N-Heterocycles Scaffolds as Quorum Sensing Inhibitors. Design, Synthesis, Biological and Docking Studies

Quorum sensing is a communication system among bacteria to sense the proper time to express their virulence factors. Quorum sensing inhibition is a therapeutic strategy to block bacterial mechanisms of virulence. The aim of this study was to synthesize and evaluate new bioisosteres of N-acyl homoserine lactones as Quorum sensing inhibitors in Chromobacterium violaceum CV026 by quantifying the specific production of violacein. Five series of compounds with different heterocyclic scaffolds were synthesized in good yields: thiazoles, 16a–c, thiazolines 17a–c, benzimidazoles 18a–c, pyridines 19a–c and imidazolines 32a–c. All 15 compounds showed activity as Quorum sensing inhibitors except 16a. Compounds 16b, 17a–c, 18a, 18c, 19c and 32b exhibited activity at concentrations of 10 µM and 100 µM, highlighting the activity of benzimidazole 18a (IC50 = 36.67 µM) and 32b (IC50 = 85.03 µM). Pyridine 19c displayed the best quorum sensing inhibition activity (IC50 = 9.66 µM). Molecular docking simulations were conducted for all test compounds on the Chromobacterium violaceum CviR protein to gain insight into the process of quorum sensing inhibition. The in-silico data reveal that all 15 the compounds have higher affinity for the protein than the native AHL ligand (1). A strong correlation was found between the theoretical and experimental results.


