ZA200202412B - Methods of preparing substituted tetracyclines with transition metal-based chemistries. - Google Patents

Description

METHODS OF PREPARING SUBSTITUTED TETRACYCLINES WITH

TRANSITION METAL-BASED CHEMISTRIES

RELATED APPLICATIONS

This application is related to copending U.S. Provisional Application No(s). 60/154,701, filed on September 14, 1999, and U.S. Provisional Application, Attorney Docket No. PKZ-018- 2, filed September 12, 2000, the entire contents of both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result of a systematic screening of soil specimens collected from many parts of the world for evidence of microorganisms capable of producing bacteriocidal and/or bacteriostatic compositions. The first of these novel compounds was introduced in 1948 under the name chlortetracycline. Two years later oxytetracycline became available. The detailed elucidation of the chemical structure of these agents confirmed their similarity and furnished the analytical basis for the production of a third member of this group in 1952, tetracycline. By 1957, a new family of tetracycline compositions characterized chemically by the absence of the position 6 ring-attached OH group present in the earlier compositions was prepared and became publicly available in 1967; and » minocycline was in use by 1972. Individual tetracycline-type agents are structurally compared - within Table I below, with reference made the following structural formula: :

HC, OHH H N(CH); . : OH

LLL

CONH,

OH

OH lo} OH 0

AE EN

Table I

Congener Substituent(s) At Carbon Position

Nos.

Chlortetracycline -Cl @)

Oxytetracycline -OH,-H (5)

Demeclocycline -OH,-H:;-Cl (6,7)

Methacycline -OH,-H;=CH, (5:6)

Doxycycline -OH.-H;-CHj3,-H (5:6)

Minocycline -H,-H;-N(CH3), (6:7)

More recent research efforts have focused on developing new tetracycline antibiotic compositions effective under varying therapeutic conditions and routes of administration; and for developing new tetracycline analogues which might prove to be equal or more effective then the originally introduced tetracycline families beginning in 1948. Representative of such developments include U.S. Patent Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280; 4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. It will be understood that these issued patents are merely representative of the range of diversity of investigations seeking tetracycline and tetracycline analogue compositions which are pharmacologically active.

Historically, soon after their initial development and introduction, the tetracyclines, ) regardless of specific formulation or chemical structure, were found to be highly effective - pharmacologically against rickettsiae; a number of gram-positive and gram-negative bacteria; ) and the agents responsible for lymphogranuloma venereum, inclusion conjunctivitis, and psittacosis. Hence, tetracyclines became known as "broad spectrum” antibiotics. With the subsequent establishment of their in vitro antimicrobial activity. effectiveness in experimental infections, and pharmacological properties. the tetracyclines as a class rapidly became widely used for therapeutic purposes. However, this widespread use of tetracyclines for both major and minor illnesses and diseases led directly to the emergence of resistance to these antibiotics even among highly susceptible bacterial species both commensal and pathogenic - as for example pneumococci and Salmonella. The rise of tetracycline-resistant organisms has resulted in a general decline in use of tetracyclines and tetracycline analogue compositions as antibiotics of choice.

SUMMARY OF THE INVENTION

The present invention relates to novel chemistries which allow for the production of substituted tetracycline compounds including substituted tetracycline compounds which exhibit significant antibacterial activity. The methods disclosed herein utilize reactive tetracycline- based precursor compounds, reactive organic substituent precursors and transition metals or transition metal catalysts under conditions such that a tetracycline compound substituted with the desired organic substituent is formed. In one embodiment of the invention, a substituted tetracycline compound may be prepared by combining a reactive tetracycline-based precursor : compound such as an arene tetracycline diazonium salt, and a reactive organic substituent precursor, e.g., alkenes, substituted alkenes, vinyl monomers, aromatics and heteroaromatics, in the presence of a transition metal catalyst, such as palladium chloride, under conditions such that a tetracycline compound substituted with the organic substituent is formed. In another embodiment, a substituted tetracycline compound may be prepared by contacting a reactive tetracycline chemical complex comprising a reactive tetracycline-based precursor compound and a transition metal or transition metal catalyst forming a reactive chemical intermediate with a reactive organic substituent precursor under conditions such that a tetracycline compound substituted with the organic substituent is formed.

