An Efficient UPLC-MS/MS Method for the Determination of Pyrroloquinoline Quinone in Rat Plasma and Its Application to a Toxicokinetic Study

Pyrroloquinoline quinone (PQQ) is a powerful antioxidant coenzyme existing in diet, benefiting growth, development, cognition function, and the repair of damaged organs. However, a method for detecting PQQ in vivo was rarely described, limiting the research on the bioanalysis and metabolic properties of PQQ. In this study, a novel, simple, and efficient ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify the concentration of PQQ in rat plasma. Detection through mass spectrometry was operated by multiple reaction monitoring (MRM) in negative electrospray ionization mode with ion transitions m/z 328.99→197.05 for PQQ and m/z 280.04→195.04 for the internal standard. The calibration curves were linear up to 10,000 ng/mL, with a lower limit of quantitation of 10 ng/mL. Inter-run and intra-run precision ranged from 1.79% to 10.73% and accuracy ranged from −7.73% to 7.30%. The method was successfully applied to a toxicokinetic study in Sprague–Dawley rats after the oral administration of PQQ disodium salt at doses of 250 mg/kg, 500 mg/kg, and 1000 mg/kg. The toxicokinetic parameters were subsequently analyzed, which may provide valuable references for the toxicokinetic properties and safety evaluation of PQQ.

Introduction
Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was proven to function as a redox cycle coenzyme in bacteria and other eukaryotes [1]. The primary source of PQQ in the human body is diet, including vegetables, meat, and milk [2]. To date, PQQ was proven to play an essential role in mammals, influencing numerous physiological and biochemical processes [3,4]. A deficiency of PQQ results in growth impairment, abnormal immune responses, and reproductive dysfunction in mice [5]. Due to the versatile roles of PQQ, it is classified as a newcomer to vitamin B [6]. In addition, studies also showed the effect of PQQ in disease prevention. PQQ exhibits therapeutic effects on cognitive impairments and liver injury through multiple aspects, including ameliorating oxidative stress, inhibiting lipopolysaccharide-induced inflammation, and reducing DNA damage [7][8][9][10]. Dietary supplements containing PQQ, generally in the form of water-soluble PQQ disodium salt (PQQ Na 2 salt), are currently approved in Canada and the United States for health maintenance [11].
As a natural health product, the safety of PQQ Na 2 salt is noteworthy. The histopathological and hematological biochemical results for PQQ Na 2 salt were reported [2,12] after oral administration to rats. However, toxicokinetic data for PQQ Na 2 salt, which could quantitatively describe exposure and the relationship between internal and external doses, were never clarified [13,14]. In addition, there is limited in vivo research reporting analytical methods for PQQ Na 2 salt. Natsumi Noji et al. developed a method combining high-performance liquid chromatography (HPLC) with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS) to rapidly detect PQQ in solid and liquid food after extraction [15]. Another research report, published by Chikara Kato et al., also developed a method for quantifying PQQ in foods using enzymatic and LC-MS/MS methods [16]. Several new methods were also developed to analyze PQQ in other substrates, including specific neurotransmitter amino acid reaction mixtures and PQQ-derivative mixtures [17][18][19]. However, these methods were not validated for in vivo analysis. A gas chromatography/mass spectrometry method, established by Takeshi Kumazawa, analyzed PQQ in human and rat samples using a complicated pretreatment [20]. Mizuho Fukuda et al. optimized and validated a method for determining PQQ in human plasma via HPLC with chemiluminescence detection (CL) and solid-phase (SPE) extraction [18]. However, the extraction process was complicated and run time was up to 15 min, which is time-consuming for multiple sample analysis. Thus, a more efficient and convenient method is still required for rapid detection of PQQ concentration in plasma.
In this study, a rapid and efficient Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the concentration of PQQ Na 2 salt in rat plasma. Using the multiple reaction monitoring (MRM) scan mode, triple quadrupole tandem mass spectrometry can achieve high selectivity and sensitivity [21]. The plasma was pre-treated using the protein precipitation method, which was simple and convenient. The linearity of this method ranged from 10 to 10,000 ng/mL and the run time was 3 min. Subsequently, this method was successfully applied to a toxicokinetic study of PQQ Na 2 salt after oral administration to rats, providing possible perspective for explaining the in vivo toxicity of PQQ [22]. In the current study, ESI (electrospray ionization) was used to generate ions because of its high sensitivity and fragmentation. Positive (ESI+) and negative (ESI−) ion-detection modes were both explored to optimize the ESI conditions for the detection of PQQ Na 2 salt and the internal standard (IS), whose chemical structures are shown in Figure 1. The compounds showed a good response and low background signal in the negative (ESI−) mode, which was applied in the detection. Ion response is related to collision energy, cone voltage, ion source temperature, and desolvation gas flow. Thus, these parameters were optimized via manual manipulation to obtain a high response and sensitivity [23]. As shown in Table 1, the mass transition for PQQ was m/z 328.99→197.05; for IS, it was m/z 280.04→195.04. The collision energies of PQQ and IS were 26 eV and 18 eV, respectively. The cone voltages of PQQ and IS were 31 V and 34 V. The source temperature was optimized to 150 • C. The desolvation temperature was 600 • C and gas flow was 1000 L/h.  [2,12] after oral administration to rats. However, toxicokinetic data for PQQ Na2 salt, which could quantitatively describe exposure and the relationship between internal and external doses, were never clarified [13,14]. In addition, there is limited in vivo research reporting analytical methods for PQQ Na2 salt. Natsumi Noji et al. developed a method combining high-performance liquid chromatography (HPLC) with electrosprayionization (ESI) tandem mass spectrometry (MS/MS) to rapidly detect PQQ in solid and liquid food after extraction [15]. Another research report, published by Chikara Kato et al., also developed a method for quantifying PQQ in foods using enzymatic and LC-MS/MS methods [16]. Several new methods were also developed to analyze PQQ in other substrates, including specific neurotransmitter amino acid reaction mixtures and PQQderivative mixtures [17][18][19]. However, these methods were not validated for in vivo analysis. A gas chromatography/mass spectrometry method, established by Takeshi Kumazawa, analyzed PQQ in human and rat samples using a complicated pretreatment [20]. Mizuho Fukuda et al. optimized and validated a method for determining PQQ in human plasma via HPLC with chemiluminescence detection (CL) and solid-phase (SPE) extraction [18]. However, the extraction process was complicated and run time was up to 15 min, which is time-consuming for multiple sample analysis. Thus, a more efficient and convenient method is still required for rapid detection of PQQ concentration in plasma.

Results and Discussion
In this study, a rapid and efficient Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the concentration of PQQ Na2 salt in rat plasma. Using the multiple reaction monitoring (MRM) scan mode, triple quadrupole tandem mass spectrometry can achieve high selectivity and sensitivity [21]. The plasma was pre-treated using the protein precipitation method, which was simple and convenient. The linearity of this method ranged from 10 to 10,000 ng/mL and the run time was 3 min. Subsequently, this method was successfully applied to a toxicokinetic study of PQQ Na2 salt after oral administration to rats, providing possible perspective for explaining the in vivo toxicity of PQQ [22].

Conditions of Mass Spectrometry
In the current study, ESI (electrospray ionization) was used to generate ions because of its high sensitivity and fragmentation. Positive (ESI+) and negative (ESI−) ion-detection modes were both explored to optimize the ESI conditions for the detection of PQQ Na2 salt and the internal standard (IS), whose chemical structures are shown in Figure 1. The compounds showed a good response and low background signal in the negative (ESI−) mode, which was applied in the detection. Ion response is related to collision energy, cone voltage, ion source temperature, and desolvation gas flow. Thus, these parameters were optimized via manual manipulation to obtain a high response and sensitivity [23]. As shown in Table 1, the mass transition for PQQ was m/z 328.99→197.05; for IS, it was m/z 280.04→195.04. The collision energies of PQQ and IS were 26 eV and 18 eV, respectively. The cone voltages of PQQ and IS were 31 V and 34 V. The source temperature was optimized to 150 °C. The desolvation temperature was 600 °C and gas flow was 1000 L/h.   To better separate the compounds in the chromatographic column, several columnsmobile phase, flow rate, and sample injection volume-were tested in the study. Firstly, two types of columns were taken into consideration. The peak using Waters Xbridge C18 (2.1 mm × 50 mm, 3.5 µm) was wider than that using Waters ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 µm); the latter was, therefore, chosen for the experiment.
The mobile phase was then tested, including water/acetonitrile, water/methanol, and water/acetonitrile-methanol (1:1, v/v). However, the methanol had little polarity and there was no peak under this condition. The water/acetonitrile mobile phase exhibited a bifurcated peak that required improving. As an ion-pairing agent, dibutylamine acetate possesses the ability to improve molecular retention. After changing the mobile phase to 10 mM dibutylamine acetate-water solution/acetonitrile, the peak shape improved significantly. Finally, the flow rate and sample injection volume were investigated at 0.5 mL/min and 5 µL; this resulted in interference by a bifurcated peak for the former and mixed peaks for the latter. Under the conditions of a 0.3 mL/min flow rate and 2 µL injection volume, the shape of the peak was excellent.

