A flexible route to (5R)-thiolactomycin, a naturally occurring inhibitor of fatty acid synthesis.

Article abstract of DOI:10.1021/ol026685k

[formula: see text] A new and efficient asymmetric synthesis of naturally occurring (5R)-thiolactomycin (1) using D-alanine as the source of chirality is described.

Full text of DOI:10.1021/ol026685k

ORGANIC
LETTERS
2002
Vol. 4, No. 22
3859-3862
A Flexible Route to (5R)-Thiolactomycin,
a Naturally Occurring Inhibitor of Fatty
Acid Synthesis
Jill M. McFadden, Gojeb L. Frehywot, and Craig A. Townsend*
Department of Chemistry, The Johns Hopkins UniVersity, 3400 North Charles Street,
Baltimore, Maryland 21218
ctownsend@jhu.edu
Received August 6, 2002
ABSTRACT
A new and efficient asymmetric synthesis of naturally occurring (5R)-thiolactomycin (1) using D-alanine as the source of chirality is described.
Naturally occurring thiolactomycin is a unique molecule that
exerts selective activity against only the dissociable type II
fatty acid synthase (FAS) enzymes.1 Isolated from Nocardia
sp., thiolactomycin is the most well studied of the reported
naturally occurring thiotetronic acids.2 (Figure 1) It is active
and can be used to treat urinary tract and intraperitoneal
bacterial infections.2
Thiolactomycin (1, TLM) is a reversible inhibitor1a of the
â-ketoacyl synthase (KAS) of bacterial FAS systems includ-
ing KAS I-III in E. coli. Recently, a crystal structure of
KAS I (FabB from E. coli) with thiolactomycin bound
reveals the essential enzyme-ligand binding interactions and
establishes the existence of hydrophobic and pantetheine
binding pockets that are both unoptimally filled.3 Indeed,
structure-activity studies demonstrate that TLM analogues
with an extended C5 hydrocarbon chain exhibit enhanced
antimycobacterial activity4a and effective inhibition of pea
(Pisum satiVum) FAS.4b Also, C3-acetyl analogues of thio-
lactomycin with C5 aryl or alkyl functionality display
(2) (a) Sasaki, H.; Oishi, H.; Hayashi, T.; Matsuura, I.; Ando, K. Sawada,
M. J. Antiobiotics 1982, 35, 396-400. (b) Noto, T.; Miyakawa, S.; Oishi,
H.; Endo, H.; Okazaki, H. J. Antibiotics 1982, 35, 401-410. (c) Miyakawa,
S.; Suzuki, K.; Noto, T.; Harada, Y.; Okazaki H. J. Antibiotics 1982, 35,
411-419. (d) Oishi, H.; Noto, T.; Sasaki, H.; Suzuki, K. J. Antibiotics 1982,
35, 391-395. (e) Sato, T.; Suzuki, K.; Kadota, S.; Abe, K.; Takamura, S.;
Iwanami, M. J. Anibiotics 1989, 890-896. (f) Omura, S.; Nakagawa, A.;
Iwata, R.; Hatano, A. J. Antibiotics 1983, 36, 1781-1782. (g) Sato, T.;
Suzuki, K.; Kadota, S.; Abe, K.; Takamura, S.; Iwanami, M. J. Antibiotics
1989, 42, 890-896. (h) Omura, S.; Iwai, Y.; Nakagawa, A.; Iwata, R.;
Takahashi, Y.; Shimizu, H.; Tanaka, H. J. Antibiotics 1983, 36, 109-114.
(i) Nishida, I.; Kawaguchi, A.; Yamada, M. J. Biochem. 1986, 99, 1447-
1454.
Figure 1.
in vitro against Gram-positive, Gram-negative, and anaerobic
bacteria. Furthermore, thiolactomycin is nontoxic in mice
(1) (a) Nishida, I.; Kawaguchi, A.; Yamada, M.; J. Biochem. 1986, 99,
1447-1454. See reviews: (b) Heath, R. J.; White, S. W.; Rock, C. O. Prog.
Lipid Res. 2001, 40, 467-497. (c) Campbell, J. W.; Cronan, J. E. Annu.
ReV. Microbiol. 2001, 55, 305-332.
(3) Price, A. C.; Choi, K. H.; Heath, R. J.; Li, Z.; White, S.; Rock, C.
O. J. Biol. Chem. 2001, 276, 9, 6551-6559.
10.1021/ol026685k CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/08/2002

