The structure, texture, surface composition, and adsorption properties of the as-prepared catalysts were extensively characterized. Then, the obtained samples were evaluated for their catalytic performance in hydrochlorination of acetylene. The results provided by the X-ray photoelectron spectroscopy, acetylene temperature-programmed desorption, and HCl adsorption confirmed the nature of the active sites (i.e. Sn-Nx) involved in the reactant adsorption, and hence in the improved catalytic performance. These active sites were also related to the improved lifetime of alkylorganotin-based catalysts in the hydrochlorination of acetylene. At a constant reaction temperature of 200 °C with an acetylene gas hourly space velocity (C2H2 -GHSV) of 30 h-1 , Sn-g1 -C3N4 /AC-550 exhibited the highest acetylene conversion (~98.0%) and selectivity toward the vinyl chloride monomer (>98.0%). From the catalytic test results, it was reasonably concluded that the hexamethylenetetramine is the most suitable N precursor, as compared to the dicyandiamide and urea, to prepare high-performance catalysts. From the BET specific surface area of fresh and used catalysts, it was suggested that, in contrast to dicyandiamide and urea, hexamethylenetetramine could delay the deposition of coke on alkylorganotin-based catalysts, which is reflected by the extended lifetime.A series of alkylorganotin-based catalysts (Sn-g-C Owing to the widespread applications of polyvinyl chloride (PVC) in all human activities, there is an increased demand for vinyl chloride monomer (VCM) as precursor for PVC manufacture . The hydrochlorination of acetylene is the main technology for producing VCM in several countries, especially in China, due to their rich coal reserves. However, there are several drawbacks of the use of carbon-supported mercuric chloride catalysts for the synthesis of VCM via acetylene hydrochlorination. Specifically, stringent government policies and severe mercury pollution have urged the researchers to explore alternative catalysts [3]. Hence, the design and development of mercury-free catalysts is extensively investigated [4].4 -CuCl2 -BiCl3 -CeCl3 /AC) and reported an optimal acetylene conversion of 95.1% . Deng et al. [12] have used a trimetallic supported catalyst (SnCl2 -BiCl3 -CuCl/AC) for the hydrochlorination of acetylene and reported that the deactivation of SnCl2 -BiCl3 -CuCl/AC is mainly attributed to the loss of tin(IV) chloride. In addition, Guo et al. [13] have prepared a SnCl2 -ZnCl2 -Tb4O7 /AC catalyst and reported the highest acetylene conversion of 67.7%  . Moreover, researchers have reported that nonmetal elements, including N [21], P [22], S [23], and B [24] can act as active catalysts for acetylene hydrochlorination.In recent years, remarkable progress has been achieved in the development of new catalysts, mainly gold-[5–10], nonprecious-metal- [11-20], and nonmetal-based catalysts [21–24]. Researchers have found that Au-based catalysts with a superior hydrochlorination activity are mainly deactivated due to the reduction of Au3+ to Au0 during acetylene hydrochlorination [5–10]. In addition, the high cost of gold limits the application of Au-based catalysts for the large-scale production of VCM. Among non-precious metal catalysts, the mercury-free catalysts have been developed for VCM production. Carbon-supported tin catalysts with a high catalytic performance [11–14] have attracted little attention compared to copper-based catalysts [15–20]. For instance, Xiong et al. [11] have prepared a tetrametallic supported catalyst (SnCl3N4) has attracted considerable attention for heterogeneous catalysis, photocatalysis, gas adsorption, and storage due to its high chemical stability and excellent acid and alkali resistance . Particularly, the Dai’s group has investigated the catalytic performance of g-C3N4 /AC for acetylene hydrochlorination and reported that the nitrogen enrichment of catalysts can promote the adsorption of hydrogen chloride and thus, significantly increase the activity of this metal-free catalyst [21]. However, the manner in which different g-C3N4 precursors affect the physicochemical properties of organotin is still not clear. Thus, it is imperative to further examine the use of alkylorganotin-based catalysts for acetylene hydrochlorination.Alkylorganotin compounds have been widely used as stabilizers and catalysts, as well as used in cancer treatment . Previous studies have revealed that organotin can catalyze the hydrochlorination of acetylene to obtain vinyl chloride. Moreover, additional performance enhancement of organotin compounds has been achieved by using dicyandiamide additives [14]. Carbon nitride , N2 adsorption-desorption isotherms, thermogravimetric analysis (TGA), and derivative thermogravimetric (DTG) analysis, X-ray photoelectron spectroscopy (XPS), HCl adsorption, and acetylene temperature-programmed desorption (C2H2 -TPD). The results indicated that hexamethylenetetramine is the best g-C3N4 precursor as Sn-Nx in alkylorganotin serves as catalytically active sites.In this study, hexamethylenetetramine, urea, and dicyandiamide were selected as precursors of g-C3N4 . Figure 1a shows the XRD patterns of g1 -C3N4 -550, g2 -C3N4 -550, and g3 -C3N4 -550. Two peaks were observed at 13.2°and 27.5°, respectively . In the XRD patterns of g1 -C3N4 /AC-550, g2 -C3N4 /AC-550, and g3 -C3N4 /AC-550 , 3 peaks at around 24.4°, 26.7°, and 43.7°can be observed, which correspond to the (002), (103), and (101) diffraction planes of the graphitic carbon, respectively (PDF#50-0926). Figure 1c displays the N2 physisorption isotherms of AC, g1 -C3N4 /AC-550, g2 -C3N4 /AC-550, and g3 -C3N4 /AC-550 samples. According to the IUPAC classification, they belong to the type I isotherms . Figure 1d illustrates the pore size distribution curves for g-C3N4 /AC-550 and AC. They reveal the coexistence of micro- and mesopores in all samples. Based on the isotherms and applying specific equations, the values of the textural parameters were calculated, and they are listed in Table 1. Compared with those of bare AC  , the BET specific surface area and total pore volume of g1 -C3N4 /AC-550  , g2 - C3N4 /AC-550  , and g3 -C3N4 /AC-550  decreased, indicating the successful loading of g-C3N4 on the AC support (Table 1). The XRD and N2 physisorption results revealed that g-C3N4 is well dispersed on the AC surface.According to the previous reports , the hexamethylenetetramine, dicyandiamide and urea were calcinated at 550 °C for 4 h under nitrogen atmosphere to successfully prepare g-C3N4 /AC catalysts exhibited a significantly increased acetylene conversion under the same reaction conditions. That is, conversions of 67.2%, 62.5%, and 61.7% were obtained for g1 -C3N4 /AC-550, g2 -C3N4 /AC-550, and g3 -C3N4 /AC-550, respectively. Notably, high acetylene conversion was obtained for g1 -C3N4 /AC-550, g2 -C3N4 /AC-550, and g3 -C3N4 /AC-550 catalysts, but the values were still less than 90%.An acetylene conversion of 8.9% was obtained over the AC support at 200 °C . The g-C3N4 precursor in the range of 400 °C–650 °C had a positive effect on the hydrochlorination activity of organotin. Thus, to investigate the effect of various g-C3N4 precursors on the performance of alkylorganotin-based catalysts for acetylene hydrochlorination, the calcination of Sn-g-C3N4 /AC catalyst precursors was carried out at 400, 450, 500, 550, and 650 °C.In our previous study [14], the change in the calcination temperature of the g-C3N4 /AC catalysts. Clearly, the BET specific surface areas and total pore volumes of Sn-g-C3N4 /AC catalysts were lower than those of g-C3N4 /AC  , indicating that the pores of g-C3N4 /AC were filled with alkylorganotin. Hence, BET specific surface areas between 210 and 653 m2 g-1 are obtained depending on the calcination temperature. First, with the increase in the calcination temperature, the values of the textural properties increased and then decreased, demonstrating the effect of the temperature on the textural properties of Sn-g-C3N4 /AC. To identify the optimal calcination temperature of Sn-g-C3N4 /AC, the acetylene hydrochlorination over all samples was performed. Figure 2b depicts the acetylene conversion obtained over Sn-g1 -C3N4 /AC-400, Sn-g1 -C3N4 /AC-450, Sn-g1 -C3N4 /AC-500, Sn-g1 -C3N4 /AC-550, and Sn-g1 - C3N4 /AC-650. With the increase in the reaction temperature from 150 °C to 200 °C, the acetylene conversion over all catalysts clearly increased . At an optimal reaction temperature of 200 °C, Sn-g1 -C3N4 /AC-550 exhibited the highest acetylene conversion (97.8%). In addition, the acetylene conversion over g2 -C3N4 /AC obtained at different calcination temperatures decreased in the order of Sn-g2 -C3N4 /AC-450 (97.2%) >Sn-g2 -C3N4 /AC-500 (81.2%) >Sn-g2 -C3N4 /AC-550 (72.1%) >Sn-g2 -C3N4 /AC-650 (63.1%) >Sn-g2 -C3N4 /AC-400 (62.9%) . These results indicated