A robust platform for the synthesis of new tetracycline antibiotics

Article abstract of DOI:10.1021/ja806629e

Tetracyclines and tetracycline analogues are prepared by a convergent, single-step Michael-Claisen condensation of AB precursor 1 or 2 with D-ring precursors of wide structural variability, followed by removal of protective groups (typically in two steps). A number of procedural variants of the key C-ring-forming reaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursor followed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situ lithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed by warming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robust across a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with a high degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derived from a given D-ring precursor to enones 1 or 2 is quite rapid at -78 °C, while Claisen cyclization of the enolate produced is rate-determining, typically occurring upon warming to 0 °C. The AB plus D coupling strategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable by latter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assays and preliminary data obtained from a murine septicemia model show that many of the novel tetracyclines synthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform for tetracycline synthesis described gives access to a broad range of molecules that would be inaccessible by semisynthetic methods (presently the only means of tetracycline production) and provides a powerful engine for the discovery and, perhaps, development of new tetracycline antibiotics.

Full text of DOI:10.1021/ja806629e

Published on Web 12/03/2008
A Robust Platform for the Synthesis of New Tetracycline
Antibiotics
Cuixiang Sun, Qiu Wang, Jason D. Brubaker, Peter M. Wright, Christian D. Lerner,
Kevin Noson, Mark Charest, Dionicio R. Siegel, Yi-Ming Wang, and
Andrew G. Myers*
Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
Received August 21, 2008; E-mail: myers@chemistry.harvard.edu
Abstract: Tetracyclines and tetracycline analogues are prepared by a convergent, single-step Michael-Claisen
condensation of AB precursor 1 or 2 with D-ring precursors of wide structural variability, followed by removal
of protective groups (typically in two steps). A number of procedural variants of the key C-ring-forming
reaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursor
followed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situ
lithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed by
warming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robust
across a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with a
high degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derived
from a given D-ring precursor to enones 1 or 2 is quite rapid at -78 °C, while Claisen cyclization of the
enolate produced is rate-determining, typically occurring upon warming to 0 °C. The AB plus D coupling
strategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable by
latter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assays
and preliminary data obtained from a murine septicemia model show that many of the novel tetracyclines
synthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform for
tetracycline synthesis described gives access to a broad range of molecules that would be inaccessible by
semisynthetic methods (presently the only means of tetracycline production) and provides a powerful engine
for the discovery and, perhaps, development of new tetracycline antibiotics.
Introduction
widely variant in the left-hand or D-ring portion of the
molecule.2 Here, we expand upon our original findings, describ-
In prior research we showed that cyclohexenones 1 and 2
can be transformed into 6-deoxytetracycline antibiotics using a
sequence of as few as three chemical steps.1
ing the synthesis of more than 50 different tetracyclines and
tetracycline analogues, many of which are active in inhibiting
the growth of cultured Gram-positive and Gram-negative
bacteria, including tetracycline-resistant strains. We provide
detailed protocols for the key cyclization reaction in its various
forms and discuss features of stereochemistry, chemical ef-
ficiency, and mechanism. We also report minimum inhibitory
concentration values for selected analogues in a number of
different bacterial strains, as well as preliminary in vivo data
obtained in a mouse septicemia model using a tetracycline-
sensitive strain of Staphylococcus aureus.
The first and key step of the sequence forms the C ring of
the tetracyclines by a Michael-Claisen cyclization reaction, a
potentially general means for constructing tetracycline analogues
Background
The first tetracycline antibiotic was discovered in 1948, when
Benjamin Duggar of Lederle Laboratories isolated the natural
product chlorotetracycline (Aureomycin, 3) from the culture
broth of a novel species of Streptomyces.3 Within two years, a
research team from Chas. Pfizer and Co. had isolated a second
natural tetracycline, oxytetracycline (Terramycin, 4),4 and in
(1) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers,
A. G. Science 2005, 308, 395–398.
(2) As pointed out by a reviewer, the second step of the key C-ring-forming
reaction sequence is more properly designated as an internal Claisen
reaction, rather than a Dieckmann reaction, as we previously described
it; for discussion, see: (a) Hauser, C. R.; Swamer, F. W.; Adams, J. T.
Org. React. (N.Y.) 1954, 8, 59. (b) Schaefer, J. P.; Bloomfield, J. J.
Org. React. (N.Y.) 1967, 15, 1. For brevity, this sequence is referred
to here as a Michael-Claisen cyclization.
(3) (a) Duggar, B. M Ann. N.Y. Acad. Sci. 1948, 51, 177–181. (b) Duggar,
B. M. Aureomycin and Preparation of Same. U.S. Patent 2,482,055,
Sept 13, 1949.
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A R T I C L E S
Sun et al.
raising concern among public health officials, particularly with
the emergence of antibiotic-resistant bacterial strains in com-
munity settings. While careful management of the use of existing
antibiotics in society is warranted, it would seem unwise to
abandon the search for new agents, given the diversity of
bacteria and their capacity to evolve rapidly. Attempts to develop
antibacterial agents with novel targets have met with little
success.13 As a consequence, many research programs seeking
to discover new antibiotics have been refocused toward the
modification of agents in proven classes, such as the tetracy-
clines, with an emphasis on overcoming bacterial resistance.
While the innovations of chemists seeking to modify the
structure of naturally occurring tetracyclines have been extraor-
dinary, the slowing pace of discovery in this area is evident.
From the time that the structures of the tetracycline antibiotics
were first revealed by Woodward and collaborators,14 many
laboratories have sought to develop a practical route for their
synthesis. In 2003, an expert opinion of the patent literature
summarized the state of the art, concluding, “the original effort
of Woodward has survived as the basic strategy for the total
synthesis of this series and at greater than 25 steps is clearly
not to be considered as practical. ... we believe there is ample
justification to explore new total synthetic and convergent
synthetic routes that take full advantage of the body of chemistry
that has become available since Woodward’s original effort.” 15
Among the many developments since Woodward and co-
workers first synthesized sancycline (6-deoxy-6-demethyltetra-
1953, tetracycline itself (5) was prepared from chlorotetracycline
by catalytic hydrogenolysis of the carbon-chlorine bond, a
transformation discovered by Lloyd Conover of Pfizer.5 Sub-
sequently, tetracycline was found to be a natural product,6 and
later still, Lederle researchers isolated 6-demethyltetracyclines
(see structure 6) from culture broths of a mutant strain of
Streptomyces.7 Conover has provided detailed and insightful
accounts of research efforts leading to new tetracyclines
specifically and antibiotics more generally.8 All tetracyclines
approved for human or veterinary use are fermentation products
or are derived from fermentation products by semisynthesis.
