New: N -(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,2-benzothiazole-3-carboxamides and acetamides as antimicrobial agents

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RESEARCH ARTICLE

New N-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,2-

benzothiazole-3-carboxamides and acetamides as

antimicrobial agents†

Cite this: DOI: 10.1039/c7md00334j

a

b

c

Matteo Incerti, Paola Vicini, Athina Geronikaki, * Phaedra Eleftheriou,

c

d

Athanasios Tsagkadouras, Panagiotis Zoumpoulakis, Charalmpos Fotakis,

Ana Ćirić, Jasmina Glamočlija and Marina Soković

e

A

series of 21 novel N-[2-phenyl-4-oxo-1,3-thiazolidin-3-yl]-1,2-benzothiazole-3-carboxamides/

acetamides (4a–4p) as well as a series of N′-(halophenylmethylidene)-1,2-benzothiazole-3-acetohydrazides

3h–3p) have been synthesized and evaluated for their antimicrobial activity against eight bacterial and eight

(

fungal species, among them plant, animal and human pathogens and food contaminating species. All com-

pounds appeared to be potent and the best activity was exhibited by compound 4d with MIC in the range

−

1

−2

−1

−2

Received 30th June 2017,

Accepted 9th October 2017

of 10.7–21.4 μmol mL × 10 and MBC of 21.4–40.2 μmol mL × 10 . The best antifungal activity was ob-

served for compounds 4p and 3h. Elucidation of the relationship between the antimicrobial activity and

molecular properties of the synthesized compounds was also performed. Synthetic intermediates were also

tested with several exhibiting good antimicrobial activities. Docking studies for some compounds were

performed.

DOI: 10.1039/c7md00334j

rsc.li/medchemcomm

bial resistance is most evident. Thus, the development of

1

. Introduction

new antimicrobial agents with different modes of action to

2

Antimicrobial resistance is a growing problem worldwide, re-

quiring a concerted international approach. In fact, since

their discovery during the 20th century, antimicrobial agents

have substantially reduced the threat posed by infectious

avoid cross-resistance is increasingly important.

Five-membered heterocycles with two or three hetero-

atoms, such as imidazoles, thiazoles, triazoles and others,

are key structural units in many pharmaceutical prepara-

tions. Moreover, the potential of 4-thiazolidinone ring sys-

tems, as core structures in various synthetic compounds

displaying antibacterial and antifungal activity, is well known

1

diseases.

The use of these “wonder drugs”, combined with improve-

ments in sanitation, housing, and nutrition as well as the ad-

vent of widespread immunization programmes, has led to a

dramatic drop in deaths from diseases that were previously

widespread, untreatable, and frequently fatal.

These gains are now seriously jeopardized by another recent

development: the emergence and spread of microbes that are

resistant to cheap and effective first-choice or “first-line” drugs.

The bacterial infections that contribute most to human

disease are also those in which emerging and existent micro-

3

–9

and was confirmed by our recent findings.

In addition, benzo-fused 1,2-thiazole derivatives have been

investigated over a period of time by our group, discovering

many compounds possessing interesting biological properties

1

0–16

including antimicrobial activity.

We previously designed and synthesized some 2-benzothiazole/

benzoisothiazole 5-benzylidene-4-thiazolidinones as antimicro-

bial agents. It was found that, in most cases, benzisothiazole

derivatives appeared to be less active than 2-benzothiazole-5-

benzylidene-4-thiazolidinones. Furthermore, the position of

the substituent exerts, in general, a certain effect among

a

Dipartimento di Scienze degli Alimenti e del Farmaco, Universita' degli Studi di

Parma, Italy

b

Aristotle University, School of Pharmacy, Thessaloniki, 54124, Greece.

1

5

the isomeric chloro-substituted compounds. It was ob-

served that the introduction of the bulky electron-

withdrawing chloro-group led to enhanced antimicrobial

activity.

E-mail: geronik@pharm.auth.gr; Fax: +302310 998559; Tel: +30 2310 997616

c

Department of Medical Laboratory Studies, School of Health and Medical Care,

Alexander Technological Educational Institute of Thessaloniki, Greece

d

Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic

Research Foundation, 48 Vas. Constantinou Ave., 11635, Athens, Greece

Our aim now, supported by our previous findings, was to

address the synthesis of a new class of N-(2-phenyl-4-oxo-1,3-

thiazolidin-3-yl)-1,2-benzothiazole-3-carboxamides and acet-

amides derived from N′-phenylmethylidene-1,2-benzothiazole-

e

Mycological Laboratory, Department of Plant Physiology, Institute for Biological

Research, Siniša Stanković, University of Belgrade, Bulevar, Serbia

†

Electronic supplementary information (ESI) available. See DOI: 10.1039/

c7md00334j

This journal is © The Royal Society of Chemistry 2017

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3

-carbohydrazides and acetohydrazides, respectively, and

are presented in Tables 1 and 2, respectively. The minimal in-

hibitory and minimal bactericidal/fungicidal concentrations

were determined using a microdilution method.

their evaluation as antibacterial and antifungal agents.

The novel structures of six of the intermediate hydrazides

encouraged us to also test their antimicrobial properties.

The synthetic compounds showed antibacterial activity

against all tested bacteria but on different levels. The mini-

mal inhibitory concentrations (MIC) ranged between 10.2–

2

. Results and discussion

−

1

−2

7

8.9 μmol mL × 10 , while the minimal bactericidal con-

2

.1. Chemistry

The target compounds N-[2-phenyl-4-oxo-1,3-thiazolidin-3-yl]-

,2-benzothiazole-3-carboxamides/acetoamides (4a–p), mostly

−

1

−2

centrations (MBC) were 14.7–115.2 μmol mL × 10 . The

antibacterial potential can be presented as following: 4d >

1

4h > 4f > 4m > 4c > 4a > 4p > 4i > 4b > 4k > 4o > 4l > 4e

halophenyl substituted, were synthesized using a step-wise

reaction protocol (Scheme 1), starting from 1,2-benzothiazole-3-

carbohydrazide 1 or 1,2-benzothiazole-3-acetohydrazide 2, syn-

thesized using procedures reported by us previously.

Condensation of the amino group of carbohydrazide 1

and its higher homologue 2 with the carbonyl group of com-

>

4n > 4j > 4g. The best antibacterial activity was obtained

−1

for compound 4d, which exhibited a 10.7–21.4 μmol mL

×

−2

−1

−2

10

range of MIC and a 21.4–40.2 μmol mL × 10 range of

10

MBC. The lowest antibacterial activity among all the com-

pounds tested was obtained for 4g (MIC 20.5–78.9 μmol ×

−1

−2

1

0–2 mL and MBC 38.4–115.2 μmol ml × 10 ). The most

mercially

available

phenylmethylidene)-1,2-benzothiazole-3-carbo/acetohydrazides

3a–q) in good yields. The reaction of the hydrazides 3a–p

aldehydes

afforded

the

N′-

sensitive bacterial species among Gram-positive bacteria was

Staphylococcus aureus, while the most resistant species was

Listeria monocytogenes. For Gram-negative bacteria, the most

sensitive was Escherichia coli, while Salmonella typhimurium

was the most resistant. The standard drugs used as positive

controls, streptomycin and ampicillin, were also active

(

with sulfanylacetic acid in refluxing toluene afforded the final

compounds 4a–p. The structure and purity of the novel com-

pounds 3h–j, 3o–q and 4a–p were confirmed by H NMR, MS

and IR spectroscopy and elemental analyses. The characteri-

zation of previously reported hydrazides 3a–g and 3k–n was

consistent with the literature data.

1

against all the bacteria.

−1

The MIC range for streptomycin was 4.3–25.8 μmol mL

×

9

−2

−1

−2

10

and the range of MBC was 8.6–51.6 μmol mL × 10 ,

while ampicillin showed a slightly lower antibacterial poten-

2

.2. Biological evaluations

.2.1. Antibacterial activity. The results of antimicrobial

−1

−2

tial with a range of MIC 24.8–74.4 μmol mL × 10 and

−

1

−2

2

MBC 37.2–124.0 μmol mL × 10 . All the tested compounds

possessed strong antibacterial activity and showed similar or

higher effects than ampicillin and, in many cases, were better

than streptomycin. It should be noted that all compounds

activity of synthetic compounds (3h, i, j, o, and p and 4a–p)

and standard antibiotics/antimycotics against a panel of se-

lected Gram-positive and Gram-negative bacteria and fungi

Athina Geronikaki graduated from Tashkent State University in 1971 and gained a specialty in organic chemistry. Since 1971 till 1977,

she had been a senior researcher at the Institute of Chemistry of Natural Products of the Uzbek Academy of Science. In 1977, she

defended her PhD thesis and received her Ph.D. in Chemistry (Ph.D., Doctor of Philosophy in Chemistry). In 1984, she graduated from

the School of Pharmacy of the Aristotelian University of Thessaloniki. From 2006–2013, she was the Head of the Department of Pharm.

Chemistry. Since 2010, she has been a Full Professor of Medicinal Chemistry of the School of Pharmacy of the Aristotle University of

Thessaloniki. During 2009–2011, she was Vice President of the School of Pharmacy of the Aristotle University of Thessaloniki. In July

2013, Prof. Geronikaki was elected as a full member of the Mediterranean Academy of Science and Arts. Her scientific interests are the

following: chemistry of natural products isolation, determination of structure; chemistry of biologically active compounds and evaluation

of their activity using different computational methods. She organized two international conferences: Computational Methods in Toxicol-

ogy and Pharmacology, Integrating Internet Resources (2003) Thessaloniki; 4th Eurasian Meeting on Heterocyclic Chemistry, 2006,

Thessaloniki. Prof. Geronikaki is a member of the organizing committee of more than 20 conferences, an international editor in the jour-

nal Revista de Ciências Farmacêuticas Básica e Aplicada/Journal of Basic and Applied Pharmaceutical Sciences and a member of the Ad-

visory Board of the Jordan Journal of Chemistry. Prof. Geronikaki has published 88 papers and made 140 announcements in interna-

tional conferences in most cases as an invited speaker or plenary lecturer. Published a chapter in “Targets in Heterocyclic Systems.

Chemistry and Properties”, Chemistry. Athina Geronikaki, Phaedra Eleftheriou and Vladimir Poroikov. Anti-HIV agents. Current status

and recent trends. In: Reverse Transcriptase Inhibitors in HIV/AID Therapy. Edited by Gail Skowron and Richard Ogden, Humania Press,

Totowa, New Jersey, 2015. Athina Geronikaki, Petrou Anthi, Phaedra Eleftheriou, and Fliur Macaev. Protein tyrosine phosphatase 1B: an

attractive target for treatment diabetes type II. In: Springer Series in Material Science. Published 112 papers. An invited speaker in 31

international conferences and a member of the Advisory Board of 25 conferences. Awarded a medal for the development of international

collaboration by the Scientific Partnership Foundation, awarded a diploma for the investment in the development of science and interna-

tional collaboration by the Scientific Partnership Foundation. 2013 – Awarded a gold medal for the investment in the development of sci-

ence and international collaboration by the Scientific Partnership Foundation. Citations: a) Scopus 2262; b) Google 3023, h-index 27,

i10-index 52.

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6 4 3 2 3 6 5

Scheme 1 Synthesis of title compounds. Reagents and conditions: (a) R-C H CHO, CH COOH, EtOH, reflux, 3 h; (b) HSCH COOH, CH C H ,

reflux, 48 h.

showed very good antibacterial effects against Pseudomonas

aeruginosa, being much better than ampicillin.

upon the nature of each aromatic substituent and also on the

number of methylene groups. Thus, among compounds with

n = 0 it seems that the most favourable substitution for anti-

bacterial activity is the 2,6-di-chloro-(3j), followed by the di-

fluoro-(3h) and the less favorable appeared to be 2-chloro-6-

fluoro-(3i) substitution. On the other hand, for compounds

with n = 1 the best activity was observed for the 2-chloro-6-

fluoro substituted derivative.

Referring to compounds 4a–p, analyses of structure–activ-

ity relationships revealed that antibacterial activity depends,

as in the case of compounds 3, not only on the nature of the

substituents but also on their position at the aromatic ring.

Thus, for chloro-derivatives (n = 0) position 3 is the most

favourable and the relative activity of these compounds fol-

lows 3-Cl > 2-Cl > 4-Cl. This order was also observed for

chloro-derivatives with n = 1, showing that the addition of

one more methylene group between the benzoisothiazole and

CO group does not affect the activity.

On the other hand, with fluoro-derivatives the most active

derivative appeared to be 4-F, followed by 3-F and then 2-F.

In the case of double halogen substitution with n = 0, the or-

der of activity was as follows: 2,6-di-F > 2-Cl, 6-F > 2,6-di-Cl.

The 2,6-di-fluoro-substitution results in better activity com-

pared to 2-fluoro-6-chloro-substitution. Almost the same was

observed when n = 1. In general, the most active compounds

appeared to be fluoro-derivatives.

It should be noted that the order of activity for intermedi-

ates 3h, 3i, 3j, 3o and 3p is the opposite of that of the title

compounds where the more preferable substitution was 2,6-

difluoro- and the less the 2,6-dichloro-derivative.

