U.S. patent application number 10/921580 was filed with the patent office on 2005-01-27 for substituted tetracycline compounds as antifungal agents.
This patent application is currently assigned to Paratek Pharmaceuticals, Inc.. Invention is credited to Draper, Michael, Nelson, Mark L..
Application Number | 20050020545 10/921580 |
Document ID | / |
Family ID | 23054478 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020545 |
Kind Code |
A1 |
Draper, Michael ; et
al. |
January 27, 2005 |
Substituted tetracycline compounds as antifungal agents
Abstract
Methods and compositions for treating fungal associated
disorders in subjects are discussed.
Inventors: |
Draper, Michael; (Plaistow,
NH) ; Nelson, Mark L.; (Norfolk, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Paratek Pharmaceuticals,
Inc.
Boston
MA
02111
|
Family ID: |
23054478 |
Appl. No.: |
10/921580 |
Filed: |
August 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10921580 |
Aug 18, 2004 |
|
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10097457 |
Mar 14, 2002 |
|
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60275948 |
Mar 14, 2001 |
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Current U.S.
Class: |
514/152 |
Current CPC
Class: |
A61K 31/65 20130101;
A61P 43/00 20180101; A61P 31/10 20180101 |
Class at
Publication: |
514/152 |
International
Class: |
A61K 031/65 |
Claims
1. A pharmaceutical composition comprising an effective amount of a
substituted tetracycline compound to treat a fungal associated
disorder in a subject and a pharmaceutically acceptable
carrier.
2. The pharmaceutical composition of claim 110, wherein said
effective amount is effective to treat histoplasmosis, systemic
candidiasis, aspergillosis, blastomycosis, coccidioidomycosis,
paracoccidioidomycosis, cryptococcosis, dermatophyte infections,
tinea pedis, tinea cruris, candidiasis, actinomycosis, mycoses,
aspergillosis, candidosis, chromomycosis, entomophthoromycosis,
epizootic lymphangitis, geotrichosis, histoplasmosis, mucormycosis,
mycetoma, north american blastomycosis, oomycosis, paecilimycosis,
penicilliosis, rhinosporidiosis, or sprotrichiosis.
3. The pharmaceutical composition of claim 110, wherein said
substituted tetracycline compound is of the formula: 144X is
CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or O; R.sup.2,
R.sup.2', R.sup.4', and R.sup.4" are each independently hydrogen,
alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,
heteroaromatic or a prodrug moiety; R.sup.4 is NR.sup.4'R.sup.4",
alkyl, alkenyl, alkynyl, hydroxyl, halogen, or hydrogen; R.sup.3,
R.sup.11 and R.sup.12 are each hydrogen, or a pro-drug moiety;
R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9 to
form a ring; R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy; 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;
R.sup.7is hydrogen, halogen, nitro, alkyl, alkenyl, alkynyl, aryl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl, amino,
arylalkenyl, arylalkynyl, or
--(CH.sub.2).sub.0-3NR.sup.7cC(.dbd.W')WR.sup.7a; R.sup.9 is
hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,
arylalkynyl, thionitroso(e.g., --N.dbd.S), or
--(CH.sub.2).sub.0-3NR.sup.9cC(.dbd.Z')Z- R.sup.9a; Z is
CR.sup.9dR.sup.9e, S, NR.sup.9bor O; Z' is O, S, or NR.sup.9f; W is
CR.sup.7dR.sup.7e, S, NR.sup.7b or O; W' is O, NR.sup.7f S;
R.sup.7a,R.sup.7b,R.sup.7c,R.sup.7d,R.sup.7e,R.sup.9a,R.sup.9b,R.sup.9-
c,R.sup.9d, and R.sup.9e are each independently hydrogen, acyl,
alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,
heteroaromatic or a prodrug moiety; R.sup.8 is hydrogen, hydroxyl,
halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; R.sup.13
is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and Y'
and Y are eah independently hydrogen, halogen, hydroxyl, cyano,
sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, and
pharmaceutically acceptable salts thereof.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
10/097,457, filed Mar. 14, 2002, entitled "Substituted Tetracycline
Compounds as Antifungal Agents," Issuing; which claims priority to
U.S. Provisional Application Ser. No. 60/275,948, filed Mar. 14,
2001, entitled "Substituted Tetracycline Compounds as Antifungal
Agents". The entire contents of each of these applications is
hereby incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] For many years, the development of effective therapeutic
agents for fungal diseases (mycoses) has lacked the attention
devoted to drugs effective against other infective organisms. The
most common mycotic infections are superficial in nature, are not
life threatening, and provide little medical impetus to
pharmaceutical companies to develop novel treatments. This scenario
is changing, however, and while death from fungal disease is not
new, the incidence of systemic fungal infections that cause these
fatalities is increasing. Ironically, advances in modern medical
techniques in other fields (immunosuppressive and/or cytotoxic
therapy) and the advent of disease such as Acquired Immuno
Deficiency Syndrome (AIDS) are major contributing causes to the
increased number of serious fungal infections.
[0003] Fungal associated disorders can, thus, be divided into the
life-threatening systemic infections, such as histoplasmosis,
systemic candidiasis, aspergillosis, blastomycosis,
coccidioidomycosis, paracoccidioidomycosis, and cryptococcosis, and
the more common superficial ones, such as dermatophyte (ringworm)
infections, for example, tinea pedis (athlete's foot) and tinea
cruris (jock itch), candidiasis, and actinomycosis. The
life-threatening fungal infections are a growing problem not only
for immunosuppressed or immunocompromised individuals as noted
above but individuals with other viral infections, such as
cytomegalovirus (CMV), and influenza, for cancer patients receiving
chemotherapy or radiotherapy, for transplant patients receiving
antirejection agents, and for patients that have received toxic
chemicals, metals and radiation exposure.
[0004] Mycoses are often caused by fungi which are opportunists,
rather than pathogens. Candidiasis, aspergillosis, phycomycosis,
nocardiosis, and cryptococcosis are typically opportunistic fungal
infections. For example, Candida albicans, is normally found in the
alimentary tract as a commensal, yet it is a major cause of
systemic fungal infections in immunocomprised patients and topical
infections in healthy individuals.
[0005] Most drugs currently available for the treatment of mycoses
have limited efficacy or are poorly tolerated. A persistent and
vexatious problem with antifungal agents, largely unattended by the
prior art, is the lack of an agent that is easy and economical to
synthesize, and possesses high activity and broad spectrum activity
against organisms, low toxicity and limited adverse effects.
[0006] Moreover, many known agents merely have fungistatic
properties, rather than fungicidal properties. Fungistatic activity
is the ability to prevent growth of fungi, while fungicidal
(fungitoxic) activity is the ability to kill the fungi. Many agents
used in the treatment of superficial mycoses are virtually devoid
of either fungistatic or fungicidal actions in the concentrations
used, and their beneficial effects probably depend upon factors not
related to any direct effect on fungi.
[0007] Despite a plethora of agents which have or are alleged to
have antifungal properties, most are simply fungistatic and not
fungitoxic. For those that are fungicidal, for example,
amphotericin B, there are severe adverse side effects which limit
their use and their chemical properties, e.g., solubility, limit
drug delivery method.
SUMMARY OF THE INVENTION
[0008] Although opportunistic systemic fungal infections have a
high morbidity and mortality and their incidence is increasing, the
art has yet to provide a safe, effective water soluble,
simple-to-synthesize, fungitoxic agent with a broad antifungal
spectrum of activity coupled with limited adverse effects and low
toxicity.
