U.S. patent application number 14/068450 was filed with the patent office on 2014-02-27 for oxazolidinone containing dimer compounds, compositions and methods to make and use.
This patent application is currently assigned to TRIUS THERAPEUTICS, INC.. The applicant listed for this patent is TRIUS THERAPEUTICS, INC.. Invention is credited to Robert J. Duguid, John Finn, David Keith Hester, II.
Application Number | 20140057874 14/068450 |
Document ID | / |
Family ID | 42333294 |
Filed Date | 2014-02-27 |
United States Patent
Application |
20140057874 |
Kind Code |
A1 |
Hester, II; David Keith ; et
al. |
February 27, 2014 |
OXAZOLIDINONE CONTAINING DIMER COMPOUNDS, COMPOSITIONS AND METHODS
TO MAKE AND USE
Abstract
Dosage forms or pharmaceutical compositions comprise a compound
having the structure of Formula IV: ##STR00001## wherein n is a
non-negative integer; wherein each Z is an oxazolidinone-containing
moiety having antibiotic activity in vivo upon cleaving, wherein M
is independently OR.sub.1 or NR.sub.1R.sub.2; wherein R.sub.1 and
R.sub.2 are independently selected from the group consisting of H,
an optionally-substituted hydrocarbyl residue or a pharmaceutically
acceptable cation; wherein the compound in the dosage form or a
pharmaceutical composition is present in an amount effective for
treating or preventing an antibacterial infection in a mammalian
subject. Methods of preparing and using these dosage forms or
pharmaceutical compositions are also disclosed.
Inventors: |
Hester, II; David Keith;
(Delmar, NY) ; Duguid; Robert J.; (Glenmont,
NY) ; Finn; John; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRIUS THERAPEUTICS, INC. |
San Diego |
CA |
US |
|
|
Assignee: |
TRIUS THERAPEUTICS, INC.
San Diego
CA
|
Family ID: |
42333294 |
Appl. No.: |
14/068450 |
Filed: |
October 31, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12787293 |
May 25, 2010 |
8580767 |
|
|
14068450 |
|
|
|
|
61181955 |
May 28, 2009 |
|
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Current U.S.
Class: |
514/92 ;
548/119 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 31/04 20180101; A61K 31/675 20130101; C07F 9/65583
20130101 |
Class at
Publication: |
514/92 ;
548/119 |
International
Class: |
C07F 9/6558 20060101
C07F009/6558 |
Claims
1. A dosage form or a pharmaceutical composition comprising a
therapeutically effective amount of compound having the structure
of Formula IV: ##STR00028## wherein n is 0, 1 or 2; wherein each Z
is an oxazolidinone-containing moiety having antibiotic activity in
vivo upon cleaving, wherein M is independently OR.sub.1 or
NR.sub.1R.sub.2; wherein R.sub.1 and R.sub.2 are independently
selected from the group consisting of H, an optionally-substituted
hydrocarbyl residue that may contain one or more heteroatoms or a
pharmaceutically acceptable cation.
2. The dosage form or the pharmaceutical composition of claim 1,
wherein each Z is ##STR00029## wherein * is the point of attachment
of Z to P; wherein R.sup.1a and R.sup.1b are independently selected
from H and F, provided that at least one of R.sup.1a and R.sup.1b
is F, and Het is an optionally-substituted five- or six-membered
heterocycle comprising at least one N, O, or S atom.
3. The dosage form or the pharmaceutical composition of claim 2,
wherein the compound has the structure ##STR00030## wherein M is
OR.sub.1 and R.sub.1 is a pharmaceutically-acceptable cation.
4. The dosage form or the pharmaceutical composition of claim 3
wherein R.sub.1 is a nitrogen-containing cation.
5. The dosage form or the pharmaceutical composition of claim 3
wherein R.sub.1 is an imidazolium cation.
6. The dosage form or the pharmaceutical composition of claim 3
wherein each Z is ##STR00031##
7. The dosage form or the pharmaceutical composition of claim 6,
wherein each Z is ##STR00032##
8. The dosage form or the pharmaceutical composition of claim 1,
wherein the dosage form or the pharmaceutical composition is a
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier, diluent or excipient.
9. The dosage form or the pharmaceutical composition of claim 1,
wherein each Z has R stereochemistry.
10. The dosage form or the pharmaceutical composition of claim 5,
wherein each Z is ##STR00033##
11. The dosage form or the pharmaceutical composition of claim 2,
which is ##STR00034## or pharmaceutically acceptable salt
thereof.
12. The dosage form or the pharmaceutical composition of claim 11,
wherein Het is tetrazolyl.
13. A method of preparing the compound in the dosage form or
pharmaceutical composition of claim 1, comprising the step of
treating a compound of the formula Z--H with a phosphorylating
agent.
14. A method of preparing the compound in the dosage form or
pharmaceutical composition of claim 1, comprising the step of
treating with a dehydrating agent of the compound Z--P', wherein P'
is a mono- or dihydrogen phosphate group.
15. The method of claim 13, wherein the phosphorylating agent is
POCl.sub.3.
16. A method of treating a bacterial infection comprising
administering the dosage form or the pharmaceutical composition of
claim 1 to a subject in need thereof.
17. A method of treating a bacterial infection comprising
administering the dosage form or the pharmaceutical composition of
claim 12 to a subject in need thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/787,293, filed May 25, 2010, which claims
priority to U.S. Provisional Application No. 61/181,955, filed May
28, 2009. The content of each of these applications is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present disclosure relates to pharmaceutically-useful
oxazolidinone-containing dimer compounds, compositions, and methods
of making and using them for treatment and prophylaxis of diseases
in mammals.
[0004] 2. Description of the Related Art
[0005] Various oxazolidinone-containing compounds have been
disclosed for use as antibiotics. For example,
oxazolidinone-containing compounds have been described in U.S.
patent application Ser. No. 10/596,412 (filed Dec. 17, 2004), and
WO 04/048350, WO 03/022824 and WO 01/94342, which are incorporated
herein by reference.
[0006] U.S. patent application Ser. No. 12/577,089 (filed Oct. 9,
2009) and U.S. patent application Ser. No. 12/699,864 (filed Feb.
3, 2010), which are assigned to the same assignee as in the present
application, disclose phosphate dimer impurities made during the
process of making of the compounds disclosed therein. Surprisingly,
it has been found that compounds containing at least two phosphates
binding two oxazolidinone-containing moieties, such as dimers of
oxazolidinone-containing compounds have antibacterial activity
similar to their dihydrogen monophosphate analog, but have an
advantageously different release profile, as discussed below in
more detail.
