Fusaric acid and analogues as Gram-negative bacterial quorum sensing inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.11.044

Taking advantage of microwave-assisted synthesis, efficient and expedite procedures for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum sensing and one compound inhibited activity of the las quorum sensing system. To our delight, none of the compounds showed growth inhibitory effects in the tested concentration ranges.

Full text of DOI:10.1016/j.ejmech.2016.11.044

Accepted Manuscript
Fusaric acid and analogues as gram-negative bacterial quorum sensing inhibitors
Truong Thanh Tung, Tim Holm Jakobsen, Trong Tuan Dao, Anja Thoe Fuglsang,
Michael Givskov, Søren Brøgger Christensen, John Nielsen
PII:
S0223-5234(16)30982-5
10.1016/j.ejmech.2016.11.044
EJMECH 9077
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 September 2016
Revised Date: 27 October 2016
Accepted Date: 21 November 2016
Please cite this article as: T.T. Tung, T.H. Jakobsen, T.T. Dao, A.T. Fuglsang, M. Givskov, S.B.
Christensen, J. Nielsen, Fusaric acid and analogues as gram-negative bacterial quorum sensing
inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.044.
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ACCEPTED MANUSCRIPT
Table of Contents/ Graphical Abstracts
R2
O
Homologous
O
O
OH
R1
O
N
N
N
N
structure
H
H
O
Fusaric acid
O
O
O
QS signal molecule C6-HSL
- Microwave-assisted synthesis
- QS inhibitors, SAR

ACCEPTED MANUSCRIPT
Fusaric acid and analogues as gram-negative
bacterial quorum sensing inhibitors
Truong Thanh Tung,a Tim Holm Jakobsen,b Trong Tuan Dao,a,c Anja Thoe Fuglsang,c Michael
Givskov,b,d Søren Brøgger Christensena,* and John Nielsena,*
aDepartment of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken
2, 2100 Copenhagen Ø, Denmark.
bCosterton Biofilm Center, Faculty Of Health Sciences, Department of Immunology and
Microbiology, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
cDepartment of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej
40, 1871 Frederiksberg C, Denmark.
dSingapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang
Technological University, Singapore 637551, Singapore.
*Corresponding author
E-mail: soren.christensen@sund.ku.dk, Tel.: +45 3533 6253 (S. B. Christensen);
john.nielsen@sund.ku.dk, Tel.: +45 3533 6706 (J. Nielsen).
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Abstract: Taking advantage of microwave-assisted synthesis, efficient and expedite procedures
for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid
analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum
sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio
fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum
sensing and one compound inhibited activity of the las quorum sensing system. To our delight,
none of the compounds showed growth inhibitory effects in the tested concentration ranges.
Keywords: fusaric acid, quorum sensing inhibitors, microwave-assisted synthesis.
1. Introduction
The mycotoxin fusaric acid (FA, Fig. 1) was originally isolated by Yabuta in 1934 from the
fungus Fusarium heterosporum [1]. Later studies have revealed that the compound is produced
by a large number of species belonging to the Fusarium genus and maybe even by all Fusarium
species. In some cultures, more than 1 mg of FA per gram medium is produced. The agent was
the first compound shown to have a pivotal role in plant diseases such as tomato, cucumber and
banana wilt [2, 3]. In addition to the involvement in plant diseases [2-4], FA has a number of
pharmacological properties even though only in moderate to high doses [5, 6] and possesses the
ability to augment the effects of other mycotoxins. Despite a number of effects in ranging from
alteration of membrane activity, decreased mitochondrial activity, inhibition of ATP synthesis
and reduced production of polyphenol oxidase and peroxidases, the mechanism of FA is still not
fully delineated [2]. FA is also reported to chelate divalent cations and inhibit of dopamine β-
hydroxylase [7a-b]. Over the year, FA has undergone several preclinical and clinical trials for
treatment of diseases such as mania, cancer and hypertension [7].
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We hypothesized that FA can be considered a bioequivalent to N-hexanoyl-L-homoserine
lactone, a signal molecule involved in qorum sensing (QS) in bacteria. In addition, FA like some
QS inhibitors (QSIs) possesses a pyridine ring (Fig. 1c). QSIs are very encouraging for design of
new antimicrobial drugs acting through a hitherto untapped bacterial pathway. No new molecular
entities are currently in clinical trials for interfering with QS in patients. Notably however,
azithromycin, one of the best-selling antibiotics, has been demonstrated to inhibit virulence and
cooperation of Pseudomonas aeruginosa through QS mechanism [8a]. Recently, hamamelitannin
analogues were suggested to increase the susceptibility of a methicillin-resistant Streptococcus
aureus strain through QS inhibition [8b].
Fig. 1
During QS bacteria use small signal molecules or autoinducers for cell-cell communication.
QS enables bacteria to coordinate a collective response such as biofilm formation as a protection
towards external factors. Therefore, compounds possessing QS inhibitory ability but void of
bactericidal or bacteriostatic activity inactivate bacteria defense systems, reduce the production
of toxins and most importantly are less prone to selection for resistance than traditional cytotoxic
remedies [9, 10]. In Gram-negative bacteria, the QS signal compounds are N-Acyl-L-
homoserine lactones (AHLs) [9-11]. Many QSIs based on structure of AHLs have been designed
and tested [12]. As shown in Fig. 2 FA might be considered bioequivalent to the AHL.
