U.S. patent application number 11/679744 was filed with the patent office on 2007-06-28 for tetracycline derivatives and methods of use thereof.
Invention is credited to Richard J. Ablin, Joseph J. Hlavka.
Application Number | 20070149488 11/679744 |
Document ID | / |
Family ID | 23276795 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070149488 |
Kind Code |
A1 |
Hlavka; Joseph J. ; et
al. |
June 28, 2007 |
Tetracycline Derivatives and Methods of Use Thereof
Abstract
A treatment for inhibiting microbial or tumor growth is
disclosed, which comprises adding to a subject in need of treatment
an effective amount of one or more tetracycline derivatives.
Inventors: |
Hlavka; Joseph J.; (Tuxedo
Park, NY) ; Ablin; Richard J.; (Ringwood,
NJ) |
Correspondence
Address: |
DIAMOND LAW OFFICE LLC
1605 JOHN STREET, SUITE 102
FORT LEE
NJ
07024
US
|
Family ID: |
23276795 |
Appl. No.: |
11/679744 |
Filed: |
February 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10264454 |
Oct 4, 2002 |
7214669 |
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11679744 |
Feb 27, 2007 |
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60327502 |
Oct 5, 2001 |
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Current U.S.
Class: |
514/152 |
Current CPC
Class: |
C07C 237/26 20130101;
A61P 35/00 20180101; A61P 31/04 20180101; A61K 31/65 20130101; C07C
2603/46 20170501 |
Class at
Publication: |
514/152 |
International
Class: |
A61K 31/65 20060101
A61K031/65 |
Claims
1-6. (canceled)
7. A method for treating breast cancer, melanoma, myeloma or
prostate cancer, the method comprising: administering a compound or
a salt thereof to a subject in need of treatment for a cancer
selected from the group consisting of breast cancer, melanoma,
myeloma and prostate cancer, the compound being selected from the
group consisting of: ##STR13##
8. The method of claim 7, wherein the administering step comprises
administering the compound: ##STR14## or a salt thereof to the
subject.
9. The method of claim 7, wherein the administering step comprises
administering the compound: ##STR15## or a salt thereof to the
subject.
10. The method of claim 7, wherein the administering step comprises
administering the compound: ##STR16## or a salt thereof to the
subject.
11. The method of claim 7, wherein the administering step comprises
administering the compound: ##STR17## or a salt thereof to the
subject.
Description
FIELD OF INVENTION
[0001] The present invention relates to novel tetracycline
derivatives, methods for producing the novel derivatives and
methods of using these derivatives.
BACKGROUND OF THE INVENTION
[0002] The compound tetracycline, exhibits the following general
structure ##STR1##
[0003] The numbering system of the ring nucleus is as follows:
##STR2##
[0004] Tetracycline as well as the 5-OH (Terramycin) and 7-Cl
(Aureomycin) derivatives exist in nature, and are well known
antibiotics. Natural tetracycline may be modified without losing
their antibiotic properties, although certain elements of the
structure must be retained. The modifications that may and may not
be made to the basic tetracycline structure have been reviewed by
Mitscher in The Chemistry of Tetracyclines, Chapter 6, Marcel
Dekker, Publishers, New York (1978). According to Mitscher, the
substituents at positions 5-9 of the tetracycline ring system may
be modified without the complete loss of antibiotic properties.
Changes to the basic ring system or replacement of the substituents
at positions 1-4 and 10-12, however, generally lead to synthetic
tetracyclines with substantially less or effectively no
antimicrobial activity. Some examples of chemically modified
non-antimicrobial tetracyclines (hereinafter CMT) are
4-dedimethylaminotetraccyline, 4-dedimethylaminotetraccyline
(6-demethyl-6 -deoxy-4-dedimethylaminotetracycline),
4-dedimethylaminominocycline
(7-dimethylamino4-dedimethylaminotetracycline), and
4-dedimethylaminodoxycycline
(5-hydroxy-6-deoxy-4-dedimethyaminotetracycline).
[0005] Some 4-dedimethylaminotetracycline derivatives are disclosed
in U.S. Pat. Nos. 3,029,284 and 5,122,519. They include
6-demethyl-6-deoxy-4-dedimethylaminotetraccyline and
5-hydroxy-6-deoxy-4-dedimethylaminotetracycline with hydrogen and
other substituents at C7, and the C9 positions on the D ring. These
substitutents include amino, nitro, di (lower alkyl) amino, and
mono (lower alkyl) amino or halogen. The
6-demethyl-6-deoxy-4-dedimethylaminotetracycline derivatives and
5-hydroxy-6-deoxy-4-dedimethylaminotetracycline derivatives are
said to be useful as antimicrobial agents.
[0006] Other 4-dedimethylaminotetracycline derivatives with an
oxime group at the C4 position on the A ring are disclosed in U.S.
Pat. Nos. 3,622,627 and 3,824,285. These oxime derivatives have
hydrogen and halogen as substitutents at the C7 position and
include
7-halo-6-demethyl-6-deoxy-4-dedimethylamnino-4-oximinotetracycline,
and
7-halo-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximninotetracycline.
[0007] Alkylamino (NH-alkyl), and alkylhydrazone (N--NH-alkyl)
groups have been substituted on the A ring at the C4 position of
4-dedimethylaminotetracycline. These compounds are known for their
antimicrobial properties. See U.S. Pat. Nos. 3,345,370, 3,609,188,
3,622,627, 3,502,660, 3,509,184, 3,502, 696, 3,515,731, 3,265,732,
5,122,519, 3,849,493, 3,772,363, and 3,829,453.
[0008] In addition to their antimicrobial properties, tetracyclines
have been described as having a number of other uses. For example,
tetracyclines are also known to inhibit the activity of collagen
destructive enzymes, such as matrix metalloproteinases (MMP),
including collagenases (MMP-1), gelatinase (MMP-2) and stromelysin
(MMP-3). Golub et al., J Periodont. Res. 20:12-23 (1985); Golub et
al. Crit. Revs. Oral Biol. Med. 2:297-322 (1991); U.S. Pat. Nos.
4,666,897; 4,704,383; 4,935,411; 4,935,412. Also, tetracyclines
have been known to inhibit wasting and protein degradation in
mammalian skeletal muscle, U.S. Pat. No. 5,045,538, and to enhance
IL-10 production in mammalian cells.
[0009] Furthermore, tetracyclines were reported to enhance bone
protein synthesis in U.S. Pat. No. Re. 34,656, and to reduce bone
resorption in organ culture in U.S. Pat. No. 4,704,383.
[0010] Similarly, U.S. Pat. No. 5,532,227 to Golub et al, discloses
that tetracyclines can ameliorate the excessive glycosylation of
proteins. In particular, tetracyclines inhibit the excessive
collagen cross linking which results from excessive glycosylation
of collagen in diabetes.
[0011] Tetracyclines are known to inhibit excessive phospholipase
A.sub.2 activity involved in inflammatory conditions such as
psoriasis as disclosed in U.S. Pat. No. 5,532,227. In addition,
tetracyclines are also known to inhibit cycloxygenase-2 (COX-2),
tumor necrosis factor (TNF), nitric oxide and IL-1
(interleukin-1).
[0012] These properties cause the tetracyclines to be useful in
treating a number of diseases. For example, there have been a
number of suggestions that tetracyclines, including
non-antimicrobial tetracyclines, are effective in treating
arthritis. See, for example, Greenwald, et al. "Tetracyclines
Suppress Metalloproteinase Activity in Adjuvant Arthritis and, in
Combination with Flurbioprofen, Ameliorate Bone Damage," Journal of
Rheumatology 19:927-938 (1992); Greenwald et al., "Treatment of
Destructive Arthritic Disorders with MMP Inhibitors; Potential Role
of Tetracyclines in Inhibition of Matrix Metalloproteinases:
Therapeutic Potential," Annals of the New York Academy of Sciences
732:181-198 (1994); Kloppenburg, et al. "Minocycline in Active
Rheumatoid Arthritis," Arthritis Rheum 37:629-636 (1994); Ryan et
al., "Potential of Tetracycline to Modify Cartilage Breakdown in
Osteoarthritis," Current Opinion in Rheumatology 8:238-247 (1996);
O'Dell et al, "Treatment of Early Rheumatoid Arthritis with
Minocycline or Placebo," Arthritis Rheum 40:842-848 (1997).
[0013] Tetracyclines have also been suggested for use in treating
skin diseases. For example, White et al., Lancet, Apr. 29, p. 966
(1989) report that the tetracycline minocyline is effective in
treating dystrophic epidermolysis bullosa, which is a
life-threatening skin condition believed to be related to excess
collagenase.
[0014] Furthermore, studies have also suggested that tetracyclines
and inhibitors of metalloproteinases inhibit tumor progression,
DeClerck et al., Annals N.Y. Acad. Sci., 732:222-232 (1994), bone
resorption, Rifkin et al., Annals N.Y. Acad. Sci., 732:165-180
(1994), angiogenesis, Maragoudakis et al., Br. J. Pharmacol.,
111:894-902 (1994), and may have anti-inflammatory properties,
Ramamurthy et al., Annals N.Y. Acad. Sci., 732, 427-430 (1994).
[0015] Based on the foregoing, tetracyclines have been found to be
effective in treating numerous diseases and conditions. Therefore,
there is a need for new and even more useful derivatives.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] Compounds disclosed herein are derivatives of tetracycline,
which exhibit antimicrobial and/or anti-cancer activity.
[0017] In a preferred embodiment, the present invention relates to
a treatment for inhibiting microbial or tumor growth which
comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR3## wherein R is selected from
CH.sub.2N(R.sub.aR.sub.b) or ##STR4## [0018] R.sub.aR.sub.b are
C.sub.1 to C.sub.6 alkyl; [0019] R.sub.c and R.sub.d are
(CH.sub.2).sub.n CHR.sub.c, n is 0 or 1 and R.sub.e is H, C.sub.1
to C.sub.6 alkyl, or NH.sub.2, and X is NH, S, or CH.sub.2; [0020]
R.sub.1 is H or OH; [0021] R.sub.2 is H, OH, .dbd.O, or OCOR.sub.8,
where R.sub.8 is C.sub.1 to C.sub.6 alkyl, or alternatively,
R.sub.2 is N(R.sub.9).sub.2, where R.sub.9 is hydrogen or C.sub.1
to C.sub.6 lower alkyl, or R.sub.9 CO, with the proviso that when
R.sub.2 is keto or N(R.sub.9).sub.2, then R.sub.3 and R.sub.4 are H
or CH.sub.3, and R.sub.3 and R.sub.4 are not both CH.sub.3 or H;
[0022] 5a hydrogen is .alpha. or .beta.; [0023] R.sub.3 and R.sub.4
are H, CH.sub.3 or F, with the proviso that when R.sub.3 is
CH.sub.3, then R.sub.4 is H or F, and when R.sub.4 is CH.sub.3,
then R.sub.3 is H or F and R.sub.4 and R.sub.5 are not both F;
[0024] R.sub.5 and R.sub.7 are halogen, H, NO.sub.2, N.sub.3,
N.sub.2.sup.+, C.sub.2H.sub.5OC(S)S--, CN, NR.sub.10R.sub.11, where
R.sub.10 and R.sub.11 are H, C.sub.1 to C.sub.10 alkyl, and
R.sub.12(CH.sub.2).sub.nCO--, where n is 0 to 5 and R.sub.9 is H,
NH.sub.2, mono or disubstituted amino selected from straight,
branched or cyclized C.sub.1 to C.sub.10 alkyl groups, with the
proviso that [0025] R.sub.10 and R.sub.11 are not both
R.sub.12(CH.sub.2).sub.nCO--; [0026] R.sub.7, alternatively, is
tertiary butyl; and [0027] R.sub.6 is halogen, acetylene,
R.sub.13-C.sub.6H.sub.5, where R.sub.13 is NO.sub.2, halogen,
acetylamino, amino, phenyl, alkyl, or alkoxy.
