Ethylenic conjugated coumarin thiazolidinediones as new efficient antimicrobial modulators against clinical methicillin-resistant Staphylococcus aureus

Title page

Title:

Ethylenic conjugated coumarin thiazolidinediones as new efficient antimicrobial modulators against clinical

methicillin-resistant Staphylococcus aureus

Author Names and Affiliations:

a

a,†

a,‡

Chun-Fang Hu , Peng-Li Zhang , Yan-Fei Sui , Jing-Song Lv , Mohammad Fawad Ansari , Narsaiah Battini ,

b,

a,

Shuo Li , Cheng-He Zhou and Rong-Xia Geng

a

Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Luminescent and Real-Time Analytical Chemistry

Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University,

(

Tiansheng street 2, Beibei district,Chongqing 400715, PR China.

b

School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China.

Corresponding Address:



Tel.: +86-23-68254967; Fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);

geng0712@swu.edu.cn (Rong-Xia Geng); Lishuo@cqut.edu.cn ( Shuo Li).

†

Postdoctoral fellow from Jamia Millia Islamia, Delhi, India

‡

Postdoctoral fellow from CSIR-Indian Institute of Integrative Medicine, Jammu, India

Abstract:

In an effort for the development of novel antimicrobial agents, ethylenic conjugated coumarin thiazolidinediones

as potential multi-targeting new antimicrobial compounds were synthesized through convenient procedures from

commercially available resorcinol and were evaluated for their antimicrobial potency. Bioactive evaluation

revealed that some of the prepared compounds showed strong antimicrobial activities towards the tested

microorganisms including clinically drug-resistant strains. Especially, propargyl derivative 12b exhibited effective

anti-MRSA potency with MIC value of 0.006 μmol/mL, which was highly advantageous over clinical

antibacterial drug norfloxacin. Compound 12b showed rapid killing effect, low toxicity against hepatocyte LO2

cells line, and no obvious drug resistance development against MRSA. Preliminary exploration of action

mechanism manifested that molecule 12b acted upon MRSA through forming stable supramolecular complex

with bacterial DNA which might impede DNA replication. Molecular docking showed that compound 12b could

bind with DNA-gyrase through hydrogen bonds.

Keywords: Coumarin, Thiazolidinedione, Antimicrobial, MRSA, DNA

1

. Introduction

In recent years, antibiotic-resistant pathogens have spread rapidly since its emergence, which is one of the

greatest threats to modern medicinal development and mankind [1]. Staphylococcus aureus (S. aureus), notorious

for its ability to produce a series of virulence factors, is categorized as kind of fatal pathogen responsible for

community- and hospital-acquired infections [2,3]. Particularly, the complicated treatments of

methicillin-resistant S. aureus (MRSA) infections have increased the morbidity and mortality of patients [4].

Isolated strains of MRSA have developed resistance to a series of antibacterial drugs, including β-lactams,

macrolides, fluoroquinolones, glycopeptides and oxazolidinones [5]. However, the emergence of multi-resistant

MRSA strain and complicated treatments mean that it is urgent for researchers to explore new treatment strategies

[6].

Poor penetration of compounds across the microbial membrane is the greatest challenge for the discovery of

novel antimicrobial drugs. Natural products have been used to treat disease for thousands of years and play an

essential role in the discovery and development of drugs [7]. Some natural products, which can break through the

penetration barriers, are historically significant as lead compounds for antimicrobials [8]. Coumarins are a vast

class of natural products widely occurring as secondary plant metabolites [9]. More than 1800 different natural

and synthetic coumarins have been discovered and applied to date, and many of them exhibited high level of

biological activities including insecticidal, antioxidant, anticancer, anti-HIV, antimicrobial, and antibiotic [10–12].

Series of naturally occurring coumarin based antibiotics such as novobiocin, chlorobiocin and coumermycin A1

have been used successfully in clinic (Fig. 1). Especially, novobiocin has a good inhibitory effect on DNA gyrase,

which exerts antibacterial ability via inhibition of ATPase activity of bacterial DNA gyrase in competition with

ATP binding to the B subunit of the enzyme [13,14]. In addition, some investigators have focused on structural

modification of the coumarin skeleton in the benzene ring, lactone ring or both, and found that the resulted

coumarin derivatives showed good antimicrobial potential [15]. Consequently, the in-depth researches toward

coumarin compounds for potential antimicrobial agents have gained much more attentions.

Fig. 1

Thiazolidinedione is an important heterocyclic fragment in design and development of novel medicinal agents

concerning biological activity, including antihyperglycemic, anticancer, antiarthritic, antiinflammatory and

antimicrobial [16,17]. The unique five membered structure of thiazolidinedione ring with carbonyl groups at 2-

and 4-positions which acts as electron-accepting group along with NH and –S– as electron-donating groups is

favorable to effectively interact with biologically important species such as DNA, enzymes and receptors [18].

The representative exploration of clinical thiazolidinedione drugs like pioglitazone and its analogues as

antidiabetic agents, and GSK1059615 as first clinical PI3K inhibitor [19,20], have been motivating medicinal

chemists to develop more effective bioactive molecules from the thiazolidinedione moiety. Especially, the

substitution at 5-position of thiazolidinedione brings out the greatest change in structure and properties of

thiazolidinedione with potent antimicrobial activity [21]. Furthermore, the introduction of different substituents on

2

NH group of thiazolidinedione, which might be beneficial to improve solubility and interaction with biomolecular

targets, has been an important and prevalent strategy to modify thiazolidinedione analogues for the potential

clinical drugs [22].

Molecular hybridization strategy is to integrate two or more pharmacophoric units into one molecular scaffold

with possibly different modes of action [23]. The multifunctional attributes of these hybrids offer multiple

biological activities, high selectivity and favorable pharmacokinetics, which renders hybrid molecules a rationally

attractive source for current drug discovery [24]. In our previous work, we found that the introduction of

thiazolidinedione to berberine showed great capacity to treat microorganisms, especially MRSA. The antibacterial

mechanism showed that the suppression was accounted for dual roles of blocking bacterial DNA replication and

intercalating into DNA with excellent antimicrobial activity [25]. In view of the above factors, it is quite

interesting for us to combine the thiazolidinedione moiety and coumarin ring via an ethylenic bond bridge to

develop a new structural framework of potentially antimicrobial agents (Fig. 2).

Fig. 2

The design of target ethylenic conjugated coumarin thiazolidinediones were mainly based on the following

three considerations: (1) the ethylenic bond-bridged new π-conjugated skeleton expanded the conjugated system

of coumarin ring and helpfully enhanced the DNA binding affinity of target molecules; (2) the different coupled

positions of coumarin ring with thiazolidinedione were considered with aim to evaluate their contribution to

antimicrobial activities; (3) various alkyl and unsaturated hydrocarbon chains with different lengths were

introduced in order to regulate the binding affinity as well as lipid-water partition coefficient, while the

substituents like chloro- and fluoro- on phenyl groups were considered to affect the pharmacological properties by

modulating lipophilicity and cell membrane permeability, thus influencing the rate of transport and absorption of

drugs.

All the synthesized ethylenic conjugated coumarin thiazolidinediones were characterized by H NMR, ¹³

1

C

NMR and HRMS spectral analysis, and were screened for their antibacterial and antifungal activities in vitro.

Compound 12b was selected to be further investigated for its interaction with DNA to explore the DNA binding

efficacy and antimicrobial mechanism. To identify the effect of 12b on cell viability, the cytotoxicity was tested

against human hepatocyte LO2 cell line. Bacterial membrane permeabilization was implemented to evaluate drug

uptake. Further experiments including bactericidal kinetic assay, drug resistance development, and theoretical

investigation of molecular docking were also performed to verify the prospect of the most active compound.

2

. Results and discussion

2

.1. Chemistry

The commercial resorcinol, malic acid, ethylacetoacetate and 4-chloroacetoacetate were employed as starting

materials to synthesize the target ethylenic conjugated coumarin thiazolidinediones, which were prepared via

multistep reactions including cyclization, formylation, hydrolysis, oxidation, electrophilic substitution and

Knoevenagel condensation reaction, as outlined in Schemes 1 and 2. Condensation of resorcinol 1 with malic

3

o

acid, ethylacetoacetate or 4-chloroacetoacetate ethyl ester in concentrated sulfuric acid at 0 C respectively

afforded coumarin derivatives 2ab and 5 with high yields (81.290.0%). Compounds 2ab were easily

formylated by hexamethylenetetramine (HMTA) using glacial acetic acid as solvent to produce the corresponding

coumarin aldehydes 3ab in yields of 10.024.0%. The hydrolysis of 4-chloromethyl-7-hydroxycoumarin 5 in

boiling water produced 7-hydroxy-4-(hydroxymethyl) coumarin 6 with 92.0% yield, which was further oxidized

by manganese dioxide to give 4-formyl-7-hydroxycoumarin 7 in yield of 41.0%. Intermediates 3ab and 7

underwent condensation reaction with thiazolidinedione in anhydrous ethanol using piperidine as base to afford

the target compounds 4ab and 8 with moderate yields of 51.966.0%.