that the optimal calcination temperatures are 450 °C and 550 °C for Sn-g2 -C3N4 /AC and Sn-g1 -C3N4 /AC, respectively. The hydrochlorination activities of Sn-g1 -C3N4 /AC-550 (97.8%), Sn-g2 -C3N4 /AC-450 (97.2%), and Sn-g3 -C3N4 /AC-400 (92.1%) [14] were higher than that of Sn/AC (89.1%)  [14]. Therefore, the enhancement in activity of alkylorganotinbased catalysts correlates to the g-C3N4 precursor, and hexamethylenetetramine is obviously the best g-C3N4 precursor. Usually, XRD is used to investigate the dispersion of metal species and eliminate the interference of metal agglomeration . As shown in Figure 2e, the XRD patterns of Sn-g1 -C3N4 -550, Sn-g2 -C3N4 -450, and Sn-g3 -C3N4 -400 display 3 obvious peaks at 26.7°, 33.9°, and 51.7°, which are characteristic to Sn-g-C3N4 . However, there are no typical peaks of tin metal (PDF#18-1380) and SnO2 phases (PDF#33-1374) in Sn-g-C3N4 , inferring that the high temperature favoured the formation of Sn-g-C3N4 . Figure 2f displays the XRD pattern of Sn-g1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, and Sn-g3 -C3N4 /AC-400, which are similar to that of AC. Furthermore, 3 peaks at around 24.4°, 26.7°, and 43.7°can be observed, which correspond to the (002), (103), and (101) diffraction planes of the graphitic carbon, respectively (PDF#50-0926). Particularly, the diffraction peaks of Sn-g-C3N4  were not observed in the diffractograms of Sn-g-C3N4 /AC samples , suggesting that Sn-g1 -C3N4 -550, Sn-g2 -C3N4 -450, and Sn-g3 -C3N4 -400 are well dispersed on the AC surface. In line with the conditions used to perform acetylene hydrochlorination at the industrial level, the reaction temperature and acetylene gas hourly space velocity were controlled in the range of 130 °C–180 °C and 30–50 h-1 , respectively [42]. However, when the reaction temperature was 180 °C , the acetylene conversion over the Sn-g-C3N4 /AC did not achieve ~98%. When the temperature increased to 200 °C, the acetylene conversion over Sn-g-C3N4 /AC increased to ~98%, which is close to the activity of HgCl2/AC. Therefore, the stability of catalysts was tested in acetylene hydrochlorination at 200 °C.Tables 2 and 3 summarize the BET specific surface areas and total pore volumes of Sn-g-C1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, and Sn-g3 -C3N4 /AC-400. Figure 3a shows the obtained results. After 14 h of reaction, the acetylene conversion of Sn-g1 -C3N4 /AC-550 was 97.8%, which decreased to 82.1% after 40 h. The acetylene conversion of Sn-g2 -C3N4 /AC-450 and Sn-g3 -C3N4 /AC-400 gradually decreased from 97.2% and 92.1% to 77.5 and 72.1%, respectively, after 40 h of reaction. By comparison, Sn/AC only exhibited an acetylene conversion of 49.5% after 40 h of reaction [14]. Sn-g1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, and Sn-g3 -C3N4 /AC-400 were highly selective toward VCM . Moreover, the selectivity did not change over the entire reaction period. After the reaction, the BET specific surface area and pore volume of the used catalysts decreased in comparison with those of the fresh catalysts (Table 4) due to the deposition of coke on the catalyst surface. Thus, the percentage decrease in the BET specific surface area of Sn-g1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, Sn-g3 -C3N4 /AC-400, and Sn/AC are 54, 68, 77, and 79%, respectively. This phenomenon is mainly attributed to the polymerization of acetylene and vinyl chloride during acetylene hydrochlorination [43–48]. Consequently, the g-C3N4 precursor may prevent the loss of the BET specific surface area of alkylorganotin-based catalysts in the hydrochlorination of acetylene, thereby extending the lifetime of the alkylorganotin-based catalysts. Furthermore, from this point of view, among the 3 g-C3N4 precursors, the hexamethylenetetramine is proved to be the optimum one.Next, stability experiments were performed for Sn-g1 -C3N4 -550, Sn-g2 -C3N4 -450, and Sn-g3 -C3N4 -400 catalyst surfaces. Sn3d , C1s , N1s , and Cl1s signals were detected in all 3 samples .XPS was employed to investigate the chemical states of Sn, N, C, and Cl on the Sn-gx (486.7eV) [51] and/or Sn-Clx (486.9 eV) in Sn-g-C3N4 /AC, which indicates bond formation between Sn4+ and N/Cl [52–54]. According to the relative peak areas (Table 6), the relative contents of Sn-Nx /Sn-Clx in the fresh Sn-g1 -C3N4 -550, Sn-g2 -C3N4 -450, and Sn-g3 -C3N4 -400 catalysts were 2.25%, 2.00%, and 1.64%, respectively. Table 6 summarizes the quantitative results for the N species, and they suggest that the alkylorganotins, which promote the formation of Sn-Nx bonds, hinder the loss of Sn during the preparation of catalysts. Compared with the Sn-g2 -C3N4 -450 and Sn-g3 -C3N4 -400, Sn-g1 -C3N4 -550 has a higher content of Sn-Nx /Sn-Clx . The four individual peaks observed at ~397.7, 398.4, 399.4, and 401.0 eV  correspond to the coexistence of Nx-Sn, pyridinicN, graphitic N, and pyrrolicN, respectively in all 3 catalysts  . The Nx-Sn content decreased in the order of Sn-g1 -C3N4 -550 (9.17 wt%) >Sn-g2 -C3N4 -450 (6.65 wt%) >Sn-g3 -C3N4 -400 (3.06 wt%), which is in well agreement with the catalytic activity trend (Table 7). The XPS results confirmed that Sn-Nx sites play a significant role in the improvement of the hydrochlorination performance of the catalysts. Moreover, compared with the catalysts prepared with urea and dicyandiamide, the catalyst prepared with hexamethylenetetramine (Sn-g1 -C3N4 -550) displays the highest Nx-Sn content.The Sn3d5/2 signal was fitted with 2 peaks . The peak at 487.5 eV corresponds to Sn-C  while the one at 486.8 eV corresponds to Sn-N1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, and Sn-g3 -C3N4 /AC- 400, TGA curves were recorded under nitrogen. Figures 5a, 5b, and 5c show the TG-DTG curves recorded between 25 °C and 800 °C for the investigated catalysts. All 3 catalysts exhibited a similar weight loss trend. DTG peaks observed at ~684.9 °C (Sn-g1 -C3N4 /AC-550), 517.1 °C (Sn-g2 -C3N4 /AC-450), and 299.0 °C (Sn-g3 -C3N4 /AC-400) suggested that the thermal stability of Sn-g1 -C3N4 /AC-550 is higher than those of Sn-g2 -C3N4 /AC-450 and Sn-g3 -C3N4 /AC-400.To investigate the thermal stability of Sn-g4+ and HCl as electron-acceptors do not react with each other [58], inferring that alkylorganotin firstly prefers to interact with C2H2 in acetylene hydrochlorination and then reacts with HCl to generate vinyl chloride. Therefore, reactant adsorption of catalysts behaves a significant impact on catalytic performance. As shown in Figure 6a, the acetylene adsorption capacity follows the order of Sn-g1 -C3N4 -550 >Sn-g2 -C3N4 -450 >Sn-g3 - C3N4 -400, suggesting that compared to Sn-g2-C3N4/AC-450 and Sn-g3-C3N4/AC-400, Sn-g1-C3N4/AC-550 exhibits the highest acetylene adsorption capacity. The temperature of acetylene adsorption on Sn-g1-C3N4/AC-550 (145.7 °C) is higher than those at which the acetylene was adsorbed on Sn-g2-C3N4/AC-450 (138.5 °C) and Sn-g3-C3N4/AC-400 (138.4 °C) . Therefore, the strength of acetylene adsorption on Sn-g1-C3N4/AC-550 is higher as compared to those on Sn-g2-C3N4/AC-450 and Sn-g3-C3N4/AC-400. The C2H2 - TPD profiles of Sn-g1 -C3N4 /AC-550, g1 -C3N4 /AC-550, and Sn/AC are illustrated in Figure 6b. Although Sn-C and Sn-Clx co-exist in Sn/AC and Sn-g1 -C3N4 /AC-550, the last sample exhibits higher acetylene adsorption capacity than Sn/AC-550. Furthermore, Figure 6b shows that the acetylene adsorption capacity and the adsorption strength both increased in Sn-g1 -C3N4 /AC-500 samples, improvement was associated with the existence of Sn-Nx in these catalysts. As shown in Table 6, hexamethylenechloride as nitrogen precursor can stabilize the content of Sn and thus, an enhanced catalytic performance was obtained for this sample in comparison with those of the catalysts obtained with urea and dicyandiamide. The corresponding catalytic results are depicted in Figure 2d.Both Sn1 -C3N4 /AC-550, Sn-g2 -C3N4 /AC-450, and Sn-g3 -C3N4 /AC-400 were 0.59, 0.51, and 0.40 mmol g-1 , respectively . Moreover, the amount of hydrogen chloride adsorbed on the catalysts with various compositions decreased in the order of Sng 1 -C3N4 /AC-550 (0.59 mmol g-1) >g1 -C3N4 /AC-550 (0.41 mmol g-1) >Sn/AC (0.37 mmol g-1) . The higher hydrogen chloride adsorption capacity of Sn-g1 -C3N4 /AC-550 is attributed to the coexistence of pyridinic N [24] and Sn-Nx sites.A previous study [59] has reported that hydrogen chloride adsorption is the rate determining step of the reaction. Thus, the adsorption of hydrogen chloride on the catalyst surface is the key factor in acetylene hydrochlorination. The amounts of hydrogen chloride adsorbed on Sn-g3N4 /AC exhibited acetylene conversion of 89.1% and 76.5%, respectively, in acetylene hydrochlorination . To meet the industrial requirement of activity and the green route development of chemical industry and further study the properties of Sn-based