Bile Acid Oligomers and Their Combination with Antibiotics To Combat

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Bile Acid Oligomers and their Combination with Antibiotics to Combat Bacterial Infections Poonam Singla, Priyanka Dalal, Mahaldeep Kaur, Geeta Arya, Surendra Nimesh, Rachna Singh, and Deepak B Salunke J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01433 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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.

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Journal of Medicinal Chemistry

Bile Acid Oligomers and their Combination with Antibiotics to Combat Bacterial Infections

Poonam,1 Priyanka,2 Mahaldeep Kaur,2 Geeta Arya,3 Surendra Nimesh,3 Rachna Singh,2,* and Deepak B. Salunke1,*

1Department

of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University, Chandigarh, 160014, India.

2Department

of Microbial Biotechnology, Panjab University, Chandigarh, 160014, India.

3Department

of Biotechnology, Central University of Rajasthan, Ajmer, 305817, India.

*Corresponding authors: [email protected]; [email protected]

1 ACS Paragon Plus Environment


ABSTRACT O

Polar Surface

Hydrophobic Surface

OH

Hydrophobic Surface

OH

Polar Surface

OH

Hydrophobic Surface

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

Page 2 of 43

OH Bile acid

The ever-growing risk of bacterial resistance is a critical concern. Among the various antimicrobial resistant bacterial strains, methicillin and vancomycin resistant Staphylococcus aureus are among the most dreadful, causing serious complications. Based on the hypothesis that microbes have reduced ability to develop resistance against membrane targeting antibiotics, bile acid oligomers having unique facially amphiphilic topologies were designed and synthesized. The oligomers with specific linkers, exhibited potent and selective antibacterial activity against Gram-positive bacteria. The lead compounds also improved the efficacy of a range of known antibiotics belonging to different classes, when tested in combination. The active dimers were found to be effective against antibiotic-resistant clinical isolates of S. aureus, including multidrug resistant isolates. A significant inhibitory activity against S. aureus biofilm, a highly drugresistant bacterial phenotype often unresponsive to antibiotic therapy, was also noticed. No adverse effects were observed by these dimers in a cell viability assay against HEK293 cells.


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INTRODUCTION The rise in drug resistance amongst bacteria has led to the emergence of life-threatening infections that are difficult to treat with the presently available antibiotics. Synthesis of new antimicrobials with increased potency towards microbes and lower toxicity is a major challenge these days. Staphylococcus aureus, a Gram-positive opportunistic pathogen, has remarkably developed resistance to most of the commonly used antibiotics, such as penicillin, methicillin and even vancomycin. The diseases caused by this organism are common in both developed and developing countries, involving organs ranging from skin, soft tissue, respiratory system, bone, joints to endovascular tissues.1,2 So, there is an immediate need for the development of novel antimicrobial drugs or formulations that can work well against such drug-resistant microbes to combat the infections caused by them. Membrane targeting antibiotics are generally considered favorable over the other drugs because of the reduced ability of microbes to develop resistance mechanisms against them. These include daptomycin 1, polymyxinB 2 and amphotericin B (Amp B) 3 (Figure1).3 The other interesting membrane targeting molecules include Cationic Steroid Antibiotics (CSA) such as ceragenin 4 as well as bile acid based molecular umbrellas which are known to complex with phospholipids of the bacterial cell membranes.4-6 An inherent facial amphiphilicity is the crucial element responsible for the membrane disrupting ability of these antimicrobials.7-10 For instance, Amp B (Figure 1), a facially amphiphilic molecule widely used as an antifungal agent to treat systemic mycoses, is known to kill fungi by punching holes in their cell membranes.11-15


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H 3N

NH2

O NH O C9H19

NH

N H

N H

CONH2 O NH O H N N H O O COOH

O

O

O HOOC HOOC O

NH2

OH O

O

O HO

N H HN

O

O

OH

O N H

N H

O

O

O

N H

N H

NH2 HN H O N

O

OH

O

H N O NH2 O O O

NH NH

N H NH2

2. Polymixin B

O

OH OH O

H N

NH2

NH

H 2N

O OH

H N

OH

O

OH

OH H

HOOC

HO H N

O

1. Daptomycin

HO H 2N

H N

N H

HO

O

O H

OH

H

H H 2N

O 3. Amphotericin B

N H

O

H

H O

NH2

4. Ceragenin CSA-13

Figure 1. Membrane targeting antibiotics. The self-assembled supramolecular structures facilitated by the hydrophilic interactions in Amp B and its specific interaction with sterols in the fungal cell membranes are responsible for the observed activity of the Amp B.16-18 The dimeric and tetrameric structures of Amp B thus formed orient and localize themselves in such a way that they can penetrate in to the membrane hydrophobic core of the fungus.19,20 We earlier reported the synthesis of bile acid dimers with different amphiphilic topology having antifungal and antiproliferative activities.21 A typical facial amphiphilic conformation of these dimers forming a sandwich of hydrophilic face between two hydrophobic faces was suggested to be responsible for the observed activity. The active dimer was found to be selectively toxic to fungus and was not explored for its antibacterial activity. Such membrane-active compounds are expected to show synergistic activity with antibiotics and this component was also not explored in past. Furthermore, to understand the structure activity relationship in the lead structure, 4 ACS Paragon Plus Environment

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additional bile acid oligomers were synthesized in one step and screened for their antibacterial activity against Gram-positive as well as Gram-negative bacteria. RESULTS AND DISCUSSION We previously reported the synthesis of steroid dimers by two step procedures wherein, activation of carboxylic acid was carried out using N-hydroxysuccinimide/DCC and the final dimers were prepared on reaction with di- and tri-amines.21 In the present work (Scheme 1), bile acid oligomers were synthesized by following a typical one step peptide coupling protocol. Scheme 1. Synthesis of steroidal dimers using one step protocol.a O O

OH

OH

H HO

H

H R

n

OH

O

H

NH

NH R OH

a

HO

R HO

HN

OH R

NH

HO HN

O HO

R HO

O

5, R = OH 6, R = H 7a, R = OH, n = 1 8a, R = OH, n = 2 8b, R = H, n = 2 7b, R = H, n = 1

aReagents

9a, R = OH 9b, R = H

and conditions: (a) DMF, EDCI, HOBt, Et3N, 25 C, 24h (for compounds 7a and 7b,

ethylenediamine; for 8a and 8b, 1,3-diaminopropane and for 9a and 9b, diethylenetriamine was used in the coupling reactions). Ethylenediamine, 1,3-diaminopropane and diethylenetriamine were selected to couple with cholic acid 5 and deoxycholic acid 6 to form six steroidal dimers (Scheme 1). For the efficient one step synthesis of these amphiphatic molecules, the reactions were performed in DMF using



EDCI, HOBt as coupling reagents in the presence of triethylamine as a base to form amide linkage between bile acids and oligoamines. The reactions were completed within 24 h and the progress of the reaction was monitored by TLC. After the completion of reaction, chilled water was added to the reaction mixture and the precipitated product was filtered and washed with cold water. The crude products obtained were further purified by column chromatography to furnish desired oligomers in good yields. All the products were characterized by 1H NMR, 13C NMR, IR and mass spectroscopy analysis. In the newly formed steroid oligomers, distinct peaks in the 1H NMR were observed in the region of δ 3.5 ppm attributed to the α-methylene groups of the oligoamines whereas, the amide carbonyls appeared at δ 170-175 ppm in the 13C NMR spectra. The formation of these compounds was further confirmed by mass spectroscopy revealing [M+H]+ and [M+Na]+ peak in each case. We investigated the antimicrobial properties of all the synthesized amphiphatic oligomers as these molecules can best mimic the Amp B, ceragenins, polyhydroxylated ionophoric steroids as well as bile acid based molecular umbrellas.4,6,8,22 We hypothesized that the specific conformation of these oligomers in the appropriate aggregate form (Figure 2) could be responsible for their antimicrobial activity. O

Polar Surface

Hydrophobic Surface

OH

Hydrophobic Surface

OH

Polar Surface

OH

Hydrophobic Surface

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

Page 6 of 43

OH Bile acid

Figure 2. A pictorial representation of facial amphiphilic conformations of bile acid oligomers.


