Synthesis of Long-Chain β-Lactones and Their Antibacterial Activities against Pathogenic Mycobacteria

Article abstract of DOI:10.1002/cmdc.201800720

In the quest for new antibacterial agents, a series of novel long- and medium-chain mono- and disubstituted β-lactones was developed. Their activity against three pathogenic mycobacteria—M. abscessus, M. marinum, and M. tuberculosis—was assessed by the resazurin microtiter assay (REMA). Among the 16 β-lactones synthesized, only 3-hexadecyloxetan-2-one (VM005) exhibited promising activity against M. abscessus, whereas most of the β-lactones showed interesting activities against M. marinum, similar to that of the classical antibiotic, isoniazid. Regarding M. tuberculosis, six compounds were found to be active against this mycobacterium, with β-lactone VM008 [trans-(Z)-3-(hexadec-7-en-1-yl)-4-propyloxetan-2-one] being the best growth inhibitor. The promising antibacterial activities of the best compounds in this series suggest that these molecules may serve as leads for the development of much more efficient antimycobacterial agents.

Full text of DOI:10.1002/cmdc.201800720

Accepted Article
Title: Synthesis of long chain β-lactones and their antibacterial
activities against pathogenic mycobacteria
Authors: Pierre Santucci, Christina Dedaki, Alexandros Athanasoulis,
Laura Gallorini, Anaïs Munoz, Stéphane Canaan, Jean-
François Cavalier, and Victoria Magrioti
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800720
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800720
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Synthesis of long chain β-lactones and their antibacterial
activities against pathogenic mycobacteria
Pierre Santucci§[a], Christina Dedaki§[b], Alexandros Athanasoulis[b], Laura Gallorini[a], Anaïs Munoz[a],
Stéphane Canaan[a], Jean-François Cavalier*[a] and Victoria Magrioti*[b]
[a]
[b]
Pierre Santucci, Laura Gallorini, Anaïs Munoz, Dr. Stéphane Canaan, Dr. Jean-François Cavalier
Aix-Marseille Univ
CNRS, LISM, IMM FR3479
Marseille, France
Christina Dedaki, Alexandros Athanasoulis, Dr. Victoria Magrioti
Department of Chemistry
National and Kapodistrian University of Athens
Panepistimiopolis, Athens, 15771, Greece
E-mail: jfcavalier@imm.cnrs.fr (J.-F. Cavalier), vmagriot@chem.uoa.gr (V. Magrioti)
§These authors have contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: In the quest for new antibacterial agents, a series of novel
long and medium chain mono- and disubstituted β-lactones has
been developed. Their respective antibacterial activity using the
resazurin microtiter assay (REMA) was further assessed against
three pathogenic mycobacteria: M. abscessus, M. marinum and M.
tuberculosis. The promising antibacterial activities of the best
compounds suggest that these molecules may serve as potential
leads for the development of much more efficient anti-mycobacterial
agents.
Figure 1. Structure of Orlistat and related β-lactone analogs. The carbonyl
reactive sites on the β-lactone ring, where nucleophilic attack by catalytic
serine or cysteine residue occurs, are indicated by an arrow.[1a, 1b]
The physician Robert Koch identified M. tb as the causative
agent of tuberculosis (TB) in 1882. Today, more than a century
later, TB remains the leading cause of death due to a single
infectious agent according to the World Health Organization.[7]
Moreover, the continuous emergence of resistant strains
highlights the pressing need for new therapeutic approaches.[8]
In this context, lipolytic enzymes represent novel and promising
targets to combat the disease.[9] Indeed, M. tb survival and
persistence in vivo require the utilization of distinct host-derived
lipids as energy source suggesting a major role of these
enzymes in the tubercle bacilli life cycle.[10] Many serine/cysteine
hydrolases, which are involved in lipid metabolism, are playing
an essential role in cell wall and mycolic acid biosynthesis /
maintenance.[11] Therefore, the use of inhibitors of these
enzymes to impair such activity is becoming an interesting
alternative strategy not only in the fight against TB,[12] but also
against other chronic mycobacterial infections like those caused
by M. marinum and M. abscessus.[13]
Introduction
Among the potent lipolytic enzyme inhibitors, β-lactones
represent an important class of compounds bearing the strained
2-oxetanone 4-membered ring responsible for potent inhibitory
activity against enzymes from the serine/cysteine hydrolase
class.[1] Chemical profiling studies have indeed revealed that β-
lactones target more than 20 different enzymes of four classes
comprising oxidoreductases, transferases, hydrolases, and
ligases. The labelled enzymes contained either an active site
serine or cysteine residue, and belonged to different classes,
including lipases, proteases, esterases or thioesterases.[1b,
1c]
Most natural occurring and synthetic β-lactones that have been
explored as antibacterials or enzyme inhibitors are trans
diastereoisomers,[2] however cis-β-lactones have been also
shown to be as potent in some cases.[2c, 2d] Recently, β-lactones
have also emerged as attractive probes for activity-based
protein profiling (ABPP) of the larger serine hydrolase family
thus making them exemplary structures for the identification of
novel biological targets.[3] The most representative member of
this family of inhibitors, is the FDA approved anti-obesity drug
Orlistat (also known as tetrahydrolipstatin, THL, 1 – Figure 1),
that inhibits the human digestive lipases by covalently binding to
their active site.[4] Functioning as a versatile serine/cysteine
hydrolase inhibitor, Orlistat, or its recent β-lactone EZ120
analog,[5] has been reported to block Mycobacterium
tuberculosis (M. tb) growth via covalent inhibition of various
endogenous lipolytic enzymes involved in bacterial lipid
metabolism.[6]
M. marinum is an opportunist pathogenic mycobacteria, typically
found in aquatic environments where it causes a systemic
tuberculosis-like infection and disease in ectotherms such as
frogs and fish. In humans, this mycobacterium is typically limited
to lesions of skin and soft tissues extremities, which established
M. marinum as
a
relevant human pathogen albeit
opportunistic.[14] M. abscessus is responsible for cutaneous and
pulmonary infections, representing up to 90% of non-tuberculous
mycobacteria (NTM) infections in cystic fibrosis (CF)
populations.[15] M. abscessus displays two distinct morphotypes:
a smooth (S) and a rough (R) one.[16] The major difference
between both variants resides in the total loss of surface-
1
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10.1002/cmdc.201800720
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FULL PAPER
associated glycopeptidolipids (GPL) in the R form.[17] Moreover,
several studies have clearly established the hypervirulence
phenotype of the R variant in severe infection caused by M.
abscessus.[18] Accordingly, M. abscessus is intrinsically resistant
to a broad range of antibiotics including most antitubercular
drugs, and is considered the most pathogenic and
chemotherapy-resistant rapidly growing mycobacterium.[19]
In our quest for discovering novel anti-mycobacterial agents that
might react covalently with catalytic serine/cysteine-based
enzymes of the mycobacteria, we herein report the synthesis of
a series of long and medium chain substituted β-lactones (i.e.,
VM compounds, Figure 1) together with their antibacterial
activities against the latter three pathogenic mycobacteria. The
rationale behind the present design was to create structures
which incorporate i) lipidic chains, such as the oleyl chain, that
appear in naturally occurring lipids and resemble natural lipolytic
enzyme substrates; but also ii) the β-lactone group which will
interact with the catalytic serine or cysteine residue of
hydrolases that might be essential for the growth and/or survival
of the mycobacteria.[6]
Scheme 1. Synthesis of compounds VM001-3. Reagents and conditions: (i) a)
LDA, dry THF, 0 °C, 1 h; b) butyraldehyde, dry THF, 0 °C, 1 h, then r.t. o.n.; (ii)
p-TsCl, dry pyridine, 0 °C, 1 h, then 4 °C, 3 days.
These three β-lactones were tested for their anti-mycobacterial
activity against three pathogenic mycobacterial strains as a
preliminary test to identify a lead. The antibacterial activity was
evaluated by determining their respective MIC using the
21]
resazurin microtiter assay (REMA).[12b,
Therefore,
susceptibility testing of the above molecules was performed
towards M. marinum M; M. tb mc26230 (H37Rv ΔRD1 ΔpanCD)
a derivative of H37Rv which contains a deletion of the RD1
region and panCD, resulting in a pan(−) phenotype strain;[22] and
both S and R variants of M. abscessus (Table 1).
