Appendix A. dithioloquinolinethiones as new potential multitargeted antibacterial and antifungal agents: Synthesis, biological evaluation and molecular docking studies

Accepted Manuscript Appendix A. dithioloquinolinethiones as new potential multitargeted antibacterial and antifungal agents: Synthesis, biological evaluation and molecular docking studies V. Kartsev, Khidmet S. Shikhaliev, A. Geronikaki, Svetlana M. Medvedeva, Irina V. Ledenyova, Mikhail Yu Krysin, A. Petrou, A. Ciric, J. Glamoclija, M. Sokovic PII:

S0223-5234(19)30364-2

DOI:

https://doi.org/10.1016/j.ejmech.2019.04.046

Reference:

EJMECH 11281

To appear in:

European Journal of Medicinal Chemistry

Received Date: 7 March 2019 Revised Date:

11 April 2019

Accepted Date: 16 April 2019

Please cite this article as: V. Kartsev, K.S. Shikhaliev, A. Geronikaki, S.M. Medvedeva, I.V. Ledenyova, M.Y. Krysin, A. Petrou, A. Ciric, J. Glamoclija, M. Sokovic, Appendix A. dithioloquinolinethiones as new potential multitargeted antibacterial and antifungal agents: Synthesis, biological evaluation and molecular docking studies, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/ j.ejmech.2019.04.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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Journal homepage: ht tp: //www .el se vier . com/lo cate /ejme ch

Research paper

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Dithioloquinolinethiones as new potential multitargeted antibacterial and antifungal agents: synthesis, biological evaluation and molecular docking studies V.

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V.Kartseva, Khidmet S. Shikhalievb, A. Geronikakic, Svetlana M. Medvedevab,Irina Ledenyovab,Mikhail Yu. Krysinb, A. Petrouc, A. Ciricd, J. Glamoclijad, M. Sokovicd* a

InterBioscreen, Moscow, Russia Department of organic chemistry, Faculty of chemistry, Voronezh State University, Voronezh, 394018, Russian Federation c Aristotle University, School of Pharmacy, Thessaloniki, 54124, Greece d Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research, Siniša Stanković, University of Belgrade, Bulevar Despota Stefana, Serbia b

ABSTRACT

Article history:

Herein we report the design, synthesis, molecular docking study and evaluation of antimicrobial activity of ten new dithioloquinolinethiones. The structures of compounds were confirmed by 1H-NMR, 13C-NMR

Received 00 December 00 Received in revised form 00 January 00 Accepted 00 February 00

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ARTICLE INFO

and HPLC-HRMS. Before evaluation of their possible antimicrobial activity prediction of toxicity was performed. All compounds showed antibacterial activity against eight Gram positive and Gram negative bacterial species. All compounds appeared to be more active than ampicillin and almost all than

Dithioloquinolinethiones

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Antibacterial Antifungal Molecular docking -E.coli GyrB -CYP51ca

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streptomycin. The best antibacterial activity was observed for compound 8c 4,4,8-trimethyl-5-{[(4-

Keywords:

1. Introduction

phenyl-5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)thio]acetyl}-4,5-dihydro-1H-[1,2]dithiolo[3,4c]quino lone-1-thione). The most sensitive bacterium En.cloacae followed by S. aureus, while L.monocytogenes

was the most resistant. All compounds were tested for antifungal activity also against eight fungal species. The best activity was expressed by compound 8d (5-[(4,5-Dihydro-1,3-thiazol-2-ylthio)acetyl]-4,4dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione). The most sensitive fungal was T. viride, while P. verrucosum var. cyclopium was the most resistant one. All compounds were more potent as antifungal agent than reference compound bifonazole and ketoconazole. The docking studies indicated a probable involvement of E. coli DNA GyrB inhibition in the anti-bacterial mechanism, while CYP51ca inhibition is probably responsible for antifungal activity of tested compounds. It is interesting to mention that docking results coincides with experimental.

1,2-Dithiole-3-thiones (DTTs, 1) are an interesting and unusual class of polythiaheterocyclic compounds with diverse biological activity. As

* Corresponding author. Tel.: +0-000-000-0000 ; fax: +0-000-000-0000. E-mail address: [email protected] Peer review under responsibility of Holy Spirit University of Kaslik. 2214-4234/$ – see front matter © 2013 Holy Spirit University of Kaslik. Hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.rgo.2013.10.012

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Fig 1. Structure of 1,2-dithiole-3-thiones.

Hydroquinolines (3), especially the 6-ethoxy derivative (R = H, R1 = EtO, ethoxyquine), industrially are widely used as antioxidants for polymeric materials and food products [31]. As for dithioloquinolinethiones (4), the pharmacophoric fragment of DTT in their structure is associated with the initial hydroquinoline cycle, which also is known for the diverse biological activity. In particular, in the series of simple dihydroquinoline 3 antagonists of progesterone receptors [32], mycobacterium bovis BCG [33], agonists of glucocorticoid receptors [34,35], antioxidants for neurodegenerative processes [36], substances possessing antitripanosomal activity [37] were found. Polycyclic fused heterocyclic analogs of dihydroquinoline 3 were, in contrast, agonists of progesterone receptors [38-40], as well as selective glucocorticoid modulators [41-43] and exhibited an antibacterial effect by inhibition of E. coli dehydrofolate reductase [44]. The dihydroquinoline fragment in DTT-DHQ 4 has several directions for diversifying the structure, in particular, the variation of substituents at C-8 when selecting the appropriate initial dihydroquinolines 3, production of derivatives substituted at C-6 or nitrogen, annelated at the (N)-(C-5a)(C-6) bonds (see below, Chemistry). Thus, DTT-DHQs can be considered as new promising multi-target agents [45-50] with high flexibility of structural modification. Based on literature reports regarding antimicrobial activity of DTT derivatives the purpose of this study was to see whether a series of dithioloquinolinethiones possess antibacterial and antifungal activity. Therefore, we selected 8-EtO-DTT-DHQ, obtained from commercially available ethoxyquine, as well as a number of DTT-DHQs, with substituents in the benzene ring or functionally modified acyl groups at the nitrogen atom of the quinoline core. In addition, it was interesting to study these types of biological activity for polycyclic fused dithiolo[3,4c]pyrrolo[3,2,1-ij]quinolines, for some representatives of which we previously found an inhibitory effect on blood coagulation system factor FXa [51]. All compounds were tested for antibacterial activity against some Gram positive and Gram negative bacteria, as well as against a panel of fungal strain.

