WO2005082860A1 - Tetracyclines and their use as calpain inhibitors - Google Patents

Abstract

Tetracyclines are useful as calpain inhibitors, particularly inhibitors of calpain I and II, as demonstrated in enzymatic assays as well as at the cellular and animal levels. Tetracyclines may be used in the treatment of a wide range of conditions implicated by or associated with calpain activity or activation, including cellular protection from apoptosis and necrosis, particularly n eu ro protection, prevention of cell motility (e.g. anti-metastasis of cancer) and treatment of certain infectious diseases (e.g. malaria and AIDS). Some tetracyclines are particularly useful as calpain inhibitors since they are also antioxidants, oxidative stress often being associated with conditions where calpain is activated.

Description

TETRACYCLINES AND THEIR USE AS CALPAIN INHIBITORS

CROSS-REFERENCE APPLICATIONS

This application claims the benefit of United States Provisional Application 60/547,780 filed February 27, 2004 and United States Provisional Application 60/590,345 filed July 23, 2004, both herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to tetracyclines and their use as calpain inhibitors, particularly their uses in treating conditions implicated by or associated with calpain activity or activation.

BACKGROUND OF THE INVENTION

Calpains are cysteine proteases and are activated by Ca²⁺. At least 15 calpains (calpain 1-15) have been reported in mammals and are classified as 9 typical and 6 atypical calpains. Typical calpains have EF-hand Ca²⁺-binding domain at the C-terminal, while atypical calpains do not have such EF-hand domain. Calpains hydrolyze specific proteins at specific sites as Ca²⁺-dependent modulators and are often involved in signaling pathways at irreversible steps. The physiological roles of calpains are not fully understood, some of them play critical roles in cell proliferation, cell cycle progression, cell differentiation, gene expression, cytoskeletal reorganization, cell motility, apoptosis, necrosis and etc. Especially, calpains I and II are expressed in all tissues and are activated by Ca²⁺ at ~50 μM and ~200 μM, respectively. In neural cells, neural damage such as ischemia induces calcium influx into the neural cells and activates calpain, resulting in cell death.

In vitro and in vivo effects of calpain inhibitors have been examined for several diseases or disease models. Calpains are capable of degrading critical cytoskeletal and regulatory proteins, mainly causing post-ischemic neuronal necrosis. Calpain inhibitors protect neurons from various damages such as traumatic brain injury, traumatic spinal cord injury, stroke, Wallerian degeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease, damage to motoneurons from exocitotoxic cell death, axonal degeneration and peripheral neuropathy.

Calpain inhibitors also protect other cells from apoptosis and necrosis and reduce acute and chronic injuries or dysfunctions of organs, such as muscular dystrophy, Duchenne muscular dystrophy, rheumatoid arthritis, diabetic retinopathy, acoustic trauma, virus-induced myocardial injury, acute myocardial infarction, testicularlorsion, liver ischemia, kidney ischemia, inflammatory bowel disease and sinusoidal endothelial cell apoptosis in liver transplantation.

Cell motility occurs in various events such as repair from injury. Cell motility is also involved in diseases such as cancer metastasis and angiogenesis. Calpains are required for deadhesion of the tail during both haptokinesis and chemokinesis. Thus, calpain inhibitors reduce cancer metastasis.

Calpains are involved in certain infectious diseases and calpain inhibitors block the replication of HIV-1 and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) as well as propagation of Creutzfeldt Jacob disease, Calpain inhibitors also treat malaria.

Contrary to their protective effects against cell apoptosis and necrosis, calpain inhibitors induce apoptosis in leukemia and in tumor cell lines in gene therapy.

Tetracyclines are a family of compounds that are structurally related to a natural product tetracycline from Streptomyces species. Most of natural or semi- synthetic tetracyclines share a core structure of four linear fused rings, as labeled the ABCD rings, where only the D-ring is aromatized. The numbering system for the core structure is shown below with tetracycline:

Tetracyclines have excellent properties of absorption, distribution, metabolism, and excretion as well as very low toxicity and side effects. They are readily absorbed orally and distributed throughout the body including brain with a half-life of several hours. Over 7,000 natural and semi-synthesized tetracyclines have been reported. Most of them are aimed at improving the antimicrobial activity, and minocycline and doxycycline are the successful examples being used clinically. A series of 4-dedimethylamino tetracyclines (CMTs) were reported as non-antimicrobial MMP inhibitors, except CMT-5, an 11 ,12-pyrazolo derivative of tetracycline.

Some tetracyclines show bacteriostatic activity and inhibit protein synthesis. More specifically, tetracyclines inhibit the binding of aminoacyl-tRNA to bacterial ribosomes. Tetracyclines can also inhibit bacterial growth by affecting membrane integrity and mechanical properties. Some tetracyclines are bacteriocidal by inhibiting all cellular processes and macromolecular synthesis pathways. Several activities and therapeutic applications of tetracyclines have been reported [Nelson et al., 2002]. At the molecular level, some tetracyclines inhibit MMPs, stromelysin, α protease inhibitor degradation, phospholipases A₂, prostaglandins/cytokines, plasminogen activator, protein kinase C, macrophage/polymorphonuclear elastase, oxy radicals, and protein glycation, and affect NO production and collagen metabolism. Some tetracyclines inhibit/enhance cytokine production. At the cellular systems, some tetracyclines inhibit angiogenesis, cell proliferation, cell invasion/migration, cell attachment, cellular metabolism, bone resorption/osteoclasts, cartilage degradation, leukocyte function, collagen synthesis, cathepsin L, and affect T-cell activation, non-specific proteolysis, and polymorphonuclear leukocytes cell function. At animal models/diseases, some tetracyclines are indicated to be effective on diabetic rat, arthritic rat, rat caries, rabbit cornea, osteoporosis, osteoarthritis, lung injury, wound healing, ischemia/neurologic, apoptosis, ischemia/reperfusion, aneurysm/vascular, periodontal disease, metabolic bone disease, and cancer. At clinical studies, some tetracyclines show therapeutic effects on adult periodontal disease, rheumatoid arthritis, osteoarthritis, aneurysm, and cancer.

SUMMARY OF THE INVENTION According to the present invention, there is provided a use of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof for inhibiting calpain activity or activation.

There is further provided a use of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof for treating or preventing a condition associated with calpain activation or activity.

There is yet further provided a novel tetracycline derivative of formula I:

)

wherein:

Y is CH-C(R°ZW), or CR >9^S _DR10.

Z and W are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-Cβ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Cis aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, hydroxyl, CrC₈ alkoxy, sulfhydryl, Cι-C₈ alkylthio, C C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, CrC₈ alkylamino, cyano or halogen;

R⁴ is NR⁵R⁶, hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C7-C1₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl or halogen;

R¹, R², R⁵ and R⁶ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, CrC₈ alkoxy, Cι-C₈ alkylthio, C-ι-C₈ alkylsulfynyl, C-ι-C₈ alkylamino, C₆-C-ι₈ aryl, C₇-C₁₉ arylalkyl or C₂-Cι₈ heteroaryl, or R¹ and R² form a 5- or 6-membered nitrogen-containing ring, or R⁵ and R⁶ form a 5- or 6-membered nitrogen-containing ring;

R³, R¹³ and R¹⁴ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Cis aryl, C7-C1₉ arylalkyl or C₂-Cι₈ heteroaryl;

R⁷ is hydrogen, Cι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C6-C-ι₈ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl, CrC₈ alkoxy, sulfhydryl, Cι-C₈ alkylthio, CrC₈ alkylsulfynyl, C C₈ alkylsulfonyl, C C₈ alkylamino, halogen, CrC₈ alkanoyl, C₇-C₁₉ aralkanoyl, C₇-C₁₉ alkaroyl, Cβ-Ciβ aroyl, C₂-C-ι₈ heteroaroyl, C₂-C₈ alkylcarbonyloxy or C7-C₁₉ arylcarbonyloxy;

R⁸ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C-|₈ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, CrC₈ alkoxy, sulfhydryl, C C₈ alkylthio, C-i-C₈ alkylsulfynyl, C-ι-C₈ alkylsulfonyl or CrC₈ alkylamino;

R⁹ and R¹⁰ are each independently hydrogen, =CH₂, absent, hydroxyl, halogen, sulfhydryl, Cι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Ciβ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, Cι-C₈ alkoxy, CrC₈ alkylthio, CrC₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl or C C₈ alkylamino;

R¹¹ and R¹² are each independently hydrogen, C C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₇-C₁₉ arylalkyl, C₈-C₂o aralkenyl, C_S-C₂₀ aralkynyl, C₆-Cι₈ aryl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, C-i-C₈ alkoxy, sulfhydryl, CrC₈ alkylthio, C-ι-C₈ alkylsulfynyl, C-ι-C₈ alkylsulfonyl, amino, Cι-C₈ alkylamino, C₂-Cιβ dialkylamino, Cι-C₈ acyl, nitro, cyano, carboxyl or CrC₈ alkoxycarbonyl; dotted lines between C11 and C12 are each independently bonds within the tetracycline ring structure or bonds from the tetracycline ring structure to X², X³ or X⁴;

X¹ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, d-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, halogen, nitro, C-ι-C₈ alkoxy, C₈-Cι₈ aryloxy, amino, CrC₈ alkylamino, C₂-C₁₆ dialkylamino, Cι-C₈ alkylamido or Cι-C₈ acyl;

X² is hydrogen, halogen, hydroxyl, CrC₈ alkoxy, mercapto, C-ι-C₈ alkylthio, phenoxy, C₆-C-ι₂ aryloxy, phenyl, amino, CrC₈ alkylamino, CrC₈ amido or Cι-C₈ acyl, or X² is oximino or oxo when the dotted line at C11 forms a bond with X²;

X³ and X⁴ are each independently hydrogen, halogen, hydroxyl, C-ι-C₈ alkoxy, mercapto, C C₈ alkylthio, phenoxy, phenyl, C₆-Cι₂ aryloxy, amino, C-ι-C₈ alkylamino, Cι-C₈ amido or C C₈ acyl, or X³ and X⁴ are joined to form oxo, or X⁴ is absent when the dotted line at C12 is a bond within the tetracycline ring structure, or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

X⁵ and X⁶ are independently hydrogen, halogen, hydroxyl, CrC₈ alkoxy, mercapto, Cι-C₈ alkylthio, C₆-Cι₂ aryloxy, C₆-C1₂ aryl, C₂-Cι₈ heteroaryl, amino, C C₈ alkylamino, CrC₈ amido, Cι-C₈ acyl, or X⁵ and X⁶ are joined to form oxo, or X⁶ is a bond from the tetracycline ring to X⁵ when X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

and pharmaceutically acceptable salts thereof,

with the proviso that either: X² is oximino or Cι-C₈ alkylamino; or X⁴ is amino; or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring. DETAILED DESCRIPTION

Compounds:

Preferred tetracyclines for use as calpain inhibitors are 7-substituted tetracyclines, 11 -substituted tetracyclines, 12-substituted tetracyclines and their pharmaceutically acceptable salts. Of the 7-substituted tetracyclines, 7-halo tetracyclines (e.g. 7-chloro, 7-bromo, 7-iodo and 7-fluoro) are preferred, with 7-chloro tetracyclines more preferred. Of the 11- or 12-substituted tetracyclines, substitution groups that block binding of cations between the 11- and 12-positions are preferred. Particularly preferred are tetracyclines having an amino group in the 12-position, an oximino group or a CrC₈ alkylamino group in the 11 -position, and/or bridging groups between the 1- and 12-positions.

Tetracyclines of formula II are particularly preferred calpain inhibitors:

wherein:

Y is CH-C(R°ZW), .

or CR j9^a _DR1 π0u.

Z and W are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, hydroxyl, CrC₈ alkoxy, sulfhydryl, C C₈ alkylthio, CrC₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, C C₈ alkylamino, cyano or halogen; R⁴ is NR⁵R⁶, hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-C s aryl, C7-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl or halogen;

R¹, R², R⁵ and R⁶ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C-ι-C₈ alkoxy, C-i-C₈ alkylthio, C C₈ alkylsulfynyl, Cι-C₈ alkylamino, C₆-Cι₈ aryl, C₇-C₁₉ arylalkyl or C₂-Cιs heteroaryl, or R¹ and R² form a 5- or 6-membered nitrogen-containing ring, or R⁵ and R⁶ form a 5- or 6-membered nitrogen-containing ring;

R³, R¹³ and R¹⁴ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C7-C1₉ arylalkyl or C₂-Cι₈ heteroaryl;

R⁷ is hydrogen, C C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Ciβ aryl, C7-C₁₉ arylalkyl, C2-C-ι₈ heteroaryl, hydroxyl, C-i-C₈ alkoxy, sulfhydryl, Cι-C₈ alkylthio, Cι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, C C₈ alkylamino, halogen, C C₈ alkanoyl, C7-C₁₉ aralkanoyl, C₇-C₁₉ alkaroyl, Cβ-C-iβ aroyl, C₂-C-ι₈ heteroaroyl, C₂-C₈ alkylcarbonyloxy or C₇-C-₁₉ arylcarbonyloxy;

R⁸ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-C-iβ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, CrC₈ alkoxy, sulfhydryl, C C₈ alkylthio, C C₈ alkylsulfynyl, C-ι-C₈ alkylsulfonyl or Cι-C₈ alkylamino;

R⁹ and R¹⁰ are each independently hydrogen, =CH₂, absent, hydroxyl, halogen, sulfhydryl, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-ι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C-ι₈ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, CrC₈ alkoxy, C C₈ alkylthio, CrC₈ alkylsulfynyl, CrC₈ alkylsulfonyl or Cι-C₈ alkylamino;

R¹¹ and R¹² are each independently hydrogen, C-ι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C7-C₁₉ arylalkyl, C₈-C₂o aralkenyl, C₈-C₂o aralkynyl, Cβ-Ciβ aryl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, C-ι-C₈ alkoxy, sulfhydryl, C C₈ alkylthio, C-ι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, C C₈ alkylamino, C₂-C16 dialkylamino, CrC₈ acyl, nitro, cyano, carboxyl or C-ι-C₈ alkoxycarbonyl;

dotted lines between C11 and C12 are independently bonds within the tetracycline ring structure or bonds from the tetracycline ring structure to X², X³ or X⁴;

X¹ is hydrogen, Cι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C-ι₈ aryl, C₇-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, halogen, nitro, CrC₈ alkoxy, C₆-C₁₈ aryloxy, amino, Cι-C₈ alkylamino, C₂-Cι₆ dialkylamino, CrC₈ alkylamido or C C₈ acyl; X² is hydrogen, halogen, hydroxyl, C C₈ alkoxy, mercapto, CrC₈ alkylthio, phenoxy, C6-C12 aryloxy, phenyl, amino, CrC₈ alkylamino, Cι-C₈ amido or C-ι-C₈ acyl, or X² is oximino or oxo when the dotted line at C11 forms a bond with X², or X² is joined with X³ to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure when the dotted line at C11 forms a bond with X²;

X³ and X⁴ are independently hydrogen, halogen, hydroxyl, C C₈ alkoxy, mercapto, Cι-C₈ alkylthio, phenoxy, phenyl, C6-C12 aryloxy, amino, C-ι-C₈ alkylamino, CrC₈ amido or C-ι-C₈ acyl, or X³ and X⁴ are joined to form oxo, or X³ is joined with X² to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure when the dotted line at C11 forms a bond with X², or X⁴ is absent when the dotted line at C12 is a bond within the tetracycline ring structure, or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

X⁵ and X⁶ are independently hydrogen, halogen, hydroxyl, CrC₈ alkoxy, mercapto, Cι-C₈ alkylthio, C₆-C12 aryloxy, C₆-C-i₂ aryl, C₂-Cι₈ heteroaryl, amino, C-ι-C₈ alkylamino, Cι-C₈ amido, C-ι-C₈ acyl, or X⁵ and X⁶ are joined to form oxo, or X⁶ is a bond from the tetracycline ring to X⁵ when X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

and pharmaceutically acceptable salts thereof. In both compounds of formula I and formula II, the following preferred definitions are considered.

