The Rational Design, Synthesis, and Antimicrobial Properties of Thiophene Derivatives That Inhibit Bacterial Histidine Kinases

Article abstract of DOI:10.1021/acs.jmedchem.6b00580

The emergence of multidrug-resistant bacteria emphasizes the urgent need for novel antibacterial compounds targeting unique cellular processes. Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli

Full text of DOI:10.1021/acs.jmedchem.6b00580

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Article
The rational design, synthesis and antimicrobial properties of
thiophene derivatives that inhibit bacterial histidine kinases.
Thibaut Boibessot, Christopher P. Zschiedrich, Alexandre Lebeau, David Bénimèlis, Catherine
Dunyach-Rémy, Jean-Philippe Lavigne, Hendrik Szurmant, Zohra Benfodda, and Patrick Meffre
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00580 • Publication Date (Web): 30 Aug 2016
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Title: The rational design, synthesis and
antimicrobial properties of thiophene derivatives
that inhibit bacterial histidine kinases
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Authors: Thibaut Boibessot†#
‡#
†
,
Christopher P. Zschiedrich , Alexandre Lebeau , David
†
§
§
‡Ψ
Bénimèlis , Catherine DunyachꢀRémy , JeanꢀPhilippe Lavigne , Hendrik Szurmant∗ , Zohra
†
ϖ
Ψ
ϖ
† Ψ
Benfodda∗
, Patrick Meffre
†
UNIV. NIMES, EA7352 CHROME, Rue du Dr G. Salan, 30021 Nîmes cedex 1, France.
Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University
‡
of Health Sciences, Pomona, CA 91766, USA and Department of Mol. & Exp. Medicine, The
Scripps Research Institute, La Jolla, CA 92037, USA
ϖIBMM, UMRꢀCNRS5247, Université de Montpellier, Place Eugène Bataillon, 34095
Montpellier, France.
§
Institut National de la Santé et de la Recherche Médicale, U1047, Montpellier University,
CHU de Nîmes, Place du Pr R. Debré, 30029 Nîmes.
Keywords: Twoꢀcomponent system, histidine kinase, thiophene derivatives, rational design,
molecular docking, antibacterial agents, bacterial resistance
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ABSTRACT
The emergence of multiꢀdrugꢀresistant bacteria emphasizes the urgent need for novel
antibacterial compounds targeting unique cellular processes. Twoꢀcomponent signal
transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to
adaptive responses, are absent in mammals, and are embedded in various pathogenic pathways.
To attenuate these signaling pathways, we aimed to target the TCS signal transducer histidine
kinase (HK) by focusing on their highly conserved adenosine triphosphate (ATP)ꢀbinding
domain. We used a structureꢀbased drug design strategy that begins from an inhibitorꢀbound
crystal structure and includes a significant number of structurally simplifiying “intuitive”
modifications to arrive at the simple achiral, biaryl target structures. Thus, ligands were
designed, leading to a series of thiophene derivatives. These compounds were synthesized and
evaluated in vitro against bacterial HKs. We identified eight compounds with significant
inhibitory activities against these proteins, two of which exhibited broadꢀspectrum
antimicrobial activity. The compounds were also evaluated as adjuvants for the treatment of
resistant bacteria. One compound was found to restore the sensivity of these bacteria to the
respective antibiotics.
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1. INTRODUCTION
Antibiotics are considered among the most important medical discoveries of the 20th century.
Unfortunately, the clinical use of conventionally available antibiotics has led to the rapid
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development of resistance . One of the first clinical antibiotics, penicillin, was introduced in
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the midꢀ1940s and resistant mutants appeared within 2 years of its introduction. After 75
years of extensive antimicrobial use against strains of Salmonella enterica, a leading cause of
bacterial gastroenteritis, several bacteria have become resistant to front line antibiotics, and
the emergence of methicillinꢀresistant Staphylococcus aureus (MRSA) strains has increased
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in nosocomial and community settings. The high level of inherent antibiotic resistance in
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Pseudomonas aeruginosa makes the treatment of cystic fibrosis problematic. In contrast, the
pharmaceutical companies' investments in the discovery and development of new antibiotics
have stagnated compared with their investments in drugs combatting chronic diseases such as
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cancer and diabetes. Antimicrobial resistances is not only a major health problem but is also
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an economic issue. Hence, innovative research to develop antiꢀinfective agents with novel
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modes of action that circumvent the current resistance mechanisms is urgently needed.
Bacteria have evolved a variety of mechanisms to respond to environmental changes. Among
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the most commonly utilized are twoꢀcomponent signal transduction systems (TCSs). TCSs
were proposed as attractive targets because they are absent in mammals and essential or
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conditionally essential for viability in several important bacterial pathogens.
to diverse environmental changes, a bacterium typically possesses multiple TCSs.
TCSs are implicated in survival roles and pathogenic mechanisms, such as nutrient acquisition,
To respond
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These
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sporulation, biofilm formation and antibiotic resistance.
TCS inhibitors are expected not
only to work as antibacterial agents but also to be developed as adjuvants with known
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antimicrobials to target drug resistance, colonization or virulence factor expression.
Most commonly, a TCS consists of a membraneꢀspanning sensor HK and a cytosolic
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transcription factor, termed the response regulator (RR); however many variations including
soluble HK and nonꢀtranscription factor RR proteins, exist. In response to an environmental
or cellular signal, HKs autophosphorylate a conserved histidine residue in the dimerization
domain and the phosphoryl group is subsequently transferred to a conserved aspartic acid in
the regulatory domain of its paired RR. The phosphorylated RR typically binds to the
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promoter regions of target genes modulating their expression (Figure 1). Interest in
deactivating TCS transduction by targeting the catalytic and adenosine triphosphate (ATP)ꢀ
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binding (CA) domain of the HK has increased.
The catalytic core within HKs has been
reported to exhibit a high degree of homology in both Gramꢀpositive and Gramꢀnegative
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bacteria.
This degree of homology suggests that a single agent targeting this CA domain
could inhibit multiple TCSs simultaneously. Consequently, bacterial resistance would be less
likely to develop.
The search for inhibitors capable of interrupting TCS has yielded several classes of effective
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HK inhibitors. Unfortunately many of them suffer from poor bioavailability stemming from
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their highly hydrophobic properties.
Some other inhibitors have demonstrated poor
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selectivity and appear to cause protein aggregation. Finally, some inhibitors lead to
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hemolysis. More recently, several interesting reports have described the experimental or in
silico identification of specific inhibitors against the essential cell wall homeostasis regulator
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kinase WalK with antimicrobial activity against some Gramꢀpositive organisms.
However,
currently whether these compounds are of clinical value and whether the focus on a single
kinase might greatly reduce the spectrum of these compounds are unclear. An approach to
identify broad spectrum inhibitors of HK proteins has been published while this manuscript
was in preparation employing a combination of fragment based screening and in silico
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docking technology. Also of note, HK activation rather than inhibition has also recently
been described as a strategy to control virulence of Gramꢀnegative bacteria, since avirulent
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Salmonella and Haemophilus species commonly have mutations that lead to constitutive
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activation of the conjugative plasmid expression TCS CpxRA. The development of new
inhibitors capable of disrupting TCS signaling remains a challenging. In the present study, we
used a structureꢀbased drug design strategy, based on the crystal structure of the ATP pocket
of essential cell wall homeostasis regulator kinase WalK (Protein Data Bank [PDB]: 3SL2),
and PLANTS as a virtual screening software to identify new inhibitors of HK. To this end, we
utilized the fact that HKs share a related CA domain with DNAꢀgyrases and topoisomerases.
The latter represent known antimicrobials targets, and highꢀresolution structures of inhibitorꢀ
bound proteins exist. HK ligands were designed leading to the development of a series of
thiophene derivatives (6aꢀ6u, 7aꢀ7c, 8aꢀi). These compounds were synthesized, and their
inhibitory properties were evaluated against three bacterial HK proteins, B. subtilis phosphate
limitation sensor kinase PhoR, respiration sensor kinase ResE, and kinase WalK. Their
antimicrobial activities against nine bacterial strains were assessed. Moreover, the compounds
were evaluated as adjuvants against antibioticꢀresistance bacterial strains. We identified
several compounds with desirable activities against HKs. These lead compounds represent
potential starting points for future HK inhibitor development.
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2. RESULTS AND DISCUSSIONS
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.1 Design of new compounds.
To generate HK inhibitors, we utilized a structureꢀbased design methodology. Inhibitors are
expected to exhibit antibacterial properties when a single essential TCS is targeted or when
multiple systems whose activities function not necessarily alone but in combination are
targeted simultaneously. Our aim was to inhibit multiple HKs simultaneously and to make
evolving resistance more difficult. We decided to target the ATPꢀbinding domain because this
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strategy was very recently validated . The ATPꢀbinding domain is characterized by a
Bergerat fold, which is a domain shared with DNAꢀgyrases and topoisomerase, heat shock
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proteins and the mutator protein MutL protein. The Bergerat fold consists of a sandwich of αꢀ
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helices in one layer, and mixed βꢀstrands in another, and a discrete and flexible ATP lid
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To design the inhibitors, we used the available Xꢀray structure of the ATPꢀbinding domain of
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WalK (PDB: 3SL2 ) at a resolution of 1.61 Å. This structure comprises a twoꢀlayer α/β
sandwichꢀfold with a large ATP pocket (Figure 2A). One layer is composed of a fiveꢀstranded
βꢀsheet. Strands 1 and 2 run parallel, whereas the directions of strands 2, 3, 5 and 4 alternate.