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To date, it has been established that the DTT ability to show chemoprevention and cytoprotective effects is associated with activation the response via the Nrf2 – KEAP1 complex [5,6], accelerating the transcription of cytoprotective phase II enzymes [5-7]. Several classes of DTTs efficiently modulate the activities of proteins that play crucial roles in normal and cancer cells, including glutathione S-transferase, cyclooxygenases and master regulator NF-κB [5]. DTTs also exhibit anticancer effects due to inhibition of tubulin polymerization [9], inactivation of protein tyrosine phosphatase [10]. An interesting mechanism by which dithiolethiones exhibit anticancer activities is through the release of hydrogen sulfide, an important gaseous signaling molecule (gasotransmitter) in the human body [5,11,12]. DTTs as H2Sreleasing compounds, have not only anti-cancer activity, but also show analgesic activity [13], protect the cardiomyocytes from ischemic cell death [14], and prevent oxidative stress-induced cellular injury [15]. Hybrids DTTs and nonsteroidal anti-inflammatory drugs (aspirin, diclophenac) are being developed as promising drugs for the treatment of inflammatory diseases of various etiologies [16,17]. For some DTTs, as inhibitors of cyclooxygenase and lipoxygenas, the possibility of manifestation of neuroprotective activity and suppression of neuroinflammation has been established, including those observed in the pathogenesis of neurodegenerative Alzheimer's and Parkinson's diseases [18-22]. DTTs also suppresses neuroinflammation and ameliorates disease severity in experimental autoimmune encephalomyelitis [23,24]. For a number of DTT derivatives, antiretroviral activity [25], prevention of insulin resistance by inhibiting glucose production in hepatocyte-derived cell lines [26] protective effect during endotoxic shock by activation of Nrf2 [27] has been demonstrated. Recently DTT-based fibrates were synthesized and evaluated for their antihyperlipidemic and hepatoprotective potentials [28]. Antimicrobial activity of DTT was also shown against Staphylococcus aureus [29]. It should be noted that the above-mentioned numerous studies of the DTT biological activity were carried out exclusively for monocyclic DTT. 4,5-Dihydro-4,4-dimethyl-1H-[1,2]dithiolo[3,4-c]quinolon e-1-thiones (DTT-DHQ, 4), in which DTT's and hydroquinoline cycles are annulated to bond c of the latter, were synthesized by Brown according to the reaction of the corresponding 1,2-dihydro-2,2,4-trimethylquinolines (3) with sulfur [30].

Scheme 1. Synthesis of [1,2]dithiolo[3,4-c]quinoline-1-thiones (4).

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protective phytochemicals, they have been isolated from cruciferous vegetables [1]. Oltipraz (4-methyl-5- (2-pyrazinyl) -1,2-dithiole-3-thione 2) was the first DTT derivative introduced into the clinical practice as antishistosomal agent against Schistosoma mansoni [2]. Further studies have shown that oltipraz and other simple DTTs also exhibit a chemoprevention action and can be used in the treatment of cancer [3-5].

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2. Results and discussion 2.1. Prediction of Toxicity The prediction of toxicity of compounds is very important step in design and development of new drug candidates. More rapid and less expensive process is to use in silico toxicity study instead of in vivo toxicity testing in animals, since it is helpful to significantly decrease the number of animals for the experiment. Several online programs were developed using in silico models to access toxicity, that predict average lethal dose, carcinogenicity, mutagenicity etc. Two computer programs, ToxPredict (OPEN TOX) and PROTOX, were used in this work 52-54. The ToxPredict program predicts the probability of carcinogenicity of the compounds in various organisms, as well as the probability of mutagenesis using an in silico model corresponding to the Ames test. The results are presented in Table 1(SI). It should be mentioned that the accuracy of prediction increases as the confidence values rises. In particular, reliable estimates are considered to be more than 0.025.

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The general routes for the synthesis of the starting compounds and the target compounds are shown in Schemes 2, 3, respectively. By condensation of the corresponding arylamines 5a-d with acetone in the presence of iodine as catalyst by the known procedure [57] substituted on the aromatic ring of 2,2,4-trimethyl-1,2-dihydroquinolines 3a-d were prepared. From the obtained dihydroquinolines 3a-d by refluxing in dimethylformamide with a 5-fold excess of sulfur according to the previously described method [30], the corresponding 4,4-dimethyl-4,5dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thiones 4a-d were synthesized (Scheme 1). Further molecular design of all condensed dithiolothiones 4ad was carried out using the methods developed earlier by us [58-62] based on the reactions of electrophilic reagents for hydrogen atoms in position 6 of the phenyl ring or/and the secondary amino group of the dihydroquinoline ring. By nitration of compound 4c with acetyl nitrate in

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2.2. Chemistry

chloroform under mild conditions, the 6-nitro-[1,2]dithiolo[3,4c]quinoline-1-thione 6 was synthesized [58]. By interaction of dithioloquinolines 4a-c with various carbonyl chlorides (including chloroacetyl chloride) by refluxing in toluene N-acyl-[1,2]dithiolo[3,4c]quinoline-1-thiones 7a-e [59] were obtained. By acylation of dithioloquinolines 4a-d with chloracetyl chloride intermediate Nchloroacetyl-derivatives 7с-e were synthesized. These compounds, containing a mobile chlorine atom, were further used to alkylate succinimide and some heterocyclic mercaptans to form N-heterylacetyl[1,2]dithiolo[3,4-c]quinoline-1-thione 8a and N-(heterylthio)acetyl[1,2]dithiolo[3,4-c]quinoline-1-thiones 8b-d, respectively [60,61]. By refluxing dithioloquinolines 4a,c with oxalyl chloride in dry toluene intermediate pyrrolo[3,2,1-ij]quinoline-1,2-diones 9a,b [62 ] were obtained, which were underwent selective condensation with thiosemicarbazide or rhodanine leading to 1,2-dithiolo[3,4c]pyrrolo[3,2,1-ij]quinoline-4,5-dione 4-thiosemicarbazone 10a and 4-(4oxo-2-thioxo-1,3-thiazolidin-5-ylidene)-[1,2]dithiolo[3,4-c]pyrrolo[3,2,1ij]quinolin-5(4H)-one 10b, respectively.

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Another program used is PROTOX [55], predicting the average lethal dose (LD50) in rodents. According to this program all chemical compounds can be classified into six Globally Harmonized System of Classification and Labeling of Chemicals(GHS) categories,[56] depending on the toxicity of the compounds and the LD50 values (Table 2SI). Category I: LD50 ≤ 5 mg/kg Category II: 5 < LD50 ≤ 50 mg/kg Category III: 50 < LD50 ≤ 300 mg/kg Category IV: 300 < LD50 ≤ 2000 mg/kg Category V: 2000 < LD50 ≤ 5000 mg/kg Category VI: LD50 > 5000 mg/kg All compounds tested except of one were in category IV.

i

NH2

5a-d

S ii

N H 3a-d

S S

R N H 4a-d

R = H(a), Me(b), MeO(c), EtO(d).

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Scheme 2. Synthesis of the key intermediates (4a-d). Reagents and conditions: (i) (CH3)2CO, I2, 175 ºC, 5 h; (ii) elemental sulfur, DMF, reflux, 15 h.

Scheme 3. Synthesis of the key intermediates (7c-e and 9a,b) and target compounds (6, 7a,b, 8a-d, and 10a,b). Reagents and conditions: (a) CHCl3, fuming HNO3, (CH3CO)2O, 0 ºC, 0.5 h + 20 ºC, 1 h; (b1) dry toluene, R-carbonyl chloride, reflux, 8 h; (b2) dry dioxane, K2CO3, succinimide or various heterocyclic mercaptans, 50 ºC, 4 h; (c1) dry toluene, oxalyl chloride, reflux, 2 h; (c2); butanol-1, thiosemicarbazide or rhodanine, reflux, 4 h. The structures of compounds were confirmed by 1H-NMR, 13C-NMR and HRMS (See experimental part)

The structure of compounds 4d, 6, 7a,b, 8a-d, and 10a,b were unambiguously confirmed by 1H and 13C NMR spectroscopy and HPLC–

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3. Biological evaluation 3.1. Antibacterial activity

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of the carbonyl group of thiazolinone was closely to C(7)-H and thus Zconfiguration was confirmed. The 13C NMR spectrum of compounds 6 could not be acquired of good quality due to the low solubility in DMSO. In the spectra of compounds 4d, 7a,b, 8a-d, and 10a,b there were characteristic signals of the thiocarbonyl carbon atom at 210.7-211.1 ppm. In the mass spectra of compounds 4d, 6, 7a,b, 8a-d, and 10a,b, peaks of protonated molecular ions were observed in accordance with their elemental composition.

