Further investigation of inhibitors of MRSA pyruvate kinase: Towards the conception of novel antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.018

Several novel series of compounds were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as a highly interconnected essential ‘hub’ protein in MRSA, with structural features distinct from the human homologs which makes it a novel antimicrobial target. Several MRSA PK inhibitors (including the hydrazide 1) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to human PK isoforms. Structure–activity relationship (SAR) studies were carried out on the replacement of the hydrazide linker with 3-atoms, 2-atoms and 0-atom linkers and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1?μg/mL.

Full text of DOI:10.1016/j.ejmech.2016.09.018

Accepted Manuscript
Further investigation of inhibitors of MRSA pyruvate kinase: Towards the conception
of novel antimicrobial agents
Christophe Labrière, Huansheng Gong, B. Brett Finlay, Neil E. Reiner, Robert N.
Young
PII:
S0223-5234(16)30746-2
10.1016/j.ejmech.2016.09.018
EJMECH 8884
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 July 2016
Revised Date: 4 September 2016
Accepted Date: 6 September 2016
Please cite this article as: C. Labrière, H. Gong, B.B. Finlay, N.E. Reiner, R.N. Young, Further
investigation of inhibitors of MRSA pyruvate kinase: Towards the conception of novel antimicrobial
agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.018.
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Graphical Abstract
Further investigation of inhibitors of MRSA pyruvate kinase:
Towards the conception of novel antimicrobial agents
Christophe Labrière1&*, Huansheng Gong2, B. Brett Finlay2,3, Neil E. Reiner2,4, Robert N.
Young1*
1 Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
2 Department of Medicine, Division of Infectious Diseases, University of British Columbia,
Vancouver, BC, Canada.
3 Department of Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, BC, Canada.
4 Department of Microbiology and Immunology, Michael Smith Laboratories, University of
British Columbia, Vancouver, BC, Canada.
*Corresponding authors:
Christophe Labriere, Phone: +33 (0) 6 86 54 72 74, E-mail: christophe.labriere@gmail.com
Robert Young, Phone: +1 778 782 3351, Fax: +1 778 782 3765, E-mail: roberty@sfu.ca
Present Address
& C. L.: Garenn Sant Houardon, 29690 La Feuillée, France.
1

ACCEPTED MANUSCRIPT
Further investigation of inhibitors of MRSA pyruvate kinase:
Towards the conception of novel antimicrobial agents
Christophe Labrière1&*, Huansheng Gong2, B. Brett Finlay2,3, Neil E. Reiner2,4, Robert N.
Young1*
1 Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
2 Department of Medicine, Division of Infectious Diseases, University of British Columbia,
Vancouver, BC, Canada.
3 Department of Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, BC, Canada.
4 Department of Microbiology and Immunology, Michael Smith Laboratories, University of
British Columbia, Vancouver, BC, Canada.
*Corresponding authors:
Christophe Labriere, Phone: +33 (0) 6 86 54 72 74, E-mail: christophe.labriere@gmail.com
Robert Young, Phone: +1 778 782 3351, Fax: +1 778 782 3765, E-mail: roberty@sfu.ca
Present Address
& C. L.: Garenn Sant Houardon, 29690 La Feuillée, France.
Abstract
Several novel series of compounds were synthesized and evaluated as inhibitors of methicillin-
resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as a highly
interconnected essential ‘hub’ protein in MRSA, with structural features distinct from the human
homologs which makes it a novel antimicrobial target. Several MRSA PK inhibitors (including
the hydrazide 1) were identified using in silico screening combined with enzyme assays and were
found to be selective for bacterial enzyme compared to human PK isoforms. Structure–activity
relationship (SAR) studies were carried out on the replacement of the hydrazide linker with 3-
atoms, 2-atoms and 0-atom linkers and led us to discover more potent compounds with enzyme
inhibiting activities in the low nanomolar range and some were found to effectively inhibit
bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 µg/mL.
Keywords
Methicillin-resistant Staphylococcus aureus, Antimicrobial, Pyruvate kinase inhibitor
Structure–activity relationships
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1. Introduction
Infectious diseases caused by bacterial and eukaryotic pathogens continue to be a threat to
human health. In particular, many bacteria are developing antibiotic resistance. It is not
surprising that the effectiveness of the available antimicrobial drugs against bacteria such as
Staphylococcus aureus is diminishing at a rapid pace and resistant strains such as methicillin-
resistant Staphylococcus aureus (MRSA) are now escaping into the general population. Little
progress has been made in the past several decades to identify new antibacterial targets that could
be less prone to the development of resistance. A general principle of antibiotic target selection
has been to select pathogen-specific proteins without human homologs so as to avoid mechanism-
based toxicity. This has severely limited the options available.
Protein kinases constitute a large family of structurally related enzymes involved in a
wide range of signal transduction processes [1]. Pyruvate kinase (PK) is responsible for catalyzing
the final step of glycolysis, which involves the transfer of the phosphoryl group of
phosphoenolpyruvate (PEP) to ADP to produce pyruvate and ATP [2]. PKs exist as
homotetramers of identical subunits of 50-60 KDa depending on species, each consisting of three
to four domains: A, B, C, and N-terminal domains. While there are four mammalian PK
isoenzymes, M1, M2, L (liver), and R (red blood cell), with different primary structures, kinetic
properties, and tissue distributions to satisfy the metabolic requirements of various tissues, most
bacteria and lower eukaryotes have only one PK isoenzyme.
Thus, pyruvate kinase (PK) has been identified as a highly interconnected essential hub
protein in MRSA, with structural features distinct from the human homologs which makes it a
novel antimicrobial target [3]. In silico library screening, initially directed to putative binding sites
unique to MRSA PK and combined with enzyme assays, has allowed us to identify several active
MRSA PK inhibitors including compound 1 (Figure 1). This benzimidazole derivative was
highly selective for the bacterial enzyme compared to four human PK isoforms (M1, M2, R and
L). While non active on MRSA, a close analog 2 was found to inhibit both PK and bacteria
growth [4].
Figure 1. Structure and activities of molecules 1 and 2
X-ray crystal structures for 1 and 2 bound to MRSA282 PK were obtained [4] which
revealed that both compounds bind to a flat lipophilic pocket at the minor interfaces in the
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homotetrameric enzyme structure (between monomers A and B, and between monomers C and
D). This pocket was found to be modified and not accessible in the human PK isozymes.
In the X-ray structure, the ligands 1 and 2 exist in a planar conformation constrained with a
hydrogen bond between the hydrazide NH and the phenol oxygen. The 5-bromo atom is oriented
towards the interior of the site in a deep lipophilic pocket formed by Ala 358-A and Ile 351-A.
The benzimidazole/indole NH, hydrazide nitrogen and carbonyl all appear to form hydrogen
bonds to serine 362-A and B which are also interior to the site. The phenol and methyl/ethyl
group of 1/2 are oriented towards the outside (water side) of the binding site (Figure 2).
Figure 2. Scheme of the interactions between 1/2 and the MRSA PK
Based on these results, a new series of hydrazide inhibitors of MRSA PK was designed,
synthesized and biologically evaluated [5].
While we were investigating the hydrazide series, two natural marine compounds cis-3-4-
dihydrohamacanthin B (3) and bromodeoxytopsentin (4) were identified as potent inhibitors of
MRSA PK and with 166−600-fold selectivity for the bacterial PK over the four human PK
isoforms (M1, M2, R, and L). They also exhibited anti-bacterial activity with respective MIC of
12.5 µg/mL and 6.25 µg/mL (tested against S. aureus stains RN4220 and MRSA252) (Figure 3).
An X-ray crystal structure of 3 bound to MRSA PK showed that it binds to the same site as the
hydrazides compounds 1 and 2 [6].
Figure 3. Structure of cis-3-4-dihydrohamacanthin B (3) and bromodeoxytopsentin (4)
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These results suggested that some bis-indolic species might show some interesting biological
activities and based on these observations several other series of bis-indoles have been reported to
have potent activity [7].
A flat elongated structure was shown to be essential to fit within the pocket and the presence of
an appropriately situated aryl bromine atom to occupy the lipophilic pocket was found to be
important for potent inhibitory activity. The binding site of these compounds is far removed from
the enzyme active site and they have been shown to be non-competitive allosteric inhibitors and
are thought to inhibit the enzyme activity by rigidizing and elongating the enzyme tetramer
complex [3].