Results and Discussion
First, in all cases, both S & R variants of M. abscessus remained
unaffected by all three compounds up to
a 100 µg/mL
In order to investigate the effects of various α-substitutions on
the β-lactone ring, we first used at the α-position either i) an
unsaturated C16 alkenyl chain that derives from oleic acid, ii) a
long saturated C16 alkyl chain, or iii) a medium saturated C6
alkyl chain. In each case, a C3 small chain was introduced at the
β-position (i.e., compounds VM001, VM002 and VM003,
respectively). The general route for the synthesis of the
designed α,β-disubstituted β-lactones is depicted in Scheme 1.
The appropriate carboxylic acids 2a-c chosen as starting
materials were first deprotonated by treatment with LDA. After
reaction with commercial butyraldehyde, the corresponding β-
hydroxy acids 3a-c were obtained in 30-85% yields. Finally,
cyclization of the intermediate α,β-disubstituted β-hydroxy acids
3a-c by treatment with p-toluenesulfonyl chloride led to α,β-
disubstituted β-lactones VM001-3 in 42-67% yields (Scheme
1).[20] Both β-hydroxy acids 3a-c and β-lactones VM001-3 were
obtained as mixtures of diastereoisomers whose ratio was
concentration (Table 1); a result which proves that this bacteria
well deserves its nickname of “antibiotics nightmare”.[23]
Regarding M. marinum, VM003 exhibited moderate activity
(MIC50 = 15.6 µg/mL), while VM001 showed a good activity with
a MIC50 of 4.7 µg/mL similar to that of isoniazid (Table 1). With
regards to M. tb susceptibility, although Orlistat was able to
block M. tb mc26230 growth with quite good MIC50 of 11.0 µg/mL,
only VM001 displayed an interesting but moderate activity
(MIC50 = 31.8 µg/mL) towards M. tb; whereas VM003 appeared
as a weak inhibitor (MIC50 = 58.4 µg/mL) and VM002 was not
active.
Table 1. Antibacterial activities of Orlistat and analogs VM001-3 compared to
standard antimicrobial agents against three pathogenic mycobacterial strains.a
MIC50 (µg/mL) a
M. abscessus
1
determined by H NMR spectroscopy, and, in particular, by the
Cpds
CIP 104536T
M. marinum
ATCC BAA-535/M
M. tb
mc26230
relative peak integration of the characteristic α- and/or β-
methinic protons (for a detailed analysis on the diastereomeric
ratio, see Supplementary Material). According to the 1H NMR
spectra, the β-hydroxy acids 3a-c were formed in an anti:syn
isomer ratio varying from 6:4 to 7:3, while the corresponding
isolated β-lactones VM001-3 were in a trans:cis isomer ratio of
around 7:3. At this initial stage of our research, it was preferred
to synthesize the diastereoisomeric mixtures in order to identify
potential leads that may later be prepared in diastereomerically
or optically pure form. The chromatographic resolution of the
diastereoisomers of the β-lactones is quite difficult as they
present similar solubility and polarity.
Smooth
Rough
57.0
>100
>100
>100
-
Orlistat
VM001
VM002
VM003
INH
44.4
>100
>100
>100
-
6.8
4.7
11.0
31.8
>100
58.4
0.07
0.24
>100
15.6
2.8
AMK
2.3
4.3
0.62
a Minimum inhibitory concentration leading to 50% growth inhibition (MIC50) as
determined by the resazurin microtiter assay (REMA), are expressed as mean
values of two independent assays performed in triplicate (CV% < 5%). INH:
isoniazid; AMK: amikacin.
Taking into account these preliminary results and by identifying
VM001 with the oleyl chain at the α-position as the most
promising one, we further investigated the effects of different
substituents on the β-position. Therefore, α,β-disubstituted β-
2
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10.1002/cmdc.201800720
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lactones were synthesized (Scheme 2) in order to examine the
influence of such side chains on the antibacterial activity of the
corresponding compounds.
and prolonged reaction times (overnight r.t. stirring for 6a,b in
comparison to 2 days reflux for 6c) were applied.
Scheme 3. Synthesis of compounds VM004-6. Reagents and conditions: (i) a)
NaH 60%, dry THF; b) RBr , reflux, o.n.; (ii) KOH , EtOH; (iii) LiBH4 2.0 M, i-
PrOH/dry THF 1:2, 0 °C, 1 h, then r.t., o.n.; (iv) DEAD, PPh3, 0 °C, 1 h, then
r.t., o.n.
Scheme 2. A) Synthesis of compounds VM009-12. Reagents and conditions:
(i) a) LDA, dry THF, 0 °C, 1 h; b) RCHO, dry THF, 0 °C, 1 h, then r.t. o.n.; (ii)
p-TsCl, dry pyridine, 0 °C, 1 h, then 4 °C, 3 days. B) Racemic cis and trans
pure stereoisomers obtained.
Finally, we also synthesized the racemic β-monosubstituted β-
lactones VM014-16 to study the effect of the lack of the α-side
chain on the compound activity (Scheme 4). The appropriate
carboxylic acids 2a, 8a,b were converted to the corresponding
β-keto esters 9a-c using Masamune’s method with 1,1΄-carbonyl
diimidazole (CDI) and monoethyl malonic acid magnesium
salt.[27] Reduction of the ketone using NaBH4, followed by
saponification yielded the β-monosubstituted β-hydroxy acids
11a-c.
The cyclization into the β-lactone was not successful using
either p-toluenesulfonyl chloride/pyridine or DEAD/PPh3. So we
explored other routes in order to achieve the cyclization and the
most efficient reaction proved to be the use of 2,2΄-dipyridyl
disulphide with PPh3 as a first step and then mercuric triflate at
50°C as a second step, that produced β-lactones VM014-16 in
28-66% yields.[28]
A methyl, ethyl, hexyl or a nonyl chain was then incorporated as
β-side chain (Scheme 2). The synthetic procedure was similar to
that of the initial compounds. Preparation of the desired β-
hydroxy acid using LDA and the appropriate aldehyde, was
followed by cyclization using p-toluenesulfonyl chloride in
pyridine. Thus, β-lactones VM009-12 were obtained in 36-63%
yields (Scheme 2A) with a trans:cis isomer ratio of 7:3.
Furthermore, from the lead of the initial screening, VM001, the
two racemic mixtures of cis-VM001 and trans-VM001, namely
VM007 and VM008, respectively, were successfully isolated via
column chromatography in order to be tested (Scheme 2B). The
1
geometry of these isolated stereoisomers was determined by H
NMR spectroscopy based on the chemical shifts of the
characteristic peaks of α- and β-protons that were compared to
24]
the corresponding reference peaks in the literature.[2d,
The
racemic mixture of trans-VM009 assigned as VM013 was also
chromatographically isolated in pure form and tested.
As we were also interested in investigating the influence of the
absence of the β-side chain on the compound activity, racemic
α-monosubstituted β-lactones, i.e. compounds VM004 and
VM005, were synthesized (Scheme 3). These two compounds
were synthesized by using diethyl malonate as starting material.
The appropriate alkyl or alkenyl bromide was used in order to
prepare the substituted diethyl malonates 5a,b that were then
hydrolyzed under controlled conditions into the corresponding
acids 6a,b bearing one ester group. The ester groups were
reduced using LiBH4[25] and finally the β-hydroxy acids 7a,b were
Scheme 4. Synthesis of compounds VM014-16. Reagents and conditions: (i)
a) CDI, dry THF, r.t., 6 h; b) MgCl2, CH3CH2OCOCH2COOK, r.t., 18 h; (ii)
NaBH4, EtOH, r.t.; (iii) NaOH, 1,4-dioxane, r.t., 16h; (iv) a) 2,2΄-dipyridyl
disulphide, PPh3, dry CH2Cl2; b) Hg(CF3SO3)2, dry MeCN, 50 °C, 20 min.