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Compounds 4d, 6, 7a,b, 8a-d, 10a,b were tested for antibacterial activity against four Gram positive and four Gram negative bacteria (Table 1). From the obtained results it is obvious that all compounds showed antibacterial activity. The antibacterial potency could be presented in the following order: 8c > 4d>8d >7b> 8a > 10a> 8b >7a > 10b >6. The best antibacterial potency against all bacteria tested was observed for compound 7a with minimal inhibitory (MIC) and minimal bactericidal concentration (MBC) at 2.75 µΜ x 10-2 and 5.50 mmol/ml x 10-2 respectively. The lowest activity was possessed by compound 6 with MIC in the range of 4.41-17.63 µΜ x 10-2 and MBC at 8.81-35.25 µΜ x 10-2. It should be mentioned that bacteria showed almost the same sensitivity to compounds tested with very small differences. Thus the antibacterial potency against M.flavus, S.aureus and S.typhimirium can be presented as follows: 8c >4d > 8d > 7b > 8a > 10a > 10b > 8b > 7a > 6, while against L. monocytogenes and P.aeruginosa as: 8c >4d > 8d > 7b > 8a > 10a > 8b > 7a> 6 > 10b. E.coli and En.cloacae showed the same sensitivity to compounds tested which is the following: 7 >4d > 8d > 7b > 8a > 10a > 10b> 8b> 7a > 6. A little different is the antibacterial potency of compounds against B.cereus 8c >4d > 8d > 7b > 8a > 10a > 6 > 10b> 8b > 7a. The general observation is that compounds 4d, 7b, 8a, 8d, and 10a showed the same sensitivity to all bacteria tested. Differences were observed in the potency of compounds 6, 7a, 8c and 10b.

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HRMS-ESI analyses. In 1H NMR spectra of all compounds, the signals of gem-dimethyl protons appeared in their respective fields – at 1.4-2.1 ppm. The signals of the proton C(9)-H were shifted to a weak field (8.2–9.5 ppm) due to the anisotropic effect of the thioketo-group. The signals of other quinoline protons were observed in the aromatic field [57]. For the compound 4d singlet of the hydroquinolinic NH proton was in the characteristic region at 6.0 ppm, while in the spectrum of nitro-derivative 6, the downfield chemical shift (7.8 ppm) of this proton was observed. Another characteristic feature of the spectrum of compound 6 was the splitting of the aromatic protons signals into two doublets with the characteristic for meta-protons spin coupling constant 2.9 Hz [58]. Compared with the spectra of the starting dithiolothiones 4a-d, in the spectra of compounds 7a, b, 8a-d and 10a,b the signals of the NH-proton were disappeared. For compounds 10a,b in the aromatic protons region one of the signals was also missing due to the closure of the pyrrolinedione cycle. In the spectra of compounds 7a, b, and 8a-d, signals of the corresponding N-acyl- or N-heteryl(thio)acetyl fragments appear in characteristic regions [59,61 ]. Furthermore, in the spectra of compounds 8a–d the signals of two protons of the substituted acetyl fragment appeared as singlets at 3.9–4.3 ppm [61 ]. A characteristic feature of the spectra of compound 10a was the presence of a pair of signals of protons of the C(S)NH2 group at 7.6 and 8.7 ppm, which were not equivalent due to hindered rotation through the thioamide bond. Furthermore, the signal offset of the proton NH to the low field region (up to 12.3 ppm) was observed due to the intramolecular hydrogen bond of this proton with the oxygen atom. Analysis of the 1H NMR spectrum showed that compound 10b was in the form of a mixture of E- and Z-isomers with different arrangement of the carbonyl groups of the thiazolidinone and pyrrolone fragments relative to the exocyclic double bond. Moreover, the Z-isomer was the main product. In the spectrum of this compound, the broadened singlet of the NH proton is observed in the low-field region at 13.8-14.4 ppm. In the aromatic protons region two sets of signals of protons C(7)-H (at 7.1 and 8.3 ppm) and C(9)-H (at 9.3 and 9.4 ppm) with the integral intensity ratios approximately 2 : 1 were observed. In this case, the signal with a higher intensity corresponded to a proton C(7)-H was observed in a rather weak-field region of 8.3 ppm. This indicated that the oxygen atom

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Table 1. Antibacterial activity (MIC/MBC in µΜ x 10-2) R.br Structure S