Selectivity
The MRM chromatography of PQQ and IS in blank plasma, spiked plasma, and real plasma samples is shown in Figure 2. The PQQ response of double blank (DB) samples at the retention time did not exceed 20% of the LLOQ response, while the IS response of the DB samples did not exceed 5% of the LLOQ response. The results show that there was no significant peak in the retention time of PQQ and IS in the DB samples, indicating no endogenous inference in the plasma.

Linearity and Carry-over
The linearity of the method was evaluated by analyzing the calibration curve in plasma, using least squares regression of the peak area ratios of PQQ and IS (y) versus the standard concentration of PQQ (x) with a weight factor. Linearity demonstrated a wide linear range (from 10 to 10,000 ng/mL), with an excellent correlation coefficient (r > 0.99). The LLOQ of the method was 10 ng/mL, representing the sensitivity of the method as 10 ng/mL. Carry-over samples were also evaluated in this assay, with less than 20% for PQQ and 5% for IS in each separate batch, respectively.

Precision and Accuracy
Four concentrations of PQQ were assessed to evaluate precision and accuracy. As listed in Table 2, the intra-day and inter-day precision of the QC samples ranged from 1.79 to 10.73% and the accuracy of the QC samples ranged from −7.73 to 7.30%, which were all within 15%, indicating the method was reliable and accurate.  The matrix effect was assessed at LQC and HQC levels by comparing the peak area ratios of the post-extract blank plasma to the same level solution in pure solvent. From the experimental data, the mean matrix effects at LQC and HQC levels were 90.73% and 100.73%. The coefficient of variation of the matrix effect data from six batches did not exceed 15%. The results show that the different sources of the blank matrix had no effect on the analytes.
Recovery was evaluated by comparing the response of PQQ between the post-extract samples and the extracted samples at the LQC, MQC, and HQC levels. The mean recovery of QCs was from 65.26 to 73.26% and the RSD% was less than 15%, indicating the recovery was consistent. The results are shown in Table 3.

Stability
As shown in Table 4, the stability results illustrate that PQQ was stable in plasma samples for 19.5 h at room temperature, for 106 days at −80 • C, and for three freeze-thaw cycles. It was also stable after being processed for 19.5 h at 4 • C and room temperature. The average deviation of HQC and LQC between the evaluated values and accurate values did not exceed 15%. Therefore, the method was proved to be stable throughout the experiment. The results of dilution integrity show that the accuracy and precision of the samples were within 15% after a five-fold dilution, indicating that PQQ plasma samples were stable after a five-fold dilution. The results are shown in Table 5.

Application to Toxicokinetic Study
After a single oral administration of PQQ Na 2 salt to rats, the developed and validated method was applied to the toxicokinetic experiment. The plasma-concentration of PQQ Na 2 salt varies with time: the calculated toxicokinetic parameters are shown in Figure 3 and Table 6. The results show that PQQ Na 2 salt was rapidly absorbed after oral administration, the average T max for doses of 250 mg/kg, 500 mg/kg, and 1000 mg/kg on day 1 being 1.50 ± 0.55 h, 1.29 ± 1.36 h, and 4.83 ± 2.71 h, respectively. Rapid absorption was also observed for repeat doses of PQQ Na 2 salt to rats on day 28, with a T max of 1.00 ± 0.00 h, 1.21 ± 1.40 h, and 4.67 ± 1.63 h, respectively. The average T 1/2 for different doses of PQQ Na 2 salt was 2.18 ± 0.32 h, 3.26 ± 1.01 h, and 10.23 ± 8.87 h on day 1, and 2.58 ± 1.19 h, 7.46 ± 5.94 h, and 8.74 ± 2.70 h on day 28, indicating that the elimination of PQQ Na 2 salt was slow, especially in the high-dose group. In addition, with the increasing dose of PQQ Na 2 salt, both the AUC (0-t) and C max increased in all groups, providing a reference for the dose-exposure relationship. Twenty-four hours after administration, PQQ Na 2 salt was rarely detected in the rat plasma in the low-and medium-dose groups, whereas it could still be detected in the high-dose group; it was, however, completely eliminated forty-eight hours after administration. These results suggest that high-dose administration of PQQ Na 2 salt may have a longer retention time in the body compared with that of low-and medium-dose administration.     The relations between toxicokinetic parameters and other factors, including single or repeat dosing and gender, were also considered. From the plasma concentration data for D28 at pre-dose, PQQ can be detected at dozens of ng/mL, demonstrating it was not completely eliminated following the previous administration. After repeated intragastric administration for four weeks, the AUC (0-t) D28/D1 for different doses was 1.38, 2.88, and 2.06, respectively, and the C max D28/D1 for different doses was 1.48, 1.71 and 1.98, indicating that the systemic exposure to PQQ in rats on D28 was a little higher than that on D1. Thus, our results indicate that there might be a slight accumulation of PQQ Na 2 salt in rats following repeated administration for 28 days, leading to the different systemic exposure to PQQ between a single dose and 28 days' administration. Comparing the parameters in male and female rats, the C max for females was more than twice as high as the C max for males on day 1. However, on day 28, the C max for females/males was 1.03, 1.00, and 0.78 for the different doses, respectively; the same results were also observed for the AUC (0-t) .
These results indicate that the exposure to PQQ Na 2 salt for males and females on day 1 might be different, and it may be necessary to design rational dosage regimens for men and women.

Chemicals and Reagents
Standard PQQ (98.0%) was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). IS reference Levofloxacin Impurity 27 (94.37%) was obtained from SINCO PHARMACHEM Inc. (Middletown, DE, USA). The structures of PQQ and IS are shown in Figure 1. HPLC grade methanol and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). Dibutylamine acetate was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ammonium hydroxide was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Purified water was prepared with a Milli-Q water purification system from Merck KGaA (Darmstadt, Germany).
Mass spectrometric analysis was carried out in negative ion mode with electrospray ionization. Multiple reactions monitoring (MRM) was utilized in the study and the parameters were optimized as summarized in Table 1. Mass transition for PQQ was m/z 328.99→197.05 and IS was m/z 280.04→195.04. The source temperature was 150 • C. The desolvation temperature was 600 • C and gas flow was 1000 L/h.

Preparation of Calibration Standard and Quality Control Solution
Two copies of stock solution of PQQ were dissolved in methanol to 1 mg/mL. The IS stock solution was first prepared in ammonium hydroxide and then dissolved in methanol. One PQQ stock solution was diluted with methanol-water (1:1, v/v) solution to a series of standard working solutions of different concentrations (0.20, 0.40, 2.0, 6.0, 20, 50, 100, 200 µg/mL). Four levels of quality control working solution (0.20, 0.60, 10, 160 µg/mL) were prepared with the other PQQ stock solution. The IS stock solution was also diluted with the same diluent to 50 ng/mL. All the stock and working solutions were stored at 4 • C.

