New N-phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATPase inhibitors of DNA gyrase

Accepted Manuscript New N-Phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATPase Inhibitors of DNA Gyrase Nace Zidar, Tihomir Tomašič, Helena Macut, Anja Sirc, Matjaž Brvar, Sofia Montalvão, Päivi Tammela, Janez Ilaš, Danijel Kikelj PII:

S0223-5234(16)30263-X

DOI:

10.1016/j.ejmech.2016.03.079

Reference:

EJMECH 8504

To appear in:

European Journal of Medicinal Chemistry

Received Date: 5 January 2016 Revised Date:

25 February 2016

Accepted Date: 26 March 2016

Please cite this article as: N. Zidar, T. Tomašič, H. Macut, A. Sirc, M. Brvar, S. Montalvão, P. Tammela, J. Ilaš, D. Kikelj, New N-Phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATPase Inhibitors of DNA Gyrase, European Journal of Medicinal Chemistry (2016), doi: 10.1016/ j.ejmech.2016.03.079. 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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New N-Phenyl-4,5-dibromopyrrolamides and NPhenylindolamides as ATPase Inhibitors of DNA

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Gyrase Nace Zidar, a,* Tihomir Tomašič, a Helena Macut, a Anja Sirc, a Matjaž Brvar, b Sofia Montalvão, c Päivi Tammela, c Janez Ilaš, a Danijel Kikelj a

Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia

b

National Institute of Chemistry, Laboratory for Biocomputing and Bioinformatics, 1001 Ljubljana,

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a

Slovenia

Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of

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c

Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland

ABSTRACT

Following the withdrawal of novobiocin, the introduction of a new ATPase inhibitor of

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DNA gyrase to the clinic would add the first representative of this mechanistic class to the antibacterial pipeline. This would be of great importance because of the well-known problems associated with antibacterial resistance. Using structure-based design and starting from the recently determined crystal structure of the N-phenyl-4,5-dibromopyrrolamide inhibitor-DNA

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gyrase B complex, we have prepared 28 new N-phenyl-4,5-dibromopyrrolamides and Nphenylindolamides and evaluated them against DNA gyrase from Escherichia coli. The most compound

was

2-((4-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-2-

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potent

oxoacetic acid (9a), with an IC50 of 0.18 µM against E. coli gyrase. A selected set of compounds was evaluated against DNA gyrase from Staphylococcus aureus and against topoisomerase IV from E. coli and S. aureus, but the activities were weaker. The binding affinity of 2-((4-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-2-oxoacetic acid (9a) to E. coli gyrase was studied using surface plasmon resonance. In the design of the present series, the focus was on the optimisation of biological activities of compounds – especially by varying their size, the position and orientation of key functional groups, and their acid-base properties. The structure-activity relationship (SAR) was examined and the results were rationalised with molecular docking.

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ACCEPTED MANUSCRIPT KEYWORDS:

antibacterial;

4,5-dibromopyrrole;

DNA

gyrase;

GyrB;

inhibitor;

topoisomerase IV ABBREVIATIONS ATCC, American type culture collection; ATR, attenuated total reflectance; CFU, colonyunit;

CLSI,

Clinical

diazabicycloundec-7-ene;

and

DTT,

Laboratory

Standards

dithiothreitol;

Institute;

EDC,

DBU,

1,8-

N-ethyl-N′-(3-

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forming

dimethylaminopropyl)carbodiimide hydrochloride; GyrA, DNA gyrase A; GyrB, DNA gyrase B; Kd, dissociation constant; MH, Mueller Hinton; NHS, N-hydroxysuccinimide; NMM, Nmethylmorpholine; ParC, topoisomerase IV subunit A; ParE, topoisomerase IV subunit B;

(benzotriazol-1-yl)uronium

tetrafluoroborate;

trifluoroacetic

acid;

topo

IV,

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topoisomerase IV.

TFA,

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RA, residual activity; SPR, surface plasmon resonance; TBTU, N,N,N′,N′-tetramethyl-O-

INTRODUCTION

The European Centre for Disease Prevention and Control (ECDC) estimates that: “Each year, antimicrobial resistance results in 25,000 deaths at a cost of over 1.5 billion EUR due

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to healthcare expenses and productivity losses in the EU” [1]. The mortality in patients infected with resistant bacterial strains is up to twice as high as in patients infected with nonresistant bacteria [2]. It is becoming more and more evident that the pharmaceutical industry today is not able to keep up with the growing need for effective antimicrobial drugs,

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especially against the Gram negative pathogens. As recently discussed in a review by Bisacchi and Manchester [3], important reasons for this situation are associated with

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economics, including the growing cost of antibacterial R&D, strict regulatory requirements, and the pricing of antibiotics. To bypass the increasing resistance problems, combined action of all sectors of government and society is needed. DNA gyrase (gyrase) is a member of type IIA bacterial topoisomerases and is a well-known and clinically validated pharmacological target for antibacterial drugs. The function of DNA gyrase is to catalyse the transient break and reunion of the DNA double strand, a process crucial for negative supercoiling or relaxation of positive supercoils in the DNA molecule during its replication. DNA gyrase is a tetrameric protein composed of two GyrA and two GyrB subunits that form the active A2B2 complex. GyrA subunit binds the DNA molecule, while the GyrB subunit possesses ATPase activity and provides energy for the supercoiling 2

ACCEPTED MANUSCRIPT process. Topoisomerase IV (topo IV), an enzyme structurally and functionally similar to gyrase, forms a C2E2 complex composed of two ParC and two ParE subunits that are homologous to the GyrA and GyrB of gyrase, respectively. The main function of topo IV is to decatenate the two linked daughter chromosomes following DNA replication. As opposed to GyrA/ParC functions, which are inhibited by a clinically relevant group of fluoroquinolones,

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none of the known inhibitors of GyrB/ParE subunits are currently in the clinical pipeline. After several decades of research in the field of ATPase inhibitors of gyrase/topo IV, novobiocin, a natural coumarin antibiotic, remains the only GyrB inhibitor that has ever progressed to the clinic. Although it was later withdrawn from the market because of toxicity

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issues, its main value – validation of the mechanism of action of ATPase inhibitors of GyrB/ParE – still stands [4-6].

In recent years, our understanding of the molecular factors governing GyrB/ParE inhibition

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has increased considerably due to the resolution of several enzyme-inhibitor co-crystal structures. Furthermore, there is emerging awareness of the structural properties that, in addition to strong on-target activity, endow the compounds with physicochemical and pharmacokinetic properties that enable them to penetrate the bacterial cell wall and thus exhibit good antibacterial effects. The accumulated knowledge of the essential structural

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factors governing the activity of compounds, combined with the advances in high-throughput screening (HTS) and structure-based design (SBD), has resulted in many new promising chemical classes of GyrB/ParE inhibitors, such as pyrazoles [7], indazoles [8], indolin-2-ones [9, 10], 4,5’-bithiazoles [11], arylaminopyrimidines [12], pyrrolopyrimidines [13, 14],

[20],

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imidazo[1,2-a]pyridines [15], benzimidazole ureas [16-18], pyrazolthiazoles [19], azaindoles pyrrolamides

[21-23],

tetrahydrobenzothiazoles

[24],

and

N-phenyl-4,5-

dibromopyrrolamides [25]. Several clinical candidates have been identified, but because of

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unfavourable physicochemical properties (e.g. chemical instability, poor aqueous solubility), pharmacokinetic issues (e.g. low oral bioavailability, high protein binding), or because of the adverse economics associated with antibacterial R&D in general, many of the existing industrial antibacterial drug-discovery programs have recently been terminated [3]. This fact highlights the need for a renewed and more coordinated research effort in the fight against antibacterial resistance by both pharmaceutical industry and academia. In the present study, in continuation of our recent work [25], we describe the design, synthesis and evaluation of a new series of N-phenyl-4,5-dibromopyrrolamides and Nphenylindolamides as ATPase inhibitors of DNA gyrase. We aimed at optimizing the inhibitory potency of our previous series by (i) modifying the central core of the molecules, 3

ACCEPTED MANUSCRIPT (ii) adjusting the distance between key pharmacophoric elements, (iii) introducing different right-hand side substituents to interact with Arg76 and Arg136 of the gyrase binding site, and by (iv) constraining the conformation of the inhibitors. The structure-activity relationship (SAR) of the prepared compounds, supported by molecular docking experiments, enabled us to analyse the essential structural elements for GyrB/ParE inhibition, and to study the factors

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important for translating the enzymatic inhibitory activity into an antibacterial effect.

RESULTS AND DISCUSSION

Design. Based on our recently reported class of N-phenyl-4,5-dibromopyrrolamide gyrase B

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inhibitors [25], a critical analysis of their SAR enabled us to identify key interaction points of the enzyme. The design of the present series, aimed at improving the enzymatic inhibitory and antibacterial activity, was guided by the co-crystal structure of our inhibitor within the GyrB

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active site (compound A, Figure 1). In compound A, in the new series, the central amide bond is seen to be inverted and/or rigidified, resulting in compounds with aminobenzene (I, II, IV; Figure 1) or benzoyl moieties (III, Figure 1) as a central core connecting the pyrrolamide/indolamide moieties on the left-hand side and various functional groups on the right-hand side of the compounds. The benzene ring is able to form hydrophobic contacts with

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amino acid residues Gly77, Ile78 and Pro79, and allows the introduction of functionalities that project toward other key interaction regions, e.g. Arg76 and Arg136. To the left-hand side of the central aminobenzene or benzoyl scaffold, 4,5-dibromopyrrolamide or the indolamide moieties were retained to reach into the hydrophobic pocket formed by amino acid residues

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Val43, Ala47, Val71, Val120, and Val167. Both moieties possess the ring NH and the adjacent C=O groups that are essential for forming a network of hydrogen bonds with Asp73 and a conserved water molecule. Since this interaction is crucial for the binding of the adenine

