Synthesis and Structure–Activity Relationship of Thioacetamide-Triazoles against Escherichia coli

Infections due to Gram-negative bacteria are increasingly dangerous due to the spread of multi-drug resistant strains, emphasizing the urgent need for new antibiotics with alternative modes of action. We have previously identified a novel class of antibacterial agents, thioacetamide-triazoles, using an antifolate targeted screen and determined their mode of action which is dependent on activation by cysteine synthase A. Herein, we report a detailed examination of the anti-E. coli structure–activity relationship of the thioacetamide-triazoles. Analogs of the initial hit compounds were synthesized to study the contribution of the aryl, thioacetamide, and triazole sections. A clear structure–activity relationship was observed generating compounds with excellent inhibition values. Substitutions to the aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems. Substitutions to the central thioacetamide linker section were more nuanced; the introduction of a methyl branch to the thioacetamide linker substantially decreased antibacterial activity, but the isomeric propionamide and N-benzamide systems retained activity. Changes to the triazole portion of the molecule dramatically decreased the antibacterial activity, further indicating that 1,2,3-triazole is critical for potency. From these studies, we have identified new lead compounds with desirable in-vitro ADME properties and in-vivo pharmacokinetic properties.


Introduction
The usage of antibiotics to treat and prevent bacterial infections has saved millions of people since their introduction. However, the overuse of antibiotics and the bacteria's natural ability to develop resistance has led to a loss of effectiveness [1]. Antimicrobial resistance significantly increases the morbidity and mortality associated with bacterial infections in humans [2]. One reason for such a high mortality rate is the lack of alternative antibiotics to treat drug-resistant pathogens [3]. Therefore, there is a growing concern over antibiotic resistance and a need for the discovery of new drugs with novel mechanisms of action to treat resistant pathogens [4]. Previously, we have identified a thioacetamidetriazole hit series via a metabolically biased high-throughput screen against E. coli K12 in a search for novel antifolates (Figure 1) [5]. We demonstrated that these compounds function as prodrugs and are activated by the cysteine synthase A (CysK) enzyme. CysK plays a fundamental role in several key metabolic processes including cysteine biosynthesis and sulfur assimilation, and is also linked to the folate biosynthetic pathway through the downstream product homocysteine [5,6]. Thioacetamide triazoles (TATs) form a false product with the CysK substrate O-acetyl-L-serine [7] and thus inhibit the growth of E. coli. In continuation of our interest in identifying novel compounds for Gram-negative bacteria, Thioacetamide triazoles (TATs) form a false product with the CysK substrate O-acetyl-Lserine [7] and thus inhibit the growth of E. coli. In continuation of our interest in identifying novel compounds for Gram-negative bacteria, in this study we explore the structure-activity relationships of the TAT series with the goal to develop potent lead compounds with excellent pharmacological properties.

Synthesis
To expand the structure-activity relationship (SAR) of the TAT series, we proposed a stepwise study to evaluate the contributions of the functional motifs found within the TAT scaffold. These series also allowed for the improvement of the physiochemical and pharmacological properties of our initial hits as we navigated our design between the competing requirements of CysK activation, Gram-negative intracellular accumulation, and pharmacokinetic exposure. Therefore, we focused on sequentially generating modifications to the aryl (right hand side), thioacetamide (middle) and triazole (left hand side) sections of the initial leads ( Figure 2). First, analogs were synthesized to probe the aryl region by incorporating various electron-donating, electron-withdrawing and sterically bulky groups on the phenyl aryl group (Scheme 1). The synthesis commenced from suitably substituted amines 4a-w, which were converted to chloroacetamide derivatives 5a-w [8]. Finally, the compounds were treated with sodium 1H-1,2,3-triazole-5-thiolate 6 (Scheme 1) to afford target TAT analogs 7-22 and 31-33. In addition, N-methyl amide 23 and N-ethyl amide 24 derivatives were synthesized from N-methyl 4-fluoro aniline and N-ethyl-2-fluoroaniline via a similar scheme of coupling with chloroacetyl chloride and nucleophilic displacement with 6. Using a similar approach, analogs with an alteration to the thioacetamide linker were generated using 2-chloropropionyl chloride and 3-chloropropionyl chloride, to give corresponding methyl branched derivatives 28 and 29 and the propionamide derivative 30.