The invention relates in another embodiment to reactive tetracycline chemical complexes comprising a reactive tetracycline-based precursor compound and a transition metal catalyst forming a chemical intermediate, which can advantageously be used in the methods of - the invention. -

In yet another embodiment substituted tetracycline analogs are disclosed, wherein the substituent (denoted herein as “Z”") at the desired position, e.g., 7, 9, 13, is connected with a -C-C- linkage, and wherein the substituent comprises an aromatic or heteroaromatic moiety.

The substituent may also comprise a -C=C- bond adjacent to the -C-C- linkage, e.g., han (2)

R> , wherein R, and R, are each independently hydrogen, alkyl. alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl. alkoxycarbonyl. aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl; or R, and R.. taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring having 5 to 15 atoms in the ring.

y . ® .

The methods and chemical intermediates disclosed herein allow for novel substituted tetracycline-type compounds and therapeutic methods and pharmaceutical compositions that comprise such compounds.

The method of the invention includes providing Z substituents, above, on the basic tetracycline ring structure through a process involving forming a reactive intermediate (comprising a tetracycline arenediazonium salt in a preferred embodiment) at the desired position and adding a reactive compound, e.g., a n-bond containing compound in the presence of a transition metal catalyst to that position. The reactive intermediate may be formed in situ.

In an advantageous embodiment such substituents are provided on the D ring of the basic tetracycline ring structure, e.g., positions 7 and/or 9. In another advantageous embodiment, such substitutions may be made at position 13. Such synthetic schemes are heretofore new in this art and advantageously allow for direct substitution of different and/or heretofore complex substituent groups at desired positions.

Compounds of the invention are active against susceptible microorganisms, including tetracycline-sensitive bacteria as well as tetracycline-resistant bacteria. Particularly preferred compounds of the invention exhibit 24-hr minimum inhibitory concentration (MIC) values of about 10 pg/mL or less, more preferably about 1 pg/mL or less, against tetracycline-resistant £. coli, S. aureus and E. faecalis strains such as E. coli pHCM1, S. aureus RN4250 and E. faecalis ’ pMV158. Preferred compounds of the invention also include those that exhibit such MIC values against tetracycline-sensitive E. coli, S. aureus and E. faecalis strains such as E. coli

D31m4, S. aureus RN450 and E. faecalis ATCC9790.

The invention provides methods of treatment against susceptible microorganisms such as bacteria, fungi, rickettsia, parasites and the like, and diseases associated with such microorganisms. These therapeutic methods in general comprise administration of a therapeutically effective amount of one or more compounds of the invention to a living subject that is suffering from or susceptible to infection by a susceptible microorganism such as bacteria, fungi, rickettsia and the like. Suitable subjects for treatment include animals, particularly a mammal such as human, or plants.

Pharmaceutical compositions comprising one or more compounds of the invention and a suitable carrier are also provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be more fully illustrated by reference to the definitions set forth below. “Tetracycline” or "tetracycline-type" is intended to include tetracycline and other tetracycline family members such as oxytetracycline; chlortetracycline; demeclocycline; doxycycline; chelocardin; minocycline; rolitetracycline; lymecycline; sancycline; methacycline; apicycline; clomocycline; guamecycline; meglucycline; mepylcycline; penimepicycline; pipacycline; etamocycline; penimocycline, etc. as well as other tetracycline compounds having the characteristic naphthacene A-B-C-D ring structure noted in the Background Of The

Invention. Additionally, numbered tetracycline ring positions as referred to herein are the same as designated in the above structural formula. “Reactive tetracycline-based precursor compound” or “RT-based precursor compound” includes tetracyclines which have a reactive position on the tetracycline ring structure, e.g., at 7, 9 or 13, such that substitution of the reactive tetracycline-based precursor compound may be accomplished as disclosed herein to form a substituted tetracycline compound. Examples of

