In Silico Driven Design and Synthesis of Rhodanine Derivatives as Novel Antibacterials Targeting the Enoyl Reductase InhA

Article abstract of DOI:10.1021/acs.jmedchem.5b01620

Here, we report on the design, synthesis, and biological evaluation of 4-thiazolidinone (rhodanine) derivatives targeting Mycobacterial tuberculosis (Mtb) trans-2-enoyl-acyl carrier protein reductase (InhA). Compounds having bulky aromatic substituents at position 5 and a tryptophan residue at position N-3 of the rhodanine ring were the most active against InhA, with IC₅₀ values ranging from 2.7 to 30 μM. The experimental data showed consistent correlations with computational studies. Their antimicrobial activity was assessed against Mycobacterium marinum (Mm) (a model for Mtb), Pseudomonas aeruginosa (Pa), Legionella pneumophila (Lp), and Enterococcus faecalis (Ef) by using anti-infective, antivirulence, and antibiotic assays. Nineteen out of 34 compounds reduced Mm virulence at 10 μM. 33 exhibited promising antibiotic activity against Mm with a MIC of 0.21 μM and showed up to 89% reduction of Lp growth in an anti-infective assay at 30 μM. 32 showed high antibiotic activity against Ef, with a MIC of 0.57 μM.

Full text of DOI:10.1021/acs.jmedchem.5b01620

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Article
In silico driven design and synthesis of rhodanine derivatives
as novel antibacterials targeting the enoyl reductase InhA
Liudas Slepikas, Gianpaolo Chiriano, Remo Perozzo, Sébastien Tardy, Agata Kranjc-Pietrucci, Ophélie
Patthey-Vuadens, Hajer Ouertatani-Sakouhi, Sébastien Kicka, Christopher F. Harrison, Tiziana Scrignari,
Karl Perron, Hubert Hilbi, Thierry Soldati, Pierre Cosson, Eduardas Tarasevicius, and Leonardo Scapozza
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01620 • Publication Date (Web): 05 Jan 2016
Downloaded from http://pubs.acs.org on January 11, 2016
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In silico driven design and synthesis of rhodanine
derivatives as novel antibacterials targeting the enoyl
reductase InhA
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Liudas Slepikas,a,b† Gianpaolo Chiriano,a† Remo Perozzo,a Sébastien Tardy,a Agata KranjcꢀPietrucci,a
Ophélie PattheyꢀVuadens,a Hajer OuertataniꢀSakouhi,c Sébastien Kicka,d Christopher F. Harrison,e
Tiziana Scrignari,f Karl Perron,f Hubert Hilbi,e,g Thierry Soldati,d Pierre Cosson,c Eduardas Taraseꢀ
viciusb and Leonardo Scapozzaa*
aSchool of Pharmaceutical Sciences, Department of Pharmaceutical Biochemistry, University of Geneꢀ
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va and University of Lausanne, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland; Faculty of
Pharmacy, Lithuanian University of Health Sciences, LT 44307 Kaunas, Lithuania; cDepartment of cell
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physiology and metabolism, CMU, Rue MichelꢀServet 1 1211 Geneva, Switzerland; Department of
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Biochemistry, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland; Max von
Pettenkofer Institute, Department of Medicine, LudwigꢀMaximilians University Munich, 80336 Muꢀ
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nich, Germany; Microbiology Unit, Department of Botany and Plant Biology, University of Geneva,
Switzerland; gInstitute of Medical Microbiology, Department of Medicine, University of Zürich, Gloriꢀ
astrasse 30/32, 8006 Zürich, Switzerland.
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ABSTRACT
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Here, we report on the design, synthesis and biological evaluation of 4ꢀthiazolidinone (rhodanine) deꢀ
rivatives targeting Mycobacterial tuberculosis (Mtb) transꢀ2ꢀenoylꢀacyl carrier protein reductase (Inꢀ
hA). Compounds having bulky aromatic substituents at position 5 and a tryptophan residue at position
Nꢀ3 of the rhodanine ring were the most active against InhA with IC50 values ranging from 2.7 µM to
30 µM. The experimental data showed consistent correlations with computational studies. Their antiꢀ
microbial activity was assessed against Mycobacterium marinum (Mm) (a model for Mtb), Pseudomoꢀ
nas aeruginosa (Pa), Legionella pneumophila (Lp) and Enterococcus faecalis (Ef), by using antiꢀ
infective, antiꢀvirulence and antibiotic assays. 19 out of 34 compounds reduced Mm virulence at 10
µM. 33 exhibited promising antibiotic activity against Mm with a MIC of 0.21 µM and showed up to
89% reduction of Lp growth in an antiꢀinfective assay at 30 µM. 32 showed high antibiotic activity
against Ef with a MIC of 0.57 µM.
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INTRODUCTION
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Infectious diseases are a serious threat worldwide as emerging bacterial resistances to antibiotics raises
concerns about the use of antibacterial agents in clinical practice.1 Thus, the search for new drugs effecꢀ
tive for the treatment of bacterial infections is of high priority. Focusing on known validated intracelluꢀ
lar targets remains a valid approach to identify new drug candidates with novel chemical structures.2
One of the strategies adopted to control bacterial pathogens is to target the biosynthesis pathway of
fatty acids (FASꢀII), in which the highly conserved enoylꢀacyl carrier protein reductase (InhA) plays a
key role.3 The suitability of targeting InhA for combating tuberculosis has been validated by the firstꢀ
line antitubercular drug isoniazid, a very powerful mycobacterial InhA inhibitor.4 Furthermore, the catꢀ
alytic domain of InhA is well characterized and amenable for structureꢀbased drug design. Since isoꢀ
niziad, a proꢀdrug converted by catalaseꢀperoxidase (KatG) into an InhA inhibitor, was introduced in
1952, the rising resistance in clinical practice due to KatG modifications calls for the discovery of new
drugs acting directly against InhA.5 Indeed, isoniazidꢀrelated Mycobacterium resistance has been reꢀ
ported to be mainly associated with the KatG and not with the InhA protein.6 These findings indicate
that targeting mycobacterial InhA and its orthologs in other pathogens remains a suitable strategy for
developing compounds against tuberculosis and other bacterial infections. Based on several ligandꢀ or
structureꢀbased approaches, different classes of InhA inhibitors have been developed, such as diphenyl
ethers related to triclosan,7 pyrrolidine carboxamides,8 INHꢀNAD analogs9 and pyridomycin.10 Howevꢀ
er, only a few of them inhibited Mtb growth in vitro.11
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In this article, we describe the in silico analysisꢀbased rational design, synthesis and characterization of
InhA inhibitors bearing rhodanine as a privileged scaffold. The inhibitory effect of the rationally deꢀ
signed rhodanine derivatives against Mm was analyzed using both antiꢀvirulent and antibiotic assays.
Interestingly, these compounds also inhibited the growth of other Gramꢀpositive (Ef) and Gramꢀ
negative (Lp) pathogenic bacteria.
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RESULTS AND DISCUSSION
Design and Chemical Space Exploration
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As a first step towards the discovery of novel InhA inhibitors, we identified a moiety chemically accesꢀ
sible for designing and synthesizing a consistent library of compounds to be biologically evaluated. We
focused on the rhodanine moiety for the following reasons: i) rhodanine represents a privileged strucꢀ
ture as several derivatives have been reported to be pharmacologically active against various bacterial
targets, such as UDPꢀgalactopyranose mutase,12 UDPꢀNꢀacetylmuramoylꢀLꢀalanine synthetase,13 nuꢀ
cleoside diphosphoꢀglycosyltransferase,14 penicillinꢀbinding proteins,15 C class βꢀlactamase,16 RNA
polymerase,17 dihydrolipoamide acyltransferase,18 anthrax lethal factor,19 botulinum neurotoxins20 and
the bacterial oxyreductase class of enzymes;21 ii) the rhodanine scaffold is amenable to parallel syntheꢀ
sis of differently polysubstituted derivatives. While many rhodanine derivatives have been designed
previously, none of them has been reported to target bacterial InhA. However, inhibition of the homolꢀ
ogous Plasmodium falciparum FabI has been studied using compounds bearing the rhodanine scafꢀ
fold.22
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Based on these considerations, we designed, synthesized and screened a first series of compounds (1ꢀ
20) (see Chart 1). The initial screening of derivatives 1ꢀ20 was performed at 30 µM to reveal comꢀ
pounds with a promising inhibitory profile against InhA (the data of the most active derivatives are
shown in Figure 1, and the complete data set can be found in Table S1 of the supporting material). The
results clearly show that several of the designed compounds strongly inhibit InhA (Figure 1). The reꢀ
sults suggest some trends in term of structureꢀactivity relationship. In this respect, the first observation
is that increased lipophilicity probably plays an important role in modulating InhA inhibitory activity,
as all tested derivatives bearing bulky lipophilic substituents at position 5 (derivatives 13ꢀ18) and Nꢀ3
of the rhodanine ring (derivatives 12ꢀ14, 16ꢀ17) show higher or even complete inhibition of the enzyme
at 30 ꢁM (Figure 1). In particular, the presence of a tryptophan residue at position Nꢀ3 of the rhodanine
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ring, e.g. in derivatives 12 and 17, seems to be very favorable for the InhA inhibitory activity (Figure
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Rationale of StructureꢀBased Design
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Compounds with the highest inhibitory activity were investigated by molecular docking simulations,
carried out with GOLD software (5.2.2 version),23 to identify their putative binding modes within the
active site of InhA and help to further design the series of compounds. The binding mode of derivative
12 at the InhA binding pocket is representative of the first series of compounds and is reported in Figꢀ
ure 2. The oxygen of the carbonyl group (Cꢀ4) within the rhodanine ring interacts via Hꢀbond with the
phenolic hydroxyl group of Tyr158 and the 2′ꢀhydroxyl group of the nicotinamide ribose (Figure 2).