This is also true of most ꢀ-lactam and all macrolide antibiotics.
Tracing the paths of human efforts to produce new antibiotics
from natural products not accessible by synthesis reveals an
evolutionary process marked by specific, impactful discoveries.
In the case of the tetracyclines, Pfizer scientists achieved a major
enabling advance approximately 10 years after the class had
been identified when they demonstrated that the C6-hydroxyl
group of the natural products oxytetracycline (4), tetracycline
(5), and 6-demethyltetracycline (6) could be removed reduc-
tively.9 The 6-deoxytetracyclines produced, including 6-deox-
ytetracycline itself (7), were found to be more stable than the
parent compounds, yet they retained broad-spectrum antibacte-
rial activity. The important and now generic antibiotics doxy-
cycline (8; Pfizer, 1967) and minocycline (9; Lederle, 1972)
followed as a consequence, the latter arising from the additional
discovery that electrophilic aromatic substitution at C7 becomes
possible when the more stable 6-deoxytetracyclines are used
as substrates.10 Decades later, a team of Wyeth scientists led
by Frank Tally synthesized 7,9-disubstituted tetracycline deriva-
tives, leading to the discovery of the antibiotic tigecycline
(Tigacyl, 10; U.S. approval 2005).11
(4) (a) Finlay, A. C.; Hobby, G. L.; P’an, S. Y.; Regna, P. P.; Routien,
J. B.; Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.;
Vinson, J. W.; Kane, J. H. Science 1950, 111, 85. (b) Sobin, B. A.;
Finlay, A. C. Terramycin and its Production. U.S. Patent 2,516,080,
July 18, 1950.
(5) (a) Booth, J. H.; Morton, J.; Petisi, J. P.; Wilkinson, R. G.; Williams,
J. H. J. Am. Chem. Soc. 1953, 75, 4621. (b) Conover, L. H.; Moreland,
W. T.; English, A. R.; Stephens, C. R.; Pilgrim, F. J. J. Am. Chem.
Soc. 1953, 75, 4622–4623.
(6) Minieri, P. P.; Sokol, H.; Firman, M. C. Process for the Preparation
of Tetracycline and Chlorotetracycline. U.S. Patent 2,734,018, Feb 7,
1956.
(7) McCormick, J. R. D.; Sjolander, N. O.; Hirsch, U.; Jensen, E. R.;
Doerschuk, A. P. J. Am. Chem. Soc. 1957, 79, 4561–4563.
(8) (a) Conover, L. H. Res. Technol. Management 1984, 17–22. (b)
Conover, L. H. ACS AdV. Chem. Ser. 1971, 108, 33–80.
(9) (a) Stephens, C. R.; Murai, K.; Rennhard, H. H.; Conover, L. H.;
Brunings, K. J. J. Am. Chem. Soc. 1958, 80, 5324–5325. (b)
McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P.
J. Am. Chem. Soc. 1960, 82, 3381–3386. (c) Stephens, C. R.;
Beereboom, J. J.; Rennhard, H. H.; Gordon, P. N.; Murai, K.;
Blackwood, R. K.; Schach von Wittenau, M. J. Am. Chem. Soc. 1963,
85, 2643–2652. (d) Blackwood, R. K.; Stephens, C. R. J. Am. Chem.
Soc. 1962, 84, 4157–4159.
(10) (a) Martell, M. J.; Boothe, J. H. J. Med. Chem. 1967, 10, 44–46. (b)
Church, R. F. R.; Schaue, R. E.; Weiss, M. J. J. Org. Chem. 1971,
36, 723–725. (c) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski,
H. M.; Boothe, J. H. J. Med. Chem. 1963, 6, 405–407. (d) Zambrano,
R. T. U.S. Patent 3,483,251, Dec 9, 1969.
(11) (a) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.;
Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–
188. (b) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9,
1459–1462.
(12) Listing of Newly Approved Drug Therapies (2008)http://www.
centerwatch.com/patient/drugs/druglist.html, 2008 (accessed August
2008).
(13) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat.
ReV. Drug DiscoVery 2007, 6, 29–40.
(14) (a) Hochstein, F. A.; Stephens, C. R.; Conover, L. H.; Regna, P. P.;
Pasternack, R.; Gordon, P. N.; Pilgrim, F. J.; Brunings, K. J.;
Woodward, R. B. J. Am. Chem. Soc. 1953, 75, 5455–5475. (b)
Stephens, C. R.; Conover, L. H.; Hochstein, F. A.; Regna, P. P.;
Pilgrim, F. J.; Brunings, K. J. J. Am. Chem. Soc. 1952, 74, 4976–
4977.
Tigacyl is one of only three new antibiotics to be brought to
market in the United States in the past three years, and the only
broad-spectrum agent.12 Diminishing economic incentives and
increasing regulatory hurdles have led many pharmaceutical
companies to discontinue efforts to develop new antibiotics,
(15) Podlogar, B. L.; Ohemeng, K. A.; Barrett, J. F. Expert Opin. Ther.
Patents 2003, 13, 467–478.
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
cycline) in 1962,16 one of particular note, and of relevance to
the results described herein, is Stork and Hagedorn’s strategy
for protection of the vinylogous carbamic acid group of the A
ring of the tetracyclines as a 3-benzyloxyisoxazole group;
subsequent deprotection occurs under mild (hydrogenolytic)
conditions.17
research groups first reported that simple o-toluate ester anions
(unsubstituted at the benzylic position) undergo Michael-Claisen
cyclization reactions with ꢀ-methoxycyclohexenones and γ-py-
rones to form naphthyl ketones (see eq 4 for one example), a
sequence sometimes referred to as the Staunton-Weinreb
annulation.25
Strategically, the original route developed by Woodward and
collaborators for the synthesis of sancycline employed a “left-
to-right” or DfA mode of construction. The Shemyakin and
Muxfeldt research groups adopted a similar directionality in their
remarkable syntheses of tetracycline (5; 1967) and terramycin
(4; 1968), respectively, using a bicyclic CD-ring precursor as
starting material.18,19 With the benefit of more than 50 years of
structure-activity relationship data, as well as X-ray crystal
structures of tetracycline bound to the bacterial ribosome (its
putative target),20 the left-to-right mode of construction used
in these pioneering synthetic efforts can be seen to present a
practical disadvantage to the discovery of new tetracycline
antibiotics, for the D ring has emerged as one of the most
promising sites for structural variation. This was a primary
consideration guiding our initial retrosynthetic analysis of the
tetracycline class, leading us to focus upon disconnection of
the C ring. Thus, we envisioned assembling tetracyclines by a
convergent coupling of D- and AB-ring precursors. Although
model studies suggested that both Diels-Alder and Michael-
Claisen cyclization reactions might be used to form the C ring,21
only the latter proved successful with the AB precursors that
we later targeted and synthesized (1 and 2).