Synthesis
To expand the structure-activity relationship (SAR) of the TAT series, we proposed a stepwise study to evaluate the contributions of the functional motifs found within the TAT scaffold. These series also allowed for the improvement of the physiochemical and pharmacological properties of our initial hits as we navigated our design between the competing requirements of CysK activation, Gram-negative intracellular accumulation, and pharmacokinetic exposure. Therefore, we focused on sequentially generating modifications to the aryl (right hand side), thioacetamide (middle) and triazole (left hand side) sections of the initial leads ( Figure 2). Thioacetamide triazoles (TATs) form a false product with the CysK substrate O-acetyl-Lserine [7] and thus inhibit the growth of E. coli. In continuation of our interest in identifying novel compounds for Gram-negative bacteria, in this study we explore the structure-activity relationships of the TAT series with the goal to develop potent lead compounds with excellent pharmacological properties.

Synthesis
To expand the structure-activity relationship (SAR) of the TAT series, we proposed a stepwise study to evaluate the contributions of the functional motifs found within the TAT scaffold. These series also allowed for the improvement of the physiochemical and pharmacological properties of our initial hits as we navigated our design between the competing requirements of CysK activation, Gram-negative intracellular accumulation, and pharmacokinetic exposure. Therefore, we focused on sequentially generating modifications to the aryl (right hand side), thioacetamide (middle) and triazole (left hand side) sections of the initial leads ( Figure 2). First, analogs were synthesized to probe the aryl region by incorporating various electron-donating, electron-withdrawing and sterically bulky groups on the phenyl aryl group (Scheme 1). The synthesis commenced from suitably substituted amines 4a-w, which were converted to chloroacetamide derivatives 5a-w [8]. Finally, the compounds were treated with sodium 1H-1,2,3-triazole-5-thiolate 6 (Scheme 1) to afford target TAT analogs 7-22 and 31-33. In addition, N-methyl amide 23 and N-ethyl amide 24 derivatives were synthesized from N-methyl 4-fluoro aniline and N-ethyl-2-fluoroaniline via a similar scheme of coupling with chloroacetyl chloride and nucleophilic displacement with 6. Using a similar approach, analogs with an alteration to the thioacetamide linker were generated using 2-chloropropionyl chloride and 3-chloropropionyl chloride, to give corresponding methyl branched derivatives 28 and 29 and the propionamide derivative 30. First, analogs were synthesized to probe the aryl region by incorporating various electron-donating, electron-withdrawing and sterically bulky groups on the phenyl aryl group (Scheme 1). The synthesis commenced from suitably substituted amines 4a-w, which were converted to chloroacetamide derivatives 5a-w [8]. Finally, the compounds were treated with sodium 1H-1,2,3-triazole-5-thiolate 6 (Scheme 1) to afford target TAT analogs 7-22 and 31-33. In addition, N-methyl amide 23 and N-ethyl amide 24 derivatives were synthesized from N-methyl 4-fluoro aniline and N-ethyl-2-fluoroaniline via a similar scheme of coupling with chloroacetyl chloride and nucleophilic displacement with 6. Using a similar approach, analogs with an alteration to the thioacetamide linker were generated using 2-chloropropionyl chloride and 3-chloropropionyl chloride, to give corresponding methyl branched derivatives 28 and 29 and the propionamide derivative 30.
Next, we planned to incorporate a methylene group between the aryl and amide groups (Scheme 2). For this synthesis, the left hand side and linker section 36 was first assembled by reacting ethyl bromoacetate with 6 to produce compound 35. Compound 35 was converted to free acid 36 with base hydrolysis [9]. Then, the acid was coupled with various benzyl amines using EDC, HOBt coupling to give compounds 38-46, allowing for the facile introduction of structural diversity in the last step.