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The antibacterial activity of all the synthesized oligomers 7-9 (Scheme 1) was explored against representative Gram-positive (S. aureus ATCC 29213) and Gram-negative (Escherichia coli ATCC 25922) bacterial strains by disk diffusion method. Amongst the synthesized derivatives, the compound 9a obtained by using diethylenetriamine and cholic acid exhibited substantial antibacterial activity against the Gram-positive strain S. aureus ATCC 29213, whereas all the other compounds were found to be inactive. The mean zone of inhibition (ZOI) diameter after exposure to 30 µg of compound 9a was 14±1 mm. By reviewing the structures of all the six synthesized dimers, the appropriate chain length connecting the two cholic acid scaffolds might be responsible for the observed activity. To confirm, one additional analogue (10, Figure 3) was synthesized using 1,5-diaminopentane (Figure 3).

O

O

NH

NH

HO HO

HO

HO

HO O

HO

N R

HN HO

HO HO

HN O

HO

HO HO

O

11, R = CH3 12, R = (CH2)14CH3

10

Figure 3. N1,N2-1,5-diaminopentanebis[cholic acid amide] 10 and N-acyl derivatives 11, 12 of compound 9a. Compound 10 was found to be inactive against S. aureus, suggesting a specific role of free secondary amine functionality in the linker region for the observed activity by compound 9a. To confirm the requirement of central free amine group for the activity, the N-acylation in compound 9a was carried out using acetic anhydride and palmitoyl chloride respectively, to yield 7 ACS Paragon Plus Environment


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compounds 11 and 12 (Figure 3). Interestingly, both the compounds 11 and 12 were found to be inactive, confirming the fundamental requirement of the central free amine in dimer 9a for the desired activity. Bile acid oligomers are also described as molecular umbrellas and are known to be amphomorphic in nature. Such molecular umbrellas composed of two or more facial amphiphiles are known to produce hydrophobic or hydrophilic exterior when exposed to a hydrophobic or hydrophilic microenvironment. Few of the bile acid derived molecular umbrellas are capable of transporting certain biomacromolecules across liposomal membranes and were explored as drug carriers.4 Based on the structural similarity and to further improve the understanding of Structure Activity Relationship (SAR) in the present investigation, additional steroidal oligomers were synthesized using tris(2-aminoethyl)amine and 1,4,8,11-tetraazacyclotetradecane oligoamines as linkers to furnish tri-walled oligomers 13a, 13b and tetra-walled oligomer 14, respectively (Scheme 2). None of these molecules were found to be active against S. aureus. Scheme 2. Synthesis of tri- and tetrameric bile acid oligomers.a


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O

R

HO HO

R

NH

HO

HO

HO HO

OH

N HN HN O

5 or 6

O

13a, R = OH 13b, R = H

a

OH O

O

HO HO

HO

HO

HO HO

OH

OH

N

N

HO N

N

HO

O

O

OH

14

aReagents

and conditions: (a) DMF, EDCI, HOBt, Et3N, 25 C, 24h. (for compounds 13a and

13b, tris(2-aminoethyl)amine and for compound 14, 1,4,8,11-tetraazacyclotetradecane were used). So far, the only steroidal dimer (9a) synthesized using cholic acid as umbrella “walls” and diethylenetriamine as “linker” was capable of showing the desired antibacterial activity. The chain length was then increased by keeping the amine functionality in the linker region. These desired analogs (15a and 15b) were synthesized using bis(hexamethylene)triamine via the same one step amide coupling protocol (Scheme 3). Interestingly, the dimer synthesized using cholic acid (15a) retained the antibacterial activity (131 mm at 30 g of disk content) and 15b was found to be inactive. For further confirmation of the need of central free amine for the activity, the N-acylation of 15a with another molecule of cholic acid was carried to yield oligomer 16 (Scheme 3). As expected, the acylation of free amino functionality in 15a also abolished the



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observed activity, clearly emphasizing the importance of central secondary free amine and the particular conformation/interaction of the overall dimer derived from cholic acid scaffold. Scheme 3. Synthesis of bile acid derived dimers and trimer.a

O

O HO 5 or 6

R

HO

HN 5

a

HO

HO

HN

HO

O 5 N

NH HO HO

R

HO

HN

5

HO

NH HO O

5

HO

O 15a, R = OH 15b, R = H

aReagents

HO 16

HO

and conditions: (a) Bis(hexamethylene)triamine, DMF, EDCI, HOBt, Et3N, 25 C,

24h. Having observed the specific need of amino functionality for the biological activity, we were keen to explore the effect of additional amino functionality in the linker region on the observed activity. Accordingly, the synthesis of cholic acids dimer 17 was achieved using triethylenetetramine (Scheme 4). The newly synthesized dimer 17 was found to be significantly active against Gram-positive bacterial strain. The mean zone of inhibition (ZOI) diameter after exposure to 30 µg of compound 17 was 11±1 mm. Scheme 4. Synthesis of triethylenetetramine derived steroidal dimers.a


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O

O

NH HO

NH

HO HO

HO

HO

O

HO

HN

O

N O

5

a

NH

b

N

HN HO

HO HO

HN O

HO

HO HO

17

aReagents

O

O

18

and conditions: (a) Triethylenetetramine, DMF, EDCI, HOBt, Et3N, 25 C, 24h. (b)

Boc2O, DMF, Et3N, 25 C, 30 min. Further masking of the free amino functionalities in compound 17 using Boc2O resulted in compound 18 (Scheme 4), which was found to be inactive. Thus, it can be concluded from the above SAR studies that the basic requirement for the desired activity is the free amine group in the linker region and particular conformation adopted by the oligomer 9a, 15a and 17 synthesized using cholic acid. The specific conformational flexibility by the cholic acid dimer 9a was recently described by Zhu and coworkers by studying the behavior of three guest molecules of different polarity. Using molecular modeling it was suggested that the molecular conformations and interactions with the guest molecule may depend on the length and the functionality of the linker between the two cholic acid moieties in the dimer.23 The complete SAR study also demands the analysis for the necessity of two cholic acid moieties along with the appropriate linker containing free amine. The need of cholic acid scaffold to adopt specific conformation leading to the biological activity was confirmed by synthesizing analogue 22 wherein one of the cholic acid moiety was replaced by palmitoyl group. The desired analogue 11 ACS Paragon Plus Environment


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was synthesized using multistep synthetic procedure starting from diethylenetriamine (Scheme 5). Scheme 5. Stepwise synthesis of asymmetric bile acid derived palmitamide.a

H N

H 2N

a

NH2

O

O O

N H

O N

NH2

O

O

b O

N H

19 O

c

H 2N

H N

N H

O N H

d 14

OH

21

OH

O N 20 H N

O N H

14

O N H

14

22

OH

aReagents

and conditions: (a) i. F3CCOOEt, MeOH, 0 C, 1h. ii. Boc2O, 0 C to rt, 1h. iii. Aq.