Michael-Claisen and Michael-Dieckmann reaction se-
quences have been widely employed to construct naphthalene
derivatives since 1978, when three different cyclization protocols
were introduced by independent research groups. Hauser and
Rhee used a sulfoxide-stabilized o-toluate ester anion as the
nucleophilic component in a Michael-Dieckmann cyclization
reaction with methyl crotonate (eq 1). In this case, aromatization
occurred upon thermal elimination of phenylsulfenic acid.22 The
use of phthalide and cyanophthalide anions as nucleophilic
components was described by Broom and Sammes (eq 2)23 and
Kraus and Sugimoto (eq 3),24 respectively. Formal loss of water
and hydrogen cyanide, respectively, led to naphthoate ester
products in these procedures. In 1979, the Weinreb and Staunton
There is also precedent for the formation of non-aromatic
six-membered rings by Michael-Claisen and Michael-
Dieckmann reaction sequences.23,26,27 With few exceptions,27
stereochemical features of these cyclization reactions have not
been discussed, frequently because they were of little conse-
quence (aromatization followed cyclization). The Michael-Claisen
cyclizations detailed below are unusual in their stereochemical
complexity, stereocontrol, and efficiency. In 2000, while our
studies were in progress, Tatsuta and co-workers reported a
synthesis of (-)-tetracycline (34 steps, 0.002% yield) that
employed an early stage Michael-Claisen cyclization reaction
to form an aromatic C-ring precursor, which was dearomatized
later in the sequence.28 Since 2005, our laboratory has reported
two different routes to synthesize the AB precursor 1 in optically
active form; the more recent of these was scaled to prepare >40
g of crystalline product in one batch.29 Here we provide details
of the different protocols that can be used to construct the C
(16) (a) Conover, L. H.; Butler, K.; Johnston, J. D.; Korst, J. J.; Woodward,
R. B. J. Am. Chem. Soc. 1962, 84, 3222–3224. (b) Woodward, R. B.
Pure Appl. Chem. 1963, 6, 561–573. (c) Korst, J. J.; Johnston, J. D.;
Butler, K.; Bianco, E. J.; Conover, L. H.; Woodward, R. B. J. Am.
Chem. Soc. 1968, 90, 439–457.
(17) Stork, G.; Hagedorn, A. A., III. J. Am. Chem. Soc. 1978, 100, 3609–
3611.
(18) (a) Gurevich, A. I.; Karapetyan, M. G.; Kolosov, M. N.; Korobko,
V. G.; Onoprienko, V. V.; Popravko, S. A.; Shemyakin, M. M.
Tetrahedron Lett. 1967, 8, 131–134. (b) Kolosov, M. N.; Popravko,
S. A.; Shemyakin, M. M. Liebigs Ann. 1963, 668, 86–91.
(19) (a) Muxfeldt, H.; Hardtmann, G.; Kathawala, F.; Vedejs, E.; Mooberry,
J. B. J. Am. Chem. Soc. 1968, 90, 6534–6536. (b) Muxfeldt, H.; Haas,
G.; Hardtmann, G.; Kathawala, F.; Mooberry, J. B.; Vedejs, E. J. Am.
Chem. Soc. 1979, 101, 689–701.
(25) (a) Dodd, J. H.; Weinreb, S. M. Tetrahedron Lett. 1979, 38, 3593–
3596. (b) Dodd, J. H.; Starrett, J. E.; Weinreb, S. M. J. Am. Chem.
Soc. 1984, 106, 1811–1812. (c) Leeper, F. J.; Staunton, J. J. Chem.
Soc., Chem. Commun. 1979, 5, 206–207. (d) Leeper, F. J.; Staunton,
J. J. Chem. Soc., Perkin Trans. 1 1984, 1053–1059.
(26) (a) Tarnchompoo, B.; Thebtaranonth, C.; Thebtaranonth, Y. Synthesis
1986, 9, 785–786. (b) Boger, D. L.; Zhang, M. J. Org. Chem. 1992,
57, 3974–3977. (c) Nishizuka, T.; Hirosawa, S.; Kondo, S.; Ikeda,
D.; Takeuchi, T. J. Antibiot. 1997, 50, 755–764. (d) Hill, B.; Rodrigo,
R. Org. Lett. 2005, 7, 5223–5225.
(20) (a) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-
Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103,
1143–1154. (b) Pioletti, M.; Schlu¨nzen, F.; Harms, J.; Zarivach, R.;
Glu¨hmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.;
Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F EMBO J. 2001, 20,
1829–1839.
(27) For examples of stereocontrol in Michael-Claisen and Michael-
Dieckmann cyclization reactions, see: (a) Franck, R. W.; Bhat, V.;
Subramaniam, C. S. J. Am. Chem. Soc. 1986, 108, 2455–2457. (b)
Tatsuta, K.; Yamazaki, T.; Mase, T.; Yoshimoto, T. Tetrahedron Lett.
1998, 39, 1771–1772. (c) White, J. D.; Demnitz, F. W. J.; Qing, X.;
Martin, W. H. C. Org. Lett. 2008, 10, 2833–2836.
(21) Parrish, C. A. Ph.D. Thesis, California Institute of Technology,
Pasadena, CA, 1999.
(22) Hauser, F. M.; Rhee, R. P. J. Org. Chem. 1978, 43, 178–180.
(23) Broom, N. J. P.; Sammes, P. G. J. Chem. Soc., Chem. Commun. 1978,
162–164.
(28) Tatsuta, K.; Yoshimoto, T.; Gunji, H.; Okado, Y.; Takahashi, M. Chem.