[0007] Bacterial infections pose a continuing medical problem
because anti-bacterial drugs eventually engender resistance in the
bacteria on which they are used. Consequently, a need exists for
new drugs with efficacy against pathogenic bacteria for use in the
therapy and prophylaxis of bacterial infections.
[0008] In particular, a need exists for anti-bacterial drugs with a
variety of pharmacokinetic properties, such as drugs with a slow
release profile, to enable physicians to employs different dosing
schedules in therapeutic and prophylactic regimens in different
clinical contexts, and also a variety of physicochemical properties
to facilitate preparation of different formulations of drugs with
similar modes of action.
SUMMARY OF THE INVENTION
[0009] The dosage forms and compositions herein fulfill these
needs.
[0010] In some aspects, a dosage form or a pharmaceutical
composition comprises a therapeutically effective amount of
compound having the structure of Formula IV:
##STR00002##
[0011] wherein n is a non-negative integer;
[0012] wherein each Z is an oxazolidinone-containing moiety having
antibiotic activity in vivo upon cleaving,
[0013] wherein M is independently OR.sub.1 or NR.sub.1R.sub.2;
[0014] wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of H, an optionally-substituted hydrocarbyl
residue that may contain one or more heteroatoms or a
pharmaceutically acceptable cation.
[0015] In some embodiments, each Z of the dosage form or the
pharmaceutical composition described herein, is
##STR00003##
[0016] wherein * is the point of attachment of Z to P;
[0017] wherein R.sup.1a and R.sup.1b are independently selected
from H and F, provided that at least one of R.sup.1a and R.sup.1b
is F, and
[0018] Het is an optionally-substituted five- or six-membered
heterocycle comprising at least one N, O, or S atom.
[0019] In some embodiments, the compound in the dosage form or
composition described herein has the structure
##STR00004##
[0020] wherein M is OR.sub.1 and R.sub.1 is a
pharmaceutically-acceptable cation, such as a nitrogen-containing
cation, e.g., an imidazolium cation. In some aspects of these
embodiments, each Z is
##STR00005##
[0021] The dosage form or the pharmaceutical composition may also
further comprise a pharmaceutically acceptable carrier, diluent or
excipient.
[0022] In some embodiments, each Z in the compound in the dosage
form or the pharmaceutical composition has R stereochemistry, such
as wherein each Z is
##STR00006##
[0023] In some embodiments, the compound in the dosage form or the
pharmaceutical composition is
##STR00007##
In some aspects, Het is tetrazolyl.
[0024] Some embodiments also include methods of preparing the
compound in the dosage form or pharmaceutical composition described
herein, comprising the step of treating o the compound f the
formula Z--H with a phosphorylating such as POCl.sub.3.
[0025] Some embodiments include methods of preparing the compound
described herein in the dosage form or pharmaceutical composition,
comprising the step of treating with a dehydrating agent of the
compound Z--P', wherein P' is a mono- or dihydrogen phosphate
group.
[0026] Embodiments also include methods of and uses for treating a
bacterial infection comprising administering the dosage form or the
pharmaceutical composition described herein to a subject in need
thereof.
[0027] These and other embodiments are described in greater detail
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows a plot of the pharmacokinetics of a Formula IV
compound (as illustrated in Example 2) when administered either
orally or intravenously showing both the concentration of the
prodrug Formula IV or the active antibacterial agent of Formula
Ia.
[0029] FIG. 2 shows a plot of the pharmacokinetics of a Formula III
compound (as illustrated in Example 1) when administered
intravenously showing both the concentration of the prodrug Formula
III and the active antibacterial agent Formula Ia.
[0030] FIG. 3 charts the in vivo efficacy of TR-701 and the dimer
Formula IV at 24 and 48 hour intervals at various dosage levels in
accordance with Example 5.
[0031] FIG. 4 shows a graph of the Staphylococcus aureus in vivo
efficacy of TR-701 and the dimer Formula IV at 24 and 48 hour
intervals in accordance with Example 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0032] In some embodiments, the present disclosure describes new
prodrug compounds having antibiotic activity and pharmaceutical
compositions containing the compounds as well as methods of making
and using them. In some embodiments, the compounds are prodrugs
that are cleaved after administration to release an active
oxazolidinone-containing metabolite, in vivo. In some embodiments,
the compound has the structure of Formula IV:
##STR00008##
[0033] wherein n is a non-negative integer;
[0034] wherein Z is an oxazolidinone-containing moiety having
antibiotic activity in vivo upon cleaving,
[0035] wherein M is independently OR.sub.1 or NR.sub.1R.sub.2;
[0036] wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of H, an optionally-substituted hydrocarbyl
residue or a pharmaceutically acceptable cation.
[0037] In some embodiments, the compound is in a pharmaceutical
composition or a dosage form, wherein the pharmaceutical
composition or dosage form provides an effective
antibiotic-treating or -preventing amount of the compound.
[0038] In some embodiments, the oxazolidinone-containing moiety, Z,
has the structure
##STR00009##
[0039] wherein * is the point of attachment of Z to P;
[0040] wherein R.sup.1a and R.sup.1b are independently selected
from H and F, provided that at least one of R.sup.1a and R.sup.1b
is F, and
[0041] Het is an optionally-substituted five- or six-membered
heterocycle comprising at least one N, O, or S atom, such as
tetrazolyl or oxadiazolyl.
[0042] In some aspects, R.sup.1a is F and R.sup.1b is H and Het is
2-methyl-tetrazol-5-yl. For example, in some embodiments Z is
##STR00010##
[0043] In some aspects, the active metabolite results after in vivo
cleaving of the Z moiety from the compounds described herein. For
example, an active metabolite may have the following formula:
##STR00011##
[0044] In some embodiments, the active drug of Formula I is
(5R)-3-[3-fluoro-4-[6-(2-methyl-2H-tetrazol-5-yl)-3-pyridinyl]phenyl]-5-(-
hydroxymethyl)-2-oxazolidinone, i.e.,
##STR00012##
[0045] These active compounds have been disclosed in WO 05/058886
and US Patent Publication No. 20070155798, while processes for
making these and related compounds have been disclosed in U.S.
patent application Ser. No. 12/577,089 (filed Oct. 9, 2009), and a
crystalline form of the phosphate ester and related salts of the
above compound has been disclosed in U.S. patent application Ser.
