Note: Descriptions are shown in the official language in which they were submitted.
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OXAZOLIDINONE PHOTOAFFINITY PROBES, USES AND COMPOUNDS
FIELD OF THE INVENTION
The present invention is directed, in part, to novel methods of using
photoaffinity probes
for locating relevant antibiotic binding sites within sensitive cells. In
particular, the photoaffinity
probes are oxazolidinone photoaffinity probes that are used to identify
biological targets of
oxazolidinone class of antibiotics. The invention is also directed, in part,
to methods of
identifying compounds that inhibit binding of a probe to a biological target.
BACKGROUND OF THE INVENTION
A number of compounds have been recently developed and have been shown to act
as
antimicrobial or antibacterial agents. International Publication WO 99/41244
discloses
substituted aminophenyl isoxazoline compounds useful as antimicrobial agents.
U.S. Patent No.
5,910,504 describes hetero-aromatic ring substituted phenyloxazolidinone
antimicrobial agents.
In addition, International Publication WO 00/10566 discloses isoxazolinone
antibacterial agents.
An important step in the development of new antimicrobial or antibacterial
agents, such as those
disclosed above, is the elucidation of a mechanism of action. The specific
site of interaction of
non selective antibiotics/antitumor agents, such as sparsomycin, that inhibit
protein translation by
a different, less useful, and direct mechanism, has been described. Porse et
al., PYOC. Natl. Acad.
Sci. USA,1999, 96, 9003-9008. Previous studies with chemical probes using
isolated, cell-free
systems have failed to define the relative sites of interaction of these types
of antibiotic
compounds of the oxazolidinone class. Matassova, et al., RNA, 1999, S, 939-
946. This is, in part,
because the previous methods were incapable of defining the sites of the
particular and specific
mechanism of action of this important class of antibiotics. Probes that help
to elucidate the
mechanism of action of antimicrobial and/or antibacterial agents and methods
of using the same
are highly desired.
The present invention is directed, if2ter alia, to novel methods of
identifying biological
targets of an oxazolidinone-type antibiotic, as well as to methods of
identifying compounds that
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inhibit binding of a probe to a biological target thereof. The present
invention comprises use of
compounds/probes by a novel mechanism of study that allows the identification
of the specific
oxazolidinone interaction sites) within sensitive cells. Applicants' methods
comprise using
particular compounds in intact cells using competition for the cross-linl~ing
to specific sites by
active and inactive enantiomers of relevant compounds. These and other aspects
of the invention
are described below.
SUMMARY OF THE INVENTION
The present invention is directed to, inter alia, identification of
oxazolidinone binding
sites within a cell, such as gram-positive and gram-negative bacteria, as well
as mammalian cells.
The present invention is also directed to screening compounds for
antimicrobial activity.
In particular, the present invention is directed to methods for identifying a
biological
target of an oxazolidinone-type antibiotic comprising the steps of contacting
a susceptible cell
with an oxazolidinone photoaffinity probe, exposing the photoaffinity probe to
light to form a
complex between the photoaffinity probe and at least one biological target,
and detecting the
complex.
Another embodiment of the invention is directed to methods of identifying a
compound
that inhibits binding of a probe to a biological target thereof comprising the
steps of contacting
the biological target with a probe, wherein the biological target is ribosomal
RNA, tRNA, LepA,
L27, or any combination thereof, contacting the biological target with a test
compound, and
comparing the amount of binding between the probe and the biological target in
the presence and
absence of the test compound, wherein a decrease in the amount of binding
between the probe
and the biological target in the presence of the test compound indicates that
the test compound
inhibits binding of the probe to the biological target.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Various definitions are made throughout this document. Most words have the
meaning
that would be attributed to those words by one skilled in the art. Words
specifically defined either
below or elsewhere in this document have the meaning provided in the context
of invention as a
whole and as are typically understood by those skilled, in the art.