[0009] In one embodiment, the invention pertains, at least in part
to a method for inhibiting the growth of a fungus. The method
includes contacting the fungus with an effective amount of a
substituted tetracycline compound, such that the growth of said
fungus is inhibited. In a further embodiment, the substituted
tetracycline compound is of formula I: 1
[0010] X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or
O;
[0011] R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0012] R.sup.4 is NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl,
hydroxyl, halogen, or hydrogen;
[0013] R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety;
[0014] R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9
to form a ring;
[0015] R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy;
[0016] 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;
[0017] R.sup.7 is hydrogen, halogen, nitro, alkyl, alkenyl,
alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
arylalkyl, amino, arylalkenyl, arylalkynyl, or
--(CH.sub.2).sub.0-3NR.sup.7cC(.dbd.W')WR.su- p.7a;
[0018] R.sup.9 is hydrogen, halogen, nitro, alkyl, alkenyl,
alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
arylalkyl, amino, arylalkenyl, arylalkynyl, thionitroso(e.g.,
--N.dbd.S), or --CH.sub.2).sub.0-3NR.sup.9cC(.dbd.Z')ZR.sup.9a;
[0019] Z is CR.sup.9dR.sup.9e, S, NR.sup.9b or O;
[0020] Z' is O, S, or NR.sup.9f;
[0021] W is CR.sup.7dR.sup.7e, S, NR.sup.7b or O;
[0022] W' is O, NR.sup.7f S;
[0023] R.sup.7a, R.sup.7b, R.sup.7c, R.sup.7d, R.sup.7e, R.sup.9a,
R.sup.9b, R.sup.9c, R.sup.9d, and R.sup.9e are each independently
hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0024] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl;
[0025] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; and
[0026] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl, and pharmaceutically acceptable salts thereof.
[0027] The invention also pertains to a method for treating a
fungal associated disorder in a subject. The method includes
administering to the subject an effective amount of a substituted
tetracycline compound such that the subject is treated for the
fungal associated disorder.
[0028] The invention also pertains to pharmaceutical compositions,
which contain an effective amount of a substituted tetracycline
compound to treat a fungal associated disorder in a subject and a
pharmaceutically acceptable carrier.
[0029] The invention also pertains to a method of killing fungus,
by contacting the fungus with a substituted tetracycline compound
of the invention, such that the fungus is killed.
DETAILED DESCRIPTION OF THE INVENTION
[0030] In addition to their well known antibacterial properties,
minocycline and doxycycline have been shown to possess limited
antifungal activity both alone and in synergy with amphotericin B
(Antimicrob. Agents Chemother. (1984), 26(6)837-40; Pathol Biol.
(1975) 23(9):725-8). The invention pertains, at least in part, to
methods and pharmaceutical compositions comprising substituted
tetracycline compounds with enhanced antifungal activity.
[0031] In an embodiment, the invention pertains to methods for
inhibiting the growth of a fungus. The method includes contacting
the fungus with an effective amount of a substituted tetracycline
compound, such that the growth of the fungus is inhibited.
[0032] The terms "fungus" or "fungi" include a variety of
nucleated, sporebearing organisms which are devoid of chlorophyll.
The term includes all fungi whose growth can be inhibited by the
compounds of the invention. Examples include, but are not limited
to, yeasts, mildews, molds, rusts, and mushrooms. Examples of fungi
also include, but are not limited to Aspergillus fumigatus,
Aspergillus flavus, Aspergillus nidulans, Candida albicans, Candida
glabrata, Candida guilliermondii, Candida krusei, Candida
lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus
neoformans, Issatchenkia orientalis, Coccidioides,
Paracoccidioides, Histoplasma, Blastomyces, and Neurospora crassa.
In one embodiment, the fungi of the invention includes fungi of the
genus Candida (e.g., C. tropicalis, C. parapsilosis, C. lusitaniae,
C. krusei, C. guilliermondii, C. glabrala, C. dubliniensis, and C.
albicans).
[0033] The term "inhibiting the growth of a fungus" includes both
fungistatic and fungicidal activity. Fungistatic activity includes
any decrease in the rate of growth of a fungal colony. Fungistatic
activity may be manifested by a fungus maintaining its present size
or failing to colonize the surrounding areas. Fungistatic activity
may be a result of inhibition of the fungal reproductive processes.
Fungicidal activity generally includes, for example, irraditication
of a fungus or fungal colony, killing a fungus or fungal colony or,
in one embodiment, a decrease in the mass or size of a fungus or
fungal colony.
[0034] The term "tetracycline compounds" includes tetracycline
family members such as methacycline, sancycline, apicycline,
clomocycline, guamecycline, meglucycline, mepylcycline,
penimepicycline, pipacycline, etamocycline, penimocycline, etc. as
well as other tetracycline compounds having the characteristic
naphthacene A-B-C-D ring structure. Additional tetracycline
compounds can be found, for example, in U.S. patent application
Ser. No.: 09/234,847, and U.S. Pat. Nos. 5,834,450; 5,532,227;
5,789,395; 5,639,742 and German patents DE 28 14 974 and DE 28 20
983. The entire contents of the aforementioned applications and
patents are hereby expressly incorporated herein by reference.
[0035] Recent research efforts have focused on developing new
tetracycline compositions effective under varying therapeutic
conditions and routes of administration; and for developing new
tetracycline analogues which might prove to be equal or more
effective as antibiotics than the originally introduced
tetracycline families (See, U.S. Pat. Nos. 3,957,980; 3,674,859;
2,980,584; 2,990,331; 3,062,717; 3,557,280; 4,018,889; 4,024,272;
4,126,680; 3,454,697; and 3,165,531).
[0036] The term "substituted tetracycline compounds" includes
tetracycline compounds which have at least one substitution, e.g.,
at the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11a, 12 and 12a position,
which allows the compound to perform its intended function, e.g.,
inhibit the growth of fungus. In an embodiment, the term
"substituted tetracycline compounds" does not include unsubstituted
tetracycline, minocycline, or doxycycline. In an embodiment, the
substituted tetracycline compounds of the invention have lower MIC
for Candida fungus (as measured in the assay given in Example 2)
than doxycycline or minocycline. In one embodiment, the substituted
tetracycline compounds of the invention have MIC's for a fungus
which is about 95% or less, about 90% or less, about 85% or less,
about 80% or less, about 75% or less, about 70% or less, about 65%
or less, about 60% or less, about 55% or less, about 50% or less,
about 45% or less, about 40% or less, about 35% or less, about 30%
or less, about 25% or less, about 20% or less, about 15% or less,
about 10% or less, about 5% or less, or about 1% or less than the
MIC of unsubstituted tetracycline, unsubstituted doxycycline, or
unsubstituted minocycline for that particular fungus. Values and
ranges included and/or intermediate within the ranges set forth
herein are also intended to be within the scope of the present
invention. For example, a MIC of less than 10% includes MIC's of
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc., which are intended to be
included within the range of less than 10%.
[0037] Furthermore, the substituted tetracycline compounds also may
advantageously be less cytotoxic than unsubstituted tetracycline,
minocycline, or doxycycline. In one embodiment, the cytotoxicity of
the substituted tetracycline compounds is about 95% or less, about
90% or less, about 85% or less, about 80% or less, about 75% or
less, about 70% or less, about 65% or less, about 60% or less,
about 55% or less, about 50% or less, about 45% or less, about 40%
or less, about 35% or less, about 30% or less, about 25% or less,
about 20% or less, about 15% or less, about 10% or less, about 5%
or less, or about 1% or less than the cytotoxicity of unsubstituted
tetracycline, unsubstituted doxycycline, or unsubstituted
minocycline. Values and ranges included and/or intermediate within
the ranges set forth herein are also intended to be within the
scope of the present invention. For example, a cytoxicity of less
than 10% includes cytotoxicities of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, etc. are intended to be included within the range of less than
10%.
[0038] The term "substituted tetracycline compound" includes, for
example, substituted sancycline compounds, substituted minocycline
compounds and substituted doxycycline compounds.
