U.S. patent application number 11/870317 was filed with the patent office on 2013-05-02 for substituted tetracycline compounds for treatment of bacillus anthracis infections.
The applicant listed for this patent is Michael N. Alekshun, S. Ken Tanaka. Invention is credited to Michael N. Alekshun, S. Ken Tanaka.
Application Number | 20130109617 11/870317 |
Document ID | / |
Family ID | 39148587 |
Filed Date | 2013-05-02 |
United States Patent
Application |
20130109617 |
Kind Code |
A1 |
Alekshun; Michael N. ; et
al. |
May 2, 2013 |
SUBSTITUTED TETRACYCLINE COMPOUNDS FOR TREATMENT OF BACILLUS
ANTHRACIS INFECTIONS
Abstract
Methods and compositions for the treatment of Bacillus anthracis
infections are described.
Inventors: |
Alekshun; Michael N.;
(Marlboro, NJ) ; Tanaka; S. Ken; (Needham,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alekshun; Michael N.
Tanaka; S. Ken |
Marlboro
Needham |
NJ
MA |
US
US |
|
|
Family ID: |
39148587 |
Appl. No.: |
11/870317 |
Filed: |
October 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60851211 |
Oct 11, 2006 |
|
|
|
Current U.S.
Class: |
514/2.9 ;
514/152; 514/154; 514/24; 514/29 |
Current CPC
Class: |
A61K 31/65 20130101;
A61K 38/14 20130101; A61P 31/04 20180101; A61K 31/69 20130101; A61K
31/7056 20130101; A61K 45/06 20130101; A61K 31/43 20130101 |
Class at
Publication: |
514/2.9 ;
514/152; 514/154; 514/24; 514/29 |
International
Class: |
A61K 31/65 20060101
A61K031/65; A61K 31/7056 20060101 A61K031/7056; A61K 31/43 20060101
A61K031/43; A61K 45/06 20060101 A61K045/06; A61K 38/14 20060101
A61K038/14 |
Claims
1-4. (canceled)
5. A method for treating a bacillus anthracis infection in a
subject, comprising administering to said subject an effective
amount of a substituted tetracycline compound, such that said
bacillus anthracis infection in said subject is treated, wherein
said substituted tetracycline compound is of the formula I:
##STR00026## wherein R.sup.2'' is C(.dbd.O)--NR.sup.2R.sup.2';
R.sup.2, R.sup.2', and R.sup.3 are hydrogen; R.sup.4a and R.sup.4b
are each alkyl; R.sup.10, R.sup.11, and R.sup.12 are each hydrogen;
R.sup.4 and R.sup.4' are each independently NR.sup.4aR.sup.4b;
R.sup.5 and R.sup.5' are each hydrogen; R.sup.6 and R.sup.6' are
each hydrogen; R.sup.7 is hydrogen, dialkylamino, alkyl, aryl,
heterocyclic, or alkyl-O--N.dbd.C--CR.sup.7gR.sup.7h, wherein
R.sup.7g and R.sup.7h are each independently hydrogen or alkyl;
R.sup.8 is hydrogen; R.sup.9 is of the formula: ##STR00027##
wherein: J.sup.5 and J.sup.6 are each independently hydrogen,
alkyl, alkenyl, or linked to form a ring; and J.sup.7 and J.sup.8
are each alkyl, halogen, or hydrogen; and X is CR.sup.6'R.sup.6; or
a pharmaceutically acceptable salt, ester or enantiomer
thereof.
6-9. (canceled)
10. The method of claim 5, wherein R.sup.7 is substituted or
unsubstituted heteroaryl.
11. The method of claim 10, wherein R.sup.7 is substituted or
unsubstituted pyrimidinyl, pyridinyl, or furanyl.
12-13. (canceled)
14. The method of claim 5, wherein R.sup.7 is hydrogen.
15-18. (canceled)
19. The method of claim 5, wherein J.sup.7 and J.sup.8 are each
hydrogen.
20. The method of claim 19, wherein J.sup.6 is hydrogen.
21. The method of claim 19, wherein J.sup.5 is substituted or
unsubstituted alkyl.
22. The method of claim 21, wherein J.sup.5 is propyl.
23-27. (canceled)
28. The method of claim 19, wherein J.sup.5 and J.sup.6 are linked
to form a ring.
29. The method of claim 28, wherein J.sup.5 and J.sup.6 are linked
to form a substituted or unsubstituted piperidinyl ring or fused
ring.
30. The method of claim 29, wherein said fused ring is
2,3-dihydro-indole or decahydro-isoquinoline.
31. The method of claim 29, wherein said piperidinyl ring is
substituted with one or more halogens or one or more heterocyclic
groups.
32. The method of claim 5, wherein said substituted tetracycline
compound is selected from the group consisting of: ##STR00028##
##STR00029## ##STR00030##
33. The method of claim 5, wherein said substituted tetracycline
compound is administered in combination with a second agent.
34. The method of claim 33, wherein said second agent is an
antibiotic.
35. The method of claim 34, wherein said second agent is selected
from the group consisting of rifampin, vancomycin, ampicillin,
chloramphenicol, imipenem, clindamycin, and clarithromycin.
36. The method of claim 5, wherein said bacillus anthracis is
multidrug resistant.
37-57. (canceled)
58. The method of claim 5, wherein R.sup.7 is dimethylamino.
59. The method of claim 22, wherein R.sup.7 is substituted or
unsubstituted heteroaryl, dimethylamino, or hydrogen.
60. The method of claim 28, wherein R.sup.7 is substituted or
unsubstituted heteroaryl, dimethylamino, or hydrogen.
61. The method of claim 29, wherein R.sup.7 is substituted or
unsubstituted heteroaryl, dimethylamino, or hydrogen.
62. The method of claim 30, wherein R.sup.7 is substituted or
unsubstituted heteroaryl, dimethylamino, or hydrogen.
63. The method of claim 31, wherein R.sup.7 is substituted or
unsubstituted heteroaryl, dimethylamino, or hydrogen.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/851,211, filed on Oct. 11, 2006, the entire
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] In the fall of 2001, letters intentionally contaminated with
Bacillus anthracis were mailed to individuals in Florida,
Washington, D.C., and New York City. These events resulted in
exposures both at the sites of delivery and also at sites the
letters passed through in New Jersey, Pennsylvania, Virginia,
Maryland, and Connecticut. In total, there were 11 cases of
documented inhalation anthrax infections, including 5 deaths, and
11 cases of documented cutaneous anthrax infections. Antimicrobial
prophylaxis for at least 60 days was recommended for about 10,000
individuals; ultimately, about 32,000 people actually received
prophylactic therapy.
[0003] The public health crisis in antibiotic resistance generally
focuses on nosocomial and community-acquired infections with
organisms that have naturally become resistant to multiple agents.
This situation has developed due to a combination of antibiotic use
(including overuse and misuse) and the emergence of freely
transmissible resistance determinant(s). Organisms that might be
(or have been) used by bioterrorists could acquire antibiotic
resistance not only naturally, but also as a result of intentional
manipulation.
[0004] Ciprofloxacin, doxycycline, and penicillin G procaine
(penicillin) are the three drugs currently approved for intravenous
therapy of all forms of anthrax (cutaneous (skin), inhalation, and
gastrointestinal) infection. Mobile elements that confer resistance
to tetracyclines and penicillins can be introduced into B.
anthracis and are functional; resistance to ciprofloxacin can be
induced by passage in vitro. Thus, there is a real possibility of
multiple drug resistant (MDR) anthrax and alternative agents
effective against such strains are needed.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the invention pertains to novel,
narrow-spectrum, orally bioavailable substituted tetracycline
compounds that are active against B. anthracis, including strains
expressing resistance to known tetracycline resistance
elements.
[0006] In a further embodiment, the invention pertains to a method
for treating a Bacillus anthracis infection in a subject. The
method includes administering to the subject an effective amount of
a substituted tetracycline compound, such that the Bacillus
anthracis infection in the subject is treated.
[0007] In another embodiment, the invention also pertains to a
pharmaceutical composition comprising an effective amount of a
substituted tetracycline compound for the treatment of a Bacillus
anthracis infection and a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION:
[0008] In one embodiment, the invention pertains to a method for
treating a Bacillus anthracis infection in a subject. The method
includes administering to the subject an effective amount of a
substituted tetracycline compound, such that the Bacillus anthracis
infection in the subject is treated.