Fig. 2
FA comprises of a pyridine nucleus decorated with a carboxylic group at C-2 and an n-butyl
moiety protruding from C-5. Overlay of the ester carbonyl of the two molecules, C-3 of
homoserine lactone with C-2 of FA, and the methylene groups in the side chains of the two
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molecules gives a good match indicating that these groups might be bioequivalent (Fig. 2). In
addition, molecules containing a pyridine ring (4-NPO, IC50 of 24 µg/ml or 171 µM [12c, 13])
and these structural features are found to be QSIs (Fig. 1b, c). Bloemberg et al. [14] have also
reported that FA prevents the production of the signaling molecule N-hexanoyl-L-homoserine
lactone (C6-HSL) but no FA analogues were synthesized and evaluated to get structure-activity
relationship and thereby maybe hint to understand the mechanism of action. Based on these
arguments, FA was chosen as a suitable template for construction of a library of potential QSIs
[15].
2. Results and discussion
2.1. Strategy for design of library
Fig. 3
The strategy behind the design of the library to get structure-activity relationship and
thereby to identify pharmacophore descriptors is illustrated in Fig. 3. To further elaborate on
these structural features the compounds shown in Table 1 were designed, prepared and screened
as QSI. The importance of the substituent at C-5 was investigated by decorating with a broad
plethora of substituents with different lengths, aromatic rings and functional groups.
2.2. Microwave assisted synthesis of fusaric acid and analogues
Only a few analogues of FA have been reported [16-19]. Common for all reported protocols,
however, are that they only afford a limited spectrum of analogues. No biological studies have
been performed on these compounds and none of the reported methods describe microwave-
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assisted synthesis. Taking advantage of microwave methodology, we have developed a protocol
for fast preparation of FA and a diverse library of analogues (Scheme 1).
Scheme 1
o
Treatment of 2,5-dibromopyridine (1) with TMSCH2Li-LiDMAE in toluene at 0 C for 30
min afforded the 2-lithiated intermediate selectively [20]. Addition of methyl or ethyl formate at
-78 oC and stirring for 3h followed by oxidation with iodine in methanol or ethanol introduced an
ester group at C-2 in medium yield to give methyl 5-bromopicolinate (2a) or ethyl 5-
bromopicolinate (2b), respectively.
FA and analogues can be prepared from key intermediate 2a or 2b. In pathway 1, compound
2a or 2b is converted into methyl or ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)picolinate (3a or 3b, respectively) by treatment with bis(pinacolato)diboron. The reaction was
accelerated by irradiation with microwave and catalyzed with 10 mol % of Pd(dppf)Cl2. After
just 15 min, all starting material was converted into the target compound 3 (in situ) as observed
by TLC and UPLC (compared to 12-36 h in traditional heating). Crude compound 3a or 3b
(without purification) was subsequently treated with different bromo- or chloroalkanes or with
halogenated heterocycles to afford esters 4 in excellent yield. An alternative successful pathway
(Pathway 2) consisted in treatment of key intermediate 2 with various boronate esters using the
same condition as stated above for Pathway 1, under which conditions esters 4 were formed in
good yield. All esters 4 were converted to corresponding acids 5 by saponification (Scheme 1).
The alkoxy groups at C-5 (7) were installed via a Williamson ether synthesis from the
reaction of methyl 5-hydroxypicolinate (6) with an alkyl halide under basic condition at room
temperature for 18 - 48 h. The use of microwave-assisted synthesis reduced the reaction time to
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only 30 to 180 min. Saponification 7 afforded the carboxylic acids 8. The ester methyl 5-(2-oxo-
2-(phenylamino)ethoxy)picolinate (7c) was completely hydrolyzed in 4 h to give 5-(2-oxo-2-
(phenylamino)ethoxy)picolinic acid (8c). Remarkably, the instability of 8c was illustrated by
complete conversion into 5-(carboxymethoxy)picolinic acid (8c') if work up of the reaction was
postponed until the next day. Compound N-propyl-5-(propylamino)picolinamide (10a) was
obtained directly from 2a by reaction with propylamine in the presence of 6 mol % Pd(dppf)Cl2
under microwave irradiation. Under these conditions substitution of the ester as well as the 5-
bromo group occurred to give 10a in good yield. Ethyl 5-(chloromethyl)picolinate (9) was
successfully converted into ethyl 5-((ethylthio)methyl)picolinate (10b) by treatment with
ethanethiol at room temperature (Scheme 2).
Scheme 2
Overall, the broad spectrum of FA and analogues can be prepared from the key intermediate
2a/2b using our microwave-assisted synthesis method. This protocol can be used for fast
expansion of the library compounds for biological studies in the future.
2.3. Synthesis of fusaric acid
The microwave irradiation condition enabled synthesis of ester of FA in 90 % yield via
Pathway 2 using n-butylboronic acid pinacol ester and 2b as starting materials to give ethyl 5-
butylpicolinate (FAE). Saponification of FAE afforded 75 % of FA (Scheme 3).
Scheme 3
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Pathway 1 using 2b converted into ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)picolinate and n-butyl bromide as starting materials for preparation of FAE succeeded in 70 %
yield from 2b over 2 steps (Scheme 3).
In summary, FA and a library of 39 analogues was prepared by an efficient microwave-assisted
synthesis. The members of the library are depicted in Table 1.
Table 1
2.4. Biological assays
Fig. 4
Table 2
Three cell-based biological screens were used to test for QSI activity. The first two reporter
systems, lasB-gfp [21] and rhlA-gfp [22] in P. aeruginosa background contain fusions of the QS-
controlled lasB promoter or rhlA promoter to Green Fluorescence Protein ((gfp) (ASV)) encoding
an unstable GFP variant. The third QS reporter system, luxI-gfp [23] in an Escherichia coli
background contains the promoter region of the luxI fused to gfp (ASV) and a luxR gene. With
these reporter systems activation of QS can be measured by an increase in fluorescence. Presence
of a QSI will therefore results in a decrease in GFP expression. Bacterial growth was measured
simultaneously to be certain the test compounds do not affect the growth rate.