No. 12/699,864 (filed Feb. 3, 2010). The latter two applications
are assigned to the same assignee as in the present application.
Each of the references cited herein is hereby incorporated by
reference in its entirety.
[0046] Compounds of Formula IV may occur as an impurity, along with
other impurities, during the phosphorylation of the active
metabolite of an oxazolidinone-containing antibiotic (e.g., Formula
I). Therefore, these "impurities" have not been contemplated for
use as pharmaceutical drugs. As an illustration, phosphorylation of
Formula I forms the phosphate dihydrogen monoester prodrug of
Formula II
##STR00013##
[0047] wherein M is described herein, such as a compound with the
following formula
##STR00014##
[0048] Likewise, in a similar illustration, phosphorylation of the
compound of Formula Ia (TR-700) forms the phosphate dihydrogen
monoester of Formula IIa,
[(5R)-3-{3-fluoro-4-[6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl]phenyl}-2--
oxo-5-oxazolidinyl]methyl]phosphate,
##STR00015##
[0049] In addition, salts of Formula II such as the disodium salts
of Formula IIa may also be formed such as Formula IIb below:
##STR00016##
[0050] Examples of impurities that may result during such
phosphorylation of an active metabolite of an
oxazolidinone-containing antibiotic include compounds of Formula
III ("bis" compounds),
##STR00017##
[0051] such as the phosphate monohydrogen diester (when M=OH),
wherein Z is
##STR00018##
and wherein * is the point of attachment of Z to P.
[0052] The side products may also include Formula IV
##STR00019##
[0053] wherein n is a non-negative integer such as 0, 1, or 2. In
some embodiments, n is 0 and has the following formula:
##STR00020##
[0054] wherein Z is as defined above, such as, the diphosphate
dihydrogen diester of Formula I (when M=OH).
[0055] In some embodiments, Formula IV is an acid or an acid
derivative, such as a salt, ester, or amide. In some embodiments, M
is independently OR.sub.1 or NR.sub.1R.sub.2; wherein R.sub.1 and
R.sub.2 are independently selected from the group consisting of H,
optionally-substituted hydrocarbyl residue that may contain one or
more heteroatoms, a pharmaceutically-acceptable cation, and a
positively charged nitrogen-containing ion. In some embodiments,
the presence of M may not substantially destroy the antibiotic
activity of the active metabolite of the compound of Formula IV. In
some embodiments, however, the presence of M may alter the degree
of antibiotic activity.
[0056] In some embodiments, an optional substituent on the
hydrocarbyl residue mentioned above, likewise may not substantially
destroy antibiotic activity of the active metabolite of the
compound of Formula IV. In some embodiments, the substituent may
alter the degree of antibiotic activity, however. Antibiotic
activity may be measured by various methods known in the art, such
as the mouse septicemia model as described in Example 5.
[0057] As used herein, "hydrocarbyl residue" refers to a residue
which contains only carbon and hydrogen. The residue may be
aliphatic or aromatic, straight-chain, cyclic, branched, saturated
or unsaturated. The hydrocarbyl residue, when indicated, may
contain heteroatoms over and above the carbon and hydrogen members
of the substituent residue. Thus, when specifically noted as
containing such heteroatoms, the hydrocarbyl residue may also
contain carbonyl groups, amino groups, hydroxyl groups and the
like, or contain heteroatoms within the "backbone" of the
hydrocarbyl residue. In some embodiments, the hydrocarbyl residue
has 1-20C containing 0-5 heteroatoms selected from O, S and N.
[0058] As used herein, the term "alkyl," "alkenyl" and "alkynyl"
include straight- and branched-chain and cyclic monovalent
substituents. Examples include methyl, ethyl, isobutyl, cyclohexyl,
cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. Typically,
the alkyl, alkenyl and alkynyl substituents contain 1-10C (alkyl)
or 2-10C (alkenyl or alkynyl), such as 1-6C (alkyl), 2-6C (alkenyl
or alkynyl) or 3-6C cycloalkyl. Heteroalkyl, heteroalkenyl and
heteroalkynyl are similarly defined but may contain 1-2 O, S or N
heteroatoms or combinations thereof within the backbone
residue.
[0059] As used herein, "acyl" encompasses the definitions of alkyl,
alkenyl, alkynyl and the related hetero-forms which are coupled to
an additional residue through a carbonyl group.
[0060] "Aromatic" moiety refers to a monocyclic or fused bicyclic
moiety such as phenyl or naphthyl; "heteroaromatic" also refers to
monocyclic or fused bicyclic ring systems containing one or more
heteroatoms selected from O, S and N. The inclusion of a heteroatom
permits inclusion of 5-membered rings as well as 6-membered rings.
Thus, typical aromatic systems include pyridyl, pyrimidyl, indolyl,
benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl,
oxazolyl, imidazolyl, tetrazolyl, oxadiazolyl and the like. Any
monocyclic or fused ring bicyclic system which has the
characteristics of aromaticity in terms of electron distribution
throughout the ring system is included in this definition.
Typically, the ring systems contain 5-12 ring member atoms.
[0061] Similarly, "arylalkyl" and "heteroarylalkyl" refer to
aromatic and heteroaromatic systems which are coupled to another
residue through a carbon chain, including substituted or
unsubstituted, saturated or unsaturated, carbon chains, typically
of 1-6C. These carbon chains may also include a carbonyl group,
thus making them able to provide substituents as an acyl
moiety.
[0062] Optional substituents may be selected from the group
consisting of optionally substituted alkyl, alkenyl, alkynyl, aryl,
N-aryl, NH-aroyl, halo, OR, NR.sub.2, SR, --OOCR, --NROCR, RCO,
--COOR, --CONR.sub.2, SO.sub.2NR.sub.2, CN, CF.sub.3, and NO.sub.2,
wherein each R is independently H or alkyl (1-4C).
[0063] In some embodiments where M=OR.sub.1, R.sub.1 may be a H or
a metal cation such as an alkali metal cation or an alkaline earth
metal cation. In some embodiments, the metal cation is sodium,
calcium, potassium, magnesium, aluminum, zinc, or lithium
cation.