catalysts during acetylene hydrochlorination, in this study, Sn-g-C3N4 /AC as novel nonprecious metal-based catalyst was prepared with alkylorganotin and g-C3N4 precursors by wet impregnation, as well as exhibited higher acetylene conversion (97.8%). The excellent performance was mainly attributed to the coexistence of Sn-Nx and pyridinic N in Sn-g-C3N4 /AC catalysts. The results of XPS, TG-DTG, C2H2 -TPD, HCl adsorption, N2 physisorption, and stability tests confirmed that the g-C3N4 precursors can stabilize the Sn species provided by the alkylorganotin precursors, improve the thermal stability of Sn species and adsorption capacity of the resulted catalysts. In addition, the coke deposition is delayed over these alkylorganotin-based catalysts, which is favourable for a longer lifetime, as compared with Sn/AC. Among the 3 g-C3N4 precursors, hexamethylenetetramine, having higher nitrogen content, proved to be the best g-C3N4 precursor.According to the previous study, MF-600 with 94.5% acetylene conversion was prepared using melamine and toxic formaldehyde [57]. In addition, Sn/AC and g-C2 , 99.0%), and dicyandiamide  were purchased from the Tianjin Guangfu Fine Chemical Industry Research Institute. Carbon supports were purchased from Shanxi Xinhua Chemical Company. All reagents were used as received without any further purification.Tin(IV) chloride (99.0%) was purchased from Aladdin Co., Ltd. Dioctyldichlorotin (98.0%) was purchased from Energy Chemical Co., Ltd. Hexamethylenetetramine , urea  were prepared in a similar way.N1 (2.0 g) and AC (14.0 g) were mixed with ethanol (100 mL) and then stirred at 80 °C for 3.5 h. Afterwards, the mixture was dried overnight at 100 °C. Finally, the sample was subjected to calcination at 550 °C for 4 h to obtain the g3N4 /AC was prepared according to one of our previous studies by using an optimum molar ratio of 1.6:1.0 (SnCl4 to C16H34Cl2Sn) [14]. First, organotin compounds  and 2.0 g N1 were dissolved in 100 mL of ethanol and stirred at 80 °C for 30 min. Secondly, AC (14.0 g) was added to the solution and magnetically stirred for 3 h. Next, the obtained sample was dried overnight at 100 °C in an oven. Finally, solids were subjected to calcination at 450 °C for 4 h under nitrogen to obtain the final catalyst, labelled as Sn-g1 -C3N4 /AC-450. The other 2 catalysts  were prepared in a similar way.Sn-g-CPowder X-ray diffraction patterns were recorded on a Shimadzu XRD-6000 instrument with Cu-Kα radiation operated at 40kV.The textural properties of samples were analysed by nitrogen physisorption on a Nova2000e instrument (Quantachrome) after degassing the samples at 150 °C for 3h.-1 under air at a flow rate of 30 mL min-1 .Thermogravimetric analysis was performed on a NETZSCH STA 449F3 analyser. The temperature was increased from room temperature to 800 °C at a heating rate of 15°C minX-ray photoelectron spectroscopy was performed with an EscaLab 250Xi spectrometer using a monochromatic Al Kα source.2H2 -TPD) was performed with a FINESORB-3010 chemisorption analyser. TPD experiments were carried out with ~50 mg of sample, which was first treated at 200 °C for 1.5 h under Ar. After cooling, it was continually flushed with C2H2 at a flow rate of 25 mL min-1 for 1 h and then heated from room temperature to 500 °C at a heating rate of 10 °C min-1 .Acetylene temperature-programmed desorption . The amount of hydrogen chloride in the final solution was evaluated by titration [60].Hydrogen chloride adsorption experiments were performed in a fixed-bed reactor. Catalysts were initially pretreated at 200 °C for 1 h under Ar. Then, hydrogen chloride was fed into the reactor at a flow rate of 30 mL minHClV/C2H2V= 1.1) was passed through the reactor  . The unreacted hydrogen chloride in the product gas was adsorbed on limestone. The clean gas was then analysed online using a gas chromatograph equipped with a GDX-301column and TCD.The performance of catalysts (4.0 mL) was tested in a fix-bed reactor (d = 10mm). To activate the catalyst and remove the air and physisor bed water, hydrogen chloride gas was initially passed through the reaction system for 40 min. Then, a mixture of hydrogen chloride and acetylene (


Escherichia coli is one of the main pathogens responsible for veterinary and human infections, and it is associated with significant economic losses in the livestock, as it causes severe diseases to humans, particularly in children. For that reason, there is a need for introducing new drugs to treat E. coli diseases. The Brazilian species richness is a source of potential new antibacterial natural products. The study aimed at the biological and chemical investigation of the organic extract obtained from the stem of Microplumeria anomala (Apocynaceae), EB127, as it was identified as a potential source of new antibacterial compounds to be used in Veterinary.The antibacterial activity was evaluated by disk diffusion and microdilution assays; chromatography, nuclear magnetic resonance spectrometry, and mass spectrometry were used in the isolation and identification of compounds.E. coli ATCC25922, and against three E. coli strains that were isolated from frigarte’s cloaca, named 31/1A, 35A, and 51A. Lupeol, 3-acetyl-11-oxo-β-amyrin, 3-acetyl-11-oxo-α-amyrin, sitosterol, stigmasterol, 3β,7α-dihydroxy-cholest-5-ene, 3β-hydroxy-cholest-5-en-7-one, and 3β-hydroxy-cholest-5,22-dien-7-one were identified in fraction Hex/CHCl3, while loganin, loganic acid, methylanomaline, and anomaline were all identified in EB127 and protocatechuic acid hexoside, ferulic acid, secoxyloganin, feruloylquinic acid, vanillic acid hexoside, protocatechuic acid-4-O-β-hexoside, and rosmarinic acid were tentatively identified in fraction 10%ACN/H2O. E. coli 51A (virulent/non-resistant) showed sensitivity to the antibacterial action of fraction Hex/CHCl3 which contains alkaloids, triterpenes, and steroids, while E. coli 35A (resistant/non-virulent) were more susceptible to 10%ACN/H2O, which contains iridoids as loganin and loganic acid, and glycosylated and non-glycosylated caffeic acids.EB127 showed activity against 2O is of interest in pursuing new drugs to treat resistant E. coli, in veterinary. All compounds were isolated from the plant for the first time and have shown potential as new antibacterial natural products from Amazon plants to be used in veterinary and human diseases.Fraction 10%ACN/H Escherichia coli is one of the most important micro-organisms in veterinary pathology due to its impact on animal production and in pet’s disease. E. coli is a Gram-negative bacterium with a short rod shape, facultative anaerobe that does not sporulate . After being dried under 40°C and ground in a hammer mill , the plant material was submitted to a 24h-maceration with methanol and dichloromethane (1:1) . The organic extract, named EB127, (2.55% yield) had the solvents evaporated under reduced pressure (Buchi) before being stored at −27°C (Revco) until its use [The stem of  its use ,16. The E. coli (Migula) Castellani and Chalmers ATCC® 25922™  and strains 31/1A, 35A, and 51A, obtained from frigate (Fregata magnificens) cloaca, were used in the biological assays. Isolated bacteria were previously classified according to their virulence/resistance phenotypes  Petri dishes . Fresh bacteria suspensions were prepared at 0.5 McFarland (or 1.5 × 108 CFU/mL). Petri dishes containing 15 mL of MHA, prepared according to the manufacturer’s instructions, were surface-inoculated [All oculated  using stThe assays were performed with EB127, its residues and fractions, as well as with the isolated substances, in triplicate, in Petri dishes of 120 mm diameter. Two diameters of each inhibition zone generated were measured, so, six measurements were obtained for each treatment. The same procedure was repeated for all analyzes.