[0039] Substituted tetracycline compounds used in the methods and
compositions of the invention include compounds of Formula I: 2
[0040] X is CHC(R.sup.13Y'Y), CR.sup.6'R.sup.6, S, NR.sup.6, or
O;
[0041] R.sup.2, R.sup.2', R.sup.4', and R.sup.4" are each
independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0042] R.sup.4 is NR.sup.4'R.sup.4", alkyl, alkenyl, alkynyl,
hydroxyl, halogen, or hydrogen;
[0043] R.sup.3, R.sup.11 and R.sup.12 are each hydrogen, or a
pro-drug moiety;
[0044] R.sup.10 is hydrogen, a prodrug moiety, or linked to R.sup.9
to form a ring;
[0045] R.sup.5 is hydroxyl, hydrogen, thiol, alkanoyl, aroyl,
alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,
alkyl carbonyloxy, or aryl carbonyloxy;
[0046] 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;
[0047] R.sup.7 is hydrogen, halogen, nitro, alkyl, alkenyl,
alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
arylalkyl, amino, arylalkenyl, arylalkynyl, or
--(CH.sub.2).sub.0-3NR.sup.7cC(.dbd.W')WR.su- p.7a;
[0048] R.sup.9 is hydrogen, halogen, nitro, alkyl, alkenyl,
alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
arylalkyl, amino, arylalkenyl, arylalkynyl, thionitroso(e.g.,
--N.dbd.S), or --CH.sub.2).sub.0-3NR.sup.9cC(.dbd.Z')ZR.sup.9a;
[0049] Z is CR.sup.9d, R.sup.9e, S, NR.sup.9b or O;
[0050] Z' is O, S, or NR.sup.9f;
[0051] W is CR.sup.7dR.sup.7e, S, NR.sup.7b or O;
[0052] W' is O, NR.sup.7f S;
[0053] R.sup.7a, R.sup.7b, R.sup.7c, R.sup.7d, R.sup.7e, R.sup.9a,
R.sup.9b, R.sup.9c, R.sup.9d, and R.sup.9e are each independently
hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety;
[0054] R.sup.8 is hydrogen, hydroxyl, halogen, thiol, alkyl,
alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl;
[0055] R.sup.13 is hydrogen, hydroxy, alkyl, alkenyl, alkynyl,
alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl; and
[0056] Y' and Y are each independently hydrogen, halogen, hydroxyl,
cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an
arylalkyl, and pharmaceutically acceptable salts thereof.
[0057] In an embodiment, the substituted tetracycline compounds
used in the methods and compositions of the invention are
substituted sancycline compounds, e.g., with substitution at the,
for example, 2, 5, 6, 7,8, 9, 10, 11, 11a, 12, 12a position and/or,
in the case of minocycline, 13. In substituted sancycline compounds
of the invention, R.sup.2', R.sup.3, R.sup.10, R.sup.11, and
R.sup.12 are each hydrogen or a prodrug moiety; R.sup.4' and
R.sup.4" are each alkyl (e.g., lower alkyl, e.g., methyl); X is
CR.sup.6R.sup.6'; and R.sup.2, R.sup.5, R.sup.6, R.sup.6', and
R.sup.8 are each, generally, hydrogen. In other embodiments, In an
embodiment, the substituted tetracycline compound is a substituted
tetracycline (e.g., generally, wherein R.sup.4 is
NR.sup.4'R.sup.4", R.sup.4' and R.sup.4" are methyl, R.sup.5 is
hydrogen and X is CR.sup.6R.sup.6', wherein R.sup.6 is methyl and
R.sup.6' is hydroxy); substituted doxycycline (e.g., wherein
R.sup.4 is NR.sup.4'R.sup.4", R.sup.4' and R.sup.4" are methyl,
R.sup.5 is hydroxyl and X is CR.sup.6R.sup.6', wherein R.sup.6 is
methyl and R.sup.6' is hydrogen); substituted minocycline (e.g.,
wherein R.sup.4 is NR.sup.4'R.sup.4", R.sup.4' and R.sup.4" are
methyl; R.sup.5 is hydrogen and X is CR.sup.6R.sup.6' wherein
R.sup.6 and R.sup.6' are hydrogen atoms and R.sup.7 is
dimethylamino) or substituted sancycline (wherein R.sup.4 is
NR.sup.4'R.sup.4", R.sup.4' and R.sup.4" are methyl; R.sup.5 is
hydrogen and X is CR.sup.6R.sup.6' wherein R.sup.6 and R.sup.6' are
hydrogen atoms).
[0058] In one embodiment, R.sup.5is substituted, e.g., not hydrogen
or hydroxy. In a further embodiment R.sup.5 is an ester
(alkcarbonyloxy). In an embodiment, R.sup.5 is an alkyl ester.
Examples of R.sup.5 include alkyl esters such as C.sub.1-C.sub.12
alkyl, alkenyl, alkynyl, or aryl esters. The alkyl groups may be
straight chains, branched chains, and/or contain rings. Examples of
esters include, but are not limited to, tetracycline esters of
ethanoic acid, propanoic acid, pentanoic acid, hexanoic acid,
2-cyclopentane ethanoic acid, cyclopentanoic acid, cycloheptanoic
acid, 2-methyl propanoic acid, cyclohexanoic acid, and adamantane
2-carboxylic acid. In other embodiments, R.sup.5 is hydrogen.
[0059] For 7-substituted tetracycline compounds, R.sup.9 may be
hydrogen. In one embodiment, R.sup.7 is substituted or
unsubstituted phenyl. Examples of R.sup.7 substituents include all
substituents which allow the tetracycline compound to perform its
intended function, such as but are not limited to, alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
alkyloxycarbonyl, carboxy, arylcarbonyloxy, alkoxycarbonylamino,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0060] In an embodiment, the phenyl is substituted with at least
one alkyl group, which itself may be, branched, straight chain or
alkyl, unsubstituted or substituted (e.g., halogenated). Examples
of alkyl groups include, but are not limited to, methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, etc.
[0061] In another embodiment R.sup.7 is a halogen, e.g., chlorine,
bromine, or iodine.
[0062] In another embodiment, R.sup.7 is substituted or
unsubstituted heteroaryl. Examples of heteroaromatic groups include
both monocyclic and polycyclic (e.g., multicylic rings), such as,
but not limited to, furanyl, imidazolyl, benzothiophenyl,
benzofuranyl, quinolinyl, isoquinolinyl, pyridinyl, pyrazolyl,
benzodioxazolyl, benzoxazolyl, benzothiazolyl, benzoimidazolyl,
methylenedioxyphenyl, indolyl, thienyl, pyrimidyl, pyrazinyl,
purinyl, pyrazolyl, oxazolyl, isooxazolyl, naphthridinyl,
thiazolyl, isothiazolyl, and deazapurinyl. In an embodiment,
R.sup.7 is benzofuranyl. Examples of substituents include all
substituents which allow the tetracycline compound to perform its
intended function, such as but are not limited to, alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
alkyloxycarbonyl, carboxy, arylcarbonyloxy, alkoxycarbonylamino,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0063] In another embodiment, R.sup.7 is substituted or
unsubstituted, branched, straight chain or cyclic alkyl. Examples
of substituents include those which allow the substituted
tetracycline compound to perform its intended function, e.g.,
inhibit the growth of a fungus. Examples of substitutents include,
but are not limited to, alkenyl, alkynyl, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, trialkylsilyl,
arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,
phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,
alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, cyano, azido,
heterocyclyl, alkylaryl, aryl and heteroaryl.
[0064] Examples of alkyl R.sup.7 groups include C.sub.1-C.sub.15
groups and C.sub.1-C.sub.10 groups. Examples include 2-ethyl
pentyl, methyl, ethyl, propyl, pentyl, hexyl, heptyl, etc.
[0065] In one embodiment, R.sup.7 is substituted or unsubstituted
alkenyl. Examples of substituents include all substituents which
allow the tetracycline compound to perform its intended function,
such as but are not limited to, alkyl, alkenyl, alkynyl, halogen,
hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0066] In one embodiment, an alkenyl R.sup.7 moiety is substituted
with a substituted or unsubstituted cyclic moiety. Cyclic moieties
include both carbocyclic, heterocyclic, aryl, heteroaryl,
cycloalkenyl, and cycloalkyl groups. Examples of cyclic moieties
include, for example, cyclobutane, cylopentane, cyclohexane,
phenyl, etc. The cyclic moiety can be substituted, e.g., with any
substituent listed above for alkenyl R.sup.7 moieties.