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of ATP, no mutations at Asp73 have so far been reported, which makes the development of bacterial resistance to such compounds less probable. The amide NH of the 4,5dibromopyrrolamide/indolamide moiety is important for making possible indirect contacts with Asn46 through a crystal water molecule, and was thus left unsubstituted. Because the central and the right-hand sides of the designed inhibitors do not lie in the ATP binding site, their interactions with the gyrase are important to ensure selectivity against other ATPases, especially those with structurally similar nucleotide binding folds, such as those from the GHKL (gyrase, Hsp90, histidine kinase, MutL) superfamily. Based on the para or meta substituents on the central benzene ring, the designed analogues can be divided into five different sets, (i) compounds with N-oxalyl, -malonyl, or -succinyl groups (I, Figure 1), (ii) 4

ACCEPTED MANUSCRIPT compounds with piperidinecarboxylic acid or 2-oxopiperidinecarboxylic acid substituents (II, Figure 1), (iii) compounds with L- or D-proline side chains (III, Figure 1), (iv) compounds possessing an oxazolidin-2-one substituent (IV, Figure 1), and (v) a compound with an acetophenone substructure (V, Figure 1). In type I compounds, oxalic (compounds 8a, 8d, 9a, 9d), malonic (compounds 8b, 9b), or

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succinic acid (compound 8c) substituents were selected for interaction with the Arg76 and Arg136 region of the protein. The number of methylene groups in the substituent chains was varied to determine the optimal length of the molecules for binding. Compounds 7 and 9c were prepared to assess the effect of meta substitution. In type II compounds possessing (2-

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oxo)piperidinecarboxylic acid groups on the right-hand side, the effect of rigidification and the difference in activity between analogues with a COOH group on position 3 (compounds 15a, 15d, 16a, 16d) and on position 4 (compounds 15b-c, 15e-f, 16b-c, 16e-f) of the (2-

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oxo)piperidin-1-yl fragment, were studied. Additionally, the differences in potency between cyclic amides (compounds 15c, 15f, 16c, 16f) and compounds with the reduced carbonyl group (15a-b, 15d-e, 16a-b, 16d-e) were evaluated. Four compounds of the type III series containing L-Pro (compounds 21a, 22a) and D-Pro (compounds 21b, 22b) were prepared in order to study the effect of rigidification of the terminal region of the molecule, and also to

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evaluate the influence of stereochemistry on inhibition of the enzymatic activity. Type IV compounds, with the oxazolidin-2-one heterocycle (26a-b), and type V compound, with an acetophenone substructure (28), are shorter than compound A and not ionized under physiological conditions, and are thus expected to penetrate more easily the cytoplasmic

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membrane. From the enzyme inhibitor co-crystal structure (Figure 1) it can be seen that the terminal acetate group of compound A does not form any direct hydrogen bonds with Arg136 and that it projects toward the bulk solvent, while the benzoyl carbonyl is in contact with

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Arg76. By omitting the terminal acetate group of compound A (compound 28), we aimed to retain all the interactions with the protein observed in the X-ray structure, at the same time aiming to improve the physicochemical properties and consequently antibacterial activity.

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Figure 1. Design of a new class of N-phenyl-4,5-dibromopyrrolamides and Nphenylindolamides as ATPase inhibitors of DNA gyrase, based on the enzyme-inhibitor A cocrystal structure (PDB code: 4ZVI [25]).

Chemistry. Type I compounds (Figure 1) were synthesized according to Schemes 1 and 2. First, 4-nitroaniline (1) was reacted with ethyl oxalyl chloride (2a), methyl malonyl chloride

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(2b) or methyl succinyl chloride (2c) to obtain compounds 3a-c. The nitro groups of 3a-c were then reduced by catalytic hydrogenation, and the obtained amines 4a-c coupled with 4,5dibromopyrrole-2-carboxylic acid or indole-2-carboxylic acid to give compounds 8a-c.

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Compound 7, with a meta substituted central benzene ring, was prepared in two reaction steps, first by an N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU)-promoted coupling of benzene-1,3-diamine (5) with 4,5-dibromo-pyrrole-2-

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carboxylic acid, and then by the reaction of compound 6 with 2b in the presence of triethylamine. To prepare the final products 9a-d, the alkyl esters 7 and 8a-d were hydrolysed with aqueous lithium hydroxide. Because of the in situ formation of a succinimide ring, the hydrolysis of the methyl ester 8c was not successful.

Scheme 1. Synthesis of N-phenyl-4,5-dibromopyrrolamides and N-phenylidolamides 9ada

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a

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Reagents and conditions: (a) K2CO3, CH3CN, rt, 15 h (for the synthesis of 3a), K2CO3,

CH3CN, 40 °C, 30 h (for the synthesis of 3b), Et3N, CH3CN, 50 °C, 30 h (for the synthesis of

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3c); (b) H2, Pd-C, THF, rt, 3 h; (c) 4,5-dibromopyrrole-2-carboxylic acid (for the synthesis of 7 and 8a-c) or indole-2-carboxylic acid (for the synthesis of 8d), TBTU, NMM, CH2Cl2, rt, 15 h; (d) 2b, Et3N, CH2Cl2/THF, rt, 2 h; (e) LiOH, THF/H2O, rt, 15 h.

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The synthesis of (2-oxo)piperidinecarboxylic acids 16a-f (II, Figure 1) is presented in Schemes 2 and 3. The first three steps were accomplished using described procedures [26].

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First, 1-(4-nitrophenyl)piperidinecarboxylates 12a and 12b were prepared by reacting methyl nipecotate (11a) or ethyl isonipecotate (11b) with 1-fluoro-4-nitrobenzene (10) in the presence of potassium carbonate in dimethylsulfoxide. Ethyl

1-(4-nitrophenyl)-2-

oxopiperidine-4-carboxylate (13) was obtained by oxidizing a methylene group at position 2 of

the

piperidine

ring

of

compound

12b

with

potassium

permanganate

and

benzyltriethylammonium chloride (BTEAC) in refluxing dichloromethane [27]. After the reduction of the nitro groups of compounds 12a, 12b and 13, the obtained amines 14a-c were coupled with 4,5-dibromopyrrole-2-carboxylic acid or indole-2-carboxylic acid to obtain compounds 15a-f. Alkaline hydrolysis of esters 15a-f gave the target compounds 16a-f.

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Reagents and conditions: (a) K2CO3, DMSO, 55 °C, 15 h; (b) KMnO4, BTEAC, CH2Cl2,

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reflux, 7 d; (c) H2, Pd-C, MeOH, rt, 5 h.

Scheme 3. Synthesis of 1-(4-(4,5-dibromo-pyrrole-2-carboxamido)phenyl)piperidinecarboxylic acids 16a-c and 1-(4-(indole-2-carboxamido)phenyl)piperidine-carboxylic

a

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acids 16d-fa

Reagents and conditions: (a) 4,5-dibromo-1H-pyrrole-2-carboxylic acid, TBTU, NMM,

CH2Cl2, reflux, 15 h; (b) 1H-indole-2-carboxylic acid, TBTU, NMM, CH2Cl2, reflux, 15 h; (c) 2M LiOH, THF/H2O, rt, 5 h. (4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)benzoyl)prolines 22a and 22b (III, Figure 1) were synthesized according to Scheme 4. First, L- or D-Pro methyl ester was reacted with 4nitrobenzoyl chloride to give amides 19a-b. After the reduction of the nitro group, the

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ACCEPTED MANUSCRIPT obtained amines 20a-b were coupled with 4,5-dibromopyrrole-2-carboxylic acid to give compounds 21a-b, which were converted to products 22a-b upon hydrolysis of the methyl esters.

Scheme 4. Synthesis of (4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoyl)prolines 22a

a

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and 22ba

Reagents and conditions: (a) K2CO3, CH3CN, rt, 15 h; (b) H2, Pd-C, THF/EtOH, rt, 6 h; (c) i)

4,5-dibromo-1H-pyrrole-2-carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 20a or

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20b, pyridine, CH2Cl2, rt, 6 h; (d) 2M NaOH, MeOH/THF, rt, 15 h. 4,5-Dibromo-N-(4-(2-oxooxazolidin-3-yl)phenyl)-1H-pyrrole-2-carboxamide (26a) and N(4-(2-oxooxazolidin-3-yl)phenyl)-1H-indole-2-carboxamide (26b) (IV, Figure 1) were prepared according to Scheme 5. First, 3-(4-nitrophenyl)oxazolidin-2-one (24) was

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synthesized by heating 4-nitroaniline (23) with ethylene carbonate in the presence of 1,8diazabicycloundec-7-ene (DBU) as base at 100 °C. After the reduction of the nitro group of

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24, the obtained 3-(4-aminophenyl)oxazolidin-2-one (25) was coupled with 4,5-dibromopyrrole-2-carboxylic acid or indole-2-carboxylic acid, to give target compounds 26a and 26b. N-(4-acetylphenyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (28) was prepared by coupling 4aminoacetophenone (27) with 4,5-dibromo-pyrrole-2-carboxylic acid chloride (Scheme 6).

Scheme 5. Synthesis of 4,5-dibromo-N-(4-(2-oxo-oxazolidin-3-yl)phenyl)-1H-pyrrole-2carboxamide (26a) and N-(4-(2-oxo-oxazolidin-3-yl)phenyl)-1H-indole-2-carboxamide (26b)a

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Reagents and conditions: (a) ethylene carbonate, DBU, 100 °C, 3 h; (b) H2, Pd-C, THF, rt, 5

h; (c) 4,5-dibromo-pyrrole-2-carboxylic acid, TBTU, NMM, CH2Cl2, 50 °C, 15 h; (d) indole-

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2-carboxylic acid, TBTU, NMM, CH2Cl2, 50 °C, 15 h.

a

Reagents and conditions: i)

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Scheme 6. Synthesis of N-(4-acetylphenyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (28) a

4,5-dibromo-1H-pyrrole-2-carboxylic acid, oxalyl chloride,

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CH2Cl2, rt, 15 h, then ii) 27, pyridine, CH2Cl2, rt, 6 h.