AC C N H

S

S.a

L.m

En.cl

P.a

S.t

E.coli

MIC

2.95

2.95

2.95

2.95

2.95

2.95

2.95

2.95

MBC

5.90

5.90

5.90

5.90

5.90

5.90

5.90

5.90

MIC

4.41

13.22

8.81

17.63

8.81

8.81

17.63

13.22

MBC

8.81

17.63

17.63

35.25

17.63

17.63

35.25

17.63

MIC

1.21

1.21

0.03

0.03

0.03

0.03

0.03

1.21

MBC

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

S

S

6

M.f

S

O

4d

B.c

NH N

O

+

O

O

S

S

S

7a

N

N O

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S

S

S

O

7b

MIC

3.04

3.04

3.04

3.04

3.04

3.04

3.04

3.04

MBC

6.09

6.09

6.09

6.09

6.09

6.09

6.09

6.09

MIC

3.71

3.71

3.71

3.71

3.71

3.71

3.71

3.71

MBC

7.42

7.42

7.42

7.42

7.42

7.42

7.42

7.42

MIC

10.60

10.60

10.60

10.60

7.07

7.07

10.60

10.60

MBC

14.13

14.13

14.13

14.13

14.13

14.13

14.13

MIC

2.75

MBC

5.50

MIC

2.98

MBC

5.97

N Cl O +

N O O

N

8a

O

O

N O

S

N

O

8b S

N

S S S

N

N N

8c

S S

S O

S S S

8d

N N

S

S

O

O

S

N O

10a N H N S

S

S

S

N

strep tomy cin

2.75

2.75

2.75

2.75

5.50

5.50

5.50

5.50

5.50

5.50

5.50

2.98

2.98

2.98

2.98

2.98

2.98

2.98

5.97

5.97

5.97

5.97

5.97

5.97

5.97

3.82

3.82

3.82

3.82

3.82

3.82

3.82

3.82

MBC

7.64

7.64

7.64

7.64

7.64

7.64

7.64

7.64

MIC

6.46

9.69

6.46

25.83

6.46

12.91

6.46

9.69

MBC

12.91

12.91

12.91

25.83

12.91

12.91

MIC

24.80

24.80

24.80

37.20

74.40

37.20

24.80

74.40

MBC

37.20

37.20

37.20

74.40

124.00

49.20

38.00

124.00

MIC

4.30

8.60

17.20

25.80

27.00

17.20

4.30

27.00

MBC

8.60

17.20

34.40

51.60

34.40

34.40

8.60

34.40

O

S

O

ampi cillin

2.75

O

AC C

10b

2.75

MIC

EP

H 2N

2.75

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S S

14.13

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S S

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S S S

N H

S

51.66

12.91

B.c.-B.cereus, M.f.-M.flavus, S.a.-S.aurues, l.m.L.monocytogenes, E.c.-E.coli, En.c.-En.cloacae, P.a.-P.aeruginosa, S.t.-S.typhimurium. Relative standard deviations were all < 2.0 The most sensitive bacterium appeared to be En.cloacae followed by respectively, whereas for Gram-negative bacteria the MIC and MBC S. aureus, while L.monocytogenes was the most resistant. ranged from 2.75-17.63 × 10-2 milliM, and 5.50 to 35.25 × 10-2 milliM In particular, for the Gram-positive bacteria the range of MIC and respectively. It seems that the tested compounds are more potent against MBC was 2.75-25.83 × 10-2 milliM and 5.50-51.66 × 10-2 milliM Gram negative bacteria than against Gram positive.

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3.2. Antifungal activity All compounds were tested for their antifungal activity against a panel of fungal strain (Table 2).The minimum inhibitory concentration (MIC) was in range of 0.4-25.83 milliM x 10-2 and minimum fungicidal 0.851.65 milliM x 10-2. The order of antifungal activity could be presented as followes: 8d > 6> 4d > 8b > 7b > 8c > 7a > 8a > 10a > 10b. The most active compound appeared to be compound 2 with MIC at 0.4-1.59 milliM x 10-2 and MFC 0.8-2.98 milliM x 10-2, while compound 10b was the less active with MIC and MFC at 1.72-25.83 milliM x 10-2 and 3.2351.65 milliM x 10-2 respectively.

Table 2. Antifungal activity (MIC/MFC in milliM x 10-2)

7a 7b 8a 8b 8c 8d 10a

A.n 4.32 11.79 4.41

MFC MIC

35.25 7.80

8.81 7.80

4.41 14.63

8.81 1.56

MFC MIC MFC MIC MFC MIC

15.60 4.46 12.17 11.12 14.83 3.53

15.60 12.17 24.34 14.83 29.67 3.53

31.21 1.62 3.04 14.83 29.67 1.88

MFC MIC

7.06 8.24

7.06 21.99

MFC MIC MFC MIC

21.99 1.59 2.98 7.64

MFC

15.23

ketoconazo le bifonazole

T.v 1.18 2.95 3.23

P.o 0.79 1.57 4.41

P.f 11.79 23.59 4.41

Pvc 8.85 23.59 6.46

4.41 1.04

8.81 7.80

8.81 15.60

17.62 7.80

7.80 2.23 6.09 7.42 14.83 3.53

2.08 1.22 1.62 1.48 1.98 1.88

15.60 1.22 1.62 0.99 1.98 3.53

31.21 3.04 6.09 11.12 29.67 3.53

15.60 24.34 48.68 14.83 29.67 3.53

3.53 1.47

7.06 2.02

3.53 1.1

7.06 2.75

7.06 2.75

7.06 21.99

43.97 1.59 2.98 11.46

2.75 1.59 2.98 15.23

5.50 1.59 2.98 15.23

1.47 0.80 1.59 1.52

5.50 0.40 0.8 3.82

5.50 1.59 2.98 11.46

43.97 1.59 2.98 11.46

30.57

SC

A.o 11.79 23.59 2.35

30.57

30.57

3.82

7.64

30.57

30.57

MIC

25.83

12.91

1.72

25.83

1.72

3.23

12.91

25.83

MFC

51.65

25.83

3.23

51.65

3.23

6.46

25.83

51.65

MIC MFC

38.0 95.0

285. 380.

38.0 95.0

38.00 95.00

475.00 570.00

38.0 95.0

380. 380.

38.00 57

MIC MFC

48.0 64.0

48.0 64.0

48.0 80.0

48.00 64.00

64.00 80.00

64.0 80.0

48.0 64.0

48 64

AC C

10b

A.v 5.90 11.79 4.41

M AN U

6

A.f 5.90 11.79 17.62

EP

4d

MIC MFC MIC

TE D

Comp/ds

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It should be noticed that in general all compounds showed better activity than ampicillin and all compounds except of 7a and 10b against B.cereus, M flavus and L.monocytogenes than streptomycin. The study of structure-activity relationships revealed that the presence of 4-phenyl-5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioacetyl part in quinoline moiety nitrogen of (8c) was very beneficial for antibacterial activity. 1,3-Thiazol-2-ylthio)acetyl unit (8d) was also favorable for activity. In the presence of 4-chloro-3-nitrobenzoyl group (7b) activity decreased. Replacement of the latter by 2-(2,5-dioxopyrrolidin-1-yl)acetyl fragment as well as removal of 8-ethoxy group on benzene ring led to compound 8a with moderate activity. The presence of 6-NO2 group in 8methoxy-4,4 dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1thione 6 was detrimental causing negative effect to antibacterial activity.

A.fum.-A.fumigatus, A.v.-A.versicolor, A.o.-A.ochraceus, A.n.-A.niger, T.v.-T.viride, P.f.-P.funiculosum, P.o.-P.ochrochloron, C.a.-C.albicans, P.v.c.P.cyclpoium var verucosum. Despite the fact that all compounds appeared to be active against all fungi tested, the sensitivity of the fungi was different. No similarity in activity of compounds tested against fungi species was not observed in the contrary with bacteria. Thus, the order of activity against T. viride , the most sensitive fungal can be presented as : 8d >8c > 7b > 7a > 8a >4d >10b > 10a > 8b > 6, while against P.v.c. , the most resistant fungal as: 8d > 8b > 7a >6 > 4d > 10a > 8a > 8c > 7b > 10b. The sensitivity of A. versicolor, the second most resistant fungal to compounds tested have an order 8d > 8b > 6 > 4d > 7a > 7b > 10b > 10a >8a >8c. Compounds (4d,

7b, 8d), (7a, 8b, 8c, 10a, 10b) exhibited good activity against T.viride with MIC milliM and MFC at 0.80-1.88 x 10-2 and 1.59-3.82 milliM x 10-2 respectively, while some of them (4d, 7b, 8a, 8d) with MIC and MFC at 0.40-1.22 milliM x 10-2 and 0.80-1.98 milliM x 10-2 respectively were also potent against P. ochrochloron . Good activity was also shown by compound 8b and 8c against A. ochraceus with MIC at 1.47-1.88 milliM x 102 and MFC at 2.75-3.53 milliM x 10-2 . The most sensitive fungal was T. viride, while P. verrucosum var. cyclopium was the most resistant one.

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4. Molecular docking 4.1. Docking to antibacterial targets

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To evaluate the antibacterial mechanism of compounds action, molecular docking studies were performed in the active sites of the enzymes E. coli DNA GyrB, DNA Topoisomerase IV and Thymidylate kinase. Docking studies revealed that the Free energy of Binding to DNA TopoIV and Thymidylate kinase were higher than that to E. coli DNA GyrB. Therefore, it may be concluded that E. coli DNA GyrB is probably the enzyme involved in the mechanism of antibacterial activity. (Table 3). Furthemore, the docking results are coincide with experimental.

