Development of cyclobutene- and cyclobutane-functionalized fatty acids with inhibitory activity against mycobacterium tuberculosis

Article abstract of DOI:10.1002/cmdc.201402067

Eleven fatty acid analogues incorporating four-membered carbocycles (cyclobutenes, cyclobutanes, cyclobutanones, and cyclobutanols) were investigated for the ability to inhibit the growth of Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). A number of the analogues displayed inhibitory activity against both mycobacterial species in minimal media. Several of the molecules displayed potent levels of inhibition against Mtb, with MIC values equal to or below those observed with the anti-tuberculosis drugs D-cycloserine and isoniazid. In contrast, two of the analogues that display the greatest activity against Mtb failed to inhibit E. coli growth under either set of conditions. Thus, the active molecules identified herein may provide the basis for the development of anti-mycobacterial agents against Mtb.

Full text of DOI:10.1002/cmdc.201402067

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201402067
Development of Cyclobutene- and Cyclobutane-
Functionalized Fatty Acids with Inhibitory Activity against
Mycobacterium tuberculosis
Wantanee Sittiwong,[a] Denise K. Zinniel,[b] Robert J. Fenton,[b] Darrell D. Marshall,[a]
Courtney B. Story,[a] Bohkyung Kim,[c] Ji-Young Lee,[c] Robert Powers,[a] Raffll G. Barletta,[b] and
Patrick H. Dussault*[a]
Eleven fatty acid analogues incorporating four-membered car-
bocycles (cyclobutenes, cyclobutanes, cyclobutanones, and cy-
clobutanols) were investigated for the ability to inhibit the
growth of Mycobacterium smegmatis (Msm) and Mycobacterium
tuberculosis (Mtb). A number of the analogues displayed inhibi-
tory activity against both mycobacterial species in minimal
media. Several of the molecules displayed potent levels of in-
hibition against Mtb, with MIC values equal to or below those
observed with the anti-tuberculosis drugs d-cycloserine and
isoniazid. In contrast, two of the analogues that display the
greatest activity against Mtb failed to inhibit E. coli growth
under either set of conditions. Thus, the active molecules iden-
tified herein may provide the basis for the development of
anti-mycobacterial agents against Mtb.
Introduction
Tuberculosis (TB) resulting from Mycobacterium tuberculosis
(Mtb) infection remains one of humankind’s most significant
disease threats. Based on skin test reactivity, it is estimated
that one-third of the world’s population has been exposed, re-
sulting in approximately nine million incident cases annually
and 1.4 million deaths (2010 data).[1] A number of therapeutic
agents have been developed, but current treatment regimens
require patients to take multiple drugs over a period of
months and are associated with significant side effects.[2] The
result is frequent patient noncompliance, leading to relapses
and the emergence of drug resistance, with a high fraction of
active cases now involving multidrug-resistant (MDR, XDR)
strains.[1] There is, therefore, a need for new classes of thera-
peutics for TB.[3] Importantly, the lethal target and/or mecha-
nism of action should also be novel to avoid established mech-
anisms of resistance and to provide synergy with current treat-
ments.
branched-chain fatty acids.[4] A number of existing treatments
for Mtb, exemplified by isoniazid and ethionamide, disrupt my-
colic acid biosynthesis by inhibiting important biosynthetic en-
zymes.[5] Mycobacteria incorporate intact C16 and C18 fatty acid
skeletons as biosynthetic feed stocks, and given the prior evi-
dence for the incorporation of modified fatty acids into mycol-
ic acid biosynthesis,[6] we hypothesized that hijacking this path-
way could provide a unique approach for the development of
potential therapeutics.[4] Toward this end, we proposed to in-
vestigate specifically functionalized fatty acids for their ability
to limit mycobacterial growth.[7] As our initial steps toward vali-
dating this hypothesis, we investigated two series of fatty acid
analogues (Figure 1). One series (compounds 1–4) is based on
a decenoic acid (10:1) framework, and a second series (com-
pounds 5–11) is based on the frameworks of oleic or elaidic
acids (18:1). The analogues preserve the approximate lengths
and cross-sections of the “parent” fatty acids while incorporat-
ing four-membered carbocycles into the backbone. Herein we
report that several of these analogues display significant anti-
mycobacterial activity against Mtb.
Much of the hardiness and drug resistance of mycobacteria
is due to an unusually thick lipid cell wall that contains a signifi-
cant proportion of mycolic acids, a unique class of C54–C63
[a] W. Sittiwong, D. D. Marshall, C. B. Story, Prof. R. Powers, Prof. P. H. Dussault
Department of Chemistry
Results and Discussion
University of Nebraska-Lincoln, Lincoln, NE 68588-0304 (USA)
E-mail: pdussault1@unl.edu
Substrate preparation
The only previous description of molecules included in this
study was the preparation of a mixture of the methyl esters of
5 and 10 via a nonselective radical decarboxylation.[8] In search
of an efficient, general, and stereospecific approach to incorpo-
ration of four-membered rings onto a fatty acid backbone, we
were drawn to methodology for modification of dichlorocyclo-
butanones.[9] Our approach is illustrated in detail for the prepa-
[b] D. K. Zinniel, R. J. Fenton, Prof. R. G. Barletta
School of Veterinary Medicine and Biomedical Sciences
University of Nebraska-Lincoln, Lincoln, NE 68583-0905 (USA)
[c] Dr. B. Kim, Prof. J.-Y. Lee
Department of Nutritional Sciences
University of Connecticut, Storrs, CT 06269-4017 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402067.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1838

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
in acetic acid, followed by deprotection of the benzyl ester
with Pd(C)/H2, furnished cyclobutanone 3. Hydride reduction of
the intermediate ketone with hydride prior to hydrogenolysis
generated cyclobutanol 4 as a mixture of diastereomers at the
newly created alcohol stereocenter.
The preparation of analogues incorporating the four-mem-
bered ring carbocycles onto a cis-C18 framework is illustrated
in Scheme 2. In this case, the initial cycloaddition with benzyl
oleate produced an inseparable mixture of regioisomeric di-
chlorocyclobutanones 19. The cycloadducts, which were prone
to decomposition during purification, were directly reduced to
furnish a mixture of regioisomeric 2,2-dichloro-1-cyclobutanols.
The corresponding methanesulfonate (mesylate) esters 20 un-
derwent reduction/fragmentation as described above to fur-
nish cyclobutene 5,[8] which could be hydrogenated to cyclo-
butane 6. The related cyclobutanone 7 and cyclobutanol 8
were prepared from the dichlorocyclobutanone in a similar
manner as described earlier for the C10 series. Alternatively,
controlled dehalogenation of the dichlorocyclobutanone cyclo-
adduct with stoichiometric Zn or Zn(Cu) in acetic acid fur-
nished a mixture of regioisomeric monochlorocyclobutanones
22 contaminated by small amounts of dichloroketone (starting
material) and cyclobutanone 3 (over-reduction).[11] Hydrogenol-
ysis of the monochlorocyclobutanone as before, furnished ana-
logue 9.
The ready availability of elaidic acid, a C18 fatty acid contain-
ing a trans-9,10 alkene, made it possible to investigate the in-
fluence of stereoisomers (Scheme 3). Beginning with elaidic
acid (14) we were able to synthesize cyclobutene 10 and mon-
ochlorocyclobutanone 11 in a manner analogous to that de-
scribed previously for oleate-derived substrates 5 and 9. The
only major difference from the route employed in Scheme 2
was the need to use activated Zn or Zn/Cu for the initial
ketene generation/cycloaddition.[12]
Figure 1. Analogues investigated, along with structures of oleic (cis-18:1),
elaidic (trans-18:1) and decenoic (10:1) acids; R=octyl.
ration of analogues 1–4 (Scheme 1). The benzyl ester of 9-de-
cenoic acid underwent cycloaddition with dichloroketene to
afford one major dichlorobutanone, 15,[10] which was only
moderately stable toward purification, and was therefore di-
rectly reduced with sodium borohydride to furnish a mixture
of stereoisomeric 2,2-dichloro-1-cyclobutanols. The choice of
sodium borohydride was for convenience, as a similar product
mixture is available using other reducing reagents such as
BH3·THF and Na(OAc)3BH. The alcohols were converted into
the corresponding methanesulfonate (mesylate) esters 16. Re-
action with sodium in ammonia resulted in simultaneous re-
ductive cleavage of the benzyl ester and fragmentation/reduc-
tion of the b-chloro methanesulfonate to furnish cyclobutene
1. Hydrogenation of 1 cleanly generated cyclobutane 2. Alter-
natively, dehalogenation of the initial cycloadduct 15 with zinc
Inhibitor stability and solubility
Analogues 1–11 were each tested for thermal stability using
differential scanning calorimetry (DSC) in tandem with thermal
gravimetric analysis (TGA); details are provided in the Support-
ing Information. Although we originally had concerns for the
stability of the cyclobutene-containing analogues (1, 5, and
10) toward thermally induced cycloreversion,[13] we observed
no decomposition of any analogue below 1008C; most of the
new analogues were stable to ꢀ1508C.
One-dimensional (1D) 1H NMR spectroscopy was used to
verify the aqueous stability and solubility for each of the
eleven analogues. An example series is illustrated in Figure 2;
more detailed data are provided in the Supporting Informa-
tion. The results demonstrate that the analogues are stable in
aqueous buffer. All of the analogues based on a C18 scaffold
(compounds 5–11) display evidence of aggregation at all con-
centrations tested (down to 100 mm). In contrast, analogues 1–
4, based on the C10 backbone, were not observed to aggregate
even at millimolar concentrations (Table 1).
Scheme 1. Preparation of analogues 1–4 in the C10 series. Reagents and con-
ditions: a) DMAP, DCC, BnOH; b) Zn dust (5 equiv), Cl3CCOCl (2.5 equiv), RT,
Et2O; c) NaBH4, 2-propanol; d) MsCl, Et3N; e) Na/NH3, À78 to À338C; f) H2,
Pd/C; g) Zn (5 equiv), AcOH; h) NaBH4, MeOH. See Experimental Section for
reaction details.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1839

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
trol for E. coli. Isoniazid (Inh), one of the main first-
line drugs used to treat TB, and d-cycloserine (DCS),
a second-line clinically used TB drug, were employed
as controls that would allow benchmarking against
other inhibitors. Results for the fatty acid analogues
are listed in Table 2.
Neither of the two analogues, 1 (a C10-cyclobutene)
nor 6 (a C18-cyclobutane), tested against E. coli were
inhibitory in rich (LB broth) or minimal media. Inter-
estingly, the susceptibility of E. coli toward the con-
trol drugs (DCS, Inh, and Kan) was greater in minimal
media, as indicated by the significantly lower MIC
values. Preliminary studies indicated that Msm was
not susceptible to compounds 7 and 10 in complete
Middlebrook 7H9 broth supplemented with either
Tween or Tyloxapol (data not shown). In this context,
a potential interfering compound is bovine serum al-
bumin (BSA), which has been shown to bind to fatty
acids.[14] Indeed, preliminary tests using Msm and
compound 7 indicate that increasing concentrations
of BSA significantly increase the MIC value (unpub-
Scheme 2. Synthesis of analogues 5–9. Reagents and conditions: a) DMAP, DCC, BnOH;
b) Zn dust (5 equiv), Cl3CCOCl (2.5 equiv), Et2O; c) NaBH4, 2-propanol; d) MsCl, Et3N;
e) Na/NH3, À78 to À338C; f) H2, Pd/C EtOAc; g) Zn (5 equiv), AcOH; h) NaBH4, MeOH,
À108C; i) Zn (1.1 equiv), AcOH. R=octyl. See Experimental Section for reaction details.
lished results). Thus, the entire set of compounds synthesized
was tested in minimal media against Msm and the two Mtb
strains. Under these conditions, none of the compounds dem-
onstrated significant activity against Msm. However, four (1, 2,
Scheme 3. Synthesis of analogues 10 and 11. Reagents and conditions:
a) DMAP, DCC, BnOH; b) Zn(Cu) (5 equiv), Cl3CCOCl (2.5 equiv), RT, Et2O;
c) NaBH4, 2-propanol; d) MsCl, Et3N; e) Na/NH3, À78 to À338C; f) Zn
(1.1 equiv), AcOH; g) H2, Pd/C EtOAc. See Experimental Section for reaction
details.
Table 1. Critical micellar coefficient (CMC) of analogues under assay con-
ditions.[a]
CMC
ꢁ100 mm
5–11
ꢀ1000 mm
1–4
analogues
[a] Designated concentration or range indicates onset of aggregation ob-
served in 1H NMR spectra of buffer solutions; see Experimental Section
for details.
MIC determination
MICs were determined against the following bacteria: Escheri-
chia coli (E. coli) wild-type strain G58-1; M. smegmatis (Msm)
1
Figure 2. 1D H NMR spectra of compounds A) 5 and B) 1 were inspected for
strain mc2155,
a non-pathogenic mycobacteria used as
evidence of micelle formation (peak broadening) by comparing peak widths
relative to TMSP (standard). Peak widths for 5 demonstrated potential mi-
celle formation or aggregation over the range of tested concentrations
(boxed region). No micelle formation was observed for compounds 1–4 be-
tween 100 and 1000 mm.
a model for Mtb to analyze processes that are likely to be con-
served in the genus; and two Mtb strains (CDC1551 and
H37Rv). Kanamycin (Kan) was used as a positive inhibition con-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1840

