Design, synthesis, and cytotoxicity of stabilized mycolactone analogs

Article abstract of DOI:10.1016/j.bmcl.2017.01.036

On exposure to visible light, mycolactone A/B, the causative toxin of Buruli ulcer, rearranges to a mixture of four photo-mycolactones apparently via a rare photochemically-induced [₄π_s?+?₂π_a] cycloaddition. In

Full text of DOI:10.1016/j.bmcl.2017.01.036

Bioorganic & Medicinal Chemistry Letters 27 (2017) 1274–1277
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and cytotoxicity of stabilized mycolactone analogs
⇑
Vaddela Sudheer Babu, Ya Zhou, Yoshito Kishi
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
On exposure to visible light, mycolactone A/B, the causative toxin of Buruli ulcer, rearranges to a mixture
Received 5 December 2016
Revised 11 January 2017
Accepted 12 January 2017
Available online 17 January 2017
of four photo-mycolactones apparently via a rare photochemically-induced [4ps
+ 2pa] cycloaddition. In
order to prevent the rearrangement, two C60-C70 dihydromycolactone analogs 60a-15 and 60b-15 were
designed and synthesized. 60a-15 and 60b-15 were shown to be stable under not only photochemical,
but also acidic and basic conditions. Cytotoxicity was tested against arbitrarily chosen four cell lines
(human Hek-293, human lung carcinoma A-549, human melanoma LOX-IMVI, and mouse L-929), thereby
revealing that: (1) both analogs maintain potent cytotoxicity; (2) 60b-15 exhibits significantly higher
potency against human cell lines than 60a-15; (3) in comparison with parent mycolactone A/B, 60b-15
exhibits equal potency against human Hek-293, whereas significantly lower potency against human lung
carcinoma A-549 and human melanoma LOX-IMVI.
Keywords:
Buruli ulcer
Mycolactones
Cytotoxicity
Dihydromycolactones
Ó 2017 Elsevier Ltd. All rights reserved.
Photochemical [4ps
+ 2pa] cycloaddition
Mycolactone A/B, the causative toxin of Buruli ulcer, was iso-
lated from Mycobacterium ulcerans by Small and co-workers in
1999.1 This devastating disease results in progressive necrotic
lesions that, if untreated, can extend up to 15% of a patient’s skin
surface. Surgical intervention was the only practical curative ther-
apy for Buruli ulcer. Encouragingly, combination treatments with
rifampicin and either streptomycin or amikacin have recently been
reported to prevent the growth of the bacteria, especially in early
lesions.2 Evidence from animal studies suggests that mycolactone
A/B is directly responsible for the observed pathology, and recent
studies have shed light on the mode of action of mycolactone A/
B.3–5 The gross structure of mycolactones A and B was elucidated
with spectroscopic methods, whereas the stereochemistry was
predicted via the universal NMR database approach and confirmed
by total synthesis.6–9 Under standard laboratory conditions, myco-
lactones A and B exist as a rapidly equilibrating 3:2 mixture of
mixture of 40E, 60E, 80E, 100E, 40Z, 60E, 80E, 100E, 40E, 60Z, 80E, 100E,
and 40Z, 60Z, 80E, 100E geometrical isomers. The facile E¡Z isomer-
ization is then followed by a slower photochemically induced [4-
ps + 2pa] cyclization, to furnish the four photo-mycolactones (see:
the structures depicted in the bracket in Scheme 1). According to
the proposed mechanism, the C60 double bond is required for the
photochemical cyclization to proceed, thereby suggesting a possi-
bility of synthesizing a photochemically-stabilized mycolactone
analog with a replacement of the C60-C70 double bond for a CAC
single bond. This operation of structure-modification results in
two dihydromycolactones 60a-15 and 60b-15 (Scheme 2). In this
letter, we report a synthesis of these mycolactone-A/B analogs
and their photochemical and chemical stability, as well as cytotox-
icity, in comparison with parent mycolactone A/B.
