U.S. patent application number 12/608227 was filed with the patent office on 2010-05-06 for methods for synthesizing and purifying aminoalkyl tetracycline compounds.
This patent application is currently assigned to Paratek Pharmaceuticals, Inc.. Invention is credited to Sean Johnston, Tadeusz Warchol.
Application Number | 20100113401 12/608227 |
Document ID | / |
Family ID | 39739603 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100113401 |
Kind Code |
A1 |
Johnston; Sean ; et
al. |
May 6, 2010 |
METHODS FOR SYNTHESIZING AND PURIFYING AMINOALKYL TETRACYCLINE
COMPOUNDS
Abstract
Methods for the synthesis and purification of 9-amino alkyl
tetracycline compounds are described.
Inventors: |
Johnston; Sean; (Doylestown,
PA) ; Warchol; Tadeusz; (Northborough, MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Paratek Pharmaceuticals,
Inc.
Boston
MA
|
Family ID: |
39739603 |
Appl. No.: |
12/608227 |
Filed: |
October 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12110627 |
Apr 28, 2008 |
|
|
|
12608227 |
|
|
|
|
60926461 |
Apr 27, 2007 |
|
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Current U.S.
Class: |
514/152 ;
552/205 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 19/10 20180101; A61P 25/18 20180101; A61P 1/04 20180101; A61P
13/02 20180101; A61P 9/10 20180101; A61P 17/02 20180101; A61P 25/20
20180101; C07C 231/24 20130101; A61P 31/00 20180101; A61P 33/06
20180101; A61P 25/22 20180101; A61P 25/24 20180101; A61P 3/04
20180101; A61P 11/16 20180101; A61P 31/12 20180101; A61P 25/00
20180101; A61P 35/04 20180101; A61P 9/12 20180101; A61P 19/08
20180101; A61P 31/04 20180101; A61P 31/10 20180101; A61P 1/02
20180101; A61P 3/10 20180101; A61P 11/02 20180101; A61P 25/08
20180101; A61P 25/14 20180101; A61P 43/00 20180101; A61P 9/14
20180101; A61P 35/00 20180101; A61P 25/02 20180101; A61P 29/00
20180101; A61P 25/16 20180101; A61P 21/02 20180101; A61P 25/06
20180101; A61P 25/28 20180101; A61P 27/02 20180101; C07C 231/12
20130101; C07C 2603/46 20170501; A61P 1/12 20180101; A61P 19/02
20180101; A61P 11/00 20180101; C07C 231/12 20130101; C07C 237/26
20130101 |
Class at
Publication: |
514/152 ;
552/205 |
International
Class: |
A61K 31/65 20060101
A61K031/65; C07C 237/26 20060101 C07C237/26; A61P 31/00 20060101
A61P031/00 |
Claims
1. A method of purifying an alkylaminomethyl minocycline compound,
comprising: a) injecting a low pH aqueous solution of said
alkylaminomethyl minocycline compound into a liquid chromatography
device in a polar organic solvent gradient, and combining the
product fractions; b) adjusting the pH of said product fractions to
4.0-4.5; c) washing said product fractions with a first non-polar
organic solvent to form a first organic layer and a first aqueous
layer, and discarding said first organic layer; d) adjusting the pH
of said first aqueous layer to 7.5-8.5; and e) washing said first
aqueous layer with a second non-polar organic solvent to form a
second organic layer and a second aqueous layer, and discarding
said second aqueous layer, such that said alkylaminomethyl
minocycline compound is purified.
2. The method of claim 1, wherein said low pH aqueous solution has
a pH of 2-3.
3. The method of claim 1, wherein said low pH aqueous solution
comprises methyl sulfonic acid.
4. The method of claim 1, wherein said polar organic solvent is
acetonitrile.
5. The method of claim 1, wherein said pH in step b) or d) is
adjusted with a base.
6. The method of claim 5, wherein said base is selected from the
group consisting of metal hydroxide, metal carbonate, metal
bicarbonate, ammonia, organic primary amine, organic secondary
amine and organic tertiary amine
7. The method of claim 6, and wherein said metal is selected from
the group consisting of lithium, sodium, potassium, calcium,
magnesium and aluminum.
8. The method of claim 6, wherein said base is sodium hydroxide or
ammonia.
9. The method of claim 1, wherein said first non-polar organic
solvent is methylene chloride.
10. The method of claim 1, wherein said first organic layer
comprises by-products, hydrophobic impurities and oxidative
degradents of said alkylaminomethyl minocycline compound.
11. The method of claim 1, wherein said second non-polar organic
solvent is methylene chloride.
12. The method of claim 1, wherein said second aqueous layer
comprises by-products and .beta. epimer of said alkylaminomethyl
minocycline compound.
13. The method of claim 1, wherein an antioxidant is added.
14. The method of claim 13, wherein said antioxidant is ammonium
sulfite, sodium sulfite, bisulfite or meta bisulfite.
15. The method of claim 1, wherein said alkylaminomethyl
minocycline compound is: ##STR00015## wherein R.sup.A is alkyl, and
R.sup.B is hydrogen or alkyl.
16. The method of claim 15, wherein R.sup.B is hydrogen.
17. The method of claim 16, wherein R.sup.A is alkyl.
18. The method of claim 17, wherein said alkyl is
(CH.sub.3).sub.3CCH.sub.2--.
19. The method of claim 1, wherein said alkylaminomethyl
minocycline compound is: ##STR00016##
20. The method of claim 1, wherein hydrophobic impurities and
oxidative degradents are removed from said alkylaminomethyl
minocycline compound.
21. The method of claim 1, wherein by-products and .beta.-C-4
epimer are removed from said alkylaminomethyl minocycline
compound.
22. The method of claim 1, wherein said alkylaminomethyl
minocycline compound is essentially free of hydrophobic impurities
and oxidative degradents.
23. The method of claim 1, wherein said alkylaminomethyl
minocycline compound is essentially free of by-products, .beta.-C-4
epimer, hydrophobic impurities and oxidative degradents.
24. The method of claim 1, wherein said alkylaminomethyl
minocycline compound comprises at least 50% .alpha.-C-4 epimer.
25. The method of claim 24, wherein said alkylaminomethyl
minocycline compound comprises at least 95% .alpha.-C-4 epimer.
