Synthesis and Antichlamydial Activity of Molecules Based on Dysregulators of Cylindrical Proteases

Abstract

Chlamydia trachomatis is the most common sexually transmitted bacterial disease globally and the leading cause of infertility and preventable infectious blindness (trachoma) in the world. Unfortunately, there is no FDA-approved treatment specific for chlamydial infections. We recently reported two sulfonylpyridines that halt the growth of the pathogen. Herein, we present a SAR of the sulfonylpyridine molecule by introducing substituents on the aromatic regions. Biological evaluation studies showed that several analogues can impair the growth of C. trachomatis without affecting host cell viability. The compounds did not kill other bacteria, indicating selectivity for Chlamydia. The compounds presented mild toxicity towards mammalian cell lines. The compounds were found to be non-mutagenic in a Drosophila melanogaster assay and exhibited a promising stability in both plasma and gastric fluid. The presented results indicate this scaffold is a promising starting point for the development of selective antichlamydial drugs.

Graphical Abstract

Introduction

Chlamydia trachomatis is a Gram-negative bacterium that infects 1.7 million people in the US with a ~8.8% increase in infection rates since 2013.^{1, 2} It is the most common reportable bacterial sexually transmitted infection (STI) worldwide according to recent surveillance by the Centers for Disease Control and Prevention.^{2, 3} Chlamydia, which primarily targets epithelial cells, is considered the leading cause of infertility and preventable infectious blindness (trachoma) in the world.^{4, 5} Perhaps the most serious issue with chlamydial infections, and a chief cause of its deleterious consequences, is the asymptomatic nature of the infection.^{6, 7} Untreated chlamydial infections can result in chronic sequelae, such as pelvic inflammatory disease, which can lead to ectopic pregnancy and tubal factor infertility.⁴

The chlamydial developmental cycle commences by attachment of an infective, non-replicative elementary body (EB) to the plasma membrane of the host cell. Within the host cell, the EB remains within a host-derived vesicle, termed an inclusion. Later the EB differentiates into its replicative form, the reticulate body (RB), which begins the replication process.^{8, 9} After multiple rounds of polarized division¹⁰ within the inclusion, RBs undergo a secondary differentiation to EBs, and the pathogen is released from the host cell starting another round of infection^{9, 11}

Currently, there is no vaccine nor a selective drug approved by the FDA to treat Chlamydia trachomatis.^{12, 13} The first-line antibiotics used for chlamydia infections (i.e., Azithromycin (AZM) and Doxycycline) are broad spectrum drugs that can affect regular functions of the commensal microbiota and encourage the development of bacterial resistance.¹⁴ In addition, C. trachomatis recurrence after antibiotic treatment remains a considerable issue that may eventually lead to treatment failure and the chronic sequelae associated with this pathogen.^15-17 For example, repeated chlamydial infections at rates of ~25% for women and ~20% for men have been reported after AZM treatment.¹⁸ Taking into consideration the steady increase in infection cases, the risk of contagion, and the rise of general bacterial resistance due to untargeted treatments, the development of a selective chlamydial drug is needed to meet the challenges posed by this STI.

Several approaches are being pursued to develop specific treatments against chlamydial infections. Noteworthy is the seminal work of Almqvist et al. that sought to inhibit chlamydial growth by blocking the glucose-6-phosphate pathway.^{19, 20} This group has developed a novel class of thiazolino-2-pyridones that shown remarkable inhibitory activity and low toxicity towards mammalian cells. Elofsson and co-workers also reported another intriguing approach focused on blocking the type II fatty acid synthesis pathway (FAS II).²¹ The same group has prepared compounds with dual activity by combining key features from active compounds into hybrid systems.²² These important works highlight the importance of designing compounds that affect nontraditional bacterial targets to eradicate this pathogen.

Another antimicrobial target that has gathered considerable attention recently are the cylindrical proteases.^{23, 24} It has been suggested that dysregulation of proteolytic enzymes is a novel approach to treat bacterial infections^25-28 because the indiscriminate degradation of proteins can damage the physiology, pathogenicity, and cellular processes of the organism.^{29, 30} Previously, Brötz-Oesterhelt et al. reported some cyclic acyldepsipeptides (ADEP) (Figure 1) activate ClpP, leading to the death of Escherichia coli.²⁵ Others expanded their findings showing that Clp activation can kill other bacteria, including Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Pseudomonas aeruginosa, and Staphylococcus aureus, through various in vitro and in vivo assays.^31-35 Although promising, ADEPs possess some inherent limitations such as poor solubility, metabolic instability, fast clearance in animal studies, and challenging chemical synthesis.^36-38

Figure 1.

Structures of ADEP and ACP compounds.

Building upon that precedent, Leung and coworkers combined computational chemistry and biochemical experiments to identify new scaffolds that can activate ClpP.³⁵ Two of the synthesized molecules, named ACP1a and b (1 and 2, Figure 1), showed moderate antibacterial activity against N. meningitidis (64 μg/mL and 16 μg/mL, respectively) and H. influenzae (32 μg/mL and 8 μg/mL, respectively). Both molecules were 10-20-fold less potent than ADEP1. However, ACP1b was found to activate E. coli ClpP in an enzymatic assay, suggesting its potential as an antibiotic against this target.³⁵

The biological functions of Chlamydia are also hypothesized to be regulated by cylindrical proteases, which degrade proteins and peptides to maintain homeostasis and perhaps to regulate differentiation of the developmental forms.^39-41 Recent work by us and others highlights the critical role of this degradation machinery in Chlamydia.^{41, 42} Four core Clp proteins have been identified in this organism: the caseinolytic proteases, ClpP1 and ClpP2, and two associated ATPase chaperones (AAA+) with diverse cellular activities ⁴³, ClpC and ClpX.^{27, 44, 45} These chaperones bind to the axial faces of ClpP regulating its proteolytic activity. Using the work of Leung as a starting point, we synthesized molecules 1 and 2 and studied their ability to affect C. trachomatis as well as their effects on chlamydial growth and viability. ⁴¹ We found both compounds cause a drastic decrease in the formation of infectious EBs, although we could not definitively assign this effect to a disruption in ClpP activity in Chlamydia. Based on our early report, we decided to optimize compounds 1 and 2 to establish structure-activity relationships (SAR).

In the present work, we report the antichlamydial activity of new antichlamydial agents. One of the new molecules (compound 40) was found to affect both chlamydial inclusion numbers, size, and morphology in infected HEp-2 cells. In addition, we performed preliminary mechanism of action studies to understand the eradication process. We also investigated the antimicrobial activity of the compounds against several Gram-positive and Gram-negative bacteria, as well as fungi, to establish their spectrum of activity. Finally, we assessed the toxicity of various compounds towards human cells, their mutagenicity in Drosophila melanogaster, and their plasma, simulated gastric, and metabolic stability utilizing human liver microsomes. The results indicate these new compounds are selective for Chlamydia and can be used as a starting point to develop new drugs selective towards this pathogen.

Results and discussion

Chemistry

Our synthetic strategy was based on reacting a substituted sulfonyl aryl carboxylic acid with the desired amine.^{35, 41} The carboxylic acid derivatives (7a-f, Scheme 1) were prepared by reacting 2-thioaryl derivatives (3) with an appropriate α-halo ester derivative (4).³⁵ The obtained thioether derivatives 5a-f were reacted, without further purification, with potassium peroxymonosulfate (OXONE^®) in a dioxane-water mixture to provide the corresponding sulfone derivatives 6a-f. Hydrolysis of the ester group under mild basic condition provides the corresponding carboxylic acid derivatives 7a-f. The monomethyl acid derivative 7d was alternatively prepared, in two steps, by the reaction of 5-(trifluoromethyl)pyridine-2-thiol with 2-bromopropanoic acid followed by oxidation (Scheme S1).⁴⁶

Scheme 1.

Synthesis of key carboxylic acid intermediates 7 a-f

The carboxylic acid derivatives 7a-f were activated using PYBOP and DIPEA in tetrahydrofuran (THF) and then reacted with 3-phenylpropylamine to yield six derivatives, 9-14 (Scheme 2).^{41, 47, 48} All the prepared molecules satisfy the Lipinski rule of five (Table S1).⁴⁹ PYBOP proved to be the best choice for this type of reaction while other coupling agents, such as HBTU and HATU (with or without Oxyma), only gave trace amounts of the product. The selection of THF was also critical because using DMF resulted in a Smiles rearrangement reaction due to the presence of a sulfonyl moiety (Scheme S2).⁵⁰

Scheme 2.

Synthesis of ACP1 analogues with modified the western part

Then, five derivatives (16-20, Scheme 3) were synthesized to understand the importance of the thiol and chloro atoms on the eastern part of the molecule. Accordingly, the amine precursors 15a-b (a = 2-((2-chlorophenyl)thio)ethan-1-amine; b = 2-((4-chlorophenyl)thio)ethan-1-amine) were prepared as reported previously (Scheme S4)^{51, 52} and reacted with the carboxylic acids 7a-d.

Scheme 3.

Synthesis of ACP1b analogues by modification in the western fragment

To investigate the importance of the thiol on the eastern part, we synthesized derivatives 23-31 (Scheme 4). Firstly, we converted 2-bromoethylamine (21) into the functionalized 3-phenoxyethylamines 22a-c (a = 2-phenoxyethan-1-amine; b = 2-(2-chlorophenoxy)ethan-1-amine; c = 2-(4-chlorophenoxy)ethan-1-amine). These molecules were coupled to the carboxylic acid intermediates 7a,c,f using the conditions described above.

Scheme 4.

ACP derivatives with oxygen in the western part

To further develop SAR, we synthesized a set of compounds with restricted rotation by including piperazine linkers in the middle part (33-45). The carboxylic acid derivatives 7d,f were reacted with various piperazines (32a-j) to evaluate the effect of this substitution on the eastern region of the molecules, as shown in Scheme 5. This rigid linker restricts the flexibility of the side chain and reduces the entropic penalty of binding of the compounds to their target.^{53, 54}

Scheme 5.

Synthesis of ACP1 analogues with a rigid linker

Biological evaluation

Antichlamydial activity.

As mentioned, once EBs are internalized, they differentiate into RBs and remain within a membrane-bound vesicle called an inclusion. This protects the pathogen during its developmental cycle. Because the inclusions are linked to the growth and replication of the pathogen,^{55, 56} we determined antichlamydial activity by analyzing the number and size of the inclusions using an Immunofluorescence assay (IFA).^{57, 58} Briefly, HEp-2 cells were infected with C. trachomatis serovar L2, and the synthesized molecules were added at 50 μg/mL, 8 hours post infection (hpi). After 16 h (total incubation of the cells for 24 h), the inclusions were analyzed and compared with the control, untreated cells. The morphology and viability of the cells were examined under phase contrast microscopy to ensure the eradication of the inclusions was not the result of a reduction in viable cells due to compound toxicity (Figure 3b and SI). Table 1 shows the results of this pre-screening assay.

Figure 3.

(a) Immunofluorescence images of the effects of selected compounds on chlamydial inclusion growth. In blue, HEp-2 cells nuclei; in green, chlamydial inclusions. Images were acquired at 24h postinfection. (b) Cell morphology images using an EVOS FL auto cell imaging microscope.

Table 1.

Initial antichlamydial activity screening of ACP derivatives vs. C. trachomatis (serovar LGV-L2)

Compound No.	Inhibition activity*
ACP1	−
1	++
2	+
9	−
10	−
11	++
12	−
13	+
14	Toxic
16	−
17	−
1859	++
19	++
2059	−
23	−
2459	++
25	++
26	−
27	++
2859	+
29	−
30	−
31	−
33	−
34	−
35	+
36	++
37	++
38	+
39	−
40	++
41	++
42	+
43	−
44	−
45	−

Compounds were tested at 50 μg/mL.

^*The compound’s effectiveness is a measure of its ability to inhibit chlamydial inclusions and can be divided into three categories: [−] = Not effective; [+] = intermediate effect; [++] = effective. One compound demonstrated clear toxicity to the host cells and is noted.

We found that analogues 9 and 10, molecules lacking the trifluoromethyl group, were inactive, which highlighted the importance of electron withdrawing groups at position 4. Meanwhile, the presence of the trifluoromethyl-substituent (compound 11) provided antichlamydial activity. Next, we replaced the trifluoromethyl group with a chlorine atom (13), obtaining an analogue with moderate activity. Two other derivatives containing an unsubstituted phenyl group (16, 17) and a chlorine atom on the eastern part of the molecule did not exhibit activity. However, the addition of the trifluoromethyl group, while keeping with the same substitution pattern on the eastern part (18, 19), yielded antichlamydial compounds.

