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

Article abstract of DOI:10.1021/acs.jmedchem.0c00371

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 toward mammalian cell lines. The compounds were found to be nonmutagenic 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.

Full text of DOI:10.1021/acs.jmedchem.0c00371

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Article
Synthesis and Antichlamydial Activity of Molecules
Based on Dysregulators of Cylindrical Proteases
Mohamed A. Seleem, Nathalia Rodrigues de Almeida, Yashpal Singh S. Chhonker, Daryl J Murry,
Z. R. Guterres, Amanda M Blocker, Shiomi Kuwabara, Derek J Fisher, Emilse S Leal, Manuela
R Martinefski, Mariela Bollini, Maria Eugenia Monge, Scot Ouellette, and Martin Conda-Sheridan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00371 • Publication Date (Web): 31 Mar 2020
Downloaded from pubs.acs.org on April 4, 2020
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Synthesis and Antichlamydial Activity of Molecules Based on Dysregulators of
Cylindrical Proteases
a
b
c
Mohamed A. Seleem, Nathalia Rodrigues de Almeida, Yashpal Singh Chhonker, Daryl J.
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Murry, Zaira da Rosa Guterres, Amanda M. Blocker, Shiomi Kuwabara, Derek J. Fisher,
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Emilse S. Leal, Manuela R. Martinefski, Mariela Bollini, María Eugenia Monge, Scot P.
g,*
a,
Ouellette, Martin Conda-Sheridan. *
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center,
Omaha, NE 68198, USA.
b
Department of Chemistry, College of Arts and Sciences, University of Nebraska at Omaha, Omaha, NE,
68182, USA
c
Clinical Pharmacology Laboratory, Department of Pharmacy Practice and science, College of Pharmacy,
University of Nebraska Medical Center, Omaha, NE 68198, USA
d
Laboratory of Cytogenetics and Mutagenesis, State University of Mato Grosso do Sul, Mundo Novo,
Matto Grasso do Sul, Brazil
e
School of Biological Sciences, Southern Illinois University Carbondale, Carbondale, IL 62901, USA
f
Centro de Investigaciones en BioNanociencias (CIBION), Consejo Nacional de Investigaciones Científicas
y Técnicas (CONICET), Godoy Cruz, 2390 Ciudad de Buenos Aires, Argentina
g
Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical
Center, Omaha, NE 68198, USA.
Key words: Chlamydia trachomatis, ClpP, activators of cylindrical proteases, sexually transmitted
diseases, heterocycles
* Corresponding authors
scot.ouellette@unmc.edu
martin.condasheridan@unmc.edu
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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 presnted 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 anti-chlamydial drugs.
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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
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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
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rounds of polarized division within the inclusion, RBs undergo a secondary differentiation to
EBs, and the pathogen is released from the host cell starting another round of infection9, 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, and
Doxycycline) are broad spectrum drugs that can affect regular functions of the commensal
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microbiota and encourage the development of bacterial resistance. 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
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AZM treatment. 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.
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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 have 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
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systems. 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 infections25-28 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
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to the death of Escherichia coli. 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
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Building upon that precedent, Leung and coworkers combined computational chemistry and
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biochemical experiments to identify new scaffolds that can activate ClpP. Two of the synthesized
molecules, named ACP1a and b (1 and 2, Figure 1), showed moderate antibacterial activity against
N. meningitidis (64 g/mL and 16 g/mL, respectively) and H. influenzae (32 g/mL and 8 g/mL
respectively). Both molecules were 10-20-fold less potent than ADEP1. However, ACP1b was
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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
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chaperones (AAA+) with diverse cellular activities , ClpC and ClpX.
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
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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.
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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-
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thioaryl derivatives (3) with an appropriate -halo ester derivative (4). 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
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Scheme 1. Synthesis of key carboxylic acid intermediates 7 a-f
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The carboxylic acid derivatives 7a-f were activated using PYBOP and DIPEA in tetrahydrofuran
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THF) and then reacted with 3-phenylpropylamine to yield six derivatives, 9-14 (Scheme 2).41, 47,
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All the prepared molecules satisfy the Lipinski rule of five (Table S1). 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-
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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
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To investigate the importance of the thiol on the eastern part, we synthesized derivatives 23-31
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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
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Scheme 5. Synthesis of ACP1 analogues with a rigid linker
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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.
Table 1. Initial antichlamydial activity screening of ACP derivatives vs. C. trachomatis (serovar LGV-L2)
Compound
Inhibition
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activity*
No.
