Published on Web 12/03/2008 A Robust Platform for the Synthesis of New Tetracycline
Cuixiang Sun, Qiu Wang, Jason D. Brubaker, Peter M. Wright, Christian D. Lerner, Kevin Noson, Mark Charest, Dionicio R. Siegel, Yi-Ming Wang, and Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138 Received August 21, 2008; E-mail: [email protected] 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.
widely variant in the left-hand or D-ring portion of themolecule.2 Here, we expand upon our original findings, describ- In prior research we showed that cyclohexenones 1 and 2
ing the synthesis of more than 50 different tetracyclines and can be transformed into 6-deoxytetracycline antibiotics using a tetracycline analogues, many of which are active in inhibiting sequence of as few as three chemical steps.1 the growth of cultured Gram-positive and Gram-negativebacteria, including tetracycline-resistant strains. We providedetailed protocols for the key cyclization reaction in its variousforms and discuss features of stereochemistry, chemical ef-ficiency, and mechanism. We also report minimum inhibitoryconcentration values for selected analogues in a number ofdifferent bacterial strains, as well as preliminary in vivo dataobtained 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 The first tetracycline antibiotic was discovered in 1948, when potentially general means for constructing tetracycline analogues Benjamin Duggar of Lederle Laboratories isolated the natural
product chlorotetracycline (Aureomycin, 3) from the culture
(1) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, broth of a novel species of Streptomyces.3 Within two years, a A. G. Science 2005, 308, 395–398.
(2) As pointed out by a reviewer, the second step of the key C-ring-forming research team from Chas. Pfizer and Co. had isolated a second reaction sequence is more properly designated as an internal Claisen natural tetracycline, oxytetracycline (Terramycin, 4),4 and in
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.
(3) (a) Duggar, B. M Ann. N.Y. Acad. Sci. 1948, 51, 177–181. (b) Duggar,
Org. React. (N.Y.) 1967, 15, 1. For brevity, this sequence is referred
B. M. Aureomycin and Preparation of Same. U.S. Patent 2,482,055, to here as a Michael-Claisen cyclization.
Sept 13, 1949.
10.1021/ja806629e CCC: $40.75 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 17913–17927 9 17913
Sun et al. raising concern among public health officials, particularly withthe emergence of antibiotic-resistant bacterial strains in com-munity settings. While careful management of the use of existingantibiotics in society is warranted, it would seem unwise toabandon the search for new agents, given the diversity ofbacteria and their capacity to evolve rapidly. Attempts to developantibacterial agents with novel targets have met with littlesuccess.13 As a consequence, many research programs seekingto discover new antibiotics have been refocused toward themodification 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 thestructure 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 manylaboratories have sought to develop a practical route for theirsynthesis. In 2003, an expert opinion of the patent literaturesummarized the state of the art, concluding, "the original effortof Woodward has survived as the basic strategy for the totalsynthesis of this series and at greater than 25 steps is clearlynot to be considered as practical. we believe there is amplejustification to explore new total synthetic and convergentsynthetic routes that take full advantage of the body of chemistrythat has become available since Woodward's original effort." 15Among 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
(4) (a) Finlay, A. C.; Hobby, G. L.; P'an, S. Y.; Regna, P. P.; Routien, transformation discovered by Lloyd Conover of Pfizer.5 Sub- J. B.; Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.; sequently, tetracycline was found to be a natural product,6 and 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,
later still, Lederle researchers isolated 6-demethyltetracyclines July 18, 1950.
(see structure 6) from culture broths of a mutant strain of
(5) (a) Booth, J. H.; Morton, J.; Petisi, J. P.; Wilkinson, R. G.; Williams, Streptomyces.7 Conover has provided detailed and insightful J. H. J. Am. Chem. Soc. 1953, 75, 4621. (b) Conover, L. H.; Moreland,
accounts of research efforts leading to new tetracyclines W. T.; English, A. R.; Stephens, C. R.; Pilgrim, F. J. J. Am. Chem.