Lett. 2000, 646–647.
(24) Kraus, G. A.; Sugimoto, H. Tetrahedron Lett. 1978, 26, 2263–2266.
(29) Brubaker, J. D.; Myers, A. G. Org. Lett. 2007, 9, 3523–3525.
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A R T I C L E S
Sun et al.
ring of the tetracyclines and exemplify these protocols with the
preparation of a number of novel substances with antibacterial
properties.
to the enone) and/or steric effects (addition of the nucleophile
to the π-face opposite the tert-butyldimethylsilyloxy substituent,
which is also axial; see the space-filling model of enone 1
depicted in Figure 1).
Directed by this key finding, we proceeded to strategically
modify the D-ring precursor, with three objectives: (1) to activate
the ester toward Claisen cyclization (which we did not observe
with the methyl ester substrate 11), (2) to obviate the use of
organotin intermediates, and (3) to mask the C10 phenoxy
substituent with a protective group more labile than methyl.
These objectives were achieved using the tert-butoxycarbonyl-
protected phenyl ester substrate 13 (Scheme 2).31,32
Results
A critical early experiment in our attempts to assemble the
C ring of the tetracyclines by a Michael-Claisen cyclization
reaction provided both direction and mechanistic insight. As
depicted in Scheme 1, treatment of a solution of organostannane
1130 in tetrahydrofuran (THF) with n-butyllithium at -78 °C,
followed by transfer of this mixture to a solution of enone 1,
also at -78 °C, and subsequent quenching with tert-butyl-
dimethylsilyl triflate (TBSOTf), afforded the Michael addition
product 12 as a single stereoisomer in 98% yield.
Scheme 2. Synthesis of 6-Deoxytetracycline (7) by
Michael-Claisen Cyclization Using the
tert-Butoxycarbonyl-Protected Phenyl Ester 13 as the D-Ring
Precursor and a Stepwise Protocol for Addition of the Base and
Enone 1, Followed by Deprotection
Scheme 1. Michael Addition of a Benzylic Anion (Generated by
Tin-Lithium Exchange) to Enone 1, Followed by Trapping of the
Resultant Enolate with tert-Butyldimethylsilyl Triflate
Crystallization of the chromatographically purified product
from hexanes at -30 °C provided a single crystal suitable for
X-ray analysis (mp 146 °C). Inspection of the crystal structure
(Figure 1) revealed that the stereochemistry of positions “C5a”
and “C6” of the adduct 12 corresponded to that of 6-deoxytet-
racycline (7) and doxycycline (8), which was fortuitous given
that as many as four diastereomers could have been formed in
the Michael addition reaction. The product that is formed
apparently arises from selective addition of the nucleophile to
the concave face of the enone. This selectivity may arise as a
consequence of stereoelectronic factors (pseudoaxial addition
Deprotonation of 13 (3 equiv) with lithium diisopropylamide
(LDA, 4 equiv) at -78 °C in the presence of N,N,N′,N′-
tetramethylethylenediamine (TMEDA, 4 equiv) afforded a deep
red solution of the corresponding o-toluate ester anion; addition
of a solution of the enone 1 (1 equiv) and slow warming of the
resulting mixture to 0 °C over 3 h provided the Michael-Claisen
cyclization product 14 in 81% yield in diastereomerically pure
form after isolation by reverse-phase high-performance liquid
chromatography (rp-HPLC). A minor diastereomer (<4%),
believed to be epimeric at C6, was isolated separately.
Thus, Michael addition occurs with >20:1 stereoselectivity
at C6, in the sense indicated in Scheme 2, and appears to proceed
with complete stereocontrol at C5a (attack upon a single
diastereoface of the enone). These observations have held
consistently in more than 40 different C-ring-forming cyclization
reactions examined to date, with two (closely related) exceptions,
discussed below.
There is little question that the transformation of enone 1 to
the 6-deoxytetracycline precursor 14 proceeds by a stepwise
mechanism, involving sequential Michael and Claisen reactions,
for the intermediate Michael adduct can be intercepted by
protonation with acetic acid at -78 °C to give the keto ester
16 in 88% yield (eq 5).33
Figure 1. Crystal structures of the Michael addition product 12 and the
enone 1, presented as ball and stick and space-filling models, respec-
tively.
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
Scheme 4. Synthesis of 6-(S)-Phenylsancycline (21) by
Indeed, we have observed across a range of different D-ring
precursors that Michael addition is relatively rapid at -78 °C,
while Claisen cyclization proceeds more slowly, typically upon
warming to 0 °C. Thus, we view Claisen cyclization and not
Michael addition as rate-determining. It is evident from these
observations that C-ring formation cannot occur by Diels-Alder
cycloaddition of an o-quinonemethide intermediate, which is
hypothetically a mechanistic alternative to Michael-Claisen
cyclization.
Michael-Claisen Cyclization Using the
tert-Butoxycarbonyl-Protected Phenyl Ester 19 as the D-Ring
Precursor and a Stepwise Protocol for Addition of the Base and
Enone 1, Followed by Deprotection
With the establishment of an effective protocol for construc-
tion of the C ring, a two-step deprotection sequence was
employed to transform the cyclization product 14 into 6-deox-
ytetracycline (Scheme 2). The tert-butoxycarbonyl and tert-
butyldimethylsilyl protective groups were removed upon treat-
ment of 14 with hydrofluoric acid in acetonitrile at 23 °C (2
days). Hydrogenolysis of the crude reaction product (15) in the
presence of a palladium catalyst under an atmosphere of
hydrogen in methanol-dioxane at 23 °C and subsequent
purification by rp-HPLC then afforded 6-deoxytetracycline (7)
in 85% yield.17 As the examples below will demonstrate, this
two-step deprotection protocol has been found to be widely
applicable, though in some instances it is advantageous to invert
the ordering of the two steps. In general, the tert-butoxycarbonyl
group is cleaved relatively rapidly in the deprotection step
employing hydrofluoric acid, while the tertiary tert-butyldim-
ethylsilyl ether undergoes protodesilylation more slowly.
4),32 followed by warming of the resulting mixture to -15 °C,
provided the Michael-Claisen cyclization product 20 in 97%
yield after purification by rp-HPLC. Standard deprotection of
20 then afforded the novel tetracycline analogue 6-(S)-phenyl-
sancycline (21), which was found to effectively inhibit the
growth of a number of Gram-positive bacteria, including
tetracycline-resistant strains (vide infra), prompting the synthesis
of a series of 6-aryl-substituted tetracyclines (see Table 1).35 It
is worthy of note that more than half of the cyclization reactions
presented in the tables below were attempted only once.