[0028] In a further preferred embodiment, the present invention
relates to a treatment for inhibiting microbial or tumor growth
which comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR5## wherein R is selected from
CH.sub.2N(R.sub.aR.sub.b) or ##STR6## [0029] where R.sub.aR.sub.b
are C.sub.1 to C.sub.6 alkyl; [0030] R.sub.c and R.sub.d are
(CH.sub.2).sub.nCHR.sub.e, n is 0 or 1 and R.sub.e is H, C.sub.1 to
C.sub.6 alkyl, or NH.sub.2, and X is NH, S, or CH.sub.2; [0031]
R.sub.1 is H or OH; [0032] R2 is H or OH; [0033] R.sub.3 is H or
CH.sub.3; [0034] R.sub.4 and R.sub.6 are halogen, H, NO.sub.2,
N.sub.3, N.sub.2.sup.+, C.sub.2H.sub.5OC(S)S--, CN,
NR.sub.7R.sub.8, where R.sub.7 and R.sub.8 are H, C.sub.1 to
C.sub.10 alkyl, and R.sub.9(CH.sub.2),CO--, where n is 0 to 5 and
R.sub.9 is H, NH.sub.2, mono or disubstituted amino selected from
straight, branched or cyclic C.sub.1-C.sub.10 alkyl, with the
proviso that R.sub.7 and R.sub.8 are not both
R.sub.9(CH.sub.2).sub.nCO--; and [0035] R.sub.6 is H or halogen,
acetylene, RIO-C.sub.6H.sub.5, where RIO is N0.sub.2, halogen,
acetylamino, amino, phenyl, alkyl, or alkoxy.
[0036] In a further preferred embodiment, the present invention
relates to a treatment for inhibiting microbial or tumor growth
which comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR7## wherein R is selected from
CH.sub.2N(R.sub.aR.sub.b) or ##STR8## [0037] where R.sub.aR.sub.b
are C.sub.1-C.sub.6 alkyl; [0038] R.sub.c and Rd are
(CH.sub.2).sub.nCHR.sub.e, n is 0 or 1 and R.sub.e is H. C.sub.1 to
C.sub.6 alkyl, or NH.sub.2, and X is NH, S, or CH.sub.2; [0039]
R.sub.1 and R.sub.2 are H or N(R.sub.9).sub.2, where R.sub.9 is H
or C.sub.1 to C.sub.6 alkyl with the proviso that R, and R.sub.2
are not both N(R.sub.9).sub.2, R.sub.1 and R2 are also
N(R.sub.10).sub.3 I, where R.sub.10 is C.sub.1 to C.sub.6 alkyl
with the proviso that R.sub.1 and R.sub.1 are not both
N(R.sub.10).sub.3 I; [0040] R.sub.3 is H; [0041] R.sub.4 and
R.sub.5 are H, CH.sub.3 or F, with the proviso that when R.sub.4 is
CH.sub.3, then R.sub.5 is H or F, and when R.sub.5 is CH.sub.3,
then R.sub.4 is H or F, and R.sub.4 and R.sub.5 are not both F;
[0042] R.sub.6 and R.sub.8 are halogen, H, NO.sub.2, N.sub.3,
N.sub.2.sup.+, C.sub.2H.sub.5OC(S)S--, CN, NR.sub.11R.sub.12, where
R.sub.11 and R.sub.12 are H, C.sub.1 to C.sub.10 alkyl, and
R.sub.13(CH.sub.2).sub.nCO--, where n is 0 to 5 and R.sub.13 is H,
NH2, mono or disubstituted amino selected from straight, branched
or cyclized C.sub.1-C.sub.10 alkyl groups, with the proviso that
R.sub.10 and R.sub.11 are not both R.sub.12 (CH.sub.2).sub.nCO--;
and [0043] R.sub.8 can also be tertiary butyl and R.sub.7 is H or
halogen.
[0044] In an additional preferred embodiment, the present invention
relates to a treatment for inhibiting microbial or tumor growth
which comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR9## wherein R is selected from
CH.sub.2N(R.sub.aR.sub.b) or ##STR10## [0045] R.sub.a R.sub.b are
C.sub.1-C.sub.6; [0046] R.sub.c and R.sub.d are (CH.sub.2)n CH, n
is 0 or 1, R.sub.e is H, alkyl (C.sub.1 to C.sub.6), NH.sub.2, and
X is NH, S, or CH.sub.2; [0047] R.sub.1 and R.sub.2 are H or
N(R.sub.9).sub.2 where R.sub.9 is hydrogen or lower alkyl (C.sub.1
to C.sub.6 ), with the proviso that R.sub.1 and R.sub.2 can not
both be N(R.sub.9).sub.2. [0048] R.sub.3 is H, OH, .dbd.O,
OCOR.sub.10, where R.sub.10 is C.sub.1 to C.sub.6, or
alternatively, R.sub.3 is N(R.sub.9 ).sub.2 where R.sub.9 is
hydrogen or C.sub.1 to C.sub.6, with the proviso that when R.sub.23
is .dbd.O or N(R.sub.9 ).sub.2, then R.sub.4 and R.sub.5 are H or
CH.sub.3 and R.sub.4 and R.sub.5 are not both CH.sub.3 or H; [0049]
5a hydrogen is .alpha. or .beta.; [0050] R.sub.4 and R.sub.5 are H,
CH.sub.3 or F, with the proviso that when R.sub.4 is CH.sub.3, then
R.sub.5 is H or F, and when R.sub.5 is CH.sub.3 then R.sub.4 is H
or F and R.sub.4 and R.sub.5 are not both F; [0051] R.sub.6 and
R.sub.8 are halogen, H, NO.sub.2, N.sub.3, N.sub.2.sup.+,
C.sub.2H.sub.5OC(S)S--, CN, NR.sub.10R.sub.11, where R.sub.10 and
R.sub.11 are H, alkyl (C.sub.1 to C.sub.10), and R.sub.12
(CH.sub.2).sub.nCO--, where n is 0 to 5 and R.sub.12 is H,
NH.sub.2, mono or disubstituted amino selected from straight,
branched or cyclized C.sub.1 to C.sub.10 groups, with the proviso
that R.sub.10 and R.sub.11 are both R.sub.12 (CH.sub.2).sub.nCO--,
or, alternatively, R.sub.8 is tertiary butyl; [0052] R.sub.7 is H
or halogen, acetylene, R.sub.12--C.sub.6H.sub.5, where R.sub.12 is
NO.sub.2, halogen, acetylamino, amino, phenyl, alkyl, or
alkoxy.
[0053] In a further preferred embodiment, the present invention
relates to a treatment for inhibiting microbial or tumor growth
which comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR11## wherein R.sub.1 is H or
N(CH.sub.3).sub.2; R.sub.2 is H, CH.sub.3 or OCOCH.sub.3; R.sub.3
is H or CH.sub.3; R.sub.4 is H; R.sub.5 N(CH.sub.3).sub.2,
NH.sub.2, N(C.sub.4H.sub.9).sub.2, N(C.sub.6Hl.sub.3).sub.2, N(3,3-
dimethylbutyl).sub.2, N.sub.3, N.sub.2.sup.+, NHCOCH.sub.3,
NH(n-propyl).sub.2, NH-isobutyl, NH-isobutylmethyl, NH(cyclobutyl),
NH(cyclobutyl methyl); and R.sub.6 is H, NO.sub.2, NH.sub.2,
(CH.sub.3).sub.2 CHNH, N.sub.3, NHCOCH.sub.3, N2.sup.+, N.sub.3 or
(CH.sub.3).sub.3C.
[0054] In a further preferred embodiment, the present invention
relates to a treatment for inhibiting microbial or tumor growth
which comprises adding to a subject in need of said treatment an
effective amount of a tetracycline composition or salt thereof,
comprising a general formula: ##STR12## wherein R.sub.1 is H or
N(CH.sub.3).sub.2; R.sub.2 is H, OH or OCOCH.sub.3; R.sub.3 is H or
CH.sub.3; R.sub.4 is H; R.sub.5 is H, N(CH.sub.3).sub.2;
N(C.sub.4H.sub.9).sub.2, N(C.sub.6H.sub.13).sub.2,
N(3,3-dimethylbutyl).sub.2; N.sub.3, N.sub.2.sup.+, NHCOCH.sub.3,
NH(n-propyl).sub.2, NH-isobutyl, NH-isobutylmethyl,
N(CH.sub.3CO)(isobutyl), NH(cyclobutyl), NH(cyclobutyl methyl), or
NH.sub.2; and R.sub.6 is H, NO.sub.2, NH2, (CH.sub.3).sub.2 CHNH,
N.sub.3, NHCOCH.sub.3, N.sub.2.sup.+, or (CH.sub.3).sub.3C.
[0055] The following examples describe the preparation of various
substances in accordance with the present invention.
[0056] HPLC analyses were performed on Waters reverse phase
C.sub.18-symmetry columns using water (containing 0.1% TFA) and
acetonitrile as eluents
[0057] Analytical LC/MS runs were performed on a Hewlett Packard
Series 1100 liquid chromatrographer equipped with a series 1100 MSD
detector, with ES ionization mode and UV detection at 270 nm, using
a 4.6.times.100 mm column with a flow rate of 0.4 mL/min.
Preparative HPLC was performed on a Gilson serial HPLC with WV
detection at 270 nm, using a 19.times.150 mm column and a flow rate
of 15 ml/min .sup.1H NMR were recorded on a Bruker 300 MHz
spectrometer in deuterated methanol.
9-Nitro-doxycycline Sulfate
[0058] To a stirred solution of doxycycline hydrochloride (1 g,
2.08 mmol) in 30 mL of concentrated sulfuric acid at 0.degree. C.
was added potassium nitrate (252 mg, 2.49 mmol). The resulting
mixture was stirred for 20 min at 0.degree. C., then poured into
1.2 L cold ether with stirring. The solid which precipitated was
filtered out, washed with cold ether and dried under vacuum, and
1.22 g of cream colored product was obtained. If needed, the
product was purified by dissolving in methanol, filtering and
pouring the filtrate into ether. The solid which separated was
filtered out and dried.
9-Amino-doxycycline Sulfate
[0059] To a stirred solution of 9-nitro-doxycycline sulfate (1 g,
1.7 mmol) in 35 mL of ethylene glycol monomethyl ether and 5 mL of
methanol was added 700 mg of 10% Pd/C. The reaction mixture was
hydrogenated at atmospheric pressure for 6 hrs. The catalyst was
filtered off through celite washing with methanol. The filtrate was
concentrated, then added dropwise to 800 mL cold ether with
stirring. The solid that separated was filtered out, washed with
cold ether and dried under vacuum, and 843 mg of cream colored
product was obtained.
9-Isopropylamino-doxycycline Sulfate
[0060] To a stirred solution of 9-amino-doxycycline sulfate (100
mg, 0.18 mmol) on 10 mL of ethylene glycol monomethyl ether was
added 0.05 ml of concentrated sulfuric acid, 0.5 mL acetone and 100
mg of 10% Pd/C. The resulting mixture was hydrogenated at
atmospheric pressure for 1 h 15 min, then the catalyst was filtered
through celite, washing with methanol. The filtrate was
concentrated, then poured into 150 mL of cold ether with stirring.