ACP1
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Compound
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++
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
2
3-29, 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
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that this 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.
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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
4
2 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
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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
6
3
of our compounds, 128 μg/mL of spectinomycin (2x MIC), and 5 units/mL of penicillin (3x
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63
MIC) 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
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2
(IFU) assay as described elsewhere. 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
6
4
(
5 U/mL ~ 3 μg/mL). 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).
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
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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.
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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 testedcompounds elicited a notable
impact on the size as well as the number of the inclusions (Figure 5b). Despite the reported
6
9
treatment efficacy of both drugs, 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
7
0
complications associated with chlamydial infections. 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.
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Mechanism of action. Antibiotics can be bacteriostatic, stop growth or reproduction by targeting
78
essential functions such as cell wall growth, or bactericidal, which kills the bacteria. 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.
0
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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,
5
9
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
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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
41
absence of ACPs (Figure 7). Briefly, 20 µM of FITC-labeled casein was incubated with 1 or 0.1
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(
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 also performed
and measured by resolved 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. Surprinsingly, we did not observe
activation of chlamydial ClpP1, ClpP2 or the ClpP1/2 dimer (other than some residual
decomposition triggerer 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.
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
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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,
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5, 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 6) 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 no toxic towards HeLa 229 cells but
affect ~50% of the HaCaT cells. Compounds 24, 37, and 42 were more tolerated by the HaCaT
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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 is 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.
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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 genome81-83, 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.
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
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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).
0
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Table 3 - Frequency of mutant spots in the wings of marked trans-heterozygous descendants (mwh/flr ) of D.
3
melanogaster using the standard cross (ST) and marked trans-heterozygous descendants (mwh/flr ) 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
3
mwh/flr of D. melanogaster - Standard cross (ST)
Small single
spots (1-2 cell)
m = 2
Large single
spots (>2 cell)
m = 2
Spots with
mwh clone
Comp
ID
Concentration
(mM)
Nº of
flies (N)
Twin spots Total spots
b
b
c
m = 5
m = 2
(n)
Control
20
20
20
20
20
20
20
20
20
0.40 (8)
0.25 (5) -
0.20 (4) -
0.35 (7) -
0.20 (4) -
0.15 (3) -
0.30 (6) -
0.10 (2) -
0.15 (3) -
0.20 (4) -
0.15 (3)
0.0 (0)-
0.0 (0)
0.0 (0) -
0.0 (0) -
0.0 (0) -
0.0 (0) -
0.0 (0) -
0.0 (0) -
0.0 (0) -
0.05 (1) -
0.0 (0) -
0.55 (11)
0.25 (5) -
0.20 (4) -
0.40 (8) -
0.20 (4) -
0.15 (3) -
0.35 (7) -
0.15 (3) -
0.35 (7) -
0.25 (5) -
10
5
4
8
4
3
7
3
7
5
0
.25
1
4
1
0
0.50
1.00
0.0 (0) -
0.05 (1) -
0.0 (0) -
0.0 (0) -
0.05 (1) -
0.05 (1) -
0.15 (3) -
0.05 (1) -
0.25
0.50
1
0
.00
.25
41
0.50
1.00
20
3
mwh/flr of D. melanogaster - High Bioactivation (HB) cross
Control
20
20
20
20
20
20
20
20
0.25 (5)
0.15 (3) -
0.10 (2) -
0.20 (4) -
0.45 (9) i
0.25 (5) -
0.50 (10) i
0.40 (8) -
0.10 (2)
0.05 (1) -
0.15 (3) -
0.05 (1) -
0.0 (0) -
0.05 (1)
0.05 (1) -
0.0 (0) -
0.05 (1) -
0.0 (0) -
0.10 (2) -
0.05 (1) -
0.0 (0) -
0.40 (8)
0.25 (5) -
0.25 (5) -
0.30 (6) -
0.45 (9) -
0.35 (7) -
0.60 (12) -
0.45 (9) -
8
5
0
.25
11
0.50
1.00
5
6
0
.25
0.50
.00
0.25
9
4
4
0
1
0.0 (0) -
7
1
0.05 (1) -
0.05 (1) -
12
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0
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.50
.00
20
20
0.40 (8) -
0.35 (7) -
0.05 (1) -
0.10 (2) -
0.05 (1) -
0.0 (0) -
0.50 (10) -
0.45 (9) -
10
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a
Statistical diagnoses according to Frei and Würgler [1988]. U test, two-sided; probability levels: -, negative; +,
positive; i, inconclusive; P<0,05 vs, untreated control. Including flr single spots. Considering mwh clones from
mwh single and twin spots. NC: Negative control.
b
3
c
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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 (t1/2) and the intrinsic clearance (CLint) 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 T1/2, intrinsic clearance, and hepatic clearance.
