Chemoenzymatic approaches toward dechloroansamitocin P-3

Article abstract of DOI:10.1021/ol0702270

Equation Presented The enantioselective total synthesis of proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin P-3, is described which bears a diene-ene RCM as the key macrocyclization step. Feeding of proansam

Full text of DOI:10.1021/ol0702270

ORGANIC
LETTERS
2007
Vol. 9, No. 8
1489-1492
Chemoenzymatic Approaches toward
Dechloroansamitocin P-3†
Axel Meyer,‡ Marco Bru
1
njes,‡ Florian Taft,‡ Thomas Frenzel,‡ Florenz Sasse,§ and
Andreas Kirschning*,‡
Institut fu¨r Organische Chemie, Leibniz UniVersita¨t HannoVer, Schneiderberg 1B,
30167 HannoVer, Germany, and Helmholtzzentrum fu¨r Infektionsforschung (HZI),
Mascheroder Weg 1, 38124 Braunschweig, Germany
andreas.kirschning@oci.uni-hannoVer.de
Received January 29, 2007
ABSTRACT
The enantioselective total synthesis of proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin P-3,
is described which bears a diene-ene RCM as the key macrocyclization step. Feeding of proansamitocin to an AHBA block mutant Actinosynnema
pretiosum (HGF073) yielded ansamitocin P-3 as well as dechloroansamitocin P-3, the latter also being formed upon fermentation in the presence
of 3-amino-5-methoxybenzoic acid.
Maytansine, first isolated from the Ethiopian plant Maytenus
serrata,1,2 and the related ansamitocins P-1 to P-4,3-5 which
are of microbial origin (Actinosynnema pretiosum), consist
of a 19-membered macrolactam ring and differ in the side
chain at C-3. They inhibit growth of different leukaemia cell
lines as well as human solid tumors at very low concentra-
tions (10-3 to 10-7 µg/mL) by inhibiting tubulin polymeri-
zation. However, the clinical development of maytansinoids
had to be stopped in phase II2a,6 due to gastrointestinal side
effects and neurotoxicities.4b,7
† Dedicated to E. Winterfeldt on the occasion of his 75th birthday.
‡ Leibniz University Hannover.
§ Helmholtzzentrum fu¨r Infektionsforschung (HZI).
(1) (a) Kupchan, S. M.; Komoda, Y.; Court, W. A.; Thomas, G. J.; Smith,
R. M.; Karim, A.; Gilmore, C. J.; Haltiwanger, R. C.; Bryan, R. F. J. Am.
Chem. Soc. 1972, 94, 1354-1356. (b) Kupchan, S. M.; Komoda, Y.;
Branfman, A. R.; Sneden, A. T.; Court, W. A.; Thomas, G. J.; Hintz, H. P.
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C. R., Jr.; Powell, R. G. In Alkaloids; Pelletier, S. W., Ed.; John Wiley and
Sons: New York, 1984; Vol. 2, pp 149-204.
Total synthesis approaches5,8 contributed little to our
knowledge of the structure-activity relationships; this
(4) A mutant of A. pretiosum spp. auranticum provided 15 additional
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(b) A recent review of the synthetic approaches is given in ref 5b.
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10.1021/ol0702270 CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/23/2007