Heteroatoms in the substituted alkyl, alkenyl or alkynyl groups are preferably nitrogen, oxygen, sulfur or a combination thereof.

Halogens are preferably fluoro, bromo, iodo or chloro, more preferably chloro.

The fused rings are preferably a pyrazole or a pyridine, more preferably a pyrazole. Non-limiting examples of alkyl are methyl, ethyl, n-propyl, i-propyl, n-butyl, t- butyl, n-pentyl, etc. Non-limiting examples of alkenyl are ethenyl, propenyl, but-1- enyl, etc. Non-limiting examples of alkynyl are ethynyl, propynyl, but-1-ynyl, etc. Example of aryl are phenyl and naphthyl, etc. Non-limiting examples of heteroaryl are pyrazolyl, pyridinyl, thienyl, furyl, etc.

Pharmaceutically acceptable salts include acid and base addition salts. Acid addition salts are preferred. Acid addition salts include protic acids having anions such as, for example, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate.

More preferred are 7-substituted, 11 -substituted and/or 12-substituted tetracyclines and their pharmaceutically acceptable salts. Of the 7-substituted tetracyclines, 7-halo tetracyclines (e.g. where X¹ is chloro, bromo, iodo or fluoro) are preferred, with 7-chloro tetracyclines more preferred. Of the 11 - or 12- substituted tetracyclines, substitution groups that block binding of cations between the 11- and 12-positions are preferred. Preferred 11 -substituted tetracyclines are compounds in which X² is oximino or C-ι-C₈ alkylamino. A preferred 12-substituted tetracycline is a compound in which X³ is amino and the broken line at the 12- position is a bond in the tetracycline ring. Another preferred 12-substituted tetracycline is a compound in which X⁴ and X⁵ are joined to form a 5- or 6- membered nitrogen-containing ring fused to the tetracycline ring structure (i.e. there is a bridging group between the 1- and 12-positions. The fused ring is preferably a pyrazole ring where three carbon atoms in the tetracycline ring form a ring with a nitrogen atom bonded at the 1 -position and another nitrogen atom bonded at the 12-position.

Y is preferably CR⁹R¹⁰ or C=CR⁸Z. R⁹ and R¹⁰ are preferably each independently hydrogen, hydroxyl or Cι-C₈ alkyl, or R⁹ is =CH₂ and R¹⁰ is absent. CrC₈ alkyl is preferably methyl. R⁸ and Z are preferably hydrogen. R¹ and R² are preferably hydrogen, or R¹ and R² together form a 5-membered nitrogen-containing ring. The 5-membered nitrogen-containing ring is preferably pyrrolidino.

R³, R¹³ and R¹⁴ are preferably hydrogen. R⁴ is preferably NR⁵R⁶ or hydrogen. R⁵ and R⁶ are prefereably hydrogen or

Cι-C₈ alkyl, more preferably methyl.

R⁷ is preferably hydrogen or hydroxyl.

R¹¹ and R¹² are preferably each independently hydrogen or halogen. More preferably, R¹¹ is hydrogen and R¹² bromo. Compounds of the formula II include either the (+) or (-) stereoisomers or a mixture of both the (+) and (-) stereoisomers.

Some specific tetracyclines are as follows:

12-aminochlortetracycline rolitetracycline

meclocycline 12-aminodemeclocycline

chlortetracycline (CTC) 11-oximinochlortetracycline

12-aminodoxycycline CMT-3

H₃

12-aminominocycline 12-aminosancycline

,12-pyrazolochlortetracycline (CTP) 11-(N-methylamino)-minocycline

doxycycline oxytetracycline

CMT-1 demeclocycline (DMC)

tetracycline minocycline

sancycline 11 ,12-pyrazolotetracycline (CMT-5)

,12-pyrazolominocycline 12-hydroxy-1 ,12-pyrazolotetracycline

12-hydroxy-1 ,12-pyrazolominocycline 12-aminotetracycline

7-aminosancycline 7-nitrosancycline

7,9-dibromosancycline 7-bromosancycline

CMT-4 7-iodosancycline

7-(2-thienyl)sancycline 7-phenylsancycline

7-chlorosancycline CMT-10 Semi-synthetic tetracyclines are commercially available or may be prepared according to known methods. The synthesis of various semi-synthetic tetracyclines is extensively documented, for example, Mitscher L A, The Chemistry of the Tetracycline Antibiotics. Ch. 6, Marcel Dekker, New York (1978) and Nelson, M. et al, eds., Tetracyclines in Biology. Chemistry and Medicine. Veriag, Boston (2001 ), pp. 3-64, the disclosures of which are herein incorporated by reference.

Substituted tetracycline compounds can be synthesized by using methods described in the Synthetic Methods section below, by using techniques readily recognized in the art, and/or by methods of the following Schemes 1-9.

Scheme 1

As shown in Scheme 1 , 4-dedimethylaminotetracyclines (1 B) may be synthesized by treating a tetracycline compound with iodomethane to form a methiodide (1A), the methiodide being subsequently treated with zinc and acetic acid. Scheme 2

As shown in Scheme 2, 12-aminotetracyclines (2A) can be synthesized by reacting ammonia gas with a tetracycline.

For example, anhydrous ammonia is reacted with a tetracycline and the product isolated and purified using known procedures. In particular, anhydrous ammonia is bubbled into a stirred solution or suspension of a known tetracycline (either as hydrochloride or free base) in absolute ethanol under reflux. Stirring is continued at room temperature, then the reaction mixture is directly filtered to remove insoluble material, or is brought to pH 7 with dilute acetic acid and then filtered. Solvents in the filtrate are partially removed under reduced pressure, and the resulting solid is separated using a combination of column chromatography and high performance liquid chromatography (HPLC) or high performance displacement chromatography (HPDC). Alternatively, the solid material is extracted with dichloromethane and recrystallized from an appropriate solvent or mixture of solvents.

Scheme 3

3A

3B

As presented in Scheme 3, tetracyclines having a pyrazole ring fused to the tetracycline ring structure may be synthesized by treating a tetracycline with hydrazine to form a 11 ,12-pyrazolotetracycline (3A) and/or a 12-hydroxy-1 ,12- pyrazolinotetracycline (3B).

For example, a tetracycline hydrochloride is reacted with hydrazine hydrate. Both 1 ,12- and 11 ,12- pyrazolotetracyclines are produced and may be isolated and purified by known techniques. In particular, a tetracycline hydrochloride dissolved in a lower alcohol or water is treated with hydrazine hydrate in excess (2-10 mmole) under reflux or at room temperature for several hours or overnight, respectively. The alcoholic solvent is removed in vacuo to give a material that is treated with water under stirring to afford a precipitate that is filtered. Alternatively, the evaporation of water in reactions conducted in this particular solvent leads to a solid material. Preparative HPLC separation of the resulting solid material affords both 11 ,12-pyrazolotetracyclines and 12-hydroxy-1 ,12-pyrazolinotetracyclines. Scheme 4

4A

As shown in Scheme 4, 11-oximinotetracyclines (4A) may be synthesized by reacting hydroxylamine hydrochloride with a tetracycline compound.

For example, a tetracycline hydrochloride is reacted with hydroxylamine hydrochloride in the presence of a base (e.g. triethylamine). The resulting product is isolated and purified using known techniques. In particular, a tetracycline hydrochloride and hydroxylamine hydrochloride are dissolved in water and treated with triethylamine. The reaction mixture is stirred at 60°C under nitrogen for several hours. The crude mixture is filtered and the solvent removed to give a solid that is separated using preparative HPLC.

Scheme 5

5A

As shown in Scheme 5, 11-methylaminotetracyclines (5A) may be synthesized by reacting methylamine with a tetracycline compound.

For example, a tetracycline hydrochloride is reacted with methylamine and the resulting product isolated and purified by known techniques. In particular, a tetracycline hydrochloride in absolute ethanol is treated with methylamine (30-100- fold excess, 33% solution in absolute ethanol) under reflux for several hours. The solvent is removed in vacuo, and the resulting solid separated using preparative HPLC.

Scheme 6

6B

As shown in Scheme 6, 7-aryl or 7-heteroaryl tetracyclines (6B) may be synthesized through a Suzuki coupling of an arylboronic (or heteroarylboronic) acid with a 7-iodotetracycline. A 7-iodotetracycline (6A) may be synthesized by treating a tetracycline compound with at least one equivalent of N-iodosuccinimide under acidic conditions. To form an aryl derivative, a 7-iodotetracycline (6A) is treated with a base (e.g. Na₂CO₃) and the appropriate boronic acid in the presence of a palladium catalyst (e.g. Pd(OAc)₂). Scheme 7

As shown in Scheme 7, 9- and 7-substituted tetracyclines may be synthesized by treating a tetracycline compound with sulfuric acid and sodium nitrate. The resulting product is a mixture of 7-nitro and 9-nitro isomers (7A and 7B, respectively). The 7-nitro (7A) and 9-nitro (7B) tetracyclines are treated by hydrogenation using hydrogen gas and a palladium catalyst to yield 7-amino (7C) and 9-amino (7D) tetracyclines. Isomers are separated by conventional methods. Scheme 8

8A

As shown in Scheme 8, 7-chloro substituted tetracyclines may be synthesized by treatiing a 7-amino substituted tetracycline with butyl nitrite and copper (I) chloride in anhydrous acetonitrile. The product (8A) may be purified by known methods (e.g. HPLC).

Scheme 9

9B

As shown in Scheme 9, 7-bromo and 7,9-dibromo substituted tetracyclines (9A and 9B, respectively) may be synthesized by treating a tetracycline compound with N-bromosuccinimide under acidic conditions. The ratio of the tetracycline compound to N-bromosuccinimide determines the ratio of 7-bromo and 7,9-dibromo substituted tetracyclines. Uses:

Conditions associated with calpain activation or activity that are treatable or preventable by tetracyclines include neurological and non-neurological conditions. Neurological conditions include, for example, traumatic brain injury, traumatic spinal cord injury, stroke, Wallerian degeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease, damages of motoneurons from exocitotoxic cell death, axonal degeneration, and peripheral neuropathy. Non- neurological conditions include, for example, inflammation, severe hemorrhage, muscular dystrophy, Duchenne muscular dystrophy, rheumatoid arthritis, diabetic retinopathy, acoustic trauma, virus-induced myocardial injury, acute myocardial infarction, testicular torsion, liver ischemia, kidney ischemia, inflammatory bowel disease and sinusoidal endothelial cell apoptosis liver transplantation.

Tetracycline calpain inhibitors may also prevent cell motility, which is useful for metastasis of prostate cancer, lung cancer, and renal cancer as well as for inhibiting angiogenesis. Calpain inhibitors may also help prevent infectious diseases such as HIV-1 replication, replication of severe acute respiratory syndrome-associated coronavirus, invasion of erythrocytes by P. falciparum, and prion propagation in Creutzfeldt Jacob disease. Calpain inhibitors may also induce caspase-dependent apoptosis in human acute lymphoblastic leukemia and non- Hodgkin's lymphoma cells, activate p53-dependent apoptosis in tumor cell as well as retard cataractogenesis, and block platelet secretion, aggregation, and spreading during platelet activation events.

Tetracyclines are particularly useful in treating or preventing calpain- mediated physiological damage induced by calcium activation of calpain in a subject. Preferably, tetracyclines are useful in the inhibition of calpain I, calpain II, or both calpain I and calpain II.

Antioxidant tetracyclines are particularly useful calpain inhibitors since calpain activation is often associated with oxidative stress. Therefore, calpain inhibitors having additional antioxidant properties would be advantageous since administration of a separate antioxidant would not be required. Therefore, in the treatment or prevention of calpain-mediated physiological damage, antioxidant tetracyclines advantageously permit the effective use of fewer drugs. In addition, lower doses of tetracyclines may be used since the anti-oxidant activity may work synergistically with the calpain inhibition. In chronic diseases, glycation occurs under oxidative stress and damages cells and tissues. Thus, the anti-glycation activity of certain tetracyclines may protect cells and tissues synergistically with calpain inhibition. Tetracyclines having a bridging group between the 1- and 12-positions are particularly noteworthy in this regard.

Certain tetracyclines have no antibiotic, no MMP inhibitory and/or no Ca²⁺ binding properties. Such tetracyclines are particularly preferred since they present fewer side effects. Tetracyclines substituted in the 11- and/or 12-positions are particularly noteworthy. These tetracyclines include those in which only the 11 -position is substituted, in which only the 12-position is substituted, in which the 11 -position is joined to the 12-position and in which the 1 -position is joined to the 12-position (e.g. where X² and X³ are joined or where X⁴ and X⁵ are joined). Of the tetracyclines in which the 11-, 12- and 1 -positions are not joined, 12-amino tetracyclines are of particular note.

Subjects that may benefit from tetracycline therapy for calpain-mediated physiological damage are preferably mammals, for example, primates (humans, monkeys, gorillas, etc., canines (domestic dogs, wolves, etc.), felines (e.g. domestic cats, lions, tigers, etc.) and rodents (e.g. rats, mice, etc.). Subjects are more preferably humans.

Calpain activation or activity can be inhibited by contacting at least one calpain with a calpain-inhibiting effective amount of a tetracycline. The methods may be carried out in vivo or in vitro with purified enzymes, cells, tissues or whole animals; with one or more calpains; and with one or more tetracyclines. Additionally, the methods may employ a tetracycline in combination with other therapeutic agents, such as other tetracyclines, other calpain inhibitors or other therapeutic agents. In a clinical setting, a subject at risk of suffering calpain-mediated physiological damage is first identified and then provided with a calpain inhibiting effective amount of a tetracycline. The tetracycline may be administered as a prophylactic (i.e. preventive) measure or as a post-event treatment. Identification of an at-risk subject may include diagnosing a subject with a condition or an impending condition associated with calpain induced physiological damage.

As a prophylactic measure, a human subject demonstrating signs of an impending stroke may be administered a tetracycline disclosed herein. Identification of an at-risk subject may also include choosing an individual research subject for experimental purposes. For example, a rat may be selected to receive treatment intended to induce a stroke and then administered a calpain inhibitor disclosed herein.

As a treatment, a tetracycline may be administered to a subject following an actual event implicating activation of calpain (e.g. angina, cataract, myocardial infarction, stroke, or recognition of calcium activation of calpain) within the subject, thus putting the subject at risk of suffering calpain-mediated physiological damage. For example, a human subject who recently suffered a cardiovascular ischemic event (e.g., heart attack or stroke) may be administered a therapeutically effective amount of pharmaceutical composition that includes a tetracycline calpain inhibitor. The composition may be administered within several hours of the event precipitating calpain-mediated pathologies.

The pharmaceutical composition may include one or more tetracycline calpain inhibitors, or pharmaceutically acceptable salts thereof, together with a pharmaceutically compatible carrier, agent, excipient, adjuvant, vehicle or combination thereof, a calpain inhibitor of a different class (in addition to the tetracycline calpain inhibitor), a drug having a different therapeutic indication, or a combination thereof.