The present invention has identified a specific site of interaction of these
specific
antibiotics as near the peptidyl transfer center of the ribosome but involving
interaction in a
manner different than that described for other inhibitors of protein
translation. Interaction of
these oxazolidinone antibiotics involves the central region V of the 23S RNA
together with
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tRNA, and two proteins of 64 kDa (LepA) and 11 kDa (L27). Identification of
relevant sites with
this novel approach (compounds and technique) now allows for directed
discovery mechanisms
using structure-based design together with interaction screens with these
specific targets. In
particular, the methods of the present invention can be used to identify
cellular components that
bind to oxazolidinone-type antibiotics and, thus, serve as targets for
oxazolidinone-type
antibiotics. In addition, the methods of the present invention can be used to
screen, for example,
libraries of compounds, in order to identify compounds that inhibit binding of
a photoaffinity
probe, for example, to a biological target thereof. Such compounds can have
antibacterial or
antimicrobial activity themselves or can be used to design compounds having
antibacterial or
antimicrobial activity.
As used herein, the phrase "biological target" means any protein, nucleic
acid, lipid, etc.
within a cell. Biological targets include, but are not limited to, the
contents of the cytoplasm,
nucleus, cell membrane, cell wall, and the like. In particular, biological
targets include, but are
not limited to, ribosomal RNA, tRNA, LepA protein and L27. Biological targets
are capable of
binding a probe.
As used herein, the term "contacting" means either direct or indirect,
application of a
probe or test compound within a cell, on or to a cell, or to biological
targets from a cell ifz vitro or
in vivo or ex vivo. The test compound and probe can be present within a
buffer, salt, solution, etc.
As used herein, the term "cross-linking" or "binding" means the physical
interaction P
between a probe and at least one biological target within a cell or from a
cell or combinations
thereof. Binding includes ionic, non-ionic, Hydrogen bonds, Van der Waals,
hydrophobic
interactions, etc. The physical interaction, the binding, can be either direct
or indirect through or
because of another protein or compound. Direct binding refers to interactions
that do not take
place through or because of another protein or compound but instead are
without other
substantial chemical intermediates.
As used herein, the term "oxazolidinone" means a compound of the class known
as
oxazolidinones, including the compounds described in U.S. Serial Numbers
07/438,759,
07/553,795, 07/786,107, 07/831,213, 08/329,717, 07/909,387, 60/015,499,
09/138,209,
60/008,554, 60/064,738, 60/065,376, 60/067,830, 60/089,498, 60/100,185,
60/088,283,
60/092,765, 07/244,988, 07/253,850; European Patents EP 0500686, EP 0610265,
EP 0673370;
PCT Application Numbers PCT/LTS90/06220, PCT/US94108904, PCT/LTS94/10582,
PCT/LTS95/02972, PCT/US95/10992, PCT/LTS93/04850, PCT/LTS95/12751,
PCT/LTS96/00718,
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PCT/LTS93/03570, PCTlUS93/09589, PCT/LTS96/05202, PCT/LTS97103458,
PCT/LTS96/12766,
PCT/LTS97/01970, PCT/US96/14135, PCT/LJS96/19149, PCT/LTS96/17120,
PCT/LTS98/09889,
PCT/LTS98/13437;and U.S. Patent Numbers 5,700,799, 5,719,154, 5,547,950,
5,523,403,
5,668,286, 5,652,238, 5,688,792, 5,247,090, 5,231,188, 5,654,428, 5,654,435,
5,756,732,
5,164,510, 5,182,403, 5,225,565, 5,618,949, 5,627,197, 5,534,636, 5,532,261,
5,776,937,
5,529,998, 5,684,023, 5,627,181, 5,698,574, 5,220,011, 5,208,329, 5,036,092,
4,965,268,
4,921,869, 4,948,801, 5,043,443, 5,130,316, 5,254,577, 4,877,892, 4,791,207,
4,642,351,
4,665,171, 4,734,495, 4,775,752, 4,870,169, 4,668,517, 4,340,606, 4,362,866,
4,193,918,
4,000,293, 3,947,465, 4,007,168, 3,674,780, 3,686,170, 3,906,101, 3,678,040,
3,177,114,
3,141,889, 3,149,119, 3,117,122, 5,719,154, 5,254,577, 4,801,600, 4,705,799,
4,461,773,
4,243,801, 3,794,665, 3,632,577, 3,598,830, 3,513,238, 3,598,812, 3,546,241,
3,318,878,
3,322,712, 5,565,571, 5,880,118, 5,952,324, 5,910,504, 6,166,056, 5,968,962,
6,090,820,
5,736,545, 6,277,985, 5,955,460, 5,922,7076,255,304, 6,218,413, 5,977,373,
6,251,869,
5,929,248, and 5,801,246; the disclosures of each of which are incorporated
herein by reference
in their entirety. Preferred oxazolidinones include linezolid and eperezolid.