[0009] The term "Bacillus anthracis infection" includes any state,
diseases, or disorders caused or which result from exposure or
alleged exposure to Bacillus anthracis or another member of the
Bacillus cereus group of bacteria.
[0010] The Bacillus cereus group of bacteria is composed of B.
anthracis (the etiologic agent of anthrax), B. cereus and B.
weihenstephanensis (food borne pathogens), B. thuringiensis (an
insect pathogen), and B. mycoides (non-pathogenic). B. anthracis is
associated with three different clinical forms of infection.
Inhalation anthrax is rare, with only 18 cases reported in the US
from 1900-1976 and none from 1976-2001. The mortality rate of
inhalation anthrax has been reported to range from 40% to 89%;
however, many cases are from the pre-antibiotic era {Inglesby, 2002
#1942}. Patients that died following the accidental dissemination
of B. anthracis from a bioweapons facility in Sverdlovsk, Russia in
1976 exhibited hemorrhagic thoracic lymphadenitis, hemorrhagic
mediastinitis, and pleural effusions. This experience confirmed
that typical bronchopneumonia is not a characteristic of pulmonary
anthrax.
[0011] The most common infection due to B. anthracis is cutaneous
anthrax, which is rarely fatal when treated with appropriate
antibiotics. Gastrointestinal anthrax may develop after eating
improperly prepared, contaminated meat; these infections are
typically encountered in developing countries in Africa and
Asia.
[0012] The term "subject" includes animals (e.g., mammals, e.g.,
cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels,
bears, primates (e.g., chimpanzees, gorillas, and humans)) which
are capable of (or currently) suffering from a Bacillus anthracis
infection. It also includes transgenic animal models.
[0013] The term "treated," "treating" or "treatment" includes
therapeutic and/or prophylactic treatment of a Bacillus anthracis
infection. The treatment includes the diminishment or alleviation
of at least one symptom associated or caused by a Bacillus
anthracis infection. For example, treatment can be diminishment of
one or several symptoms of a Bacillus anthracis infection or
complete eradication.
[0014] The language "effective amount" of the tetracycline compound
is that amount necessary or sufficient to treat or prevent a
Bacillus anthracis infection in a subject, e.g. prevent the various
morphological and somatic symptoms of multiple sclerosis. 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.
[0015] The term "tetracycline compound" does not include
minocycline, doxycycline, or tetracycline. The term includes
substituted tetracycline compounds or compounds with a similar ring
structure to tetracycline. Examples of tetracycline compounds
include: chlortetracycline, oxytetracycline, demeclocycline,
methacycline, sancycline, chelocardin, rolitetracycline,
lymecycline, apicycline; clomocycline, guamecycline, meglucycline,
mepylcycline, penimepicycline, pipacycline, etamocycline,
penimocycline, etc. Other derivatives and analogues comprising a
similar four ring structure are also included (See Rogalski,
"Chemical Modifications of Tetracyclines," the entire contents of
which are hereby incorporated herein by reference). Table 1 depicts
tetracycline and several known other tetracycline derivatives.
TABLE-US-00001 TABLE 1 ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
[0016] Other tetracycline compounds which may be modified using the
methods of the invention include, but are not limited to,
6-demethyl-6-deoxy-4-dedimethylaminotetracycline;
tetracyclino-pyrazole; 7-chloro-4-dedimethylaminotetracycline;
4-hydroxy-4-dedimethylaminotetracycline;
12.alpha.-deoxy-4-dedimethylaminotetracycline;
5-hydroxy-6.alpha.-deoxy-4-dedimethylaminotetracycline;
4-dedimethylamino-12.alpha.-deoxyanhydrotetracycline;
7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;
tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline
4,6-hemiketal; 4-oxo-11a
Cl-4-dedimethylaminotetracycline-4,6-hemiketal;
5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;
4-hydroxyimino-4-dedimethylamino tetracyclines;
4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;
4-amino-4-dedimethylamino-5a, 6 anhydrotetracycline;
4-methylamino-4-dedimethylamino tetracycline;
4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylamino
tetracycline; tetracycline quaternary ammonium compounds;
anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides;
4-keto tetracyclines; 5-keto tetracyclines; 5a, 11a dehydro
tetracyclines; 11a Cl-6, 12 hemiketal tetracyclines; 11a
Cl-6-methylene tetracyclines; 6, 13 diol tetracyclines;
6-benzylthiomethylene tetracyclines; 7,
11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro
(.alpha.)-6-demethyl-6-deoxy tetracyclines; 6-fluoro
(.beta.)-6-demethyl-6-deoxy tetracyclines; 6-.alpha.
acetoxy-6-demethyl tetracyclines; 6-.beta. acetoxy-6-demethyl
tetracyclines; 7, 13-epithiotetracyclines; oxytetracyclines;
pyrazolotetracyclines; 11a halogens of tetracyclines; 12a formyl
and other esters of tetracyclines; 5, 12a esters of tetracyclines;
10, 12a-diesters of tetracyclines; isotetracycline;
12-a-deoxyanhydro tetracyclines;
6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines;
B-nortetracyclines; 7-methoxy-6-demethyl-6-deoxytetracyclines;
6-demethyl-6-deoxy-5a-epitetracyclines;
8-hydroxy-6-demethyl-6-deoxy tetracyclines; monardene;
chromocycline; 5a methyl-6-demethyl-6-deoxy tetracyclines; 6-oxa
tetracyclines, and 6 thia tetracyclines.
[0017] The term "substituted tetracycline compound" includes
tetracycline compounds with one or more additional substituents,
e.g., at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12, 12a or 13
position or at any other position which allows the substituted
tetracycline compound of the invention to perform its intended
function, e.g., treat B. anthracis infections.
[0018] In a further embodiment, the substituted tetracycline
compound has an MIC less than that of doxycycline for at least one
strain of Bacillus anthracis. The MIC of the substituted
tetracycline compound can be tested using the method described in
the Examples. In a further embodiment, the substituted tetracycline
compound has an MIC less than 32 .mu.g/ml for a doxycycline
resistant strain of Bacillus anthracis. In a further embodiment,
the MIC of the substituted tetracycline has an MIC that is 90% or
less, 50% or less, 20% or less, 10% or less, 5% or less than the
MIC of doxycycline for a particular strain of Bacillus
anthracis.
[0019] In a further embodiment, the substituted tetracycline
compound has an MIC less than that of ciproflaxin for at least one
strain of Bacillus anthracis. The MIC of the substituted
tetracycline compound can be tested using the method described in
the Examples. In a further embodiment, the substituted tetracycline
compound has an MIC less than 32 .mu.g/ml for a ciproflaxin
resistant strain of Bacillus anthracis. In a further embodiment,
the MIC of the substituted tetracycline has an MIC that is 90% or
less, 50% or less, 20% or less, 10% or less, 5% or less than the
MIC of ciproflaxin for a particular strain of Bacillus
anthracis.