26. The method of claim 25, wherein said alkylaminomethyl
minocycline compound comprises at least 99.9% .alpha.-C-4
epimer.
27. The method of claim 1, wherein said alkylaminomethyl
minocycline compound comprises less than 7% .beta.-C-4 epimer.
28. The method of claim 27, wherein said alkylaminomethyl
minocycline compound comprises less than 3% .beta.-C-4 epimer.
29. A pharmaceutical composition comprising alkylaminomethyl
minocycline compound purified by the method of claim 1 and a
pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 12/110,627, filed Apr. 28, 2008, pending,
which claims priority to U.S. Pro. Appl. No. 60/926,461; filed Apr.
27, 2007, each of which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] The development of the tetracycline antibiotics was the
direct result of a systematic screening of soil specimens collected
from many parts of the world for evidence of microorganisms capable
of producing bactericidal and/or bacteriostatic compositions. The
first of these novel compounds was introduced in 1948 under the
name chlortetracycline. Two years later, oxytetracycline became
available. The elucidation of the chemical structure of these
compounds confirmed their similarity and furnished the analytical
basis for the production of a third member of this group in 1952,
tetracycline. A new family of tetracycline compounds, without the
ring-attached methyl group present in earlier tetracyclines, was
prepared in 1957 and became publicly available in 1967; and
minocycline was in use by 1972.
[0003] Recently, research efforts have focused on developing new
tetracycline antibiotic compositions effective under varying
therapeutic conditions and routes of administration. New
tetracycline analogues have also been investigated which may prove
to be equal to or more effective than the originally introduced
tetracycline compounds. Examples include U.S. Pat. Nos. 2,980,584;
2,990,331; 3,062,717; 25 3,165,531; 3,454,697; 3,557,280;
3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These
patents are representative of the range of pharmaceutically active
tetracycline and tetracycline analogue compositions.
[0004] Historically, soon after their initial development and
introduction, the tetracyclines were found to be highly effective
pharmacologically against rickettsiae; a number of gram-positive
and gram negative bacteria; and the agents responsible for
lymphogranuloma venereum, inclusion conjunctivitis, and
psittacosis. Hence, tetracyclines became known as "broad spectrum"
antibiotics. With the subsequent establishment of their in vitro
antimicrobial activity, effectiveness in experimental infections,
and pharmacological properties, the tetracyclines as a class
rapidly became widely used for therapeutic purposes. However, this
widespread use of tetracyclines for both major and minor illnesses
and diseases led directly to the emergence of resistance to these
antibiotics even among highly susceptible bacterial species both
commensal and pathogenic (e.g., pneumococci and Salmonella). The
rise of tetracycline-resistant organisms has resulted in a general
decline in use of tetracyclines and tetracycline analogue
compositions as antibiotics of choice.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the invention pertains, at least in part,
to a method of synthesizing an aminoalkyl tetracycline compound.
The method includes contacting a tetracycline compound with an
N-hydroxymethyl-phthalimide in the presence of a water scavenger
and an acid under appropriate conditions, such that an aminomethyl
tetracycline intermediate compound is formed.
[0006] In another embodiment, the invention pertains, at least in
part, to a method for the synthesis of an aminoalkyl tetracycline
compound. The method includes: contacting a tetracycline compound
with a N-hydroxymethyl-phthalimide in the presence of a water
scavenger and an acid under appropriate conditions to form an
aminomethyl tetracycline intermediate compound; treating the
aminomethyl tetracycline intermediate compound with methylamine
under second appropriate conditions to form a second aminomethyl
tetracycline intermediate; and treating the second aminomethyl
tetracycline intermediate under appropriate hydrogenation
conditions, such that an aminomethyl tetracycline compound is
formed.
[0007] In another embodiment, the invention pertains, at least in
part, to a method of purifying alkylaminomethyl minocycline
compounds by injecting an aqueous low pH solution of the compound
into an HPLC in a polar organic solvent gradient, and combining the
product fractions containing the alkylaminomethyl minocycline
compound.
[0008] In yet another embodiment, the invention pertains, at least
in part, to a method of removing hydrophobic impurities and
oxidative degradents from an alkylaminomethyl minocycline compound.
The invention includes dissolving the minocycline compound in an
aqueous solution of a pH of 4.0-4.5, washing the aqueous solution
with a non-polar organic solvent, and retaining the aqueous
solution, such that hydrophobic impurities and oxidative degradents
are removed from the alkylaminomethyl minocycline compound.
[0009] In yet another embodiment, the invention also pertains, at
least in part, to a method of removing the .beta. epimer and by
products from an alkylaminomethyl minocycline compound. The method
includes dissolving the minocycline compounds in an aqueous
solution of a pH of 7.5-8.5, washing the aqueous solution with a
non-polar organic solvent, and retaining the organic solution, such
that the .beta. epimer and by products are removed from the
alkylaminomethyl minocycline compound.
[0010] In yet another embodiment, the invention includes
pharmaceutical compositions comprising a tetracycline compound
synthesized and/or purified by the methods of the invention and a
pharmaceutically acceptable carrier.
[0011] In yet another embodiment, the invention includes a method
for treating a tetracycline responsive state in a subject, by
administering to the subject an effective amount of a tetracycline
compound synthesized and/or purified by the methods of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
1. Methods for Synthesizing Amino-Methyl Tetracycline Compounds
[0012] In an embodiment, the invention pertains to an improved
synthesis of 4-.alpha.-9-amino methyl tetracyclines with low
4-.beta.-epimer content. Epimerization of tetracycline compounds at
the C-4 position has been a challenge for chemists working to
synthesize new tetracycline derivatives.
[0013] In one embodiment, method of synthesizing an aminoalkyl
tetracycline compound, comprising contacting a tetracycline
compound with a N-hydroxymethyl-phthalimide in the presence of an
acid and a water scavenger under appropriate conditions, such that
an aminomethyl tetracycline intermediate compound is formed.
[0014] The term "tetracycline compound" includes many compounds
with a similar ring structure to tetracycline. Examples of
tetracycline compounds include: tetracycline, chlortetracycline,
oxytetracycline, demeclocycline, methacycline, sancycline,
doxycycline, and minocycline. Other derivatives and analogues
comprising a similar four ring structure are also included. Table 1
depicts tetracycline and several known tetracycline derivatives.
