HK1170218B - Tetracycline derivatives for the treatment of bacterial, viral and parasitic infections - Google Patents
Tetracycline derivatives for the treatment of bacterial, viral and parasitic infections Download PDFInfo
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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 bactericidal 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 elucidation of the chemical structure of these compounds confirmed their similarity and furnished the analytical basis for the production of a third member of this group in 1952, tetracycline. A new family of tetracycline compounds, without the ring-attached methyl group present in earlier tetracyclines, was prepared in 1957 and became publicly available in 1967; and minocycline was in use by 1972.
Recently, research efforts have focused on developing new tetracycline antibiotic compositions effective under varying therapeutic conditions and routes of administration. New tetracycline analogues have also been investigated which may prove to be equal to or more effective than the originally introduced tetracycline compounds. Examples include U.S. Patent Nos. 2,980,584 ; 2,990,331 ; 3,062,717 ; 3,165,531 ; 3,454,697 ; 3,557,280 ; 3,674,859 ; 3,957,980 ; 4,018,889 ; 4,024,272 ; and 4,126,680 . These patents are representative of the range of pharmaceutically active tetracycline and tetracycline analogue compositions.
Historically, soon after their initial development and introduction, the tetracyclines 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 (e.g., pneumococci and Salmonella). The rise of tetracycline-resistant organisms has resulted in a general decline in use of tetracyclines as antibiotics of choice.
In WO-A-2004064728 methods and compounds for treating diseases with tetracycline compounds having a target therapeutic activity are described.
The present invention pertains, at least in part, to substituted tetracycline compounds of Formula VIIa:
wherein
- R9m* is unsubstituted or substituted oxazolyl, unsubstituted or substituted oxadiazolyl, unsubstituted or substituted isoxazolyl, unsubstituted or substituted pyrazolyl, unsubstituted or substituted thiazolyl, methylimidazolyl, or -CONR9maR9mb;
- R9ma is hydrogen and R9mb is hydroxyl, alkyl, hydroxyalkyl, aryl or alkoxy; or R9ma is alkyl and R9mb is alkyl or hydroxyl; or a pharmaceutically acceptable salt thereof.
In one embodiment, the invention pertains, at least in part, to a pharmaceutical composition comprising a therapeutically effective amount of a substituted tetracycline compound of Formula VIIa, and a pharmaceutically acceptable carrier.
In another further embodiment, the invention pertains, at least in part, to a substituted tetracycline compound of Formula VIIa for use in methods for treating subjects for tetracycline responsive states, e.g., by administering to them an effective amount of said substituted tetracycline compound.
The present invention pertains, at least in part, to novel substituted tetracycline compounds as described above. These tetracycline compounds can be used to treat numerous tetracycline compound-responsive states, such as bacterial infections, inflammation, and neoplasms, as well as other known applications for minocycline and tetracycline compounds in general, such as blocking tetracycline efflux and modulation of gene expression. The term "tetracycline compound" includes many compounds with a similar ring structure to tetracycline. Examples of tetracycline compounds include: chlortetracycline, oxytetracycline, demeclocycline, methacycline, sancycline, chelocardin, rolitetracycline, lymecycline, apicycline; clomocycline, guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline, etamocycline, penimocycline, etc. Other derivatives and analogues comprising a similar four ring structure are also included (See Rogalski, "Chemical Modifications of Tetracyclines,"). Table 1 depicts tetracycline and several known other tetracycline derivatives.
The substituted tetracycline compounds of the invention include compounds of Formula VIIa:
wherein
R9m* is is unsubstituted or substituted oxazolyl, unsubstituted or substituted oxadiazolyl, unsubstituted or substituted isoxazolyl, unsubstituted or substituted pyrazolyl, unsubstituted or substituted thiazolyl, methylimidazolyl, or -CONR9maR9mb; and
R9ma is hydrogen and R9mb is hydroxyl, alkyl, hydroxyalkyl, aryl or alkoxy; or R9ma is alkyl and R9mb is alkyl or hydroxyl; or a pharmaceutically acceptable salt thereof.
In yet another embodiment, R9m* is unsubstituted or substituted oxazolyl, unsubstituted or substituted oxadiazolyl (e.g., alkyl substituted oxadiazolyl, such as methyl or isopropyl substituted oxazolyl), unsubstituted or substituted isoxazolyl, unsubstituted or substituted pyrazolyl, unsubstituted or substituted thiazolyl, methylpyrazolyl, or methylimidazolyl.
In yet another embodiment, R9m* is -CONR9maR9mb-; R9ma is hydrogen and R9mb is hydroxyl, hydroxyalkyl (e.g., hydroxyethyl), alkoxy (e.g., methoxy), aryl (e.g., phenyl) or alkyl (e.g., t-butyl). In a further embodiment, R9ma is alkyl (e.g., methyl, ethyl or propyl) and R9mb is alkyl (e.g., methyl, ethyl or propyl) or hydroxyl.
Examples of substituted tetracycline compounds of Formulae VIIa include:
and pharmaceutically acceptable salts thereof.
The substituted tetracycline compounds of the invention can be synthesized using the methods described in the following schemes and by using art recognized techniques. All novel substituted tetracycline compounds described herein are included in the invention as compounds.
In Scheme 1, a general synthetic scheme for synthesizing 7-substituted tetracyclines is shown. A palladium catalyzed coupling of an iodosancycline (1) is performed to form a 7-substituted aldehyde intermediate (2). The aldehyde intermediate is reduced in the presence of a hydroxylamine to give the desired product (3).
7- and 9-substituted tetracycline compounds may be synthesized by reacting the 7-iodo-9-aminoalkyl sancycline derivative (4) with trimethylsilylethyne in the presence of a palladium catalyst to yield a 7-substituted alkynyl intermediate. Subsequent acid hydrolysis yields the 7-acyl intermediate (5). Further derivitization of the 9-position may be accomplished by reductive alkylation of the amino group with t-butyl aldehyde, hydrogen and palladium on carbon to form compound 6, which can then be reacted with a primary hydroxylamine to form the oxime 7.
7- and 9-substituted tetracycline compounds may also be prepared as shown in Scheme 3. Beginning with a 7-iodo-9-nitro substituted sancycline derivative (8), a Hiyama coupling followed by acid hydrolysis yields a 7-acyl-9-nitro intermediate (9). The nitro moiety may then be reduced to the amino group by hydrogen gas in the presence of a palladium catalyst (10). Reaction of the acyl group with a primary hydroxylamine provides the product 11.
Scheme 4 also provides a method for synthesizing 7-substituted tetracyclines. As described above, a palladium catalyzed carbonylation of an iodosancycline (1) is performed to form a 7-substituted aldehyde intermediate (2). The aldehyde intermediate is reduced in the presence of a hydroxylamine to give compound 12, which may then be reacted with formaldehyde and triethylamine, followed by reduction to give the desired product (3).
Scheme 5 details the synthesis of substituted tetracyclines with hydroxy in the 10-position. A 7-substituted tetracycline compound may be reacted with N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonimide) to form a trifluoromethane substituted intermediate (14), which can then be reacted with ammonium formate in the presence of a palladium catalyst to form the desired product (15).
Scheme 6 outlines the general synthesis of 7-substituted tetracyclines. A 7-iodo sancycline derivative (1) may undergo a Stille coupling or a Suzuki coupling by reacting with an alkyl tin derivative or a boronic acid derivative in the presence of a palladium catalyst to form the desired product (16).
The 7-substituted oxime derivatives may also be prepared as shown in Scheme 7. An 7-iodo sancycline derivative (1) can be reacted with a substituted alkyne in the presence of palladium to synthesize the alkenyl derivative 17. Compound 17 may be converted to the acyl substituted compound 18 by any technique known in the art. The desired oxime product 19 can be obtained by reacting the acyl moiety with a primary hydroxylamine.
Scheme 8 is a general synthetic scheme showing the synthesis of 7-substitued hydrazone compounds. A 7-substituted aldehyde tetracycline derivative, prepared as described above in Scheme 4, is combined with a primary hydrazone to form the desired product 20.
