Synthesis and Antimicrobial Activity of Burkholderia-Related 4-Hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and Their N-Oxide Counterparts

Article abstract of DOI:10.1021/acs.jnatprod.0c00171

The Burkholderia genus offers a promising potential in medicine because of the diversity of biologically active natural products encoded in its genome. Some pathogenic Burkholderia spp. biosynthesize a specific class of antimicrobial 2-alkyl-4(1H)-quinolo

Full text of DOI:10.1021/acs.jnatprod.0c00171

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Article
Synthesis and Antimicrobial Activity of Burkholderia-Related
4‑Hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and Their
N‑Oxide Counterparts
́
Marianne Piochon, Pauline M. L. Coulon, Armand Caulet, Marie-Christine Groleau, Eric Deziel,*
and Charles Gauthier*
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ABSTRACT: The Burkholderia genus offers a promising potential in
medicine because of the diversity of biologically active natural products
encoded in its genome. Some pathogenic Burkholderia spp. biosynthesize
a specific class of antimicrobial 2-alkyl-4(1H)-quinolones, i.e., 4-
hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and their N-oxide
derivatives (HMAQNOs). Herein, we report the synthesis of a series
of six HMAQs and HMAQNOs featuring a trans-Δ2 double bond at the
C2-alkyl chain. The quinolone scaffold was obtained via the Conrad−
Limpach approach, while the (E)-2-alkenyl chain was inserted through
Suzuki−Miyaura cross-coupling under microwave radiation without
noticeable isomerization according to the optimized conditions.
Subsequent oxidation of enolate-protected HMAQs cleanly led to the formation of HMAQNOs following cleavage of the ethyl
carbonate group. Synthetic HMAQs and HMAQNOs were evaluated in vitro for their antimicrobial activity against different Gram-
negative and Gram-positive bacteria as well as against molds and yeasts. The biological results support and extend the potential of
HMAQs and HMAQNOs as antimicrobials, especially against Gram-positive bacteria. We also confirm the involvement of HMAQs
in the autoregulation of the Hmq system in Burkholderia ambifaria.
he Burkholderia genus includes a vast group of Gram-
Burkholderia spp. biosynthesize a specific class of AQs,
namely, 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs, 1−
3) or 2-alkenyl-3-methylquinolin-4(1H)-ones according to
IUPAC, which feature a methyl group at C3 and a trans-Δ2
unsaturation at the C2-alkyl chain (Figure 1).17−27 HMAQs 1
1,2
Tnegative bacteria found in diverse ecological niches.
Some Burkholderia spp. are of serious pathogenic concerns,
such as the Centers for Disease Control and Prevention
(CDC) Tier 1 select agents3 Burkholderia pseudomallei and B.
malleithe infectious agents of melioidosis4,5 and glanders,6
respectivelyand the devastating plant crop pathogens B.
glumae and B. gladioli, which cause major yield losses in rice
productions.7,8 Others, like those forming the B. cepacia
complex, include species that can both live in beneficial
associations with their eukaryotic hosts (mammals, plants, and
fungi)2 and cause several hard-to-treat opportunistic infections,
such as the cepacian syndrome in individuals suffering from
cystic fibrosis.9
Burkholderia spp. offer a tantalizing potential in medicine
because of their capacity to produce highly potent and
structurally diverse metabolites.10,11 A plethora of natural
products exhibiting various biological functions have been
identified from Burkholderia spp.10 To name a few examples for
B. pseudomallei, cytotoxic siderophores (e.g., malleilactone),12
proteasome inhibitors (e.g., deoxyglidobactin C),13 tensioac-
tive lipopeptides (e.g., malleipeptin A),13 and rhamnolipids,14
as well as quorum sensing signals and modulators such as N-
acyl homoserine lactones (AHLs),15 and 2-alkyl-4(1H)-
quinolones (AQs)16,17 have been reported.
Figure 1. Chemical structures of Burkholderia-related HMAQs 1−3
and HMAQNOs 4−6. The ketorather than the enolform of the
quinolone ring is arbitrarily drawn throughout the paper.
Received: February 15, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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and 3, respectively the heptenyl and nonenyl congeners, exhibit
potent antifungal19,20,22,24,25 and antibacterial19,27 activities
against diverse microorganisms as well as plant growth
promoting activities.22 Furthermore, HMAQs 1 and 3 act as
potent modulators of quorum sensing in B. ambifaria HSJ1 by
decreasing the production of AHLs.17 HMAQ 2 (burkholone),
the 2-octenyl congener, is cytotoxic against a murine
hemopoietic cell line devoid of the insulin receptor substrate
1.28 HMAQs are also found in the form of their N-oxide
counterparts (HMAQNOs 4−6, Figure 1).29,30 HMAQNO 6
(YM-30059), identified from both Burkholderia and Arthro-
bacter spp., is a potent antibiotic agent against multi-drug-
resistant Staphylococcus aureus, S. epidermidis, and Bacillus
subtilis strains.31 Interestingly, 2-heptyl-4(1H)-quinolone N-
oxide (HQNO)the unmethylated and saturated analogue of
HMAQNO 4is able to act synergistically with HMAQ 3,
enabling inhibition of bacterial growth while showing divergent
biological functions when tested alone.32 Taken together, these
studies show that subtle structural modifications can
profoundly impact the biological activities of AQs.
Although an obvious evolutionary advantage for bacteria, the
large number of closely related AQ congeners (>29)17 of
similar polarities can complicate the purification process from
bacterial cultures. Highly potent AQs could be found as minor
contaminants within samples of other less potent AQs isolated
from bacterial extracts, generating experimental bias regarding
the biological activity of single compounds. Chemical synthesis
has a long-standing record of success regarding the preparation
of pure and homogeneous natural products.33−35 Well-
designed synthetic pathways can allow the preparation of
natural products and analogues in sufficient amounts for
enabling the confirmation of structural identities and biological
activities as well as for sustaining drug discovery and
pharmaceutical development.35,36 Within this framework, we
herein report the synthesis, antifungal, and antibacterial
activities of HMAQs 1−3 and HMAQNOs 4−6 identified
from Burkholderia spp. We also confirm their involvement in
the autoregulation of the Hmq system, which is responsible for
synthesizing HMAQs in B. ambifaria.37,38
Figure 2. Retrosynthetic analysis of HMAQs 1−3 and HMAQNOs
4−6.
regioisomer could also be formed during the formation of the
quinolone core III, i.e., the alkyl chain at C2 rather than at
C3,42 a second route was planned in which HMAQ scaffold IV
would be obtained via the Conrad−Limpach approach.
Functionalization of derivative IV into 2-methyl chloride II
followed by Suzuki−Miyaura cross-coupling with boronic ester
alkenyl chains would provide HMAQs. Finally, regioselective
oxidation of HMAQs would yield HMAQNOs.