The antibacterial activities of all the ethylenic conjugated coumarin thiazolidinediones (4a-b and 8) were tested,

and we found that 4-methyl-8-thiazolidinedionyl coumarin derivative 4b showed more efficient antimicrobial

ability than compounds 4a and 8. On the purpose of investigating the essential influence of various substituents on

the antimicrobial activity, the structure of compound 4b was further modified by various substituents including

aliphatic bromides, benzyl halides and 2-bromoethanol. Much effort in modifying the 7-hydroxyl group of

compound 4b is confronted with the side reaction of NH substitution. According to previous researches, NH

group should be preserved to increase aqueous solubility and enhance DNA affinity. Therefore, another synthetic

route was adopted starting from compound 3b as shown in Scheme 2. The 7-hydroxyl group of compound 3b was

substituted by a variety of saturated or unsaturated alkyl bromides using potassium carbonate as base in

o

N,N-dimethylformamide (DMF) at 80 C to give corresponding aliphatic derivatives 9ag and 11ab with good

yields (73.185.8%). They were further reacted with thiazolidinedione to produce the various desirable ethylenic

conjugates of coumarin thiazolidinediones 10ag and 12ab in 48.675.4% yields, respectively. A series of target

phenyl coumarin thiazolidinediones derivatives 15ae were also synthesized under similar reaction conditions

from intermediate 3b by reacting with the substituted benzyl chlorides and then condensing with

thiazolidinedione. In order to investigate whether the substituents at NH group would decrease, retain or improve

the antimicrobial activity, the target compounds 13ab were effectively prepared by similar reaction starting from

intermediates 12ab with the same aliphatic bromides. All the newly synthesized ethylenic conjugated coumarin

1

13

thiazolidinediones were confirmed by H NMR, C NMR and HRMS spectra.

Scheme 1

Scheme 2

2

.2. Analysis of configuration (E or Z)

When thiazolidinedione was reacted with coumarin aldehydes 3, 7, 9, 11 and 14 respectively by the

Knoevenagel condensation, theoretically there are two isomers on tautomeric equilibrium. In order to identify the

structure of the isomers, the single crystal of the compound 13a was cultured and the single crystal X-ray

diffraction analysis (Fig. 3) showed that the thiazolidinedione fragment and coumarin ring via an ethylenic bond

bridge generated a new structural type with Z configuration. It was possible that the E configuration was generally

unstable, and it easily converted to the Z configuration.

4

2

.3. Biological activity

All newly synthesized ethylenic conjugated coumarin thiazolidinediones were evaluated for in vitro

antibacterial and antifungal activity against five Gram-positive bacteria, six Gram-negative bacteria and four

fungal strains recommended by the Clinical and Laboratory Standard Institute (CLSI) using the referenced

antimicrobial drugs norfloxacin and fluconazole as positive controls.

2

.3.1. Antibacterial activity

The result for antibacterial activity (Table 1) showed that some of the newly synthesized ethylenic conjugated

coumarin thiazolidinediones exhibited potent efficacy comparable to reference drug norfloxacin against the tested

bacterial stains.

We sought to establish the structure-activity relationship (SAR) for this class of compounds by introducing

thiazolidinedione on the three different coumarin rings, and then choosen the best antimicrobial activity coumarin

ring and modified the C-7 hydroxyl group of coumarin skeleton by different length of “C-linker” aliphatic chains,

hydroxyethyl fragment and substituted aromatic rings to investigated their antimicrobial effects. Preliminary

biological screening showed that 8-thiazolidinedionyl coumarin derivatives 4a and 4b possessed slightly better

antibacterial efficacy than 4-thiazolidinedionyl coumarin derivative 8. At the same time,

4

8

7

-methyl-8-thiazolidinedionyl coumarin 4b was superior to coumarin 4a without substitution at the C-4 position of

-thiazolidinedionyl group in antibacterial activity, especially against Gram-negative bacteria. Most of the

-hydroxyl thiazolidinedione coumarin derivatives exhibited moderate to good antibacterial activities against

MRSA, K. pneumonia and A. baumanii, demonstrating the rationality of our design strategy.

Compounds 10ab and 12ab displayed excellent antibacterial activity with MIC (0.006 to 0.012 µmol/mL)

against MRSA in comparison with norfloxacin with MIC = 0.025 µmol/mL. Particularly, compounds 12a and 12b

were the most potent inhibitors of MRSA (MIC = 0.006 µmol/mL), which showed 4-fold higher inhibitory

activity than norfloxacin. Ethylenic conjugated coumarin thiazolidinediones 12a-b with short unsaturated alkyl

chain showed better inhibitory activity than short saturated alkyl derivatives 10a and 10b against MRSA.

Compounds with long saturated alkyl chain (i.e., 10c, 10d, 10e, 10f) didn’t suppress the growth of MRSA

effectively. The above results demonstrated that the introduction of the short unsaturated hydrocarbons and

aliphatic chains could significantly strengthen antibacterial activity, which might regulate the lipid-water partition

coefficient and binding affinity to target enzymes. Furthermore, when the NH position of compounds 12a and 12b

were substituted by the same unsaturated hydrocarbons, the antibacterial abilities decreased about 56 folds with

MIC values of 0.167 and 0.338 µmol/mL, indicating that the NH group could strengthen antibacterial ability by its

weak interactions with biological targets.

Most of derivatives with MIC (0.047 to 0.387 µmol/mL) were found to improve antibacterial activities against

K. pneumonia in comparison with norfloxacin (MIC = 0.401 µmol/mL). Especially, compound 12b was identified

as the most potent inhibitor of K. pneumonia (MIC = 0.047µmol/mL), around 9-fold higher inhibitory activity

than norfloxacin. Noticeably, coumarin halophenyl derivatives 15bd also had higher levels of bacterial growth

inhibition activities against K. pneumonia with MIC values of 0.311, 0.139 and 0.278 µmol/mL, respectively. This

5

manifested that the lipophilicity and membrane permeability might be increased by the introduction of halogen

atoms, which could be helpful for the rate of absorption as well as transportation of drugs to strengthen the

antibacterial activities.

The antibacterial evaluation showed that MRSA was sensitive to most of the ethylenic conjugated coumarin

thiazolidinediones, especially compounds 12a and 12b. Most of target compounds could not inhibit the growth of

S. aureus ATCC 25923, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. coli, E. faecalis, P. aeruginosa,

E. coli ATCC 25922 effectively. Modification of compound 4b indicated that the short unsaturated hydrocarbon

and alkyl chain derivatives at the C-7 of coumarin ring were much more potent than the other substituents.

Table 1

2

.3.2. Antifungal activity

The data for antifungal activity listed in Table S1 displayed that most of the target ethylenic conjugated

coumarin thiazolidinediones showed comparable or better antifungal activity than reference drug. Compounds

0ab and 12ab (MIC = 0.0060.012 µmol/mL) exhibited superior inhibition against the growth of A.

1

fumigates, at least 139 folds more potent than fluconazole (MIC = 1.672 µmol/mL). It was found that the short

unsaturated hydrocarbon and alkyl chains significantly strengthened antifungal activity. Similarly, benzyl

derivatives had better activities than ethylenic conjugated coumarin thiazolidinediones unsubstituted at C-7

hydroxyl group. In the series of compounds 15ae, compound 15e showed the best anti-A. fumigates activity. It

implied that strong electron-withdrawing groups on phenyl ring were beneficial to antifungal activities. Notably,

C. tropicals was more valuable to compounds 4b, 10ab, 12ac and 15ae (MICs = 0.023–0.371 µmol/mL) than

fluconazole (MIC = 0.836 µmol/mL).

All the antimicrobial results revealed that the antimicrobial efficacies of ethylenic conjugated coumarin

thiazolidinediones is due to not only the modification of thiazolidinedione on coumarin ring but also the

substitution at 7-position of coumarin skeleton. In particular, ethylenic conjugated coumarin thiazolidinedione

compound 12a bearing a propenyl chain and compound 12b with a propinyl chain exhibited unprecedentedly

effective inhibition toward MRSA with the same low MIC (0.006 µmol/mL), and they could be further studied as

potentially antimicrobial agents.

2

.4. Bactericidal kinetics

It is well-known that antibacterial effect can be evaluated through the rate of bactericidal activity [26]. To

explore the antibacterial potency of compound 12b, the kinetics of killing MRSA were investigated for compound

2b bearing a propinyl chain. As shown in Fig. 4, compound 12b (4 × MIC) was superior to norfloxacin with 5

1

log CFU/mL reduction within 3 h, suggesting that it possessed bacteriostatic and bactericidal effects.