1953, 75, 4622–4623.
specifically and antibiotics more generally.8 All tetracyclines (6) Minieri, P. P.; Sokol, H.; Firman, M. C. Process for the Preparation approved for human or veterinary use are fermentation products of Tetracycline and Chlorotetracycline. U.S. Patent 2,734,018, Feb 7, or are derived from fermentation products by semisynthesis.
(7) McCormick, J. R. D.; Sjolander, N. O.; Hirsch, U.; Jensen, E. R.; This is also true of most -lactam and all macrolide antibiotics.
Doerschuk, A. P. J. Am. Chem. Soc. 1957, 79, 4561–4563.
Tracing the paths of human efforts to produce new antibiotics (8) (a) Conover, L. H. Res. Technol. Management 1984, 17–22. (b)
from natural products not accessible by synthesis reveals an Conover, L. H. ACS AdV. Chem. Ser. 1971, 108, 33–80.
evolutionary process marked by specific, impactful discoveries.
(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)
In the case of the tetracyclines, Pfizer scientists achieved a major McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P.
enabling advance approximately 10 years after the class had J. Am. Chem. Soc. 1960, 82, 3381–3386. (c) Stephens, C. R.;
been identified when they demonstrated that the C6-hydroxyl Beereboom, J. J.; Rennhard, H. H.; Gordon, P. N.; Murai, K.;
Blackwood, R. K.; Schach von Wittenau, M. J. Am. Chem. Soc. 1963,
group of the natural products oxytetracycline (4), tetracycline
85, 2643–2652. (d) Blackwood, R. K.; Stephens, C. R. J. Am. Chem. (5), and 6-demethyltetracycline (6) could be removed reduc-
Soc. 1962, 84, 4157–4159.
tively.9 The 6-deoxytetracyclines produced, including 6-deox- (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,
ytetracycline itself (7), were found to be more stable than the
36, 723–725. (c) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, parent compounds, yet they retained broad-spectrum antibacte- H. M.; Boothe, J. H. J. Med. Chem. 1963, 6, 405–407. (d) Zambrano,
rial activity. The important and now generic antibiotics doxy- R. T. U.S. Patent 3,483,251, Dec 9, 1969.
cycline (8; Pfizer, 1967) and minocycline (9; Lederle, 1972)
(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–
followed as a consequence, the latter arising from the additional 188. (b) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9,
discovery that electrophilic aromatic substitution at C7 becomes possible when the more stable 6-deoxytetracyclines are used (12) Listing of Newly Approved Drug Therapies (2008)http://www.
centerwatch.com/patient/drugs/druglist.html, 2008 (accessed August as substrates.10 Decades later, a team of Wyeth scientists led by Frank Tally synthesized 7,9-disubstituted tetracycline deriva- (13) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat. tives, leading to the discovery of the antibiotic tigecycline ReV. Drug DiscoVery 2007, 6, 29–40.
