Influence of Open Chain and Cyclic Structure of Peptidomimetics on Antibacterial Activity in E. coli Strains

An efficient method for the synthesis of functionalized peptidomimetics via multicomponent Ugi reaction has been developed. The application of trifluoroethanol (TFE) as a reaction medium provided desired products with good yields. Further, using the developed cyclisation reaction, the obtained peptidomimetics were transformed into the cyclic analogues (diketopiperazines, DKPs). The goal of the performed studies was to revised and compare whether the structure of the obtained structurally flexible acyclic peptidomimetics and their rigid cycling analogue DKPs affect antimicrobial activity. We studied the potential of synthesized peptidomimetics, both cyclic and acyclic, as antimicrobial drugs on model E. coli bacteria strains (k12, R2–R4). The biological assays reveal that DKPs hold more potential as antimicrobial drugs compared to open chain Ugi peptidomimetics. We believe that it can be due to the rigid cyclic structure of DKPs which promotes the membrane penetration in the cell of studied pathogens. The obtained data clearly indicate the high antibiotic potential of synthesized diketopiperazine derivatives over tested antibiotics.


Introduction
Ugi reaction is a one pot multicomponent reaction for peptidomimetics synthesis. This transformation was already successfully engaged as a crucial step for diketopiperazines (DKPs) synthesis [1,2]. Diketopiperazine derivatives are known for their well-recognized biological activities. These small, cycling and conformationally rigid molecules have multiple functional groups which can interact with receptors and so show a broad spectrum of biological actions [3]. There are plentiful reports on bioactive compounds containing DKP moiety. This scaffold is quite important due to its interesting medicinal properties such as PDE5 inhibitors [4], oxytocin antagonists [5], cancer inhibitors [6], metalloproteinase inhibitors [7], antivirals [8], antibiotics [9], antibacterial [10], neuroprotective reagents [11], anxiolytic agents [12], anti-inflammatory inhibitors [13], bio herbicides [14] and natural products [15] (Figure 1). This work is a continuation of earlier work reported by us, in which we paid attention to the structure-activity relationship of a library of peptidomimetics synthesized via Ugi reaction to check their antimicrobial potential against E. coli strains [16]. However, here, we are more interested in assessing the effect of cyclisation with respect to the open chain Ugi peptidomimetics. In order to study the influence of structure, particularly its rigidity, it is necessary to develop the method for the synthesis of these two groups of compounds. We have exploited the diversity of Ugi reaction using several aldehydes, isocyanides, amines and acrylic acid to make a library of compounds. Reported peptidomimetics are a class of alkylating agents which should aid its antimicrobial activity but our research has shown otherwise, which may be related to cell membrane permeability in pathogens. 2-chloropropionic acid derivatives are also proven to be biologically active. For example, piracetam derivatives are known as cognitive enhancers [17] and 3CL pro inhibitors [18]. Due to antimicrobial resistance, there is a need to find new potent antimicrobial drugs. There are very few reports on the antimicrobial activities of DKPs against E. coli [19]. The goal of the presented studies is to design a method for the synthesis of DKPs and to substantiate their antimicrobial potential against model E. coli strains.

Chemicals
Starting materials and all other reagents were purchased from Sigma-Aldrich. All solvents were of analytical grade and were used without prior distillation. Merck silica gel plates 60 F254 were used for TLC (thin layer chromatography) analysis. Crude reaction mixtures were purified using column chromatography on Merck silica gel This work is a continuation of earlier work reported by us, in which we paid attention to the structure-activity relationship of a library of peptidomimetics synthesized via Ugi reaction to check their antimicrobial potential against E. coli strains [16]. However, here, we are more interested in assessing the effect of cyclisation with respect to the open chain Ugi peptidomimetics. In order to study the influence of structure, particularly its rigidity, it is necessary to develop the method for the synthesis of these two groups of compounds. We have exploited the diversity of Ugi reaction using several aldehydes, isocyanides, amines and acrylic acid to make a library of compounds. Reported peptidomimetics are a class of alkylating agents which should aid its antimicrobial activity but our research has shown otherwise, which may be related to cell membrane permeability in pathogens. 2-chloropropionic acid derivatives are also proven to be biologically active. For example, piracetam derivatives are known as cognitive enhancers [17] and 3CL pro inhibitors [18]. Due to antimicrobial resistance, there is a need to find new potent antimicrobial drugs. There are very few reports on the antimicrobial activities of DKPs against E. coli [19]. The goal of the presented studies is to design a method for the synthesis of DKPs and to substantiate their antimicrobial potential against model E. coli strains.