The C-4 position is marked by an arrow.
TABLE-US-00001 TABLE I ##STR00001## ##STR00002## ##STR00003##
##STR00004##
[0015] The tetracycline compound may be substituted at any position
of the tetracycline ring. For example, the tetracycline compound
may further be substituted at the 1, 2, 3, 4, 5, 6, 7, 8, 10, 10a,
11, 11a, 12, 12a, and/or 13 position. The term tetracycline
compound also includes compounds of the formula (I):
##STR00005##
wherein
[0016] X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or
O;
[0017] 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;
[0018] R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety;
[0019] R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9
to form a ring;
[0020] 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;
[0021] 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;
[0022] R.sup.7 is hydrogen, halogen, nitro, alkyl, alkenyl,
heterocyclic, acyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,
arylalkynyl, or
--(CH.sub.2).sub.0-3(NR.sup.7c).sub.0-1C(.dbd.W')WR.sup.7a;
[0023] R.sup.9 is hydrogen;
[0024] W is CR.sup.7dR.sup.7e, S, NR.sup.7b or O;
[0025] W' is O, NR.sup.7f S;
[0026] R.sup.7a, R.sup.7b, R.sup.7c, R.sup.7d, R.sup.7e, and
R.sup.7f are each independently hydrogen, acyl, alkyl, alkenyl,
alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a
prodrug moiety;
[0027] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl;
[0028] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; and
[0029] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl, and pharmaceutically acceptable salts thereof.
[0030] In a further embodiment, the tetracycline compound is
minocycline. The structure of minocycline is shown below:
##STR00006##
[0031] The term "N-hydroxymethyl phthalimide" includes compounds of
the formula:
##STR00007##
[0032] wherein
[0033] R.sup.1 and R.sup.2 are each hydrogen, alkyl, halogen,
alkenyl, alkynyl, aryl, cyano, amino, amidino, alkoxy, and acyl. In
a further embodiment, R.sup.1 and R.sup.2 are each alkyl or
hydrogen.
[0034] The term "acid" includes organic acids such as triflic acid,
methane sulfonic acid, and fuming sulfuric acid. Other acids known
in the art are also included such as hydrochloric, sulfuric,
phosphoric, hydrobromic, etc.
[0035] The term "water scavenger" includes agents which remove
moisture from the reaction. These agents include acid chlorides and
acid anhydrides (e.g., methyl sulfonic anhydride).
[0036] In certain embodiments, the reaction is conducted at a
temperature of about 20-25.degree. C. In certain embodiments, the
reaction is stirred for about 1-2 hours after the addition of a
first portion of N-hydroxymethylphthalimide. An additional portion
of N-hydroxyphthalimide may be added, keeping the temperature
between 20-35.degree. C. The reaction may be continued until HPLC
analysis of an aliquot of the reaction confirms that less than 4
area percent of mono-alkylated minocycline is remaining. The
reaction conditions may further comprise a work up of adding the
solution to an ice chilled flask of water, where the temperature is
kept below 25.degree. C. The product may be filtered, washed with
water, and brought to a neutral pH. In a further embodiment, the
appropriate conditions include an inert atmosphere (e.g., nitrogen
or argon). In addition, the aminomethyl tetracycline intermediate
may further be dissolved in acetone or acetonitrile to effect a
more precise neutralization.
[0037] In another further embodiment, the resulting aminomethyl
tetracycline intermediate comprises a 9:1 ratio of bis substituted
(e.g., substituted at the 2 and 9 position) to tris substituted
(e.g., substituted at the 2, 9 and 10 position). In a further
embodiment, the amount of bis substituted amino methyl tetracycline
compound is greater than about 50%, greater than about 60%, greater
than about 70%, greater than about 80%, or greater than about
90%.
[0038] In a further embodiment, the aminomethyl tetracycline
intermediate compound may be of the formula:
##STR00008##
[0039] The aminomethyl tetracycline intermediate may be further
treated with methylamine to form an aminomethyl tetracycline
compound. The treatment with methylamine may be performed in an
alkyl alcohol (e.g., EtOH, MeOH, etc.) solvent. The phthalamide
by-product which forms may be removed by precipitation and the
product may be precipitated from a mixture of cyclic/alkyl ether
and alkyl alcohol in a ratio of about 1.5:1. In a further
embodiment, the appropriate amount of the alkyl alcohol is an
amount sufficient to prevent the precipitation of an aminomethyl
tetracycline intermediate from the reaction.
[0040] In a further embodiment, the invention pertains to a method
for the synthesis of an aminoalkyl tetracycline compound. The
method includes contacting a tetracycline compound with a
N-hydroxymethyl-phthalimide in the presence of an acid and a water
scavenger under appropriate conditions to form an aminomethyl
tetracycline intermediate compound; treating said aminomethyl
tetracycline intermediate compound with methylamine under second
appropriate conditions to form a second aminomethyl tetracycline
intermediate; treating the second aminomethyl tetracycline
intermediate under appropriate hydrogenation conditions, such that
an amino methyltetracycline compound is formed. In a further
embodiment, the tetracycline compound is minocycline.
[0041] In a further embodiment, the second appropriate conditions
include dissolving the aminomethyl tetracycline intermediate
compound in cyclic or alkyl ether and an appropriate amount of an
alkyl alcohol. The alkyl alcohol may be used, for example, to
selectively precipitate the by products and the cyclic or alkyl
ether may be used to selectively precipitate the aminoalkyl
tetracycline intermediate.
[0042] The resulting second aminomethyl tetracycline intermediate
can then be isolated as a free base. Different salts of the free
base can also be formed to effect a purification. Examples of acids
which can be used to convert the free base to a salt include, but
are not limited to, HCl, trifluoroacetic acid, methylsulfonic acid,
and acetic acid. For example, when precipitating a hydrochloride
salt, hydrochloric acid in isopropanol is added to a suspension of
the compound in methanol to a pH of about 3.0. The mixture is
stirred and filtered if necessary to remove insoluble constituents.
The salt of the second aminomethyl tetracycline may be precipitated
with t-butylmethylether and isolated by filtration. The salts of
the second aminomethyl tetracycline intermediates may then directly
undergo hydrogenation.