[26]
cyclized using DEAD/PPh3
to yield the desired β-lactones
VM004 and VM005 in around 17% overall yield. In order to
conclude whether the stereochemical hindrance and/or the
absence of the α-proton will make a significant difference in the
activity, we also prepared the α,α’-disubstituted β-lactone
VM006 bearing a second small C4 chain at the α-position. Here,
diethyl butylmalonate 4c was used as starting material and
similar reactions were performed, although stronger conditions
(more equivalents of NaH and KOH, for 5c and 6c, respectively)
All these 12 new β-lactone derivatives were tested for their
antibacterial
activities
against
the
three
pathogenic
mycobacterial strains using the validated and routine REMA
method with the use of resazurin as a marker for bacterial
viability to determine their respective MIC50[12b, 13, 21] (Table 2).
3
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Table 2. Antibacterial activities of Orlistat and its 16 analogs (VM001-16)
compared to standard antimicrobial agents against three pathogenic
mycobacterial strains.a
VM005 and VM015-16. Conversely, the second substituent was,
however, not found to play a significant role in the activity.
Regarding M. tb susceptibility (Table 2), VM008 (trans-VM001)
displayed the most interesting antitubercular activity
(MIC50 = 19.7 µg/mL), of the same order of magnitude than that
of Orlistat. VM010 and VM013 showed only low inhibitory activity
against M. tb growth with similar MIC50 of 33.5 and 33.6 µg/mL,
respectively. The enhanced results of VM008 and VM013 (trans-
VM009) on M. tb when compared to the cis/trans isomeric
mixtures VM001 (MIC50 = 31.8 µg/mL) and VM009 (MIC50 >100
µg/mL), suggest that the trans-β-lactones are more active than
the corresponding cis isomers. The replacement of the propionyl
group at the β-position of VM001 by the ethyl group (VM010) led
to similar results. Conversely, the replacement by a methyl
group (VM009) or by longer chains (VM011-12) resulted in
inactive compounds. Finally, VM015, a β-monosubstituted β-
lactone bearing a saturated C15 long chain, exhibited a modest
but interesting MIC50 value of 64.6 µg/mL, and could be used as
a second lead in another series of β-lactones.
MIC50 (µg/mL)a
M. abscessus
CC50
M. marinum
ATCC BAA-
535/M
Cpds
CIP 104536T
(µg/mL)b
M. tb
mc26230
Smooth
44.4
Rough
57.0
Orlistat
VM001
VM002
VM003
VM004
VM005
VM006
VM007
VM008
VM009
VM010
VM011
VM012
VM013
VM014
VM015
VM016
INH
6.8
4.7
11.0
31.8
>100
58.4
>100
>100
>100
>100
19.7
>100
33.5
>100
>100
33.6
>100
64.6
>100
0.07
0.24
ND
>125
>125
113 ±1.5
>125
>125
114 ±1.8
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>20 c
>90 c
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
-
>100
>100
>100
>100
62.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
-
>100
15.6
18.4
>100
>100
4.4
Finally, in view of testing our compounds in future ex vivo
experiments, their toxicity against murine Raw264.7
macrophages was further investigated using a classical dose-
5.6
response assay.[30] The calculated response parameter was
6.5
21c, 29, 31]
CC50,[12b,
which corresponds to the concentration
13.8
7.6
required for a 50% reduction of cell viability. Except for
compounds VM003 and VM006, in all other cases, no
cytotoxicity against Raw264.7 macrophages was observed at
the highest concentration tested (125 µg/mL); yielding
a
16.4
13.8
9.8
selectivity index (SI = CC50/MIC50) for VM008 being in a range
from >6.3 and up to >22.3 for M. tb and M. marinum,
respectively (Table 2).
>100
>100
2.8
Conclusion
In conclusion, among the 16 β-lactones synthesized, only
VM005 exhibited promising activity against M. abscessus; while
most of the β-lactones showed interesting activities against M.
marinum, similar to those of both INH and Orlistat. Regarding M.
AMK
2.3
4.3
0.62
a Minimum inhibitory concentration leading to 50% growth inhibition (MIC50) as
determined by the resazurin microtiter assay (REMA), are expressed as mean
values of two independent assays performed in triplicate (CV% < 5%). The
best MIC50 obtained are highlighted in bold. b Cytotoxic concentration of VM
compound leading to 50% macrophage (Raw264.7) cell death (CC50) is the
tb, only
6 compounds were found active against this
mycobacterium, with β-lactone VM008 (trans-VM001) being the
most promising one. Despite the fact that at this stage their
exact mode of action as well as their enzymatic targets remain
unknown, the leads of these compounds may serve as potential
scaffolds for the generation of new anti-mycobacterial agents.
Moreover, based on their good antibacterial activities against M.
marinum together with their low toxicity towards host cells, these
compounds may represent potential promising candidates to
treat cutaneous infections related to this extracellular bacteria.
These various aspects are currently under study in our
laboratories and will be reported in due course.
c
mean for a triplicated dose-response. Data from[29]. INH: isoniazid; AMK:
amikacin.
Concerning M. abscessus, one interesting result was displayed
by VM005, a α-monosubstituted β-lactone bearing a saturated
long chain in the α position. This compound was found as active
as Orlistat towards the R variant only, but with MIC50 values
around 14 times higher than that of amikacin (4.3 µg/mL) used
as reference drug. VM005 being the sole compound with
antibacterial activity against M. abscessus, these data thus
suggest more an isolated activity rather than a class effect.
Most of the β-lactones showed good activity against M. marinum
(Table 2) with β-lactones VM001 and VM007 (cis-VM001)
showing the most potent activity (MIC50 of 4.7 and 4.4 µg/mL,
respectively), similar to those of both Orlistat and INH. It should
be noted that the oleyl chain seems to be important in the β-
lactone structure. Both α-monosubstituted (VM004) and β-
monosubstituted (VM014) β-lactones bearing this side chain
indeed displayed significant antibacterial activity against M.
marinum, in contrast to other monosubstituted β-lactones
Experimental Section
Chemistry
General: Air- or moisture-sensitive reactions were carried out under an
inert gas atmosphere. Thin-layer chromatography (TLC) was performed
on Silica Gel 60 F254 aluminum plates. TLC spots were visualized with
UV light and/or phosphomolybdic acid in EtOH and/or ninhydrin in EtOH.
Chromatographic purification of products was accomplished using Merck
Silica Gel 60 (230-400 mesh). Melting points were determined using a
4
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10.1002/cmdc.201800720
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FULL PAPER
Büchi 530 apparatus and were uncorrected. 1H and 13C spectra were
recorded on a Varian Mercury (200 MHz and 50 MHz respectively) in
CDCl3. Chemical shifts are given in ppm using solvent as an internal
standard and coupling constants (J) in Hz. Peak multiplicities are
described as follows: s, singlet, d, doublet, t, triplet, q, quartet and m,
multiplet. Electron spray ionization (ESI) mass spectra were recorded on
a Finnigan, Surveyor MSQ Plus spectrometer. HRMS spectra were
(Z)-2-(1-hydroxypropyl)octadec-9-enoic acid (3e): Use of propanal.
Mixture of diastereoisomers (d.r. 7:3). Yield 88% (900 mg); Yellowish oil;
1H NMR (200 MHz, CDCl3): δ=5.41-5.20 (m, 2H), 3.80-3.68 (m, 0.3H),
3.68-3.53 (m, 0.7H), 2.51-2.33 (m, 1H), 2.11-1.85 (m, 4H), 1.77-1.07 (m,
24H), 0.95 (t, J = 7 Hz, 3H), 0.85 (t, J = 7 Hz, 3H); 13C NMR (50 MHz,
CDCl3): δ=179.8, 129.8, 129.5, 73.6, 50.7, 31.8, 29.7, 29.6, 29.5, 29.4,
29.2, 29.1, 29.0, 27.8, 27.7, 27.2, 27.1, 26.8, 22.6, 14.0, 10.2, 9.8; HRMS
-
recorded on
a
Bruker Maxis Impact QTOF Spectrometer.
m/z [M-H]- calcd for C21H39O3 : 339.2899, found: 339.2894; Anal. calcd
Dichloromethane was dried by standard procedures and stored over
molecular sieves. Extra dry THF over molecular sieves was purchased
from Acros. All the products gave satisfactory elemental analysis. The
diastereoisomeric ratio (d.r.) of the α,β-substituted β-hydroxy acids and
for C21H40O3: C, 74.07, H 11.84, found: C, 73.87, H 11.93.