Chemicals
Starting materials and all other reagents were purchased from Sigma-Aldrich. All solvents were of analytical grade and were used without prior distillation. Merck silica gel plates 60 F254 were used for TLC (thin layer chromatography) analysis. Crude reaction mixtures were purified using column chromatography on Merck silica gel 60/230-400 mesh, with an appropriate mixture of hexane and ethyl acetate as a solvent. THF was dried according to standard procedure. Nuclear magnetic resonance spectra (NMR) were performed on a Bruker Avance 400 and Varian 500MHz instrument. Chemical shifts are expressed in ppm and coupling constant (J) in Hz using TMS as an internal standard. High-resolution mass spectra were acquired on a Maldi SYNAPT G2-S HDMS (Waters) apparatus with a QqTOF analyser.
The bacterial tests used, and MIC and MBC were accurately described in detail in the previous work [20][21][22][23][24][25][26]. An MTT test to assess the metabolic activity of cells was performed on the basis of [27][28][29][30], with THLE-5b as the control and the caco-2 cell line derived from human adenocarcinoma.

General Procedure for Synthesis of Compounds Va-Vh
To the mixture of benzyl amine (0.25 mmol) in 2,2,2-trifluoroethanol (1 mL, 0.25 mmol) corresponding aldehyde was added and the reaction mixture was stirred for 20 min at room temperature followed by the addition of 2-chloropropionic acid (0.25 mmol). After 20 min, isocyanide (0.25 mmol) was added to the reaction mixture and was stirred continuously for 18 h at 50 • C. Then, the solvent was evaporated and the crude products were purified by column chromatography on silica gel using hexane/AcOEt as the eluent.           13

General Procedure for Synthesis of Compounds VIa-VIh
Compound V (0.25 mmol) obtained in step 1 was dissolved in THF (3 mL) and cooled to 0 • C in an ice bath. After 5 min, sodium hydride (NaH 3 eq.) was added to it portion wise and the reaction mixture was refluxed for 3 hrs. The solvent was evaporated and the crude mixture was purified by column chromatography on silica gel using hexane/AcOEt as the eluent.

Chemistry
Diketopiperazines possess a variety of biological activities. They are found in numerous natural products and are also obtained from the degradation of polypeptides in foodstuff. Their small, conformationally strained structure makes them more attractive along with the possibility of instigating several substituents at six different positions.
Due to their rigid backbone, they are quite popular in drug discovery as an important pharmacophore. Post-Ugi transformation for the synthesis of diketopiperazines has been reported [31,32] but these transformations were using reagents such as PPh 3 which make the purification process hard. In this study, we synthesized a library of differently substituted diketopiperazines varying the substrates for the Ugi multicomponent reaction. To see the effect of substituents on cyclic and acyclic peptidomimetics we used different isocyanides (p-methoxybenzylisocyanide and isocyanocyclohexane) and eight different aldehydes. Diketopiperazines were synthesized in a two-step process. First, target peptidomimetics were obtained via Ugi multicomponent reaction (Scheme 1). A model Ugi reaction was carried out using 2-chloropropionic acid, benzyl amine, benzaldehyde and p-methoxybenzylisocyanide as substrates (Scheme 2) in methanol at room temperature following the same procedure reported by us [33], which resulted in product Va with 30% yield. To check the effect of temperature on reaction yield we performed the reaction with methanol at 30 • C and obtained product Va with 32% yield. A further increase in temperature to 50 • C led to a gradual increase in yield but when the temperature was increased to 60 • C, the reaction yield remained the same which indicates 50 • C as an optimal temperature for this reaction. Since we know that solvent can also affect the progress of the reaction and hence the yield, so we screened various polar protic solvents known for the Ugi reaction and we found Trifluoroethanol to be an efficient solvent for this transformation ( Table 1, entry 8). Then we further increased the temperature to 60 • C (entry 9) and yield dropped down to 42%. The mechanistic reason is unclear but it can be that the non-nucleophilic nature of TFE is suppressing the side reactions [34] and increasing the overall yield of product. So, we took the best conditions found in optimization ( Table 1, entry 8) for the synthesis of compounds Va-Vh with good yield (Figure 2). This Ugi reaction led to the formation of a diastereomeric mixture of peptidomimetics.