7-substituted hydrazines may also be synthesized as shown in Scheme 9. Starting with compound 2, synthesized as described in Scheme 4 above, may be reacted with a secondary hydrazine in the presence of a reducing agent to form compound 21.
Scheme 10 further depicts a method of synthesizing a 7-substituted aminoalkyl tetracycline compound. Compound 2 is reacted with a primary amine in the presence of a reducing agent to form the secondary amine intermediate (22), which is then mixed with an acid chloride to form compound 23.
Scheme 11 describes a general method for preparing 9-substituted aminoalkyl substituted tetracycline compounds. Compound 24 may be reacted directly with a secondary amine to form compounds similar to 26. Alternatively, compound 24 may be mixed with a primary amine to yield the substituted imine 25, which may be further reduced to produce the aminoalkyl compound 26.
7-substituted tetracycline may also be prepared as shown in Scheme 12. Starting again with compound 2, reductive alkylation with a dioxalanyl secondary amine yields the intermediate 27. Subsequently exposing 27 to acidic conditions removes the protecting group to form intermediate 28, which may then be reacted with a primary amine to form product 29.
Schemes 13 and 14 illustrate the general synthesis of cyclobutene 7-substituted tetracycline compounds. Beginning with 30, tin reagent 31 is synthesized by reacting 30 with a trimethylsilyl substituted alkyltin derivative.
Scheme 14 continues to show the synthesis of cyclobutenedione 7-substituted tetracycline compounds, by reacting building block 31 with 7-iodo substituted sancycline (1) in a Stille coupling reaction to form 32. The amino substitution of product 33 is accomplished by reacting 32 with a primary amine in methanol.
Scheme 15 depicts generally the synthesis of substituted aromatic 7-substituted tetracycline compounds. Beginning with 1 and performing a Suzuki coupling reaction in the presence of a boronic acid and a palladium catalyst, compounds of general formula 34 are formed.
Scheme 16 also depicts the synthesis of substituted aromatic 7-substituted tetracycline compounds. Again, starting from 7-iodo substituted sancycline (1), a Suzuki coupling reaction is performed with a boronic acid in the presence of a palladium catalyst to provide intermediate 35 in which R7tb is either an amine or a carboxylic acid. If R7tb is a carboxylic acidic moiety, a coupling to a secondary amine in the presence of base and a typical coupling reagent to form 7-substituted tetracyclines similar to 36a. If R7tb is an amino moiety, a coupling to an acid chloride or carboxylic acid in the presence of a base and a typical coupling reagent to form 7-substituted tetracyclines similar to 36b.
Scheme 17 depicts a method for synthesizing aromatic substituted 9-substituted tetracycline compounds. A 9-iodo tetracycline derivative is reacted under Suzuki conditions by mixing with a boronic acid in the presence of the appropriate palladium catalyst to give compounds similar to compound 38.
Scheme 18 also depicts a method for synthesizing 9-substituted tetracycline compounds by reacting 37 with a palladium catalyst and carbon monoxide in the presence of N-hydroxysuccinimide to generate an activated ester intermediate. Reaction of this intermediate with nucleophilic compounds such as alcohols, hydroxylamines or amines yields the desired ester, hydroxamic acid or amide, respectively, similar to 39.
9-substituted oxime tetracycline compounds may be prepared as shown in Scheme 19. A 9-iodo substituted tetracycline derivative is subjected to Heck conditions to form an alkyne intermediate which is converted to 40 by acid hydrolysis. Intermediate 40 is subsequently reacted with an appropriate hydroxylamine to yield the desired product 41.
As shown in Scheme 20, 7-substituted tetracycline compounds can also be prepared by reacting the acyl intermediate 41 with hydrogen bromide to form the α-bromoketone substituted tetracycline 42. By reacting the brominated tetracycline with a secondary amine, followed by exposure to an acid chloride, the desired product 43 can be formed.
As shown in Scheme 21, 9-substituted-4-dedimethylamino tetracycline compounds may be synthesized starting from minocycline, which is exposed to the dedimethylamino conditions of methyl iodide and zinc to form 4-dedimethylsancycline 44. Intermediate 44 is halogenated at the 9-position to form intermediate 45, and upon exposing 45 to the appropriate palladium conditions, compounds similar to 46 are formed. Compounds BQ, BR, BW, BX, BY, CA, CC, CD, EI, EJ, CI, CJ, CK, EN, EO, ES, FM and FN may be synthesized as shown in Scheme 21.
As shown in Scheme 22, 9-substituted-4-dedimethylamino doxycycline compounds may be synthesized starting from doxycycline, which is exposed to the dedimethylamino conditions of methyl iodide and zinc to form 4-dedimethyldoxycycline 47. Intermediate 47 is halogenated at the 9-position to form intermediate 48, and upon exposing 48 to the appropriate palladium conditions, compounds similar to 49 are formed.
Scheme 23 illustrates the synthesis of 10-substituted tetracycline compounds. Starting with minocycline, the 10-position hydroxide is deprotonated in the presence of a strong base, followed by the addition of triflate to form intermediate 50, which then undergoes either Stille or Suzuki conditions or carbonylation conditions to form compounds similar to 10-substituted compounds 51.
Scheme 24 illustrates the synthesis of 7-aminomethyl substituted tetracycline compounds. Compound 1 is exposed to a secondary amine in the presence of a reducing agent to from compounds similar to 52.
Scheme 25 provides a method for synthesizing 9-aminomethyl substituted tetracycline compounds. 9-formyl substituted compound 54 is reacted with a secondary amine in the presence of a reducing agent to provide the 9-aminomethyl substituted tetracycline compounds 55. Compounds FD, FE, FF, FG, FH, FI, FJ, FK and FL may be synthesized as illustrated in Scheme 25.
Scheme 26 illustrates the methods for synthesizing 9-alkyl or 9-carbonyl substituted tetracycline compounds starting with the 9-iodominocycline or 9-iodo-4-dedimethylminocycline compound 56, followed by palladium catalyzed alkynylation to form intermediate 57. Intermediate 57 may undergo either hydrogenolysis to form 9-alkyl substituted tetracycline compounds (58) or acid catalyzed hydrolysis to form 9-carbonyl substituted tetracycline compound (59).
9-Alkyl substituted tetracycline compounds may also be synthesized as shown in Scheme 27. Again starting with 9-iodominocycline or 9-iodo-4-dedimethylminocycline compound 56, either Suzuki or Stille coupling conditions may produce the 9-alkyl substituted tetracycline compounds (60) or reaction with copper iodide with a fluorinated ester compound yields 9-trifluoroalkyl substituted compound 61.
7-Furanyl substituted tetracycline compounds may be synthesized as illustrated in Scheme 28. 7-Iodosancycline (1) is subjected to a formyl substituted furanyl boronic acid in the presence of palladium (II) acetate and sodium carbonate to yield intermediate 62. A reductive amination is then performed in the presence of an appropriate reducing agent and a secondary amine to convert the formyl moiety to a tertiary alkylamine (63). The substituents of the tertiary amine may be further derivitized, as shown by the reaction of compound 63 with methylchloroformate to form compound 64.
7-Pyridinyl-9-aminocarbonyl substituted tetracyclines may be synthesized as shown in Scheme 29. The 7-position of the 7-iodo-9-nitro tetracycline compound (8) is reacted under Stille conditions to form the 7-pyridinyl intermediate 64, which is then subjected to reducing conditions to form the 7-pyridinyl-9-amino tetracycline compound 65. The amino moiety of compound 65 is then reacted with a chloroformate to form the desired aminocarbonyl substituent in the 9 position (66).
