Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: Study of 6-substituted pyridine-3-boronic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.02.056

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors.

Full text of DOI:10.1016/j.ejmech.2015.02.056

Accepted Manuscript
Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux
pump: study of 6-substituted pyridine-3-boronic acid derivatives
Fanny Fontaine, Arnaud Héquet, Anne-Sophie Voisin-Chiret, Alexandre Bouillon,
Aurélien Lesnard, Thierry Cresteil, Claude Jolivalt, Sylvain Rault
PII:
S0223-5234(15)00152-X
10.1016/j.ejmech.2015.02.056
EJMECH 7738
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2015
Revised Date: 26 February 2015
Accepted Date: 28 February 2015
Please cite this article as: F. Fontaine, A. Héquet, A.-S. Voisin-Chiret, A. Bouillon, A. Lesnard, T.
Cresteil, C. Jolivalt, S. Rault, Boronic species as promising inhibitors of the Staphylococcus aureus NorA
efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.056.
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ACCEPTED MANUSCRIPT
Boronic species as promising inhibitors of the
Staphylococcus aureus NorA efflux pump: study of
6
-substituted pyridine-3-boronic acid derivatives
†
,♯
‡
†,♯,*
∫
Fanny Fontaine, Arnaud Héquet, Anne-Sophie Voisin-Chiret,
Alexandre Bouillon, Aurélien
†
,♯
§
‡,‼
†,♯
Lesnard, Thierry Cresteil, Claude Jolivalt, Sylvain Rault
GRAPHICAL ABSTRACT
B(OH)2
B(OH)2
(
)
n
O
N
O
N
R
I
3
i, MIC >128 µg/mL, MMC =4 µg/mL
4
MIC >128 µg/mL
MMC
4
=16 µg/mL
4
3j, MIC=32µg/mL, MMC =4 µg/mL

ACCEPTED MANUSCRIPT
Boronic species as promising inhibitors of the
Staphylococcus aureus NorA efflux pump: study of
6
-substituted pyridine-3-boronic acid derivatives
†
,♯
‡
†,♯,*
∫
Fanny Fontaine, Arnaud Héquet, Anne-Sophie Voisin-Chiret,
Alexandre Bouillon, Aurélien
†
,♯
§
‡,‼
†,♯
Lesnard, Thierry Cresteil, Claude Jolivalt, Sylvain Rault
†
Normandie Univ, France
♯
UNICAEN, CERMN (Centre d’Etudes et de Recherche sur le Médicament de Normandie) - FR
CNRS INC3M, Boulevard Becquerel, 14032 Caen, France
‡
Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel,
UMR-7223, CNRS, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France
‼
Sorbonne Universités, UPMC Univ Paris 06, CNRS UMR 7197 Laboratoire de Réactivité de
Surface, F-75005, Paris, France
∫BoroChem S.A.S., 10 rue du Professeur Zarifian, F-14200 Hérouville Saint Clair, France
Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, avenue de la Terrasse, F-91198
Gif sur Yvette, France
§
KEYWORDS
Boronic acids; Pyridine derivatives; Antimicrobial agents; Efflux pump inhibitors; Staphylococcus
aureus.
HIGHLIGHTS
•
A series of 6-(aryl)alkoxypyridine-3-boronic acids was designed and synthesized

ACCEPTED MANUSCRIPT
•
•
•
They were evaluated as efflux pump inhibitors against the S. aureus 1199B strain
Compounds 3i and 3j potentiate the activity of ciprofloxacin by a 4-fold increase
They represent a new chemotype for the development of compounds with improved potency
ABSTRACT. In response to the extensive use of antibiotics, bacteria have evolved numerous
mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial
agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to
one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-
resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms.
Emergence of these pathogens requires development of novel therapeutic strategies and inhibition
of efflux pumps appears to be a promising strategy that could restore the potency of existing
antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is
known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide
range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-
benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the
NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we
describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic
acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic
acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent
compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr)
accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors.
B(OH)2
B(OH)2
(
)
n
O
N
O
N
R
I
3
i, MIC >128 µg/mL, MMC =4 µg/mL
4
MIC >128 µg/mL
MMC =16 µg/mL
3j, MIC=32µg/mL, MMC =4 µg/mL
4
4
Graphical abstract

ACCEPTED MANUSCRIPT
INTRODUCTION
Enhanced efflux through overexpressed pumps is an important mechanism of resistance by
which bacteria efficiently extrude antimicrobial agents outside the cell [1,2,3]. Many of these
transporters can export a broad range of structurally unrelated compounds leading to multidrug-
resistant (MDR) microorganisms with cross-resistance between various antibiotics [4,5].
Structural pharmacomodulation of existing antibacterial classes is proving its limits in the
quest for new antimicrobial agents [6]. The fight against resistance mechanisms, and particularly
against efflux pumps, appears to be a promising approach that could restore the potency of existing
antibiotics. This strategy relies on the combination between an efflux pump inhibitor (EPI) and the
extruded antibiotic to prevent its efflux and recover its intracellular concentration [7,8]. Therefore,
EPIs are expected to have added clinical benefits in increasing antibiotics potency, expanding their
spectrum of activity and reducing the rate of resistance development [9].
Methicillin-resistant Staphylococcus aureus (MRSA) strains are of particular concern among
MDR microorganism and are responsible for both community- and hospital-acquired infections
[
10]. NorA-overexpressing strains have been shown to be the most common among MRSA strains
11]. This pump belongs to the Major Facilitator Superfamily (MFS) and confers resistance to a
[
wide range of unrelated substrates such as hydrophilic fluoroquinolones (ciprofloxacin,
norfloxacin), biocides (acriflavine, cetrimide, benzalkonium chloride) and dyes (ethidium bromide)
[12,13]. Over the past decade, a number of NorA inhibitors have been identified [14]. The alkaloid
reserpine is commonly used as a positive control in pump inhibitory assays but it is not clinically
relevant due to neurotoxicity at concentrations required for activity [15]. Other natural-based EPIs
[16] include flavonolignan and flavone compounds [17,18], N-caffeoylphenalkylamide derivatives
[19], piperine and piperine analogs [20], citral and citronellal derivatives [21], and polyacylated
oligosaccharides [22]. Synthetic inhibitors are mainly based on heterocyclic scaffolds, such as
indoles [23,24], phenylpiperidines [25], benzothiophenes [26], phenothiazines and thioxanthenes
[27,28], quinolines and quinolones [29,30] and tricyclic compounds [31].

ACCEPTED MANUSCRIPT
For several years, our laboratory has focused on the synthesis and physicochemical study of
various heterocyclic compounds bearing boronic functions. Organoboron compounds are very well
described for their key role in cross-coupling reactions [32]. Their therapeutic relevance is yet to be
fully recognized but launch of Bortezomib, a boroaminoacid proteasome inhibitor in 2003 [33], and
of Tavaborole, an antifungal benzoxaborole in 2014 [34] opened challenging perspectives.
Biological activity of boronated compounds relies on their ability to shift from a neutral and
2
3
trigonal planar sp configuration to an anionic tetrahedral sp one, through nucleophilic attack on
the boron open shell. These compounds are thus able to form reversible covalent complexes with
nucleophilic molecules such as amino acids, nucleic acids, sugars…[35] In this way, organoboron
compounds have been described as insecticide [36], antifungal [37], antiparasitic [38], antiviral
[39], anti-inflammatory [40] and as protease inhibitors [41]. Of particular interest are the
antibacterial properties of various organoboron compounds such as diazaborines [42],
benzoxaboroles [43,44], oxazaborolidines [45], borinic [46] and boronic esters [47] and boronic
acids [48,49].
In our quest to explore the biological properties of a series of boronic species that were
prepared in our laboratory over the past decade, we investigated their antibacterial activity. As part
of this program, we previously reported the first identification of heterocyclic boronic acids as
potential inhibitors of the NorA efflux pump of Staphylococcus aureus [50]. Starting from a
chemical library of about 150 compounds, we previously identified 12 pyridine-3-boronic acids as
interesting compounds, from which structure-activity relationship studies revealed that: i) the boron
atom was required for biological activity as carbonylated and hydroxymethyl analogs were found to
be inactive; ii) weaker activity were achieved when the boronic group was esterified; iii) best results
were obtained when substituents were in para-position with respect to the boronic moiety; iv)
activity was retained when a C-5 group was added to 6-substituted compounds or when cyclisation
occurred at the C-5/C-6 positions (Figure 1) [50].