The importance of the trifluoromethyl substituent was further highlighted in derivatives 23-29, which also possess an aryl ether group. Surprisingly, three molecules contained a trifluoromethyl group and were still inactive against Chlamydia: compounds 20, 30, and 31. Note that these inactive compounds possess a chlorine atom at the para position on the eastern portion of the molecules. Collectively, these data suggest a detrimental effect of substitution at the para position. To test the importance of the sulfonyl group, a thioether derivative 14 was synthesized by skipping the oxidation step in scheme 2. The obtained derivative showed prominent host cell toxicity. Compound 12, which has one methyl group instead of the gem-dimethyl group, was found to be inactive.

Next, we decided to replace the amide bond at the middle part of the core molecule with rigid cyclic amine (piperazine) groups to generate a series of derivatives without hydrogen bond donor groups (Table 1 and Table S1). Compounds 33 and 34, possessing phenyl or pyridyl groups attached to the piperazine moiety, lacked antichlamydial activity. In contrast, replacement of the phenyl group with an o-tolyl (35), 2,5-dimethyl phenyl (36), or 3,4-dichloro phenyl (37) enhanced antichlamydial activity. The inclusion of a methyl spacer between the piperazine and the eastern part, gave molecules 38 and 42, which displayed moderate activity. The fluorinated analogue of 38, molecule 39, did not possess activity, but the chlorinated molecule (40) was active. Thus, the data suggest that a larger group with electron withdrawing properties is needed. Molecules 41 and 42 possess substituents at the meta position, and, once again, the electron withdrawing group enhanced activity. Finally, three derivatives (43-45) with a monomethyl group in the middle part exhibited no antichlamydial activity.

After the initial assessment of antichlamydial activity, we determined the impact of the most active compounds in vitro by determining the number and morphology of infectious units (the EB), and compared their activity against two antibiotics: spectinomycin and penicillin, which are among the FDA approved antibiotics used to treat STDs.^{60, 61} We understand penicillin is not commonly used to treat Chlamydia since it blocks cell division and growth without eradicating the bacteria but we were looking for a comparison against a common drug.⁶² HEp-2 cells were infected with C. trachomatis serovar L2, and then the selected molecules were added; 50 μg/mL of our compounds, 128 μg/mL of spectinomycin (2x MIC),⁶³ and 5 units/mL of penicillin (3x MIC)⁶³ 8 h after infection. To accurately determine the “infectious progeny” by quantifying the number of chlamydiae from treated cultures as well as controls we used an inclusion forming units (IFU) assay as described elsewhere.⁶² Briefly, after 24 h, the infected cells were scraped, collected in chlamydia transport medium, and used to re-infect a fresh HEp-2 cell monolayer. The chlamydial infectivity, reported as IFUs, was determined by counting the number of fluorescent inclusions after immunofluorescent staining, 24 h post-secondary infection (Figures 2 and 3a) from a minimum of 15 fields of view using an epifluorescence microscope. We also assessed the morphology, size, and appearance of the chlamydial inclusions after exposure to the tested compounds. Among the designated compounds, derivatives 11, 24, 25, 35, 37, 40, and 41 showed a remarkable impact on chlamydiae. Compound 40 showed superior activity than spectinomycin, which was used at a concentration roughly 2.5 fold higher (128 μg/mL) and on par with penicillin (5 U/mL ~ 3 μg/mL).⁶⁴ The immunofluorescent staining (Figure 3a) revealed the inclusions were relatively small and irregular in cells treated with our compounds when compared with the untreated sample and the reference molecules. The cell morphology investigation (Figure 3b) supported that the tested compounds did not show any toxicity during the assay. We reasoned the high activity of this derivative is due to the combination of amide bond restriction in the middle part, the electron withdrawing group in the eastern part, and the acceptable LogP and HBD/HBA values (Table S1).

Figure 2.

Quantification of Chlamydial growth in the presence of selected ACP derivatives (numbered) and antibiotics spectinomycin (Spec) and penicillin (Pen) as compared to an untreated (UTD) control at 24h post-infection. Results are reported as an average with standard deviation on a log10 scale and represent a minimum of two biological replicates.

Dose-response curve.

The dose-response effect was investigated for compound 40 on Chlamydia at six concentrations using the IFU assay described previously. As expected, compound 40 was found to exhibit inhibition activity in a dose-dependent manner with an IC₅₀ (the concentration, which shows 50% inclusion inhibition) of 5.2 μg/mL (Figure 4a). The dose-response curve revealed a high reduction of chlamydial progeny in a consistent way (Figure 4a). Besides affecting progeny yields, the immunofluorescence images showed an increase in the number and size of chlamydial inclusions as the concentration of the compound decreased (Figure 4). Compound 40 reduced the chlamydial infection at both 100 and 50 μg/mL and still maintained good activity up to a concentration of 12.5 μg/mL, with inclusion yields around 60% lower than the untreated cells. We did not observe cell toxicity at 100 μg/mL, which supports the tolerability of 40.

Figure 4.

(a) Dose-response curve for the effect of 40 reported on a log10 scale, (b) Immunofluorescence analysis of 40 inhibitory effect. In green, chlamydial inclusions; in blue, HEp-2 cell nuclei.

Antichlamydial effect of ACP derivatives in comparison with two marketed drugs.

We further tested the activity of compound 40 at 50 μg/mL and 5.20 μg/mL (IC₅₀ value) against our lead compound (ACP1b) at 50 μg/mL, and two frontline antibiotics, Azithromycin and Doxycycline, at their reported minimal chlamydicidal concentrations (MCCs), (4 μg/mL and at 1 μg/mL, respectively).^65-68 We confirmed our lead compound (ACP1b) and compound 40 showed good inhibitory activity against chlamydiae (Figure 5a). The four tested compounds elicited a notable impact on the size as well as the number of the inclusions (Figure 5b). Despite the reported treatment efficacy of both drugs,⁶⁹ these antibiotics are associated with two major concerns. The first is treatment failure rate, which can reach 22% and lead to repeat infections, and existing complications associated with chlamydial infections.⁷⁰ The second concern involves bacterial resistance that can be triggered by these broad spectrum therapies (in both Chlamydia and other normal flora bacteria)^71-74 and can lead to chlamydial persistence.^75-77 Given these concerns and the promising activity of our compounds, we consider ACP a new scaffold with a unique mechanism of action that can serve as a starting point to generate new antichlamydial agents.

Figure 5.

(a) Quantification of Chlamydial growth in the prescence of selected ACP derivatvies (40 and rnr!trni^> af a nti biotics Azithromycin (Azith) and Doxycycline (Dox) as compareed to an untreated (UTD) control at 24h post-infection. (b) Immunofluorescence analysis of the tested compounds inhibitory effect in green, chlamydial inclusions; in blue, HEp-2 cells muclei.

Mechanism of action.

Antibiotics can be bacteriostatic, stopping growth or reproduction by targeting essential functions such as cell wall growth, or bactericidal, which kills the bacteria.⁷⁸ To classify compound 40, we treated human cells 8 hpi with 50 μg/mL of the molecule, followed by incubation for an additional 16 h. Subsequently, the drug was washed out before the infected cells were further incubated for another 24 h. As seen in Figure 6a, compound 40 reduced the inclusion yield as expected but, 24h after drug washout, the number of inclusions increased by ~1 log in comparison with untreated cells, suggesting a bacteriostatic mechanism of action. Further, incubation of the infected cells with 40 for 48 h led to higher inclusion output suggesting a loss of the drug activity via metabolic degradation, development of resistance (unlikely on the time frame of the experiment) or an increase on the total number of inclusions at the end of the cycle, which cannot be eradicated by the drug, or a combination of the three. Additional studies are needed to understand this result.

Figure 6.

(a) Investigation of the bacteriostatic or bactericidal activity of 40 as measured by IFU output and reported on a log scale, (b) Immunofluorescence analysis of 40 impact 24h after its removal (Reactivation) in comparison with untreated (UTD) samples at 24h post-infection. In green chlamydial inclusions; in blue, HEp-2 cell nuclei.

Although the EB numbers increased after drug removal or incubation for additional 24 h, we noticed a reduction in the inclusion size and loss in inclusion morphology reflecting a lack of development of the pathogen, which suggests the impact of our compounds on chlamydial growth (Figure 6b). Generally, the static effect of these compounds was consistent with a general mechanism depending on inhibition of protein turnover inside the chlamydial organisms. Bacteriostatic drugs can help in treatment of chlamydia infections since the progression of Chlamydia into a latent form and the clearance of bacteria are not only dependent on the antibiotic category but also on the capability of the host cells to eliminate the bacteria.⁷⁹

In vitro protease activity.

The presented compounds are analogues to known ClpP activators.^{41, 59} Therefore, we tested the ability of selected compounds to stimulate ClpP-dependent protein degradation in vitro in the absence of a AAA+ chaperone. The protease activities of recombinant ClpP1 and ClpP2 from C. trachomatis L2, recombinant ClpP from E. coli, and recombinant ClpP from human and mouse cells (mitochondrially localized) were measured in the presence and absence of ACPs (Figure 7).⁴¹ Briefly, 20 μM of FITC-labeled casein was incubated with 1 or 0.1 (40 only) μM ClpP from E. coli, 1 μM mammalian ClpP, or 6 μM of C. trachomatis ClpP1 and ClpP2 at 32°C for 3 h with or without compounds. Fluorescence owing to FITC-casein degradation was measured every 3 minutes. In parallel, degradation of unlabeled casein was performed and assessed using SDS-PAGE followed by staining with Coomassie Brilliant Blue (Figure S4). Derivatives 11 and 40 enabled some species of ClpP to degrade casein in the absence of a chaperone while little or no degradation was observed for compounds 9, 16, or 17. Importantly, 40 did not activate human or mouse ClpP, suggesting possible selectivity of the compounds for the bacterial ClpP at least in reference to the ClpP from E. coli. Surprisingly, we did not observe activation of chlamydial ClpP1, ClpP2 or the ClpP1/2 dimer (other than some residual decomposition triggered by 9 and 11). We assessed the activity of the chlamydial ClpP1/P2 preparations using Native PAGE to confirm oligomerization (Figure S2) and digestion of the fluorescent peptide Suc-Luc-Tyr-AMC (Figure S3). This prompted us to consider the killing effect may be the result of ClpP inhibition. However, we did not observe inhibition of ClpP1/P2 activity using the Suc-Luc-Tyr-AMC fluorescent peptide assay. Also, under the conditions tested, we did not observe alterations in ClpP1/P2 activity in the presence of the compounds and the chaperones (data not shown). The results suggest that while E. coli ClpP is activated chlamydial ClpP function is not altered, suggesting the molecules may have a mechanism of action independent of this target.

Figure 7.

In vitro protease assay shows the degradation of casein (unlabeled) by three different recombinant ClpP preparations with or without the compounds as assessed by SDS-PAGE analysis. Ec = Escherichia coli, Ctr = Chlamydia trachomatis, Hs = Homo sapiens, Mm = Mus musculus. DMSO is a negative control. Note the mammalian ClpP orthologues migrate slightly lower than casein in the gels. The top band in all images is casein and the lower band is the respective ClpP. Molecular weight markers (in kDa) are present in lane 1 of each gel.

Other antimicrobial activity.

To evaluate the selectivity of our molecules, we tested their biological activity against other lab strains and ESKAPE microorganisms and selected fungal pathogens: Staphylococcus aureus JE 2, S. aureus ATCC 43300 MRSA; E. coli ATCC 25922; E. coli K12, P. aeruginosa ATCC 27853; K. pneumoniae ATCC 700603; Acinetobacter baumannii ATCC 19606; Candida albicans ATCC 90028, and Cryptococcus neoformans var. grubii H99 ATCC 20882. Notably, none of our compounds showed inhibitory activity against these species in a preliminary screening at 32 μg/mL (Table S2). To further investigate their selectivity, we subjected S. aureus JE2 and E. coli K12 to concentrations up to 256 μg/mL of compounds 11, 21, 25, 28, 35, 37, 40, 42 , and once again, bacterial death was not observed. These results indicate that the compounds may be selective for Chlamydia. These data also suggest the molecules may not affect the normal gut flora, which in turn suggests development of widespread resistance is unlikely. We think the lack of activity against other microorganisms is due to the unique cell wall structure of C. trachomatis when compared to other bacteria and the unique developmental cycle of Chlamydia, which may favor the selectivity of the compounds. In support of these points, 40 was a strong activator of the E. coli ClpP in vitro (Figure 7) without showing antibacterial properties towards either tested E. coli strain.