Sample Preparation
A method of protein precipitation was used in this study to extract the compound from the plasma. An amount of 5 µL standard or quality control working solution was mixed with 95 µL rat plasma as the plasma sample. The concentrations of standard samples were 10, 20, 100, 300, 1000, 2500, 5000, 10,000 ng/mL and quality control samples at low, medium, high were 30, 500, 8000 ng/mL, respectively. IS stock solution was diluted to 500 ng/mL with 50% methanol-water (1:1, v/v) as the IS solution. Quantities of 50 µL IS solution and 100µL acetonitrile were added to a 50 µL plasma sample and vortexed at 1200 rpm for 5 min. Double blank (DB) samples did not contain PQQ or IS; these were prepared with a 50 µL blank plasma sample, 50µL 50% methanol-water (1:1, v/v), and 100 µL acetonitrile. Blank samples only contained IS without PQQ and were prepared with a 50µL blank plasma sample, 50 µL IS, and 100 µL acetonitrile. The mixture was centrifuged at 13,000 rpm for 5 min. Next, 50 µL supernatant and 50µL mobile phase A (10 mM DBAA) were added to plates. The samples were injected for LC-MS/MS analysis after mixing.

Method Validation
The validation in this study included selectivity, linearity and carry-over, precision and accuracy, matrix effect, recovery, stability, and dilution integrity.

Selectivity
Blank plasma from six different individual sources and one blank hemolysis plasma (plasma:hemolysis blood = 100:2, v/v) were prepared as DBs and lower limit of quantification (LLOQ) samples. The samples were treated as described above (3.5 Sample preparation) and then injected for analysis. The response to PQQ and IS of the blank samples were compared to that of the LLOQ samples. Correspondence analysis was performed for each source sample.

Linearity and Carry-Over
The linear calibration curve was established by plotting the peak area ratio of PQQ to IS and the standard concentration (from 10 to 10,000 ng/mL) of PQQ, with weighted least squares (W = 1/X 2 ) to calculate the linear regression. The LLOQ of the calibration curve was determined on a signal to noise ratio of at least 10:1. The acceptable criterion was a correlation coefficient of the calibration curve of no less than 0.99 (r ≥ 0.99). The accuracy of the LLOQ was within 20% of the labeled value, and other levels of concentration were within 15% of the labeled value.
Carry-over samples were DB samples after the injection of the ULOQ samples, normally following the ULOQ in the calibration curve. It was acceptable that the peak area of PQQ in the DBs did not exceed 20% of that in the LLOQ. The peak area of IS in the DBs also did not exceed 5% of that in the LLOQ.

Precision and Accuracy
The inter-and intra-day precision and accuracy were investigated by analyzing six LLOQ samples, six LQC samples, six MQC samples, and six HQC samples, in three separated batches, on at least two days. Precision was expressed as relative standard deviation (RSD%) and accuracy was expressed as relative error (RE%).

Matrix Effect and Recovery
The matrix effect was evaluated by comparing the peak areas of the post-extract blank samples with those of the pure solvent. The blank plasma from six different individual sources and one hemolysis unit (plasma:hemolysis blood = 100:2, v/v) was tested with LQC and HQC samples, with three parallel samples at each concentration.
Recovery was prepared by analyzing the differences in peak areas between extracted QC samples and post-extraction spiked samples of LQC, MQC, and HQC.

Stability
The stability of PQQ in plasma exposed to different conditions was examined. LQC and HQC samples were tested under the following conditions: plasma-sample short-term storage at room temperature; processed samples at room temperature for 19.5 h; processed samples at 2-8 • C for 19.5 h; plasma-sample long-term storage at −80 • C for 106 days; and three freeze-thaw cycles from −80 • C to room temperature. The stability of samples was evaluated by RE% between estimated and exact values.

Dilution Integrity
The concentration of PQQ in plasma may be above the ULOQ of the calibration curve. Therefore, the samples with concentrations higher than ULOQ were diluted to be within the range of the calibration curve. An amount of 800 µg/mL working solution of PQQ was first diluted to 40 µg/mL with rat plasma. Next, 20 µL of the above samples was added to 80 µL blank plasma to obtain five-fold diluted samples of PQQ with a concentration of 8000 ng/mL. The final samples were detected after pretreatment.

Animals
The animal studies were approved by Zhejiang University Animal Research Committee (Approval number IACUC-20-201), and the experiments were conducted in line with the institutional guidelines. Sprague-Dawley rats (200-220 g, 6-8 weeks, 24 males and 24 females) were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China). The rats were kept at a temperature of 20-26 • C and humidity of 40-70% with a 12 h light/dark cycle. All the animals were acclimated for 7 days; they fasted for 12 h and had free access to water before the experiments were performed [24].

Toxicokinetic Study
According to the ICH guidelines for dose settings and the results of the preliminary experiment, no dose-related toxicity was observed at 250 mg/kg; feces were green when the dose ≥500 mg/kg; and kidney weight in females increased at a dose of 1000 mg/kg. The maximum tolerated dose (MTD) of repeated administration of PQQ Na 2 salt was greater than 1000 mg/kg. Therefore, 250 mg/kg, 500 mg/kg, and 1000 mg/kg were set as the low, medium, and high doses, respectively. Forty-eight rats were randomly assigned into four groups, each group having an equal number of males and females. The four groups were set as the control, the PQQ low-dose group (250 mg/kg), the PQQ medium-dose group (500 mg/kg), and the PQQ high-dose group (1000 mg/kg), respectively. PQQ Na 2 salt was dissolved in normal saline. Half of the rats in the four groups (3 males and 3 females in each group) were administered a single dose of different concentrations of PQQ Na 2 salt, as above. The day of the first administration was named day 1. Blood samples were then collected into heparinized tubes using the tail-bleed method before administration and, thereafter, at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. Each time, 0.25 mL blood was collected. The remaining rats were administered with repeated dosing for four weeks and their blood samples were also taken on day 28 at pre-dose, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. In addition, blood samples of rats in the control group were collected at two time points only-pre-dose and 2 h after administration. The blood samples were then immediately centrifuged at 3000 rpm for 10 min. The supernatant plasma was collected and then stored at −80 • C until analysis.

Data Analysis
The UPLC-MS/MS data were collected and processed with Unifi 1.9.3 and then analyzed with Microsoft Excel 2007 (Microsoft Co., Redmond, Washington, DC, USA). The toxicokinetic parameters were calculated using DAS 3.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). SPSS software (IBM, Chicago, IL, USA) was also used in the study to analyze the kinetic parameters.

Conclusions
In this study, a rapid and simple UPLC-MS/MS method was developed and applied in toxicokinetic experiments with PQQ Na 2 salt in rat plasma. The linear range of the method was from 10 to 10,000 ng/mL and the method validation proved that the method is accurate, precise, stable, and reliable. After the successful application of this method in toxicokinetic studies in rats, we showed that PQQ was rapidly absorbed and slowly eliminated, especially in high-dose groups. The method and toxicokinetic data may be further applied to the in vivo detection and safety evaluation of PQQ, to provide data support and an analytical method for the development of PQQ.


Ag@TiO2 Nanocomposite as an Efficient Catalyst for Knoevenagel Condensation

In the present study, a new series of different heterocycles was synthesized through base-free Knoevenagel condensation of various aldehydes and active methylene-containing compounds using the hydrothermal developed Ag@TiO2 as a heterogeneous catalyst. The catalyst was synthesized by mixing TiO2 (P25) with AgNO3 and hydrothermally treated in ethanol at 180 °C for 12 h. The developed Ag@TiO2 catalyst was directly applied for Knoevenagel condensation, and the optimized procedure involved stirring the aldehydes and active methylene-containing compounds with Ag@TiO2 in ethanol at 65 °C. The reaction scope was investigated for various aromatic and heterocyclic aldehydes with active methylene-containing compounds, and the isolated yields were significantly high. The reusability of the catalyst was investigated for up to five cycles, where an insignificant decrease in the catalyst’s reactivity was observed. Also, the reaction could proceed in water as a solvent, and the isolated yield was 40%. Hence, this protocol features mild reaction conditions, a facile procedure, and clean reaction profiles.