The second layer is made of three αꢀhelices. These two layers are linked by a large flexible
loop (Asp533–Gly567) containing a short helical segment that is crucially involved in ATP
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binding . This loop is designated as the ATPꢀlid. The interactions among ATP, Asp533 and
Water822 are known to be critical for proteinꢀcofactor binding. Additionally, we observed πꢀ
stacking between the adenine ring and Tyr507 (Figure 2B). Thus, our aim was to design
ligands that can form hydrogen bonds and hydrophobic interactions with these elements.
As mentioned above, the ATPꢀbinding domain of the TCS, a Bergerat fold, is also found in
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Gyrase B, heat shock protein Hsp90, histidine kinases and mutator protein MutL . The
PDB was probed for structures of ligandꢀbound domains of these proteins to investigate
inhibitor binding modes. Gyrase B was singled out because several Gyrase Bꢀligand
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complexes are available with affinities in the nanomolar range.
Moreover, a Gyrase B
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ligand has been reported to bind to the ATPꢀdomain of HKs.
We decided to focus on the
complex PDB: 3TTZ because the ligand 2ꢀ[(3S,4R)ꢀ4ꢀ{[(3,4ꢀdichloroꢀ5ꢀmethylꢀ1Hꢀpyrrolꢀ2ꢀ
yl)carbonyl]amino}ꢀ3ꢀfluoropiperidinꢀ 1ꢀyl]ꢀ1,3ꢀthiazoleꢀ5ꢀcarboxylic acid (pyrrolamide,
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Figure 3 D) has an 50% inhibitory concentration (IC50) value of 4 nM. Comparative docking
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was performed according to the typical protein kinase protocol.
Known ligands were
transferred from the template structure into the target structure. The deduced protein–ligand
complexes were visualized and the protein–ligand interactions were evaluated. To transfer the
ligand of Gyrase B into the HK CA domain, we first superimposed the two structures with a
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root mean square deviation (RMSD) of 0.740 Å using ViTO (Figure 3A). The Gyrase B
pyrrolamide ligand (Figure 3D) was then transferred into histidine kinase CA domain and
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evaluated with DSX (Figure 3B and 3C). We observed that the ligand forms a hydrogen
bond with Asp533 in the bottom of the pocket; however, significant steric hindrance exists at
the edge of the pocket with Ile536 and Tyr507. Furthermore, the electronic effects in the edge
of the pocket are dissimilar in Gyrase B and HK WalK. Indeed, in Gyrase B, the edge of the
pocket appear to be charged. Arg84 and Glu58 form a salt bridge, and Arg144 interacts with
the acidic moeity of the pyrrolamide inhibitor. In contrast, in the HK WalK, the edge of the
pocket appears to be relatively hydrophobic as noted in the presence of Tyr507, Leu568 and
Ile536.
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These observations guided our strategy for designing the HK ATPꢀbinding domain inhibitors
of type 6. Our thought process is visualized in Figure 4. We noted that after scoring the
pyrrolamide ligand the ꢀNH of the pyrrole moiety forms a hydrogen bond with Asp533, and
the C=O of the amide bond establishes a hydrogen bond with Water822. Consequently, we
rigidified the amide bond viaa cyclization to produce structure A in Figure 4. We added an
aromatic ring to maintain hydrophobicity because the edge of the pocket in the HK WalK
(
and others) is hydrophobic and because doing to imbues pyrrolamide with a planeꢀlike
geometry, generating structure B (Figure 4). As mentioned above, steric hindrance occurs at
the edge of the pocket, and thus, the size of requiring size ligand must be reduced.Therefore,
we removed the piperidine ring and the chirality, which is often difficult to control in
chemical synthesis, which produced a subtituted phenyl ring (Figure 4, structure C). Finally,
we chose a thiophene ring instead of a thiazole to gain easy and quick access to a series of
compounds of type 6 as shown in Figure 4.
Initially, a virtually focused chemical library was designed based on general structure 6
(
Figure 5) to form hydrogen bonds with Asp533 and hydrophobic interactions with Tyr507.
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This library was constructed to be easily accessible chemically. Thus, in positions R1, R2 and
R3, we preferred to derivatize the phenyl ring with only one substituent, which can be either a
hydrogen bond donor or acceptor. In position R’, we chose to add polar substituents to
increase the compounds' solubility. We docked this virtual chemical library into the ATPꢀ
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binding site of WalK (PDB: 3SL2) using PLANTS . Protein and ligands were prepared with
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SPORES
before docking and the docking poses were reevaluated using the DSX scoring
function. When inspecting the docking poses, the phenyl ring can be observed at the bottom
of the pocket, and the substituent points in the direction of Asp533. The thiophene ring
participates in πꢀstaking with Tyr507, and the polar substituents note of the pocket towards
the solvent (Figure 6A and 6B). When comparing the binding poses and the scores obtained
from both the docking and reevaluation experiments (Table 1), 21 compounds in the virtual
library were considered promising and singled out for synthesis.
2
.2 Synthesis
Thiophene derivatives (6aꢀu) were synthesized using the palladiumꢀcatalyzed SuzukiꢀMiyaura
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crossꢀcoupling reaction (Scheme 1). Various commercially available phenyl boronic acid
derivatives (5aꢀ5g) were reacted with commercially available 2ꢀbromoꢀ5ꢀnitrothiophene (1),
2ꢀacetylꢀ5ꢀbromothiophene (2), and easily generated 2ꢀacetamideꢀ5ꢀbromothiophene (3). 3
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was prepared according to the literature. Indeed, the reaction between 2ꢀacetylꢀ5ꢀ
bromothiophene (2) and hydroxylamine hydrochloride gave a mixture of two isomeric oximes
(
4aꢀb), which were separated by column chromatography, yielding the respective isomers E
a (54%) and Z 4b (45%) (Scheme 2). Then, isomer E (4a) was used in a Beckmann
rearrangement to give the acetamide 3 in 88% yield.
For the Suzuki reaction (Scheme 1), Pd(PPh or PdCl
Na CO , or K CO or Cs CO was used as the base; and typical solvents were used (for more
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(PPh ) was used as the catalyst;
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details see the Supporting Information). The reactions were conducted at 70°Cꢀ90°C, and
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moderate to good yields of the desired compounds (6aꢀu) were obtained (Table 2). The
Suzuki coupling gave complete conversion in all cases, although the work up and purification
were cumbersome. Compounds 6aꢀc reacted with 2ꢀiodoethanol at 90°C under inert
conditions in N,Nꢀdimethylformamide (DMF) or without a solvent to produce compounds
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(
7aꢀc) in moderate yields (Scheme 3, Table 2).
To overcome the solubility problems encountered during biological evaluations, we decided
to synthesize the hydrochloride salts of the amines. Treating the amines (6aꢀc, gꢀi, and mꢀo)
with 12ꢀN HCl in tetrahydrofuran (THF) or with in situꢀgenerated HCl afforded the amines as
their HCl salts 8aꢀi in moderate to good yields (Scheme 4).
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The final compounds 6aꢀu, 7aꢀc, and 8aꢀi were fully characterized by H nuclear magnetic
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resonance (NMR),
C NMR, infrared (IR) spectroscopy, highꢀperformance liquid
chromatography (HPLC) and highꢀresolution mass spectrometry (HRMS). The mass spectra
displayed the correct molecular ions peaks because the measured HRMS data are in good
agreement with the calculated values (see theSupporting Information).
The designed compounds 6aꢀu and 7aꢀc were then evaluated against different HK proteins.
2
.3 Biological evaluation of compounds
.3.1 Several compounds exhibit inhibitory activity against the HK WalK
2
In an initial screening, compounds 6aꢀu and 7aꢀc were evaluated for their ability to inhibit the
autophosphorylation activity of HK WalK from Bacillus subtilis. To this end, two distinct
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04ꢀ612
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cytoplasmic WalK constructs were utilized, WalK
and WalK
. Both constructs
share a catalytic core, including the ATPꢀbinding Bergerat fold or CA domain of the kinase,
but the longer construct contains an additional Nꢀterminal HAMP domain. We argued that
both fragments should behave similarly with regard to potential compound inhibition
suggesting that these compounds indeed target the catalytic portion of the proteins. When
screening with equimolar concentrations of the compounds and ATP, eight (6c, 6d, 6e, 6h, 6i,
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6k, 6s, and 7c) of the 24 compounds were observed to significantly inhibit the kinase activity
(Figure 7). Six of the eight compounds inhibited both fragments of the kinase to similar
extents, whereas two compounds, 6h and 6i, inhibit the longer WalK fragment to a greater
extent than the shorter one. Regardless, these eight compounds were considered as potentially
interesting HK inhibitors and were subjected to moreꢀdetailed downstream evaluations.
