TW548257B - Methods of preparing substituted tetracyclines with transition metal-based chemistries - Google Patents

Abstract

The present invention relates to novel chemistries which allow for heretofore unobtainable substituted tetracycline compounds which exhibit significant antibacterial activity. The methods disclosed herein utilize reactive tetracycline-based precursor compounds, reactive organic substituent precursors and 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. Such compounds may optionally act as intermediates for making other compounds, e.g., hydrogenation of unsaturated groups on the substituent.

Description

548257 V. Description of the invention (1) Related applications This application is related to the pending US provisional application 6 0/1 5 4, 7 01, which was filed on September 14, 1999. The entire contents are hereby incorporated as reference. BACKGROUND OF THE INVENTION The development of tetracycline antibiotics is the result of contaminated samples collected from many parts of the world, systematically screened to prove that microbes that can produce bactericidal and / or bacteriostatic compositions. These novel compounds were first introduced in 1984 under the name chlorotetracycline. Two years later, tetracycline hydrochloride was introduced. A detailed description of the chemical structures of these reagents confirmed their similarity, and the analytical basis for the manufacture of the third member of the family, tetracycline, was completed in 1952. By 1975, a new family of tetracycline compositions was prepared, which was chemically characterized by the absence of the 0H group at the linking ring 6 position in earlier compositions, and was announced in 1967; and diamine Tetracycline was used in 1972. With reference to the following structural formulas, the individual tetracycline-type reagents are structurally compared in Table I below:

Substituents-C1 -OH, -H -OH, -H; -C1 are all gas tetracycline tetracycline hydrochloride dechlorotetracycline at the carbon position number (7) (5) (6; 7)

Page 6 548257 V. Description of the invention (2) pinene tetracycline-0H, -H = CH2 (5; 6) deoxytetracycline -OH, -H; -CH3, -fluorene (5; 6) diamine tetracycline- Η,-Η; -N (CH3) 2 (6; 7) Recent research has focused on the development of novel tetracycline antibiotic compositions that are effective under different treatment conditions and routes of administration; and development has proven to be equal to or greater than The first tetracycline family introduced in 1984 was a more effective novel tetracycline homologue. Representatives of such developments include U.S. patents 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, 4 5 4, 6 9 7; and 3, 1 6 5, 5 3 1. It should be understood that these issued patents are merely representative of various scope surveys seeking pharmacologically active tetracycline and tetracycline homologous compositions. Historically, regardless of the specific formula or chemical structure, tetracycline has been found to target Rickettsia shortly after its initial development and introduction; many species of Gram-positive and Gram-negative bacteria; and responsible for inguinal lymph granuloma (including Conjunctivitis) and parrot fever are highly effective pharmacologically. Therefore, tetracycline is well known as a π wide range π antibiotic. Tetracyclines were quickly and widely used for therapeutic purposes in the subsequent establishment of antimicrobial activity, experimental infection effects, and pharmacological properties in their test tubes. However, the widespread use of tetracycline for small and large diseases directly contributes to resistance to these antibiotics, and even symbiotic and pathogenic highly susceptible bacterial species-for example, pneumococcus and salmonella. The rise of tetracycline-resistant organisms has led to a decline in the use of tetracycline and tetracycline 0 homologous compositions as an antibiotic of choice. Summary of invention

Page 7 548257 V. Description of the invention (3) The present invention relates to a novel chemistry that can produce substituted tetracycline compounds including substituted tetracycline compounds exhibiting significant bactericidal activity. The method disclosed herein utilizes a reactive tetracycline-based precursor compound, a reactive organic substituent precursor, and a transition metal catalyst under conditions such that a tetracycline compound substituted with a desired organic substituent is formed. In a specific embodiment of the present invention, a substituted tetracycline compound can be formed by combining a reactive tetracycline base in the presence of a transition metal catalyst (such as palladium chloride) under conditions that allow the formation of a tetracycline compound substituted with an organic substituent. Precursor compounds (such as aromatic tetracycline diazonium salts) and reactive organic substituent precursors (eg, olefins, substituted olefins, ethylene monomers, aromatics, and heteroaromatics) are prepared. In another specific embodiment, a substituted tetracycline compound may be obtained by contacting a reactive organic substituent precursor with a reactive organic substituent precursor under conditions such that an organic substituent-substituted tetracycline compound is formed. Or a reactive tetracycline chemical complex of a transition metal catalyst to form a reactive chemical intermediate. In another embodiment, the present invention relates to a reactive tetracycline chemical complex comprising a reactive tetracycline-based precursor compound and a transition metal catalyst, which can be advantageously used in the method of the present invention. In another specific embodiment, a substituted tetracycline homologue is disclosed in which a substituent (represented herein as π Z π) at a desired position (eg, 7, 9, 1 3) is linked by a -CC- linkage, and Wherein the substituent includes an aromatic or heteroaromatic moiety. Substituents may also include -C = C-bonds of adjacent -C-C _ linkages, such as ’

(Z)

Bill

Page 8 548257 V. Description of the invention (4) where r2 and 1 to 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkyl, aryl, aryl, etc. Oxyweiyl, aryloxyweiyl, amine, mesityl, cyano, carboxy, aryloxy, weiyl, carboxy, aryloxycarbonyl; or 1? 2 and 1? 3 -Form a substituted or unsubstituted carbocyclic or heterocyclic ring having 5 to 15 atoms in the ring. The methods and chemical intermediates disclosed herein can produce novel substituted tetracycline-type compounds and therapeutic methods, as well as pharmaceutical compositions containing such compounds. The methods of the present invention involve the preparation of reactive intermediates (in a preferred embodiment) The example includes a tetracycline arene diazonium salt method), providing the above Z substituent at a desired position on the basic tetracycline ring structure, and adding a reactive compound to this position in the presence of a transition metal catalyst, for example, containing 7Γ -Bond compounds. Reactive intermediates can be formed in situ. In advantageous embodiments, this substituent is provided on the D ring of the basic tetracycline ring structure, e.g., the 7 and / or 9 positions. In another advantageous embodiment, this substituent can be made at the 13 position. Such synthetic methods are heretofore novel in this art and can advantageously directly substitute different and / or previous complex substituents at the desired positions. The compounds of the present invention are effective against susceptible microorganisms, including tetracycline-sensitive bacteria and anti-tetracycline-resistant bacteria. The particularly preferred compounds of the present invention are targeted to tetracycline-resistant E. coli, S. aureus, and E. faecalis strains, such as E. coli pHCMl, S. aureus RN 4 2 50, and E. faecal is pMV158, showing about 10 micrograms / Milliliter or less, more preferably a minimum inhibitory concentration (M IC) value of about 1 μg / ml or less for 24 hours. The best of this invention

Page 9 548257 V. Description of the invention (5) The compounds also include tetracycline-sensitive E. coli, S. aureus and E. faecalis strains, such as E. coli D 31m4, S. aureus RN 4 5 0, and E. faecalis ATCC9790, presents this MIC value. The present invention provides a method for treating susceptible microorganisms, such as bacteria, fungi, rickettsial bodies, parasites, and the like, and diseases accompanying the microorganisms. These treatments usually involve the administration of a therapeutically effective amount of one or more compounds of the invention to a living patient suffering from or susceptible to infection by susceptible microorganisms such as bacteria, fungi, rickettsial, etc., suitable for such treatment. This includes animals, especially lactating animals such as humans, or plants. Pharmaceutical compositions comprising one or more compounds of the invention and suitable carriers are also provided. DETAILED DESCRIPTION OF THE INVENTION The present invention is described in more detail with reference to the definitions described below. "Tetracycline π or π tetracycline type π is intended to include tetracycline and other members of the tetracycline family, such as tetracycline hydrochloride; chlorotetracycline; norchlorotetracycline; deoxytetracycline; chelomycin; diamine tetracycline; Tetracycline; Tetracycline; Tetracycline; Tetracycline; Tetracycline; Metetracycline; Penicillin V Tetracycline; Tetracycline; Tetracycline; Tetracycline and the like, and have the characteristics shown in the background of the invention Other tetracycline compounds of the naphthalene ABCD ring structure. In addition, the numbered tetracycline ring positions are the same as those mentioned in the above structural formula. Π reactive tetracycline-based precursor compounds π or n RT-based precursor compounds The ring structure has a reactive position, for example, at 7, 9, or 1 3, so that the substitution of a reactive tetracycline-based precursor compound can be as here