Scheme 30 illustrates methods for preparing both 9-aminomethyl substituted sancycline compounds and 7-substituted-9-aminomethyl tetracycline compounds. Sancycline is bromonated at the 7-position with N-bromosuccinimide and iodated at the 9-position with N-iodosuccinimide to form the dihalogenated reactive intermediate 67, which then undergoes formylation at the 9-position to yield a 7-bromo-9-formyl substituted tetracycline compound (68). Compound 68 may then undergo a reductive amination procedure in the presence of an appropriate secondary amine and a reducing agent to form compound 69. The bromo moiety at the 7-position may then be removed by exposing 69 to palladium on carbon in the presence of hydrogen gas to provide 9-aminomethyl sancycline compounds (71). Alternatively, the reactive intermediate 68 may first be exposed to reductive amination conditions as described above, followed by a pallium-indium cross coupling reaction to form 7-substituted-9-aminomethyl tetracycline compounds (70).
7-Substituted tetracycline compounds may be generally synthesized as shown in Scheme 31. A 7-iodo tetracycline compound (71) may be reacted under Suzuki, Stille or indium-palladium cross coupling reactions to form 7-substituted tetracycline compounds.
The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1 to 6 carbon atoms.
Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkynyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
The term "aryl" includes groups, including 5- and 6-membered single-ring aromatic groups that, may include from zero to four heteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles," "heterocycles," "heteroaryls" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 or straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.
Moreover, the term alkenyl includes both "unsubstituted alkenyls" and "substituted alkenyls," the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
For example, the term "alkynyl," includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing 2 to 6 carbon atoms.
Moreover, the term alkynyl includes both "unsubstituted alkynyls" and "substituted alkynyls," the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. "Lower alkenyl" and "lower alkynyl" have chain lengths of, for example, 2-5 carbon atoms.
The term "acyl" includes compounds and moieties which contain the acyl radical (CH3CO-) or a carbonyl group. It includes substituted acyl moieties. The term "substituted acyl" includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term "acylamino" includes moieties wherein an acyl moiety is bonded to an amino group. For example, the term includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
The term "aroyl" includes compounds and moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
The terms "alkoxyalkyl," "alkylaminoalkyl" and "thioalkoxyalkyl" include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
The term "amine" or "amino" includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term includes "alkyl amino" which comprises groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term "dialkyl amino" includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term "arylamino" and "diarylamino" include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. The term "alkylarylamino," "alkylaminoaryl" or "arylaminoalkyl" refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term "alkaminoalkyl" refers to an alkyle, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
The term "amide," "amido" or "aminocarbonyl" includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes "alkaminocarbonyl" or "alkylaminocarbonyl" groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms "alkylaminocarbonyl," "alkenylaminocarbonyl," "alkynylaminocarbonyl," "arylaminocarbonyl," "alkylcarbonylarnino," "alkenylcarbonylamino," "alkynylcarbonylamino," and "arylcarbonylamino" are included in term "amide." Amides also include urea groups (aminocarbonylamino) and carbamates (oxycarbonylamino).
The term "carbonyl" or "carboxy" includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. The carbonyl can be further substituted with any moiety which allows the compounds of the invention to perform its intended function. For example, carbonyl moieties may be substituted with alkyls, alkenyls, alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
The term "thiocarbonyl" or "thiocarboxy" includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
The term "ether" includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes "alkoxyalkyl" which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
The term "ester" includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term "ester" includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are as defined above.
The term "thioether" includes compounds and moieties which contain a sulfur atom bonded to two different carbon or hetero atoms. Examples of thioethers include alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term "alkthioalkyls" include compounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls" refer to compounds or moieties wherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
The term "hydroxy" or "hydroxyl" includes groups with an -OH or -O-.
The term "halogen" includes fluorine, bromine, chlorine, iodine, etc. The term "perhalogenated" generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
The terms "polycyclyl" or "polycyclic radical" refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings." Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, cyano, amido, amino (including alkyl, amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term "heteroatoms" includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term "prodrug moiety" includes moieties which can be metabolized in vivo to a hydroxyl group and moieties which may advantageously remain esterified in vivo. Preferably, the prodrugs moieties are metabolized in vivo by esterases or by other mechanisms to hydroxyl groups or other advantageous groups. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts" J. Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkylamino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters.
It will be noted that the structure of some of the tetracycline compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof.
The invention also pertains to the substituted tetracycline compounds of the invention for treating a tetracycline responsive state, such as a bacterial infection, a viral infection, a parasitic infection, malaria, an inflammatory process, acne, rosacea, or multiple sclerosis. The method comprises administering to a subject an effective amount of a substituted tetracycline compound of the invention such that the tetracycline responsive state is treated.
The term "treating" includes curing as well as ameliorating at least one symptom of the state, disease or disorder, e.g., the tetracycline compound responsive state.
The language "tetracycline compound responsive state" or "tetracycline responsive state" includes states which can be treated, prevented, or otherwise ameliorated by the administration of a tetracycline compound of the invention Tetracycline compound responsive states include bacterial, viral, parasitic, and fungal infections (including those which are resistant to other tetracycline compounds), cancer (e.g., prostate, breast, colon, lung melanoma and lymph cancers and other disorders characterized by unwanted cellular proliferation, including those described in U.S. 6,100,248 ), arthritis, osteoporosis, diabetes, stroke, AMI, aortic aneurysm, neurodegenerative diseases and other states for which tetracycline compounds have been found to be active (see, for example, U.S. Patent Nos. 5,789,395 ; 5,834,450 ; 6,277,061 and 5,532,227 ). Compounds of the invention can be used to prevent or control important mammalian and veterinary diseases such as diarrhea, urinary tract infections, infections of skin and skin structure, ear, nose and throat infections, wound infection, mastitis and the like. In addition, methods for treating neoplasms using tetracycline compounds of the invention are described (van der Bozert et al., Cancer Res., 48:6686-6690 (1988)). In a further embodiment, the tetracycline responsive state is not a bacterial infection. In another embodiment, the tetracycline compounds of the invention are essentially non-antibacterial. For example, non-antibacterial tetracycline compounds of the invention may have MIC values greater than about 4 µg/ml (as measured by assays known in the art and/or the assay given in Example 2).
Tetracycline compound responsive states also include inflammatory process associated states (IPAS). The term "inflammatory process associated state" includes states in which inflammation or inflammatory factors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins, plasma proteins, cellular defense systems, cytokines, lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.), are involved or are present in an area in aberrant amounts, e.g., in amounts which may be advantageous to alter, e.g., to benefit the subject. The term "inflammatory process associated state" also includes states in which there is an increase in acute phase proteins (e.g., C-reactive protein). The inflammatory process is the response of living tissue to damage. The cause of inflammation may be due to physical damage, chemical substances, micro-organisms, tissue necrosis, cancer or other agents. Acute inflammation is short-lasting, lasting only a few days. If it is longer lasting however, then it may be referred to as chronic inflammation.
Tetracycline responsive states also include inflammatory disorders. Inflammatory disorders are generally characterized by heat, redness, swelling, pain and loss of function. Examples of causes of inflammatory disorders include microbial infections (e.g., bacterial and fungal infections), physical agents (e.g., bums, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis and various types of immunologic reactions.
Examples of inflammatory disorders include osteoarthritis, rheumatoid arthritis, acute and chronic infections (bacterial and fungal, including diphtheria and pertussis); acute and chronic bronchitis, sinusitis, and upper respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis and urethritis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis); uremic pericarditis; acute and chronic cholecystis; acute and chronic vaginitis; acute and chronic uveitis; drug reactions; animal bites (e.g., spider bites, snake bites, insect bites and the like); bums (thermal, chemical, and electrical); inflammatory bowel disorder (IBD); common obstructive pulmonary disease (COPD); acute respiratory distress syndrome (ARDS); vasculitis; asthma; sepsis; nephritis; pancreatitis; hepatitis; lupus; viral infections; parasitic infections; and sunburn.
Tetracycline compound responsive states also include NO associated states. The term "NO associated state" includes states which involve or are associated with nitric oxide (NO) or inducible nitric oxide synthase (iNOS). NO associated state includes states which are characterized by aberrant amounts of NO and/or iNOS. Preferably, the NO associated state can be treated by administering tetracycline compounds of the invention. The disorders, diseases and states described in U.S. Patents Nos. 6,231,894 ; 6,015,804 ; 5,919,774 ; and 5,789,395 are also included as NO associated states.
