New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2018.05.011

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC₅₀ values against DNA gyrase, and submicromolar IC₅₀ values against topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compound in the series has an IC₅₀ value of 13 nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC₅₀ value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 μM against Enterococcus faecalis, and 3.13 μM against wild type S. aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) is 4.6 μM.

Full text of DOI:10.1016/j.ejmech.2018.05.011

Accepted Manuscript
New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimisation of efficacy and
antibacterial activity
Martina Durcik, Denise Lovison, Žiga Skok, Cristina Durante Cruz, Päivi Tammela,
Tihomir Tomašič, Davide Benedetto Tiz, Gábor Draskovits, Ákos Nyerges, Csaba Pál,
Janez Ilaš, Lucija Peterlin Mašič, Danijel Kikelj, Nace Zidar
PII:
S0223-5234(18)30415-X
10.1016/j.ejmech.2018.05.011
EJMECH 10421
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2018
Revised Date: 27 March 2018
Accepted Date: 7 May 2018
Please cite this article as: M. Durcik, D. Lovison, Ž. Skok, C.D. Cruz, Pä. Tammela, T. Tomašič, D.B.
Tiz, Gá. Draskovits, Á. Nyerges, C. Pál, J. Ilaš, L.P. Mašič, D. Kikelj, N. Zidar, New N-phenylpyrrolamide
DNA gyrase B inhibitors: Optimisation of efficacy and antibacterial activity, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.011.
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GRAPHICAL ABSTRACT:

ACCEPTED MANUSCRIPT
New N-phenylpyrrolamide DNA gyrase B inhibitors:
optimisation of efficacy and antibacterial activity
Martina Durcik, a Denise Lovison, a Žiga Skok, a Cristina Durante Cruz, b Päivi Tammela, b
Tihomir Tomašič, a Davide Benedetto Tiz, a Gábor Draskovits, c Ákos Nyerges, c Csaba Pál, c
Janez Ilaš, a Lucija Peterlin Mašič, a Danijel Kikelj, a and Nace Zidar *,a
a Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
b
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), 00014 Helsinki, Finland
c
Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of
the Hungarian Academy of Sciences, Szeged H-6726, Hungary
ABSTRACT
The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the
development of new antibacterial agents. In recent decades, several small-molecule inhibitor
classes have been discovered but none has so far reached the market. We present here the
discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values
against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia
coli and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13
nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive
bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC50 value of
85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC
values of 1.56 µM against Enterococcus faecalis, and 3.13 µM against wild type S. aureus,
methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The
activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine
β-naphthylamide (PAβN) is 4.6 µM.
KEYWORDS: antibacterial; DNA gyrase; GyrB; inhibitor; N-phenylpyrrolamide; ParE;
topoisomerase IV

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ABBREVIATIONS
ATCC, American type culture collection; ATR, attenuated total reflectance; CDI, 1,1'-
carbonyldiimidazole; CFU, colony-forming unit; CLSI, Clinical and Laboratory Standards
Institute; DIAD, diisopropyl azodicarboxylate; DTT, dithiothreitol; FBS, fetal bovine serum;
GyrA, DNA gyrase A; GyrB, DNA gyrase B; HepG2, human hepatocellular carcinoma cell line;
HUVEC, human umbilical vein endothelial cells; MH, Mueller Hinton; MRSA, methicillin-
resistant
Staphylococcus
aureus;
MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NMM, N-methylmorpholine; ParC,
topoisomerase IV subunit A; ParE, topoisomerase IV subunit B; PAβN, phenylalanine-arginine
β-naphthylamide; RA, residual activity; TBTU, N,N,N′,N′-tetramethyl-O-(benzotriazol-1-
yl)uronium tetrafluoroborate; topo IV, topoisomerase IV; VRE, vancomycin-resistant
Enterococci.
INTRODUCTION
The discovery of antibacterials is considered to be one of the greatest medical achievements of all
time. However, since the 1960s only a small number of new-class antibacterial agents have
reached clinical practice while, on the other hand, the number of multi-drug resistant (MDR)
bacteria is rising [1, 2]. Nowadays, we are increasingly faced with life-threatening infections due
to resistant Gram-positive and Gram-negative pathogens that belong to the “ESKAPE” group
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species). Thus, the ESKAPE pathogens
were included by the World Health Organisation in the »WHO priority pathogens list for R&D of
new antibiotics« [3, 4].
DNA gyrase (gyrase) is a member of bacterial type IIA topoisomerase enzymes [5] that control
the topology of DNA during processes of transcription, replication and recombination by
introducing transient breaks to both DNA strands [6, 7]. DNA gyrase helps relieve torsional
tension by introducing negative supercoils to the DNA molecule during replication. It is a
heterotetrameric protein composed of two GyrA subunits where the DNA cleavage site is located,
and two GyrB subunits that provide the energy necessary for the catalytic function of the enzyme
through ATP hydrolysis [6, 8, 9]. The four subunits form a functional tetramer A2B2. The other

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member of bacterial type IIA topoisomerases, topoisomerase IV (topo IV), is composed of two
ParC and two ParE subunits that possess homologous structures to GyA and GyrB subunits of
DNA gyrase. The main function of topo IV is decatenation of two daughter chromosomal DNA
molecules after replication [8]. The structural and functional similarities of the two enzymes
indicate that there is a possibility of designing inhibitors that target active sites of both gyrase and
topo IV. This could reduce the capacity of bacteria to develop target-based drug resistance, since
the probability of concurrent mutations on both targets is low [10]. Drugs targeting bacterial type
IIA topoisomerases act by two main mechanisms, either by stabilizing the complex between a
DNA molecule and the ParC/GyrA active site of the enzyme (e.g. quinolones), or by inhibiting
the ATPase activity of the ParE/GyrB subunit (e.g. aminocoumarin class of inhibitors) [5, 6].
Novobiocin, a representative of the natural aminocoumarins, is the only ATP-competitive
inhibitor to have been used in the clinic but it has been withdrawn due to its toxicity and low
effectiveness [11]. Studies of many co-crystal structures of ParE and GyrB subunits with small
ligands and fragment-based design campaigns have led to several new classes of GyrB/ParE
inhibitors being discovered (Figure 1) [8, 10]. However, none of them have advanced beyond
phase I clinical trials, and most are only active against Gram-positive bacteria [10-12].

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Figure 1. Representative DNA gyrase B inhibitors: benzothiazole A [13], pyrrolopyrimidine B
[14], indazole C [15], pyrrolamide D [16], pyrimidoindole E [17], azaindole urea F [18], and their
IC50 or Ki, and MIC values.
RESULTS AND DISCUSSION
Design. The design of the presented set of compounds was based on our previous N-
phenylpyrrolamide inhibitors which had low nanomolar inhibitory activities against E. coli
gyrase (IC50 < 100 nM). An example is compound D (Figure 2a) [16]. The aim was to improve
the GyrB/ParE binding affinity of compounds and their antibacterial activity by structural
modifications, resulting in type I (Schemes 1-3) and type II (Scheme 4) compounds (Figure 2b).
For easier discussion, we have divided the structures into three parts: Part A, Part B and Part C
(Figure 2a).
To Part A, we introduced either a 4,5-dibromo-1H-pyrrole or a 3,4-dichloro-5-methyl-1H-pyrrole
group. The pyrrole NH and pyrrolamide C=O groups are important hydrogen bond donor and
acceptor groups that interact with Asp73 (E. coli numbering) and with a conserved water
molecule (Figure 2c) in the GyrB binding site. Halogen atoms on the pyrrole moiety lower the
pKa of the NH group and thus strengthen the formed H-bond. Chlorine and bromine atoms also
increase the lipophilicity of the pyrrole moiety and thus increase hydrophobic interactions with
the amino acid residues in the hydrophobic pocket of the enzyme (Val43, Ala47, Val71, Val167).
On the Part B benzene ring, compound D contains a lipophilic isopropoxy substituent that can
form hydrophobic interactions with residues Ile78 and Ile94 in the lipophilic floor of the enzyme.
Since these interactions were found to be favourable for the binding affinity, in some type I
compounds (7a-f, 8a-f, 10a, 11a and 15a-b) the isopropoxy group on the 3-position of the
benzene ring was retained. To other type I compounds (9a-e and 12) we introduced a 2-
aminoethoxy substituent at this position, with the aim of improving the solubility of the
compounds and/or broadening their antibacterial spectrum. Recently, the presence of an amino
group, especially a primary amino group, was found to contribute to the ability of compounds to
accumulate in Gram-negative E. coli [19]. With the type II compounds, we further explored the
lipophilic floor of the enzyme by changing the position of substituents on the benzene ring from
position 3- to 2-position. Similarly as in some type I compounds, in some type II compounds
(22c-d and 23c-d) we introduced the isopropoxy substituent to the 2-position of the benzene ring.

