U.S. patent application number 15/899535 was filed with the patent office on 2019-02-07 for methods of using substituted tetracycline compounds to modulate rna.
The applicant listed for this patent is Paratek Pharmaceuticals, Inc.. Invention is credited to Michael P. Draper, Graham Jones, Stuart B. Levy, Mark L. Nelson.
Application Number | 20190040001 15/899535 |
Document ID | / |
Family ID | 32176687 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190040001 |
Kind Code |
A1 |
Levy; Stuart B. ; et
al. |
February 7, 2019 |
METHODS OF USING SUBSTITUTED TETRACYCLINE COMPOUNDS TO MODULATE
RNA
Abstract
A method for modulating RNA with tetracycline compounds is
described.
Inventors: |
Levy; Stuart B.; (Boston,
MA) ; Draper; Michael P.; (Windham, NH) ;
Nelson; Mark L.; (Midway, UT) ; Jones; Graham;
(Blue Bell, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Paratek Pharmaceuticals, Inc. |
Boston |
MA |
US |
|
|
Family ID: |
32176687 |
Appl. No.: |
15/899535 |
Filed: |
February 20, 2018 |
Related U.S. Patent Documents
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15668055 |
Aug 3, 2017 |
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15899535 |
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15384694 |
Dec 20, 2016 |
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15668055 |
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14790179 |
Jul 2, 2015 |
9562003 |
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15384694 |
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14201401 |
Mar 7, 2014 |
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14790179 |
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13426408 |
Mar 21, 2012 |
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14201401 |
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10692764 |
Oct 24, 2003 |
8173624 |
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13426408 |
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60421248 |
Oct 24, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
31/14 20180101; A61P 43/00 20180101; A61P 9/00 20180101; A61P 35/00
20180101; A61P 25/28 20180101; A61P 7/06 20180101; A61P 9/10
20180101; A61P 19/02 20180101; A61P 25/14 20180101; A61P 31/12
20180101; A61P 3/06 20180101; A61K 31/65 20130101; A61P 11/00
20180101; A61P 11/06 20180101; A61P 3/10 20180101; C07C 237/26
20130101 |
International
Class: |
C07C 237/26 20060101
C07C237/26; A61K 31/65 20060101 A61K031/65 |
Claims
1. A method for treating a subject for a DTMR, comprising:
administering to said subject an effective amount of a tetracycline
compound, such that said DTMR is treated.
2-56. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/201,401, filed Mar. 7, 2014; which is a
continuation of U.S. patent application Ser. No. 13/426,408, filed
Mar. 21, 2012; which is a continuation of U.S. patent application
Ser. No. 10/692,764, filed Oct. 24, 2003, now U.S. Pat. No.
8,173,624, issued May 8, 2012; which claims the benefit of U.S.
Provisional Application No. 60/421,248, filed Oct. 24, 2002. Each
of the foregoing applications is incorporated herein by reference
in its entirety.
BACKGROUND
[0002] Molecules of RNA are transcribed from DNA. RNA molecules are
relatively short compared to DNA molecules. RNA transcripts that
direct the synthesis of protein molecules are called messenger RNA
(mRNA) molecules; while other RNA transcripts serve as transfer
RNAs (tRNAs) or form the RNA components of ribosomes (rRNA) or
smaller ribonucleoprotein particles.
[0003] The amount of RNA-made from a particular region of DNA is
controlled by gene regulatory proteins that bind to specific sites
on DNA close to the coding sequence of a gene. In any cell at any
given time, some genes are used to make RNA in very large
quantities while other genes are not transcribed at all. For an
active gene thousands of RNA transcripts can be made from the same
DNA segment in each cell generation. Because each mRNA molecule can
be translated into many thousands of copies of a polypeptide chain,
the information contained in a small region of DNA can direct the
synthesis of millions of copies of a specific protein.
[0004] In eukaryotes, a primary RNA transcript is synthesized; this
transcript contains both introns and exons. Intron Sequences are
cut out and exon sequences on either side of an intron are joined
together by RNA splicing.
[0005] The translation of mRNA into protein depends on a set of
small RNA molecules known as transfer RNAs (tRNAs), each about 80
nucleotides in length. A tRNA molecule has a folded
three-dimensional conformation that is held together in part by
noncovalent base-pairing interactions like those that hold together
the two strands of the DNA helix. In the single-stranded tRNA
molecule, however, the complementary base pairs form between
nucleotide residues in the same chain, which causes the tRNA
molecule to fold up in a unique way.
[0006] The codon recognition process by which genetic information
is transferred from mRNA via tRNA to protein depends on the same
type of base-pair interactions that mediate the transfer of genetic
information from DNA to DNA and from DNA to RNA. The mechanics of
ordering the tRNA molecules on the mRNA require a ribosome. Each
ribosome is a large protein-synthesizing machine on which tRNA
molecules position themselves so as to read the genetic message
encoded in an mRNA molecule. The ribosome first finds a specific
start site on the mRNA that sets the reading frame and determines
the amino-terminal end of the protein. Then, as the ribosome moves
along the mRNA molecule, it translates the nucleotide sequence into
an amino acid sequence one codon at a time, using tRNA molecules to
add amino acids to the growing end of the polypeptide chain. When a
ribosome reaches the end of the message, both it and the freshly
made carboxyl end of the protein are released from the 3' end of
the mRNA molecule, into the cytoplasm.
[0007] Although most tRNAs are initially synthesized as a larger
precursor RNA, an RNA molecule has been shown to play the major
catalytic role in an RNA-protein complex that recognizes these
precursors and cleaves them at specific sites. A catalytic RNA
sequence also plays an important part in the life cycle of many
plant viroids. Most remarkably, ribosomes are now suspected to
function largely by RNA-based catalysis, with the ribosomal
proteins playing a supporting role to the ribosomal RNAs (rRNAs),
which make up more than half the mass of the ribosome. The large
rRNA by itself, for example, has peptidyl transferase activity and
catalyzes the formation of new peptide bonds:
[0008] The development of compositions and methods for modulation
of RNA would be of great benefit in modulating numerous cellular
processes and in the treatment of disorders.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the invention pertains at least in part,
to methods for modulating RNA. The method includes contacting an
RNA molecule or a cellular component with a substituted
tetracycline, such that modulation of RNA occurs.
[0010] In yet another embodiment, the invention includes a method
for treating a subject for a disorder treatable by modulation of
RNA or by modulation of RNA in combination with a second agent
(DTMR). The method includes administering to the subject an
effective amount of a tetracycline compound, or with a tetracycline
compound in combination with a second agent such that the DTMR is
treated. In further embodiments, the effective amount is effective
to modulate translation, the half-life, message translocation, the
binding of proteins, or splicing of the subject's RNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a bar graph depicting variations of iNOS mRNA,
protein, and nitrite levels in LPS stimulated mouse
macrophages.
DETAILED DESCRIPTION OF THE INVENTION
I. Methods for Modulating RNA
[0012] In one embodiment, the invention pertains at least in part,
to methods for modulating RNA. The method includes contacting an
RNA molecule, or a cellular component, with a tetracycline
compound, such that modulation of RNA occurs. In certain
embodiments, the RNA molecule is located within a subject.
[0013] The term "modulate," "modulating" or "modulation" includes
increasing, decreasing, or otherwise changing the level, amount,
function, structure, and/or activity of a particular molecule of
RNA.
[0014] The term "modulating RNA" or "modulation of RNA" includes
modulation of all functions, structures, amounts, and/or activities
of RNA which can be modulated by substituted tetracycline compounds
of the invention. Modulation of RNA includes, for example,
modulation of transcription, translation, translocation, catalysis,
secondary structure, splicing, stability, etc. The term also
includes modulations of the half-life of RNA. RNA can be modulated
within an organism, cell, intracellular space, nucleus and/or other
cellular compartment.
[0015] In one embodiment, a specific RNA molecule can be modulated,
e.g., a specific type of RNA (such as mRNA or rRNA) and/or an mRNA
specifying a particular protein can be modulated, while other mRNA
molecules are not affected. In another embodiment, RNA molecules in
general, e.g., several different types of RNA can be modulated
and/or a plurality of mRNA molecules specifying different proteins,
can be modulated according to the invention.
[0016] Modulation of RNA can occur directly, e.g., by modulation of
RNA itself, for example by binding of tetracycline to the RNA (for
example to alter its secondary structure) or indirectly, e.g., by
binding of a molecule to a component of a cell, e.g., a protein
with which the RNA interacts. For example, the tetracycline
compound may interact with a particular protein necessary for the
synthesis of an RNA molecule or with a protein molecule with which
the RNA molecule interacts (e.g., a ribosomal protein), and thus
modulate the RNA without directly binding to the RNA itself.
[0017] Examples of RNA molecules which may be modulated using the
methods of the invention include, but are not limited to, hnRNA,
mRNA, tRNA, ribosomal RNA, nuclear RNA, snRNA, and small RNA
aptamers.
[0018] The RNA may also be e.g., RNA from a prokaryotic cell, a
eukaryotic cell or may be viral RNA.
[0019] The term "cellular component" includes cells (in vivo and in
vitro), cellular organelles (e.g., ribosomes, nuclei, mitochondria,
chloroplasts, etc.), cytoplasm, etc. In a further embodiment, the
cells are located within a subject. In another embodiment, the
cells are in vitro. In another further embodiment, the cellular
component comprises RNA. In a further embodiment, the cellular
component is a cell which is associated with (e.g., derived from a
subject having or present in a subject having) a particular
disorder treatable by modulation of RNA or by modulation of RNA in
combination with a second agent (DTMR). For example, when the DTMR
is a tumor, the cellular component may be a cell of the tumor.
[0020] RNA modulation can occur via a variety of different
mechanisms. Exemplary mechanisms are listed below.
[0021] In one embodiment, RNA is modulated by direct interaction
with a tetracycline molecule (e.g., Berens. 2001. Tetracyclines in
Biology, Chemistry and Medicine ed. By M. Nelson, W. Hillen and R.
A. Greenwald, pp 177-196). Preferably, "modulation of RNA" as used
herein excludes interaction of a tetracycline molecule with the 30S
ribosomal subunit of a bacterial cell. In another preferred
embodiment, binding to 16S RNA and the proteins S4, S7, S9, and S17
are preferentially excluded from the term "modulation of RNA".
[0022] In embodiment, the RNA is modulated by altering RNA
transcription. For example, tetracycline compound may inhibit or
decrease the transcription of an mRNA. In another embodiment, a
tetracycline compound may inhibit transcription.
[0023] Levels of transcription can be measured in the presence and
the absence of a tetracycline compound using techniques that are
known in the art. Transcription of a specific gene can be measured
or genome-wide transcription (transcription of many genes) can be
detected. For example, in one embodiment, transcription levels can
be detected by performing nascent-chain run-on analysis. This
technique is known in the art and requires using P.sup.32 labeled
nucleotides; genes with high transcription levels can be detected
by intensity. In another example, transcription of a reporter gene,
e.g., luciferase which is easily detectable, can be operably linked
to a gene of interest. Detection of light will indicate
transcription of the gene of interest. Other exemplary methods for
measuring transcription include Northern blots and in situ
hybridization. Detection of transcription levels of more than one
gene can be performed using, e.g., microarrays (e.g., cDNA or
synthetic oligonucleotide arrays) or PCR.
[0024] In one embodiment, the RNA is modulated by altering RNA
translation. For example, tetracycline compound may inhibit or
decrease the translation of a mRNA. In one embodiment, a
tetracycline compound may inhibit RNA translation by inhibiting its
initiation. In another embodiment, a tetracycline compound may
inhibit translation by altering the point at which translation
terminates. For example, in one embodiment a tetracycline compound
can cause a ribosome to skip a termination codon and continue
translation.
[0025] In one embodiment, the level of a specific protein
translated from mRNA can be measured using standard techniques. For
example, in vitro or in situ analysis of enzyme activity can be
measured, if the protein is an enzyme. In vitro analysis can
include activity in bulk protein extracts, or after electrophoresis
to partially separate the enzyme from other proteins. In another
example, in vitro or in situ analysis can be performed using
immunochemical methods, i.e., employing a labeled antibody specific
for the protein. Quantification/visualization of the antibody can
the be performed. Western blots can be performed after
electrophoresis or cellular extracts or components can be assayed
directly, e.g., by ELISA or immunoprecipitation. If the protein is
sufficiently abundant, it can also be directly visualized after 1D
or 2D electrophoresis if it can be separated sufficiently from
other proteins by this method.
[0026] In one embodiment, the level of mRNA specifying a particular
protein can be measured. In another embodiment, the level of total
mRNA can be measured. Such measurements can be made using
techniques described herein or other techniques known in the
art.
[0027] In another embodiment, the half-life of RNA is modulated by
contacting the cellular component with the tetracycline compound.
For example, in one embodiment, the half-life of mRNA is increased.
In one embodiment, a tetracycline compound of the invention
increases the binding of RNA to a ribosome, thereby increasing the
stability of the RNA (Wei and Bechhofer. 2002. J. of Bacteriology
184: 889). In another embodiment, the half-life of mRNA is
decreased. In a further embodiment, the tetracycline compound is
not tetracycline or otherwise described in Wei and Bechhofer. 2002.
J. of Bacteriology 184:889.
[0028] For example, in one embodiment, a tetracycline molecule of
the invention increases the degradation of a specific mRNA
molecule. For example, the half-life of mRNA specifying a protein
such as iNOS (Amin et al. 1997. FEBS Letters 410:259) can be
measured. In a further embodiment, the tetracycline compound is not
doxycycline, minocycline, or a tetracycline compound described in
Amin et al. 1997. FEBS Letters 410:259.