Fig. 4

2

.5. Development of resistance to compound 12b

The ever-growing risk of bacterial resistance is a critical concern, and it is the most dreadful that the various

6

antimicrobial resistant bacterial strains caused serious complications [27]. However, antibacterial agents that

induce the death of resistant bacterial strains are deficient [28]. Thus, researches on the resistance of the highly

active compound 12b against the microbial strains were significantly essential. Due to the excellent effect of

compound 12b against MRSA, the susceptible MRSA was chosen as an experimental strain for measuring drug

resistance, and norfloxacin was taken as control. The bacterial resistance of compound 12b remained unchanged

after 16 passages, whereas MRSA developed resistance against norfloxacin after six passages, suggesting that

compound 12b was harder to induce drug resistance than norfloxacin (Fig. 5).

Fig. 5

2

.6 Cytotoxicity

Cytotoxicity is an important consideration for the development of potential drug candidates. Therefore,

compound 12b was further examined for their cytotoxicity by MTT assay against the growth of human hepatocyte

LO2 cells [29]. It was evident from the results (Fig. 6) that the compound 12b didn’t show any appreciable

cytotoxicity and cells were significantly alive even after 24 h or 48 h, and more than 95% of cells were viable at

the lowest concentration (0.8 µM). Therefore, compound 12b showed the low toxicity, implying no obvious harm

to the human body.

Fig. 6

2

.7. Bacterial membrane permeabilization

Bacterial membrane active agents are topical antibacterial compounds that permeabilize bacterial membranes,

thus enhancing drug uptake. While Gram-positive bacteria are surrounded by an inner membrane and a thick layer

of cell wall [30], this structure forms a physical barrier that renders Gram-positive bacteria naturally resistant to

many antibacterial agents. These facts adequately illustrate the significance of membrane active molecules.

Herein, the disruption of bacterial membrane of compound 12b was evaluated by utilizing propidium iodide (PI)

dye. If it can target or interact with cell membrane, compound 12b will pass through the compromised cell

membranes and increase fluorescence with a complex formed by DNA. The fluorescence intensity of

experimental group (in the presence of compound 12b) gradually increased with time and then stabilized, while

the control group almost showed no change (Fig. 7). It indicated that compound 12b could effectively permeate

the membrane of MRSA.

Fig. 7

2

.8. Interactions of compound 12b with MRSA DNA

Previous work revealed that coumarins could effectively interact with DNA through its excellent DNA binding

affinity [31]. As is well known to us, DNA is used for storing genetic information to continue the life and can also

be considered as a multiple-site target for the design and synthesis of efficient antimicrobial drugs [32]. Therefore,

to investigate the possible antimicrobial mechanism, the binding behavior of compound 12b with isolated

7

drug-resistant MRSA DNA were investigated by UV-vis spectroscopy using spectral probe of neutral red.

2

.8.1. Absorption spectra of DNA in the presence of compound 12b

The UV-Vis absorption in Fig. 8 showed a red-shift peak for the maximum absorption of DNA with

proportional increasing concentrations of compound 12b. It was observed that the absorption value of 12b-DNA

complex was a little smaller than the simply sum of free DNA and free compound 12b, in line with previous

studies [33,34], which suggested the hypochromism of the complex of DNA and compound 12b. This implied the

intercalation of the aromatic chromophore of compound 12b into the helix and the aromatic chromophore was in

close proximity to the DNA bases.

Fig. 8

2

.8.2. Absorption spectra of NR interaction with DNA

In order to further explore the mode of action of molecule 12b and MRSA DNA, this work used neutral red

(NR) as spectral probe. As shown in Fig. S2, with the increasing concentration of MRSA DNA, the NR absorption

peak around 460 nm gradually decreased and a new band at around 530 nm appeared, which accounted for the

formation of DNA–NR complex. At the same time, the formation of DNA-NR complex was also revealed by the

signal occurrence of an isosbestic point at 504 nm.

2

.8.3. Absorption spectra of competitive interaction of compound 12b and NR with DNA

The competitive binding of NR and compound 12b with DNA was shown in Fig. 9. With the increasing

concentration of compound 12b, the maximum absorption peak of DNA-NR complex at 530 nm decreased, which

was opposite with the absorption of free NR as the concentrations of DNA increased. This showed that compound

1

2b replaced NR and intercalated in the double helices of MRSA DNA. Therefore, it was further manifested that

compound 12b could exert the antibacterial activities by intercalating into DNA to block its replication.

Fig. 9

2

.9. Molecular docking

To further rationalize the observed antimicrobial activity, a flexible ligand-receptor docking investigation

was undertaken against gyrase-DNA complex (PDB code: 2XCS) [35,36]. Molecular docking (Fig. 10)

demonstrated that the carbonyl group of thiazolidinedione moiety could bind with the active site ARG1122 in a

non-covalent binding mode. The oxygen atom of carbonyl group in thiazolidinedione ring was adjacent to the

ARG1122, forming two hydrogen bonds with distances of 2.8 and 3.3 Å, respectively. The hydrogen bonds could

be helpful for the stability of the 12b-DNA complex, which might be responsible for the good inhibitory efficacy

of compound 12b against MRSA.

Fig. 10

3

. Conclusion

8

A class of new ethylenic conjugated coumarin thiazolidinediones as potential antimicrobial agents were

1

13

developed via some appropriate and productive synthetic routes. Their structures were confirmed by H NMR, C

NMR and HRMS spectra. The antimicrobial evaluation in vitro revealed that some target compounds manifested

better efficacy toward the tested strains of bacteria and fungi in comparison with the reference drugs norfloxacin

and fluconazole. Moreover, the SAR study indicated that the type of substituents on the 7-hydroxyl group of

coumarin skeleton was an important influencing factor on antimicrobial potency and most of the aliphatic and

benzyl substituents exerted relatively potent antimicrobial activities. Especially, compound 12b, the ethylenic

conjugated coumarin thiazolidinediones with propargyl group, exhibited excellent unexpected interference and

inhibitory ability toward MRSA (MIC = 0.006 µmol/mL) and drug-resistant A. fumigates (MIC=0.012 µmol/mL).

In addition, it showed no toxicity against LO2 cell line and did not prompt resistance development in MRSA.

Preliminary mechanism studies demonstrated that the active molecule 12b might target on cell membrane and

DNA by intercalating into MRSA DNA to form a steady 12b-DNA complex, which might shut off DNA

replication to inhibit the growth of MRSA. Moreover, molecular calculation study showed the binding between

compound 12b and gyrase-DNA complex though hydrogen bonds. These results strongly demonstrated that

compound 12b bearing propargyl group at C-7 position of coumarin ring would have enormous possibilities as

new bactericidal compound, especially as a multi-targeting anti-MRSA agent.

4

. Experimental protocols

4

.1. General methods

All commercially available solvents and reagents were purchased without further purification. Melting points

1

were determined with X–6 melting point apparatus. TLC analysis was done using pre-coated silica gel plates. H

NMR and ¹³C NMR spectra were recorded on AVANCE III 600 MHz spectrometer using tetramethylsilane

(TMS) as an internal standard. The chemical shifts were reported in parts per million (ppm), the coupling

constants (J) were expressed in hertz (Hz) and signals were described as singlet (s), doublet (d), triplet (t), quartet

(

q) as well as multiplet (m). Mass spectra were recorded on an LCMS-2010A, the high-resolution mass spectra

HRMS) were analyzed by an IonSpec FTICR mass spectrometer with ESI resource.

(

4

.1.1. General procedures for the preparation of hydroxycoumarin and coumarin aldehydes (2ab, 3ab and

7)

5

Compound 2ab, 3ab and 57 were obtained according to the synthetic methods reported in literature [37,38].

4

.1.2. General procedures for the preparation of aliphatic and aromatic coumarin derivatives (9af, 11ac and

4ae)

1

Compound 9af, 11ac and 14ae were obtained through the previously reported methods in literature [39].

.1.3. Synthesis of 7-ethoxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (9a)

4

Compound 3b (350 mg, 1.7 mmol) and potassium carbonate (355 mg, 2.6 mmol) were stirred in

o

N,N-Dimethylformamide (50 mL) at 80 C for 1 h, then the bromoethane (280 mg, 2.6 mmol) was added and the

9

o

temperature maintained at 80 C for 6 h. After cooling at room temperature, the reaction mixture was diluted with

water (25 mL) and extracted with chloroform (3 × 20 mL). The organic phase was concentrated under vacuum.

The crude product was purified by gel column chromatography (eluent, petroleum ether/chloroform (20/1, V/V))

o

1

to afford the desired compound 9a (321 mg, 80.6%) as yellow solid. Mp: 210212 C; H NMR (600 MHz,

CDCl ) δ 10.65 (s, 1H, CHO), 7.72 (d, J = 8.9 Hz, 1H, coumarin-5-H), 6.93 (d, J = 8.9 Hz, 1H, coumarin-6-H),

3

6

.19 (s, 1H, coumarin-3-H), 4.24 (q, J = 6.9 Hz, 2H, OCH CH ), 2.41 (s, 3H, coumarin-4-CH ), 1.52 (t, J = 6.9

2 3 3

1

3

Hz, 3H, OCH CH ) ppm; C NMR (151 MHz, CDCl ) δ 187.1 (CHO), 162.6 (OC=O), 159.6, 155.2, 151.9, 130.6,

2

3

1

13.5, 112.9, 112.6, 108.6, 65.4 (OCH ), 18.8 (coumarin-4-CH ), 14.5 (OCH CH ) ppm.

