Enantioselective synthesis of pactamycin, a complex antitumor antibiotic

Article abstract of DOI:10.1126/science.1234756

Medicinal application of many complex natural products is precluded by the impracticality of their chemical synthesis. Pactamycin, the most structurally intricate aminocyclopentitol antibiotic, displays potent antiproliferative properties across multiple phylogenetic domains, but it is highly cytotoxic. A limited number of analogs produced by genetic engineering technologies show reduced cytotoxicity against mammalian cells, renewing promise for therapeutic applications. For decades, an efficient synthesis of pactamycin amenable to analog derivatizations has eluded researchers. Here, we present a short asymmetric total synthesis of pactamycin. An enantioselective Mannich reaction and symmetry-breaking reduction sequence was designed to enable assembly of the entire carbon core skeleton in under five steps and control critical three-dimensional (stereochemical) functional group relationships. This modular route totals 15 steps and is immediately amenable for structural analog synthesis.

Full text of DOI:10.1126/science.1234756

Enantioselective Synthesis of Pactamycin, a Complex Antitumor
Antibiotic
Justin T. Malinowski et al.
Science 340, 180 (2013);
DOI: 10.1126/science.1234756
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REPORTS
Acknowledgments: Additional pseudo–first-order rate
at Lawrence Berkeley National Laboratory. Sandia is a
multiprogram laboratory operated by Sandia Corporation,
a Lockheed Martin Company, for the National Nuclear Security
Administration under contract DE-AC04-94-AL85000.
D.K.W.M., J.M.D., C.J.P., and E.P.F.L. acknowledge support
from the Research Grant Council of the Hong Kong Special
Administrative Region (grant no. Polyu 5019/11P) and
the National Service for Computational Chemistry Software
(UK) for computational resources. The experiments were
conceived by C.A.T., C.J.P., and D.E.S.; designed by C.A.T.,
D.L.O., C.J.P., O.W., and A.J.E.; and carried out by O.W.,
J.D.S., A.J.E., A.M.S., B.R., C.J.P., C.A.T., and D.L.O. E.P.F.L.,
D.K.W.M., and J.M.D. were responsible for the quantum
chemistry and Franck-Condon calculations. All authors
participated in the data analysis and interpretation and
contributed to the manuscript.
constants, details of the experiments and calculations, and
spectroscopic data underpinning this work are presented in
the supplementary materials. The participation of O.W.,
A.J.E., J.D.S., D.L.O., and C.A.T. and the development of the
experimental kinetics apparatus were funded by the Division
of Chemical Sciences, Geosciences, and Biosciences, the
Office of Basic Energy Sciences, the U.S. Department
of Energy. D.E.S., J.M.D., and C.J.P. thank the Natural
Environment Research Council for funding, and J.M.D. thanks
the Leverhulme Trust for a senior fellowship. The Advanced
Light Source is supported by the Director, Office of
Supplementary Materials
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Materials and Methods
Supplementary Text
Figs. S1 to S19
Schemes S1 and S2
Tables S1 to S8
References (31–49)
Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under contract DE-AC02-05CH11231
2 January 2013; accepted 15 February 2013
10.1126/science.1234689
a compelling case can be made that a more prac-
tical synthesis solution is needed.
Enantioselective Synthesis of Pactamycin,
In this Report, we disclose a 15-step total
synthesis of pactamycin, which has, in the initial
pass, produced the natural product on a milligram
scale and a key branch-point intermediate on a
gram scale. We emphasized both modular con-
struction and introduction of functionality in the
final desired form of pactamycin, enabling an ap-
proach amenable to derivatization for analog syn-
thesis. Late-stage introduction of the aniline- and
salicylate-binding elements provides an opportu-
a Complex Antitumor Antibiotic
Justin T. Malinowski,* Robert J. Sharpe,* Jeffrey S. Johnson†
Medicinal application of many complex natural products is precluded by the impracticality of their
chemical synthesis. Pactamycin, the most structurally intricate aminocyclopentitol antibiotic, displays
potent antiproliferative properties across multiple phylogenetic domains, but it is highly cytotoxic. A
limited number of analogs produced by genetic engineering technologies show reduced cytotoxicity
against mammalian cells, renewing promise for therapeutic applications. For decades, an efficient
synthesis of pactamycin amenable to analog derivatizations has eluded researchers. Here, we present nity for future SAR studies.