Page 10 548257 V. Completion as disclosed in (6) of the invention,-Basic precursor compound 4 derivatives of the family. Cyclin, deoxytetracycline, π-dimethylamine tetracycline: The nucleus of the cyclin core structure exists. The structure for description is shown below: to form a tetracycline of a substituted tetracycline compound. Examples of R T 7 include tetracycline compounds obtained from this technology. This family of tetracycline compounds includes, but is not limited to, tetramine, and tetracycline compounds. The t-base precursor compound π is intended to include a tetramidine tetramer, which is different from the tetracycline core structure in that it does not have a fluorenyl group and a hydroxyl group at the 7 position and at the 6 position, and has no hydroxyl group at the 5 position. N (r) 2

n (ch3) 2 4 OH

It should be understood that other fluorene and other positions other than the 6 position in the nuclear structure and other acceptable groups include, for example, nitrogen, alkyl, arylweilyl, cyano, alkyloxy, carbonyl, aryloxycarbonyl It can be recognized that other than fluorenyl groups, amine tetracycline-based precursor compounds can be, for example, 5 l, substituted, unsubstituted or derived. For example, the position may be, for example, the 8 position, substituted or unsubstituted, substituted or derived, such as the 2-position fluorenyl. Suitable substituents, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonylalkoxycarbonyl, aryloxycarbonyl, amine, hydroxyl, aryloxy, carboxyl, carbaminyl, carboxylate, Alkoxy, carbocyclic or heterocyclic groups, and combinations thereof. Familiar with other substituents. In addition, R in the above formula may represent, for example, a low-carbon alkyl group such as ethyl, propyl, and the like. Reaction 548257 V. Description of the invention (7) Basic diamine tetracycline basic precursor compounds include, but are not limited to, 9-diazodiamidamine tetracycline basic compounds, 9-iododiamine tetracycline basic compounds, 9-bromodimethylamine tetracycline Basic compounds, and 9-Chlorodiamine tetracycline basic compounds. "Deoxytetracycline-based precursor compound" is intended to include compounds having a deoxytetracycline core structure which differs from the tetracycline core structure in that the hydroxyl group is replaced with hydrogen at the 6 position and the hydrogen is replaced with hydroxyl at the 5 position. For the purpose of description, the nuclear structure of the deoxytetracycline-based precursor compound is shown below:

It should be understood that the deoxytetracycline-based precursor compound may be substituted, unsubstituted, or derived at, for example, the 7, 8, and / or 9 positions. For example, it may be substituted or unsubstituted at other positions in the nuclear structure, for example, the 8 position, and others may be substituted or derived, such as a 5-position hydroxyl group or a 2-position amine. Suitable substituents include, for example, hydrogen, alkyl, dialkyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxyl, cyano, Oxyoxy, aryloxy, ammonium, ammonium carbonate, sulfonyl, alkoxycarbonyl, aryloxycarbonyl, carbocyclic or heterocyclic groups, and combinations thereof. Those skilled in the art will recognize other substituents. In addition, R in the above formula may represent a group other than a fluorenyl group, for example, a low-carbon alkyl group such as ethyl, propyl, and the like. Reactive deoxytetracycline-based precursor compounds include but are not limited to 7-and

Page 12 548257 V. Description of the invention (8) / or 9-diazodeoxytetracycline basic compound, 7- and / or 9-iododeoxytetracycline basic compound, 7- and / or 9-bromodeoxytetracycline basic compound , 7- and / or 9-chlorodeoxytetracycline base compounds. "Basic tetracycline basic precursor compound" is intended to include compounds having a tetracycline core structure, which is different from the tetracycline core structure in that the fluorenyl group is replaced with hydrogen at the 6 position and the hydroxyl group is replaced with hydrogen. For the purpose of description, the nuclear structure of the deoxytetracycline-based precursor compound is shown below:

CONHo should be substituted with hydrogen at other positions, carbonyl, oxy, oxy, and oxycarbonyl, but not 9-Eshan solution, shanzhi, jingjia, or derivatives, dilute oxyaryl aryloxy Alkyl, carbon, and other 7-tetracycline tetracycline-based precursor compounds may be, for example, 7, 8 and substituted, unsubstituted, or derived. For example, it may be substituted or unsubstituted in the nuclear structure, such as the 8 position, and others may be, for example, a 2-amino group. Suitable substituents include, e.g., alkynyl, cycloalkyl, aryl, alkynyl, arylcarbonyl, aryloxycarbonyl, amine, hydroxy, cyano, alkane, carboxyl, carboamido, carboxyl Esters, alkoxycarbonyl, aromatic or heterocyclic groups, and combinations thereof. Familiar with this skill. Reactive Tetracycline Basic Precursor Compounds and / or 9-Diazotetracycline Basic Compounds, 7- and / or Basic Compounds, 7- and / or 9-Bromotetracycline Basics

Page 13 548257 V. Description of the invention (π)

-ΝΗο where Zi, Z2, and Z3 are each Η or

Where R2 and 1-3 are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkynyl, arylWeiyl, alkoxyweiyl, aryloxychiyl, Amine, mesityl, cyano, alkoxy, aryloxy, alkyl, alkoxy, aryloxyweiyl; or R2 and I together form a substituted or substituted 5 to 15 atom or Unsubstituted carbocyclic or heterocyclic ring; and Ri is Η or 0H — In another embodiment, R2 is hydrogen, and R3 is R4 is hydrogen, cyano, or Ci-G alkoxy. In another specific embodiment, h and 1? 2 together form a substituted or unsubstituted carbocyclic or heterocyclic ring having 5 to 15 atoms in the ring; the ring may be a conjugated or unconjugated aromatic ring system. Good for (: 5 to (: 8 -r4 where

Page 16 548257 V. Description of the invention (12) Suitable substituents for Z include

In another state, the present invention also provides a method for preparing a substituted tetracycline # compound, desirably a 7, 9 or 13-substituted compound, and in another state, a tetracycline compound prepared by this method. Such compounds can be prepared substantially as described in the schematics described below. In the discussion of the sketch, various substituents are the same as defined above; n Rn includes R2 and 1-3. For the purpose of illustration, deoxytetracycline is also described as the base π tetracycline compound, although it should be understood that a wide variety of tetracycline compounds can be used in the same manner. For example, the basic tetracycline compound substituted at the 7-, 9-and / or 1 3-positions may suitably be tetracycline hydrochloride; chlorotetracycline; dechlorotetracycline; deoxytetracycline; chelomycin; dimethylamine tetracycline; Tetracycline; Intermediates, etc.

Page 17 548257 V. Description of the invention (13) The tetracycline compounds of the present invention can be easily prepared by following the methods outlined below. Scheme I relates to the preparation of a tetracycline compound, which is prepared from the starting compound of Formula 1. It is a tetracycline antibiotic commonly used in clinical practice and is called deoxytetracycline. It has been found that 6- (substituted) 5-hydroxy-6-deoxytetracycline (1, r2 = ch3, deoxytetracycline) or its mineral acid salt is soluble in concentrated acids as suitable solvents, such as H2S04, and A confirmatory reagent reaction, such as sodium wall acid or If, to make 7 and 9-nitrotetracycline derivatives (male, this). These compounds are separated by a variety of techniques. The preferred method is preparative HPLC on C18-reverse phase silica gel using a binary gradient system containing a phosphate buffered ethylenediaminetetraacetic acid, sodium salt (EDTA), and a methanol gradient. , Or an acetonitrile gradient of 0.1% trifluoroacetic acid. These barrier compounds are easily reduced to typical amine functional groups using typical reducing agents such as hydrogen, and transition metal catalysts, starting oxides, carbon, or the like, to produce 7-NH2 and 9- Off 2 tetracycline (4, beans). Alternatively, 7-NH2 tetracycline (deoxytetracycline) can be prepared by reductive alkylation of 7- (N, N dicarboxybenzyloxyhydrazine) tetracycline (as detailed in US Patent 3, 4 83, 2 51, which Incorporated herein by reference). Compounds exhibiting aniline functional groups can use nitrous acid (Η Ο N 0) or organic reagents such as butyl nitrite, which are liable to form diazonium salts (such as hydrochloride or tetrafluoroborate) (beans). The diazotization reaction proceeds near quantitative yield. Suitable reactive tetracycline-based precursor compounds in the form of diazonium salts can now be used with organic palladium catalysts, and species that cause carbon-carbon bond formation between tetracycline reactant intermediates and selected reactive organic substituent precursors, chemistry Ground mismatch. Use transition metal catalysts such as CuCl2 (Meerwin reaction) and palladium catalysts such as chloride, palladium acetate, or other above-mentioned catalysts to make the 7 and 9 positions of tetracycline

Page 18 548257 V. Description of the invention (14) The substituted derivative is preferably palladium acetate. The reaction is generally carried out in a polar solvent with a residual amount of mineral acid- (HC 1, 0.1%), such as DMS 0, water, and alcohol, to react substituted or unsubstituted aromatic or heteroaromatic, alkyl, Alkenyl, or alkynyl substructures to make the desired substituted compounds. Non-polar solvents can also be used for the reaction. It is known that transition metal compounds such as Krypton and copper halides react with aromatic diazonium salts to form complexes which can be further reacted. Transition metal halides are used as catalysts to facilitate the formation of carbon-carbons through carbon-carbon additions-oxidation-reduction addition of carbon substructures (double bonds and other structures that exhibit 7Γ-bonds), and become electron-deficient nitrogen diazo reactive groups. For example, when a suitable olefinic hydrocarbon forms a reactive coordination complex in a reaction system, palladium-catalyzed carbon-carbon bond formation is liable to occur. This is followed by the insertion of f into the carbon α-bond to produce a tertiary complex. Catalysts such as Ibarth are recycled and regenerated via, for example, / 3-hydride removal, thereby forming carbon-carbon covalent bonds. Using these conditions, molecular substructures exhibiting a 7Γ-bond system, such as olefins or acrylates or any of many other compounds exhibiting double bonds, are susceptible to reactive tetracycline-based precursor compounds (e.g., tetracycline aromatic diazonium salts). ) Aromatization. Other transition metal-catalyzed reactions such as transmetallation and, for example, carbon monoxide insertion reactions are also intended (for example, transition metal-catalyzed reactions can be found in Hegedus above). Using palladium complexes and suitable reactive species, homogeneous catalysis of carbon-carbon bond formation is possible. The use of tetracycline, for example, deoxytetracycline or diamine tetracycline, produces a reactive diazo functional group in the D ring, and the reactive plus stomach> products can be obtained from structurally different chemical families. Therefore, reactive tetracycline-based precursor compounds, such as