Other examples of tetracycline compound responsive states includemalaria, senescence, diabetes, vascular stroke, hemorrhagic stroke, neurodegenerative disorders (Alzheimer's disease & Huntington's disease), cardiac disease (reperfusion-associated injury following infarction), juvenile diabetes, inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute, recurrent and chronic infections (bacterial, viral and fungal); acute and chronic bronchitis, sinusitis, and respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis and urethritis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendonitis); uremic pericarditis; acute and chronic cholecystis; cystic fibrosis, acute and chronic vaginitis; acute and chronic uveitis; drug reactions; insect bites; bums (thermal, chemical, and electrical); and sunburn.
The term "inflammatory process associated state" also includes matrix metalloproteinase associated states (MMPAS). MMPAS include states characterized by aberrant amounts of MMPs or MMP activity. These are also included as tetracycline compound responsive states.
Examples of other tetracycline compound responsive states include arteriosclerosis, angiogenesis, corneal ulceration, emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann. Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol.1997, 72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronic pulmonary obstructive disease, skin and eye diseases, periodontitis, osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatory disorders, tumor growth and invasion (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)), metastasis, acute lung injury, stroke, ischemia, diabetes, aortic or vascular aneurysms, skin tissue wounds, dry eye, bone and cartilage degradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr. Op. Rheumatol. 1996, 8;238-247). Other MMPAS include those described in U.S. Pat. Nos. 5,459,135 ; 5,321,017 ; 5,308,839 ; 5,258,371 ; 4,935,412 ; 4,704,383 , 4,666,897 , and RE 34,656 .
Another tetracycline compound responsive state is cancer. Examples of cancers which the tetracycline compounds of the invention may be useful to treat include all solid tumors, i.e., carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas are carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures. Sarcomas broadly include tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue. Examples of carcinomas which may be treated using the methods of the invention include carcinomas of the prostate, breast, ovary, testis, lung, colon, and breast. The methods described herein are not limited to the treatment of these tumor types, but extend to any solid tumor derived from any organ system. Examples of treatable cancers include colon cancer, bladder cancer, breast cancer, melanoma, ovarian carcinoma, prostatic carcinoma, lung cancer, and a variety of other cancers as well. The methods described herein may also enable the inhibition of cancer growth in adenocarcinomas, such as, for example, those of the prostate, breast, kidney, ovary, testes, and colon.
As noted above, another tetracycline responsive state of is cancer. Described herein is a method for treating a subject suffering or at risk of suffering from cancer, by administering an effective amount of a substituted tetracycline compound, such that inhibition cancer cell growth occurs, i.e., cellular proliferation, invasiveness, metastasis, or tumor incidence is decreased, slowed, or stopped. The inhibition may result from inhibition of an inflammatory process, down-regulation of an inflammatory process, some other mechanism, or a combination of mechanisms. Alternatively, the tetracycline compounds may be useful for preventing cancer recurrence, for example, to treat residual cancer following surgical resection or radiation therapy. The tetracycline compounds useful according to the invention may be substantially non-toxic compared to other cancer treatments. The compounds of the invention may be administered in combination with standard cancer therapy, such aschemotherapy.
Other examples of tetracycline compound responsive states include neurological disorders which include both neuropsychiatric and neurodegenerative disorders such as Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis (e.g., including relapsing and remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, Korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affective neurological disorders,e.g., migraine and obesity. Further neurological disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.
Other examples of tetracycline compound responsive states are described in WO 03/005971A2 , U.S.S.N. 60/421,248 , and U.S.S.N. 60/480,482
The language "in combination with" another therapeutic agent or treatment includes co-administration of the tetracycline compound, (e.g., inhibitor) and with the other therapeutic agent or treatment, administration of the tetracycline compound first, followed by the other therapeutic agent or treatment and administration of the other therapeutic agent or treatment first, followed by the tetracycline compound. The other therapeutic agent may be any agent that is known in the art to treat, prevent, or reduce the symptoms of a particular tetracycline responsive state. Furthermore, the other therapeutic agent may be any agent of benefit to the patient when administered in combination with the administration of a tetracycline compound. Cancers that may be treated by the methods described herein include those described in U.S. Patent Nos. 6,100,248 ; 5,843,925 ; 5,837,696 ; or 5,668,122 .
Another tetracycline compound responsive state is diabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I, or diabetes type II. Protein glycosylation may not be affected by the administration of the tetracycline compounds of the invention. The tetracycline compound may be administered in combination with standard diabetic therapies, such as insulin therapy. The IPAS includes disorders as described in U.S. Patents Nos. 5,929,055 ; and 5,532,227 .
Another tetracycline compound responsive state is a bone mass disorder. Bone mass disorders include disorders are disorders and states where the formation, repair or remodeling of bone is advantageous. For examples bone mass disorders include osteoporosis (e.g., a decrease in bone strength and density), bone fractures, bone formation associated with surgical procedures (e.g., facial reconstruction), osteogenesis imperfecta (brittle bone disease), hypophosphatasia, Paget's disease, fibrous dysplasia, osteopetrosis, myeloma bone disease, and the depletion of calcium in bone, such as that which is related to primary hyperparathyroidism. Bone mass disorders include all states in which the formation, repair or remodeling of bone is advantageous to the subject as well as all other disorders associated with the bones or skeletal system of a subject which can be treated with the tetracycline compounds of the invention. Bone mass disorders include those described in U.S. Patents Nos. 5,459,135 ; 5,231,017 ; 5,998,390 ; 5,770,588 ; RE 34,656 ; 5,308,839 ; 4,925,833 ; 3,304,227 ; and 4,666,897 .
Another tetracycline compound responsive state is acute lung injury. Acute lung injuries include adult respiratory distress syndrome (ARDS), post-pump syndrome (PPS), adelectasis (e.g., collapsed lung) and trauma. Trauma includes any injury to living tissue caused by an extrinsic agent or event. Examples of trauma include crush injuries, contact with a hard surface, or cutting or other damage to the lungs.
Described herein is a method for treating acute lung injury by administering a substituted tetracycline compound of the invention.
Other tetracycline responsive states include chronic lung disorders. Described herein are methods for treating chronic lung disorders by administering a tetracycline compound, such as those described herein. The method includes administering to a subject an effective amount of a substituted tetracycline compound such that the chronic lung disorder is treated. Examples of chronic lung disorders include to asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and emphesema. The tetracycline compounds of the invention may be used to treat acute and/or chronic lung disorders such as those described in U.S. Patents No. 5,977,091 ; 6,043,231 ; 5,523,297 ; and 5,773,430 .
Another tetracycline compound responsive state is ischemia, stroke, hemorrhagic stroke or ischemic stroke. Described herein is a method for treating ischemia, stroke, hemorrhagic stroke or ischemic stroke by administering an effective amount of a substituted tetracycline compound of the invention. The tetracycline compounds of the invention may be used to treat such disorders as described in U.S. Patents No. 6,231,894 ; 5,773,430 ; 5,919,775 or 5,789,395 .
Another tetracycline compound responsive state is a skin wound. Described herein is a method for improving the healing response of the epithelialized tissue (e.g., skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape, etc.). The method may include using a tetracycline compound of the invention (which may or may not have antibacterial activity) to improve the capacity of the epithelialized tissue to heal acute wounds. The method may increase the rate of collagen accumulation of the healing tissue. The method may also decrease the proteolytic activity in the epthithelialized tissue by decreasing the collagenolytic and/or gellatinolytic activity of MMPs. The tetracycline compound of the invention may be administered to the surface of the skin (e.g., topically). The tetracycline compound of the invention may be used to treat a skin wound, and other such disorders as described in, for example, U.S. Patent Nos. 5,827,840 ; 4,704,383 ; 4,935,412 ; 5,258,371 ; 5,308,8391 5,459,135 ; 5,532,227 ; and 6,015,804 .