[0067] In another embodiment, R.sup.7 is substituted or
unsubstituted alkynyl. Examples of substituents include all
substituents which allow the tetracycline compound to perform its
intended function, such as but are not limited to, alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
alkyloxycarbonyl, carboxy, arylcarbonyloxy, alkoxycarbonylamino,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0068] The R.sup.7 alkynyl moiety may be substituted with a
substituted or unsubstituted cyclic moiety. Cyclic moieties include
both carbocyclic, heterocyclic, aryl, heteroaryl, cycloalkenyl, and
cycloalkyl groups. Examples of cyclic moieties include, for
example, cyclobutane, cylopentane, cyclohexane, phenyl, etc. The
cyclic moiety can be substituted, e.g., with any substituent listed
above for alkynyl R.sup.7 moieties.
[0069] Examples of cyclic substituents for alkynyl R.sup.7 moieties
include, but are not limited to, phenyl, cyclohexyl, p-nitro
phenyl, p-methyl phenyl, and 1-hydroxy cyclohexane.
[0070] In another embodiment. R.sup.7 is substituted or
unsubstituted alkoxy. Examples of substituents include all
substituents which allow the tetracycline compound to perform its
intended function, such as but are not limited to, alkenyl,
alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
alkyloxycarbonyl, carboxy, arylcarbonyloxy, alkoxycarbonylamino,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl. In one embodiment, the alkoxy group
is C.sub.1-C.sub.10. In another embodiment, it is methoxy, ethoxy,
propoxy, butoxy, pentoxy, hexoxy, etc.
[0071] In another embodiment, the invention pertains to substituted
doxycycline compounds wherein R.sup.5 is hydroxy or
alkylcarbonyloxy; X is CHR.sup.6; R.sup.6 is alkyl (e.g., lower
alkyl, e.g., methyl); and R.sup.8 is hydrogen. R.sup.7 may be
hydrogen or alkyl. R.sup.2 may be hydrogen or alkyl.
[0072] In one embodiment, R.sup.9 is substituted or unsubstituted
aryl, e.g., phenyl, biaryl, heteroaryl (e.g., pyridine, etc.), etc.
Examples of substituents include all substituents which allow the
tetracycline compound to perform its intended function, such as but
are not limited to, alkyl, alkenyl, alkynyl, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0073] Other examples of R.sup.9 include substituted or
unsubstituted alkyl (e.g., methyl, ethyl, propyl, t-butyl, n-butyl,
i-butyl, pentyl, etc.)or N.sub.3.
[0074] In another embodiment, the invention pertains to methods and
compositions which the substituted tetracycline compound is a
substituted minocycline compound. Examples of these compounds
include compounds wherein X is CR.sup.6R.sup.6'; R.sup.2, R.sup.5,
R.sup.6, R.sup.6', and R.sup.8 are each hydrogen, and R.sup.7 is
dimethyl amino.
[0075] In an embodiment, R.sup.9 is substituted or unsubstituted
aryl (e.g., phenyl, biaryl (naphthyl, benzofuranyl), heteroaryl,
etc.) or araalkyl. Examples of substituents include, but are not
limited to, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,
alkylcarbonyloxy, alkyloxycarbonyl, carboxy, arylcarbonyloxy,
alkoxycarbonylamino, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkylaminoacarbonyl, arylalkyl
aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,
aminoalkyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
silyl, aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl,
phosphonato, phosphinato, cyano, amino, acylamino, amido, imino,
sulfhydryl, alkylthio, sulfate, arylthio, thiocarboxylate,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano,
azido, heterocyclyl, alkylaryl, aryl and heteroaryl. In one
embodiment, the aryl R.sup.9 moiety is substituted or unsubstituted
phenyl.
[0076] Other examples of R.sup.9 moieties include substituted and
unsubstituted, cyclic, branched or straight chain alkyl (e.g.,
C.sub.1-C.sub.15, C.sub.1-C.sub.10, e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, 2-cyclopentane ethyl, etc.). Examples of
substituents include those listed above for aryl R.sup.9
moieties.
[0077] In another embodiment, R.sup.9 is substituted or
unsubstituted, branched, straight chain or cyclic alkyl. Examples
of substituents include those which allow the substituted
tetracycline compound to perform its intended function, e.g.,
inhibit the growth of a fungus. Examples of substitutents include,
but are not limited to, alkenyl, alkynyl, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, trialkylsilyl,
arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, silyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, aralkyl, phosphonato,
phosphinato, cyano, amino, acylamino, amido, imino, sulfhydryl,
alkylthio, sulfate, arylthio, thiocarboxylate, alkylsulfinyl,
sulfonato, sulfamoyl, sulfonamido, nitro, cyano, azido,
heterocyclyl, alkylaryl, aryl and heteroaryl.
[0078] Examples of alkyl R.sup.9 groups include C.sub.1-C.sub.15
groups and C.sub.1-C.sub.10 groups. Examples include 2-ethyl
pentyl, methyl, ethyl, propyl, pentyl, hexyl, heptyl, etc.
[0079] In one embodiment, R.sup.9 is substituted or unsubstituted
alkenyl. Examples of substituents include all substituents which
allow the tetracycline compound to perform its intended function,
such as but are not limited to, alkyl, alkenyl, alkynyl, halogen,
hydroxyl, alkoxy, alkylcarbonyloxy, alkyloxycarbonyl, carboxy,
arylcarbonyloxy, alkoxycarbonylamino, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0080] In one embodiment, an alkenyl R.sup.9 moiety is substituted
with a substituted or unsubstituted cyclic moiety. Cyclic moieties
include both carbocyclic, heterocyclic, aryl, heteroaryl,
cycloalkenyl, and cycloalkyl groups. Examples of cyclic moieties
include, for example, cyclobutane, cylopentane, cyclohexane,
phenyl, etc. The cyclic moiety can be substituted, e.g., with any
substituent listed above for alkenyl R.sup.9 moieties.
[0081] In another embodiment, R.sup.9 is substituted or
unsubstituted alkynyl. Examples of substituents include all
substituents which allow the tetracycline compound to perform its
intended function, such as but are not limited to, alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
alkyloxycarbonyl, carboxy, arylcarbonyloxy, alkoxycarbonylamino,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,
alkylcarbonyl, arylcarbonyl, aminoalkyl, arylalkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, silyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, aralkyl, phosphonato, phosphinato,
cyano, amino, acylamino, amido, imino, sulfhydryl, alkylthio,
sulfate, arylthio, thiocarboxylate, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, cyano, azido, heterocyclyl,
alkylaryl, aryl and heteroaryl.
[0082] The R.sup.9 alkynyl moiety may be substituted with a
substituted or unsubstituted cyclic moiety. Cyclic moieties include
both carbocyclic, heterocyclic, aryl, heteroaryl, cycloalkenyl, and
cycloalkyl groups. Examples of cyclic moieties include, for
example, cyclobutane, cylopentane, cyclohexane, phenyl, etc. The
cyclic moiety can be substituted, e.g., with any substituent listed
above for alkynyl R.sup.9 moieties.
[0083] Furthermore, the substituted tetracycline compounds of the
invention can be substituted with combinations of any one of the
substituents described above or shown in Table 2.
[0084] Examples of cyclic substituents for alkynyl R.sup.9 moieties
include, but are not limited to, phenyl, cyclohexyl, p-nitro
phenyl, p-methyl phenyl, and 1-hydroxy cyclohexane.
[0085] In a further embodiment, R.sup.9 is substituted or
unsubstituted aminoalkyl. Examples of substituents include, but are
not limited to, alkyl, alkenyl, alkynyl, and aryl substituents.
[0086] In one embodiment, the substituted tetracycline compounds
for use in the methods and compositions of the invention have a
greater antifungal activity than unsubstituted doxycycline or
minocycline. Both of these compounds have Minimum Inhibitory
Concentrations (MIC) for C. Albicans, C. giabrata, C. tropicalis,
and C. parapsilosis in excess of 64 (See example 2, for the
procedure for measuring the MIC).
[0087] In another embodiment, the substituted tetracycline
compounds of the invention may have anti-inflammatory activity,
e.g., as measured in art recognized assays.
[0088] In another embodiment, the substituted tetracycline
compounds of the invention may be non-antibacterial, e.g., have
little or no antibacterial activity. The antibacterial activity of
the compounds of the invention can be measured using the assay
given in Example 5. In an embodiment, a compound is considered to
be non-antibacterial if it has a MIC (against bacteria) of 4 .mu.M
or greater.
