DNA Structural Alteration Leading to Antibacterial Property of 6

Subscriber access provided by RUTGERS UNIVERSITY

Article

DNA Structural Alteration Leading to Antibacterial Property of 6-Nitroquinoxaline Derivatives Khondakar Sayef Ahammed, Ritesh Pal, Jeet Chakraborty, Ajay Kanungo, Polnati Sravani Purnima, and Sanjay Dutta J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00599 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

DNA Structural Alteration Leading to Antibacterial Property of 6-Nitroquinoxaline Derivatives Khondakar Sayef Ahammed,§,† Ritesh Pal,§,†, ‡ Jeet Chakraborty,§,† Ajay Kanungo,†,‡ Polnati Sravani Purnima,† Sanjay Dutta*,†,‡ † Organic and Medicinal Chemistry Division, CSIR- Indian Institute of Chemical Biology 4, Raja S.C.Mullick Road, Kolkata- 700032, West Bengal, India. E-mail: [email protected] ‡ Academy of Scientific and Innovative Research (AcSIR), Kolkata-700032, West Bengal, India. § These authors contributed equally.

* Corresponding author.

1

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Structural integrity of bacterial genome plays an important role in bacterial survival. Cellular consequences of intolerable amount of change in DNA structure are not well understood in bacteria. Here we have stated that, binding of synthetic 6-nitroquinoxaline derivatives with DNA led to change in its global structure, subsequently ended up with over-supercoiled form through in-path intermediates. This structural change results in induction of programmed cell death like physiological hallmarks, which is dependent on substitution driven structural modulation properties of the scaffold. Sub-lethal dose of a representative derivative 3a significantly inhibits DNA synthesis, produce fragmented nucleoid and alter membrane architecture. We have also shown that exposure of the compound changes native morphology of S. aureus cells and significantly disrupt preformed biofilm. Thus, our study gives a new insight on bacterial responses to local or global DNA structural changes induced by 6nitroquinoxaline small molecules. Keywords: 6-nitroquinoxaline, Antibacterial, DNA structural change, Induced Circular Dichroism (ICD), Bacterial response, Biofilm disruption.

2


Page 2 of 49

Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Continuous emergence of bacterial resistance to conventional antibiotics is a serious global health concern. Bacteria that adopt resistance to antibiotics, use several mechanisms to reduce antibiotic effectiveness like drug target modifications, drug efflux from the cell, chemical modifications of the drug or by some other means.1 More importantly some of the pathogenic bacteria can form biofilms that worsen the problem of drug resistance.2 Biofilm formation limits drug effectiveness by reducing the penetration of antibiotics, altering their microenvironments and neutralizing antibiotics thereby forming a multilayered defense system that is contributing towards the global problem of antibiotic resistance.3 The increasing tolerance of antibacterial drug insists the reinforcement of developing new antibacterial compounds with distinctive mechanism. Bacterial DNA is far lengthier than the average size of a bacterial cell. Inside the cell, bacteria maintain the structural architecture of its genome in a finely regulated compact form.4 Many genome structuring proteins such as histone like protein HU and H-NS play crucial role in maintaining the structure of bacterial nucleoid.5,6 Recent research indicates local supercoiling and dynamics of bacterial chromosome associated proteins regulate the fundamental processes like replication, gene expression etc.5-7 Cellular responses to intolerable amount of local or global structural alteration in DNA induced by small molecules has not been explored well. We have probed the physiological responses during the shock of small molecule induced structural alteration of DNA in bacterial cells. Small molecules which bind to DNA may influence the survival of a cell by interfering with essential cellular functions. Quinoxaline based small molecules are biologically active compounds, widely known as potent antiproliferative-antimicrobial agents. Quinoxaline antibiotics like Triostin A (Figure 1A) are known to intercalate into dsDNA, preferentially active against gram positive bacteria.8-10 Several synthetic scaffolds using the quinoxaline moiety have been developed that have antibacterial property against a wide range of pathogenic bacterial strains.11-13 Quinoxaline N-oxides (QNOs) exhibit antibacterial activity by targeting DNA and induction of reactive oxygen species (ROS).14 For a long time, quinoxaline containing antibiotic have been used as veterinary medicines for the treatment of bacterial infection in animal husbandry. Considering the moderate selectivity index, subtherapeutic dose of QNOs (for example; OLA; 2-(N-2’-hydroxyethyl-carbamoyl)-3-methyl quinoxaline 1,4-dioxide, CBX; hydrazine carboxylic acid (2-quinoxalinyl-methylene) methyl ester 1,4-dioxide, CYA; 2-formylquinoxaline-N1, N4-dioxide cyanocetylhydrazone) with feed 3



additives are used as growth promoting and antibacterial agents for poultry and aquatic animals.15 There are couple of reports on the indole-quinoxaline fused system which showed antiviral as well as potent DNA binding activities.16,17 Quinoxaline metal complexes are also known to have DNA binding and antibacterial properties.18 In addition, a number of small molecules with mono-quinoxaline core have been reported as potent antiproliferative agents with excellent activities against cancer cells.19 Because of the diverse range of biological activities, quinoxaline compounds play major role in medicinal chemistry research. In our previous study we have shown that, a 6-nitroquinoxaline scaffold 3g (Figure 1B) intercalates into DNA preferentially in GC rich sequence through complex cooperative binding events.20 Considering the importance of the structural integrity of bacterial genome in its survival, we have synthesized a series of mono-quinoxaline based derivatives by substitutional tuning of 3g that possesses the distinct ability to induce change in native DNA structure. These small molecules can cause intercalation induced structural change which ultimately lead to over-supercoiled form in a dose dependent manner.21 The DNA structural changing property is dependent on the substitution at C2 and C3 positions of the 6nitroquinoxaline scaffold (Figure 1C) which correlate with the antibacterial properties of these derivatives against gram positive bacteria. Though, some of the 6-nitroquinoxaline derivatives exhibit potential activity against mammalian cancer cells, however in this study we have only focused on the DNA structural change guided antibacterial responses by this scaffold. Exposure to sub minimal inhibitory concentration (MIC) of 3a (a representative molecule of this class) induces major physiological and morphological responses in gram positive Staphylococcus aureus. At sub-MIC, this compound inhibits cellular DNA synthesis, induces DNA break and nucleoid fragmentation in bacterial cell. Compound 3a has an effect on bacterial membrane integrity and induces morphology plasticity in S. aureus bacterial cell. In addition, 3a also has the ability to disrupt preformed biofilms in S. aureus and S. epidermidis. Overall, we report potent antibacterial activities and cellular responses of a new class of 6nitroquinoxaline small molecules which induces structural alteration in bacterial DNA. The structural change has been characterized as topological alteration in bacterial genomic DNA which ultimately leads to DNA condensation. This study gives new insights to understand a cellular system with topologically altered DNA.

4


Page 4 of 49

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. A) Structure of Triostin A, a known DNA intercalator. B) N2-benzyl-N3-(3(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine (3g), a previously reported DNA intercalator.20 C) Two sets of compounds were evaluated for the antibacterial activity. Set-1: para-substituted benzylamine group at C2 (3a, 3h-l); positional substitution in benzylamine moiety at C2 (3m-r); di-substituted benzylamine group at C2 (3s-u) and N, Ndimethylpropane-1, 3-diamine at C3 position. Set-2: p-trifluoromethylbenzyamine moiety at C2 and different amine derivatives at C3 position (3b-f).

5



RESULTS AND DISCUSSION: Design and synthesis: Here we have designed a series of mono-quinoxaline based derivatives (Figure 1C) from the modification of the scaffold 3g (Figure 1B).20 In order to achieve this, two sets of compounds were designed by changing the substituents at C-2 and C-3 positions of the 6nitroquinoxaline moiety, all of which were subjected to antibacterial evaluation (Figure 1C). The set-1 derivatives (3a, 3h-u, Figure 1C) comprises of variously substituted benzylamine group at C-2 position of the quinoxaline scaffold by keeping C-3 position intact with N,Ndimethylaminopropylamine tail. To understand the stereoelectronic effects at different positions of this system, we have altered the position of the substituents in the benzyl ring. In order to check the electronic effect of the substituents, we have used electron donating groups like -Me, -OMe and electron withdrawing groups such as –CF3, -Br, -Cl, -F on the benzyl moiety. We have also examined the stereoelectronic effect by substituting the ortho position of the benzyl moiety with some of the electron donating (3o-p, Figure 1C) and withdrawing substituents (3m-n, Figure 1C). Other than mono-substituted derivatives, set-1 also includes some of the di-substituted derivatives (3s-u, Figure 1C). In contrast to set-1 derivatives, the set-2 derivatives (3b-f, Figure 1C), consist of different amine substituents at C-3 position of the quinoxaline core without changing the p-trifluoromethylbenzylamine substituent at C-2 position. In order to explore the effect of amine substituents, here we have used different cyclic and acyclic amine group with various chain lengths. All the derivatives were synthesized (Figure 2) by following the previously reported procedure22 for 3g, from 2,3-dichloro-6-nitroquinoxaline (1, Figure 2) by simple two-step synthesis. In the first step, a simple nucleophilic substitution reaction by a benzylamine or substituted benzylamine on the C-2 position of the 2,3-dichloro-6-nitroquinoxaline resulted in the formation of mono substituted 6-nitroquinoxaline derivative (2a-p, Figure 2). The product obtained from the first step on further treatment with amine or substituted amine (primary or secondary amine) under Buchwald condition resulted in 2,3 di-substituted 6-nitroquinoxaline derivatives ( 3a-f, 3g-u, Figure 2).

6


Page 6 of 49

Page 7 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synthetic scheme of 6-nitroquinoxaline derivatives: O 2N

N

Cl

N

N H

2a (40 - 70%)

(i-a)

N 3 Cl

O 2N

O 2N

(i-b)

N 2 Cl

N

Cl

N

N H R1

(1)

CF 3

2b - p (40 - 81%)

R3 R2

(ii-b)

(ii-a) :R1 =

O 2N

R4

N

R5

N

N H

3a - f (52 - 74%)

CF 3

3a : R5 = HN(CH 2)3N(CH 3)2 3b : R5 = HN(CH2) 3N(CH2CH 2)2NCH3 3c : R5 = HN(CH 2)4N(CH 3)2 3d : R5 = N(CH 2CH2) 2CH2 3e : R5 = N(CH 2CH 2)2NCH3 3f : R5 = N(CH2CH 2)2NH

H, R2 = H,

R3 =

H, R4 = H

3g 3h :R1 = H, R2 = H, R3 =Br, R4 = H 3i : R1 = H, R2 = H, R3 = Cl, R4 = H 3j : R1 = H, R2 = H, R3= F, R4 = H O 2N 3k :R1 = H, R2 = H, R3= Me, R4 = H 3l : R1 = H, R 2= H, R3 = OMe, R4 = H 3m: R1 = CF3, R2 = H, R3 = H, R4 = H 3n : R1 = Br, R2 = H, R3 = H, R4 = H 3o : R1 = Me, R2 = H, R3 = H, R4 = H 3p : R1 = OMe, R2 = H, R3 = H, R4 = H 3q : R1 = H, R2 = Br, R3 = H, R4 = H 3r : R1 = H, R2 = CF3, R3 = H, R4 = H 3s : R1 = H, R2 = CF 3, R3= Cl, R4 = H 3t : R1 = H, R 2 = CF3, R3 = H, R4 = F 3u : R1 = H, R2 = CF3, R3 = H, R4 = CF 3

N

H N

N

N H

3g - u R1 (51 - 87%)

N R4

2b : R1 = H, R2 = H, R3 = H, R4 = H 2c : R1 = H, R2 = H, R3 = Br, R4 = H 2d : R1 = H, R2 = H, R3 = Cl, R4 = H 2e : R1 = H, R2 = H, R3= F, R4 = H 2f : R1 = H, R2 = H, R3= Me, R4 = H 2g : R1 = H,R2 = H, R3 = OMe, R4 = H 2h : R1 = CF3, R2 = H, R3 = H, R4 = H 2i : R1 = Br, R2 = H, R3 = H, R4 = H 2j : R1 = Me, R2 = H, R3 = H, R4 = H 2k : R1 = OMe, R2 = H, R3 = H, R4 = H 2l : R1 = H, R2 =Br, R3 = H, R4 = H 2m: R1 = H, R2 = CF3, R3 = H, R4 = H 2n : R1 = H, R2 = CF3, R3= Cl, R4 = H 2o : R1 = H, R2 = CF3, R3 = H, R4 = F 2p : R1 = H, R2 = CF 3, R3 = H, R4 = CF3

R3 R2

Figure 2. Reagents and conditions: (i-a) 4-(Trifluoromethyl)benzylamine (1.475 mmol), CaCO3 (3.442 mmol), DCM, rt, overnight; (ii-a) substituted amines (0.375 mmol), Cs2CO3 (0.375 mmol), Xantphos (0.025 mmol), Pd2(dba)3 (0.0125 mmol), Dioxane, 110 oC, 4 h; (i-b) substituted benzyl amines (1.475 mmol), CaCO3 (3.442 mmol), DCM, rt, overnight; (iib) 3-(Dimethylamino)-1-propylamine (0.375 mmol), Cs2CO3 (0.375 mmol), Xantphos (0.025 mmol), Pd2(dba)3 (0.0125 mmol), Dioxane, 110 oC, 4 h. Antibacterial evaluation of mono-quinoxaline derivatives: Table 1 shows minimal inhibitory concentration (MIC) values of rationally synthesized two sets of mono-quinoxaline derivatives (set-1 and set-2), which were determined against gram positive (Staphylococcus aureus MTCC737, Staphylococcus epidermidis MTCC3615, Arthrobacter chlorophenolicus A6 MTCC3706) as well as gram negative bacteria (Escherichia coli MTCC1916, Pseudomonas aeruginosa MTCC1688). Previously reported DNA intercalator 3g (Figure 1B) exhibit limited antibacterial activities against three gram positive bacteria (MIC ranging from 7.6 to 13.3 µg/mL). Positional substitution in the benzyl moiety (set-1; 3a, 3h-u) and substitution with different amine tail at C3 position (set-2; 3b-f) improves the antibacterial activity of the scaffold. Among the set-1 derivatives, 4-trifluoromethylbenzylamine substituted 6-nitroquinoxaline derivative 3a was observed to have increased activity (MIC - 1.79 µg/mL) (Figure S1A) when compared to 3g.