Activities as inhibitors of the Lux QS system were found for compound 4b, 4g, 7a, 7b, 7c,
FAE and FA (Fig. 4, Table 2). To our delight, none of the compounds affected the bacterial
growth in the tested concentration ranges except for FA. No QSI activity was found against two
QS reporter strains lasB-gfp and rhlA-gfp in concentrations up to 100 µg/ml. Three compounds
7

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5c, 8a, 8b reduced the GFP expression, however that reduction was potentially generated by a
simultaneous growth inhibition meaning that it cannot be regarded as a QSI (Fig. 5). FA seem to
delay the bacterial growth in concentrations ranging from 6.2 to 100 µg/ml, whereas at a
concentration of 3.1 µg/ml a small reduction in GFP expression was seen with no simultaneous
inhibition of growth. The ester FAE showed clearly QSI activity in concentrations from 6.2 to 50
µg/ml. All compounds with QSI activity are shown in Fig. 4. (See supporting information for
screening results of other compounds). The larger number of compounds targeting the lux QS
system compared to the las and rhl systems are potentially because of a higher binding affinity
for the Lux receptor protein and the use of different background strains in the monitor systems.
Fig. 5
2.5. Structure-activity relationship
Except for our lead compound FA, all the QSIs 4b, 4g, 7a, 7b, 7c, and FAE are esters. The
ester group might simulate the intermolecular interaction solicited by the lactone moiety in
structures of current QSIs (Fig. 1b). Another possibility is that the zwitterionic character of the
free acids prevents penetration over the cell wall of plasma membrane. This latter hypothesis,
however, is unsubstantiated by the observation that all the growth inhibitors 5c, 8a, 8b are acids
(Fig. 5).
The QS inhibitory activities of 4b, 4g and 7c possessing 5-methoxy-2-pyridyl-, phenyl- and
2-oxo-2-(phenylamino)- ethoxy-, respectively, reveals that a lipophilic alkyl group in the 5
position as in the case for FA as well as AHL is not mandatory (Fig. 4). In contrast the ester
moiety at C-2 position seems to be favorable for QS inhibition. In addition, FA is delaying the
growth of bacterial as did some of the other carboxylic acids.
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Compounds 2a, 4j, 5e, 5k, 5l, 8c, 8c´ did not show QS inhibitory activity or growth
inhibitory effect in concentrations up to 100 µg/ml. At concentrations above 125 µg/ml
compound 2a inhibited lasB expression in the lasB-gfp reporter without affecting growth (Fig.
6).
Fig. 6
In summary, the observed structure-activity relationship for inhibition of QS suggested by
this study is depicted in Fig. 7.
Fig. 7
2.6. Screening for the inhibition of plasma membrane H+-ATPase (Pma1)
Reduced stomatal [24] conductance has been observed for banana plants infected by
Fursarium spp. [4, 25] suggesting that the agent might stimulate the H+-ATPase. The possibility
of inhibition of H+-ATPase [26, 27] inspired us to test our library towards the pump, but no
compounds showed any significant activity (data not shown).
3. Conclusions
An efficient, facile and expedite microwave-assisted synthesis of FA and analogues have
been developed and used to prepare a library of 40 analogues. The compounds were tested in
three QS screening systems, lasB-gfp (P. aeruginosa), rhlA-gfp (P. aeruginosa) and luxI-gfp (E.
coli). In the luxI-gfp system compounds 4b, 4g, 7a, 7b, 7c and FAE revealed QS inhibitory
activity in the concentration range 6.25 to 100 µg/ml. Compound 2a showed good inhibitory
activity in concentration from 125 µg/ml toward lasB-gfp. In the luxI-gfp system, FA showed a
little QS inhibition activity at 3.13 µg/ml while at higher concentrations from 6.25 to 100 µg/ml
9

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the delaying of cell growth were observed along with QS inhibitory activity. Structure-activity
relationship studies suggest that the ester group at C-2 is essential for QS inhibition. In contrast,
the alkyl substituent at the 5-position can be exchanged with an alkoxy or aromatic/heterocyclic
aromatic ring. Even though the compounds reveal a modest QS inhibitory activity, FA analogues
have not previously been found to possess this system making it a new scaffold for developing
QSIs. Moreover, we have demonstrated that a natural product scaffold with homologous
structure toward the QS signals could serve successfully as a starting point for design of a
focused library of new compounds with QS activities.
4. Experimental section
4.1. Biology
4.1.1. QSI screening Assay
The QS reporter strains lasB-gfp
(P. aeruginosa) [21], rhlA-gfp (P. aeruginosa) [22]
and luxI-gfp E. coli) [23] were used for measuring inhibition of QS. The following
(
growth medium was used for bacterial growth; BT minimal medium (B medium plus
2.5 mg thiamine L−1) supplemented with 10% A10, 0.5% (wt/vol) glucose, 0.5% (wt/vol)
Casamino Acids. An overnight culture of each reporter strain was grown for 16 h at 37 °C
with shaking at 180 rpm. The overnight cultures were diluted to a final optical density at
450 nm (OD450) of 0.1. The biological assays were conducted in 96-well microtiter
dishes (Black Isoplate®, Perkin Elmer®, Waltham Massachusetts, USA). Test compounds,
growth media and reporter strains were added to the microtiter dishes. The corresponding
signal molecule 3-oxo-C6-HSL was added in a final concentration of 10 nM to the screen
with the reporter strain, luxI-gfp. Growth and green fluorescent protein (GFP) expression
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was monitored using Victor™ X4 multilabel plate reader (Perkin Elmer®, Waltham
Massachusetts, USA) set at a constant temperature of 34 °C measuring every 15 min over
a time course of 14 h. GFP expression was measured as fluorescence at an excitation
wavelength of 485 nm and an emission wavelength of 535 nm. Please see [28] for full
protocol.