(Tigacyl, 10; U.S. approval 2005).11
(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.; Tigacyl is one of only three new antibiotics to be brought to Woodward, R. B. J. Am. Chem. Soc. 1953, 75, 5455–5475. (b)
market in the United States in the past three years, and the only Stephens, C. R.; Conover, L. H.; Hochstein, F. A.; Regna, P. P.; broad-spectrum agent.12 Diminishing economic incentives and Pilgrim, F. J.; Brunings, K. J. J. Am. Chem. Soc. 1952, 74, 4976–
increasing regulatory hurdles have led many pharmaceutical (15) Podlogar, B. L.; Ohemeng, K. A.; Barrett, J. F. Expert Opin. Ther. companies to discontinue efforts to develop new antibiotics, Patents 2003, 13, 467–478.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
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 research groups first reported that simple o-toluate ester anions the results described herein, is Stork and Hagedorn's strategy (unsubstituted at the benzylic position) undergo Michael-Claisen for protection of the vinylogous carbamic acid group of the A cyclization reactions with -methoxycyclohexenones and γ-py- ring of the tetracyclines as a 3-benzyloxyisoxazole group; rones to form naphthyl ketones (see eq 4 for one example), a subsequent deprotection occurs under mild (hydrogenolytic) sequence sometimes referred to as the Staunton-Weinreb 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 There is also precedent for the formation of non-aromatic derivatives since 1978, when three different cyclization protocols six-membered rings by Michael-Claisen and Michael- were introduced by independent research groups. Hauser and Dieckmann reaction sequences.23,26,27 With few exceptions,27 Rhee used a sulfoxide-stabilized o-toluate ester anion as the stereochemical features of these cyclization reactions have not nucleophilic component in a Michael-Dieckmann cyclization been discussed, frequently because they were of little conse- reaction with methyl crotonate (eq 1). In this case, aromatization quence (aromatization followed cyclization). The Michael-Claisen occurred upon thermal elimination of phenylsulfenic acid.22 The cyclizations detailed below are unusual in their stereochemical use of phthalide and cyanophthalide anions as nucleophilic complexity, stereocontrol, and efficiency. In 2000, while our components was described by Broom and Sammes (eq 2)23 and studies were in progress, Tatsuta and co-workers reported a Kraus and Sugimoto (eq 3),24 respectively. Formal loss of water synthesis of (-)-tetracycline (34 steps, 0.002% yield) that and hydrogen cyanide, respectively, led to naphthoate ester employed an early stage Michael-Claisen cyclization reaction products in these procedures. In 1979, the Weinreb and Staunton to form an aromatic C-ring precursor, which was dearomatizedlater in the sequence.28 Since 2005, our laboratory has reported (16) (a) Conover, L. H.; Butler, K.; Johnston, J. D.; Korst, J. J.; Woodward, two different routes to synthesize the AB precursor 1 in optically
R. B. J. Am. Chem. Soc. 1962, 84, 3222–3224. (b) Woodward, R. B.
active form; the more recent of these was scaled to prepare >40 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.
g of crystalline product in one batch.29 Here we provide details Chem. Soc. 1968, 90, 439–457.
of the different protocols that can be used to construct the C (17) Stork, G.; Hagedorn, A. A., III. J. Am. Chem. Soc. 1978, 100, 3609–
(18) (a) Gurevich, A. I.; Karapetyan, M. G.; Kolosov, M. N.; Korobko, V. G.; Onoprienko, V. V.; Popravko, S. A.; Shemyakin, M. M.
(25) (a) Dodd, J. H.; Weinreb, S. M. Tetrahedron Lett. 1979, 38, 3593–
Tetrahedron Lett. 1967, 8, 131–134. (b) Kolosov, M. N.; Popravko,
3596. (b) Dodd, J. H.; Starrett, J. E.; Weinreb, S. M. J. Am. Chem. S. A.; Shemyakin, M. M. Liebigs Ann. 1963, 668, 86–91.
Soc. 1984, 106, 1811–1812. (c) Leeper, F. J.; Staunton, J. J. Chem.
(19) (a) Muxfeldt, H.; Hardtmann, G.; Kathawala, F.; Vedejs, E.; Mooberry, Soc., Chem. Commun. 1979, 5, 206–207. (d) Leeper, F. J.; Staunton,
J. B. J. Am. Chem. Soc. 1968, 90, 6534–6536. (b) Muxfeldt, H.; Haas,
J. J. Chem. Soc., Perkin Trans. 1 1984, 1053–1059.
G.; Hardtmann, G.; Kathawala, F.; Mooberry, J. B.; Vedejs, E. J. Am. (26) (a) Tarnchompoo, B.; Thebtaranonth, C.; Thebtaranonth, Y. Synthesis Chem. Soc. 1979, 101, 689–701.