Page 19 548257 V. Description of the invention (15) Nitrogen salts, that is, for example, reactive and functional groups at positions 7 and 9 of the tetracycline molecular group can be similarly reacted with olefins in the presence of appropriate transition metal catalysts , Substituted olefinic hydrocarbons, ethylene monomers, aromatic and heteroaromatic reactive groups (unsubstituted or substituted) react to produce 7-(substituted) and · 9-(substituted) tetracyclines in good yields ( Dad, once, sketch I). For example, the 7-position via the reaction sequence (11) produced by the nitration of dimethylamine tetracycline (M) into a 9-N02 derivative (j_i), followed by catalytic reduction to a 9-NH2 'derivative (11) and then diazotization Instead of the tetracycline 9-diazonium salt, it can also react with double bond compounds such as olefins and reactive-products and reagents to produce diamidine tetracycline derivatives of formula (II) (VII, sketch II). In a specific embodiment, the reaction products of the formula I and I I can further generate reagents and reagents, as described in the schematic II I-VI I, so as to act as an intermediate for the manufacture of other compounds that are not easily accessible. The 9-dilute substituted deoxytetracycline of formula I (this, 9) can be hydrogenated with a palladium or palladium on carbon catalyst under low pressure hydrogen to form a 9-alkyl derivative of deoxytetracycline ( U, sketch III). Similarly, the 9-alkenyl derivative (fluorene) of diamine tetracycline can also be reduced to an alkyl derivative () using a catalytic argonization method as shown in the schematic IV. 7 or 9 derivatives of deoxytetracycline of formula I (schemes I and III) can also be made by reacting with carboxylic acids when dissolved in strong acids such as anhydrous hydrofluoric acid or pinanesulfonic acid or trifluoromethanesulfonic acid. 7 and 9 substituted 5-ester derivatives of deoxytetracycline Qi, 19 (schematic V). 7 or 9 derivatives of deoxytetracycline of formula I (schemes I, II I and V) can also be reacted by 7 or 9 derivatives with formaldehyde and appropriate base (pyrrolidine) to form Mannich basic derivatives, And manufacturing Mannich test addition products (where, ^ 1,

Page 20 548257 V. Description of the invention (16) (Picture V I). The 9 derivative (II, sketch II) of the diamine tetracycline of formula II can also be prepared by reacting the 7 or 9 derivative with formaldehyde and an appropriate base (pyrrolidine) to form a Mannich test derivative, and to make a Mannich test Addition product (foot ^, sketch VI I). The tetracycline diazo reactive functional group produced in the sketch I can also be reacted with carbon monoxide in an alcohol in the presence of a transition metal catalyst such as palladium acetate to produce 7 and 9-carboxylic acid derivatives (0U in good yield). ), Which is easily esterified to produce 9-position tetracycline esters (schematic VIII). The diamine tetracycline diazo-reactive functional group produced in sketch II can also react with carbon monoxide in an alcohol in the presence of a transition metal catalyst such as palladium acetate to produce 9-carboxylic acid derivatives in good yield. It is easy to esterify and produce 9-position diamidine tetracycline carboxylate (U, sketch IX). Other reactions of 7 and 9 amine tetracycline via diazo functional groups are possible. Tetracycline arene diazonium salts also react with active fluorenyl compounds such as acetamidine acetate esters and their derivatives, active aromatic rings and unsubstituted and substituted olefins, acetylene, substituted acetylene, arylethylene, styrene , Conjugated diene, isoprene, vinyl ether, α, stone-unsaturated aldehydes and ketones, arylethylene and arylisoprene ketones, quinones, α, 10,000-unsaturated acids and their derivatives . All multiple-bond compounds are easily coupled to aromatic diazonium salts and are nucleophilic. Reactive tetracycline-based precursor compounds at positions 7 and 7 and 9 (hydrogenated derivatives of tetracycline, as shown in sketch X) also make tetracycline 7 and 9 derivatives. Aromatic substitution reaction of 7 and 9 halogen derivatives of deoxytetracycline (M, M) or tetracycline (^ _ ^, pull) with good yield by iodination, desertification or chlorination

Page 21 548257 V. Description of the invention (17) is described in, for example, J. Am. Chem. Soc., Hlavka, J.J., et al., '84, 1961, 1426-1430. The 7 and 9-position halogenated derivatives of tetracycline can be further produced in N-methylpyrrolidone by coupling iodoolefins or iodoaromatic hydrocarbons with thiophenone chloride or other transition metal catalysts, and in good yields. · Deoxytetracycline 7 or 9 position derivatives (M, or deoxytetracycline 7 or * 9 position derivatives (M, M). Tetracycline 1 3 position derivatives can be used in palladium chloride or other transition metal catalysts In the presence of alcohols such as fluorenol, phenylboronic acid and limonene tetracycline (11 of the · outer ring double bond reaction is prepared (schematic XI)), and tetracycline is produced in good yield. Prime 1 1 -benzene Derivatives (M). The following synthetic scheme is a description of the present invention: Read. Scheme I 7- (Substituted) -6-fluorenyl-6-deoxy5-Cycyltetracycline and 9- (Substituted) -6 -Methyl-6-Deoxy-5 -Ethyl tetracycline sketch II 9-(Substituted) difluorenamine tetracycline sketch 1117- (alkyl substituted) -6-fluorenyl-6-deoxy5 -hydroxytetracycline with 9- (Carbonyl Substitution) -6-methyl-6-deoxy 5-Hydroxytetracycline Outline IV 9-(Alkyl Substituted) Dimethylamine Tetracycline Outline V 7- (Carbonyl or Aryl Substitution 6-methyl Base-6 -Deoxy 5 -propoxytetracycline with 9- (alkyl or aryl substituted) -6-methyl-6 -Deoxy 5 -propoxytetracycline sketch VI 7- (alkyl or aryl Substituted) -6-methyl-6-deoxy5-hydroxytetracycline and 9- (alkyl or aryl substituted) -6-fluorenyl-6-deoxy5-hydroxy2- -carbohydrazone Amine substitution) Tetracycline sketch VII 9- (Carbonyl substitution) -2- (Carbonamino substitution) Diamine tetracycline

Page 22 548257 V. Description of the invention (18) Outline drawing VIII7- (Weiji or Weiji vinegar) -6-methyl-6-deoxy 5-Aminotetracycline and 9- (Retyl or Weiji ester)- 6-fluorenyl-6-deoxy 5-via radical 2-(carboxamino substituted) tetracycline sketch IX 9- (carboxy or carboxy ester) diamido tetracycline sketch X 7- (alkenyl or aryl)- 6-methyl-6-deoxy5-hydroxytetracycline and 9- (alkenyl or aryl) -6-fluorenyl-6-deoxy5-hydroxytetracycline, X 9- (alkenyl or aryl) -6_Defluorenyl-6-deoxytetracycline, and 9- (alkenyl or aryl) -6-defluorenyl-6-deoxytetracycline Schematic XI 13- (substituted) -6-fluorenylene -5-hydroxy-6-deoxytetracycline

Page 23 548257

Page 24 548257 V. Description of the invention (20) Sketch II H3c, / CH3

13 Sketch III

Page 548 257

Page 548 257

Page 548 257

Page 548 257

Page 548257 V. Description of the invention (25) The compound of the present invention is targeted at susceptible microorganisms, such as bacteria, fungi, rickettsia, parasites, etc., and combines such microorganisms (including tetracycline-sensitive, Bacteria and tetracycline bacteria) are effective. The specially optimized 5 substances of the present invention are directed against tetracycline-sensitive E. coli, S. aureus and E. faecalis strains, ^ E. Col i pHCMl, S. aureus RN4 2 50, and E. faeca 1 isp Μ V 1 5 8 'presents a minimum inhibitory concentration (μIC) value of about 10 μg / ml or less, more preferably about 1 μg / ml or less for 24 hours. Preferred compounds of the invention also include tetracycline sensitive E. col i ^ S. "