7



The other para-substitutions in the benzyl ring with halides like -Br, -Cl (3h-i) also show significant activities (MIC ranging from at 2.29 to 6.22 µg/mL). This indicates that halosubstituent, most imortantly -CF3 (3a) group in the benzyl ring plays a significant role in the enhancement of antibacterial activity of this scaffold. Interestingly, substitution with electron donating group like -OMe (3l, 3p), decreases the activity in comparison to electron withdrawing substituents. On the other hand, meta (3r) and ortho (3m-p) substituted derivatives show more or less comparable activities. Amongst the mono substituted compounds (3a, 3h-r), meta-substituted 3q exhibits better antibacterial activity (ranging between 0.9-1.6 µg/mL) against the three gram positive bacteria. The ortho effect of electron withdrawing group (-CF3) vs electron donating group (-OMe, -Me) shows different behavior. When we determined the steric effect by introducing the -CF3 group at the ortho position of benzyl ring (3m) the activity was retained, but substitution with electron donating groups like -OMe or -Me at ortho position (3o and 3p) decreases the activity. Again positional substitution of electron donating substituents like -Me or -OMe from para position (3k, 3l) to ortho position (3o, 3p) in the benzyl ring, shows comparable antibacterial activity. The di-substituted compounds (3s-u) exhibited significant antibacterial activity. Compound 3u (with 2,5-trifluoromethylbenzylamine substituted) exhibited highest antibacterial potency against S. epidermidis (MIC -0.52 µg/mL). The result indicates that, the presence of two substituents in the benzyl ring enhances the activity as compared to mono- substituted derivatives. In set-2, the effect of amine tail part at C2 position of the scaffold has been clarified. The MIC values of set-2 (3b-f) derivatives show that compound 3b is the most effective (MIC1.76 µg/mL) against high GC content A. chlorophenolicus. In case of low GC content S. aureus, 3f is the most effective one (MIC- 3.03 µg/mL). The rest of the compounds in set-2 (except 3d) showed more or less similar activity against gram positive bacteria. Whereas, 3d did not show antibacterial activity even at high concentrations (200 µg/mL) (Figure S1A). MIC values also indicate that increasing the linker length between the quinoxaline moiety and the N-methylpiperazine ring (3b vs 3e) doesn’t cause much change in the antibacterial properties of the compounds. These results conclude that the piperazine ring (3b, 3e, 3f) or the dimethylamino group (3c) is very crucial for the antibacterial activity of the quinoxaline compounds in set-2. Thus removal of the terminal nitrogen from the piperazine ring abrogates the antibacterial activity for 3d.

8


Page 8 of 49

Page 9 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition, gram positive bacteria are more susceptible to these mono-quinoxaline based derivatives than gram negative bacteria. The table-1 shows that in case of gram negative bacteria, 3c is the most effective compound among set-2 derivatives (MIC-2.31 µg/mL) against E. coli and is comparable to a well-known antibiotic kanamycin in terms of MIC. Other than 3c, all the derivatives exhibited low or moderate activities against two of the gram negative bacteria. The notable difference between 3a and 3c is of increase in linker length between N,N-dimethylamino group and the quinoxaline ring which plays an important role for the antibacterial activity. We have also compared the antibacterial activities of the designed quinoxaline compounds with other reported synthetic quinoxaline derivatives and natural products (Echinomycin) against gram positive and gram negative bacterial strains as shown in supporting (Table-S1). We have determined mammalian cell cytotoxicity (Table-1) of the compounds against Human embryonic kidney cell line (HEK 293) to identify relatively less cytotoxic 6nitroquinoxaline compounds. For 3q, which has the highest antibacterial activity against S. aureus (MIC- 0.89 µg/mL), exhibits IC50 ~7.99 µg/mL and is almost 9 fold greater than its MIC. Whereas, 3n shows antibacterial activity at 15 fold less concentration than its cytotoxicity against HEK 293 cell line. Morphology of HEK 293 cells were retained when exposed to 3a at higher than MIC (in S. aureus) dose (5 µM or 2.24 µg/mL) (Figure S3).

9



Page 10 of 49

Table 1. Minimal Inhibitory Concentration (MIC) of compounds against different bacteria and mammalian cell cytotoxicity against HEK 293 cell line.

Compounds S.aureus

SET 1

SET 2

MIC (µg/mL) Gram positive bacteria S. epidermidis A. chlorophenolicus

Gram negative bacteria E. coli P. aeruginosa

IC50 (µg/mL) HEK 293

3a

1.79 ± 0.25

2.24 ± 0.64

0.897 ± 0.38

3.36 ± 0.64

6.73 ± 0.65

5.66 ± 0.77

3h

3.45 ± 0.60

3.45 ± 0.57

2.29 ± 0.26

4.59 ± 1.14

>230

9.63 ± 1.16

3i

3.11 ± 1.29

6.22 ± 2.58

2.49 ± 1.03

>207

>207

10.74 ± 4.45

3j

7.97 ± 1.99

5.98 ± 1.15

3.98 ± 1.61

86.32 ± 6.08

>200

4.28 ± 0.46

3k

5.92 ± 1.08

3.94 ± 0.22

2.76 ± 0.19

110 ± 5.21

>200

15.78 ± 1.64

3l

10.26 ± 2.21

5.13 ± 2.10

4.1 ± 1.68

>200

>200

38.58 ± 2.83

3m

3.36 ± 0.34

2.24 ± 0.90

2.91 ± 2.50

68.31 ± 5.23

>224

24.66 ± 2.49

3n

3.45 ± 0.22

4.59 ± 0.70

2.76 ± 0.35

>230

>230

51.91 ± 2.02

3o

4.93 ± 0.52

7.89 ± 0.61

6.9 ± 0.13

75.6 ± 4.10

>200

27.61 ± 2.58

3p

8.21 ± 0.83

6.16 ± 1.13

6.16 ± 0.62

>200

>200

36.12 ± 1.85

3q

0.89 ± 0.34

0.89 ± 0.22

1.57 ± 0.34

34.3 ± 3.40

>200

7.99 ± 1.29

3r

4.59 ± 0.66

2.29 ± 0.60

2.29 ± 0.92

4.59 ± 1.65

13.78 ± 1.61

3.66 ± 0.95

3s

1.69 ± 0.24

0.96 ± 0.24

1.69 ± 0.50

7.24 ± 0.83

>240

3.26 ± 0.73

3t

0.93 ± 0.35

1.63 ± 0.13

2.33 ± 0.71

35 ± 1.88

>235

2.67 ± 0.53

3u

1.03 ± 0.14

0.52 ± 0.19

1.81 ± 0.64

>250

>250

4.32 ± 1.03

3b

3.78 ± 0.66

3.78 ± 0.88

1.76 ± 0.43

3.78 ± 1.24

>250

9.53 ± 1.45

3c

3.47 ± 0.48

3.47 ± 1.15

3.47 ± 0.13

2.31 ± 0.46

>230

9.73 ± 1.75

3d

>200

>200

>200

>200

>200

>200

3e

4.46 ± 1.46

4.46 ± 0.89

4.46 ± 0.64

>220

>220

6.99 ± 1.56

3f

3.03 ± 0.24

4.32 ± 0.97

4.32 ± 0.99

4.32 ± 0.54

10.80 ± 2.21

9.61 ± 1.19

3g

11.41 ± 1.50

13.31 ± 1.50

7.6 ± 1.41

114 ± 5.49

57 ± 5.02

2.74 ± 0.92

Kanamycin

1..85 ± 0.13

1.98 ± 0.22

3.2 ± 0.25

2.08 ± 0.38

>125

>500

Echinomycin

0.04 ± 0.005

0.02 ± 0.0043

0.07 ± 0.01

21 ± 3.60

>200

0.014 ±0.005

Control

The experiment was performed in triplicate. ± indicates the standard deviation of MIC values.

10


Page 11 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DNA-molecule interaction: DNA structural change: DNA intercalators are known to unwind negatively supercoiled DNA. The structural change of DNA by intercalator is reflected in the mobility of supercoiled plasmid DNA in agarose gel.23 Previously we have shown that compound 3g (with mandatory benzyl moiety) shows gel shift of pBR322 plasmid DNA indicating DNA intercalation.20 To investigate the structural change of DNA induced by designed quinoxaline molecules, we have studied the agarose gel shift assay of supercoiled plasmid DNA (pCDNA3.1) by the most potent analogues (3a, 3u, 3q) and compound 3d, which does not show any antibacterial activity. For 4-trifluoromethylbenzylamine substituted compound 3a, unwinding of plasmid DNA begins at 2:1 [compound]:[DNA base pair] ratio, leading to significant structural change at 4:1 ratio (Figure 3A1). Most significantly, for 3,5-bis(trifluoromethyl)benzylamine substituted derivative (3u), gel shift begins at low ratio (1:1) with DNA condensation at higher ratios (Figure 3A2). 3-trifluoromethylbenzylamine substituted derivative 3q also shows gel shift at similar ratios, indicating positional substitution from para to meta does not affect the activity (Figure 3A3). In contrast, the less active -F substituted compound 3j initiates gel shift at higher (5:1) ratios (Figure S4). For the compounds with electron donating substituents like Me or -OMe, gel shift occurs but not significantly as that of electron withdrawing halo substituted derivatives (Figure S4). Changing the position of substituent in the benzyl ring does not reflect any major change in gel shift and retains the activity in a moderate range. In case of 3d (piperidine substituted), which does not show any antibacterial activity even upto higher concentration, no such gel shift was observed up to 6:1 ratios (Figure 3A4). This result indicates that the DNA structural changing properties have an important role on the antibacterial efficacy of these derivatives. Agarose gel shift assay was also performed with S. aureus genomic DNA and compound 3a, which exhibited DNA structural changes with increasing concentration of 3a (Figure 3A5).

11



Figure 3: Agarose gel shift assay of supercoiled plasmid DNA (pCDNA3.1) with (A1) 3a, (A2) 3u, (A3) 3q, and (A4) 3d. Lane number indicates [compound]/ [DNA base pair] ratio. A5) Gel shift assay with S. aureus genomic DNA treated with 3a in indicated [compound]/ [DNA base pair] ratio. S denotes supercoiled DNA, M denotes structurally altered DNA (mixed population) and C denotes condensed DNA as indicated in the panel. DNA structural change induced by small molecule can also be determined by change in the circular dichroism (CD) spectra of DNA.24 Appearance of an induced CD (ICD) has a direct relation with structural change and superstructure formation which is mediated by close association of DNA bound nitroquinoxaline scaffold.20, 21 Titrating calf thymus DNA (CTDNA) with increasing concentrations of 3a, 3q, 3u lead to decrease in the typical B-DNA CD peak at 265 nm (Figure 4A-4C). A negative ICD also appeared at 320-325 nm wavelength indicating DNA intercalation induced structural change. Both the ICD appearance and decrease in 265 nm band intensity started from 1:1 [compound]:[DNA bp] ratio, changes in an concentration dependent manner. A plot of CD spectra at 265 nm band in an increasing concentration of 3a, 3q and 3u yielded a sigmoidal pattern, which indicates a cooperative