Other notable features of the cyclization reactions of D-ring
precursors with benzylic anion-stabilizing groups include the
fact that additives such as TMEDA were typically not required
and that stoichiometries of just over 1 equiv of a given D-ring
precursor frequently sufficed to achieve a high yield of cycliza-
tion product. We also found that benzylic deprotonation could
be conducted with the weaker base lithium bis(trimethylsilyl)-
amide (LHMDS) in lieu of the standard base LDA, and, using
N-imidazoyl substrate 22 (Scheme 5), the important observation
was made that condensation could be achieved with high
efficiency by an in situ deprotonation protocol, which is to say
by addition of base to the D-ring precursor in the presence of
Scheme 3. Synthesis of Minocycline (9) by Michael-Claisen
Cyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester
17 as the D-Ring Precursor and a Stepwise Protocol for Addition
of the Base and Enone 1, Followed by Deprotection
(30) Carpenter, T. A.; Evans, G. E.; Leeper, F. J.; Staunton, J.; Wilkinson,
M. R. J. Chem. Soc., Perkin Trans. I 1984, 1043–1051.
The conditions developed for the cyclization reaction depicted
in Scheme 2, sequential deprotonation of a D-ring precursor
followed by addition of the enone 1, have been effective for
the synthesis of a number of known tetracyclines as well as
novel tetracycline analogues variant within or near the D ring.
For example, treatment of phenyl ester 1734 (3 equiv, Scheme
3) with LDA (3 equiv) in the presence of TMEDA (6 equiv) at
-78 °C, followed by addition of enone 1 (1 equiv) and warming
of the resulting mixture to -10 °C, provided the Michael-Claisen
cyclization product 18 in 83% yield after purification by rp-
HPLC. Two-step deprotection and purification by rp-HPLC then
afforded minocycline (9) in 74% yield.
Among the modified D-ring precursors we have investigated,
substrates with benzylic anion-stabilizing groups were found
to undergo particularly efficient cyclization reactions. For
example, addition of enone 1 (1 equiv) to a solution of the
stabilized anion derived from substrate 19 (3 equiv, Scheme
(31) Phenyl ester activation in toluate ester condensations is precedented:
(a) White, J. D.; Nolen, E. G., Jr.; Miller, C. H. J. Org. Chem. 1986,
51, 1150–1152 (see also ref 27c).
(32) Bennetau, B.; Mortier, J.; Moyroud, J.; Guesnet, J.-L. J. Chem. Soc.,
Perkin Trans. 1 1995, 1265–1271.
(33) For other examples of the isolation of uncyclized Michael adducts,
see refs 23, 25c, 25d, and 26b.
(34) Phenyl ester 17 was synthesized in six steps from ethyl 6-methylsali-
cylate; see: (a) Olah, G. A.; Malhotra, R.; Narang, S. C. Nitration,
Methods and Mechanisms; VCH Publishers Inc.: New York, 1980.
(b) Bellamy, F. D.; Ou, K. Tetrahedron Lett. 1984, 26, 839–842. (c)
Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.;
Shah, R. D. J. Org. Chem. 1996, 61, 3849–3862.
(35) Leading references for the synthesis of D-ring precursors to 6-aryl-
substituted tetracyclines: (a) Miyaura, N.; Suzuki, A Chem. ReV. 1995,
95, 2457–2483. (b) Kotha, S.; Mandal, K. Eur. J. Org. Chem. 2006,
23, 5387–5393. (c) Wright, S. W.; Hageman, D. L.; McClure, L. D.
J. Org. Chem. 1994, 59, 6095–6097. (d) Osyanin, V. A. ; Purygin,
P. P.; Belousova, Z. P. Russ. J. Gen. Chem. 2005, 75, 111–117. (e)
Appukkuttan, P.; Dehaen, W.; Fokin, V. V.; Van der Eycken, E. Org.
Lett. 2004, 6, 4223–4225.
9
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A R T I C L E S
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Table 1. Synthesis of 6-Substituted Tetracyclines by Michael-Claisen Cyclization Using D-Ring Precursors with Anion-Stabilizing
Substituents in the Benzylic Position and a Stepwise Protocol for Addition of the Base and Enone 1, Followed by Deprotection [(i) HF (aq),
CH3CN; (ii) H2, Pd/C (or Pd black), CH3OH-dioxane]
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
Scheme 5. Synthesis of 6-(R)-N-Imidazoylsancycline (24) by
Michael-Claisen Cyclization Using the
tert-Butoxycarbonyl-Protected Phenyl Ester 22 and Enone 1 as
Substrates, with Deprotonation in Situ, Followed by Deprotection
reactions we have studied (these providing the two exceptions
referred to above), which we attribute to epimerization at C6
after cyclization. We have not yet conducted the rigorous studies
necessary to establish if the product ratios were kinetically or
thermodynamically determined in these examples.
The in situ deprotonation protocol has proven to be effective
for the Michael-Claisen cyclization reactions of a number of
different D-ring precursors containing benzylic anion-stabilizing
substituents (see Table 2). Because of its greater experimental
convenience, this has become the preferred method for the
synthesis of tetracycline analogues substituted at the C6-position.
Scheme 7. Synthesis of 7-Aza-10-deoxysancycline (30) by
Michael-Claisen Cyclization Using Phenyl Ester 28 and Enone 1
as Substrates, with Deprotonation in Situ, Followed by
Deprotection
Scheme 6. Synthesis of 6-(S)-Carbomethoxysancycline (26) by
Michael-Claisen Cyclization Using the Methyl Phenyl Diester 25
and Enone 1 as Substrates, with Deprotonation in Situ, Followed
by Deprotectiona
D-ring heterocyclic analogues of tetracyclines had not been
made before, so far as we are aware, and their construction by
semisynthesis cannot be easily imagined. In targeting analogues
of this type, we initially chose to explore the synthesis of the
D-ring pyridine derivative 7-aza-10-deoxysancycline (30, Scheme
7). As we previously reported, in situ deprotonation of phenyl
ester 28 (4 equiv) with LDA (5 equiv) at -95 °C in the presence
of enone 1 (1 equiv) and hexamethylphosphoramide (HMPA,
10 equiv), followed by warming to -50 °C, afforded the
Michael-Claisen cyclization product 29 in 76% yield (Scheme
7).1 Claisen ring-closure was notably more facile in this example
than in others we have studied, proceeding to completion in
less than 1 h upon warming to -50 °C. 7-Aza-10-deoxysan-
cycline (30) was then obtained in 79% yield after removal of
protective groups.