Inhibitory activities against DNA gyrase and topoisomerase IV. A total of 28 compounds were prepared and evaluated against DNA gyrase from Escherichia coli in the

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DNA supercoiling assay. The results are presented in Tables 1-3 as residual activities (RA = % activity of the enzyme in the presence of 100 µM of compound) or as IC50 values

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(concentration of compound that inhibits the enzyme activity by 50%) for the active compounds (RA < 50% at 100 µM). Most potent compounds were also tested against DNA gyrase from Staphylococcus aureus, and against topoisomerases IV from E. coli and S. aureus. Molecular docking experiments of all the tested compounds in the E. coli DNA gyrase ATP-binding site were conducted to rationalize the observed differences in inhibitory activities. Docking was performed using FlexX [28, 29], as available in LeadIT (BioSolveIT GmbH) [30]. Two of the prepared compounds (9a and 9b) displayed a promising, lower than 0.5 µM IC50 value on E. coli gyrase, and three additional compounds had IC50 values lower than 5 µM (8b, 21a, 28). Most active compounds were representatives of the type I series, with oxalic, malonic, or succinic acid functional groups on the right-hand side of the scaffolds. 4,510

ACCEPTED MANUSCRIPT Dibromopyrrole containing compounds were generally more active than compounds with indole groups, as can be seen e.g. by comparing the activities of 9a (E. coli gyrase IC50 = 0.18 µM) and its indole analogue 9d (E. coli gyrase IC50 = 26 µM). Seemingly, steric occlusions lead to less optimal binding of the indole moiety to the hydrophobic binding pocket of the protein ATP-binding site than that of a smaller 4,5-dibromopyrrole. Furthermore, in type I

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series, the potencies of compounds with free carboxylic acid groups were higher than those of the corresponding esters (e.g. comparison of 8b with IC50 = 1.5 µM, and 9b with IC50 = 0.47 µM). Moreover, a 1,4 substitution pattern on the central benzene ring was more favorable (compound 9b with IC50 = 0.47 µM) than the 1,3 substitution pattern (compound 9c with RA

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= 65% at 100 µM) that apparently offers a less optimal 3D orientation of the key functional groups for interaction with the enzyme. Based on molecular docking studies, the amide carbonyl group of the malonomonoamide substituent of 1,4-substituted compound 9b forms a

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hydrogen bond with Arg76 side chain, like that observed in the crystal structure of compound A in complex with E. coli GyrB. In addition, a salt bridge is formed between the terminal carboxylate of the malonomonoamide substituent of inhibitor 9b and the Arg136 side chain (Figure 2a). In contrast, the docking studies suggest that the malonomonoamide carbonyl group of 1,3-substituted compound 9c is rotated in a way such that the interaction with Arg76

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is lost, but that there is a possibility that a hydrogen bond can be formed with Gly77. The terminal carboxylate group of 9c is positioned in the space between Arg76 and Arg136 side chains, where it might form two hydrogen bonds, but its orientation is less optimal than that of 9b (Figure 2b). An additional parameter that was optimized in the type I series was the

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length of the right-hand side-chain that is most appropriate to enable the interaction of the terminal carboxylic group with Arg76 or Arg136. The length of the compounds increases from oxalic, through malonic, to succinic acid containing analogues. Based on the inhibition

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of E. coli gyrase, in the methyl ester series, the malonic acid analog 8b (IC50 = 1.5 µM) is more potent than the corresponding oxalic acid analog 8a (RA = 68%), or succinic acid analogue 8c (RA = 65%). On the contrary, the higher potency of the oxalic acid derivative was seen in the carboxylic acid series by comparing the potencies of compound 9b (IC50 = 0.47 µM) and its oxalic acid containing counterpart 9a (IC50 = 0.18 µM). Docking studies show that the amide carbonyl of 9a interacts with Arg136 instead of Arg76. Two additional hydrogen bonds are formed between carboxylate group of 9a and Arg76 and Arg136 (Figure 2c).

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Figure 2. Binding modes of inhibitors a) 9b, b) 9c, c) 9a and d) 28 (in yellow sticks) in the E. coli gyrase ATP-binding site (PDB code: 4DUH [11], in cyan) predicted by molecular docking with FlexX. For clarity, only key amino acid residues forming hydrogen bonds with inhibitors are presented. Hydrogen bonds are presented as black dashed lines. The figure was

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prepared by PyMOL [31].

Type II compounds with piperidinecarboxylic or 2-oxopiperidinecarboxylic substituents were found to be weaker inhibitors of gyrase and topo IV enzymes than type I compounds. As

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in the type I series, 4,5-dibromopyrrole analogues were more potent on E. coli gyrase than indole-based analogues, as can be seen by comparing the potencies of 16a with a value of 7.0 µM IC50 with that of the corresponding indole analogue, 16d, with 76% residual activity at

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100 µM. Additionally, the carboxylic acid compounds (e.g. 16a with IC50 = 7.0 µM) were more active than their methyl or ethyl ester precursors (e.g. 15a with RA = 89%). There was no clearly measurable difference in activities between piperidinecarboxylic acid- and 2oxopiperidinecarboxylic acid-based derivatives. The importance of the position of the carboxylate group on the piperidine ring was established by comparing the 1,3-substituted compound 16a (IC50 = 7.0 µM) and its corresponding 1,4-substituted analogue 16b (RA = 88%) – 1,3-substitution was found to be more favorable. In the type III series we analyzed the activities of compounds with an L- or D-Pro residue that imposes rigidity in the side chain of compound A (Figure 1). With IC50 values of 7 and 14 µM for 22a and 22b, these compounds were less potent than compound A (IC50 = 0.45 µM) 12

ACCEPTED MANUSCRIPT [25]. The L analogues 21a and 22a, with IC50 values of 2.9 µM and 7.0 µM, were more potent than the

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analogues 21b and 22b, each with an IC50 value of 14 µM, highlighting the

importance of the correct spatial orientation of the carboxylic group for optimal interaction with the protein. Interestingly, in contrast to the type I series, the potency of compound 22a, with a free carboxylic group, was less than that of its methyl ester precursor 21a.

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While compounds 26a-b, possessing oxazolidin-2-one directly bound to the phenyl ring, were not active, an interestingly high activity was observed for the acetophenone 28 (IC50 = 1.6 µM). The latter, with a very short COCH3 substituent on the position 4 of the aminobenzene ring, was, surprisingly, more potent than all the compounds of the type II, type

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III and type IV series, more potent than the methyl ester of compound A (IC50 = 23 µM) but weaker than compound A (IC50 = 0.45 µM). From a detailed analysis of docking poses of all the tested compounds it can be concluded that a hydrogen bond between the amide carbonyl

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of the right-hand side groups of inhibitors and the Arg76 or Arg136 residue is important for potent E. coli GyrB inhibition, since this interaction is observed in the binding modes of the most potent inhibitors 9a, 9b and 28 (Figures 2c, 2a and 2d), as well as in the binding mode of inhibitor A (Figure 1). Furthermore, the X-ray structure (PDB code: 4ZVI, [25]) and molecular dynamics simulation [25] indicate that the methylenecarboxylate side chain of

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inhibitor A is very flexible and that the terminal carboxylate is in contact with Arg136 only for a short period of time, being mainly in contact with the solvent. Based on these results it appears that the high entropic penalty, due to the flexibility of the right-hand side groups of type I-IV compounds, is only partly compensated by additional interactions formed by the

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terminal carboxylate groups.

In general, for the entire series, the inhibitory potencies of compounds on gyrase from S. aureus (the most active was compound 9a with IC50 value of 51 µM), and on topo IV from E.

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coli and S. aureus, were weaker than those on gyrase from E. coli. This can be explained by the more occluded active sites of S. aureus gyrase and topo IV enzymes than of E. coli gyrase, because of the differences in certain key active site amino acids, as discussed recently [24, 25].

Surface Plasmon Resonance (SPR) Experiments. For the most active compound, 9a, surface plasmon resonance (SPR) experiments [32] were performed with a 24 kDa large Nterminal part of the B subunit of E. coli gyrase (G24) immobilized on a CM5 sensor chip. The sensograms are presented in Figure 1S of the Supplementary Information. The Kd value obtained from the concentration-response curve fitted to a 1:1 steady state binding model was 13

ACCEPTED MANUSCRIPT 2.7 µM. Together with the fact that the present series of compounds was designed based on information derived from the crystal structure of compound A in complex with gyrase B from E. coli [25], the results of the SPR measurements for which G24 protein, a smaller fragment of the GyrB subunit incorporating the ATP binding site, was used, suggest that our

RI PT

compounds bind to DNA gyrase B.

Table 1. Inhibitory activities of type I compounds against DNA gyrase and topoisomerase IV.

R2

4,5-Dibromo-

7

pyrrol-2-yl

8c 8d 9a

9b

9c 9d

pyrrol-2-yl 4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl

4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl 4,5-Dibromo-

Indol-2-yl

1,3

1

Et

1,4

0

Me

1,4

1

Me

1,4

2

Et

Indol-2-yl

pyrrol-2-yl

Me

H

H

E. coli

S. aureus

E. coli

S. aureus

gyrase

gyrase

topo IV

topo IV

98%

n.d.d

n.d.

n.d.

68%

n.d.

n.d.

n.d.

1.5 ± 0.8 µM

75%

96%

104%

65%

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

51± 10 µM

81%

85%

1,4

0

1,4

0

1,4

1

0.47 ± 0.09 µM

119%

70%

95%

EP

8b

4,5-Dibromo-

n

103%

0.18 ± 0.06 µM Kd = 2.7 µMc

H

1,3

1

65%

n.d.

n.d.

n.d.

H

1,4

0

26 ± 6 µM

n.d.

n.d.

n.d.

AC C

8a

Subst.

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R1

TE D

Compd.

SC

IC50 (µM)a or RA (%)b

a

Concentration of compound that inhibits the enzyme activity by 50%.

b

Residual activity of the enzyme at 100 µM of compound.

c

Dissociation constant determined with surface plasmon resonance experiments.

d

n.d. = not determined

Table 2. Inhibitory activities of type II compounds against DNA gyrase and topoisomerase IV.

14

ACCEPTED MANUSCRIPT IC50 (µM)a or RA (%)b

15a

15b

15c

R1

R2

4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl

X

Subst.