[0043] Examples of second aminomethyl tetracycline intermediates
include compounds of the formula:
##STR00009##
[0044] In a further embodiments, the appropriate hydrogenation
conditions include transferring the second aminomethyl tetracycline
intermediate to a hydrogenation flask and charging the flask with
Pd/C or Pd/C/S either wet or as a dry powder. The reaction may be
conducted using solvent such as methanol and an aldehyde.
Appropriate hydrogenation conditions also include the use of
hydrogen gas, which may be incorporated by conducting the reaction
under a pressure of about 30 psi. The hydrogenation may occur for
an appropriate length of time, such as about 24 hours. An example
of an amino methyl tetracycline compound is:
##STR00010##
[0045] In a further embodiment, the aminomethyl tetracycline
compound is contacted with an aldehyde or ketone under appropriate
conditions, such that a substituted aminomethyl tetracycline
compound is formed. Examples of substituted aminomethyl
tetracycline compounds, include compounds of the formula:
##STR00011##
[0046] wherein R.sup.A and R.sup.B are each independently hydrogen,
alkyl, alkenyl, alkynyl or aryl.
[0047] In one embodiment, R.sup.B is hydrogen and R.sup.A is alkyl
(e.g., (CH.sub.3).sub.3CCH.sub.2--). In another embodiment, the
substituted aminomethyl tetracycline compound is:
##STR00012##
[0048] The 4-.alpha. and the 4-.beta. epimer of an aminomethyl
minocycline compound are shown below. The arrow marks the C-4
position of the tetracycline ring system.
##STR00013##
[0049] The term "epimeric purity" refers to the % of the
tetracycline compounds (e.g., a tetracycline compound of the
invention, (e.g., a substituted aminoalkyl tetracycline compound,
an aminomethyl tetracycline compound, or an alkylaminomethyl
minocycline compound) in a given sample with a particular desired
epimeric configuration. In one embodiment, the epimeric purity of a
tetracycline compound of the invention is greater than 95% of the
.alpha.-epimer of a tetracycline compound at the C-4 position. In a
further embodiment, the epimeric purity of the tetracycline
compound is at least 96%, at least 97%, at least 98%, at least 99%,
at least 99.5%, or at least 99.9% the .alpha.-epimer at the C-4
position of the tetracycline compound.
[0050] Preferably, the substituted aminoalkyl tetracycline
compound,aminomethyl tetracycline compound, or alkylaminomethyl
minocycline compound of the invention comprises mostly (e.g., at
least 50%) .alpha.-C-4 epimer. In a further embodiment, the
compound is about 60% .alpha.-C-4 epimer, about 70% .alpha.-C-4
epimer, about 80% .alpha.-C-4 epimer, about 90% .alpha.-C-4 epimer,
at least about 95% .alpha.-C-4 epimer, or at least about 97%
.alpha.-C-4 epimer. In a further embodiment, the substituted
aminoalkyltetracycline compound, aminomethyl tetracycline compound,
or alkylaminomethyl minocycline compound of the invention comprises
less than about 7% .beta.-C-4 epimer, less than about 5% .beta.-C-4
epimer, or less than about 3% .alpha.-C-4 epimer.
[0051] The epimeric purity of a particular tetracycline compound
made by the methods of the invention can be determined by using
methods known in the art. For example, epimeric purity can be
determined by HPLC or high field NMR.
[0052] In one embodiment, any hydrophobic impurities and oxidative
degradents of the aminoalkyl tetracycline compound, aminomethyl
tetracycline compound, or alkylaminomethyl minocycline compound of
the invention (e.g., late-eluting hydrophobic impurities such as
4-carbonyl by products and other oxidative degradents from the
acidic aqueous solution of particular compound of the invention)
may be removed by washing the aqueous solution with a non-polar
organic solvent (e.g., CH.sub.2Cl.sub.2). The organic layers may be
discarded and the aqueous layers may then be combined and
retained.
2. Methods for the Isolation and Purification of 9-Alkyl Amino
Methyl Tetracycline Compounds
[0053] In another embodiment, the invention pertains to methods of
purifying 9-alkyl amino methyl tetracyclines (or any tetracycline
compound capable of being purified by the present methods) from
impurities, .beta.-epimer, and by products.
[0054] In one embodiment, the invention pertains to a method of
purifying alkylaminomethyl minocycline compounds using
chromatography. The method includes injecting an aqueous low pH
solution of the compound into an HPLC in a polar organic solvent
gradient, and combining the product fractions, such that the
alkylaminomethyl minocycline compound is purified.
[0055] It has been found that selection of suitable acidic mobile
phases enhances process stability and selectivity. Organic and
mineral acid mobile phases may be effective for separating
by-products including epimer impurities and closely-eluting by
products through pH control or choice of acid. Acidic mobile phases
may also protect against oxidative degradation of the minocycline
compound.
[0056] In a further embodiment, the low pH solution has a pH of
between about 2-3. Examples of solutions that may be used include
0.1% aqueous solutions of methane sulfonic acid. In certain
embodiments, an isocratic gradient of 94% of the aqueous solution
and 6% acetonitrile or another polar organic solvent may be used to
purify the minocycline compound from epimeric and closely eluting
by-products.
[0057] The resulting aqueous product fractions may be combined and
the pH may be adjusted to between about 4.0-4.5 using a base (e.g.,
NaOH). Hydrophobic impurities and oxidative degradents of the
minocycline compound may be removed by washing the aqueous solution
with a non-polar organic solvent (e.g., CH.sub.2Cl.sub.2). The
organic layers may then be discarded and the aqueous layers are
combined and retained.
[0058] It should be noted that the organic solvents, such as
methylene chloride, may be used to selectively remove late-eluting
hydrophobic impurities such as 4-carbonyl by products and other
oxidative degradents from the acidic aqueous solution of the
minocycline compound.
[0059] The pH of the combined aqueous layers may then be adjusted
to neutral pH, e.g., about 7.5 to about 8.5. The pH may be adjusted
by the addition of a base, such as NaOH. The neutral solution is
then washed with a non-polar organic solvent, such as methylene
chloride. It should be noted that selective pH adjustment to
neutral pH ranges also allows the minocycline compound to be
extracted into the organic solvent while retaining undesired
.beta.-epimer and by products are dissolved the aqueous phase.