[0064] In some embodiments where M=OR.sub.1, R.sub.1 may be a
positively charged nitrogen-containing group that forms a salt with
the negatively charged oxygen of Formula IV, such as ammonium and
the quaternized or positively charged salts of imidazole,
N-methylglucamine, choline, piperazine, tromethamine, diethylamine,
4-phenylcyclohexylamine, or benzathine. In this context
"quaternized" refers to a nitrogen atom having four bonds and
therefore having a net positive charge, such as nitrogen with four
pendant groups or bonds, such as one with a double bond. In some
embodiments, pendant groups may include one or more hydrogens. In
some embodiments, M is O-imidazolium salt, that is,
##STR00021##
[0065] Pharmaceutically-acceptable cations, the identities of which
are well-known in the art, have been compiled in P. Heinrich Stahl
and Camille G. Wermuth, Handbook of Pharmaceutical Salts:
Properties, Selection and Use. International Union of Pure and
Applied Chemistry, Wiley-VCH 2002, and L. D. Bighley, S. M. Berge,
D. C. Monkhouse, in "Encyclopedia of Pharmaceutical Technology`.
Eds. J. Swarbrick and J. C. Boylan, Vol. 13, Marcel Dekker, Inc.,
New York, Basel, Hong Kong 1995, pp. 453-499.
[0066] In some aspects, the compound is not the dimer disclosed in
U.S. patent application Ser. No. 12/577,089 (filed Oct. 9, 2009)
having the following structure or a pharmaceutically acceptable
salt of the dimer
##STR00022##
[0067] wherein R.sup.1a and R.sup.1b are independently selected
from H and F, provided that at least one of R.sup.1a and R.sup.1b
is F,
[0068] Het is an optionally-substituted five- or six-membered
heterocycle comprising at least one N, O, or S atom, such as
tetrazolyl or oxadiazolyl.
[0069] In some aspects, the compound is not the dimer disclosed in
U.S. patent application Ser. No. 12/699,864 (filed Feb. 3, 2010),
i.e.,
##STR00023##
[0070] Compounds of Formula IV can be prepared by a variety of
methods, including phosphorylation of an active metabolite of an
oxazolidinone-containing antibiotic, such as the compound of
Formula I. When the phosphorylation reaction is conducted with two
or more equivalents of the active metabolite such as the Formula I
compound, formation of the bis-esters such as Formula III is
favored. However, in some embodiments, the bis-esters are
ineffective antibiotic agents, thus showing that not all
phosphate-containing compounds are effective. Therefore, in some
embodiments, less than two equivalents of the active metabolite
such as Formula I compound are used. Phosphorylation methods
typically involve use of an electrophilic phosphorus (V) compound,
such as phosphorus oxychloride, POCl.sub.3, or a phosphate in the
presence of a dehydrating agents, such as a carbodiimide, but other
methods can be used. For example, the compounds of Formula IV can
be prepared by transesterification of a phosphate-containing ester
such as a diphosphate ester with an active metabolite such as the
compound of Formula I. Such transesterification reactions are
well-known. Similarly, compounds of Formula IV can be prepared in
high yield from oxazolidinone-containing phosphates, such as a
monophosphate of Formula II by treatment with a dehydrating agent
such as carbonyldiimidazole (CDI).
[0071] In some embodiments, compounds of Formula IV have utility as
water soluble prodrugs of antibacterial agents of
oxazolidinone-containing antibiotics, such as Formula I. In some
embodiments, Formula IV phosphate dimers have a longer half life in
vivo than the oxazolidinone-containing phosphates, such as the
monophosphate of Formula II. Consequently, in some embodiments
compounds of Formula IV release the active metabolite (e.g.,
Formula I) over a longer period, which extends the in vivo
half-life of the antibacterial agent. Longer biological half-life
increases the time the active compound remains above the minimum
inhibitory concentration (MIC), and thereby promotes efficacy at a
lower drug dose. Longer half-life also reduces the maximum
concentration, C.sub.max, of some embodiments of the antibacterial
drug, thereby reducing side-effects in which C.sub.max is the
pharmacodynamic driver.
[0072] Those skilled in the art will appreciate that a variety of
prodrugs, salts, hydrates, solvates, and polymorphs can be produced
from the compounds disclosed here, and that various
isotopically-substituted variants (through, e.g., substitution of
deuterium for hydrogen, .sup.13C for carbon, .sup.15N for nitrogen,
or .sup.32P for phosphorus) can also be readily produced. All such
derivatives are contemplated within the scope of this
disclosure.
[0073] In another aspect, the present disclosure relates to a
pharmaceutical composition comprising one or more physiologically
acceptable surface active agents, additional carriers, diluents,
excipients, smoothing agents, suspension agents, film forming
substances, and coating assistants, or a combination thereof; and a
composition disclosed herein. Acceptable additional carriers or
diluents for therapeutic use are well known in the pharmaceutical
art, and are described, for example, in Remington's Pharmaceutical
Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), which
is incorporated herein by reference in its entirety. Preservatives,
stabilizers, dyes, sweeteners, fragrances, flavoring agents, and
the like may be provided in the pharmaceutical composition. For
example, sodium benzoate, ascorbic acid and esters of
p-hydroxybenzoic acid may be added as preservatives. In addition,
antioxidants and suspending agents may be used. In various
embodiments, alcohols, esters, sulfated aliphatic alcohols, and the
like may be used as surface active agents; sucrose, glucose,
lactose, starch, microcrystalline cellulose, crystallized
cellulose, mannitol, light anhydrous silicate, magnesium aluminate,
magnesium metasilicate aluminate, synthetic aluminum silicate,
calcium carbonate, sodium acid carbonate, calcium hydrogen
phosphate, calcium carboxymethyl cellulose, and the like may be
used as excipients; magnesium stearate, talc, hardened oil and the
like may be used as smoothing agents; coconut oil, olive oil,
sesame oil, peanut oil, soya may be used as suspension agents or
lubricants; cellulose acetate phthalate as a derivative of a
carbohydrate such as cellulose or sugar, or
methylacetate-methacrylate copolymer as a derivative of polyvinyl
may be used as suspension agents; and plasticizers such as ester
phthalates and the like may be used as suspension agents.
[0074] The term "pharmaceutical composition" refers to a mixture of
a compound disclosed herein with other chemical components, such as
diluents or additional carriers. The pharmaceutical composition
facilitates administration of the compound to an organism. Multiple
techniques of administering a pharmaceutical composition exist in
the art including, but not limited to, oral, injection, aerosol,
parenteral, and topical administration. Pharmaceutical compositions
can also be obtained by reacting the free acid dihydrogen phosphate
with inorganic or organic bases such as sodium hydroxide or
magnesium hydroxide. In some embodiments, pharmaceutically
acceptable salts of the compounds disclosed herein (e.g., as made
in situ during the manufacture of an intravenous formulation) are
provided.
