.84 , 7.01 , 7.12–7.16 , 7.26–7.28 , 7.33–7.37 , 7.79 , 8.44–8.47 , 9.87 ; 13C NMR  δ: 35.1 (Aliph-C), 50.8 (Aliph-C), 56.2 (-OCH3), 114.4 (2 × Ar-C), 120.9 (2 × Ar-C), 124.9 (Ar-C), 127.1 (Ar-C), 129.9 (2 × Ar-C), 130.2 (2 × Ar-C), 138.9 (Ar-C), 162.6 (Ar-C), 166.8 (C=O). FT-IR υmaks (cm–1): 3339, 3258 (N-H stretching), 1632 (amide I band), 1504 (amide II band), 1301 (SO2 asymmetric stretching), 1138 (SO2 symmetric stretching) cm–1. Exact mass: 334.10, MS (EI): m/z (%); 335 , 135 (100). Elemental analysis calculated (%) for C16H18N2O4S: C, 57.47; H, 5.43; N, 8.38; S, 9.59. Found: C, 57.21; H, 5.29; N, 8.19; S, 9.60.A solution of 2-amino-N-phenylethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methylbenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:1) and crystallized from isopropanol to afford the title compound 4 as a white solid ; Rf = 0.43 (ethyl acetate:hexane 1:1); m.p. 149.7–150.7 °C; 1H NMR  δ: 2.38 , 3.36–3.39 , 3.64–3.67 , 7.12–7.16 , 7.25–7.30 , 7.33–7.37 , 7.72 , 8.50–8.53 , 9.88 . 13C NMR  δ: 21.8 (-CH3), 35.1 (Aliph-C), 50.7 (Aliph-C), 120.9 (2 × Ar-C), 124.9 (Ar-C), 128.0 (2 × Ar-C), 129.7 (2 × Ar-C), 130.2 (2 × Ar-C), 132.1 (Ar-C), 138.9 (Ar-C), 142.1 (Ar-C), 167.1 (C=O). FT-IR υmaks (cm–1): 3399 (N-H stretching), 1640 (amide I band), 1542 (amide II band), 1313 (SO2 asymmetric stretching), 1142 (SO2 symmetric stretching) cm–1. Exact mass: 318.10, MS (EI): m/z (%); 319 , 119 (100). Elemental analysis calculated (%) for C16H18N2O3S: C, 60.36; H, 5.70; N, 8.80; S, 10.07. Found: C, 60.37; H, 5.50; N, 8.68; S, 10.18.A solution of 2-amino-D2, 1.0 mmol) in water (10 mL) was treated with a solution of benzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:3) to afford the title compound 5 as a white solid ; Rf = 0.13 (ethyl acetate:hexane 2:1); m.p. 107–108 °C; 1H NMR  δ: 3.20–3.22 , 3.36–3.38 , 3.37–3.70 , 7.50–7.53 , 7.56–7.60 , 7.86–7.89 , 8.68–8.71 . 13C NMR  δ: 34.8 (Aliph-C), 46.2 (2 × Aliph-C), 47.7 (Aliph-C), 66.7 (2 × Aliph-C), 128.0 (2 × Ar-C), 129.2 (2 × Ar-C), 132.3 (Ar-C), 134.9 (Ar-C), 167.2 (C=O). FT-IR υmaks (cm-1): 3328 (N-H stretching), 1639 (amide I band), 1536 (amide II band), 1318 (SO2 asymmetric stretching), 1144 (SO2 symmetric stretching) cm–1. Exact mass: 298.10, MS (EI): m/z (%); 299 , 212 (100). Elemental analysis calculated (%) for C13H18N2O4S: C, 52.33; H, 6.08; N, 9.39; S, 10.75. Found: C, 52.24; H 6.24; N, 9.42; S,10.45 4.1.2.6. 4-Chloro-N-(2-(morpholinosulfonyl)ethyl)benzamide (6): CAS: 1216690-49-1.A solution of 2-(morpholinosulfonyl)ethan-1-amine.HCl  in water (10 mL) was treated with a solution of 4-chlorobenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:5) and crystallized from isopropanol:ethanol to afford the title compound 6 as a white solid ; Rf = 0.14 (ethyl acetate:hexane 2:1); m.p. 162–163 °C; 1H NMR : δ: 3.20–3.22 , 3.36–3.38 , 3.66–3.69 , 7.59 , 7.89 , 8.81-8.88 . 13C NMR  δ: 34.8 (Aliph-C), 46.2 (2 × Aliph-C), 47.6 (Aliph-C), 66.7 (2 × Aliph-C), 129.4 (2 × Ar-C), 130.0 (2 × Ar-C), 133.7 (Ar-C), 137.1 (Ar-C), 166.2 (C=O). FT-IR υmaks (cm–1): 3341 (N-H stretching), 1635 (amide I band), 1544 (amide II band), 1319 (SO2 asymmetric stretching), 1102 (SO2 symmetric stretching) cm–1. Exact mass: 332.06, MS (EI): m/z (%); 333 , 41 (100). Elemental analysis calculated (%) for; C13H17ClN2O4S: C, 46.92; H, 5.15; N, 8.42; S, 9.63. Found: C, 46.80; H, 5.29; N, 8.12; S, 9.65.A solution of 2-(morpholinosulfonyl)ethan-1-amine.HCl  in water (10 mL) was treated with a solution of 4-methoxybenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:3) and crystallized from isopropanol:water to afford the title compound 7 as a white solid ; Rf = 0.14 (ethyl acetate:hexane 2:1); m.p. 183.5–184.5 °C; 1H NMR : δ: 3.20–3.21 , 3.66–3.67 , 3.84 , 7.03–7.05 , 7.84–7.86 , 8.53–8.56 . 13C NMR  δ: 34.7 (Aliph-C), 46.2 (2 × Aliph-C), 47.8 (Aliph-C), 56.2 (-OCH3), 66.7 (2 × Aliph-C), 114.4 (2 × Ar-C), 127.2 (Ar-C), 129.9 (2 × Ar-C), 162.6 (Ar-C), 166.7 (C=O). FT-IR υmaks (cm-1): 3290 (N-H stretching), 1605 (amide I band), 1543 (amide II band), 1332 (SO2 asymmetric stretching), 1111 (SO2 symmetric stretching) cm–1. Exact mass: 328.11, MS (EI): m/z (%); 329 , 115 (100). Elemental analysis calculated (%) for; C14H20N2O5S·0.1H2O: C, 50.93; H, 6.17; N, 8.48; S, 9.71 Found: C, 50.77; H, 6.10; N, 8.439; S, 9.65.A solution of 2-(morpholinosulfonyl)ethan-1-amine.HCl  in water (10 mL) was treated with a solution of 4-methylbenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:3) and crystallized from isopropanol:ethanol to afford the title compound 8 as a white solid ; Rf = 0.12 (ethyl acetate:hexane 2:1); m.p. 174.2–175.2 °C; 1H NMR : δ: 2.39 , 3.20–3.21 , 3.33–3.35 , 3.67–3.68 , 7.31 , 7.78 , 8.60–8.62 . 13C NMR  δ: 21.8 (-CH3), 34.7 (Aliph-C), 46.1 (2 × Aliph-C), 47.7 (Aliph-C), 66.7 (2 × Aliph-C), 128.1 (2 × Ar-C), 129.8 (2 × Ar-C), 132.1 (Ar-C), 142.2 (Ar-C), 167.1 (C=O). FT-IR υmaks (cm-1): 3338 (N-H stretching), 1663 (amide I band), 1548 (amide II band) 1322 (SO2 asymmetric stretching), 1109 (SO2 symmetric stretching) cm–1. Exact mass: 312.11, MS (EI): m/z (%); 313 , 226 (100). Elemental analysis calculated (%) for C14H20N2O4S: C, 53.83; H, 6.45; N, 8.97; S, 10.26. Found: C, 53.75; H, 6.09; N, 8.83; S, 10.30.A solution of 2-(morpholinosulfonyl)ethan-1-amine.HCl  in water (10 mL) was treated with a solution of benzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (3:1) to afford the title compound 9 as a white solid ; Rf = 0,19 (ethyl acetate:hexane 3:1); m.p. 162–163 °C (previously reported as 165–166 °C [35]); 1H NMR : δ: 3.27–3.30 , 3.67–3.72 , 6.97 , 7.49–7.59 , 7.87 , 8.62 . 13C NMR  δ: 35.6 (Aliph-C), 54.5 (Aliph-C), 128.1 (2 × Ar-C), 129.2 (2 × Ar-C),132.2 (Ar-C), 135.1 (Ar-C), 167.3 (C=O). FT-IR υmaks (cm-1): 3382, 3331, 3266 (N-H stretching); 1635 (amide I band); 1529 (Amide II band); 1294 (SO2 asymmetric stretching); 1133 (SO2 symmetric stretching) cm–1. Exact mass: 228.06, MS (EI): m/z (%); 229 , 115 (100), 74 (18), 55 (5). Elemental analysis calculated (%) for C9H12N2O3S: C, 47.36; H, 5.30; N, 12.27; S, 14.05. Found: C, 47.07; H, 5.21; N, 12.34; S, 13.64.A solution of 2-aminoethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-chlorobenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane:methanol (3:1:0.5) and triturated from ethyl acetate:hexane to afford the title compound 10 as a white solid ; Rf = 0.63 (ethyl acetate:hexane:methanol 3:1:0.5); m.p. 158–159 °C; 1H NMR  δ: 3.20–3.24 , 3.59–3.65 , 6.90 , 7.53 , 7.82 , 8.64 . 13C NMR  δ: 35.7 (Aliph-C), 54.4 (Aliph-C), 129.3 (2 × Ar-C), 129.9 (2 × Ar-C), 133.8 (Ar-C), 137.1 (Ar-C), 166.3 (C=O). FT-IR υmaks (cm–1): 3397, 3314, 3189 (N-H stretching); 1636 (amide I band); 1532 (Amide II band); 1278 (SO2 asymmetric stretching), 1143 (SO2 symmetric stretching) cm–1. Exact mass: 262.02, MS (EI): m/z (%); 263 , 115 (100), 74 (15), 55 (7). Elemental analysis calculated (%) for C9H11ClN2O3S·0.15C4H8O2: C, 41.79; H, 4.46; N, 10.15; S, 11.62. Found: C, 42.06; H, 4.26; N, 10.49; S, 11.40.A solution of 2-aminoethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methoxybenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane:methanol (3:1:0.2) and crystallized from acetonitrile:water to afford the title compound 11 as a white solid ; Rf = 0.5 (ethyl acetate:hexane 3:1:0.2); m.p. 176–177 °C; 1H NMR : δ: 3.25–3.29 , 3.65–3.70 , 3.79 , 6.95 , 7.03 , 7.84 , 8.47 . 