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Table 2. MIC values against E. coli, Msm, and Mtb.
Compd
Scaffold
Functionality
MIC [mgmLÀ1/(mm)][a]
E. coli[b]
Msm[c]
Mtb CDC1551
Mtb H37Rv, week 7
LB[d]
MM[e]
MM[e]
MM[e]
MM[e]
1
2
3
4
5
6
7
8
9
10
11
12
C10
C10
C10
C10
cis-C18
cis-C18
cis-C18
cis-C18
cis-C18
trans-C18
trans-C18
C10
alkene
alkane
ketone
alcohol
alkene
alkane
ketone
alcohol
chloroketone
alkene
chloroketone
alkene (acyclic)
>512/(>2608)
>512/(2608)
512/(2608)
512/(2582)
256/(1206)
128/(597)
128/(415)
256/(824)
128/(394)
256/(784)
256/(713)
256/(830)
128/(357)
512/(3007)
64/(627)
32/(233)
–
16/(82)
4/(20)
128/(603)
64/(299)
128/(415)
8/(26)
16/(82)
4/(20)
–
–
–
–
–
–
–
–
256/(1206)
128/(597)
16/(52)
16/(52)
32/(99)
2/(6)
16/(45)
16/(52)
256/(713)
32/(188)
8/(78)
>512/(>1649)
>512/(1649)
–
–
–
–
–
–
–
–
–
–
–
–
64/(197)
8/(24)
256/(713)
128/(415)
256/(713)
32/(188)
4/(39)
DCS
isoniazid
kanamycin
256/(2508)
>512/(>3733)
64/(110)
16/(157)
16/(117)
<8/(14)
4/(29)
–
8/(58)
–
[a] MIC values were determined as described in the Experimental Section. [b] G58-1, day 2. [c] mc2155, day 4. [d] Luria–Bertani broth. [e] Minimal media.
6, and 8) of the eleven compounds yielded MIC values
<100 mm for both Mtb strains, with two of the compounds (2
and 8) proving superior to DCS on a molar basis. Importantly,
compound 2 yielded MIC values equal (CDC1551 strain) or su-
perior (H37Rv strain) to those obtained with Inh. In summary,
these compounds were poor inhibitors of Msm, but quite ef-
fective against Mtb grown in minimal media.
stable. All of the analogues based on a C18 backbone aggre-
gate at concentrations overlapping the MIC values observed
for Mtb or Msm. Correspondingly, the biological mechanism of
action for analogues 5–11 is suspect, and these analogues are
unlikely to be viable drug leads. The propensity for these com-
pounds to form micelles or aggregate at concentrations lower
than their MIC values must also be considered in any attempt
to extract structure–activity relationships. In contrast, the ana-
logues based on a C10 backbone (compounds 1–4) displayed
no sign of aggregation at concentrations up to 1000 mm, levels
well above the measured MICs (Table 1). The observation of
significant and superior inhibition of Mtb with compounds
from the shorter-chain series provides strong evidence that the
inhibition is specific and not simply due to the general toxicity
often observed with very hydrophobic substrates.[16] Although
some short-chain fatty acids have also demonstrated limited
antimicrobial activity,[7] Mtb is also reported to use decanoic
acid (10:0) as a carbon source.[17] Moreover, decenoic acid (12),
the parent 10:1 framework of our shorter-chain analogues,
demonstrated very modest levels of inhibition in Mtb. Finally,
we note that the extremely hydrophobic oleic acid (13), the
parent 18:1 framework of analogues 5–9, is used as a standard
nutrient for Mtb at 0.05 gLÀ1 (177 mm), a concentration higher
than the MICs of the best inhibitors. Nonetheless, oleic acid is
always used in media containing BSA, which may ameliorate
its toxicity.[14]
Nonspecific toxicity
A sampling of six analogues, along with decanoic acid (12) and
the Mtb therapeutics Inh and DCS, were investigated for toxici-
ty in RAW 264.7 macrophages. SDS was employed as a positive
control for maximum toxic effect. As illustrated in Table S1
(Supporting Information), three of the tested analogues (4, 5,
and 6), as well as DCS and Inh showed little or no toxicity at
concentrations <250 mm;[15] toxicity for 12 was only slightly
greater. Modest or significant toxicity was observed for ana-
logues 1, 2, and 8.
Discussion
The MIC results demonstrate promising levels of inhibition
with a variety of four-membered-ring carbocycles (cyclobuta-
nol, cyclobutanone, cyclobutene, and cyclobutane). A critically
important component of evaluating new and potentially prom-
ising therapeutic entities is validation of a drug-like mode of
action. Unfortunately, there are numerous undesirable mecha-
nisms resulting from poor physical behavior of a compound
that will result in a false positive in a biological assay. These
poor properties may include insolubility, reactivity, micelle for-
mation, impurities, aggregation, instability and nonspecific
binding. This has resulted in numerous compounds erroneous-
ly reported as chemical leads that are not acceptable drug can-
didates because of these undesirable physiochemical proper-
ties.[16] The fatty acid analogues discussed herein are thermally
The nature of the functionality has a clear impact on activity.
Within the less hydrophobic short-chain series (compounds 1–
4), the measured MICs vary by >60-fold against Mtb H37Rv
and >30-fold against strain CDC1551. In contrast, a <10-fold
difference is observed for MICs measured against Msm. Al-
though the potential for micelle formation is cause for caution
in making structure–activity comparisons involving the long-
chain series, the different influences of a given functional
group within the C18 and C10 series are interesting. For exam-
ple, the short-chain cyclobutanone 3 and cyclobutanol 4 are
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1841

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
much less potent toward Mtb than the corresponding long-
chain analogues 7 and 8, whereas the values for the cyclobu-
tenes (1 versus 5 or 10) are similar for Mtb (H37Rv). The influ-
ence of stereochemistry varies by analogue. The MICs for the
cis and trans isomers of the C18 monochloroketones (9 and 11)
differ by >10-fold for Mtb (H37Rv); in contrast, no difference is
observed between the cis- and trans-cyclobutenes 5 and 10
with either Mtb strain. Finally, we note that in both the C18 and
C10 series, the saturated analogues (cyclobutanes 6 and 2) are
considerably more potent than the corresponding cyclobu-
tenes 5 and 1.
harsh chemical environments.[20] Thus, as an alternative to dis-
rupting mycolic acid biosynthesis, it is also plausible that our
four-membered ring analogues are converted into analogues
of mycolic acids and that the physical properties of these un-
natural cell wall constituents results in cell death. However, it
must be noted that while mycobacteria are known to directly
incorporate structurally modified fatty acid feedstocks into my-
colic acid biosynthesis,[6] there is as yet no evidence that the
same is true for the specific fatty acid analogues described
herein.[7] Indeed, further studies are required to determine an
in vivo mechanism of inhibition for our compounds.
Comparing MIC values obtained against Mtb versus Msm,
ten of eleven analogues demonstrate lower MIC values in at
least one strain of Mtb compared with Msm; for six of the
eleven, lower MIC values are obtained against both strains of
Mtb. The ratio of MIC values (Msm/Mtb) ranges from less than
one to >600; viewed across the eleven analogues, the average
ratio of MICs for Mtb versus Msm is 68 (CDC1551) and nearly
90 (H37Rv). The different levels of inhibition produced in Msm
and Mtb may result from a mechanism of action specific to
Mtb. Isoniazid, for example, demonstrates 100-fold more
potent inhibition of Mtb compared with Msm and possesses
low toxicity toward other mycobacteria and prokaryotic patho-
gens.[18] The lack of inhibition observed in E. coli using two of
the analogues (1 and 6) most potent against Mtb could indi-
cate specific inhibition of a metabolic pathway unique to the
latter.
In this context, it is worth noting that our initial choice of
molecular scaffold was based on the availability of low-cost
precursors. If the modified fatty acid chains are indeed taken
up into the mycobacterial biosynthetic pathways, greater activ-
ity or different specificities might be identified through the use
of different molecular backbones or alternative positioning of
the four-membered-ring carbocycles.
Conclusions
Analogues of fatty acids incorporating four-membered-ring
carbocycles may provide a starting point for the development
of new anti-mycobacterials. Four of eleven analogues tested
yielded MIC values <100 mm for both Mtb strains, with two of
the analogues proving more active than DCS and one ana-
logue proving equally potent or superior to isoniazid, one of
the main first-line drugs used for the treatment of TB.
The initial hypothesis behind the design of the molecules re-
ported herein was that uptake of a fatty acid analogue bearing
a reactive functional group (for example, cyclobutene, cyclobu-
tanone, chloroketone) might disrupt an enzymatic process re-
lated to cell wall synthesis. We imagined that one such target
might be methyltransferases, which are common in mycobac-
teria, but relatively uncommon in other organisms.[19] In an ear-
lier example of a related approach, the inhibition of growth in
Mtb observed in the presence of alkyne-containing fatty acids
(MICs of 20–25 mm) was attributed to irreversible modification
of an enoyl reductase within the mycobacterial FASII
system.[4,7] However, the significant inhibition produced by cy-
clobutanes 2 and 6, which lack a chemically reactive group, is
more suggestive of a noncovalent mode of inhibition. The rela-
tively low MIC values indicate our fatty acid analogues have
promise as anti-mycobacterial agents, but there is still a need
to confirm cellular uptake and to establish the in vivo target.
A variety of carbocyclic fatty acids including cyclopropyl, cy-
clopentyl, cyclohexyl, and cycloheptyl fatty acids are found in
bacteria and plants.[20] Their roles, while not always completely
understood, may be related to the control of membrane fluidi-
ty.[21] In the case of mycobacteria, the presence of cyclopro-
pane subunits in mycolic acids is known to be associated with
the structural integrity of the cell wall and the ability of the tu-
bercle bacillus to resist oxidative stress inside macrophages. In
contrast, cyclobutane fatty acids are rare. An unusual fatty acid
incorporating a ladder-type structure of fused cyclobutanes, re-
cently isolated from anammox bacteria found near undersea
vents, is believed to contribute to the formation of dense and
impermeable lipid membranes that protect the bacteria from
Experimental Section
General procedures: All reagents and solvents were used as pur-
chased except for pyridine and CH2Cl2 (distilled from CaH2 and
kept under N2) and THF (distilled from Na/benzophenone under
N2). Reactions were performed under N2 atmosphere unless stated
otherwise. Thin-layer chromatography (TLC) was performed on
0.25 mm hard-layer silica G plates; developed plates were visual-
ized with a hand-held UV lamp or by staining: 1% Ce(SO4)2 and
10% (NH4)2MoO4 in 10% aq. H2SO4 (general stain, after heating);
1% aq. KMnO4 (for unsaturated compounds); 3% vanillin in 3%
H2SO4 in EtOH (general stain, after heating). Unless otherwise
noted, NMR spectra were recorded at 400 (1H) or 100 MHz (13C) in
CDCl3; peaks are reported as: chemical shift (multiplicity, J cou-
plings in Hz, number of protons); “app” and “br” refer to apparent
and broad signals, respectively. IR spectra were recorded as neat
films (ZnSe, ATR mode) with selected absorbances reported in
wavenumbers (cmÀ1). Flash chromatography was performed on
32–60 mm silica gel. Preparative HPLC was performed on a 21”
250 mm normal-phase Si (8 mm) column at
a flow rate of
10 mLminÀ1 of the indicated solvent, unless otherwise noted. Ana-
lytical purity of compounds was checked by using an analytical
column (250 mm”4.6 mm; Microsorb) with 20% EtOAc/hexane at
1 mLminÀ1; detection was carried out with a differential refractom-
eter interfaced with a data module. All compounds tested for bio-
logical activity showed >97% purity by HPLC analysis except for 5
and 7. Melting points are uncorrected.
3-(Octanoic acid-8-yl-benzyl ester)-2,2-dichlorocyclobutanone
(15) was prepared similarly to compound 23 and was afforded
(13.8 g, 80%) as a brown oil from benzyl 9-decenoate (12.4 g,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1842