Introduction
Quorum sensing (QS), a mechanism of cell-to-cell communication in bacteria and fungi, involves self-produced chemical signals called autoinducers that function as semiochemicals [1]. Bacteria use this mechanism to communicate among themselves through the recognition and measurement of extracellular autoinducers, which accumulate in the local environment until reaching a certain level. At such a point, the bacterial population is sufficient to allow for group actions and therefore signaling pathways are activated and specific genes (some related to virulence factors) are transcribed [2][3][4][5].
Since the disruption of QS could plausibly attenuate or halt bacterial virulence and overcome bacterial resistance, it is an attractive target for drug discovery [6,7]. In Gram-negative bacteria the autoinducers for QS are N-acyl homoserine lactones (AHLs 1). They are synthesized and released to the extracellular medium, diffuse freely through the bacterial membrane, and bind to specific cytoplasmic receptors. Once reaching a certain level, AHLs 1 promote specific gene expression After synthesizing AHL bioisosteres, it is important to demonstrate their capacity to inhibit QS. An excellent model for this purpose is Chromobacterium violaceum due to it is a QS biosensor [19]. The aim of the present study was to synthesize new bioisosteres of AHL and evaluate them in C. violaceum CV026 as QSIs by quantifying the specific production of violacein. Five series of compounds were synthesized, each containing a distinct heterocyclic scaffold in its structure: thiazoles (16a-c), thiazolines (17a-c), benzimidazoles (18a-c), pyridines (19a-c) and imidazolines (32a-c) to be assayed experimentally. Finally, molecular docking simulations were conducted for all 15 test compounds on C. violaceum CviR protein to provide insights into the process of QS inhibition. After synthesizing AHL bioisosteres, it is important to demonstrate their capacity to inhibit QS. An excellent model for this purpose is Chromobacterium violaceum due to it is a QS biosensor [19]. The aim of the present study was to synthesize new bioisosteres of AHL and evaluate them in C. violaceum CV026 as QSIs by quantifying the specific production of violacein. Five series of compounds were synthesized, each containing a distinct heterocyclic scaffold in its structure: thiazoles (16a-c), thiazolines (17a-c), benzimidazoles (18a-c), pyridines (19a-c) and imidazolines (32a-c) to be assayed experimentally. Finally, molecular docking simulations were conducted for all 15 test compounds on C. violaceum CviR protein to provide insights into the process of QS inhibition.
To obtain 2-amine thiazol 22, Hantzsch synthesis was carried out with an α-halo carbonyl compound, α-bromo ethyl pyruvate (20) and thiourea as raw materials and EtOH as solvent according to the methodology published by Kaushik [25]. The reaction mixture was stirred for 16 h at room temperature (rt), furnishing the desired product in good yields. (Scheme 1.) Subsequently, intermediary 22 was acylated with the corresponding carboxylic acid, a reaction described by various authors [26][27][28][29]. Such a reaction occurs in 2-amine thiazole with electron with drawing groups in position 4, for which heterogeneous yields have been documented in the literature. For instance, low yields (17-47%) were provided when Parish performed the acylation reaction with acyl chlorides in the presence of bases [26]. The acylation reaction has also been carried out with carboxylic acids and coupling reagents CDI or EDCI, affording moderate to good yields (54-69%) [27]. Considering, this background, distinct conditions were herein investigated but two procedures were followed: MW energy, as reported by You [28], and DCC as a coupling reagent with conventional heating as the energy source [29] (Table 1). Data in the literature [27] indicate that the acylation reaction of compounds like 22 depends on the coupling reagents, the solvent and the mixture of these two factors. Additionally, it is necessary Scheme 1. Synthetic route to obtain the N-(thiazol-2-yl)-amine (22). Reagents and conditions: a: EtOH, rt, 16 h.
To obtain 2-amine thiazol 22, Hantzsch synthesis was carried out with an α-halo carbonyl compound, α-bromo ethyl pyruvate (20) and thiourea as raw materials and EtOH as solvent according to the methodology published by Kaushik [25]. The reaction mixture was stirred for 16 h at room temperature (rt), furnishing the desired product in good yields. (Scheme 1.) Subsequently, intermediary 22 was acylated with the corresponding carboxylic acid, a reaction described by various authors [26][27][28][29]. Such a reaction occurs in 2-amine thiazole with electron with drawing groups in position 4, for which heterogeneous yields have been documented in the literature. For instance, low yields (17-47%) were provided when Parish performed the acylation reaction with acyl chlorides in the presence of bases [26]. The acylation reaction has also been carried out with carboxylic acids and coupling reagents CDI or EDCI, affording moderate to good yields (54-69%) [27]. Considering, this background, distinct conditions were herein investigated but two procedures were followed: MW energy, as reported by You [28], and DCC as a coupling reagent with conventional heating as the energy source [29] (Table 1). Pyridines 19a-c. Although the pyridines contain two elements of AHL, they are regarded as non-classical bioisosteres because the pyridine ring is completely different from the lactone ring. Consequently, they are based on an exchange of one functional group for another [12].
To obtain 2-amine thiazol 22, Hantzsch synthesis was carried out with an α-halo carbonyl compound, α-bromo ethyl pyruvate (20) and thiourea as raw materials and EtOH as solvent according to the methodology published by Kaushik [25]. The reaction mixture was stirred for 16 h at room temperature (rt), furnishing the desired product in good yields. (Scheme 1.) Subsequently, intermediary 22 was acylated with the corresponding carboxylic acid, a reaction described by various authors [26][27][28][29]. Such a reaction occurs in 2-amine thiazole with electron with drawing groups in position 4, for which heterogeneous yields have been documented in the literature. For instance, low yields (17-47%) were provided when Parish performed the acylation reaction with acyl chlorides in the presence of bases [26]. The acylation reaction has also been carried out with carboxylic acids and coupling reagents CDI or EDCI, affording moderate to good yields (54-69%) [27]. Considering, this background, distinct conditions were herein investigated but two procedures were followed: MW energy, as reported by You [28], and DCC as a coupling reagent with conventional heating as the energy source [29] (Table 1). Data in the literature [27] indicate that the acylation reaction of compounds like 22 depends on the coupling reagents, the solvent and the mixture of these two factors. Additionally, it is necessary to consider the low nucleophilicity of the amine group conjugated to an electron withdrawing group. When Toyoshima [30] carried out the synthesis of compounds 16, the yields were lower than those found presently. In the acylation reaction of an amine, the excess concentration of the acid is important due autocatalysis [31]. Compounds 16 have been tested as antivirals and anti-tuberculosis drugs [30] but never as QSIs.
The synthetic pathway began with the reaction between 4-amino benzonitrile and cysteamine hydrochloride to prepare intermediate 25.
To obtain 25, 4-amine benzonitrile and cysteamine hydrochoride were reacted by using EtOH: H 2 O 1:1 as solvent ( Table 2). to consider the low nucleophilicity of the amine group conjugated to an electron withdrawing group. When Toyoshima [30] carried out the synthesis of compounds 16, the yields were lower than those found presently. In the acylation reaction of an amine, the excess concentration of the acid is important due autocatalysis [31]. Compounds 16 have been tested as antivirals and anti-tuberculosis drugs [30] but never as QSIs.
The synthetic pathway began with the reaction between 4-amino benzonitrile and cysteamine hydrochloride to prepare intermediate 25.
To obtain 25, 4-amine benzonitrile and cysteamine hydrochoride were reacted by using EtOH: H2O 1:1 as solvent ( Table 2). Cazin conditions [32] provided a low yield for 25 (Table 2, experiment 1), observing an incomplete reaction. Lengthening the reaction time (experiment 2) did not improve the outcome, nor did a greater amount of base (experiment 3), which caused the yield to drop sharply due to the hydrolysis of benzonitrile to carboxylic acid. The combination of a weaker base (K2CO3 instead of NaOH) and a higher reaction temperature, as described by Hintermann [33], gave better results (experiment 4). Since the raw material was detected in TLC, the reaction time was increased (experiment 5), leading to a high yield with the complete reaction of the raw material.