INTRODUCTION
Knoevenagel condensation is a reaction involving nucleophilic addition of a hydrogen active species to a carbonyl compound generally aldehyde or ketone followed by dehydration, resulting in olefin compounds, and this reaction commonly occurs in the presence of a weakly basic amine catalyst. 1−3 This reaction has originally been described by the German chemist Emil Knoevenagel, and it can be described as a modification of the aldol condensation. 4 The importance of this reaction originated from its feasibility route for the synthesis of various biologically active compounds containing C�C bonds. 3 The α,β-unsaturated products produced from the Knoevenagel reaction had mostly been used as potential intermediates in the synthesis of many naturally occurring compounds, 5 therapeutic agents, 6,7 adequate chemicals, 8 and polymers containing different functional groups. 9 As mentioned above, the Knoevenagel reaction is common in academics and industry, with broad applications in pharmaceutical synthesis. 7,10 Usually, the reaction is homogeneously catalyzed by organic bases such as piperidine, and this convention method suffers from many difficulties like timeconsuming work-up procedures, undesired side products, applying high temperatures, difficult catalyst isolation, and hazardous reaction residues. 11 Because of these limitations, many efforts have been devoted in this area to find alternative routes to perform this reaction under base-free conditions. Previous studies have reported the use of different heterogeneous catalysts containing zeolites, mesoporous silica, ionic liquids, and graphitic carbon nitride. 12,13 Also, composites containing metal oxides showed high reactivity as heterogeneous catalysts for Knoevenagel condensation, and the metal oxides in these composites were prior chemically modified or doped with metals. 14−17 Titanium dioxide (TiO 2 )-based catalysts are considered promising and recyclable catalysts for organic synthesis, and metal or nonmetal dopants would enhance the catalytic stability and activity. 18 Noble metal-doped TiO 2 catalysts are widely used for the synthesis of Schiff bases, 19 reduction of nitrobenzenes to hydrazobenzens, 20 and dehydrogenation of nheterocycles. 21 One of these promising catalysts is Ag@TiO 2 , which is used as an effective catalyst in the synthesis of bis(pyrazol-5-ol) and dihydropyrano [2,3-c]pyrazole, pyrido-[2,3-d]pyrimidine, benzimidazole, and benzoxazole derivatives. 22−25 For the Knoevenagel condensation, composites of TiO 2 -La 2 O 2 CO 3 , Fe 3 O 4 @SiO 2 @TiO 2 , and mesoporous TiO 2 −CeO 2 were reported to be effective catalysts. 15,26,27 Inspired by these studies, Ag@TiO 2 was utilized in this study as the catalyst for the reactions between 3-chloro-1H-indole-2-carbaldehydes and other aromatic aldehydes with different compounds containing active methylene. Indolecontaining compounds represent an important class of heterocyclic compounds because of their use as alkaloid precursors, 28 and their biological activities, such as antitumor, 29 antiviral, 30 and anti-inflammatory activity. 31 Besides, the 3-chloro-1H-indole-2-carbaldehyde is an easily accessible source for indole carbaldehyde, which can be functionalized at the 2-position to produce various heterocyclic compounds with the chlorine atom at the 3-position, which may boost their biological activities. 32−36 Reaction optimization and detailed scope are comprehensively studied. Also, the recyclability of the catalyst is investigated, and the reaction mechanism is suggested. Such a comprehensive study of Knoevenagel condensation on Ag@TiO 2 has not been previously reported to the best of our knowledge. Successful completion of Knoevenagel condensation using such recyclable Ag@TiO 2 catalyst without applying other hazardous homogenous catalysts would pave the way for a facile, green, and environmental-friendly synthetic method of heterocyclic compounds.

Synthesis of Ag@TiO 2 .
One gram of TiO 2 (P25, Degussa, 20 nm, the Brunauer−Emmett−Teller (BET) surface area is 50 ± 15 m 2 /g), 20 mL of water, and 20 mL of ethanol were transferred into a Teflon vessel. After that, a Teflon container was sonicated in an ultrasonic cleaning bath (40 kHz) and magnetically stirred for 20 min each. The calculated amounts of AgNO 3 (Sigma, ≥99%) were added into the Teflon container, sonicated, and stirred for 20 min for homogenous mixing with TiO 2 to prepare Ag(5%)@TiO 2 . After that, the Teflon container was sealed and transferred into a hydrothermal synthesis autoclave reactor and heated at 180°C for 12 h. After completing the hydrothermal process, the reactor was left to be cooled down in an ambient environment, and the Ag@TiO 2 was separated using a centrifuge at 4000 rpm. The product was washed three times using deionized water and dried at 60°C for 24 h.

General Method for Knoevenagel Condensation.
3-Chloro-1H-indole-2-carbaldehyde (1a, 0.5 g, 3 mmol) was introduced into a 50 mL reaction vessel with ethyl cyanoacetate (0.41 g, 3.9 mmol), 20 mL of solvent, and the developed catalyst (50 mg). The resulting mixture was warmed and stirred at 60°C in an oil bath for 1 h. Ethyl acetate (20 mL) was added to the reaction mixture after TLC confirmed that the reaction had been completed. The catalyst was then filtered out of the mixture. Under reduced pressure, the solvent was evaporated, disclosing the desired final product.

Characterization Instruments.
The XRD measurements of the developed catalysts were investigated by A Philips 1700 version diffractometer with Cu Kα radiation. The morphology and crystallinity of the developed catalyst were examined by TEM (JEOL, JEM-2100F, Japan), where an accelerating voltage of 200 kV was applied. Also, the element mapping was investigated by applying energy-dispersive X-ray spectroscopy (EDX). The XPS analysis was performed using a Kratos Axis Supra spectrometer with a monochromatic Al Kα radiation (1486.7 eV) at low pressure (<10 −6 Pa). The collected spectra were analyzed by CasaXPS software. The BET method was applied to estimate the surface area and average pore size of Ag@TiO 2 samples using NOVA 2200e (Quantachrome Instruments). The degasification was per-formed prior to the measurements under vacuum at 100°C for 12 h. Fourier transform infrared (FTIR) spectra of the developed catalysts were collected using a Bruker Tensor 37 spectrometer, where an ATR accessory was used with 4 cm −1 resolution. NMR spectra were collected using an INOVA-500 MHz instrument (Varian, Palo Alto, CA, USA), and trimethylsilyl propanoic acid (TSP) was used as an internal standard. Figure 1a Figure 1b shows the XRD spectra of normalized Ag(5%)@TiO 2 after subtracting the normalized XRD spectrum of TiO 2 to resolve the overlapping between the characteristic peaks of Ag and TiO 2 . A characteristic peak at 2θ of 38°was observed, which is indexed to the (111) crystallographic planes of Ag nanoparticles. 41,42 Peaks attributed to the (200), (220), and (311) crystallographic planes of Ag were also found at the same position. These XRD data illustrated that the Ag(0) was successfully formed on TiO 2 without changing the phases of TiO 2 . 40−42 Figure 2a shows the XPS Ag3d spectrum, where two peaks were observed at 368.4 and 374.4 eV, which are attributed to 3d 5/2 and 3d 3/2 , respectively. The energy separation between the two peaks is 6 eV, which illustrates the reduction of Ag + to metallic Ag nanoparticles. 40−42 The XRD spectrum of Ti2p is shown in Figure 2b, where two peaks corresponding to 2p 3/2 and 2p 1/2 were observed at 458.8 and 464.5 eV. 40,42 The separation between the two peaks is 5.7 eV which is the characteristic of Ti +4 of TiO 2 . Figure S1 shows the TEM image after the hydrothermal deposition of Ag nanoparticles on TiO 2 , where the sizes of Ag and TiO 2 nanoparticles were almost <10 nm and >20 nm, respectively. Besides, Ag nanoparticles with a size of <5 nm were observed and well distributed. Figure 2c shows the high resolution (HR)TEM image, where the d spacing  (111) and anatase TiO 2 (101), respectively. 19,42 The polycrystallinity of Ag@TiO 2 due to the presence of Ag, anatase TiO 2 , and rutile TiO 2 nanoparticles was also indicated from the SAED image illustrated in Figure S2. The EDX mapping of TiO 2 and Ag(5%)@TiO 2 components is illustrated in Figure S3, where the distribution of titanium, oxygen, and silver elements is shown. The BET surface area of TiO 2 (P25) increased to 78 m 2 /g after 5% Ag doping, which indicates the increase in the catalyst reactivity after doping.

