Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles

Accepted Manuscript Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles Peng-Yan Bai, Shang-Shang Qin, Wen-Chao Chu, Yi Yang, De-Yun Cui, Yong-Gang Hua, Qian-Qian Yang, En Zhang PII:

S0223-5234(18)30545-2

DOI:

10.1016/j.ejmech.2018.06.054

Reference:

EJMECH 10524

To appear in:

European Journal of Medicinal Chemistry

Received Date: 11 November 2017 Revised Date:

10 May 2018

Accepted Date: 22 June 2018

Please cite this article as: P.-Y. Bai, S.-S. Qin, W.-C. Chu, Y. Yang, D.-Y. Cui, Y.-G. Hua, Q.-Q. Yang, E. Zhang, Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

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Synthesis and antibacterial bioactivities of cationic deacetyl linezolid amphiphiles Peng-Yan Bai#a, Shang-Shang Qin#a, Wen-Chao Chua, Yi Yanga, De-Yun Cuia, Yong-Gang Huaa,

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Qian-Qian Yanga, En Zhanga, b* a

School of Pharmaceutical Sciences; Institute of Drug Discovery and Development; Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; Zhengzhou University, Zhengzhou 450001, PR China. bCollaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China. # These authors contributed equally.

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E-mail: [email protected]; ABSTRACT

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Bacterial infections cause various life-threatening diseases and have become a serious public health problem due to the emergence of drug-resistant strains. Thus, novel antibiotics with excellent antibacterial activity and low cytotoxicity are urgently needed. Here, three series of novel cationic deacetyl linezolid amphiphiles bearing one lipophilic alkyl chain and one non-peptidic amide bond were synthesized and

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tested for antimicrobial activities. Several compounds showed excellent antibacterial activity toward drug-sensitive bacteria such as gram-negative bacteria Escherichia coli (E. coli), Salmonella enterica (S. enterica) and gram-positive Staphylococcus

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aureus (S. aureus), Enterococcus faecalis (E. faecalis). Moreover, these amphiphilic molecules also exhibited strong activity against drug-resistant species such as

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methicillin-resistant S. aureus (MRSA), KPC (Klebsiella pneumoniae carbapenemase) and NDM-1 (New Delhi metallo-β-lactamase 1) producing carbapenem-resistant Enterobacteriaceae

(CRE).

For

example,

the

MICs

(minimum

inhibitory

concentrations) of the best compound 6e, ranged from 2 to 16 µg/mL and linezolid ranged from 2 to >64 µg/mL against these strains. Therefore, 6e is a broad-spectrum antimicrobial compound that may be a suitable lead as an antibiotic. The molecule 6e were found to function primarily by permeabilization and depolarization of bacterial membranes. Importantly, bacterial resistance against compound 6e was difficult to induce, and 6e was stable under plasma conditions and showed suitable activity in

ACCEPTED MANUSCRIPT mammalian plasma. Thus, these compounds can be further developed into a potential new class of broad-spectrum antibiotics. Keywords: Antimicrobial; deacetyl linezolid; amphiphilic; drug-resistant 1. Introduction

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Infectious diseases caused by pathogenic bacteria is widespread and the use of current antibiotics to treat bacterial infections is becoming ever more problematic with the emergence of multiple antibiotic-resistant bacteria [1]. The overuse and misuse of antimicrobial agents has given rise to multidrug-resistance bacteria, and

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these strains now represent a severe threat to public health and antibiotic therapy [2]. There are many pathogenic bacteria that show resistances to antibiotics, notably the

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“ESKAPE” bacteria: Enterococcus faecium (E. faecium), S. aureus, Klebsiella pneumoniae (K. pneumonia), Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species [3]. Penicillin-resistant S. aureus was first reported in the 1940s [4] and MRSA appeared in the 1960s [5]. Currently, MRSA is the most commonly identified drug-resistant bacterium worldwide [6, 7]. Quaternary ammonium

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compounds (QACs) were widely used as antibiotic agents because of their biofilm-eradicating properties, but the presence of qac genes in MRSA led to the emergence of resistance to QACs.[8-12] The carbapenem-resistant strains are also

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very problematic. For example, KPC-1 and KPC-2 from a carbapenem-resistant strain of K. pneumoniae exhibit resistance to all β-lactam compounds and other antimicrobials [13, 14], and the NDM-producing isolates are highly resistant to most

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antibiotics [15-17]. Although the number of bacteria showing antibiotic resistance continues to grow, the number of new systemic antibacterial agents approved by the US FDA between 1983 and 2012 has continued to decrease [18]. Thus, prevailing bacterial resistance to antibiotics combined with stagnant antibiotic discovery has created an urgent need for the development of new antimicrobial agents that exert novel mechanisms of action [19, 20]. Linezolid was the first oxazolidinone antimicrobial agent [21] and this compound represented a new synthetic class of antibacterial agents with activity against gram-positive bacteria [22]. Linezolid inhibits initiation of protein synthesis in

ACCEPTED MANUSCRIPT bacteria [23]. Previous studies reported that linezolid showed antibacterial activity against several microorganisms such as S. aureus, penicillin-resistant Streptococcus pneumoniae, MRSA and vancomycin-resistant E. faecium [24-28]. However, recent studies reported that E. faecium, S. aureus, vancomycin-resistant E. faecium and S.

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cohnii have developed resistance against linezolid [29-32]. Furthermore, linezolid is not well suited for combating gram-negative pathogenic bacteria that are intrinsically resistant because of efflux pumps that efficiently excrete linezolid from the cell [33, 34]. Resistance to linezolid by bacteria is caused mainly by modification of the

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linezolid binding site on the ribosome [35, 36]. Thus, there is an extremely urgent need to develop new antibacterial drugs that tackle drug-resistant bacteria. Besides

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linezolid, Eperezolid and Tedizolid were also approved antibiotics, and some compounds (Sutezolid, AZD5847 and Radezolid) were under clinical investigation as potential antibacterial agents (Fig. 1) [37]. Moreover, many studies reported linezolid derivatives with excellent antibacterial activities. For example, torezolid showed higher in vitro antibacterial activities and better in vivo protective effects in mice [38].

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agents [39].

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In addition, 1,2,4-triazolo[4,3-a]pyrimidine oxazolidinones are potential antibacterial

Fig.1. Structures of approved and developmental antibacterial oxazolidinone. Antimicrobial peptides (AMPs) have received substantial attention in recent years owing to their membrane targeting mechanism, broad-spectrum activity against

ACCEPTED MANUSCRIPT bacteria and low cytotoxicity [40-43]. Infections caused by antimicrobial-resistant bacteria are currently treated with several AMPs in preclinical and clinical settings [44]. Encouraged by the huge achievement of AMPs, a variety of synthetic membrane-active mimics have been developed as an alternative to antibacterial

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peptides. Binaphthyl-1,2,3-triazole peptidomimetics [45], nonpeptidic xanthone derivatives [46, 47] and other molecules with potent antibacterial and antibiofilm activities have been developed [48-53]. Additionally, several amphiphilic derivatives based on approved antibiotics have potent antimicrobial activity, such as vancomycin

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[54-56] and kanamycin B [57, 58]. S-galactosyl oligo(Arg) conjugates have been shown to overcome the serious problem of passage through the E. coli cell membrane

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[59]. Antibacterial diamines can also target bacterial membranes[60]. Moreover, guanidine and biguanide compounds had potency in preventing biofilms formation and breaking down existing biofilms[61]. Importantly, there are currently many antibacterial peptidomimetics, including LTX 109 [62], CSA-13 [63] and brilacidin [64] (Fig. 2) undergoing clinical trials. Thus, exploring the diversity of the structures

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of these molecules should offer new compound leads [65].

Fig. 2. Structures of some representative AMPs.

During the course of our recent investigations for new structures with good biological activity [66, 67], we discovered several molecules that have good antimicrobial activity and excellent activity against drug-resistant strains[68-71]. Inspired by the antibacterial activity of linezolid and antibacterial peptide mimics, we sought to find more effective antibacterial deacetyl linezolid derivatives. Recently,

ACCEPTED MANUSCRIPT three series of cationic deacetyl linezolid derivatives with broad-spectrum antibacterial activity were designed and synthesized. The final compounds were obtained through two or three step syntheses. Moreover, these compounds possessed positive charges, lipophilic alkyl chains and a non-peptidic amide bond. In this report,

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the activity of these compounds against drug-sensitive bacteria, including S. aureus, E. coli, E. faecalis and S. enterica, and drug-resistant bacteria such as MRSA, NDM and

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KPC were studied.

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(a) K2CO3, chloroacetyl chloride, acetone, r. t., 0.5-1 h. (b) N, N-dimethylalkylamine, CH3CN, high-pressure reactor, 85 oC, 24 h

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Scheme 1. Synthesis of the deacetyl linezolid derivatives 2a–2f.

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(a) RCHO, NaBH(OAc)3, 1,2-Dichloroethane, over night, r.t.. (b) 4a or 4b, DIPEA, HBTU, DMF/CHCl3(5:2), r. t., 24 h; (c) CH3COCl, CH3OH, 0oC-r. t., 24 h;

Scheme 2. Synthesis of the deacetyl linezolid derivatives 6a–6j. 2. Results and discussion 2.1. Synthesis and characterization The molecules 2a–2f and 6a–6j were easily synthesized by either two or three step syntheses using the deacetyl linezolid as the starting material (Schemes 1 and 2). Five or six compounds in each series were synthesized to assess the importance of the length of the lipophilic alkyl chain. To further fine-tune the structure-activity relationship (SAR) of compounds 6a–6j, hydrophilic moieties (amino acids) were

ACCEPTED MANUSCRIPT also varied on these lipophilic frameworks. The deacetyl linezolid was acetylated through chloroacetyl chloride to obtain intermediate 1a. Next, compounds 2a–2f were synthesized

through

quaternization

by

using

intermediate

1a

and

N,N-dimethyl-n-alkylamines. To synthesize molecules 6a–6j, the intermediates 4a

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[53] and 4b [72] were prepared by known methods. The intermediates of N-alkylamines 3a–3e were accomplished by a conventional procedure. Key amide coupling compounds 5a–5j were synthesized using N-Boc-protected amino acids 4a, 4b and 3a–3e. Finally, target compounds 6a–6j were obtained individually from 5a–

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5j with acetyl chloride in methanol. The products were obtained without further chromatography purification in high yield. Finally, we have synthesized 16 cationic

13

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deacetyl linezolid derivatives. All final compounds were characterized by 1H NMR, C NMR and HRMS and were purified by reversed-phase HPLC to more than 95%

purity.

2.2. Antibacterial activity

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The antibacterial efficacy of these compounds was determined in suitable culture medium and expressed as the MIC (minimum inhibitory concentration) (Tables 1-4). The glycopeptide antibiotic vancomycin, the β-lactam antibiotic meropenem,

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linezolid and deacetyl linezolid were used to compare the MIC results with our compounds. These compounds displayed preferable antibacterial potency against a wide spectrum of drug-sensitive bacteria such as S. aureus, E. coli, E. faecalis and S.

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enterica (Table 1, and Figs. 3 and 4), and drug-resistant bacteria such as MRSA (Table 2), KPC (Table 3) and NDM-1 (Table 4). Information describing MRSA, NDM-1 and KPC strains is summarized in Table S1. Table 1. MICs of small molecules 2a–2f, 6a–6j against drug-sensitive bacteria. MIC (µg/mL) Compound

S. aureus

E. faecalis

E. coli

S. enterica

2a 2b 2c 2d

>128 128 8 8

>128 64 16 4

>128 128 32 16

>128 128 32 16

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16 >128 >128 128 64 32 8 128 >128 64 16 8 >128 >64 64 <0.125

Da-LIN (deacetyl linezolid); b LIN (linezolid); c VAN (vancomycin); d MEM (meropenem); All

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a

4 16 128 64 16 8 4 128 32 32 8 8 >128 2 2 4

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4 4 128 32 16 4 2 64 16 16 8 4 >128 2 2 0.125

2e 2f 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j Da-LINa LINb VANc MEMd

experimental data was repeated twice (If the results were different, take the larger MIC).

In general, compounds 2a–2f, 6c–6e and 6i–6j (MIC: 2–16 µg/mL) showed good activities against gram-positive S. aureus and E. faecalis (Table 1), whereas

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compounds 2d–2e, 6e and 6j (MIC: 2–16 µg/mL) showed good activities against both gram-positive and gram-negative bacteria. Moreover, compound 6e was even comparable to vancomycin and linezolid against all bacteria tested. Compounds 2c–2e, 6d–6e and 6i–6j (MIC: 8–32 µg/mL) were found to be more active than vancomycin

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(MIC: >64 µg/mL) and linezolid (MIC: 32–64 µg/mL) against gram-negative bacteria E. coli and S. enterica. These cationic small molecules 2a–2f, 6c–6e and 6i–6j were

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found to be active against both gram-positive and gram-negative bacteria, with activity toward gram-positive bacteria strongest. The MICs for deacetyl linezolid were >128 µg/mL against drug-sensitive bacteria, whereas MICs were <0.125–4 µg/mL for meropenem. Clearly, these cationic deacetyl linezolid amphiphiles were more active than deacetyl linezolid but less active than meropenem. Accordingly, these compounds showed broad-spectrum activities, whereas vancomycin and linezolid showed selective activities against gram-positive bacteria. Fig. 3 presents the MICs of compounds 2a–2f and several control agents against drug-sensitive bacteria, and clearly shows that the small molecules synthesized were

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more active against gram-positive bacteria S. aureus and E. faecalis.