Furthermore, the docking reveals that the arylidene substituent at position 5 of the rhodanine ring is
directed towards the end of the InhA binding pocket and interacts hydrophobically with the side chains
of Met199, Ile215, and Leu218 (Figure 2). Remarkably, the enhanced bulkiness and hydrophobicity of
the substituent of 17 at position 5 (4ꢀdimethylaminoꢀnaphtylidene ) compared to 12 (4ꢀdimethylaminoꢀ
benzylidene) induces a better occupancy of the pocket. This correlates well with increased inhibitory
activity of 17 when compared with that of 12. The tryptophan residue of compound 12 is directed toꢀ
wards the solventꢀexposed entry site of InhA with its carboxyl moiety pointing towards the NAD+
phosphate bridge. Depending on the protonation state of the ligand or the NAD+ within the binding
site, the observed position of the carboxyl moiety could eventually lead to the formation of an additionꢀ
al hydrogen bond.
Based on the aboveꢀmentioned findings, we designed and synthesized a second series of rhodanine
derivatives (21ꢀ30, 32ꢀ34) starting from compounds 12 and 17 to explore the lipophilic interactions
within the accessible hydrophobic cavity.
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Chemistry
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The synthesis of the designed compounds, 1ꢀ34 (Chart 1), was performed as presented in Scheme 1.
Selected amino acids with different polarity and size were used as starting materials for synthetizing
derivatives 1ꢀ5, 19ꢀ20 and 6ꢀ18, 21ꢀ34. The reaction of the carbon disulfide with the amino acids in the
presence of potassium hydroxide in aqueous solution yielded amino acid dithiocarbamates. Further
reaction of the latest with potassium chloroacetate and cyclization with hydrochloric acid led to
rhodanine ring formation with the amino acid moiety attached to the nitrogen at position 3 (Nꢀ3) . All
intermediate rhodanine derivatives bearing amino acids at the position Nꢀ3 were synthesized as deꢀ
scribed in the literature24 and used directly, without prior purification, for the synthesis of the final
compounds 1ꢀ34 by Knoevenagel condensation between Nꢀsubstituted rhodanine derivatives and difꢀ
ferent aldehydes in acetic acid in the presence of a catalytic amount of sodium acetate. The identity of
the final compounds was verified by FTIR, 1HꢀNMR, 13CꢀNMR and HRMS.
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Improved InhA Inhibition for the Designed Second Series of Rhodanine Derivatives
Based on the initial screening data of the first series of rhodanineꢀcontaining compounds, 1ꢀ20, and
driven by molecular docking studies we then designed and synthesized a second series of derivatives,
21ꢀ34 (Chart 1). Subsequent evaluation of their inhibitory activity at a fixed concentration of 30 µM
showed an improved Mtb InhA inhibitory profile (Figure 1).
The screening results revealed that compounds carrying bulky substituents at position 5 of the
rhodanine ring 32ꢀ34 were more active, in agreement with what has been predicted by modeling. In
general, 10 out of the 14 newly synthesized compounds (21ꢀ34), showed complete InhA inhibition at
30 ꢁM. Notably, a strong correlation between in silico prediction and in vitro analysis was observed for
the derivatives bearing a tryptophan moiety at position Nꢀ3 of the rhodanine scaffold, i.e. compounds
24ꢀ30 and 32ꢀ34; This finding is in line with the observation that tryptophan plays an important role for
the activity against InhA compared to the synthetic derivatives 1ꢀ11, 13ꢀ16, 18 and 20ꢀ23, which all
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harbor other amino acids at position Nꢀ3. The IC50 values for the most active InhA inhibitors (12, 16,
21ꢀ22, 25ꢀ30, 32ꢀ34) were determined and found to be in the range of ~3ꢀ28 µM (see Table 1). The
most potent inhibitor, compound 32, showed an IC50 of 2.9 ± 0.7 ꢁM which is about twice as potent as
the reference compound triclosan (IC50 of 6.1 ± 2.1 ꢁM).
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StructureꢀActivity Relationship (SAR) Studies
Molecular modeling studies allowed us to improve and potentially correlate the results from the inhibiꢀ
tion studies with the dockingꢀbased binding poses. In particular, the computational analysis of comꢀ
pound 33 revealed that the introduction of more bulky 4ꢀmethylꢀnaphthylidene substituent at the posiꢀ
tion 5 of the rhodanine ring does not affect the key Hꢀbond interactions between the carbonyl group of
the rhodanine with the hydroxyl group of Tyr158 and with the 2′ꢀhydroxyl group of the nicotinamide
ribose (Figure 2, Figure 3). Furthermore, the carboxyl group of tryptophan is positioned in a favorable
manner to eventually form additional hydrogen bonds with NAD+ phosphate (Figure 3). As with the
first series of derivatives, 1ꢀ20, arylidene or more bulky naphthylidene substituents occupy the fatty
acid binding pocket of the InhA binding site. Moreover, derivatives bearing bulky lipophilic substituꢀ
ents form hydrophobic interactions with the side chains of Phe149, Met199, Ile215, Leu218, and
Met155 located in the InhA binding pocket (Figures 2 and 3).
The SAR of the synthesized compounds revealed the importance of substituents at positions 5 and Nꢀ3
of rhodanine in InhA inhibition. Derivatives 12, 17 and 24ꢀ30, 32ꢀ34 with tryptophan linked to Nꢀ3
were very active, and hence the indolyl moiety appeared to be the most effective substituent (Figure 1).
In contrast, derivatives 1ꢀ3 and 20 bearing small (short chain) amino acids, e.g. αꢀalanine, βꢀalanine, or
αꢀaminobutyric acid, or a second carboxyl group (compound 11) did not show any significant inhibiꢀ
tion (Chart 1, Figure 1). Similarly, derivatives 4ꢀ5 and 6ꢀ8 harboring small lipophilic side chains (e.g.,
leucine, norꢀleucine) displayed low inhibitory activity. In general, derivatives consisting of amino acids
with branched (valine, leucine, isoleucine, 3ꢀ6) or nonꢀbranched side chains (norꢀvaline, norꢀleucine, 7ꢀ
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8) did not exhibit significantly different inhibitory activity towards InhA. However, the side chain
length of amino acid residues appeared to be important in modulating the activity of derivatives 13ꢀ16
harboring the 1ꢀdimethylꢀnaphthylidene group as substituent in position 5 of rhodanine (Figure 1). In
particular compound 16 carrying a norꢀleucine moiety was very potent against InhA while the analog
15 bearing a norꢀvaline side chain was inactive. Furthermore, replacing the sulfur atom in methionine
with the more bulky selenium (derivatives 9 and 10) did not lead to any significant change in activity.
In agreement with the single dose inhibition data (Figure 1) the InhA inhibition doseꢀresponse experiꢀ
ments (see Table 1) data show that the presence of the tryptophan substituent at Nꢀ3 of rhodanine (deꢀ
rivatives 12, 17, 24ꢀ30, 32ꢀ34) correlates with an increase of the InhA inhibitory activity of the comꢀ
pounds. Thus, the most potent inhibitors presented in this work share the tryptophan moiety at Nꢀ3.
Further investigations with respect to the impact of aromatic substituents at the position 5 of rhodanine
showed that the presence of an extended bulky aliphatic chain in para position of the benzylidene
group (compounds 21, 24ꢀ28) is important for the InhA activity of the compounds while a smaller subꢀ
stitution leads to a decreased activity (compounds 22ꢀ23).
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In agreement with the in silico postulated binding mode, the in vitro data indicate that the improved
inhibitory activity of the synthesized derivatives is strongly related to the possibility of increasing hyꢀ
drophobic interactions in the binding pocket of InhA (Figure 2B, Figure 3). This was clearly confirmed
by comparing compounds carrying the dimethylamino group (derivatives 4, 21) with those having the
more lipophilic diethylamino moiety (derivatives 12, 24) in para position of the benzylidene. The more
lipophilic substituent in the para position shows a higher inhibitory activity at 30 µM (Figure 1). Howꢀ
ever, replacing the diethylamino group with more lipophilic longer aliphatic substituents (compounds
25ꢀ26) did not significantly improve the inhibitory activity suggesting that the increase of flexibility
due to longer aliphatic chain is reducing the impact of the enhanced hydrophobic interactions. In line
with the postulated binding mode, the compound with a bulky and hydrophobic chlorine in para posiꢀ
tion (derivative 29) showed similar activity compared to the one having the diethylamino group (IC50
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of 19.7 ± 1.4 µM). This activity could be improved about twofold to reach an IC50 value of 9.5 ± 2.2
µM with the introduction of a second chlorine atom in ortho (derivative 30). The trend observed with
compound 30 and the modelling showing that a proximal rigid bulky group at position 5 of the
rhodanine is favorable for the inhibition activity led us to synthetize derivatives 32ꢀ24 where the benꢀ
zylidene moiety is replaced by the bulkier and hydrophobic anthracenylidene or naphthylidene group.