As used herein, the term "oxazolidinone-type antibiotic" means any compound
having
antimicrobial activity and which binds to or interacts with a biological
target (proteins, nucleic
acids, etc.) of an oxazolidinone antibiotic. Thus, the oxazolidinone-type
antibiotic may have the
same mechanism of action as an oxazolidinone antibiotic. Alternately, the
oxazolidinone-type
antibiotic may interact with or bind to some of the same biological targets as
does an
oxazolidinone antibiotic. Further, the oxazolidinone-type antibiotic may have
a chemical
structure that is different from an oxazolidinone antibiotic.
As used herein, the term "probe" means any compound, protein, nucleic acid
molecule,
small organic molecule, and the like, that can bind to a biological target.
Probes include, but are
not limited to, photoaffinity compounds, antibodies, oligonucleotides,
oxazolidinone antibiotics,
and the like. Probes can be labeled or unlabeled.
As used herein, the phrase "susceptible cell" means any cell in which a
photoaffinity
probe can bind to a biological target. Susceptible cells include, but are not
limited to, bacteria,
fungi, and mammalian cells.
As used herein, the term "test compound" means any identifiable chemical or
molecule,
small molecule, peptide, protein, sugar, natural or synthetic, that is
suspected to potentially
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interact with or compete with a probe for cross-linking to a biological target
within a cell or from
a cell.
The present invention is directed to methods of using oxazolidinone
photoaffinity
probes to elucidate and/or identify biological targets of oxazolidinone
antibiotics. The present
invention is also directed to izz vitro assays for determining whether a
particular test compound is
able to interact with a biological target of oxazolidinone antibiotics. Such
methods allow, inter
alia, generation of molecular-based drug discovery approaches for novel
antibiotics based on the
mechanism of antibacterial activity of oxazolidinone antibiotics.
One embodiment of the present invention is directed to methods for identifying
a
biological target of an oxazolidinone-type antibiotic. A susceptible cell is
contacted with a probe,
such as an oxazolidinone photoaffinity probe. Susceptible cells of the present
invention include,
but are not limited to, gram positive bacterial pathogens, including, for
example, Staphylococcus
aureus; Staphylococcus epidermidis (A, B, C biotypes); Staphylococcus
caseolyticus;
Staphylococcus gallinarum; Staphylococcus haemolyticus; Staphylococcus
lzominis;
Staphylococcus saproplzyticus; Streptococcus agalactiae (group B);
Streptococcus mutanslrattus;
Streptococcus pneunzozziae; Streptococcus pyogenes (group A); Streptococcus
salivarius;
Streptococcus sazzguis; Streptococcus sobrinus; Actinonzyces spps.;
Arthrobacter
lzistidinolovorans; Corynebacteriuzn diptheriae; Clostridium diffzcle;
Clostridium spps.;
Enterococcus casseliflavus; Enterococcus durarzs; Enterococcus faecalis;
Enterococcus faeciunz;
Enterococcus gallinarum; Erysipelotlzrix rlZUSiopathiae; Fusobacterium spps.;
Listeria
monocytogenes; Prevotella spps.; Propionibacterium aches; and Porplzyromozzas
gingivalis.