Next, we planned to incorporate a methylene group between the aryl and amide groups (Scheme 2). For this synthesis, the left hand side and linker section 36 was first assembled by reacting ethyl bromoacetate with 6 to produce compound 35. Compound 35 was converted to free acid 36 with base hydrolysis [9]. Then, the acid was coupled with various benzyl amines using EDC, HOBt coupling to give compounds 38-46, allowing for the facile introduction of structural diversity in the last step. For the introduction of further right hand side aryl substitutions, we sought inspiration from the SAR of other antibiotic families. In fluoroquinolones, C-7 cycloalkyl amine substitutions are used to improve the antibacterial spectrum, solubility, and pharmacokinetic profiles of the antibiotic class, all properties we hoped to improve in the TAT series [10,11]. The C-7 fluoroquinolone substitution motifs are most commonly comprised of 5-or 6-membered cycloalkyl amines that contain an additional basic functional group, and consequently, we wanted to see if analogous functionalization could be beneficial to our compound series [12]. To test this hypothesis, we derivatized our initial hits with the 3-amino pyrrolidine, pyrrolo-oxazinyl, and N-methyl piperazine moieties. Compounds 50, 53 and 56 were synthesized from 4-fluoro-nitrobenzene, as shown in Scheme 3. The nucleophilic displacement reaction of 4-fluoronitrobenzene 47 with boc-protected 3-aminopyrrolidine 48 in the presence of triethylamine yielded compound 49 [13]. Then, the nitro derivative 49 was reduced using Raney Ni to give amine, then treated with chloroacetyl chloride to provide the chloroacetamide derivative. Finally, the treatment of the chloroacetamide with thiolate 6 produced the final compound 50. Similarly, compounds 53 and 56 were synthesized from amino derivatives 51 and 54, respectively [14]. For the introduction of further right hand side aryl substitutions, we sought inspiration from the SAR of other antibiotic families. In fluoroquinolones, C-7 cycloalkyl amine substitutions are used to improve the antibacterial spectrum, solubility, and pharmacokinetic profiles of the antibiotic class, all properties we hoped to improve in the TAT series [10,11]. The C-7 fluoroquinolone substitution motifs are most commonly comprised of 5-or 6-membered cycloalkyl amines that contain an additional basic functional group, and consequently, we wanted to see if analogous functionalization could be beneficial to our compound series [12]. To test this hypothesis, we derivatized our initial hits with the 3-amino pyrrolidine, pyrrolo-oxazinyl, and N-methyl piperazine moieties. Compounds 50, 53, and 56 were synthesized from 4-fluoro-nitrobenzene, as shown in Scheme 3. The nucleophilic displacement reaction of 4-fluoronitrobenzene 47 with boc-protected 3-aminopyrrolidine 48 in the presence of triethylamine yielded compound 49 [13]. Then, the nitro derivative 49 was reduced using Raney Ni to give amine, then treated with chloroacetyl chloride to provide the chloroacetamide derivative. Finally, the treatment of the chloroacetamide with thiolate 6 produced the final compound 50. Similarly, compounds 53 and 56 were synthesized from amino derivatives 51 and 54, respectively [14]. For the introduction of further right hand side aryl substitutions, we sought inspiration from the SAR of other antibiotic families. In fluoroquinolones, C-7 cycloalkyl amine substitutions are used to improve the antibacterial spectrum, solubility, and pharmacokinetic profiles of the antibiotic class, all properties we hoped to improve in the TAT series [10,11]. The C-7 fluoroquinolone substitution motifs are most commonly comprised of 5-or 6-membered cycloalkyl amines that contain an additional basic functional group, and consequently, we wanted to see if analogous functionalization could be beneficial to our compound series [12]. To test this hypothesis, we derivatized our initial hits with the 3-amino pyrrolidine, pyrrolo-oxazinyl, and N-methyl piperazine moieties. Compounds 50, 53, and 56 were synthesized from 4-fluoro-nitrobenzene, as shown in Scheme 3. The nucleophilic displacement reaction of 4-fluoronitrobenzene 47 with boc-protected 3-aminopyrrolidine 48 in the presence of triethylamine yielded compound 49 [13]. Then, the nitro derivative 49 was reduced using Raney Ni to give amine, then treated with chloroacetyl chloride to provide the chloroacetamide derivative. Finally, the treatment of the chloroacetamide with thiolate 6 produced the final compound 50. Similarly, compounds 53 and 56 were synthesized from amino derivatives 51 and 54, respectively [14]. To probe the importance of the central TAT phenylamide group, compounds 58 and 59, in which the amide group was inverted, were synthesized. To achieve this, the thiolate 6 was treated with tert-butyl (2-bromoethyl)carbamate in THF to give the protected thiotriazole ethylamine, which was converted to the free amine 57 with acid. Compound 57 was then coupled with 2-fluorobenzoic acid to give 58, and using a similar coupling protocol, 59 was synthesized from 3,4-dihydroxybenzoic acid (Scheme 4).