Furthermore, some novel intermediate compounds (3h, i,

j, o and p) were also evaluated for their antimicrobial activity.

The results of antimicrobial activity of intermediates 3h–j

and 3o and p and standard antibiotics/antimycotics against a

panel of selected Gram-positive and Gram-negative bacteria

and fungi are presented in Table 1. The minimal inhibitory

and minimal bactericidal/fungicidal concentrations were de-

termined using a microdilution method.

The synthetic compounds showed antibacterial activity

against all the tested bacteria but on different levels to com-

pounds 4a–p. Minimal inhibitory concentrations (MIC)

−

1

−2

ranged between 2.9–36.2 μmol mL × 10 , while minimal

bactericidal concentrations (MBC) were between 6.3–96.4

−

1

−2

μmol mL × 10 . The antibacterial potential follows the or-

der: 3o > 3j > 3h > 3i > 3p.

The best antibacterial activity was obtained for com-

−

1

−2

pound 3o, with a MIC range of 2.9–11.6 μmol mL × 10 and

−1

−2

an MBC at 23.26 μmol ml × 10 . The lowest antibacterial ac-

tivity among all compounds tested in this study was obtained

−

2

−1

for 3p (MIC 24.1–36.2 μmol × 10 mL and MBC 48.2–96.4

−2

−1

μmol × 10 mL ). The most sensitive bacterial species were

Staphylococcus aureus and Salmonella typhimurium, while the

most resistant species were Listeria monocytogenes and Pseudo-

monas aeruginosa. The standard drugs used as positive controls,

streptomycin and ampicillin, were also active against all the

bacteria.

All the tested compounds possessed strong antibacterial

activity and showed similar or better effects compared to

streptomycin and higher activities than ampicillin.

Concerning compounds 3, analyses of structure–activity

relationships revealed that antibacterial activity depends

2.2.2. Antifungal activity. The antifungal activities of com-

pounds 3h, i, j, o, and p and 4a–p are presented in Table 2.

All the compounds showed good antifungal activities in the

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Table 1 Antibacterial activity of compounds 3h, i, j, o, p and 4a–4p (MIC and MBC in μmol mL × 10

MedChemComm

−1

−2

)

Compounds

B.c.

M.f.

S.a.

L.m.

25.2 ± 0.8

E.c.

En.cl.

6.3 ± 0.1

25.2 ± 0.6

6.0 ± 0.0

P.a.

25.2 ± 2.1

S.t.

3

4

h

i

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

12.6 ± 0.3

25.2 ± 1.2

12.0 ± 0.6

24.0 ± 2.1

11.6 ± 0.6

23.2 ± 0.8

11.6 ± 0.8

23.2 ± 0.0

24.1 ± 1.2

48.2 ± 2.8

22.5 ± 0.9

42.2 ± 0.6

21.4 ± 0.2

40.2 ± 0.9

10.7 ± 0.6

40.2 ± 1.2

21.4 ± 0.6

40.2 ± 1.2

15.4 ± 0.6

38.4 ± 0.6

10.2 ± 0.6

38.4 ± 1.2

20.5 ± 0.6

38.4 ± 1.3

10.2 ± 0.8

20.4 ± 0.8

19.6 ± 0.3

36.8 ± 1.2

39.5 ± 0.9

79.0 ± 2.2

21.6 ± 0.6

40.5 ± 1.0

19.8 ± 0.9

37.2 ± 0.6

19.8 ± 0.6

37.2 ± 1.2

10.0 ± 0.6

19.8 ± 0.6

37.0 ± 1.2

14.7 ± 0.6

36.7 ± 1.2

4.3 ± 0.06

8.6 ± 0.03

24.8 ± 0.2

37.2 ± 0.2

25.2 ± 2.1

50.4 ± 1.2

12.0 ± 0.8

24.0 ± 1.2

11.6 ± 0.3

23.2 ± 1.2

11.6 ± 0.6

23.2 ± 0.3

36.2 ± 0.2

48.2 ± 1.2

22.5 ± 0.6

42.2 ± 0.9

29.5 ± 0.6

40.2 ± 1.2

21.4 ± 0.9

40.2 ± 1.3

21.4 ± 0.3

40.2 ± 0.9

38.4 ± 0.9

76.8 ± 1.0

20.5 ± 0.3

38.4 ± 1.2

38.4 ± 0.9

76.8 ± 1.0

10.2 ± 0.6

20.4 ± 0.6

27.0 ± 0.6

36.8 ± 0.6

54.3 ± 1.2

79.0 ± 2.0

29.7 ± 0.6

40.5 ± 2.0

27.3 ± 0.6

37.2 ± 0.6

19.8 ± 0.3

37.2 ± 0.9

27.3 ± 1.0

37.2 ± 0.6

27.2 ± 0.3

37.0 ± 0.6

27.0 ± 0.6

36.7 ± 0.9

8.6 ± 0.03

17.2 ± 0.6

24.8 ± 0.3

37.2 ± 0.1

12.6 ± 0.6

25.2 ± 1.2

6.0 ± 0.0

12.0 ± 0.2

5.8 ± 0.5

25.2 ± 0.6

50.4 ± 1.2

12.0 ± 0.0

24.0 ± 0.6

11.6 ± 0.8

23.2 ± 0.3

11.6 ± 0.5

23.2 ± 0.3

36.2 ± 0.2

48.2 ± 1.2

11.2 ± 0.3

22.5 ± 0.2

21.4 ± 0.2

40.2 ± 0.9

16.0 ± 0.3

21.4 ± 0.6

10.7 ± 0.6

21.4 ± 0.6

28.2 ± 0.3

38.4 ± 1.2

10.2 ± 1.2

20.5 ± 0.6

58.9 ± 2.0

76.8 ± 2.1

10.2 ± 0.6

20.4 ± 1.0

27.0 ± 0.6

36.8 ± 2.1

54.3 ± 1.0

79.0 ± 1.0

21.6 ± 1.0

40.5 ± 1.8

19.8 ± 0.3

37.2 ± 1.2

19.8 ± 0.3

37.2 ± 0.3

27.3 ± 1.0

37.2 ± 1.0

27.2 ± 1.2

37.0 ± 1.2

27.0 ± 0.5

36.7 ± 0.3

17.2 ± 0.6

34.4 ± 0.9

37.2 ± 0.3

49.2 ± 0.6

3.15 ± 0.1

6.3 ± 0.2

6.0 ± 0.0

50.4 ± 1.2

24.0 ± 0.8

48.0 ± 0.6

23.2 ± 0.6

46.4 ± 1.2

11.6 ± 0.6

23.2 ± 0.8

48.2 ± 0.8

96.4 ± 1.2

22.5 ± 0.3

42.2 ± 0.3

21.4 ± 0.3

40.2 ± 0.9

21.4 ± 0.6

40.2 ± 0.8

21.4 ± 0.9

40.2 ± 0.6

58.9 ± 1.2

76.8 ± 0.6

2.05 ± 0.06

3.84 ± 0.1

58.9 ± 2.1

76.8 ± 1.2

20.4 ± 0.6

40.8 ± 1.2

27.0 ± 0.9

36.8 ± 2.1

39.5 ± 1.0

79.0 ± 2.0

21.6 ± 1.0

40.5 ± 1.6

37.2 ± 0.3

74.4 ± 1.2

19.8 ± 0.6

37.2 ± 0.3

74.4 ± 1.2

111.6 ± 3.1

54.3 ± 1.2

74.0 ± 1.2

36.7 ± 0.3

73.4 ± 1.2

25.8 ± 0.3

51.6 ± 0.8

37.2 ± 0.3

74.4 ± 0.8

50.4 ± 2.1

24.0 ± 0.8

48.0 ± 2.1

23.2 ± 0.2

46.4 ± 1.2

11.6 ± 0.6

23.2 ± 0.3

36.2 ± 0.2

48.2 ± 1.2

22.5 ± 0.6

42.2 ± 0.3

21.4 ± 0.6

40.2 ± 1.2

21.4 ± 0.9

80.4 ± 2.1

10.7 ± 0.3

21.4 ± 0.9

28.2 ± 0.3

38.4 ± 0.6

20.5 ± 0.6

38.4 ± 1.0

20.5 ± 0.8

76.8 ± 2.1

20.4 ± 0.8

40.8 ± 1.2

19.6 ± 0.6

36.8 ± 0.9

18.9 ± 1.0

39.5 ± 1.2

21.6 ± 0.6

40.5 ± 2.1

37.2 ± 0.6

74.4 ± 2.0

19.8 ± 1.0

37.2 ± 0.6

37.2 ± 0.9

74.4 ± 1.5

27.2 ± 0.6

37.0 ± 0.6

27.0 ± 0.3

36.7 ± 0.3

17.2 ± 0.6

34.4 ± 0.3

74.4 ± 0.8

124.0 ± 1.2

12.0 ± 0.1

11.6 ± 0.3

23.2 ± 0.8

11.6 ± 0.1

23.2 ± 0.3

36.2 ± 0.2

48.2 ± 0.6

11.2 ± 0.2

22.5 ± 0.8

21.4 ± 0.9

40.2 ± 0.6

21.4 ± 0.9

21.4 ± 0.6

10.7 ± 0.00

21.4 ± 0.3

20.5 ± 0.9

38.4 ± 0.6

20.5 ± 0.9

38.4 ± 0.9

78.9 ± 1.8

115.2 ± 3.2

10.2 ± 0.3

20.4 ± 0.8

36.8 ± 1.0

73.5 ± 2.5

54.3 ± 0.9

79.0 ± 0.9

10.8 ± 0.6

21.4 ± 0.6

27.3 ± 0.3

37.2 ± 0.9

19.8 ± 0.6

37.2 ± 0.6

57.0 ± 2.1

74.4 ± 2.0

19.8 ± 0.6

37.0 ± 1.0

14.7 ± 0.3

19.6 ± 0.3

4.3 ± 0.03

8.6 ± 0.6

12.0 ± 0.6

11.6 ± 1.8

23.2 ± 0.6

11.6 ± 0.5

23.2 ± 0.8

24.1 ± 0.3

48.2 ± 1.2

22.5 ± 1.0

42.2 ± 0.6

21.4 ± 0.6

40.2 ± 1.0

21.4 ± 1.0

40.2 ± 1.2

10.7 ± 0.2

21.4 ± 0.6

20.5 ± 0.6

38.4 ± 0.3

20.5 ± 0.3

38.4 ± 1.2

76.8 ± 1.6

115.2 ± 3.5

20.4 ± 0.6

40.8 ± 1.0

19.6 ± 0.6

36.8 ± 0.6

39.5 ± 1.2

79.0 ± 1.0

21.6 ± 0.6

40.5 ± 2.0

19.8 ± 0.00

37.2 ± 0.6

10.0 ± 0.0

37.2 ± 0.8

37.2 ± 0.9

74.4 ± 1.2

19.8 ± 0.3

37.0 ± 0.3

19.6 ± 0.6

36.7 ± 0.3

17.2 ± 0.3

34.4 ± 0.5

24.8 ± 0.6

49.2 ± 0.8

j

17.6 ± 0.8

2.9 ± 0.1

o

p

a

b

c

d

e

f

23.2 ± 0.3

24.1 ± 0.8

48.2 ± 1.2

22.5 ± 0.3

42.2 ± 0.6

21.4 ± 0.6

40.2 ± 1.5

10.7 ± 0.3

21.4 ± 0.9

10.7 ± 0.1

21.4 ± 0.3

10.2 ± 0.6

38.4 ± 0.3

10.2 ± 0.8

20.5 ± 0.6

20.5 ± 1.2

38.4 ± 0.9

10.2 ± 0.3

20.4 ± 0.6

19.6 ± 1.2

36.8 ± 0.9

18.9 ± 0.6

39.5 ± 1.0

21.6 ± 0.9

40.5 ± 2.1

19.8 ± 0.6

37.2 ± 0.3

10.0 ± 0.2

37.2 ± 0.6

19.8 ± 0.6

37.2 ± 0.9

19.8 ± 0.3

37.0 ± 0.3

14.7 ± 0.3

19.6 ± 0.3

17.2 ± 0.1

34.4 ± 0.3

24.8 ± 0.1

37.2 ± 0.3

g

h

i

j

k

l

m

n

o

p

Streptomycin

Ampicillin

24.8 ± 0.2

37.2 ± 0.3

B.c.: Bacillus cereus (clinical isolate), M.f.: Micrococcus flavus (ATCC 10240), S.a.: Staphylococcus aureus (ATCC 6538), L.m.: Listeria monocytogenes

NCTC 7973), E. coli: Escherichia coli (ATCC 35210), En.Cl.: Enterobacter cloacae (human isolate). P.a.: Pseudomonas aeruginosa (ATCC 27853), S.

(

t.: Salmonella typhimurium (ATCC 13311).