[0020] In a further embodiment, the substituted tetracycline
compound of the invention is of the formula I:
##STR00010##
wherein
[0021] R.sup.1 is hydrogen, alkyl, alkenyl, alkynyl, aryl,
arylalkyl, amido, alkylamino, amino, arylamino, alkylcarbonyl,
arylcarbonyl, alkylaminocarbonyl, alkoxy, alkoxycarbonyl,
alkylcarbonyloxy, alkyloxycarbonyloxy, arylcarbonyloxy, aryloxy,
thiol, alkylthio, arylthio, alkenyl, heterocyclic, hydroxy, or
halogen, optionally linked to R.sup.2 to form a ring;
[0022] R.sup.2'' is cyano or C(.dbd.)--NR.sup.2R.sup.2';
[0023] R.sup.2 is hydrogen, alkyl, halogen, alkenyl, alkynyl, aryl,
hydroxyl, thiol, cyano, nitro, acyl, formyl, alkoxy, amino,
alkylamino, heterocyclic, or absent, optionally linked to R.sup.1
to form a ring;
[0024] R.sup.2', R.sup.3, R.sup.4a, and R.sup.4b are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0025] R.sup.10, R.sup.11, and R.sup.12 are each independently
hydrogen, alkyl, aryl, benzyl, arylalkyl, or a pro-drug moiety;
[0026] R.sup.4 and R.sup.4' are each independently
NR.sup.4aR.sup.4b, alkyl, acyl, alkenyl, alkynyl, hydroxyl,
halogen, hydrogen, or taken together .dbd.N--OR.sup.4a;
[0027] R.sup.5 and R.sup.5' are each independently hydroxyl,
hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic,
alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy, or aryl
carbonyloxy;
[0028] 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;
[0029] R.sup.7 is hydrogen, dialkylamino, hydroxyl, halogen, thiol,
nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,
arylalkynyl, acyl, aminoalkyl, heterocyclic, boronic ester,
alkylcarbonyl, thionitroso, or
--(CH.sub.2).sub.0-3(NR.sup.7c).sub.0-1C(.dbd.W')WR.sup.7a;
[0030] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, amino, arylalkenyl, arylalkynyl, acyl,
aminoalkyl, heterocyclic, thionitroso, or
--(CH.sub.2).sub.0-3(NR.sup.8c).sub.0-1C(=E')ER.sup.8a;
[0031] R.sup.9 is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, arylalkyl, amino, arylalkenyl, arylalkynyl, acyl,
aminoalkyl, heterocyclic, thionitroso, or
--(CH.sub.2).sub.0-3(NR.sup.9c).sub.0-1C(=Z')ZR.sup.9a;
[0032] R.sup.7a, R.sup.7b, R.sup.7c, R.sup.7d, R.sup.7e, R.sup.7f,
R.sup.8a, R.sup.8b, R.sup.8c, R.sup.8d, R.sup.8e, R.sup.8f,
R.sup.9a, R.sup.9b, R.sup.9c, R.sup.9d, R.sup.9e, and R.sup.9f are
each independently hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
aryl, heterocyclic, heteroaromatic or a prodrug moiety;
[0033] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, aryl, alkylsulfinyl, alkylsulfonyl, alkylamino,
or an arylalkyl;
[0034] E is CR.sup.8dR.sup.8e, S, NR.sup.8b or O;
[0035] E' is O, NR.sup.8f, or S;
[0036] W is CR.sup.7dR.sup.7e, S, NR.sup.7b or O;
[0037] W' is O, NR.sup.7f, or S;
[0038] X is CHC(R.sup.13Y'Y), C.dbd.CR.sup.13Y, CR.sup.6'R.sup.6,
S, NR.sup.6, or O;
[0039] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl;
[0040] Z is CR.sup.9dR.sup.9e, S, NR.sup.9b or O;
[0041] Z' is O, S, or NR.sup.9f, and pharmaceutically acceptable
salts, esters and enantiomers thereof.
[0042] In a further embodiment, R.sup.2'' is C(.dbd.O)NH.sub.2;
R.sup.3, R.sup.10, R.sup.11, and R.sup.12 are each hydrogen or a
prodrug moiety; R.sup.4 is NR.sup.4aR.sup.4b; R.sup.4a and R.sup.4b
are each methyl; R.sup.5 is hydrogen; R.sup.8 is hydrogen; X is
CR.sup.6R.sup.6'; R.sup.6 is hydrogen; and R.sup.5' and R.sup.6'
are hydrogen.
[0043] In another further embodiment, R.sup.8 and R.sup.9 are
hydrogen.
[0044] In yet another further embodiment, R.sup.7 is substituted
phenyl, a boronic ester, alkylcarbonyl, heterocyclic, aminoalkyl,
or arylalkynyl. Examples of substituents for phenyl R.sup.7 groups
include, but are not limited to, alkoxy,
alkyl-O--N.dbd.C--CR.sup.7gR.sup.7h, alkylaminoalkyl,
alkenylaminoalkyl, alkoxyalkylaminoalkyl, substituted alkyl, and
substituted carbonylamino, wherein R.sup.7g and R.sup.9h are each
independently hydrogen or alkyl.
[0045] In another further embodiment, R.sup.7 is substituted or
unsubstituted heteroaryl, e.g., substituted or unsubstituted
pyrimidinyl, pyridinyl, or furanyl.
[0046] In another further embodiment, R.sup.7 is substituted or
unsubstituted piperdinyl-alkyl. In other embodiments, R.sup.7 is
pyridinyl-alkynyl or substituted or unsubstituted
phenyl-alkynyl.
[0047] In other embodiment, R.sup.7 is hydrogen and R.sup.9 is
substituted carbonylamino.
[0048] In other embodiments, R.sup.8 is hydrogen; R.sup.7 is
heterocyclic, alkyl, alkyl-O--N.dbd.C--CR.sup.7gR.sup.7h, or
dimethylamino, wherein R.sup.7g and R.sup.9h are each independently
hydrogen or alkyl.
[0049] In a further embodiment, R.sup.9 is aminoalkyl. Examples of
aminoalkyl R.sup.9 moieties include aminomethyl moieties and
moieties of the formula:
##STR00011##
[0050] wherein:
[0051] J.sup.5 and J.sup.6 are each independently hydrogen, alkyl,
alkenyl, alkynyl, aryl, sulfonyl, acyl, alkoxycarbonyl,
alkaminocarbonyl, alkaminothiocarbonyl, substituted thiocarbonyl,
substituted carbonyl, alkoxythiocarbonyl, or linked to form a ring;
and
[0052] J.sup.7 and J.sup.8 are each alkyl, halogen, or
hydrogen.
[0053] In a further embodiment, J.sup.7 and J.sup.8 are each
hydrogen.
[0054] In another further embodiment, J.sup.6 is hydrogen and
J.sup.5 is substituted or unsubstituted alkyl, e.g., methyl, ethyl,
propyl, butyl, pentyl, 2-methyl-propyl, hexyl, and/or cyclohexyl.
Examples of substituents of J.sup.5 include one or more fluorines
or substituted or unsubstituted phenyl groups.
[0055] In another embodiment, J.sup.5 and/or J.sup.6 is substituted
or unsubstituted alkyl or alkenyl. Examples of J.sup.5 and/or
J.sup.6 include methyl, ethyl, propyl, propenyl, 2-methyl-propyl,
butyl, butenyl, pentyl, pentenyl, hexyl, and hexenyl. In a further
embodiment, J.sup.5 is substituted with one or more fluorines or
substituted or unsubstituted phenyl groups.
[0056] In another further embodiment, J.sup.5 and J.sup.6 are
linked to form a ring, e.g., a piperdinyl ring or a fused ring,
e.g., 2,3-dihydro-indole or an decahydro-isoquinoline. In another
further embodiment, the piperidinyl ring is substituted with one or
more halogens, one or more heterocyclic groups or one or more
halogenated alkyl groups (e.g., trifluoromethyl).
[0057] In one embodiment, R.sup.2'' is C(.dbd.O)NH.sub.2; R.sup.4',
R.sup.5', R.sup.3, R.sup.10, R.sup.11, and R.sup.12 are each
hydrogen or a prodrug moiety; R.sup.4 is NR.sup.4aR.sup.4b;
R.sup.4a and R.sup.4b are each methyl; R.sup.5 is hydroxyl; R.sup.8
is hydrogen; X is CR.sup.6R.sup.6'; R.sup.6 is hydrogen and
R.sup.6' is alkyl (e.g., methyl).
[0058] In a further embodiment, R.sup.7 is hydrogen and R.sup.9
aminoalkyl (e.g., piperidinyl alkyl, such as halogenated alkyl
substituted piperidinyl alkyl, for example, trifluoromethyl
substituted piperidinylalkyl).
[0059] In another embodiment, the substituted tetracycline compound
is selected from the group consisting of:
##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016##
[0060] and pharmaceutically acceptable salts thereof.
[0061] The tetracycline compounds of this invention can be
synthesized using the methods described in the Schemes and/or by
other techniques known to those of ordinary skill in the art.
[0062] The substituted tetracycline compounds of the invention can
be synthesized using the methods described in the following schemes
and by using art recognized techniques. All novel substituted
tetracycline compounds described herein are included in the
invention as compounds.
##STR00017##
[0063] 9- and 7-substituted tetracyclines can be synthesized by the
method shown in Scheme 1. As shown in Scheme 1, 9- and
7-substituted tetracycline compounds can be synthesized by treating
a tetracycline compound (e.g., doxycycline, 1A), with sulfuric acid
and sodium nitrate. The resulting product is a mixture of the
7-nitro and 9-nitro isomers (1B and 1C, respectively). The 7-nitro
(1B) and 9-nitro (1C) derivatives are treated by hydrogenation
using hydrogen gas and a platinum catalyst to yield amines 1D and
1E. The isomers are separated at this time by conventional methods.