[0089] In another embodiment, the invention also pertains to
4-dedimethylaminotetracycline compounds with the substituents
described herein or shown in Table 2 (e.g., compounds with the same
substituents as described herein or in Table 2, except at the
R.sup.4 position where the shown dimethylamino group is a
hydrogen.)
[0090] Examples of substituted tetracycline compounds for use in
the methods and compositions of the invention include the compounds
shown in Table 2, as well as those shown below: 345678910
[0091] The substituted tetracycline compounds of the invention can
be synthesized using the methods described in Example 1 and in the
following schemes. Any novel tetracycline compounds described
herein are included in the invention as compounds, in addition to
methods of using them and pharmaceutical compositions containing
them.
[0092] 9-substituted tetracyclines such as 9-cyclopentenyl
doxycycline can be synthesized by the method shown in Scheme 1. As
in Scheme 1,9- and 7-substituted tetracycline compounds can be
synthesized by treating a tetracycline compound (e.g., doxycycline,
1 A), with sulfuric acid and sodium nitrate. The resulting product
is a mixture of the 7-nitro and 9-nitro isomers (1 B and 1 C,
respectively). The 7-nitro (1 B) and 9-nitro (1 C) derivatives are
treated by hydrogenation using hydrogen gas and a platinum catalyst
to yield amines 1 D and 1 E. 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 (1 E and 1 F,
respectively) is treated with HONO, to yield the diazonium salt (1
G and 1 H). The salt (1 G and 1 H) is treated with an appropriate
halogenated reagent (e.g., R.sup.9Br, wherein R.sup.9 is an aryl,
alkenyl, or alkynyl moiety) to yield the desired compound(e.g., in
Scheme 1, 7-cyclopent-1-enyl doxycycline (1 H) and
9-cyclopent-1-enyl doxycycline (1 I)). 1112 13
[0093] 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 (2 A) is
treated with NaNO.sub.2 under acidic conditions forming 7-nitro
sancycline (2 B) in a mixture of positional isomers.
7-nitrosancycline (2 B) is then treated with H.sub.2 gas and a
platinum catalyst to form the 7-amino sancycline derivative (2 C).
To form the urea derivative (2 E), isocyanate (2 D) is reacted with
the 7-amino sancycline derivative (2 C). To form the carbamate (2
G), the appropriate acid chloride ester (2 F) is reacted with 2 C.
14
[0094] 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 (3 A) is reacted with
Fmoc-isothiocyanate (3 B) to produce the protected thiourea (3 C).
The protected thiourea (3 C) is then deprotected yielding the
active sancycline thiourea (3 D) compound. The sancycline thiourea
(3 D) is reacted with an .alpha.-haloketone (3 E) to produce a
thiazole substituted 7-amino sancycline (3 F). 15
[0095] 7-alkenyl tetracycline compounds, such as 7-alkynyl
sancycline (4 A) and 7-alkenyl sancycline (4 B), can be
hydrogenated to form alkyl 7-substituted tetracycline compounds
(e.g., 7-alkyl sancycline, 4 C). 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. 16
[0096] 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 (5 B) can be synthesized from sancycline by
treating sancycline (5 A) with at least one equivalent
N-iodosuccinimide (NIS) under acidic conditions. The reaction is
quenched, and the resulting 7-iodo sancycline (5 B) can then be
purified using standard techniques known in the art. To form the
aryl derivative, 7-iodo sancycline (5 B) is treated with an aqueous
base (e.g., Na.sub.2CO.sub.3) and an appropriate boronic acid (5 C)
and under an inert atmosphere. The reaction is catalyzed with a
palladium catalyst (e.g., Pd(OAc).sub.2). The product (5 D) can be
purified by methods known in the art (such as HPLC). Other 7-aryl
and alkynyl tetracycline compounds can be synthesized using similar
protocols.
[0097] 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. 17
[0098] 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., 6-iodosancycline, 6 A) 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 (6 B) or alkyne (6
D), and triethylamine are then added and the mixture is heated for
several hours, before being cooled to room temperature. The
resulting 7-substituted alkenyl (6 C) or 7-substituted alkynyl (6
E) tetracycline compound can then be purified using techniques
known in the art. 18
[0099] To prepare 7-(2'-Chloro-alkenyl)-tetracycline compounds, the
following procedure can be used. 7-(alkynyl)-sancycline (7 A) is
dissolved in saturated methanol and hydrochloric acid and stirred.
The solvent is then removed to yield the product (7 B). 19
[0100] As depicted in Scheme 8,5-esters of 9- substituted
tetracycline compounds can be formed by dissolving the 9-
substituted compounds (8 A) in strong acid (e.g. HF,
methanesulphonic acid, and trifluoromethanesulfonic acid) and
adding the appropriate carboxylic acid to yield the corresponding
esters (8 B).
[0101] As shown in Scheme 9 below, 7 and 9 aminomethyl
tetracyclines may be synthesized using reagents such as
hydroxymethyl-carbamic acid benzyl ester. 20
[0102] The term "alkenyl" includes unsaturated aliphatic groups,
including straight-chain alkenyl groups, branched-chain alkenyl
groups, cycloalkenyl (alicyclic) groups, alkenyl substituted
cycloalkyl or cycloalkenyl groups, and cycloalkenyl substituted
alkyl or alkenyl groups. The term alkenyl further includes alkenyl
groups, which can further include oxygen, nitrogen, sulfur or
phosphorous atoms replacing one or more carbons of the hydrocarbon
backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In
preferred embodiments, a straight chain or branched chain alkenyl
group has 10 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for branched
chain), and more preferably 6 or fewer. Likewise, preferred
cycloalkenyl groups have from 4-7 carbon atoms in their ring
structure, and more preferably have 5 or 6 carbons in the ring
structure, e.g., cyclopentene or cyclohexene.
[0103] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. The term alkyl further
includes alkyl groups, which can further include oxygen, nitrogen,
sulfur or phosphorous atoms replacing one or more carbons of the
hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous
atoms. In preferred embodiments, a straight chain or branched chain
alkyl has 10 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for branched
chain), and more preferably 6 or fewer. Likewise, preferred
cycloalkyls have from 4-7 carbon atoms in their ring structure, and
more preferably have 5 or 6 carbons in the ring structure.
[0104] 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, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. It will be understood by those
skilled in the art that the moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls
can be further substituted, e.g., with the substituents described
above. An "alkylaryl" moiety is an alkyl substituted with an aryl
(e.g., phenylmethyl (benzyl)).
[0105] The term "aryl" includes aryl groups, including 5- and
6-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, benzoxazole, benzothiazole, triazole,
tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Aryl groups also include polycyclic fused aromatic
groups such as naphthyl, quinolyl, indolyl, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl 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, 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,
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).
[0106] The terms "alkenyl" and "alkynyl" include unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond, respectively. Examples of substituents of alkynyl
groups include, for example alkyl, alkenyl (e.g., cycloalkenyl,
e.g., cyclohenxenyl), and aryl groups.
[0107] 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 three carbon atoms in its backbone structure.
Likewise, "lower alkenyl" and "lower alkynyl" have similar chain
lengths.
[0108] The terms "alkoxyalkyl", "polyaminoalkyl" 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.
[0109] 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, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino (including alkyl
amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
[0110] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0111] The term "alkylsulfinyl" include groups which have one or
more sulfinyl (SO) linkages, typically 1 to about 5 or 6 sulfinyl
linkages. Advantageous alkylsulfinyl groups include groups having 1
to about 12 carbon atoms, preferably from 1 to about 6 carbon
atoms.
[0112] The term "alkylsulfonyl" includes groups which have one or
more sulfonyl (SO.sub.2) linkages, typically 1 to about 5 or 6
sulfonyl linkages. Advantageous alkylsulfonyl groups include groups
having 1 to about 12 carbon atoms, preferably from 1 to about 6
carbon atoms.
[0113] The term "alkanoyl" includes groups having 1 to about 4 or 5
carbonyl groups. The term "aroyl" includes aryl groups, such as
phenyl and other carbocyclic aryls, which have carbonyl
substituents. The term "alkaroyl" includes aryl groups with
alkylcarbonyl substituents, e.g., phenylacetyl.