2 3 2 3

4

.1.4. Synthesis of 4-methyl-2-oxo-7-propoxy-2H-chromene-8-carbaldehyde (9b)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol), 1-bromopropane

320 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were employed to produce the pure product 9b

(

o

1

342 mg, 81.7%) as light yellow solid. Mp: 158159 C; H NMR (600 MHz, CDCl ) δ 10.67 (s, 1H, CHO), 7.72

3

d, J = 9.0 Hz, 1H, coumarin-5-H), 6.93 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.19 (s, 1H, coumarin-3-H), 4.12 (t, J

=

6.4 Hz, 2H, OCH CH CH ), 2.41 (s, 3H, coumarin-4-CH ), 1.94–1.88 (m, 2H, OCH CH CH ), 1.09 (t, J = 7.4

2 2 3 3 2 2 3

1

3

Hz, 3H, O(CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 187.1 (CHO), 162.8 (OC=O), 159.6, 155.3, 151.8,

2

3

1

30.6, 113.5, 113.0, 112.6, 108.6, 71.1 (OCH ), 22.4, 18.8 (coumarin-4-CH ), 10.4 ppm.

2 3

4

.1.5. Synthesis of 7-butoxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (9c)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol), 1-bromobutane (356

mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were employed to produce the pure product 9c (380

o

1

mg, 85.8%) as yellow solid. Mp: 154155 C; H NMR (600 MHz, CDCl ) δ 10.65 (s, 1H, CHO), 7.72 (d, J = 9.0

3

Hz, 1H, coumarin-5-H), 6.93 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.18 (s, 1H, coumarin-3-H), 4.16 (t, J = 6.4 Hz,

2

H, OCH (CH ) CH ), 2.41 (s, 3H, coumarin-4-CH ), 1.89–1.83 (m, 2H, OCH CH CH CH ), 1.58–1.51 (m, 2H,

2 2 2 3 3 2 2 2 3

1

3

O(CH ) CH CH ), 1.00 (t, J = 7.4 Hz, 3H, O(CH ) CH ) ppm; C NMR (151 MHz, CDCl ) δ 187.1 (CHO),

2

3

2 3

3

1

62.9 (OC=O), 159.6, 155.2, 151.9, 130.6, 113.5, 113.0, 112.6, 108.6, 69.4 (OCH ), 30.9, 19.1, 18.8

2

(

coumarin-4-CH ), 13.7 ppm.

3

4

.1.6. Synthesis of 7-(decyloxy)-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (9f)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol), 1-bromodecane

(

575 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were employed to produce the pure product 9f

o

1

476 mg, 81.3%) as yellow solid. Mp: 201202 C; H NMR (600 MHz, DMSO-d ) δ 10.46 (s, 1H, CHO), 7.97

6

d, J = 9.0 Hz, 1H, coumarin-5-H), 7.22 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.30 (s, 1H, coumarin-3-H), 4.21 (t, J

=

6.4 Hz, 2H, OCH (CH ) CH ), 2.42 (s, 3H, coumarin-4-CH ), 1.80–1.74 (m, 2H, OCH CH (CH ) CH ), 1.44

2 2 8 3 3 2 2 2 7 3

(

dd, J = 15.0, 7.5 Hz, 2H, O(CH ) CH (CH ) CH ), 1.33 (t, J = 10.7 Hz, 2H, O(CH ) CH (CH ) CH ), 1.28–1.22

2 2 2 2 6 3 2 3 2 2 5 3

1

3

(

m, 10H, O(CH ) (CH ) CH ), 0.85 (t, J = 6.9 Hz, 3H, O(CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2 4 2 5 3 2 9 3 6

1

3

87.46 (CHO), 163.21 (OC=O), 159.62, 154.08, 153.71, 132.48, 113.64, 112.65, 112.16, 109.88, 69.86 (OCH₂),

1.74, 31.13, 29.37, 29.10, 28.82, 25.77, 22.53, 18.8 1(coumarin-4-CH ), 14.55, 14.38 ppm.

3

1

0

4

.1.7. Synthesis of 7-(allyloxy)-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (11a)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol), 3-bromoprop-1-ene

(315 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were employed to produce the pure product 11a

o

1

(

327 mg, 78.7%) as yellow solid. Mp: 172173 C; H NMR (600 MHz, CDCl ) δ 10.68 (s, 1H, CHO), 7.73 (d, J

3

=

9.0 Hz, 1H, coumarin-5-H), 6.94 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.20 (s, 1H, coumarin-3-H), 6.06 (ddd, J =

1

5.5, 10.2, 4.8 Hz, 1H, CH=CH ), 5.54 (d, J = 17.2 Hz, 1H, CH=CH ), 5.37 (d, J = 10.7 Hz, 1H, CH=CH ), 4.75

2

1

3

(

d, J = 4.7 Hz, 2H, OCH ), 2.42 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, CDCl ) δ 185.2 (CHO),

2

3

1

6

60.2 (OC=O), 157.7, 153.7, 150.1, 129.8 (CHC=CH ), 128.8, 116.8 (CHC=CH ), 112.0, 111.4, 111.0, 107.3,

2 2

8.2 (OCH ), 17.0 ppm.

2

4

.1.8. Synthesis of 4-methyl-2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromene-8- carbaldehyde (11b)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol), 3-bromoprop-1-yne

(309 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were employed to produce the pure product

o

1

8

1b (301 mg, 73.1%) as yellow solid. Mp: 206207 C; H NMR (600 MHz, DMSO-d ) δ 10.45 (s, 1H, CHO),

6

.02 (d, J = 9.0 Hz, 1H, coumarin-5-H), 7.27 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.32 (s, 1H, coumarin-3-H), 5.08

1

3

(

s, 2H, OCH ), 3.70 (s, 1H, CCH), 2.43 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 187.3

2

3

6

CHO), 161.3 (OC=O), 159.4, 154.2, 153.5, 132.2, 114.4, 113.1, 112.6, 110.3, 79.9 (CCH), 78.6 (CCH), 57.6

OCH ), 18.8 (coumarin-4-CH ) ppm.

2

3

4

.1.9. Synthesis of 7-((2-fluorobenzyl)oxy)-4-methyl-2-oxo-2H-chromene-8- carbaldehyde (14a)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol),

1

-(chloromethyl)-2-fluorobenzene (376 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were

o

1

employed to produce the pure product 14a (411 mg, 77.5%) as yellow solid. Mp: 255257 C; H NMR (600

MHz, DMSO-d ) δ 10.51 (s, 1H, CHO), 8.03 (d, J = 9.0 Hz, 1H, coumarin-5-H), 7.77 (d, J = 5.1 Hz, 1H, Ph-4-H),

6

7

1

.53 (d, J = 2.8 Hz, 1H, coumarin-6-H), 7.44–7.41 (m, 2H, Ph-3,5-2H), 7.35 (d, J = 9.0 Hz, 1H, Ph-6-H), 6.34 (s,

1

3

H, coumarin-3-H), 5.43 (s, 2H, OCH ), 2.43 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2

3

6

87.4 (CHO), 162.1 (OC=O), 159.5, 154.4, 153.6, 133.9, 132.8, 132.6, 130.5, 130.3, 129.9, 127.9, 114.3, 112.5,

10.3, 68.8 (OCH ), 18.8 (coumarin-4-CH ) ppm.

2

3

4

.1.10. Synthesis of 7-((4-fluorobenzyl)oxy)-4-methyl-2-oxo-2H-chromene-8- carbaldehyde (14b)

According to the similar preparation of compound 9a, compound 3b (350 mg, 1.7 mmol),

1

-(chloromethyl)-4-fluorobenzene (376 mg, 2.6 mmol) and potassium carbonate (355 mg, 2.6 mmol) were

o

1

employed to produce the pure product 14b (424 mg, 79.9%) as yellow solid. Mp: 248249 C; H NMR (600

MHz, CDCl ) δ 10.71 (s, 1H, CHO), 7.72 (d, J = 8.9 Hz, 1H, coumarin-5-H), 7.47 (dd, J = 8.5, 5.4 Hz, 2H,

3

Ph-2,6-2H), 7.09 (t, J = 8.6 Hz, 2H, Ph-3,5-2H), 6.98 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.21 (d, J = 0.9 Hz, 1H,

1

3

1

H, coumarin-3-H), 5.25 (s, 2H, OCH ), 2.41 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, CDCl ) δ

2 3 3

1

86.9 (CHO), 163.5 (OC=O), 161.9, 161.6, 159.4, 155.7, 151.8, 131.2, 130.6, 128.8, 115.8, 115.7, 114.0, 113.4,

13.0, 109.2, 70.5 (OCH ), 18.8 (coumarin-4-CH ) ppm.