Providing a pharmaceutical composition to a subject includes methods of administering that composition. Routes of administration include, but are not limited to, oral and parenteral routes, such as intravenous (IV), intraperitoneal (IP), rectal, topical, ophthalmic, nasal, and transdermal. For oral administration, the pharmaceutical compositions are generally provided or administered in the form of a unit dose in solid, semi-solid, or liquid dosage forms such as tablets, pills, powders, liquid solutions, or liquid suspensions. However, the drugs also may be administered intravenously in any conventional medium for intravenous injection, such as an aqueous saline medium, or in a blood plasma medium. The medium also may contain conventional pharmaceutical adjunct materials, such as pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers (e.g., cyclodextrins), proteins (e.g., serum albumin), hydrophilic agents (e.g., methyl cellulose), detergents, buffers, preservatives and the like. A more detailed explanation of acceptable pharmaceutical adjunct materials can be found in Remington: The Science and Practice of Pharmacy (19^th Edition, 1995) in chapter 95.

Therapeutically effective amounts of tetracycline calpain inhibitors may be determined in different ways. For example, the effective amount can be determined based on (1 ) in vitro inhibition of calpain using Ac-Leu-Leu-Tyr-AFC as the substrate for calpain activity with fluorescence measured with a 400 nm excitation filter and 505 nm emission filter with or without isolating the product AFC by HPLC, (2) effective protection of cultured cerebellar granule neurons against glutamate toxicity, (3) effective reduction of brain damage caused by the occlusion of the middle cerebral artery, and (4) effective improvement of neurological behavior of ischemic animals. In other embodiments, the effective amount may be reduced further due to the anti-oxidant activities of the tetracycline.

Therapeutically effective doses of the calpain inhibitors disclosed herein may be provided to a subject for a short period of time. This period of time may be measured after a diagnosis that the subject is at risk for calpain-mediated physiological damage, or after a particular ischemic event, such as a cardiovascular ischemic event. The duration of treatment may be, for example, less than about a month, two weeks, one week, or even less than about 72 hours. For example, a patient suffering a stroke can be provided a therapeutically effective dose of a tetracycline for about 72 hours or less. Alternatively, the therapeutically effective dosage may be provided for a period of time from about 6 to about 72 hours. However, the duration of therapy with the calpain inhibitors disclosed herein can also be prolonged, for example, in the treatment of chronic angina or recurrent transient ischemic attacks (TIA's). Moreover, administration can be repeated, but intermittent (for example, following an episode of angina or TIA), even though intermittent or episodic administration would be avoided in an antiviral treatment because it could lead to the development of viral drug resistance.

The specific dose level, frequency of dosage, and duration of treatment for any particular subject may be varied and will depend upon a variety of factors, including: the activity of the specific pharmaceutical composition; the metabolic stability and length of action of that composition; the age, body weight, general health, gender, diet, and other characteristics of the subject; mode and time of administration; the rate of excretion; drug combination parameters; and severity of the condition of the subject undergoing treatment.

Oral administration is one of the preferred routes of delivery. Liquid or solid (e.g., tablets, gelatin capsules) formulations can be employed. Parenteral delivery (e.g., intravenous, intramuscular, subcutaneous injection) is another preferred route of delivery. Preferably, oral and parental compositions comprise the active tetracycline, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable carrier, agent, excipient, adjuvant, vehicle, or combination thereof.

Alternatively, delivery of the active tetracycline may be by topical application. Suitable topical applications include, for example, gels, salves, lotions, ointments, drops and the like.

Another preferred route of delivery is via a slow-release delivery vehicle, e.g., a polymeric material, surgically implanted at or near the lesion sites.

According to an advantageous embodiment of the invention, tetracyclines and/or tetracycline compositions (i.e. the active agent) may be used as a part of a combination therapy with other therapeutic agents or therapeutic techniques to achieve the optimal result to prevent and suppress the negative results of a brain stroke and/or spinal injury. For example, the active agent can be administered during an ambulance ride to a hospital. This early administration can be combined, for example, with administration of a thrombolytic drug (like Activase™, a recombinant tissue plasminogen activator, tPA, available from Genentech). The administration of the active agent can then be continued in combination with hospital procedures, such as neurointerventional procedures, when, for example, a rheolytic or laser-based clot removal device is used for the treatment of occlusive stroke. Such devices are, for example, a pulsed-dye laser system of LATIS™ (available from Horsham in Pennsylvania, USA) and ANGIOJET™ rheolytic thrombectomy system of Possis Medical (of Minneapolis, Minn., USA). After clot removal, the treated artery can be equipped with a stent to keep the treated artery open. Advantageously, the stent can be coated with a polymer layer releasing the active agent into the wall of the artery and through it into the ischemic tissue. The stent can also be manufactured of a bioabsorbable polymer releasing the active agent and possibly other therapeutic agents into the wall of the artery. The active agent can be administered also in combination with Retrograde Transvenous Neuroperfusion (RTN) (of Neuroperfusion, Irvine, Calif., USA) where oxygenated blood from the femoral artery in the leg is pumped back into the brain via the jugular vein. Ultimately, this blood reaches the ischemic tissue affected by the stroke, providing there oxygen and the active agent.

Calpain inhibiting tetracyclines may be used to manufacture a medicament for inhibiting calpain activity or for treating or preventing a condition associated with calpain activation or activity. A calpain inhibiting tetracycline, or a composition comprising the tetracycline, may be packaged in a commercial package comprising the tetracycline, or a composition comprising the tetracycline, together with instructions for its use for inhibiting calpain activity or for treating or preventing a condition associated with calpain activation or activity. Further features of the invention will be described or will become apparent in the course of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, preferred embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which: Figures 1 A-1 F provide graphical evidence showing that chlortetracycline

(CTC) and demeclocycline (DMC) inhibit activity of calpain I and calpain II in vitro (Fig 1A and Fig 1B), in glutamate-treated cultured cerebellar granule neurons (CGNs) (Fig 1 C and Fig 1 D), and in ischemic brain (Fig 1 E and Fig 1 F) (NB, normal brain; c, contralateral side; I, ischemic side of the brain); Figure 1G is a graph showing inhibition of calpain II by various tetracyclines;

Figure 1 H is a graph showing inhibition of calpain I by various tetracyclines

Figure 2 provides graphical and pictorial evidence that tetracyclines provide neuroprotection in glutamate-treated cultured cerebellar granule neurons (CGNs);

Figure 3 provides graphical and pictorial evidence that chlortetracycline (CTC) and demeclocycline (DMC) reduce cerebral infarction (Fig 3A-3D) and improve neurological behaviour in middle cerebral artery occlusion (MCAO) mice (Fig 3E);

Figure 4 provides graphical evidence showing that chlortetracycline (CTC) and demeclocycline (DMC) weakly antagonize NMDA receptor (Fig 4A) and calcium influx (Fig 4B) in cultured CGNs; and,

Figure 5 provides graphical evidence of non-antibiotic activity of 11- and/or 12-substituted tetracyclines. EXAMPLES

Example 1 : Enzymatic assays of the inhibitors of calpains I and II by tetracyclines

The effects of tetracyclines on calpain activity were evaluated by an in vitro assay. Calpain activity was measured using a calpain activity assay kit (Calbiochem, Mississauga, ON, Canada) following the manufacturer's instructions. The assay is based on fluorometric detection of cleavage of calpain substrate Ac- Leu-Leu-Tyr-AFC using a Cytofluor™ 2350 Fluorescence Measurement System (Millipore). Cleavage results in the release of AFC that can be measured in a fluorometer. Briefly, constitutive calpain I (0.1 U/ml) or calpain II (0.2 U/ml) (purchased from Calbiochem) was activated by Ca²⁺ (500 μM in the final assay solution) and was mixed with chlortetracycline (CTC) (150 μM), demeclocycline (DMC) (150 μM), minocycline (150 μM) or calpain inhibitor ALLN (10 μM) and 5 μl of calpain substrate to a final volume of 100 μl. The mixture was incubated at 37°C for 1 h in the dark. The cleavage of the substrate resulted in the release of AFC that can be detected by a fluorometer at an excitation of 400 nm and emission of 505 nm.

Referring to Fig 1 , in vitro experiments were performed using purified exogenous calpains. Purified exogenous calpain I (0.1 U/ml) and calpain II (0.2 U/ml) were mixed with calpain specific inhibitor ALLN or the compounds as indicated at 150 μM concentration. After 1 hour incubation with the calpain substrate, the release of fluorescent AFC was recorded. The fluorescent unit per μg protein per h was calculated from at least three independent experiments and plotted in Fig 1A (calpain I) and Fig 1 B (calpain II) (mean ± SEM). Inhibitions of calpian II in vitro by other tetracyclines (150 μm each) are shown in Fig 1G. Inhibitions of calpian I in vitro by other tetracyclines (150 μm each) are shown in Fig 1 H.

CTC and DMC significantly inhibited the activities of active calpain I (Fig 1 A) and calpain II (Fig 1 B). Specific inhibitor to calpains (ALLN) was used as a positive control. As evidenced by Figs 1G and 1 H, calpain I and calpain II were also inhibited completely or at some degree by sancycline, doxycycline, meclocycline, oxytetracycline, rolitetracycline, 7-aminosancycline, 7-chlorosancycline, 7-bromosancycline, 7-iodosancycline, 7-nitrosancycline, 7-phenylsancycline, 7-(2-thienyl)sancycline, 7,9-dibromosancycline, 4-de(dimethylamino)tetracycline (CMT-1 ), 4-de(dimethylamino)sancycline (CMT-3),

4-de(dimethylamino)chlotetracycline (CMT-4), 4-de(dimethylamino)minocycline (CMT-10), 11 ,12-pyrazolominocycline, 12-aminotetracycline, 12-aminochlortetracycline, 12-aminodemeclocycline, 12-aminominocycline, 12-aminodoxycycline, 12-aminosancycline, 11-N-methylaminominocycline, and 11-oximechlortetracycline.

Example 2: Cell-based assays of the inhibitions of calpain I and II by tetracyclines

Calpain I, activated at about 50 μM concentrations of Ca²⁺, and calpain II, activated at about 200 μM Ca²⁺ concentrations, have similar proteolytic specificities. For example, both calpains cleave the cytoskeletal element spectrin. Calpain-mediated proteolysis of spectrin leads to the immediate production of breakdown products (SBP) of -150 kDa in size. Rapid (within 30 min) and sustained activation of calpain occurs following focal neocortical ischemia and global ischemia and following glutamate treatment of cultured hippocampal and cerebellar neurons. Direct inhibition of calpain reduces calpain-mediated proteolysis of spectrins and decreases brain infarction in ischemic rats and gerbils, and protects cultured hippocampal and cerebellar neurons against glutamateinduced toxicity.

Primary cultures of mouse (C57/B6) cerebellar granule neurons (CGNs) were prepared from 6 - 9 day postnatal mice. Cerebella were explanted and cleaned free of meninges. Mechanical and enzymatic dissociation in a 0.025% w/v trypsin solution for 25 min followed. Trypsin inhibitor was then added to block the enzyme and 0.05% w/v DNase was added to break DNAs from dead cells. A series of trituration and mild centrifugation steps were included to disperse the neurons prior to resuspension in medium and to remove undissociated debris prior to plating in Eagle's minimum essential medium containing 0.8 mM glutamine, 27 mM glucose, 0.01 % gentamycin, 9% FBS and supplemented with K⁺ to a final concentration of 23 mM. Cells were plated onto 24-well dishes containing poly- lysine coated coverslips at a density of 6 x 105 per well. After approximately 18 h, cytosine alpha-D-arabinofuranoside (AraC) was added to a final concentration of 5 μM, to prevent glial cell proliferation. 100 mm dish cultures were seeded with 21 x 10⁶ cells in 10ml of culture medium.

All procedures using animals were approved by the local Animal Care Committee following the guidelines established by the Canadian Council on Animal Care. The C57B/6 mice (20-23 g) were obtained from Charles River and bred locally. Under temporary isoflurane anesthesia, mice were subjected to MCAO using an intraluminal filament. After 1 h of MCAO, the filament was withdrawn, blood flow restored, and wounds sutured. Mice were ip injected with CTC or DMC at 90 mg/kg body weight 4 h before ischemia followed by two injections per day. Control group included a no treatment control and a vehicle control in which the animal was injected with the same volume of water. Brains were removed after 24 h reperfusion and the spectrin breakdown products (SBP) were measured as follows.

Referring to Fig 1 , Fig 1C to Fig 1 F show Western blotting and its quantification of spectrin breakdown products (SBP) produced by the activation of calpain. Glutamate-treated CGNs in the presence or absence of calpain inhibitor or compounds was collected after the time indicated in Fig 1 C and the protein extract was subjected to Western blotting with a primary antibody against SBP. An antibody to GAPDH was used as protein loading control. The production of SBP was normalized against GAPDH and the mean ± SEM is presented in Fig 1 D. Similarly, ischemic brains were collected for Western blotting to detect the production of SBP and the level of it was normalized against GAPDH (Fig 1 E and Fig 1 F). The mean ± SEM is presented in Fig 1 F.

As shown in Fig 1C and Fig 1 D, after 20 min treatment with 50 μM glutamate, the level of SBP increased significantly (p < 0.001 ) and the level of SBP reached a peak after 2.5 h (Fig 1 D). Calpain inhibitor, ALLN, CTC or DMC was applied to cultured CGNs 40 min prior to glutamate treatment. Calpain inhibitor, ALLN, CTC and DMC significantly reduced the level of SBP induced by glutamate treatment in comparison with glutamate alone treated sample at 2.5 h (Fig 1 D). Moreover, the two compounds CTC and DMC also inhibited the calpain activity in the middle cerebral artery occlusion (MCAO) mouse brain as shown by the reduced level of SBP on Western blot (Fig 1 E and Fig 1 F). SBP level increased sharply in the ischemic mouse brain of vehicle-treated mouse, but the level of SBP was significantly reduced in CTC and DMC-treated mouse brains (Fig 1 E and Fig 1 F, p < 0.05). Taken together, CTC and DMC are selective inhibitors to calpains activated in response to excitotoxicity and cerebral ischemia.

Results of this example illustrate that 7-halo tetracyclines, particularly 7-chloro tetracyclines are expected to be particularly useful in the treatment of calpain-associated neural ischemia, such as that caused by stroke.

Example 3: Tetracyclines protect neural cells against glutamate toxicity

It has been validated that some calpain inhibitors protect neural cells from apoptosis or necrosis. The neuroprotective effect of tetracyclines is studied against glutamate toxicity, which is a model of stroke at cellular level.

Tetracyclines were added to 8 day-in-vitro (DIV) cultured CGNs at 37°C for 15 - 20 min prior to treatment with 50 μM glutamate or NMDA. The plates were then incubated for 6 h at 37°C. Untreated controls were also included. At the end of the treatment period, neuronal viability was measured using the 5-(6)- Carboxyfluorescein diacetate (CFDA) assay. The CFDA stock solution was diluted using 0.01 M phosphate-buffered saline (PBS) to a final concentration of 5 μg/ml. Cultures were incubated with 500 μL of the CFDA solution at 37°C for 30 min. The intensities of fluorescence was quantified using a Cytofluor™ 2350 Fluorescence Measurement System (Millipore) at λ_ext = 480 nm and λ_em = 530 nm. Cellular viability was normalized against the fluorescent reading from the control cells. Neurons were also fixed in 4% formaldehyde and mounted in Dako^R fluorescent mounting medium containing 5 μg/μl Hoechst 33258 for examination of cell morphology with a fluorescent microscope. Duplicate assessment of each treatment was made on each plate in at least three separate experiments per treatment. Referring to Fig 2, cultured CGNs were treated with or without prior treatment with the indicated compound at the dose and time indicated in Fig 2A and Fig 2B followed by the addition of 50 μM glutamate. The concentration of both CTC and DMC in Fig 2B was 150 μM. Neuronal viability was determined by a CFDA assay. Data in Fig 2A and Fig 2B represents the mean ± SEM of at least five independent experiments. Treated CGNs were also fixed with 4% formaldehyde and nuclei stained with Hoechst 33258. Representative morphologies of neurons were taken by a digital camera and presented in Fig 2C to Fig 2F. The concentration of both CTC and DMC in Fig 2E and Fig 2F was 150 μM. Arrows indicate glutamate-induced death CGNs, while arrowheads show live CGNs. Scale bar = 80 μm.

