AU2014250722B2 - Synthesis of tetracyclines and analogues thereof - Google Patents
Synthesis of tetracyclines and analogues thereof Download PDFInfo
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Abstract
H:Ada.1nicnvov.nNRPorbl\DCC\DAR689S12_L DC-17/101lin The tetracycline class of antibiotics has played a major role in the treatment of infectious diseases for the past 50 years. However, the increased use of the tetracyclines in human and veterinary medicine has led to resistance among many organisms previously susceptible to tetracycline antibiotics. The modular synthesis of tetracyclines and tetracycline analogs described provides an efficient and enantioselective route to a variety of tetracycline analogs and polycyclines previously inaccessible via earlier tetracycline syntheses and semi-synthetic methods. These analo gs may be used as anti-microbial agents or anti-proliferative agents in the treatment of diseases of humans or other animals.
Description
SYNTHESIS OF TETRACYCLINES AND ANALOGUES THEREOF This application is a divisional of Australian Patent Application No. 2012202559, the entire content of which is incorporated herein by reference. Related Applications [0001] The present application claims priority to U.S. provisional patent applications, USSN 60/660,947, filed March 11,2005, and USSN 60/573,623, fled May 21,2004, each of which is incorporated herein by reference. GovermnWt Support [002] The work described herein was supported, in part, by gants from the National Institutes of Health (R01 A148825) and the National Science Foundaion (predoctoral fellowship R10964). The United States.government may have certain rights in the invention. Background of the Invendon [0001] The tetracyclines are broad spectrum anti-microbial agents that arc widely used in human and veterinary medicine (Schappinger et a, "Tetracyclines: Antibiotic Action, Uptake, and Resistance Mechanisam"Arch Afcrobiol 165:359-69, 1996; Mitscher, Medicinal Research Sedes, Vol.9, The Chemistry of the Tetracycline Antibiotics, Marcel Dekker Inc. New York, 1978). The total production of tetracyclines by frmentation or semi-synthesi is measured inths thousands of metric tons per year. The first tetracycline, cldorotetracycline (1) (Aurcomycin), was isolated from the soil bacterium Streptomyces auruofaciem by Lederle Laboratories (Wyeth-Ayerst Research) inthe 1945 (Duggar, Amr N Acad SUt 51:177-181, 1948; Duggar, Aureamycin and Preparation of Some, U.S. Patent 2,482,055, 1949; incorporated herein by refence). Oxyteiracycline (2)was isolated soon after from S rfmasus by scientists at Pfizer Laboratories (Finlay etat. Science 111:85, 1950). The structures of chlorotetracycline and oxytetracycline were elucidated by scientists at Pfizer in collaboration with R. B. Woodward and co-workers at Harvard University (Hochstein at al. Am. Chem. Soc. 74:3708-3709,1952; Hocbstein etd J. Aft Cem. Soc. 75:5455-75, 1953; Stephens et a J Am. Chem Soc. 74:4976-77, 1952; Stephens et al I Am. Chem. Soc. 76:3566-75,1954). Tetracycline (3) was later prepared by the - 1 hydrogenolysis of chlorotetracycline and was found to retain the anti-microbial activity of chlorotetracycline and oxytetracycline and had increased stability (Boothe et al. J Am. Chem. Soc. 75:4621, 1953; Conover et al J Am. Chem. Soc. 75:4622-23, 1953). Tetracycline was later found to be a natural product of S. aureofaciens, S. viridofaciens, and S. rimosus. OHC H3 H3CN CH 3 CI HOtC3 H HO C OHH - -OH OH D C BD A NH2 NH2 OH =6H OH 0 OH 0 0 OH 0 OH 0 0 Chlorotetracycine (1) Oxytetracycline (2) H3C. .CH3 HO CN 6 4 OH 8 6a 5a 4a 3 9 10 111 ta 12a 2 NH2 OH 0 OH 0 0 Tetracycline (3) [0002] The primary tetracyclines of clinical importance today include tetracycline (3) (Boothe et al. J Am. Chem. Soc. 75:4621, 1953), oxytetracycline (2, TerramycinTm) (Finlay et al Science 111:85, 1950), doxycycline (Stephens et at £ Am. Chem. Soc. 85:2643, 1963), and minocycline (Martefl et al. Med Chem. 10:44, 1967; Martell et al. J Med. Chem. 10:359, 1967). The tetracyclines exert their anti-microbial activity by inhibition of bacterial protein synthesis (Bentley and O'Hanlon, Eds., Anti Infectives Recent Advances in Chemistry and Structure-Activity Relationships The Royal Society of Chemistry: Cambridge, UK, 1997). Most tetracyclines are bacteriostatic rather than bactericidal (Rasmussen et al. Antirnicrob. Agents Chemother. 35:2306-11, 1991; Primrose and Wardlaw, Ed. "The Bacteriostatic and Bacteriocidal Action of Antibiotics" Sourcebook of Experiments for the Teaching of Microbiology Society for General Microbiology, Academic Press Ltd., London, 1982). It has been proposed that after tetracycline passes through the cytoplasmic membrane of a bacterium it chelates Mg+ 2 , and this tetracycline-Mg+ 2 complex binds the 30S subunit 2 of the bacterial ribosome (Goldman et al. Biochemistry 22:359-368, 1983). Binding of the complex to the ribosome inhibits the binding of aminoacyl-tRNAs, resulting in inhibition of protein synthesis (Wissmann et al. Forum Miroblol 292-99, 1998; Epe et al. EMBO J 3:121-26, 1984). Tetracyclines have also been found to bind to the 40$ subunit of eukaryotic ribosome; however, they do not achieve sufficient concentrations in eukaryotic cells to affect protein synthesis because they are not actively transported in eukaryotic cells (Epe et al. FEBS Lett 213:443-47, 1987). [0003] Structure-activity relationships for the tetracycline antibiotics have been determined empirically from 50 years of semi-synthetic modification of the parent structure (Sum et al. Curt. Pharm. Design 4:119-32, 1998). Permutations with the upper left-hand portion of the natural product, also known as the hydrophobic domain, have provided new therapeutically active agents, while modifications of the polar hydrophobic domain result in a loss of activity. However, semi-synthesis by its very nature has limited the number of tetracycline analogs that can be prepared and studied. H3CN CH3 H-OH
OHNH
2 OH OH 0 OH 0 0 Tetracycline (3) [0004] The tetracyclines are composed of four linearly fused six-membered rings with a high density of polar functionality and stereochemical complexity. In 1962, Woodward and co-workers reported the first total synthesis of racemic 6 desmethyl-6-deoxytetracycline (sancycline, 4), the simplest biologically active tetracycline (Conover et al. J. Am. Chem. Soc. 84:3222-24, 1962). The synthetic route was a remarkable achievement for the time and proceeded by the stepwise construction of the rings in a linear sequence of 22 steps (overall yield -0.003%). The first enantioselective synthesis of (-)-tetracycline (3) from the A-ring precursor D glucosamine (34 steps, 0.002% overall yield) was reported by Tatsuda and co-workers in 2000 (Tatsuta et al Chem. Lett. 646-47, 2000). Other approaches to the synthesis of tetracycline antibiotics, which have also proceeded by the stepwise assembly of the 3 ABCD ring system beginning with D or CD precursors, include the Shemyakin synthesis of (±)-12a-deoxy-5a,6-anhydrotetracycline (Gurevich et al. Tetrahedron Lett. 8:131, 1967; incorporated herein by reference) and the Muxfeldt synthesis of(t)-5 oxytetracycline (terramycin, 22 steps, 0,06% yield) (Muxfeldt et al. J Am. Chem. Soc. 101:689, 1979; incorporated herein by reference). Due to the length and poor efficiency of the few existing routes to tetracyclines, which were never designed for synthetic variability, synthesis of tetracycline analogs is still limited. HSOC% N.CHS - H H OH
NH
2 OH 0 OH 0 0 Sancycline (4) [0005] There remains a need for a practical and efficient synthetic route to tetracycline analogs, which is amenable to the rapid preparation of specific analogs that can be tested for improved antibacterial and potentially antitumor activity. Such a route would allow the preparation of tetracycline analogs which have not been prepared before. Summary of the Invention [0006] The present invention centers around novel synthetic approaches for preparing tetracycline analogs. These synthetic approaches are particularly useful in preparing 6-deoxytetracyclines, which are more stable towards acid and base than 6 hydroxytetracyclines. Doxycycline and minocycline, the two most clinically important tetracyclines, as well as tigecycline, an advanced clinical candidate, are members of the 6-deoxytetracycline class. 4 H3C,,N .ICH3 H30s%%N OWCH3 O OH t-BuHN NH2 OH 0 =OH 0 igecyclins HCN .ICH 3 H3 H OH N H HO= =5SH oH 0 On H (S)-Ydocycline H3CNIC CH3 HaCN NICH3 H H T zON NH2 OH OH 0 OH 0 0 (s)-minacycline The approaches are also useful in preparing 6-hydroxytetracyclines, pentacyclines, hexacyclines, CS-substituted tetracyclines, CS-unsubstituted tetracyclines, tetracyclines with heterocyclic D-rings, and other tetracycline analogs. [0007] These novel synthetic approaches to tetracycline analogs involve a convergent synthesis of the tetracycline ring system using a highly fumctionalized chiral enone (5) as a key intermediate. The first approach involves the reaction of the enone with an anion formed by the deprotonation of a toluate (6) or metallation of a benzylic halide as shown below. The deprotonation of a toluate is particularly useful in preparing 6-deoxytetracyclines with or without a C5-substituent. The metallation (e.g., metal-halogen exchange (e.g., lithium-balogen exchange), metal-metalloid exchange (e.g., lithium-metalloid exchange)) is particularly useful in preparing 6 deoxytetracyclines with or without a C5-substituent as well as pentacyclines. Any organometallic reagent may be used in the cyclization process. Particularly useful reagents may include lithium reagents, Grignard reagents, zero-valent metal reagents,
S
and ate complexes, In certain embodiments, milder conditions for the cyclization reaction may be preferred. H HCSN .CH 3 HSCN CH3 H-H O- OH
NH
2 OR' Y OH 0 OH 0 0 OR 0 0 0 O0" 6-deoxytetracycline 6 5 Os H 3 SC% 0CH 3
H
3 C .H 3 H H H H -OH Hal O + N
NH
2 OR' 5H O0P n OH 0 OH 0 0 OR 0 O 0 OBn 6-deoxytetracycline [00081 The second approach involves reacting the enone (5) in a Diels-Alder type reaction with a diene (7) or a benzocyclobutenol (8). H3CN CH3 H3CN .CH3
H
3 0 N R N SH H S F H S -H- OH 2 = - O NH2 OH 0 OH 0 0 OP 0 0 OBn 6-deoxytetracycline 5 HSCN NCH 3 H3CN CH3 H H .:H OH CH3 Y+ \ N NH2 OPY YH OP P OH 0 OH 0 0 OR O OBn 6-deoxyietracycline 5 In both these approaches, the chiral enone provides the functionalized A and B rings of 6 H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016 the tetracycline core, and the D-ring is derived from the toluate (6), benzylic halide, or benzocyclobutenol (8). In bringing these two portions of the molecule together in a stereoselective manner the C-ring is formed. These approaches not only allow for the stereoselective and efficient synthesis of a wide variety of tetracycline analogs never before prepared, but they also allow for preparation of tetracycline analogs in which the D-ring is replaced with a heterocycle, 5-membered ring, or other ring system. They also allow the prepartion of various pentacyclines or higher cyclines containing aromatic and non-aromatic carbocycles and heterocycles. [0009] Through the oxidation at C6 of 6-deoxytetracycline analogs, 6-oxytetracycline analogs may be prepared as shown in the scheme below:
H
3 Cs ACH3 H 3 Cs ,CH 3
H
3 Cs~ CH3 HO CH 3 HN CH 3 HN H 3 HN z-'H H~ HH HE OH .\ N U\N N H 2 - I OH -H OH OH 0 OH 0 0 OP OH OH 0 OP 0 OH 0 OP (-)-Tetracycline (3) 6-deoxytetracycline The 6-deoxytetracycline is transformed into an aromatic napthol intermediate which undergoes spontaneous autoxidation to form the hydroperoxide. Hydrogenolysis of the hydroperoxide results in the 6-oxytetracycline. This oxidation of 6-deoxytetracycline analogs can be used to prepare tetracyclines in which the D-ring is replaced with a heterocycle, 5 membered ring, or other ring system as well as pentacyclines and other polycyclines containing aromatic and non-aromatic carbocycles and heterocycles. [0010] The present invention not only provides synthetic methods for preparing these tetracycline analogs but also the intermediates, including chiral enones (5), toluates (6), dienes (7), benzylic halides, and benzocyclobutenol (8), used in these syntheses, and novel derivatives accessed by them. [0011] Some of the broad classes of compounds available through these new approaches and considered to be a part of the present invention include tetracyclines and various analogs. Accordingly, in one aspect, the present invention provides a compound of Formula: 7 H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016
R
2
R
3
R
4
R
5 R1, H -. H F ~ O (R7)n | N OP O OHi OP OP or a salt, isomer, or tautomer thereof; wherein:
R
1 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORA; -C(=O)RA; -CO2RA; -CN; -SCN; -SRA; -SORA; -S02RA; -NO 2 ; -N(RA)2; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio;
R
2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORB; -C(=O)RB; -CO 2 RB; -CN; -SCN; -SRB; -SORB; -SO2RB; -NO 2 ; -N(RB) 2 ; -NHC(O)RB; or -C(RB)3; wherein each occurrence of RB is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio;
R
3 hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORc; -C(=O)Rc; -CO 2 Rc; -CN; -SCN; -SRc; -SORc; -SO2Rc; -NO 2 ; -N(Rc) 2 ; -NHC(O)Rc; or -C(Rc) 3 ; wherein each occurrence of Rc is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio;
R
4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORD; -C(=O)RD; -CO2RD; -CN; -SCN; -SRD; -SORD; -SO2RD; -NO 2 ; -N(RD)2; -NHC(O)RD; or 7a H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016 -C(RD)3; wherein each occurrence of RD is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio;
R
5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORE; -CN; -SCN; -SRE; or -N(RE) 2 ; wherein each occurrence of RE is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; each occurrence of R 7 is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; -ORG; -C(=O)RG; -CO2RG; -CN; -SCN; -SRG; -SORG; -SO2RG; -NO 2 ; -N(RG)2; -NHC(O)RG; or -C(RG)3; wherein each occurrence of RG is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; P is independently selected from the group consisting of hydrogen, lower alkyl group, acyl group, or a protecting group; each occurrence of P' is independently selected from the group consisting of hydrogen or a protecting group; and n is an integer in the range of 0 to 3, inclusive: wherein each instance of aliphatic, heteroaliphatic, aryl, and heteroaryl is optionally and independently substituted with one or more groups selected from the following listing of substituents: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; -CHCl 2 ; -CH 2 OH; -CH 2
CH
2 OH; -CH 2
NH
2 ; CH 2
SO
2
CH
3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; S(O) 2 Rx; and -NRx(CO)Rx, wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl; wherein each instance of aliphatic, heteroaliphatic, arylalkyl, and heteroarylalkyl provided in the listing of substituents is independently branched or unbranched, cyclic or acyclic, and unsubstituted or substituted with one or more moieties selected from the group 7b H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016 consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; -CHCl 2 ; -CH 2 OH; -CH 2
CH
2 OH; -CH 2
NH
2 ; CH 2
SO
2
CH
3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; S(O) 2 Rx; and -NRx(CO)Rx; wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl; and wherein each instance of aryl and heteroaryl provided in the listing of substituents is independently unsubstituted or substituted with one or more moieties selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; -CHCl 2 ; -CH 2 OH; -CH 2
CH
2 OH; -CH 2
NH
2 ; CH 2
SO
2
CH
3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; S(O) 2 Rx; and -NRx(CO)Rx; wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl. Important subclasses of tetracyclines include 6-deoxytetracyclines with or without a C5 hydroxyl group, and 6-hydroxytetracyclines with or without a C5-hydroxyl group. Many of the analogs available through these new approaches have never been synthesized before given the limitations of semi-synthetic approaches and 7c earlier total syntheses. For example, certain substitutions about the D-rihg become accessible using the present invention's novel methodologies. In certain classes of compounds of the invention, the D-ring of the tetracyclines analog, which is usually a phenyl ring, is replaced with a heterocyclic moiety, which may be bicyclic or tricyclic. In other classes, the D-ring is replaced with a non-aromatic ring. The size of the D-ring is also not limited to six-membered rings, but instead it may be three-membered, four membered, five-membered, seven-membered, or larger. In the case of pentacyclines, the five rings may or may not be linear in arrangement. Each of the D- and E-rings may be heterocyclic or carbocyclic, may be aromatic or non-aromatic, and may contain any number of atoms ranging from three to ten atoms. In addition, higher cyclines such as hexacyclines may be prepared. In certain classes, the C-ring may not be fully formed, leading to dicyclines with the A-B fused ring system intact. The compounds of the invention include isomers, stereoisomers, enantiomers, diastereomers, tautomers, protected forms, pro-drugs, salts, and derivatives of any particular compound. R RR Rt 5 C H (Ry)nNH2 OG H 0 R RR R NRs)2 OH OHH 0 OH 0 O R7 R, RO R R OH NH2 =6 H Ry 0 OH 0 RR R R5 OH NH2 R7 OH R-1 0 OH 0 0 R~ RfR3 R RS OH s..N.-NH2 6H 0 OH 0 0 [0012] The present invention also includes intermediates useful in the synthesis of compounds of the present invention. These intermediates include chiral enones, toluates, benzylic halides, and benzocyclobutenol. The intermediates includes various substituted forms, isomers, tautomers, stereoisomers, salts, and derivatives thereof. [0013] In another aspect, the present invention provides methods of treatment and pharmaceutical composition including the novel compounds of the present invention. The pharmaceutical compositions may also include a pharmaceutically acceptable excipient. The methods and pharmaceutical compositions may be used to treat any infection including cholera, influenza, bronchitis, acne, malaria, urinary tract infections, sexually transmitted diseases including syphilis and gonorrhea, Legionnaires' disease, Lyme disease, Rocky Mountain spotted fever, Q fever, typhus, bubonic plague, gas gangrene, leptospirosis, whooping cough, and anthrax. In certain embodiments, the infections are caused by tetracycline-resistant organisms. In certain instances, the compounds of the invention exhibit anti-neoplastic or anti-proliferative activity, in which case the compounds may be useful in the treatment of diseases such as cancer, autoimmune diseases, inflammatory diseases, and diabetic retinopathy. The methods and compositions may be used to treat disease in humans and other animals including domesticated animals. Any mode of administration including oral and parenteral administration of the pharmaceutical composition may be used. [0014] Given past work in the synthesis of tetracyclines, the present inventive strategies represent a breakthrough, providing new synthetic routes to tetracyclines and various analogs. The ability to prepare a wide variety of tetracycline analogs and the 9 use of some of these compounds in the treatment of diseases such as cancer and infectious diseases marks an advance not only in synthetic organic chemistry but also in medicine. The tetracycline class of antibiotics has played a major role in the treatment of infectious diseases in human and veterinary medicine for the past .50 years; however, with the high use of these antibiotics over many years resistance has become a major problem. The present invention fortunately allows for the development of tetracycline analogs with activity against tetracycline-resistant organisms. Therefore, the developments described herein will allow the tetracycline class of antibiotics to remain part of a physician's armamentarium against infection diseases. Definitions [00151 Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75' Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference. [0016] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. [0017] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios 10 are contemplated for more complex isomer mixtures. [0018] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. [00191 One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group", as used herein, it is meant that a particular functional moiety, e.g., 0, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Hydroxyl protecting groups include methyl, methoxylnethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4 methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4 pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2 trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4 methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl 5,S-dioxide, 1-[(2 11i chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMPI 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl 4,7-methanobenzofuran-2-y, I-ethoxyethyl, 1-(2-chloroethoxy)ethy, 1-methyl-I methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-I-benzyloxy-2-flooroethyl, 2,2,2 trichloroethyl, 2-trimethylsifylethyl, 2-(phenylselcnyl)ethyl, t-butyl, allyl, p chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl,p-methoxybenzyl, 3,4 dimethoxybcnzyl, o-nitrobenzyl, p-nitiobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p oyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4' bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5 dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4" tris(benzoyloxypheftyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9 phenyl-1 0-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEPS), dimethylthexylsilyl, t butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p xylylsilyl, triphenylsilyI, diphenylnethylsilyl (DPMS), t-butylnethoxyphenylsilyl (TBMPS), format, benzoylfornate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6 trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-flubrenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2 (trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2 (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkylp-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o 12 nitrobenzyl carbonate, alkylp-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4 ethoxy-1 -napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4 azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2 formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6 dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1 dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl N,N,V',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,44initrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1--butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2 trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4 dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1 -ethoxyethylidine ortho ester, 1,2 dimethoxyethylidene ortho ester, a-methoxybenzylidene ortho ester, 1-(NN dimethylamino)ethylidene derivative, a-(N'-dimethylamnino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3 tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3 diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate. Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbanate, 2,7-di-t-butyl-[9-(I 0,10 dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4 methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2 trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(I-adamantyl) 1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl 2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate 13 (TCBOC), 1-methyl-i -(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl) 1-nethylethyl carbamate (t-Bumeac), 2-(2'- and 4'-pyridyi)ethyl carbamate (Pyoc), 2 (N,N-dicyclohexylcarboxamido)ethyl carbanate, t-butyl carbamate (BOC), 1 adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbanate (Alloc), 1 isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz),p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate,p-bromobenzyl carbamatep-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(I,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbanate (Bmpc), 2-phosphonioethyl carbamate (Peac), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1 -dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyi carbamate, 5-benzisoxazolylmethyl carbamate, 2 (trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N' phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p cyanobenzyl carbamate, cyclobutyl carbainate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamatep-decyloxybenzyl carbamate, 2,2 dimethoxycarbonylvinyl carbamate, o-(NN-dimethylcarboxamido)benzyl carbamate, 1,1 -dimethyl-3-(NN-dimethylcarboxamido)propy carbamate, 1,1 -dimethylpropynyl carbamnate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoboryni carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p' methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, I methylcyclohexyl carbamate, 1-methyl- 1-cyclopropylmethyl carbamate, 1-methyl-I (3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-i -(p-phenylazophenyl)ethyl carbamate, 1 -methyl- I -phenylethyl carbanate, 1-methyl- 1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzy carbamate, 2,4,6-tri-r-butylphenyl carbamate, 14 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formwnide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N' dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propananide, 3-(o nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o (benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4 tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl 1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2 (trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l-isopropyl 4-nitro-2-oxo-3-pyroolin-3-yl)amine, quatemary ammonium salts, N-benzylamine, N di(4-methoxyphenyl)nethylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N 2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2 pyridyl)mesityllmethyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N' isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5 chlorosalicylidencamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N cyclohexylideneamine, N-(5,5-dimethyl-3-oxo- I -cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, NV-[pheny(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4 15 dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4 methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesuffenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4 methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6 dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4 methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6 trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmie), mnethanesulfonamide (Ms), p-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8' dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.. Exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T.W. and Wuts, P.O., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference. [00201 It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein 16 which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for asufficient period of time to be useful for the purposes detailed herein. [00211 -The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term alkyll" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl", and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms. [0022] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkylalkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH 2 -cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert 17 butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, I -methyl-2 buten-1 -yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. [0023] The term "alkoxy", or "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4 aliphatic carbon atoms. Examples of alkoxy, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like. [0024] The term "alkylamino" refers to a group having the structure -NR', wherein R' is aliphatic, as defined herein. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic group employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n propylamino, iso-propylamino, cyclopropylamino, n-butyiamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like. [0025] The term "dialkylamino" refers to a group having the structure -NRR', wherein R and R' are each an aliphatic group, as defined herein. R and R' may be the same or different in an dialkyamino moiety. In certain embodiments, the aliphatic 18 groups contains 1-20 aliphatic carbon atoms, In certain other embodiments, the aliphatic groups contains 1-10 aliphatic carbon atoms. in yet other embodiments, the aliphatic groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups contains 1-4 aliphatic carbon atoms. Examples of dialkylamino groups include, but are not limited to, dimethylarmino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R' are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or non-aromatic. Examples of cyclic diaminoulkyl groups include, but are not listed to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl. [0026] Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; -CHCl 2 ; -CH 2 OH; -CH 2
CH
2 OH; -CH 2
NH
2 ; CH 2
SO
2
CH
3 ; -C(O)Rx; -CO2(Rx); -CON(Rx) 2 ; -OC(O)Rg; -OCO 2 R,; -OCON(Rx)2; N(Rx) 2 ; -S(O) 2
R
1 ; -NR,(CO)Rx wherein each occurrence of R,, independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein. [0027] In general, the terms "aryl" and "heteroaryl", as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously 19 mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from 8, 0, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from 8, 0, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyloxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like. [0028] It will be appreciated that aryl and heteroaryl groups can be unsubstituted or substituted, wherein substitution includes replacement of one, two, three, or more of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroatyloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO 2 ; -CN; CF 3 ; -CH 2
CF
3 ; -CHC1 2 ; -CH 2 OH; -CH 2 CH2OH; -CH 2
NH
2 ; -CH12SO2CH3; -C(O)Rx; C02(%(); -CON(RX) 2 ; -OC(O)R,; -OCO 2 R,; -OCON(R)2; -N(R0) 2 ; -S(0) 2 R.; NR(CO)R., wherein each occurrence of R, independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein. [0029] The term "cycloalkyl", as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls 20 include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic, or hetercyclic moieties, may optionally be substituted with substituents including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; -CHC 2 ; -CH 2 OH; CH 2
CH
2 OH; -CH 2
NH
2 ; -CH 2
SO
2
CH
3 ; -C(O)R.; -CO2(R,); -CON(RX) 2 ; -OC(O)R; OCO 2 R,; -OCON(R.)2; -N(R) 2 ; -S(0) 2
R
1 ; -NR,(CO)R,, wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the ary) or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein. [00301 The term "heteroaliphatic", as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthid; -F; -Cl; -Br; -1; -OH; -NO 2 ; -CN; -CF 3 ; CH 2
CF
3 ; -CHCl 2 ; -CH 2 OH; -CH 2
CH
2 0H; -CH 2
NH
2 ; -CH 2
SO
2
CH
3 ; -C(Q)R.; CO2(R,); -CON(Rx)Z; -OC(O)R; -OCO 2 RX; -OCON(R4) 2 ; -N(R) 2 ; -S(0)2R,; NRx(CO)R, wherein each occurrence of R, independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and 21 herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein. [0031] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine. [0032] The term "haloalkyl" denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, ftifluoromethyl, and the like. [0033] The term "heterocycloalkyl" or "heterocycle", as used herein, refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6 membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,.isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" group is utilized and as used herein, refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2
CF
3 ; CHC 2 ; -CH 2 0H; -CH 2
CH
2 OH; -CI 2
NH
2 ; -CH 2
SO
2
CH
3 ; -C(O)R.; -CO 2 (R); CON(R.) 2 ; -OC(O)R,; -OCO 2 Rx; -OCON(Rx) 2 ; -N(R.) 2 ; -S(0) 2 Rx; -NRx(CO)Rx, wherein each occurrence of R. independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, 22 and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein. [0034] "Carbocycle": The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is a carbon atom. [0035 "Independently selected": The term independently selected"is used herein to indicate that the R groups can be identical or different. [0036] "Labeled": As used herein, the term "labeled" is intended to mean that a compound has at least one element, isotope, or chemical compound attached to enable the detection of the compound. In general, labels typically fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes, including, but not limited to, 2 H, 3 H, ' 2 P, I-IS, Ga, ""'Tc (Tc-99m), "In, 3, 125, 69 1Yb and Is6Re; b) immune labels, which may be antibodies or antigenswhich may be bound to enzymes (such as horseradish peroxidase) that produce detectable agents; and c) colored, luminescent, phosphorescent, or fluorescent dyes. It will be appreciated that the labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound that is being detected. In certain embodiments, hydrogen atoms in the compound are replaced with deuterium atoms
(
2 H) to slow the degradation of compound in vivo. Due to isotope effects, enzymatic degradation of the deuterated tetracyclines may be slowed thereby increasing the half life of the compound in vivo. In certain embodiments of the invention, photoaffinity labeling is utilized for the direct elucidation of intermolecular interactions in biological systems. A variety of known photophores can be employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (See. Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam.), the entire contents of which are hereby incorporated by reference. In certain embodiments of the invention, the photoaffinity labels employed are o-, m- and p-azidobenzoyls, substituted with one or more halogen moieties, including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid. [00371 "Tautoners": As used herein, the term "tautomers" are particular 23 isomers of a compound in which a hydrogen and double bond have changed position with respect to the other atoms of the molecule. For a pair of tautomers to exist there must be a mechanism for interconversion. Examples of tautomers include keto-enol forms, imine-enamine forms, amide-imino alcohol forms, amidine-aminidine forms, nitroso-oxime forms, thio ketone-enethiol forms, N-nitroso-hydroxyazo forms, nitro aci-nitro forms, and pyridione-hydroxypyridine forms. [0038] Definitions of non-chemical terms used throughout the specification include: [0039] "Animal": The term animal, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). A non-human animal may be a transgenic animal. [00401 "Associated with": When two entities are "associated with"one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. [00411 "Effective amount": In general, the "effective amount" of an active agent or the microparticles refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient For example, the effective amount of a tetracycline analog antibiotic is the amount that results in a sufficient concentration at the site of the infection to kill the microorganism causing the infection (bacteriocidal) or to inhibit the reproduction of such microorganisms (bacteriostatic). In another example, the effective amount of tetracycline analog antibiotic is the amount sufficient to reverse clinicals signs and symptoms of the infection, including fever, redness, warmth, pain, chills, cultures, and pus production. 24 Brief Description of the Drawing [00421 Figure 1 shows the modular synthesis of tetracycline and tetracycline analogs starting from benzoic acid. [0043] Figure 2 depicts the total synthesis of (-)-tetracycline starting from benzoic acid and involving an o-quinone dimethide Diels-Alder reaction between the chiral enone 10 and the benzocyclobutenol 11. The overall yield for the 17 step syntheis was 1.1%. [0044] Figure 3 is the total synthesis of(-)-doxycycline in 18 steps (overall yield 8.2%). The synthesis includes the reaction of the chiral none 23 with the anion 24 to yield the tetracycline core. The first seven steps are identical to the first seven steps in the synthesis of (-).tetracycline shown in Figure 2. [0045] Figure 4 shows a first and second generation synthesis of isoxazole 4 used in the synthesis of (-)-tetracycline and (-)-doxycycline as shown in Figure 2.. [0046] Figure 5 shows the synthesis of benzocyclobutenol 11 used in the synthesis of (-)-tetracycline as shown in Figure 2. [00471 Figure 6 shows the synthesis of dicyclines. Dicyclines preserve the hydrophilic region thought to be important for the antimicrobial activity of tetracyclines. [0048] Figure 7 depicts the synthesis of tricyclines via a Diels-Alder reaction with the chiral enone 10 and a diene (41). Tricyclines preserve the hydrophobic region thought to be important for antimicrobial activity. [00491 Figure 8 shows the synthesis of pentacyclines. [0050] Figure 9 shows the synthesis of bridge pentacyclines by reacting anion 47 with a chiral enone. [0051] Figure 10 shows five compounds that may be used as analog platforms for the synthesis of tetracycline analogs. [0052] Figure 11 is a scheme showing the synthesis of a pyridone/hydroxypyridine analog of sancycline. 25 [00531 Figure 12 shows the total synthesis of 6-deoxytetracycline from benzoic acid in 14 steps (overall yield 8%). The first ten steps are identical to the first 10 steps in the synthesis of(-)-tetracycline shown in Figure 2. [0054] Figure 13A shows the synthesis of a pyridine analog of sancycline, 7 aza-10-deoxysancycline. Figure 13B shows the synthesis of 10-deoxysancycline. [00551 Figure 14A and 140 show a number of examples of heterocyclines, tetracycline analogs, pentacyclines, and polycyclines potentially accessible via the inventive method. [0056] Figure 15 shows the chemical structures of various tetracycline antibiotics. (-)-Tetracycline (1) was first produced semi-synthetically, by hydrogenolysis of the fermentation product aureomycin (7-chlorotetracycline), but later was discovered to be a natural product and is now produced by fermentation (M\4. Nelson, W. Hillen, R. A. Greenwald, Eds., Tetracyclines in Biology, Chemistry and Medicine (Birkhauser Verlag, Boston, 2001); incorporated herein by reference). (-) Doxycycline (2) and minocycline (3) are clinically important non-natural antibiotics and'are both manufactured by multi-step chemical transformations of fermentation products (semi-synthesis) (M. Nelson, W. Hillen, R. A. Greenwald, Eds., Tetracyclines in Biology, Chemistry and Medicine (Birkhauser Verlag, Boston, 2001); incorporated herein by reference). Structures 4-6 are representative of tetracycline-like molecules that cannot be prepared by any known semi-synthetic pathway, but which are now accessible by the convergent assembly depicted in Figure 153. Figure 15B depicts a generalized Michael-Dieckmann reaction sequence that forms the C-ring of tetracyclines from the coupling of structurally varied carbanionic D-ring precursors with either of the AB precursors 7 or 8. [0057] Figure 16 shows the transformation of benzoic acid in 7 steps to the key bicyclic intermediate 14. This product is then used to prepare the AB precursor enone 7 by the 4-step seqUence shown, or to enone 8, AB precursor to 6-deoxy-5 hydroxytetracycline derivatives, by the 8-step sequence shown. [0058] Figure 17 shows the synthesis of the clinically important antibiotic (-) doxycycline (2) by the convergent coupling of the o-toluate anion derived from 18 and the AB precursor enone 8. 26 [0059] Figure 18 shows the synthesis of structurally diverse 6 deoxytetracyclines by coupling of structurally diverse D-ring precursors and AB precursors 7 or S. The number of steps and overall yields from benzoic acid are shown in parentheses below each structure synthesized. MIC values (gg/mL) are also shown for whole-cell antibacterial testing of each analog against 5 Gram-positive and 5-Gram negative microorganisms. Corresponding MICs for tetracycline (1), a testing control, appear at bottom. [0060] Figure 19 shows a crystalline Michael adduct as the product of a lithium anion and a chiral enone. [00611 Figure 20 shows the synthesis of a pentacycline via a Michael Dieckman reaction sequence. [0062] Figure 21 shows the synthesis of various novel tetracycline analogs and their corresponding D-ring precursor. These compounds represent significant gaps in the tetracycline fields, likely missing from the literature for lack of a viable synthesis. [00631 Figure 22 shows alternative sequences to AB none precursors from lS,2R-cis-dihydroxybenzoic acid. [0064] Figure 23 shows novel routes to AB precursors. These routes do not involve the microbial dihydroxylation of benzoic acid. Detailed Description of Certain Preferred Embodiments of the Invention [00651 The present invention provides a strategy for the synthesis of tetracycline analogs via a convergent synthesis using as an intermediate, the highly functionalized chiral enone 9 as shown below: %3%= 0 0 OP (g) wherein R 3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or 27 unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORc; =0; -C(=O)Rc; -CO2Rc; CN; -SCN; -SRc; -SORc; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(0)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORD; =0; -C(=0)Ru; -CO 2 RD; CN; -SCN; -SR; -SORD; -SO2RD; -NO 2 ; -N(Ro) 2 ; -NHC(O)RD; or -C(RD) 3 ; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbrancbed aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SR; or N(Rs) 2 ; wherein each occurrence of R E is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups; 28 P is independently selected from the group consisting of hydrogen or a protecting group. The chiral enone 9 can be reacted with anions of phthalides, anions of toluates, benzocyclobutenole, or dienes to yield tetracycline analogs including heterocyclic tetracyclines, dicyclines, tricyclines, pentacyclines, heterocyclic pentacyclines, polycyclines, and heterocyclic polycyclines. These new compounds are tested for anti-microbial activity against microbes including traditionally tetracycline sensitive organisms as well-as organisms known to be tetracycline-resistant. Compounds found to be bacteriocidal or bacteriostatic are used in formulating pharmaceutical for the treatment of infections in human and veterinary medicine. The compounds are also tested for anti-proliferative activity. Such compounds are useful in the treatment of antiproliferative diseases including cancer, anti-inflammatory diseases, autoimmune diseases, benign neoplasms, and diabetic retinopathy. The inventive approach to the synthesis of tetracycline analogs allows for the efficient synthesis of many compounds never before prepared or available using earlier routes and semi synthetic techniques. Compounds [0066] Compounds of the present invention include tetracycline analogs, heterocyclic tetracycline analogs, dicyclines, tricyclines, pentacyclines, heterocylic pentatcyclines, bridged pentacyclines, heterocyclic polycyclines, bridged polycyclines, and other polycyclines. Particularly useful compounds of the present invention include those with biological activity. In certain embodiments, the compounds of the invention exhibit antimicrobial activity. For example, the compound may have a mean inhibitory concentration, with respect to a particular bacteria, of less than 50 pg/mL, preferably less than 25 pg/mL, more preferably less than 5 pg/mL, and most preferably less than 1 pg/mL. For example, infection caused by the following organisms may be treated with antimicrobial compounds of the invention: Gram-positivives--Staphylocococcus aureus, Streptococcus Group A, Streptococcus viridans, Streptococcus pneumoniae; Gram-negatives-Neisseria meningitidis, Netsseria gonorrhoeae, Haemophilus influenza, Escherichia coil, Bacteroidesfragilis, other Bacteroides; and Others 29 Mycoplasma pneumoniae, Treponemapallidum, Rickettsia, and Chlamydia. In other embodiments, the compounds of the invention exhibit antiproliferative activity. [0067] In certain embodiments, the tetracycline analogs of the present invention are represented by the formula: R1 RfiR 3 R5 . Ra OH (Ry).-- D - NH2 0 OH O O (10). The D-ring of 10 may include one, two, or three double bonds. In certain embodiments, the D-ring is aromatic. In other embodiments, the D-ring includes only one double bond, and in yet other embodiments, the D-ring includes two double bonds which may or may not be in conjugation. The D-ring may be substituted with various groups R7, R 6 , and R8 as defined below. In 10, R, can be hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched hetervaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=O)RA;
-CO
2 RA; -CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)z; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R, is hydrogen, In othe embodiments, R 1 is lower alkyl, alkenyl, or alkynyl. In yet other embodiments, Ri is methyl, ethyl, n-propyl, cyclopropyl, or isopropyl. In still other embodiments Ri is methyl. R% may be hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alipliatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted 30 or unsubstituted, branched or unbranched heteroaryl; -ORB; =0; -C(=O)Ra; -CO2Ra; CN; -SCN; -SR; -SOR 8 ; -S0 2
R
0 ; -NO 2 ; -N(Ra) 2 ; -NHC(O)Rn; or -C(R)]; wherein each occurrence of R1 is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthia; arylthio; amino, alkylamino, dialkylarino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R 2 is hydrogen. In other embodiments, R 2 is hydroxyl or a protected hydroxyl group. In certain embodiments,
R
2 is alkoxy. In yet other embodiments, R 2 is a lower alkyl, alkenyl, or alkynyl group. In certain embodiments, R, is methyl, and R 2 is hydroxyl. In other embodiments, R, is methyl, and R 2 is hydrogen. In certain embodiments, R, and R 2 are taken together to form a carbocyclic or heterocyclic ring system spiro-linked to 10.