E. coli

S. aureus

E. coli

S. aureus

gyrase

gyrase

topo IV

topo IV

Me

CH2

1,3

89%

n.d.c

n.d.

n.d.

Et

CH2

1,4

94%

n.d.

n.d.

n.d.

Et

C=O

1,4

142%

n.d.

RI PT

Compd.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

Indol-2-yl

Me

CH2

1,3

132%

n.d.

15e

Indol-2-yl

Et

CH2

1,4

95%

n.d.

15f

Indol-2-yl

Et

C=O

1,4

131%

n.d.

H

CH2

1,3

7.0 ± 0.2 µM

420 ± 80 µM

100%

102%

H

CH2

1,4

88%

n.d.

n.d.

n.d.

H

C=O

1,4

16b

16c

4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl 4,5-Dibromopyrrol-2-yl

16d

Indol-2-yl

H

CH2

1,3

16e

Indol-2-yl

H

CH2

1,4

16f

Indol-2-yl

H

C=O

1,4

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16a

SC

15d

50%

n.d.

n.d.

n.d.

76%

n.d.

n.d.

n.d.

143%

n.d.

n.d.

n.d.

90%

n.d.

n.d.

n.d.

Concentration of compound that inhibits the enzyme activity by 50%.

b

Residual activity of the enzyme at 100 µM of compound.

c

n.d. = not determined

TE D

a

Table 3. Inhibitory activities of type III, type IV, and type V compounds against DNA gyrase

AC C

EP

and topoisomerase IV.

A

Compd.

Str.

*

R1

R2

B IC50 (µM)a or RA (%)b R3

E. coli

S. aureus

E. coli

S. aureus

gyrase

gyrase

topo IV

topo IV

21a

A

L

Me

/

/

2.9 ± 0.4 µM

128%

74%

100%

21b

A

D

Me

/

/

14 ± 3 µM

101%

97%

105%

22a

A

L

H

/

/

7.0 ± 0.7 µM

116%

108%

116%

22b

A

D

H

/

/

14 ± 2 µM

129%

106%

113%

15

ACCEPTED MANUSCRIPT B

/

/

26b

B

/

/

28

B

/

/

a

4,5-Dibromo-

104%

n.d. c

n.d.

n.d.

110%

n.d.

n.d.

n.d.

1.6 ± 0.2 µM

63%

115%

99%

pyrrol-2-yl

Indol-2-yl 4,5-Dibromopyrrol-2-yl

COCH3

Concentration of compound that inhibits the enzyme activity by 50%.

b

Residual activity of the enzyme at 100 µM of compound.

c

n.d. = not determined

RI PT

26a

Antibacterial Activity. Due to observed activity against E. coli gyrase, minimum

SC

inhibitory concentration (MIC) experiments for ten selected compounds against E. coli (ATCC 25922) were carried out, but the compounds did not show any activity (MICs >200

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µM, Table 1S). In addition, the antibacterial activity of the selected compounds was further evaluated against two Gram-positive (E. faecalis ATCC 29212, S. aureus ATCC 25923) and one Gram-negative (Pseudomonas aeruginosa ATCC 27853) bacterial strain at 50 µM concentration (Table 1S). Most active was analogue 9d with 21% inhibition of growth of Gram-negative P. aeruginosa. Neither compound 9a with the highest gyrase inhibitory activity, nor the less acidic derivative 28, displayed any considerable antibacterial effect.

TE D

Negative antibacterial results against the E. coli bacteria, for which the gyrase inhibitory activities were promising, can possibly be attributed to the problems associated with bacterial cell entry and/or the activity of efflux pumps. Therefore, further optimization of the physicochemical properties of compounds is probably needed for reaching sufficiently high

AC C

CONCLUSION

EP

intracellular concentrations.

If judging by the huge amount of experimental data, numerous filed patents and enormous investments by governments and the pharmaceutical industry in the last decades, the introduction of a first “new-age” ATPase inhibitor of gyrase/topo IV to the clinic would appear to be a matter of time. However, because of different economic factors, strict regulatory requirements, and problems associated with antibacterial resistance, this goal remains further away than it appears. New research, including a contribution from the academia, is urgently needed. We have designed and prepared 28 new N-phenyl-4,5-dibromopyrrolamides and Nphenylindolamides as ATPase inhibitors of DNA gyrase and evaluated them against DNA 16

ACCEPTED MANUSCRIPT gyrase from E. coli, and the most potent of them also against DNA gyrase from S. aureus and against topoisomerase IV from E. coli and S. aureus. The most potent compound was 2-((4(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-2-oxoacetic acid (9a) with an IC50 value of 0.18 µM on E. coli gyrase. Four other compounds had IC50 values lower than 5 µM. The activities against gyrase from S. aureus, and topo IV from E. coli and S. aureus were

RI PT

generally weaker. Important SAR considerations resulting from this study can be summarized as follows: (i) the size of the proton donor-proton acceptor-containing moiety interacting with Asp73 is important because of possible occlusions in the hydrophobic binding pocket – larger groups, such as the indolamide group, reduce the activity, especially against gyrase from

SC

Gram-positive S. aureus and against topo IV. (ii) Derivatives with free carboxylic acid groups are generally more potent than their ester precursors, but the differences in activities are not so pronounced, (iii) reducing the molecular size can be favourable for the activity if it does

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not involve groups that are crucial for binding to the protein, (iv) the hydrogen bond between the amide carbonyl of the right-hand side groups of inhibitors and Arg76 or Arg136 residue is important for potent E. coli GyrB inhibition. The results of this study further elucidate the SAR of this interesting structural class of GyrB inhibitors and make a valuable contribution to

TE D

the discovery of improved ATPase inhibitors of DNA gyrase.

EXPERIMENTAL SECTION

Determination of Inhibitory Activities on E. coli and S. aureus DNA Gyrase. The assay for the determination of IC50 values (Inspiralis) was performed on the black streptavidin-

EP

coated 96-well microtiter plates (Thermo Scientific Pierce). The plates were first rehydrated with the supplied wash buffer [20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.01% (w/v) BSA, 0.05% (v/v) Tween 20] and biotinylated oligonucleotide was immobilized onto the wells. The

AC C

excess of oligonucleotide was then washed off, and the enzyme assay was carried out in the wells. The final reaction volume of 30 µL in buffer [35 mM Tris × HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT (dithiothreitol), 1.8 mM spermidine, 1 mM ATP, 6.5 % (w/v) glycerol, 0.1 mg/mL albumin] contained 1.5 U of gyrase from E. coli or S. aureus, 0.75 µg of relaxed pNO1 plasmid, and 3 µL of inhibitor solution in 10% DMSO and 0.008% Tween 20. Reactions were incubated for 30 min at 37 °C and after the addition of the TF buffer [50 mM NaOAc (pH 5.0), 50 mM NaCl and 50 mM MgCl2] which terminated the enzymatic reaction for another 30 min at rt to allow the triplex formation (biotin-oligonucleotide-plasmid). Afterwards, the unbound plasmid was washed off using TF buffer and the solution of SybrGOLD stain in T10 buffer [10 mM Tris × HCl (pH 8.0) and 1 mM EDTA] was added. 17

ACCEPTED MANUSCRIPT After mixing the fluorescence (excitation: 485 nm, emission: 535 nm) was read using a BioTek's Synergy H4 microplate reader. Preliminary screening was performed at inhibitor concentrations of 100 and 10 µM. For most potent compounds IC50 was determined with 7 concentrations of the inhibitors. IC50 values were calculated using GraphPad Prism software and represent the concentration of inhibitor lowering the residual activity of the enzyme to

RI PT

50% of original. All compounds were assayed in three independent measurements and a final result is given as their average value. Novobiocin [IC50 = 0.168 µM (lit. [33] 0.08 µM) for E. coli gyrase and IC50 = 0.041 µM (lit. [33] 0.01 µM) for S. aureus gyrase] was used as the internal standard.

SC

Determination of Inhibitory Activities on E. coli and S. aureus Topoisomerase IV. The assay for the determination of IC50 values (Inspiralis) was performed on the black streptavidin-coated 96-well microtiter plates (Thermo Scientific Pierce). The plates were first

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rehydrated with the supplied wash buffer [20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.01% (w/v) BSA, 0.05% (v/v) Tween 20] and biotinylated oligonucleotide was immobilized onto the wells. The excess of oligonucleotide was then washed off, and the enzyme assay was carried out in the wells. The final reaction volume of 30 µL in buffer [40 mM HEPES KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM

TE D

ATP, 0.05 mg/mL albumin] contained 1.5 U of topoisomerase IV from E. coli or S. aureus, 0.75 µg of supercoiled pNO1 plasmid, and 3 µL of inhibitor solution in 10% DMSO and 0.008% Tween 20. Reactions were incubated for 30 min at 37 °C and after the addition of the TF buffer [50 mM NaOAc (pH 5.0), 50 mM NaCl and 50 mM MgCl2] which terminated the

EP

enzymatic reaction for another 30 min at rt to allow the triplex formation (biotinoligonucleotide-plasmid). Afterwards, the unbound plasmid was washed off using TF buffer and the solution of SybrGOLD stain in T10 buffer [10 mM Tris-HCl (pH 8.0) and 1 mM

AC C

EDTA] was added. After mixing the fluorescence (excitation: 485 nm, emission: 535 nm) was read using a BioTek's Synergy H4 microplate reader. Preliminary screening was performed at inhibitor concentrations of 100 and 10 µM. For most potent compounds IC50 was determined with 7 concentrations of the inhibitors. IC50 values were calculated using GraphPad Prism software and represent the concentration of inhibitor lowering the residual activity of the enzyme to 50% of original. The assays for all compounds were run in triplicate and a final result is given as their average value. Novobiocin [IC50 = 11.1 µM (lit. [33] 10 µM) for E. coli topoisomerase IV and IC50 = 26.7 µM (lit. [33] 20 µM) for S. aureus topoisomerase IV] was used as the internal standard.