ACCEPTED MANUSCRIPT
Figure 1. Previous SAR studies for boronic species of the primary screening [50].
6
-benzyloxypyridine-3-boronic acid I (Figure 2) was identified as the most promising
compound among the tested test since it was able to potentiate the activity of ciprofloxacin by a 4-
fold increase at a concentration of 16µg/mL against the NorA-overexpressing SA-1199B strain.
This promising result led us to deepen our study and start different chemical pharmacomodulations
in order to obtain more efficient derivatives. In this paper, we describe the chemical exploration of
the C-6 position of the pyridine-3-boronic scaffold by replacing the 6-benzyloxy group of I with
various length (aryl)alkoxy chains to explore the putative hydrophobic binding site of NorA [51].
We also study the effect of the introduction of substituents on the phenyl ring of compound I, as
well as the modification of the ether linkage between the pyridine ring and the initial 6-benzyloxy
group (Figure 2).
B(OH)2
B(OH)2
(
)
n
O
N
Y
N
R
I
Y = O, S, NH
n = 0 to 9
R = Cl, F, OMe, Piperonyl
Figure 2. Considered pharmacomodulations on hit compound I.
RESULTS AND DISCUSSION
Chemistry. We first decided to explore the chemical space around the C-6 position of the pyridine-
-boronic scaffold, with the goal of obtaining more potent derivatives. 6-Substituted pyridine-3-
boronic acids 3a-t were expected to be readily available using the classical halogen-lithium
3

ACCEPTED MANUSCRIPT
exchange methodology followed by boronation of the intermediate lithiated species. We prepared
starting bromopyridines 2a-t through aromatic nucleophilic substitution of 5-bromo-2-
fluoropyridine with commercially available alcohols 1a-r, and benzyl mercaptan 1s. The procedure
first involved deprotonation of the alcohol or the thiol in the presence of sodium hydride in
anhydrous THF with subsequent addition of the dihalogenated pyridine, leading to compounds 2a-s
with high yields (76%-quant.). Attempts to synthesize 5-bromo-2-(2,4-dichlorophenoxy)pyridine 2t
with this procedure only resulted in the unmodified starting materials. 2t was therefore prepared in
good yields (81%) using a slightly modified procedure involving KOH and DMF in place of NaH
and THF (Scheme 1) [52].
Bromopyridines 2a-m and 2p-t were then subjected to bromine-lithium exchange with n-
BuLi in anhydrous ether at -78°C, followed by the reaction with triisopropylborate B(OiPr) [53].
3
Specific attention must be given to the boronic acid work-up because of the concomitant presence
of the boronic acid and the pyridine moieties. Thus, the desired product with protonated boronic
acid and free amine is only predominant in aqueous phase in a narrow range of pH that was
theoretically determined using Marvin Sketch® protonation calculator [54]. Optimized work-up
involved slow addition of 4% aqueous NaOH solution to the reaction mixture allowing
solubilization of the boronate sodium salt in the aqueous layer while retaining organic by-products
in the ethereal phase. The resulting aqueous phase was then carefully acidified by HCl 3N until
complete precipitation of the expected boronic acid. In the special case of 6-dodecanyloxypyridine-
3-boronic acid 3f, complete solubiliszation of the boronate salt in the aqueous basic phase was
prevented by the presence of the long hydrophobic dodecanyle chain. Optimized procedure
involved direct acidification of the reaction medium with acetic acid until theoretical pH, followed
by aqueous work-up and recryistallisation of the desired product. Pyridine-3-boronic acids 3a-m
and 3p-t were thus obtained as stable white solids in moderate to good yields, yet with unmatched
purity (47-86%, Scheme 1).

ACCEPTED MANUSCRIPT
Attempts to synthesize 6-(4-chlorobenzyloxy)pyridine-3-boronic acid 3n using the above
methodology only resulted in the dehalogenated 2-(4-chlorobenzyloxy)pyridine in 79% yield.
Introducing the borate prior to n-BuLi in order to overpass the eventual instability of the lithiated
intermediate prevented the dehalogenation reaction but only gave unmodified starting 2-(4-
chlorobenzyloxy)-5-bromopyridine 2n. Optimized procedure finally involved the introduction of
use of in situ trimethyl borate B(OMe) in the reaction medium prior to the followed by addition of
3
n-BuLi in anhydrous THF at -78°C [55]. Subsequent hydrolysis led to the expected boronic acid 3n
with 60% yield. This procedure was then successfully applied to 2-(4-fluorobenzyloxy)-5-
bromopyridine 2o, leading to the corresponding boronic acid 3o in 48% yield (Scheme 1).
a
Scheme 1. Synthesis of boronic acids 3a-t.
Br
B(OH)2
(
)
i, ii
iii, iv, v
n
( )n
( )n
R
YH
R
Y
N
R
Y
N
1
a-t
2a-t
3a-t
Br
Br
B(OH)2
F
N
(
)
i
( )n
iii, iv, v
n
( )n
( )n
R
YH
R
Y
ii
R
Y
N
R
Y
N
1a-t
2a-t
3a-t
R
n
2
3
4
5
6
11
0
2
3
4
5
6
7
1
1
1
Y
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
a
Me
Me
Me
Me
Me
Me
Ph
b
c
d
e
f
g
h
i
Ph
Ph
j
Ph
k
l
Ph
Ph
m
Ph
c
n
4-Cl-Ph
4-F-Ph
c
o
p
3,4-Cl-Ph

ACCEPTED MANUSCRIPT
q
r
s
3,4,5-OMe-Ph
1,3-benzodioxol
Ph
1
1
1
0
O
O
S
b
t
2,4-Cl-Ph
O
a
Reagents and conditions: i) Alcohols 1a-r or thiol 1s 1.5 equiv., 60% NaH 2 equiv., anhyd. THF,
0°C to rt, 0.5h then reflux, 1h; ii) 5-bromo-2-fluoropyridine 1 equiv., reflux, 12h; iii) n-BuLi 1.25
b
equiv., anhyd. ether, -78°C, 1h; iv) B(OiPr) 1.25 equiv., -78°C to rt, 1h; v) Hydrolysis. Reagents
3
and conditions: i) Alcohol 1t 2 equiv., KOH 2.5 equiv., anhyd. THF, reflux, 1h; ii) 5-bromo-2-
c
fluoropyridine 1 equiv., DMF, reflux, 12h. Reagents and conditions: iii) B(OMe) 2.5 equiv.,
3
d
anhyd. THF, -78°C, 10min.; iv) n-BuLi 2.5 equiv., -78°C to rt, 1h.; v) Hydrolysis. Isolated yields.
6
-Benzylaminopyridine-3-boronic acid 7 was synthesized in a 4-step procedure as shown in
Scheme 2. Treatment of 5-bromo-2-fluoropyridine with benzylamine and sodium hydride in DMF
afforded 6-benzylamino-5-bromopyridine 4 which was then Boc-protected with di-tert-butyl
dicarbonate to give compound 5 [56]. Bromine-lithium exchange and boronylation of 5 was
performed using the in situ borate procedure previously described for compounds 3n-o and the non-
isolated boronic acid 6 was finally deprotected to obtain the expected boronic acid 7 in 59% yield
over 2 steps.
a
Scheme 2. Synthesis of 6-benzylaminopyridine-3-boronic acid 7.
Br
Br
Br
i
ii
N
N
N
N
F
N
H
Boc 5, 43%
4
, 50%
iii, iv, v
B(OH)2
B(OH)2
vi
N
N
N
N
H
7
, 59%
Boc
6
a
Reagents and conditions : i) Benzylamine 1.2 equiv., K CO 1.5 equiv., DMF, reflux, 12h.; ii)
2
3
Boc O 1.2 equiv., Et N 1.2 equiv., DMAP cat., CH Cl , reflux, 12h.; iii) B(OiPr) 1.25 equiv., THF,
2
3
2
2
3

ACCEPTED MANUSCRIPT
-
78°C, 10min.; iv) n-BuLi 1.25 equiv., -78°C to rt, 1h.; v) Hydrolysis; vi) EtOH/HCl 3N 1:1, reflux,
1
2h.
Finally, using previously described procedures, 6-benzyloxy-5-methylpyridine-3-boronic
acid 9 and 5-benzyloxypyridine-3-boronic acid 11 were prepared in good yields from
bromopyridines 8 and 10. (Scheme 3). The unfavorable nucleophilic substitution at the 3-position of
the pyridine ring accounts for the poor yield obtained for compound 10.
a
Scheme 3. Synthesis of boronic acids 9 and 11.
Br
B(OH)2
OH
OH
i, ii
iii, iv, v
BnO
BnO
N
BnO
BnO
N
8
, 95%
9, 86%
Br
B(OH)2
vi, vii
iii, iv, v
N
N
10, 26%
11, 61%
a
Reagents and conditions : i) Benzyl alcohol 1.5 equiv., 60% NaH 2 equiv., anhyd. THF, 0°C to rt,
0.5h then reflux, 1h; ii) 2,5-dibromo-3-methylpyridine 1 equiv., reflux, 12h; iii) n-BuLi 1.25 equiv.,
anhyd. ether, -78°C, N , 1h; iv) B(OiPr) 1.25 equiv., -78°C to rt, 1h; v) Hydrolysis ; vi) Benzyl
2
3
alcohol 0,95 equiv., 60% NaH 1.5 equiv., DMF, rt, 1h.; vii) 3,5-dibromopyridine 1.0 equiv., reflux,
8h.
4
Microbiological assays. In a first screening assay, the intrinsic antibacterial activity of compounds
against susceptible and resistant strains of S. aureus was assessed and measured by their minimum
inhibitory concentration (MIC). Chosen strains were the susceptible S. aureus ATCC25923 strain
and the fluoroquinolone-resistant S. aureus 1199B strain that overexpresses the NorA efflux pump
and is derived from a methicillin-susceptible S. aureus bloodstream isolate from a patient with
endocarditis [57]. Ciprofloxacin has a MIC of 0.5 µg/mL against the parent susceptible S. aureus
1
199 strain and of 8 µg/mL against the resistant strain. Compounds that were inactive alone were
then investigated for their ability to restore the potency of ciprofloxacin against the resistant SA-
199B strain in order to identify potential NorA efflux pump inhibitors. Therefore, compounds
1