[0114] The structures of some of the tetracycline compounds of this
invention include asymmetric carbon atoms. The isomers arising from
the chiral atoms (e.g., all enantiomers and diastereomers) are
included within the scope of this invention, unless indicated
otherwise. Such isomers can be obtained in substantially pure form
by classical separation techniques and by stereochemically
controlled synthesis.
[0115] The invention also pertains to methods of treating fungal
associated disorders in a subject, by administering to the subject
an effective amount of a substituted tetracycline compound such
that the subject is treated. The substituted tetracycline compound
may, in one embodiment, be a compound of formula (I) or any one of
the compounds depicted in Table 2.
[0116] The language "effective amount" of the tetracycline compound
is that amount necessary or sufficient to inhibit the growth of
fungus or treat a fungus associated disorder, e.g., in an animal or
in a plant, e.g., and prevent the various morphological and somatic
symptoms of a fungal associated disorder. The effective amount can
vary depending on such factors as the size and weight of the
subject, the type of disorder, or the particular substituted
tetracycline compound. For example, the choice of the substituted
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 substituted tetracycline compound without
undue experimentation. An in vivo assay as described in Example 4
below or an assay similar thereto (e.g., differing in choice of
cell line or type of illness) also can be used to determine an
"effective amount" of a tetracycline compound. The ordinarily
skilled artisan would select an appropriate amount of a
tetracycline compound for use in the aforementioned in vivo assay.
In an embodiment, the effective amount of the tetracycline is
effective to treat a mammal suffering from a fungal associated
disorder which is associated with a fungus from the genus
Candida.
[0117] The term "subject" any organism which may benefit from the
inhibition of a fungus or which is capable of having a fungal
associated disorder. Examples of subjects include not only animals,
such as mammals, birds, fish, etc., but plants which may be
adversely effected by the presence of a fungus.
[0118] The term "mammal" includes, but is not limited to, ruminants
(e.g., cattle and goats), mice, rats, hamsters, dogs, cats, horses,
pigs, sheep, lions, tigers, bears, monkeys, chimpanzees, and, in a
preferred embodiment, humans. The mammal may be immunocompetent or
immunocompromised, e.g., suffering from an immunodeficiency. For
example, the mammal may have AIDS or may have previously or
concurrently undergone chemotherapy. In another embodiment, the
mammal may be elderly or young. The mammal may or may not be
suffering from a fungal associated disorder. The tetracycline
compounds may be administered to a mammal susceptible to a fungal
associated disorder to prevent the occurrence of the disorder.
[0119] The language "fungal associated disorder" includes disorders
which are related to the presence of fungus in a subject. Examples
of fungal associated disorders include both topical fungal
infections caused by, e.g., Candida, and dermatophytes such as
Trichophyton, Microsporum or Epidermophyton, or in mucosal
infections caused by Candida albicans (e.g., oral thrush and
vaginal candidiasis). The substituted tetracycline compounds of the
invention are also useful for treatment of systemic fungal
infections caused by, for example, Candida albicans, Cryptococcus
neoformans, Aspergillus flavus, Aspergillus funigatus,
Coccidioides, Paracoccidioides, Histoplasma or Blastomyces. The
substituted tetracycline compounds of the invention may be useful
for treating fungal infections in immunocompromised patients such
as patients with viral infections such as AIDS, CMV, and influenza,
cancer patients receiving chemotherapy or radiotherapy, transplant
patients receiving antirejection agents, and patients that have
received toxic chemicals, metals and radiation exposure.
[0120] Examples of fungal associated disorders in animals include
systemic infections, such as histoplasmosis, systemic candidiasis,
aspergillosis, blastomycosis, coccidioidomycosis,
paracoccidioidomycosis, and cryptococcosis, and superficial fungal
disorders, such as dermatophyte (ringworm) infections, for example,
tinea pedis (athlete's foot) and tinea cruris (jock itch),
candidiasis, and actinomycosis. Another example of a fungal
associated disorder include mycoses, which may be caused by fungi
which are opportunists, rather than pathogens. Examples of fungi
which may cause mycoses include candidiasis, aspergillosis,
phycomycosis, nocardiosis, and cryptococcosis.
[0121] Other fungal associated disorders include aspergillosis,
candidosis, chromomycosis, coccidioidiocycosis, cryptocococcosis,
entomophthoromycosis, epizootic lymphangitis, geotrichosis,
histoplasmosis, mucormycosis, mycetoma, north american
blastomycosis, oomycosis, paecilimycosis, penicilliosis,
rhinosporidiosis, and sprotrichiosis in animals. In an embodiment,
the substituted tetracycline compounds of the invention can be
included in feed for the livestock, such that normal consumption of
said feed provides about 1 mg to about 200 mg of at least one of
the substituted tetracycline compounds of the invention per kg of
animal per day.
[0122] The invention also pertains to a pharmaceutical composition
containing an effective amount of a tetracycline compound to treat
or prevent a fungal associated disorder in a subject, e.g., a
mammal, and a pharmaceutically acceptable carrier. The tetracycline
compound may be a substituted tetracycline, a compound of formula
(I), or a compound depicted in Table 2.
[0123] The language "pharmaceutically acceptable carrier" includes
substances capable of being coadministered with the substituted
tetracycline compound(s), and which allows the substituted
tetracycline compounds to perform their intended function, e.g.,
treating a fungal associated state or preventing a fungal
associated disorder. Examples of such carriers include solutions,
solvents, dispersion media, delay agents, emulsions and the like.
The use of such media for pharmaceutically active substances are
well known in the art. Any other conventional carrier suitable for
use with the tetracycline compounds of the present invention are
included.
[0124] For example, one or more substituted tetracycline compounds
of the invention may be administered alone to a subject, or more
typically a compound of the invention will be administered as part
of a pharmaceutical composition in mixture with conventional
excipient, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for parenteral, oral or other desired
administration and which do not deleteriously react with the active
compounds and are not deleterious to the recipient thereof.
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, hydroxymethylcellulose, 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.
[0125] At least many of the substituted tetracycline compounds of
the invention suitably may be administered to a subject in a
protonated and water-soluble form, e.g., as a pharmaceutically
acceptable salt of an organic or inorganic acid, e.g.,
hydrochloride, sulfate, hemi-sulfate, phosphate, nitrate, acetate,
oxalate, citrate, maleate, mesylate, etc. Also, where an
appropriate acidic group is present on a compound of the invention,
a pharmaceutically acceptable salt of an organic or inorganic base
can be employed such as an ammonium salt, or salt of an organic
amine, or a salt of an alkali metal or alkaline earth metal such as
a potassium, calcium or sodium salt.
[0126] Therapeutic compounds can be administered to a subject in
accordance with the invention by any of a variety of routes.
Topical (including transdermal, buccal or sublingual), oral, and
parenteral (including intraperitoneal, subcutaneous, intravenous,
intradermal or intramuscular injection) are generally
preferred.
[0127] 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 tetracycline compounds may also be formulated such that the
compound is released over an extended period of time.
[0128] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Substituted tetracycline 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.
[0129] 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.
[0130] For topical applications, the substituted tetracycline
compound(s) 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.
[0131] The actual preferred amounts of active substituted
tetracycline compounds used in a given therapy will vary according
to the specific compound being utilized, 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.
[0132] 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.
[0133] 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.