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
1
48 mmol), trichloroacetyl chloride (10.7 mL, 95 mmol), and Zn(Cu)
(15.55 g, 238 mmol) in Et2O 0.1m (340 mL). Consistent with a previ-
ous report, only one isomer was observed.[22] The assignment was
confirmed by observation of the pair of geminal hydrogen atoms
adjacent to the ketone at d=3.33 and 2.94 ppm by 1H NMR and
by the observation of 11 rather than 12 carbon atoms (symmetry)
upon dechlorination to cyclobutanone 3: Rf =0.34 (10% EtOAc/
308C; IR: n˜ =3035 (br), 2916, 2849, 1695, 909, 733 cmÀ1; H NMR:
d=11.34 (brs, 1H, COOH), 2.37 (t, J=7.2, 2H), 2.25 (sept., J=7.2,
1H), 2.10–1.98 (m, 2H), 1.90–1.75 (m, 2H), 1.70–1.52 (m, 2H), 1.40–
1.12 ppm (brm, 10H); 13C NMR: d=180.3, 37.0, 36.2, 34.1, 29.4,
29.2, 29.0, 28.4, 27.1, 24.7, 18.5 ppm; HRCIMS calcd for C12H22O2:
[M+H]+ 199.1698, found: 199.1695; purity >99% by HPLC (tR:
4.92 min).
Hex); IR: n˜ =2935, 2852, 1814, 1730, 905, 725 cmÀ1 1H NMR: d=
;
3-(Octanoic acid benzyl ester-8-yl)cyclobutanone (17) was pre-
pared in a similar manner as 21, via reaction of dichloroketone 15
(1.05 g, 2.69 mmol), 6 mL glacial acetic acid, and Zn(Cu) (1.76 g,
26.9 mmol). The product was purified by flash chromatography
(10% EtOAc/Hex) to afford a colorless oil (0.4859 g, 59%), which
was a mixture of regioisomers: Rf =0.55 (20% EtOAc/Hex); IR: n˜ =
7.39–7.29 (m, 5H), 5.12 (s, 2H), 3.33 (dd, J=9.4, 17.2, Hz, 1H), 2.94
(dd, J=9.0, 17.2 Hz, 1H), 2.88–2.81 (m, 1H), 2.37/2.36 (overlapping
t, J=7.5 Hz, 2H), 1.94–1.84 (m, 1H), 1.70–1.72 (m, 3H), 1.46–
1.28 ppm (m, 8H); 13C NMR: d=192.8,173.4, 136.0, 128.4, 128.0,
88.8, 65.9, 47.7, 45.8, 34.1, 31.2, 29.0, 28.9, 28.8, 27.2, 24.7 ppm;
HREIMS calcd for C19H25O3Cl2: [M+H]+ 371.1181, found: 371.1188.
1
2923, 2854, 2780, 2732, 1163, 730, 697 cmÀ1; H NMR: d=7.35–7.28
3-(Octanoic acid-8-yl-benzyl ester)-2,2-dichlorocyclobutanol
methanesulfonate ester (16) was prepared similarly to compound
20 from the previous dichloroketone 15 (10.0 g, 26.9 mmol),
NaBH4 (1.53 g, 40.4 mmol), and iPrOH (350 mL). The crude product
was purified by flash column chromatography with 10% EtOAc/
Hex to furnish the dichlorobutanol (5.76 g, 57%) as a colorless oil:
Rf =0.39 (20% EtOAc/Hex); IR: n˜ =3440 (br), 2927, 2854, 1733,
(brm, 5H), 5.09 (s, 2H), 3.13–3.03 (m, 2H), 2.65–2.57 (m, 2H), 2.33
(t, J=7.2, 2H), 2.33–2.26 (m, 1H), 1.68–1.58 (brquint., J=6.4, 2H),
1.58–1.48 (brq, J=7.2, 2H), 1.29 ppm (brs, 8H); 13C NMR: d=208.2,
173.3, 134.0, 128.3, 127.9, 65.8, 52.3, 36.1, 34.0, 28.9, 28.8, 28.0,
24.7, 23.6 ppm; HRESIMS calcd for C19H26O3Na [M+Na]+: 325.1780,
found: 325.1775.
1164, 960, 697 cmÀ1 1H NMR: d=7.40–7.30 (m, 5H), 5.12 (s, 2H),
;
3-(Octanoic acid-8-yl)cyclobutanone (3) was prepared by a similar
procedure as for 7 through reaction of benzyl ester 17 (0.0346,
0.1 mmol), 10% Pd/C (0.020 g, 0.02 mmol) and EtOAc (1 mL). The
product was purified by flash column chromatography on silica gel
(step gradient of 20:40% EtOAc/Hex) to afford acid 3 (0.0189 g,
90%) as a low-melting white solid: Rf =0.3 (Hex/EtOAc/AcOH
6:4:0.05); mp: 35–378C; IR: n˜ =3094 (br), 2923, 2853, 1780,
4.73–4.64 (m, 0.1H) 4.34–4.27 (m, 0.9H), 2.97 (d, J=8.2 Hz, 0.1H,
OH is trans to backbone), 2.87 (d, J=10.3 Hz, 0.9H, OH is cis to
backbone), 2.48–2.34 (m, 1H), 2.36 (t, J=7.6 Hz, 2H), 1.78–1.60 (m,
3H), 1.53–1.28 ppm (m, 11H); 13C NMR: d=173.7, 136.0, 128.5,
128.1, 92.9, 75.4, 66.1, 45.6, 34.5, 34.2, 29.8, 29.2, 29.0, 28.9, 26.5,
24.8 ppm; HRESIMS calcd for C19H23O3Cl2 [M+Na]+: 395.1157,
found: 395.1151.
1
1705 cmÀ1; H NMR: d=10.61 (brs, 1H, COOH), 3.17–3.08 (m, 2H),
2.69–2.61 (m, 2H), 2.35 (brt, J=7.6, 2H), 2.39–2.31 (br, 1H), 1.69–
1.53 (brm, 4H), 1.32 ppm (brs, 8H); 13C NMR: d=208.8, 179.9, 52.5,
36.3, 34.0, 29.2, 29.1, 28.9, 28.2, 24.6, 23.8 ppm; HRCIMS calcd for
C12H20O3Na [M+Na]+: 213.1491, found: 213.1490; purity >97% by
HPLC (tR: 10.75 min).
Conversion into the methanesulfonate was performed as described
for compound 20, using methanesulfonyl chloride (20 mL,
29.4 mmol), the dichlorobutanol (5.50 g, 14.7 mmol), and Et3N
(9.20 mL, 66.3 mmol) in CH2Cl2 (70 mL). The crude product was pu-
rified by flash chromatography (10% EtOAc/Hex) to afford 16 as
a light-yellow oil (6.13 g, 92%): Rf =0.36 (20% EtOAc/Hex); IR: n˜ =
3-(Octanoic acid benzyl ester)cyclobutanol (18) was prepared by
a similar procedure as compound 8 via reaction of cyclobutanone
17 (0.0509 g, 0.17 mmol) and NaBH4 (0.013 g, 0.34 mmol) in anhy-
drous MeOH (2.5 mL). Compound 18 (after purification by flash
column chromatography with 20% EtOAc/Hex) was isolated as
a low-melting white solid (0.040 g, 76%). The product was a 9:1
mixture of cis/trans stereoisomers based on the previous assign-
ments for cyclobutanol 8; Rf =0.29 (20% EtOAc/Hex); mp: 30–
1
2927, 2858, 1731, 1364, 1179, 952 cmÀ1; H NMR: d=7.36–7.31 (m,
5H), 5.11 (s, 2H), 5.13–5.09 (m, 1H), 3.18 (s, 3H), 2.56–2.50 (m, 2H),
2.35 (t, J=7.6 Hz, 2H), 1.93–1.28 ppm (m, 13H); 13C NMR: d=173.5,
136.0, 128.4, 128.1, 88.4, 78.3, 66.0, 45.8, 39.3, 34.1, 31.6, 29.8, 29.1,
28.87, 28.85, 26.2, 24.8 ppm; HRESIMS calcd for C20H28O5Cl2SNa
[M+Na]+: 473.0932, found: 473.0930.
1
8-(2-Cyclobuten-1-yl)octanoic acid (1) was prepared by a similar
procedure as employed for 5 via reaction of Na metal (2.80 g,
120 mmol) with a solution of 5.40 g (12.0 mmol) of the mixture of
methanesulfonate 16 in THF (30 mL) and NH3 (~250 mL). The
crude product was purified by flash column chromatography (2%
EtOAc/Hex) to afford 9,10-ethenooctadecanoic acid 1 (1.64 g, 52%)
as a low-melting white solid: Rf =0.47 (Hex/EtOAc/AcOH 6:4:0.05);
318C; IR: n˜ =3356 (br), 2922, 1734, 1154, 735, 696 cmÀ1; H NMR:
d=7.40–7.30 (m, 5H), 5.11 (s, 2H), 4.42–4.34 (brm, 0.1H), 4.14–4.03
(brm, 0.9H), 2.48–2.40 (m, 0.18H), 2.35 (t, J=7.2, 0.18H), 2.16–2.04
(m, 0.2H), 1.95 (t, J=6.6, 0.2H), 1.70–1.56 (brm, 3H), 1.48–
1.13 ppm (brm, 12H); 13C NMR: d=173.6, 136.0, 128.4, 128.1, 66.0,
63.7, 39.7, 37.0, 34.2, 29.2, 29.1, 29.0, 27.3, 25.4, 24.82 ppm;
HRCIMS calcd for C19H29O3Na [M+Na]+: 305.2117, found: 305.2116.
mp: 20–218C; IR: n˜ =3112 (br), 3043, 2920, 2853, 1705, 697 cmÀ1
;
1H NMR: d=10.27 (brs, 1H, COOH), 6.11 (brd, J=2.4 Hz, 1H), 6.04
(brdd, J=0.8, 2.0 Hz, 1H), 2.77 (m, 1H), 2.66 (ddd, J=0.8, 4,
13.2 Hz, 1H), 2.35 (t, J=7.2 Hz, 2H), 2.04 (d, J=13.2 Hz, 1H), 1.64
(quint., J=7.2 Hz, 2H), 1.51–1.38 (brm, 2H), 1.38–1.23 ppm (brm,
8H); 13C NMR: d=180.0, 141.1, 135.1, 44.2, 36.9, 34.6, 34.0, 29.5,
29.2, 29.0, 27.9, 24.7 ppm; HRESIMS calcd for C12H20O2Na [M+Na]+:
219.1361, found: 219.1355; purity 100% by HPLC (tR: 5.01 min).
3-(Octanoic acid)cyclobutanol (4) was prepared by a similar pro-
cedure as employed for 8 through reaction of benzyl ester 18
(0.034 g, 0.110 mmol), 10% Pd/C (0.024 g, 0.022 mmol) and EtOAc
(1 mL). The crude product was purified by flash column chroma-
tography (40% EtOAc/Hex) to afford acid 4 (0.013 g, 54%) as
a white solid: Rf =0.24 (Hex/EtOAc/AcOH 6:4:0.05); mp: 74–758C;
1
IR: n˜ =3341 (br), 2916, 2849, 1695, 1064 cmÀ1; H NMR (MeOD): d=
4.30 (quint., J=6.8, 0.1H), 4.01 (quint., J=6.4, 0.9H), 2.45–2.35
(brm, 2H), 2.29 (t, J=7.2, 2H), 1.75–1.55 (brm, 3H), 1.50–1.15 ppm
(brm, 12H); 13C NMR (MeOD): d=176.3, 62.8, 38.9, 36.9, 36.8, 33.6,
29.1, 29.0, 28.8, 27.1, 25.4, 24.7 ppm; HRESIMS calcd for C12H22O3Na
[M+Na]+: 237.1467 found: 237.1460; purity >97% by HPLC (tR:
9.65 and 11.36 min).
8-Cyclobutyloctanoic acid (2) was prepared by a similar procedure
as for
6 from the hydrogenation of cyclobutene 1 (0.034,
0.170 mmol), over 10% Pd/C (0.034 g, 0.030 mmol) in EtOAc
(1.7 mL) to afford the unprotected acid 2 (0.030 g, 89%) as a low-
melting white solid: Rf =0.47 (Hex/EtOAc/AcOH 6:4:0.05); mp: 28–
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1843