MeOH, NH3, 25 C, 15h; (b) palmitoyl chloride, DCM, Et3N, 30 min; (c) TFA:DCM (1:1), 25 C, 10 min; (d) Compound 5, EDCI, HOBt, Et3N, 25 C, 8h. For the synthesis of compound 22, firstly one of the primary amino group of diethylenetriamine was selectively protected with ethyl trifluoroacetate (Scheme 5).24 The reaction led to the formation of mono-trifluoroacetamide as the major product. Boc2O was added to this reaction mixture to protect the remaining two free amino functionalities which was monitored by TLC. After completion of the reaction, concentrated aqueous ammonia was added to the reaction mixture until the pH was increased to 11 to cleave the trifluoroacetate protecting group to yield intermediate 19. The synthesized compound 19 was isolated, column purified and confirmed by MS analysis (304.22 corresponding to the [M+H]+ was observed). The primary amine group of compound 19 was allowed to react with palmitoyl chloride in the presence of triethylamine as 12 ACS Paragon Plus Environment

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base to afford the compound 20 (confirmed by 1H NMR and MS analysis). Compound 20 was reacted with TFA:DCM (1:1) for the deprotection of Boc groups to afford the compound 21 which on further coupling reaction with cholic acid resulted in desired analogue 22. The formation of compound 22 was confirmed using 1H NMR and mass spectroscopy analysis. The inactivity of compound 22 against S. aureus bacterial strain confirmed the requirement of two cholic acid scaffolds for the activity. Conclusively, the bis-cholic amide with linker containing free amine is the fundamental requirement for the desired activity. None of the synthesized oligomers showed any inhibitory activity against E. coli ATCC 25922. To further confirm the specificity of these compounds against Gram-positive bacteria, the compounds were also screened against another Gram-negative bacterium Pseudomonas aeruginosa and no antibacterial activity was noted. The selective antibacterial activity of compounds 9a, 15a and 17 against Gram-positive but not against Gram-negative bacteria may be attributed to the differences in their cell-wall architecture and composition. Apart from containing peptidoglycan that is a cell-wall constituent in all bacteria, Gram-negative bacteria also harbor a layer of outer membrane.25 This outer membrane comprises of lipopolysaccharide and serves as an additional barrier to the entry of compounds into the bacterial cell, thereby conferring increased protection to drug penetration and efficacy.26 Apart from testing the antibacterial activity, all the synthesized compounds were also screened for antifungal activity. Only the compounds 9a and 15a were found to be active against fungi by disk diffusion, albeit to a lesser extent than bacteria. The mean ZOI diameters obtained after exposure of C. albicans to 30 μg of these compounds were 10 ± 1 and 7 ± 0 mm respectively. These results further demonstrate that differences in cell-wall structure and composition are 13 ACS Paragon Plus Environment


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likely to influence the penetrability and thereby the activity of the tested compounds against different microbial cell types. The minimum inhibitory concentrations (MIC) of compounds 9a, 15a and 17 against S. aureus ATCC 29213 were found to be 4, 4 and 16 µg/mL, respectively. Their IC50 values were 2.87, 2.36 and 12.08 µg/mL, respectively. Based on the results obtained, compound 9a and compound 15a were selected for further study against S. aureus. Scanning electron micrographs revealed bacterial cell disruption upon exposure to these compounds, possibly by damage to the cell surface. Morphological alterations such as bleb formation and shriveling were observed (Figure 4).

Figure 4. Scanning electron micrographs of S. aureus ATCC 29213 treated with compounds 9a and 15a at 0.5  MIC (2 μg/mL) and 1  MIC (4 μg/mL), respectively. A, Untreated control; B and C, Cells treated with Compound 9a; D, Cells treated with Compound 15a.


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The compounds were also found to be effective against drug-resistant clinical isolates of S. aureus by both disk diffusion and broth microdilution assay (Table 1). Table 1: Susceptibility of drug-resistant clinical isolates of S. aureus to compounds 9a and 15a Strain

Compound 9a

Compound 15a

ZOI

MIC

IC50

ZOI

MIC

IC50

(mm)

(µg/mL)

(µg/mL)

(mm)

(µg/mL)

(µg/mL)

S. aureus 1704

13±1

8

4.36

10±1

8

5.32

S. aureus 2673

13±1

4

2.18

11±1

8

3.75

The interaction of the active compounds 9a and 15a with four commonly used antibiotics, belonging to different classes, namely cefotaxime (beta-lactams), amikacin (aminoglycosides), vancomycin (glycopeptides) and ciprofloxacin (fluoroquinolones) was tested by broth microdilution checkerboard assay at concentrations ranging from 0.03 to 32 µg/mL. An additive effect of both the compounds was observed with all the four classes of antibiotics (Table 2), with Fractional Inhibitory Concentration (FIC) indices ranging from 0.51 to 1. At 2 µg/mL of the test compound, the MIC of cefotaxime, amikacin and ciprofloxacin was appreciably reduced (Table 2). A 33-fold reduction in the MIC of cefotaxime and 67-fold reduction in the MIC of amikacin was observed when combined with 2 µg/mL of the test compound 9a. The interaction appeared to be weaker for vancomycin. This may be attributed to its bigger size (1449.265 g/mol) and incompatibility to be accommodated in the hydrophilic cavity formed by the steroid dimers. Table 2. Additive interaction of the test compounds 9a and 15a with known antibiotics against S. aureus ATCC 29213. 15 ACS Paragon Plus Environment


Antibiotic

Cefotaxime

Amikacin

Antibiotic Antibiotic MIC (µg/mL) with 2 FIC index MIC

µg/mL of test compound*

(µg/mL)

Compound 9a

Compound 15a

Compound 9a

Compound 15a

1

0.03

0.03

0.53

0.56

(33)

(33)

0.06

0.5

0.51

0.56

(67)

(8)

0.03

0.25

0.56

1

(17)

(2)

1

2

1

1

(2)

(1)

4

Ciprofloxacin 0.5

Vancomycin

*values

Page 16 of 43

2

in parenthesis indicate fold change in the MIC of the antibiotic when used with 2 µg/mL

of the test compounds. The MIC of both compounds 9a and 15a against S. aureus ATCC 29213 was 4 µg/mL. Most of the bacterial infections involve formation of thick, multilayered biofilms, which are highly hydrated cell clusters irreversibly associated with a biotic (e.g. damaged tissue) or abiotic surface (e.g. indwelling medical devices like catheters) and encased in a self-produced extracellular polymeric substance matrix. These biofilms are highly resistant to the action of the most of the commercially available antibiotics; consequently, biofilm-associated infections are difficult to treat and often exhibit relapsing symptoms.27 The activity of compounds 9a and 15a was therefore evaluated against S. aureus biofilms, a highly drug-resistant bacterial phenotype commonly implicated in device-associated and chronic infections that are often unresponsive to antibiotic therapy. The steroid oligomers 9a and 15a demonstrated a significant inhibitory 16 ACS Paragon Plus Environment

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activity against S. aureus biofilm where the minimum biofilm inhibitory concentrations (MBIC) were found to be 32 µg/mL and 64 µg/mL, respectively. At these concentrations, complete inhibition of biofilm growth (MBIC100) was observed. In contrast, the known antibiotics tested (cefotaxime, amikacin, ciprofloxacin and vancomycin) were ineffective and were unable to cause even a 50% of reduction in biofilm growth at concentration as high as 128 µg/mL. The in-vitro cell viability of all the synthesized compounds was estimated via alamar blue assay using HEK293 cells which is one of the most sensitive and preferred assay and have no toxic effect on cells itself.28 It was evident from the results (Figure 5) that the synthesized compounds did not show any appreciable cytotoxicity and cells were significantly viable even after 24 h of treatment with test compounds as compared to the control. Only the compound 16 was found to be slightly toxic with 35% decrease in viability at very high concentration (200 µM) in which more than 92% of cells were viable at lowest concentration (1 µM).

16 (15a) 14

13b 13a

(9a)

8b

8a

7b

7a

100

80

% (Cell Viabiity)

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


60

40

20

0 control

1µM

5µM

10µM

50µM

100µM

200µM

Concentration

Figure 5. Cell viability assay via Alamar Blue against HEK293 cells with different concentrations of test compounds and untreated cells as positive control. 17 ACS Paragon Plus Environment


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CONCLUSION A series of steroid oligomers having facially amphiphilic topologies were designed and synthesized. Among all the synthesized compounds, three dimers 9a, 15a and 17 having free amine in the linker displayed antibacterial activity against Gram-positive bacterium i.e. S. aureus and towards its antibiotic-resistant strains. No antibacterial activity was observed against Gram negative bacteria i.e. E. coli and P. aeruginosa. The lead compounds 9a and 15a also showed additive effect with four known antibiotics. A 33-fold reduction in the MIC of cefotaxime and 67-fold reduction in the MIC of amikacin was observed when combined with 2 µg/mL of the test compound 9a. Interestingly, the lead compounds also demonstrated biofilm inhibitory activity. A complete inhibition of biofilm growth was observed at 32 µg/mL concentration of compound 9a. The known antibiotics were found to be ineffective at this concentration and were unable to cause even 50% reduction in biofilm growth at concentration as high as 128 µg/mL. A very specific Gram-positive antibacterial activity of the lead bile acid dimers against S. aureus, the additive effect with known antibiotics and the ability of these compounds to inhibit the biofilm formation along with their non-cytotoxic nature against HEK293 cell lines demonstrate therapeutic potential of these amphiphilic steroids to be used either alone or in combination with known antibiotics to treat deadly infections. The exact mechanism of action of these steroid oligomers is still not clear and work is in progress to understand the exact mechanism, structural requirements for the biological activity and preparation of mixed formulations with known antibiotics. EXPERIMENTAL SECTION


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Chemistry.