13C NMR  δ: 35.6 (Aliph-C), 54.7 (Aliph-C), 56.3 (-OCH3), 114.5 (2 × Ar-C), 127.3 (Ar-C), 129.9 (2 × Ar-C), 162.6 (Aliph-C), 166.8 (C=O). FT-IR υmaks (cm-1): 3378, 3264 (N-H stretching); 1613 (amide I band); 1551, 1509 (Amide II band); 1322 (SO2 asymmetric stretching); 1140 (SO2 symmetric stretching) cm–1. Exact mass: 258.07, MS (EI): m/z (%); 259 , 204 (9), 135 (47), 74 (12). Elemental analysis calculated (%) for C10H14N2O4S·0.2C2H3N: C, 46.87; H, 5.52; N, 11.56; S, 12.03. Found: C, 47.00; H, 5.13; N, 11.17; S, 11.65.A solution of 2-aminoethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methylbenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane:methanol (3:1:0.5) and crystallized from ethyl acetate:hexane to afford the title compound 12 as a white solid ; Rf = 0.68 (ethyl acetate:hexane:methanol 3:1:0.5); m.p. 181–182 °C; 1H NMR  δ: 2.39  3.25–3.29 , 3.66–3.70 , 6.96 , 7.31 , 7.77 , 8.55 . 13C NMR  δ: 21.9 (-CH3), 35.6 (Aliph-C), 54.6 (Aliph-C), 128.1 (2 × Ar-C), 129.8 (2 × Ar-C), 132.3 (Ar-C), 142.1 (Ar-C), 167.2 (C=O). FT-IR υmaks (cm–1): 3373, 3323, 3266 (N-H stretching); 1634 (amide I band); 1533 (Amide II band); 1297 (SO2 asymmetric stretching); 1133 (SO2 symmetric stretching) cm–1. Exact mass: 242.07, MS (EI): m/z (%); 243 , 225 (62), 115 (100), 74 (13), 55 (7). Elemental analysis calculated (%) for C10H14N2O3S·0.05C4H8O2: C, 49.66; H, 5.88; N, 11.36; S, 13.00. Found: C, 49.53; H, 5.66; N, 11.53; S, 12.68.A solution of 2-aminoethane-1-sulfonamide.HCl (N-(4-methoxyphenyl)ethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of benzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:1) and crystallized from ethyl acetate:diethyl ether to afford the title compound 13 as a white solid ; Rf = 0.26 (ethyl acetate:hexane 1:1); m.p. 110–111 °C; 1H NMR  δ: 3.27–3.31 , 3.65–3.70 , 3.76 , 6.93 , 7.22 , 7.48–7.51 , 7.55–7.58 , 7.82–7.84 , 8.59–8.62 , 9.56 . 13C NMR  δ: 34.7 (Aliph-C), 49.5 (Aliph-C), 55.7 (-OCH3), 114.9 (3 × Ar-C), 123.9 (2 × Ar-C), 127.6 (2 × Ar-C), 130.8 (2 × Ar-C), 131.8 (Ar-C), 134.4 (Ar-C), 157.1 (Ar-C), 166.8 (C=O). FT-IR υmaks (cm–1): 3325 (N-H stretching), 1625 (amide I band), 1537 (amide II band), 1313 (SO2 asymmetric stretching), 1141 (SO2 symmetric stretching) cm–1. Exact mass: 334.10, MS (EI): m/z (%); 335 , 271 (33), 266 (22), 253 (12), 225 (16), 212 (23), 184 (6), 148 (12), 115 (30), 100 (67). Elemental analysis calculated (%) for C16H18N2O4S: C, 57.47; H, 5.43; N, 8.38; S, 9.59. Found: C, 57.67; H, 5.20; N, 8.42; S, 9.52.A solution of 2-amino-N-(4-methoxyphenyl)ethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-chlorobenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (1:1) and crystallized from isopropanol:water to afford the title compound 14 as a white solid ; Rf = 0.29 (ethyl acetate:hexane 1:1); m.p. 160–161 °C; 1H NMR  δ: 3.27–3.30 , 3.64–3.69 , 3.76 , 6.93 , 7.21 , 7.57 , 7.84 , 8.68–8.71 , 9.56 . 13C NMR  δ: 34.7 (Aliph-C), 49.6 (Aliph-C), 55.7 (-OCH3), 114.9 (2 × Ar-C), 123.9 (2 × Ar-C), 128.9 (2 × Ar-C), 129.5 (2 × Ar-C), 130.8 (Ar-C), 133.2 (Ar-C), 136.7 (Ar-C), 157.1 (Ar-C), 165.7 (C=O). FT-IR υmaks (cm–1): 3310, 3272 (N-H stretching), 1643 (amide I band), 1547 (amide II band), 1309 (SO2 asymmetric stretching), 1144 (SO2 symmetric stretching) cm–1. Exact mass: 368.06, MS (EI): m/z (%); 369 , 305 (10), 287 (6), 165 (5), 143 (29), 115 (100), 100 (50), 74 (8), 55 (20). Elemental analysis calculated (%) for C16H17ClN2O4S·0.15H2O: C, 51.72; H, 4.69; N, 7.54; S, 8.63. Found: C, 51.63; H, 4.50; N, 7.62; S, 8.53.A solution of 2-amino-N-(4-methoxyphenyl)ethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methoxybenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (3:1) and crystallized from aceton:diethyl ether to afford the title compound 15 as a white solid ; Rf = 0.5 (ethyl acetate:hexane 3:1); m.p. 112–113°C; 1H NMR  δ: 3.25–3.28 , 3.62–3.67 , 3.76 , 3.84 , 6.92–6.94 , 7.01–7.03 , 7.20–7.22 , 7.80 , 8.46 , 9.54 . 13C NMR  δ: 35.0 (Aliph-C), 50.3 (Aliph-C), 56.1 (-OCH3), 56.2 (-OCH3), 114.4 (2 × Ar-C), 115.4 (2 × Ar-C), 124.4 (2 × Ar-C), 127.1 (2 × Ar-C), 129.9 (Ar-C), 131.2 (Ar-C), 157.5 (Ar-C), 162.6 (Ar-C), 166.7 (C=O). FT-IR υmaks (cm–1): 3346, 3206 (N-H stretching), 1630 (amide I band), 1540 (amide II band), 1301 (SO2 asymmetric stretching), 1128 (SO2 symmetric stretching) cm–1. Exact mass: 364.11, MS (EI): m/z (%); 365 , 301 (10), 242 (6), 178 (3.5), 135 (11), 115 (90), 83 (77), 55 (13), 41 (5). Elemental analysis calculated (%) for C17H20N2O5S·0.15C3H6O: C, 56.17; H, 5.65; N, 7.51; S, 8.59. Found: C, 56.49; H, 5.59; N, 7.80; S, 8.37.A solution of 2-amino-N-(4-methoxyphenyl)ethane-1-sulfonamide.HCl  in water (10 mL) was treated with a solution of 4-methylbenzoic acid (1.0 mmol), CDI (1.0 mmol), and DIPEA (2.0 mmol) in dry ACN (8.0 mL) according to the general procedure B. The crude product was purified by silica gel column chromatography eluting with ethyl acetate:hexane (2:1) and crystallized from ethyl acetate:diethyl ether to afford the title compound 16 as a white solid ; Rf = 0.26 (ethyl acetate:hexane 2:1); m.p. 127–128 °C; 1H NMR  δ: 2.38 , 3.25–3.29 , 3.63–3.68 , 3.76 , 6.93 , 7.21 , 7.29 , 7.73 , 8.51–8.54 , 9.50 . 13C NMR  δ: 21.8 (Aliph-C), 35.1 (Aliph-C), 50.2 (Aliph-C), 56.1 (-OCH3), 115.4 (2 × Ar-C), 124.4 (2 × Ar-C), 128.1 (2 × Ar-C), 129.7 (2 × Ar-C), 131.3 (Ar-C), 132.1 (Ar-C), 142.2 (Ar-C), 157.6 ( Ar-C), 167.1 (C=O). FT-IR υmaks (cm–1): 3377, 3325 (N-H stretching), 1625 (amide I band), 1540 (amide II band), 1314 (SO2 asymmetric stretching), 1143 (SO2 symmetric stretching) cm–1. Exact mass: 348.11, MS (EI): m/z (%); 349 , 285 (10), 226 (16), 162 (10), 115 (90), 83 (86), 55 (15), 41 (5). Elemental analysis calculated (%) for C17H20N2O4S: C, 58.60; H, 5.79; N, 8.04; S, 9.20. Found: C, 58.34; H, 5.49; N, 8.04; S, 8.92.A solution of 2-amino-The human neuroblastoma cells (SH-SY5Y), triple-negative breast cancer cells (MDA-MB-231), and human pancreatic cancer cells (Panc1) used in this study were purchased from the American Type Culture Collection (ATCC). The cells were suspended in complete Dulbecco’s modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin  and plated in cell culture dishes. The cultures were maintained at 37 °C in a 5% CO2 95% humidified atmosphere. After reaching 85% confluence, the cells were transferred to 96-well plates or culture dishes. For MTT assays, the cells were seeded into 96-well cell culture plates at a density of 2 × 103 cells per well and allowed to adhere for 24 h. The cells were treated with fresh medium containing experimental compounds  and incubated in a 5% CO2 incubator for 48 and 72 h. For protein analysis, the cells were seeded into 6-well cell culture plates at a density of 0.5 x 106 cells/well and allowed to adhere to the surface for 24 h. The stock solutions of the test compounds (5 mM) were prepared in sterile DMSO, and these stocks were then appropriately diluted with the complete culture medium. DMSO levels were maintained below 1% in the test concentrations.Briefly, following exposures, MTT  was added to each well, and plates were incubated for 2 h at 37 °C in a 5% CO2 humidified incubator. The reaction mixture was removed, and DMSO was added to each well. The plates were shaken at room temperature, and the absorbance was measured at 570 nm and 630 nm using a microplate reader . Percent survival was plotted relative to vehicle control cells, which were normalized to 100% survival.The cells (0.5 × 106 cell/well) were seeded into 6 well plates and incubated for 24 h, and then treated with selected experimental compounds for 24 h and 48 h. Following incubation, the cells were washed with ice-cold 1X PBS and lysed in 1X cell lysis buffer. These lysates were quantified using the BCA protein assay. Then, the cell lysates were analyzed by western blot.On a 12% (w/v) tris-glycine