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
2,2-Dichloro-4-(octanoic acid benzyl ester-8-yl)-3-octylcyclobuta-
none-; 2,2-Dichloro-3-(octanoic acid benzyl ester-8-yl)-4-octylcy-
clobutanone (19) was prepared by a modification of a known pro-
cedure.[22–24] To a mixture of zinc dust (0.879 g, 13.4 mmol) and
benzyl oleate 19 (1.02 g, 2.73 mmol) in 20 mL anhydrous Et2O was
First-eluting cis: Rf =0.33; IR: 3457, 2924, 2854, 1736, 1456, 1170,
1
697 cmÀ1; H NMR: d=7.37–7.32 (m, 5H), 5.12 (s, 2H), 4.55 (dd, J=
10.8, 8.8 Hz, 1H, CH-OH, cis to the backbone assigned based on
2D-NOESY analysis and 4JdiagonalH =0), 2.72 (dt, J=9.2, 5.6 Hz, 1H),
2.59 (d, J=10.8 Hz, 1H, -OH), 2.55 (appdq, J=9.2, 5.6 Hz, 1H), 2.36
(t, J=7.6 Hz, 2H), 1.67–1.51 (brm, 3H), 1.52–1.13 (m, 24H),
0.88 ppm (appt, J=6.8 Hz, 3H); 13C NMR: d=173.6, 136.1, 128.5
(two overlapping signals), 128.2 (two overlapping signals), 93.5,
77.2, 66.1, 49.5, 40.9, 34.3, 31.8, 29.9, 29.4, 29.4, 29.3, 29.2, 29.0,
27.0, 25.7, 24.9, 23.2, 22.7, 14.1 ppm; HRFABMS (3-NBA matrix):
calcd for C27H43O3Cl2 [M+H]+: 485.2589, found: 485.2572.
slowly added
a solution of trichloroacetyl chloride (0.75 mL,
6.8 mmol) in 7 mL Et2O via dropping funnel over a period of
45 min. The reaction was stirred until determined complete by the
absence of starting material (4 h, TLC). The reaction mixture was fil-
tered through a plug of Celite, and the residue was washed with
Et2O (2”75 mL). The organic solution was stirred a minimum with
50% aq. NaHCO3, and the separated aqueous layer back-extracted
with Et2O (2”20 mL). The combined organic layers were dried over
Na2SO4 and concentrated under vacuum to give the product
(1.30 g, 99%) as a yellow oil. The unpurified material was generally
used directly for the next reaction because of a tendency to de-
compose during chromatography. However, small quantities could
be purified by rapid flash chromatography (5% EtOAc/Hex) to
afford a 1:1 mixture of regioisomeric dichlorocyclobutanones 19 as
a colorless oil: Rf =0.53 (10% EtOAc/Hex); IR: n˜ =2926, 2855, 1800,
First-eluting trans: Rf =0.28; IR: n˜ =3449, 3028, 2920, 2850, 1736,
1
1456, 1157, 696 cmÀ1; H NMR: d=7.37–7.32 (5H), 5.12 (s, 2H), 3.99
(ddd, J=10.0, 8.4, 1.2 Hz, 1H, CH-OH, trans to the backbone as-
signed based on 2D-NOESY analysis and 4JdiagonalH =1.2 Hz), 2.68–
2.64 (m, 1H), 2.52 (d, J=10.0 Hz, 1H, -OH), 2.34 (t, J=7.6 Hz, 1H),
2.29–2.20 (m, 1H), 1.67–1.26 (brm, 27H), 0.88 ppm (appt, J=
6.8 Hz, 3H); 13C NMR: d=173.6, 136.1, 128.5 (two overlapping sig-
nals), 128.2 (two overlapping signals), 91.1, 82.6, 66.1, 50.3, 44.4,
34.3, 31.9, 29.7, 29.5, 29.2, 29.04, 29.02, 28.4, 28.23, 28.15, 27.7,
24.9, 22.7, 14.1 ppm; ESIMS calcd for C27H42O3Cl2Na [M+Na]+:
507.2409, found: 507.2422.
1735, 1456, 1162, 696 cmÀ1 1H NMR: d=7.36–7.32 (5H), 5.120/
;
5.116 (overlapping s, 2H), 3.55–3.49 (m, 1H) 2.97–2.93 (m, 1H),
2.37/2.35 (overlapping t, J=7.6 Hz, 2H), 1.74–1.62 (m, 4H), 1.66–
1.48 (m, 4H), 1.45–1.27 (brm, 18H), 0.89/0.88 ppm (overlapping
triplets, J=6.9 Hz, total 3H); 13C NMR: d=197.5, 173.5, 136.1,
128.5, 128.1, 88.1, 66.0, 58.0, 57.9, 49.4, 49.4, 34.2, 31.8, 31.80,
29.60, 29.3, 29.3, 29.2, 29.18, 29.15, 29.12, 29.0, 28.9, 28.9, 28.04,
27.96, 26.72, 26.68, 26.3 ppm; HRFABMS (3-NBA+Li matrix): calcd
for C27H40O3Cl2Li [M+Li]+: 489.2515, found: 489.2499.
Second-eluting cis: Rf =0.20; IR: n˜ =3464, 2924, 2854, 1736, 1456,
;
1169, 696 cmÀ1 1H NMR: d=7.37–7.32 (m, 5H), 5.11 (s, 2H), 4.55
(dd, J=10.8, 8.8 Hz, 1H CH-OH, cis to the backbone assigned
based on 2D-NOESY analysis and 4JdiagonalH =0), 2.72 (dt, J=9.2,
5.6 Hz, 1H), 2.65 (d, J=10.8 H z, 1H, -OH), 2.54 (appdq, J=9.2,
5.6 Hz, 1H), 2.35 (t, J=7.2 Hz, 2H), 1.68–1.60 (brm, 3H), 1.52–1.13
(m, 24H), 0.89 ppm (appt, J=6.8 Hz, 3H); 13C NMR: d=173.7,
136.1, 128.5 (two overlapping signals), 128.2 (two overlapping sig-
nals), 93.5, 77.2, 66.1, 49.5, 40.9, 34.3, 31.8, 29.6, 29.4, 29.2, 29.1,
29.04, 29.00, 27.0, 25.8, 24.8, 23.2, 22.7, 14.1 ppm; HRFABMS (3-
NBA matrix): calcd for C27H43O3Cl2 [M+H]+: 485.2589, found:
485.2581
2,2-Dichloro-4-(octanoic acid benzyl ester-8-yl)-3-octylcyclobuta-
nol methanesulfonate ester; 2,2-Dichloro-3-(octanoic acid
benzyl ester-8-yl)-4-octylcyclobutanol methanesulfonate ester
(20): Reduction of the dichlorocylobutanone 19 was achieved
using a modification of a known procedure.[9,24] To a 0 8C solution
of dichlorocyclobutanones 19 (1.5 g, 3.1 mmol) in iPrOH (45 mL)
was added NaBH4 (0.176 g, 4.65 mmol). The reaction was allowed
to slowly warm to RT and stirred until starting material had disap-
peared (TLC, ~2.5 h; by-products tended to accumulate upon pro-
longed reaction). The reaction was then cooled to 08C and
quenched with 1n HCl (40 mL). The solution was stirred for an ad-
ditional 30 min, concentration to one third the original volume,
and then extracted with EtOAc (3”100 mL). The organic layer was
washed sequentially with H2O/sat. NaHCO3/brine and then dried
over Na2SO4. Evaporation of the organic solvent and flash chroma-
tography of the residue on silica gel, using a step gradient of
5:10% EtOAc/Hex, gave (0.821 g, 55% yield) of a 1:1 mixture of re-
gioisomeric cyclobutanols as a colorless oil. The products were pre-
dominantly a regioisomeric mixture of the cis,cis diastereomers[22]
based on 1H NMR, HSQC, 2D-COSY, and 2D-NOESY analysis:[25] Rf
(10% EtOAc/Hex)=0.33 (major, OH is cis to backbone) 0.28 (minor,
OH is trans to backbone), 0.2 (major, OH is cis to backbone) and
0.15 (minor, OH is trans to backbone); IR: n˜ =3473, 3036, 2924,
Second-eluting trans: Rf =0.15; IR: n˜ =3464, 3033, 2925, 2854, 1738,
1
1498, 1160, 697 cmÀ1; H NMR: d=7.37–7.32 (m, 5H), 5.11 (s, 2H),
3.99 (ddd, J=10.0, 8.4, 1.2 Hz, 1H, CH-OH, trans to the backbone
4
assigned based on 2D-NOESY analysis and JdiagonalH =1.2 Hz), 2.69–
2.64 (m, 1H), 2.56 (d, J=10.0 Hz, 1H, -OH), 2.35 (t, J=7.6 Hz, 2H),
2.26–2.22 (m, 1H), 1.66–1.26 (m, 27H), 0.88 ppm (appt, J=6.8 Hz,
3H); 13C NMR: d=173.7, 136.1, 128.5 (two overlapping signals),
128.2 (two overlapping signals), 91.1, 82.6, 66.1, 50.3, 44.3, 34.3,
31.8, 29.7, 29.4, 29.2, 29.04, 28.97, 28.5, 28.21, 28.18, 27.5, 24.8,
22.7, 14.1 ppm; HRESIMS calcd for C27H42O3Cl2Na [M+Na]+:
507.2409, found: 507.2426.
The mixture of regioisomeric cyclobutanols were converted into
the methanesulfonate using a variant of published procedures.[9,24]
Methanesulfonyl chloride (62 mL, 0.80 mmol) was slowly added to
a stirred solution of the cyclobutanols at 08C (0.193 g, 0.398 mmol)
and Et3N (0.25 mL, 8.0 mmol) in CH2Cl2 (2 mL) over 15 min. The re-
action was allowed to slowly warm to RT and was stirred for 17 h
or until no starting material remained (TLC). The reaction was dilut-
ed with CH2Cl2 (75 mL) and washed sequentially with 1n HCl (2”
25 mL), sat. aq. NaHCO3 (2”25 mL) and H2O (2”25 mL). The organ-
ic solution was then dried with Na2SO4 and concentrated under re-
duced pressure. The residue was purified by flash chromatography
using 10% EtOAc/Hex to give 20 (0.206 g, 92%) as a light-yellow
oil consisting of a 1:1 mixture of regioisomers, each a 92:8 mixture
1
2854, 1733, 1456, 1168, 696 cmÀ1; H NMR: d=7.36–7.32 (m, 5H),
5.116/5.112 (apps, total 2H), 4.55 (dd, J=10.8, 8.8 Hz, 0.92H, CH-
OH, cis to the backbone assigned based on 2D-NOESY analysis and
4JdiagonalH =0), 3.99 (brt, J=10 Hz, 0.08H), 2.75–2.69 (m, 1H), 2.64/
2.62 (overlapping d, J=10.8 Hz, total 1H, -OH proton assigned
based on the disappearance of this peak upon D2O addition),
2.60–2.55 (m, 1H), 2.36/2.35 (overlapping t, J=7.6 Hz, total 2H),
1.67–1.62 (brm, 3H), 1.52–1.19 (24H), 0.89/0.88 ppm (overlapping
t, J=6.8 Hz, total 3H). Small quantities of the individual isomers
could be isolated by semipreparative HPLC (10% EtOAc/Hex, RI de-
tection):
1
of cis/trans stereoisomers based on H NMR: Rf (10% EtOAc/Hex)=
0.31 (first trans), 0.26 (first cis/second trans), 0.21 (second cis); IR:
;
n˜ =3032, 2925, 2855, 1735, 1458, 1180, 696 cmÀ1 1H NMR: d=
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1844