[0134] In a still further aspect, the substituted tetracycline
compounds of the present invention (e.g., substituted tetracycline
compounds, e.g., compounds of formula (I), or compounds shown in
Table 2) can also be advantageously used in agricultural
compositions, for example, compositions for plants and seeds to
treat or prevent a variety of plant pathogenic fungi, including
rusts, mildews, and molds. Generally, the compounds of the present
invention are dispensed in the form of dusting powders, granules,
seed dressings, aqueous solutions, dispersions or emulsions, dips,
sprays, aerosols or smokes. Compositions may also be supplied in
the form of dispersible powders, granules or grains, or
concentrates for dilution prior to use. Such compositions may
contain such conventional carriers, diluents or adjuvants as are
known and acceptable in agriculture and horticulture, and they are
manufactured in accordance with conventional procedures. The
compositions typically contain from 0.01 to 10 wt %, preferably 0.1
to 1 wt. % of the active ingredient. The compositions may also
incorporate other active ingredients, for example, compounds having
herbicidal or insecticidal activity or a further fungicide. The
compounds and compositions can be applied in a number of ways, for
example, they can be applied directly to the plant foliage, stems,
branches, seeds or roots or to the soil or other growing medium and
they may be used not only to eradicate disease, but also
prophylactically to protect the plants or seeds from attack. For
field use, likely application rates of active ingredient are about
100 to 10,000 g/acre. The substituted tetracycline compounds of the
invention can also be formulated for use in cleaning supplies,
e.g., to prevent a growth of a fungus, or to kill or stop the
growth of a fungus
[0135] The present invention is further illustrated by the
following examples. These examples are provided to aid in the
understanding of the invention and are not to be construed as
limitations thereof.
EXEMPLIFICATION OF THE INVENTION
Example 1
[0136] Synthesis of Tetracycline Compounds
[0137] The following example discusses methods of synthesizing the
substituted tetracycline compounds of the invention. One of
ordinary skill in the art will be able to use the presented
examples and/or art recognized techniques to synthesize the
compounds of the invention.
[0138] Experimental
[0139] Melting points were taken on a Mel-Temp capillary melting
point apparatus and are uncorrected. Nuclear magnetic resonance
(.sup.1H NMR) spectra were recorded at 300 MHz on a Bruker Avance
spectrometer. The chemical shift values are expressed in .delta.
values (ppm) relative to tetramethylsilane or
3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt, as either
an internal or external standard using CDCl.sub.3, DMSO-d.sub.6, or
MeOH-d.sub.4 as the solvent. Column chromatography was performed
according to the method of Still using Baker "flash" grade silica
gel (40 .mu.m) that was treated with a saturated solution of
Na.sub.2EDTA, washed with water, filtered and dried in an oven at
130.degree. C. for three hours prior to use. Analytical TLC
separations employed the use of 0.25 mm silica gel plates with
florescence indicator obtained from J.T. Baker Chemical Co.,
Phillipsburg, N.J., that were pretreated by immersion into a
saturated solution of Na.sub.2EDTA for five minutes and reactivated
at 130.degree. C. for three hours. Solvent systems used were as
follows: 50:50:5 CHCl.sub.3/MeOH/5% Na.sub.2EDTA (lower phase) (I),
65:20:5, CHCl.sub.3/MeOH/Na.sub.2EDTA (lower phase) (II).
Visualization of TLC was accomplished by 0.5% aqueous Fast Blue BB
salt and heating at 130.degree. C. for 5 minutes. Analytical HPLC
was performed on a Waters Bondapak C18 reverse phase column by
using two Varian SD 100 HPLC pumps at a 1.6 mL/min flow rate
controlled by software. Detection was by UV absorption with Model
441 absorbance detector operating at 280 nm. Mobile phases used
followed a linear gradient from 30% to 100% methanol over 30
minutes at 1.6 mL/min flow rate followed by isocratic elution with
MeOH; solvent system A: 0.02 M Na.sub.2HPO.sub.4+0.001 M
Na.sub.2EDTA adjusted to pH 4.5 with H.sub.3PO.sub.3; solvent
system B: 100% MeOH. Semipreparative HPLC separations used a Waters
semipreparative C18 reverse-phase column at a flow rate of 6.4
mL/min. Low and high resolution mass spectra were performed on a PE
Mariner spectrometer (Nelson el al., J. Med. Chem. (1993)
36(3):374).
[0140] 7 lodo Sancycline
[0141] One gram of sancycline was dissolved in 25 mL of TFA
(trifluoroacetic acid) that was cooled to 0.degree. C. (on ice).
1.2 equivalents of N-iodosuccinimide (NIS) was added to the
reaction mixture and reacted for forty minutes. The reaction was
removed from the ice bath and was allowed to react at room
temperature for an additional five hours. The mixture was then
analyzed by HPLC and TLC, was driven to completion by the stepwise
addition of NIS. After completion of the reaction, the TFA was
removed in vacuo and 3 mL of MeOH was added to dissolve the
residue. The methanolic solution was the added slowly to a rapidly
stirring solution of diethyl ether to form a greenish brown
precipitate. The 7-iodo isomer of sancycline was purified by
treating the 7-iodo product with activated charcoal, filtering
through Celite, and subsequent removal of the solvent in vacuo to
produce the 7-isomer compound as a pure yellow solid in 75%
yield.
[0142] MS(M+H) (formic acid solvent) 541.3.
[0143] .backslash.Rt: Hypersil C18 BDS Column, 11.73
[0144] .sup.1H NMR (Methanol d4-300 MHz) .delta.7.87-7.90 ( d, 1H),
6.66-6.69 (d, 1H), 4.06 (s, 1H), 2.98 (s, 6H), 2.42 (m, 1H), 2.19
(m, 1H), 1.62 (m, 4H), 0.99 (m, 2H)
[0145] 7-(2',5' Dimethyl-Phenyl Sancycline
[0146] 7-iodosancycline (0.28 mM), Pd(OAc).sub.2 and 10 mL of MeOH
are added to a flask with a stir bar and the system degassed
3.times. using argon. Na.sub.2CO.sub.3 (0.8 mM) dissolved in water
and argon degassed is added via syringe is added along with
2,5-dimethylphenyl boronic acid (0.55 mM) in MeOH that was also
degassed. The reaction was followed by HPLC for 2 hours and cooled
to room temperature. The solution was filtered, and dried to
produce a crude mixture. The solid was dissolved in
dimethylformamide and injected onto a preparative HPLC system using
C18 reverse-phase silica. The solvent was removed in vacuo to yield
the product plus salts. The salts were removed by extraction into
50:25:25 water, butanol, ethyl acetate and dried in vacuo. This
solid was dissolved in MeOH and the HCl salt made by bubbling in
HCl gas.
[0147] 7-(Hexynyl)-Sancycline
[0148] 7-1-Sancycline (1 gm, 1.86 mmol ), taken in 25 mL of
acetonitrile was degassed and purged with nitrogen (three times).
To this suspension Pd(OAc).sub.2 (20 mg, 0.089 mmol), CuI (10 mg,
0.053 mmol), (o-tolyl).sub.3P (56 mg, 0.183 mmol) were added and
purged with nitrogen for few minutes. 1-Hexyne (3.72 mmol) and
triethylamine (1 mL) were added to the suspension. It was turned
into a brown solution upon addition of Et.sub.3N. The reaction
mixture was then heated to 70.degree. C. for 3 hours. Progress of
the reaction was monitored by HPLC. It was then cooled down to room
temperature and was filtered through celite. Evaporation of the
solvent gave a brown solid, which was then purified on preparative
HPLC to give a yellow solid. The structure of this compound has
been characterized using 1H NMR, HPLC, and MS.
[0149] 9-(4'-Fluorophenylethyl)-Minocycline
[0150] 9-(4-Fluorophenylethynyl)-minocycline (1 mmol) was taken in
saturated solution of MeOH/HCl. To this solution 10% Pd/C was added
and was subjected to hydrogenation at 50 psi for 12 hrs. It was
then filtered through celite. The solvent was evaporated to give a
yellow powder. Finally, it was precipitated from MeOH/diethylether.
The structure of this compound has been characterized using 1H NMR,
HPLC, and MS.
[0151] 9-(2', 5'-Dimethylphenyl) Minocycline
[0152] In a clean, dry reaction vessel, was placed
9-iodominocycline (0.762 mmoles) bis HCl salt, palladium (II)
acetate (0.076 mmoles) along with 10 ml of reagent grade methanol.
The solution was immediately purged, with stirring, with a stream
of argon gas for approximately 5 minutes. The reaction vessel was
brought to reflux and to it was sequentially added via syringe 2M
potassium carbonate solution, followed by a solution of
2,5-dimethylphenyl boronic acid (1.53 mmoles) in 5 ml of reagent
DMF. Both of these solutions were previously degassed with argon
gas for approximately 5 minutes. The reaction was heated for 45
minutes, the progress was monitored via reverse phase HPLC. The
reaction was suctioned filtered through a pad of diatomaceous earth
and washed the pad with DMF. The filtrates were reduced to an oil
under vacuum and residue treated with t-butylmethyl ether. Crude
material was purified via reverse phase HPLC on DVB utilizing a
gradient of water and methanol/acetonitrile containing 1.0%
trifluoroacetic acid.