18

ACCEPTED MANUSCRIPT Surface Plasmon Resonance (SPR) Measurements. Surface plasmon resonance (SPR) measurements were performed on a Biacore T100 apparatus using CM5 sensor chip (Biacore, GE Healthcare). The system was primed twice with running buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4). The 24 kDa part of E. coli GyrB subunit (G24 protein) was immobilized on the second flow cell of a sensor chip CM5 using standard

RI PT

amino coupling method. The carboxymethylated dextran layer was activated with 7 min pulse of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mixed in a 1:1 ratio. Protein was diluted to the final concentration of 50 µg/mL in 10 mM sodium acetate (pH 4.5) and injected in two short pulses to reach the final immobilization

SC

level of around 18.000 response units. Finally, the rest of the surface was deactivated with 7 min injection of ethanolamine. The first flow cell was activated with EDC/NHS and deactivated with ethanolamine and served as a reference cell for subtraction of nonspecific

M AN U

binding. The activity of the chip was tested and confirmed using novobiocin as a standard. Measured Kd for novobiocin was 28 nM (lit. [29] 19 nM). Analytes were prepared as DMSO 100× stock solutions and were diluted with a running buffer prior to the injection. They were injected at a flow rate of 30 µL/min for 90 s, and dissociation was monitored for additional 120 s. Since the dissociation of analytes from the ligand was rapid, no regeneration protocol

TE D

was needed. For the titration of analytes, the 1% of the DMSO was added to the running buffer in order to diminish the difference in a refractive index between the samples and running buffer. Inhibitor 9a was tested in eight different concentrations in three parallel titrations. The sensorgrams (Supplementary Information, Figure 1S) were analysed using

EP

BiaEval software (Biacore, GE Healthcare). The equilibrium binding responses were determined from the binding levels 5 s before the stop of the injection. Kd values were determined with Origin software by the fitting of the data to 1:1 steady state binding model.

AC C

Determination of Antibacterial Activity. Clinical control strains of Enterococcus faecalis (Gram-positive,

ATCC

29212),

Escherichia

coli

(Gram-negative,

ATCC

25922),

Pseudomonas aeruginosa (Gram-negative, ATCC 27853), and Staphylococcus aureus (Grampositive, ATCC 25923) were obtained from Microbiologics Inc. (St. Cloud, Minnesota, USA). Bacterial cultures were initiated on cation-adjusted MH (Mueller Hinton) agar (Becton Dickinson, Franklin Lakes, NJ, USA) slants and prior to the assays, suspensions were prepared into cation-adjusted MH broth (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 37 °C for 16–20 h at 100 rpm. Antimicrobial assays were performed by the broth microdilution method in 96-well plate format according to the CLSI guidelines [34]. Briefly, bacterial suspension was diluted with MH broth to obtain a final inoculum of 5×105 CFU/mL 19

ACCEPTED MANUSCRIPT in the assay. An equal volume of bacterial suspension and test compound solution diluted into assay media were mixed together in the plate and incubated for 24 h at 37 °C. Absorbance values measured at 620 nm were used for evaluating the antimicrobial effects of test compounds by comparing to untreated controls and expressed as percentage inhibition of growth. Ciprofloxacin was used as positive control on every assay plate (see Table 1S for

RI PT

details). MIC experiments against E. coli were carried out by using 8 different concentrations in the range of 6.25-200 µM (n = 3). Compounds were additionally assayed against E. faecalis, P. aeruginosa and S. aureus at final concentration of 50 µM (n = 3).

Molecular modelling. Ligand and protein preparation. Three-dimensional models of

SC

designed compounds were built in ChemBio3D Ultra 13.0 [35]. Their geometries were optimized using MMFF94 [36] force field and partial atomic charges were added. Energy was minimized until the gradient value was smaller than 0.001 kcal/(mol Å). The optimized

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structure was further refined with GAMESS interface in ChemBio3D Ultra 13.0 using the semiempirical PM3 method, QA optimization algorithm and Gasteiger Hückel charges for all atoms for 100 steps [36]. Molecular docking calculations were performed using FlexX, [28, 29] as available in LeadIT [30], running on four octal core AMD Opteron CPU processors, 16 GB RAM, two 750 GB hard drives, running 64-bit Scientific Linux 6.0. Receptor was

TE D

prepared in a LeadIT graphical user interface using the Receptor wizard. Amino acid residues within a radius of 7 Å around the ligand from the X-ray structure (PDB entry: 4DUH [11]) were defined as the binding site. Hydrogen atoms were added to the binding site residues and correct tautomers and protonation states were assigned. Water molecules, except HOH614,

EP

and the ligand were deleted from the crystal structure. Ligand docking. The FlexX molecular docking program, as available in LeadIT [30], was used for ligand docking. A hybrid algorithm (enthalpy and entropy driven ligand binding) was

AC C

used to place the ‘base fragment’. The maximum number of solutions per iteration and the maximum number of solutions per fragmentation parameter values were increased to 1000, while other parameters were set at their default values. Proposed binding modes and scoring function scores of the top five highest scored docking poses per ligand were evaluated and the highest ranked binding pose was used for graphical representation in PyMOL [32]. General Procedures - Chemistry. Chemicals were obtained from Acros Organics (Geel, Belgium), Sigma-Aldrich (St. Louis, MO, USA) and Apollo Scientific (Stockport, UK) and used without further purification. Analytical TLC was performed on silica gel Merck 60 F254 plates (0.25 mm), using visualization with UV light and spray reagents. Column chromatography was carried out on silica gel 60 (particle size 240–400 mesh). HPLC analyses 20

ACCEPTED MANUSCRIPT were performed on an Agilent Technologies 1100 instrument (Agilent Technologies, Santa Clara, CA, USA) with a G1365B UV-Vis detector, a G1316A thermostat and a G1313A autosampler using a Phenomenex Luna 5-µm C18 column (4.6 × 150 mm or 4.6 × 250 mm, Phenomenex, Torrance, CA, USA) and a flow rate of 1.0 mL/min. The eluent consisted of trifluoroacetic acid (0.1% in water) as solvent A and methanol or acetonitrile as solvent B.

and

13

RI PT

Melting points were determined on a Reichert hot stage microscope and are uncorrected. 1H C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker

AVANCE III 400 spectrometer (Bruker Corporation, Billerica, MA, USA) in DMSO-d6 or CDCl3 solutions, with TMS as the internal standard. IR spectra were recorded on a

SC

PerkinElmer Spectrum BX FT-IR spectrometer (PerkionElmer, Inc., Waltham, MA, USA) or Thermo Nicolet Nexus 470 ESP FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Mass spectra were obtained using a VG Analytical Autospec Q mass

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spectrometer (Fisons, VG Analytical, Manchester, UK). Optical rotations were measured on a Perkin-Elmer 241 MC polarimeter. The reported values for specific rotation are average values of 10 successive measurements using an integration time of 5 s. The purity of the tested compounds was established to be ≥95%. Synthetic procedures.

TE D

Ethyl 2-((4-nitrophenyl)amino)-2-oxoacetate (3a). To a suspension of 4-nitroaniline (1, 0.75 g, 5.43 mmol) and potassium carbonate (1.50 g, 10.87 mmol) in acetonitrile (40 mL) cooled on an ice bath, a solution of ethyl oxalyl chloride (2a, 0.61 mL, 0.75 g, 5.43 mmol) in acetonitrile (10 mL) was added dropwise. The mixture was stirred at rt for 15 h upon which

EP

the solvent was evaporated under reduced pressure, the residue dissolved in ethyl acetate (100 mL) and washed with water (2 × 30 mL) and brine (2 × 20 mL). The organic phase was dried over Na2SO4, filtered and the solvent evaporated under reduced pressure to afford 3a (1.06 g)

AC C

as yellow crystals. Yield 82% (1.06 g); mp 143-145 °C (144-145 °C, lit [37]); IR (ATR) ν = 3342, 3203, 3126, 3078, 1704, 1599, 1495, 1343, 1297, 1170, 858, 751 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.33 (t, 3H, 3J = 7.2 Hz, CH3), 4.31-4.36 (m, 2H, CH2), 8.04 (d, 2H, 3J = 9.2 Hz, Ar-H-2,6), 8.28 (d, 2H, 3J = 9.2 Hz, Ar-H-3,5), 11.34 (s, 1H, NH). General Procedure A. Synthesis of Compounds 4a-c (with 4a as an Example). Compound 3a (1.00 g, 4.20 mmol) was dissolved in tetrahydrofuran (50 mL), Pd-C (200 mg) was added and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The catalyst was filtered off and the solvent removed under reduced pressure to give 4a (0.80 g) as a brown solid.

21

ACCEPTED MANUSCRIPT Ethyl 2-((4-aminophenyl)amino)-2-oxoacetate (4a). Brown solid; yield 92% (0.80 g); mp 110-112 °C (110-113 °C, lit [38]); IR (ATR) ν = 3476, 3317, 3348, 3217, 2981, 2931, 1728, 1672, 1516, 1282, 1177, 824 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.30 (t, 3H, 3J = 7.2 Hz, CH3), 4.25- 4.31 (m, 2H, CH2), 5.06 (s, 2H, NH2), 6.52 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 7.37 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5), 10.39 (s, 1H, NH).

RI PT

General Procedure B. Synthesis of Compounds 6 and 8a-d (with 6 as an Example). To a suspension of 4,5-dibromopyrrole-2-carboxylic acid (400 mg, 1.49 mmol) and TBTU (521 mg, 1.62 mmol) in dichloromethane (15 mL) N-methylmorpholine (377 µL, 4.06 mmol) was added and the mixture stirred at rt for 0.5 h upon which a clear solution formed. Benzene-1,3-

SC

diamine (5, 144 mg, 0.69 mmol) was added and the mixture stirred at 50 °C for 15 h. The solvent was evaporated under reduced pressure, the residue dissolved in ethyl acetate (30 mL), and washed with water (2 × 15 mL) and brine (2 × 10 mL). The organic phase was dried

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over Na2SO4, filtered and the solvent evaporated under reduced pressure. Crude product was purified with flash column chromatography using ethyl acetate/petroleum ether (1:2 to 1:1) as solvent, to obtain 6 (522 mg) as brown solid.