12


Page 12 of 49

Page 13 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


phenomena (Figure 4H). It can also be inferred that 3u (3,5-trifluoromethylbenzylamine substituted compound) has higher ability to change the structure of DNA than 3q or 3a which is correlated with other experimental evidence of DNA-molecule interaction. In case of 3d, no significant ICD as well as change in the B-DNA peak was observed even up to high [3d]:[DNA bp] (4:1) ratio (Figure 4D). GC content of bacterial genome varies from less than 20% to greater than 70%.25 We have investigated the DNA sequence preferentiality of compound 3a and whether this property is reflected in the activity (MIC) against low GC content S. aureus vs high GC content A. Chlorophenolicus. Titration of Poly (dA-dT) · Poly (dA-dT) (10 µM) and Poly (dG-dC) · Poly (dG-dC) (10 µM) DNA sequence with 3a (20 µM to 60 µM) clearly indicates GC preferentiality of that compound (Figure S5). In case of poly GC, significant change in BDNA peak as well as appearance of ICD was observed at 2:1 [3a]:[DNA bp] ratio and which increased with increasing compound concentrations. Whereas, in case of poly AT, negligible changes in both the CD peaks were noticed up to 6:1 [3a]:[DNA bp] ratio. Differences in CD spectra were also observed in case of S. aureus (low GC content) and A. chlorophenolicus (high GC content) bacterial genomic DNA when titrated with increasing concentration of 3a (Figure 4E-4F). Ellipticity plot at 320 nm band of S. aureus and A. chlorophenolicus genomic DNA titrated with 3a resulted in a sigmoidal pattern. The change in intensities of ICD spectra at 320 nm (Figure 4G) in case of GC rich bacterial genomic DNA is higher than in AT rich one. These two results indicate that 3a has higher preference for GC rich DNA sequences, induces more structural change in high GC DNA and is correlated with MIC values against two bacteria (Figure S1B). The possible reason for lesser MIC of 3a againt A. Chlorophenolicus is due to higher structural changes arising for high GC content genome in comparison to S. aureus. Other para-substituted derivatives also showed significantly low MIC against high GC containing bacteria (Figure S1B). Meta and di-substituted compounds do not follow the trends, though the sequence preferentiality of these two groups of molecules may change with substitution and is subjected to further investigation. The B-DNA CD ellipticity change by the compound 3a (15 µM to 180 µM) was also observed in mammalian genomic DNA (isolated from HEK 293 cells).( Figure S6) The 262 nm band of CD spectra decreased in concentration dependent manner and significant decrease was found at relatively higher [3a]:[DNA bp] ratios. Compared to bacterial genomic DNA, the appearance of ICD is less in mammalian genomic DNA. 13



Figure 4: (A-D) Circular dichroism (CD) spectra of 15 µM CT-DNA with 3a, 3u, 3q, 3d at increasing concentrations of 15, 30, 45, 60 µM. (E-F) Circular dichroism (CD) spectra of 15 µM S. aureus genomic DNA (E) and A. chlorophenolicus genomic DNA (F) with 3a (15-60 µM).(G) Ellipticity plot at 320 nm wavelength obtained from S. aureus (red) and A. chlorophenolicus (green) titrated with 3a (0-60 µM). (H) Plot of ellipticity at 265 nm band with increasing concentration (0-60 µM) of indicated compound (3a, 3u, 3q) and CT-DNA.

14


Page 14 of 49

Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Probing the path of structural change: Using Atomic Force Microscopy (AFM), the path of DNA conformational change has been tracked in pBR322 supercoiled plasmid DNA. Visualization of DNA structures treated with increasing concentration of 3a indicates that it is a local to global structural change phenomena (Figure 5A-5F). At lower [compound]:[DNA base pair], i.e. 0.5:1 and 1:1 ratios (Figure 5B-5C), the compound binds locally, unwind the supercoiling and partially alter DNA native structure. At 1.5:1 ratio (Figure 5D) the structural change becomes more prominent by bringing the strands in close proximity and looping of the strands. Upon further increase of compound concentration, the structural change becomes highly significant; the height of the DNA increases gradually from 0.32 ± 0.02 nm to 1.30 ± 0.11 nm (Figure 5H). At higher ratio (2:1) transition of local to global DNA structural change occurs (Figure 5E) which ultimately (at 3:1 ratio) ended up with oversupercoiled compact form (Figure 5F). This type of superstructure formation was previously observed in case of 3g molecule and a known intercalator doxorubicin.21 The inactive compound 3d which has no antibacterial activity at higher concentration, does not show such DNA compaction or intermediate structures even up to 5:1[compound]:[DNA base pair] ratio (Figure 5G).

15



Figure 5: AFM images of (A) pBR322 supercoiled plasmid DNA without compound, (B-F) pBR322 with increasing [compound]:[DNA base pair] ratio of 3a i.e. 0.5:1(B); 1:1(C); 1.5:1(D); 2:1(E); 3:1(F) respectively. (G) Treated with 3d in 5:1 [compound]:[DNA base pair] ratio. (H) A plot of pBR322 height with or without treatment of 3a. DNA binding affinity: The competitive binding of small molecules with DNA leads to displacement of ethidium bromide (EtBr) from EtBr bound DNA and quenching of its fluorescence intensity.26 The DNA binding property of 3a, 3q and 3u were measured using EtBr displacement assay (Figure 6A-6C). Titrating the EtBr bound genomic DNA of S. aureus with an increasing concentration (2.5 – 50 µM) of 3a, 3q and 3u led to a quenching of the fluorescence intensity which was studied using the Stern Volmer (SV) quenching plot {F0/F = 1 + Ksv[Q]} where F0, F are the fluorescence intensity observed in the absence and presence of quencher respectively, Ksv is the SV constant and Q is the concentration of the quencher.27 From the 16


Page 16 of 49

Page 17 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SV plot, the SV constant (Ksv) reflects the potency of the quencher molecule. The Ksv values for the 3a, 3q and 3u were 1 x 104 M-1, 1.7 x 104 M-1 and 2 x 104 M-1 respectively. Whereas, the compounds 3a, 3q and 3u do not quench the fluorescence intensity of EtBr in the absence of DNA (Figure S11). At optimum growth temperature of bacteria i.e. 37 0 C, 3a effectively binds with DNA (Figure S7). The DNA binding was retained at different pH (pH 6 to 8) (Figure S7), whereas, a slight higher fluorescence quenching was observed at pH 6. Since fluorescence quenching may not always be a true reflection of binding interaction therefore the Kd (dissociation constant) for the binding of 3a, 3q and 3u were plotted from Isothermal Titration Calorimetry study.28 From the ITC experiments, the binding affinity (Ka), enthalpy change (∆H) and binding stoichiometry (n) were measured to determine the interaction between DNA and compounds (Figure S12). The binding affinity (Ka) values of compound 3a, 3q and 3u were observed as 2.27 x 104 M-1, 3.06 x 104 M-1 and 9.9 x 104 M-1 respectively. No significant binding was observed in case of 3d. The Ka values obtained from the ITC experiment also follow the same trend (3u >3q >3a) as observed in case of fluorescence intercalator displacement assay which reflects that the compounds bind with dsDNA with great affinity.

17



Figure 6: Fluorescence intercalator displacement assay (FID) with 10 µM S. aureus genomic DNA and 5 µM EtBr (λex = 480 nm) titrated with 3a, 3u, 3q (A-C) in an increasing concentration ranging 2.5 µM to 50 µM (the fluorescence spectra were corrected for inner filter effect and the assay was done in triplicates). Fluorescence quenching was studied using the Stern Volmer (SV) quenching plot (inset). Correlation between DNA structural change and antibacterial property of the molecules: The structural alteration of DNA induced by the synthetic 6-nitroquinoxaline derivatives influences the antibacterial properties of the compounds. 3a, 3q, 3u exhibit potent antibacterial activities (Table 1) and display gel shift with pCDNA3.1 plasmid at low compound/DNA bp ratios (Figure 3A) as well as change in B-DNA CD peak at 265 nm (Figure 4A-4C). At low Drug/DNA ratio of 3a (1:2) significant structural change of plasmid

18


Page 18 of 49

Page 19 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DNA was also visualized by AFM (Figure 5). Binding experiments like FID (Figure 6) and ITC (Figure S7) also supports the results. Whereas, compounds like 3j, which shows minimal gel shift of pCDNA3.1 plasmid up to 5:1 compound/DNA bp ratio (Figure S4) exhibits antibacterial activity at higher concentration. These results indicate that DNA structural change is an important factor for the antibacterial activities of these compounds. In case of 3d, no gel shift was observed even up to higher [3d]: [DNA base pair] ratio (6:1) (Figure 3A). Similarly, B-DNA CD peak also does not alter at 4:1 ratio. 3d treated plasmid DNA does not induce over-supercoiled structure at 5:1 ratio as observed in AFM images. These results clearly indicate that 3d does not induce characteristic structural change in DNA. The antibacterial property of 3d also correlates with the results, which does not exhibits antibacterial activity even up to higher concentration >200 µg/mL. UV data shows that 3d is soluble even at higher concentrations (Figure S10), albeit 6-nitroquinoxaline derivatives are known to cause aggregation.20 The loss of activity for 3d is due to the absence of dimethylaminopropyl group at C3 position of the scaffold which prevents interaction of the molecules with DNA. According to our previous study, the quinoxaline part of the scaffold 3g intercalates into the DNA bases and C3 amine group is involved in electrostatic interaction with DNA and most importantly, benzyl group helps in cooperative inter-molecular interactions. Increasing the salt concentrations (50, 200 mM NaCl) result in the reduction of fluorescence quenching in a typical FID assay of compound 3a with S. aureus genomic DNA (Figure S8). This indicates that electrostatic interaction plays a major role in the DNA binding of the 6-nitroquinoxaline molecules. This phenomenon is reflected in the activity of the compounds. Bacterial chromosome condensation Alike supercoiled plasmid DNA (pBR322), the condensation event through path intermediates has also been observed in bacterial genomic DNA. AFM images (Figure 7A) explore the path of intercalation induced structural changes in S. aureus genomic DNA (2.8 Mbp). Increasing concentration of 3a leads to formation of local to global structural alteration which ultimately forms DNA superstructures. At 1:1 [3a]: [DNA base pair] ratio DNA strand relaxation occurs and regional nucleation starts by bringing the strands at close proximity. Cooperative binding nature of the molecule further progresses the superstructure formation with increasing concentration of 3a (at 2:1 ratio). Most of the DNA was found to be in condensed form at 3:1 ratio. This indicates 3a can also induce structural change in large DNA 19



like bacterial genomic DNA. The oversupercoiled DNA, including structurally altered intermediates seems to be intolerable and lethal for the bacterial cells. One of the possible outcomes can be that these structures might hinder the function of DNA binding protein machineries inside the cell and ultimately lead to bacterial cell death. Bacterial chromosome dynamically interacts with various DNA binding proteins and occupies a sub cellular area inside a bacterial cell.4,6 This structural dynamicity of bacterial nucleoid (which is maintained by a number of nucleoid structural proteins) is crucial for maintaining the normal cellular functions of the bacteria.5,29,30 We further investigated the DNA condensation event by monitoring the nucleo-cytoplasmic (N/C) ratio in the DAPI (4′,6-diamidino-2-phenylindole) stained 3a treated or untreated S. aureus cells. An overall shrinkage of nucleoid (DAPI stained) area was observed in 3a (10µM) treated S. aureus cells when compared to control (DMSO) cells (Figure 7B). A significant decrease in nucleoid areas of S. aureus cell population (~ 30%) was observed when the average nucleoid area of 3a treated cells was plotted against control cells (Figure S13B). This result suggests that DNA condensation event also occurs in cellular condition, which is in accordance with in-vitro conditions. To strengthen the findings by considering the size of individual bacterial cells, we have calculated the [Nucleoid]/ [cytoplasmic] ratios (n=35) and analyzed in density contour plots (Figure 7C-7D). A plot of N/C ratios of 3a treated and untreated S. aureus cells indicate, nucleo-cytoplasmic ratios significantly decreases in 3a treated cells (Figure 7B). The correlation pattern of nucleoid, cytoplasmic surface area (Figure 7D) and frequency density plot (Figure S13A) for 3a treated and untreated S. aureus cell population also supports the significance of nucleoid condensation event. These results also supports the occurrence of the global DNA structural change (which was observed in AFM images) in the form of nucleoid condensation event in the 3a treated S. aureus cell population.

20


Page 20 of 49

Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7: (A) AFM images of S. aureus genomic DNA (untreated) and treated with indicated [compound]:[DNA base pair] ratio of 3a. (B) Untreated (0.5% DMSO) and treated (with 10µM of 3a) S. aureus cells in phase contrast (for cytoplasmic area) and stained with DAPI (to determine the area of bacterial nucleoid). FFT (Fast Fourier Transform) represents the merge image of cytoplasmic area (circles) and nucleoid area (white regions inside the circles). (C) N/C ratios were plotted for both control and treated samples (n=35) that showed significant reduction in case of treated samples as compared to control. Values are presented as mean ± SD, * P< 0.05, ** P< 0.01, *** P< 0.005. (D) Nucleoid and cytoplasmic surface area showed similar correlation pattern (𝜏1 = 0.84, 𝜏2 = 0.79) for both control and treated samples but treated cell showed overall compaction of nucleoid surface area. Inhibition of DNA synthesis in S. aureus DNA replication is very rapid in the log phase of bacterial growth. During the replication process, a number of DNA binding proteins dynamically interact with bacterial DNA which is required for opening up the DNA strands and polymerization process. Intercalation guided structural change in DNA may influence the DNA replication machineries which can lead to accumulation of non-replicative cells and inhibition of growth. Here, we have investigated

21



the effect of 3a induced DNA-structural change on global replication by monitoring the level of newly synthesized DNA in S. aureus cells. To test this hypothesis, we assessed the ability of 3a exposed S. aureus in synthesizing DNA during its growth phase by incorporating BrdU (bromodeoxyuridine) and identified actively replicating cells using FITC-tagged anti-BrdU antibody.31 Our results show that S. aureus treated with sub-minimal concentration of 3a (3.5 µM or 1.56 µg/mL) failed to retain the anti-BrdU antibody stain whereas majority of the untreated cells were found to be positively stained (Figure 8A). To further confirm our hypothesis, treated or untreated S. aureus in the log phase was pulsed with 3HThymidine

3HThymidine

for 30 minutes and cellular incorporation of

was measured.32,33 Our results clearly indicate that there is a decrease in

radioactive signal (of

3HThymidine)

in treated bacteria with respect to untreated bacteria

(Figure 8B). Previously we have shown that 3g binds to dsDNA, alters the native structure of DNA and induces DNA superstructure formation which leads to eviction of histone from in vitro assembled nucleosome.21 This phenomenon indicates that, 3g has the ability to interfere with the binding of DNA interacting proteins. Accumulation and binding of replication responsive machinery can be blocked due to DNA structural change. DNA condensation and intercalation properties of the active small molecules may greatly influence the binding process of proteins responsible for DNA synthesis. Though the actual molecular mechanism – how the replication machinery fails to synthesize the DNA in S. aureus cells remains unexplored, but the DNA binding, intercalation, structural change are the most likely reasons for the global DNA synthesis stress in a bacterial cell population.