®, Thermo, USA), in 96-well microplates , with inoculum adjusted to a concentration of 1 × 108 colony-forming unit per mL (CFU/mL) grown on sterilized Müeller-Hinton agar medium . A 190 μL aliquot of the inoculum was dispensed into each well, and 10 μL aliquots of extract/fractions were added to the inoculum. Microplates were incubated at 36°C for 24 h. Inhibition of bacterial growth was assessed by subculturing each well of the microplate on sterile agar medium, so as to evaluate bacterial growth. Following a procedure similar to that described above in sterile MBA, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were obtained for the samples, against the four E. coli strains. Solvent dimethyl sulfoxide  were tested as vehicle control [The extract and its fractions were tested in the MBA; Oxoid, London, England), using sterilized Müeller-Hinton broth medium , for 24 h. The samples were dissolved in DMSO-H20 (1:1 v/v) and tested, in sextuplicates, against positive control doxorubicin .The cytotoxicity was tested by the sulforhodamine B  assay against human breast carcinoma (MCF-7) and prostate carcinoma (PC-3) cell lines -21. The 3, BuOH, and water, which resulted in three residues named CHCl3, BuOH, and H2O, respectively. Organic residues had the solvents evaporated (Buchi) while the aqueous fraction was lyophilized (Virtis). The residue CHCl3 was subjected to a new fractionation using column chromatography (CC) packed with Sephadex LH-20. The solvents hexane (Hex), dichloromethane (DCM), and MeOH were subsequently used as the mobile phase, to obtain three new fractions, as shown in 2O were subjected to CC packed with C-18 reversed-phase silica as the stationary phase and the mixture composed of acetonitrile (ACN):water (H2O) acidified with 0.1% trifluoroacetic acid  in the proportion of 10% and 50%, and MeOH100% as the subsequent mobile phases to perform the elution. Three new fractions were obtained for each residue and were named Hex/CHCl3, DCM/CHCl3, MeOH/CHCl3, 10%ACN/BuOH, 50% CAN/BuOH, MeOH/BuOH, 10%ACN/H2O, 50% CAN/H2O, and MeOH/H2O .EB127 was fractionated using a liquid-liquid partition system , as desc3, 10%ACN/BuOH, 50%ACN/BuOH, MeOH/BuOH, 10% ACNH2O, 50%ACNH2O, and MeOH/H2O . Each sample was weighed (1 mg), solubilized in MeOH (2 mL) and filtered with 0.45 μM Millex™ JBR13LCR1 filters . The eluent system was A: 0.1% TFA water, B: HPLC grade ACN, filtered with 47 mm diameter Nylon (0.45 μM) Phenex Filter Membranes  filter. The chromatographic run was performed in gradient system: T0 → 5 = 5% B, T5 → 35 = 5% B up to 100% B, T35 → 40 = 100% B, T40 → 50 = 100% B up to 5 % of B, T50 → 55 = 5% of B, in Agilent’s HPLC  and DAD detector , injection volume was 20 μL.Reversed-phase HPLC fingerprints were obtained for fractions MeOH/CHCl2O was submitted to analysis performed in high-performance liquid chromatography coupled to electrospray ionization mass spectrometer . Equipment was set at T0-10 = 5% ACN, T10-11 = 10% ACN, T11-20 = 10% ACN, T20-21 = 20% ACN, T21-40 = 20% of ACN, T40-41 = 27% of ACN, T41-60 = 27% of ACN, T60-61 = 32% of ACN, T61-68 = 32% of ACN, T68-69 = T69-85 = 39% ACN, T85-86 = 50% ACN, T86-96 = 50% ACN, T96-106 = 100% ACN, T106-110 = 100% ACN, and T110-120 = 5 % ACN.Fraction 10%ACN/H1H-NMR, 300 MHz, CDCl3; 13C NMR, 75 MHz, CDCl3, respectively) analyses to carry out structural elucidation of the samples.The purified fractions were subjected to hydrogen and carbon NMR spectrometry in Bruker AIII spectrometer .E. coli. The strains that were used in the assays are displayed in =10.52; p=0.0002). E. coli ATCC25922. There was a clear effect of fraction 10%ACN/H2O, which was as effective as EB127, in relation to the other treatments =997.9; p<0.0001). E. coli 31/1A, a virulent and relatively resistant E. coli strain. Although there was significance in the growth inhibition zone of EB127 in relation to the fractions =8,97; p<0.0001), it is possible to observe that fractions Hex/CHCl3 and 10%ACN/H2O were more active than the other ones. E. coli 35A, a non-virulent but resistant E. coli strain. Fraction 10%ACN/H2O was significantly more active than EB127 or the other fractions =3260; p<0.0001). E. coli 51A, a virulent but not resistant E. coli strain. Fraction Hex/CHCL3 was significantly more active than EB127 or the other fractions =1478; p<0.0001). E. coli ATCC25922. Although all purified fractions performed better than EB127, fraction UN-251 showed to be as active as CHX 1% =24.58; p<0.0001).E. coli strains which suspensions were prepared at different concentrations. Reports are displayed in EB127 was tested in the MBA model to determine MIC and MBC against the four different Microplumeria anomala. Microplumeria anomala.Compounds had the structure elucidated based on data obtained from hydrogen and carbon NMR and from mass spectrometry.3) shows the characteristic signals of a triterpene, such as two doublets in δ 4.69 and 4.57 with J = 4.69Hz, both doublets corresponding to the hydrogens bound to C-29, a double-doublet in δ3, 19 with J = 10.94, 5.14 Hz corresponding to H-3. Six singlets between δ0.76 and 1.035 corresponding to the methyl groups 23, 24, 25, 26, 27, and 28 and a singlet at δ 1.69 of methyl C-30. The 13C-NMR spectrum (CDCl3) shows 29 carbons: 14,52 (C-27), 15,35 (C-24), 15,95 (C-26), 17,98 (C-28), 18.29 (C-6), 19,28 (C-30), 20,90 (C-11), 25,10 (C-12), 27,38 (C-15), 27,42 (C-2), 27,96 (C-23), 29.82 (C-21), 34,25 (C-7), 35,55 (C-16), 37,14 (C-10), 38,02 (C-13), 38,68 (C-1), 38,83 (C-4), 39.97 (C-22), 40,79 (C-8), 42,80 (C-14), 42,97 (C-17), 47,95 (C-19), 48,27 (C-18), 50,41 (C-9), 55.27 (C-5), 78,97 (C-3), 109,30 (C-29), and e 150,93 (C-20), data were compared with the literature [Lupeol (A) was identified in two fractions called UN-217 (10 mg) and UN-250 (9.8 mg). The 1H-NMR spectrum  shows the characteristic signals of an acetylated oleane and ursane skeleton such as the singlets at δ 5.69 and 5.55, each corresponding to the H-12 of the ursane and oleane skeleton, respectively. A double-doublet sign at δ 4.52 with J = 11.79 and 4.61 Hz can be seen and corresponds to the H-3 of both skeletons. The singlet signs at δ 2.36 and 2.35 corresponds to the H-9 of the ursane and oleane skeleton, respectively. A singlet sign at δ 2.06 corresponds to methyl acetate. Also, the 14 singlets ranging from δ 0.82 to 1.37 correspond to the methyl groups of both compounds, and the two doublets at δ 0.81 and 0.90 with J = 6 Hz corresponds to the methyl groups attached to the C-19 and C-20 of the ursane skeleton. The 13C-NMR spectrum (CDCl3) reported for 11-oxo-3-β-methylamine shows the following signs: 16.39 (C-25), 16.67 (C-24), 18.49 (C-6), 21.29 (CH 3 -COO), 24.47 (C-30), 23.55 (C (C-15), 27.21 (C-2), 28.04 (C-23), 28.74 (C-28), 30 (C-21), 34.42 (C-21), 36.48 (C-20), 36.48 (C- 23), 36.91 (C-10), 38.03 (C-4), 38.78 (C-1), 43.38 (C-8), 45.10 (C- 13 (C-14), 47.59 (C-18), 54.97 (C-5), 61.66 (C-9), 80.64 (C-3), 128.04 (C-13), 171.00 (COO), and 200.17 (C-11). 13C-NMR spectrum (CDCl3) and for 11-oxo-3α-methylamine, the following signs: 16.54 (C-24 and C-25), 17.45 (C-26), 18.69 (C-6), 20.47 (C-30), 21.29 (CH 3 COO), 23.36 (C- 27), 27.48 (C-2), 28.04 (C-23), 28.48 (C-15) (C-10), 38.03 (C-7), 36.78 (C-10), 38.03 (C-7), 38.86 (C-1), 39.19 (C-20), 39.26 (C-19), 40.89 (C-22), 43.62 (C-8), 45.13 (C (C-3), 130.37 (C-12), 164 (C-3), 99 (C-13), 171.00 (COO), and 199.71 (C-11). The data were compared with the literature [The mixture of 3-acetyl-11-oxo-β-amyrin (B) and 3-acetyl-11-oxo-α-amyrin (C) was identified in the sample UN-257 = 10.1 mg. The 3) showed the characteristic signals of these steroids as two broad singlets at δ 5.36 and 5.35 corresponding to the stigmasterol and sitosterol H-6, respectively, a double-doublet at δ 5.16 with J = 15.22, 8.8 Hz corresponding to the H-20 of stigmasterol, a double-doublet at δ 5.02 with J = 15.05, 8.62 Hz corresponding to the H-19 of stigmasterol, and a multiplet in δ 3.53 corresponding to the H-3 of both steroids. The signals corresponding to the steroid methyl groups are present in δ 0.69-1.26. The 13C-NMR spectrum (CDCl3) for sitosterol: 11.85 (C-29), 11.96 (C-18), 18, (C-19), 19.79 (C-27), 21.07 (C-11), 23.04 (C-28)), 24.28 (C-15), 26.06 (C-25), 28.23 (C-16), 29.68 (C-23), 31.63 (C-2), 31.88 (C-7 and C-8), 33.93 (C-22), 36.13 (C-20), 36.16 (C-10), 37.23 (C-1), 39.75 C-12), 42.19 (C-13), 42.28 (C-4), 50.13 (C-9 and C-24), 56.04 (C-17), 56.74 (C-6), and C (C-5), for stigmasterol, 12.02 (C-29), 12.23 (C-18), 18.97 (C-26), 19.38 (C-19), 21.07 (C-11), 21.20 (C-21 and C-27), C-15), 25.39 (C-28), 28.89 (C-16), 31.44 (C-2), 31.88 , 36 (C-10), 37.23 (C-1), 39.66 (C-12), 40.47 (C-20), 42.19 (C-13), 42.28 (C- 4), 50.13 (C-9), 51.22 (C-24), 55.93 (C-17), 56.85 (C-14), 71.78 (C- (C-6), 129.25 (C-23), 138.31 (C-22), and 140.73 (C-5), data were compared with the literature [The mixture of sitosterol (D) and stigmasterol (E) was identified in the sample UN-271 = 17.5 mg. The 1H-NMR spectrum (CDCl1H-NMR spectrum (CDCl3) shows the following signals, a broad doublet at δ 5.61 with J = 5.3 Hz corresponding to H-6. A multiplet in δ 3.59 corresponding to H-3 and the methyl groups corresponding to the proposed structure are present between δ 0.69 and 1.10. The 13C-NMR spectrum (CDCl3): δ 11.79 (C-18), 18.23 (C-19), 18.89 (C-21), 20.68 (C-11), 22 (C-16), 67 (C-26), 23.04 (C-23 and C-27), 24.28 (C-15), 28.26 (C-34 (C-2), 35.83 (C-20), 36.08 (C-22), 36.99 (C8), 37.38 (C-1), 37.49 39.15 (C-12 and C-24), 42.12 (C-4), 42.26 (C-9 and C13), 49.47 (C-14), 65.35 (C-7), 71.33 (C-3), 123.84 (C-6), and 146.23 (C-5), the data were compared with the literature [Sample UN-274 = 5.3 mg contained the steroid 3β,7α-dihydroxy-cholest-5-ene (F). The terature .1H-NMR spectrum (CDCl3) shows the characteristic signals of these steroids as δ 5.69 of H-6 of the two steroids: A double-doublet in δ 5.18 with J = 15.26, 8.85 Hz of H-23  and a double-doublet in δ 5.03 with J = 15.16, 8.54 Hz of