Table 3 Molecular docking binding affinities to antibacterial targets.

4d 6

Binding affinity score E. coli DNA GyrB

M AN U

N/N

Est. binding energy (kcal/mol) E. coli DNA Thymidyl DNA topoIV ate kinase GyrB PDB ID: PDB ID: PDB ID: 1S16 4QGG 1KZN -6.17 -4.02 -11.59

SC

The structure-activity relationships revealed that the presence of 1,3thiazol-2-ylthio)acetyl fragment on the of quinoline moiety nitrogen (8d) was favorable for antifungal activity. In the contract with antibacterial activity the presence of 6-NO2 group on 8-methoxy-4,4 dimethyl-4,5dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione (6) had positive influence on activity. Also good activity was shown by 8-ethoxy-4,4dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione (4d), unsubstituted at the nitrogen atom. It should be mentioned that compound 8d exhibited very good antibacterial activity as well. Good influence to antifungal activity was observed also for 2-((4,5-dihydrothiazol-2yl)thio)acetyl derivative (8b) while 2-thioxothiazolidin-5-ylidene group in dithiolo[3,4-c]pyrrolo[3,2,1-ij]quinolinone (10b) had negative effect on activity. Recently, a similar dual antimicrobial and antifungal effect was discovered by us for a number of adamantane derivatives containing linear and condensed thiazole, thiadiazole or triazole cycles [63, 64].

I-H

Residues E. coli DNA GyrB

-34.85

3

Asn46, Arg136, Gly117

-6.14

-22.97

-

-

-2.28

-3.22

-7.15

-26.66

1

Asn46

7b

-4.17

-3.12

-10.55

-32.18

3

Asn46, Asp73, Gly117

8a

-4.07

8b -6.86

8d

-6.00

10a

-3.16

10b

-2.78

-9.46

-31.13

2

Asn46, Gly117

-2.66

-8.63

-28.41

2

-4.58

-12.26

-36.51

4

-3.19

-10.88

-32.29

3

Asp73, Arg136 Asn46, Arg76, Arg136, Gly117 Asn46, Asp73, Gly117

-8.78

-28.45

2

Asn46, Asp73

-7.02

-24.13

1

Asn46

EP

8c

TE D

7a

-2.11

AC C

The most active compound 8c binds to E. coli DNA GyrB throughout four favorable H-bonding interactions. The first one between the O atom of carboxyl group of the compound and the H atom of the side chain of Asn46 (distance 2.98 Å), the second one between the exocyclic S atom of 1,3,4-thiadiazole ring and the O of the side chain of Gly117 (distance 3.54 Å), another one H-Bond between the S atom of fused rings and the H

of the side chain of Arg76 (distance 3.19 Å) and the last one between the S atom of C=S group of 1,2-dithiole and the H of the side chain of Arg136 (distance 3.75 Å). The fused rings form hydrophobic contacts with Glu50, Asp73, Gly77, Pro79, Ile90, Ile78 and Thr165, while the benzene ring interacts hydrophobically with Asp45 and Asp49 (Fig 2A2).

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EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY 100 (2019) 000–000

Fig. 2. (A1-2) Docked conformation of the most active compound 8c in E. coli DNA GyrB.

EP

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M AN U

SC

It is remarkable to say that compound 8c binds DNA GyrB in a way that it is similar to clorobiocin, showing the same H-bonding interactions with the residues Asn46 and Arg136 (fig. 3). Furthermore, compound 8c binds deep in the active centre of the enzyme forming hydrogen bonds just like clorobiocin (fig. 4) indicating its high inhibitory level.

AC C

Fig. 3: Docked conformation of Clorobiocin in E. coli DNA GyrB.

Fig. 4: Docked conformation of the most active compound 8c (green) and Clorobiocin (yellow) in E. coli DNA GyrB.

Docking to antifungal targets As we don’t know the exact mechanism of antifungal action of the compounds, molecular docking studies were performed in the active sites of the enzymes 14-alphademethylase of C. albicans (CYP51ca), alpha-beta tubulin and dihydrofolate reductase. Docking studies revealed that the Free energies of Binding to dihydrofolate reductase and alpha-beta tubulin were higher than that to CYP51ca. Thus, probably inhibition of CYP51ca is the most appropriate mechanism of antifungal activity of compounds tested.

Table 4. Molecular docking binding affinities to antifungal targets. Est. binding energy (kcal/mol) N/N

DNA topoIV PDB ID: 1S16

Alphabeta Tubulin: PDB ID: 1JFF

CYP51 of C. albicans: PDB ID: 5V5Z

Binding affinity score CYP51ca

I-H

Residues CYP51ca

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-5.05

-7.02

-8.76

-26.49

1

Met508

6

-5.24

-8.54

-9.13

-28.46

1

Tyr64

7a

-2.31

-6.11

-6.21

-22.41

-

-

7b

-3.28

-6.63

-7.69

-25.44

1

Met508

-5.42

-6.14

-22.15

-

-

-7.15

-8.17

-26.03

1

Tyr131

-6.35

-7.55

-25.41

1

Met508

-8.77

-10.25

-31.42

1

Tyr131

-3.94

-5.49

-20.73

-

-

-2.56

-5.12

-20.29

-

-

-8.87

-29.46

1

Tyr64

8b

-4.05

8c 8d

-6.12

10a 10b

-1.88

Ket.

compound and the H of the side chain of the residue Tyr131 (distance 2.11 A). Hydrophobic interactions between the residues Ile377, Ph234, Leu310, Tyr315 and the fused rings of the compound 2 were detected. The benzothiazole core interacts also hydrophobically with the residues The139, Ala144, Vla143, Leu159, Phe152 and Ala1311.

AC C

EP

TE D

M AN U

Docking results revealed that all the synthesized compounds may bind to CYP51Ca in a way that it is similar to the binding of ketoconazole. Compound 2 take place inside the enzyme alongside to heme group, forming a plenty of positive ionisable interactions between the heme of CYP51Ca and the benzothiazole ring of the compound. An H-bonding interaction was observed between the O of -OCH3 substituent of the

SC

8a

RI PT

4d

Fig. 5: Docked conformation of ketoconazole in lanosterol 14alpha-demethylase of C. albicans (CYP51ca).