ACCEPTED MANUSCRIPT
were evaluated in combination with a subinhibitory concentration of the antimicrobial agent:
MIC/2, i.e. 4µg/mL or MIC/4, i.e. 2µg/mL of ciprofloxacin. Inhibitor potentiating activities are
reported as minimum modulatory concentration (MMC) MMC or MMC values, which stand for
4
2
the lowest concentration of the inhibitor required to achieve antibacterial activity in combination
with corresponding subinhibitory concentrations of ciprofloxacin. Reserpine, which is a known
inhibitor of the NorA efflux pump, was used as a positive control and displayed a MMC of 4-8
4
µg/mL. Results are shown in Table 1.
Active antibacterial agents should display no or limited detrimental side effect upon the
infected host cells: to evaluate this aspect the cytotoxicity of compounds was ascertained on KB
cells at a concentration of 10 µM. KB are epithelial cells originated from an epidermal carcinoma of
the mouth sensitive to a variety of structurally different agents and lacking the efflux pump P-gp
(
ABC B1). Docetaxel was used as positive control at its IC concentration (0.15 nM). The results
50
are shown in Table 1 as percentage of cellular growth inhibition.
Table 1. Evaluation of antibacterial activity and cytotoxicity of boronic acids 3a-t, 7, 9 and 11.
R'
B(OH)2
BnO
B(OH)2
(
)
n
R
Y
N
N
3
a-t, 7, 9
11
a
b
d
Activity against Se and Rt S. aureus
pKa
Rt
Rt
Rt
Rt
Se
MIC
µg/
c
log
P
Id.
R
n
Y
R’
MIC
(µg/
MMC2 MMC4 MMC4 Cyt.
(µg/
mL)
d
N
OH
OH
(
(µg/
mL)
(µM)
mL)
mL)
>128
>128
>128
>128
>16
3
3
a
Me
Me
Me
Me
Me
Me
Ph
2
3
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
>128
>128
>128
N.D.
>16
4
2
>128
64
64
16
8
-
0.0
1.0
1.58 1.70
1.98 1.70
8.53 10.76
8.53 10.76
8.53 10.76
8.53 10.76
8.53 10.76
8.53 10.76
8.48 10.71
8.52 10.76
8.53 10.76
8.53 10.76
b
328.17
306.15
71.72
33.74
-
3
c
4
2
16.0 2.38 1.70
46.0 2.77 1.70
73.0 3.17 1.70
13.0 5.15 1.70
14.0 2.46 0.73
3
d
5
2
3
e
6
0.5
>16
2
3
f
11
0
>16
>16
>16
64
16
16
4
3
g
>128
>128
>128
>128
>128
>128
>128
>128
297.54
69.85
65.82
15.56
I
Ph
1
1
2.0
2.55 1.65
3
h
Ph
2
1
28.0 2.80 1.69
83.0 3.20 1.70
3
i
Ph
3
0.5
6
4-
3
j
Ph
4
O
H
32
0.25
4
14.75
13.0 3.59 1.70
8.53 10.76
1
28
3
k
Ph
Ph
5
6
O
O
H
H
N.D.
>16
16
2
4
4
14.03
13.37
60.0 3.99 1.70
46.0 4.39 1.70
8.53 10.76
8.53 10.76
3
l
>16
0.5

ACCEPTED MANUSCRIPT
3
m
Ph
7
1
1
1
O
O
O
O
H
H
H
H
>16
≥128
>128
N.D.
>16
128
>128
16
0.5
1
4
32
32
1
12.77
121.45
129.54
3.35
62.0 4.78 1.70
33.0 3.07 1.65
8.53 10.76
8.52 10.76
8.52 10.76
8.52 10.76
3
n
o
p
4-Cl-Ph
4-F-Ph
3
2
8.0
2.69 1.65
3
3
3,4-Cl-Ph
0.125
44.0 3.59 1.65
3
,4,5-OMe-
Ph
q
1
O
H
>128
>128
>128
8
N.D.
-
3.0
1.79 1.65
8.52 10.76
1
,3-
3
r
s
1
1
O
S
H
H
>128
64
64
N.D.
16
-
0.0
8.0
2.23 1.65
3.35 2.26
8.52 10.76
8.42 10.63
benzodioxol
6
4-
3
Ph
0.5
65.28
1
28
3
t
2,4-Cl-Ph
0
1
1
O
NH
O
H
H
N.D.
>128
N.D.
128
1
32
4
16
128
16
56.36
561.28
65.82
-
1.0
0.0
3.49 0.69
2.38 5.42
8.48 10.71
8.74 11.01
8.55 10.79
8.28 10.47
7
Ph
Ph
>128
>128
>128
9
Me
15.0 3.02 2.19
0.0 1.85 3.51
1
1
>128
0.25
8
>128
Ampicillin
Ciprofloxacin
8
6
.57-
Reserpine
a
4-8
1
3.14
b
Se: susceptible S. aureus ATCC25923. Rt : resistant S. aureus 1199B (NorA).
c
Cytotoxicity as % of inhibition of cellular growth of KB cells in the presence of 10µM of the tested
d
compound. Calculated values [58]. N.D.: No Data.
Structural characterization of NorA, using in silico approaches, suggests that it is made of
twelve transmembrane helices and intercalated hydrophilic loops and possesses a large hydrophobic
binding site. Thus, pharmacomodulations first involved the replacement of the 6-benzyloxy group
of hit compound I with alkoxy chains with various lengths (compounds 3a-f) to explore the size of
the putative hydrophobic pocket of NorA. We observed an overall increase of the potentiative
activity in correlation with the increase of the side chain length and the increase of the logP values,
which is in agreement with the binding site putative geometry. However, for compound with the
longest alkyl side chain 3f, no activity was observed. In that case, the stock solution used for the
serial dilution in the MIC determination experiment was lowered to 16 µg/mL which was the
highest value allowing complete solubilisation of the compound. This low solubility is incidentally
correlated to a high logP calculated value, namely 5.15.
In an attempt to enhance hydrophobicity without extending the chain length of the boronic
derivatives, a phenyl ring was added at the end of the side chain (compounds 3g-m). As previously,
we observed an increase in the potentiative activity with the increase of the chain length. Besides,

ACCEPTED MANUSCRIPT
compounds with the phenyl ring were found to be more efficient than those with an alkyl chain.
Compounds I and 3h-i showed a 4-fold decrease in their MMC (16 or 4 µg/mL) compared to
4
compounds 3b-d (64 or 16 µg/mL), whereas 3j showed a 2-fold decrease in MMC (4 µg/mL)
4
compared to 3e (8 µg/mL). For both alkoxy and arylalkoxy compounds 3a-m evaluated in this
study, the best activity was observed for a total-length chain of seven carbons, phenyl included
(compounds 3e and 3j). 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-
phenylbutoxy)pyridine-3-boronic acid 3j showed the highest activities and potentiated ciprofloxacin
by a 4-fold increase at MMC of 4 µg/mL. No significant antibacterial activity was observed except
4
for 6-(5-phenylpentoxy)pyridine-3-boronic acid 3k that displayed mild activity with a MIC of 16
µg/mL. This was the first identified compound of our library with intrinsic antibacterial activity. As
mentioned above for 3f, we faced solubility problems with compounds 3e-f and 3l-m harboring the
longest (aryl)alkoxy chains. These compounds were not soluble at the usual highest tested
concentration of 128 µg/mL and were thus evaluated at most at 16 µg/mL. Therefore, possible
antibacterial activity could not be evaluated above this concentration.
Then, we studied the effect of the introduction of substituents on the phenyl ring of hit
compound I but mostly faced unsatisfactory results: the 4-chloro (3n) and 4-fluoro (3o) derivatives
showed a 2-fold decrease in the potentiative activity of ciprofloxacin while the 3,4,5-trimethoxy
(3q) and 3,4-piperonyl (3r) substituents led to a total loss of activity. Better results were obtained
with the 3,4-dichloro derivative 3p but its moderate intrinsic antibacterial activity (MIC=16µg/mL)
invalidates its foreseeable action as an EPI. Interestingly, modification of 6-phenoxypyridine-3-
boronic acid 3g to give the triclosan-like 3t led to a 4-fold increase in the potentiative activity of
ciprofloxacin, suggesting that introduction of two chlorine atoms may be important for both
antibacterial and EPI activity.
Modification of the ether linkage between the pyridine ring and the 6-benzyloxy group of I
revealed opposite effects: conservation of a H-bond acceptor linkage with the sulfanyl compound 3s
led to similar potentiative activity (MMC =16 µg/mL), whereas introduction of the H-bond donor
4

ACCEPTED MANUSCRIPT
amine linkage (7) was detrimental for activity with a 8-fold decrease in activity (MMC =128
4
µg/mL). Further pharmacomodulations showed that shifting the benzyloxy group from the C-6 to
the C-5 position of pyridine-3-boronic acid (compound 11 vs. I) resulted in a total loss of activity.
Finally, introduction of a methyl group in the C-5 position (compound 9) had little influence on the
activity.
Except for compound 3i, all molecules inhibited less of 80% of the growth of KB cells and
were to be considered as poorly cytotoxic. In particular, the most promising compound 3j as a
potential NorA inhibitor showed a low level of growth inhibition (13%). With an 83% inhibition of
-5
cellular growth, the other active molecule 3i displayed a moderate level of toxicity at 10 M, but a
-6
low level of toxicity (9.0%) at 10 M. Similarly, compounds 3k and 3p with intrinsic antibacterial
-5
activity showed a moderate level of toxicity at 10 M, and a low level of toxicity (respectively 6.0
-6
and 0.0%) at 10 M.
To investigate whether compounds have a specific synergistic activity with ciprofloxacin or
could be used in combination with other antibacterial agents that are substrate of the NorA efflux
pump, we tested their ability to potentiate the activity of norfloxacin. Norfloxacin has a MIC of 64
µg/mL against the SA 1199B strain. Fifteen compounds (I, 3a-e, 3g-j, 3n-o, 3s-t and 9) were
evaluated in combination with the fluoroquinolone at a subinhibitory concentration of 16 µg/mL
(MIC/4). Data reported in Table 2 show that compounds displayed similar activity in the presence
of ciprofloxacin or norfloxacin.
Table 2. Evaluation of antibacterial activity in combination with norfloxacin.
Antibacterial activity against SA 1199B + Nor 16 µg/mL
Id.
MMC (µg/mL)
Id.
3g
I
MMC (µg/mL)
Id.
3n
3o
3s
3t
9
MMC (µg/mL)
4
4
4
3
a
> 128
64
128
32
16
8
16
64
16
32
8
3
b
3
c
64
3h
3i
3j
3
d
16
3
e
8
8