22


Page 22 of 49

Page 23 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8: (A) Global DNA synthesis in S. aureus cells. 3a (3.5µM) treated or untreated cells was pulsed with BrdU and stained with anti BrdU antibody. Images were captured in FITC channel at 60X magnification; scale bar-20 µm. (B) The level of global replication was measured in untreated or 3a (3.5µM, 5µM) treated S. aureus cells by incorporation of 3HThymidine

in the replicating DNA. 5-fluorouracil (5FU-2 µM) taken as a positive control.

Bacterial DNA fragmentation and DNA breaks: Programmed cell death (PCD) in higher eukaryotic cells is a well-known phenomenon, where a series of events lead to cell death. Recent studies suggest similar types of events are observed in antibiotic treated bacteria.34 A very well characterized hallmark in PCD is DNA fragmentation. Massive DNA breaks, resulting from treatment of antibiotics, DNA damaging agents or other factors can produce fragmented chromosomal DNA directly or indirectly, ultimately resulting cell death. Antibiotic induced bacterial DNA fragmentation has been well studied using a DNA fluorochrome SYBR Gold.35 In this study, we have used EtBr to probe the ability of 3a in generating fragmented DNA in S. aureus, by single cell nucleoid diffusion based techniques. Unexposed S. aureus genomic DNA appears as a dense, single nucleoid structure with surrounding DNA fibers (Figure 9A). In 3a (sub-MIC) exposed cells, this nucleoid structure is heavily distorted with a significant increase in chromosomal DNA fragmentation (Figure 9A). To determine the presence of DNA breaks in S. aureus, terminal deoxynucleotidyltransferase (TdT) has been used that can incorporate fluorescent nucleotides at the free 3’-OH end of DNA strand-break. This assay is commonly used to determine DNA breaks in mammalian cells (TUNEL).36 In sub-MIC 3a treated cells, a significant increase in the fluorescence intensity has been recorded as compared to untreated cells (Figure 9B Left pannel), implying 3a induced DNA breaks in S. aureus. Thus, our study indicates massive DNA break and genomic DNA fragmentation occurring on exposure to 3a which correlates with the antibacterial outcome of the compound. This may be a direct or indirect effect of DNA targeting nature of the molecules and replication stress in S. aureus cells. As discussed earlier, 3a induces structural change in mammalian genomic DNA. Extensive cellular DNA damage was also found in mammalian cell (HEK 293) when treated with slight higher concentration (15 µM) of 3a (Figure 9B Left pannel).

23



Figure 9: DNA break in S. aureus: (A) Single cell nucleoid fragmentation was detected in sub-lethal concentration (3.5 µM) of 3a followed by EtBr staining. Images were captured at 60X magnification; scale bar-10µm. (B) Cells were treated with 3a (3.5 µM) or 0.5 % DMSO (control) and subjected to incorporation of fluorescence nucleotides by terminal deoxynucleotidyltransferase (TdT). Images were captured at 60X magnification; scale bar-5 µm. (C) DNA break in mammalian cells detected by TUNNEL assay; HEK 293 cells were treated with 3a (15 µM) or 0.5 % DMSO (control) and subjected to incorporation of fluorescence nucleotides by terminal deoxynucleotidyltransferase (TdT). Images were captured at 60X magnification; scale bar-20 µm. Morphology plasticity induced by 3a in S. aureus: Morphology alteration of bacteria is an interesting phenomenon, which can be observed under different stressful conditions.37 In such cases, some bacteria adapts survival strategies by changing size and shape to increase its surface area.38 Filamentation is one of such reported events observed in uropathogenic E. coli (UPEC) to escape phagocytic killing events of host defense.39-41 S. aureus are normally clustered round shaped cells. Here, the change in

24


Page 24 of 49

Page 25 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the morphology of S. aureus cells has been observed upon treatment of 3a at 3.5 µM (which is lower than the MIC value against S. aureus) and 5 µM (higher than the MIC value) concentrations. AFM images (Figure 10A) indicate that the untreated cells are mostly round shaped clustered with a mean length of around 1.37 ±0.09 µm. Whereas, upon exposure to 3a at sub-MIC (3.5 µM) and greater than MIC dose (5 µM), the length becomes 2.30 ±0.22 µm and 2.36 ±0.22 µm respectively (Figure 10A). The bacterial cells become aggregated as observed under AFM. Notably, the increase in cell length for both the concentrations was more or less similar. Morphological alteration has also been observed by light microscopy wherein the bacterial cells were stained with acridine orange (AO) (Figure 10B). The population of cells which respond to morphological changes after exposure to 3a has been quantified by flow cytometry42 which indicates that the percentage of morphologically altered cells increases in a concentration dependent manner (Figure 10C). Targeting DNA by small molecules or production of ROS can cause DNA damage, which induces SOS response, resulting in an important impact on bacterial cell shape alteration.43 sulA (in E. coli), is an SOS responsive gene that is responsible for inhibiting cell division by blocking FtsZ polymerization and Z ring formation, resulting cell size and shape alteration.44 It was previously reported, that morphology change induced by quinoxaline 1,4-di-N-oxides (QNOs) were broadly dependent on DNA damage responsive SOS activation and sulA overexpression in E.coli cells.45 lexA, a DNA damage retrieval gene was also up regulated in QNOs treated E. coli. Quinoxaline 1,4-di-N-oxides also induce morphology alteration in Clostridium perfringens and Brachyspira hyodysenteriae. DNA damage and replication stress might be responsible and determining factor for the change in morphology of 3a exposed S.aureus cells.46 However, SOS response in this case is yet to be explored. Inhibition of DNA replication may result in incomplete cell division and DNA damage may activate SOS response-implicated by elongation of cells.

25



Figure 10. Morphology alteration of S. aureus cells under compound stress.(A) AFM images of S. aureus cells untreated or treated with 3a-3.5 µM (1.56 µg/mL), 5 µM (2.24 µg/mL) visualized under 100 mm scanner. Cell lengths (µm) were plotted versus concentration of 3a. (B) Visualization of S.aureus cells treated or untreated with 3.5 µM of 3a visualized under a semi-confocal microscopy stained with acridine orange (AO) under 60X magnification, scale bar-5 µm. (C) Quantification of morphologically altered bacterial cell population treated with 3a (3.5 µM, 5 µM, 7.5 µM) determined by flow cytometry. Membrane integrity and permeability measurement DNA replication inhibition may induce incomplete cell division. Peptidoglycan synthesis (spatial PG) is greatly dependent on cell division machinery.37 Faulty cell division may create stress in PG synthesis resulting in the loss of cell wall integrity in bacteria. Cell wall plays a crucial role in maintaining cell shape and resisting osmotic pressure. Healthy bacterial cells with intact membrane are impermeable to propidium iodide (PI).47 Bacterial cells with altered membrane architecture are more permeable to PI than normal cells and this feature is used to differentiate between a normal cell from a damaged one in flow cytometry. When S. 26


Page 26 of 49

Page 27 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aureus cells were treated with sub-MIC or higher concentration with 3a (Figure 11A), population of PI-positive cells increased significantly. In sub-MIC dose, the PI uptake was in ~ 8% of cell population whereas for higher than MIC concentration, the value was in ~ 20%. This demonstrates that higher uptake of PI in treated cells in comparison to untreated cells (~ 1.7 %), suggesting significant disruption of bacterial membrane. To study the membrane integrity upon exposure to 3a, extracellular alkaline phosphatase (ALP) activity was measured in E. coli cells. ALP is an enzyme, present in E. coli periplasmic space. Loss of membrane integrity in bacterial cell may result in exposure of ALP in extracellular condition, which can be determined in a colorimetric based assay.14 In this assay, treatment of cells with sub-MIC or higher concentration of 3a resulted in an increase in ALP activity with respect to untreated cells (Figure 11B), indicating significant disruption of membrane architecture.

27



Figure 11. (A) Membrane permeability detected by measuring propidium iodide (PI) penetration inside the bacterial cells treated with 3a (3.5 µM, 5 µM) as compared to untreated cells. (B) Effect on membrane integrity upon exposure to 3a was detected by extracellular ALP level in 3a (3.5 µM, 5 µM) exposed bacteria. Purified ALP enzyme with substrate pnitrophenylphosphate (PNPP) was taken as a positive control (ALP + PNPP). Disruption of preformed biofilm in S. aureus One of the greatest challenges for any antibiotic is penetration inside the biofilms that are associated with persistent infections.48 Mostly comprising of extracellular polysaccharides, proteins and DNA, biofilms pose a strong challenge to antibiotics in comparison to Planktonic bacterial cells. Cells that form the biofilm, experience very stringent conditions for growth and their survival strategies are based on their ability to exchange genetic information horizontally with other species present in the biofilm. S. aureus is a gram positive pathogen responsible for various infections ranging from skin infection to serious bacteraemia. S. epidermis cells are opportunistic pathogens responsible for nosocomial infection in patients undergoing various medical implants.49 Both of the bacteria are well known for biofilm formation which reduces the effectiveness of antibiotic treatment. As discussed earlier, 3a has antibacterial activity on planktonic S. aureus (1.79 μg/mL). Preformed S. aureus biofilms that were treated with different concentrations of 3a indicates disruption of biofilm started at minimal inhibitory concentration of the compound. The biofilms were almost completely disrupted at four fold MIC, as observed under a light microscope (Figure 12A-12B). Similarly, 3a also has potential to disrupt preformed S. epidermis biofilm (Figure S14).

28


Page 28 of 49

Page 29 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 12. Biofilm disruption assay. (A) Visualization of compound 3a treated (1 MIC, 2 MIC, 4 MIC) or untreated S. aureus biofilm under light microscope at 20X. (B) Quantification and visualization of biofilm formation in bacteria using crystal violet stain. PT- represents pre-treatment. (C) Images of the well of biofilms without (control) or with treatment. CONCLUSION We have successfully designed and synthesized a series of 6-nitroquinoxaline derivatives, with potent antibacterial activities, primarily against gram positive bacteria. Intercalation induced structural change of bacterial genomic DNA is likely responsible factor for the activity of these derivatives which, in turn are dependent on the substituents in benzyl moiety and amino group. These molecules probably act on DNA and exhibit DNA responsive cellular responses. Cell membrane damage and change of cellular morphology was also observed. The molecules also have the potential to disrupt preformed biofilms. There is a future prospect to design more potent mono-quinoxaline based compounds to target GC rich bacteria like Mycobacterium tuberculosis (GC ~ 66%) and multi drug resistant (MDR) pathogens.