2

3

4

.1.11. Synthesis of 7-((3,4-dichlorobenzyl)oxy)-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (14c)

According to the similar preparation of compound 9a, compound 3b (300 mg, 1.47 mmol),

1

,2-dichloro-4-(chloromethyl)benzene (431 mg, 2.21 mmol) and potassium carbonate (305 mg, 2.21 mmol) were

o

1

employed to produce the pure product 14c (230 mg, 43.2%) as white solid. Mp: 248249 C; H NMR (600 MHz,

DMSO-d ) δ 10.54 (s, 1H, CHO), 8.02 (d, J = 9.0 Hz, 1H, coumarin-5-H), 7.85 (s, 1H, Ph-5-H), 7.69 (d, J = 8.3

6

Hz, 1H, Ph-2-H), 7.53 (d, J = 8.2 Hz, 1H, coumarin-6-H), 7.27 (d, J = 9.0 Hz, 1H, Ph-6-H), 6.34 (s, 1H,

1

3

coumarin-3-H), 5.40 (s, 2H, OCH ), 2.43 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2

3

6

1

87.52 (CHO), 132.52, 131.25, 129.70, 127.94, 112.46, 110.24, 69.46 (OCH ), 18.82 (coumarin-4-CH ) ppm.

2

3

4

.1.12. Synthesis of (Z)-5-((7-hydroxy-2-oxo-2H-chromen-4-yl)methylene)thiazolidine-2,4-dione (8)

Compound 7 (200 mg, 1.06 mmol), thiazolidine-2,4-dione (186 mg, 1.59 mmol) and piperidine (15 mg) were

o

stirred in ethanol (20 mL) at 80 C for 12 h. Upon completion of the reaction, the residue was filtered and washed

with ethanol, and the crude product was further purified by column chromatography (eluent, petroleum ether

o

1

/

chloroform (1/1, V/V)) to afford the desired compound 8 (200 mg, 65.3%) as yellow solid. Mp: > 250 C; H

NMR (600 MHz, DMSO-d ) δ 12.88 (s, 1H, NH), 10.76 (s, 1H, OH), 7.88 (s, 1H, C=CH), 7.64 (d, J = 8.8 Hz, 1H,

6

coumarin-5-H), 6.83 (dd, J = 8.7, 2.3 Hz, 1H, coumarin-6-H), 6.78 (d, J = 2.3 Hz, 1H, coumarin-8-H), 6.22 (s,

1

3

1

H, coumarin-3-H) ppm; C NMR (151 MHz, DMSO-d ) δ 167.44 (SC=O), 166.66 (CC=ONH), 162.46 (OC=O),

6

60.16, 155.70, 148.07, 133.93, 127.18, 124.29, 113.85, 110.46, 109.99, 103.25 ppm; HRMS (ESI) calcd. for

+

C H NO S [M-H] , 287.9967, found, 287.9966.

1

3

7

5

4

.1.13. Synthesis of (Z)-5-((7-hydroxy-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione ( 4a )

According to the similar preparation of compound 8, compound 3a (190 mg, 1.0 mmol), thiazolidine-2,4-dione

(176 mg, 1.5 mmol) were employed to produce the pure product 4a (150 mg, 51.9%) as orange solid. Mp: > 250

o

1

C; H NMR (600 MHz, DMSO-d ) δ 7.89–7.83 (m, 2H, C=CH, coumarin-4-H), 7.44 (d, J = 8.7 Hz, 1H,

6

coumarin-5-H), 6.65 (s, 1H, coumarin-6-H), 6.09–6.05 (m, 1H, coumarin-3-H) ppm; ¹³C NMR (151 MHz,

DMSO-d ) δ 171.09 (SC=O), 170.70 (CC=ONH), 165.44 (OC=O), 160.64, 154.85, 145.42, 131.45, 126.48,

6

+

1

23.88, 115.67, 109.25, 108.91 ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 290.0123, found, 290.0125;

1

3

7

5

+

[

M+Na] , 311.9943, found, 311.9940.

4

.1.14. Synthesis of (Z)-5-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (4b)

According to the similar preparation of compound 8, compound 3b (204 mg, 1.0 mmol), thiazolidine-2,4-dione

(175 mg, 1.5 mmol) were employed to produce the pure product 4b (200 mg, 66.0%) as orange solid. Mp: > 250

o

1

C; H NMR (600 MHz, DMSO-d ) δ 12.09 (s, 1H, NH), 7.93 (s, 1H, C=CH), 7.71 (d, J = 8.8 Hz, 1H,

6

coumarin-5-H), 6.94 (d, J = 8.8 Hz, 1H, coumarin-6-H), 6.22 (s, 1H, coumarin-3-H), 2.39 (s, 3H, coumarin-4-CH₃)

ppm; ¹³C NMR (151 MHz, DMSO-d ) δ 169.03 (SC=O), 167.98 (CC=ONH), 159.67 (OC=O), 154.21, 152.96,

6

1

2

1

28.80, 127.84, 124.27, 113.01, 112.49, 110.95, 108.48, 18.76 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for

3

+

C H NO S [M+H] , 304.0280, found, 304.0282.

1

4

9

5

4

.1.15. Synthesis of (Z)-5-((7-ethoxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (10a)

According to the similar preparation of compound 8, compound 9a (334 mg, 1.44 mmol),

thiazolidine-2,4-dione (253 mg, 2.16 mmol) were employed to produce the pure product 10a (256 mg, 53.7%) as

o

1

yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.52 (s, 1H, NH), 7.88 (s, 1H, C=CH), 7.84 (d, J

6

=

9.0 Hz, 1H, coumarin-5-H), 7.18 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.29 (d, J = 0.8 Hz, 1H, coumarin-3-H),

4

.31 (d, J = 7.0 Hz, 2H, OCH CH ), 2.42 (d, J = 0.6 Hz, 3H, coumarin-4-CH ), 1.42 (t, J = 7.0 Hz, 3H,

2

3

1

3

OCH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.60 (SC=O), 167.72 (CC=ONH), 159.49 (OC=O),

2

3

6

1

59.09, 153.95, 152.32, 128.99, 123.76, 113.77, 111.97, 109.79, 109.16, 65.50 (OCH ), 18.71 (coumarin-4-CH ),

2 3

+

4.78 ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 332.0593, found, 332.0592.

1

6

13

5

4

.1.16. Synthesis of (Z)-5-((4-methyl-2-oxo-7-propoxy-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (10b)

According to the similar preparation of compound 8, compound 9b (224 mg, 0.99 mmol),

thiazolidine-2,4-dione (175 mg, 1.49 mmol) were employed to produce the pure product 10b (170 mg, 49.7%) as

o

1

white solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.53 (s, 1H, NH), 7.88 (s, 1H, C=CH), 7.84 (d, J =

6

9

.0 Hz, 1H, coumarin-5-H), 7.19 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.29 (s, 1H, coumarin-3-H), 4.20 (t, J = 6.7

Hz, 2H, OCH CH CH ), 2.43 (s, 3H, coumarin-4-CH ), 1.82 (dt, J = 14.2, 7.1 Hz, 2H, OCH CH CH ), 0.99 (t, J

2

3

2

3

1

3

=

7.4 Hz, 3H, OCH CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.60 (SC=O), 167.69 (CC=ONH),

2 2 3 6

1

59.49 (OC=O), 159.34, 153.97, 152.22, 129.06, 123.79, 113.80, 111.99, 109.90, 109.30, 71.22 (OCH ), 22.26,

2

+

8.73 (coumarin-4-CH ), 10.68 ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 346.0749, found, 346.0750.

3

17 15

5

4

.1.17. Synthesis of (Z)-5-((7-butoxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (10c)

According to the similar preparation of compound 8, compound 9c (128 mg, 0.49 mmol),

thiazolidine-2,4-dione (87 mg, 0.74 mmol) were employed to produce the pure product 10c (105 mg, 59.3%) as

o

1

light yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.52 (s, 1H, NH), 7.88 (d, J = 1.3 Hz, 1H,

6

C=CH), 7.86–7.83 (m, 1H, coumarin-5-H), 7.19 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.29 (s, 1H, coumarin-3-H),

4

.24 (t, J = 6.6 Hz, 2H, OCH (CH ) CH ), 2.41 (d, J = 12.1 Hz, 3H, coumarin-4-CH ), 1.82–1.75 (m, 2H,

2 2 2 3 3

1

3

OCH CH CH CH ), 1.7–1.40 (m, 2H, O(CH ) CH CH ), 0.94 (dd, J = 12.3, 4.9 Hz, 3H, O(CH ) CH ) ppm; C

2

3

2 2

2

3

2 3

3

NMR (151 MHz, DMSO-d ) δ 168.58 (SC=O), 167.67 (CC=ONH), 159.49 (OC=O), 159.35, 153.99, 152.22,

6

1

29.06, 123.82, 113.81, 111.98, 109.90, 109.31, 69.53 (OCH ), 30.84, 19.08, 18.74 (coumarin-4-CH ), 14.07

2 3

+

ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 360.0906, found, 360.0905.