Knoevenagel Condensation. 3.2.1. Optimal Reaction Conditions.
As previously stated, 3-chloro-1H-indole-2carbaldehyde (1a) represents an easily accessible source for the synthesis of 3-chloro-2-substituted indoles, so compound 1a was selected as a model substrate for this transformation, which then was allowed to react with ethyl cyanoacetate as the active hydrogen-containing compound, as illustrated in Table  1. Carrying out the reaction in the absence of the catalyst gave no product at all. However, the addition of TiO 2 (P25) nanoparticles to the reaction slightly enhanced the reaction activity, and product 3a was separated in a 10% yield (entry 2). Following the optimal conditions that we have previously obtained for the synthesis of Schiff bases using Au (5%)@ TiO 2 , 19 we successfully isolated the Knoevenagel product in a 70% yield (entry 3). The use of the Pt@TiO 2 catalyst gave a 50% yield product (entry 4). It has been reported that higher loadings of Au or Pt on TiO 2 would reduce the catalytic activity because of the increase in the sizes of the nanoparticles leading to a decrease in the active sites. 43,44 Interestingly, the Ag (5%)@TiO 2 catalyst smoothly delivered the product in a 90% yield as the best reaction yield (entry 5). Moreover, trying different Ag doping amounts on TiO 2 represented in 1% and 3% successfully delivered the product, but the yields were 60% and 65%, respectively (entries 6 and 7). Previous studies showed that the Ag loadings on TiO 2 with up to 10% would increase the adsorption capacity and photocatalytic activities of TiO 2 . 45,46 In Figure S1, the Ag nanoparticles with <5 nm were also observed and well distributed on TiO 2 , which could illustrate the reactivity increase with increasing Ag loadings.
Furthermore, optimization using different solvents (entries 8− 10) suggested that ethanol is the optimal solvent. Besides, increasing the catalyst load to 15 or 20 mg did not result in high yields, but when the load was attenuated to 0.5 or 5 mg, it led to the diminishing of the isolated yield (entries 11 −14). Reaction time and temperature were finally screened showing that the product smoothly obtained in a good yield when the reaction was conducted at 65°C for 1 h (entries 15−19).

Reaction Scope.
After identifying the optimal reaction conditions, the scope of this transformation using different aldehydes and active methylene-containing compounds was investigated. The target of this study is to find an eco-friendly approach for synthesizing different heterocyclic moieties through Knoevenagel condensation because of their biological importance. Hence, we turned our attention to the screening of different heterocyclic carbaldehydes, as shown in Table 2. The reaction proceeded smoothly with a wide range of heterocyclic aldehydes providing an interesting class of α,βunsaturated heterocycles. Both monocyclic and fused heterocyclic aldehydes were tolerated in this transformation under the optimal conditions. Aldehydes containing thiophene and pyrazole moieties gave the products 3e, 3j with an excellent yield up to 99%, while furan-2-carbaldehyde gave the product 3d in only 50% yield after 1 h; however, for a prolonged time of 3 h, 3d was isolated in 90% yield. Thiophene is well known to be adsorbed on noble metals like Au or Ag, which might facilitate the condensation and enhance the isolated yield. 47,48 Fused heterocyclic aldehydes underwent the reaction smoothly to afford Knoevenagel derivatives in good yields (up to 95%), where coumarin, quinoline, and naphthalene-containing carbaldehydes have fascinatedly tolerated in this conversion  with acceptable yields. Notably, tricyclic benzoquinoline aldehyde 1g underwent Knoevenagel condensation to give the product 3g in a 90% yield after 3 h. Interestingly, the reaction of 2-hydroxy-1-naphthaldehyde (1h) with ethyl cyanoacetate under the optimal conditions led to the formation of 3-oxo-3H-benzo[f ]chromene-2-carbonitrile (3h) instead of α,β-unsaturated product, which resulted from the cyclic condensation of the Knoevenagel intermediate by loss of the ethanol molecule, which has been further assigned by mass spectroscopy.
The scope of active methylene-containing compounds with 3-chloro-1H-indole-2-carbaldehyde was also examined under the standard reaction conditions, as shown in Table 3; various active methylene derivatives were found to be suitable substrates in this transformation with a good yield (between 80 and 99%). The use of cyanoacetamide and cyanothioacetamide produced derivatives 3m and 3o in 85 and 80%, respectively, while malononitrile and acetylacetone gave the 3k and 3n products in 82 and 99% yields, respectively. Moreover, the condensation of aldehyde 1a with malonic acid under the standard conditions gave the acrylic acid derivative 3l in 98%, which resulted from the decarboxylation of the Knoevenagel intermediate. 32 Furthermore, the substrates of indole-bearing active hydrogen were compatible enough in this methodology, producing 3p in 85%.
The generality of the current catalytic system has also extended to include different aromatic aldehydes that make the catalytic system more effective, as shown in Table 4. To achieve this goal, benzaldehyde was allowed to react with ethyl cyanoacetate under the standard conditions, and the product 3na was delivered in a 95% yield. Moreover, different aromatic aldehydes with electron-donating or electron-withdrawing substituents in o-, p-, and m-positions have been examined. p-Tolualdehyde reacted smoothly to give the product 3oa in 95% yield, whereas benzaldehyde with an electron deficiency NO 2 group in the p-position exhibited more activity, and the product 3pa separated in 98% yield. Also, p-chlorobenzaldehyde has successfully reacted to produce the product 3qa in a 95%yield. Furthermore, different aromatic aldehydes with osubstituents, namely, o-nitrobenzaldehyde and o-anisaldehyde have been examined, and the 3ra and 3sa products were isolated in 94 and 95%, respectively. Moreover, m-substituted  benzaldehyde including m-chlorobenzaldehyde and m-anisaldehyde have also successfully checked in this reaction, which gave products 3ta and 3ua in 95 and 97% yields, respectively. Finally, more substituted 2,4,5-trimethoxy benzaldehyde and cinnamaldehyde have also been tolerated, which afforded the products 3va and 3wa in excellent yields.
To show the synthetic utility of the Ag(5%)@TiO 2 , the yield of the 3na product and the applied procedures of some catalysts that have been reported are listed in Table 5. The present protocol offers advantages in the isolated yields, reaction time, and reaction conditions compared to the other catalysts.
To explore the synthetic practicality of our designed reaction, a scale-up protocol was carried out by the reaction of benzaldehyde (1n) with ethylcyano acetate (2a) under the optimal reaction conditions ( Figure 3). It proceeded smoothly to deliver the product 3na in a 95% yield. Given the versatile reactivity of the Knoevenagel products, in addition, indolestethered pyrazole or pyrimidine are considered one of the essential scaffolds in the medicinal chemistry field and found to exhibit a variety of biological activities, 63,64 a variety of further transformations can be carried out to generate new structures. For example, the hydrazinolysis of 3c in ethanol gave an interesting aminopyrazolidenone derivative 4. 65 Moreover, the reaction of compound 3c with thiourea in ethanol delivered the indolyl thioxopyrimidine derivative 5. Furthermore, the reaction of compound 3c with urea in ethanol and potassium carbonate gave the Biginelli product dihydropyrimidine 6.
3.2.3. Reaction Mechanism. According to the abovementioned findings and based on the previously reported studies concerning Knoevenagel condensation, the suggested mechanism for Ag@TiO 2 catalyzed Knoevenagel condensation is illustrated in Figure 4. 66,67 Among various condensation reactions, Knoevenagel condensation is a well-known reaction, and it can be catalyzed by different kinds of organic bases, acids, or catalysts containing both acid and basic sites. 15,68 The mechanism can be explained as, in the first step, the acidic centers of Ag@TiO 2 would activate the carbonyl group of aldehydes to coordinate between these centers and the oxygen of carbonyl groups. 15,19,24 The active hydrogen species would attach to the carbon of the carbonyl group, which then protonated and underwent water elimination, resulting in the product. The coordination environment surrounding the Ag centers is anticipated to prevent the sterically congested Z-type product.
3.2.4. Recyclability. Catalyst recycling was performed by separating the catalysts from the reaction mixture. Then, the separated catalyst was washed in ethanol three times and dried at 80°C for 12 h. The recycled catalyst was characterized by FTIR and XRD. The XRD pattern ( Figure S4) of the recycled catalyst, where the characteristic peaks of Ag nanoparticles, rutile TiO 2, and anatase TiO 2 were still observed without significant changes compared to the as-prepared Ag@TiO 2   Figure S5, where the absorption bands at 1400 and <700 cm −1 were ascribed to Ti−O−Ti (TiO 2 ). 19,69 Bands at 1615 and 3413 cm −1 were attributed to the absorbed water. 19,69 No significant changes were observed after recycling. As previously described, the development of the Ag@TiO 2 catalyst is facile without requiring complicated steps or careful handling. Besides, the use of this catalyst for performing Knoevenagel condensation did not require harsh conditions or applying hazardous materials. Also, the catalyst was effective alongside 5 cycles without a significant decrease in the isolated yield, as shown in Figure 5. These results illustrate the reactivity of Ag@TiO 2 in catalyzing the Knoevenagel condensation for several cycles. Also, green, and eco-friendly conditions can be applied during Knoevenagel condensation in the presence of Ag@TiO 2 , and the yield approaches up to 40%. Besides, the reuse of the catalyst does not require specific treatments.