2.3.2 Some compounds inhibit multiple HKs
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A desirable trait for any HK inhibitor is to be capability to specifically inhibit multiple HKs of
in organism, rather than a single HK, because it is more difficult for a bacterium to acquire
resistance against a multiꢀtarget inhibitor as such inhibitors could be expected to have a
broader antimicrobial spectrum. To assess whether the eight compounds that inhibited WalK
in vitro also inhibited other HKs, we utilized B. subtilis HKs PhoR, which is involved in the
detection of phospholimitation, and HK ResE, which is involved in the transition to anaerobic
conditions. The IC50 values were determined against all three HKs. Because the kinase serves
as both, the substrate and enzyme in this assay, the concentration of the enzyme is higher than
those of most other proteins, and as a result, the lower limit for the IC50 values is in the low
ꢁM range rather than in the nM range. A typical assay for compound 6e is depicted in Figure
8A, demonstrating that this compound inhibits all three HKs to varying extents. Similar
results were obtained for the other seven compounds and the IC50 values are summarized in
Table 4. Screens were also performed with the B. subtilis sporulation HK KinA, resulting in
similar inhibition patterns: the IC50 values were not determined for this kinase (data not
shown). Of note, one compound 6s showed significantly better inhibition of PhoR (IC : 1.6
50
ꢁM) than ResE and WalK, demonstrating that selectivity can be achieved by varying phenyl
group substituents. At this point we can only speculate why this compound would be
selectively inhibiting PhoR. The docking pose in Figure 6 suggests that the phenyl group
substituents are in contact with conserved Asp533 deep within the ATP binding pocket. This
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pocket might be more accommodating for increased size of substituents in PhoR or might
make additional contacts with these substituents. In summary, the eight compounds could
inhibit the autophosphorylation activity of multiple different HKs in vitro.
2.3.3 The compounds do not inhibit the serine/threonine kinase IreK
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Based on the results thus far, the eight compounds possibly inhibited enzymatic activity nonꢀ
specifically or inhibited protein kinases in general rather than only HKs. This trait would be
an undesirable because kinases are abundant and essential proteins in all domains of life. To
ensure that the eight compounds did not inhibit kinases with different structures and activities,
we utilized the structurally unrelated eukaryoticꢀlike serine/threonine kinase IreK (intrinsic
resistence of enterococci kinase) of E. faecalis, which also utilizes ATP as a phosphorylꢀ
group donor. The IC50 values were determined for concentrations of up to 800 ꢁM of each
compound, analogous to the HK inhibition assays. A typical assay is shown for compound 6e
is shown in Figure 8B. The results for all compounds were identical and are summarized in
Table 4. We observed no inhibition of the serine/threonine kinase IreK, suggesting that the
eight compounds target HKs specifically and do not merely inhibit any enzyme or any kinase.
2.3.4 The compounds do not significantly inhibit E. coli DNAꢀgyrase
ATPꢀdependent DNAꢀgyrases and topoisomerases catalyze essential bacterial DNAꢀpacking
processes and utilize an ATPꢀbinding domain that is structurally related to that in HKs. This
fact was utilized to design the current compound series starting with a known gyrase inhibitor.
Thus, the novel compounds possibly not only inhibited HKs but also targeted DNAꢀgyrase.
To investigate whether the eight compounds with activity against HKs also inhibited DNAꢀ
gyrase activity, we utilized the commercially available E. coli DNAꢀgyrase. To this end, a
standard gyrase assay was utilized, where relaxed plasmid DNA was exposed to DNAꢀgyrase
in the absence or presence of varying amounts of the eight compounds. As a control, we
utilized a known gyrase inhibitor and antibiotic, ciprofloxacin at a concentration of 200 ꢁM.
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Whereas ciprofloxacin completely inhibited the ability of DNAꢀgyrase to induce the
supercoiling of relaxed plasmid DNA, the eight compounds showed no or little inhibitory
activity up to a concentrations of 800 ꢁM. A typical assay of compound 6e is shown in Figure
8C. The IC50 values for all eight compounds are summarized in Table 4. Although all
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compounds exhibited IC50 values for DNAꢀgyrase that were much larger than those observed
for the HKs, some compoundsꢀ6c, 6d, 6k and 7cꢀ inhibited DNAꢀgyrase at the highest tested
concentrations. In summary, the eight compounds show a preference for HKs but we cannot
conclude that the gyrases and topoisomerases from some organisms will not also be subject to
inhibition by some of these compounds.
2.3.5 The compounds 6d and 6e exhibit significant antimicrobial activity
Given these promising in vitro HK inhibition data, we wondered whether any of these tested
compounds exhibited antimicrobial activity. To this end, we screened the eight compounds
against HK activity in a standard KirbyꢀBauer disk diffusion assay against MRSA TCH1516
USA300. Paper disks containing 100 ꢁg of each of the eight respective compounds were
placed on plates with growing lawns of Staphylococcus aureus. Following incubation clear
rings of growth inhibition could be observed for two of the eight compounds ꢀ6d and 6eꢀ
analogous to a vancomycin control; no growth inhibition was observed for the other six
compounds (Figure 9A). To further investigate whether this growth inhibition was significant
a similar assay was perfomed with decreasing concentrations of compounds 6d and 6e (Figure
9B). The diameter of the ring of growth inhibition did not change even when only 20 ꢁg of
compound was used per disk. A reduced ring of growth inhibition was identified using 4 ꢁg of
compound 6e.
These promising results prompted us to quantify the antimicrobial activity of compounds 6d
and 6e against nine selected bacteria including several important human pathogens. To this
end, we utilized the Clinical & Laboratory Standards Institute (CLSI) twoꢀfold serial brothꢀ
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dilution method to determine the minimum inhibitory concentration (MIC), which is defined
as the concentration at which no bacterial growth can be observed, and the minimum
bactericidal concentration (MBC), which is defined as the concentration at which 99.99% of
bacteria are killed. The data from these assays are summarized in Table 5. Clearly both,
compounds 6d and 6e exhibit activity against Gramꢀpositive and Gramꢀnegative bacteria. E.
faecalis appeared to be the least susceptible of the tested bacterial strains. In general, both
compounds showed very similar inhibition spectra, but compound 6d seemed to be slightly
more potent than 6e.
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In summary, of the eight identified HK inhibitors, two exhibited broadꢀspectrum antimicrobial
activity against different significant pathogens.
2.3.6 The compounds 6d and 6e are bactericidal rather than bacteriostatic
Antimicrobials are usually classified as either bacteriostatic or bactericidal. Whereas the
former merely inhibit growth, the latter induce the lysis and killing of cells. It is typically
thought that bacteriostatic antimicrobials inhibit cellular processes such as transcription or
translation, whereas bactericidal antibiotics target the cell envelope. To test whether
compounds 6d and 6e were bacteriostatic or bactericidal, we grew cultures of B. subtilis (Fig.
9C) and MRSA (not shown) in the absence or presence of 50 ꢁg/ml compounds 6d or 6e. As a
control bacteriostatic antibiotic, we utilized chloramphenicol and as a control bactericidal
compound we utilized vancomycin. We observed that compound 6d behaved very similarly to
vancomycin in this assay, inducing slow bacterial lysis over a time course of 9 h. In contrast,
compound 6e induced more rapid cell lysis and clearing of the culture than either vancomycin
or compound 6d (Fig. 9C). In summary, compounds 6d and 6e appear to be bactericidal rather
than bacteriostatic.
2.3.7 The compounds do not exhibit hemolytic activity against sheep red blood cells
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Several previously identified HK inhibitors were demonstrated to not be useful because they
exhibited significant hemolytic activity. Thus, the bactericidal effects of compounds 6d and
6e possibly were attributable to unspecific lytic activity. To ensure that the compounds of
interest, primarily compounds 6d and 6e, but also the other HK inhibitors, did not exhibit
hemolytic activity, we utilized sheep red blood cells and a standard hemolysis protocol
(material and methods). When sheep red blood cells were exposed to large concentrations (up
to 1 mM) of the compounds, no hemolysis was observed (not shown), demonstrating that
none of the compounds were hemolytic. This finding suggests that the antimicrobial activities
of compounds 6d and 6e are not merely attributable to the unspecific induction of cell lysis.
2.3.8 The relatively soluble HCl salts of amines do not improve antimicrobial
activities or HK inhibition
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Several of the eight tested compounds showed very low solubility, which we suspected may
have contributed to the lack of antimicrobial activity among some of them. To test whether
increasing solubility of any of these compounds would positively affect their antimicrobial
activities, we evaluated the HCl salts of the amines of compounds 6c, 6h and 6i. These
compounds did indeed show higher solubility in aqueous solutions. However, antimicrobial
activity was not observed for any of the HCl salts according to the Kirby Bauer disk diffusion
method (data not shown). Exposing PhoR kinase to these compounds also revealed that the
HCl salts were significantly lessꢀpotent HK inhibitors than their lessꢀsoluble counterparts
(data not shown). This finding is consistent with the idea that the amino groups of compounds
6c, 6h and 6i contribute significantly to their interactions with and inhibition of kinases.