[0060] In addition, antioxidants may also be added to the aqueous
solutions of minocycline compounds described herein. The
antioxidants may be provided to prevent oxidative degradation of
the minocycline compounds. Suitable antioxidants include, for
example, sulfites (e.g., meta bisulfite, bisulfite, ammonium
sulfite, etc.), citric acid, etc.
[0061] 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.
[0062] 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.
[0063] The term "aryl" includes groups, including 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, phenyl, pyrrole, furan,
thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole,
pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine, and the like. Furthermore, the term "aryl" includes
multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g.,
naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, napthridine, indole, benzofuran, purine, benzofuran,
deazapurine, or indolizine. Those aryl groups having heteroatoms in
the ring structure may also be referred to as "aryl heterocycles",
"heterocycles," "heteroaryls" or "heteroaromatics". The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Aryl groups can also be fused or bridged with alicyclic or
heterocyclic rings which are not aromatic so as to form a polycycle
(e.g., tetralin).
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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 "alkylaminocarboxy,"
"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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0083] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.- X.sup.+, where X.sup.+ is a counter ion.
[0084] 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.
[0085] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0086] 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.
[0087] The term "prodrug moiety" includes moieties which can be
metabolized in vivo to a hydroxyl group and moieties which may
advantageously remain esterified in vivo. Preferably, the prodrugs
moieties are metabolized in vivo by esterases or by other
mechanisms to hydroxyl groups or other advantageous groups.
Examples of prodrugs and their uses are well known in the art (See,
e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19). The prodrugs can be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent. Hydroxyl groups can be converted
into esters via treatment with a carboxylic acid. Examples of
prodrug moieties include substituted and unsubstituted, branch or
unbranched lower alkyl ester moieties, (e.g., propionoic acid
esters), lower alkenyl esters, di-lower 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.
3. Pharmaceutical Compositions Comprising Tetracycline Compounds of
the Invention
[0088] In a further embodiment, the invention pertains to
pharmaceutical compositions comprising a tetracycline compound of
the invention (e.g., synthesized, or purified by the methods of the
invention) or a pharmaceutically acceptable salt, prodrug or ester
thereof. The pharmaceutical compositions may comprise a
pharmaceutically acceptable carrier.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal 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 per cent, this amount will range from about 1 per cent to
about ninety-nine percent of active ingredient, preferably from
about 5 per cent to about 70 per cent, most preferably from about
10 per cent to about 30 per cent.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0108] 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.
[0109] 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.
[0110] 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 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.
[0111] 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.
[0112] 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.
[0113] 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 is
preferred.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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, when used for the indicated analgesic
effects, 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. 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.
[0122] 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.
[0123] 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).
[0124] 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.
[0125] 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.).
[0126] The invention also pertains to tetracycline compounds, which
are synthesized and/or purified by the methods of the invention,
and pharmaceutically acceptable salts thereof.
4. Methods of Using the Tetracycline Compounds of the Invention
[0127] The invention also pertains to a method for treating a
tetracycline responsive state in a subject, by administering to the
subject an effective amount of a tetracycline compound synthesized
and/or purified by the method the invention or a pharmaceutically
acceptable salt thereof, such that the state is treated.
[0128] The term "treating" includes curing as well as ameliorating
at least one symptom of the state, disease or disorder, e.g., the
tetracycline compound responsive state.
[0129] The language "tetracycline compound responsive state" or
"tetracycline responsive state" includes states which can be
treated, prevented, or otherwise ameliorated by the administration
of a tetracycline compound of the invention. Tetracycline compound
responsive states include bacterial, viral, and fungal infections
(including those which are resistant to other tetracycline
compounds), cancer (e.g., prostate, breast, colon, lung melanoma
and lymph cancers and other disorders characterized by unwanted
cellular proliferation, including, but not limited to, those
described in U.S. Pat. No. 6,100,248), arthritis, osteoporosis,
diabetes, and other states for which tetracycline compounds have
been found to be active (see, for example, U.S. Pat. Nos.
5,789,395; 5,834,450; 6,277,061 and 5,532,227, each of which is
expressly incorporated herein by reference). Compounds of the
invention can be used to prevent or control important mammalian and
veterinary diseases such as diarrhea, urinary tract infections,
infections of skin and skin structure, ear, nose and throat
infections, wound infection, mastitis and the like. In addition,
methods for treating neoplasms using tetracycline compounds of the
invention are also included (van der Bozert et al., Cancer Res.,
48:6686-6690 (1988)). In a further embodiment, the tetracycline
responsive state is not a bacterial infection. In another
embodiment, the tetracycline compounds of the invention are
essentially non-antibacterial. For example, non-antibacterial
tetracycline compounds of the invention may have MIC values greater
than about 4 .mu.g/ml (as measured by assays known in the art
and/or the assay given in Example 2).
[0130] Tetracycline compound responsive states also include
inflammatory process associated states (IPAS). The term
"inflammatory process associated state" includes states in which
inflammation or inflammatory factors (e.g., matrix
metalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins,
plasma proteins, cellular defense systems, cytokines, lipid
metabolites, proteases, toxic radicals, adhesion molecules, etc.)
are involved or are present in an area in aberrant amounts, e.g.,
in amounts which may be advantageous to alter, e.g., to benefit the
subject. The inflammatory process is the response of living tissue
to damage. The cause of inflammation may be due to physical damage,
chemical substances, micro-organisms, tissue necrosis, cancer or
other agents. Acute inflammation is short-lasting, lasting only a
few days. If it is longer lasting however, then it may be referred
to as chronic inflammation.
[0131] IPAF's include inflammatory disorders. Inflammatory
disorders are generally characterized by heat, redness, swelling,
pain and loss of function. Examples of causes of inflammatory
disorders include, but are not limited to, microbial infections
(e.g., bacterial and fungal infections), physical agents (e.g.,
burns, radiation, and trauma), chemical agents (e.g., toxins and
caustic substances), tissue necrosis and various types of
immunologic reactions.
[0132] Examples of inflammatory disorders include, but are not
limited to, osteoarthritis, rheumatoid arthritis, acute and chronic
infections (bacterial and fungal, including diphtheria and
pertussis); acute and chronic bronchitis, sinusitis, and upper
respiratory infections, including the common cold; acute and
chronic gastroenteritis and colitis; acute and chronic cystitis and
urethritis; acute and chronic dermatitis; acute and chronic
conjunctivitis; acute and chronic serositis (pericarditis,
peritonitis, synovitis, pleuritis and tendinitis); uremic
pericarditis; acute and chronic cholecystis; acute and chronic
vaginitis; acute and chronic uveitis; drug reactions; insect bites;
burns (thermal, chemical, and electrical); and sunburn.