Aureus and E. faecalis strains, such as E. coli D31m4, Saureus RN450, and E. faecalis ATCC9790, are presented here. As discussed above, the present invention provides a method for treating microbial infections in human diseases. This method generally comprises the administration of a therapeutically effective amount of a compound of the invention to a patient. An animal is preferably a ^ λ 4 thing, and a., ^ Αα " * 1 a horse is a human primate. In the method of treatment of the present invention,-it is administered to the patient alone, or more often, the compound of the present invention can be suspended as the compound of the present invention. A, M 1 early-formers (that is, suitable for parenteral administration) Tian Wei 'Yi He Xi 4d ax 丄-intestines, oral or other hope # ~ consciously endanger harmful compounds with active compounds; 5 Temple η μ * ^ ^ music, but π ^ Λ ^ ^ ri ^ Its recipients are harmless shovel and no hot air carrier substance), a part of the pharmaceutical composition / learning acceptable medicines including but not injurious. Oil, polyethylene. Alcohol, gelatin, Α 丨 = a solitary cold night, alcohol, select \ a #. ^ Lingli sugar, amylose, hard 胪 # broken vegetable stone evening acid: seed chain burning classic, flavor oil, fatty acid : J, talc, western purpose, petroleum ether fatty acid purpose, methyl_cellulose,

548257 V. Description of Invention (26) and so on. Pharmaceutical products can be sterilized and, if necessary, adjuvants that do not react with the active compound, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, Seasonings and / or aromatic substances. At least many of the compounds of the present invention can be suitably administered to patients in deprotonated and water-soluble forms, for example, pharmaceutically acceptable salts of organic or inorganic acids, such as hydrochloride, sulfate, hemisulfate, phosphate, nitric acid Salt, acetate, oxalate, citrate, maleate, mesylate and the like. Where appropriate, where acid groups are present on the compounds of the present invention, pharmaceutically acceptable salts of organic or inorganic bases, such as ammonium salts, or organic amine salts, or metal or earth metal salts, such as potassium, can also be used. , Word or sodium salt. In accordance with the present invention, a therapeutic compound can be administered to a patient by any route. Local (including intracerebral, oral or sublingual) and parenteral (including intraperitoneal, subcutaneous, intravenous or submuscular injections) are usually preferred. For gastrointestinal applications, it is particularly suitable as a solution, preferably an oily or aqueous solution, and as a suspension, emulsion or implant, including suppositories. Therapeutic compounds are formulated in sterilized form in multiple or single-dose formats, such as dispersed in a fluid carrier, such as a sterile saline solution or a 5% saline dextrin solution commonly used for injection. For enteral applications, it is particularly suitable for tablets, sugar balls or capsules with talc and / or carbohydrate carrier binders, etc. The carrier is preferably lactose and / or corn starch and / or sweet potato starch. Syrups, alchemists and the like in which a sweetening vehicle is used can be used. Sustained release compositions can be formulated that include the active ingredient in which they are protected by different degradable coatings, for example, by microencapsulation, multiple coatings, and the like.

Page 31 548257 V. Description of the invention (27) For topical topical carriers, including water, glycerol, or mineral oil isopropyl ester, polywater, laurel, such as antioxidants in addition to human applications, such as chickens, ducks, it should be understood, Known for the specific application site used, the most suitable application rate is usually, the reference dose of the drug used for vegetarian treatment is per kilogram per kilogram per kilogram, and the local dose is administered. For example, the application and the therapeutic compound should be understood. It may be suitably incorporated in a pharmacologically inert such as a gel, ointment, lotion or cream. This topical carrier pack contains alcohol, propylene glycol, fatty alcohol, triglyceride, and fatty acid. Other possible topical carriers are liquid paraffin, ethylene glycol palmitate, 95% ethanol, 5% polyethylene oxide monoglyceride in sodium sulfate and 5% water. In addition, if necessary, additives, humectants, viscosity stabilizers and the like can be added. For the treatment, the treatment method of the present invention also has significant veterinarians for treating livestock such as cattle, sheep, goats, dairy cows, pigs, etc .; poultry such as goose, turkey; horses; The actual preferred amount of active compound for a particular treatment will vary depending on the particular compound, the particular composition being formulated, the mode of application, the particular features, and the like. Those skilled in the art use the above-mentioned dosimetry test to easily determine the rate for a specific application agreement. The compound of the invention under treatment can be used to administer a prior art quadruple dose to a patient. For example, see Physicians' Desk 3 For example, one or more compounds of the present invention are suitable for the effective weight of the recipient in the range of 0.01 to 100 mg per day, which is better than the range of 0.1 to 50 mg per day in the weight of the recipient. Body weight ranges from 1 to 20 mg per day. The required dose may be administered as appropriate, or as many doses at appropriate intervals as the day, such as 2 to 5 doses, or other suitable schedules. , Should pay attention to the normal and customary matters about the administration of tetracycline

Page 32 548257 V. Description of the invention (28) Efficiency under the use environment. Especially when used in humans, the physician should take all reasonable precautions and toxic effects. Therefore, conventional methods and negative reactions recognized by inflammation, nephrotoxic blood changes, and items due to aluminum, calcium, and magnesium ions should be used to determine their biological treatment items in normal classes and animals to avoid habituation. The knowledge of each other appropriately considers gastrointestinal factors, high sensitivity reactions, and damage to absorption. Antibiotics in vitro of various compounds manufactured by Bio-Life Shishengming, inhibit the growth of bacteria at an appropriate temperature for 18 hours by using L-broth or M ue 1 1 er-H int ο η broth ί thus adjusting Mueller-Hinton Decoction of Yang Yang. National Commission for y Standards Document M 7-A 2, p. 10, 2nd edition, Vi 1 lanova (19 9 0) in Pennsylvania. Due to the ability to shed tetracycline and confer resistance, the tested organisms appeared to be susceptible to infection with four blue-positive and gram-negative bacterial species. Tetracycline-susceptible to efflux or ribosome protection or in vitro evaluation method The activity according to the present invention is evaluated as follows. Minimum inhibitory concentration The lowest drug concentration is determined by the soup dilution method. And, as described in Waititz, JA Clinical Laborator Volume, No. 8, Nos. 13-20, all the clinical strains used to protect against cyclin or tetracycline-resistant leather by the ribosome-protection mechanism are counteracted by drugs. . Table I-Description of the compounds Compound name Deoxytetracycline [4S- (4a, 12a α)]-4- (Diamido) -l, 4,4a, 5,5a, 6,11,12a-octahydro- 3,5,10,12,12a-pentahydroxy

__Figure

Page 33 548257 V. Description of the invention (29) -6-Amidino-1,11-dioxy-2-tetracarboxamidine diamidamine tetracycline [48- (43,123 «)]-4,7 -Fluorene- (diamidoamino) -1,4,4a, 5,5a, 6,11,12a-octahydro-3,10,12,12a-tetramethyl-1,11-dioxy-2 ~ • Teshin Carboamine A [43- (43,123 «)]-9- (nitro) -4- (diamido) -1,2,4a, 5,5a, 6,11,12a -Octahydro-3,5,10,12,12a -pentaethyl-6-fluorenyl-1,11-dioxy-2 -tetracarboxamide (9-nitro-6-deoxy-5 -Hydroxytetracycline) B [43- (43,123 ")]-9- (Amino) -4- (diamidoamino)-1,4, 4a, 5, 5a, 6, 11, 12a-octahydro -3, 5, 10, 12, φ 12a -pentahydroxy-6-fluorenyl-1,11-dioxy-2 -tetracarboxamide (9-amino-6-deoxy-5- Tetracycline) C [4S- (4a, 12aa)]-9- (diazo) -4- (dimethylamino) -1,4, 4a, 5, 5a, 6, 11, 12a-octahydro- 3, 5, 10, 12, 12a -pentahydroxy-6-fluorenyl-1,11-dioxy-2-tetracarboxamide (9-diazo-6-deoxy-5 -hydroxytetracycline ) D [4S- (4a, 12aa)]-9- [3,-(E) -acrylic acid] -4- (dimethylamino) -1,2,4a, 5,5a , 6, 11,12a-octahydro-3,5,10,12,12a-pentaecyl-6-fluorenyl-1,11-dioxy-2-tetrahydrocarbamidine (9- [3 '- (E) -Ethylacrylic acid] -6-deoxy-5 -hydroxyl

Page 548 257

V. Description of the invention (30) E Tetracycline) [4S- (4a, 12a α)]-9-[3 '-(E) -butyl acrylate]-4- (Diamido) -1,4,4a , 5,5a, 6,11,12a-octazine_3,5,10,12,12a-pentamentyl-6-fluorenyl-1,11-dioxy-2-tetracarboxamide (9- [3 '-(E) -Butyl Acrylate] -6-Deoxy-5-Cyclocycline) [4S-(4a, 12a α)]-9- [3'-(E) -Acrylic acid Butyl ester]-4, 7-fluorene (diamidoamino) -1, 4, 4a, 5, 5a, 6, 11

