Synthesis and Biological Evaluation of Chlorinated Analogs of Leukotoxin Diol

Article abstract of DOI:10.1021/acs.orglett.5b02814

This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This sugge

Full text of DOI:10.1021/acs.orglett.5b02814

Letter
pubs.acs.org/OrgLett
Synthesis and Biological Evaluation of Chlorinated Analogs of
Leukotoxin Diol
Christian Nilewski, Camille Le Chapelain, Susanne Wolfrum, and Erick M. Carreira*
Vladimir Prelog Weg 3, Laboratory of Organic Chemistry, ETH-Zurich, CH-8093 Zurich, Switzerland
̈
S
* Supporting Information
ABSTRACT: This study documents that chlorinated analogs of
leukotoxin diol 1, in which the vic-diol has been replaced with vic-
chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in
a dose-dependent manner in analogy to the parent diol. This suggests
that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.
s part of our ongoing studies on chlorinated natural
Aproducts, we became interested in investigating the effects
of chlorides in lipids vis-a-
̀
vis their biological activity.1 Chloride
atoms and hydroxyl groups are regarded as classical isosteres,2
and isosteric displacement of substituents is an approach widely
employed to fine tune the biological, physicochemical, and
pharmacokinetic properties of a bioactive molecule.3 Herein,
we document that chlorinated analogs of leukotoxin diol 1,
wherein the vic-diol has been replaced with vic-chlorides to give
dichloride 2, induce caspase 3 activity and apotosis on HepG2
cells in a dose-dependent manner in analogy to the parent diol
(Figure 1).
Figure 2. Structures of sucrose (3) and its chlorinated analog sucralose
(4).
In analogy, we wondered whether there may be situations in
which substitution of vic-diols in certain lipids with the
corresponding vic-chlorides might lead to structures which
reproduce the biological responses observed with the parent
vic-diols in the naturally occurring biomolecule. As an initial
approach, our attention turned to leukotoxin diol 1, and we
were eager to see whether its biological effects could be
retained in the chloride analog 2. Leukotoxin diol 1 and its
regioisomer iso-1 are biosynthesized from linoleic acid in
mammalian neutrophils (Scheme 1).6 Linoleic acid is oxidized
Figure 1. Structures of leukotoxin diol 1 and its chlorinated analog.
Scheme 1. Biosynthesis of Leukotoxin Diol 1 in Mammals
Despite their consideration as isosteres, there are prominent
differences between chlorides and alcohols. First, the
substitution of a hydroxyl by a chloride leads to marked
increase in lipophilicity (i-PrOH clog P = 0.38 vs 2-
chloropropane clog P = 1.61). Second, alcohols can participate
in hydrogen bonding as acceptors and donors, while organo-
chlorides, however, cannot, but instead, they can engage in
halogen bonding.4 A noteworthy example of hydroxyl−chloride
exchange has been provided by the replacement of three of the
hydroxyl groups of sucrose 3 by chlorides to furnish the
noncaloric artificial sweetener sucralose (4) (Figure 2).5
Sucralose is known to be considerably sweeter than sucrose,
and it constitutes the major ingredient of several commercial-
ized artificial sweeteners. This difference has been correlated
with the increase of lipophilicity and enhanced permeability of
chlorinated derivative 4 compared to natural sugar 3 (Figure 2).
Received: September 29, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02814
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Letter
to leukotoxin 5 by cytochrome P-450 dependent monoox-
ygenase or by reactive oxygen species (ROS) such as hydroxyl
radicals generated by respiratory burst oxidase.7 The epoxide is
then converted to the threo diol by action of epoxide hydrolase,
mostly soluble epoxide hydrolase (sEH).8
Yoshimitsu and co-workers.21,22 Facile saponification of
dichloroester 11 completed the synthesis of dichloride 2.