N-(3-Aminophenyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (6). Brown solid; yield 95% (522 mg); mp 193-195 °C; IR (ATR) ν = 3412, 3367, 3339, 3185, 1642, 1606, 1547, 1523, 1493, 1443, 1409, 1387, 1316, 1215, 1167, 972, 844, 814, 776, 745 cm-1. 1H NMR (400 MHz,

TE D

DMSO-d6) δ 5.13 (s, 2H, NH2), 6.28-6.31 (m, 1H, Ar-H), 6.83-6.84 (m, 1H, Ar-H), 6.93-6.98 (m, 2H, 2 × Ar-H), 7.21 (s, 1H, pyrr-CH), 9.54 (s, 1H, NH), 12.81 (s, 1H, pyrr-NH);

13

C

NMR (100 MHz, DMSO-d6) δ 98.01, 105.44, 105.68, 108.02, 109.62, 113.52, 128.19, 128.87,

EP

139.29, 148.85, 157.04; MS (ESI) m/z (%) = 355.9 ([M-H]-). HRMS for C11H8Br2N3O: calculated 355.9034, found 355.9031. Methyl 3-((3-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-3-oxopropanoate

AC C

(7). To the solution of compound 6 (50 mg, 0.13 mmol) in a mixture of dichloromethane (5 mL) and tetrahydrofuran (2 mL) triethylamine (22 µL, 0.16 mmol) and 2b (16 µL, 0.15 mmol) were added and the mixture was stirred at rt for 2 h. The solvent was evaporated under reduced pressure, the residue dissolved in ethyl acetate (10 mL) and washed with water (2 × 5 mL), saturated solution of NaHCO3 (2 × 5 mL) and brine (5 mL). The organic phase was dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure. Off-white solid; yield 83% (58 mg); mp 214-216 ºC; 1H NMR (400 MHz, DMSO-d6) δ 3.49 (s, 2H, CH2), 3.67 (s, 3H, CH3), 7.27-7.28 (m, 3H, 3 × Ar-H), 7.45-7.48 (m, 1H, Ar-H), 8.05 (s, 1H, pyrr-CH), 9.88 (s, 1H, NH), 10.24 (s, 1H, NH), 12.89 (s, 1H, pyrr-NH); 13C NMR (100 MHz, DMSO-d6) δ 43.46 (CH3), 51.95 (CH2), 98.13, 105.88, 110.83, 113.90, 114.29, 115.24, 22

ACCEPTED MANUSCRIPT 127.86, 128.91, 138.99, 139.15, 157.25, 163.96, 168.17; IR (ATR) ν = 3390, 3202, 2954, 1732, 1660, 1641, 1604, 1534, 1486, 1423, 1391, 1327, 1285, 1225, 1200, 1171, 1145, 813, 792, 771, 749 cm-1; MS (ESI) m/z (%) = 455.9 ([M-H]-), HRMS for C15H12Br2N3O4: calculated 455.9141, found 455.9183; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile phase: 30–90% acetonitrile in TFA (0.1%) in 16 min, 90% acetonitrile to

RI PT

20 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 9.560 min (96.1% at 280 nm).

General Procedure C. Synthesis of Compounds 9a-d (with 9b as an Example). To a stirred solution of 8b (20 mg, 0.04 mmol) in THF/water (5:1, 10 mL) 2 M LiOH (45 µL, 0.06

SC

mmol) was added. The mixture was stirred at rt for 15 h, neutralized with 1 M HCl and concentrated under reduced pressure. The residual aqueous solution was acidified to pH 2 with 1 M HCl and the product extracted with ethyl acetate (3 × 10 mL). The combined

M AN U

organic phases were washed with water (2 × 10 mL) and brine (2 × 10 mL), dried over Na2SO4, filtered and the solvent evaporated under reduced pressure to afford 9b as white solid (14 mg).

3-((4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-3-oxopropanoic acid (9b). White solid; yield 72% (14 mg); mp 280-282 °C; IR (ATR) ν = 3379, 3291, 3169, 3113, 2973, 2614, 2517, 1707, 1562, 1516, 1415, 1241, 1212, 826 cm-1. 1H NMR (400 MHz,

TE D

DMSO-d6) δ 3.36 (s, 2H, CH2, overlapping with the signal for water), 7.21 (s, 1H, pyrr-CH), 7.55 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6/3,5), 7.64 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6/3,5), 9.84 (s, 1H, NH), 10.13 (s, 1H, NH), 12.65 (br s, 1H, COOH), 12.89 (s, 1H, pyrr-NH);

13

C NMR (100

EP

MHz, DMSO-d6) δ 43.87 (CH2), 98.07, 105.70, 113.57, 119.33, 120.42, 127.95, 134.25, 134.63, 157.10, 164.31, 169.31; MS (ESI) m/z (%) = 441.9 ([M-H]-). HRMS for C14H10Br2N3O4: calculated 441.9038, found 441.9029. HPLC: Phenomenex Luna 5 µm C18

AC C

column (4.6 mm × 150 mm); mobile phase: 10-90% of MeOH in TFA (0.1%) in 20 min, 90% MeOH to 25 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 17.965 min (98.1% at 280 nm).

General Procedure D. Synthesis of Compounds 12a and 12b (with 12a as an Example). To the solution of 1-fluoro-4-nitrobenzene (10, 0.73 mL, 6.84 mmol) and (S)-methyl nipecotate hydrochloride (11a, 1.23 g, 6.84 mmol) in dimethylsulfoxide (20 mL) potassium carbonate (2.84 g, 20.5 mmol) was added and the suspension was heated at 55 °C for 15 h. Water was added (20 mL) and the obtained solution extracted with dichloromethane (2 × 20 mL), the organic phase was dried over Na2SO4, filtered and the solvent evaporated under

23

ACCEPTED MANUSCRIPT reduced pressure. The crude product was purified with flash column chromatography using ethyl acetate/petroleum ether (2:1) as eluent, to obtain 12a (1.63 g) as yellow crystals. Methyl 1-(4-nitrophenyl)piperidine-3-carboxylate (12a) [26]. Yellow crystals; yield 90% (1.63 g); [α]D25 +180 (c 0.307, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 1.48-1.59 (m, 1H, CH), 1.67-179 (m, 2H, CH2), 1.94-2.00 (m, 1H, CH), 2.60-2.67 (m, 1H, CH), 3.16-3.23 (m,

RI PT

1H, CH), 3.32-3.40 (m, 1H, CH, overlapping with the signal for water), 3.63 (s, 3H, CH3), 3.78-3.83 (m, 1H, CH), 3.97-4.01 (m, 1H, CH), 7.03 (d, 2H, 3J = 9.6 Hz, Ar-H-2,6), 8.05 (d, 2H, 3J = 9.6 Hz, Ar-H-3,5).

Ethyl 1-(4-nitrophenyl)-2-oxopiperidine-4-carboxylate (13) [26]. To the solution of

SC

compound 12b (1.50 g, 5.39 mmol) in dichloromethane (30 mL) benzyltriethylammonium chloride (7.37 g, 32.4 mmol) and potassium permanganate (5.11 g, 32.4 mmol) were added in three equal portions at the beginning, after 3 days, and after 7 days of 14 days long refluxing

M AN U

of the suspension. To the reaction mixture water (50 mL) and 20% solution of sodium sulphite (120 ml) were added and the mixture stirred for 1 h at 0 °C. The obtained suspension was filtered off, the phases were separated and the organic phase was dried over Na2SO3, filtered and the solvent evaporated under reduced pressure. The residue was purified with flash column chromatography using ethyl acetate/petroleum ether (2:1) as eluent, to obtain 13 (0.866 g) as yellow crystals. Yield 55% (0.866 g); 1H NMR (400 MHz, DMSO-d6) δ 1.22 (t,

TE D

3H, 3J = 7.2 Hz, CH3), 1.97-2.06 (m, 1H, CH), 2.19-2.26 (m, 1H, CH), 2.64-2.66 (m, 2H, CH2), 3.07-3.14 (m, 1H, CH), 3.63-3.69 (m, 1H, CH), 3.79-3.85 (m, 1H, CH), 4.14 (q, 2H, 3J = 7.2 Hz, CH2), 7.61 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 8.25 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5).

EP

General Procedure E. Synthesis of Compounds 14a-c (with 14a as an Example). Compound 12a (1.50 g, 5.68 mmol) was dissolved in methanol (30 mL), Pd-C (200 mg) was added and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The catalyst

oil.

AC C

was filtered off and the solvent removed under reduced pressure to give 14a (1.20 g) as brown

Methyl 1-(4-aminophenyl)piperidine-3-carboxylate (14a) [26]. Brown oil; yield 90% (1.20 g); [α]D25 +19.5 (c 0.341, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 1.48-1.62 (m, 2H, CH2), 1.70-174 (m, 1H, CH), 1.83-1.86 (m, 1H, CH), 2.51-2.68 (m, 2H, CH2), 2.75-2.80 (m, 1H, CH), 3.08-3.11 (m, 1H, CH), 3.28-3.33 (m, 1H, CH, overlapping with the signal for water), 3.63 (s, 3H, CH3), 4.63 (s, 2H, NH2), 6.59 (d, 2H, 3J = 8.4 Hz, Ar-H-3,5), 6.67 (d, 2H, 3J = 8.4 Hz, Ar-H-2,6). General Procedure F. Synthesis of Compounds 15a-f (with 15a as an Example). To a suspension of 4,5-dibromopyrrole-2-carboxylic acid (316 mg, 1.17 mmol) and TBTU (411 24

ACCEPTED MANUSCRIPT mg, 1.28 mmol) in dichloromethane (15 mL) N-methylmorpholine (352 µL, 3.20 mmol) was added and the mixture stirred at rt for 0.5 h upon which a clear solution formed. Compound 14a (250 mg, 1.07 mmol) was added and the mixture stirred at 50 °C for 15 h. The solvent was evaporated under reduced pressure, the residue dissolved in ethyl acetate (50 mL), and washed with water (2 × 20 mL) and brine (2 × 15 mL). The organic phase was dried over