1986, 9, 785–786. (b) Boger, D. L.; Zhang, M. J. Org. Chem. 1992,
(20) (a) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan- 57, 3974–3977. (c) Nishizuka, T.; Hirosawa, S.; Kondo, S.; Ikeda, Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103,
D.; Takeuchi, T. J. Antibiot. 1997, 50, 755–764. (d) Hill, B.; Rodrigo,
1143–1154. (b) Pioletti, M.; Schlu¨nzen, F.; Harms, J.; Zarivach, R.; R. Org. Lett. 2005, 7, 5223–5225.
Glu¨hmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.; (27) For examples of stereocontrol in Michael-Claisen and Michael- Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F EMBO J. 2001, 20,
Dieckmann cyclization reactions, see: (a) Franck, R. W.; Bhat, V.; Subramaniam, C. S. J. Am. Chem. Soc. 1986, 108, 2455–2457. (b)
(21) Parrish, C. A. Ph.D. Thesis, California Institute of Technology, Tatsuta, K.; Yamazaki, T.; Mase, T.; Yoshimoto, T. Tetrahedron Lett. Pasadena, CA, 1999.
1998, 39, 1771–1772. (c) White, J. D.; Demnitz, F. W. J.; Qing, X.;
(22) Hauser, F. M.; Rhee, R. P. J. Org. Chem. 1978, 43, 178–180.
Martin, W. H. C. Org. Lett. 2008, 10, 2833–2836.
(23) Broom, N. J. P.; Sammes, P. G. J. Chem. Soc., Chem. Commun. 1978,
(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.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Sun et al. ring of the tetracyclines and exemplify these protocols with the to the enone) and/or steric effects (addition of the nucleophile preparation of a number of novel substances with antibacterial 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 A critical early experiment in our attempts to assemble the modify the D-ring precursor, with three objectives: (1) to activate C ring of the tetracyclines by a Michael-Claisen cyclization the ester toward Claisen cyclization (which we did not observe reaction provided both direction and mechanistic insight. As with the methyl ester substrate 11), (2) to obviate the use of
depicted in Scheme 1, treatment of a solution of organostannane organotin intermediates, and (3) to mask the C10 phenoxy 1130 in tetrahydrofuran (THF) with n-butyllithium at -78 °C,
substituent with a protective group more labile than methyl.
followed by transfer of this mixture to a solution of enone 1,
These objectives were achieved using the tert-butoxycarbonyl- also at -78 °C, and subsequent quenching with tert-butyl- protected phenyl ester substrate 13 (Scheme 2).31,32
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
Scheme 1. Michael Addition of a Benzylic Anion (Generated by
Tin-Lithium Exchange) to Enone 1, Followed by Trapping of the
Precursor and a Stepwise Protocol for Addition of the Base and Resultant Enolate with tert-Butyldimethylsilyl Triflate Enone 1, Followed by Deprotection
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
Deprotonation of 13 (3 equiv) with lithium diisopropylamide
consequence of stereoelectronic factors (pseudoaxial addition (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 proceedwith complete stereocontrol at C5a (attack upon a singlediastereoface of the enone). These observations have heldconsistently in more than 40 different C-ring-forming cyclizationreactions 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-
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Synthesis of New Tetracycline Antibiotics A R T I C L E S
Indeed, we have observed across a range of different D-ring Scheme 4. Synthesis of 6-(S)-Phenylsancycline (21) by
precursors that Michael addition is relatively rapid at -78 °C, Michael-Claisen Cyclization Using the
tert-Butoxycarbonyl-Protected Phenyl Ester 19 as the D-Ring
while Claisen cyclization proceeds more slowly, typically upon Precursor and a Stepwise Protocol for Addition of the Base and warming to 0 °C. Thus, we view Claisen cyclization and not Enone 1, Followed by Deprotection
Michael addition as rate-determining. It is evident from theseobservations that C-ring formation cannot occur by Diels-Aldercycloaddition of an o-quinonemethide intermediate, which ishypothetically a mechanistic alternative to Michael-Claisencyclization.