denaturing gel, 20 μg protein was separated by electrophoresis, and then transferred to a PVDF membrane. After blocking, the membrane was incubated with primary antibodies, anti-Bax rabbit monoclonal antibody, anti-Bcl-2 rabbit monoclonal antibody, anti-Bcl-xL rabbit monoclonal antibody, anti-caspase-3 rabbit monoclonal antibody, and anti-caspase-9 rabbit monoclonal antibody  at 4 °C overnight. Following the incubation, the membrane was incubated with an anti-β-actin mouse monoclonal antibody  at room temperature for 1 h. After washing, the membrane was incubated with peroxidase-conjugated secondary antibodies for 1 h to visualize labeled proteins by enhanced chemiluminescence.Th e cells were treated with cisplatin or compound 7, 14, or 15 at their respective cytotoxic doses against three cancer cell lines. The cells were harvested after 48 h, washed with PBS, and analyzed using the Annexin V FITC/PI Apoptosis Detection kit  with a benchtop flow cytometer . While the lower right quadrant shows the early apoptotic cells (FITC positive and PI negative) indicating Annexin V binding and cytoplasmic membrane integrity, the upper right quadrant shows the late apoptotic cells (positive for FITC and PI). The upper left quadrant displays the necrotic cells, negative for FITC, and showing PI uptake.Data were expressed as means ± standard deviation (SD) for five (MTT) or three (western blotting) independent experiments. Comparisons of means between groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Statistical significance was assigned at P < 0.05.


The series was structurally characterized based on their spectral data and HRMS, and the structures of xanthone derivatives 1, 7, and 8 were determined by single-crystal X-ray diffraction. This series, along with an in-house series of aminated xanthones 16–33, was tested for in-vitro antimicrobial activity against seven bacterial (including two multidrug-resistant) strains and five fungal strains. 1-(Dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (7) and 1-(dibromomethyl)-3,4,6-trimethoxy-9H-xanthen-9-one (8) exhibited antibacterial activity against all tested strains. In addition, 3,4-dihydroxy-1-methyl-9H-xanthen-9-one (3) revealed a potent inhibitory effect on the growth of dermatophyte clinical strains , with a MIC of 16 µg/mL for all the tested strains. Compounds 3 and 26 showed a potent inhibitory effect on two C. albicans virulence factors: germ tube and biofilm formation.A series of thirteen xanthones  H-xanthene-1,6-dicarboxylate . For. For1]. boxylate ,9  and 3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one (2) 2 system under mild conditions produced 2-bromo-3,4-dimethoxy-1-methyl-9H-xanthen-9-one (5) and 2-bromo-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one (6)  are equivalent for each methoxyl group. In spite of their similarity, the H-4 protons present a slightly higher chemical shift than the H-3 protons. This evidence was confirmed by heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) techniques. The 13C-NMR spectra of the synthesized xanthones 3–6, 8, 10, 12–15 (C-9 176.0–178.0 ppm). It was also possible to visualize signals corresponding to the remaining twelve carbons of the xanthone scaffold (δC 99.7–165.5 ppm). The two (or three) signals corresponding to the resonance of the protons of the methoxyl groups  and one regarding the resonance of the protons of the methyl group (C-1′) presented the lowest chemical shifts. 13C-NMR assignments of directly bound nuclei were determined by HSQC and, on the other hand, nuclei separated from each other with two or more chemical bonds were deduced by HMBC experiments.The structural elucidation of compounds terature ,20. In g0, 12–15  show the0, 12–15  revealedH-xanthen-9-one 8 are represented in H-1′ 8.98 ppm) can be identified through the correlation with C-2 and C-9a. Between the aromatic protons, H-8 (δH-8 8.20 ppm) appears in the form of a doublet and presents a correlation with C-6, C-9, and C-10a. On the other hand, the signals corresponding to the resonance of protons H-2 (δH-2 7.74 ppm) and H-5 (δH-5 6.97–6.92 ppm) are easily distinguished through the correlations presented: while H-2 correlates with C-1′, C-3, C-4 and C-9a, H-5 presents correlations with C-6, C-7, C-8a, and C-10a.As an example, the main correlations between protons and carbons for 1-(dibromomethyl)-3,4,6-trimethoxy-91, 7, and 8, were determined by single-crystal X-ray diffraction and are shown in 3 is well out of the aromatic plane due to a rotation along the C-OCH3 bond.The structures of three xanthone derivatives, 3–15, along with a series of 18 in-house 3,4-dioxygenated xanthones 16–33 , with a MIC of 16 µg/mL for 3 and 32 µg/mL for 26 for all the tested strains. The fungicidal activity at the same concentration at that MIC was observed only for compound 3 with M. canis FF1 and compound 27 with T. rubrum FF5.Nevertheless, compounds 3, 4, 13, 23, 26, 27 and 31 were also evaluated for synergistic effects for T. rubrum. No synergy was observed with fluconazole (data not shown).Compounds C. albicans virulence was assessed, namely on germ tube and biofilm formation. Only 3 and 26 had an inhibitory effect on germ tube formation of C. albicans ATCC 10,231 , while 3 had a significant impact at concentrations ranging between 128 and 32 μg/mL , ensuing a similar inhibition zone to that of the reference strain . Regarding Gram-positive bacteria, compounds 7, 8, 20 and 26 displayed an inhibitory effect against E. faecalis ATCC 29212, with inhibition halos of 9, 10, 9, 9 and 8.5 mm, whereas compounds 7, 8, 26 and 27 were active against S. aureus ATCC 29,213 with inhibition halos of 9, 11, 9 and 9.5 mm, respectively. Similarly, 7, 8, and 26 inhibited the growth of either methicillin-resistant S. aureus (MRSA) or vancomycin-resistant Enterococci (VRE), resulting in an inhibition zone of 10, 11 and 9 mm for MRSA and 9, 8 and 8.5 mm for VRE.In order to evaluate the antimicrobial activity of compounds 12 inhibited MRSA growth, presenting an inhibition halo of 8 mm, and compound 20 inhibited VRE, with an inhibition halo of 9 mm. Despite these encouraging results, it was not possible to determine an MIC for any compound in any of the strains in the range of concentrations tested. This might be related to the fact that some compounds are poorly soluble in the culture media used for the determination of the MIC, and the amount of available compound in the solution was probably lower than intended. Regarding the screening for potential synergies with multidrug-resistant bacterial strains and the tested compounds in combination with clinically relevant antibiotics, none of the compounds revealed a synergistic association with antibiotics (data not shown).Additionally, 3) at C-3 and C-4 seems to be important, since compound 4, with an additional hydroxyl group at C-6, was not as potent as 3; on the other hand, the presence of activating groups (CH3) at C-1 (3) is more favorable than the presence of deactivating groups (COOCH3) at the same position (13). Moreover, we observed some tolerance to variations in the substituent groups at C-1 position, namely amine moieties , with aminated xanthones with halogenated aromatic rings being the most promising concerning antifungal activity  against all the tested dermatophyte clinical strains . Regarding antibacterial activity, SAR suggests that the presence of two bromine atoms (7 and 8) plays an important role towards this activity. Interestingly, compounds with a halogen atom at the amine moiety  exhibited potent antibacterial activity, suggesting once more that the halogen atoms are important for activity.Concerning SAR analysis, the obtained results were consistent with data previously reported for some natural ,28,29 an1H- and 13C-NMR spectra were taken in CDCl3 at room temperature on a Bruker Avance 300 instrument  (300.13 or 500.13 MHz for 1H- and 75.47 or 125.77 MHz for 13C-). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. Coupling constants are reported in hertz (Hz). 13C-NMR assignments were made by 2D HSQC and HMBC experiments . HRMS mass spectra were measured on a Bruker Daltonics micrOTOF Mass Spectrometer , recording in ESI (electrospray) mode in Centro de Apoio Científico e Tecnolóxico á Investigation (C.A.C.T.I.), University of Vigo, Galicia, Spain.All reagents and solvents were purchased from TCI , Acros , Sigma Aldrich , or Alfa Aesar  and had no further purification process. Solvents were evaporated using a rotary evaporator  under reduced pressure, Buchi Waterchath B-480. Microwave (MW) reactions were performed using an Ethos MicroSYNTH 1600 Microwave Labstation from Milestone . The internal reaction temperature was controlled by a fiber-optic probe sensor. All reactions were monitored by TLC carried out on precoated plates with 0.2-mm thickness using Merck silica gel 60 (GF254) with appropriate mobile phases and detection at 254 and/or 365 nm. Purification of the synthesized compounds was performed by chromatography flash column using Merck silica gel 60 (0.040–0.063 mm). Melting points (m.p.) were measured in a Köfler microscope  and are uncorrected. H-xanthen-9-one  was dissolved in dry toluene (15 mL), and aluminum chloride (6.660 mmol) was carefully added. The reaction mixture was refluxed, with magnetic stirring under a nitrogen atmosphere, for 1.5 h. After cooling the reaction mixture to room temperature, excess conc. HCl  was added and the mixture was extracted with ethyl acetate (4 × 50 mL) and the organic layers were evaporated and dried with Na2SO4 and the solvent was removed under reduced pressure to obtain the crude product. Purification by preparative TLC  gave the pure products 3,4-dihydroxy-1-methyl-9H-xanthen-9-one  or 3,4,6-trihydroxy-1-methyl-9H-xanthen-9-one  as brown solids.The appropriate methoxy-1-methyl-93,4-Dihydroxy-1-methyl-9H-xanthen-9-one (3); Brown solid ; m.p. = 265–267 °C. 1H-NMR : δ = 10.34 , 9.18 , 8.11 , 7.78 , 7.57 , 7.40 , 6.68 , 2.68  ppm. 13C-NMR : δ = 176.9 (C-9), 154.7 (C-10a), 150.4 (C-3), 147.5 (C-4a), 134.4 (C-6), 131.2 (C-1), 130.7 (C-4), 126.0 (C-8), 123.7 (C-7), 121.8 (C-8a), 117.5 (C-5), 115.2 (C-2), 112.8 (C-9a), 22.4 (1-CH3) ppm. HRMS (ESI+): m/z [C14H10O4 + H]+ calcd. for [C14H11O4]: 243.06519; found 243.06505.3,4,6-Trihydroxy-1-methyl-9H-xanthen-9-one (4); Brown solid ; m.p. >300 °C. 1H-NMR : δ = 7.93 , 6.84–6.77 , 6.62 , 2.65  ppm. 13C-NMR : δ = 176.2 (C-9), 162.9 (C-6), 156.5 (C-10a), 149.8 (C-3), 147.4 (C-4a), 130.7 , 127.9 (C-8), 114.9 , 113.3 (C-7), 112.6 (C-9a), 101.6 (C-5), 22.4 (1-CH3) ppm. HRMS (ESI+): m/z [C14H10O5 + H]+ calcd. for [C14H11O5]: 259.06010; found 259.05987.2 (1.110 mmol) was suspended in anhydrous CH2Cl2 (2 mL) under a nitrogen atmosphere at room temperature. Bu4NBr (1.110 mmol) was added and the mixture was stirred at room temperature for 30 min. The appropriate methoxy-1-methyl-9H-xanthen-9-one (0.370 mmol) in anhydrous in CH2Cl2 (2 mL) was added and the mixture was stirred at 40 °C for 7 days. The reaction mixture was quenched with saturated aqueous ammonium chloride, and the aqueous portion was separated and extracted with CH2Cl2. The organic layer was dried over MgSO4 and evaporated in vacuo. The crude product was purified by preparative TLC  to give the pure products 2-bromo-3,4-dimethoxy-1-methyl-9H-xanthen-9-one  or 2-bromo-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one  as white solid.PhI(OAc)2-Bromo-3,4-dimethoxy-1-methyl-9H-xanthen-9-one (5); White solid ; m.p. 171–173 °C. 1H-NMR : δ = 8.27 , 7.70 , 7.52 , 7.37 , 4.07 , 4.04 , 3.05  ppm.13C-NMR : δ = 177.8 (C-9), 154.9 (C-10a), 154.3 (C-3), 151.6 (C-4a), 139.8 (C-4), 137.0 (C-1), 134.6 (C-6), 127.1 (C-8), 124.4 (C-7), 122.7 (C-8a), 118.0, 117.8 , 117.6 (C-5), 62.0 (3-OCH3), 61.4 (4-OCH3), 21.7 (1-CH3) ppm. HRMS (ESI+): m/z [C16H13BrO4 + H]+ calcd. for [C16H14BrO4]: 349.00700; found 349.00692.2-Bromo-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one (6); White solid ; m.p. 171–173 °C. 1H-NMR : δ = 8.18 , 6.94 , 7.52 , 4.06 , 4.03 , 3.94 , 3.06  ppm. 13C-NMR : δ = 176.9 (C-9), 164.8 (C-6), 156.6 (C-10a), 153.8 (C-3), 151.4 (C-4a), 139.7 (C-4), 136.8 (C-1), 134.6 (C-6), 128.5 (C-8), 118.0 (C-9a), 117.7 (8a), 116.5 (C-2), 113.6 (C-7), 99.7 (C-5), 61.9 (3-OCH3), 61.2 (4-OCH3), 55.9 (6-OCH3), 21.5 (1-CH3) ppm. HRMS (ESI+): m/z [C17H15BrO5 + H]+ calcd. for [C17H16BrO5]: 379.0176; found 379.0161.1-(Dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (7)  was synthesized from 3,4-dimethoxy-1-methyl-9H-xanthen-9-one (1) and characterized according to the previously described procedures + calcd. for [C17H15Br2O5]: 456.9281; found 456.9275.3,4-Dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (9)  was synthesized from 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (7) and characterized according to the previously described procedure   mixed with H2O, was heated at 100 °C with stirring for 3 h. The reaction mixture was allowed to cool and extracted with ethyl acetate (3 × 5 mL). The organic layer was dried with anhydrous sodium sulfate, concentrated under reduced pressure, and the crude material was purified by flash column chromatography  to give the pure product 3,4,6-trimethoxy-9-oxo-9H-xanthene-1-carbaldehyde  as a fluffy white solid; m.p. 221–223 °C. 1H-NMR : δ = 11.23 , 8.20 , 7.52 , 6.99–6.94 , 4.09 , 4.05 , 3.95  ppm. 13C-NMR : δ = 193.1 (CHO), 177.1 (C-9), 165.5 (C-6), 157.5 (C-10a), 156.0 (C-3), 150.9 (C-4a), 140.7 (C-4), 133.6 (C-1), 128.3 (C-8), 116.4 (C-9a), 115.9 (C-8a), 114.2 (C-7), 108.3 (C-2), 100.1 (C-5), 61.9 (4-OCH3), 56.7 (3-OCH3), 56.1 (6-OCH3) ppm. HRMS (ESI+): m/z [C17H14O6 + H]+ calcd. for [C17H15O6]: 315.0863; found 315.0860.3,4-Dimethoxy-9-oxo-9H-xanthene-1-carboxylic acid (11)  was synthesized from 3,4-Dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (9) and characterized according to the previously described procedure + calcd. for [C17H15O7]: 331.0812; found 331.0810.H-xanthene-1-carbaldehyde (0.176 mmol) in methanol (2 mL) was added Oxone® , and it was stirred under reflux for 5 h. After solvent removal under vacuum, 1N HCl was used to dissolve the salts, and EtOAc was added to extract the products. The organic extract was washed with 1N HCl (3x) and brine, dried over Na2SO4, and the solvent was removed under reduced pressure to obtain the crude product. Products were purified by preparative plates TLC  to give the pure products methyl 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde  or methyl 3,4,6-trimethoxy-9-oxo-9H-xanthene-1-carbaldehyde  as white solid.To a mixture of the corresponding methoxy-9-oxo-9Methyl 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (13); White solid ; m.p. 169–171 °C. 1H-NMR : δ = 8.28 , 7.75 , 7.59 , 7.40 , 6.99 , 4.06 , 4.04 , ppm. 13C-NMR : δ = 176.0 (C-9), 170.4 (C-1′), 157.3 (C-10a), 156.3 (C-3), 150.5 (C-4a), 138.0 (C-4), 135.5 (C-6), 130.8 (C-8), 124.9 (C-8a), 121.9 (C7), 118.5 (C-5), 114.3 (C-9a), 108.4 (C-2), 62.3 (3-OCH3), 57.2 (4-OCH3), 53.8 (COOCH3) ppm. HRMS (ESI+): m/z [C16H10O5 + H]+ calcd. for [C16H11O5]: 283.06010; found 283.06000.Methyl 3,4,6-trimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (14); White solid ; m.p. 169–171 °C. 1H-NMR : δ = 8.17 , 6.98–6.90 , 4.03 , 