Cytotoxicity.

The previous assays not only indicated antichlamydial activity, but also low toxicity toward the HEp-2 host cells (Figure 5b and S1). To further examine the tolerability of the synthesized compounds, we evaluated their in vitro toxicity against epithelial cervix adenocarcinoma (HeLa 229) and human keratinocyte (HaCaT) cells. Molecules were evaluated at a concentration of 50 μg/mL, i.e., the highest tested concentration used in antichlamydial assays, and results are summarized in Figure 8. The compounds were tolerated by HeLa 229 cells (only 20 and 24 presented viabilities less than 65%) while the normal HaCaT proved to be more sensitive to some of the molecules. For example, compound 40 was not toxic towards HeLa 229 but affected ~50% of the HaCaT cells. Compounds 24, 37, and 42 were more tolerated by the HaCaT cells. This result is not necessarily consistent with the in vitro casein degradation data. For example, 11 presented toxicity towards both cells and activated mammalian ClpP but 40 did not activate these ClpP orthologs. Future studies will be performed to assess if the observed degradation data are related to a limit of detection issue with the casein assay in combination with an increased need for ClpP/mitochondrial function in the HaCaT cells versus the HeLa cells or to other off-target mechanisms of action.

Figure 8.

Toxicity analysis of the most active compounds against HeLa 229 and human keratinocytes (HaCaT) at 50 pg/mL

Mutagenic studies.

One major concern of potential antibiotics is their mutagenesis potential, which can damage the host cells and facilitate the adaptation of the bacteria to the antibiotic pressure and other types of stress. Accordingly, we evaluated whether the ACP derivatives were mutagenic towards eukaryotes using the Somatic Mutation and Recombination Tests (SMART) in wing-somatic cells of Drosophila melanogaster.⁸⁰ This in vivo assay simultaneously detects mutational and mitotic recombination events and quantifies the recombinogenic activity of chemicals and drugs. Some antimicrobial drugs have been reported to promote DNA damage because of oxidative stress in the mammalian genome^81-83, leading to severe side effects such as bone marrow depression, aplastic anemia, and leukemia. We analyzed marked trans-heterozygous descendants (mwh/flr3) resulting from Standard (ST) and High Bioactivation (HB) crossings, which were chronically exposed to ACP derivatives at three different concentrations. The frequency of different mutant clones was scored and the total spots, which indicate the final genotoxicity of each compound at different concentrations, are shown in Table 3 and Table S3.

Table 3 -.

Frequency of mutant spots in the wings of marked trans-heterozygous descendants (mwh/flr³) of D. melanogaster using the standard cross (ST) and marked trans-heterozygous descendants (mwh/flr³) of D. melanogaster using the high bioactivation (HB) after chronic treatment of larvae with 11, 40 and 41 derivatives

Spots per fly (number of spots) statistical diagnosisa
mwh/flr3 of D. melanogaster - Standard cross (ST)
Comp ID	Concentration (mM)	N° of flies (N)	Small single spots (1-2 cell)b m = 2	Large single spots (>2 cell)b m = 2	Twin spots m = 5	Total spots m = 2	Spots with mwh clonec (n)
Control		20	0.40 (8)	0.15 (3)	0.0 (0)	0.55 (11)	10
11	0.25	20	0.25 (5) -	0.0 (0)-	0.0 (0) -	0.25 (5) -	5
	0.50	20	0.20 (4) -	0.0 (0) -	0.0 (0) -	0.20 (4) -	4
	1.00	20	0.35 (7) -	0.05 (1) -	0.0 (0) -	0.40 (8) -	8
40	0.25	20	0.20 (4) -	0.0 (0) -	0.0 (0) -	0.20 (4) -	4
	0.50	20	0.15 (3) -	0.0 (0) -	0.0 (0) -	0.15 (3) -	3
	1.00	20	0.30 (6) -	0.05 (1) -	0.0 (0) -	0.35 (7) -	7
41	0.25	20	0.10 (2) -	0.05 (1) -	0.0 (0) -	0.15 (3) -	3
	0.50	20	0.15 (3) -	0.15 (3) -	0.05 (1) -	0.35 (7) -	7
	1.00	20	0.20 (4) -	0.05 (1) -	0.0 (0) -	0.25 (5) -	5
mwh/flr3 of D. melanogaster - High Bioactivation (HB) cross
Control		20	0.25 (5)	0.10 (2)	0.05 (1)	0.40 (8)	8
11	0.25	20	0.15 (3) -	0.05 (1) -	0.05 (1) -	0.25 (5) -	5
	0.50	20	0.10 (2) -	0.15 (3) -	0.0 (0) -	0.25 (5) -	5
	1.00	20	0.20 (4) -	0.05 (1) -	0.05 (1) -	0.30 (6) -	6
40	0.25	20	0.45 (9) i	0.0 (0) -	0.0 (0) -	0.45 (9) -	9
	0.50	20	0.25 (5) -	0.0 (0) -	0.10 (2) -	0.35 (7) -	7
	1.00	20	0.50 (10) i	0.05 (1) -	0.05 (1) -	0.60 (12) -	12
41	0.25	20	0.40 (8) -	0.05 (1) -	0.0 (0) -	0.45 (9) -	9
	0.50	20	0.40 (8) -	0.05 (1) -	0.05 (1) -	0.50 (10) -	10
	1.00	20	0.35 (7) -	0.10 (2) -	0.0 (0) -	0.45 (9) -	9

^aStatistical diagnoses according to Frei and Würgler [1988]. U test, two-sided; probability levels: −, negative; +, positive; i, inconclusive; P≤0,05 vs, untreated control.

^bIncluding flr³ single spots.

^cConsidering mwh clones from mwh single and twin spots. NC: Negative control.

We found that flies treated with 11, 20, 24, 26, 40 and 41 displayed frequencies of clone formation (per individual) for the ST and HB crosses ranging from 0.15 to 0.45 (P <0.05) and 0.20 to 0.60 (P <0.05), respectively, at 0.25, 0.5, and 1 mM. The flies treated with compound 26 at 1 mM showed a higher mutation frequency of 0.70 and 0.75 in descendants from both crossings, although these were not statistically significant. Overall, the results were negative, indicating that the compounds are non-genotoxic in somatic cells of D. melanogaster at the concentrations tested, even in HB cross, which has high metabolic bioactivation. However, 26 indicated that the mutant spot frequencies are directly dependent on the concentration, and it is possible that concentrations higher than 1 mM may present mutagenic potential. Compounds 11, 20, 24, 26, 40 and 41 did not show toxicity against D. melanogaster at the tested concentrations, and it was observed that the survival rate in the treated groups did not differ statistically from the negative control (P <0.05).

Stability studies.

We investigated the in vitro metabolic stability of the compounds using liver microsomes and mouse plasma. The incubation with human liver microsomes (HLM) is considered a relevant pharmacokinetic indicator for a compound in vivo.^{84, 85} The metabolic half-life (t_1/2) and the intrinsic clearance (CL_int) of compounds 11, 24, 25, 37, and 40 are summarized in Table 4. The assay revealed that all tested compounds underwent rapid modification with approximately 90% of the drug modified in 10 minutes as shown in Figure 9. All tested compounds were stable in the negative control experiment (without NADPH) up to 60 minutes. For compound 40 the main metabolite presented a M+16 peak (Figure S5), which may correspond to the N-oxidated adduct. Fragmentation spectra of both neat and 10 min incubated samples indicated no chemical change in the eastern part (Figure S5). This compound persisted 60 min post incubation with HLM. The metabolic site and the complete chemical structure of this metabolite is still under investigation.

Table 4.

In vitro experimental Values of T_1/2, intrinsic clearance, and hepatic clearance.

Comp. ID	T1/2 (min)		CL(int) uL/(min*mg protein)		CL(int,H) mL/(min*kg bodywgt.)
	Mean	SD	Mean	SD	Mean	SD
11	6.13	0.16	113.09	3.02	890.58	23.77
24	2.98	0.17	233.77	13.74	1840.91	108.17
25	3.73	0.03	185.60	1.72	1461.60	13.51
37	6.25	0.16	111.96	2.85	873.80	22.45
40	< 2.00	NA	NA		NA

NA: Not applicable

Figure 9.

Time-dependent metabolic stability of the tested compounds in human liver microsomes fortified with NADPH and without NADPH (negative control). Metabolic elimination profiles (% turnover or amount remaining vs. incubation time). Data shown as mean±S.D (n=3).

Then, we explored the stability of some representative compounds in mouse plasma and simulated gastric fluid (SGF, pH 1.2) by calculating the percentage of drug remaining after contact with both media. The tested compounds were incubated with diluted mouse plasma and SGF at 37 °C and the samples were analyzed by HPLC-MS. As shown in Figure 10, all the tested compounds exhibited a high stability profile and their concentrations remained constant for up to 120 minutes. No modification or degradation was detected after the different incubation periods.

Figure 10.

Stability profile in mouse plasma (a) and stimulated gastric fluid (b). Relative concentration is represented as a function of incubation time between the tested compound and mouse plasma and simulated gastric fluid (SGF, pH 1.2). Error bars represent SD of three independent experiments.

Conclusion

Caseinolytic protease P (ClpP) activators have been shown to eradicate bacteria and prevent bacterial resistance. C. trachomatis is among the few bacterial species that possess two caseinolytic protease paralogues, ClpP1 and ClpP2.⁴¹ The Clp protein machinery system is essential for the chlamydial developmental cycle, which includes differentiation between the EB and RB forms.⁸⁶ In this study, we synthesized compounds based on known ClpP activators to kill C. trachomatis. We found some interesting lead compounds that were able to eradicate the pathogen. The biological results allowed us to conduct initial SAR studies to identify key regions that promote biological action (Figure 11). Noteworthy is the fact that compounds did not present activity against other types of bacteria, suggesting a degree of selectivity for Chlamydia. While the the active compounds were able to activate the E. coli ClpP, activation of chlamydial ClpP1/2 was not observed as determined by diverse assays. This suggests the compounds may impact Chlamydia by affecting a different target. In vitro metabolic stability assays revealed these ACP derivatives were enzymatically transformed by liver microsomes in the first 10 minutes of incubation. Conversely, the compounds demonstrated good stability in mouse plasma and simulated gastric fluid. Our results indicate the ACP derivatives represent a promising scaffold that can be further developed to obtain a specific treatment for C. trachomatis infection. Studies are currently undergoing to understand the target of the molecules.

Figure 11.

SAR graphical summary

Experimental Procedures

Chemistry:

General.

All reagents and solvents were used as received from commercial suppliers unless otherwise noted. All used solvents were dried and stored with activated molecular sieves to ensure dryness on the long run. All coupling reactions were carried out in dried glassware under N₂ atmosphere. Reaction progression was detected using TLC, which was performed on Merck silica gel IB2-F plates (0.25 mm thickness), and the spots detected using UV light source at 254 nm. ¹H and ¹³CNMR spectra were run at 500 MHz in deuterated chloroform (CDCl₃), or dimethyl sulfoxide (DMSO-d₆) on a BRUKER- 500 NMR spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (d) scale. Chemical shifts were calibrated relative to those of the solvents. Flash chromatography was performed on RF 200i Flash Chromatography System from Teledyne ISCO. Low resolution mass spectra was obtained on an Agilent 6120 or 6150 mass spectrometer with an electrospray ionization (ESI) source. The tested compounds possessed purities above 95%. The purity tests were performed on an Agilent 1200 HPLC system equipped with a multiple wavelength absorbance UV detector set for 254 nM and using 5 mM C-18 reversed-phase column with methanol and water as a mobile phase. All reported yields refer to isolated compounds. Marvin was used for characterizing of the physicochemical charecters of the synthesized compounds, Marvin 20.4, ChemAxon (https://www.chemaxon.com).

General procedure for the synthesis of acid derivatives 7a-f:

2-(arylthio)propanoate derivatives 5a-f.

To a stirred solution of an appropriated aryl thiol 3 (3.00 mmol) in ethanol (10 mL), were added, potassium hydroxide pellets (0.25g, 4.40 mmol) followed by ethyl α-bromo ester 4 (3.00 mmol) and the reaction mixture was heated at reflux for 18 h. After completion of the reaction, the flask content was allowed to cool down to room temperature, and the formed inorganic salt was removed by filtration and washed with cold ethanol. The filtrate was evaporated under reduced pressure, and the oily residue was dissolved in DCM (20 mL) and washed with deionized water (3×10 mL) and brine solution (1×10 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the desired product as yellow oil which used in the next step without further purification.

2-(arylsulfonyl)propanoate derivatives 6a-f.