CONCLUSIONS
In   ■ REFERENCES


s2fieldsofstudy: Chemistry Biology

text:
Theoretical Investigation of Repurposed Drugs Potentially Capable of Binding to the Catalytic Site and the Secondary Binding Pocket of Subunit A of Ricin

Recently, we reported a library of 82 compounds, selected from different databanks through virtual screening and docking studies, and pointed to 6 among them as potential repurposed dual binders to both the catalytic site and the secondary binding pockets of subunit A of ricin (RTA). Here, we report additional molecular modeling studies of an extended list of compounds from the original library. Rounds of flexible docking followed by molecular dynamics simulations and further rounds of MM-PBSA calculations using a more robust protocol, enabled a better investigation of the interactions of these compounds inside RTA, the elucidation of their dynamical behaviors, and updating the list of the most important residues for the ligand binding. Four compounds were pointed as potential repurposed ricin inhibitors that are worth being experimentally investigated.

■ INTRODUCTION
Ricin is a highly toxic plant toxin present in the seeds of Ricinus communis, a bush native from the Mediterranean area, Eastern Africa and India, which can also be found in all tropical regions today. Also known as castor oil plant, R. communis is the source of the ricin oil, a raw material extensively used in the production of lubricants, green fuel, drugs, and cosmetics. 1 However, the high toxicity of ricin occasionally causes accidental intoxication of workers involved in the oil extraction. Also, the castor oil cake, a byproduct of the ricin oil extraction, is rich in minerals and other nutrients, being, for this reason, used as a fertilizer and to feed livestock. As this byproduct is not always totally free of ricin, intoxication or even death of livestock also happens as well.
Due to its easiness of obtention, water solubility, and toxicity higher than that of the nerve agents, ricin has also been used as a chemical-biological warfare agent for assassination and terrorism purposes. Many reports of this misuse can be found in the literature, 2−4 with the most notorious being the assassination, in London, of the Bulgarian journalist Georgi Markov in September 1978. The murderer used an umbrella adapted with a hypodermic needle at its extremity to inject a tiny sphere stuffed with around 0.4 mg of ricin, and covered with wax, in Markov's leg, causing his death 4 days later. Literature also reports the tentative of aerolization or mass production of ricin by terrorist groups in 1989 2 and 2010, 4 besides several cases of letters contaminated with ricin being delivered to public authorities in the USA in the decades 2000 and 2010. 2,5 Ricin is a N-glycosidase classified as a type II ribosomeinactivating protein (RIP). This family of proteins found in bacteria and plants is capable of inhibiting the protein synthesis in eukaryotic cells, playing an important defense role against pathogens and insects. 6 Despite sharing similar mechanisms of action and being structurally related, the RIP types I and III are not as cytotoxic as ricin and the other RIP type II, such as abrin. This happens because those RIPs lack the B domain responsible to enable their entrance into the cells. 6 The mechanism of action of ricin comprises a permanent damage to the rRNA due to the abstraction of the adenine 4324 (A-4324) from the loop GAGA of the 28S rRNA, which is highly conserved in eukaryotic cells. This causes an interruption in the protein synthesis and lead to cell death. 7,8 The ricin unit responsible for this is known as RTA (or Ricin Toxin A) which unites to RTB (Ricin Unit B) through a disulfide bond to compose the whole ricin structure. Once inside the endoplasmic reticulum of the cell, RTA is separated from RTB, due to the action of the enzyme disulfide isomerase, and moves to the cytosol where it will promote the rRNA damage. To date, there is no antidote against such action yet and the vaccines developed so far to prevent ricin intoxication are not effective. 8,9 The search for antidotes against ricin has already afforded many compounds capable of binding to the catalytic site of RTA (comprising residues Val81, Gly121, Glu177, and Arg180) and work as competitive inhibitors. 10,11 However, no activity below the micromolar range was achieved yet, with the most promising compounds reported so far (Figure 1) presenting IC 50 between 6 and 20 μM. 11 The publication of the crystallographic structure of NNCP (Figure 1) complexed at the catalytic site of RTA in the Protein Data Bank (PDB) (https://www.rcsb.org/) under the ID: 4HUO, elucidated the binding mode (see Figure S1a) of those compounds. 11 Besides it also revealed that they are not capable of also binding to the secondary binding pocket of RTA (comprising residues Asp75, Asn78, Asp96, and Asp100). This pocket had been previously revealed by Ho et al. (2009) 12 through the crystallographic structure of RTA complexed with the model cyclic tetranucleotide inhibitor C2X [ Figure S1b], which mimics the recognition loop of the 28S rRNA [PDB (https://www.rcsb.org/) ID: 3HIO]. 12 This cyclic tetranucleotide was designed by Ho et al. (2009) 12 with the goal of establishing the catalytic site features contributing to the RTA catalytic activity. It is also a transition state structure meant to guide the design and synthesis of potent RIP inhibitors. Analysis of 4HUO [ Figure S1b] shows clearly that there is still enough  room for the drug design/discovery of new and more potent inhibitors, targeting at the same time the catalytic site and the secondary binding pocket of RTA.
Recently, we proposed the repurposing approach as a promising strategy to follow in the search for more effective antidotes against ricin intoxication capable of binding into both the catalytic site and the secondary pocket of RTA. 13 On this line, we performed virtual screening (VS) searches in the FDAapproved drugs data set, available at Cheminfo (https:// chemoinfo.ipmc.cnrs.fr/), the approved drugs library available at DrugBank (https://www.drugbank.ca/), and the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). This search enabled the selection of 6795 potential binders to RTA which, after additional refinements, including drug-likeness, afforded a library of 82 compounds. Further molecular dynamics (MD) simulations on the first 15 compounds selected from this library enabled pointing 6 ( Figure S2) as potential antidotes against RTA. 13 Here, we moved forward on the investigation of this library through additional theoretical studies on the 6 molecules shown in Figure S2 plus 9 more selected compounds ( Figure 2). Flexible docking of these compounds enabled drawing their fingerprints inside RTA and plotting the most important residues for the ligand binding. Also, longer MD simulations of 500 ns of the best poses of these compounds inside RTA enabled reevaluating the former results and selecting the ones that should keep the interactions inside the catalytic site and the secondary pocket, besides ranking them according to the potential of inhibiting RTA.
■ METHODOLOGY Protein Preparation. The receptor used to perform our studies was the three-dimensional structure of RTA in complex with the cyclic tetranucleotide inhibitor C2X available in the PDB (https://www.rcsb.org/) under the code: 3HIO. 12 This structure was downloaded and optimized using the default configuration of the quickprep tool of the MOE package (https://www.chemcomp.com/Products.htm) in order to remove gaps, optimize bond lengths and angles, calculate charges, and properly protonate residues and ligand according to the physiologic pH. Crystallographic water molecules were also removed, and only ligand and RTA were kept in the structure for the theoretical studies.
Ligand Preparation. The 3D structures of the ligands studied here (Figures 2 and S2) were constructed using the builder tool of the MOE package (https://www.chemcomp. com/Products.htm) and added to a MOE databank named as "ligands.mdb". Afterward, each entry of the databank was "washed", using the compute/molecule/wash tool of the MOE package (https://www.chemcomp.com/Products.htm), in order to optimize bond lengths, angles, and charges and also to afford the dominant species of each ligand under physiologic pH.