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
7.36–7.33 (m, 5H), 5.33 (d, J=9.6 Hz, 0.92H), 5.114/5.109 (two s,
total 2H), 4.84 (d, J=7.6 Hz, 0.08H), 3.208/3.205 (two s, total 3H),
2.76–2.74 (m, 2H), 2.36/2.34 (two overlapping t, J=7.6 Hz, total
2H), 1.66–1.60 (m, 4H), 1.46–1.26 (m, 22H), 0.88/0.87 ppm (overlap-
ping t, J=6.8 Hz, total 3H). Small quantities of individual isomers
could be separated for analysis by HPLC using 10% EtOAc/Hex.
The stereochemical assignments were confirmed by 2D NMR ex-
periments and by preparation of individual methanesulfonates
from individual stereoisomers of alcohol 19, prepared as described
in the previous step.
pension was extracted with CH2Cl2 (3”50 mL). The organic layers
were combined and washed sequentially with 1n aq. HCl/H2O/sa-
turated aq. NaHCO3/H2O/brine. The organic solution was then
dried with NaSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography using 10%
EtOAc/Hex to afford the cyclobutene carboxylic acid 5 (0.131 g,
87%) as a low-melting white solid: Rf =0.50 (Hex/EtOAc/AcOH
80:20:1); mp: 33–358C; IR: n˜ =3493, 3036, 2923, 2853, 1708, 1467,
1275, 728 cmÀ1 1H NMR: d=10.94 (brs, 1H, COOH), 6.17–6.15
;
(brm, 2H), 2.83–2.75 (brs, 2H), 2.35 (t, J=7.6 Hz, 2H), 1.68–1.60
(brm, 2H), 1.52–1.40 (brm, 2H), 1.35–1.20 (23H), 0.88 ppm (t, J=
6.8 Hz, 3H); 13C NMR (found 18 C) d=180.6, 140.0, 139.9, 46.8,
46.7, 34.1, 31.9, 29.9, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 29.3, 29.2,
29.0, 28.3, 28.2, 24.6, 22.7, 14.1 ppm; HRFABMS (Gly matrix) calcd
for C20H35O2 [MÀH]À: 307.2637, found: 307.2636; purity >99% by
HPLC (tR: 4.63 min).
First eluting trans: Rf =0.31; IR: n˜ =3032, 2926, 2855, 1737, 1733,
1
1456, 1180, 697 cmÀ1; H NMR: d=7.37–7.32 (5H), 5.12 (s, 2H), 4.84
(d, J=7.6 Hz, 1H), 3.20 (s, 3H), 2.74–2.68 (m, 2H), 2.36 (t, J=7.6 Hz,
2H), 1.67–1.32 (brm, 26H), 0.88 ppm (t, J=6.8 Hz, 3H); 13C NMR:
d=173.6, 136.1, 128.5 (two overlapping signals), 128.2 (two over-
lapping signals), 86.4, 85.8, 66.1, 50.5, 41.1, 39.5, 34.3, 31.8, 29.5,
29.4, 29.3, 29.2, 29.01, 28.98, 28.3, 28.1, 27.6, 27.1, 24.9, 22.6,
14.1 ppm; HRESIMS calcd for C28H44O5Cl2S [M+Na]+: 585.2184,
found: 585.2188.
cis-9,10-Ethanooctadecanoic acid (6): A solution of cyclobutene 5
(0.031 g, 0.100 mmol) and 10% Pd/C (0.020 g, 0.020 mmol) in
EtOAc (1 mL) was placed in a vial under an atmosphere of H2 and
stirred at RT overnight (16 h). The mixture was then filtered
through a plug of Celite, which was washed with ~10 mL EtOAc.
The solution was then evaporated under vacuum to afford acid 6
(0.030 g, 96%) as a colorless oil, which was sufficiently pure to use
without chromatography: Rf =0.50 (Hex/EtOAc/AcOH 80:20:1); IR:
Second-eluting trans: Rf =0.26; IR: n˜ =3040, 2927, 2855, 1734, 1456,
;
1179, 697 cmÀ1 1H NMR: d=7.37–7.32 (m, 5H), 5.11 (s, 2H), 4.84
(dd, J=8.4, 0.8 Hz, 1H), 3.19 (s, 3H), 2.76–2.65 (m, 2H), 2.35 (t, J=
7.6 Hz, 2H), 1.67–1.32 (26H), 0.89 ppm (t, J=6.8 Hz, 3H); 13C NMR
d=173.6, 136.1, 128.5 (two overlapping signal), 128.2 (two overlap-
ping signal), 86.4, 85.7, 66.1, 50.5, 41.1, 39.5, 34.3, 31.8, 29.6, 29.31,
29.25, 29.2, 29.0, 28.3, 28.1, 27.6, 27.0, 24.9, 22.7, 14.1 ppm: HRE-
SIMS; calcd for C28H44O5Cl2S [M+Na]+: 585.2184, found: 585.2205.
n˜ =3051 (br), 2921, 2852, 1708 cmÀ1 1H NMR: d=10.93 (brs, 1H,
;
COOH), 2.35 (t, J=7.3, 2H), 2.24 (brs, 2H), 2.00–1.90 (brm, 1H),
1.64 (quint., J=7.3 Hz, 2H), 1.60–1.50 (m, 2H), 1.45–1.10 (brm,
24H), 0.88 ppm (brt, J=7.0 Hz, 3H); 13C NMR: d=180.2, 37.7, 37.6,
34.1, 31.9, 30.11, 30.05, 29.9, 29.7, 29.7, 29.4, 29.3, 29.1, 27.64,
27.55, 24.8, 24.7, 22.7, 14.1 ppm; HRCIMS calcd for C20H38O2 [M+
H]+: 311.2950, found: 311.2949; purity 100% by HPLC (tR:
4.56 min).
First-eluting cis: Rf =0.26; IR: n˜ =2926, 2855, 1736, 1456, 1182,
;
697 cmÀ1 1H NMR: d=7.37–7.32 (m, 5H), 5.33 (appd, J=9.6 Hz,
1H), 5.11 (s, 2H), 3.19 (s, 3H), 2.80–2.72 (brm, 2H), 2.36 (appt, J=
7.6 Hz, 2H), 1.70–1.63 (m, 4H), 0.87 ppm (t, J=6.8 Hz, 3H);
13C NMR: d=173.6, 136.0, 128.5 (two overlapping signal), 128.1
(two overlapping signal), 88.9, 80.7, 66.0, 49.5, 40.2, 39.2, 34.2, 31.8,
29.7, 29.3, 29.3, 29.2, 29.0, 28.8, 26.7, 25.7, 24.8, 23.7, 22.6,
14.1 ppm: HRFABMS (3-NBA matrix): calcd for C28H45O5Cl2S [M+H]+
: 563.2365, found: 563.2349.
2-(Octanoic acid benzyl ester-8-yl)-3-octylcyclobutanone and 3-
(Octanoic acid benzyl ester-8-yl)-2-octylcyclobutanone (21) was
prepared by a modification of a known procedure.[22–24] To a solu-
tion of the regioisomeric dichloroketones 19 (0.670 g, 1.40 mmol)
in glacial acetic acid (3 mL) was added Zn(Cu) (0.450 g, 6.90 mmol).
The mixture was stirred at RT under N2 until TLC showed complete
disappearance of starting material (~5 h). The reaction mixture was
then filtered through a plug of Celite and washed with Et2O. The
filtrate was washed sequentially with saturated aq. NaHCO3 and
H2O, and dried over Na2SO4. The residue obtained upon concentra-
tion was purified by flash chromatography on silica gel (5% EtOAc
in Hex) to afford (0.388 g, 87%) of a mixture of cyclobutanones 21
as a colorless oil: Rf =0.41 (10% EtOAc/Hex); IR: n˜ =3034, 2923,
Second-eluting cis: Rf =0.21; IR: n˜ =2925, 2855, 1735, 1457, 1181,
;
697 cmÀ1 1H NMR: d=7.36–7.32 (m, 5H), 5.33 (appd, J=9.6 Hz,
1H), 5.11 (s, 2H), 3.20 (s, 3H), 2.80–2.69 (brm, 2H), 2.34 (appt, J=
7.6 Hz, 2H), 1.74–1.60 (4H), 1.50–1.19 ppm (22H); 13C NMR: d=
173.6, 136.1, 128.5 (two overlapping signal), 128.1 (two overlapping
signal), 89.0, 80.7, 66.0, 49.5, 40.2, 39.3, 34.2, 31.8, 29.5, 29.5, 29.3,
29.2, 28.9, 28.9, 28.7, 26.8, 25.7, 24.8, 23.7, 22.6, 14.1 ppm;
HRFABMS (3-NBA matrix): calcd for C28H45O5Cl2S [M+H]+: 563.2365,
found: 563.2360.
2853, 1774, 1735, 1161, 696 cmÀ1 1H NMR: d=7.36–7.26 (brm,
;
5H), 5.115 (s, 2H), 3.28–3.17 (brm, 1H), 3.15–3.05 (brm, 1H), 2.51–
2.43 (m, 1H), 2.42–2.32 (brm, 1H), 2.35/2.34 (overlapping t, J=
7.53 Hz, 2H), 1.70–1.50 (brm, 4H), 1.45–1.14 (brm, 22H), 0.88/
0.87 ppm (overlapping t, J=7.19 Hz, 3H); 13C NMR: d=212.0,
173.6, 136.1, 128.5, 128.1, 66.0 (brs), 61.9/61.8 (two apps), 50.1/
50.1 (two apps), 34.2, 31.8, 30.1, 30.0, 29.6, 29.6, 29.5, 29.4, 29.4,
29.23, 29.17, 29.0, 28.2, 28.08, 28.05, 27.98, 27.6, 24.9, 24.3, 24.3,
22.6, 14.1 ppm; HREIMS calcd for C27H41O3Na [M+Na]+: 437.3032,
found: 437.3029.
1-(Octanoic acid-8-yl)-2-octylcyclobutene (5) was prepared from
the regioisomeric mixture of dichloromethanesulfonates 20 using
a reported procedure.[9,24] NH3 (~15 mL, liquid) was condensed into
a 100 mL three-necked round-bottom flask fitted with a dry ice/
acetone condenser. Sodium (0.113 g, 4.90 mmol, sliced under Hex)
was added in small pieces. The resulting deep-blue solution was
stirred for 20 min. A solution of 0.276 g (0.49 mmol) of 20 in anhy-
drous THF (1.5 mL) was added over 10 min. The cooling bath was
removed, and the reaction was allowed to stir at ~À358C (NH3 at
reflux) 5–30 min (depending on the scale of preparation). The reac-
tion was then re-cooled to À788C (dry ice/acetone) and stirred for
2 h or until the starting material was consumed by TLC. Saturated
aq. NH4Cl was slowly added until the blue color was no longer visi-
ble. The condenser was removed, and the reaction mixture was
slowly allowed to warm to 08C with evaporation of NH3. The re-
maining reaction mixture was diluted with 30 mL H2O, and the sus-
2-(Octanoic acid-8-yl)-3-octylcyclobutanone and 3-(octanoic
acid-8-yl)-2-octylcyclobutanone (7): A solution of cyclobutanones
21 (0.039 g, 0.09 mmol) and 10% Pd/C (0.020 g, 0.18 mmol) in an-
hydrous MeOH (1 mL) in a vial was placed under an atmosphere of
H2 and stirred at RT overnight (16 h). The mixture was then concen-
trated under vacuum. The residue was resuspended in EtOAc (~
20 mL) and filtered through a plug of Celite. The residue obtained
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1845