To probe the importance of the central TAT phenylamide group, compounds 58 and 59, in which the amide group was inverted, were synthesized. To achieve this, the thiolate 6 was treated with tert-butyl (2-bromoethyl)carbamate in THF to give the protected thiotriazole ethylamine, which was converted to the free amine 57 with acid. Compound 57 was then coupled with 2-fluorobenzoic acid to give 58, and using a similar coupling protocol, 59 was synthesized from 3,4-dihydroxybenzoic acid (Scheme 4). Finally, we prepared a series of compounds bearing various triazole analogs to explore structure-activity relationships on the triazole motif of the original hits. Compounds 62-67 were synthesized using a similar protocol as used in Scheme 1 (Scheme 5). Amines 60a-b were converted to chloroacetyl derivatives 61a-b and coupled with respective thiazoles to give compounds 62-67.  Finally, we prepared a series of compounds bearing various triazole analogs to explore structure-activity relationships on the triazole motif of the original hits. Compounds 62-67 were synthesized using a similar protocol as used in Scheme 1 (Scheme 5). Amines 60a-b were converted to chloroacetyl derivatives 61a-b and coupled with respective thiazoles to give compounds 62-67.
To probe the importance of the central TAT phenylamide group, compounds 58 and 59, in which the amide group was inverted, were synthesized. To achieve this, the thiolate 6 was treated with tert-butyl (2-bromoethyl)carbamate in THF to give the protected thiotriazole ethylamine, which was converted to the free amine 57 with acid. Compound 57 was then coupled with 2-fluorobenzoic acid to give 58, and using a similar coupling protocol, 59 was synthesized from 3,4-dihydroxybenzoic acid (Scheme 4). Finally, we prepared a series of compounds bearing various triazole analogs to explore structure-activity relationships on the triazole motif of the original hits. Compounds 62-67 were synthesized using a similar protocol as used in Scheme 1 (Scheme 5). Amines 60a-b were converted to chloroacetyl derivatives 61a-b and coupled with respective thiazoles to give compounds 62-67. To examine the contribution of the triazole N-H, the corresponding N-Me derivatives 68 and 69 were prepared by the methylation of 10 using cesium carbonate and iodomethane. Similarly, compounds 70 and 71 were synthesized in good yields (Scheme 6).

Antibacterial Studies
To explore the SAR around the right hand aryl portion of the TAT series (Table 1), compounds were evaluated for their in vitro antibacterial activity against E. coli in the M9-based minimal media ATCC 2511. Growth inhibition was also evaluated in methionine supplemented media, as it has been shown to rescue cellular inhibition of the initial hit compounds [5] as well as other antifolates [15,16]. An MIC shift between the two medias was used to indicate that antibacterial activity of the compounds remained on target, and this was happily seen with all of the most MIC active compounds generated in this study. Within the first series of compounds in Table 1, modifications to the phenyl ring are generally well tolerated. MICs remain within 2-fold of the initial hit 1 for the fluoro-and cyano-substituted compounds 1-8. The installation of a 2-pyridyl nitrogen 9, 13, 14, 16 was generally well tolerated, with the exception of the 4-pyridyl analog 10, which showed an 8-fold loss in MIC activity. Incorporating the electron-donating group -OMe and sterically bulky iodine group in both ortho and para position, 17-20, maintained activity, whereas the introduction of the strong electron-withdrawing -CF 3 group, 21 and 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity.