1

8

17

5

4

.1.18. Synthesis of (Z)-5-((4-methyl-2-oxo-7-(pentyloxy)-2H-chromen-8-yl)methylene)thiazolidine2,4-dione (10d)

According to the similar preparation of compound 8, compound 9d (217 mg, 0.79 mmol),

thiazolidine-2,4-dione (139 mg, 1.19 mmol) were employed to produce the pure product 10d (175 mg, 59.4%) as

o

1

yellow solid. Mp: 250 C; H NMR (600 MHz, DMSO-d ) δ 12.53 (s, 1H, NH), 7.87 (s, 1H, C=CH), 7.84 (d, J =

6

1

3

8

.9 Hz, 1H, coumarin-5-H), 7.19 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.29 (s, 1H, coumarin-3-H), 4.22 (t, J = 6.6

Hz, 2H, OCH (CH ) CH ), 2.42 (s, 3H, coumarin-4-CH ), 1.84–1.77 (m, 2H, OCH CH (CH ) CH ), 1.42–1.31

2

2 3

3

2

2 2

3

1

3

(

m, 4H, O(CH ) (CH ) CH ), 0.88 (t, J = 7.2 Hz, 3H, O(CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2 2 2 2 3 2 4 3 6

1

67.74 (SC=O), 159.52 (CC=ONH), 159.35 (OC=O), 154.02, 152.24, 129.01, 123.81, 113.84, 111.99, 109.97,

09.37, 69.80 (OCH ), 28.43, 28.00, 22.22, 18.74 (coumarin-4-CH ), 14.28 ppm; HRMS (ESI) calcd. for

2

3

+

C H NO S [M+Na] , 366.0882, found, 366.0880.

1

9

19

5

4

.1.19. Synthesis of (Z)-5-((7-(hexyloxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (10e)

According to the similar preparation of compound 8, compound 9e (195 mg, 0.68 mmol),

thiazolidine-2,4-dione (119 mg, 1.02 mmol) were employed to produce the pure product 10e (128 mg, 48.6%) as

o

1

white solid. Mp: 240–242 C; H NMR (600 MHz, DMSO-d ) δ 12.52 (s, 1H, NH), 7.87 (s, 1H, C=CH), 7.84 (d, J

6

=

9.0 Hz, 1H, coumarin-5-H), 7.18 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.29 (d, J = 1.1 Hz, 1H, coumarin-3-H),

4

.22 (t, J = 6.6 Hz, 2H, OCH (CH ) CH ), 2.42 (d, J = 0.9 Hz, 3H, coumarin-4-CH ), 1.82–1.77 (m, 2H,

2

2 4

3

OCH CH (CH ) CH ), 1.44–1.38 (m, 2H, O(CH ) CH (CH ) CH ), 1.33–1.27 (m, 4H, O(CH ) (CH ) CH ), 0.86

2

2 3

3

2 2

2

2 2

3

2 3

2 2

3

1

3

(

t, J = 7.1 Hz, 3H, O(CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.53 (SC=O), 167.64 (CC=ONH),

2 5 3 6

1

3

59.49 (OC=O), 159.33, 153.97, 152.24, 129.16, 128.98, 123.81, 113.81, 111.99, 109.94, 109.34, 69.80 (OCH₂),

1.72, 29.34, 29.08, 28.70, 25.79, 22.53, 18.73 (coumarin-4-CH ), 14.36 ppm; HRMS (ESI) calcd. for

3

+

C H NO S [M+Na] , 410.1038, found, 410.1037.

2

0

21

5

4

.1.20. Synthesis of (Z)-5-((7-(decyloxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (10f)

According to the similar preparation of compound 8, compound 9f (215 mg, 0.62 mmol), thiazolidine-2,4-dione

(109 mg, 0.93 mmol) were employed to produce the pure product 10f (205 mg, 74.5%) as light yellow solid. Mp:

o

1

2

25–227 C; H NMR (600 MHz, DMSO-d ) δ 12.52 (s, 1H, NH), 7.88 (s, 1H, C=CH), 7.84 (d, J = 8.8 Hz, 1H,

6

coumarin-5-H), 7.19 (d, J = 8.9 Hz, 1H, coumarin-6-H), 6.29 (s, 1H, coumarin-3-H), 4.23 (t, J = 6.1 Hz, 2H,

OCH (CH ) CH ), 2.42 (s, 3H, coumarin-4-CH ), 1.82–1.76 (m, 2H, OCH CH (CH ) CH ), 1.39 (d, J = 6.6 Hz,

2

2 8

3

2

2 7

3

2

H, O(CH ) CH (CH ) CH ), 1.27 (d, J = 44.9 Hz, 12H, O(CH ) (CH ) CH ), 0.85 (t, J = 6.5 Hz, 3H,

2 2 2 2 6 3 2 3 2 6 3

1

3

O(CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.53 (SC=O), 167.64 (CC=ONH), 159.49 (OC=O),

2

9

3

6

1

2

4

59.33, 153.97, 152.24, 129.16, 128.98, 123.81, 113.81, 111.99, 109.94, 109.34, 69.80 (OCH ), 31.72, 29.34,

2

+

9.08, 28.70, 25.79, 22.53, 18.73 (coumarin-4-CH ), 14.36 ppm; HRMS (ESI) calcd. for C H NO S [M+H] ,

3

24 29

5

44.1845, found, 444.1846.

.1.21. Synthesis of (Z)-5-((7-(2-hydroxyethoxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-

4

dione (10g)

According to the similar preparation of compound 8, compound 9g (51 mg, 0.21 mmol), thiazolidine-2,4-dione

(37.5 mg, 0.32 mmol) were employed to produce the pure product 10g (41 mg, 56.3%) as white solid. Mp: > 250

o

1

C; H NMR (600 MHz, DMSO-d ) δ 7.71 (d, J = 8.9 Hz, 1H, C=CH), 7.32 (s, 1H, coumarin-5-H), 7.14 (d, J =

6

8

.9 Hz, 1H, coumarin-6-H), 6.24 (s, 1H, coumarin-3-H), 4.19 (t, J = 5.2 Hz, 2H, OCH CH OH), 3.78 (t, J = 5.2

2 2

1

4

1

3

Hz, 2H, OCH CH OH), 2.42 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 181.36 (SC=O),

2

3

6

1

3

75.98 (CC=ONH), 160.11 (OC=O), 159.25, 153.93, 151.65, 141.96, 126.42, 114.71, 113.76, 113.52, 111.75,

+

09.23, 71.06 (OCH ), 59.92, 22.91 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H NO S [M+H] ,

2

3

16 13

6

48.0536, found, 348.05301.

4

.1.22. Synthesis of (Z)-5-((7-(allyloxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-dione (12a)

According to the similar preparation of compound 8, compound 11a (200 mg, 0.82 mmol),

thiazolidine-2,4-dione (144 mg, 1.23 mmol) were employed to produce the pure product 12a (212 mg, 75.4%) as

o

1

yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.53 (s, 1H, NH), 7.90 (s, 1H, C=CH), 7.85 (d, J

6

=

9.0 Hz, 1H, coumarin-5-H), 7.18 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.31 (d, J = 1.1 Hz, 1H, coumarin-3-H),

6

.13–6.06 (m, 1H, OCH CH=CH ), 5.43 (dd, J = 17.3, 1.6 Hz, 1H, CH=CH ), 5.33 (dd, J = 10.6, 1.4 Hz, 1H,

2

CH=CH ), 4.86 (d, J = 5.3 Hz, 2H, OCH CH=CH ), 2.43 (d, J = 0.9 Hz, 3H, coumarin-4-CH ) ppm; ¹³C NMR

2

3

(

151 MHz, DMSO-d ) δ 168.63 (SC=O), 167.75 (CC=ONH), 159.46 (OC=O), 158.77, 153.97, 152.22, 133.04

6

CCH=CH), 129.28, 128.93, 123.62, 118.94 (CCH=CH), 114.00, 112.12, 110.01, 109.60, 70.06 (OCH ), 18.74

2

+

coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 344.0593, found, 344.0595.

3

17 13

5

4

.1.23. Synthesis of (Z)-5-((4-methyl-2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromen-8-yl)methylene)thiazolidine-2,4-

dione (12b)

According to the similar preparation of compound 8, compound 11b (320 mg, 1.32 mmol),

thiazolidine-2,4-dione (232 mg, 1.98 mmol) were employed to produce the pure product 12b (268 mg, 59.5%) as

o

1

white solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.54 (s, 1H, NH), 7.92 (d, J = 9.0 Hz, 1H,

6

coumarin-5-H), 7.89 (s, 1H, C=CH), 7.26 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.34 (s, 1H, coumarin-3-H), 5.12 (d,

1

3

J = 2.1 Hz, 2H, OCH ), 3.70 (s, 1H, C≡CH), 2.44 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d )

2

3

6

δ 168.61 (SC=O), 167.67 (CC=ONH), 159.40 (OC=O), 157.54, 153.94, 152.23, 129.52, 128.87, 123.31, 114.50,

1

12.43, 110.31, 109.76, 79.94 (C≡CH), 78.56 (C≡CH), 56.99, 49.07, 18.76 (coumarin-4-CH ) ppm; HRMS (ESI)

3

+

calcd. for C H NO S [M+Na] , 364.0256, found, 364.0258.