Synthesis of HMAQs. The synthesis of quinolone scaffold
S6 via the Luo et al.42 procedure was first attempted (see
Scheme S1 in the SI file). Oxazolinylphenylamine S3 was
synthesized beforehand through zinc(II) chloride-promoted
condensation of isatoic anhydride (S1) with 2-amino-2-
methylpropanol (S2).43 Then, oxazoline S3 was subjected to
acid-catalyzed condensation−cyclization with ethyl 3-oxopen-
tanoate (S4) in refluxing n-butanol,42 which indeed led to a
quinolone derivative. However, NMR analyses proved that the
opposite regioisomer S544 was exclusively formed instead of
target derivative S6. This result was rationalized by the
formation of an imine, which, once tautomerized into the most
stable enamine intermediate, would favor the 6-exo-trig
cyclization.42 In our case, as regioisomer S5 was formed, the
most stable enamine would come from proton transfer
tautomerization at the C2 rather than at the C4 position of
the pentanoate chain.
RESULTS AND DISCUSSION
■
Retrosynthetic Analysis. Two important challenges had
to be addressed for the total synthesis of HMAQs,39,40 i.e., the
formation of the 2,3-dialkylatedquinolin-4(1H)-one core along
with the presence of an unconjugated trans-Δ2 unsaturation at
the 2-alkyl chain. Synthetic routes in which the 2-alkenyl chain
would be attached to the molecule prior to the acid- or base-
catalyzed cyclization for the formation of the quinolone core41
would possibly result in failure because of the thermodynami-
cally favorable isomerization of the double bond occurring
from the trans-Δ2 to the trans-Δ1 position.
As depicted in our retrosynthetic strategy (Figure 2), we
envisioned to study two alternative approaches to construct the
quinolone scaffold. In both synthetic routes, the (E)-2-alkenyl
chain would be attached to the quinolone core at the final step
via either a Wittig reaction or a Suzuki−Miyaura cross-
coupling, theoretically avoiding the risk of double-bond
isomerization. For the first route, aldehyde I would serve as
an advanced intermediate for the Wittig reaction. It would
come from the reduction of corresponding ester III, which
would be generated via the para-toluenesulfonic acid (PTSA)-
catalyzed reaction of 2-(4,4-dimethyl-1,3-oxazolin-2-yl)-
phenylamine with ethyl 3-oxopentanoate. As the opposite
We then turned our attention to the Conrad−Limpach
approach.45−47 This route was straightforward and rather high-
yielding for the formation of the quinolone scaffold (Scheme
1). Aniline (7) was condensed with diethyl 2-methyl-3-
oxosuccinate (8) in the presence of acetic acid to furnish
diester 9, which underwent polyphosphoric acid (PPA)-
promoted intramolecular Friedel−Crafts acylation.48 Resulting
quinolone ester 10 was then reduced under the action of
B
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HMAQ 319,20,26,27 were in agreement with those published
for the isolated natural products.
Scheme 1. Synthesis of 2-(Chloromethyl)quinolone 11 via
Conrad−Limpach Approach
1
Table 2. H NMR Data [600 MHz, δ (ppm)] for HMAQs
1−3 in CDCl3
H no.
1
2
3
N−H
9.60 (1H, br s)
10.17 (1H, br s)
9.89 (1H, br s)
Ar−H 8.40−8.34 (1H, m) 8.41−8.34 (1H, m) 8.40−8.34 (1H, m)
7.32−7.23 (1H, m) 7.30−7.24 (1H, m) 7.32−7.23 (1H, m)
7.56−7.79 (1H, m) 7.56−7.45 (2H, m) 7.56−7.49 (2H, m)
7.43−7.36 (1H, m)
3.48 (2H, d, J = 6.5 3.50 (2H, d,(J = 6.4 3.49 (2H, d, J = 6.6
Hz) Hz) Hz)
5.47−5.72 (2H, m) 5.48−5.68 (2H, m) 5.48−5.69 (2H, m)
7.46−7.42 (1H, m)
1′
2′
3′
4′
2.06 (2H, dd,
2.02 (2H, dd,
J = 14.3, 7.1 Hz)
2.03 (2H, dd,
J = 14.2, 7.0 Hz)
J = 13.8, 7.0 Hz)
5′
6′
7′
1.39−1.25 (4H, m) 1.38−1.17 (6H, m) 1.39−1.19 (8H, m)
LiAlH4 and the primary alcohol chlorinated with SOCl2. Using
this synthetic route, ∼5 g of chloride 11 in convenient yields
and purity (as indicated by NMR) was prepared. It is
noteworthy that partial insolubility of most of the inter-
mediates in common organic solvents rendered the workups
and purification procedures of this synthetic route challenging
(see experimental procedure for details).
0.89 (3H, t,J = 7.1
Hz)
8′
9′
1″
n.a.
0.87 (3H, t, J = 7.1
Hz)
n.a.
n.a.
0.87 (3H, t, J = 7.0
Hz)
With large amounts of 2-methylchloridequinolone 11 in
hand, we next investigated the synthesis of the target HMAQs
1−3 via Suzuki−Miyaura cross-coupling.47,49 Commercially
available trans-alkenylboronic acid pinacol esters 12−14 were
reacted with chloride 11 under the catalytic action of
tetrakis(triphenylphosphine)palladium(0) using sodium car-
bonate as a base in a microwave-heated 1,4-dioxane−water
solution.47 As depicted in Table 1, although coupling yields
were better in microwave-heated than in classic refluxing
conditions (entry 1), isomerization occurred when prolonged
reaction times were employed (entries 2 and 3). As revealed by
1H NMR, the thermodynamically stable trans-Δ1 (15) and cis-
Δ1 (16) derivatives were formed through isomerization of
trans-Δ2 HMAQ 1. Pleasingly, we found that decreasing the
reaction time and temperature to 1 min and 70 °C,
respectively, avoided isomerization while still allowing full
conversion of the starting material (entries 4 to 6). Analytical
(HRMS) and spectral (1H and 13C NMR, Tables 2 and 3) data
of HMAQ 1,18−22,24,25,27 HMAQ 2 (burkholone),28 and
2.17 (3H, s)
2.18 (3H, s)
2.18 (3H, s)
Synthesis of HMAQNOs. To enable the vinylogous amine
oxidation of HMAQs, the quinolone ring needed to be locked
into its hydroxyquinoline tautomer. This was performed by
converting HMAQs 1−3 into their corresponding ethyl
carbonates 17−19 using potassium tert-butoxide as a base in
the presence of ethyl chloroformate.29,50 The latter derivatives
were then oxidized with mCPBA at low temperature (0 °C) to
afford N-oxide ethyl carbonates 20−22 (Scheme 2).50 The
moderate yields obtained for this reaction can be explained by
the concomitant peroxidation of the unconjugated trans-Δ2
double bond, leading to epoxide byproducts. Methyltrioxo-
rhenium was used as another oxidation system to improve the
reaction yields, but it mainly led to degradation. Deprotection
of ethyl carbonates 20−22 was cleanly performed under the
action of potassium hydroxide in ethanol, and, upon
acidification, HMAQNOs 4−6 precipitated in good yields
from the reaction mixture (Scheme 3). Analytical (HRMS)
Table 1. Synthesis of Target 2-Alkenylquinolones via Suzuki−Miyaura Reaction
a
b
entry
Bpin alkene
temp (°C)
time (min)
product (yield, %)
alkene selectivity ratio (E-Δ2:E-Δ1:Z-Δ1)
c
1
2
3
4
5
6
14
14
14
14
12
13
120
120
120
70
70
70
120
120
10
1
1
1
3 (32)
3 (78)
3 (58)
1:0:0
d
2.6:1.0:1.5
2.7:1.0:1.4
1:0:0
1:0:0
1:0:0
d
3 (78)
1 (70)
2 (74)
a
b
c
d
1
Isolated yields. Determined by H NMR analysis of the crude reaction mixture. Classical refluxing conditions. Isolated as an inseparable
mixture of three isomers.