(Z)-2-(1-hydroxyheptyl)octadec-9-enoic acid (3f): Use of heptanal.
Mixture of diastereoisomers (d.r. 6:4). Yield 84% (1.1 g); Yellowish oil; 1H
NMR (200 MHz, CDCl3): δ=5.44-5.23 (m, 2H), 3.92-3.77 (m, 0.4H), 3.77-
3.58 (m, 0.6H), 2.54-2.30 (m, 1H), 2.14-1.85 (m, 4H), 1.82-1.09 (m, 32H),
0.95-0.75 (m, 6H); 13C NMR (50 MHz, CDCl3): δ=180.1, 129.8, 129.5,
72.2, 72.1, 51.2, 50.9, 35.1, 33.9, 31.8, 31.7, 29.7, 29.5, 29.4, 29.2, 29.1,
29.0, 27.7, 27.3, 27.1, 26.7, 25.9, 25.6, 22.6, 22.5, 14.0, 13.9; HRMS m/z
β-lactones was determined by 1H NMR spectroscopy. Compounds 5a[32]
,
5b[33], 6a[32], 6b[34], 9a[35], 9b[36], 9c[37], 10b[38] and 10c[39] were prepared
according to the literature or to the methods described below and their
spectroscopic data were in accordance with those in the literature.
-
General method for the synthesis of α,β-substituted β-hydroxy
acids (3a-g): To a stirred solution of diisopropylamine (303 mg, 3 mmol)
in dry THF (2 mL), under argon at 0 °C, a solution of 1.6M n-BuLi in
hexane (1.9 mL, 3 mmol) was slowly added via syringe and the solution
of LDA was stirred at 0 °C for 10 min. The carboxylic acid (2a-c) (1
mmol) in dry THF (3 mL) was then added and the solution was stirred at
0 °C for 1 h. Then, the appropriate aldehyde (1.3 mmol) in dry THF (2
mL) was added and the solution was stirred at 0 °C for 1 h and at room
temperature overnight. The solvent was removed under reduced
pressure. The reaction mixture was acidified with 1N HCl and extracted
with Et2O (3 x 30 mL). The organic layers were combined, washed with
brine (30 mL) and dried. The solvent was removed and the product was
[M-H]- calcd for C25H47O3 : 395.3525, found: 395.3520; Anal. calcd for
C25H48O3: C, 75.70, H 12.20, found: C, 75.55, H 12.29.
(Z)-2-(1-hydroxydecyl)octadec-9-enoic acid (3g): Use of decanal.
Mixture of diastereoisomers (d.r. 7:3). Yield 83% (1.10 g); Yellowish oil;
1H NMR (200 MHz, CDCl3): δ=5.44-5.23 (m, 2H), 3.90-3.76 (m, 0.3H),
3.76-3.55 (m, 0.7H), 2.54-2.31 (m, 1H), 2.14-1.85 (m, 4H), 1.80-1.03 (m,
38H), 0.95-0.75 (m, 6H); 13C NMR (50 MHz, CDCl3): δ=180.4, 129.9,
129.6, 72.2, 72.1, 51.2, 50.8, 35.2, 33.8, 31.9, 29.7, 29.6, 29.5, 29.4,
29.3, 29.1, 27.8, 27.3, 27.2, 26.5, 26.0, 25.7, 22.6, 14.0; HRMS m/z [M-
-
H]- calcd for C28H53O3 : 437.3995, found: 437.3994; Anal. calcd for
C28H54O3: C, 76.65, H 12.41, found: C, 76.47, H 12.52.
purified by column chromatography eluting with
a gradient of
CHCl3/MeOH 97:3 to 95:5 (v/v).
General method for the cyclization of α,β-substituted β-hydroxy
acids to β-lactones (VM001-VM003, VM007-VM013): To a stirred
solution of β-hydroxy acid (3a-g) (1 mmol) in dry pyridine (2 mL), under
argon at 0 °C, p-toluenesulfonyl chloride (381 mg, 2 mmol) in dry pyridine
(1 mL) was added slowly via syringe. The solution was stirred at 0 °C for
1 h and kept at 4 °C for 3 days. Then, Et2O (30 mL) was added and the
organic layer was washed with 10% Na2CO3 (2 x 30 mL), 1N HCl (2 x 30
mL) and brine (30 mL). The organic layer was dried and the solvent was
removed in vacuo. The product was purified by column chromatography
eluting with a gradient of petroleum ether (bp 40-60 °C) /AcOEt 97:3 to
95:5 (v/v).
(Z)-2-(1-Hydroxybutyl)octadec-9-enoic acid (3a): Use of butyraldehyde.
Mixture of diastereoisomers (d.r. 6:4). Yield 85% (900 mg); Yellowish oil;
1H NMR (200 MHz, CDCl3): δ=5.44-5.23 (m, 2H), 3.95-3.80 (m, 0.4H),
3.80-3.61 (m, 0.6H), 2.54-2.36 (m, 1H), 2.14-1.85 (m, 4H), 1.83-1.07 (m,
26H), 1.03-0.77 (m, 6H); 13C NMR (50 MHz, CDCl3): δ=180.6, 129.9,
129.6, 71.9, 71.8, 51.2, 50.8, 37.3, 35.9, 31.8, 29.7, 29.6, 29.5, 29.3,
29.1, 27.8, 27.3, 27.1, 26.6, 22.6, 22.5, 19.1, 18.8, 18.1, 14.0, 13.9;
HRMS m/z [M+Na]+ calcd for C22H42O3Na+: 377.3026, found: 377.3025;
Anal. calcd for C22H42O3: C, 74.52, H 11.94, found: C, 74.31, H 12.02.
2-(1-Hydroxybutyl)octadecanoic acid (3b): Use of butyraldehyde.
Mixture of diastereoisomers (d.r. 6:4). Yield 30% (310 mg); White Solid;
mp: 25-30 °C; 1H NMR (200 MHz, CDCl3): δ=3.94-3.81 (m, 0.4H), 3.81-
3.63 (m, 0.6H), 2.54-2.36 (m, 1H), 1.80-1.17 (m, 34H), 1.03-0.80 (m, 6H);
13C NMR (50 MHz, CDCl3): δ=180.3, 72.0, 71.9, 51.3, 50.9, 37.3, 35.9,
31.9, 29.7, 29.5, 29.3, 27.8, 27.4, 26.6, 22.6, 19.1, 18.8, 14.0, 13.9;
HRMS m/z [M+Na]+ calcd for C22H44O3Na+: 379.3183, found: 379.3181;
Anal. calcd for C22H44O3: C, 74.10, H 12.44, found: C, 73.98, H 12.51.
(Z)-3-(Hexadec-7-en-1-yl)-4-propyloxetan-2-one (VM001): Mixture of
diastereoisomers (d.r. 7:3). Yield 67% (330 mg); Colorless oil; 1H NMR
(200 MHz, CDCl3): δ=5.45-5.24 (m, 2H), 4.63-4.48 (m, 0.3H), 4.30-4.15
(m, 0.7H), 3.67-3.52 (m, 0.3H), 3.24-3.09 (m, 0.7H), 2.14-1.90 (m, 4H),
1.90-1.55 (m, 4H), 1.55-1.20 (m, 22H), 0.99 (t, J = 7 Hz, 3H), 0.88 (t, J =
7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=172.3, 171.6, 130.1, 130.0,
129.6, 129.5, 77.9, 75.4, 56.1, 52.6, 36.4, 32.1, 31.9, 29.7, 29.5, 29.4,
29.3, 29.2, 28.9, 27.8, 27.5, 27.2, 27.0, 26.9, 23.9, 22.6, 18.9, 18.4, 14.1,
13.7; HRMS m/z [M+Na]+ calcd for C22H40O2Na+: 359.2921, found:
359.2927; Anal. calcd for C22H40O2: C, 78.51, H 11.98, found: C, 78.30, H
12.09.