Antibacterial Studies
To explore the SAR around the right hand aryl portion of the TAT series (Table 1), compounds were evaluated for their in vitro antibacterial activity against E. coli in the M9-based minimal media ATCC 2511. Growth inhibition was also evaluated in methionine supplemented media, as it has been shown to rescue cellular inhibition of the initial hit compounds [5] as well as other antifolates [15,16]. An MIC shift between the two medias was used to indicate that antibacterial activity of the compounds remained on target, and this was happily seen with all of the most MIC active compounds generated in this study. Within the first series of compounds in Table 1, modifications to the phenyl ring are generally well tolerated. MICs remain within 2-fold of the initial hit 1 for the fluoro-and cyano-substituted compounds 1-8. The installation of a 2-pyridyl nitrogen 9, 13, 14, 16 was generally well tolerated, with the exception of the 4-pyridyl analog 10, which showed an 8-fold loss in MIC activity. Incorporating the electron-donating group -OMe and sterically bulky iodine group in both ortho and para position, 17-20, maintained activity, whereas the introduction of the strong electron-withdrawing -CF3 group, 21 and 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity. Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide substitutions 38, 39, 41 maintained good MIC activity with only a two-fold MIC shift from the values of the corresponding phenyl amides 1, 3, 7. However, the other analogs, 40 and 42-46, showed decreased antibacterial activity against E. coli. This series indicates that benzamides are slightly less preferable from an MIC activity standpoint, but do offer optional extra chemical diversity that could be utilized in future series.
The introduction of the fluoroquinolone C7 basic motif into the para-position of the right hand side phenyl ring in compounds 50, 53, and 56, in an effort to improve the pharmacokinetic profile of the series, was not successful. None of these compounds show any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which the amide group was inverted, were synthesized to determine if modification could be made to the central TAT phenylamide group. These compounds only had moderate activity. To assess whether or not the lack of antibacterial activity accompanying the modifications of the phenyl ring is due to poor cellular uptake, we examined the accumulation of 50, 53, 56, 58, 59 and 1 in an LC-MS/MS based assay (Table 3 and Table S3) [17]. This assay indicated that the lack of MIC activity of 50, 53 and 56 was not due to a much lower drug accumulation within the cell than 1, and that incompatibility with the biochemical target CysK is most likely responsible for poor antimicrobial activity. In terms of general accumulation, all these analogs tested were lower than the ciprofloxacin control, suggesting room for improvement. The catechol 59, designed to capitalize on outer membrane siderophore iron-dependent transport mechanisms, showed the lowest accumulation of the whole series, suggesting outer membrane permeability may not be limiting for the series [18]. 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity. 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity. 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity. 22, led to poorer MIC activity. Blocking the linker amide NH with the Me or Et (23 and 24) groups also reduced activity. The phenyl ring isosteres-thiazoles 25 and 26-both retained good MIC activity. Adding a methyl branch to the thioacetamide linker 28 and 29 led to a striking loss of activity. Replacement of the aryl group with the aliphatic groups cyclohexyl 31, cyclopentyl 32, tert-butyl groups 33 all reduced activity. Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 3 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 25 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 25 n.d. Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 25 n.d.