ACCEPTED MANUSCRIPT
Effects on EtBr accumulation in the SA 1199B strain. As compounds 3i and 3j, in association
with ciprofloxacin, showed a strong effect against the SA 1199B strain, we carried out their
characterization as potential NorA inhibitors by measuring their effect on the accumulation of
ethidium bromide (EtBr, a DNA-fluorescing agent substrate of the NorA pump). EtBr becomes
fluorescent only when intercalated to bacterial DNA. Thus, when a compound is an EPI, its activity
results in intracellular accumulation of EtBr and is measured by the increase in fluorescence [59].
Compounds were evaluated at a concentration of 20 µg/mL, as compared to that of the positive
control reserpine. Figure 3 illustrates the effect of boronic acids 3i and 3j on the uptake of EtBr as a
function of time. In the absence of inhibitors, the fluorescence intensity remained constant at about
800 arbitrary units (a.u.). Upon addition of reserpine, 3i or 3j, fluorescence reached about 1450,
1200 and 1300 a.u. respectively, after 30 minutes, resulting in a 73%, 43% and 42% increase in the
EtBr uptake inside the cells. These results suggested that compounds promoted accumulation of
EtBr, thus acting as inhibitors of the NorA pump, but to a lesser extent that reserpine.
Figure 3. Effect of compounds 3i (ꢀ) and 3j (ꢀ) on EtBr accumulation of SA-1199B. The tested
compound was added after 10 min (ꢁ) of bacterial incubation with EtBr. The positive control was
reserpine at 5 (ꢀ) and 20 (ꢁ) µg/m and the negative control (ꢂ) was the bacteria in the presence of
1.6% DMSO. Vertical bars are the mean ± standard deviations for two or three independent
experiments.

ACCEPTED MANUSCRIPT
Effect in the human P-gp efflux pump. Although the bacterial NorA and the human P-gp efflux
pumps differ in their structure and mode of action, a partial overlapping of the substrates/inhibitors
had been demonstrated [60,61]. To evaluate the selectivity of the molecules 3i and 3j upon Nor A,
we next examined their capacity to inhibit of the human P-gp efflux pump. Evaluation was carried
out on the breast cancer cell line NCI/ADR-RES which overexpresses the P-gp pump. Compounds
were evaluated at concentrations of 100, 10 and 1 µM for their ability to promote accumulation of
Rhodamine-123, a fluorescent substrate of the pump, in NCI/ADR-RES cells. Cyclosporine A, a
reference inhibitor of the P-gp pump was used as positive standard at a concentration of 50 µM.
Figure 4 shows the cellular accumulation of rhodamine as a function of the concentration of P-gp
potential inhibitors. The effect of the known P-gp inhibitor Cyclosporine A (CyA) was noticeable,
with a 5-fold increase in fluorescence, whereas no significant increase in the uptake of Rhodamine-
123 was observed upon addition of compounds 3i and 3j. Therefore, these compounds were not
dual inhibitors for P-gp and NorA and could be used as antimicrobial agents without alteration of
drug distribution and pharmacokinetics under the control of P-gp.
Figure 4. Uptake of Rhodamine-123 by the NCI/ADR-RES cell line.

ACCEPTED MANUSCRIPT
CONCLUSION
In this study, we investigated the antibacterial properties of a series of boronic derivatives
against susceptible and resistant S. aureus strains. Starting from a chemical library of about 150
compounds, we selected pyridine-3-boronic acid compounds for their ability to restore the activity
of ciprofloxacin against the NorA-overexpressing SA1199B strain. Structure-activity relationships
revealed the importance of the presence of an (aryl)alkoxy side chain at the C-6 position. Chemical
pharmacomodulation of the hit compound I led to 6-(3-phenylpropoxy)pyridine-3-boronic acid 3i
and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j that showed a 4-fold increase in activity
compared to compound I. They emerged as potential NorA inhibitors as they potentiate the activity
of ciprofloxacin and norfloxacin, have no significant intrinsic antibacterial activity, promote
accumulation of EtBr, show reduced cytotoxicity and do not inhibit the mammalian P-gp efflux
pump. In the course of our study, we also identified 6-(5-phenylpentoxy)pyridine-3-boronic acid 3k
and 6-(3,4-dichlorobenzyloxy)pyridine-3-boronic acid 3p for their moderate antibacterial activity.
Although the EPI activity of 3i and 3j appears to be lower than that of reserpine, they represent a
promising chemotype for the development of new compounds with improved potency.
EXPERIMENTAL SECTION

ACCEPTED MANUSCRIPT
Synthesis. All chemical products, reagents and solvents were purchased from commercial sources
and used without further purification except THF, which was distilled from Na/benzophenone.
Chromatography was carried out on a column using flash silica gel 60 Merck (0.063-0.200 mm) as
the stationary phase. The eluting solvent indicated for each purification was determined by thin-
layer chromatography (TLC) performed on 0.2 mm precoated plates of silica gel 60F254 (Merck)
and spots were visualized using an ultraviolet-light lamp. Melting points were determined on a
Kofler melting point apparatus. IR spectra were recorded on KBr disks using a Perkin-Elmer BX
-1
1
FT-IR spectrophotometer. The band positions are given in reciprocal centimeters (cm ). H NMR
1
3
(
400 MHz) and C NMR (100 MHz) spectra were recorded on a JEOL Lambda 400 spectrometer.
1
13
H NMR (500 MHz) and C NMR (125 MHz) spectra were recorded on a Bruker 500 Avance III
spectrometer. Chemical shifts (δ) are expressed in parts per million downfield from
tetramethylsilane as an internal standard and coupling constants (J) in hertz. The abbreviations used
are as follows: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.
The purities of all tested compounds were analyzed by LC-MS, with the purity all being higher than
95%. Analyses were performed using a Waters alliance 2695 and MS detection was performed with
a SQDetector.
General procedure A for the synthesis of compounds 2a-s. To a solution of alcohol 1a-r or
benzyl mercaptan 1s (1.5 equiv.) in anhydrous THF (100 mL) was slowly added 60% sodium
hydride (2.0 equiv.). The mixture was allowed to react at room temperature for 30 min then it was
heated to reflux for 1 h. 5-bromo-2-fluoropyridine (1 equiv.) was added and the reaction was
continued for 12 h. After cooling down to room temperature, the mixture was poured into water and
extracted with ethyl acetate. The organic phase was washed with brine, dried on MgSO , filtered,
4
evaporated and purified by silica gel chromatography.
5-Bromo-2-propoxypyridine (2a). Starting from 1a (3.19 mL, 42.62 mmol) using general
procedure A and cyclohexane/CH Cl 98:2 as the eluent for chromatography, 2a was obtained as a
2
2
-1
1
colorless oil. Yield 97%. IR (KBr): ν (cm ) 2966, 1586, 1460, 1301, 1243, 1009, 971, 824, 671. H

ACCEPTED MANUSCRIPT
NMR (400 MHz, CDCl ): δ 1.01 (3H, t, J=7.1 Hz, CH ), 1.74-1.83 (2H, m, OCH CH CH ), 4.21
3
3
2
2
3
(
2H, t, J=7.1 Hz, OCH C H ), 6.65 (1H, d, J=8.8 Hz, H-3), 7.63 (1H, dd, J=8.8 and 2.9 Hz, H-4),
2 2 5
1
3
8
6
5
.17 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.9, 147.4, 141.0, 112.7, 111.4,
3
8.0, 22.2, 10.5.
-Bromo-2-butoxypyridine (2b) [62]. Starting from 1b (2.34 mL, 25.57 mmol) using general
procedure A and cyclohexane/CH Cl 99:1 as the eluent for chromatography, 2b was obtained as a
2
2
-1
1
colorless oil. Yield 93%. IR (KBr): ν (cm ) 2959, 2873, 1585, 1461, 1364, 1281, 824, 671. H
NMR (400 MHz, CDCl ): δ 0.96 (3H, t, J=7.1 Hz, CH ), 1.42-1.51 (2H, m, OC H CH CH ), 1.71-
3
3
2
4
2
3
1
.78 (2H, m, OCH CH C H ), 4.24 (2H, t, J=7.1 Hz, OCH C H ), 6.63 (1H, d, J=8.8 Hz, H-3), 7.62
2 2 2 5 2 3 7
1
3
(
1H, dd, J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ
3
162.9, 147.4, 141.0, 112.7, 111.3, 66.2, 31.0, 19.2, 13.8.
5-Bromo-2-pentoxypyridine (2c). Starting from 1c (3.71 mL, 34.09 mmol) using general
procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2c was obtained as a
-1
colorless oil. Yield 77%. IR (KBr): ν (cm ) 2956, 1586, 1463, 1358, 1243, 1090, 1003, 824, 674.
1
H NMR (400 MHz, CDCl ): δ 0.92 (3H, t, J=6.8 Hz, CH ), 1.37-1.44 (4H, m, OC H C H CH ),
3
3
2
4
2
4
3
1
7
1
5
.73-1.79 (2H, m, OCH CH C H ), 4.24 (2H, t, J=6.8 Hz, OCH C H ), 6.64 (1H, d, J=8.8 Hz, H-3),
2
2
3
7
2
4
9
13
.62 (1H, dd, J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ
3
62.9, 147.4, 141.0, 112.7, 111.3, 66.5, 28.6, 28.1, 22.4, 14.0.
-Bromo-2-hexyloxypyridine (2d). Starting from 1d (3.21 mL, 25.57 mmol) using general
procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2d was obtained as a
-1
1
colorless oil. Yield 88%. IR (KBr): ν (cm ) 2956, 2858, 1586, 1461, 1281, 1003, 824, 673, 515. H
NMR (400 MHz, CDCl ): δ 0.90 (3H, t, J=6.8 Hz, CH ), 1.32-1.45 (6H, m, OC H C H CH ), 1.71-
3
3
2
4
3
6
3
1
7
1
.79 (2H, m, OCH CH C H ), 4.24 (2H, t, J=6.8 Hz, OCH C H ), 6.64 (1H, d, J=8.8 Hz, H-3),
2 2 4 9 2 5 11
13
.62 (1H, dd, J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ
3
62.8, 147.4, 141.0, 112.7, 11.3, 66.5, 31.5, 28.9, 25.7, 22.6, 14.0.