[0075] The term "carrier" refers to a chemical compound that
facilitates the incorporation of a compound into cells or
tissues.
[0076] The term "diluent" refers to chemical compounds diluted in
water that will dissolve the composition of interest as well as
stabilize the biologically active form of the compound. Salts
dissolved in buffered solutions are utilized as diluents in the
art. One commonly used buffered solution is phosphate buffered
saline because it mimics the salt conditions of human blood. Since
buffer salts can control the pH of a solution at low
concentrations, a buffered diluent rarely modifies the biological
activity of a compound. As used herein, an "excipient" refers to an
inert substance that is added to a composition to provide, without
limitation, bulk, consistency, stability, binding ability,
lubrication, disintegrating ability, etc., to the composition. A
"diluent" is a type of excipient.
[0077] The term "physiologically acceptable" refers to a carrier or
diluent that does not abrogate the biological activity and
properties of the compound.
[0078] The pharmaceutical compounds described herein can be
administered to a human patient per se, or in pharmaceutical
compositions where they are mixed with other active ingredients, as
in combination therapy, or suitable carriers or excipient(s). In
some embodiments, a dosage form includes those forms in which the
compound is administered per se. In addition, a dosage form may
include a pharmaceutical composition. In any case, the dosage form
may comprise a sufficient amount of the dimer compound to treat a
bacterial infection as part of a particular administration
protocol, as would be understood by those of skill in the art.
Techniques for formulation and administration of the compounds of
the instant application may be found in "Remington's Pharmaceutical
Sciences," Mack Publishing Co., Easton, Pa., 18th edition,
1990.
[0079] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, topical, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intravenous, intramedullary injections, as well as intrathecal,
direct intraventricular, intraperitoneal, intranasal, or
intraocular injections. The compound can also be administered in
sustained or controlled release dosage forms, including depot
injections, osmotic pumps, pills, transdermal (including
electrotransport) patches, and the like, for prolonged and/or
timed, pulsed administration at a predetermined rate.
[0080] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or tabletting
processes.
[0081] Pharmaceutical compositions may be formulated in any
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Proper formulation is dependent upon the
route of administration chosen. Any of the well-known techniques,
diluents, carriers, and excipients may be used as suitable and as
understood in the art; e.g., in Remington's Pharmaceutical
Sciences, above.
[0082] Injectables can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Suitable excipients are, for example, water, saline, dextrose,
mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. Physiologically compatible buffers include,
but are not limited to, Hanks's solution, Ringer's solution, or
physiological saline buffer. If desired, absorption enhancing
preparations may be utilized.
[0083] For transmucosal administration, penetrants appropriate to
the barrier to be permeated may be used in the formulation.
[0084] Pharmaceutical formulations for parenteral administration,
e.g., by bolus injection or continuous infusion, include aqueous
solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared
as appropriate oily injection suspensions. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers or agents that increase the solubility of the compounds
to allow for the preparation of highly concentrated solutions.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0085] For oral administration, the composition can be formulated
readily by combining the compositions of interest with
pharmaceutically acceptable carriers well known in the art. Such
carriers, which may be used in addition to the cationic polymeric
carrier, enable the compositions of the invention to be formulated
as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like, for oral ingestion by a patient
to be treated. Pharmaceutical preparations for oral use can be
obtained by combining the active compound with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP),
e.g., Povidone. If desired, disintegrating agents may be added,
such as the cross-linked polyvinylpyrrolidone (e.g. Crospovidone),
agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses.
[0086] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0087] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in a conventional
manner.
[0088] For administration by inhalation, the composition can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0089] Further disclosed herein are various pharmaceutical
compositions well known in the pharmaceutical art for uses that
include intraocular, intranasal, and intraauricular delivery.
Suitable penetrants for these uses are generally known in the art.
Such suitable pharmaceutical formulations are most often and
preferably formulated to be sterile, isotonic and buffered for
stability and comfort. Pharmaceutical compositions for intranasal
delivery may also include drops and sprays often prepared to
simulate in many respects nasal secretions to ensure maintenance of
normal ciliary action. As disclosed in Remington's Pharmaceutical
Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990), which
is incorporated herein by reference in its entirety, and well-known
to those skilled in the art, suitable formulations are most often
and preferably isotonic, slightly buffered to maintain a pH of 5.5
to 6.5, and most often and preferably include antimicrobial
preservatives and appropriate drug stabilizers. Pharmaceutical
formulations for intraauricular delivery include suspensions and
ointments for topical application in the ear. Common solvents for
such aural formulations include glycerin and water.
[0090] The compositions may also be formulated in rectal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0091] In addition to the formulations described previously, the
compositions may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0092] For hydrophobic compounds, a suitable pharmaceutical carrier
may be a cosolvent system comprising benzyl alcohol, a nonpolar
surfactant, a water-miscible organic polymer, and an aqueous phase.
A common cosolvent system used is the VPD co-solvent system, which
is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar
surfactant Polysorbate 80.TM., and 65% w/v polyethylene glycol 300,
made up to volume in absolute ethanol. Naturally, the proportions
of a co-solvent system may be varied considerably without
destroying its solubility and toxicity characteristics.
Furthermore, the identity of the co-solvent components may be
varied: for example, other low-toxicity nonpolar surfactants may be
used instead of POLYSORBATE 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0093] Methods for treating bacterial infections may include
administering a therapeutically effective amount of the therapeutic
compounds as described herein. Treating a bacterial infection may
also include prophylactically administering the therapeutic
compounds to prevent infection or the spread of an infection in a
subject at imminent risk of infection, such as a subject receiving
or about to undergo surgery, an immunocompromised subject, or
subject otherwise at risk of an infection if the compound was not
administered. The compounds show inhibitory activity against a
broad spectrum of bacteria, against methicillin resistant
Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci
(VRE) and have excellent relative antibiotic activity with a
relatively low concentration thereof or in vivo. Further, the
compounds of the present invention may exert potent antibacterial
activity versus various human and animal pathogens, including
Gram-positive bacteria such as Staphylococci, Enterococci and
Streptococci, anaerobic microorganisms such as Bacteroides and
Clostridia, and acid-resistant microorganisms such as Mycobacterium
tuberculosis and Mycobacterium avium. In an embodiment, the
bacterial infection that may be treated or ameliorated is MRSA.
[0094] The compositions or pharmaceutical compositions described
herein may be administered to the subject by any suitable means.