To better understand the scope of structures that can take advantage of this novel
mechanism, we explored other analogous compounds capable of occupying the binding site and
we present herein a detailed account of structure-activity relationships for enzyme inhibitory and
optimization of antibacterial activity for new series biaryl non-indolic, indolic and bis-indolic
derivatives with several distinct linkers.
2. Results
2.1. Chemistry
2.1.1. Preparation of the 3-atoms linkers
2.1.1.1. Chalcones
Chalcones 7a-t were synthesized by basic aldol condensation of commercially available aromatic
aldehydes 5 with methyl ketone derivatives 6 (Scheme 1 and Table 1 & 2). The base used was
either piperidine, benzyltrimethylammonium hydroxide (Triton B) or sodium hydroxide.
Scheme 1. Aldol condensation reaction allowing modifications on the two aromatic parts
A rigidified analog of 7j (9) was obtained by reaction between 2,3,4,9-tetrahydro-1H-
carbazol-1-one 8 with the corresponding aldehyde (i.e. 5-bromo-2-methoxybenzaldehyde)
(Scheme 2). Attempts to remove the methyl protection were unsuccessful.
Scheme 2. Synthesis of the rigidified analog 9
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Ar1
Ar2
IC50a (nM)
MICb (µg/mL)
Conditions
Product
7a
piperidine,
10% @ 1 µM
NDc
reflux, 16h (23%)
piperidine, RT,
24h (44%)
553
2% @ 10 µM
343
> 64
ND
7b
7c
7d
7e
7f
piperidine, RT,
4h (31%)
Br
Br
piperidine,
> 64
> 64
> 64
> 64
reflux, 20h (10%)
N
H
piperidine,
15% @ 1 µM
132
N
H
reflux, 20h (4%)
Br
Br
piperidine, RT,
24h (69%)
N
H
piperidine, RT,
5h (40%)
41% @ 1 µM
7g
N
H
Triton B, reflux,
24h (33%)
355
24
8
7h
7i
7j
2.0
1.0
Triton B, reflux,
14h (20%)
Triton B, reflux,
12h (55%)
> 64
Triton B, reflux,
48h (8%)
> 64
> 64
7k
7l
14
Triton B, reflux,
3 days (11%)
403
Table 1. Structure, preparation (yield) and biological evaluation of chalcones bearing one indole ring
a IC50 values are calculated from a triplicate 15 points titration. Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
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Ar1
Ar2
IC50a (nM)
Conditions
MICb
Product
7m
piperidine, µW,
180°C (30%)
1% @ 1 µM
NDc
piperidine, µW,
190°C (9%)
28% @ 1 µM
1778
ND
7n
7o
piperidine,
> 64
reflux, 48h (16%)
piperidine,
45% @ 1 µM
5
ND
> 64
> 64
ND
7p
7q
7r
7s
reflux, 48h (29%)
NaOH, RT, 5h
(48%)
Br
Br
NaOH, RT, 6h
(52%)
38% @ 1 µM
6% @ 1 µM
N
H
NaOH, RT, 4h
(44%)
N
H
Br
NaOH, RT, 1h30
(16%)
21% @ 1 µM
> 64
7t
N
H
Table 2. Structure, preparation (yield) and biological evaluation of chalcones bearing two indole rings
a IC50 values are calculated from a triplicate 15 points titration. Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
2.1.1.2. Other analogs
Compounds with interposed aryl groups that could mimic the chalcones linkers have also been
synthesized. Suzuki coupling between the 2,6-dibromopyridine 11 and the boronic acid of the
Boc-protected indole specie 10 afforded compound 12 that was further reacted, under the same
conditions, with the boronic acid 13 to give, after deprotection with TFA, the final product 14
(Scheme 3). Similarly, a double Suzuki coupling carried on 2-bromo-4-iodopyridine 15 with the
boronic acids 16 and 18 afforded the compound 19, mimetic of 7i (Scheme 4).
The carbohydrazide 20 was condensed with the benzoic acid 21 to afford the oxadiazole 22
(Scheme 5). The methyl was removed classically using boron tribromide to give 23.
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Scheme 3. Preparation of product 14
Scheme 4. Preparation of product 19
Scheme 5. Preparation of product 22 and 23
2.1.2. Preparation of the 2-atoms linkers
2.1.2.1. Secondary amides
Several families of aromatic secondary amides 26a-z were synthesized by reacting the primary
amines 24 with the carboxylic acid derivatives 25 under amide coupling conditions (Scheme 6
and Table 3 & 4). The carboxylic acid was either converted to an active specie or an acyl chloride
via PCl3 or SOCl2, either reacted with HBTU, HATU or EDC.HCl.
Scheme 6. Amide coupling reaction allowing modifications on the two aromatic parts
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Ar1
Ar2
IC50a (nM)
MICb (µg/mL)
Conditions
Product
neat, 200°C,
80 min (43%)
26a [8]
13% @ 1 µM
48% @ 1 µM
> 64
neat, 200°C,
80 min (43%)
> 64
26b
HBTU, RT,
24h (13%)
> 64
> 64
26c
26d
51
8
HATU, RT,
48h (2%)
neat, 200°C,
80 min (63%)
> 64
> 64
> 64
4.0
26e
26f
26g
26h
26i
26j
3
PCl3, reflux,
3h (39%)
65% @ 1 µM
PCl3, reflux,
3h (31%)
214
PCl3, reflux,
3h (9%)
43
HATU, RT,
24h (23%)
22% @ 1 µM
> 64
> 64
4.0
PCl3, reflux,
3h (61%)
8
PCl3, reflux,
4h (26%)
43% @ 0.1 µM
26k
26l
SOCl2, reflux,
12h (54%)
4
1.0
HBTU, RT,
24h (10%)
> 64
> 64
> 64
26m
26n
26o
11
23
HATU, RT,
48h (3%)
HBTU, RT,
48h (10%)
41% @ 1 µM
Table 3. Structure, preparation (yield) and biological evaluation of amides bearing a benzothiazole ring
a IC50 values are calculated from a triplicate 15 points titration. Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213
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Ar1
Ar2
IC50a (nM)
MICb (µg/mL)
4.0
Conditions
Product
26p
neat, 200°C,
80 min (41%)
11
HBTU, RT,
24h (9%)
> 64
> 64
NDc
> 64
> 64
> 64
> 64
> 64
26q
26r
3
HBTU, RT,
24h (7%)
2
28% @ 1 µM
41% @ 1 µM
12
HBTU, RT,
12h (11%)
26s
26t
HBTU, RT,
24h (33%)
HBTU, RT,
12h (15%)
26u
26v
26w
26x
HBTU, RT,
36h (31%)
10
HBTU, RT,
36h (26%)
116
EDC.HCl, RT,
48h (31%)
43% @ 0.1 µM
Table 4. Structure, preparation (yield) and biological evaluation of amides bearing a benzimidazole, indole
or benzothiophene ring
a IC50 values are calculated from a triplicate 15 points titration. Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
2.1.2.1. Other analogs
Some amide-related and 2-atoms linkers compounds have also been synthesized. An analog of
26e (29) was obtained by reacting the amine 27 with the carboxylic acid 28 converted into the
acyl chloride by reaction with PCl3 (Scheme 7).
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Scheme 7. Preparation of product 29
Reaction of the amine 30 with the anhydride 31 afforded the dione 32 (Scheme 8).
Scheme 8. Preparation of product 32
The Schiff base 34 was synthesized by condensation with the aldehyde 33 (Scheme 9).
OH
Br
CHO
a
N
S
N
S
+
NH2
N
Br
HO
Br
Br
33
30
34
Reagents and conditions: (a) piperidine, EtOH, reflux, 50 min (73%)
Scheme 9. Preparation of product 34
Reductive amination undergone on an isomer of 34 gave the amine 36 (Scheme 10).
HO
Br
OH
a
N
S
Br
CHO
N
S
+
NH2
NH
Br
Br
30
36
35
Reagents and conditions: (a) (1) piperidine, EtOH, reflux, 1h, (2) NaBH4, EtOH, 10 min (63%)
Scheme 10. Preparation of product 36
2.1.3. Preparation of the 0-atom linkers
Herein, we have investigated the direct attachment of the bromophenyl moiety to the indole ring.
To do so, we have used specific C-H activation conditions [9]. Thus, we have synthesized the
three derivatives 39, 41 and 43 (Scheme 11-13).
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Scheme 11. Preparation of product 39
Scheme 12. Preparation of product 41
Scheme 13. Preparation of product 43
2.2. Biological results and discussion
2.2.1. Biological evaluation of the 3-atoms linkers (Table 1-2 & 5)
For purpose of clarity we will discuss the SAR derived for the in vitro enzyme inhibition first and
separately from the cellular antibacterial activity.