[0133] Tetracycline compound responsive states also include NO
associated states. The term "NO associated state" includes states
which involve or are associated with nitric oxide (NO) or inducible
nitric oxide synthase (iNOS). NO associated state includes states
which are characterized by aberrant amounts of NO and/or iNOS.
Preferably, the NO associated state can be treated by administering
tetracycline compounds of the invention. The disorders, diseases
and states described in U.S. Pat. Nos. 6,231,894; 6,015,804;
5,919,774; and 5,789,395 are also included as NO associated states.
The entire contents of each of these patents are hereby
incorporated herein by reference.
[0134] Other examples of NO associated states include, but are not
limited to, malaria, senescence, diabetes, vascular stroke,
neurodegenerative disorders (Alzheimer's disease & Huntington's
disease), cardiac disease (reperfusion-associated injury following
infarction), juvenile diabetes, inflammatory disorders,
osteoarthritis, rheumatoid arthritis, acute, recurrent and chronic
infections (bacterial, viral and fungal); acute and chronic
bronchitis, sinusitis, and respiratory infections, including the
common cold; acute and chronic gastroenteritis and colitis; acute
and chronic cystitis and urethritis; acute and chronic dermatitis;
acute and chronic conjunctivitis; acute and chronic serositis
(pericarditis, peritonitis, synovitis, pleuritis and tendonitis);
uremic pericarditis; acute and chronic cholecystis; cystic
fibrosis, acute and chronic vaginitis; acute and chronic uveitis;
drug reactions; insect bites; burns (thermal, chemical, and
electrical); and sunburn.
[0135] The term "inflammatory process associated state" also
includes, in one embodiment, matrix metalloproteinase associated
states (MMPAS). MMPAS include states characterized by aberrant
amounts of MMPs or MMP activity. These are also include as
tetracycline compound responsive states which may be treated using
compounds of the invention.
[0136] Examples of matrix metalloproteinase associated states
("MMPAS's") include, but are not limited to, arteriosclerosis,
corneal ulceration, emphysema, osteoarthritis, multiple sclerosis
(Liedtke et al., Ann. Neurol. 1998, 44:35-46; Chandler et al., J.
Neuroimmunol. 1997, 72:155-71), osteosarcoma, osteomyelitis,
bronchiectasis, chronic pulmonary obstructive disease, skin and eye
diseases, periodontitis, osteoporosis, rheumatoid arthritis,
ulcerative colitis, inflammatory disorders, tumor growth and
invasion (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993,
9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987,
907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),
metastasis, acute lung injury, stroke, ischemia, diabetes, aortic
or vascular aneurysms, skin tissue wounds, dry eye, bone and
cartilage degradation (Greenwald et al., Bone 1998, 22:33-38; Ryan
et al., Curr. Op. Rheumatol. 1996, 8;238-247). Other MMPAS include
those described in U.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839;
5,258,371; 4,935,412; 4,704,383, 4,666,897, and RE 34,656,
incorporated herein by reference in their entirety.
[0137] In another embodiment, the tetracycline compound responsive
state is cancer. Examples of cancers which the tetracycline
compounds of the invention may be useful to treat include all solid
tumors, i.e., carcinomas e.g., adenocarcinomas, and sarcomas.
Adenocarcinomas are carcinomas derived from glandular tissue or in
which the tumor cells form recognizable glandular structures.
Sarcomas broadly include tumors whose cells are embedded in a
fibrillar or homogeneous substance like embryonic connective
tissue. Examples of carcinomas which may be treated using the
methods of the invention include, but are not limited to,
carcinomas of the prostate, breast, ovary, testis, lung, colon, and
breast. The methods of the invention are not limited to the
treatment of these tumor types, but extend to any solid tumor
derived from any organ system. Examples of treatable cancers
include, but are not limited to, colon cancer, bladder cancer,
breast cancer, melanoma, ovarian carcinoma, prostatic carcinoma,
lung cancer, and a variety of other cancers as well. The methods of
the invention also cause the inhibition of cancer growth in
adenocarcinomas, such as, for example, those of the prostate,
breast, kidney, ovary, testes, and colon.
[0138] In an embodiment, the tetracycline responsive state of the
invention is cancer. The invention pertains to a method for
treating a subject suffering or at risk of suffering from cancer,
by administering an effective amount of a substituted tetracycline
compound, such that inhibition cancer cell growth occurs, i.e.,
cellular proliferation, invasiveness, metastasis, or tumor
incidence is decreased, slowed, or stopped. The inhibition may
result from inhibition of an inflammatory process, down-regulation
of an inflammatory process, some other mechanism, or a combination
of mechanisms. Alternatively, the tetracycline compounds may be
useful for preventing cancer recurrence, for example, to treat
residual cancer following surgical resection or radiation therapy.
The tetracycline compounds useful according to the invention are
especially advantageous as they are substantially non-toxic
compared to other cancer treatments. In a further embodiment, the
compounds of the invention are administered in combination with
standard cancer therapy, such as, but not limited to,
chemotherapy.
[0139] Examples of tetracycline responsive states also include
neurological disorders which include both neuropsychiatric and
neurodegenerative disorders, but are not limited to, such as
Alzheimer's disease, dementias related to Alzheimer's disease (such
as Pick's disease), Parkinson's and other Lewy diffuse body
diseases, senile dementia, Huntington's disease, Gilles de la
Tourette's syndrome, multiple sclerosis, amylotrophic lateral
sclerosis (ALS), progressive supranuclear palsy, epilepsy, and
Creutzfeldt-Jakob disease; autonomic function disorders such as
hypertension and sleep disorders, and neuropsychiatric disorders,
such as depression, schizophrenia, schizoaffective disorder,
Korsakoff's psychosis, mania, anxiety disorders, or phobic
disorders; learning or memory disorders, e.g., amnesia or
age-related memory loss, attention deficit disorder, dysthymic
disorder, major depressive disorder, mania, obsessive-compulsive
disorder, psychoactive substance use disorders, anxiety, phobias,
panic disorder, as well as bipolar affective disorder, e.g., severe
bipolar affective (mood) disorder (BP-1), bipolar affective
neurological disorders, e.g., migraine and obesity. Further
neurological disorders include, for example, those listed in the
American Psychiatric Association's Diagnostic and Statistical
manual of Mental Disorders (DSM), the most current version of which
is incorporated herein by reference in its entirety.