To synthesize 7- or 9-substituted alkenyl derivatives, the 7- or
9-amino tetracycline compound (1E and 1F, respectively) is treated
with HONO, to yield the diazonium salt (1G and 1H). The salt (1G
and 1H) is treated with an appropriate reactive reagent to yield
the desired compound (e.g., in Scheme 1, 7-cyclopent-1-enyl
doxycycline (1H) and 9-cyclopent-1-enyl doxycycline (1I)).
##STR00018##
[0064] As shown in Scheme 2, tetracycline compounds of the
invention wherein R.sup.7 is a carbamate or a urea derivative can
be synthesized using the following protocol. Sancycline (2A) is
treated with NaNO.sub.2 under acidic conditions forming 7-nitro
sancycline (2B) in a mixture of positional isomers.
7-nitrosancycline (2B) is then treated with H.sub.2 gas and a
platinum catalyst to form the 7-amino sancycline derivative (2C).
To form the urea derivative (2E), isocyanate (2D) is reacted with
the 7-amino sancycline derivative (2C). To form the carbamate (2G),
the appropriate acid chloride ester (2F) is reacted with 2C.
##STR00019##
[0065] As shown in Scheme 3, tetracycline compounds of the
invention, wherein R.sup.7 is a heterocyclic (i.e., thiazole)
substituted amino group can be synthesized using the above
protocol. 7-amino sancycline (3A) is reacted with
Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).
The protected thiourea (3C) is then deprotected yielding the active
sancycline thiourea (3D) compound. The sancycline thiourea (3D) is
reacted with an .alpha.-haloketone (3E) to produce a thiazole
substituted 7-amino sancycline (3F).
##STR00020##
[0066] 7-alkenyl tetracycline compounds, such as 7-alkynyl
sancycline (4A) and 7-alkenyl sancycline (4B), can be hydrogenated
to form 7-alkyl substituted tetracycline compounds (e.g., 7-alkyl
sancycline, 4C). Scheme 4 depicts the selective hydrogenation of
the 7-position double or triple bond, in saturated methanol and
hydrochloric acid solution with a palladium/carbon catalyst under
pressure, to yield the product.
##STR00021##
[0067] In Scheme 5, a general synthetic scheme for synthesizing
7-position aryl derivatives is shown. A Suzuki coupling of an aryl
boronic acid with an iodosancycline compound is shown. An iodo
sancycline compound (5B) can be synthesized from sancycline by
treating sancycline (5A) with at least one equivalent
N-iodosuccinimide (NIS) under acidic conditions. The reaction is
quenched, and the resulting 7-iodo sancycline (5B) can then be
purified using standard techniques known in the art. To form the
aryl derivative, 7-iodo sancycline (5B) is treated with an aqueous
base (e.g., Na.sub.2CO.sub.3) and an appropriate boronic acid (5C)
and under an inert atmosphere. The reaction is catalyzed with a
palladium catalyst (e.g., Pd(OAc).sub.2). The product (5D) can be
purified by methods known in the art (such as HPLC). Other 7-aryl,
alkenyl, and alkynyl tetracycline compounds can be synthesized
using similar protocols.
[0068] The 7-substituted tetracycline compounds of the invention
can also be synthesized using Stille cross couplings. Stille cross
couplings can be performed using an appropriate tin reagent (e.g.,
R--SnBu.sub.3) and a halogenated tetracycline compound, (e.g.,
7-iodosancycline). The tin reagent and the iodosancycline compound
can be treated with a palladium catalyst (e.g.,
Pd(PPh.sub.3).sub.2Cl.sub.2 or Pd(AsPh.sub.3).sub.2Cl.sub.2) and,
optionally, with an additional copper salt, e.g., CuI. The
resulting compound can then be purified using techniques known in
the art.
##STR00022##
[0069] The compounds of the invention can also be synthesized using
Heck-type cross coupling reactions. As shown in Scheme 6, Heck-type
cross-couplings can be performed by suspending a halogenated
tetracycline compound (e.g., 7-iodosancycline, 6A) and an
appropriate palladium or other transition metal catalyst (e.g.,
Pd(OAc).sub.2 and CuI) in an appropriate solvent (e.g., degassed
acetonitrile). The substrate, a reactive alkene (6B) or alkyne
(6D), and triethylamine are then added and the mixture is heated
for several hours, before being cooled to room temperature. The
resulting 7-substituted alkenyl (6C) or 7-substituted alkynyl (6E)
tetracycline compound can then be purified using techniques known
in the art.
##STR00023##
[0070] To prepare 7-(2'-chloro-alkenyl)-tetracycline compounds, the
appropriate 7-(alkynyl)-sancycline (7A) is dissolved in saturated
methanol and hydrochloric acid and stirred. The solvent is then
removed to yield the product (7B).
##STR00024##
[0071] As depicted in Scheme 8, 5-esters of 9-substituted
tetracycline compounds can be formed by dissolving the
9-substituted compounds (8A) in strong acid (e.g., HF,
methanesulphonic acid, and trifluoromethanesulfonic acid) and
adding the appropriate carboxylic acid to yield the corresponding
esters (8B).
[0072] As shown in Scheme 9 below, 7 and 9 aminomethyl
tetracyclines may be synthesized using reagents such as
hydroxymethyl-carbamic acid benzyl ester.
##STR00025##
[0073] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. The term alkyl
further includes alkyl groups, which can further include oxygen,
nitrogen, sulfur or phosphorus atoms replacing one or more carbons
of the hydrocarbon backbone. In certain embodiments, a straight
chain or branched chain alkyl has 6 or fewer carbon atoms in its
backbone (e.g., C.sub.1-C.sub.6 for straight chain, C.sub.3-C.sub.6
for branched chain), and more preferably 4 or fewer. Likewise,
preferred cycloalkyls have from 3-8 carbon atoms in their ring
structure, and more preferably have 5 or 6 carbons in the ring
structure. The term C.sub.1-C.sub.6 includes alkyl groups
containing 1 to 6 carbon atoms.
[0074] 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.
[0075] 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).
[0076] 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.
[0077] 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
phosphorus atoms replacing one or more carbons of the hydrocarbon
backbone. In certain embodiments, a straight chain or branched
chain alkenyl group has 6 or fewer carbon atoms in its backbone
(e.g., C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6 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.6 includes
alkenyl groups containing 2 to 6 carbon atoms.
[0078] 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.
[0079] 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.
[0080] 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 phosphorus atoms replacing one
or more carbons of the hydrocarbon backbone. In certain
embodiments, a straight chain or branched chain alkynyl group has 6
or fewer carbon atoms in its backbone (e.g., C.sub.2-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain). The term
C.sub.2-C.sub.6 includes alkynyl groups containing 2 to 6 carbon
atoms.
[0081] Moreover, the term alkynyl includes both "unsubstituted
alkynyls" and "substituted alkynyls," the latter of which refers to
alkynyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0082] 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.
[0083] The term "acyl" includes compounds and moieties which
contain the acyl radical (CH.sub.3CO--) or a carbonyl group. It
includes substituted acyl moieties. The term "substituted acyl"
includes acyl groups where one or more of the hydrogen atoms are
replaced by for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] The term "amine" or "amino" includes compounds where a
nitrogen atom is covalently bonded to at least one carbon or
heteroatom. The term includes "alkyl amino" which comprises groups
and compounds wherein the nitrogen is bound to at least one
additional alkyl group. The term "dialkyl amino" includes groups
wherein the nitrogen atom is bound to at least two additional alkyl
groups. The term "arylamino" and "diarylamino" include groups
wherein the nitrogen is bound to at least one or two aryl groups,
respectively. The term "alkylarylamino," "alkylaminoaryl" or
"arylaminoalkyl" refers to an amino group which is bound to at
least one alkyl group and at least one aryl group. The term
"alkaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group bound
to a nitrogen atom which is also bound to an alkyl group.