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Molecules 2022, 27 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 12 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide 12 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2 Substituting the phenylaniline TAT motif with a benzylamine to remove the aniline motif was explored in a second series (38-46, Table 2). The simple fluorine benzamide substitutions 38, 39, 41 maintained good MIC activity with only a two-fold MIC shift from the values of the corresponding phenyl amides 1, 3, 7. However, the other analogs, 40 and   The introduction of the fluoroquinolone C7 basic motif into the para right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these co any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which t was inverted, were synthesized to determine if modification could be ma TAT phenylamide group. These compounds only had moderate acti whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulati 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This assa the lack of MIC activity of 50, 53 and 56 was not due to a much lower dru within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general ac these analogs tested were lower than the ciprofloxacin control, sugge N   The introduction of the fluoroquinolone C7 basic motif into the par right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be ma TAT phenylamide group. These compounds only had moderate act whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulat 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general a these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the para right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these co any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which t was inverted, were synthesized to determine if modification could be mad TAT phenylamide group. These compounds only had moderate acti whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulati 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This assa the lack of MIC activity of 50, 53 and 56 was not due to a much lower dru within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general ac these analogs tested were lower than the ciprofloxacin control, sugge N   The introduction of the fluoroquinolone C7 basic motif into the par right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be ma TAT phenylamide group. These compounds only had moderate act whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulat 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general a these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the par right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be ma TAT phenylamide group. These compounds only had moderate act whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulat 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general a these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the pa right hand side phenyl ring in compounds 50, 53, and 56, in an effor pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be m TAT phenylamide group. These compounds only had moderate ac whether or not the lack of antibacterial activity accompanying the mo phenyl ring is due to poor cellular uptake, we examined the accumula 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical tar likely responsible for poor antimicrobial activity. In terms of general these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the par right hand side phenyl ring in compounds 50, 53, and 56, in an effor pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be ma TAT phenylamide group. These compounds only had moderate ac whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulat 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical tar likely responsible for poor antimicrobial activity. In terms of general a these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the par right hand side phenyl ring in compounds 50, 53, and 56, in an effor pharmacokinetic profile of the series, was not successful. None of these c any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which was inverted, were synthesized to determine if modification could be m TAT phenylamide group. These compounds only had moderate ac whether or not the lack of antibacterial activity accompanying the mo phenyl ring is due to poor cellular uptake, we examined the accumulat 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This ass the lack of MIC activity of 50, 53 and 56 was not due to a much lower dr within the cell than 1, and that incompatibility with the biochemical tar likely responsible for poor antimicrobial activity. In terms of general a these analogs tested were lower than the ciprofloxacin control, sugg N   The introduction of the fluoroquinolone C7 basic motif into the para right hand side phenyl ring in compounds 50, 53, and 56, in an effort pharmacokinetic profile of the series, was not successful. None of these co any notable anti-E.coli activity (Table 3). Compounds 58 and 59, in which t was inverted, were synthesized to determine if modification could be mad TAT phenylamide group. These compounds only had moderate acti whether or not the lack of antibacterial activity accompanying the mod phenyl ring is due to poor cellular uptake, we examined the accumulati 58, 59 and 1 in an LC-MS/MS based assay (Tables 3 and S3) [17]. This assa the lack of MIC activity of 50, 53 and 56 was not due to a much lower dru within the cell than 1, and that incompatibility with the biochemical targ likely responsible for poor antimicrobial activity. In terms of general ac these analogs tested were lower than the ciprofloxacin control, sugge N  To complete our SAR analysis, a small series of compounds with various triazole analogs were evaluated to determine if any modifications to the left hand side could be tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole 64 analogs were inactive (Table 4). Similarly, compounds 65-71 were found to be inactive. These results are consistent with our prior mode of action studies that indicate the triazole NH is required for CysK activation [5].