2-(1-Hydroxybutyl)octanoic acid (3c): Use of butyraldehyde. Mixture of
diastereoisomers (d.r. 7:3). Yield 33% (200 mg); White solid; mp: 20-
1
25 °C; H NMR (200 MHz, CDCl3): δ=3.94-3.80 (m, 0.3H), 3.80-3.63 (m,
0.7H), 2.53-2.35 (m, 1H), 1.85-1.15 (m, 14H), 1.03-0.77 (m, 6H); 13C
NMR (50 MHz, CDCl3) δ 180.7, 72.0, 71.8, 51.1, 50.9, 37.4, 36.0, 31.6,
29.4, 29.3, 29.2, 27.7, 27.3, 26.6, 22.5, 19.1, 18.8, 14.0, 13.9; HRMS m/z
[M+Na]+ calcd for C12H24O3Na+: 239.1618, found: 239.1619; Anal. calcd
for C12H24O3: C, 66.63, H 11.18, found: C, 66.45, H 11.27.
3-Hexadecyl-4-propyloxetan-2-one
(VM002):
Mixture
of
diastereoisomers (d.r. 7:3). Yield 42% (100 mg); White solid; mp: 41-
1
44 °C; H NMR (200 MHz, CDCl3): δ=4.61-4.47 (m, 0.3H), 4.29-4.15 (m,
0.7H), 3.67-3.50 (m, 0.3H), 3.24-3.08 (m, 0.7H), 1.96-1.13 (m, 34H), 0.98
(t, J = 7 Hz, 3H), 0.87 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3):
δ=172.3, 171.6, 77.9, 75.4, 56.1, 52.6, 36.4, 32.1, 31.9, 29.6, 29.5, 29.4,
29.3, 29.2, 27.8, 27.5, 26.9, 23.9, 22.6, 18.9, 18.4, 14.1, 13.7; HRMS m/z
[M+Na]+ calcd for C22H42O2Na+: 361.3077, found: 361.3079; Anal. calcd
for C22H42O2: C, 78.05, H 12.50, found: C, 77.89, H 12.60.
(Z)-2-(1-hydroxyethyl)octadec-9-enoic acid (3d): Use of acetaldehyde.
Mixture of diastereoisomers (d.r. 7:3). Yield 52% (500 mg); Yellowish oil;
1H NMR (200 MHz, CDCl3): δ=5.43-5.23 (m, 2H), 4.12-3.99 (m, 0.3H),
3.99-3.85 (m, 0.7H), 2.52-2.25 (m, 1H), 2.11-1.85 (m, 4H), 1.77-1.55 (m,
2H), 1.55-1.03 (m, 23H), 0.87 (t, J = 7 Hz, 3H); 13C NMR (50 MHz,
CDCl3) δ 179.9, 179.6, 129.9, 129.6, 68.5, 68.2, 52.9, 51.9, 34.1, 31.8,
29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 29.0, 27.7, 27.1, 24.8, 22.6, 21.2, 19.8,
3-Hexyl-4-propyloxetan-2-one (VM003):[20] Mixture of diastereoisomers
(d.r. 7:3). Yield 60% (70 mg); Colorless oil; 1H NMR (200 MHz, CDCl3):
δ=4.62-4.48 (m, 0.3H), 4.29-4.17 (m, 0.7H), 3.68-3.53 (m, 0.3H), 3.24-
3.11 (m, 0.7H), 1.97-1.17 (m, 14H), 0.99 (t, J = 7 Hz, 3H), 0.89 (t, J = 7
Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=172.3, 171.6, 77.9, 75.5, 56.1,
52.6, 36.4, 32.2, 31.4, 29.0, 28.9, 27.8, 27.5, 26.9, 23.9, 22.5, 22.4, 18.9,
-
14.0; HRMS m/z [M-H]- calcd for C20H37O3 : 325.2743, found: 325.2740;
Anal. calcd for C20H38O3: C, 73.57, H 11.73, found: C, 73.38; H 11.81.
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800720
ChemMedChem
FULL PAPER
18.4, 14.0, 13.8, 13.7; HRMS m/z [M+Na]+ calcd for C12H22O2Na+:
221.1512, found: 221.1513; Anal. calcd for C12H22O2: C, 72.68, H 11.18,
found: C, 72.50, H 11.31.
trans-(Z)-3-(hexadec-7-en-1-yl)-4-nonyloxetan-2-one (VM013): Yield
20% (20 mg), Colorless oil; H NMR (200 MHz, CDCl3): δ=5.44-5.23 (m,
1
2H), 4.40 (dq, J = 6 Hz, 4 Hz, 1H), 3.16 (ddd, J = 9 Hz, 7 Hz, 4 Hz, 1H),
2.11-1.90 (m, 4H), 1.89-1.60 (m, 2H), 1.56 (d, J = 6 Hz, 3H), 1.50-1.13
(m, 20H), 0.89 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=171.3,
130.1, 129.6, 74.6, 57.6, 31.9, 29.7, 29.5, 29.3, 29.2, 29.1, 28.9, 27.7,
27.2, 27.1, 26.8, 22.7, 20.3, 14.1; HRMS m/z [M+Na]+ calcd for
C20H36O2Na+: 331.2607, found: 331.2611; Anal. calcd for C20H36O2: C,
77.87, H 11.76, found: C, 77.65, H 11.87.
cis-(Z)-3-(Hexadec-7-en-1-yl)-4-propyloxetan-2-one (VM007): Yield
10% (10 mg); Colorless oil; H NMR (200 MHz, CDCl3): δ=5.45-5.24 (m,
1
2H), 4.63-4.48 (m, 1H), 3.67-3.52 (m, 1H), 2.14-1.90 (m, 4H), 1.90-1.42
(m, 6H), 1.55-1.20 (m, 20H), 0.99 (t, J = 7 Hz, 3H), 0.88 (t, J = 7 Hz, 3H);
13C NMR (50 MHz, CDCl3): δ=172.3, 130.0, 129.6, 75.4, 52.6, 32.1, 31.9,
29.7, 29.5, 29.4, 29.3, 28.9, 27.5, 27.2, 27.0, 23.9, 22.6, 18.9, 14.1, 13.7;
HRMS m/z [M+Na]+ calcd for C22H40O2Na+: 359.2921, found: 359.2927;
Anal. calcd for C22H40O2: C, 78.51, H 11.98, found: C, 78.32, H 12.08.
Diethyl (Z)-2-butyl-2-(octadec-9-en-1-yl)malonate (5c): A solution of
diethyl butyl malonate (583 mg, 2.7 mmol) in dry THF (2.7 mL) was
slowly added via syringe to a stirred mixture of 60% NaH (140 mg, 3.5
mmol) in dry THF (5.4 mL) and the solution was stirred under argon at
room temperature for 10 min. Then, a solution of the oleyl bromide (894
mg, 2.7 mmol) in dry THF (1.6 mL) was slowly added via syringe, and the
mixture was stirred at room temperature for 10 min and then refluxed
overnight. The solvent was removed under reduced pressure, water (80
mL) was added to the reaction mixture which was extracted with Et2O (3
x 80 mL). The organic layers were combined, washed with brine and
dried. The solvent was removed and the product was purified by column
chromatography eluting with a gradient of petroleum ether (bp 40-
60 °C)/AcOEt 97:3 to 90:10 (v/v). Yield 58% (730 mg); Colorless oil; 1H
NMR (200 MHz, CDCl3): δ=5.43-5.23 (m, 2H), 4.17 (q, J = 7 Hz, 4H),
2.10-1.93 (m, 4H), 1.93-1.76 (m, 4H), 1.45-1.01 (m, 34H), 0.96-0.80 (m,
6H); 13C NMR (50 MHz, CDCl3): δ=171.6, 129.6, 129.5, 60.5, 57.2, 31.9,
31.7, 31.6, 29.7, 29.6, 29.3, 29.2, 29.1, 29.0, 27.0, 25.9, 23.7, 22.8, 22.5,
13.9, 13.6; MS (ESI) m/z (%): 484.5 [(M+NH4+), 100]; Anal. calcd for
C29H54O4: C, 74.63, H 11.66, found: C, 74.44, H 11.75.
trans-(Z)-3-(Hexadec-7-en-1-yl)-4-propyloxetan-2-one (VM008): Yield
37% (50 mg); Colorless oil; H NMR (200 MHz, CDCl3): δ=5.45-5.24 (m,
1
2H), 4.23 (dd, J = 7 Hz, 6 Hz, 4 Hz, 1H), 3.17 (ddd, J = 9 Hz, 7 Hz, 4 Hz,
1H), 2.14-1.90 (m, 4H), 1.90-1.65 (m, 4H), 1.55-1.20 (m, 22H), 0.99 (t, J
= 7 Hz, 3H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=171.6,
130.1, 129.5, 77.9, 56.1, 36.4, 31.9, 29.7, 29.5, 29.4, 29.3, 29.2, 28.9,
27.8, 27.2, 27.0, 26.9, 22.6, 18.4, 14.1, 13.7; HRMS m/z [M+Na]+ calcd
for C22H40O2Na+: 359.2921, found: 359.2927; Anal. calcd for C22H40O2: C,
78.51, H 11.98, found: C, 78.35, H 12.11.