[0089] The term "amide," "amido" or "aminocarbonyl" includes
compounds or moieties which contain a nitrogen atom which is bound
to the carbon of a carbonyl or a thiocarbonyl group. The term
includes "alkaminocarbonyl" or "alkylaminocarbonyl" groups which
include alkyl, alkenyl, aryl or alkynyl groups bound to an amino
group bound to a carbonyl group. It includes arylaminocarbonyl and
arylcarbonylamino groups which include aryl or heteroaryl moieties
bound to an amino group which is bound to the carbon of a carbonyl
or thiocarbonyl group. The terms "alkylaminocarbonyl,"
"alkenylaminocarbonyl," "alkynylaminocarbonyl,"
"arylaminocarbonyl," "alkylcarbonylamino," "alkenylcarbonylamino,"
"alkynylcarbonylamino," and "arylcarbonylamino" are included in
term "amide." Amides also include urea groups (aminocarbonylamino)
and carbamates (oxycarbonylamino).
[0090] The term "carbonyl" or "carboxy" includes compounds and
moieties which contain a carbon connected with a double bond to an
oxygen atom. The carbonyl can be further substituted with any
moiety which allows the compounds of the invention to perform its
intended function. For example, carbonyl moieties may be
substituted with alkyls, alkenyls, alkynyls, aryls, alkoxy, aminos,
etc. Examples of moieties which contain a carbonyl include
aldehydes, ketones, carboxylic acids, amides, esters, anhydrides,
etc.
[0091] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0096] 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.
[0097] 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, amido, amino
(including alkyl amino, dialkylamino, arylamino, diarylamino, and
alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0098] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0099] 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.
[0100] It will be noted that the structure of some of the
tetracycline compounds of this invention includes asymmetric carbon
atoms. It is to be understood accordingly that the isomers arising
from such asymmetry (e.g., all enantiomers and diastereomers) are
included within the scope of this invention, unless indicated
otherwise. Such isomers can be obtained in substantially pure form
by classical separation techniques and by stereochemically
controlled synthesis. Furthermore, the structures and other
compounds and moieties discussed in this application also include
all tautomers thereof.
[0101] In another further embodiment, the substituted tetracycline
compound is administered in combination with a second agent.
[0102] The language "in combination with" a second agent includes
co-administration of the tetracycline compound, and with the second
agent, administration of the tetracycline compound first, followed
by the second agent and administration of the second agent,
followed by the tetracycline compound. The second agent may be any
agent which is known in the art to treat, prevent, or reduce the
symptoms of a Bacillus anthracis infection. Furthermore, the second
agent may be any agent of benefit to the subject when administered
in combination with the administration of an tetracycline
compound.
[0103] Examples of second agents include antibiotics, such as
rifampin, vancomycin, ampicillin, chloramphenicol, imipenem,
clindamycin, and clarithromycin.
[0104] In another embodiment, the invention pertains to
pharmaceutical compositions comprising an effective amount of a
substituted tetracycline compound of the invention for the
treatment of a Bacillus anthracis infection and a pharmaceutically
acceptable carrier.
[0105] The language "pharmaceutically acceptable carrier" includes
substances capable of being coadministered with the tetracycline
compound(s), and which allow both to perform their intended
function, e.g., treat or prevent a Bacillus anthracis infection.
Suitable pharmaceutically acceptable carriers include but are not
limited to water, salt solutions, alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, petroethral fatty acid
esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The
pharmaceutical preparations can be sterilized and if desired mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, colorings, flavorings and/or aromatic
substances and the like which do not deleteriously react with the
active compounds of the invention.
[0106] The tetracycline compounds of the invention that are basic
in nature are capable of forming a wide variety of salts with
various inorganic and organic acids. The acids that may be used to
prepare pharmaceutically acceptable acid addition salts of the
tetracycline compounds of the invention that are basic in nature
are those that form non-toxic acid addition salts, i.e., salts
containing pharmaceutically acceptable anions, such as the
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, acid phosphate, isonicotinate, acetate,
lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,
bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,
gluconate, glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate and palmoate [i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)] salts. Although such
salts must be pharmaceutically acceptable for administration to a
subject, e.g., a mammal, it is often desirable in practice to
initially isolate a tetracycline compound of the invention from the
reaction mixture as a pharmaceutically unacceptable salt and then
simply convert the latter back to the free base compound by
treatment with an alkaline reagent and subsequently convert the
latter free base to a pharmaceutically acceptable acid addition
salt. The acid addition salts of the base compounds of this
invention are readily prepared by treating the base compound with a
substantially equivalent amount of the chosen mineral or organic
acid in an aqueous solvent medium or in a suitable organic solvent,
such as methanol or ethanol. Upon careful evaporation of the
solvent, the desired solid salt is readily obtained. The
preparation of other tetracycline compounds of the invention not
specifically described in the foregoing experimental section can be
accomplished using combinations of the reactions described above
that will be apparent to those skilled in the art.
[0107] The tetracycline compounds of the invention that are acidic
in nature are capable of forming a wide variety of base salts. The
chemical bases that may be used as reagents to prepare
pharmaceutically acceptable base salts of those tetracycline
compounds of the invention that are acidic in nature are those that
form non-toxic base salts with such compounds. Such non-toxic base
salts include, but are not limited to those derived from such
pharmaceutically acceptable cations such as alkali metal cations
(e.g., potassium and sodium) and alkaline earth metal cations
(e.g., calcium and magnesium), ammonium or water-soluble amine
addition salts such as N-methylglucamine-(meglumine), and the lower
alkanolammonium and other base salts of pharmaceutically acceptable
organic amines. The pharmaceutically acceptable base addition salts
of tetracycline compounds of the invention that are acidic in
nature may be formed with pharmaceutically acceptable cations by
conventional methods. Thus, these salts may be readily prepared by
treating the tetracycline compound of the invention with an aqueous
solution of the desired pharmaceutically acceptable cation and
evaporating the resulting solution to dryness, preferably under
reduced pressure. Alternatively, a lower alkyl alcohol solution of
the tetracycline compound of the invention may be mixed with an
alkoxide of the desired metal and the solution subsequently
evaporated to dryness.
[0108] The tetracycline compounds of the invention and
pharmaceutically acceptable salts thereof can be administered via
either the oral, parenteral or topical routes. In general, these
compounds are most desirably administered in effective dosages,
depending upon the weight and condition of the subject being
treated and the particular route of administration chosen.
Variations may occur depending upon the species of the subject
being treated and its individual response to said medicament, as
well as on the type of pharmaceutical formulation chosen and the
time period and interval at which such administration is carried
out.
[0109] The tetracycline compounds of the invention may be
administered alone or in combination with pharmaceutically
acceptable carriers or diluents by any of the routes previously
mentioned, and the administration may be carried out in single or
multiple doses. For example, the novel therapeutic agents of this
invention can be administered advantageously in a wide variety of
different dosage forms, i.e., they may be combined with various
pharmaceutically acceptable inert carriers in the form of tablets,
capsules, lozenges, troches, hard candies, powders, sprays (e.g.,
aerosols, etc.), creams, salves, suppositories, jellies, gels,
pastes, lotions, ointments, aqueous suspensions, injectable
solutions, elixirs, syrups, and the like. Such carriers include
solid diluents or fillers, sterile aqueous media and various
non-toxic organic solvents, etc. Moreover, oral pharmaceutical
compositions can be suitably sweetened and/or flavored. In general,
the therapeutically-effective compounds of this invention are
present in such dosage forms at concentration levels ranging from
about 5.0% to about 70% by weight.
[0110] For oral administration, tablets containing various
excipients such as microcrystalline cellulose, sodium citrate,
calcium carbonate, dicalcium phosphate and glycine may be employed
along with various disintegrants such as starch (and preferably
corn, potato or tapioca starch), alginic acid and certain complex
silicates, together with granulation binders like
polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,
lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc are often very useful for tabletting purposes.
Solid compositions of a similar type may also be employed as
fillers in gelatin capsules; preferred materials in this connection
also include lactose or milk sugar as well as high molecular weight
polyethylene glycols. When aqueous suspensions and/or elixirs are
desired for oral administration, the active ingredient may be
combined with various sweetening or flavoring agents, coloring
matter or dyes, and, if so desired, emulsifying and/or suspending
agents as well, together with such diluents as water, ethanol,
propylene glycol, glycerin and various like combinations thereof.
The compositions of the invention may be formulated such that the
tetracycline compositions are released over a period of time after
administration.