To examine the potential for the further development of the newly synthesized analogs, in vitro ADME properties of promising compounds 9 and 25, and our original hits 1, 2, and 3 were examined (Table 5 and Table S1). Both compounds 9 and 25 demonstrated improved solubility, and plasma and metabolic stability over compound 1. Encouraged by the improved mouse plasma and microsomal stability, the mouse pharmacokinetic profile of 25 by intravenous dosing was determined. Compound 25 showed a moderate exposure (AUC inf 2807 h·ng/mL) and a mean elimination half-life of 0.55 h (Table S2).  To complete our SAR analysis, a small series of compounds with vari analogs were evaluated to determine if any modifications to the left hand si tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be ina results are consistent with our prior mode of action studies that indicate the is required for CysK activation. [5]  improvement. The catechol 59, designed to capitalize on outer membrane iron-dependent transport mechanisms, showed the lowest accumulation o series, suggesting outer membrane permeability may not be limiting for the To complete our SAR analysis, a small series of compounds with var analogs were evaluated to determine if any modifications to the left hand s tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazol were inactive (Table 4). Similarly, compounds 65-71 were found to be ina results are consistent with our prior mode of action studies that indicate the is required for CysK activation. [5]   To complete our SAR analysis, a small series of compounds with vari analogs were evaluated to determine if any modifications to the left hand si tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be ina results are consistent with our prior mode of action studies that indicate the is required for CysK activation. [5]  improvement. The catechol 59, designed to capitalize on outer membrane s iron-dependent transport mechanisms, showed the lowest accumulation of series, suggesting outer membrane permeability may not be limiting for the se To complete our SAR analysis, a small series of compounds with vario analogs were evaluated to determine if any modifications to the left hand sid tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be inac results are consistent with our prior mode of action studies that indicate the t is required for CysK activation. [5]  improvement. The catechol 59, designed to capitalize on outer membrane iron-dependent transport mechanisms, showed the lowest accumulation o series, suggesting outer membrane permeability may not be limiting for the s To complete our SAR analysis, a small series of compounds with vari analogs were evaluated to determine if any modifications to the left hand si tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be ina results are consistent with our prior mode of action studies that indicate the is required for CysK activation. [5]    To complete our SAR analysis, a small series of compounds with vario analogs were evaluated to determine if any modifications to the left hand sid tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be inac results are consistent with our prior mode of action studies that indicate the tr is required for CysK activation. [5]  To complete our SAR analysis, a small series of compounds with vario analogs were evaluated to determine if any modifications to the left hand sid tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be inact results are consistent with our prior mode of action studies that indicate the tr is required for CysK activation. [5]  To complete our SAR analysis, a small series of compounds with vario analogs were evaluated to determine if any modifications to the left hand sid tolerated. The N-methyl tetrazole 62, thiadiazole 63, and methyl-thiadiazole were inactive (Table 4). Similarly, compounds 65-71 were found to be inac results are consistent with our prior mode of action studies that indicate the tr is required for CysK activation. [5]  To examine the potential for the further development of the newly s analogs, in vitro ADME properties of promising compounds 9 and 25, and o hits 1, 2, and 3 were examined (Tables 5 and S1). Both compounds 9 and 25 dem improved solubility, and plasma and metabolic stability over compound 1. E by the improved mouse plasma and microsomal stability, the mouse pharm profile of 25 by intravenous dosing was determined. Compound 25 showed a exposure (AUCinf 2807 h·ng/mL) and a mean elimination half-life of 0.55 h (Tab To examine the potential for the further development of the newly s analogs, in vitro ADME properties of promising compounds 9 and 25, and o hits 1, 2, and 3 were examined (Tables 5 and S1). Both compounds 9 and 25 dem improved solubility, and plasma and metabolic stability over compound 1. E by the improved mouse plasma and microsomal stability, the mouse pharm profile of 25 by intravenous dosing was determined. Compound 25 showed a exposure (AUCinf 2807 h·ng/mL) and a mean elimination half-life of 0.55 h (Tab To examine the potential for the further development of the newly sy analogs, in vitro ADME properties of promising compounds 9 and 25, and o hits 1, 2, and 3 were examined (Tables 5 and S1). Both compounds 9 and 25 dem improved solubility, and plasma and metabolic stability over compound 1. E by the improved mouse plasma and microsomal stability, the mouse pharm profile of 25 by intravenous dosing was determined. Compound 25 showed a exposure (AUCinf 2807 h·ng/mL) and a mean elimination half-life of 0.55 h (Tab >200

Discussion
In this study, the structure-activity relationship of the TAT series was closely examined by targeted synthesis and anti-E.coli testing. The results, joined with observations from our initial publication, [5] provide a full picture of this novel anti-Gram negative chemotype. The combined SAR is summarized in Figure 3, showing a clear structure-activity relationship with the tightest structural requirements for the thio-linked triazole ring. This is consistent with our prior finding that triazole NH is a critical acceptor for the CysK activation and consequential false product formation. Substitutions to the right-hand side aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems to increase the pharmacological properties. Substitutions to the central acetamide linker section were more nuanced, the introduction of a methyl branch substantially decreased antibacterial activity, but the isomeric propionamide and N-benzamide systems retained activity.