Another novel class of tetracyclines that we have explored
is the pentacyclines (Scheme 8). This required that we develop
a In one experiment conducted with the corresponding benzyl phenyl
diester 27 as the D-ring precursor, a diastereomeric mixture of cyclization
products was obtained.
Scheme 8. Synthesis of the Pentacycline 33 by Michael-Claisen
Cyclization Using the Phenyl Bromomethylnaphthoic Acid Ester 31
and Enone 1 as Substrates, and an in Situ Protocol for
Lithium-Halogen Exchange, Followed by Deprotection
enone 1. Thus, treatment of a mixture of phenyl ester 22 (1.3
equiv) and enone 1 (1 equiv) with an excess of LHMDS (3.3
equiv) at -78 °C, followed by warming of the resulting mixture
to -30 °C over 90 min, provided the cyclization product 23 in
94% yield (Scheme 5); removal of protective groups then
provided 6-(R)-N-imidazoylsancycline (24, 42% yield over two
steps).
In situ deprotonation with LHMDS was also effective in
bringing about cyclization of the methyl phenyl diester substrate
25 with enone 1, as well as cyclization of the corresponding
benzyl phenyl diester substrate 27, as depicted in Scheme 6. In
both cases, the major product of cyclization was epimeric at
C6 relative to the major products of all other cyclization
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A R T I C L E S
Sun et al.
Table 2. Synthesis of 6-Substituted Tetracyclines by Michael-Claisen Cyclization Using D-Ring Precursors with Anion-Stabilizing
Substituents in the Benzylic Position and Enone 1 as Substrates, with Deprotonation in Situ, Followed by Deprotection [(i) HF (aq), CH3CN;
(ii) H2, Pd/C, CH3OH-dioxane]37
chemistry suitable for Michael-Claisen cyclization of naph-
thoate ester DE-ring precursors,22-24 as the protocols that we
had used to this point were found to be largely ineffective with
these substrates.
Lithium-halogen exchange is rarely employed to form
benzyllithium reagents in organic synthesis, due to the propen-
sity of the benzyl halide substrates to engage in Wurtz-type
coupling reactions in the presence of an alkyllithium reagent.36
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
Scheme 9. Synthesis of a Pyrazole Analogue (36) by
Michael-Claisen Cyclization Using the Benzylic Bromide 34 and
Enone 1 as Substrates, and an in Situ Protocol for
The in situ lithium-halogen exchange protocol developed
for the synthesis of the pentacycline 33 has proven to be
generally effective for the synthesis of a number of quite
different tetracycline analogues. In many cases we have adopted
the procedural modification of substituting the less reactive
reagent phenyllithium for n-butyllithium in the lithium-halogen
exchange reaction. For example, addition of phenyllithium to a
solution of pyrazole 34 (3 equiv) and enone 1 (1 equiv)
containing HMPA (6 equiv) at -90 °C, followed by warming
to 0 °C over 2 h, provided the Michael-Claisen cyclization
product 35 in 81% yield (Scheme 9). Removal of the protective
groups led to concomitant cleavage of the heteroaryl carbon-
chlorine bond during hydrogenolysis, affording the tetracycline
analogue 36 containing a D-ring pyrazole (87% yield over two
steps).
Lithium-Halogen Exchange, Followed by Deprotection
A very similar procedure produced 8-fluorosancycline (38)
from the benzyl bromide 37 (Scheme 10). Like the D-ring
pyrazole analogue 36, the 8-fluorotetracycline analogue 38
Scheme 10. Synthesis of 8-Fluorosancycline (38) by
Michael-Claisen Cyclization Using Benzylic Bromide 37 and
Enone 1 as Substrates, and an in Situ Protocol for
Lithium-Halogen Exchange, Followed by Deprotection
Scheme 12. Synthesis of Pentacyclines with Heterocyclic E Rings
by Michael-Claisen Cyclizations Using Bromomethylquinolines
and Enone 1 as Substrates, and an in Situ Protocol for
Lithium-Halogen Exchange, Followed by Deprotection
Scheme 11. Synthesis of 10-Deoxysancycline (41) by
Michael-Claisen Cyclization Using Phenyl
2-(Bromomethyl)benzoate (39) and Enone 1 as Substrates, with
Deprotonation in Situ, Followed by Deprotection
In a surprising transformation, treatment of a solution of phenyl
bromomethylnaphthoic acid ester 31 (4 equiv) and enone 1 (1
equiv) with n-butyllithium (4 equiv) at -100 °C, followed by
warming to 0 °C, provided the Michael-Claisen cyclization
product 32 in 75% yield (Scheme 8).1 A three-step deprotection
sequence then afforded pentacycline 33 in 74% yield.
(36) (a) Parham, W. E.; Jones, L. D.; Sayed, Y. A. J. Org. Chem. 1978,
41, 1184–1186. (b) Berk, S. C.; Yeh, M. C. P.; Jeong, N.; Knochel,
P. Organometallics 1990, 9, 3053–3064.
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A R T I C L E S
Sun et al.
Scheme 13. Synthesis of 6-Aryl-Substituted Heterocyclic
Pentacyclines by Michael-Claisen Cyclizations Using Benzylically
Substituted Quinolines as DE-Ring Precursors and a Stepwise
Protocol for Addition of the Base and Enone 1, Followed by
Deprotection
0 °C over 30 min, provided the Michael-Claisen cyclization
product 40 in 81% yield.1,38 The typical two-step deprotection
sequence then transformed the cyclized product 40 into 10-
deoxysancycline (41) in 84% yield.
Bromomethylquinoline derivatives39 were also employed as
DE-ring precursors in Michael-Claisen cyclization reactions
with enone 1, providing pentacycline precursors with hetero-
cyclic E rings (Scheme 12). The typical two-step deprotection
sequence afforded tetrahydroquinoline products in these ex-
amples, while the use of modified conditions led to deprotection
without reduction of the pyridine E-ring. Cyclization reactions
of quinoline substrates with 6-aryl substituents were also
investigated,35b using stepwise deprotonation conditions (Scheme
13).