T
1/2 (min)
CL(int) uL/(min*mg protein)
CL(int,H) mL/(min*kg bodywgt.)
Comp. ID
Mean
6.13
2.98
3.73
6.25
SD
Mean
113.09
233.77
185.60
111.96
NA
SD
3.02
13.74
1.72
2.85
Mean
890.58
1840.91
1461.60
873.80
NA
SD
1
2
2
3
4
1
4
5
7
0
0.16
0.17
0.03
0.16
NA
23.77
108.17
13.51
22.45
< 2.00
NA: Not applicable
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
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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.
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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
4
1
protease paralogues, ClpP1 and ClpP2. 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 suggest 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.
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Experimental Procedures
Chemistry:
General. All reagents and solvents were used as received from commercial suppliers unless
1
1
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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 N
2
atmosphere. Reaction progression was detected using TLC, which was performed on Merck silica
1
gel IB2-F plates (0.25 mm thickness), and the spots detected using UV light source at 254 nm. H
and 13CNMR spectra were run at 500 MHz in deuterated chloroform (CDCl
sulfoxide (DMSO-d ) on a BRUKER- 500 NMR spectrometer. Chemical shifts are given in parts
), or dimethyl
3
6
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
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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
Na
oil which used in the next step without further purification.
-(arylsulfonyl)propanoate derivatives 6a-f. Potassium mono persulfate (OXONE ) (2.2 eq)
2
SO
4
, filtered, and concentrated under reduced pressure to afford the desired product as yellow
0
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9
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9
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®
2
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
1
8 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 Na SO
2
4
,
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
1
mg, 77%). H NMR (500 MHz, CDCl
3
) 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).
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2
-Methyl-2-(Pyridin-2-ylsulfonyl)propanoic acid (7b). Starting with 6b (925 mg), White solid
1
(
280 mg, 41%). H NMR (500 MHz, CDCl
3
) 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).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-Methyl-2-((4-(trifluoromethyl)phenyl)sulfonyl)propanoic acid (7c). Starting with 6c (1160
1
mg), White solid (840 mg, 82%). H NMR (500 MHz, CDCl
3
) δ 8.04 (d, J = 8.5 Hz, 2H), 7.83 (d,
1
9
J = 8.5 Hz, 2H), 1.66 (s, 6H); F NMR δ −63.27 (s).
2
-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanoic acid (7d). Starting with 6d (1120 mg),
1
White solid (750 mg, 85%). H NMR (500 MHz, CDCl
3
) δ 9.01 (s, 1H), 8.253-8.250 (m, 1H), 4.75
1
9
(
q, J = 7.5 Hz, 1H), 1.74 (d, J = 7.5 Hz, 3H); F NMR δ −62.45 (s).
2-Methyl-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanoic acid (7e). Starting with 6e
1
(
1170 mg), White fluffy solid (890 mg, 83%). H NMR (500 MHz, CDCl
3
) δ 9.02 (s, 1H), 8.27–
1
9
19
8
.22 (m, 2H), 1.76 (s, 6H); F NMR δ −62.68 (s); F NMR δ −62.68 (s).
2-((5-Chloropyridin-2-yl)sulfonyl)-2-methylpropanoic acid (7f). Starting with 6f (1050 mg),
1
White solid (700 mg, 79%). H NMR (500 MHz, CDCl
3
) δ 8.73-8.72 (m, 1H), 8.07 (d, J = 8.5 Hz,
1
H), 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
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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),
0
1
2
3
4
5
6
7
8
9
0
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2
3
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7
8
9
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8
9
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1
white solid (46 mg, 84%). H NMR (500 MHz, CDCl
3
) 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,
1
3
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) ; C NMR (125
MHz, CDCl
3
) δ 167.44, 141.18, 135.10, 134.34, 130.04, 129.03, 128.54, 128.40, 126.10, 68.01,
+
3
3
9.90, 33.19, 30.79, 20.93; HPLC purity, 97.1%; HRMS (m/z): [M+H] calcd for C19
H
24NO S,
3
46.1399; 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
1
mg), white solid (46 mg, 83%). H NMR (500 MHz, CDCl
3
) 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
1
3
(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) ; C NMR (125
MHz, CDCl
3
) δ 167.76, 154.89, 150.25, 141.44, 137.94, 128.47, 128.42, 127.76, 125.99, 124.71,
+
6
7.40, 39.94, 33.13, 30.65, 20.73; HPLC purity, 98.9%; HRMS (m/z): [M+H] calcd for
C
18
H
22
N
2
3
O S, 347.1351; found, 347.1423.exact mass(monoisotopic) from spectrum 346.1350.