R
3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORc; =0; -C(=O)Rc; -CO2Rc; CN; -SCN; -SRc; -SORc; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(0)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R 3 is hydrogen. In other embodiments, R 3 is a hydroxyl group or a protected hydroxyl group. In yet other embodiments, R 3 is alkoxy. In still further embodiments, R 3 is lower alkyl, alkenyl, or alkynyl.
R
4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORo; 0; -C(=0)RD; -CO 2
R
0 ; CN; -SCN; -SRD; -SORD; -SO 2
R
0 ; -NO 2 ; -N(RD)z; -NHIC(O)Rp; or -C(RD)3; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic 31 moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R4 is hydrogen. In other embodiments, R4 is a hydroxyl group or a protected hydroxyl group. In yet other embodiments, R4 is alkoxy. In still further embodiments, R4 is lower alkyl, alkenyl, or alkynyl. In certain embodiments, both R and R4 are hydrogen. In other embodiments, R3 and R4 are taken together to form a carbocyclic or heterocyclic ring system spiro linked to the B-ring of 10. Rs may be hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SRE; or N(RE) 2 ; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, Rs is amino, alkylamino, or dialkylamino; preferably dimethylamino, diethylamino, methyl(ethyl)amino, dipropylamino, methyl(propyl)amino, or ethyl(propyl)amino. In other embodiments, R5 is hydroxyl, protected hydroxyl, or alkoxy. In yet other embodiments, R 5 is sulfhydryl, protected sulhydryl, or alkylthioxy. R7 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORo; =0; -C(=0)RO; -CO 2
R
2 ; CN; -SCN; -SRa; -SORG; -S0 2
R
0 ; -NO2; -N(Ro) 2 ; -NHC(O)Roj; or -C(Ro) 3 ; wherein each occurrence of R 0 is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylanino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R7 is hydroxyl, protected hydroxyl, 32 alkoxy, lower alkyl, lower alkenyl, lower alkynyl, or halogen. R6 and Rs are absent if the dashed line between the carbon atoms which R6 and Rs are attached to represents a bond, or are each selected independently from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, SH, alkylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups. In certain embodiments, R6 and R 8 are absent, In other embodiments, R6 or R8 is absent. [00681 The variable n is an integer in the range of 0 to 8, inclusive. As will be appreciated by one of skill in the art, when the D-ring is aromatic n is an integer between 0 and 4, preferably between 1 and 3, more preferable between 1 and 2. In certain embodiments, when n is 2, the substituents R7 are in the ortho configuration. In other embodiments, when n is 2, the substituents R7 are in the para configuration. And in yet other embodiments, when n-is 2, the substituents R7 are in the meta configuration. [00691 A dashed line in formula 10 may represent a bond or the absence of a bond. [00701 As will be appreciated by one of skill in this art, compounds of fonnula 10 include derivatives, labeled forms, salts, pro-drugs, isomers, and tautomers thereof. Derivatives include protected forms. Salts include any pharmaceutically acceptable salts including HC, HBr, HI, acetate, and fatty acid (e.g., lactate, citrate, myristoleate, oleate, valerate) salts. In certain embodiments, the inventive compound exists in zwitterionic form at neutral pH with the R5 being a protonated amino group and the C-3 hydroxyl group deprotonated as shown in formula 10a. R1 RR 3 H(RE)2 (Rr)n NH 0 OH 0 0 (10a) Isomers include geometric isomers, diastereomers, and enantiomers. Tautomers include both keto and enol forms of carbonyl moieties as well as various tautomeric forms of substituted and unsubstituted heterocycles. For example, the B-ring as shown 33 in formula 10 includes an enol moiety as drawn, but the enol may exist as the keto form in certain compounds as shown below in formula 10b and 10c: R3 R R R R5 OH (Ry)n- -H OH 0 0 0 (10b) R, R R R R5 OH (Ry)n- NH R NH2 OH 0 0 O (10C) Other tautomeric forms will be appreciated by one of skill in the art and will depend on the substitution patten of the core ring structure. The formulae drawn are only given as examples and do not in any way represent the full range of tautomers that may exist for a particular compound. [0071] Various subclasses of compounds of the formula 10 which include a substituted or unsubstituted aromatic D-ring are shown below. These subclasses include unsustituted, monosubstituted, disubstituted, and trisubstituted D-ring. R R RR 3 RR 5 - R 3 R R 5 - -5 OH OH NH2 NH2 6H OH 0 OH 0 0 R7 0 OH 0 O RI RRe R% Rs RI RhR R R OH R7 OH NH2 NH2 R7 6H O 0 OH 0 O 0 OH 0 O 34 7 Rl R Rs Rl R R R R6 ON OH OH NH2 OH R7 NH2 OH 0 0 0 nm n n R7 OH 0 0 F j R R R Rs 7 R R5 R7 RI R R DH NH2 OH NH2 5H SH OH R? a OH 0 R7 0 OH 6H 0 Rl RhR3 R5 Ry R7 Rl R R R Rs OH : o OH 7 . - I NH2 I I OH R7--- NH2 0 OH 0 0 0 OH OH 0 0 7 Rl R R R RS Ri R % % RS R?, ON Rl OH R" NH2 R7'---- NH2 OH 0" a OH' 0 0 R7 0 OH 6H 0 7 F j R R R R5 7 Rs Rl R R R R7 ON ON R7 NH2 R7 NH2 OH 0 ON 0 OH y 0 R7 0 H 0 0 7 F j R R R5 R7 R R R Rs R7 ON R7 OH OH z NH2 R7 NH2 ON 0 OH 0 0 0H 7 R7 0 OH 0 0 35 wherein the definitions of RI,, R2, R3, R 4 , and Rs are as described above, and R7 is halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted; branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORa; =0; -C(=O)R; -CO 2
R
0 ; -CN; -SCN; -SR 0 ; -SORo; S02R; -NO2; -N(Ro) 2 ; -NHC(0)Rg; or -C(R) 3 ; wherein each occurrence of R is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R 7 is hydroxyl, protected hydroxyl, alkoxy, lower alkyl, lower alkenyl, lower alkynyl, or halogen. In other embodidments, R 7 is cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; or cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic. In yet other embodiments, R 7 is amino, alkylamino, or dialkylamino. In other embodiments, R 7 is substitued or unsubstituted cyclic, heterocyclic, aryl, or heteroaryl. In certain embodiments, R7 is branched or unbranched acyl. 100721 Various subclasses of compounds of the formula 10 which include a hydroxyl group at C10 are shown: R1R R R N (RE)2 OH NH2 OH OH 0 OH 0 O R7 R, R3R R N(RE)2 NH2 =0 H 36 RI R R R N(RE)2 T OH Ry NH2 6H OH 0 OH 0 R, RfR R tt(RE)2 Ry OH NH2 P=OH OH 0 OH 0 0 R~ RfP 0 N(RE)2 OH NH2 O0H R7 0 OH 0 O H3C O R(RE)2 OH (Ry)n--H OH OH 0 OH 0 0 R-1 R N(RE)2 - H : OH (Ry)nII NH2 OH. OH 0 OH 0 0 H3C H N(RE)2 OH (Ry)nII NH2 5H OH O OH 0 0 37 R RR 3 R SRE OM (Ry)n HNR OH OH 0 OH 0 0 R R RR R (RE OH (Ry)n-NH 8NH OH OH 0 OH 0 0 wherein the definitions of RI, R2, R3, R4, R5, RE, and R7 are as described above. In certain embodiments, the compounds are 6-deoxytetracyclines as shown in the HR R3 (RE)2 HO NH2 OH formulae below: OH 0 OH 0 O R7 R1 R3 (RE)2 HO NH2 OH OH 0 OH 0 Rl HR3 R N(RE)2 OH R NH2.
OH
OH 0 OH 0 O R1 HR3 R N(RE)2 R7 OH NH2 OH 0 OH 0 O 38 R1 H.Rs N(RE)2 HC R ~± N(R) 2 HOH
NH
2 ~5H R O0 OH 0 0 H3 R3 R (RE)2 OH (R)n
NH
2 NNH2 6H OH 0 OH 0 0 R1 N(RE)2 = H H E OH (Ry)n H OH -OH. O OH 0 0 HsO N(RE)2 V OH (R7)n NH2 OH OH 0 OH 0 0 !11 HR3 R QRE OH (Ry)n- -H OH OH 0 OH 0 0 Rl HR3 Rf SRE 0 H (R7)n NH2 OH 0 OH O 0 wherein R 2 is hydrogen, and the definitions of RI, R,, R4. Rs, RE, and R 7 are as described above. 39 f0073] In another aspect of the invention, the carbocyclic D-ring of tetracycline is replaced with a heterocyclic or carbocyclic moiety as shown in formula (11): RI R R Rt R5 ... ?- i ^ OH oHH 0 OH 0 0 (11). The definitions of Ri, R2, R 3 , R4, and Rs are as described above for formula 10. The D ring represented by ---- can be a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic moiety, in which each occurrence ofX is selected from the group consisting of-C-, -S-, -NR-, -C(R 7 )z-; n is an integer in the range of I to 5, inclusive; and the bonds between'adjacent X moieties are either single or double bonds. In certain embodiments, ' -- is a polycyclic ring system such as a bicyclic or tricyclic moiety. In other embodiments, '--- is a monocyclic moiety. In yet other embodiments, -- is a substituted or unsubstituted heterocyclic moiety. In certain embodiments, --- is not a substituted or unsubstituted phenyl ring. In other embodiments, --- is a pyridinyl moiety as shown:
(R
7 )-( ((Rny)n(R (R) NN 4 4 In another embodiment, ' -- is selected from the group consisting of 40 H NN (Ra)n ) (R)n (R8)n- (R hr ) HNN 0 OH 0 0 N N (Rs)n (Ra)n Rn-- (RO)n N N (Ry)n (Ry)n OH OH. In yet another embodiment, s-- a five-membered heterocyclic ring selected from the group consisting of: (R)n Nk (R7)n (Ry)n (Ry)n (R 7 )n RZN R7 Ry (Ry)n (Ry)n (Ry)nt (Ryn (7n Ry (Ry)n (R7)n N (Ry)n OD (Ryf)n KS) (Ry)nN S 0 N N N Various tetracyclines (heterocyclines) of the invention are also shown in Figure 14. [00741 Other compounds of the invention include pentacyclines of the formula: 41 R R 3 R R .T OH -..-- N-.NH2 OH 0 OH 0 O wherein RI, R 2 , R 3 , R 4 , R5, and --- are as defined above. In certain embodiments, the rings of the compound are linear. In other embodiments, the ring system is not linear. Each occurrence of the ring ---- ,in certain embodiments, is a monocyclic ring system. Each occurrence of '---- is heterocylic or carbocyclic. '---- is three membered, four-membered, five-membered, six-membered, or seven-membered; preferably, five-membered or six-membered. Other classes of pentacyclines include compounds of the formulae (12), (13), and (14):
R
7
R
1 R R R NH2 OH
R
7 0 OH 0 0 (12)
R
7 0 OH 0 0 (13)
R
7
R
1 RkR 3 Rh t5 2OH NH2 5H O Ry O OH O O (13) 4NH wherein Ri, R 2 , R3,14, Rs, and R7 are as defined above. In formulae 12,13, and 14, --- represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic moiety, in which each occurrence of X is selected from the group consisting of -0-, -S-, -NR 8 -, -C(Rs)2-; n is an integer in the range of I to 5, inclusive; and the bonds between adjacent X moieties are either single or double bonds. In certain embodiments, '---- is a polycyclic ring system such as a bicyclic or tricyclic moiety. In other embodiments, ---- is a monocyclic moiety. In other embodiments, 'is a substituted or unsubstituted, aromatic or nonaromatic carbocyclic moiety, for example a phenyl ring. In yet other embodiments, '--- is a substituted or unsubstituted heterocyclic moiety. In certain embodiments, '--- is not a substituted or unsubstituted phenyl ring. In other embodiments, '---- is a pyridinyl moiety as shown: NN (Rsn--- (s~n(RS)n (Ro)n In another embodiment, '--- is selected from the group consisting of 43 H ( R (R)n(R r o OH 0 0 0 0 (RB)n (Rs)n RS)n RnI (Ra)n (R)n OH OH. In yet another embodiment - is a five-membered heterocyclic ring selected from the group consisting of: (RO)n N (RSa (RS)n (Ra)n(R n Na R (Rs)n% 0 (Rs)r (R) 0 (Ra)nN
(R
8 )n ( R a ) n N ( Ra n ( Ra)n 0 ( R ) n N ( R 5 ) n N
-
N NN [0075] Various subclasses of the formula (12) include: 44
R
7
R
1 RR R R OH 's. NH2 OH OH 0 OH 0 0 RR R 1
RR
3 Rs OH NH2 OOH H 0 OH 0 0 R7 R R 1
NR
3 (RE)2 - - 'OH NH2 R7 0 OH 0 0 R7 RI RjR3 N(RE)2 OH 's. NH2 OH OH 0 OH 0 O 45 R7 Ri RORR R - - -OH (Rs)n- NH2 OH R7 0 OH 0 O anid R7 R RR R5 OH (Ra)n NH2 OH OH 0 OH 0 O0 [0076] Various subclasses of the formula (13) include:, 45 F j R Rs OH NH2 R7 OH OH 0 OH 0 0 RI R?,R3 R5 OH NH2 z 0 H 0H 0 OH 0 0 R, t!(RE)2 RR*j % OH NH2 F)H R7 0 OH N(RE)2 F I" RT T OH NH2 R7 OH OH 0 OH 0 0 F j, RfiR3 R R5 (RO)n OH NH2 7 OH R7 0 OH 0 0 and R5 Rl RfiR3 (Ra)n OH NH2 R7 OH OH 0 OH 0 0 46 10077] Various subclasses of the formula (14) include:
R
1 _ R R 3
R
1 , Rs R OH OH
R
7
R
1 RRN NH2 H O OHO O
R
7
R
1 RhR R E R OH NH2 OH 0 OH 0 O R7 RR R N(RE)2 R7R OH .NH2 0 OH O 0 O R7 R3 R RE R R7 NH2 --.. EOH (R~n | 0 OH 0 O0n Ry7 OH NH2 "R~ n 0 O H 0 On R47 [0078] In certain embodiments, the tetracycline analogs of the present invention are represented by the formula: RR1 HR3 R R5 +X OH (Rr);- DI H Re OH O 0 OH 0 0 wherein X is nitrogen, sulfur, and oxygen, and RI, R3, R4, Rs, R6, R7, Rs, and n are defined as above with the caveat that when X is S or 0, R, is absent. [00791 Other classes of compounds of the invention include dicyclines of the formula (15). H R9 OH R1s NH2 OP O OP 0 O0 15 wherein R 3 , R4, and R, are as defined above. P is hydrogen or a protecting group. R, is hydrogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR 1 ; -CN; -SCN; -SRI; or -N(RI) 2 ; wherein each occurrence of R is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R9 is hydrogen or lower (Ci-C6) alkyl, alkenyl, or alkynyl. In other embodiments, R, is a vinyl group. In yet other embodiments, R, is a substituted or unsubstituted aryl group. In still other embodiments, R, is a substituted or unsubstituted heterocyclic group. 48 Rio is cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstitued, branched or unbranched aryl; or substituted or unsubstituted, branched or unbranched heteroaryl moiety. In certain embodiments, Rio is a substituted or unsubstituted phenyl ring. In certain embodiments, Rio is a substituted or unsubstituted heterocyclic ring. In certain embodiments, Rio is a substituted or unsubstituted aryl ring. In other embodiments, RIO is a lower (C-C 6 ) alkyl, alkenyl, or alkynyl group. Methods of Synthesis [0080] The present invention also includes ali steps and methodologies used in preparing the compounds of the invention as well as intermediates along the synthetic route. The present invention provides for the modular synthesis of tetracyclines and its various analogs by joining a highly functionalized chiral enone, which will become the A- and B-rings of the tetracycline core, with a molecule which will become the D-ring of the tetracycline core. The joining of these two intermediates results in the formation of the C-ring, preferably in an enantioselective manner. This methodology also allows for the synthesis of pentacyclines, hexacyclines, or higher ring systems as well as the incorporation of heterocycles into the ring system. In particular, the joining of these two fragments includes various nucleophilic addition reactions and cycloaddition reactions with enone (9) as described above. [0081] The synthesis begins with the preparation of the enone (9) starting from benzoic acid. As shown in Figure 2, the first step of the synthesis involves the microbial dihydroxylation of benzoic acid using Alcaligenes eutrophus. The diol (1 in Figure 2), which is preferably optically pure, then undergoes hydroxyl-directed epoxidation to yield the allylic epoxide (2 in Figure 2). Protection and rearrangement of allylic epoxide 2 yielded the isomeric allylic epoxide (3 in Figure 2). The metalated isoxazole (4 in Figure 2) was added to the isomeric allylic epoxide to yield 5 (Figure 2), which was subsequently metalated to close the six-membered ring by nucleophilic attack of the epoxide. The intermediate 6 (Figure 2) was then rearranged, deprotected, and oxidized to yield the chiral enone 9 (Figure 2). As will be appreciated by one of 49 skill in this art, functionalization and rearrangement of intermediates 6, 7, 8, and 9 in Figure 2 will allow for the preparation of different class of compounds of the invention. [0082] In one embodiment, enone (9) is reacted with an anion resulting from the deprotonation of toluate (6). The toluate of formula:
R
1 (R)n R OP 0 wherein R 1 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=0)RA; -CO 2 RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA) 2 ; -NHC(0)RA; or -C(RA) 3 ; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
7 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORo; 0; -C(=O)Ro; -CO 2 Ro; CN; -SCN; -SRo; -SOR 0 ; -SO2R 0 ; -NO 2 ; -N(Ra)2; -NHC(0)Ro; or -C(Ro) 3 ; wherein each occurrence of Ro is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and n is an integer in the range of 0 to 3, inclusive; R is -- OR,; -CN; -SCN; -SRI; or -N(Ri) 2 ; wherein each occurrence of RI is 50 independently a hydrogen, a protecting group; a cyclic or acyclic, substituted or unsubstituted aliphatic moiety; a cyclic or acyclic, substituted or unsubstituted aliphatic heteroaliphatic moiety; a substituted or unsubstituted aryl moiety; or a substituted or unsubstituted heteroaryl moiety; and P is selected from the group consisting of hydrogren, lower (C -C 6 ) alkyl group, an acyl group, and a protecting group; is deprotonated under basic conditions (e.g., LDA, HMDS), and the resulting anion is reacted with an enone of formula: RR RS
-
0 R6 0 OP OP wherein R 3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORc; -0; -C(=0)Rc; -CO2Rc; CN; -SCN; -SRc; -SORc; -SOzRc; -NO 2 ; -N(Rc) 2 ; -NHC(O)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORD; =0; -C(0)Rr,; -CO 2 Rp; CN; -SCN; -SRD; -SOR; -SO 2 RD; -NO 2 ; -N(RD)2; -NHC(0)RD; or -C(RD) 3 ; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; 51 alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, beteroaryloxy; or heteroarylthio moiety; R, is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SRE; or N(RS)2; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroary moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroaryltbio moiety; R6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups; and P is independently selected from the group consisting of hydrogen or a protecting group; to form the product: R R R5 OP\ OP 0 OH 0 wherein RI, R3, R 4 , R5, R 7 , P, and n are as defied above; R2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORB; ;tO -C(=O)Rs; -CO 2 Ra; CN; -SCN; -SR; -SORB; -SO2RB; -NO2; -N(Rs) 2 ; -NHC(O)ka; or -C(Rs) 3 ; wherein each occurrence of Ra is independently a hydrogen, a protecting group, an aliphatic 52 moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. As will be appreciated by one of skill in this art, the toluate may be further substituted in certain embodiments. In addition, the phenyl ring of the toluate may be substituted for an aromatic heterocyclic ring such as as pyridine ring as shown in Figures 11 and 13. Other examples of carbocyclic and heterocyclic analogs of toluate (6) include: R1 R1 R1 R1 .7 KN (Ry)n (Ry) 7)n-i (R)n R9 N/ A N 9 R R9 o 0 0 0 RI R- R1 R1 N N (Ry)n) (R7)n I 4 Rg N /R R9 o OP 0 OP 0 OP 0 R1 R1 R1 R1 N N (Ry)0-;r ( I (Ry),-r N R aK n RAR 7 N R O R N o 0 0 0 0 0 R1 R 1 R I R 1 (R?)nJ <N 0 (R7)n 1Re N (Ry)n (R7),- Rg OR Rg RS
N
7 0 0 0 0 0 0 Other toluates are shown in Figure 21. In certain embodiments, polycyclic toluates are used in the Michael-Dieckmann reaction sequence to form pentacyclines, hexacyclines, or higher cyclines. Toluates useful in preparing pentacyclines are exemplified by-the 53 formula: R R 1 - Rr O
R
7 0 wherein R 1 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=O)RA; -COzRA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)2; -NHC(0)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each R 7 is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR; =0; -C(=0)Ra;
-CO
2
R
0 ; -CN; -SCN; -SR 0 ; -SORG; -SO 2
R
0 ; -NO 2 ; -N(Ro)2; -NHC(0)Ro; or -C(Ro) 3 ; wherein each occurrence of Rr is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; -- represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic moiety, in which each occurrence of X is selected from the group consisting of -0-, -S-, -NR 8 -, -C(Rs)2-; Rs is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, 54 branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatie; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORe; =0; -C(=0)RH; -CO2RH; CN; -SCN; -SRH; -SORH; -SO2RH; -NO 2 ; -N(RH)2; -NHC(O)Rn; or -C(RH)3; wherein each occurrence of RH is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; n is an integer in the range of 1 to 5, inclusive; and the bonds between adjacent X moieties are either single or double bonds; and R9 is selected from the group consisting of substituted or unsubstituted aryl or heteroaryl groups. [00831 In another embodiment, enone (9) is reacted with an anion, which is generated through metallation (e.g., metal-halogen exchange, metal-metalloid exchange, lithium-halogen exchange, lithium-tin exchange, etc. by reacting the toluate with the appropriate metal reagent) of a toluate of the the following formula: .R1 Y (Ry)n. Rs 0 wherein R, is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or imbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=Q)RA; -CO2RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA) 2 ; :NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; 55 alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R7 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORG; =0; -C(=0)Ro; -CO2Ro; CN; -SCN; -SRo; -SORG; -SO2R; -NO 2 ; -N(Rr) 2 ; -NHC(O)Ro; or -C(RG) 3 ; wherein each occurrence of&u is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; n is an integer in the range of 0 to 3, inclusive; R9 is selected from the group consisting of substituted or unsubstituted aryl or heteroaryl groups; and Y is a halogen or Sn(Ry)3, wherein Ry is alkyl. The anion generated is reacted with an enone of formula:
R
3 R R O\ N Re 0 OP 0 . wherein R3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbmnched heteroaryl; -ORc; =0; -C(=O)Rc; -CO2Rc; CN; -SCN; -SRc; -SORc; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(O)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; 56 or heteroarylthio moiety; R4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR,; =0; -C(=O)RD; -CO2RD; CN; -SCN; -SRD); -SORD; -SO 2 Rn; -NO 2 ; -N(RD) 2 ; -NHC(O)RD; or -C(RD) 3 ; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; aryithio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio.moiety; R5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or branched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SRE; or N(RE)2; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups; and P is independently selected from the group consisting of hydrogen or a protecting group; to generate the product of formula:
R
1 R R R R5 0 OH O 57 wherein R 1 , R3, R4, ItR7, P, and n are as defined above; and
R
2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORB; =0; -C(=O)Rg; -CO 2 RB; CN; -SCN; -SRB; -SORB; -SO 2 RB; -NO 2 ; -N(R)2; -NHC(O)Ra; or -C(RB)3; wherein each occurrence of R is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. [0084] Any metal may be used in the metallation reaction to generate the metal anionic reagent to be reacted with the enone. In certain embodiments, the metal is a Group I element on the periodic chart. In other embodiments, the metal is a Group II element on the periodic chart. in other embodiments, the metal is a transition metal. Exemplary metals useful in the metallation reaction include sodium, lithium, calcium, aluminium, cadmium, copper, beryllium, arsenic, antimony, tin, magnesium, titanium, zinc, manganese, iron, cobalt, nickel, zinc, platinum, palladium, mercury, and ruthenium. In certain prefared embodiments, the metal is chosen from lithium, magnesium, titanium, zinc, and copper. In yet other embodiments, the metal is magnesium, lithium, sodium, beryllium, zinc, mercury, arsenic, antimony, or tin, in certain particular embodiments, a lithium-halogen exchange is used. The lithium halogen exchange may be performed in situ in the presence of the enone. The lithium halogen exchange may be preformed using any lithium reagent including, for example, alkyllithium reagents, n-butyllithium, t-butyllithium, phenyl lithium, mesityl lithium, and methyllithium. In certain embodiments, other organometallics reagents are generated and reacted with the enone. Examples include Grignard reagents, zero-valent metal complexes, ate complexes, etc. In certain embodiments, the metal reagent is a magnesium reagent including, but not limited to, magnesium metal, magnesium anthracene, activated magnesium turnings, etc. In certain embodiments, the reagent is zinc-based. The reagent may be generated in situ in the presence of the enone, or the 58 reagent may be generated separately and later contacted with the enone. In certain embodiments, milder conditions for the cyclization are used (e.g., a zinc reagent). [0085] As will be appreciated by one of skill in this art, the toluate may be further substituted in certain embodiments. In addition, the phenyl ring of the toluate may be substituted for an aromatic heterocyclic ring or ring system such as a pyridine ring. Examples of carbocyclic and heterocyclic analogs of toluate include: R1 R1 R1 R1 Y Y Y NY (RyOn (R~-R7)n-- R7)n--9 R9,N 4/ R R9 N t o 0 0 0 R1 R1 R1 R1 NY Y Y NY (Ry)7R)n R7)n-g (Ry~n R N N R9 R9 O OP O OP O OP O R1 R1 R1 R1 N N (Ry)n- (Ry),
(R
7
).-
N 9 R R 8 0 R N o 0 0 0 o 0 R1 R1 R7 R1 R1 N (Ry)n N y (Ry7)n N (Ry~n (Ry).