The solid that separated was filtered out, washed with cold ether
and dried under vacuum, and 109 mg of cream colored product was
obtained. The product was purified by dissolving in methanol,
filtering and pouring the filtrate into ether. The solid which
formed was filtered out and dried.
9-Acetamido-doxycycline Sulfate
[0061] To a stirred solution of 9-amino-doxycycline sulfate (72 mg,
0.13 mmol) in 1.5 mL of 1,3-dimethyl-2-imidazolidinone were added
70 mg sodium bicarbonate followed by 0.05 mL of acetyl chloride.
The mixture was stirred at room temperature for 1 hour and then
filtered. The filtrate was concentrated and poured into 100 ml cold
ether with stirring. The solid that separated was filtered out and
dried under vacuum, and 92 mg was obtained.
9-Diazonium-doxycycline Sulfate Hydrochloride
[0062] To a stirred solution of 9-amino-doxycycline sulfate (300
mg, 0.54 mmol) in 9 mL of 0.1N hydrochloric acid in methanol cooled
in an ice bath was added 0.3 mL of n-butyl nitrite. The solution
was stirred at 0.degree. C. for 30 minutes then poured into 300 mL
of cold ether with stirring. The solid that separated was filtered
out and dried under vacuum, and 256 mg of an orange-brown product
was obtained.
9-Azido-doxycycline Sulfate
[0063] To a stirred solution of 9-diazonium-doxycycline sulfate
hydrochloride (80 mg, 0.14 mmol) in 4.5 mrL of 0.1N hydrochloric
acid in methanol was added 10 mg of sodium azide. The solution was
stirred at room temperature for 2 hours, then poured into 100 mL of
cold ether with stirring. The solid that separated was filtered out
and dried under vacuum, and 43 mg of product was obtained.
9-(4-Flurophenyl)-doxycycline
[0064] To a solution of 9-diazonium-doxycycline sulfate
hydrochloride (358 mg, 0.59 mmol) and 4-flurophenyl boronic acid
(107 mg, 0.76 mmol) in 4 mL of methanol was added 18 mg of
palladium(II)acetate and the resulting mixture was stirred at room
temperature for 16 hours. The catalyst was filtered off and the
residue was concentrated and purified by preparative HPLC. The
collected fractions from BPLC were concentrated and poured into 20
ml of cold ether. The solid that was separated was filtered out and
dried under vacuum, and 32 mg of product was obtained.
7-[1,2-Bis(carbobenzyloxy)hydrazino]-doxycycline
[0065] To a stirred solution of doxycycline hydrochloride (300 mg,
0.62 mmol) in 2.5 ml of TUF and 3.2 mL of methanesulfonic acid at
0.degree. C. was added 230 mg of dibenzyazodicarboxylate. The
resulting mixture was stirred at 0.degree. C. for 2 hours, then
poured into 400 mL of cold ether. The solid that separated was
filtered out and dried under vacuum, and 479 mg of an off-white
product was obtained.
7-Amino-doxycycline
[0066] To a solution of
7-[1,2-bis(carbobenzyloxy)hydrazino]-doxycycline (478 mg) in 30 mL
of ethylene glycol monomethyl ether was added 200 mg of 10% Pd/C
and the resulting mixture was hydrogenated for 3 hours at room
temperature. The catalyst was filtered through celite washing with
methanol and the filtrate was concentrated and poured into 500 mL
cold ether. The solid that separated was filtered out and dried
under vacuum, and 268 mg of product was obtained.
7-Dimethylamino-doxycycline Sulfate
[0067] To a solution of 7-amino-doxycycline (100 mg) in 8 ML of
ethylene glycol monomethyl ether were added 1 mL of a 37% aqueous
formaldehyde solution, 0.05 mL of concentrated sulfuric acid and
100 mg of 10% Pd/C. The resulting mixture was hydrogenated at
atmospheric pressure for 6 hours, then the catalyst was filtered
through celite, washing with methanol. The residue was concentrated
and poured into 100 ml cold ether with stirring. The solid that
separated was filtered out with difficulty, being very gluey. The
solid was immediately redissolved in methanol and poured into 100
ml cold ether with stirring. The solid that separated was filtered
out and dried under vacuum, and 89 mg of product was obtained.
7-Dipropyl-doxycycline Sulfate
[0068] To a solution of 7-amino-doxycycline (90 mg) in 8 mL of
ethylene glycol monomethyl ether were added 0.5 mL of
propionaldehyde, 0.05 mL of concentrated sulfuric acid and 80 mg of
10% Pd/C. The resulting mixture was hydrogenated at atmospheric
pressure for 5 hours, then the catalyst was filtered through
celite, washing with methanol. The residue was concentrated and
poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 415 mg of
product was obtained.
7-Dibutylamino-doxycycline Sulfate
[0069] To a solution of 7-amino-doxycycline (100 mg, 0.2 mmol) in 6
mL of ethylene glycol monomethyl ether were added 0.6 mL of
butyraldehyde, 0.05 mL of concentrated sulfuric acid and 100 mg of
10% Pd/C. The resulting mixture was hydrogenated at atmospheric
pressure for 4 hours, then the catalyst was filtered through
celite, washing with methanol. The residue was concentrated and
poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 103 mg of
product was obtained.
7-Di(3,3-dimethyl)butylamino-doxycycline Sulfate
[0070] To a solution of 7-amino-doxycycline (100 mg, 0.2 mmol) in 6
mL of ethylene glycol monomethyl ether were added 0.5 mL of
3,3-dimethyl butyraldehyde, 0.05 mL of concentrated sulfuric acid
and 100 mg of 10% Pd/C. The resulting mixture was hydrogenated at
atmospheric pressure for 4 hrs, then the catalyst was filtered
through celite, washing with methanol. The residue was concentrated
and poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 122 mg of
product was obtained.
7-Dihexylamino-doxycycline Sulfate
[0071] To a solution of 7-amino-doxycycline (100 mg, 0.2 mmol) in 6
mL of ethylene glycol monomethyl ether were added 0.6 mL of
hexanal, 0.05 mL of concentrated sulfuric acid and 100 mg of 10%
Pd/C. The resulting mixture was hydrogenated at atmospheric
pressure for 4 hours, then the catalyst was filtered through
celite, washing with methanol. The residue was concentrated and
poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 129 mg of
product was obtained.
7-Isopropylamino-doxycycline Sulfate
[0072] To a solution of 7-amino-doxycycline (90 mg, 0.2 mmol) in 8
mL of ethylene glycol monomethyl ether were added 0.5 mL of
isobutyraldehyde, 0.05 mL of concentrated sulfuric acid and 90 mg
of 10% Pd/C. The resulting mixture was hydrogenated at atmospheric
pressure for 6 hours, then the catalyst was filtered through
celite, washing with methanol. The residue was concentrated and
poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 142 mg of
product was obtained.
7-Methyl-7-isopropylamino-doxycycline Sulfate
[0073] To a solution of 7-isopropylamino-doxycycline (60 mg) in 4
mL of ethylene glycol monomethyl ether were added 0.4 mL of a 37%
aqueous solution of formaldehyde, 0.05 mL of concentrated sulfuric
acid and 50 mg of 10% Pd/C. The resulting mixture was hydrogenated
at atmospheric pressure for 2 hours, then the catalyst was filtered
through celite, washing with methanol. The residue was concentrated
and poured into 80 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 52 mg of
product was obtained.
7-Cyclobutylamino-doxycycline Sulfate
[0074] To a solution of 7-amino-doxycycline (80 mg) in 7 mL of
ethylene glycol monomethyl ether were added 0.3 mL of
cyclobutanone, 0.04 mL of concentrated sulfuric acid and 60 mg of
10% Pd/C. The resulting mixture was hydrogenated at atmospheric
pressure for 24 hours, then the catalyst was filtered through
celite, washing with methanol. The residue was concentrated and
poured into 100 ml cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 85 mg of
product was obtained.
7-Methyl-7-cyclobutylamino-doxycycline Sulfate
[0075] To a solution of 7-cyclobutylamino-doxycycline (40 mg) in
3.5 mL of ethylene glycol monomethyl ether were added 0.3 mL of a
37% aqueous solution of formaldehyde, 0.03 mL of concentrated
sulfuric acid and 40 mg of 10% Pd/C. The resulting mixture was
hydrogenated at atmospheric pressure for 6 hrs, then the catalyst
was filtered through celite, washing with methanol. The residue was
concentrated and poured into 80 ml cold ether with stirring. The
solid that separated was filtered out and dried under vacuum, and
35 mg of product was obtained.
7-Acetamido-doxycycline Sulfate
[0076] To a stirred solution of 7-amino-doxycycline (200 mg) in 3
mL of 1,3-dimethyl-2-imidazolidinone were added 200 mg sodium
bicarbonate followed by 0.09 mL of acetyl chloride. The mixture was
stirred at room temperature for 1 hr and then filtered. The
filtrate was concentrated and poured into 200 ml cold ether with
stirring. The product which precipitated was filtered out and dried
under vacuum. Obtained 202 mg of product.
7-Acetamido-7-isopropyl-doxycycline Sulfate
[0077] To a stirred solution of 7-isobutylamino-doxycycline (60 mg)
in 1.5 mL of 1,3-dimethyl-2-imidazolidinone were added 60 mg sodium
bicarbonate followed by 0.05 mL of acetyl chloride. The mixture as
stirred at room temperature for 1 hr 30 min and then filtered. The
filtrate was concentrated and poured into 80 ml cold ether with
stirring. The product which precipitated was filtered out and dried
under vacuum.
7-Diazonium-doxycycline hydrochloride or Tetrafluoroborate
[0078] To a stirred solution of 7-amino-doxycycline (200 mg, 0.4
mmol) in 4 mL of 0.1N hydrochloric acid in methanol cooled in an
ice bath was added 0.2 mL of n-butyl nitrite. The solution was
stirred at 0.degree. C. for 30 minutes, then poured into 200 mL of
cold ether with stirring. The solid that separated was filtered out
and dried under vacuum, and 173 mg of product was obtained. The
diazonium tetrafluoroborate salt was prepared by using
tetrafluoroboric acid (54% solution in ether) instead of
hydrochloric acid.
7-Azido-doxycycline
[0079] To a stirred solution of 7-diazonium-doxycycline
hydrochloride (80 mg, 0.14 mmol) in 4.5 mL of O. lN hydrochloric
acid in methanol cooled on an ice bath was added 12 mg of sodium
azide. The solution was stirred at 0.degree. C. for 2 hours, then
poured into 100 mL of cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 43 mg of
product was obtained.
7-Amino-9-nitro-doxycycline Sulfate
[0080] To a stirred solution of 7-amino-doxycycline (220 g, 0.44
mmol) in 4 mL of concentrated sulfuric acid at 0.degree. C. was
added potassium nitrate (52 mg, 0.51 mmol). The resulting mixture
was stirred for 15 minutes at 0.degree. C., then poured into 300 mL
cold ether with stirring. The solid that separated was filtered
out, washed with cold ether and dried under vacuum. The product was
redissolved in methanol, filtered and the filtrate was poured into
ether. The solid that separated was filtered out and dried under
vacuum, and 222 g of product was obtained.