RT-based precursor compounds include derivatives from art-recognized tetracycline compound families. Without limitation, such tetracycline compound families include minocycline, - doxycycline and sancycline compounds. “Minocycline-based precursor compound” is intended to include compounds having the ) core structure of minocycline, which differs from the core structure of tetracycline by the presence of a dimethylamino group at position 7. and the absence of methyl and hydroxyl groups at position 6, and the absence of a hydroxyl group at position 5. The core structure of minocycline-based precursor compounds is shown below for the purposes of illustration:

N(R)2 N(CHa), i, OH ¢

LT

CONH,

OH

OH lo} OH o)

It should be understood that minocycline-based precursor compounds can be substituted. unsubstituted or derivatized, e.g.. at positions other than positions 5 and 6. For example, other positions in the core structure, e.g., position 8, can be substituted or unsubstituted and others can be substituted or derivatized, such as the 2-position amido group.

Suitable substituents include moieties such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano. alkoxy, aryloxy, carboxyl, carboxamido, carboxy ester, alkoxycarbonyl, aryloxycarbonyl, carbocyclic or heterocyclic groups, and combinations thereof. Other substituent groups will be recognized by those of skill in the art. Further, R in the above formula can represent a group other than methyl, e.g., lower alkyl such as ethyl, propyl, etc. Reactive minocycline-based precursor compounds include, without limitation, 9-diazonium minocycline-based compounds, 9-iodo minocycline-based compounds, 9-bromo minocycline-based compounds, and 9-chloro minocycline-based compounds. “Doxycycline-based precursor compound” is intended to include compounds having the core structure of doxycycline, which differs from the core structure of tetracycline by the substitution of a hydrogen for a hydroxyl at position 6, and the substitution of a hydroxyl for a hydrogen at position 5. The core structure of doxycycline-based precursor compounds is shown below for the purposes of illustration: i R HHO H N(CH), . { {_, OH

LTE

CONH;,

OH

OH fo) OH 0

It should be understood that doxycycline-based precursor compounds can be substituted, unsubstituted or derivatized, e.g.. at positions 7, 8 and/or 9. For example, other positions in the core structure, e.g., position 8. can be substituted or unsubstituted and others can be substituted or derivatized, such as the 5-position hydroxyl group or the 2-position amido group. Suitable substituents include moieties such as hydrogen, alkyl, alkenyl, alkynyl. cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl. alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl. carboxamido, carboxy ester. alkoxycarbonyl, aryloxycarbonyl, carbocyclic or heterocyclic groups, and combinations thereof. Other substituent groups will be recognized by those of skill in the art. Further, R in the above formula can represent a group other than methyl, e.g.. lower alkyl such as ethyl. propyl. etc.

Reactive doxycycline-based precursor compounds include, without limitation, 7- and/or 9- diazonium doxycycline compounds, 7- and/or 9-iodo doxycycline compounds, 7- and/or 9- bromo doxycycline compounds, and 7- and/or 9-chloro doxycycline compounds. “Sancycline-based precursor compound” is intended to include compounds having the core structure of sancycline, which differs from the core structure of tetracycline by the substitution of a hydrogen for a methyl group and hydrogen for a hydroxyl at position at position 6. The core structure of sancycline-based precursor compounds is shown below for the purposes of illustration:

N(CHa)p 4 {, OH

BOVE! s CONH,

OH

OH lo} OH 0]

It should be understood that sancycline-based precursor compounds can be substituted, unsubstituted or derivatized, e.g., at positions 7, 8 and/or 9. For example. other positions in the core structure, e.g., position 8, can be substituted or unsubstituted and others can be substituted or derivatized, such as the 2-position amido group. Suitable substituents include moieties such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbony!l, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, carboxamido, carboxy ester, alkoxycarbonyl, aryloxycarbonyl, carbocyclic or heterocyclic groups, and combinations thereof. Other substituent groups will be recognized by those of skill in the art. Reactive sancycline-based precursor compounds include, without limitation, 7- and/or 9-diazonium sancycline compounds, 7- and/or 9-iodo sancycline compounds, 7- and/or 9-bromo sancycline compounds, and 7- and/or 9-chloro sancycline compounds.