[0029] In one embodiment, the half-life of RNA can be measured
using in vitro nuclear run-on transcription assays known in the
art. Nuclei can be isolated from cells and incubated in vitro with
radioactive precursors under conditions where nascent RNA pol II
will continue elongation off of the native gene, but will not
initiate transcription. The fraction of total incorporated
radioactivity in a specific transcript can be measured and a
degradation rate constant can be generated. In another embodiment,
a kinetic analysis can be performed. For example, radioactive
precursors can be provided and, over time, amounts of radioactivity
(specific activity) in a particular mRNA can be measured by
hybridization with unlabeled cloned DNA. The concentration of mRNA
can be followed over time using this method.
[0030] In another embodiment, the Kd can be independently assayed
by performing a pulse-chase experiment where radioactive precursor
is chased out of the cell, and then the decline in radioactivity of
mRNA molecules made during the pulse is followed. In yet another
example, synthesis and/or degradation rates can be estimated using
transcription reporters.
[0031] In another embodiment, the RNA can be modulated by
modulating the translocation of the RNA. For example, in one
embodiment, a tetracycline molecule may interfere with the
translocation of an RNA molecule to or from the nucleus of a
cell.
[0032] Translocation of RNA to the nucleus can be measured, e.g.,
by nuclear translocation assays in which the emission of two or
more fluorescently-labeled species is detected simultaneously. For
example, the cell nucleus can be labeled with a known fluorophore
specific for DNA, such as Hoechst 33342. The RNA can be directly or
indirectly labeled, e.g., fluorescently-labeled antibody specific
for RNA. The amount of RNA that translocates to or from the nucleus
can be determined by determining the amount of a first
fluorescently-labeled species, i.e., the nucleus, that is
distributed in a correlated or anti-correlated manner with respect
to a second fluorescently-labeled species, i.e., the RNA as
described in U.S. Pat. No. 6,400,487, the contents of which are
hereby incorporated by reference.
[0033] Modulation of RNA also includes modulation of the processing
of a particular RNA molecule by splicing. The tetracycline compound
may affect the arrangement, or the inclusion, or the exclusion of
sections of the RNA by affecting the mechanisms governing splicing.
For example, in the case of mRNAs, the tetracycline compound may,
for example, promote the inclusion of a particular exon, or promote
the exclusion of a particular exon, or cause a particular exon size
to become altered, for example, by inclusion of a sequence at the
5' or the 3' ends of the exon. The tetracycline compound may
promote the inclusion or the exclusion of an exon containing, for
example, a premature stop codon. The tetracycline compound may
modulate splicing by, for example, activating cryptic splice sites,
or silencing consensus splice sites, or silencing exonic or
intronic splicing enhancers (ESEs or ISEs) or by silencing exonic
or inronic splicing silencers (ESSs or ISSs), or altering the
binding orf a component of the splicing machinery to the RNA, or by
affecting the intermolecular interactions between components of the
splicing machinery. Examples of RNA splicing are discussed in Stoss
et al. (2000), Gene Ther. Mot Biol. 5:9-30; Liu et al. (1994) J.
Euk. Microbiol. 41:31; Hertweck et al., 2002. Eur. J. Biochem
269:175.
[0034] In another embodiment, the amount of spliced mRNA specifying
a particular protein can be measured. In another embodiment, the
effect of a tetracycline compound on splicing of RNA can be
measured, e.g., using standard assays such as .beta. globin
splicing assays (Hertweck et al. 2002. Eur. J. Biochem. 269:175).
In one embodiment, a particular form of RNA (e.g., an mRNA molecule
comprising a particular exon) can be measured in a cell. In a
further embodiment, the tetracycline compound is not tetracycline,
chlortetracycline, or other tetracycline compounds described in
Hertweck et al. 2002. Eur. J. Biochem. 269:175 or Liu et al. 1994.
J. Euk. Microbiol. 41(1):31.
[0035] Various spliced forms of mRNA can be detected in a cell
using techniques known in the art. For example, in one embodiment,
PCR can be performed using primer sets that specifically amplify
the products to be detected (see, e.g., Lim and Hertel. 2001 J.
Biol. Chem 276:45476). In another embodiment, a reporter cell line
is used to detect changes in RNA splicing. For example, a cell line
such as the 654 EGFP reporter cell line (which comprises a C to T
mutation at nucleotide 654 of the human .beta.-globin intron 2
(see, e.g., Sazani et al. 2001. Nucleic Acids Research 29:3965).
Treatment of these cells with an agent that modulates RNA splicing
can restore proper splicing and translation of EGFP, thereby
providing a rapid and positive readout for identification of such
agents.
[0036] In another embodiment, the RNA is modulated by altering the
interactions of proteins with the RNA molecule. Examples of
proteins which interact with RNA include hnRNP proteins, snRNP
proteins, ribosomal proteins, endonucleases, and other enzymes. The
substituted tetracycline compound may either promote or inhibit the
interactions of particular proteins with RNA. In certain
embodiments, the interaction of RNA with another nucleic acid
molecule may also be modulated by the interaction of the
tetracycline compound.
[0037] The ability of the tetracycline compound to modulate binding
of an RNA molecule to one or more proteins can also be determined.
Determining the ability of the test compound to binding can be
accomplished, for example, by coupling the RNA molecule or the
protein(s) with a radioisotope or enzymatic label such that binding
of the RNA to the protein can be determined by detecting the
labeled molecule in a complex. For example, RNA or protein can be
labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product. Alternatively,
if the protein with which the RNA interacts is an enzyme, it can be
detected without labeling.
[0038] In one embodiment, the amount of binding of RNA to the
protein target molecule in the presence of the tetracycline
compound is greater than the amount of binding of RNA to the target
molecule in the absence of the tetracycline compound, in which case
the tetracycline compound is identified as a compound that enhances
binding of RNA. In another embodiment, the amount of binding of the
RNA to the target molecule in the presence of the tetracycline
compound is less than the amount of binding of the RNA to the
target molecule in the absence of the tetracycline compound, in
which case the tetracycline compound is identified as a compound
that inhibits the binding of RNA to protein.
[0039] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either RNA
or a target protein molecule, for example, to facilitate separation
of complexed from uncomplexed forms of one or both of the
molecules, or to accommodate automation of the assay. Binding of a
tetracycline compound to an RNA molecule, or interaction of an RNA
molecule with a protein molecule in the presence and absence of a
test compound, can be accomplished in any vessel suitable for
containing the reactants. Examples of such vessels include
microtitre plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a
matrix.
[0040] In one embodiment, RNA modulation can be detected in a cell
by measuring the effect of a tetracycline compound on the amount or
activity of one or more proteins in a cell. Preferably the protein
is one associated with a particular disorder in a subject, i.e., is
a therapeutically relevant protein.
[0041] In another aspect, the invention pertains to a method for
treating a subject for a disorder treatable by modulation RNA or by
modulation of RNA in combination with a second agent (DTMR). The
method includes administering to the subject an effective amount of
a substituted tetracycline compound or an effective amount of a
substituted tetracycline compound and a second agent (e.g., a
chemotherapeutic agent) such that the DTMR is treated.
[0042] The term "disorders treatable by modulation of RNA" or
"DTMR" includes viral, neurodegenerative and other disorders which
are caused or related to RNA function, structure, amounts and/or
other activities of RNA which are lower or higher than desired and
those disorders treatable by compounds described herein. Examples
of DTMR include viral disorders (e.g., retroviral disorders (e.g.,
HIV, etc.), disorders caused by human rhinovirus RNA and proteins,
VEE virus, Venezuelan equine encephalitis virus, eastern X disease,
West Nile virus, bacterial spot of peach, camelpox virus, potato
leafroll virus, stubborn disease and infectious variegations of
citrus seedlings, viral protein synthesis in Escherichia coli
infected with coliphage MS2, yellow viruses, citrus greening
disease, ratoon stunting disease, European yellows of plants,
inclusion conjunctivitis virus, meningopneumonitis virus, trachoma
virus, hog plague virus, ornithosis virus, influenza virus, rabies
virus, viral abortion in ungulates, pneumonitis, and cancer.
[0043] Other exemplary DTMRs include disorders caused by, or
associated with splicing. For example, some disorders associated
with defects in pre-mRNA processing result from a loss of function
due to mutations in regulatory elements of a gene. Examples of such
mutations are described in Krawczak et al. (1992) Hum. Genet,
90:41-54; and Nakai et al. (1994) Gene 14:171-177. Other DTMR
include disorders which have been attributed to a change in
trans-acting factors. Examples of DTMRs which are associated with
splicing include those described in Philips et al. (2000), Cell.
Mol. Life Sci, 57:235-249), as well as, FTDP-17 (frontotemporal
dementia with parkinsonism) and 3-thalassemia.
[0044] Certain DTMRs associated with splicing include those which
are generated by point mutations that either destroy splice-sites
or generate new cryptic sites in the vicinity of normally used
exons. Examples of such DTMRs include cystic fibrosis (Friedman et
al. (1999) J. Biol. Chem. 274:36193-36199), muscular dystrophy
(Wilton et al. (1999) Neuromuscul. Disord. 9:330-338), and
eosinophilic diseases (Karras et al., (2000) Mol. Pharamcol.
58:380-387).
[0045] Other DTMRs include cancers which may change splicing
patterns during cancer formation and progression. Example of such
cancers include, but are not limited to leukemia, colon/rectal
cancer, myeloid leukemia, breast cancer, gastric carcinomas, acute
leukemia, multiple myeloma, myeloid cell leukemia, lung cancer,
prostate cancer, etc. Addition DTMRs associated with splicing are
discussed in Stoss et al., (2000), Gene Ther. Mol. Blot
5:9-30).
[0046] Another example of a DTMR is a cancer in which treatment of
the cancer cells with a tetracycline compound results in the
modulation of RNA, where the modulation of RNA increases the
susceptability of the cell to a second agent, e.g., a
chemotherapeutic agent. Such DTMRs can be treated using a
combination of the tetracycline compound and a chemotherapeutic
agent. Exemplary cancers include those in which the tetracycline
compound modulates the form of BCL expressed by the cells.
[0047] Other DTMRs include disorders wherein particular ribozymes
are present in aberrant quantities. Examples include breast cancer,
hepatitis C virus (HCV), liver cirrhosis, and heptacellular
carcinoma.
[0048] In a further embodiment, the tetracycline compounds for
treating cancer do not include, for example, the tetracycline
compounds described in U.S. Pat. Nos. 6,100,248; 5,843,925;
5,837,696; 5,668,122; WO 98/31224; US 20020045603; WO 99/49871; WO
01/87823; WO 00/28983; U.S. Pat. No. 5,574,026; incorporated herein
by reference in their entirety.
[0049] Other DTMRs include, but are not limited to, asthma,
arthritis, anemia, Alzheimer's, Huntington's disease, aortic
aneurysm, diabetes, ischemia, hyperlipidemia, and obesity.
[0050] In an embodiment, when the DTMR is an aortic aneurysm, the
tetracycline compound is not doxycycline. In another embodiment,
when the DTMR is Huntington's disease, the tetracycline compound is
not minocycline. In another embodiment, when the DTMR is cerebral
ischemia, the tetracycline compound is not tetracycline. In other
embodiments, when the DTMR is asthma, the tetracycline compound is
not minocycline or doxycycline.
[0051] In other embodiments, the DTMRs of the invention do not
include aortic aneurysm, Huntington's disease, asthma or cerebral
ischemia.
[0052] The term "subject" with reference to treatment includes
humans and other organisms and viruses which have RNA such as
plants, animals (e.g., mammals, e.g., cats, dogs, horses, pigs,
cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g.,
chimpanzees and gorillas)).
[0053] The language "effective amount" of the tetracycline compound
is that amount necessary or sufficient to treat or prevent a DTMR
or modulate RNA in a subject. The effective amount can vary
depending on such factors as the size and weight of the subject,
the particular DTMR, or the particular tetracycline compound. For
example, the choice of the tetracycline compound can affect what
constitutes an "effective amount". One of ordinary skill in the art
would be able to study the aforementioned factors and make the
determination regarding the effective amount of the tetracycline
compound without undue experimentation.
[0054] The regimen of administration can affect what constitutes an
effective amount. The tetracycline compound can be administered to
the subject either prior to or after the onset of a disease which
is treatable. Further, several divided dosages, as well as
staggered dosages, can be administered daily or sequentially, or
the dose can be continuously infused, orally administered,
administered by inhalation, or can be a bolus injection. Further,
the dosages of the tetracycline compound(s) can be proportionally
increased or decreased as indicated by the exigencies of the
therapeutic or prophylactic situation.
[0055] The term "treated," "treating" or "treatment" includes
therapeutic and/or prophylactic treatment. The treatment includes
the diminishment or alleviation of at least one symptom associated
or caused by the DTMR. For example, treatment can be diminishment
of one or several symptoms of a disorder or complete eradication of
the DTMR.
[0056] In another aspect, the invention pertains to methods for
identifying tetracycline compounds for treating DTMR, comprising:
contacting a cellular component with a tetracycline compound;
measuring the ability of the tetracycline compound to modulate RNA,
to thereby identify a tetracycline compound for treating DTMR,
either alone or in combination with a second agent.