−

1

−2

MIC range of 1.58–48.2 μmol mL

× 10

and in the

The synthetic fungicide bifonazole possessed an inhibitory

activity of 48.0–64.0 μmol mL × 10 and a fungicidal activ-

−

1

−2

−1

−2

MFC range of 6.0–96.4 μmol mL × 10 . The antifungal

potential can be presented as follows: 4p > 4m > 4o >

−

1

−2

ity of 64.0–80.0 μmol mL × 10 , while ketoconazole showed

−

1

−2

4

c > 4b > 4g > 4f > 4a > 4k > 4l > 4n > 4e > 4h >

d > 4j > 4i. Compound 4p exhibited the best fungistatic

a lower range of MIC of 38.0–475.0 μmol mL × 10 and

−

1

2

4

MFC of 95.0–570.0 μmol mL × 10 (Table 2). However, all

the compounds tested showed much stronger antifungal activity

than commercial mycotics, particularly ketoconazole (Table 2).

The results of antifungal activity of intermediate com-

pounds 3h, 3i, 3j, 3o and 3p are presented in Table 2. All the

compounds showed good antifungal activities in the MIC

−

1

−2

activity at 3.8–9.8 μmol mL × 10 , and fungicidal activ-

−

1

−2

ity was achieved at 4.9–36.7 μmol mL × 10 . The lowest

antifungal effect was observed for compound 4i with MIC

−

1

−2

1

8.4–73.5 μmol mL

× 10

and MFC 18.4–85.8 μmol

−

1

−2

mL × 10 . The majority of compounds showed the best

activity against T. viride, while the most resistant was A.

fumigatus.

−

1

−2

range of 1.58–48.2 μmol mL × 10 and MFC range of 6.0–

−

1

−2

96.4 μmol mL × 10 .

Med. Chem. Commun.

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Table 2 Antifungal activity of compounds 3h–3p and 4a–4p (MIC and MFC in μmol mL × 10

Research Article

−1

−2

)

Compounds

A.fum.

A.v.

A.o.

A.n.

T.v.

12.6 ± 1.2

25.2 ± 0.3

24.0 ± 1.2

48.0 ± 1.2

11.6 ± 1.2

46.4 ± 1.2

6.1 ± 0.6

P.f.

P.o.

12.6 ± 0.8

C.a.

3

4

h

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MBC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

1.58 ± 0.3

25.2 ± 0.3

24.0 ± 0.8

30.0 ± 2.0

23.2 ± 0.1

46.4 ± 0.2

48.2 ± 2.1

96.4 ± 1.6

46.4 ± 1.2

92.8 ± 3.2

11.2 ± 0.6

22.5 ± 0.3

10.7 ± 0.9

21.4 ± 0.1

5.4 ± 0.03

10.7 ± 0.3

10.7 ± 0.6

21.4 ± 0.6

10.25 ± 0.1

20.5 ± 0.6

38.4 ± 0.3

76.8 ± 1.2

20.5 ± 0.6

38.4 ± 0.9

40.8 ± 2.1

51.0 ± 1.2

36.8 ± 1.2

73.5 ± 2.3

39.5 ± 1.2

79.0 ± 2.1

10.8 ± 0.00

40.5 ± 2.1

37.2 ± 0.8

74.4 ± 2.1

10.0 ± 0.00

37.2 ± 1.0

74.4 ± 2.5

29.6 ± 0.9

37.0 ± 1.0

9.8 ± 0.1

1.58 ± 0.3

12.6 ± 0.2

6.0 ± 0.3

12.0 ± 0.8

46.4 ± 0.0

58.0 ± 2.2

9.1 ± 0.6

12.1 ± 0.6

11.6 ± 0.6

23.2 ± 0.8

22.5 ± 1.0

42.2 ± 2.1

21.4 ± 0.3

40.2 ± 1.2

5.4 ± 0.03

10.7 ± 0.6

40.2 ± 2.1

80.4 ± 2.1

2.56 ± 0.1

5.1 ± 0.1

1.58 ± 0.1

12.6 ± 0.1

6.0 ± 0.6

25.2 ± 0.1

50.4 ± 0.6

6.0 ± 1.0

3.15 ± 0.6

12.6 ± 0.3

3.0 ± 0.3

25.2 ± 0.2

50.4 ± 1.2

24.0 ± 0.9

30.0 ± 1.2

23.2 ± 1.0

46.4 ± 1.2

12.1 ± 0.3

24.1 ± 0.6

46.4 ± 2.0

58.0 ± 1.2

25.3 ± 0.5

42.2 ± 2.0

10.7 ± 0.6

21.4 ± 1.2

10.7 ± 0.3

21.4 ± 0.6

8.0 ± 0.1

10.7 ± 0.6

38.4 ± 0.9

76.8 ± 2.3

10.2 ± 0.3

20.5 ± 0.6

10.2 ± 0.1

20.5 ± 0.3

40.8 ± 1.2

51.0 ± 1.0

73.5 ± 2.2

85.8 ± 2.3

39.5 ± 1.0

79.0 ± 1.2

21.6 ± 0.6

40.5 ± 2.2

19.8 ± 0.1

37.2 ± 1.0

10.0 ± 0.8

19.8 ± 0.3

19.8 ± 0.6

37.2 ± 0.8

9.9 ± 1.0

25.2 ± 0.2

i

6.0 ± 0.6

12.0 ± 0.3

23.2 ± 1.2

46.4 ± 1.0

18.1 ± 0.6

24.1 ± 0.8

11.6 ± 0.3

23.2 ± 0.3

11.2 ± 0.6

22.5 ± 0.9

5.4 ± 0.03

10.7 ± 0.9

10.7 ± 0.6

21.4 ± 0.3

40.2 ± 2.1

80.4 ± 1.2

38.4 ± 1.0

76.8 ± 2.1

20.8 ± 0.3

38.4 ± 1.2

10.2 ± 0.2

20.5 ± 0.3

10.2 ± 0.3

51.0 ± 1.0

36.8 ± 0.6

85.8 ± 2.5

39.5 ± 1.0

79.0 ± 1.0

16.2 ± 0.6

21.6 ± 2.0

14.4 ± 0.6

19.8 ± 0.9

7.4 ± 0.06

10.0 ± 1.0

10.0 ± 0.00

19.8 ± 0.3

14.8 ± 0.9

19.8 ± 0.6

4.9 ± 0.03

9.8 ± 0.03

190.0 ± 1.6

285.0 ± 3.2

64.0 ± 1.0

80.5 ± 2.0

12.0 ± 0.6

23.2 ± 1.0

46.4 ± 1.0

12.1 ± 0.3

24.1 ± 1.0

11.6 ± 0.8

23.2 ± 0.6

2.80 ± 0.06

5.60 ± 0.06

8.00 ± 0.03

21.4 ± 0.9

29.2 ± 0.9

40.2 ± 2.3

40.2 ± 2.0

80.4 ± 2.0

10.2 ± 0.3

20.5 ± 0.6

10.2 ± 0.1

20.5 ± 0.3

10.2 ± 0.1

20.5 ± 1.2

5.1 ± 0.06

10.2 ± 0.6

36.8 ± 0.6

73.5 ± 2.0

9.45 ± 0.1

18.9 ± 0.9

5.4 ± 0.03

10.8 ± 0.1

10.0 ± 0.00

19.8 ± 0.3

7.4 ± 0.06

10.0 ± 1.0

10.0 ± 0.9

19.8 ± 0.6

7.4 ± 0.06

9.9 ± 0.03

9.8 ± 0.03

19.6 ± 1.0

28.0 ± 0.2

38.0 ± 0.6

48.0 ± 1.0

80.0 ± 0.9

12.0 ± 0.3

46.4 ± 1.2

58.0 ± 0.6

12.1 ± 0.6

24.1 ± 0.8

11.6 ± 1.0

23.2 ± 1.2

11.2 ± 0.1

22.5 ± 0.4

10.7 ± 0.6

21.4 ± 0.1

10.7 ± 0.6

21.4 ± 1.2

40.2 ± 2.1

80.4 ± 2.3

10.2 ± 0.4

20.5 ± 0.6

7.7 ± 0.06

10.2 ± 0.3

10.2 ± 0.8

20.5 ± 0.9

20.4 ± 0.3

40.8 ± 1.0

36.8 ± 0.3

73.5 ± 1.2

39.5 ± 0.9

79.0 ± 1.2

16.2 ± 0.1

21.6 ± 0.3

14.4 ± 0.3

19.8 ± 0.3

10.0 ± 1.0

19.2 ± 0.6

19.8 ± 0.6

37.2 ± 1.0

14.8 ± 0.6

19.8 ± 0.9

19.6 ± 1.0

36.7 ± 0.9

38.0 ± 0.6

95.0 ± 1.0

48.0 ± 0.9

64.0 ± 0.9

6.0 ± 0.6

j

46.4 ± 2.1

58.0 ± 2.0

9.1 ± 0.6

o

p

a

b

c

d

e

f

12.1 ± 0.8

11.6 ± 0.8

23.2 ± 0.6

5.60 ± 0.06

11.2 ± 0.6

2.7 ± 0.06

5.4 ± 0.06

5.4 ± 0.00

10.7 ± 0.9

40.2 ± 2.0

80.4 ± 1.2

10.2 ± 0.2

20.5 ± 0.6

5.1 ± 0.03

10.2 ± 0.6

2.56 ± 0.03

5.12 ± 0.06

5.1 ± 0.03

10.2 ± 0.6

13.8 ± 0.6

18.4 ± 0.9

13.2 ± 0.6

18.9 ± 0.3

5.4 ± 0.03

10.8 ± 0.3

7.4 ± 0.03

10.0 ± 0.6

5.0 ± 0.3

12.1 ± 0.6

11.6 ± 0.6

23.2 ± 0.6

22.5 ± 0.6

42.2 ± 1.2

10.7 ± 0.1

21.4 ± 0.6

10.7 ± 0.6

21.4 ± 0.3

21.4 ± 0.9

53.6 ± 1.0

20.5 ± 0.6

38.4 ± 1.2

10.2 ± 0.9

20.5 ± 0.5

10.2 ± 0.3

20.5 ± 0.1

10.2 ± 0.1

20.4 ± 0.3

36.8 ± 0.3

73.5 ± 2.2

39.5 ± 1.2

79.0 ± 1.2

21.6 ± 0.3

21.6 ± 2.1

10.0 ± 0.00

19.8 ± 0.8

10.0 ± 0.3

19.8 ± 0.9

14.4 ± 1.0

19.8 ± 0.6

7.4 ± 0.00

9.9 ± 0.3

5.1 ± 0.1

10.2 ± 0.6

10.2 ± 0.3

20.5 ± 1.0

20.4 ± 0.9

40.8 ± 0.9

18.4 ± 0.3

73.5 ± 2.1

18.9 ± 0.9

70.8 ± 2.5

21.6 ± 0.9

40.5 ± 2.0

7.4 ± 0.1

10.0 ± 0.1

19.2 ± 0.6

10.0 ± 0.9

19.8 ± 0.9

7.4 ± 0.06

9.9 ± 0.03

4.9 ± 0.03

9.8 ± 0.6

g

h

i

j

k

l

m

n

o

p

10.0 ± 0.3

19.8 ± 0.6

7.4 ± 0.03

9.9 ± 0.6

3.8 ± 0.06

4.9 ± 0.03

190.0 ± 4.5

285.0 ± 2.5

64.0 ± 1.2

80.0 ± 1.0

19.8 ± 0.6

4.9 ± 0.03

9.8 ± 0.06

38.0 ± 0.3

56.0 ± 2.0

32.0 ± 0.6

64.0 ± 2.5

4.9 ± 0.05

9.8 ± 0.1

38.0 ± 1.2

95.0 ± 1.8

64.0 ± 1.0

80.0 ± 2.1

19.6 ± 1.0

38.0 ± 0.1

95.0 ± 2.1

48.0 ± 1.0

64.0 ± 1.0

Ketoconazole

Bifonazole

38.0 ± 0.3

95.0 ± 2.1

32.0 ± 0.9

64.0 ± 0.6

A.fum: Aspergillus fumigatus (plant isolate), A.v.: Aspergillus versicolor (ATCC 11730), A.o.: Aspergillus ochraceus (ATCC 12066), A.n.: Aspergillus

niger (ATCC 6275), T.v.: Trichoderma viride (IAM 5061), P.f.: Penicillium funiculosum (ATCC 36839), P.o. Penicillium ochrochloron (ATCC 9112),

C.a.: Candida albicans (human isolate).

The antifungal potential order is: 3h > 3i > 3j > 3o > 3p.

Compound 3h exhibited the best fungistatic activity at 1.58–

Studies of structure–activity relationships confirmed the

previous observations that the antifungal activity of the

studied compounds depends not only on the nature of

the substituent but also on its position on the aromatic

ring.

Thus, in the case of compounds 3h, i, j, o, and p it seems

that the absence of a methylene group (n = 0) has a positive

influence on antifungal activity. In these compounds, 2,6-di-

fluoro substitution appeared to be the most favorable. Thus,

the only similarity to antibacterial activity was observed for

compound 3o, which showed the lowest antibacterial as well

as antifungal activity.