Fig. 3. MICs of cationic deacetyl linezolid derivatives 2a–2f, Da-LIN, LIN, VAN

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and MEM against drug-sensitive bacteria. The black, blue and yellow star represents

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MICs >128, > 64 and <0.125 µg/mL. All experimental data was repeated twice.

Fig. 4. MICs of cationic deacetyl linezolid derivatives with varying alkyl chain lengths: (a), (b) and (c) MICs of small molecules 2a–2f, 6a–6e and 6f–6j. The black star represents MIC >128 µg/mL. All experimental data was repeated twice. The length of the alkyl chain of compounds 2a–2f, 6a–6e and 6f–6j (Fig. 4) was

ACCEPTED MANUSCRIPT extremely important in defining the antibacterial activity of the compounds prepared. The MICs of compounds 2a–2f (Fig. 4a) were lower when the lipophilic chain was C9H19, C12H25 and or C14H29. Compounds 2c–2e were found to show much higher activity against drug-sensitive bacteria than 2a, 2b and 2f. However, MICs of 6a–6e

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(Fig. 4b) and 6f–6j (Fig. 4c) were lower when the lipophilic chain was C9H19, C10H21 or C11H23. Additionally, when the lipophilic chain was C11H23, molecules 6e and 6j displayed the strongest activities. These results were similar to those reported by other groups [51, 53]. Furthermore, compounds 6e and 6j with the same lipophilic alkyl

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chain but different hydrophilic amino acids displayed negligible differences in their antibacterial activity. Molecules 6e and 6j had similar MICs against drug-resistant

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bacteria. The SAR studies of this series of cationic deacetyl linezolid derivatives 6e and 6j revealed that an amphiphilic structure was critical for broad-spectrum activity, especially against gram-negative bacteria. Thus, the antibacterial activity of these compounds was affected strongly by the length of the alkyl chain, whereas the types of amino acids (lysine and arginine) had only a small effect on defining the

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antibacterial activity of the compounds.

Table 2. MIC of small molecules against MRSA.

a

M-2

M-3

M-4

M-5

M-6

M-7

M-8

M-9

M-10

4 4 4 2 4 1 64

8 16 4 8 4 1 64

2 2 2 4 4 1 32

16 4 4 4 4 1 32

4 4 4 8 4 1 64

4 2 2 4 4 2 32

4 2 2 4 4 1 64

8 4 4 4 4 2 64

8 2 4 4 4 1 64

8 8 4 4 4 1 32

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2d 2e 6e 6j b LIN c VAN d MEM

MRSAa

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Compound

MRSA (10 different clinical isolated strains); b LIN (linezolid); c VAN (vancomycin); d MEM (meropenem); All

experimental data was repeated twice (If the results were different, take the larger MIC).

The antibacterial activity (Table 2) of selected small molecules against MRSA was further investigated. All selected molecules had good MICs. Compound 6e was the most effective among the four compounds examined. In addition, this series of small cationic amphiphiles were extremely active against MRSA (MICs: 2–16

ACCEPTED MANUSCRIPT µg/mL). The efficiency activity of compound 6e (MICs: 2–4 µg/mL) was comparable to linezolid (MICs: 4 µg/mL) and better than meropenem (MICs: 32–64 µg/mL), but lower than vancomycin (MICs: 1–2 µg/mL). Table 3. MIC of small molecules against bacteria producing KPC. K-2

K-3

K-4

K-5

K-6

K-7

K-8

K-9

K-10

32 32 16 16 >64 >64 >64

32 32 16 16 >64 >64 >64

64 16 16 8 >64 >64 2

64 32 16 8 >64 64 32

64 32 16 16 >64 64 32

32 32 16 16 >64 >64 >64

64 32 16 32 >64 >64 >64

32 32 16 8 >64 >64 32

64 32 16 8 >64 64 >64

64 32 16 16 >64 >64 64

KPC (10 different clinical isolated strains); b LIN (linezolid); c VAN (vancomycin); d MEM (meropenem); All

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a

K-1

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2d 2e 6e 6j LINb VANc MEMd

KPCa

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Compound

experimental data was repeated twice (If the results were different, take the larger MIC).

The antibacterial activity (Table 3) of small molecules against KPC was tested. The MICs of these compounds ranged from 8 to 64 µg/mL. The MICs of the most effective compounds 6e and 6j were 8–16 µg/mL. Most importantly, the activities of

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compounds 2d, 2e, 6e and 6j (MICs: 8–64 µg/mL) were better than linezolid (MICs: >64 µg/mL), vancomycin (MICs: 64 or >64 µg/mL) and meropenem (MICs: 2–64 or >64 µg/mL), but worse than meropenem (MIC: 2 µg/mL) against K-3 strain.

Compound

a

NDM-1a

N-1

N-2

N-3

N-4

N-5

N-6

N-7

N-8

N-9

N-10

>64 >64 >64 >64 >64 >64 32

>64 >64 >64 >64 >64 >64 16

64 32 16 16 >64 >64 64

64 64 16 64 >64 >64 64

32 32 8 16 >64 64 8

16 32 16 16 >64 64 2

32 16 16 16 >64 64 64

16 16 16 16 >64 64 >64

16 16 16 16 >64 64 >64

32 16 16 8 >64 >64 >64

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2d 2e 6e 6j b LIN c VAN d MEM

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Table 4. MIC of small molecules against bacteria producing NDM-1.

NDM-1 (10 different clinical isolated strains); b LIN (linezolid); c VAN (vancomycin);

d

MEM (meropenem);

All experimental data was repeated twice (If the results were different, take the larger MIC).

The antibacterial activity (Table 4) of small molecules against NDM-1-producing bacteria was investigated. The MICs ranged from 8 to 64 µg/mL and even >64 µg/mL.

ACCEPTED MANUSCRIPT In addition, compounds 2d–2e, 6e and 6j (MICs: 8–64 µg/mL) showed higher activity than linezolid (MICs: >64 µg/mL), vancomycin (MICs: 64 or >64 µg/mL) and meropenem (MICs: 2–64 or >64 µg/mL) against N-3, N-4, N-7 to N-10, but displayed lower activity than meropenem (MIC: 2 µg/mL) against N-5 and N-6 strains.

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Meropenem showed better activity against N-1 and N-2 (MICs: 16–32 µg/mL) than 2d–2e, 6e, 6j, linezolid and vancomycin with MICs >64 µg/mL.

From the MICs (Tables 1–4), compounds 2d–2e, 6e and 6j had potent activity against both drug-sensitive bacteria and drug-resistant bacteria. Furthermore, the

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antibacterial activities of compounds 2d–2e, 6e and 6j were better than the corresponding activity of linezolid and vancomycin. Compounds 2d–2e, 6e and 6j

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showed broad-spectrum antibacterial activity and 2d–2e are QACs with alkyl chains (-C12H25, –C14H29) and a positive charge. Additionally, molecules 6e and 6j have the same alkyl chain (-C11H23) with an L-lysine or L-arginine residue. The positive charges and the positively charged amino acids may be essential for achieving the

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maximum antibacterial activity of these small cationic molecules.

Fig. 5. % of Hemolysis by cationic small molecules at different concentrations: (a)

ACCEPTED MANUSCRIPT compound 2e; (b) compound 6e; (c) Linezolid. All experimental data were averaged after three repetitions.

2.3. Hemolytic activity

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The hemolytic activity of the cationic small molecules 2e, 6e and linezolid are presented in Fig. 5. The hemolysis rate of other compounds was not determined. The ability of compounds to lyse red blood cells (RBCs) was used to evaluate the toxicity of the compounds toward mammalian cells. The 50% hemolysis concentration (HC50)

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of 2e, 6e and linezolid were approximately 60, 100 and 550 µg/mL, respectively. In general, the cationic small molecules 2e and 6e showed low toxicity toward RBCs. In

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addition, compound 6e, one of the most potent molecules, displayed good selectivity (S = HC50/MIC) against S. aureus (S = 50) and E. coli (S = 12.5). The hemolytic toxicity study revealed that these compounds had low toxicity toward mammalian RBCs with linezolid showing lower toxicity toward mammalian cells than compounds

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2e and 6e.

Fig. 6. Plasma stability and antibacterial activity in complex mammalian fluids of small molecules: (a) antibacterial efficacy of compound 6e against S. aureus after preincubating in 50% plasma for different periods of time (0, 3 and 6 h); (b) minimum bactericidal concentrations (MBCs) of 6e in 50% plasma, serum and blood against MRSA. The black star represents MIC >128 µg/mL. All experimental data were averaged after two repetitions.

2.4. Plasma stability

ACCEPTED MANUSCRIPT Protease degradation of AMPs is a major disadvantage for their use as antibiotics [73]. To determine the stability of the cationic deacetyl linezolid derivatives under plasma conditions, antibacterial efficacy of compound 6e against S. aureus after preincubating in 50% plasma for different periods of time (0, 3 and 6 h) was evaluated.

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The MBCs of compound 6e did not increase (MBC: 64 µg/mL) after 0, 3 and 6 h treatment in 50% plasma (Fig. 6a). The above results indicate that compound 6e was stable upon pretreatment in 50% plasma. 2.5. Antibacterial activity in complex mammalian fluids

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The loss of antibacterial activity of the compounds in the presence of complex mammalian fluids is another serious problem when considering their suitability as

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antimicrobial compounds. Antibacterial activity of compound 6e was investigated by determining the MBCs in 50% serum, plasma and blood supplemented, respectively, with 50% MHB against MRSA. (Fig. 6b). Compound 6e was found to be active in 50% plasma, but was inactive in 50% serum and 50% blood. The MBCs of compound 6e were 128 µg/mL in media and 50% plasma, whereas the MBCs were >128 µg/mL in

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50% serum and 50% blood. The increase of the MBCs could be due to negatively charged proteins and macromolecules in human serum or blood that bind tightly to the cationic molecules, thereby quenching their activity toward bacterial membranes. The

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above results indicate that compound 6e is active in complex mammalian fluids like plasma, but this compound was not able to retain its antibacterial activity in serum

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and blood.

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Fig. 7. Time-dependent killing. (a, c) S. aureus were grown to early exponential phase and challenged with 6e at 4×, 6×MIC (8, 12 µg/mL) and vancomycin at 4×MIC (8 µg/mL); (b, d) E.coli was grown to early exponential phase and

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challenged with 6e at 2×, 4×MIC (16, 32 µg/mL) and moxalactam at 6×MIC (6 µg/mL). The control was treatment with sterile water. Pictures (c, d) taken after treatment with compounds for 6 h. All experimental data were averaged after two

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repetitions.

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2.6. Bactericidal time-kill kinetics The time-kill kinetics of compound 6e was performed to determine the rate of

bactericidal action. S. aureus and E. coli, the two tested bacteria, were grown to early exponential phase and challenged with compound 6e (2×, 4× and 6× MIC), vancomycin (S. aureus) and moxalactam (E. coli). Compound 6e had excellent bactericidal activity against S. aureus, showing superior activity when compared with that of vancomycin in killing early exponential phase populations (Fig. 7a). Moreover, 6e (6× MIC: 12 µg/mL) killed early exponential phase S. aureus at 6 h, whereas vancomycin ( 4× MIC: 8 µg/mL) did not kill S. aureus at 6 h. Compound 6e displayed

ACCEPTED MANUSCRIPT rapid bactericidal activity against E. coli and was superior to moxalactam in killing early phase bacteria (Fig. 7b). Clearly, compound 6e (4× MIC: 32 µg/mL) killed E. coli at 2 h, whereas moxalactam did not kill early exponential phase E. coli at 6 h. Thus, compound 6e had excellent antibacterial activity against S. aureus and E. coli

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(Fig. 7c,d). The vancomycin and moxalactam samples were cloudy after 6 h treatment, but the samples treated with 6e were both clear. Thus, the results suggest that the

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cationic molecule 6e kills both gram-positive and gram-negative bacteria.

Fig. 8. Antibiofilm activity of small molecule 6e. (a) cell viability in non-treated and

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treated biofilms of S. aureus grown on cover slips for 24 h; (b) cell viability in non-treated and treated biofilms of E. coli grown on cover slips for 72 h; (c) images of the treated and non-treated biofilms of S. aureus after staining with crystal violet; (d) images of the treated and non-treated biofilms of E. coli after staining with crystal violet. All experimental data were averaged after three or more repetitions.

2.7. Biofilm disruption activity To evaluate the efficiency of this class of compounds to eradicate preformed biofilms, compound 6e was used against established S. aureus and E. coli biofilms.