N9 R Rs(7) Re R9 Ry 00 0 0 0 0 In certain embodiments, the halogen Y is bromine. In other embodiments, Y is iodine. In yet other embodiments, Y is chloride. In certain embodiments, Y is a metalloid (e.g., tin, selenium, tellurium, etc.). In certain embodiments, Y is -SnR 3 , wherein each occurrence of R is independently alkyl (e.g., -Sn(CH 3 )3). After the metallation 59 reaction, Y is a metal such as lithium, magnesium, zinc, copper, antimony, sodium, etc. In certain embodiments, R, is hydrogen or lower alkyl (C.C). In certain particular embodiments, R, is hydrogen. Other toluates are shown in Figure 21. [00861 In other embodiments, polycyclic toluates may be used to prepare pentacyclines, hexacyclines, or highe cyclines. Toluates useful in the preparation of such cyclines are of the formula: Rr R 1
R
7 0 wherein RI is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=O)RA; -CO2RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)2; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each R7 is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORO; =0; -C(=0)R0; -CO2R; -CN; -SCN; -SR; -SORG; -SO2R; -NO 2 ; -N(Ro)2; -NHC(O)Ro; or -C(&)3; wherein each occurrence of Ro is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; 60 -- represents a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic moiety, in which each occurrence of X is selected from the group consisting of -O-, -S-, -Nl,-, -C(Rs)-; Rs is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORH; =0; -C(='O)RH; -CO2RH; CN; -SCN; -SRH; -SORH; -SO 2 RH; -NO 2 ; -N(RH)2; -NHC(O)RH; or -C(RH) 3 ; wherein each occurrence of RH is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; n is an integer in the range of 1 to 5, inclusive; and the bonds between adjacent X moieties are either single or double bonds;
R
9 is selected from the group consisting of substituted or unsubstituted aryl or heteroaryl groups; and Y is a halogen or Sn(Ry)3, wherein Ry is alkyl. In certain embodiments, the halogen Y is bromine. In certain embodiments, the halogen Y is bromine. In other embodiments, Y is iodine. In yet other embodiments, Y is chloride. In certain embodiments, Y is a metalloid (e.g., tin, selenium, tellurium, etc.). In certain embodiments, Y is -SnR 3 , wherein each occurrence of R is independently alkyl (e.g., Sn(CH 3
)
3 ). After the metallation reaction, Y is a metal such as lithium, magnesium, zinc, copper, sodium, mercury, antimony, etc. In certain embodiments, R, is hydrogen or lower alkyl (CI-C 6 ). In certain particular embodiments, R, is hydrogen. In certain embodiments, R 9 is phenyl or substituted phenyl. In certain embodiments, ortho-R 7 is alkoxy such as methoxy. In other embodiments, R 7 is hydrogen. Exemplary polycyclic toluates include: 61
R
7
R
1 R R, R 7
R
1 (R),-- (R), N (Rs)n N 9
R
7 0- R 7 0 R 7 0 Ry R 1 Ry R 1
R
7
R
1 Y Y N Y (Ra)n (RS) - (R) A n R9 N R9 N R9
R
7 0 R 7 Ry 0
R
7 Rt Ry R 1
R
7
R
1 N N (RS)n (RA)n (Ra)nT ( NYj NRs' OR9 NORN R9 N R1
R
7 0 R 7 0 0 R 7 0
R
7
R
1
R
7 R R 7
R
1 an N y (R)n Ny (RS) \ Y (Ra)- 0 (RR)N N / 0 RO
R
7 0 R 7 0 R 7 0 [0087] Compounds of the formula below with a heterocyclic C-ring: RI R13 R R5s RS I H X OH' (Ry)- D IC NH2 R6 OH 6H 0 O may be prepared by Michael-Dieckmann closure of a D-ring precursor derived from the corresponding anilide, phenol, or thiophenol. A representative example using anthranilic acid (i.e., anilide as the nucleophile in the Michael addition reaction) is shown below: 62 H3CN .,CH 3
H
3 C N.CH 3 H T H H H NH 2 p t. Base N T OH F T V , 2. Dapwotection N/ II NH 2
CO
2 Ph O83P O)H oP o 0 OH 0 OH 0 0 [0088] In another embodiment, the enone (9) is reacted with a benzocyclobutenol in an o-quinone dimethide Diels-Alder reaction. The enone of formula:
R
3 Rf N(Rs) 2 O N RG j OP 0 0 OP wherein R 3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORc; =0; -C(=0)Rc; -CO2Rc; CN; -SCN; -SRk; -SORc; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(O)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylanino, heteroaryloxy; or heteroarylthio moiety; R4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORD; =0; -C(=0)Ro; -CO2Ro; CN; -SCN; -SRD; -SORD; -SO2RD; -NO 2 ; -N(RD)2; -NHC(O)R-D; or -C(RD)3; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic 63 moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR; -CN; -SCN; -SRE1; or N(RE) 2 ; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a 'heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups; P is independently selected from the group consisting of hydrogen or a protecting group; is reacted under suitable conditions (e.g., heat) with a benzocyclobutenol of formula: .,R1 OP
OP
wherein R 1 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=O)RA; -CO2RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)2; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; 64 alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R7 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR 0 ; =0; -C(=0)Ro; -CO 2 RO; CN; -SCN; -SRO; -SOR; -SO2RO; -NO 2 ; -N(Ra)2; -NHC(0)Rg; or -C(Ro) 3 ; wherein each occurrence of Ra is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; P are each selected independently from the group consisting of hydrogen or a protecting group; and n is an integer in the range of 0 to 3, inclusive; to form the product of formula: (Ry)n- N Rs OP OP oP Op 0 o wherein R 1 , R3, R 4 , Rs, R 6 , R7, and P are defined as above; and R2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORB; =0; -C(=O)Ra; -CO2Ra; CN; -SCN; -SRa; -SORB; -S02A; -NO 2 ; -N(R)2; -NHC(0)RB; or -C(RB)3; wherein each occurrence of R is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio;. amino, alkylamino, dialkylamino, heteroaryloxy; 65 or heteroarylthio moiety. As will be appreciate by one of skill in this art, the reactants may be substituted further and still fall within the claimed invention. For example, the phenyl ring of the benzocyclobutenol ring may be futher substituted. [0089] In another embodiment, the enone is reacted with a diene in a Diels Alder reaction to yield a tricycline. The enone of formula:
R
3 R R 5 N o O wherein R 3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched beteroalipliatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORc; =0; -C(=O)Rc; -CO 2 Rc CN; -SCN; -SRc; -SORC; -SO2Re; -NO 2 ; -N(Rc)2; -NHC(O)Rc; or -C(Rc)3; wherein each occurrence of Re is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORD; tQ; -C(=O)RD; -CO2R; CN; -SCN; -SRD; -SORD; -SO 2 Rn; -NO 2 ; -N(Ro) 2 ; -NHC(O)RD; or -C(Rn) 3 ; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylanino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, 66 branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SRE; or N(RE)2; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthioxy, -NO 2 , amino, alkyl amino, and dialkyl amino groups; are as defined above; and P is independently selected from the group consisting of hydrogen or a protecting group; is reacted under suitable conditions (e.g., heat) with a diene of formula: R1 OP wherein R is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(=O)RA; -CO2RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)z; -NHC(0)RA; or -C(RA)s; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; aryithio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and P are each selected independently from the group consisting of hydrogen and 67 protecting groups; to yield a protected tricycline of formula: R RR 3 R I | N wherein R 2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroary]; -OR; =0; -C(=O)RB; -CO 2
R
9 ; CN; -SCN; -SRa; -SORB; -SO2Rs; -NO 2 ;--N(Ra) 2 ; -NHC(0)Ra; or -C(R)3; wherein each occurrence of RB is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. As will be appreciated by one of skill in this art, the enone and diene may be further substituted and still be encompassed within the present invention. [0090] In yet another embodiment, the enone is reacted with an anion of a phthalide or cyano-phthalide. The enone of formula: R3R~ Rs N Rs 5P O 0 OP wherein R 3 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORC; =0; -C(=O)RC; -CO 2 Rc; CN; -SCN; -SRC; -SOk; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(O)Rc; or -C(Rc)3; wherein 68 each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR,; =0; -C(=0)RD; -CO 2 RD; CN; -SCN; -SRo; -SORD; -SOzRD;. -NO 2 ; -N(RD)2; -NHC(O)RD; or -C(Rn) 3 ; wherein each occurrence of RD is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORE; -CN; -SCN; -SRE; or N(RE)2; wherein each occurrence of RE is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R6 is selected from the group consisting of hydrogen, halogen, substituted or unsubstitued aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted alkoxy, -OH, -CN, -SCN, -SH, alkylthio, arylthio, -NO 2 , amino, alkyl amino, and dialkyl amino groups; and P is independently selected from the group consisting of hydrogen or a protecting group; is reacted under basic conditions (e.g., LDA, Ph 3 CLi) with the anion of the phthalide of formula: 69 R1 (Rr)n OP 0 wherein RI is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -ORA; =0; -C(-0)RA; -CO2RA; CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA)2; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;
R
7 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR 0 ; =0; -C(=O)Ro; -CO2RJ; CN; -SCN; - 51 o; -SOR 0 ; -SO2Ro; -NO 2 ; -N(RG) 2 ; -NHC(O)Ro; or -C(Ro)3; wherein each occurrence of Ro is independently a hydrogen, aprotecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; P are each selected independently from the group consisting of hydrogen, lower alkyl group, acy group, or a protecting group; and n is an integer in the range of 0 to 3, inclusive; to yield a product of formula: 70 R RR
R
S (Rr)n- N OP 0 OH 0 OP wherein R2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR 8 ; =0; -C(=O)R 3 ; -CO 2 RB; CN; -SCN; -SRB; -SORB; -SO2Rn; -NO 2 ; -N(Ra) 2 ; -NHC(O)Rs; or -C(Rs)3; wherein each occurrence of R 3 is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylanino, heteroaryloxy; or heteroarylthio moiety. [0091] The products of the above reactions are then further finctionalized, reduced, oxidized, rearranged, protected, and deprotected to yield the final desired product. Various exemplary reactions used in the final syntheses of the compounds of the invention are shown in Figure 2, 3, 11, 12, and 13. As will be appreciated by one of skill in the art, various isolation and purification techniques including flash chromatography, crystallization, distillation, HPLC, thin layer chromatography, extraction, filtration, etc. may be used in the course of synthesizing compounds of the invention. These techniques may be used in the preparation or purification of intermediates, reagents, products, starting materials, or solvents. Pharmaceutical Compositions [0092] This invention also provides a pharmaceutical preparation comprising at least one of the compounds as described above and herein, or a pharmaceutically acceptable derivative thereof, which compounds inhibit the growth of or kill microorganisms, and, in certain embodiments of special interest are inhibit the growth of or kill tetracycline-resistant organisms including chlortetracycline-resistant 71 organisms, oxytetracycline-resistant organisms, demeclocycline-resistant organisms, doxycycline-resistant organisms, minacycline-resistant organisms, or any organisms resistant to antibiotics of the tetracycline class used in human or veterinary medicine. In other embodiments, the compounds show cytostatic or cytotoxic activity against neoplastic cells such as cancer cells. In yet other embodiments, the compounds inhibit the growth of or kill rapidly dividing cells such as stimulated inflammatory cells. [0093] As discussed above, the present invention provides novel compounds having antimicrobial and antiproliferative activity, and thus the inventive compounds are useful for the treatment of a variety of medical conditions including infectious diseases, cancer, autoiimune diseases, inflammatory diseases, and diabetic retinopathy. Accordingly, in another aspect of the present invention, pharmaceutical compositions are provided, wherein these compositions comprise any one of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents, e.g., another anti-microbial agent or another anti proliferative agent. In other embodiments, these compositions further comprise an anti inflammatory agent such as aspirin, ibuprofen, acetaminophen, etc., pain reliever, or anti-pyretic. [0094] It will also be appreciated that certain of the compounds of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof, e.g., a prodrug. (00951 As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. 72 Berge, et al. describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19, 1977; incorporated herein by reference, The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base functionality with a suitable organic or inorganic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid,.sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fiumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate. [00961 Additionally, as used herein, the term "pharmaceutically acceptable ester" refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include fornates, acetates, 73 propionates, butyrates, acrylates and ethylsuccinates. In certain embodiments, the esters are cleaved by enzymes such as esterases. [0097] Furthermore, the term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference. [0098J As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the anti-cancer compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; tale; Cremophor; Solutol; 74 excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Uses of Compounds and Pharmaceutical Compositions [0099] The invention further provides a method of treating infections and inhibiting tumor growth. The method involves the administration of a therapeutically effective amount of the compound or a pharmaceutically acceptable derivative thereof to a subject (including, but not limited to a human or animal) in need of it. [001001 The compounds and pharmaceutical compositions of the present invention may be used in treating or preventing any disease or conditions including infections (e.g., skin infections, GI infection, urinary tract infections, genito-urinary infections, systemic infections), proliferative diseases (e.g., cancer), and autoimmune diseases (e.g., rheumatoid arthritis, lupus). The compounds and pharmaceutical compositions may be administered to animals, preferably mammals (e.g., domesticated animals, cats, dogs, mice, rats), and more preferably humans. Any method of administration may be used to deliver the compound of pharmaceutical compositions to the animal. In certain embodiments, the compound or pharmaceutical composition is administered orally. In other embodiments, the compound or pharmaceutical composition is administered parenterally. [00101] In yet another aspect, according to the methods of treatment of the present invention, bacteria are killed, or their growth is inhibited by contacting the bacteria with an inventive compound or composition, as described herein. Thus, in still another aspect of the invention, a method for the treatment of infection is provided comprising administering a therapeutically effective amount of an inventive compound, 75 or a pharmaceutical composition comprising an inventive compound to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a "therapeutically effective amount" of the inventive compound or pharmaceutical composition is that amount effective for killing or inhibiting the growth of bacteria. The compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for killing or inhibiting the growth of bacteria. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular compound, its mode of administration, its mode of activity, and the like. The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. [001021 Furthermore, after formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 76 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about I mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other'day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). [00103] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, beIzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofirfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the compounds of the invention are mixed with solubilizing agents such an Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof. [00104] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any 77 bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. [00105] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. [00106] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in tum, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. [00107 Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. [00108] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, 78 c) humectants such as glycerol, d) disintegrating agents such as agar--agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. [001091 Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like. [001101 The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active 79 ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. [001111 Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. [00112] It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anticancer agent), or they may achieve different effects (e.g., control of any adverse effects). [001131 In still another aspect, the present invention also provides ' pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container(s),can be a notice in 80 the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration. [001141 These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims. Examples Example 1-Synthesis of (-)-Tetracvcline [00115] General Procedures. All reactions were performed in flame-dried round bottomed or modified Schlenk (Kjeldahl shape) flasks fitted with rubber septa under a positive pressure of argon, unless otherwise noted. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Where necessary (so noted), solutions were deoxygenated by alternative freeze (liquid nitrogen)/evacuation/ thaw cycles (?: three iterations). Organic solutions were concentrated by rotary evaporation at -25 Torr (house vacuum). Flash column chromatography was performed on silica gel (60 A, standard grade) as described by Still et al. (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-2925; incorporated herein by reference). Analytical thin-layer chromatography was performed using glass plates pre-coated with 0.25 mm 230-400 mesh silica gel impregnated with a fluorescent indicator (254 nm). Thin layer chromatography plates were visualized by exposure to ultraviolet light and/or exposure to ceric ammonium molybdate or an acidic solution ofp-anisaldehyde followed by heating on a hot plate. [00116] Materials. Commercial reagents and solvents were used as received with the following exceptions. Chlorotrimethylsilane, triethylamine, diisopropylamine, 2,2,6,6-tetramethylpiperidine, NN, N',N'-tetramethylethylenediamine, DMPU, HMPA, and NN-diisopropylethylamine were distilled from calcium hydride under dinitrogen atmosphere. Benzene, dichloromethane, ethyl ether, methanol, pyridine, 81 tetrahydrofuran, hexane, acetonitrile, NN-dimethylformanide, and toluene were purified by the method of Pangborn et al. (Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996,15, 1518-1520; incorporated herein by reference). The molarity of n-butyliithium, s-butyllithium, and t-butyllithium were determined by titration with a tetrahydrofuran solution of 2-butanol using triphenylmethane as an indicator (Duhamel, L.; Palquevent, J.-C. J Org. Chem. 1979, 44, 3404-3405; incorporated herein by reference). [00117 Instrumentation. Proton nuclear magnetic resonance ('H NMR) spectra and carbon nuclear magnetic resonance ( 13 C NMR) were recorded with Varian Unity/Inova 600 (600 MHz), Varian Unity/Inova 500 (500 MHz/125 MHz), or Varian Mercury 400 (400 MHz/100 MHz) NMR spectrometers. Chemical shifts for protons are reported in parts per million scale (5 scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvents (CHC13: 5 7.26, CDH: 6 7.15,
D
2 HCOD: 5 3.31, CDHC 2 : 8 5.32, (CD 2
H)CD
3 SO: 32.49). Chemical shifts for carbon are reported in parts per million (8 scale) downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent (CDC 3 : 877.0, CD 6 : 8 128.0,
D
3 COD: 5 44.9, CD 2 Cl 2 : 5 53.8, (CD 3
)
2 SO: 8 39.5). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t= triplet, q = quartet, m = multiplet, br = broad), integration, coupling constant in Hz, and assignment. Infrared (IR) spectra were obtained using a Perkin-Elmer 1600 FT-IR spectrophotometer referenced to a polystyrene standard. Data are represented as follows: frequency of the absorption (cn-), intensity of absorption (s = strong, sb = strong broad, m= medium, w = weak, br =broad), and assignment (where appropriate). Optical rotations were determined on a JASCO DIP-370 digital polarimeter equipped with a sodium lamp source using a 200-pL or 2-mL solution cell. High resolution mass spectra were obtained at the Harvard University Mass Spectrometry Facilities. Microbial Dihydroxylation Product DRSJ: A. remphus Q1co 2 H 14% O Inzoicacid 1R4% 82 Preparation of Glycerol Stock Solutions [00118] Alcaligenes eutrophus B9 cells lyophilizedd powder, 20 mg, generously supplied by Prof. George D. Hegeman (Indiana University); Reiner, A. M.; Hegeman, G. D. Biochemistry 1971, 10, 2530.) were suspended in nutrient broth (5 mL, prepared by dissolving 8 g of Difco Bacto@ Nutrient Broth in I L of nanopure water followed by sterilization in an autoclave at 125 *C) in a 20-mL sterile culture tube. Aqueous sodium succinate solution (16.7 yL of a 2.5 M aqueous solution, S mM final concentration) was added, and the culture tube was shaken at 250 rpm at 30 "C until cell growth became apparent (3 d). An aliquot (250 pL) of the cellular suspension was then transferred to 5 mL of Hutner's mineral base medium (HMB, see paragraph below) containing sodium succinate (16.7 pL of a 2.5 M aqueous solution, 5 mM final concentration) in a 20-mL sterile culture tube. The culture tube was shaken at 250 rpm for 2 d at 30 "C, whereupon an aliquot (250 gL) of the fermentation solution was subcultured in a sterile Erlenmeyer flask containing 50 mL of HMB and aqueous sodium succinate solution (167 pL of a 2.5 M solution, 5 mM final concentration). The flask was shaken at 250 rpm for 24 h at 30 "C. The resulting solutionwas used directly for the preparation of glycerol stock solutions. Thus, a portion of the subcultured cellular suspension (5 mL) was diluted with an equal volume of sterile glycerol, and the resulting solution was divided equally into ten 2-mL sterile Eppendorf tubes. The individual stock solutions were then stored at -80 "C. Hutner's Mineral Base Medium [001191 Hutner's mineral base medium (HMB) was prepared as follows. Solid potassium hydroxide (400 mg) was dissolved in 500 mL of nanopure water in a 2-L Erlenmeyer flask. Nitrilotriacetic acid (200 mg), magnesium sulfate (283 mg), calcium chloride dihydrate (67 mg), ammonium molybdate (0.2 mg), iron (II) sulfate (2.0 mg), Hutner's Metals 44 solution (1 mL, see paragraph below), ammonium sulfate (1.0 g), potassium dihydrogen phosphate (2.72 g) and sodium monohydrogen phosphate heptahydrate (5.36 g) were added sequentially. The solution was diluted to a total volume of I L and the pH was adjusted to 6.8 with concentrated hydrochloric acid. The medium was sterilized by filtration or by heating in an autoclave. 83 [00120] Hutner's Metals 44 solution was prepared as follows. Concentrated sulfuric acid (100 pL) was added to nanopure water (50 mL) in a 250-mL Erlenmeyer flask. Solid EDTA (0.50 g), zinc sulfate heptahydrate (2.20 g), iron (II) sulfate heptahydrate (1.0 g), copper (I) sulfate (0.39 g), cobalt (II) nitrate hexahydrate (50 mg) and sodium tetraborate decahydrate (36 mg) were then added in sequence, followed by 50 mL of nanopure water. Cellular Dihydroxylation of Sodium Benzoate [001211 A sterile pipette tip was streaked across the surface of a frozen glycerol stock solution to produce small shards (ca. 10 mg). The frozen shards were added to a sterile 125 mL Erlenmeyer flask containing HMB (25 mL) and aqueous sodium succinate solution (140 pL of a 15 M solution, 5 mM final concentration). The flask was shaken at 250 rpm for 2 days at 30 "C. An aliquot (10 nL) of the white, heterogeneous solution was transferred using a sterile pipette to a mammalian cell growth jar containing HMB (6 L) and aqueous sodium succinate solution (20 mL of a 1.5 M solution, 5 mM final concentration). The jar was warmed on a hot plate to an internal temperature of 30 *C; cotton-filtered air was sparged through the medium. After 2 days, the white, heterogeneous solution was treated with aqueous sodium benzoate solution (18 mL of a 1.0 M solution) and aqueous sodium succinate solution (10 mL of a 1.5 M solution), inducing dihydroxylation. The resulting mixture was aerated vigorously for 6 hours at an internal temperature of 30 "C. After induction, sufficient aqueous sodium benzoate solution (24 to 48 mL of a 1.0 M solution, depending on the rate of consumption) was added hourly to maintain a concentration of 10-20 mM (determined by UV absorbance at 225 nm). Aqueous sodium succinate solution (10 mL of a 1.5 M solution) was added every fourth hour. These additions proceeded over 18 hours, then the solution was aerated overnight at an internal temperature of30 *C, to ensure complete conversion. The fermentation broth was centrifuged, in portions, at 6000 rpm (Sorvall GS-3 rotor, model SLA-3000) to remove cellular material. The supernatant was concentrated to a volume of 400 mL using a rotary evaporator (bath temperature <45 *C). The concentrate was cooled to 0 *C and then acidified to pH 3.0 using concentrated aqueous hydrochloric acid. The acidified 84 aqueous solution was extracted repeatedly with ethyl acetate (8 x 500 mL, 4 x 800 mL, 8 x 1 L). The ethyl acetate extracts were dried over sodium sulfate before concentration, using a rotary evaporator (bath temperature <45 *C), providing a pale yellow solid residue. Trituration of the residue with dichloromethane (2 x 200 mL) followed by drying in vacuo afforded pure (1S,2R)-1,2-dihydroxycyclohexa-3,5-diene 1-carboxylic acid (DRS1) as a white powder mp 95-96 *C dec (38 g, 74%, [a] 0 -114.8 (c 0.5 in EtOH), lit., (aID -106 (c 0.5 in EtOH) Jenkins, G. N.; Ribbons, D. W.; Widdowson, D. A.; Slawin, A. M. Z.; Williams, D. J. J Chem. Soc. Perkin Trans. 1 1995,2647.). Epoxide DRS2: m-CPBA, EtOAr C2H 023 C 00OH a 6H HO 83% H DRS1 DMIs2 [00122] m-Chloroperoxybenzoic acid (mCPBA was purified as follows: 50 g of 77% mCPBA (Alrich) was dissolved in benzene (1 L), the benzene solution was then washed with pH 7.4 phosphate buffer (3 x 1 L) and dried over Na 2
SO
4 for 3 hours and concentrated (<40 *C, thermal detonation hazard) to provide pure mCPBA as a white solid; 10.7 g, 62.3 mmol, 1.2 equiv) was added in three equal portions over 30 min. to a suspension of the microbial dihydroxylation product DRSI (8.10 g, 51.9 mmol, 1.0 equiv) in ethyl acetate (400 mL) at 23 "C. The heterogeneous solution was stirred for 10 h, then was diluted with benzene (80 mL) and stirred for 1 h The supernatant was decanted and the solid residue was triturated with benzene (2 x 15 nL). The resulting pasty solid was dried in vacuo to provide the epoxide DRS2 as an amorphous white powder (7.36 g, 83%). [001231 mp 87-91 "C; 'H NMR (400 MHz, CD 3 0D) 8 6.23 (dd, 1H, J= 9.6, 3.9 Hz, =CHC(OCH)), 5.92 (dd, lH, J= 9.6, 1.9 Hz, =CHC(COzH)), 4.40 (d, 1H, J= 1.3 Hz, CHOH), 3.58 (dd, 1H, J= 4.4, 1.3 Hz, CHCHOH), 3.49 (in, 1H, =CCHO); "C NMR (100 MHz, CD 3 0D) 8 175.8, 135.1, 128.8, 75.4, 70.9, 57.5, 50.3; FTIR (neat), om-' 3381 (s, OH), 1738 (s, C=0), 1608 (m), 1255 (m), 1230 (m), 1084 (m, C-0); HRMS (CO m/z calod for (C 7 HsO5+NH 4 ) 190.0715, found 190.0707. 85 Epoxide DJB1: L TMSCHN 2 ,.eCo± H 2. TBSOTf Et 3 N TS O 0 2 cH 3 Ha DcM,-6O-*23 0 C T856 DRS2 DJBI 70% [001241 A solution of trimethylsilyldiazomethane in hexanes (2.0 M, 25.5 mL, 51.0 mmol, 1.2 equiv) was added to a solution of the epoxide DRS2 (7.36 g, 42.8 mmol, 1.0 equiv) in methanol-benzene (1:3, 160 mL) at 23 *C. Extensive gas evolution was observed upon addition. The yellow solution was stirred for 5 min, then was concentrated, affording a light yellow solid. The solid was dried by azeotropic distillation from benzene (2 x 25 mL), and the dried solid was suspended in dichloromethane (200 mL). Triethylamine (20.8 ml, 149 mmol, 3.5 equiv) and tert butyldimethylsilyl trifluoromethanesulfonate (29.4 ml, 128 mmol, 3.0 equiv) were then added in sequence, providing a homogeneous solution. The reaction solution was stirred at 23 *C for 30 min. An aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 300 mL) was added followed by dichloromethane (100 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oil. The product was purified by flash column chromatography (5:95 ethyl acetate-hexanes), affording the epoxide DJB1 as a light yellow oil (12.4 g, 70% over 2 steps). Rf0.50 (1:4 ethyl acetate-hexanes); 'H NMR (400 MHz, CDCI 3 ) 8 5.95 (dd, 1H, J= 9.8, 3.4 Hz, =CHCOTBS), 5.89 (ddd, 1H, J= 9.8, 2.9, 1.5 Hz, =CHCHOCCO 2 ), 4.63 (d, 1H, J= 3.9 Hz, O 2 CCCHOTBS), 4.42 (m, 1H, =CCHOTBS), 3.78 (s, 3H, OCH 3 ), 3.31 (d, 1H, J =2.0 Hz, CHOCCO 2 ), 0.90 (s, 9H, C(CH 3
)
3 ), 0.89 (s, 9H, C(CH 3
)
3 ), 0.09 (s, 3H, SiCH 3 ), 0.08 (s, 6H, SiCH 3 ), 0.07 (s, 3H, SiCH 3 ); "C NMR (100 MHz, CDC1 3 ) 8 170.2, 138.7, 122.6, 69.3, 68.4, 59.7, 52.5, 52.0, 25.9, 25.7, 18.3, 18.2, -4.18, -4.27, -4.45, -5.21; FTIR (neat), em 1759 (m, C=0), 1736 (s, C=O), 1473 (m), 1256 (w), 1253 (s), 1150 (s, C-0), 1111 (m, C-0), 1057 (s, C-0), 940 (m); HRMS (ES) m/z called for (C 2 aH 3 sOsSi 2 ) 414.2258, found 414.2239. Isoxazole MGC2 (Method A): 86 OH . MsCi, stN, DMAP, DCM,0-+23 , 2. (CH 3
)