Indeed, according to the design and docking studies, the amino group is expected to create a
hydrogen bond with the conserved Asp533 residue in the HK, which is usually involved in
binding of ATP (Figure 6). In summary, the HCl modification of this group is not a useful
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strategy to improve the potency of the HK inhibitors, and thus, other strategies to increase
solubility should be explored.
2.3.9 Adjuvant activity
Because TCSs are involved in increasing the fitness and adaptability of bacteria, we wondered
whether sublethal concentrations of some of the compounds might exhibit adjuvant activity,
rendering various antimicrobial resistant strains sensitive to the relevant antibiotic. All
compounds were evaluated in association with several antibiotics, and one compound showed
clear evidence of adjuvant activity. Table 6 shows the results obtained for compound 6d. The
use of 6d in association with several antibiotics restored the sensitivity of the resistant isolates
tested. Indeed, 6d exerted this effect in both species evaluated (E. coli and S. aureus),
regardless of the resistance mechanism involved. For example, for the extended spectrum beta
lactamase (ESBL) E. coli isolate (ARS 108), the MIC values were >8 ꢁg/mL when
cefotaxime was used alone and <0.03 ꢁg/mL after the addition of 6d as an adjuvant. For S.
aureus, identical results were found for the C1P15 penicillin Gꢀresistant isolate and the
C1BP13 MRSA isolate. However, the addition of 6d did not restore the bactericidal effect of
ofloxacin in the G02ꢀresistant strain. These results show that TCS inhibitor 6d presented
potent bactericidal activity when used alone at concentrations over 32 µg/mL or as adjuvant
combined with an antibiotic at the sublethal concentration of 25 µg/mL for all isolates and
tested antibiotics.
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In the current context, with the emergence of multiꢀresistant bacteria, such as MRSA and
ESBL E. coli, becoming a serious public health problem in terms of therapeutic management,
these results showing that one HK inhibitor (6d) could able to restore the sensitivity of
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resistant isolates, regardless of the mechanism involved, are encouraging.
This effect may
be attributable to the inhibition of HK, which could prevent the expression of the genes
responsible for the development of resistance mechanisms.
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3. CONCLUSIONS
In this study, we designed and synthesized a series of thiophene derivatives as potential HK
inhibitors. Eight of the compounds target HK WalK and inhibit the autophosphorylation
activity of this essential HK. Among these eight compounds, all were able to inhibit multiple
HKs, and the IC50 values were determined. Moreover, they do not inhibit the serine/threonine
kinase IreK or E. coli DNAꢀgyrase. Two of the compounds exhibit significant antimicrobial
activity, and none of the eight compounds have hemolytic activity against sheep red blood
cells. Finally, the use of one of the HK inhibitors (6d) as an adjuvant in association with the
antibiotics tested (amoxicillin or cefotaxim) restored the sensitivity of drugꢀresistant isolates.
As the number of multidrug resistant bacteria, such as MRSA and ESBL E. coli, increases, the
need to develop different strategies to fight these infections is becoming more urgent. The
compounds identified here can be considered as initial leads for the development of improved
antibacterial agents.
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4. EXPERIMENTAL SECTION
.1. Computational Studies: Preparation of Enzyme and Compounds for Docking
The crystal structure of the ATPꢀbinding domain of Histidine kinase WalK was extracted
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from Protein Data Bank (PDB code: 3SL2). All the water molecules except water822 and
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heteroatoms were removed. The protein was completed, hydrogen atoms were added, and
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reprotonated with SPORES. The chemical structures of all the synthesized compounds were
generated using Marvin sketch and were subsequently converted into 3D format using Open
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Babel and prepared with SPORES. A serie of docking experiment were carried out with all
the designed compounds against the ATPꢀbinding domain of WalK using PLANTS for
possible Histidine kinase inhibition activities. The compounds were selected on the basis of
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their scores and their pose, all compounds were reꢀscored with DSX. As a parameter for the
molecular docking, the ant algorithm was set to 1 (highest reliability). The pocket is a 30Å
radius center on Asp533 making sure those inhibitors can fit in the active site. The number of
poses was set to 10 with a clustering RMSD of 2Å and the pyrrole and amide bond of the
pyrrolamide inhibitor was used as a shape constraint. Tyr507, Ile536 and Asn503 were set as
flexible side chains. The PLANT scoring function was set to chemplp and sulfur acceptors
were enabled. The finally obtained docked complexes were subsequently visualized using
PyMol.
4.2. Chemistry.
General. All reagents were purchased from the Aldrich Chemical Co. and used without any
purification. Solvents were distilled from the appropriate drying agents immediately prior to
use. NMR spectra were recorded with a Bruker Avance 300 spectrometer (300 and 75 MHz
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for H and C NMR, respectively). Chemical shifts (δ) and coupling constants (J) are given in
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ppm and Hz, respectively, using residual solvent signals as reference for the H and C. The
following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, br s = broad
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signal, dd = double doublet, dt = double of triplet, m = multiplet. High resolution mass spectra
(HRMS) were obtained by electrospray using a TOF analyzer Platform. IR spectra were
obtained using a Jasco FTꢀIR 410 instrument as a thin film on NaCl disc as stated; only
ꢀ1
structurally important peaks (ῡ) are presented in cm . Reactions were monitored with Merck
Kieselgel 60F254 precoated aluminium silica gel plates (0.25 mm thickness). Melting points
were determined on a Stuart scientific SMP10 apparatus and are uncorrected. Flash
chromatography were performed on a Grace Reveleris X2 using 40µm packed silica cartridge.
Flash chromatography experiments were carried out on Silica Gel premium Rf grade (40ꢀ63
µm) or were performed on a Grace Reveleris X2 using 40µm packed silica cartridge. HPLC
analyses were obtained on the Waters Alliance 2795 using the following conditions: thermo
Hypersil C18 column (3 µm, 50 mm L x 2.1 mm ID), 20°C column temperature, 0.2 mL/min
flow rate, photodiodearray detection (210ꢀ400 nm), mobile phase consistent of a gradient of
water and acetonitrile (each containing 0.1% trifluoroacetic acid). UPLC analyses were
obtained on the Waters Acquity HꢀClass using the following conditions: Waters Acquity BEH
C18 column (1.7µm, 50 mm x 2.1 mm), 25°C column temperature, 0.5 mL/min flow rate,
photodiodearray detection (TUV – 214nm), mobile phase consistent of a gradient of water and
acetonitrile (each containing 0.1% of formic acid). The purity of all synthetic compounds was
determined by HPLC analysis and was greater than 95%.
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(E) and (Z)ꢀ2ꢀ(5ꢀbromoꢀ2ꢀthienyl)propꢀ1ꢀenꢀ1ꢀol (4a and 4b). Compounds 4a and 4b were
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prepared according to a procedure described by Lucas or minor modifications thereof.
solution of 2ꢀacetylꢀ5ꢀbromothiophene (2) (4.0 g, 19.5 mmol), hydroxylamine hydrochloride
1.63 g, 23.4 mmol) and sodium acetate trihydrate (3.18 g, 23.4 mmol) in a mixture of
A
(
absolute ethanol and water (55 mL, 4/1, v/v) was heated under refluxed for 3 hours. The
solvent was removed in vacuo and the residue was purified by column chromatography (40ꢀ
2 2
63 µm) on silica using a mixture of CH Cl /MeOH (98/2, v/v) to give the oxime 4a (2.33 g,
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54%) as white solid; mp 134ꢀ136 °C; R 0.35 (CH Cl ). H NMR (DMSOꢀd ) δ: 2.12 (s, 3H,
f
2
2
6
13
CH
3
), 7.16 (d, 1H, J = 4.0 Hz), 7.18 (d, 1H, J = 4.0 Hz), 11.34 (s, 1H, NꢀOH). C NMR
DMSOꢀd ) δ: 11.2 (CH ), 112.4 (C), 126.9 (CH), 130.6 (CH), 142.6 (C), 149.3 (C). IR: 3233
OH), 1428 (ῡC=N), 781 (ῡCꢀBr). Moreover, using a mixture of CH
(
(
6
3
ῡ
Cl
2 2
/MeOH (80/20, v/v) to
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give the oxime 4b (1.90 g, 45%) as a white solid; mp 175ꢀ177 °C; R
f
0.06 (CH
2
Cl
2
). H NMR
(DMSOꢀd ) δ: 2.23 (s, 3H, CH ), 7.27 (d, 1H, J = 4.1 Hz), 7.31 (d, 1H, J = 4.1 Hz), 11.89 (s,
6
3
13
1
H, NꢀOH). C NMR (DMSOꢀd
6 3
) δ: 18.4 (CH ), 117.4 (C), 128.6 (CH), 129.1 (CH), 133.1
(
C), 144.9 (C). IR: 1411 (ῡC=N), 791 (ῡCꢀBr) (The IR spectra show no absorption band for the –
6
3
OH stretching in according to the litterature).