Non-limiting examples of methods of administration include, among
others, (a) administration though oral pathways, which
administration includes administration in capsule, tablet, granule,
spray, syrup, or other such forms; (b) administration through
non-oral pathways such as rectal, vaginal, intraurethral,
intraocular, intranasal, or intraauricular, which administration
includes administration as an aqueous suspension, an oily
preparation or the like or as a drip, spray, suppository, salve,
ointment or the like; (c) administration via injection,
subcutaneously, intraperitoneally, intravenously, intramuscularly,
intradermally, intraorbitally, intracapsularly, intraspinally,
intrasternally, or the like, including infusion pump delivery; as
well as (d) administration topically; as deemed appropriate by
those of skill in the art for bringing the active compound into
contact with living tissue.
[0095] Pharmaceutical compositions suitable for administration
include compositions where the active ingredients are contained in
an amount effective to achieve its intended purpose. In some
embodiments, a therapeutically effective amount of a compound is an
amount effective to treat a bacterial infection, for example, in a
mammalian subject (e.g., a human). The therapeutically effective
amount of the compounds disclosed herein required as a dose will
depend on the route of administration, the type of animal,
including human, being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize. More specifically, a
therapeutically effective amount means an amount of compound
effective to prevent, alleviate or ameliorate symptoms of disease
or prolong the survival of the subject being treated. Determination
of a therapeutically effective amount is well within the capability
of those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0096] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0097] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved adverse
side effects disappear. The dosage may range broadly, depending
upon the desired effects and the therapeutic indication. Typically,
dosages may be about 10 microgram/kg to about 100 mg/kg body
weight, preferably about 100 microgram/kg to about 10 mg/kg body
weight. Alternatively dosages may be based and calculated upon the
surface area of the patient, as understood by those of skill in the
art.
[0098] The exact formulation, route of administration and dosage
for the pharmaceutical compositions of the present invention can be
chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl et al. 1975, in "The Pharmacological
Basis of Therapeutics", which is hereby incorporated herein by
reference in its entirety, with particular reference to Ch. 1, p.
1). In some embodiments, the dose range of the composition
administered to the patient can be from about 0.5 to about 1000
mg/kg of the patient's body weight. The dosage may be a single one
or a series of two or more given in the course of one or more days,
as is needed by the patient. In instances where human dosages for
compounds have been established for at least some condition, the
present invention will use those same dosages, or dosages that are
about 0.1% to about 500%, more preferably about 25% to about 250%
of the established human dosage. Where no human dosage is
established, as will be the case for newly-discovered
pharmaceutical compositions, a suitable human dosage can be
inferred from ED.sub.50 or ID.sub.50 values, or other appropriate
values derived from in vitro or in vivo studies, as qualified by
toxicity studies and efficacy studies in animals.
[0099] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity or organ dysfunctions. Conversely, the attending
physician would also know to adjust treatment to higher levels if
the clinical response were not adequate (precluding toxicity). The
magnitude of an administrated dose in the management of the
disorder of interest will vary with the severity of the condition
to be treated and to the route of administration. The severity of
the condition may, for example, be evaluated, in part, by standard
prognostic evaluation methods. Further, the dose and perhaps dose
frequency will also vary according to the age, body weight, and
response of the individual patient. A program comparable to that
discussed above may be used in veterinary medicine.
[0100] Although the exact dosage will be determined on a
drug-by-drug basis, in most cases, some generalizations regarding
the dosage can be made. The daily dosage regimen for an adult human
patient may be, for example, an oral dose of about 0.1 mg to 2000
mg of the active ingredient, preferably about 1 mg to about 500 mg,
e.g. 5 to 200 mg. In other embodiments, an intravenous,
subcutaneous, or intramuscular dose of the active ingredient of
about 0.01 mg to about 100 mg, preferably about 0.1 mg to about 60
mg, e.g. about 1 to about 40 mg is used. In cases of administration
of a pharmaceutically acceptable salt, dosages may be calculated as
the free acid. In some embodiments, the composition is administered
1 to 4 times per day. Alternatively the compositions of the
invention may be administered by continuous intravenous infusion,
preferably at a dose of up to about 1000 mg per day. As will be
understood by those of skill in the art, in certain situations it
may be necessary to administer the compounds disclosed herein in
amounts that exceed, or even far exceed, the above-stated,
preferred dosage range in order to effectively and aggressively
treat particularly aggressive diseases or infections. In some
embodiments, the compounds will be administered for a period of
continuous therapy, for example for a week or more, or for months
or years.
[0101] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the antibiotic effects, or minimal effective concentration
(MEC). The MEC will vary for each compound but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
However, HPLC assays or bioassays can be used to determine plasma
concentrations.
[0102] Dosage intervals can also be determined using MEC value.
Compositions should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
[0103] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0104] The amount of composition administered may be dependent on
the subject being treated, on the subject's weight, the severity of
the infection, the manner of administration and the judgment of the
prescribing physician.
[0105] Compositions disclosed herein can be evaluated for efficacy
and toxicity using known methods. For example, the toxicology of
the compound may be established by determining in vitro toxicity
towards a cell line, such as a mammalian, and preferably human,
cell line. The results of such studies are often predictive of
toxicity in animals, such as mammals, or more specifically, humans.
Alternatively, the toxicity of particular compounds in an animal
model, such as mice, rats, rabbits, or monkeys, may be determined
using known methods. The efficacy of a particular compound may be
established using several recognized methods, such as in vitro
methods, animal models, or human clinical trials. Recognized in
vitro models exist for nearly every class of condition. Similarly,
acceptable animal models may be used to establish efficacy of
chemicals to treat such conditions. When selecting a model to
determine efficacy, the skilled artisan can be guided by the state
of the art to choose an appropriate model, dose, and route of
administration, and regime. Of course, human clinical trials can
also be used to determine the efficacy of a compound in humans.
[0106] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied with
a notice associated with the container in form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the drug for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the approved product insert. Compositions comprising a
compound of the invention formulated in a compatible pharmaceutical
carrier may also be prepared, placed in an appropriate container,
and labeled for treatment of an indicated condition.
[0107] In some embodiments, in the pharmaceutical industry, it
standard practice to provide substantially pure material when
formulating pharmaceutical compositions. Therefore, in some
embodiments, "substantially pure" refers to the amount of purity
required for formulating pharmaceuticals, which may include, for
example, a small amount of amorphous material or other material,
wherein the material may still achieve sufficient pourability, lack
of hygroscopicity, and purity suitable for pharmaceutical use. In
some embodiments, the substantially pure compound contains at least
about 96% of the compound by weight, such as at least about 96.1%,
96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%,
97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%,
98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of
the compound.