All the chemicals, reagents and solvents were purchased from commercial

suppliers. The bulk solvents (Hexane, DCM, MeOH) were distilled before use. The organic solvents were dried by literature procedures wherever necessary. The reactions were performed under nitrogen atmosphere. All the reactions were monitored by thin-layer chromatography (TLC) carried out on Merck silica gel aluminium sheets and visualized with different stains such as phosphomolybdic acid and ninhydrin made in ethanol. Column chromatography was carried out using 60-120, 230-400 mesh silica gel or neutral alumina as per the requirement. 1H NMR were recorded on Bruker 300 MHz or Bruker Advance II 400 MHz or JEOL JNM ECS400 or JEOL 500 MHz spectrometer with DMSO-d6, CDCl3 or CD3OD as solvents and TMS as internal standard. The splitting patterns in NMR are designated as s, singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet. The chemical shift values were reported in units of δ. The mass spectra were recorded on Waters Q-TOF or Thermo LTQ-XL Mass spectrometer. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was performed on Waters HPLC system attached to waters 1525 binary HPLC pump linked with Waters 2424 ELSD detector to determine the purity of active compound. Elution was carried on C18 column by isocratic conditions for 30 minutes where methanol:water (1:1) were used as eluents. The HPLC data displayed >95% purity of the active compound. Experimental Details and Analytical Data for Compounds 7-22. (4R,4'R)-N,N'-(ethane-1,2-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (7a). To a solution of cholic acid 5 (500 mg, 1.22 mmol) in dry DMF (10 mL), ethylenediamine (41µL, 0.61 mmol), HOBt (83 mg, 0.61 mmol) and triethylamine (341 µL, 2.45 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 oC, EDCI 19 ACS Paragon Plus Environment


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(352 mg, 1.83 mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for overnight and monitored by TLC. After completion of the reaction, water was added to the reaction mixture and the precipitated crude product was collected through filtration. The crude solid obtained was further purified by column chromatography on silica gel using methanol/DCM as eluents to obtain compound 7a as White solid. Yield: 78%; IR (neat, cm-1) υ: 3279 (m), 1629 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.76 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.15-3.20 (m, 2H), 3.05 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H), 0.79 (s, 6H), 0.56 (s, 6H).

13C

NMR (75 MHz, DMSO-d6) δ 172.9(2C), 71.0(2C), 70.5(2C),

66.3(2C), 46.1(2C), 45.8(4C), 41.5(2C), 41.4(2C), 38.4(2C), 35.3(2C), 35.1(2C), 34.9(2C), 34.4(4C), 32.6(2C), 31.6(2C), 30.4(2C), 28.6(2C), 27.3(2C), 26.1(2C), 22.8(2C), 22.6(2C), 17.1(2C), 12.3(2C). (HRMS) m/z calculated for C50H85N2O8+ [M+H]+: 841.6300, found: 841.6333. The 1H NMR and

13C

NMR spectra were consistent with that reported in the

literature.21 (4R,4'R)-N,N'-(ethane-1,2-diyl)bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide) (7b). To a solution of deoxycholic acid 6 (500 mg, 1.27 mmol) in dry DMF (10 mL), ethylenediamine (43 µL, 0.64 mmol), HOBt (86 mg, 0.64 mmol) and triethylamine (351 µL, 2.54 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 oC, EDCI (366 mg, 1.91 mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for overnight and monitored by TLC. The follow up of the reaction was done using the similar procedure as described for compound 7a to obtain compound 7b as white solid. Yield 83%; IR (neat, cm-1) υ: 3309 (m), 1634 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.66 (m, 2H), 4.36 (m, 2H), 4.01 (m, 2H), 3.79 (m, 2H), 3.09 (m, 4H), 0.92 (d, J = 5.8 Hz, 6H), 0.83 20 ACS Paragon Plus Environment

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(s, 6H), 0.58 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ 172.8(2C), 71.0(2C), 69.9(2C), 47.5(2C), 46.2(2C), 46.0(2C), 41.6(2C), 38.4(2C), 36.3(2C), 35.7(2C), 35.1(2C), 35.1(2C), 33.8(2C), 32.9(2C), 32.6(2C), 31.6(2C), 30.2(2C), 28.6(2C), 27.2(2C), 26.9(2C), 26.1(2C), 23.5(2C), 23.1(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C50H85N2O6+ [M+H]+: 809.64, found: 809.46, calculated for [M+Na]+: 831.62, found: 831.36. The 1H NMR and 13C NMR spectra were consistent with that reported in the literature.21 (4R,4'R)-N,N'-(propane-1,3-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (8a). The synthesis of compound 8a was achieved using the same protocol as described for compound 7a except the amine used was 1,3-diaminopropane. White solid; Yield: 74%; IR (neat, cm-1) υ: 3292 (m), 1628 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.76 (t, J = 5.6 Hz, 2H), 4.37 (d, J = 4.2 Hz, 2H), 4.12 (d, J = 3.5 Hz, 2H), 4.03 (d, J = 3.3 Hz, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.18 (m, 2H), 2.98-3.02 (m, 4H), 0.91 (d, J = 6.4 Hz, 6H), 0.80 (s, 6H), 0.56 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 172.5(2C), 71.0(2C), 70.4(2C), 66.2(2C), 46.1(2C), 45.7(4C), 41.5(2C), 41.3(2C), 36.2(2C), 35.3(2C), 35.1(2C), 34.8(2C), 34.3(4C), 32.5(2C), 31.7(2C), 30.4(2C), 29.3(1C), 28.5(2C), 27.3(2C), 26.2(2C), 22.8(2C), 22.6(2C), 17.0(2C), 12.3(2C). (ESI-MS) m/z calculated for C51H87N2O8+ [M+H]+: 855.64, found: 855.59, calculated for [M+Na]+: 877.62, found: 877.50. (4R,4'R)-N,N'-(propane-1,3-diyl)bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (8b). The synthesis of compound 8b was achieved using the same protocol as described for compound 7b except the amine used was 1,3-diaminopropane. White solid; yield 79%; IR (neat, cm-1) υ: 3292 (m), 1652 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.73 (t, J = 5.6 21 ACS Paragon Plus Environment


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Hz, 2H), 4.47 (d, J = 4.3 Hz, 2H), 4.19 (d, J = 4.1 Hz, 2H), 3.78 (m, 2H), 2.98-3.03 (m, 4H), 0.91 (d, J = 6.4 Hz, 6H), 0.84 (s, 6H), 0.58 (s, 6H).