ACCEPTED MANUSCRIPT
5
-Bromo-2-heptyloxypyridine (2e). Starting from 1e (3.63 mL, 25.57 mmol) using general
procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2e was obtained as a
-1
1
yellow oil. Yield 79%. IR (KBr): ν (cm ) 2928, 2857, 1586, 1460, 1281, 1009, 824, 673, 515. H
NMR (400 MHz, CDCl ): δ 0.89 (3H, t, J=6.8 Hz, CH ), 1.28-1.43 (8H, m, OC H C H CH ), 1.72-
3
3
2
4
4
8
3
1
7
1
5
.79 (2H, m, OCH CH C H ), 4.23 (2H, t, J=6.8 Hz, OCH C H ), 6.64 (1H, d, J=8.8 Hz, H-3),
2 2 5 11 2 6 13
13
.62 (1H, dd, J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ
3
62.9, 147.5, 141.0, 112.7, 111.3, 66.5, 31.8, 29.0, 28.9, 26.0, 22.6, 14.1.
-Bromo-2-dodecanyloxypyridine (2f). Starting from 1f (9.53 mL, 42.62 mmol) using general
procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2f was obtained as a
-1
beige solid. Yield 91%. Mp < 50°C. IR (KBr): ν (cm ) 2917, 2851, 1587, 1459, 1279, 1024, 819,
1
6
1
77. H NMR (400 MHz, CDCl ): δ 0.88 (3H, t, J=6.8 Hz, CH ), 1.26 (18H, bs, OC H C H CH ),
3
3
2
4
9
18
3
.71-1.78 (2H, m, OCH CH C H ), 4.23 (2H, t, J=6.8 Hz, OCH C H ), 6.64 (1H, d, J=8.8 Hz,
2
2
10 21
2 11 23
1
3
H-3), 7.62 (1H, dd, J=8.8 and 2.9 Hz, H-4), 8.17 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz,
CDCl ): δ 162.9, 147.5, 141.0, 112.7, 111.4, 66.5, 31.9, 29.7, 29.64, 29.6, 29.57, 29.38, 29.36, 28.9,
3
26.0, 22.7, 14.1.
5-Bromo-2-phenoxypyridine (2g) [63]. Starting from 1g (3.21 g, 34.09 mmol) using general
procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2g was obtained as a
-1
1
colorless oil. Yield 95%. IR (KBr): ν (cm ) 3066, 1576, 1456, 1367, 1271, 1091, 758, 693. H
NMR (400 MHz, CDCl ): δ 6.83 (1H, d, J=8.8 Hz, H-3), 7.12 (2H, m, H-2’), 7.22 (1H, m, H-4’),
3
1
3
7
.41 (2H, m, H-3’), 7.75 (1H, dd, J=8.8 and 2.7 Hz, H-4), 8.22 (1H, d, J=2.7 Hz, H-6). C NMR
(
100 MHz, CDCl ): δ 162.5, 153.7, 148.4, 141.9, 129.7, 125.0, 121.1, 113.4, 113.0.
3
5-Bromo-2-(2-phenylethoxy)pyridine (2h). Starting from 1h (3.06 mL, 25.57 mmol) using
general procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2h was
-1
obtained as a yellow oil. Yield 97%. IR (KBr): ν (cm ) 3028, 2955, 1584, 1456, 1361, 1280, 1012,
1
8
25, 699. H NMR (400 MHz, CDCl ): δ 3.07 (2H, t, J=7.1 Hz, OCH CH ), 4.48 (2H, t, J=7.1 Hz,
3
2
2
OCH CH ), 6.63 (1H, d, J=8.8 Hz, H-3), 7.22-7.32 (5H, m, H-Ph), 7.62 (1H, dd, J=8.8 and 2.9 Hz,
2
2

ACCEPTED MANUSCRIPT
1
3
H-4), 8.17 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.5, 147.4, 141.0, 138.3,
3
129.0, 128.4, 126.4, 112.8, 111.6, 66.8, 35.3.
5-Bromo-2-(3-phenylpropoxy)pyridine (2i). Starting from 1i (5.80 mL, 42.62 mmol) using
general procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2i was
-1
obtained as a yellow oil. Yield 93%. IR (KBr): ν (cm ) 2951, 1586, 1462, 1363, 1282, 1027, 825,
1
6
99. H NMR (400 MHz, CDCl ): δ 2.05-2.12 (2H, m, OCH CH CH ), 2.77 (2H, t, J=6.8 Hz,
3
2
2
2
OC H CH ), 4.27 (2H, t, J=6.8 Hz, OCH C H ), 6.65 (1H, d, J=8.8 Hz, H-3), 7.17-7.31 (5h, m, H-
2
4
2
2
2
4
1
3
Ph), 7.63 (1H, dd, J=8.8 and 2.9 Hz, H-4), 8.17 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz,
CDCl ): δ 162.7, 147.4, 141.5, 141.0, 128.4, 125.9, 112.6, 111.5, 65.6, 32.2, 30.5.
3
5-Bromo-2-(4-phenylbutoxy)pyridine (2j). Starting from 1j (4.05 mL, 25.57 mmol) using general
procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2j was obtained as a
-1
white solid. Yield 85%. Mp 60-62°C. IR (KBr): ν (cm ) 2925, 1587, 1484, 1366, 1295, 1022, 830,
1
6
99. H NMR (400 MHz, CDCl ): δ 1.74-1.80 (4H, m, OCH C H CH ), 2.68 (2H, t, J=6.8 Hz,
3
2
2
4
2
OC H CH ), 4.27 (2H, t, J=6.8 Hz, OCH C H ), 6.63 (1H, d, J=8.8 Hz, H-3), 7.16-7.30 (5H, m, H-
3
6
2
2
3
6
1
3
Ph), 7.62 (1H, dd, J=8.8 and 2.9 Hz, H-4), 8.17 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz,
CDCl ): δ 162.7, 147.4, 142.2, 141.0, 128.4, 128.3, 125.7, 112.7, 111.4, 66.1, 35.5, 28.5, 27.8.
3
5-Bromo-2-(5-phenylpentoxy)pyridine (2k). Starting from 1k (2.87 mL, 17.05 mmol) using
general procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2k was
-1
obtained as a colorless oil. Yield 87%. IR (KBr): ν (cm ) 2935, 1585, 1162, 1281, 1002, 825, 699.
1
H NMR (400 MHz, CDCl ): δ 1.44-1.52 (2H, m, OC H CH C H ), 1.65-1.73 (2H, m,
3
2
4
2
2
4
OC H CH CH ), 1.75-1.83 (2H, m, OCH CH C H ), 2.63 (2H, t, J=7.2 Hz, OC H CH ), 4.23 (2H,
3
6
2
2
2
2
3
6
4
8
2
t, J=7.2 Hz, OCH C H ), 6.62 (1H, d, J=8.8 Hz, H-3), 7.16-7.29 (5H, m, H-Ph), 7.61 (1H, dd, J=8.8
2
4
8
1
3
and 2.9 Hz, H-4), 8.16 (1H, d, J=2.9 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.8, 147.4, 142.5,
3
141.0, 128.4, 128.2, 125.6, 112.7, 111.4, 66.3, 35.8, 31.2, 28.8, 25.7.
5-Bromo-2-(6-phenylhexyloxy)pyridine (2l). Starting from 1l (1.59 mL, 8.52 mmol) using general
procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2l was obtained as a