Potassium mono persulfate (OXONE^®) (2.2 eq) was added in one portion to a solution of the 2-(arylthio)propanoate derivatives 5a-f (3.4 mmol) in dioxane-water 5:1 (25 mL). The formed white suspension was vigorously stirred at 23 °C for 18 h. The white solid was filtered off, washed with dioxane, and the combined filtrate was concentrated under reduced pressure to remove the organic layer. The resulting aqueous solution was extracted with DCM (3×15 mL). The combined organic solution was dried over Na₂SO₄, filtered, and the organic solvent was removed under reduced pressure to afford the desired product as white solids which were used directly in the next step without further purification.

2-(arylsulfonyl) acid derivatives 7a-f.

2-(arylsulfonyl)propanoate derivative 6a-f (3.6 mmol) was dissolved in a mixture of THF/Water (4:1, 20 mL), lithium hydroxide monohydrate (7.2 mmol) was milled and added portion wise over 30 minutes and the reaction was stirred at room temperature for 18 h. The organic solvent was evaporated under reduced pressure, and the aqueous solution was washed with DCM (1×20 mL) to remove byproducts. Then, the aqueous layer was cooled in an ice bath and treated with 1 N HCl to pH 2. The formed precipitate was filtered off to afford the desired products. The physical characters and the spectral data of separated product are listed below:

2-methyl-2-(Phenylsulfonyl)propanoic acid (7a).

Starting with 6a (925 mg), White solid (520 mg, 77%).¹H NMR (500 MHz, CDCl₃) δ 7.89 (d, J = 7.0 Hz, 2H), 7.71-7.68 (m, 2H), 7.70 (t, J = 7.5 Hz, 1H), 1.63 (s, 6H).

2-Methyl-2-(Pyridin-2-ylsulfonyl)propanoic acid (7b).

Starting with 6b (925 mg), White solid (280 mg, 41%). ¹H NMR (500 MHz, CDCl₃) δ 8.77 (m, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.03 (t, J = 8.0 Hz, 1H), 7.63–7.61 (m, 1H), 1.70 (s, 6H).

2-Methyl-2-((4-(trifluoromethyl)phenyl)sulfonyl)propanoic acid (7c).

Starting with 6c (1160 mg), White solid (840 mg, 82%). ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.5 Hz, 2H), 1.66 (s, 6H); ¹⁹F NMR δ −63.27 (s).

2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanoic acid (7d).

Starting with 6d (1120 mg), White solid (750 mg, 85%). ¹H NMR (500 MHz, CDCl₃) δ 9.01 (s, 1H), 8.253-8.250 (m, 1H), 4.75 (q, J = 7.5 Hz, 1H), 1.74 (d, J = 7.5 Hz, 3H); ¹⁹F NMR δ −62.45 (s).

2-Methyl-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanoic acid (7e).

Starting with 6e (1170 mg), White fluffy solid (890 mg, 83%). ¹H NMR (500 MHz, CDCl₃) δ 9.02 (s, 1H), 8.27–8.22 (m, 2H), 1.76 (s, 6H); ¹⁹F NMR δ −62.68 (s); ¹⁹F NMR δ −62.68 (s).

2-((5-Chloropyridin-2-yl)sulfonyl)-2-methylpropanoic acid (7f).

Starting with 6f (1050 mg), White solid (700 mg, 79%). ¹H NMR (500 MHz, CDCl₃) δ 8.73-8.72 (m, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.97-7.95 (m, 1H), 1.71 (s, 6H).

General procedures for synthesis of 2-Methyl-N-(3-Phenylpropyl)-2 (arylsulfonyl) propanamide (9-14):

A solution of 2-methyl-2-(arylsulfonyl)propanoic acid 7a-f (0.16 −0.17 mmol) in dry THF (10 mL) was treated with a solution of PyBOP (87 mg, 0.17 mmol) in THF (1 mL) followed by DIPEA (83 μL). Then, the reaction mixture was stirred at room temperature for 10 min. A solution of the 3-phenylpropan-1-amine (0.16 mmol) in THF (1 mL) was then added dropwise. The formed yellow solution stirred at room temperature for 1 h. The solvent was removed under vaccum, and the crude product was absorbed onto silica gel and purified by flash column chromatography eluting with a 0–100% gradient of EtOAc in hexanes. The physical characters and the spectral data of the obtained products are listed below:

2-Methyl-N-(3-Phenylpropyl)-2-(phenylsulfonyl)propanamide (9).

Starting with 7a (37 mg), white solid (46 mg, 84%). ¹H NMR (500 MHz, CDCl₃) δ 7. 81 (d, J = 7.5 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.31–7.26 (m, 2H), 7.21–7.18 (m, 3H), 7.05 (br s, 1H), 3.33 (q, J = 6.5 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 1.90 (q, J = 6.5 Hz, 2H), 1.54 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.44, 141.18, 135.10, 134.34, 130.04, 129.03, 128.54, 128.40, 126.10, 68.01, 39.90, 33.19, 30.79, 20.93; HPLC purity, 97.1%; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₂₄NO₃S, 346.1399; found, 346.1470,exact mass(monoisotopic) from spectrum 345.1397.

2-Methyl-N-(3-Phenylpropyl)-2-(pyridin-2-ylsulfonyl)propanamide (10).

Starting with 7b (37 mg), white solid (46 mg, 83%). ¹H NMR (500 MHz, CDCl₃) δ 8.66-8.65 (m, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.92(t, J = 7.5 Hz, 1H), 7.53-7.51 (m, 1H), 7.29–7.26 (m, 1H), 7.19–7.18 (m, 4H), 3.33 (q, J = 6.5 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 1.91 (q, J = 7.5 Hz, 2H), 1.60 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.76, 154.89, 150.25, 141.44, 137.94, 128.47, 128.42, 127.76, 125.99, 124.71, 67.40, 39.94, 33.13, 30.65, 20.73; HPLC purity, 98.9%; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₂N₂O₃S, 347.1351; found, 347.1423.exact mass(monoisotopic) from spectrum 346.1350.

2-Methyl-N-(3-Phenylpropyl)-2-((4-(trifluoromethyl)phenyl)sulfonyl)propanamide (11).

Starting with 7c (48 mg), white solid (53 mg, 81%).¹H NMR (500 MHz, CDCl₃) δ 7.94 (d, J = 8 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.32–7.29 (m, 2H), 7.22–7.20 (m, 3H), 6.87 (br s, 1H), 3.33 (q, J = 6.5 Hz, 2H ), 2.71 (t, J = 6.5 Hz, 2H ), 1.93 (q, J = 6.5 Hz, 2H), 1.55 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 166.94, 141.03, 138.7, 136.06, 135.80, 130.68, 128.60, 128.37, 126.11(q, J =3.75 Hz), 126.19,121.93, 68.32, 40.02, 33.19, 30.71, 20.67; HPLC purity, 97.4%; HRMS (m/z): [M+H]⁺ calcd for C₂₀H₂₂F₃NO₃S, 414.1272; found, 414.1345, exact mass(monoisotopic) from spectrum 413.1727.

N-(3-Phenylpropyl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanamide (12).

Starting with 7d (50 mg), white solid (48 mg, 68%); ¹H NMR (500 MHz, CDCl₃) δ: 8.97 (s, 1 H), 8.19 (s, 2H), 7.30-7.26 (m, 2H), 7.21-7.16 (m, 3H), 6.58 (br s, 1H), 4.33 (q, J = 7.5 Hz, 1H), 3.32-3.28 (m, 2H), 2.66 (t, J = 7.5 Hz, 2H), 1.85 (p, J = 7.5 Hz, 2H), 1.55 (d, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 163.62, 158.79, 147.39, 147.36, 141.13, 135.81, 135.78, 130.22, 128.53, 128.35, 126.12, 123.22, 121.28, 62.55, 39.84, 33.02, 30.70, 11.47; HPLC purity, 98.3; ESIMS calcd for C₁₈H₁₉F₃N₂O₃S 401.42 [M+H]⁺. Found 401.01; HRMS (m/z): [M+H] calcd for C₁₈H₁₉F₃N₂O₃S, 401.1068; found, 401.1139, exact mass(monoisotopic) from spectrum 400.1067.

Alternative method for the synthesis of molecule 12:

N-(3-Phenylpropyl)-2-((5-(trifluoromethyl)pyridin-2-yl)thio)propanamide.

To a solution of 2-((5-(trifluoromethyl)pyridin-2-yl)thio)propanoic acid (0.05 g, 0.20 mmol) in dry THF (7 mL), PYBOP (0.08 g, 0.18 mmol) and DIPEA (70 μL, 0.5 mmol) were added and the mixture was stirred at room temperature for 15 min, Phenyl propylamine (28 μL, 0.20 mmol) was added. The reaction mixture was stirred at room temperature for 1h, thereafter, the organic solvent was evaporated under reduced pressure and the crude residue was purified by flash column chromatography to yield the desired product as yellow oil (55 mg, 75% ), ¹H NMR (500 MHz, CDCl₃) δ: 8.69 (s, 1 H), 7.73 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.28-7.24 (m, 2H), 7.19-7.16 (m, 1H), 7.08-7.06 (m, 2H), 4.47 (q, J = 7.5 Hz, 1H), 3.34-3.22 (m, 2H), 2.54 (t, J = 7.5 Hz, 2H), 1.82-1.76 (m, 2H), 1.60 (d, J = 7.5 Hz, 3H).

N-(3-Phenylpropyl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanamide (12).

To a stirred solution of 3-phenylpropyl 2-((5-(trifluoromethyl)pyridin-2-yl) thio) propanoate (55 mg, 0.15 mmol) in dioxane/water (5:1, 3 mL) in a 10 mL scintillation vial, was added in one portion, (160mg, 0.3 mmol) potassium mono persulfate (OXON^®). The obtained white suspension was stirred at room temperature for 16 h. After the reaction was completed as seen by TLC, the white solid was filtered off, and dioxane was removed under reduced pressure. Then, the aqueous solution was extracted with DCM (3×5mL). The collected organic layers were dried over sodium sulfate, removed under reduced pressure, and purified with flash column chromatography to afford the desired oxidized derivative as white crystals (33 mg, 56%).

2-((5-Chloropyridin-2-yl)sulfonyl)-2-methyl-N-(3-phenylpropyl)propanamide (13).

Starting with 6f (42 mg), white solid (55 mg, 88%). ¹H NMR (500 MHz, CDCl₃) δ 8.58 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.88–7.86 (dd, J = 8.5 Hz, J = 1.5 Hz, 1H), 7.29–7.26 (m, 2H), 7.20–7.17 (m, 3H), 7.04 (br s, 1H), 3.30 (q, J = 6.5 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H), 1.90 (q, J = 7.5 Hz, 2H), 1.59 (6H, s) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.46, 152.64, 149.31, 141.33, 137.51, 137.03, 128.52, 128.40, 126.06, 125.70, 67.65, 39.98, 33.12, 30.60, 20.62; HPLC purity, 98.5%; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₁ClN₂O₃S, 381.0961; found, 381.1033, exact mass(monoisotopic) from spectrum 380.0960.

2-Methyl-N-(3-phenylpropyl)-2-((5-(trifluoromethyl)pyridin-2-yl)thio)propanamide (14).

Starting with thioacid (50 mg), White solid (47 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 8.65 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.34 (br s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 2H), 3.26 (q, J = 7.5 Hz, 2H), 2.54 (t, J = 6.5 Hz, 2H), 1.76 (q, J = 7.5 Hz, 2H), 1.68 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 173.85, 162.41, 146.06, 146.01, 141.36, 133.14, 133.11, 128.43, 128.24, 125.97, 124.59, 123.39, 123.12, 122.85, 52.77, 39.59, 33.13, 30.91, 26.61; HPLC purity, 98.9%; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₂₁F₃N₂OS, 383.1327; found, 383.1397, exact mass(monoisotopic) from spectrum 382.1324. N.B: For this derivative, we hydrolyzed 100 mg of 5e using the same condition, followed by the reaction with phenyl propylamine.

General procedures for synthesis of 2-((Chlorophenyl)thio]ethan-1-amine (15 a-b).^{51, 52}

To a solution of sodium metal (97.0 mg, 4.22 mmol) in i-PrOH (15 mL), was added, an appropriate chlorothiophenol derivative (475 mg, 3.30 mmol), the reaction mixture was stirred at room temperature for 30 minutes. Then, 2-oxazolidinone (100 mg, 1.14 mmol) was added and the reaction was heated at reflux for 6 h. After reaction completion, the organic solvent was evaporated under vacuum, and the crude product was purified using flash column chromatography (DCM: Methanol 90:10) to afford the desired product as the following:

2-((2-Chlorophenyl)thio)ethan-1-amine (15a).