effective activity against Staphylococcus aureus and Pas-
teurella multocida.4c
Retrosynthetic analysis envisioned thiolactomycin (1) to
be derived through a thio-Dieckman condensation of 10
(Scheme 1). Carefully controlled conditions would enable
Importantly, inhibition of mammalian type I FAS by C75
(2) and cerulenin (3) leads to selective cytotoxicity against
various cancer cells in vitro.5 Xenografts of MCF7 breast
cancer cells in nude mice treated with C75 show FAS
inhibition, followed by apoptosis and reduction in tumor size.
Reports also demonstrate that C75 causes significant weight
loss by reducing hypothalamic NPY expression and stimulat-
ing CPT-1 activity and fatty acid (FA) oxidation.6
Scheme 1. Retrosynthesis of (5R)-Thiolactomycin
Indeed these studies suggest that inhibition of FAS can
be used not only as a strategy to develop new antibacterial
agents (e.g., thiolactomycin) but also as a selective route to
treat cancer and obesity (e.g., C75 and cerulenin). Perhaps,
even more striking and not understood is the high specificity
of TLM for only the â-ketoacyl synthases of type II FAS,
while C75 and cerulenin are inactivators of both type I and
II FAS systems. Intrigued by TLM’s unique selectivity and
the inherent potential for analogues of TLM to be potent
therapeutic agents, we have developed an efficient asym-
metric synthesis of naturally occurring (5R)-thiolactomycin.
There have been only a few reports on the synthesis of
thiolactomycin. Salvino et al. described the first racemic total
synthesis of thiolactomycin, which involved the alkylation
of a thiotetronic acid dianion with an isoprene cation
equivalent (3-ethoxy-2-methyl-2-propenal).7a Treatment of
the resulting aldehyde with methylene triphenyl-phosphorane
afforded thiolactomycin. Thomas and co-workers developed
an asymmetric synthesis of (5S)-thiolactomycin and other
thiotetronic acids. The key step in this route used a
stereoselective [3,3]-rearrangement of an allyl xanthate to
the corresponding dithiocarbonate.7b-d To our knowledge,
these are the only reported syntheses of thiolactomycin, with
only the latter addressing enantiomeric purity.
10 to be synthesized from the corresponding oxathiolanone
8. Conversion of allylic alcohol 7 through a sulfenate-
sulfoxide [2,3]-sigmatropic rearrangement accompanied by
a thermal syn-elimination was anticipated to provide diene
8. The allylic alcohol 7 can be obtained directly and likely
with complete 1,3-diastereoinduction from addition of opti-
cally pure oxathiolanone 5. Oxathiolanone 5 of high enan-
tiomeric purity can be prepared from (2S)-thiolactic acid 4.8,9b
Optically pure (2S)-thiolactic acid was obtained by the
method of Kellogg as previously described (Scheme 2).9
Our synthesis employs Seebach’s self-regeneration of
chirality method, which utilizes amino acids as the chiral