[0057] In one embodiment, the ability of the compound to modulate
RNA translation is measured. In another embodiment, the ability of
the compound to modulate the half-life of RNA is measured. In
another embodiment, the ability of the compound to modulate
translocation of RNA is measured. In another embodiment, the
ability of the compound to modulate the interaction of RNA with
proteins is measured. In another embodiment, modulation of RNA
splicing is measured. Modulation of RNA can be detected using any
of the methods described herein or other art recognized
methods.
II. Substituted Tetracycline Compounds
[0058] In one embodiment, the tetracycline compound is a
substituted tetracycline compound.
[0059] The term "tetracycline compound" includes substituted
tetracycline compounds and compounds with a similar ring structure
to tetracycline, including minocycline, doxycycline, tetracycline,
chlortetracycline, oxytetracycline, demeclocycline, methacycline,
sancycline, chelocardin, rolitetracycline, lymecycline,
apicycline;
[0060] clomocycline, guamecycline, meglucycline, mepylcycline,
penimepicycline, pipacycline, etamocycline, penimocycline, etc.
Other derivatives and analogues comprising a similar four ring
structure are also included (See Rogalski, "Chemical Modifications
of Tetracyclines," the entire contents of which are hereby
incorporated herein by reference). Table 1 depicts tetracycline and
several known other tetracycline derivatives.
TABLE-US-00001 TABLE 1 ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
[0061] Other tetracycline compounds which may be modified using the
methods of the invention include, but are not limited to,
6-demethyl-6-deoxy-4-dedimethylaminotetracycline;
tetracyclino-pyrazole; 7-chloro-4-dedimethylaminotetracycline;
4-hydroxy-4-dedimethylaminotetracycline;
12.alpha.-deoxy-4-dedimethylaminotetracycline;
5-hydroxy-6.alpha.-deoxy-4-dedimethylaminotetracycline;
4-dedimethylamino-12.alpha.-deoxyanhydrotetracycline;
7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;
tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline
4,6-hemiketal; 4-oxo-11a
C1-4-dedimethylaminotetracycline-4,6-hemiketal;
5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;
4-hydroxyimino-4-dedimethylamino tetracyclines;
4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;
4-amino-4-dedimethylamino-5a, 6 anhydrotetracycline;
4-methylamino-4-dedimethylamino tetracycline;
4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylamino
tetracycline; tetracycline quaternary ammonium compounds;
anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides;
4-keto tetracyclines; 5-keto tetracyclines; 5; 11a dehydro
tetracyclines; 11a C1-6, 12 hemiketal tetracyclines; 11a
C1-6-methylene tetracyclines; 6, 13 diol tetracyclines;
6-benzylthiomethylene tetracyclines; 7,
11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro
(.alpha.)-6-demethyl-6-deoxy tetracyclines; 6-fluoro
(.beta.)-6-demethyl-6-deoxy tetracyclines; 6-.alpha.
acetoxy-6-demethyl tetracyclines; 6-.beta. acetoxy-6-demethyl
tetracyclines; 7, 13-epithiotetracyclines; oxytetracyclines;
pyrazolotetracyclines; 11a halogens of tetracyclines; 12a formyl
and other esters of tetracyclines; 5, 12a esters of tetracyclines;
10, 12a-diesters of tetracyclines; isotetracycline;
12-a-deoxyanhydro tetracyclines;
6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines;
B-nortetracyclines; 7-methoxy-6-demethyl-6-deoxytetracyclines;
6-demethyl-6-deoxy-5a-epitetracyclines;
8-hydroxy-6-demethyl-6-deoxy tetracyclines; monardene;
chromocycline; 5a methyl-6-demethyl-6-deoxy tetracyclines; 6-oxa
tetracyclines, and 6 thia tetracyclines. In certain embodiments,
the term tetracycline compound does not include
7-chlorotetracycline, minocycline, doxycycline, or
tetracycline.
[0062] The term "tetracycline compounds" includes substituted
tetracycline compounds as defined below, and as described in the
specification. The tetracycline compounds may or may not have
antibacterial or antiinfective activity. In certain embodiments of
the invention, the tetracycline compound has antiinfective,
antiinflammatory and/or antibacterial activity. In other
embodiments of the invention, the tetracycline compound does not
have significant antiinfective, antiinflammatory or antibacterial
therapeutic activity.
[0063] Examples of substituted tetracycline compounds include
compounds described in U.S. Pat. Nos. 6,165,999; 5,834,450;
5,886,175; 5,567,697; 5,567,692; 5,530,557; 5,512,553; 5,430,162
each of which is incorporated herein by reference in its entirety.
Other examples of substituted tetracycline compounds include those
described in, for example, WO 99/37307, WO 02/12170, WO 02/04407,
WO 02/04406, WO 02/04404, WO 01/98260, WO 01/98259, WO 01/98236, WO
01/87824, WO 01/74761, WO 01/52858, WO 01/19784, WO 84/01895, U.S.
Ser. No. 60/367,050, U.S. Ser. No. 09/895,797, U.S. Ser. No.
60/305,546, U.S. Ser. No. 60/346,930, U.S. Ser. No. 60/346,929,
U.S. Ser. No. 60/347,065, U.S. Ser. No. 60/346,956, U.S. Ser. No.
60/367,049, U.S. Ser. No. 10/097,095, U.S. Ser. No. 10/097,135,
U.S. Ser. No. 60/362,654, U.S. Ser. No. 60/367,045, U.S. Ser. No.
60/366,915, U.S. Ser. No. 60/367,048, and Ser. No. 10/196,010.
Other examples of substituted tetracycline compounds are described
in EP 0582810 B1; EP 0536 515B1; EP 0582 78981; EP 0582 829B1; EP
058278881; U.S. Pat. No. 5,530,117; U.S. Pat. No. 5,495,030; U.S.
Pat. No. 5,495,018; U.S. Pat. No. 5,494,903; U.S. Pat. No.
5,466,684; EP 0535 346B1; U.S. Pat. No. 5,457,096; U.S. Pat. No.
5,442,059; U.S. Pat. No. 5,430,162; U.S. Pat. No. 5,420,272; U.S.
Pat. No. 5,401,863; U.S. Pat. No. 5,401,729; U.S. Pat. No.
5,386,041; U.S. Pat. No. 5,380,888; U.S. Pat. No. 5,371,076; EP 618
190; U.S. Pat. No. 5,326,759; EP 582 829; EP 528 810; EP 582 790;
EP 582 789; EP 582 788; U.S. Pat. No. 5,281,628; EP 536 515; EP 535
346; WO 96/34852; WO 95122529A1; U.S. Pat. No. 4,066,694; U.S. Pat.
No. 3,862,225; U.S. Pat. No. 3,622,627; WO 01/87823A1; and WO
00/28983A1. Each of these aforementioned applications and patents
are hereby incorporated herein by reference in its entirety. In
addition, the invention pertains to each of the compounds described
herein, methods of using each of the compounds, and pharmaceutical
compositions comprising each of the compounds.
[0064] The term "substituted tetracycline compound" includes
tetracycline compounds with one or more additional substituents,
e.g., at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a or 13
position or at any other position which allows the substituted
tetracycline compound of the invention to perform its intended
function, e.g., to modulate RNA or treat a DTMR. In certain
embodiments, the substituted tetracycline compound is a
7-substituted sancycline compound, a 9-substituted minocycline
compound, or a 7,9-substituted sancycline compound. In certain
embodiments, the term "substituted tetracycline compound" does not
include tetracycline compounds with a chlorine, hydrogen or
dimethylamino substituent at the 7-position. In other embodiments,
the term "substituted tetracycline compound" does not include
compounds with a hydrogen as a 9-position substituent. In other
embodiments, the term substituted tetracycline does not include
5-hydroxy tetracycline, 7-chlorotetracycline,
6-demethyl-7-chlorotetracycline, anhydrochlorotetracycline,
4-epi-anhydrochlorotetracycline, or .beta.-chelocardin.
[0065] The term "substituted tetracycline compound" also includes
substituted tetracycline compounds of the formula (I):
##STR00010##
wherein [0066] R.sup.2, R.sup.2', R.sup.4', and R.sup.4'' are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety; [0067] R.sup.2',
R.sup.3, R.sup.10, R.sup.11 and R.sup.12 are each hydrogen, alkyl,
alkenyl, alkynyl, aryl, substituted carbonyl, or a pro-drug moiety;
[0068] R.sup.4 is NR.sup.4'R.sup.4'', alkyl, alkenyl, alkynyl,
hydroxyl, halogen, or hydrogen; [0069] R.sup.5 is hydroxyl,
hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic,
alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy, or aryl
carbonyloxy; [0070] R.sup.6 and R.sup.6' are each independently
hydrogen, methylene, absent, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; [0071] R.sup.7 is
hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl, alkynyl,
aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,
amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl, heterocyclic,
thionitroso, or --(CH.sub.2).sub.0-3NR.sup.7cC(.dbd.W')WR.sup.7a;
[0072] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, amino, arylalkenyl, arylalkynyl, acyl,
aminoalkyl, heterocyclic, thionitroso, or
--(CH.sub.2).sub.0-3NR.sup.8cC(=E')ER.sup.8a; [0073] R.sup.9 is
hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl, alkynyl,
aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl,
amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl, heterocyclic,
thionitroso, or --(CH.sub.2).sub.0-3NR.sup.9cC(.dbd.Z')ZR.sup.9c;
[0074] R.sup.7a, R.sup.7b, R.sup.7c, R.sup.7d, R.sup.7e, R.sup.7d,
R.sup.8a, R.sup.8b, R.sup.8c, R.sup.8d, R.sup.8e, R.sup.8f,
R.sup.9a, R.sup.9b, R.sup.9c, R.sup.9d, R.sup.9e, and R.sup.8f are
each independently hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
aryl, heterocyclic, heteroaromatic or a prodrug moiety; [0075]
R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; [0076] E is CR.sup.8dR.sup.8e, S, NR.sup.8b or O; [0077]
E' is O, NR.sup.8f, or S; [0078] W is CR.sup.7dR.sup.7e, S,
NR.sup.7b or O; [0079] W' is O, NO, or S; [0080] X is
CHC(R.sup.13Y'Y), C.dbd.CR.sup.13Y, R.sup.6'R.sup.6, NR.sup.6, or
O; [0081] Y' and Y are each independently hydrogen, halogen,
hydroxyl, cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; [0082] Z is CR.sup.9dR.sup.9e, S, NR.sup.9b or O; [0083]
Z' is O, S, or NR.sup.9f, and pharmaceutically acceptable salts,
esters and enantiomers thereof.
[0084] In a further embodiment, the substituted tetracycline
compounds of formula (I) comprise compounds wherein R.sup.2,
R.sup.2', R.sup.8, R.sup.10, R.sup.11, and R.sup.12 are each
hydrogen, X is CR.sup.6R.sup.6', and R.sup.4 is NR.sup.4'R.sup.4'',
wherein R.sup.4' and R.sup.4'' are each methyl. In addition,
R.sup.9 may be hydrogen.
[0085] In one embodiment, R.sup.7 is substituted or unsubstituted
aryl, e.g., phenyl or heteroaryl. In a further embodiment, R.sup.7
is substituted with one or more substituents which allow the
substituted tetracycline compound to perform its intended function,
e.g., treat a DTMR or modulate RNA. Examples of such substituents
include alkyl, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, aryl or
heterocyclic moiety.
[0086] In another embodiment, R.sup.7 is substituted or
unsubstituted alkenyl. Examples of substituents for alkenyl R.sup.7
groups include alkyl, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino, acylamino, amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, aryl or
heterocyclic moiety.
[0087] In another embodiment, R.sup.7 is substituted or
unsubstituted heteroaryl and R.sup.9 is alkyl.
[0088] In another further embodiment, the substituted tetracycline
compound is a substituted minocycline compound, e.g., R.sup.7 is
dialkylamino. In a further embodiment, R.sup.9 is alkylamino. In
another embodiment, R.sup.9 is --NR.sup.9cC(.dbd.Z')ZR.sup.9a,
wherein R.sup.9c is hydrogen, Z' is nitrogen or oxygen, Z is NH,
and R.sup.9a is aryl or aralkyl.
[0089] Examples of tetracycline compounds include:
##STR00011## ##STR00012## ##STR00013## ##STR00014##
and pharmaceutically acceptable salts, esters, and prodrugs
thereof. Other examples of substituted tetracycline compounds are
shown in Table 2, below.