Susceptible cells also include, but are not limited to, gram negative
bacterial pathogens,
including, for example, Acinetobacter calcoaceticus; Acinetobacter
haemolyticus; Aeromonas
hydrophila; Bordetella pertusszs; Bordetella parapertussis; Bordetella
brozzchiseptica;
Bacteroides fragilis; Bartonella bacilliforYrais; Brucella abortus; Brucella
nzelitezzsis;
Caznpylobacter fetus; Campylobacter jejuni; Clzlanzydia pzzeumoniae;
Chlaznydza psittaci;
Chlamydia traclzomatzs; Citrobacter freundii; Coxiella burnetti; Edwardsiella
tarda;
Edwardsiella hoshinae; Enterobacter aerogenes, Enterobacter cloacae (groups A
and B);
Escherichia coli (to include all pathogenic subtypes) Ehrlicia spps.;
Francisella tularensis;
Haejyzophilus actinoyrzycetefzzconzitans; Haemophilus ducreyi; Haemophilus
lzaemolyticus;
Haemophilus influezzzae; Haemophilus parahaemolyticus; Haemophilus
paraizzfluenzae; Hafnia
alvei; Helicobacter pylori; Kingella kingae; Klebsiella oxytoca; Klebsiella
pneumoniae;
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Legionella pneumophila; Legiofzella spps.; Morgahella spps.; Moraxella
cattarlzalis; Neisseria
gotZOrrhoeae; Neisseria nzehifzgitidis; Plesiomonas shigelloides; Proteus
fzzirabilis; Proteus
pefzfzeri; Providerzcia spps.; Pseudomoyzas aeruginosa; Pseudonzonas species;
Rickettsia
prowazekii; Rickettsia rickettsii; Rickettsia tsutsugamuslzi; Rochalimaea
spps.; Salmonella
subgroup 1 serotypes (to include S. paratyphi and S. typlzi); Salmonella
subgroups 2, 3a, 3b, 4,
and 5; Serratia marcesans; Serratia spps.; SlZigella boydii; Slzigella
flexueri; Shigella
dyserzteriae; Shigella sohnei; Yersinia enterocolitica; Yersitzia pestis;
Yersiyzia
pseudotuberculosis; Vibrio cholerae; Vibrio vulrzificus; and Vibrio
parahaemolyticus.
Susceptible cells also include, but are;not limited to, Mycobacterial species,
including,
for example, Mycobacterium tuberculosis; Mycobacterium aviurn; and other
Mycobacterium
spps.
Susceptible cells also include, but are not limited to, Mycoplasmas (or
pleuropneumonia-like organisms), including, for example, Mycoplaszfza
gerzitalium; Mycoplasma
pheumoniae; and other Mycoplasma spps.
Susceptible cells also include, but are not limited to, Treponemataceae
(spiral
organisms) including, for example, Borrelia burgdorferi; other Borrelia
species; Leptospira
spps.; Treporzema pallidum.
Susceptible cells also include, but are not limited to, mammalian cells.
After a susceptible cell is contacted with a photoaffinity probe, the
photoaffinity probe
is exposed to light, preferably ultraviolet light, in order to form a complex
between the
photoaffinity probe and the biological target, e.g. cross-link the
photoaffinity probe to at least one
biological target within the susceptible cell. The complex formed between the
photoaffinity
probe and the biological target is detected by standard methodology.
Preferably, the photoaffinity
probe is detectably labeled. Detectable labels include, but are not limited
to, enzymatic,
fluorescent, chemiluminescent, or radioactive labels, many of which are
commercially available.
Preferred radioactive labels include, but are not limited to, 3H, 3sS, and
lasl. The complex
between the photoaffinity probe and the biological target is detected by any
number of well
known detection methods including, for example, autoradiography, enzymatic
activity detection,
chemical shifts/measurement, fluorescence intensity, ELISA with anti-
photoaffinity probe
antibodies, and the like, depending upon the particular detectable label that
is used. A plurality of
photoaffinity probes can also be used at the same time. Applicants have
identified several
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biological targets of oxazolidinone compounds by the methods described above,
including, 23S
RNA, tRNA, LepA and L27.