Another tetracycline compound responsive state is an aortic or vascular aneurysm in vascular tissue of a subject (e.g., a subject having or at risk of having an aortic or vascular aneurysm, etc.). The tetracycline compound may by effective to reduce the size of the vascular aneurysm or it may be administered to the subject prior to the onset of the vascular aneurysm such that the aneurysm is prevented. In one embodiment, the vascular tissue is an artery, e.g., the aorta, e.g., the abdominal aorta. In a further embodiment, the tetracycline compounds of the invention are used to treat disorders described in U.S. Patent Nos. 6,043,225 and 5,834,449 .
Bacterial infections may be caused by a wide variety of gram positive and gram negative bacteria. Some of the compounds of the invention are useful as antibiotics against organisms which are resistant and/or sensitive to other tetracycline compounds. The antibiotic activity of the tetracycline compounds of the invention may by using the in vitro standard broth dilution method described in Waitz, J.A., CLSI, Document M7-A2, vol. 10, no. 8, pp. 13-20, 2nd edition, Villanova, PA (1990).
The tetracycline compounds may also be used to treat infections traditionally treated with tetracycline compounds such as, for example, rickettsiae; a number of gram-positive and gram-negative bacteria; and the agents responsible for lymphogranuloma venereum, inclusion conjunctivitis, or psittacosis. The tetracycline compounds may be used to treat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A. baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E. coli, S. aureus or E. faecalis. In one embodiment, the tetracycline compound is used to treat a bacterial infection that is resistant to other tetracycline antibiotic compounds. The tetracycline compound of the invention may be administered with a pharmaceutically acceptable carrier.
The language "effective amount" of the compound is that amount necessary or sufficient to treat or prevent a tetracycline compound responsive state. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular tetracycline compound. For example, the choice of the tetracycline compound can affect what constitutes an "effective amount." One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the tetracycline compound without undue experimentation.
The invention also enables methods of treatment against microorganism infections and associated diseases. The methods include administration of an effective amount of one or more tetracycline compounds to a subject. The subject can be either a plant or, advantageously, an animal, e.g., a mammal, e.g., a human.
In the therapeutic methods, one or more tetracycline compounds of the invention may be administered alone to a subject, or more typically a compound of the invention will be administered as part of a pharmaceutical composition in mixture with conventional excipient, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, oral or other desired administration and which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof.
The invention also pertains to pharmaceutical compositions comprising a therapeutically effective amount of a tetracycline compound of Formula VIIa and a pharmaceutically acceptable carrier.
The language "pharmaceutically acceptable carrier" includes substances capable of being coadministered with the tetracycline compound(s), and which allow both to perform their intended function, e.g., treat or prevent a tetracycline responsive state. Suitable pharmaceutically acceptable carriers include water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds of the invention.
The tetracycline compounds of the invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of the tetracycline compounds of the invention that are basic in nature are those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and palmoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts. Although such salts must be pharmaceutically acceptable for administration to a subject, e.g., a mammal, it is often desirable in practice to initially isolate a tetracycline compound of the invention from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The preparation of other tetracycline compounds of the invention not specifically described in the foregoing experimental section can be accomplished using combinations of the reactions described above that will be apparent to those skilled in the art.
The preparation of other tetracycline compounds of the invention not specifically described in the foregoing experimental section can be accomplished using combinations of the reactions described above that will be apparent to those skilled in the art.
The tetracycline compounds of the invention that are acidic in nature are capable of forming a wide variety of base salts. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those tetracycline compounds of the invention that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include those derived from such pharmaceutically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. The pharmaceutically acceptable base addition salts of tetracycline compounds of the invention that are acidic in nature may be formed with pharmaceutically acceptable cations by conventional methods. Thus, these salts may be readily prepared by treating the tetracycline compound of the invention with an aqueous solution of the desired pharmaceutically acceptable cation and evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, a lower alkyl alcohol solution of the tetracycline compound of the invention may be mixed with an alkoxide of the desired metal and the solution subsequently evaporated to dryness.
The preparation of other tetracycline compounds of the invention not specifically described in the foregoing experimental section can be accomplished using combinations of the reactions described above that will be apparent to those skilled in the art.
The tetracycline compounds of the invention and pharmaceutically acceptable salts thereof can be administered via either the oral, parenteral or topical routes. In general, these compounds are most desirably administered in effective dosages, depending upon the weight and condition of the subject being treated and the particular route of administration chosen. Variations may occur depending upon the species of the subject being treated and its individual response to said medicament, as well as on the type of pharmaceutical formulation chosen and the time period and interval at which such administration is carried out.
The pharmaceutical compositions of the invention may be administered alone or in combination with other known compositions for treating tetracycline responsive states in a subject, e.g., a mammal. Preferred mammals include pets (e.g., cats, dogs, ferrets, etc.), farm animals (cows, sheep, pigs, horses, goats, etc.), lab animals (rats, mice, monkeys, etc.), and primates (chimpanzees, humans, gorillas). The language "in combination with" a known composition is intended to include simultaneous administration of the composition of the invention and the known composition, administration of the composition of the invention first, followed by the known composition and administration of the known composition first, followed by the composition of the invention.
The tetracycline compounds of the invention may be administered alone or in combination with pharmaceutically acceptable carriers or diluents by any of the routes previously mentioned, and the administration may be carried out in single or multiple doses. For example, the novel therapeutic agents of this invention can be administered advantageously in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays (e.g., aerosols, etc.), creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, oral pharmaceutical compositions can be suitably sweetened and/or flavored. In general, the therapeutically-effective compounds of this invention are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight.
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof. The compositions of the invention may be formulated such that the tetracycline compositions are released over a period of time after administration.
For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection), solutions of a therapeutic compound of the present invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parenteral application, examples of suitable preparations include solutions, preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories. Therapeutic compounds may be formulated in sterile form in multiple or single dose formats such as being dispersed in a fluid carrier such as sterile physiological saline or 5% saline dextrose solutions commonly used with injectables.
Additionally, it is also possible to administer the compounds of the present invention topically when treating inflammatory conditions of the skin. Examples of methods of topical administration include transdermal, buccal or sublingual application. For topical applications, therapeutic compounds can be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion or a cream. Such topical carriers include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other possible topical carriers are liquid petrolatum, isopropylpalmitate, polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5% in water, and the like. In addition, materials such as anti-oxidants, humectants, viscosity stabilizers and the like also may be added if desired.
For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch. A syrup, elixir or the like can be used wherein a sweetened vehicle is employed. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
In addition to treatment of human subjects, the therapeutic methods will have significant veterinary applications, e.g., for treatment of livestock such as cattle, sheep, goats, cows, swine and the like; poultry such as chickens, ducks, geese, turkeys and the like; horses; and pets such as dogs and cats. Also, the compounds of the invention may be used to treat non-animal subjects, such as plants.
It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.
In general, compounds of the invention for use in treatment can be administered to a subject in dosages used in prior tetracycline therapies. See, for example, the Physicians' Desk Reference. For example, a suitable effective dose of one or more compounds of the invention will be in the range of from 0.01 to 100 milligrams per kilogram of body weight of recipient per day, preferably in the range of from 0.1 to 50 milligrams per kilogram body weight of recipient per day, more preferably in the range of 1 to 20 milligrams per kilogram body weight of recipient per day. The desired dose is suitably administered once daily, or several sub-doses, e.g., 2 to 5 sub-doses, are administered at appropriate intervals through the day, or other appropriate schedule.
It will also be understood that normal, conventionally known precautions will be taken regarding the administration of tetracyclines generally to ensure their efficacy under normal use circumstances. Especially when employed for therapeutic treatment of humans and animals in vivo, the practitioner should take all sensible precautions to avoid conventionally known contradictions and toxic effects. Thus, the conventionally recognized adverse reactions of gastrointestinal distress and inflammations, the renal toxicity, hypersensitivity reactions, changes in blood, and impairment of absorption through aluminum, calcium, and magnesium ions should be duly considered in the conventional manner.
Also described herein is the use of a tetracycline compound of Formula VIIafor the preparation of a medicament. The medicament may include a pharmaceutically acceptable carrier and the tetracycline compound is an effective amount, e.g., an effective amount to treat a tetracycline responsive state.