29



EXPERIMENTAL SECTION General Information: All chemicals and solvents were purchased commercially as a reagent grade and used without further purification unless especially mentioned. Solvents were distilled well before use. All reactions were carried out in oven-dried glassware at anhydrous condition under nitrogen atmosphere unless otherwise noted and monitored by thin layer chromatography (TLC) (Merck silica gel 60 F254) under UV (254 and 365 nm) lamp. Column chromatography was carried out using silica gel (100-200 and 230-400 mesh).1H nmr spectra were recorded at 300, 400 and 600 MHz frequency, and

13C

nmr spectra were recorded at 100 and 150 MHz

frequency by using CDCl3, CD3OD and DMSO-d6 as solvents. Chemical Shifts are given in parts per million using TMS as an internal standard .Coupling constants (J) are given in Hz with signals are abbreviated as: singlet, s; broad singlet, br s; doublet, d; double-doublet, dd; triplet, t; multiplet, m. High resolution mass spectra were collected by using EI and ESI techniques (JEOL-JMS 700 and XEVO G2-XS Q-Tof mass spectrometer respectively). The purities (> 95 %) of all compounds were determined by analytical HPLC (1 mL/min flow, 0100 % linear gradient of solvent B (either methanol/ Acetonitrile) in A (0.05 % TFA in water) over 16 min) on Shimadzu SCL-10A VP instrument. General Synthesis: All compounds were synthesized following the procedure for the synthesis of 3g as reported earlier,22 by using 2,3-Dichloro-6-nitroquinoxaline as starting material in two steps. In the first step , to a solution of 2,3-Dichloro-6-nitroquinoxaline (1.229 mmol, 1 equiv) in DCM (20 ml) , a benzyl amine derivative (1.475 mmol, 1.2 equiv) was added , followed by the addition of CaCO3 (3.442 mmol, 2.8 equiv) which was stirred for 18 h at rt. After the consumption of starting material as indicated by TLC, water (50 ml) was added and product was extracted with DCM, dried over Na2SO4 to procure the crude product. Using column chromatography with ethyl acetate and pet ether as eluents, intermediates 2a-p were purified (with 40-81 % isolated yield). Next, to a solution of N-benzyl-3-chloro-6-nitroquinoxaline or its derivative (2a-j) (0.25 mmol, 1 equiv) in dry dioxane (10 ml), XantPhos (0.025 mmol, 0.1 equiv), Cs2CO3 (0.375 mmol, 1.5 equiv) and Pd2(dba)3 (0.0125 mmol, 0.05 equiv) were added. Finally amine or substituted amine (0.375 mmol, 1.5 equiv) was added and the reaction mixture was refluxed for 4 h. After the completion of the reaction (as indicated by TLC), water was added and the product was extracted with DCM. Then, organic phase was concentrated and the purified 30


Page 30 of 49

Page 31 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


products (3a-f, 3h-u) were obtained by column chromatography using MeOH and CHCl3 as eluents. N2-(4-(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3diamine(3a). Compound 3a was synthesized according to the general procedure by using 4(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1propylamine (0.375 mmol) in the last step. Reddish solid; mp 130-132 oC; yield 55 %. 1H NMR (600 MHz, DMSO-d6) δ: 8.43 (br s, 1H), 8.14 (d, J = 2.4 Hz, 1H), 7.93 (dd, J = 2.4, 9 Hz, 1H), 7.69 (d, J = 7.8 Hz, 2H), 7.64 (br s, 1H), 7.62 (d, J = 7.8 Hz, 2H), 7.44 (d, J = 9 Hz, 1H), 4.82 (d, J = 5.4 Hz, 2H), 3.52 (dd, J = 6.6, 12 Hz, 2H), 2.63 (t, J = 6.9 Hz, 2H), 2.34 (s, 6H), 1.88 (m, 2H);

13C

NMR (150 MHz, DMSO-d6) δ: 145.6, 145.2, 144.2, 143.1, 142.1,

136.3, 129.0 (4C), 128.1 (q, JC-F = 31 Hz, 1C), 125.7 (dd, JC-F = 3, 7.5 Hz, 1C), 125.5, 120.1, 118.3, 56.5, 44.6 (2C), 44.2, 39.3, 25.6; HRMS (EI+): m/z calculated for C21H23F3N6O2 [M+]: 448.1835; found 448.1838; HPLC purity: 100 %. N2-(4-(trifluoromethyl)benzyl)-N3-(3-(4-methylpiperazin-1-yl)propyl)-6-nitroquinoxalin e-2,3-diamine(3b). Compound 3b was synthesized according to the general procedure by using 4-(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3-(4-methylpiperazin -1-yl)propan-1-amine (0.375 mmol) in the last step. Yellowish solid; mp 155-157 oC; yield 65 %. 1H NMR (600 MHz, CDCl3) δ: 8.44 (d, J = 2.4 Hz, 1H), 8.04 (dd, J = 2.4, 9 Hz, 1H), 7.60 (d, J = 7.8 Hz, 2H), 7.55 (m, 3H), 5.71 (br s, 1H), 4.95 (d, J = 5.4 Hz, 2H), 3.67 (t, J = 5.1 Hz, 2H), 2.68 (t, J = 5.7 Hz, 2H), 2.61 (br s, 4H), 2.38 (br s, 4H), 2.14 (s, 3H), 1.94(m, 2H); 13C NMR (150 MHz, CDCl3) δ: 145.2, 144.9, 144.1, 142.6, 141.4, 136.6, 129.9 (q, JC-F = 32 Hz, 1C), 128.3 (4C), 125.9, 125.7 (dd, JC-F = 3, 7.5 Hz, 1C), 121.3, 118.6, 58.2, 54.8, 53.2 (2C), 45.8 (2C), 44.7, 42.5, 23.1; HRMS (ESI): m/z calculated for C24H28F3N7O2 + H+ [M+H+]: 504.2335; found 504.2338; HPLC purity: 98.1 %. N2-(4-(trifluoromethyl)benzyl)-N3-(4-(dimethylamino)butyl)-6-nitroquinoxaline-2,3diamine(3c). Compound 3c was synthesized according to the general procedure by using 4(trifluoromethyl)benzylamine (1.475 mmol) in the first step and N,N-dimethylbutane-1,4diamine (0.375 mmol) in the last step. Reddish solid; mp 116-117 oC; yield 52 %; 1H NMR (400 MHz, CDCl3) δ: 8.41 (d, J = 2.8 Hz, 1H), 8.03 (dd, J = 2.6, 9 Hz, 1H), 7.57 (s, 4H), 7.53 (d, J = 8.8 Hz, 1H), 6.96 (br s, 1H), 4.90 (d, J = 5.6 Hz, 2H), 3.63 (t, J = 5.4 Hz, 2H), 3.23 (br s, 1H), 2.70 (t, J = 6.2 Hz, 2H), 2.44 (s, 6H), 1.86 (m, 4H); 13C NMR (100 MHz, CDCl3) δ: 31



145.3, 145.0, 143.8, 143.1, 141.8, 136.5, 129.6 (q, JC-F = 32 Hz, 1C), 128.6 (4C), 125.6, 125.5 (dd, JC-F = 4, 7 Hz, 1C), 121.1, 118.5, 58.8, 44.6, 44.3 (2C), 40.6, 25.9, 24.1; HRMS (ESI): m/z calculated for C22H25F3N6O2 + H+ [M+H+]: 463.2069; found 463.2080; HPLC purity: 97.3 %. N-(4-(trifluoromethyl)benzyl)-6-nitro-3-(piperidin-1-yl)quinoxalin-2-amine(3d). Compound 3d was synthesized according to the general procedure by using 4(trifluoromethyl)benzylamine (1.475 mmol) in the first step and piperidine (0.375 mmol) in the last step. Light yellow solid; mp 143-145 oC ; yield 74 %; 1H NMR (600 MHz, CDCl3) δ: 8.61 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 3.0, 9 Hz, 1H), 7.64 (m, 3H), 7.52 (d, J = 7.8 Hz, 2H), 5.98 (br s, 1H), 4.87 (d, J = 6 Hz, 2H), 3.25 (t, J = 5.1 Hz, 4H), 1.76 (m, 4H), 1.69 (m, 2H); 13C

NMR (150 MHz, CDCl3) δ: 150.9, 148.4, 143.8, 143.8, 142.4, 135.5, 129.9 (q, JC-F = 33

Hz, 1C), 128.0 (4C), 126.1, 125.7 (dd, JC-F = 3, 7.5 Hz, 1C), 123.4, 121.4, 50.3 (2C), 44.9, 25.8 (2C), 24.3; HRMS (ESI): m/z calculated for C21H20F3N5O2 + H+ [M+H+]: 432.1647; found 432.1656; HPLC purity: 96.7 %. N-(4-(trifluoromethyl)benzyl)-3-(4-methylpiperazin-1-yl)-6-nitroquinoxalin-2amine(3e). Compound 3e was synthesized according to the general procedure by using 4(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 1-methylpiperazine (0.375 mmol) in the last step. Light yellow solid; mp 83-85 oC; yield 70 %; 1H NMR (600 MHz, CDCl3) δ: 8.62 (d, J = 2.4 Hz, 1H), 8.23 (dd, J = 2.4, 9 Hz, 1H), 7.66 (d, J = 9 Hz, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 5.90 (br s, 1H), 4.87 (d, J = 6 Hz, 2H), 3.38 (br s, 4H), 2.64 (br s, 4H), 2.38 (s, 3H);

13C

NMR (150 MHz, CDCl3) δ: 149.8, 148.0, 144.0,

143.8, 142.3, 135.4, 130.0 (q, JC-F = 32 Hz, 1C), 128.0 (4C), 126.2, 125.8 (dd, JC-F = 3, 7.5 Hz, 1C), 123.5, 121.6, 54.6 (2C), 48.8 (2C), 46.1, 44.9; HRMS (ESI): m/z calculated for C21H21F3N6O2 + H+ [M+H+]: 447.1756; found 447.1762; HPLC purity: 97.2 %. N-(4-(trifluoromethyl)benzyl)-6-nitro-3-(piperazin-1-yl)quinoxalin-2-amine(3f). Compound 3f was synthesized according to the general procedure by using 4(trifluoromethyl)benzylamine (1.475 mmol) in the first step and piperazine (0.375 mmol) in the last step. Light yellow solid; mp 81-83 oC; yield 62 %; 1H NMR (600 MHz, CDCl3) δ: 8.62 (d, J = 1.8 Hz, 1H), 8.23 (dd, J =2.4, 9 Hz, 1H), 7.66 (d, J = 9 Hz, 1H) 7.63 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H), 5.98 (br s, 1H), 4.87 (d, J = 5.4 Hz, 2H), 3.34 (br s, 4H), 3.11 (br s, 4H);

13C

NMR (150 MHz, CDCl3) δ: 150.1, 148.1, 144.0, 143.8, 142.3, 135.4, 32


Page 32 of 49

Page 33 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


130.0 (q, JC-F = 32 Hz, 1C), 128.1 (4C), 126.2, 125.8 (dd, JC-F = 3, 7.5 Hz, 1C), 123.6, 121.7, 50.1 (2C), 45.6 (2C), 44.9; HRMS (ESI): m/z calculated for C20H19F3N6O2 + H+ [M+H+]: 433.1600; found 433.1620; HPLC purity: 96.8 %. N2-(4-bromobenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3h). Compound 3h was synthesized according to the general procedure by using 4bromobenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 152-154 oC; yield 75 %; 1H NMR (600 MHz, CDCl3) δ: 8.46 (d, J = 9.0 Hz, 1H), 8.25 (br s, 1H), 8.06 (dd, J = 2.4, 9.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 5.00 (br s, 1H), 4.72 (d, J = 4.8 Hz, 2H), 3.66 (t, J = 5.4 Hz, 2H), 2.55 (t, J = 5.1 Hz, 2H), 2.12 (s, 6H), 1.85 (m, 2H); 13C NMR (150 MHz, CDCl3) δ: 145.3, 145.0, 144.1, 141.5, 137.2, 136.8, 132.0 (2C), 130.1 (2C), 125.8, 121.9, 121.2, 118.4, 59.9, 45.4, 45.1 (2C), 42.9, 23.8; HRMS (ESI): m/z calculated for C20H23BrN6O2 + H+ [M+H+]: 459.1138; found 459.1142; HPLC purity: 98.4 %. N2-(4-chlorobenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3i). Compound 3i was synthesized according to the general procedure by using 4chlorobenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 158-160 oC; yield 71 %; 1H NMR (600 MHz, DMSO-d6) δ: 8.13 (s,1H), 8.05 (br s, 1H), 7.93 (d, J = 9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.41 (q, J = 8.4 Hz, 4H), 7.33 (br s, 1H), 4.73 (d, J = 4.8 Hz, 2H), 3.49 (m, 2H), 2.30 (t, J = 6.6 Hz, 2H), 2.14 (s, 6H), 1.77 (m, 2H);

13C

NMR (150 MHz, DMSO-d6) δ: 145.5, 145.1,

143.1, 142.1, 138.1, 136.3, 132.2, 130.3 (2C), 128.8 (2C), 125.5, 120.1, 118.2, 57.2, 45.7 (2C), 44.1, 40.5, 26.6; HRMS (ESI): m/z calculated for C20H23ClN6O2 [M+]: 414.1571; found 414.1574; HPLC purity: 100 %. N2-(4-fluorobenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3j). Compound 3j was synthesized according to the general procedure by using 4fluorobenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 148-150 oC; yield 59 %; 1H NMR (300 MHz, CD3OD in CDCl3) δ: 8.43 (d, J = 2.4 Hz, 1H), 8.07 (dd, J = 2.4, 8.7 Hz, 1H), 7.59 (d, J = 9 Hz, 1H), 7.45 (dd, J = 5.7, 8.1 Hz, 2H), 7.05 (t, J = 8.7 Hz, 2H), 6.08 (br s, 1H), 4.76 ( d, J = 4.8 Hz, 2H), 3.70 (t, J = 5.6 Hz, 2H), 2.81 (t, J = 5.6 Hz, 2H), 2.38 (s, 6H), 2.03 (m, 2H); 13C NMR (150 MHz, DMSO-d6) δ: 161.8 (d, JC-F = 241.5 Hz, 1C), 145.5, 145.1, 143.0, 142.2, 33



Page 34 of 49

136.3, 135.2 (d, JC-F = 3 Hz, 1C), 130.5 (d, JC-F = 9 Hz, 2C), 125.5, 120.1, 118.3, 115.6 (d, JC-F = 3 Hz, 2C), 56.8, 45.0 (2C), 44.0, 39.5, 26.0; HRMS (EI+): m/z calculated for C20H23FN6O2 [M+]: 398.1867; found 398.1871; HPLC purity: 97.6 %. N2-(4-methylbenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3k). Compound 3k was synthesized according to the general procedure by using 4methylbenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Yellow solid; mp 187-189 oC; yield 82 %; 1H NMR (600 MHz, CD3OD in CDCl3) δ: 8.34 (d, J = 2.4 Hz, 1H ), 7.99 (dd, J = 8.7, 2.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 7.8 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 6.95 (br s, 1H), 4.70 (s, 2H), 3.57 (t, J = 6.3 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.39 (s, 6H), 2.28 (s, 3H), 1.92 (m, 2H);