Docking Studies. The entries of the ligands.mdb file described above were submitted to rounds of docking calculations on the active site of the crystallographic structure of RTA (PDB ID: 3HIO) using the dock module of the MOE package (https://www.chemcomp.com/Products.htm). The docking grid was defined to include all residues present in the whole binding pocket of C2X and the docking placement method used was Triangle Matcher with induced fit refinement and the generate f ingerprints options active. First, the 100 best ranked poses (lowest energies) of each ligand were collected using the London dG scoring function, and afterward, those poses were re-scored using the GBVI/WSA dG function with the 20 best results being collected for further analysis of the fingerprints of each ligand inside RTA. This docking protocol was validated by re-docking of the crystallographic structure of C2X inside 3HIO, where the five best ranked poses were collected. The docking energy of NNCP inside RTA was obtained after energy minimization of the crystallographic structure 4HUO. Regarding NNCT, once it is reported in the literature that it binds the same way as NNCP, 11 the docking energy was calculated through energy minimization of 4HUO after changing the structure of NNCP to NNCT by adding an −OH group in the para position of the NNCP phenyl ring.
MD Simulations. The dynamics tool of the MOE package (https://www.chemcomp.com/Products.htm) was used to run the MD simulations. The Compute/Simulations/Dynamics path was used to prepare each system according to the parameters of the NAMD 14 software and using the forcefield AMBER10:EHT, 15 with a cutoff of 10 for electrostatic and 8, 10 for VdW interactions. Each complex was centered in a cubic box containing around 9000 water molecules and neutralized with NaCl ions. The rounds of simulations involved first 10 ps of an energy minimization step followed by 100 ps of NPT and 200 ps of NVT. Afterward, production steps of 500 ns of free MD simulation were performed. The MD simulation results were analyzed using the md_analysis tool and the database viewer (DBV) menu of MOE (https://www.chemcomp.com/ Products.htm). The trajectory files of each system, used for analysis of the MD simulations, were generated after superposition of the initial and simulated frames of protein and ligand.
MM-PBSA Calculations. Hybrid sequential QM/MM− MD methods are among the most accurate ways to estimate the free energy associated to the protein−ligand binding. 16 However, as many configurations are generated in large MD simulations, the number of QM calculations required is too high because in every step, an energy evaluation of the system is needed. Therefore, a great computational effort is necessary to carry out this kind of simulation. Aiming, then, to reduce the number of QM calculations without the loss of the relevant information from the simulation, new methods based on the statistical inefficiency and wavelet analysis for selecting MD conformations had been reported in the literature. 17 However, the computational cost involved is still high. One alternative to reduce this computational cost with an acceptable impact on the accuracy of the calculations is the use of the MM-PBSA approach, 18 which has become one of the most used methods for the estimation of free energy in biological systems. 19 Despite not explicitly calculating the entropy effect, the MM-PBSA method considers bonded and nonbonded interactions (vacuum potential energy), as well as polar and nonpolar terms (free energy of solvation). 18 The Poisson−Boltzmann equation is used to estimate the solvation energy term (usually with dielectric constant set to 1), while the surface area (SASA) method 18 is used to calculate the nonpolar solvation energy term.
Here, the MM-PBSA calculations were performed as before 13 using the g_mmpbsa tool, compatible with GROMACS 2019.4 software. 18 For this, it was necessary first to run 100 ns of MD simulation of the best poses of the ligands inside RTA obtained in the docking studies in order to afford the trajectory frames needed to feed the g_mmpbsa tool. Those MD simulations were performed following the same protocol used before 13 and described in detail in the Supporting Information. The g_mmpbsa tool was used to predict the binding free energy of the ligands that showed capable of keeping interactions in both pockets of RTA during the 500 ns of MD simulations with NAMD. 14 ■ RESULTS AND DISCUSSION As mentioned in the Introduction, we studied in this work the 6 molecules pointed before as potential antidotes against ricin ( Figure S2) 13 plus 9 additional compounds ( Figure 2) originally selected by VS and pointed by docking studies as potential dual binders to RTA, but not investigated through MD simulations. By extending the study to these 9 compounds, we advanced one more step toward the ultimate goal of investigating through MD simulations the whole library of 82 compounds formerly selected by VS. 13 These 9 compounds were selected based on a visual inspection to ensure a structural diversity that represents the most of the whole library.
Docking Study. The total energy values of the five poses returned from the re-docking of C2X over its crystallographic structure (3HIO) ranged from −15.23 to −15.62 kcal·mol −1 , while the root-mean-square deviation (RMSD) values ranged from 1.66 to 2.01 Å. Because a RMSD < 2.0 Å is considered valid according to the literature recommendation, 20 the poses obtained during our re-docking studies were good enough to validate the docking protocol used. The superposition with the lowest RMSD observed (1.66 Å) is shown in Figure S3.
It is well known that an appropriate theoretical strategy for selecting docking poses is crucial for the subsequent steps of MD simulations in order to avoid conformational changes in the time scale of few nanoseconds, which could compromise the MD simulation viability. For this reason, distinct selecting poses approaches have been reported in literature. 21 In the current work, as we are looking for potential dual binders to RTA, we selected the best ranked pose (lowest energy) of each ligand showing interactions with at least one residue of both the catalytic site and the secondary pocket of RTA. The only exception was C2X which the pose selected was the one with lowest RMSD from the re-docking, shown in Figure S3. The docking results obtained for the selected poses are summarized in Table 1.
As shown in Table 1, no ligand presented more negative energy value compared to C2X. This was already expected once this compound mimics the sarcin−ricin recognition loop of the 28S rRNA. However, the best poses of most ligands presented total energy more negative than NNCP and NNCT. This suggests a higher affinity for the RTA active site than the reference ligands and, therefore, a higher potential to act as more effective RTA inhibitors. Regarding the 9 additional compounds included in this work, all ranked in the same range of values (between −7.44 and −9.74 kcal·mol −1 ) as the 6 compounds repurposed before, 13 with the most promising among all these molecules being CID 135977982 and CID 136132835 ranking, respectively, −9.74 and −9.72 kcal·mol −1 . This means that they might also be good options of repurposed ligands against RTA.
Results in Table 1 also show that among the residues not belonging to the catalytic or the secondary pockets, Arg213 and Arg258 showed up in interactions with most of the ligands. These two residues are located in the border of the active site of RTA which includes the catalytic site and the secondary pocket,  but in opposition to them, as illustrated in Figure S4, which shows the best pose of CID 135977982 inside RTA. As far as we know Arg213 and Arg258 have not been reported yet in the literature as important residues for the binding of inhibitors to RTA.