1 NH, DMF MGCI 7MGC2 [001251 Triethylamine (37.5 mnL, 0.269 mol, 1.15 equiv), 4 (dimethylamino)pyridine (289 mg, 2.34 mmol, 0.01 equiv), and methanesulfonyl chloride (20.8 mL, 0.269 mol, 1.15 equiv) were added in sequence to a solution of the alcohol MGC1 (prepared in two steps from commercially available methyl 3-hydroxy 5-isoxazolecarboxylate as previously reported by: Reiss, R.; Schtn, M.; Laschat, S.; Jiger, V. Eur. J Org. Chem. 1998,473-479.) (48.0 g, 0.234 mol, L. equiv) in dichloromethane (450 mL) at 0 "C. The reaction mixture was stirred at 0 *C for 2.5 h, then was concentrated, affbrding an orange oil. Chilled dimethylamine (condensed using a cold finger with dry ice/acetone, 26.2 mL, 0.480 mol, 2.0 equiv) was added to a mixture of the orange oil prepared above and NN-dimethylformamide (150 mL) at 0 *C, providing a homogenous solution. The solution was stirred at 0 *C for 2 h, then was allowed to warm to 23 *C; stirring was continued at that temperature for 24 h. The solution was partitioned between saturated aqueous sodium bicarbonate solution-brine (2:1, 300 mL) and ethyl acetate-hexanes (1:1, 500 mL). The organic phase was separated and washed with brine (2 x 200 mL), and dried over anhydrous sodium sulfate The dried solution was filtered and the filtrate was concentrated, furnishing a brown residue. The product was purified by flash column chromatography (1:4 to 1:1 ethyl acetate-hexanes), affording the isoxazole MGC2 as a light yellow oil (40.1 g, 74%).. Rf 0.34 (1:1 ethyl acetate-hexanes); 'H NMR (500 MHz, CDCI 3 ) 6 7.43-7.31 (m, 5H, ArH), 5.82 (s, 1H, =CH), 5.23 (s, 2H, OCH 2 Ar), 3.48 (., 2H, CH 2 N(CH3) 2 ), 2.27 (s, 6H, N(CH3) 2 ); "C NMR (125 MHz, CDCl 3 ) 8 171.9, 171.2,136.1, 128.8, 128.5, 128.7, 94.8, 71.7, 55.1, 45.3; FTIR (neat), cm' 2950 (s, CH), 1615 (s), 1494 (s), 1452 (s), 1136 (m); HRMS (ES) m/z calcd for (C13HiN22)232.1212, found 232.1220. Isoxazole MGC4: 87 'N (CH) 2 Nt DMF O-+ 23"OC Br MG 79% MC4 1001261 Chilled dimethylamine (condensed into a reaction vessel submerged in a 0 "C bath using a cold finger with dry ice/acetone, 106 mL, 1.94 mol, 2.2 equiv) was added dropwise via cannula to a solution of the isoxazole MGC3 (prepared in two steps from glyoxylic acid as reported by: Pevarello, P.; Varasi, M. Synth, Commun. 1992, 22, 1939.) (174 g, 0.884 mol, 1.0 equiv) in acetonitrile (2 L) at 0 *C. The reaction mixture was stirred at 0 *C for 2h, then the cooling bath was removed. The reaction mixture was allowed to warm to 23 *C; stirring was continued at that temperature for 8 h. The mixture was partitioned between brine-saturated aqueous sodium bicarbonate solution (1:1, 1.5 L) and ethyl acetate (1.5 L). The organic phase was separated and the aqueous phase was further extracted with ethyl acetate (3 x 400 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated to a volume of 500 mL, resulting in the formation of a white precipitate. The concentrate was filtered and the filtrate was concentrated, providing the isoxazole MGC4 as an orange oil (143 g, 79%). An analytical sample was prepared by flash column chromatography (1:9 to 2:8 ethyl acetate-hexanes), affording the isoxazole MGC4 as a light yellow oil. [00127] Rf 0.30 (1:4 ethyl acetate-hexanes); 'H N.MR (300 MHz, CDCI 3 ) 66.26 (s, 1H, vinyl), 3.63 (s, 2H, CH 2
N(CH
3
)
2 ), 2.30 (s, 6H, N(CH3)2); 3 C NMR (100 MHz, CDC1 3 ) 8 172.1, 140.5, 106.8, 54.5, 45.3; FTIR (neat), cm - 3137 (w), 2945 (m), 2825 (m), 2778 (m), 1590 (s), 1455 (m), 1361 (m), 1338 (s), 1281 (s), 1041 (m); HRMS (ES) m/z calcd for (CJHgBrNzO+H) 204.9976, found 204.9969, Isoxazole MGC2 (Method B):
N(CH
3
)
2 N(CHa)2 K4 benzyl alcohol Na. 120C 63% MGC4 MCC2 [00128] Sodium metal (32.63 g, 1.42 mol, 2.03 equiv) was added portionwise 88 over 8 h to benzyl alcohol (1 L) at 23 *C. The resulting mixture was stirred vigorously for 24 h, then was transferred via large bore cannula to the neat isoxazole MGC4 (143 g, 0.700 moil, 1.0 equiv) at 23 *C. The resulting light brown mixture was placed in an oil bath preheated to 120 *C and was stirred for 20 h at that temperature. Ethyl acetate (2 L) was added to the cooled reaction mixture and stirring was continued for 15 min. Aqueous hydrochloric acid (1.0 M, 2 L) was added and the aqueous phase was separated. The organic phase was further extracted with two 300-mL portions of 1.0 M aqueous hydrochloric acid. The aqueous phases were combined and the pH adjusted to 9 by slow addition of aqueous sodium hydroxide (6.0 M, approx. 350 mL). The resulting mixture was extracted with dichloromethane (3 x 500 mL). The organic extracts were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, yielding the isoxazole MGC2 as a yellow oil (102 g, 63%). An analytical sample was prepared by flash column chromatography (3:7 ethyl acetate-hexanes, then 5:95 methanol in ethyl acetate), affording the isoxazole MGC2 as a light yellow oil (spectroscopic data was identical to that obtained for material prepared by Method A). Ketone MGC5: N(CH3)2 1,n-BuU,THP,-78-C TBSOO K TesIo , -,,IoCH Tesff o J MGC2 TBSO DiB1 MGC 73% [00129] A solution of n-butyllithium in hexanes (2.47 M, 16.0 mL, 39.5 mmol, 1.0 equiv) was added to a solution of the isoxazole MGC2 (9.16 g, 39.5 mmol, 1.0 equiv) in tetrahydrofuran (150 mL) at -78 *C. The resulting rust-colored solution was stirred at -78 *C for I h whereupon a solution of the methyl ester DJB1 (9.82 g, 23.7 mmol, 0.6 equiv) in tetrahydrofuran (6 mL) was added dropwise via cannula. The transfer was quantitated with two 1-mL portions of tetrahydrofuran. The resulting brown solution was stirred at -78 *C for 1 h, then an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 250 mL) was added. The biphasic mixture was allowed to warm to 23 *C, then was extracted with dichloromethane (2 x 300 mL). The organic 89 extracts were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by flash column chromatography (1:9 to 1:3 ethyl acetate-hexanes), affording the ketone MGC5 as a light yellow solid (10.6 g, 73%). [001301 Rf0.59 (1:3 ethyl acetate-hexanes); 'H NMR (500 MHz, CDCl 3 ) 67.44 7.35 (m, 5H, ArH), 5.90 (ddd, II, J= 9.8, 5.9, 2.0 Hz, =CHCHOSi), 5.82 (dd, 1H, J 9.8, 3.4 Hz, =CHCHOCC), 5.31 (m, 2H, OCU 2 Ar), 4.58 (d, 1H, J= 4.2 Hz, (O)CCCHOSi), 4.27 (in, H, =CHCHOSi), 3.94 (d, 1H, J= 15.6 Hz, CHH'N), 3.77 (d, 1H, J =15.6 Hz, CHH'N), 3.17 (dd, 1H, J=3.4, 1.5 Hz, HCOCC(O)), 2.35 (s, 6H, N(Ci 3
)
2 ), 0.89 (s, 9H, C(CH 3
)
3 ), 0.83 (s, 9H, C(CH 3
)
3 ), 0.06 (s, 3H, SiCH 3 ), 0.05 (S, 3H, SiCH3), 0.04 (s, 3H, SiCH 3 ), -0.07 (s, 3H, SiCH 3 ); "C NMR (125 MHz, CDC 3 ) 5 191.8, 176.3, 168.9, 136.5, 135.5, 128.8, 128.7, 125.0,106.9,72.4,69.6,67.8, 67.4, 55.3, 52.6, 45.9, 26.2, 26.0,18.5, 18.3, -3.1, -3.8, -3.8, -5.1; FTIR (neat), cm' 2952 (s, CH), 1682 (s, C=O), 1594 (s), 1502 (s), 1456 (in), 1097 (s, C-0), 774 (s); HRMS (FAB) m/z caled for (C32H5oN20 6 Si 2 +Na) 637.3105, found 637.3097. Ketones MGC6 and MGC7: N(CH3)2 U(CH (c aN o-N
N(CH
3 2 tJC~sh Cci 3 )w, 4 0Gm 1.LicTf, tokuee,0C+ H TBso" 2.TFA:DCM(9:1) Ho . Tsd o Oat G-23- 9C Ts8 o T0S sU' MCS MGC,62% MGC7,28% [001311 Solid lithium trifluoromethanesulfonate (76.0 mg, 0.490 mmol, 0.05 equiv) was added to a solution of the ketone MGC5 (6.02 g, 9.80 mmol, 2.0 equiv) in toluene (500 mL) at 23 "C. The resulting heterogeneous light yellow mixture was placed in an oil bath preheated to 65 "C and was stirred at that temperature for 3 h. The reaction mixture was cooled to 23 "C and was filtered. The solids were washed with toluene (50 mL) and the filtrate was concentrated, providing a yellow oil. The oil was covered with dichloromethane-trifluoroacetic acid (10:1, 165 nL) and the resulting mixture was stirred at 23 "C for 18 h. Aqueous sodium bicarbonate solution (300 mL) was added and extensive gas evolution was observed upon addition. The biphasic mixture was extracted with diethyl ether (4 x 300 mL) and the organic extracts were 90 combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oil. The product was purified by flash column chromatography (1:9 to 1:5 ethyl acetate-hexanes), affording the ketone MGC6 as a white foam (3.20 g, 62%) and the ketone MGC7 as a viscous yellow oil (1.68 g, 28%). Ketone MGC6: [001321 Rf 0.52 (1:3 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC1 3 ) 8 7.45 (m, 2H, ArH), 7.36-7.30 (m, 3H, ArH), 5.96 (bs, 1H, =CH), 5.45 (bs, 1H, =CH), 5.32 (m, 2H, OCHH'Ar), 5.33 (bs, 1H, CHOSi), 4.15 (d, IH, J= 8.8 Hz, CHOSi), 3.59 (d, IH, J= 3.9 Hz, CHN(CH 3 )2), 3.34 (bs, 1I-, C3CH), 2.57 (bs, 1H, O), 2.39 (s, 6H, N(CH3)2), 0.90 (s, 9H, C(CH3)3), 0.16 (s, 3H, SiCHs), 0.11 (s, 3H, SiCH 3 ); '3C NMR (100 MHz, C 6
D
6 )8 189.2, 178.3, 168.6, 135.3, 128.5, 128.4, 128.3, 125.4, 106.4, 79.8, 72.3, 72.2, 67.1, 63.6, 42.9, 26.1, 18.5,-4.0, -4.8; FTIR (neat), cm- 1 3549 (bs, OH), 3455 (bs, OH), 2942 (s, CH), 1698 (s, C=0), 1617 (m), 1508 (s), 1032 (s, C-0), 906 (s); HRMS (ES) m/z calod for (C2sH36N206Si+H) 501.2421, found 501.2422. Ketone MGC7: [00133] Rf 0.64 (1:5 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC 3 ) 6 7.50 (d, 2H, J = 1.5 Hz, ArH), 7.40-7.32 (m, 3H, ArH), 5.94 (dd, 1H, J= 9.7, 6.4 Hz, =CHCHCHOSi), 5.76 (d, 1H, J = 9.7 Hz, =CHCOH), 5.37 (d, 1H1, J= 12.2 Hz, OCHH'Ph), 5.32 (d, 1H, J = 12.2 Hz, OCHH'Ph), 4.09 (d, IH, J = 2.9 Hz, HOCCHOSi), 4.03 (s, IH, OH), 3.88 (m, 1H, NCHCHCHOSi), 3.74 (d, 1H, J= 3.9 Hz, (CH 3
)
2 NCH), 2.46 (s, 6H, N(CH 3 )2), 0.91 (s, 9H, C(CH 3
)
3 ), 0.87 (s, 9H, C(CH3)3), 0.06 (s, 3H, SiCH 3 ), 0.05 (s, 3H, SiCH 3 ), 0.04 (s, 3H, SiCH 3 ), 0.03 (s, 3H, SiCH 3 ); '3C NMR (125 MHz, CDCl 3 ) 8 194.9, 173.9, 170.5, 135.8, 132.6, 128.8, 128.5, 128.3, 127.9, 106.2, 81.6, 74.8, 72.0, 71.7, 69.5, 44.6, 43.2, 26.1, 25.9, 18.7, 18.2, -3.6, -4.1, -4.3, -4.3; FTIR (neat), cm~' 3461 (bs, OH), 2940 (s; CH), 1693 (s, C=0), 1663 (s), 1647 (m), 1503 (m), 1080 (s, C-0), 774 (s); HRMS (ES) m/z called for (C32HsoN 2
O
6 Si 2 +H) 615.3285, found 615.3282. Alkene DRS3: 91 H theM,) 2 L PAh3. DEAD I 2. NBsH TBSN 0 03n 74% TBS(5o " MGC6 DRS3 [00134] Diethyl azodicarboxylate (472 pL, 3.00 mmol, 3.0 equiv) was added to a solution of the ketone MGC6 (500 mg, 1.00 mmol, 1.0 equiv) and triphenylphosphine (789 mg, 3.00 mmol, 3.0 equiv) in toluene (6.0 mL) at 0 "C. The mixture was stirred at 0 "C for 90 min whereupon a solution of 2-nitrobenzenesulfonyl hydrazine (651 mg, 3.00 mmol, 3.0 equiv) in tetrahydrofuran (3 mL) was added dropwise via cannala. The resulting mixture was stirred at 0 "C for 10 min, then was allowed to warm to 23 *C; stirring was continued at that temperature for 23 h. An aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) was added and the resulting biphasic mixture was extracted with dichloromethane (2 x 50 mL). The organic extracts were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow sludge. The product was purified by flash column chromatography (95:5 to 1:9 ethyl acetate-hexanes), affording the alkene DRS3 as a white solid (356 mg, 74%). [00135] Rf0.65 (1:3 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC 3 ) 57.46 (d, 2H, J= 6.8 Hz, ArH), 7.39-7.34 (m, 3H, ArH), 5.81 (m, 1H, =CHCH2), 5.55 (dd, lH, J= 10.3,2.0 Hz, =CHCOSi), 5.39 (d, 1H, J= 12.2 Hz, OCHH'Ph), 5.35 (d, 1H, J = 12.2 Hz, OCHH'Ph), 4.15 (s, 1H, CHOSi), 4.04 (bs, 1H, OH), 3.76 (d, 1H, J= 10.7 Hz, CHN(CH 3 )2), 2.58 (dd, 1H, J= 10.7, 3.9 Hz, C3CH), 2.47 (m, SH, N(CH 3
)
2 ,
=CCH
2 ), 0.86 (s, 9H, C(CH3)3), -0.05 (s, 3H, SiCH 3 ), -0.13 (s, 3H, SiCH 3 ); '3C NMR (125 MHz, CDCl 3 ) 8 191.5, 183.3, 167.9, 135.3, 128.8, 128.7, 128.5, 127.4, 106.8, 78.3, 72.6, 72.0,67.9, 60.7, 43.0,42.1, 26.0, 25.8,23.6, 18.2, -4.6,-5.0; FTIR (neat), omF 3528 (w, OH), 2933 (s, QH), 1702 (s, C=0), 1600 (M), 1507 (s), 1092 (S; C-0), 1061 (s, C-0); HRMS (ES) m/z called for (C 26
H
36
N
2 0sSi+H)*485.2472, found 485.2457. Diol DRS4: 92 THP C- I R/N TDAFHOAo TBSO 76% K DRs3 DRs4 [00136] Acetic acid (83.0 gL, 1.44 mmol, 2.0 equiv) and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1.0 M, 1.44 mL, 1.44 mmol, 2.0 equiv) were added in sequence to a solution of the olefin DRS3 (350 mg, 0.723 mmol, 1.0 equiv) in tetrahydrofuran (7.0 mL) at 0 *C. The resulting light gray solution was stirred at 0 "C for 30 min, then was allowed to warm to 23 "C; stirring was continued at that temperature for 5 h. The reaction mixture was concentrated, providing a brown oil. The product was purified by flash column chromatography (1:4 to 1:1 ethyl acetate hexanes), affording the diol DRS4 as a waxy white solid (202 mg, 76%). [001371 Rfj0.38 (1:1 ethyl acetate-hexanes); 'H NMR (500 MHz, CDCl 3 ) 57.51 7.48 (m, 2H, ArH), 7.42-7.36 (M, 3H, ArH), 5.84 (m, 1H, =CHCH 2 ), 5.55 (m, 1H, =CHCOH), 5.36 (m, 2H, OCH2Ph), 4.15 (d, 1H, J= 8.1 Hz, CHOH), 3.69 (d, IH, J= 8.8 Hz, CHN(CHs)2), 2.67 (m, IH, C 3 CH), 2.47 (s, 6H, N(CH 3 )z), 2.43 (dd, IH, J= 7.7, 1.5 Hz, =CCHH'), 2.36 (m, IH, =CCHH'); FTIR (neat), cn~' 3492 (w, OH), 3272 (s, OH), 1703 (s, C=0), 1606 (m), 1509.(s), 1008 (s, C-0), 732 (s); HRMS (ES) m/z called for (C 2 oH22N205+H) 371.1607, found 371.1601. Cyclohexenone DRSS: N(cHab2 U(CHB)h H H .B DMS0 n n 84%6 DRs4 DRSS [001381 Solid o-iodoxybenzoic acid (558 mg, 1.99 mmol, 3.0 equiv) was added to a solution of the diol DRS4 (246 mg, 0.665 mmol, 1.0 equiv) in dimethylsulfoxide (5.0 mL) at 23 "C. The resulting heterogeneous mixture was stirred for 5 min whereupon it became homogeneous. The brown reaction mixture was stirred at 23 *C for 36 h. Water (10 mL) was added resulting in the precipitation of excess o iodoxybenzoic acid. The mixture was filtered and the filtrate was partitioned between saturated aqueous sodium bicarbonate solution-brine (1:1, 20 mL) and ethyl acetate 93 hexanes (2:1, 45 mL). The organic phase was separated and the aqueous phase was further extracted with a 45-mL portion of ethyl acetate-hexanes (2:1). The organic extracts were combined and washed with aqueous sodium sulfite solution (2.0 M, 50 mL), brine (50 mL), and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing the cyclohexenone DRS5 as a light brown foam (206 mg, 84%). [001391 Rf 0.15 (1:3 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC3) 8 7.48 (d, 2H, J= 7.3 Hz, ArH), 7.40-7.34 (m, 3H, ArH), 6.98 (m, 1H, =CHCH2),6.12 (ddd, 1H, J= 122, 2.0,2.0 Hz, =CHC(O)), 5.35 (m, 2H, OCH 2 Ar), 4.75 (bs, 1H, OH), 3.85 (d, iH, J= 9.8 Hz, CHN(CH 3
)
2 ), 2.82 (m, 3H, C 3 CH, CCH 2 ), 2.48 (s, 6H, N(CH 3 )2); "C NMR (125 MHz, CDCl 3 ),5 192.8, 188.2, 182.8, 167.6, 149.7, 135.0, 128.9, 128.8, 128.6, 128.3, 107.9, 79.7, 72.8, 60.4, 45.5, 42.4, 25.4; FTIR (neat), cmf 3447 (w, OH), 1707 (s, C=0), 1673 (s, C=O), 1600 (m), 1512 (s), 1018 (s, C-0), 730 (s); HRMS (ES) m/z called for (C 20
H
2
N
2 0 5 +H)* 369.1450, found 369.1454. Silyl-Cyclohexenone DRS6: H U(H12H NJCN* TBSOT, 2,6-uddine 0 DCM BnB a91% DEs5 DMs6 100140] 2,6-Lutidine (75.0 pL, 0.640 mmol, 5.0 equiv) and tert butyldimethylsilyl trifluoromethanesulfonate (88.0 pL, 0.380 mmol, 3.0 equiv) were added in sequence to a solution of the cyclohexenone DRS 5 (47.0 mg, 0.130 mmol, 1.0 equiv) in dichloromethane (3 mL) at 23 *C. The mixture was stirred at 23 *C for 3 h, then an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) was added. The biphasic mixture was extracted with dichloromethane (2 x 20 mL) and the organic extracts were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording the silyl cyclohexenone DRS6 as a white crystalline solid (56.0 mg, 91%). [00141 Mp 157-158 "C (dec); Rf 0.54 (1:3 ethyl acetate-hexanes); 'HNMR (500 MHz, CDCJ,) 8 7.51 (d, 2H, J= 1.5 Hz, ArH), 7.50-7.34 (m, 3H, ArH), 6.94 (m, 1H, 94
=CHCH
2 ), 6.10 (ddd, iH, J= 10.3, 1.5, 1.5 Hz, =CHC(O)), 5.36 (m, 2H, OCH2Ar), 3.79 (d, 1H, J= 10.7 Hz, CHN(CH 3
)
2 ), 2.83 (m, 2H, =CCH 2 ), 2.78 (m, 1H, CCH), 2.46 (s, 6H, N(CH3) 2 ), 0.84 (s, 9H, C(CH3)3), 0.27 (s, 3H, SiCH 3 ), 0.06 (s, 3H, SiCH3); '3C NMR (125 MHz, CDCbs) 8 193.4, 187.9, 181.6, 167.7, 149.5, 135.2, 128.8, 128.8, 128.8, 128.6, 108.6, 83.5, 72.8, 59.8,48.1, 42.2,26.3, 25.8, 19.3, -2.2, -3.8; FTIR (neat), cm- 2942 (s), 1719 (s, CO), 1678 (s, C=0), 1602 (m), 1510 (s), 1053 (s, C-0), 733 (s); HRMS (ES) m/z calcd for (C2H34N20sSi+H)* 483.23 15, found 483.2321. Ketone MGC9: CHO 1. CHjMgBr, THF, SC
(
5 9Br 2. TEMPO, NOOCJ,NaB Br NaHCO 3 , THE, H 2 0, 0 -C MGO MGC9 M80%(2sleps) [00142] A solution of methylmagnesium bromide in ether (3.15 M, 11.6 mL, 36.7 mmol, 1.07 equiv) was added to a solution of the aldehyde MGC8 (synthesized in 2 steps from commercially available 3-benzyloxy benzyl alcohol as reported by: Hollinshed, S. P.; Nichols, J. B.; Wilson, J. W. J Org. Chem 1994, 59, 6703.) (10.0 g, 34.3 mmol, 1.0 equiv) in tetrahydrofuran (90 nL) at-5 *C (NaCL/ice bath). The light brown solution was stirred at -5 *C for 60 min, then was partitioned between saturated aqueous ammonium chloride solution (400 mL) and ethyl acetate (400 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a light yellow oil (10.1 g, 95% crude). The product was used without further purification. [00143] Sodium bromide (846 mg, 8.22 mmol, 0.25 equiv) and 2,2,6,6 tetramethyl- I-piperidinyloxyl (51.0 mg, 0.329 mmol, 0.01 equiv) were added in sequence to a solution of the light yellow oil prepared above (10.1 g, 32.8 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) at 0 "C. A freshly prepared solution of sodium bicarbonate (690 mg, 8.22 mmol, 0.25 equiv) in commercial Clorox bleach (90 mL) was cooled to 0 *C and was added in one portion to the mixture prepared above at 0 *C The resulting bright yellow mixture was stirred vigorously at 0 "C for 1.5 h whereupon sodium sulfite (1.0 g) was added. The resulting mixture was stirred for 15 min at 23 95 *C, then was partitioned between water (400 mL) and ethyl acetate (400 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a light brown oil. The product was crystallized from ethanol, punishing the ketone MGC9 as a white solid (8.08 g, 80% over 2 steps). [001441 Rf 1 0.80 (3:7 ethyl acetate-hexanes); 'H NMR(400 MHz, CDCI 3 ) 5 7.26 7.48 (m, 6H, ArH), 6.98 (m, 2H, ArH), 5.19 (s, 2H, OCH2Ph), 2.62 (s, 3H,
C(=O)CH
3 ); ' 3 C NMR (100 MHz, CDCla) 5 202.4, 155.5,144.4, 136.3, 128.9, 128.7, 128.3, 127.2, 120.3, 115.2, 109.1, 71.3, 30.9; FTIR (neat), em - 1 3065 (w), 3032 (w), 2918 (m), 1701 (s, C=0), 1565 (m), 1426 (m), 1300 (s), 1271 (s), 1028 (in); HRMS (ES) miz called for (CisHisO2Br+H)* 304.0099, found 304.0105. Epoxide MGC1A: 0- H CH 3 5 (CHHS(O)CHE Br OBn DMso, 23 'C 94% MGC9 9MGCI0 [00145] Dimethylsulfoxide (90 mL) was added dropwise via syringe to a mixture of solid trimethylsulfoxonium iodide (694 mg, 3.15 mmol, 1.3 equiv) and solid sodium hydride (60% in oil, 126 mg, 3.15 mmol, 1.3 equiv, washed with three 2-mL portions of n-hexane) at 23 "C. Vigorous gas evolution was observed upon addition. The resulting cloudy gray mixture was stirred at 23 "C for 40 mim, then a solution of the ketone MGC9 (8.08 g, 26.5 mmol, 1.0 equiv) in dimethylsulfoxide (30 mL) was added dropwise via cannula. The transfer was quantitated with a 2-mL portion of dimethylsulfoxide. The resulting orange mixture was stirred at 23 *C for 35 h, then was partitioned between brine (1 L) and ether (500 mL). The organic phase was separated and the aqueous phase was further extracted with one 500-mL portion of ether. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. rhe product was purified by flash column chromatography (5:95 ethyl acetate bexanes), affording the epoxide MGC1O as a clear oil (7.94 g, 94%). 96 [001461 Rf 0.90 (3:7 ethyl acetate-hexanes); 'H NMR (300 MHz, CDC1I) 57.20 7.52 (m, 6H, ArH), 7.10 (dd, 1H, J=7.5, 1.2 Hz, o-ArH), 6.88 (dd, IH, J= 8.1, 1.2 Hz, o-ArH), 5.16 (s, 2H, OCH 2 Ph), 3.03 (d, IH, J=4.8 Hz, CHH'OCCH3), 2.87 (d, 1H, J = 4.8 Hz, CHH'OCCH 3 ), 1.67 (s, 3H, COCH 3 ); 13C NMR (100 MHz, CDC 3 ) 8 155.0, 143.4, 136.7, 128.8, 128.4, 128.2, 127.2, 121.2, 112.8, 112.3,71.2,59.7,55.9,22.9; FTIR (neat), cm -' 3034 (w), 2981 (w), 2925 (w), 1595 (w), 1567 (s), 1469 (S), 1423 (s), 1354 (s), 1300 (s), 1266 (s), 1028 (s); HRMS (ES) mz called for (CioHIs0 2 Br+H)* 318.0255, found 318.0254. Benzocyclobutenol MGC11: r 1. n-BuLi, THF,-78 *C 2. MgBrz-78 -+23 "C 1L. Bn 67%(+7%cis) oai MGCI MGCII [00147] A solution of n-butyllithium in hexanes (1.60 M, 8.25 mL, 13.6 mrnol, 1.4 equiv) was added dropwise via syringe down the side of a reaction vessel containing a solution of the epoxide MGC10 (3.11 g, 9.74 mmol, 1.0 equiv) in tetrahydrofuran (90 mL) at -78 *C. The resulting yellow solution was stirred at -78 *C for 20 min whereupon a suspension of magnesium bromide (3.95 g, 21.4 mmol, 2.2 equiv) in tetrahydrofuran (25 mL) was added dropwise via cannula.. The transfer was quantitated with two 2.5-mL portions of tetrahydrofuran. The resulting cloudy mixture was stirred at -78 0C for 60 min, then the cooling bath was removed and the reaction mixture was allowed to warm to 23 "C. The mixture became clear upon warming and was stirred at 23 0C for 1 h. The reaction mixture was poured into aqueous Rochelle's salt solution (10% wt/wt, 1 L) and the resulting mixture was extracted with ethyl acetate (2 x 400 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing an off-white solid. The product was purified by flash column chromatography (1:9 to 2:9 ethyl acetate-hexanes), affording the trans benzocyclobutenol MGC11 as a white solid (1.57 g, 67%). [00148] Rf0.50 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC 3 ) 8 7.44 97 (br d, 2H, J= 7.5 Hz, ArH), 7.38 (br t, 2H, J=7.5 Hz, ArH), 7.22-7.34 (m, 2H, ArH), 6.82 (d, 1H, J= 8.5 Hz, o-Arff), 6.75 (d, IH, J= 7.5 Hz, o-ArH), 5.35 (d, 1H, J =12.0 Hz, OCHH'Ph), 5.25 (d, IH, J= 12.0 Hz, OCHH'Ph),), 4.71 (br d, IH, J=5.5 Hz, CHOH), 3.31 (br q, iH, J=7.0 Hz, CHCH 3 ), 2.21 (br d, 1H, J= 7.0 Hz, OH), 1.38 (d, 3H, J=7.0 Hz, CHCH3); ' 3 C NMR (100 MHz, CDC,,) 6 154.0, 148.9, 137.4, 131.5, 128.5, 128.4, 127.8, 127.3, 115.2, 114.6,77.6, 71.2,50.6, 16.5; FTIR (neat), cm 3249 (m, OH), 2958 (w), 1602 (m), 1580 (s), 1453 (s), 1261 (s), 1039 (s); HRMS (ES) m/z called for (C1H, 6 0 2 +H) 240.1150, found 240.1154. Benzocyclobutenol MGC12: -^kJ s TSrfNCH3 OBn 100% Gin MGCli MGC12 [001491 Triethylamine (336 pL, i41 mmol, 1.4 equiv) and triethylsilyl triflucromethanesulfonate (468 gL, 2.07 mmol, 1.2 equiv) were added in sequence to a solution of the benzocyclobutenol MGC11 (500 mg, 1.72 mmol, 1.0 equiv) in dichloromethane (10 mL) at 23 "C. The light yellow solution was stirred at 23 "C for 15 min, then was partitioned between water (30 mL) and dichloromethane (30 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by flash column chromatography (5:95 ethyl acetate-hexanes), affording the benzocyclobutenol MGC12 (609 mg, 99%) as a clear oil. [001501 Rf 0.85 (1:4 ethyl acetate-hexanes); 'H NMR (400 MHz, CDC 3 ) 8 7.48 7.32 (m, 5H, ArM), 7.24 (m, 2H, Art), 6.82 (d, IH, J= 8.4 Hz, o-ArH), 6.74 (d, 1H, J = 7.2 Hz, o-ArH), 5.37 (d, 1H, J= 11.2 Hz, CHH'Ph),), 5.20 (d, 1H, J= 11.2 Hz, CH'Ph),), 4.87 (d, 1H, J= 1.6 Hz, CHOTES), 3.45 (dq, 1HJ=7.2, 1.6 Hz, CHCHa), 1.42 (d, 3H, =7.2 Hz, CHCH 3 ), 0.98 (t, 9H, J=7.6 Hz, TES), 0.56 (q, 6H, J = 7.6 Hz, TES); ' 3 C NMR (100 lHz, CDC1 3 ) 5 154.2, 148.8, 137.6, 131.3, 129.0, 128.7, 128.1, 127.8, 115.1, 114.7, 71.7, 49.9, 16.9, 7.1, 5.2, 5.1; FTIR (neat), cm - 2952 (w), 2923 (w), 2854 (w), 1606 (w), 1469 (w), 1371 (in), 1265 (s), 1086 (s), 1057 (s), 1048 (s); HRMS (ES) m/z caled for (C22H 3 oO 2 Si+H) 354.2015, found 354.2006. 98 Vinyl Sulfide MGC13: b(CH3)2 U(CHS)2 v ~(C~ak .Pyr4I~r3, DCMH 2. PbSH, DBU DMF,Gc PhN DRS 660/.(2 steps) MGC13 [00151] Solid pyridinium hydrobromide perbromide (293 mg, 0.917 mmol, 2.5 equiv) was added to a solution of the cyclohexenone DRS5 (135 mg, 0.367 mmol, 1.0 equiv) in dichloromethane (4 mL) at 23 *C. The brown solution was stirred vigorously at 23 "C for 17 h whereupon sodium sulfite (150 mg, 1.19 mmol, 3.25 equiv) was added. The resulting mixture was partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) and dichloromethane (30 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a light brown foamy solid. The product was used immediately without fRther purification.
R
1 0.45 (2:3 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 ) 8 7.24 (d, 2H, J = 7.0 Hz, o-ArH), 7.02 (t, 2H, J = 7.0 Hz, m-ArH), 6.99 (d, 1H, J= 7.0 Hz, p-ArH), 6.42 (ddd, 1H, J= 6.0, 3.5, 2.0 Hz, CH=CBr), 5.12 (d, 1HI, J= 12.5 Hz, CHH'Ph),), 5.03 (d, 1H, J = 12.5 Hz, CHH'Ph),), 4.00 (br s, 1HI, OH), 3.25 (d, 1H, J = 11.0 Hz,
CHN(CH
3
)
2 ), 2.28-2.22 (m, 2H, CH 2 CH, CH 2 CH), 2.16 (dd, 1H, J = 18.0, 6.0 Hz,
CH
2 CH), 1.83 (s, 6H, N(CH3) 2 ); FTIR (neat), cn' 3397 (m, OH), 3063 (m), 2943 (m), 1714 (s, C=O), 1606 (s), 1514 (s), 1477 (s), 1371 (m), 1022 (m); HRMS (ES) /z called for (C2oHwOsBrNz)* 447.0555, found 447.0545. [001521 Benzenethiol (39.0 giL, 0.378 mmol, 1.03 equiv) and 1,8 diazabicyclo[5,4,0]undec-7-ene (56.0 pL, 0.378 mmol, 1.03 equiv) were added in sequence to a solution of the product prepared above (164 mg, 0.367 mmol, 1.0 equiv) in N,N-dimethylformanide (4 mL) at 0 "C. The resulting dark brown mixture was stirred vigorously at 0 *C for 25 min, then was partitioned between ethyl acetate hexanes (1:1, 30 mL) and an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL). The organic phase was separated and the aqueous phase was further extracted with two 15-mL portions of ethyl acetate-hexanes (1:1). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was 99 filtered and the filtrate was concentrated, providing a brown oil. The product was purified by flash column chromatography (15:85 to 1:4 ethyl acetate-hexanes), furnishing the vinyl sulfide MGC13 as a white foam (116 mg, 66% over two steps). [00153] Rf 0.47 (2:3 ethyl acetate-hexanes); 'H NMR(500 MHz, C 6
D
6 ) 57.34 (dd, 2H, J= 7.0, 1.0 Hz, o-ArH!), 7.23 (d, 2H, J= 6.5 Hz, o-ArH), 6.85-7.04 (m, 6H, ArH), 6.27 (ddd, 1H, J= 6.0, 3.0, 1.0 Hz, CR=CSPh), 5.11 (d, 1H, J= 12.0 Hz, OCRH'Ph), 5.02 (d, 1H, J= 12.0 Hz, OCHH'Ph), 4.62 (br s, 1H, OH), 3.42 (d, 111, J= 10.5 Hz, CHN(CH3) 2 ), 2.44 (ddd, 1H, J= 20.0, 5.5,3.0 Hz, CH 2 CH), 2.27-2.34 (i, 2H, CH 2 CH, CH 2 CI), 1.87 (s, 6H, N(CH3)2); ' 3 C NMR (100 MHz, CDCbs) a 188.9, 187.4, 182.5, 167.6, 145.4, 135.3, 135.2, 132.8, 132.6, 129.5, 128.6, 128.4, 128.3, 128.0, 127.8, 108.1, 80.3, 72.5, 59.8, 45.7, 41.4,25.9; FTIR (neat), ce' 3445 (w, OH), 3056 (w), 2943 (m), 2800 (w), 1711 (s, C=O), 1682 (s), 1600 (m), 1507 (s), 1471 (s), 1451 (m), 1333 (m), 1020 (m); HRMS (ES) m/z called for (C 26
H
2 4 OsN 2 S+H)* 477.1484, found 447.1465. Diel-Alder Addition Product MGC14 and Lactone MGC15: H(CH) H lCHah H ) H2 H nea AaHUW(CHa)2 POhn T Sn PhS U H n TEs15 SMh MGCI3 MGCnZ MGC14,64% MGCI5,9% [00154] A reaction vessel containing a mixture of the vinylsulfide MGC13 (131 mg, 0.275 mmol, 1.0 equiv) and the benzocyplobutenol MGC12 (750 mg, 2.11 nmol, 7.7 equiv) was placed in an oil bath preheated to 85 *C. The light yellow solution was stirred at 85 *C for 48 h, then was allowed to cool to 23 *C. The cooled mixture was purified by flash column chromatography (1:19 to 1:4 ethyl acetate-hexanes), affording the Diels-Alder addition product MGC14 as an off-white foamy solid (145 mg, 64%), the lactone MGC15 as a clear oil (20.0 mg, 9%), and the recovered benzocyclobutcnol MGC12 as a clear oil (650 mg). Diels-Alder Addition Product MGC14: [00155] mp 178-179 *C; Rf 0.55 (2:3 ethyl acetate-hexanes); 'H NMR (600 MHz, CD) 8 7.27 (d, 2H, J= 7.2 Hz, o-ArH), 7.06-7.22.(m, 8H, ArH), 6.92-6.96 (m, 100 3H, ArH), 6.85 (d, 1H, J=7.2 Hz, ArH), 6.70-6.75 (m, 3H, ArH), 6.55 (d, 1H, J= 8.4 Hz, a-ArH), 5.75 (s, 1H, CHOTES), 5.29 (br s, 1H, OH), 5.16 (d, 1H,= 12.0 Hz, OCHH'Ph), 5.10 (d, 1H, J= 12.0 Hz, OCHH'Ph), 4.66 (d, 1H, J= 10.8 Hz, OCHH'Ph'), 4.63 (d, 1H, J= 10.8 Hz, OCHH'Ph'), 4.36 (d, 1H, J= 6.6 Hz,
CHN(CH
3 )2), 3.02 (dq, 1H, J=7.8, 6.0 Hz, CH 3 CH), 2.77 (ddd, 1H, J =6.6, 6.0, 4.2 Hz, CHCIIN(CH 3
)
2 ), 2.41-2.52 (m, 2H, CHCHH'CH, CH 3 CHCHCH2), 2.08 (s, 6H,
N(CH
3 )2), 1.83 (ddd, 1H, J= 13.2,4.2,4.2 Hz, CHCHF'CH), 1.34 (d, 3H, J=7.8 Hz,
CH
3 CH), 0.70 (t, 9H, J=7.8 Hz, Si(CH2CH 3
)
3 ), 0.48 (d, 6H, J= 7.8 Hz, Si(CH 2
CH
3 )3); ' 3 C NMR (100 MHz, CDC3) a 196.3, 186.1, 181.4, 168.3, 156.3, 143.9, 137.6, 136.6, 135.4, 130.6, 129.8, 129.3, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8, 125.4, 121.1, 109.3, 108.4, 80.6, 72.4, 70.2, 66.0, 62.5, 61.7,43.2, 42.0, 38.1, 37.2, 27.4, 20.5, 6.9, 4.9; FTIR (neat), cm7 3490 (w, OH), 3063 (w), 3023 (w), 2951 (m), 2871 (m), 1715 (s, C=0), 1602 (m), 1589 (m), 1513 (s), 1457 (s), 1366 (m), 1260 (s), 1065 (s), 1012 (s); HRMS (FAB) m/z called for (C4sH5407N2SSi+Na)* 853.3318, found 853.3314. Lactone MGC1 5: [00156] R 0.55 (3:7 ethyl acetate-hexanes); 'H NMR (600 MHz, C 6
D
6 ) 8 7.34 (d, 2H, J=7.2 Hz, o-ArE), 7.02-7.18 (m, 11H, ArH), 6.72-6.84 (m, 4H, ArH), 6.54 (d, 1H, J=7.8 Hz, o-ArH), 5.73 (s, 1H, CHOTES), 5.49 (d, 1H, J=6.6 Hz, (C=O)OCHC=O), 5.20 (s, 2H, OCH 2 Ph), 4.60 (d, 1H, J= 11.4 Hz, OCHH'Ph'), 4.57 (d, 1H, J= 11.4 Hz, OCHH'Ph'), 3.49 (d, 1H, J= 11.4 Hz, CRN(CH 3
)
2 ), 3.23 (dq, 1H, J= 9.0, 7.2 Hz, CH 3 CH), 2.49 (m, 1H, CH 3 CHCHCHH'), 2.30-2.40 (m, 2H,
CHCHN(CH
3
)
2 , CH 3
CHCHCH
2 ), 2.16 (dd, 1H, J= 12.0,0.6 Hz, CH 3 CHCHCHH'), 1.96 (s, 6H, N(CH 3
)
2 ), 1.33 (d, 3H, J=7.2 Hz, CH3CH), 0.73 (t, 9H, J=7.8 Hz, Si(CH2CH 3
)
3 ), 0.46-0.62 (m, 6H, Si(CH2CH 3
)
3 ); "C NMR (100 MHz, CDCl 3 ) 6 196.4, 176.0, 170.0, 157.9, 156.0, 144.0, 136.6, 136.5, 135.6, 129.8, 129.7, 129.4, 128.9, 128.6, 128.4, 128.3, 128.2, 128.1, 127.8, 125.1, 121.2, 108.8, 101.9, 75.9,72.1, 70.1, 64.7, 64.6, 62.9, 41.4, 36.7, 35.6,27.7, 21.7, 6.9, 4.9; FTIR (neat), cmf- 3062(w), 3033 (w), 2950 (m), 2874 (m), 1731 (s, C=0), 1599 (m), 1590 (m), 1514 (s), 1453 (s), 1365 (m), 1259 (s), 1120 (s), 1059 (s), 1010 (s); HRMS (ES) m/z called for 101
(C
4 aHS40 7 NZSSi+H 831.3499, found 831.3509. Alcohol MGC16: Ha t(CHa) Hg H U(CH 3 N 3HF N THE' Bn6 g 76%A BrIGOi H7 Rn TESd ~P Ph MGC14 MGCE {001571 Triethylanine trihydrofluoride (200 FL, 1.23 mmol, 8.5 equiv) was added to a solution of the Diels-Alder addition product MGC14 (120 mg, 0.144 nmol, 1.0 equiv) in tetrahydrofuran (6 mL) at 23 "C. The mixture was stirred vigorously at 23 "C for 12 h, then was partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) and ethyl acetate (30 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a light brown solid. The product was purified by flash column chromatography (1:4 to 1:1 ethyl acetate-hexanes), affording the alcohol MGC16 as a colorless oil (78.3 mg, 76%). [00158] Rf 0.20 (2:3 ethyl acetate-hexanes); 'H NMR (600 MHz, C 6 D)8 7.69 (dd, 2H, J= 7.2, 0.6 Hz, o-ArH), 7.24 (d, 21, J= 7.2 Hz, ArH), 6.92-7.06 (m, 12H, ArH), 6.76 (d, 1H, J= 7.8 Hz, ArHl), 6.47 (d, IH, J= 8.4 Hz, o-ArH), 5.44 (br s, iH, CHOH), 5.18 (d, LH, J =12.0 Hz, OCHH'Ph), 5.16 (d, 1H, J =12.0 Hz, OCHH'Ph), 4.57 (d, IfH, J= 12.6 Hz, OCH.H'Ph), 4.52 (d, 1H, J= 12.6 Hz, OCHH'Ph'), 3.44 (dq, 1H, J= 6.6, 5.4 Hz, CH3CH), 2.98 (d, IH, J= 3.0 Hz, CHN(CH 3
)
2 ), 2.90 (in, 1H,
CIICHN(CH
3
)
2 ), 2.76 (br s, 1H, OH), 2.32 (m, IH, CH 3
CHCHCH
2 ), 1.94 (m, IH1,
CH
3 CHCHCH2), 1.79 (s, 611, N(CH3)2), 1.07 (m, I H, CH 3
CHCHCH