4.00 , 3.93 , ppm. 13C-NMR : δ = 174.7 (C-9), 170.1 (C-1′), 165.3 (C-6), 157.8 (C-10a), 156.5 (C-3), 150.5 (C-4a), 137.5 (C-4), 129.7 (C-1), 128.3 (C-8), 115.3 (C-8a), 113.9 , 107.7 (C-2), 100.4 (C-5), 61.8 (3-OCH3), 56.7 (4-OCH3), 56.1 (6-OCH3), 53.3 (COOCH3) ppm. HRMS (ESI+): m/z [C18H16O7 + H]+ calcd. for [C18H17O7]: 345.0969; found 345.0965.H-xanthene-1-carbaldehyde (0.14 mmol) in pyridine (1.48 mL), NH2OH.HCl (0.77 mmol) was added. After stirring for 1.5 h at 50 °C, the reaction mixture was evaporated under reduced pressure, and the result residue was extracted with ethyl acetate (3 × 50 mL) from water (20 mL). The resulting organic phase was washed with 5% HCl aqueous (50 mL), water (50 mL), and 10% NaCl aqueous solution (50 mL), dried over Na2SO4, filtrated, and concentrated to dryness. The crude residue was purified by crystallization from ethyl acetate to give (Z)-3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde oxime (15) as a white solid.To a stirred mixture of 3,4-dimethoxy-9-oxo-9(Z)-3,4-Dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde oxime (15); White solid ; m.p. 204–205 °C. 1H-NMR : δ 11.44 , 9.31 , 8.14 , 7.85 , 7.67 , 7.46 , 7.43 , 3.99 , 3.94 , ppm. 13C-NMR : δ = 177.0 (C-9), 156.1 (C-3), 154.7 (C-10a), 150.7 (C-4a), 147.9 (C-1′), 136.9 (C-4), 135.3 (C-6), 129.7 (C-1), 126.1 (C-8), 124.4 (C-7), 121.4 (C-5), 117.8 (C-8a), 113.2 (C-9a), 106.8 (C-2), 61.1 (4-OCH3), 56.3 (3-OCH3) ppm. HRMS (ESI+): m/z [C18H13NO5 + H]+ calcd. for [C18H14NO5]: 300.08665; found 300.08639.Single crystals were mounted on cryoloops using paratone. X-ray diffraction data were collected at 290 K with a Gemini PX Ultra equipped with CuKα radiation (λ = 1.54184 Å). The structures were solved by direct methods using SHELXS-97 and refined with SHELXL-97 .Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were either placed at their idealized positions using appropriate HFIX instructions in SHELXL and included in subsequent refinement cycles or were directly found from difference Fourier maps and were refined freely with isotropic displacement parameters.Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre (CCDC codes below).3 and unit cell dimensions a = 7.2661(4) Å, b = 13.3692(7) Å and c = 13.5206(7) Å, and β = 100.347(5)°. The refinement converged to R  = 5.37% and wR2  = 13.77%. CCDC 1910877.Crystal was monoclinic, space group P21/c, cell volume 1292.06(12) Å3 and unit cell dimensions a = 9.7287(7) Å, b = 14.5814(9) Å and c = 11.7638(8) Å, and β = 112.510(8)° (uncertainties in parentheses). The refinement converged to R  = 8.52% and wR2  = 21.22%. CCDC 1910874.Crystal was monoclinic, space group P21/n, cell volume 1541.66(18) Å3 and unit cell dimensions a = 9.6176(3) Å, b = 17.2880(3) Å and c = 11.0362(3) Å, and β = 111.702(3)°. The refinement converged to R  = 8.59% and wR2  = 21.46%. CCDC 1910875.Crystal was monoclinic, space group P21/n, cell volume 1704.91(8) ÅStaphylococcus aureus ATCC 29,213 and Enterococcus faecalis ATCC 29212—and two Gram-negative bacteria—Escherichia coli ATCC 25,922 and Pseudomonas aeruginosa ATCC 27,853—reference strains were used. Multidrug-resistant bacterial strains isolated from public buses (MRSA S. aureus 66/1) [E. faecalis B3/101) [E. coli SA/2) were also used when there was a minimum inhibitory concentration (MIC) value for ATCC strains or an inhibition halo. Frozen stocks of all strains were grown in Mueller–Hinton agar  at 37 °C. All bacterial strains were sub-cultured in MH agar and incubated overnight at 37 °C before each assay. For antifungal activity screening, a yeast reference strain Candida albicans ATCC 10231, a filamentous fungi reference strain Aspergillus fumigatus ATCC 46645, and tree dermatophyte clinical strains Trichophyton rubrum FF5, Microsporum canis FF1 and Epidermophyton floccosum FF9 were used. Frozen stocks of all fungal strains were sub-cultured in Sabouraud dextrose agar  before each test, to ensure optimal growth conditions and purity.In the present study, two Gram-positive bacteria—us 66/1) , river w B3/101) , and a c5, 10, 11 and 28, which were less soluble in DMSO than the other compounds, a stock solution of 2 mg/mL was prepared. In the case of compounds 9, 12 and 22, the stock solution prepared was 1 mg/mL. Two-fold serial dilutions of the compounds were prepared in Mueller–Hinton broth 2  within the concentration range of 0.062–64 µg/mL. The highest concentration tested was chosen in order to maintain DMSO in-test concentration below 1% (v/v), as recommended by the CLSI [5 CFU/mL). The MIC was determined as the lowest concentration at which no visible growth was observed. The minimum bactericidal concentration (MBC) was assessed by spreading 10 µL of culture collected from wells showing no visible growth on MH agar plates. The MBC was determined as the lowest concentration at which no colonies grew after 16–18-h incubation at 37 °C. These assays were performed in duplicate.An initial screening of the antibacterial activity of the compounds was performed by the disk diffusion method as previously described ,33. Briethe CLSI . At thisC. albicans, A. fumigatus, T. rubrum, M. canis and E. floccosum. The MIC of each compound was determined by the broth microdilution method according to CLSI guidelines . The inoculum of C. albicans was adjusted to 0.5–2.5 × 103 CFU/mL. Two-fold serial dilutions of the compounds were prepared in RPMI-1640 broth medium supplemented with MOPS within the concentration range of 4–128 µg/mL, with maximum DMSO concentration not exceeding 2.5% (v/v). Sterility and growth controls were also included in each assay. The plates were incubated for 48 h at 35 °C  or during 5–7 days at 25 °C . MICs were recorded as the lowest concentrations resulting in 100% growth inhibition in comparison to the compound-free controls. Voriconazole MIC for Candida krusei ATCC 6258 was determined as quality control [C. albicans and A. fumigatus) or 5–7 days incubation at 25 °C . All the experiments were repeated independently at least two times.The antifungal activity of the test compounds was evaluated against r yeasts  and M38-r yeasts  for fila control . The resE. coli SA/2, oxacillin  for S. aureus 66/1, and vancomycin  for E. faecalis B3/101. Antibiotic disks alone (controls) and antibiotic disks impregnated with 15 µg of each compound were placed on MH agar plates seeded with the respective bacteria. Sterile 6-mm blank paper, impregnated with 15 µg of each compound, alone, was also tested. A blank disk with DMSO was used as a negative control. MH-inoculated plates were incubated for 18–20 h at 37 °C. Potential synergism was recorded when the halo of an antibiotic disk impregnated with a compound was greater than the halo of the antibiotic or compound-impregnated blank disk alone.In order to evaluate the combined effect of the compounds and clinically relevant antimicrobial drugs, a screening was conducted using the disk diffusion method, as previously described ,33. A seIn order to evaluate the combined effect of the compounds and clinically relevant antifungal drugs, a checkerboard assay was conducted, as previously described . Flucona3, 4, 13, 23, 24, 26, 27 and 31 in germ tube formation of C. albicans was determined as follows: cell suspensions from overnight SDA cultures of C. albicans ATCC 10,231 were prepared in NYP medium  and adjusted to a density of (1.0 ± 0.2) × 106 CFU/mL, as determined by cell counts using a hemocytometer. An appropriate volume of compound stock solution at 10 mg/mL was added in order to obtain final concentrations ranging between 128 and 16 μg/mL. Filamentation controls were