information was basically collected from semisynthetic work
starting with the natural products.2a,e
highly potent secondary metabolites like the ansamitocins.
As part of these studies we disclosed the total synthesis of
the N-acetylcysteamine derivative of seco-proansamitocin
1b,15 which is the SNAC-analogue for the natural substrate
of the cyclizing amide synthase (asm9).16
We now report on the first synthesis of proansamitocin
1b the product of the amide synthase which we wish to utilize
in screening for the amide synthase and in chemoenzymatic
studies with strain HGF073.
Analysis of 2 led us to consider three strategies for
macrocyclization. While macrolactamization (strategy I) is
a biomimetic approach the other two concepts (intramolecular
Heck-reaction (strategy II)17 and diene-ene ring closing
metathesis18 (RCM) (strategy III)) are based on transition
metal catalysis and would, compared to the first approach,
provide more synthetic novelty (Scheme 2). In fact, we found
As a result of detailed biosynthetic studies on ansamycin
antibiotics9-13 including ansamitocin P-3, Floss and co-
workers designed a block mutant (HGF073) of Actinosyn-
nema pretiosum which is unable to biosynthesize the starter
unit, 3-amino-5-hydroxybenzoic acid (AHBA),9 of the type
I modular polyketide synthase. This synthase is responsible
for assembling the carbon framework (through chain exten-
sion by one “glycolate”, three propionate, and three acetate
units). The last PKS module holds the seco-proansamitocin
1a, which is released and cyclized by an amide synthase
(gene asm9)12 to yield the cyclic 19-membered macrocyclic
lactam, proansamitocin 2 (Scheme 1).13
Scheme 1. Biosynthesis of Maytansin 3 and Ansamitocins 4,
5 via seco-Proansamitocin 1 and Proansamitocin 2
Scheme 2. Macrocyclization Strategies (I-III) and
Retrosynthetic Analysis for Strategies II and IIIa
Recently, we initiated a research program dedicated to
synthetically exploit genetically engineered microorganisms
such as the AHBA block mutant (HGF073)14 for chemoen-
zymatically generating new analogues of pharmaceutically
a PG ) protecting group. TBS ) tert-butyldimethylsilyl.
(9) Yu, T.-W.; Bai, L.; Clade, D.; Hoffmann, D.; Toelzer, S.; Trinh, K.
Q.; Xu, J.; Moss, S. J.; Leistner, E.; Floss, H. G. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 7968-7973.
that macrolactamization of related acyclic aniline precursors
(ansamitocin and ansatrienine) was not successful and
proceeded only in low yields.19 A suitable retrosynthetic
(10) (a) Hopwood, D. A. Chem. ReV. 1997, 97, 2465-2497. (b) Khosla,
C.; Gokhale, R. S.; Jacobsen, J. R.; Cane, D. E. Annu. ReV. Biochem. 1999,
68, 219-253. (c) Staunton, J.; Weisman, K. J. Nat. Prod. Rep. 2002, 18,
380-416 and references cited therein.
(11) Hatano, K.; Akiyama, S.-I.; Asai, M.; Rickards, R. W. J. Antibiot.
1982, 35, 1415-1417.
(14) (a) Yu, T.-W.; Bai, L.; Clade, D.; Hoffmann, D.; Toelzer, S.; Trinh,
K. Q.; Xu, J.; Moss, S. J.; Leistner, E.; Floss, H. G. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 7968-7973. (b) Kubota, T.; Bru¨njes, M.; Frenzel, T.; Xu,
J.; Kirschning, A.; Floss, H. G. ChemBioChem 2006, 7, 1221-1225.
(15) Fenzel, T.; Bru¨njes, M.; Quitschalle, M.; Kirschning, A. Org. Lett.
2006, 8, 135-138.
(12) Yu, T.-W.; Shen, Y.; Doi-Katayama, Y.; Tang, L.; Park, C.; Moore,
B. S.; Hutchinson, C. R.; Floss, H G. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 9051-9056.
(13) Stratmann, A.; Toupet, C.; Schilling, W.; Traber, R.; Oberer, L.;
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1490
Org. Lett., Vol. 9, No. 8, 2007