CTC and DMC showed potent neuroprotection against glutamate-mediated toxicity to CGNs in a dose- and time-dependent manner as measured by CFDA assay (Fig 2A and Fig 2B). More than 85% of the CGNs were protected by CTC and DMC at doses between 80 -150 μM. This protection lasted up to 8 h following glutamate treatment when more than 50% of the control CGNs were killed by glutamate. The appearance of dead neurons was visualized by Hoechst staining of the nuclei (Fig 2C to Fig 2F). CTC and DMC were not toxic to CGNs at the ranges of doses tested.

Example 4: Tetracyclines reduce the middle cerebral artery occlusion (MCAO)- induced brain damage

It has been validated that some calpain inhibitors effectively protect brain from the damages by stroke. The brain protection of tetracyclines is studied against MCAO-induced brain damage, which is a stroke model. All procedures using animals were approved by the local Animal Care Committee following the guidelines established by the Canadian Council on Animal Care. The C57B/6 mice (20 - 23 g) were obtained from Charles River and bred locally. Under temporary isoflurane anesthesia, mouse was subjected to MCAO using an intraluminal filament. After 1 h of MCAO, the filament was withdrawn, blood flow restored and wounds sutured. Mice were ip injected with CTC or DMC at 90 mg/kg body weight 4 h before ischemia followed by twice injection per day. Control group included no treatment control and vehicle control in which animal was injected with the same volume of water. Brains were removed after 24 h reperfusion and the brain infarction was measured by a colormetric staining method using 2,3,5-triphenyltetrazolium chloride (TTC). Briefly, brains were dissected out and cut into four 2 mm thick coronal slices which were stained with 5 ml of 2% TTC for 90 min at 37°C. Afterwards, the tissue was rinsed with saline followed with a mixture of ethanol/dimethylsulfoxide (1 :1 ) which was to solubilize the formazan. After 24 h incubation in the dark, the red solvent extracts were diluted 1 :20 with fresh ethanol/DMSO solvent in three tubes and placed in cuvettes. Absorbance was measured at 485 nm in a spectrophotometer and the values were averaged. Percentage loss in brain TTC staining (and apparent tissue injury) in the ischemic side of the brain was compared to the contralateral side of the brain of the same animal using the following equation:

% Loss = [1 - (absorbance of ischemic hemisphere / absorbance of contralateral hemisphere) X 100]

Both compounds significantly reduced the infarction size in the cerebral cortex by almost 50% in comparison with the non-treated ischemic control and vehicle-treated brain (Fig 3A). Fig 3B to Fig 3D are repetitive images of coronal sections of brains from MCAO mouse (Fig 3B), CTC-treated MCAO mouse (Fig 3C) and DMC-treated MCAO mouse (Fig 3D). The numbers 1-4 in Fig 3B, Fig 3C and Fig 3D indicate the first to the last slice of the MCAO brain and arrows indicate ischemic infarction (white-colored region on the brain slice). Most of the infarctions occurred in the first two brain slices in the cerebral cortex and striatum as indicated by arrows in Fig 3B. The infarction was significantly reduced in the same areas of CTC- and DMC-treated brains (Fig 3C and Fig 3D).

Example 5: Tetracyclines improve neurological behaviour in a mouse model of focal ischemia

This example examines if tetracyclines protect not only brain physically, but also have therapeutic effects in neurological behaviour after focal ischemia.

Behavioral assessments were made at 0 and 24 h after reperfusion by an individual blinded to the treatment of the mice. The neurological deficits were scored as follows: 0, normal; 1 , mild turning behavior with or without inconsistent curling when picked up by tail, <50% attempts to curl to the contralateral side; 2, mild consistent curling, >50% attempts to curl to contralateral side; 3, strong and immediate consistent curling, mouse holds curled position for more than 1-2 sec, mouse's nose almost reaches tail; 4, severe curling progressing into barreling, loss of walking or righting reflex; 5, comatose or moribund. At least eight groups of mice were evaluated and scores were averaged for statistical analysis. Using this six-point valuation system, the scores of the neurological behavior of ischemic animals were compared with those of vehicle-treated or ischemic animals immediately after surgery and after 24 h reperfusion. As shown in Fig 3E, mice treated with the two compounds showed significant improvement after 24 h of reperfusion (p < 0.05) compared with the vehicle-treated or ischemic animals, demonstrating that CTC and DMC reduced MCAO-induced neurological deficits.

Example 6: Tetracyclines weakly anatgonize NMDA receptors

Glutamate-mediated toxicity over-activates NMDA receptors, causing increases in intracellular Ca²⁺ levels leading to the accumulation of toxic levels of intracellular calcium ions. Elevation in intracellular Ca²⁺ concentrations activates Ca²⁺-dependent proteases, such as calpains, which break down critical structural proteins causing neuronal death. Thus, chemical compounds directly blocking glutamate toxicity to neurons, i.e., NMDA receptor blockers, may have the potential to be developed as therapeutics to stroke. But, NMDA receptor blockers, such as MK801 , have failed in human stroke clinical trials due to the severe side effects of interference with the normal physiological functions of the NMDA receptors. Consequently, it is desired to eliminate the activity to block NMDA receptors from neuroprotecting agents.

Whole-cell recording using electrophysiological methods was performed as follows. CGNs, cultured on 35 mm culture dishes, were perfused continuously at 1 ml/min at 22°C using a solution containing 140 mM NaCl, 5 mM KCI, 2 mM CaCI₂, 10 mM HEPES, 3 mM glucose, pH 7.4. The perfusion solution also contained 1 μM TTX, 30 μM glycine, and 1 /nμM strychnine. Patch pipettes (2 - 4 M /n resistance) were constructed from 1.5 mm outer diameter /1.0 mm inner diameter Pyrex 7740 glass (Corning, Big Flats, MN, USA). A modified DAD-12 perfusion system (ALA Scientific Inst., Westbury, NY, USA) was used to rapidly apply NMDA (2 s duration) followed by co-application of NMDA and the test compound (5 s duration). The pipette solution contained 140 mM CsCI, 1.1 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP at pH 7.2. Whole-cell currents were acquired using an Axopatch 1-D amplifier equipped with a CV-4 head stage with a 1 G feedback resistor. Voltage command and current acquisition were accomplished using a Digidata 1200 interface and pClamp 6.0 software (Axon Inst). Neurons were held at a membrane potential of -60 mV. The fractional block of NMDA-evoked currents was calculated according to the formula: B = I - IB / 1, where I is the steady-state current evoked by NMDA and IB is the current evoked by NMDA in the presence of the test compound at the end of the co-application.

Both CTC and DMC at 150 μM showed weak, but rapid, antagonism to 50 μM NMDA-induced currents (Fig 4A). A 5 s co-application of NMDA plus 150 μM CTC resulted in a 14 ± 1 % (n=5) reduction in NMDA-induced current. A 5 s co- application of NMDA plus 150 μM DMC produced a 16 ± 2% (n=5) reduction in NMDA-induced currents. Thus, CTC and DMC are weak antagonists of NMDA receptors. Since NMDA activation induces intracellular Ca²⁺ influx, we next tested whether CTC and DMC affect glutamate-induced intracellular Ca²⁺ levels. Intracellular mitochondrial calcium concentration was measured as follows. Culture medium in the 24-well plate was replaced with 250 μL of Fluo-4 dye to a final concentration of 40 μg/ml. After 30 min incubation, the dye was removed and cells were incubated with the original medium at 37°C for 15 min. Intracellular calcium fluorescent intensities were quantified using a Cytofluor™ 2350 Fluorescence Measurement System (Millipore) at λ_ex = 485 nm and λ_em = 530 nm. NMDA (50 μM) was then added to the wells and changes in calcium florescence were recorded after 5, 10, 20, 30 and 40 min. Fold induction in intracellular calcium concentration was normalized against non-treated cells and plotted.

As shown in Fig 4B, NMDA receptor-mediated intracellular calcium influx increased rapidly after 5 min. After 5 min of addition of NMDA, the two compounds showed slight blockade of Ca²⁺ Influx. But the intracellular Ca²⁺ level eventually increased to the same level as that of NMDA-treated CGNs. MK801 , an antagonist to NMDA receptor, completely blocked NMDA-induced Ca²⁺ influx (Fig 4B). Taken together, these data demonstrated that CTC and DMC are extremely weak and transient blockers of the NMDA receptor currents and NMDA receptor-mediated Ca²⁺ influx. Such a weak and transient reduction in NMDA receptor current and Ca²⁺ influx is not sufficient to account for the more than 90% neuroprotection conferred by these two compounds, suggesting that CTC and DMC have novel intracellular targets.

Example 7: Anti-oxidant activity of tetracyclines

Since calpain activation is often associated with oxidative stress, antioxidant activities of tetracyclines may show synergistic effect with the calpain inhibition and reduce the effective amount of a tetracycline to treat or inhibit calpain-medicated physiological damages.

Three antioxidant activities were measured. These are superoxide radical scavenging activity, DPPH radical scavenging activity, and ABTS cation radical scavenging activity. Superoxide radical scavenging activity was measured as follows. The mixture consisted of 140 μL of 0.030 mM riboflavin, 1 mM EDTA, 0.60 mM methionine and 0.030 mM NBT solution in 50 mM potassium phosphate buffer (pH 7.8) and 10 μL of a sample solution, which includes the test compounds and the reference compounds at various concentrations in DMSO, as well as DMSO as a control. The solutions of the tested compounds had concentrations ranging from 3 μg/ml to 1000 μg/ml, whereas the concentrations of the solutions of the reference compounds varied from 0.1 μg/ml to 1000 μg/ml. The photoinduced reactions to generate superoxide anion were carried out in an aluminum foil-lined box with two 20 W fluorescent lamps. The distance between reactant and lamp was adjusted until the intensity of illumination reached 1000 lux. The reactant was illuminated at 25°C for 8 min. The photochemically reduced riboflavin generated superoxide anion, which reduced NBT to form the blue formazan. The un-illuminated reaction mixture was used as a blank. Reduction of NBT was measured by the absorbance change at 560 nm before and after irradiation using a microplate. Scavenging activity was calculated from the absorbance changes of control and test samples:

Scavenging activity (%) = (1 - ΔA_sampie / ΔA_COntroi) x 100,

where ΔA_SamPi_e is the change of the absorbance in the wells containing the tested compounds, and _controi is the change of the absorbance in the wells containing the reference compounds). The EC₅o value is defined as the concentration of substrate that causes 50% loss of the reduced NBT. The assays were performed in triplicate and the absorbance changes were averaged before calculation.

DPPH radical scavenging activity was determined as follows. The solution of the sample (10 μL) in ethanol was added to 90 μL of a 0.15 mM DPPH radical in ethanol in a 96-well plate. The sample solution refers to the tested compounds and the reference antioxidants at various concentrations, as well as ethanol as a control. The solutions of the tested compounds had concentrations ranging from 3 μg/ml to 1000 μg/ml, whereas the concentrations of the solutions of the reference compounds varied from 0.1 μg/ml to 1000 μg/ml. The reaction leading to the scavenging of DPPH radical was complete within 10 min at 25°C. The absorbance of the mixture was then measured at 517 nm using a microplate reader. The reduction of DPPH radical was expressed as percentage:

Scavenged DPPH (%) = (1 - A_test / Acontroi) x 100

where A._est is the absorbance of a sample at a given concentration after 10 min reaction time, and A_controi is the absorbance recorded for 10 μL ethanol. The EC₅o value is defined as the concentration of sample that causes 50% loss of the DPPH radical.

The ABTS cation radical was produced by the reaction between 7.0 mM ABTS/water and 2.45 mM potassium persulfate for 12 h in the dark at room temperature. The ABTS solution was diluted with PBS until A₇₃ = 0.7. The reaction was initiated by adding 190 μL of ABTS to 10 μL sample solution at 25°C. The percentage of reduction of A734 was recorded and was plotted as a function of the sample's concentration. The antioxidant activities of some tetracyclines are listed in Table 1. Table 1 : In vitro antioxidant activity of tetracyclines (EC₅₀ in μM)

Example 8: Anti-glycation activity of tetracyclines

Since calpain activation is often associated with oxidative stress, glycation may be involved in chronic diseases associated with calpain activation. Thus, anti- glycation activity of tetracyclines may show synergistic effect with the calpain inhibition.

Anti-glycation activity of tetracyclines was measured by the inhibition of the fluorescence of glycated protein. Stock solutions of bovine serum albumin (BSA, 67kDa) and d-ribose were prepared separately into Dulbecco's Phosphate Buffered Saline (D-PBS). All tetracyclines were dissolved D-PBS to prepare the appropriate concentrations. Then, stock solutions of BSA, d-ribose and tetracyclines were mixed in a 96 well plate and incubated for 5 days at 37°C under mild shaking, in the dark. The final concentrations of BSA and d-ribose were 0.075mM (4.5mM Lys residue) and 50mM, respectively. The range of tested concentrations of tetracycline analogs were 15 nM to 4 mM. After incubation, aliquots of the sample solutions were applied to a size exclusion chromatographic column (Superose™ 12PC 3.2/30 (Amersham Biosciences, England, UK), 100 mM phosphate buffer pH7.4) to separate the proteins and the small molecules and to measure the intensity of fluorescence from the glycated BSA. The excitation and emission wavelength values were 375nm and 440nm, respectively.

The inhibitory activity of tested compounds was calculated using the equation:

Inhibition (%) = 100-(F-F₀)/(F₁₀o-Fo) x 100

where F₀ is the fluorescence represented by the peak area of incubated BSA alone, F₁₀o is the fluorescence represented by the peak area of incubated mixture BSA - d-ribose, F is the fluorescence represented by the peak area of incubated mixture BSA - d-ribose - tetracycline. The IC₅₀ values were calculated by curve fitting using software Origin™ 7.0 (OriginLab, Northampton, MA, USA). The anti-glycation IC₅₀ values of some tetracyclines are listed in the following Table 2.

Table 2: In vitro anti-glycation activity of tetracyclines

Example 9: Non-antibiotic activity of 11 - and/or 12-substituted tetracyclines

As chronic use of antimicrobial tetracyclines may induce drug resistant microorganisms, non-antibiotic tetracyclines such as 12-amino-tetracyclines may have an advantage for long-term use such as prevention.