In a preferred embodiment, the reactive tetracycline-based precursor compound is an arene tetracycline diazonium salt, and alternately iodo derivatized tetracycline compounds, or tetracycline compounds that possess a double bond and are reactive with boronic acid derivatives, e.g., at position 13. In one embodiment, the reactive tetracycline-based precursor compound and a transition metal catalyst form a reactive chemical intermediate useful in making novel tetracyclines, through techniques known in the art (see, for example, Hegedus,

Transition Metals in the Synthesis of Complex Organic Molecules, University Science Books.

Mill Valley, CA, 1994. incorporated herein by reference). The reactive chemical intermediate are preferably formed in situ with the reactive organic substituent precursor. “Transition metal catalyst” is an art-recognized term which includes transition metals and catalysts comprising a transition metal, e.g., including elements 21 through 29, 39 through 47, 57 through 79, and 89 on. Exemplary transition metal catalysts include CuCl.. copper (I) triflate, copper thiophene chloride, palladium (II) chloride, organopalladium catalysts such as palladium acetate, Pd(PPh,),, Pd(AsPh;),, PdCL(PhCN),, PdCl, (Ph;P),, Pd,(dba),-CHCI, (“dba”=dibenzylacetone); and combinations thereof. Other transition metal catalysts include those containing metals such as rhodium (e.g. rhodium (II) acetate and Rh,(CO),). iron, iridium, chromium, zirconium, and nickel. A skilled artisan will be able to select the appropriate transition metal catalyst to perform the desired reaction, based on the existing literature (see, for example, Lipshutz, B.H. Org. React. 1992, 41:135, incorporated herein by reference.) © “Reactive organic substituent precursor” includes organic substituents having a reactive ) group that allows for addition to the reactive tetracycline-based precursor compound as disclosed herein. Preferably the reactive organic substituent precursor comprises at least one ) reactive group. In an embodiment, the reactive organic substituent precursor may include 7- bonded species such as methylene compounds, aryl boronic acids, active aromatic rings and unsubstituted and substituted olefins and alkynes, nitriles, acetylenes, substituted acetylenes, arylethylenes, styrenes. conjugated dienes, isoprenes, vinyl ethers, a, B-unsaturated aldehydes and ketones, aryl vinyl and arylisoprenyl ketones, iodoalkenes and iodoarenes, quinones, a, B- unsaturated acids and their derivatives. “Reactive organic substituent precursors” also include compounds (which may be formed in situ) which react with the reactive intermediate to form a desired tetracycline analog.

For example, the reactive intermediate can be transmetallated to form a wide variety analogs through reactions with other organometal complexes such as tributyltin compounds and lithium diorganocuprates (see for example, Kalanin, Synthesis, 1992, 413; Sawamuru, Chem. Rev. 1992, 92:857; Negeishi. Acct. Chem. Res., 1982, 15:340, incorporated herein by reference).

Other precursors include those suitable for transition metal catalyzed reactions include compounds with bonds which are reactive with the transition metal containing intermediates.

Such precursors include. for example. compounds with halogen groups, hydroxyl groups, triflate groups, thiol groups, amino groups. Intramolecular reactions are also included wherein the reactive organic substituent precursor is bonded or associated with the reactive chemical intermediate (see Hegedus, supra).

Compounds of the invention include 7-substituted tetracycline analogs, 9-substituted tetracycline analogs, and 13-substituted tetracycline analogs. These compounds may be illustrated by the general formula

I

Z; Z4 0 N(CHg), 4

OH

8 ® » » _

Z3 ! c— NH, on [1]

OH (6) OH oO oO wherein Z,, Z, and Z, are individually H or ) — )

Ra wherein R, and R, are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl; or R, and R,, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring having 5 to 15 atoms in the ring; and R, is H or OH.