included in each assay with and without 1.28% DMSO. Following a 3-h incubation at 37 °C, 100 cells from each sample were counted, using a hemocytometer, and the percentage of germ tubes was determined. Germ tubes were considered when they were at least as long as the blastospore. Protuberances showing a constriction at the point of connection to the mother cell, typical for pseudohyphae, were not considered [The effect of compounds nsidered . At leas3 and 27 on biofilm formation was evaluated through quantification of total biomass by crystal violet staining [C. albicans ATCC 10,231 suspensions prepared in RPMI-1640 broth medium supplemented with MOPS, from overnight SDA cultures, at a final concentration of (1.0 ± 0.2) × 106 CFU/mL, as determined by cell counts using a hemocytometer. A control with appropriate concentration of DMSO, as well as a negative control , was included. Sterile 96-well flat-bottomed untreated polystyrene microtiter plates were used. After a 48-h incubation at 37 °C, the biofilms were stained with 1% (v/v) crystal violet  for 5 min. The stain was solubilized with 33% (v/v) acetic acid , and the biofilm biomass was quantified by measuring the absorbance of each sample at 570 nm in a microplate reader . The background absorbance (RPMI-1640 without inoculum) was subtracted from the absorbance of each sample and the data are presented as percentage of control. Three independent assays were performed in triplicate for each experimental condition.The effect of staining ,39. Brie3–15) and evaluate their antimicrobial activity, along with a series of 19 in-house 3,4-dioxygenated xanthones 16–33. The results of antimicrobial screening revealed the potential of some of the obtained compounds either as new antibacterial  or antifungal  agents, with compound 3 exhibiting a potent inhibitory effect on the growth of dermatophyte clinical strains , with a MIC of 16 µg/mL for all the tested strains. Compounds 3 and 26 also showed a potent inhibitory effect of C. albicans ATCC 10,231 germ tube and biofilm formation, important virulence factors. In general, it was observed that compounds with halogen atoms were the most promising in terms of antibacterial activity, with some of the representatives being highly active against either Gram-positive or Gram-negative strains. However, although some of the aminated xanthones exhibited low solubility, their conversion to hydrochloride salts increase their potential as antibacterial or antifungal agents.Using several straightforward transformations, it was possible to synthetize a series of structurally diverse xanthones (


D2 and D3 symmetries, is described. They have been obtained from aromatic dicarboxylic acids, in combination with an axially-chiral, suitable dibenzylic alcohol, derived from 1,1′-binaphthyl-2,2′-diol (BINOL) using one-pot esterification reactions in good isolated yields. NMR and circular dichroism (CD) spectroscopies detect the structural and shape variability in the scaffolds, reflected both in terms of the changes in chemical shifts and the shape of selected proton resonances, and in terms of the variation of the CD signature related to the dihedral angle defined by the binaphthyl units embedded in the rigid cyclic skeleton. The D2 cyclic adducts are able to form stable complexes with aromatic diphenols, with binding strengths that are dependent on small variations in the spacing units, and therefore on the shapes of the internal cavities of the cyclic structures.The synthesis of several rigid, homochiral organic macrocycles possessing, respectively, average molecular  Large, shape-persistent macrocyclic structures are of increasing interest for applications in the field of supramolecular chemistry, molecular recognition, and nanoscience ,2,3,4. SChirality has been exploited in combination with shape-persistent macrocyclic structures both for sensing and recognition, and for the assembly of chiral nanotubes . Chiropt60. In this paper, we report on the synthesis of novel macrocycles, in which the phenolic functionalities of the binaphthyl units are protected by t-butyloxycarbonyl groups, and on their unexpected binding of π-electron rich aromatic diphenols.We have previously reported, in a sequence of papers, our approach to imparting chirality to covalent cyclic structures for chiroptical sensing and the assembly of chiral nanotubes, based on the use of 1,1′-binaphthyl-2,2′-diol (BINOL)-based molecular synthons ,27,28,29In our molecular design, and in line with the literature and our own previous work, a modification of the binaphthyl synthons followed the elaboration of the 2,2′ and 3,3′ positions, amongst the most reactive and easy to functionalize on the naphthyl skeletons, in order to insert suitable functionalities for both cyclization and molecular recognition. The use of high-yielding, well established synthetic protocols for the rapid covalent construction of the cyclic moieties was also an unavoidable premise. The synthesis of the key precursors and of the macrocycle is shown in 5 and 6 could be precursors, via the unmasking of the phenolic functionalities of very interesting supramolecular receptors with acidic hydroxyl groups pointing directly inside the chiral cavities. We have previously described the synthesis of resolved, optically pure BINOL derivatives bearing dimethyl-tbutylsilyl(TBS)-protected benzylic diols in the 3,3′-positions of the binaphthyl moieties (compound (R)-1) rtD (c = g (100 mL solvent) −1). CD spectroscopy was performed at 25 °C at a scanning speed of 50 nm·min−1, and the spectra were background corrected.All commercially available compounds were purchased from commercial sources and used as received. Aromatic phenols and methyl ethers were recrystallized before use. Compounds PTSA-DMAP  and (R)-R)-1  were preR)-1 . THF (Na1H-NMR) were conducted as follows. To a stock solution of the host molecule (solution A) in pure CH3CN  were added several aliquots of the guest compound (solution B). Solution B was formed by the guest molecule at a higher concentration dissolved in solution A, in order to maintain the ligand always at the same, constant concentration.The titration experiments , and a.c is the molar absorptivity of the complex, εs is the molar absorbivity of the substrate at the desired wavelength (which could be directly determined), C is the total concentration of the titrate , and ka is the association constant for the 1:1 complex.In the case of a 1:1 binding isotherm, by employing a nonlinear fitting curve program, the plot of the absorbance against the metal concentration x was fitted by Equation (1), thus affording the value of the association constant kCompound(R)-2. (R)-1  was added to a solution of DMAP  and THF dry (10 mL). After 10 min, the iPr2NEt  and di-BOC anhydride  were added to the mixture. After 15 h of stirring at R.T. and a TLC (hexane/ethyl acetate 98:2), the reaction mixture was concentrated in vacuo, and the crude product was purified by column chromatography (hexane/ethyl acetate 98:2) to yield compound 2 . [α]rtD = −69° . 1H-NMR  δ = 8.15 , 7.90 , 7.42 , 7.24 , 5.00 , 4.89 , 1.04 3), 1.01 3), 0.18 2tBu). 13C-NMR  δ = 150.3 (C=O), 144.5 (Cq), 133.2 (Cq), 132.1 (Cq), 131.7 (Cq), 127.6 (CH), 126.7 (CH), 126.3 (CH), 125.7 (CH), 125.5 (CH), 123.7 (Cq), 82.7 (Cq), 77.1 (Cq), 60.2 (CH2), 26.8 (CH3), 25.9 (CH3), −5.4 (CH3). MS(ESI): m/z = 797 [M + Na]+ (100%). Anal. calcd. for C44H62O8Si2: C 68.2%, H 8.1%; found C 68.3%, H 8.0%.Compound(R)-3. Bu4NF solution 1M in hexane  was added to a solution of compound 2 , THF (20 mL), and H2O (2 mL), and stirred overnight at R.T. After checking by TLC (hexane/ethyl acetate 9:1) the full conversion of compound 2, the reaction mixture was quenched with water (20 mL), and THF was removed in vacuo, extracted with Et2O (3 × 30 mL), and dried over Na2SO4. The solution was filtered and concentrated in vacuo and the crude product was purified by column chromatography (hexane/ethyl acetate 9:1) to yield compound 3 . [α]rtD = +12° . 