Scheme 2 outlines a retrosynthetic analysis of 60a-15 and 60b-
15. This analysis largely relies on our previous work, including:
(1) final esterification step11; (2) choice of protecting groups for
five hydroxyl groups8; (3) use of the two previous synthetic inter-
mediates, i.e., the protected form of mycolactone core 14 and alde-
hyde 12.8 Thus, the major remaining question is concerned with
the coupling of the C10-C80 building block with aldehyde 12, to
form the E-olefin 13. For this case, Julia-Kocienski olefination12
0
0
0
0
D
4 ,5 -Z (major) and 4 ,5 -E (minor) isomers, and are referred to
D
as mycolactone A/B in this paper (Scheme 1).
We have been interested in the chemical and biological
properties of mycolactones and recently reported the photochem-
ically-induced rearrangement of mycolactone A/B into four
photo-mycolactones A1, A2, B1, and B2 (Scheme 1).10 Interestingly,
all four photo-mycolactones were found to exhibit significantly
reduced cytotoxicity, compared with parent mycolactone A/B.
On exposure to light through a 365 nm filter at 30 °C in acetone,
mycolactone A/B (1) rapidly yields an approximately 2:7:1:1
appears to be an obvious choice, i.e., 60a-10/60b-10 + 12 ? 60a
13/60b-13, respectively.
-
Scheme 3 summarizes the synthesis of sulfones 60a-10 and 60b-
10 from commercially available (S)- and (R)-glycidols, respectively.
For this synthesis, we planned to incorporate a chiral methyl group
at C60 into the fatty-acid backbone. Among several options, we
chose to adopt the method developed for the synthesis of the
⇑
Corresponding author.
E-mail address: kishi@chemistry.harvard.edu (Y. Kishi).
http://dx.doi.org/10.1016/j.bmcl.2017.01.036
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

V.S. Babu et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1274–1277
1275
Scheme 3. Stereoselective synthesis of sulfones 60b-10 and 60a-10. Reagents and
conditions: (a) 1. TBDPS-Cl, imidazole, CH2Cl2 (96%); 2. MeCH2CO2Bu-t, LiHMDS,
AlEt3, THF (95%). (b) 1. PTSA, CHCl3, reflux (96%); 2. LDA, 2,6-di-tert-butylphenol,
THF (dr = 8–10:1), then recrystallization (dr = 50–100:1; 65%). (c) 1. LiBH4, THF,
MeOH (97%); 2. Pv-Cl, Py, CH2Cl2 (90%). (d) 1. TBAF, THF (96%); 2. NaIO4, aqꢀTHF
(93%); 3. NaBH4, MeOH (96%); 4. TBS-Cl, imidazole, CH2Cl2. (94%) (e) 1. DIBAL,
CH2Cl2 (92%); 2. SO3ꢀPy, (i-Pr)2(Et)N, DMSO, CH2Cl2 (90%); 3. Ph3P = C(Me)CO2Et,
CH2Cl2 (90%). (f) 1. DIBAL, CH2Cl2 (94%); 2. MnO2, CH2Cl2 (92%); 3. (EtO)2P(O)
CH2CO2Et, n-BuLi, THF (93%); (g) 1. PPTS, EtOH (90%); 5. 1-phenyl-1H-tetrazole-5-
thiol, DIAD, TPP, THF (94%); 2. H2O2, (NH4)6Mo7O24ꢀ4H2O, EtOH (90%). With use of
the same sequence of reactions, sulfone ester 60b-10 was prepared from (R)-
glycidol.