It is known that the transformation of benzonitrile to thiazoline was established in accordance with the computational studies by Mor and Cavalli [34], (Figure 3). The synthesis of 17 was completed by an acylation reaction of 25 with the corresponding carboxylic acids. Two possibilities were taken into account when planning the synthetic route of 17: the acylation of amine thiazole 22 and the acylation of anilines, the latter frequently reported. The different conditions that have been adopted for the amine acylation reaction include carbodiimides as coupling reagents [26,27], MW and SiO2 [35], zeolitas as catalyst [31] and transamidation with  Cazin conditions [32] provided a low yield for 25 (Table 2, experiment 1), observing an incomplete reaction. Lengthening the reaction time (experiment 2) did not improve the outcome, nor did a greater amount of base (experiment 3), which caused the yield to drop sharply due to the hydrolysis of benzonitrile to carboxylic acid. The combination of a weaker base (K 2 CO 3 instead of NaOH) and a higher reaction temperature, as described by Hintermann [33], gave better results (experiment 4). Since the raw material was detected in TLC, the reaction time was increased (experiment 5), leading to a high yield with the complete reaction of the raw material.
It is known that the transformation of benzonitrile to thiazoline was established in accordance with the computational studies by Mor and Cavalli [34], (Figure 3). to consider the low nucleophilicity of the amine group conjugated to an electron withdrawing group. When Toyoshima [30] carried out the synthesis of compounds 16, the yields were lower than those found presently. In the acylation reaction of an amine, the excess concentration of the acid is important due autocatalysis [31]. Compounds 16 have been tested as antivirals and anti-tuberculosis drugs [30] but never as QSIs.
To obtain 25, 4-amine benzonitrile and cysteamine hydrochoride were reacted by using EtOH: H2O 1:1 as solvent ( Table 2). Cazin conditions [32] provided a low yield for 25 ( Table 2, experiment 1), observing an incomplete reaction. Lengthening the reaction time (experiment 2) did not improve the outcome, nor did a greater amount of base (experiment 3), which caused the yield to drop sharply due to the hydrolysis of benzonitrile to carboxylic acid. The combination of a weaker base (K2CO3 instead of NaOH) and a higher reaction temperature, as described by Hintermann [33], gave better results (experiment 4). Since the raw material was detected in TLC, the reaction time was increased (experiment 5), leading to a high yield with the complete reaction of the raw material.
It is known that the transformation of benzonitrile to thiazoline was established in accordance with the computational studies by Mor and Cavalli [34], (Figure 3). The synthesis of 17 was completed by an acylation reaction of 25 with the corresponding carboxylic acids. Two possibilities were taken into account when planning the synthetic route of 17: the acylation of amine thiazole 22 and the acylation of anilines, the latter frequently reported. The different conditions that have been adopted for the amine acylation reaction include carbodiimides as coupling reagents [26,27], MW and SiO2 [35], zeolitas as catalyst [31] and transamidation with The synthesis of 17 was completed by an acylation reaction of 25 with the corresponding carboxylic acids. Two possibilities were taken into account when planning the synthetic route of 17: the acylation of amine thiazole 22 and the acylation of anilines, the latter frequently reported. The different conditions that have been adopted for the amine acylation reaction include carbodiimides as coupling reagents [26,27], MW and SiO 2 [35], zeolitas as catalyst [31] and transamidation with zirconocene dichloride [36]. The methodology chosen, based on the acylation of thiazole 22 (Table 3), furnished the novel thiazolines 17a-c in good yields. zirconocene dichloride [36]. The methodology chosen, based on the acylation of thiazole 22 (Table 3), furnished the novel thiazolines 17a-c in good yields.
Experiment 1 was carried out under the conditions described by Micheva [37], who obtained very good yields at 3 h of reaction. In our hands, however, the outcome was different, detecting the presence of phenylenediamine after 3 h (experiment 1). A change to MW energy (experiment 2) was also unsuccessful. By using conventional energy and increasing the reaction time from 3 to 5 h (experiment 3), the total consumption of raw material was observed and thione 27 was produced in a higher yield. In accordance with the work of Sartori [38], the proposed reaction sequence is the formation of ethyl(2-amino-5-chlorophenyl)carbamothioate 29 followed by its conversion into isothiocyanonitrile 30 and finally into thione 27 (Scheme 3). This intermediate 27 was then subjected to an acylation reaction [25] with the bromoacetamides 28a, 28b or alkylation with n-bromononane 28c, to obtain 18a-c, Scheme 3. Bromoacetamides 28a-b were synthesized with 2-bromo acetyl bromide and 3-Cl aniline 31 or 4-Cl benzylamine 32 (Scheme 4). Finally 28 were reacted with 27 to achieve 18a-c. Scheme 2. There are reports of benzimidazole acetamides as antidiabetic drugs [39], antimicrobial agents [40], and QSI in P. aeruginosa [41] Experiment 1 was carried out under the conditions described by Micheva [37], who obtained very good yields at 3 h of reaction. In our hands, however, the outcome was different, detecting the presence of phenylenediamine after 3 h (experiment 1). A change to MW energy (experiment 2) was also unsuccessful. By using conventional energy and increasing the reaction time from 3 to 5 h (experiment 3), the total consumption of raw material was observed and thione 27 was produced in a higher yield. In accordance with the work of Sartori [38], the proposed reaction sequence is the formation of ethyl(2-amino-5-chlorophenyl)carbamothioate 29 followed by its conversion into isothiocyanonitrile 30 and finally into thione 27 (Scheme 3).  Reactions conditions: Phenylenediamine (1 eq), CS2 (1.3 eq), KOH (3 eq), EtOH/H2O 7:3 (5 mL), 80 °C.
Experiment 1 was carried out under the conditions described by Micheva [37], who obtained very good yields at 3 h of reaction. In our hands, however, the outcome was different, detecting the presence of phenylenediamine after 3 h (experiment 1). A change to MW energy (experiment 2) was also unsuccessful. By using conventional energy and increasing the reaction time from 3 to 5 h (experiment 3), the total consumption of raw material was observed and thione 27 was produced in a higher yield. In accordance with the work of Sartori [38], the proposed reaction sequence is the formation of ethyl(2-amino-5-chlorophenyl)carbamothioate 29 followed by its conversion into isothiocyanonitrile 30 and finally into thione 27 (Scheme 3). This intermediate 27 was then subjected to an acylation reaction [25] with the bromoacetamides 28a, 28b or alkylation with n-bromononane 28c, to obtain 18a-c, Scheme 3. Bromoacetamides 28a-b were synthesized with 2-bromo acetyl bromide and 3-Cl aniline 31 or 4-Cl benzylamine 32 (Scheme 4). Finally 28 were reacted with 27 to achieve 18a-c. Scheme 2. There are reports of benzimidazole acetamides as antidiabetic drugs [39], antimicrobial agents [40], and QSI in P. aeruginosa [41]. However, benzimidazoles 18 are new compounds that open a broad range of opportunities for research into QSIs. This intermediate 27 was then subjected to an acylation reaction [25] with the bromoacetamides 28a, 28b or alkylation with n-bromononane 28c, to obtain 18a-c, Scheme 3. Bromoacetamides 28a-b were synthesized with 2-bromo acetyl bromide and 3-Cl aniline 31 or 4-Cl benzylamine 32 (Scheme 4).  Reactions conditions: Phenylenediamine (1 eq), CS2 (1.3 eq), KOH (3 eq), EtOH/H2O 7:3 (5 mL), 80 °C.
Experiment 1 was carried out under the conditions described by Micheva [37], who obtained very good yields at 3 h of reaction. In our hands, however, the outcome was different, detecting the presence of phenylenediamine after 3 h (experiment 1). A change to MW energy (experiment 2) was also unsuccessful. By using conventional energy and increasing the reaction time from 3 to 5 h (experiment 3), the total consumption of raw material was observed and thione 27 was produced in a higher yield. In accordance with the work of Sartori [38], the proposed reaction sequence is the formation of ethyl(2-amino-5-chlorophenyl)carbamothioate 29 followed by its conversion into isothiocyanonitrile 30 and finally into thione 27 (Scheme 3). This intermediate 27 was then subjected to an acylation reaction [25] with the bromoacetamides 28a, 28b or alkylation with n-bromononane 28c, to obtain 18a-c, Scheme 3. Bromoacetamides 28a-b were synthesized with 2-bromo acetyl bromide and 3-Cl aniline 31 or 4-Cl benzylamine 32 (Scheme 4). Finally 28 were reacted with 27 to achieve 18a-c. Scheme 2. There are reports of benzimidazole acetamides as antidiabetic drugs [39], antimicrobial agents [40], and QSI in P. aeruginosa [41]. However, benzimidazoles 18 are new compounds that open a broad range of opportunities for research into QSIs. Finally 28 were reacted with 27 to achieve 18a-c. Scheme 2. There are reports of benzimidazole acetamides as antidiabetic drugs [39], antimicrobial agents [40], and QSI in P. aeruginosa [41]. However, benzimidazoles 18 are new compounds that open a broad range of opportunities for research into QSIs.