Thus far, each different cyclization reaction described has
produced a single tetracycline analogue. It is worth noting
explicitly that the convergent coupling strategy employed to
prepare individual tetracycline analogues can also be used to
target structures that serve as branch points to large numbers
of analogues, versatile structures such as the aryl bromide 43
or the aldehyde 44 (Scheme 14). Both of these pentacycline
precursors were targeted for synthesis.
Scheme 14. Synthesis of the Diversifiable Pentacycline Precursors
43 and 44 by Michael-Claisen Cyclization Using the Benzylic
Bromide 42 and Enone 1 as Substrates, with an in Situ Protocol
for Selective Lithium-Halogen Exchangea
a Further transformation of 44 by a reductive amination reaction provides
a route to alkylaminomethylpentacyclines, as illustrated by the synthesis
of the tert-butylaminomethylpentacycline 45.
Michael-Claisen cyclization of the benzylic bromide 4240
withenone1wassuccessfullyachievedbyinsitulithium-halogen
exchange using phenyllithium, but not n-butyllithium. Thus,
treatment of a mixture of the bis-bromide 42 (3 equiv) and
enone 1 (1 equiv) with phenyllithium (3 equiv) at -100 °C,
would have been difficult, if not impossible, to prepare by
semisynthetic methods.
Lithium-halogen exchange was also employed for the
synthesis of 10-deoxysancycline (41, Scheme 11). As we
reported previously, treatment of a solution of phenyl 2-(bro-
momethyl)benzoate (39, 4 equiv) and enone 1 (1 equiv) with
n-butyllithium (4 equiv) at -100 °C, followed by warming to
(38) Both the stepwise and in situ protocols for deprotonation-cyclization
were also effective for the synthesis of the Michael-Claisen cyclization
product 40 from phenyl o-toluate and enone 1, though yields were
lower.
(39) Van Leusen, A. M.; Terpstra, J. W. Tetrahedron Lett. 1981, 22, 5097–
5100.
(37) The use of a methyl ether protecting group in the penultimate entry
of Table 2 required one additional deprotection step (BBr3, CH2Cl2,-
78 °C f 23 °C) following the typical two-step sequence.
(40) Synthesized from 6-bromophthalide and methyl crotonate: Broom,
N. J. P.; Sammes, P. G. J. Chem. Soc., Perkin Trans. 1 1981, 465–
470.
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
Chart 1. Alkylaminomethylpentacyclines Synthesized from
Chart 2. 7-Dimethylamino-alkylaminomethylpentacyclines
Aldehyde 44 by Reductive Amination Followed by Deprotectiona
Synthesized from Aldehyde 48 by Reductive Amination Followed
by Deprotection43
a See Supporting Information for experimental details.42
Scheme 15. Synthesis of the Diversifiable Pentacycline Precursors
47 and 48 by Michael-Claisen Cyclization Using the Phenyl
Naphthoate Ester 46 and Enone 1 as Substrates, with
Deprotonation in Situa
of product in this cyclization reaction was moderate, it
remained consistent during scale-up and allowed for sufficient
quantities of 43 to be produced to explore late-stage
diversification. The aryl bromide intermediate 43 was treated
sequentially with phenyllithium and n-butyllithium to effect
deprotonation and lithium-halogen exchange, respectively,
and the resulting dianion was formylated with N,N-dimeth-
ylformamide (DMF) to give the aldehyde 44; reductive
amination with tert-butylamine and subsequent deprotection
afforded tert-butylaminomethylpentacycline 45 in 61% yield
after purification by rp-HPLC. The use of a number of
different amines in the reductive amination reaction led to
the synthesis of various alkylaminomethylpentacycline ana-
logues from the common intermediate 44 (Chart 1).
We also successfully pursued the synthesis of a parallel
series of diversifiable pentacycline precursors containing a
dimethylamino substituent at C7, as in minocycline (9) and
tigecycline (10, see Scheme 15). In the case of the dimethy-
lamino-substituted naphthoate ester 46, it was possible to
conduct cyclization by in situ deprotonation, in the presence
of enone 1, forming the dimethylamino-substituted aryl
bromide 47 in 57% yield. Subjection of 47 to deprotonation
a Further transformation of 48 by a reductive amination reaction provides
a route to 7-dimethylamino-alkylaminomethylpentacyclines, as illustrated
by the synthesis of the 7-dimethylamino-azetidinylmethylpentacycline 49.
followed by addition of LHMDS (1 equiv) and warming of
the resulting mixture to -10 °C, provided the cyclization
product 43 in 44% yield (Scheme 14).41 Although the yield
(43) 7-Dimethylamino-alkylaminomethylpentacyclines were initially syn-
thesized from a DE-ring precursor in which the phenolic hydroxyl
group was protected as a methyl ether (necessitating a three-step
deprotection sequence); it was later found that the corresponding DE-
ring precursor containing a tert-butoxycarbonyl protecting group
(compound 46) was also an effective cyclization substrate (allowing
for standard two-step deprotection, see Supporting Information for
details).
(41) The yield of the Michael-Claisen cyclization product 43 was
somewhat lower (28%) when LHMDS was omitted from the reaction.
(42) The final compound in Chart 1 was prepared by N-acetylation after
reductive amination with methylamine.
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A R T I C L E S
Sun et al.
Table 3. Synthesis of 5-Hydroxytetracyclines by Michael-Claisen Cyclization Using a Number of Different D-Ring Precursors and Enone 2
as Substrates, Followed by Deprotectiona
a Deprotection conditions: A, (i) HF (aq), CH3CN; (ii) H2, Pd-black, CH3OH-dioxane; B, (i) HF (aq), CH3CN; (ii) H2, Pd/C, CH3OH-dioxane; C,
(i) H2, Pd-black, CH3OH-dioxane; (ii) HF (aq), CH3CN.
and lithium-halogen exchange, and formylation of the
resulting dianion with DMF, gave the pentacycline precursor
aldehyde 48 in 80% yield; reductive amination with azetidine
and subsequent deprotection afforded azetidinylmethylpen-
tacycline 49 in 74% yield after purification by rp-HPLC
(Scheme 15). As with aldehyde 44 above, reductive amination
of 48 could be conducted using a number of different amines,
providing various 7-dimethylamino-alkylaminomethylpenta-
cyclines upon deprotection (see Chart 2).