Having established a synthetic route to leukotoxin diol (1)
and its parent dichloride 2, we focused on the synthesis of their
erythro-diastereoisomeric counterparts (Scheme 3). Thus,
Shortly after its discovery, leukotoxin 5 was associated with
numerous toxic effects, including pulmonary edema, vaso-
dilatation, acute respiratory distress syndrome (ARDS),
cardiovascular toxicity, mitochondrial dysfunction, nitric oxide
elevation, and more recently, immunosuppression.9 Interest-
ingly, in vitro studies on sEH were conducted that reported
leukotoxin to act only as a protoxicant, with its hydrolyzed
metabolites 1 and iso-1 being responsible for the observed
adverse effects.10 This appeared as a surprise as hydrolysis of
epoxides contributes usually to detoxification processes.11 Later
reports shed more critical light on this conclusion.12 However,
in vivo assays on Swiss webster mice supported the hydrolysis
of the epoxide to be the trigger for toxicity.13 The role of
leukotoxin and its diol on mitochondria has been documented
in early reports;14 however, the exact mechanism of action of
these compounds has not yet been elucidated.15 In order to
gain insights on the effects of the Cl/OH exchange, we set out
to synthesize leukotoxin diol 1, its chlorinated analog 2, as well
as the corresponding erythro diastereomers.
Scheme 3. Synthesis of Diols 14 and 16 and Dichlorides 15
and 17
Our synthetic endeavors commenced with alcohol 6
(Scheme 2), which was converted to sulfone 7 in two steps
Scheme 2. Synthesis of Diols 1 and 10 and Dichlorides 2 and
11.
erythro diol 14 and its chlorinated analog 15, as well as their
corresponding methyl esters 16 and 17, were prepared via a
similar sequence of reactions starting from (Z)-enyne 18
(Scheme 3). Enyne 18 was obtained as a single diastereoisomer
by a Wittig reaction between aldehyde 8 and triphenylphos-
phine iodide 19.23,24 Subsequent dihydroxylation followed by
Lindlar reduction led to dihydroxy methyl ester 16, which was
readily hydrolyzed to erythro-leukotoxin diol 14. Gratifyingly,
enyne 18 could also be readily converted to epoxide 20 in two
steps, including epoxidation with m-CPBA and subsequent
Lindlar reduction. Homoallylic epoxide 20 was then trans-
formed into erythro-dichloride 17 by applying again Yoshi-
mitsu’s methodology. Ester hydrolysis of 17 proceeded as
expected and provided erythro-dichloride 15.
With both diastereoisomers of leukotoxin diol (1 and 14)
and their chlorinated counterparts (2 and 15) in hand, the stage
was set for the biological evaluation of their cytotoxicities
against different cell lines. In this regard, we measured the
activation of caspase 3, the key mediator of programmed cell
death (apoptosis) following exposure to the leukotoxin diol
analogs.25 None of the analogs triggered activation of caspase 3
in human breast and lung carcinoma cells (MCF-7/HTB-22; A-
549/CCL-185). In contrast, some of the synthesized
compounds induced activation of caspase 3 in human liver
carcinoma cells (HepG2/HB8065) and murine hepatoma cells
(Hepa 1−6/CRL-1830) (Figure 3a,b). In Hepa cells, the threo
form (dichloride 2 and diol methyl ester 10) seemed to be
more active than the erythro form (dichloride 15) (Figure 3a).
In HepG2cells, both leukotoxin diol 1 and its chlorinated
counterpart 2 showed a strong increase in caspase 3 activity,
while the corresponding methyl esters 10 and 11 showed a
reduced pro-apoptotic effect compared to the free acids. threo-
Dichloride 15 also induced moderate apoptosis (Figure 3b).