Scheme 1. Photochemical rearrangement of mycolactone A/B (1) into photo-
mycolactones A1, A2, B1, & B2 (only one structure of the photo-mycolactones
shown).
protection, TBDPS-deprotection, NaIO4-oxidation, NaBH4-reduc-
tion, and then TBS-protection treatment straightforwardly gave
C50-C80 building block 60a-7. We then followed the step-wise chain
elongation route used in the previous work16 to transform 60a-7 to
6
0a-9 via 60a-8. TBS-deprotection of 60a-9, followed by treatment
with 1-phenyl-1H-tetrazole-5-thiol under the Mitsunobu condi-
tion,17 gave the sulfide, which was oxidized with (NH4)6Mo7O24
-
ꢀ4H2O/30% H2O2,17 to furnish sulfone ester 60a-10, required for
the proposed Julia-Kocienski olefination.
Using the same sequence of reactions, 60b-10 was also prepared
from (R)-glycidol.
Scheme 2. Retrosynthetic analysis of C60-C70 dihydromycolactones 60b-15 and 60a
15.
-
C23-C26 building block of halichondrins.13 Thus, after protection of
the primary alcohol with TBDPS ether, (S)-glycidol was treated
with tert-butyl propionate under the condition reported by Taylor,
to give tert-butyl ester 4 which, upon treatment with PTSA,
resulted in the
with LDA, then with 2,6-di-tert-butylphenol, gave an 8–10:1
diastereomeric mixture of -lactones as a white solid. On single
recrystallization from hexanes, the diastereomeric ratio was
improved up to 50–100:1, to give -lactone 5. Based on the consid-
c
-lactone as a diastereomeric mixture.14 Treatment
c
c
eration that the protonation took place preferentially from the
direction opposite to the CH2OTBDPS group, we assigned, and
proved, the stereochemistry of the major diastereomer as indi-
Scheme 4. Completion of the synthesis. Reagents and conditions: (a) MnO2, CH2Cl2
(96%); (b) KHMDS, THF, ꢁ78 °C (90%); (c) 1. LiOH, THF, MeOH, Water (92%), 2. 14,
2,4,6-Trichlorobenzoyl chloride, DMAP, DIPEA, Toluene (86%), 3. TBAF, THF (80%);
Prepared 60b-15 using the same series of reaction sequence starting from 60b-10.
cated.15 LiBH4-reduction of
c-lactone 5, selective primary alcohol

1276
V.S. Babu et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1274–1277
Synthesis of the unsaturated fatty acid ester is summarized in
We then studied the acidic, basic, and thermal stability of dihy-
dromycolactones 60a-15 and 60b-15, relative to parent mycolac-
tone A/B. For the acid-stability test, we chose the condition of
methanol containing 0.1 M aq. HCl (15 eq) at rt, and the stability
was monitored by 1H NMR spectroscopy. For 2 h, no significant
decomposition was detected for dihydro-mycolactones 60a-15
and 60b-15, as well as mycolactone A/B. However, for 8 h, parent
mycolactone A/B extensively decomposed (ꢂ80%), whereas dihy-
dromycolactones 60a-15 and 60b-15 were stable. Similarly, the
base-stability under the condition of methanol containing 0.1 M
aq. NaOH (15 eq) at rt was tested. Once again, for 2 h, no significant
decomposition was detected for dihydromycolactones 60a-15 and
60b-15, as well as mycolactone A/B. However, for 8 h, parent myco-
lactone A/B extensively decomposed (ꢂ90%), whereas dihydro-
mycolactones 60a-15 and 60b-15 were stable. Lastly, thermal stabil-
ity was studied in acetone at 60 °C; mycolactone A/B existed as a
the upper half of Scheme 4. MnO2-oxidation of allylic alcohol 11
gave aldehyde 12, which was then subjected to the one-pot version
of Julia-Kocienski olefination with 60a-10, to furnish E-olefin ester
6
0a-13. The product was chromatographically isolated in 90% yield
and fully characterized (HR-MS, 1H NMR, 13C NMR, UV, and IR). The
stereochemistry of newly introduced olefin was established as E
0
0
from J8 ,9 = 15.7 Hz.