7-Dimethylamino-9-nitro-doxycycline Sulfate
[0081] To a stirred solution of 7-dimethylamino-doxycycline (40 g,
0.07 mmol) in 1.5 mL of concentrated sulfuric acid at 0.degree. C.
was added potassium nitrate (12 mg, 0.12 mmol). The resulting
mixture was stirred for 30 min at 0.degree. C., then poured into 60
mL cold ether with stirring. The solid that separated was filtered
out, washed with cold ether and dried under vacuum. LC/MS shows
mostly one product containing two nitro groups (possibly a nitrate
ester of the desired product).
7-Dimethylamino-9-diazonium-doxycycline Sulfate Hydrochloride
[0082] To a stirred solution of 7-dimethylamino-9-amino-doxycycline
(50 mg) in 1.5 mL of 0.1N hydrochloric acid in methanol cooled in
an ice bath was added 0.05 mL of a n-butyl nitrite. The solution
was stirred at 0.degree. C. for 30 minutes, then poured into 80 mL
of cold ether with stirring. The solid that separated was filtered
out and dried under vacuum, and 38 mg of product was obtained.
7-Acetamido-9-nitro-doxycycline Sulfate
[0083] To a stirred solution of 7-acetamino-doxycycline (60 g,
0.095 mrmol) in 2 mL of concentrated sulfuric acid at 0.degree. C.
was added potassium nitrate (11 mg, 0.11 mmol). The resulting
mixture was stirred for 5 minutes at 0.degree. C., then poured into
80 mL cold ether with stirring. The solid that separated was
filtered out, washed with cold ether and dried under vacuum.
7-Acetamido-9-amino-doxycycline Sulfate
[0084] To a stirred solution of 7-acetamido-9-nitro-doxycycline
sulfate (77 mg, 0.12 mmol) in 7 mL of ethylene glycol monomethyl
ether was added 60 mg of 10% Pd/C. The reaction mixture was
hydrogenated at atmospheric pressure for 7 hours. The catalyst was
filtered off through celite washing with methanol. The filtrate was
concentrated, then added dropwise to 100 mL cold ether with
stirring. The solid that separated was filtered out, washed with
cold ether and dried under vacuum, and 62 mg of product was
obtained.
7-Acetamido-9-diazonium-doxycycline Sulfate Hydrochloride
[0085] To a stirred solution of 7-acetamido-9-amino-doxycycline
(100 mg) in 3 mL of 0.1N hydrochloric acid in methanol cooled in an
ice bath was added 0.1 mL of n-butyl nitrite. The solution was
stirred at 0.degree. C. for 30 minutes, then poured into 100 mL of
cold ether with stifling. The solid that separated was filtered out
and dried under vacuum, and 92 mg of product was obtained.
7-Acetamido-9-azido-doxycycline Sulfate
[0086] To a stirred solution of 7-acetamido-9-diazonium-doxycycline
(112 mg, 0.19 mmol) in 6 mL of 0.1N hydrochloric acid in methanol
cooled in an ice bath was added 14 mg (0.21 mmol) of sodium azide.
The solution was stirred at 0.degree. C. for 35 minutes, then
poured into 150 mL of cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 94 mg of
product was obtained.
Doxycycline Methiodide
[0087] Doxycycline free base (315 mg) was dissolved in 2 mL of
methanol and 10 mL of THF, then 1 mL of methyl iodide was added.
The reaction mixture was kept for 5 days; no crystallization of the
product occurred. The reaction mixture was concentrated and poured
into 300 mL of cold ether with stirring. The product that separated
was filtered out and dried under vacuum, and 269 mg was obtained;
LC/MS shows pure product.
5-Acetoxy-doxycycline
[0088] A solution of doxycycline (100 mg) in 3 mL of a 30% HBr in
acetic acid solution was stirred at room temperature for 3.5 days;
the reaction mixture was then poured into 100 mL of cold ether with
stirring and the product that separated was filtered out. LC/MS
showed incomplete conversion of starting material, so the product
(86 mg) was resubjected to the reaction conditions for 9 hrs, and
then isolated in the same way.
9-Tert-butyl-doxycycline
[0089] A solution of doxycycline (100 mg) in 2 mL of tert-butanol
and 3 mL of methanesulfonic acid was stirred at room temperature
for 18 hours, then poured into 100 mL of cold water and extracted
with n-butanol. The organic extracts were basified with a 1N sodium
hydroxide solution and the pH was quickly adjusted to 6.5-7 with
concentrated hydrochloric acid. A solid precipitated which was
filtered out, then dissolved in methanol, concentrated and added
dropwise to 30 mL of an ether/PET ether cold solution with
stirring. The solid that separated was filter out and dried under
vacuum.
9-Tert-butyl-7-nitro-doxycycline
[0090] A solution of doxycycline hydrochloride (2 g) in 10 mL of
tert-butanol and 30 mL of methanesulfonic acid was stirred at room
temperature for 3 hours, then 500 mg of potassium nitrate was added
and the resulting mixture stirred for an additional hour. The
reaction mixture was poured in 100 ml cold solution of water
containing 23 g of sodium hydroxide; more dilute NaOH solution was
added dropwise until the reaction mixture became basic. The pH was
quickly adjusted to 6.5-7 with concentrated hydrochloric acid. The
gluey dark solid that separated was filtered out. The aqueous
filtrate was extracted once with n-butanol to recover more product.
The crude product was dissolved in the minimum amount of methanol
and purified by HPLC. The combined fractions from HPLC were
concentrated to dryness, dissolved in methanol and added dropwise
to 50 mL of a 3:1 mixture of ether/PET ether. The solid that
separated was filtered out, and 788 mg of pale yellow product was
obtained.
9-Tert-butyl-7-amino-doxycycline
[0091] To a solution of 9-tert-butyl-7-doxycycline (500 mg) in 25
mL of ethylene glycol monomethyl ether was added 350 mg of 10% Pd/C
and the resulting mixture was hydrogenated at atmospheric pressure
for 17 hours. The catalyst was filtered through celite, washing
with methanol. The residue was concentrated, dissolved in THF and
added dropwise to a cold 1:1 solution of ether/PET ether with
stirring. The product that precipitated was filtered out and dried
under vacuum, and 452 mg of product was obtained.
9-Tert-butyl-7-diazonium-doxycycline
[0092] To a stirred solution of 9-tert-butyl-7-amino-doxycycline
(93 mg, 0.18 mmol) in 2.5 mL of 0.1N hydrochloric acid in methanol
cooled in an ice bath was added 0.1 mL of n-butyl nitrite. The
solution was stirred at 0.degree. C. for 30 minutes, and then half
was poured into 30 mL of a 3:1 cold mixture of ether/PET ether with
stirring. The product stuck to the bottom of the beaker as a gum.
It was redissolved in methanol and concentrated, the residue was
poured into 20 mL of cold ether and the solid that separated was
filtered out immediately and dried under vacuum; 42 mg of product
was obtained. The other half of the reaction mixture was used
directly for the 9-tert-butyl material below.
9-Tert-butyl-7-azido-doxycycline
[0093] To a stirred solution of
9-tert-butyl-7-diazonium-doxycycline reaction mixture (1.5 mL) at
0.degree. C. were added 8 mg (0.12 mmol) of sodium azide. The
solution was stirred for 3 hours while allowed to warm to room
temperature. After concentration of the reaction mixture, the
residue was poured into a cold 3:1 mixture of ether/PET ether with
stirring. No solid separated, so the solution was concentrated to
dryness to obtain 35 mg of product.
9-Tert-butyl-7-dimethylamino-doxycycline
[0094] To a stirred reaction mixture from
9-tert-butyl-7-amino-doxycycline containing 167 mg of
9-tert-butyl-7-amino-doxycycline in 10 mL of ethylene glycol
monomethyl ether and 115 mg of 10% Pd/C was added 1.2 ml of a 37%
aqueous solution of formaldehyde. The resulting mixture was
hydrogenated for 3.5 hours at atmospheric pressure, then the
catalyst was filtered through celite washing with methanol. The
filtrate was concentrated and the residue was poured into 10 mL of
cold ether with stirring. The product that separated was filtered
out and dried under vacuum. LC/MS showed the expected product, but
was contaminated by baseline impurity.
9-Tert-butyl-7-acetamido-doxycycline
[0095] To a stirred solution of 9-tert-butyl-7-amino-doxycycline
(100 mg) in 2 mL of 1,3-dimethyl-2-imidazolidinone were added 100
mg of sodium bicarbonate followed by 0.07 mL of acetyl chloride.
The mixture was stirred at room temperature for 1.5 hours and then
filtered. The filtrate was concentrated and poured into 100 mL of
cold ether with stirring. The product that separated was filtered
out. The crude product was redissolved in methanol, filtered and
added dropwise to a cold 3:1 mixture of ether/PET ether with
stirring. The product was collected by filtration and dried under
vacuum, and 76 mg of product was obtained.
4-Dedimethylamnino-doxycycline
[0096] To a stirred solution of tetracycline methiodide (860 mg) in
12 mL of acetic acid and 12 mL of water were added 432 mg of zinc
dust. After stirring for 15 min the zinc powder was filtered out.
The filtrate was diluted with 86 mL of water containing 0.86 mL of
concentrated hydrochloric acid. The product that separated was
filtered out and dried under vacuum, and 419 mg of product was
obtained.
4-Dedimethylamino-9-nitro-doxycycline
[0097] To a stirred solution of 4-dedimethylamino-doxycycline (402
mg, 1 mmol) in 20 mL of concentrated sulfuric acid at 0.degree. C.
was added 111 mg of potassium nitrate. After stirring for 20
minutes, the reaction mixture was poured into 100 mL of cold water.
n-Butanol was added to the water containing the product and the
organic layer was separated. Extraction with n-butanol was repeated
twice. The combined organic extracts were concentrated and added to
cold water with stirring. The product that separated was filtered
out; more product was recovered by re-extracting the aqueous
filtrate with n-butanol, concentrating, pouring into cold water and
filtering. After drying under vacuum, 353 mg of product were
obtained.
4-Dedimethylamino-9-amino-doxycycline
[0098] To a stirred solution of
4-dedimethylamino-9-nitro-doxycycline (353 mg) in 17 mL of ethylene
glycol monomethyl ether were added 0.1 mL of concentrated sulfuric
acid and 200 mg of 10% Pd/C, and the resulting mixture was
hydrogenated at atmospheric pressure for 10 hours. The catalyst was
filtered through celite, washing with methanol. The filtrate was
concentrated and poured into 100 mL of ether. The solid that
separated was filtered out and dried under vacuum, and 292 mg of
product was obtained.
4-Dedimethylamino-9-diazonium-doxycycline Hydrochloride
[0099] To a stirred solution of
4-dedimethylamino-9-amino-doxycycline (220 mg) in 5 mL of 0.1N
hydrochloric acid in methanol cooled in an ice bath was added 0.2
mL of n-butyl nitrite. The solution was stirred at 0.degree. C. for
30 minutes, then poured into 150 mL of a 2:1 cold mixture of
ether/PET ether with stirring. The solid that separated was
filtered out and dried under vacuum, and 191 mg of product was
obtained.
4-Dedimethylamino-9-azido-doxycycline
[0100] To a stirred solution of
4-dedimethylamino-9-diazonium-doxycycline (150 mg, 0.32 mmol) in
6.5 mL of 0.1N hydrochloric acid in methanol cooled in an ice bath
were added 23 mg of sodium azide, and the resulting mixture was
stirred for 1 hour at 0.degree. C. The mixture was then poured into
120 mL of cold ether with stirring. Some solid formed, which was
filtered out and found not to be the expected product. The filtrate
was concentrated to dryness, taken up in methanol and poured into
50 mL of cold water with stirring. The solid that separated was
filtered out and dried under vacuum; more product was obtained by
extracting the filtrate with n-butanol, concentrating, pouring into
water and filtering out the solid, and 84 mg of product was
obtained.