TABLE-US-00002 TABLE 2 ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## `
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112##
##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117##
##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122##
##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127##
##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132##
##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137##
##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142##
##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147##
##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152##
##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157##
##STR00158## ##STR00159## ##STR00160## ##STR00161## ##STR00162##
##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167##
##STR00168## ##STR00169## ##STR00170## ##STR00171## ##STR00172##
##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177##
##STR00178## ##STR00179## ##STR00180## ##STR00181## ##STR00182##
##STR00183## ##STR00184## ##STR00185## ##STR00186## ##STR00187##
##STR00188##
##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193##
##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198##
##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203##
##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208##
##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213##
##STR00214## ##STR00215## ##STR00216## ##STR00217## ##STR00218##
##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223##
##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228##
##STR00229## ##STR00230## ##STR00231## ##STR00232## ##STR00233##
##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238##
##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243##
##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248##
##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253##
##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258##
##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263##
##STR00264## ##STR00265## ##STR00266## ##STR00267## ##STR00268##
##STR00269## ##STR00270## ##STR00271## ##STR00272## ##STR00273##
##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278##
##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283##
##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288##
##STR00289## ##STR00290## ##STR00291## ##STR00292## ##STR00293##
##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298##
##STR00299## ##STR00300## ##STR00301## ##STR00302## ##STR00303##
##STR00304## ##STR00305## ##STR00306## ##STR00307## ##STR00308##
##STR00309## ##STR00310## ##STR00311## ##STR00312## ##STR00313##
##STR00314## ##STR00315## ##STR00316## ##STR00317## ##STR00318##
##STR00319## ##STR00320## ##STR00321## ##STR00322## ##STR00323##
##STR00324## ##STR00325## ##STR00326## ##STR00327## ##STR00328##
##STR00329## ##STR00330## ##STR00331## ##STR00332## ##STR00333##
##STR00334## ##STR00335## ##STR00336## ##STR00337## ##STR00338##
##STR00339## ##STR00340## ##STR00341## ##STR00342## ##STR00343##
##STR00344## ##STR00345## ##STR00346## ##STR00347## ##STR00348##
##STR00349## ##STR00350## ##STR00351## ##STR00352##
##STR00353## ##STR00354## ##STR00355## ##STR00356## ##STR00357##
##STR00358## ##STR00359## ##STR00360## ##STR00361## ##STR00362##
##STR00363## ##STR00364## ##STR00365## ##STR00366## ##STR00367##
##STR00368## ##STR00369## ##STR00370## ##STR00371## ##STR00372##
##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377##
##STR00378## ##STR00379## ##STR00380## ##STR00381## ##STR00382##
##STR00383## ##STR00384## ##STR00385## ##STR00386## ##STR00387##
##STR00388## ##STR00389## ##STR00390## ##STR00391## ##STR00392##
##STR00393## ##STR00394## ##STR00395## ##STR00396## ##STR00397##
##STR00398## ##STR00399## ##STR00400## ##STR00401## ##STR00402##
##STR00403## ##STR00404## ##STR00405## ##STR00406## ##STR00407##
##STR00408## ##STR00409## ##STR00410## ##STR00411## ##STR00412##
##STR00413## ##STR00414## ##STR00415## ##STR00416## ##STR00417##
##STR00418## ##STR00419## ##STR00420## ##STR00421## ##STR00422##
##STR00423## ##STR00424## ##STR00425## ##STR00426## ##STR00427##
##STR00428## ##STR00429## ##STR00430## ##STR00431## ##STR00432##
##STR00433## ##STR00434## ##STR00435## ##STR00436## ##STR00437##
##STR00438## ##STR00439## ##STR00440## ##STR00441## ##STR00442##
##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447##
##STR00448## ##STR00449## ##STR00450## ##STR00451## ##STR00452##
##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457##
##STR00458## ##STR00459## ##STR00460## ##STR00461## ##STR00462##
##STR00463## ##STR00464## ##STR00465## ##STR00466## ##STR00467##
##STR00468## ##STR00469## ##STR00470## ##STR00471## ##STR00472##
##STR00473## ##STR00474## ##STR00475## ##STR00476## ##STR00477##
##STR00478## ##STR00479## ##STR00480## ##STR00481## ##STR00482##
##STR00483## ##STR00484## ##STR00485## ##STR00486## ##STR00487##
##STR00488## ##STR00489## ##STR00490## ##STR00491## ##STR00492##
##STR00493## ##STR00494## ##STR00495## ##STR00496## ##STR00497##
##STR00498## ##STR00499## ##STR00500## ##STR00501## ##STR00502##
##STR00503## ##STR00504## ##STR00505## ##STR00506## ##STR00507##
##STR00508## ##STR00509## ##STR00510## ##STR00511## ##STR00512##
##STR00513## ##STR00514## ##STR00515## ##STR00516## ##STR00517##
##STR00518## ##STR00519## ##STR00520## ##STR00521## ##STR00522##
##STR00523## ##STR00524## ##STR00525## ##STR00526## ##STR00527##
##STR00528## ##STR00529## ##STR00530## ##STR00531## ##STR00532##
##STR00533## ##STR00534## ##STR00535## ##STR00536## ##STR00537##
##STR00538## ##STR00539## ##STR00540##
##STR00541## ##STR00542## ##STR00543## ##STR00544## ##STR00545##
##STR00546## ##STR00547## ##STR00548## ##STR00549## ##STR00550##
##STR00551## ##STR00552## ##STR00553## ##STR00554## ##STR00555##
##STR00556## ##STR00557## ##STR00558## ##STR00559## ##STR00560##
##STR00561## ##STR00562## ##STR00563## ##STR00564## ##STR00565##
##STR00566## ##STR00567## ##STR00568## ##STR00569## ##STR00570##
##STR00571## ##STR00572## ##STR00573## ##STR00574## ##STR00575##
##STR00576## ##STR00577## ##STR00578## ##STR00579## ##STR00580##
##STR00581## ##STR00582## ##STR00583## ##STR00584## ##STR00585##
##STR00586## ##STR00587## ##STR00588## ##STR00589## ##STR00590##
##STR00591## ##STR00592## ##STR00593## ##STR00594## ##STR00595##
##STR00596## ##STR00597## ##STR00598## ##STR00599## ##STR00600##
##STR00601## ##STR00602## ##STR00603## ##STR00604## ##STR00605##
##STR00606## ##STR00607## ##STR00608## ##STR00609## ##STR00610##
##STR00611## ##STR00612## ##STR00613## ##STR00614## ##STR00615##
##STR00616## ##STR00617## ##STR00618## ##STR00619## ##STR00620##
##STR00621## ##STR00622## ##STR00623## ##STR00624## ##STR00625##
##STR00626## ##STR00627## ##STR00628## ##STR00629## ##STR00630##
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##STR02137## ##STR02138## ##STR02139## ##STR02140## ##STR02141##
##STR02142## ##STR02143## ##STR02144## ##STR02145## ##STR02146##
##STR02147## ##STR02148## ##STR02149## ##STR02150## ##STR02151##
##STR02152## ##STR02153## ##STR02154## ##STR02155## ##STR02156##
##STR02157## ##STR02158## ##STR02159## ##STR02160## ##STR02161##
##STR02162## ##STR02163## ##STR02164## ##STR02165## ##STR02166##
##STR02167## ##STR02168## ##STR02169## ##STR02170## ##STR02171##
##STR02172## ##STR02173## ##STR02174## ##STR02175## ##STR02176##
##STR02177## ##STR02178## ##STR02179## ##STR02180## ##STR02181##
##STR02182## ##STR02183## ##STR02184## ##STR02185## ##STR02186##
##STR02187## ##STR02188## ##STR02189## ##STR02190## ##STR02191##
##STR02192## ##STR02193## ##STR02194## ##STR02195## ##STR02196##
##STR02197## ##STR02198## ##STR02199## ##STR02200## ##STR02201##
##STR02202## ##STR02203## ##STR02204## ##STR02205## ##STR02206##
##STR02207## ##STR02208## ##STR02209## ##STR02210## ##STR02211##
##STR02212## ##STR02213## ##STR02214## ##STR02215## ##STR02216##
##STR02217## ##STR02218## ##STR02219## ##STR02220## ##STR02221##
##STR02222## ##STR02223## ##STR02224## ##STR02225## ##STR02226##
##STR02227## ##STR02228## ##STR02229## ##STR02230## ##STR02231##
##STR02232## ##STR02233## ##STR02234## ##STR02235##
[0090] In certain embodiments, the substituted tetracycline
compounds of the invention have antibacterial activity against gram
+ and/or gram - bacteria. In certain embodiments, the tetracycline
compounds of the invention do not have antibacterial activity
against gram + and/or gram - bacteria. In other embodiments,
compounds with MIC of greater than about 2 .mu.g/ml, greater than
about 3 .mu.g/ml, greater than about 4 .mu.g/ml, greater than about
5 .mu.g/ml, greater than about 6 .mu.g/ml, greater than about 8
.mu.g/ml, greater than about 9 .mu.g/ml, greater than about 10
.mu.g/ml, greater than about 11 .mu.g/ml, greater than about 12
.mu.g/ml, greater than about 13 .mu.g/ml, greater than about 14
.mu.g/ml, greater than about 15 .mu.g/ml, greater than about 16
.mu.g/ml, greater than about 17 .mu.g/ml, greater than about 18
.mu.g/ml, greater than about 19 .mu.g/ml, greater than about 20
.mu.g/ml, greater than about 25 .mu.g/ml, greater than about 30
.mu.g/ml, greater than about 40 .mu.g/ml, or greater than about 50
.mu.g/ml for gram + and/or gram - bacteria are considered not to
have anti-bacterial activity.
[0091] In other embodiments, compounds with MIC of less than about
50 .mu.g/ml, less than about 40 .mu.g/ml, less than about 30
.mu.g/ml, less than about 25 .mu.g/ml, less than about 20 .mu.g/ml,
less than about 15 .mu.g/ml, less than about 14 .mu.g/ml, less than
about 13 .mu.g/ml, less than about 12 .mu.g/ml, less than about 11
.mu.g/ml, less than about 10 .mu.g/ml, less than about 9 .mu.g/ml,
less than about 8 .mu.g/ml, less than about 6 .mu.g/ml, less than
about 5 .mu.g/ml, less than about 4 .mu.g/ml, less than about 3
.mu.g/ml, less than about 2 .mu.g/ml, less than about 1 .mu.g/ml,
or less than about 0.5 .mu.g/ml for gram + and/or gram - bacteria
are considered to have anti-bacterial activity.
[0092] In one embodiment, the tetracycline compound of the
invention may retain antibiotic, antibacterial, or antimicrobial
activity, it may have decreased antibiotic, antibacterial, or
antimicrobial activity, or, it may have little to no antibiotic,
antibacterial or antimicrobial activity. In an embodiment, the
substituted tetracycline compound is substituted at the 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a and/or 13 position. In certain
embodiments, the tetracycline compounds of the invention are 7
and/or 9 substituted, e.g., 7 and/or 9-substituted tetracycline
compounds (e.g., compounds wherein R.sup.7 and/or R.sup.9 are not
both hydrogen). In yet a further embodiment, the tetracycline
compounds of the invention are 7 and/or 9 substituted sancycline
compounds. Other examples of tetracycline compounds which may be
used in the methods of the invention include those otherwise
described herein or incorporated by reference.
[0093] The substituted tetracycline compounds of the invention can
be synthesized using the methods described in Example 1, in the
following schemes and/or by using art recognized techniques. All
novel substituted tetracycline compounds described herein are
included in the invention as compounds.
##STR02236##
[0094] 9- and 7-substituted tetracyclines can be synthesized by the
method shown in Scheme 1. As shown in Scheme 1, 9- and
7-substituted tetracycline compounds can be synthesized by treating
a tetracycline compound (e.g., doxycycline, 1A), with sulfuric acid
and sodium nitrate. The resulting product is a mixture of the
7-nitro and 9-nitro isomers (1B and 1C, respectively). The 7-nitro
(1B) and 9-nitro (1C) derivatives are treated by hydrogenation
using hydrogen gas and a platinum catalyst to yield amines 1D and
1E. The isomers are separated at this time by conventional methods.
To synthesize 7- or 9-substituted alkenyl derivatives, the 7- or
9-amino tetracycline compound (1E and 1F, respectively) is treated
with HONO, to yield the diazonium salt (1G and 1H). The salt (1G
and 1H) is treated with an appropriate reactive reagent to yield
the desired compound (e.g., in Scheme I, 7-cyclopent-1-enyl
doxycycline (1H) and 9-cyclopent-1-enyl doxycycline (1I)).
##STR02237##
[0095] As shown in Scheme 2, tetracycline compounds of the
invention wherein R.sup.7 is a carbamate or a urea derivative can
be synthesized using the following protocol. Sancycline (2A) is
treated with NaNO.sub.2 under acidic conditions forming 7-nitro
sancycline (2B) in a mixture of positional isomers.
7-nitrosancycline (2B) is then treated with H.sub.2 gas and a
platinum catalyst to form the 7-amino sancycline derivative (2C).
To form the urea derivative (2E), isocyanate (2D) is reacted with
the 7-amino sancycline derivative (2C). To form the carbamate (2G),
the appropriate acid chloride ester (2F) is reacted with 2C.
##STR02238##
[0096] As shown in Scheme 3, tetracycline compounds of the
invention, wherein R.sup.7 is a heterocyclic (i.e. thiazole)
substituted amino group can be synthesized using the above
protocol. 7-amino sancycline (3A) is reacted with
Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).
The protected thiourea (3C) is then deprotected yielding the active
sancycline thiourea (3D) compound. The sancycline thiourea (3D) is
reacted with an .alpha.-haloketone (3E) to produce a thiazole
substituted 7-amino sancycline (3F).
##STR02239##
[0097] 7-alkenyl tetracycline compounds, such as 7-alkynyl
sancycline (4A) and 7-alkenyl sancycline (4B), can be hydrogenated
to form 7-alkyl substituted tetracycline compounds (e.g., 7-alkyl
sancycline, 4C). Scheme 4 depicts the selective hydrogenation of
the 7-position double or triple bond, in saturated methanol and
hydrochloric acid solution with a palladium/carbon catalyst under
pressure, to yield the product.