In another embodiment R, is hydrogen, and R; is Rs. where R, is hydrogen. cyano, or a C,-C; alkoxy group. In another embodiment R; and R», taken together, form a substituted

Claims (72)

CLAIMS What is claimed is:

1. A method of preparing a substituted tetracycline compound, comprising contacting a reactive tetracycline chemical complex comprising a reactive tetracycline-based precursor compound and a transition metal catalyst forming a reactive chemical intermediate with a reactive organic substituent precursor under conditions such that a tetracycline compound substituted with said organic substituent is formed.

2. A method of preparing a substituted tetracycline compound, comprising combining a reactive tetracycline-based precursor compound and a reactive organic substituent precursor in the presence of a transition metal catalyst under conditions such that a tetracycline compound substituted with said organic substituent is formed.

3. The method of claim 1 or 2, wherein said transition metal catalyst comprises an organopalladium catalyst.

4. The method of claim 3, wherein said organopalladium catalyst comprises palladium chloride, palladium acetate, PdCL,(PhCN),, PdCl, (Ph,P),, Pd,(dba),-CHCI,, or a combination thereof.

5. The method of any one of claims 1-4, wherein said transition metal catalyst comprises copper, rhodium, iron, iridium, chromium, zirconium, or nickel.

6. The method of claim 5, wherein said transition metal catalyst comprises CuCl, Cul, thodium (II) acetate, Rh,(CO),,), or combinations thereof.

7. The method of any one of claims 1-6, wherein said reactive tetracycline-based precursor compound is oxytetracycline, chlortetracycline, demeclocycline, doxycycline, chelocardin, minocycline, roliteteracycline, lymecycline, sancycline, methacycline, apicycline,

clomocycline, guamecycline. meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline, or penimocycline based precursor compound.

8. The method of any one of claims 1-6, wherein said reactive tetracycline-based precursor compound is selected from the group consisting of reactive minocycline-based precursor compounds, reactive doxycycline-based precursor compounds, and reactive sancycline-based precursor compounds.

9. The method of any one of claims 1-8, wherein said reactive tetracycline-based precursor compound is an arenediazonium salt, iodo derivative, or a boronic acid derivative of a tetracycline compound.

10. The method of any one of claims 1-9, wherein said reactive organic substituent precursor has at least one reactive n-bond containing group. }

11. The method of claim 10, wherein said reactive organic substituent precursor is alkenyl, alkynyl, or aromatic.

12. The method of claim 11, wherein said reactive organic substituent precursor is alkenyl.

13. The method of claim 12, wherein said alkenyl reactive organic substituent precursor is a vinyl monomer.

14. The method of claim 12, wherein said alkenyl reactive organic substituent precursor is substituted.

15. The method of claim 12 or 14, wherein said alkenyl reactive organic substiuent precursor is a methylenyl compound, conjugated diene, isoprene, vinyl ether, iodoalkene, or a derivative thereof.

16. The method of claim 11, wherein said aryl reactive organic substituent precursor is heteroaromatic.

17. The method of claim 11 or 16, wherein said aryl reactive organic substitutent precursor is an aryl boronic acid, iodoaryl, quinone, arylethylene, or styrene.

18. The method of claim 10, wherein said reactive organic substituent precursor comprises a carbonyl or thiocarbony! group.

19. The method of claim 18, wherein said reactive orgainc substituent precursor is an aryl viny! ketone, arylisoprenyl ketone, «, B-unsaturated aldehyde, o, B-unsaturated ketone, . a, B-unsaturated acid, or a derivative thereof.

20. The method of claim 11, wherein said alkynyl reactive organic substituent precursor is substituted or unsubstituted acetylene.

21. A reactive tetracycline chemical complex comprising a reactive tetracycline- based precursor compound and a transition metal catalyst.

22. The reactive tetracycline chemical complex of claim 21, wherein said catalyst comprises an organopalladium catalyst.

23. The reactive tetracycline chemical complex of claim 22, wherein said organopalladium catalyst comprises palladium chloride, palladium acetate, PdCL,(PhCN),, PdCl, (Ph,P),, Pd,(dba),-CHCl,, or a combination thereof.