13C

NMR (100 MHz, DMSO-d6) δ

172.5(2C), 71.0(2C), 69.9(2C), 47.5(2C), 46.2(2C), 45.9(2C), 41.6(2C), 36.3(2C), 36.2(2C), 35.7(2C), 35.1(2C), 34.9(2C), 33.8(2C), 32.9(2C), 32.6(2C), 31.7(2C), 30.2(1C), 29.4(2C), 28.6(2C), 27.2(2C), 26.9(2C), 26.1(2C), 23.5(2C), 23.1(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C51H87N2O6+ [M+H]+: 823.66, found: 823.67 [M+H]+, calculated for [M+Na]+: 845.63, found: 845.68. (4R,4'R)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (9a). The synthesis of compound 9a was achieved using the same protocol as described for compound 7a except the amine used was diethylenetriamine. White solid; yield: 77%; IR (neat, in cm-1) υ: 3292 (m), 1632 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.72 (t, J = 5.5 Hz, 2H), 4.35 (m, 2H), 4.11 (m, 2H), 4.02 (m, 2H) 3.77 (m, 2H), 3.60 (m, 2H), 3.18 (m, 2H), 3.06-3.09 (m, 4H), 0.91-0.92 (d, J = 6.4 Hz, 6H), 0.80 (s, 6H), 0.57 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ 173.2(2C), 71.5(2C), 70.9(2C), 66.7(2C), 48.7(2C), 46.6(2C), 46.2(4C), 42.0(2C), 41.8(2C), 38.8(2C), 35.8(2C), 35.6(2C), 35.4(2C), 34.9(4C), 33.0(2C), 32.1(2C), 30.9(2C), 29.0(2C), 27.8(2C), 26.7(2C), 23.3(2C), 23.1(2C), 17.4(2C), 12.8(2C). (ESI-MS) m/z calculated for C52H90N3O8+ [M+H]+: 884.67. found: 884.62, calculated for [M+Na]+: 906.65, found: 906.54. (HRMS) m/z calculated for C52H90N3O8+ [M+H]+: 884.6738, found: 884.6722, calculated for [M+Na]+: 906.6490, found: 906.6542. The 1H NMR, 13C NMR spectra were consistent with that reported in the literature.23 (4R,4'R)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1722 ACS Paragon Plus Environment

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yl)pentanamide) (9b). The synthesis of compound 9b was achieved using the same protocol as described for compound 7b except the amine used was diethylenetriamine. White solid; Yield: 83%; IR (neat, cm-1) υ: 3273 (m), 1646 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.70 (t, J = 5.5 Hz, 2H), 4.47 (m, 2H), 4.19 (m, 2H), 3.77 (m, 2H), 3.04-3.09 (m, 4H), 2.52-2.53 (m, 4H), 0.90 (d, J = 6.4 Hz, 6H), 0.83 (s, 6H), 0.57 (s, 6H).

13C

NMR (100 MHz, DMSO-d6) δ 172.6(2C),

71.0(2C), 69.9(2C), 48.4(2C), 47.4(2C), 46.2(2C), 45.9(2C), 41.6(2C), 38.6(2C), 36.2(2C), 35.6(2C), 35.1(2C), 35.1(2C), 33.8(2C), 32.9(2C), 32.5(2C), 31.6(2C), 30.2(2C), 28.6(2C), 27.2(2C), 26.9(2C), 26.1(2C), 23.5(2C), 23.1(2C), 17.0(2C), 12.4(2C). (HRMS) m/z calculated for C52H90N3O6+ [M+H]+: 852.6824, found: 852.6857. (4R,4'R)-N,N'-(pentane-1,5-diyl)bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (10). The synthesis of compound 10 was achieved using the same protocol as described for compound 7a except the amine used was 1,5-diaminopentane. White solid; yield 82%; IR (neat, cm-1) υ: 3292 (m), 1627 (m).1H NMR (400 MHz, DMSO-d6) δ 7.75 (t, J = 5.4 Hz 2H), 4.48 (m, 2H), 4.10 (m, 2H), 4.01 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.16 (m, 2H), 2.953.00 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H), 0.79 (s, 6H), 0.56 (s, 6H). 13C NMR (100 MHz, DMSOd6) δ 172.5(2C), 70.9(2C), 70.4(2C), 66.2(2C), 46.1(2C), 45.7(4C), 41.5(2C), 41.3(2C), 38.2(2C), 35.3(2C), 35.1(2C), 34.8(2C), 34.3(4C), 32.5(2C), 31.7(2C), 30.3(2C), 28.8(2C), 28.5(2C), 27.3(2C), 26.2(2C), 23.6(1C), 22.8(2C), 22.6(2C), 17.1(2C), 12.3(2C). (ESI-MS) m/z calculated for C53H90N2O8Na+ [M+Na]+: 905.65, found: 905.58. N,N-bis(2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)ethyl)acetamide (11). To a solution of 9a (320 mg, 0.36 mmol) in dry DMF (1 mL), acetic anhydride (34 µL, 0.36 23 ACS Paragon Plus Environment


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mmol), and triethylamine (51 µL, 0.36 mmol) were added at room temperature and stirred for 1 h and monitored by TLC. After completion of the reaction, solvent was evaporated under vacuum and the crude product obtained was column purified using DCM/MeOH as eluents. White solid; Yield: 81%; IR (neat, cm-1) υ: 3275 (m), 1624 (m). 1H NMR (400 MHz, CD3OD-d4) δ 3.94 (m, 2H), 3.79 (m, 2H), 3.44-3.47 (m, 4H), 3.34-3.37 (m, 6H), 2.11 (s, 3H), 1.02 (d, J = 6.2 Hz, 6H), 0.90 (s, 6H), 0.71 (s, 6H).

13C

NMR (100 MHz, , CD3OD-d4) δ 177.2 (1C), 177.1(1C),

173.9(1C), 74.0(2C), 72.9(2C), 69.0(2C), 49.9(1C), 48.0(1C), 47.9(1C), 47.5(2C), 46.5(1C), 43.2(2C), 43.0(2C), 41.0(2C), 40.5(2C), 38.7(1C), 38.3(1C), 36.9(2C), 36.5(2C), 35.9(4C), 34.1(1C), 34.0(1C), 33.2(2C), 31.2(2C), 29.6(2C), 28.7(2C), 27.9(2C), 24.3(2C), 23.2(2C), 21.5(1C), 17.7(2C), 13.1(2C). (ESI-MS) m/z calculated for C54H92N3O9+ [M+H]+: 926.68, found: 926.75, calculated for [M+Na]+: 948.66, found: 948.73. N,N-bis(2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamido)ethyl)palmitamide (12). To a solution of compound 9a (200 mg, 0.23 mmol) in dry DMF (1 mL), palmitoyl chloride (62 mg, 0.23 mmol), and triethylamine (68 µL, 0.23 mmol) were added at room temperature and stirred for 1 h and monitored by TLC. After completion of the reaction, solvent was evaporated under vacuum and the crude product obtained was column purified using DCM/MeOH as eluents. White solid; Yield: 86%; IR (neat, cm-1) υ: 3273 (m), 1642 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.95 (t, J = 5.9 Hz, 1H), 7.77 (t, J = 5.6 Hz, 1H), 4.32 (m, 2H), 4.09 (m, 2H), 4.00 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.19 (m, 2H), 3.10 (m, 8H), 0.91 (d, J = 6.2 Hz, 6H), 0.85 (t, J = 6.8 Hz, 3H), 0.80 (s, 6H), 0.57 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ 173.6(1C), 173.3(1C), 172.9(1C), 71.5(2C), 70.9(2C), 66.7(2C), 46.6(2C), 46.2(2C), 41.9(2C), 41.8(2C), 35.8(2C), 35.5(2C), 35.3(2C), 34.8(2C), 32.9(2C), 24 ACS Paragon Plus Environment

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32.0(3C), 31.8(2C), 30.8(2C), 29.6(10C), 29.2(2C), 28.9(2C), 27.7(1C), 26.6(2C), 25.5(1C), 23.3(3C), 23.0(4C), 22.6(2C), 17.5(3C), 14.4(2C), 12.8(2C). (ESI-MS) m/z calculated for C68H120N3O9+ [M+H]+: 1122.90, found: 1123.01, calculated for [M+Na]+: 1144.88, found: 1145.06. (4R,4'R,4''R)-N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(4((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro1H-cyclopenta[a]phenanthren-17-yl)pentanamide) (13a). To a solution of cholic acid 5 (500 mg, 1.22 mmol) in dry DMF (10 mL), tris(2-aminoethyl)amine (61 µL, 0.40 mmol), HOBt (83 mg, 0.61 mmol) and triethylamine (511 µL, 3.67 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 oC, EDCI (352 mg, 1.83 mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for overnight and monitored by TLC. The follow up of the reaction was done using the similar procedure described for compound 7a to obtain compound 13a as white solid. Yield 68%; IR (neat, cm-1) υ: 3308 (m), 1627 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.68 (m, 3H), 4.32 (d, J = 4.3 Hz, 3H), 4.05 (d, J = 3.5 Hz, 3H), 3.96 (d, J = 3.3 Hz, 3H), 3.78 (m, 3H), 3.61 (m, 3H), 3.18 (m, 3H), 3.06 (m, 6H), 2.45 (m, 6H), 0.91 (d, J = 6.3 Hz, 9H), 0.80 (s, 9H), 0.57 (s, 9H).