[0108] The terms "approximately, "about," and "substantially" as
used herein represent an amount close to the stated amount that
still performs the desired function or achieves the desired result.
For example, the terms "approximately," "about" and "substantially"
may refer to an amount that is within less than 10% of, within less
than 5% of, within less than 1% of, within less than 0.1% of, and
within less than 0.01% of the stated amount.
EXAMPLES
Example 1
Preparation of the Phosphate Monohydrogen Diester, Formula III
[0109] In this and the following Examples, "Formula III" refers to
a compound wherein Z is
##STR00024##
and M=OH.
[0110] A 1-L, three-neck round-bottom flask equipped with a
magnetic stirrer, nitrogen inlet/outlet and thermocouple was
charged with the compound of Formula Ia below (16.0 g, 0.0499 mol],
THF (320 mL, 20 vol) and Et.sub.3N (21.9 g, 0.216 mol, 5.0
equiv.).
##STR00025##
[0111] POCl.sub.3 (3.31 g, 0.0216 mol, 0.5 equiv.) was added
dropwise via syringe over 5 minutes. The reaction temperature was
maintained below 25.degree. C. The batch was aged for 16 hours at
room temperature at which point HPLC analysis (XBridge, C18)
indicated that the reaction was complete. The reaction vessel was
then immersed in an ice-water bath and a 500-mL addition funnel
charged with 320 mL of H.sub.2O was attached to the reaction
vessel. When the temperature of the reaction reached 2.7.degree.
C., H.sub.2O was added drop wise over 30 minutes. The temperature
of the reaction was maintained below 10.degree. C. Upon completion
of the H.sub.2O addition, the ice-water bath was removed and the
batch was aged for 3 hours. The solution was transferred to a 2-L
round-bottom flask and concentrated under reduced pressure on a
rotary evaporator. After removal of most of the THF from the
solution, the aqueous mixture was extracted with 5 1-L portions of
CH.sub.2Cl.sub.2:MeOH (9:1). The CH.sub.2Cl.sub.2 layers were
combined and concentrated to a dark oil. This crude material was
purified on 200 g of silica gel, eluting with 10%
MeOH/CH.sub.2Cl.sub.2 to 20% 2 N NH.sub.3 in MeOH/CH.sub.2Cl.sub.2.
Fractions containing mostly the bis-ester (as judged by TLC Rf=0.3
eluting with 20% 2 N NH.sub.3 in MeOH/CH.sub.2Cl.sub.2) were
combined and concentrated under reduced pressure on a rotary
evaporator, during which time a white precipitate was observed. The
flask containing the slurry was removed from the rotary evaporator
and equipped with a magnetic stir bar and allowed to stir while
cooling to room temperature over 3 hours, during which time the
slurry thickened. The solid was filtered and dried in a vacuum oven
at 45.degree. C. for 16 hours to give 3.55 g of bis-ester as an
off-white solid (20% yield). HPLC analysis (Method A): 99.0% (AUC),
tR=16.3 min. This reaction was repeated and the combined lots of
the compound of Formula III (6.7 g) were slurried in 100 mL of MeOH
(15 vol). The slurry was heated to 40.degree. C. for 30 minutes and
then allowed to cool to room temperature over 1 hour. The off-white
solid was filtered and dried in a vacuum oven at 40.degree. C. for
16 hours to give 6.15 g of the compound of Formula III (92% yield).
The .sup.1H NMR analysis of the product was consistent with the
assigned structure. HPLC analysis (Method A): 99.0% (AUC), tR=16.3
min.
Example 2
Preparation of the Diphosphate Dihydrogen Diester, Formula IV
[0112] In Examples 2-5, "Formula IV" refers to a compound wherein Z
is
##STR00026##
n=0 and M=O-imidazolium salt.
[0113] A 250-mL 3-neck round-bottom flask equipped with a magnetic
stirrer, nitrogen inlet/outlet and thermocouple was charged with
the compound of Formula IIa below (5.0 g, 11.1 mmol),
carbonyldiimidazole (890 mg, 5.55 mmol, 0.5 equiv.) and DMF (100
mL, 20 vol).
##STR00027##
[0114] The suspension was heated to 50.degree. C. and held at that
temperature for 4 hours at which point HPLC analysis (XBridge, C18)
indicated that the reaction was complete. The reaction was filtered
at 50.degree. C. and dried in a vacuum oven at 50.degree. C. for 24
hours to give 5.15 g of the imidazolium salt (i.e., the compound of
Formula IV) as an off-white solid (98% yield). The .sup.1H NMR
analysis of the product was consistent with the assigned structure.
HPLC analysis (Method A): 94.5% (AUC), tR=14.6 min.
TABLE-US-00001 TABLE 1 Method A (Waters XBridge C18 Column) Time
(min) Flow (mL/min) % A % B 0.0 1.0 98.0 2.0 15.0 1.0 5.0 95.0 25.0
1.0 5.0 95.0 27.0 1.0 98.0 2.0 30.0 1.0 98.0 2.0 A = 87% 25 mM
ammonium bicarbonate solution in water/13% Acetonitrile B =
Acetonitrile Wavelength = 300 nm
Example 3
Pharmacokinetic Determinations
[0115] Balb/c female mice, 6-7 weeks old (.about.20 g) were
randomized into treatment groups of three animals. Pharmacokinetics
of each of the test compounds was evaluated. The test compounds (10
mg/kg) were administered via tail vein or orally to study mice.
Blood was collected by cardiac puncture at 5
(intravenously-administered compounds only), 15, 30 minutes and 1,
4, 6, 8, 12 and 24 hours (n=3 at each timepoint). Plasma
concentration of each of the compounds of Formula Ia, III (as
defined in Example 1), and IV (as defined in Example 2) was
analyzed through use of a validated HPLC/UV method. Data are
plotted in FIG. 1 and FIG. 2.