##STR02240##
[0098] In Scheme 5, a general synthetic scheme for synthesizing
7-position aryl derivatives is shown. A Suzuki coupling of an aryl
boronic acid with an iodosancycline compound is shown. An iodo
sancycline compound (5B) can be synthesized from sancycline by
treating sancycline (5A) with at least one equivalent
N-iodosuccinimide (NIS) under acidic conditions. The reaction is
quenched, and the resulting 7-iodo sancycline (5B) can then be
purified using standard techniques known in the art. To form the
aryl derivative, 7-iodo sancycline (5B) is treated with an aqueous
base (e.g., Na.sub.2CO.sub.3) and an appropriate boronic acid (5C)
and under an inert atmosphere. The reaction is catalyzed with a
palladium catalyst (e.g., Pd(OAc).sub.2). The product (5D) can be
purified by methods known in the art (such as HPLC). Other 7-aryl,
alkenyl, and alkynyl tetracycline compounds can be synthesized
using similar protocols.
[0099] The 7-substituted tetracycline compounds of the invention
can also be synthesized using Stille cross couplings. Stille cross
couplings can be performed using an appropriate tin reagent (e.g.,
R-SnBu.sub.3) and a halogenated tetracycline compound, (e.g.,
7-iodosancycline). The tin reagent and the iodosancycline compound
can be treated with a palladium catalyst (e.g.,
Pd(PPh.sub.3).sub.2Cl.sub.2 or Pd(AsPh.sub.3).sub.2Cl.sub.2) and,
optionally, with an additional copper salt, e.g., CuI. The
resulting compound can then be purified using techniques known in
the art.
##STR02241##
[0100] The compounds of the invention can also be synthesized using
Heck-type cross coupling reactions. As shown in Scheme 6, Heck-type
cross-couplings can be performed by suspending a halogenated
tetracycline compound (e.g., 7-iodosancycline, 6A) and an
appropriate palladium or other transition metal catalyst (e.g.,
Pd(OAc).sub.2 and CuI) in an appropriate solvent (e.g., degassed
acetonitrile). The substrate, a reactive alkene (6B) or alkyne
(6D), and triethylamine are then added and the mixture is heated
for several hours, before being cooled to room temperature. The
resulting 7-substituted alkenyl (6C) or 7-substituted alkynyl (6E)
tetracycline compound can then be purified using techniques known
in the art.
##STR02242##
[0101] To prepare 7-(2'-Chloro-alkenyl)-tetracycline compounds, the
appropriate 7-(alkynyl)-sancycline (7A) is dissolved in saturated
methanol and hydrochloric acid and stirred. The solvent is then
removed to yield the product (7B).
##STR02243##
[0102] As depicted in Scheme 8, 5-esters of 9-substituted
tetracycline compounds can be formed by dissolving the
9-substituted compounds (8A) in strong acid (e.g. HF,
methanesulphonic acid, and trifluoromethanesulfonic acid) and
adding the appropriate carboxylic acid to yield the corresponding
esters (8B).
##STR02244##
[0103] As shown in Scheme 9, methacycline (9A) can be reacted with
a phenylboronic acid in the presence of a palladium catalyst such
as Pd(OAc).sub.2 to form a 13 aryl substituted methacycline
compound. The resulting compound can then be purified using
techniques known in the art such as preparative HPLC and
characterized.
[0104] As shown in Scheme 10 below, 7 and 9 aminomethyl
tetracyclines may be synthesized using reagents such as
hydroxymethyl-carbamic acid benzyl ester. The resulting aminomethyl
tetracycline compounds may be further derivatized
##STR02245##
[0105] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (e.g., cyclopropyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. The term alkyl further includes alkyl groups, which can
further include oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone. In
certain embodiments, a straight chain or branched chain alkyl has
20 or fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.20
for straight chain, C.sub.3-C.sub.20 for branched chain), and more
preferably 4 or fewer. Cycloalkyls may have from 3-8 carbon atoms
in their ring structure, and more preferably have 5 or 6 carbons in
the ring structure. The term C.sub.1-C.sub.6 includes alkyl groups
containing 1 to 6 carbon atoms.
[0106] Moreover, the term alkyl includes both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" or an "arylalkyl" moiety is an
alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The
term "alkyl" also includes the side chains of natural and unnatural
amino acids.
[0107] The term "aryl" includes groups, including 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, phenyl, pyrrole, furan,
thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine, and the like. Furthermore, the term "aryl" includes
multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g.,
naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxophenyl, quinoline,
isoquinoline, naphthridine, indole, benzofuran, purine, benzofuran,
deazapurine, or indolizine. Those aryl groups having heteroatoms in
the ring structure may also be referred to as "aryl heterocycles",
"heterocycles," "heteroaryls" or "heteroaromatics". The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,
arylalkyl aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Aryl groups can also be fused or
bridged with alicyclic or heterocyclic rings which are not aromatic
so as to form a polycycle (e.g., tetralin).
[0108] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond.
[0109] For example, the term "alkenyl" includes straight-chain
alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl,
hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain
alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or
alkenyl substituted cycloalkenyl groups, and cycloalkyl or
cycloalkenyl substituted alkenyl groups. The term alkenyl further
includes alkenyl groups which include oxygen, nitrogen, sulfur or
phosphorous atoms replacing one or more carbons of the hydrocarbon
backbone. In certain embodiments, a straight chain or branched
chain alkenyl group has 20 or fewer carbon atoms in its backbone
(e.g., C.sub.2-C.sub.20 for straight chain, C.sub.3-C.sub.20 for
branched chain). Likewise, cycloalkenyl groups may have from 3-8
carbon atoms in their ring structure, and more preferably have 5 or
6 carbons in the ring structure. The term C.sub.2-C.sub.20 includes
alkenyl groups containing 2 to 20 carbon atoms.
[0110] Moreover, the term alkenyl includes both "unsubstituted
alkenyls" and "substituted alkenyls", the latter of which refers to
alkenyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0111] The term "alkynyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one triple bond.
[0112] For example, the term "alkynyl" includes straight-chain
alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl,
hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain
alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl
groups. The term alkynyl further includes alkynyl groups which
include oxygen, nitrogen, sulfur or phosphorous atoms replacing one
or more carbons of the hydrocarbon backbone. In certain
embodiments, a straight chain or branched chain alkynyl group has
20 or fewer carbon atoms in its backbone (e.g., C.sub.2-C.sub.20
for straight chain, C.sub.3-C.sub.20 for branched chain). The term
C.sub.2-C.sub.6 includes alkynyl groups containing 2 to 6 carbon
atoms.
[0113] Moreover, the term alkynyl includes both "unsubstituted
alkynyls" and "substituted alkynyls", the latter of which refers to
alkynyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including, e.g., alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0114] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to five carbon atoms in its backbone structure.
"Lower alkenyl" and "lower alkynyl" have chain lengths of, for
example, 2-5 carbon atoms.
[0115] The term "acyl" includes compounds and moieties which
contain the acyl radical (CH.sub.3CO--) or a carbonyl group. The
term "substituted acyl" includes acyl groups where one or more of
the hydrogen atoms are replaced by for example, alkyl groups,
alkenyl, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0116] The term "acylamino" includes moieties wherein an acyl
moiety is bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido
groups.
[0117] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of
substituted alkoxy groups include halogenated alkoxy groups. The
alkoxy groups can be substituted with groups such as alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
[0118] The terms "alkoxyalkyl", "alkylaminoalkyl" and
"thioalkoxyalkyl" include alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or
sulfur atoms.
[0119] The term "amide" or "aminocarboxy" includes compounds or
moieties which contain a nitrogen atom which is bound to the carbon
of a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups which include alkyl, alkenyl, or alkynyl
groups bound to an amino group bound to a carboxy group. It
includes arylaminocarboxy groups which include aryl or heteroaryl
moieties bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. The terms "allcylaminocarboxy,"
"alkenylaminocarboxy," "alkynylaminocarboxy," and
"arylaminocarboxy" include moieties wherein alkyl, alkenyl, alkynyl
and aryl moieties, respectively, are bound to a nitrogen atom which
is in turn bound to the carbon of a carbonyl group.
[0120] The term "amine" or "amino" includes compounds where a
nitrogen atom is covalently bonded to at least one carbon or
heteroatom. The term "alkyl amino" includes groups and compounds
wherein the nitrogen is bound to at least one additional alkyl
group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups. The
term "arylamino" and "diarylamino" include groups wherein the
nitrogen is bound to at least one or two aryl groups, respectively.
The term "alkylarylamino," "alkylaminoaryl" or "arylaminoalkyl"
refers to an amino group which is bound to at least one alkyl group
and at least one aryl group. The term "alkaminoalkyl" refers to an
alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is
also bound to an alkyl group.
[0121] The term "aroyl" includes compounds and moieties with an
aryl or heteroaromatic moiety bound to a carbonyl group. Examples
of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
[0122] The term "carbonyl" or "carboxy" includes compounds and
moieties which contain a carbon connected with a double bond to an
oxygen atom. Examples of moieties which contain a carbonyl include
aldehydes, ketones, carboxylic acids, amides, esters, anhydrides,
etc.
[0123] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0124] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0125] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
wherein all hydrogens are replaced by halogen atoms.
[0126] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0127] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-X.sup.+, where X.sup.+ is a counterion.
[0128] The terms "polycyclyl" or "polycyclic radical" refer to two
or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls) in which two or more
carbons are common to two adjoining rings, e.g., the rings are
"fused rings". Rings that are joined through non-adjacent atoms are
termed "bridged" rings. Each of the rings of the polycycle can be
substituted with such substituents as described above, as for
example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, alkylaminoacarbonyl, arylalkylaminocarbonyl,
alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl
carbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl,
alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino
(including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0129] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0130] The term "thioether" includes compounds and moieties which
contain a sulfur atom bonded to two different carbon or hetero
atoms. Examples of thioethers include, but are not limited to
alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term
"alkthioalkyls" include compounds with an alkyl, alkenyl, or
alkynyl group bonded to a sulfur atom which is bonded to an alkyl
group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls"
refer to compounds or moieties wherein an alkyl, alkenyl, or
alkynyl group is bonded to a sulfur atom which is covalently bonded
to an alkynyl group.
[0131] The term "oximyl" includes moieties which comprise an oxime
group.
[0132] The term "dimeric moiety" includes moieties which comprise a
second tetracycline four ring structure. The dimeric moiety may be
attached to the substituted tetracycline through a chain of from
1-30 atoms. The chain may be comprised of atoms covalently linked
together through single, double and triple bonds. The tetracycline
ring structure of the dimeric moiety may further be substituted or
unsubstituted. It may be attached at the 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 11a, 12, 12; and/or 13 position.
[0133] The term "prodrug moiety" includes moieties which can be
metabolized in vivo. Generally, the prodrugs moieties are
metabolized in vivo by esterases or by other mechanisms to hydroxyl
groups or other advantageous groups. Examples of prodrugs and their
uses are well known in the art (See, e.g., Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). The prodrugs can
be prepared in situ during the final isolation and purification of
the compounds, or by separately reacting the purified compound in
its free acid form or hydroxyl with a suitable esterifying agent.
Hydroxyl groups can be converted into esters via treatment with a
carboxylic acid. Examples of prodrug moieties include substituted
and unsubstituted, branch or unbranched lower alkyl ester moieties,
(e.g., propionoic acid esters), lower alkenyl esters, di-lower
alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester),
acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy
lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters
(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),
substituted (e.g., with methyl, halo, or methoxy substituents) aryl
and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower
alkyl amides, and hydroxy amides. Preferred prodrug moieties are
propionoic acid esters and acyl esters. Prodrugs which are
converted to active forms through other mechanisms in vivo are also
included.
[0134] The structures of some of the substituted tetracycline
compounds used in the methods and compositions of the invention
include asymmetric carbon atoms. The isomers arising from the
chiral atoms (e.g., all enantiomers and diastereomers) are included
within the scope of this invention, unless indicated otherwise.
Such isomers can be obtained in substantially pure form by
classical separation techniques and by stereochemically controlled
synthesis. Furthermore, the structures and other compounds and
moieties discussed in this application also include all tautomers
thereof.
[0135] The method may further comprise administering the
tetracycline compound in combination with a second agent, e.g., an
agent which may enhance treatment of the DTMR, enhance the
modulation of RNA, or the second agent may be selected for treating
a different DTMR or a different disease state not related to the
RNA modulation.
[0136] The language "in combination with" a second agent includes
co-administration of the tetracycline compound, and with the second
agent, administration of the tetracycline compound first, followed
by the second agent and administration of the second agent first,
followed by the tetracycline compound. The second agent may be any
agent which is known in the art to treat, prevent, or reduce the
symptoms of a DTMR. Furthermore, the second agent may be any agent
of benefit to the patient when administered in combination with the
administration of an tetracycline compound. Examples of second
agents include neuroprotective agents and chemotherapeutic
agents.
[0137] The language "chemotherapeutic agent" includes chemical
reagents which inhibit the growth of proliferating cells or tissues
wherein the growth of such cells or tissues is undesirable or
otherwise treat at least one resulting symptom of such a growth.
Chemotherapeutic agents are well known in the art (see e.g., Gilman
A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed.,
Sec 12:1202-1263 (1990)), and are typically used to treat
neoplastic diseases. Examples of chemotherapeutic agents include:
bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate,
etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine
(Gemzar), goserelin acetate (Zoladex), granisetron (Kytril),
irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel
(Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride
(Salagen), porfimer sodium (Photofrin), interleukin-2 (Proleukin),
rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin),
tretinoin (Retin-A), Triapine, vincristine, and vinorelbine
tartrate (Navelbine).