Colorless oil with fruity odor. (125 mg, 36%); ¹H NMR (500 MHz, CDCl₃) δ: 7.38-7.31 (dd, J = 7.0 Hz, 1H 2H), 7.22-7.18 (m, 1H), 7.13-7.10 (m, 1H), 3.03 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 1.70 (s, 2H).

2-((4-Chlorophenyl)thio)ethan-1-amine (15b).

Yellow oil with characteristic odor. (305 mg, 88.6%); ¹H NMR (500 MHz, CDCl₃) δ: 7.28 (d, J = 8.5 Hz, 2H), 7.229-7.23 (m ,4H), 2.90-2.89 (m, 2H), 1.54 (br s, 2H).

N-(2-((chlorophenyl)thio)ethyl)-2-methyl-2-(arylsulfonyl)propanamide (16-20).

To a solution of the appropriate carboxylic acid derivative (7a-e 0.16 – 0.22 mmol) in dry THF (5 mL), PYBOP (1.05 eq) and DIPEA (2.7 eq) were added followed by stirring at room temperature for 10 min under nitrogen atmosphere. The reaction mixture was then charged with an appropriate chlorophenyl thioethan-1-amine derivative (15 a-b 0.16 mmol) and stirred for 1h, after reaction completion, THF was evaporated under reduced pressure and the crude product was purified by flash column chromatography to yield the desired product:

N-(2-((2-chlorophenyl)thio)ethyl)-2-methyl-2-(phenylsulfonyl)propanamide (16).

Starting with 7a (50 mg) and 15a (40 mg), white solid (70 mg, 82%); ¹H NMR (500 MHz, CDCl₃) δ: 7.84 (d, J = 8.0 Hz, 2H), 7.64-7.52 (m, 1H), 7.50-7.44 (m, 2H), 7.44 - 7.37 (m, 3H include br s peak), 7.23-7.14 (m, 1H), 7.14-7.12 (m, 1H), 3.51 (q, J = 6.5 Hz, 2H), 3.11 (t, J = 6.5 Hz, 2H), 1.55 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.91, 135.00, 134.56, 134.41, 134.12, 130.12, 129.99, 129.85, 129.06, 127.43, 68.06, 39.19, 31.95, 20.85; HPLC purity, 95.3%; ESIMS calcd for C₁₈H₂₀ClNO₃S, 397.06, Found mass [M+H]⁺: 398.00; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₀ClNO₃S, 398.0573; found, 398.0647, exact mass(monoisotopic) from spectrum 397.0574.

N-(2-((4-Chlorophenyl)thio)ethyl)-2-methyl-2-(phenylsulfonyl)propanamide (17).

Starting with 7a (50 mg) and 15b (40 mg), white solid (67 mg, 77%) ESIMS calcd for C₁₈H₂₀ClNO₃S, 397.06, Found mass [MH+]: 398.00; ¹H NMR (500 MHz, CDCl₃) δ: 7.85 (d, J = 7.5 Hz, 2H), 7.68 (t, J = 7.5 Hz, 2H), 7.53 (t, J = 8.0 Hz, 2H), 7.40 (br s, 1H), 7.36 (d, J = 9.0 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 3.49 (q, J = 6.5 Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H), 1.56 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 167.85, 134.98, 134.43, 133.30, 132.77, 131.31, 130.08, 129.33, 129.07, 68.03, 39.26, 33.25, 20.87; HPLC purity, 98.7; ESIMS calcd for C₁₈H₂₀ClNO₃S, 397.06, Found mass [M+H]⁺: 398.00; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₀ClNO₃S, 398.0573; found, 398.0647, exact mass(monoisotopic) from spectrum 397.0575.

N-(2-((2-Chlorophenyl)thio)ethyl)-2-methyl-2((4-(trifluoromethyl)phenyl)sulfonyl) propanamide (18).

Starting with 7c (50 mg) and 15a (33 mg), white solid (69 mg, 87%) ¹H NMR (500 MHz, CDCl₃) δ: 7.98 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.43-7.38 (m, 2H), 7.30 (br s, 1H), 7.26-7.22 (m, 1H), 7.18-7.14. (m, 1H), 3.51 (q, J = 6.5 Hz, 2H), 3.12 (t, J = 6.5 Hz, 2H), 1.57 (s, 6H) ; ¹³C NMR (DMSO-d6) δ 167.46, 138.69, 134.85, 133.86, 130.78, 130.18, 130.09, 127.47, 126.15, 126.12, 68.35, 39.13, 32.27, 20.65; HPLC purity, 97%; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₁₉ClF₃NO₃S₂, 466.0447; found, 466.0522, exact mass(monoisotopic) from spectrum 465.0449.

N-(2-((4-Chlorophenyl)thio)ethyl)-2-methyl-2-((4-(trifluoromethyl)phenyl)sulfonyl) Propanamide (19).

Starting with 7c (50 mg) and 15b (33 mg), white crystals ( 65 mg, 82%); ¹H NMR (500 MHz, CDCl₃) δ: 7.96 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.24 (br s, 1H), 3.49 (q, J = 6.0 Hz, 2H), 3.08 (t, J = 6.5 Hz, 2H), 1.56 (s, 6H); ¹⁹F NMR δ −63.31 (s); ¹³C NMR (125 MHz, CDCl₃) δ 167.36, 138.64, 135.98 (q, J= 32.5 Hz), 133.14, 132.92, 131.36, 130.74, 129.38, 126.15 (q, J= 3.75 Hz), 124.08, 121.90, 68.32, 39.26, 33.33, 20.65; ¹⁹F NMR δ −63.31 (s) ; HPLC purity, 99.2%; ESIMS calcd for C₁₉H₁₉ClF₃NO₃S₂: 465.04, Found mass [M+H]⁺: 466.0; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₁₉ClF₃NO₃S₂, 466.0447; found, 466.0523, exact mass(monoisotopic) from spectrum 465.0450.

N-(2-((4-chlorophenyl)thio)ethyl)-2-methyl-2-)(5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propanamide (20).

Starting with 7e (50 mg) and 15b (31 mg), white solid ( 53 mg, 68%) ¹H NMR (500 MHz, CDCl₃) δ: 8.91 (s, 1H), 8.21-8.17 (m, 2H), 7.37 (br s, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 3.48 (q, J = 6.5 Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H), 1.62 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.09, 158.12, 147.16, 135.58, 133.44, 132.73, 131.25, 130.45, 130.18, 129.28, 124.47, 121.27, 67.59, 39.47, 33.09, 20.71; HPLC purity, 95%; ESIMS calcd for C₁₉H₁₉ClF₃NO₃S₂: 466.04, Found mass [M+H]⁺: 467.19; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₁₉ClF₃NO₃S₂, 467.0447; found, 467.1023.

Synthesis of 2-(4-Chlorophenoxy)ethan-1-amine (22c).

Starting from 2-bromo ethan-1-amine 21 (500 mg, 2.45 mmol), the synthesis of 23c was carried out over three successive steps:

Step 1: synthesis of tert-butyl (2-Bromoethyl)carbamate.

To a stirred solution of 2-bromo ethan-1-amine 21 (500 mg, 2.45 mmol) in DCM (15 mL) was added triethylamine (0.45 mL). The solution was cooled with ice bath before a solution of di-tert-butyl dicarbonate (640 mg, 2.9 mmol) in DMC (5mL) was added dropwise over 15 min. The mixture was stirred at 23 °C for 12 h. After reaction completion (as seen by TLC) , the system was quenched with brine (15 mL), the organic layer washed with water (3×15mL), dried over anhydrous Na₂SO_4, and evaporated under reduced pressure to afford the N-protected molecule as a yellowish oil (480 mg, 52%) ¹H NMR (500 MHz, CDCl₃) δ: 5.01 (br s, 1H), 3.50-3.41 (m, 2H), 3.42 (t, J = 5.5 Hz, 2H), 1.41 (s, 12H).

Step 2: synthesis of tert-butyl (2-(4-Chlorophenoxy)ethyl)carbamate.

p-Chlorophenol (250 mg, 1.95 mmol) in DMF (1mL) was added gradually to a stirred solution of tert-butyl (2-bromoethyl) carbamate (300 mg, 1.33 mmol) followed by oven-dried potassium carbonate (280 mg, 2.0 mmol) in DMF (3mL). The mixture was stirred at 23 °C for 24 h before the flask content was poured over ice-cold ether (20 mL). The ether layer was washed with 2M NaOH solution (15 mL), Water (15 mL), and brine solution (15 mL). The ether layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography to afford the desired product as colorless oil (155 mg, 43%). ¹H NMR (500 MHz, CDCl₃) δ: 7.44 (d, J = 12 Hz, 2H), 6.79 (d, J = 12 Hz, 2H), 5.02 (br s, 1H), 3.96 (t, J = 5 Hz, 2H), 3.50-3.43 (m, 2H), 1.43 (s, 12H).

2-(4-Chlorophenoxy)ethan-1-amine (22c).

To a stirred solution of the tert-butyl (2-(4-chlorophenoxy)ethyl)carbamate (155 mg, 0.5 mmol) in DMC (3 mL) was added dropwise over 30 min, trifluoroacetic acid (2.5 mL). The reaction mixture was stirred at 23 °C for 18 h. Then, the solvent was evaporated, and the crude product was dissolved in ethyl acetate (15 mL) and washed with 1M NaOH aqueous solution (10 mL). The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to afford the desired product as white crystals (80 mg, 82%). ¹H NMR (500 MHz, CDCl₃) δ: 7.22 (d, J = 9 Hz, 2H), 6.82 (d, J = 9 Hz, 2H),3.95 (t, J = 5 Hz, 2H), 3.07 (t, J = 5 Hz, 2H), 1.96 (br s, 2H).

General procedures for the synthesis of 2-methyl-N-(2-aryloxyethyl)-2-(arylsulfonyl)propanamide (23-31).

A solution of 2-methyl-2-(arylsulfonyl)propanoic acid 7a,c,f (0.16 – 0.22 mmol) in dry THF (10 mL) in a 50 mL foil-wrapped rounded-bottom flask (to protect the amine derivatives from light as recommended), was treated with a solution of PyBOP (87 mg, 0.17 mmol) in THF (1 mL) followed by addition of DIPEA (83 μL). The mixture was stirred at room temperature for 10 min. A solution of the appropriate phenoxyethan-1-amine derivative (0.16 mmol) in THF (1 mL) was added and the reaction mixture was allowed to stir at the same temperature for 1 h. The solvent was removed in vacuo, and the crude product was purified by flash column chromatography using ethyl acetate-hexanes (1:3) as eluent. The physical characters and the spectral data of the separated products are listed below:

2-Methyl-N-(2-phenoxyethyl)-2-(phenylsulfonyl)propanamide (23).

Starting with 7a (50 mg, 0.22 mmol), white crystals (44 mg, 57%); ¹H NMR (500 MHz, CDCl₃) δ: 7.82 (d, J = 7.5 Hz, 2H), 7.63 (br s, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.34-7.32 (m, 2H), 7.29-7.27 (m, 2H), 6.97 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 4.06 (t, J = 5.0 Hz, 2H), 3.70 (q, J = 5.5 Hz, 2H), 1.60 (s, 6H); ¹³C NMR (500 MHz, CDCl₃) δ 168.09, 158.39, 135.09, 134.27, 130.06, 129.69, 129.00, 121.36, 114.56, 67.97, 65.98, 40.01, 20.71; HPLC purity, 100%; ESIMS calcd for C₁₈H₂₁NO₄S: 347.12, Found mass [M+H]⁺: 348.08, [M+Na]⁺: 370.06; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₁NO₄S, 348.1191; found, 348.1263, exact mass(monoisotopic) from spectrum 347.1190.

2-Methyl-N-(2-Phenoxyethyl)-2-))4-(trifluoromethyl)phenyl(sulfonyl(propenamide (24).

Starting with 7c (50 mg, 0.16 mmol), white solid (38 mg, 54%); ¹H NMR (500 MHz, CDCl₃) δ: 7.94 (d, J = 8.0 Hz, 2H), 7.52 (br s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.0 Hz, 2H), 4.06 (t, J = 5.0 Hz, 2H), 3.70 (q, J = 5.5 Hz, 2H), 1.64 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.64, 158.26, 138.69, 135.91 (q, J = 32.5 Hz), 130.68, 129.81, 126.10 (q, J = 3.75 Hz), 124.47, 67.59, 39.47, 33.09, 20.71; HPLC purity, 100%; ESIMS calcd for C₁₉H₂₀F₃NO₄S: 415.11, Found mass [M+Na]⁺: 438.05; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₂₀F₃NO₄S, 416.1065; found, 416.1137, exact mass(monoisotopic) from spectrum 415.1064.