4.1.2. Pma1 ATPase Screening Assay
Yeast plasma membrane protein Pma1 was prepared from Saccharomyces cerevisiae
as previously described [29a]. The protein activity was measured based on Baginski
method [29b], by using 0.5 µg of purified Pma1 protein in 300 µL of reaction mixture
containing 5 mM NaN3, 3.5 mM sodium molybdate, 50 mM KNO3, 10 mM MgSO4,
5mM ATP, 20 mM MES-KOH adjusted to pH 5.9. Pma1 was first pre-incubated with 100
µM of compounds for 30 min at room temperature. Reactions were stopped after 30 min
by adding 300 µL of an ice-cold stop solution (containing 93 mM ascobic acid, 0.273 N
HCl, 0.059 % SDS, 5 mM ammonium heptamolybdate). The tubes were continuously
incubated on ice for 10 min to allow formation of the phosphate-molybdate complex.
Excess molybdate was complexed by the addition of 450 µL of an arsenite-solution
(containing 154 mM NaAsO2, 68 mM trisodium citrate, 350 mM acetic acid). After 60
min of incubation at room temperature, the absorbance was determined
spectrophotometrically at 860 nm.
4.2. Chemistry
4.2.1. General information
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Commercially available reagents (Aldrich) were used without further purification
unless otherwise noted. Microwave reactions were carried out in an automatic Biotage®
Initiator equipped with robot sixty (an automated 60-position system) and using high
precision microwave vials and aluminum seals. NMR-spectra were acquired using a 400
or 600 MHz Bruker Avance III HD equipped with a cryogenically cooled 5 mm dual
13
1
probe optimized for C and H. Samples were dissolved in one of following solvent:
1
DMSO-d6, CDCl3, MeOH-d4 and analyzed at 300K. H, COSY, HMBC, and HSQC
13
spectra were acquired at 400 or 600 MHz. C spectra were recorded at 151 MHz or 101
MHz. Chemical shifts (
NMR) or the solvent peak (13C NMR) as the internal standard. The coupling constant (
δ
) are reported in ppm relative to the residual solvent peak (1H
J)
are reported in Hertz. Solvents used for the synthesis were of analytical grade, dried over
activated 4 Å molecular sieves when necessary (all solvents used under dry conditions
had a water content <25 ppm). Analytical TLC was performed using pre-coated silica gel
60 F254 plates and visualized using UV light. Flash column chromatography was
performed using Merk silica 60. Melting points were determined on a Mettler Toledo
MP70 Melting Point system. All tested compounds possess ≥95% purity (purified by
preparative RP-HPLC for compounds less than 90% purity). Purity determinations were
performed on a Waters 2795 system equipped with a Waters 996 PDA detector and a
Waters Symmetry C18 Column (2.1 × 50 mm, 3.5 lm) with a flow of 0.2 ml min−1. 100%
→ 0% A 0–10 min (A: 0.1% aq formic acid, B: 95% CH3CN in 0.05% aq formic acid).
High-resolution mass spectral (HRMS) data were obtained on an electrospray (ESI) or a
MALDI-TOF mass spectrometer.
4.2.2. General synthetic procedure for compounds 2a–b
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N,
N-Dimethyl aminoethanol (0.85 ml, 8.5 mmol,
1
equiv.) and
(trimethylsilyl)methyllithium 1M (25.3 ml, 25.3 mmol, 3 equiv.) was added dropwise to
25 ml of dry toluene at 0 °C and the solution was stirred for 30 min to form TMSCH2Li-
LiDMAE (in situ). Then, A solution of 2,5-dibromopyridine
1 (2 g, 8.5 mmol, 1 equiv.) in
9 ml of dry toluene was added dropwise. The reaction mixture was stirred for 30 min,
cooled to -78 °C and added methyl formate (5.2 ml, 85 mmol, 10 equiv.) or ethyl formate
(6.9 ml, 85 mmol, 10 equiv.) in 10 ml of toluene. The reaction mixture stirred for 3
hours, and I2 (6.4 g, 25.5 mmol, 3 equiv.), K2CO3 (5.8 g, 42.5 mmol, 5 equiv.) and 20 ml
methanol or ethanol were added. The mixture was stirred at room temperature for 18
hours and added 10 ml of a sat. aqueous Na2SO3 solution. Water (10 ml) was added and
the mixture extracted with dichloromethane (3 x 40 ml), washed with water (1 x 25 ml)
and brine (1 x 25 ml). The residue was purified by silica-gel flash column
chromatography (eluent: heptane/diethyl ether = 2 : 1) to obtain 2a–b
.
4.2.2.1. Methyl 5-bromopicolinate (2a).
◦
From
1 (2 g) gave 2a (997 mg, 55%); white solid; >99% purity; mp 103 – 104 C
(reported 98.0-102.0 ◦C); 1H-NMR (600 MHz, CDCl3)
δ
8.77 (dd,
J = 0.74, 0.71 Hz, 1H,
H2), 8.01 (dd,
(100 MHz, CDCl3):
J = 0.84, 0.79 Hz, 1H, H5), 7.98 (dd, J
= 2.3, 2.2 Hz, 1H, H6);13C NMR
δ
165.0(C=O, C-7), 151.0(C-2), 146.3(C-4), 139.7(C-6), 126.3(C-5),
+
125.1(C-1); HRMS (ESI) m/z [M+H]+ calcd for C7H7BrNO2 215.9660, found 215.9659.
Data was in accordance with those reported [30a].
4.2.2.2.