While the closest chalcone analog to 2 (7a) shows only some very weak activity at 1 µM, one
isomer with bromine in meta position to the hydroxyl (7b) exhibits a moderate potency for
inhibition of the PK (Table 1). Introduction of a bromine in the 6- position of the indole moiety
gives a gain in activity and we can observe the same pattern as above with the meta compound 7f
showing the best inhibition. It is interesting to note that the absence of bromine on the phenol
part (7d) is not detrimental for the activity towards the enzyme.
Unlike 7a, the reverse linker compound 7h showed a significant inhibition of the PK. Simple
modelling energy calculations (MM2, ChemDraw) suggested that the structure of 7a is non-
planar with a strong hydrogen bond between the phenolic hydroxyl and the ketone, whereas the
scaffold of 7h is totally flat, which may explain the difference in activities.
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As above, introduction of a bromine in the 6- position of the indole is favorable and provides a
family of nanomolar inhibitors (7i to 7l) with the best activities obtained when the phenolic
hydroxyl is capped with a methyl group (7j) and, as previously, with the meta arrangement (7k).
Among the bis-indolic chalcones (Table 2), 7m and 7n, bis-functionalized in the 3- position of the
indole, could be considered as mimetics of the potent inhibitors 3 and especially 4. They turned
out to be totally inactive. Monofunctionalization in the 2- position of the indole ring brings some
activity (7o, 7p) while the best result is obtained with the linker attached to the two rings in the 2-
position (7q). Introduction of a bromine on both indoles appears to be detrimental for the
inhibition of the PK (7s, 7t) which suggests that such compounds are too long to fit in the PK
pocket, a conclusion in accordance with the results obtained in the bis-indolic series.7
Among the other compounds (Table 5), the rigidified but non-planar chalcone 9 did not show the
same range of activity as its linear analog, 7j. The derivative 19 exhibits an average inhibition at
0.1 µM, confirming that it could mimic the active compound 7i. The two oxadiazoles derivatives
22 and 23 were inactive.
Considering now the antimicrobial activity, two compounds 7h and 7i of the first family
of chalcones (Table 1) have significant MICs, 7i showing the best activity. The SAR is tight as the
analogs 7j and 7h, active on the enzyme, do not inhibit bacterial growth, demonstrating that the
phenolic hydroxyl and the para arrangement between the latter and the bromine are necessary for
cellular activity. None of the bis-indolic chalcones were found to give significant MICs for MRSA
(Table 2), as was the case for the chalcone mimetics (Table 5).
Overall, the best results were obtained with a flat structure and when a bromine was
attached to both the phenol and indole moieties, probably to better fit the two lipophilic pockets
identified with the X-ray. The phenolic oxygen atom is presumably involved as a H-bond
acceptor, but the OH group is essential for the cellular activity. Replacement of the phenol moiety
with an indole is possible, but limited as such bis-indolic compounds do not exert antibacterial
growth inhibition (Table 2).
a
Product
IC50
MICb (µg/mL)
NDc
22% @ 1 µM
6% @ 1 µM
41% @ 0.1 µM
19% @ 1 µM
8% @ 1 µM
9
> 64
14
19
22
23
> 64
> 64
> 64
Table 5. Biological evaluation of chalcone mimetics
a Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
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2.2.2. Biological evaluation of the 2-atoms linkers (Table 3-4 & 6)
As above, we will discuss the SAR derived for the in vitro enzyme inhibition first and separately
from the cellular antibacterial activity.
The closest benzothiazole analog to 2 (26a) shows only a weak PK inhibition (Table 3).
Introduction of a bromine atom in the 5- position of the benzothiazole moiety (26b) results in a
small gain in activity while its introduction in the 6- position (26e) afforded a very potent
inhibitor. This result led us to synthesize several analogs bearing the same 6-bromobenzothiazole
ring. Abstraction of the bromine on the phenol moiety results in a significant drop in activity
(26f). When a bromine atom is attached to the phenol, the para position is preferred to ortho and
meta (26e vs 26h & 26g), showing a significant difference between the amides and the chalcones.
In the ortho position, replacement of the bromine atom with a chlorine results in a loss of potency
(26k), as is the case when the hydroxyl group is capped with a methyl (26i). Simple modelling
energy calculations (MM2, ChemDraw) suggested that the structure of 26i is non-planar, whereas
the scaffold of 26h is totally flat with a strong hydrogen bond between the phenolic hydroxyl and
the carbonyl, which may explain the difference in activities. Capping the phenolic hydroxyl in
para to the bromine with a methyl group (26j vs 26e) did not provoke a such loss of activity; the
latter was found to have a flat structure (MM2, ChemDraw). It is interesting to note that the
introduction of two atoms of bromine in ortho and para positions (26l) afforded a compound
equipotent to the one with only one bromine in para position (26e). We next investigated the
replacement of the phenol moiety with an indole ring. This replacement provides good inhibition
of the PK, even with the bromine in 5- position of the benzothiazole (26c & 26d), the best result
being obtained when a bromine atom is also introduced in 5- position of the indole (26d). The
same range of activities was observed with the bromine in 6- position of the benzothiazole (26m
& 26n), capping the indolic nitrogen with a methyl group resulting in a dramatic loss of activity
(26o vs 26m).
We have also explored the replacement of the benzothiazole moiety and synthetized analogs
bearing a benzimidazole, indole or benzothiophene ring (Table 4). The bromobenzimidazole ring
was attached, through the amide linker, either to the phenol with a bromine in para position
(26p), either to an indole ring with a bromine in 5- (26q) or 6- position (26r). Each of three
compounds exhibit nanomolar activities. We have used a reverse linker for the analogs with an
indole or a benzothiophene ring as they are linked to the carbonyl. While activities are poor when
no bromine is attached to the aforementioned ring (26s, 26t), nanomolar inhibitions are obtained
with a bromine introduced in the 6- position (26u to 26x), with a preference for the indole (26v vs
26x). On the phenol moiety, para substitution appears to be preferred to ortho (26u vs 26w).
Capping the amide NH of the linker with a methyl provokes an important loss of potency (29 vs
26e) (Table 6). This result could be explained by modelling energy calculations (MM2,
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ChemDraw) which indicated that a non-flat structure for 29 is preferred. Lesser potency may also
be due to the lack of ability to form a H-bond between the compound and the binding site. The
other amide-related and two atoms linker compounds did not exhibit any strong activities.
Considering now the antimicrobial activity, three compounds 26h, 26k and 26l of the first
benzothiazole family have significant MICs, with 26l showing the best activity (Table 3). It
appears that the halogen atom in ortho position to the phenolic hydroxyl brings the anti-MRSA
activity, the one in the para position bringing the potency towards the PK. None of the
benzothiazole-indole derivatives, active on the enzyme, inhibit bacterial growth.
Among the second family, one benzimidazole derivative shows an antibacterial activity (26p)
showing that an additional H-bond donor is beneficial for the cellular activity (Table 4). In that
case, the halogen atom is in para position to the hydroxyl, a significant difference with the
benzothiazole series. None of the series of indole or benzothiophene tested showed measurable
MIC values, as it was the case for the amide-related compounds. Only 34 showed a weak activity
(Table 6).