In one embodiment, the substituted tetracycline compound is a compound of Table 2.
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| CJ | |||
| CK |
Minocycline/HCl salt (200 g, 0.406 mol) was suspended in 3 L water and an amount of NaHCO3 (34 g, 0.406 mol) was added in 3 portions and the pH was adjusted to 6.5-7.0. The solution was then extracted with 2 x 1.5 L CH2Cl2. The solution was concentrated to dryness to give minocycline as the freebase, then redissolved in THF (1.6 L) and was charged in a 3 L 3-necked flask equipped with an over-head stirrer and a temperature probe while under argon. An amount of methyl iodide (289 g, 2.03 mol) was added and the solution was heated at 40-45°C for approximately 16 hours, at which point it was verified by HPLC that no minocycline was left in solution. The solution was then poured into 6 L of heptane while on ice bath and stirred for at least 20 minutes at < 5°C. The precipitate was filtered and washed with hexane (400 mL). The solid was dried under reduced pressure to a constant weight. An amount of 186 g methylammonium salt of minocycline was isolated.
In a 3 L, 3-necked RBF equipped with overhead stirrer and a temperature probe a mixture of 200 ml DMF, 50 ml TFA, and 15 ml water was cooled on an ice bath to <5 °C. An amount of 4-methylammonium minocycline (100 g) was added to the flask. Upon dissolution, Zn powder (14g, 100 mesh) was added in 6 portions approximately every 30 minutes (∼2.33 g each addition). The reaction was monitored by HPLC. When less than approximately 10% of 4-trimethyammonium minocycline remained, the solution was filtered through a bed of Celite and was washed with 500 mL water. The solution was then poured into 2 L of water and the pH was adjusted with aqueous ammonia to a pH of 3.5. The aqueous solution was extracted first with 1 L dichloromethane (two times) and the combined organic layers were back-washed with 1 L water, dried on sodium sulfate, filtered and concentrated under reduced pressure down to an oil, to yield 4-dedimethylamino minocycline (48 g).
An amount of 4-dedimethyl minocycline (48 g, 0.115 mol) was charged in a flask under argon atmosphere and methanesulfonic acid (350 mL) was added. Ag2SO4 (75 g, 0.24 mol) and iodine (61.5 g, 0.24 mol) were added and the mixture was stirred for 3 hours. Upon HPLC confirming completion of the reaction, the mixture was poured into 4% aqueous sodium sulfite (3.5 L) and was stirred for at least one hour. The solution was filtered through a bed of Celite, then washed with 200 ml of water. The aqueous layer was loaded onto a column containing divinylbenzyl resin. A gradient of 20-60% organic (1:1 methanol:acetonitrile) in water with an overall trifluoroacetic acid of 1.0% was used to elute compound 4-dedimethylamine-9-iodo minocycline. The combined fractions were reduced of organic, the pH adjusted with aqueous NaHCO3 to a pH of 7 and extracted with methylene chloride to give 20 g of 4-dedimethylamine-9-iodo minocycline as the freebase.
To a 500 mL flask was added (2.00 g) 4-dedimethylamino-9-iodo minocycline free base and NMP (37 mL), N-hydroxysuccinimide (3.9 g). To remove residual water from the above reactants, toluene was added (37 mL), the flask was placed on the rotary evaporator (35 mm Hg, 45 °C) until all the toluene was evaporated. The flask was backfilled with argon and the contents were then transferred via cannula to a dry 500 L flask. To the 500 mL flask was added tetrakis-(triphenylphosphine)palladium(O) (2.00 g) and DIEA (2.60 mL). The flask was placed under vacuum (20 mm Hg) and purged 3 times with carbon monoxide. The flask was then heated to 60 °C under 1.0 atm of carbon monoxide and let stir for 1 hour. Subsequently, methylhydroxylamine (1.7 mL) and DIEA (0.5 mL) was added and the reaction was heated in a microwave reactor for 1 minute at 100°C. The reaction was added to water (1.0 L) and the pH was lowered to 2 using trifluoroacetic acid. The solution was then filtered through celite, loaded onto a reverse phase column and the crude product was purified by reverse phase HPLC (C18, linear gradient 10-30% MeCN in water with 0.1% TFA). The fractions containing the final product were loaded onto DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with acetonitrile to give the HCl salt of 4-dedimethylamino-9-N-methylhydroxamic acid minocycline (1000 mg, 44%). 1H-NMR (300 MHz, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.5-1.7 (m, 1H), 2.1-2.25 (m, 1H), 2.35-2.7 (m, 3H), 2.9-3.1 (m, 1H), 3.2-3.3 (brs, 7H), 3.35 (s, 2H), 3.45 (s, 2H), 7.91 (s, 1H). MW calcd for C23H25N3O9 487.46, ESIMS obs. m/z 488.25 (MH+). Compounds BQ, BR, BW, BX, BY, CA, CD and EJ, were prepared as described above.
To a 500 mL flask was added (2.00 g, 4.30 mmol) 4-dedimethylamine-9-iodo minocycline free base (see synthesis of compound CC), NMP (37 mL), N-hydroxysuccinimide (3.9 g, 38 mmol). To remove residual water from the above reactants toluene was added (37 mL), the flask was placed on the rotary evaporator (5 mm Hg, 45 °C) until all the toluene was evaporated. The flask was backfilled with argon and the contents were then transferred via cannula to a dry 500 L flask. To the 0.5 L flask was added tetrakis(triphenylphosphine)palladium(O) (2.00 g, 1.67 mmol) and DIEA (2.60 mL, 1.48 mmol). The flask was placed under vacuum (20 mm Hg) and purged 3 times with carbon monoxide. The flask was then heated to 60 °C under 1.0 atm of carbon monoxide and let stir for 1 hour until all 4-dedimethlyamino-9-iodo minocycline was consumed and a peak for the corresponding NHS-ester intermediate (M+1) of 556 M/Z was formed as determined via LCMS. Subsequently, tert-butylamine (4.0 mL, 38 mmol) and DIEA (4.0 mL, 38 mmol) was added and the reaction was heated in a microwave reactor for 1 minute at 100 C. The reaction was added to acetonitrile (150 mL) followed by water (0.8 L) and the pH was lowered to pH 2 using trifluoroacetic acid. The solution was then filtered through celite to remove the catalyst, loaded onto a reverse phase column and the crude product was purified by HPLC (C 18, linear gradient 30-45% acetonitrile in water with 0.2% Formic acid). The fractions containing the final product were loaded onto DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give the HCl salt of 4-dedimethyl-9-tertbutylcarboxalido minocycline (500 mg, 0.91 mmol, 20%). 1H-NMR (300 MHz, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.4 (s, 9H), 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H), 2.35-2.7 (m, 3H), 2.9-3.1 (m, 1H), 3.15-3.3 (m, 1H), 3.38 (s, 1H), 8.45 (s, 1H). MS (electron spray) calcd for C26H31N3O8 513.54, found (MH+) 514.25. Compounds BQ, BR, BW, BX, BY, CA, CD and EJ were prepared as described above.
To 9-iodo-4-dedimethylaminominocycline (2.0 g) was added a DMF (15 mL) previously purged with argon to remove any oxygen, a previously prepared solution of Na2CO3 (784 mg) in water (5.0 mL), dichloro(1,1'bis-diphenylphosphine)(Ferrocene)Pd(0) complexed with DCM (541mg) and 4-isoxazoleboronic acid pinacol ester (1.08 g). The reaction was subject to microwave irradiation for duration of 1 minute at temperature of 100 C. Following, the reaction was added to an aqueous solution containing acetonitrile (20%) and TFA (0.2%). The solution was then filtered through celite to remove the catalyst, loaded onto a C18 reverse phase column and the crude product was purified by reverse phase HPLC (C 18, linear gradient 20-40% MeCN in water with 0.1% TFA). The fractions containing the final product were loaded onto DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give the HCl salt of 4-dedimethylamino-9-(isoxazol-4-yl)-minocycline (1000 mg, 1.93 mmol, 51%).1H-NMR (Bruker DPX300 300 MHz spectrometer, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H), 2.35-2.7 (m, 3H), 2.9-3.1 (m, 1H), 3.18-3.3 (m, 2H), 3.35-3.45 (m, 6H), 8.3 (s, 1H), 9.15 (s, 1H), 9.35 (s, 1H). MW calcd for C24H23N3O8 481.47, ESIMS found m/z 482 (MH+). Compounds CI, CK and ES were prepared in this manner.