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In other type II compounds we introduced a sterically larger benzyloxy substituent (compounds
22a and 23a) or a basic 2-(dimethylamino)ethoxy substituent (compounds 22b and 23b), with the
aim of enabling additional interactions with the enzyme and improving the water solubility of the
compounds.
To Part C of type I compounds, groups that are able to form either ionic interactions with Arg136
side chain or π-stacking interactions with the Glu50-Arg76 salt bridge were attached. The
influence of different α-amino acids attached to Part C was studied, aiming to determine how the
stereochemistry and the size of the amino acid side chain affect the binding affinity. Thus,
glycine, L- and D-alanine, L-valine, and L-phenylalanine derivatives were prepared. The activities
of these derivatives, in the form of methyl esters, free carboxylic acids, and hydrazides, were
compared. Additionally, three compounds (11a, 11b and 12), each with a 1,3,4-oxadiazol-2-one
ring as bioisosteric replacement for the carboxylic acid functionality, were prepared to reduce the
acidity and polarity and thus improve bacterial cell membrane penetration of the compounds [20,
21]. Furthermore, two compounds, each with a pyridine containing substituent in Part C, pyridin-
2-ylmethanamine (n = 1, compound 15a) and 2-(pyridin-2-yl)ethan-1-amine (n = 2, compound
15b) were synthesized, and the optimal length for the activity determined. The pyridine moiety
was intended to enable π-stacking interactions with the Glu50-Arg76 salt bridge.

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a)
Part B
c)
Part C
OH
O
Part A
O
N
H
N
H
O
N
H
O
Cl
Cl
D
E. coli gyrase: IC50 = 47 nM
S. aureus gyrase: IC50 = 2.3 µM
E. coli topo IV: IC50 = 1.4 µM
S. aureus topo IV: IC50 = 2.3 µM
E. faecalis: MIC = 50 µM
E. coli: no inhibition
S.aureus: no inhibition
b)
type I
type II
X = 4,5-diBr,
X = 4,5-diBr,
3,4-diCl-5-Me
R1
n
n = 1, 2
O
O
N
3,4-diCl-5-Me
Y
Y =
OH
R2
O
N
H
O
N
,
H
N
H
N
H
O
N
H
R1 = H, CH3, iPr, Bn
R2 = COOH, CONHNH2,
N
O
H
O
O
X
X
Z
Z
O
N
N
H
Z = iPr, CH2CH2NH3+Cl-
Z = iPr, Bn, CH2CH2NH(Me)2+Cl-
Figure 2. a) A representative N-phenylpyrrolamide D and its inhibitory activities on DNA gyrase
and topoisomerase IV [16]; b) Structures of type I and type II N-phenylpyrrolamide inhibitors; c)
Docking binding mode of inhibitor D coloured according to the atom chemical type (C, yellow;
N, blue; O, red; Cl, green) in the ATP binding site of E. coli GyrB (in cyan, PDB code: 4DUH
[22]). The water molecule is presented as a red sphere.
Chemistry. The synthesis of type I compounds (8a-i, 9a-e, 11a-b, 12, 15a-b) is presented in
Schemes 1 to 3. 3-Hydroxy-4-nitrobenzoic acid (1) was reacted with thionyl chloride in methanol
to give methyl ester 2, which was converted to 3a in a Mitsunobu reaction using isopropanol, and
to 3b in the presence of potassium carbonate using tert-butyl (2-chloroethyl)carbamate as an
alkylating agent. Compounds 3a-b were hydrolysed with 1 M sodium hydroxide to give
carboxylic acids 4a-b, which were coupled with different amino acid methyl esters using TBTU
(N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate) in order to prepare
amides 5a-g. The nitro groups of 5a-g were reduced by catalytic hydrogenation to give amines
6a-g. Compounds 6a-g were then coupled with 4,5-dibromopyrrole-2-carboxylic acid (to give 7a-
c and 7g-h) or with 3,4-dichloro-5-methylpyrrole-2-carboxylic acid to give 7d-f and 7i in a two-
step reaction. Oxalyl chloride was used to form pyrrole-2-carboxylic acid chloride in the first step

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followed by its aminolysis in pyridine in the second step. Products 8a-i were prepared by alkaline
hydrolysis of 7a-i. Removal, by acidolysis, of the Boc protecting group from compounds 7h-i and
8g-i led to the final compounds 9a-e. Compounds 7e and 7i were additionally reacted with
hydrazine monohydrate under reflux to prepare hydrazides 10a-b. These were converted to 11a-b
using 1,1'-carbonyldiimidazole (CDI) at 100 °C. The Boc protecting group was removed from
11b to yield the target compound 12.
To prepare products 15a-b, 4a was first coupled with pyridin-2-ylmethanamine (to prepare
13a) or 2-(pyridin-2-yl)ethan-1-amine (to prepare 13b) using TBTU. Reducing the nitro groups
of 13a-b by catalytic hydrogenation led to compounds 14a-b. The final products 15a-b were
prepared by coupling amines 14a-b with 4,5-dibromopyrrole-2-carboxylic acid.

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O
O
O
O
OH
O
O
OH
a
b
c
O2N
O2N
O2N
O2N
OH
OH
OR1
3a (R1 = iPr)
OR1
4a (R1 = iPr)
1
2
3b (R1 = CH2CH2NHBoc)
4b (R1 = CH2CH2NHBoc)
O
R2
O
R2
O
O
d
N
e
N
H
H
O
O
O2N
H2N
OR1
OR1
5a (R1 = iPr, R2 = iPr, L isomer)
5b (R1 = iPr, R2 = Bn, L isomer)
5c (R1 = iPr, R2 = CH3, L isomer)
5d (R1 = iPr, R2 = CH3, D isomer)
5e (R1 = CH2CH2NHBoc, R2 = H)
6a (R1 = iPr, R2 = iPr, L isomer)
6b (R1 = iPr, R2 = Bn, L isomer)
6c (R1 = iPr, R2 = CH3, L isomer)
6d (R1 = iPr, R2 = CH3, D isomer)
6e (R1 = CH2CH2NHBoc, R2= H)
5f (R1 = CH2CH2NHBoc, R2 = Bn, L isomer)
5g (R1 = CH2CH2NHBoc, R2 = CH3, L isomer)
6f (R1 = CH2CH2NHBoc, R2 = Bn, L isomer)
6g (R1 = CH2CH2NHBoc, R2 = CH3, L isomer)
O
R2
O
R2
O
OH
O
N
O
N
g
H
H
f
H
H
O
O
N
N
R3
R3
N
N
H
H
OR1
OR1
R5
R5
R4
R4
7a (R1 = iPr, R2 = iPr, R3 = R4 = Br, R5 = H, L isomer)
7b (R1 = iPr, R2 = Bn, R3 = R4 = Br, R5 = H, L isomer)
7c (R1 = iPr, R2 = CH3, R3 = R4 = Br, R5 = H, L isomer)
7d (R1 = iPr, R2 = Bn, R3 = CH3, R4 = R5 = Cl, L isomer)
8a (R1 = iPr, R2 = iPr, R3 = R4 = Br, R5 = H, L isomer)
8b (R1 = iPr, R2 = Bn, R3 = R4 = Br, R5 = H, L isomer)
8c (R1 = iPr, R2 = CH3, R3 = R4 = Br, R5 = H, L isomer)
8d (R1 = iPr, R2 = Bn, R3 = CH3, R4 = R5 = Cl, L isomer)
7e (R1 = iPr, R2 = CH3, R3 = CH3, R4 = R5 = Cl, L isomer) 8e (R1 = iPr, R2 = CH3, R3 = CH3, R4 = R5 = Cl, L isomer)
7f (R1 = iPr, R2 = CH3, R3 = CH3, R4 = R5 = Cl, D isomer) 8f (R1 = iPr, R2 = CH3, R3 = CH3, R4 = R5 = Cl, D isomer)
7g (R1 = CH2CH2NHBoc, R2 = H, R3 = R4 = Br, R5 = H)
7h (R1 = CH2CH2NHBoc, R2 = Bn, R3 = R4 = Br, R5 = H,
L isomer)
8g (R1 = CH2CH2NHBoc, R2 = H, R3 = R4 = Br, R5 = H)
8h (R1 = CH2CH2NHBoc, R2 = Bn, R3 = R4 = Br, R5 = H,
L isomer)
7i (R1 = CH2CH2NHBoc, R2 = CH3, R3 = CH3, R4 = R5 = Cl, 8i (R1 = CH2CH2NHBoc, R2 = CH3, R3 = CH3, R4 = R5 = Cl,
L isomer)
L isomer)
O
R2
9a (R1 = CH3, R2 = Bn, R3 = R4 = Br, R5 = H, L isomer)
9b (R1 = CH3, R2 = CH3, R3 = CH3, R4 = R5 = Cl, L isomer)
9c (R1 = H, R2 = H, R3 = R4 = Br, R5 = H)
9d (R1 = H, R2 = Bn, R3 = R4 = Br, R5 = H, L isomer)
9e (R1 = H, R2 = CH3, R3 = CH3, R4 = R5 = Cl, L isomer)
O
O
N
R1
H
H
7h-i
8g-i
h
O
N
R3
N
H
O
R5
Cl
NH3
R4
Scheme 1. Reagents and conditions: (a) thionyl chloride, MeOH, 0 °C
isopropanol, triphenylphosphine, diisopropyl azodicarboxylate, THF, rt, 15 h (for the synthesis of
3a), tert-butyl (2-chloroethyl)carbamate, K2CO3, KI, DMF, rt 60 °C, 15 h (for the synthesis of
→ rt, 15 h; (b)
→
3b); (c) 1 M NaOH, MeOH, rt, 15 h; (d) corresponding amino acid methyl ester hydrochloride,
TBTU, NMM, CH2Cl2, rt, 15 h; (e) H2, Pd-C, MeOH, rt, 2-4 h; (f) corresponding pyrrole