Ethyl 5-bromopicolinate (2b)
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◦
From
1 (2 g) gave 2b (889 mg, 46%); yellow solid; >99% purity; mp 59.3–60.4 C
(reported 63-64 °C [102]); 1H NMR (600 MHz, CDCl3)
δ
8.77 (dd,
J
J
= 2.2 Hz,
J = 0.79,
0.75 Hz, 1H, H2), 7.99 (dd,
H6), 4.45, (q, = 7.1 Hz, 2H, H11), 1.40 (t,
CDCl3):
J = 0.73, 0.74 Hz, 1H, H5), 7.95 (dd,
= 2.2, 2.2 Hz, 1H,
J
J
= 7.1 Hz, 3H; H12);13C NMR (100 MHz,
δ
164.6(C=O, C-8), 151.1(C-2), 146.7(C-4), 139.7(C-6), 126.3(C-5), 124.9(C-1),
+
62.2(C-11), 14.3(C-12); HRMS (ESI) m/z [M+H]+ calcd for C8H9BrNO2 229.9817,
found 229.9807. Data was in accordance with those reported [30a-b].
4.2.3. General synthetic procedure for compounds 4a
4.2.3.1. Method
4.2.3.1.1. Method 1.
–m.
A mixture of 2a or 2b (1 g, 1 equiv.), bis(pinacolato)diboron (1.2 equiv.), [1,1′-
bis(diphenylphosphino)ferrocene] dichloropalladium(II) complex with dichloromethane
(10 mol %), potassium acetate (2 equiv.), 1,4-dioxane (15 ml) and water (7.5 ml) was
sealed in a 20 ml microwave reaction vial (Biotage). The vial was irradiated in a
microwave apparatus at 110 °C, high absorption for 15 min. The reaction mixture was
cooled to room temperature (afforded crude 3a or 3b) and used to the next step without
purification. An appropriate alkyl halide (0.95 equiv.), cesium carbonate (2.0 equiv) and
water (4 ml) were added directly to the reaction mixture. The vial was sealed and
irradiated in a microwave apparatus at 110 °C, normal absorption for 30-90 min. After
cooling to room temperature, water (100 ml) was added and the mixture was extracted
with ethyl acetate (4 x 100 ml). The organic layer was washed with brine (200 ml), dried
with anhydrous Na2SO4(s) and concentrated. The residue was purified by silica-gel flash
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column chromatography (eluent: ethyl acetate/heptane = 1:1) to obtain the esters 4a and
4j-m.
4.2.3.1.2. Method 2.
A mixture 2a or 2b (1 g, 1 equiv.), an appropriate pinacol boronate ester (1.2 equiv.),
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex
with
dichloromethane (10 mol %), cesium carbonate (2.0 equiv.), 1,4-dioxane (8 ml) and water
(4 ml) was sealed in a 20 ml microwave reaction vial (Biotage). The vial was irradiated
in a microwave apparatus at 110 °C, normal absorption for 30-90 min. The reaction
mixture was cooled to room temperature and work up was performed as described in
method 1 to obtain the esters 4b
-i.
4.2.3.2. Ethyl 5-butylpicolinate (FAE).
Method 2; 2b (1 g) gave FAE (810 mg, 90%). If method 1 was used the yield was
70% over 2 steps; yellow oil; >98% purity; 1H NMR (600 MHz, CDCl3)
8.57 (d, J = 2.2
δ
Hz, 1H, H2), 8.05 (d,
J = 7.8 Hz, 1H, H5), 7.63 (dd, J = 2.2, 7.8 Hz, 1H, H6), 4.47 (q, J
=7.1 Hz, 2H, H14), 2.69 (t,
J
= 7.6 Hz, 2H, H10), 1.62 (m, 2H, H11), 1.44 (t,
J = 7.1, 3H,
H15), 1.37 (qt,
MHz, CDCl3)
J
=7.3, 7.6 Hz, 2H, H12), 0.94 (t, J
= 7.3 Hz, 3H, H13);13C NMR (151
δ
165.4(C=O, C-7), 150.1(C-2), 145.9(C-4), 141.9(C-1), 136.5(C-6),
124.8(C-5), 61.7(C-14), 32.9(C-10), 32.7(C-11), 22.2(C-12), 14.4(C-15), 13.8(C-13);
+
HRMS (ESI) m/z [M+H]+ calcd for C12H18NO2 208.1338, found 208.1340. Data was in
accordance with those reported [30c].
4.2.3.3.
Methyl 5-benzylpicolinate (4a).
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Method 1; 2a (1 g) gave 4a (925 mg, 88%); white solid; >98% purity; mp 66–67◦C;
1H NMR (600 MHz, CDCl3)
H12), 7.59 (dd,
H2), 7.15-7.17 (d,
(151 MHz, CDCl3)
δ 8.63 (dd, J = 1.6, 1.6 Hz, 1H, H9), 8.05 (d, J = 7.3 Hz, 1H,
J
= 2.4, 2.4 Hz, 2H, H13), 7.30-7.32 (m, 2H, H4, H6), 7.23-7.27 (m, 1H,
= 7.3 Hz, 2H, H1, H3), 4.05 (s, 2H, H7), 3.99 (s, 3H, H17); 13C NMR
J
δ
165.7(C=O, C-14), 150.2(C-9), 146.0(C-8), 140.6(C-11), 138.8(C-
5), 137.2(C-13), 128.8(C-4, C-6), 128.8(C-1, C-3), 126.8(C-2), 125.1(C-12), 52.8(C-7),
+
39.0(C-17); HRMS (ESI) m/z [M+H]+ calcd for C14H14NO2 228.1025, found 228.1025.
4.2.3.4.