Another embodiment of the invention is directed to methods of identifying
compounds
that inhibit binding of a probe to a biological target thereof. Such methods
can be used, for
example, to identify antimicrobial compounds having a mechanism of action
similar to
oxazolidinones. A biological target is contacted with a probe,~such as an
oxazolidinone
compound, preferably in vitro. Preferably, the probe is linezolid or
eperezolid, or a derivative
thereof. The biological target is ribosomal RNA, tRNA, LepA protein, L27
ribosomal protein, or
any combination thereof. The biological target is also contacted with a test
compound. The
amount of binding between the probe and the biological target in the presence
and absence of the
test compound is compared. A decrease in the amount of binding between the
probe and the
biological target in the presence of the test compound indicates that the test
compound inhibits
binding of the probe to the biological target.
In some embodiments of the invention, the biological target is bound to a
solid phase
including, but not limited to, controlled pore glass, a microtiter plate, a
column, a scintillation
proximity bead, sepharose, polyacrylamide, and the like. Thus, for example,
LepA from a
susceptible source, or a biologically active fragment of LepA, is attached to
scintillation
proximity beads (SPA beads), for example, by antibody attached to the beads
and directed
against an antibody that recognizes the LepA peptide sequence. The test target
can also be
attached by other standard means such as His-copper, strep-avidin, etc.
Microtiter plates
containing the SPA beads are then incubated with or without additional targets
(as described
herein) with a labeled reference probe such as 3H-eperezolid. Test compounds
are evaluated for
their ability to reduce binding of the labeled probe to the target or
collection of targets for the
oxazolidinone antibiotics. Compounds able to recognize the same sites) on the
targets) can
have useful antibiotic or antimicrobial activity. Similarly the other targets
described herein (e.g.
ribosomal RNA, tRNA or L27) can be attached first to the SPA beads and the
assay can be
constructed and conducted in the same fashion. Unknown compounds that reduce
the binding of
the labeled probe (e.g. in this example 3H-eperezolid) in a manner similar to
unlabeled probe
(e.g., in this example eperezolid) can have useful antibiotic or antimicrobial
activity.
In some embodiments of the invention, the biological target is 23S ribosomal
RNA or a
fragment thereof. Preferably, the fragment of the 23S RNA comprises the
central peptidyl
transferase loop. Fragments of 23S RNA can be from about 10 nucleotides to
about 1000
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nucleotides, more preferably from about 25 nucleotides to about 750
nucleotides, more
preferably from about 50 nucleotides to about 500 nucleotides, and more
preferably from about
100 nucleotides to about 250 nucleotides in length. Preferably, the fragments
comprise
contiguous nucleotides from within the nucleotide sequence for the 23S
ribosomal RNA. RNA
comprising the central peptidyl transferase region and the contacts identified
by these techniques
include analogous 23S RNA regions, with their respective sequences, that are
isolated from any
of the organisms recited above. 23S ribosomal RNA can be isolated by the
methods described
above, or can be isolated or constructed by standard methodology. The
nucleotide sequence of S.
aureus 23S RNA is described in, for example, Ludwig et al., Syst. Appl.
Microbiol.,1992, IS,
487-501 and Brosius et al., Proc. Natl. Acad. Sci. USA,1980, 77, 201-4, each
of which is
incorporated herein by reference in its entirety. 23S RNA can be isolated as
described in, for
example, Moazed et al., J. Mol. Biol., 1986, 187, 399-416, which is
incorporated herein by
reference in its entirety.
In some embodiments of the invention, the biological target is a tRNA
molecule.
Preferably, the tRNA is tRNA~et. The tRNA can be isolated by the methods
described above, or
can be isolated from cells, such as E. coli, or constructed by standard
methodology. The
nucleotide sequence and isolation of fmet tRNA is described in, for example,
Seong et al., Proc.
Natl. Acad. ,Sci. USA, 1987, 84, 334-8, which is incorporated herein by
reference in its entirety.
In some embodiments of the invention, the biological target is LepA protein,
or a
fragment thereof. Lep A is also known as the protein product of the YqeQ gene.