[0111] For parenteral administration (including intraperitoneal,
subcutaneous, intravenous, intradermal or intramuscular injection),
solutions of a therapeutic compound of the present invention in
either sesame or peanut oil or in aqueous propylene glycol may be
employed. The aqueous solutions should be suitably buffered
(preferably pH greater than 8) if necessary and the liquid diluent
first rendered isotonic. These aqueous solutions are suitable for
intravenous injection purposes. The oily solutions are suitable for
intraarticular, intramuscular and subcutaneous injection purposes.
The preparation of all these solutions under sterile conditions is
readily accomplished by standard pharmaceutical techniques well
known to those skilled in the art. For parenteral application,
examples of suitable preparations include solutions, preferably
oily or aqueous solutions as well as suspensions, emulsions, or
implants, including suppositories. Therapeutic compounds may be
formulated in sterile form in multiple or single dose formats such
as being dispersed in a fluid carrier such as sterile physiological
saline or 5% saline dextrose solutions commonly used with
injectables.
[0112] Additionally, it is also possible to administer the
compounds of the present invention topically when treating
inflammatory conditions of the skin. Examples of methods of topical
administration include transdermal, buccal or sublingual
application. For topical applications, therapeutic compounds can be
suitably admixed in a pharmacologically inert topical carrier such
as a gel, an ointment, a lotion or a cream. Such topical carriers
include water, glycerol, alcohol, propylene glycol, fatty alcohols,
triglycerides, fatty acid esters, or mineral oils. Other possible
topical carriers are liquid petrolatum, isopropylpalmitate,
polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5%
in water, sodium lauryl sulfate 5% in water, and the like. In
addition, materials such as anti-oxidants, humectants, viscosity
stabilizers and the like also may be added if desired.
[0113] For enteral application, particularly suitable are tablets,
dragees or capsules having talc and/or carbohydrate carrier binder
or the like, the carrier preferably being lactose and/or corn
starch and/or potato starch. A syrup, elixir or the like can be
used wherein a sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings,
e.g., by microencapsulation, multiple coatings, etc.
[0114] In addition to treatment of human subjects, the therapeutic
methods of the invention also will have significant veterinary
applications, e.g., for treatment of livestock such as cattle,
sheep, goats, cows, swine and the like; poultry such as chickens,
ducks, geese, turkeys and the like; horses; and pets such as dogs
and cats.
[0115] It will be appreciated that the actual preferred amounts of
active compounds used in a given therapy will vary according to the
specific compound being used, the particular compositions
formulated, the mode of application, the particular site of
administration, etc. Optimal administration rates for a given
protocol of administration can be readily ascertained by those
skilled in the art using conventional dosage determination tests
conducted with regard to the foregoing guidelines.
[0116] In general, compounds of the invention for treatment can be
administered to a subject in dosages used in prior tetracycline
therapies. See, for example, the Physicians' Desk Reference. For
example, a suitable effective dose of one or more compounds of the
invention will be in the range of from 0.01 to 100 milligrams per
kilogram of body weight of recipient per day, preferably in the
range of from 0.1 to 50 milligrams per kilogram body weight of
recipient per day, more preferably in the range of 1 to 20
milligrams per kilogram body weight of recipient per day. The
desired dose is suitably administered once daily, or several
sub-doses, e.g. 2 to 5 sub-doses, are administered at appropriate
intervals through the day, or other appropriate schedule.
[0117] It will also be understood that normal, conventionally known
precautions will be taken regarding the administration of
tetracyclines generally to ensure their efficacy under normal use
circumstances. Especially when employed for therapeutic treatment
of humans and animals in vivo, the practitioner should take all
sensible precautions to avoid conventionally known contradictions
and toxic effects. Thus, the conventionally recognized adverse
reactions of gastrointestinal distress and inflammations, the renal
toxicity, hypersensitivity reactions, changes in blood, and
impairment of absorption through aluminum, calcium, and magnesium
ions should be duly considered in the conventional manner.
[0118] Furthermore, the invention also pertains to the use of a
substituted tetracycline of the invention, for the preparation of a
medicament. The medicament may include a pharmaceutically
acceptable carrier and the tetracycline compound is an effective
amount, e.g., an effective amount to treat a Bacillus anthracis
infection.
EXEMPLIFICATION OF THE INVENTION
Example 1
Antibacterial Activity of Tetracycline Compounds Against
Susceptible and (Multiple) Antibiotic Resistant Organisms
[0119] Efflux. The tetracycline efflux proteins, in general, confer
resistance to both tetracycline and doxycycline. S. aureus RN4250
bears a TetK efflux mechanism and is resistant to both agents, but
susceptible to minocycline (Table 2). A number of tetracyclines
that overcome gram-positive efflux (Table 2) have been
identified.
TABLE-US-00002 TABLE 2 MICs (.mu.g/ml) of novel TCs against strains
with efflux resistance determinants. S. aureus S. aureus
RN450.sup.a RN4250.sup.b Compound MIC (ug/ml) Doxycycline 0.06 4
Minocycline 0.25 0.5 Tetracycline 0.06 64 O 0.06 0.06 M 0.06 0.06 Q
0.06 0.06 P 0.06 0.06 S 0.06 0.06 T 0.06 0.06 U 0.06 0.06 V 0.06
0.06 W 0.06 0.06 X 0.06 0.06 Y 0.06 0.06 .sup.aWild type S. aureus.
.sup.bContains a TetK (efflux) resistance determinant.
[0120] Ribosome protection. The ribosome protection determinants,
which confer resistance to tetracycline, doxycycline and
minocycline, are predominantly found in gram-positive bacteria and
are probably the most widespread tetracycline resistance
determinant in these organisms. A number of tetracycline compounds
that can overcome this mechanism of resistance in a variety of
gram-positive bacteria including S. aureus, E. faecium, and S.
pneumoniae (Table 3).
TABLE-US-00003 TABLE 3 MICs (.mu.g/ml) of tetracycline compounds
against strains with ribosome protection resistance determinants S.
S. S. aureus S. aureus E. faecium pneumoniae pneumoniae Com-
RN450.sup.a MRSA5.sup.b 494.sup.c 157E.sup.a 700905.sup.d pound MIC
(ug/ml) Doxy- 0.06 4 8 0.06 4 cycline Mino- 0.25 2 16 0.06 8
cycline Tetra- 0.06 32 64 0.06 32 cycline Z 0.13 0.5 2 0.06
ND.sup.e AA 1 0.5 1 0.5 1 AB 0.06 1 0.06 0.06 4 AD 0.06 1 2 0.13
0.5 AE 0.06 0.5 2 0.13 1 AK 1 2 1 0.5 0.5 .sup.aWild type.
.sup.bMethicillin resistant S. aureus; contains TetM (ribosome
protection); also multi-drug resistant. .sup.cContains TetL
(efflux) and TetM (ribosome protection); is also resistant to
vancomycin and erythromycin. .sup.dContains TetM (ribosome
protection); is also resistant to penicillin and erythromycin.
.sup.eND, not determined.
[0121] Efflux and ribosome protection concurrently. A number of
tetracycline compounds were tested against gram-positive bacteria
possessing both tetracycline efflux and ribosome protection
determinants as well as other non-tetracycline resistance
mechanisms. Compounds with substitutions at both R.sup.7 and
R.sup.9 position in Formula I e.g., substituted
7-dimethylamino-9-aminomethylcyclines and 7-aryl or heteroaryl
sancyclines) demonstrated activity against both tetracycline
sensitive isolates and tetracycline resistant gram-positive
bacteria containing efflux and ribosome protection determinants
(Table 4).
TABLE-US-00004 TABLE 4 MICs (.mu.g/ml) of tetracycline compounds
against strains with ribosome protection and efflux resistance
determinants. E. faecium E. faecalis S. aureus S. pneumoniae
494.sup.a 29212.sup.b MRSA5.sup.c 700905.sup.d Compound MIC (ug/ml)
Doxycycline 16 4 4 4 Minocycline 16 4 2 8 Tetracycline 64 16 32 32
A 1 1 1 0.25 B 1 1 1 0.5 C 0.25 0.5 1 0.25 D 1 0.25 1 0.25 E 1 0.25
0.25 0.06 F 1 0.5 0.5 0.25 G 1 0.5 0.5 0.06 H 0.5 0.5 0.35 0.06 I 1
1 1 0.5 J 1 0.25 0.5 0.06 K 1 1 0.5 0.25 L 1 1 0.5 0.75 R 1 2 1
0.13 N 0.5 1 1 0.13 AH 0.25 0.25 1 0.06 .sup.aHas TetM (ribosome
protection) and TetL (efflux); is resistant to vancomycin and
erythromycin. .sup.bHas TetM (ribosome protection).