building blocks.8 This approach is potentially quite versatile,
enabling selective functionalization of both the C3 and C5
positions of the thiolactone ring.
Scheme 2. Synthesis of (2S)-Thiolactic Acid9
(4) (a) Kremer, L.; Douglas, J. D.; Baulard, A. R.; Morehouse, C.; Guy,
M. R.; Alland, D.; Dover: L. G.; Lakey, J. H.; Jacobs, W. R.; Brennan, P.
J.; Minnikin, D. E.; Besra, G. S. J. Biol. Chem. 2000, 275, 22, 16857-
16864. (b) Jones, A. L.; Herbert, D.; Rutter, A. J.; Dancer, J. E.; Harwood:
J. L. Biochem. J. 2000, 347, 205-209. (c) Sakya, S. M.; Suarez-Contreras,
M.; Dirlam, J. P.; O′Connell, T. N.; Hayashi, S. F.; Santoro, S. L.; Kamicker,
B. J.; George, D. M.; Ziegler, C. B. Bioorg. Med. Chem. Lett. 2001, 11,
2751-2754.
(5) (a) Pizer, E. S.; Thupari, J.; Han, W. F.; Pinn, M. L.; Chrest, F. J.;
Frehywot, G. L.; Townsend, C. A.; Kuhajda, F. P. Cancer Res. 2000, 60,
213-218. (b) Kuhajda, F. P.; Pizer, E.; Li, J. N.; Mani, N. S.; Frehywot,
G. L.; Townsend, C. A. PNAS 2000, 97, 3450-3454. (c) Pizer, E. S.; Lax,
S. F.; Kuhajda, D. P.; Pasternack, G. R.; Kurman, R. J. Cancer 1998, 528-
537.
Briefly, (2R)-alanine was converted to (2R)-chloropropionic
acid (11, 65%) with retention of configuration using the
classical diazotization-chlorination protocol.9c Chloride
displacement with clean inversion of stereochemistry was
achieved with cesium thioacetate in DMF 12 (62%, Scheme
2). Deacylation was carried out without loss of optical purity
in 1 N NH3, providing (2S)-thiolactic acid (4, 84%).
A mixture of cis and trans (2.5:1) (S)-oxathiolanones 5
and 6 (99%) was prepared from the acid-catalyzed acetal-
ization of (2S)-thiolactic acid (4) with pivalaldehyde as
previously reported.8,9 Recrystallization (8:1 pentane/ether)
(6) (a) Thupari, J. N.; Landree, L. E.; Ronnett, G. V.; Kuhajda, F. P.;
PNAS 2002, 99, 14, 9498-9502. (b) Loftus, T. M.; Jaworsky, D. E.;
Frehywot, Gojeb L.; Townsend, Craig, A.; Ronnett, Gabriele, V.; Lane,
Daniel M.; Kuhajda, Francis P. Science 2000, 288, 2379-2381.
(7) (a) Wang, C. J.; Salvino, J. M. Tetrahedron Lett. 1984, 25, 46, 5243-
5246. (b) Chambers, M. S.; Thomas, E. J.; Williams, D. J. J. Chem. Soc.,
Chem. Commun. 1987, 1228-1230. (c) Chambers, M. S.; Thomas, E. J. J.
Chem. Soc. Chem. Commun. 1989, 23-24. (d) Chambers, M. S.; Thomas,
E. J. J. Chem. Soc., Perkin Trans. 1 1997, 417-431.
(9) (a) Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664-
3671. (b) Strijtveen, B.; Kellogg, R. M. Tetrahedron 1987, 43, 21, 5039-
5054. (c) Fu, S. C. J.; Birnbaum, S. M.; Greenstein, J. P. J. Am. Chem.
Soc. 1954, 6054-6058.
(8) (a) Seebach, D.; Naef, R.; Calderari, G. Tetrahedron 1984, 40, 8,
1313-1324. (b) Seebach, D.; Sting, A. R.; Hoffman, M. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 2708-2748.
3860
Org. Lett., Vol. 4, No. 22, 2002