13C

NMR (150 MHz, CD3OD in CDCl3) δ: 145.3, 144.9, 143.4, 141.9, 137.1, 135.7, 135.1, 129.1 (2C), 128.3 (2C), 125.2, 120.5, 118.5, 56.8, 45.0, 44.2 (2C), 39.1, 25.1, 20.9; HRMS (ESI): m/z calculated for C21H26N6O2 + H+ [M+H+]: 395.2195; found 395.2194; HPLC purity : 97.1 %. N2-(4-methoxylbenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3diamine(3l). Compound 3l was synthesized according to the general procedure by using 4methoxylbenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Yellow solid; mp 158-160 oC; yield 87 %; 1H NMR (600 MHz, CD3OD in CDCl3) δ: 8.36 (d, J = 2.4 Hz, 1H ), 8.01 (dd, J = 2.4, 9 Hz, 1H), 7.54 (d, J = 9 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 9 Hz, 2H), 6.95 (br s, 1H), 4.69 (s, 2H), 3.76 (s, 3H), 3.59 (t, J = 6.3 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.39 (s, 6H), 1.94 (m, 2H); 13C NMR (150 MHz, CD3OD in CDCl3) δ: 158.9, 145.2, 144.8, 143.4, 141.98, 135.8, 130.3, 129.8 (2C), 125.2, 120.6, 118.5, 113.9 (2C), 57.0, 55.2, 44.8, 44.2 (2C), 39.2, 25.0; HRMS (ESI): m/z calculated for C21H26N6O3 + H+ [M+H+]: 411.2145; found 411.2142; HPLC purity: 96.9 %. N2-(2-(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3diamine(3m). Compound 3m was synthesized according to the general procedure by using 2(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1propylamine (0.375 mmol) in the last step. Yellow solid; mp 189-191 oC; yield 76 %; 1H NMR (600 MHz, CD3OD in CDCl3) δ: 8.41 (d, J = 2.4 Hz, 1H ), 8.03 (dd, J = 2.4, 9 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.5 (t, J = 7.2 Hz, 34


Page 35 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1H), 7.39 (t, J = 7.5 Hz, 1H), 6.48 (br s, 1H), 4.99 (s, 2H), 3.65 (t, J = 5.7 Hz, 2H), 2.66 (t, J = 6 Hz, 2H), 2.34 (s, 6H), 1.96 (m, 2H); 13C NMR (100 MHz, CD3OD in CDCl3) δ: 145.2, 144.9, 143.8, 141.8, 136.9, 136.2, 132.1, 130.5, 128.5 (q, JC-F = 32 Hz, 1C), 127.6, 126.0 (dd, JC-F = 6, 12 Hz, 1C), 125.6, 123.2, 120.8, 118.5, 57.3, 44.5 (2C), 41.9, 39.7, 25.2; HRMS (ESI): m/z calculated for C21H23F3N6O2 + H+ [M+H+]: 449.1913; found 449.1910; HPLC purity: 96.7 %. N2-(2-bromobenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3n). Compound 3n was synthesized according to the general procedure by using 2bromobenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 153-154 oC; yield 56 %; 1H NMR (400 MHz, CDCl3) δ: 8.43 (d, J = 2.4 Hz, 1 H ), 8.06 (dd, J = 2.6, 9 Hz, 1H), 7.59 (m, 3H), 7.29 (t, J = 7.4 Hz, 1H), 7.17 (t, J = 7.0 Hz, 1H), 5.85 (br s, 1H), 4.89 (d, J = 4.8 Hz, 2H), 3.69 (t, J = 5.6 Hz, 2H), 2.74 (t, J = 5.4 Hz, 2H), 2.34 (s, 6H), 1.96 (m, 2H); 13C NMR (150 MHz, CDCl3) δ: 145.2, 144.9, 143.9, 141.7, 137.4, 136.5, 132.9, 131.1, 129.4, 127.7, 125.7, 124.2, 121.0, 118.4, 59.0, 45.8, 44.7 (2C), 41.7, 23.7; HRMS (ESI): m/z calculated for C21H23BrN6O2 + H+ [M+H+]: 459.1144; found 459.1143; HPLC purity: 96.3 %. N2-(2-methylbenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3o). Compound 3o was synthesized according to the general procedure by using 2methybenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 182-184 oC; yield 77 %; 1H NMR (600 MHz, CD3OD in CDCl3) δ: 8.39 (d, J = 2.4 Hz, 1H ), 8.02 (dd, J = 8.7, 2.7 Hz, 1H), 7.56 (d, J = 9 Hz, 1 H), 7.35 (d, 7.2 Hz, 1 H), 7.19 (m, 3 H), 6.35 (br s, 1 H), 4.74 (s, 2 H), 3.60 (t, 6.3 Hz, 2H), 2.58 (t, J = 6.3 Hz, 2H), 2.39 (s, 3H), 2.26 (s, 6H), 1.90 (m, 2H); 13C NMR (600 MHz, CD3OD in CDCl3) δ: 145.2, 144.8, 143.6, 141.9, 136.9, 136.0, 135.9, 130.4, 129.3, 127.9, 126.1, 125.3, 120.8, 118.4, 57.5, 44.4 (2C), 43.8, 43.7, 24.8, 19.0; HRMS (ESI): m/z calculated for C21H26N6O2 + H+ [M+H+]: 395.2195; found 395.2195; HPLC purity: 97.8 %. N2-(2-methoxybenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3diamine(3p). Compound 3p was synthesized according to the general procedure by using 2methoxybenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 184-186 oC; yield 72%; 1H NMR (400 MHz, CD3OD in CDCl3) δ: 8.34 (d, J = 2.4 Hz, 1H ), 7.99 (dd, J = 2.6, 9 Hz, 1H), 7.54 (d, J = 8.8 35



Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.89 (t, J = 8.2 Hz, 2H), 6.58 (br s, 1H), 4.77 (s, 2H), 3.85 (s, 3H), 3.63 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 7.0 Hz, 2H), 2.44 (s, 6H), 1.99 (m, 2H);

13C

NMR (100 MHz, CD3OD in CDCl3) δ: 157.8, 145.7, 145.0, 143.5,

142.2, 135.9, 130.2, 129.0, 126.2, 125.5, 120.8, 120.5, 118.6, 110.5, 57.2, 55.5, 44.1 (2C), 40.9, 39.5, 24.6; HRMS (ESI): m/z calculated for C21H26N6O3 + H+ [M+H+]: 411.2145; found 411.2140; HPLC purity: 97.8 %. N2-(3-(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3diamine(3q). Compound 3q was synthesized according to the general procedure by using 3(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1propylamine (0.375 mmol) in the last step. Orange solid; mp 132-134 oC; yield 61 %; 1H NMR (600 MHz, CDCl3) δ: 8.40 (d, J = 2.4 Hz, 1H), 8.03 (dd, J = 2.4, 9 Hz, 1H), 7.74 (s, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.55 (m, 2H), 7.47 (t, J = 7.8 Hz, 1H), 6.39 (br s, 1H), 4.85 (d, J = 5.4 Hz, 2H), 3.70 (t, J = 6 Hz, 2H), 2.84 (t, J = 6 Hz, 2H), 2.41 (s, 6H), 2.03 (m, 2H); 13C NMR (150 MHz, CDCl3) δ: 145.2, 144.8, 143.9, 141.7, 139.6, 136.3, 132.0, 130.9 (q, JC-F = 32 Hz, 1C), 129.1, 125.7, 125.2 (dd, JC-F = 3, 7.5 Hz, 1C), 124.4 (dd, JC-F = 3, 7.5 Hz, 1C), 123.2, 121.1, 118.6, 58.3, 45.0, 44.3 (2C), 40.7, 23.7; HRMS (ESI): m/z calculated for C21H23F3N6O2 + H+ [M+H+]: 449.1913; found 449.1918; HPLC purity: 96.7 %. N2-(3-bromobenzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine(3r). Compound 3r was synthesized according to the general procedure by 3-bromobenzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 149-151 oC; yield 72 %; 1H NMR (600 MHz, CDCl3) δ: 8.42 (d, J = 2.4 Hz, 1H), 8.05 (dd, J = 2.4, 9 Hz, 1H), 7.60 (br s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.01 (br s, 1H), 4.76 (d, J = 4.8 Hz, 2H), 3.68 (t, J = 5.7 Hz, 2H), 2.74 (t, J = 6 Hz, 2H), 2.34 (s, 6H), 1.98 (m, 2H); 13C NMR (150 MHz, CDCl3) δ: 145.2, 144.9, 143.9, 141.7, 140.8, 136.4, 131.3, 130.8, 130.3, 127.1, 125.7, 122.7, 121.0, 118.5, 58.6, 45.0, 44.5 (2C), 41.3, 23.8; HRMS (ESI): m/z calculated for C20H23BrN6O2 + H+ [M+H+]: 459.1144; found 459.1147; HPLC purity: 96.3 %. N2-(4-chloro-3-(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxali ne-2,3-diamine(3s). Compound 3s was synthesized according to the general procedure by using 4-chloro-3-(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Orange solid; mp 130-132 oC; 36


Page 36 of 49

Page 37 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


yield 62 %; 1H NMR (400 MHz, DMSO-d6) δ: 8.77 (br s, 1H), 8.15 (s, 1H), 7.95 (d, 9.6 Hz, 2H), 7.73 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.35 (br s, 1H), 4.78 (d, J = 4.8 Hz, 2H), 3.55 (d, J = 4.4 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.54 (s, 6H), 1.97 (m, 2H);

13C

NMR (100 MHz, DMSO-d6) δ: 145.6, 145.3, 143.2, 142.1, 139.6, 136.3,

134.2, 132.1, 129.6, 128.0 (dd, JC-F = 5, 10 Hz, 1C), 126.9 (q, JC-F = 30 Hz, 1C), 125.5, 124.8, 120.2, 118.3, 56.1, 43.9 (2C), 43.6, 39.0, 24.9; HRMS (ESI): m/z calculated for C21H22ClF3N6O2 + H+ [M+H+]: 483.1523; found 483.1554; HPLC purity: 96.6 %. N2-(3-fluoro-5-(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxali ne-2,3-diamine(3t). Compound 3t was synthesized according to the general procedure by using 3-fluoro-5-(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3(dimethylamino)-1-propylamine (0.375 mmol) in the last step. Reddish solid; mp 157-158 oC; yield 51 %; 1H NMR (600 MHz, CDCl3) δ: 8.41 (d, J = 2.4 Hz, 1H), 8.04 (dd, J = 2.7, 8.7 Hz, 1H), 7.54 (m, 2H), 7.38 (d, J = 9 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 6.89 (br s, 1H), 4.86 (d, J = 5.4 Hz, 2H), 3.69 (t, J = 5.7 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.49 (s, 6H), 2.07 (m, 2H);

13C

NMR (100 MHz, DMSO-d6) δ: 162.5 (d, JC-F = 245 Hz, 1C), 145.7, 145.3, 144.4,

144.3, 143.3, 142.0, 136.4, 131.7 (q, JC-F = 33 Hz, 1C), 125.6, 121.4 (d, JC-F = 2 Hz, 1C), 120.2, 119.5 (d, JC-F = 22 Hz, 1C), 118.3, 111.9 (d, JC-F = 22 Hz, 1C) 56.4, 44.4 (2C), 43.9, 39.2, 25.4; HRMS (ESI): m/z calculated for C21H22F4N6O2 + H+ [M+H+]: 467.1819; found 467.1830; HPLC purity: 97.9 %. N2-(3,5-bis(trifluoromethyl)benzyl)-N3-(3-(dimethylamino)propyl)-6-nitroquinoxaline2,3-diamine(3u). Compound 3u was synthesized according to the general procedure by using 3,5-bis(trifluoromethyl)benzylamine (1.475 mmol) in the first step and 3-(dimethylamino)-1propylamine (0.375 mmol) in the last step. Reddish solid; mp 179-181 oC; yield 57 %; 1H NMR (600 MHz, CD3OD in CDCl3) δ: 8.33 (d, J = 2.4 Hz, 1H), 7.98 (dd, J = 2.4, 9 Hz, 1H), 7.92 (s, 2H), 7.71(br s, 1H), 7.47 (d, J = 9 Hz, 1H), 4.84 (s, 2H), 3.62 (t, J = 6 Hz, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.57 (s, 6H), 2.04 (m, 2H);

13C

NMR (150 MHz, DMSO-d6) δ: 145.6,

145.3, 143.2, 142.9, 141.9, 136.3, 130.4 (q, JC-F = 33 Hz, 2C), 129.5 (dd, JC-F = 4.5, 7.5 Hz, 2C), 125.4, 124.7, 122.9, 120.2, 118.3, 56.2, 44.1 (2C), 43.9, 39.1, 25.1. HRMS (ESI): m/z calculated for C22H22F6N6O2 + H+ [M+H+]: 517.1787; found 517.1788; HPLC purity: 97.4 %.