With use of the protocol developed for the synthesis of myco-
lactone A/B,8 E-olefin ester 60a-13 was uneventfully converted to
dihydromycolactone 60a-15 in 3 steps in 63% overall yield. The
final product was isolated with preparative TLC (500 lM silica
gel; 5% MeOH/EtOAc) and fully characterized (HR-MS, 1H NMR,
13C NMR, UV, and IR).
Similarly, dihydromycolactone 60b-15 was synthesized from 60b-
10 and was fully characterized. As expected, 60a-15 and 60b-15
exhibited very similar, but distinctly different 1H NMR properties.
With dihydromycolatones 60a-15 and 60b-15 in hand, we stud-
ied their stability, relative to parent mycolactone A/B, under the
photochemical, as well as acidic, basic, and thermal conditions
(Table 1). It should be noted that mycolactone A/B is stable to iso-
late and fully characterize. However, it gradually decomposes, par-
ticularly in neat. For this reason, we store mycolactone A/B as an
EtOAc solution in a sealed brown ampoule at ꢁ20 °C; under this
condition, mycolactone A/B has been shown to be stable for at least
five years.19
0
0
0
0
rapidly equilibrating 3:2 mixture of
D D
4 ,5 -Z and 4 ,5 -E isomers,
whereas 60a-15 and 60b-15, were stable even after 10 days.
With photochemically and chemically stabilized mycolactone
analogs in hand, we studied their biological activity. For this pur-
pose, we chose to measure their cytotoxicity against arbitrarily
chosen Hek-293, A-549, LOX-IMVI, and L-929 cell lines as the first
step (Table 2). It is worth noting that: (1) both 60a-15 and 60b-15
preserve potent cytotoxicity; (2) 60b-15 exhibits a practically same
potency against human Hek-293 as parent mycolactone A/B, but
significantly less potency against human cancer cell lines; (3)
Mycolactone A/B shows UV absorption at 362 nm (log
e: 4.35 in
6
0a-15 and 60b-15 exhibit a significant difference in cytotoxicity
MeOH) and, on exposure to light through a 365 nm filter, it is
cleanly transformed into a mixture of 4 photo-mycolactones (vide
ante). Dihydromycolactones 60a-15 and 60b-15 exhibit an expected
against human-cell lines, but not mouse-cell lines.
We are interested in the observation that an inversion in the
stereochemistry of the C60 methyl group caused a significant differ-
ence in cytotoxicity against human cell lines, thereby hinting an
importance of a specific stereostructure presented by the unsatu-
rated fatty acid moiety. In this connection, it is worthwhile men-
tioning recent reports on the cytotoxicity of mycolactone analogs
by Altmann and by Blanchard21; changes in the unsaturated fatty
acid moiety affected the cytotoxic activity against mammalian cells
more profoundly than changes in the mycolactone core moiety.
In summary, we have synthesized dihydromycolactones 60a-15
and 60b-15, and shown their stability under not only the
photochemical, but also acidic, basic, and thermal conditions.
Using arbitrarily chosen 4 cell lines, we have shown that both
blue shift in UV absorption (UV (MeOH) kmax 268 nm (log
e 4.53),
kmax 238 nm (log 4.58), kmax 230 nm (log 4.59)). Importantly,
e
e
they show virtually no UV absorption at the region uncovered by
a 365 nm filter. As expected, on exposure to light through a
365 nm filter, dihydromycolactones 60a-15 and 60b-15 were found
to be stable. Sunlight is also known to induce the transformation of
mycolactone A/B to photomycolactones.10a Therefore, the
photochemical stability of dihydromycolactones 60a-15 and 60b-
15 was also tested under sunlight; on exposure to sunlight in
acetone at rt, for 2 days, dihydromycolactones 60a-15 and 60b-15
exhibited only geometrical isomerization of double bonds, but no
skeletal rearrangement.18,20
6
0a-15 and 60b-15 exhibit potent cytotoxicity. In addition, we have
Table 1
Stability comparison of mycolactone A/B vs. dihydro-mycolactones.