To 9-iodo-4-dedimethyl minocycline (1.5 g, 2.78 mmol, see synthesis of compound CC) was added N-methylpyrrolidone (10 mL, previously purged with argon to remove any oxygen), a previously prepared solution of NaCO3 (584 mg, 5.56 mmol) in water (4.0 mL, also previously purged with argon), 1,1'-Bis(diphenylphosphino)-ferrocene)dichloro-palladium(II)complex with dichloromethane 1:1 (400 mg, 0.556 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (867 mg, 4.17 mmol). The reaction was subject to microwave irradiation for duration of 2 minutes at temperature of 100 °C. Following, the reaction was added to an aqueous solution containing acetonitrile (10%) and TFA (0.2%). The solution was then filtered through celite to remove the catalyst, loaded onto a reverse phase column and purified by HPLC (C 18, linear gradient 15-25% acetonitrile in water with 0.1 % TFA). The fractions containing the final product were loaded onto DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give the HCl salt of 4-dedimethyl-9-(1-methyl-pyrazole) minocycline (510 mg, 0.96 mmol, 35%). 1H-NMR (Bruker DPX300 300 MHz spectrometer, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H), 2.35-2.65 (m, 3H), 2.8-3.2 (m, 6H), 3.2-3.3 (m, 1H), 3.79 (s, 3H), 6.4 (s, 1H), 7.55 (s, 1H), 7.75 (brs, 1H). MS (electron spray) calcd for C25H26N4O7 494.50, found (M+1) 495.20. Compounds CI, CK and ES were prepared in this manner.
To a 100 mL round bottom flask was added 9-iodo 4-dedimethyl minocycline (1.5 g, 2.78 mmol, see synthesis of compound CC), N-methylpyrrolidone (10 mL) and toluene (10 mL). The toluene and residual water of the stock solution containing the 9-iodo 4-dedimethyl minocycline was then removed by rotary evaporation (5.0 mm Hg, 45 °C) and backfilled with argon to give 10 mL of a 0.28 M stock solution of 9-iodo 4-dedimethyl minocycline in NMP. To a 20 mL microwave vial was added tris(dibenzyldieneacetone)dipalladium(O) (250 mg, 0.28 mmol), tri-2-furylphosphine (645 mg, 2.8 mmol), Cul (53 mg, 0.28 mmol), and the stock solution of 9-iodo-4-dedimethyl minocycline followed by 1-methyl-5-tributylstannanyl-1H-imidazole (2.0 g, 5.56 mmol). The microwave vial was capped, heated to 100 °C for 15 minutes using a microwave reactor. The reaction mixture was then cooled to room temperature, added to water (1.0 L) and the pH was lowered to pH 2 using trifluoroacetic acid. The solution was then filtered through celite to remove the catalyst, loaded onto a reverse phase column and the crude product was purified by HPLC (C 18, linear gradient 10-25% acetonitrile in water with 0.1 % TFA). The fractions containing the final product were loaded onto a DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with acetonitrile to give the HCl salt (510 mg, 0.96 mmol, 35%). 1H-NMR (300 MHz spectrometer, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H), 2.3-2.65 (m, 3H), 2.8-3.0 (m, 7H), 3.4-3.5 (m, 2H), 3.89 (s, 3H), 7.7 (d, J = 7.4 Hz, 2H), 9.1 (s, 1H). MS (electron spray) calcd for C26H25N3O7 494.50, found (M+1) 495.20. Compounds CI, CK, and ES, were prepared in this manner.
To a 500 mL flask was added (4.00 g, 8.60 mmol) 4-dedimethylamine-9-iodo minocycline free base (see the synthesis of compound CC), NMP (50 mL), N-hydroxysuccinimide (3.9 g, 38 mmol). To the reaction flask was added a stir bar, tetrakis(triphenylphosphine)palladium(O) (2.00 g, 1.67 mmol) and DIEA (3.0 mL, 1.7 mmol). The flask was placed under vacuum (20 mm Hg) and purged 3 times with carbon monoxide. The flask was then heated to 60 °C under 1.0 atm of carbon monoxide and was stirred for 1 hour until the 4-dedimethlyamine-9-iodo minocycline was consumed and a peak for the corresponding 9-NHS-ester 4-dedimethylamino minocycline intermediate [(M+1) of 556 M/Z] was formed as observed by LCMS. The NHS-ester intermediate was then reacted with N'-hydroxy-2-methylpropanimidamide (2.0 g, 19.6 mmol) at room temperature for 2 hours to give the noncyclized intermediate [(M+1) of 543 M/Z] as determined by LCMS. The noncyclized intermediate was isolated by adding it to 50 mL acetonitrile followed by dilution of the reaction mixture with water to a total volume of 2.0 L. Subsequently, the water was adjusted to a pH of 2 using trifluoroacetic acid. The aqueous solution was then filtered and loaded onto a plug of divinylbenzene resin and purified (10-60% MeCN, 0.1% TFA) to give 1 g of crude noncyclized intermediate. To noncyclized-intermediate (2.0 g, 3.7 mmol) in a 500 mL round bottom flask was added NMP (80 mL) and toluene (80 mL). To prevent hydrolysis during the subsequent cyclization step, residual water was removed from the noncyclized intermediate by subjecting it to rotary evaporator (5 mm Hg, 45 °C) until all the toluene/water was evaporated. Next, the flask was backfilled with argon and diisopropylamine (2 mL, 1.13 mmol) was added. To facilitate cyclization the contents were heated to 125 °C for 8 minutes via microwave irradiation. The reaction was then added to acetonitrile, diluted with water to a final volume of two liters and trifluoroacetic acid was added to a final pH of 2. The solution was then filtered through celite to remove the catalyst, loaded onto a reverse phase column and the crude product was purified by HPLC (C18, linear gradient 30-40% acetonitrile in water with 0.1% TFA). The fractions containing the final product were loaded onto a DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give the HCl salt of 9-(3-isopropyl-1,2,4-Oxadiazoyl)-4-dedimethylamino minocycline (280 mg, 0.53 mmol, 12%). 1H-NMR (300 MHz, chemical shifts in ppm with TMS as internal reference at 0 ppm) δ 1.47 (d, J = 7.5 Hz, 6H), 1.55-1.75 (m, 1H), 2.0-2.2 (m, 1H), 2.15-2.6 (m, 3H), 2.6-2.9 (m, 7H), 3.05-3.19 (m, 1H), 3.20-3.45 (m, 3H), 8.00 (s, 1H). ESI-MS (electron spray) calcd. for C26H28N4O8 524.54, found (M+1), 525.25. Compound FN was also synthesized in a similar manner.
The gram (+) and gram (-) antibacterial activities of the tetracycline compounds used in the methods of the invention may be assessed in accordance with the following method.
Gram (-) and gram (+) antibacterial minimum inhibitory concentration (MIC) values (µg/mL) are obtained using CLSI methodology for anti-bacterial susceptibility testing. On each day of testing, serial dilutions of compounds are prepared in microdilution plates using a Tecan robotic workstation. Mueller Hinton broth cultures of representative sensitive and resistant gram negative strains are grown or adjusted to match the turbidity of a 0.5 McFarland standard. 1:200 dilutions are made in an appropriate broth (cation supplemented Mueller Hinton broth) to allow a final inoculum of 1 x 105 cfu. Plates are incubated at 35 °C in ambient air for 18-24 hours, are read spectrophotometrically and checked manually for evidence of bacterial growth. The lowest dilution of compound that inhibited growth is recorded as the MIC. Lysed horse blood is used to supplement broth for testing S. pneumoniae. The MIC's for each compound are assessed against S. aureus, S. pneumoniae, P. acnes, E. coli and B. theta. The results form minocycline and doxycylcine are shown in Table 3. Good antibacterial activity (e.g., less than about 4 µg/mL) is indicated by "***," modest antibacterial activity (between about 4 and 8 µg/mL) is indicated by "**," or weak or no antibacterial activity (greater than about 8 µg/mL) is indicated by "*." The symbol "-" indicates that no data was obtained.