13C

NMR (100 MHz, DMSO-d6) δ 172.7(3C), 71.0(3C), 70.5(3C), 66.3(3C), 53.6(3C), 46.3(3C), 45.8(6C), 41.5(3C), 41.4(3C), 36.9(3C), 35.3(3C), 35.2(3C), 34.9(3C), 34.4(6C), 32.7(3C), 31.7(3C), 30.4(3C), 28.6(3C), 27.3(3C), 26.2(3C), 22.9(3C), 22.6(3C), 17.1(3C), 12.4(3C). (ESIMS) m/z calculated for C78H133N4O12+ [M+H]+, 1317.99, found: 1317.76, calculated for [M+Na]+: 1339.97, found: 1339.74. The 1H NMR and

13C

NMR spectra were consistent with

that reported in the literature.29



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(4R,4'R,4''R)-N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(4((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1Hcyclopenta[a]phenanthren-17-yl)pentanamide) (13b). To a solution of deoxycholic acid 6 (500 mg, 1.27 mmol) in dry DMF (10 mL), tris(2-aminoethyl)amine (63 µL, 0.42 mmol), HOBt (86 mg, 0.64 mmol) and triethylamine (533 µL, 3.82 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 oC, EDCI (366 mg, 1.91 mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for overnight and monitored by TLC. The follow up of the reaction was done using the similar procedure described for compound 7a to obtain compound 13b as white solid. Yield 71%; IR (neat, in cm-1) υ: 3313 (m), 1634 (m).1H NMR (400 MHz, DMSO-d6) δ 7.68 (t, J = 5.3 Hz, 3H), 4.48 (d, J = 4.2 Hz, 3H), 4.18 (m, 3H), 3.77 (m, 3H), 3.05 (m, 6H), 2.43 (m, 6H), 0.91 (d, J = 6.3 Hz, 9H), 0.84 (s, 9H), 0.58 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 172.5(3C), 71.0(3C), 70.0(3C), 53.7(3C), 47.4(3C), 46.3(3C), 46.0(3C), 41.6(3C), 37.0(3C), 36.3(3C), 35.7(3C), 35.2(6C), 33.8(3C), 32.9(3C), 32.6(3C), 31.7(3C), 30.2(3C), 28.6(3C), 27.2(3C), 27.0(3C), 26.2(3C), 23.6(3C), 23.1(3C), 17.1(3C), 12.5(3C). (ESI-MS) m/z calculated for C78H133N4O9+ [M+H]+: 1270.00, found: 1270.63 [M+H]+. The 1H NMR spectra was consistent with that reported in the literature.29 (4R,4'R,4''R,4'''R)-1,1',1'',1'''-(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrayl)tetrakis(4((3R,5S,7R,8R,9R,10S,12S,13R,14R,17R)-3,7,12-trihydroxy-8,9,10,13,14pentamethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentan-1-one) (14). To a solution of cholic acid 5 (500 mg, 1.22 mmol) in dry DMF (10 mL), 1,4,8,11tetraazacyclotetradecane (61 mg, 0.31 mmol), HOBt (83 mg, 0.61 mmol) and triethylamine (682 µL, 4.89 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 26 ACS Paragon Plus Environment

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oC,

EDCI (352 mg, 1.83 mmol) was added to the reaction mixture. The reaction mixture was

allowed to warm to room temperature and stirred for overnight and monitored by TLC. The follow up of the reaction was done using the similar procedure described for compound 7a to obtain compound 14 as white solid. Yield: 67%; IR (neat, cm-1) υ: 3350 (m), 1622 (m).1H NMR (400 MHz, DMSO-d6): 4.36 (m, 4H),4.12 (m, 4H), 4.02 (m, 4H), 3.78 (m, 4H), 3.60 (m, 4H), 3.16 (m, 2H), 0.93 (m, 12H), 0.79 (s, 12H), 0.58 (s, 12H).

13C


172.8(4C), 71.1(4C), 70.5(4C), 66.3(4C), 55.0(4C), 46.7(4C), 46.4(4C), 45.7(8C), 41.6(4C), 41.4(4C), 35.8(4C), 35.4(4C), 35.2(4C), 34.9(4C), 34.4(12C), 30.4(4C), 28.5(4C), 27.5(4C), 26.2(6C), 22.9(4C), 22.6(4C), 17.3(4C), 12.4(4C). ESI-MS calculated for C106H177N4O16+ m/z 1762.31 [M+H]+, found: 1762.28, calculated for [M+Na]+: 1784.29, found: 1785.26. (4R,4'R)-N,N'-(azanediylbis(hexane-6,1-diyl))bis(4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (15a). The synthesis of compound 15a was achieved using the same protocol as described for compound 7a except the amine used was bis(hexamethylene)triamine. White solid; yield: 72%; IR (neat, cm-1) υ: 3296 (m), 1635 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.72 (t, J = 5.2 Hz, 2H), 4.35 (m, 2H), 4.11 (m, 2H), 4.03 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.16 (m, 2H), 2.99 (m, 4H), 2.46 (m, 4H), 0.91 (d, J = 6.2 Hz, 6H), 0.80 (s, 6H), 0.57 (s, 6H).

13C

NMR (100 MHz, DMSO-d6) δ 172.6(2C), 71.0(2C), 70.5(2C), 66.3(2C), 49.3(2C), 46.2(2C), 45.7(4C), 41.5(2C), 41.4(2C), 38.3(2C), 35.3(2C), 35.1(2C), 34.9(2C), 34.4(4C), 32.6(2C), 31.8(2C), 30.4(2C), 29.3(2C), 29.2(2C), 28.6(2C), 27.3(2C), 26.6(2C), 26.4(2C), 26.2(2C), 22.8(2C), 22.6(2C), 17.1(2C), 12.3(2C). (ESI-MS) m/z calculated for C60H106N3O8+ [M+H]+: 996.79, found: 996.54 [M+H]+, calculated for [M+Na]+: 1018.77, found: 1018.63. (HRMS) m/z



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calculated for C60H105N3O8 [M+H]+: 996.7974, found: 996.7908, calculated for [M+Na]+: 1018.7794, found: 1018.7691. (4R,4'R)-N,N'-(azanediylbis(hexane-6,1-diyl))bis(4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamide) (15b). The synthesis of compound 15b was achieved using the same protocol as described for compound 7b except the amine used was bis(hexamethylene)triamine. White solid; Yield: 79%; IR (neat, cm-1) υ: 3276 (m), 1637 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.72 (t, J = 5.6 Hz, 2H), 4.49 (m, 2H), 4.20 (m, 2H), 3.78 (m, 2H), 2.99 (m, 4H), 2.43 (t, J = 7.0 Hz, 4H), 0.91 (d, J = 6.2 Hz, 6H), 0.84 (s, 6H), 0.58 (s, 6H).

13C


172.4(2C), 71.1(2C), 70.0(2C), 49.4(2C), 47.5(2C), 46.3(2C), 46.0(2C), 41.6(2C), 38.3(2C), 36.3(2C), 35.7(2C), 35.2(2C), 35.0(2C), 33.8(2C), 32.9(2C), 32.6(2C), 31.8(2C), 30.2(2C), 29.6(2C), 29.2(2C), 28.6(2C), 27.2(2C), 27.0(2C), 26.7(2C), 26.4(2C), 26.2(2C), 23.5(2C), 23.1(2C), 17.1(2C), 12.4(2C). (HRMS) m/z calculated for C60H106N3O6+ [M+H]+: 964.8076, found: 964.8113. (R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N,N-bis(6-((R)-4((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro1H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexyl)pentanamide (16). The synthesis of compound 16 was achieved using the same protocol as described for compound 13a except the amine used was bis(hexamethylene)triamine. White solid; yield: 62%; IR (neat, cm-1) υ: 3297 (m), 1622 (m).1H NMR (400 MHz, DMSO-d6) δ 7.73 (m, 2H), 4.34 (d, J = 4.2 Hz, 3H), 4.11 (m, 3H), 4.02 (m, 3H), 3.77 (m, 3H), 3.60 (m, 3H), 3.17 (m, 7H), 2.99 (m, 4H), 0.92 (m, 9H), 0.80 (s, 9H), 0.56 (s, 9H).