The human HepG2 cell line was chosen for a more thorough
dose-dependent analysis of the synthesized leukotoxin diol
through a Mitsunobu reaction with 1-phenyl-1H-tetrazole-5-
thiol and subsequent oxidation of the intermediate sulfide with
m-CPBA.16 Julia−Kocienski coupling between aldehyde 817
and sulfone 7 using KHMDS in DME enabled the formation of
(E)-enyne 9 as a single diastereoisomer.18 Upjohn dihydrox-
ylation of the (E) alkene followed by Lindlar reduction of the
alkyne moiety delivered ester 10,19,20 which underwent facile
hydrolysis under standard conditions to afford leukotoxin diol
(1). The corresponding dichlorides 2 and 11 could be derived
from enyne 9 as well. Epoxidation of the alkene moiety in 9
with m-CPBA and subsequent Lindlar reduction of the
intermediate trans-epoxide led to the formation of epoxide
12. The epoxide moiety was then converted into a trans
dichloride motif by implementation of a method developed by
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these results correlate with a previous report in which
chlorinated analogs of stearic and myristic acids were
documented to exhibit cytotoxic effects toward HepG2
cells.26 The relatively high concentration of compounds (250
and 125 μM) needed to induce apoptosis could be the
consequence of low intracellular concentrations due to
inefficient uptake of the leukotoxin diols and/or a natural
mechanism that ensures that cell death is only induced in
criticial situations indicated by high abundance of leukotoxin
diols. A plausible explanation for the noninduction of caspase 3
activity in lung and breast cancer cells could be their high levels
of resistance to endogenous toxins, a key feature of cancer cells.
In summary, we have conducted a comparative study of both
diastereoisomers (threo/erythro) of leukotoxin diol as well as
their chlorinated counterparts. Cytotoxicity assays were
performed on a variety of cell lines by measuring caspase 3
activity. Leukotoxin diol and its chlorinated analog, and to a
lesser extent their methyl esters, were found to induce apotosis
in HepG2 cells in a dose-dependent manner. It is noteworthy
that the biological effect of leukotoxin diol on HepG2 cells was
retained in the parent dichloride, which indicates that it may be
possible to generate analogs of leukotoxin diols with chlorides
while retaining biological readout.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b02814.
Experimental procedures and characterization data for all
1
reactions and products, including H NMR and 13C
NMR spectra of new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: carreira@org.chem.ethz.ch.
Notes
Figure 3. Relative caspase 3 activity measured on (a) Hepa 1−6 and
on (b) HepG2 cells at 250 μM for each compound. The significance of
each result is shown above the bar: p < 0.05 = *; p < 0.01 = **.
The authors declare no competing financial interest.
The work is taken in part from undergraduate research work
analogs. As shown in Figure 4, a dose-dependent decrease of
caspase 3 activity was seen for all active compounds, indicating
that this is a genuine and not an unspecific effect. In summary,
the polar terminal function as well as the configuration of the
diol motif appeared to influence the bioactivity. Interestingly,
(Semesterarbeit) conducted by C.L.C. at ETH-Zurich.
̈
ACKNOWLEDGMENTS
We thank the ERC for support of this work in part through
grant 320666_CHLIP.
■
REFERENCES
■
(1) (a) Nilewski, C.; Geisser, R. W.; Carreira, E. M. Nature 2009,
457, 573−576. (b) Nilewski, C.; Geisser, R. W.; Ebert, M.-O.; Carreira,
E. M. J. Am. Chem. Soc. 2009, 131, 15866−15876. (c) Nilewski, C.;
Deprez, N. R.; Fessard, T. C.; Li, D. B.; Geisser, R. W.; Carreira, E. M.
Angew. Chem., Int. Ed. 2011, 50, 7940−7943. (d) Huwyler, N.;
Carreira, E. M. Angew. Chem., Int. Ed. 2012, 51, 13066−13069.
(e) Shemet, A.; Sarlah, D.; Carreira, E. M. Org. Lett. 2015, 17, 1878−
1881.
(2) Burger, A. Medicinal Chemistry, 3rd ed.; Wiley: New York, 1970;
p 127.
(3) Naumann, K. Pest Manag. Sci. 2000, 56, 3−21 and references
cited therein.