The cytotoxicity of the tetracycline compounds used in the methods of the invention may be assessed in accordance with the following method.
Mammalian cell cytotoxicity is assessed to evaluate potential in vivo risks associated with the tetracycline compounds of the invention. A soluble, non-toxic redox dye ("Resazurin"; Alamar Blue) is used to assess a tetracycline compound's effect on cellular metabolism. At the onset of the experiment, cultures of mammalian COS-1 or CHO cells are washed, trypsinized, and harvested. Cell suspensions are prepared, seeded into 96-well black-walled microtiter plates, and incubated overnight at 37 °C, in 5% CO2 and approximately 95% humidity. On the next day, serial dilutions of test drug are prepared under sterile conditions and transferred to cell plates. Plates are then incubated under the above conditions for 24 hours. Following the incubation period, the media/drug is aspirated, and 50 µL of resazurin is added. Plates are then incubated under the above conditions for 2 hours and then in the dark at room temperature for an additional 30 minutes. Fluorescence measurements are taken (excitation 535 nm, emission 590 nm) and toxic effects in treated versus control cells are compared based on the degree of fluorescence in each well. The results for minocycline and doxycycline are shown in Table 4. Compounds which show cytotoxicity at concentrations of less than about 35 µg/mL are indicated by "***," compounds which show cytoxicity at concentrations between about 35 and 75 µg/mL are indicated by "**," and compounds that show minimal or no cytoxicity are indicated by "*" (e.g., at concentrations greater than about 75 µg/mL).
The phototoxic potential of the tetracycline compounds used in the methods of the invention may be assessed in accordance with the following method. In particular, 3T3 fibroblast cells are harvested and plated at a concentration of 1 x 105 cells/mL and the plates are incubated overnight at 37°C, in 5% CO2 and approximately 95% humidity. On the following day the medium is removed from the plates and replaced with Hanks' Balanced Salt Solution (HBSS). Drug dilutions are made in HBSS and added to the plates. For each compound tested, a duplicate plate is prepared that is not exposed to light as a control for compound toxicity. Plates are then incubated in a dark drawer (for controls), or under UV light (meter reading of 1.6-1.8 mW/cm2) for 50 minutes. Cells are then washed with HBSS, fresh medium is added, and plates are incubated overnight as described above. The following day neutral red is added as an indicator of cell viability. The plates are then incubated for an additional 3 hours. Cells are then washed with HBSS and blotted on absorbent paper to remove excess liquid. A solution of 50% EtOH, 10% glacial acetic acid is added and after 20 minutes incubation, and the plate's absorbance at 535 nm is read using a Wallace Victor 5 spectrophotometer. The phototoxicity reflects the difference between the light-treated and control cultures. Results for the tetracycline derivative COL-3, as well doxycycline and minocycline are shown in Table 5 for comparison. Compounds which show phototoxicity are indicated by "****" (e.g., less than 5 µg/mL), compounds which show moderate phototoxicity are indicated by "***" (e.g., greater than about 5 µLg/mL and less than about 25 µg/mL), compounds which show some phototoxicity are indicated by "**" (e.g., greater than about 25 µg/mL and less than about 75 µg/mL) and compounds that show minimal or no phototoxicity are indicated by "*" (e.g., greater than about 75 µg/mL).
The half-life of minocycline and a tetracycline compound of the invention are assessed under oxidative conditions, as described in Nilges, et al. (Nilges M, Enochs W, Swartz H. J. Org. Chem. 1991, 56,-5623-30). Not to be limited by theory, it is believed that the tissue staining may be caused oxidative instability. The tetracycline compounds are subjected to accelerated oxidation in a continuous-flow microreactor using a 15 molar excess of sodium periodate at pH 11 and 22 °C. Aliquots of each reaction mixture are quenched at various time points with ascorbic acid and the disappearance of each compound is determined by RP-HPLC. Pseudo first-order rate constants and t1/2 values are obtained from the plots of log (Ao-At/Ao) versus time, where Ao is the HPLC area determined for each compound at time = 0 and At is the HPLC area at time = t. The results indicated that minocycline has a half-life for oxidation of 8.2 seconds.
The in vivo anti-bacterial activity of the tetracycline compounds used in the methods of the invention may be assessed in accordance with the following method.
Groups of five mice are injected intraperitoneally with a lethal dose of S. aureus RN450 in a medium of mucin. Mice are evaluated at 24 hours to determine survival. Untreated animals experience 100% mortality. Subcutaneous treatment with a single dose of minocycline or doxycycline results in 100% survival. In some instances, a dose response study is performed with the compound such that a PD50 (a dose of compound that protects 50% of the animals) could be calculated. The results for minocycline and doxycycline are shown in Table 6.
| Untreated | - | 0(0/5) | -- |
| Minocycline | 5 | 100 (5/5) | 0.72 |
| Doxycycline | 5 | 100 (5/5) | 0.13 |
To asses the anti-inflammatory potential of the tetracycline compounds used in the methods of the invention, the tetracycline compounds may be assessed in a model of carrageenan induced rat paw inflammation. The model uses a sub-plantar injection of carrageenan in the rat to induce an inflammatory response. The test compound or saline (control) is administered IP 30 minutes before a subplantar injection of carrageenan (1.5 mg/0.1 mL). Paw volume is measured (mm2) before subplantar injection and again 3 hours after the injection of carrageenan using a plethysmometer. Significant differences as determined by a Kruskal-Wallis One Way ANOVA are noted between the inflammation of the untreated controls versus treated animals (p = 0.5).
Claims (13)
- A substituted tetracycline compound of formula (VIIa): wherein R9m* is unsubstituted or substituted oxazolyl, unsubstituted or substituted oxadiazolyl, unsubstituted or substituted isoxazolyl, unsubstituted or substituted pyrazolyl, unsubstituted or substituted thiazolyl, methylimidazolyl, or -CONR9maR9mb; and R9ma is hydrogen and R9mb is hydroxyl, alkyl, hydroxyalkyl, aryl or alkoxy; or R9ma is alkyl and R9mb is alkyl or hydroxyl; or a pharmaceutically acceptable salt thereof.
- The compound of claim 1, wherein R9m* is unsubstituted or substituted oxazolyl.
- The compound of claim 1, wherein R9m* is unsubstituted or substituted oxadiazolyl.
- The compound of claim 1, wherein R9m* is unsubstituted or substituted isoxazolyl.
- The compound of claim 1, wherein R9m* is unsubstituted or substituted pyrazolyl.
- The compound of claim 1, wherein R9m* is unsubstituted or substituted thiazolyl.
- The compound of claim 1, wherein R9m* is methylimidazolyl.
- The compound of claim 1, wherein R9m* is -CONR9maR9mb, in which R9ma is hydrogen and R9mb is hydroxyl, hydroxyalkyl, alkoxy, phenyl, or alkyl.
- The compound of claim 1, wherein R9m* is -CONR9maR9mb, in which R9ma is alkyl and R9mb is alkyl or hydroxyl.
- The compound of claim 1, wherein said compound is selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
- The compound of claim 1, wherein said compound is: or a pharmaceutically acceptable salt thereof.
- A substituted tetracycline compound according to any preceding claim for use in a method for treating a tetracycline responsive state in a subject, optionally wherein the tetracycline responsive state is a baterial infection, a viral infection, a parasitic infection, malaria, an inflammatory process, acne, rosacea, or multiple sclerosis.
- A pharmaceutical composition comprising a therapeutically effective amount of a tetracycline compound according to any one of claims 1-11 and a pharmaceutically acceptable carrier.
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| US94300307P | 2007-06-08 |
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