Synthesis of N-Substituted-2-[4-(imidazolin-2-yl)-phenoxy]-acetamides (32a-c)
In addition to designing bioisosters 16-19, three other novel imidazoline acetamides were synthesized (32a-c) because of the similarity of their structure to that of imidazoline, the QS inhibitory activity of which is well recognized [20]. Moreover, their structural elements coincide with those considered presently. In a previous theoretical study, the ΔG of the complexes formed by 32ac and the CviR protein were calculated, finding much lower values than for the complex of AHL with the same protein [44].
Based on the method described by our group [20], imidazolines 32 were synthesized with different substituents on the amide group. Obtaining compounds 32 began with the synthesis of the aldehydes 35, for this it was necessary to obtain acetamide 34 (Scheme 5). To prepare aldehydes 35, a previous report was considered [20] (Table 6) Although compounds 19 are known and their use has been reported [42,43], their possible application as QSIs is unexplored. Characterization of pyridines 19 was not found.

Synthesis of
In addition to designing bioisosters 16-19, three other novel imidazoline acetamides were synthesized (32a-c) because of the similarity of their structure to that of imidazoline, the QS inhibitory activity of which is well recognized [20]. Moreover, their structural elements coincide with those considered presently. In a previous theoretical study, the ∆G of the complexes formed by 32a-c and the CviR protein were calculated, finding much lower values than for the complex of AHL with the same protein [44].
Based on the method described by our group [20], imidazolines 32 were synthesized with different substituents on the amide group. Obtaining compounds 32 began with the synthesis of the aldehydes 35, for this it was necessary to obtain acetamide 34 (Scheme 5).

Synthesis of N-Substituted-2-[4-(imidazolin-2-yl)-phenoxy]-acetamides (32a-c)
In addition to designing bioisosters 16-19, three other novel imidazoline acetamides were synthesized (32a-c) because of the similarity of their structure to that of imidazoline, the QS inhibitory activity of which is well recognized [20]. Moreover, their structural elements coincide with those considered presently. In a previous theoretical study, the ΔG of the complexes formed by 32ac and the CviR protein were calculated, finding much lower values than for the complex of AHL with the same protein [44].
Based on the method described by our group [20], imidazolines 32 were synthesized with different substituents on the amide group. Obtaining compounds 32 began with the synthesis of the aldehydes 35, for this it was necessary to obtain acetamide 34 (Scheme 5). To prepare aldehydes 35, a previous report was considered [20] (Table 6). To prepare aldehydes 35, a previous report was considered [20] (Table 6).  Compared to the report on the preparation of aldehyde 35a, a shorter time was herein achieved ( Table 6) by using 2-bromoacetyl bromide rather than the previously employed α-bromoacetic acid and DCC [20]. Compounds 35 were subjected to cyclization with ethylenediamine and subsequent oxidation with NBS under ultrasound irradiation, as reported by Torres [29]. The corresponding imidazolines 32a-c were provided in good yields (85-90%) (Scheme 6).

Characterization of Compounds
All compounds were characterized by spectroscopic methods utilizing IR, NMR, and DIP-MS. Spectra were assigned with the help of heteronuclear correlation (gHSQC, gHMBC) and in some cases with homonuclear correlation (gCOSY). Mass spectra were recorded with positive and negative ions. Spectra for all compounds are attached in the Supplementary Materials (Figures S1-S104).

Evaluation of QS Inhibition on Chromobacterium violaceum CV026
The synthesized compounds were added (at 10, 100 and 1000 µM) to the bacteria (OD600 of 0.12) in thioglycolate broth supplemented with C6-AHL, followed by incubation for 16 h. The determination of the specific production of violacein, calculated as the ratio of detectable pigment (OD577) per amount of bacteria (OD720), demonstrated the existence of QS inhibitory activity for almost all test compounds. (Figures 4-8). There is a clear difference between quorum sensing and antibacterial activity. In this research, growth inhibition by the evaluated heterocycles results in a Compared to the report on the preparation of aldehyde 35a, a shorter time was herein achieved ( Table 6) by using 2-bromoacetyl bromide rather than the previously employed α-bromoacetic acid and DCC [20]. Compounds 35 were subjected to cyclization with ethylenediamine and subsequent oxidation with NBS under ultrasound irradiation, as reported by Torres [29]. The corresponding imidazolines 32a-c were provided in good yields (85-90%) (Scheme 6).  Compared to the report on the preparation of aldehyde 35a, a shorter time was herein achieved ( Table 6) by using 2-bromoacetyl bromide rather than the previously employed α-bromoacetic acid and DCC [20]. Compounds 35 were subjected to cyclization with ethylenediamine and subsequent oxidation with NBS under ultrasound irradiation, as reported by Torres [29]. The corresponding imidazolines 32a-c were provided in good yields (85-90%) (Scheme 6).

Characterization of Compounds
All compounds were characterized by spectroscopic methods utilizing IR, NMR, and DIP-MS. Spectra were assigned with the help of heteronuclear correlation (gHSQC, gHMBC) and in some cases with homonuclear correlation (gCOSY). Mass spectra were recorded with positive and negative ions. Spectra for all compounds are attached in the Supplementary Materials (Figures S1-S104).