Two bacterial strains were used for antibiotic assay. One is the tetracycline-sensitive strain BL21 (DE3), E. coli B F^" dcm ompT hsdSfo. me.) gal λ(DE3) and the other is the tetracycline-resistant strain XL2-Blue, recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacPZAMIδ Tn10(Tet^r) Amy Cam^r]. The bacterial strains were plated fresh from -80°C glycerol stocks onto 2xYT media agar plates (16g/L bacto-tryptone, 10g/L bacto-yeast extract, 5g/L NaCl, 15g/L bacto-agar. pH was adjusted to 7.0 with NaOH) and grown overnight at 37°C. Single colonies of BL21(DE3) and XL2-Blue were picked and used to inoculate 7 mL flasks of LB liquid media (10g/L bacto-tryptone, 5g/L bacto-yeast extract, 10g/L NaCl. pH adjusted to 7.0 with NaOH. Plates contained 15g/L bacto-agar). Cultures were grown for 6 h at 250 rpm at 37°C and then for 14 h at 250 rpm at 30°C. Bacterial concentrations were determined with an absorbance at 600 nm and approximately 2,000 CFU's of bacteria were used to incubate LB plates containing variable concentrations of tetracyclines in 50% ethanol/water. Plates were incubated at 37°C for 20 h and then examined for bacterial growth. Fig 5 shows the growth of BL21 (DE3) bacterial strain in the presence of a fixed concentration (80 μM) of antibiotic minocycline and demeclocycline, and non- antibiotic 11 ,12-pyrazolominocycline and 12-aminodemeclocycline. Table 3 shows the antibiotic activity of some tetracyclines. It is apparent that 1 ,12-bridged tetracyclines and 12-amino tetracyclines are not as effective at inhibiting the growth of bacteria, therefore, these tetracyclines lack significant antibiotic activity. The lack of antibiotic activity is preferable when tetracyclines are used to inhibit calpain activity or activation.

Table 3: In vitro antibiotic activity of tetracyclines

Relative growth of bacterial strains when incubated in the presence of tetracyclines. (++++) indicates 100% growth and (-) indicates no growth as compared to tetracycline-free plates.

Example 10: No inhibition of matrix metalloproteases (MMPs) of 11- and/or 12- substituted tetracyclines

Matrix metalloproteinases (MMPs) are medicinal targets due to the activity of these enzymes associated with diseases such as cancer, cardiovascular diseases, pulmonary diseases, osteoarthritis, and rheumatoid arthritis. Almost all MMP inhibitors extensively developed failed in clinical trials because they lack specificity to disease-related MMPs and some caused musculoskeletal pain and inflammation. Thus, tetracyclines such as 12-amino-tetracyclines which lack MMP inhibition activity may have an advantage to reduce side effects. Zn²⁺ chelation is required for tetracyclines to inhibit MMPs and the chelation of Zn²⁺ shifts the absorbance of tetracyclines at around 280-380 nm to longer wavelength. Thus, a tetracycline that shows no absorbance change by adding Zn²⁺ does not inhibit MMPs.

The solutions of compounds required for measuring their UV-Vis absorption were obtained by mixing 0.10 mL solution of each compound (1 mM in water) with 0.80 mL methanol and 0.10 mL of 50 mM Tris buffer (pH 7.4) in a 1 cm quartz cuvette (1.0 mL volume). After the absorption curves of the compounds were obtained, an aliquot (10 μL) of aqueous solution containing zinc sulfate (10 mM) was added to them to record their absorption spectra in the presence of Zn²⁺ (0.1 mM). The change in volume resulted from the addition of 10 μL zinc sulfate solution to 1.0 mL sample was ignored. The baseline absorption spectrum was recorded with 0.10 mL water, 0.80 mL methanol and 0.10 mL 50 mM Tris buffer (pH 7.4), and was subtracted from the absorption spectra of the samples. An absorbance at around 280-380 nm, which arises from the chromophore composed of beta-diketone at the ring B and C and of aromatic ring D is sensitive to the binding of cations at 11 ,12-beta-diketone, resulting red-shift. Thus, the binding of Zn²⁺ was monitored by the red-shift of the absorbance band at about 280-380 nm. Binding of Zn²⁺ to tetracyclines is listed in Table 4 with "+" means that the absorbance band at around 350 - 380 nm red-shifted more than 10 nm and "-" means that the shift of the absorbance band is less than 3 nm.

Table 4: Chelation of a tetracycline to Zn 2+ ion

Example 11 : No calcium binding to 11 and/or 12-substituted tetracyclines

Tetracyclines that chelate Ca²⁺ are limited not to be administered to children and pregnant woman, and are incorporated into teeth, cartilage and bone, resulting in discoloration of both the primary and permanent dentitions. Ca²⁺ also reduces the intestinal absorption of tetracyclines. Thus, tetracyclines such as 12aminotetracyclines which lack calcium binding may have an advantage to improve the absorption and to reduce the side effects. Since the chelation of a tetracycline to Ca²⁺ shift the absorbance at around 280-380 nm to longer wavelength, the absorbance change at around 280-380 nm was monitored for Ca²⁺ binding.

The solutions of compounds required for measuring their UV-Vis absorption were obtained by mixing 0.10 mL solution of each compound (1 mM in water) with 0.80 mL methanol and 0.10 mL of 50 mM Tris buffer (pH 7.4) in a 1 cm quartz cuvette (1.0 mL volume). After the absorption curves of the compounds were obtained, an aliquot (10 μL) of aqueous solution containing calcium chloride (10 mM) was added to them to record their absorption spectra in the presence of Ca²⁺ (0.1 mM). The change in volume resulted from the addition of 10 μL calcium chloride solution to 1.0 mL sample was ignored. The baseline absorption spectrum was recorded with 0.10 mL water, 0.80 mL methanol and 0.10 mL 50 mM Tris buffer (pH 7.4), and was subtracted from the absorption spectra of the samples. An absorbance at around 280-380 nm arises from the chromophore composed of beta-diketone at the ring B and C and of aromatic ring D is sensitive to the binding of cations at 11 ,12-beta-diketone, resulting red-shift. Thus, the binding of Ca²⁺ was monitored by the red-shift of the absorbance band at around 280-380 nm.

Binding of Ca²⁺ to tetracyclines is listed in Table 5 with "+" means that the absorbance band at around 350 - 380 nm red-shifted more than 10 nm and "-" means that the shift of the absorbance band is less than 3 nm. Table 5: Chelation of a tetracycline to Ca 2+ ion

Example 12: Stability of 12-aminotetracyclines in acidic pH

12-Amino group of 12-aminotetracyclines can be replaced with hydroxyl group in acidic water environment such as in stomach, converting them to antibiotic and MMP-inhibitory tetracyclines. Thus, the acid stability of 12-aminotetracyclines was examined at pH = 2 and 37°C. Each 12-aminotetracycline (2 mM) was incubated in 100 mM Gly.HCI buffer (pH 2.0) or in 100 mM sodium acetate (pH 4.0) at 37°C for up to 6 h. The hydrolysis was quenched by increasing the pH to 7 by taking aliquots at appropriate time interval from the reaction solution. The 12-aminotetracycline and its hydrolyzed product were separated by HPLC (Waters, symmetry column 50 x 4.6 mm, 2 mL/min flow rate, 0-80% acetonitrile gradient/0.1 % TFA in 7 min). A diode array detector (Waters™ model 2996) was used to record their UV spectra. The half-life was estimated as the time to hydrolyze 50% of the 12-aminotetracycline. The half-life of 12-aminotetracylines is listed in Table 6. Oral administration of 12-aminotetracyclines may be recommended when the pH of the stomach is around 4.

Table 6: Stability of 12-aminotetracyclines against acid hydrolysis.

Synthetic Methods: All synthetic products were purified using various types of column chromatography with different solvents as eluents and/or by recrystallization from various solvents according to the procedures. The purity of compounds was established using an analytical Waters HPLC (Symmetry™ 3.5 by 50 mm C-iβ reverse-phase column, gradient 0-90% acetonitrile in water, 0,1 %TFA; flow rate 0.8 mL/min, 15 min). The compounds were characterized by mass spectrometry using an electrospray ionization mass spectrometer (ESI-MS) (Sciex API III mass spectrometer) and by ¹H NMR (Bruker-DRX-500 MHz).

The purity of all synthetic products (> 98%) was established by HPLC using an analytical C18-reverse phase SymmetryShield™ column (3.5μm; 4.6 x 50 mm). The methods used either a binary gradient of 0.1% trifluoroacetic acid (TFA) in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 0% phase B at time 0 to 80% phase B in 10 min at a flow rate of 1.5 mL/min (Method A), or a binary gradient of phase A and phase B on a gradient from 0% phase B at time 0 to 80% phase B in 7 min at a flow rate of 2.0 mL/min (Method B). ¹H NMR spectra were recorded on a Bruker Advance™ 500 (500 MHz) instrument. Mass spectra were recorded on an API III mass spectrometer (Sciex, Concord, ON, Canada).

Reaction between tetracyclines and reducing agents. Modification of the substituent in position 4.

(12aa)-3,6, 10, 12, 12a-Pentahydroxy-6-methyl-1 , 11-dioxo-1,4,4a,5,5a,6, 11, 12a- octahydro-naphthacene-2-carboxylic acid amide.

Tetracycline hydrate (5.0 g, 11 mmol) in tetrahydrofuran (300 mL) was reacted with iodomethane (12 mL) at room temperature for 6 days. The precipitated tetracycline methiodide was filtered and washed with cold tetrahydrofuran and diethyl ether. R_t (method A) = 5.6 min. ESI-MS m/z (M + H) 459.2.

Tetracycline methiodide (2.5 g, 4.3 mmol) was dissolved in 50% acetic acid (40 mL), then zinc dust (1.2 g) was added to the solution in one portion. The reaction mixture was stirred at room temperature for 15 min. After the excess zinc dust had been filtered off, the mixture was cooled to 0°C and treated with cold dilute HCI. The precipitated yellowish (12aα)-3,6,10,12,12a-pentahydroxy-6- methyl-1 ,11-dioxo-1 ,4,4a, 5,5a,6,11 ,12a-octahydro-naphthacene-2-carboxylic acid amide was filtered, washed with cold dilute HCI, and recrystallized from a mixture of ethyl acetate and diethyl ether. R_t (method A) = 7.4 min. ESI-MS m/z (M + H) 402.0. ¹H NMR (CD₃OD) δ 1.61 (s, 3H), 1.97 (q, 1 H, J = 12 Hz), 2.06 (m, 1 H), 2.46 (m, 1 H), 2.90 (m, 1 H), 3.29 (m, 1 H), 6.93 (d, 1 H, J = 8.2 Hz), 7.16 (d, 1 H, J = 7.4 Hz), 7.51 (dd, 1 H, J = 7.4 and 8.2 Hz).

(12a )-3, 10, 12, 12a-Tetrahydroxy-1 , 11-dioxo-1,4,4a,5,5a,6, 11, 12a-octahydro- naphthacene-2-carboxylic acid amide.

Sancycline (5.0 g, 12.1 mmol) was stirred with methyl iodide (7.5 mL) in acetone (50 mL) for 3 days. The resulting precipitate of sancycline methiodide was filtered off and air-dried. R_t (method A) = 6.5 min.

Sancycline methiodide (7.0 g, 12.6 mmol) was dissolved in 50% acetic acid (200 mL) and treated with zinc dust (4.0 g). Stirring was continued for 30 min, then the unreacted zinc was removed by filtration, and the filtrate was acidified with 1 N HCI (200 mL). The precipitate was filtered off after 2 h and purified by column chromatography (silica gel, dichloromethane-chloroform) followed by recrystallization from acetone-water to afford (12aα)-3,10,12,12a-tetrahydroxy- 1 ,11-dioxo-1 ,4,4a,5,5a,6,11 ,12a-octahydro- naphthacene-2-carboxylic acid amide as a yellowish powder. R_t (method A) = 8.9 min. ESI-MS m/z (M + H) 371.9. ¹H NMR (CD₃OD) δ 1.52 (q, 1 H, J = 15 Hz), 2.05 (d, 1 H, J = 10 Hz), 2.40 (d, 1 H, J = 10 Hz), 2.47 (t, 2H, J = 15 Hz), 2.83 (dd, 1 H, J = 15 and 6 Hz), 2.88 (tt, 1 H, J = 15 and 6 Hz), 3.24 (dd, 1 H, J = 15 and 6 Hz), 6.74 (d, 1 H, J = 7.8 Hz), 6.76 (d, 1 H, J = 7.8 Hz), 7.39 (t, 1 H, J = 7.8 Hz).

(12aα)-7-Chloro-3,6,10,12,12a-pentahydroxy-6-methyl-1 ,11 -dioxo-1 ,4,4a,5, 5a,6,11 ,12a-octahydro-naphthacene-2-carboxylic acid amide. Chlortetracycline (1.01 g, 2.0 mmol) was stirred for 2 h with zinc dust (640 mg) in 50% acetic acid (32 mL). Unreacted zinc was removed by filtration, then the filtrate was acidified with 1 N HCI (150 mL). The precipitated product was collected by filtration after stirring for 30 min. Extraction of the filtrate with dichloromethane provided further amounts of CMT-4. The combined solid fractions were purified by column chromatography (silica gel, chloroform-acetonitrile), followed by recrystallization from methanol afforded (12aα)-7-chloro-3,6,10,12,12a- pentahydroxy-6-methyl-1 ,11-dioxo-1 ,4,4a, 5, 5a,6,11 ,12a-octahydro-naphthacene- 2-carboxylic acid amide as a yellowish powder. R_t (method B) = 5.9 min. ESI-MS m/z (M + H) 436.1. ¹H NMR (CD₃OD) δ 1.98 (s, 3H), 2.10-2.19 (m, 2H), 2.54 (tt, 1 H, J = 19 and 3 Hz), 2.66 (dd, 1 H, J = 19 and 3 Hz), 2.98 (t, 1 H, J = 8 Hz), 3.22 (dd, 1 H, J = 18.5 and 5.5 Hz), 6.92 (d, 1 H, J = 9 Hz), 7.49 (d, 1 H, J = 9 Hz).

(12a )-7-Dimethylamino-3, 10, 12, 12a-tetrahydroxy-1, 11-dioxo- 1,4,4a,5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide.

Minocycline (1.86 g, 3.8 mmol) was stirred with zinc dust (1.2 g) in 30% acetic acid (60 mL) for 50 min. Unreacted zinc was removed by filtration, then the filtrate was brought to neutral pH with 10N NaOH (31 mL). After stirring for 10 min, the precipitated product was collected by filtration. Purification by high performance displacement chromatography (HPDC) (C18-reverse phase Shiseido Capcell Pak™ AQ 4.6 x 250 mm, Λ/-cetylpyridinium trifluoroacetate as displacer, 0.1 % TFA in water as mobile phase, flow rate 1 mL/min) gave, after the collected fractions had been made neutral with triethylamine, (12aα)-7-dimethylamino- 3,10,12,12a-tetrahydroxy-1 ,11-dioxo-1 ,4,4a,5,5a,6,11 ,12a-octahydro- naphthacene-2-carboxylic acid amide as a free base. Rt (method A) = 5.5 min. ESI-MS m/z (M + H) 415.0. ¹H NMR (CD₃OD) δ 1.70 (q, 1 H, J = 13 Hz), 2.08 (dd, 1 H, J = 13 and 3 Hz), 2.18 (d, 1 H, J = 8 Hz), 2.22 (d, 1 H, J = 16 Hz), 2.50 (d, 1 H, J = 16 Hz), 2.58 (s, 6H), 2.85 (tt, 1 H, J = 16 and 4 Hz), 3.34 (td, 2H, J = 16 and 4 Hz), 6.83 (d, 1 H, J = 9.0 Hz), 7.31 (d, 1 H, J = 9.0 Hz). Reaction between tetracyclines and neucleophiles. Modification of the carbonyl moieties.