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
4),32 followed by warming of the resulting mixture to -15 °C, group is cleaved relatively rapidly in the deprotection step provided the Michael-Claisen cyclization product 20 in 97%
employing hydrofluoric acid, while the tertiary tert-butyldim- yield after purification by rp-HPLC. Standard deprotection of ethylsilyl ether undergoes protodesilylation more slowly.
20 then afforded the novel tetracycline analogue 6-(S)-phenyl-
sancycline (21), which was found to effectively inhibit the
Scheme 3. Synthesis of Minocycline (9) by Michael-Claisen
growth of a number of Gram-positive bacteria, including Cyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester tetracycline-resistant strains (vide infra), prompting the synthesis 17 as the D-Ring Precursor and a Stepwise Protocol for Addition
of the Base and Enone 1, Followed by Deprotection
of a series of 6-aryl-substituted tetracyclines (see Table 1).35 Itis worthy of note that more than half of the cyclization reactionspresented 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
(30) Carpenter, T. A.; Evans, G. E.; Leeper, F. J.; Staunton, J.; Wilkinson, The conditions developed for the cyclization reaction depicted M. R. J. Chem. Soc., Perkin Trans. I 1984, 1043–1051.
in Scheme 2, sequential deprotonation of a D-ring precursor (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,
followed by addition of the enone 1, have been effective for
51, 1150–1152 (see also ref 27c).
the synthesis of a number of known tetracyclines as well as (32) Bennetau, B.; Mortier, J.; Moyroud, J.; Guesnet, J.-L. J. Chem. Soc., novel tetracycline analogues variant within or near the D ring.
Perkin Trans. 1 1995, 1265–1271.
(33) For other examples of the isolation of uncyclized Michael adducts, For example, treatment of phenyl ester 1734 (3 equiv, Scheme
see refs 23, 25c, 25d, and 26b.
3) with LDA (3 equiv) in the presence of TMEDA (6 equiv) at (34) Phenyl ester 17 was synthesized in six steps from ethyl 6-methylsali-
-78 °C, followed by addition of enone 1 (1 equiv) and warming
cylate; see: (a) Olah, G. A.; Malhotra, R.; Narang, S. C. Nitration, of the resulting mixture to -10 °C, provided the Michael-Claisen Methods and Mechanisms; VCH Publishers Inc.: New York, 1980.
(b) Bellamy, F. D.; Ou, K. Tetrahedron Lett. 1984, 26, 839–842. (c)
cyclization product 18 in 83% yield after purification by rp-
Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; HPLC. Two-step deprotection and purification by rp-HPLC then Shah, R. D. J. Org. Chem. 1996, 61, 3849–3862.
afforded minocycline (9) in 74% yield.
(35) Leading references for the synthesis of D-ring precursors to 6-aryl- substituted tetracyclines: (a) Miyaura, N.; Suzuki, A Chem. ReV. 1995,
Among the modified D-ring precursors we have investigated, 95, 2457–2483. (b) Kotha, S.; Mandal, K. Eur. J. Org. Chem. 2006,
substrates with benzylic anion-stabilizing groups were found 23, 5387–5393. (c) Wright, S. W.; Hageman, D. L.; McClure, L. D.
to undergo particularly efficient cyclization reactions. For 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)
example, addition of enone 1 (1 equiv) to a solution of the
Appukkuttan, P.; Dehaen, W.; Fokin, V. V.; Van der Eycken, E. Org. stabilized anion derived from substrate 19 (3 equiv, Scheme
Lett. 2004, 6, 4223–4225.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Sun et al. 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]
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Synthesis of New Tetracycline Antibiotics A R T I C L E S
Scheme 5. Synthesis of 6-(R)-N-Imidazoylsancycline (24) by
reactions we have studied (these providing the two exceptions Michael-Claisen Cyclization Using the referred to above), which we attribute to epimerization at C6 tert-Butoxycarbonyl-Protected Phenyl Ester 22 and Enone 1 as
after cyclization. We have not yet conducted the rigorous studies Substrates, with Deprotonation in Situ, Followed by Deprotection necessary to establish if the product ratios were kinetically orthermodynamically determined in these examples.