RI PT

Na2SO4, filtered and the solvent evaporated under reduced pressure. After evaporation of the solvent from the reaction mixture dichloromethane (20 mL) and methanol (5 mL) were added to the residue, the obtained suspension was sonicated, filtered, washed with dichloromethane (2 × 5 mL), and dried to afford 15a (300 mg) as a grey solid. Methyl

(S)-1-(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)piperidine-3-

SC

carboxylate (15a). Grey solid; yield 58% (300 mg); mp 187-192 ºC; [α]D25 +49.0 (c 0.311, MeOH); IR (ATR) ν = 3387, 3206, 3118, 2950, 2814, 2656, 1706, 1645, 1595, 1552, 1519,

M AN U

1431, 1417, 1386, 1328, 1310, 1225, 1214, 1194, 1166, 1031, 974, 820 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.54-1.65 (m, 2H, CH2), 1.68-1.78 (m, 1H, CH), 1.85-1.95 (m, 1H, CH), 2.64-2.70 (m, 1H, CH), 2.75-2.81 (m, 1H, CH), 2.93-2.98 (m, 1H, CH), 3.30-3.40 (m, 1H, CH, overlapping with the signal for water), 3.57-3.64 (m, 1H, CH), 3.64 (s, 3H, CH3), 6.93 (d, 2H, 3J = 9.2 Hz, Ar-H-2,6), 7.18 (s, 1H, Pyrr-CH), 7.54 (d, 2H, 3J = 9.2 Hz, Ar-H-3,5), 9.69 13

C NMR (100 MHz, DMSO-d6) δ 23.43, 26.34, 40.32,

TE D

(s, 1H, NH), 12.84 (s, 1H, NH);

49.32, 51.50, 51.67, 97.98, 105.32, 113.25, 116.49, 121.17, 128.16, 130.65, 147.39,156.92, 173.59; MS (ESI) m/z (%) = 482.0 ([M-H]-), HRMS for C18H18Br2N3O3: calculated 481.9715, found 481.9723; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile

EP

phase: 10-90% of methanol in TFA (0.1%) in 20 min, 90% methanol to 25 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 18.303 min (99.3% at 280 nm). General Procedure G. Synthesis of Compounds 16a-f (with 16b as an Example). To a

AC C

stirred solution of 15a (100 mg, 0.206 mmol) in THF/methanol/water (3:3:1, 15 mL) 2 M LiOH (206 µL, 0.412 mmol) was added. The mixture was stirred at rt for 15 h, neutralized with 1 M HCl and concentrated under reduced pressure. The residual aqueous solution was acidified to pH 2 with 1 M HCl and the product extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with water (2 × 20 mL) and brine (2 × 15 mL), dried over Na2SO4, filtered and the solvent evaporated under reduced pressure. To the residue ether (10 mL) was added, the obtained suspension was sonicated, filtered, washed with ether (2 × 5 mL), and dried to afford 16a (87 mg) as an brown solid. (S)-1-(4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)phenyl)piperidine-3-carboxylic (16a). Brown solid; yield 90% (87 mg); mp 221-224 ºC;

[α]D25

acid

+36.5 (c 0.337, MeOH); IR 25

ACCEPTED MANUSCRIPT (ATR) ν = 3124, 2949, 2857, 1704, 1637, 1602, 1552, 1512, 1412, 1387, 1328, 1228, 1179, 1105, 973, 826 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.52-1.64 (m, 2H, CH2), 1.69-1.76 (m, 1H, CH), 1.87-1.92 (m, 1H, CH), 2.56 (m, 1H, CH, overlapping with the signal for DMSOd5), 2.74-2.81 (m, 1H, CH), 2.90-2.95 (m, 1H, CH), 3.40 (m, 1H, CH, overlapping with the signal for water), 3.57-3.61 (m, 1H, CH), 6.95 (br d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 7.18 (d, 1H, J = 2.8 Hz, Pyrr-CH), 7.55 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5), 9.70 (s, 1H, NH), 12.35 (s, 1H,

COOH), 12.84 (s, 1H, NH);

13

RI PT

4

C NMR (100 MHz, DMSO-d6) δ 23.59, 26.41, 40.40, 49.41,

51.82, 97.99, 105.30, 113.27, 116.47, 121.16, 128.15, 130.59, 147.49,156.91, 174.84; MS (ESI) m/z (%) = 468.0 ([M-H]-), HRMS for C17H16Br2N3O3: calculated 467.9558, found

SC

467.9570; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile phase: 10-90% of methanol in TFA (0.1%) in 20 min, 90% methanol to 25 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 17.117 min (98.4% at 280 nm).

M AN U

General Procedure H. Synthesis of Compounds 19a-b (with 19a as an Example). To a suspension of L-Pro methyl ester (18a, 3.00 g, 23.2 mmol) and K2CO3 (6.42 g, 46.5 mmol) in acetonitrile (40 mL) 4-nitrobenzoyl chloride (17, 4.31 g, 23.2 mmol) was added and the mixture stirred at rt for 15 h. The solvent was evaporated, the residue dissolved in ethyl acetate (40 mL) and water (40 mL), the layers were separated and the organic phase washed

TE D

with water (2 × 20 mL) and brine(20 mL), dried over Na2SO4 and evaporated under reduced pressure. To the residue petroleum ether (20 mL) was added, the obtained suspension was sonicated, filtered, washed with petroleum ether (2 × 5 mL), and dried. The crude product was recrystallized from cyclohexane to afford 19a (3.23 g) as white crystals.

EP

Methyl (4-nitrobenzoyl)-L-prolinate (19a). White crystals; yield 50% (3.23 g); mp 107 ºC; [α]D25 -72.4 (c 0.333, MeOH); IR (ATR) ν = 3113, 2980, 2862, 1744, 1626, 1594, 1520, 1425, 1354, 1317, 1199, 1161, 867, 850, 731, 710 cm-1; 1H NMR (400 MHz, DMSO-d6) δ

AC C

1.81-2.02 (m, 3H, CH2, HA from CH2), 2.26-2.36 (m, 1H, HB from CH2), 3.46-3.51 (m, 2H, CH2), 3.69 (s, 3H, CH3), 4.51-4.54 (m, 1H, CH), 7.79 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 8.31 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5);

13

C NMR (100 MHz, DMSO-d6) δ 25.37, 29.36, 49.79, 52.47,

59.33, 124.19, 128.94, 142.50, 148.64, 167.01, 172.52. General Procedure I. Synthesis of Compounds 20a-b (with 20a as an Example). To the solution of compound 19a (1.60 g, 5.75 mmol) in tetrahydrofuran (80 mL) Pd-C (500 mg) was added and the reaction mixture was stirred under hydrogen atmosphere for 15 h. The catalyst was filtered off and the solvent removed under reduced pressure to give 20a (1.47 g) as light brown crystals.

26

ACCEPTED MANUSCRIPT Methyl (4-aminobenzoyl)-L-prolinate (20a). Light brown crystals; yield 92% (1.47); mp 137-138 ºC; [α]D25 -50.6 (c 0.380, MeOH); IR (ATR) ν = 3429, 3330, 3221, 2972, 2878, 1728, 1563, 1438, 1360, 1308, 1202, 1173, 840, 787, 761, 711 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.80-1.93 (m, 3H, CH2, HA from CH2), 2.20-2.26 (m, 1H, HB from CH2), 3.63 (br s, 5H, CH2N, CH3), 4.41-4.44 (m, 1H, CH), 5.61 (s, 2H, NH2), 6.54 (d, 2H, 3J = 8.4 Hz,

RI PT

Ar-H-3,5), 7.32 (br d, 2H, Ar-H-2,6); 13C NMR (100 MHz, DMSO-d6) δ 25.37, 28.77, 49.76, 51.67, 59.22, 112.40, 121.90, 129.42, 150.97, 168.31, 172.72; MS (ESI) m/z (%) = 249.1 ([MH]+), HRMS for C13H17N2O3: calculated 249.1239, found 249.1233.

General Procedure J. Synthesis of Compounds 21a-b (with 21a as an Example).

SC

To the solution of 4,5-dibromo-1H-pyrrole-2-carboxylic acid (150 mg, 0.57 mmol) in anhydrous dichloromethane (5 mL) oxalyl chloride (2 M solution in dichloromethane, 0.723 mL, 1.43 mmol) was added and the mixture stirred under an argon atmosphere for 15 h. The

M AN U

solvent was removed under reduced pressure, to the residue anhydrous dichloromethane (3 mL), pyridine (3 mL) and compound 20a (92 mg, 0.37 mmol) were added and stirred at rt. After 6 h the solvent was removed in vacuo, and the residue dissolved in ethyl acetate (10 mL) and water (10 mL). Organic phase was washed with 1M HCl (2 × 7 mL), saturated solution of NaHCO3 (2 × 7 mL) and brine (5 mL), dried over Na2SO4 and evaporated under

TE D

reduced pressure. The crude product was purified with flash column chromatography using ethyl acetate/petroleum ether (1:1 to 2:1) as eluent, to obtain 21a (117 mg) as an off-white solid.

Methyl (4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoyl)-L-prolinate (21a). Off-

EP

white solid; yield 40% (117 mg); mp 222-225 ºC; [α]D25 -29.0 (c 0.331, MeOH); IR (ATR) ν = 3198, 3116, 2950, 2871, 1657, 1631, 1597, 1521, 1424, 1406, 1329, 1247, 1223, 1186, 834, 750 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.84-1.96 (m, 3H, CH2, HA from CH2), 2.23-2.32

AC C

(m, 1H, HB from CH2), 3.57-3.63 (s, 2H, CH2N), 3.67 (s, 3H, CH3), 4.46-4.49 (m, 1H, CH), 7.28 (d, 1H, 4J = 2.4 Hz, Pyrr-CH), 7.57 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5), 7.81 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 10.04 (s, 1H, NH), 12.98 (d, 1H, 4J = 2.4 Hz, Pyrr-NH); 13C NMR (100 MHz, DMSO-d6) δ 25.10, 28.84, 49.59, 51.80, 59.03, 98.22, 106.35, 114.16, 118.99, 127.66, 128.27, 130.44, 140.64, 157.41, 167.76, 172.44; MS (ESI) m/z (%) = 497.9 ([M-H]+), HRMS for C18H18Br2N3O4: calculated 497.9664, found 497.9671; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile phase: 30-90% of acetonitrile in TFA (0.1%) in 16 min, 90% acetonitrile to 20 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 9.924 min (97.0% at 280 nm).