C
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Table 3. 13C NMR Data [150 MHz, δ (ppm)] for HMAQs
1−6
Scheme 3. Final Step in the Synthesis of HMAQNOs
a
a
a
b
b
b
C no.
1
2
3
4
5
6
Ar−C
147.0
139.0
131.3
126.3
123.8
117.3
115.9
146.8
139.0
131.3
126.3
123.8
123.1
117.2
115.9
178.2
35.6
123.2
136.8
32.7
31.5
29.0
147.2
139.1
131.3
126.2
123.8
117.4
118.9
155.9
140.1
134.7
128.0
125.3
122.5
117.0
116.4
169.3
33.3
122.9
136.4
33.0
32.4
23.2
152.2
140.8
133.2
126.2
125.3
124.5
116.3
116.0
175.6
33.5
124.2
135.2
32.6
32.5
30.0
152.0
140.8
133.1
126.2
125.2
124.6
116.3
116.0
n.d.
1
Table 4. H NMR Data [600 MHz, δ (ppm)] for
CO
1′
2′
3′
4′
5′
6′
7′
8′
178.2
35.6
123.2
136.6
32.4
31.4
22.4
14.0
n.a.
178.2
35.6
123.2
136.5
32.7
31.8
29.3
29.0
22.7
14.2
10.7
HMAQNOs 4−6 in CD3OD
33.5
124.3
135.1
32.8
32.6
30.3
29.8
23.6
14.4
11.5
H no.
4
5
6
Ar−H 8.45−8.39 (1H, m)
8.32−8.26 (1H,
8.32−8.26 (1H,
m)
m)
7.73−7.68 (1H, m)
7.48−7.40 (1H,
7.48−7.38 (1H,
m)
m)
8.24−8.19 (1H, m)
8.01−7.95 (1H,
8.01−7.94 (1H,
22.6
14.2
n.a.
10.6
14.2
n.a.
n.a.
11.8
23.5
14.4
n.a.
11.5
m)
m)
8.01−7.94 (1H, m)
8.79−7.71 (1H,
8.79−7.71 (1H,
9′
1″
n.a.
10.6
m)
m)
1′
2′
3.96 (2H, dd, J = 5.7,
3.81−3.74 (2H,
3.83−3.73 (2H,
a
b
0.8 Hz)
m)
m)
In CDCl3. In CD3OD.
5.69−5.57 (2H, m)
5.64−5.55 (2H,
5.64−5.54 (2H,
m)
m)
and spectral (1H and 13C NMR, Tables 3 and 4) data of
HMAQNO 631 were in agreement with those published for the
isolated natural product. Analytical and spectral data for
HMAQNOs 4 and 5 had not been reported until now.
3′
4′
2.05 (2H, dd, J = 13.4, 2.06−1.98 (2H,
2.08−1.99 (2H,
6.5 Hz)
m)
m)
5′
1.46−1.21 (4H, m)
1.39−1.21 (6H,
1.41−1.18 (8H,
m)
m)
Antimicrobial Evaluation of HMAQs and HMAQNOs
1−6. Several studies have proposed that HMAQs act as potent
antimicrobial molecules.24,25,51 However, these studies focused
only on the extraction of active molecules from bacterial
culture supernatants. As previously mentioned, the isolated
metabolites could contain contaminants affecting the biological
results. To validate previous data and better characterize the
biological activity, nine bacteria and 10 fungi were tested for
their susceptibility to HMAQs 1−3 and HMAQNOs 4−6
using two different antimicrobial methods, i.e., the Kirby−
Bauer disk diffusion test and the broth microdilution assay.
Our results using the Kirby−Bauer method showed that
HMAQs 1−3 did not affect any of the bacteria grown on solid
medium (Figure 3 and Figure S1). Among the Gram-negative
bacteria, HMAQNOs 4−6 only strongly inhibited the growth
of Actinobacillus pleuropneumoniae, and some activity was also
noted against Xanthomonas campestris. On the other hand, all
the tested Gram-positive bacteria, i.e., B. subtilis, S. aureus,
Streptococcus agalactiae, and Paenibacillus peoriae, were sensitive
to HMAQNOs 4−6, but sometimes with significant differ-
ences between congeners (Figure 3). Furthermore, both
HMAQs 1−3 and HMAQNOs 4−6 were able to inhibit the
6′
7′
8′
0.88 (3H, t, J = 7.2 Hz)
n.a.
0.86 (3H, t, J = 7.0
Hz)
9′
n.a.
n.a.
0.85 (3H, t, J = 7.0
Hz)
1″
2.39 (3H, s)
2.22 (3H, s)
2.21 (3H, s)
growth of Cryptococcus neoformans, whereas they had no effect
against Candida albicans (Figure 3 and Figure S1). The length
of the alkyl chain of HMAQs did not impact the activity
against C. neoformans, whereas the yeast was more sensitive
toward HMAQNOs 5 and 6 than toward HMAQNO 4.
Surprisingly, all eight molds were resistant to HMAQs 1−3
and HMAQNOs 4−6, meaning that previous reports showing
activity of HMAQs against different molds were presumably
due to other copurified metabolites (Figure 3 and Figure
S2).24,25,51
As we were not able to detect any antimicrobial activity for
HMAQs 1−3 against bacteria and molds, we decided to use
the more sensitive broth microdilution method. Importantly, as
Scheme 2. Synthesis of Protected N-Oxide Quinolones
D
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For A. pleuropneumoniae, B. subtilis, and S. aureus, the
observed activity increased with the length of the chain,
suggesting that the activity of HMAQs 1−3 is probably linked
to the alkyl chain portion and not to the quinolone ring (Table
5 and Figure S4). Wu and Seyedsayamdost32 demonstrated by
bacterial cytological profiling and primary metabolite analysis
in Escherichia coli that HMAQ 3 acts like monensin on the
proton gradient of the proton motive force. They also showed
that both HQNO and HMAQ 3 act synergistically by
competing with coenzyme Q and inhibiting the synthesis of
pyrimidines.32 Hypothesizing that HMAQNOs 4−6 act
following the same mechanism as HQNO, HMAQs 1−3 and
HMAQNOs 4−6 could also play a synergic role together or
with clinically used antibiotics; this needs to be further
confirmed.
As expected, C. neoformans was more sensitive to HMAQs
1−3 than HMAQNOs 4−6 also in liquid medium, while we
found that C. albicans was resistant to HMAQs 1−3 and
HMAQNOs 4−6 in liquid medium (Table 5 and Figure S5).
F. oxyporum, F. solani, and Mucorales were slightly sensitive to
HMAQs 1−3 and resistant to HMAQNOs 4−6, while A. niger
was resistant to HMAQs 1−3 and HMAQNOs 4−6 in
microdilution (Table 5 and Figure S5).