Nꢀ(5ꢀbromoꢀ2ꢀthienyl)acetamide (3). Compounds 3 was prepared according to a procedure
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described by Lucas or minor modifications thereof. To a solution of 4a (2.330 g, 10.6 mmol)
in a mixture of acetone and water (54 mL, 3.5/1, v/v) was added 4ꢀmethylbenzeneꢀ1ꢀsulfonyl
chloride (2.231 g, 11.7 mmol) and sodium hydroxide (0.468 g, 11.7 mmol) at 0°C. The
reaction was allowed to warm to room temperature and heated to 60 °C during 1.5 hours.
After cooling to room temperature, the solvent was removed in vacuo. The residue is
dissolved in ethyl acetate (150 mL) and washed with water (3*40 mL), aqueous saturated
NaHCO
3 4
(3*40 mL) and brine (3*40 mL). The organic layer was dried over MgSO , filtered
and concentrated. The residue was purified by flash chromatography (silica gel, PE/EtOAc;
10/90 ramping to 100/0) provided 3 (2.054 g, 88%) as a pale grey solid; mp 135ꢀ137 °C; R
f
1
0
.20 (PE/EtOAc: 40/60; v/v). H NMR (DMSOꢀd
6
) δ: 2.06 (s, 3H, CH
3
), 6.41 (d, 1H, J = 4.1
13
Hz), 6.94 (d, 1H, J = 4.1 Hz), 11.40 (s, 1H, NH). C NMR (DMSOꢀd ) δ: 22.2 (CH ), 102.4
6
3
(
C), 109.8 (CH), 126.6 (CH), 140.4 (C), 166.7 (CO). IR: 3242, 3202 (ῡNH), 1562 (ῡC=O), 781
(ῡCꢀBr).
General Synthetic Procedure A for the synthesis of 6aꢀ6c and 6gꢀ6i. Compounds 6aꢀ6c and
gꢀ6i were prepared according to a procedure described by Slevin et al. or minor
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4
modifications thereof. To a solution of the thiophene (2.30 mmol), the corresponding
aminophenylboronic acid (HCl salt) (2.30 mmol) and Pd(PPh (5 mol%) in a mixture of
3 4
)
toluene/ethanol (16 mL, 2.3/1, v/v) was added 2M aqueous sodium carbonate (4.5 mL). The
mixture was refluxed during 16 hours. The cold solution was diluted with ethyl acetate (50
mL), filtered through a Celite pad and the filtrate was diluted with water (60 mL). The
aqueous solution was extracted with ethyl acetate (3*50 mL). The organic phases were
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
4
combined, dried over MgSO , filtered, and concentrated under reduced pressure to give the
crude compound. The residue was purified by flash column chromatography (silica gel,
AtOAc/PE, 0/100 ramping to 100/0, v/v) to give the desired compound.
2ꢀ(5ꢀnitroꢀ2ꢀthienyl)benzenamine (6a). The desired product was dissolved in a small
amount of ethyl acetate and 150 mL of cold pentane (ꢀ20°C) was added, resulting in
precipitation of a solid. The solid was filtered, washed twice with 20 mL of cold pentane and
1
suctionꢀdried to yield 6a as a red solid; R
f
0.30 (PE/EtOAc: 80/20; v/v). H NMR (DMSOꢀd
6
)
δ: 5.47 (s, 2H, NH
2
), 6.68 (t, 1H, J = 7.5Hz), 6.86 (d, 1H, J = 8.1Hz), 7.16 (t, 1H, J = 7.7Hz),
1
3
7.33 (d, 1H, J = 7.7Hz), 7.42 (d, 1H, J = 4.4Hz), 8.15 (d, 1H, J = 4.4Hz). C NMR (DMSOꢀ
d ) δ: 116.5 (CꢀNH ), 117.1 (CH), 117.4 (CH), 125.7 (CH), 130.0 (CH), 130.8 (CH), 130.9
6
2
+
(
CH), 146.4 (C), 148.5 (C), 150.7 (C). IR: 3434, 3356 (ῡNH2). HRMS: [M + H] calcd for
S, 221.0385; found, 221.0384.
3ꢀ(5ꢀnitroꢀ2ꢀthienyl)benzenamine (6b). The desired product was dissolved in a small
10 9 2 2
C H N O
amount of ethyl acetate and 150 mL of cold pentane (ꢀ20°C) was added, resulting in
precipitation of a solid. The solid was filtered, washed twice with 20 mL of cold pentane, and
1
suctionꢀdried to yield 6b as an orange solid; R
DMSOꢀd ) δ: 5.40 (s, 2H, NH
7.49 (d, 1H, J = 4.4Hz), 8.12 (d, 1H, J = 4.4Hz). C NMR (DMSOꢀd
f
0.24 (PE/EtOAc: 70/30; v/v). H NMR
(
6
2
), 6.65ꢀ6.69 (m, 1H), 6.93ꢀ6.96 (m, 2H), 7.11ꢀ7.16 (m, 1H),
1
3
6
) δ: 110.8 (CH), 113.7
(
CH), 116.0 (CH), 123.2 (CH), 130.1 (CH), 131.4 (CH), 132.0 (CꢀNH ), 148.6 (C), 149.6 (C),
2
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+
152.9 (C). IR: 3439, 3342 (ῡNH2). HRMS: [M + H] calcd for C H N O S, 221.0385; found,
10
9
2
2
221.0384.
4
ꢀ(5ꢀnitroꢀ2ꢀthienyl)benzenamine (6c). The desired product was dissolved in a small
amount of ethyl acetate and 150 mL of cold pentane (ꢀ20°C) was added, resulting in
precipitation of a solid. The solid was filtered, washed twice with 20 mL of cold pentane, and
10
11
12
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16
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20
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46
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48
49
50
51
52
53
54
55
56
57
58
59
60
1
suctionꢀdried to yield 6c as a red solid; R 0.20 (PE/EtOAc: 70/30; v/v). H NMR (DMSOꢀd )
f
6
δ: 5.92 (s, 2H, NH
2
), 6.62 (d, 2H, J = 8.6Hz), 7.36 (d, 1H, J = 4.5Hz), 7.52 (d, 2H, J = 8.6Hz),
13
8
.06 (d, 1H, J = 4.5Hz). C NMR (DMSOꢀd
6
) δ: 113.9 (2CH), 118.5 (CꢀNH
2
), 120.3 (CH),
1
27.8 (2CH), 132.1 (CH), 145.8 (C), 151.6 (C), 154.9 (C). IR: 3464, 3371 (ῡNH2). HRMS: [M
+
+ H] calcd for C10
H
9
2
N O
2
S, 221.0385; found, 221.0386.
1
ꢀ(5ꢀ(2ꢀaminophenyl)thiophenꢀ2ꢀyl)ethanone (6g). Compound 6g was obtained as a yellow
1
powder; R
f
0.41 (PE/EtOAc: 60/40; v/v). H NMR (DMSOꢀd
6
) δ: 2.54 (s, 3H, CH
= 1.1Hz), 6.82 (dd, 1H, J = 8.1Hz, J = 0.9Hz), 7.08ꢀ
= 1.4Hz), 7.38 (d, 1H, J = 3.9Hz), 7.93 (d, 1H,
), 116.3 (CH), 116.9 (CH), 117.3 (CꢀNH ),
26.6 (CH), 129.8 (CH), 130.0 (CH), 134.7 (CH), 142.0 (C), 145.7 (C), 150.2 (C), 190.5 (CO).
3
), 5.27 (s,
2
H, NH
2
), 6.64 (td, 1H, J
1
= 7.7Hz, J
2
1
2
7
.13 (m, 1H, HAR), 7.23 (dd, 1H, J
1
= 7.7Hz, J
2
1
3
J = 3.9Hz). C NMR (DMSOꢀd
6
) δ: 26.4 (CH
3
2
1
+
IR: 3401, 3333 (ῡNH2), 1633 (ῡC=O). HRMS: [M + H] calcd for C12
18.0632.
H12NOS, 218.0640; found,
2
1ꢀ(5ꢀ(3ꢀaminophenyl)thiophenꢀ2ꢀyl)ethanone (6h). Compound 6h was obtained as a pale
1
yellow powder; R
f
0.30 (PE/EtOAc: 60/40; v/v). H NMR (DMSOꢀd
6
) δ: 2.52 (s, 3H, CH
3
),
5
1
1
.31 (s, 2H, NH ), 6.59 (d, 1H, J = 7.2 Hz), 6.88ꢀ6.91 (m, 2H), 7.09 (t, 1H, J = 7.7Hz), 7.46 (d,
2
13
H, J = 4.0Hz), 7.90 (d, 1H, J = 4.0Hz). C NMR (DMSOꢀd
6
) δ: 26.3 (CH
3
), 110.9 (CH),
13.5 (CH), 114.9 (CH), 124.2 (CH), 129.8 (CH), 133.2 (CꢀNH
2
), 135.1 (CH), 142.0 (C),
+
149.4 (C), 152.7 (C), 190.5 (CO). IR: 3372, 3218 (ῡNH2), 1642 (ῡC=O). HRMS: [M + H] calcd
for C12 12NOS, 218.0640; found, 218.0641.