27

ACCEPTED MANUSCRIPT (4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)benzoyl)-L-proline

(22a).

Synthesized

according to general procedure G from 21b (45 mg, 0.12 mmol). Light brown solid; yield 66% (46 mg); mp 207-210 ºC; [α]D25 -27.8 (c 0.260, MeOH); IR (ATR) ν = 3113, 2957, 2873, 2357, 1716, 1597, 1523, 1436, 1406, 1328, 1242, 1306, 1242, 1180, 832, 763 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 1.80-1.96 (m, 3H, CH2, HA from CH2), 2.23-2.30 (m, 1H, HB

RI PT

from CH2), 3.54-3.62 (m, 2H, CH2), 4.38-4.41 (m, 1H, CH), 7.28 (d, 1H, 4J = 2.4 Hz, PyrrCH), 7.56 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5), 7.80 (d, 2H, 3J = 8.8 Hz, Ar-H-2,6), 10.03 (s, 1H, NH), 12.53 (s, 1H, COOH), 12.98 (d, 1H, 4J = 2.4 Hz, Pyrr-NH);

13

C NMR (100 MHz,

DMSO-d6) δ 25.06, 28.90, 49.62, 59.09, 98.27, 106.31, 114.11, 119.06, 127.63, 128.19,

SC

130.76, 140.44, 157.41, 167.80, 173.44; MS (ESI) m/z (%) = 481.9 ([M-H]-), HRMS for C17H14Br2N3O4: calculated 481.9351, found 481.9361; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile phase: 30-90% of acetonitrile in TFA (0.1%) in 16 min,

8.029 min (98.4% at 280 nm).

M AN U

90% acetonitrile to 20 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time:

3-(4-Nitrophenyl)oxazolidin-2-one (24). 4-Nitroaniline (23, 1.00 g, 7.24 mmol), ethylene carbonate (3.19 g, 36.2 mmol) and DBU (1.08 mL, 7.24 mmol) were mixed together in a 25 mL flask and heated at 100 °C for 3 h. The mixture was cooled down to rt, water (50 mL) was added, the yellow precipitate was filtered off, washed with water (20 mL) and dried, to obtain

TE D

24 as a yellow solid (1.22 g). Yield 81% (1.22 g); mp 142-145 °C; IR (ATR) ν = 3127, 2991, 2930, 2627, 1759, 1746, 1593, 1512, 1502, 1479, 1398, 1319, 1299, 1205, 1188, 1133, 1109, 1050, 998, 957, 857, 824, 747 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.17 (dd, 2H, 3J1 = 8.8 Hz, J2 = 7.2 Hz, CH2), 4.59 (dd, 2H, 3J1 = 8.8 Hz, 3J2 = 7.2 Hz, CH2), 7.76 (d, 2H, 3J = 9.2 Hz,

EP

3

Ar-H-2,6), 8.29 (d, 2H, 3J = 9.2 Hz, Ar-H-3,5);

13

C NMR (100 MHz, DMSO-d6) δ 44.71,

61.84, 117.55, 124.80, 142.17, 144.40, 154.62.

AC C

3-(4-Aminophenyl)oxazolidin-2-one (25). Synthesized according to General Procedure I from compound 24 (1.17 g, 5.64 mmol) with 5 h reaction time. Grey solid; yield 98% (0.98 g); mp 146-148 °C; IR (ATR) ν = 3438, 3346, 3230, 2997, 2924, 1712, 1652, 1631, 1515, 1480, 1418, 1287, 1231, 1136, 1117, 1036, 995, 952, 829, 754 cm-1; 1H NMR (400 MHz, CDCl3) δ 4.02 (s, 2H, NH2), 4.47 (dd, 2H, 3J1 = 8.8 Hz, 3J2 = 7.2 Hz, CH2), 4.59 (dd, 2H, 3J1 = 8.8 Hz, 3J2 = 7.2 Hz, CH2), 6.72 (d, 2H, 3J = 8.8 Hz, Ar-H-3,5), 7.31 (d, 2H, 3J = 8.8 Hz, ArH-2,6). 4,5-Dibromo-N-(4-(2-oxooxazolidin-3-yl)phenyl)-1H-pyrrole-2-carboxamide

(26a).

Synthesized according to General Procedure F from 4,5-dibromo-1H-pyrrole-2-carboxylic acid (91 mg, 0.337 mmol) and compound 25 (60 mg, 0.337 mmol). After the completion of 28

ACCEPTED MANUSCRIPT the reaction, the solvent was evaporated under reduced pressure and to the residue ethyl acetate (10 mL) and water (10 mL) were added. The two-phase system was shaken vigorously, the undissolved suspension was filtered off, washed with water (5 mL) and ethyl acetate (5 mL), and dried, to obtain 26a as an off-white solid (83 mg). Yield 57% (83 mg); mp > 300 °C; IR (ATR) ν = 3370, 3199, 3111, 2975, 2919, 2893, 1736, 1648, 1606, 1556, 1528, 1

RI PT

1516, 1478, 1408, 1333, 1308, 1251, 1222, 1129, 1113, 1041, 991, 970, 820, 806, 754 cm-1; H NMR (400 MHz, DMSO-d6) δ 4.06 (dd, 2H, 3J1 = 8.8 Hz, 3J2 = 7.2 Hz, CH2), 4.42-4.46

(m, 2H, CH2), 7.23 (s, 1H, Pyrr-CH), 7.54 (d, 2H, 3J = 9.2 Hz, Ar-H), 7.72 (d, 2H, 3J = 9.2 Hz, Ar-H), 9.88 (s, 1H, NH), 12.91 (s, 1H, Pyrr-NH);

13

C NMR (100 MHz, DMSO-d6) δ

44.82, 61.41, 98.10, 105.79, 113.64, 118.40, 120.41, 127.88, 134.11, 134.44, 154.94, 157.17.

SC

MS (ESI) m/z (%) = 425.9 ([M-H]-), HRMS for C14H10Br2N3O3: calculated 425.9089, found 425.9097; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm × 150 mm); mobile phase:

M AN U

30-90% of acetonitrile in TFA (0.1%) in 16 min, 90% acetonitrile to 20 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 9.504 min (95.1% at 280 nm). N-(4-Acetylphenyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (28). Synthesized according to General Procedure F from 4,5-dibromo-1H-pyrrole-2-carboxylic acid (109 mg, 0.407 mmol) and 4-aminoacetophenone (27, 50 mg, 0.370 mmol). Off-white solid; yield 60% (143 mg); mp 280-282 °C; IR (ATR) ν = 3330, 3195, 3125, 2966, 1656, 1592, 1543, 1522, 1503,

TE D

1411, 1391, 1337, 1274, 1249, 1228, 1176, 1186, 974, 964, 838, 822, 750 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 2.55 (s, 3H, CH3), 7.31 (d, 1H, 4J = 2.4 Hz, Pyrr-H), 7.88 (d, 2H, 3J = 8.8 Hz, Ar-H), 7.97 (d, 2H, 3J = 8.8 Hz, Ar-H), 10.14 (s, 1H, NH), 13.02 (s, 1H, Pyrr-NH);

13

C

EP

NMR (100 MHz, DMSO-d6) δ 26.44, 98.31, 106.86, 114.43, 118.96, 127.52, 129.39, 131.73, 143.29, 157.48, 196.53. MS (ESI) m/z (%) = 382.9 ([M-H]-), HRMS for C13H10Br2N2O2: calculated 382.9031, found 382.9022; HPLC: Phenomenex Luna 5 µm C18 column (4.6 mm

AC C

× 150 mm); mobile phase: 30-90% of acetonitrile in TFA (0.1%) in 16 min, 90% acetonitrile to 20 min; flow rate 1.0 mL/min; injection volume: 10 µL; retention time: 10.681 min (96.9% at 280 nm).

ASSOCIATED CONTENT Supplementary Information. Full description of antibacterial activity of compounds, surface plasmon resonance sensograms, detailed experimental procedures, analytical data and NMR spectra. This material is available free of charge via the internet.

AUTHOR INFORMATION 29

ACCEPTED MANUSCRIPT Corresponding Author *E-mail: [email protected]. Phone: +386-1-4769561. Fax: +386-1-4258031. Author Contributions The manuscript was written through contributions of all authors. All authors have given

Funding Sources

RI PT

approval to the final version of the manuscript.

The work was funded by the Slovenian Research Agency (Grant No. P1-0208 and Grant No. Z1-5458), by the EU FP7 Integrated Project MAREX (Project No. FP7-KBBE-2009-3-

Conflict of Interest

SC

245137) and by the Academy of Finland (Grant no. 277001).

The authors declare no conflict of interest including any financial, personal or other

M AN U

relationships with other people or organizations.

ACKNOWLEDGMENT

This work was supported by the Slovenian Research Agency (Grant No. P1-0208 and Grant No. Z1-5458), by the EU FP7 Integrated Project MAREX (Project No. FP7-KBBE-2009-3-

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245137), and by the Academy of Finland (Grant no. 277001). We thank Dr. Dušan Žigon (Mass Spectrometry Center, Jožef Stefan Institute, Ljubljana, Slovenia) for recording mass spectra, Klemen Čamernik and Francesca Magari for the help with chemical synthesis, and Michaela Barančokova for the help with performing the enzymatic assays. The authors thank

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Prof. Roger Pain for proofreading the manuscript. We thank OpenEye Scientific Software,

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Santa Fe, NM., for free academic licenses for the use of their software.

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HIGHLIGHTS

Twenty eight new compounds were designed and prepared as ATPase inhibitors of DNA gyrase.

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Compounds were evaluated against gyrase and topoisomerase IV from E. coli and S. aureus.

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The most potent compound had an IC50 of 0.18 µM against E. coli gyrase.

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Five compounds had lower than 5 µM IC50 values against E. coli gyrase.

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The binding affinity to E. coli gyrase was studied using surface plasmon resonance.

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