Chemistry
Diketopiperazines possess a variety of biological activities. They are found in numerous natural products and are also obtained from the degradation of polypeptides in foodstuff. Their small, conformationally strained structure makes them more attractive along with the possibility of instigating several substituents at six different positions. Due to their rigid backbone, they are quite popular in drug discovery as an important pharmacophore. Post-Ugi transformation for the synthesis of diketopiperazines has been reported [31][32][33] but these transformations were using reagents such as PPh3 which make the purification process hard. In this study, we synthesized a library of differently substituted diketopiperazines varying the substrates for the Ugi multicomponent reaction. To see the effect of substituents on cyclic and acyclic peptidomimetics we used different isocyanides (p-methoxybenzylisocyanide and isocyanocyclohexane) and eight different aldehydes. Diketopiperazines were synthesized in a two-step process. First, target peptidomimetics were obtained via Ugi multicomponent reaction (Scheme 1). A model Ugi reaction was carried out using 2-chloropropionic acid, benzyl amine, benzaldehyde and p-methoxybenzylisocyanide as substrates (Scheme 2) in methanol at room temperature following the same procedure reported by us [34], which resulted in product Va with 30% yield. To check the effect of temperature on reaction yield we performed the reaction with methanol at 30 °C and obtained product Va with 32% yield. A further increase in temperature to 50 °C led to a gradual increase in yield but when the temperature was increased to 60 °C, the reaction yield remained the same which indicates 50 °C as an optimal temperature for this reaction. Since we know that solvent can also affect the progress of the reaction and hence the yield, so we screened various polar protic solvents known for the Ugi reaction and we found Trifluoroethanol to be an efficient solvent for this transformation ( Table 1, entry 8). Then we further increased the temperature to 60 °C (entry 9) and yield dropped down to 42%. The mechanistic reason is unclear but it can be that the non-nucleophilic nature of TFE is suppressing the side reactions [35][36][37][38][39][40] and increasing the overall yield of product. So, we took the best conditions found in optimization (Table 1, entry 8) for the synthesis of compounds Va-Vh with good yield (Figure 2). This Ugi reaction led to the formation of a diastereomeric mixture of peptidomimetics.       Naliapara et. al. reported the cyclisation of Ugi peptidomimetic using a transition metal catalyst [35]. We modified the conditions for our reaction to eliminate the usage of a metal catalyst (Table 2, Scheme 3, Entry 1). This reaction yielded 25% product. Then, the same reaction was performed at a lower temperature (90 • C; Table 2, entry 2) as well as a high temperature (110 • C; Table 2, entry 3) but yield remained the same. Since the solvent is known to be a crucial parameter in terms of modulating the yield, several different organic solvents were applied (Table 2, entry 4). Further model reaction was conducted at different temperatures ( Table 2, entries 4-6); however, it was observed that at high temperatures, the yield decreases (Table 2, entry 6), which may be due to product decomposition. It indicates 65 • C as the optimal temperature for the studied cyclisation reaction. The application of various solvents (Table 2, entry 9-11) resulted in maximum yield (49%) with THF. Having optimized the solvent and temperature for the model reaction, we screened various organic and inorganic bases ( Table 2, entry [12][13][14][15][16] and observed that NaH in THF at 65 • C gives product VIa with a good yield of 70%. These conditions were further used to obtain DKPs VIa-VIh with the yields ranging from 44% to 70%. Upon cyclisation, we observed the change in diastereomeric ratios varying with the attached substituents in the peptidomimetic scaffold.

Cytotoxic Studies of the Synthesized Compounds
The obtained results indicate that all tested Diketopiperazines show cytotoxic activity in all analyzed E.coli strains differing in LPS length. Different inhibitory activity was found depending on the nature of the R1 and R2 substituents attached to the chlorine atom of the tested compounds. Among all tested compounds, the compounds from VIa-VIh showed a stronger antibacterial effect than Va-Vh. It is worth noting that the introduction of the chlorine atom into the structure of the tested compounds had a significant impact on their activity and cytotoxicity and high selectivity against selected E. coli model strains in the MIC and MBC tests, which is often observed in various types of compounds showing strong microbiological activity on cells [12]. These compounds showed higher activity against strains R2, R3 and R4 than commonly used antibiotics (Figures 3-7). The values of the MIC and MBC tests for each model of E. coli R2-R4 and K12 strains were visible on all analyzed growth microplates after the addition of resazurin.