2 ), 0.84 (d, 3H, J 6.6 Hz, CH3CH); ' 3 C NMR (100 MHz, CDC 3 ) 6 202.5, 185.6, 179.2, 168.9, 156.9, 139.4, 139.1, 137.1, 136.5, 135.3, 130.5, 129.6, 128.8, 128.7, 128.6, 128,5, 128.4, 128.3, 127.8, 126.9, 124.7, 119.3, 110.0, 106.8, 82.3, 72.5, 69.9, 66.4,64.2, 59.3, 43.0, 39.1, 37.8, 32.6,25.3, 16.8; FTIR (neat), cnf-' 3435 (w, OH), 3066 (w), 2964 (w), 2933 (w), 2871 (w), 1738 (s, C=O), 1698 (s, C=0), 1614 (m), 1583 (m), 1513 (S), 1471 (s), 1453 (s), 1369 (in), 1263 (m), 1035 (m), 1014 (m); HRMS (ES) i/z calcd for
(C
42 H4oO 7
N
2 S+H)* 717.2634, found 717.2631. 102 Triketone MGC 17: Ha 3 H N(CHab HjQ MhcHa lax0 os Nn SPI 79% SPh MGC16 GC7 [00159] Solid o-iodoxybenzoic acid (459 mg, 1.64 mmol, 15.0 equiv) was added in one portion to a solution of the alcohol MGC16 (78.3 mg, 0.109 mmol, 1.0 equiv) in dimethylsulfoxide (3.0 mL) at 23 *C. The resulting heterogeneous mixture was stirred for 5 min whereupon it became homogeneous. The reaction vessel was protected from light and was placed in an oil bath preheated to 35 *C. The brown solution was stirred vigorously at 35 *C for 18 h, then was partitioned between saturated aqueous sodium bicarbonate solution-brine-water (2:1:1, 75 mL) and ethyl acetate-ether (1:2, 35 mL). The organic phase was separated and the aqueous phase was further extracted with two 25-mL portions ethyl acetate-ether (1:2). The organic phases were combined and dried over anhydrous sodium sulfate. Thb dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by flash column chromatography (1:2 ethyl acetate-hexanes), affording the ketone MGC17 as a yellow oil (61.7 mg, 79%). [00160] Rf0.45 (2:3 ethyl acetate-hexanes); 'H NMR (600 MHz, C 6
D
6 ) 8 7.57 (d, 2H, J= 7.2 Hz, o-ArH), 7.40 (d, 2H, J= 7.2 Hz, ArH), 7.18-7.23 (m, 3H, ArH), 6.94-7.06 (m, 6H, ArH), 6.76-6.84 (m, 3H, ArH), 6.59 (d, 1H, J= 7.8 Hz, ArH), 6.53 (d, 111, J= 8.4 Hz, o-ArH), 5.09 (d,I 1H, J= 12.6 Hz, OCHH'Ph), 4.96 (d, 1H, J= 12.6 Hz, OCHH'Ph), 4.77 (d, IH, = 12.0 Hz, OCHH'Ph'), 4.72 (d, 1H, J= 12.0 Hz, OCHH'Ph'), 4.48 (br s, 1H, OH), 4.06 (dq, 1H, J= 7.2, 3.0 Hz, CH 3 CH), 3.15 (d, 1H, J= 12.0 Hfz, CHN(CH 3
)
2 ), 2.20 (ddd, 1H, J= 12.6, 5.4, 3.0 Hz, CH3CHCHCH 2 ), 2.13 (ddd, 1H, J =12.0, 3.0, 0.6 Hz, CHCHN(CH3)2), 1.81-1.88 (m, 7H, N(CH 3
)
2 ,
CH
3 CHCHCHH'), 1.78 (ddd, 1H, J= 13.8, 5.4,0.6 Hz, CH 3 CHCHCHH'), 1.01 (d, 3H, J= 7.2 Hz, CHaCH); 13 CNMR(J00MHz,CDCWa)8200.3, 187.5,183.1,167.8,160.6, 146.4, 138.2, 137.1, 135.3, 134.3, 131.7, 129.6, 128.9, 128.6, 128.5,128.4, 128.3, 127.7, 126.7,121.3, 118.0, 112.8, 108.3, 82.9, 77.5, 72.4,70,3,58.1,47.0,44.1,32.4, 103 18.7, 18.0, 16.3; FTIR (neat), cm 3457 (w, OH), 3063 (w), 2939 (w), 2878 (w), 2795 (w), 1727 (s, C=0), 1704 (s, C=O), 1667 (m, C=O), 1593 (s), 1513 (s), 1471 (s), 1453 (s), 1371 (m), 1276 (m), 1044 (m); FIRMS (ES) m/z caled for (C 42 Haa0 7
N
2 S+H)* 715.2478, found 715.2483. Peroxide MGC 18:
H
3 9 H V(cH32 1.m-CPBA, TFA Ha 0CH b (cwz DCM-78-+35 9 2.03g, 2111 no 0 o tn o o o O MGC1? MGC18 [00161] A solution of trifluoroacetic acid in dichloromethane (1.0 M, 0.189 mL, 0.189 mmol, 2.5 equiv) and a solution of m-chloroperoxybenzoic acid in dichloromethane (0.5 M, 0.228 mL, 0.114 mmol, 1.5 equiv) were added in sequence to a solution of the sulfide MGC17 (54.2 mg, 0.0758 mmol, 1.0 equiv) in dichloromethane (4.0 mL) at -78 *C. The resulting cloudy mixture was stirred at -78 "C for 10 min, then the -78 *C bath was replaced with a 0 "C bath. The mixture became homogeneous upon warming. The solution was stirred at 0 aC for 30 min, then was partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a bright yellow oil. The oil was taken up in toluene (1 mL) and dried by azeotropic distillation at 40 T under high vacuum. The resulting yellow oil was dissolved in chloroform (2 mL) and the reaction vessel was exposed to atmospheric oxygen. The mixture was allowed to stand until oxidation was complete as evidenced by 'H NMR spectroscopy. The mixture was filtered and the filtrate was concentrated, providing the peroxide MGC18 as a brown oil. The product was reduced immediately to tetracycline. [001621 The peroxide MGC18 can also be prepared by following the procedure reported by Wasserman (J. Am. Chem, Soc. 1986, 108, 4237-4238.): [00163] A solution of trifluoroacetic acid in dichloromethane (1.0 M, 24.5 iaL, 104 0.0245 mmol, 2.5 equiv) and a solution of m-chloroperoxybenzoic acid in dichloromethane (0.5 M, 29.4 pL, 0.0147 mmol, 1.5 equiv) were added in sequence to a solution of the sulfide MGC17 (7.00 mg, 0.00979 mmol, 1.0 equiv)-in dichloromethane (0.5 mL) at -78 "C. The resulting cloudy mixture was stirred at -78 "C for 10 min, then the -78 "C bath was replaced with a 0 C bath. The mixture became homogeneous upon warming. The solution was stirred at 0 *C for 30 min, then was partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 8 mL) and dichloromethane (8 mL). The organic phase was separated and dried over anhydrous sodium sulfate. 'The dried solution was filtered and the filtrate was concentrated, providing a bright yellow oil. The oil was taken up in toluene (1 mL) and dried by azeotropic distillation at 40 *C under high vacuum. The resulting yellow oil was dissolved in chloroform (2 mL) and meso-tetraphenylporphine (0.6 mg, 0.979 pmol, 0.10 equiv) was added in one portion. Oxygen gas was bubbled through the resulting mixture under UV irradiation (200 W Hg lamp) for 10 min. The mixture was concentrated to 0.5 mL and was diluted with methanol (5 mL) resulting in precipitation of meso-tetraphenylporphine. The resulting mixture was filtered and the filtrate was concentrated, providing the peroxide MGC18 a light yellow solid. [00164] Rf 0.10 (2:3 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 , keto tautomer reported) 58.95 (br s, IH, OOH), 7.48 (d, 2H, J= 7.0 Hz, o-ArH), 7.28 (d, 211, J= 7.0 Hz, ArH), 6.96-7.16 (m, SH, ArH), 6.53 (d, 1H, J= 8.0 Hz, ArH), 5.14 (d, 1H, J= 12.0 Hz, OCHH'Ph), 5.03 (d, 1H, J= 12.0 Hz, OCHW'Ph), 4.83 (d, 1H, J= 12.5 Hz, OCHH'Ph'), 4.74 (d, iH, J= 12.5 Hz, OCHH'Ph'), 4.60 (br s, IH, OH), 3.54 (d, IH, J= 11.0 Hz, CHCHN(CH 3
)
2 ), 3.12 (dd, Il, J =18.0, 0.5 Hz, CHCHH'CH), 2.82 (dd, iN, J= 18.0, 4.5 Hz, CHCHH'CH), 2.44 (ddd, 1H, J= 11.0, 4.5, 0.5 Hz,
CHCHN(CH
3
)
2 ), 1.86 (s, 6H, N(CH 3
)
2 ), 1.01 (s, 3H, CH 3 ); "C NMR (100 MHz, CD 6 , enol and keto tautomers reported) 5 194.4, 188.6, 187.8, 187.2, 182.3, 178.4,171.9, 167.7, 165.6, 159.5, 158.4, 147.9, 145.9, 137.0, 136.8, 136.6, 135.4, 135.3, 134.5, 134.3, 133.5, 133.4, 133.1, 12.9, 131.0, 130.8, 130.2, 129.9, 129.7, 129.2, 128.9, 126.8,126.7,124.5,124.3,122.2,118.6, 116.9,116.5, 113.4, 113.3,113.2,108.2, 107.9, 103.3, 83.7, 81.7, 80.1, 79.1, 72.4, 70.7, 70.4, 63.9, 59.1, 46.1,44.9,41.4,40.8, 31.5,30.0,26.8, 22.9,21.4; FTIR (neat film), cm t 3035 (w), 2946 (w), 1907 (w), 1731 105 (s, C=O), 1410 (s), 1379 (m), 1235 (m), 1170 (m), 1136 (m); HRMS (ES) m/z called for
(C
6 H32OgN 2 +H)~ 637.2186, found 637.2190. (-)-etracycline (MGC29):
H
3 Q. OH H (CH H H(Cak 1k, Nd black done NH2 n " f(44tamthe HO 0 HOHO 0 ketone MtUCI7) MGC18 (-)-tctmoycline [00165] Pd black (14.1 mg, 0.133 mmol, 1.75 equiv) was added in one portion to a solution of the peroxide MGC18 (48.2mg, 0.0758 mmol, 1.0 equiv) in dioxane (3 mL) at 23 *C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The Pd catalyst was initially present as a fine dispersion, but aggregated into clumps within 5 min. The yellow heterogeneous mixture was stirred at 23 "C for 2 h, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 piM, 250 x 10 mm, flow rate 4.0 mL/min, Solvent A: methanol-0.005 N aq. HCI (1:4), Solvent B: acetonitrile) using an injection volume of solvent A (500 pL) containing oxalic acid (10 mg) and an isochratic elution of 5% B for 2 min, then a gradient elution of 5-50% B for 20 min. The peak eluting at 11-16 min was collected and concentrated, affording ( )-tetracycline hydrochloride as a yellow powder (16.0 mg, 44% from triketone MGC17), which was identical with natural (-)-tetracycline hydrochloride in all respects. [00166] 'H NMR (600 MHz, CD 3 0D, hydrochloride) 8 7.50 (dd, IH, J= 8.4, 7.8 Hz, ArH), 7.15 (d, 1H, J=7.8 Hz, ArH), 6.91 (d, 1H, J= 8.4 Hz, ArH), 4.03 (s, 1H,
CHN(CH
3 )2), 2.96-3.04 (m, 7H, HOC(CH 3 )CH, N(CH 3
)
2 ),2.91 (br dd, 1H1, J= 12.6, 2.4 Hz, (CH 3
)
2 NCHCH), 2.18 (ddd, 1H, J= 12.6, 6.0, 2.4 Hz, CHCHH'CH), 1.90 (ddd, 1H, J= 12.6, 12.6,12.0 Hz, CHCH'CH), 1.60 (s, 3H, CH 3 ); 3 C NMR (100 MHz,
CD
3 0D) 8 195.4, 174.5, 163.8, 148.3, 137.8, 118.7, 116.4,116.0, 107.5, 96.5, 74.7, 71.2, 70.1, 43.5, 43.0, 35.9, 27.8, 22.9; UV max (0.1 N HCI), run 217, 269, 356; [a]D = -251" (c = 0.12 in 0.1 M H1C4); lit. (The Merck Index: An Encyclopedia of Chemicals, 106 Drugs, and Biologicals, 12' ed. Budavati, S.; O'Neal, M. J.; Smith, A.; Heckelnan, P. E.; Kinneary, J. F, Eds.; Merck & Co.: Whitehouse Station, NJ, 1996; entry 9337.) UV max (0.1 N HC1), mu 220,268, 355; [a] 0 = -257.9" (c = 0.5 in 0.1 M HCI); HRMS (ES) m/z caled for (C22H240sN2+H) 445.1611, found 445.1608. Example 2-Synthesis of (--Doxveveline Allylic Bromide MGC19: L(oHah (CHah2 CBr 4 , Ph 1 P CoH3CN, 23"C Br TqS" 90% TBS$ R MGC6 MGC19 [00167 Triphenylphosphine (297 mg, 1.13 mmol, 3.5 equiv) and carbon tetrabromide (376 mg, 1.13 mmol, 3.5 equiv) were added in sequence to a solution of the allylic alcohol MGC6 (162 mg, 0.324 mmol, 1.0 equiv) in acetonitrile (2.5 mL) at 0 *C. The resulting brown suspension was stirred at 0 "C for 10 min, then the cooling bath was removed. The mixture was allowed to warm to 23 "C and stirring was continued at that temperature for 10 min. The mixture was partitioned between ethyl acetate (50 mL) and saturated aqueous sodium bicarbonate solution (40 mL). The organic phase was separated and the aqueous phase was further extracted with an additional 50 mL-portion of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oily solid. The product was purified by flash column chromatography (1:9 to 2:8 ethyl acetate-hexanes), yielding the allylic bromide MGC19 (164 mg, 90%) as a white solid. [001681 Rf 0.30 (3:7 ethyl acetate-hexanes);'H NMR (500 MHz, C 6
D
6 ) 8 7.30 (d, 2H, J= 7.0, o-ArE), 7.06 (dd, 2H, J= 7.0, 6.0 Hz, m-ArH), 7.01 (d, 1H, J= 6.0,p ArH), 5.75 (dd, 1H, J= 10.5, 2.5 Hz, =CHCHBr), 5.71 (in, IHl, CR=CHCHBr), 5.17 (d, IH, J=11.5 Hz, OCHH'Ph), 5.07 (d, 1H, J 11.5 Hz, OCHH'Ph), 4.69 (m, 1H, =CHCHBr), 4.43 (br s, lH, OH), 4.24 (d, 1H, J= 7.0 Hz, CHOTBS), 3.57 (d, 1H, J= 10.0 Hz, CHN(CH 3
)
2 ), 2.69 (ddd, 1H, J= 10.0, 4.5, 0.5 Hz, CHCHN(CH 3
)
2 ), 1.92 (s, 6H, N(CH 3
)
2 ), 0.99 (s, 91, SiC(CH 3 )3), 0.22 (s, 3H, SiCH 3 ),-0.02 (s, 3H, SiCH 3 ); ' 3 C NMR (125 MHz, CAJK) 5 189.3, 181.3, 167.8, 135.2, 129.5, 128.6,128.6, 128.5, 128.2, 107 127.6, 107.3, 80.8, 76.9, 72.4, 64.8, 54.6,46.3,41.5,26.2, 18.4, -2.9, -4.2; FTIR (neat), cm~' 3499 (m, OH), 2930 (m), 2856 (m), 2799 (w), 1704 (s, C=O), 1605 (s), 1514 (s), 1471 (s), 1362 (s), 1255 (s), 1144 (s), 1053 (s); HRMS (ES) m/ called for (C2H 3 SBrN 2 05Si+H) 4 563.1577, found 563.1575. Allylic Sulfide MGC20: (CHala H(CHs)2 PhSH, Et 3 N CHICN Tas a n 97% Tpsh " MGC19 MGC2D [00169] Triethylamine (0.229 mL, 1.64 mmol, 1.3 equiv) and benzenethiol (0.150 mL, 1.45 mmol, 1.15 equiv) were added in sequence to a solution of the allylic bromide MGC19 (712 mg, 1.26 mmol, 1.0 equiv) in acetonitrile (17 mL) at 0 *C. The mixture was stirred at 0 "C for 20 min, then the cooling bath was removed. The reaction mixture was allowed to warm to 23 *C and stirring was continued at that temperature for 10 min. The reaction mixture was partitioned between ethyl acetate (100 mL) and an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 100 mL). The organic phase was separated and the aqueous phase was further extracted with an additional 30-mL portion of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, finishing a clear oil. The product was purified by flash column chromatography (0.01:2:8 to 0.013:7 triethylamine-ethyl acetate-hexanes), affording the allylic sulfide MGC20 as a white foamy solid (728 mg, 97%). [00170] Rf0.65 (3:7 ethyl acetate-hexanes); 'H NMR (400 MHz, CD 6 ) 8 7.35 (d, 2H, J= 7.2 Hz, o-ArH), 7.19 (m, 2H, o-ArH), 6.95 (m, 3H,p,m-Arfl), 6.89 (m, 2H, p,m-ArH), 6.83 (d, 1H, J= 7.2 Hz,p-ArH), 5.51 (m, I1H, CH=CHCHSPh), 5.12 (m, 2H, CROTBS, OCHH'Ph), 5.05 (d, 1H, J= 12.4 Hz, OCHH'Ph), 4.73 (dt, IH, J= 10.0, 2.0 Hz, CH=CHCHSPh), 4.38 (m, 1H, CH=CHCHSPh), 3.47 (m, 1H,
CHCHN(CH
3 )2), 2.92 (d, 1H, J= 2.0 Hz, CHCHN(CH3)2), 1.75 (s, 6H, N(CH 3
)
2 ), 1.14 (s, 9H, SiC(CH 3 )3), 0.35 (s, 3H, SiCH 3 ), 0.31 (s, 3H, SiCH3); "C NMR (125 MHz,
C
6
D
6 ) 8 189.9, 177.0, 168.9,136.7, 135.2, 131.3, 130.3, 129.2, 128.5, 128.4, 128.3, 126.2,124.0, 106.2, 79.2, 72.4, 71.7, 63.2,49.8,43.4, 39.0,26.6, 19.1, -2.9, -4.5; FTIR 108 (neat), cm - 3310 (m, OH), 2927 (m), 2854 (m), 2792 (w), 1697 (s, C=O), 1621 (s), 1505 (s), 1470 (s), 1365 (s), 1254 (s), 1145 (s), 1089 (s); HRMS (ES) r/z caled for (C32H 4 oN 2 05SSi+H) 593.2505, found 593.2509. LoLer I Sulfoxide MGC21: U(CHab2 Ho 5(CHa Phn DCM r,8 n C PhTe Ssn MGQ MGeI (*Wc Rk) [001711 (-)-[(8,8)-(Dichlorocamphoryl)sunfonyl]oxaziridine (118 mg, 0.395 mmol, 1.5 equiv) was added to a solution of the allylic sulfide MGC20 (156 mg, 0.263 mmol, 1.0 equiv) in dichloromethane (2 mL) at 23 "C. The mixture was stirred at 23 *C for 20 h, then was concentrated, providing a light brown solid. The product was purified by flash column chromatography (0.001:2:8 to 0.001:3:7 triethylamine-ethyl acetate-hexanes), affording the lower R, allylic sulfoxde MGC21 as a white solid (165 mg, 99%). [001721 Rf 0. 18 (3:7 ethyl acetate-hexanes); 'H NMR (400 MHz, C 6
D
6 ) 87.43 (dd, 2H, J= 8.0, 1.5 Hz, o-ArH), 7.16 (m, 2H, o-ArH), 6.92 (m, 6H,pm-ArH), 5.43 (m, 1H, CH=CHCHS(O)Ph), 5.33 (d, 1H, J= 5.0 Hz, CHOTBS), 5.09 (d, 111, J= 11,5 Hz, OCHH'Ph), 5.02 (m, 2H, CH=CHCHS(O)Ph, OCHHPh), 3.73 (m, IH, CH=CHCHS(O)Ph), 3.41 (m, 1H, CHCHN(CH 3 )2), 2.85 (d, 1H, J 2.5 Hz, CHCHN(CH)2), 1.70 (s, 6H, N(CI 3
)
2 ), 1.12 (s, 9H, SiC(CH 3
)
3 ), 0.39 (s, 3H, SiCH3), 0.36 (s, 3H, SiCH 3 ); ' 3 C NMR (125 MIHz, CA]s) 8 189.5, 176.9, 168.8, 145.5, 135.2, 130.2, 129.9, 129.0, 128.5, 128.4, 128.3, 127.8, 124.3, 122.9, 106.1, 79.3, 72.4, 70.6, 67.8, 63.1,43.4,38.5, 26.6, 19.2, -2.6, -4.7; FTIR (neat), m ' 3310 (m, OH), 2927 (m), 2854 (m), 2792 (w), 1697 (s, C=O), 1621 (s), 1505 (s), 1470 (s), 1365 (s), 1254 (s), 1145 (s), 1089 (s); HRMS (ES) n/z called for (C32H 4 oN 2 0sSSi+H) 609.2455, found 609.2452. Rearranged Allylic Alcohol MGC22: 109 H (C H Kb" ~Bn
CH
3 OH, 70 C 76% (2 sOps) MGC2I (*Lower Rrdiast) MGC22 [001731 Trimethylphosphite (0.620 mL, 5.26 mmol, 20.0 equiv) was added to a solution of the lower Rr allylic sulfoxide MGC21 (160 mg, 0.263 mmol, 1.0 equiv) in methanol (5 mL) at 23 *C. The solution was placed in an oil bath preheated to 65 "C and was stirred at that temperature for 36 h. The solution was concentrated, providing a light yellow oil. The product was purified by flash column chromatography (0.001:1:9 to 0.001:2:8 triethylamine-ethyl acetate-hexanes), affording the allylic alcohol MGC22 as a white solid (100 mg, 76%). Rf 0.40 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CD 6 ) 8 7.30 (d, 2H, J=7.0 Hz, o-ArH), 7.06 (dd, 2H, J= 7.5, 7.0 Hz, m-ArH!), 7.00 (d, 1H, J= 7.5 Hz,p-ArH), 5.85 (m, 1H,=CHCHOH), 5.42 (br d, 1H, J= 10.5 Hz, =CHCHOTBS), S.16 (d, 1H, J = 12.5 Hz, OCHH'Ph), 5.06 (d, 1H, J= 12.5 Hz, OCHH'Ph), 4.44 (m, 1H1, =CHCHOH), 4.31 (br s, 1H, OH), 4.07 (br s, 1H, =CHCHOTBS), 3.34 (br s, 1H, OH), 3.33 (di, IH, J= 11.5 Hz, CHCHN(CH 3 )),2.75 (br d, 111, J= 11.5 Hz,
CHCHN(CH
3 )2), 2.03 (, 6H, N(CH 3
)
2 ), 0.89 (, 9W, SiC(CH) 3 ),-0.1 (s, 311, SiCH 3 ), -0.13 (s, 3H, SiCH 3 ); "C NMR (100 MHz, CD 6 ) 6 189.7,182.2, 167.7, 135.2, 129.2, 128.8, 128.3, 128.2, 106.6,78.6, 71.9, 68.1, 64.1, 59.6, 48.8,41.2, 25.5, 17.8, -5.2, 5.6; FTIR (neat), cm ' 3515 (m, OH), 2917 (m), 2852 (m), 1708 (s, C=O), 1601 (s), 1511 (s), 1471 (m), 1369 (m), 1254 (m), 1100 (m), 1022(m); HRMS (ES) m/z calcd for (C2 6
H
3 sN 2 0 6 Si+H) 501.2421, found 501.2424. Benz] Carbonate MGC23: H b(CHS)2 Lno C H32 BnoCCt DMA P DCM MGC2 MGC23 [001741 Benzyl chloroformate (120 pL, 0.841 mmol, 2.95 equiv) and 4 (dimethylamino)pyridine (104 mg, 0.852 mmol, 3.0 equiv) were added in sequence to a solution of the allylic alcohol MGC22 (142 mg, 0.284 mmol, 1.0 equiv) in 110 dichloromethane (3 mL) at 23 "C. The reaction mixture was stirred at 23 *C for 2 h, then was partitioned between ethyl acetate (50 mL) and saturated aqueous sodium bicarbonate solution (50 mL). The organic phase was separated and the aqueous phase was further extracted with an additional 30-mL portion of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a clear oil (180 mg, 99%). The product was used in the next step without further purification. An analytical sample was prepared by purification of the crude reaction mixture by flash column chromatography (0.001:2:8 to 0.001:3:7 triethylamine-ethyl acetate-hexanes), affording the benzyl carbonate MGC23 as a white solid. [00175] Rf0.60 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 ) 8 7.26 (d, 2H, J= 7.0 Hz, o-ArH), 7.02 (m, SH, ArH), 5.75 (br dd, 1H, J =10.5, 3.0 Hz,
=CHCHOCO
2 Bn), 5.70 (br dd, 1H, J= 10.5, 2.5 Hz, =CHCHOTBS), 5.37 (m, 1H,
=CHCHOCO
2 Bn), 5.10 (d, 1H, J= 12.5 Hz, OCIH'Ph), 5.06 (d, 1H, J= 12.5 Hz, OCHH'Ph), 4.91 (d, 1H, J= 12.0 Hz, OCHH'Ph'), 4.88 (d, 1H, J= 12.0 Hz, OCHU'Ph'), 4.41 (m, 1H, =CHCHOTBS), 3.38 (d, 1H, J= 7.5 Hz, CHCHN(CH 3 )2), 3.11 (m, 1 H, CHCHN(CH 3
)
2 ), 1.92 (s, 6H, N(CHI 3
)
2 ), 0.92 (s, 9H, SiC(CH3)3), 0.02 (s, 3H, SiCH3), -0.02 (s, 3H, SiCH3); 3 C NMR (100 MHz, C 6
D
6 ) b 188.9, 179.9, 168.3, 155.2, 135.6, 135.4, 133.2, 128.6, 128.5, 128.4, 128.3, 127.7, 124.9, 107.0, 77.3, 72.2, 71.6, 69.6, 66.6,60.3,44.4,42.2,25.9, 18.2, -4.8,-4.8; FTIR (neat), cm -13532 (w, OH), 2948 (in), 2842 (m), 1738 (s, C=0), 1708 (s, C=O), 1608 (s), 1512 (s), 1471 (m), 1383 (m), 1258 (s), 1101 (m); HRMS (ES) m/z called for (C 34 N42N 2 0sSi+H) 635.2789, found 635.2786. Di MGC24: BrO 2 CQ H N(cH 3
)
2 On0 2 0e N(CH3)2 TBAP, HOAc THF TBB8 H 08" 92%(2 steps) HO H MGC23 MGC24 [001761 Acetic acid (40.0 gL, 0.709 mmol, 2.5 equiv) and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1.0 M, 0.709 mL, 0.709 mmol, 2.5 equiv) were added in sequence to a solution of the benzyl carbonate MGC23 (180 mg, 111 0.284 mmol, 1.0 equiv) in tetrahydrofuran (3 mL) at 23 "C. The resulting yellow solution was stirred at 23 "C for 4 h, then was partitioned between ethyl acetate (50 mL) and an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 50 mL). The organic phase was separated and the aqueous phase was further extracted with two 20 mL portions of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oil. The product was purified by flash column chromatography (2:8 to 1:1 ethyl acetate-hexanes), affording the diol MGC24 as a white solid (135 mg, 92% over 2 steps). [00177] R 1 0.15 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 ) 57.24 (d, 2H, J= 7.0 Hz, o-ArH), 7.02 (m, 8H, ArH), 5.68 (br dd, 1H, J=10.5, 2.5 Hz, =CHCHOCO2Bn), 5.63 (br dd, 1H, J= 10.5,3.9 Hz, =CICHOH), 5.26 (in, 1H,
=CHCHOCO
2 Bn), 5.09 (d, 1H, J=12.0 Hz, OCHH'Ph), 5.05 (d, 1H, J=12.0 Hz, OCHH'Ph), 4.89 (d, 1H, J= 12.0 Hz, OCHH'Ph'), 4.86 (d, 1H, J= 12.0 Hz, OCHH'Ph'), 4.16 (m, 1H, =CHCHOH), 3.24 (d, 1H, J=6.5 Hz, CHCHN(CH3) 2 ), 2.94 (in, 1H, CHCHN(CH3)2), 2.25 (br s, IH, OH), 1.82 (s, 6H, N(CH3) 2 ); 1 3 C NMR (100 MHz, CDCl 3 ) 8 168.1, 154.8, 135.1, 134.9, 132.2, 128.9, 128.9, 128.8, 128.7, 128.6, 126.4, 106.7, 76.6, 72.9, 71.3, 70.3, 64.9, 60.3, 44.4, 43.3; FT]R (neat), cm -' 3468 (m, OH), 3034 (w), 2949 (m), 2798 (m), 1738 (s, C=0), 1705 (s, C=O), 1606 (s), 1513 (s), 1475 (m), 1379 (m), 1261 (s), 1022 (m); HRMS (ES) m/z called for (C 2 gH 2
&N
2 0s+H)* 521.1929, found 521.1926. Zyclohexenone MGC25: BnOCQ W(CHa)2 Bn(3cO H a)2 0'a0 MGC4 MGC2S 00178] Solid o-iodoxybenzoic acid (79.0 mg, 0.281 mmol, 6.5 equiv) was added n one portion to a solution of the diol MGC24 (22.5 mg, 0,0433 mmol, 1.0 equiv) in limethylsulfoxide (0.7 mL) at 23 *C. The reaction mixture was initially heterogeneous, nit became homogeneous within 5 min. The brown reaction mixture was protected 'rom light and was stirred vigorously at 23 *C for 12 h. The resulting orange reaction 112 mixture was partitioned between ether (20 mL) and water (20 m). The organic phase was separated and the aqueous phase was further extracted with two 10 mL-portions ether. The organic phases were combined and washed with saturated aqueous sodium bicarbonate solution (8 mL, containing 30 mg of sodium bisulfite) and brine (10 mL). The washed solution was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated, yielding the cyclohexenone MGC25 as a white oily solid (22.2 mg, 99%). [00179] Rf0.33 (2:3 ethyl acetate-hexanes); 'H NMR (400 MHz, C 6
D
6 ) 57.22 (d, 2H, J= 6.8 Hz, o-ArH), 6.99 (m, 8H, ArH), 6.12 (ddd, 1H, J= 10.4,4.0, 1.2 Hz,
CH=CHCHOCQ
2 Bn), 5.74 (dd, 1H, J= 10.4, 1.2 Hz, CH=CHCHOCO 2 Bn), 5.41 (ddd, 1IH, J= 4.0, 1.2, 1.2 Hz, CH=CHCHOCO 2 Bn), 5.18 (br s, IH, OH), 5.08 (d, 1H, J= 12.0 Hz, OCHH'Ph), 5.01 (d, 1H, J= 12.0 Hz, OCHH'Ph), 4.89 (d, 1H, J= 12.4 Hz, OCHH'Ph'), 4.83 (d, IH, J= 12.4 Hz, OCHH'Ph'), 3.28 (d, IH, J= 8.4 Hz,
CHCHN(CH
3
)
2 ), 2.85 (ddd, 1H, J= 8.4, 4.0, 1.2 Hz, CHCHN(CH 3
)
2 ), 1.92 (, 6H,
N(CH
3 )2); ' 3 C NNR (100 MHz, C 6
D
6 ) 5 192.3, 186.2, 180.5,167.8, 154.8, 141.8, 135.3, 135.2, 129.9, 128.6, 128.6, 128.5, 128.4, 127.8, 107.7, 78.9, 72.5, 69.9, 59.9, 48.4, 41.9; FTIR (neat), cm - 3442 (m, OH), 3030 (w), 2948 (m), 2793 (m), 1742 (s, C=O), 1711 (s, C=O), 1608 (s), 1510 (s), 1448 (m), 1376 (m), 1258 (s), 1056 (m); HRMS (ES) m/z caled for (C2gH 2 6
N
2 0s+)* 519.1767, found 519.1773. Silyl-Cyclhexenone MGC26; BnO 2 CQ H U(CH)2 BnO2CQ t(CH)2 YY N TBSOTf, Et 3 N THF, O 0 C H 93%(2steps) o o n MGC2S 668 MGC26 [001801 Triethylamine (172 pL, 1.24 mmol, 3.5 equiv) and tert butyldimethylsilyl trifluoromethanesulfonate (243 pL, 1.06 mmol, 3.0 equiv) were added in sequence to a solution of the cyclohexenone MGC25 (183 mg, 0.353 mmol, 1.0 equiv) in tetrahydrofuran (8 mL) at 0 "C. The reaction mixture was stirred at 0 *C for 40 min, then was partitioned between ethyl acetate (50 mL) and an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 50 nL). The organic phase was 113 separated and the aqueous phase was further extracted with a 25-mL portion of ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oily solid. The product was purified by flash column chromatography (1:9 to 2:8 ethyl acetate-hexanes), affording the silyl-cyclohexenone MGC26 as a clear oil (207 mg, 93%). 1001811 R 1 0.50 (3:7 ethyl acetate-hexanes); 'H NMR (400 MHz, C 6
D
6 ) 87.21 (dd, 2H, J =7.5, 1.0 Hz, o-ArH), 7.15 (d, 2H, J=8.0 Hz, o-ArH),.7.05 (t, 2H, J= 8.0 Hz, m-ArH), 6.98 (m, 4H, mp-ArH), 6.30 (ddd, 1H, J= 10.5, 5.0,2.0 Hz, CH=CHCHOCO2Bn), 5.68 (dd, 1H, J=10.5, 1.0 Hz, CH=CHCHOCO 2 Bn), 5.65 (br d, 1H, J= 5.0 Hz, CH=CHCHOCO 2 Bn), 5.10 (d, 1H, J= 12.5 Hz, OCHH'Ph), 5.01 (d, 1H, J= 12.5 Hz, OCHH'Ph), 4.95 (d, 1H, J= 12.5 Hz, OCHH'Ph'), 4.82 (d, 1H, J= 12.5 Hz, OCHH'Ph'), 3.11 (d, IH, J= 11.0 Hz, CHCHN(CH3)2), 2.94 (br d, IH, J= 11.0 Hz, CHCHN(CH 3
)
2 ), 1.96 (s, 6H, N(CH 3
)
2 ), 1.08 (s, 9H, SiC(CH 3
)
3 ), 0.59 (s, 3H, SiCH 3 ), 0.29 (s, 3H, SiCH 3 ); ' 3 C NMR (100 MHz, C 6
D
6 ) 8193.3, 186.7, 180.3, 167.8, 154.9, 140.9; 135.6, 135.3, 129.9, 128.6, 128.5, 128.5, 128.4, 128.0, 127.8, 108.6, 82.4, 72.4, 69.6, 69.3, 59.7, 50.2, 41.4, 26.5, 19.6, -1.9, -3.4; FTIR (neat), cm -12930 (m), 2855 (m), 1745 (s, C=0), 1722 (s, C=O), 1691 (m), 1613 (m), 1513 (s), 1473 (in), 1455 (m), 1378 (m), 1264 (s), 1231 (s), 1046 (in); HRMS (ES) in/ calcd for
(C
34
H
4
ON
2 0s+H) 633.2632, found 633.2620. Michael-Dieckmann addition Product MGC27: $013 ~-OBn WHLDATMEDA, THF, -78"C 0 C2h 2,-78 -+0 T / EnO2CQ Bo o HO I n CDIA-28Q NTB5 OTB MGC26 6TSS IGCZ7 80% [00182] A solution of n-butyllithium in hexanes (1.55 M, 155 pL, 0.241 inmol, 5.1 equiv) was added to a solution of N,N,N',N'-tetramethylethylenediamine (39.0 jL, 114 0.261 mmol, 5.5 equiv) and diisopropyl amine (34.0 pL, 0.249 nmol, 5.25 equiv) in tetrahydrofuran (1. mL) at -78 "C. The resulting mixture was stirred vigorously at -78 *C for 30 min whereupon a solution of the ester CDL-I-280 (73.0 mg, 0.213 mmol, 4.5 equiv) in tetrahydrofuran (I mL) was added dropwise via cannula. The resulting deep red mixture was stirred vigorously at -78 "C for 75 min, then a solution of the silyl cyclohexenone MGC26 (30.0 mg, 0.0474 mmol, 1.0 equiv) intetrahydrofuran (1 mL) was added dropwise via cannula. The resulting light red mixture was allowed to warm slowly to 0 *C over 2 h, then was partitioned between an aqueous potassium phosphate buffer solution (pH 7.0,0.2 M, 10 mL) and dichloromethane (10 mL). The organic phase was separated and the aqueous phase was further extracted with two 10-mL portions of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column (10 pM, 250 x 10 mm, flow rate 3.5 mL/min, Solvent A: methanol, Solvent B: water) using an injection volume of 400 pL (methanol) and an isochratic elation of 10% B for 75 min. The peak eluting at 36-42 min was collected and concentrated, affording the Michael-Dieckmann addition product MGC27 (33.0 mg, 80%) as a light yellow solid. [00183] Rf 0.35 (1:4 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 ) 5 16.55 (br s, 1H, enol), 7.26 (d, 2H, J= 7.0 Hz, o-ArfH), 7.14 (d, 2H, J= 7.5 Hz, ArH), 6,85 7.05 (m, 6H, ArH), 6.66-6.74 (in, 2H, ArM), 6.51 (dd, 1H, J= 9.0, 1.5 Hz, ArH), 5.73 (br d, 1H, J=4.0 Hz, BnOCO 2 CH), 5.17 (d, 1H, J= 12.5 Hz, OCHH'Ph), 5.03 (d, IH, J= 12.5 Hz, OCHH'Ph), 4.99 (d, 1H, J= 12.5 Hz, OCHH'Ph'), 4.93 (d, 1H, J= 12.5 Hz, OCHH'Ph'), 3.58 (d, 1H, J= 11.5 Hz, CHCHN(CH 3
)
2 ), 3.35 (dd, 1H, J= 12.5, 4.0 Hz, CH 3 CHCH), 2.99 (d, 1H, J= 11.5 Hz, CHCHN(CH 3 )2),2.56 (dq, IH, J= 12.5, 7.0 Hz, CH 3 CH), 2.18 (s, 6H, N(CH3)2), 1.33 (s, 9H, C(CH 3
)
3 ), 1.16 (d, 3H, J =7.0 Hz,
CH
3 CH), 1.11 (s, 9H, C(CH3)3), 0.61 (s, 3H, CH3), 0.36 (s, 3H, CH3); '3C NMR (100 MHz, CDC13)8 189.7, 186.3, 180.9,178.4, 167.9, 154.7, 152.1, 150.8, 145.9, 136.1, 135.5, 133.9, 128.7, 128.6, 128.5, 127.3, 123.8, 122.7, 122.6,108.9, 105.5, 83.0, 82.9, 74.8, 72.4, 69.2, 60.8, 52.7, 43.2, 38.4, 27.5, 26.6, 19.5, 16.3, -1.8, -2.7; FTIR (neat film), cm~' 2974 (w), 2933 (w), 2851 (w), 1760 (s, C=0), 1748 (s, C=0), 1723 (s, 115 C=O), 1606 (m), 1513 (m), 1471 (m), 13 70 (m). 1260 (s), 1232(s), 1148 (s); HRMS (ES) m/z calcd for (C4HssOzzN 2 Si) t 881.3681, found 881.3684. Initial Deprotection of Michael-Dieckmann Addition Product MGC28: H& Q (CHs)2 HasQ . c )2 HF,
CH
3 CN O Hrqs Ho Hog MGC27 MGCIS [00184] Hydrofluoric acid (1.2 mL, 48% aqueous) was added to a polypropylene reaction vessel containing a solution of the Michael-Diecknann addition product MGC27 (33.0 mg, 0.0375 mmol, 1.0 equiv) in acetonitrile (7.0 mL) at 23 *C. The resulting mixture was stirred vigorously at 23 "C for 60 h, then was poured into water (50 mL) containing K2HPO 4 (7.0 g). The resulting mixture was extracted with ethyl acetate (3 x 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, furnishing the pentacyclic phenol MGC28 as a yellow oil (25.0 mg, 99%). The product was used in the next step without fiuther purification. 1001851 Rf 0.05 (1:4 ethyl acetate-hexanes); 'H NMR (600 MHz, C 6 DA, crude) 8 14.86 (br s, 1H, enol), 11.95 (s, lH, phenol), 7.23 (d, 2H, J= 7.8 Hz, o-ArH), 7.14 (d, 2H, J= 7.2 Hz, o-ArH), 6.94-7.02 (m, 6H, ArH), 6.86 (t, 1H, J= 8.4 Hz, ArH), 6.76 (d, 1H, J= 8.4 Hz, ArH), 6.28 (d, 1H, J= 7.8 Hz, ArH), 5.46 (dd, IH, J= 3.6, 3.0 Hz, BnOCO 2 CH), 5.12 (d, 1H,J= 12.0 Hz, OCHH'Ph), 5.04 (d, 1H, J =12.0 Hz, OCHH'Ph), 4.92 (s, 2H, OCH 2 Ph), 3.41 (d, 1H, J= 9.6 Hz, CHCHN(CH 3
)
2 ), 2.82 (dd, 1H, J= 9.6, 3.0 Hz, CHCHN(CH3)z), 2.65 (dd, 1H, J= 13.2, 3.6 Hz, CHSCHCR), 2.78 (dq, 1H, J= 13.2, 7.2 Hz, CH 3 CH), 2.05 (s, 6H, N(CH 3
)
2 ), 1.04 (d, 3H, J= 7.2 Hz,
CH
3 CH); ' 3 C NMR (100 MHz, C 6
D
6 , crude) 8 193.4, 186.2, 181.3, 172.3, 167.9, 163.3, 154.6, 145.8, 136.6, 135.8, 128.6, 128.4, 127.2, 116.8, 116.0, 115.6, 107.6, 104.7, 76.8, 73.9, 72.5, 69.5, 60.3, 48., 43.0, 41.8, 37.5, 15.3; FTIR (neat filn), cm- 1 3424 (m, OH), 3059, 3030, 2925, 2857, 1744 (s, C=0), 1713 (s, C=0), 1614(s), 1582 (s), 1455 (s), 1252 (s); HRMS (ES) iz calcd for (C37H340ioN2+H)* 667.2292, found 667.2300. 116 (-)-Dxycycline (MGC30): H3Q bl(.HkOH Hz Pd black H
THF-CH
3 OH NH2 HO O HO Oa) N VH o MGC2 (-)-doxycyclina [001861 Pd black (7.00 mg, 0.0657 mmol, 1.75 equiv) was added in one portion to a solution of the pentacyclic phenol MGC28 (25.0 mg, 0.0375 mmol, 1.0 equiv) in tetrahydrofuran-methanol (1:1, 2.0 mL) at 23 *C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The Pd catalyst was initially present as a fine dispersion, but aggregated into clumps within 5 min. The yellow heterogeneous mixture was stirred at 23 "C for 2 h, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil (>95% doxycycline based on 'H NMR analysis). The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 M, 250 x 10 mm, flow rate 4.0 mL/min, Solvent A: methanol-0.005 N aq. HCI (1:4), Solvent B: acetonitrile) using an injection volume of solvent A (400 p) containing oxalic acid (10 mg) and an isochratic elution of 5% B for 2 min, then a gradient elution of 5-50% B for 20 min. The peak elating at 12-17 min was collected and concentrated, affording (-) doxycycline hydrochloride as a yellow powder (16.2 mg, 90%), which was identical with natural (-)-doxycycline hydrochloride in all respects. [001871 'H NMR (600 MHz, CD 3 OD, hydrochloride) & 7.47 (t, IH, J= 8.4 Hz, ArH), 6.93 (d, 1H, J= 8.4 Hz, ArH), 6.83 (d, 1H, J= 8.4 Hz, ArH), 4.40 (s, IH,
(CH
3
)
2 NCH), 3.53 (dd, 1H, J= 12.0, 8.4 Hz, CHOH), 2.95 (s, 3H, N(CR 3
)CH
3 '), 2.88 (s, 3H, N(CH 3 )CH3'), 2.80 (d, 1H, J= 12.0 Hz, CHCHN(CH 3
)
2 ), 2.74 (dq, 1H, J= 12.6,6.6 Hz, CH 3 CH), 2.58 (ddl, H, J= 12.6, 8.4 Hz, CI3CHCH), 1.55 (d, 3H, J= 6.6 Hz, CH 3 CHCH); "C NMR (100 MHz, CD 3 0D) 8 195.3, 188.2, 173.8, 172.1, 163.2, 149.0, 137.7, 117.1, 116.9, 116.6, 108.4, 96.0, 74.5, 69.8,66.9,47.5,43.4, 43.0,41.9, 40.0, 16.3; UV max (0.01 N methanolic HCI), nm 218,267,350; [a] 0 =-1090(c= 0.16 in 0.01 M methanolic HCi); lit. (The Merck Index: An Encyclopedia of Chemicals, 117 Drugs, andBiologicals, 12 " ed. Budavari, S.; O'Neal, M. L; Simith, A.; Heckelman, P. E.; Kinneary, J. F., Eds.; Merck & Co.: Whitehouse Station, NJ, 1996; entry 3496.) UV max (0.01 N methanolic HCl, inn 267, 351; [aD = -110 (c = 1 in 0.01 M methanolic HCI); HRMS (ES) m/z caled for (C22H2OsN2+H)* 445.1611, found 445.1603. Example 3-Synthesis of 6 -Deoxytetracycline Ester CDL-I-280: 1. sec-BuLi, TMEDA, 1. (CC01)2, DMF (cat.), THF, -90 "C
.-