Conditions
Time
Mycolactone A/B
Dihydro- mycolactones
Stable
Photolysis at 365 nm in acetone
2 h ꢂ 3 days
Unstable (initial E/Z double- bond isomerization,
followed by photocyclization to [3.1.0]-products)
Stable
Acid (0.1 M HCl (15 eq) in MeOH)
Base (0.1 M NaOH (15 eq) in MeOH)
Thermal (60 °C in acetone)
2 h
8 h
2 h
14 h
1 day
10 days
Stable
Stable
Stable
Stable
Stable
Stable
ꢂ80% decomposition
Stable
ꢂ90% decomposition
Stable (E/Z double-bond Isomerisation)
Stable (E/Z double-bond Isomerisation)
Table 2
Cytotoxicity of mycolactone A/B and dihydro-mycolactones 60a-15 and 60b-15 (quadruple experiments for each case).
Compound
Cell line
IC50 (nM)
Human lung carcinoma A-549
Human Hek-293
Human mela-noma LOX-IMVI
Mouse L-929
Mycolactone A/B (1)
C60a-Me dihydro-mycolactone 60a-15
C60b-Me dihydro-mycolactone 60b-15
3.2 0.9
83 31
3.0 0.6
0.77 0.15
400 229
77 29
6.9 0.7
120 14
13
63
53
4
4
4
29
4

V.S. Babu et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1274–1277
1277
7. (a) Benewoitz AB, Fidanze S, Small PLC, Kishi Y. J. Am. Chem. Soc.
2001;123:5128;
(b) Fidanze S, Song F, Szlosek-Pinaud M, Small PLC, Kishi Y. J. Am. Chem. Soc..
2001;123:10117.
8. (a) Song F, Fidanze S, Benowitz AB, Kishi Y. Tetrahedron. 2007;63:5739;
(b) Jackson KL, Li W, Chen C-L, Kishi Y. Tetrahedron. 2010;66:2263.
9. (a) For synthetic studies from: Negishi group:, Wang G, Yin N, et al. Chem. Eur. J.
2011;17. 4118 and references cited therein.;
(b) Altmann group, Gersbach P, Jantsch A, et al. Chem. Eur. J. 2011;17. 13017 and
references cited therein.;
made an interesting observation that an inversion in the stereo-
chemistry of the C60 methyl group caused a significant difference
in cytotoxicity against human cell lines, hinting an importance of
a specific stereostructure presented by the unsaturated fatty acid
moiety. Overall, the chemically stabilized mycolactone analogs
might serve us as a valuable tool for study on the biology of
mycolactone A/B.
(c) Blanchard group, Chany AC, Veyron-Churlet R, et al. Med. Chem. 2014;57.
7382 references cited therein;
Acknowledgements
(d) Burkart group, Ko KS, Alexander MD, et al 2010;8 :5159. and references
cited therein;
(e) van Summeren RP, Feringa BL, Minnaard AJ. Org. Biomol. Chem. 2005;3:2524;
(f) Wang Y, Dai W-M. Eur. J. Org. Chem. 2014;323;
Financial support from the Eisai USA Foundation is gratefully
acknowledged.
(g) Brown CA, Aggarwal VK. Chem. Eur. J. 2015;21:13900. and references cited
therein.
10. (a) Xing Y, Hande SM, Kishi Y. J. Am. Chem. Soc. 2012;134:19234;
(b) Li X, Babu VS, Kishi Y. Tetrahedron Lett. 2015;56:3220.
11. (a) Inanaga J, Hirata K, Saeki H, Katsuki T, Yamaguchi M. Bull. Chem. Soc. Jpn.
1979;52:1989;
(b) Hikota M, Sakurai Y, Horita K, Yonemitsu O. Tetrahedron Lett. 1990;31:6367.
12. (a) Kocienski PJ, Lythgoe B, Waterhouse I. J. Chem. Soc. Perkin. Trans.
1980;1:1045;
A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.01.
036.
(b) Blakemore PR, Cole WJ, Kocienski PJ, Morley A. Synlett. 1998;26.