Cytotoxic Studies of the Synthesized Compounds
The obtained results indicate that all tested Diketopiperazines show cytotoxic activity in all analyzed E.coli strains differing in LPS length. Different inhibitory activity was found depending on the nature of the R1 and R2 substituents attached to the chlorine atom of the tested compounds. Among all tested compounds, the compounds from VIa-VIh showed a stronger antibacterial effect than Va-Vh. It is worth noting that the introduction of the chlorine atom into the structure of the tested compounds had a significant impact on their activity and cytotoxicity and high selectivity against selected E. coli model strains in the MIC and MBC tests, which is often observed in various types of compounds showing strong microbiological activity on cells [12]. These compounds showed higher activity against strains R2, R3 and R4 than commonly used antibiotics (Figures 3-7). The values of the MIC and MBC tests for each model of E. coli R2-R4 and K12 strains were visible on all analyzed growth microplates after the addition of resazurin. significant impact on their activity and cytotoxicity and high selectivity against selected E. coli model strains in the MIC and MBC tests, which is often observed in various types of compounds showing strong microbiological activity on cells [12]. These compounds showed higher activity against strains R2, R3 and R4 than commonly used antibiotics (Figures 3-7). The values of the MIC and MBC tests for each model of E. coli R2-R4 and K12 strains were visible on all analyzed growth microplates after the addition of resazurin.      The analyzed bacterial strains used in the experiments were used in 48-well plates; which were treated with the analyzed compounds in the MIC and MBC assays. On the basis of their analysis, color changes were observed for all tested compounds, but at different levels and at different dilutions. The most sensitive to the effects of the analyzed compounds were the bacterial strains R3 and R4 due to the increasing length of their LPS (visible dilutions 10 −2 corresponding to a concentration of 0.0015 µM); more than strains K12 and R2 (visible dilutions of 10 −6 corresponding to a concentration of 0.0015 µM). Strain R4 was the most sensitive, possibly due to the longest length of lipopolysaccharide (LPS) in the bacterial membrane. In all analyzed cases, the MBC test values were approximately 75 times higher than the MIC test values in eight analyzed compounds including Cl (Figures 3-5 and Table 3). The y-axis shows the MIC value in μg/mL −1 . Investigated strains of E. coli K12 as control (blue), R2 strains (orange), R3 strain (grey), and R4 strain (yellow). The y-axis shows the MBC value in μg/mL −1 . The order in which the compounds were applied to the plate is shown in Supplementary Materials Figure S1.  and R4 strain (yellow). The y-axis shows the MBC value in μg/mL −1 . The order in which the compounds were applied to the plate is shown in Supplementary Materials Figure S1.  , and R2-R4 strains (x-axis). All analyzed compounds numbered were statistically significant at <0.05 (see Table 2 and Supplementary Materials Figure S2 panel A and B). , and R2-R4 strains (x-axis). All analyzed compounds numbered were statistically significant at <0.05 * (see Table 2 and Supplementary Materials Figure S2 panel A and B).

Analysis of R2-R4 E. coli Strains Modified with Tested Compounds diketopiperazines
The obtained MIC values, as well as our previous studies with various types of the analyzed compounds [20][21][22][23][24][25][26][27][28][29][30], indicate that derivatives of diketopiperazines also show a strong toxic effect of the analyzed model strains of bacteria. The three compounds analyzed were selected for further analysis by modifying their DNA. Modified bacterial DNA was digested with Fpg as previously described [36][37][38][39][40][41]. All selected analyzed derivatives of diketopiperazines including various types of alkoxy groups, substituents located at aromatic rings and the length of the alkyl chain can strongly change the bacterial DNA topology. After Fpg digestion, approximately 3.5% of the oxidative damage was identified, which, similar to previous observations, indicates very strong oxidative damage in bacterial DNA [7][8][9]. Different types of alkoxy groups, substituents located on the aromatic ring and the length of the alkyl chain, may determine the toxicity of the analyzed E. coli strains, including in particular R4, as evidenced by the obtained MIC, MBC and MTT values. The obtained results for individual compounds were statistically significant at the level of p < 0.05 (Figures 6-8). The analyzed bacterial strains used in the experiments were used in 48-well plates; which were treated with the analyzed compounds in the MIC and MBC assays. On the basis of their analysis, color changes were observed for all tested compounds, but at different levels and at different dilutions. The most sensitive to the effects of the analyzed compounds were the bacterial strains R3 and R4 due to the increasing length of their LPS (visible dilutions 10 −2 corresponding to a concentration of 0.0015 μM); more than strains K12 and R2 (visible dilutions of 10 −6 corresponding to a concentration of 0.0015 μM). Strain R4 was the most sensitive, possibly due to the longest length of lipopolysaccharide (LPS) in the bacterial membrane. In all analyzed cases, the MBC test values were approximately 75 times higher than the MIC test values in eight analyzed compounds including Cl (Figures 3-5 and Table 3).