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
upon concentration was purified by flash column chromatography
on silica gel (10:25% EtOAc/Hex) to afford the regioisomeric mix-
ture of carboxylic acids 7 (0.027 g, 87%) as a colorless oil: Rf =0.16
(25% EtOAc/Hex); IR: n˜ =3322 (br), 2926, 2856, 1776, 1709, 913,
349.2719, found 349.2715; purity >99% by HPLC (tR: 11.8 and
15.9 min).
2-Chloro-4-(octanoic acid benzyl ester-8-yl)-3-octylcyclobuta-
none; 2-Chloro-3-(octanoic acid benzyl ester-8-yl)-4-octylcyclo-
butanone (22) was prepared based on a modification of a known
procedure.[9,22,24] To a solution of dichloroketone 19 (0.992 g,
2.100 mmol) in 4 mL glacial acetic acid was added Zn dust
(0.148 g, 2.30 mmol). [Note: The amount of Zn required depends
on the purity of the dichlorocyclobutanone; it was generally neces-
sary to add a second equivalent after ~12 h of reaction.] The mix-
ture was stirred at RT until complete disappearance of the starting
material was observed (16 h, TLC). The reaction mixture was then
cooled in an ice bath and diluted with H2O (20 mL). The resulting
solution was extracted with EtOAc (3”50 mL) and the combined
organic layers were washed sequentially with H2O (2”100 mL) and
saturated NaHCO3 solution (2”50 mL). The resulting organic layer
was dried over Na2SO4 and concentrated under reduced pressure
to furnish a colorless oil (0.895 g, 97% crude yield) as a 1:1 mixture
of regioisomers, each a 9:1 mixture of cis/trans stereoisomers
(1H NMR); the mixture was used directly in the next step. Analysis
1
749 cmÀ1; H NMR: d=3.28–3.18 (brm, 1H), 3.13/3.09 (overlapping
dd, J=9.6, 3.1 and 9.0, 3.5, 0.56H/0.44H), 2.50/2.46 (two app ab-
normal-shape quints., 0.55H/0.52H), 2.42 (brdd, J=9.6, 4.5 Hz, 1H),
2.35/2.34 (two appt, J=7.45 Hz, 2H), 1.63–1.54 (brm, 4H), 1.43–
1.18 (brm, 22H), 0.88/0.87 ppm (overlapping br t, J=7.0 Hz, 3H);
13C NMR: d=212.1, 179.9, 62.0/61.9 (two apps), 50.20/50.17 (two
apps), 34.0, 31.9, 30.1, 30.1, 29.7, 29.6, 29.6, 29.4, 29.3, 29.2, 29.02,
28.98, 28.2, 28.1, 28.0, 27.6, 24.6, 24.3, 24.3, 22.7, 14.1 ppm; HREIMS
calcd for C20H36O3Na [M+Na]+: 347.2557, found: 347.2557; purity
>97% by HPLC (tR: 6.82 min).
2-(Octanoic acid-8-yl)-3-octylcyclobutanol and 3-(Octanoic acid-
8-yl)-2-octylcyclobutanol (8): To a À108C (ice/NaCl bath) solution
of the cyclobutanone 21 (0.037 g, 0.070 mmol) in anhydrous
MeOH (1.5 mL) was added NaBH4 (0.005 g, 0.150 mmol) under N2.
The reaction mixture was stirred at À108C until no starting materi-
al remained on TLC (~20 min). The reaction was diluted with
CH2Cl2 (~5 mL) and quenched with sat. NaHCO3 (2 mL). The mix-
ture was stirred for 10 min. The organic layer was removed and the
aqueous layer was extracted with CH2Cl2 (3”5 mL). The organic
layers were combined, dried over with Na2SO4, and evaporated to
afford the alcohol as a colorless oil (0.029 g, 99%). Although the
1
of the H NMR indicated that the expected product was accompa-
nied by recovered starting material 19 (~14%) and the cyclobuta-
none 21 (over-reduction product, ~10%). These by-products could
not be removed easily by flash chromatography. Rf =0.58/0.53
(major), 0.48/0.43 (minor) (10% EtOAc in Hex); IR: n˜ =3036, 2925,
1
1
2854, 1789, 1735, 1456, 1162, 696 cmÀ1; H NMR: d=7.36–7.25 (m,
crude product appears pure by H NMR, it is actually a mixture of
5H), 5.12/5.11 (overlapping s, 2H), 4.97 (dd, J=2.6, 9.5, 0.1H), 4.51
(dd, J=2.8, 7.6 Hz, 0.9H), 3.35–3.20 (m, 1.1H), 2.75–2.65 (m, 0.1H),
2.53–2.40 (m, 0.9H), 2.36/2.35 (overlapping t, J=7.2 Hz, 2H), 1.75–
1.20 (m, 26H), 0.92–0.85 ppm (m, 3H); 13C NMR: d=203.48/203.45,
173.5, 136.1, 128.5, 128.1, 66.0, 65.7, 58.3, 58.2, 40.74/40.72, 34.18,
31.77/31.75, 29.5, 29.3, 29.24, 29.16, 29.14, 29.13, 29.02, 28.97,
28.90, 28.86, 28.02, 27.96, 27.92, 27.87, 25.64/25.57, 24.80/24.78,
22.59/22.58, 14.0 ppm; HRESIMS calcd for C27H41O3ClNa [M+Na]+:
471.2642, found 471.2635.
two regioisomers, each a 70:30 mixture of the 1,2,3-cis/cis and the
1,2-trans-2,3-cis isomers (identities established through 2D NMR).
The individual isomers can be detected by TLC in 20% EtOAc/Hex:
Rf =0.50 (major, all cis), 0.45 (major, all cis), 0.39 (minor, OH is trans
to backbone), and 0.37 (minor, OH is trans to backbone); IR: n˜ =
1
3396 (br), 2922, 2852, 1737, 1156, 732, 696 cmÀ1; H NMR: d=7.39–
7.28 (brm, 5H), 5.14 (s, 2H), 4.23 br q, J=7.3, 0.7H), 3.93 (brq, J=
7.3, 0.3H), 2.42–2.36 (overlapping signal, 1H), 2.37 (t, J=7.6, 2H),
2.15–1.95 (brm, 1H), 1.90–1.78 (brm, 1H), 1.70–1.60 (brm, 2H),
1.60–1.48 br m, 2H), 1.45–1.20 (brm, 22H), 0.90 ppm (brt, J=7.6,
2H); 13C NMR: d=173.68/173.65 (overlapping), 136.1, 128.5, 128.1,
72.2, 66.09/66.06 (two overlapping s), 66.0, 43.97/43.95 (overlap-
ping), 37.3, 34.3, 31.9, 30.7, 30.6, 30.2, 29.94, 29.87, 29.8, 29.7,
29.62, 29.58, 29.5, 29.33, 29.29, 29.2, 29.14, 29.08, 29.0, 27.7, 27.6,
24.90, 24.88, 23.4, 23.3, 22.7, 14.1 ppm; HRESIMS calcd for
C27H44O3Na [M+Na]+: 439.3188, found: 439.3174.
2-Chloro-4-(octanoic acid-8-yl)-3-octylcyclobutanone-; 2-Chloro-
3-(octanoic acid-8-yl)-4-octylcyclobutanone (9) was prepared in
a similar manner as compound 6 from reaction of benzyl ester 22
(0.085 g, 0.19 mmol), 10% Pd/C (0.040 g, 0.038 mmol) and EtOAc
(1.9 mL). The product, acid 9 (0.048 g, 70%), was obtained as a col-
orless oil after purification by flash column chromatography (20%
EtOAc/Hex). The product, which was predominantly the cis,cis ste-
reoisomer on the cyclobutanone, included ~5% of an inseparable
trans-chlorocyclobutanone 11 (1H NMR signal at d=4.37 ppm),
probably arising from the epimerization of 9: Rf =0.19 (Hex/EtOAc
Using a procedure similar to that described for preparation of 6,
the cyclobutanol benzyl esters (0.025 g, 0.060 mmol) were subject-
ed to hydrogenation in the presence of 10% Pd/C (0.013 g,
0.012 mmol) and EtOAc (0.6 mL) to afford cyclobutanoic acid 8
(0.016 g, 81%) as a colorless oil. Although the product is assumed
to consist of a similar mixture of regio- and stereoisomers as was
present in the benzyl ester precursor, the individual isomers are
not separable: Rf =0.32 (EtOAc/Hex/AcOH 4:6:0.05); IR: n˜ =3309
1
4:1); IR: n˜ =3036, 2924, 2854, 1788, 1706, 1461 cmÀ1; H NMR: d=
10.74 (brs, 1H, COOH), 4.96 (dd, J=9.4, 2.6, 0.05H), 4.50 (dd, J=
7.6, 2.8, 0.95H), 3.35–3.20 (m, 1.05H), 2.46 (quint., J=8.1, 0.95H),
2.74–2.62 (m, 0.05H), 2.35/2.33 (overlapping t, J=7.3, 2H), 1.80–
1.15 (m, 22H), 0.88/0.87 ppm (overlapping t, J=6.9, 3H); 13C NMR:
d=203.62/203.56, 180.1, 65.7, 58.30/58.26, 401.8, 34.0, 31.8, 29.5,
29.4, 29.3, 29.18/29.15, 29.04/29.00, 28.9, 28.04/27.98, 27.95/27.90,
25.67/25.59, 24.5, 22.6, 14.1 ppm; HRESIMS calcd for C20H35O3ClNa
[M+Na]+: 381.2172, found 381.2191.
1
(br), 2922, 2853, 1709 cmÀ1; H NMR: d=4.22 (q, J=7.6, 0.7H, as-
sumed -OH is cis to backbone from the characterization of starting
material), 3.92 (q, J=7.6, 0.3H, assumed OH is trans to backbone),
2.43–2.33 (brm, 1H), 2.34 (t, J=7.3, 2H), 2.18–1.92 (m), 1.84
(brsept., J=8.0, 1H), 1.68–1.58 (brquint., J=7.0, 2H), 1.58–1.48
(brm, 2H), 1.43–1.18 (brm, 22H), 0.88 ppm (brt, J=7.0 Hz, 3H);
13C NMR: d=179.50/179.46 (two apps), 72.28, 66.16/66.12 (two
apps), 48.87/48.79 (two apps), 43.97, 37.26, 35.06, 33.98, 31.89,
30.69/30.62, 30.24, 30.18, 29.90, 29.87, 29.79, 29.74, 29.63, 29.60,
29.51, 29.46, 29.34, 29.31, 29.21, 29.11, 29.06, 29.01, 28.99, 28.97,
28.67, 28.60, 28.05, 27.95, 27.92, 27.73, 27.60, 24.66/24.63, 23.38/
23.34, 22.67, 14.10 ppm; HRESIMS calcd for C20H38O3Na [M+Na]+:
2,2-Dichloro-4-(octanoic acid benzyl ester-8-yl)-3-octylcyclobuta-
none-; 2,2-Dichloro-3-(octanoic acid benzyl ester-8-yl)-4-octylcy-
clobutanone (23) was prepared based on a known procedure.[22–24]
To a mixture of Zn(Cu) (1.84 g, 28.0 mmol) and benzyl elaidate
(2.07 g, 5.60 mmol) in 11 mL of anhydrous Et2O was added over
2 h (syringe pump) trichloroacetyl chloride (2.0 mL, 18 mmol). The
reaction mixture was stirred at RT for 5 h (or until the starting ma-
terial is consumed by TLC) and filtered through a plug of Celite.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1846