The inhibitory activity data confirmed the hypothesis as compound 32 is the strongest InhA of the seꢀ
ries with an IC50 value of 2.9 ± 0.7µM. In line with the established SAR the introduction of a small
fluorine atom in the para position (23) caused a loss of activity (Figure 1).
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Overall, on the basis of inhibition data at 30 µM (Figure 1) confirmed by the IC50 value of the doseꢀ
response experiments (Table 1), among the synthesized compounds those with the polycyclic aromatic
substituents at position 5 of the rhodanine ring were the most active ones (see 13ꢀ14, 16ꢀ18, 32ꢀ34).
These results strongly correlate with the in silico data showing bicyclic (derivatives 13ꢀ18, 33ꢀ34) and
tricyclic substituents (derivative 32) to interact strongly via their large surface area with the hydrophoꢀ
bic pocket of the InhA binding site (Figure 2 and Figure 3). Interestingly, the absence of the carboxyl
group in the tryptophan moiety (compound 31) led to loss of activity.
Antibiotic and AntiꢀInfective Effects on Pathogenic Bacteria
The antimicrobial potential of the compound series was further tested towards the pathogenic bacteria
Mm, Pa, Lp and Ef, using cellꢀbased antibiotic and antiꢀinfection assays. To this end we further investiꢀ
gated the most powerful InhA inhibitors, compounds 12, 16ꢀ18, 21, 24ꢀ30, and 32ꢀ34, by determining
their antibiotic effect expressed measuring MIC against Mm at concentrations ranging from submiꢀ
cromolar to submillimolar. These experiments show that most of the derivatives possessed antiꢀMm
activity with MIC ranging from 0.2 to 185 µM (Table 1). Compound 24 bearing a longer aliphatic
chain (diethylamino) at the para position was more active than 12 with the shorter dimethylamino
group. Replacement of the diethylamino group with the aliphatic propyl chain (derivatives 25 and 27)
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did not increase antiꢀMm activity. Moreover, introduction of chlorinated substituents (derivatives 29
and 30) did not increase activity. Yet, lipophilic substituents at position 5 of the rhodanine seemed to
play an important role, as the derivatives 15 and 33ꢀ34, bearing the naphthylidene fragment, were most
active against Mm. Notably, the chirality of the tryptophan residue introduced on the Nꢀ3 position of the
rhodanine moiety was particularly relevant when comparing compounds 25, 26 and 33 (Lꢀtryptophan)
with their corresponding R enantiomers used to synthesize 27, 28 and 34 (Dꢀtryptophan). Indeed, comꢀ
pounds 26 and 33 showed a 10ꢀfold higher antiꢀMm activity than 28 and 34 (Table1). If we compare
compounds having similar logP, we clearly see that an increased inhibitory activity on the isolated enꢀ
zyme results in lower MIC values against Mm. This supports the idea that InhA is a relevant target for
the antibiotic activity of the rhodanine derivatives.
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Derivative 32 bearing a tricyclic 10ꢀchloroꢀanthracenylidene ring did not show a significant effect
against Mm most likely due to very high logP (calculated LogP = 5.98; http://www.molinspiration.com)
causing the trapping of the molecule into the membrane lipid of Mm.
In addition, the compounds were tested against Mm in a recently developed assay using Dictyostelium
discoideum amoebae growth as an inverse measure of the pathogen virulence.25 Dictyostelium cannot
grow on a mixed bacterial lawn of Mm and Klebsiella pneumoniae (Kp). However, 19 out of 34
rhodanine derivatives (~56%) were able to reduce Mm virulence at a concentration of 10 µM. The reꢀ
sults are shown in Table 1 whereas the complete data set is presented in supplementary Table S2. Inꢀ
deed, upon treatment with, compounds such as 17, 18, 21, 24ꢀ30 and 32ꢀ34 (Table 1), Dictyostelium
grew by feeding on Kp and formed phagocytic plaques in presence of Mm. Those compounds were not
toxic to the host and allowed Dictyostelium to proliferate. Therefore, these rhodanine compounds speꢀ
cifically attenuated the virulence of Mm without affecting Kp that feeds Dictyostelium.
Recently, gemfibrozil was reported to inhibit the intracellular growth of Lp in macrophages by targeting
in a noncompetitive manner FabI, the Lp orthologue of Mtb InhA.26 These findings prompted us to test
the rhodanine derivatives against Lp (Figure 4).27 29 compounds (~85%) possessed the capacity to deꢀ
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crease axenic Lp growth at 30 ꢁM. Interestingly, the same SAR pattern as found for Mm was observed
for Lp, strongly suggesting LpFabI to be the target of these rhodanine derivatives. Accordingly, the imꢀ
pact of different amino acids at position Nꢀ3 could clearly be identified. Synthetic compounds having a
βꢀamino acid (20), methionine (10), phospholeucine (18) or derivatives without a carboxylic group (19
and 31) were less active than the others. Also in the case of LpFabI, the indolyl moiety appeared to be
the most effective substituent. With the exception of compound 28, all the ligands bearing this group
strongly inhibited axenic Lp growth. Furthermore, by increasing the bulkiness of substituents at posiꢀ
tion 5 of rhodanine, as in derivatives 13ꢀ18 and 32ꢀ34, the inhibitory activity against Lp was retained.
The compounds series was also tested against the intracellular growth of Lp using the established Acanꢀ
thamoeba castellanii ꢀ Lp hostꢀpathogen system.27 Using this system, the compounds 25, 27, and 33ꢀ
34, all of them bearing the tryptophan residue, showed remarkable activity against intracellular replicaꢀ
tion of Lp. In particular, the most active compound, derivative 33, reduced the growth of Lp by 89%.
Moreover, compounds 33ꢀ34 bearing naphthylidene, were more active if compared to the ones with the
arylidene substituent, and the bulky naphthylidene substituent (33) was the most effective against Lp
growing in the host cell. On the other hand, inactivity of all other synthetic compounds in this infection
assay might be due to poor penetration through the amoeba cell membrane, possible inactivation inside
or activity of pumps transporting the drugs out of the cell.
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Antibacterial activity against Ef and Pa was also investigated via the serial broth dilution antibiotic
assay (Table 1). The synthetic rhodanine derivatives 1ꢀ34 were all inactive against the Gramꢀnegative
pathogen Pa. In contrast, the compounds 32ꢀ34 were effective against Ef with MIC values ranging
from 0.57 µM to 5.2 µM. The growth inhibition of the derivative with the lowest IC50 value against
InhA, a homolog of Ef FabI (62% homology), compound 32 (harboring the tricyclic 10ꢀchloroꢀ
anthracenylidene fragment), was also the most active molecule against Ef in the in vitro assay (Table 1).
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Thirtyꢀfour rhodanine derivatives, characterized by carrying amino acid residues at the position Nꢀ3,
were synthesized and their inhibitory activity was evaluated against Mtb InhA. Enzymatic data together
with molecular modeling results supported InhA to be a possible target of the rhodanine derivatives.
The potential putative binding modes/affinities between selected ligands and the target enzyme InhA
were identified. The most promising compounds showed both antibiotic and antiꢀvirulent activity
against Mm and, in particular, the compounds 32ꢀ34, also exhibited remarkable activity against Ef.
Several compounds showed a strong antibiotic effect, and some of them reduced the intracellular repliꢀ
cation of Lp. Furthermore, derivative 33, the most potent compound, showed broad range activity in all
in vitro experiments and might represent a good starting point for optimizing the structure to develop
more active antibacterials.
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In conclusion, the results obtained in this work highlight amino acid substituted rhodanines as very
promising antibacterial scaffolds acting through enoylꢀacyl carrier protein reductases inhibition. This
study suggests that the development of more potent enoylꢀacyl carrier protein reductases inhibitors
could lead to novel broadꢀspectrum antibacterial drug candidates.
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EXPERIMENTAL PROCEDURES
Reagents and Analytical Procedures
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Solvents and reagents were used without further purification. The progress of all reactions was moniꢀ
tored by TLC on precoated silica gel plates (Merck Silica gel 60 F254). The developed chromatograms
were viewed under UV light at 254 nm. For preparative thin layer chromatography 20×20 cm TLC
plates (1 mm) from Analtech were used. All 1H NMR and 13C NMR spectra were acquired on a Varian
300 operating at 300 and 75 MHz, respectively. The proton spectra were referenced to the respective
residual solvent peaks (DMSOꢀd6, 2.50 ppm) except for those recorded in CDCl3 (7.26 ppm). Carbon
spectra were referenced to the central peak of the respective residual solvents (DMSOꢀd6 39.50; CDCl3
77.0 ppm). Highꢀresolution mass spectra were obtained using electrospray ionization (ESI) and QSTAR
Pulsar (AB/MDS Sciex) mass spectrometer. Infrared (IR) spectra were measured on a Perkin Elmer
FTIR 100 spectrophotometer using ATR. Melting points were determined in a Büchi Bꢀ540 melting
point apparatus and are uncorrected. To assess the purity of the reported compounds, 1 mg of each one
of them was weighed and dissolved in 50 ml of acetonitrile. The sample was filtered and analysed by
HPLC analysis using the Agilent 1200 series with Agilent Zorbax RRHD column C18 column (1.8 µm,
2.1 mm x 50 mm) and applying a flow rate of 0.4 mL/min and an injection volume of 1 µL. The mobile
phase was composed of solution A (0.1% formic acid in water) and solution B (acetonitrile), and differꢀ
ent gradient conditions were applied (details are shown in the SI material). DADꢀUV detection was
performed at the wavelength of 410 nm or 474 nm. The purity of the active compounds is ≥95%, being
the Z isomer the most prevalent one accordingly to the 1HꢀNMR analysis.