E CH2C2 ? Et C02H 2. EtI, -78 C -> 23 "C 0 2H 2. phenol, DMAP, 2Ph Oq eCM pyndilis OMe OMe EId" Me anisic acid CDL-1-279 50% from anisic acid CDL-1-280 [00188J A solution of sec-butyllithiun in cyclohexane (1.40 M, 24.0 mL, 33.6 mmol, 2.6 equiv) was added to a solution of N,N,NN-tetramethylethylenediamine (4.9 mL, 33 mmol, 2.5 equiv) in tetrahydrofuran (25 mL) at -78 "C. The resulting yellow solution was cooled to -90 *C (internal temperature) in a liquid nitrogen-ethanol bath. A solution of o-anisic acid (2.00 g, 13.1 mmol, 1.0 equiv) in tetrahydrofuran (10 mL) was added dropwise via cannula over a period of 30 min to the yellow solution. The resulting orange suspension was stirred for an additional 30 min at -90 "C, then was allowed to warm to -78 "C over 15 min, whereupon iodoethane (4.2 mL, 52 mmol, 4.0 equiv) was added. The mixture was allowed to warm to 23 *C over 15 min, then was partitioned between water (50 mL) and ether (50 mL). The aqueous layer was separated and diluted with aqueous hydrochloric acid (1.0 M, 100 mL). The resulting mixture was extracted with ethyl acetate (4 x 80 mL). The organic layers were combined and then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oil (1.8 g). 'H NMR (500 MHz, CDC 3 ) analysis of the crude product showed an 8:2 ratio of the carboxylic acid CDL-I-279 (8 3.89, OCH 3 ) and unreacted anisic acid (8 4.07, 0CH 3 ). Oxalyl chloride (1.0 mL, I I mmol, 0.8 equiv) and NN-dimethylfonnamide (100 gL) were added in sequence to a solution of the residue in dichloromethane (20 ML) at 23 *C. Vigorous gas evolution was observed upon addition of NN-dimethyfonnauide. The reaction 118 mixture was stirred for 2 h at 23 *C, whereupon phenol (1.4 g, 15 mmol, 1.1 equiv), pyridine (2.4 mL, 30 mmol, 2.3 equiv), and 4-(dimethylamino)pyridine (10 mg, 0.081 mmol, 0.006 equiv) were added in sequence at 23 "C. The resulting brown reaction mixture was then stirred for 2 h at 23 "C. Aqueous hydrochloric acid (1 M, 50 mL) was added and the resulting mixture was extracted with ethyl acetate (2 x 50 mL). The organic layers were combined, then washed with an aqueous sodium hydroxide solution (0.1 M, 50 mL), followed by brine (50 mL), and were then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a clear oil. The product was purified by flash column chromatography (5:95 ethyl acetate-hexanes), affording the ester CDL-I-280 as a colorless oil (1.7 g, 50%). [00189] Rf 0.28 (5:95 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC1 3 ) 8 7.56 (t, 2H, = 7.8 Hz, ArM), 7.37 (t, IHK, =7.8 Hz, ArH), 7.31-7.26 (m, 3H, ArH), 6.93 (d, 1H, J= 7.8 Hz, ArH), 6.85 (d, 1H, J= 8.3 Hz, ArH), 3.91 (s, 3H, OCH 3 ), 2.79 (q, 2H, J=7.8 Hz, CH 2
CH
3 ), 1.33 (t, 3H, J=7.8 Hz, CH 2 CH3); "C NMR (125 MHz, CDCIs) 8 166.9, 156.5, 150.8, 142.8, 130.9, 129.5, 125.9, 122.5, 121.6, 120.9, 108.5, 55.9,26.6, 15.6; FTIR (neat film), cm-' 2970 (m), 1740 (s, C=O), 1583 (s), 1488 (s), 1471 (s), 1438 (m), 1298 (w), 1270 (s), 1236 (s), 1186 (s), 1158 (m), 1091 (m), 1046 (s), 1001 (w); HRMS (ES)m/z called for (C 16
H
16
O
3 +H)* 257.1178, found 257.1183. Phenol CDL-I-298: CEt BBr 3 , CH 2
CI
2 Et CO2Ph 0 "C K(CO 2 Ph OMe 97% OH CDL-1-280 CDL--298 [001901 A solution of boron tribromide in dichloromethane (1.0 M, 5.2 mL, 5.2 minmol, 2.0 equiv) was added to a solution of the ester CDL-I-280 (662 mg, 2.58 mmol, 1.0 equiv) in dichloromethane (10 mL) at 0 *C. The resulting yellow solution was stirred for 70 min at 0 "C, whereupon saturated aqueous sodium bicarbonate solution (50 mL) was added. The resulting biphasic mixture was stirred for 20 min at 0 "C, dichloromethane (50 mL) was added, the layers were separated, and the aqueous phase 119 was further extracted with dichloromethane (50 mL). The organic layers were combined and then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing the phenol CDL-I-298 as a colorless oil (605 mg, 97%). [00191] RJ 0.47 (5:95 ethyl acetate-hexanes); 'H NMR (500 MHz, CDCla) 6 10.94 (s, 1H, OH), 7.49 (t, 2H, J= 7.8 Hz, ArH), 7.41 (t, IH, J= 7.8 Hz, ArM), 7.35 (t, 1H, J= 7.3 Hz, ArH), 7.24 (d, 2H, J= 7.8 Hz, ArH), 6.93 (d, IH, J=8.3 Hz, ArH), 6.85 (d, 1H, J=8.3 Hz, ArH), 3.13 (q, 2H, J= 7.8 Hz, CH 2
CH
3 ), 1.34 (t, 3H, J= 7.8 Hz, CH 2
CH
3 ); "C NMR (125 MHz, CDCl 3 ) 6 170.3, 163.2, 149.8, 147.8, 135.1, 129.7, 126.4,122.0, 121.6, 115.9, 111.1, 29.8, 16.4; FTIR (neat film), cmf 2973 (w), 1670 (s, C-O), 1609 (in), 1588 (m), 1490 (w), 1444 (m), 1311 (m), 1295 (m), 1234 (m), 1187 (s), 1162 (s), 1105 (m); HRMS (ES) Wz calcd for (CisHw03+H) 243.1021, found 243.1014. Ester CDL-I-299: y Et Boc 2 0, i-Pr 2 NEt yEt 2 Ph DMAP, CH 2
C
2
CO
2 Ph OH 86% OBoc CDL-I-298 CDL--299 [001921 NA-diisopropylethylamine (520 gL, 2.99 mmol, 1.2 equiv), di-t-butyl dicarbonate (645 mg, 2.96 mmol, 1.2 equiv), and 4-(dimethylamino)pyridine (31 mg, 0.25 mmol, 1.5 equiv) were added in sequence to a solution of the phenol CDL-I-298 (605 mg, 2.50 mmol, 0.1 equiv) in dichloromethane (10 mL) at 23 *C. The reaction mixture was stirred for 1 h at 23 *C, whereupon saturated aqueous ammonium chloride solution (50 mL) was added. Dichloromethane (50 mL) was added, the layers were separated, and the aqueous phase was extracted with dichloromethane (50 mL). The organic layers were combined and then dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a brown oil. The product was purified by flash column chromatography (1:9 ether-hexanes), affording the ester CDL 1-299 as a colorless oil, which crystallized upon standing overnight at -14 "C (733 mg, 120 86%), mp 58 *C. [001931 Rf 0.23 (1:9 ether-hexanes); 'H NMR (500 MHz, CDCl 3 )6 7.46-7.42 (in, 3H, ArH), 7.31-7.26 (m, 3H, ArH), 7.22 (d, 1H, J= 7.3 Hz, ArH), 7.15 (d, 1H, J= 7.3 Hz, ArH), 2.86 (q, 2H, J=7.3 Hz, CH 2
CH
3 ), 1.46 (s, 9H, Boc), 1.31 (t, 3H, J= 7.3 Hz, CH2CH 3 ); ' 3 C NMR (125 MHz, CDC 3 ) 6 165.1, 151.6, 150.6, 148.7, 144.5, 131.3, 129.4, 126.8, 126.1, 125.4, 121.7, 120.5, 83.8, 27.5, 26.8, 15.6; FTIR (neat film), cnf 2964 (w), 1754 (s, C=O), 1586 (w), 1491 (w), 1467 (w), 1457 (w), 1368 (w), 1278 (s), 1234 (s), 1190 (s), 1145 (s), 1051 (m); HRMS (ES) m/z caled for (C2oH 22 Os+NH4) 360.1811, found 360.1808. Michael-Die a A tion Product CDL-I-287: 1. LDA, TMEDA, H 3 ' H V3 l(CH3)2 THF, -78 *C
CO
2 Ph 2. -78 "C - 0 * HO OBn OBoc _N(CH 3
)
2 BocO 0 HO OBS b 0 OTBS CDL-1-299 CDL-i-287 6 O OBn OTBS DRSO 83% [001941 A solution of n-butyllithium in hexanes (1.45 M, 47 RL, 0.068 mmol, 6.8 equiv) was added to a solution of diisopropylamine (10 pL, 0.071 mmol, 7.1 equiv) and N,N',N-tetramethylethylenediamine (10 pL, 0.066 mmol, 6.6 equiv) in tetrahydrofuran (300 pL) at -78 "C. The resulting solution was stirred at -78 "C for 30 min whereupon a solution of the ester CDL-1-299 (17 mg, 0.050 mmol, 5.0 equiv) in tetrahydrofuran (200 pL) was added, forming a deep red solution. The solution was stirred at -78 *C for 75 min, then a solution of the enone DRS6 (5.0 mg, 0.010 mmol, 1.0 equiv) in tetrahydrofuran (100 JL) was added at -78 *C. The color of the reaction mixture remained deep red following the addition. The mixture was allowed to warm to 0 *C over 150 min. Upon reaching 0 *C, an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) was added. The resulting yellow mixture was extracted with dichloromethane (3 x 15 mL). The organic layers were combined and 121 then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column'(5 pim, 250 x 10 mm, flow rate 3.5 mL/min, Solvent A: water, Solvent B: methanol, UV detection at 350 un) using an injection volume of 500 gL methanol with an isochratic elation of 89.5% B. The peak eluting at 31-40 min was collected and concentrated affording the Michael Dieckmann product CDL-I-287 as a light yellow solid (6.1 mg, 83%), mp 114 C. [00195] Rf0.37 (2:8 tetrahydrofuran-hexanes); 'H NMR (500 MHz, CDClb) 5 (s, 1H, 16.24, enol-OH), 7.55-7.50 (m, 3H, ArH), 7.40-7.35 (m, 4H, ArH), 7.10 (d, 1H, J = 7.8 Hz, ArM), 5.39-5.34 (m, 2H, OCH 2 Ph), 3.92 (d, 1H, J= 10.7 Hz, CHN(CH 3 )2), 2.81-2.71 (m, 2H, CH 3 CH, CH 3 CHCH), 2.55 (dd, 1H, J=10.7,5.7 Hz,
CHICHN(CH
3 )2), 2.48 (s, 6H, N(CH 3
)
2 ), 2.40 (d, 1H, J= 14.7 Hz,
CHH'CHCHN(CH
3
)
2 ), 2.31 (ddd, 1H, J= 14.7, 9.3, 5.7, CHH'CHCHN(CH3)2), 1.56 (s, 3H, CR 3 ), 1.55 (s, 9H, Boc), 0.84 (s, 9H, TBS), 0.27 (s, 3H, TBS), 0.13 (s, 3H, TBS);
'
3 C NMR (125 MHz, CDCI 3 ) 8 187.4, 183.1, 182.8, 181.6,167.6, 151.7,150.2, 147.4, 135.0, 134.0, 128.5, 128.5, 123.4, 123.0, 122.4, 108.3, 107.4,94.8,83.9,81.5,72.5, 61.5, 46.4, 41.9, 39.5, 34.9, 27.7, 26.0, 20.7, 19.0, 16.0, -2.6, -3.7; FTIR (neat film), cm' 2923 (m), 2841 (m), 1759 (s, C=0), 1718 (s, C=0), 1605 (s), 1508 (s), 1467 (m), 1456 (m), 1369 (m), 1277 (s), 1262 (m), 1231 (s), 1144 (s), 1005 (w); HRMS (ES) m/z calcd for (C 4 oHsoN 2 OSi+H 731.3364, found 731.3370. 6-Deoxytetracycline CDL-I-322 HQ l(CH)2 HQ N(CHa)2 0 1.Bsq. HFCH 3 OH N H 2 , Pd black HNH 2 BocO O HO O OBn THF-CHaOH HO O HO 0 0 OTBS 81% OH CDL-i-287 CDL-1-322 [00196j Hydrofluoric acid (0.6 mL, 48% aqueous) was added to a polypropylene reaction vessel containing a solution of the Michael-Dieckmann addition product CDL 1-287 (15 mg, 0.021 mmol, 1.0 equiv) in acetonitrile (3.5 mL) at 23 C. The reaction mixture was stirred at 23 "C for 55 h, then was poured into water (20 mL) containing 122
K
2
HPO
4 (4.0 g). The resulting mixture was extracted with ethyl acetate (4 x 20 mL). The organic phases were combined and then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a light yellow oil. Pd black (7.6 mg, 0.071 mmol, 3.4 equiv) was added in one portion to a solution of the residue in methanol-tetrahydrofuran (1:1, 2 mL). An atmosphere of hydrogen gas was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The mixture was stirred at 23 0C for 2 h. Within 5 min, the color changed from light yellow to dark yellow. The reaction mixture was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil (10 mg). The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 pm, 250 x 10 mm, flow rate 5 mnL/min, Solvent A: methanol-0.02 N HCl (1:4), Solvent B: acetonitrile, UV detection at 365 mu) using an injection volume of 400 gL methanol containing oxalic acid monohydrate (10 mg) and an isochratic elution of 18% B for 15 min, then a linear gradient elution of 18-60% B in 15 min. The peak eluting at 17.5-22.5 min was collected and concentrated to give 6-deoxytetracycline hydrochloride (CDL-I-322-HCl) as a yellow powder (8.1 mg, 81%). [001971 'H NMR (500 MHz, CD 3 0D, hydrochloride) 8 7.49 (t, 111, J= 7.8 Hz, ArM), 6.95 (d, IH, J = 7.8 Hz, ArH), 6.84 (d, 1H, J = 7.8 Hz, ArH), 4.09 (s, 1H,
CHN(CH
3
)
2 ), 3.03 (br s, 3H, N(CH 3 )), 2.97 (br s, 311, N(CH 3 )), 2.90 (br d, 1H, J= 12.7 Hz, CHCHN(CH3) 2 ), 2.67 (ddd, 11, J= 12.7, 12.7,5.2 Hz, CH 3 CHCH), 2.61-2.56 (m, 1H, CH 3 CH), 2.30 (ddd, J= 13.7, 5.2, 2.9 Hz, CHH'CHCHN(CH 3
)
2 ), 1.54 (ddd, J= 13.7, 12.7, 12.7 Hz, CHH'CHCHN(CH 3 )2), 1.38 (d, 3H, J= 6.8 Hz, CH 3 CH). HRMS (ES) m/z calcd for (C22H24N207+H)* 429.1662, found 429.1660. Example 4-Synthesis of a Pyridone Saneeline Analog Phen IEster CDL-H-464: 123 0 CI 1. ClCC H3C CH3 HAC CH3 H IC NOHB Et 3 N, THF HCx C N ./ C0H N CO 2 Ph C0 2 H OBn 2. PhOH, DMAP 0n CDL-I1417 85% CDL-11-464 [00198 2,4,6-Trichlorobenzoyl chloride (356 PL, 2.28 mmol, 1.1 equiv) was added to a solution of the carboxylic acid CDL-H-417 (reported by Alt Osman, M.M Ismail, M.A. Barakat, Revue Roumaine de Chime 1986,31,615-624) (534 mg, 2.08 mmol, 1.0 equiv) and triethylamine (320 pL, 2.28 mmol, I1 equiv) in tetrahydrofuran (25 mL) at 23 "C. A white precipitate was formed upon addition. The reaction mixture was stirred for 30 min at 23 "C.. A solution ofphenol (489 mg, 5.20 mmol, 2.5 equiv) and 4-(dimethylamino)pyridine (583 mg, 5.20 mmol, 2.5 equiv) in tetrahydrofuran (10 mL) was added via cannula to the reaction mixture prepared above at 0 "C. The resulting mixture was allowed to warm to 23 "C over 10 min, and was stirred for 90 min at that temperature. An aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 30 mL) was then added and the resulting mixture was extracted with dichloromethane (3 x 30 mL). The organic extracts were combined and then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate concentrated, providing a colorless oil. The product was purified by flash column chromatography (6:94 ethyl acetate-hexanes), affording the phenyl ester CDL-II-464 as a white solid (590 mg, 85%), mp 65 *C. [00199] RW 0.33 (1:9 ethyl acetate-hexanes); 'H NMR (500 MHz, CDC1 3 ) 87.49 (d, 2H, J =7.3 Hz, ArH), 7.40-7.24 (m, 6H, ArH), 7.14 (d, 2H, J= 7.3 Hz, ArH), 6.69 (s, IH, pyr-H), 5.49 (s, 2H, CHI 2 Ph), 2.47 (s, 3H, CH3), 2.43 (s, 3H, CH3); "C NMR (125 MHz, CDC 3 ) 8 165.9, 160.1, 157.8, 150.7,148.5, 137,3, 129.4, 128.3, 127.7, 127.6, 125.9, 121.7, 118.1, 113.4, 67.8, 24.1, 19.2; FTIR (neat film), cm' 1738 (s, C=0), 1600 (s), 1569 (s), 1492 (m), 1441 (m), 1400 (m), 1333 (s), 1272 (s), 1185 (s), 1159 (m), 1097 (m), 1051 (s); HRMS (ES) n/z called for (C 21 HIgNO 3 +fH) 334.1443, found 334.1442. 124 Michael-Dieckmann Addition Product CDL-1l-466: 1. LDA, DMPU, H 3 (H) H3C CH13 Ph 2.THF, -78 TCC H3C N N2~~Ph 2. -78 C-00'C n OBn N(CH 3
)
2 BnO 0 HO 0 OBn C OTS CDL-11-464 0CDL-11-46 O i OOBn OTBS DRS6 67% [00200] A solution of n-butyllithium in hexanes (1.67 M, 80 pL, 0.13 mmol, 4.2 equiv) was added to a solution of diisopropylamine (20 pL, 0.14 mmol, 4.5 equiv) in tetrahydrofuran (2.5 mL) at -78 00. The resulting solution was allowed to warm to 0 00 over 15 min. N-dimethylpropyleneurea (17 pL, 0.14 mmol, 4.5 equiv) was added to the mixture prepared above at 0 "C, whereupon the mixture was cooled to -78 *C. A solution of the ester CDL-H-464 (31 mg, 0.093 nmol, 3.0 equiv) in tetrahydrofuran (250 1L) was then added at-78 "C. The resulting yellow solution was stirred for 5 min at -78 *C, then a solution of the enone DRS6 (15 mg, 0.031 mmol, 1.0 equiv) in tetrahydrofuran (250 gL) was added at -78 *C. The resulting deep red mixture was allowed to warm to 0 C over 4 h. Acetic acid (40 pL) was added at to the deep red mixture at 0 "C, followed by an aqueous potassium phosphate buffer solution (pH 7.0, ).2 M, 15 mL). The resulting yellow mixture was extracted with dichloromethane (3 x 15 mL). The organic extracts were combined and then dried over anhydrous sodium iulfate. The dried solution was filtered and the filtrate was concentrated, providing a ellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere )DS column (5 pm, 250 x 10 mmun, flow rate 3.5 L/mmin, Solvent A: water, Solvent B: nethanol, UV detection at 350 nm) using an injection volume of 500 gL DMSO and a gradient elution of 92-100% B over 30 min. The peak eluting at 21-29 min was collectedd and concentrated to give enol CDL-I-466 as a light yellow solid (15.0 mg, i7%). 002011 Rf 0.55 (3:7 ethyl acetate-hexanes); 'H NMR (600 MHz, CD2C12) 6 .6.05 (a, 1H, enol-OH), 7.52-7.26 (m, 10H, AMR), 6.66 (s,,1H, pyr-H), 5.57 (d, 1H, J= 125 12.7 Hz, OCHH'Ph), 5.43 (d, J= 12.7 Hz, 1H, OCHH'Ph), 5.33-5.28 (m, 2H,
OCH
2 Ph), 3.99 (d, 2H, J =10.5 Hz, CHN(CH 3
)
2 ), 3.04-3.00 (m, 1H,
CHCH
2
CHCHN(CH
3
)
2 ), 2.84 (dd, 1H, J= 16.1,4.9 Hz, CHH'CHCH 2 CHCHN(CH3) 2 ), 2.74 (dd, 1H, J= 16.1, 16.1 Hz, CHH'CHCH 2 CHCHN(CHb) 2 ), 2.53 (dd, 1H, J= 10.5, 3.9 Hz, CHCHN(CH 3
)
2 ), 2.51-2.43 (m, 10H, N(CH3)2, Ar-CH3, CHH'CHCHN(CH3) 2 ), 2,07 (d, 1H, J= 14.2 Hz, CHHCHCHN(CH 3 )), 0.82 (s, 911, TBS), 0.22 (s, 3H, TBS), 0.10 (s, 3H, TBS); "C NMR (100 MHz, CD202) 8 187.9, 185.2, 182.5, 178.8, 167.9, 161.9, 161.8, 154.8, 137.9, 135.6, 129.1, 129.0, 129.0, 128.7, 127.9, 127.9, 116.4, 111.6, 108.6, 107.5, 82.0, 73.0, 68.1, 61.7, 46.9,420, 39.2, 28.6,26.1, 24.6, 23.0, 19.3, -2.4, -3.5; FTIR (neat film), cm~' 2939 (m), 2857 (w), 1720 (s, C=0), 1593 (s), 1510 (s), 1469 (m), 1449 (m), 1326 (s), 1254 (m), 1187 (w), 1157(m), 1090 (m), 1064 (m), 1007 (m); HRMS (ES) m/z caled for (C 4 1 4 7
N
3 0,Si+H)* 722.3262, found 722.3261. Pyridone Sancycline Analog CDL-l-460: Hi(CH3) 2 1. H 2 , Pd(OH) 2 /C
N(CH
3
)
2 - HCI
H
3 C dioxane, CH 3 0H HC - OH 2. aq. HCI, MeOH HN NH BnO 0 OH= 0 OBn 74% 0 0 Oi _O 0 OTBS OH CDL-I-466 CDL-1-400 [00202] Palladium hydroxide on carbon (20 wL % Pd, wet, water max. 50%, 10 mg, 0.0094 mmol, 0.7 equiv) was added to a solution of the Michael-Dieckmann addition product CDL-H-466 (10 mg, 0.014 mnmol, 1.0 equiv) in dioxane-methanol (1:1, 10 mL) at 23 *C. An atmosphere of hydrogen gas was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The resulting mixture was stirred at 23 *C for 2 h. The color turned green after 5 min and then gradually to yellow within the reaction time. The mixture was filtered through a plug of cotton and then concentrated to a yellow oil. Aqueous hydrochloric acid (37%, 100 .L) was added to a solution of the residue in methanol (10 mL) at 23 *C, The reaction was monitored by analytical HPLC on a Coulter Ultrasphere ODS column (5 gm, 250 x 4.6 mm, flow rate 1 ml/min, Solvent A: 0.1% TFA in water, Solvent B: 0.1% TFA in acetonitrile, UV detection at 395 un) with a gradient elution of 10-100% B over 15 126 min. The peak at 7.0 min indicated the desired product. After stirring for 3 h at 23 "C the deprotection was complete and the mixture was concentrated to a yellow oil. The crude mixture was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 pm, 250 x 10 mm, flow rate 4 ml/min, Solvent A: 0.01 N aqueous hydrochloric acid, Solvent B: acetonitrile, UV detection at 365 nm) using an injection volume of 500 pL methanol containing oxalic acid monohydrate (30 mg) and a linear gradient of 0-20% B over 40 min. The peak eluting at 20-29 min was collected and concentrated to give the hydrochloride of CDL-I-460 as a yellow powder (4.8 mg, 74%). [002031 'H NMR (500 MHz, CD 3 0D, hydrochloride) 66.37 (s, 1H, ArH), 4.06 (s, IH, CHN(CH3)2), 3.05-2.95 (m, 8H, N(CH3) 2 , CHCHN(CH3)z,
CHCH
2 CHCHN(CH3)2), 2.79 (dd, 1H, J= 16.1, 3.9 Hz, CHH'CHCH 2 CHCHN(CH3) 2 ), 2.55 (dd, 1H, J= 16.1, 16.1 Hz, CHH'CHCH 2
CHCHN(CH
3
)
2 )), 2.40 (s, 3H, Ar-CH3), 2.18 (br. d, 1H, J= 12.7 Hz, CUH'CHCHN(CH3)2), 1.59 (ddd, IH, J= 12.7, 12,7, 12.7 Hz, CHH'CHCHN(CH3)2); "C NMR (100 MHz, (CD3) 2 SO) 8 187.3, 183.5, 177.8, 172.1,160.6, 159.8, 153.3, 115.3, 107.2, 106.9,95.6, 74.2,68.4,41.5,35.7,34.5,33.9, 31.0, 19.2; IRMS (ES) mz called for (C2H23N 3 0 7 +H) 430.1614, found 430.1607. Example5-Synthesis of Pyridine Sancycline Analog (7-Aza-10-Deoxysancycline) . eq. NaOH, ethanol, reflux N CH3 2. (COCl) 2 , DMF, CH 2 Cl2 N CH3 3. phenolDMAP, pyridine,
CO
2 Et 66% '0 2 Ph JDBI-67-SM JOBI-67 [002041 A solution of 2-methyl-nicotinic acid ethyl ester JDB1-67-SM (0.589 g, 3.56 mmol, 1.0 equiv), aqueous sodium hydroxide (1.0 M, 3.9 mL, 3.9 mmol, 1.1 equiv), and ethanol (5 nL) was heated at reflux for 18 h. The reaction mixture was allowed to cool to 23 *C, and was concentrated, affording the carboxylate salt (710 mg) as a white solid. Oxalyl chloride (357 gL, 4.09 mmol, 1.15 equiv) was added to a mixture of the carboxylate salt in dichloromethane (20 mL) at 23 "C. Vigorous gas evolution was observed upon addition. The reaction mixture was stirred at 23 aC for 30 min, then N,N-dimethylformamide (20 sL) was added. After stirring for an additional 127 30 min at 23 "C, phenol (837 mg, 8.90 mmol, 2.5 equiv), pyridine (864 pL, 10.7 mmol, 3.0 equiv), and dimethylaminopyridine (3 mg) were added in sequence. The resulting solution was stirred for 90 min at 23 "C, whereupon an aqueous potassium phosphate buffer solution (pH 7.05, 0.2 M, 5.0 mL) was added. The resulting mixture was partitioned between water (30 mL) and ethyl acetate (50 mL). The aqueous phase was extracted with an additional 50-mL portion of ethyl acetate. The organic layers were combined and washed with an aqueous sodium hydroxide solution (50 mL, IM), brine (50 mL), and then dried over anhydrous sodium sulfate. The dried solution was decanted and concentrated, affording a colorless oil (900 nug). The product was purified by flash column chromatography (25:75 ethyl acetate-hexanes), providing the ester JDB1-67 as a colorless oil (500 mg, 66%). [00205] Rf 0.15 (3:7 ethyl acetate-hexanes); 'H NMR (300 MHz, CDCl 3 )8 8.70 (dd, 1H, J- 1.7,4.95 Hz, pyr-H), 8.44 (dd, 1H, J= 1.7, 7.8 Hz, pyr-H), 7.48-7.43 (m, 2H, ArH), 7.33-7.20 (m, 4H, ArH, pyr-H), 2.93 (s, 1H, CH,); "C NMR (100 MHz, CDCl 3 ) 8 164.8, 160.8, 152.4, 150.5, 138.9, 129.5, 126.1, 124.5,121.6, 121.0,25.0; FTIR (neat film), cm-' 3406 (m), 1948 (w), 1747 (s), 1578 (s), 1487 (s), 1435 (s), 1273 (s), 1237 (s), 1191 (s), 1046 (s), 915 (m), 822 (m), 749 (s), 689 (s); HRMS (ES) m/z caled for (CuHIINO 2 +H) 214.0868, found 214.0866. N(CH3)2 N(CH3)2 H H I NjC§Ii1, LDA, HMPA O, + N N QXCO2Ph -95 C -O HO 0 C OTBS 72% OTBS JDBi-67 DRS6 JDB1-87 [00206] A solution of n-butyllithium in hexanes (1.47 M, 136 pL, 0.200 mmol, 8.03 equiv) was added to a solution of diisopropylamine (26.5 , 0.202 mmol, 8.05 equiv) in tetrahydrofuran (0.750 mL) at -78 "C. The reaction mixture was briefly (10 min) transferred to an ice bath, with stirring, then was cooled to -78 *C. Hexamethylphosphoramide (49.0 FL, 0.399 mmol, 16.0 equiv) was added to the mixture prepared above at -78 "C. The resulting mixture was stirred for 5 minutes 128 whereupon a colorless solution was formed. The resulting solution was added dropwise via cannula to a solution of the ester JDB1-67 (36.0 mg, 0.169 mmol, 6.79 equiv), and the enone DRS6 (12.2 mg, 0.0249 mmol, 1.00 equiv) in tetrahydrofuran (1 mL) at -95 "C dropwise via cannula. The light red mixture was allowed to warm to -50 *C over 50 min and was then partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 5.0 mL) and dichloromethane (25 mL). The organic phase was separated and the aqueous phase was further extracted with dichloromethane (3 x 15 mnL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was decanted and concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column (10 pm, 250 x 10 mm, 3.5 mL/min, Solvent A: water, Solvent B: methanol, UV detection at 350 nm) using an injection volume of 500 pL methanol and a linear gradient elution of 85-100% B over 30 min. The peak at 21-27 min was collected and concentrated to give enol JDB1-87 as a white solid (11.0 mg, 72%). [00207] Rf 0.07 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CD 2 Cl 2 ) 8 15.21 (s, 1H, enol), 8.63 (d, IN, J= 4.5 Hz, pyr-H), 8.19(d 1H, J= 7.5 Hz, pyr-H), 7.54-7.43 (m, 5H, ArH), 7.34 (d, lH, J= 4.5, 7.5 Hz, pyr-H), 5.36 (d, 1H, J= 12.0 Hz, OCHIH'Ph), 5.33 (d, 1H, J =12.0 Hz, OCHH'Ph), 4.03 (d, 1H, J= 10.7 Hz,
CHN(CH
3
)
2 ), 3.36-3.31 (m, 1H, CHCH 2
CHCHN(CH
3
)
2 ), 3.23 (dd, 1H, J= 16.3, 5.6 Hz, CJH'CHCH 2
CHCHN(CH
3
)
2 ), 2.99 (dd, 1H, J= 16.3, 16.3 Hz, CHH'CHCH2CHCHN(CH3)2), 2.63 (ddd, lH, J= 1.6,4.4,10.7 Hz, CHCHN(CH 3
)
2 ), 2.54-2,48 (m, 7H, N(CH 3 )2, CHH'CHCHN(CH 3 )2), 2.19 (dd, 1H, J= 1.6, 14.5 Hz,
CHH'CHCHN(CH
3 )2), 0.87 (s, 9H, TBS), 0.26 (s, 3H, TBS), 0.13 (s, 3H, TBS); "C NMR (100 MHz, CD 2 Cl 2 )8 187.7, 183.5, 182.6, 182.2, 167.9, 161.2, 153.4, 137.6, 134.1, 129.2, 129.1, 129.1, 126.8, 123.0, 108.7, 106.9, 82.2, 73.0, 61.8, 47.0, 42.1, 41.4, 30.1, 28.4, 26.1, 23.2, 19.3, -2.4, -3.5; HRMS (ES) m/z calod for (C33Ha 9 N306Si+H) 602.2686, found 602.2686. 129 U (CHs) 1 H2, Pd black H H t(CHs) 2 N 0 dioxane-CH 3 0H N - OH N 2. HF(aq), CH 3 CN, 35 C jNH 0 HO o OBn 86% 0 HO 0 O OTBS OH JDBI-87 JDBI-109 [00208] - Pd black (3.0 mg, 0.028 mmol, 2.6 equiv) was added in one portion to a solution of the enol JDBI-87 (6.5 mg, 0.011 mmol, 1.0 equiv) in dioxane-methanol (7:2, 9.0 mL) at 23 *C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The green mixture was stirred for 7 hr, and then filtered through a plug of cotton. The filtrate was concentrated, providing the carboxamide as a yellow oil (7.0 mg). Aqueous hydrofluoric acid (48%, 0.5 mL) was added to a polypropylene reaction vessel containing a solution of the carboxamide in acetonitrile (4.5 mL) at 23 *C. The reaction mixture was heated to 35 *C and was stirred at that temperature for 27 hr. The excess hydrofluoric acid was quenched with methoxytrimethylsilane (3.5 mL, 25 mmol). The reaction mixture was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 pum, 250 x 10 mm, 4 mUL/min, Solvent A: 0.5% trifluoroacetic acid in water, Solvent B: 0.5% trifluoroacetic acid in methanol-acetonitrile (1:1), UV detection at 350 nm) using an injection volume of 500 pL methanol and a linear gradient of 0-20% B over 40 min. The peak at 35-45 min was collected and concentrated to give a yellow oil. The oil was dissolved in 1 mL methanol, treated with concentrated hydrochloric acid (20 pL), and then concentrated to give the hydrochloride of JDBI-109 as a yellow powder (3.7 mg, 86%). [002091 'H NMR (500 MHz, CD 3 OD, hydrochloride) 6 8.79-8.77 (m, 2H, pyr H)7.91 (dd, 1H, J=6.8, 6.8 Hz, pyr-H), 4.12 (s, 1H, CHN(CH3) 2 ), 3.41-3.22 (m, 2H,
CHH'CHCH
2
CHCHN(CH
3
)
2 , CHCH 2 CHCHN(CH3) 2 ), 3.11-3.00 (m, 8H,
CHH'CHCH
2
CHCHN(CH
3
)
2 , CHCHN(CH 3
)
2 , N(CH 3
)
2 ), 2.34 (ddd, IH, J= 12.9, 4.4, 2.4 Hz, CHH'CHCHN(CH 3
)
2 ), 1.77 (ddd, 1HI, J= 12.9, 12.9, 12.9 Hz,
CHH'CHCHN(CH
3
)
2 ); HRMS (ES) n/z calcd for (C 2 oH 2 iNO 6 +H)* 400.1508, found 130 400.1504. Example 6-Synthesis of 1-Deorysancycline CH 1. (COCI) 2 , DMF, CHI- 2 C1 2 2. phenol, DMAP, pyridine CO2H 99% CO 2 Ph JDB1-113-SM JD81-113 (002101 NN-dimethylformrnamide (20 L) was added was added to a solution of the carboxylic acid JDB1-113-SM (500 mg, 3.67 mmol, 1.0 equiv) and oxalyl chloride (367 pl, 4.22 mmol, 1.15 equiv) in dichloromethane (20 mL) at 23 C. Vigorous gas evolution was observed. After stirring for 80 min at 23 *C, phenol (863 mg, 9.18 mmol, 2.5 equiv), pyridine (890 gL, 11.0 mmol, 3.0 equiv), and dimethylaminopyridine (3 mg) were added in sequence. The resulting solution was stirred for 90 min at 23 *C, whereupon an aqueous potassium phosphate buffer solution (pH 7.05, 0.2 M, 5.0 mL) was added. The resulting mixture was partitioned between water (30 mL) and ethyl acetate (50 mL). The aqueous phase was extracted with an additional 50-mL portion of ethyl acetate. The organic layers were combined and washed with an aqueous sodium hydroxide solution (50 mL, I M), brine (50 mL), and then dried over anhydrous sodium sulfate. The dried solution was decanted and concentrated, affording a colorless oil (850 mg). The product was purified by flash column chromatography (25:75 ethyl acetate-hexanes), providing the ester JDB1-113 as a colorless oil (774 mg, 99%). [00211] Rf 0.43 (3:7 ethyl acetate-hexanes); 'H NMR (300 MHz, CDC 3 ) 88.18 (d, 1H, J = 8.1 Hz, ArH), 7.49-7.20 (m, 8H; ArH, OArH), 2.69 (s, 3H, ArCH 3 ); "C NMR (100 MHz, CDCI 3 ) 5 165.8, 150.9, 141.3, 132.7, 132.0, 131.2, 129.5, 128.5, 125.9, 125.8, 121.8, 22.0; FTIR (neat film), cm' 3046 (w), 2923 (w), 1739 (s), 1594 (m), 1487 (m), 1287 (m), 1241 (s), 1189 (s), 1159 (m), 1041 (s), 733 (s); KRMS (ES) m/z called for (C 1 4H 2 0 2 +NH4) 230.1181, found 230.1187. 131 H _N(CHa)2 h L a(CHA CH Y; 3 I DA, KMPA0 +N THE C -95C 70C 0n HO Q OTBS 85% OTBS JDBI-113 DRS6 JDBI-114 [00212] A solution of n-butyllithium in hexanes (1.47 M, 38.0 pL, 0.0565 mmol, 8.26 equiv) was added to a solution of diisopropylamine (7.4 sL, 0.057 mmol, 8.3 equiv) in tetrahydrofuran (0.50 mL) at -78 "C. The reaction mixture was briefly (10 min) transferred to an ice bath, with stirring, then was cooled to -78 "C. Hexamethylphosphoramide (13.9 gL, 0.113 mmol, 16.5 equiv) was added to the mixture prepared above at -78 "C. The resulting mixture was stirred for 5 minutes whereupon a colorless solution was formed. The resulting solution was added dropwise via cannula to a solution of the ester JDB1-113 (10.0 mg, 0.0471 mmol, 6.88 equiv), and the enone DRS6 (3.3 mg, 0.00684 mmol, 1.00 equiv) in tetrahydrofuran (0.50 mL) at -95 *C dropwise via cannula. The light red mixture was allowed to warm to -70 *C over 30 min and was then partitioned between an aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 5.0 mL) and dichloromethane (20 mL). The organic phase was separated and the aqueous phase was further extracted with an additional 20-mL portion of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was decanted and concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column (10 pm, 250 x 10 mm, 3.5 mL/min, Solvent A: water, Solvent B: methanol, UV detection at 350 nm) using an injection volume of 500 pL methanol and a linear gradient elution of 85-100% B over 30 min. The peak at 25 30 min was collected and concentrated to give enol JDB1-87 as a white solid (3.5 mg, 85%). [002131 Rf 0.46 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CD 2
C
2 ) 8 15.53 (s, IH, enol), 7.94 (d, 1H, J= 7.9 Hz, ArH), 7.54 - 7.28 (m, 8H, ArH, OCH2ArH), 5.37-5.34 (m, 211, OCH 2 Ph), 4.05 (d, 1H, J= 10.7 Hz, CHN(CH 3
)
2 ), 3.24 3.18 (m, 1HI, CHCH 2
CHCHN(CH
3
)
2 ), 2.99 (dd, 1H, J= 15.5,5.6 Hz, CHH'CHCH2CHCHN(CH3) 2 ), 2.88 (dd, 1H, J=15.5, 15.5 Hz, 132
CHH'CHCH
2
CHCHN(CH
3
)
2 ), 2.61 (dd, 1H, J= 4.4, 10.7 Hz, CHCHN(CH3)2), 2.54 2.44 (m, 7H, N(CH 3
)
2 , CHH'CHCHN(CH 3
)
2 ), 2.14 (d, 1H, J= 14.3 Hz,
CHH'CHCHN(CH
3
)
2 ), 0.86 (s, 9H, TBS), 0.25 (s, 3H, TBS), 0.12 (s, 3H, TBS); '3C NMR (100 MHz, CD2C 2 )6 187.8, 183.0, 182,8, 182.4, 167.7, 141.7,135.4, 133.4, 130.9, 129.0, 128.9, 128.9, 128.1, 127.5, 126.5, 108.5, 106.8, 82.1, 72.8, 61.5, 58.5, 46.9, 41.9, 38.6, 29.0,25.9, 23.1, 19.1,-2.6, -3.7; HRMS (ES) m/z caled for
(C
3 4H4ONsQ 6 Si+H) 601.2734, found 601.2730. H N(CH 3
)
2 - 1. HF(aq), CH 3 CN, 35 "C j H NL(CHs)2 ' 2 H2, HPdNbackH [CI~ hJIII)INdioxane-CH 3 O4
INH
2 0 HO O OBn 0 HO O 0 OTBS OH JDBI-114 JDBI.130 [00214] Hydrofluoric acid (1.1 mL, 48% aqueous) was added to a polypropylene reaction vessel containing a solution of the enol JDBI-114 (15.1 mg, 0.0251 mmol, 3.0 equiv) in acetonitrile (10 mL) at 23 "C. The resulting mixture was stirred vigorously at 23 *C for 12 hr, then was poured into water (50 mL) containing K 2
HPO
4 (4.7 g). The resulting mixture was extracted with ethyl acetate (3 x 25 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, furnishing the intermediate alcohol as a yellow solid (12.2 mg, 99%). Pd black was added in one portion to a solution of the residue in methanol-dioxane (1:1, 3.0 mL). An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushitg wAth pure hydrogen (I atm). The mixture was stirred at 23 "C for 20 min. Within 5 min, the color changed from light yellow to green. The reaction mixture was filtered through a plug of cotton. The filtrate was concentrated to a yellow solid (13 mg). The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 pm, 250 x 10 mm, flow rate 5 mL/min, Solvent A: 0.01 N HC, Solvent B: acetonitrile, UV detection at 350 nm) using an injection volume of 450 RL methanol containing oxalic acid monohydrate (10 mg) in two injections and a linear gradient elution of 5:-50% B in 30 min, The peak eluting at 16-22 mi was collected and concentrated-to give 10 133 deoxysancycline hydrochloride (JDB1-130-HCl) as a white powder (9.1 mg, 91%). (002151 'H NMR (500 MHz, CD 3 0D, hydrochloride) 8 7.96 (d, 1H, J= 7.3 Hz, ArH) 7.51 (dd, 1H, J= 7.3, 7.3 Hz, ArH), 7.39 (dd, 1HI, J- 7.3, 7.3 Hz, Ar!!), 7.30 (d, IH, J= 7.3 Hz, ArH), 4.04 (s, 1H, CHN(CH3)2), 3.31-2.99 (m, 8H,