[0140] The language "in combination with" another therapeutic agent
or treatment includes co-administration of the tetracycline
compound, (e.g., inhibitor) and with the other therapeutic agent or
treatment, administration of the tetracycline compound first,
followed by the other therapeutic agent or treatment and
administration of the other therapeutic agent or treatment first,
followed by the tetracycline compound. The other therapeutic agent
may be any agent which is known in the art to treat, prevent, or
reduce the symptoms of an IPAS. Furthermore, the other therapeutic
agent may be any agent of benefit to the patient when administered
in combination with the administration of an tetracycline compound.
In one embodiment, the cancers treated by methods of the invention
include those described in U.S. Pat. Nos. 6,100,248; 5,843,925;
5,837,696; or 5,668,122, incorporated herein by reference in their
entirety.
[0141] In another embodiment, the tetracycline compound responsive
state is diabetes, e.g., juvenile diabetes, diabetes mellitus,
diabetes type I, or diabetes type II. In a further embodiment,
protein glycosylation is not affected by the administration of the
tetracycline compounds of the invention. In another embodiment, the
tetracycline compound of the invention is administered in
combination with standard diabetic therapies, such as, but not
limited to insulin therapy. In a further embodiment, the IPAS
includes disorders described in U.S. Pat. Nos. 5,929,055; and
5,532,227, incorporated herein by reference in their entirety.
[0142] In another embodiment, the tetracycline compound responsive
state is a bone mass disorder. Bone mass disorders include
disorders where a subjects bones are disorders and states where the
formation, repair or remodeling of bone is advantageous. For
examples bone mass disorders include osteoporosis (e.g., a decrease
in bone strength and density), bone fractures, bone formation
associated with surgical procedures (e.g., facial reconstruction),
osteogenesis imperfecta (brittle bone disease), hypophosphatasia,
Paget's disease, fibrous dysplasia, osteopetrosis, myeloma bone
disease, and the depletion of calcium in bone, such as that which
is related to primary hyperparathyroidism. Bone mass disorders
include all states in which the formation, repair or remodeling of
bone is advantageous to the subject as well as all other disorders
associated with the bones or skeletal system of a subject which can
be treated with the tetracycline compounds of the invention. In a
further embodiment, the bone mass disorders include those described
in U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE
34,656; 5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of
which is hereby incorporated herein by reference in its
entirety.
[0143] In another embodiment, the tetracycline compound responsive
state is acute lung injury. Acute lung injuries include adult
respiratory distress syndrome (ARDS), post-pump syndrome (PPS), and
trauma. Trauma includes any injury to living tissue caused by an
extrinsic agent or event. Examples of trauma include, but are not
limited to, crush injuries, contact with a hard surface, or cutting
or other damage to the lungs.
[0144] The invention also pertains to a method for treating acute
lung injury by administering a substituted tetracycline compound of
the invention.
[0145] The tetracycline responsive states of the invention also
include chronic lung disorders. The invention pertains to methods
for treating chronic lung disorders by administering a tetracycline
compound, such as those described herein. The method includes
administering to a subject an effective amount of a substituted
tetracycline compound such that the chronic lung disorder is
treated. Examples of chronic lung disorders include, but are not
limited, to asthma, cystic fibrosis, and emphysema. In a further
embodiment, the tetracycline compounds of the invention used to
treat acute and/or chronic lung disorders such as those described
in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and 5,773,430,
each of which is hereby incorporated herein by reference in its
entirety.
[0146] In yet another embodiment, the tetracycline compound
responsive state is ischemia, stroke, or ischemic stroke. The
invention also pertains to a method for treating ischemia, stroke,
or ischemic stroke by administering an effective amount of a
substituted tetracycline compound of the invention. In a further
embodiment, the tetracycline compounds of the invention are used to
treat such disorders as described in U.S. Pat. Nos. 6,231,894;
5,773,430; 5,919,775 or 5,789,395, incorporated herein by
reference.
[0147] In another embodiment, the tetracycline compound responsive
state is a skin wound. The invention also pertains, at least in
part, to a method for improving the healing response of the
epithelialized tissue (e.g., skin, mucusae) to acute traumatic
injury (e.g., cut, burn, scrape, etc.). The method may include
using a tetracycline compound of the invention (which may or may
not have antibacterial activity) to improve the capacity of the
epithelialized tissue to heal acute wounds. The method may increase
the rate of collagen accumulation of the healing tissue. The method
may also decrease the proteolytic activity in the epthithelialized
tissue by decreasing the collagenolytic and/or gellatinolytic
activity of MMPs. In a further embodiment, the tetracycline
compound of the invention is administered to the surface of the
skin (e.g., topically). In a further embodiment, the tetracycline
compound of the invention used to treat a skin wound, and other
such disorders as described in, for example, U.S. Pat. Nos.
5,827,840; 4,704,383; 4,935,412; 5,258,371; 5,308,839; 5,459,135;
5,532,227; and 6,015,804; each of which is incorporated herein by
reference in its entirety.
[0148] In yet another embodiment, the tetracycline compound
responsive state is an aortic or vascular aneurysm in vascular
tissue of a subject (e.g., a subject having or at risk of having an
aortic or vascular aneurysm, etc.). The tetracycline compound may
by effective to reduce the size of the vascular aneurysm or it may
be administered to the subject prior to the onset of the vascular
aneurysm such that the aneurysm is prevented. In one embodiment,
the vascular tissue is an artery, e.g., the aorta, e.g., the
abdominal aorta. In a further embodiment, the tetracycline
compounds of the invention are used to treat disorders described in
U.S. Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by
reference in their entirety.
[0149] Bacterial infections may be caused by a wide variety of gram
positive and gram negative bacteria. The compounds of the invention
are useful as antibiotics against organisms which are resistant to
other tetracycline compounds. The antibiotic activity of the
tetracycline compounds of the invention may be determined using the
in vitro standard broth dilution method described in Waitz, J. A.,
National Commission for Clinical Laboratory Standards, Document
M7-A2, vol. 10, no. 8, pp. 13-20, 2.sup.nd edition, Villanova, Pa.