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General Procedure for Preparation of (4ꢀoxoꢀ2ꢀthioxothiazolidinꢀ3ꢀyl)ꢀAmino Acids Derivatives.
2ꢀ(4ꢀoxoꢀ2ꢀthioxothiazolidinꢀ3ꢀyl)ꢀamino acid derivatives were synthesized according to the general
procedures described in the literature.24 In a roundꢀbottom flask equipped with a magnetic stirrer, amiꢀ
no acid (30 mmol) was dissolved with potassium hydroxide (30 mmol) in water (30 mL). Carbon diꢀ
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sulfide CS2 (30 mmol) was added to the reaction mixture, which was stirred for 2ꢀ6 hours. An aqueous
solution of potassium chloroacetate (30 mmol) was added and stirring was continued at room temperaꢀ
ture for 30 minutes. Then the reaction mixture was acidified with 2N HCl until pH 2.0ꢀ3.0 and heated
to 90 °C for 5ꢀ30 minutes. The reaction mixture was poured in cold water and precipitated intermediate
was used for the synthesis of targeted compounds without any further purification.
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General Procedure for Preparation of 5ꢀyliden (4ꢀoxoꢀ2ꢀthioxothiazolidinꢀ3ꢀyl)ꢀAmino Acids Deꢀ
rivatives by Knoevenagel Condensation. 10 mmol of 2ꢀ(4ꢀoxoꢀ2ꢀthioxothiazolidinꢀ3ꢀyl)ꢀamino acid
derivative were solubilized with 10 mL acetic acid and 300 mg of AcONa and 11 mmol of appropriate
aldehyde were added and heated overnight. The reaction mixture was poured in cold water, precipitated
solid was filtered, washed with a small amount of AcOH, EtOH, Et2O and dried. The derivatives were
purified by crystallization from acetic acid or by preparative thin layer chromatography.
(2S)ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}propanoic acid
1
(1).27 Red solid, 48% yield. M.p. 248ꢀ252 °C, from acetic acid. HꢀNMR (DMSOꢀd6) δ 7.63 (s, 1H),
7.43 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.55 (q, J = 7.1 Hz, 1H), 3.01 (s, 6H), 1.50 (d, J =
13
7.1 Hz, 3H). CꢀNMR (DMSOꢀd6) δ 170.4, 166.93, 166.93, 152.70, 135.73, 133.97, 120.43, 113.53,
113.22, 52.99, 40.20, 14.22. FTIR ꢀ 2880, 1693, 1611, 1568, 1522, 1437, 1376, 1340, 1305, 1249,
1229, 1213, 1186, 1115, 1076, 997, 923, 808, 741, 681 cmꢀ1. HRMS (ESI+): calcd. 337.0675 for
C15H17N2O3S2 [M+H]+; Found: 337.0676.
(2S)ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}butanoic
acid
1
(2). Red solid, 40% yield. M.p. 189ꢀ191 °C, from acetic acid. HꢀNMR (DMSOꢀ d6) δ 7.64 (s, 1H),
7.44 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 5.43 (t, J = 7.3 Hz, 1H), 3.01 (s, 6H), 2.27ꢀ2.07 (m,
2H), 0.78 (t, J = 7.3 Hz, 3H). 13CꢀNMR (DMSOꢀ d6) δ 193.73, 169.97, 167.26, 152.70, 135.79, 133.54,
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120.45, 113.28, 112.91, 59.42, 40.42, 21.73, 11.34. FTIR ꢀ 2976, 2890, 2698, 1610, 1574, 1525, 1436,
1374, 1328, 1274, 1231, 1205, 1180, 1127, 1063, 952, 908, 806, 788, 740 cmꢀ1. HRMS (ESI+): calcd
351.0831 for C16H19N2O3S2 [M+H]+; Found: 351.0837.
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(2R,S)ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ3ꢀ
methylbutanoic acid (3). Red solid, 60% yield. M.p. 210ꢀ212 °C, from acetic acid. 1HꢀNMR (DMSOꢀ
d6) δ 7.7 (s; 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.16 (d, J = 8.5 Hz, 1H), 3.04 (s,
6H), 2.84ꢀ2.63(m, 1H), 1.19 (d, J = 6.3 Hz, 3H), 0.72 (d, J = 6.7 Hz, 3H). 13CꢀNMR (DMSOꢀd6) δ
193.45, 169.54, 167.33, 152.80, 136.28, 134.13, 120.37, 112.92, 112.79, 62.54, 27.75, 22.45, 19.54.
FTIR ꢀ 2967, 2888, 1726, 1686, 1616, 1573, 1528, 1470, 1441, 1377, 1326, 1286, 1254, 1230, 1211,
1186, 1127, 897, 800, 744, 688 cmꢀ1. HRMS (ESI+): calcd 365.0988 for C17H21N2O3S2 [M+H]+; Found:
365.0988.
(2R)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ4ꢀ
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methylpentanoic acid (4). Red solid, 52% yield. M.p. 100ꢀ102 °C, from acetic acid. HꢀNMR
(DMSOꢀd6) δ 7.68 (s, 1H), 7.47 (d, J = 9 Hz, 2H), 6.83 (d, J = 9 Hz, 2H), 5.56 (m, 1H), 3.05 (s, 6H),
13
2.22ꢀ1.99 (m, 2H), 1.51ꢀ1.33 (m, 1H), 0.92 (d, J = 6,6 Hz, 3H), 0.85 (d, J = 6.6 Hz; 3H). CꢀNMR
(DMSOꢀd6) δ 193.56, 170.25, 167.32, 152.77, 136.01, 134.07, 120.41, 111.75, 111.70, 56.54, 37.17,
25.46, 23.61, 22.67. FTIR ꢀ 2954, 1696, 1611, 1563, 1519, 1437, 1338, 1273, 1213, 1183, 1131, 1096,
944, 809, 744 cmꢀ1. HRMS (ESI+): calcd 379.1144 for C18H23N2O3S2 [M+H]+; Found: 379.1143.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ4ꢀ
1
methylpentanoic acid (5). Red solid, 49% yield. M.p. 180ꢀ182 °C, from acetic acid. HꢀNMR
(DMSOꢀd6) δ 7.68 (s, 1H), 7.48 (d, J = 9.0 Hz, 2H), 6.84 (d, J = 9.0 Hz, 2H), 5.58ꢀ5.47 (m, 1H), 3.04
13
(s, 6H), 2.22ꢀ1.99 (m, 2H), 1.51ꢀ1.33 (m, 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). Cꢀ
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NMR (DMSOꢀd6) δ 193.50, 170.26, 167.30, 152.79, 136.10, 134.10, 120.39, 113.0, 112.95, 56.36,
37.17, 25.53, 23.60, 22.67. FTIR ꢀ 2955, 1695, 1611, 1561, 1519, 1437, 1338, 1272, 1212, 1169, 1126,
1095, 944, 807, 742, 680 cmꢀ1. HRMS (ESI+): calcd 379.1144 for C18H23N2O3S2 [M+H]+; Found:
379.1153.
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(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ3ꢀ
methylpentanoic acid (6). Red solid, 55% yield. M.p. 193ꢀ195 °C, from acetic acid. 1HꢀNMR (CDCl3)
δ 7.65 (s, 1H), 7.39 (d, J = 9.0 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 5.46 (d, J = 9.5 Hz, 1H), 3.08 (s, 6H),
13
2.75ꢀ2.56 (m, 1H), 1.34ꢀ0.94 (m, 2H), 1.26 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H). CꢀNMR
(CDCl3) δ 193.37, 172.45, 168.13, 152.29, 135.83, 133.39, 120.97, 112.22, 114.32, 62.07, 40.29, 22.83,
25.33, 17.71, 11.31. FTIR ꢀ 2954, 1693, 1611, 1562, 1519, 1436, 1332, 1271, 1211, 1167, 1094, 943,
809 cmꢀ1. HRMS (ESI+): calcd 379.1144 for C18H23N2O3S2 [M+H]+; Found: 379.1142.
(2R,S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}pentanoic
acid (7). Red solid, 53% yield. M.p. 250ꢀ251 °C, from acetic acid. 1HꢀNMR (DMSOꢀd6) δ 7.65 (s, 1H),
7.44 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 9 Hz, 2H), 5.59ꢀ5.45 (m, 1H), 3.02 (s, 6H), 2.27ꢀ1.99 (m, 2H),
13
1.32ꢀ1.06 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H). CꢀNMR (DMSOꢀd6) δ 193.49, 169.94, 167.21, 152.74,
136.01, 134.05, 120.41, 113.11, 113.01, 57.68, 28.50, 27.95, 22.52, 14.44. FTIR ꢀ 2882, 1689, 1616,
1572, 1525, 1437, 1374, 1329, 1266, 1209, 1184, 1124, 1059, 944, 801, 745 cmꢀ1. HRMS (ESI+): calcd
365.0988 for C17H21N2O3S2 [M+H]+; Found: 365.0979.