H-22 . A δ 3.68 multiplet of the H-3 of both steroids and the steroid methyl groups are present between δ 0.68 and 1.21.The mixture of the 3β-hydroxy-cholest-5-en-7-one (G) and 3β-hydroxy-cholest-5,22-dien-7-one (H) steroids was identified in the UN-283 sample. The 13C-NMR spectrum (CDCl3): δ 11.96 (C-18 and C-29), 17.31 (C-19), 18.98 (C-26) (C-11), 22.69 (C-28), 23.04 (C-15), 26.31 (C-23), 28.55 (C- C-16), 29.36 (C-25), 31.18 (C-2), 33.72 (C-22), 36.08 (C-20), 36.34 (C-1), 36.27 (C-10), 38.68 (C-12), 41.79 (C-4), 43.09 (C-13), 45.41 (C-8), (C-9), 54.69 (C-17), 70.52 (C-3), 126.11 (C-6), 165.06 (C- 5), and 202.33 (C-7), for 3β-ol-choleste-5,19-dien-7-one: δ 11.96 (C-18), 12.25 (C-29) 31 (C-19), 19.03 (C-27), 19.78 (C-26), 20.49 (C-21), 21.2 ), 23.04 (C-15), 28.55 (C-16), 31.18 (C-2), 31.9 (C-25), 36.34 (C-1), 36.27 (C-10), 38.68 (C-12), 40.25 (C-20), 41.79 (C-4), 43.09 (C-13), 45.41, C-17, C-14, C-14, C-14, C-14, (C-22), 138.08 (C-22), 165.06 (C-5), and 202.33 (C-7), data were compared with the literature [The  18.98 C- (C-11), 3 and DCM/CHCl3 fractions, by CC, analytical thin-layer chromatography and preparative thin-layer chromatography. Methylanomaline (I) was identified by means of 1H-NMR and 13C-NMR [The sample named UN-244 was obtained from the Hex/CHCl 13C-NMR   was identified by means of 1H-NMR and 13C-NMR [The sample named UN-374 was obtained from the Hex/CHCl 13C-NMR   was identified by means of 1H-NMR and 13C-NMR [Loganin was identified in the sample named UN-365, which was obtained from the 10%ACN/H 13C-NMR   was identified in the sample named UN-365, which was obtained from the 10%ACN/H 13C-NMR   cloaca [E. coli 31/1A, characterized by a virulent and resistant profile, was classified in the B2 phylogenetic group, and was named O2:H7. E. coli 35A, characterized by a non-virulent and resistant profile, was classified in the A phylogenetic group, and was named NP. E. coli 51A, characterized by a virulent and non-resistant profile, was classified in the B2 phylogenetic group, and was named ONT:H7.The present study aimed at the evaluation of the antibacterial activity of M. anomala against four ) cloaca . These b) cloaca , the chaE. coli ATCC® 25922™ was used in the initial screening made with more than 1300 plant extracts obtained from the Amazon Forest, which resulted in four active extracts, being EB127 one of them [E. coli that were obtained from frigate’s cloaca were introduced in the analysis, to verify whether the antimicrobial potential of the plant extract would be the same, or if it would be specific to each kind of micro-organism, considering their characteristics of virulence and resistance.2O was more active against E. coli ATCC and E. coli 35A, which is a non-virulent and resistant strain, while fraction Hex/CHCl3 is active against E. coli 51A, which is a virulent and non-resistant strain. E. coli 31/1A, which is a virulent and resistant strain, showed sensibility against all the fractions, particularly the two fractions mentioned before. The interpretation can be related to the chemicals present in each of the fractions.According to the antibacterial findings, it was possible to observe that EB127 and its fractions showed specificity in antibacterial activity, as the fraction 10%ACN/HM. anomala belongs to the Apocynaceae family, which is known to have compounds widely used in therapeutics, as the vinca alkaloids vinblastine and vincristine, and some cardioactive glycosides, as those occurring in Thevetia peruviana [M. anomala were reported, the presence of alkaloids such as aspidocarpine, anomaline, methylanomaline, and demethoxyanomaline has been previously described in the species [eruviana . Bioactieruviana . Althoug species .3 and 10%ACN/H2O were selected to be chemically studied due to their significant antibacterial activity against the E. coli strains that were used in the present evaluation. Lupeol, 3-acetyl-11-oxo-β-amyrin, and 3-acetyl-11-β-amyrin, sitosterol, and stigmasterol, 3β, 7α-dihydroxy-cholest-5-ene, 3β-hydroxy-cholest-5-en-7-one and 3β-hydroxy-cholest-5,22-dien-7-one, anomaline, and methylanomaline were isolated from fraction Hex/CHCL3, while protocatechuic acid hexoside [2O.EB 127 was fractionated and nine fractions of different polarities were obtained. These fractions were submitted to DDA and two active fractions, named Hex/CHCLhexoside , ferulichexoside , secoxylhexoside , feruloyhexoside , vanillihexoside , protocahexoside , rosmarihexoside , loganichexoside , and loghexoside  were ide3, which has been active against E. coli 51A, the virulent and non-resistant strain, is formed by lupeol, stigmasterol, sitosterol, 3-acetyl-11-oxo-β-amyrin and 3-acetyl-11-β-amyrin, sitosterol and stigmasterol, 3β, 7α-dihydroxy-cholest-5-ene, 3β-hydroxy-cholest-5-en-7-one and 3β-hydroxy-cholest-5,22-dien-7-one, anomaline, and methylanomaline. Lupeol was isolated and tested in the DDA model against the E. coli strain ATCC 25922 and in the cytotoxicity models against breast and prostate cancer cell lines, showing no significant antimicrobial or cytotoxic activity. The literature provides controversial data regarding the antibacterial activity of lupeol. While some authors report the activity of this compound against Gram-positive bacteria and fungi [3, as some derivatives of amyrin, sitosterol, and stigmasterol. Studies done with Aspidosperma pyrifolium from the caatinga forest, in Brazil, showed the occurrence of some alkaloids, lupeol, and amyrin, but there are no studies to prove that these isolated molecules are related to the biological activities. The Chinese plant Ilex asprella is commonly used as anticancer drugs, anti-flu, and anti-inflammatory drugs. Its main components are the α- and β-amyrin triterpenes [Alstonia scholaris also have shown antibacterial activity [Rauvolfia caffra and showed some activity against Mycobacterium tuberculosis [The group of compounds that are reported to occur in fraction Hex/CHCLnd fungi , others nd fungi . Triterpterpenes . Pentacyactivity . A sitosrculosis .3. Although these alkaloids have been identified in M. anomala before [M. anomala, followed by methylanomaline, although no activity was seen against E. coli strains, in the DDA assay, and no antibacterial activity against was observed to any of the E. coli strains tested in the present work. We figured that the antimicrobial activity of fraction Hex/CHCl3 is due to a synergistic activity of the compounds that were identified in the fraction.Alkaloids anomaline and methylanomaline were also identified in fraction Hex/CHCla before , no repo2O is formed by protocatechuic acid hexoside, ferulic acid, secoxyloganin, feruloylquinic acid, vanillic acid hexoside, protocatechuic acid-4-O-β-hexoside, rosmarinic acid, loganic acid, and loganin. The iridoids belong to the terpene class and can be found in more than 50 plant families. Up to 2,500 different iridoids have been cataloged so far, and they can be found in both plants and animals. Iridoids were identified to occur in several Angiosperms and have a function of eliminating herbivorous predators through the production of poisons and for having a bitter taste. Its biological properties are related to anti-inflammatory, vasoconstricting, antiviral, antitumor, and antibacterial activities [2O gathers more active compounds than fraction Hex/CHCl3 and was found to be more active against E. coli 35A, which is non-virulent and resistant. Further work concerning the evaluation of the antimicrobial activity of the isolated compounds is required, but present findings have shown that the enriched fraction named 10%ACN/H2O is of interest in pursuing new drugs to treat resistant E. coli, in veterinary.The group of compounds that were identified in fraction 10%ACN/Htivities ,44. Fractivities , and alstivities . FractioE. coli strains. Fractions obtained from EB127, named Hex/CHCl3, containing the triterpenes, steroids, and alkaloids, and 10%ACN/H2O, containing the iridoids and phenolic acids, showed a specific antibacterial activity against the virulent or the resistant strains, respectively. None of the extract or fractions have shown cytotoxicity against breast and prostate cancer cell lines. The present findings indicate that the crude extract obtained from the stem of Microplumeria anomala can be considered as a potential source of new veterinary antibacterial products to be used in the treatment of E. coli diseases. Nineteen Microplumeria anomala compounds were described and their occurrence is being reported in the species for the first time.EB127, an organic extract containing triterpenes, steroids, iridoids, and alkaloids, showed activity against four LRPC collected data. MLBP, SAF, and IECD collected plant samples. VMC, RNY, LFLR and ADV analyzed data. IBS designed and managed the study, analyzed data and wrote the manuscript. All authors have read and approved the final manuscript.