ACCEPTED MANUSCRIPT Mature S. aureus biofilms in a 96-well plate (grown for 24 h) with an initial count of 16.0 log10 CFU/mL per well of bacteria were treated with 6e at seven different concentrations. Molecule 6e was found to reduce cell viability of the biofilms (13.9, 12.9, 9.9, 8.7, 7.5, 5.9 and 4.7 log10 CFU per well at 2, 4, 8, 16, 32, 64 and 128 µg/mL,

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respectively), whereas the cell viability of the non-treated biofilm increased to 18.9 log10 CFU per well (Fig. 8a). Compound 6e was also able to reduce the cell viability of mature E. coli biofilms (developed for 72 h) from an initial count of 14.5 log10 CFU per well to 10.2, 8.5, 8.0, 7.1, 6.1, 6.0 and 5.3 log10 CFU per well at 2, 4, 8, 16,

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32, 64 and 128 µg/mL, respectively, whereas the cell viability of the non-treated biofilm increased to 16.1 log10 CFU per well (Fig. 8b). Disruption of biofilm growth

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by compound 6e was visually observed by crystal violet staining (Fig. 8c,d and Fig. 9). The MBIC (minimal bacterial inhibition concentration) and MBEC (minimal bacterial eradicated concentration) values of compound 6e and linezolid were tested (Table 5). The results of these tests using 6e gave MBIC80 (minimum concentration required to inhibit 80% biofilm formation) values of 25.9 µg/mL against S. aureus and 61.9

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µg/mL against E. coli, and the MBEC values of 6e were 64 µg/mL and 128 µg/mL against S. aureus and E. coli, respectively. Fig. 9 shows the results of the MBEC assay using 6e. Linezolid only inhibited S. aureus biofilm formation, and was not as

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active in eradicating established biofilms when compared with the results using 6e. The results indicate that compound 6e can inhibit bacterial biofilm formation and

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eradicate established biofilms at 64 and 128 µg/mL.

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Fig. 9. Antibiofilm activity of small molecule 6e. Images of the small molecule treated and non-treated biofilms of S. aureus and E. coli after staining with crystal

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violet. All experimental data was repeated twice. Table 5. MBIC and MBEC of compound 6e.

S. aureus 25.9/34.1 26.8/36.1

E. coli 61.9/72.6 >128

MBEC80 / MBEC90 / MBEC (µg/mL) S. aureus 50.2/59.8/64 >128

E. coli 112.8/120.3/128 >128

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Compound 6e Linezolid

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MBIC80 / MBIC90 (µg/mL)

All experimental data were averaged after two repetitions.

2.8. Mechanism of action The molecular mechanism of action was investigated to determine whether these

cationic molecules functioned by disrupting the integrity of the bacterial cell membrane. Gram-positive S. aureus and gram-negative E. coli were used to assess the action mechanism of compound 6e. We also used three antibiotics (linezolid, vancomycin and meropenem) for comparison. The mechanism of action can be

ACCEPTED MANUSCRIPT confirmed by the following three experiments: membrane depolarization, and inner

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membrane and outer membrane permeabilization (Fig. 10).

Fig. 10. Mechanism of antibacterial action of the cationic small molecule 6e. Inner membrane permeabilization of S. aureus (a) and E. coli (b). Cytoplasmic membrane depolarization of S. aureus (c) and E. coli (d). (e) Outer membrane permeabilization of E. coli in the presence of amphiphilic small molecules at 10 µg/mL. All experimental data were averaged after three repetitions.

2.8.1. Inner membrane permeabilization Bacterial cytoplasmic membrane permeabilization was studied using the

ACCEPTED MANUSCRIPT fluorescent probe propidium iodide (PI). PI enters bacteria only through compromised membranes and fluoresces upon binding to cellular DNA. After treatment with the molecule 6e (final concentration was 20 µg/mL), an enhancement in the fluorescence intensity was observed in S. aureus and E. coli (Fig. 10a,b). However, there was no

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increase when treated with other antibiotics. Thus, the result indicate that compound 6e can permeabilize the membrane of both gram-positive and gram-negative bacteria. 2.8.2. Cytoplasmic membrane depolarization

The deacetyl linezolid derivatives were found to dissipate the membrane

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potential of both gram-positive and gram-negative bacteria, as monitored by the membrane-potential fluorescence sensitive dye diSC35. Furthermore, compound 6e

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and the three antibiotics showed different levels of dissipation of the membrane potential. Compounds 6e (final concentration was 20 µg/mL) showed maximum membrane depolarization, whereas the other three antibiotics did not exhibit membrane depolarization activity against S. aureus and E. coli (Fig. 10c,d). The results further indicated that compound 6e is able to dissipate the membrane.

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2.8.3. Outer membrane permeabilization

Outer membrane permeabilization was studied using the hydrophobic dye N-phenyl naphthylamine (NPN). NPN is generally excluded from the outer membrane

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of gram-negative bacteria. NPN can access the impaired outer membrane when this membrane is damaged and exhibits an increase in fluorescence intensity. After treatment with 6e and the three antibiotics (final concentration was 20 µg/mL), no

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enhancement in the fluorescence intensity was observed (Fig. 10e). The above results indicated that compound 6e was not able to permeabilize the outer membrane of E. coli.

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Fig. 11. Inverted microscopy images of HeLa cells following treatment with

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compounds for 24 h. (a), (b) and (c) cells treated with 6e (1, 2 and 4 µg/mL); (d) non-treated cells (negative control); (e) cells treated with 0.1% Triton-X (positive

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control). Scale bar is 10 µm. All experiments were repeated three times.

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Fig. 12. Fluorescence microscopy images of HeLa cells after treatment with compounds for 24 h and staining with calcein AM and propidium iodide (PI). (a-c) Non-treated cells (negative control); (d-f), (g-i) and (j-l) treated with 6e (1, 2 and 4 µg/mL); (m-o) treated with 0.1% Triton-X (positive control). Scale bar is 200 nm. All experiments were repeated three times.

2.9. Fluorescence and inverted microscopy images HeLa cells were seeded into the wells of a 12-well plate and then treated with

ACCEPTED MANUSCRIPT compound 6e at various concentrations (1, 2 and 4 µg/mL). The treated cell lines were imaged by optical microscopy to visualize the morphology (Fig. 11). The treated cells were found to have normal morphology at 1 µg/mL, 2µg/mL and 4 µg/mL (Fig. 11a–c), and they were similar to the untreated cell lines (Fig. 11d). In contrast, cells

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treated with Triton-X were found to have completely damaged cell morphologies (Fig. 11e). Fluorescence microscopy studies using the live/dead staining method showed that cells treated with compound 6e showed green fluorescence at 1, 2 and 4 µg/mL (Fig. 12d–f, g–i, j–l) and were similar to the untreated cells (Fig. 12a–c). In

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contrast, cells treated with Triton-X were found to have red fluorescence (Fig. 12m– o). The above results indicate that 6e was not toxic to mammalian cells at 2× the

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MIC value (4 µg/mL).

Fig. 13. Propensity of development of bacterial resistance against compound 6e. (a) For S. aureus where antibiotic norfloxacin and linezolid were used as the control; (b) for E. coli where lipopeptide colistin was used as the control; (c) for MRSA where linezolid was used as the control. All experiments was repeated twice. 2.10. Propensity to induce bacterial resistance It is important to evaluate the potential emergence of bacterial resistance against

ACCEPTED MANUSCRIPT these small molecules. Compound 6e was selected as a representative compound to evaluate the ability of these compounds to suppress the development of resistance against both gram-positive S. aureus, MRSA and gram-negative E. coli. Three control antibiotics norfloxacin, colistin and linezolid were chosen for S. aureus, E. coli and

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MRSA, respectively. In the case of norfloxacin, colistin and linezolid, the initial MICs were 1, 0.5 and 4 µg/mL, respectively. After the initial MIC experiment, serial passaging was initiated by transferring the growing bacterial suspension at 1/2 MIC of the compound or antibiotics, and was subjected to another MIC assay and the process

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was repeated for 20 passages. The cationic compound 6e and linezolid showed no change in the MIC against S. aureus even after 20 passages, whereas a 256-fold

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increase in the MIC was observed for norfloxacin (Fig. 13a). Moreover, compound 6e showed only a two-fold increase in MIC against E. coli and MRSA, whereas 32-fold and 8-fold increases in the MIC were observed for colistin against E. coli and linezolid against MRSA, respectively (Fig. 13b,c). The results indicate that induced bacterial resistance by continual exposure to these small compounds is difficult and

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they represent potential compounds to combat drug-resistant bacteria. In contrast, resistance to the two positive control antibiotics norfloxacin and colistin by S. aureus

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and E. coli, respectively, was easily developed (Fig. 13a,b).

3. Conclusions

Three series of small molecules were synthesized from deacetyl linezolid. These

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compounds displayed potent broad-spectrum and membrane-active antibacterial activity against various drug-sensitive and drug-resistant bacteria. Variation of the alkyl chain length had a great effect on their activity. The SAR studies of these cationic small molecules revealed that the positive charges and amphiphilic structure were critical for broad-spectrum activity, especially against gram-negative bacteria. Additionally, the small molecules were found to be stable under plasma conditions and showed acceptable levels of activity in mammalian plasma, but these compounds were not able to retain antibacterial activity when incubated in serum and blood. Moreover, these small molecules acted on bacteria rapidly and did not allow bacteria

ACCEPTED MANUSCRIPT to develop resistance. Various spectroscopic and microscopic studies revealed that depolarization and disruption of the bacterial cell membrane are the primary mechanisms describing the bactericidal action of the compounds. Furthermore, an optimized molecule showed negligible toxicity toward mammalian RBCs and HeLa

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cells. Therefore, these molecules can combat drug-resistance pathogens, inhibit bacterial biofilm formation and can eradicate established biofilms. Thus, these compounds are potential agents that can be used to tackle infections caused by various

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bacteria.

4. Experimental section

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General: Reagents and solvents were purchased from commercial sources and were used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz and 100 MHz spectrometer respectively, and TMS as internal standard reference. Coupling constants (J) are given in hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass

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spectrometer. The compounds were purified by reverse phase HPLC (Waters Symmetry C-18 4.5×250 mm, 5 µm) using sodium dihydrogen phosphate buffer (pH = 6-8)/acetonitrile (0−100%) as mobile phase to more than 95% purity. Analytical

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thin layer chromatography (TLC) was performed on glass plates pre-coated with silica gel (5–40 um, Qingdao Marine Chemical Factory (China)) to monitor the reactions. Visualization was accomplished using UV light. Column chromatography was

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performed on silica gel. For optical density and fluorescence measurements, Tecan Infinite Pro series M200 Microplate Reader was used. The biological experiments were performed with a 1300 series A2 Biological Safety Cabinet. TDL-5M Desktop Low-speed Refrigerated Centrifuge was used in antibacterial studies. All the solvents were of reagent grade. Drug-sensitive bacterial strains, E. coli (ATCC 25922), S. aureus (ATCC 29213), E. faecalis (ATCC 29212) and S. enterica (ATCC 8387). Drug-resistant bacteria, MRSA, KPC and NDM-1 were clinical isolated strains. Sheep RBCs were used for hemolytic assay and HeLa cells were used for mammalian cytotoxicity study.

ACCEPTED MANUSCRIPT General procedure for the synthesis o f compound (1 a ) : To the mixture of deacetyl linezolid (1 g, 3.39 mmol) and K2CO3 (562 mg, 4.06 mmol) in 25 mL acetone was added chloroacetyl chloride (306 µL) dropwise (about 0-3 min). The resulting solution was stirred at room temperature for 0.5-1 h. Upon completion, the

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mixture was poured into 25 mL of ice water. The precipitate formed was filtrated, washed with water and dried under vacuum to obtain compound 1a without further purification.[74]

(S)-2-chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5

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yl)methyl)acetamide (1a): White solid, yield 64.5%. 1H NMR (400 MHz, CDCl3) δ 7.41 (dd, J = 14.3, 2.6 Hz, 1H), 7.19 (s, 1H), 7.12 – 7.03 (m, 1H), 6.92 (t, J = 9.1 Hz,

3H), 3.13 – 2.98 (m, 4H).

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1H), 4.85 – 4.73 (m, 1H), 4.12 – 3.98 (m, 3H), 3.91 – 3.83 (m, 4H), 3.82 – 3.60 (m, C NMR (100 MHz, CDCl3) δ 166.20, 155.70, 153.25,

153.12, 135.70, 135.61, 131.85, 131.75, 117.87, 117.83, 113.05, 113.01, 106.73, 106.47, 70.43, 65.93, 49.98, 49.95, 46.77, 41.53, 41.39.

General procedure for the synthesis o f f i n a l compounds (2 a - 2 f ) : Compound

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1a was dissolved in CH3CN, then N, N-dimethylalkylamines were added to the organic solutions (1a: N, N-dimethylalkylamines = 1 : 3, molar ratio) separately in screw top pressure tubes and the reaction mixtures were stirred at 85 °C for about 24

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h. [75] After the reaction, the mixtures were allowed to cool down to room temperature and transferred to round bottom flask. Then, the organic solvents were removed and volume of the reaction mixtures was reduced to 1/10 to its original

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volume. Finally, the products were precipitated in excess of dry diethyl ether. The precipitates were filtered and washed repeatedly with diethyl ether. The compounds were vacuum dried.