[5S-(5a,5aa,6a , 12ba, 12ca)]-5-Dimethylamino-1,5,5a,6,6a, 7, 12, 12a, 12b, 12c- decahydro-4, 7, 11, 12b, 12c-pentahydroxy-7-methyl-12-oxo-1 ,2-diaza- cyclopenta[de]-naphthacene-3-carboxylic acid amide

and

[6S-(2bα,6α,6aα, 7aα)]-6-dimethylamino-1,2b,3,6,6a, 7, 7a,8-octahydro-2b,5,8, 12- tetrahydroxy-8-methyl-3-oxo-1,2-diaza-cyclopenta[fg]naphthacene-4-carboxylic acid amide. Tetracycline hydrochloride (0.2 g, 0.42 mmol) was suspended in 8.0 mL absolute ethanol. Hydrazine hydrate (0.050 mL, 1.04 mmol) was then added, and the reaction mixture was refluxed for 3 h. The crude mixture was evaporated to dryness and subjected to high vacuum to remove any traces of the solvent. Water (0.80 mL) was added to dissolve the dry residue. After a few minutes, a beige precipitate formed. Stirring continued for 1 h, then the precipitate was collected and dried (0.160 g). The material was then dissolved in 3.0 mL of 20% acetic acid and separated using a C18-reverse phase Vydac™ column (50 x 250 mm) and employing 0.6% acetic acid in water at a flow rate of 20 mL/min as mobile phase. After freeze-drying of the collected fractions, the solid materials were dissolved in the minimum amount of water and treated with the required amount of triethylamine. The tetracycline derivatives were separated from the water-soluble salts using PrepSep™-C18 disposable extraction columns to give the compounds as free bases.

[5S-(5α,5aα,6aα, 12b , 12c )]-5-dimethylamino- 1 ,5,5a,6,6a,7,12,12a,12b,12c-decahydro-4,7,11 ,12b,12c-pentahydroxy-7-methyl- 12-oxo-1 ,2-diaza-cyclopenta[de]-naphthacene-3-carboxylic acid amide. R_t (method A) = 4.6 min. ESI-MS m/z (M + H) 459.2. ¹H NMR (D₂O) δ 1.48 (q, 1 H, J = 3 Hz), 1.76 (s, 3H), 2.35 (m, 2H), 2.76 (d, 1 H, J = 8.8 Hz), 2.96 (s, 6H), 3.07 (d, 1 H, J = 11.8 Hz), 3.75 (s, 1 H), 7.04 (d, 1 H, J = 8.3 Hz), 7.31 (d, J = 7.6 Hz), 7.67 (dd, 1 H, J = 7.6 and 8.3 Hz).

[6S-(2bα,6α,6aα,7aα)]-6-Dimethylamino-1 ,2b,3,6,6a,7,7a,8-octahydro- 2b,5,8,12-tetrahydroxy-8-methyl-3-oxo-1 ,2-diaza-cyclopenta[ g]naphthacene-4- carboxylic acid amide. R_t (method A) = 4.7 min. ESI-MS m/z (M + H) 441.2. ¹H NMR (CD₃OD) δ 1.57 (s, 3H), 1.95 (m, 1 H), 2.07 (m, 1 H), 2.53 (s, 1 H), 2.92-3.12 (m, 3H), 7.04 (d, J = 5.7 Hz, 1 H), 7.12 (bs, 2H).

[5S-(5a,5aa,6aa, 12b , 12c )]-5,8-Bis(dimethylamino)- 1,5,5a,6,6a, 7, 12, 12a, 12b, 12c-decahydro-4, 11, 12b, 12c-tetrahydroxy-12-oxo-1 ,2- diaza-cyclopenta[de]naphthacene-3-carboxylic acid amide

and

[6S-(2b ,6α,6aα, 7aα)]-6,9-bis(dimethylamino)-1,2b,3,6,6a, 7, 7a,8-octahydro- 2b, 5, 12-trihydroxy-3-oxo-1,2-diaza-cyclopenta[fg]naphthacene-4-carboxylic acid amide.

Minocycline hydrochloride (0.050 g, 0.10 mmol) was suspended in water (1.25 mL). Hydrazine hydrate (0.0175 mL, 0.36 mmol) was then added and the reaction mixture was stirred at room temperature overnight under nitrogen. After the solvent had been removed by freeze-drying, the crude product was separated using a C18-reverse phase Vydac™ column (50 x 250 mm). By applying a binary gradient of 0.1% TFA in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 0% phase B to 5% phase B in 100 min at a flow rate of 20 mL/min, [5S-(5α,5aα,6a , 12bα, 12cα)]-5,8-bis(dimethylamino)- 1 ,5,5a,6,6a,7,12,12a,12b,12c-decahydro-4,11 ,12b,12c-tetrahydroxy-12-oxo-1 ,2- diaza-cyclopenta[ /e]naphthacene-3-carboxylic acid amide separated as a trifluoroacetate. [6S-(2bα,6α,6aα,7aα)]-6,9-Bis(dimethylamino)-1 ,2b,3,6,6a,7,7a,8- octahydro-2b,5,12-trihydroxy-3-oxo-1 ,2-diaza-cyclopenta[ g]naphthacene-4- carboxylic acid amide eluted soon afterwards under isocratic conditions (95% phase A, 5% phase B, 20 mL/min). After freeze-drying the collected fractions, the solid materials were dissolved in the minimum amount of water and treated with the required amount of triethylamine. The tetracycline derivatives were separated from the water-soluble salts using PrepSep™C18 disposable extraction columns. [5S-(5α,5aα,6aα,12bα,12cα)]-5,8-Bis(dimethylamino)-

1 ,5,5a,6,6a,7,12,12a,12b,12c-decahydro-4,11 ,12b,12c-tetrahydroxy-12-oxo-1 ,2- diaza-cyclopenta[cte]naphthacene-3-carboxylic acid amide. R_t (method A) = 2.8 min. ESI-MS m/z (M + H) 472.2. ¹H NMR (D₂O) δ 1.55 (q, 1 H, J = 12.7 Hz), 2.22 (d, 1 H, J = 10.9 Hz), 2.47 (m, 1 H), 2.74 (dd, 1 H, J = 13.0 and 15.3 Hz), 2.87 (m, 1 H), 3.02 (s, 6H), 3.03 (m, 1 H), 3.19 (d, 1 H, J = 13.6 Hz), 3.28 (s, 6H), 3.86 (s, 1 H), 7.12 (d, 1 H, J = 9.1 Hz), 7.91 (d, 1 H, J = 9.1 Hz).

[6S-(2bα,6α,6aα,7aα)]-6,9-Bis(dimethylamino)-1 ,2b,3,6,6a,7,7a,8- octahydro-2b,5,12-trihydroxy-3-oxo-1 ,2-diaza-cyclopenta[fg]naphthacene-4- carboxylic acid amide. R_t (method A) = 4.0 min. ESI-MS m/z (M + H) 454.2. ¹H NMR (D₂O) δ 1.53 (q, 1 H, J = 11.5 Hz), 2.12 (t, 1 H, = 15.1 Hz), 2.24 (d, 1 H, J = 11.4 Hz), 2.96 (s, 6H), 3.06 (m, 2H), 3.20 (bs, 7H), 3.95 (s, 1 H), 6.93 (d, 1 H, J = 8.9 Hz), 7.42 (d, 1 H, J = 8.9 Hz).

[6S-(2ba,6a,6aa, 7a )]-9-Chloro-6-dimethylamino-1,2b,3,6,6a, 7, 7a,8-octahydro- 2b, 5, 8, 12-tetrahydroxy-8-methyl-3-oxo- 1 , 2-diaza-cyclopenta[fg]naphthacene-4- carboxylic acid amide.

Chlortetracycline hydrochloride (2.0 g, 3.9 mmol) was reacted with hydrazine hydrate (2.0 mL) in methanol (400 mL) at room temperature for 2 h. The resulting precipitate was filtered and treated with water (50 mL) with stirring for 1 hr at room temperature to give, after filtration and thoroughly washing with water, [6S- (2bα,6α,6aα,7aα)]-9-chloro-6-dimethylamino-1 ,2b,3,6,6a,7,7a,8-octahydro- 2b,5,8,12-tetrahydroxy-8-methyl-3-oxo-1 ,2-diaza-cyclopenta[fg]naphthacene-4- carboxylic acid amide as a white precipitate. R_t (method A) = 5.7 min. ESI-MS m/z (M + H) 475.7. ¹H NMR (CD₃OD/CF₃CO₂D) δ 2.05 (q, 1 H, J = 12.0 Hz), 2.11 (s, 3H), 2.34 (d, 1 H, J = 10.1 Hz), 3.10 (s, 6H), 3.19 (m, 2H), 4.00 (s, 1 H), 6.93 (d, 1 H, J = 8.7 Hz), 7.73 (d, 1 H, J = 8.7 Hz).

[4S-(4a, 12a )]-12-Amino-4-dimethylamino-3,6, 10, 12a-tetrahydroxy-6-methyl- 1, 11-dioxo-1,4,4a,5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide.

Anhydrous ammonia was bubbled into a stirred suspension of tetracycline hydrated (0.50 g, 1.1 mmol) in absolute ethanol (100 mL) under reflux for 6 h. Stirring continued at room temperature overnight, then the mixture was filtered, and the solvent was removed under reduced pressure. The residue, after a primary separation by column chromatography (silica gel, dichloromethane- ethanol), was further purified by high performance displacement chromatography (HPDC) (C18-reverse phase Shiseido Capcell Pak™ AQ 4.6 x 250 mm, N- cetylpyridinium trifluoroacetate as displacer, flow rate 1 mL/min). After neutralizing with NaHCO₃, the collected fractions were freeze-dried, and the tetracycline derivative as a free base was separated from the water-soluble salts using

PrepSep™-C18 disposable extraction columns. R_t (method A) = 5.4 min. ESI-MS m/z (M + H) 443.9. ¹H NMR (D₂O) δ 1.66 (s, 3H), 1.87-1.90 (m, 1 H), 2.27-2.30 (m, 1 H), 2.80 (d, 1 H, J = 13.2 Hz), 2.92 (s, 6H), 3.06-3.09 (m, 1 H), 3.79 (s, 1 H), 7.00 (d, 1 H, J = 8.1 Hz), 7.23 (d, 1 H, J = 7.4 Hz), 7.51-7.54 (m, 1 H).

4S-(4α, 12aα)]-12-Amino-7-chloro-4-dimethylamino-3,6, 10, 12a-tetrahydroxy-6- methyl-1, 11-dioxo-1 ,4,4a, 5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide.

Chlortetracycline hydrochloride (500 mg, 0.97 mmol) was dissolved in absolute ethanol (40 mL), then anhydrous ammonia was bubbled into the refluxing solution for 2 h. The crude mixture was afterwards evaporated to dryness. Primary column chromatography (silica gel, ethyl acetate-ethanol), followed by preparative HPLC (C18-reverse phase Vydac™ column, 250 x 50 mm, using a binary gradient of 0.1% TFA in water (phase A) and 0.1 % TFA in acetonitrile (phase B) on a gradient from 20% phase B at time 0 to 40% phase B at 120 min at a flow rate of 10 mL/min) gave 4S-(4α,12aα)]-12-amino-7-chloro-4-dimethylamino- 3,6,10,12a-tetrahydroxy-6-methyl-1 ,11-dioxo-1 ,4,4a,5,5a,6,11 ,12a-octahydro- naphthacene-2-carboxylic acid amide as trifluoroacetate. After neutralizing the collected fractions with Na₂CO₃ and freeze-drying, the solid material was dissolved in the minimum amount of water and separated from the water-soluble salts using PrepSep™-C18 disposable extraction column to give the pure compound as a free base. R_t (method A) = 6.3 min. ESI-MS m/z (M + H) 478.1. ¹H NMR (D₂O) δ 1.76 (q, 1 H, J = 12.3 Hz), 1.95 (s, 3H), 2.16 (m, 1 H), 2.56 (s, 6H), 2.68 (d, 1 H, J = 12.8 Hz), 2.92 (bs, 1 H), 3.11 (s, 1 H), 6.92 (d, 1 H, J = 8.6 Hz), 7.50 (d, 1 H, J = 8.6 Hz).

4S-(4a, 12aa)]-12-Amino-7-chloro-4-dimethylamino-3,6, 10, 12a-tetrahydroxy-1, 11- dioxo-1 ,4,4a, 5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide. Anhydrous ammonia was bubbled into a stirred suspension of demeclocycline hydrochloride (0.400 g, 0.80 mmol) in absolute ethanol (130 mL) under reflux for 7 h. Stirring continued at room temperature overnight, then the reaction mixture was brought to pH 7 with diluted acetic acid, and filtered. The solvents in the filtrate were partially removed under reduced pressure, then the remaining solution was freeze-dried. After a primary purification by column chromatography (silica gel, dichloromethane-ethanol), the residue was separated by HPLC (C18-reverse phase Vydac™ column, 50 x 250 mm, by applying a binary gradient of 0.1 % TFA in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 20% phase B to 30% phase B in 100 min at a flow rate of 15 mL/min). After neutralizing with NaHCO₃, the collected fractions were freeze-dried, and the tetracycline derivative as a free base was separated from the water- soluble salts using PrepSep™-C18 disposable extraction columns. R_t (method A) = 5.9 min. ESI-MS m/z (M + H) 463.9. ¹H NMR (CD₃OD) δ 1.62-1.64 (m, 1 H), 1.85- 1.88 (m, 1 H), 2.26 (s, 6H), 2.45-2.47 (m, 1 H), 2.67-2.74 (m, 2H), 4.59 (s, 1 H), 6.62-6.64 (m, 2H), 7.18-7.20 (m, 2H). [4S-(4a, 12aa)]-12-Amino-4-dimethylamino-3,5, 10, 12a-tetrahydroxy-6-methyl-1 , 11- dioxo-1 ,4,4a, 5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid.

Doxycycline hyclate (512 mg, 1.00 mmol) was dissolved in absolute ethanol (20 mL). Ammonia gas was bubbled into the solution under reflux for 6 h. Upon treatment of the cooled reaction mixture with 0.1 M acetic acid (50 mL), a solid impurity separated that was filtered off. After neutralizing the filtrate with NaHCO₃₎ the solvent was removed under reduced pressure. The residue was purified twice on HPLC C18-reverse phase Vydac™ column, 50 x 250 mm, by applying a binary gradient of 0.1 % TFA in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 20% phase B to 40% phase B in 200 min at a flow rate of 15 mL/min). After freeze-drying the collected fractions, the solid material was dissolved in the minimum amount of water and treated with the required amount of triethylamine. The tetracycline derivative was separated from the water-soluble salts using PrepSep™-C18 disposable extraction columns to give [4S-(4α,12aα)]- 12-amino-4-dimethylamino-3,5, 10,12a-tetrahydroxy-6-methyl-1 , 11 -dioxo- 1 ,4,4a,5,5a,6,11 ,12a-octahydro-naphthacene-2-carboxylic acid as pale yellow powder. R_t (method B) = 4.6 min. ESI-MS m/z (M + H) 443.9. ¹H NMR (CD₃OD) δ 1.47(dd, 3H, J = 7.4 Hz), 2.41 (dd, 1 H, J = 11.6 and 8.1 Hz), 2.49 (bs, 7H), 2.64 (m, 1 H), 3.46 (bs, 1 H), 3.72 (bs, 1 H), 6.70 (d, 1 H, J = 8.3 Hz), 6.82 (d, 1 H, J = 7.3 Hz), 7.28 (dd, 1 H, J = 8.3 and 7.3 Hz).