[0138] Other examples of chemotherapeutic agents include alkylating
drugs such as Nitrogen Mustards (e.g., Mechlorethamine (HN.sub.2),
Cyclophosphamide, Ifosfamide, Melphalan (L-sarcolysin),
Chlorambucil, etc.); ethylenimines, methylmelamines (e.g.,
Hexamethylmelamine, Thiotepa, etc.); Alkyl Sulfonates (e.g.,
Busulfan, etc.), Nitrosoureas (e.g., Carmustine (BCNU), Lomustine
(CCNU), Semustine (methyl-CCNU), Streptozocin (streptozotocin),
etc.), triazenes (e.g., Decarbazine (DTIC;
dimethyltriazenoimi-dazolecarboxamide)), Alkylators (e.g.,
cis-diamminedichloroplatinum II (CDDP)), etc.
[0139] Other examples of chemotherapeutic agents include
antimetabolites such as folic acid analogs (e.g., Methotrexate
(amethopterin)); pyrimidine analogs (e.g., fluorouracil
('5-fluorouracil; 5-FU); floxuridine (fluorode-oxyuridine); FUdr;
Cytarabine (cyosine arabinoside), etc.); purine analogs (e.g.,
Mercaptopurine (6-mercaptopurine; 6-MP); Thioguanine
(6-thioguanine; TG); and Pentostatin (2'-deoxycoformycin)),
etc.
[0140] Other examples of chemotherapeutic agents also include vinca
alkaloids (e.g., Vinblastin (VLB) and Vincristine); topoisomerase
inhibitors (e.g., Etoposide, Teniposide, Camptothecin, Topotecan,
9-amino-campotothecin CPT-11, etc.); antibiotics (e.g.,
Dactinomycin (actinomycin D), adriamycin, daunorubicin,
doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin
(mitomycin C), Taxol, Taxotere, etc.); enzymes (e.g.,
L-Asparaginase); and biological response modifiers (e.g.,
interferon-; interleukin 2, etc.). Other chemotherapeutic agents
include cis-diaminedichloroplatinum II (CDDP); Carboplatin;
Anthracendione (e.g., Mitoxantrone); Hydroxyurea; Procarbazine
(N-methylhydrazine); and adrenocortical suppressants (e.g.,
Mitotane, aminoglutethimide, etc.).
[0141] Other chemotherapeutic agents include adrenocorticosteroids
(e.g., Prednisone); progestins (e.g., Hydroxyprogesterone caproate;
Medroxyprogesterone acetate, Megestrol acetate, etc.); estrogens
(e.g., diethylstilbestrol; ethenyl estradiol, etc.); antiestrogens
(e.g. Tamoxifen, etc.); androgens (e.g., testosterone propionate,
Fluoxymesterone, etc.); antiandrogens (e.g., Flutamide); and
gonadotropin-releasing hormone analogs (e.g., Leuprolide).
III. Pharmaceutical Compositions for the Treatment of DTMR
[0142] The invention also pertains at least in part to
pharmaceutical compositions for the treatment of DTMR. The
pharmaceutical compositions comprise a tetracycline compound of the
invention in combination with a pharmaceutical acceptable carrier.
The composition may further comprise a second agent for the
treatment of a DTMR.
[0143] The language "pharmaceutical composition" includes
preparations suitable for administration to mammals, e.g., humans.
When the compounds of the present invention are administered as
pharmaceuticals to mammals, e.g., humans, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0144] The phrase "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material,
composition or vehicle, suitable for administering compounds of the
present invention to mammals. The carriers include liquid or solid
filler, diluent, excipient, solvent or encapsulating material,
involved in carrying or transporting the subject agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations.
[0145] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0146] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0147] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal, pulmonary and/or parenteral administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will generally be
that amount of the compound which produces a therapeutic effect.
Generally, out of one hundred percent, this amount will range from
about 1 percent to about ninety-nine percent of active ingredient,
preferably from about 5 percent to about 70 percent, most
preferably from about 10 percent to about 30 percent.
[0148] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0149] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0150] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0151] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0152] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0153] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluent commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0154] Besides inert dilutents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0155] Suspensions, in addition to the active, compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0156] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0157] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0158] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0159] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0160] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and propane.
Sprays also can be delivered by mechanical, electrical, or by other
methods known in the art.
[0161] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active compound in a polymer
matrix or gel.
[0162] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0163] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0164] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0165] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial, antiparasitic and
antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into
the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0166] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form may be
accomplished by dissolving or suspending the drug in an oil
vehicle. The compositions also may be formulated such that its
elimination is retarded by methods known in the art.
[0167] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0168] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administration or
administration via inhalation is preferred.
[0169] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0170] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0171] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and sublingually.
Other methods for administration include via inhalation.
[0172] The tetracycline compounds of the invention may also be
administered to a subject via stents. The compounds may be
administered through the stent or be impregnated in the stent
itself.
[0173] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0174] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0175] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0176] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0177] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous and subcutaneous doses of the compounds of
this invention for a patient will range from about 0.0001 to about
100 mg per kilogram of body weight per day, more preferably from
about 0.01 to about 50 mg per kg per day, and still more preferably
from about 1.0 to about 100 mg per kg per day.
[0178] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0179] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition. Compounds or
pharmaceutical compositions can be administered in combination with
other agents.
[0180] As set out above, certain embodiments of the present
compounds can contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically acceptable salts" is art recognized and includes
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or by separately reacting a purified compound of
the invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Farm. SCI. 66:1-19).
[0181] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances includes relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like.
[0182] The term "pharmaceutically acceptable esters" refers to the
relatively non-toxic, esterified products of the compounds of the
present invention. These esters can be prepared in situ during the
final isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent. Carboxylic acids can be
converted into esters via treatment with an alcohol in the presence
of a catalyst. Hydroxyls can be converted into esters via treatment
with an esterifying agent such as alkanoyl halides. The term also
includes lower hydrocarbon groups capable of being solvated under
physiological conditions, e.g., alkyl esters, methyl, ethyl and
propyl esters. (See, for example, Berge et al., supra.)
[0183] The invention also pertains, at least in part, to packaged
compositions comprising a tetracycline compound of the invention
and instructions for using said compound for the treatment of a
DTMR.
[0184] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXEMPLIFICATION OF THE INVENTION
[0185] Compounds of the invention may be made as described below,
with modifications to the procedure below within the skill of those
of ordinary skill in the art.
Example 1: Synthesis of 7-Substituted Tetracyclines
7 Iodo Sancycline
[0186] One gram of sancycline was dissolved in 25 mL of TFA
(trifluoroacetic acid) that was cooled to 0 C (on ice). 1.2
equivalents of N-iodosuccinimide (NIS) was added to the reaction
mixture and reacted for forty minutes. The reaction was removed
from the ice bath and was allowed to react at room temperature for
an additional five hours. The mixture was then analyzed by HPLC and
TLC, was driven to completion by the stepwise addition of NIS.
After completion of the reaction, the TFA was removed in vacuo and
3 mL of MeOH was added to dissolve the residue. The methanolic
solution was the added slowly to a rapidly stirring solution of
diethyl ether to form a greenish brown precipitate. The 7-iodo
isomer of sancycline was purified by treating the 7-iodo product
with activated charcoal, filtering through Celite, and subsequent
removal of the solvent in vacuo to produce the 7-isomer compound as
a pure yellow solid in 75% yield.
[0187] MS (M+H) (formic acid solvent) 541.3.
[0188] \Rt: Hypersil C18 BDS Column, 11.73
[0189] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 7.87-7.90 (d,
1H), 6.66-6.69 (d, 1H), 4.06 (s, 1H), 2.98 (s, 6H), 2.42 (m, 1H),
2.19 (m, 1H), 1.62 (m, 4H), 0.99 (m, 2H)
7-Phenyl Sancycline
[0190] 7-iodosancycline, 150 mg (0.28 mM), Pd(OAc).sub.2 and 1 0 mL
of MeOH are added to a flask with a stir bar and the system
degassed 3x using argon. Na.sub.2CO.sub.3 (87 mg, 0.8 mM) dissolved
in water and argon degassed is added via syringe is added along
with phenylboronic acid (68 mg, 0.55 mM) in MeOH that was also
degassed. The reaction was followed by HPLC for 2 hours and cooled
to room temperature. The solution was filtered, and dried to
produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The fraction at 36-38 minutes was
isolated, and the solvent removed in vacuo to yield the product
plus salts. The salts were removed by extraction into 50:25:25
water, butanol, ethyl acetate and dried in vacuo. This solid was
dissolved in MeOH and the HCl salt made by bubbling in HCl gas. The
solvent was removed to produce the product in 42% yield as a yellow
solid.
[0191] Rt 21.6 min: MS (M+H, formic acid solvent): 491.3
[0192] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 7.87 (d,
J=8.86 Hz, 1H), 7.38 (m, 5H), 6.64 (d, 8.87 Hz, 1H), 4.00 (s, 1H),
3.84 (s, 2H), 3.01 (s, 6H), 2.46 (m, 2H), 1.63 (m, 4H), 0.95 (m,
2H)
7-(4'-Chlorophenyl) Sancycline
[0193] 7-iodosancycline, 500 mg (0.91 mM), Pd(OAc).sub.2 21 mg, and
20 mL of MeOH are added to a flask with a stir bar and the system
degassed 3x using argon. Na.sub.2CO.sub.3 (293 mg, 2.8 mM)
dissolved in water and argon degassed is added via syringe is added
along with 4-Cl-phenylboronic acid (289 mg, 1.85 mM) in MeOH that
was also degassed. The reaction was followed by HPLC for 45 minutes
and cooled to room temperature. The solution was filtered, and
dried to produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The fraction at 39 minutes was isolated,
and the solvent removed in vacuo to yield the product plus salts.
The salts were removed by extraction into 50:25:25 water, butanol,
ethyl acetate and dried in vacuo. This solid was dissolved in MeOH
and the HCl salt made by bubbling in HCl gas. The solvent was
removed to produce the product in 57% yield as a yellow solid.
[0194] Rt 20.3 min: MS (M+H, formic acid solvent): 525.7
[0195] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 7.49-7.52 (d,
J=8.54 Hz, 1H), 6.99-7.01 (d, 8.61 Hz, 1H), 4.12 (s, 1H), 3.67 (m,
1H), 3.06 (s, 6H), 2.58 (m, 2H), 1.62 (m, 4H), 1.01 (m, 2H)
7-(4'-Fluorophenyl) Sancycline
[0196] 7-iodosancycline, 200 mg (0.3 mM), Pd(OAc).sub.2 8.3 mg, and
10 mL of MeOH are added to a flask with a stir bar and the system
degassed 3x using argon. Na.sub.2CO.sub.3 (104 mg, 1.1 mM)
dissolved in water and argon degassed is added via syringe is added
along with 4-F-phenylboronic acid (104 mg, 0.7 mM) in MeOH that was
also degassed. The reaction was followed by HPLC for 20 minutes and
cooled to room temperature. The solution was filtered, and dried to
produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The fraction at 19-20 minutes was
isolated, and the solvent removed in vacuo to yield the product
plus salts. The salts were removed by extraction into 50:25:25
water, butanol, ethyl acetate and dried in vacuo. This solid was
dissolved in MeOH and the HCl salt made by bubbling in HCl gas. The
solvent was removed to produce the product in 47% yield as a yellow
solid.
[0197] Rt 19.5 min: MS (M+H, formic acid solvent): 509.4
[0198] .sup.1H NMR (Methanol d.sub.4-300 MHz) .delta. 6.92-6.95 (d,
1H), 7.45-7.48 (d, 1H), 7.15-7.35 (m, 4H), 4.05 (s, 111), 3.62 (m,
1H), 3.08 (s, 611), 2.55 (m, 2H), 1.65 (m, 4H), 1.00 (m, 2H)
7-(4'-Iodo-1',3'-carboethoxy-1',3'-butadiene) Sancycline
[0199] 7-I-Sancycline (1 gm, 1.86 mmol), was dissolved in 25 mL of
acetonitrile and was degassed and purged with nitrogen (three
times). To this suspension Pd(OAc).sub.2 (20 mg, 0.089 mmol), CuI
(10 mg, 0.053 mmol), (o-tolyl).sub.3P (56 mg, 0.183 mmol) were
added and purged with nitrogen. Ethyl propiolate (1 mL) and
triethylamine (1 mL) were added to the suspension. It turned to a
brown solution upon addition of Et.sub.3N. The reaction mixture was
then heated to 70 degrees C. for two hours. Progress of the
reaction was monitored by HPLC. It was then cooled down to room
temperature and was filtered through celite. Evaporation of the
solvent gave a brown solid, which was then purified on preparative
HPLC to give a yellow solid.
7-(2'-Chloroethenyl)-Sancycline
[0200] To a solution/suspension of 0.65 g (1 mmol) of 7-iodo
sancycline, 0.05 g tetrakis triphenyl phosphinato palladate, 0.012
g palladium acetate, 0.05 g copper (I) iodide in 10 mL
acetonitrile, 2 mL triethylamine and 0.5 g trimethylsilyl acetylene
was added at room temperature. The reaction proceeded for two hours
before being filtered through a celite bed and concentrated. The
crude product was purified by preparative HPLC. The collected
fractions were concentrated and the residue was taken up in about 1
mL of methanol and 2 mL of MCI saturated methanol. The product was
precipitated with ether. The solids were filtered off and dried
under reduced pressure. NMR spectroscopy and LC-MS showed that the
compound was 7-(2-chloroethenyl) sancycline.