In this study, Ech A degradation products formed during oxidation by O2 in air-equilibrated aqueous solutions were identified, isolated, and structurally characterized. An HPLC method coupled with diode-array detection (DAD) and mass spectrometry (MS) was developed and validated to monitor the Ech A degradation process and identify the appearing compounds. Five primary oxidation products were detected and their structures were proposed on the basis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) as 7-ethyl-2,2,3,3,5,7,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone (2), 6-ethyl-5,7,8-trihydroxy-1,2,3,4-tetrahydronaphthalene-1,2,3,4-tetraone (3), 2,3-epoxy-7-ethyl-2,3-dihydro-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone (4), 2,3,4,5,7-pentahydroxy-6-ethylinden-1-one (5), and 2,2,4,5,7-pentahydroxy-6-ethylindane-1,3-dione (6). Three novel oxidation products were isolated, and NMR and HR-ESI-MS methods were used to establish their structures as 4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid (7), 4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid (8), and 4-ethyl-2,3,5-trihydroxybenzoic acid (9). The known compound 3-ethyl-2,5-dihydroxy-1,4-benzoquinone (10) was isolated along with products 7–9. Compound 7 turned out to be unstable; its anhydro derivative 11 was obtained in two crystal forms, the structure of which was elucidated using X-ray crystallography as 7-ethyl-5,6-dihydroxy-2,3-dioxo-2,3-dihydrobenzofuran-4-carboxylic acid and named echinolactone. The chemical mechanism of Ech A oxidative degradation is proposed. The in silico toxicity of Ech A and its degradation products 2 and 7–10 were predicted using the ProTox-II webserver. The predicted median lethal dose (LD50) value for product 2 was 221 mg/kg, and, for products 7–10, it appeared to be much lower (≥2000 mg/kg). For Ech A, the predicted toxicity and mutagenicity differed from our experimental data.Echinochrome A (Ech A,  Histoch1) is one of the main pigments of various sea urchin species −, which was 18 Da less than mass of compound 7. The absorption band at 385 nm in the absorption spectrum of compound 11 indicated a longer π → π* transition chain in its molecule compared to compound 7. In the 1H-NMR spectrum of compound 11, we observed a triplet (δH 1.24) and a quartet (δH 2.78) of an ethyl substituent, a broadened singlet of two hydroxyl groups (δH 5.33), and a singlet of the hydroxyl group bound to carbonyl (δH 12.84) , three quaternary carbons , and seven quaternary carbons bound to oxygen   showed correlations with δC 121.4, 159.0, and 161.6 . Recrystallization from these solvents simultaneously provided crystals as dark-red plates (α-form) and as orange prisms (β-form) (11 (It turned out that compound H 12.84) . The 13Cd 177.8) . In the nd 161.6 . These s(β-form) . Red cryorm)  Å, b = 7.9520(9) Å, c = 14.4705(17) Å, and Z = 4, and the final R-value was found to be 0.0512  Å, b = 7.9520(9) Å, c = 14.4705(17) Å, and Z = 2, and the final R-value was found to be 0.0407  at a temperature of T = 173(2) K than the monoclinic α-form (1.642 g/cm3) and could formally be considered as more stable.In the α- and β-forms of molecule me plane . The devme plane ; in the e values . The torramework . The cooirection . Ribbonsirection . The coo11 was assigned the structure of 7-ethyl-5,6-dihydroxy-2,3-dioxo-2,3-dihydrobenzofuran-4-carboxylic acid, and this compound was named echinolactone.On the basis of X-ray data, 7 and 8 turned out to be unstable under conditions of repeated chromatographic separation; therefore, to establish their structures, their stable methyl derivatives with m/z [M − H]− 297 and 239, respectively, were obtained by methylation with diazomethane.Compounds 1H- and 13C-NMR spectra of the dimethyl ether of compound 7, methyl ether of compound 8, and of compound 9  was hydrogen-bonded to the carbonyl of the ester group at C-9 (δH 169.1), and the proton of the hydroxyl group at C-3 (δH 11.30) was hydrogen-bonded to the carbonyl of the methylcarboxy group at C-10 (δC 186.8)  . The sig13C-NMR spectrum of methyl ether of compound 8 contained a signal in a low field at δC 195.2, the chemical shift of which was characteristic for the aldehyde carbon atom , which corresponded to a signal at δC 104.8 ppm in the 13C-NMR spectrum (H 7.54) indicated that it was not bound by an intramolecular hydrogen bond as in compound 8. Thus, the structure of compound 9 was established as 4-ethyl-2,3,5-trihydroxybenzoic acid , the result of which is bis-gem-diol 2 formation, were described in detail in previous studies −; HR-ESI-MS m/z: 299.0399 [M − H]− .4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid (7); C11H10O8; UV (CH3CN–H2O) λmax 219, 271, 320 nm; ESI-MS m/z: 269 [M − H]−; HR-ESI-MS m/z: 269.0304 [M − H]− .Dimethyl ether of compound7: light-yellow powder; C13H14O8; UV (CH3CN–H2O) λmax 224, 250, 324, 384 nm; IR (CDCl3) νmax 3520, 3156, 2975, 2956, 2937, 2878, 2855, 1734, 1683, 1632, 1595 cm−1; ESI-MS m/z: 297 [M − H]−; 1H- and 13C-NMR ; C10H10O6; light-yellow powder; UV (CH3CN–H2O) λmax 270, 320 nm. ESI-MS m/z: 225 [M − H]−; HR-ESI-MS m/z: 225.0405 [M − H]− .Methyl ether of compound8: C11H12O6; light-yellow powder; UV (CH3CN–H2O) λmax 238, 303, 369 nm; IR νmax (CDCl3) 3011, 2959, 2935, 2878, 2865, 1668, 1634, 1611 cm−1. ESI-MS m/z: 239 [M − H]−; HR-ESI-MS m/z: 239.0570 [M − H]− ; 1H- and 13C-NMR ; C9H10O5; light-yellow powder; UV (CH3CN–H2O) λmax 228, 251, 333 nm; IR νmax (CCl4) 3352, 2960, 2928, 2875, 2855, 1642 cm−1. ESI-MS m/z: 197 [M − H]−; HR-ESI-MS m/z: 197.0452 [M − H]− ; 1H- and 13C-NMR ; C8H8O4; light-yellow powder; UV (CH3CN–H2O) λmax 228 nm, literature 228 nm −; HR-ESI-MS m/z: 167.0343 [M − H]− . 1H-NMR  δ, ppm : 1.21 , 2.49 , 6.01 , 7.54 ; 1H- and 13C-NMR data of dimethyl ether of 10 : C11H8O7; UV (ethanol) λmax (logε ): 215 (3.4), 331 (2.8), 385 (2.8) nm; IR νmax (CDCl3) 3483, 1838, 1698, 1581 cm−1; ESI-MS m/z: 251 [M − H]−; EI-MS m/z: 252 [M]+. 1H-NMR , δ, ppm: 1.24 , 2.78 , 5.24, , 12.88 . 13C-NMR , δ, ppm: 13.3, 17.8, 106.9, 108.8, 121.9, 151.7, 158.6, 160.5, 162.2, 171.5, 178.4.11 crystal forms are given in The main crystal data, data collection, and refinement parameters are presented in The potential acute toxicity, hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity of Ech A and its degradation products via oral administration were assessed using ProTox-II .2 in air-equilibrated aqueous solutions were identified, isolated, and structurally characterized. During the oxidation of Ech A, transformation was found to occur only in the quinonoid ring, with the formation of a hydrogenated quinonoid cycle with two carbonyl groups and two pairs of geminal hydroxyl groups. The further oxidation of bis-gem-diol occurred with the cleavage of the dihydroquinonoid ring and a chain of subsequent decarboxylations.In this study, Ech A degradation products formed during oxidation by OThe HPLC method with DAD and MS detection was developed and validated to monitor the Ech A degradation process and to identify the appearing compounds. The structural studies and obtained HPLC–MS parameters of the main Ech A oxidation products are of great interest from the point of view of investigation of the chemical properties of drug substances and for developing methods for monitoring the quality of drugs and food additives obtained from sea urchin pigments, in addition to their stability.In recent years, there has also been a growing interest in the environmental significance of methods to characterize the destruction of pharmaceutical compounds and study their toxic properties in order to avoid the accumulation of these compounds in nature.The in silico toxicity studies performed using ProTox-II webserver revealed that Ech A oxidative degradation products do not exhibit mutagenic properties, and their toxicity values were much lower than that of Ech A. This means that the spontaneous formation of these degradation products in preparations using Ech A would not be harmful to patients.