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carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 6a-g, pyridine, CH2Cl2, rt, 15 h; (g) 1
M NaOH, MeOH/THF, rt, 15 h (for the synthesis of 8a-e and 8g-i) or 1 M LiOH, MeOH/THF, rt,
4 h (for the synthesis of 8f); (h) 4 M HCl in 1,4-dioxane, THF, rt, 2 h.
Scheme 2. Reagents and conditions: (a) hydrazine monohydrate, MeOH/THF, 65 °C, 15 h (for
the synthesis of 10a) or 4 d (for the synthesis of 10b); (b) CDI, 1,4-dioxane/DMF, 100 °C, 15 h;
(c) 4 M HCl in 1,4-dioxane, THF, rt, 5 h.
Scheme 3. Reagents and conditions: (a) pyridin-2-ylmethanamine (for the synthesis of 13a) or 2-
(pyridin-2-yl)ethan-1-amine (for the synthesis of 13b), TBTU, NMM, CH2Cl2, rt, 15 h; (b) H2,
Pd-C, MeOH, rt, 5 h; (c) 4,5-dibromopyrrole-2-carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h,
then ii) 14a-b, pyridine, CH2Cl2, rt, 15 h.
The synthesis of type II compounds (23a-d) is outlined in Scheme 4. 2-Hydroxy-4-nitrobenzoic
acid (16) was converted to its methyl ester 17 using thionyl chloride in methanol. Compounds
18a-b were prepared by reacting 17 with benzyl bromide or
β-dimethyl-aminoethylchloride
hydrochloride using potassium carbonate, while 18c was synthesized under Mitsunobu
conditions, using isopropyl alcohol. Alkaline hydrolysis of methyl esters 18a-c led to carboxylic
acids 19a-c, which were coupled with glycine methyl ester hydrochloride, using TBTU, to give

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20a-c. The amines 21a-c obtained after reduction of nitro groups of 20a-c were coupled with
either 4,5-dibromopyrrole-2-carboxylic acid to give 22a-c, or 3,4-dichloro-5-methylpyrrole-2-
carboxylic acid to give 22d. Finally, methyl esters of 22a-d were hydrolysed with 1 M sodium
hydroxide to give target compounds 23a-d.
Scheme 4. Reagents and conditions: (a) thionyl chloride, MeOH, 0 °C
→ rt, 15 h; (b) K2CO3,
benzyl bromide, CH3CN, 60 °C, 15 h (for the synthesis of 18a), -dimethyl-aminoethylchloride
β
hydrochloride, K2CO3, THF, 60 °C, 72 h (for the synthesis of 18b), isopropanol,
triphenylphospine, DIAD, THF, rt, 15 h (for the synthesis of 18c); (c) 1 M NaOH, MeOH, rt, 15
h; (d) glycine methyl ester hydrochloride, TBTU, NMM, CH2Cl2, rt, 15 h; (e) SnCl2,
EtOAc/MeOH, 55 °C, 15 h (for the synthesis of 21a), H2, Pd-C, MeOH, rt, 3 h, (for the synthesis
of 21b-c); (f) the corresponding pyrrole carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii)
21a-c, pyridine, CH2Cl2, rt, 15 h; (g) 1 M NaOH, MeOH/THF, rt, 15 h.
Inhibitory Activities against DNA Gyrase and Topoisomerase IV. All final compounds
were evaluated for their inhibitory activity against E. coli DNA gyrase in a supercoiling assay.

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Results are presented as residual activities (RAs) of the enzyme at 1 µM of compounds or as IC50
values for compounds with RA < 50% (Tables 1-2). Compounds with submicromolar IC50 values
were evaluated against S. aureus DNA gyrase, and E. coli and S. aureus topoisomerase IV (Table
3). To determine the possible binding modes of compounds, molecular docking of all tested
compounds to the E. coli GyrB binding site was performed, using GOLD software [23].
Compounds 8e, 8f, 9b, 9e, 11a, 12, 23a and 23d showed inhibitory activities stronger than that
of novobiocin (IC50 = 170 nM) against E. coli DNA gyrase, with low nanomolar inhibitory values
(IC50
≤ 91 nM). Seven of the eight most active compounds contained the 3,4-dichloro-5-methyl-
1H-pyrrole moiety in Part A of the molecules, which thus proved to be more suitable than the
4,5-dibromo-1H-pyrrole moiety. The reason for this is probably the slightly smaller size of the
chloro and methyl substituents than of the bromo substituents which bind to the hydrophobic
pocket of the enzyme more strongly [16]. Compounds with free carboxylic acid groups in the
eastern part of the molecules showed more potent activities than did their methyl ester analogues.
For example, methyl ester 7f was almost inactive at 1 µM concentration (RA = 87%), while its
carboxylic acid derivative 8f had an IC50 value of 41 nM and was among the most potent
compounds of the series. Free carboxylic acids are able to form ionic interactions and/or
hydrogen bonds with the Arg136 side chain in the binding site of the enzyme, while esters can
only form hydrogen bonds, thus resulting in their weaker activity. A similar trend can be
observed for other carboxylic acid – methyl ester pairs presented in Tables 1 and 2. The only
methyl ester derivative with activity in the low nanomolar range was compound 9b, with an IC50
value of 34 nM. Further, in some type I compounds (compounds 7e and 7i), methyl ester groups
were converted to hydrazides (compounds 10a and 10b), which were then further converted to
oxadiazolone rings (compounds 11a-b and 12). The activities of hydrazides (10a, IC50 = 280 nM)
were weaker than those of the corresponding carboxylic acids (8e, IC50 = 38 nM) or
oxadiazolones (11a, IC50 = 85 nM), probably because they cannot form ionic interactions with
Arg136. On the other hand, both compound 8e with a carboxylic acid group (IC50 = 38 nM) and
its oxadiazolone containing analogue 11a (IC50 = 85 nM) inhibited the enzyme in the low
nanomolar range, although compound 8e was approximately two-fold more potent. A different
result was observed when oxadiazolone 12 (IC50 = 13 nM) was compared with its carboxylic acid
analogue 9e (IC50 = 28 nM), where the former was more potent. The only compound containing
an oxadiazolone ring that did not show low nanomolar potency was 11b, with an IC50 value of

ACCEPTED MANUSCRIPT
1.6 µM, probably because it contains a side chain with the sterically larger tert-butyl carbamate
group.
As reported recently [16], lipophilic substituents at the 3-position of the Part B benzene ring
(e.g. methoxy, isopropoxy and benzyloxy substituents) form favourable interactions with the
hydrophobic floor of the enzyme. In type I compounds, three different substituents have thus
been introduced at this position: isopropoxy, 2-aminoethoxy and N-Boc-2-aminoethoxy
substituents. Analogues with either an isopropoxy (8e, IC50 = 38 nM) or a 2-aminoethoxy
substituent (9e, IC50 = 28 nM) had greater activity than analogues with an N-Boc-2-aminoethoxy
substituent (8i, IC50 = 590 nM), probably because the tert-butyl carbamate group is too large to fit
into the binding site of the enzyme. When compounds with isopropoxy and 2-aminoethoxy
substituents were compared, the latter were generally more potent, as seen by comparing the
isopropoxy derivatives 8d (RA = 91% at 1 µM) and 7e (RA = 66% at 1 µM) with their 2-
aminoethoxy counterparts 9d (IC50 = 370 nM) and 9b (IC50 = 34 nM). Based on the docking
experiments, it was predicted that the amino group of the 2-aminoethoxy substituent could form
an H-bond with Ala100 that is part of the flexible loop formed by Gly97-Ser108 (compound 9e,
Figure 3), thus increasing the inhibitory potency. Similarly, in the crystal structure of a bithiazole
inhibitor in complex with E. coli GyrB reported by Brvar et al. (PDB code: 4DUH [22]), a
hydrogen bond was seen with Gly101 that is also a part of this loop. These interactions could
stabilize the loop, reduce its flexibility and lead to stronger binding of the inhibitor.
Figure 3. The GOLD-predicted binding pose of inhibitor 9e (in orange sticks) in the E. coli GyrB
ATP-binding site (PDB entry: 4DUH [22], in grey). Hydrogen bonds are presented as green
dashed lines. The water molecule is shown as a red sphere. The figure was prepared with PyMOL
[24].