(Z)-3-(hexadec-7-en-1-yl)-4-methyloxetan-2-one (VM009): Mixture of
diastereoisomers (d.r. 7:3). Yield 36% (110 mg); Colorless oil; 1H NMR
(200 MHz, CDCl3): δ=5.44-5.23 (m, 2H), 4.81-4.66 (m, 0.3H), 4.46-4.33
(m, 0.7H), 3.67-3.52 (m, 0.3H), 3.22-3.10 (m, 0.7H), 2.11-1.90 (m, 4H),
1.89-1.60 (m, 2H), 1.56 (d, J = 6 Hz, 2.1H), 1.46 (d, J = 6 Hz, 0.9H),
1.42-1.13 (m, 20H), 0.89 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3):
δ=172.0, 171.3, 130.1, 130.0, 129.6, 129.5, 74.6, 71.7, 57.6, 52.8, 31.9,
29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 28.9, 27.7, 27.3, 27.2, 27.1, 26.8, 23.9,
22.7, 20.3, 15.6, 14.1; HRMS m/z [M+Na]+ calcd for C20H36O2Na+:
331.2607, found: 331.2611; Anal. calcd for C20H36O2: C, 77.87, H 11.76,
found: C, 77.62, H 11.85.
(Z)-2-Butyl-2-(ethoxycarbonyl)icos-11-enoic acid (6c): To a stirred
solution of substituted malonate 5c (700 mg, 1.5 mmol) in EtOH (3 mL), a
solution of KOH (2.1 mmol) in EtOH (3 mL) was added and the solution
was refluxed for 2 days. The solvent was removed under reduced
pressure, water (45 mL) was then added to the reaction mixture which
was extracted with CH2Cl2 (45 mL). The aqueous layer was acidified with
1N HCl and extracted with CH2Cl2 (3 x 45 mL). The organic layers were
combined, washed with brine and dried. The solvent was removed and
the product was purified by column chromatography eluting with a
gradient of CHCl3/MeOH 98:2 to 95:5 (v/v). Yield 40% (260 mg);
Yellowish oil; 1H NMR (200 MHz, CDCl3): δ=5.44-5.25 (m, 2H), 4.31 (q, J
= 7 Hz, 2H), 2.12-1.73 (m, 8H), 1.45-1.00 (m, 31H), 0.96-0.81 (m, 6H);
13C NMR (50 MHz, CDCl3): δ=176.7, 173.6, 129.9, 129.7, 61.7, 57.6,
34.1, 33.9, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.0, 26.6, 24.4,
22.8, 22.6, 14.1, 14.0, 13.8; MS (ESI) m/z (%): 437.4 [(M-H)-, 100]; Anal.
calcd for C27H50O4: C, 73.92, H 11.49, found: C, 73.78, H 11.61.
(Z)-4-ethyl-3-(hexadec-7-en-1-yl)oxetan-2-one (VM010): Mixture of
diastereoisomers (d.r. 7:3). Yield 52% (420 mg); Colorless oil; 1H NMR
(200 MHz, CDCl3): δ=5.42-5.20 (m, 2H), 4.49-4.35 (m, 0.3H), 4.19-4.06
(m, 0.7H), 3.64-3.47 (m, 0.3H), 3.20-3.05 (m, 0.7H), 2.09-1.89 (m, 4H),
1.89-1.55 (m, 4H), 1.53-1.10 (m, 20H), 1.08-0.92 (m, 3H), 0.85 (t, J = 7
Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=172.1, 171.4, 130.0, 129.9, 129.5,
129.4, 79.0, 76.8, 55.6, 52.4, 31.8, 29.7, 29.5, 29.4, 29.2, 29.1, 28.8,
27.8, 27.5, 27.4, 27.1, 27.0, 26.9, 23.8, 23.4, 22.6, 14.0, 9.8, 9.0; HRMS
m/z [M+Na]+ calcd for C21H38O2Na+: 345.2764, found: 345.2764; Anal.
calcd for C21H38O2: C, 78.20, H 11.88, found: C, 78.01, H 11.98.
(Z)-3-(hexadec-7-en-1-yl)-4-hexyloxetan-2-one (VM011): Mixture of
diastereoisomers (d.r. 7:3). Yield 56% (530 mg); Colorless oil; 1H NMR
(200 MHz, CDCl3): δ=5.43-5.22 (m, 2H), 4.57-4.42 (m, 0.3H), 4.25-4.12
(m, 0.7H), 3.64-3.48 (m, 0.3H), 3.21-3.07 (m, 0.7H), 2.11-1.89 (m, 4H),
1.89-1.58 (m, 4H), 1.58-1.10 (m, 28H), 0.95-0.78 (m, 6H); 13C NMR (50
MHz, CDCl3): δ=172.1, 171.4, 130.0, 129.9, 129.5, 129.4, 78.0, 75.6,
56.0, 52.5, 34.4, 31.8, 31.5, 30.1, 29.7, 29.5, 29.4, 29.2, 29.1, 28.9, 28.8,
27.8, 27.5, 27.1, 27.0, 26.9, 25.4, 24.9, 23.8, 22.6, 22.4, 14.0, 13.9;
HRMS m/z [M+Na]+ calcd for C25H46O2Na+: 401.3390, found: 401.3396;
Anal. calcd for C25H46O2: C, 79.30, H 12.25, found: C, 79.11, H 12.35.
Synthesis of α-substituted β-hydroxy acids (7a-c): To a stirred
solution of ethoxycarbonyl acids (6a-c) (1 mmol) in iPrOH/dry THF 1:2
(2.4 mL), a solution of LiBH4 2.0 M in THF (2 mL, 4 mmol) was added
under argon at 0 °C and was stirred at 0 °C for 1 h and at room
temperature overnight. The solvents were removed under reduced
pressure, water (30 mL) was added to the reaction mixture and was
extracted with Et2O (30 mL). The aqueous layer was acidified with HCl
1Ν and extracted with CH2Cl2 (3 x 30 mL). The organic layers were
combined, washed with brine and dried. The solvent was removed and
the product was purified by column chromatography eluting with a
gradient of CHCl3/MeOH 95:5 to 90:10 (v/v).
(Z)-3-(hexadec-7-en-1-yl)-4-nonyloxetan-2-one (VM012): Mixture of
diastereoisomers (d.r. 7:3). Yield 63% (600 mg), Colorless oil; 1H NMR
(200 MHz, CDCl3): δ=5.44-5.23 (m, 2H), 4.59-4.45 (m, 0.3H), 4.26-4.13
(m, 0.7H), 3.65-3.50 (m, 0.3H), 3.22-3.07 (m, 0.7H), 2.14-1.91 (m, 4H),
1.91-1.56 (m, 4H), 1.56-1.10 (m, 34H), 0.89 (t, J = 7 Hz, 6H); 13C NMR
(50 MHz, CDCl3): δ=172.2, 171.5, 130.0, 129.9, 129.5, 129.4, 78.0, 75.6,
56.0, 52.5, 34.4, 31.8, 31.7, 30.1, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1,
28.9, 27.8, 27.5, 27.1, 27.0, 26.9, 25.5, 25.0, 23.9, 22.6, 14.0; HRMS m/z
[M+Na]+ calcd for C28H52O2Na+: 443.3859, found: 443.3861; Anal. calcd
for C28H52O2: C, 79.94, H 12.46, found: C, 79.77, H 12.57.