(2R,S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}hexanoic
acid (8). Red solid, 61% yield. M.p. 196ꢀ198 °C, from acetic acid. 1HꢀNMR (DMSOꢀd6) δ 7.65 (s, 1H),
7.44 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.60ꢀ5.42 (m, 1H), 3.02 (s, 6H), 2.26ꢀ2.04 (m, 2H),
13
1.34ꢀ1.00 (m, 4H), 0.79 (t, J = 7.0 Hz, 3H). CꢀNMR (DMSOꢀd6) δ 193.49, 169.94, 167.21, 152.74,
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136.01, 134.05, 120.41, 113.11, 113.01, 57.68, 28.50, 27.96, 22.52, 14.44, 14.41. FTIR ꢀ 2915, 1856,
1693, 1612, 1563, 1520, 1441, 1372, 1337, 1298, 1253, 1222, 1185, 1118, 1095, 942, 905, 820, 806,
681 cmꢀ1. HRMS (ESI+): calcd 379.1144 for C18H23N2O3S2 [M+H]+; Found: 379.1136.
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(2R,S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ4ꢀ
(methylsulfanyl)butanoic acid (9). Red solid, 56% yield. M.p. 152ꢀ154 °C, from acetic acid. 1HꢀNMR
(DMSOꢀd6) δ 7.65 (s, 1H), 7.45 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 5.79ꢀ5.61 (m, 1H), 3.02
(s, 6H), 2.51ꢀ2.30 (m, 4H), 1.99 (s, 3H). 13CꢀNMR (DMSOꢀd6) δ 193.55, 169.85, 167.36, 152.74,
135.84, 134.02, 120.44, 113.46, 112.44, 56.72, 40.28, 30.83, 27.84, 15.27. FTIR ꢀ 2910, 1722, 1691,
1611, 1567, 1521, 1436, 1316, 1252, 1252, 1228, 1189, 1102, 942, 803, 685 cmꢀ1. HRMS (ESI+): calcd
397.0708 for C17H21N2O3S3 [M+H]+; Found: 397.0704.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ4ꢀ
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(methylselenyl)butanoic acid (10). Red solid, 40% yield. M.p. 88ꢀ90 °C, from acetic acid. HꢀNMR
(DMSOꢀd6) δ 7.62 (s, 1H), 7.44 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.70ꢀ5.48 (m, 1H), 3.02
(s, 6H), 2.57ꢀ2.35 (m, 3H, overlaping with DMSO) 1.96ꢀ1.82 (m, 4H). 13CꢀNMR (DMSOꢀd6) δ 193.79,
169.87, 167.41, 152.77, 135.49, 134.03, 120.52, 113.92, 112.93, 58.42, 40.28, 29.09, 22.08, 4.31. FTIR
ꢀ 2919, 1694, 1611, 1562, 1519, 1437, 1338, 1293, 1338, 1293, 1226, 1170, 1148, 1098, 943, 810 cmꢀ1.
HRMS (ESI+): calcd 445.0153 for C17H21N2O3S2Se [M+H]+; Found: 445.0163.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}butanedioic
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acid (11). Red solid, 45% yield. M.p. 274ꢀ275 °C, from acetic acid. HꢀNMR (DMSOꢀd6) δ 7.58 (s,
1H), 7.45 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 5.52ꢀ5.38 (m, 1H), 3.62ꢀ3.44 (m, 1H), 3.03 (s,
13
6H), 2.29 (dd, J = 13.2, 2.1 Hz, 1H). CꢀNMR (DMSOꢀd6) δ 193.39, 172.79, 169.33, 167.33, 152.55,
134.76, 133.71, 120.64, 112.95, 112,90, 55.18, 40.29, 38.00. FTIR ꢀ 2922, 1719, 1676, 1563, 1524,
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1436, 1412, 1366, 1343, 1283, 1254, 1168, 1104, 1045, 1003, 962, 943, 893, 800, 764 cmꢀ1. HRMS
(ESI+): calcd 381.0573 for C16H17N2O5S2 [M+H]+; Found: 381.0578.
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(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ
3ꢀyl)propanoic acid (12). Red solid, 73% yield. M.p. 148ꢀ150 °C, from toluene. 1HꢀNMR (DMSOꢀd6)
δ 10.77 (s, 1H), 7.62 (s, 1H), 7.48 (d, J = 7.8, Hz, 1H), 7.41 (d, J = 9 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H),
7.1ꢀ6.98 (m, 2H), 6.90 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 9.0 Hz, 2H), 5.96ꢀ5.73 (m, 1H), 3.85ꢀ3.67 (m,
1H), 3.59 (dd, J = 14.9, 4.9 Hz, 1H), 3.03 (s, 6H). 13CꢀNMR (DMSOꢀd6) δ 192.99, 169.91, 167.30,
152.69, 136.62, 135.56, 133.96, 127.85, 124.25, 121.55, 120.39, 119.01, 118.59, 113.33, 112.91,
112.01, 109.91, 58.66, 40.26, 23.79. FTIR ꢀ 3407, 2902, 1690, 1611, 1556, 1517, 1437, 1374, 1340,
1277, 1221, 1165, 1098, 943, 885, 810, 738 cmꢀ1. HRMS (ESI+): calcd 452.1097 for C23H22N3O3S2
[M+H]+; Found: 452.1113.
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(2S)ꢀ4ꢀMethylꢀ2ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀ
thiazolidinꢀ3ꢀyl]pentanoic acid (13). Red solid, 72% yield. M.p. 165ꢀ167 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 8.33 (s, 1H), 8.16 (d, J = 7.8 Hz, 2H), 7.67ꢀ7.50 (m, 3H),
7.16 (d, J = 7.8 Hz, 1H), 5.58ꢀ5.10 (m, 1H), 2.92 (s, 6H), 2.43ꢀ1.27 (m, 3H), 0.88 (d, J = 13.4 Hz, 6H).
13CꢀNMR (DMSOꢀd6) δ 195.21, 167.28, 154.46, 133.63, 129.62, 129.19, 128.2, 127.94, 126.33,
125.93, 124.29, 123.57, 121.66, 121.64, 113.81, 58.75, 45.02, 26.33, 23.93, 22.58. FTIR ꢀ 2952, 1695,
1556, 1514, 1386, 1332, 1270, 1200, 1134, 1012, 761, 736 cmꢀ1. HRMS (ESI+): calcd 429.1301 for
C22H25N2O3S2 [M+H]+; Found: 429.1313.
(2R)ꢀ4ꢀMethylꢀ2ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀ
thiazolidinꢀ3ꢀyl]pentanoic acid (14). Red solid, 73% yield. M.p. 152ꢀ155 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 8.32 (s, 1H), 8.15 (d, J = 7.8 Hz, 2H), 7.71ꢀ7.40 (m, 3H),
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7.14 (d, J = 7.8 Hz, 1H), 5.59ꢀ5.30 (m, 1H), 2.94 (s, 6H), 2.41ꢀ1.27 (m, 3H), 0.88 (d, J = 12.5 Hz, 6H).
13CꢀNMR (DMSOꢀd6) δ 195.06, 167.18. 154.59. 133.66, 130.08, 129.32, 128.06, 127.87, 125.94,
126.14, 124.30, 123.42, 121.31, 121.27, 113.76, 58.75, 45.00, 26.02, 23.81, 22.69. FTIR ꢀ 2953, 1696,
1554, 1514, 1452, 1387, 1332, 1270, 1199, 1134, 1038, 1012, 909, 854, 761, 736, 687 cmꢀ1. HRMS
(ESI+): calcd 429.1301 for C22H25N2O3S2 [M+H]+; Found: 429.1300.
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(2R,S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(Dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀ
thiazolidinꢀ3ꢀyl]pentanoic acid (15). Red solid, 70% yield. M.p. 157ꢀ160 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 8.29 (s, 1H), 8.16 (d, J = 7.8 Hz, 2H), 7.72ꢀ7.48 (m, 3H),
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7.16 (d, J = 7.8 Hz, 1H), 5.48ꢀ5.10 (m, 1H), 2.92 (s, 6H), 2.42ꢀ1.0 (m, 6H), 0.87 (s, 3H). CꢀNMR
(DMSOꢀd6) δ 195.37, 167.21, 154.36, 133.59, 129.18, 129.09, 128.19, 127.96, 126.35, 125.91, 124.28,
123.68, 121.86, 121.43, 113.84, 61.31, 45.03, 31.27, 20.60, 14.55. FTIR ꢀ 3391, 2955, 2869, 1698,
1553, 1514, 1452, 1387, 1335, 1268, 1198, 1124, 1096, 1038, 1012, 908, 817, 761, 687 cmꢀ1. HRMS
(ESI+): calcd 415.1144 for C21H23N2O3S2 [M+H]+; Found: 415.1142.
(2R,S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(Dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀ
thiazolidinꢀ3ꢀyl]hexanoic acid (16). Red solid, 70% yield. M.p. 159ꢀ163 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 8.32 (s, 1H), 8.15 (d, J = 7.8 Hz, 2H), 7.7ꢀ7.52 (m, 3H),
7.15 (d, J = 7.8 Hz, 1H), 5.54ꢀ5.21 (m, 1H), 2.92 (s, 6H), 2.44ꢀ1.81 (m, 2H), 1.46ꢀ0.98 (m, 4H), 0.78
(s, 3H). 13CꢀNMR (DMSOꢀd6) δ 195.19, 167.09, 154.51, 133.63, 129.54, 129.08, 128.19, 127.96,
126.14, 125.91, 124.27, 123.48, 121.81, 121.43, 113.77, 60.55, 44.67, 29.51, 29.25, 22.52, 14.10. FTIR
ꢀ 2951, 1867, 1697, 1553, 1514, 1452, 1387, 1334, 1221, 1191, 1126, 1039, 1012, 914, 819, 761, 735,
687 , 687 cmꢀ1. HRMS (ESI+): calcd 429.1301 for C22H25N2O3S2 [M+H]+; Found: 429.1300.