[4S-(4 , 12a )]-12-Amino-4, 7-bis(dimethylamino)-3, 10, 12a-trihydroxy-1, 11-dioxo- 1, 4,4a,5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide. Minocycline hydrochloride (100 mg, 0.20 mmol) was dissolved in absolute ethanol (20 mL). Ammonia gas was bubbled into the solution under refluxed for 2 h, then the reaction mixture was further stirred at room temperature for 2 h under nitrogen. The reaction mixture was then evaporated to dryness, water (5.0 mL) was added, and the solid material was filtered off. The filtrate was extracted with dichloromethane (3 x 5 mL), the organic phase was dried over anhydrous sodium sulfate, and treated with hexane (15 mL) prior to leaving it at -20°C for 1 h. The precipitated material was filtered off, and the filtrate was evaporated to dryness to give, after crystallization from ethanol-hexane, the [4S-(4α,12aα)]-12-amino-4,7- bis(dimethylamino)-3,10,12a-trihydroxy-1 ,11-dioxo-1 , 4,4a, 5,5a,6,11 ,12a- octahydro-naphthacene-2-carboxylic acid amide as pale yellowish powder. R_t (method A) = 4.8 min. ESI-MS m/z (M + H) 456.9. ¹H NMR (D₂O) δ 1.63 (q, 1 H, J = 13.0 Hz), 2.14 (d, 1H, J = 13.4 Hz), 2.19 (t, 1 H, J = 14.5 Hz), 2.58 (s, 6H), 2.69 (d, 1 H, J = 12.2 Hz), 2.75-2.95 (m, 1 H), 2.85 (s, 6H), 2.98 (d, 1 H, J = 15.1 Hz), 3.73 (s, 1 H), 6.80 (d, 1 H, J = 8.7 Hz), 7.35 (d, 1 H, J = 8.7 Hz).

[4S-(4a, 12aa)]-12-Amino-4-dimethylamino-3, 10, 12a-trihydroxy-1 , 11-dioxo- 1,4,4a, 5,5a, 6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide.

Sancycline (0.83 g, 2.0 mmol) was dissolved in absolute ethanol (40 mL). Ammonia gas was bubbled into the refluxing solution for 5 h. After water (1.0 mL) had been added, the crude mixture was evaporated to dryness under reduced pressure. The dry crude material was extracted with dichloromethane (100 mL) with vigorous stirring, then the solid material was filtered off, and the filtrate was treated with charcoal (1.0 g). After the solvent had been removed under reduced pressure, the residue was recrystallized from ethanol to give [4S-(4α,12aα)]-12- amino-4-dimethylamino-3, 10,12a-trihydroxy-1 , 11 -dioxo-1 ,4,4a,5,5a,6, 11 ,12a- octahydro-naphthacene-2-carboxylic acid amide as pale yellow powder. R_t (method A) = 6.6 min. ESI-MS m/z (M + H) 413.7. ¹H NMR (CD₃OD/CF₃COOD) δ 1.02 (dd, 1 H, J = 6.6 and 6.7 Hz), 1.36 (dd, 1 H, J = 12.3 and 12.1 Hz), 1.94 (d, 1 H, J = 10.9 Hz), 2.30 (d, 1 H, J = 13.9 Hz), 2.60 (d, 1 H, J = 14.6 Hz), 2.73 (d, 1 H, J = 13.2 Hz), 2.85 (s, 6H), 3.83 (s, 1 H), 6.47 (d, 1 H, J = 6.9 Hz), 6.53 (d, 1 H, J = 8.0 Hz), 7.09 (dd, 1 H, J = 8.0 and 6.9 Hz). [4S-(4 , 12aa)]-7-Chloro-4-dimethylamino-3,6, 10, 12, 12a-pentahydroxy-11- hydroxy-imino-6-methyl-1-oxo-1,4,4a,5,5a,6, 11, 12a-octahydro-naphthacene-2- carboxylic acid amide.

Chlortetracycline hydrochloride (0.50 g, 0.97 mmol) and hydroxylamine hydrochloride (0.135 g) were dissolved in water (15 mL) and treated with triethylamine (0.41 mL). The reaction mixture was stirred at 60°C under nitrogen for 1.5 h. The crude mixture was filtered and the solvent was removed to give a residue which was purified using a preparative C18-reverse phase Vydac™ HPLC column (50 x 250 mm) and a method employing a binary gradient of 0.1% TFA in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 10% phase B at time 0 to 20% phase B in 100 min at a flow rate of 20 mL/min. The fractions collected were freeze-dried, and the tetracycline derivative as a trifluoroacetate was dissolved in the least volume of water and neutralized with triethylamine. The solution was passed through a PrepSep™-C18 column, which was then washed with water. The retained [4S-(4α,12aα)]-7-chloro-4- dimethylamino-3,6, 10,12,12a-pentahydroxy-11 -hydroxy-imino-6-methyl-1 -oxo- 1 ,4,4a,5,5a,6,11 ,12a-octahydro-naphthacene-2-carboxylic acid amide as a free base was eluted with methanol, the methanolic solution was treated with hydrochloric acid in a small excess and freeze-dried to give the desired compound. R_t (method A) = 5.8 min. ESI-MS m/z (M + H) 494.1. ¹H NMR (CD₃OD) δ 1.18 (t, 1 H, J = 14.4 Hz), 1.66 (q, J = 12.7 Hz), 1.81 (s, 3H), 2.44 (d, 1 H, J = 12.4 Hz), 2.75-2.78 (m, 2H), 3.07 (s, 1 H), 6.99 (d, 1 H, J = 8.5 Hz), 7.60 (d, 1 H, J = 8.5 Hz).

Reaction between tetracyclines and electrophile. Modification of aromatic substituents.

[4S-(4α, 12a )]-7-Nitro-3, 10, 12, 12a-tetrahydroxy-1, 11-dioxo 1,4,4a,5;5a,6, 11, 12a octahydro-naphthacene-2-carboxylic acid amide.

Sodium nitrite (1.07 g, 12.5 mmol) was added in one portion to a solution of sancycline (5.0 g, 12.2 mmol) in concentrated sulfuric acid (120 mL) at 0°C. The reaction mixture was stirred for 30 min. at 0°C, and then poured onto crashed ice. The mixture was extracted with t7-butanol (4 x 150 mL), and the n-butanol phase was washed with water (3 x 100 mL). The solvent was evaporated under reduced pressure to 200 mL and allowed to stand in refrigerator overnight. The precipitated product, comprising a mixture of 7- and 9-nitrosancycline, was collected by filtration. The separation of 7-nitrosancycline from 9-nitrosancycline was achieved by HPLC (Symmetry C18™ column, 19 x 50 mm, binary gradient of 0.1% TFA in water (phase A) and 0.1% TFA in acetonitrile (phase B) on a gradient from 16% phase B at time 0 to 18% phase B in 45 min at a flow rate of 10 mL/min). The obtained trifluoroacetate was converted into hydrochloride by addition of HCI, which was recrystallized from methanol-ether gave yellow solid of [4S-(4 ,12aα)]- 7-nitro-3, 10,12, 12a-tetrahydroxy-1 ,11-dioxo-1 ,4,4a,5,5a,6, 11 , 12a-octahydro- naphthacene-2-carboxylic acid amide. R_t (method A) = 6.75 min. ESI-MS m/z (M + H) 460.1. ¹H NMR (CD₃OD) δ 1.50 (q, 1 H, J = 7.5 Hz), 2.26 (d, 2H, J = 7.5 Hz), 2.65 (t, 2H, J = 7.5 Hz), 2.87 (dd, 1 H, J = 14.5 and 3.5 Hz), 3.06 (s, 6H), 4.11 (s, 1 H), 6.94 (d, 1 H, J = 8.5 Hz), 8.08 (d, 1 H, J = 8.5 Hz).

[4S-(4 , 12a )]-7-Amino-3, 10, 12, 12a-tetrahydroxy-1, 11-dioxo- 1,4,4a,5,5a,6, 11, 12a-octahydro-naphthacene-2-carboxylic acid amide.

A mixture of 7- and 9-nitrosancycline (3.8 g, 8.3 mmol), resulted from the nitration of sancycline, was dissolved in methanol (330 mL) containing concentrated HCI (8.6 mL). To the resulting solution, 10% palladium on activated carbon (380 mg) was added, and the reaction mixture was hydrogenated under a hydrogen atmosphere (40 psi.) for 1 h. After filtration through a Celite™ plug, the filtrate was evaporated to dryness. HPLC separation (Symmetry C18™ column, 19 x 50 mm, isocratic conditions, 5% acetonitrile containing 0.1 % TFA in water containing 0.1 % TFA at a flow rate of 5 mL/min), and subsequent anion exchange by treatment with hydrochloric acid gave, after recrystallization from methanol-ether, [4S-(4α,12aα)]- 7-amino-3, 10,12, 12a-tetrahydroxy-1 ,11-dioxo-1 ,4,4a, 5,5a,6, 11 , 12a-octahydro- naphthacene-2-carboxylic acid amide as hydrochloride. R_t (method A) = 4.56 min. ESI-MS m/z (M + H) 430.1. ¹H NMR (CD₃OD) δ 1.40 (q, 1 H, J = 14 Hz), 1.97 (d, 1 H, J = 14 Hz), 2.13 (t, 1 H, J = 14 Hz), 2.58-2.88 (m, 10H), 6.66 (d, 1 H, J = 9 Hz), 7.22 (d, 1 H, J = 9 Hz).

[4S-(4a, 12a )]-7,9-Dibromo-4-dimethylamino-3, 10, 12, 12a-tetrahydroxy-1 , 11- dioxo-1 ,4,4a,5,5a,6, 11 , 12a-octahydronaphthacene-2-carboxylic acid amide (7,9-di- Br-sc).

Sancycline (414 mg, 1.00 mmole) was dissolved in concentrated sulfuric acid (5 mL) under efficient stirring at 0°C, and treated with /V-bromosuccinimide (392 mg. 2.2 mmoles). Stirring was continued at the same temperature for 30 min, then the reaction mixture was added dropwise to cold diethylether (250 mL). The solid that precipitated was filtered, dried and separated on a C18-reverse phase Vydac column (50 x 250 mm) by applying a binary gradient of 0.1 % TFA in water (phase A) and 0.1 % TFA in acetonitrile (phase B) on a gradient from 20% phase B to 50% phase B in 150 min at a flow rate of 20 mL/min. After freeze-drying the collected fractions, the solid material was dissolved in the minimum amount of water and treated with the required amount of triethylamine. The tetracycline derivative as a free base was separated from the water-soluble salts using PrepSep™-C18 disposable extraction columns. R_t (method A) = 7.8 min. ESI-MS m/z (M + H) 573.0. ¹H NMR (CD₃OD) δ 1.77 (q, 1 H, J = 11.7 Hz), 2.33 (d, 1 H, J = 12.7 Hz), 2.48 (t, 1 H, J = 14.8 Hz), 3.00-3.45 (m, 3H), 4.22 (s, 1 H), 8.13 (s, 1 H). From the foregoing, it will be seen that this invention is one well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. REFERENCES

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Tillmanns H, Neuhof H. Reduction of myocardial infarction by calpain inhibitors A- 705239 and A-705253 in isolated perfused rabbit hearts. Biol. Chem. 2004, 385, 1077-1082.

Ryan ME, Usman A, Ramamurthy NS, Golub LM, Greenwald RA. Excessive matrix metalloproteinase activity in diabetes: inhibition by tetracycline analogues with zinc reactivity. Curr. Med. Chem. 2001 , 8, 305-316. Schumacher PA, Siman RG, Fehlings MG. Pretreatment with calpain inhibitor CEP-4143 inhibits calpain I activation and cytoskeletal degradation, improves neurological function, and enhances axonal survival after traumatic spinal cord injury. J. Neurochem. 2000, 74, 1646-1655 Wang CH, Chen YJ, Lee TH, Chen YS, Jawan B, Hung KS, Lu CN, Liu JK.

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Claims

Claims:

1. Use of a calpain-inhibiting effective amount of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof for inhibiting calpain activity or activation.

2. Use according to claim 1 , wherein the calpain is one or more of calpains 1- 15.

3. Use according to claim 1 , wherein the calpain is calpain I, calpain II, or both calpain I and calpain II.

4. Use according to any one of claims 1 to 3 for treating or preventing a condition associated with calpain activation or activity in a subject in need of treatment or prevention of the condition.

5. Use according to claim 4, wherein the condition is selected from the group consisting of traumatic brain injury, traumatic spinal cord injury, stroke, Wallerian degeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease, damages of motoneurons from exocitotoxic cell death, axonal degeneration, peripheral neuropathy, inflammation, severe hemorrhage, muscular dystrophy, Duchenne muscular dystrophy, rheumatoid arthritis, diabetic retinopathy, acoustic trauma, virus-induced myocardial injury, acute myocardial infarction, testicular torsion, liver ischemia, kidney ischemia, inflammatory bowel disease, liver transplantation, prostate cancer, lung cancer, renal cancer, platelet secretion, platelet aggregation, platelet spreading, HIV-1 replication, replication of severe acute respiratory syndrome-associated coronavirus, invasion of erythrocytes by P. falciparum, prion propagation in Creutzfeldt Jacob disease, human acute lymphoblastic leukemia, non-Hodgkin's lymphoma cells, tumor cell, cataractogenesis and combinations thereof.

6. Use according to claim 4, wherein the condition is a neurological condition or disease.

7. Use according to claim 4, wherein the condition is an acute or chronic condition or disease.

8. Use according to any one of claims 1 to 3, for treating or preventing calpain- mediated physiological damage induced by calcium activation of calpain in a subject.

9. Use according to claim 8, wherein the calpain-mediated physiological damaged is induced by ischemia.

10. Use according to claim 9, wherein the ischemia is a neural ischemic event which is identified prior to using the tetracycline.

11. Use according to any one of claims 4 to 10, wherein the subject is a mammal.

12. Use according to any one of claims 4 to 10, wherein the subject is a primate, a canine, a feline, or a rodent.

13. Use according to any one of claims 4 to 10, wherein the subject is a human.

14. Use according to any one of claims 1 to 13, wherein the calpain is located within a cell.

15. Use according to any one of claims 1 to 14, wherein the effective amount provides measurable levels of calpain inhibition as measured by fluorescence using Ac-Leu-Leu-Tyr-AFC as a substrate for calpain activity, with fluorescence measured with a 400 nm excitation filter and a 505 nm emission filter.

16. Use according to any one of claims 1 to 15, wherein the tetracycline is also an antioxidant.

17. Use according to any one of claim 1 to 16, wherein the tetracycline is not an antibiotic.

18. Use according to any one of claim 1 to 17, wherein the tetracycline is not an MMP inhibitor.

19. Use according to any one of claims 1 to 18, wherein the tetracycline has anti-glycation activity.

20. Use according to any one of claims 1 to 19, wherein the tetracycline does not bind Ca²⁺.

21. Use according to any one of claim 1 to 15, wherein the tetracycline is a 7-substituted tetracycline or a pharmaceutically acceptable salt thereof.

22. Use according to claim 21 , wherein the 7-substituted tetracycline is substituted by a halogen in the 7-position.

23. Use according to claim 22, wherein the halogen is chloro.

24. Use according to any one of claims 21 to 23 for treating or preventing stroke.

25. Use according to any one of claims 1 to 15 or 21 to 24, wherein the tetracycline is an 11 -substituted tetracycline, a 12-substituted tetracycline, an 11- and 12-substituted tetracycline, or a pharmaceutically acceptable salt thereof.

26. Use according to claim 25, wherein the tetracycline is substituted in the 12-position by a group that blocks binding of cations between the 11- and 12-positions.