1H-NMR  δ = 8.06 , 7.90 , 7.39 , 7.33 , 7.11 , 5.43 , 1.54 3). 13C-NMR  δ = 153.7 (C=O), 150.7 (Cq), 133.3 (Cq), 130.9 (CH), 128.9 (Cq), 128.4 (CH), 127.5 (CH), 124.3 (Cq), 124.1 (CH), 124.1 (CH), 111.9 (Cq), 82.6 (Cq), 77.1 (Cq), 64.5 (CH2), 27.7 (CH3). MS(ESI): m/z 1114.5 [2 M + Na]+ (65%), 569 [M + Na]+ (35%). Anal. calcd. for C32 H34O8: C 70.3%, H 6.3%; found C 70.3%, H 6.0%Macrocycles (RR)-5a and (RRR)-6a. Compound 3  and PTSA-DMAP  were mixed in 15 mL of CH2Cl2 under N2. Then, a solution of diacid 4a  in 15 mL of CH2Cl2 was added to the above solution. After 15 min DIC  was added. The solution was stirred under N2 overnight (16 h). After checking by TLC (hexane/ethyl acetate 8:2) the full conversion of compound 2, the reaction mixture was quenched with water (30 mL), extracted with CH2Cl2 (3 × 50 mL), and dried over Na2SO4. The solution was filtered and concentrated in vacuo, and the crude product was purified by column chromatography (hexane/CH2Cl2 2:8 for 5a and CH2Cl2 for 6a) to yield macrocycle 5a  and 6a . For 5a: [α]25D = +79° . 1H NMR  δ = 8.08 , 8.05 , 7.89 , 7.45 , 7.27 , 7.18 , 7.08 , 5.35–5.20 , 1.32 . 13C NMR  δ = 163.7 (Cq), 161.0 (Cq), 153.4 (Cq), 144.9 (Cq), 133.4 (Cq), 132.2 (2CH), 131.7 (Cq), 130.6 (CH), 128.3 (CH), 127.5 (CH + Cq), 126.7 (CH), 126.4 (CH), 125.1 (Cq), 124.3 (Cq), 119.2 (2CH), 82.5 (Cq t-But), 64.9 (CH2), 27.7 (3CH3). MS(ESI): m/z 1560.5 . Anal. calcd. for C92H80O22: C 71.9%, H 5.2%; found C 71.6%, H 5.0%. For 6a: [α]25D = +45° . 1H NMR  δ = 8.01 , 7.90 , 7.65 , 7.49 , 7.41–7.29 , 7.25 , 5.18 , 1.35 . 13C NMR  δ = 167.3 (Cq), 160.4 (Cq), 152.8 (Cq), 144.3 (Cq), 132.8 (Cq), 131.6 (2CH), 131.1 (Cq), 130.0 (CH), 127.7 (CH), 126.9 (CH + Cq), 126.1 (CH), 125.8 (CH), 124.5 (Cq), 123.7 (Cq), 118.6 (2CH), 81.9 (Cq t-Bu), 64.3 (CH2), 27.1 (3CH3). MS(ESI): m/z 2336.3 . Anal. calcd. for C138H120O33: C 71.9%, H 5.2%; found C 71.6%, H 5.0%.Macrocycles (RR)-5b and (RRR)-6b. They were prepared with the same procedure of 5a and 6a, but with compound 4b instead of 4a. The crude product was purified by the column chromatography to yield (RR)-5b (26%) and (RRR)-6b (10%). [α]25D = +79° . 1H NMR  δ = 8.12 , 7.92 , 7.47 , 7.35 , 7.27 , 6.96 , 6.51 , 5.55 , 4.56 . 13C NMR  δ = 165.5 (Cq), 160.2 (Cq), 154.5 (Cq), 149.0 (Cq), 145.5 (Cq), 134.2 (Cq), 132.5 (CH), 131.6 (2CH), 130.4 (Cq), 129.0 (Cq), 128.4 (CH), 127.5 (CH), 125.6 (CH), 125.4 (CH), 125.1 (Cq), 118.7 (2CH), 73.8 (CH2), 63.3 (CH2). MS(ESI): m/z 1864.5 . Anal. calcd. for C98H80F12O20: C 65.2%, H 4.5%; found C 65.6%, H 4.8%. For 6b: [α]25D = +39° . 1H NMR  δ = 8.11 , 8.09 , 7.91 , 7.47 , 7.29 , 7.20 , 6.91 , 5.17 , 1.34 . 13C NMR  δ= 167.3 (Cq), 160.4 (Cq), 152.8 (Cq), 144.3 (Cq), 132.8 (Cq), 131.6 (2CH), 131.1 (Cq), 130.0 (CH), 127.7 (CH), 126.9 (CH + Cq), 126.1 (CH), 125.8 (CH), 124.5 (Cq), 123.7 (Cq), 118.6 (2CH), 81.9 (Cq t-But), 64.3 (CH2), 27.1 (3CH3). MS(ESI): m/z 2796.3 .D2 and D3 molecular symmetries, respectively. The larger macrocycles reach 3000 Da as molecular mass. We have demonstrated that NMR and CD spectroscopies are essential in order to detect the structural and shape variability in the molecular scaffolds. The D2 cyclic adducts are able to form stable complexes with aromatic diphenols, with binding strengths that are dependent on small variations in the spacing units, and therefore on the shapes of the internal cavities of the cyclic structures. In the case of methyl-protected hydroquinone, a weak chiroptical readout of the titration is possible, reinforcing the usefulness of the introduction of atropoisomerically-chiral CD reporting moieties in supramolecular receptors.We have reported the synthesis of homochiral organic macrocycles, starting from elaborated optically-active binaphthyl-based synthons and suitable, commercially available dicarboxylic acids. The syntheses are relatively straightforward and proceed affording cyclized products in high yields, also considering that no template is used. The isolated cyclic adducts comprise two or three binaphthyl units, affording molecular entities possessing overall


G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds -(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of  Skeletal muscle is composed of multinucleated myofibers and accounts for about half of the total body weight . MyogeneLife expectancy is increasing globally, and thus the incidences of age-associated chronic diseases and associated treatment-related costs are increasing. Sarcopenia is generally characterized by age-associated gradual decreases in skeletal muscle mass, strength, and function . In addiVarious negative regulators increase protein degradation in muscular dystrophy and sarcopenia, and, of these, myostatin (MSTN) effectively inhibits muscle growth, and thus is regarded as a potential curative target for the treatment of muscle wasting associated diseases . MSTN inGlycyrrhiza uralensis is a familiar medicinal herb belonging to the family Leguminoceae. The main bioactive constituents of G. uralensis are triterpene saponins and different flavones such as licochalcone A, glycyrrhizic acid, isoliquiritigenin, liquiritigenin, and liquiritin which have been reported to exhibit different pharmacological activities, including anti-inflammatory, anti-allergic, antioxidant, antiulcer, hepatoprogenic, and neuroprotective activities .,28.4-Hyd15H12O4; 1H NMR  δ 7.68 , 7.28 , 6.77 , 6.45 , 6.31 , 5.32 , 3.00 , 2.64 . 13C NMR  δ 192.1 (C=O), 165.3 (C-7), 164.1 (C-9), 157.5 (C-4′), 129.9 (C-1′), 128.4 (C-5), 127.6 , 114.9 , 113.5 (C-10), 110.3 (C-6), 102.4 (C-8), 79.6 (C-2), 43.5 (C-3) (Liquiritigenin (2): Colorless needle crystals; C.5 (C-3)  29,30].,30.15H12R)-(-)-Vestitol (3): Colorless crystals; C16H16O4; 1H NMR  δ 7.05 , 6.89 , 6.50 , 6.42 , 6.36 , 6.28 , 4.23 , 3.98 , 3.72 , 3.47 , 2.99 , 2.82 . 13C NMR  δ 160.3 (C-4′), 157.4 (C-7), 156.6 (C-2′), 156.1 (C-9), 131.0 (C-5), 128.7 (C-6′), 120.9 (C-1′), 114.3 (C-10), 108.7 (C-6), 105.6 (C-5′), 103.6 (C-8), 102.4 (C-3′), 70.4 (C-2), 55.3 (-OCH3), 32.6 (C-3), 31.0 (C-4) ((.0 (C-4)  31]..R)-(-)-V15H12O4; 1H NMR  δ 7.90 , 7.73 , 7.56 , 6.79 , 6.36 , 6.24 . 13C NMR  δ 193.5 (C=O), 167.5 (C-4′), 166.3 (C- 2′), 161.5 (C-4), 145.6 (C-β), 133.3 (C-6′), 131.8 , 127.8 (C-1), 118.2 (C-α), 116.9 , 114.7 (C-1′), 109.1 (C-5′), 103.8 (C- 3′) (Isoliquiritigenin (4):Yellow crystals; C (C- 3′)  32,33].,33.15H1216H14O4;1H NMR  δ 7.39 , 7.13 , 6.55 , 6.45 , 6.42 , 5.50 , 4.24 , 3.77 , 3.62 , 3.54 .13C NMR  δ 161.2 (C-9), 160.7 (C-10a), 157.1 (C-3), 156.8 (C-4a), 132.3 (C-1), 124.9 (C-7), 119.2 (C-6b), 112.8 (C-11b), 109.9 (C-2), 106.5 (C-8), 103.8 (C-4), 97.0 (C-10), 78.6 (C-11a), 66.6 (C-6), 55.6 (-OCH3), 39.6 (C-6a) (Medicarpin (5): White amorphous powder; C6 (C-6a)  34]..16H14O4;16H14O6;1H NMR  δ 7.89 , 7.55 , 7.48 , 7.45 , 7.14 , 6.82 , 6.59 , 3.78 . 13C NMR  δ 191.4 (C=O), 152.1 (C-4′), 150.7 (C-4), 149.9 (C-2), 146.5 (C-3′), 140.9 (C-3), 139.6 (C-β), 131.8 (C-1′), 123.4 (C-6′), 121.4 (C-1), 120.5 (C-α), 120.3 (C-6), 116.3 (C-2′), 115.9 (C-5′), 112.7 (C-5), 61.7 (-OCH3) : Yellow needles; C (-OCH3)  35]..16H14O6;16H14O5;1H NMR  δ 7.97–7.91 , 7.61 , 7.19 , 6.85 , 6.61 , 3.81 . 