.sup.cMethicillin resistant S. aureus; contains TetM, ribosome
protection; also multi-drug resistant. .sup.dHas TetM (ribosome
protection).
[0122] Bacillus cereus. In order to prevent the unnecessary use of
the anthrax pathogen, a group of tetracycline resistant B. cereus
was obtained. In this panel, B. cereus 95/3032 and 98/2658 were
classified as tetracycline susceptible whereas B. cereus 98/2620
and 97/4144 were tetracycline resistant (Table 6). Preliminary MICs
were determined for common antibiotics against the B. cereus
isolates (Table 5).
[0123] B. cereus containing natural tetracycline resistance
determinants were chosen rather than creating isogenic tetracycline
resistant B. anthracis strains since it would be a violation of
International Bioweapons Treaty to purposefully create an
antibiotic resistant category A agent. In addition, B. cereus are
generally more tetracycline resistant than B. anthracis.
TABLE-US-00005 TABLE 5 Activity of tetracycline compounds against
tetracycline susceptible and tetracycline resistant Bacillus
cereus. B. cereus B. cereus B. cereus B. cereus 98/2620.sup.a
95/3032.sup.b 98/2658.sup.c 97/4144.sup.d Compound MIC (ug/ml)
Doxycycline 4 .ltoreq.0.06 .ltoreq.0.06 4 Minocycline 0.5
.ltoreq.0.06 .ltoreq.0.06 0.5 Tetracycline 32 .ltoreq.0.06
.ltoreq.0.06 64 Cefotaxime 64 >64 >64 32 Penicillin 32 >64
>64 >64 Vancomycin 1 1 2 1 Erythromycin .ltoreq.0.06 0.125 1
0.125 Clindamycin 0.25 0.5 0.5 0.5 .sup.aAn industrial fermenter
isolate, serotype 1. .sup.bIsolated from an orthopedic-related
area, serotype 24. .sup.cNon-typeable. .sup.dIsolated from an
individual with food poisoning, serotype AA.
[0124] Bacillus anthracis. The panel of B. anthracis isolates
(n=27) that was available for susceptibility studies included two
organisms that exhibit reduced susceptibility to doxycycline (Table
6). B. anthracis V770 was 4.fwdarw.33-fold less susceptible to
doxycycline than 25 other B. anthracis and strain V770NPIR was
fully doxycycline-resistant.
[0125] The group of organisms listed in Table 6 all possessed the
same tetracycline resistance determinants that would be found in B.
anthracis and the majority were multi-drug resistant. The criteria
for selecting compounds for subsequent testing in B. anthracis were
(a) the compounds must not possess cytotoxicity in vitro (Table 9)
and (b) the compounds were required to possess a MIC of .ltoreq.0.5
.mu.g/ml against this panel of resistant isolates (Table 7). At
least five tetracycline compounds were identified (Table 7).
[0126] The activities of these tetracyclines were tested against B.
anthracis (n=5), including the tetracycline resistant strains V770
and V770NPIR (Table 7). As illustrated, these compounds possessed
exceptional activity against tetracycline susceptible and resistant
B. anthracis isolates in vitro (Table 7). Compounds AI, H, and AJ
all contain substituents at the R.sup.9 position of the
tetracycline core while compounds AM and AF bear substitutions at
the R.sup.7 and R.sup.9 positions. Without being bound by theory,
these data support the hypothesis that tetracycline compounds
directed against common tetracycline resistant organisms, e.g., S.
aureus, S. pneumoniae, and Enterococcus spp. may also target
tetracycline resistant B. anthracis.
TABLE-US-00006 TABLE 6 Activity of tetracycline compounds against
susceptible and doxycycline resistant B. anthracis. Vollum1B Sterne
Ames V770 V770NPIR Compound MIC (ug/ml) Ciprofloxacin 0.25 0.25
0.25 0.12 0.25 Doxycycline.sup.a 0.06 0.12 <0.03 1 32 AI
<0.03 <0.03 0.06 0.12 0.06 AM 0.06 0.06 0.06 0.12 0.06 AF
<0.03 <0.03 <0.03 0.06 0.06 H <0.03 <0.03 <0.03
<0.03 0.06 AJ <0.03 <0.03 <0.03 <0.03 <0.03
.sup.aThe activity of doxycycline against the entire B. anthracis
panel (n = 27) is as follows: MIC50 = 0.06 .mu.g/ml; MIC90 = 0.25
.mu.g/ml; MIC Range = <0.03-32 .mu.g/ml.
TABLE-US-00007 TABLE 7 Activity of tetracycline compounds against
common susceptible and tetracycline resistant bacteria. S. aureus
E. faecium E. faecalis S. pneumoniae MRSA5.sup.a 494.sup.b
29212.sup.c 700905.sup.d Compound MIC (ug/ml) Doxycycline 4 16 4 4
Minocycline 2 16 4 8 Tetracycline 32 64 16 32 AI 0.5 0.5 0.5 0.06
AM 0.25 0.25 0.13 0.06 AF 0.13 0.25 0.13 0.06 H 0.35 0.5 0.5 0.06
AJ 0.5 0.5 0.5 0.06 .sup.aMethicillin resistant S. aureus; contains
TetM, ribosome protection; also multi-drug resistant. .sup.bHas
TetM (ribosome protection) and TetL (efflux); is resistant to
vancomycin and erythromycin. .sup.cHas TetM (ribosome protection).
.sup.dHas TetM (ribosome protection); is resistant to penicillin
and erythromycin.
Example 2
Additional Potential Mechanisms of Antibacterial Activity by
Tetracycline Compounds
[0127] In addition to inhibiting protein synthesis, molecules
within the tetracycline family are reported to affect peptidoglycan
biosynthesis. Using cell-free macromolecular synthesis assays early
studies divided the tetracycline compounds into two classes based
on these additional activities. Class 1 compounds (tetracycline,
minocycline, and doxycycline) were potent inhibitors of protein
synthesis compared to the weak effects of class 2 molecules
(chelocardin, anhydrotetracycline, and 4-epi-anhydrochlorotet).
[0128] Using chemical footprinting assays, minocycline,
doxycycline, and tetracycline were shown to affect the reactivity
of nucleotides known to mediate binding of the antibiotics within
the 16S rRNA. Tigecycline exhibited a chemical footprint similar to
that of tetracycline. A similar effect was not seen with
chelocardin or anhydrotetracycline, which correlates with their
poor activity against the purified ribosome in vitro.
[0129] As illustrated in Table 8, these previous findings were
confirmed and methods for deriving IC50 values (i.e., compound
concentration necessary to inhibit a biological process by 50%)
were developed. In particular, compounds AA, O, and tigecycline
have a profile similar to class I compounds. Compounds AA and A
also affect peptidoglycan biosynthesis.
TABLE-US-00008 TABLE 8 Effect of tetracycline compounds on
macromolecular synthesis of S. aureus RN450. Protein Peptidoglycan
MIC synthesis.sup.a synthesis Antibiotic (.mu.g/ml) IC50 IC90 IC50
IC90 Tetracycline 0.06 <0.03 0.11 >32 >32 Minocycline 0.19
<0.03 0.1 4.6 20.9 Doxycycline 0.06 <0.03 <0.03 3.9 18.23
Anhydrotetracycline 2 <0.03 <0.03 19.2 >32 Tigecycline 2
0.12 0.68 >32 >32 AA 1 0.14 1.4 3.8 23 AB 0.06 0.19 1.8 18.3
>32 A 0.25 1.9 5 2.1 3.8 AE 0.06 0.07 0.62 6.0 >32 O 0.06
0.33 0.92 >32 >32 .sup.aCompounds were assayed against S.
aureus RN450, a TC susceptible organism and IC50 and IC90 values
are reported in .mu.g/ml.
Example 3
In Vitro and in Vivo Toxicity
[0130] An in vitro determination of the cytotoxicity of the
compounds of the invention was performed using standard mammalian
cell assays and in vivo using mice. Specifically, African green
monkey kidney (COS-1) and Chinese hamster ovary (CHO-K1) cell lines
were used according to standard methods (see Zhi-Jun, Y., N.