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
The residue was washed with Et2O (20 mL). The combined ether
layers were transferred to a round-bottom flask and cooled over
ice. The black solution was then diluted with 40 mL H2O and
40 mL sat. NaHCO3. The mixture was allowed to warm to RT and
stirred overnight. The aqueous layer was extracted with Et2O (3”
50 mL), and the combined organic layers were dried over Na2SO4
and evaporated to dryness. The residue obtained upon concentra-
tion could be used for the next step without further purification or,
alternatively, could be purified by rapid flash column chromatogra-
phy through a short plug of silica (2.5%/5% EtOAc/Hex) to afford
a yellow oil (1.69 g, 63%) consisting of a 1:1 mixture of regioisom-
ers: Rf =0.63 (20% EtOAc/Hex); IR: n˜ =2926, 2855, 1802, 1735,
J=6.4 Hz, 3H); 13C NMR, 75 MHz: d=173.7, 136.1, 128.5, 128.2,
90.2, 78.4, 66.1, 57.0, 42.0, 34.3, 31.9, 31.3, 29.6, 29.5, 29.41, 29.39,
29.2, 29.1, 29.0, 27.6, 27.5, 26.7, 24.9, 22.7, 14.1 ppm; HRESIMS
calcd for C27H40O3Cl2 [M+Na]+: 507.2409, found: 507.2398.
Second-eluting H1-H3 cis: Rf =0.51; IR: n˜ =3444 (br), 2924, 2854,
1
1734, 1162, 734, 696 cmÀ1; H NMR: d=7.37–7.31 (m, 5H), 5.11 (s,
2H), 3.90 (dd, 3JH1,OH =10.8 Hz, 3JH1,H4(trans) =8.0 Hz, 4JH1,H3(cis) =0 Hz,
1H, CH-OH), 2.52 (OH, d, J=10.8 Hz, 2H), 2.35 (t, J=7.6 Hz, 2H),
2.13–2.02 (m, 1H, CH-CCl2), 1.75–1.18 (m, 27H), 0.89 ppm (t, J=
6.8 Hz, 3H); 13C NMR: (75 MHz): d=173.7, 136.1, 128.5, 128.1, 91.0,
81.6, 66.1, 51.9, 48.0, 34.2, 33.0, 31.8, 30.0, 29.6, 29.4, 29.3, 29.2,
29.02, 28.97, 28.9, 27.2, 26.8, 24.9, 24.8, 22.6, 14.1 ppm; HRESIMS
calcd for C27H40O3Cl2: [M+Na]+ 507.2409, found:507.2409.
1456, 1162, 696 cmÀ1 1H NMR: d=7.47–7.23 (m, 5H), 5.120/5.117
;
(overlapping s, 2H), 3.158/3.126 (overlapping dt, J=7.2, 1.5 Hz,
1H), 2.56–2.46 (m, 1H), 2.37/2.34 (overlapping t, J=7.6 Hz, 2H),
1.96–1.18 (m, 26H), 0.90/0.87 ppm (overlapping t, J=7.2 Hz, 3H);
13C NMR, 75 MHz: d=196.3, 173.5, 136.0, 128.4, 128.1, 87.1, 66.0,
60.7, 60.6, 52.3, 34.2, 34.1, 31.8, 31.7, 31.37, 31.36, 29.42, 29.40,
29.3, 29.23, 29.20, 29.14, 29.12, 29.08, 28.93, 28.90, 28.85, 27.5, 27.4,
27.12, 27.05, 24.80, 24.78, 22.59, 22.57, 14.04, 14.03 ppm; HRESIMS
calcd for C27H40O3Cl2Na [M+Na]+: 505.2252, found: 505.2248.
The mixture of dichlorocyclobutanols was converted into the
methanesulfonates (mesylate) in a similar manner as for 20. Reac-
tion of methanesulfonyl chloride (0.05 mL, 0.70 mmol), the dichlor-
ocyclobutanols (0.166 g, 0.300 mmol), and Et3N (0.20 mL, 1.7 mmol)
in CH2Cl2 (2 mL), followed by purification using flash chromatogra-
phy (5% EtOAc/Hex) provided dichloromesylate 24 as a light-
yellow oil (0.146 g, 86%) in a mixture of inseparable regio- and ste-
reoisomers: Rf =0.40/0.38 (20% EtOAc/Hex); IR: n˜ =2925, 2851,
2,2-Dichloro-4-(octanoic acid benzyl ester-8-yl)-3-octylcyclobuta-
nol, methanesulfonate ester; 2,2-Dichloro-3-(octanoic acid
benzyl ester-8-yl)-4-octylcyclobutanol, methanesulfonate ester
(24) was prepared by a similar procedure as 20 through reaction
of dichlorocyclobutanone 23 (0.210 g, 0.400 mmol), NaBH4
(0.055 g, 1.50 mmol), and iPrOH (6 mL). The 1:1 mixture of regioiso-
meric dichlorocyclobutanols (0.166 g, 85%) was obtained as a color-
less oil following flash chromatography (5% EtOAc/Hex): Rf =
0.66(minor)/0.61(major)/0.56(minor)/0.51(major) (10% EtOAc/Hex);
1734, 1368, 1180, 964, 697 cmÀ1 1H NMR: d=7.38–7.30 (m, 5H),
;
5.20 (dd, J=7.1, 1.2 Hz, 0.4H), 5.115/5.112 (two overlapping s, total
2H), 4.84 (d, J=8.8 Hz, 0.6H), 3.202/3.199 (two overlapping s,
1.8H), 3.166/3.163 (two overlapping s, total 1.2H), 2.66–2.58 (m,
0.4H), 2.40–2.30 (m, 2H), 2.21–2.12 (m, 0.6H), 2.10–1.98 (m, 0.6H),
1.80–1.15 (m, 26.4H), 0.91–0.86 ppm (m, 3H); 13C NMR: d=173.6,
136.10/136.08, 128.5/128.1, 86.45/86.38, 84.09, 83.86/83.82, 66.03/
66.01, 57.18, 51.81/51.80, 44.51/44.50, 41.71, 39.5, 39.0, 34.2, 32.21/
32.18, 31.78/31.77, 31.41/31.36, 30.1, 29.49 29.46, 29.42, 29.34,
29.32, 29.27, 29.22, 29.16, 28.95, 28.90, 28.2, 27.0, 26.9, 26.8, 26.7,
26.6, 26.5, 26.4, 26.3, 24.8, 22.6, 14.1 ppm; HRESIMS C28H44O5Cl2SNa
[M+Na]+: 585.2184, found: 585.2186.
IR: n˜ =3459 (br), 2924, 2853, 1736, 1456, 1161, 734, 696 cmÀ1
;
1H NMR: d=7.36–7.31 (m, 5H), 5.12 (s, 2H), 4.37 (brd, J=6.4 Hz,
0.4H), 3.91 (brd, J=8.0 Hz, 0.6H), 2.80–2.45 (brm, 2H), 2.37/2.36
(overlapping t, J=7.6 Hz, 2H), 1.80–1.05 (brm, 26H), 0.89/0.88 ppm
(overlapping t, J=6.7 Hz, 3H). Small quantities of individual iso-
mers could be separated for analysis by flash column chromatogra-
phy using 10% EtOAc/Hex. Stereochemical assignments are based
on 2D NMR experiments (COSY, NOESY, and HSQC).
1-(Octanoic acid-8-yl)-2-octylcyclobutene (10) was prepared from
reductive fragmentation and deprotection of dichloromethansulfo-
nate 24 (0.143 g, 0.250 mmol) in THF (1 mL), NH3 (~10 mL) and Na
(~0.090 g, 4.10 mmol) by a procedure similar to that employed for
5. The crude product was purified by flash chromatography (10%
EtOAc/Hex) to afford the acid 10 (0.035 g, 46%) as a colorless oil:
Rf =0.35 (Hex/EtOAc 60:40); IR: n˜ =3124, 2923, 2852, 1708, 910,
First-eluting H1-H3 trans: Rf =0.66; IR: n˜ =3463 (br), 2927, 2855,
1
1738, 1453, 1158 cmÀ1; H NMR: d=7.37–7.31 (m, 5H), 5.12 (s, 2H),
3
3
4
4.38 (ddd, JH1,H4 =6.8 Hz, JH1,OH =5.1 Hz, JH1,H3(trans) =1.3 Hz, 1H, CH-
1
736 cmÀ1; H NMR: d=6.12 (brs, 2H), 2.35 (t, J=7.2 Hz, 2H), 2.26
3
OH), 2.62–2.55 (m, 1H, CH-CCl2), 2.44 (d, JH1,OH =5.1 Hz, -OH), 2.36
(t, J=7.2 Hz, 2H), 1.64 (quint., J=7.2 Hz, 2H), 1.44 (brm, 4H), 1.35–
1.20 (23H), 0.88 ppm (t, J=6.8 Hz, 3H); 13C NMR: d=179.7, 139.2,
139.1, 50.4, 50.3, 34.02, 33.97, 31.9, 29.9, 29.6, 29.3, 29.2, 29.0, 28.4,
28.3, 24.7, 22.7, 14.1 ppm; HRFABMS (3-NBA matrix): calcd for
C20H35O2Li2 [MÀH+2Li]+: 321.2957, found: 321.2969; purity 100%
by HPLC (tR: 4.73 min).
(t, J=7.6 Hz, 2H), 2.18–2.08 (m, 1H, CH-CHOH), 1.80–1.10 (brm,
26H), 0.89 ppm (t, J=6.7 Hz, 3H); 13C NMR (NOESY): d=173.6,
136.1, 128.5, 128.2, 90.2, 78.5, 66.1, 57.0, 42.0, 34.3, 31.9, 31.3, 29.7,
29.5, 29.4, 29.3, 29.0, 27.7, 27.5, 26.6, 24.9, 22.7, 14.1 ppm; HRESIMS
calcd for C27H40O3Cl2 [M+Na]+: 507.2409, found: 507.2409.
First-eluting H1-H3 cis: Rf =0.61; IR: n˜ =3448 (br), 2925, 2854, 1737,
1
1456, 1163, 734, 697 cmÀ1; H NMR: d=7.37–7.31 (m, 5H), 5.12 (s,
2-Chloro-4-(octanoic acid-8-yl)-3-octylcyclobutanone-; 2-Chloro-
3-(octanoic acid-8-yl)-4-octylcyclobutanone (11) was prepared by
a similar procedure as described for compound 9. Reaction of
benzyl ester 23 (0.500 g, 1.000 mmol), Zn(Cu) (0.074 g, 1.10 mmol)
and glacial acetic acid (2 mL) furnished, after flash chromatography
(5% EtOAc/Hex), trans-monochlorocyclobutanone (0.313 g, 70%)
as a colorless oil, which included an equal mix of the 2-chloro-4-
octyl and 2-chloro-3-octyl regioisomers, each of which included
both epimers at the chloride-bearing carbon: Rf =0.42/0.39 (minor)
and 0.33 (major) (10% EtOAc/Hex). The amount of Zn dust varied
from 1.1 to 2.2 equivalents; generally, a second equivalent of zinc
dust was added to the incomplete reaction mixture after stirring
for ~12 h. The residual dichlorocyclobutanone was easily removed
by flash column chromatography. However, the cyclobutanone
2H), 3.91 (dd, 3JH1,OH =10.8 Hz, 3JH1,H4(trans) =8.0 Hz, 4JH1,H3(cis) =0 Hz,
1H, CH-OH), 2.46 (d, J=10.8 Hz, 2H, OH), 2.36 (t, J=7.6 Hz, 2H),
2.11–2.04 (m, 1H, CH-CCl2), 1.75–1.18 (m, 27H), 0.88 ppm (t, J=
6.8 Hz, 3H); 13C NMR, 75 MHz (NOESY): d=173.6, 136.1, 128.5,
128.2, 91.0, 81.6, 66.1, 51.9, 48.1, 34.3, 33.0, 31.8, 30.0, 29.6, 29.41,
29.38, 29.2, 29.0, 27.3, 26.8, 24.9, 22.6, 14.1 ppm; HRESIMS calcd for
C27H40O3Cl2 [M+Na]+: 507.2409, found: 507.2415.
Second-eluting H1-H3 trans: Rf =0.56; IR: n˜ =3463 (br), 2925, 2854,
1
1736, 1456, 1163, 696 cmÀ1; H NMR: d=7.36–7.31 (m, 5H), 5.11 (s,
2H), 4.40–4.34 (m, 1H), 2.64–2.55 (m, 1H, CH-OH), 2.64–2.55 (m,
1H, CH-CCl2), 2.46 (d, J=4.8 Hz, 1H, OH), 2.35 (t, J=7.6 Hz, 2H),
2.20–2.03 (m, 1H, CH-CHOH), 1.80–1.10 (brm, 26H), 0.88 ppm (t,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1847