a short asymmetric total synthesis of pactamycin. An enantioselective Mannich reaction and
The recognition of a hidden symmetry in the
symmetry-breaking reduction sequence was designed to enable assembly of the entire carbon core skeleton northeast quadrant of pactamycin (1) was critical
in under five steps and control critical three-dimensional (stereochemical) functional group relationships. to our synthetic plan. Depicted in Fig. 2A, the car-
This modular route totals 15 steps and is immediately amenable for structural analog synthesis.
bon chain connecting C-4 and C-8 can be extracted
to a symmetrical a-ureido-2,4-pentanedione 2.
omplex organic molecules produced by to high cytotoxicity (median inhibitory concen- We envisaged simplified formation of the fully
bacteria have been used to treat numerous tration of 95 nM against human diploid embryonic substituted C-1 center via a Mannich reaction.
disease types for nearly a century (1–4). cell line MRC-5) (11). Pactamycin is a proto- Due to the symmetrical methyl ketone substitu-
C
However, many naturally derived compounds typical example of a promising bioactive natural ents at C-1, diastereoselectivity considerations are
that exhibit interesting bioactivities are practical- product whose complexity hampers the investi- obviated, allowing for a focus on the enantio-
ly inaccessible via synthetic organic chemistry. A gation of structure-activity relationships (SARs) selective C-2–amino incorporation during the
natural product’s structural complexity can create that might lead to a serviceable therapeutic ap- C-1–C-2 bond construction. The nascent C-2
an insurmountable impediment to the preparation plication and/or a better understanding of intrin- stereocenter would then need to direct a site- and
of analogs that might exhibit improved character- sic bioactivity.
diastereoselective diketone monoreduction, setting
istics. An ongoing challenge in the field of syn- Genetic engineering studies have reignited the C-2/C-1/C-7 stereotriad (red dashed arrows in
thetic chemistry is the development of methods promise for medicinal application, as 7-deoxy– Fig. 2B). This sequence would provide the entire
that close the gap between the efficiency of bio- and 8″-hydroxy–derivatives were isolated and pactamycin carbon-core skeleton from which mod-
synthetic machinery and laboratory synthesis. Be- displayed diminished cytotoxicity (11–14). In the ular delivery of various functionality (Fig. 2C)
cause of the inherent flexibility of the latter, context of the work described herein, it is worth could provide 1 and/or its congeners in rapid
success in this endeavor could provide access to noting that Lu et al. contend that the structural fashion.
useful structural variants that might otherwise be complexity of 1 renders these and related struc-
The first challenge we faced was implementa-
inaccessible. tural modifications “inaccessible by synthetic tion of the Mannich reaction with an appropriately
Pactamycin (1; Fig. 1) was isolated from organic chemistry” (12). Conversely, we have pro- configured imine electrophile. We were encour-
Streptomyces pactum var. pactum in 1961 by ceeded from the hypothesis that the genetic en- aged by results reported by Schaus and co-workers,
Argoudelis et al. (5). The bioactivity profile of gineering approach to pactamycin analogs might
this natural product is especially notable, as it dis- be inherently limited by the biosynthetic machin-
plays antitumor, antimicrobial, antiviral, and anti- ery (15, 16). Though pactamycin is commercially
protozoal properties by acting as a universal inhibitor available, a chemical approach to its synthesis
of translocation (6–9). Within the ribosomal sub- could, in principle, provide far greater opportu-
unit in which it interacts, pactamycin mimics an nity and flexibility for discovering and advancing
RNA dinucleotide through interactions of its an- useful compounds. However, this tactic will only
iline and salicylate moieties with stem loops in be feasible in the presence of an efficient synthe-
the 16S RNA (10). Unfortunately, pactamycin’s sis platform that rapidly develops the appropriate
therapeutic benefits have yet to be realized, due level of structural complexity. In fact, synthetic
interest in pactamycin has recently flourished,
culminating in the landmark 32-step total synthe-
Department of Chemistry, University of North Carolina at
sis from Hanessian and co-workers (17, 18), as
well as numerous partial synthetic studies (19–23).