7-(4'-aminophenyl) Sancycline
[0201] To a solution of 200 mg of 7-(4-nitrophenyl) sancycline in
50 mL methanol, 10 mg of 10% palladium on charcoal catalyst was
added. The reaction mixture was shaken under 40 psi hydrogen
pressure for 2 hours and was then filtered followed by
concentration. The residue was further purified by preparative
HPLC. 35 mg was isolated as the HCl salt and the structure was
proved by NMR and LC-MS to be 7-(4-aminophenyl) sancycline.
7-(NN-Dimethylpropynyl)-Sancycline
##STR02246##
[0203] 7-I-Sancycline (1 gm, 1.86 mmol), taken in 25 mL of
acetonitrile was degassed and purged with nitrogen (three times).
To this suspension Pd(OAc).sub.2 (20 mg, 0.089 mmol), CuI (10 mg,
0.053 mmol), (o-tolyl).sub.3P (56 mg, 0.183 mmol) were added and
purged with nitrogen for few minutes. NN-Dimethylpropyne (308 mg,
3.72 mmol) and triethylamine (1 mL) were added to the suspension.
It was turned into a brown solution upon addition of Et.sub.3N. The
reaction mixture was then heated to 70.degree. C. for 3 hours.
Progress of the reaction was monitored by HPLC. It was then cooled
down to room temperature and was filtered through celite.
Evaporation of the solvent gave a brown solid, which was then
purified on preparative HPLC to give a yellow solid. The structure
of this compound has been characterized using 1H NMR, HPLC, and
MS.
7-(2'-Chloro-3-Hydroxypropenynl)-Sancycline
##STR02247##
[0205] 7-(alkynyl)-sancycline (100 mg) was taken in 20 ml of
saturated MeOH/HCl and stirred for 20 min. The solvent was then
evaporated to give a yellow powder. The structure of this compound
has been characterized using 1H NMR, HPLC, and MS.
7-(3'-Methoxyphenylethyl)-Sancycline
##STR02248##
[0207] 7-(3'-Methoxyphenylethynyl)-sancycline (1 mmol) was taken in
saturated solution of MeOH/HCl. To this solution 10% Pd/C was added
and was subjected to hydrogenation at 50 psi for 12 hrs. It was
then filtered through celite. The solvent was evaporated to give a
yellow powder. Finally, it was precipitated from MeOH/diethylether.
The structure of this compound has been characterized using 1H NMR,
HPLC, and MS.
(2-Dimethylamino-Acetylamino)-Sancycline
##STR02249##
[0209] NN-Dimethylglycine (1.2 mmol) was dissolved in DMF (5 mL)
and 0-Benzotriazol-1-yl-N, N, N', N',-tetramethyluronium
hexafluorophosphate (HBTU, 1.2 mmol) was added. The solution was
then stirred for 5 minutes at room temperature. To this solution,
7-aminosancycline (1 mmol) was added, followed by the addition of
diisopropylethyl amine (DIEA, 1.2 mmol). The reaction was then
stirred at room temperature for 2 hours. The solvent, DMF, was
removed on vacuum. The crude material was dissolved in 5 mL of MeOH
and filtered using autovials and purified using preparative HPLC.
The structure of the product has been characterized using 1H NMR,
HPLC, and MS.
7-(N-Methylsulphonamidopropargylamine) Sancycline
##STR02250##
[0211] To a mixture of 7-iodosancycline mono trifluoroacetic acid
salt (1 g; 1.53 mmoles), palladium II acetate (17.2 mg; 0.076
mmoles), tetrakis triphenylphosphine palladium (176.8 mg; 0.153
mmoles), and copper (I) iodide (49 mg; 0,228 mmoles) was added 15
ml of reagent grade acetonitrile in a clean dry 2 necked round
bottom flask. The reaction was purged with a slow steam of argon
gas, with stirring, for 5 minutes before the addition (in one
portion as a solid) of N-methylsulphonamidopropargyl amine. The
sulphonamide was prepared by a method known in the art (J. Med.
Chem 31(3) 1988; 577-82). This was followed by one milliliter of
triethylamine (1 ml; 0.726 mg; 7.175 mmoles) and the reaction was
stirred, under an argon atmosphere, for approximately 1.0 hour at
ambient temperature. The reaction mixture was suctioned filtered
through a pad of diatomaceous earth and washed with acetonitrile.
The filtrates were reduced to dryness under vacuo and the residue
was treated with a dilute solution of trifluroroacetic acid in
acetonitrile to adjust the pH to approximately 2. The residue was
treated with more dilute trifluoroacetic acid in acetonitrile,
resulting in the formation of a precipitate, which was removed via
suction filtration. The crude filtrates were purified utilizing
reverse phase HPLC with DVB as the solid phase; and a gradient of
1:1 methanol/acetonitrile 1% trifluoroacetic acid and 1%
trifluoroacetic acid in water. The appropriate fractions were
reduced to dryness under reduced pressure and solid collected. The
product was characterized via .sup.1H NMR, mass spectrogram and LC
reverse phase.
7-(2'-methoxy-5'-formylphenyl)sancycline
##STR02251##
[0213] 7-iodo-sancycline (1 g, 1.5 3 mmol), Pd(OAc).sub.2 (34 mg,
0.153 mmol), and MeOH (50 mL) were combined in a 250 mL 2 neck
round bottom flask equipped with a condenser and argon line. The
solution was then purged with argon (15 min) while heated in an oil
bath to approximately 70.degree. C. Sodium carbonate (482 mg, 4.58
mmol) was dissolved in water (3-5 mL) and added to reaction flask.
The flask was then purged with argon for another 5 minutes.
2-Methoxy-5-formylphenyl boronic acid (333 mg, 1.83 mmol) was
dissolved in MeOH (5 mL) and added to reaction flask. The flask was
then purged again with argon for 10 minutes. The reaction was
monitored to completion within 3 hours. The contents of the flask
were filtered through filter paper and the remaining solvent was
evacuated. To make the hydrochloric acid salt, the residue was
dissolved in MeOH (sat. HCl) to make the HCl salt. The solution was
then filtered and the solvent was evacuated. The product was then
characterized by .sup.1H NMR, LC-MS.
7-(2'-Methoxy-5'-N,N'-Dimethylaminomethylphenyl)Sancycline
##STR02252##
[0215] 7-(2'-methoxy-5'-formylphenyl)sancycline (1 g, 1.82 mmol),
dimethylamine HCl (297 mg, 3.64 mmol), triethylamine (506 .mu.L,
3.64 mmol), and 1,2-DCE (7 mL) were combined in a 40 mL vial. The
contents were dissolved within several minutes of shaking or
stirring. Sodium triacetoxyborohydride (772 mg, 3.64 mmol) was then
added as a solid. The reaction was monitored by HPLC and LC-MS and
was complete within 3 hours. The reaction was quenched with MeOH (2
0 mL) and the solvent was subsequently evacuated. The residue was
redissolved in 3 mL DMF and separated on a C-18 column. Fractions
from the prep column dried down in-vacuo and the HCl salt was made
by dissolving contents in methanol (sat. HCl). The solvent was
reduced and a yellow powder obtained. Characterized by .sup.1H NMR,
LC-MS, HPLC.
7-Furanyl Sancycline
[0216] 7-iodo sancycline (1.3 mg) and Pd(OAc).sub.2 were taken in
100 mL of methanol and purged with argon for five minutes at
70.degree. C. To this solution was added a solution of sodium
carbonate (44 mg) in water (previously purged with argon). A yellow
precipitate was obtained and the mixture was heated for another ten
minutes. 3-Furanyl boronic acid (333 mg, solution in DMF, purged
with argon) was then added and the mixture was heated for another
two hours at 70.degree. C., The reaction was monitored by MPLC/MS.
When the reaction was complete, the mixture was filtered through
celite and the solvent was removed to give a crude material. The
crude material was purified by precipitating it with ether (200
ml). The yellow precipitate was filtered and purified using
preparative HPLC. The hydrochloride salt was made by dissolving the
material in MeOH/HCl and evaporating to dryness. The identity of
the resulting solid was confirmed using HPLC, MS, and NMR.
4S-(4.alpha.,12a.alpha.)]-4-Dimethylamino-7-ethynyl-3,10,12,12a-tetrahydro-
xy-1,11-dioxo-1,4,
4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxamide
[0217] 300 mg of 7-iodosancycline 6A was dissolved in 20 mL of
acetonitrile and 2.0 mL triethylamine, 50.0 mg Pd(PPh.sub.3).sub.4,
50 mg CuI, 12.5 mg Pd(OAc).sub.2 was added followed by 0.5 mL of
trimethylsilylacetylene. The reaction was stirred at room
temperature for 4 hours, filtered through a divinyl-benzene
cartridge (25 g), and concentrated in vacuo to yield 280 mg of the
crude material (monitored by LC/MS). The TMS group was removed by
dissolving the crude material in methanol, and adding 250 mg of
K.sub.2CO.sub.3 while stirring for 4 hours at room temperature to
yield compound 6E (Scheme 11). The mixture was filtered through a
divinylbenzene cartridge. The solvent was removed in vacuo to yield
the 7-ethynyl sancycline 6E (Scheme 11) in 60% yield by HPLC.
General Method for Synthesis of 7-acetyl sancycline and
7-carbonylalkyl Derivatives of Sancycline
[0218] 7-ethynyl sancycline 6E (Scheme 11, 300 mg) or ethynyl
substituted derivatives of 7-ethynyl sancycline are dissolved in
0.1 mL water, 2 mL of H.sub.2SO.sub.4, optionally with HgSO.sub.4
(170 mg) and stirred overnight at room temperature. The aqueous
layer is extracted into butanol, CH.sub.2Cl.sub.2 or an equivalent
and the solvent is removed to yield the crude compound 11A (Scheme
11). 7-acetyl sancycline (11A, Scheme 11) is isolated via C18
reverse-phase HPLC or by other methods in the art to yield pure
compound in good yield. M+H=457.4
Conversion of 7-acetyl or 7-carbonylalkyl Derivatives of Sancycline
to Oximes or O-alkyl oximes
[0219] 1 gram of 7-acetyl or 7-carbonylalkyl derivatives of
sancycline 11A (Scheme 11, 2 mmol) and hydroxylamine HCl are
dissolved in methanol or ethanol and stirred at room temperature
for 2 or more hours. The compounds are isolated as the syn and anti
isomers appropriately by preparative C18-HPLC or by other methods
in the art to yield 7-oximecarbonyl alkyl derivatives of sancycline
or 7-O-substituted oximecarbonyl derivatives in good yield.
7-acetyl-oxime (Scheme 11, 11B); M+H=473.5. 11C
7-acetyl-oxime-O-methyl ether; M+H=487.5. The syn or anti isomers
are both attainable by fractionation of HPLC solvent volumes.
General Methods and Conversion of 7-acetyl or 7-carbonylalkyl
Derivatives of Sancycline to 7-carbonyl-.alpha.-amino
Derivatives
[0220] 1 gram of 7-acetyl or 7-carbonylalkyl derivatives of
sancycline 11A (Scheme 11, 2 mmol) is reacted with bromine (4 mmol)
or typical halogenating agent (NBS, NCS or equivalent, 2-4 mmol) to
produce the .alpha.-halogenated derivative 11D (Br, Cl) as crude
solid. This compound is isolated by extraction or other methods in
the art and may be reacted with nucleophilic amines (2-4 mmol) or
other nucleophiles (C or O-based) to yield .alpha.-amino
derivatives of 7-acetyl 11E or other 7-carbonylalkyl derivatives of
sancycline.
##STR02253##
Example 2: Preparation of 9--Substituted Minocyclines
Preparation of 9-Iodominocycline
[0221] To 200 ml of 97% methanesulfonic acid was slowly added, at
ambient temperature, portionwise [30 g; 56.56 mM] of
minocycline-bis-hydrochloride salt. The dark yellow brown solution
was then stirred at ambient temperature while [38 g; 169.7 mM] of
N-iodosuccinimide was added, in six equal portions, over 3.0 hours
time. The reaction was monitored via analytical LC, noting the
disappearance of the starting material.
[0222] The reaction was slowly quenched into 2 L of ice cold water
containing [17.88 g; 1134.1 mM] of sodium thiosulfate with rapid
stirring. This quench was stirred for approximately 30 minutes at
ambient temperature. The aqueous layer was then extracted with
6.times.200 ml of ethyl acetate before the aqueous was poured onto
[259.8 g; 3.08M] of sodium hydrogen carbonate containing 300 ml of
n-butanol. The phases were split and the aqueous extracted with
4.times.250 ml of n-butanol. The organic fractions were combined
and washed with 3.times.250 ml of water and once with 250 ml of
saturated brine. The resulting organic phase was reduced to dryness
under reduced pressure. The residue was suspended in methanol
(.about.600 ml) and anhydrous HCl gas was bubbled into this mixture
until solution occurred This solution was reduced to dryness under
reduced pressure. The filtrates were reduced to dryness under
reduced pressure. The resulting material was triturated with 300 ml
of methyl t-butyl ether and isolated via filtration. This material
was redissolved in 300 ml of methanol and treated with 0.5 g of
wood carbon, filtered and filtrates reduced to dryness under
reduced pressure. The material was again powdered under methyl
t-butyl ether, isolated via suction filtration and washed with more
ether, and finally hexanes. The material was vacuum dried to give
22.6 g of a light yellow brown powder.