A primary challenge in Gram-negative drug discovery is understanding the SAR rules of MIC activity, which is a combination of the ability of the inhibitor to penetrate and accumulate into the bacterial cytoplasm, and its ability to inhibit the molecular target [19]. In this case, the TAT series has many desirable properties previously reported for Gramnegative entry, including a low molecular weight and polarity range [20]. To further understand the SAR, we performed a retrospective analysis of the physiochemical and MIC properties of all the compounds generated in the series that had the optimal thioacetamidetriazole motifs required and diverging only in the aryl section substitution ( Figure S1). Compounds 1, 7, 9, and 25, with the best MIC values (MIC ≤ 3.1 µg/mL), all clustered with LogD 0.54-2.25 and TPSA 70-110 Å 2 ranges, consistent with the properties of other Gram-negative drugs that are less susceptible to efflux [21,22]. triazole ring. This is consistent with our prior finding that triazole NH is a critical acceptor for the CysK activation and consequential false product formation. Substitutions to the right-hand side aryl ring were generally best tolerated, including the introduction of thiazole and pyridine heteroaryl systems to increase the pharmacological properties. Substitutions to the central acetamide linker section were more nuanced, the introduction of a methyl branch substantially decreased antibacterial activity, but the isomeric propionamide and N-benzamide systems retained activity. A primary challenge in Gram-negative drug discovery is understanding the SAR rules of MIC activity, which is a combination of the ability of the inhibitor to penetrate and accumulate into the bacterial cytoplasm, and its ability to inhibit the molecular target [19]. In this case, the TAT series has many desirable properties previously reported for To complement this study a subseries of compounds was generated, designed to increase intracellular accumulation by the incorporation of basic cycloalkyl rings to the aryl motif. This strategy was not successful in increasing intracellular accumulation for the TAT series and was likely incompatible with the intracellular molecular target, resulting in a loss in MIC activity. In this series, smaller TAT analogs appear to be advantageous.
The range of TAT analogs generated allows for the selection of compounds with desirable pharmacological properties and the potential for future development. The plasma stability of our initial lead 1 was low, likely due to inactivation by mouse carboxyesterases, a known issue with similar amide systems [23]. Though plasma stability was less of an issue for human plasma, the development path required mouse efficacy experiments and better compounds were sought, this analysis led us to prioritize compound 25 for further development due to its excellent MIC, human and mouse plasma stabilities, and microsomal stabilities. Pharmacokinetic profiling of 25 suggested it may be suitable for further evaluation.

General Experimental Procedure
All solvents used for chromatography and liquid chromatography were purchased from Aldrich. Flash column chromatography silica cartridges were obtained from Biotage Inc (Biotage, LLC, Charlotte, NC, USA). Reactions were monitored by thin-layer chromatography (TLC) on pre-coated Merch 60 F254 silica gel plates and visualized using UV light (254 nm). A Biotage FLASH column chromatography system (Biotage, LLC, Charlotte, NC, USA) was used to purify mixtures. 1 H NMR spectra were recorded on a Varian INOVA-500 spectrometer or on a Bruker 400 MHz NMR spectrometer (Bruker Scientific LLC, Billerica, MA, USA). Chemical shifts (δ) are reported in parts per million, relative to the residual solvent peak or internal standard (tetramethylsilane), and coupling constants (J) are reported in hertz (Hz). Purity of the products was confirmed by UPLC/MS (the Waters Acquity) (Waters Corporation, Milford, MA, USA). Optical rotations were analyzed on a Jasco P-1010 polarimeter instrument with a path length of 1 dm (589 nm) and reported as follows: [α] D T (c in grams per 100 mL of solvent). Melting points were recorded using Büchi melting point B-545 instrument (Büchi Corporation, New Castle, DE, USA). Analytical data are given for active compounds, and data for all other compounds are provided in the Supplementary Information. calculated using the non-compartmental analysis tool of Phoenix WinNonlin (Version 7.0). All procedures of the study were in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) as published in The Gazette of India, 15 December 1998. Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained before initiation of the study.
Supplementary Materials: The following are available online. Analytical data are given for synthesized compounds, Table S1. Solubility of Lead compounds 1, 2, 3, 9 and 25, Table S2. In vivo pharmacokinetics profile of compound 25, Table S3. LC-MS/MS methods for whole cell accumulation, Figure S1. LogD vs. TPSA of TATs. References [30,31] are cited in the supplementary materials.  Data Availability Statement: All the data generated in the current research work has been included in the manuscript.