2-Methyl-N-(2-Phenoxyethyl)-2-))5-(trifluoromethyl)pyridin-2-yl(sulfonyl(propenamide (25).

Starting with 7e (50 mg, 0.16 mmol), white solid (60 mg, 85%); ¹H NMR (500 MHz, CDCl₃) δ: 8.79 (s, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.51 (br s, 1H), 7.30-7.27 (m, 2H), 7.00-6.97 (m, 1H), 6.88 (d, J = 8.0 Hz, 2H), 4.01 (t, J = 5.0 Hz, 2H), 3.69 (q, J = 5.5 Hz, 2H), 1.70 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.84, 158.46, 158.19, 147.05, 147.02 135.47, 147.02, 130.14 (q, J = 33.75 Hz), 124.35, 121.33, 114.51, 67.83, 66.05, 39.98, 20.55; HPLC purity, 98.5%; ESIMS calcd for C₁₈H₁₉F₃N₂O₄S: 416.10, Found mass [M+H]⁺: 417.10, [M+Na]⁺: 439.04; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₁₉F₃N₂O₄S, 417.1018; found, 417.1090, exact mass(monoisotopic) from spectrum 416.1018.

N-(2-(2-Chlorophenoxy)ethyl)-2-methyl-2-(Phenylsulfonyl)propanamide (26).

Starting with 7a (50 mg, 0.22 mmol), white solid ( 40 mg, 48%); ¹H NMR (500 MHz, CDCl₃) δ: 7.85 (d, J = 7.5 Hz, 2H), 7.60 (br s, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.40 (d, J = 7.5 Hz, 1H), 7.35 (m, 2H), 7.24 (m, 1H), 6.97-6.93 (m, 2H), 4.12 (t, J = 5.0 Hz, 2H), 3.74 (q, J = 5.0 Hz, 2H), 1.60 (s, 6H); ¹³C NMR (500 MHz, CDCl₃) δ 168.13, 153.95, 135.15, 134.26, 130.53, 130.14, 128.93, 127.85, 123.26, 122.14, 113.69, 68.02, 67.34, 39.88, 20.72; HPLC purity, 100%; ESIMS calcd for C₁₈H₂₀ClNO₄S: 381.08, Found mass [M+H]⁺: 382.05, [M+Na]⁺: 404.03; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₀ClNO₄S, 382.0802, exact mass(monoisotopic) from spectrum 381.0800.

N-(2-(2-Chlorophenoxy)ethyl)-2-methyl-2-((4-(trifluoromethyl)phenyl)sulfonyl) propanamide (27).

Starting with 7c (50 mg, 0.16 mmol), white solid (53 mg, 70%); ¹H NMR (500 MHz, CDCl₃) δ: 7.98 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.52 (br s, 1H), 7.42-7.40 (m, 1H), 7.27-7.23 (m, 1H), 6.99-6.96 (m, 1H), 6.94-6.92 (m, 1H), 4.12 (t, J = 5.0 Hz, 2H), 3.73 (q, J = 5.0 Hz, 2H), 1.62 (s, 6H) ; ¹³C NMR (500 MHz, CDCl₃) δ 167.69, 153.82, 138.81, 135.90 (q, J = 32.5 Hz), 130.77, 130.59, 127.95, 125.97, 124.05, 123.17, 122.31, 113.66, 68.31, 67.24, 40.03, 20.52; HPLC purity, 97.4%; ESIMS calcd for C₁₉H₁₉ClF₃NO₄S: 449.07, Found mass [M+H]⁺: 450.01, [M+Na]⁺: 472.00; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₁₉ClF₃NO₄S, 450.0675; found, 450.0751, exact mass(monoisotopic) from spectrum 449.0678.

N-(2-(2-Chlorophenoxy)ethyl)-2-methyl-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propanamide (28).

Starting with 7e (50 mg, 0.16 mmol), white solid (64 mg, 84%); 1H NMR (500 MHz, CDCl3) δ: 8.75 (s, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.51 (br s, 1H), 7.38-7.36 (m, 2H), 7.22 (t, J = 7.5 Hz, 1H), 6.96-6.92 (m, 1H), 6.81 (d, J = 8.0 Hz, 1H), 4.09 (t, J = 5.0 Hz, 2H), 3.73 (q, J = 5.5 Hz, 2H), 1.70 (s, 6H) ; ¹³C NMR (DMSO-d₆) δ 167.94, 158.44, 153.93, 146.97, 146.94, 135.41, 130.49, 129.91, 127.87, 124.40, 123.16, 122.17, 113.70, 67.95, 67.35, 39.86, 20.56; HPLC purity, 97.8%; ESIMS calcd for C₁₈H₁₈ClF₃N₂O₄S: 450.06, Found mass [M+H]⁺: 451.05; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₁₈ClF₃N₂O₄S, 451.0704; found, 450.0751, exact mass(monoisotopic) from spectrum 450.0631.

N-(2-(4-Chlorophenoxy)ethyl)-2-methyl-2-(phenylsulfonyl)propanamide (29).

Starting with 7a (50 mg, 0.22 mmol), white solid ( 21 mg, 25%); ¹H NMR (500 MHz, CDCl₃) δ: 8.12 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 7.5 Hz, 2H), 7.33 (d, J = 7.5 Hz, 2H), 7.28-7.26 (m, 2H), 6.88 (d, J = 10.5 Hz, 2H), 4.03 (t, J = 5.0 Hz, 2H), 3.68 (q, J = 5.0 Hz, 2H), 1.58 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 168.11, 157.01, 135.05, 134.36, 130.03, 129.57, 129.02, 126.30, 115.85, 67.97, 66.44, 39.87, 20.77; HPLC purity, 99.5%; ESIMS calcd for C₁₈H₂₀ClNO₄S: 381.08, Found mass [M+H]⁺: 382.20; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₀ClNO₄S, 382.0802; found, 382.0873, exact mass(monoisotopic) from spectrum 381.0800.

N-(2-(4-Chlorophenoxy)ethyl)-2-methyl-2-((4-(trifluoromethyl)Phenyl)sulfonyl) propanamide (30).

Starting with 7c (50 mg, 0.16 mmol), white solid ( 54 mg, 71%); ¹H NMR (500 MHz, CDCl₃) δ: 7.94 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.45 (br s, 1H), 7.29-7.26 (m, 2H), 6.90-6.87 (m, 2H), 4.03 (t, J = 5.0 Hz, 2H), 3.68 (q, J = 5.5 Hz, 2H), 1.60 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 167.60, 156.90, 138.69, 135.65 (q, J = 32.5 Hz), , 130.67, 129.64, 126.52, 126.11, 124.01, 115.84, 68.27, 66.37, 39.99, 20.58; ¹⁹F NMR δ −63.41 (s); HPLC purity, 98.7%; ESIMS calcd for C₁₉H₁₉ClF₃NO₄S: 449.07, Found mass [M+H]⁺: 450.03; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₁₉ClF₃NO₄S, 450.0675; found, 450.0750, exact mass(monoisotopic) from spectrum 449.0677.

N-(2-(4-Chlorophenoxy)ethyl)-2-methyl-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propenamide (31).

Starting with 7e (50 mg, 0.16 mmol), white solid ( 43 mg, 57%) ¹H NMR (500 MHz, CDCl₃) δ: 8.76 (s, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.49 (br s, 1H), 7.22 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 4.02 (t, J = 5.0 Hz, 2H), 3.68 (q, J = 5.0 Hz, 2H), 1.66 (s, 6H) ; ¹³C NMR (125 MHz, CDCl₃) δ 167.90, 158.22, 157.11, 147.04, 135.47, 129.87 (q, J = 33.75 Hz), 129.46, 126.25, 124.25, 123.38 115.85, 67.64, 66.51, 39.84, 20.54; HPLC purity, 97.8%; ESIMS calcd for C₁₈H₁₈ClF₃N₂O₄S: 450.06, Found mass [M+H]⁺: 451.01, [M+Na]⁺: 473.01; HRMS (m/z): [M+H]⁺ calcd for C₁₈H₁₈ClF₃N₂O₄S, 451.0628; found, 451.0703, exact mass(monoisotopic) from spectrum 450.0630.

General procedure for synthesis of 1-(4- arylpiperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)alkyl-1-one (33-45).

To a stirred solution of the appropriate carboxylic acid derivative ( 7d,e 50 mg, 0.16 – 0.17 mmol) in dry THF (10 mL) were added, a solution of PyBOP (87 mg, 0.17 mmol) in THF (1 mL) followed by DIPEA (83 μL). Then, the mixture was stirred at room temperature for 10 min under nitrogen gas before a solution of the appropriate 4-aryl piperazine (0.16 – 0.17 mmol) in THF (1 mL) was added. The result yellow solution was allowed to stir at room temperature for 1 h. The solvent was removed under reduced pressure, and the crude product was absorbed onto silica gel and purified by flash column chromatography using ethyl acetate/ hexane as eluent to afford the desired product as the following:

2-methyl-1-(4-phenylpiperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propan-1-one (33).

Starting with 7e (50 mg, 0.16 mmol) and 1-phenylpiperazine (25 μL, 0.16 mmol), off-white solid (42 mg, 61%); ¹H NMR (500 MHz, CDCl₃) δ: 8.94 (s, 1 H), 8.23 (d, J = 8.5 Hz, 1H), 8.19 (d, J = 12.0 Hz, 1H), 7.29 (d, J = 7.5 Hz, 2H), 6.95-6.92 (m, 3H), 3.91 (s, 4H), 3.26 (s, 4H), 1.87 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.62, 160.05, 146.83, 135.41, 135.40, 129.98, 129.71, 124.57, 123.75, 121.57, 120.68, 116.61, 71.54, 49.48, 45.75, 22.90; HPLC purity, 97.49%; ESIMS calcd for C₂₀H₂₂F₃N₂O₃S: 441.13, Found mass [M+H]⁺: 442.50; HRMS (m/z): [M+H]⁺ calcd for C₂₀H₂₂F₃N₂O₃S, 442.1334; found, 442.1404, exact mass(monoisotopic) from spectrum 441.1331.

2-methyl-1-(4-(pyridin-2-yl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (34).

Starting with 7e (50 mg, 0.16 mmol) and 1-(pyridin-2-yl)piperazine (28 μL, 0.16 mmol), off-white solid (57 mg, 81%); ¹H NMR (500 MHz, CDCl₃) δ: 8.94 (s, 1 H), 8.24-8.17 (m, 3H), 7.53 (d, J = 7.0 Hz, 1H), 6.70-6.66 (m, 2H), 3.88 (s, 4H), 3.66-3.65 (m, 4H), 1.87 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.62, 159.93, 158.76, 147.62, 146.70, 137.98, 135.30, 129.71 (q, J = 33.75 Hz), 124.45, 123.64, 114.00, 107.45, 71.44, 45.19, 22.73; HPLC purity, 97.69%; ESIMS calcd for C₁₉H₂₁F₃N₄O₃S: 442.13, Found mass [M+H]⁺: 443.39; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₂₁F₃N₄O₃S, 443.1286; found, 443.1357, exact mass(monoisotopic) from spectrum 442.1284.

2-methyl-1-(4-(o-tolyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propan-1-one (35).

Starting with 7e (50 mg, 0.16 mmol) and 1-(o-tolyl)piperazine (30 μL, 0.16 mmol), white solid (40 mg, 56%); ¹H NMR (500 MHz, CDCl₃) δ: 8.95 (s, 1 H), 8.26 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 7.20-7.15 (m, 2H), 7.03-6.99 (m, 2H), 3.87 (s, 4H), 2.95 (s, 3H), 2.32 (s, 3H), 1.90 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.75, 160.47, 150.60, 146.66, 135.26, 132.71, 131.25, 129.75, 129.48, 126.73, 124.40, 123.88, 119.16, 71.84, 51.77, 22.76, 17.83; HPLC purity 97.9%; ESIMS calcd for C₂₁H₂₄F₃N₂O₃S: 455.16, Found mass [M+H]⁺: 456.53; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₄F₃N₂O₃S, 456.1490; found, 456.1561, exact mass(monoisotopic) from spectrum 455.1489.

1-(4-(2,5-dimethylphenyl)piperazin-1-yl)-2-methyl-2-((5(trifluoromethyl)pyridin-2-yl)sulfonyl)propan-1-one (36).