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(2S)ꢀ3ꢀ(1HꢀIndolꢀ3ꢀyl)ꢀ2ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀ
thioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]propanoic acid (17). Red solid, 68% yield. M.p. 189ꢀ192 °C, from preparꢀ
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ative TLC hexane/acetone 3:1. HꢀNMR (CDCl3) δ 8.39 (s, 1H), 8.21 (d, J = 8.7 Hz, 1H), 8.13 (d, J =
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8.1 Hz, 1H), 7.97 (s, 1H), 7.67ꢀ7.45 (m, 4H), 7.30 (d, J = 7.8 Hz, 1H), 7.20ꢀ6.99 (m, 2H), 6.21ꢀ6.05 (m,
1H), 3.95ꢀ3.71 (m, 2H), 2.99 (s, 6H). 13CꢀNMR (CDCl3) δ 193.59, 172.37, 167.19, 154.59, 136.22,
133.81, 130.93, 128.76, 128.28, 127.60, 127.57, 125.74, 125.64, 124.01, 123.97, 123.34, 122.35,
121.24, 119.87, 118.76, 113.12, 111.29, 110.65, 57.63, 44.93, 23.96. FTIR ꢀ 3406, 2938, 1698, 1553,
1514, 1455, 1387, 1334, 1275, 1176, 1038, 1012, 734 cmꢀ1. HRMS (ESI+): calcd 502.1253 for
C27H24N2O3S2 [M+H]+; Found: 502.1254.
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{3ꢀMethylꢀ1ꢀ[(5Z)ꢀ5ꢀ{[4ꢀ(dimethylamino)naphthaleneꢀ1ꢀyl]methylidene}ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀ
thiazolidinꢀ3ꢀyl]ꢀ3ꢀmethylbutyl}phosphonic acid (18). Red solid, 65% yield. M.p. 231ꢀ233 °C, puriꢀ
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fied by washing several times with acetone. HꢀNMR (DMSOꢀd6) δ 8.35 (s, 1H), 8.21ꢀ8.11 (m, 2H),
7.74ꢀ7.50 (m, 3H), 7.16 (d, J = 8.2 Hz, 1H), 5.46ꢀ5.29 (m, 1H), 2.93 (s, 6H), 2.46ꢀ1.79 (m, 2H), 1.41ꢀ
1.1 (m, 4H), 0.81 (t, J = 6.9 Hz, 3H). 13CꢀNMR (DMSOꢀd6) δ 194.95, 166.58, 154.63, 133.65, 130.03,
129.29, 128.09, 127.80, 126.17, 126.00, 124.20, 123.35, 120.39, 113.78, 55.79 (d, J = 146 Hz), 45.01,
28.54 (d, J = 11.2 Hz), 26.93, 22.47, 14.43. FTIR ꢀ 2930, 2864, 1698, 1562, 1514, 1451, 1387, 1359,
1319, 1249, 1202, 1125, 1013, 914, 824, 761, 727 cmꢀ1. HRMS (ESI+): calcd 465.1066 for
C21H26N2O4PS2 [M+H]+; Found: 465.1065.
Nꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ3ꢀ
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methylbutanamide (19). Orange solid, 68% yield. M.p. 250ꢀ251 °C, from acetic acid. HꢀNMR
(DMSOꢀd6) δ 11.00 (s, 1H), 7.71 (s, 1H), 7.46 (d, J = 9.1 Hz, 2H), 6.81 (d, J = 9.1 Hz, 2H), 3.02 (s,
13
6H), 2.18 (d, J = 6.9 Hz, 2H), 2.12ꢀ1.95 (m, 1H), 0.94 (d, J = 7.4 Hz, 6H). CꢀNMR (DMSOꢀd6) δ
190.84, 170.51, 164.09, 152.80, 136.55, 134.17, 120.35, 112.92, 111.34, 42.81, 26.98, 22.89. FTIR ꢀ
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3214, 2952, 2869, 1719, 1664, 1612, 1579, 1514, 1435, 1369, 1265, 1242, 1190, 1147, 1112, 988, 947,
807, 711 cmꢀ1. HRMS (ESI+): calcd 364.1147 for C17H22N3O2S2 [M+H]+; Found: 364.1153.
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3ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Dimethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}propanoic
acid
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(20). Red solid, 46% yield. M.p. 260ꢀ262 °C, from acetic acid. HꢀNMR (DMSOꢀ d6) δ 12.50 (s, 1H),
7.64 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 4.31ꢀ4.10 (m, 2H), 3.04 (s, 6H), 2.70ꢀ
2.54 (m, 2H). 13CꢀNMR (DMSOꢀ d6) δ 193.1, 172.43, 167.33, 152.56, 135.29, 133.85, 120.49, 114.48,
112.85, 40.48, 40.26, 31.55. FTIR ꢀ 2894, 1691, 1610, 1561, 1519, 1431, 1360, 1329, 1303, 1262,
1223, 1160, 1092, 1160, 1092, 1064, 947, 810, 794, 739, 668 cmꢀ1. HRMS (ESI+): calcd. 337.0675 for
C15H17N2O3S2 [M+H]+; Found: 337.0683.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Diethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}ꢀ4ꢀ
methylpentanoic acid (21). Red solid, 70% yield. M.p. 109ꢀ110 °C, from preparative TLC hexꢀ
ane/acetone 3:1. 1HꢀNMR (DMSOꢀd6) δ 7.62 (s, 1H), 7.42 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H),
5.63ꢀ5.49 (m, 1H), 3.41 (q, J = 6.9 Hz, 4H), 2.28ꢀ1.89 (m, 2H), 1.52ꢀ1.33 (m, 1H), 1.10 (t, J = 6.9 Hz,
6H), 0.90 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H) 13CꢀNMR (DMSOꢀd6) δ 193.38, 170.26, 167.30,
150.58, 136.02, 134.47, 119.86, 112.52, 112.51, 56.34, 44.70, 37.18, 25.54, 23.55, 22.68, 13.10. FTIR ꢀ
2960, 1695, 1610, 1561, 1515, 1438, 1409, 1338, 1269, 1213, 1183, 1157, 1096, 1074, 811 cmꢀ1.
HRMS (ESI+): calcd 407.1457 for C20H27N2O3S2 [M+H]+; Found: 407.1467.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Methylsulfanyl)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl}methyl pentaꢀ
noic acid (22). Yellow solid, 75% yield. M.p. 79ꢀ81 °C, from preparative TLC hexane/acetone 3:1. 1Hꢀ
NMR (DMSOꢀd6) δ 7.78 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H), 5.61ꢀ5.48 (m, 1H),
2.52 (s, 3H), 2.24ꢀ1.92 (m, 2H), 1.54ꢀ1.38 (m, 1H), 0.9 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H).
13CꢀNMR (DMSOꢀd6) δ 193.94, 170.04, 167.28, 144.48, 134.45, 131.92, 129.54, 126.48, 119.93,
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56.54, 37.09, 25.50, 23.55, 22.62, 14.64. FTIR ꢀ 2957, 1709, 1578, 1491, 1332, 1273, 1238, 1206,
1188, 1137, 1088, 810, 742 cmꢀ1. HRMS (ESI+): calcd 382.0599 for C17H20N1O3S3 [M+H]+; Found:
382.0603.
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(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ(4ꢀFluorobenzylidene)ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ4ꢀmethylpentanoic acid
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(23). Yellow solid, 80% yield. M.p. 72ꢀ73 °C, from preparative TLC hexane/acetone 3:1. HꢀNMR
(DMSOꢀd6) δ 7.82 (s, 1H), 7.75ꢀ7.64 (m, 2H), 7.37 (t, J = 8.7 Hz, 2H), 5.63ꢀ5.46 (m, 1H), 2.25ꢀ1.90
(m, 2H), 1.56ꢀ1.33 (m, 1H), 0.88 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H). 13CꢀNMR (DMSOꢀd6) δ
194.17, 169.99, 167.20, 164.45 (d, J = 250 Hz), 133.99 (d, J = 8.8 Hz), 133.48, 130.21 (d, J = 3.2 Hz),
121.44, 117.41 (d, J = 21.8 Hz), 56.76, 37.12, 25.52, 23.54, 22.59. FTIR ꢀ 3541, 2958, 1696, 1592,
1505, 1468, 1410, 1336, 1279, 1226, 1197, 1158, 1108, 1022, 918, 826, 742 cmꢀ1. HRMS (ESI+): calcd
354.0628 for C16H17FNO3S2 [M+H]+; Found: 354.0629.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[4ꢀ(Diethylamino)benzylidene]ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ3ꢀ
yl)propanoic acid (24). Red solid, 74 % yield. M.p. 198ꢀ201 °C, from preparative TLC hexane/acetone
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4:1. HꢀNMR (DMSOꢀd6) δ 10.88 (s, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.64 (s, 1H), 7.43 (d, J = 9.0 Hz,
2H), 7.33 (d, J = 7.9 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 7.10ꢀ6.95 (m, 2H), 6.79 (d, J = 9.0 Hz, 2H),
4.34ꢀ4.16 (m, 2H), 3.42 (q, J = 6.8 Hz, 4H), 3.11ꢀ2.95 (m, 2H), 1.11 (t, J = 7.0 Hz, 6H). 13CꢀNMR
(DMSOꢀd6) δ 193.12, 174.70, 169.97, 167.45, 150.37, 136.63, 135.15, 134.29, 129.58, 128.89, 127.92,
124.10, 121.50, 119.90, 119.11, 112.25 112.23, 110.50, 59.50, 44.67, 23.82, 13.11. FTIR ꢀ 3406, 2970,
1699, 1562, 1515, 1340, 1271, 1221, 1176, 1099, 1010, 812, 738 cmꢀ1. HRMS (ESI+): calcd 480.1410
for C25H26N3O2S2 [M+H]+; Found: 480.1418.