alkyne,21 followed by iodination; Scheme 3). Vinyl iodide
14 was then transformed into the two free aniline derivatives
15 and 16, respectively. 15 was simply prepared after O-silyl-
ation while the latter sequence included exhaustive Teoc-
protection, a Stille coupling for constructing the diene unit
in 16, and deprotection followed by O-silylation. Now the
stage was set to achieve intermolecular amide formation.
BOP-chloride22 turned out to be the best coupling reagent for
coupling carboxylic acid 11 with both anilines 15 and 16,
respectively, to yield the corresponding amides 17 and 18.
Despite the fact that intermolecular Heck coupling between
fragments 11 and 14 proceeds well under Jeffery condi-
tions,23,24 we were unable to obtain the expected Heck cross-
coupling product by Pd(0)-catalyzed macrocyclization of
vinyl iodide 17, while the Jeffery conditions (K2CO3, Bu4-
NCl, cat. Pd(OAc)2 , NEt3, DMF, rt) led to migration of the
terminal olefinic double bond furnishing the (E)-configured
R,â-unsaturated ketone 19, perhaps because of the basic
conditions. However, the Fu conditions (Pd2(dba)3, P(t-Bu)3,
Cy2NMe, dioxane, 110 °C)25 generated the (Z)-stereoisomer
of 19, so that a simple base-mediated isomerization most
likely has to be excluded.
Scheme 3 a
We were delighted to find that the diene-ene RCM
concept (strategy III) turned out to be successful when
Grubbs 1 catalyst 20 was employed (Scheme 4). The Grubbs
Scheme 4. Diene-Ene RCM of 19a
a dppf ) bis(diphenylphosphinoyl)ferrocene, TMS ) trimeth-
ylsilyl, TBAF ) tetra-n-butylammonium fluoride, Cp ) cyclopen-
tadienyl, TBDPS) tert-butyldiphenylsilyl, Teoc ) trimethylsi-
lylethoxycarbonyl, BOP-Cl ) N,N-bis(2-oxo-3-oxazolidinyl)-
phosphonic chloride.
precursor for the Heck-strategy II is vinyl iodide 6, which
derives from the advanced ketide fragment 11. This had been
prepared by us before as part of our total synthesis of the
SNAC-ester 1b,15 and aniline 7. Vinyl iodide 7 is also the
starting material for the diene-ene RCM strategy III, which
relies on the intermediate Stille coupling product 10. This
compound is planned to be coupled with fragment 11 so that
amide 9 serves as the RCM precursor.
In principle, the two required aromatic building blocks 15
and 16 had to be prepared from the same starting benzyl
bromide 12. It was transformed into vinyl iodide 14 (after
Pd-catalyzed alkynylation with alkynylindium 13,20 desily-
lation, Negishi-type methyl metalation of intermediate
a Cy ) cyclohexyl.
2 catalyst did not afford RCM products. Besides unreacted
starting material (∼30%), we isolated the RCM products as
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G. J. Am. Chem. Soc. 2003, 125, 14236-14237.
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1663.
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1985, 107, 6639-6647. (b) Negishi, E.-i.; Kondakov, S. Y.; Choueiry, D.;
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Org. Lett., Vol. 9, No. 8, 2007
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a mixture of stereoisomers, the (E,E-)-isomer being favored
(∼3:1). Removal of the silyl protection finally afforded
proansamitocin 226 and the (E,Z)-isomer 21.27
We then conducted preliminary feeding experiments to test
whether complex substrates such as the synthetic proansami-
tocin 2 are accepted and processed by Actinosynnema
pretiosum. Compound 2 (2.6 µmol) was fed in three equal
portions 72, 96, and 120 h after inoculation to a 25 mL
culture of Actinosynnema pretiosum mutant HGF073, which
lacks the ability to synthesize AHBA (Scheme 5).14 Parallel
troscopy. Therefore, we tested whether dechloroansamitocin
P-3 22 can be prepared directly by feeding 3-amino-5-
methoxybenzoic acid 23 (37.5 µmol) to a culture of Acti-
nosynnema pretiosum mutant HGF073. After workup and
HPLC purification, 1.5 mg (2.5 µmol) (from 250 mL of
fermentation broth) of 22 was isolated as pure material. In
tests with cultured human tumor cell lines it showed strong
antiproliferative activity with IC50 values down to 10 pg/
mL (Table 1).
Table 1. Antiproliferative Activity IC50 [ng/mL]28
cell line
origin
4
22
Scheme 5. Feeding Experiments (Functional Groups
Introduced Are Labeled in Red)a
KB-3-1
U-937
PC-3
A-431
A-498
SK-OV-3
cervix carcinoma
lymphoma
prostate carcinoma
epidermoid carcinoma
kidney carcinoma
ovarian carcinoma
0.11
0.0035
0.035
0.05
1.1
0.5
0.01
0.08
0.25
9
0.03
0.1
In conclusion, we achieved the first total synthesis of
proansamitocin 2 and showed that such a complex biosyn-
thetic intermediate can successfully be fed to a AHBA block
mutant of Actinosynnema pretiosum thereby reestablishing
AP-3 production. Formation of the biologically highly active
byproduct dechloroansamitocin P-3 22 was independently
confirmed by mutasynthesis feeding 3-amino-5-methoxy-
benzoic acid.
In principle, these results pave the way to prepare many
new AP-3 derivatives by feeding simple as well as advanced
derivatives of biosynthetic intermediates. Additionally, with
proansamitocin in hand, we will be able to screen for the
amidase (gene asm9).
a DDQ ) dichlorodicyanoquinone, DCC ) dicyclohexylcarbo-
diimide, DMAP ) 4-dimethylaminopyridine, Pyr ) pyridine.
fermentations were carried out with the wild-type strain and
with mutant HGF073 supplemented with AHBA and without
supplementation. The cultures were harvested after 7 days
and extracted with ethyl acetate. The extract was subjected
to electrospray ionization mass spectrometry (ESI-MS) and
revealed formation of AP-3 4 along with a new metabolite
(parent ions at m/z 601 (M + H)+ and m/z 624 (M + Na)+)
that is consistent with dechloroansamitocin P-3 22. We could
not obtain confirmation of the structure using NMR spec-
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft (grant Ki 397/7-1) and
by the Fonds der Chemischen Industrie. We thank H. G.
Floss (University of Washington, Seattle) for providing strain
HGF073, and Birte Engelhardt and Bettina Hinkelmann
(HZI, Braunschweig, Germany) for performing the cell
proliferation assays.
Supporting Information Available: Descriptions of
experimental procedures for compounds and analytical
characterization as well as details on the cell proliferation
assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
(26) 1H and 13C NMR data were identical in every respect with those
reported for proansamitocin 2, a fermentation byproduct (ref 16).
(27) Yields refer to isolated yields of pure isomers 2 and 21. These were
collected after several chromatographic runs, which included preparative
HPLC.
(28) Proansamitocin showed no antiproliferative activity. Like AP-3 (IC50
) 0.015 ng/ml) dechloroansamitocin P-3 22 (IC50 ) 0.15 ng/ml) also
showed strong antiproliferative activity for primary human endothel cells.
OL0702270
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