−

1

2

5.2 μmol mL × 10–2, and fungicidal activity was achieved

−

1

−2

at 12.6–50.4 μmol mL × 10 . Compound 3p exhibited the

lowest antifungal effect with MIC of 6.1–48.2 μmol mL

−

1

×

−

2

−1

−2

1

0

and MFC of 12.1–96.4 μmol mL × 10 . The majority of

the compounds showed high activity against A. ochraceus,

while the most resistant was A. fumigatus.

It should be mentioned that all the compounds tested

showed much stronger antifungal activity than commercial

mycotics, particularly ketoconazole (MIC of 38.0–475.0 μmol

−1

−2

−1

−2

mL × 10 and MFC of 95.0–570.0 μmol mL × 10 ) (Table 2).

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respect to their antifungal properties, with the three most ac-

tive antifungal compounds 4p, 4m and 4o constituting group

(

4-i).

This suggests that 4p, 4m and 4o probably exhibit simi-

larly potent biological activities explained by some common

descriptors.

A supervised OPLS-DA was then extracted between groups

(

4-i) and (4-ii) (Fig. 1B). The volcano plot highlights the de-

scriptors that contribute to the clustering between the two

groups. From these, the most significant parameters for

group (4-i) are (a) the hydrophobic component of the solvent

accessible surface area – SASA (FOSA), (b) the hydrophilic

component of the SASA (FISA), (c) the van der Waals surface

area of polar nitrogen and oxygen atoms and carbonyl carbon

atoms (PSA), (d) the predicted aqueous solubility (QPlogS),

Fig. 1 (A) PCA-X (A = 6, N = 37, R2XĲcum) = 0.99, Q2Ĳcum) = 0.97) of

synthesized compounds 4a–p and 3h, i, j, o, and p based only on the

predicted physicochemical properties; (B) OPLS-DA-X (A = 1 + 1, N =

(

e) the computed dipole moment of the molecule (dipole), (f)

32, R2XĲcum) = 0.65, R2YĲcum) = 0.98, Q2Ĳcum) = 0.97) of synthesized

compounds 4a–p.

the square of the dipole moment divided by the molecular

volume which is the key term in the Kirkwood–Onsager equa-

tion for the free energy of solvation of a dipole with volume V

(dip^2/V), (g) the globularity descriptor (glob), (h) the PM3

calculated ionization potential in eV (IP), (i) the predicted

maximum transdermal transport rate (Jm) and (j) the pre-

dicted IC50 value for blockage of HERG K+ channels

(QPlogHERG). As can be observed, these physicochemical pa-

rameters are related to the shape, lipophilicity, charge distri-

bution and ionization potential of the molecules. These pro-

duced values can be further used as descriptors for QSAR

and in silico screening techniques.

The structure–activity relationship studies of compounds

4

a–p showed that the antifungal activity of chloro-derivatives

with n = 0 is stronger for the 4-chloro-derivative, followed by

-chloro- and 2-chloro – which is the opposite of what was

3

observed for antibacterial activity. The addition of one more

methylene group (n = 1) to the side chain led to changes in

the activity of compounds, with the order of activity being

3

-Cl > 2-Cl > 4-Cl.

In the series of fluoro derivatives with n = 0, the activity

follows the order mentioned above for chloro derivatives with

n = 1.

In the case where n = 0, disubstitution of fluorine is

preferable (2,6-difluoro- > 2,6-dichloro- > 2-chloro-6-fluoro-)

while substitution with two different halogens seems to be

more favourable when n = 1.

Regarding the antibacterial activity of the studied com-

pounds, no clear evidence could be extracted from the PCA

model.

2

.4. Docking-based prediction of the mechanism of

antimicrobial activity

.4.1. Antibacterial activity. Antibacterials may act on sev-

2

.3. Elucidating the relationship between antimicrobial

eral drug targets inhibiting cell wall biosynthesis, protein

synthesis, and nucleic acid biosynthesis or disturbing the cell

membrane normal function. According to the literature,

thiazolyl derivatives have been found to act both on cell wall

activity and molecular properties of synthesized compounds

The predicted values for the physically significant descriptors

and pharmaceutically relevant properties are provided in

ESI† Table S1. Multivariate data analysis was implemented to

elucidate the relation between selected molecular properties

of the synthesized compounds and their antimicrobial activi-

ties. A PCA model was extracted (Fig. 1) showing three dis-

tinct groups of compounds. More specifically, intermediate

compounds 3h, i, j, o, and p were located at the second quad-

rant close to the edge of the model due to their significantly

different scaffolds compared to compounds 4a–p. Interest-

ingly, compounds 4a–p were divided into two groups: one at

the third and one at the first quadrant. The first group (4-i)

included both the R and S enantiomers of 4m, 4o, and 4p

and only the S enantiomers of 4k, 4l and 4n.The second

group (4-ii) included both the R and S enantiomers of 4a, 4b,

1

7,18

biosynthesis and DNA replication.

Meanwhile, MurA, the

first of the six enzymes of the family to be involved in the

biosynthesis of cell wall peptidoglycan, is the drug target of

the antibiotic fosfomycin; thiazolidinones have been found

1

8,19

to inhibit the second enzyme of this process, MurB.

On

the other hand, several benzothiazole derivatives have been

1

7

mentioned in the literature as gyrase inhibitors. Based on

the structural similarities of our compounds to these inhibi-

tors, we theoretically evaluated the probability of our com-

pounds to act as MurB or gyrase inhibitors, using docking

analysis. The crystal structures of E. coli MurB 2Q85 and E.

coli gyrase 4TMA were used for the analysis, and the results

are shown in Table 3.

4

c, 4d, 4e, 4f, 4g, 4h, 4i, and 4j and only the R enantiomers

According to prediction results, all compounds could act

as E. coli MurB or E. coli gyrase inhibitors. The estimated

binding energy to MurB varied between −9.1 Kcal per mole

of 4k, 4l and 4n. At first glance, there is a spontaneous dis-

crimination between more and less active compounds with

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Research Article

and −6.1 kcal per mole and to E. coli gyrase between −8.2

Kcal per mole and −6.5 Kcal per mole. The two lowest esti-

mated energies to E. coli MurB (−9.1 kcal per mole and −8.1

kcal per mole, respectively) were calculated for compounds 4f

and 4h which exhibited the best antimicrobial activity against

E. coli (MIC = 10.2). The best estimated binding energy to E.

coli gyrase (−8.2 kcal per mole) was also calculated for com-

pound 4f. However, the antimicrobial activity cannot be

explained for all the compounds by means of the estimated

binding energy to MurB and gyrase.

Although in some cases, such as the third and fourth most

active compounds 4a and 4c (MIC 11.2 and 16.0, respec-

tively), the poor binding to MurB (est. binding energy −6.9

kcal per mole and −7.5 kcal per mole, respectively) may be

balanced by the stronger binding to gyrase (estimated bind-

ing energies −7.8 kcal per mole and −8.1 kcal per mole, re-

spectively); this cannot explain the placement of some less

potent antimicrobials in the activity range. For example, com-

pound 4i with est. binding energies to MurB and gyrase of

exhibited lower antimicrobial activity in vitro, although some

exceptions were observed (Table 3).

An attempt to explore a probable correlation of the esti-

mated binding energy with both enzymes and antimicrobial

activity for the compounds of the thiazolidinone series re-

vealed that such a correlation could poorly follow an expo-

2

nential curve. The best correlation curve (R = 0.6829) for the

estimated binding energy against E. coli MurB and E. coli

MIC was obtained for compounds with lower estimated bind-

ing energy to MurB compared to gyrase (Fig. S1†). A curve

2

with R = 0.5934 was obtained for the correlation of the low-

est estimated binding energy against E. coli MurB or gyrase

(which is lowest) with E. coli MIC, for compounds with clog P

2

4.000. Another curve with R = 0.5571 and p = 0.02529 was

obtained for the correlation of the lowest estimated binding

energy against E. coli MurB or gyrase (which is lowest) with

E. coli MIC, for all compounds with no clog P restrictions.

The results enforced the conception that multiple factors af-

fect activity but suggest a serious involvement mainly of MurB

inhibition in the mechanism of action of the most active

compounds.

−

7.7 kcal per mole and −8.2 kcal per mole, respectively, was

−

1

−2

found to be less active (MIC 27.0 μmol mL × 10 ) than

−

1

−2

compound 4d (MIC 21.4 μmol mL × 10 ) with est. binding

energies to MurB and gyrase of −7.7 kcal per mole and −7.0

kcal per mole, respectively. The probable interaction with

other targets as well as the permeability of the compound

may be responsible for these differences. According to pre-

dicted clog P values, compound 4i with a clog P 4.335 may ex-

hibit lower permeability than compound 4d with a clog P

For most of the compounds, the estimated binding energy

to MurB was lower than the estimated binding energy to gyr-

ase. Also among the general observations was that carboxa-

mide bridged derivatives, 4a–j, of the thiazolidinone series,

4a–p, exhibited better estimated binding energy to MurB than

the acetamide bridged compounds, 4k–p, of this series. In

addition, better results were obtained for all substituted carb-

oxamide bridged derivatives compared to the non-substituted

one (4a), indicating that the presence of halogen atoms at

the benzyl ring may favor complex stabilization. Docking

analysis revealed that carboxamide bridged derivatives obtain

a conformation that enables the aromatic moieties of the

molecule to be placed in proximity with the aminoacids

Tyr254, Tyr190 and Tyr125 with which they form pi interac-

tions. In addition, hydrogen bonds are formed between halo-

gens of the benzyl ring and Tyr125 or Tyr190 but also with

FAD or Pro 252 that further stabilize the complex. The N

atom of the thiazolyl ring and the N and O atoms of the

thiazolidinone moiety also form hydrogen bonds with Tyr190

3

.622. In general, compounds with calculated clog P 4.000

Table 3 In vitro measurement of minimal inhibitory concentration, MIC,

against E. coli and estimated binding energy (kcal per mole) to E. coli

MurB and E. coli gyrase according to docking analysis

|Estimated binding

energy

E. coli E. coli

E. coli

clog

Compound

n

Substituent MIC

MurB

gyrase

P

4

3

f

0

1

0

1

0

1

0

1

0

1

3-Cl

2,6-F

H

3-F

3-Cl

2-Cl

4F

H

2-F

2-F,6-Cl

10.2

11.2

16.0

19.8

21.4

21.6

22.5

27.0

27.2

27.3

28.2

54.3

58.9

11.6

12.0

25.2

36.2

−9.1

−8.1

−6.9

−7.5

−7.2

−6.1

−7.7

−6.7

−7.4

−7.7

−7.8

−7.2

−6.6

−7.9

−7.6

−8.0

−7.7

−7.0

−7.5

−8.0

−8.2

−7.3

−7.8

−8.1

−6.8

−7.8

−7.0

−7.5

−8.2

−7.6

−6.9

−6.5

−7.5

−7.2

−7.4

−7.9

−7.5

−7.7

−7.4

3.623

3.766

3.470

3.622

4.183

3.622

3.470

3.622

4.335

4.326

4.695

4.182

4.193

4.906

4.193

3.658

3.851

3.726

3.156

3.088

h

a

c

m

l

d

k

b

i

p

o

n

e

j

g

o

j

2

2,6-F

4-Cl

2-Cl

2

2,6-Cl

4-Cl

2

2,6-F

2

2,6-Cl2

2-F,6-Cl

i

h

p

Fig. 2 Docking of compounds 4f and 4c in the active site of E. coli

MurB. Tyr254 and carbon 2 of the benzothiazolyl moiety with which it

forms pi-pi interactions are in dark yellow.

2,6-F

2-F,6-Cl

2

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and Gly123 in many cases, while the O and N atoms of the

carboxamide bridge participate in hydrogen bond formation

mainly with Ser229 and Arg159. Polar and other interactions

mostly involve Asn233, Tyr158 or Leu218.

acids which, probably, play an essential role in complex

stabilization (Fig. S2†).

The most active compound in the group of N′-

(phenylmethylidene)-1,2-benzothiazole-3-carbo/acetohydrazides

According to docking analysis, the greatest number of hy-

drogen bonds and pi interactions are observed in the pre-

dicted complexes of the most active compounds (4f and 4h)

against E. coli.

(3) was the 2,6-F derivative (3o). Hydrogen bond interactions

2

between N atoms of the acetamide bridge and the OH group

of Tyr190 as well as pi–pi interactions between the 2,6-F2

benzyl ring and Tyr158, and between the benzothiazolyl moi-

ety and Tyr256 seem to play the main role in complex stabili-

zation (Fig. 3).

More precisely, in the complex stabilization of compound

4

f, the N atoms of the thiazolyl moiety and the carboxamide

bridge are predicted to form hydrogen bonds with Gly123

and the O atom of the thiazolidinone is predicted to form hy-

drogen bonds with Ser229 while the Cl substituent of the

benzyl ring interacts with Tyr125. Pi–pi interactions are ob-

served between carbon atoms of the benzothiazole moiety

and Tyr190 and Tyr254 and between carbon atoms of the 3Cl-

benzyl moiety and Tyr 190, Tyr125 and Tyr254. Ten such pi–

pi interactions may be formed (Fig. 2).