H
2
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1ꢀ(5ꢀ(4ꢀaminophenyl)thiophenꢀ2ꢀyl)ethanone (6i). Compound 6i was obtained as a dark
1
yellow powder; R
f
0.25 (PE/EtOAc: 60/40; v/v). H NMR (DMSOꢀd
6
) δ: 2.48 (s, 3H, CH
3
),
5
7
1
2
.62 (s, 2H, NH ), 6.60 (d, 2H, J = 8.5Hz), 7.33 (d, 1H, J = 4.0Hz), 7.44 (d, 2H, J = 8.5Hz),
13
.83 (d, 1H, J = 4.0Hz). C NMR (DMSOꢀd
6
3 2
) δ: 26.1 (CH ), 113.9 (2CH), 120.1 (CꢀNH ),
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
21.5 (CH), 127.2 (2CH), 135.5 (CH), 139.7 (C), 150.3 (C), 153.9 (C), 190.0 (CO). IR: 3327,
+
3234 (ῡNH2), 1626 (ῡC=O). HRMS: [M + H] calcd for C H NOS, 218.0640; found, 218.0640.
12
12
General Synthetic Procedure B for the synthesis of 6dꢀ6f and 6jꢀ6l. Compounds 6dꢀ6f, 6jꢀ
l and 6sꢀ6t were prepared according to a procedure described by Bugge et al. or minor
6
65
modifications thereof. To a solution of the thiophene (2.90 mmol), the corresponding
hydroxyphenylboronic acid (2.90 mmol) and Pd(PPh (5 mol%) in a mixture of 1,4ꢀ
dioxane/water (22 mL, 1/1, v/v) was added fine powder K CO (11.6 mmol). The reaction
3 4
)
2
3
was then stirred at 80°C during 6 hours. The cold solution was diluted with ethyl acetate (50
mL), filtered through a Celite pad and the filtrate was diluted with water (60 mL). The
aqueous solution was extracted with ethyl acetate (3*50 mL). The organic phases were
combined, dried over MgSO
crude residue.
4
, filtered, and concentrated under reduced pressure to give the
2
ꢀ(5ꢀnitrothiophenꢀ2ꢀyl)phenol (6d). The crude residue was purified by flash column
chromatography (silica gel, CH Cl /Cyclohexane, 0/100 ramping to 100/0) to give the desired
compound. The desired product was dissolved in CH Cl (100 mL). The organic phase was
2
2
2
2
ꢀ1
washed five times with aqueous solution of NaOH (6 mol.L ). Then, the aqueous phases were
combined, chilled on an ice bath with stirring, acidified to pH 2ꢀ3 and extract with CH Cl
2
2
(
4*50 mL). The organics phases were combined, dried over MgSO
4
, filtered, and concentrated
1
under reduced pressure to give 6d as an orange powder; R
f
0.20 (CH
2
Cl
2
). H NMR (DMSOꢀ
d
6
) δ: 6.95 (t, 1H, J = 7.6Hz), 7.04 (d, 1H, J = 8.0Hz), 7.28ꢀ7.33 (m, 1H), 7.77 (d, 1H, J =
4
.6Hz), 7.95 (dd, 1H, J = 8.0Hz, J = 1.1Hz), 8.12 (d, 1H, J = 4.6Hz), 11.32 (br s, 1H, OH).
1
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C NMR (DMSOꢀd ) δ: 116.5 (CH), 118.4 (CꢀOH), 119.9 (CH), 123.3 (CH), 127.5 (CH),
6
+
1
29.5 (CH), 131.2 (CH), 147.3 (C), 149.4 (C), 154.5 (C). IR: 3433 (ῡOH). HRMS: [M + H]
NO S, 222.0225; found, 222.0226.
ꢀ(5ꢀnitrothiophenꢀ2ꢀyl)phenol (6e). The crude residue was purified by flash column
chromatography (silica gel, CH Cl /Cyclohexane, 0/100 ramping to 100/0) to give the desired
compound. The desired product was dissolved in CH Cl (100 mL). The organic phase was
calcd for C10
H
8
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
2
2
ꢀ1
washed five times with aqueous solution of NaOH (6 mol.L ). Then, the aqueous phases were
combined, chilled on an ice bath with stirring, acidified to pH 2ꢀ3 and extract with CH Cl
, filtered, and concentrated
2
2
(4*50 mL). The organics phases were combined, dried over MgSO
4
2
1
under reduced pressure to give 6e as a brown powder; R 0.15 (CH
f
2 6
Cl ). H NMR (DMSOꢀd )
δ: 6.88ꢀ6.91 (m, 1H), 7.14ꢀ7.16 (m, 1H), 7.23ꢀ7.33 (m, 2H), 7.60 (d, 1H, J = 4.4Hz), 8.15 (d,
13
1
H, J = 4.4Hz), 9.89 (br s, 1H, OH). C NMR (DMSOꢀd
6
) δ: 112.7 (CH), 117.1 (CH), 117.5
(CH), 123.9 (CH), 130.7 (CH), 131.4 (CH), 132.7 (CꢀOH), 149.1 (C), 151.7 (C), 158.2 (C).
+
IR: 3414 (ῡOH). HRMS: [M + H] calcd for C10
H
8
NO
3
S, 222.0225; found, 222.0223.
4ꢀ(5ꢀnitrothiophenꢀ2ꢀyl)phenol (6f). The crude residue was purified by flash column
chromatography (silica gel, CH Cl /Cyclohexane, 0/100 ramping to 100/0) to give the desired
2
2
compound. The desired product was dissolved in CH
washed five times with aqueous solution of NaOH (6 mol.L ). Then, the aqueous phases were
combined, chilled on an ice bath with stirring, acidified to pH 2ꢀ3 and extract with CH Cl
4*50 mL). The organics phases were combined, dried over MgSO , filtered, and concentrated
under reduced pressure to give 6f as a red powder; R 0.34 (PE/EtOAc: 80/20; v/v). H NMR
2 2
Cl (100 mL). The organic phase was
ꢀ1
2
2
(
4
1
f
(DMSOꢀd
6
) δ: 6.87 (d, 2H, J = 8.7Hz), 7.49 (d, 1H, J = 4.4Hz), 7.68 (d, 2H, J = 8.7Hz), 8.11
13
(d, 1H, J = 4.4Hz), 10.19 (br s, 1H, OH). C NMR (DMSOꢀd
6
) δ: 116.3 (2CH), 122.1 (CH),
122.5 (CꢀOH), 128.1 (2CH), 131.7 (CH), 147.6 (C), 152.9 (C), 159.8 (C). IR: 3342 (ῡOH).
+
HRMS: [M + H] calcd for C10
H
8
NO
3
S, 222.0225; found, 222.0221.
2
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1ꢀ(5ꢀ(2ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)ethanone (6j). The crude residue was purified by
flash column chromatography (silica gel, EtOAc/PE, 0/100 ramping to 100/0, v/v) to give the
desired compound. The desired product was dissolved in a small amount of ethyl acetate and
150 mL of cold pentane (ꢀ20°C) was added, resulting in precipitation of a solid. The solid was
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
filtered, washed twice with 20 mL of cold pentane, and suctionꢀdried to yield 6j as a beige
1
powder; R 0.50 (PE/EtOAc: 60/40; v/v). H NMR (DMSOꢀd ) δ: 2.52 (s, 3H, CH ), 6.90 (t,
f
6
3
1H, J = 7.5Hz), 6.99 (d, 1H, J = 7.9Hz), 7.19ꢀ7.25 (m, 1H), 7.71 (d, 1H, J = 4.1Hz), 7.77 (dd,
13
1H, J
1
= 7.8Hz, J
2
= 1.2Hz), 7.88 (d, 1H, J = 4.1Hz), 10.66 (br s, 1H, OH). C NMR
(DMSOꢀd
6
3
) δ: 26.5 (CH ), 116.5 (CH), 119.5 (C), 119.6 (CH), 125.4 (CH), 127.9 (CH), 130.0
(CH), 133.6 (CH), 142.2 (C), 147.7 (C), 154.2 (C), 190.9 (CO). IR: 3168 (ῡOH), 1614 (ῡC=O).
+
HRMS: [M + H] calcd for C H O S, 219.0480; found, 219.0480.
1
2
11
2
1ꢀ(5ꢀ(3ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)ethanone (6k). The crude residue was purified by
flash column chromatography (silica gel, EtOAC/PE, 0/100 ramping to 100/0, v/v) to give 6k
1
as a pale yellow powder; R
CH
f
0.50 (PE/EtOAc: 50/50; v/v). H NMR (DMSOꢀd
6
) δ: 2.53 (s, 3H,
3
), 6.80ꢀ6.83 (m, 1H), 7.11 (t, 1H, J = 2.0Hz), 7.18ꢀ7.29 (m, 2H), 7.56 (d, 1H, J = 4.0Hz),
1
3
7
1
1
.92 (d, 1H, J = 4.0Hz), 9.75 (br s, 1H, OH). C NMR (DMSOꢀd ) δ: 26.4 (CH ), 112.6 (CH),
6 3
16.3 (CH), 116.9 (CH), 124.9 (CH), 130.4 (CH), 133.9 (CꢀOH), 135.1 (CH), 142.5 (C),
+
51.6 (C), 158.0 (C), 190.6 (CO). IR: 3251 (ῡOH), 1619 (ῡC=O). HRMS: [M + H] calcd for
C
12
H
11
O
2
S, 219.0480; found, 219.0479.