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10

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Fig. 6: Docked conformation of compound 2 in lanosterol 14alpha-demethylase of C. albicans (CYP51ca). Interaction with the heme group was also observed with the benzene ring of ketoconazole which forms positive ionisable interactions (fig. 5 and 6). However, compound 2 forming more stable complex of ligand with enzyme due to its plenty van der Walls interactions. This is probably the reason why compound 2 have better free energy of binding, and as a consequence, better antifungal activity than ketoconazole. 5. Experimental

TE D

5.1. Chemistry

AC C

EP

5.1.1. General Melting points were determined on a PTP-M apparatus. The 1H and 13 C NMR spectra were recorded on a Bruker DRX-500 spectrometer in DMSO-d6 at 500 and 125 MHz, respectively. TMS was used as the internal standard. HPLC–HRMS analyses were performed on an Agilent Infinity 1260 liquid chromatograph equipped with an Agilent 6230 TOF mass selective detector. The conditions of chromatographic separation were the following: mobile phase 0.1% formic acid in MeCN (eluent А) / 0.1% formic acid in water (eluent В), gradient 0–100%: А, 3.5 min, 50%; А, 1.5 min, 50–100%; В, 3.5 min, 50%; В, 1.5 min, 50–0%; flow rate 0.4 ml/min, column – Poroshell 120 EC-C18 (4.6 × 50 mm, 2.7 µm), thermostat at 28°С, electrospray ionization (ESI, capillary voltage –3.5 kV; fragmentor voltage +191 V; OctRF +66 V – positive polarity). The reactions were monitored and the purity of the products was checked by TLC with Merck TLC Silica gel 60 F254 plates using chloroform as eluent. The solvents were purified according to standard methods. Commercially available reagents from Lancaster were used in the syntheses. 5.1.2. Synthesis of compounds 3a-d, 4a-d, 7c-e and 9a,b. Compounds 3a-d, 4a-c, 7c-e and 9a,b were prepared according to the literature procedure [30, 57, 59 , 60, 62,]. 5.1.2.1. Synthesis of 8-Ethoxy-4,4-dimethyl-4,5-dihydro-1H[1,2]dithiolo[3,4-c]quinoline-1-thione 4d. A mixture of quinoline 3d (50mmol) and elemental sulfur (250 mmol) in DMF (30 ml ) was refluxed for 15 h. The reaction mixture was poured into water, and the precipitate was recrystallized from toluene to furnish the desired product 4d.

Orange powder (yield 15.45 g, 63%), m.p. 152-153 °C (lit. m.p. 153154 °C [30]); 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.31 (t, J = 6.96 Hz, 3H, CH3), 1.49 (br s, 6H, (CH3)2), 3.93 (q, J = 6.96 Hz, 2H, CH2), 6.01 (s, 1H, NH), 6.76 (d, J = 8.63 Hz, 1H, H-6(7) quinoline), 6.80 (d, J = 8.63 Hz, 1H, H-7(6) quinoline), 8.93 (s, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 14.9, 27.2, 55.9, 63.3, 108.2, 115.5, 116.6, 118.5, 134.2, 136.9, 150.0, 177.5, 211.4; HPLC-HRMS (ESI) calcd for C14H15NOS3 +H+, 310.0389; found, 310.0394. 5.1.3. Synthesis of 8-Methoxy-4,4-dimethyl-6-nitro-4,5-dihydro-1H[1,2]dithiolo[3,4-c]quinoline-1-thione 6. To the starting compound 4c (5 mmol), dissolved in chloroform (3 ml), acetyl nitrate previously prepared from acetic anhydride (0.1 ml) and fuming HNO3 (d = 1.48) (0.06 ml) in the chloroform (5 ml) solution was added dropwise with stirring at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then at 20 °C for 1 h until the complete conversion as monitored by TLC. After that, mixture was diluted with cold water (10 ml). The organic layer was separated, washed with solution Na2CO3 and water. The chloroform was distilled off and the residue was crystallized from toluene to furnish the desired product 6. Red powder (yield 0.95 g, 56%), m.p. 217-218 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.65 (s, 6H, (CH3)2), 3.77 (s, 3H, CH3O), 7.47 (s, 1H, H-7 quinoline), 7.82 (s, 1H, NH), 8.21 (s, 1H, H-9 quinoline); 13 C NMR (DMSO-d6, 125 MHz) δ ppm: 28.4, 55.6, 55.7, 106.6, 118.0, 119.7, 120.8, 131.9, 134.4, 149.6*; HPLC-HRMS (ESI) calcd for C13H12N2O3S3+H+, 341.0084; found, 341.0081. *The 13C NMR spectra of compounds 6 could not be acquired of good quality due to the low solubility in DMSO. 5.1.4. General procedure for synthesis of substituted N-acyl[1,2]dithiolo[3,4-c]quinoline-1-thione 7a,b To a solution of the starting compounds 4b, d (5 mmol) in dry toluene (10 ml) a solution of corresponding acylchloride (5.5 mmol) in toluene (10 ml) was added dropwise under cooling . The reaction mixture was refluxed 8 h to complete dissolution of S-methylthiolium salt monitored by TLC. Toluene was distilled off under reduced pressure, the precipitate was filtered and recrystallized from toluene to furnish the desired products 7a, b.

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5.1.5.4. 5-[(4,5-Dihydro-1,3-thiazol-2-ylthio)acetyl]-4,4-dimethyl-4,5dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione (8d). Orange powder (yield 1.86 g, 74%), m.p. = 160-161 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.70 (br s, 6H, (CH3)2), 3.82 (s, 3H, CH3), 4.28 (s, 2H, CH2), 7.05 (d, J = 7.61 Hz, 1H, H-Ar); 7.33 (t, J = 7.61 Hz, 1H, H-Ar); 7.41 (t, J = 7.61 Hz, 1H, H-Ar); 7.53 (d, J = 8.39 Hz, 1H, H6(7) quinoline); 7.65 (d, J = 8.39 Hz, 1H, H-7(6) quinoline); 7.89 (d, J = 7.61 Hz, 1H, H-Ar); 8.82 (s, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 26.2, 55.5, 61.8, 108.9, 114.3, 121.0, 121.6, 124.54, 126.3, 127.1, 127.3, 128.7, 134.5, 134.7, 152.5, 157.3, 165.7, 168.4, 181.1, 211.2; HPLC-HRMS (ESI) calcd for C22H18N2O2S5+H+, 503.0046; found, 503.0040.

M AN U

5.1.4.2. 5-(4-Chloro-3-nitrobenzoyl)-8-ethoxy-4,4-dimethyl-4,5-dihydro1H-[1,2]dithiolo[3,4-c]quinoline-1-thione (7b). Orange powder (yield 2.0 g, 81%), m.p. 163-164 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.30 (t, J = 6.96 Hz, 3H, CH3), 1.92 (br s, 6H, (CH3)2), 3.95 (q, J = 6.96 Hz, 2H, CH2), 6.67-6.72 (m, 2H, H-Ar), 7.47 (d, J = 8.42 Hz, 1H, H-6(7) quinoline), 7.70 (d, J = 8.42 Hz, 1H, H6(7) quinoline), 8.13 (s, 1H, H-Ar);8.88 (s, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 14.6, 25.4, 62.1, 63.4, 108.8, 114.5, 124.7, 126.8, 127.4, 127.6, 128.8, 131.6, 133.7, 134.0, 137.0, 147.3, 155.2, 155.3, 166.4, 179.9, 211.5; HPLC-HRMS (ESI) calcd for C21H17ClN2O4S3+H+, 493.0113; found, 493.0112.

4.11 (br s, 2H, CH2CO), 7.23 (d, J = 8.25 Hz, 1H, H-6(7) quinoline); 7.43 (d, J = 8.25 Hz, 1H, H-6(7) quinoline) 7.49 (t, J = 7.29 Hz, 1H, H-Ar), 7.77 (t, J = 7.29 Hz, 2H, H-Ar), 7.65 (d, J = 7.29Hz, 2H, H-Ar), 8.96 (s, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 20.1, 25.9, 39.7, 39.8, 40.2, 61.8, 123.4, 125.7, 125.9, 126.0, 129.1, 129.3, 129.8, 133.0, 134.4, 135.9, 138.0, 155., 168.2, 180.3, 185.5, 211.1; HPLCHRMS (ESI) calcd for C23H19N3OS6+H+, 545.9926; found, 545.9925.