Docking of the compound (5z)-3-(4-chlorophenyl)-4-

hydroxy-5-(1-naphthylmethylene)furan-2Ĳ5H)-one, with the li-

gand bound to MurB in the 2q85 crystal structure and used

for docking analysis, showed an estimated binding energy of

−8.23 kcal per mole. The main amino acids involved in com-

plex stabilization were Tyr190, Asn233, Pro252 and Lys262,

which participated in hydrogen bond formation, and Tyr254,

which participated in pi interactions (Fig. S3†).

In an analogue way, five hydrogen bonds can be formed

between atoms of the thiazolyl moiety and the carboxamide

bridge of compound 4f and aminoacids Gly123, Ser229,

Ala124 and Arg159, while Tyr 254, Tyr190 and Tyr125 may

participate in six pi–pi interactions.

Replacement of the Cl substituent of compound 4f with

the F atom in compound 4c leads to a different orientation

with the F atom to interact with Pro254 and the carbon

atoms of the 3F-benzyl moiety to form three pi-pi interactions

with Tyr254. Two hydrogen bonds are now formed between

the N atoms of the thiazolidinone moiety and the carboxa-

mide bridge and hydroxyl group of Tyr190. A much lower

number of hydrogen and pi–pi interactions are now formed

Docking analysis using E. coli gyrase (crystallographic

structure, PDB ID: 4TMA) revealed the implication of the

aminoacids Val90, Gln94, Ser97, Asp36 and Arg46 in the sta-

bilization of the most potent compounds which is in accor-

dance with the literature (Fig. S4†). Docking of novobiocin,

a known competitive inhibitor of DNA gyrase, showed an esti-

mated binding energy of −8.4 and the main interactions in-

volved the aminoacids Gln94, Arg91, Phe96 and Ile112.

2

0

2.4.2. Anti-fungal activity. Antifungals can be divided into

several categories according to the drug targets. Most antifun-

gals target ergosterol synthesis, inhibiting specific enzymes

of the biosynthetic pathway. Among these, allylamides and

thiocarbamates have been found to inhibit the squalene

epoxidase ERG1, several morpholines are known to inhibit

ERG2 and ERG24–27 enzymes, polyenes inhibit the last en-

zyme of the pathway ERG4 while azoles mostly act by

inhibiting 14a-lanosterol demethylase encoded by the ERG11

(Fig. 2).

The additional methylene group at the acetamide bridged

derivatives, 4k–p, of the thiazolidinone series forces them to

take a different conformation compared with the carboxa-

mide bridged derivatives. The compounds, either taking a

more linear conformation (4k) or a more curved conforma-

tion (4o, 4l), failed to simultaneously interact with Tyr 254,

Tyr190 and Tyr125 which is observed in the case of the best

compounds of the carboxamide series. The 2-F,6-Cl deriva-

tive, 4p, with a lower estimated binding energy in the acet-

amide series, seems to interact with all three tyrosine amino-

2

1

gene.

The Candida albicans ERG11 enzyme, which is available in

the NCBI database (PDB ID: 5v5z), was used in a first attempt

to explore the probable mechanism of antifungal action of

our compounds using docking analysis. The results are

shown in Table 4.

Table 4 Estimated binding energy (kcal per mole) to 14a-lanosterol de-

methylase (ERG11) of Candida albicans

C.a. MIC

(μM × 10

Estimated binding

energy ERG11

−2)

Compound

n

Substituent

4

3

p

d

o

m

c

a

j

h

i

j

1

0

1

0

2-F,6-Cl

4-F

4.9

8.0

9.9

−8.59

−7.41

−9.15

−8.30

−7.35

−9.30

−10.13

−8.88

−8.09

−9.74

−9.38

2,6-F

3-Cl

3-F

2

10.0

10.7

25.3

39.5

40.8

73.5

23.2

24.0

H

2,6-Cl

2,6-F

2-F,6-Cl

2,6-Cl

2-F,6-Cl

2

Fig. 3 Docking of compound 3o in the active site of E. coli MurB.

3i

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−

1

According to the results, all the analyzed compounds may

exhibit inhibitory action against 14a-lanosterol demethylase

68ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3153

2 2

1

(NH), 1660 (CO). H NMR (DMSO-d6, δ, ppm): 12.54 (s, 1H,

NH); 8.78–8.74 (m, 2H, CH) 8.35 (d, 1H, J = 9, Ar); 7.74–7.49

(m, 3H, Ar); 7.30–7.19 (m, 2H, Ar); C NMR (DMSO-d6, δ,

ppm): 172.68 (CO); 163.10 (CN); 152.09 (C); 141.96 (C);

137.65 (CH); 135.26 (C); 132.84 (C); 131.36 (C); 128.39 (CH);

126.68 (CH); 124.52 (CH), 123.48 (CH); 120.94 (CH).

(

ERG11). The estimated binding energy obtained for the ana-

lyzed compounds varied between −7.35 kcal per mole and

10.13 kcal per mole. Interestingly, the estimated binding en-

1

3

−

ergy of the known antifungal fluconazole according to the

docking analysis at the same enzyme structure was −7.52 kcal

per mole. Docking of compound 4j and fluconazole is shown

in Fig. S4.† However, there is no correlation between experi-

mental antifungal activity of the compounds and predicted

3.2.2. N′-[(2-Chloro-6-fluorophenyl)methylidene]-1,2-benzo-

thiazole-3-carbohydrazide (3i). Mp: 275 °C (DMSO), yield

−

1

94ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3176

2

1

4a-lanosterol demethylase (ERG11) inhibition. Although

(NH), 1662 (CO). H NMR (DMSO-d6, δ, ppm): 12.65 (s,

1H, NH); 8.87 (s, 1H, CH); 8.74 (d, 1H, J = 9, Ar); 8.35 (d,

1H, J = 9, Ar); 7.74–7.61 (m, 2H, Ar); 7.54–7.30 (m, 3H, Ar);

C NMR (DMSO-d6, δ, ppm): 170.58 (CO); 161.82 (CN);

150.64 (C); 138.44 (C); 136.75 (C); 135.04 (C); 134.09 (CH);

32.34 (C); 124.54 (CH); 123.09 (CH); 120.40 (CH), 112.10

CH).

.2.3. N′-[(2,6-Dichlorophenyl)methylidene]-1,2-benzo-

in vitro inhibition assay is needed to elucidate the implica-

tion of ERG11 inhibition in the mechanism of antifungal ac-

tivity, the results indicate that ERG11 may be one but proba-

bly not the only target of these compounds.

1

3

1

(

3

. Experimental

thiazole-3-carbohydrazide (3j). Mp: 263–265 °C (DMSO), yield

3

.1. Materials and methods

−1

64ꢀ. TLC: CH Cl

2 2

/EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3170

1

Unless otherwise noted, reagents were obtained from com-

mercial suppliers and were used without purification. Anhy-

drous toluene was obtained by distillation from Na, and

anhydrous CH Cl was obtained by distillation from cal-

(NH), 1660 (CO). H NMR (DMSO-d6, δ, ppm): 12.70 (s, 1H,

NH); 8.81 (s, 1H, CH); 8.74 (d, 1H, J = 9, Ar); 8.35 (d, 1H, J = 9,

1

3

Ar); 7.74–7.58 (m, 4H, Ar); 7.47 (t, 1H, J = 9, Ar); C NMR

(DMSO-d6, δ, ppm): 171.12 (CO); 167.88 (CN); 151.12 (C);

137.45 (C); 135.68 (C); 132.28 (C); 130.75 (CH); 127.80 (CH);

125.43 (CH); 124.33 (CH); 123.89 (CH), 120.64 (CH).

2

cium hydride. Melting points were measured using Büchi

12 apparatus (Büchi Italia, Milano, Italia) and are

5

uncorrected. Progress of reaction was monitored by TLC

with F254 silica-gel precoated sheets (Merck, Darmstadt,

Germany). UV light was used for detection. Flash chroma-

tography was performed using Merck silica gel 60 (Si 60,

3.2.4. N′-[(2,6-Difluorophenyl)methylidene]-1,2-benzo-

thiazol-3-ylacetohydrazide (3o). Mp: 190–192 °C (THF), yield

−

1

90ꢀ. TLC: CH Cl /EtOH, 95 : 5. FTIR (KBr, ν, cm ): 3183

2

1

(NH), 1670 (CO). H NMR (DMSO-d6, δ, ppm): 11.82 (s, 1H,

NH); 8.29 (s, 1H, CH); 8.17 (d, 1H, J = 9 Ar); 8.10 (d, 1H, J = 9

4

0–63 μm, 230–400 mesh ASTM). Elemental analyses for C,

1

3

H, and N were performed using a ThermoQuest Flash 1112

elemental analyser (Thermoquest Italia, Milano, Italy). The

percentages found were within ±0.4ꢀ of the theoretical

values. IR spectra were recorded on a Jasco FT-IR 300E

Ar); 7.65–7.28 (m, 5H, Ar); 4.50 (s, 2H, CH₂); C NMR

(DMSO-d6, δ, ppm): 170.75 (CO); 161.09 (CN); 152.12 (C);

140.69 (C); 138.66 (CH); 135.26 (C); 133.86 (C); 132.09 (C);

128.39 (CH); 126.77 (CH); 125.34 (CH), 124.65 (CH); 120.87

(CH); 35.66 (CH₂).

1

spectrometer (Jasco Europe, Carpi MO, Italia). H-NMR

1

3

and C NMR spectra were recorded on a Bruker Avance

00 spectrometer (Bruker Italia, Milano, Italia). The spectra

3.2.5. N′-[(2-Chloro-6-fluorophenyl)methylidene]-1,2-benzo-

thiazol-3-ylacetohydrazide (3p). Mp: 203–205 °C (EtOH), yield

4

−

1

were acquired from samples in DMSO-d . Chemical shifts

2 2

60ꢀ. TLC: CH Cl /EtOH, 95 : 5. FTIR (KBr, ν, cm ): 3187,

6

1

are reported as δ (ppm) relative to tetramethylsilane; cou-

pling constants ( J) are expressed in Hz.

(NH), 1668 (CO). H NMR (DMSO-d6, δ, ppm): 11.77 (s, 1H,

NH); 8.21–8.17 (m, 2H, CH); 8.11 (d, 1H, J = 9 Ar); 7.65–7.43

1

3

(

m, 3H, Ar); 7.21–7.14 (m, 2H, Ar); 4.51 (s, 2H, CH

2

);

C

NMR (DMSO-d6, δ, ppm): 170.87 (CO); 164.71 (CN);

3

(

3

.2. General procedure for the synthesis of N′-

halophenylmethylidene)-1,2-benzothiazole-3-carbohydrazides

h–j and N′-(halophenylmethylidene)-1,2-benzothiazole-3-

1

51.90 (C); 138.90 (C); 137.77 (C); 135.28 (C); 134.06 (CH);

32.03 (C); 125.45 (CH); 124.62 (CH); 120.92 (CH), 112.90

(

CH); 35.86 (CH₂).

.2.6. N′-[(2,6-Dichlorophenyl)methylidene]-1,2-benzo-

thiazol-3-ylacetohydrazide (3q). Mp: 230–232 °C (THF), yield

acetohydrazides 3o–q

3

A solution of 1,2-benzothiazole-3-carbohydrazide 1 or 1,2-

benzothiazole-3-acetohydrazide 2 (6 mmol) and an equimolar

amount of the requisite aldehyde in anhydrous ethanol (60

mL) was heated to reflux for 3 hours in the presence of acetic

acid (1 mL). The mixture was cooled to room temperature,

and the white precipitate that formed was filtered off, washed

with water and then recrystallized.

−

1

93ꢀ. TLC: CH Cl /EtOH, 95 : 5. FTIR (KBr, ν, cm ): 3183

(NH), 1668 (CO). H NMR (DMSO-d6, δ, ppm): 11.86 (s, 1H,

NH); 8.29 (s, 1H, CH); 8.17 (d, 1H, J = 9 Ar); 8.09 (d, 1H, J = 9

Ar); 7.65–7.36 (m, 5H, Ar); 4.28 (s, 2H, CH₂); C NMR

(DMSO-d6, δ, ppm): 171.01 (CO); 169.91 (CN); 151.90 (C);

138.90 (C); 135.21 (C); 134.35 (C); 131.44 (CH); 129.80 (CH);

128.26 (CH); 125.33 (CH); 124.38 (CH), 120.88 (CH); 39.37

2

1

3

.2.1. N′-[(2,6-Difluorophenyl)methylidene]-1,2-benzo-

thiazole-3-carbohydrazide (3h). Mp: 298 °C (DMSO), yield

2

(CH ).

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3

1

.3. General procedure for the synthesis of N-(4-oxo-2-phenyl-

,3-thiazolidin-3-yl)-1,2-benzothiazole-3-carboxamides 4a–j

(s, 1H, CH); 3.98 (dd, 1H, J = 16 CH–H′); 3.83 (d, 1H, J = 16.1

1

3

CH–H). C NMR (DMSO-d6, δ, ppm): 169.34 (CO); 159.99,

(CO); 154.49 (C); 153.31 (C); 134.70 (C); 134.11 (C); 130.72

(C); 128.88 (CH); 126.81 (CH); 125.60 (C); 121.28 (CH), 115.92

and N-(4-oxo-2-phenyl-1,3-thiazolidin-3-yl)-1,2-benzothiazole-

-ylacetamides 4k–p

3

(

CH); 115.71 (CH); 61.65 (CH); 29.85 (CH₂).