(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ(4ꢀPropylbenzylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ3ꢀ
yl)propanoic acid (25). Yellow solid, 83% yield. M.p. 146ꢀ148 °C, from preparative TLC hexꢀ
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ane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 10.75 (s, 1H), 7.73 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.49 (s,
1H), 7.41ꢀ7.17 (m, 3H), 7.14ꢀ6.78 (m, 4H), 5.90ꢀ5.75 (m, 1H), 3.87ꢀ3.57 (m, 2H), 2.58 (t, J = 7.5 Hz,
2H), 1.66ꢀ1.44 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). 13CꢀNMR (DMSOꢀd6) δ 193.63, 169.92, 167.30,
146.83, 136.64, 134.14, 131.53, 131.06, 130.23, 127.82, 124.23, 121.57, 120.63, 119.02, 118.58,
112.05, 110.11, 59.39, 37.83, 24.42, 23.90, 14.31. FTIR ꢀ 3402, 2928, 1750, 1592, 1451, 1338, 1278,
1238, 1177, 1115, 869, 738 cmꢀ1. HRMS (ESI+): calcd 451.1144 for C24H23N2O3S2 [M+H]+; Found:
451.1143.
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(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ{4ꢀ[bis(2ꢀChloroethyl)amino]benzylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ
(1Hꢀindolꢀ3ꢀyl)propanoic acid (26). Red solid, 82% yield. M.p. 172ꢀ174 °C, from preparative TLC
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DCM/MeOH 5:1. HꢀNMR (DMSOꢀd6) δ 10.66 (s, 1H), 7.55 (s, 1H), 7.46ꢀ7.35 (m, 3H), 7.23 (d, J =
7.8 Hz, 1H), 7.01ꢀ6.84 (m, 5H), 5.72ꢀ5.57 (m, 1H), 4.33ꢀ4.03 (m, 2H), 3.85ꢀ3.55 (m, 8H). 13CꢀNMR
(DMSOꢀd6) δ 193.75, 170.96, 167.72, 149.58, 136.66, 136.50 133.81, 127.98, 123.48, 121.95, 121.65,
121.45, 118.84, 118.68, 113.22, 113.16, 111.96, 61.44, 52.33, 41.62, 21.34. FTIR ꢀ 3396, 2968, 1698,
1567, 1514, 1395, 1342, 1275, 1224, 1170, 1101, 948, 813, 740 cmꢀ1. HRMS (ESIꢀ): calcd 546.0485 for
C25H22Cl2N3O3S2 [MꢀH]ꢀ. Found: 546.0473.
(2R)ꢀ2ꢀ[(5Z)ꢀ4ꢀOxoꢀ5ꢀ(4ꢀpropylbenzylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ3ꢀ
yl)propanoic acid (27). Yellow solid, 81% yield. M.p. 165ꢀ167 °C, from preparative TLC hexꢀ
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ane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 10.79 (s, 1H), 7.71 (s, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.46 (s,
1H), 7.41ꢀ7.15 (m, 3H), 7.11ꢀ6.79 (m, 4H), 5.90ꢀ5.77 (m, 1H), 3.91ꢀ3.62 (m, 2H), 2.57 (t, J = 7.4 Hz,
2H), 1.64ꢀ1.47 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). 13CꢀNMR (DMSOꢀd6) δ 193.80, 169.88, 167.36,
146.73, 136.66, 133.89, 131.30, 130.21, 127.86, 124.08, 121.54, 120.78, 118.98, 118.6, 112.04, 110.45,
59.99, 37.83, 24.41, 24.05, 14.31. FTIR ꢀ 3407 2958, 1704, 1591, 1455, 1338, 1277, 1237, 1177, 1115,
870, 737 cmꢀ1. HRMS (ESI+): calcd 451.1144 for C24H23N2O3S2 [M+H]+; Found: 451.1145.
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(2R)ꢀ2ꢀ[(5Z)ꢀ5ꢀ{4ꢀ[bis(2ꢀChloroethyl)amino]benzylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ
(1Hꢀindolꢀ3ꢀyl)propanoic acid (28). Red solid, 80% yield. M.p. 181ꢀ184 °C, from preparative TLC
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DCM/MeOH 5:1. HꢀNMR (DMSOꢀd6) δ 10.69 (s, 1H), 7.58 (s, 1H), 7.48ꢀ7.35 (m, 3H), 7.24 (d, J =
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8.0 Hz, 1H), 7.01ꢀ6.81 (m, 5H), 5.79ꢀ5.62 (m, 1H), 4.20ꢀ3.96 (m, 2H), 3.88ꢀ3.53 (m, 8H). 13CꢀNMR
(DMSOꢀd6) δ 193.35, 170.79, 167.28, 149.51, 136.49, 136.44, 133.68, 127.73, 123.57, 121.68, 121.39,
121.27, 118.70, 118.42, 113.04, 112.97, 111.76, 61.38, 52.14, 41.41 21.11. FTIR ꢀ 3400, 2912, 1698,
1566, 1513, 1456, 1395, 1340, 1276, 1222, 1170, 1101, 1036, 812, 739 cmꢀ1. HRMS (ESIꢀ): calcd
`546.0485 for C25H22Cl2N3O3S2 [MꢀH]ꢀ. Found: 546.0472.
(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ(4ꢀChlorobenzylidene)ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ3ꢀ
yl)propanoic acid (29). Yellow solid, 83% yield M.p. 181ꢀ183 °C, from preparative TLC hexꢀ
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ane/acetone 3:1. HꢀNMR (DMSOꢀd6) δ 10.79 (s, 1H), 7.78 (s, 1H), 7.6ꢀ7.56 (m, 2H), 7.54ꢀ7.34 (m,
2H), 7.33ꢀ7.15 (m, 2H), 7.14ꢀ6.8 (m, 4H), 5.93ꢀ5.72 (m, 1H), 3.91ꢀ3.59 (m, 2H). 13CꢀNMR (DMSOꢀd6)
δ 193.47, 169.86, 167.18, 136.65, 136.39, 132.95, 132.54, 132.36, 130.23, 129.18, 127.82, 124.21,
122.57, 121.58, 119.24, 119.03, 118.57, 112.05, 67.03. FTIR ꢀ 3398, 2914, 1710, 1610, 1591, 1489,
1456, 1339, 1279, 1236, 1177, 1116, 1090, 1010, 869, 821, 738 cmꢀ1. HRMS (ESIꢀ): calcd. 441.0139
for C21H14ClN2O3S2 [MꢀH]ꢀ. Found: 441.0153.
(2S)ꢀ2ꢀ[(5Z)ꢀ5ꢀ(2,4ꢀDichlorobenzylidene)ꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀindolꢀ3ꢀ
yl)propanoic acid (30). Yellow solid, 81% yield. M.p. 167ꢀ169 °C, from preparative TLC hexꢀ
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ane/acetone 3:1. HꢀNMR (DMSOꢀd6) δ 10.78 (s, 1H), 7.87 (s, 1H), 7.82ꢀ7.63 (m, 1H), 7.62ꢀ7.55 (m,
1H), 7.39ꢀ7.40 (m, 2H), 7.38ꢀ7.20 (m, 2H), 7.12ꢀ6.86 (m, 3H), 5.88ꢀ5.64 (m, 1H), 3.86ꢀ3.59 (m, 2H).
13CꢀNMR (DMSOꢀd6) δ 193.38, 169.67, 166.96, 136.66, 136.36, 131.25, 130.84, 130.71, 130.42,
129.14, 127.83, 124.07, 118.98, 118.57, 112.03, 111.95, 110.64, 110.57, 67.03. FTIR ꢀ 3399, 2915,
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1713, 1579, 1456, 1339, 1235, 1105, 1047, 869, 739 cmꢀ1. HRMS (ESIꢀ): calcd. 474.9750 for
C21H14ClN2O3S2 [MꢀH]ꢀ; Found: 474.9750.