Sriranganathan, T. Vaught, S. K. Arastu, and S. A. Ahmed. 1997. A
dye-based lymphocyte proliferation assay that permits multiple
immunological analyses: mRNA, cytogenetic, apoptosis, and
immunophenotyping studies. J Immunol Methods 210:25-39). Briefly,
suspensions of tissue culture cells were grown overnight in the
presence of serial dilutions of drug up to a maximum concentration
of 50 or 100 .mu.g/ml. The metabolism of the tissue culture cells
was monitored with resazurin, a soluble non-toxic dye. Control
cytotoxic and non-cytotoxic compounds were routinely included in
all assays. Tox100 values represented the concentration of compound
necessary to inhibit cellular proliferation by 100%. Compounds
without measurable cytotoxicity in vitro were assigned a Tox100
value of greater than the highest concentration assayed (e.g., 50
or 100 .mu.g/ml). The results are shown in Table 9.
TABLE-US-00009 TABLE 9 Cytotoxicity of tetracycline compounds.
Tox50 (.mu.g/ml).sup.a Compound COS-1 CHO-K1 AI >100 >100 AM
>100 92.85 AF >100 >100 H >100 >100 AJ >100
>100 .sup.aRepresents the concentration necessary to cause 50%
inhibition of cell growth in tissue culture cells.
Example 4
Efficacy of Tetracycline Compounds Against Susceptible and
Resistant Organisms in Animal Infection Models
[0131] In vivo efficacy as well as oral bioavailability of the
tetracycline compounds were assessed in murine models of infection
and compared to control tetracyclines and other currently available
antibiotics. In the standard screening assay of acute systemic
infection (Table 10), mice were given a lethal intraperitoneal
inoculum of S. pneumoniae strain 157E (tetracycline susceptible) or
700905 (tetracycline resistant), followed by a single dose of drug,
and then observed for survival over 48 hours. Each experiment
routinely included an untreated group (n=5; expected
survival<5%) and a group (n=5) treated with a conventional
antibiotic (e.g., minocycline, ciprofloxacin, and ampicillin;
expected survival>80%). The results are tabulated in Table
10.
TABLE-US-00010 TABLE 10 Efficacy of selected tetracyclines in the
screening assay of acute systemic infection due to S. pneumoniae
157E. SC PO Compound dose % survival dose % survival B 5 mg/kg 100%
5 mg/kg 40% C 5 mg/kg 100% 10 mg/kg 60% D 5 mg/kg 0% 10 mg/kg 0% AI
5 mg/kg 40% 10 mg/kg 0% E 5 mg/kg 40% 10 mg/kg 0% AM 5 mg/kg 100%
50 mg/kg 0% F 5 mg/kg 100% 50 mg/kg 100% AJ 5 mg/kg 100% 50 mg/kg
80% ND, not determined.
[0132] Compounds providing .gtoreq.60% survival at 10 mg/kg were
further assessed in a dose response study to determine the PD50
(the drug concentration necessary to prevent death in 50% of the
mice in a treatment group). These experiments involved an untreated
group, a group treated with a control antibiotic (e.g.,
minocycline, ciprofloxacin, and ampicillin), and up to five groups
each receiving a different doses of an active experimental
compound; all groups included 5 animals (Table 11). Compounds B and
C were efficacious following tetracycline administration and
compounds H and I exhibited oral activity (Tables 10 and 11).
Compound H, which exhibited potency against tetracycline resistant
B. anthracis (Table 6), exhibits IV and PO efficacy against
infections caused by tetracycline susceptible and resistant
organisms (Table 11), and is efficacious in a model of lung
infection following IV and PO drug administration (Table 12).
TABLE-US-00011 TABLE 11 Efficacy (PD50) of selected tetracycline
compounds in mice with acute systemic infection due to S.
pneumoniae. S. pneumoniae S. pneumoniae 157E 700905 IV PO IV PO
Compound PD50 (mg/kg) Minocycline 0.53 1.5 >50 >50
Ciprofloxacin >50 ND ND >50 Ampicillin 0.6 1.1 43.7 >50 H
1.1 5 2.2 12.7 I 1 13.4 2.2 31.6 AN 0.54 2.3 1.4 8.4
[0133] A chronic model of murine S. pneumoniae lung infection was
also established and the efficacy of a variety of currently
available tetracycline compounds were tested in this model (Table
12).
TABLE-US-00012 TABLE 12 Efficacy (PD50) of tetracycline compounds
in mice with acute pulmonary infection due to S. pneumoniae
PBS1339. PD50 (mg/kg) Compound IV PO Minocycline 4.5 3.6
Doxycycline 7.1 35.3 Ampicillin ND 3.2 H <5.0 3.6
Example 5
In Vivo Murine Model of B. Anthracis Infection
[0134] In this example, mice were exposed to B. anthracis using
whole body aerosol challenge, which approximated the mode of
pathogen dissemination that would be expected during a bioterrorist
event and was therefore preferred over other models (e.g.,
intratracheal inoculation). Animals were challenged with 75-100
LD50 of B. anthracis Ames strain spores (LD50 3.4.times.104
CFU/ml), which has been demonstrated repeatedly to cause death in
90-100% of untreated animals. Treatment with the substituted
tetracycline compounds of the invention began 24 hours after
challenge and continued for 21 days. Due to the persistence of
ungerminated anthrax spores in the lungs of challenged animals, a
treatment duration of 21 days was used regardless of the antibiotic
class. Antibiotics were administered by parenteral injection or
oral administration. Treatment groups were followed for an
additional 30 days after cessation of antibiotic treatment. In
addition to monitoring survival in each treatment group, animals
were sacrificed at selected time points to monitor microbiological
burdens. Tissues, including brain, spleen, lungs, heart, and liver
were excised and pathogens were enumerated using agar plates.
Additionally, emergence of resistance was monitored by culturing
organs on antibiotic containing media (usually at 3.times. the
baseline MIC).
[0135] Individual treatment groups consisted of 15 animals and the
study endpoint was death after infectious challenge. Ciprofloxacin
(30 mg/kg, q12h) or doxycycline (at experimentally determined
doses) was included as the active control in all experiments.
Moribund animals exhibiting labored breathing, showing signs of
paralysis, or that are unresponsive were humanely euthanized.
Challenged survivors were humanely euthanized at the conclusion of
the example.
[0136] Throughout the study the mice were observed three to four
times daily and mortality was recorded with each inspection. All
moribund mice were euthanized and the deaths were recorded as the
day of sacrifice. All mice that died or were sacrificed had their
lungs and spleens quantitatively cultured on drug-free and
antibiotic supplemented agar (3.times. MIC) to determine the effect
of the treatment regimen on the total and drug-resistant bacterial
populations, respectively.
[0137] Twenty-four hours after the last dose was given, a group of
surviving mice (n=5) were sacrificed and the lungs and spleens were
aseptically harvested. The homogenized specimens were washed with
saline to prevent drug carryover and bacteria were quantitatively
cultured on drug-free and antibiotic-supplemented agar (3.times.
MIC). The remaining animals were observed for survival for 14 days
after the last dose of drug is given. Those that were alive after
the 2-week observation period were sacrificed and their lungs and
spleens were quantitatively cultured for total and drug-resistant
populations. Portions of the homogenates were "heat shocked" for
spore determination and bacterial load was determined by plating
onto culture media and incubated at 36.degree. C.
[0138] Differences in survival between treatment and control groups
was assessed by the Fisher exact test and by survival analysis
techniques (Kaplan-Meier analysis and Cox proportional hazards
modeling). Differences in bacterial concentrations in the lungs
were determined by Student's t-test or by ANOVA. A P value<0.05
is considered statistically significant.
[0139] The results of the in vivo assay indicate that untreated
mice exposed to B. anthracis survived approximately 4 days, all
mice treated with 10 mg/kg compound AN survived the entire 21 days
and 75% of mice treated with 25 mg/kg of compound AN survived the
entire 21 days.
Equivalents
[0140] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the present
invention and are covered by the following claims. The contents of
all references, patents, and patent applications cited throughout
this application are hereby incorporated by reference. The
appropriate components, processes, and methods of those patents,
applications and other documents may be selected for the present
invention and embodiments thereof.
* * * * *