Action of HMAQs 1−3 on B. ambifaria Quorum
Sensing. As reported in a previous study, the activity of an
hmqA-lacZ reporter was higher in an hmqA- mutant than in the
WT strain. Adding purified HMAQs restored the activity to
the WT levels. We thus repeated this experiment with the
addition of synthetic HMAQs (Figure 4). The results
confirmed that adding HMAQs lowered the expression from
the hmqABCDEFG promoter in the hmqA− mutant.
CONCLUSION
■
In summary, we have accomplished the total synthesis of
HMAQs 1−3 via the Conrad−Limpach approach for the
formation of the quinolone core followed by a microwave-
heated Suzuki−Miyaura cross-coupling for the insertion of the
(E)-2-alkenyl chain while avoiding isomerization of the
nonconjugated trans-Δ2 double bond. Subsequent oxidation
of the protected enolate form of HMAQs 1−3 cleanly
provided HMAQNOs 4−6. This series of AQs (1−6)
produced by Burkholderia spp. was obtained in pure and
homogeneous forms, which allowed us to confirm and extend
their roles as antimicrobials. Strikingly, HMQANOs 4−6 were
found to be highly active against Gram-positive as compared to
Gram-negative bacteria. Future investigations are required to
determine if HMAQs 1−3 and HMAQNOs 4−6 have the
same targets in both Gram-negative and Gram-positive bacteria
than HQNO and PQS. According to previous studies, we
believe that HMAQs and HMAQNOs are promising
metabolites to be used in synergy with other antibiotics.
Figure 3. Antimicrobial activity of HMAQs 1−3 and HMAQNOs 4−
6 using the Kirby−Bauer method. (A) Antimicrobial activity of
HMAQNOs 4−6 tested against different Gram-negative and Gram-
positive bacteria. (B) Antimicrobial activity of HMAQs 1−3 and
HMAQNOs 4−6 against C. neoformans H99. MeOH was used as the
control in assays with HMAQs 1−3 and DMSO with HMAQNOs 4−
6. Means with standard error to the mean are shown. A one-way
ANOVA statistical test was performed (**p value <0.01).
HMAQs 1−3 and HMAQNOs 4−6 are relatively poorly
soluble in water, the highest possible tested concentration was
100 μM, corresponding to 25.6, 27, 28.4, 27.2, 28.6, and 30
μg/mL for respectively HMAQ 1, HMAQ 2, HMAQ 3,
HMAQNO 4, HMAQNO 5, and HMAQNO 6. In general, the
bacteria were once again more sensitive to HMAQNOs 4−6
than to HMAQs 1−3 (Table 5). The growth was completely
inhibited at 100 μMor 27.2, 28.6, and 30 μg/mL for
HMAQNO 4, HMAQNO 5, and HMAQNO 6 respectively
for S. aureus and B. subtilis (Figure S4). These results correlate
with the already reported antimicrobial effect of HQNO and
the growth inhibition effect of 2-heptyl-3-hydroxy-4-quinolone
(PQS) from Pseudomonas aeruginosa.52−55 HMAQNOs 4−6
could act like HQNO and target (a) the bacterial respiration
by inhibiting the cytochrome b1 in Gram-positive; (b) the Na
+− pumping activity of the NADH−quinone reductase system
in Gram-negative; and (c) the cytochrome c in eukaryotes, as it
has already been shown in S. aureus, B. subtilis, and Vibrio
alginolyticus as well as on beef heart mitochondria par-
ticles.53,56,57
EXPERIMENTAL SECTION
■
General Experimental Procedures. All chemicals and starting
materials were purchased from commercial sources and used without
further purification. Air- and water-sensitive reactions were performed
in heat gun-dried glassware under an argon atmosphere. Anhydrous
solvents (dichloromethane, THF) were dried for at least 3 days over 4
Å molecular sieves previously activated by heating with a heat-gun for
∼15 min under high vacuum. Reactions in a microwave were
performed on a CEM Discover SP microwave coupled with an
Explorer 12 Hybrid autosampler (Matthews, NC, USA). All reactions
were monitored by thin-layer chromatography (TLC) with silica gel
60 F2540.25 mm precoated glass plates. Compounds were visualized
E
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Table 5. Minimal Inhibitory Concentration of HMAQs 1−3 and HMAQNOs 4−6 against Microorganisms
minimal inhibitory concentration [μg/mL] (μM)
microorganism
HMAQ 1
HMAQ 2
HMAQ 3
HMAQNO 4
HMAQNO 5
HMAQNO 6
Gram-negative bacteria
A. pleuropneumoniae
E. coli
P. aeruginosa
P. fluorescens
X. campestris
Gram-positive bacteria
B. subtilis
P. peoriae
S. aureus
S. agalactiae
Fungi
0.80 (3.13)
25.60 (100)
NA
6.40 (25)
25.60 (100)
0.42 (1.56)
27.00 (100)
13.50 (50)
6.75 (25)
0.44 (1.56)
28.40 (100)
NA
14.20 (50)
28.40 (100)
0.42 (1.56)
6.80 (25)
13.60 (50)
13.60 (50)
1.70 (6.25)
0.45 (1.56)
1.79 (6.25)
7.15 (25)
7.15 (25)
0.90 (3.13)
0.47 (1.56)
1.88 (6.25)
3.75 (12.5)
7.50 (25)
a
27.00 (100)
3.75 (12.5)
0.40 (1.56)
3.20 (12.5)
0.40 (1.56)
0.40 (1.56)
0.42 (1.56)
3.38 (12.5)
0.42 (1.56)
0.42 (1.56)
0.44 (1.56)
3.55 (12.5)
0.44 (1.56)
0.44 (1.56)
0.42 (1.56)
0.42 (1.56)
0.42 (1.56)
0.42 (1.56)
0.45 (1.56)
0.45 (1.56)
0.45 (1.56)
0.45 (1.56)
0.94 (3.13)
0.47 (1.56)
0.47 (1.56)
0.47 (1.56)
C. neoformans
C. albicans
0.80 (3.13)
NA
0.42 (1.56)
NA
0.44 (1.56)
NA
13.60 (50)
NA
3.58 (12.5)
NA
1.88 (6.25)
NA
A. niger
NA
NA
NA
NA
NA
NA
F. oxyporum
F. solani
Mucorales
3.20 (12.5)
NA
1.60 (6.25)
NA
NA
3.38 (12.5)
3.55 (12.5)
1.78 (6.25)
3.55 (12.5)
NA
NA
NA
NA
NA
NA
NA
NA
NA
a
NA: nonactive at the maximum tested concentration (MIC > 100 μM for each molecule or 25.6, 27, 28.4, 27.2, 28.6, and 30 μg/mL for HMAQ 1,
HMAQ 2, HMAQ 3, HMAQNO 4, HMAQNO 5, and HMAQNO 6, respectively).
cooling to rt, water (100 mL) was gently poured and the mixture was
transferred into a separatory funnel. The aqueous phase was extracted
with DCM (3 × 100 mL), and the combined organic layers were
dried over anhydrous MgSO4 and evaporated under reduced pressure.
The residue was purified by silica gel flash chromatography (hexanes−
EtOAc, 100:0 to 95:5) to give diester 9 as a yellow oil (13.9 g, 68%).