4.2.4. General synthetic procedure for compounds FA
Ester (FAE 4a
Characterization data of compounds 4b
-
4m
:
see supporting information.
and 2´.
,
5a
–
m
,
-
m, 2a or 2b) (500 mg scale, 1 equiv.) was dissolved in 6 ml THF at
0°C in a 25 ml round-bottom flask. Then NaOH(aq) (5 equiv.) was added dropwise and
stirred for 15h at room temperature. After starting materials were consumed (by TLC),
water (20 ml) was added. The reaction mixture was washed with ethyl acetate (2 x 20 ml).
The aqueous solution was acidified (pH 2-3) with 1 M HCl(aq) causing precipitation of a
solid, which was filtered and dried under vacuum. Recrystallization in ethanol afford
clean compound.
4.2.4.1.
Fusaric acid (FA).
FAE (500 mg) gave FA (324 mg, 75%); slightly yellow solid; >99% purity after
1
recrystallization from ethanol; mp 96.9-98.0°C (reported 96-99°C[172]); H NMR (600
MHz, DMSO-d6):
= 8.1, 2.2 Hz, 1H, H6), 2.74 (t,
7.3x(3), 7.7x(2) Hz, 2H, H12), 0.96 (t,
δ
8.46 (d,
J
= 2.2 Hz, 1H, H2), 8.16 (d,
J
= 8.1 Hz, 1H, H5), 7.76 (dd,
J
J
= 7.7x(2) Hz, 2H, H10), 1.66 (m, 2H, H11), 1.4 (tq,
J
=
13
J
= 7.3, 7.3 Hz, 3H, H13); C NMR (151 MHz,
16

ACCEPTED MANUSCRIPT
DMSO-d6)
δ
164.2(C=O, C-7), 148.2(C-2), 143.8(C-4), 143.5(C-1), 138.1(C-6), 123.4(C-
5), 32.9(C-11), 32.8(C-10), 22.2(C-12), 13.8(C-13); HRMS (ESI) m/z [M+H]+ calcd for
+
C10H14NO2 180.1025, found 180.1025. Data was in accordance with those reported [19].
4.2.4.2.
5-Benzylpicolinic acid (5a).
4a (500 mg) gave 5a (432 mg, 92%); white solid; >98% purity; mp 147–148.9 ◦C; 1H
NMR (600 MHz, DMSO-d6)
= 7.9 Hz, 1H, H12), 7.79 (dd,
δ
13.09 (s, 1H, OH), 8.64 (d,
J = 2.2 Hz, 1H, H9), 7.98 (d, J
J
= 2.3, 2.2 Hz, 2H, H13), 7.31-7.34 (m, 2H, H4, H6), 7.27-
7.28 (br, 2H, H2, H3), 7.21 (br, 1H, H2), 4.07 (s, 2H, H7); 13C NMR (151 MHz, DMSO-
d6) 166.5(C=O, C-14), 150.1(C-9), 146.8(C-8), 141.0(C-11), 140.3(C-5), 137.7(C-13),
δ
129.2(C-4, C-6), 129.1(C-1, C-3), 126.9(C-2), 125.0(C-12), 38.3(C-7); HRMS (ESI) m/z
+
[M+H]+ calcd for C13H12NO2 214.0868, found 214.0868.
4.2.4.3.
Characterization data of compounds 5b-m and 2´: see supporting
information.
4.2.5. General synthetic procedure for compounds 7a
–c.
A mixture of methyl 5-hydroxypicolinate (1.0 g, 4.6 mmol, 1 equiv.), the
6
appropriate alkyl halide (5.06 mmol, 1.1 equiv.) potassium carbonate (1.3 g, 9.2 mmol, 2
equiv.) and DMF (5 ml) was stirred at room temperature for 18 - 48 hours. Water (100
ml) was added and the mixture was extracted with EtOAc (4 x 100 ml). The organic layer
was washed with brine (200 ml) dried with anhydrous Na2SO4(s) and concentrated. The
residue was purified by silica-gel flash column chromatography (eluent: ethyl
acetate/heptane = 1:1) to give 7a–c
.
17

ACCEPTED MANUSCRIPT
4.2.5.1.
Methyl 5-propoxypicolinate (7a).
◦
From
6 (1 g) gave 7a (1.13 g, 89%); yellow semi-solid; >98% purity; mp 38–40 C;
1H NMR (600 MHz, CDCl3)
δ
8.39 (d, J = 2.8 Hz, 1H; H2), 8.10 (d , J = 8.7 Hz, 1H, H5),
7.25 (dd,
J
= 2.9, 2.9 Hz, 1H, H6), 4.03 (t,
J = 6.5 Hz, 2H, H12), 3.98 (s, 3H, H10), 1.83-
1.88 (m, 2H, H13), 1.07 (t,
J
= 7.4, 3H, H14); 13C NMR (151 MHz, CDCl3)
δ 165.5(C=O,
C-7), 157.8(C-4), 139.9(C-1), 138.6(C-2), 126.5(C-6), 120.2(C-5), 70.2(C-12), 52.6(C-
+
10), 22.4(C-13), 10.4(C14); HRMS (ESI) m/z [M+H]+ calcd for C10H14NO3 196.0974,
found 196.0974.
4.2.5.2.
From
NMR (600 MHz, CDCl3) δ 8.39 (d,
Methyl 5-(cyclopropylmethoxy)picolinate (7b).