As above, with the chalcone series, the best results were obtained with a flat structure and
when a bromine was attached to both the phenol and indole moieties. To interact with the PK,
the only mandatory H-bond donor appears to be the NH of the amide, the phenolic O being
presumably involved as a H-bond acceptor. The OH group seems to be mandatory for the cellular
activity, however. Replacement of the phenol part with an indole, and even a 5- or 6-
bromoindole, is possible, but such compounds do not exert antibacterial growth inhibition.
a
Product
IC50
MICb (µg/mL)
5% @ 1 µM
34% @ 1 µM
41% @ 1 µM
17% @ 1 µM
NDc
> 64
64
29
32
34
36
ND
Table 6. Biological evaluation of amide-related compounds
a Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
2.2.3. Biological evaluation of the 0-atom linkers
Linking the bromophenyl moiety to the 2- position of the indole ring to generate a derivative of
26v with abstraction of the amide linker did not afford an active product (39) while
functionalization in the 3- position of the indole brings inhibition of both PK and MRSA growth,
whether the bromine is attached to the 5- or 6- position of the indole (41 & 43, resp.). As in the
case of 2- and 3-atoms linkers, the best result for the inhibition of PK was obtained with a
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bromine in the 6- position (43). However, due to the reduced dimensions of these compounds,
these results indicate that interaction with only one of the two lipophilic pockets is sufficient to
impart antibacterial and PK inhibitory potency.
a
Product
IC50
MICb (µg/mL)
21% @ 1 µM
20% @ 0.1 µM
102
NDc
1.0
39
41
43
2.0
Table 7. Biological evaluation of the 0-atom linkers
a Alternatively, the % inhibition at a specific concentration is presented.
b Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213; c Non Determined
3. Conclusion
The inhibitory potency of the 55 final products against the MRSA PK was evaluated. For the best
inhibitors, evaluation of their antibacterial properties was also investigated. For each of the three
families of compounds (3-atoms, 2-atoms and 0-atom linkers), we have identified, at least, one
product with better activities than the original compound, thus validating our approach in the
replacement of the hydrazide linker. As with the previous series, hydrazide [4] and bis-indolic [7],
there was, overall, a poor correlation between the in vitro enzyme inhibitory activity and anti-
bacterial activity against S. aureus ATCC 29213. Earlier studies indicated that this lack of
correlation is due, at least in part, to selective active efflux from the bacteria as shown when the
use of a known efflux inhibitor, verapamil, produced a pronounced enhancement of MIC values
for selected compounds in the bis-indolic series [7].
The lead compounds with best MIC values are currently being evaluated to determine in vivo
absorption and tissue exposure in preparation for evaluation in in vivo animal infection models.
4. Experimental section
4.1. Chemistry
4.1.1. General
1H and 13C NMR spectra were recorded with either Bruker Avance II™ 600 MHz, Bruker
Avance III™ 500 MHz or Bruker Avance III™ 400 MHz. The high-resolution mass spectra were
recorded in positive ion-mode with an ESI ion source on an Agilent™ Time-of-Flight LC/MS
mass spectrometer. Analytical thin layer chromatography (TLC) was performed on aluminum
plates pre-coated with silica gel 60F-254 as the absorbent. The developed plates were air-dried,
exposed to UV light and/or dipped in KMnO4 solution and heated. Column chromatography
16

ACCEPTED MANUSCRIPT
was performed with silica gel 60 (230–400 mesh). Purity of >95% for all final compounds
was confirmed by analytical reverse-phase HPLC utilizing a Dikma Technologies™ Inspire C18
reverse-phase analytical column (4.6 x 150 mm).
4.1.2. Synthesis
Specific procedures are described below. All the experimental procedures and detailed attribution
of the different 1H and 13C signals are available in the Supplementary material section.
4.1.2.1. (2E)-1-(5-bromo-2-hydroxyphenyl)-3-(1H-indol-2-yl)prop-2-en-1-one (7a)
To a mixture of 1H-indole-2-carbaldehyde (290 mg, 2.0 mmol, 1.0 eq.) and 1-(5-bromo-2-
hydroxyphenyl)ethanone (430 mg, 2.0 mmol, 1.0 eq.) in ethanol (10 mL) was added piperidine (1
mL). The mixture was refluxed during 16 hours and then diluted with ethyl acetate after cooling
to room temperature. The organic layer was washed with a 1M aqueous hydrochloric acid
solution and water, dried over Na2SO4 and then evaporated under reduced pressure. The residue
was purified by silica gel flash-column chromatography (eluent: heptane/EtOAc, 90/10 to
80/20) to afford, after trituration with diethyl ether, 7a as a red solid (160 mg, 23 %). 1H
NMR (DMSO-d6, 600 MHz): δ (ppm): 7.02 (1H, d, J= 8.9 Hz), 7.07 (1H, tm, J= 7.8 Hz), 7.14
(1H, bs), 7.27 (1H, tm, J= 7.8 Hz), 7.47 (1H, d, J= 7.8 Hz), 7.63 (1H, d, J= 8.6 Hz), 7.72 (1H,
dd, J= 8.9 Hz, J= 2.4 Hz), 7.86 (1H, d, J= 15.4 Hz), 7.89 (1H, d, J= 15.4 Hz), 8.21 (1H, d, J
= 2.4 Hz), 11.75 (1H, s). 13C NMR (DMSO-d6, 150 MHz): δ (ppm): 110.2, 111.2, 111.6, 118.7,
120.1, 120.3, 121.5, 122.8, 124.9, 128.0, 131.9, 134.4, 135.5, 138.2, 138.6, 160.6, 191.8. HRMS
calcd for C17H1379BrNO2: m/z = 342.0124, found: 342.0160.
4.1.2.2. (2E)-3-(5-bromo-2-hydroxyphenyl)-1-(1H-indol-2-yl)prop-2-en-1-one (7h)
To a mixture of 1-(1H-indol-2-yl)ethanone (114 mg, 0.7 mmol, 1.0 eq.) and 5-bromo-2-
hydroxybenzaldehyde (144 mg, 0.7 mmol, 1.0 eq.) in ethanol (3 mL) was added a solution of
benzyl trimethylammonium hydroxide in water (40 %, 0.5 mL, 1.3 mmol, 1.8 eq.). The mixture
was refluxed during 24 hours and then diluted with ethyl acetate after cooling to room
temperature. The organic layer was washed with a 1M aqueous hydrochloric acid solution and
water, dried over Na2SO4 and then evaporated under reduced pressure. The residue was purified
by silica gel flash-column chromatography (eluent: heptane/EtOAc, 90/10 to 30/70) to afford,
after trituration with diethyl ether, 7h as a yellow solid (80 mg, 33 %). 1H NMR (DMSO-d6, 400
MHz): δ (ppm): 6.91 (1H, d, J= 8.8 Hz), 7.11 (1H, tm, J= 7.8 Hz), 7.31 (1H, tm, J= 7.8 Hz),
7.42 (1H, dd, J= 8.6 Hz, J= 2.5 Hz), 7.48 (1H, d, J= 7.8 Hz), 7.73 (1H, d, J= 7.8 Hz), 7.80 (1H,
m), 7.93 (1H, d, J= 15.8 Hz), 8.01 (1H, d, J= 15.8 Hz), 8.19 (1H, d, J= 2.5 Hz), 10.57 (1H, s),
11.88 (1H, s). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 110.5, 111.4, 113.2, 118.8, 120.8, 123.0,
17

ACCEPTED MANUSCRIPT
123.2, 124.2, 126.1, 127.6, 130.5, 134.5, 135.8, 137.5, 138.7, 156.7, 181.2. HRMS calcd for
C17H1379BrNO2: m/z = 342.0124, found: 342.0109.
4.1.2.3. (2E)-3-(5-bromo-1H-indol-3-yl)-1-(1H-indol-3-yl)prop-2-en-1-one (7m)
To a mixture of 5-bromo-1H-indole-3-carbaldehyde (112 mg, 0.5 mmol, 1.0 eq.) and 1-(1H-indol-
3-yl)ethanone (80 mg, 0.5 mmol, 1.0 eq.) in ethylene glycol (0.8 mL) was added piperidine (0.08
mL). The reaction mixture was irradiated with microwaves for 2 hours at 180°C. The mixture
was extracted with ethyl acetate and then, the organic layer was washed with a saturated
ammonium chloride aqueous solution and dried over Na2SO4. The solvent was removed under
reduced pressure and the residue purified by silica gel flash-column chromatography (eluent:
CH2Cl2/EtOH, 99/1 to 97/3) to afford, after trituration with diethyl ether and heptane, 7m as a
dark yellow powder (55 mg, 30 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.21 (1H, tm, J=
7.6 Hz), 7.24 (1H, tm, J= 7.6 Hz), 7.35 (1H, dd, J= 8.6 Hz, J= 1.8 Hz), 7.46 (1H, d, J= 8.6 Hz),
7.51 (1H, d, J = 7.6 Hz), 7.57 (1H, d, J= 15.7 Hz), 7.90 (1H, d, J= 15.7 Hz), 8.07 (1H, m), 8.29
(1H, d, J= 1.8 Hz), 8.37 (1H, d, J= 7.6 Hz), 8.73 (1H, m), 11.91 (1H, s), 11.96 (1H, s). 13C NMR
(DMSO-d6, 100 MHz): δ (ppm): 112.5, 112.8, 113.9, 114.6, 118.1, 119.7, 122.0, 122.3, 122.9,
123.3, 125.5, 126.6, 127.5, 132.5, 133.9, 134.3, 136.5, 137.3, 184.7. HRMS calcd for
C19H1479BrN2O: m/z = 365.0284, found: 365.0261.