Evaluation of QS Inhibition on Chromobacterium violaceum CV026
The synthesized compounds were added (at 10, 100 and 1000 µM) to the bacteria (OD600 of 0.12) in thioglycolate broth supplemented with C6-AHL, followed by incubation for 16 h. The determination of the specific production of violacein, calculated as the ratio of detectable pigment (OD577) per amount of bacteria (OD720), demonstrated the existence of QS inhibitory activity for almost all test compounds. (Figures 4-8). There is a clear difference between quorum sensing and antibacterial activity. In this research, growth inhibition by the evaluated heterocycles results in a  Compared to the report on the preparation of aldehyde 35a, a shorter time was herein achieved ( Table 6) by using 2-bromoacetyl bromide rather than the previously employed α-bromoacetic acid and DCC [20]. Compounds 35 were subjected to cyclization with ethylenediamine and subsequent oxidation with NBS under ultrasound irradiation, as reported by Torres [29]. The corresponding imidazolines 32a-c were provided in good yields (85-90%) (Scheme 6).

Characterization of Compounds
All compounds were characterized by spectroscopic methods utilizing IR, NMR, and DIP-MS. Spectra were assigned with the help of heteronuclear correlation (gHSQC, gHMBC) and in some cases with homonuclear correlation (gCOSY). Mass spectra were recorded with positive and negative ions. Spectra for all compounds are attached in the Supplementary Materials (Figures S1-S104).

Evaluation of QS Inhibition on Chromobacterium violaceum CV026
The synthesized compounds were added (at 10, 100 and 1000 µM) to the bacteria (OD600 of 0.12) in thioglycolate broth supplemented with C6-AHL, followed by incubation for 16 h. The determination of the specific production of violacein, calculated as the ratio of detectable pigment (OD577) per amount of bacteria (OD720), demonstrated the existence of QS inhibitory activity for almost all test compounds. (Figures 4-8). There is a clear difference between quorum sensing and antibacterial activity. In this research, growth inhibition by the evaluated heterocycles results in a  Compared to the report on the preparation of aldehyde 35a, a shorter time was herein achieved ( Table 6) by using 2-bromoacetyl bromide rather than the previously employed α-bromoacetic acid and DCC [20]. Compounds 35 were subjected to cyclization with ethylenediamine and subsequent oxidation with NBS under ultrasound irradiation, as reported by Torres [29]. The corresponding imidazolines 32a-c were provided in good yields (85-90%) (Scheme 6).

Characterization of Compounds
All compounds were characterized by spectroscopic methods utilizing IR, NMR, and DIP-MS. Spectra were assigned with the help of heteronuclear correlation (gHSQC, gHMBC) and in some cases with homonuclear correlation (gCOSY). Mass spectra were recorded with positive and negative ions. Spectra for all compounds are attached in the Supplementary Materials (Figures S1-S104).

Evaluation of QS Inhibition on Chromobacterium violaceum CV026
The synthesized compounds were added (at 10, 100 and 1000 µM) to the bacteria (OD600 of 0.12) in thioglycolate broth supplemented with C6-AHL, followed by incubation for 16 h. The determination of the specific production of violacein, calculated as the ratio of detectable pigment (OD577) per amount of bacteria (OD720), demonstrated the existence of QS inhibitory activity for almost all test compounds. (Figures 4-8). There is a clear difference between quorum sensing and antibacterial activity. In this research, growth inhibition by the evaluated heterocycles results in a , 10 min.

Characterization of Compounds
All compounds were characterized by spectroscopic methods utilizing IR, NMR, and DIP-MS. Spectra were assigned with the help of heteronuclear correlation (gHSQC, gHMBC) and in some cases with homonuclear correlation (gCOSY). Mass spectra were recorded with positive and negative ions. Spectra for all compounds are attached in the Supplementary Materials (Figures S1-S104).

Evaluation of QS Inhibition on Chromobacterium violaceum CV026
The synthesized compounds were added (at 10, 100 and 1000 µM) to the bacteria (OD600 of 0.12) in thioglycolate broth supplemented with C6-AHL, followed by incubation for 16 h. The determination of the specific production of violacein, calculated as the ratio of detectable pigment (OD577) per amount of bacteria (OD720), demonstrated the existence of QS inhibitory activity for almost all test compounds. (Figures 4-8). There is a clear difference between quorum sensing and antibacterial activity. In this research, growth inhibition by the evaluated heterocycles results in a decrease in OD720. While this decrease was statistically significant, taking cultures grown in the absence of inhibitors as a basis of comparison, this is denoted with a letter 'a' on the top of the corresponding bar and stated as 'a' the footnote of the figure.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 26 decrease in OD720. While this decrease was statistically significant, taking cultures grown in the absence of inhibitors as a basis of comparison, this is denoted with a letter 'a' on the top of the corresponding bar and stated as 'a' the footnote of the figure.

Thiazolines
The results for thiazoline series are shown in Figure 5. Thiazoline 17a displayed a moderate QS inhibition at 10 and 100 µM being of 44% and 47% respectively, while at 1000 µM it behaved as antimicrobial. Thiazoline 17b exhibited a limited to good inhibition at 10, 100 and 1000 µM being of 23%, 37% and 65% respectively, in addition 17b gave an IC50 value of 517.86 µM. Compound 17c reduced violacein production 23%, 24% and 48% at 10, 100 and 1000 µM respectively. Figure 5. Violacein specific production by Chromobacterium violaceum CV026 in the presence of the synthesized thiazolines at 10, 100 and 1000 µM concentrations. Data are expreseed as the percentage of the specific production of violacein (mean ± SEM) and normalized on the violacein production without compound addition and this was considered as 100%. Significance was confirmed by the Student's t-test with an accuracy of * p < 0.05 at the marked bars; a represents antimicrobial effect of the tested compound. Experiments were performed with n = 6.
For the benzimidazole series (Figure 6), 18a gave 27% and 85% QS inhibition at 10 and 100 µM respectively, presented a value of IC50 of 36.67 µM, and behaved as an antimicrobial at 1000 µM. For 18b at 100 and 1000 µM, good and excellent activity as a QSI was found (31% and 95%, respectively). The IC50 value of 18b is 376.92 µM. For 18c the inhibitory effect was 17%, 24% and 23% at 10, 100 and 1000 µM, respectively. percentages of violacein specific production (mean ± SEM) and normalized on the violacein production without compound addition and this was considered as 100%. Significance was confirmed by the Student´s t-test with an accuracy of * p < 0.05 at the labelled bars; a Indicates an antimicrobial effect of the compound. Experiments were performed with n = 6.