Spectral and analytical data of diester 9 were in accordance with those
published.47 Diester 9 (13.6 g, 49.1 mmol) and polyphosphoric acid
(68 g) were refluxed at 130 °C for 2 h under argon with strong
stirring. The resulting slurry was poured slowly into a large beaker
containing an iced solution of saturated aqueous NaHCO3. The
precipitate was filtered and washed with cold water and cold diethyl
ether. The residue was then recovered with acetone and concentrated
under reduced pressure to give ester 10 (10.5 g, 93%) as a beige
amorphous powder. Spectral and analytical data of ester 10 were in
accordance with those published.47 Ester 10 (8.58 g, 37.1 mmol) was
dissolved in anhydrous THF (408 mL), and the mixture was cooled at
0 °C. The reducing agent LiAlH4 (2.81 g, 74.2 mmol) was then slowly
added, and the reaction was stirred at rt for 2 h. To quench the
reaction, the mixture was cooled in an ice/water bath, and cold
EtOAc (40 mL) was poured very slowly using an addition funnel.
MeOH (10 mL) was then added dropwise, and the mixture was
evaporated under reduced pressure. The residue (dry pack) was
purified on silica gel flash chromatography (DCM−MeOH, 10:0 to
8:2) to obtain the desired alcohol as a yellow amorphous powder
(5.58 g, 78%). The latter compound (5.58 g, 29.5 mmol) was
dissolved in anhydrous DCM (221 mL) and cooled to 0 °C. Thionyl
chloride (21.4 mL, 295 mmol) was then added dropwise. The
reaction was stirred at rt for 90 min. The mixture was evaporated
under reduced pressure and resuspended in MeOH. Toluene was
added, and the mixture was coevaporated three times under reduced
pressure in the presence of silica (ratio 3:1). The resulting dried pack
was purified by silica gel flash column chromatography (DCM−
MeOH, 10:0 to 9:1) to afford chloride 11 as a white amorphous solid
(5.06 g, 83%). Spectral and analytical data of chloride 11 were in
accordance with those published.47
Figure 4. β-Galactosidase activity of an hmqA-lacZ reporter in B.
ambifaria HSJ1 hmqA- following the addition of HMAQs. DMSO was
added in controls. ANOVA statistical tests were performed giving
****p value < 0.0001, ***p value < 0.001, **p value < 0.01, and *p
value < 0.05.
by UV254. Flash column chromatography was carried out using silica
1
gel 60 Å (15−40 μm). H and 13C NMR spectra were recorded on
600 MHz Bruker Advance III NMR spectrometers. Elucidations of
chemical structures were based on 1H and 13C and spectral
assignments, which were confirmed by 2D experiments (COSY and
HSQC). Chemical shifts (δ) are reported in parts per million (ppm)
relative to residual solvent peaks or tetramethylsilane (TMS, δH = δC
= 0 ppm), and coupling constants are expressed in hertz (Hz). The
labile OH and NH signals appearing sometimes were not listed. High-
resolution mass spectra (HRMS) were recorded on an ESI-Q-TOF
General Procedure for the Synthesis of HMAQs. To a
solution of chloride 11 (1.0 equiv) and corresponding trans-
alkenylboronic acid pinacol ester (1.3 equiv) in 1,4-dioxane (8.3
mL mmol−1) was added aqueous sodium carbonate (2 M, 8.3 mL
mmol−1). After bubbling with nitrogen for 5 min, Pd(PPh3)4 (0.10
equiv) was added, and the resulting suspension was heated at 120 °C
́
mass spectrometer (Department of Chemistry, Universite de
́
Montreal).
Synthesis of 2-(Chloromethyl)-3-methylquinolin-4(1H)-one
(11). A mixture of aniline (7, 6.8 mL, 74 mmol, 1 equiv) and diethyl
2-methyl-3-oxosuccinate (8, 14 mL, 74 mmol, 1 equiv) in glacial
acetic acid (37 mL) was refluxed at 50 °C for 24 h under argon. After
F
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under microwave irradiation for 1 min. After cooling, the mixture was
filtered through Celite to remove the catalyst, and the cake was rinsed
with EtOAc. The organic phase was then washed twice with brine and
dried over anhydrous MgSO4, and the solution was evaporated under
reduced pressure. The crude material was purified by flash
chromatography to give HMAQs 1−3.
flash column chromatography (hexanes−EtOAc, 100:0 to 80:20) to
give ethyl carbonate 18 (207 mg, 54%) as a yellow oil: 1H NMR (600
MHz, CDCl3) δ (ppm) 8.08−8.01 (m, 1H, HAr), 7.83−7.79 (m, 1H,
HAr), 7.69−7.62 (m, 1H, HAr), 7.53−7.48 (m, 1H, HAr), 5.72−5.45
(m, 2H, H-2′ and H-3′), 4.42−4.34 (m, 2H), 3.75 (m, 2H, H-1′),
2.34 (s, 3H, H-1″), 2.01 (dd, J = 14.0 Hz, J = 7.1 Hz, 2H, H-4′),
1.53−1.40 (m, 3H), 1.38−1.19 (m, 4H, H-5′, H-6′, and H-7′), 0.83−
0.91 (m, 3H, H-8′); 13C NMR (150 MHz, CDCl3) δ (ppm) 161.9,
152.5, 151.9, 147.6, 133.3 (C-2′), 129.2 (CAr), 129.1 (CAr), 126.6
(CAr), 125.8 (C-3′), 121.8 (CN), 121.5 (CN), 120.6 (CAr), 65.7
(CH2), 40.8 (C-1′), 32.7 (C-4′), 31.5 (CH2), 29.1 (CH2), 22.7
(CH2), 14.4 (CH3), 14.2 (C-9′), 12.0 (C-1″); HRMS (ESI-TOF) m/
z [M + H]+ calcd for C21H28NO3 342.20637; found 342.20514.
Synthesis of (E)-2-(Non-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one Ethyl Carbonate (19). According to the general
procedure for the protection of HMAQs, quinolone 3 (290 mg,
1.03 mmol) was reacted with t-BuOK (150 mg, 1.34 mmol) and ethyl
chloroformate (244 μL, 2.55 mmol). The crude material was purified
by silica gel flash column chromatography (hexanes−EtOAc, 100:0 to
Synthesis of (E)-2-(Hept-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one (1). According to the general procedure for the synthesis
of HMAQs, chloride 11 (160 mg, 0.771 mmol) was reacted with
trans-hexen-1-ylboronic acid pinacol ester 12 (257 μL, 1.00 mmol).
The crude material was purified by silica gel flash column
chromatography (100% hexanes to 100% EtOAc) to give HMAQ 1
(399 mg, 70%) as a white amorphous powder. 1H and 13C NMR data
are reported in Tables 2 and 3, respectively. HRMS (ESI-TOF) m/z
[M + H]+ calcd for C17H22NO 256.16959; found 256.16938; m/z [M
+ Na]+ calcd for C17H21NNaO 278.15154; found 278.15152.