SC

5.1.4.1. 4,4,8-Trimethyl-5-(pyridin-3-ylcarbonyl)-4,5-dihydro-1H[1,2]dithiolo[3,4-c]quinoline-1-thione (7a). Orange powder (yield 1.4 g, 73%), m.p. 177-178 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.85 (s, 6H, (CH3)2), 2.23 (s, 3H, CH3), 6.61 (d, J = 8.12 Hz, 1H, H-6(7) quinoline); 6.88 (d, J = 8.12 Hz, 1H, H6(7) quinoline) 7.36 (dd, d, J1 = 4.82 Hz, J2 = 1.65 Hz 1H, H-Ar), 7.77 (d, J = 7.96 Hz, 1H, H-Ar), 9.04 (s, 1H, H-Ar), 8.55 (d, J = 4.82 Hz, 1H, HAr) 9.04 (s, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm:20.7, 25.4, 61.9, 123.1, 123.4, 123.5, 126.4, 129.4, 132.4, 133.6, 134.0, 134.2, 137.2, 150.2, 151.6, 168.2, 179.3, 211.5; HPLC-HRMS (ESI) calcd for C19H16N2OS3+H+, 385.0498; found, 385.0496.

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5.1.5. General procedure for synthesis of substituted N-heterylacetyl[1,2]dithiolo[3,4-c]quinoline-1-thione 8a and N-(heterylthio)acetyl[1,2]dithiolo[3,4-c]quinoline-1-thione 8b-d. A mixture of N-chloroacetyl derivatives 7c-e (5 mmol), K2CO3 (11 mmol) and succinimide or the corresponding mercaptans (5mmol) in dry dioxane (30 ml ) was stirred at 40-50 ° C for 4 h until the complete conversion as monitored by TLC. The reaction mixture was poured into water, and the precipitate was recrystallized from toluene to furnish the desired products 8a-d.

AC C

EP

5.1.5.1. 1-[2-(4,4-Dimethyl-1-thioxo-1,4-dihydro-5H-[1,2]dithiolo[3,4c]quinolin-5-yl)-2-oxoethyl]pyrrolidine-2,5-dione (8a). Orange powder (yield 1.67 g, 83%), m.p. > 300 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.72 (br s, 6H, (CH3)2), 2.49-2.89 (m, 4H, CH2CH2), 4.12 (s, 2H, CH2CO), 7.40 – 7.46 (m, 2H, H-7, H-8 quinoline), 7.60 (d, J = 7.51 Hz, 1H, H-6 quinoline) 9.12 (d, J = 7.51 Hz, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 25.8, 27.8, 35.9, 42.8, 61.6, 123.0, 125.6, 126.1, 126.4, 129.7, 134.3, 135.0, 167.7, 176.6, 180.7, 211.1; HPLC-HRMS (ESI) calcd for C18H16N2O3S3+H+, 405.0397; found, 405.0397. 5.1.5.2. 5-[(4,5-Dihydro-1,3-thiazol-2-ylthio)acetyl]-4,4-dimethyl-4,5dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione (8b). Orange powder (yield 1.84 g, 87%), m.p. = 153-154 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.70 (br s, 6H, (CH3)2), 3.34 (t, J = 7.86 Hz, 2H, CH2), 3.85 (t, J = 7.86 Hz, 2H, CH2), 3.95 (s, 2H, CH2CO), 7.40 – 7.46 (m, 3H, H-6, H-7, H-8 quinoline), 9.13 (d, J = 7.41 Hz, 1H, H-9 quinoline); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 25.9, 35.6, 38.1, 61.4, 63.5, 122.8, 125.7, 126.0, 129.0, q134.5, 136.0, 162.1, 168.7, 180.0, 211.1; HPLC-HRMS (ESI) calcd for C17H16N2OS5+H+, 494.9940; found, 494.9937.

5.1.6. General procedure for synthesis of 7,7-dimethyl-10-thioxo-7,10dihydro[1,2]dithiolo[3,4-c]pyrrolo[3,2,1-ij]quinoline-4,5-dione 4thiosemicarbazone 10a and 2-methoxy-7,7-dimethyl-4-(4-oxo-2-thioxo1,3-thiazolidin-5-ylidene)-10-thioxo-7,10-dihydro[1,2]dithiolo[3,4c]pyrrolo[3,2,1-ij]quinolin-5(4H)-one 10b. A mixture of corresponding pyrrolo[3,2,1-ij]quinoline-1,2-diones 9a,b (5mmol) and thiosemicarbazide or rhodanine (5 mmol) in butanol-1 (20 ml ) was refluxed for 4 h. The precipitate formed during cooling was filtered, washed with methanol. The formed products 10a,b did not need recrystallization. 5.1.6.1. 7,7-Dimethyl-10-thioxo-7,10-dihydro[1,2]dithiolo[3,4c]pyrrolo[3,2,1-ij]quinoline-4,5-dione 4-thiosemicarbazone (10a). Orange powder (yield 1.70 g, 87%), m.p. = 272-273 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 2.13 (s, 6H, (CH3)2), 7.14 (t, J = 7.82 Hz, 1H, H-2 quinoline); 7.61 (d, J = 7.82 Hz, 1H, H-3 quinoline), 8.76 (s, 1H, NH2), 9.15 (s, 1H, NH2), 9.50 (d, J = 7.82 Hz, 1H, H-9 quinoline), 12.33 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 28.5, 61.2, 114.5, 117.4, 120.8, 121.8, 122.1, 130.7, 137.5, 160.4, 178.2, 178.6, 210.9; HPLC-HRMS (ESI) calcd for C15H12N4OS4+H+, 392.9967; found, 392.9971. 5.1.6.2. 2-Methoxy-7,7-dimethyl-4-(4-oxo-2-thioxo-1,3-thiazolidin-5ylidene)-10-thioxo-7,10-dihydro[1,2]dithiolo[3,4-c]pyrrolo[3,2,1ij]quinolin-5(4H)-one (10b). Dark brown powder (yield 1.37 g, 59%), m.p. > 300 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 2.08 (s, 6H, (CH3)2), 3.75 (s, 3H, CH3), 7.10 + 8.3 (both s, 1H, H-3 quinoline); 9.28 + 9.43 (both s, 1H, H-1 quinoline), 13.80-14.40 (br s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 28.2, 55.4, 55.9, 60.8, 61.1, 108.2, 109.6, 113.2, 114.8, 115.2, 117.7, 123.6, 130.4, 154.3, 157.6, 165.9, 210.7; HPLC-HRMS (ESI) calcd for C18H12N2O3S5+H+, 464.9525; found, 464.9521.