24. The reactive tetracycline chemical complex of any one of claims 21-23, wherein said transition metal catalyst comprises copper, rhodium, iron, iridium, chromium, zirconium, or nickel.

25. The reactive tetracycline chemical complex of claim 24, wherein said transition metal catalyst comprises CuCl,, Cul,, rhodium (II) acetate, Rhy(CO),,), or combinations thereof.

26. The reactive tetracycline chemical complex of any one of claims 21-25, wherein said reactive tetracycline-based precursor compound is oxytetracycline, chlortetracycline, demeclocycline, doxycycline, chelocardin, minocycline, roliteteracycline, lymecycline, sancycline, methacycline, apicycline, clomocycline, guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline, or penimocycline based precursor compound.

27. The reactive tetracycline chemical complex of any one of claims 21-25, wherein said reactive tetracycline-based precursor compound is selected from the group consisting of reactive minocycline-based precursor compounds, reactive doxycycline-based precursor compounds, and reactive sancycline-based precursor compounds.

28. The reactive tetracycline chemical complex of any one of claims 21-27, wherein said reactive tetracycline-based precursor compound is an arenediazonium salt, iodo derivative, or a boronic acid derivative of a tetracycline compound.

29, A 7-substituted tetracycline compound, wherein the substituent at the 7 position is connected with a -C-C- linkage, and wherein said substituent comprises an aromatic or heteroaromatic moiety.

30. The 7-substituted tetracycline compound of claim 29, wherein said compound is 7-4’-Cl-phenyl sancycline.

31. The 7-substituted tetracycline compound of claim 29, wherein said compound is 7-(4-fluorophenyl)sancycline.

32. The 7-substituted tetracycline compound of claim 29, wherein said compound is 7-(4-nitrophenyl)sancycline.

33. The 7-substituted tetracycline compound of claim 29, wherein said compound is 7-(2-pyridyl)doxycycline.

34. A 7-substituted tetracycline compound, wherein the substituent at the 7 position is connected with a -C-C- linkage, and wherein said substituent comprises a -C=C- bond adjacent to said -C-C- linkage. .

35. The 7-substituted tetracycline compound of claim 34, wherein said substituent is of the formula 3 — Ow (Z) R2 wherein R, and R; are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl; or R, and R,, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring.

36. The 7-substituted tetracycline compound of claim 35, wherein R, is hydrogen, L and R, is Ry , and R, is hydrogen, cyano, or a C,-C; alkoxy group.

37. The 7-substituted tetracycline compound of claim 35, wherein R, and R,, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring.

38. The 7-substituted tetracycline compound of claim 35 or 37, wherein said ring comprises 5 to 15 atoms.

39. The 7-substituted tetracycline compound of any one of claims 35, 37 or 38, wherein said ring is a conjugated or unconjugated aromatic ring system.

40. The 7-substituted tetracycline compound of claim 35, wherein said compound is 7-ethylenylsancycline. ] 15

41. A 9-substituted tetracycline compound, wherein the substituent at the 9 position is connected with a -C-C- linkage, and wherein said substituent comprises an aromatic or heteroaromatic moiety.

42. A 9-substituted tetracycline compound, wherein the substituent at the 9 position is connected with a -C-C- linkage, and wherein the substituent comprises a -C=C- bond adjacent to said -C-C- linkage.

43. The 9-substituted tetracycline compound of claim 42, wherein the substituent is of the formula

NY —_ J Ry Z) R2 wherein R, and R, are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl; or R, and R;, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring. 44, The 9-substituted tetracycline compound of claim 43, wherein R, is hydrogen, LL and R; is Ry , where R, is hydrogen, cyano, or a C,-C, alkoxy group.

45. The 9-substituted tetracycline compound of claim 43, wherein R, and R5, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring.

46. The 9-substituted tetracycline compound of claim 43 or 45, wherein said ring has from 5 to 15 atoms in the ring. }

47. The 9-substituted tetracycline compound of any one of claims 43, 45, or 46, wherein said ring is a conjugated or unconjugated aromatic ring system.