, 12a-octahydro-3, 10, 12, 12a-tetrahydroxy-1,11-dioxy-2-tetracarboxamide I

G (9-[3 '-(E) -butyl acrylate] -diamine tetracycline) [4S- (4a, 12a α)]-7-[4, -C phenylphenyl] -4- (dimethylformate Amine)-1,4,4a, 5,5a, 6,11,12a-octahydro-3, 10, 12, 12a-tetrahydroxy-1,11-dioxy_2-tetracarbinol 7-[4'-Cl -Phenyl) tetracycline [4S-(4a, 12a α)]-7 -phenyl-9-phenyl-4- (diamidoamino) -1,4,4a, 5 , 5a, 6,11,12a -octahydro-3, 10, 12, 12a_ tetrahydroxy-1,11-dioxy-2 -tetrahydrocarbonamine 7, 9 -diphenyltetracycline 1 [4S- (4a , 12aa)]-13- [4'-fluorenylphenyl] -4- (diamidoamino) -1,4,4a, 5,5a, 6,11,12a-octahydro

Page 35 548257 V. Description of the invention (31) -3, 5, 10, 12, 12a-pentahydroxy-6-methylene-1,11-j oxy_2 ~ tetrashenyl carbonamine 13-(4 '-Fluorenylphenyl) -6-deoxy-6-methylene-5-meryltetracycline [4S- (4a, 12a α)]-13- (3, -Weiylphenyl) -4- (:) Methylamine-1, 4, 4a, 5, 5a, 6, 11, 12a- octahydro-3, 5, 10, 12, 12a-pentahydroxy-6-methylene-1,11-dioxy Carboxyl-2 -tetracarboxanamine 13- (3'-kilylphenyl) -6-deoxy-6-methylene-5-tetracycline [4S- (4a, 12aa)]-13- [4'_ Ethoxyphenyl]-4- (diamidoamino) -1,4,4a, 5,5a, 6,11,12a -octahydro-3,5,10,12,12a_ pentamer group -6_ methylene-1,11-dioxy-2 -carboxamidine 13- (4'-ethoxyphenyl) -6-deoxy-6-fluorenylene-5 -meridyl tetracycline

Page 36 548257 V. Description of the invention (32)

Table II Antibacterial activity of tetracycline derivatives catalyzed by transition metals DEFGHIJK E.coli ML308-225 Tcs 0.78 25 6.25 > 50 > 50 > 50 > 50 > 50 12.5 E.coli D1-299 Tcr 25 > 50 > 50 > 50 > 50 > 50 > 50 > 50 > 50 E.C0 // D1-209 Tcr 50 > 50 > 50 > 50 > 50 > 50 > 50 > 50 > 50 E.coli D31m4Tcs 1.56 > 50 3.12 3.12 0.78 1.56 6.25 > 50 12.5 E.coli D 31m4 pHCM1 Tcr 25 > 50 6.25 6.25 0.78-25 > 50 > 50 S. aureus RN450 Tcs < 0.098 3.12 0.78 1.56 < 0.098 1.56 < 0.098 0.39 0.195 S. wamerii Tcr ATCC12715 50 > 50 6.25 3.12 < 0.098 0.78 12.5 > 50 12.5 S. aureus RN4250 Tcr 25 > 50 6.25 3.12 < 0.098 0.78 12.5 > 50 6.25 S. aureus MRSA5 Tcr 6.25 > 50 0.39 3.12 0.195 0.78 6.25 > 50 6.25 E. htrae ATCC9790 Tcs 0.195 3.12 3.12 3.12 < 0.098 0.78 0.39 3.12 0.39 E. hirae 9790 with PMV158 Tcr 6.25 12.5 6.25 3.12 < 0.098 0.39 3.12 > 50 6.25 E. hirae 9790 with PAM211 Tcr 6.25 > 50 6.25 3.12 < 0.098 1.56 12.5 > 50 3.1 2

Tcs = susceptible to tetracycline Tcr = anti-tetracycline Experiment The compounds of the present invention can be prepared as shown in sketches I to I X above, and / or as described below.

Page 37 548257 V. Description of the invention (33) In sketch I, deoxytetracycline is dissolved in cold concentrated sulfuric acid, and an equal amount of potassium nitrate is added. The reaction temperature is maintained in the range of 0 to 5 ° C for 1 to 3 hours, and 7 and 9-nitro-6-substituted-5 -hydroxytetracycline of formula IV are produced. These intermediates with appropriate chemically reactive functional groups can be reacted with a wide range of reducing agents, such as Pt02 or hydrogen, and palladium or platinum catalysts, to produce compounds of general formula IV. The diazonium salts of 7 and 9 amine derivatives are manufactured by the action of nitrite (sodium nitrite, butyl nitrite or the like) and intermediates used without further purification. Example 1 [4S-(4 a, 12a α)]-9- (nitro)-4- (dimethylamino) -l, 4,4a, 5,5a, 6,11,12a-octazine-3 , 5,10,12,12a -pentadecyl-6-fluorenyl-1,11-dioxy-2 -tetracarbasamine in 1.0 g of deoxytetracycline hydrochloride in 10 ml of concentrated sulfuric acid To the ice bath solution, 0.23 1 g of potassium picoate was added. The reaction was stirred under ambient atmosphere for 1 hour. The mixture was then poured into 150 grams of ice and extracted with n-butanol to produce a solid, and dried to provide 0.9 grams of the desired product such as a yellow-green solid. MS (FAB): m / z 490 (M + H). NMR (CD3OD): 5 7.50 (d, 1H, J = 8.07 Hz, Η-8); 6.86 (d, 1H, J = 8. 07 Hz, H-7); 4.44 (bs, 1 Η, H-4 ); 3.62 (dd, 1H, J = 11. 42; 8.35 Hz, H-5); 2.95 (bs, 6H, NMe2); 2.81 (d, 1H, J-11.45 Hz, H-4a); 2.71 £ ( dq, 1H, Jl 2. 41; 6.5 Hz, H-6); 2.53 (dd, 1H, JII '12.23; 8.20 Hz, H-5a); 1.51 (d, 3H, J = 6.78 Hz, CH3

Page 38 548257 V. Description of the invention (34) Example 2 [4S- (4 a, 12a α)]-9- (amino)-4- (diamidoamino) -l, 4,4a, 5,5a , 6,11,12a-octazine-3,5,10,12,12a_ pentadecyl-6-methyl-1,11-dioxy-2 -tetracarbadonamine in a 200 ml hydrogenation bottle 1.0 g of the product obtained from Example 1, 40 ml of methanol, 1 ml of concentrated HC1, and 100 mg of 10% palladium were added to the carbon. The mixture was subjected to 30 p s i of hydrogen for 3 hours using an argonization device. The catalyst was filtered and the filtrate was dried to provide 0.9 g of dihydrochloride as a yellow solid. MS (FAB): m / z 460 (M + H). NMR (CD3OD): d 7. 54 (d, 1 H, J = 8.08 Hz, H-8); 6.88 (d, 1 H, J = 8. 08 Hz, H-7); 5.16 (dd, J = 1 0.44; 7 • 94 i Hz, H -5); 4. 44 (bs, 1H, H-4); 3.74 (d, 1H, J = 2.07 Hz, H-4); 3 04 (bs, 6H, NM e 2); \ \ 90 (dd, 1 H ,. J = 7.94; 2. 07 H z, H-4a); 2. 72 (dq, 1H, J = 1 2. 3 1; 6.56 Hz, H -6); 2. 6 1 (dd, 1H, J = 12.31; 10. 44 Hz, H-5a); 2. 54 (Q, 2H, J 2 7.48 Hz, CH 2-.); 1.44 (bs, 9H, CM 6 3); 1. 29 (d, 3H, J = 6. 56 H z, CH〇);; 1.20 (t, 3H, J- 7.48 Hz, C-CH3). Example 3 [4S- (4 a, 12a α)]-9- (diazo) -4- (dimethylamine) -l, 4,4a, 5,5a, 6, 11,12a-octahydro-3, 5, 10, 12, 12a -Pentahydroxy-6-methyl-1,11-dioxy-2-tetramethylene amine carbonate 10 ml round bottom flask has 100 mg of the product obtained from Example 2, And dissolved in 4 ml of 0.1 N methanolic hydrochloric acid. The solution was cooled to 0 ° C and added with stirring

Page 39 548257 V. Description of the invention (35) 35 microliters of butyl nitrite. After 1 hour, the bright red reaction mixture was added dropwise to 100 ml of cold anhydrous diethyl ether. The product was collected by transition, washed with ether, and dried in a vacuum dryer to produce 73 mg of a diazonium salt such as an orange solid. MS (FAB): m / z 472 (M + H) ° lH NMR (CD3OD): d 7,55 (d, 1H, J = 8.08 Hz, H-8); 6.86 (d, 1H, J-8.08 Hz , H-7); 5.13 (dd, J = 10.44; 7.94

Hz, H-5); 4.41 (bs, 1H, H-4); 3.72 (d, 1H, J = 2.07

Hz, H-4); 3.04 (bs, 6H, NCH3); 2.90 (dd, 1H, J = 7.94; 2.07 Hz, H-4a); 2.70 (dq, 1H, J = 12.31; 6.56