2
-Methyl-N-(3-Phenylpropyl)-2-((4-(trifluoromethyl)phenyl)sulfonyl)propanamide
(11).
1
Starting with 7c (48 mg), white solid (53 mg, 81%). H NMR (500 MHz, CDCl
3
) 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,
1
3
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) ; C NMR (125
MHz, CDCl ) δ 166.94, 141.03, 138.7, 136.06, 135.80, 130.68, 128.60, 128.37, 126.11(q, J =3.75
3
Hz), 126.19,121.93, 68.32, 40.02, 33.19, 30.71, 20.67; HPLC purity, 97.4%; HRMS (m/z): [M+H]+
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calcd for C20
13.1727.
H
22
F
3
NO
3
S, 414.1272; found, 414.1345, exact mass(monoisotopic) from spectrum
4
N-(3-Phenylpropyl)-2-((5-(trifluoromethyl)pyridin-2-yl)sulfonyl)propanamide (12). Starting
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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9
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1
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9
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1
with 7d (50 mg), white solid (48 mg, 68%); H NMR (500 MHz, CDCl
3
) δ: 8.97 (s, 1 H), 8.19 (s,
2
H), 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,
1
3
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); C NMR (125
MHz, CDCl
3
) δ 163.62, 158.79, 147.39, 147.36, 141.13, 135.81, 135.78, 130.22, 128.53, 128.35,
1
26.12, 123.22, 121.28, 62.55, 39.84, 33.02, 30.70, 11.47; HPLC purity, 98.3; ESIMS calcd for
+
C
18
H
19
F
3
N
2
O
3
S 401.42 [M+H] . Found 401.01; HRMS (m/z): [M+H] calcd for C18
H
19
F
3
N
2
3
O S,
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
1
chromatography to yield the desired product as yellow oil (55 mg, 75% ), H NMR (500 MHz,
CDCl ) 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),
3
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,
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.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%).
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2
-((5-Chloropyridin-2-yl)sulfonyl)-2-methyl-N-(3-phenylpropyl)propanamide (13). Starting
1
with 6f (42 mg), white solid (55 mg, 88%). H NMR (500 MHz, CDCl
3
) 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),
.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
7
13
(
6H, s) ; C NMR (125 MHz, CDCl ) δ 167.46, 152.64, 149.31, 141.33, 137.51, 137.03, 128.52,
3
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 C18
H21ClN
2
3
O S, 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).
1
Starting with thioacid (50 mg), White solid (47 mg, 76%). H NMR (500 MHz, CDCl
3
) δ 8.65 (s,
1
7
H), 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),
.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
3
1.76 (q, J = 7.5 Hz, 2H), 1.68 (s, 6H) ; C NMR (125 MHz, CDCl
3
) δ 173.85, 162.41, 146.06,
1
3
46.01, 141.36, 133.14, 133.11, 128.43, 128.24, 125.97, 124.59, 123.39, 123.12, 122.85, 52.77,
+
9.59, 33.13, 30.91, 26.61; HPLC purity, 98.9%; HRMS (m/z): [M+H] calcd for C19
H
21
F
3
2
N OS,
383.1327; found, 383.1397, exact mass(monoisotopic) from spectrum 382.1324. N.B: For this
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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:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
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3
4
5
6
7
8
9
0
2-((2-Chlorophenyl)thio)ethan-1-amine (15a). Colorless oil with fruity odor. (125 mg, 36%);
1H NMR (500 MHz, CDCl
m, 1H), 3.03 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 1.70 (s, 2H).