The in situ deprotonation protocol has proven to be effective for the Michael-Claisen cyclization reactions of a number ofdifferent D-ring precursors containing benzylic anion-stabilizingsubstituents (see Table 2). Because of its greater experimentalconvenience, this has become the preferred method for thesynthesis 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
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 Scheme 8. Synthesis of the Pentacycline 33 by Michael-Claisen
diester 27 as the D-ring precursor, a diastereomeric mixture of cyclization
Cyclization Using the Phenyl Bromomethylnaphthoic Acid Ester 31
products was obtained.
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
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
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
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- Lithium-halogen exchange is rarely employed to form thoate ester DE-ring precursors,22-24 as the protocols that we benzyllithium reagents in organic synthesis, due to the propen- had used to this point were found to be largely ineffective with sity of the benzyl halide substrates to engage in Wurtz-type these substrates.
coupling reactions in the presence of an alkyllithium reagent.36 J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Synthesis of New Tetracycline Antibiotics A R T I C L E S
Scheme 9. Synthesis of a Pyrazole Analogue (36) by
The in situ lithium-halogen exchange protocol developed Michael-Claisen Cyclization Using the Benzylic Bromide 34 and
for the synthesis of the pentacycline 33 has proven to be
Enone 1 as Substrates, and an in Situ Protocol for
Lithium-Halogen Exchange, Followed by Deprotection
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
A very similar procedure produced 8-fluorosancycline (38)
from the benzyl bromide 37 (Scheme 10). Like the D-ring
Scheme 10. Synthesis of 8-Fluorosancycline (38) by
pyrazole analogue 36, the 8-fluorotetracycline analogue 38
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.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Sun et al. Scheme 13. Synthesis of 6-Aryl-Substituted Heterocyclic
0 °C over 30 min, provided the Michael-Claisen cyclization Pentacyclines by Michael-Claisen Cyclizations Using Benzylically product 40 in 81% yield.1,38 The typical two-step deprotection
Substituted Quinolines as DE-Ring Precursors and a Stepwise sequence then transformed the cyclized product 40 into 10-
Protocol for Addition of the Base and Enone 1, Followed by
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
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
with enone 1 was successfully achieved by in situ lithium-halogen
would have been difficult, if not impossible, to prepare by exchange using phenyllithium, but not n-butyllithium. Thus, treatment of a mixture of the bis-bromide 42 (3 equiv) and
Lithium-halogen exchange was also employed for the enone 1 (1 equiv) with phenyllithium (3 equiv) at -100 °C,
synthesis of 10-deoxysancycline (41, Scheme 11). As we
reported previously, treatment of a solution of phenyl 2-(bro-
(38) Both the stepwise and in situ protocols for deprotonation-cyclization were also effective for the synthesis of the Michael-Claisen cyclization momethyl)benzoate (39, 4 equiv) and enone 1 (1 equiv) with
product 40 from phenyl o-toluate and enone 1, though yields were
n-butyllithium (4 equiv) at -100 °C, followed by warming to (39) Van Leusen, A. M.; Terpstra, J. W. Tetrahedron Lett. 1981, 22, 5097–
(37) The use of a methyl ether protecting group in the penultimate entry (40) Synthesized from 6-bromophthalide and methyl crotonate: Broom, of Table 2 required one additional deprotection step (BBr3, CH2Cl2,- N. J. P.; Sammes, P. G. J. Chem. Soc., Perkin Trans. 1 1981, 465–
78 °C f 23 °C) following the typical two-step sequence.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Synthesis of New Tetracycline Antibiotics A R T I C L E S
Chart 1. Alkylaminomethylpentacyclines Synthesized from
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-
a Further transformation of 48 by a reductive amination reaction provides
lamino-substituted naphthoate ester 46, it was possible to
a route to 7-dimethylamino-alkylaminomethylpentacyclines, as illustrated conduct cyclization by in situ deprotonation, in the presence by the synthesis of the 7-dimethylamino-azetidinylmethylpentacycline 49.