Synthesis of (E)-2-(Oct-2′-en-1′-yl)-3-methylquinolin-4(1H)-
one (2). According to the general procedure for the synthesis of
HMAQs, chloride 11 (184 mg, 0.886 mmol) was reacted with trans-
hepten-1-ylboronic acid pinacol ester 13 (296 μL, 1.15 mmol). The
crude material was purified by silica gel flash column chromatography
(100% hexanes to 100% EtOAc) to give HMAQ 2 (176 mg, 74%) as
1
80:20) to give ethyl carbonate 19 (238 mg, 65%) as a yellow oil: H
NMR (600 MHz, CDCl3) δ (ppm) 8.11−8.00 (m, 1H, HAr), 7.86−
7.76 (m, 1H, HAr), 7.69−7.62 (m, 1H, HAr), 7.54−7.47 (m, 1H, HAr),
5.76−5.45 (m, 2H, H-2′ and H-3′), 4.38 (dd, J = 14.2 Hz, J = 7.1 Hz,
2H), 3.75 (br d, J = 6.1 Hz, 2H, H-1′), 2.34 (s, 3H, H-1″), 2.01 (dd, J
= 14.0 Hz, J = 7.1 Hz, 2H, H-4′), 1.48−1.40 (m, 3H), 1.37−1.18 (m,
8H, H-5′, H-6′, H-7′, and H-8′), 0.90−0.81 (m, 3H, H-9′); 13C NMR
(150 MHz, CDCl3) δ (ppm) 161.9, 152.5, 151.9, 147.6, 133.3 (C-2′),
129.2 (CAr), 129.0 (CAr), 126.6 (CAr), 125.8 (C-3′), 121.8 (CN),
121.5 (CN), 120.6 (CAr), 65.7 (CH2), 40.8 (C-1′), 32.7 (C-4′),
31.8 (CH2), 29.4 (CH2), 28.9 (CH2), 22.7 (CH2), 14.4 (CH3), 14.2
(C-9′), 12.0 (C-1″); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C22H30NO3 356.22202; found 356.22144.
General Procedure for the Oxidation of HMAQ Ethyl
Carbonates. Protected quinolone (17−19, 1.0 equiv) was reacted
with mCPBA (1.1 equiv) in anhydrous DCM (40 μL mg−1) at rt for 3
h. The reaction was washed twice with aqueous sodium carbonate
(0.5 M) and once with water. The organic phase was dried over
anhydrous MgSO4, filtered through Celite, and evaporated under
reduced pressure. The crude material was purified by silica gel flash
chromatography to give ethyl carbonate N-oxides (20−22).
1
a white amorphous powder. H and 13C NMR data are reported in
Tables 2 and 3, respectively. HRMS (ESI-TOF) m/z [M + H]+ calcd
for C18H24NO 270.18524; found 270.18416.
Synthesis of (E)-2-(Non-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one (3). According to the general procedure for the synthesis
of HMAQs, chloride 11 (307 mg, 1.48 mmol) was reacted with trans-
octen-1-ylboronic acid pinacol ester 14 (519 μL, 1.92 mmol). The
crude material was purified by silica gel flash column chromatography
(100% hexanes to 100% EtOAc) to give HMAQ 3 (316 mg, 75%) as
1
a white amorphous powder. H and 13C NMR data are reported in
Tables 2 and 3, respectively. HRMS (ESI-TOF) m/z [M + H]+ calcd
for C19H26NO 284.20089; found 284.20121; m/z [M + Na]+ calcd for
C19H25NNaO 306.18284; found 306.18269.
General Procedure for the Protection of HMAQs. HMAQ
(1−3, 1.0 equiv) was reacted with t-BuOK (1.25 equiv) in anhydrous
THF (7.0 mL.mmol−1) at rt for 1 h. Ethyl chloroformate (2.15 equiv)
was then added, and the reaction was left stirring at rt for 1.0−1.5 h.
When the reaction was completed (as revealed by TLC), water was
added, and the mixture was evaporated under reduced pressure to
remove THF. The aqueous phase was diluted in water and extracted
with EtOAc (3×). The combined organic phases were dried over
anhydrous MgSO4, filtered through Celite, and evaporated under
reduced pressure. The crude material was purified by silica gel flash
chromatography to give HMAQ ethyl carbonates 17−19.
Synthesis of (E)-2-(Hept-2′-en-1′-yl)-3-methylquinolin-
4(1H)-yl) Ethyl Carbonate N-Oxide (20). According to the general
procedure for the oxidation of HMAQ ethyl carbonates, quinolone 17
(192 mg, 0.586 mmol) was reacted with mCPBA (126 mg, 87% NMR
purity, 0.645 mmol). The crude material was purified by flash silica
gel flash column chromatography (hexanes−EtOAc, 95:5 to 70:30) to
1
give N-oxide 20 (51 mg, 26%) as a yellow oil: H NMR (600 MHz,
Synthesis of (E)-2-(Hept-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one Ethyl Carbonate (17). According to the general
procedure for the protection of HMAQs, quinolone 1 (20 mg,
0.078 mmol) was reacted with t-BuOK (11 mg, 0.098 mmol) and
ethyl chloroformate (16 μL, 0.17 mmol). The crude material was
purified by silica gel flash column chromatography (hexanes−EtOAc,
90:10 to 80:20) to give ethyl carbonate 17 (22 mg, 85%) as a yellow
CDCl3) δ (ppm) 8.80−8.75 (m, 1H, HAr), 7.86−7.80 (m, 1H, HAr),
7.77−7.70 (m, 1H, HAr), 7.66−7.59 (m, 1H, HAr), 5.72−5.57 (m, 2H,
H-2′ and H-3′), 4.39 (dd, J = 14.2 Hz, J = 7.1 Hz, 2H), 3.95 (br d, J =
5.8 Hz, 2H, H-1′), 2.35 (s, 3H, H-1″), 2.04−1.95 (m, 2H, H-4′), 1.44
(t, J = 7.1 Hz, 3H), 1.37−1.18 (m, 4H, H-5′ and H-6′), 0.86 (t, J =
7.1 Hz, 3H, H-7′); 13C NMR (150 MHz, CDCl3) δ (ppm) 152.6,
148.6, 142.0, 140.9, 134.1 (C-3′), 130.1 (CAr), 128.7 (CAr), 123.4
(CN), 123.1 (CN), 122.0 (C-2′), 121.4 (CAr), 120.6 (CAr), 66.0
(CH2), 33.3 (C-4′), 32.1 (C-1′), 31.5 (CH2), 22.4 (CH2), 14.4
(CH3), 14.1 (C-7′), 12.6 (C-1″); HRMS (ESI-TOF) m/z [M + H]+
calcd for C20H26NO4 344.1856; found 344.1861.