An alternative approach to diversification involved reduc-
tion of aldehyde 44 with sodium triacetoxyborohydride,
mesylation of the resulting primary alcohol with methane-
sulfonic anhydride, and then nucleophilic displacement (see
Scheme 16). When imidazole was used as the nucleophile,
substitution afforded the N-imidazoylmethyl product 50 (59%
yield over two steps); removal of protective groups afforded
the corresponding pentacycline analogue 51 in 40% yield.
As we have previously shown, by variation of our original
synthetic route to the enone 1 the corresponding C5-benzyl-
oxycarbonyloxy substituted enone 2 (see Introduction) can
be prepared in gram amounts.1 This in turn has enabled the
synthesis of a number of C5-hydroxytetracyclines (Scheme
17 and Table 3). Like the corresponding cyclization reactions
with enone 1, Michael-Claisen condensations with enone 2
proceed with uniformly high stereoselectivity. For example,
addition of enone 2 (1 equiv) to a solution of the o-toluate
ester anion derived from phenyl ester 13 (4.5 equiv) at -78
°C, followed by warming of the resulting mixture to 0 °C
over 2 h, provided the Michael-Claisen cyclization product
52 in 79% yield in diastereomerically pure form after
purification by rp-HPLC (Scheme 17). A minor diastereomer,
believed to be 6-epi-52, was isolated separately (<7% yield).
Doxycycline (8) was obtained in 90% yield after removal of
protective groups and purification by rp-HPLC.1
Antibacterial Activities
Minimum inhibitory concentrations (MICs) were deter-
mined in whole-cell antimicrobial assays using a panel of
tetracycline-sensitive and tetracycline-resistant Gram-positive
and Gram-negative bacteria. The results for selected com-
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
Table 4. Minimum Inhibitory Concentration (MIC) Values for Selected Tetracycline Analogues (µg/mL)a
a Organisms in red are antibiotic-resistant. Abbreviations: A, ampicillin; M, methicillin; P, penicillin; V, vancomycin; T, tetracycline; Mult,
multiple. Organisms: SA, S. aureus; EF, E. faecium; SP, S. pneumoniae; EC, E. coli; AB, A. baumanii; PA, P. aeruginosa; HI, H. influenzae.
pounds are shown in Table 4. Two promising new series of
tetracycline antibiotics emerge from this study: 6-aryltetra-
cyclines, which are active in both tetracycline-sensitive and
tetracycline-resistant Gram-positive strains, and alkylami-
nomethylpentacyclines, which show good activity in both
tetracycline-sensitive and tetracycline-resistant Gram-positive
and Gram-negative organisms.
mg/kg. Mortality was monitored once daily for 7 days. All mice
survived at the highest level of dosing (30 mg/kg) for each of the
compounds tested, indicating that the threshold of acute toxicity
in mice is greater than this value. The 6-phenyltetracycline analogue
showed some efficacy in this murine model (ED50 10.7 mg/kg),
while the alkylaminomethylpentacyclines were more potent, with
efficacy similar to that of tetracycline (ED50 1.7-2.8 mg/kg).
Several compounds were selected for further study in a mouse
septicemia model to determine efficacy in vivo (Table 5). Mice
were inoculated intraperitoneally with an LD90-100 bolus of S.
aureus (Smith). One hour later, tetracycline analogues were
administered intravenously at dose levels ranging from 0.3 to 30
Conclusion
The results described illustrate the application of a general
AB plus D strategy for the synthesis of more than 50
tetracyclines and tetracycline analogues. C-ring formation was
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A R T I C L E S
Sun et al.
Table 5. In Vivo Efficacy of Selected Tetracycline Analogues in a Tetracycline-Sensitive Strain of S. aureusa
a Mice were inoculated intraperitoneally with an LD90-100 bolus of S. aureus (Smith) (3.5 × 105 CFU/mouse) in 0.5 mL of BHI broth
containing 5% mucin. Tetracycline analogues were administered intravenously to test animals at 1 h after bacterial inoculation. Mortality was
monitored once daily for 7 days.
Scheme 16. An Alternative Route to Diversification of the Aldehyde
44, Involving Reduction, Activation, and Nucleophilic
Displacement, As Exemplified by the Synthesis of the
N-Imidazoylmethylpentacycline 51
Scheme 17. Synthesis of Doxycycline (8) by Michael-Claisen
Cyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester
13 as the D-Ring Precursor and a Stepwise Protocol for Addition
of the Base and Enone 2, Followed by Deprotection
modification of both AB and D-ring components allows for
the preparation of individual tetracyclines, as well as for the
synthesis of tetracycline precursors that are readily diversified
by late-stage transformations. In many cases a single cy-
clization attempt provided, after deprotection, sufficient
material for antibacterial screening against a panel of Gram-
positive and Gram-negative organisms. For compounds of
interest, reactions could then be readily scaled to provide
amounts necessary for further evaluation in assays such as a
achieved by a stereocontrolled Michael-Claisen cyclization
reaction employing one or more of the protocols detailed
herein. The examples discussed demonstrate that structural
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Synthesis of New Tetracycline Antibiotics
A R T I C L E S
toral fellowship support (C.D.L.). We acknowledge Vivisource
for execution of in vivo studies, Micromyx, LLC for execution
of in vitro studies, and Tetraphase Pharmaceuticals for sponsor-
ship of these. We thank Dr. Richard Staples and Dr. Andrew
Haidle for conducting the X-ray crystallographic analyses.
murine septicemia model. A number of novel structural
classes were explored, including D-heteroaryl tetracyclines,
pentacyclines, and E-heteroaryl pentacyclines. The platform
for tetracycline synthesis described gives access to a broad
range of molecules that would be inaccessible by semisyn-
thetic methods (presently the only means of tetracycline
production) and provides a powerful engine for the discovery
and, perhaps, development of new tetracycline antibiotics.
Supporting Information Available: Detailed experimental
procedures for Michael-Claisen cyclization reactions and
deprotection sequences, and characterization data for all
tetracycline analogues. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was generously supported by
the National Institutes of Health (AI048825). We thank the NSF
for predoctoral graduate fellowship support (J.D.B. and M.G.C.)
and the Deutscher Akademischer Austausch Dienst for postdoc-
JA806629E
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