4.1.2.4. (2E)-3-(1H-indol-2-yl)-1-(6-bromo-1H-indol-2-yl)prop-2-en-1-one (7q)
To a mixture of 1H-indole-2-carbaldehyde (67 mg, 0.46 mmol, 1.0 eq.) and 1-(6-bromo-1H-indol-
2-yl)ethanone (110 mg, 0.46 mmol, 1.0 eq.) in ethanol (3 mL) and water (1 mL) was added
sodium hydroxide (15 mg, 0.37 mmol, 0.8 eq.). The reaction was stirred at room temperature
during 5 hours and quenched with a 1M aqueous hydrochloric acid solution. The mixture was
extracted with ethyl acetate and then, the organic layer was washed with a saturated ammonium
chloride aqueous solution and dried over Na2SO4. The solvent was removed under reduced
pressure and the residue purified by silica gel flash-column chromatography (eluent:
heptane/EtOAc, 95/5 to 65/35) to afford, after trituration with methyl tert-butyl ether, 7q as an
orange powder (80 mg, 48 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.06 (1H, s), 7.06 (1H,
m), 7.25 (1H, tm, J= 7.8 Hz), 7.26 (1H, dd, J= 8.6 Hz, J= 1.8 Hz), 7.46 (1H, d, J= 7.8 Hz), 7.55
(1H, bs), 7.61 (1H, d, J= 7.8 Hz), 7.65 (1H, bs), 7.77 (1H, d, J= 8.6 Hz), 7.78 (1H, d, J=
15.6 Hz), 7.86 (1H, d, J= 15.6 Hz), 11.70 (1H, s), 12.07 (1H, s). 13C NMR (DMSO-d6, 100 MHz):
δ (ppm): 109.3, 109.9, 112.0, 115.6, 118.9, 120.4, 120.6, 121.7, 123.9, 124.8, 125.1, 126.5, 128.6,
132.9, 135.1, 138.3, 138.6, 139.2, 180.7. HRMS calcd for C19H1279BrN2O: m/z = 363.0138,
found: 363.0080.
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ACCEPTED MANUSCRIPT
4.1.2.5. tert-butyl 2-(6-bromopyridin-2-yl)-1H-indole-1-carboxylate (12)
To a solution of [1-(tert-butoxycarbonyl)-1H-indol-2-yl]boronic acid (4.0 g, 0.015 mol, 1.0 eq.) in
anhydrous and degassed THF (30 mL) were added, under an argon atmosphere, 2,6-
dibromopyridine (4.4 g, 0.018 mol, 1.2 eq.), 2M aqueous potassium carbonate solution (30 mL)
and Pd(PPh3)4 (867 mg, 0.75 mmol, 0.05 eq.). The reaction was stirred at reflux temperature for 6
hours and then, quenched with brine after cooling to room temperature. The mixture was
extracted with ethyl acetate and the organic layer was dried over Na2SO4 and evaporated under
reduced pressure. The residue was purified by silica gel flash-column chromatography (eluent:
heptane/CH2Cl2, 80/20 to 40/60) to afford 12 as a white solid (3.0 g, 54 %). 1H NMR (DMSO-
d6, 400 MHz): δ (ppm): 1.33 (9H, s), 7.06 (1H, s), 7.30 (1H, t, J= 7.2 Hz), 7.41 (1H, t, J=
7.2 Hz), 7.67 (1H, m), 7.69 (1H, m), 7.77 (1H, d, J= 7.6 Hz), 7.88 (1H, t, J= 7.6 Hz), 8.07 (1H,
d, J= 7.2 Hz). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 27.0, 83.6, 111.6, 114.0, 121.5, 122.0,
123.1, 125.5, 126.7, 128.1, 137.1, 137.2, 140.1, 140.4, 149.2, 153.0. HRMS calcd for
C18H1779BrN2NaO2: m/z = 395.0366, found: 395.0327.
4.1.2.6. 2-[6-(5-bromo-2-methoxyphenyl)pyridin-2-yl]-1H-indole (14)
To a solution of 12 (0.70 g, 1.88 mmol, 1.2 eq.) in anhydrous and degassed THF (20 mL) were
added, under an argon atmosphere, (5-bromo-2-methoxyphenyl)boronic acid (0.36 g, 1.57 mmol,
1.0 eq.), 2M aqueous potassium carbonate solution (10 mL) and Pd(PPh3)4 (91 mg, 0.079 mmol,
0.05 eq.). The reaction was stirred at reflux temperature for 10 hours and then, quenched with
brine after cooling to room temperature. The mixture was extracted with ethyl acetate and the
organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude was
taken up in a mixture of dichloromethane (5 mL) and TFA (5 mL), and then the reaction mixture
was stirred at room temperature for 8 hours and quenched with a 1M aqueous sodium hydroxide
solution. The mixture was extracted with ethyl acetate and the organic layer was washed with
water, dried over Na2SO4 and evaporated. The residue was purified by silica gel flash-column
chromatography (eluent: heptane/EtOAc, 90/10 to 60/40) to afford 14 as a white solid (0.5 g, 84
%). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 3.88 (3H, s), 7.03 (1H, tm, J= 7.2 Hz), 7.15 (1H,
tm, J= 7.2 Hz), 7.18 (1H, d, J= 9.0 Hz), 7.20 (1H, m), 7.53 (1H, d, J= 7.2 Hz), 7.60 (1H, d, J=
7.2 Hz), 7.62 (1H, dd, J= 9.0 Hz, J= 2.5 Hz), 7.79 (1H, dd, J= 7.6 Hz, J= 1.2 Hz), 7.90 (1H, t,
J= 7.6 Hz), 7.95 (1H, dd, J= 7.6 Hz, J= 1.2 Hz), 8.15 (1H, d, J= 2.5 Hz), 11.60 (1H, s). 13C
NMR (DMSO-d6, 100 MHz): δ (ppm): 56.0, 100.8, 112.1, 112.5, 114.3, 118.6, 119.5, 120.6,
122.3, 123.2, 128.4, 130.1, 132.5, 133.1, 136.9, 137.1, 137.2, 150.3, 153.1, 156.2. HRMS calcd for
C20H1679BrN2O: m/z = 379.0441, found: 379.0437.
4.1.2.7. 4-bromo-2-[2-(6-bromo-1H-indol-2-yl)pyridin-4-yl]phenol (19)
19

ACCEPTED MANUSCRIPT
To a mixture of 2-bromo-4-iodopyridine (1.0 g, 3.45 mmol, 1.0 eq.), (5-bromo-2-
hydroxyphenyl)boronic acid (748 mg, 3.45 mmol, 1.0 eq.), K2CO3 (1.44 g, 10.35 mol, 3.0 eq.) in
degassed toluene/EtOH/H2O (10/5/5 mL) was added, under an argon atmosphere,
PdCl2(PPh3)2 (246 mg, 0.35 mmol, 0.1 eq.). The reaction was stirred at 85°C for 20 hours and
then, quenched with a saturated aqueous ammonium chloride solution after cooling to room
temperature. The mixture was extracted with ethyl acetate and the organic layer was dried over
Na2SO4 and evaporated under reduced pressure. Unreacted pyridine was removed by silica gel
flash-column chromatography (eluent: heptane/EtOAc, 90/10 to 70/30). To the pure dibromo
compound thus obtained and taken up in degassed toluene/EtOH/H2O (10/5/5 mL) were added
[6-bromo-1-(tert-butoxycarbonyl)-1H-indol-2-yl]boronic acid (599 mg, 1.76 mmol), K2CO3 (735
mg, 5.28 mol) and PdCl2(PPh3)2 (126 mg, 0.18 mmol, 0.1 eq.). The reaction was stirred at 85°C
for 20 hours and then, quenched with a saturated aqueous ammonium chloride solution after
cooling to room temperature. The mixture was extracted with ethyl acetate and the organic layer
was dried over Na2SO4 and evaporated under reduced pressure. The crude was purified by silica
gel chromatography chromatography (eluent: heptane/EtOAc, 90/10 to 60/40) and the Boc-
protected compound was taken up in a mixture of dichloromethane (10 mL) and TFA (5 mL).
The reaction mixture was stirred at room temperature for 8 hours and quenched with a 1M
aqueous sodium hydroxide solution. The mixture was extracted with ethyl acetate and the
organic layer was dried over Na2SO4 and filtered through a silica gel pad to afford, after
evaporation and trituration with methyl tert-butyl ether, 19 as a dark yellow solid (220 mg, 45 %).