4-Dedimethylamino-7-nitro-9-tert-butyl-doxycycline
[0101] A solution of 4-dedimethylamino-doxycycline (500 mg 1.25
mmol) in 3 mL of tert-butanol and 15 mL of methanesulfonic acid was
stirred at room temperature for 15 hours, then 140 mg of potassium
nitrate was added and the resulting mixture stirred for an
additional hour. The reaction mixture was poured in 200 ml of cold
water with stirring. The solid that separated was filtered out and
air dried. The crude product was dissolved in the minimum amount of
methanol and purified by BPLC. The combined fractions from HPLC
were concentrated to dryness, dissolved in methanol and added
dropwise to 50 mL of cold water. The solid that separated was
filtered out and dried under vacuum, and 217 mg of product was
obtained.
4-Dedimethylamino-7-amino-9-tert-butyl-doxycycline
[0102] To a stirred solution of
4-dedimethylamino-7-nitro-9-tert-butyl-doxycycline (217 mg, 0.42
mmol) in 12 mL of ethylene glycol monomethyl ether were added 170
mg of 10% Pd/C, and the resulting mixture was hydrogenated at
atmospheric pressure for 13 hours. The catalyst was filtered
through celite, washing with methanol. The filtrate was
concentrated and poured into 20 of a 3:1 mixture of ether/PET
ether, and 40 mg of product was obtained. The orange filtrate was
concentrated to dryness to obtain additional 138 mg of product.
4-Dedimethylamino-7-diazonium-9-tert-butyl-doxycycline
[0103] To a stirred
4-dedimethylamino-7-amino-9-tert-butyl-doxycycline (165 mg) in 5 mL
of 0.1N hydrocholirc acid in methanol cooled in an ice bath was
added 0.15 mL of n-butyl nitrite. The solution was stirred at
0.degree. C. for 30 minutes; half of the material was poured into
50 mL of cold ether with stirring. The solid that separated was
filtered out and dried under vacuum, and 50 mg of product was
obtained.
4-Dedimethylamino-7-amino-doxycycline
[0104] To a stirred solution of 4-dedimethylamino-doxycycline (400
mg) in 4 mL of THF and 4.5 mL of methanesulfonic acid at 0.degree.
C. were added 418 mg (1.4 mmol) of dibenzyl azodicarboxylate, and
the resulting mixture was stirred for 4 hours while warming to room
temperature. Water and n-butanol were added and two layers
separated. The aqueous layer was extracted with a mixture of
ether/n-butanol. The combined organic extracts were concentrated to
dryness, and 10 mL of ethylene glycol monomethyl ether were added.
Then, 430 mg of 10% Pd/C were added and the resulting mixture
hydrogenated at atmospheric pressure for 12 hours. The catalyst was
filtered off through celite, washing with methanol. The filtrate
was concentrated and poured into a 3:1 cold mixture of ether/PET
ether with stirring; the product that separated was filtered out
and dried under vacuum, and 114 mg of product was obtained.
9-Nitro-minocycline Sulfate
[0105] To a stirred solution of minocycline hydrochloride (1 g,
2.02 mmol) in 30 mL of concentrated sulfuric acid at 0.degree. C.
was added potassiumnitrate (246 mg). The resulting mixture was
stirred for 45 minutes at 0.degree. C., then poured into 1.2 L cold
ether with stirring. The solid that separated was filtered out,
washed with cold ether and dried under vacuum, and 1.33 g of
product was obtained.
9-Amino-minocycline Sulfate
[0106] To a stirred solution of 9-nitro-minocycline sulfate (500 g,
0.83 mmol) in 10 mL of ethylene glycol monomethyl ether and 7 mL of
methanol were added 250 mg of 10% Pd/C. The reaction mixture was
hydrogenated at atmospheric pressure for 4 hours. The catalyst was
filtered off through celite, washing with methanol. The filtrate
was concentrated, then added dropwise to 600 mL cold ether with
stirring. The solid that separated was filtered out, washed with
cold ether and dried under vacuum, and 465 mg of product was
obtained.
9-Diazonium-minocycline Sulfate Hydrochloride
[0107] To a stirred solution of 9-amino-minocycline sulfate (100
mg) in 3 mL of 0.1N hydrochloric acid in methanol cooled in an ice
bath was added 0.1 mL of tert-butyl nitrite (or n-butyl nitrite).
The solution was stirred at 0.degree. C. for 30 minutes, then
poured into 100 mL of cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 87 mg of an
orange-brown product was obtained.
9-Azido-minocycline Sulfate
[0108] To a stirred solution of 9-diazonium-minocycline sulfate
hydrochloride (485 mg, 0.83 mmol) in 18 mL of 0.1N hydrochloric
acid in methanol were added 59 mg of sodium azide. The solution was
stirred at room temperature for 2 hours, then filtered, and the
filtrate was poured into 500 mL of cold ether with stirring. The
solid that separated was filtered out and dried under vacuum, and
480 mg of beige product was obtained.
9-Acetamido-minocycline
[0109] To a stirred solution of 9-amino-minocycline sulfate (100
mg, 0.175 mmol) in 1.5 mL of 1,3-dimethyl-2-imidazolidinone were
added 100 mg of sodium bicarbonate followed by 0.05 mL of acetyl
chloride. The mixture was stirred at room temperature for 45
minutes and then poured into 150 mL of cold ether with stirring.
The solid that separated was filtered out and dried under vacuum,
and 165 mg of product was obtained.
4-Dedimethylamino-minocycline
[0110] Minocycline free base (175 mg, 0.38 mmol) was dissolved in 2
mL of THF, then 0.43 mL of methyl iodide was added. No
crystallization of the product occurred. The reaction mixture was
concentrated and poured into 200 mL of cold ether/PET ether 40:60
with stirring. The product that separated was filtered out and
dried under vacuum; the product was resubjected to the reaction
conditions for 3 additional days, then precipitated from ether as
before, and 123 mg of minocycline methiodide was obtained. The
crude product was dissolved in 2 mL of acetic acid, and 2 mL of
water and 60 mg of zinc dust were added with stirring. After 15
minutes, the zinc powder was filtered out. The filtrate was diluted
with 15 mL of water containing concentrated hydrochloric acid. The
combined organic extracts were dried over magnesium sulfate and
concentrated. The solid that separated was filtered out and dried
under vacuum, and 150 mg of a yellow product was obtained.
4-Dedimethylamino-9-nitro-minocycline
[0111] 4-Dedimethylamino minocycline (415 mg, 1 mmol) was dissolved
in 30 mL of concentrated sulfuric acid at room temperature. The
solution was cooled on an ice bath and potassium nitrate (110 mg,
1.09 mmol) was added with stirring. The resulting mixture was
stirred for 15 minutes, then poured into 300 mL cold ether with
stirring. The solid that separated was filtered out, washed with
cold ether and dried under vacuum. LC/MS showed incomplete
conversion of starting material so the product was redissolved in
15 mL of concentrated sulfuric acid, 50 mg of potassium nitrate
were added at 0.degree. C. and the resulting mixture was stirred
for 45 minutes. The product was isolated as before, and 542 mg of
product was obtained.
7,9-Dibromo Sancycline
[0112] To a stirred solution of sancycline (50 mg, 0.12 mmol) in
1.5 mL of concentrated sulfuric acid at 0.degree. C. were added 42
mg of N-bromosuccinimide. After 30 minutes, the reaction mixture
was poured into 60 mL of cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 70 mg of
yellow product was obtained.
Mix of 7-nitro and 9-nitro-sancycline
[0113] To a stirred solution of sancycline (40 mg, 0.09 mmol) in
1.5 mL of concentrated sulfuric acid at 0.degree. C. was added 10
mg of potassium nitrate. After 20 minutes, the reaction mixture was
poured into 50 mL of cold ether with stirring. The solid that
separated was filtered out and dried under vacuum, and 59 mg of
product was obtained. LC/MS shows a 1.6:1 ratio of two
mono-nitrated products.
[0114] In further testing of the present invention, the Minimum
Inhibitory Concentrations (MIC) of five test products when
challenged with ten different microorganism strains were evaluated.
The study was an MIC evaluation for five test products, performed
using a modification of the Macrodilution Broth Method outlined in
NCCLS document M7-A5, Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically, Fifth
Edition. Each test product was evaluated, in duplicate, against
challenge suspensions of ten microorganism strains. Prior to using
each product for testing, it was diluted (w/v) in sterile
Mueller-Hinton Broth (MHB) to achieve an initial concentration,
based on a potency of 1000 .mu.g/mg, of 160 .mu.g/mL.
TABLE-US-00001 Product 1 - Tetracycline Derivative 14.8 mg of this
product was dissolved in 92.5 mL of Lot Number: INN01182 (MW 615)
MHB to achieve the initial concentration of 160 .mu.g/mL Product 2
- Tetracycline Derivative 13.6 mg of this product was dissolved in
85.0 mL of Lot Number: INN01183 (MW 627) MHB to achieve the initial
concentration of 160 .mu.g/mL Product 3 - Tetracycline Derivative
11.9 mg of this product was dissolved in 74.4 mL of Lot Number:
INN01185 (MW 655) MHB to achieve the initial concentration of 160
.mu.g/mL Product 4 - Tetracycline Derivative 9.6 mg of this product
was dissolved in 60.0 mL of Lot Number: INN01189 (MW 625) MHB to
achieve the initial concentration of 160 .mu.g/mL Product 5 -
Tetracycline Derivative 13.8 mg of this product was dissolved in
86.25 mL of Lot Number: INN01195 (MW 643) MHB to achieve the
initial concentrations of 160 .mu.g/mL
[0115] Approximately 48 hours prior to testing, separate sterile
tubes of Tryptic Soy Broth were inoculated from lyophilized vials
containing each of the challenge microorganisms, as found in Table
I, below. The broth cultures were incubated at
35.degree..+-.2.degree. C. for approximately 24 hours. The broth
cultures prepared as described above were inoculated onto the
surface of Tryptic Soy Agar contained in Petri plates, and
incubated at 35.degree..+-.2.degree. C. for approximately 24 hours.
This produced "lawns" of the microorganisms on the surface of the
agar plates, which were used to prepare the challenge
suspensions.
[0116] Prior to initiating testing, an initial challenge suspension
containing approximately 1.0.times.10.sup.9 CFU/mL was prepared for
each microorganism by inoculating a test tube of Sodium Chloride
Irrigation with microorganisms taken from the plates of solid
media, prepared as described above. The final challenge suspensions
containing approximately 1.0.times.10.sup.6 CFU/mL were prepared
for each microorganism species, by placing a 0.2 mL aliquot of the
1.0.times.10.sup.9 CFUI/mL suspension into a sterile 250 mL
polypropylene bottle, containing 200 mL of Mueller-Hinton Broth.
The final challenge suspensions were mixed thoroughly prior to use
in testing. The final plated dilutions were 10.sup.3, 10.sup.-4 and
10.sup.-5. These plates were incubated at 35.degree..+-.2.degree.