6. Biological evaluation 5.1.5.3. 4,4,8-Trimethyl-5-{[(4-phenyl-5-thioxo-4,5-dihydro-1,3,4thiadiazol-2-yl)thio]acetyl}-4,5-dihydro-1H-[1,2]dithiolo[3,4c]quinoline-1-thione (8c). Orange powder (yield 1.88 g, 69%), m.p. = 162-163 °C; 1H NMR (DMSO-d6, 500 MHz) δ ppm: 1.49 (br s, 6H, (CH3)2), 2.23 (s, 3H, CH3)

6.1. Antibacterial activity The following Gram-negative bacteria: Escherichia coli (ATCC 35210), Enterobacter cloacae (clinical isolate), Pseudomonas aeruginosa

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6.3. Statistical analysis

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microplates were incubated at rotary shaker (160 rpm) for 72 h at 28° C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 µL of tested fractions 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 MFC indicating 99.5% killing of the original inoculum. The fungicides bifonazole and ketoconazole were used as positive controls (13500 µg/mL). Three independent experiments were performed in duplicate.

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All the assays were carried out in triplicate and the results are expressed as mean values and standard deviation (SD). The results were analyzed using oneway analysis of variance (ANOVA) followed by Tukey’s HSD Test with α = 0.05. This treatment was carried out using SPSS v. 18.0 programs. 6.4. Docking studies

All docking calculations were carried out using the AutoDock 4.2® software. The free binding energy (∆G) of lanosterol 14alphademethylase of C. albicans (CYP51ca), alpha-beta tubulin in complex with paclitaxel and dihydrofolate reductase (PDB ID: 5V5Z, 1JFF and 4HOF respectively), in complex with the inhibitors 1-10 were generated using this molecular docking program. The crystal structures of these enzymes were obtained from the protein data bank (PDB). For the docking, the grid size was set to 50 × 50 × 50 xyz points with grid spacing of 0.375Å. The grid centers were calculated for CYP51ca (x=−47.731, y=−13.422, z=22.982), and dihydrofolate reductase (x=−0.895, y=0.131, z=32.109). For the preparation of ligand structures, 2D structure was sketched in chemdraw12.0; hydrogens were added and converted to mol2 format. For the docking simulation, default values of quaternation, translation and torsion steps were applied. The number of docking runs was 100. The graphical depictions of all ligand-protein complexes were achieved LINGASCOUT.

6.2. Antifungal activity

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(ATCC 27853), Salmonella typhimurium (ATCC 13311), E.coli as well as Gram-positive bacteria: Listeria monocytogenes (NCTC 7973), Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC 10240), and Staphylococcus aureus (ATCC 6538) were used. The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research ‘Siniša Stankovic, Belgrade, Serbia The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined by the microdilution method. Briefly, fresh overnight culture of bacteria was adjusted by the spectrophotometer to a concentration of 1×105 CFU/mL. Dilutions of inocula were cultured on solid medium to verify the absence of contamination and check the validity of the inoculum. Tested compounds were dissolved in 5% DMSO and added in broth Triptic Soy broth (TSB) medium (100 µL) with bacterial inoculum (1.0×104 CFU per well) to achieve the wanted concentrations (0.001-1.0 mg/ml) in dilution order. The microplates were incubated for 24 h at 370C. The MIC of the samples was detected following the addition of 40 µL of iodonitrotetrazolium chloride (INT) (0.2 mg/mL) and incubation at 37°C for 30 min. The lowest concentration that produced a significant inhibition of the growth of the bacteria in comparison with the positive control was identified as the MIC. The optical density of each well was measured at a wavelength of 655 nm by Microplate manager 4.0 (Bio-Rad Laboratories) and compared with a blank and the positive control. The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to tested extracts were determined also by a colorimetric microbial viability assay based on reduction of a INT color and compared with positive control for each bacterial strains. MBC was determined by serial subcultivation of 10 µL into microplates containing 100 µL of TSB. The lowest concentration that shows no growth after this sub-culturing was read as the MBC indicating 99.5% death of the original inoculum. Standard drugs, namely streptomycin and ampicillin were used as positive controls. Five % of DMSO was used as negative controls. All experiments were performed in duplicate and repeated three times. The antibacterial assay was carried out by the microdilution method as previously reported.[55, 65, 66].All experiments were performed in duplicate and repeated three times.

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For the antifungal bioassays, eight fungi were used: Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC 12066), Aspergillus fumigatus (1022), Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM 5061), and Penicillium verrucosum var. cyclopium (food isolate). The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research ‘‘Siniša Stankovic,’’ Belgrade, Serbia. All experiments were performed in duplicate and repeated three times.[ 67,68] The micromycetes were maintained on malt agar and the cultures were stored at 4° C and sub-cultured once a month. The antifungal assay was carried out by modified microdilution technique. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0×105 in a final volume of 100 µL per well. The inocula were stored at 4° C for further use. Dilutions of the inoculum were cultured on solid malt agar to verify the absence of contamination and to check the validity of the inoculum. MIC determinations were performed by a serial dilution technique using 96-well microtiter plates. The examined compounds were diluted in 5% of DMSO (0.001-1.0 mg/ml) and added in broth Malt medium (MA) with inoculum. The

Conclusion Ten compounds were synthesized and evaluated for antimicrobial activity. All tested compounds showed good antibacterial activity against all bacteria tested with MIC at 0.045-12.9 milliM x10-2 and MBC at 1.0451.659milliM x10-2, being more active than ampicillin and except of 7a and 10b against B.cereus, M flavus and L.monocytogenes than streptomycin drugs. Among bacteria tested most sensitive appeared to be En.cloacae followed by S. aureus, while L.monocytogenes was the most resistant. It should be mentioned that some differences regarding sensitivity to our compounds was observed. It should be mentioned that compounds 4d, 7b, 8a, 8d and 10a showed the same sensitivity to all bacteria tested. According to structure-activity relationships study the presence of 4phenyl-5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioacetyl part in quinoline moiety nitrogen of (8c) was very beneficial for antibacterial activity while the presence of 6-NO2 group in 8-methoxy-4,4 dimethyl4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-thione 6 was detrimental. As far as antifungal activity is concerned all tested compounds showed good antifungal activity against all fungi tested with MIC at 1.04-25.83 x10-2 milliM and MFCat 2.08-51.65 milliM x10-2 being much more active than reference drugs ketoconazole and bifonazole. Among all fungi the most sensitive appeared to be T. viride, while the most resistant was P. verucosum v.cyclopium. As in case of bacteria fungi too showed different sensitivity towards our compounds.

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In the chemistry part this work was financially supported by the Russian Science Foundation, Agreement no. 18-74-10097. The research results were partially obtained on the equipment of The collective use Center of Voronezh State University (URL: http://ckp.vsu.ru)

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It was found that activity of compounds depends on the nature of substituents. Thus, the presence of 1,3-thiazol-2-ylthio)acetyl fragment on the of quinoline moiety nitrogen (8d) was favorable for antifungal activity, while 2-thioxothiazolidin-5-ylidene group in dithiolo[3,4c]pyrrolo[3,2,1-ij]quinolinone (10b) had negative effect on activity.Docking analysis indicate that gyrase inhibition may be the putative mechanism of their antibacterial action while the inhibition of 14α-demethylase may be responsible for antifungal action. From the obtained results it is obvious that tested compounds are promising multitargeted agents against bacteria and fungi.

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Highlights Antimicrobial and antifungal activities of novel steroids Molecular docking studies on E.coli MurB enzyme Molecular docking studies on C.albicans lanosterol 14alpha-demethylase (CYP51).

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1. 2. 3.