General Procedure for Preparation of 9-Alkynyl Minocycline
Compounds
[0223] 1 mmol 9-iodo minocycline, 50 mg tetrakis
triphenylphosphinato palladate, 12 mg palladium acetate, 32 mg
copper (1) iodide are dissolved/suspended in 10 ml acetonitrile. 2
to 5 ml triethylamine and 3 to 5 mmol alkynyl derivative is added.
The reaction mixture is vigorously stirred between ambient
temperature to 70.degree. C. The reaction time is 2-24 hours. When
the reaction is completed the dark suspension is filtered through a
celite bed and concentrated. The crude product is purified by prep
HPLC. The combined fractions are concentrated and taken up in
.about.1 ml methanol. .about.3 ml HCl saturated methanol is added,
and the product is precipitated with ether.
General Procedure for Preparation of 9-Aryl Minocycline
Compounds
[0224] 0.15 mmol of 9-iodominocycline, PdOAc (3.2 mg), 229 .mu.l 2M
Na.sub.2CO.sub.3 and 2 equivalents of phenyl boronic acid were
dissolved/suspended in 10 ml methanol. The reaction flask was
purged with argon and the reaction run for a minimum of four hours
or until HPLC monitoring shows consumption of starting material
and/or the appearance of products. The suspension was filtered
through celite, and subject to purification by prep HPLC on a
divinylbenzene or CIE reverse-phase column.
9-(4-Trifluoromethoxyphenylureido)-Methyl Minocycline
##STR02254##
[0226] To 3 mL of dimethylformamide was added 150 mg (0.25 mmol) of
9-methyl aminominocyline trihydrochloride and 67 mL (0.50 mmol) of
triethylamine at 25.degree. C. With stirring, 75 mL (0.50 mmol) of
4-trifluoromethoxyphenylisocyanate was added and the resulting
reaction mixture was stirred at 25.degree. C. for two hours. The
reaction was monitored by analytical HPLC (4.6.times.50 mm reversed
phase Luna C18 column, 5 minute linear gradient 1-100% B buffer, A
buffer was water with 0.1% trifluoroacetic acid, B buffer was
acetonitrile with 0.1% trifluoroacetic acid). Upon completion, the
reaction was quenched with 1 mL of water and the pH adjusted to
approximately 2.0 with concentrated HCl. The solution was filtered
and the compound purified by preparative HPLC. The product yield
was 64 mg (37% yield). The purity of the product was 95%, as
determined by LCMS (M+1=690).
9-(4'Carboxy phenyl) Minocycline
##STR02255##
[0228] In a clean, dry reaction vessel, was placed
9-iodominocycline [500 mg; 0.762 mmoles]bis HCl salt, palladium
(II) acetate [17.2 mg; 0.076 mmoles] along with 10 ml of reagent
grade methanol. The solution was immediately purged, with stirring,
with a stream of argon gas for approximately 5 minutes. The
reaction vessel was brought to reflux and to it was sequentially
added via syringe 2M potassium carbonate solution [1.91 ml; 3.81
mmoles], followed by a solution of p-carboxyphenyl boronic acid
[238.3 mg; 1.53 mmoles] in 5 ml of reagent DMF. Both of these
solutions were previously degassed with argon gas for approximately
5 minutes. The reaction was heated for 45 minutes, the progress was
monitored via reverse phase HPLC. The reaction was suctioned
filtered through a pad of diatomaceous earth and washed with DMF.
The filtrates were reduced to an oil under vacuum and residue
treated with t-butylmethyl ether. Crude material was purified via
reverse phase HPLC on DVB utilizing a gradient of water and
methanol/acetonitrile containing 1.0% trifluoroacetic acid. Product
confirmed by mass spectrum: found M+1 578.58; the structure
corroborated with 1H NMR.
Example 3: Modulation of Murine Macrophage mRNAs Using
Tetracyclines
Materials and Methods
[0229] Two murine macrophage cell lines were used: J774.2 (gift
from Peter Lambert, Aston University, UK), and RAW 264.7 (ATCC item
number TIB-71). Cells were harvested from nearly confluent culture
flasks and seeded into 6 well plates at a density of
5.times.10.sup.6 cells in a volume of 3 ml Dulbecco's modified
essential medium supplemented with 10% fetal calf serum. After 2
hours, cells were exposed to the following conditions:
[0230] 1) control (J774 cells on two separate occasions, and RAW
264.7 cells)
[0231] 2) 50 .mu.g/ml minocycline (J774 cells on two separate
occasions, and RAW 264.7 cells)
[0232] 3) 100 ng/ml LPS (J774 cells on two separate occasions, and
RAW 264.7 cells)
[0233] 4) 50 .mu.g/ml minocycline+100 ng/ml LPS (J774 cells on two
separate occasions, and RAW 264.7 cells)
[0234] 5) 50 .mu.g/ml Compound A+100 ng/ml LPS (J774 cells
only)
[0235] 6) 50 .mu.g/ml Compound B+100 ng/ml LPS (J774 cells
only)
[0236] The tetracycline compounds were added 1.5 hours post
seeding, thirty 15 minutes before the addition of LPS. The plates
were incubated at 37.degree. C. at 5% CO.sub.2 in a humidified
incubator.
Sample Processing, Hybridization, and Scanning
[0237] 24 hours after incubation under experimental conditions,
media was removed from the wells, and total RNA was purified from
each sample using QIAGEN RNeasy.RTM. Mini columns. The
manipulations which were then performed on the total RNA samples
were as outlined in The Affymetrix.RTM. GeneChip.RTM. Expression
Analysis technical manual, sections 2, chapter 1, entitled
Eukaryotic Target Preparation. Briefly, RNA was reverse transcribed
into double stranded cDNA with an oligo dT primer containing a T7
promoter. The product was then purified by
phenol:chloroform:isoamyl extraction and ethanol precipitation, and
then used in an in vitro translation reaction to synthesize
biotin-labelled antisense cRNA (Affymetrix controls of B. subtilis
genes excised from pBluescript plasmid with Xho I digestion were
added at this stage to control for correct translation and biotin
incorporation). The cRNA was then cleaned using QIAGEN RNeasy.RTM.
Mini columns, and the resulting cRNA solution fragmented using
metal-induced hydrolysis.
[0238] Samples were prepared for hybridization with the Affymetrix
murine genome chips U74AV2 according to the directions in the
Affymetrix.RTM. GeneChip.RTM. Expression Analysis technical manual,
sections 2, chapters 3 and 4, entitled Eukaryotic Target
Hybridization and Eukaryotic Arrays: Washing, Staining and
Scanning. Briefly, 15 ug Fragmented cRNA was mixed with Affymetrix
hybridization controls, herring sperm DNA, BSA, and concentrated
hybridization buffer, boiled for 5 minutes, centrifuged at
14000.times.g for 5 minutes to obtain a precipitated-free solution,
and hybridized with the array for 16 hours. Following
hybridization, the Affymetrix washing and staining procedure was
used entitled Washing and Staining Procedure 2: Antibody
Amplification for Eukaryotic Targets.
Data Analysis
[0239] I. Finding mRNAs which are Up-Regulated or Down-Regulated by
Minocycline
[0240] For both J774.2 and RAW264.7 cells, two lists were
generated, one of mRNAs which were increased at lease 2-fold by
minocycline, and one of mRNAs which were decreased at least 2-fold
by minocycline. In the case of mRNAs which were increased, the
mRNAs had to be statistically `Present` in the samples which
contained minocycline (`Present` as determined by the Affymetrix
microarray suite software). In the case of mRNAs which were
decreased, the mRNAs had to be statistically `Present` in the
samples which did not contain minocycline.
[0241] The three experimental conditions produced three lists of
increased mRNAs, and three lists of decreased mRNAs. The mRNAs
common to all three lists are tallied in Table 2, below.
II. Finding mRNAs which are Up-Regulated or Down-Regulated by
Minocycline in Samples which are Stimulated with LPS
[0242] For both J774.2 and RAW264.7 cells stimulated by LPS, two
lists were generated, one of mRNAs which were increased at lease
2-fold by minocycline, and one of mRNAs which were decreased at
least 2-fold by minocycline. In the case of mRNAs which were
increased, the mRNAs had to be statistically `Present` in the
samples which contained minocycline (`Present` as determined by the
Affymetrix microarray suite software). In the case of mRNAs which
were decreased, the mRNAs had to be statistically `Present` in the
samples which did not contain minocycline.
[0243] The three experimental conditions produced three lists of
increased mRNAs, and three lists of decreased mRNAs. The mRNAs
common to all three lists are tallied in Table 2, below.
III. Finding mRNAs which are Up-Regulated or Down Regulated by
Compounds A and B in Samples Also Stimulated with LPS
[0244] For J774.2 cells stimulated by LPS, two lists were
generated, one of mRNAs which were increased at lease 2-fold by
Compounds A and/or B, and one of mRNAs which were decreased at
least 2-fold by Compounds A and/or B. In the case of mRNAs which
were increased, the mRNAs had to be statistically `Present` in the
samples which contained Compounds A and/or B (`Present` as
determined by the Affymetrix microarray suite software). In the
case of mRNAs which were decreased, the mRNAs had to be
statistically `Present` in the samples which did not contain
Compounds A and/or B. The structures of compounds A and B are shown
beneath Table 3.
[0245] mRNAs were found which were up-regulated by both Compounds A
and B, and mRNAs were found which were down-regulated by both
compounds. Results are tallied in Table 3, below.
TABLE-US-00003 TABLE 3 Numbers of mRNAs with Numbers of mRNAswith
Numbers of mRNAs with levels significantly altered by levels
significantly altered levels significantly altered minocycline, in
the presence by both Compounds A and B, by minocycline (in J774.2
of LPS (in J774.2 and in the presence of LPS and RAW264.7 cells)
RAW264.7 cells) (in J774.2 only) Increased 21 28 133 Decreased 4 9
108
##STR02256##
Example 4: Modulation of Inducible Nitric Oxide Synthase (iNOS)
with Minocycline
Materials and Methods
[0246] Mouse macrophage J774.2 cells were seeded into 6 well plates
as described above, and exposed to either minocycline alone, or in
combination with LPS as above (untreated and LPS-alone conditions
were used as controls). Data representing the modulation of iNOS
mRNA was extracted from the Affymetrix data, described above.
[0247] Nitrite levels were measured in the supernatants of the
samples using the Greiss reaction. Briefly, 100 .mu.l singlicates
of supernatant were incubated in the dark for 10 minutes with
sulfanilamide solution (I % sulfanilamide in 5% H.sub.2PO.sub.4).
Then 50 .mu.l of NED (0.1% N-1-napthylethylenediamine
dihydrochloride in water) was added, and the samples incubated for
a further 10 minutes in the dark. Samples were read in a Wallac
Victor V plate reader at 535 nm.
[0248] Protein levels were measured by Western analysis. Cells were
lysed in 10 mM Tris HCl, pH 7.4, 1 mM EDTA, 0.5% SDS, protease
inhibitors and DNAse. The antibody used to detect the iNOS protein
was an anti iNOS antibody from Transduction laboratories. The
results of the experiment are shown in FIG. 1.
Example 5: Mammalian Cytotoxicity Assay
[0249] COS-1 and CHO-K1 cell suspensions were prepared, seeded into
96-well tissue culture treated black-walled microliter plates
(density determined by cell line), and incubated overnight at
37.degree. C., in 5% CO.sub.2 and approximately 95% humidity. The
following day, serial dilutions of drug were prepared under sterile
conditions and transferred to cell plates. Cell/Drug plates were
incubated under the above conditions for 24 hours. Following the
incubation period, media/drug was aspirated and 50 .mu.l of
Resazurin (0.042 mg/ml in PBS w/Ca and Mg) was added. The plates
were then incubated under the above conditions for 2 hours and then
in the dark at room temperature for an additional 30 minutes.
Fluorescence measurements were taken (excitation 535 nm, emission
590 nm). The IC.sub.50 (concentration of drug causing 50% growth
inhibition) was then calculated. The cytotoxicity of both
unsubstituted minocycline and doxycycline were found to be greater
than 25 .mu.g/ml. Each of the compounds tested was found to have an
acceptable cytotoxicity.
Example 6: In Vitro Anti-Bacterial Activity Assay
[0250] The following assay was used to determine the efficacy of
the tetracycline compounds against gram positive (S. aureus RN450)
and gram negative (E. coli ML308 225) bacteria. 2 mg of each
compound was dissolved in 100 .mu.l of DMSO. The solution was then
added to cation-adjusted Mueller Hinton broth (CAMHB), which
resulted in a final compound concentration of 200 .mu.g per ml. The
tetracycline compound solutions were diluted to 50 .mu.L volumes,
with a test compound concentration of 0.098 .mu.g/ml. Optical
density (OD) determinations were made from fresh log-phase broth
cultures of the test strains. Dilutions were made to achieve a
final cell density of 1.times.10.sup.6 CFU/ml. At OD=1, cell
densities for different genera were approximately:
TABLE-US-00004 E. coli 1 .times. 10.sup.9 CFU/ml S. aureus 5
.times. 10.sup.8 CFU/ml
[0251] 50 .mu.l of the cell suspensions were added to each well of
microtiter plates. The final cell density was approximately
5.times.10.sup.5 CFU/ml. These plates were incubated at 35.degree.
C. in an ambient air incubator for approximately 18 hours. The
plates were read with a microplate reader and were visually
inspected when necessary. The MIC was defined as the lowest
concentration of the tetracycline compound that inhibits
growth.
EQUIVALENTS
[0252] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
[0253] All patents, patent applications, and literature references
cited herein are hereby expressly incorporated by reference.
* * * * *