(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2 -oxoethyl)-N,N-dimethylbutan-1-aminium (2a): Dark yellow oily liquid, yield 78.6%. 1

H NMR (400 MHz, DMSO-d6) δ 9.56 (t, J = 5.6 Hz, 1H), 7.49 (dd, J = 15.0, 2.5 Hz,

1H), 7.19 (dd, J = 8.8, 2.1 Hz, 1H), 7.06 (t, J = 9.3 Hz, 1H), 4.80 (dd, J = 7.6, 5.5 Hz, 1H), 4.24 (s, 1H), 4.11 (t, J = 9.0 Hz, 1H), 3.86 (dd, J = 9.0, 6.6 Hz, 1H), 3.77 – 3.69 (m, 4H), 3.54 (dd, J = 10.0, 5.3 Hz, 1H), 3.46 (dd, J = 11.6, 5.4 Hz, 2H), 3.39 (s, 4H),

ACCEPTED MANUSCRIPT 3.20 (s, 4H), 2.98 – 2.93 (m, 4H), 1.73 – 1.62 (m, 2H), 1.26 (dd, J = 14.7, 7.4 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.95, 155.74, 153.92, 153.31, 135.62, 135.53, 133.36, 133.26, 119.21, 119.17, 114.27, 114.24, 106.91, 106.65, 70.96, 66.13, 64.36, 61.80, 51.14, 50.68, 50.66, 47.37, 41.21, 23.82, 19.09,

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13.37. HR-MS (ESI) Calcd for C22H34ClFN4O4 [M-Cl] +: 437.2559, found: 437.2563. (S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2 -oxoethyl)-N,N-dimethyloctan-1-aminium (2b): Yellow sticky solid, yield 97.0%. 1H NMR (400 MHz, DMSO-d6) δ 9.24 (t, J = 5.7 Hz, 1H), 7.49 (dd, J = 15.0, 2.4 Hz,

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1H), 7.22 – 7.17 (m, 1H), 7.07 (t, J = 9.3 Hz, 1H), 4.85 – 4.74 (m, 1H), 4.16 – 4.08 (m, 2H), 3.79 (dd, J = 9.1, 6.8 Hz, 1H), 3.76 – 3.71 (m, 4H), 3.59 – 3.47 (m, 2H), 3.42 (dd,

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J = 9.9, 7.0 Hz, 4H), 3.17 (s, 5H), 2.99 – 2.94 (m, 4H), 1.67 (s, 2H), 1.26 (s, 10H), 0.87 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.94, 153.90, 135.63, 133.36, 119.23, 114.20, 106.85, 106.60, 71.07, 66.13, 64.57, 61.73, 51.20, 50.69, 47.26, 41.23, 31.11, 28.37, 25.65, 21.99, 21.82, 13.89. HR-MS (ESI) Calcd for C26H42ClFN4O4 [M-Cl] +: 493.3185, found: 493.3192.

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(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2 -oxoethyl)-N,N-dimethylnonan-1-aminium (2c): Yellow sticky solid, yield 77.4%. 1

H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.50 (dd, J = 15.0, 2.5 Hz, 1H), 7.23 –

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7.15 (m, 1H), 7.06 (t, J = 9.3 Hz, 1H), 4.20 (s, 1H), 4.10 (s, 1H), 3.87 – 3.81 (m, 1H), 3.76 – 3.70 (m, 4H), 3.55 – 3.51 (m, 1H), 3.49 – 3.40 (m, 6H), 3.24 (dd, J = 10.2, 6.7 Hz, 1H), 3.18 (s, 4H), 3.00 (s, 2H), 2.98 – 2.93 (m, 4H), 1.69 – 1.60 (m, 2H), 1.25 (d,

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J = 5.9 Hz, 12H), 0.86 (dd, J = 6.8, 3.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.98, 153.91, 119.20, 114.21, 106.86, 71.01, 66.12, 64.47, 62.80, 61.72, 51.13, 50.68, 49.93, 47.30, 41.16, 31.20, 28.71, 28.55, 28.44, 25.73, 25.67, 22.05, 21.82, 21.63, 13.91. HR-MS (ESI) Calcd for C27H44ClFN4O4 [M-Cl] +: 507.3341, found: 507.3345. (S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2 -oxoethyl)-N,N-dimethyldodecan-1-aminium (2d): Yellow sticky solid, yield 69.8%. 1

H NMR (400 MHz, DMSO-d6) δ 7.50 (dd, J = 15.0, 2.4 Hz, 1H), 7.18 (dd, J = 8.8,

1.7 Hz, 1H), 7.05 (t, J = 9.3 Hz, 1H), 4.83 – 4.76 (m, 1H), 4.24 (s, 1H), 4.09 (t, J =

ACCEPTED MANUSCRIPT 9.0 Hz, 1H), 3.85 (dd, J = 7.6, 5.3 Hz, 1H), 3.77 – 3.71 (m, 5H), 3.69 (d, J = 4.8 Hz, 1H), 3.56 – 3.49 (m, 2H), 3.17 (s, 5H), 2.98 – 2.92 (m, 5H), 2.82 (d, J = 4.2 Hz, 1H), 2.56 – 2.48 (m, 1H), 1.67 (s, 2H), 1.23 (s, 18H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.07, 153.92, 135.57, 135.48, 133.38, 133.28, 119.15,

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114.16, 106.82, 106.56, 70.97, 66.12, 51.06, 50.67, 47.30, 31.27, 29.00, 28.92, 28.76, 28.69, 28.45, 25.69, 22.06, 21.80, 13.91. HR-MS (ESI) Calcd for C30H50ClFN4O4 [M-Cl] +: 549.3811, found: 549.3816.

(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2

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-oxoethyl)-N,N-dimethyltetradecan-1-aminium (2e): Dark yellow sticky solid, yield 81.8%. 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 7.50 (dd, J = 14.9, 2.4 Hz, 1H),

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7.19 (dd, J = 8.7, 1.9 Hz, 1H), 7.06 (t, J = 9.4 Hz, 1H), 4.79 (s, 1H), 4.17 (s, 1H), 4.10 (t, J = 9.1 Hz, 1H), 3.82 (dd, J = 9.0, 6.8 Hz, 1H), 3.76 – 3.70 (m, 4H), 3.56 – 3.50 (m, 1H), 3.45 – 3.39 (m, 2H), 3.34 (s, 3H), 3.18 (s, 4H), 2.99 – 2.94 (m, 3H), 2.93 (d, J = 3.4 Hz, 1H), 2.81 (d, J = 8.1 Hz, 1H), 1.67 (s, 2H), 1.24 (s, 22H), 0.85 (t, J = 6.6 Hz, 3H).

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C NMR (100 MHz, DMSO-d6) δ 163.95, 153.88, 153.33, 135.52, 119.19,

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114.17, 106.84, 106.58, 71.04, 66.13, 64.47, 61.71, 51.19, 50.66, 47.28, 42.38, 41.19, 31.26, 29.03, 28.99, 28.92, 28.82, 28.75, 28.67, 28.57, 28.44, 26.10, 25.67, 22.05, 21.83, 13.90. HR-MS (ESI) Calcd for C32H54ClFN4O4 [M-Cl] +: 577.4124, found:

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577.4130.

(S)-N-(2-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amino)-2 -oxoethyl)-N,N-dimethyloctadecan-1-aminium (2f): Yellow foamed solid, yield

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81.6%. 1H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H), 7.50 (dd, J = 14.8, 2.2 Hz, 1H), 7.17 (t, J = 12.1 Hz, 1H), 7.09 – 7.00 (m, 1H), 4.79 (s, 1H), 4.12 (dd, J = 17.6, 8.3 Hz, 2H), 3.83 – 3.77 (m, 1H), 3.75 – 3.71 (m, 4H), 3.53 (dd, J = 10.4, 4.8 Hz, 1H), 3.49 – 3.39 (m, 4H), 3.17 (s, 4H), 2.94 (dd, J = 11.0, 4.1 Hz, 5H), 1.66 (s, 2H), 1.23 (s, 30H), 0.85 (t, J = 6.6 Hz, 3H), 0.63 – 0.63 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.95, 153.88, 135.52, 133.36, 119.19, 114.14, 106.81, 106.55, 71.06, 66.12, 64.44, 61.67, 51.22, 50.66, 47.24, 41.96, 41.20, 31.25, 29.00, 28.96, 28.92, 28.75, 28.66, 28.43, 25.95, 25.66, 22.05, 21.82, 13.90. HR-MS (ESI) Calcd for C36H62ClFN4O4 [M-Cl] +: 633.4750, found: 633.4757.

ACCEPTED MANUSCRIPT General procedure for the synthesis of intermediate molecules (3a-3e): Alkylaldehyde (RCHO) (1.1 equiv) was added into a solution of deacetyl linezolid (1 equiv., 300 mg) and NaBH(OAc)3 (1.3 equiv.) in 7.5 mL 1,2-dichloroethane in a round-bottom flask. The mixture solution with N2 protected was stirred at room

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temperature for 12-24 h. Then saturated NaHCO3 was used to quench the reaction and alkalified to pH = 8. The reaction solution was then extracted with dichloromethane (DCM). The organic layer was subsequently washed with H2O (2-3 times) and brine (1-2 times). Then the organic layer was dried over anhydrous Na2SO4, filtrated, and

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concentrated under reduced pressure and the residue was purified using column chromatography (petroleum ether: ethyl acetate= 5:1 to 1:1 v/v and with 0.5%

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triethylamine) to obtain the product.

(S)-3-(3-fluoro-4-morpholinophenyl)-5-((heptylamino)methyl)oxazolidin-2-one (3a): Yellow solid, yield 28.0%. 1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 14.4, 2.6 Hz, 1H), 7.17 – 7.10 (m, 1H), 6.92 (t, J = 9.1 Hz, 1H), 4.75 (s, 1H), 4.00 (t, J = 8.6 Hz, 1H), 3.90 – 3.85 (m, 4H), 3.82 (dd, J = 8.5, 6.9 Hz, 1H), 3.08 – 3.02 (m, 4H), 2.94 (dd,

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J = 12.8, 5.3 Hz, 2H), 2.65 (dd, J = 13.6, 7.0 Hz, 2H), 1.52 (s, 1H), 1.47 (dd, J = 12.8, 5.7 Hz, 2H), 1.32 – 1.24 (m, 8H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.78, 154.56, 154.33, 136.30, 136.21, 133.55, 133.44, 118.84, 118.80,

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113.82, 113.78, 107.52, 107.26, 72.35, 66.97, 52.57, 51.07, 51.04, 50.10, 48.30, 31.78, 30.08, 29.69, 29.18, 27.17, 22.60, 14.06. (S)-3-(3-fluoro-4-morpholinophenyl)-5-((octylamino)methyl)oxazolidin-2-one (3b):

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Yellow solid, yield 26.4%. 1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 14.4, 2.6 Hz, 1H), 7.14 (dd, J = 8.8, 1.6 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 4.80 – 4.70 (m, 1H), 4.00 (t, J = 8.6 Hz, 1H), 3.89 – 3.85 (m, 4H), 3.82 (dd, J = 8.4, 6.9 Hz, 1H), 3.10 – 3.01 (m, 4H), 2.93 (qd, J = 12.9, 5.2 Hz, 2H), 2.65 (dd, J = 13.5, 6.9 Hz, 2H), 1.50 – 1.42 (m, 3H), 1.28 (d, J = 5.6 Hz, 10H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.79, 154.55, 154.34, 136.32, 136.23, 133.56, 133.46, 118.85, 118.81, 113.83, 113.79, 107.53, 107.27, 72.35, 66.98, 52.57, 51.08, 51.05, 50.11, 48.30, 31.81, 30.09, 29.48, 29.23, 27.22, 22.64, 14.07.

ACCEPTED MANUSCRIPT (S)-3-(3-fluoro-4-morpholinophenyl)-5-((nonylamino)methyl)oxazolidin-2-one (3c): Yellow solid, yield 28.6%.1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 14.4, 2.6 Hz, 1H), 7.17 – 7.10 (m, 1H), 6.92 (t, J = 9.1 Hz, 1H), 4.82 – 4.69 (m, 1H), 4.00 (t, J = 8.6 Hz, 1H), 3.90 – 3.77 (m, 5H), 3.08 – 3.01 (m, 4H), 2.93 (dd, J = 13.0, 5.3 Hz, 2H),

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2.65 (dd, J = 13.7, 7.0 Hz, 2H), 1.53 (s, 1H), 1.50 – 1.44 (m, 2H), 1.27 (d, J = 7.3 Hz, 12H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.81, 154.57, 154.36, 136.33, 136.24, 133.59, 133.48, 118.86, 118.82, 113.84, 113.81, 107.55, 107.28, 72.37, 67.00, 52.59, 51.10, 51.07, 50.12, 48.32, 31.89, 30.11, 29.55, 29.54, 29.28,

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27.24, 22.67, 14.11.