ACCEPTED MANUSCRIPT
To Part C of the type I compounds, we attached various amino acids: glycine, L- and D-alanine,
L
-valine and L-phenylalanine. Additionally, two compounds (15a and 15b) were prepared with
side chains containing a pyridine ring bound through either a methylene or an ethylene linker to
the central benzamide core. Compounds with alanine side chains displayed the strongest
inhibitory activity of the series while
For example, -alanine analogue 8c exhibited an IC50 value of 370 nM, while its
containing counterpart 8b was inactive at 1 µM concentration (RA = 100%). From these results it
can be concluded that the benzyl group of the -Phe side chain does not form favourable
hydrophobic or interactions with the enzyme. Furthermore, the size of the -Phe group may
L-phenylalanine containing compounds were the weakest.
L
L-phenylalanine
L
π
-π
L
force the molecule to take up an unfavourable conformation. Because of its size and flexibility,
the benzyl group might be oriented out of the binding site towards the solvent, as was predicted
with molecular docking of compound 8b into the E. coli GyrB ATP-binding site (Figure 1Sa,
Supporting information). There were no marked differences in the activities of
L-alanine
(compound 8e, IC50 = 38 nM) or -alanine containing compounds (compound 8f, IC50 = 41 nM).
D
Compounds 15a and 15b, with pyridine containing groups, were not active against E. coli gyrase
(RA = 100% or 87%). Even though the interaction between the pyridine nitrogen of compound
15a and Arg76 was predicted by molecular docking, this side chain is probably too flexible to
form a stable contact with Arg76 (Figure 1Sb, Supporting information).
To further explore the binding site, type II compounds with substituents at the 2-position of the
Part B benzene ring were prepared. Compounds with isopropoxy, benzyloxy and 2-
dimethylaminoethoxy groups at this position were evaluated. As in type I compounds, only
compounds with free carboxylic acid groups in the eastern part of the molecules showed
nanomolar enzymatic inhibition. Compound 23b, with a 2-dimethylaminoethoxy substituent (RA
= 82% at 1 µM), showed the weakest activity of the series. The benzyloxy analogue 23a (IC50 =
88 nM) was the most active type II compound, being 4-fold more active than its isopropoxy
analogue 23c (IC50 = 400 nM), indicating that it can form stronger hydrophobic interactions with
the enzyme. A second very potent compound in the type II series was compound 23d (IC50 = 91
nM) with an isopropoxy substituent on the benzene ring and a 3,4-dichloro-5-methylpyrrole
group attached as Part A.

ACCEPTED MANUSCRIPT
In general, the activities on DNA gyrase from S. aureus and on topo IV from E. coli and S.
aureus were weaker than those on E. coli gyrase, but compounds 8e, 9e and 12 that were among
the most potent inhibitors of E. coli gyrase also displayed promising results on these three
enzymes (Table 3). Compounds 8e, 9e and 12 had IC50 values in the submicromolar range (< 1
µM) against E. coli topo IV, which is much lower than that for novobiocin (IC50 = 11 µM).
Compound 8e, with an isopropoxy substituent, and compound 9e, with aminoethoxy substituent
on the 3-position of the benzene ring, additionally showed nanomolar IC50 values (93 nM and
110 nM, respectively) against S. aureus gyrase. Compound 8e displayed the most balanced
activities against all four tested enzymes, with a low IC50 of 280 nM also against S. aureus topo
IV (IC50 of novobiocin is 27 µM) and thus appears to be a promising dual-target inhibitor.
Interestingly, compound 8f, a D-alanine analogue of 8e, that showed an activity comparable to
that of 8e on DNA gyrase from E. coli, did not display the same potency on the other three
enzymes. Since there are certain structural differences between the enzymes, the stereochemistry
could be more important in the latter. A difference between 9e and its methyl ester analogue 9b
was also observed, with weaker activities of the latter on all enzymes. These results correlate well
with those for E. coli gyrase, leading to the assumption that an acidic functional group is also
important for the binding to S. aureus gyrase and to topo IV from E. coli and S. aureus. Similarly,
the carboxylic acid analogue 8e displayed stronger activity than either the corresponding
hydrazide 10a or the oxadiazolone 11a on these enzymes. Furthermore, the activity of the 3,4-
dichloro-5-methyl-pyrrolamides (8e, 8f, 8i, 9b, 9e, 10a, 11a, 12 and 23d) was stronger than that
of the 4,5-dibromopyrrolamides (8c, 9d, 23a and 23c). The reason probably lies in the smaller
size of the binding pockets of S. aureus DNA gyrase and of topo IV that results from differences
in certain amino acid residues in the ATP binding site [25, 26]. 3,4-Dichloro-5-
methylpyrrolamides with smaller groups on the pyrrole ring bind more effectively to the enzyme.
Table 1. Inhibitory activity of type I compounds 7a-i, 8a-i, 9a-e, 10a-b, 11a-b, 12 and 15a-b
against DNA gyrase from E. coli.
Compd.
R1
R2
R3
R4
*
IC50 (nM)a or RA (%)b

ACCEPTED MANUSCRIPT
E. coli gyrase
94%
4,5-diBr
4,5-diBr
iPr
iPr
Bn
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOH
L
L
L
L
L
D
/
7a
7b
7c
7d
7e
7f
iPr
100%
4,5-diBr
iPr
CH3
Bn
93%
3,4-diCl-5-Me
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
iPr
100%
iPr
CH3
CH3
H
66%
iPr
87%
CH2CH2NHBoc
53%
7g
7h
7i
4,5-diBr
CH2CH2NHBoc
Bn
L
L
L
L
L
L
L
D
/
100%
3,4-diCl-5-Me
4,5-diBr
CH2CH2NHBoc
CH3
iPr
100%
iPr
70%
8a
8b
8c
8d
8e
8f
4,5-diBr
iPr
Bn
COOH
100%
4,5-diBr
iPr
iPr
CH3
Bn
COOH
370 ± 160 nM
91%
3,4-diCl-5-Me
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
COOH
iPr
CH3
CH3
H
COOH
38 ± 9 nM
41 ± 16 nM
96%
iPr
COOH
CH2CH2NHBoc
CH2CH2NHBoc
CH2CH2NHBoc
COOH
8g
8h
8i
4,5-diBr
Bn
COOH
L
L
L
100%
3,4-diCl-5-Me
4,5-diBr
CH3
Bn
COOH
590 ± 20 nM
85%
CH2CH2NH3 Cl-
COOMe
+
9a
+
3,4-diCl-5-Me
4,5-diBr
CH2CH2NH3 Cl-
CH3
H
COOMe
COOH
COOH
L
L
L
34 ± 1 nM
50%
9b
9c
9d
+
CH2CH2NH3 Cl-
+
4,5-diBr
CH2CH2NH3 Cl-
Bn
370 ± 10 nM
+
3,4-diCl-5-Me
3,4-diCl-5-Me
3,4-diCl-5-Me
CH2CH2NH3 Cl-
CH3
CH3
CH3
COOH
L
L
L
28 ± 7 nM
280 ± 80 nM
68%
9e
iPr
CONHNH2
CONHNH2
10a
10b
CH2CH2NHBoc
3,4-diCl-5-Me
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
iPr
CH3
CH3
CH3
H
L
L
L
/
85 ± 7 nM
1600 ± 200 nM
13 ± 4 nM
100%
11a
11b
12
CH2CH2NHBoc
CH2CH2NH3 Cl-
+
iPr
iPr
15a
4,5-diBr
H
/
87%
15b
novobiocin
170 ± 20 nM
a Concentration of compound that inhibits the enzyme activity by 50%. b Residual activity of the enzyme at 1 µM of
the compound.

ACCEPTED MANUSCRIPT
Table 2. Inhibitory activity of type II compounds 22a-d and 23a-d against DNA gyrase from E.
coli.
IC50 (nM)a or RA (%)b
Compd.
R1
R2
R3
E. coli gyrase
100%
4,5-diBr
4,5-diBr
Bn
Me
Me
Me
Me
H
22a
22b
CH2CH2N(Me)2
88%
4,5-diBr
iPr
iPr
Bn
91%
22c
3,4-diCl-5-Me
4,5-diBr
59%
22d
88 ± 0 nM
82%
23a
+
4,5-diBr
CH2CH2NH(Me)2 Cl-
H
23b
4,5-diBr
iPr
iPr
H
400 ± 90 nM
91 ± 29 nM
170 ± 20 nM
23c
3,4-diCl-5-Me
H
23d
novobiocin
a Concentration of compound that inhibits the enzyme activity by 50%. b Residual activity of the
enzyme at 1 µM of the compound.
Table 3. Inhibitory activity of selected compounds against DNA gyrase from S. aureus and
topoisomerase IV from E. coli and S. aureus.
IC50 (µM)a or RA (%)b
Compd.
S. aureus gyrase
5.9 ± 0.7 µM
0.093 ± 0.065 µM
1.6 ± 0.6 µM
1.3 ± 0.0 µM
0.39 ± 0.09 µM
89%
E. coli topo IV
78%
S. aureus topo IV
98%
8c
8e
0.45 ± 0.12 µM
7.4 ± 1.5 µM
100%
0.28 ± 0.20 µM
1.3 ± 0.1 µM
99%
8f
8i
89%
3.7 ± 0.3 µM
98%
9b
96%
9d
0.11 ± 0.04 µM
22 ± 10 µM
0.75 ± 0.15 µM
1.0 ± 0.3 µM
79%
0.53 ± 0.06 µM
100%
3.2 ± 0.4 µM
92%
9e
10a
11a
12
11 ± 0 µM
0.57 ± 0.16 µM
42 ± 11 µM
100 %
12 ± 0 µM
0.96 ± 0.09 µM
39 ± 0 µM
96%
23a
23c
23d
85%
0.14 ± 0.02 µM
100%
12 ± 0 µM