To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike active cytokinins-bases, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2′-deoxyribo- and 5′-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2′-deoxyribo- and 5′-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5′-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be “activated” by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid  and aromatic  cytokinins indicate a significant difference in the biosynthesis of these compounds.The biosynthesis of aromatic cytokinins  N6 position. CKs are divided into two groups depending on the structure of the N6 substituent: (i) aliphatic or isoprenoid, including N6-isopentenyladenine (iP) and zeatins (N6-benzyladenine (BA), topolins, and N6-furfuryladenine (kinetin) (Cytokinins (CKs) are a group of phytohormones that play a crucial role in many processes of plant growth and development. One of the most important effects of CKs is the stimulation of plant cell division and growth. CKs promote the formation of shoots, control the root development, stimulate seed germination and the formation of pigments, activate the chloroplast formation, etc. ,2. Natur zeatins A, and (ikinetin) B 1,2].,2.N6 posin vivo is obviously due to complex pathways of their biosynthesis.Since the discovery of kinetin in 1955 by Miller, Skoog et al. , a largeN6 position of adenosine-5′-monophosphate (AMP), adenosine-5′-diphosphate (ADP) or adenosine-5′-triphosphate (ATP), with the formation of the respective 5′-nucleotides  (N6-isopentenyladenosine (iPR), followed by hydrolysis of the N-glycosidic bond catalyzed by adenosine nucleosidase. The reverse process of ribosylation with the formation of nucleosides is catalyzed by purine nucleoside phosphorylase, and the process of reverse phosphorylation of nucleosides with the formation of nucleotides is catalyzed by adenosine kinase; (ii) iPRm.p. can be directly cleaved to iP by specific enzyme phosphoribohydrolase (LOG). Importantly, LOG specifically binds only the 5′-monophosphates of cytokinin nucleosides, but not di- or triphosphates, Am.p. or cytokinin ribosides. The reverse process of iPRm.p. formation from iP is catalyzed by adenosine phosphoribosyltransferase undec-7-ene (DBU) (1 mmol), and corresponding bromide (1 mmol) in dry acetonitrile (5 mL) was kept at ambient temperature for 24 h. The reaction was monitored by TLC . After 24 h the reaction mixture was concentrated in vacuo to dryness. The residue was diluted with ethyl acetate (20 mL) and washed successively with brine (2 × 20 mL), 10% aqueous sodium bicarbonate (20 mL) and water (2 × 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel. The column was washed with methylene chloride, the product was eluted with CH2Cl2-EtOH, 98:2. Purified acetyl-protected compound was dissolved in 4M n-PrNH2 in MeOH solution (50 mmol) and was left for 24 h, after which the mixture was concentrated in vacuo and the residue was purified by column chromatography on silica gel. The column was washed with CH2Cl2-EtOH, 95:5 and the product was eluted with CH2Cl2-EtOH, 90:10. The resulting product was dried for 24 h in a vacuum desiccator over phosphorous pentaoxide (P2O5).A mixture of N6-benzyladenosine (4). Yield for two steps was 72% as a powder. Rf = 0.32 . m.p. 164–165 °C. 1H-NMR : δ = 8.40 br s , 8.37 s , 8.20 br s , 7.37–7.17 m , 5.89 d , 5.41 d , 5.34 dd , 5.15 d , 4.72 br s , 4.61 ddd , 4.15 ddd , 3.97 ddd , 3.67 ddd , 3.56 ddd . 13C NMR : 154.62 (C-6), 152.39 (C-2), 148.48 (C-4), 139.96 (C-8), 128.26 (Ph), 127.16 (Ph), 126.68 (Ph), 119.78 (C-5), 88.05 (C-1′), 85.96 (C-4′), 73.58 (C-2′), 70.70 (C-3′), 61.72 (C-5′), 42.99 (NHCH2). HRMS: m/z [M + H]+ calculated C17H20N5O4+ 358.1510, found 358.1510.N6-isopentenyladenosine (5). Yield for two steps was 91% as a powder. Rf = 0.45 . m.p. 144–146 °C. 1H-NMR : δ = 8.31 s , 8.19 br s , 7.82 br s , 5.87 d , 5.40 d , 5.39 dd , 5.33–5.27 m , 5.15 d , 4.60 ddd , 4.14 ddd , 4.08 br s , 3.96 ddd , 3.67 ddd , 3.55 ddd . 13C NMR : 154.93 (C-6), 153.14 (C-2), 148.63 (C-4), 140.49 (C-8), 135.41 (=CMe2), 121.73 (CH=), 120.14 (C-5), 88.79 (C-1′), 86.57 (C-4′), 74.22 (C-2′), 71.20 (C-3′), 62.22 (C-5′), 38.57 (NHCH2), 26.02 (Me), 18.49 (Me). HRMS: m/z [M + H]+ calculated C15H22N5O4+ 336.1666, found 336.1666.N6-benzyl-2′-deoxyadenosine (8). Yield for two steps was 55% as a foam. Rf 0.08 . 1H NMR : δ = 8.41 , 8.35 , 8.19 , 7.37–7.16 , 6.35 , 5.30 , 5.20 , 4.70 , 4.40 , 3.88 , 3.62 , 3.51 , 2.74 , 2.25 . 13C NMR : δ = 154.90 (C6), 152.86 (C2), 148.61 (C4), 140.10 (C8), 128.82 (Ph), 127.58 (Ph), 127.32 (Ph), 119.55 (C5), 88.28 (C1′), 84.62 (C4′), 71.34 (C3′), 62.20 (C5′), 43.38 (NHCH2), 39.49 (C2′ overlapping with DMSO). HRMS: m/z [M + H]+ calculated C17H20N5O3+ 342.1561, found 342.1562; m/z [M–deoxyribosyl]+ calculated C12H12N5+ 226.1087, found 226.1083.N6-isopentenyl-2′-deoxyadenosine (9). Yield for two steps was 43% as a foam. Rf 0.33 . 1H NMR : δ = 8.30 s , 8.18 br s , 7.79 br s , 6.34 dd , 5.30 dd , 5.27 d , 5.18 dd , 4.40 dddd , 4.07 br s , 3.88 ddd , 3.62 ddd , 3.52 ddd , 2.71 , 2.25 , 1.69 , 1.66 . 13C NMR : δ = 154.32 (C6), 152.26 (C2), 148.09 (C4), 139.21 (C8), 133.14 (=CMe2), 122.07 (CH=), 119.57 (C5), 87.96 (C1′), 83.92 (C4′), 70.93 (C3′), 61.86 (C5′), 39.49 , 37.67 (NHCH2), 25.32 (CH3 isopentenyl), 17.77 (CH3 isopentenyl). HRMS: m/z [M + H]+ calculated C15H22N5O3+ 320.1717, found 320.1720; m/z [M–deoxyribosyl]+ calculated C12H12N5+ 204.1244, found 204.1236.N6-(2-phenylethyl)-2′-deoxyadenosine (10). Yield for two steps was 58% as a foam. Rf 0.06 . 1H NMR : δ = 8.32 , 8.22 , 7.83 , 7.35–7.15 , 6.35 , 5.27 , 5.18 , 4.44–4.36 , 3.88 , 3.82–3.60 , 3.62 , 3.52 , 2.92 , 2.72 , 2.72 . 13C NMR : δ = 156.21 (C6), 153.51 (C2), 148.86 (C4), 140.93 (C8), 140.47 (Ph), 129.88 (Ph), 129.46 (Ph), 127.31 (Ph), 121.30 (C5), 89.91 (C1′), 87.15 (C4′), 73.07 (C3′), 63.67 (C5′), 43.19 (NHCH2), 41.58 (C2′), 36.68 (CH2Ph). HRMS: m/z [M + H]+ calculated C18H22N5O3+ 356.1717, found 356.1724; m/z [M–deoxyribosyl]+ calculated C13H14N5+ 240.1244, found 240.1241.N6-benzyl-5′-deoxyadenosine (12). Yield for two steps was 77% as a powder. Rf 0.32 . m.p. 198–201 °C. 1H NMR : δ = 8.29–8.37 , 8.21 , 7.16–7.37 , 5.85 , 5.38 , 5.10 , 4.60–4.83 , 3.93–4.10 , 1.30 . 13C NMR : δ = 154.47 (C6), 152.54 (C2), 148.79 (C4), 140.04 (Ph), 139.82 (C8), 128.14 (Ph), 127.08 (Ph), 126.54 (Ph), 119.51 (C5), 87.92 (C1′), 79.71 (C4′), 74.59 (C2′), 73.05 (C3′), 42.89 (CH2Ph), 16.89 (CH3). HRMS: m/z [M + H]+ calculated C17H20N5O3+ 342.1561, found 342.1566; m/z [M–deoxyribosyl]+ calculated C12H12N5+ 226.1087, found 226.1086.N6-isopentenyl-5′-deoxyadenosine (13). Yield for two steps was 47% as a powder. Rf 0.31 . m.p. 117-120 °C. 1H NMR : δ = 8.29 , 8.21 , 7.77 , 5.84 , 5.38 , 5.30 , 5.10 , 4.65 , 4.08 , 3.93–4.01 , 1.70 , 1.67 , 1.30 . 13C NMR : δ = 154.30 (C6), 152.53 (C2), 148.43 (C4), 139.53 (C8), 133.11 (CMe2), 122.13 (CH=), 119.52 (C5), 87.84 (C1′), 79.63 (C4′), 74.57 (C2′), 73.05 (C3′), 37.67 (NHCH2), 25.33 (CH3), 18.86 (CH3 isopentenyl), 17.79 (CH3 isopentenyl). HRMS: m/z [M + H]+ calculated C15H22N5O3+ 320.1717, found 320.1723; m/z [M–deoxyribosyl]+ calculated C12H12N5+ 204.1244, found 204.1240.N6-acetyl-2′,3′,5′-tri-O-acetyladenosine (1) or N6-acetyl-3′,5′-di-O-acetyl-2′-deoxyadenosine (2) or N6-acetyl-2′,3′-di-O-acetyl-5′-deoxyadenosine (3) (1 mmol) with triphenylphosphine (Ph3P) (2 mmol) and corresponding alcohol (2 mmol) in tetrahydrofuran (THF) (5 mL) diethyl azodicarboxylate (DEAD) (2 mmol) was added in one portion and the solution was kept at r.t. for 48 h. The reaction was monitored by TLC . After 48 h the reaction mixture was concentrated in vacuo and the residue was dissolved in CH2Cl2 and washed with brine (3 × 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography . Partially purified compound was dissolved in 4M n-PrNH2 in MeOH solution (50 mmol) and left at r.t. for 24 h, after which the mixture was concentrated in vacuo and the residue was purified by column chromatography on silica gel. The column was washed with CH2Cl2-EtOH, 95:5, the product was eluted with CH2Cl2-EtOH, 90:10. The resulting product was dried for 24 hrs in a vacuum desiccator over phosphorous pentaoxide (P2O5).To the solution of N6-(2-phenylethyl)-adenosine (6). Yield for two steps was 65% as a powder. Rf 0.28 . m.p. 169–171 °C. 1H NMR : δ = 8.34 , 8.23 , 7.88 , 7.16–7.33 , 5.89 , 5.41 , 5.36 , 5.15 , 4.60 ,4.15 , 3.97 , 3.63–3.81 , 3.55 , 2.93 . 13C NMR : δ = 154.61 (C6), 152.35 (C2), 148.27 (C4), 139.70 (C8), 139.48 (Ph), 128.63 (Ph), 128.27 (Ph), 126.00 (Ph), 119.74 (C5), 87.94 (C1′), 85.88 (C4′), 73.49 (C2′), 70.63 (C3′), 61.66 (C5′), 41.23 (NCH2), 34.39 (CH2Ph). HRMS: m/z [M + H]+ calculated C18H22N5O4+ 372.1666, found 372.1666.N6-furfuryladenosine (7). Yield for two steps was 64% as a powder. Rf = 0.33 . m.p. 150–155 °C. 1H NMR : δ = 8.36 s , 8.23 br s , 8.23 s , 7.52 dd , 6.35 dd , 6.23 dd , 5.89 d , 5.41 d , 5.34 dd , 5.15 d , 4.71 br s , 4.61 ddd , 4.15 ddd , 3.97 ddd , 3.67 ddd , 3.56 ddd . 