(S)-5-((decylamino)methyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one (3d):

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Yellow solid, yield 25.3%. 1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 14.4, 2.5 Hz, 1H), 7.19 – 7.08 (m, 1H), 6.92 (t, J = 9.1 Hz, 1H), 4.75 (dt, J = 13.0, 6.5 Hz, 1H), 3.99 (d, J = 8.6 Hz, 1H), 3.89 – 3.84 (m, 4H), 3.84 – 3.79 (m, 1H), 3.10 – 3.01 (m, 4H), 2.94 (dd, J = 13.1, 5.2 Hz, 2H), 2.65 (d, J = 6.7 Hz, 2H), 1.79 (s, 1H), 1.51 – 1.43 (m, 2H), 1.27 (d, J = 8.8 Hz, 14H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz,

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CDCl3) δ 156.79, 154.54, 154.34, 136.32, 136.23, 133.55, 133.44, 118.85, 118.81, 113.83, 113.80, 107.54, 107.28, 72.31, 66.97, 52.55, 51.07, 51.04, 50.08, 48.31, 31.88, 30.04, 29.68, 29.57, 29.56, 29.51, 29.30, 27.21, 22.66, 14.09. (S)-3-(3-fluoro-4-morpholinophenyl)-5-((undecylamino)methyl)oxazolidin-2-one

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(3e): Yellow solid, yield 24.3%. 1H NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 14.4, 2.5 Hz, 1H), 7.13 (dd, J = 8.7, 2.0 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 4.81 – 4.69 (m, 1H),

AC C

4.00 (t, J = 8.6 Hz, 1H), 3.91 – 3.78 (m, 5H), 3.09 – 3.02 (m, 4H), 2.94 (ddd, J = 19.0, 12.9, 5.3 Hz, 2H), 2.66 (dd, J = 13.1, 6.9 Hz, 2H), 1.56 (s, 1H), 1.51 – 1.45 (m, 2H), 1.27 (d, J = 9.9 Hz, 16H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.79, 154.55, 154.34, 136.33, 136.24, 133.55, 133.45, 118.85, 118.81, 113.83, 113.80, 107.54, 107.28, 72.33, 66.98, 52.56, 51.08, 51.05, 50.11, 48.31, 31.90, 30.06, 29.68, 29.61, 29.58, 29.52, 29.32, 27.22, 22.67, 14.09. General procedure for the synthesis of intermediate molecule (4a): L-lysine (5 g, 1 equiv.) was dissolved in H2O (100 mL), and NaOH (3 equiv) was added to this solution and stirred. Di-tert-butyl dicarbonate (Boc2O) (2.4 equiv) in 50 mL of

ACCEPTED MANUSCRIPT tetrahydrofuran (THF) was added at 0 °C.[53] Then the reaction mixture was stirred at room temperature for 24 h. At the end of the reaction, THF was removed under reduced pressure and the aqueous layer was washed with diethyl ether to remove organic impurities. Then the aqueous layer was acidified to pH 4~5 using 1M H2SO4

RI PT

aqueous solution. The aqueous layer was then extracted with dichloromethane (DCM). The organic layer was then washed with brine and dried over anhydrous Na2SO4. The organic layer was removed under reduced pressure to obtain the compound.

SC

Boc-Lys(Boc)-OH (4a): Light yellow solid, yield 64.3%. 1H NMR (400 MHz, CDCl3) δ 5.30 (s, 1H), 4.73 (s, 1H), 4.21 (d, J = 73.0 Hz, 1H), 3.12 (d, J = 5.1 Hz, 2H), 1.78 13

C NMR (100 MHz, CDCl3) δ 155.90,

M AN U

(d, J = 51.3 Hz, 2H), 1.55 – 1.37 (m, 22H).

125.01, 80.08, 79.36, 53.32, 40.08, 32.02, 29.56, 28.43, 28.36, 22.42. General procedure for the synthesis of intermediate molecules (4b): L-arginine (8.7 g, 50 mmol) was added into a solution of tert-butanol (150 mL) and water (150 mL) in a 500 mL round-bottom flask. The mixture was cooled to 0 °C in

TE D

an ice bath and NaOH (7.0 g, 175 mmol) was added. The solution was stirred for 5 min at 0 °C and was added di-tert-butyl dicarbonate (43.7 g, 200 mmol) in portions.[72] The reaction mixture was stirred for 48 h at room temperature and

EP

monitored by TLC (CH2Cl2/MeOH/CH3CO2H, 40:1:0.2). The organic solvent (150 mL) was evaporated under reduced pressure and the residue was extracted with ethyl ether. The extracted solution was divided into three layers in the separating funnel, the

AC C

middle layer was collected and carefully acidified with citric acid to pH 3~4 and was extracted with ethyl acetate (3 × 60 mL). The extract was dried with anhydrous sodium sulfate and evaporated in a vacuum. The white solid was obtained after dried in a vacuum oven.

Boc-Arg(Boc)2-OH (4b): White solid, yield 61.1%. 1H NMR (400 MHz, CDCl3) δ 5.71 (s, 1H), 4.33 (s, 1H), 3.88 (s, 2H), 3.42 (s, 1H), 1.73 (dd, J = 37.7, 31.2 Hz, 5H), 1.52 (d, J = 5.0 Hz, 9H), 1.49 (s, 9H), 1.44 (s, 9H).

13

C NMR (100 MHz, CDCl3) δ

171.29, 156.20, 154.75, 153.17, 84.32, 83.19, 79.71, 79.45, 77.39, 77.07, 76.75, 60.43, 44.35, 40.51, 29.02, 28.36, 28.18, 28.02, 27.96, 27.89, 24.67, 21.02, 14.16.

ACCEPTED MANUSCRIPT General procedure for the synthesis of amide coupling compounds (5a-5j): To a stirred solution of intermediates ( 3a-3e) (200mg, 1 equiv.) in 5:2 DMF/CHCl3 (8.4 mL) was added N, N-di-isopropyl-ethylamine (DIPEA, 3 equiv.) at 0 °C. To this solution was then added HBTU (1.25 equiv.). This mixture was stirred for 5 min at

RI PT

0 °C, and subsequently the Boc-protected amino acids (4a-4b) (1.25 equiv.) were added to it. The mixture was stirred at 0 °C for 30 min and subsequently at room temperature for 24 h typically [53]. At the end, CHCl3 was evaporated under reduced pressure and the resulting solution was diluted to 2 times of its original volume by

SC

addition of ethyl acetate. This mixture was subsequently washed with 0.5 M KHSO4, H2O (three times), and brine. The combined organic layer was dried over anhydrous

M AN U

Na2SO4, filtrated, and concentrated under reduced pressure and the residue was purified using column chromatography (petroleum ether: ethyl acetate= 5:1 v/v and with 5%triethylamine) to obtain the products. di-tert-butyl

((5S)-6-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(heptyl)am

TE D

ino)-6-oxohexane-1,5-diyl)dicarbamate (5a): Brown oily liquid, yield 79.6%. 1H NMR (400 MHz, CDCl3) δ 7.79 (dd, J = 41.3, 7.9 Hz, 1H), 7.45 (s, 1H), 7.44 – 7.40 (m, 1H), 7.10 (d, J = 8.8 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 5.26 (s, 1H), 4.83 (dt, J =

EP

8.0, 5.2 Hz, 1H), 4.62 (d, J = 46.4 Hz, 2H), 4.36 – 4.25 (m, 1H), 4.03 (d, J = 8.9 Hz, 2H), 3.92 – 3.81 (m, 4H), 3.71 – 3.62 (m, 1H), 3.46 (dd, J = 17.2, 10.0 Hz, 1H), 3.24 (dd, J = 14.3, 7.8 Hz, 1H), 3.11 (d, J = 5.6 Hz, 2H), 3.08 – 3.02 (m, 4H), 1.60 (dd, J =

AC C

8.4, 5.5 Hz, 4H), 1.45 – 1.42 (m, 18H), 1.29 (dt, J = 9.2, 7.5 Hz, 10H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 154.22, 126.69, 118.87, 118.83, 114.02, 107.72, 107.46, 71.87, 66.95, 51.02, 50.99, 50.03, 49.89, 48.44, 38.62, 33.39, 31.70, 29.54, 29.15, 28.98, 28.42, 28.33, 26.66, 22.60, 22.51, 14.03. di-tert-butyl ((5S)-6-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(octyl)ami no)-6-oxohexane-1,5-diyl)dicarbamate (5b): Brown oily liquid, yield 96.9%. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 14.1 Hz, 1H), 7.10 (d, J = 6.2 Hz, 1H), 6.93 (dd, J = 11.8, 6.0 Hz, 1H), 5.24 (s, 1H), 4.84 (s, 1H), 4.61 (d, J = 31.6 Hz, 2H), 4.09 –

ACCEPTED MANUSCRIPT 3.99 (m, 2H), 3.87 (s, 4H), 3.67 (s, 1H), 3.47 (s, 2H), 3.24 (d, J = 6.8 Hz, 1H), 3.12 (s, 2H), 3.06 (d, J = 3.3 Hz, 4H), 2.97 (d, J = 5.5 Hz, 1H), 2.89 (d, J = 5.5 Hz, 1H), 1.66 – 1.54 (m, 4H), 1.43 (s, 18H), 1.35 – 1.23 (m, 12H), 0.88 (t, J = 6.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 118.82, 113.96, 107.45, 71.86, 66.95, 50.99, 49.98, 48.44,

RI PT

31.71, 29.18, 28.43, 28.34, 26.71, 22.60, 14.05. di-tert-butyl

((5S)-6-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(nonyl)am ino)-6-oxohexane-1,5-diyl)dicarbamate (5c): Brown oily liquid, yield 89.8%. 1H

SC

NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 14.3, 2.5 Hz, 1H), 7.09 (d, J = 1.6 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 5.27 (s, 1H), 4.84 (ddd, J = 10.4, 8.0, 3.1 Hz, 1H), 4.70 (s,

M AN U

1H), 4.54 (dd, J = 24.3, 4.3 Hz, 1H), 4.04 (t, J = 8.8 Hz, 2H), 3.90 – 3.83 (m, 4H), 3.67 (s, 1H), 3.47 (s, 2H), 3.25 (dd, J = 14.2, 7.7 Hz, 1H), 3.12 (s, 2H), 3.07 – 3.01 (m, 4H), 1.73 – 1.50 (m, 6H), 1.43 (d, J = 3.2 Hz, 18H), 1.34 – 1.19 (m, 14H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.49, 156.73, 154.28, 154.18, 136.52, 136.43, 133.14, 133.03, 118.84, 118.80, 113.99, 113.95, 107.68, 107.42, 79.70, 79.06,

TE D

71.86, 66.93, 51.00, 50.97, 49.98, 49.86, 48.41, 40.21, 33.38, 31.79, 29.50, 29.46, 29.31, 29.16, 28.42, 28.32, 26.70, 22.60, 14.06. di-tert-butyl

((5S)-6-(decyl((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)amin

EP

o)-6-oxohexane-1,5-diyl)dicarbamate (5d): Brown oily liquid, yield 96.7%. 1H NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 14.3, 2.5 Hz, 1H), 7.17 – 7.07 (m, 1H), 6.92 (t, J =

AC C

9.1 Hz, 1H), 5.20 (dd, J = 24.6, 8.5 Hz, 1H), 4.84 (dd, J = 8.1, 2.4 Hz, 1H), 4.61 (d, J = 30.0 Hz, 2H), 4.07 – 4.00 (m, 2H), 3.89 – 3.84 (m, 4H), 3.71 – 3.64 (m, 1H), 3.46 (d, J = 7.1 Hz, 2H), 3.24 (dd, J = 14.0, 7.6 Hz, 1H), 3.12 (s, 2H), 3.08 – 3.03 (m, 4H), 1.70 – 1.54 (m, 6H), 1.43 (d, J = 3.0 Hz, 18H), 1.28 (d, J = 16.8 Hz, 16H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.45, 154.18, 118.86, 118.82, 114.00, 107.70, 107.44, 71.86, 66.95, 51.02, 50.99, 49.99, 49.86, 48.45, 31.84, 29.52, 29.47, 29.33, 29.26, 29.17, 28.43, 28.34, 26.72, 22.65, 22.59, 14.08. di-tert-butyl ((5S)-6-(((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(undecyl)a

ACCEPTED MANUSCRIPT mino)-6-oxohexane-1,5-diyl)dicarbamate (5e): Yellow oily liquid, yield 67.8%. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 14.2 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.92 (t, J = 9.0 Hz, 1H), 5.25 (d, J = 7.9 Hz, 1H), 4.84 (d, J = 7.0 Hz, 1H), 4.65 (s, 2H), 4.09 – 3.99 (m, 2H), 3.87 (s, 4H), 3.68 (d, J = 7.8 Hz, 1H), 3.44 (d, J = 10.5 Hz, 2H), 3.22

RI PT

(d, J = 7.2 Hz, 1H), 3.11 (s, 2H), 3.05 (s, 4H), 1.57 (d, J = 14.5 Hz, 6H), 1.43 (s, 18H), 1.28 (d, J = 16.5 Hz, 18H), 0.88 (t, J = 6.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 154.19, 118.87, 113.99, 107.46, 71.87, 66.95, 60.38, 51.00, 49.87, 48.45, 31.89, 29.57, 29.53, 29.34, 29.29, 28.43, 28.34, 26.73, 22.66, 21.01, 14.18, 14.09.

SC

(5f): Yellow oily liquid, yield 73.8%.1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 14.3, 2.5 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 5.34 (d, J = 8.2

M AN U

Hz, 1H), 4.87 – 4.76 (m, 1H), 4.56 (s, 1H), 4.06 – 4.01 (m, 1H), 3.96 – 3.82 (m, 6H), 3.76 – 3.66 (m, 1H), 3.54 – 3.38 (m, 2H), 3.28 (dd, J = 14.4, 7.1 Hz, 1H), 3.06 – 3.04 (m, 3H), 2.80 (s, 2H), 1.71 – 1.57 (m, 6H), 1.52 (d, J = 3.8 Hz, 9H), 1.47 (s, 9H), 1.41 (s, 9H), 1.39 – 1.11 (m, 12H), 0.88 (t, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 163.79, 154.13, 125.00, 118.80, 113.97, 107.40, 83.81, 78.77, 71.94, 66.97, 51.00,

14.05.