1
oil: H NMR (600 MHz, CDCl3) δ (ppm) 8.10−8.01 (m, 1H, HAr),
7.83−7.76 (m, 1H, HAr), 7.68−7.62 (m, 1H, HAr), 7.55−7.48 (m, 1H,
HAr), 5.74−5.44 (m, 2H, H-2′ and H-3′), 4.39 (dd, J = 14.2 Hz, J =
7.1 Hz, 2H), 3.75 (br d, J = 5.8 Hz, 2H, H-1′), 2.34 (s, 3H, H-1″),
2.01 (dd, J = 13.5 Hz, J = 6.8 Hz, 2H, H-4′), 1.46−1.41 (m, 3H),
1.39−1.24 (m, 4H, H-5′ and H-6′), 0.93−0.84 (m, 3H, H-7′); 13C
NMR (150 MHz, CDCl3) δ (ppm) 161.9, 152.5, 151.9, 147.6, 133.3
(C-3′), 129.2 (CAr), 129.1 (CAr), 126.6 (CAr), 125.8 (C-2′), 121.8
(CN), 121.5 (CN), 120.6 (CAr), 65.7 (CH2), 40.8 (C-4′), 32.5
(C-1′), 31.6 (C-5′), 22.3 (C-6′), 14.4 (CH3), 14.1 (C-7′), 12.0 (C-
1″); HRMS (ESI-TOF) m/z [M + H]+ calcd for C20H26NO3
328.19072; found 328.18946.
Synthesis of (E)-2-(Oct-2′-en-1′-yl)-3-methylquinolin-4(1H)-
yl) Ethyl Carbonate N-Oxide (21). According to the general
procedure for the oxidation of HMAQ ethyl carbonates, quinolone 18
(207 mg, 0.607 mmol) was reacted with mCPBA (130 mg, 87% NMR
purity, 0.668 mmol). The crude material was purified by silica gel
flash column chromatography (hexanes−EtOAc, 95:5 to 70:30) to
1
give N-oxide 21 (61 mg, 28%) as a yellow oil: H NMR (600 MHz,
CDCl3) δ (ppm) 8.80−8.74 (m, 1H, HAr), 7.86−7.79 (m, 1H, HAr),
7.77−7.70 (m, 1H, HAr), 7.65−7.59 (m, 1H, HAr), 5.72−5.57 (m, 2H,
H-2′ and H-3′), 4.39 (dd, J = 14.2 Hz, J = 7.1 Hz, 2H), 3.95 (br d, J =
5.8 Hz, 2H, H-1′), 2.35 (s, 3H, H-1″), 2.03−1.95 (m, 2H, H-4′), 1.44
(t, J = 7.1 Hz, 3H), 1.39−1.18 (m, 6H, H-5′, H-6′, and H-7′), 0.85 (t,
Synthesis of (E)-2-(Oct-2′-en-1′-yl)-3-methylquinolin-4(1H)-
one Ethyl Carbonate (18). According to the general procedure for
the protection of HMAQs, quinolone 2 (302 mg, 1.12 mmol) was
reacted with t-BuOK (175 mg, 1.56 mmol) and ethyl chloroformate
(293 μL, 3.06 mmol). The crude material was purified by silica gel
G
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J = 7.1 Hz, 3H, H-8′); 13C NMR (150 MHz, CDCl3) δ (ppm) 152.6,
148.6, 142.0, 140.9, 134.1 (C-3′), 130.1 (CAr), 128.7 (CAr), 123.4 (C
= N), 123.1 (CN), 121.9 (C-2′), 121.4 (CAr), 120.6 (CAr), 66.0
(CH2), 32.6 (C-4′), 32.1 (C-1′), 31.5 (CH2), 29.0 (CH2), 22.6
(CH2), 14.4 (CH3), 14.2 (C-8′), 12.6 (C-1″); HRMS (ESI-TOF) m/
z [M + H]+ calcd for C21H28NO4 358.2013; found 358.2013.
Synthesis of (E)-2-(Non-2′-en-1′-yl)-3-methylquinolin-
4(1H)-yl) Ethyl Carbonate N-Oxide (22). According to the general
procedure for the oxidation of HMAQ ethyl carbonates, quinolone 19
(92 mg, 0.26 mmol) was reacted with mCPBA (110 mg, 87% NMR
purity, 0.563 mmol). The crude material was purified by silica gel
flash column chromatography (hexanes−EtOAc, 100:0 to 70:30) to
Antimicrobial Disk Susceptibility Tests. Antimicrobial suscept-
ibility disks were used to determine the antimicrobial activity of
HMAQs on bacterial and fungal strains using the Kirby−Bauer
method.58,59 Cultures of bacteria and yeasts were grown overnight at
30 °C with shaking in tryptic soy broth (TSB). Then, cells were
spread onto Mueller-Hinton agar (MHA) plates using a sterile cotton
swab. A total of 10 μL (10 000 μg.mL−1 in MeOH for HMAQs or
DMSO for HMAQNOs) for each HMAQ was added on blank
antimicrobial susceptibility disks. A 10 μL MeOH or DMSO blank
antimicrobial susceptibility disk was used as control. Once MeOH or
DMSO was evaporated, the disks were deposited to the surface of an
agar plate. Molds were first grown on PDA plates at 25 °C for 4 days.
An agar plug was transferred in the middle of a new PDA plate
cultured at 25 °C for 4 days. Dried antimicrobial susceptibility disks
containing 100 μg of compounds and the control were added around
the plug of fungus on the PDA plate. The plates were incubated at 25
°C for 7 more days.
Antimicrobial Susceptibility Assay by Broth Microdilution.
Based on the Clinical and Laboratory Standards Institute protocol,
bacterial cells were suspended into Mueller-Hinton broth at an
OD600 nm of 0.02. A stock of 20 mM of each HMAQ was prepared in
DMSO. Serial dilutions were performed to reach concentrations of
HMAQs ranging from 100 to 0.78 μM. Bacteria were added at a final
OD600 of 0.01. The plates were incubated for 48 h at 30 °C with
shaking at 250 rpm. The OD600 was measured using a Cytation 3 plate
reader (Biotek). A. niger, F. oxyporum, F. solani, and Mocurales TM29
were grown at 25 °C for 4 days on PDA plates and harvested by
adding sterile water onto the plate and transferring into a sterile tube.
Then, fungal spores were counted using a hemacytometer cell
counting chamber. As described by Pujol et al.,60 a 2 × 104 spores
mL−1 suspension was prepared into RPMI 1640 medium containing
L-glutamine and buffered to pH 7.0 with 3-(N-morpholino)-
propanesulfonic acid. The spores were added to RPMI 1640 medium
containing serially diluted HMAQs, and the plates were incubated for
2 days at rt. The OD600 was measured using a Cytation 3 plate reader.
Measurement of hmqA-lacZ Activity. As previously described
by Chapalain et al.,38 the levels of expression from the hmqABCDEFG
promoter were assessed using B. ambifaria HSJ1 strains carrying the
chromosomal hmqA-lacZ transcriptional fusion. The β-galactosidase
assays were performed as previously described.61 HMAQs were added
to cultures to a final concentration of 50 μM from stocks prepared in
DMSO. DMSO was added as a control.