(4) For a seminal review on halogen bonding, see: Metrangolo, P.;
Neukirch, H.; Pilati, T.; Resnati, G. Acc. Chem. Res. 2005, 38, 386−395.
(5) Mathlouthi, M.; Seuvre, A.-M; Birch, G. G. Carbohydr. Res. 1986,
152, 47−61.
Figure 4. Dose−response for HepG2 cells.
C
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Organic Letters
Letter
(6) (a) Hayakawa, M.; Sugiyama, S.; Takamura, T.; Yokoo, K.; Iwata,
M.; Suzuki, K.; Taki, F.; Takahashi, S.; Ozawa, T. Biochem. Biophys.
Res. Commun. 1986, 137, 424−430. (b) Ozawa, T.; Sugiyama, S.;
Hayakawa, M.; Taki, F.; Hanaki, Y. Biochem. Biophys. Res. Commun.
1988, 152, 1310−1318.
(7) (a) Hayakawa, M.; Ogawa, T.; Sugiyama, S.; Ozawa, T. Biochem.
Biophys. Res. Commun. 1989, 161, 1077−1085. (b) Iwase, H.; Takatori,
T.; Aono, K.; Iwadate, K.; Takahashi, M.; Nakajima, M.; Nagao, M.
Biochem. Biophys. Res. Commun. 1995, 216, 483−488. (c) Iwase, H.;
Takatori, T.; Niijima, H.; Nagao, M.; Amano, T.; Iwadate, K.; Matsuda,
Y.; Nakajima, M.; Kobayashi, M. Biochim. Biophys. Acta, Lipids Lipid
Metab. 1997, 1345, 27−34.
(23) Skaanderup, P. R.; Poulsen, C. S.; Hyldtoft, L.; Jørgensen, M. R.;
Madsen, R. Synthesis 2002, 2002, 1721−1727.
(24) For a review of the Wittig olefination, see: Maryanoff, B. E.;
Reitz, A. B. Chem. Rev. 1989, 89, 863−927.
(25) Nicholson, D. W.; Ali, A.; Thornberry, N. A.; Vaillancourt, J. P.;
Ding, C. K.; Gallant, M.; Gareau, Y.; Griffin, P. R.; Labelle, M.;
Lazebnik, Y. A.; Munday, N. A.; Raju, S. M.; Smulson, M. E.; Yamin,
T.-T.; Yu, V. L.; Miller, D. K. Nature 1995, 376, 37−43.
(26) Lystad, E.; Hostmark, A. T.; Jebens, E. Pharmacol. Toxicol. 2001,
89, 85−91.
(8) Moghaddam, M. F.; Motoba, K.; Borhan, B.; Pinot, F.;
Hammock, B. Biochim. Biophys. Acta, Gen. Subj. 1996, 1290, 327−339.
(9) (a) Fukushima, A.; Hayakawa, M.; Sugiyama, S.; Ajioka, M.; Ito,
T.; Satake, T.; Ozawa, T. Cardiovasc. Res. 1988, 22, 213−218.
(b) Sakai, T.; Ishizaki, T.; Ohnishi, T.; Sasaki, F.; Ameshima, S.; Nakai,
T.; Miyabo, S.; Matsukawa, S.; Hayakawa, M.; Ozawa, T. Am. J. Physiol.
1995, 269, L326−331. (c) Ishizaki, T.; Shigemori, K.; Nakai, T.;
Miyabo, S.; Ozawa, T.; Chang, S.-W.; Voelkel, N. F. Am. J. Physiol.
1995, 269, L65−70. (d) Ishizaki, T.; Shigemori, K.; Yamamura, Y.;
Matsukawa, S.; Nakai, T.; Miyabo, S.; Hayakawa, M.; Ozawa, T.;
Voelkel, N. F. Biochem. Biophys. Res. Commun. 1995, 210, 133−137.
(e) Ishizaki, T.; Takahashi, H.; Ozawa, T.; Chang, S.-W.; Voelkel, N. F.