of enone 1, forming the dimethylamino-substituted aryl
bromide 47 in 57% yield. Subjection of 47 to deprotonation
followed by addition of LHMDS (1 equiv) and warming ofthe resulting mixture to -10 °C, provided the cyclization (43) 7-Dimethylamino-alkylaminomethylpentacyclines were initially syn- product 43 in 44% yield (Scheme 14).41 Although the yield
thesized from a DE-ring precursor in which the phenolic hydroxylgroup was protected as a methyl ether (necessitating a three-stepdeprotection sequence); it was later found that the corresponding DE- (41) The yield of the Michael-Claisen cyclization product 43 was
ring precursor containing a tert-butoxycarbonyl protecting group somewhat lower (28%) when LHMDS was omitted from the reaction.
(compound 46) was also an effective cyclization substrate (allowing
(42) The final compound in Chart 1 was prepared by N-acetylation after for standard two-step deprotection, see Supporting Information for reductive amination with methylamine.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
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 be prepared in gram amounts.1 This in turn has enabled the resulting dianion with DMF, gave the pentacycline precursor synthesis of a number of C5-hydroxytetracyclines (Scheme aldehyde 48 in 80% yield; reductive amination with azetidine
17 and Table 3). Like the corresponding cyclization reactions and subsequent deprotection afforded azetidinylmethylpen- with enone 1, Michael-Claisen condensations with enone 2
tacycline 49 in 74% yield after purification by rp-HPLC
proceed with uniformly high stereoselectivity. For example, (Scheme 15). As with aldehyde 44 above, reductive amination
addition of enone 2 (1 equiv) to a solution of the o-toluate
of 48 could be conducted using a number of different amines,
ester anion derived from phenyl ester 13 (4.5 equiv) at -78
°C, followed by warming of the resulting mixture to 0 °C cyclines upon deprotection (see Chart 2).
over 2 h, provided the Michael-Claisen cyclization product An alternative approach to diversification involved reduc- 52 in 79% yield in diastereomerically pure form after
tion of aldehyde 44 with sodium triacetoxyborohydride,
purification by rp-HPLC (Scheme 17). A minor diastereomer, mesylation of the resulting primary alcohol with methane- believed to be 6-epi-52, was isolated separately (<7% yield).
sulfonic anhydride, and then nucleophilic displacement (see Doxycycline (8) was obtained in 90% yield after removal of
Scheme 16). When imidazole was used as the nucleophile, protective groups and purification by rp-HPLC.1 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.
Minimum inhibitory concentrations (MICs) were deter- As we have previously shown, by variation of our original mined in whole-cell antimicrobial assays using a panel of synthetic route to the enone 1 the corresponding C5-benzyl-
tetracycline-sensitive and tetracycline-resistant Gram-positive oxycarbonyloxy substituted enone 2 (see Introduction) can
and Gram-negative bacteria. The results for selected com- J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
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 mg/kg. Mortality was monitored once daily for 7 days. All mice tetracycline antibiotics emerge from this study: 6-aryltetra- survived at the highest level of dosing (30 mg/kg) for each of the cyclines, which are active in both tetracycline-sensitive and compounds tested, indicating that the threshold of acute toxicity tetracycline-resistant Gram-positive strains, and alkylami- in mice is greater than this value. The 6-phenyltetracycline analogue nomethylpentacyclines, which show good activity in both showed some efficacy in this murine model (ED50 10.7 mg/kg), tetracycline-sensitive and tetracycline-resistant Gram-positive while the alkylaminomethylpentacyclines were more potent, with and Gram-negative organisms.