ACCEPTED MANUSCRIPT
novobiocin
0.041 ± 0.07 µM
11 ± 2 µM
27 ± 7 µM
a Concentration of compound that inhibits the enzyme activity by 50%. b Residual
activity of the enzyme at 1 µM of the compound.
Antibacterial Activity. Compounds were tested for their antibacterial activity against two
Gram-positive (S. aureus ATCC 25923 and E. faecalis ATCC 29212) and two Gram-negative (E.
coli ATCC 25922 and P. aeruginosa ATCC 27853) wild type bacterial strains and, additionally,
against two E. coli mutant strains (JW5503
∆tolC and JD17464 ∆lpxC) at inhibitor
concentrations of 50 µM. The tolC deletion mutant represents Gram-negative bacteria with
defective efflux mechanisms and the lpxC deletion mutant represents a Gram-negative strain with
disrupted bacterial cell wall. Results are presented in Table 1S (Supporting information) as
percentages of growth inhibition. For compounds that inhibited bacterial strain growth by
MIC values were also determined (Table 4).
≥80%,
Overall, the compounds displayed stronger inhibitory activity against Gram-positive than
against Gram-negative bacteria. Nine compounds (8a, 8d, 8e, 8f, 9a, 9b, 10a, 11a and 11b)
showed more than 80% growth inhibition of E. faecalis, and three (9a, 9b and 11a) inhibited the
growth of S. aureus by more than 80%. Compound 9b, with an aminoethoxy substituent on the 3-
position of the central benzene ring, inhibited the growth of wild type E. coli by 67% (Table 1S)
and completely suppressed the E. coli ∆tolC deletion mutant at 50 µM. For compounds 7c, 8e, 8f,
9a, 11a and 12, significant differences in growth inhibition between wild type E. coli and the E.
coli mutant strain having a defective efflux pump were also observed, while growth inhibition of
the E. coli strain with impaired outer membrane was similar to that of the wild type for all
compounds (Table 1S). These results suggest that some compounds are subject to bacterial
efflux, which is probably the main reason for their weaker activity against Gram-negative
bacteria.
Compound 11a exhibited the strongest antibacterial activity of the series, with an MIC value of
1.56 µM against E. faecalis, which is almost two-fold lower than that of ciprofloxacin (3 µM),
and of 3.13 µM against S. aureus. Compound 11a, that contains an oxadiazolone ring in the
eastern part of the molecule, was twice as active as its carboxylic acid analogue 8e (E. faecalis
MIC = 3.13 µM) and four times more active than the corresponding hydrazide 10a (E. faecalis
MIC = 6.25 µM). This result is in agreement with our design strategy that compounds with a less
acidic and less polar bioisostere for the carboxylic acid group could permeate bacterial membrane

ACCEPTED MANUSCRIPT
more easily and display stronger antibacterial activity. Compounds with an aminoethoxy
substituent at the 3-position of the benzene ring (9a, 9b and 12) exhibited no or weaker activity
against E. faecalis (MIC values >25 µM for 9a and 9b and no growth inhibition for compound
12) than isopropoxy derivatives, presumably because the polarity of the amino group reduces the
ability of compounds to enter the Gram-positive bacteria. Compounds containing both a free
carboxylic acid group at the eastern part of the molecule and an aminoethoxy substituent at the 3-
position of the benzene ring (9c, 9d and 9e) were inactive against bacteria (less than 36% of
growth inhibition, Table 1S) although compound 9e was among the most active in enzyme assays
(S. aureus gyrase IC50 = 110 nM). On the other hand, compound 9b, a methyl ester analogue of
9e with a higher IC50 value (S. aureus gyrase IC50 = 390 nM), inhibited the growth of Gram-
positive bacteria more strongly than 9e, with MIC values of 50 µM against S. aureus and 25 µM
against E. faecalis (Table 4). Additionally, compound 9b showed a MIC value of 25 µM against
an E. coli strain with defective efflux pump. Similar activities against an E. coli strain with a
defective efflux pump were obtained for compound 9a (MIC = 25 µM) and for compound 12
(MIC = 50 µM) that also contain an aminoethoxy substituent. These results suggest that the
aminoethoxy substituent improves activity against Gram-negative bacteria, but the higher activity
is not observed because the compounds are possible substrates for efflux pumps.
Table 4. Minimum inhibitory concentrations (MICs) of compounds 7c, 8a, 8d, 8e, 8f, 9a, 9b,
10a, 11a, 11b and 12 against S. aureus (ATCC 25923), E. faecalis (ATCC 29212) and E. coli
(JW5503, a tolC deletion mutant).
MIC (µM)a
Compd.
S. aureus (ATCC 25923)
E. faecalis (ATCC 29212)
n.d.
E. coli (JW5503) ∆tolC b
n.d.c
25 µM
n.d.
7c
8a
n.d.
>75 µM
3.13 µM
3.13 µM
6.25 µM
50 µM
n.d.
n.d.
8d
8e
n.d.
50 µM
n.d.
n.d.
8f
50 µM
50 µM
n.d.
25 µM
25 µM
n.d.
9a
25 µM
9b
10a
11a
11b
12
6.25 µM
1.56 µM
100 µM
3.13 µM
n.d.
n.d.
n.d.
n.d.
n.d.
50 µM

ACCEPTED MANUSCRIPT
ciprofloxacin
1.5 µM
3.0 µM
0.015 µM
a
MIC (minimum inhibitory concentration that inhibits the growth of bacteria by ≥ 90%) values
against E. faecalis, S. aureus and E. coli JW5503 (∆tolC). Ciprofloxacin was used as a positive
control. b E. coli strain with mutated efflux pump. c Not determined.
Advanced antibacterial evaluation of compound 11a. Compound 11a was selected as the
most promising of the series, since it had the low nanomolar IC50 values in enzyme tests and the
lowest MIC values against Gram-positive strains of all the compounds. For this reason,
compound 11a was further evaluated against a diverse panel of Gram-positive and Gram-
negative bacterial strains (Table 5). In line with our previous observations, compound 11a
displayed an MIC value of 4.17 µM against the Gram-positive human pathogen S. aureus ATCC
29213. Furthermore, it was active against methicillin-resistant Staphylococcus aureus (MRSA,
ATCC 43300) and vancomycin-resistant Enterococcus (VRE, ATCC 70022), with MICs of 3.13
µM (Table 5). On the other hand, compound 11a displayed no bioactivity against wild type E.
coli strains (ATCC 25922, MG1655 and BW 25113). We therefore sought to explore
systematically the limiting factors for lack of anti-Gram-negative activity of compound 11a. To
investigate possible difficulties with its penetration into the bacterial cell, we tested compound
11a on a diverse panel of mutant E. coli strains carrying loss-of-function mutations in either
dapF, mrcB or surA genes that disrupt the bacterial cell wall integrity [27]. As a parallel
approach, to test the susceptibility of compounds to efflux, we treated the parental strains and
their corresponding mutants with an efflux pump inhibitor phenylalanine-arginine
naphthylamide (PA N), which is in fact not a typical inhibitor but a substrate for efflux pumps
that is preferentially effluxed. Furthermore, we tested compound 11a against E. coli BW 25113
acrB and tolC deletion mutants with defective efflux pumps. Mutations in the genes involved
in the formation of the cell wall (dapF, mrcB and surA) did not increase the antibacterial activity
(MIC > 50 µM) but, in the case of surA strain, stronger inhibition was observed in the presence
of PA N (MIC = 13.8 µM). The presence of PA N also increased the activity against wild type
E. coli strains ATCC 25922 (MIC = 4.6 µM) and MG1655 (MIC = 10.2 µM). Compound 11a did
not inhibit the efflux pump deficient E. coli BW 25113 strains acrB and tolC (MIC > 50 µM,
Table 5), but it displayed weak activity against the E. coli JW5503 tolC strain (51% inhibition
β-
β
∆
∆
∆
β
β
∆
∆
∆
at 50 µM, Table 1S). These results indicate that the most probable reason for the inactivity of
compound 11a against Gram-negative strains lies in bacterial efflux.