13. (a) Guo H, Xu H, Kishi Y. Unpublished results.(b) Reference 10b.
14. Taylor SK, Fried JA, Grassl YN, et al. J. Org. Chem. 1993;58:7304.
15. The structure of the major diastereomer was confirmed by NOE studies in
comparison with the minor diastereomer synthesized in the separate route. See
supporting information for more details.
References
1. (a) For general reviews on Buruli ulcer and mycolactones, see: Asiedu K,
Scherpbier R, Raviglione M, eds. Buruli ulcer: mycobacterium ulcerans
infection. Geneva, Switzerland: World Health Organization; 2000;
(b) Hong H, Demangel C, Pidot SJ, Leadlay PF, Stinear T. Nat. Prod. Rep.
2008;25:447;
16. Kim H-J, Kishi Y. J Am. Chem. Soc. 2008;130:1842.
17. Smith III AB, Dong S, Brenneman JB, Fox RJ. J. Am. Chem. Soc. 2009;131:12109.
18. On exposure to sunlight in acetone at rt for 2 days yields an approximately
2:1:1 mixture of 20E, 40E, 80E, 100E, 20E, 40E, 80E, 100Z and 20E, 40Z, 80E, 100Z
geometrical isomers.
19. Approximately 20–30% mycolactone A/B decomposed in neat at rt for 10 days.
Mycolactone A/B was also stored as a DMSO or EtOH solution in sealed brown
ampoules at ꢁ20 °C. Under this condition, mycolactone A/B was stable at least
for 1 month. However, it was found that ꢂ20% mycolactone A/B in DMSO and
EtOH decomposed over three years.
20. Mycolactone A/B is insoluble in water. However, it can be solubilized by
addition of 0.1% Triton X-100 or NP-40. The stability of mycolactone A/B,
dihydromycolactones 60a-15 and 60b-15 in water containing 0.1% Triton X-100
and 1 N HCl (15 eq) or IN NaOH (15 eq) was found to be very similar to that in
methanol, respectively.
(c) Kishi Y. Proc. Natl. Acad. Sci. USA. 2011;108:6703;
(d) Chany A-C, Tresse C, Casarotto V, Blanchard N. Nat. Prod. Rep. 2013;30:1527.
2. Fact Sheet Number 199, 2007, World Health Organization (WHO), reports that
treatment with rifampicin and streptomycin for eight weeks leads to complete
healing of nearly 50% of Buruli lesions.
3. (a) George KM, Chatterjee D, Gunawardana G, et al. Science. 1999;283:854;
(b) George KM, Pascopella L, Welty DM, Small PLC. Infect. Immun. 2000;68:877;
(c) Rohr J. Angew. Chem. Int. Ed. 2000;39:2847.
4. For biological activities of the mycolactones, see also Dobos KM, Small PL,
Deslauriers M, Quinn FD, King CH. Infect. Immun. 2001;69:7182. and references
cited therein.
5. (a) Guenin-Macé L, Romain Veyron-Churlet R, Thoulouze MI, et al. J. Clin. Invest.
2013;123:1501;
(b) Baron L, Paatero AO, Morel JD, et al. J. Exp. Med. 2016. http://dx.doi.org/
10.1084/jem.20160662;
(c) McKenna M, Simmonds RE, High S. J. Cell. Sci. 2016;129:1404;
(d) Sarfo FS, Phillips R, Wansbrough-Jones M, et al. Cell. Microbiol. 2016;18:17.
6. Gunawardana G, Chatterjee D, George KM, Brennan P, Whittern D, Small PLC. J.
Am. Chem. Soc. 1999;121:6092.
21. (a) Scherr N, Gersbach P, Dangy J-P, et al. PLoS Neglected Trop. Dis. 2013;7:
e2143;
(b) Guenin-Macé L, Baron L, Chany AC, et al. Sci. Transl. Med. 2015;7. 289ra85
and references cited therein.