ACCEPTED MANUSCRIPT
-1
1
colorless oil. Yield 91%. IR (KBr): ν (cm ) 2931, 1585, 1462, 1281, 1090, 1002, 825, 699. H
NMR (400 MHz, CDCl ): δ 1.37-1.50 (4H, m, OC H C H C H ), 1.60-1.68 (2H, m,
3
2
4
2
4
2
4
OC H CH CH ), 1.72-1.79 (2H, m, OCH CH C H ), 2.61 (2H, t, J=7.2 Hz, OC H CH ), 4.23 (2H,
4
8
2
2
2
2
4
8
5
10
2
t, J=7.2 Hz, OCH C H ), 6.63 (1H, d, J=8.8 Hz, H-3), 7.16-7.29 (5H, m, H-Ph), 7.62 (1H, dd,
2
5
10
1
3
J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.8, 147.4,
3
142.7, 141.0, 128.4, 128.2, 125.6, 112.7, 111.4, 66.4, 35.9, 31.4, 29.0, 28.8, 25.9.
5-Bromo-2-(7-phenylheptyloxy)pyridine (2m). Starting from 1m (1.70 mL, 8.52 mmol) using
general procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2m was
-1
obtained as a colorless oil. Yield 76%. IR (KBr): ν (cm ) 2930, 1585, 1463, 1281, 1090, 1004, 825,
1
6
99. H NMR (400 MHz, CDCl ): δ 1.37-1.41 (6H, m, OC H C H C H ), 1.57-1.64 (2H, m,
3
2
4
3
6
2
4
OC H CH CH ), 1.71-1.76 (2H, m, OCH CH C H ), 2.60 (2H, t, J=7.2 Hz, OC H CH ), 4.23
5
10
2
2
2
2
5
10
6
12
2
(
2H, t, J=7.2 Hz, OCH C H ), 6.63 (1H, d, J=8.8 Hz, H-3), 7.16-7.29 (5H, m, H-Ph), 7.62 (1H, dd,
2 6 12
1
3
J=8.8 and 2.4 Hz, H-4), 8.17 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.8, 147.4,
3
142.8, 141.0, 128.4, 128.2, 125.6, 112.7, 111.4, 66.4, 35.9, 31.4, 29.2, 28.9, 26.9, 25.9.
5-Bromo-2-(4-chlorobenzyloxy)pyridine (2n). Starting from 1n (6.08 g, 42.62 mmol) using
general procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2n was
-1
obtained as a white solid. Yield 98%. Mp 93-95°C. IR (KBr): ν (cm ) 1454, 1286, 1012, 822, 800,
1
6
42. H NMR (400 MHz, CDCl ): δ 5.31 (2H, s, OCH ), 6.72 (1H, d, J=8.8 Hz, H-3), 7.32-7.39
3
2
1
3
(
4H, m, H-2’, H-3’), 7.66 (1H, dd, J=8.8 and 2.9 Hz, H-4), 8.19 (1H, d, J=2.9 Hz, H-6). C NMR
(
100 MHz, CDCl ): δ 162.3, 147.4, 141.3, 135.4, 133.8, 129.3, 128.6, 112.9, 112.0, 67.1.
3
5-Bromo-2-(4-fluorobenzyloxy)pyridine (2o) [63]. Starting from 1o (4.65 mL, 42.62 mmol) using
general procedure A and cyclohexane/AcOEt 98:2 as the eluent for chromatography, 2o was
-1
obtained as a white solid. Yield 98%. Mp 66-68°C. IR (KBr): ν (cm ) 1455, 1292, 1090, 810, 668.
1
H NMR (400 MHz, CDCl ): δ 5.30 (2H, s, OCH ), 6.71 (1H, d, J=8.8 Hz, H-3), 7.03-7.08 (2H, m,
3
2
H-3’), 7.40-7.43 (2H, m, H-2’), 7.66 (1H, dd, J=8.8 and 2.9 Hz, H-4), 8.20 (1H, d, J=2.9 Hz, H-6).

ACCEPTED MANUSCRIPT
1
3
C NMR (100 MHz, CDCl ): δ 162.2, 147.4, 141.2, 132.7, 132.6, 130.0, 129.9, 115.5, 115.3,
3
112.9, 112.0, 67.2.
5-Bromo-2-(3,4-dichlorobenzyloxy)pyridine (2p). Starting from 1p (4.53 g, 25.57 mmol) using
general procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2p was
-1
obtained as a white solid. Quant. yield. Mp 88-90°C. IR (KBr): ν (cm ) 1585, 1474, 1348, 1284,
1
1
7
7
1
5
029, 802, 645. H NMR (400 MHz, CDCl ): δ 5.29 (2H, s, OCH ), 6.73 (1H, d, J=8.8 Hz, H-3),
3
2
.26 (1H, dd, J=8.3 and 2.0 Hz, H-6’), 7.43 (1H, d, J=8.3 Hz, H-5’), 7.53 (1H, d, J=2.0 Hz, H-2’),
13
.67 (1H, dd, J=8.8 and 2.4 Hz, H-4), 8.19 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ
3
61.8, 147.4, 141.4, 137.2, 132.5, 131.9, 130.4, 129.8, 127.1, 112.8, 112.3, 66.3.
-Bromo-2-(3,4,5-trimethoxybenzyloxy)pyridine (2q). Starting from 1q (2.74 mL, 17.05 mmol)
using general procedure A and cyclohexane/AcOEt 8:2 as the eluent for chromatography, 2q was
-1
obtained as a white solid. Quant. yield. Mp 76-78°C. IR (KBr): ν (cm ) 2995, 1594, 1449, 1354,
1
1
238, 1123, 1003, 833, 706. H NMR (400 MHz, CDCl ): δ 3.86 (9H, s, OCH ), 5.26 (2H, s,
3
3
OCH ), 6.68 (2H, s, H-2’), 6.74 (1H, d, J=8.8 Hz, H-3), 7.67 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.22
2
1
3
(
1H, d, J=1.9 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.3, 153.3, 147.4, 141.3, 137.7, 132.3,
3
112.9, 112.0, 105.3, 68.3, 60.8, 56.1.
2-(1,3-Benzodioxol-5-ylmethoxy)-5-bromopyridine (2r). Starting from 1r (3.89 g, 25.57 mmol)
using general procedure A and cyclohexane/AcOEt 95:5 as the eluent for chromatography, 2r was
-1
obtained as a white solid. Yield 98%. Mp 87-89°C. IR (KBr): ν (cm ) 2888, 1585, 1466, 1283, 931,
1
8
28, 807. H NMR (400 MHz, CDCl ): δ 5.23 (2H, s, OCH ), 5.96 (2H, s, OCH O), 6.69 (1H, d,
3
2
2
J=8.8 Hz, H-3), 6.80 (1H, d, J=7.8 Hz, H-2’), 6.90-6.94 (2H, m, H-5’, H-6’), 7.64 (1H, dd, J=8.8
1
3
and 1.9 Hz, H-4), 8.20 (1H, d, J=1.9 Hz, H-6). C NMR (100 MHz, CDCl ): δ 162.3, 147.7,
3
147.41, 147.38, 141.2, 130.6, 121.9, 112.9, 111.9, 108.9, 108.2, 101.1, 67.9.
2-Benzylsulfanyl-5-bromopyridine (2s) [64]. Starting from 1s (3.0 mL, 25.57 mmol) using
general procedure A and cyclohexane/AcOEt 99:1 as the eluent for chromatography, 2s was
-1
obtained as a beige solid. Quant. yield. Mp < 50°C. IR (KBr): ν (cm ) 1560, 1452, 1350, 1115,

ACCEPTED MANUSCRIPT
1
8
21, 715, 693, 486. H NMR (400 MHz, CDCl ): δ 4.40 (2H, s, SCH ), 7.05 (1H, d, J=7.8 Hz, H-3),
3 2
1
3
7
.24-7.40 (5H, m, H-Ph), 7.57 (1H, dd, J=7.8 and 1.9 Hz, H-4), 8.50 (1H, d, J=1.9 Hz, H-6). C
NMR (100 MHz, CDCl ): δ 157.5, 150.2, 138.5, 137.6, 128.9, 128.5, 127.2, 123.1, 116.1, 34.5.
3
5-Bromo-2-(2,4-dichlorophenoxy)pyridine (2t). To a solution of 1t (3.70 g, 22.72 mmol) in
anhydrous THF (100 mL) was slowly added potassium hydroxide (1.59 g, 28.41 mmol). The
mixture was allowed to react at room temperature for 30 min then it was heated to reflux for 1 h.
The reaction was concentrated to dryness and diluted in anhydrous DMF (100 mL). 5-bromo-2-
fluoropyridine (2.0 g, 11.36 mmol) was added and the reaction was continued for 12 h. After
cooling down to room temperature, the mixture was poured in water and extracted with ethyl
acetate. The crude product was purified by silica gel chromatography (cyclohexane/AcOEt 98:2)
-1
affording 2t as a white solid. Yield 81%. Mp 82-84°C. IR (KBr): ν (cm ) 1479, 1369, 1279, 1093,
1
8
04. H NMR (400 MHz, CDCl ): δ 6.93 (1H, d, J=8.8 Hz, H-3), 7.14 (1H, d, J=8.8 Hz, H-6’), 7.29
3
(1H, dd, J=8.8 and 2.4 Hz, H-5’), 7.48 (1H, d, J=2.4 Hz, H-3’), 7.80 (1H, dd, J=8.8 and 2.4 Hz, H-
1
3
4
), 8.15 (1H, d, J=2.4 Hz, H-6). C NMR (100 MHz, CDCl ): δ 164.5, 148.1, 148.0, 142.2, 131.2,
3
130.4, 128.2, 128.1, 124.7, 114.0, 112.7.
General procedure B for the synthesis of boronic acids 3a-m, 3p-t, 9 and 11. To a slurry of
.5M n-BuLi (1.25 equiv.) in anhydrous diethyl ether (80 mL) cooled at -78°C was added a solution
2
of 5-bromopyridine (1.0 equiv.) in anhydrous THF (10 mL). The mixture was allowed to react at
this temperature for over 60 min. A solution of triisopropyl borate (1.25 equiv.) was then added and
left to react for 60 min. Unless otherwise specified, treatment of the reaction proceeded as follows:
the mixture was allowed to warm to room temperature and was quenched by slow addition of 4%
aqueous NaOH solution (50 mL). The resulting aqueous layer was collected and acidified to
adequate pH by dropwise addition of 3N HCl. The resulting precipitate was filtered on a sintered-
glass filter, washed with diethyl ether and dried to give corresponding boronic acids.
6-Propoxypyridine-3-boronic acid (3a). Starting from 2a (1.0 g, 4.63 mmol) using general
-
procedure B (pH 5), 3a was obtained as a white solid. Yield 77%. Mp 118-120°C. IR (KBr): ν (cm