ACCEPTED MANUSCRIPT
Additionally, in order to validate the interaction between the oxadiazolone ring of 11a and
Arg136 in the DNA gyrase binding site, compound 11a was tested, in the presence of PAβN, on
E. coli MG1655 strain with the Arg136 to Cys mutation.. The compound was not active on the
mutated bacteria (MIC > 50 µM), but displayed activity against wild type E. coli in the presence
of PAβN (MIC = 10.2 µM), indicating that the compound interacts with Arg136.
Table 5. Minimum inhibitory concentrations (MICs) of compound 11a against a broad panel of
Gram-positive and Gram-negative bacterial strains.
Compound 11a
MIC (µM)a
Gram-positive bacteria
S. aureus (ATCC 29213)
4.17 µM
3.13 µM
3.13 µM
S. aureus (MRSA, ATCC 43300)
E. faecium (VRE, ATCC 70022)
Gram-negative bacteria
E. coli (ATCC 25922)
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
No inhibition up to 50 µM
4.6 µM
E. coli (MG1655)
E. coli (BW 25113)
E. coli (BW 25113) ∆tolCb
E. coli (BW 25113) ∆acrBb
E. coli (BW 25113) ∆dapFc
E. coli (BW 25113) ∆mrcBc
E. coli (BW 25113) ∆surAc
E. coli (ATCC 25922) + 50 µg/ml PAβN
E. coli (MG1655) + 50 µg/ml PAβN
E. coli (BW 25113) + 50 µg/ml PAβN
E. coli (BW 25113) ∆dapFc + 50 µg/ml PAβN
E. coli (BW 25113) ∆mrcBc + 50 µg/ml PAβN
E. coli (BW 25113) ∆surAc + 50 µg/ml PAβN
10.2 µM
No inhibition up to 20 µM
No inhibition up to 20 µM
No inhibition up to 20 µM
13.8 µM
E. coli (MG1655) R136-to-C mutant + 50 µg/ml PAβN
No inhibition up to 50 µM
a
MIC (minimum inhibitory concentration that inhibits the growth of bacteria by ≥ 90%)
measurements were performed according to the EUCAST guidelines in 3 independent
measurements. b E. coli strain with mutated efflux pump. c E. coli strain with loss-of-function
mutation in the gene involved in the cell wall formation.
Cytotoxic activity of compound 11a. The cytotoxicity of compound 11a was determined by
MTS assay against a human hepatocellular carcinoma cancer cell line (HepG2) and against
human endothelial cells (HUVEC). The decrease in cell proliferation after the treatment with 11a

ACCEPTED MANUSCRIPT
was compared with that after treatment with 50 µM of etoposide, as positive control. Compound
11a showed no cytotoxicity against HepG2 cells at 50 µM (85% cell proliferation), while it
showed an IC50 value of 40.7 µM against HUVEC cells (Table 2S, Supporting information).
However, the concentration at which the compound was active against HUVEC cells was much
higher than that at which it is active against Gram-positive bacteria.
CONCLUSIONS
Overall, thirty-eight compounds were designed, synthesized and evaluated against DNA gyrase
and topoisomerase IV, and against several Gram-positive and Gram-negative bacterial strains.
The most active compound in enzymatic assays was 3,4-dichloro-5-methyl-pyrrolamide 8e, with
an isopropoxy substituent at the 3-position of the benzene ring and a free carboxylic acid
functionality in the eastern part of the molecule. Compound 8e had an IC50 value of 38 nM
against E. coli gyrase and 93 nM against S. aureus gyrase, and was also active against E. coli
topo IV (IC50 = 0.45 µM) and S. aureus topo IV (IC50 = 0.28 µM). Compound 11a, an
oxadiazolone analogue of 8e, possessed low micromolar antibacterial activity against Gram-
positive bacteria, with MIC values of 1.56 µM and 3.13 µM against E. faecalis and S. aureus,
respectively. Further, compound 11a inhibited the growth of MRSA and VRE, key targets for
antibiotic research, with MIC values of 3.13 µM [28]. Compound 11a was not active against wild
type E. coli strains, but was active in the presence of PAβN (MIC = 4.6 µM). These results
suggest that, in Gram-negative bacteria, the compounds are sensitive to the efflux mechanism. In
recent years, however, new compounds that can be used as adjuvants to potentiate the activity of
antibiotics against Gram-negative strains are becoming available [29]. In short, low nanomolar
enzymatic activity against DNA gyrase and topo IV, low micromolar MIC values against drug-
resistant Gram-positive bacteria, and low cytotoxicity make compound 11a a promising starting
point for further optimisation.
EXPERIMENTAL SECTION
Determination of Inhibitory Activities on E. coli and S. aureus DNA Gyrase. The assay for
the determination of IC50 values was performed according to previously reported procedures [16].

ACCEPTED MANUSCRIPT
Determination of Inhibitory Activities on E. coli and S. aureus Topoisomerase IV. The
assay for the determination of IC50 values was performed according to previously reported
procedures [16].
Bacterial strains used in the study.
Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 25923, S. aureus ATCC
43300, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 70022, Escherichia
coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 have been obtained from the
American Type Culture Collection (ATCC) via Microbiologics Inc. (St. cloud, MN), E. coli
MG1655 originated from the laboratory collection of Dr. Csaba Pál. E. coli BW 25113, and E.
coli BW 25113
∆acrB,
∆dapF,
∆mrcB and
∆surA mutant strains originated from the KEIO
collection [30]. Single-gene knock-out strains of E. coli, JW5503
∆
tolC [30] and JD17464 lpxC
∆
were obtained from the NBRP-E.coli collection at the National Institute of Genetics (NIG,
Japan).
Determination of Antibacterial Activity.
The antibacterial activities against S. aureus ATCC 25923, E. faecalis ATCC 29212, E. coli
ATCC 25922, P. aeruginosa ATCC 27853, E. coli JW5503, E. coli JD17464, S. aureus ATCC
43300, and E. faecium ATCC 70022 were determined following the CLSI guidelines and
performed according to previously reported procedures [16].
MIC values against S. aureus ATCC 29213, E. coli ATCC 25922, E. coli MG1655, E. coli BW
25113, and E. coli BW 25113
∆acrB, ∆dapF, ∆mrcB and ∆surA were determined using a
standard broth microdilution technique according to the EUCAST guidelines and ISO 20776-
1:2006 [31]. In brief, 12-step serial dilutions of the test compound were prepared in 100 µL of
cation adjusted Mueller-Hinton II Broth (Catalog n.o. 90922 from Merck KGaA, Darmstadt,
Sigma) in 96-well microtiter plates. Approximately 5x104
well. Plates were incubated at 37 °C and shaken at 300 r.p.m. Measurements were performed in 3
ꢀbacteria were inoculated onto each
ꢀ
replicates. After 18 h of incubation, optical density values were measured in a Biotek Synergy
microplate reader at 600 nm wavelength for each well. MIC values were defined as the lowest
concentration of the test substance where no visible growth can be observed, i.e. the background-
normalized optical density of the culture at 600 nm was below 0.05.
In vitro cytotoxicity measurements. Cytotoxicity of compound 11a was determined in MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)

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assay [32] with a few modifications. HepG2 (ATCC) and HUVEC (ATCC) cells were cultured in
Eagle's MEM medium supplemented with L-glutamine (2 mM), penicillin/streptomycin (100
UI/mL/100 µg/mL), and 10% FBS. The cells were incubated in a humidified atmosphere with 5%
CO2 at 37 °C.
The cells were seeded in 96-well plates at densities 2000 cells per well in 100 µL of growth
medium and incubated for 24 h to attach onto the wells. 50 µL of compounds at 50 µM in DMSO
(0.5% final concentration) were added and incubated for 72 h. After 72 h 10 µL of CellTiter96®
Aqueous One Solution Reagent, (Promega) [33] was added to determine the number of viable
cells. The plates were incubated for another 3 h and absorbance (490 nm) was read with a
BioTek's Synergy H4 microplate reader. Etoposide [IC50 = 20.1 µM (Ref. [34]: 30.2 µM) for
HepG2 and IC50 = 5.21 µM (Ref. [35]: 1.25 µM) for HUVEC] at 50 µM was used as a positive
control and 0.5% DMSO as a vehicle control. To determine cell viability, results from the wells
that contained test compound-treated cells were compared to those with cells incubated in 0.5%
DMSO. Independent experiments were run in triplicate and repeated two times. Statistical
significance (p < 0.05) was calculated with two-tailed Welch's t test between treated groups and
DMSO. On HUVEC cell line, where compound showed higher activity, IC50 value (concentration
of compound that inhibits cell proliferation by 50%) was determined using six concentrations of
the tested compound. GraphPad Prism 5.0 software [36] was used to calculate the IC50 value.
Molecular Modeling.
Protein and Ligand Preparation. 3D compound models were built using ChemBio3D Ultra 16.0
[37]. MMFF94 force field [38] was used for the optimization of geometries and partial atomic
charges were added. Energy was minimized to less than 0.001 kcal/(mol Å) gradient value. The
structure was refined with GAMESS interface using PM3 method, QA optimization algorithm
and Gasteiger Hückel charges for all atoms for 100 steps [37]. GOLD Suite v5.4 [23, 39] was
used for molecular docking calculations. GOLD graphical user interface was used for receptor
preparation. To the protein hydrogen atoms were added and correct tautomers and protonation
states were assigned. Except for HOH614, all water molecules and ligands were deleted from the
crystal structure. Amino acid residues within 7 Å around the ligand (PDB entry: 4DUH [22])
were selected as the binding site.
Ligand Docking. Compounds were docked to the defined binding site in 25 independent
genetic algorithm (GA) runs by applying different GA parameters (population size = 100,