MD Simulations.
The plots of total energy for the 18 complexes studied by MD simulation (Figures S5−S10) show that all systems stabilized since the beginning of the simulation time, with an average energy ranging between −0.9 × 10 −6 and −1.3 × 10 −6 kcal·mol −1 . Also, the RMSD plots calculated after    (Figure 3), show that the fluctuations never passed 6.0 and 2.5 Å for the ligands and protein, respectively, except for plazomicin that was not capable of stabilizing inside RTA and left after 50 ns of MD simulation (see also movie: Plazomicin_RTA.mpg in the Supporting Information). For the other 17 systems, the total energy and RMSD results point to stability over the simulated time and accommodation of the ligands inside RTA. Table 2 lists all H-bonds formed during the MD simulations with their respective percentages of occupancy, while the plots for the ligands capable of dual biding to RTA during more than 10% of the simulated time are shown in Figures S11−S14. In those Figures, the graphs in red lines represent the number of Hbonds formed with residues of the catalytic site of RTA, while the graphs in blue lines represent H-bonds formed with residues of the secondary pocket. As can be seen, most of the interactions pointed in the docking studies (Table 1) were confirmed by the MD simulations, with some additional interactions showing up during the MD simulations. It can also be seen in Table 2 that both Arg213 and Arg258 show up h-bonding to C2X with occupancies > 10%. Arg258 also shows up h-bonding to many other ligands with high percentages of occupancy. This corroborates the docking findings and suggests that these two residues might be key for the binding into RTA.
Analysis of Table 2 and Figure S11 show that C2X was capable of maintaining H-bonds with the residues of both the catalytic site and the secondary pocket during the whole simulated time, showing occupancies over 10% with at least three residues from each site. Also, the RMSD plot of C2X (see its red line in Figure  3) shows stabilization around 3.5 Å, what means a good accommodation inside RTA. This is well illustrated in the movie: C2X_RTA.mpg in Supporting Information and by the frames shown in Figure 4 (where the external loop of C2X was omitted for better clarity). As can be seen, the cytosine and guanosine portions of C2X, found, respectively, in the catalytic site and the secondary pocket of RTA in the crystallographic structure (PDB code 3HIO), stay inside those cavities during the whole simulation time. Therefore, our theoretical results confirmed that C2X is capable of binding and stabilizing inside RTA, mimicking the recognition loop of 28S rRNA. This corroborates the results reported by Ho et al. (2009) 12 and the mechanism of action reported in the literature for RTA. 12 Only CID 20044260, CID 22659428, naldemedine, CID 135977982, and CID 136132835 kept H-bonds with residues of both the catalytic site and the secondary pocket during the whole simulated time (see Figures S11−S14). Analysis of the dynamical behavior of those compounds (see the respective movies supplied as the Supporting Information) revealed that only naldemedine, CID 135977982, and CID 136132835 showed a good accommodation inside RTA as can be also observed in the superposition of frames collected during the MD simulations shown in Figure 4. This is also reflected in Figure 3 which show very little RMSD fluctuations for these compounds. CID 20044260 and CID 22659428, besides showing larger RMSD fluctuations (Figure 3), also did not accommodate well inside RTA as can be seen in the superposition of frames collected during the MD simulations shown in Figure S15 and the respective movies in the Supporting Information.
The large RMSD fluctuations observed in some systems are due to unstable interactions between protein and ligand which make both to move more. Larger fluctuation means worse behavior and, therefore, not good accommodation of the ligand inside RTA. They reflect changes in positions of the ligands during the MD simulations and the corresponding effect in the RTA behavior. These movements can be observed in the movies of the MD simulations supplied as Supporting Information.
Analysis of Figure S15 and the movies supplied as Supporting Information show that most of the no dual binders did not stabilize well inside RTA during the MD simulations. Regarding NNCP and NNCT, while the pterinic rings stabilized inside the catalytic site, the other moiety of those molecules did not find where to anchor and remained unstable the whole simulation. This is a possible reason for the IC 50 in the range of μM reported for these compounds. 11 A similar behavior was also observed for CID 136023163, CID 18498053, CID 20044260, deferoxamine, CID 18309602, ertapenem, leucovorin, and CID 22659482. Ceftaroline, on the other hand, moved from the active site since the beginning of the simulation and despite staying close to RTA, did not accommodate well inside it. The only ligands in Figure S15 that showed stable dynamic behavior inside RTA were nilotinib and CID 18493267 which, despite not behaving as dual binders, stabilized during the MD simulation, keeping interactions inside the secondary pocket and at the entrance of the catalytic site (see movies: Nilotinib_RTA.mpg and CID18493267_RTA.mpg supplied as the Supporting Information). This means that these two ligands might also bind strongly to RTA, avoiding the interactions with the loop GAGA of the rRNA 28S.
MM-PBSA Calculations. The bars plot in Figure 5 show the values of binding energies predicted through MM-PBSA calculations for the reference compounds and the potential dual binders pointed in the MD simulations. As expected, all of them presented negative binding energies, with a good correlation with the docking and MD results. This fact confirms their affinities for RTA. It is interesting to notice, however, that CID 135977982 and CID 136132835 showed better (more negative) binding energies than C2X. This is probably due to the cyclic part of C2X which did not establish stable interactions inside RTA and fluctuated during the MD simulation (see movie C2X_RTA.mpg in the Supporting Information). Those fluctuations might be adding unfavorable interactions which contribute to increase the binding energy value.
Comparison of Results for the 6 Potential Dual Binders Pointed Before. The flexible docking studies performed with MOE corroborated the rigid docking results   Table 3). This might be due to the higher flexibility of this compound that would favor finding a more energetically favorable pose during the flexible docking in comparison to the rigid docking. Despite this, in general, the results suggest that both docking protocols might be suitable for finding appropriate poses for the further MD simulations steps. Differently from the docking studies, not much corroboration was found when comparing the MD protocols applied to the 6 first potential dual binders. The results obtained for the extended MD simulations suggest that the 50 ns of free MD simulation ran before might not be enough to fully characterize the dynamical behavior of these compounds inside RTA. As shown in the H-bond plots in Figures 4 and S11, none of the 6 compounds was capable of keeping H-bonds with residues of the catalytic site and the secondary pocket during the whole simulated time. Deferoxamine was the one with best performance but lost consistency after 200 ns. CID 18498053, CID 18309602, and leucovorin established H-bonds mainly with the secondary pocket, while CID 136023163 and plazomicin lost the H-bonds with the RTA during the first third of the simulated time. Plazomicin was not even capable of stabilizing in another position inside RTA and left before 100 ns of simulation, as can be seem in the RMSD plots shown in Figure 3 and in the movie Plazomicin_RTA.mpg in the Supporting Information.
Pterin Derivatives. It is important to notice that the compounds CID 135977982, CID 136023163, and CID 136132835 (Figures 2 and S2) are all pterin derivatives, differing from each other only by the substitution pattern in the phenol ring. CID 136023163 brings two iodine atoms in the ortho positions to the OH group, while CID 136132835 brings one fluorine in one ortho position and an iodine in the other, and CID 135977982 has only one fluorine atom in ortho to the OH group. Despite this single difference, the best pose of CID 136023163 was not capable of stabilizing inside RTA forming H-bonds with the catalytic site and secondary pocket (see Figure  S15 and the movie CID136023163-RTA.mpg supplied as Supporting Information), while the other two analogues displayed the best binding results of our study, showing a binding mode very similar to the reference compound C2X (see Figure S14 and the movies CID135977982-RTA.mpg and CID136132835_RTA.mpg supplied as Supporting Information). This probably happened because the two large iodine atoms did not allow the stabilization of the phenol ring of CID 136023163 in the secondary pocket. Such result adds some valuable structure activity relationship (SAR) information to this project that certainly will help in the selection/design of the ideal ring size on this part of the molecule to achieve an effective and stable binding.

■ CONCLUSIONS
Our theoretical study allowed a refinement of the protocol used before 13 for the theoretical investigation of repurposed drugs selected by VS, as potential antidotes against ricin and also moving forward on the theoretical investigation of the library selected before. 13 Despite no significant changes were observed using the new docking protocol, the extension of the time of MD simulation for 500 ns showed a fundamental step to corroborate the docking results and filter the ligands which are really capable of performing stable interactions inside RTA. This refinement enabled pointing with more confidence four compounds to further in vitro assays: CID 135977982, CID 136132835, and naldemidine, as potential dual binders to RTA, and nilotinib and CID 18493267, as single binders to the secondary pocket of RTA. Despite not interacting directly with residues of the catalytic site, these compounds seem to be capable of interacting with residues at its entrance, blocking the catalytic activity of RTA. This might trigger a stronger inhibition. We believe that Table 3. Rigid and Flexible Docking Results for the 6 Potential Dual Binders Pointed Before ACS Omega http://pubs.acs.org/journal/acsodf Article these five compounds will present IC 50 values in the nM range after experimentally evaluated. Our study also corroborated the complex RTA/C2X as a consistent model of the RTA binding to the loop GAGA of the 28S rRNA. This validates the PDB structure 3HIO reported by Ho and con-workers 12 for the drug design against RTA. On this sense also the discovery of consistent H-bonds with residues Arg213 and Arg258 observed for most of the ligands in both the docking and MD simulations, opens new opportunities for the drug design of new antidotes against ricin. As far as we know, this was not described in the literature before. Still on the context of drug design, the limitation imposed by the two iodine atoms in the phenol ring of the pterin derivatives adds useful information for the design of more effective pterin derivatives as antidotes against ricin.