TE D

49.69, 48.47, 44.30, 38.62, 31.72, 29.16, 28.98, 28.34, 28.31, 28.07, 26.70, 22.52,

(5g): Yellow oily liquid, yield 77.5%. 1H NMR (400 MHz, CDCl3) δ 7.50 – 7.39 (m,

EP

1H), 7.10 (d, J = 8.9 Hz, 1H), 6.96 – 6.88 (m, 1H), 5.32 (d, J = 8.8 Hz, 1H), 4.85 – 4.75 (m, 1H), 4.56 (s, 1H), 4.03 (dd, J = 11.8, 5.7 Hz, 1H), 3.88 (dd, J = 10.5, 6.0 Hz, 6H), 3.74 – 3.67 (m, 1H), 3.46 (dd, J = 13.7, 7.0 Hz, 2H), 3.28 (dd, J = 14.3, 7.0 Hz,

AC C

1H), 3.06 – 3.04 (m, 3H), 2.80 (s, 11H), 1.73 – 1.57 (m, 6H), 1.52 (d, J = 4.0 Hz, 9H), 1.47 (s, 9H), 1.41 (s, 9H), 1.39 – 1.14 (m, 14H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.28, 165.76, 163.77, 160.52, 156.75, 154.89, 154.30, 154.12, 136.51, 133.08, 118.84, 118.80, 113.95, 107.67, 107.40, 83.81, 79.64, 78.77, 71.93, 66.96, 51.03, 51.00, 50.26, 49.68, 48.47, 44.30, 38.60, 31.71, 30.60, 29.69, 29.28, 29.19, 28.33, 28.31, 28.06, 26.75, 25.00, 22.60, 14.05. (5h): Yellow oily liquid, yield 69.2%. 1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 14.3, 2.5 Hz, 1H), 7.10 (d, J = 7.0 Hz, 1H), 6.91 (t, J = 9.1 Hz, 1H), 5.43 – 5.29 (m, 1H), 4.80 (ddd, J = 15.6, 7.2, 3.7 Hz, 1H), 4.56 (s, 1H), 4.03 (s, 1H), 3.88 (dd, J =

ACCEPTED MANUSCRIPT 10.9, 6.3 Hz, 6H), 3.75 – 3.64 (m, 1H), 3.46 (dd, J = 13.5, 7.0 Hz, 2H), 3.27 (dt, J = 22.2, 11.1 Hz, 1H), 3.06 – 3.03 (m, 3H), 1.72 – 1.59 (m, 6H), 1.52 (d, J = 4.1 Hz, 9H), 1.47 (s, 9H), 1.42 (d, J = 4.7 Hz, 9H), 1.30 (s, 4H), 1.26 (dd, J = 7.0, 4.1 Hz, 12H), 0.88 (d, J = 6.5 Hz, 3H).

RI PT

(5i): Yellow oily liquid, yield 74.3%. 1H NMR (400 MHz, CDCl3) δ 7.50 – 7.38 (m, 1H), 7.14 – 7.04 (m, 1H), 6.92 (s, 1H), 5.32 (d, J = 8.5 Hz, 1H), 4.80 (qd, J = 7.0, 3.6 Hz, 1H), 4.56 (s, 1H), 4.04 (d, J = 9.1 Hz, 1H), 3.88 (dd, J = 10.9, 6.4 Hz, 6H), 3.76 – 3.64 (m, 1H), 3.46 (dd, J = 13.9, 6.9 Hz, 2H), 3.27 (dt, J = 21.6, 10.8 Hz, 1H), 3.06 –

SC

3.03 (m, 3H), 1.74 – 1.56 (m, 6H), 1.52 (d, J = 4.1 Hz, 9H), 1.47 (s, 9H), 1.41 (s, 9H), 1.34 – 1.21 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.25,

M AN U

171.12, 163.77, 160.52, 155.31, 154.89, 154.29, 154.11, 118.84, 118.80, 113.97, 107.66, 107.40, 83.80, 79.63, 78.75, 71.93, 66.96, 60.37, 51.03, 51.00, 50.25, 49.68, 48.47, 44.30, 38.60, 31.84, 30.61, 29.54, 29.47, 29.33, 29.27, 29.16, 28.33, 28.31, 28.05, 26.76, 24.99, 22.65, 21.03, 14.19, 14.09.

(5j): Yellow oily liquid, yield 75.6%. 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 14.4

TE D

Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 9.1 Hz, 1H), 5.41 (dd, J = 28.9, 8.8 Hz, 1H), 4.80 (d, J = 3.9 Hz, 1H), 4.56 (s, 1H), 4.07 – 4.01 (m, 1H), 3.87 (dd, J = 10.9, 6.5 Hz, 6H), 3.71 (d, J = 7.8 Hz, 1H), 3.46 (d, J = 5.5 Hz, 2H), 3.29 (dd, J = 14.3, 7.1 Hz, 1H), 3.05 (d, J = 4.2 Hz, 3H), 1.77 – 1.60 (m, 6H), 1.51 – 1.39 (m, 27H), 1.30 (s,

EP

4H), 1.26 (t, J = 7.0 Hz, 16H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 171.12, 154.82, 154.15, 136.50, 133.20, 118.86, 114.01, 107.69, 107.43, 83.98,

AC C

79.67, 71.91, 66.94, 60.36, 51.02, 51.00, 49.70, 48.47, 44.37, 31.87, 30.54, 29.56, 29.53, 29.51, 29.32, 29.27, 29.15, 28.37, 28.33, 28.28, 28.13, 28.04, 27.94, 27.85, 26.75, 24.92, 22.65, 22.59, 21.00, 14.17, 14.07. General procedure for the synthesis of final compounds (6a-6j): This was the reaction about deprotection of Boc Groups. The amide coupling compounds (5a-5j) (1 equiv.) were dissolved in methanol and then stirred at 0 °C, then dropwise addition of a known amount of acetyl chloride (6 equiv.) to an ice cold solution of excess amount of methanol.[76] Ice cold solutions are used in order to increase the solubility of the HCl and prevent its escape, the initial generation of the HCl being exothermic. In

ACCEPTED MANUSCRIPT cases where simple esterifications are carried out, excess acetyl chloride may be used without detrimental effects, since the workup involves simple evaporation of the solvent and excess HCl. The solutions were allowed to warm to room temperature and the reactions were completed within 24 h. The final compounds were characterized by H NMR, 13C NMR, and mass spectrometry.

RI PT

1

(S)-6-((((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin

5yl)methyl)(heptyl)

amino)-6-oxohexane-1,5-diaminium chloride (6a): Light yellow solid, yield 84.2%. 1

H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 28.2 Hz, 3H), 8.23 (s, 3H), 7.52 (s, 1H),

SC

7.20 (s, 1H), 7.12 (td, J = 9.3, 3.4 Hz, 1H), 4.85 (s, 3H), 4.25 (dd, J = 42.2, 35.7 Hz, 1H), 4.12 (s, 1H), 3.74 (s, 6H), 3.47 (dd, J = 14.3, 8.3 Hz, 1H), 3.22 (ddd, J = 20.0,

M AN U

18.8, 7.8 Hz, 1H), 2.99 (s, 3H), 2.74 (d, J = 5.5 Hz, 2H), 1.88 – 1.69 (m, 2H), 1.53 (d, J = 62.4 Hz, 6H), 1.31 – 1.17 (m, 8H), 0.84 (t, J = 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.75, 169.13, 168.83, 155.75, 153.88, 153.77, 153.32, 142.74, 133.81, 127.94, 127.09, 124.36, 119.51, 118.94, 114.21, 109.84, 106.85, 106.59, 71.73, 71.08, 65.95, 52.73, 51.58, 50.83, 49.14, 47.65, 38.22, 38.08, 31.16, 30.17, 29.96, 29.19,

TE D

28.45, 28.36, 26.52, 26.36, 26.19, 26.10, 25.88, 21.99, 21.17, 20.74, 13.90. HR-MS (ESI) Calcd for C27H46Cl2FN5O4 [M-2H-2Cl+H]+: 522.3450, found: 522.3453.

EP

(S)-6-((((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(octyl) amino)-6-oxohexane-1,5-diaminium chloride (6b): Light yellow foamed solid, yield 85.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 30.5 Hz, 3H), 8.25 (d, J = 4.6

AC C

Hz, 3H), 7.53 (ddd, J = 14.8, 12.2, 2.3 Hz, 1H), 7.22 (d, J = 8.9 Hz, 1H), 7.13 (td, J = 9.3, 3.7 Hz, 1H), 4.30 (dd, J = 68.8, 5.2 Hz, 1H), 4.14 (q, J = 8.7 Hz, 1H), 3.82 – 3.70 (m, 6H), 3.59 – 3.44 (m, 1H), 3.36 – 3.19 (m, 1H), 3.00 (s, 3H), 2.75 (d, J = 8.1 Hz, 2H), 1.76 (dd, J = 33.8, 16.0 Hz, 2H), 1.61 (d, J = 6.8 Hz, 3H), 1.47 (dd, J = 15.2, 7.6 Hz, 3H), 1.26 (t, J = 12.7 Hz, 10H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.13, 168.83, 155.76, 153.87, 153.77, 153.33, 133.75, 133.65, 119.47, 114.20, 106.85, 106.59, 71.69, 71.07, 65.98, 50.81, 49.13, 48.59, 48.52, 47.65, 38.06, 33.91, 31.20, 31.18, 30.17, 29.95, 28.77, 28.68, 28.61, 28.49, 26.51, 26.37, 26.25,

ACCEPTED MANUSCRIPT 25.94, 22.03, 20.73, 13.91. HR-MS (ESI) Calcd for C28H48Cl2FN5O4 [M-2H-2Cl+H]+: 536.3607, found: 536.3389. (S)-6-((((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(nonyl) amino)-6-oxohexane-1,5-diaminium chloride (6c): Light yellow foamed solid, yield

RI PT

88.9%. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 27.8 Hz, 3H), 8.11 (s, 3H), 7.52 (t, J = 13.0 Hz, 1H), 7.21 (d, J = 8.9 Hz, 1H), 7.10 (dt, J = 13.3, 6.7 Hz, 1H), 4.90 (d, J = 34.6 Hz, 1H), 4.30 (d, J = 62.8 Hz, 1H), 4.16 – 4.09 (m, 1H), 3.74 (s, 6H), 3.69 (d, J = 13.0 Hz, 1H), 3.60 – 3.52 (m, 1H), 3.48 (dd, J = 14.2, 8.3 Hz, 1H), 3.33 – 3.19 (m,

SC

1H), 2.97 (s, 3H), 2.74 (s, 2H), 1.76 (s, 2H), 1.52 (dd, J = 47.1, 18.5 Hz, 6H), 1.25 (s, 12H), 0.86 (t, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 66.08, 50.70, 40.03,

M AN U

39.82, 39.62, 39.41, 39.20, 38.99, 38.78, 31.23, 28.64, 22.05, 13.93. HR-MS (ESI) Calcd for C29H50Cl2FN5O4 [M-2H-2Cl+H]+: 550.3763, found: 550.3767. (S)-6-(decyl(((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) amino)-6-oxohexane-1,5-diaminium chloride (6d): Light yellow foamed solid, yield 87.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 26.5 Hz, 3H), 8.23 (d, J = 4.9

TE D

Hz, 3H), 7.59 – 7.48 (m, 1H), 7.22 (dd, J = 8.8, 2.5 Hz, 1H), 7.13 (dd, J = 9.4, 4.0 Hz, 1H), 5.00 – 4.84 (m, 1H), 4.43 – 4.19 (m, 1H), 4.14 (d, J = 6.9 Hz, 1H), 3.86 (s, 1H), 3.85 – 3.65 (m, 6H), 3.58 (d, J = 11.0 Hz, 1H), 3.52 – 3.43 (m, 1H), 3.26 (ddd, J =

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22.7, 11.9, 5.1 Hz, 1H), 3.00 (d, J = 3.8 Hz, 3H), 2.81 – 2.70 (m, 2H), 1.77 (s, 2H), 1.60 (d, J = 5.6 Hz, 3H), 1.47 (dd, J = 15.1, 7.5 Hz, 3H), 1.25 (s, 14H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.13, 168.84, 153.87, 119.43, 114.19,

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106.84, 106.58, 66.00, 50.80, 50.77, 31.24, 28.95, 28.92, 28.66, 22.05, 13.92. HR-MS (ESI) Calcd for C30H52Cl2FN5O4 [M-2H-2Cl+H]+: 564.3920, found: 564.3926. (S)-6-((((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)(undecyl) amino)-6-oxohexane-1,5-diaminium chloride (6e): Dark yellow foamed solid, yield 95.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 102.0 Hz, 3H), 8.07 (s, 3H), 7.51 (dd, J = 9.9, 7.4 Hz, 1H), 7.20 (s, 1H), 7.09 (td, J = 9.4, 4.8 Hz, 1H), 4.91 (dd, J = 35.7, 9.1 Hz, 1H), 4.26 (dd, J = 39.0, 32.7 Hz, 1H), 4.13 (dd, J = 16.7, 8.6 Hz, 1H), 3.86 (dd, J = 18.9, 10.0 Hz, 1H), 3.74 (dd, J = 13.2, 8.5 Hz, 6H), 3.49 (d, J = 8.6 Hz, 2H), 3.33 – 3.20 (m, 1H), 2.97 (s, 3H), 2.76 (d, J = 5.9 Hz, 2H), 1.80 (dd, J = 19.1,