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selection pressure = 1.1, number of operations = 100,000, niche size = 2, number of islands = 5,
mutation frequency = 95, crossover frequency = 95, migration frequency = 10) and scoring
functions (GoldScore, ChemScore, CHEMPLP). The most representative results were obtained
using GoldScore as a scoring function. Ligands with RMSD value less than 1.5 Å were joined in
clusters and early termination was allowed if the top 3 solutions were within 1.0 Å of the RMSD
value. Proposed binding modes of the top 5 highest scored docking poses were evaluated for each
ligand and the highest scored pose was used for graphical representation with PyMOL [24].
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 were performed on an Agilent Technologies
1100 instrument (Agilent Technologies, Santa Clara, CA, USA) with a G1365B UV-Vis detector,
a G1316A thermostat, a G1313A autosampler, and a ChemStation data system or on a Thermo
Scientific Dionex Ultimate 3000 Binary Rapid Seperation LC System (Thermo Fisher Scientific,
Waltham, MA, USA) with an autosampler, a binary pump system, a photodiode array detector, a
thermostated column compartment, and a Chromeleon Chromatography Data System. The eluent
for consisted of trifluoroacetic acid (0.1% in water) or 20 mM phosphate buffer (pH 6.8) as
solvent A and acetonitrile as solvent B. Two methods were used, method A: Agilent Eclipse Plus
C18 column (5 µm, 4.6 mm × 150 mm), mobile phase of 30-90% of acetonitrile in TFA (0.1%) in
16 min, 90% acetonitrile to 20 min, a flow rate of 1.0 mL/min and a sample injection volume of
10
of 50-80% of acetonitrile in 20 mM phosphate buffer (pH 6.8) in 30 min, a flow rate of 1.0
mL/min and a sample injection volume of 10 L. Melting points were determined on a Reichert
µL, and method B: Phenomenex Luna C18 column (5 µm, 4.6 mm × 250 mm), mobile phase
µ
hot stage microscope and are uncorrected. 1H and 13C 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, CDCl3 or acetone–d6 solutions, with TMS as the internal standard. IR
spectra were recorded on a Thermo Nicolet Nexus 470 ESP FT-IR spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA). Mass spectra were obtained using a Q-TOF Premier mass
spectrometer (Micromass, Waters, Manchester, UK) or ADVION expression CMSL mass
spectrometer (Advion Inc., Ithaca, USA). Optical rotations were measured on a Perkin-Elmer 241

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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
≥ 95% as
established by HPLC.
Synthetic Procedures.
General procedure A. Synthesis of Compounds
2 and 17 (with 2 as an Example). To a suspension
of 3-hydroxy-4-nitrobenzoic acid (1) (5.00 g, 27.3 mmol) in methanol (150 mL) cooled on ice
bath thionyl chloride (5.90 mL, 81.9 mmol) was added dropwise. The mixture was stirred at rt for
15 h upon which a clear solution formed. The solvent was evaporated under reduced pressure. To
the residue petroleum ether (50 mL) was added, the obtained suspension was sonicated, filtered
and washed with petroleum ether (20 mL). The purification was repeated twice and the residue
was dried to give compound 2 (4.92) as yellow crystals.
Methyl 3-hydroxy-4-nitrobenzoate (2) [40]. Yellow crystals; yield 92 % (4.92 g); mp 87-88 °C
(86-88 °C, lit.[40]). IR (ATR)
ν
= 3309, 3053, 2961, 1720, 1623, 1586, 1434, 1321, 1220, 1146,
1
1097, 966, 842, 798, 743, 667 cm-1. H NMR (400 MHz, CDCl3)
δ 3.99 (s, 3H, CH3), 7.64 (dd,
1H, 3J = 8.8 Hz, 4J = 1.6 Hz, Ar-H-6), 7.86 (d, 1H, 4J = 1.6 Hz, Ar-H-2), 8.20 (d, 1H, 3J = 8.8 Hz,
Ar-H-5), 10.53 (s, 1H, OH).
General procedure B. Synthesis of Compounds 3a and 18c (with 3a as an Example). To a stirred
solution of compound 2 (3.00 g, 15.2 mmol) and triphenylphosphine (5.40 g, 20.6 mmol) in
anhydrous tetrahydrofuran (30 mL) isopropanol (1.5 mL, 19.8 mmol) was added and the mixture
was stirred at rt for 10 minutes. Diisopropyl azodicarboxylate (DIAD, 3.90 mL, 19.8 mmol) was
added dropwise and the mixture was stirred at rt for 15 h under argon atmosphere. The solvent
was evaporated under reduced pressure and the crude product was purified with flash column
chromatography using ethyl acetate/petroleum ether (1/6) as an eluent to give 3a (3.50 g) as
yellow solid.
Methyl 3-isopropoxy-4-nitrobenzoate (3a). Yellow solid; yield 97% (3.50 g); mp 42-44 °C
(39-41 °C, lit.[41]). IR (ATR)
942, 835, 741 cm-1. 1H NMR (400 MHz, DMSO-d6)
ν
= 3117, 2989, 2950, 1724, 1610, 1525, 1422, 1284, 1103, 1004,
1.30 (d, 6H, 3J = 6.0 Hz, CH(CH3)2), 3.91
δ
(s, 3H, COOCH3), 4.93 (spt, 1H, 3J = 6.0 Hz, CH(CH3)2), 7.64 (dd, 1H, 3J = 8.4 Hz, 4J = 1.6 Hz,
Ar-H-6), 7.78 (d, 1H, 4J = 1.2 Hz, Ar-H-2), 7.95 (d, 1H, 3J = 8.0 Hz, Ar-H-5).
General procedure C. Synthesis of Compounds 4a-b and 19a-c (with 4a as an Example). To the
solution of compound 3a (3.50 g, 14.6 mmol) in methanol (30 mL) 1 M NaOH (28 mL, 29.3

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mmol) was added. The mixture was stirred at rt for 15 h. The solvent was removed under reduced
pressure and the residue acidified with 1M HCl (10 mL). Water phase was extracted with ethyl
acetate (3 × 20 mL). The combined organic phases were washed with water (3 × 20 mL) and
brine (2 × 20 mL), dried over Na2SO4, filtered and the solvent removed under reduced pressure to
afford 4a (3.00 g) as yellow solid.
3-Isopropoxy-4-nitrobenzoic acid (4a). Yellow solid; yield 91% (3.00 g); mp 170-173 °C
(173-175°C, lit.[41]). IR (ATR)
ν
= 1987, 2838, 2603, 1689, 1522, 1427, 1296, 110, 937, 837,
3
742 cm-1. 1H NMR (400 MHz, DMSO-d6)
δ
0.86 (d, 6H, J = 6.0 Hz, CH(CH3)2), 4.46 (spt, 1H,
3J = 6.0 Hz, CH(CH3)2), 7.17 (dd, 1H, 3J = 8.0 Hz, 4J = 1.6 Hz, Ar-H-6), 7.32 (d, 1H, 4J = 1.6 Hz,
Ar-H-2), 7.48 (d, 1H, 3J = 8.4 Hz, Ar-H-5), 13.21 (s, 1H, COOH).
General procedure D. Synthesis of Compounds 5a-g, 13a-c and 20a-b (with 5a as an Example).
To the suspension of 4a (800 mg, 3.55 mmol) and TBTU (1.48 g, 4.62 mmol) in dichloromethane
(40 mL) N-methylmorpholine (1.2 mL, 10.7 mmol) was added. The reaction mixture was stirred
at rt for 30 minutes upon which a clear solution formed. L-valine methyl ester hydrochloride (715
mg, 4.26 mmol) was added and the mixture was stirred at rt for 15 h. The solvent was removed
under reduced pressure and the residue was dissolved in ethyl acetate (30 mL) and water (30
mL). The organic phase was washed with saturated aqueous NaHCO3 solution (3 × 20 mL) and
brine (2 × 20 mL), dried over Na2SO4, filtered and the solvent evaporated under reduced pressure
to give 5a (1.11 g) as yellow oil.
25
Methyl (3-isopropoxy-4-nitrobenzoyl)-
-8.32 (c 0.263, MeOH). IR (ATR)
842, 748 cm-1. 1H NMR (400 MHz, CDCl3)
6 Hz, OCH(CH3)2), 2.27-2.35 (m, 1H, CHCH(CH3)2), 3.81 (s, 3H, COOCH3), 4.75-4.82 (m, 2H,
L-valinate (5a). Yellow oil; yield 92% (1.11 g); [α]
D
ν
= 3279, 2974, 1744, 1640, 1533, 1317, 1256, 1103, 1021,
1.00-1.04 (m, 6H, CHCH(CH3)2), 1.42 (d, 6H, 3J =
δ
3
3
4
CHCH(CH3)2, OCH(CH3)2), 6.71 (d, 1H, J = 8.4 Hz, NH), 7.31 (dd, 1H, J = 8.4 Hz, J = 1.6
Hz, Ar-H-6), 7.62 (d, 1H, 4J = 1.2 Hz, Ar-H-2), 7.81 (d, 1H, 3J = 8.0 Hz, Ar-H-5). 13C NMR (100
MHz, DMSO-d6)
δ 19.01 (CH3), 19.10 (CH3), 21.50 (CH(CH3)2), 21.56 (CH(CH3)2), 29.60 (CH),
51.75 (COOCH3), 58.77 (CH), 72.43 (CH), 115.32, 119.76, 124.62, 138.42, 142.29, 149.61,
165.28 (CONH), 171.97 (COOCH3).
General procedure E. Synthesis of Compounds 6a-g
, 14b-c and 21a-b (with 6a as an Example).
Compound 5a (1.11 g, 3.28 mmol) was dissolved in methanol (40 mL), Pd/C (111 mg) was