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). Micewere inoculated intraperitoneally with an LD90-100 bolus of S. The results described illustrate the application of a general aureus (Smith). One hour later, tetracycline analogues were AB plus D strategy for the synthesis of more than 50 administered intravenously at dose levels ranging from 0.3 to 30 tetracyclines and tetracycline analogues. C-ring formation was J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
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 wasmonitored once daily for 7 days.
Scheme 16. An Alternative Route to Diversification of the Aldehyde
Scheme 17. Synthesis of Doxycycline (8) by Michael-Claisen
44, Involving Reduction, Activation, and Nucleophilic
Cyclization Using the tert-Butoxycarbonyl-Protected Phenyl Ester Displacement, As Exemplified by the Synthesis of the 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 forthe preparation of individual tetracyclines, as well as for thesynthesis of tetracycline precursors that are readily diversifiedby late-stage transformations. In many cases a single cy-clization attempt provided, after deprotection, sufficientmaterial for antibacterial screening against a panel of Gram- achieved by a stereocontrolled Michael-Claisen cyclization positive and Gram-negative organisms. For compounds of reaction employing one or more of the protocols detailed interest, reactions could then be readily scaled to provide herein. The examples discussed demonstrate that structural amounts necessary for further evaluation in assays such as a J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008
Synthesis of New Tetracycline Antibiotics A R T I C L E S
murine septicemia model. A number of novel structural toral fellowship support (C.D.L.). We acknowledge Vivisource classes were explored, including D-heteroaryl tetracyclines, for execution of in vivo studies, Micromyx, LLC for execution pentacyclines, and E-heteroaryl pentacyclines. The platform of in vitro studies, and Tetraphase Pharmaceuticals for sponsor- for tetracycline synthesis described gives access to a broad ship of these. We thank Dr. Richard Staples and Dr. Andrew range of molecules that would be inaccessible by semisyn- Haidle for conducting the X-ray crystallographic analyses.
thetic methods (presently the only means of tetracyclineproduction) and provides a powerful engine for the discovery Supporting Information Available: Detailed experimental
and, perhaps, development of new tetracycline antibiotics.
procedures for Michael-Claisen cyclization reactions anddeprotection sequences, and characterization data for all Acknowledgment. This work was generously supported by
tetracycline analogues. This material is available free of the National Institutes of Health (AI048825). We thank the NSF charge via the Internet at http://pubs.acs.org.
for predoctoral graduate fellowship support (J.D.B. and M.G.C.)and the Deutscher Akademischer Austausch Dienst for postdoc- J. AM. CHEM. SOC. 9 VOL. 130, NO. 52, 2008

Source: http://faculty.chemistry.harvard.edu/files/myers/files/128_2008_sun_jacs_130_17913.pdf

Kujawa-roeleveld_2011_removal of pharmaceuticals compounds from concentrated wastewater

018530 - SWITCH Sustainable Water Management in the City of the Future Integrated Project Global Change and Ecosystems Training material Pharmaceutical compounds in environment Removal of pharmaceuticals from concentrated wastewater streams in source oriented sanitation Prepared by: dr. ir. Katarzyna Kujawa-Roeleveld Wageningen University, Wageningen, The Netherlands LeAF (Lettinga Associates Foundation) Based on deliverables of SWITCH project, other overlapping projects and literature Material to be used with PowerPoint presentations I-VIII


Arch Dis Child 2007;92:251–256. doi: 10.1136/adc.2006.106120 bone exceeds the ability of the bone to box). It is easier to show ligamentous laxity in this absorb the force by deforming. Fractures in older group than in infants. children are common—approximately one third ofchildren will have a fracture by 16 years of age, PATHOPHYSIOLOGY OF BONE DISEASES with more boys experiencing fracture than girls.1