ACCEPTED MANUSCRIPT 11.7 Hz, 2H), 1.60 (s, 3H), 1.43 (s, 3H), 1.24 (s, 16H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.79, 168.84, 153.89, 153.32, 119.29, 106.58, 66.09, 51.61, 50.69, 47.61, 38.16, 31.24, 29.24, 28.96, 28.82, 28.66, 26.43, 26.16, 25.93, 22.04, 21.16, 13.91. HR-MS (ESI) Calcd for C31H54Cl2FN5O4 [M-2H-2Cl+H]+:

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578.4076, found: 578.4082. (6f): Dark yellow foamed solid, yield 86.7%. 1H NMR (400 MHz, DMSO-d6) δ 9.08 – 8.94 (m, 1H), 8.44 (d, J = 22.3 Hz, 3H), 8.27 – 8.00 (m, 1H), 7.52 (ddd, J = 12.1, 8.6, 2.5 Hz, 1H), 7.33 (d, J = 50.8 Hz, 1H), 7.21 (dd, J = 10.8, 4.6 Hz, 1H), 7.11 (dt, J

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= 13.8, 7.0 Hz, 1H), 4.91 (d, J = 43.9 Hz, 1H), 4.41 – 4.10 (m, 19H), 3.90 (dd, J = 14.4, 3.6 Hz, 1H), 3.65 – 3.43 (m, 2H), 3.35 (t, J = 11.5 Hz, 2H), 3.22 – 3.09 (m, 1H),

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2.98 (s, 4H), 1.80 (s, 2H), 1.68 – 1.55 (m, 3H), 1.49 (dd, J = 13.7, 5.7 Hz, 6H), 1.35 – 1.19 (m, 9H), 0.86 (t, J = 5.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 157.14, 153.44, 135.11, 124.50, 119.39, 114.19, 106.58, 83.51, 66.00, 50.75, 48.52, 40.04, 39.83, 39.62, 39.42, 39.21, 39.00, 38.79, 38.21, 34.09, 31.19, 28.45, 27.51, 25.87, 22.01, 13.92. HR-MS (ESI) Calcd for C27H47Cl3FN7O4 [M-3H-3Cl+H]+: 550.3512,

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found: 550.3515.

(6g): Dark yellow foamed solid, yield 95.2%. 1H NMR (400 MHz, DMSO-d6) δ 9.09 – 8.91 (m, 1H), 8.44 (d, J = 23.3 Hz, 2H), 8.09 – 8.00 (m, 1H), 7.53 (dd, J = 13.7,

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10.7 Hz, 1H), 7.23 (dd, J = 17.3, 10.6 Hz, 1H), 7.11 (dd, J = 10.6, 6.5 Hz, 1H), 4.91 (d, J = 44.4 Hz, 1H), 4.44 (s, 1H), 4.18 (d, J = 26.4 Hz, 17H), 3.96 – 3.85 (m, 1H), 3.75 (d, J = 4.4 Hz, 4H), 3.66 – 3.55 (m, 1H), 3.48 (dd, J = 14.2, 7.9 Hz, 1H), 3.32 (d,

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J = 15.5 Hz, 1H), 3.17 (s, 1H), 2.99 (s, 3H), 2.69 (s, 2H), 2.54 – 2.44 (m, 1H), 1.80 (s, 1H), 1.70 – 1.53 (m, 2H), 1.50 (d, J = 18.0 Hz, 4H), 1.36 – 1.17 (m, 8H), 0.92 – 0.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 153.84, 119.32, 106.57, 83.53, 66.05, 50.71, 40.07, 39.86, 39.65, 39.44, 39.23, 39.02, 38.82, 38.21, 31.22, 28.63, 27.52, 25.93, 22.04, 21.55, 13.93. HR-MS (ESI) Calcd for C28H49Cl3FN7O4 [M-3H-3Cl+H]+: 564.3668, found: 564.3673. (6h): Dark yellow foamed solid, yield 83.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 19.8 Hz, 3H), 8.05 – 7.96 (m, 1H), 7.56 – 7.46 (m, 2H), 7.20 (d, J = 6.6 Hz, 1H), 7.09 (td, J = 9.3, 5.2 Hz, 1H), 5.30 (s, 1H), 4.94 (s, 1H), 4.66 (d, J = 4.4 Hz, 1H),

ACCEPTED MANUSCRIPT 4.17 (dd, J = 32.7, 25.7 Hz, 2H), 3.94 – 3.68 (m, 39H), 3.60 (dd, J = 14.4, 7.2 Hz, 1H), 3.47 (dd, J = 14.3, 8.0 Hz, 1H), 3.37 – 3.25 (m, 1H), 3.15 (dd, J = 14.6, 7.6 Hz, 2H), 2.97 (s, 4H), 1.79 (d, J = 6.1 Hz, 2H), 1.55 (t, J = 19.0 Hz, 4H), 1.25 (d, J = 4.9 Hz, 15H), 0.85 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 157.08, 127.19,

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109.74, 66.06, 50.70, 45.26, 40.07, 39.86, 39.65, 39.44, 39.24, 39.03, 38.82, 38.21, 33.99, 31.24, 28.65, 25.93, 22.05, 13.93, 8.37. HR-MS (ESI) Calcd for C29H51Cl3FN7O4 [M-3H-3Cl+H]+: 578.3825, found: 578.3832.

(6i): Dark yellow foamed solid, yield 85.9%. 1H NMR (400 MHz, DMSO-d6) δ 8.59

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(s, 1H), 8.30 (d, J = 21.7 Hz, 2H), 7.73 (dd, J = 103.9, 8.4 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.28 (dd, J = 15.0, 7.1 Hz, 1H), 7.13 – 7.06 (m, 1H), 6.98 (td, J = 9.3, 4.8 Hz,

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1H), 4.73 (s, 1H), 4.16 (d, J = 42.3 Hz, 14H), 4.07 – 3.87 (m, 4H), 3.78 (dd, J = 14.4, 3.7 Hz, 1H), 3.68 – 3.58 (m, 6H), 3.36 (dd, J = 14.4, 7.6 Hz, 1H), 3.20 (s, 1H), 3.03 (dd, J = 11.5, 5.4 Hz, 2H), 2.97 – 2.89 (m, 2H), 2.86 (s, 3H), 2.42 – 2.35 (m, 4H), 1.77 – 1.59 (m, 3H), 1.47 (d, J = 8.0 Hz, 4H), 1.21 – 1.04 (m, 15H), 0.73 (t, J = 6.6 Hz, 3H). HR-MS (ESI) Calcd for C30H53Cl3FN7O4 [M-3H-3Cl+H]+: 592.3981, found:

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592.3985.

(6j): Light yellow foamed solid, yield 97.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.47 (d, J = 25.3 Hz, 3H), 8.05 (d, J = 5.6 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.45

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– 7.38 (m, 1H), 7.24 – 7.19 (m, 1H), 7.13 (dd, J = 11.3, 6.9 Hz, 1H), 5.46 – 4.80 (m, 9H), 4.15 (s, 1H), 4.04 (s, 1H), 3.76 (s, 5H), 3.68 (d, J = 11.0 Hz, 2H), 3.59 (t, J = 8.9 Hz, 2H), 3.48 (dd, J = 12.5, 7.2 Hz, 1H), 3.35 (d, J = 6.5 Hz, 1H), 3.16 (d, J = 5.7 Hz,

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3H), 3.00 (s, 3H), 1.91 – 1.79 (m, 2H), 1.70 – 1.53 (m, 4H), 1.48 – 1.47 (m, 2H), 1.32 – 1.16 (m, 16H), 0.85 (t, J = 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 169.69, 157.18, 127.08, 124.34, 118.95, 109.83, 65.99, 54.07, 54.01, 52.75, 51.46, 50.81, 48.44, 40.08, 39.87, 39.66, 39.46, 39.25, 39.04, 38.83, 38.22, 31.25, 28.97, 28.82, 28.72, 28.67, 27.52, 27.09, 25.94, 24.22, 22.05, 13.90. HR-MS (ESI) Calcd for C31H55Cl3FN7O4 [M-3H-3Cl+H]+: 606.4138, found: 606.4145. Microorganisms and culture conditions: The antibacterial activity of all the small molecules was evaluated against both Gram-positive bacteria (S. aureus, E. faecalis and MRSA) and Gram-negative bacteria (E. coli, S.enterica, KPC, NDM-1). All the

ACCEPTED MANUSCRIPT bacteria were cultured in Muller-Hinton broth (5.0 g of beef extract, 17.5 g of casein hydrolysate, and 1.5 g of starch in 1000 mL of distilled water) Brain-heart infusion broth (5.0 g beef heart infusion form, 12.5 g of calf brains infusion form, 2.5 g Na2HPO4, 2.0 g D-glucose, 10 g of peptone and 5.0 g NaCl in 100 mL of sterile distilled

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water) was used for stock samples of bacteria, the freeze dried stock samples of bacteria in 33.3% glycerol were stored at -80 ºC. For solid media, Mueller-Hinton agar (5.0 g of beef extract, 17.5 g of casein hydrolysate, 1.5 g of starch and 12.5 g of agar in 1000 mL of distilled water) was used as growth medium.

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Cell culture: Human cervical carcinoma cell line (HeLa cell), maintained in complete DMEM media (Bioind) supplemented with 10% FBS (Zeta Life), at 37 ºC in a

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humidified atmosphere of 5% CO2 in air. All the cells were mycoplasma free. The cells were trypsinized, counted and seeded in 96-well plates for viability studies or in 12-well plates for other studies. The cells were allowed to adhere overnight before they were used for experiments.

Antibacterial assay: MIC of all the small molecular compounds (2a-2f, 6a-6j) were

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determined by broth microdilution method according to CLSI guidelines. The test medium for most species was cation-adjusted Muller-Hinton broth (MHB). The 4-6 h grown culture as described in the microorganism and culture condition section gives about 108 CFU/mL of bacteria. The bacterial cultures were then diluted to give

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approximately 106 CFU/mL in Muller-Hinton broth media which were then used for determining antibacterial efficacy. All the final compounds were water soluble at room

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temperature. Stock solutions of the final compounds were prepared with sterile Milli-Q water. Then the stock solutions were serially diluted to different concentration (256, 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 µg/mL) by using Muller-Hinton broth media. These dilutions (100 µL) were added to the wells of 96 well plate followed by the addition of 100 µL of bacterial suspension (106 CFU/mL). Two controls were made: one containing 200 µL of media (negative contrast) and the other containing 200 µL of bacterial solution (106 CFU/mL, positive contrast). The plates were then incubated at 37 ºC for 16-20 h. After the incubation, read the results. Each concentration was determined in twice and the whole experiment was repeated at least twice. The

ACCEPTED MANUSCRIPT antibacterial activity was thus expressed as MIC. To determine the MBC, the bacterial suspension that appeared to have less/little turbidity in the MIC experiment was plated (20 µL) and the agar plates were incubated for 20-24 h at 37 °C. Concentration at which no bacterial growth (no bacterial colony) was observed was taken as the MBC of the

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respective compounds. Antibacterial activity in plasma (Plasma stability): Bacteria (S. aureus) was grown in a similar way as mentioned in the microorganism and culture condition and finally diluted in the respective media to give 106 CFU/mL. The fresh sterile defiber sheep

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blood (from commercial resource) was centrifuged at 3500 rpm for 10 min. The plasma, separated from the blood cells after centrifugation, was carefully collected. The

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test compound 6e was dissolved in 50% sterile Milli-Q water and 50% plasma at a concentration of 512 µg/mL. Three such test samples were preincubated at 37 °C in 50% plasma for 0, 3 and 6 h respectively. Then the three samples were serially diluted to several concentration (256, 128, 64, 32, 16, 8 and 4 µg/mL). After that, 50 µL of the above solutions was added to wells of a 96-well plate and 150 µL of the bacterial

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suspension (105 CFU/mL) was added to wells. The plate was then incubated for 20-24 h at 37 °C and MBCs of the compound were determined as described in the previous section (antibacterial assay).

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Antibacterial assay in complex mammalian fluids: The fresh sterile defiber sheep blood was bought from a biochemical reagent company (China). Plasma was obtained as mentioned above. Serum was isolated by using serum tube containing sheep blood

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and then centrifuging the blood at 3,500 rpm for 10 min. MRSA was grown in way as mentioned in the microorganism and culture conditions. Finally, MRSA was diluted with 50% Muller-Hinton broth (MHB) medium and 50% mammalian media (serum, plasma, blood), individually in a way to give 105 CFU/mL of MRSA in 50% serum, 50% plasma, and 50% blood (having 50% MHB medium). The test molecule 6e was dissolved in sterile water with the serial dilution method at the concentration of (512, 256, 128, 64, 32, 16, 8 and 4 µg/mL). Then 50 µL of the dilutions was added to the wells of a 96-well plate and 150 µL of the bacterial suspension (105 CFU/mL) was added separately to the wells containing the dilutions of the compound 6e. The plate was then

ACCEPTED MANUSCRIPT incubated for 20-24 h at 37 °C and MBC of the test compound was determined by plating the bacterial suspension (20 µL) directly from the wells onto Muller-Hinton agar (MHA) plate. The agar plates were incubated at 37 °C for 20-24 h and colonies were observed to determine the MBC.