Hz, H-6); 2.61 (dd, 1H, J = 12.31; 10.44 Hz, H-5a); # 2.2 (m, 6 H, J 2. 7.48 Hz, Acetyl); 1.44 (bs, 9 H, C ( CH3) 3); 1.29 (d, 3H, J-6.56 Hz, CH3); 1.20 (t, 3H, J = 7.48 Hz, C-CH3). General procedure for olefination. To a solution of 0.1 g of 9-diazo compound in (wet or anhydrous) methanol, 0.05 equivalent of acetic acid was added. The reaction mixture was stirred at room temperature for 5 minutes, and 2 equivalents of the desired olefin was added. Stir in the surrounding atmosphere for 18 hours or then HPL C. Stirring can also be continued in an N2 atmosphere. When completed, the catalyst was filtered and the filtrate was dried to produce a crude product. The purified product was isolated by preparative reverse-phase HPLC using a gradient of methanol and a phosphate buffer. Example 4 ❹ [4S-(4 a, 12a α)]-9-[3,-(E) -Acrylic acid] -4- (dimethylamino) -1,4,4a, 5,5a, 6 , 1 1,12a -octahydro-3,5,10,12,12a -pentacyl-6-

Page 40 548257 V. Description of the invention (36) fluorenyl-1,11-dioxy-2 -tetracarboxamidine MS (FAB): m / z 515 (M + H). Example 5 [4S- (4 (2,12a 6〇]-9- [1,-(E)-(2, -phenyl) ethenyl]-4- (diamidoamino) -1,4, 4a, 5,5a, 6, 11, 12a-octahydro-3, 5, 10, 12, 12a-pentahydroxy-6-fluorenyl-1,11-dioxy-2-carboxamidine MS ( FAB): m / z 54 7 (M + H). Example 6 [4S- (4 a, 12a α)]-7- [3 '-(E)-Butyl acrylate] -4- (dimethylamino ) -1,4,4a, 5,5a, 6,11,12a_octazine-3,5,10,12,12a-pentamentyl-6-methyl-1,11-dioxy-2-tetra Examine the general procedure of carbamidine aromatization. To a solution of the 9-diazo compound in methanol is added 0.10 equivalents of palladium acetate. The mixture is stirred at room temperature for 5 minutes, and 2 equivalents of arylboronic acid are added. After 6 hours, the catalyst was filtered and the filtrate was dried. The crude product was purified by preparative reverse-phase HPLC using a gradient of methanol phosphate buffer. General procedure for carboxylation. Included 2 rubber septa, vacuum source, and stirred A 3-neck round-bottomed flask was charged with 100 mg of diazonium compound, 6.0 mg of palladium acetate, and 10 ml of anhydrous dimethylformamide. The reaction vessel was evacuated and CO was passed through a syringe. mixing 1 hour. The mixture was stirred for another 2 hours, and then the solvent was removed in vacuo to give a crude product. The title compound was isolated by preparative C18 reverse phase HPLC using a binary solvent gradient. Example 7 [48- (4 ^ 123 ^ 〇] -9- (Carboxy) -4- (diamidoamino) -1,4,43,5,

Page 41 548257 V. Description of the invention (37) 5a, 6,11,12a_ Octa-3,5,10,12,12a-Pentadecyl-6-fluorenyl-1,11 -dioxy-2-tetra Carbenamine MS (FAB): m / z 489 (M + H). General procedure for hydrogenation. The compound was prepared by dissolving 0.1 g of Example 4 in 10 ml of methanol, adding 0.1% of concentrated HC1 and 10 mg of 10% palladium on carbon. The mixture was hydrogenated at 40 psi for 6 hours on a Parr device at room temperature and monitored by HPLC. The resulting crude product was subjected to semi-preparative binary solvent chromatography on the C18 reverse phase to produce the desired product. General procedure for 7-position olefination. To a solution of 0.1 g of the 7-diazo compound produced in a similar manner as described in Examples 1 and 2 in wet methanol, 0.05 equivalent of palladium acetate was added. The reaction mixture was stirred at room temperature for 5 minutes, and 2 equivalents of the desired olefin was added. Stirring was continued in the surrounding atmosphere for 18 hours, followed by ΗPLC. Upon completion, the catalyst was filtered through C e 1 ite and the filtrate was dried to produce a crude product. The crude product was purified by preparative reverse phase LLC using a gradient of methanol and phosphate buffer. Example 8 9-Phenyldiamidamine tetracycline [43- (4〇:, 1230:)]-9- (phenyl) -4,7-fluorene (diamidino) -1,4,4a, 5 , 5a, 6, 11,12a-octahydro-3, 5, 10, 12, 12a-tetrahydroxy-6-methyl-1,11-dioxy-2-tetracarboxamide use 0.1 1 0 0 g of 9-aminodimethylamine tetracycline and similar reagents and conditions found in Example 5 were used to prepare the compound. The reaction was stirred under a nitrogen atmosphere overnight, and the solvent was removed in vacuo to produce 0.06 g of a crude product. Chromatography using C18 reverse phase preparation and binary solvent system, followed by extraction of the product into butanol

Page 42 548257 V. Description of the invention (38) and the product was evaporated in vacuo to give 0.07 g of the desired product as a yellow solid. MS (FAB): m / z 571 (M + H). Example 9 7- Etooxytetracycline 30.0 ml of concentrated sulfuric acid was added with stirring to 1000 g of deoxytetracycline hydrochloride hemihydrate, and the solution was cooled to 0 ° C. 0.93 g of N-iodosuccinimine was added to the solution in portions over 1 hour, and the reaction was monitored with ΗPLC and TLC to confirm completion. The solution was poured into 250 ml of ice water, extracted three times with butanol, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to provide 1.13 g (89%) of the title compound and dark yellow crystals. MS (FAB): m / z 5 8 7 (M + H) LH NMR (methanol d-4, 3 0 0 MHz) 5 7.94 (d, J = 8.19 Hz, 1H), 6.78 (d, J-8.18 Hz, 1H), 4.13 (s, 1H), 3.53 (m, 3H), 2.85 (s, 7H), 2. 66 (m, 4H), 2.41 (s, ih), 1.49 (d, J-6.52 Hz , 3H), 0.95 (t, 1 = 1.21 Hz, 2H) 〇 Example 1 〇 and 11 17-iodine tetracycline and 7, 9-diiodine tetracycline 3.0 ml of concentrated sulfuric acid was added to the stirring with 1 · 〇 〇g of mountain tetracycline hydrochloride hemihydrate, and the solution was cooled to 0 ° C. 1.09 g of N-discurcum white sulfonimine was added to the solution in portions over 1 hour, and the reaction mixture was monitored with HCl and LC. The reaction mixture was poured into 250 ml of ice water, extracted three times with butanol, and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to yield 787 mg (61%) of 7-iodotetracycline and 291 mg (22%) of 7 l

Page 43 548257 V. Description of the invention (39) Diiodine tetracycline, such as yellow and dark yellow crystals. · MS (FAB): m / z 587 (M + H). 7-iodine tetracycline 4 NMR (methanol d-4, 300 MHz) 57.89 (d, J-8. 86 Hz, 1H), 6.67 (d, 8.87 Hz, 1H), 3.56 (s, 1H), 3.03 (s, 2H w), 2.84 (s, 6H), 2.46 (m, 2H), 1.63 (m, 4H), 0.95 (m, 2H) ° with 8 (? 8 6): 111/2 667 (^ 1 +} 〇7,9-Diiodine tetracycline 1 H NMR (shared d-4, 300 MHz) 5 8.35 (s, 1H), 3.78 · (s, 1H), 3.33 (s, 2H), 2. 88 (s, 7H), 2.41 (m, 2H), 1 · 41 (m, 5H). Example 1 2--General coupling step Φ 7-4'-Cl -phenyltetracycline will be 100 mg of 7- Iodosan tetracycline or 7-iododeoxytetracycline (0.18 mM) and 4 mg of it? (1 (0 eight (:) 2 was added argon degassed solution of methanol, then 200 μl of 2M Na2C03. The resulting solution was in the chamber Stir gently for 10 minutes. Dissolve 4'-C1-benzylboronic acid (58 mg, 0.37 mM) in 1 ml of methanol, add iodotetracycline and degas the reaction flask 3 times with argon. The reaction is stirred at room temperature for 15 minutes And then heated to reflux for 18 hours. The solution was cooled, filtered, and the solvent was removed under reduced pressure. The crude product was purified by C 1 8 reverse phase chromatography to produce 23 mg of the product as dark yellow crystals. MS (FAB): m / z ( M + H) 5 2 5.1 8 5 2 NMR (methanol d4, 3 0 0 MHz) 5 7.3 5-7. 44 (m, 4H), see

7.21-7.24 (d, 1H), 6.8 5 -6.8 8 (d, 1H), 3.55 (s, 1H), 2.88 (s, 6H), 2.47 (m, 2H), 1.52 (m, 2H)