48. A substituted tetracycline compound made by a method comprising contacting a reactive tetracycline chemical complex comprising a reactive tetracycline-based precursor compound and a transition metal catalyst forming a reactive chemical intermediate with a reactive organic substituent precursor under conditions such that a tetracycline compound substituted with said organic substituent is formed.

49. The substituted tetracycline compound of claim 48, wherein said reactive tetracycline-based precursor compound is substituted at positions 7, 9 or 13 with said organic substituent.

50. The substituted tetracycline compound of claim 48 or 49, wherein said catalyst comprises an organopalladium catalyst.

51. The substituted tetracycline compound of claim 50, wherein said organopalladium catalyst comprises palladium chloride, palladium acetate, PdCl,(PhCN),, PdCl, (Ph,P),, Pd,(dba),-CHCI,, or a combination thereof.

52. The substituted tetracycline compound of any one of claims 48-51, wherein said transition metal catalyst comprises copper, rhodium, iron, iridium, chromium, zirconium, or nickel. ’

53. The substituted tetracycline compound of any one of claims 48-52, wherein said transition metal catalyst comprises CuCl,, Cul,, rhodium (II) acetate, Rhy(CO),,), or . combinations thereof.

54. The substituted tetracycline compound of any one of claims 48-53, wherein said reactive tetracycline-based precursor compound is oxytetracycline, chlortetracycline, demeclocycline, doxycycline, chelocardin, minocycline, roliteteracycline, lymecycline, sancycline, methacycline, apicycline, clomocycline, guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline, or penimocycline based precursor compound.

55. The substituted tetracycline compound of any one of claims 48-53, wherein said reactive tetracycline-based precursor compound is selected from the group consisting of reactive minocycline-based precursor compounds, reactive doxycycline-based precursor compounds, and reactive sancycline-based precursor compounds.

56. The substituted tetracycline compound of any one of claims 48-55, wherein said reactive tetracycline-based precursor compound is an arenediazonium salt, iodo derivative, or a boronic acid derivative of a tetracycline compound.

57. The substituted tetracycline compound of any one of claims 48-56, wherein said reactive organic substituent precursor has at least one reactive n-bond containing group.

58. The substituted tetracycline compound of any one of claims 48-57, wherein said reactive organic substituent precursor is selected from the group consisting of alkenes, substituted alkenes, vinyl monomers, aromatic and heteroaromatic reactive groups.

59. The substituted tetracycline compound of any one of claims 48-58, wherein said organic substituent is of the formula : = SE (2) R, wherein R, and R, are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl; or R, and R;, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring.

60. The substituted tetracycline compound of claim 59, wherein R, is hydrogen, and L R; is Ry , where R, is hydrogen, cyano, or a C,-C; alkoxy group.

61. The substituted tetracycline compound of claim 59, wherein R; and Rj, taken together, form a substituted or unsubstituted carbocyclic or heterocyclic ring.

62. The substituted tetracycline compound of claim 59 or 61, wherein said ring has from 5 to 15 atoms in the ring.

63. The substituted tetracycline compound of any one of claims 59, 61, or 62, wherein said ring is a conjugated or unconjugated aromatic ring system. :

64. The substituted tetracycline compound of any one of claims 59, or 61-63, wherein said ring has 5 to 8 atoms in the ring.

65. A method according to claim 1, substantially as herein described with reference to any one of the illustrative examples.

66. A method according to claim 2, substantially as herein described with reference to any one of the illustrative examples.

67. A reactive tetracycline chemical complex according to claim 21 as specifically described herein.

68. A 7T-substituted tetracycline compound according to claim 29, other than the ’ compounds of claims 30 to 33, as specifically described herein.

69. A 7-substituted tetracycline compound according to claim 34 as specifically described herein.

70. A 9-substituted tetracycline compound according to claim 41 as specifically ’ described herein.

71. A 9-substituted tetracycline compound according to claim 42 as specifically described herein.

72. A substituted tetracycline compound according to claim 48 as specifically described herein. -44- Amended Sheet 2003-07-07