(Z)-2-(hydroxymethyl)icos-11-enoic acid (7a): Yield 71% (300 mg);
Colorless oil; 1H NMR (200 MHz, CDCl3): δ=5.44-5.24 (m, 2H), 3.77 (d, J
= 6 Hz, 2H), 2.68-2.49 (m, 1H), 2.14-1.88 (m, 4H), 1.78-1.13 (m, 26H),
0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=180.4, 129.9, 129.8,
62.9, 47.5, 31.9, 29.7, 29.5, 29.4, 29.3, 28.2, 27.2, 22.7, 14.1; HRMS m/z
[M+Na]+ calcd for C21H40O3Na+: 363.2870, found: 363.2868; Anal. calcd
for C21H40O3: C, 74.07, H 11.84, found: C, 73.90, H 11.95.
2-(Hydroxymethyl)octadecanoic acid (7b)[40] : Yield 50% (120 mg);
White solid; mp: 78-81 °C; mp of literature: 85.5-87 °C;[40] 1H NMR (200
MHz, CDCl3): δ=3.80 (d, J = 6 Hz, 2H), 2.71-2.52 (m, 1H), 1.79-1.47 (m,
6
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10.1002/cmdc.201800720
ChemMedChem
FULL PAPER
2H), 1.47-1.03 (m, 28H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz,
CDCl3): δ=179.4, 62.8, 47.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.2, 27.2,
22.7, 14.1; HRMS m/z [M+Na]+ calcd for C19H38O3Na+: 337.2713, found:
337.2711.
mmol) and H2O (15 mL) was then added and the mixture was extracted
with Et2O (3 x 30 mL). The organic layer was washed with brine, dried
and concentrated under vacuum.
(Z)-3-Hydroxyicos-11-enoic acid (11a): Yield 84% (411 mg); White
solid; mp: 58-60 °C; 1H NMR (200 MHz, CDCl3): δ=5.44-5.28 (m, 2H),
4.12-3.96 (m, 1H), 2.65-2.37 (m, 2H), 2.12-1.87 (m, 4H), 1.70-1.00 (m,
24H), 0.88 (t, J = 6 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=178.0, 130.0,
129.8, 68.0, 41.1, 36.4, 31.9, 29.7, 29.5, 29.4, 29.3, 29.2, 27.2, 25.4,
22.6, 14.1; Anal. calcd for C20H38O3: C, 73.57, H 11.73, found: C, 73.35,
H 11.86.
(Z)-2-Butyl-2-(hydroxymethyl)icos-11-enoic acid (7c): Yield 73% (150
mg); Colorless oil; 1H NMR (200 MHz, CDCl3): δ=5.44-5.25 (m, 2H), 3.68
(s, 2H), 2.17-1.84 (m, 4H), 1.77-1.45 (m, 4H), 1.45-1.07 (m, 28H), 0.99-
0.78 (m, 6H); 13C NMR (50 MHz, CDCl3): δ=183.1, 129.9, 129.8, 64.9,
50.6, 33.1, 32.8, 31.9, 30.3, 29.8, 29.6, 29.5, 29.3, 27.2, 26.1, 24.0, 23.3,
22.7, 14.1, 14.0; HRMS m/z [M+Na]+ calcd for C25H48O3Na+: 419.3496,
found: 419.3493; Anal. calcd for C25H48O3: C, 75.50, H 12.20, found: C,
75.39, H 12.28.
3-Hydroxyoctadecanoic acid (11b)[41]: Yield 75% (338 mg); White
solid; mp: 89-90 °C; mp of literature: 88-90 °C;[42] 1H NMR (200 MHz,
CDCl3): δ=4.05-3.87 (m, 1H), 2.60-2.25 (m, 4H), 1.60-1.00 (m, 26H), 0.85
(t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=176.0, 68.3, 41.2, 36.7,
32.1, 29.9, 29.8, 29.7, 29.6, 25.7, 22.9, 14.3.
Cyclization of α-substituted β-hydroxy acids to β-lactones (VM004-
VM006): To a stirred solution of PPh3 (525 mg, 2 mmol) in dry THF (10
mL), DEAD (348 mg, 2 mmol) was slowly added under argon at 0 °C and
the solution was stirred at 0 °C for 1 h. Then, solution of β-hydroxy acid
7a-c (1 mmol) in dry THF (8 mL) was added at 0 °C and the solution was
stirred at 0 °C for 1 h and at room temperature overnight. The solvent
was removed under reduced pressure and the product was purified by
column chromatography eluting with a gradient of petroleum ether (bp
40-60 °C)/AcOEt 95:5 to 90:10 (v/v).
3-Hydroxyhexanoic acid (11c)[43]: Yield 81% (160 mg); Colorless oil; 1H
NMR (200 MHz, CDCl3): δ=7.00 (s, 1H), 4.20-3.90 (m, 1H), 2.65-2.32 (m,
2H), 1.65-1.20 (m, 4H), 0.93 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3):
δ=177.7, 67.8, 41.1, 38.4, 18.6, 13.9.
General method for the synthesis of β-substituted β-lactones
(VM014-VM016): In a dry flask 2,2’-dipyridyl disulphide (330 mg, 1.5
mmol) and triphenylphosphine (420 mg, 1.6 mmol) were dissolved in dry
CH2Cl2 (10 mL) at room temperature under argon. The hydroxy acid
11a-c (1 mmol) was slowly added. After 30 min, the resulting yellow
(Z)-3-(Octadec-9-en-1-yl)oxetan-2-one (VM004): Yield 52% (55 mg);
Colorless oil; H NMR (200 MHz, CDCl3): δ=5.44-5.24 (m, 2H), 4.36 (t, J
1
= 6 Hz, 1H), 4.01 (t, J = 6 Hz, 1H), 3.78-3.61 (m, 1H), 2.13-1.91 (m, 4H),
1.91-1.63 (m, 2H), 1.55-1.16 (m, 24H), 0.88 (t, J = 7 Hz, 3H); 13C NMR
(50 MHz, CDCl3): δ=171.8, 129.9, 129.7, 65.0, 52.0, 31.9, 29.7, 29.6,
29.5, 29.4, 29.3, 29.2, 28.1, 27.2, 27.1, 26.8, 22.6, 14.1; HRMS m/z
[M+Na]+ calcd for C21H38O2Na+: 345.2764, found: 345.2767; Anal. calcd
for C21H38O2: C, 78.20, H 11.88, found: C, 78.02, H 11.99.
solution was added dropwise to
a vigorously stirred solution of
mercury(II) trifluoromethanesulfonate (2 mmol) in dry MeCN (24 mL) at
50 °C under argon. After 20 min at 50 °C, the reaction mixture was
filtered and the precipitate was washed several times with CH2Cl2. The
solvent was removed under reduced pressure and the product was
purified by column chromatography eluting with a gradient of petroleum
ether (bp 40-60 °C)/AcOEt 85:15 to 80:20 (v/v) to give the β-lactone.
3-Hexadecyloxetan-2-one (VM005): Yield 74% (20 mg); White solid;
1
mp: 48-50 °C; H NMR (200 MHz, CDCl3): δ=4.37 (t, J = 6 Hz, 1H), 4.01
(t, J = 6 Hz, 1H), 3.79-3.63 (m, 1H), 1.99-1.65 (m, 2H), 1.54-1.15 (m,
28H), 0.89 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=171.8, 65.0,
52.1, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 28.1, 26.8, 22.7, 14.1;
HRMS m/z [M+Na]+ calcd for C19H36O3Na+: 319.2608, found: 319.2612;
Anal. calcd for C19H36O2: C, 76.97, H 12.24, found: C, 76.78, H 12.32.