ACCEPTED MANUSCRIPT
1
1
)
3400, 2963, 1611, 1332, 1301. H NMR (400 MHz, DMSO-d ): δ 0.94 (3H, t, J=7.3 Hz, CH ),
6 3
1
7
.65-1.74 (2H, m, OCH CH CH ), 4.19 (2H, t, J=7.3 Hz, OCH C H ), 6.73 (1H, d, J=8.8 Hz, H-5),
2 2 3 2 2 5
1
3
.97 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.11 (2H, s, OH), 8.48 (1H, d, J=1.9 Hz, H-2). C NMR (100
MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 109.7, 66.8, 21.9, 10.4. LC-MS (ESI): t = 5.34 min;
6
R
+
[
M+H] 182,48.
6-Butoxypyridine-3-boronic acid (3b). Starting from 2b (1.0 g, 4.35 mmol) using general
-
procedure B (pH 5), 2b was obtained as a white solid. Yield 79%. Mp 109-111°C. IR (KBr): ν (cm
1
1
)
3409, 2955, 1611, 1335, 1304. H NMR (400 MHz, DMSO-d ): δ 0.91 (3H, t, J=7.3 Hz, CH ),
6 3
1
.35-1.44 (2H, m, OC H CH CH ), 1.63-1.70 (2H, m, OCH CH C H ), 4.23 (2H, t, J=7.3 Hz,
2 4 2 3 2 2 2 5
OCH C H ), 6.71 (1H, d, J=8.8 Hz, H-5), 7.97 (1H, d, J=8.8 Hz, H-4), 8.10 (2H, s, OH), 8.48 (1H,
2
3
7
1
3
s, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 109.7, 65.0, 30.6, 18.8, 13.7. LC-
6
+
MS (ESI): t = 6.38 min; [M+H] 196.50.
R
6-Pentoxypyridine-3-boronic acid (3c). Starting from 2c (1.0 g, 4.10 mmol) using general
-
procedure B (pH 5), 3c was obtained as a white solid. Yield 86%. Mp 139-141°C. IR (KBr): ν (cm
1
1
)
3410, 2954, 1611, 1334, 1303. H NMR (400 MHz, DMSO-d ): δ 0.87 (3H, t, J=6.8 Hz, CH ),
6 3
1
.33-1.37 (4H, m, OC H C H CH ), 1.65-1.72 (2H, m, OCH CH C H ), 4.23 (2H, t, J=6.8 Hz,
2 4 2 4 3 2 2 3 7
OCH C H ), 6.72 (1H, d, J=8.8 Hz, H-5), 7.97 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.10 (2H, s, OH),
2
4
9
1
3
8
2
6
.47 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 109.7, 65.3,
6
+
8.2, 27.8, 21.9, 13.9. LC-MS (ESI): t = 5.71 min; [M+H] 209.16.
R
-Hexyloxypyridine-3-boronic acid (3d). Starting from 2d (1.0 g, 3.87 mmol) using general
-1
procedure B (pH 6), 3d was obtained as a white solid. Yield 73%. Mp 90-92°C. IR (KBr): ν (cm )
1
3
393, 2944, 2917, 1609, 1335, 1301. H NMR (400 MHz, DMSO-d ): δ 0.87 (3H, t, J=6.9 Hz,
6
CH ), 1.28-1.34 (6H, m, OC H C H CH ), 1.65-1.72 (2H, m, OCH CH C H ), 4.24 (2H, t, J=6.9
3
2
4
3
6
3
2
2
4
9
Hz, OCH C H ), 6.73 (1H, d, J=8.3 Hz, H-5), 7.98 (1H, dd, J=8.3 and 2.0 Hz, H-4), 8.10 (2H, s,
2
5
11
1
3
OH), 8.49 (1H, d, J=2.0 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 109.7,
6
+
6
5.3, 31.1, 28.5, 25.2, 22.1, 13.9. LC-MS (ESI): t = 7.20 min; [M+H] 224.56.
R

ACCEPTED MANUSCRIPT
6
-Heptyloxypyridine-3-boronic acid (3e). Starting from 2e (1.0 g, 3.67 mmol) using general
-1
procedure B (pH 6), 3e was obtained as a white solid. Yield 61%. Mp 97-99°C. IR (KBr): ν (cm )
1
3
393, 2951, 2921, 2852, 1612, 1334, 1302. H NMR (400 MHz, DMSO-d ): δ 0.85 (3H, t, J=6.7
6
Hz, CH ), 1.33-1.36 (8H, m, OC H C H CH ), 1.64-1.71 (2H, m, OCH CH C H ), 4.23 (2H, t,
3
2
4
4
8
3
2
2
5
11
J=6.7 Hz, OCH C H ), 6.72 (1H, d, J=8.3 Hz, H-5), 7.97 (1H, dd, J=8.3 and 1.9 Hz, H-4), 8.10
2
6
13
13
(
2H, s, OH), 8.47 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5,
6
+
1
09.7, 65.3, 31.3, 28.53, 28.49, 25.5, 22.1, 14.0. LC-MS (ESI): t = 7.82 min; [M+H] 238.57.
R
6-Dodecanyloxypyridine-3-boronic acid (3f) [65]. Starting from 2f (1.0 g, 2.92 mmol), we used
general procedure B and treatment proceeded as follows: after cooling down to room temperature,
the mixture was acidified to pH 5 by dropwise addition of acetic acid. The organic phase was
washed with brine, dried on MgSO , filtered and evaporated. Recristallisation of the crude product
4
-1
from diethyl ether gave product 3f as a white solid. Yield 58%. Mp 98-100°C. IR (KBr): ν (cm )
1
3
400, 2918, 2850, 1611, 1336, 1303. H NMR (400 MHz, DMSO-d ): δ 0.84 (3H, t, J=6.6 Hz,
6
CH ), 1.23 (18H, bs, OC H C H CH ), 1.63-1.70 (2H, m, OCH CH C H ), 4.23 (2H, t, J=6.6 Hz,
3
2
4
9
18
3
2
2 10 21
OCH C H ), 6.71 (1H, d, J=8.8 Hz, H-5), 7.972 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.10 (2H, s,
2
11 23
1
3
OH), 8.47 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 109.7,
6
6
5.3, 31.3, 29.04, 29.01, 29.0 (2 C), 28.8, 28.7, 25.5, 22.1, 14.0. LC-MS (ESI): t = 9.62 min;
R
+
[
M+H] 308.68.
6-Phenoxypyridine-3-boronic acid (3g). Starting from 2g (1.0 g, 4.00 mmol) using general
-
procedure B (pH 5), 3g was obtained as a white solid. Yield 65%. Mp 147-149°C. IR (KBr): ν (cm
1
1
)
3418, 1586, 1242, 757, 690. H NMR (400 MHz, DMSO-d ): δ 6.95 (1H, d, J=8.1 Hz, H-5), 7.11
6
(2H, m, H-2’), 7.21 (1H, m, H-4’), 7.41 (2H, m, H-3’), 8.13 (1H, d, J=8.1 Hz, H-4), 8.24 (2H, s,
13
OH), 8.45 (1H, s, H-2). C NMR (100 MHz, DMSO-d ): δ 164.4, 153.7, 153.5, 145.6, 129.7,
6
+
1
24.6, 121.3, 110.4. LC-MS (ESI): t = 6.48 min; [M+H] 216.48.
R
6-(2-Phenylethoxy)pyridine-3-boronic acid (3h). Starting from 2h (1.0 g, 3.60 mmol) using
general procedure B (pH 5), 3h was obtained as a white solid. Yield 79%. Mp 111-113°C. IR

ACCEPTED MANUSCRIPT
-1
1
(
KBr): ν (cm ) 3400, 1610, 1302, 1013, 696. H NMR (400 MHz, DMSO-d ): δ 3.01 (2H, t, J=6.8
6
Hz, OCH CH ), 4.46 (2H, t, J=6.8 Hz, OCH CH ), 6.72 (1H, d, J=8.8 Hz, H-5), 7.18-7.30 (5H, m,
2
2
2
2
13
H-Ph), 7.98 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.12 (2H, s, OH), 8.49 (1H, d, J=1.9 Hz, H-2). C
NMR (100 MHz, DMSO-d ): δ 164.5, 153.3, 144.6, 138.5, 128.9, 128.3, 126.3, 109.8, 65.9, 34.7.
6
+
LC-MS (ESI): t = 5.84 min; [M+H] 243.13.
R
6-(3-Phenylpropoxy)pyridine-3-boronic acid (3i). Starting from 2i (1.0 g, 3.42 mmol) using
general procedure B (pH 5), 3i was obtained as a white solid. Yield 76%. Mp 98-100°C. IR (KBr):
-1
1
ν (cm ) 3418, 1606, 1329, 1285, 1023, 697. H NMR (400 MHz, DMSO-d ): δ 1.97-2.04 (2H, m,
6
OCH CH CH ), 2.71 (2H, t, J=6.8 Hz, OC H CH ), 4.23 (2H, t, J=6.8 Hz, OCH C H ), 6.75 (1H, d,
2
2
2
2
4
2
2
2
4
J=8.8 Hz, H-5), 7.17-7.29 (5h, m, H-Ph), 7.99 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.12 (2H, s, OH),
1
3
8
1
6
.47 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.6, 153.3, 144.5, 141.4, 128.4,
6
+
28.3, 125.9, 109.8, 64.7, 31.6, 30.2. LC-MS (ESI): t = 6.08 min; [M+H] 257.21.
R
-(4-Phenylbutoxy)pyridine-3-boronic acid (3j). Starting from 2j (1.0 g, 3.27 mmol) using
general procedure B (pH 5), 3j was obtained as a white solid. Yield 72%. Mp 95-97°C. IR (KBr): ν
-1
1
(
cm ) 3401, 1607, 1334, 1300, 1017. H NMR (400 MHz, DMSO-d ): δ 1.66-1.71 (4H, m,
6
OCH C H CH ), 2.61 (2H, t, J=6.8 Hz, OC H CH ), 4.26 (2H, t, J=6.8 Hz, OCH C H ), 6.72 (1H,
2
2
4
2
3
6
2
2
3
6
d, J=8.8 Hz, H-5), 7.14-7.280 (5H, m, H-Ph), 7.97 (1H, dd, J=8.8 and 1.9 Hz, H-4), 8.11 (2H, s,
1
3
OH), 8.47 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.5, 142.0,
6
+
1
28.3, 128.2, 125.7, 109.7, 65.1, 34.8, 28.2, 27.5. LC-MS (ESI): t = 6.42 min; [M+H] 271.20.
R
6-(5-Phenylpentoxy)pyridine-3-boronic acid (3k). Starting from 2k (1.0 g, 3.12 mmol) using
general procedure B (pH 5), 3k was obtained as a white solid. Yield 69%. Mp 164-166°C. IR
-1
1
(
KBr): ν (cm ) 3400, 2927, 1609, 1334, 1300, 1016, 737, 639. H NMR (500 MHz, DMSO-d ): δ
6
1
.40-1.44 (2H, m, OC H CH C H ), 1.59-1.65 (2H, m, OC H CH CH ), 1.71-1.75 (2H, m,
2 4 2 2 4 3 6 2 2
OCH CH C H ), 2.59 (2H, t, J=7.1 Hz, OC H CH ), 4.25 (2H, t, J=7.1 Hz, OCH C H ), 6.73 (1H,
2
2
3
6
4
8
2
2
4
8
d, J=8.3 Hz, H-5), 7.15-7.29 (5H, m, H-Ph), 7.99 (1H, dd, J=8.3 and 2.0 Hz, H-4), 8.12 (2H, s, OH),