37



Determination of Minimal Inhibitory Concentration (MIC) Broth micro dilution assay was performed to determine MIC of all compounds (as per CLSI protocol)

50

against gram positive (S. aureus MTCC737, S. epidermidis MTCC3615, A.

chlorophenolicus A6 MTCC3706) as well as gram negative bacteria (E. coli MTCC1916, P. aeruginosaMTCC1688). Bacterial cultures containing 5×105 cfu/mL in Luria-Bertani (LB) broth were added to each well of a 96 well plate; compounds were dissolved in DMSO and added at respective concentration keeping the final concentration of DMSO under 0.6% and DMSO control was taken as well. The plates were incubated at recommended growth temperature; time for each individual bacterial culture and OD was recorded at 600 nm. Mammalian cell cytotoxicity determination Mammalian cell toxicity of all compounds were determined in HEK 293 (Human Embryonic Kidney) cell line containing 4000 cells/well in a 96 well plate using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT). Cells were seeded in a 96 well plate in DMEM medium supplemented with 10% Fetal Bovine Serum and kept at 37°C overnight in a CO2 incubator until all the cells have properly attached to the substratum. The cells were treated with increasing concentration of the compounds keeping the final DMSO percent less that 0.5% and the control cells were treated with DMSO only. Cells were treated for 24 hours in the CO2 incubator. MTT solution was added at a final concentration of 1 mg/mL and cells were further kept in the incubator for 3 hours until the formazan crystals have properly formed. The plates were then taken out; medium was discarded and 150 μl DMSO was added. The plates were then kept in an orbital shaker for 15-20 mins away from light so that the formazan crystals are completely dissolved.51 Results were obtained by measuring absorbance at 595 nm in a multi-channel plate reader. IC50 values were determined from the OD value obtained from the experimental results done in triplicates. EtBr fluorescence quenching assay S. aureus genomic DNA (10 µM) and EtBr (5 µM) were mixed in a cuvette in buffer containing 10 mM NaP of pH 7 with 10 mM NaCl and 1% DMSO at 25 and 37 °C, titrated with increasing concentrations (2.5-100) µM of respective compound. Fluorescence intensity scan was recorded using a Horiba PTI QuantaMasterTM 8000 fluorescence spectrometer using excitation wavelength at 480 nm and emission wavelength scan from 520 to 700 nm was measured. Results were plotted using normalized fluorescence intensity vs compound concentration. 38


Page 38 of 49

Page 39 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Circular Dichroism (CD) 15 µM CT-DNA in 10 mM CP buffer pH 7.0 with 10 mM NaCl and 1% DMSO were treated with increasing concentration of 3a ranging from 15 µM to 60 µM in a CD compatible quartz cuvette with 10 mm path length. CD spectra were recorded from 230 to 500 nm at 100 nm/min scan speed. For genomic DNA CD experiment, S. aureus and A. chlorophenolicus genomic DNA was isolated from an overnight grown culture using GenElute bacterial genomic DNA isolation kit from Sigma-Aldrich, genomic DNA concentrations was measured using a Cary 50 Bio UV-VIS spectrophotometer and further experiments were performed following previously described experimental condition. Gel-shift assay pCDNA 3.1 plasmid was amplified in E.coliDH5α, isolated using NucleoSpin® Plasmid QuickPure miniprep Kit from MACHEREY-NAGEL, Germany. A reaction condition containing 50 mM NaP buffer of pH 7, 10mMNaCl and 40 μM pCDNA 3.1 plasmid DNA were mixed with different concentration of compounds (calculated as compound concentration :DNA base pair ratio) and incubated for 16 hours at 37°C. Samples were run on 1% agarose gel in 50 V for ~ 3 hours. Gels were stained with EtBr (1μg/mL) at room temperature for 5 mins, developed in a ChemiDoc™ MP Imaging Systemfrom Bio-Radand results were analysed using Image LabTM software. For genomic DNA gel-shift experiments, E.coli genomic DNA was isolated from overnight grown culture using GenElute Bacterial genomic DNA isolation kit from Sigma-Aldrich. Reaction was performed as described earlier with slight modifications wherein the final gDNA concentration of 20 μM per reaction was used, samples were run in 0.6% agarose gel solution and gel electrophoresis was performed at 50 V for ~ 4 hours. Gels were stained with EtBr(1 μg/mL) and visualized in gel doc. DNA condensation in S. aureus cells S.aureus cells (~105 CFU/mL) were grown for 6 hour at 37°C. Cell pellet was washed and dissolved in PBS. Cells were then treated with 10 µM of compound 3a or 0.5% of DMSO (control) for 1 hour at 37°C in shaking condition. Cells were centrifuged at 5000 x g for 10 mins, pellet was collected and gently washed in PBS. The treated and untreated cells were fixed by resuspending in 16% paraformaldehyde solution for 30 mins at 4°C, washed resuspended in PBS. Cells were stained with DAPI for 5 mins at 37°C, washed with PBS, smeared on top of a clear slide, mounted with coverslip using mounting solution and sealed 39



with DPX. Slides were visualized using DAPI filter set in Carl-Ziess Axio Observer LSM 880 confocal laser scanning fluorescence microscope. Cell Surface area and nucleoid surface area were calculated using density threshold function using Image J (Fiji) software. Images were taken from three independent experiments. Total 35 data points were taken of cytoplasmic and Nucleoid Surface area from both Control and treated samples. DNA synthesis stress induced by 3a in S. aureus Log phase S.aureus cell suspension (105to 106 CFU/mL) was treated with 3a for 1 hour at 37°C. 30 min pulse of BrdU (Sigma-Aldrich Stock concentration of 10µM) was added under similar condition. Bacterial cells were fixed with 16 % paraformaldehyde solution for 30 mins at 4°C and permeabilized with (0.5 % Triton X-100 and lysostaphin 0.05 mg/mL) at 4°C for 10 mins. Samples were incubated in 2N HCl for 10 mins to denature the DNA and neutralizing buffer (50 mM Citrate Phosphate buffer pH 7.4) was added and incubated for 10 mins at room temperature. Cells were incubated with FITC tagged anti-BrdU mouse monoclonal antibody (BD Biosciences) for 30 mins in dark at room temperature. Cells were washed with PBS, smeared in L-lysine pre-coated glass slides and observed via FITC channel in Olympus FLUOVIEW FV-10i confocal laser scanning fluorescence microscope. For radioactive DNA synthesis assay 105 to106 CFU/mL log phase S. aureus cells were incubated at different concentration of 3a. After a 30 mins pulse of 3HThymidine (1µCi/well), cells were harvested and radioactive counts were measured after addition of scintillation liquid (Cocktail O) using a Perkin Elmer Tri-Carb 2810TR liquid scintillation counter. DNA fragmentation Treated or untreated S.aureus cells were mixed with low melting agarose, placed on top of a clear glass slide pre-coated with 1% agarose and covered with a coverslip for 30 mins in a moist chamber at 4°C. Coverslips were removed and slides were coated with 1% agarose solution and dried for 20 mins in a moist chamber so that the agarose solidifies and the cells get entrapped within the agarose layer. Slides were then submerged in lysis buffer (1.2 M NaCl, 100 mM Na2-EDTA, 1% Triton X-100, 0.2 M NaOH (pH > 10) overnight in dark at 4°C. Slides were submerged in alkaline electrophoresis buffer (30 mM NaOH, 2 mM Na2EDTA) for 30 mins and electrophoresis were performed at 15 V (0.6 V/cm) for 25 mins. Slides were then neutralized with 50 mM Tris-HCl buffer pH 7.5 and washed with deionised water. Slides were stained with EtBr(1μg/mL) for 3-5 mins and visualised in Olympus 40


Page 40 of 49

Page 41 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FLUOVIEW FV-10i confocal laser scanning fluorescence microscope using a Texas Red filter (~ 620 nm). TUNEL Assay S. aureus cells in suspension treated with compounds were centrifuged at 5000 x g for 10 mins. Cell pellet was washed with PBS and fixed using 16 % paraformaldehyde at 4°C for 30 mins and washed with PBS. Cells were permeabilised with pre-chilled 0.2 % Triton X-100 and lysostaphin (0.05 mg/mL) and incubated at 4°C for 5 mins. Cells were then resuspended in equilibration buffer, nucleotide mix and TdT Enzyme, incubated in humidified dark 37°C incubator for 60 mins for the tailing reaction to occur. The reaction was stopped by incubating with 2X SSC buffer (Saline Sodium Citrate) for 15 mins at room temperature. Cells were centrifuged and pellet resuspended in PBS. The cells were then spreaded over a clean slide, covered by mounting a coverslip using mounting solution and then sealed using DPX (Dibutylphthalate Polystyrene Xylene). Slides were visualized using a standard fluorescein filter set (520 +/- 20 nm) in Olympus FLUOVIEW FV-10i confocal laser scanning fluorescence microscope. Atomic Force Microscopy of bacteria: Control and treated S. aureus cells in LB broth were centrifuged at 5000 x g for 5 mins and resuspended in PBS (pH 7.4). Cells were fixed with 16% paraformaldehyde for 30 mins at 4°C and washed with PBS. Cells were smeared on top of a clear glass coverslip and another coverslip was placed on top of it such that the cells are equally spread and adhere across the glass surface. The coverslips were kept for 2-3 mins, removed and washed by dropwise addition of sterile water using a 2 mL syringe. Any residual water present was air dried. Coverslips were then placed inside a Pico plus 5500 ILM AFM (Agilent Technologies USA) operating in AAC mode. AFM (contact mode) was performed using a Pico plus 5500 AFM (Agilent Technologies USA) using a piezoscanner with a maximum range of 100 μm. Micro fabricated silicon cantilevers of length 450 μm containing a nominal spring force constant of (0.02-0.77) N/m and a resonance frequency of (6 – 21) kHz was used. Images were captured at a scan speed rate of 0.5 lines/second and size ranging from (0.5 – 5) μm. Images were processed using Pico view 1.10.1. Bacterial cell length, height and width were measured manually using Pico scan 5 software.

41



Acrydine Orange Staining: S. aureus cells were treated with 3a and incubated at 37°C for 6 hours. Cells were centrifuged at 5000 x g for 10 mins and fixed by resuspending in 16% paraformaldehyde solution for 30 mins at 4°C. Centrifuged at 5000 x g for 10 mins and resuspended in PBS (pH 7.4). Cells were permeabilised with pre-chilled 0.2% Triton X-100 solution and lysostaphin (0.05 mg/mL) at 4°C for 10 mins, centrifuged and resuspended in PBS. Cells were stained with acrydine orange (3 µM) for 5 mins at 37°C, washed with PBS, smeared on top of a clear slide, mounted with coverslip using mounting solution and sealed with DPX. Slides were visualized using acrydine orange filter set in Olympus FLUOVIEW FV-10i confocal laser scanning fluorescence microscope. Flow Cytometry Analysis: S. aureus cells in LB broth were treated with 3a and incubated at 37°C for 2hours. Cells were centrifuged at 5000 x g for 10 mins, fixed with pre-chilled 80% methanol for 20 mins at 4°C and resuspended in PBS (pH 7.4). Cells were visualized in BD LSRFotressaTM cell analyzer. Results were analyzed using the software FlowJoTM. Membrane permeability determination: Propidium iodide permeability assay: Treated and untreated S. aureus cells in the log phase were centrifuged at 5000 x g for 10 mins and resuspended in PBS. Cells were stained with propidium iodide (4 μg/mL) for 10 mins at room temperature in dark and analyzed using PE/Texas red channel (610 +/- 10) nm in BD LSRFotressaTM cell analyzer. Results were analyzed using FlowJoTM software. Alkaline Phosphatase assay: S. aureus cells in the log phase (3 x 105) were treated with 3a for 2 hours. pnitrophenylphosphate (PNPP) was added and incubated for 1 hour at 37°C. OD was measured in an Elisa plate reader at 410 nm. Biofilm disruption assay: S. aureus and S. epidermidis cells (3 x 105) in LB broth were seeded in a 96 well plate and incubated in an incubator at 37°C for 24 hours for the biofilm to develop. Cells were treated with 3a for 12 hours at 37°C. Plates were washed thoroughly by immersing in sterile water and inverting them over a dry paper towel to remove all planktonic bacterial cells that were 42


Page 42 of 49

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


not involved in biofilm formation. Plates were stained with 1% crystal violet solution, incubated for 10 mins at room temperature in a shaker, washed thoroughly by immersing in water and inverting over a dry paper towel to remove any excess crystal violet not bound to the biofilm formed at the bottom of the plate. Plates were kept overnight to dry and 30% acetic acid solution was added and incubated for 20 mins at room temperature to solubilize the crystal violet. Absorbance was measured at 550 nm using a plate reader. Plates were also placed under a light microscope and images were taken at 20X magnification. The images of the crystal violet stained 96 well plates containing the treated and untreated samples were captured using a digital camera later.