Analysis of R2-R4 E. coli Strains Modified with Tested Compounds diketopiperazines
The obtained MIC values, as well as our previous studies with various types of the analyzed compounds [20,21,22,23,24,25,26,27,28,29,30], indicate that derivatives of diketopiperazines also show a strong toxic effect of the analyzed model strains of bacteria. The three compounds analyzed were selected for further analysis by modifying their DNA. Modified bacterial DNA was digested with Fpg as previously described   [36,37,38,39,40,41]. All selected analyzed derivatives of diketopiperazines including various types of alkoxy groups, substituents located at aromatic rings and the length of the alkyl chain can strongly change the bacterial DNA topology. After Fpg digestion, approximately 3.5% of the oxidative damage was identified, which, similar to previous observations, indicates very strong oxidative damage in bacterial DNA [7,8,9]. Different types of alkoxy groups, substituents located on the aromatic ring and the length of the alkyl chain, may determine the toxicity of the analyzed E. coli strains, including in particular R4, as evidenced by the obtained MIC, MBC and MTT values. The obtained results for individual compounds were statistically significant at the level of p < 0.05 (Figures 6-8).  Figure S3).

R2-R4 E. coli Strains with Tested Peptidomimetics
The performed studies support the concept that the synthesized compounds can be considered drug candidates for further studies (Supplementary Materials Figure S3).

R2-R4 E. coli Strains with Tested Peptidomimetics
The performed studies support the concept that the synthesized compounds can be considered drug candidates for further studies (Supplementary Materials Figure S3).

Conclusions
An efficient method for the synthesis of peptidomimetics was developed using the Ugi reaction; these were then used to synthesis diketopiperazines. The established protocol was used to synthesize a series of target products containing differently substituted aldehydes and isocyanides. This protocol ensures the efficient, gentle and metal-free synthesis of the target products with good yields (49%-70%). The cytotoxic effect of the obtained cyclic and acyclic peptidomimetics was assessed on model E. coli strain and its mutants. The analyzed diketopiperazines derivatives are able to modify all model strains of E. coli (R2-R4) and their bacterial DNA, changing the spatial structure of LPS contained in their cell membranes. Compared to the derivatives, the most active among the tested derivatives turned out to be those with cyclic structures VI-VIh. We have found that the stiffening of the peptidomimetic structure is responsible for increase in their antimicrobial activity. Figure 3 reveals that compounds VIa, VIb, VIc and VIg have the highest potential as antibacterial drug candidates among the tested DKPs. Our studies also show that synthesized DKPs have a lower MIC value compared to well-known antibiotics, which allows us to say that DKPs hold more potential as antibiotic drug candidates due to high anti-bacterial activity for all the tested mutants. The results of the presented research are important for understanding the biological properties of the studied derivative diketopiperazines as a function of potential new antibiotics and their toxic effects on gram-negative bacteria and cancer cell lines in the face of the growing drug resistance pandemic. Referring to our previous work related to the characteristics of the E. coli K12 and R2-R4 models it is worth continuing this research on the separation of diastereomers of the analyzed compounds and their studies on E. coli strains and on human cell lines in various types of cancer.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27113633/s1, Figure S1. Examples of MIC and MBC on microplates with different concentration of studied compounds (mg L −1 ). Figure S2. An example of an agarose gel electrophoresis separation of isolated plasmids DNA on R4 strains modified with selected compounds (Panel A) from 8 selected compounds, as shown in Figure 3, and digested with repair Fpg protein (Panel B). M = marker. Figure S3. Example of an agarose gel electrophoresis separation of isolated plasmids DNA from R2-R4 strains modified with antibiotics: bleomycin, ciprofloxacin, and cloxacillin digested with repair enzymes Fpg. M = marker. Figure S4: Structure of antibiotics. Figure