1H NMR (DMSO-d6, 400 MHz):
δ (ppm): 6.99 (1H, d, J= 8.6 Hz), 7.15 (1H, dd, J= 8.4 Hz, J= 1.8 Hz), 7.26 (1H, m), 7.46 (1H,
dd, J= 8.6 Hz, J= 2.3 Hz), 7.55 (1H, d, J= 8.4 Hz), 7.57 (1H, dd, J= 5.3 Hz, J= 1.6 Hz), 7.64
(1H, m), 7.65 (1H, d, J= 2.3 Hz), 8.16 (1H, m), 8.65 (1H, d, J= 5.3 Hz), 10.28 (1H, s), 11.86
(1H, s). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 101.3, 111.2, 114.9, 115.4, 118.9, 120.3,
122.8, 122.9, 123.2, 127.5, 127.9, 132.7, 133.1, 138.4, 138.6, 146.0, 149.4, 150.5, 154.7. HRMS
calcd for C19H1379Br81Br N2O: m/z = 444.9374, found: 444.9385.
4.1.2.8. 2-(5-bromo-2-methoxyphenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (22)
A mixture of 1H-indole-2-carbohydrazide (175 mg, 1.0 mmol, 1.0 eq.) and 5-bromo-2-
methoxybenzoic acid (347 mg, 1.5 mmol, 1.5 eq.) in POCl3 (3 mL) was stirred at 100°C during
10 hours. The mixture was poured into ice cold water and extracted with ethyl acetate. The
organic layer was washed with 1M hydrochloric acid and 1M sodium hydroxide aqueous
solutions, dried over Na2SO4 and then evaporated under reduced pressure. The residue was
purified by by silica gel flash-column chromatography (eluent: heptane/EtOAc, 80/20 to 20/80)
to afford 22 as a light brown solid (70 mg, 19 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 3.97
20

ACCEPTED MANUSCRIPT
(s, 3H), 7.13 (tm, J= 7.0 Hz, 1H), 7.29 (tm, J= 7.0 Hz, 1H), 7.30 (d, J= 9.0 Hz, 1H), 7.33 (m, 1H),
7.52 (d, J= 7.0 Hz, 1H), 7.70 (d, J= 7.0 Hz, 1H), 7.82 (dd, J= 9.0 Hz, J= 2.5 Hz, 1H), 8.14 (d,
J= 2.5 Hz, 1H), 12.31 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 57.0, 105.7, 112.3,
112.8, 114.6, 115.7, 120.9, 121.4, 122.0, 124.8, 127.8, 132.5, 136.3, 138.3, 157.3, 159.8, 161.2.
HRMS calcd for C17H1379BrN3O2: m/z = 370.0186, found: 370.0220.
4.1.2.9. 2-(5-bromo-2-hydroxyphenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (23)
To a mixture of 22 (80 mg, 0.22 mmol, 1.0 eq.) in anhydrous dichloromethane (5 mL) cooled
to -78°C (dry ice/ethanol bath) was added, under an argon atmosphere, a 1M boron tribromide
solution in dichloromethane (0.65 mL, 0.65 mmol, 3.0 eq.). The reaction mixture was stirred at
ambient temperature for 14 hours and then quenched with ethanol. The mixture was extracted
with ethyl acetate and the organic layer was washed with water, dried over Na2SO4 and
evaporated under reduced pressure. The residue was triturated with diethyl ether to afford 23 as a
light purple solid (70 mg, 89 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.10 (d, J= 9.0 Hz,
1H), 7.13 (tm, J= 7.0 Hz, 1H), 7.29 (tm, J= 7.0 Hz, 1H), 7.34 (m, 1H), 7.52 (d, J= 7.0 Hz, 1H),
7.65 (dd, J= 9.0 Hz, J= 2.5 Hz, 1H), 7.71 (d, J= 7.0 Hz, 1H), 8.08 (d, J= 2.5 Hz, 1H), 10.65 (s,
1H), 12.32 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 105.9, 110.9, 112.2, 112.8, 119.9,
120.9, 121.3, 122.0, 124.8, 127.8, 131.3, 136.2, 138.3, 156.1, 159.4, 161.9. HRMS calcd for
C16H1179BrN3O2: m/z = 356.0029, found: 356.0031.
4.1.2.10. 5-bromo-N-(5-bromo-1,3-benzothiazol-2-yl)-2-hydroxybenzamide (26b)
A mixture of 5-bromo-1,3-benzothiazol-2-amine (200 mg, 0.87 mmol, 1.0 eq.) and 4-nitrophenyl
5-bromo-2-hydroxybenzoate (325 mg, 0.96 mmol, 1.1 eq.) was melted at 200°C for 80 minutes.
The solid was taken up in ethanol and heated at reflux temperature during 20 minutes. The solid
product was filtered and then, triturated with diethyl ether to afford 26b as an off-white solid (160
mg, 43 %). 1H NMR (DMSO-d6, 600 MHz): δ (ppm): 7.03 (1H, d, J= 8.7 Hz), 7.52 (1H, dd, J=
8.5 Hz, J= 1.4 Hz), 7.64 (1H, dd, J= 8.7 Hz, J= 2.3 Hz), 7.94 (1H, bs), 8.00 (1H, d, J= 8.5 Hz),
8.04 (1H, d, J= 2.3 Hz), 12.25 (1H, bs). 13C NMR (DMSO-d6, 150 MHz): δ (ppm): 111.0, 119.7,
119.9, 120.1, 124.5, 127.1, 132.8, 137.3. HRMS calcd for C14H979Br81Br N2O2S: m/z = 428.8726,
found: 428.8774.
4.1.2.11. N-(5-bromo-1,3-benzothiazol-2-yl)-1H-indole-2-carboxamide (26c)
To a mixture of 1H-indole-2-carboxylic acid (84 mg, 0.52 mmol, 1.0 eq.) and HBTU (237 mg,
0.62 mmol, 1.0 eq.) in NMP (6 mL) were added 5-bromo-1,3-benzothiazol-2-amine (120 mg, 0.52
mmol, 1.0 eq.) and DIPEA (180 µL, 1.04 mmol, 2.0 eq.). The reaction mixture was stirred at
room temperature during 24 hours, quenched with water and extracted with ethyl acetate. The
21

ACCEPTED MANUSCRIPT
organic layer was washed with 1M hydrochloric acid and 1M sodium hydroxide aqueous
solutions, and then was dried over Na2SO4. The solvent was evaporated under reduced pressure
and the residue purified by silica gel flash-column chromatography (eluent: heptane/EtOAc,
90/10 to 70/30) to afford 26c as a white solid (25 mg, 13 %). 1H NMR (DMSO-d6, 500 MHz): δ
(ppm): 7.11 (1H, t, J= 7.6 Hz), 7.29 (1H, t, J= 7.6 Hz), 7.50 (1H, d, J= 7.6 Hz), 7.51 (1H, dd, J=
8.5 Hz, J= 1.8 Hz), 7.71 (1H, d, J= 7.6 Hz), 7.77 (1H, s), 8.00 (1H, bs), 8.02 (1H, d, J= 8.5 Hz),
12.03 (1H, s), 13.11 (1H, s). 13C NMR (DMSO-d6, 150 MHz): δ (ppm): 107.3, 113.1, 119.4,
120.8, 122.9, 123.2, 124.1, 125.4, 126.7, 127.4, 129.2, 131.4, 138.1, 150.7, 160.4, 160.6. HRMS
calcd for C16H1179BrN3OS: m/z = 371.9806, found: 371.9839.
4.1.2.12. 5-bromo-N-(5-bromo-1,3-benzothiazol-2-yl)-1H-indole-2-carboxamide (26d)
To a mixture of 5-bromo-1H-indole-2-carboxylic acid (264 mg, 1.10 mmol, 1.0 eq.) and HATU
(500 mg, 1.32 mmol, 1.2 eq.) in NMP (30 mL) were added 5-bromo-1,3-benzothiazol-2-amine
(250 mg, 1.10 mmol, 1.0 eq.) and DIPEA (0.38 mL, 2.18 mmol, 2.0 eq.). The reaction mixture
was stirred at room temperature during 48 hours, then quenched with water and extracted with
ethyl acetate. The organic layer was dried over Na2SO4, the solvent evaporated under reduced
pressure and the residue purified by silica gel flash-column chromatography (eluent:
heptane/EtOAc, 90/10 to 70/30) to afford 26d as a pale green solid (8 mg, 2 %). 1H
NMR (DMSO-d6, 400 MHz): δ (ppm): 7.39 (1H, dd, J= 8.8 Hz, J= 1.4 Hz), 7.45 (1H, d, J=
8.8 Hz), 7.29 (1H, t, J= 7.6 Hz), 7.51 (1H, dd, J= 8.8 Hz, J= 1.4 Hz), 7.72 (1H, s), 7.95 (1H, s),
7.99 (1H, s), 8.01 (1H, d, J= 8.8 Hz), 12.21 (1H, s), 13.17 (1H, s). 13C NMR (DMSO-d6, 100
MHz): δ (ppm): 106.5, 113.2, 115.1, 119.4, 124.1, 124.9, 126.7, 127.9, 129.1, 136.6. HRMS calcd
for C16H879Br81BrN3OS: m/z = 449.8734, found: 449.8657.