C. until sufficient growth was observed (Table I).
[0117] Following incubation, the colonies on the plates were
counted manually using a hand-tally counter. Counts in the 30 to
300 CFU range were used in the data calculations. The Initial
Population (CFU/mL) was calculated for each challenge suspension as
follows: CFU/nmL=(C.sub.i.times.10.sup.-D) Where: [0118]
C.sub.i=Average of the 2 Plates Counted
[0119] D=Dilution Factor of the Plate Counts Used The population
(CFU/mL) per tube of product/broth following inoculation was
calculated for each challenge suspension as follows: Population
.times. .times. per .times. .times. Tube .times. .times. ( CFU
.times. / .times. mL ) = ( C i .times. 10 - D ) 2 ##EQU1## Where:
[0120] C.sub.i=Average of the Two (2) Plates Counted [0121]
D=Dilution Factor of the Plate Counts Used [0122] 2=Total Volume
(mL) present in each product/broth tube following inoculation
[0123] The testing procedure was as follows: 30 mL aliquots of
Mueller-ffinton Broth were dispensed into sterile bottles. A 30 mL
aliquot of product (initial concentration of 160 .mu.g/mL) was
dispensed into the first of a series of 11 sterile bottles, each
containing 30 mL of Mueller-Hinton Broth, and mixed thoroughly in
order to achieve the 1:2 (v/v) dilution of product. A 30 mL aliquot
was removed from the bottle prepared as described above, and used
to initiate a 1:2 (v/v) dilution series through the remaining 10
bottles (1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1,024
and 1:2,048 product dilutions), each containing 30 mL of
Mueller-Hinton Broth. Each bottle was mixed thoroughly prior to
removing the 30 mL aliquot required for the next dilution in the
series. 1.0 mL aliquots of each product dilution prepared and 1.0
mL aliquots of the initial product preparation (160 .mu.g/mL
product concentration) were transferred to separate sterile test
tubes. A series of 12 tubes, each containing 1.0 mL of the
appropriate product dilution (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64,
1:128, 1:256, 1:512, 1:1,024, and 1:2,048) was prepared for each
microorganism evaluated against each product (Table I). This
resulted in product concentrations ranging from 160 .mu.g/mL to
0.078 .mu.g/mL.
[0124] A 1.0 mL aliquot of a suspension containing approximately
1.0.times.10.sup.6 CFU/mL was introduced into each product dilution
tube in the series, thereby resulting in a final product dilution
series of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512,
1:1,024, 1:2,048, and 1:4,096, with each dilution tube containing
approximately 5.0.times.10.sup.5 CFU/mL of the challenge
microorganism. This resulted in final product concentrations
ranging from 80 .mu.g/mL to 0.039 .mu.g/mL. The test procedure
described above was performed, in duplicate, for each microorganism
species tested (Table I) against each test product. A positive
control tube (growth control) containing a 1.0 mL aliquot of
Mueller-Hinton Broth and a 1.0 mL aliquot of the challenge
suspension was prepared for each microorganism (Table I). A
negative (media) control tube (sterility control; no microbial
inoculation) of Mueller-Hinton Broth was also prepared.
[0125] Product turbidity controls were prepared for each product by
transferring 1.0 mL aliquots of each product dilution into separate
sterile test tubes. A 1.0 mL aliquot of sterile Mueller-Hinton
Broth was introduced into each product dilution tube created and
mixed thoroughly using a vortex mixer, thereby resulting in a final
product dilution series identical to that described above. The
challenge suspension/product dilution tubes and the controls were
incubated at 35.degree..+-.2.degree. C. for 20 hours, until good
growth was apparent in the positive control tubes.
[0126] Following incubation, the tubes were examined for growth of
the microorganisms, determined visually on the basis of
turbidity.
[0127] The Minimum Inhibitory Concentration (MIC) for each test
product versus each challenge microorganism was recorded as the
highest dilution of test product that completely inhibited growth
of the microorganism, as detected by the unaided eye. The results
of the duplicate runs for each test product versus each
microorganism were recorded and then averaged together to provide
the final reported values. TABLE-US-00002 TABLE I Incubation Time*
Incubation Microorganism Species ATCC # (MIC Tubes) Temp.* Media
Enterococcus faecalis 29212 20 Hours 35.degree. .+-. 2.degree. C.
TSB/TSA/MHB Escherichia coli 25922 20 Hours 35.degree. .+-.
2.degree. C. TSB/TSA/MHB Escherichia coli (Tetracycline-Resistant)
51657 20 Hours 35.degree. .+-. 2.degree. C. TSB/TSA/MHB Escherichia
coli (Tetracycline-Resistant) 51658 20 Hours 35.degree. .+-.
2.degree. C. TSB/TSA/MHB Escherichia coli (Tetracycline-Resistant)
51659 20 Hours 35.degree. .+-. 2.degree. C. TSB/TSA/MHB
Staphylococcus aureus (Tetracycline- 27217 20 Hours 35.degree. .+-.
2.degree. C. TSB/TSA/MHB Resistant) Staphylococcus aureus
(Tetracycline- 27659 20 Hours 35.degree. .+-. 2.degree. C.
TSB/TSA/MHB Resistant) Staphylococcus aureus (Tetracycline- 27660
20 Hours 35.degree. .+-. 2.degree. C. TSB/TSA/MHB Resistant)
Staphylococcus aureus 29213 20 Hours 35.degree. .+-. 2.degree. C.
TSB/TSA/MHB *= Initial Population plates were inadvertently
incubated at room temperature (20.degree.-27.degree. C.) for
approximately 48 hours prior to being incubated at 35.degree. .+-.
2.degree. C. for approximately 72 hours.
[0128] Table II presents the Minimum Inhibitory Concentrations,
expressed as product dilution, for each test product versus each of
the 10 microorganisms tested. Table In presents the Minimum
Inhibitory Concentrations, expressed as product concentration
(.mu.g/mL), for each test product versus each of the 10
microorganisms tested. The initial solution prepared for each
product had a concentration, based on a potency of 1000 .mu.g/mg,
of 160 .mu.g/mL. TABLE-US-00003 TABLE II Minimum Inhibitory
Concentrations (Expressed as Product Dilution) Microorganism
Species ATCC # Product #1 Product #2 Product #3 Product #4 Product
#5 Enterococcus faecalis 29212 <1:2 1:16 1:16 1:2 1:8
Escherichia coli 25922 1:2 <1:2 <1:2 <1:2 1:2 Escherichia
coli (Tetracycline- 51657 <1:2 <1:2 <1:2 <1:2 <1:2
Resistant) Escherichia coli (Tetracycline- 51658 <1:2 <1:2
<1:2 <1:2 <1:2 Resistant) Escherichia coli (Tetracycline-
51659 <1:2 <1:2 <1:2 <1:2 <1:2 Resistant)
Pseudomonas aeruginosa 27853 <1:2 <1:2 <1:2 <1:2
<1:2 Staphylococcus aureus 27217 <1:2 1:32 1:32 1:8 1:16
(Tetracycline-Resistant) Staphylococcus aureus 27659 <1:2 1:32
1:24 1:8 1:16 (Tetracycline-Resistant) Staphylococcus aureus 27660
<1:2 1:32 1:16 1:8 1:32 (Tetracycline-Resistant) Staphylococcus
aureus 29213 1:8 1:32 1:32 1:8 1:32
[0129] TABLE-US-00004 TABLE III Minimum Inhibitory Concentrations
(Expressed as Product Dilution) Microorganism Species ATCC #
Product #1 Product #2 Product #3 Product #4 Product #5 Enterococcus
faecalis 29212 >80 10 10 80 20 Escherichia coli 25922 80 >80
>80 >80 80 Escherichia coli 51657 >80 >80 >80 >80
>80 (Tetracycline-Resistant) Escherichia coli 51658 >80
>80 >80 >80 >80 (Tetracycline-Resistant) Escherichia
coli 51659 >80 >80 >80 >80 >80
(Tetracycline-Resistant) Pseudomonas aeruginosa 27853 >80 >80
>80 >80 >80 Staphylococcus aureus 27217 >80 5 5 20 10
(Tetracycline-Resistant) Staphylococcus aureus 27659 >80 5 6.67
20 10 (Tetracycline-Resistant) Staphylococcus aureus 27660 >80 5
10 20 5 (Tetracycline-Resistant) Staphylococcus aureus 29213 20 5 5
20 5
[0130] In additional studies, the activity of the tetracycline
derivatives of the present invention as inhibitors of matrix
degrading metalloproteinases (MMPs) that are involved in malignant
tumor growth and metastasis were investigated. The studies used the
Dunning MAT LyLu tumor model which was developed in Copenhagen rats
from a transplantable tumor, transferred from an original prostate
tumor from the dorsal prostate of an aged Copenhagen male rat. The
MAT LyLu tumor is spontaneously metastatic to lymph nodes and lungs
when injected, and produces bone metastases in approximately
80-100% of the recipient animals. Fresh dosing solutions containing
10 mg/ml of each test article in 2% carboxymethylcellulose were
prepared daily and used in the testing.
[0131] The studies initially evaluated toxicity in two groups of
five non-tumor bearing animals, each receiving the two tetracycline
analogs (9-amino-doxycycline and 9-nitro-doxycycline) at a dose of
40 mg/kg daily for seven days.
[0132] Three groups of animals (10/group) were dosed by gavage with
either vehicle, or one of the two analogs daily for three weeks,
and then three times per week until death or sacrifice. Dosing was
begun seven days prior to implantation of the MAT LyLu tumor cells
by tail vein injection. Approximately 0.1 ml of tumor cell
suspension was injected into one of the lateral tail veins. The
dose to be chosen was based on two factors: (1) efficacy and (2)
morbidity associated with the analog, if any. The following table
(Table IV below), demonstrates survival time and weekly body weight
of animals in control and treated groups. As shown in the table,
9-amino-doxycycline and 9-nitro-doxycycline demonstrated a marked
increase in surviving number after tumor implantation, while also
resulting in indistinguishable body weight changes as compared to
the control group. This latter finding is particularly significant,
in that conventional anti-cancer treatments often result in
significant weight loss. TABLE-US-00005 TABLE IV Number of Days
Post Tumor Surviving Number Implantation 9-amino-doxycycline
9-nitro-doxycycline Control 14 10 8 9 17 10 8 8 24 10 8 7 27 10 8 6
28 9 8 6 29 9 6 5 30 7 4 5 34 7 4 3 42 7 4 3
[0133] In further studies, the inhibitory activity of MMPs, as well
as percent inhibition of various cancers by various tetracycline
derivative were evaluated. Results appear in Table V, below, and
demonstrate the efficacy of the treatments of the present
invention. One objective of the studies was to assess a series of
tetracycline derivatives as inhibitors of purified human matrix
metalloproteinases including MMP1, 2, 3, 7, 9 and 14. The
tetracycline derivatives were effective inhibitors in the mM-.mu.M
range.
Experimental Protocols
1. Protocol for Measuring MMP1 (Collagenase-1) Inhibition by
Tetracycline Derivatives
[0134] MMP1 activity was determined by the release of soluble
.sup.14C labelled collagen fragments from .sup.14C acetylated rat
skin type I collagen (Methods in Enzymology 80 711, 1981).
Assay Constituents:
[0135] 100 .mu.g .sup.14C labelled rat skin type I collagen (5-6000
dpm) [0136] 50 mM Tris HCl pH 7.9 [0137] 15 mM CaCl.sub.2 0.02%
azide [0138] Human recombinant MP1 3-4 nM [0139] .+-.Tetracycline
(up to 1 mM) in a total volume of 300 .mu.l.