Am. J. Physiol. 1995, 268, L123−128. (f) Ozawa, T.; Nishikimi, M.;
Sugiyama, S.; Taki, F.; Hayakawa, M.; Shionoya, H. Biochem. Int. 1988,
16, 369−373. (g) Edwards, L. M.; Lawler, N. G.; Nikolic, S. B.; Peters,
J. M.; Horne, J.; Wilson, R.; Davies, N. W.; Sharman, J. E. J. Lipid Res.
2012, 53, 1979−1986.
(10) (a) Moghaddam, M.; Grant, D. F.; Cheek, J. M.; Greene, J. F.;
Williamson, K. C.; Hammock, B. D. Nat. Med. 1997, 3, 562−566.
(b) Moran, J.; Weise, R.; Schnellmann, R.; Freeman, J.; Grant, D.
Toxicol. Appl. Pharmacol. 1997, 146, 53−59. (c) Greene, J.; Newman,
J.; Williamson, K.; Hammock, B. Chem. Res. Toxicol. 2000, 13, 217−
226.
(11) (a) Aran, M.; Oesch, F. Mammalian xenobiotic epoxide
hydrolases. Enzyme Systems that Metabolize Drugs and Other
Xenobiotics; Wiley: New York, 2002; pp 459−483. (b) Hammock, B.
D.; Storms, D. H.; Grant, D. F. Epoxide hydrolases. Biotransformations;
Pergamon Press: Oxford, 1997; Vol. 3, pp 283−305.
(12) (a) Moran, J. H.; Mitchell, L. A.; Bradbury, J. A.; Qu, W.; Zeldin,
D. C.; Schnellmann, R. G.; Grant, D. F. Toxicol. Appl. Pharmacol.
2000, 168, 268−279. (b) Mitchell, L. A.; Moran, J. H.; Grant, D. F.
Toxicol. Lett. 2002, 126, 187−196 and references cited therein.
(13) Zheng, J.; Plopper, C.; Lakritz, J.; Storms, D.; Hammock, B. Am.
J. Respir. Cell Mol. Biol. 2001, 25, 434−438.
(14) Ozawa, T.; Hayakawa, M.; Takamura, T.; Sugiyama, S.; Suzuki,
K.; Iwata, M.; Taki, F.; Tomita, T. Biochem. Biophys. Res. Commun.
1986, 134, 1071−1078.
(15) (a) Sisemore, M. F.; Zheng, J.; Yang, J. C.; Thompson, D. A.;
Plopper, C. G.; Cortopassi, G. A.; Hammock, B. D. Arch. Biochem.
Biophys. 2001, 392, 32−37. (b) Moran, J. H.; Mon, T.; Hendrickson,
T. L.; Mitchell, L. A.; Grant, D. F. Chem. Res. Toxicol. 2001, 14, 431−
437.
(16) Bonini, C.; Chiummiento, L.; Videtta, V. Synlett 2005, 3067−
3070.
(17) Gung, B. W.; Dickson, H. Org. Lett. 2002, 4, 2517−2519.
́
(18) Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morley, A. Synlett
1998, 1998, 26−28.
(19) VanRheenen, V.; Kelly, R. C.; Cha, D. Y. Tetrahedron Lett. 1976,
17, 1973−1976.
(20) Lindlar, H. Helv. Chim. Acta 1952, 35, 446.
(21) Yoshimitsu, T.; Fukumoto, N.; Tanaka, T. J. Org. Chem. 2009,
74, 696−702.
(22) The relative configuration of the vicinal dichloride motif of 11
was supported by selective conversion of 11 to the corresponding
diene followed by analysis of the 1H NMR spectrum: (a) Sonnet, P. E.;
Oliver, J. E. J. Org. Chem. 1976, 41, 3279−3283. (b) Frost, D. J.;
Gunstone, F. D. Chem. Phys. Lipids 1975, 15, 53−85.
D
DOI: 10.1021/acs.orglett.5b02814
Org. Lett. XXXX, XXX, XXX−XXX