Imidazolines
For the series of imidazolines (Figure 8), 32a and 32c at 100 µM exhibited 24% and 34% activity as QSI, respectively. Imidazoline 32b was QSI at all concentrations and displayed IC50 = 65.09 µM. The QS inhibition activity of 32b is comparable to other synthesized bioisosteres [21]. Imidazoline 32c at 1000 µM afforded 46% activity and compound 32a behaved as an antimicrobial at the latter concentration. Compound 32b is one of the top three in the series of compounds synthesized in this work, its QS inhibition activity is lower than that of ethyl (4-fluorobenzoyl) acetate (IC50 = 23 µM) the best QSI of a library with 26 compounds [46], but it is similar to 2-[4′-(pentyloxy)phenyl]-4,5-dihydro-1H-imidazole (IC50 = 56.38 µM) and more active than QSI 4-nitro-pyridine-N-oxide (active at 100 µM) [11,45]. inhibition activity of 32b is comparable to other synthesized bioisosteres [21]. Imidazoline 32c at 1000 µM afforded 46% activity and compound 32a behaved as an antimicrobial at the latter concentration. Compound 32b is one of the top three in the series of compounds synthesized in this work, its QS inhibition activity is lower than that of ethyl (4-fluorobenzoyl) acetate (IC50 = 23 µM) the best QSI of a library with 26 compounds [46], but it is similar to 2-[4′-(pentyloxy)phenyl]-4,5-dihydro-1H-imidazole (IC50 = 56.38 µM) and more active than QSI 4-nitro-pyridine-N-oxide (active at 100 µM) [11,45]. Data are presented as percentages of violacein specific production (mean ± SEM), and normalized on the violacein production without compound addition and this was considered as 100%. Significance was confirmed by the Student's t-test with an accuracy of * p < 0.05 at the marked bars. a Denotes antimicrobial effect of the tested compound. Experiments were performed with n = 6.

Thiazoles
The QS inhibitory activity of the series of thiazoles is illustrated in Figure 4. No such effect was found for 16a at any concentration. Thiazole 16b at 10, 100 and 1000 µM showed low-moderate QS inhibitory activity (32%, 39% and 51%, respectively) with an IC 50 = 925.0 µM. The thiazole 16c was QSI only at 10 µM, causing limited inhibition (22%).

Docking Results
Molecular docking was carried out to provide insight into the non-bonding interactions of the test compounds with the CviR protein (PDB code: 3QP6). The binding modes were observed and the affinity energy of each compound was calculated and expressed as ∆G (kcal/mol). The Docking simulations were performed with AutoDock 4.2, utilizing the blind docking method. The grid box size was adjusted to 126 Å 3 with a grid spacing of 0.375 Å 3 . The Lamarckian genetic algorithm was employed with a randomized initial population of 100 individuals and a maximum number of energy evaluations of 1 × 10 7 . [47] The docking results were analyzed and visualized on AutoDockTools and PyMOL [48], represented as 3D plot conformations. The affinity energies of the evaluated compounds are shown in Table 7.
The theoretical calculations reveal that all tested compounds had greater affinity for the protein than the native ligand C6-AHL (1, R 1 = H, R 2 = C 3 H 7 ). In addition, a strong correlation exists between the in silico and experimental results. For example, 17b-c, 18a-c and 19c displayed good experimental inhibition and had the greatest affinity energies in silico.
The amino acids residues of the protein involved in the interaction with the C6-AHL, the native ligand, also participate in the binding of the most test compounds. There are hydrogen bond interactions of the polar groups of the compounds with the TRP84, ASP97 and SER155 residues. Additionally, there are π-π interactions of the phenyl ring on the thiazolines 17a and 17b with the phenyl group of TYR88, of the benzimidazoles 18a-b with TYR80, and of the pyridine ring of compound 19c with TRP111. Morever, hydrophobic interactions took place with most of the residues at the binding site, including ILE57, VAL59, MET72, LEU85, ILE99, PHE126, ALA130 and MET135. (Figure 9). Table 7. Affinity energies (∆G) and amino acid residues of the binding site for the ligands evaluated. Hydrogen bond interaction (*), π-π interaction ( ).

Ligand
Additionally, there are π-π interactions of the phenyl ring on the thiazolines 17a and 17b with the phenyl group of TYR88, of the benzimidazoles 18a-b with TYR80, and of the pyridine ring of compound 19c with TRP111. Morever, hydrophobic interactions took place with most of the residues at the binding site, including ILE57, VAL59, MET72, LEU85, ILE99, PHE126, ALA130 and MET135. (Figure 9).  Additionally, there are π-π interactions of the phenyl ring on the thiazolines 17a and 17b with the phenyl group of TYR88, of the benzimidazoles 18a-b with TYR80, and of the pyridine ring of compound 19c with TRP111. Morever, hydrophobic interactions took place with most of the residues at the binding site, including ILE57, VAL59, MET72, LEU85, ILE99, PHE126, ALA130 and MET135. (Figure 9).