1

7

11

5

4

.1.24. Synthesis of (Z)-3-allyl-5-((7-(allyloxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-2,4-

dione (13a)

According to the similar preparation of compound 9a, compound 12a (88 mg, 0.26 mmol), 3-bromoprop-1-ene

(35 mg, 0.29 mmol) and potassium carbonate (40 mg, 0.29 mmol) were employed to produce the pure product 13a

o

1

(

50 mg, 50.2%) as yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.06 (s, 1H, C=CH), 7.88 (d, J =

6

9

.0 Hz, 1H, coumarin-5-H), 7.20 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.32 (d, J = 0.9 Hz, 1H, coumarin-3-H), 6.09

(

ddd, J = 15.9, 10.6, 5.3 Hz, 1H, OCH CH=CH ), 5.90–5.84 (m, 1H, NCH CH=CH ), 5.43 (dd, J = 17.3, 1.5 Hz,

2

1

H, OCH CH=CH ), 5.33 (dd, J = 10.6, 1.3 Hz, 1H, OCH CH=CH ), 5.20–5.16 (m, 2H, NCH CH=CH ), 4.88 (d,

2 2 2 2 2 2

J = 5.3 Hz, 2H, OCH CH=CH ), 4.26 (d, J = 5.4 Hz, 2H, NCH CH=CH ), 2.43 (d, J = 0.6 Hz, 3H,

2

1

3

coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 167.69 (SC=O), 165.74 (CC=ONH), 159.40 (OC=O),

3

6

1

5

1

58.83, 153.99, 152.34, 133.00, 131.70, 129.27, 126.81, 124.88, 119.03, 117.97, 114.06, 112.17, 109.76, 109.67,

+

09.53, 70.13, 43.89, 18.76 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H NO S [M+H] , 384.0906,

3

20 17

5

+

found, 384.0906; [M+Na] , 406.0725, found, 406.0726.

.1.25. Synthesis of (Z)-5-((4-methyl-2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromen-8-yl)methylene)-3-(prop-2-yn-1-

4

yl)thiazolidine-2,4-dione (13b)

According to the similar preparation of compound 9a, compound 12b (150 mg, 0.44 mmol),

3

-bromo-1-propyne (58 mg, 0.484 mmol) and potassium carbonate (61 mg, 0.44 mmol) were employed to

o

1

produce the pure product 13b (98 mg, 58.8%) as yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ

6

8

.07 (s, 1H, C=CH), 7.95 (d, J = 9.0 Hz, 1H, coumarin-5-H), 7.28 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.35 (s, 1H,

coumarin-3-H), 5.14 (d, J = 2.1 Hz, 2H, OCH CCH), 4.43 (d, J = 2.4 Hz, 2H, NCH CCH), 3.71 (t, J = 2.1 Hz, 1H,

2

OCH CCH), 3.33 (t, J = 2.4 Hz, 1H, NCH CCH), 2.45 (s, 3H, coumarin-4-CH ) ppm; ¹³C NMR (151 MHz,

2

3

DMSO-d ) δ 167.14 (SC=O), 165.03 (CC=ONH), 159.31 (OC=O), 157.60, 153.95, 152.37, 129.33, 126.67,

6

1

25.21, 114.56, 112.49, 110.01, 109.82, 80.04, 78.48, 77.65, 74.98, 57.09, 31.00, 18.76 (coumarin-4-CH ) ppm;

3

+

HRMS (ESI) calcd. for C H NO S [M+H] , 380.0593, found, 380.0594.

2

0

13

5

4

2

.1.26. Synthesis of (Z)-5-((7-((2-fluorobenzyl)oxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-

,4-dione (15a)

According to the similar preparation of compound 8, compound 14a (232 mg, 0.74 mmol),

thiazolidine-2,4-dione (130 mg, 1.11 mmol) were employed to produce the pure product 15a (205 mg, 67.4%) as

o

1

yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.44 (s, 1H, NH), 7.87 (d, J = 8.8 Hz, 2H,

6

coumarin-5-H, C=CH), 7.55 (dd, J = 7.5, 6.4 Hz, 1H, ph-4-H), 7.46–7.41 (m, 1H, ph-3-H), 7.32 (d, J = 9.0 Hz, 1H,

coumarin-6-H), 7.28–7.22 (m, 2H, ph-5,6-2H ), 6.30 (s, 1H, coumarin-3-H), 5.43 (s, 2H, OCH ), 2.43 (s, 3H,

2

1

3

coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.54 (SC=O), 167.72 (CC=ONH), 161.72 (OC=O),

3

6

1

4

60.08, 159.42, 158.85, 153.96, 152.13, 131.23, 129.65, 128.99, 125.05, 123.45, 123.17, 116.02, 115.88, 114.37,

+

12.30, 110.44, 109.78, 65.59, 18.74 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H FNO S [M+H] ,

3

21 14

5

12.0655, found, 412.0653.

4

2

.1.27. Synthesis of (Z)-5-((7-((4-fluorobenzyl)oxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-

,4-dione (15b)

According to the similar preparation of compound 8, compound 14b (87 mg, 0.27 mmol),

thiazolidine-2,4-dione (48 mg, 0.41 mmol) were employed to produce the pure product 15b (56 mg, 50.5%) as

o

1

light yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.51 (s, 1H, NH), 7.90 (s, 1H, C=CH), 7.85

6

(d, J = 8.9 Hz, 1H, coumarin-5-H), 7.54–7.50 (m, 2H, ph-2,6-2H), 7.24 (dd, J = 16.5, 8.4 Hz, 3H, ph-3,5-2H,

coumarin-6-H), 6.31 (s, 1H, coumarin-3-H), 5.39 (s, 2H, OCH ), 2.42 (s, 3H, coumarin-4-CH ) ppm; ¹³C NMR

2

3

(

151 MHz, DMSO-d ) δ 168.55 (SC=O), 167.69 (CC=ONH), 163.22 (OC=O), 161.60, 159.43, 158.92, 153.97,

6

1

52.14, 132.56, 130.50, 129.48, 128.94, 123.62, 115.91, 115.77, 114.18, 112.21, 110.32, 109.86, 70.38, 18.73

+

(

coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H FNO S [M+Na] , 434.0474, found, 434.0474.

3

21 14

5

1

6

4

2

.1.28. Synthesis of (Z)-5-((7-((2,4-dichlorobenzyl)oxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-

,4-dione (15c)

According to the similar preparation of compound 8, compound 14c (276 mg, 0.76 mmol),

thiazolidine-2,4-dione (134 mg, 1.14 mmol) were employed to produce the pure product 15c (185 mg, 52.9%) as

o

1

yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.48 (s, 1H, NH), 7.87 (d, J = 7.4 Hz, 2H, ph-3-H,

6

C=CH), 7.70 (d, J = 2.0 Hz, 1H, coumarin-5-H), 7.59 (d, J = 8.3 Hz, 1H, ph-5-H), 7.48 (dd, J = 8.3 Hz, 1H,

ph-6-H), 7.28 (d, J = 9.1 Hz, 1H, coumarin-6-H), 6.32 (d, J = 1.0 Hz, 1H, coumarin-3-H), 5.42 (s, 2H, OCH₂),

1

3

2

1

.43 (s, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 168.47 (SC=O), 167.65 (CC=ONH),

3 6

59.38 (OC=O), 158.62, 153.91, 152.09, 134.48, 134.33, 132.83, 132.07, 129.75, 129.53, 129.00, 128.07, 123.44,

14.51, 112.39, 110.52, 109.83, 68.29, 18.73 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H Cl NO S

3

21 13

2

5

+

[

M+H] , 461.9970, found, 461.9969.

4

2

.1.29. Synthesis of (Z)-5-((7-((3,4-dichlorobenzyl)oxy)-4-methyl-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-

,4-dione (15d)

According to the similar preparation of compound 8, compound 14d (230 mg, 0.64 mmol),

thiazolidine-2,4-dione (113 mg, 0.96 mmol) were employed to produce the pure product 15d (178 mg, 60.3%) as

o

1

yellow solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.46 (s, 1H, NH), 7.85–7.80 (m, 2H, ph-5-H,

6

C=CH), 7.74 (d, J = 1.7 Hz, 1H, coumarin-5-H), 7.66 (d, J = 8.3 Hz, 1H, ph-2-H), 7.44 (dd, J = 8.3, 1.8 Hz, 1H,

ph-6H), 7.20 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.30 (d, J = 0.8 Hz, 1H, coumarin-3-H), 5.40 (s, 2H, OCH ), 2.42

2

(

s, 3H, coumarin-4-CH ) ppm; ¹³C NMR (151 MHz, DMSO-d ) δ 159.62 (SC=O), 158.42 (CC=ONH), 153.91

3 6

(OC=O), 151.95, 137.83, 131.65, 131.15, 129.90, 128.10, 114.34, 112.25, 109.78, 69.37, 18.71 (coumarin-4-CH₃)

+

ppm; HRMS (ESI) calcd. for C H Cl NO S [M+H] , 461.9964, found, 461.99619.