13C

NMR (100 MHz, DMSO-d6) δ 172.44 (1C), 172.40(1C), 171.9(1C), 28 ACS Paragon Plus Environment

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71.0(3C), 70.4(3C), 66.3(3C), 46.2(3C), 45.8(3C), 45.7(3C), 41.5(3C), 41.4(3C), 38.3(3C), 35.3(3C), 35.1(3C), 34.9(3C), 34.4(6C), 32.6(3C), 31.8(3C), 30.4(3C), 29.2(3C), 28.8(1C), 28.6(3C), 27.4(3C), 26.2(3C), 26.1(2C), 22.8(3C), 22.6(3C), 17.2(3C), 17.1(3C), 12.3(3C). (ESIMS) m/z calculated for C84H144N3O12+ [M+H]+: 1387.07, found: 1386.84, Calculated for [M+Na]+: 1409.05, found: 1408.81. (4R,4'R)-N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))bis(4((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro1H-cyclopenta[a]phenanthren-17-yl)pentanamide) (17). The synthesis of compound 17 was achieved using the same protocol as described for compound 7a except the amine used was triethylenetetramine. White solid; Yield: 61%; IR (neat, cm-1) υ: 3273 (m), 1642 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.73 (t, J = 5.2 Hz, 2H), 4.34 (m, 2H), 4.10 (m, 2H), 4.02 (m, 2H), 3.77 (m, 2H), 3.60 (m, 2H), 3.06-3.22 (m, 10H), 2.66 (m, 1H), 2.53 (m, 4H), 0.91 (d, J = 6.3 Hz, 6H), 0.80 (s, 6H), 0.57 (s, 6H).

13C

NMR (75 MHz, DMSO-d6) δ 172.7(2C), 71.1(2C), 70.5(2C),

66.3(2C), 48.8(4C), 46.2(2C), 45.8(4C), 41.6(2C), 41.4(2C), 38.8(2C), 35.3(2C), 35.2(2C), 34.9(2C), 34.4(4C), 32.6(2C), 31.7(2C), 30.4(2C), 28.6(2C), 27.3(2C), 26.2(2C), 22.8(2C), 22.6(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C54H95N4O8+ [M+H]+: 927.71, found: 927.69. di-tert-butyl

ethane-1,2-diylbis((2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-

trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamido)ethyl)carbamate) (18). Compound 17 (500 mg, 0.54 mmol) was dissolved in 1.5 mL of DMF, to it triethylamine (151 µL, 1.08 mmol) and Boc2O (371 µL, 1.62 mmol) were added and reaction was stirred for 1 h. Completion of the reaction was monitored by TLC. The solvent was evaporated under vacuum. The crude product obtained was purified by column 29 ACS Paragon Plus Environment


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chromatography. White solid; Yield: 79%; IR (neat, cm-1) υ: 3340 (m), 1652 (m). 1H NMR (400 MHz, DMSO-d6) δ 7.87 (m, 2H), 4.33 (m, 2H), 4.10 (m, 2H), 4.01 (m, 2H), 3.78 (m, 2H), 3.62 (m, 2H), 3.14- 3.22 (m, 13H), 1.40 (s, 18H), 1.25 (m, 16H), 0.93 (m, 6H), 0.82 (s, 6H), 0.59 (s, 6H).

13C

NMR (100 MHz, DMSO-d6) δ 172.8(2C), 154.6(2C), 78.7(2C), 71.0(2C), 70.5(2C),

66.3(2C), 46.1(4C), 45.7(4C), 41.6(4C), 41.4(4C), 35.3(2C), 35.2(2C), 34.9(2C), 34.4(4C), 32.5(2C), 31.7(2C), 30.4(2C), 28.6(2C), 28.0(6C), 27.3(2C), 26.2(2C), 22.8(2C), 22.6(2C), 17.1(2C), 12.4(2C). (ESI-MS) m/z calculated for C64H111N4O12+ [M+H]+: 1127.82, found: 1128.00, Calculated for [M+Na]+: 1149.80, found: 1150.06. tert-butyl (2-aminoethyl)(2-((tert-butoxycarbonyl)amino)ethyl)carbamate (19). Selective protection of one of the primary amine group of diethylenetriamine was achieved by the literature reported procedure25 using ethyl trifluoroacetate. Ethyl trifluoroacetate (231 µL, 1.94 mmol) was added to the solution of diethylenetriamine (200 mg, 210 µL, 1.94 mmol) in methanol at 0 oC and stirred for 2 h at 0 oC and the progress of the reaction was monitored by TLC which afforded mono-trifluoroacetate as a major product. Boc2O (4.45 mL, 19.38 mmol) was added to the reaction mixture and the reaction mixture was stirred for 30 min. Aqueous ammonia was added to the reaction mixture till the pH of the solution was increased to 11 (to achieve the cleavage of trifluoroacetate functionality). The completion of the reaction was monitored by TLC and after completion of the reaction, the solvent was evaporated under vacuum and crude product obtained was purified by flash column chromatography using methanol/DCM as eluents to yield the compound 19 as white viscous oil. (HRMS) m/z calculated for C14H30N3O4+ [M+H]+: 304.2231, found: 304.2231. tert-butyl (2-((tert-butoxycarbonyl)amino)ethyl)(2-palmitamidoethyl)carbamate (20). To a solution of compound 19 (120 mg, 0.40 mmol) in DCM (3 mL), palmitoyl chloride (144 µL, 30 ACS Paragon Plus Environment

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0.48 mmol) and triethylamine (61 µL, 0.44 mmol) were added and the reaction mixture was stirred for 30 min and completion of the reaction was monitored by TLC. The solvent was evaporated under vacuum and crude product obtained was purified by column chromatography using methanol/DCM as eluents to yield compound 20 as a white viscous oil. 1H NMR (400 MHz, CDCl3) δ 6.48 (m, 1H), 4.88-4.98 (m, 1H), 3.26-3.39 (m, 8H), 2.16 (m, 2H), 1.68-1.74 (m, 2H), 1.59-1.64 (m, 2H), 1.47 (s, 9H), 1.43 (s, 9H), 1.25 (m, 24H), 0.81 (t, J = 6.8 Hz, 3H). (HRMS) m/z calculated for C30H60N3O5+ [M+H]+: 542.4527, found: 542.4540. Calculated for [M+Na]+: 564.4347, found: 564.4357. N-(2-((2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17yl)pentanamido)ethyl)amino)ethyl)palmitamide (22). TFA:DCM (2 mL,1:1) was added to compound 20 (160 mg, 0.30 mmol) and the reaction mixture was stirred for 15 min and completion of the reaction was monitored by TLC. The solvent was evaporated under reduced pressure and the crude product N-(2-((2-aminoethyl)amino)ethyl)palmitamide (21) was used as such for further reaction. To a solution of compound 21 (100 mg, 0.29 mmol) in DMF, cholic acid (120 mg, 0.29 mmol), HOBt (20 mg, 0.15 mmol) and triethylamine (82 µL, 0.59 mmol) were added at 0 oC under nitrogen atmosphere. After 10 minutes of stirring at 0 oC, EDCI (113 mg, 0.59 mmol) was added to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for overnight. After completion of the reaction, solvent was evaporated under vacuum and crude solid product obtained was purified using silica gel column chromatography using methanol/DCM as eluents. White solid; Yield: 30%; IR (neat, cm-1) υ: 3286 (m), 1663 (m). 1H NMR (400 MHz, CDCl3) δ 6.97 (m, 1H), 3.98 (m, 1H), 3.85 (m, 1H),