Structure-Activity Relationship
The elements present in the bioisosteres of this work were a heterocycle, the amide group and the side chain. In one of the 5 bioisosteres, a phenyl ring was introduced as a linker between the amide and the heterocycle (17).
The chain length is important for the activity, not because of the interactions that it establishes, since they are all hydrophobic, but rather because of the size that permits other interactions; thus the compound 16a that has an aromatic ring, the thiazol, and the amide group but a chain of only 3C, did not display activity at any concentration. According to the Docking analysis this compound does not satisfy the size required to give all interactions like its homologous 16b and 16c. The latter compounds showed hydrogen bonding between the C=O of the amido group and TYR 80 and SER 155.
Thiazolines 17 contain a thiazoline ring, a phenyl as a connector, the amido group, and propyl, pentyl and heptyl chains. They exhibited some degree of QS inhibition, except for 17a at 1000 µM, showing antimicrobial activity. In an earlier work, our group synthesized thiazolines, 33a-c, (Figure 13) like the thiazolines 17 but without the phenyl conector; all those thiazolines were inactive as QSI [29,49]. Thiazoline 17b afforded a low activity as QSI, 23% and 37% at 10 and 100 µM, respectively, as well as 65% at 1000 µM. The phenyl linker enables a greater number of interactions than the aliphatic chains alone, and increases the length of the compound. The π-π interactions of the complex formed by compound 17b and tyrosine 88 at the active site the CviR protein is illustrated in Figure 12.

Structure-Activity Relationship
The elements present in the bioisosteres of this work were a heterocycle, the amide group and the side chain. In one of the 5 bioisosteres, a phenyl ring was introduced as a linker between the amide and the heterocycle (17).
The chain length is important for the activity, not because of the interactions that it establishes, since they are all hydrophobic, but rather because of the size that permits other interactions; thus the compound 16a that has an aromatic ring, the thiazol, and the amide group but a chain of only 3C, did not display activity at any concentration. According to the Docking analysis this compound does not satisfy the size required to give all interactions like its homologous 16b and 16c. The latter compounds showed hydrogen bonding between the C=O of the amido group and TYR 80 and SER 155.
Thiazolines 17 contain a thiazoline ring, a phenyl as a connector, the amido group, and propyl, pentyl and heptyl chains. They exhibited some degree of QS inhibition, except for 17a at 1000 µM, showing antimicrobial activity. In an earlier work, our group synthesized thiazolines, 33a-c, (Figure 13) like the thiazolines 17 but without the phenyl conector; all those thiazolines were inactive as QSI [29,49]. Thiazoline 17b afforded a low activity as QSI, 23% and 37% at 10 and 100 µM, respectively, as well as 65% at 1000 µM. The phenyl linker enables a greater number of interactions than the aliphatic chains alone, and increases the length of the compound. The π-π interactions of the complex formed by compound 17b and tyrosine 88 at the active site the CviR protein is illustrated in Figure 12. All benzimidazoles (18) with a thiol functionality display good QSI activity, 18a and 18b, so the compound 18a exhibited 85% QSI at 100 µM and 18b 95% at 1000 µM. Benzimidazol 18c displayed only 17, 24 and 23% inhibitory activity at 10, 100 and 1000 µM, respectively. According to the Docking simulations, the benzimidazole ring occupies the space of the lactone and the amide of AHL, while thioacetamide engages the space of the aliphatic chain of AHL. The interactions of the protein with the benzimidazole rings were similar to those found with the lactone ring and the amido group of AHL Moreover, the phenyl ring of 18a y 18b presents π-π interactions with the receptor. Conversely, no π-π interactions were observed for 18c because there is no phenyl ring in its structure, and the benzimidazol ring, which could give these interactions, establishes the interactions mentioned above. Benzimidazoles 18a-b do not contain an aliphatic chain; but have a thioacetamide group, giving them greater length and flexibility.
Since 18a is QSI at 100 µM and antimicrobial at 1000 µM, it is possible to find a concentration that is able to restore sensitivity to an antibiotic. It is known that the QSI addition to an antibiotic can restore the sensitivity to the drug and increase its potency [11,50], therefore we consider that benzimidazol 18a that behaves as very good QSI at one concentration and antimicrobial at another is very promising.
Pyridine 19a with a propyl chain exhibited little QSI activity, 17% at 100µM and behaved as an antimicrobial at 1000 µM suggesting that chain was too short to properly couple to the protein.
Among the pyridines, the best activity as a QSI was produced by 19c, the compound with a nonyl chain. Its inhibitory activity was surprisingly similar at all three concentrations (52, 55 and 58% at 10, 100 and 1000 µM, respectively), which has no apparent explanation. Pyridines 19a and 19b generate the same hydrogen bond as AHL between NH and Asp 97. With 19b, there is in an additional hydrogen bond of C=O of the amide group with SER 155 and TYR 80. It was observed that 19c is rotated 180° in relation to AHL and forms two hydrogen bonds: one between the compound 19c carbonyl and SER 155, and the other between N and TRP 84. All imidazolines displayed activity as QSI at 100 µM and 1000 µM, although only 32b (at 1000 µM) showed good activity. Compound 32c, containing a propyl lateral chain, gave rise to 34 and 46% QSI at 100 and 1000 µM respectively. All benzimidazoles (18) with a thiol functionality display good QSI activity, 18a and 18b, so the compound 18a exhibited 85% QSI at 100 µM and 18b 95% at 1000 µM. Benzimidazol 18c displayed only 17, 24 and 23% inhibitory activity at 10, 100 and 1000 µM, respectively. According to the Docking simulations, the benzimidazole ring occupies the space of the lactone and the amide of AHL, while thioacetamide engages the space of the aliphatic chain of AHL. The interactions of the protein with the benzimidazole rings were similar to those found with the lactone ring and the amido group of AHL Moreover, the phenyl ring of 18a y 18b presents π-π interactions with the receptor. Conversely, no π-π interactions were observed for 18c because there is no phenyl ring in its structure, and the benzimidazol ring, which could give these interactions, establishes the interactions mentioned above. Benzimidazoles 18a-b do not contain an aliphatic chain; but have a thioacetamide group, giving them greater length and flexibility.
Since 18a is QSI at 100 µM and antimicrobial at 1000 µM, it is possible to find a concentration that is able to restore sensitivity to an antibiotic. It is known that the QSI addition to an antibiotic can restore the sensitivity to the drug and increase its potency [11,50], therefore we consider that benzimidazol 18a that behaves as very good QSI at one concentration and antimicrobial at another is very promising.
Pyridine 19a with a propyl chain exhibited little QSI activity, 17% at 100µM and behaved as an antimicrobial at 1000 µM suggesting that chain was too short to properly couple to the protein. Among the pyridines, the best activity as a QSI was produced by 19c, the compound with a nonyl chain. Its inhibitory activity was surprisingly similar at all three concentrations (52, 55 and 58% at 10, 100 and 1000 µM, respectively), which has no apparent explanation. Pyridines 19a and 19b generate the same hydrogen bond as AHL between NH and Asp 97. With 19b, there is in an additional hydrogen bond of C=O of the amide group with SER 155 and TYR 80. It was observed that 19c is rotated 180 • in relation to AHL and forms two hydrogen bonds: one between the compound 19c carbonyl and SER 155, and the other between N and TRP 84. All imidazolines displayed activity as QSI at 100 µM and 1000 µM, although only 32b (at 1000 µM) showed good activity. Compound 32c, containing a propyl lateral chain, gave rise to 34 and 46% QSI at 100 and 1000 µM respectively.