Example 2
[0153] Antifungal Activity of Substituted Tetracycline
Compounds
[0154] Antifungal activity of the tetracyclines was determined by a
broth microdillution technique following NCCLS (1997) Standards.
Assays were setup using a Tecan Genesis robotic workstation. All
drugs were dissolved in 10% DMSO. Drug concentration ranged from
0.125 to 64 .mu.g/mL in 2 fold serial dilutions. Each tetracycline
was tested at 10 concentrations ranging from 0.125 to 64 .mu.g/mL.
The compounds were tested for their antifungal activity against
Candida albicans (ATCC#90028). The final concentration of DMSO was
kept below 1%. Checkerboard analysis of the initial hits will be
performed to better determine the activity of the compound.
[0155] The strains tested include those listed in Table 1.
1 TABLE 1 Genus Species ATCC/FGSC # Aspergillus fumigatus ATCC
13073 (Fresenius) Aspergillus nidulans FGSCA991 (wt) Candida
albicans ATCC90028 Candida albicans PCI-1 Candida albicans PCI-17
Candida albicans ATCC 36082 Candida glabrata ATCC 90030 Candida
guilliermondii ATCC 14242 Candida krusei ATCC 96685 Candida krusei
ATCC 90878 Candida lusitaniae ATCC 24347 Candida parapsilosis ATCC
22109 Candida tropicalis ATCC 14246 Candida tropicalis ATCC 28707
Cryptococcus neoformans ATCC 90012 Cryptococcus neoformans ATCC
90013 Issatchenkia orientalis ATCC 6258 Neurospora crassa
FGSC853
[0156] The results are shown in Table 2. For each compound, *
represents good antifungal activity against the particular fungus,
** represents very good inhibition of the fungus, and ***
represents excellent inhibition of a particular fungus.
2TABLE 2 As- As- Sac- In As- per- per- C. Can- Can- Can- Can- Can-
Can- Crypto- charo- Vitro per- gillus gillus dub- dida dida dida
Can- dida dida dida coccus I. myces Cyto- gillus fumi- fer- lin-
albi- gla- guiller- dida lusi- para- tropic- neo- orient- cere-
toxi- ID STRUCTURE flavus getus reus iensis cans brata mondil
krusei tanise psilosis alis formans sils visies city A 21 * ** B 22
** ** C 23 * ** D 24 * ** E 25 * * * ** F 26 ** ** G 27 ** ** ** **
*** ** ** H 28 ** ** I 29 ** ** J 30 * ** K 31 ** ** L 32 * ** M 33
** ** N 34 ** ** O 35 ** ** P 36 * * ** Q 37 * * ** R 38 ** ** S 39
** ** T 40 ** ** U 41 * ** V 42 ** ** W 43 * ** X 44 * ** Y 45 ***
** Z 46 * ** AA 47 * ** AB 48 ** ** AC 49 * ** AD 50 ** * AE 51 ***
** AF 52 ** ** AG 53 * ** AH 54 *** ** AI 55 * ** AJ 56 ** ** AK 57
* ** AL 58 * ** AM 59 ** ** AN 60 ** ** AO 61 * ** AP 62 * ** AQ 63
** ** AR 64 ** ** *** ** *** *** *** ** AS 65 ** *** ** AT 66 ** **
AU 67 * ** AV 68 * * AW 69 ** ** ** *** ** ** ** ** ** *** ** AX 70
** ** ** AY 71 ** ** AZ 72 ** ** BA 73 * ** ** * * ** BB 74 ** **
** ** ** * ** BC 75 ** *** ** ** ** BD 76 ** ** BE 77 ** ** ** ***
*** ** *** *** ** ** BF 78 * ** * *** *** ** *** *** ** ** BG 79 **
** BH 80 * * * ** ** BI 81 ** ** * ** *** *** ** ** ** ** BJ 82 **
** BK 83 ** ** BL 84 ** ** *** ** ** BM 85 ** ** BN 86 ** ** ** BO
87 * ** BP 88 ** * BQ 89 ** ** BR 90 ** ** ** ** ** * ** * *** **
BS 91 ** ** BT 92 ** ** ** ** *** ** *** *** ** BU 93 ** ** ** ***
** ** ** ** BV 94 * ** BW 95 *** ** BX 96 ** ** ** *** ** ** ** ***
** ** ** BY 97 * ** ** * * * ** BZ 98 * ** CA 99 * ** CB 100 ** **
CC 101 ** ** ** CD 102 * * CE 103 ** ** ** ** ** ** ** CF 104 ** *
CG 105 ** ** CH 106 ** * ** * ** ** ** CI 107 * ** CJ 108 ** *** **
** * ** ** ** ** ** CK 109 * ** CL 110 * ** ** ** *** *** *** ** **
CM 111 ** * * * ** * ** ** CN 112 * ** CO 113 ** ** CP 114 *** **
CQ 115 * * * ** * ** CR 116 *** ** CS 117 * ** CT 118 * ** CU 119 *
** CV 120 *** ** CW 121 ** ** CX 122 ** ** CY 123 ** ** CZ 124 * **
DA 125 * ** DB 126 * ** DC 127 * ** DD 128 ** ** DE 129 * ** DF 130
** *** ** DG 131 ** *** ** DH 132 *** *** ** DI 133 ** ** DJ 134 *
* DK 135 * ** DL 136 * ** DM 137 * * DN 138 ** ** ** DO 139 * ** DP
140 * * * DQ 141 ** ** DR 142 *** ** DS 143 * ** **
Example 3
[0157] Mammalian Cytotoxicity Assay
[0158] COS-1 and CHO cell suspensions are prepared, seeded into
96-well tissue culture treated black-walled microtiter plates
(density determined by cell line), and incubated overnight at
37.degree. C., in 5% CO.sub.2 and approximately 95% humidity. The
following day serial dilutions of drug are prepared under sterile
conditions and transferred to cell plates. Cell/Drug plates are
incubated under the above conditions for 24 hours. Following the
incubation period, media/drug is aspirated and 50 .mu.l of
Resazurin is added. Plates are then incubated under the above
conditions for 2 hours and then in the dark at room temperature for
an additional 30 minutes. Fluorescence measurements are taken
(excitation 535 nm, emission 590 nm). The IC.sub.50 (concentration
of drug causing 50% growth inhibition) is then calculated. The
cytotoxcity of both unsubstituted minocycline and doxycycline were
found to be greater than 25.
Example 4
[0159] In vitro Anti-Bacterial Activity Assay
[0160] The following assay is used to determine the efficacy of the
tetracycline compounds against common bacteria. 2 mg of each
compound is dissolved in 100 .mu.l of DMSO. The solution is then
added to cation-adjusted Mueller Hinton broth (CAMHB), which
results in a final compound concentration of 200 .mu.g per ml. The
tetracycline compound solutions are diluted to 50 .mu.L volumes,
with a test compound concentration of 0.098 .mu.g/ml. Optical
density (OD) determinations are made from fresh log-phase broth
cultures of the test strains. Dilutions are made to achieve a final
cell density of 1.times.10.sup.6 CFU/ml. At OD=1, cell densities
for different genera should be approximately:
3 E. coli 1 .times. 10.sup.9 CFU/ml S. aureus 5 .times. 10.sup.8
CFU/ml Enterococcus sp. 2.5 .times. 10.sup.9 CFU/ml
[0161] 50 .mu.l of the cell suspensions are added to each well of
microtiter plates. The final cell density should be approximately
5.times.10.sup.5 CFU/ml. These plates are incubated at 35.degree.
C. in an ambient air incubator for approximately 18 hr. The plates
are read with a microplate reader and are visually inspected when
necessary. The MIC is defined as the lowest concentration of the
tetracycline compound that inhibits growth.
[0162] Equivalents
[0163] 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.
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