(Z)-4-(Heptadec-8-en-1-yl)oxetan-2-one (VM014): Yield 28% (87 mg);
Yellowish oil; 1H NMR (200 MHz, CDCl3): δ=5.44-5.26 (m, 2H), 4.60-4.42
(m, 1H), 3.51 (dd, J = 16 Hz, 6 Hz, 1H), 3.06 (dd, J = 16 Hz, 6 Hz, 1H),
2.10-1.95 (m, 4H), 1.90-1.65 (m, 2H), 1.55-1.15 (m, 22H), 0,88 (t, J = 7
Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=168.4, 130.0, 129.7, 71.3, 42.8,
29.7, 29.6, 29.5, 29.3, 29.1, 29.0, 27.2, 24.9, 22.7, 14.1; HRMS m/z
[M+Na]+ calcd for C20H36O2Na+: 331.2607, found: 331.2609; Anal. calcd
for C20H36O2: C, 77.87, H 11.76, found: C, 77.68, H 11.89.
(Z)-3-Butyl-3-(octadec-9-en-1-yl)oxetan-2-one (VM006): Yield 70% (50
mg); Colorless oil; 1H NMR (200 MHz, CDCl3): δ=5.45-5.25 (m, 2H), 4.10
(s, 2H), 2.12-1.89 (m, 4H), 1.80-1.61 (m, 4H), 1.54-1.15 (m, 28H), 1.02-
0.78 (m, 6H); 13C NMR (50 MHz, CDCl3): δ=174.6, 130.0, 129.8, 69.2,
61.5, 32.5, 32.2, 31.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 27.2, 27.1,
26.5, 24.3, 22.9, 22.7, 14.1, 13.9; HRMS m/z [M+Na]+ calcd for
C25H46O2Na+: 401.3390, found: 401.3389; Anal. calcd for C25H46O2: C,
79.30, H 12.25, found: C, 79.19, H 12.33.
4-Pentadecyloxetan-2-one (VM015): Yield 66% (187 mg); White solid;
mp: 47.5-49 °C; 1H NMR (200 MHz, CDCl3): δ=4.58-4.40 (m, 1H), 3.51
(dd, J = 16 Hz, 6 Hz, 1H), 3.06 (dd, J = 16 Hz, 6 Hz, 1H), 1.95-1.65 (m,
2H), 1.64-1.10 (m, 26H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz,
CDCl3): δ=168.4, 71.3, 42.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 14.1;
HRMS m/z [M+Na]+ calcd for C18H34O2Na+: 305.2451, found: 305.2450;
Anal. calcd for C18H34O2: C, 76.54, H 12.13, found: C, 76.32, H 12.19.
Ethyl (Z)-3-hydroxyicos-11-enoate (10a): NaBH4 (38 mg, 1 mmol) was
added to a solution of β-keto ester 9a (704 mg, 2 mmol) in EtOH (8 mL)
under argon. The mixture was stirred at room temperature until complete
conversion. 1N HCl (10 mL) was then added at 0 °C and the mixture was
extracted with CH2Cl2 (3 x 30 mL). The organic layer was washed with
brine, dried and concentrated under vacuum. The product was purified by
column chromatography eluting with a gradient of petroleum ether (bp
40-60 °C)/AcOEt 90:10 to 80:20 (v/v). Yield 78% (276 mg); Colorless oil;
1H NMR (200 MHz, CDCl3): δ=5.41-5.27 (m, 2H), 4.17 (q, J = 7 Hz, 2H),
4.07-3.92 (m, 1H), 2.82-2.60 (m, 1H), 2.60-2.30 (m, 2H), 2.10-1.90 (m,
4H), 1.55-1.15 (m, 27H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (50 MHz,
CDCl3): δ=173.1, 129.9, 129.8, 68.0, 60.6, 41.3, 36.5, 31.9, 29.7, 29.5,
29.3, 29.2, 27.2, 25.4, 14.2, 14.1; Anal. calcd for C22H42O3: C, 74.52, H
11.94, found: C, 74.38, H 12.05.
1
4-Propyloxetan-2-one (VM016)[44]: Yield 42% (48 mg); Colorless oil; H
NMR (200 MHz, CDCl3): δ=4.60-4.45 (m, 1H), 3.52 (dd, J = 16 Hz, 6 Hz,
1H), 3.06 (dd, J = 16 Hz, 6 Hz, 1H), 2.00-1.65 (m, 2H), 1.64-1.32 (m, 2H),
0.99 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ=168.4, 71.1, 42.9,
36.7, 18.3, 13.7; HRMS m/z [M+Na]+ calcd for C6H10O2Na+: 137.0573,
found: 137.0576; Anal. calcd for C6H10O2: C, 63.14, H 8.83, found: C,
62.97, H 8.92.
In vitro anti-mycobacterial activity and cytotoxicity
Biological assay for antibacterial activity: Stock solutions of Orlistat
(10 mg/mL) or VM molecules (5 mg/mL), in which the compounds were
found to be completely soluble in dimethyl sulfoxide (DMSO), were
prepared prior to susceptibility testing.
General method for the synthesis of β-hydroxy acids (11a-c):
Aqueous solution of 1N NaOH (2 mL, 2 mmol) was added to a solution of
β-hydroxy ester 12a-c (1 mmol) in 1,4-dioxane (2 mL). The reaction
mixture was stirred at room temperature overnight. 1N HCl (3 mL, 3
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800720
ChemMedChem
FULL PAPER
Bacterial strains and growth condition: M. marinum ATCC BAA-
535/M, M. abscessus CIP104536T with either a smooth (S) or rough (R)
morphotype, and M. tuberculosis mc26230 (H37Rv RD1 panCD)[22]
strains were routinely grown in Middlebrook 7H9 broth (BD Difco)
supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma-Aldrich) and
10% oleic acid, albumin, dextrose, catalase (OADC enrichment; BD
Difco) (7H9-SOADC). In the case of M. tuberculosis mc26230, 24 µg/mL D-
panthothenate (Sigma-Aldrich) was also added in the 7H9-SOADC medium.
All cultures were kept at 37 °C without shaking, except M. marinum which
was cultured at 32 °C.
Acknowledgements
This study was supported by the Centre National de la
Recherche Scientifique (CNRS) and the Special Account for
Research Grants of the National and Kapodistrian University of
Athens (SARG/NKUA). P. Santucci received financial support for
his PhD fellowship from the Ministère de l’Enseignement
Supérieur de la Recherche et de l’Innovation, France. C. Dedaki
is indebted to the Greek National Scholarship Foundation (IKY)
for financial support. We would like to thank Dr. M. G. Kokotou
for the HRMS spectra and Dr. E. Sakki and Dr. A. Paschalidou
for the MS spectra.
Resazurin microtiter assay (REMA) for MICs determination:
Susceptibility testing was performed using the Middlebrook 7H9 broth
microdilution method. All assays were carried out at least in duplicate.
MICs were determined in 96-well flat-bottom Nunclon Delta Surface
microplates with lid (Thermo-Fisher Scientific, ref. 167008) using the
resazurin microtiter assay (REMA).[21a] Briefly, log-phase bacteria were
diluted to a cell density of 5 × 106 cells/mL in 7H9-SOADC (7H9 broth +
10% OADC + 0.2% glycerol + 0.05% Tween 80, and 24 µg/mL D-
panthothenate when needed). Then 100 µL of the above inoculum was
added to each well containing 100 µL 7H9-SOADC medium, serial two-fold
dilutions of the selected inhibitor or controls to a final volume of 200 µL
(final bacterial charge of 2.5 × 106 cells/mL per well). The final volume of
DMSO was kept under 3%. Growth controls containing no inhibitor but
with 3% DMSO (i.e., bacteria only = B), inhibition controls containing 50
µg/mL kanamycin and sterility controls (i.e., medium only = M) without
inoculation were also included. Plates were incubated at 37 °C (32 °C for
M. marinum) in a humidity chamber[45] to prevent evaporation for 3-5
days (M. smegmatis, M. abscessus) or 10-14 days (M. marinum, M.
bovis BCG, M. tuberculosis mc26230). Then, 20 µL of a 0.025% (w/v)
resazurin solution was added to each well, and the plates were incubated
at 37 °C (or 32 °C) for color change from blue to pink or violet and for a
reading of fluorescence units (FU). Fluorescence corresponding to the
resazurin reduction was quantified using a Tecan Spark 10M multimode
microplate reader (Tecan Group Ltd, France) with excitation at 530 nm
Keywords: Antibacterial activity • β-lactones • Mycobacterium
marinum • serine hydrolases • tuberculosis
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