4.1.2.13. N-(6-bromo-1,3-benzothiazol-2-yl)-2-hydroxybenzamide (26f)
A mixture of salicylic acid (72 mg, 0.52 mmol, 1.0 eq.) and PCl3 (23 µL, 0.26 mmol, 0.5 eq.) in
chlorobenzene was heated at reflux temperature during 15 minutes. After cooling at room
temperature, 6-bromo-1,3-benzothiazol-2-amine (120 mg, 0.52 mmol, 1.0 eq.) was added and the
reaction was stirred at reflux temperature during 3 hours. The mixture was diluted with ethanol,
filtered and the solid residue was triturated with methyl tert-butylether to afford 26f as an off-
white solid (70 mg, 39 %). 1H NMR (DMSO-d6, 600 MHz): δ (ppm): 7.03 (1H, t, J= 7.6 Hz), 7.08
(1H, d, J= 7.6 Hz), 7.52 (1H, t, J= 7.6 Hz), 7.62 (1H, dm, J= 8.3 Hz), 7.70 (1H, bm), 8.01 (1H,
dm, J= 7.6 Hz), 8.31 (1H, bs), 11.79 (1H, bs), 12.16 (1H, bs). 13C NMR (DMSO-d6, 150 MHz): δ
(ppm): 116.2, 117.7, 120.3, 123.0, 125.0, 129.9, 131.0, 135.3. HRMS calcd for
C14H979BrN2NaO2S: m/z = 370.9460, found: 370.9467.
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4.1.2.14. 3,5-dibromo-N-(6-bromo-1,3-benzothiazol-2-yl)-2-hydroxybenzamide (26l)
To a mixture of 3,5-dibromo-2-hydroxybenzoic acid (651 mg, 2.2 mmol, 1.0 eq.) in SOCl2 (2 mL)
were added several drops of DMF. The reaction was stirred at reflux temperature during 8 hours
and then evaporated under reduced pressure. The benzoyl chloride thus obtained was taken up in
chlorobenzene (6 mL) and 6-bromo-1,3-benzothiazol-2-amine (458 mg, 2.0 mmol, 1.0 eq.) was
added. The reaction was stirred at reflux temperature during 4 hours. After cooling, the mixture
was diluted with ethanol, stirred at room temperature during 10 minutes, filtered and triturated
with methyl tert-butyl ether to afford 26l as a dark green solid (600 mg, 54 %). 1H NMR (DMSO-
d6, 400 MHz): δ (ppm): 7.59 (1H, d, J= 8.8 Hz), 7.67 (1H, dd, J= 8.8 Hz, J= 2.1 Hz), 7.96 (1H, d,
J= 2.3 Hz), 8.09 (1H, J= 2.3 Hz), 8.28 (1H, d, J= 2.1 Hz). 13C NMR (DMSO-d6, 100 MHz): δ
(ppm): 109.9, 113.1, 116.6, 120.8, 125.8, 130.7, 131.9, 139.1, 159.9. HRMS calcd for
C14H879Br81Br2N2O2S: m/z = 508.7816, found: 508.7827.
4.1.2.15. 6-bromo-N-(4-bromophenyl)-benzothiophene-2-carboxamide (26x)
To a mixture of 6-bromo-benzothiophene-2-carboxylic acid (500 mg, 1.95 mmol, 1.0 eq.) and
EDC.HCl (1.12 g, 5.85 mmol, 3.0 eq.) in NMP (30 mL) was added 4-bromoaniline (335 mg, 1.95
mmol, 1.0 eq.). The reaction mixture was stirred at room temperature during 48 hours, quenched
with water and extracted with ethyl acetate. The organic layer was washed successively with 1M
NaOH, 1M HCl and water, and then dried over Na2SO4 and filtered through a silica gel pad. The
solvent was evaporated under reduced pressure and the residue was triturated with methyl tert-
butyl ether to afford 26x (600 mg, 31 %) as an orange solid. 1H NMR (DMSO-d6, 400 MHz): δ
(ppm): 7.57 (2H, d, J= 8.9 Hz), 7.63 (1H, dd, J= 8.5 Hz, J= 1.8 Hz), 7.74 (2H, d, J= 8.9 Hz),
7.97 (1H, d, J= 8.5 Hz), 8.34 (1H, bs), 8.38 (1H, d, J= 1.8 Hz), 10.66 (1H, s). 13C NMR (DMSO-
d6, 125 MHz): δ (ppm): 115.8, 119.9, 122.2, 125.4, 125.7, 127.1, 128.3, 131.6, 137.9, 138.0, 140.5,
142.1, 160.1. HRMS calcd for C15H1079Br81BrNOS: m/z = 411.8824, found: 411.8827.
4.1.2.16. 5-bromo-2-(6-bromo-1,3-benzothiazol-2-yl)-1H-isoindole-1,3(2H)-dione (32)
To a mixture of 6-bromo-1,3-benzothiazol-2-amine (120 mg, 0.52 mmol, 1.0 eq.) and 4-
bromophthalic anhydride (660 mg, 2.92 mmol, 2.6 eq.) in toluene (3 mL) was added
triethylamine (1 mL). The reaction mixture was irradiated with microwaves for 120 minutes at
200°C. The residue was filtered and triturated with ethanol and methyl tert-butylether to afford 32
as an off pink solid (190 mg, 50 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.70 (1H, dd, J=
8.8 Hz, J= 2.1 Hz), 7.98 (1H, d, J= 8.8 Hz), 8.00 (1H, d, J= 8.0 Hz), 8.17 (1H, dd, J= 8.0 Hz, J=
1.6 Hz), 8.30 (1H, d, J= 1.6 Hz), 8.46 (1H, d, J= 2.1 Hz). 13C NMR (DMSO-d6, 100 MHz): δ
(ppm): 118.0, 124.5, 125.0, 126.4, 127.4, 129.6, 130.2, 130.5, 133.5, 134.9, 138.7, 148.5, 153.2,
163.6, 164.3. HRMS calcd for C15H779Br81BrN2O2S: m/z = 438.8569, found: 438.8589.
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4.1.2.17. 4-bromo-2-{(E)-[(6-bromo-1,3-benzothiazol-2-yl)imino]methyl}phenol (34)
To a mixture of 6-bromo-1,3-benzothiazol-2-amine (200 mg, 0.87 mmol, 1.0 eq.) and 5-bromo-2-
hydroxybenzaldehyde (175 mg, 0.87 mmol, 1.0 eq.) in ethanol (3 mL) were added several drops
of piperidine. The reaction was stirred at reflux temperature during 50 minutes and filtered after
cooling to room temperature. The solid was taken up in ethyl acetate and the organic layer was
washed with water and dried over Na2SO4. The solvent was removed under reduced pressure to
afford 34 as a yellow solid (260 mg, 73 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.02 (1H, d,
J= 8.8 Hz), 7.66 (1H, dd, J= 8.8 Hz, J= 2.5 Hz), 7.69 (1H, dd, J= 8.8 Hz, J= 2.1 Hz), 7.90 (1H,
d, J= 8.8 Hz), 8.09 (1H, d, J= 2.5 Hz), 8.40 (1H, d, J= 2.1 Hz), 11.53 (1H, bs). 13C NMR
(DMSO-d6, 100 MHz): δ (ppm): 111.4, 118.2, 119.9, 122.3, 124.7, 125.5, 130.4, 132.1, 136.5,
138.3, 150.7, 160.1, 164.4, 171.8. HRMS calcd for C14H979Br81Br N2OS: m/z = 412.8776, found:
412.8796.