[0140] Incubations were at 35.degree. C. for 5 hours. Uncleaved
collagen fibrils (which form within the first 10 minutes) were
removed by centrifugation at 10,000 g, 4.degree. C., 10 minutes.
200 .mu.l supernatant were counted in Packard Opti Phase Supermix
scintillation fluid using a scintillation counter. Only data
derived from within the linear part of the assay (10-70% lysis of
the collagen) were utilized. Based on percentage inhibition of the
activity of MNP1 alone by a range of concentrations of each sample,
the IC50 value for each tetracycline was calculated by linear
regression analysis.
2. Protocol for Measuring MMP2 (Gelatinase A) Inhibition
[0141] MMP2 activity was determined by the release of
trichloracetic acid, TCA, soluble fragments from 14C acetylated rat
skin type I gelatin (Methods in Enzymology 248, 470, 1995. Biochem.
J. 195, 1981)
Assay Constituents:
[0142] 100 .mu.g .sup.14C labelled gelatin (type I collagen
denatured 60.degree. C. 20 min) [0143] 50 mM Tris HCl pH 7.9 [0144]
15 mM CaCl.sub.2, 0.02% azide [0145] Human recombinant MMP1,
0.1-0.5 nM [0146] .+-.Tetracycline (up to 1 MM) in a total volume
of 250 .mu.l.
[0147] Incubations were set at 37.degree. C. for 16 hours. The
reaction was stopped by cooling the incubations and addition of 50
.mu.l cold 90% (w/v) trichloracetic acid with careful mixing. TCA
precipitation was allowed to proceed for 15 minutes at 4.degree. C.
prior to cetrifugation at 10,000 g for 10 minutes at 4.degree. C.
200 .mu.l of the TCA supernatant was taken for the determination of
radioactive content by scintillation counting (Packard Opti Phase
Supermix scintillation fluid). Only data derived from within the
linear part of the assay (10-70% lysis) were utilized. The
percentage inhibition of MMP2 by each tetracycline concentration
was plotted to derive the IC50 value, using linear regression
analysis.
3. Protocol for Measuring MMP3 (Stromelysin 1) Inhibition
[0148] MMP3 activity was determined by the release of
trichloracetic acid, TCA, soluble fragments from .sup.14C
acetylated B casein (Sigma) as described in Methods in Enzymology
248, 451 (1995).
Assay Constituents:
[0149] 100 .mu.g .sup.14C casein (7000 dpm) [0150] 50 mM Tris HCl,
pH 7.9 [0151] 15 mM CaCl.sub.2, 0.02% azide [0152] Human
recombinant MMP3, 0.25-1.0 nM [0153] .+-.tetracycline (up to 1 mM)
in a total volume of 250 .mu.l.
[0154] Incubations were at 37.degree. C. for 16 hours. The reaction
was stopped by the addition of 500 .mu.l cold 18% TCA and standing
on ice for 15 minutes. TCA precipitates were removed by
centrifugation at 10,000 g for 10 minutes at 4.degree. C. and 200
.mu.l supernatant was taken for scintillation counting. Only data
derived from within the linear part of the assay (10-60% lysis)
were utilized. The percentage inhibition of MMP3 by each
concentration of the candidate tetracycline was plotted to derive
the IC50 value, using linear regression analysis.
4. Protocol for Measuring MMP7 (Matrilysin) Inhibition
[0155] MMP7 activity was determined by the release of
trichloracetic acid, TCA, soluble fragments from .sup.14C
acetylated B casein (Sigma) as described in Methods in Enzymology
248, 451 (1995).
Assay Constituents:
[0156] 100 .mu.g .sup.14C casein (7000 dpm) [0157] 50 mM Tris HCl,
pH 7.9 [0158] 15 mM CaCl.sub.2, 0.02% azide [0159] Human
recombinant MMP7, 1.5nM [0160] .+-.tetracycline (up to 1 mM) in a
total volume of 250 .mu.l.
[0161] Incubations were at 37.degree. C. for 16 hours. The
incubation was stopped by the addition of 500 .mu.l cold 18% TCA
and standing on ice for 15 minutes. TCA precipitates were removed
by centrifugation at 10,000 g for 10 minutes at 4.degree. C., and
200 .mu.l supernatant was taken for scintillation counting. Only
data derived from within the linear part of the assay (10-60%
lysis) were utilized. The percentage inhibition of MMP7 by each
concentration of the candidate tetracycline were plotted to derive
the IC50 value.
5. Protocol for Measuring MMP9 (Gelatinase B) Inhibition
[0162] MMP9 activity was determined by the release of
trichloracetic acid, TCA soluble fragments from .sup.14C acetylated
rat skin type I (Methods in Enzymology 248, 470, 1995. Biochem. J.
195 1981)
Assay Constituents:
[0163] 100 .mu.g 1.sup.4C labelled gelatin (collagen denatured
60.degree. C. 20 min) [0164] 50 mM Tris HCl pH 7.9 [0165] 15 mM
CaCl.sub.2, 0.02% azide [0166] Human recombinant MMP9, 1.2-2 nM
[0167] .+-.Tetracycline (up to 1 mM) in a total volume of 250
.mu.l.
[0168] Incubations were at 37.degree. C. for 16 hours. The
incubations were stopped by cooling the incubations and addition of
50 .mu.l cold 90% (w/v) trichloracetic acid with careful mixing.
TCA precipitation was allowed to proceed for 15 minutes at
4.degree. C. prior to centrifugation at 10,000 g for 10 minutes at
4.degree. C. 200 .mu.l of the TCA supernatant was taken for the
determination of radioactive content by scintillation counting
(Packard Opti Phase Supermix scintillation fluid). Only data
derived from within the linear part of the assay (10-70% lysis)
were utilized. The percentage inhibition of MMP9 by each
tetracycline concentration were plotted to derive the IC50
value.
6. Protocol for Measuring MMP14 (MTI-MAP) Inhibition by
Tetracycline Derivatives
[0169] MMP14 activity was determined by the release of soluble
.sup.14C labelled collagen fragments from .sup.14C acetylated rat
skin type I collagen (Methods in Enzymology 80 711, 1981).
Assay Constituents:
[0170] 100 .mu.g 1.sup.4C labelled collagen (5-6000 dpm) [0171] 50
mM Tris HCL pH 7.9 [0172] 15 mM CaCl.sub.2 0.02% azide [0173] Human
recombinant MMP14 50-100 nM [0174] .+-.Tetracycline (up to 1 mM) in
a total volume of 300 .mu.l.
[0175] Incubations were at 35.degree. C. for 15 hours. Uncleaved
collagen fibrils (which form within the first 10 minutes) were
removed by centrifugation at 10,000 g 4.degree. C. 10 minutes 200
.mu.l supernatant were counted in Packard Opti Phase Supermix
scintillation fluid using a scintillation counter. Only data
derived from within the linear part of the assay (10-70% lysis of
the collagen) were utilized. Based on percentage inhibition of the
activity of MMP1 alone by a range of concentrations of each sample,
the IC50 value for each tetracycline was calculated.
[0176] Another objective was to analyze the effects of specific,
tetracycline derived compounds on the in-vitro chemo-invasive
potential of: [0177] C8161 human melanoma cells [0178] MDA-MB-231
human breast cancer cells [0179] PC3 human prostate cancer cells
[0180] RPM18226 human myeloma cancer cells [0181] Ark human myeloma
cancer cells [0182] Arp-1 human myeloma cancer cells
[0183] The Membrane Invasion Culture System (MICS) in vitro
chemo-invasion assay was chosen to measure changes in the invasive
potential of specific human cancer cells in response to
tetracycline derived compounds Stock solutions of each compound
were hydrated in water containing 2% DMSO and pH 10.0. Upon
solubilization, HCl was added to the solution to establish a pH of
7.5-8.0. This solution was then wrapped in foil and kept at
4.degree. C. during the 24 hours of assay. Fresh compound was
prepared for each assay. The in vitro chemoinvasion analyses for
tumor cell invasiveness are performed using the Membrane Invasion
Culture System (MICS) as previously described.
[0184] The MICS system is a thermally treated plastic manifold
system consisting of two matched sets of plates containing fourteen
wells, 13 mm in diameter. Interposed between these plates was a
polycarbonate filter containing 10 .mu.m pores and coated with a
defined matrix composed of human laminin/collagen IV/gelatin that
formed a 35 .mu.m thick barrier between the top and bottom sections
of the wells in MICS. Prior to placing this barrier in place, the
lower wells were filled with serum-free RPMI culture medium made
50% in conditioned medium obtained from 2-day-old cultures of human
fibroblasts which had been cleared of cells and cellular debris by
either centrifugation, or filtering through a 0.45 .mu.m sterile
filter. The barrier was then placed on the lower wells and fresh
serum-free medium added to the upper wells after assembly of the
system. Fifty thousand tumor cells were then added to the wells
either in the presence of the compound, or the DMSO vehicle (the
controls). Of the 12 assay wells on each manifold, 3 randomized
wells acted as controls, 3 wells received 1 .mu.g/ml of compound, 3
received 10 .mu.g/ml and 3 received 25 .mu.g/ml. After 24 hours,
cells and media were removed from the lower wells and replaced by
phosphate buffered saline plus 2 mM EDTA. This solution was used to
remove all cells from the lower well, and this wash was added to
the recovered cells plus medium from the same well. These samples
were then collected onto a polylysine containing polycarbonate
membrane containing 3 .mu.m pores using a Dot-Blot manifold system.
These collection filters were then fixed in methanol, and stained
using Wright's stain (Leukostat staining kit) in situ. The filters
were then mounted on microscope slides with microscope immersion
oil (which reduced the light refraction from the pores) and 5
microscopic fields were counted to calculate the number of cells
which invaded through the membrane over the 24 hour period. These
numbers were then statistically analyzed as discussed below.