27. Use according to claim 26, wherein the group bridges the 11- and 12-positions or 1- and 12-positions of the tetracycline.

28. Use according to claim 27, wherein the bridging group forms a pyrazole ring fused with the tetracycline.

29. Use according to claim 25, wherein the tetracycline is substituted in the 12-position by an amino group.

30. Use according to any one of claims 1 to 15, wherein the tetracycline is a compound of formula II:

wherein:

Y is CH-C(R°ZW), C —= rC-oR%°>Z-. or CR _9^arR_>1 ^ι0^u.;

Z and W are each independently hydrogen, Cι-C₈ linear, branched or cyclic alkyl, C₂-Cβ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-Cβ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-C-iβ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl, CrC₈ alkoxy, sulfhydryl, CrC₈ alkylthio, CrC₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, C-ι-C₈ alkylamino, cyano or halogen;

R⁴ is NR⁵R⁶, hydrogen, Cι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C7-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, hydroxyl or halogen;

R¹, R², R⁵ and R⁶ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cι-C₈ alkoxy, CrC₈ alkylthio, C C₈ alkylsulfynyl, Cι-C₈ alkylamino, C₆-C-ι₈ aryl, C₇-C₁ arylalkyl or C₂-Cι₈ heteroaryl, or R¹ and R² form a 5- or 6-membered nitrogen-containing ring, or R⁵ and R⁶ form a 5- or 6-membered nitrogen-containing ring;

R³, R¹³ and R¹⁴ are each independently hydrogen, C C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C₁₈ aryl, C₇-C₁₉ arylalkyl or C₂-C-ι₈ heteroaryl;

R⁷ is hydrogen, C-ι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C₁₈ aryl, C₇-C₁₉ arylalkyl, C₂-C1₈ heteroaryl, hydroxyl, Cι-C₈ alkoxy, sulfhydryl, C-ι-C₈ alkylthio, Cι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, Cι-C₈ alkylamino, halogen, C-ι-C₈ alkanoyl, C₇-C₁₉ aralkanoyl, C₇-C₁₉ alkaroyl, C₆-Cι₈ aroyl, C₂-Cι₈ heteroaroyl, C₂-C₈ alkylcarbonyloxy or C₇-C₁₉ arylcarbonyloxy;

R⁸ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-C-iβ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, halogen, hydroxyl, CrC₈ alkoxy, sulfhydryl, C-ι-C₈ alkylthio,_, Cι-C₈ alkylsulfynyl, d-C₈ alkylsulfonyl or Cι-C₈ alkylamino;

R⁹ and R¹⁰ are each independently hydrogen, =CH₂, absent, hydroxyl, halogen, sulfhydryl, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C2-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C₇-C.₉ arylalkyl, C -Cι₈ heteroaryl, C C₈ alkoxy, C C₈ alkylthio, Cι-C₈ alkylsulfynyl, CrC₈ alkylsulfonyl or CrC₈ alkylamino;

R¹¹ and R¹² are each independently hydrogen, C-i-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₇-C1₉ arylalkyl, C₈-C₂o aralkenyl, C₈-C₂₀ aralkynyl, C₆-C-ι₈ aryl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, C-i-C₈ alkoxy, sulfhydryl, C-i-C₈ alkylthio, Cι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, Cι-C₈ alkylamino, C₂-C1₆ dialkylamino, C-ι-C₈ acyl, nitro, cyano, carboxyl or Cι-C₈ alkoxycarbonyl;

dotted lines between C11 and C12 are independently bonds within the tetracycline ring structure or bonds from the tetracycline ring structure to X², X³ or X⁴;

X¹ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C^C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C-ι₈ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, halogen, hydroxyl, halogen, nitro, C C₈ alkoxy, Cβ-C-is aryloxy, amino, C C₈ alkylamino, C₂-Cι₆ dialkylamino, C C₈ alkylamido or C C₈ acyl;

X² is hydrogen, halogen, hydroxyl, Cι-C₈ alkoxy, mercapto, CrC₈ alkylthio, phenoxy, C₆-C₁₂ aryloxy, phenyl, amino, CrC₈ alkylamino, C-ι-C₈ amido or CrC₈ acyl, or X² is oximino or oxo when the dotted line at C11 forms a bond with X², or X² is joined with X³ to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure when the dotted line at C11 forms a bond with X²;

X³ and X⁴ are independently hydrogen, halogen, hydroxyl, C C₈ alkoxy, mercapto, Cι-C₈ alkylthio, phenoxy, phenyl, C₆-Cι₂ aryloxy, amino, C-i-C₈ alkylamino, Cι-C₈ amido or C-i-C₈ acyl, or X³ and X⁴ are joined to form oxo, or X³ is joined with X² to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure when the dotted line at C11 forms a bond with X², or X⁴ is absent when the dotted line at C12 is a bond within the tetracycline ring structure, or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

X⁵ and X⁶ are independently hydrogen, halogen, hydroxyl, Cι-C₈ alkoxy, mercapto, CrC₈ alkylthio, C₆-Ci2 aryloxy, C₆-C12 aryl, C₂-Cι₈ heteroaryl, amino, C C₈ alkylamino, C C₈ amido, CrC₈ acyl, or X⁵ and X⁶ are joined to form oxo, or X⁶ is a bond from the tetracycline ring to X⁵ when X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

and pharmaceutically acceptable salts thereof.

31. Use according to claim 30, wherein heteroatoms in substituted alkyl, substituted alkenyl, substituted alkynyl and heteroaryl are nitrogen, oxygen, sulfur or a combination thereof.

32. Use according to any one of claims 30 to 31 , wherein X¹ is halogen.

33. Use according to any one of claims 30 to 31 , wherein X¹ is chloro.

34. Use according to any one of claims 32 to 33 for treating or preventing stroke.

35. Use according to any one of claims 30 to 34, wherein X² is CrC₈ alkylamino, or X² is oximino or oxo when the dotted line at C11 forms a bond with X², or X² and X³ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure when the dotted line at C11 forms a bond with X².

36. Use according to claim 35, wherein the fused ring is a pyrazole.

37. Use according to any one of claims 30 to 34, wherein X⁶ is a bond from the tetracycline ring to X⁵ when X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure.

38. Use according to claim 37, wherein the fused ring is a pyrazole.

39. Use according to any one of claims 30 to 34, wherein X² is oximino or C-ι-C₈ alkylamino.

40. Use according to any one of claims 30 to 34, wherein X³ is amino and the dotted line at C12 is a bond within the tetracycline ring structure.

41. Use according to any one of claims 1 to 15, wherein the tetracycline is chlortetracycline, demeclocycline, sancycline, tetracycline, minocycline, doxycycline, meclocycline, oxytetracycline, rolitetracycline, 7-aminosancycline, 7-chlorosancycline, 7-bromosancycline, 7-iodosancycline, 7-nitrosancycline, 7-phenylsancycline, 7-(2-thienyl)sancycline, 7,9-dibromosancycline,

4-de(dimethylamino)tetracycline (CMT-1 ), 4-de(dimethylamino)sancycline (CMT-3), 4-de(dimethylamino)chlortetracycline (CMT-4), 4-de(dimethylamino)minocycline (CMT-10), 11 ,12-pyrazolotetracycline (CMT-5), 11 ,12-pyrazolochlortetracycline, 11 ,12-pyrazolominocycline, 12-hydroxy-1 ,12-pyrazolotetracycline, 12-hydroxy-1 ,12-pyrazolominocycline, 12-aminotetracycline, 12-aminochlortetracycline, 12-aminodemeclocycline, 12-aminominocycline, 12-aminodoxycycline, 12-aminosancycline, 11-(N-methylamino)minocycline, 11-oximinochlortetracycline, a pharmaceutically acceptable salt thereof, or a mixture thereof.

42. Use according to any one of claims 1 to 15, wherein the tetracycline is 11-oximinochlortetracycline, 11-(N-methylamino)minocycline, 12-hydroxy-1 ,12-pyrazolotetracycline, 12-hydroxy-1 ,12-pyrazolominocycline, 12-aminochlortetracycline, 12-aminodemeclocycline, 12-aminodoxycycline, 12-aminominocycline, 12-aminosancycline, 12-aminotetracycline, a pharmaceutically acceptable salt thereof, or a mixture thereof.

43. A compound of formula I:

wherein:

Y is CH-C(R°ZW), C=CR°Z or CR^aR^1u;

Z and W are each independently hydrogen, Cι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, Cι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Cis aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl, C-ι-C₈ alkoxy, sulfhydryl, C-i-C₈ alkylthio, C-ι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, Cι-C₈ alkylamino, cyano or halogen;

R⁴ is NR⁵R⁶, hydrogen, C-ι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Cι₈ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, hydroxyl or halogen;

R¹, R², R⁵ and R⁶ are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C2-C₈ linear, branched or cyclic alkynyl, C-ι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cι-C₈ alkoxy, C-ι-C₈ alkylthio, Cι-C₈ alkylsulfynyl, C C₈ alkylamino, C₆-Cι₈ aryl, C₇-C₁₉ arylalkyl or C₂-Cι₈ heteroaryl, or R¹ and R² form a 5- or 6-membered nitrogen-containing ring, or R⁵ and R⁶ form a 5- or 6-membered nitrogen-containing ring;

R³, R¹³ and R¹⁴ are each independently hydrogen, C C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, CrC₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C₇-C₁₉ arylalkyl or C₂-Cι₈ heteroaryl;

R⁷ is hydrogen, C-ι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C7-C₁₉ arylalkyl, C₂-Cι₈ heteroaryl, hydroxyl, CrC₈ alkoxy, sulfhydryl, Cι-C₈ alkylthio, C-i-C₈ alkylsulfynyl, C-ι-C₈ alkylsulfonyl, C-i-C₈ alkylamino, halogen, CrC₈ alkanoyl, C₇-C1₉ aralkanoyl, C₇-C₁₉ alkaroyl, Cβ-Ciβ aroyl, C₂-Cι₈ heteroaroyl, C₂-C₈ alkylcarbonyloxy or C7-C₁₉ arylcarbonyloxy;

R⁸ is hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, Cβ-Ciβ aryl, C₇-C-₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, halogen, hydroxyl, Cι-C₈ alkoxy, sulfhydryl, C C₈ alkylthio, Cι-C₈ alkylsulfynyl, CrC₈ alkylsulfonyl or Cι-C₈ alkylamino;

R⁹ and R¹⁰ are each independently hydrogen, =CH2, absent, hydroxyl, halogen, sulfhydryl, C-ι-C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-i-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-Cι₈ aryl, C₇-C1₉ arylalkyl, C₂-Cι₈ heteroaryl, C C₈ alkoxy, Cι-C₈ alkylthio, C-ι-C₈ alkylsulfynyl, C C₈ alkylsulfonyl or Cι-C₈ alkylamino;

R¹¹ and R¹² are each independently hydrogen, CrC₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C7-C1₉ arylalkyl, C₈-C₂o aralkenyl, C₈-C₂o aralkynyl, C₆-Cι₈ aryl, C₂-Cι₈ heteroaryl, halogen, hydroxyl, C-ι-C₈ alkoxy, sulfhydryl, C-ι-C₈ alkylthio, Cι-C₈ alkylsulfynyl, Cι-C₈ alkylsulfonyl, amino, Cι-C₈ alkylamino, C₂-C₁₆ dialkylamino, CrC₈ acyl, nitro, cyano, carboxyl or C-ι-C₈ alkoxycarbonyl;

dotted lines between C11 and C12 are each independently bonds within the tetracycline ring structure or bonds from the tetracycline ring structure to X², X³ or X⁴;

X¹ is hydrogen, C C₈ linear, branched or cyclic alkyl, C₂-C₈ linear, branched or cyclic alkenyl, C₂-C₈ linear, branched or cyclic alkynyl, C-ι-C₈ linear, branched or cyclic alkyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkenyl substituted by a heteroatom, C₂-C₈ linear, branched or cyclic alkynyl substituted by a heteroatom, C₆-C-ι₈ aryl, C₇-C₁₉ arylalkyl, C₂-C-ι₈ heteroaryl, halogen, hydroxyl, halogen, nitro, CrC₈ alkoxy, C₆-Cι₈ aryloxy, amino, C-ι-C₈ alkylamino, C₂-C₁₆ dialkylamino, C-ι-C₈ alkylamido or CrC₈ acyl;

X² is hydrogen, halogen, hydroxyl, C C₈ alkoxy, mercapto, CrC₈ alkylthio, phenoxy, C₆-C₁₂ aryloxy, phenyl, amino, Cι-C₈ alkylamino, C-ι-C₈ amido or CrC₈ acyl, or X² is oximino or oxo when the dotted line at C11 forms a bond with X²;

X³ and X⁴ are each independently hydrogen, halogen, hydroxyl, Cι-C₈ alkoxy, mercapto, CrC₈ alkylthio, phenoxy, phenyl, C₆-Cι₂ aryloxy, amino, C C₈ alkylamino, CrC₈ amido or C-ι-C₈ acyl, or X³ and X⁴ are joined to form oxo, or X⁴ is absent when the dotted line at C12 is a bond within the tetracycline ring structure, or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

X⁵ and X⁶ are independently hydrogen, halogen, hydroxyl, C-ι-C₈ alkoxy, mercapto, Cι-C₈ alkylthio, C₆-C-ι₂ aryloxy, C₆-C₁₂ aryl, C₂-C₁₈ heteroaryl, amino, C C₈ alkylamino, C-ι-C₈ amido, C C₈ acyl, or X⁵ and X⁶ are joined to form oxo, or X⁶ is a bond from the tetracycline ring to X⁵ when X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring fused to the tetracycline ring structure;

and pharmaceutically acceptable salts thereof,

with the proviso that either: X² is oximino or Cι-C₈ alkylamino; or X⁴ is amino; or X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring.

44. The compound of claim 43, wherein X² is oximino or CrC₈ alkylamino, and pharmaceutically acceptable salts thereof.

45. The compound of claim 43, wherein X⁴ is amino, and pharmaceutically acceptable salts thereof.

46. The compound of claim 43, wherein X⁴ and X⁵ are joined to form a 5- or 6-membered nitrogen-containing ring, and pharmaceutically acceptable salts thereof.

47. The compound of claim 46, wherein X⁴ and X⁵ are joined to form a pyrazole ring, and pharmaceutically acceptable salts thereof.

48. The compound of claim 43 which is 11-oximinochlortetracycline, 11-(N-methylamino)minocycline, 12-hydroxy-1 ,12-pyrazolotetracycline, 12-hydroxy-1 ,12-pyrazolominocycline, or a pharmaceutically acceptable salt thereof.

49. The compound of claim 43 which is 12-aminochlortetracycline, 12-aminodemeclocycline, 12-aminodoxycycline, 12-aminominocycline,

12-aminosancycline or 12-aminotetracycline or a pharmaceutically acceptable salt thereof.

50. Use of a calpain-inhibiting effective amount of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof for preparing a medicament for inhibiting calpain activity or activation.

51. A commercial package comprising a calpain-inhibiting effective amount of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof together with instructions for its use for inhibiting calpain activity or activation.

52. A pharmaceutical composition comprising a calpain-inhibiting effective amount of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier, agent, excipient, adjuvant, vehicle or combination thereof, for inhibiting calpain activity or activation.

53. A method for inhibiting calpain activity or activation comprising contacting a calpain-inhibiting effective amount of a tetracycline or a pharmaceutically acceptable salt thereof or a mixture of tetracyclines or pharmaceutically acceptable salts thereof with a calpain.