(1 g) gave 7b (1.10 g, 79%); white solid; >98% purity; mp 53.9–54.8 ◦C; H
= 6 Hz, 1H, H2), 8.08 (dd, = 0.6, 0.6 Hz, 1H, H5),
1
6
J
J
7.25 (dd,
J
= 2.9, 2.9 Hz, 1H, H6), 3.98 (s, 3H, H10), 3.93 (d, J = 6.7 Hz, 2H, H12), 1.27-
13
1.33 (m, 1H, H13), 0.67-0.70 (m, 2H, H14, H16), 0.39-0.41 (m, 2H, H14, H16); C
NMR (151 MHz, CDCl3) 165.4(C=O, C-7), 157.7(C-4), 139.9(C-1), 138.6(C-2), 126.5(C-
6), 120.2(C-5), 73.4(C-12), 52.5(C-10), 9.99(C-13), 3.3(C-14, C-16); HRMS (ESI) m/z
+
[M+H]+ calcd for C11H14NO3 208.0974, found 208.0974.
4.2.5.3.
From
NMR (600 MHz, CDCl3)
Methyl 5-(2-oxo-2-(phenylamino)ethoxy)picolinate (7c).
(1 g) gave 7c (1.5 g, 83%); yellow solid; >98% purity; mp 185–186 C; H
8.54 (d, = 2.9 Hz, 1H, H13), 8.18 (d, = 8.8 Hz, 2H, H16,
◦
1
6
δ
J
J
H17), 7.59 (d,
J
= 7.9 Hz, 2H, H4, H6), 7.37 (dd,
J = 2.6, 6.5 Hz, 3H, H1, H2, H3), 7.18
(t,
J
= 7.4 Hz, 1H, NH), 4.72 (s, 2H, H9), 4.00 (s, 3H, H21); 13C NMR (151 MHz, CDCl3)
δ
165.0(C=O, C-8), 164.5(C=O, C-18), 155.6(C-12), 141.9(C-15), 138.7(C-13), 136.4,
18

ACCEPTED MANUSCRIPT
129.2 (C-4, C-6), 126.7(C-5), 125.3, 120.9(C-2), 120.3(C-4, C-6), 67.6(C-9), 52.9(C-21);
+
HRMS (ESI) m/z [M+H]+ calcd for C15H15N2O4 287.1032, found 287.1030.
4.2.6. Synthetic procedure for compounds 8a
Compounds 8a were synthesized from 7a
procedure as described for 5a
4.2.6.1. 5-Propoxypicolinic acid (8a).
From 7a (200 mg) gave 8a (149 mg, 80%); white solid; >98% purity; mp 128.9–
-c.
-
c
-c (200 mg scale) using the same
-m.
◦
1
129.2 C; H NMR (600 MHz, DMSO-d6)
δ
12.82 (s, 1H, OH), 8.35 (t,
= 2.5, 2.6 Hz, 1H, H6), 4.03 (td,
=1.7, 1.7, 1.7, 3H, H14);
J = 2.4 Hz, 1H,
H2), 8.01 (dd , = 1.7, 1.7 Hz, 1H, H5), 7.48 (dt,
J
J
J =
1.7, 1.7, 1.7 Hz, 2H, H12), 1.73-1.79 (m, 2H, H13), 0.98 (td,
13C NMR (151 MHz, DMSO-d6)
J
δ
166.2(C=O, C-11), 157.7(C-4), 140.7(C-1), 138.3(C-
2), 126.7(C-6), 121.1(C-5), 70.2(C-12), 22.3(C-13), 10.7(C-14); HRMS (ESI) m/z
+
[M+H]+ calcd for C9H12NO3 182.0817, found 182.0815.
4.2.6.2.
5-(Cyclopropylmethoxy)picolinic acid (8b).
From 7b (200 mg) gave 8b (131 mg, 70%); white solid; >98% purity; mp 138–139.5
1
◦C; H NMR (600 MHz, DMSO-d6)
δ
12.81 (s, 1H, OH), 8.36 (d,
J
= 2.7 Hz, 1H, H2),
= 7.1 Hz,
8.01 (dd, = 0.5, 0.5 Hz, 1H, H5), 7.48 (dd,
J
J
= 2.9, 2.9 Hz, 1H, H6), 3.99 (d, J
2H, H11), 1.23-1.29 (m, 1H, H12), 0.59-0.62 (m, 2H, H13, H14), 0.35-0.38 (m, 2H, H13,
H14); 13C NMR
δ
(151 MHz, DMSO-d6) 166.2(C=O, C-7), 157.7(C-4), 140.7(C-1),
138.4(C-2), 126.6(C-6), 121.2(C-5), 73.4(C-11), 10.3(C-12), 3.6 (C-13, C-14); HRMS
+
(ESI) m/z [M+H]+ calcd for C10H12NO3 194.0817, found 194.0816.
19

ACCEPTED MANUSCRIPT
4.2.6.3.
5-(2-Oxo-2-(phenylamino)ethoxy)picolinic acid (8c).
For 4 h of reaction time of hydrolysis 7c (200 mg) gave 8c (139 mg, 73%); yellow
solid; >98% purity; mp 225–226 ◦C; 1H NMR (600 MHz, DMSO-d6)
δ
12.9 (s, 1H, OH),
10.18 (s, 1H, NH), 8.45 (d,
J = 2.9 Hz, 1H, H13), 8.04 (d, J = 8.7 Hz, 1H, H16), 7.62
(dbr, 2H, H4, H6), 7.53 (dd, J = 2.9, 2.9 Hz, 1H, H17), 7.31-7.35 (m, 2H, H1, H3), 7.09
13
(m, 1H, H2), 4.91 (s, 2H, H9); C NMR (151 MHz, DMSO-d6)
δ 166.1(C=O, C-12),
166.1(C=O, C-7), 157.0(C-4), 141.5(C-1), 138.7(C-6), 138.6(C-13), 129.2(C-1, C-3),
126.6(C-16), 124.3(C-2), 121.6(C-17), 120.2(C-4, C-6), 67.6(C-9); HRMS (ESI) m/z
+
[M+H]+ calcd for C14H13N2O4 273.0875, found 273.0873.