ACCEPTED MANUSCRIPT
1
3
8
1
6
.49 (1H, d, J=2.0 Hz, H-2). C NMR (125 MHz, DMSO-d ): δ 164.6, 153.3, 144.4, 142.2, 128.3,
6
+
28.2, 125.6, 109.7, 65.2, 35.1, 30.8, 28.3, 25.2. LC-MS (ESI): t = 7.70 min; [M+H] 286.61.
R
-(6-Phenylhexyloxy)pyridine-3-boronic acid (3l). Starting from 2l (1.0 g, 2.99 mmol) using
general procedure B (pH 5), 3l was obtained as a white solid. Yield 73%. Mp 86-88°C. IR (KBr): ν
-1
1
(
cm ) 3413, 2928, 1607, 1334, 1300, 1017. H NMR (400 MHz, DMSO-d ): δ 1.31-1.44 (4H, m,
6
OC H C H C H ), 1.52-1.58 (2H, m, OC H CH CH ), 1.60-1.69 (2H, m, OCH CH C H ), 2.55
2
4
2
4
2
4
4
8
2
2
2
2
4
8
(
2H, t, J=7.1 Hz, OC H CH ), 4.22 (2H, t, J=7.1 Hz, OCH C H ), 6.71 (1H, d, J=8.6 Hz, H-5),
5 10 2 2 5 10
7.12-7.26 (5H, m, H-Ph), 7.97 (1H, dd, J=8.6 and 1.9 Hz, H-4), 8.16 (2H, s, OH), 8.47 (1H, d, J=1.9
1
3
Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.4, 144.5, 142.3, 128.3, 125.7, 109.8, 65.3,
6
+
6
5.2, 35.11, 35.10, 31.0, 28.4, 25.4. LC-MS (ESI): t = 8.05 min; [M+H] 300.62.
R
6-(7-Phenylheptyloxy)pyridine-3-boronic acid (3m). Starting from 2m (1.0 g, 2.87 mmol) using
general procedure B (pH 5), 3m was obtained as a white solid. Yield 64%. Mp 88-90°C. IR (KBr):
-1
1
ν (cm ) 3390, 2924, 1610, 1334, 1302, 1017, 745, 695, 647. H NMR (400 MHz, DMSO-d ): δ
6
1
.30-1.36 (6H, m, OC H C H C H ), 1.52-1.57 (2H, m, OC H CH CH ), 1.64-1.69 (2H, m,
2 4 3 6 2 4 5 10 2 2
OCH CH C H ), 2.54 (2H, t, J=7.1 Hz, OC H CH ), 4.22 (2H, t, J=7.1 Hz, OCH C H ), 6.71
2
2
5
10
6
12
2
2
6
12
(1H, d, J=8.6 Hz, H-5), 7.12-7.26 (5H, m, H-Ph), 7.97 (1H, d, J=8.5 Hz, H-4), 8.10 (2H, s, OH),
1
3
8
1
6
.48 (1H, s, H-2). C NMR (100 MHz, DMSO-d ): δ 164.7, 153.3, 144.49, 144.47, 142.3, 128.2,
6
+
25.6, 109.7, 65.2, 35.14, 35.1, 31.0, 28.6, 28.5, 25.5. LC-MS (ESI): t = 8.42 min; [M+H] 314.64.
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-(4-Chlorobenzyloxy)pyridine-3-boronic acid (3n). To a solution of 2n (1.0 g, 3.35 mmol) and
trimethyl borate (0.93 mL, 8.37 mmol) in anhydrous THF (80 mL) cooled at -78°C was added
dropwise a 2.5M solution of n-BuLi (3.35 mL, 8.37 mmol). The mixture was allowed to react at this
temperature for over 60 min. After cooling down to room temperature, the mixture was quenched
by slow addition of 4% aqueous NaOH solution (50 mL). The resulting aqueous layer was collected
and acidified to pH 5 by dropwise addition of 3N HCl and then extracted with ethyl acetate. The
organic phase was washed with brine, dried on MgSO , filtered and evaporated. Recrystallisation of
4
the crude product from diethyl ether gave product 3n as a white solid. Yield 60%. Mp 120-122°C.

ACCEPTED MANUSCRIPT
-1
1
IR (KBr): ν (cm ) 3429, 1610, 1497, 1334, 1297, 1015, 809, 639. H NMR (400 MHz, DMSO-d ):
6
δ 5.35 (2H, s, OCH ), 6.82 (1H, d, J=8.8 Hz, H-5), 7.41-7.47 (4H, m, H-2’, H-3’), 8.02 (1H, dd,
2
13
J=8.8 and 1.9 Hz, H-4), 8.15 (2H, s, OH), 8.50 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz,
DMSO-d ): δ 164.1, 153.2, 144.8, 136.4, 132.3, 129.7, 128.3, 109.9, 65.9. LC-MS (ESI): t = 7.42
6
R
+
min; [M+H] 264.50, 266.51.
-(4-Fluorobenzyloxy)pyridine-3-boronic acid (3o). Following the procedure for 3n synthesis, 2o
1.0 g, 3.54 mmol) was reacted with n-BuLi (3.54 mL, 8.86 mmol) and trimethyl borate (0.99 mL,
.86 mmol). The mixture was then treated as described in representative procedure (pH 5) to give
6
(
8
-1
compound 21o as a white solid. Yield 48%. Mp 139-141°C. IR (KBr): ν (cm ) 3433, 1647, 1510,
1
1
356, 1224. H NMR (400 MHz, DMSO-d ): δ 5.33 (2H, s, OCH ), 6.81 (1H, d, J=7.8 Hz, H-5),
6
2
7.17-7.21 (2H, m, H-3’), 7.47-7.51 (2H, m, H-2’), 8.02 (1H, dd, J=7.8 and 1.9 Hz, H-4), 8.14 (2H,
13
s, OH), 8.51 (1H, d, J=1.9 Hz, H-2). C NMR (100 MHz, DMSO-d ): δ 164.4, 160.4, 153.2, 144.7,
6
+
1
33.6, 130.3, 130.2, 115.3, 115.1, 109.9, 66.1. LC-MS (ESI): t = 5.94 min; [M+H] 248.16.
R
6-(3,4-Dichlorobenzyloxy)pyridine-3-boronic acid (3p). Starting from 2p (1.0 g, 3.00 mmol), we
used general procedure B and treatment proceeded as follows: after cooling down to room
temperature, the mixture was quenched by slow addition of 4% aqueous NaOH solution (50 mL).
The resulting aqueous layer was collected and acidified to pH 5 by dropwise addition of 3N HCl
and then extracted with ethyl acetate. The organic phase was washed with brine, dried on MgSO4,
filtered and evaporated. Recristallisation of the crude product from diethyl ether gave 3p as a white
-1
solid. Yield 58%. Mp 131-133°C. IR (KBr): ν (cm ) 3435, 1598, 1408, 1320, 1285, 1127, 1030,
1
7
69. H NMR (500 MHz, DMSO-d ): δ 5.37 (2H, s, OCH ), 6.86 (1H, d, J=8.3 Hz, H-5), 7.44 (1H,
6
2
dd, J=8.3 and 1.9 Hz, H-6’), 7.64 (1H, d, J=8.3 Hz, H-5’), 7.71 (1H, d, J=1.9 Hz, H-2’), 8.04 (1H,
1
3
dd, J=8.3 and 1.9 Hz, H-4), 8.17 (2H, s, OH), 8.51 (1H, d, J=1.9 Hz, H-2). C NMR (125 MHz,
DMSO-d ): δ 163.9, 153.1, 144.9, 138.7, 131.0, 130.6, 130.2, 129.7, 128.0, 110.0, 65.3. LC-MS
6
+
(
ESI): t = 6.13 min; [M+H] 298.46, 300.45, 302.45.
R