General
All reagents and solvents were purchased from Sigma Aldrich (Toluca, Mexico) and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on Merck F253 silica gel aluminum sheets, and spots were revealed with ultraviolet (UV) light (254 nm). NMR experiments were carried out in Varian NMR System (500 MHz and 125 MHz), Varian Mercury (300 MHz and 75 MHz) and Bruker ASCEND (600 MHz and 150 MHz). 1 H NMR and 13 C spectra were assigned with the help of 2-D experiments (gHSQC and gHMBC). The chemical shifts (δ) are given in ppm. Mass spectra (MS) were recorded on a Bruker Amazon Speed (ESI). Infrared (IR) spectra were obtained on a Perkin Elmer FT-IR Spectrum 2000 spectrometer from the ENCB-IPN spectroscopy instrumentation center. Melting points were determined on an Electrothermal MEL-TEMP apparatus and are uncorrected. Microwave reactions were accomplished on a CEM Discovery SP apparatus.

Procedure for the Synthesis of Ethyl 2-Aminothiazol-4-carboxylate, 22
Ethyl bromine pyruvate (7.69 mmol, 1 eq), thiourea (7.69 mmol, 1 eq) and 15 mL of EtOH were added to a flask equipped with a magnetic stirring bar and left to reaction mixture at rt for 16 h. At the end of this period, the solvent was evaporated under reduced pressure, followed by the addition of 40 mL of 20% aqueous potassium carbonate to the residue. The suspension formed was maintained under constant stirring for 30 min before subjecting it to vacuum filtration. The solid retained on the filter paper was washed with 40 mL of distilled water in two 20 mL portions and oven-dried at 40 • C.

Ethyl 2-Aminothiazol-4-Carboxylate, 22
Yield: 85%. White solid. 1  2-Amino thiazole (1.74 mmol, 1 eq) and 2 mL of carboxylic acid (butanoic acid at 150 • C, hexanoic acid at 150 • C, octanoic acid at 180 • C) were added to a reaction vial equipped with magnetic stirring, the reaction mixture was warmed for 90 min. It was then transferred to an aqueous solution of 20% potassium carbonate (40 mL) and allowed the mixture to stir for 30 min. Subsequently, the suspension was vacuum filtered and the solid obtained washed with distilled water (2 × 20 mL) and oven-dried at 40 • C.  4-aminobenzonitrile (4.23 mmol, 1 eq), cysteamine hydrochloride (6.34 mmol, 1.5 eq), potassium carbonate (21.16 mmol, 5 eq) and 5 mL of a EtOH/H 2 O (1:1) mixture were added to an ACE pressure tube equipped with a magnetic stirring bar. The reaction mixture was subjected to heating in a sand bath at 110 • C for 16 h. At the end of this period, it was transferred to a ball flask and the solvent was evaporated under reduced pressure. The residue was suspended in distilled water (20 mL) and liquid-liquid extractions were made with CH 2 Cl 2 (3 × 15 mL), the combined organic phases were dried with anhydrous Na 2 SO 4 and evaporated in a ball flask under reduced pressure. The residue was purified by chromatographic column, using silica gel as the stationary phase and an 8:2 hexane/AcOEt mixture as the mobile phase.

4-(Thiazolin-2-yl) Aniline, 25
Yield: 82%. Pink solid. 1  4-(Thiazolin-2-yl)-Aniline (1.96 mmol, 1 eq) and 2.5 mL of carboxylic acid (butanoic, hexanoic, octanoic acids) were added to a reaction vial equipped with a magnetic stirring bar. The vial was closed and the reaction mixture warmed (butanoic acid at 150 • C, hexanoic acid at 150 • C, octanoic acid at 180 • C) for 90 min. At the end of that period, the reaction mixture was transferred to an aqueous solution of 20% potassium carbonate (40 mL) and left to stand under constant stirring for 30 min. The suspension was subjected to vacuum filtration and the solid retained on the filter paper was washed with 40 mL of distilled water in two 20 mL portions, then oven-dried at 40 • C. dried with anhydrous Na 2 SO 4 and filtered, then evaporated under vacuum. Once the residue was dry, it was transferred to a 100 mL ball flask equipped with a magnetic stirring bar and reacted with 4-hydroxybenzaldehyde (4.31 mmol, 1.1 eq) and DBU (3.91 mmol, 1 eq) at 70 • C for 6 h, employing acetonitrile (20 mL) as solvent. Upon completion of that period, the solvent was evaporated under reduced pressure, and 30 mL of a 5% HCl solution was added to the residue. Afterwards liquid-liquid extractions were carried out with AcOEt (3 × 10 mL) and the organic phases were dried with anhydrous Na 2 SO 4 and filtered. The filtrate was evaporated under reduced pressure and the residue was purified by column chromatography, with silica gel as a stationary phase and a hexane/AcOEt mixture 7: 3 as the mobile phase. Funding: This research was funded by Conacyt, grants 240808 and 255420, and "IPN through grant 20201304".