CHCH
2
CHCHN(CH
3
)
2 , CHCHN(CH 3
)
2 , N(CH 3
)
2 ), 2.87 (dd, 1HI, J= 15.4,4.3 Hz,
CHH'CHCH
2
CHCHIN(CH
3 )2), 2.61 (dd, 1H, J= 15.4, 15.4 Hz,
CH'CHCH
2
CHCHN(CH
3
)
2 ), 2.21 (ddd, J= 12.8, 5.0, 2.5 Hz, CHH'CHCHN(CH 3
)
2 ), 1.66 (ddd, 1H, J= 12.8, 12.8, 12.8 Hz, CHH'CHCHN(CH 3
)
2 ). Example 7-A Convergent. Enantioselective Synthetic Raute to Structurally Diverse 6-Deoxytetracycline Antibiotics [00216] Among tetracycline, semi-synthetic approaches have led to the discovery of the 6-deoxytetracyclines doxycycline (2 in Figure 15A) and minocycline (3 in Figure 15A), clinically the most important agents in the class. 6 Deoxytetracyclines exhibit considerably improved chemical stability as compared to their 6-hydroxy counterparts and show equal or greater potencies in antibacterial assays (Stephens et al., J. Am. Chem. Soc. 85, 2643 (1963); M. Nelson, W. Hillen, R. A. Greenwald, Eds., Tetracyclines in Biology, Chemistry and Medicine (Birkhauser Verlag, Boston, 2001); each of which is incorporated herein by reference). It is evident that at present neither semi-synthesis nor modified biosynthesis is capable of addressing the great majority of novel structures that a chemist might wish to explore in pursuit of a lead structure like tetracycline; structures such as the D-ring heterocyclic analogs 4 and 5 in Figure 15A, or new ring systems such as the pentacycline 6 (Figure 15A) are exemplary. Absent a viable laboratory synthetic pathway, these structures and the regions of complex chemical space they represent must be ceded in the search for new antibiotics. Here, we report a short and efficient route for the synthesis of enantiomerically pure members of the 6-deoxytetracyclines from benzoic acid. The route we describe allows for the synthesis of 6-deoxytetracyclines (both with or without an hydroxyl group at C5) by a notably late-stage coupling reaction of the AB precursors 7 or 8 (Figure 15B) with a variety of different D-ring precursors, and has provided 134 compounds such as doxycycline (2 in Figure 15A), the heterocyclic analogs 4 and 5 (Figure 15A), the pentacycline 6 (Figure 15A), as well as other 6-deoxytetracycline analogs. [00217] The strategic advantage of a synthetic approach involving a late-stage C ring construction (AB + D -+ ABCD, Figure 15B) is that much of the polar functionality known to play a role in the binding of tetracyclines to the bacterial ribosome lies within the AB fragment (D. E. Brodersen et al., Cell 103, 1143 (2000); M. Pioletti et al., EMBO J 20,1829 (2001);each of which is incorporated herein by reference), while enormous structural variation on or near the D-ring is not only permissible, but has been cited as a means to overcome bacterial resistance. The advanced clinical candidate tigecycline (P.-E. Sum, P. Petersen, Bioorg. Med. Chem. Lett. 9, 1459 (1999); incorporated herein by reference), a minocycline derivative with a D-ring substituent, is exemplary, and is reported to be one of the most promising new antibiotics under evaluation by the FDA (K. Bush, M. Macielag, M. Weidner-Wells, Curr. Opin. Microbiol. 7, 466 (2004); incorporated herein by reference). Classically, approaches to the synthesis of the tetracycline antibiotics have proceeded by stepwise assembly of the ABCD ring system and begin with D or CD precursors, as exemplified by the Woodward synthesis of (±)-6-deoxy-6-demethyltetracycline (sancycline, 25 steps, -0.002% yield) (J. J. Korst et al., J Am. Chem. Soc. 90,439 (1968); incorporated herein by reference), the Shemyakin synthesis of (±)-1 2a-deoxy-5a,6 anhydrotetracycline (A. I. Gurevich et a., Tetrahedron Lett. 8, 131 (1967); incorporated hemin by reference), and the Muxfeldt synthesis of(±)-5-oxytetracycline (terramycin, 22 steps, 0.06% yield) (H. Muxfeldt et al., J Am. Chem. Soc. 101, 689 (1979); incorporated herein by reference). Only one published synthesis of (-) tetracycline itself has appeared, this from D-glucosamine (an A-ring precursor, 34 steps, 0.002% yield) (K. Tatsuta et at, Chem. Lett. 646 (2000); incorporated herein by reference), while the most efficient construction of the tetracycline ring system thus far is undoubtedly the synthesis of (±)-12a-deoxytetracycline by the Stork laboratory (16 steps, 18-25% yield) (G. Stork et al, J Am. Chem. Soc. 118,5304 (1996); incorporated herein by reference). The latter research served to identify Cl2a oxygenation as perhaps the greatest challenge in tetracycline synthesis (it could not be achieved with 135 12a-deoxytetracycline as substrate), a conclusion supported by the results of prior synthetic efforts (J. J. Korst et al., J Am. Chem. Soc. 90, 439 (1968); A. I. Gurevich et al., Tetrahedron Left. 8, 131 (1967); H. Muxfeldt et al., J Am. Chem. Soc. 101, 689 (1979); each of which is incorporated herein by reference). The problem is significant, for C12a oxygenation appears to greatly enhance antimicrobial activity (W. Rogalski, in Handbook of Experimental Pharmacology, J.1. Hlavka, J. H. Boothe, Eds. (Springer-Verlag, New York, 1985), vol. 78, chap. 5; incorporated herein by reference). A key feature of the synthetic approach to 6-deoxytetracyclines that we have developed is that it introduces the Cl2a hydroxyl group in the first step of the sequence (Figure 16) and uses the stereogenic center produced in that step to elaborate all others in the target molecule. To protect the vinylogous carbamic acid function of the A-ring we used the 5-benzyloxyisoxazole group developed by Stork and Haggedorn for that purpose (G. Stork, A. A. Hagedorn, III, J Am. Chem. Soc. 100, 3609 (1978); incorporated herein by reference), an innovation that proved critically enabling in the present work, while the dimethylamino group of the A-ring was incorporated without modification. [002181 Our synthesis of6-deoxytetracyclines was initiated by whole-cell, microbial dihydroxylation of benzoic acid with a mutant strain of Alcaligenes eutrophus (A. M. Reiner, G. D. Hegeman, Biochemistry 10, 2530 (1971); A. G. Myers et al., Org. Lett. 3, 2923 (2001); each of which is incorporated herein by reference), producing the diol 9(Figure 16) with >95% ee in 79% yield (90-g batch, 13 g/L, Figure 16). Hydroxyl-directed epoxidation of the microcrystalline product (9, m CPBA, EtOAc) provided the a-oriented epoxide 10 (Figure 16) in 83% yield; esterification of this product (trimethylsilyldiazomethane) followed by bis-silylation and concomitant epoxide isomerization in the presence of tert-butyldimethylsilyl triflate (3 equiv.), afforded the epoxy ester 11 (Figure 16) in 70% yield (A. G. Myers at al., Org. Lett. 3, 2923 (2001); incorporated herein by reference). Separately, 3 benzyloxy-5-dimethylaminomethylisoxazole, prepared on the mole-scale by a simple four-step sequence from glyoxylic acid (D. M. Vyas, Y. Chiang, T. W. Doyle, Tetrahedron Lett. 25, 487 (1984); P, Pevarello, M. Varasi, Synth Commun. 22, 1939 (1992); each of which is incorporated herein by reference), was deprotonated at C4 136 with n-butyllithium, and the resulting organolithium reagent (12 in Figure 16) was then added to the epoxy ester 11 (Figure 16), forming the ketone 13(73%) (Figure 16). In a noteworthy transformation, and a key step of the synthesis, exposure of the ketone 13 (Figure 16) to lithium triflate (5 mol %) at 60 "C, followed by selective removal of the allylic silyl ether of the rearranged product (TFA), afforded the tricyclic AB precursor 14 (Figure 16) in 62% yield after purification by flash column chromatography. The transformation of 13 to 14 (Figure 16) is believed to involve initial S-prime opening of the allylic epoxide by the NN-dimethylamino group followed by ylide formation and [2,3]-sigmatropic rearrangement, a process that is reminiscent of the Sommelet-Hauser rearrangement (S. H. Pine, Organic Reactions, 18, 403 (1970); incorporated herein by reference). Compound 14 (Figure 16) possesses the requisite cis stereochemistry of the AB fusion as well as an a-oriented NN-dimethylamino substituent (confirmed by X ray crystallographic analysis of a derivative), and serves as a common intermediate for the synthesis of both the AB precursor enone 7 (4 steps, 49% yield, Figure 16) and the AB precursor to 5-a-hydroxy-6-deoxytetracyclines, enone 8 (8 steps, 56% yield, Figure 16), as detailed in sequence below. [002191 To synthesize the AB precursor enone 7 (Figure 15), intermediate 14 was subjected to reductive transposition (A. G. Myers, B. Zheng, Tetrahedron Lett. 37, 4841 (1996); incorporated herein by reference) in the presence of triphenylphosphine, diethyl azodicarboxylate, and o-nitrobenzenesulfonyl hydrazide (added last, a procedural variant), affording the transposed cycloalkene 15 in 74% yield. Hydrolysis of the silyl ether group within 15 (HCI, methanol), oxidation of the resulting allylic alcohol (IBX, DMSO) (M. Frigerio, M. Santagostino, Tetrahedron Lett. 35, 8019 (1994); incorporated herein by reference), and protection of the remaining (tertiary) carbinol (TBSOTf, 2,6-lutidine) (E. . Corey et al., Tetrahedron Left. 22, 3455 (1981); incorporated herein by reference) then provided the enone 7 (Figure 15) in 66% yield (3 steps) after flash column chromatography. By a somewhat longer but slightly more efficient sequence the intermediate 14 (Figure 15) could also be transformed into the enone 8 (Figure 15), the AB precursor to 5-a-hydroxy-6-deoxytetracyclines. This sequence involved the transformation of 14 (Figure 15) into the phenylthio ether 16 (with net retention), diastereoselective sulfoxidation using a chiral.oxidant (F. A. Davis 137 et al, J Org. Chem. 57, 7274(1992); incorporated herein by reference) (99:1 selectivity), and Mislow-Evans rearrangement (E. N. Prilezhaeva, Russ. Chem. Rev. 70, 897 (2001); incorporated herein by reference), producing the allylic alcohol 17 in 66% yield (4 steps). High diastereoselectivity in the sulfoxidation step was essential, for only one diastereomer (the major isomer under the conditions specified) underwent efficient thermal rearrangement. After protection of the allylic alcohol 17 (Figure 15) using benzyl chlorofonnate, a sequence nearly identical to the final three steps of the synthesis of 7 (Figure 15) was employed to transform the resulting benzyl carbonate into the enone 8 (Figure 15) in 85% yield (56% yield and 8 steps from 14). 100220) 6-Deoxytetracyclines were assembled with all requisite functionality and stereochemistry in a single operation. In this process the AB precursors 7 or 8 (Figure 15) are coupled with a range of different carbanionic D-ring precursors in a Michael Dieckmann reaction sequence (T.-L. Ho, Tandem Organic Reactions (Wiley, New York, 1992); incorporated herein by reference) that forms two carbon-carbon bonds and the C-ring of the 6-deoxytetracyclines (Figures 15B, 17, and 18). The process is perhaps best illustrated in detail by the 3-step synthesis of (-)-doxycycline from the AB precursor 8 (Figure 17). Deprotonation of the D-ring precursor 18 (4.5 equiv, LDA, TMEDA, THF, -78 *C), synthesized in 5 steps (42% yield) from anisic acid, followed by addition of the none 8 (1 equiv, -78 ->0 *C), provided the tetracyclic coupling product 19 (Figure 17) in diastereomerically pure form in 79% yield after purification by rp-HPLC. Removal of the protective groups (2 steps, 90% yield) and purification (rp-HPLC) afforded (-)-doxycycline hydrochloride (18 steps, 8.3% yield from benzoic acid). A remarkable feature of the convergent coupling reaction that produces the tetracyclic product 19 (Figure 17) is its stereoselectivity. Although in theory four diastereomeric products can be formed, largely one was produced, corresponding in configuration (SaR, 6R) to that of known biologically active 6-deoxytetracyclines. A minor diastercomeric impurity, believed to be 6-epi-19 (Figure 17), was also isolated in separate rp-HPLC fractions (<7% yield). Michael-Dieckmann cyclization sequences (T.-L. Ho, Tandem Organic Reactions (Wiley, New York, 1992); incorporated herein reference) and condensations of o-toluate anions in particular (F. J. Leeper, J. Staunton, J.C.S. Chem. Comm., 406 (1978); F. M. Hauser, R. P. Rhee, J Org. Chem. 43, 178, 138 (1978); J. H. Dodd, S. M. Weinreb, Tetrahedron Lett. 20, 3593 (1979); each of which is incorporated herein by reference) are extensively precedented in synthesis, but we are unaware of any example exhibiting the high degree of diastereoselectivity of the present case. Phenyl ester activation in toluate condensations is also precedented, though in a system that forms a fully aromatized cyclization product (White et al., J Org. Chem. 51, 1150 (1986); incorporated herein by reference . We observed that the presence of the phenyl ester group of the D-ring precursor 18 (Figure 17) was essential for successful cyclization to occur; anions derived from simple alkyl esters and phithalide-derived anions underwent Michael addition, but the resulting adducts did not cyclize. Perhaps even more remarkable than the condensation that produces 19 (Figure 17) is the parallel transformation of 18 with the enone 7 (Figure 18, entry 1), which forms (-)-6-deoxytetracycline in protected form with >20:1 diastereoselectivity, in 81% yield after purification by rp-HPLC (diastereomerically pure; a minor diastereomer, epimeric at C6, was also isolated separately). It appears that additions to 7 and 8 proceed almost exclusively by addition to the "top" face of each enone (as drawn), producing C5a-sterochemistry corresponding to natural tetracyclines, though why this should be the case is not obvious. [00221] As the examples of entries 2-5 (Figure 18) show, efficient and stereoselective condensations are not restricted to the o-toluate anion derived from the D-ring substrate 18 (Figure 17); the novel D-ring heterocyclic analogs 4 and 5 (Figure 18) were synthesized by a related sequence from o-toluate anions of very different structures, as was- the pentacyline derivative 6 (Figure 18). In each case it was necessary to optimize the specific conditions for o-toluate anion generation and trapping. For entries 3-5 (Figure 18) anion generation was best conducted in situ, in the presence of the enone 7, either by selective deprotonation (entry 3) or by lithium halogen exchange (entries 4 and 5). A number of potentially competing non-productive reaction sequences (e.g., enolization of 7) might have occurred during in situ anion generation; the observed efficiencies of the transformations are surprising in light of this. It is also noteworthy that in situ anion generation permits the use of o-toluates lacking an o-alkoxy substituent (entries 3 and 4), substrates known to be problematic from prior studies (F. M. Hauser et al., Synthesis 72 (1980); incorporated herein by 139 reference). Finaly, o-toluate anion formation by in situ or stepwise halogen-metal exchange (entries 4 and 5) is unprecedented. f00222j The efficiencies of the synthetic sequences have allowed for the preparation of sufficient quantities of each tetracycline analog for antibacterial testing using standard serial-dilution techniques (5-20 mg amounts). Minimum inhibitory concentrations (MICs) are reported for each analog in whole-cell antimicrobial assays using five Gram-positive and five Gram-negative organisms (Figure 18). Thus far, the pentacycline derivative 6 (Figure 18) has shown the most promising antibacterial properties, with activity equal to or greater than tetracycline in each of the Gram positive strains examined, including strains with resistance to tetracycline, methicillin, and vancomycin. Experimentals 100223] General Procedures. All reactions were performed in flame-dried round bottomed or modified Schlenk (Kjeldahl shape) flasks fitted with mbber septa under a positive pressure of argon, unless otherwise noted. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation at -25 Tor (house vacuum). Flash column chromatography was performed on silica gel (60 A, standard grade) as described by Still et al. (Still, W. C.; Kahn, M.; Mitra, A. J Org. Chen. 1978, 43, 2923-2925; incorporated herein by reference). Analytical thin-layer chromatography was performed using glass plates pre-coated with 0.25 mm 230-400 mesh silica gel impregnated with a fluorescent indicator (254 un). Thin-layer chromatography plates were visualized by exposure to ultraviolet light and/or exposure to ceric ammonium molybdate or an acidic solution ofp-anisaldehyde followed by heating on a hot plate. [00224] Materials. Commercial reagents and solvents were used as received with the following exceptions. Triethylamine, diisopropylamine, N,N,N',N' tetramethylethylene-diamine, DMPU, HMPA, and N,N-diisopropylethylamine were distilled from calcium hydride under an atmosphere of dinitrogen. Dichloromethane, methanol, tetrahydrofuran, acetonitrile, and toluene were purified by the method of Pangborn et a. (Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; 140 Timmers, F. J. Organometallics 1996, 15, 1518-1520; incorporated herein by reference). [00225] Instrumentation. Proton nuclear magnetic resonance ('H NMR) spectra and carbon nuclear magnetic resonance (1 3 C NMR) spectra were recorded with Varian Unity/Inova 600 (600 MHz), Varian Unity/Inova 500 (500 Mlz/125 MHz), or Varian Mercury 400 (400 MHz/100 MHz) NMR spectrometers. Chemical shifts for protons are reported in parts per million (8 scale) and are referenced to residual protium in the NMR solvents (CHCl 3 : 8 7.26, C 6
D
5 H: 8 7.15, D 2 HCOD: 83.31, CDHCl 2 : 5 5.32, (CD2H)CD 3 SO:8 2.49). Chemical shifts for carbon are reported in parts per million (8 scale) and are referenced to the carbon resonances of the solvent (CDC 3 : 8 77.0, C 6 D6: 8 128.0, CD 3 OD: 5 44.9, CD 2
C
2 : 5 53.8, (CD3) 2 SO: 839.5). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q= quartet, m = multiplet, br = broad), integration, coupling constant in Hz, and assignment.. Infrared (IR) absorbance spectra were obtained using a Perkin-Elmer 1600 FT-IR spectrophotometer referenced to a polystyrene standard. Data are represented as follows: frequency of the absorption (cf-1), intensity of the absorption (s = strong, m medium, w = weak, br =broad), and assignment (where appropriate). Optical rotations were determined using a JASCO DIP-370 digital polarimeter equipped with a sodium lamp source. High-resolution mass spectra were obtained at the Harvard University Mass Spectrometry Facilities. 141 Synthesis of (-)-Doxycycline Cyclization Step: oBn Et c H(CH3ak 1. IDA. TMEOA, OH 1 . H cO2h .-76'C 0 c 2.,Z78:c:VT 0o Bno2cQ H N(CHB)a Boco H OTBs 0TBS OTBS /B% [002261 A solution of n-butyllithium in hexanes (1.55 M, 155 pL, 0.240 mmol, 5.1 equiv) was added to a solution of N,N,N','-tetramethylethylenediamine (39 p±L, 0.26 mmol, 5.5 equiv) and diisopropylamine (34 pL, 0.25 mmol, 5.1 equiv) in tetrahydrofuran (1 mL) at -78 *C. The resulting mixture was stirred vigorously at -78 "C for 30 min whereupon a solution of 2-(phenoxycarbony)-3-ethylpheny t-butyl carbonate (73.0 mg, 0.213 mmol, 4.5 equiv) in tetrahydrofuran (1 mL) was added dropwise via cannula; The resulting deep-red mixture was stirred vigorously at -78 "C for 75 min, then a solution of enone 8 (30.0 mg, 0.0474 mmol, 1 equiv) in tetrahydrofuran (1 mL) was added dropwise via cannula. The resulting light-red mixture was allowed to warm slowly to 0 "C over 2 h. The ice-cold product solution was then partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The organic phase was separated and the aqueous phase was further extracted with two 1 0-mL portions of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column [10 pm, 250 x 10 mm, UV detection at 350 un, injection volume: 400 yL (methanol), isochratic elution with methanol-water (9:1), flow rate: 3.5 mL/min]. Fractions eluting at 36-42 min were collected and concentrated, affording the pentacyclic addition product depicted in diastereomerically pure form (33.0 mg, 79%, a light-yellow solid). [00227] Rf 0.35 (1:4 ethyl acetate-hexanes); 'H NMR (500 MHz, C 6
D
6 ) 5 16.55 (br s, 1H, enol), 7.26 (d, 2H, J = 7.0 Hz, o-ArH), 7.14 (d, 211, J = 7.5 Hz, ArH), 6.85 142 7.05 (m, 6H, ArH), 6.66-6.74 (m, 2H, Ar), 6.51 (dd, 1H, J= 9.0, 1.5 Hz, Ar), 5.73 (br d, 1H, J= 4.0 Hz, HnOCO 2 CH), 5.17(d, 1H, J= 12.5 Hz, OCHH'Ph), 5.03 (d, IH, J= 12.5 Hz, OCHH'Ph), 4.99 (d, 1H, J= 12.5 Hz, OCHH'Ph', 4.93 (d, 1H, J= 12.5 Hz, OCHH'Ph), 3.58 (d, 1H, J= 11.5 Hz, CHCHN(CH3)2), 3.35 (dd, 1H, J= 12.5,4.0 Hz, CH 3 CHCH), 2.99 (d, 1H, J= 11.5 Hz, CHCHN(CH3)2), 2.56 (dq, 1H, J= 12.5, 7.0 Hz, CH 3 CR), 2.18 (s, 6H, N(CH 3 )2), 1.33 (s, 9H, C(CH3)), 1.16 (d, 3H, J= 7.0 Hz,
CH
3 CH), 1.11 (s, 9H, C(CH 3
)
3 ), 0.61 (s, 3H, CH3), 0.36 (s, 3H, CH 3 ); ' 3 C NMR (100 MHz, CDCI 3 ) 8 189.7, 186.3, 180.9, 178.4, 167.9, 154.7, 152.1, 150.8, 145.9, 136.1, 135.5, 133.9, 128.7, 128.6, 128.5, 127.3, 123.8, 122.7, 122.6, 108.9, 105.5, 83.0, 82.9, 74.8, 72.4, 69.2,60.8, 52.7,43.2, 38.4, 27.5, 26.6, 19.5, 16.3,-1.8, -2.7; FTIR (neat film), cmt 2974 (w), 2933 (w), 2851 (w), 1760 (s, C=O), 1748 (s, C=0), 1723 (s, C=0), 1606 (m), 1513 (m), 1471 (m), 1370 (m). 1260 (s), 1232 (s), 1148 (s); HRMS (ES) m/z calod for (C 48 H56 N2OuSi)* 881.3681, found 881.3684. 143 Deprotection Step 1: OBn e-Bn 0CN(cH, 'H(CH3) o HF, CHaCN 100% I' BotO a HO O8n O n [002281 Concentrated aqueous hydrofluoric acid (48 wt %, 1.2 mL) was added to a polypropylene reaction vessel containing a solution of the purified pentacyclic addition product from the experiment above (33.0 mg, 0.0375 mmol, I equiv) in acetonitrile (7.0 mL) at 23 *C. The resulting mixture was stirred vigorously at 23 "C for 60 h, then was poured into water (50 mL) containing dipotassium hydrogenphosphate (7.0 g). The resulting mixture was extracted with ethyl acetate (3 x 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording the product depicted as a yellow oil (25.0 mg, 100%). This product was used in the next step without further purification. [00229] Rf 0.05 (1:4 ethyl acetate-hexanes); 'H NMR (600 MHz, C 6
D
6 , crude) 8 14.86 (br s, 1H, enol), 11.95 (s, 1H, phenol), 7.23 (d, 2H, J=7.8 Hz, o-ArH), 7.14 (d, 2H, J= 7.2 Hz, o-ArH), 6.94-7.02 (m, 61, ArH), 6.86 (t, 1H, J= 8.4 Hz, AM), 6.76 (d, 1H, J= 8.4 Hz, ArH), 6.28 (d, 1H, J= 7.8 Hz, ArH), 5.46 (dd, 1H, J= 3.6, 3.0 Hz, BnOCO2CH), 5.12 (d, 1H, J=12.0 Hz, OCIH'Ph), 5.04 (d, 1H, J=12.0 Hz, OCHW'Ph), 4.92 (s, 2H, OCH 2 Ph), 3.41 (d, 1H, J= 9.6 Hz, CH-CHN(CH3) 2 ), 2.82 (dd, 1H, J= 9.6, 3.0 Hz, CHCHN(CH3)2), 2.65 (dd, 1H, J= 13.2,3.6 Hz, CH 3 CHCH), 2.78 (dq, 1H, J= 13.2, 7.2 Hz, CH 3 CH), 2.05 (s, 6H, N(CH 3
)
2 ), 1.04 (d, 3H,J= 7.2 Hz,
CH
3 CH); 1 3 C NMR (100 MHz, C 6
D
6 , crude) 8 193.4, 186.2, 181.3, 172.3, 167.9, 163.3, 154.6, 145.8, 136.6, 135.8, 128.6, 128.4, 127.2, 116.8, 116.0, 115.6,107.6, 104.7, 76.8, 73.9, 72.5, 69.5, 60.3, 48.7, 43.0, 41.8, 37.5, 15.3; FTIR (neat film), em 3424 (m, OH), 3059, 3030, 2925, 2857, 1744 (s, C-0), 1713 (s, C=0), 1614 (s), 1582 (s), 1455 (s), 1252 (s); HRMS (ES) m/z called for (C37H34N20Oo+H 667.2292, found 667.2300. Deprotection Step 2: 144 -8n C3 H 0 NCH,) H Y(CHl Hg Pd black 'c OH
THF-CH
3 OH NH 2 OH 0 HO O~n 90% HO HO 0 (-)-DUxycyllie [00230] Palladium black (7.00 mg, 0.0657 mnmol, 1.75 equiv) was added in one portion to a solution of the product from the procedure above (25.0 mg, 0.0375 mmol, I equiv) in tetrahydrofuran-methanol (1:1, 2.0 mL) at 23 "C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The palladium catalyst was initially observed to be a fine dispersion, but aggregated into clumps within 5 min. The yellow heterogeneous mixture was stirred at 23 "C for 2 h, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil: The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column (10 m, 250 x 10 mm, UV detection at 350 rnm, Solvent A: methanol-0.005 N aq. HCI (1:4), Solvent B: acetonitrile, injection volume: 400 IL (solvent A containing 10 mg oxalic acid), isochratic elution with 5% B for 2 min, then gradient elution with 5-+50% B for 20 min, flow rate: 4.0 mL/min]. Fractions eluting at 12-17 min were collected and concentrated, affording (--) doxycycline hydrochloride as a yellow powder (16.2 mg, 90%), which was identical with natural (-)-doxycycline hydrochloride [reverse-phase HPLC (co-injection), 'H NMR (including measurement of an admixture of synthetic and natural doxycycline), 'C NMR, [C]oD, UV). [00231] . 'H NMR (600 MHz, CD 3 0D, hydrochloride) 67.47 (t, lH, J= 8.4 Hz, ArH), 6.93 (d, 11, J= 8.4 Hz, ArH), 6.83 (d, lH, J= 8.4 Hz, ArH), 4.40 (s, lH,
(CH
3
)
2 NCH), 3.53 (dd, IH, J= 12.0, 8.4 Hz, CHOH), 2.95 (s, 3H, N(CH 3
)CH
3 '), 2.88 (s, 3H, N(CH3)CH3'), 2.80 (d, 1H, J= 12.0 Hz, CHCHN(CH3) 2 ), 2.74 (dq, IH, J= 12.6, 6.6 Hz, CH 3 CH), 2.58 (dd, I H, J= 12.6, 8.4 Hz, CH3CHCH), 1.55 (d, 3H,.J- 6.6 Hz, CH3CHCH); "C NMR (100 MHz, CD 3 OD) 6 195.3, 188.2, 173.8, 172.1, 163.2, 149.0,137.7,117.1, 116.9,116.6,108.4,96.0,74.5,69.8,66.9,47.5,43.4,43.0,41.9, 40.0, 16.3; UV max (0.01 M methanolic HCI), nm 218, 267, 350; [aui - -109" (c 0.16 in 0.01 M methanolic HCi); HRMS (ES) m/z called for (C22H 4
N
2 0+H)* 445.1611, found 445.1603. 145 [002321 Literature values (The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 12' ed. Budavari, S.; O'Neal, M. J.; Smith, A.; Heckelman, P. E.; Kinneary, J. F., Eds.; Merck & Co.: Whitehouse Station, NJ, 1996; entry 3496.): UV max (0.01 M methanolic HCl), nm 267, 351; [a], = -110(c = in 0.01 M methanolic HC). Synthesis of (-)-6-Deoxytetracycline Cyclizaton Step: CH;
N(CH
2
)
2 1. LDA, TMEDA, HH : THF -78 OC Nk 7COPh 2.-78'C -+ D "C oe N(0Hak Boco o H 0 T B sss 7 81% 1002331 A solution of n-butyllithium in hexanes (1.65 M, 75 pL, 0.12 mmol, 3.9 equiv) was added to a solution of diisopropylamine (17 pL, 0.12 mmol, .3.9 equiv) and N,N,N',N'-tetramethylethylenediamine (19 pL, 0.13 mmol, 4.1 equiv) in tetrahydrofuran (1 mL) at -78 *C. The resulting solution was stirred at -78 "C for 30 min whereupon a solution of 2-(phenoxycarbonyl)-3-ethylphenyl t-butyl carbonate (31.8 mg, 0.093 mmol, 3.0 equiv) in tetrahydrofuran (250 AL) was added dropwise via syringe. The resulting deep-red mixture was stirred at -78 *C for 90 min, then a solution of enone 7(15.0 mg, 0.031 mmol, I equiv) in tetrahydrofuran (250 yL) was added dropwise via syringe. The resulting deep-red mixture was allowed to warm slowly to 0 0 C over 3 h. The ice-cold product solution was then partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15 mL). The organic phase was separated and the aqueous phase was further extracted with two 15-mL portions of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column [5 gm, 250 x 10 146 mm, UV detection at 350 nm, injection volume: 500 iL (methanol), isochratic elution with methanol-water (89:11), flow rate: 3.5 mL/min]. Fractions eluting at 39-60 min were collected and concentrated, affording the pentacyclic addition product depicted in diastereomerically pure form (18.5 mg, 81%, a light-yellow foam). [002341 Rf0.37 (2:8 tetrahydrofuran-hexanes); 'H NMR (500 MHz, CDC3) 8 (s, IH, 16.24, enol-OR), 7.55-7.50 (m, 311, ArH), 7.40-7.35. (m, 411, ArH), 7.10 (d, 1H, J = 7.8 Hz, ArH), 5.39-5.34 (m, 2H, OCH 2 Ph), 3.92 (d, IH, J= 10.7 Hz, CHN(CH3) 2 ), 2.81-2.71 (m, 2H, CH 3 CH, CH3CHCH), 2.55 (dd, 111, J= 10.7, 5.7 Hz,
CHCHN(CH
3
)
2 ), 2.48 (s, 6H, N(CH3)2), 2.40 (d, 1H, J= 14.7 Hz,
CHH'CHCHN(CH
3 )2), 2.3 1- (ddd, 1H, J= 14.7, 9.3, 5.7, CHH'CHCHN(CH3) 2 ), 1.56 (s, 3, CH3), 1.55 (s, 9H, Boc), 0.84 (s, 9H, TBS), 0.27 (s, 311, TBS), 0.13 (s, 3H, TBS); '"C NMR (125 MdHz, CDCl,) 8 187.4, 183.1, 182.8, 181.6, 167.6, 151.7, 150.2, 147.4, 135.0, 134.0, 128.5, 128.5, 123.4, 123.0, 122.4, 108.3, 107.4,94.8,83.9, 81.5, 72.5, 61.5, 46.4,41.9, 39.5, 34.9,27.7,26.0, 20.7, 19.0, 16.0, -2.6, -3.7; FTIR (neat film), cm-' 2923 (m), 2841 (m), 1759 (s, C=0), 1718 (s, C=0), 1605 (s), 1508 (s), 1467(m), 1456 (m), 1369 (m), 1277 (s), 1262 (m), 1231 (s), 1144 (s), 1005 (w); HRMS (ES) m/z called for (C 4 oHsoN 2 OgSi+H) 731.3364, found 731.3370. Deprotection: CH N(CHal 2 CH H N(CH) 2 1. HF. CHaCN OH 2 H Pd back NH2 B 1 THF.CHsOH I i FI I BcO 0 HO T f 5 HO 0 HORO 0 OS 85% [002351 Concentrated aqueous hydrofluoric acid solution (48 wt %, 0.6 mL) was added to a polypropylene reaction vessel containing a solution of the purified pentacyclic addition product from the experiment above (15.0 mg, 0.0205 mmol, I equiv) in acetonitrile (3.5 mL) at 23 "C. The reaction mixture was stirred at 23 *C for 55 h, then was poured into water (20 mL) containing dipotassium hydrogenphosphate (4.0 g). The resulting mixture was extracted with ethyl acetate (4 x 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a light-yellow oil. The residue was dissolved in methanol-tetrahydrofuran (1:1, 2 mL) and to the resulting 147 solution was added palladium black (7.6 mg, 0.071 mmol, 3.5 equiv) in one portion. An atmosphere of hydrogen gas was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The yellow mixture was stirred at-23 *C for 2 h, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil (10 mg). The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column [10 pam, 250 x 10 mm, UV detection at 365 nm, Solvent A: methanol-0.02 N HCl (1:4), Solvent B: acetonitrile, injection volume: 400 pL (methanol containing 10 mg oxalic acid), isochratic elution with 18% B for 15 min, then gradient elution: with 18->60% B over 15 min, flow rate: 5 mL/min]. Fractions eluting at 17.5-22.5 min were collected.and concentrated, affording 6-deoxytetracycline hydrochloride as a yellow powder (8.1 mg, 85%). [002361 'H NMR (500 MHz, CD 3 0D, hydrochloride) 57.49 (t, lH, J= 7.8 Hz, ArH), 6.95 (d, 1H, J= 7.8 Hz, ArH), 6.84 (d, JH, J= 7.8 Hz, ArH), 4.09 (s, 1H,
CHN(CH
3 )2), 3.03 (br s, 3H, N(CH 3 )), 2.97 (br s, 3H, N(CH 3 )), 2.90 (br d, IH, J= 12.7 Hz, CHCHN(CH 3 )2), 2.67 (ddd, 1H, J= 12.7, 12.7, 5.2 Hz, CH 3 CHCH), 2.61-2.56 (m, 111, CH 3 CH), 2.30 (ddd, 1H, J= 13.7, 5.2,2.9 Hz, CHH'CHCHN(CH3) 2 ), 1.54 (ddd, lH, J= 13.7, 12.7, 12.7 Hz, CHH'CHCHN(CH 3
)