Despite these creative, state-of-the-art approaches,
Chapel Hill, Chapel Hill, NC 27599, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: jsj@unc.edu
Fig. 1. Structure of pactamycin (1). Me, methyl.
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12 APRIL 2013 VOL 340 SCIENCE www.sciencemag.org

REPORTS
wherein Cinchona alkaloids were effective in cat- lection of cinchonidine (7) as the catalyst of choice, reaction constitutes a useful advance in the syn-
alyzing the enantioselective addition of simple providing Mannich product 4 in 70% isolated thesis of differentiated, highly functionalized 1,2-
1,3-dicarbonyls to acyl imines (24, 25). However, yield and 98:2 enantiomeric ratio (er) (94% yield, diamines by adaptation of the Schaus conditions
the required asymmetric Mannich addition of 84:16 er before removal of the racemate by trit- to a new nucleophile-electrophile pair; exten-
a-amino–substituted dicarbonyls was heretofore uration). An x-ray diffraction study of a deriva- sion to other urea-carbamate combinations can
unknown. Additionally, with our goal of modular tive (26) revealed the formation of the illustrated be envisaged.
construction in mind, we planned to install the un- (R) configuration at C-2. Note that this nominally
The proposed desymmetrization of the Mannich
usual 1,1-dimethylurea in its native form early in corresponds to the incorrect configuration at C-2, adduct (4 → 5) is complicated by the fact that
our route, a tactic that was expected to obviate the but the advancement of this stereochemical mis- four diastereomeric monoreduction products are
protection, deprotection, and acylation steps that take was, in fact, critical to orchestrate downstream possible. Lithium tri(tert-butoxy)aluminum hydride
characterize all other pactamycin synthetic studies. stereochemical outcomes and efficiently complete (LTBA) emerged as a superior reducing agent for
Pronucleophile 2 was synthesized in two steps the synthesis (see below). The strategic selection the desymmetrization, affording hydroxyketone
(26) from commodity chemical acetylacetone of cinnamyl imine 3 as the Mannich electrophile 5 with high diastereoselectivity [>10:1 ratio of
(2.5 kg ~ $75) and subjected to adapted Mannich translated to the installation all five carbons of the 5:S(other diastereomers)] in 72% yield. This re-
conditions with cinnamaldehyde-derived imine 3 pactamycin core, with appropriate functional han- duction delivered the illustrated (1R,2R,7S)-
(Fig. 3). An evaluation of Lewis bases led to se- dles, in this initial C–C bond construction. This product; therefore, the incorrect C-2 isomer was
parlayed into the correct C-1/C-7 configurations.
Subsequent silyl protection of the C-7 hydroxyl
gave methyl ketone 6.
Our attention then shifted to installation of the
C-4 side chain and cyclization to complete the
cyclopentenone core (Fig. 4). We treated the lith-
ium enolate of ketone 6 with formaldehyde gas
(generated in situ by the pyrolysis of paraformal-
dehyde), which resulted in the single-aldol addi-
tion product 8 (27, 28). Alkene ozonolysis furnished
aldehyde 9 poised for intramolecular aldol con-
densation. Cyclization of the b-hydroxy ketone
(29) was effected upon treatment with sodium
methoxide to provide the five-membered pactamycin
core structure (10) in 50% yield over two steps.
Under the basic reaction conditions, the config-
urationally labile C-2 stereocenter was inverted,
and only the correct C-2 isomer was observed in
the product enone 10. This serendipitous event
corrected our initial stereochemical error, simpli-
fying subsequent core manipulation.