Starting with 7e (50 mg, 0.16 mmol) and 1-(2,5-dimethylphenyl) piperazine (32 μL, 0.16 mmol), white solid (65 mg, 88%); ¹H NMR (500 MHz, CDCl₃) δ: 8.96 (s, 1 H), 8.26 (d, J = 8.5 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 6.82 (m, 2H), 3.86 (s, 4H), 2.95 (s, 4H), 2.30 (s, 3H), 2.27 (s, 3H), 1.90 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.73, 160.51, 150.43, 146.64, 136.33, 135.26, 131.05, 129.39, 124.49, 124.39, 123.70, 119.92, 71.87, 51.78, 22.76, 21.15, 17.41; HPLC purity 98.9%; ESIMS calcd for C₂₂H₂₆F₃N₃O₃S: 469.16, Found mass [M+H]⁺: 470.24; HRMS (m/z): [M+H]⁺ calcd for C₂₂H₂₆F₃N₃O₃S, 470.1647; found, 470.1719, exact mass(monoisotopic) from spectrum 469.1647.

1-(4-(3,4-dichlorophenyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (37).

Starting with 7e (50 mg, 0.16 mmol) and 1-(3,4-dichlorophenyl) piperazine (39 mg, 0.16 mmol), white crystals (53 mg, 61.5%); ¹H NMR (500 MHz, CDCl₃) δ: 8.91 (s, 4 H), 8.21-8.19 (m, 2H), 7.30-7.28 (m, J = 9.0 Hz,1H), 6.96 (s, 1H), 6.76-6.73 (m, 1H), 3.99 (m, 4H), 3.25-3.23 (m, 4H), 1.83 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.38, 159.48, 150.12, 146.82, 135.34, 133.00, 130.63, 130.03, 129.76, 124.49, 123.10, 117.64, 115.65, 71.04, 48.78, 45.50, 22.90; HPLC purity 98.1%; ESIMS calcd for C₂₀H₂₀Cl₂F₃N₃O₃S: 509.06, Found mass [M+H]⁺: 510.16; HRMS (m/z): [M+H]⁺ calcd for C₂₀H₂₀Cl₂F₃N₃O₃S, 510.0555; found, 510.0636, exact mass(monoisotopic) from spectrum 509.0564.

1-(4-(3-methoxyphenyl)piperazin-1-yl)-2-methyl-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propan-1-one (38).

Starting with 7e (50 mg, 0.16 mmol) and 1-(m-methoxyphenyl) piperazine (30 μL, 0.16 mmol), white solid (80 mg, 97.5%); ¹H NMR (500 MHz, CDCl₃) δ: 8.93 (s, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.18 (t, J = 8.5 Hz, 1H), 6.54-6.52 (m, 1H), 6.46-6.45 (m, 2H), 3.88 (s, 4H), 3.78 (s, 3H), 3.25-3.24 (s, 4H), 1.86 (s, 6H); ¹⁹F NMR δ −62.62 (s) ; ¹³C NMR (125 MHz, CDCl₃) δ 166.51, 160.69, 159.94, 152.09, 146.73, 146.70 135.28, 135.25, 129.99, 124.44, 109.06, 105.22, 102.90, 71.42, 55.24, 49.20, 22.79; HPLC purity 97.5%; ESIMS calcd for C₂₁H₂₄F₃N₃O₄S :471.14, Found mass [M+H]⁺: 472.14; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₄F₃N₃O₄S, 472.1439; found, 472.1511, exact mass(monoisotopic) from spectrum 471.1439.

2-methyl-1-(4-(4-fluorobenzyl)piperazin-1-yl]2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (39).

Starting with 7e (50 mg, 0.16 mmol) and 1-(4-fluorobenzyl)piperazine (33 mg, 0.16 mmol), white solid (42 mg, 53%); ¹H NMR (500 MHz, CDCl₃) δ: 8.91 (s, 1 H), 8.22 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.28-7.25 (m, 2H), 7.27 (t, J = 8.5 Hz, 2H), 3.70 (s, 4H), 3.48 (s, 2H), 2.47 (s, 4H), 1.87 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.54, 161.18, 160.49, 146.58, 135.24, 133.12, 130.66, 130.59, 124.37, 115.29, 115.12, 71.83, 61.92, 52.74, 22.63; ¹⁹F NMR δ −62.61, −70.08, −71.60 ; HPLC purity, 97.9%; ESIMS calcd for C₂₁H₂₃F₄N₂O₃S: 473.14, Found mass [M+H]⁺: 474.30; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₃F₄N₂O₃S, 474.1396; found, 474.1467, exact mass(monoisotopic) from spectrum 4731394.

2-methyl-1-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (40).

Starting with 7e (50 mg, 0.16 mmol) and 1-(4-chlorobenzyl)piperazine (30 μL, 0.16 mmol), white solid (69 mg, 84.1%); ¹H NMR (500 MHz, CDCl₃) δ: 8.91 (s, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.31-7.28 (m, 4H), 3.73 (s, 4H), 3.52 (s, 2H), 2.51 (s, 3H), 2.170 (s, 2H), 1.83 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.54, 160.51, 146.57, 136.10, 135.26, 133.03, 130.36, 129.52 (q, J = 33.75 Hz), 128.51, 124.37, 123.69, 121.51, 71.84, 61.94, 52.78, 45.62, 22.61; HPLC purity, 99.3%; ESIMS calcd for C₂₁H₂₃ClF₃N₃O₃S: 489.11, Found mass [M+H]⁺: 490.14; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₃ClF₃N₃O₃S, 490.1101; found, 490.1175, exact mass(monoisotopic) from spectrum 489.1102.

2-methyl-1-(4-(3-chlorobenzyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (41).

Starting with 7e (50 mg, 0.16 mmol) and 1-(3-chlorobenzyl)piperazine (30 μL, 0.16 mmol), white solid (72 mg, 88%); ¹H NMR (500 MHz, CDCl₃) δ: 8.91 (s, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.24-7.17 (m, 3H), 3.72-3.67 (m, 4H), 3.49 (s, 2H), 2.49-2.47 (m, 4H), 1.84 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.56, 160.50, 146.59, 144.60, 139.87, 135.21, 134.34, 129.63, 129.00, 127.54, 127.13, 124.36, 71.81, 62.11, 55.91, 52.83, 22.65; HPLC purity, 98.87%; ESIMS calcd for C₂₁H₂₃ClF₃N₃O₃S: 489.11, Found mass [M+H]⁺: 490.17; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₃ClF₃N₃O₃S, 490.1101; found, 490.1173, exact mass(monoisotopic) from spectrum 489.1100.

2-methyl-1-(4-(4-methylbenzyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (42).

Starting with 7e (50 mg, 0.16 mmol) and 1-(4-methylbenzyl)piperazine (32 mg, 0.16 mmol), white solid (40 mg, 51%); ¹H NMR (500 MHz, CDCl₃) δ: 8.86 (s, 1 H), 8.22 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 3.73 (s, 4H), 3.55 (s, 2H), 2.53 (s, 4H), 2.34 (s, 3H), 1.84 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 166.59, 160.64, 146.53, 137.04, 135.22, 134.17, 129.18, 129.05, 124.37, 123.70, 121.53, 71.98, 62.47, 52.71, 45.70, 22.55, 21.11; HPLC purity, 95.9%; ESIMS calcd for C₂₂H₂₆F₃N₃O₃S: 469.16, Found mass [M+H]⁺: 470.58; HRMS (m/z): [M+H]⁺ calcd for C₂₂H₂₆F₃N₃O₃S, 470.1647; found, 470.1722, exact mass(monoisotopic) from spectrum 4691649.

1-(4-(o-tolyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propan-1-one (43).

Starting with 7d (50 mg, 0.17 mmol) and 1-(o-tolyl)piperazine (33 μL, 0.17 mmol), yellowish white solid (52 mg, 67%); ¹H NMR (500 MHz, CDCl₃) δ: 9.01 (s, 1 H), 8.24 (s, 2H), 7.19 (m, 2H), 7.01 (d, J = 7.5 Hz, 2H), 4.97 (q, J = 7.0 Hz, 1H), 3.96-3.71 (m, 4H), 3.15-2.93 (m, 4H), 2.36 (s, 3H), 1.62 (d, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 163.32, 159.20, 150.53, 147.01, 135.72, 132.72, 131.24, 130.10 (q, J = 33.75 Hz), 126.75, 123.94, 123.61, 121.43, 119.27, 58.80, 51.94, 51.53, 47.33, 43.23, 17.81, 13.37; HPLC purity, 98.3%; ESIMS calcd for C₁₉H₂₂F₃N₃O₃S: 441.13, Found mass [M+H]⁺: 442.01; HRMS (m/z): [M+H]⁺ calcd for C₁₉H₂₂F₃N₃O₃S, 442.1334; found, 442.1406, exact mass(monoisotopic) from spectrum 441.1334.

1-(4-(2,5-dimethylphenyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (44).

Starting with 7d (50 mg, 0.17 mmol) and 1-(2,5-dimethylphenyl)piperazine (35 μL, 0.17 mmol), white solid (51 mg, 64%) ¹H NMR (500 MHz, CDCl₃) δ: 9.01 (s, 1 H), 8.23 (s, 2H), 7.19 (d, J = 7.5 Hz, 1H), 7.06-7.03 (m, 2H), 4.97 (q, J = 7.0 Hz, 1H), 3.96-3.68 (m, 4H), 3.13-2.90 (m, 4H), 2.30 (s, 6H), 1.62 (d, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 163.28, 159.20, 150.38, 147.02, 136.36, 135.67, 131.04, 130.25, 129.98, 129.41, 124.59, 123.94, 120.04, 58.83, 51.93, 51.56, 47.36, 43.26, 21.13, 17.38, 13.36; HPLC purity, 99.7%; ESIMS calcd for C₂₁H₂₄F₃N₃O₃S: 455.15, Found mass [M+H]⁺: 456.17; HRMS (m/z): [M+H]⁺ calcd for C₂₁H₂₄F₃N₃O₃S, 456.1490; found, 456.1560, exact mass(monoisotopic) from spectrum 455.1487.

1-(4-(3,4-dichlorophenyl)piperazin-1-yl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl) propan-1-one (45).

Starting with 7d (50 mg, 0.17 mmol) and 1-(3,4-dichlorophenyl)piperazine (40 mg, 0.17 mmol), shiny white crystals (31 mg, 58%) ¹H NMR (500 MHz, CDCl₃) δ: 9.00 (s, 1 H), 8.42-8.20 (m, 2H), 7.30 (d, J = 9.0 Hz, 1H), 6.97 (s, 1H), 6.75 (dd, J = 6.0 Hz, 1H), 4.97 (q, J = 7.0 Hz, 1H), 4.03-3.11 (m, 8H), 1.58 (d, J = 8.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 162.96, 158.88, 150.05, 147.12, 135.78, 133.00, 130.66, 123.86, 123.54, 123.31, 121.36, 117.94, 115.92, 58.44, 49.06, 48.78, 46.33, 42.37, 13.38; HPLC purity (methanol/water, 1:1), 95.4%; ESIMS [M+H]⁺ calcd for C₁₉H₁₈C_l2F₃N₃O₃S: 497.04, Found mass [M+H]⁺: 497.15; HRMS (m/z): [M+H] calcd for C₂₁H₂₄F₃N₃O₃S, 496.0398; found, 496.0474, exact mass(monoisotopic) from spectrum 495.0401.

Biology.

Cell culture, Chlamydia trachomatis propagation, and IFU assay:

The human epithelial cell line HEp-2 was routinely propagated in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 2% L-glutamine and 10% fetal bovine serum (FBS) at 37 °C with % CO_2. All infected and uninfected cell cultures were incubated at these conditions. Compounds stocks of the synthesized compounds were prepared in sterile dimethyl sulfoxide (50 mg/mL) and frozen at −20 °C in 5 μL aliquots.⁴¹

To quantify the effect of the most active compounds on C. trachomatis, HEp-2 cells were first seeded in a 24-well plate. Cells were infected with C. trachomatis L2 at a multiplicity of infection of 1, and the tested compounds were added in triplicate 8 hours post infection (hpi). At 24 hpi, cell lysates, which contain C. trachomatis elementary bodies, were collected from infected HEp-2 cell cultures in sucrose storage medium. The resulting mixture was frozen at −80 °C and used to infect a fresh HEp2 cell monolayer in a series of 10-fold dilutions. After a further 24hpi, the recoverable EBs from the initial infection were stained using primary goat anti-MOMP antibody and a secondary donkey anti-goat antibody labelled with Alexa488. The fluorescent inclusions were counted from 15 fields of view (FOV) at 20x magnification. IFUs were calculated as the average count of inclusions in each FOV corrected for the dilution factor.^{41, 87} In mechanism of action assays, we followed the same protocol described in the IFU assay. For (24h reactivation) samples, the DMEM medium was removed after 24hpi, and the wells were washed three times with Hanks’ balanced salt solution, and fresh medium without compounds was added (0.5 mL/well).