A mixture of the requisite hydrazide 3a–p (1.6 mmol) and

sulfanylacetic acid (3.2 mmol) was heated to reflux for 48

hours in dry toluene (30 mL) with stirring. The reaction mix-

ture was then cooled to room temperature and, after concen-

tration and filtration, the solid obtained was washed with an

aqueous solution of 10ꢀ Na CO . The crude product was

3.3.5. N-[2-(2-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4e). Mp: 212–213 °C (EtOH),

−

1

yield 49ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ):

2

1

3284 (NH), 1685 (CO). H NMR (DMSO-d6 δ, ppm): 11.31

(s, 1H, NH); 8.62 (d, 1H, J = 8 Ar); 8.32 (d, 1H, J = 8 Ar); 7.86

(d, 1H, J = 7.7 Ar); 7.70–7.58 (m, 2H, Ar); 7.47–7.35 (m, 3H,

Ar); 6.32 (s, 1H, CH); 3.99 (dd, 1H, J = 1.2 J = 16.1 CH–H′);

3.83 (d, 1H, J = 16 CH–H); C NMR (DMSO-d6, δ, ppm):

169.79 (CO); 160.22 (CO); 154.54 (C); 153.30 (C); 136.05

(C); 134.13 (C); 132.52 (C); 130.77 (CH); 130.01 (CH), 128.99

(CH); 128.90 (CH); 128.31 (CH); 126.88 (CH); 125.63 (CH);

121.30 (CH); 58.89 (CH), 29.29 (CH₂).

2

3

then purified by flash chromatography and recrystallized

from an appropriate solvent or directly recrystallized.

1

3

.3.1. N-(4-Oxo-2-phenyl-1,3-thiazolidin-3-yl)-1,2-benzo-

thiazole-3-carboxamide (4a). Mp: 173–175 °C (EtOH), yield

−

1

6

(

0ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3208

2 2

1

NH), 1700 (CO). H NMR (DMSO-d6, δ, ppm): 11.14 (s, 1H,

NH); 8.56 (d, 1H, J = 8 Ar); 8.30 (d, 1H, J = 8.2 Ar); 7.69–7.64

m, 1H, Ar); 7.61–7.53 (m, 3H, Ar); 7.41–7.31 (m, 3H, Ar);

.99 (s, 1H, CH); 3.96 (dd, 1H, J = 1.6 J = 16 CH–H′); 3.83

3.3.6. N-[2-(3-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4f). Mp: 145 °C (EtOH), yield

(

5

(

1

(

1

(

−

1

2 2

72ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3218

1

3

1

d, 1H, J = 16.1 CH–H). C NMR (DMSO-d6, δ, ppm):

69.54 (CO); 160.01 (CO); 154.54 (C); 153.30 (C); 138.45

C); 134.12 (C); 129.47 (CH); 128.98 (CH); 128.87 (CH),

28.32 (CH); 126.81 (CH); 125.61 (CH); 121.26 (CH); 62.36

CH), 29.90 (CH₂).

.3.2. N-[2-(2-Fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4b). Mp: 183 °C (EtOH), yield

(NH), 1689 (CO). H NMR (DMSO-d6, δ, ppm): 11.22 (s, 1H,

NH); 8.59 (d, 1H, J = 7.9 Ar); 8.32 (d, 1H, J = 8.1 Ar); 7.70–7.57

(m, 3H, Ar); 7.52–7.47 (m, 1H, Ar); 7.42–7.39 (m, 2H, Ar); 6.00

(s, 1H, CH); 4.01 (dd, 1H, J = 1.7 J = 16.1 CH–H′); 3.82 (d, 1H, J

1

3

= 15.8 CH–H); C NMR (DMSO-d6, δ, ppm): 169.42 (CO);

160.06 (CO); 154.45 (C); 153.33 (C); 141.36 (C); 134.13 (C);

133.70 (C); 130.82 (CH); 129.40 (CH), 128.90 (CH); 128.03

(CH); 127.04 (CH); 126.84 (CH); 125.59 (CH); 121.30 (CH);

3

−

1

6

(

7ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3201

2 2

1

NH), 1685 (CO). H NMR (DMSO-d6, δ, ppm): 11.25 (s, 1H,

2

61.51 (CH), 29.75 (CH ).

NH); 8.59 (d, 1H, J = 8 Ar); 8.32 (d, 1H, J = 8.1 Ar); 7.77–7.57

3.3.7. N-[2-(4-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4g). Mp: 173–175 °C (EtOH),

(

m, 3H, Ar); 7.44–7.37 (m, 1H, Ar); 7.28–7.17 (m, 2H, Ar); 6.19

d, 1H, J = 1.2 CH); 3.99 (dd, 1H, J = 1.5 J = 15.9 CH–H′); 3.84

d, 1H, J = 15.9 CH–H); (J = 16.1 CH–H′); 3.82 (d, 1H, J = 15.9

−

1

2 2

yield 70ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ):

1

3228 (NH), 1716 (CO), 1675 (CO). H NMR (DMSO-d6, δ,

ppm): 11.15 (s, 1H, NH); 8.58 (d, 1H, J = 8.1 Ar); 8.31 (d, 1H, J

= 8.1 Ar); 7.69–7.57 (m, 4H, Ar); 7.43 (d, 2H, J = 8.4 Ar); 5.99

(s, 1H, CH); 3.99 (d, 1H, J = 16 CH–H′); 3.83 (d, 1H, J = 16

1

3

CH–H); C NMR (DMSO-d6, δ, ppm): 169.28 (CO); 160.13,

(

CO); 154.47 (C); 153.33 (C); 134.12 (CH); 131.43 (C); 129.89

CH); 128.89 (CH); 126.84 (CH); 125.87 (C); 125.60 (CH),

1

3

1

(

25.21 (C); 121.29 (CH); 116.18 (CH); 115.98 (CH); 55.98

CH); 29.62 (CH ).

.3.3. N-[2-(3-Fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4c). Mp: 179–182 °C (EtOH),

CH–H); C NMR (DMSO-d6, δ, ppm): 169.34 (CO); 159.99

(CO); 154.46 (C); 153.33 (C); 137.68 (C); 134.08 (C); 130.34

(CH); 128.97 (CH); 128.89 (CH), 126.82 (CH); 125.60 (CH);

121.29 61.59 (CH), 29.81 (CH₂).

2

3

−

1

yield 73ꢀ. TLC: CH

207 (NH), 1710 (CO). H NMR (DMSO-d6, δ, ppm): 11.21

s, 1H, NH); 8.60 (d, 1H, J = 8.1 Ar); 8.32 (d, 1H, J = 8.1 Ar);

2

Cl

2

/EtOH, 98 : 2. FTIR (KBr, ν, cm ):

3.3.8. N-[2-(2,6-Difluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-

1,2-benzothiazole-3-carboxamide (4h). Mp: 185 °C (EtOH),

1

3

(

−

1

yield 60ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν, cm ):

2 2

1

7

3

.69 (t, 1H, J = 7.2 Ar); 7.60 (t, 1H, J = 7.5 Ar); 7.46–7.35 (m,

H, Ar); 7.21–7.15 (m, 1H, Ar); 6.02 (s, 1H, CH); 4.02 (dd, 1H,

3235 (NH), 1720 (CO), 1681 (CO). H NMR (DMSO-d6, δ,

ppm): 11.33 (s, 1H, NH); 8.53 (d, 1H, J = 8.1 Ar); 8.32 (d, 1H, J

= 8.2 Ar); 7.69 (t, 1H, J = 7.5 Ar); 7.59 (t, 1H, J = 7.4 Ar); 7.50–

7.43 (m, 1H, Ar); 7.12 (t, 2H, J = 9.3 Ar); 6.22 (s, 1H, CH); 3.93

1

3

J = 1.5 J = 16.1 CH–H′); 3.82 (d, 1H, J = 15.9 CH–H); C NMR

DMSO-d6, δ, ppm): 169.47 (CO); 160.09 (CO); 154.44 (C);

(

1

3

1

53.33 (C); 141.76 (C); 134.12 (CH); 131.00 (C); 128.89 (CH);

26.84 (CH); 125.60 (CH); 124.38 (CH), 121.30 (CH); 116.40

(d, 1H, J = 16.2 CHH′); 3.84 (d, 1H, J = 15.9 CH–H);

C

NMR (DMSO-d6, δ, ppm): 168 169.19 (CO); 160.17 (CO);

154.47 (C); 153.31 (C); 134.10 (C); 132.07 (CH); 128.91 (CH);

126.85 (CH); 125.48 (CH); 121.30 (CH); 114.99 (C), 112.66

(CH); 112.47 (CH); 51.28 (CH); 29.92 (CH₂).

(

CH); 114.88 (CH); 61.53 (CH); 29.73 (CH

.3.4. N-[2-(4-Fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-1,2-

benzothiazole-3-carboxamide (4d). Mp: 208 °C (EtOH), yield

2

).

3

−

1

6

2ꢀ. TLC: CH

NH), 1714 (CO), 1673 (CO). H NMR (DMSO-d6, δ,

ppm): 11.13 (s, 1H, NH); 8.58 (d, 1H, J = 8.1 Ar); 8.32 (d, 1H, J

8.1 Ar); 7.70–7.59 (m, 4H, Ar); 7.20 (t, 2H, J = 8.9 Ar); 5.99

2

Cl

2

/EtOH, 98 : 2. FTIR (KBr, ν, cm ): 3224

3.3.9. N-[2-(2-Chloro-6-fluorophenyl)-4-oxo-1,3-thiazolidin-

3-yl]-1,2-benzothiazole-3-carboxamide (4i). Mp: 176 °C

(EtOH), yield 37ꢀ. TLC: CH Cl /EtOH, 98 : 2. FTIR (KBr, ν,

1

(

2

−

1

=

cm ): 3235 (NH), 1716 (CO), 1685 (CO). H NMR

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(

8

DMSO-d6, δ, ppm): 11.35 (s, 1H, NH); 8.54 (d, 1H, J = 8.1 Ar);

.32 (d, 1H, J = 8.1 Ar); 7.68 (td, 1H, J = 1.2 J = 7.8 Ar); 7.60

125.26 (CH); 124.37 (CH); 120.89 (CH); 61.12 (CH), 36.94

(CH ); 29.51 (CH ).

2

(

td, 1H, J = 0.9 J = 8.1 Ar); 7.49–7.42 (m, 1H, Ar); 7.36–7.27

3.3.14. N-(2-(4-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,2-

benzothiazol-3-ylacetamide (4n). Flash chromatography;

EtOAc/Hex, 4 : 6. Mp: 168–170 °C (CH C H ), yield 45ꢀ. TLC:

(m, 2H, Ar); 6.48 (s, 1H, CH); 3.95 (d, 1H, J = 15.3 CH–H′);

1

3

.86 (dd, 1H, J = 2.2 J = 15.6 CH–H); C NMR (DMSO-d6, δ,

3

6

5

−

1

ppm): 168 168.33 (CO); 160.32 (CO); 154.53 (C); 153.31

C); 134.10 (CH); 132.19 (C); 128.92 (CH); 126.86 (CH);

25.62 (CH); 121.31 (CH); 116.67 (C), 55.21 (CH); 30.00

CH ).

.3.10. N-[2-(2,6-Dichlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-

,2-benzothiazole-3-carboxamide (4j). Mp: 221–223 °C (EtOH),

EtOAc/Hex, 6 : 4. FTIR (KBr, ν, cm ): 3222 (NH), 1716 (CO),

1

(

1

(

1666 (CO). H NMR (DMSO-d6, δ, ppm): 10.62 (s, 1H, NH);

8.16 (d, 1H, J = 8.2 Ar); 7.93 (d, 1H, J = 8.1 Ar); 7.62–7.57 (m,

1H, Ar); 7.46–7.38 (m, 5H, Ar); 5.77 (d, 1H, J = 1.2 CH); 4.00

(s, 2H, CH2); 3.90 (dd, 1H, J = 1.7 J = 16 CH–H′); 3.72 (d, 1H,

2

3

1

3

1

J = 15.8 CH–H); C NMR (DMSO-d6, δ, ppm): 168 169.18

(CO); 167.13 (CO); 159.94 (C); 152.07 (C); 137.92 (C);

134.77 (CH); 134.06 (CH); 130.11 (CH); 129.10 (CH); 128.68

(CH); 128.29 (CH), 125.24 (CH); 124.37 (CH); 120.88 (CH);

−1

2 2

yield 31ꢀ. TLC: CH Cl /EtOH, 98:2. FTIR (KBr, ν, cm ): 3365

1

(NH), 1722 (CO), 1691 (CO). H NMR (DMSO-d6, δ, ppm):

1

7

1

1.33 (s, 1H, NH); 8.55 (d, 1H, J = 7.9 Ar); 8.32 (d, 1H, J = 8.2 Ar);

.71–7.55 (m, 3H, Ar); 7.47–7.38 (m, 2H, Ar); 6.82 (d, 1H, J =

.9 CH); 4.00 (dd, 1H, J = 2.2 J = 15.7 CH–H′); 3.92 (d, 1H, J =

5.6 CH–H).

124.32 (CH); 120.88 (CH); 61.20 (CH), 36.94 (CH

(CH₂).