4.2.6.4.
5-(Carboxymethoxy)picolinic acid (8c´).
Compound 8c´ was obtained from 7c after 24h of hydrolysis. 7c (200 mg) gave 8c´
◦
1
(127 mg, 92%); >98% purity; mp 263.9–264.5 C; yellow solid; H NMR (600 MHz,
DMSO-d6)
δ
13.04 (br, 2H, OH), 8.38 (d,
J = 2.9 Hz, 1H, H2), 8.02 (d, J = 8.7 Hz, 1H,
H5), 7.48 (dd,
J
= 2.9, 3.0 Hz, 3H, H6), 4.90 (s, 2H, H11); 13C NMR (151 MHz, DMSO-
d6)
δ
169.9(C=O, C-12), 166.1(C=O, C-7), 156.9(C-1), 141.3(C-4), 138.4(C-2), 126.5(C-
+
5), 121.5(C-6), 65.2(C-11); HRMS (ESI) m/z [M+H]+ calcd for C8H8NO5 198.0402,
found 198.0401.
4.2.7. N-Propyl-5-(propylamino)picolinamide (10a).
A mixture of 2a (500 mg, 2.3 mmol), propylamine (0.3 ml, 3.45 mmol, 1.5 equiv.),
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex
with
dichloromethane (168 mg, 6 mol %), cesium carbonate (2.2 g, 6.9 mmol, 2.0 equiv.) and
toluene (10 ml) were added to a 20 ml microwave reaction vial (Biotage). The vial was
20

ACCEPTED MANUSCRIPT
sealed and irradiated in a microwave apparatus at 110 °C, high absorption for 45 min.
The reaction mixture was cooled to room temperature; added 100 ml of water and the
resultant mixture was extracted with ethyl acetate (4 x 100 ml). The organic layer was
washed with brine (2 x 200 ml), dried with anhydrous Na2SO4(s). The residue after
concentration was purified by silica-gel flash column chromatography (eluent: ethyl
acetate/heptane = 1:1) to give 372 mg of 10a. Yield 73 %; colorless oil; >98% purity; 1H
NMR (600 MHz, CDCl3):
δ
7.99 (d,
J
= 8.6 Hz, 1H, H2), 7.87 (d ,
J
= 2.7 Hz, 1H, H5),
= 6.8, 6.4
7.78 (br, 1H, NH), 6.90 (dd,
J
= 2.8, 2.8 Hz, 2H, H6), 4.09 (br, 1H), 3.41 (dd,
J
Hz, 2H, H14), 3.14 (t, J = 7.1, 2H, H10) 1.60-1.71 (m, 4H, H11, H15), 0.96-1.03 (m, 6H;
13
H12, H16); C NMR (151 MHz, CDCl3)
δ 164.9(C=O, C-7), 146.3(C-1), 139.1(C-4),
133.6(C-5), 123.3(C-2), 117.7(C-6), 45.1(C-10), 41.0(C-14), 23.1(C-15), 22.5(C-11),
11.5(C-12), 11.5(C-16); HRMS (ESI) m/z [M+H]+ calcd for C12H20N3O+ 222.1606, found
222.1606.
4.2.8. Ethyl 5-((ethylthio)methyl)picolinate (10b
).
A solution of ethyl 5-(chloromethyl)picolinate
9
(500 mg, 2.5 mmol, 1 equiv.),
ethanethiol (233 mg, 3.75 mmol, 1.5 equiv.) and potassium carbonate (690 mg, 5 mmol, 2
equiv.) in 5 ml DMF was transferred to a 20 ml round-bottom flask. The mixture was
stirred overnight at room temperature. When starting materials were consumed 100 ml of
water was added and the mixture was extracted with ethyl acetate (4 x 100 ml). The
organic layer was washed with brine (2 x 200 ml) and dried with anhydrous Na2SO4(s).
The residue after concentration was purified by silica-gel flash column chromatography
(eluent: ethyl acetate/heptane = 1:1) to obtain 10b. Alternatively a microwave reactor was
used. By transferring, then sealed and irradiated in a microwave apparatus at 60 °C, high
21

ACCEPTED MANUSCRIPT
absorption for 30 min. Workup procedure is the same as solution phase stated above of
1
method 1 to obtain 366 mg of 10b. Yield 65%; yellow oil; >98% purity; H NMR (600
MHz, CDCl3):
δ
8.66 (d,
J
= 2.2 Hz, 1H, H2), 8.10 (d ,
= 7.1, 7.1 Hz, 2H, H10), 3.76 (s, 2H, H11), 2.43 (dd,
= 7.2, 3H, H15), 1.23 (t,
J
= 7.2 Hz, 1H, H5), 7.83 (dd,
J
J
=
=
2.3, 2.2 Hz, 1H, H6), 4.48 (dd,
7.4, 7.4 Hz, 2H, H13), 1.48 (t,
J
J
J
= 7.4 Hz, 3H, H14); 13C NMR
(151 MHz, CDCl3)
δ 165.1(C=O, C-7), 150.0(C-2), 147.0(C-4), 138.3(C-1), 137.2(C-6),
125.0(C-5), 61.9(C-10), 32.8(C-11), 25.4(C-13), 14.4(C-15), 14.3(C-14); HRMS (ESI)
m/z [M+H]+ calcd for C11H16NO2S+ 226.0902, found 226.0903.
Acknowledgements
This work was supported by the University of Copenhagen Excellency Program for
Interdisciplinary Excellence (KU2016) and by a grant from Kirsten & Freddy Johansen
Foundation to M.G.
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