1ꢀ(5ꢀ(4ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)ethanone (6l). The crude residue was purified by
flash column chromatography (silica gel, PE/EtOAc, 0/100 ramping to 100/0, v/v) to give 6l
1
as a dark yellow powder; R
CH
= 4.0Hz), 9.93 (br s, 1H, OH). C NMR (DMSOꢀd
CHAR), 123.8 (CꢀOH), 127.6 (2CHAR), 135.3 (CHAR), 141.1 (C), 152.4 (C), 158.6 (C), 190.3
f
0.35 (PE/EtOAc: 50/50; v/v). H NMR (DMSOꢀd
6
) δ: 2.50 (s, 3H,
3
), 6.83 (d, 2H, J = 8.6Hz), 7.44 (d, 1H, J = 4.0Hz), 7.59 (d, 2H, J = 8.6Hz), 7.87 (d, 1H, J
1
3
6
3
) δ: 26.2 (CH ), 116.0 (2CHAR), 123.1
(
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+
(CO). IR: 3117 (ῡ ), 1600 (ῡ ). HRMS: [M + H] calcd for C H O S, 219.0480; found,
OH
C=O
12 11
2
2
19.0481.
General Synthetic Procedure C for the synthesis of 6mꢀ6o. Compounds 6mꢀ6o and 6u
were prepared according to a procedure described by Valant et al. or minor modifications
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
60
6
6
thereof. To a solution of 3 (1.82 mmol), the corresponding aminophenylboronic acid (HCl
salt) (3.64 mmol) and Pd[PPh ] Cl (10 mol%) in DMF (14 mL) was added 2M aqueous
3
2
2
potassium carbonate (3.7 mL). The mixture was heated to 70°C during 1 hour. The cold
solution was diluted with ethyl acetate (50 mL), filtered through a Celite pad and the filtrate
was diluted with water (60 mL). The aqueous solution was extracted with ethyl acetate (3*50
4
mL). The organic phases were combined, washed with brine (8*40 mL), dried over MgSO ,
filtered, and concentrated under reduced pressure to give the crude compound. The residue
was purified by flash column chromatography (silica gel, EtOAC/PE, 25/75 ramping to 100/0,
v/v) to give the desired compound.
Nꢀ(5ꢀ(2ꢀaminophenyl)thiophenꢀ2ꢀyl)acetamide (6m). Compound 6m was obtained as a
1
brown powder; R
f
0.20 (PE/EtOAc: 40/60; v/v). H NMR (DMSOꢀd
6
) δ: 2.07 (s, 3H, CH
3
),
4
2
2
.98 (s, 2H, NH ), 6.56ꢀ6.63 (m, 2H), 6.75 (dd, 1H, J = 8.0Hz, J = 1.0Hz), 6.94ꢀ7.02 (m,
2
1
2
13
1 2 6
H), 7.10 (dd, 1H, J = 7.6Hz, J = 1.4Hz), 11.13 (br s, 1H, OH). C NMR (DMSOꢀd ) δ:
3
2.5 (CH ), 110.8 (CHAR), 115.6 (CHAR), 116.7 (CHAR), 118.8 (C), 122.1 (CHAR), 128.0
(CHAR), 129.6 (CHAR), 131.2 (C), 139.0 (C), 145.4 (C), 166.2 (CO). IR: 3462, 3370 (ῡNH2),
+
3
234 (ῡNH), 1570 (ῡC=O). HRMS: [M + H] calcd for C12
H
13
N
2
OS, 233.0749; found, 233.0745.
Nꢀ(5ꢀ(3ꢀaminophenyl)thiophenꢀ2ꢀyl)acetamide (6n). Compound 6n was obtained as a white
1
powder; R
H, NH ), 6.42 (dd, 1H, HAR, J
(m, 2H, HAR), 6.99 (t, 1H, HAR, J = 7.8Hz), 7.05 (d, 1H, HAR, J = 3.9Hz), 11.14 (s, 1H, NH).
f
0.20 (PE/EtOAc: 30/70; v/v). H NMR (DMSOꢀd
6
3
) δ: 2.07 (s, 3H, CH ), 5.12 (s,
2
2
1
= 7.9Hz, J
2
= 1.1Hz), 6.57 (d, 1H, HAR, J = 3.9Hz), 6.71ꢀ6.76
1
3
6 3
C NMR (DMSOꢀd ) δ: 22.5 (CH ), 110.0 (CHAR), 111.1 (CHAR), 112.4 (CHAR), 112.5
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(CH ), 119.6 (CH ), 129.4 (CH ), 134.1 (C), 134.8 (C), 138.9 (C), 149.1 (C), 166.2 (CO).
AR
AR
AR
+
IR: 3469, 3372 (ῡNH2), 3274 (ῡNH), 1562 (ῡC=O). HRMS: [M + H] calcd for C12
13 2
H N OS,
233.0749; found, 233.0749.
Nꢀ(5ꢀ(4ꢀaminophenyl)thiophenꢀ2ꢀyl)acetamide (6o). Compound 6o was obtained as a beige
0
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powder; R
f
0.15 (PE/EtOAc: 30/70; v/v). H NMR (DMSOꢀd
6
) δ: 2.05 (s, 3H, CH
3
), 5.57 (s,
2H, NH ), 6.52 (d, 1H, J = 3.9Hz), 6.60 (d, 2H, J = 8.5Hz), 6.90 (d, 1H, J = 3.9Hz), 7.24 (d,
2
13
2
6 3
H, J = 8.5Hz), 11.04 (br s, 1H, NH). C NMR (DMSOꢀd ) δ: 22.5 (CH ), 111.2 (CH),
114.7(2CH), 117.3 (CH), 122.9 (C), 125.6 (2CH), 134.7 (C), 137.3 (C), 146.8 (C), 166.0 (CO).
+
IR: 3388, 3321 (ῡNH2), 3181 (ῡNH), 1581 (ῡC=O). HRMS: [M + H] calcd for C12
H
13
N
2
OS,
233.0749; found, 233.0754.
General Synthetic Procedure D for the synthesis of 6pꢀ6r. Compounds 6pꢀ6r were prepared
67
according to a procedure described by Ashwell et al. or minor modifications thereof. To a
solution of 3 (4.54 mmol), the corresponding hydroxyphenylboronic acid (9.08 mmol) and
3 2 2
Pd[PPh ] Cl (10 mol%) in DMF (34 mL) was added fine powder of cesium carbonate (18.2
mmol) and water (10mL). The mixture was heated to 80°C during 3 hours. The cold solution
was diluted with ethyl acetate (50 mL), filtered through a Celite pad and the filtrate was
diluted with water (80 mL). The aqueous solution was extracted with ethyl acetate (3*80 mL).
4
The organic phases were combined, washed with brine (12*50 mL), dried over MgSO ,
filtered, and concentrated under reduced pressure to give the crude compound. The residue
was purified by flash column chromatography (silica gel, EtOAC/PE, 25/75 ramping to 100/0,
v/v) to give the desired compound.
Nꢀ(5ꢀ(2ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)acetamide (6p). Compound 6p was obtained as a
1
beige powder; R
f
0.45 (PE/EtOAc: 20/80; v/v). H NMR (DMSOꢀd
6
) δ: 2.06 (s, 3H, CH
= 8.1Hz, J = 1.1Hz),
= 1.6Hz), 9.99 (br s,
3
),
6.60 (d, 1H, HAR, J = 4.0Hz), 6.78ꢀ6.83 (m, 1H, HAR), 6.90 (dd, 1H, J
.01ꢀ7.06 (m, 1H), 7.31 (d, 1H, J = 4.0Hz), 7.52 (dd, 1H, J = 7.8Hz, J
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1H, OH), 11.04 (br s, 1H, NH). C NMR (DMSOꢀd ) δ: 22.6 (CH ), 110.4 (CH), 116.1 (CH),
6
3
1
19.4 (CH), 121.3 (C), 121.7 (CH), 126.9 (CH), 127.2 (CH), 129.9 (C), 140.0 (C), 153.0 (C),
+
1
66.1 (CO). IR: 3073 (ῡOH), 1563 (ῡC=O). HRMS: [M + H] calcd for C12
H
12NO
2
S, 234.0589;
found, 233.0588.
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Nꢀ(5ꢀ(3ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)acetamide (6q). Compound 6q was obtained as a
1
beige powder; R 0.35 (PE/EtOAc: 20/80; v/v). H NMR (DMSOꢀd ) δ: 2.07 (s, 3H, CH ),
f
6
3
6
.58 (d, 1H, J = 3.9Hz), 6.63 (dd, 1H, J
1
= 7.7Hz, J
2
= 2.0Hz), 6.94 (t, 1H, J = 1.9Hz), 6.99 (d,
13
1
H, J = 8.0Hz), 7.12ꢀ7.18 (m, 2H), 9.46 (br s, 1H, OH), 11.20 (br s, 1H, NH). C NMR
(
6 3
DMSOꢀd ) δ: 22.5 (CH ), 112.2 (2CH), 113.7 (CH), 115.5 (CH), 120.3 (CH), 130.0 (CH),
133.1 (C), 135.6 (C), 139.4 (C), 157.8 (C), 166.3 (CO). IR: 3248 (ῡOH), 1576 (ῡC=O). HRMS:
+
[
M + H] calcd for C12
H12NO
2
S, 234.0589; found, 233.0590.