4.1.2.18. 2-bromo-6-{[(6-bromo-1,3-benzothiazol-2-yl)amino]methyl}phenol (36)
To a mixture of 3-bromo-2-hydroxybenzaldehyde (231 mg, 1.0 mmol, 1.0 eq.) and 6-bromo-1,3-
benzothiazol-2-amine (229 mg, 1.0 mmol, 1.0 eq.) in ethanol (2 mL) were added several drops of
piperidine. The reaction was stirred at reflux temperature during 1 hour, cooled to room
temperature, diluted with methyl tert-butyl ether and the precipitate was isolated by filtration. The
imino compound thus obtained was taken up in ethanol (20 mL) and sodium borohydride (50
mg, 1.3 mmol) was added for over 5 minutes. The mixture was stirred at room temperature
during 10 minutes, and then diluted with water and extracted with ethyl acetate. The organic
layer was dried over Na2SO4, filtrated through silica gel pad and the solvent was evaporated
under reduced pressure to afford, after trituration with methyl tert-butyl ether, 36 as a beige solid
(260 mg, 63 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 4.58 (2H, d, J= 5.7 Hz), 6.80 (1H, t,
J= 7.6 Hz), 7.25 (1H, dd, J= 7.6 Hz, J= 1.4 Hz), 7.34 (1H, d, J= 8.4 Hz), 7.40 (1H, dd, J=
8.4 Hz, J= 2.0 Hz), 7.46 (1H, dd, J= 7.6 Hz, J= 1.4 Hz), 7.95 (1H, d, J= 2.0 Hz), 8.83 (1H, t, J=
5.7 Hz), 10.37 (1H, s). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 44.0, 112.1, 113.2, 119.6,
121.6, 124.2, 128.2, 129.2, 129.6, 132.5, 132.7, 150.9, 152.3, 167.9. HRMS calcd for
C15H1179Br81Br N2OS: m/z = 414.8938, found: 414.8945.
4.1.2.19. 5-bromo-2-(4-bromophenyl)-1H-indole (39)
To a mixture of 5-bromoindole (0.60 g, 3.06 mmol, 1.0 eq.), 1-bromo-4-iodobenzene (1.04 g, 3.67
mmol, 1.2 eq.), potassium acetate (0.90 g, 9.18 mmol, 3.0 eq.) in degassed water (6 mL) was
added, under an argon atmosphere, PdCl2(PPh3)2 (0.17 g, 0.24 mmol, 0.08 eq.). The reaction was
stirred at 110°C during 48 hours and then, extracted with ethyl acetate after cooling to room
24

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temperature. The organic layer was dried over Na2SO4 and evaporated under reduced pressure.
The residue was purified by silica gel flash-column chromatography (eluent: heptane/EtOAc,
95/5 to 75/25) to afford 39 as a white solid (0.11 g, 10 %). 1H NMR (DMSO-d6, 500 MHz): δ
(ppm): 6.94 (1H, bs), 7.22 (1H, dd, J= 8.5 Hz, J= 1.8 Hz), 7.36 (1H, d, J= 8.5 Hz), 7.55 (1H, bs),
7.68 (1H, d, J= 8.8 Hz), 7.73 (1H, d, J= 1.8 Hz), 7.81 (1H, d, J= 8.8 Hz), 11.82 (1H, s). 13C
NMR (DMSO-d6, 125 MHz): δ (ppm): 99.4, 112.5, 113.7, 121.3, 122.7, 124.8, 127.6, 130.9,
131.4, 132.4, 136.4, 138.4. HRMS calcd for C14H879Br81BrN: m/z = 349.9009, found: 349.8986.
4.1.2.20. 5-bromo-3-(4-bromophenyl)-1H-indole (41)
To a mixture of 5-bromoindole (0.60 g, 3.06 mmol, 1.0 eq.), 1,4-dibromobenzene (0.87 g, 3.67
mmol, 1.2 eq.), lithium hydroxide (0.22 g, 9.18 mmol, 3.0 eq.) in degassed water (6 mL) was
added, under an argon atmosphere, PdCl2(PPh3)2 (0.17 g, 0.24 mmol, 0.08 eq.). The reaction was
stirred at 110°C during 38 hours and then, diluted a 1M aqueous hydrochloric acid solution and
extracted with ethyl acetate after cooling to room temperature. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel flash-
column chromatography (eluent: heptane/EtOAc, 95/5 to 75/25) to afford 41 as a colourless oil
(0.12 g, 11 %). 1H NMR (DMSO-d6, 400 MHz): δ (ppm): 7.29 (1H, dd, J= 8.6 Hz, J= 1.8 Hz),
7.44 (1H, d, J= 8.6 Hz), 7.61 (1H, d, J= 9.0 Hz), 7.64 (1H, d, J= 9.0 Hz), 7.83 (1H, m), 7.97 (1H,
d, J= 1.8 Hz), 11.67 (1H, s). 13C NMR (DMSO-d6, 100 MHz): δ (ppm): 113.0, 114.6, 118.8,
121.5, 124.6, 126.0, 126.9, 128.9, 132.2, 134.8, 136.1. HRMS calcd for C14H879Br81BrN: m/z =
349.9009, found: 349.8937.
4.2. Biology
4.2.1. Pyruvate kinase enzymes and inhibitory activity testing
The pyruvate kinase enzymes MRSA PK were identical to and were sourced as His-tagged
constructs as described in Ref. 3c. Synthetic candidate MRSA PK inhibitors were diluted in 100%
DMSO and assayed for their abilities to inhibit enzymatic activities of MRSA PK as described in
Ref. 3c.
4.2.2. In vitro susceptibility testing Staphylococcus aureus (ATCC29213)
Determinations of in vitro susceptibility and MIC determinations in S. aureus ATCC29213 were
carried out as described in Ref. 3c except that serial (twofold) dilutions of test compounds were
done in 100% DMSO and added to give a final concentration of DMSO of 5% in cell suspensions
in brain heart infusion (BHI) broth (Difco).
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Acknowledgments
The authors acknowledge Genome British Columbia, Genome Canada, the Natural Sciences and
Engineering Council of Canada (NSERC) for financial support.
RNY acknowledges the support of Simon Fraser University, the British Columbia Government
Leading Edge Endowment Fund and Merck Frosst Canada Ltd.
Supplementary data
Supplementary data (all the experimental procedures and detailed attribution of the different 1H
and 13C signals) associated with this article can be found in the online version.
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List of captions
Page
Figure 1. Structure and activities of molecules 1 and 2…………………….…………….………….3
Figure 2. Scheme of the interactions between 1/2 and the MRSA PK…………………………….4
Figure 3. Structure of cis-3-4-dihydrohamacanthin B (3) and bromodeoxytopsentin (4)………….4
Scheme 1. Aldol condensation reaction allowing modifications on the two aromatic parts……...5
Scheme 2. Synthesis of the rigidified analog 9……………………………………………………….5
Table 1. Structure, preparation (yield) and biological evaluation of chalcones bearing one indole
ring………………………………………………………………………………………………………6
Table 2. Structure, preparation (yield) and biological evaluation of chalcones bearing two indole
rings……………………………………………………………………………………………….…….7
Scheme 3. Preparation of product 14…………………………………………………………………8
Scheme 4. Preparation of product 19…………………………………………………………………8
Scheme 5. Preparation of product 22 and 23…………………………………………………………8
Scheme 6. Amide coupling reaction allowing modifications on the two aromatic parts………….8
Table 3. Structure, preparation (yield) and biological evaluation of amides bearing a
benzothiazole ring……………………………………………………………………………………...9
Table 4. Structure, preparation (yield) and biological evaluation of amides bearing a
benzimidazole, indole or benzothiophene ring……………………………………………………..10
Scheme 7. Preparation of product 29………………………………………………………………..11
Scheme 8. Preparation of product 32………………………………………………………………..11
Scheme 9. Preparation of product 34………………………………………………………………..11
Scheme 10. Preparation of product 36………………………………………………………………11
Scheme 11. Preparation of product 39………………………………………………………………12
Scheme 12. Preparation of product 41………………………………………………………………12
Scheme 13. Preparation of product 43………………………………………………………………12
Table 5. Biological evaluation of chalcone mimetics………………………………………………13
Table 6. Biological evaluation of amide-related compounds………………………………………15
Table 7. Biological evaluation of the 0-atom linkers……………………………………………….16
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Highlights
MRSA pyruvate kinase (PK) was identified as a novel antimicrobial target.
MRSA PK inhibitors of the hydrazide family were previously identified.
Novel series of inhibitors were synthesized: 3-, 2- and 0-atom(s) linkers.