ASSOCIATED CONTENT Supporting information (SI). (PDF) NMR and HPLC spectra of the compounds. (PDF) Molecular formula strings. (CSV) AUTHOR INFORMATION Corresponding Author *S.D: e-mail, [email protected] ORCID Sanjay Dutta: 0000-0003-0435-5741 Author Contributions KSA, RP and JC contributed equally to this work. Notes The authors declare no competing financial interested. ACKNOWLEDGMENTS We thank Chandrasova Mandi for helping with HPLC purification, Dr. Abhi Das for ITC experiment, Snehasis Mishra, Dr. Krishnendu Manna for helping in FACS data analysis. Debajyoti Das for bacterial DNA condensation image analysis, we are also thankful to T. Muruganandan for AFM, Soumik Laha for ESI-Mass, Debolina Chakrabarty for FACS technical support. SD acknowledges CSIR, DBT (Grant No. BT/PR6922/BRB/10/1144/2012) and DST SERB (Grant No. EMR/2017/000659) for financial support. RP acknowledges UGC and JC acknowledges CSIR for fellowship. 43



ABBREVIATION USED HU and HNS, bacterial nucleoid associated proteins (NAPs); QNOs, Quinoxaline N-oxides; ROS, Reactive Oxygen Species; DCM, dichloromethane; Xantphos, Xantphos - 4,5Bis(diphenylphosphino)-9,9-dimethylxanthene; MIC, Minimal Inhibitory Concentration; IC50, 50% inhibitory concentration; HEK 293, Human embryonic kidney cell line; CD, Circular Dichroism; CT-DNA, calf thymus DNA; EtBr, ethidium bromide; SV, Stern Volmer; ITC, Isothermal Titration Calorimetry; Ka, association constant; FID, Fluorescence intercalator displacement; AFM, Atomic Force Microscopy; BrdU, bromodeoxyuridine; FITC, Fluorescein isothiocyanate; dsDNA, double stranded DNA; 5FU, 5-fluorouracil; PCD, Programmed cell death; TdT, terminal deoxynucleotidyltransferase; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; DMSO, dimethyl sulfoxide; UPEC, uropathogenic E. coli; AO, acridine orange; FtsZ, Filamenting temperature-sensitive mutant Z; PG, peptidoglycan; PI, propidium iodide; ALP, alkaline phosphatase; PNPP, pnitrophenylphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CFU, colony-forming unit.

REFERENCE 1. Blair, J. M.; Webber, M. A.; Baylay, A. J.; Ogbolu, D. O.; Piddock, L. J. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 2015, 13, 42-51. 2. Mah, T. F. C.; O'toole, G. A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9, 34-39. 3. Stewart P.S. Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol. 2002, 292, 107 – 113. 4. Thanbichler, M.; Wang, S. C.; Shapiro, L. The bacterial nucleoid: a highly organized and dynamic structure. J. Cell. Biochem. 2005, 96, 506-521. 5. Dillon, S. C.; Dorman, C. J. Bacterial nucleoid-associated proteins, nucleoid structure and gene expression. Nat. Rev. Microbiol. 2010, 8, 185-195. 6. Luijsterburg, M. S.; Noom, M. C.; Wuite, G. J., Dame, R. T. The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: a molecular perspective. J Struct Biol. 2006, 156, 262-272. 7. Dorman, C. J.; Dorman, M. J. DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression. Biophys. Rev. 2016, 8, 89-100. 44


Page 44 of 49

Page 45 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8. Lee, J. S.; Waring, M. J. Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid. Biochem. J. 1978, 173, 115128. 9. Waring, M. J.; Ben-Hadda, T.; Kotchevar, A. T.; Ramdani, A.; Touzani, R.; Elkadiri, S.; Hakkou, A.; Bouakka, M.; Ellis, T. 2, 3-Bifunctionalized quinoxalines: synthesis, DNA interactions and evaluation of anticancer, anti-tuberculosis and antifungal activity. Molecules 2002, 7, 641-656. 10. Bailly, C.; Waring, J. M. DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM. Biochemical Journal, 1998, 330, 81-87. 11. Pereira, J. A.; Pessoa, A. M.; Cordeiro, M. N. D.; Fernandes, R.; Prudêncio, C.; Noronha, J. P.; Vieira, M. Quinoxaline, its derivatives and applications: a state of the art review. Eur.J. Med. Chem. 2015, 97, 664-672. 12. Tariq, S.; Somakala, K.; Amir, M., Quinoxaline: an insight into the recent pharmacological advances. Eur. J. Med. Chem. 2017, 143, 542-557. 13. Seitz, L. E.; Suling, W. J.; Reynolds, R. C. Synthesis and antimycobacterial activity of pyrazine and quinoxaline derivatives. J. Med. Chem. 2002, 45, 5604-5606. 14. Xu, F.; Cheng, G.; Hao, H.; Wang, Y.; Wang, X.; Chen, D.; Peng, D.; Liu, Z.; Yuan, Z.; Dai, M. Mechanisms of antibacterial action of quinoxaline-1,4-di-N-oxides against Clostridium perfringens and Brachyspirahyodysenteriae. Front. microbiol. 2016, 7 (1948), 1-12. 15. Carta, A.; Corona, P.; and Loriga, M. Quinoxaline 1,4-dioxide: a versatile scaffold endowed with manifold activities. Curr. Med. Chem. 2005, 12, 2259–2272. 16. Wilhelmsson, L. M., Kingi, N.; Bergman, J. Interactions of antiviral indolo [2, 3-b] quinoxaline derivatives with DNA. J. Med. Chem. 2008, 51, 7744-7750. 17. Zegar, I., Gräslund, A., Bergman, J., Eriksson, M., Nordén, B. Interaction of ellipticine and an indolo [2, 3b]-quinoxaline derivative with DNA and synthetic polynucleotides. Chem biol interact., 1989, 72, 277-293. 18. Dhanaraj, C. J.; Johnson, J. Studies on some quinoxaline metal complexes: synthesis, spectral characterization, electrochemical, antimicrobial, DNA interaction, molecular docking, and DFT studies. Inorganic and Nano-Metal Chemistry, 2017, 47, 1348-1358. 19. Tseng, C.-H.; Tzeng, C.-C.; Yang, C.-L.; Lu, P.-J.; Chen, H.-L.; Li, H.-Y.; Chuang, Y.-C.; Yang, C.-N.; Chen, Y.-L. Synthesis and antiproliferative evaluation of certain indeno[1,2c]quinoline derivatives. J. Med. Chem. 2010, 53, 6164−6179.

45



20. Mahata, T.; Kanungo, A.; Ganguly, S.; Modugula, E. K.; Choudhury, S.; Pal, S. K.; Basu, G.; Dutta, S. The benzyl moiety in a quinoxaline‐based scaffold acts as a DNA intercalation switch. Angew. Chem., Int. Ed. 2016, 128, 7864-7867. 21. Mahata, T.; Chakraborty, J.; Kanungo, A.; Patra, D.; Basu, G.; Dutta, S. Intercalator-induced DNA superstructure formation: doxorubicin and a synthetic quinoxaline derivative. Biochemistry 2018, 57, 5557-5563. 22. Kanungo, A.; Patra, D.; Mukherjee, S.; Mahata, T.; Maulika, P. R.; Dutta, S. Synthesis of a visibly emissive 9-nitro-2,3-dihydro-1H-pyrimido[1,2-a]quinoxalin-5-amine scaffold with large stokes shift and live cell imaging. RSC Adv. 2015, 5, 70958-70967. 23. Xu, R.; Birke, S.; Carberry, S. E.; Geacintov, N. E.; Swenberg, C. E.; Harvey, R. G. Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo [a] pyrenediol epoxide. Nucleic Acids Res. 1992, 20, 6167-6176. 24. Garbett, N. C.; Ragazzon, P. A.; Chaires, J. B. Circular dichroism to determine binding mode and affinity of ligand–DNA interactions. Nat. Protoc. 2007, 2, 3166-3172. 25. Hildebrand, F.; Meyer, A.; Eyre-Walker, A.; Evidence of selection upon genomic GC-content in bacteria. PLoS Genet. 2010, 6(9): e1001107. 26. LePecq, J.-B.; Paoletti, C. A fluorescent complex between ethidium bromide and nucleic acids: physical—chemical characterization. J. Mol. Biol. 1967, 27, 87-106. 27. Boaz, Harold; G. K. Rollefson. The quenching of fluorescence. Deviations from the SternVolmer law. J. Am. Chem. Soc. 1950, 72, 3435-3443. 28. Buurma, Niklaas J., and Haq, I. Advances in the analysis of isothermal titration calorimetry data for ligand–DNA interactions. Methods 2007, 42, 162-172. 29. Winardhi, R.S.; Yan, J.; Kenney, L. J. H-NS regulates gene expression and compacts the nucleoid: insights from single-molecule experiments. Biophy. J. 2015, 109, 1321–1329. 30. Browning, D.F.; Grainger, D.C.; Busby, S. JW. Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression. Curr. Opin. microbiol. 2010, 13, 773780. 31. Xu, X.; Hamhouyia, F.; Thomas, S. D.; Burke, T. J.; Girvan, A. C.; McGregor, W. G.; Trent, J. O.; Miller, D. M.; Bates, P. J. Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides. J. Biol. Chem. 2001, 276, 43221-43230. 32. Opperman, T. J.; Kwasny, S. M.; Li, J. B.; Lewis, M. A.; Aiello, D.; Williams, J. D.; Peet, N. P.; Moir, D. T.; Bowlin, T. L.; Long, E. C. DNA targeting as a likely mechanism underlying

46


Page 46 of 49

Page 47 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the antibacterial activity of synthetic bis-indole antibiotics. Antimicrob. Agents Chemother. 2016, 60, 7067-7076. 33. Lacriola, C. J.; Falk, S. P.; Weisblum, B. Inhibition of DNA replication in Staphylococcus aureus by tegaserod. J. Antibiot. 2017, 70, 918-920. 34. Dwyer, D. J.; Camacho, D. M.; Kohanski, M. A.; Callura, J. M.; Collins, J. J. Antibioticinduced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis. Mol. cell. 2012, 46, 561-572. 35. Fernández, J. L.; Cartelle, M.; Muriel, L.; Santiso, R.; Tamayo, M.; Goyanes, V.; Gosálvez, J.; Bou, G., DNA fragmentation in microorganisms assessed in situ. Appl. Environ. Microbiol. 2008, 74, 5925-5933. 36. Rohwer, F.; Azam, F. Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling. Appl. Environ. Microbiol. 2000, 66, 1001-1006. 37. Yang, D. C.; Blair, K. M.; Salama, N. R. Staying in shape: the impact of cell shape on bacterial survival in diverse environments. Microbiol. MolBiol. Rev. 2016, 80, 187-203. 38. Justice, S. S.; Hunstad, D. A.; Cegelski, L.; Hultgren, S. J. Morphological plasticity as a bacterial survival strategy. Nature Rev. Microbiol. 2008, 6, 162-168. 39. Mulvey, M. A.; Lopez-Boado, Y. S.; Wilson, C. L.; Roth, R.; Parks, W. C.; Heuser, J.; Hultgren, S. J. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 1998, 282, 1494-1497. 40. Foxman, B., Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Am. J. Med. 2002, 113, 5-13. 41. Justice, S. S.; Hung, C.; Theriot, J. A.; Fletcher, D. A.; Anderson, G. G.; Footer, M. J.; Hultgren, S. J., Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1333-1338. 42. Gant, V.; Warnes, G.; Phillips, I.; Savidge, G. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 1993, 39, 147-154. 43. Kreuzer, K. N. DNA damage responses in prokaryotes: regulating gene expression, modulating growth patterns, and manipulating replication forks. Cold Spring Harb. Perspect. Biol. 2013, 5, a012674. 44. Mukherjee, A.; Cao, C.; Lutkenhaus, J. Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2885-2890.

47



45. Cheng, G.; Li, B.; Wang, C.; Zhang, H.; Liang, G.; Weng, Z.; Hao, H.; Wang, X.; Liu, Z.; Dai, M. Systematic and molecular basis of the antibacterial action of quinoxaline 1, 4-di-Noxides against Escherichia coli. PloS one. 2015, 10, e0136450. 46. Cambridge, J.; Blinkova, A.; Magnan, D.; Bates, D.; Walker, J. R. A replication-inhibited unsegregated nucleoid at mid-cell blocks Z-ring formation and cell division independently of SOS and the SlmA nucleoid occlusion protein in Escherichia coli. J. Bacteriol. 2014, 196, 36-49. 47. Konai, M. M.; Ghosh, C.; Yarlagadda, V.; Samaddar, S.; Haldar, J., Membrane active phenylalanine conjugated lipophilic norspermidine derivatives with selective antibacterial activity. J. Med. Chem. 2014, 57, 9409-9423. 48. Kwasny, S. M.; Opperman, T. J., Static biofilm cultures of gram‐positive pathogens grown in a microtiter format used for anti‐biofilm drug discovery. Curr. prot. pharmacol. 2010, 50, 13A.8.1-13A.8.23. 49. Büttner H, Mack D, Rohde H., Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions. Front Cell Infect Microbiol. 2015, 5:14. 50. Clinical Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically: approved guideline M7-A7. CLSI, Wayne, PA. 51. Reischmann, P.; Fiebeck, J.; Weiden, N.; Müller, O. Measured effects of Wnt3a on proliferation of HEK293T cells depend on the applied assay. Int. J. Cell Biol. 2015, 2015, 928502, 1-8.

48


Page 48 of 49

Page 49 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TABLE OF CONTENTS GRAPHIC

Intercalation induced DNA structural change and bacterial responses by 6-nitroquinoxaline derivatives.

49


DNA Structural Alteration Leading to Antibacterial Property of 6

Recommend Documents