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added and the reaction mixture was stirred under hydrogen atmosphere for 3 hours. The catalyst
was filtered off and the solvent removed under reduced pressure.
Methyl (4-amino-3-isopropoxybenzoyl)-
flash column chromatography using ethyl acetate/petroleum ether (1/1) as an eluent to obtain 6a
(522 mg) as yellow oil. Yield: 55% (522 mg). [
25 -5.53 (c 0.378, MeOH). 1H NMR (400 MHz,
CDCl3)
0.99-1.03 (m, 6H, CHCH(CH3)2), 1.38 (d, 6H, 3J = 6.4 Hz, OCH(CH3)2), 2.23-2.31 (m,
L-valinate (6a). The crude product was purified with
α
]
D
δ
3
1H, CHCH(CH3)2), 3.79 (s, 1H, COOCH3), 4.11-4.17 (m, 2H NH2), 4.66 (spt, 1H, J = 6.0 Hz,
3
OCH(CH3)2), 4.76-4.79 (m, 1H, CHCH(CH3)2), 6.50 (d, 1H, J = 8.8 Hz, NH), 6.70 (d, 1H, 3J =
8.0 Hz, Ar-H-5), 7.20 (dd, 1H, 3J = 8.0 Hz, 4J = 2.0 Hz, Ar-H-6), 7.40 (d, 1H, 4J = 2.0 Hz, Ar-H-
2). MS (ESI) m/z = 331.37 ([M+Na]+).
General procedure F. Synthesis of Compounds 7a-i, 15a-d and 22a-b (with 7a as an Example).
To a solution of 4,5-dibromo-1H-pyrrole-2-carboxylic acid (157 mg, 0.584 mmol) in anhydrous
dichloromethane (4 mL) oxalyl chloride (2M solution in dichloromethane, 1.46 mL, 2.92 mmol)
was added dropwise and the solution stirred at rt for 15 h under argon atmosphere. The solvent
was evaporated under reduced pressure, fresh anhydrous dichloromethane (4 mL), 6a (150 mg,
0.487 mmol) and pyridine (2 mL) were added and the reaction mixture stirred under argon
atmosphere at rt for 15 h. Solvent was removed under reduced pressure, the residue dissolved in
ethyl acetate (20 mL) and washed with water (20 mL), saturated aqueous NaHCO3 solution (3 ×
20 mL) and brine (2 × 20 mL). The organic phase was dried over Na2SO4, filtered and the solvent
removed under reduced pressure.
Methyl
(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)-3-isopropoxybenzoyl)-L-valinate
(7a). To the crude product ethyl acetate and petroleum ether (1:2, 15 mL) were added and the
precipitate was filtered off to obtain 7a (40 mg) as light brown solid. The mother liquid was
concentrated and purified with flash column chromatography using ethyl acetate/petroleum ether
(1/2) as an eluent to give another 61 mg of 7a as light brown solid. Yield 37% (101 mg); mp 145-
148 °C. [
α
]
25 +8.94 (c 0.273, MeOH). IR (ATR)
ν
= 3417, 3304, 3238, 2964, 1741, 1633, 1515,
0.94-1.00 (m, 6H,
D
1418, 1311, 1202, 1124, 978, 831, 743 cm-1. 1H NMR (400 MHz, DMSO-d6)
δ
CHCH(CH3)2), 1.32-1.35 (m, 6H, OCH(CH3)2), 2.15-2.23 (m, 1H, CHCH(CH3)2), 3.67 (s, 3H,
COOCH3), 4.30 (t, 1H, 3J = 8.0 Hz, CHCH(CH3)2), 4.72 (spt, 1H, 3J = 6.0 Hz, OCH(CH3)2), 7.18
4
3
(d, 1H, J = 2.4 Hz, Pyrr-CH), 7.53-7.55 (m, 2H, Ar-H-2,6), 7.92 (d, 1H, J = 8.4 Hz, Ar-H-5),
3
4
8.59 (d, 1H, J = 8.0 Hz, CONHCH), 9.06 (s, 1H, CONHAr), 13.02 (d, 1H, J = 2.8 Hz, Pyrr-

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NH). 13C NMR (100 MHz, DMSO-d6)
δ
19.06 (CHCH(CH3)2), 19.09 (CHCH(CH3)2), 21.59
(OCH(CH3)2), 21.67 (OCH(CH3)2), 29.45 (CHCH(CH3)2), 51.55 (COOCH3), 58.59
(CHCH(CH3)2), 71.29 (OCH(CH3)2), 98.35, 106.22, 113.18, 113.77, 120.11, 122.44, 127.50,
130.06, 130.50, 148.15, 156.95, 166.15 (Ar-CONH), 172.26 (COOCH3). MS (ESI) m/z = 556.0
([M-H]-), HRMS for C21H24Br2N3O5: calculated 556.0083, found 556.0076. HPLC: tr 14.108 min
(97.2% at 280 nm, method A).
General procedure G. Synthesis of Compounds 8a-e, 8g-i and 23a-d (with 8a as an Example). To
the solution of 7a (65 mg, 0.116 mmol) in tetrahydrofuran (8 mL) 1 M NaOH (348 µL, 348
µmol) was added and the mixture was stirred at rt for 15 h. The solvent was evaporated under
reduced pressure, the residue acidified with 1 M HCl (5 mL) to pH 1 and the water phase was
extracted with ethyl acetate (3 × 10 mL). The combined organic phases were washed with water
(3 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered and the solvent removed under
reduced pressure to obtain 8a (53 mg) as white solid.
(4-(4,5-Dibromo-1
H-pyrrole-2-carboxamido)-3-isopropoxybenzoyl)-
L-valine (8a). White
25
D
solid; yield 87% (53 mg); mp 116-120 °C; [
α]
+18.6 (c 0.288, MeOH). IR (ATR) ν = 3402,
3183, 2970, 1716, 1648, 1507, 1389, 1265, 1179, 1109, 977, 805, 751 cm-1. 1H NMR (400 MHz,
DMSO-d6) 0.96-1.00 (m, 6H, CHCH(CH3)2), 1.32-1.36 (m, 6H, OCH(CH3)2), 2.15-2.22 (m,
δ
3
3
1H, CHCH(CH3)2), 4.30 (t, 1H, J = 8.0 Hz, CHCH(CH3)2), 4.73 (spt, 1H, J = 5.6 Hz,
OCH(CH3)2), 7.18 (d, 1H, 4J = 2.4 Hz, Pyrr-CH), 7.53-7.57 (m, 2H, Ar-H-2,6), 7.92 (d, 1H, 3J =
3
8.0 Hz, Ar-H-5), 8.42 (d, 1H, J = 8.0 Hz, CONHCH), 9.05 (s, 1H, CONHAr), 12.62 (br s, 1H,
COOH), 13.02 (s, 1H, Pyrr-NH). 13C NMR (100 MHz, DMSO-d6)
δ 18.89 (CHCH(CH3)2),
19.34 CHCH(CH3)2), 21.67 OCH(CH3)2), 21.76 OCH(CH3)2), 29.52 (CHCH(CH3)2), 58.38
(
(
(
(CHCH(CH3)2), 71.36 (OCH(CH3)2), 98.41, 106.28, 113.28, 113.82, 120.16, 122.53, 127.60,
130.43, 130.46, 148.22, 157.03, 166.13 (Ar-CONH), 173.22 (COOH). MS (ESI) m/z = 542.0
([M-H]-), HRMS for C20H22Br2N3O5: calculated 541.9926, found 541.9935. HPLC: tr 12.049 min
(98.1% at 280 nm, method A).
General procedure H. Synthesis of Compounds 9a-e (with 9a as an Example). The starting
compound (7h, 40 mg, 0.057 mmol) was dissolved in 4 M HCl in 1,4-dioxane (4 mL) and
tetrahydrofuran (1 mL) and the reaction mixture stirred at rt for 2 hours. The solvent was
removed under reduced pressure, to the residue diethyl ether (10 mL) was added, the obtained