Nꢀ(5ꢀ(4ꢀhydroxyphenyl)thiophenꢀ2ꢀyl)acetamide (6r). Compound 6r was obtained as a pale
1
purple powder; R
f
0.50 (PE/EtOAc: 10/90; v/v). H NMR (DMSOꢀd
6
) δ: 2.06 (s, 3H, CH
3
),
6
.54 (d, 1H, J = 3.9Hz), 6.76 (d, 2H, J = 8.6Hz), 6.99 (d, 1H, J = 3.9Hz), 7.35 (d, 2H, J =
13
8
6 3
.6Hz), 9.49 (br s, 1H, OH), 11.09 (br s, 1H, NH). C NMR (DMSOꢀd ) δ: 22.5 (CH ), 111.2
(
CH), 115.8 (2CH), 118.5 (CH), 125.5 (C), 126.0 (2CH), 133.8 (C), 138.1 (C), 156.5 (C),
+
1
66.1 (CO). IR: 3266 (ῡ ), 1574 (ῡ ). HRMS: [M + H] calcd for C H NO S, 234.0589;
OH
C=O
12 12
2
found, 233.0589.
2ꢀ(2,3ꢀdimethoxyphenyl)ꢀ5ꢀnitrothiophene (6s). To a solution of 1 (5.49 mmol), 2,3ꢀ
Dimethoxyphenyl boronic acid (5.49 mmol) and Pd(PPh (5 mol%) in 1,4ꢀdioxane (23 mL)
3 4
)
was added potassium carbonate (22 mmol) and water (23 mL). The mixture was heated to
80°C during 6 hours. The cold solution was diluted with ethyl acetate (50 mL), filtered
through a Celite pad and the filtrate was diluted with water (80 mL). The aqueous solution
was extracted with ethyl acetate (3*80 mL). The organic phases were combined, dried over
4
MgSO , filtered, and concentrated under reduced pressure to give the crude compound. The
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residue was purified by flash column chromatography (silica gel, CH Cl /cyclohexane, 0/100
2
2
1
ramping to 100/0) to give 6s as a yellow powder; R
DMSOꢀd ) δ: 3.88 (s, 3H, OCH ), 3.89 (s, 3H, OCH
= 7.2Hz, J = 2.3Hz), 7.80 (d, 1H, HAR, J = 4.6Hz), 8.14 (d, 1H, HAR, J = 4.6Hz).
C NMR (DMSOꢀd ) δ: 56.0 (OCH ), 59.9 (OCH ), 114.3 (CH), 118.8 (CH), 124.7 (C),
f
0.40 (PE/EtOAc: 80/20; v/v). H NMR
(
6
3
3
), 7.15ꢀ7.24 (m, 2H, 2HAR), 7.61 (dd,
1H, HAR, J
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124.8 (CH), 125.0 (CH), 129.6 (CH), 145.3 (C), 145.4 (C), 150.5 (C), 152.8 (C). IR: 1258 (ῡCꢀ
+
O
). HRMS: [M + H] calcd for C12
H
12NO
4
S, 266.0487, found 266.0486.
1ꢀ(5ꢀ(2,3ꢀdimethoxyphenyl)thiophenꢀ2ꢀyl)ethanone (6t). To a solution of 2 (5.49 mmol),
2
,3ꢀDimethoxyphenyl boronic acid (5.49 mmol) and Pd(PPh (5 mol%) in 1,4ꢀdioxane (23
3 4
)
mL) was added potassium carbonate (22 mmol) and water (23 mL). The mixture was heated
to 80°C during 16 hours. The cold solution was diluted with ethyl acetate (50 mL), filtered
through a Celite pad and the filtrate was diluted with water (80 mL). The aqueous solution
was extracted with ethyl acetate (4*80 mL). The organic phases were combined, dried over
4
MgSO , filtered and concentrated under reduced pressure to give the crude compound. The
residue was purified by flash column chromatography (silica gel, EtOAC/PE, 0/100 ramping
1
to 100/0, v/v) to give 6t as a pale yellow powder; R 0.31 (PE/EtOAc: 80/20; v/v). H NMR
f
(
DMSOꢀd
.46 (dd, 1H, J
NMR (DMSOꢀd
6
) δ: 2.54 (s, 3H, CH
= 7.8Hz, J = 1.6Hz), 7.71 (d, 1H, J = 4.1Hz), 7.91 (d, 1H, J = 4.1Hz). C
) δ: 26.6 (CH ), 55.9 (OCH ), 59.8 (OCH ), 113.3 (CH), 119.3 (CH), 124.6
CH), 126.1 (C), 126.6 (CH), 133.5 (CH), 143.5 (C), 145.3 (C), 145.8 (C), 153.1 (C), 191.1
3 3 3
), 3.79 (s, 3H, OCH ), 3.86 (s, 3H, OCH ), 7.08ꢀ7.19 (m, 2H),
1
3
7
1
2
6
3
3
3
(
+
(
CO). IR: 1644 (ῡC=O), 1259 (ῡCꢀO). HRMS: [M + H] calcd for C H O S, 263.0742, found
14 15 3
2
63.0741.
Nꢀ(5ꢀ(2,3ꢀdimethoxyphenyl)thiophenꢀ2ꢀyl)acetamide (6u). To a solution of 3 (1.59 mmol),
2,3ꢀDimethoxyphenyl boronic acid (3.18 mmol) and Pd[PPh Cl (10 mol%) in DMF (12
mL) was added fine powder of potassium carbonate (6.36 mmol) and water (3.5 mL). The
3
]
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mixture was heated to 80°C during 3 hours. The cold solution was diluted with ethyl acetate
(
50 mL), filtered through a Celite pad and the filtrate was diluted with water (50 mL). The
aqueous solution was extracted with ethyl acetate (4*50 mL). The organic phases were
combined, washed with brine (6*40 mL), dried over MgSO , filtered, and concentrated under
4
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reduced pressure to give the crude compound. The residue was purified by flash column
chromatography (silica gel, EtOAC/PE, 10/90 ramping to 100/0, v/v) to give 6u as a beige
1
powder; R
f
0.30 (PE/EtOAc: 40/60; v/v). H NMR (DMSOꢀd
6
) δ: 2.07 (s, 3H, CH
= 8.1Hz, J
= 1.3Hz), 7.31 (d, 1H, J = 4.0Hz), 11.12
), 55.8 (OCH ), 59.6 (OCH ), 110.4 (CH),
10.7 (CH), 118.5 (CH), 122.7 (CH), 124.4 (CH), 127.9 (C), 128.1 (C), 141.5 (C), 144.2 (C),
3
), 3.71 (s,
3
H, OCH
3
), 3.83 (s, 3H, OCH
3
), 6.62 (d, 1H, J = 4.0Hz), 6.91 (dd, 1H, J
=8.0Hz, J
) δ: 22.6 (CH
1
2
= 1.1Hz),
7.06 (t, 1H, J = 8.1Hz), 7.25 (dd, 1H, J
1
2
13
(br s, 1H, NH). C NMR (DMSOꢀd
6
3
3
3
1
1
+
53.2 (C), 166.2 (CO). IR: 3211 (ῡNH), 1569 (ῡC=O), 1302 (ῡCꢀO). HRMS: [M + H] calcd for
S, 278.0851, found 278.0849.
14 3
C H16NO
General Synthetic Procedure E for the synthesis of 7aꢀ7c. Compounds 7aꢀ7c were prepared
according to a procedure described by Bhanu Prasad and Gilbertson or minor modifications
68
thereof. A solution of 6aꢀ6c (2.72 mmol) and 2ꢀiodoethanol (27.2 mmol) was heated to 90°C
under nitrogen atmosphere in a sealed tube during 3 hours. The cold solution was diluted with
ethyl acetate (100 mL), washed with 2M aqueous solution of NaOH (3*30 mL), brine solution
4
(3*30 mL), dried over MgSO , filtered and concentrated under reduced pressure to give the
crude compound. The residue was purified by flash column chromatography (silica gel,
EtOAC/PE, 10/90 ramping to 100/0, v/v) to give the desired compound.
2ꢀ((2ꢀ(5ꢀnitrothiophenꢀ2ꢀyl)phenyl)amino)ethanol (7a). Compound 7a was obtained as a
1
red powder; R
5.8Hz), 3.59 (q, 2H, CH
.5Hz), 6.73 (t, 1H, J = 7.4Hz), 6.82 (d, 1H, J = 8.1Hz), 7.26ꢀ7.33 (m, 2H), 7.42 (d, 1H, J =
f
0.25 (PE/EtOAc: 40/60; v/v). H NMR (DMSOꢀd
6
) δ: 3.16 (q, 2H, CH
2
O, J =
2
N, J = 5.5Hz), 4.79 (t, 1H, OH, J = 5.3Hz), 5.32 (t, 1H, NH, J =
5
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