1
give N-oxide 22 (37 mg, 38%) as a yellow oil: H NMR (600 MHz,
CDCl3) δ (ppm) 8.81−8.74 (m, 1H, HAr), 7.85−7.80 (m, 1H, HAr),
7.76−7.69 (m, 1H, HAr), 7.66−7.60 (m, 1H, HAr), 5.72−5.55 (m, 2H,
H-2′ and H-3′), 4.39 (dd, J = 14.2 Hz, J = 7.1 Hz, 2H), 3.95 (br d, J =
5.8 Hz, 2H, H-1′), 2.35 (s, 3H, H-1″), 2.03−1.95 (m, 2H, H-4′), 1.44
(t, J = 7.1 Hz, 3H), 1.37−1.17 (m, 8H, H-5′, H-6′, H-7′, H-8′), 0.85
(t, J = 7.1 Hz, 3H, H-9′); 13C NMR (150 MHz, CDCl3) δ (ppm)
152.6, 148.6, 142.0, 140.9, 134.1 (C-3′), 130.1 (CAr), 128.7 (CAr),
123.4 (CN), 123.1 (C = N), 121.9 (C-2′), 121.4 (CAr), 120.6
(CAr), 66.0 (CH2), 32.6 (C-4′), 32.1 (C-1′), 31.8 (CH2), 29.3 (CH2),
29.0 (CH2), 22.7 (CH2), 14.4 (CH3), 14.2 (C-9′), 12.6 (C-1″);
HRMS (ESI-TOF) m/z [M + H]+ calcd for C22H30NO4 372.21693;
found 372.21696; m/z [M + Na]+ calcd for C22H29NNaO4
394.19888; found 394.19824.
General Procedure for the Synthesis of HMAQNOs.
Protected quinolone N-oxide (20−22, 1.0 equiv) was dissolved in
ethanol (95%, 33 μL mg−1), and aqueous KOH was added (5.0 M, 17
equiv). The reaction was stirred at rt for 1 h and then quenched with
the addition of cold water (same volume as the solvent).
Concentrated HCl (12 N) was added until acidic pH (1−2) was
reached. A milky suspension was formed, and cold water was then
added to help the precipitation process. The product was collected by
filtration under vacuum and washed with cold water to give target
HMAQNOs (4−6).
Synthesis of (E)-2-(Hept-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one N-Oxide (4). According to the general procedure for
the synthesis of HMAQNOs, protected N-oxide 20 (47 mg, 0.14
mmol) was reacted with KOH to give HMAQNO 4 (27 mg, 72%) as
1
a beige amorphous powder: H and 13C NMR data are reported in
Tables 3 and 4, respectively. HRMS (ESI-TOF) m/z [M + Na]+ calcd
for C17H21NNaO2 272.1645; found 272.1631.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00171.
■
Synthesis of (E)-2-(Oct-2′-en-1′-yl)-3-methylquinolin-4(1H)-
one N-Oxide (5). According to the general procedure for the
synthesis of HMAQNOs, protected N-oxide 21 (56 mg, 0.16 mmol)
was reacted with KOH to give HMAQNO 5 (34 mg, 77%) as a beige
sı
1
amorphous powder. H and 13C NMR data are reported in Tables 3
Product characterization data including 1H and 13C
NMR spectra; supplementary figures for the antimicro-
bial assays (PDF)
and 4, respectively. HRMS (ESI-TOF) m/z [M + H]+ calcd for
C18H24NO2 286.18016; found 286.17984.
Synthesis of (E)-2-(Non-2′-en-1′-yl)-3-methylquinolin-
4(1H)-one N-Oxide (6). According to the general procedure for
the synthesis of HMAQNOs, protected N-oxide 22 (51 mg, 0.14
mmol) was reacted with KOH to give HMAQNO 6 (31 mg, 76%) as
AUTHOR INFORMATION
Corresponding Authors
■
1
an amorphous beige powder. H and 13C NMR data are reported in
́
Charles Gauthier − Centre Armand-Frappier Sante
Biotechnologie, Institut National de la Recherche Scientifique
Tables 3 and 4, respectively. HRMS (ESI-TOF) m/z [M + H]+ calcd
for C19H26NO2 300.19581; found 300.19555; m/z [M + Na]+ calcd
for C19H25NNaO2 322.17775; found 322.17738.
́
(INRS), Laval, Quebec, Canada H7V 1B7; orcid.org/
0000-0002-2475-2050; Phone: +1 450 687-5010 ext. 8886;
Email: charles.gauthier@iaf.inrs.ca
Bacteria and Fungi. Bacillus subtilis PY79, Paenibacillus peoriae
LMG1611, Staphylococcus aureus Newman, Streptococcus agalactiae
ATCC27956, Actinobacillus pleuropneumoniae, Burkholderia ambifaria
HSJ1 hmqA-lacZ, Burkholderia ambifaria HSJ1 hmqA-::hmqA-lacZ,
Escherichia coli ATCC25922, Pseudomonas aeruginosa PA14, Pseudo-
monas fluorescens LMG1794, Xanthomonas campestris Xg, Cryptococcus
neoformans H99, and Candida albicans were cultured in tryptic soy
broth (TSB) at 30 °C with shaking overnight. Aspergillus niger,
Fusarium solani, Fusarium oxyporum, Mocurales TM29, Moniliceae
1(22), Penicillum sp, Pythium ultimum, and Rhizoctonia solani were
grown on potato dextrose agar (PDA) plates at 25 °C for 4 days.
́
́
Eric Deziel − Centre Armand-Frappier Sante Biotechnologie,
Institut National de la Recherche Scientifique (INRS), Laval,
́
Quebec, Canada H7V 1B7; orcid.org/0000-0002-4609-
0115; Phone: +1 450 687-5010 ext. 4220;
Email: eric.deziel@iaf.inrs.ca
Authors
́
Marianne Piochon − Centre Armand-Frappier Sante
Biotechnologie, Institut National de la Recherche Scientifique
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00171
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
́
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(INRS), Laval, Quebec, Canada H7V 1B7; orcid.org/
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Pauline M. L. Coulon − Centre Armand-Frappier Sante
Biotechnologie, Institut National de la Recherche Scientifique
́
(INRS), Laval, Quebec, Canada H7V 1B7; orcid.org/
0000-0001-9482-2360
́
Armand Caulet − Centre Armand-Frappier Sante
Biotechnologie, Institut National de la Recherche Scientifique
́
(INRS), Laval, Quebec, Canada H7V 1B7
́
Marie-Christine Groleau − Centre Armand-Frappier Sante
Biotechnologie, Institut National de la Recherche Scientifique
́
(INRS), Laval, Quebec, Canada H7V 1B7; orcid.org/
́
0000-0003-4810-1030
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00171
́
Zouari, N.; Blache, Y.; Jaoua, S. World J. Microbiol. Biotechnol. 2012,
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Notes
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7266.
(27) Li, D.; Oku, N.; Hasada, A.; Shimizu, M.; Igarashi, Y. Beilstein J.
Org. Chem. 2018, 14, 1446.
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Hayakawa, Y.; Shin-Ya, K. J. Antibiot. 2007, 60, 713.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a Discovery grant from the
Natural Sciences and Engineering Research Council of Canada
(NSERC) under award number RGPIN-2016-04950 (to C.G.)
and a grant from the Canadian Institutes of Health Research
(CIHR) under award number MOP-142466 (to E.D.). C.G.
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Rantiatmodjo, R. M.; Morioka, M.; Suzuki, K. J. Antibiot. 1996, 49,
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holds a Fonds de Recherche du Quebec−Sante (FRQS)
Research Scholars Junior 2 Career Award. E.D. holds the
Canada Research Chair in Sociomicrobiology. We thank Dr.
Philippe Constant (INRS) for his help with statistical analyses.
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