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(5Z)ꢀ5ꢀ[4ꢀ(Diethylamino)benzylidene]ꢀ3ꢀ[2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl]ꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ4ꢀone
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(31). Red solid, 80% yield. M.p. 196ꢀ198 °C, from acetic acid. HꢀNMR (DMSOꢀd6) δ 10.88 (s, 1H),
7.72ꢀ7.60 (m, 2H), 7.44 (d, J = 9.1 Hz, 2H), 7.33 (d, J = 7.9 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.13ꢀ
6.93 (m, 2H), 6.81 (d, J = 9.1 Hz, 2H), 4.35ꢀ4.18 (m, 2H), 3.43 (q, J = 6.9, 4H), 3.10ꢀ2.97 (m, 2H),
1.11 (t, J = 7.2 Hz, 6H). 13CꢀNMR (DMSOꢀd6) δ 193.06, 167.51, 150.32, 136.97, 135.23, 134.28,
127.77, 123.78, 121.77, 119.98, 119.14, 118.92, 114.01, 112.42,112.18, 110.80, 45.36, 44.66, 23.21,
13.09. FTIR 3367, 2967, 1682, 1610, 1560, 1517, 1411, 1377, 1350, 1326, 1270, 1240, 1193, 1166,
1097, 1077, 1010, 810, 735 cmꢀ1. HRMS (ESI+): calcd 436.1511 for C24H26N3OS2 [M+H]+; Found:
436.1512.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[(10ꢀChloroanthracenꢀ9ꢀyl)methylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀ
indolꢀ3ꢀyl)propanoic acid (32). Yellow solid, 83% yield. M.p. 165ꢀ167 °C, from preparative TLC
hexane/acetone 4:1. 1HꢀNMR (DMSOꢀd6) δ 10.90 (s, 1H), 8.53 (s, 1H), 8.43 (d, J = 8.6 Hz, 2H), 7.85ꢀ
7.58 (m, 6H), 7.52 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.22 ꢀ 6.95(m, 3H), 5.87ꢀ5.78 (m, 1H),
3.79ꢀ3.56 (m, 2H). 13CꢀNMR (DMSOꢀd6) δ 193.40, 169.65, 165.40, 136.63, 131.71, 131.39, 130.82,
128.72, 128.58, 128.35, 128.27, 127.98,127.80, 126.12, 125.51, 125.05, 121.53, 119.28, 118.24,
112.22, 109.62, 59.58, 31.37, 23.68. FTIR ꢀ 3411, 2899, 1707, 1597, 1438, 1341, 1223, 1174, 933, 732
cmꢀ1. HRMS (ESI+): calcd 543.0598 for C29H20ClN2O3S2 [M+H]+; Found: 543.0596.
(2S)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[(4ꢀMethylnaphthalenꢀ1ꢀyl)methylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ(1Hꢀ
indolꢀ3ꢀyl)propanoic acid (33). Yellow solid, 84% yield. M.p. 143ꢀ145 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 10.80 (s, 1H), 8.38 (s, 1H), 8.23ꢀ8.05 (m, 2H), 7.74ꢀ 7.60
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(m, 2H), 7.56ꢀ7.40 (m, 2H), 7.27 (d, J = 8.2 Hz, 1H), 7.16ꢀ6.82 (m, 3H), 5.97ꢀ5.73 (m, 1H), 3.87ꢀ3.47
(m, 2H), 2.67 (s, 3H). 13CꢀNMR (DMSOꢀd6) δ 194.24, 174.65, 166.70, 139.46, 136.65, 132.97, 131.73,
130.97, 128.88, 128.11, 127.85, 127.75, 127.60, 127.19, 125.79, 124.59, 124.39, 124.19, 121.55,
119.05, 118.56, 112.04, 110.09, 62.56, 26.18, 20.19. FTIR ꢀ 3410, 2969, 1703, 1590, 1564, 1514, 1456,
1337, 1277, 1227, 1176, 1119, 1033, 943, 813, 734 cmꢀ1. HRMS (ESI+): calcd 473.0988 for
C26H21N2O3S2 [M+H]+; Found: 473.0978.
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(2R)ꢀ2ꢀ{(5Z)ꢀ5ꢀ[(4ꢀMethylnaphthalenꢀ1ꢀyl)methylideneꢀ4ꢀoxoꢀ2ꢀthioxoꢀ1,3ꢀthiazolidinꢀ3ꢀyl]ꢀ3ꢀ
(1Hꢀindolꢀ3ꢀyl)propanoic acid (34). Yellow solid, 83% yield. M.p. 138ꢀ140 °C, from preparative TLC
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hexane/acetone 4:1. HꢀNMR (DMSOꢀd6) δ 10.82 (s, 1H), 8.37 (s, 1H), 8.18ꢀ7.99 (m, 2H), 7.72ꢀ7.60
(m, 2H), 7.56ꢀ7.39 (m, 3H), 7.27 (d, J = 8 Hz, 1H), 7.16ꢀ6.85 (m, 3H), 6.01ꢀ5.84 (m, 1H), 3.85ꢀ3.58
(m, 2H), 2.66 (s, 3H). 13CꢀNMR (DMSOꢀd6) δ 194.10, 169.75, 166.66, 139.53, 136.66, 132.95, 131.72,
131.18, 128.79, 128.09, 127.85, 127.75, 127.60, 127.19, 125.76, 124.57, 124.39, 123.95, 121.60,
119.11, 118.56, 112.07, 109.71, 58.94, 23.83, 20.19. FTIR ꢀ 3404, 2894, 1705, 1590, 1563, 1335, 1279,
1251, 1178, 1033, 815, 734 cmꢀ1. HRMS (ESI+): calcd 473.0988 for C26H21N2O3S2 [M+H]+; Found:
473.0991.
All the target compounds 1ꢀ34 were obtained in good yields and characterized by the means of IR, 1H,
13C NMR and HRMS.
Expression and Purification of Mtb InhA
Mtb InhA was expressed in E. coli strain Rosetta(DE3)pLysS using the InhA expression plasmid kindly
provided by Prof. Dr. Caroline Kisker (University of Würzburg, Germany) and purified following a
slightly modified protocol published recently.28 Briefly, a 1 liter culture of TB was inoculated over
night for 14h at 37°C before reducing the temperature to 25°C. Then expression was induced with 1
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mM IPTG and cells were harvested by centrifugation after 24h. The collected bacteria were suspended
in purification buffer (20 mM Tris/HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and supplemented
with a small amount of DNase before passing the suspension twice through a French Press. The crude
extract was clarified by centrifugation (20 min, 4°C, 9800 rpm) before it was applied onto a 5 ml Niꢀ
chelating column and subsequently eluted with a gradient of 20ꢀ500 mM imidazole (for 20 CV). Fracꢀ
tions containing InhA were pooled, concentrated and further purified via a Superdex 200 column (GE
Healthcare) equilibrated with gel filtration buffer (30 mM Pipes, 150 mM NaCl, 1 mM EDTA), affordꢀ
ing highly pure (>95%) and active InhA as judged by SDSꢀPAGE (Coomassie stained) and activity
assay, respectively.
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Mtb InhA Inhibition Assay
Mtb InhA was assayed using a spectrophotometric method at 25 °C by monitoring the initial velocities
(120 seconds) of the decrease in the absorbance of NADH at 340 nm in presence of the substrate transꢀ
2ꢀdecenoylꢀNꢀacetylcysteamine, which was synthesized from transꢀ2ꢀdecenoic acid and Nꢀ
acetylcysteamine using the mixed anhydride method as described previously.29 The standard reaction
mixture, in a total volume of 1.0 mL, consisted of assay buffer (30 mM PIPES, 150 mM NaCl, 1 mM
EDTA, pH 6.8), 200 µM transꢀ2ꢀdecenoylꢀNꢀacetylcysteamine, 100 µM NADH, and 1 µg of purified
recombinant Mtb InhA. For initial screening, the compounds were dissolved in DMSO to obtain stock
solutions at a concentration of 30 mM. IC50 values were determined at increasing inhibitor concentraꢀ
tions and subsequently calculated by nonlinear fit to the sigmoidal doseꢀresponse curve using Prism 6.0
(GraphPad Software, San Diego, CA, USA). Triclosan was used as positive control and provided an
IC50 of 6.1 ± 2.1 µM which is close to values reported in the literature.7a All assays were performed in
triplicates with DMSO fixed at a concentration of 0.1%.
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Molecular Modeling
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The model of apo InhA was constructed by removing all water molecules and ligands from the Xꢀray
structure (PDBcode: 1P45),30 and by adding the Hydrogen atoms using SYBYLꢀX 2.1.1 (Tripos Assoꢀ
ciates Inc, USA).31 We selected the chain A of the PDB structure 1P45, which shows the peculiarity to
present two molecules of Triclosan in complex with the active site of InhA. This gave us the opportuniꢀ
ty to explore a higher volume of the InhA binding side together with a highly diverse and larger chemiꢀ
cal space in the active site of the InhA, and then search for additional interactions, especially in the
hydrophobic pocket, together with those “more classical” ones established with Tyr158 and NAD+.
The 3D models of the ligands were built by using the Maestro program, a tool from the Schrödinger 2013
package.32 Refinement and energy minimization of ligands were carried out by using the OPLSꢀ2005
forcefield and 10.000 iterations. Docking simulations were carried out by means of GOLD, 5.2.2 verꢀ
sion.23 GOLD 5.2.2 adopts a search genetic algorithm to generate lowest binding ligandꢀprotein comꢀ
plex energies. Genetic algorithm default parameters were set: the population size was 100, the selection
pressure was 1.1, the number of operations was 105, the number of islands was 5, the niche size was 2,
migrate was 10, mutate was 95, and crossover was 95. Docking calculations were computed to obtain
100 randomly seeded runs for each ligand. Bindingꢀsite cavity was set as a spherical region of 15 Å
radius centred on the phenolic Oxygen atom of Tyr158. To evaluate the single poses resulted by search
algorithm GoldScore scoring function was used.
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Serial Dilution Test and Determination of MIC Values Against Mm
Mm, the laboratory strain mimicking perfectly Mtb, were cultivated in a shaking culture at 32°C up to
an OD600 of 0.8ꢀ1 in 7H9 medium supplemented with OADC. 105 bacteria were placed into each well
of a 96ꢀwell white plate containing compounds in 7H9 medium (0.5% v/v DMSO). Bacterial growth at
32°C was monitored by measuring the luminescence in a platereader (Synergy H1) for 48 hours. Amiꢀ
kacin was added as a control at 10 ꢁM concentration. LuxABCDEꢀexpressing Mm33 were transferred
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