13C NMR : δ = 154.41 (C-6), 152.88 (Fur), 152.30 (C-2), 148.68 (C-4), 141.87 (Fur), 140.03 (C-8), 119.85 (C-5), 110.50 (Fur), 106.73 (Fur), 88.01 (C-1′), 85.93 (C-4′), 73.59 (C-2′), 70.66 (C-3′), 61.69 (C-5′), 36.62 (NHCH2). HRMS: m/z [M + H]‒ calculated C15H18N5O5+ 348.1302, found 348.1307.N6-furfuryl-2′-deoxyadenosine (11). Yield for two steps was 58% as a foam. Rf 0.52 . 1H NMR : δ = 8.36 s , 8.23 br s , 7.53 dd , 6.39-6.33 m , 6.23 dd , 5.29 d , 5.16 dd , 4.72 br s , 4.41 dddd , 3.89 ddd , 3.62 ddd , 3.52 ddd , 2.73 ddd , 2.25 ddd . 13C NMR : δ = 154.90 (C-6), 152.59 (C-2), 151.48 (C-4), 142.45 (C-8), 139.79 (Fur), 121.50 (C-5), 110.60 (Fur), 107.81 (Fur), 89.83 (C-1′), 87.86 (C-4′), 73.43 (C-3′), 63.56 (C-5′), 40.98 (C-2′), 37.72 (NHCH2). HRMS: m/z [M + H]+ calculated C15H18N5O4+ 332.1353, found 332.1350.N6-(2-phenylethyl)-5′-deoxyadenosine (14). Yield for two steps was 54% as a powder; Rf 0.30 . m.p. 161–163 °C. 1H NMR (DMSO-d6): δ = 8.30 , 8.24 , 7.79 , 7.15–7.40 , 5.85 , 5.38 , 5.10 , 4.66 , 3.92–4.02 , 3.72 , 2.93 , 1.31 . 13C NMR (DMSO-d6): δ = 154.50 (C6), 152.58 (C2), 148.61 (C4), 139.62 , 128.62 (Ph), 128.25 (Ph), 125.98 (Ph), 119.54 (C5), 87.85 (C1′), 79.67 (C4′), 74.59 (C2′), 73.07 (C3′), 41.25 (CH2Ph), 35.01 (NHCH2), 18.88 (CH3). HRMS: m/z [M + H]+ calculated C18H22N5O3+ 356.1717, found 356.1727; m/z [M–deoxyribosyl]+ calculated C13H14N5+ 240.1244, found 240.1243.N6-furfuryl-5′-deoxyadenosine (15). Yield for two steps was 73% as a powder; Rf 0.24 . m.p. 185–188 °C. 1H NMR (DMSO-d6): δ = 8.34 s , 8.25 s , 8.22 br s , 7.53 dd , 6.36 dd , 6.23 dd , 5.86 d , 5.41 d , 5.14 d , 4.70 br s , 4.67 ddd , 4.05-3.90 m , 1.30 d . 13C NMR : δ = 154.27 (C-6), 152.93 (C-4), 152.44 (C-2), 141.77 (C-8), 139.96 (Fur), 119.60 (C-5), 110.40 (Fur), 106.59 (Fur), 87.89 (C-1′), 79.72 (C-4′), 74.59 (C-2′), 73.05 (C-3′), 36.54 (NHCH2), 18.90 (CH3). HRMS: m/z [M + H]+ calculated C15H18N5O4+ 332.1353, found 332.1355.Investigated compounds were dissolved in 100% DMSO to a concentration of 0.1 M. Then these solutions were diluted with distilled water to concentrations of 1–10 μM. Thus, the content of DMSO in 1 μM ligand solutions was only 0.001%.Arabidopsis thaliana or Amaranthus caudatus. In the first assay, we used double mutants of Arabidopsis, in which only one of the three CK receptors  was kept active, as well as wild type (WT) Arabidopsis seedlings with all three functioning receptors [Arabidopsis plants used, including WT control, carried the reporter GUS gene under control of CK-dependent promoter ARR5P [Arabidopsis seedlings were incubated for 16 h in aqueous solutions of tested compounds [ARR5:GUSP expression [CK activities were measured in two bioassays based on seedlings of eceptors . All Araer PARR5 . 4- to 5ompounds . The levpression . Each saAmaranthus bioassay, 3- to 4-day-old etiolated seedlings with removed roots were incubated for 16 h in solutions of tested compounds in the dark. Each sample contained 10 aligned seedlings; all samples were in triplicate. The CK activity of compounds corresponds to the level of the pigment amaranthin accumulation in cotyledons, which was determined with spectrophotometer [In the case of otometer ,20.The experiments were carried out in 2–3 biological replications. In all our experiments, probes with BA were included as positive control and its activity was set as 100%. CK activity of all tested compounds was evaluated as percentage relative to the BA activity (after background subtraction) at the same concentration in the same experiment.N6-alkylation of acetyl-protected adenosine, 2′-deoxyadenosine and 5′-deoxyadenosine derivatives 1-3 either with alcohols under Mitsunobu reaction conditions . The obtained M/z values of the compounds correspond to the calculated M/z values. According to HPLC, all the obtained products are individual compounds. All spectral data are presented in full in the The structure of the compounds was confirmed by Arabidopsis thaliana [The CK activity of all synthesized compounds was studied in two plant bioassay systems. First bioassay was based on seedlings of a model plant thaliana . To studGUS gene fused to CK-dependent promoter of the ARR5 gene. The hormonal activity of the compounds was determined by the level of GUS activity reflecting the intensity of the ARR5:GUSP expression [Arabidopsis bioassay  [7, a kinetin derivative, was more active at 10−5 M for AHK2 and AHK4 and manifested comparable activity at both 10−5 M and 10−6 M for AHK3. N6-Phenylethyladenosine (6), an artificial CK derivative, demonstrated some, though weak activity at a concentration of 10−6 M only with the AHK2 receptor  . Compounreceptor . Two othArabidopsis assay makes it possible to perform a unique task, i.e. to analyze the response to CKs of individual CK receptors. In our study, the clones reacted quite uniformly, there were no case when any of CK derivatives exerted high effect with one receptor and simultaneously weak effect with another one. If WT plants or any of mutant clones demonstrated high CK activity (red color) with a defined compound, all other mutant clones showed with this compound the same (red) or at least medium (black) activity. Conversely, if WT plants or any of mutant clones demonstrated low/no CK activity (blue color) with some other compound, all other mutant clones with different receptors showed the same (blue) or medium (black) but never high (red) activity  was active at higher concentrations (10−5 M) with all individual receptors and manifested weaker activity at more physiological concentration. 2′-Deoxyribo-derivatives of aromatic CKs  were virtually inactive even at a concentration of 10−5 M except for derivative 11 which showed some activity at both concentrations, but only with AHK2.2′-Deoxyribo-derivative of iP (13) manifested high activity for WT, medium activity for AHK2 and AHK4 and weak activity for AHK3 at 10−5 M and manifested weak activity for all individual receptors at 10−6 M. On the other hand, 5′-deoxyribo-derivatives of aromatic CKs  were inactive even at a concentration of 10−5 M.5′-Deoxyribo-derivative of iP (13) cannot be phosphorylated enzymatically in vivo, thereby the LOG-mediated one-step formation of iP is not possible here. However, since this compound exerted the CK effect, the only way for this derivative conversion into active CK is the cleavage by adenosine nucleosidase  of the CK ribosides.Taking into account these results, several inferences can be made. 5′-Deoxyribo-derivative of iP  and N6-isopentenyladenosine (5) showed pronounced CK activity, while N6-phenylethyladenosine (6) demonstrated weak activity at 10−6 M nucleoside N6-furfuryladenosine (7) also showed weak activity in contrast to its medium activity in Arabidopsis bioassay. It is interesting that all 5′-deoxyribo-derivatives (12–15) as well as 2′-deoxyribo-derivatives (8–11) of both aromatic and isoprenoid CKs turned out to be essentially inactive in the Amaranthus bioassay. This peculiarity is indicative of a species specificity in the final step of active CK biosynthesis in plants.In the case of t 10−6 M . ContrarN6-benzyladenosine [N6-isopentenyladenosine [It is known that adenosine nucleosidase, isolated from various sources, has a broad substrate specificity. Substrates for this enzyme may be adenosine, guanosine, inosine, xanthosine , N6-benzdenosine , N6-isopdenosine , as welldenosine  and seveN6-isopentenyladenine and various aromatic CKs was synthesized starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using mild and efficient stereoselective N6-alkylation with further acyl deblocking, and their hormonal (cytokinin) activity was determined in two bioassays based on seedlings of Arabidopsis thaliana and Amaranthus caudatus. We demonstrated that ribo-, 2′-deoxyribo-, 5′-deoxyribo-derivatives of N6-isopentenyladenine, as well as ribosides of aromatic CKs were able to transform into active CKs and exhibited hormonal activity in Arabidopsis, while 5′-deoxyribo- and 2′-deoxyribo-derivatives of aromatic CKs were inactive. Therefore, not only ribo- but also 5′-deoxyribo- and 2′-deoxyribonucleosides may serve as precursors of active CKs, at least in Arabidopsis. However, in the latter case, the only way of biosynthesis of aromatic CKs seems to be the direct cleavage of 5′-monophosphates to active nucleobases catalyzed by phosphoribohydrolase LOG, whereas the biosynthesis of N6-isopentenyladenine may evidently proceed also by hydrolysis of CK nucleosides catalyzed by adenosine nucleosidase. As a result, it can be concluded that the biosynthesis pathways of isoprenoid (exemplified by N6-isopentenyladenine) and aromatic CKs may be different, at least in Arabidopsis, and that further research is needed to shed light on the mechanisms of biosynthesis of this class of compounds. Of course, at present it cannot be excluded that in other plant species the CK biosynthesis pathways are different. For example, in Amaranthus deoxyribo-derivatives of both N6-isopentenyladenine and aromatic CKs were equally low active. Taking into consideration the variety of biological effects of CKs on the growth, development, resistance and productivity of plants, the knowledge gained may contribute to the development of new approaches to handling the growth and development of plant species valuable to humans.A series of nucleoside derivatives of isoprenoid CK