TABLE-US-00006 TABLE V HUMAN CELL LINE EVALUATED INHIBITORY
ACTIVITY OF MATRIX METALLOPROTEINASES AND PERCENT INHIBITION
(MMPs).sup.1 Breast COMPOUND NO. MMP-1 MMP-2 MMP-3 MMP-7 MMP-9
MMP-14 MDA-MB-231 1 267 >700 258 815 43 86 7.sup.B 20.sup.B
5.sup.B 25.sup.B 25.sup.B 32.sup.B 2 479 80 348 >1000 117 119 20
15 30.sup.B 3 >1000 >1000 304 >1000 136 216 +8 +2 12 4 409
421 219 278 36 61 5 6 2 5 647 86 237 441 194 447 6 +4 46.sup.B 6
394 135 241 >1000 83 56 +5 +17.sup.B 27.sup.B 24 44.sup.B
42.sup.B 7 R (5) >1000 300 335 64 >1000 +18 +41.sup.B +27 8
>1000 599 232 388 >1000 >1000 +10 +5 +13 9 372 337 154 507
28 29 10 499 484 96 >1000 41 53 9 27.sup.B 37.sup.B +5 0 8 11
427 >1000 270 >1000 96 46 6 15 36.sup.B 12 >1000 >1000
.sup. 390.sup.2 503 270 >1000 13 126 48 206 >1000 64 14 4 0
+9 14 279 R >1000 R R 386 15 >1000 234 738 716 402 768 16 452
215 >1000 569 215 715 17 122 >1000 350 110 98 18 123 233 335
131 102 19 270 925 394 98 90 20 429 511 281 162 266 21 293 >1000
>1000 79 115 22 396 254 462 341 459 23 >1000 306 >1000
>1000 >1000 24 405 724 191 127 160 25 974 397 303 148 185 26
>1000 413 >1000 384 586 27 498 963 149 101 92 15 35 72.sup.B
5 17 48.sup.B 28 >1000 >1000 231 125 49 +24 +36.sup.B
54.sup.B +7 +15 45.sup.B 29 895 983 213 172 98 +41.sup.B 7 59.sup.B
+13 +25.sup.B 17 30 576 >1000 938 >1000 >1000 31 621 258
>1000 311 593 32 601 236 244 214 197 33 295 263 205 142 94 34
242 242 136 308 197 35 947 249 218 397 108 36 539 >1000 283
>1000 608 37 279 >1000 274 445 41 +6 1 46.sup.B 38 230 175 95
215 69 +7 37.sup.B 70.sup.B +14 +14 37 39 860 186 682 114 83
18.sup.B 29.sup.B 76.sup.B 40 560 362 91 R 29 9 15 20.sup.B 41 478
889 54 250 403 +8 3 2 42 R 606 1557 405 428 43 600 2482 174 319 44
1438 1468 224 591 45 384 2446 194 6626 46 194 3803 42 59 90
20.sup.B +5 +1 47 318 117 68 88 60 +17 +16.sup.B 0 48 12831 1929
506 424 354 49 372 1572 141 489 87 6 +5 +1 50 211 2533 48 34 40 7 0
2 51 +15.sup.B +18 25.sup.B 52 >1000 250 >1000 >1000 32 68
5 12 52.sup.B 53 9 49.sup.B 83.sup.B HUMAN CELL LINE EVALUATED AND
PERCENT INHIBITION Multiple Melanoma Myeloma Prostrate COMPOUND NO.
C8161 RPMI-8226 PC-3 ML 1 5 20 35.sup.B 20 10 15 2 47.sup.B
60.sup.B 40.sup.B 49.sup.B 56.sup.B 21.sup.B 21.sup.B 29.sup.B
13.sup.B 24.sup.B 24.sup.B 2 20.sup.B 25.sup.B 27.sup.B +8
+10.sup.B +11 20 8 15 1.sup.B +4.sup.B 7.sup.B 3 30.sup.B 22.sup.B
15.sup.B +7 +23.sup.B 27.sup.B 3 +1 25.sup.B +22 +3 +24.sup.B 4
27.sup.B 24.sup.B 17 +4 +5 +14 10 +8 3 5 15 27.sup.B 35.sup.B 6 4
36 44.sup.B 35 46.sup.B 17 37.sup.B 42.sup.B 6 18.sup.B 33.sup.B
32.sup.B +6 +25.sup.B +5 21 26 41.sup.B 8 21.sup.B 33.sup.B
12.sup.B 10 5 13 32.sup.B 30.sup.B 7 7 14 16 +14.sup.B 12 +2 0 0 3
8 13.sup.B 41.sup.B 40.sup.B 11 3 +2 0 +4 0 9 10 21 22 11 3 5
29.sup.B 40.sup.B 50.sup.B 65.sup.B 7 28.sup.B 30.sup.B 9 16 5 11
16 46.sup.B 41.sup.B +3 0 36.sup.B 12 +11 +13 +12 13 6 12 14 +11
+13 +12 8 23.sup.B 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 0
+1 73 24.sup.B 6 42.sup.B 17.sup.B 35.sup.B 70 11 +38.sup.B
+51.sup.B 20 9 11 9 56.sup.B 70 28 +20 34.sup.B 40.sup.B 4 +10
34.sup.B 7 +11.sup.B 80 +25.sup.B +53.sup.B 19 5 +3 62.sup.B
35.sup.B 28.sup.B 61 29 12 +20 61.sup.B 13 12 8 28.sup.B 50.sup.B
58 +10 +14.sup.B 1 29.sup.B 13 35.sup.B 8 8 69 30 31 32 33 34 35 36
37 +1 20 69.sup.B 10 5 46.sup.B 11 26 36 38 3 1 66.sup.B 33.sup.B
13 +11 37.sup.B 41.sup.B 41 +28 +26 39 4 +49.sup.B +8 38.sup.B
27.sup.B 52 39 10 11 24 5 10 46.sup.B 44.sup.B 67.sup.B 69.sup.B 40
+1 +5 +4 16 11 9 +1 +7 +4 41 2 8 6 7 +6 14 +4 4 4 42 43 44 45 46 21
7 10 7 +11 12 20 16 27.sup.B 47 +30 +4 +2 28 18 60.sup.B 3 3 5 48
49 27.sup.B 7 30.sup.B 11 +2 15 4 +5 6 50 5 +2 1 +14 +8 +13 +14 +17
+10 51 +9 +31.sup.B +122.sup.B 14 +26.sup.B +2 +4 +13 4 52 5 15 30
2 26.sup.B 68.sup.B 52.sup.B 68.sup.B 70.sup.B 19.sup.B 33.sup.B
46.sup.B 31.sup.B 48.sup.B 63.sup.B 53 +40.sup.B +158.sup.B
83.sup.B +95.sup.B 18 84.sup.B 29.sup.B 21 45.sup.B INHIBITORY
ACTIVITY OF MATRIX COMPOUND METALLOPROTEINASES (mmPs).sup.1 CELL
LINE EVALUATED AND PERCENT INHIBITION.sup.A NO. MMP-1 MMP-2 MMP-3
MMP-7 MMP-9 MMP-14 RPMI-8226 ARP-1 ARK 1 267 >700 258 815 43 86
20 10 15 +9 +13 1 9 +9 +8 21.sup.B 21.sup.B 29.sup.B 13 8 17.sup.B
2 479 80 348 >1000 117 119 +8 +10.sup.B +11 10 7 6 20.sup.B
9.sup.B 10 1.sup.B +4.sup.B 7.sup.B 3 >1000 >1000 304
>1000 136 216 +7 +23.sup.B +27.sup.B +13 +1 +16 +11 +6 +15 +22
+3 +24.sup.B 5 647 86 237 441 194 447 6 4 36 +25.sup.B 1 4.sup.B +7
22.sup.B 21.sup.B 17 37.sup.B 42.sup.B 6 394 135 241 <1000 83 56
+6 +25.sup.B +5 NE 12 0 8 12.sup.B 10 5 9 372 337 154 507 28 98
21.sup.B 24.sup.B 28.sup.B +14 +21.sup.B 7 17.sup.B 24.sup.B
20.sup.B 10 499 484 96 >1000 41 53 3 5 29.sup.B NE 6 +7 1 13 126
48 206 >1000 64 14 +11 +13 +12 NE 2 +7 1 For Table V:
.sup.Aconcentrations of 1, 10 and 25 ug/mL were used
.sup.Bsignificant, i.e., P < 0.05 + = increased invasiveness NE
= not evaluated R = to be repeated (number times repeated)
.sup.1IC.sub.50 in .mu.M unless specified otherwise
.sup.2Predicted
[0185] Key to Table V: TABLE-US-00007 Compound Substance 1:
9-nitro-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4 2:
9-amino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4 3:
9-Isopropylamino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4 4:
7-Dimethylamino-6-demethyl-6-deoxy-9-nitrotetracycline
H.sub.2SO.sub.4 5: Same as 2 6:
9-Azido-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4 7:
9-Amino-7-dimethylamino-6-demethyl-6-deoxytetracycline
H.sub.2SO.sub.4 8:
9-Acetamido-7-dimethylamino-6-demethyl-6-deoxytetracycline
H.sub.2SO.sub.4 9:
7-dimethylamino-6-demethyl-6-deoxytetracycline-9-diazonium
H.sub.2SO.sub.4 HCL 10:
9-Azido-7-demethylamino-6-demethyl-6-deoxytetracycline
H.sub.2SO.sub.4 11: 6-Deoxy-5-hydroxytetracycline-9-diazonium
H.sub.2SO.sub.4 12: 7-Amino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 13: 7,9-Dibromo-6-demethyl-6-deoxytetracycline
H.sub.2SO.sub.4 14: 7-Amino-6-deoxy-5-hydroxy-9-nitrotetracycline
H.sub.2SO.sub.4 15: 7-Dimethylamino-6-deoxy-5-hydroxy-tetracycline
H.sub.2SO.sub.4 16: 9-Acetamido-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 17: 7-Di-n-Butylamino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 18: 7-Di-n-Hexylamino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 19:
7-Di-(3,3-dimethylbutyl)amino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 20: 7-Azido-6-deoxy-5-hydroxy-tetracycline
H.sub.2SO.sub.4 21: 6-Deoxy-5-hydroxytetracycline-7-diazonium
H.sub.2SO.sub.4 22:
7-Acetamido-9-nitro-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
23: 7-Acetamido-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4 24:
7-Di-n-propylamino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
25: 7-Isobutylamino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
26: 7-Acetamido-9-amino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 27:
7-Isobutylmethylamino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
28: 6-Deoxy-5-acetoxy-tetracycline H.sub.2SO.sub.4 29:
7-Acetylisobutylamino-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
30: 7-Acetamido-6-deoxy-5-hydroxytetracycline-9-diazonium
H.sub.2SO.sub.4 31:
7-Dimethylamino-6-deoxy-5-hydroxytetracycline-9-diazonium
H.sub.2SO.sub.4 32: 7-Cyclobutylamino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 33:
7-Cyclobutylmethylamino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 34: 4-Dedimethylamino-6-deoxy-5-hydroxytetracycline
35: 4-Dedimethylamino-6-deoxy-5-hydroxy-9-nitrotetracycline 36:
9-animo-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate 37:
4-Dedimethylamino-6-deoxy-5-hydroxytetracycline-9-diazonium sulfate
38: 7-nitro-9-tertbutyl-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 39: 9-tertbutyl-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 40:
7-Nitro-9-tertbutyl-4-dedimethylamino-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 41:
4-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline
H.sub.2SO.sub.4 42:
7-Acetylamino-9-azido-6-deoxy-5-hydroxy-tetracycline 43:
9-tertbutyl-6-deoxy-5-hydroxytetracycline-7-diazonium
H.sub.2SO.sub.4 44:
7-Amino-9-tertbutyl-6-deoxy-5-hydroxytetracycline H.sub.2SO.sub.4
45: 7-Azido-9-tertbutyl-6-deoxy-5-hydroxytetracycline
H.sub.2SO.sub.4 46: 6-deoxy-5-hydroxytetracycline-4-Methiodide 47:
7-(1,2-benzylcarboxyhydrazine)-6-deoxy-5-hydroxy-tetracycline HCl
48: 4-Dedimethylamino-7-amino-6-deoxy-5-hydroxyltetracycline
H.sub.2SO.sub.4 49:
7-Amino-9-tertbutyl-4-dedimethyl-6-deoxy-5-hydroxytetracycline-H.sub.2-
SO.sub.4 50:
4-Dedimethylamino-9-tertbutyl-6-deoxy-5-hydroxytetracycline-7-diazoniu-
m H.sub.2SO.sub.4 51:
9-(4-fluorophenyl)-6-deoxy-5-hydroxytetracycline 52:
4-Epi-7-Chlorotetracycline hydrochloride 53:
6-Demethyl-6-deoxytetracycline HCl
[0186] While the present invention has been described with respect
to particular embodiment thereof, it is apparent that numerous
other forms and modifications of the invention will be obvious to
those skilled in the art. The appended claims and this invention
generally should be construed to cover all such obvious forms and
modifications, which are within the true spirit and scope of the
present invention.
* * * * *