2 ), 1.38 (d, 3H, J 6.8 Hz, CH 3 CH); UV max (0.01 M methanolic HC1), nm 269,353; [a],= -142"(c = 020 in 0.01 M methanolic HCI); HERMS (ES) m/z calcd for (C22H24N 2 0 7 +H)* 429.1662, found 429.1660. 148 Synthesis of a (-)-D-ring Pyridone Analog of Tetracycline Cyclization Step: 1c cHa 1.L DDPU HA ' oa Y IY THP, -?8 0 C N)0._coOPh 20-76 *C-a*C 0011 OTBS 7 87% [00237] A solution of n-butyllithium in hexanes (1.67 M,80 gL, 0.13 mmol, 4.3 equiv) was added to a solution of diisopropylamine (20 pL, 0.14 mmol, 4.6 equiv) in tetrahydrofuran (2.5 mL) at -78 "C. The resulting solution was allowed to warm to 0 *C over 15 min. N,N'-dimethylpropyleneurea (17 pL, 0.14 mmol, 4.5 equiv) was added and the resulting solution was cooled to -78 *C. A solution of phenyl 2-(benzyloxy) 4,6-dimethylpyridine-3-carboxylate (31.0 mg, 0.0930 mmol, 2.99 equiv) in tetrahydrofuran (250 yL) was then added via syringe to the cooled reaction solution. The resulting yellow solution was stirred for 5 min at -78 *C, then a solution of enone 7(15.0 mg, 0.0311 mmol, 1 equiv) in tetrahydrofuran (250 L) was added via syringe. The resulting deep-red mixture was allowed to warm to 0 "C over 4 h. Acetic acid (40 pL) was added to the deep-red mixture at 0 "C. The ice-cold product solution was then partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15 mL). The organic phase was separated and the aqueous phase was further extracted with two 15-mL portions of dichloromethane. The organic extracts were combined and then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing a yellow oil. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column (5 pm, 250 x 10 mm, UV detection at 350 nm, Solvent A: water, Solvent B: methanol, injection volume: 500 pL DMSO, gradient elution with 92-+100% B over 30 min, flow rate: 3.5 mL/min]. Fractions eluting at 21-29 min were collected and concentrated, affording the pentacyclic addition product depicted in diasteromerically pure form (15.0 mg, 67%, a light-yellow solid). 149 [002381 Rf 0.55 (3:7 ethyl acetate-hexanes); 'H NMR (600 MHz, CD 2 Cl 2 ) 8 16.05 (s, 1H, enol-OH), 7.52-7.26 (m, 10H, ArK), 6.66 (s, 1H, pyr-H), 5.57 (d, 1H, J= 12.7 Hz, OCHH'Ph), 5.43 (d, J= 12.7 Hz, 1H, OCHHPh), 5.33-5.28 (m, 2H,
OCH
2 Ph), 3.99 (d, 2H, J= 10.5 Hz, CHN(CH3)2), 3.04-3.00 (m, 1H, CHCH2CHCHN(CH 3 )2), 2.84 (dd, 1H, J= 16.1, 4.9 Hz, CHHCHCH 2
CHCHN(CH
3
)
2 ), 2.74 (dd, 1H, J= 16.1, 16.1 Hz, CHHCHCH2CHCHN(CH3)A), 2.53 (dd, 1H, J= 10.5, 3.9 Hz, CHCHN(CH3) 2 ), 2.51-2.43 (m, 10H, N(CH 3
)
2 , Ar-CH 3 , CHH'CHCHN(CH 3 )2), 2.07 (d, 1H, J= 14.2 Hz, CHH'CHCHN(CH 3
)
2 ), 0.82 (s, 9H, TBS), 0.22 (s, 3H, TBS), 0.10 (s, 3H, TBS); "C NMR (100 MHz, CD2C1 2 ) 8 187.9, 185.2, 182.5, 178.8,167.9, 161.9, 161.8, 154.8, 137.9, 135.6, 129.1, 129.0, 129.0, 128.7, 127.9,127.9, 116.4, 111.6, 108.6, 107.5, 82.0, 73.0, 68.1, 61.7, 46.9,42.0, 39.2, 28.6, 26.1, 24.6,23.0, 19.3, -2.4, -3.5; FTIR (neat film), cm 2939 (m), 2857 (w), 1720 (s, C=0), 1593 (s), 1510 (s), 1469 (m), 1449 (m), 1326 (s), 1254 (m), 1187 (w), 1157(m), 1090 (m), 1064 (m), 1007 (m); HRMS (ES) m/z calcd for (C 4
H
47
N
3 07i+H)* 722.3262, found 722.3261. 150 Deprotection:
H
3 C u uN(CHa z 1. H aC V .H /, N).r / 2. HIl, MeOH HN. NH 2 Ono Noo o 74% o 0 MO 0O 100239] Pearlman's catalyst (10 mg, 0.0094 mmol, 0.68 equiv) was added to a solution of the purified pentacyclic addition product from the experiment above (10 mg, 0.014 mnol, 1 equiv) in dioxane-methanol (1:1, 10 mL) at 23 "C. An atmosphere of hydrogen gas was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was observed to form a green color within 10 min. After stirring at 23 *C for 2 h, the reaction mixture was filtered through a plug of cotton and the filtrate was concentrated. The oily yellow residue was dissolved in methanol (10 mL) and to the resulting solution was added concentrated aqueous hydrochloric acid solution (37 wt %, 100 pL) at 23 *C. The reaction mixture was stirred at 23 *C for 3 h, then was concentrated. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column [10 pm, 250 x 10 mm, UV detection at 365 nm, Solvent A: 0.01 N aqueous hydrochloric acid, Solvent B: acetonitrile, injection volume: 500 yL (methanol containing 30 mg oxalic acid), linear gradient with 0->20% B over 40 min, flow rate: 4 ml/min]. Fractions eluting at 20-29 min were collected and concentrated, affording the D-ring pyridone hydrochloride as a yellow powder (4.8 mg, 74%). [002401 'H NMR (500 MHz, CD 3 0D, hydrochloride) 6 6.37(s, lH, ArH), 4.06 (s, IH, CHN(CH 3 )2), 3.05-2.95 (m, 8H, N(CH 3
)
2 , CHCHN(CH 3
)
2 ,
CHCH
2
CHCHN(CH
3
)
2 ), 2.79 (dd, lH,J= 16.1, 3.9 Hz, CHH'CHCH 2
CHCHN(CH
3
)
2 ), 2.55 (dd, 1H, J= 16.1, 16.1 Hz, CHH'CHCH 2
CHCHN(CH
3 )2)), 2.40 (s, 3H, Ar-CH 3 ), 2.18 (br. D, 1K J= 12.7 Hz, CHIHCHCHN(CH 3
)
2 ), 1.59 (ddd, 1H, J=12.7, 12,7, 12.7 Hz, CHH'CHCHN(CH 3
)
2 ); ' 3 C NMR (100 MHz, (CD 3
)
2 SO)8 187.3, 183.5, 177.8, 172.1,160.6, 159.8, 153.3, 115.3, 107.2, 106.9, 95.6, 74.2,68.4,41.5, 35.7, 34.5, 33.9, 31.0, 19.2; UV max (0.01 M methanolic HCI), nn 267, 370; [akD =-146* (c = 0.43 in 0.01 M methanolic HC); HRMS (ES) r/z called for (C 2 1H23N 3 0 7 +H) 430.1614, found 430.1607. 151 Synthesis of a (-)-Pentacycline Cyclization Step: N(CH3)2 N(CH 3
)
2 aCH29r .tIJfP Sa-10 "C 0# OCHS o T0 OBn 75%~ ch0 N o 3.0 O~n 7 [002411 A solution of n-butyllithium in hexanes (2.65 M, 107 IL, 0.284 mmol, 4.03 equiv) was added to a solution of phenyl 3-(bromomethyl)-1-methoxynaphthalene 2-carboxylate (105 mg, 0.283 mmol, 4.02 equiv) and enone 7 (34.0 mg, 0.0705 mmol, 1 equiv) in tetrahydrofuran (2.80 mL) at -100 *C. The resulting light-red reaction mixture was allowed to warm to 0 *C over 70 min. The ice-cold product solution was then partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) and dichloromethane (15 mL). The organic phase was separated and the aqueous phase was further extracted with two 15-mL portions of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered, and the filtrate was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Coulter Utrasphere ODS column [10 im, 250 x 10 mm, UV detection at 350 nm, Solvent A: water, Solvent B: methanol, two separate injections (750 pL each, acetonitrile), isochratic elution with 94% B for 20 min followed by a linear gradient elution with 94-+100% B over 20 min, flow rate: 3.5 mL/min]. Fractions eluting at 24-3 8 min were collected and concentrated, affording the hexacyclic addition product in diastereomerically pure form (36.1 mg, 75%, a white solid). [002421 Rf 0.37 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CDCI 3 ) 6 16.25 (s, 1H, enol-OH), 8.30 (d, 1H, J= 8.3 Hz, Ar), 7.75 (d, JH, J= 7.8 Hz, ArH), 7.59 7.34 (m, 7H, ArM), 7.26 (s, 1H, ArH), 5.38 (s, 2H, OCH 2 Ph), 4.02 (s, 3H, OCH 3 ), 3.99 (d, IH, J =10.7 Hz, CHN(CH3)2), 3.08-3.05 (m, 2H, CHCH2CHCHN(CH 3
)
2 ,
CHHCHCH
2 CHCHN(CH3) 2 ), 2.95-2.90 (m, 1H, CH'CHCH 2
CHCHN(CH
3
)
2 ), 2.58 (dd, lH, J= 10.7, 5.9 Hz, CHCHN(CH 3 )2), 2.51 (s, 6H, N(CH 3
)
2 ), 2.50-2.48 (m, 1H,
CHH'CHCHN(CH
3 )2), 2.20-2.14 (m, I H, CHH'CHCHN(CH 3 )2), 0.82 (s, 9H, TBS), 0.29 (s, 3H, TBS), 0.13 (s, 31, TBS); ' 3 C NMR (125 MHz, CDCI 3 )8 187.9, 184.1, 152 183.0, 182.0, 167.8, 159.2, 137.5, 136.7, 135.3, 129.5, 128.8, 128.7, 128.5, 127.5, 126.4, 124.2, 121.8, 119.5, 108.7, 108.7, 82.4, 72.8, 63.8,61.6, 46.8, 42.1, 40.7, 29.3, 26.2, 23.1, 19.3, -2.2, -3.5; FTIR (neat film), oE' 2934 (M), 2852 (m), 1718 (S, C=0) 1610 (s), 1513 (s), 1472 (in), 1452(m), 1369 (m), 1339 (w), 12 9 3 (m), 1252(m), 1190 (w), 1159 (m), 1067 (m), 1026(w), 1011 (w); HRMS (ES) mz called for
(C
3 H44N 2
O
7 Si+H) 681.2996, found 681.2985. Deprotection: 1. HF, CH 3 CN U(CH3)2 2. Pd, H2, H H N(CHa)k 3. BMCH0O HNH2 CHaO 0 HO 0O ca -7laO HO 0 74% 100243] Concentrated aqueous hydrofluoric acid solution (48 wt %, 1.0 mL) was added to a polypropylene reaction vessel containing a solution of the purified hexacyclic addition product from the experiment above (24.0 mg, 0.035, 1 equiv) in acetonitrile (9.0 mL) at 23 *C. The reaction mixture was stirred at 23 "C for 22 h, then was poured into water (50 nL) containing dipotassium hydrogenphosphate (12.0 g). The resulting mixture was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a yellow oil. The residue was dissolved in methanol-dioxane (1:1, 5 mL) and to the resulting solution was added palladium black (10.0 mg, 0.0940 mmol, 2.67 equiv) in one portion. An atmosphere of hydrogen gas was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The yellow mixture was stirred at 23 *C for 4 h, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil. The residue was dissolved in dichloromethane (4.5 mL) and to the resulting solution was added a solution of boron tribromide (1.0 M in dichloromethane, 0.5 mL, 14 equiv) at 78 "C. The dark-red mixture was stirred at -78 "C for 15 min, then at 23 *C for 3.5 h. Methanol (20 mL) was added and the resulting yellow solution was stirred at 23 "C for 1 h. The solution was concentrated, affording a yellow oil. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column [7 pm, 150 x 21.2 153 mm, UV detection at 350 nm, Solvent A: 0.01 N HCI, Solvent B: acetonitrile, injection volume: 500 pL (methanol containing 10 mg oxalic acid), gradient elution with 25-+50% B over 60 min, flow rate: 6 L/mmin]. Fractions eluting at 30-35 min were collected and concentrated, affording the pentacycline hydrochloride as a yellow powder (13.1 mg, 74%). [002441 'H NMR (600 MHz, CD 3 0D, hydrochloride) 6 8.36 (d, 1H, J= 7.7 Hz, ArH), 7.74 (d, 1H, J= 7.7 Hz, ArH), 7.64 (dd, 1H, J= 7.7,7.7 Hz, ArH), 7.50 (dd, 1H, J= 7.7, 7.7 Hz, ArH), 7.1 (s, 1H, ArH), 4.10 (s, 1H, CHN(CH 3 )2), 3.13-2.97 (m, 9H,
N(C
3
)
2 , CHCHN(CH 3
)
2 , CHCH2CHCHN(CHz)z, CHH'CHCH2CHCHN(CH3)2), 2.67 (dd, IH, J= 14.3, 14.3 Hz, CHH'CHCH 2
CHCHN(CH
3
)
2 ), 2.22 (ddd, 1H, J =13.6, 4.9, 2.9 Hz, CHH'CHCHN(CH 3 )2), 1.64 (ddd, 1H, J= 13.6, 13.6, 13.6 Hz,
CHH'CHCHN(CH
3 )2); UV max (0.01 M methanolic HCI), nm 268,345,402; [aD = 1130 (c = 0.18 in 0.01 M nethanolic HCl); HRMS (ES) M/z called for (C 2 sH 24
N
2 0 7 +H)* 465.1662, found 465.1656. Synthesis of (-)-7-Aza-10-Deoxysaneyeline Cyclization Step: HN(CH3ah H H N(CH3) 2
.
H3 (cH LDA, HMPA HNNCI N THE
C
2 Ph -95S"c -50 0 HO l a Bn OTBs 7% bs 7 [002451 A solution of n-butyllithium in hexanes (2.65 M, 33.0 pL, 0.0945 mmol, 5.00 equiv) was added to a solution of disopropylamine (13,2 pL, 0.0945 mmol, 5.00 equiv) in tetrahydrofuran (0.750 mL) at -78 0C. The resulting solution was briefly warmed in an ice bath (10 min), then was cooled to -78 "C. Hexamethylphosphoramide (33.0 pL, 0.189 mmol, 10.0 equiv) was added, producing a colorless solution, and this solution was then transferred (cold) dropwise via cannula to a solution containing phenyl 2-methylpyridine-3-carboxylate (16.0 mg, 0.0755 mmol, 4.00 equiv) and enone 7 (9.1 mg, 0.019 mmol, 1 equiv) in tetrahydrofuran (0.750 mL) at -95 *C, forming a light-red mixture. The reaction solution was allowed to warm to 50 "C over 50 min. The product solution was then partitioned between aqueous 154 potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (25 mL). The organic phase was separated and the aqueous phase was further extracted with three 15-mL portions of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column [10 im, 250 x 10 mm, UV detection at 350 nm, Solvent A: water, Solvent B: methanol, injection volume: 500 VL (methanol), gradient elution of 85->100% B over 30 min, flow rate: 3.5 mL/nin]. Fractions eluting at 21-27 min were collected and concentrated, affording the pentacyclic addition product in diastereomerically pure form (8.6 mg, 76%, a white solid). [00246] Rf 0.07 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CD2CI 2 ) 5 15.21 (s, 1H, enol), 8.63 (d, 1H, J= 4.5 Hz, pyr-H), 8.19 (d, IH, J= 7.5 Hz, pyr-H), 7.54-7.43 (m, 5H, ArH), 7.34 (d, lH, J= 4.5, 7.5 Hz, pyr-H), 5.36 (d, 1H, J= 12.0 Hz, OCHH'Ph), 5.33 (d, 1H, J= 12.0 Hz, OCHH'Ph), 4.03 (d, 1H, J= 10.7 Hz,
CHN(CH
3
)
2 ), 3.36-3.31 (m, 1H, CHCH 2 CHCHN(CH3) 2 ), 3.23 (dd, iH, J= 16.3, 5.6 Hz, CHH'CHCH 2
CHCHN(CH
3
)
2 ), 2.99 (dd, 1H, J= 16.3, 16.3 Hz,
CHH'CHCH
2
CHCHN(CH
3
)
2 ), 2.63 (ddd, IH, J= 1.6,4.4, 10.7 Hz, CHCHN(CH 3
)
2 ), 2.54-2.48 (m, 7H, N(CH 3
)
2 , CHH'CHCHN(CH 3
)
2 ), 2.19 (dd, iH, J= 1.6, 14.5 Hz,
CHH'CHCHN(CH
3 )2), 0.87 (s, 9H, TBS), 0.26 (s, 3M, TBS), 0.13 (s, 3H, TBS); "C NMR (100 MHz, CD 2 C2) 8 187.7, 183.5, 182.6, 182.2, 167.9, 161.2, 153.4, 137.6, 134.1, 129.2, 129.1,129.1,126.8,123.0,108.7,106.9,82.2,73.0,61.8,47.0,42.1, 41.4, 30.1, 28.4, 26.1, 23.2, 19.3, -2.4, -3.5; HRMS (ES) m/z calcd for (C33HgN30, 6 Si+H)* 602.2686, found 602.2686. 155 Deprotection: N(CHa)a 1. Hp Pd blclR N(CH 3
)
2 dtoxenh-CH 3 OH 2. HF. CHCN, 35'G
NH
2 0 Holz0 .o n 7O9% 0 6mBS [00247) Palladium black (3.0 mg, 0.028 mmol, 2.6 equiv) was added in one portion to a solution of the purified pentacyclic addition product from the experiment above (6.5 mg, 0,011 mmol, I equiv) in dioxane-methanol (7:2,9.0 mL) at 23 *C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The resulting green mixture was stirred at 23 "C for 7 hr, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow oil (7.0 mg). The residue was dissolved in acetonitrile (4.5 mL), transferred to a polypropylene reaction vessel, and concentrated aqueous hydrofluoric acid solution (48 wt %, 0.5 mL) was added to the resulting solution at 23 *C. The reaction mixture was heated to 35 C for 27 hr. Excess hydrofluoric acid was quenched by the addition of methoxytrimethylsilane (3.5 mL, 25 mmol). The reaction mixture was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column [10 pm, 250 x 10 mm, UV detection at 350 nm, Solvent A: 0.5% trifluoroacetic acid in water, Solvent B: 0,5% trifluoroacetic acid in methanol-acetonitrile (1:1), injection volume: 500 pL (methanol), gradient elution with 0-+20% B over 40 min, flow rate: 4 mnL/min]. Fractions eluting at 35-45 min were collected and concentrated to give a yellow oil. The oil was dissolved in methanolic HC1(1.0 mL, 0.10 M) and concentrated, affording 7-aza-10-deoxysancycline hydrochloride as a yellow powder (3.7 mg, 79%). 'H NMR (500 MHz, CD 3 OD, hydrochloride) 6 8.79-8.77 (m, 2H, pyr-H) 7.91 (dd, IH, J= 6.8, 6.8 Hz, pyr-H), 4.12 (s, 1H, CHN(CH 3 )z), 3.41-3,22 (m, 2H, CHH'CHCH2CHCHN(CH 3
)
2 ,
CHCH
2
CHCHN(CH
3 )2), 3.11-3.00 (m, 8H, CHH'CHCH 2
CHCHN(CH
3
)
2 ,
CHCHN(CH
3
)
2 , N(CH 3
)
2 ), 2.34 (ddd, 1H, J= 12.9, 4A, 2.4 Hz, CHH'CHCHN(CH 3 )2), 1.77 (ddd, 1H, J= 12.9, 12.9, 12.9 Hz, CHH'CHCHN(CH 3
)
2 ); UV max (0.01 M methanolic HC), rn 264, 345; [WD -154 (c =0.15 in 0.01 M methanolic HC); HRMS (ES) m/z called for (C2zH2N30O+H)* 400.1508, found 400.1504. 156 Synthesis of (-)-19-Deoxysacyeline Cyclization Step: H (CH) 2 CHah ICH 2 Br +n-BuLI, THF cP-100 *c - c CO0 2 Ph + IS ;f . : on 0T n a1% 0 H0 oan OTOS 6dMS 7 [00248] A solution of n-butyllithium in hexanes (2.65 M, 59 pL, 0.16 mmol, 4.0 equiv) was added to a solution of phenyl 2-(bromomethyl)benzoate (45.6 mg, 0.157 mmol, 3.97 equiv) and enone 7 (19.0 mg, 0.0394 mmol, 1 equiv) in tetrahydrofuran (1.57 niL) at -100 *C. The resulting light-red solution was allowed to warm to 0 *C over 30 min. The ice-cold product solution was then partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 5 mL) and dichloromethane (25 maL). The organic phase was separated and the aqueous phase was further extracted with an additional 15-mL portion of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Coulter Ultrasphere ODS column [10 pm, 250 x 10 mm, Solvent A: water, Solvent B: methanol, injection volume: 1.0 mL (methanol), gradient elution with 85-+100% B over 30 min, UV detection at 350 nm, flow rate: 3.5 mnmin]. Fractions eluting at 25-30 min were collected and concentrated, affording the pentacyclic addition product in diastereomerically pure form (19.2 mg, 81%, a white solid). [002491 Rf 0.46 (3:7 ethyl acetate-hexanes); 'H NMR (500 MHz, CD 2
C
2 ) S 15.53 (s, 1H, enol), 7.94 (d, 1H, J= 7.9 Hz, ArH), 7.54 - 7,28 (m, 8H, ArH,
OCH
2 ArH), 5.37-5.34 (m, 2H, OCH2Ph), 4.05 (d, 1H, J= 10.7 Hz, CHN(CH 3
)
2 ), 3.24 3.18 (m, 1H, CHCH 2
CHCHN(CH
3 )2), 2.99 (dd, 1H, J =15.5, 5.6 Hz, CHH'CHCH2CHCHN(CH 3
)
2 ), 2.88 (dd, 1H1, J= 15.5, 15.5 Hz, CHH'CHCH2CHCHN(CH3)2), 2.61 (dd, 1H, J= 4.4, 10.7 Hz, CHCHN(CH 3
)
2 ), 2.54 2.44 (m, 7H, N(CH 3
)
2 , CHH'CHCHN(CH 3 )2), 2.14 (d, IH,J= 14.3 Hz, CHH'CHCHN(CH3)2), 0.86 (s, 9H, TBS), 0.25 (s, 3H, TBS), 0.12 (s, 3H, TBS); ' 3 C NMR (100 MHz, CD 2 CI2) 8 187.8, 183.0, 182.8, 182.4, 167.7, 141.7, 135.4, 133.4, 157 130.9, 129.0, 128.9, 128.9, 128.1, 127.5, 126.5, 108.5, 106.8, 82.1, 72.8, 61.5, 58.5, 46.9, 41.9, 38.6, 29.0, 25.9, 23.1, 19.1, -2.6, -3.7; HRMS (ES) m/z caled for
(C
34
H
4 oN 3
O
6 Si+H)* 601.2734, found 601.2730. Deprotection: L iHU(CHa 1. HF, CHA k(CH-1 I e--cPd aoNHz O HO-O On 0 HOO0 0 OTes 1002501 Concentrated aqueous hydrofluoric acid solution (48 wt %, 1.1 mL) was added to a polypropylene reaction vessel containing a solution of the pentacyclic addition product from the experiment above (15.1 mg, 0.0251 mmol, 1 equiv) in acetonitrile (10 mL) at 23 "C. The resulting solution was stirred vigorously at 23 "C for 12 h, then was poured into water (50 mL) containing dipotassium hydrogenphosphate (4.7 g) and the product was extracted with ethyl acetate (3 x 25 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a yellow solid (12.2 mg, 99%). The residue was dissolved in methanol-dioxane (1:1, 3.0 mL) and palladium black (6.5 mg, 0.061 mmol, 2.4 equiv) was added to the resulting solution in one portion. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The resulting light-yellow mixture was stirred at 23 *C for 20 min, then was filtered through a plug of cotton. The filtrate was concentrated, affording a yellow solid. The product was purified by preparatory HPLC on a Phenomenex Polymerx DVB column [10 sm, 250 x 10 mm, UV detection at 350 rim, Solvent A: 0.01 N HCL, Solvent B: acetonitrile, injection volume: 1.0 mL (methanol containing 10 mg oxalic acid), gradient elution with 5-+50% B over 30 min, flow rate: 5 mL/min]. Fractions eluting at 16-22 min were collected and concentrated, affording 10-deoxysancycline hydrochloride as a white powder (9.1 mg, 83%). [00251] 'H NMR (500 MHz, CD 3 0D, hydrochloride) 5 7.96 (d, IH, J=7.3 Hz, Ar) 7.51 (dd, IH, J= 7.3, 7.3 Hz, AR), 7.39 (dd, 1H, J =7.3, 7.3 Hz, ArH), 7.30 (d, 1H, J= 7.3 Hz, ArH), 4.04 (s, 1H, CHN(CH 3
)
2 ),'3.31-2.99 (m, 8H, CfCH2CHCHN(CH3)2, CHCHN(CH 3
)
2 , N(CH3)2), 2.87 (dd, IH, J = 15.4, 4.3 Hz, 158 CHH'CHCHzCHCHN(CHs) 2 ), 2.61 (dd, 1H, J= 15.4, 15.4Hz, CHWCHCH2CHCHN(CH 3 )2), 2.21 (ddd, J= 12.8, 5.0,2.5 Hz, CHH'CHCHN(CH3)2), 1.66 (ddd, IH, J= 12.8, 12.8, 12.8 Hz, CHH'CHCHN(CH3)2; UV max (0.01 M methanolic HC), um 264, 348; [aI(D=-147*(c =0.15 in 0.01 M methanolic HCi); HRMS (ES) /z calcd for (C21H2N 2 0+H)* 399.1556, found 399.1554. Biological testing. [00252] Whole-cell antibacterial activity was determined according to methods recommended by the NCCLS (National Committee for Clinical Laboratory Standards. 2002. Methodsfor dilution antimicrobial susceptibiity tests for bacteria that grow aerobically: approved standard-ffth edition. NCCLS document Ml 00-S 12. National Committee for Clinical Laboratory Standards. Wayne, PA.; incorporated herein by reference). Test compounds were dissolved in dimethyl sulfoxide (DMSO) and the resulting solutions were diluted in water (1:10) to produce stock solutions with a final concentration of 256 jig tetracycline analog per mL. In a 96-well microtiter plate, 50 pL aliquots of stock solutions were diluted serially into cation-adjusted Mueller-Hinton broth (MUD; Becton-Dickinson, Cockeysville, MD). Test organisms (50 pL aliquots of solutions -5 x 10 CFU/mL) were then added to the appropriate wells of the microtiter plate. Inoculated plates were incubated aerobically at 35 *C for 18-24 h. The MIC was the lowest concentration of compound determined to inhibit visible growth. Five Gram-positive and five Gram.-negative bacterial strains were examined in minimum inhibitory concentration (MIC) assays. The Gram-positive strains were Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ACH-0016, Staphylococcus haemolyticus ACH-0013, Enterococcusfaecalis ATCC 700802 (a VRE or vancomycin-resistant enterococcus strain), and Staphylococcus aureus ATCC 700699 (carrying the tetMresistanoe gene). The Gram-negative strains were Pseudomonas aeruginosa ATCC 27853, Klebsiellapneumoniae ATCC 13883, E coli ATCC 25922, E coli ACH-0095 (multiply antibiotic-resistant), and E coli ATCC 53868::pBR322 (containing a plasmid encoding tetracycline-resistance). These strains are listed again below, along with certain other details of their origins and known resistance to antibiotics. 159 Bacterial strains Gram-Positive Organisms: Staphylococcus aureus ATCC 29213 QC strain for MIC testing Staphylococcus aureus ATCC 700699 Methicillin- and tetracycline resistant clinical isolate with intermediate resistance to vancomycin Staphylococcus epidermidis ACH-0018 Clinical isolate (Achillion strain collection) Staphylococcus haemolyticus ACH-00 13 Clinical isolate (Achillion strain collection) Enterococcusfaecalis ATCC 700802 Vancomycin-resistant clinical isolate Gram-Negative Organisms: E coli ATCC 25922 QC strain for MIC testing K col ATCC 53868::pBR322 Laboratory strain carrying a plasmid with a tetracycline resistance marker K coli ACH-0095 Multiply-resistant clinical isolate (Achillion strain collection) 160 Klebsiellapneumoniae ATCC 13883 QC stain for MIC testing Pseudomonas aeruginosa ATCC 27853 QC strain for MIC testing ATCC = American Type Culture Collection, Manassas, VA Example -Alternative Routes to Tetracycline Analogs [002531 Many of the studies described above show the generation of the carbanionic D-ring precursor by metalization of phenyl esters of o-toluate derivatives. These self-condensation reactions at times required to use of up to 4-5 equivalents of a given D-ring precursor. The presence of an electron-withdrawing substituent on the a carbon greatly improves the efficiency of metalation and coupling as described in Example 7 and elsewhere herein. Lithium-halogen exchange of benzylic bromides conducted in situ in the presence of the AB electrophile has been found to provide coupling products where benzylic metalation fails (see Example 7). These benzylic bromides can be prepared with surprising efficiencies (near quantitative yields) and are surprisingly stable. The developments may lead to a coupling reaction that could be conductable on a multi-kilo scale. Many different phenyl ester substituents (see below) may be used to optimize a coupling reaction. 161 0OH% 00HH3 OOHS OCH3 s NCg Ar 0 N Ar O Ar Ar 0 N 0 Y L O YO .00 ~ CHs H3CO OCH3 4 CHS CI Ar OCH2CF3 Ar Y Ar O A Ar 0 ONO2 C CHsC AHr 01 CI CI N
H
3 C
OH
3 CH3 Ar N Ar S% 4 Ar 0 Ar OCH3 0 0 0 0 CH 3 g O The optimal group for benzylic metalation, however, may not be the same as the optimal group for lithium-halogen exchange. In addition, for the lithium-halogen exchange process, besides ester modification, other metal reagents may be used including, but not limited to, other alkyllithium reagents (e.g., phenyllithium, mesityllithium), Grignard reagents (e.g., iso-propylmagesium chloride) and zinc-based systems. Barbier-type couplings will be explored using a variety of zero-valent metals for coupling. The AB-ring precursors may also be prepared by alternative routes. The step count for the synthesis of most 6-deoxytetracycline analogs is 14 from benzoic acid. Eleven of these 14 steps are dedicated to the synthesis of the AB-ring precursor. Any improvements in the length or efficiency of the route to these AB-ring precursors will have a substantial impact on the synthesis overall. Alternative syntheses of the AB-ring precursor are shown in Figures 22 and 23. Among the strategies for alternative A-ring closure sequences are intramolecular Michael additions, palladium-mediated processes, and iminium ion induce closures. Hypervalent iodine reagents may also be used instead of microbial dihydroxylation in the synthesis of the AB-ring precursors as shown in Figure 23. 162 Other Embodiments [00254] The foregoing has been a description of certain non-limIting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing tom the spirit oi scope of the present invention, as defined In the following claims. [00255] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise', and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or stejps but not the exclusion of any other integer or step or group of integers or steps. [002561 The' reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 163
Claims (15)
1. A compound of Formula: R 2 R 3 R 4 R 5 R14,,, H -. H~ OF OP O OHi O OP' or a salt, isomer, or tautomer thereof; wherein: R 1 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORA; -C(=O)RA; -CO 2 RA; -CN; -SCN; -SRA; -SORA; -SO2RA; -NO 2 ; -N(RA) 2 ; -NHC(O)RA; or -C(RA)3; wherein each occurrence of RA is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; R 2 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORB; -C(=O)RB; -CO 2 RB; -CN; -SCN; -SRB; -SORB; -SO2RB; -NO 2 ; -N(RB) 2 ; -NHC(O)RB; or -C(RB) 3 ; wherein each occurrence of RB is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; R 3 hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORc; -C(=O)Rc; -CO2Rc; -CN; -SCN; -SRc; -SORc; -SO2Rc; -NO 2 ; -N(Rc)2; -NHC(O)Rc; or -C(Rc) 3 ; wherein each occurrence of Rc is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; 164 H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016 R 4 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORD; -C(=O)RD; -CO 2 RD; -CN; -SCN; -SRD; -SORD; -SO 2 RD; -NO 2 ; -N(RD) 2 ; -NHC(O)RD; or -C(RD)3; wherein each occurrence of RD is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; R 5 is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; ORE; -CN; -SCN; -SRE; or -N(RE) 2 ; wherein each occurrence of RE is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; each occurrence of R 7 is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; acyl; substituted or unsubstitued aryl; substituted or unsubstituted heteroaryl; -ORG; -C(=O)RG; -CO2RG; -CN; -SCN; -SRG; -SORG; -SO2RG; -NO 2 ; -N(RG)2; -NHC(O)RG; or -C(RG)3; wherein each occurrence of RG is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, alkylamino, dialkylamino, heteroaryloxy, or heteroarylthio; P is independently selected from the group consisting of hydrogen, lower alkyl group, acyl group, or a protecting group; each occurrence of P' is independently selected from the group consisting of hydrogen or a protecting group; and n is an integer in the range of 0 to 3, inclusive: wherein each instance of aliphatic, heteroaliphatic, aryl, and heteroaryl is optionally and independently substituted with one or more groups selected from the following listing of substituents: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; CH 2 SO 2 CH 3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; 165 H:\RBR\ntrovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016 S(O) 2 Rx; and -NRx(CO)Rx, wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl; wherein each instance of aliphatic, heteroaliphatic, arylalkyl, and heteroarylalkyl provided in the listing of substituents is independently branched or unbranched, cyclic or acyclic, and unsubstituted or substituted with one or more moieties selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; CH 2 SO 2 CH 3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; S(O) 2 Rx; and -NRx(CO)Rx; wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl; and wherein each instance of aryl and heteroaryl provided in the listing of substituents is independently unsubstituted or substituted with one or more moieties selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; Cl; -Br; -I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; CH 2 SO 2 CH 3 ; -C(O)Rx; - CO 2 (Rx); -CON(Rx) 2 ; -OC(O)Rx; -OCO 2 Rx; -OCON(Rx) 2 ; -N(Rx) 2 ; S(O) 2 Rx; and -NRx(CO)Rx; wherein each Rx independently is selected from the group consisting of aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; and heteroarylalkyl.
2. The compound of claim 1, wherein each instance of R 1 , R 2 , R 3 , and R 4 is hydrogen.
3. The compound of claim 1 or 2, wherein R 5 is -N(RE) 2 , wherein each occurrence of RE is independently hydrogen, a protecting group, or lower (C 1 -C 6 ) alkyl.
4. The compound of any one of claims 1 to 3, wherein each occurrence of R 7 is independently halogen; cyclic substituted or unsubstituted heteroaliphatic; substituted or unsubstituted heteroaryl; -ORG; -CO 2 RG; -CN; -SCN; -SRG; -SORG; -SO 2 RG; or NHC(O)RG; wherein each occurrence of RG is independently hydrogen, a protecting group, aliphatic, heteroaliphatic, acyl; aryl; heteroaryl; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio.
5. The compound of claim 4, wherein at least one instance of R 7 is -NHC(O)RG 166 H:\RBR\Intrwovn\NRPortbl\DCC\RBR\0099038I.docx-6/05/2016
6. The compound of claim 4, wherein at least one instance of R 7 is halogen.
7. The compound of claim 4, wherein at least one instance of R 7 is -ORG
8. The compound of claim 4, wherein at least one instance of R 7 is cyclic substituted or unsubstituted heteroaliphatic.
9. The compound of claim 4, wherein one instance of R 7 is -ORG and one instance of R 7 is cyclic substituted or unsubstituted heteroaliphatic.
10. The compound of claim 4, wherein one instance of R 7 is halogen and one instance of R 7 is -NHC(O)RG.
11. The compound of claim 4, wherein one instance of R 7 is -ORG and one instance of R 7 is halogen.
12. The compound of any one of claims 1, 4, 6, 10, and 11, wherein halogen is -Cl.
13. The compound of any one of claims 1, 4, 6, 10, and 11, wherein halogen is -I.
14. The compound of any one of claims 1, 4, 6, 10, and 11, wherein halogen is -Br.
15. The compound of any one of claims 1, 4, 6, 10, and 11, wherein halogen is -F. 167
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| WO1995022529A1 (en) * | 1994-02-17 | 1995-08-24 | Pfizer Inc. | 9-(substituted amino)-alpha-6-deoxy-5-oxy tetracycline derivatives, their preparation and their use as antibiotics |
| WO2002004404A2 (en) * | 2000-07-07 | 2002-01-17 | Trustees Of Tufts College | 7,8 and 9-substituted tetracycline compounds |
| WO2003057169A2 (en) * | 2002-01-08 | 2003-07-17 | Paratek Pharmaceuticals, Inc. | 4-dedimethylamino tetracycline compounds |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995022529A1 (en) * | 1994-02-17 | 1995-08-24 | Pfizer Inc. | 9-(substituted amino)-alpha-6-deoxy-5-oxy tetracycline derivatives, their preparation and their use as antibiotics |
| WO2002004404A2 (en) * | 2000-07-07 | 2002-01-17 | Trustees Of Tufts College | 7,8 and 9-substituted tetracycline compounds |
| WO2003057169A2 (en) * | 2002-01-08 | 2003-07-17 | Paratek Pharmaceuticals, Inc. | 4-dedimethylamino tetracycline compounds |
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