Page 44 548257 V. Description of the invention (40) Example 1 3 7,9-Diphenyltetracycline MS (FAB) m / z (M + H) 5 6 7. 2 54 5 4 NMR (Methanol d4, 3 0 0 ΜΗ z) 5 7. 2 2-7. 8 5 (m, 1 1H), 4.02 (m, 1H), 3.53 (s, 1H), 2.86 (br s, 6H), 2.41 (m, 2H), 1.52 (m, 2H) Example 1 4 7-(4-Fluorophenyl) Tetracycline MS (FAB): m / z 509 (M + H) ^ NMR (methanol d- 4, 3 0 0 MHz) 5 7.41 (d, J-8. 61 Hz, 1H), 7.30 (td, J = 6.87, 2.16 Hz, 2H), 7.16 (td, J = 6.84, 2.11 Hz, 2H), 6.89 (d, J = 8.59 Hz, 1H), 3.56 ψ (s, 2H), 2.91 (s, 7H), 1.52 (m, 4H), 0.95 (m, H). Example 1 5 7-(4 -Nitrophenyl) tetracycline MS (FAB): m / z 536 (M + H) ^ NMR (shared d- 4, 3 0 0 MHz) 5 8.28 (d, * 1 2 8.50, 2H), 7.52 (d, J-8.52, 2H), 7.42 (d, J-8.64, 1H), 6.93 (d, J = 8. 65, 1H), 3.51 (s, 2H), 6.73 (s, 7H), 1.50 (m, 5H), 0.92 (m, 2H) ° Example 1 6 7-(2 -pyridinyl) deoxytetracycline MS (FAB): m / z 522 (M + H) M NMR (Methanol d-4, 300 MHz) 5 8. 62 (s, 1H), 7. 94 (m, 2H), 7.49 (m, 1H), 7.40 (m, 1H), 6.94 (m, 1H),

Page 45 548257 V. Description of the invention (41) 4.21 (s, 1H), 3.56 (m, 2H), 2.91 (s, 7H), 2.70 (m, 3H), 1. · 038 (s, 3H), 0 · 92 (m, 2H). Example 1 7 7-Vinyltetracycline MS (FAB): m / z 471 (M + H) ^ NMR (methanol d-4, 300 MHz) 57.65 (d, J-8. 79 Hz, 1H), 6.80 ( d, J = 8.76 Hz, 1H), 5.56 (d, J = 18.42 Hz, 1H), 5.25 (d, J-12.15 Hz, 1H), 3.84 (s, 1H), 3.19 (m, 2H), 2.98 ( s, 6H), 2. 82 (m, 1H), 2.32 (m, 2H), 0.92 (m, 1H). Equivalents Familiar with this art should understand or be able to determine the use of many types of equivalents that do not exceed the specific steps described herein for fixed experiments. These products are deemed to be within the scope of the present invention and are covered by the scope of the following patent applications. The contents of all references, issued patents, and published patent applications listed in this application are hereby incorporated by reference. < 1

Page 46

Claims (1)