2

); 29.58

3.3.15. N-(2-(3-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,2-

benzothiazol-3-ylacetamide (4m). Flash chromatography;

CH Cl /EtOH, 98 : 2. Mp: 147–148 °C (CH C H ), yield 45ꢀ.

3

.3.11. N-(4-Oxo-2-phenyl-1,3-thiazolidin-3-yl)-1,2-benzo-

thiazol-3-ylacetamide (4k). Flash chromatography; CH Cl /

EtOH, 98 : 2. Mp: 176–178 °C (CH

EtOAc/Hex, 4 : 6. FTIR (KBr, ν, cm ): 3226 (NH), 1716 (CO),

1

8

7

CH2); 3.90 (dd, 1H, J = 1.6 J = 16 CH–H′); 3.72 (d, 1H, J = 15.9

CH–H); C NMR (DMSO-d6, δ, ppm): 169.42 (CO); 160.06

(

2

3

6

5

−

1

3 6 5

C H ), yield 16ꢀ. TLC:

TLC: EtOAc/Hex, 6 : 4. FTIR (KBr, ν, cm ): 3280 (NH), 1716

(CO), 1660 (CO). H NMR (DMSO-d6, δ, ppm): 10.62 (s,

−

1

666 (CO). H NMR (DMSO-d6, δ, ppm): 10.65 (s, 1H, NH);

.16 (d, 1H, J = 8.2 Ar); 7.94 (d, 1H, J = 8.1 Ar); 7.59 (t, 1H, J =

.2 Ar); 7.43–7.38 (m, 6H, Ar); 5.76 (s, 1H, CH); 4.01 (s, 2H,

1H, NH); 8.16 (d, 1H, J = 8.2 Ar); 7.96 (d, 1H, J = 8.2 Ar); 7.59

(td, 1H, J = 1.2 J = 8.1 Ar); 7.51 (s, 1H, Ar); 7.46–7.36 (m, 4H,

Ar); 5.78 (d, 1H, J = 1.5 CH); 4.02 (s, 2H, CH2); 3.95 (dd, 1H, J

1

3

= 1.7 J = 15.9 CH–H′); 3.72 (d, 1H, J = 15.9 CH–H); C NMR

(DMSO-d6, δ, ppm): 169.27 (CO); 167.22 (CO); 159.96 (C);

156.09 (C); 141.71 (C); 134.79 (C); 133.81 (C); 130.99 (CH);

129.44 (CH); 128.30 (CH); 127.88 (CH), 126.84 (CH); 125.26

1

3

CO); 154.45 (C); 153.33 (C); 141.36 (C); 134.13 (C); 133.70

CH); 130.82 (CH); 129.40 (CH), 128.90 (CH); 128.03 (CH);

1

(

27.04 (CH); 126.84 (CH); 125.59 (CH); 121.30 (CH); 61.51

CH), 29.75 (CH ).

.3.12. N-(2-(2-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,2-

benzothiazol-3-ylacetamide (4l). Flash chromatography;

EtOAc/Hex, 4 : 6. Mp: 103–105 °C (CH /Hex), yield

5ꢀ. TLC: EtOAc/Hex, 6 : 4. FTIR (KBr, ν, cm ): 3199

(CH); 124.37 (CH); 120.89 (CH); 61.12 (CH), 36.94 (CH

29.51 (CH ).

3.3.16. N-(2-(2,6-Difluorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-

1,2-benzothiazol-3-ylacetamide (4o). Flash chromatography;

2

);

2

3

C H

3 6 5

2 2 2

CH Cl /EtOH, 98 : 2. Mp: 70–75 °C (EtOH/H O), yield 56ꢀ.

−

1

−1

4

TLC: EtOAc/Hex, 4 : 6. FTIR (KBr, ν, cm ): 3214 (NH),

1

(

NH), 1722 (CO), 1664 (CO). H NMR (DMSO-d6, δ,

ppm): 10.84 (s, 1H, NH); 8.16 (d, 1H, J = 8.2 Ar); 7.96 (d,

H, J = 8.1 Ar); 7.62–7.35 (m, 6H, Ar); 6.09 (d, 1H, J = 1.2

CH); 4.05 (s, 2H, CH2); 3.88 (dd, 1H, J = 1.5 J = 15.9

1720 (CO), 1675 (CO). H NMR (DMSO-d6, δ, ppm):

10.85 (s, 1H, NH); 8.18 (d, 1H, J = 8.2 Ar); 7.95 (d, 1H, J

= 8.1 Ar); 7.62–7.55 (m, 1H, Ar); 7.53–7.47 (m, 1H, Ar);

7.45–7.40 (m, 1H, Ar); 7.15 (t, 2H, J = 9.2 Ar); 6.08 (s, 1H,

CH); 4.03 (s, 2H, CH2); 3.82 (dd, 2H, J = 16.4 J = 20 CH–

1

3

CH–H′); 3.74 (d, 1H, J = 15 CH–H); C NMR (DMSO-d6,

δ, ppm): 169.62 (CO); 167.45 (CO); 159.93 (C); 152.09

1

3

H); C NMR (DMSO-d6, δ, ppm): 168.38 (CO); 167.51

(CO); 159.89 (C); 152.08 (C); 134.73 (CH); 132.13 (C);

128.30 (CH); 125.31 (CH); 124.14 (CH); 120.91 (CH),

(

1

(

3

C); 136.37 (C); 134.81 (CH); 132.66 (CH); 130.82 (CH);

30.29 (CH); 128.30 (CH); 128.70 (C), 128.39 (CH); 128.28

CH); 125.24 (CH); 124.32 (CH); 120.89 (CH); 58.62 (CH),

6.94 (CH ); 29.18 (CH ).

.3.13. N-(2-(3-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,2-

benzothiazol-3-ylacetamide (4m). Flash chromatography;

CH Cl /EtOH, 98 : 2. Mp: 147–148 °C (CH ), yield 45ꢀ.

TLC: EtOAc/Hex, 6 : 4. FTIR (KBr, ν, cm ): 3280 (NH), 1716

114.91 (C); 112.80 (C); 51.11 (CH), 36.89 (CH ); 29.67

2

(CH ).

3

3.3.17. N-(2-(2-Chloro-6-fluorophenyl)-4-oxo-1,3-thiazolidin-

3-yl)-1,2-benzothiazol-3-ylacetamide (4p). Flash chromatogra-

2

C H

3 6 5

2 2 3 6 5

phy; CH Cl /EtOH, 98: 2. Mp: 105 °C (CH C H /Hex), yield

−

1

−1

20ꢀ. TLC: EtOAc/Hex, 4 : 6. FTIR (KBr, ν, cm ): 3199 (NH),

1

(

CO), 1660 (CO). H NMR (DMSO-d6, δ, ppm): 10.62 (s,

H, NH); 8.16 (d, 1H, J = 8.2 Ar); 7.96 (d, 1H, J = 8.2 Ar); 7.59

td, 1H, J = 1.2 J = 8.1 Ar); 7.51 (s, 1H, Ar); 7.46–7.36 (m, 4H,

Ar); 5.78 (d, 1H, J = 1.5 CH); 4.02 (s, 2H, CH2); 3.95 (dd, 1H, J

1720 (CO), 1681 (CO). H NMR (DMSO-d6, δ, ppm): 10.85

1

(

(s, 1H, NH); 8.16 (d, 1H, J = 8.2 Ar); 7.95 (d, 1H, J = 8.1Ar);

7.59 (t, 1H, J = 7.6 Ar); 7.49–7.30 (m, 4H, Ar); 6.29 (s, 1H,

1

3

CH); 4.03 (s, 2H, CH2); 3.84–3.82 (m, 2H, CH–H);). C NMR

(DMSO-d6, δ, ppm): 168.45 (CO); 167.50 (CO); 159.85 (C);

152.09 (C); 134.75 (C); 132.17 (C); 132.06 (C); 128.28 (CH);

126.21 (CH); 125.30 (CH), 124.29 (CH); 120.89 (CH); 116.74

13

=

1.7 J = 15.9 CH–H′); 3.72 (d, 1H, J = 15.9 CH–H); C NMR

(DMSO-d6, δ, ppm): 168 169.27 (CO); 167.22 (CO); 159.96

(C); 156.09 (C); 141.71 (C); 134.79 (C); 133.81 (C); 130.99

(CH); 129.44 (CH); 128.30 (CH); 127.88 (CH), 126.84 (CH);

2 2

(CH); 116.52 (CH); 55.23 (CH), 30.89 (CH ); 29.74 (CH ).

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3

.4. Biological evaluation

.4.1. Antibacterial activity. The following Gram-negative

In order to investigate the antifungal activity of the com-

pounds, a modified microdilution technique was used.

2

2–26

3

The fungal spores were washed from the surface of agar

plates with sterile 0.85ꢀ saline containing 0.1ꢀ Tween 80 (v/v).

The spore suspension was adjusted with sterile saline to a con-

bacteria were used: Escherichia coli (ATCC 35210), Pseudomo-

nas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC

1

5

3311), and Enterobacter cloacae (human isolate); the follow-

centration of approximately 1.0 × 10 in a final volume of 100

ing Gram-positive bacteria were used: Bacillus cereus (clinical

isolate), Micrococcus flavus (ATCC 10240), Listeria mono-

cytogenes (NCTC 7973), and Staphylococcus aureus (ATCC

μL per well. The inocula were stored at 4 °C for further use. Di-

lutions of the inocula were cultured on solid malt agar to verify

the absence of contamination and to check the validity of the

inoculum. Minimal inhibitory concentration (MIC) determina-

tions were performed by a serial dilution technique using 96-

well microtiter plates. The compounds investigated were

dissolved in 5ꢀ DMSO solution containing 0.1ꢀ Tween 80 (v/

6

538). The organisms were obtained from the Mycological

Laboratory, Department of Plant Physiology, Institute for Bio-

logical Research ‘Siniša Stanković’, Belgrade, Serbia. The anti-

2

2,23

bacterial assay was carried out by a microdilution method

−1

in order to determine the antibacterial activity of compounds

tested against the human pathogenic bacteria. The bacterial

suspensions were adjusted with sterile saline to a concentra-

v) (1 mg mL ) and added in broth malt medium with inocu-

lum. The microplates were incubated in a rotary shaker (160

rpm) for 72 h at 28 °C. The lowest concentrations without visi-

ble growth (at the binocular microscope) were defined as the

MICs. The fungicidal concentrations (MFCs) were determined

by serial subcultivation of 2 μL of tested compounds dissolved

in medium and inoculated for 72 h into microtiter plates

containing 100 μL of broth per well and further incubation 72

h at 28 °C. The lowest concentration with no visible growth was

defined as the MFC indicating 99.5ꢀ killing of the original in-

oculum. 5ꢀ DMSO solution was used as a negative control,

and commercial fungicides, namely, bifonazole (Srbolek, Bel-

grade, Serbia) and ketoconazole (Zorkapharma, Šabac, Serbia),

−

1

tion of 1.0 × 105 CFU mL . The inocula were prepared daily

and stored at +4 °C until use. Dilutions of the inocula were

cultured on solid medium to verify the absence of contamina-

tion and to check the validity of the inoculum. All experi-

ments were performed in duplicate and repeated three times.

3

.4.2. Microdilution test. The minimal inhibitory and bac-

tericidal concentrations (MICs and MBCs) were determined

using 96-well microtitre plates. The bacterial suspension was

adjusted with sterile saline to a concentration of 1.0 × 105 CFU

−1

mL . Compounds to be investigated were dissolved in 5ꢀ

−1

DMSO solution containing 0.1ꢀ Tween 80 (v/v) (1 mg mL )

and added in broth (LB) medium (100 μl) with bacterial inocu-

lum (1.0 × 104 CFU per well) to achieve the desired concentra-

tions. The microplates were incubated using a rotary shaker

were used as positive controls (1–3500 μg mL ). All experi-

ments were performed in duplicate and repeated three times.

3.4.4. Statistical analysis. All the assays were carried out in

triplicate and the results are expressed as mean values and

standard deviations (SD). The results were analyzed using

one-way analysis of variance (ANOVA) followed by Tukey's

HSD Test with α = 0.05. This treatment was carried out

using the SPSS v. 18.0 program.