13C NMR  δ 191.3 (C=O), 163.9 (C-4′), 151.0 (C-4), 149.9 (C-2), 141.0 (C-β), 139.7 (C-3), 132.2 , 131.1 (C-1′), 121.3 (C-1), 120.4 (C-α), 120.3 (C-6), 116.4 , 112.7 (C-5), 61.7 (-OCH3) : Yellow needles; C (-OCH3)  .,40.21H2226H30O13; 1H NMR  δ 7.71 , 7.41 , 7.09 , 6.48 , 6.34 , 5.44 , 5.40 , 4.98 , 3.01 , 2.70 . 13C NMR  δ 193.2 (C=O), 166.8 (C-7), 165.4 (C-9), 159.1 (C-4′), 134.3 (C-1′), 129.8 (C-5), 128.8 , 117.5 , 115.0 (C-10), 111.8 (C-6), 110.7 (C-1‴), 103.8 (C-8), 100.7 (C-1″), 80.7 (C-3‴), 80.7 (C-2), 78.6 (C-2″), 78.5 (C-3″), 78.0 (C-2‴), 78.0 (C-5″), 75.4 (C-4‴), 71.3 (C-4″), 66.0 (C-5‴), 62.4 (C-6″), 44.9 (C-3) (Liquiritinapioside (9): Yellow powder; C.9 (C-3)  35,41].,41.26H3022H22O9; 1H NMR  δ 8.43 , 8.05 , 7.53 , 7.24 , 7.15 , 6.99 , 5.12 , 3.78 , 3.73 , 3.19–3.48 . 13C NMR  δ 175.1 (C-4), 161.9 (C-7), 159.4 (C-4′), 157.5 (C-9), 154.1 (C-2), 130.5 , 127.4 (C-5), 124.4 (C-1′), 123.8 (C-3), 118.9 (C-10), 116.0 (C-6), 114.0 , 103.8 (C-8), 100.4 (C-1″), 77.6 (C-5″), 76.9 (C-3″), 735. (C-2″), 70.7 (C-4″), 61.0 (C-6″), 55.6 (-OCH3) (Ononin (10): White powder; C (-OCH3)  42,43].,43.22H22G. uralensis or liquiritigenin on muscle regeneration, two-month-old mice were orally administered CWE of G. uralensis (100 mg/kg) or liquiritigenin (15 mg/kg) once a day for 9 days from one day before CTX injection. Seven days after CTX injection, mice were anesthetized with avertin i.p.  and gastrocnemius muscles were excised for Western blotting and immunohistochemistry analysis.C57BL/6 male mice were purchased from KOATECH  and kept per cage in a temperature-controlled room under a 12 h light/12 h dark cycle. Animals were provided with standard rodent chow containing 6.2% (wt/wt) total fat  and water. All experiments conformed to the guidelines and regulations issued by the Animal Care Committee of our institute, and all animal experiments were approved by the Institutional Animal Care and Use Committees of the Yeungnam University (YUMC-AEC2017–013 and YUMC-ACE2019–033). The acute skeletal muscle injury model was generated by a single intramuscular  injection of cardiotoxin . Contralateral right gastrocnemius muscles were injected with phosphate buffered saline and used as controls. To investigate the effects of the CWE of 2 incubator at 37 °C.Murine C2C12 myoblast cells  were cultured in growth media [DMEM  + 10% FBS  + 1% P/S ] in a humidified 5% COG. uralensis , G. uralensis-fractions , or EtOAc-single compounds  at 37 °C for 1 or 2 days. Non-treated and treated cells were washed with fresh DMEM and then cultured in 0.5 mg/mL of MTT reagent  at 37 °C for 1 h. After removing the MTT reagent, formazan crystals were dissolved with DMSO (500 μL /well) and absorbances were measured at 540 nm using a spectrophotometer . Percentage cell proliferations were calculated by expressing the ODs of treated cells as percentages of non-treated controls.Cells were cultured in growth media supplemented with G. uralensis , G. uralensis-fractions , or EtOAc-single compounds (0.25 ng/mL).To induce myogenic differentiation, when cells reached 100% confluence, proliferation media was switched to differentiation media [DMEM+2% FBS+1% P/S for 4 days] supplemented with the CWE of Cells were cultured in DMEM+10% FBS+1% P/S until 100% confluent. Cell layers on plates were then scratched, media was added, and cells were incubated for 1 day. Cell recovery was observed using a light microscope  and cell mobility from scratched part to recovered part was measured and then calculated as cell recovery value (%).Differentiated cells were washed with PBS, fixed with a methanol/PBS mixture for 2 min, and stained with Giemsa G250 (Sigma Aldrich). Random images were taken at three different regions using a microscope. Nuclei numbers in myotubes and total numbers of nuclei in cells were calculated in each image. Fusion indices were calculated by expressing numbers of nuclei integrated into myotubes as percentages of total numbers of nuclei. All experiments were performed in triplicate ,59.3 in cultured media were determined using commercial reagent kits  and a Cedex Bio-analyzer .Cells were cultured in differentiation media supplemented with DMSO  or EtOAc for 4 days and concentrations of NHTrizol reagent (Thermo Fisher Scientific) was used to extract total RNA from cells according to the manufacturer’s instructions. RNA (2 µg) in 20 µL of reaction mixture was used to synthesize 1st strand cDNA using random hexamer and reverse transcriptase at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. Two micro liters of cDNA and 10 pmole of gene-specific primers were used to analyze gene expression by real-time RT-PCR, which was performed using a 7500 real-time PCR system and a power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Primer information is provided in PBS was used to wash cells, which were then lysed with RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Total protein concentrations were measured using the Bradford assay. Proteins (40 µg) were electrophoresed in 10 or 12% SDS-polyacrylamide gel and then transferred to PVDF membranes . Blots were blocked using 3% skim milk or BSA in Tris-buffered saline (TBS)-Tween 20 for 1 h and then incubated overnight with protein-specific primary antibodies  in 1% skim milk or BSA in TBS at 4 °C. After washing with TBST, blots were incubated with horse radish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature and then developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).2O2 (Junsei), respectively. After blocking with 1% normal goat serum, sections were incubated with primary antibody  overnight at 4 °C, and then treated with HRP-conjugated secondary antibody  at room temperature for 1 h. Sections were then counterstained with hematoxylin, dehydrated, mounted, and examined under a light microscope . After Hematoxylin and Eosin staining , muscle tissues were examined for morphological changes under a light microscope.Muscle tissues were routinely fixed with 10% formalin and embedded in paraffin wax . ParaffiThe reduction ratio of muscle mass (%) was calculated using a given formula:Muscle tissue sections were H&E stained, and images were captured under a light microscope. Muscle fiber diameters were measured using ImageJ software .https://fafdrugs4.rpbs.univ-paris-diderot.fr/), an open-access tool for the prediction of ADMET properties that allows filtering according to Egan’s rule, Lipinski’s rule of five, and Veber’s rule for the prediction of bioavailabilities, and according to GSK and Pfizer’s rules for overall toxicity predictions [The ADMET  pharmacokinetic properties of compounds were analyzed using FAFdrugs4  was employed to analyze normalized expression mean values to categorize significant gene expression changes. Multi Gauge 3.0 software  was used to quantify band intensities in Western blots. Gene and protein expressions were normalized versus GAPDH , and the analysis was conducted using one-way ANOVA and PROC GLM in SAS, ver.9.0 .G. uralensis promotes the proliferation and differentiation of myoblasts and muscle regeneration by upregulating the expressions of the myogenic marker genes  and by downregulating the expression of MSTN (a muscle growth inhibitor). Based on the observed inhibitory potency of G. uralensis against atrophy and protein degradation-related marker genes (Atrogin1 and MuRF1), we suggest that G. uralensis be considered a therapeutic agent for the prevention and treatment of muscular dystrophy, sarcopenia, and cachexia. In addition, the observed anti-inflammatory effect of G. uralensis suggests therapeutic applications in the context of inflammatory disease.In summary, our results demonstrate that