2

1

13

2

5

4

2

.1.30. Synthesis of (Z)-5-((4-methyl-7-((4-nitrobenzyl)oxy)-2-oxo-2H-chromen-8-yl)methylene)thiazolidine-

,4-dione (15e)

According to the similar preparation of compound 8, compound 14e (254 mg, 0.75 mmol),

thiazolidine-2,4-dione (132 mg, 1.13 mmol) were employed to produce the pure product 15e (100 mg, 30.4%) was

o

1

obtained as white solid. Mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 12.49 (s, 1H, NH), 8.28–8.25 (m, 2H,

6

ph-3,5-2H), 7.94 (d, J = 21.2 Hz, 1H, C=CH), 7.85 (d, J = 9.0 Hz, 1H, coumarin-5-H), 7.73–7.70 (m, 2H,

ph-2,6-2H), 7.21 (d, J = 9.0 Hz, 1H, coumarin-6-H), 6.32 (d, J = 1.0 Hz, 1H, coumarin-3-H), 5.59 (s, 2H, OCH₂),

1

3

2

.41 (d, J = 6.2 Hz, 3H, coumarin-4-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 159.45 (SC=O), 159.05

3 6

(CC=ONH), 158.48 (OC=O), 153.95, 152.11, 147.70, 144.25, 128.76, 128.60, 125.98, 124.09, 114.43, 112.36,

+

1

4

12.02, 109.80, 109.37, 69.79, 18.73 (coumarin-4-CH ) ppm; HRMS (ESI) calcd. for C H N O S [M+H] ,

3

21 14

2

7

39.0600, found, 439.0601.

Conflicts of interest

There are no conflicts to declare.

1

7

Acknowledgments

This research was supported in part by grants from the National Natural Science Foundation of China (Nos.

2

1672173, 21971212, 21850410447), China Postdoctoral Science Foundation (2019M653821XB), Chongqing

Special Foundation for Postdoctoral Research Proposal (No. XmT2018082).

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1

Lists of table and scheme captions

Table 1. Antibacterial data as MIC (µmol/mL) for compounds 8, 4ab, 10af, 12ac, 13ab and 15ae.

Figure. 1. Drug molecules containing coumarin scaffold.

Figure. 2. Design of novel coumarin thiazolidinediones.

Figure. 3. The crystal structure of compound 13a.

Figure. 4. Time–kill kinetics of compound 12b (4 × MIC) against MRSA.

Figure. 5. Drug resistance development of compound 12b against MRSA.

Figure. 6. Cytotoxic assay of target compound 12b on human hepatocyte LO2 cells tested by MTT methodology.

Figure. 7. Membrane permeabilization of resistant bacterial MRSA toward compound 12b (12 × MIC).

Figure. 8. UV absorption spectra of MRSA DNA with different concentrations of compound 12b (pH = 7.4, T =

2

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-5

sum values of free DNA and free compound 12b. c(DNA) = 7.4 × 10 mol/L, and c(compound 12b) = 02.0

-5

×

10 mol/L for curves ai respectively at an increment 0.25 ×10 .

Figure. 9. UV absorption spectra of the competitive reaction between 12b and NR with MRSA DNA. c(DNA) =

-

5

-5

7

.4 × 10 mol/L, c(NR) = 2 × 10 mol/L, and c(compound 12b) = 04.5 ×10 mol/L for curves ai respectively

-5

at increment 0.5 × 10 . (Inset) Absorption spectra of the system with the increasing concentration of 12b in the

wavelength range of 350600 nm absorption spectra of competitive reaction between compound 12b and NR with

DNA.

Figure. 10. Molecular modeling of compound 12b docked in gyrase-DNA.

Scheme 1 Synthetic route of coumarin thiazolidinediones 1–8.

Scheme 2 Synthetic route of coumarin thiazolidinediones 9–15e.

2

Table 1. In vitro antibacterial data as MIC (µmol/mL) for target compounds 8, 4ab, 10ag, 12ab, 13ab and 15ae.^a

Gram-positive bacteria^b

Gram-negative bacteria^c

Compds

S. a.

5923

S. a.

29213

P. a.

27853

E. c.

25922

MRSA

E. f.

S. a.

K. p.

E. c.

P. a.

A. b.

2

8

0.886

1.772

0.422

0.012

1.426

0.343

0.331

1.155

0.369

0.006

0.167

0.338

0.039

0.311

0.278

0.139

0.584

0.025

0.886

0.106

0.773

0.742

1.426

0.686

0.331

1.155

0.184

0.746

0.751

0.668

0.338

0.156

0.278

0.292

0.006

0.886

0.443

0.422

0.193

0.012

1.426

1.372

0.661

1.155

0.369

0.023

0.375

0.334

0.338

0.311

0.555

0.278

0.292

0.025

1.772

0.422

0.773

0.742

1.426

1.372

0.331

1.155

0.369

0.746

0.751

0.668

0.675

0.039

0.156

0.278

0.146

0.100

1.772

0.422

0.773

0.742

1.426

0.686

0.661

1.155

0.369

0.746

0.047

0.668

0.338

0.311

0.278

0.584

0.003

0.886

1.772

0.422

0.387

0.371

1.426

0.343

0.331

1.155

0.369

0.373

0.047

0.334

0.675

0.623

0.311

0.139

0.278

0.292

0.401

1.772

0.422

0.773

0.742

1.426

1.372

0.331

1.155

0.369

0.746

0.751

0.668

0.675

0.311

0.623

0.278

0.292

0.200

0.886

1.772

0.211

0.387

0.371

1.426

0.343

0.661

1.155

0.184

0.373

0.188

0.668

0.338

0.311

0.278

0.584

0.025

1.772

0.886

0.422

0.387

0.371

1.426

0.686

0.331

1.155

0.184

0.373

0.188

0.334

0.675

0.311

0.156

0.278

0.139

0.292

0.802

0.886

1.772

0.422

0.773

0.371

1.426

1.372

0.331

1.155

0.369

0.373

0.188

0.668

0.675

0.156

0.278

0.292

0.013

0.443

0.886

0.422

0.773

0.742

1.426

1.372

0.661

1.155

0.184

0.746

0.375

0.334

0.338

0.311

0.139

0.584

0.025

4

a

4

b

1

0a

0b

0c

0d

0e

0f

1

0g

2a

1

2b

3a

3b

5a

5b

5c

5d

5e

A^d

1

^aMinimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. ^bMRSA,

methicillin-resistant Staphylococcus aureus N315; E. f., Enterococcus faecalis; S. a., Staphylococcus aureus; S. a. 25923,

c

Staphylococcus aureus ATCC 25923; S. a. 29213, Staphylococcus aureus ATCC 29213; K. p., Klebsiella pneumonia; E. c.,

Escherichia coli; P. a., Pseudomonas aeruginosa; A. b., Acinetobacter baumanii; P. a. 27853, Pseudomonas aeruginosa ATCC

d

2

7853; E. c. 25922, Escherichia coli ATCC 25922 . A = Norfloxacin

2

3

Fig. 1. Drug molecules containing coumarin scaffold.

H C

O

CH₃

3

OH

CH3

O CH₃

O

OH

H C CH3

3

H

N

O

H

N

O

H C

3

O

HO

O

O H C

HN

3

OH

NH

O

R

O

OH

Cl

CH₃

O

OH

O

CH₃

O

HO

Novobiocin, R =

N

O

NH₂

HN

CH

^C^H3

3

H

N

O

CH

3

CH

Chlorobiocin, R =

CH₃

^C^H3

Coumermycin A1

2

4

Fig. 2. Design of novel coumarin thiazolidinediones

.

2

5

Fig. 3. The crystal structure of compound 13a.

2

6

Fig. 4. Time–kill kinetics of compound 12b (4 × MIC) against MRSA.

18

16

14

12

10

8

6

4

2

0

Norfloxacin

Compound 12b

Control

0

1

2

3

4

5

6

Time (h)

2

7

Fig. 5. Drug resistance development of compound 12b against MRSA.

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

Compound 12b

Norfloxacin

0

2

4

6

8

10

12

14

16

Time (day)

2

8

Fig. 6. Cytotoxic assay of target compound 12b on human hepatocyte LO2 cells tested by MTT methodology.

100

%24h

48h

%

80

60

40

20

0

0.8 1.6 3.1 6.2 12.5 25 50 100

Concentration of compound (μmol/L)

2

9

Article Doi

Ethylenic conjugated coumarin thiazolidinediones as new efficient antimicrobial modulators against clinical methicillin-resistant Staphylococcus aureus

DOI: 10.1016/j.bioorg.2019.103434

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