548257 Preparation of the organic reactive organic substituent and transition metal chemical intermediate family, vanadium group, chromium group, aluminum group and germanium , Titanium group, group, zinc group, aluminum 3. The catalyst is organic according to the application 4. The chloride is included according to the application, (dba) 3-CHCl3 5. The copper is included according to the application, 6. The CuCl is included according to the application 2. 7. According to the application fee for cyclin-based precursors: 89118818 A method of substituted tetracycline compounds, which comprises contacting a trans-based precursor with a reactive tetracycline-based precursor in the presence of a tetracycline compound substituted with a substituent It is a reactive tetracycline chemical complex, forming a reaction, wherein the transition metal catalyst includes a town group, a number group, a titanium group, a manganese group, an iron group, a cobalt group, a nickel group, a copper group, and a zinc group. A method for substituting a tetracycline compound, which comprises combining a reactive tetracycline-based precursor compound with a substituted precursor under the condition that the substituent is substituted for the tetracycline compound, wherein the transition metal catalyst includes a magnesium group, vanadium Family, chromium group, manganese group, iron group, cobalt group, nickel group, copper group and germanium group. The method of item 1 or 2 of the patent scope, wherein the transition metal Ib catalyst. The method of item 3 of the patent, wherein the organic palladium catalyst is palladium acetate, PdCl2 (PhCN) 2, PdCl2 (Ph3P) 2, Pd2, or a combination thereof. The method of item 1 or 2 of the patent scope, wherein the transition metal is rhodium, iron, surface, complex, copper, or nickel. The method of item 5 of the patent, wherein the transition metal contacts Cul2, ammonium (II) acetate, Rh6 (CO) 16, or a combination thereof. The method of item 1 or 2 of the patent scope, wherein the reactive tetra compound is tetracycline hydrochloride, chlorotetracycline, deuterated tetracycline O: \ 66 \ 66336-920227.ptc Page 48 548257 _ Case No. 89118818_ Qiaoyue date of the previous year __ Sixth, the scope of patent applications for cycline, deoxytetracycline, chelomycin, dimethylamine tetracycline, roar nail Tetracycline, lymetracycline, tetracycline, mesotetracycline, perylenetetracycline, hydroxytetracycline, guanidinetetracycline, glucotetracycline, penicillin V, mepteracycline, piperazine, tetraethylenetetracycline, benzyltetracycline, or meptera Tetracycline-based precursor compounds. 8. The method according to claim 1 or claim 2, wherein the reactive tetracycline-based precursor compound is selected from the group consisting of reactive dimethylamine tetracycline-based precursor compounds, reactive deoxytetracycline-based precursor compounds, and reactivity Tetracycline-based precursor compounds. 9. The method according to item 1 or 2 of the scope of the patent application, wherein the reactive tetracycline-based precursor compound is an aromatic diazonium salt, an iodine derivative, or an acid derivative of a tetracycline compound. 10. The method according to item 1 or 2 of the scope of patent application, wherein the reactive organic substituent precursor has at least one reactive 7Γ-bonding group. 1 1. The method according to item 10 of the scope of patent application, wherein the precursor of the reactive organic substituent is an alkenyl group, an alkynyl group, or an aromatic group. 1 2. The method according to item 11 of the scope of patent application, wherein the precursor of the reactive organic substituent is a baking group. 13. The method according to item 12 of the scope of patent application, wherein the precursor of the alkenyl-reactive organic substituent is ethylene monomer. 14. The method according to item 12 of the scope of patent application, wherein the precursor of the alkenyl-reactive organic substituent is substituted. '1 5. The method according to item 12 or 14 of the scope of the patent application, wherein the precursor of the alkenyl reactive organic substituent is a methylene compound, a conjugated diene, isoprene O: \ 66 \ 66336-920227.ptc Page 49 548257 _Case No. 89118818_ ^ January __ VI. Scope of patent application Dilute, diethyl ether, moth hydrocarbon, or its derivative. 16. The method according to item 11 of the scope of patent application, wherein the precursor of the aryl-reactive organic substituent is heteroaromatic. 1 7. The method according to item 11 or 16 of the scope of the patent application, wherein the precursor of the aryl-reactive organic substituent is arylboronic acid, iodoaryl, quinone, arylethyl, or styrene. 18. The method according to item 10 of the scope of patent application, wherein the reactive organic substituent precursor comprises a carbonyl group or a thiocarbonyl group. 19. The method according to item 18 of the scope of patent application, wherein the precursor of the reactive organic substituent is arylketene, arylisoprenylketone, α, / 3-unsaturated acid, α, cold -Unsaturated ketone, α, fluorene-unsaturated acid, or a derivative thereof. 20. The method according to item 11 of the scope of patent application, wherein the precursor of the alkynyl-reactive organic substituent is a substituted or unsubstituted acetylene. 2 1. A reactive tetracycline chemical complex comprising a reactive tetracycline-based precursor compound and a transition metal catalyst. 22. The reactive tetracycline chemical complex according to item 21 of the patent application scope, wherein the catalyst comprises an organic Iba catalyst. 2 3. The reactive tetracycline chemical complex according to item 22 of the scope of patent application, wherein the organic palladium catalyst comprises palladium chloride, palladium acetate, PdCl2 (PhCN) 2, PdCl2 (Ph3P) 2, Pd2 (dba) 3-CHC13, or a combination thereof 2 4. The reactive tetracycline chemical complex according to any one of the claims 2 1-2 3, wherein the transition metal catalyst comprises copper, rhenium, iron, silver, O: \ 66 \ 66336-920227.ptc Page 50 548257 _Case No. 89Π8818_ February 2, 2009 Amendment_ VI. Scope of Patent Application Chromium, scandium, or nickel. 25. The reactive tetracycline chemical complex according to item 24 of the scope of the patent application, wherein the transition metal catalyst comprises CuCl2, Cul2, rhodium acetate (I I), Rh6 (CO) 16, or a combination thereof. 2 6. The reactive tetracycline chemical complex according to any one of claims 21 to 23 in the scope of the patent application, wherein the reactive tetracycline-based precursor compound is tetracycline hydrochloride, chlorotetracycline, demethyltetracycline, deoxytetracycline , Chelomycin, dimethylamine tetracycline, D than tetracycline, lymetracycline, tetracycline, lutein, tetracycline, tetrocycline, tetrocycline, tetrocycline, meglotracycline, penicillin V, mepteracycline, mepidine Tetracycline, tetraethylenetetracycline, benzyl tetracycline, or piperidine-based precursor compounds. 27. The reactive tetracycline chemical complex according to any one of claims 21 to 23 in the scope of the patent application, wherein the reactive tetracycline-based precursor compound is selected from the group consisting of a reactive dimethylamine tetracycline-based precursor compound, a reaction Serotonin-based precursor compounds, and reactive tetracycline-based precursor compounds. 28. The reactive tetracycline chemical complex according to any one of claims 21 to 23 in the scope of the patent application, wherein the reactive tetracycline-based precursor compound is an aromatic diazonium salt, a moth derivative, or an acid of a tetracycline compound. derivative. 2 9. A 7-substituted tetracycline compound in which a substituent at the 7 position is connected to a -C-C- linkage, and wherein the substituent includes an aromatic or heteroaromatic moiety. 30. A 7-substituted tetracycline compound according to item 29 of the scope of application for a patent, wherein the compound is 7-4'-C1-phenyltetracycline. 31. The 7-substituted tetracycline compound according to item 29 of the scope of the patent application, wherein the compound is 7- (4-fluorophenyl) tetracycline. O: \ 66 \ 66336-920227.ptc Page 51 548257 Case No. 89118818 Amendment VI. Patent Application Range 3 2. According to the 7-Substituted Tetracycline Compound in the Patent Application Range No. 29-9, the compound is 7-(4 -Nitrophenyl) tetracycline. 33. The 7-substituted tetracycline compound according to item 29 of the scope of the patent application, wherein the compound is 7- (2-D than pyridyl) deoxytetracycline. 34. A 7-substituted tetracycline compound in which a substituent at the 7 position is connected to a _C-C- linkage, and wherein the substituent includes a -C = C- bond adjacent to the _C-C_ linkage. 3 5. The 7-substituted tetracycline compound according to item 34 of the scope of patent application, wherein the substituent is the following formula (Z) wherein 1 and 1 to 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkynyl, arylweilyl, alkynyl, aryloxy Weyl, amine, hydroxy, cyano, alkoxy, aryloxy, carboxy, alkoxy, aryloxy, or R2 and R3 together form a substituted or unsubstituted carbocyclic or heterocyclic ring. 36. The 7-substituted tetracycline compound according to item 35 of the scope of the patent application, wherein R2 is hydrogen, and R3 is ^, wherein R4 is hydrogen, cyano, or Ci-G alkoxy. ^ — «4 3 7. According to 7-Substituted Tetracycline Compounds in Item 35 of the scope of the patent application, wherein R 2 and h together form a substituted or unsubstituted carbocyclic or heterocyclic ring. 38. 7-Substituted tetracycline compound according to item 3 5 or 37 of the scope of patent application O: \ 66 \ 66336-920227.ptc Page 52 548257 _Case No. 89118818_year > Month _ ^ _ VI. Patent applications, where the ring contains 5 to 15 atoms. 39. The 7-substituted tetracycline compound according to item 35 or 37 of the scope of patent application, wherein the ring is a co-consumption or non-co-aromatic ring system. 40. The 7-substituted tetracycline compound according to item 35 of the scope of the application for a patent, wherein the compound is 7-ethoxytetracycline. 41. A 9-substituted tetracycline compound in which a substituent at the 9 position is connected to a -C-C- linkage, and wherein the substituent comprises an aromatic or heteroaromatic moiety. 4 2. A 9-substituted tetracycline compound in which a substituent at the 9 position is connected to a -C-C-linker 'and a substituent therein includes a -C-C-bond adjacent to the -C-C-linker. 43. According to 9-Substituted Tetracycline Compounds in Item 42 of the scope of patent application, wherein the substituent is the following formula \ νΓ ^ (Z) r2 where r2 and R are each independently hydrogen, alkyl, alkenyl, alkynyl , Cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxyl, cyano, alkoxy, aryloxy, carboxy, alkoxy, Aryloxy; or R2 and R3 together form a substituted or unsubstituted carbocyclic or heterocyclic ring. 4 4. According to 9-Substituted Tetracycline Compounds in Item 43 of the scope of the patent application, wherein R2 is hydrogen and R3 is ^, wherein R4 is hydrogen, cyano, or C5 alkoxy. 4 5. 9-substituted tetracycline compounds according to item 43 of the scope of patent application, O: \ 66 \ 66336-920227.ptc Page 53 548257 _Case No. 89118818_qa year January _ifi_ VI. Scope of patent application Where R2 and R3 together form a substituted or unsubstituted carbocyclic or heterocyclic ring. 46. The 9-substituted tetracycline compound according to item 4 3 or 45 of the scope of the patent application, wherein the ring has 5 to 15 atoms in the ring. 4 7. The 9-substituted tetracycline compound according to item 43 or 45 of the scope of the patent application, wherein the ring is a conjugated or unconjugated aromatic ring system. 48. A substituted tetracycline compound comprising contacting a reactive tetracycline-based precursor compound with a transition metal by including a reactive organic substituent precursor under conditions such that a tetracycline compound substituted with the organic substituent is formed. The catalyst is made of a reactive tetracycline chemical complex and forms a reactive chemical intermediate. 49. A substituted tetracycline compound according to item 48 of the scope of application for a patent, wherein the reactive tetracycline-based precursor compound is substituted with the organic substituent at the 7, 9 or 13 position. 50. The substituted tetracycline compound according to item 48 or 49 of the scope of patent application, wherein the catalyst comprises an organic palladium catalyst. 51. The substituted tetracycline compound according to item 50 of the scope of the patent application, wherein the organic palladium catalyst comprises palladium chloride, palladium acetate, PdCl2 (PhCN) 2, PdCl2 (Ph3P) 2, Pd2 (dba) 3-CHCl3 , Or a combination thereof. 52. The substituted tetracycline compound according to claim 48 or 49, wherein the transition metal catalyst comprises copper, rhodium, iron, iridium, chromium, zirconium, or nickel. 53. The substituted tetracycline compound according to item 48 or 49 of the scope of patent application, wherein the transition metal catalyst comprises CuCl2, Cul2, rhodium acetate (I I), Rh6 (CO) 16, or a combination thereof. O: \ 66 \ 66336-920227.ptc Page 54 548257 Amendment No. 89118818 VI. Application for patent scope 5 4. According to the substituted tetracycline compound No. 48 or 49 of the scope of patent application, the reactive tetracycline base is first Substances are tetracycline hydrochloride, chlorotetracycline, dechlorotetracycline, deoxytetracycline, chelomycin, dimethylamine tetracycline, ohexyltetracycline, lyme tetracycline, tetracycline, metene tetracycline, meridine tetracycline, hydroxymethyl tetracycline, vein Methyltetracycline, meglotracycline, penicillin V-tetracycline, piperazine, tetraethylenetetracycline, benzyltetracycline, or mepteracycline-based precursor compounds. 5 5. The substituted tetracycline compound according to item 48 or 49 of the scope of the patent application, wherein the reactive tetracycline-based precursor compound is selected from the group consisting of a reactive dimethylamine tetracycline-based precursor compound, a reactive deoxytetracycline-based precursor Compounds, and reactive tetracycline-based precursor compounds. 56. The substituted tetracycline compound according to claim 48 or 49, wherein the reactive tetracycline-based precursor compound is an aromatic diazonium salt, a carbon derivative, or a monoacid derivative of a tetracycline compound. 57. The substituted tetracycline compound according to claim 48 or 49, wherein the reactive organic substituent precursor has at least one reactive 7Γ-bonding group. 58. The substituted tetracycline compound according to item 48 or 49 of the scope of the patent application, wherein the reactive organic substituent precursor is selected from the group consisting of olefins, substituted olefins, ethylene monomers, aromatics and heterocyclics. Aromatic reactive group. 59. The substituted tetracycline compound according to item 48 or 49 of the scope of application for a patent, wherein the substituent is the following formula O: \ 66 \ 66336-920227.ptc Page 55 548257 Amendment No. 89118818 6. Scope of patent application where R2 and 1-3 are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl , Alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amine, hydroxy, cyano, alkoxy, aryloxy, carboxy, alkoxyhexyl, aryloxy, or Forms a substituted or unsubstituted carbocyclic or heterocyclic ring with R3. 60. A substituted tetracycline compound according to item 59 of the scope of the patent application, wherein R2 is hydrogen, and R3 is σ, wherein R4 is hydrogen, cyano, or (M5 alkoxy group). 6 According to the scope of the patent application The substituted tetracycline compound according to item 59, wherein R2 and R3 together form a substituted or unsubstituted carbocyclic or heterocyclic ring. 6 2. The substituted tetracycline compound according to item 59 of the application, wherein the ring is in the ring Has 5 to 15 atoms in it. 6 3. The substituted tetracycline compound according to item 5 of the scope of the patent application, wherein the ring is a conjugated or unconjugated aromatic ring system. 6 4. According to the scope of patent application, 5 9 The substituted tetracycline compound of item, wherein the ring has 5 to 8 atoms in the ring. O: \ 66 \ 66336-920227.ptc Page 56