(1990).
[0150] The tetracycline compounds may also be used to treat
infections traditionally treated with tetracycline compounds such
as, for example, rickettsiae; a number of gram-positive and
gram-negative bacteria; and the agents responsible for
lymphogranuloma venereum, inclusion conjunctivitis, psittacosis.
The tetracycline compounds may be used to treat infections of,
e.g., K. pneumoniae, Salmonella, E. hirae, A. baumanii, B.
catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E. coli, S.
aureus or E. faecalis. In one embodiment, the tetracycline compound
is used to treat a bacterial infection that is resistant to other
tetracycline antibiotic compounds. The tetracycline compound of the
invention may be administered with a pharmaceutically acceptable
carrier.
[0151] The language "effective amount" of the compound is that
amount necessary or sufficient to treat or prevent a tetracycline
compound responsive state. The effective amount can vary depending
on such factors as the size and weight of the subject, the type of
illness, or the particular tetracycline compound. For example, the
choice of the tetracycline compound can affect what constitutes an
"effective amount". One of ordinary skill in the art would be able
to study the aforementioned factors and make the determination
regarding the effective amount of the tetracycline compound without
undue experimentation.
[0152] In the therapeutic methods of the invention, one or more
tetracycline compounds of the invention may be administered alone
to a subject, or more typically a compound of the invention will be
administered as part of a pharmaceutical composition in mixture
with conventional excipient, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
oral or other desired administration and which do not deleteriously
react with the active compounds and are not deleterious to the
recipient thereof.
[0153] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
Exemplification of the Invention
Example 1
Synthesis of 9-Alkyl Aminomethyl Minocycline
##STR00014##
[0155] Minocycline hydrochloride (compound 2) was dissolved in
methylsulfonic acid or hydrofluoric acid with methylsulfonic
anhydride. N-hydroxymethyl phthalimide was added to the reaction
mixture. The mixture was stirred at 20-35 C until the reaction was
complete. The acid solution was added to an ice/water mixture and
the triflic salt was readily precipitated, filtered and collected.
The salt was re-dissolved in acetone and brought to a neutral pH
with base. The product was precipitated by the addition of water.
The product was isolated as a mixture of the bis and tris alkylated
product. The isolated material of this reaction was enriched in the
desired bis ratio (90%).
[0156] The solid was suspended in the EtOH Aminolysis was carried
out by using methylamine A phthalamide by-product precipitated as
the reaction progressed and was removed by filtration. The light
yellow solid product was precipitated out by the addition of about
1.5 volumes of t-butylmethylether to the reaction mixture, and
collected through a simple filtration that left many small
impurities and methylamine reagent in the solution. Further
purification of the compound was performed through re-slurrying
with methanol.
[0157] Compound 4 as freebase was transferred to a hydrogenation
vessel which was charged with methanol and aldehyde. An inactivated
Pd/C catalyst was charged and the vessel was pressurized with
hydrogen gas. The reaction mixture was hydrogenated under hydrogen
pressure around 30 Psi for about 24 hours. When conversion of
compound 4 to 1 was complete, the solution was filtered and washed
through a Celite pad. At this point the reaction mixture contained
very low .beta. C-4 epimer, around 3-7%.
[0158] The product (1) was worked up and isolated selectively from
its impurities. The pH of the solution was adjusted to about 4.5
with concentrated HCl and the solution was washed with
dichloromethane. Sulfites were added to the aqueous layer and the
product was extracted with dichloromethane at pH of about 7 to 8 to
selectively recover the preferred epimer product (e.g., .alpha.).
The dichloromethane layers were combined and concentrated, and 2 L
of n-heptane was added to precipitate the product. Further
purification was obtained by repeating the work-up procedure with
or without t-butylmethylether to dissolve the crude product.
Example 2
Isolation and Purification of an 9-Alkyl Aminomethyl Minocycline
Compound
[0159] Crude 9-(2',2'-dimethylpropyl aminomethyl) minocycline
freebase (40 g) was dissolved in 150 mL of buffer A (0.1% aqueous
solution of methanesulfonic acid--MSA) and the pH was adjusted to
2-3 with MSA.
[0160] The solution was filtered and injected into an HPLC and the
product was eluted with an isocratic gradient of 94% buffer A and
6% acetonitrile. The product fraction collection was initiated when
the product peak was detected. Each fraction was analyzed and an
acceptance criterion of greater than 80% AUC of the main peak was
used for the early product fractions. When combining fractions, the
level of impurities and relative concentration of the pooled
fractions was factored into the selection criteria that meets the
final product specifications. To the product fractions was added a
10% aqueous solution of sodium sulfite equal to 10% of the original
volume of the collected fractions.
[0161] A product fraction volume of 3.5 liters (including sodium
sulfite) was collected and the pH was adjusted to 4.0-4.5 using a
solution of sodium hydroxide. The aqueous solution was washed with
2 liters of dichloromethane and the organic layer was separated and
discarded.
[0162] The pH of the aqueous layer was adjusted to 7.5-8.5 using
sodium hydroxide and the product was extracted four times with 2.4
liters of dichloromethane. The pH was readjusted to 7.5 to 8.5 with
sodium hydroxide, prior to each extraction.
[0163] The four dichloromethane layers were combined and
concentrated to about 200 ml, which was then added slowly (over a
period of about 10 minutes) to a vigorously stirred n-heptane (2.5
L). The suspension was stirred for about 10 minutes at room
temperature and diluted slowly (over a period of 5 minutes) with
n-heptane 1.5 L. The slurry was cooled to 0-5.degree. C. and
stirred for 1-2 hours. The suspended solid was filtered and washed
with 3.times.150 mL portions of n-heptane. The product was dried
under vacuum at 40.degree. C. for at least 24 hours until a
constant weight was achieved and the levels of all residual
solvents were within specification. Approximately 13.6 g of
9-(2',2'-dimethylpropyl aminomethyl) minocycline freebase was
isolated as a yellow solid.
[0164] The off-cuts were isolated in a similar manner and yielded
1.64 g.
Equivalents
[0165] 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.
[0166] All patents, patent applications, and literature references
cited herein are hereby expressly incorporated by reference.
* * * * *