(160 rpm) for 24 h at 37 °C. The lowest concentrations without

visible growth (at the binocular microscope) were defined as

the concentrations that completely inhibited bacterial growth

(MICs). The MBCs were determined by serial sub-cultivation of

2

μL into microtitre plates containing 100 μL of broth per well

The best curves for the correlation of the estimated bind-

ing energy with anti-bacterial activity were obtained using the

online program www.mycurvefit.com.

and further incubation for 24 h. The lowest concentration with

no visible growth was defined as the MBC, indicating 99.5ꢀ

killing of the original inoculum. The optical density of each

well was measured at a wavelength of 655 nm using Microplate

manager 4.0 (Bio-Rad Laboratories) and compared with a blank

and the positive control. Streptomycin (Sigma P 7794) and am-

picillin (Panfarma, Belgrade, Serbia) were used as positive con-

trols (1 mg mL in sterile physiological saline). 5ꢀ DMSO so-

lution was used as a negative control. All experiments were

performed in duplicate and repeated three times.

3.5. Calculation of biologically relevant physicochemical

properties

Prediction of the physically significant descriptors and phar-

maceutically relevant properties of the novel synthesized

compounds 4a–p and the new intermediates (3h, i, j, o, p)

was performed using the QikProp module of Maestro 9.8

from Schrödinger software. For compounds 4a–p both the R

and S enantiomers were evaluated.

−1

3

.4.3. Antifungal activity. For the antifungal bioassays,

eight fungi were used: Aspergillus niger (ATCC 6275), Aspergil-

lus ochraceus (ATCC 12066), Aspergillus fumigatus (human iso-

late), Aspergillus versicolor (ATCC 11730), Penicillium

funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC

3.6. Multivariate data analysis

The calculated molecular properties of the novel compounds

4a–p and 3h, i, j, o and p in relation to their antifungal and

antibacterial activities were further subjected to multivariate

data analysis. SIMCA-P version 14.0 software (Umetrics, Umeå,

Sweden) was utilized in order to implement Principal Compo-

nent Analysis (PCA). A PCA model estimates the systematic var-

iation in a data matrix by a low dimensional model plane. The

9

112), Trichoderma viride (IAM 5061), and Penicillium

versicolor var. Cyclopium. The organisms were obtained from

the Mycological Laboratory, Department of Plant Physiology,

Institute for Biological Research ‘Siniša Stanković’, Belgrade,

Serbia. The micromycetes were maintained on malt agar and

2

4

the cultures stored at 4 °C and sub-cultured once a month.

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unsupervised PCA pattern recognition method extracts the

dominant patterns in the data matrix in terms of a comple-

mentary set of scores and loading plots. This approach enables

reduction of dimensionality, data exploration finding relation-

ships between objects, estimation of the correlation structure

of the variables and investigation on the necessary components

docking. Each docking experiment was derived from 100 dif-

ferent runs that were set to terminate after a maximum of

2 500 000 energy evaluations. The population size was set to

150. During the search, a translational step of 0.2 Å and qua-

ternion and torsion steps of 5 were applied. Co-enzymes,

such as FAD, in MurB were kept in the structure. The pH for

ligand preparation was set at 7.0 in all cases.

(a linear combination of original features) in order to explain

the greater part of variance with a minimal loss of informa-

Docking analysis to E. coli gyrase (PDB ID: 4TMA) was

performed using the program PyRx based on Autodock Vina.

27

32

tion. The fraction of the variation of Y that can be predicted

by the model was extracted according to the internal cross vali-

2

8

dation default method of SIMCA-P software.

4. Conclusion

Then, classification analysis ensued by further subjecting

the data set to orthogonal partial least squares-discriminant

analysis (OPLS-DA). OPLS-DA is an extension of the super-

vised partial least squares (PLS) regression method that man-

ages to increase the quality of the classification model by sep-

arating the systematic variation in X into two parts, one that

is linearly related to Y (predictive information) and one that

is unrelated to Y (orthogonal information). The concept of

The newly synthesized N-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-

1,2-benzothiazole-3-carboxamides and acetamides exhibit re-

markable inhibition of the growth of a wide spectrum of

Gram positive and Gram negative bacteria and fungi. Almost

all our compounds exhibited better than or comparable anti-

bacterial activity to reference drugs with the most potent

among them being compound 4d followed by 4h. The most

sensitive bacterial species for these compounds were S. au-

reus and E. coli while L. monocytogenes and S. typhimurium

were the more resistant ones.

2

7

these methods has been extensively discussed.

The PCA and OPLS-DA models were UV scaled, extracted

at a confidence level of 95ꢀ and any observations at <5ꢀ

were considered to be outliers.

As far as fungi are concerned, the tested compounds pos-

sess excellent activity against all the fungal species tested, be-

ing more than 12 times more active than ketoconazole and 3

times more active than bifonazole. The most sensitive fungi

were T. viride and A. ochraceus, while A. fumigates was the

most resistant. The most promising is compound 4p followed

by 4m and 4o. A structure–antifungal activity relationship

based on predicted physicochemical properties indicated the

importance of shape together with the lipophilicity and

charge distribution parameters of the studied molecules.

It can be seen that the growth of the tested bacteria (Gram

(−) and Gram (+) bacteria) and fungi responded differently to

the tested compounds, which indicates that different compo-

nents may have different modes of action or that the metabo-

lism of some bacteria/fungi is able to better overcome the ef-

fect of the compounds or adapt to it. Gram (−) bacteria and

fungi are in general more resistant than Gram (+) bacteria.

Docking analysis using E. coli MurB and gyrase crystal

structures indicated the probable involvement mainly of

MurB inhibition and to a lesser extent gyrase inhibition in

the mechanism of action of the antibacterial activity of these

compounds.

A loading plot was extracted to reveal the variables that

have class discriminating power. The volcano plot for the

OPLS-DA model was based on feature selection, which in-

cluded scores larger than 1 for the variable importance in

projection (VIP) values and pĲcorr) > 0.4 to reveal the vari-

ables which bear class discriminating power.

The quality of the models was described by the goodness-

of-fit R2 (0 ≤ R2 ≤ 1) and the predictive ability Q2 (0 ≤ Q2 ≤

1

) values. The R2 explains the variation thus constituting a

quantitative measure of how well the data of the training set

are mathematically reproduced. The overall predictive ability

of the model is assessed by the cumulative Q2, representing

the fraction of the variation of Y that can be predicted by the

model which was extracted according to the internal cross

2

8

validation default method of SIMCA-P software.

3

.7. Docking studies

Docking analysis to the E. coli enzyme MurB (PDB ID: 2Q85)

and to the Candida Albicans 14a-lanosterol demethylase

(

ERG11) (PBD ID: 2v2z) was carried out using

2

9

DockingServer. Gasteiger partial charges were added to the

ligand atoms. Non-polar hydrogen atoms were merged, and

rotatable bonds were defined. Essential hydrogen atoms,

Kollman united atom type charges, and solvation parameters

were added with the aid of AutoDock tools. AutoDock pa-

rameter set- and distance-dependent dielectric functions were

used in the calculation of the van der Waals and the electro-

static terms, respectively. Docking simulations were

performed using the Lamarckian genetic algorithm (LGA)

Docking analysis to the antifungal target 14a-lanosterol

demethylase (ERG11) revealed that, although in vitro assays

are needed ERG11 may be one but probably not the only tar-

get of these compounds.

3

0

Conflicts of interest

The authors declare no competing interests.

3

1

and the Solis & Wets local search method. The initial posi-

tion, orientation, and torsions of the ligand molecules were

set randomly. All rotatable torsions were released during

Notes and references

1 P. Fernandes, Nat. Biotechnol., 2006, 24, 1497.

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2

D. Schillaci, V. Spanò, B. Parrino, A. Carbone, A.

Misco, J. G. Robertson, D. Stock, Y. Sun and A. W. Walsh,

Bioorg. Med. Chem. Lett., 2000, 10(8), 715.

18 M. Gjorgjieva, T. Tomašič, M. Barančokova, S. Katsamakas,

J. Ilaš, P. Tammela, L. Peterlin Mašič and D. Kikelj, J. Med.

Chem., 2016, 59, 8941.

19 S. Ahmed, M. F. Zayed, S. M. El-Messery, M. H. Al-Agamy

and H. M. Abdel-Rahman, Molecules, 2016, 21, 568, DOI:

10.3390/molecules21050568.

Montalbano, P. Barraja, P. Diana, G. Cirrincione and S.

Cascioferro, J. Med. Chem., 2017, 60(20), 8268–8297.

N. D. Amnerkar and K. P. Bhusari, J. Enzyme Inhib. Med.

Chem., 2011, 26, 22.

I. Apostolidis, K. Liaras, A. Geronikaki, D. Hadjipavlou-

Litina, A. Gavalas, M. Sokovic, J. Glamočlija and A. Ciric,

Bioorg. Med. Chem., 2013, 21, 532.

3

4

5

6

A. C. Tripathi, S. J. Gupta, F. Gul Naz, P. K. Sonar, A. Verma

and S. K. Saraf, Eur. J. Med. Chem., 2014, 72, 52.

G. H. Marques, A. Kunzler, V. D. Bareño, B. B. Drawanz,

H. G. Mastelloto, F. R. Leite, G. G. Nascimento, P. S.

Nascente, G. M. Siqueira and W. Cunico, Med. Chem.,

20 M. J. Edwards, R. H. Flatman, L. A. Mitchenall, C. E. Stevenson,

T. B. Le, T. A. Clarke, A. R. McKay, H. P. Fiedler, M. J. Buttner,

D. M. Lawson and A. Maxwell, Science, 2009, 326, 1415.

21 R. Prasad, A. H. Shah and M. K. Rawal, in Antifungals:

Mechanism of Action and Drug Resistance, ed. J. Ramos, et al.,

Yeast Membrane Transport, Advances in Experimental

Medicine and Biology, Springer International Publishing

Switzerland, 2016, vol. 892, p. 19.

22 C. Booth, Fungal Culture Media, in: Methods in Microbiology,

ed. J. R. Norris and D. W. Ribbons, Academic Press, London

and New York, 1971, pp. 49–94.

23 P. Geladi, H. Isaksson, L. Lindqvist, S. Wold and K.

Esbensen, Chemom. Intell. Lab. Syst., 1989, 5, 209.

24 K. D. Daouk, M. S. Dagger and J. E. Sattout, J. Food Prot.,

1995, 58, 1147.

2

014, 10, 355.

7

E. Pitta, E. Tsolaki, A. Geronikaki, J. Petrovic, J. Glamoclija,

M. Sokovic, E. Crespan, G. Maga, S. S. Bhunia and A. K.

Saxena, MedChemComm, 2015, 6, 319.

A. Gupta, R. Singh, P. K. Sonar and S. K. Saraf, Biochem. Res.

Int., 2016, 8.

8

9

M. Haroun, C. Tratrat, E. Tsolaki and A. Geronikaki, Comb.

Chem. High Throughput Screening, 2016, 19, 51.

1

0 P. Vicini, E. Fisicaro and M. T. Lugari, Archiv. Pharm.,

2

000, 333, 135.

1 P. Vicini, F. Zani, P. Cozzini and I. Doytchinova, Eur. J. Med.

Chem., 2002, 37, 553.

2 P. Vicini, A. Geronikaki, A. Kitka, M. Incerti and F. Zani,

Bioorg. Med. Chem., 2006, 14, 3859.

3 P. Vicini, A. Geronikaki, M. Incerti, F. Zani, J. Dearden and

M. Hewit, Bioorg. Med. Chem., 2008, 16, 3714.

4 A. Geronikaki, P. Eleftheriou, P. Vicini, A. Dixit, A. Saxena

and I. Alam, J. Med. Chem., 2008, 51, 5221.

25 H. Hanel and W. Raether, Mycoses, 1988, 31, 148.

26 A. Espinel-Ingroff, J. Clin. Microbiol., 2001, 39, 1360.

27 J. Trygg, E. Holmes and T. Lundstedt, J. Proteome Res.,

2007, 6, 469.

28 L. Eriksson, E. Johansson and N. Kettaneh-Wold, Wold S

Multi- and Megavariate Data Analysis, Umetrics, AB, Sweden,

2nd edn, 2006.

29 Z. Bikadi and E. Hazai, Application of the PM6 semi-

empirical method to modeling proteins enhances docking

accuracy of AutoDock, J. Cheminf., 2009, 1, 15.

30 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. T.

Hart, R. K. Belew and A. J. Olson, J. Comput. Chem.,

1998, 19(14), 1639.

5 A. Geronikaki, P. Vicini, N. Dabarakis, A. Lagunin, V.

Poroikov, J. Dearden, H. Modaresi, M. Hewitt and G.

Theophilidis, Eur. J. Med. Chem., 2009, 44, 473.

1

6 A. M. Panico, P. Vicini, A. Geronikaki, M. Incerti, V. Cardile,

L. Crasc, R. Messina and S. Ronsisvalle, Bioorg. Chem.,

2

011, 39, 48.

31 F. J. Solis and R. J. B. Wets, Math. Oper. Res., 1981, 6(1),

19.

32 O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455.

7 C. J. Andres, J. J. Bronson, S. V. D'Andrea, M. S. Deshpande,

P. J. Falk, K. A. Grant-Young, W. E. Harte, H. T. Ho, P. F.

Med. Chem. Commun.

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New: N -(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,2-benzothiazole-3-carboxamides and acetamides as antimicrobial agents

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