Cesium carbonate proved to be the most effective base able
to both catalyze ethanolysis and allow labile 9 to be isolated
in good yield. Immediately, 9 was acylated with propionyl
chloride to give 10 (72%, two-step yield). Enolate formation
was accomplished at -78 °C with LiHMDS, and Dieckman
condensation provided optically pure (5R)-thiolactomycin 1
(70%, ) 96% ee, Scheme 4). Deacylation of the thioester
competes with the Dieckman condensation even at low
concentration. The use of LiHMDS as the base minimized
this side reaction. Other bases, including KOtBu, NaH, LDA,
and LiTMP, all gave a significant amount of deacylation.
Scheme 3. Synthesis of 2(R),5(S)-Oxathiolanone
at -78 °C afforded optically pure 5 (54%, Scheme 3).
Formation of the lithium-enolate of 5 was achieved with
LDA at -78 °C, and addition of tiglic aldehyde (trans-2-
methyl-2-butenal) was specific to the re-face, yielding 7
(81%) as a 2:1 mixture of diastereomeric alcohols (Scheme
4).
The optical purity was assessed by formation of Mosher
esters 13 from thiol 9 and found to be 96% ee [The vinylic
C1′ hydrogens are resolved (1H NMR) and could be readily
integrated; 19F NMR displayed two resolved 19F peaks,
Scheme 5]. Recrystallization of 1 (96% ee) from 3:1 hexanes/
Scheme 4. Asymmetric Synthesis of 5(R)-Thiolactomycin
Scheme 5. Enantiomeric Purity by Mosher’s Method
24
acetone provided optically pure (5R)-thiolacomycin [R]D
+174 (c 0.6, MeOH); mp 119.5-121 °C (lit.2a [R]D20 +176
(c 1.0, MeOH), mp 120 °C).
Herein we have described an efficient and versatile
asymmetric synthesis of (5R)-thiolactomycin. This route
offers several important advantages over other existing
syntheses. First, optically enriched thiolactomycin was
synthesized in nine steps from D-alanine. Second, 5 and 9
are modular intermediates capable of selective functional-
ization at both the C3 and C5 positions of the thiolactone
ring. In comparison, Thomas et al. also used benzyl-protected
9 in their asymmetric synthesis.7c,d They assembled the C2,
C3, and C3′-CH3 carbons of the thiolactomycin skeleton
by preparation of the R-methyl-â-ketoester of benzyl-
protected 9. Unfortunately, from this intermediate it was not
possible to directly obtain TLM due to the instability of the
1,3-diene. Additional steps were required to protect the
terminal alkene as an aryl-selenide. The route described
herein enables the 1,3-diene to be obtained directly. A [2,3]-
sigmatropic rearrangement of the performed aryl sulfenate
(from 7) followed by a thermal syn elimination provided 8
with almost exclusively the trans stereochemistry at the C1′-
C2′ alkene (trans:cis ) 14:1). We installed the C2, C3, and
C3′-CH3 by preparing thiopropionate 10 from 9. Subsequent
thio-Dieckman condensation provided (5R)-thiolacomycin.
Reich and co-workers reported a method for the 1,4
dehydration of allylic alcohols to provide 1,3-dienes.10 This
sequence consists of treating the allylic alcohol with 2,4-
dinitrobenzenesulfenyl chloride to provide the sulfenate ester,
which undergoes a [2,3]-sigmatropic rearrangement to the
allylic sulfoxide. Thermal syn-elimination of the sulfoxide
gives the diene.10 Therefore, treatment of 7 with 2,4-
dinitrobenzenesulfenyl chloride and NEt3 in refluxing dichlo-
roethane provided 8 (75%) in good yield (Scheme 4).
This method is particularly advantageous in our synthesis
since the [2,3]-sigmatropic rearrangement provides almost
exclusively the trans stereochemistry at the C1′ alkene (trans:
cis ) 14:1). Dissolved in ethanol, the oxathiolanone ring of
8 was opened upon the addition of Cs2CO3. The released
thiol 9 is particularly sensitive to both acidic and basic
conditions because decomposition was commonly observed.
(10) (a) Reich, H. J.; Wollowitz, S. J. Am. Chem. Soc. 1982, 104, 7051-
7059. (b) Tang, R.; Mislow, K. J. Am. Chem. Soc. 1970, 92, 2100. (c)
Bickart, P.; Carson, F. W.; Jacobus, J.; Miller, E. G.; Mislow, K. J. Am.
Chem. Soc. 1968, 90, 4869.
Org. Lett., Vol. 4, No. 22, 2002
3861

The flexibility of this route opens the thiolactomycin class
to investigation and optimization as inhibitors of fatty acid
synthesis. The γ-thiolactone can be varied in a combinatorial
sense at C3, C5, and, in principle, at the C4-enol to develop
more potent antibacterial agents. Manipulation of functional-
ity around the thiolactone ring can be used to probe the
interaction of thiolactomycin with â-ketoacyl synthases to
gain an understanding of its unique specificity for only type
II FAS systems. These insights could reveal structural
alterations that engender inhibitory activity against type I
human FAS and enable the preparation of agents useful in
the treatment of cancers and obesity.
Acknowledgment. We are grateful to the National
Institutes of Health (CA 91634 to J.M.M. and in part AI
43846 to C.A.T.) and FASgen, Inc., for financial support.
Supporting Information Available: Spectral and ana-
lytical data for compounds for 1, 5, 7, 8, 10, and 13 and
1
copies of H NMR spectra for compounds 1, 5, 7, 8, 10,
and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL026685K
3862
Org. Lett., Vol. 4, No. 22, 2002