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Time-dependent killing: An overnight culture of bateria S. aureus (ATCC 29213) and E.coli (ATCC 25922) was diluted 1:10,000 in MHB medium and incubated at 37 °C with aeration at 225 rpm for 2 h (early exponential). Bacteria were then challenged with compounds 6e and two antibiotics (vancomycin for S.aureus and

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moxalactam for E.coli) in culture tubes at 37 °C and 225 rpm. At different intervals, 100 µL bacteria solution were removed to 96-well plate, centrifuged at 4,000 rpm for

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3 min (TDL 5M centrifuge) and resuspended in 100 µL of sterile phosphate buffered saline (1×PBS). Ten-fold serially diluted suspensions were plated on MHA plates and incubated at 37 °C overnight. Colonies were counted and CFU per mL was calculated. Experiments were performed with biological replicates.

Biofilm disruption assay (determination of viable count and imaging): The

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bacteria S. aureus and E. coli (4-6 h grown, mid-log phase) were diluted to ~105 CFU/mL into suitable media (MHB for S. aureus and M9 media supplemented with 0.02% casamino acid and 0.5% glycerol for E. coli respectively). The 96-well plates

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containing 100 µL of these suspensions were incubated under stationary conditions (for about 24 h for S. aureus and 72 h for E. coli). After incubation, the bacteria suspensions were centrifuged at 3,500 rpm for 5 min, the medium was removed and the wells were

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washed with 1×PBS once. Compound 6e (100 µL at 2, 4, 8, 16, 32, 64, and 128 µg/mL) was then added to the wells containing preformed bacterial biofilms and allowed to incubate for 24 h at 37 °C. A control was made where 100 µL of the above medium was added. After 24 h, medium was discarded and the planktonic cells were removed by washing with 1×PBS. Then 100 µL of trypsin-EDTA solution was added to the treated biofilm to make the suspension of bacterial cells present within the biofilm. Cell suspension was then assessed by plating the 10-fold serial dilutions of the suspension on suitable agar plates. After 24 h of incubation, bacterial colonies were counted and cell viability was expressed as log10 (CFU/well) along with the control. For visualizing

ACCEPTED MANUSCRIPT the disruption of biofilm by the small molecules, 100 µL of 0.1% of crystal violet (CV) was added into the wells and incubated for 10 min. Then the crystal violet solution was discarded and the plates were washed twice with 1×PBS. Finally, imaging of the stained wells was taken using a digital camera.

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Minimum biofilm eradication concentration (MBEC) assay[77]: Biofilm eradication assays involve three phases separated by wash steps, including (i) initial biofilm establishment on well surfaces without test compound; (ii) biofilm treatment with test compound; and (iii) recovery of viable biofilms in fresh medium alone. Both

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assays were used to demonstrate the biofilm eradication activities of compound 6e and linezolid. Biofilm eradication assays were performed in 96-well plates, and microtitre

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wells were inoculated with 100 µL of a 1:1000-fold exponential-phase S. aureus and E. coli (108 CFU/mL) and were incubated for 24 h at 37 °C. After 24 h, medium and planktonic cells were removed and 100 µL of two-fold serial dilutions of test compound was added to the wells in fresh medium and was incubated for 24 h at 37 °C (phase 2). After this time, the contents were removed and 100 µL of fresh medium only was added

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to allow viable biofilms to recover and to disperse planktonic bacteria into the medium resulting in a turbid microtitre well (24 h incubation at 37 °C; phase 3). After this final phase, microtitre plates were examined for visible bacterial growth (turbidity) and the

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MBEC was recorded as the lowest concentration at which no turbidity could be observed (due to eradicated biofilms). For visualizing the disruption of biofilm by the small molecules, 100 µL of 0.1% of crystal violet (CV) was added into the wells and

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incubated for 10 min. Then the crystal violet solution was discarded and the plates were washed twice with 1×PBS. Then 100 µL of ethanol was added to dissolve the crystal violet stained biofilms. Finally, imaging of the stained wells was taken using a digital camera.

Minimum biofilm inhibitory concentration (MBIC) determination [77]: In 96-well plates, two-fold serial dilutions of test compound 6e was added in MHB medium. Then, 1:1000-fold exponential-phase S. aureus and E. coli (108 CFU/mL) in MHB was added to each well and was allowed to incubate at 37 °C for 24 h. After this time, the con-tents from the 96-well plates were removed and the wells were rinsed with water, followed

ACCEPTED MANUSCRIPT by the addition of 100 µL of 0.1% crystal violet to stain the biofilms (10 min incubation at room temperature). The plates were then rinsed and 100 µL of ethanol was added to dissolve the crystal violet stained biofilms. Minimum concentrations required to inhibit 80% of biofilm formation (MBIC80) were determined (OD540) by comparing compound

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treated versus untreated wells and the resulting data were used to generate dose– response curves using Spss 20.0 (Inhibition%=1-OD540Cmpound/OD540Control). Note: these experiments were performed to determine whether the antibiofilm activities of compound 6e were dependent on or independent of their antibacterial activities.

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Cytoplasmic membrane depolarization assay: The 4-6 h grown bacteria (mid-log phase) were harvested (3,500 rpm, 5 min), washed and resuspended with 1×PBS (S.

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aureus) and 5 mM HEPES buffer, 5 mM glucose and 100 mM KCl solution at 1:1:1 ratio (E. coli). Then the bacterial suspension (~108 CFU/mL, 150 µL) was added to the wells of a 96-well plate (Black plate, clear bottom with lid). Then 3, 3′-dipropylthiadicarbocyanine iodide (diSC35) (10 µM, 50 µL) was added to the wells containing bacterial suspension and pre-incubated for about 30 min for S. aureus and

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40 min for E. coli (additional 50 µL of 200µM of EDTA was also added in case of E. coli). After the incubation, fluorescence was measured for the next 8 min at every 2 min interval at an excitation wavelength of 622 nm (slit width: 10 nm) and emission

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wavelength of 670 nm (slit width: 5 nm). Bacterial suspensions were then transferred to another well-plate containing 10 µL of 420 µg/mL of small molecule 6e and three antibiotics (linezolid, meropenem and vancomycin) and fluorescence intensity was

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monitored immediately for another 12 min at every 2 min interval, the final concentration of small molecule 6e and three antibiotics was 20 µg/mL. A control experiment was performed by treating the preincubated bacterial suspension and dye solution only with sterile Milli-Q water (50 µL). Outer membrane permeabilization assay: The outer membrane permeabilization activity of the small molecule 6e and three antibiotics (linezolid, meropenem and vancomycin) was determined by the N-phenylnapthylamine (NPN) assay. Mid-log phase bacteria (E. coli) were harvested similarly as mentioned in earlier experiments, washed and resuspended similarly as the previous method. Bacterial suspension (~108

ACCEPTED MANUSCRIPT CFU/mL, 150 µL) was transferred into the wells of a black 96-well plate. Then NPN dye (10 µM, 50 µL) was added to the wells containing bacterial suspension and pre-incubated for about 30 min for S. aureus and 40 min for E. coli. After the incubation, fluorescence was monitored for next 8 min at every 2 min interval at an

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excitation wavelength of 350 nm (slit width: 10 nm) and emission wavelength of 420 nm (slit width: 5 nm). Then, the bacterial suspensions were transferred to another black well-plate containing 10 µL of 420 µg/mL of small molecule 6e and three antibiotics and fluorescence intensity was monitored immediately for another 12 min

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at every 2 min interval, the final concentration of molecule 6e and three antibiotics was 20 µg/mL. A control experiment was performed by treating the preincubated

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bacterial suspension and dye solution only with sterile Milli-Q water (50 µL). Inner membrane permeabilization assay: The 4-6 h grown bacteria (mid-log phase) were harvested (3500 rpm, 5 min), washed and resuspended similarly as the previous method. Then the bacterial suspension (~108 CFU/mL, 150 µL) was added to the wells of a 96-well plate (Black plate, clear bottom with lid). Then propidium iodide (PI) (10

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µM, 50 µL) was added to the wells containing bacterial suspension and pre-incubated for about 30 min for S. aureus and 40 min for E. coli. After the incubation, fluorescence was measured for the next 8 min at every 2 min interval at an excitation wavelength of

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535 nm (slit width: 10 nm) and emission wavelength of 617 nm (slit width: 5 nm). Bacterial suspensions were then transferred to another well-plate containing 10 µL of

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420 µg/mL of small molecules and fluorescence intensity was monitored immediately for another 12 min at every 2 min interval, the final concentration of molecule 6e and three antibiotics (linezolid, meropenem and vancomycin) was 20 µg/mL. A control experiment was performed by treating the preincubated bacterial suspension and dye solution only with sterile Milli-Q water (50 µL). Propensity of bacterial resistance development: In order to evaluate the propensity of developing bacterial resistance towards the compounds, one of the potent compound 6e was used in the study. First, MIC of compound 6e was determined against S. aureus, E. coli and MRSA, and subsequently the compound was challenged repeatedly at the 1/2 MIC level. Three control antibiotics norfloxacin, colistin and linezolid were chosen

ACCEPTED MANUSCRIPT for S. aureus, E. coli and MRSA, respectively. In case of norfloxacin, colistin and linezolid, the initial MICs were determined against respective bacteria. After the initial MIC experiment, serial passaging was initiated by transferring bacterial suspension grown at the 1/2 MIC of the compound/antibiotics and was subjected to another MIC

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assay. After 24 h incubation period, cells grown at the 1/2 MIC of the test compound/antibiotics were once again transferred and assayed for MIC experiment. The process was repeated for 20 passages for both S. aureus, E. coli and MRSA. The MIC for test compound to the control antibiotics was plotted against days to determine

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the propensity of bacterial resistance development.

Hemolytic activity: RBCs were isolated from sheep blood and resuspended in 1×PBS

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(5%). RBCs suspension (150 µL) was then added to solutions of serially diluted small molecules 2e, 6e and linezolid at the concentration of (2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5 µg/mL) in a 96-well plate (50 µL). Two controls were prepared, one 50 µL RBCs suspension (5%) and the other with 50 µL of 0.1% solution of Triton X-100. The plate was then incubated for 1 h at 37 °C. After the incubation, the plate was centrifuged

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at 3,500 rpm for 5 minutes. Supernatant (100 µL) from each well was then transferred to a fresh 96-well plate and absorbance at 540 nm was measured. Percentage of hemolysis was determined as (A–A0) / (Atotal–A0) × 100, where A is the absorbance of

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the test well, A0 is the absorbance of the negative control (5% RBCs suspensions), and Atotal the absorbance of wells with 0.1% Triton X-100). Cytotoxicity study: Cytotoxicity of the small molecules were evaluated by the Cell

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Counting Kit-8 (CCK-8). Briefly, 5 × 103 cells in 100 µL medium were seeded to each of 96-well plates. After 24 h incubation at 37 °C, the culture medium was removed and replaced with fresh medium (100 µL) containing the candidate compounds 6e in different concentration. And only media was used as negative control. At the end of the treatment (24 h), the medium was discarded and washed twice with the new culture medium, then added 100 µL new medium (with 5% CCK-8) to each well. Cells were incubated at 37 °C for a further 4 h and then the absorbance at 450 nm was measured using a Microplate Reader. Results were expressed as percent viability = [A-A0 / Anc-A0] × 100%, where A is the absorbance of the treated cells, Anc is the absorbance of

ACCEPTED MANUSCRIPT the negative control and A0 is absorbance of the background (new medium containing 5% CCK-8). The average 50% inhibitory concentration (IC50) was determined from the dose-response curves according to the inhibition ratio for each concentration. Each concentration was analyzed in triplicate and the experiment was repeated three times.

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Fluorescence and inverted microscopy: As mentioned above for the cytotoxicity study, cells were seeded into the wells of a 12-well plate and then treated with compounds 6e at various concentrations (1, 2, and 4 µg/mL). For positive control 0.1% Triton X-100 was used. All the treated and untreated cells (as negative control) were

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washed once with 1×PBS (the images were captured with a 10× objective in inverted microscope ) and stained with 2 µM calcein AM (Fluka) and 4.5 µM propidium iodide

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(PI) (Sigma-Aldrich) (500 µL of 1:1 calcein AM:PI) for 15 min at 37 °C under 5% CO2-95% air atmosphere. Finally, the images were captured with a 10× objective in fluorescence microscope using a band-pass filter for calcein AM at 500-550 nm and a long-pass filter for PI at 590-800 nm.

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Acknowledgement

Project supported by the National Natural Science Foundation of China (No. U1204206, 81501782, 21702190), Science and Technology Department of Henan Province (No. 172102310227) and the Education Department of Henan Province (No.

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17A350004).

Appendix A. Supplementary data

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Supplementary data related to this article can be found at References

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ACCEPTED MANUSCRIPT 1) Three series of novel cationic deacetyl linezolid amphiphiles were synthesized and tested for antimicrobial activities. 2) The MIC values of the best compound 6e ranged from 2 to 16 µg/mL and linezolid ranged from 2 to >64 µg/mL against these strains.

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3) These bactericidal compounds were acted by permeabilization and depolarization of bacterial membrane.

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4) Compound 6e was difficult to induce bacterial resistance and had ability to combat drug-resistant bacteria.