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
product of over-reduction was formed in significant amounts (28%
based on 1H NMR and was nearly inseparable by flash column
chromatography: IR: n˜ =2924, 2854, 1788, 1735, 1457, 1161, 733,
Bacterial strains and culture conditions: Bacterial strains used in
this study are the E. coli wild-type strain G58–1, the genome se-
quencing Msm strain mc2155, and the Mtb strains CDC1551 and
H37Rv. Cells were grown shaking at 378C in LB broth for E. coli or
complete Middlebrook 7H9 broth supplemented with 0.05% v/v
Tween 80 to OD600 0.6–1.2 for mycobacteria. For MIC determina-
tions, cells were inoculated into LB for E. coli or a modified previ-
ously reported minimal medium for both E. coli and mycobacte-
ria.[26] The minimal media components and final concentrations are
as follows: 22 mm dibasic potassium phosphate, 16 mm monobasic
potassium phosphate, 2.8”10À5 mm ferric chloride, 8.7”10À3 mm
zinc sulfate, 8.4”10À4 mm cobalt(II) chloride, 1.0”10À2 mm manga-
nese chloride, 6.8”10À2 mm calcium chloride, 2.4 mm magnesium
sulfate, 5.0 mm ammonium chloride, 25 mm glycerol, and 0.02% v/
v Tyloxapol.
1
696 cmÀ1; H NMR: d=7.40–7.32 (m, 5H), 5.113/5.110 (overlapping
s, 2H), 4.95 (dd, J=3.1, 9.4, 0.3H), 4.37 (dt, J=2.7, 7.8 Hz, 0.7H),
2.97–2.90 (m, 0.3H), 2.84–2.74 (m, 0.7H), 2.40–2.30 (overlapping m,
2.3H), 2.08–1.97 (m, 0.7H), 1.85–1.10 (m, 26H), 0.92–0.84 ppm (m,
3H); 13C NMR: d=204.6, 202.0, 173.5, 136.1, 128.5, 128.1, 66.0,
65.52/65.46, 63.98, 63.78/63.75, 63.57/63.52, 60.74/60.69, 49.8, 43.7,
37.4, 36.63/36.59, 35.12/35.10, 34.2, 31.8, 31.3, 30.3, 30.1, 29.5,
29.42, 29.38,29.32, 29.25, 29.19, 29.03, 29.00, 28.96, 28.93, 28.88,
28.2, 27.67, 27.60, 27.55, 27.48, 27.38, 27.3, 27.2, 27.1, 24.83/24.80,
22.6, 14.0 ppm; HRESIMS calcd for C27H41O3ClNa [M+Na]+:
471.2642, found: 471.2630.
The resulting mixture of monochloroketone mixtures was subject-
ed to debenzylation described in the preparation of compound 6.
Reaction of the benzyl ester mixture (0.034 g, 0.11 mmol), with
10% Pd/C (0.024 g, 0.022 mmol) and EtOAc (1 mL) afforded the un-
protected acid (0.013 g, 54%) as a white solid: Rf =0.24 (Hex/
EtOAc/AcOH 6:4:0.05); mp: 74–758C; IR: n˜ =3062, 2924, 2854,
Drug susceptibility assays: MICs were determined by a 96-well
microplate method, as described by Chacon et al.[26] Bacteria were
harvested, washed 2” with minimal media, and inoculated to an
initial concentration of ~1.0”105 colony forming units (CFU) per
well. The initial inoculum was plated to verify retrospectively the
desired CFUmLÀ1 for each strain. Stocks of fatty acid analogue
compounds were prepared in 100% [D6]DMSO, as this solvent was
chosen to provide consistency with the NMR studies. Appropriate
doubling dilution gradients (mgmLÀ1 compound dissolved in the
corresponding percent v/v [D6]DMSO) were prepared in the follow-
ing ranges for each compound tested: E. coli, 8–512; Msm, 16–
1024; and Mtb, 0.25–256 (e.g., 64 mgmLÀ1 of compound corre-
sponded to 0.064% v/v of [D6]DMSO). Alternatively, the concentra-
tion of [D6]DMSO was adjusted in all wells to its maximum concen-
tration in the corresponding gradient (e.g., 64 mgmLÀ1 of com-
pound corresponded to 0.256% v/v of [D6]DMSO). We tested cells
in the absence of compound and [D6]DMSO to verify cell viability.
The effect of [D6]DMSO was also tested by growing cells in minimal
media with [D6]DMSO and no compound. We observed no signifi-
cant effect of [D6]DMSO on cell viability for E. coli and Msm. How-
ever for Mtb, we observed some variability with anomalous inhibi-
tory results in a few technical replicate wells containing [D6]DMSO
alone at higher concentrations. Nonetheless, most of the replicate
wells displayed no inhibition, allowing us to eliminate these anom-
alous results. Moreover, the effect of the compound was observed
at [D6]DMSO concentrations with no inhibitory action. Plates were
incubated at 378C for 2 days (E. coli), 4 days (Msm), or 7 weeks
(Mtb). MIC values were determined by the consistent results of
three biological and three technical replicates. The MIC was de-
fined by taking the mode of three independent cultures where the
MIC did not differ by more than one doubling dilution, discarding
any results that are two doubling dilutions away from the mode.
1
1789, 1708, 1464 cmÀ1; H NMR: d=8.70 (brs, 1H), 4.95 (dd, J=2.9,
9.2, 0.3H), 4.37 (ddd, J=7.8, 2.5, 1.3, 0.7H), 2.97–2.91 (m, 0.3H),
2.85–2.73 (m, 0.7H), 2.40–2.22 (m, 22H), 2.11–2.98 (m, 0.7H), 1.85–
1.10 (m, 26H), 0.92–0.82 ppm (m, 3H); 13C NMR d=211.7, 204.7,
202.2, 179.9, 65.6/65.5, 64.0, 63.85/63.82, 63.64/63.58, 60.82/60.77,
49.81, 43.8, 37.5, 36.69/36.65, 35.2, 34.0, 31.8, 31.3, 30.4, 30.2, 29.55,
29.48, 29.44, 29.38, 29.31, 29.25, 29.2, 29.06, 29.06, 28.9, 28.3, 28.2,
27.73, 27.67, 27.62, 27.44, 27.37, 27.26, 27.20, 27.17, 24.63/24.60,
22.7, 14.1 ppm; HRESIMS calcd for C20H35O3ClNa [M+Na]+:
381.2172, found: 381.2170.
Determination of inhibitor aqueous stability and solubility: 1D
1H NMR spectroscopy was used to verify the chemical purity, aque-
ous stability, and concentration-dependent micelle formation of
eleven fatty acid analogues. Each compound was dissolved in
[D6]DMSO to obtain a stock concentration of 20 mm. Six different
concentrations were prepared from the stock solutions for NMR
analysis: 1.00 mm, 750 mm, 500 mm, 100 mm, and 0 mm. All NMR
samples consisted of 600 mL of a deuterated 50 mm potassium
phosphate buffer at pH 7.2 with 50 mm of 3-(trimethylsilyl)propion-
ic-2,2,3,3-d4 acid sodium salt (TMSP). Each 600 mL NMR sample con-
tains 30 mL (5%) of [D6]DMSO and was transferred to a 5 mm NMR
tube for analysis.
A
Bruker Avance DRX 500 MHz spectrometer equipped with
a 5 mm triple-resonance cryoprobe (1 H, 13C, 15N) with a z-axis gradi-
1
ent was used for all 1D H NMR experiments. Acquisition of NMR
spectra was automated using a BACS-120 sample changer and
Icon NMR software. All spectra were acquired at 298.15 K with 16
dummy scans, 64 scans, 32 K data points, a spectral width of
5482.46 Hz, and a relaxation delay of 1.5 s. The NMR spectra were
processed and analyzed using ACD/1D NMR Manager (Advanced
Chemistry Development). The resulting 1D 1H NMR spectra were
visually inspected for evidence of micelle formation (peak broaden-
ing), or chemical instability/impurities (additional peaks).
Abbreviations: attenuated total reflection (ATR), correlation spec-
troscopy (COSY), dicyclohexyl carbodiimide (DCC), d-cycloserine
(DCS), N,N’-dimethylformamide (DMF), hexadeutero dimethyl sulf-
oxide ([D6]DMSO), differential scanning calorimetry/thermal gravi-
metric analysis (DSC/TGA), ethyl acetate (EtOAc), hexane (Hex),
high-performance liquid chromatography (HPLC), high-resolution
mass spectrometry via electrospray ionization (HRESIMS), high-res-
olution mass spectrometry via fast atom bombardment (HRFABMS),
heteronuclear single quantum coherence spectroscopy (HSQC), 4-
dimethylaminopyridine (DMAP), isopropanol (iPrOH), multidrug re-
sistant (MDR), minimum inhibitory concentration (MIC), Mycobacte-
rium smegmatis (Msm), Mycobacterium tuberculosis (Mtb), nuclear
Overhauser effect spectroscopy (NOESY), nuclear magnetic reso-
nance (NMR), tetrahydrofuran (THF), thin-layer chromatography
(TLC), ultraviolet (UV).
Measurement of nonspecific cytotoxicity: RAW 264.7 macrophag-
es were incubated with 0–100 mm of oleic acid or the C18 cyclobu-
tene fatty acid 2, each delivered as bovine serum albumin com-
plexes. After 24 h, cell viability was relative to untreated controls
was assessed using an IN CTYOTOX-CVDE Crystal violet Dye Elution
Kit. The values shown in Table S1 are means ÆSEM based on mea-
surement in triplicate.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1848

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
824; c) B. Darses, A. E. Greene, J.-F. Poisson, Org. Lett. 2010, 12, 3994–
3997. In our hands, the use of Zn and not the more active Zn/Cu was
important in obtaining cis-cyclobutene and not a cis/trans mixture.
[11] K. Blaszczyk, H. Koenig, K. Mel, Z. Paryzek, Tetrahedron 2006, 62, 1069–
1078.
[12] A. Hassner, L. R. Krepski, J. Org. Chem. 1978, 43, 2879–2882.
[13] H. M. Frey, D. C. Marshall, R. F. Skinner, Trans. Faraday Soc. 1965, 61,
861–865.
[14] B. D. Davis, R. J. Dubos, J. Exp. Med. 1947, 86, 215–228.
[15] Even simple fatty acids can display toxicity toward a macrophage line
at concentrations beyond 50–100 mm: T. M. de Lima, M. F. Cury-Boaven-
tura, G. Giannocco, M. T. Nunes, R. Curi, Clin. Sci. 2006, 111, 307–317.
[16] a) G. M. Rishton, Drug Discovery Today 1997, 2, 382–384; b) S. L. McGov-
ern, E. Caselli, N. Grigorieff, B. K. Shoichet, J. Med. Chem. 2002, 45,
1712–1722; c) S. L. McGovern, B. T. Helfand, B. Feng, B. K. Shoichet, J.
Med. Chem. 2003, 46, 4265–4272; d) J. Seidler, S. L. McGovern, T. N.
Doman, B. K. Shoichet, J. Med. Chem. 2003, 46, 4477–4486.
[17] E. E. Camargo, J. A. Kertcher, S. M. Larson, B. S. Tepper, H. N. Wagner, Jr.,
Int. J. Lepr. Other Mycobact. Dis. 1982, 50, 200–204.
[18] Y. Zhang, B. Heym, B. Allen, D. Young, S. Cole, Nature 1992, 358, 591–
593.
[19] D. Barkan, Z. Liu, J. C. Sacchettini, M. S. Glickman, Chem. Biol. 2009, 16,
499–509.
[20] a) J. S. S. DamstØ, M. Strous, W. I. Rijpstra, E. C. Hopnams, J. A. Geeneva-
sen, A. C. van Duin, L. A. van Niftrik, M. S. Jetten, Nature 2002, 419, 708–
712; b) J. S. S. DamstØ, W. I. C. Rijpstra, J. A. J. Geenevasen, M. Strous,
M. S. Jetten, FEBS J. 2005, 272, 4270–4283.
[21] B. S. Moore, H. G. Floss in Comprehensive Natural Products Chemistry,
Vol. 1 (Eds.: U. Sankawa, D. H. R. Barton, K. Nakanishi, O. Meth-Cohn),
Elsevier, New York, 1999, pp. 61–81.
Acknowledgements
This research was funded by the Life Sciences Interdisciplinary Re-
search Program (University of Nebraska-Lincoln). Portions of this
research were performed in facilities renovated with support
from the NIH (RR016544). We acknowledge useful discussions
with Prof. Ken Nickerson and Prof. Martha Morton (University of
Nebraska-Lincoln) and Dr. Chris Schwartz. We also acknowledge
technical assistance from Ms. Wendy Austin, Ms. Sara Basaiga,
and Mr. Shingo Ishihara (University of Nebraska-Lincoln).
Keywords: cyclobutanes
mycobacteria · tuberculosis
· cyclobutenes · fatty acids ·
[1] M. Raviglione, B. Marais, K. Floyd, K. Lçnroth, H. Getahun, G. B. Migliori,
A. D. Harries, P. Nunn, C. Lienhardt, S. Graham, J. Chakaya, K. Weyer, S.
Cole, S. H. E. Kaufmann, A. Zumla, Lancet 2012, 379, 1902–1913.
[2] a) A. Koul, E. Arnoult, N. Lounis, J. Guillemont, K. Andries, Nature 2011,
469, 483–490; b) Y. Zhang, Annu. Rev. Pharmacol. Toxicol. 2005, 45,
529–564.
[3] L. Ballell, R. A. Field, K. Duncan, R. J. Young, Antimicrob. Agents Chemo-
ther. 2005, 49, 2153–2163; (erratum: L. Ballell, R. A. Field, K. Duncan,
R. J. Young, Antimicrob. Agents Chemother. 2007, 51, 1888); J. van den
Boogard, G. S. Kibiki, E. R. Kisanga, M. J. Boerjee, R. E. Aarnoutse, Antimi-
crob. Agents Chemother. 2009, 53, 849–862.
[4] K. Takayama, C. Wang, G. S. Besra, Clin. Microbiol. Rev. 2005, 18, 81–101.
[5] L. Kremer, L. G. Dover, H. R. Morbidoni, C. Vilch›ze, W. N. Maughan, A.
Baulard, S. C. Tu, N. HonorØ, V. Deretic, J. C. Sacchettini, C. Locht, W. R.
Jacobs, Jr., G. S. Besra, J. Biol. Chem. 2003, 278, 20547–20554.
[6] Several reports have described the incorporation of labeled or modified
fatty acids into mycolic acid biosynthesis: a) H. R. Morbidoni, C. Vil-
cheze, L. Kremer, R. Bittman, J. C. Sacchettini, W. R. Jacobs, Chem. Biol.
2006, 13, 297–307; b) P. R. Wheeler, G. S. Besra, D. E. Minnikin, C. Rat-
ledge, Biochim. Biophys. Acta 1993, 1167, 182–188; c) R. K. Dreher, K.
Poralla, W. A. Kong, J. Bacteriol. 1976, 127, 1136–1140; d) T. Kaneda, Mi-
crobiol. Rev. 1991, 55, 288–302.
[22] a) J. P. DeprØs, F. Coelho, A. E. Greene, J. Org. Chem. 1985, 50, 1972–
1973; b) D. A. Bak, W. T. Brady, J. Org. Chem. 1979, 44, 107–110; c) A. E.
Greene, J. P. DeprØs, J. Am. Chem. Soc. 1979, 101, 4003–4005; d) W. T.
Brady, O. H. Waters, J. Org. Chem. 1967, 32, 3703–3705.
[23] Zn dust was used instead of Zn(Cu), and 2.5 equiv of Cl3CCOCl was em-
ployed.
[24] J. D. Baldwin, K. D. Belfield, J. Org. Chem. 1987, 52, 4772–4776.
[25] a) P. R. Seidl, J. F. Dias in The Chemistry of Cyclobutanes (Eds.: Z. Rappo-
port, J. F. Liebman), Wiley, Chichester, 2005, pp. 213–256; b) E. Pretsch,
P. Buhlmann, C. Affolter, Structure Determination of Organic Compounds:
Tables of Spectral Data, Springer, Berlin, 2000, p. 203.
[7] For an overview of application of fatty acids in anti-mycobacterial activ-
ity, see: N. M. Carballeira, Prog. Lipid Res. 2008, 47, 50–61.
[8] V. K. Mahindroo, V. Singh, S. Dev, Indian J. Chem. Sect. B 1988, 27, 1080–
1089.
[26] O. Chacon, Z. Feng, N. B. Harris, N. E. Cµceres, L. G. Adams, R. G. Barletta,
Antimicrob. Agents Chemother. 2002, 46, 47–54.
[9] D. C. Powers, P. A. Leber, S. S. Gallagher, A. T. Higgs, L. A. McCullough,
J. E. Baldwin, J. Org. Chem. 2007, 72, 187–194.
Received: March 12, 2014
[10] a) J. A. Hyatt, P. W. Raynolds, Org. React. 1994, 45, 159–646; b) B.
Darses, A. E. Greene, S. C. Coote, J.-F. Poisson, Org. Lett. 2008, 10, 821–
Published online on June 5, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1838 – 1849 1849