With cyclopentenone 10 in hand, three chal-
lenges remained: (i) C-5 methide addition, (ii)
C-4 hydroxylation, and (iii) C-3 aniline installa-
tion. Inspired by a related approach by Hanessian
and co-workers (17, 18), we pursued an epoxidation-
nucleophilic aniline ring-opening sequence for ac-
cess to the trans-anilinoalcohol. We anticipated
that subsequent nucleophilic methylation of the
C-5 ketone would complete the core functional-
ization. As we explored this proposed route, we
discovered the importance of both the order of
Fig. 2. Synthetic plan enabling modular introduction of functionality. (A) Hidden symmetry
recognition within the pactamycin core. Blue dots highlight a five-carbon chain from which to begin the
synthesis. Ac, acetyl. (B) Red dashed arrows illustrate the pivotal Mannich addition and symmetry-
breaking reduction steps. (C) Proposed modular construction of pactamycin. The colored components may
be varied and introduced for analog synthesis and SAR studies.
Fig. 3. Mannich reaction and diastereoselective diketone monoreduction. 7 (20 mol %), dichloromethane (CH2Cl2), –65°C; (b) LTBA, tetrahydrofuran
This two-step sequence installs all pactamycin core carbons, as well as three (THF), –40°C; (c) tert-butyldimethysilyl trifluromethanesulfonate (TBSOTf), 2,6-
contiguous stereocenters. Reagents and conditions are as follows: (a) catalyst lutidine, CH2Cl2, –78°C. Ph, phenyl.
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REPORTS
Fig. 4. Elaboration of 6 to pactamycin (1) via modular incorporation chloro(tert-butyl)diphenylsilane (TBDPSCl), triethylamine (NEt3), dimethyla-
of unprotected functionality. Reagents and conditions are as follows: (a) minopyridine (10 mol %), CH2Cl2, 0°C to rt; (f) methylmagnesium bromide
lithium diisopropylamide, formaldehyde [CH2O(g)], THF, –78° to –45°C; (b) (MeMgBr), THF, 0°C; (g) 3-acetylaniline (17), scandium(III) trifluoromethane-
ozone (O3), CH2Cl2, –78°C, then dimethylsulfide (Me2S), –78°C to room tem- sulfonate [Sc(OTf)3], toluene, 60°C; (h) TBAF, THF, 0°C; (i) potassium car-
perature (rt); (c) sodium methoxide (NaOMe), THF, 0°C; (d) hydrogen peroxide bonate (K2CO3), 18, dimethylacetamide; (j) palladium hydroxide on carbon
(H2O2), sodium hydroxide (NaOH), 7:1 CH2Cl2:methanol (MeOH), 0°C; (e) [Pd(OH)2/C], H2 (1 atm), MeOH.
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References and Notes
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overridden, at least in part, via direction by the
urea functionality, lending additional support to
the strategic decision to incorporate this function-
ality in its native form from the outset. Epoxide
12 is the crucial branch point for analog synthesis
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pass. The epoxide was subjected to a Sc(OTf )3-
promoted (OTf, trifluoromethanesulfonate) nu-
cleophilic ring opening with 3-acetylaniline (17),
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the starting material to install the C-3 aniline de-
rivative. The addition of this anilino functionality
in its desired, unprotected form completed func-
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Acknowledgments: The project described was supported by
award no. R01 GM084927 from the National Institute of General
Medical Sciences. J.T.M. acknowledges an American Chemical
Society Division of Organic Chemistry graduate fellowship.
R.J.S acknowledges an NSF graduate research fellowship. X-ray
crystallography was performed by P. White (University of North
Carolina). The Cambridge Crystallographic Data Centre (CCDC)
contains the supplementary crystallographic data for this paper,
under deposition no. 914582. These data can be obtained free
of charge from via www.ccdc.cam.ac.uk/data_request/cif. A patent
application on the route and compounds presented in this paper
has been filed through the University of North Carolina.
Deprotection of both silyl ethers was accom-
plished upon treatment with tetrabutylammonium
fluoride (TBAF) to provide tractable tetraol 15 in
90% yield, leaving a highly reactive primary al-
cohol for selective acylation. A ketene-mediated
acylation protocol developed by Shen et al. (31)
Supplementary Materials
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Materials and Methods
References (33, 34)
3 January 2013; accepted 11 February 2013
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