TABLE-US-00002 TABLE 2 Summary of pharmacokinetic data for both the
prodrug and active component when the prodrugs of Formula IV (as
defined in Example 2) and Formula III (as defined in Example 1) are
dosed in mice. Compd. .tau..sub.1/2 T.sub.max C.sub.max
AUC.sub.0-24 h Vz.sub.obs CL.sub.obs Compound Dosed Measured Rsq
(h) (h) (.mu.g/ml) (.mu.g hr/ml) (L/kg) (L/hr/kg) intravenous
Formula IV Formula 1.00 0.77 0.08 4.30 7.00 1.53 1.39 10 mg/kg IV
intravenous Formula IV Formula 0.97 3.74 4.00 4.06 36.40 1.46 0.27
10 mg/kg Ia Oral Formula 0.99 3.89 6.00 1.70 18.3 3.01 0.54 Formula
IV 10 mg/kg Ia intravenous Formula III Formula 0.93 3.05 0.08 5.79
1.14 33.43 7.61 10 mg/kg III intravenous Formula III Formula 0.58
12.04 4.00 0.17 1.16 48.65 2.80 10 mg/kg Ia
TABLE-US-00003 TABLE 3 Pharmacokinetic data for both the prodrug
parent Formula IV (as defined in Example 2) and the active
antibacterial agent Formula Ia when prodrug Formula IV is given
either orally or intravenously at 10 mg/kg (all concentrations in
ng/ml). Oral route Oral route Time Formula Ia Formula IV
Intravenous route Intravenous route (hr) conc. conc. Formula Ia
conc. Formula IV conc. 0.08 ND ND 984.41 4300 0.25 381.22 ND
1439.02 1515 0.5 821.29 ND 1588.67 3450 1 1053.56 ND 2692.21 2661 4
1520.58 ND 4059.94 157 6 1697.18 ND 2341.86 ND 8 1029.09 ND 896.47
ND 12 667.74 ND 1385.48 ND 24 65.26 ND 84.06 ND ND: Not Detected
because amount of compound is below limits of detection
TABLE-US-00004 TABLE 4 Pharmacokinetic data for both the prodrug
parent Formula III (as defined in Example 1) and the active
antibacterial agent Formula Ia when prodrug Formula III (as defined
in Example 1) is given intravenously at 10 mg/kg (all
concentrations in ng/ml). Intravenous route Intravenous route Time
(hr) Formula III conc. Formula Ia conc. 0.08 5794.47 124.18 0.25
749.02 78.46 0.5 210.06 90.85 1 81.05 123.76 2 53.78 167.77 4 39.49
174.61 6 ND 131.45 8 ND 138.71 ND: Not Detected because amount of
compound is below limits of detection
Example 4
Minimum Inhibitory Concentration Determinations
[0116] Minimum inhibitory concentrations were determined by broth
microdilution according to Clinical and Laboratory Standards
Institute (CLSI) approved methods (M7-A7) and interpreted using
Alamar Blue to visualize cell viability. Dilutions of each of the
compounds of the compounds were tested against S. aureus Smith
strain or S. aureus plus 20% mouse serum.
TABLE-US-00005 TABLE 5 MIC results (in .mu.g/ml) S. aureus Smith +
S. aureus Smith 20% mouse serum Formula IIb >64 4 (TR-701)
Formula III >64 >64 (as defined in Example 1) Formula IV
>64 32 (as defined in Example 2)
Example 5
Efficacy Testing Mouse Septicemia Model
[0117] Balb/c female mice, 6-7 weeks old (.about.20 g) were
randomized into treatment groups of 10 animals. Staphylococcus
aureus Smith strain ATCC#13709 was cultured overnight in BHI medium
at 37.degree. C. Cells were back-diluted 1:10 and allowed to grow
for 5 hours. The inoculum used for infecting mice was prepared by
diluting the culture in 5% Hog Gastric Mucin/PBS to a concentration
of 1.times.10.sup.6 cfu/ml. 100 .mu.l of culture/mucin was reserved
to quantify the starting cfu/ml by serial diluting and plating the
culture. Mice were infected with 500 .mu.l of the inoculum just
prior to drug dosing. The drugs were dosed within 15 minutes of
infection.
[0118] Mice received intravenous doses of each of the test
compounds at the concentrations described in Tables 6 and 7, based
on calculated average weight of all the mice (.about.20 g).
Forty-eight hours post-infection, the number of surviving mice was
determined.
TABLE-US-00006 TABLE 6 Survival of Balb/c Mice, female, intravenous
administration, 200 .mu.L dosage of Formual III (as defined in
Example 1), 10 mice were treated in each group. Concentration
Concentration 48 hr Group ZTreatment mg/kg mg/m.sup.2 survival 1
Formula III 10 0.2 1/10 2 Formula III 5 0.1 1/10 3 Formula III 2.5
0.05 0/10 4 Formula III 1.25 0.025 1/10 5 infection -- 1/10
control
[0119] The data in Table 6 indicate that Formula III is ineffective
as an antibiotic because the survival rates are similar to the
infection control, thus showing that not all phosphate-containing
compounds are effective.
TABLE-US-00007 TABLE 7 Survival of Balb/c Mice, female, intravenous
administration of Formula IV (as defined in Example 2), 200 .mu.L
dosage, 10 mice were treated in each group. Concentra-
Concentration 24 hr 48 hr Group Treatment tion mg/kg mg/m.sup.2
survival survival 1 Formula IV 10 0.2 10/10 10/10 2 Formula IV 5
0.1 10/10 10/10 3 Formula IV 2.5 0.05 8/10 7/10 4 Formula IV 1.25
0.025 5/10 2/10 5 infection -- 2/10 1/10 control
[0120] The data in Table 7 show 100% (i.e., 10/10) survival rate
after 24 and 48 hour intervals at dosages of 10 and 5 mg/kg.
Without being bound by theory, it appears that the presence of an
additional phosphate in Formula IV increases the space between the
Z moieties in comparison to Formula III allowing a phosphatase to
cleave the phosphate more readily. Thus, it is expected that the
presence of additional phosphate groups between Z moieties would
act similarly as the diphosphate.
TABLE-US-00008 TABLE 8 Comparison of Survival of Balb/c Mice,
female, intravenous administration of Formula IV (as defined in
Example 2) and the disodium phosphate salt of Formula Ia (TR-701),
200 .mu.L dosage, 10 mice were treated in each group. % Survival-
Concentration 10 mice per group infection mg/kg 20 10 5 2.5 1.25
control 24 hr TR701 100 100 90 60 60 24 hr dimer 100 100 80 50 0
treatment 20 48 hr TR701 100 100 90 40 50 48 hr dimer 100 100 70 20
0 treatment 48 10
[0121] The same procedure was used as in Example 5's Efficacy
Testing description. The data in Table 8 show that survival rates
were surprisingly increased by the intravenous administration of
Formula IV (as defined in Example 2) in comparison to TR-701 (the
disodium phosphate salt of Formula Ia) at dosages of 5 and 2.5
mg/kg as shown in Table 8.
* * * * *