Casein degradation assay:

All recombinant, C-terminal 6X His-tagged ClpP proteins were expressed using E. coli and purified as previously described using a clpAPX-null E. coli strain kindly provided by Dr. Peter Sass.^{41, 42} The clpP human (NM_006012.4) and mouse (NM_017393.2) mRNA sequences were used to obtain the ClpP-encoding ORF minus the mitochondrial localization signal. Genes were then codon optimized for E. coli expression and synthesized by Integrated DNA Technologies. The mouse and human clpP paralogs were cloned into the pLATE31 vector (Thermo Scientific) as previously performed for the bacterial ClpP’s. Example purified protein samples are shown in Figure S2. FITC-casein (Sigma-Aldrich, C0528) was treated with Zeba 7K cutoff spin columns (Thermo Scientific) to remove free FITC. Assays were carried out in buffer PZ (25 mM HEPES [pH 7.6], 200 mM KCl, 5 mM MgCl₂, 1 mM DTT, and 10% v/v of glycerol) using 100 μL reactions and 20 μM FITC-casein. 1 μM or 0.1 μM (for 40) of ClpP from E. coli, 1 μM mouse or human ClpP, or 6 μM of ClpP1 and ClpP2 were preincubated with either 25 μg/μl compounds or DMSO solvent at 32°C for 30 minutes before adding FITC-casein. Reactions were monitored for 3 hours with readings every 3 minutes on a Tecan m Plex plate reader using an excitation wavelength of λex = 490 nm and an emission wavelength λem = 525 nm. Reactions were run at least three times using at least two independent protein purification preparations

Antibacterial assay against additional strains:

S. aureus USA 300 JE2, and E. coli K12 were provided by Dr. Bayles’ research lab at the Department of Pathology and Microbiology of the University of Nebraska Medical Center—UNMC. The MICs of the synthesized compounds were tested in triplicate samples, using the broth microdilution method as previously reported.⁸⁸. The bacterial cultures were made in Muller Hinton Broth (MHB) using the direct colony suspension method at 1.5 X 10⁸ CFU/mL followed by dilution to ~ 10⁵ CFU/mL. The stock solutions of the tested compounds were prepared in sterile DMSO at 1 mg/mL concentration. Then, a serial dilution of the tested compounds were made in MHB, in Cellstar 96-well microtiter plates using vancomycin as a positive control and blank media as a negative control. 10 μL of bacterial culture was added per well followed by plates incubation for 16 h at optimum temperature. The MIC was categorized as the concentration at which no visible growth of bacteria was observed at 600 nm in a particular well using an AccuSkan, MultiSkan FC. The average of triplicate MIC determinations is reported (for further details please see the SI).

Cytotoxicity assay (CCK-8):

The immortal keratinocyte cell line (HaCaT) and HeLa 229 were cultured in DMEM media containing 10% FBS and 1% Penicillin/Streptomycin solution at 37 °C with 5% CO₂. The cells were seeded separately at a density of 5000 cell per well in 96-well plates. On the next day, cells were treated with 50 μg/mL of the tested drugs in triplicates and as controls we used DMSO at a concentration equivalent to the one used in drug-treated cells. Then, the plates were incubated for additional 24 h before the addition of 10 μL of the assay reagent Cell Counting Kit-8 (CCK-8) (DOJINDO laboratories) followed by incubation of the plates for 2 h. Corrected absorbance readings were determined with a 450 nm filter using a multiskan ^™ FC microplate photometer (Thermo Fischer Scientific, US).

Mutagenic Studies – SMART:

We assessed the mutagenicity of the compounds by Somatic Mutation and Recombination Test – SMART using wing-somatic cells of Drosophila melanogaster. ^{89, 90} The following 3 crosses of mutant flies were set up: 1) Standard cross (ST): flare-3 (flr³) virgin females, with genetic constituent flr³/In(3LR)TM3, ri p^p se^p I(3)89Aa bx^34e e Bd^S crossed with multiple wing hairs (mwh) males, with genetic constituent y; mwh jv and, 2) High bioactivation (HB) cross: ORR; flare (ORR; flr3) virgin female, with genetic constituent ORR; flr³ / In (3LR) TM3, ri pp sep I(3)89 Aa bx 34e Bd^S crossed with mwh males. The ORR;flr3 strain carries the chromosomes 1 and 2 from a DDT-resistant Oregon R (R) line, which contain genes responsible for the high level of metabolizing enzymes of the cytochrome P450, P(CYP)6 A2 type.⁸⁹

Eggs were collected from flies of the two different crosses in culture flasks containing a solid agar-agar base (5% w/v), covered by a layer of live baker`s yeast supplemented with sugar for 8 hours. Third instar larvae (72 ± 4 h) were washed out of the culture bottles with tap water and collected with a fine meshed strainer. Larvae groups were transferred to glass vials containing hydrated alternative medium (instant mashed potato flakes Yoki^®) and exposed to compounds 11, 20, 24, 26, 40 and 41 at 0.25, 0.5, and 1.0 mM final concentrations. Solvent (Milli-Q water, 1% of Tween-80 and 3% ethanol) was used as a negative control.

The hatched flies of the marker heterozygote (mwh +/+ flr3) and the balancer heterozygote (mwh +/+ TM3, Bds) genotypes were collected and fixed in 70% ethanol. Wings were removed, mounted on slides containing Faure’s solution (30 g of gum Arabic, 50 g of chloral hydrate, 20 mL of glycerol, and 50 mL of water) and analyzed under optical microscope (400×) for the occurrence of different types of mutant spots.⁹¹

The statistical analysis was performed as described by Frei and Würgler,⁹⁰ using the chi-squared test. Results were considered statistically significant when p < 0.05. The frequencies of each spot (single small, single large, or twin) and the total frequency of spots per fly, for each treatment, were compared in pairs (i.e. negative control versus compound-treated).

Metabolic Stability:

The in vitro metabolism stability experiment was done using human liver microsomes (XenoTech, LLC, Lenexa, KS, USA) for phase I metabolism. The results were expressed as the percentage of drug remaining (solution were 1 μM in 0.1% methanol) and the studies were performed in triplicate as described previously.⁹² For the microsomal stability test we used a solution of phosphate buffer (100 mM, pH 7.4), microsomal protein (1.0 mg/mL), magnesium chloride (10 mM) and NADPH (2 mM) at a final volume of 1.0 mL. This mixture was preincubated at 37 °C for 10 min in a water bath maintained at 60 rpm. The reaction was initialized by adding the selected compounds (1 μg/mL). Aliquots (100 μL) were collected at 0, 5, 15, 20, 30, 45 and 60 min. The reaction was quenched by adding MeOH (300 μL) containing IS (100 ng/mL). The aliquot and quenching reaction were contained in a 1.5 mL Eppendorf tube. The incubation without the addition of NADPH was used as negative control. Testosterone was incubated similarly as positive control substrates. Then, 5 μL samples of the supernatants were analyzed by LC-MS/MS.

LC-MS/MS Assay:

A: Shimadzu LC-MS/MS system (LC-MS/MS 8060, Shimadzu, Japan) was utilized for analysis. The LC system consisted of two LC-30 AD pumps and a CTO-30AS column oven plus an auto-sampler (SIL-30AC), which was used to inject 10 μL aliquots of the processed samples. The MS/MS system operated at unit resolution in the multiple reaction monitoring (MRM) mode. The following precursor ion > product ion combinations were used: 489.95>280.15, 510.05>280.05, 417.10>280.10, 416.15>98.10 AND 414.10>176.25 m/z for 40, 37, 25, 24, and 11, respectively. The compound dependent mass spectrometer parameters, such as temperature, voltage, gas pressure, etc., were optimized by auto method optimization via precursor ion search for each analyte and the internal standard (IS) using a 0.5 μg/mL solution in methanol.

B: Chromatographic separation was achieved on an ACE Excel C₁₈ column (1.7μm, 100 X 2.1 mm, from Advance Chromatography Technologies LTD., UK) with a Phenomenex C₁₈ column guard (Phenomenex, Torrance, CA). The mobile phase consisted of formic acid in water (0.1%, solvent A) and methanol (MeOH) (solvent B) using a total flow rate of 0.25 mL/min. The chromatographic separation was achieved using 5.0 min gradient elution. The initial mobile phase composition was 35% B, increasing to 90% B over 4 min, and finally brought back to initial condition of 35% B in 0.10 min followed by 1-min re-equilibration. The injection volume (5 μL) was consistent for all samples.

Mouse plasma stability:

The tested compounds were dissolved in DMSO to yield 2.5 mM solutions. Mouse plasma was diluted to 80% with PBS and heated at 37 °C before the assay. The tested compounds were incubated with the preheated plasma solution (final concentration: 50 μM) in a shaking water bath at 37 °C at six different time points: 0, 15, 30, 60, 90, and 120 min. Experiments were independently conducted in triplicate. At the end of the incubation time, 50 μL of sample were collected and mixed with 200 μL of cold acetonitrile to stop the reaction. Solutions were vortex-mixed and then centrifuged at 4 °C and 14000 rpm for 15 min. Supernatants were diluted in methanol:water (50:50 v/v) and analyzed by HPLC-PDA-ESI-SQ-MS. Peak areas of test compounds were computed for each incubation time and relative levels to time zero are reported. Enalapril was used as a positive control during incubation.

SGA stability:

The tested compounds were dissolved in DMSO to yield 1 mM. Compounds were incubated with SGF solution (final concentration: 50 μM) in a shaking water bath at 37 °C for four different time points: 0, 30, 60, and 120 min. Experiments were independently conducted in triplicate. At the end of the incubation, 125 μL of sample solution was taken and 375 μL of acetonitrile was added to stop the reaction. The solutions were vortex-mixed and centrifuged at 25 °C and 15000 rpm for 15 min. The supernatant was diluted in methanol:water (50:50 v/v) and analyzed by HPLC-MS. The percentage of compound remaining at the individual time points relative to the 0 minute sample is reported based on the peak area of the test compound.

The HPLC-PDA-ESI-SQ-MS analysis was performed using a Waters Alliance e2695 system with a Phenomenex Kinetex XB-C18 (4.6 x 150 mm, 5 μm particle size) column, coupled to a Waters 2998 photodetector array and a Waters SQD2 single quadrupole mass spectrometer with an ESI source. Gradient elution was utilized in the chromatographic separation method using 0.1% formic acid in water (mobile phase A), and methanol (mobile phase B), with the following program: 0-9 min 75 % B; 9-10 min 75-95 % B; 10 min 95% B. The flow rate was constant at 0.4 mL min⁻¹. After each sample injection, the gradient was returned to its initial condition in 16 min. The injection volume was 5 μL, and the column temperature was 40 °C. The mass spectrometer was operated in positive ion mode with a probe capillary voltage of 3.0 kV. The sampling cone voltage was set to 45.0 V. The source and desolvation gas temperatures were set at 150°C and 350°C, respectively. The nitrogen gas desolvation flow rate was 600 L h⁻¹ and the cone gas flow rate was 10 L h⁻¹. The mass spectrometer was calibrated across the range of m/z 20–2023 with a sodium and cesium iodide solution. Data were acquired in scan mode with scan duration of 0.2 sec; in SIR mode for each test compound based on the m/z value for [M+H]⁺ adduct ions, and with unit resolution. Data acquisition and processing were conducted using MassLynx, version 4.1 (Waters Corp.).

ABBREVIATIONS USED:

EB

elementary body

RB

reticulate body

STI

sextually transmitted infection

STDs

sexually transmitted diseases

ACP

Activators of Cylindrical protease

PyBOP

Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

HBTU

N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

HATU

1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

DIPEA

N, N Diisopropylethylamine

IFA

Immunofluorescence assay

IFU

Inclusion Forming Unit

Hep-2

Human epithelial type 2

HBD

hydrogen bond donor

HBA

hydrogen bond acceptor

SDS

sodium dodecyl sulfate

Suc-Luc-Tyr-AMC

Succinic acid-Leucin-Tyrosin- 7-amino-4-methyl-2H-chromen-2-one

TLC

thin layer chromatography

b rs

broad signal

DMEM

Dulbecco's Modified Eagle Medium