) δ: 7.38-7.31 (dd, J = 7.0 Hz, 1H 2H), 7.22-7.18 (m, 1H), 7.13-7.10
3
(
2-((4-Chlorophenyl)thio)ethan-1-amine (15b). Yellow oil with characteristic odor. (305 mg,
1
8
8.6%); H NMR (500 MHz, CDCl
3
) 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:
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N-(2-((2-chlorophenyl)thio)ethyl)-2-methyl-2-(phenylsulfonyl)propanamide (16). Starting
1
with 7a (50 mg) and 15a (40 mg), white solid (70 mg, 82%); H NMR (500 MHz, CDCl
3
) 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),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
6
.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,
13
H) ; C NMR (125 MHz, CDCl ) δ 167.91, 135.00, 134.56, 134.41, 134.12, 130.12, 129.99,
3
129.85, 129.06, 127.43, 68.06, 39.19, 31.95, 20.85; HPLC purity, 95.3%; ESIMS calcd for
+
+
C
18
18
H
20ClNO
20ClNO
3
S, 397.06, Found mass [M+H] : 398.00; HRMS (m/z): [M+H] calcd for
C
H
3
S, 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 C18
H
20ClNO S,
3
1
3
97.06, Found mass [MH+]: 398.00; H NMR (500 MHz, CDCl
3
) 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 =
1
3
8.5 Hz, 2H), 3.49 (q, J = 6.5 Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H), 1.56 (s, 6H); C NMR (125 MHz,
CDCl
3
) δ 167.85, 134.98, 134.43, 133.30, 132.77, 131.31, 130.08, 129.33, 129.07, 68.03, 39.26,
S, 397.06, Found mass [M+H]+:
3
3.25, 20.87; HPLC purity, 98.7; ESIMS calcd for C18
H
20ClNO
3
+
398.00; HRMS (m/z): [M+H] calcd for C18
H
20ClNO S, 398.0573; found, 398.0647, exact
3
mass(monoisotopic) from spectrum 397.0575.
N-(2-((2-Chlorophenyl)thio)ethyl)-2-methyl-2((4-(trifluoromethyl)phenyl)sulfonyl)
1
propanamide (18). Starting with 7c (50 mg) and 15a (33 mg), white solid (69 mg, 87%) H NMR
(500 MHz, CDCl ) 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,
3
(
13
2
H), 1.57 (s, 6H) ; C 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):
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+
[
M+H] calcd for C19
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%); H
NMR (500 MHz, CDCl ) 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),
H
19ClF
3
NO
3
S , 466.0447; found, 466.0522, exact mass(monoisotopic) from
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
9
13
1
.56 (s, 6H); F NMR δ −63.31 (s); C NMR (125 MHz, CDCl ) δ 167.36, 138.64, 135.98 (q, J=
3
3
2.5 Hz), 133.14, 132.92, 131.36, 130.74, 129.38, 126.15 (q, J= 3.75 Hz), 124.08, 121.90, 68.32,
9.26, 33.33, 20.65; 19F NMR δ −63.31 (s) ; HPLC purity, 99.2%; ESIMS calcd for
3
+
+
C
19
19
H
19ClF
19ClF
3
3
NO
3
S
S
2
2
: 465.04, Found mass [M+H] : 466.0; HRMS (m/z): [M+H] calcd for
C
H
NO
3
, 466.0447; found, 466.0523, exact mass(monoisotopic) from spectrum
4
65.0450.
N-(2-((4-chlorophenyl)thio)ethyl)-2-methyl-2- )( 5-(trifluoromethyl)pyridin-2-yl)sulfonyl)
1
propanamide (20). Starting with 7e (50 mg) and 15b (31 mg), white solid ( 53 mg, 68%) H NMR
(
500 MHz, CDCl
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) ; C NMR
125 MHz, CDCl ) δ 167.09, 158.12, 147.16, 135.58, 133.44, 132.73, 131.25, 130.45, 130.18,
29.28, 124.47, 121.27, 67.59, 39.47, 33.09, 20.71; HPLC purity, 95%; ESIMS calcd for
3
) δ: 8.91 (s, 1H), 8.21-8.17 (m, 2H), 7.37 (br s, 1H), 7.33 (d, J = 8.5 Hz, 2H),
1
3
(
3
1
+
+
C
19
19
H
19ClF
19ClF
3
3
NO
3
S
S
2
2
: 466.04, Found mass [M+H] : 467.19; HRMS (m/z): [M+H] calcd for
C
H
NO
3
, 467.0447; found, 467.1023.
Synthesis of 2-(4-Chlorophenoxy)ethan-1-amine (22c). Starting from 2-bromo ethan-1-amine
2
1 (500 mg, 2.45 mmol), the synthesis of 23c was carried out over three successive steps:
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