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3.65 (m, 1H), 3.45 (m, 4H), 2.93 (m, 4H), 1.25 (m, 28H), 1.01 (d, 3H), 0.86-0.90 (m, 6H), 0.67 (s, 3H). (ESI-MS) m/z calculated for C44H82N3O5+ [M+H]+: 732.62, found: 732.34. Bacterial strains Initial screening for evaluation of antibacterial activity was performed on S. aureus ATCC 29213 and E. coli ATCC 25922. S. aureus 1704 and S. aureus 2673 from our laboratory collection were used as drug-resistant clinical isolates for determining the activity of selected compounds against multidrug resistant strains. Both these isolates are methicillin resistant S. aureus (MRSA) and are also resistant to ciprofloxacin (fluoroquinolone). In addition, S. aureus 1704 is resistant to chloramphenicol (phenicol). Scanning electron microscopy (SEM), synergism testing and antibiofilm activity testing were performed on S. aureus ATCC 29213. The antifungal activity was evaluated with C. albicans ATCC 90028. Evaluation of antibacterial activity The synthesized compounds were screened for antibacterial activity by agar disk-diffusion assay according to the guidelines of Clinical Laboratory Standards Institute (CLSI).30 Briefly 5 to 100 µg of the test compounds were loaded on to sterile 6 mm paper disks. The disks were allowed to dry and then placed on Mueller Hinton agar plates that had been pre-inoculated as a lawn with bacterial suspension setto McFarland standard 0.5. A positive control (cefotaxime) and solvent only control (DMSO) were set up in parallel. The plates were incubated at 37 oC for 18 h and the diameters of the ZOI were measured up to the nearest whole millimeters. MIC of the selected compounds was determined by broth microdilution assay according to CLSI guidelines for water insoluble agents.30,31 The test compounds were prepared as a twofold serial


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dilution series in DMSO at a concentration 100 times the final desired concentration. An intermediate dilution series was then prepared in the test medium (Mueller Hinton broth), which was then subsequently diluted in the same medium such that the final DMSO concentration in the experimental set up was 1%. 5x105 cfu/mL of bacterial cells were treated with these twofold serial dilutions of the test compound in microtitre plates at 37 oC for 18 h. Positive control, untreated (growth) control and sterility control were set up in parallel. MIC was defined as the lowest concentration of test compound that completely inhibited the growth. Both turbidity measurements and XTT dye reduction test were performed to confirm that same results are obtained by the two methods. IC50 of the test compounds was calculated using AAT Bioquest web tool by plotting logarithmic concentrations of the test compound on X axis and the corresponding bacterial OD600 on Y axis. Inflection point in the resulting dose response curves, corresponding to 50% reduction in bacterial growth, was taken as IC50. Scanning electron microscopy (SEM) The bacterial cell suspension (5x105 cfu/mL) was treated with selected compounds at 0.5x and 1x MIC for 18 h at 37 oC. The untreated controls were set up in parallel. The resulting suspensions were centrifuged at 10,000 rpm for 10 minutes, the pellets were washed twice with normal saline and the samples were processed for SEM according to the method of Yasuda et al.32 Synergy testing with known antibiotics The effect of the selected compounds (9a and 15a) on the efficacy of known antibiotics (cefotaxime, amikacin, vancomycin and ciprofloxacin) was tested by fractional inhibitory concentration assay.33 Briefly 5x105 cfu/mL of bacterial cells were treated with the test agents prepared as checkerboard microdilution panels consisting of twofold serial dilutions of the test 33 ACS Paragon Plus Environment


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agents alone and in combination. The microtiter plates were incubated for 18 h at 37 oC. FIC index (∑FIC) of the combination was calculated and interpreted as synergistic when ∑FIC was ≤ 0.5; additive when ∑FIC was > 0.5 and ≤ 1; indifferent when ∑FIC was > 1 and ≤ 4; and antagonistic when ∑FIC was > 4. Evaluation of antibiofilm activity S. aureus biofilms were cultured in microtiter plates for 24 h and treated with antibiotics or selected compounds prepared as two-fold serial dilution series according to the CLSI guidelines for water soluble and water insoluble agents respectively.30,31 Briefly, 200 µL of bacterial cell suspension set to an OD600 of 0.1 in tryptic soy broth with 1% glucose was inoculated in microtiter plates and the plates were incubated for 24 h at 37 °C. The resulting biofilms were washed twice with normal saline and exposed to the test agents for 18 h at 37 °C. Untreated (growth) control and sterility control were set up in parallel. After incubation, the samples were washed twice with normal saline. 200 µL of MHB and 100 µL of XTT- menadione solution (XTT, 0.5 mg/mL; menadione, 10 µM) were added to the wells and the plates were incubated for 1 h at 37 oC in dark. The absorbance of the supernatants was measured at 450 nm. MBIC was calculated as MBIC50 and MBIC100, representing 50% and 100% inhibition in biofilm growth. Evaluation of antifungal activity Agar disk-diffusion assay was performed as described above, with certain modifications for fungi, according to the CLSI guidelines for antifungal susceptibility testing.34 For agar disk diffusion assay, MHA with 2% glucose and 0.5 µg methylene blue/mL was used as the culture medium and the plates were incubated at 37 oC for 24 h.


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Cytotoxicity assay Cell culture: Human embryonic kidney 293 (HEK293) cells were grown as monolayer in DMEM HG media supplemented with 1.85 g/L of sodium bicarbonate with 10% FBS and 1% pen-strep. The culture condition was maintained at 37 ºC with 5% CO2 in CO2 incubator. Cells having 80% confluency (passage number lower than 30) were used for in-vitro cell viability experiment. Cell viability studies: The in-vitro cell viability of the synthesized compounds was evaluated against HEK293 cells via alamar blue assay.35,36 This is a colorimetric assay having a blue colored reagent, resazurin, which is reduced to resorufin, a pink coloured compound due to the metabolic activity of viable cells that can be quantified colorimetrically or fluorimetrically. For the assay, cells were first trypsinized and seeded separately in 96-well plate at a cell density of 1×104 cells/well in 100 µL of complete media. After 24 h, the media form 96-well was discarded followed by washing twice with PBS. Cells were then treated with varying concentrations (200, 100, 50, 10, 5 and 1 µM) of compounds in complete media and incubated for 24 h. The treatment mixtures were then replaced by 90 µL fresh media and 10 µL of alamar blue reagent (0.15 mg/mL) in each well after PBS washing. The absorbance was recorded at 570 and 600 nm after 4 h using micro-plate reader. The untreated cells were taken as control with 100% viability that was used to compare the relative cell viability in the test wells. ASSOCIATED CONTENT Supporting Information



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IR, 1H NMR, 13C NMR, 2D NMR, MS, HRMS, LCMS and HPLC spectra of the synthesized compounds. Molecular Formula Strings of the synthesized compounds. AUTHOR INFORMATION *D.B.S., E-mail: [email protected] *R.S., E-mail: [email protected] AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS DBS is thankful to UGC New Delhi for start-up research grant and DBT New Delhi for the award of Ramalingaswami Fellowship. The support from UGC-CAS, DST-PURSE-II, DSTSAIF and Panjab University Development fund is gratefully acknowledged. Authors are thankful to Mohini and Dr. Rohit Sharma, Cluster Innovation Centre in Biotechnology, Panjab University, Chandigarh for providing the HPLC analysis of the lead compound. ABBREVIATIONS USED MRSA, methacillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration; Amp B, amphotericin B; CSA, cationic steroid antibiotics; HEK293, human embryonic kidney 36 ACS Paragon Plus Environment

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cells; MIC, minimum inhibitory concentration; MBIC, minimum biofilm inhibitory concentration; SEM, scanning electron microscopy; FIC, fractional inhibitory concentration; DCC, N,N'-dicyclohexylcarbodiimide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; LCMS, liquid chromatography-mass spectrometry; HRMS, high resolution mass spectrometry; OD, optical density; PBS, phosphate buffer solution; IC50, half maximal inhibitory concentration; TLC, thin layer chromatography, DMF, N,N'-dimethylformamide; TMS, tetramethylsilane. REFERENCES 1.

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Hydrophobic Surface

Hydrophobic Surface

Polar Surface

Table of Content Graphic

Hydrophobic Surface

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