The amino acid
sequence of LepA (SEQ ID NQ:1) is shown below.
1 I~KRYARSVTR FNGFRKR'.t'WY 'Y'1~l'Q~~CRVRLK 'YEAKDGN~'Y~' FHLIDTPCHV
5~. DFTYEVSR~L AACEGA~LV'V DAAQGIEAQ'.t' LANVYLALDN FLELLPVxNK
101 TDLPAAEPFR VF~QE~EDMxG T,~DQDD"~VLAS AKSi~I~~EE~ LEKIVEVVPA
~.5~. PDCDPEAPLK ALT~'DSEXDP XRCVISSIRT VDC'WKAGDK IRt~IMA:TGKE~'
20~. EV'.~E'V'GTNT~ KQLPVDEL~V GD'CtCYT~AST KhTVDDSRV~D ~I~'LASF~PAS
25~. EPLQ~YKKMN PMVYCGLFP1 DNKLV'~NDLR~ ALFKLQLNDA SL~FEPESSQ
30~. ALGFG'~RTGF LGMLi~IMEIIQ ERIEREFCIE L~ATAPSV~Y QCVLRDGSV
351 '~7NPA~MPD RDKIDFC~:E',~P ~'VR?~.~~'PN DYYGAV~~C Q~GQFTI~M
40~. DYLDDTRVN~ VYELPLA~W FDFFDQLKSN ~'KGYASFD~F FIENRESNLV
.~5~. KMDIL ~NGDK V'DALS~'~V~IR D~"AYERGKAL VEKLK~~PR. QQFEV~VQAA
X01 TGt,~~IVARTN TKSMGTCN'N'V2A KGYC~DTSRK RKLLEI~QKAG KA~CCAVGNV
5~, E~PQDAFL~.'rT L~MDD
Fragments of LepA can be from about 10 amino acids to about 550 amino acids,
more preferably
from about 25 amino acids to about 500 amino acids, more preferably from about
50 amino acids
to about 400 amino acids, more preferably from about 100 amino acids to about
300 amino acids,
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and more preferably from about 150 amino acids to about 250 amino acids in
length. Preferably,
the fragments comprise contiguous amino acids from within the amino acid
sequence for LepA.
LepA can be isolated by the methods described above, or can be isolated or
produced by standard
methodology, and can be isolated from any of the organisms recited above. LepA
can be isolated
as described in, for example, March et al., J. Biol. Chef~i., 1985, 260, 7206-
13, which is
incorporated herein by reference in its entirety.
In some embodiments of the invention, the biological target is L27, a 11 kDa
ribosomal
protein, or a fragment thereof. In S. aureus, L27 comprises the following
representative amino
acid sequence: VRCIPMLKLNLQFFASKKGVSSTKNGRDSESKRLGAKRADGQFVTGGSI
LYRQRGTKIYPGENVGRGGDDTLFAKmGVKFERKGRDKKQVSVYAVAE (SEQ a7
N0:2). In Bacillus subtilis, L27 comprises the following representative amino
acid sequence:
MLRLDLQFFASKKGVGSTKNGRDSEAKRLGAKRADGQFVTGGSILYRQRGTKIYPGEN
VGRGGDDTLFAKmGTVKFERFGRDRKKVSVY PVAQ (SEQ m N0:3). In E. coli, L27
comprises the following representative amino acid sequence:
MAIiKKAGGSTRNGRDSEAKR
LGVKRFGGESVLAGSIIVRQRGTKFHAGANVGCGRDHTLFAKADGKVKFEVKGPKN
RKFISIEAE (SEQ m N0:4). In Haemophilus influenzae, L27 comprises the following
representative amino acid sequence: MATKKAGGSTRNGRDSEAKRLGVKRFGGESVLAG
SIIVRQRGTKFHAGNNVGMGRDHTLFATADGKVKFEVKGEKSRKYVVIVTE (SEQ m
N0:5). A representative reference describing L27 is Chen et al., FEBS Lett.,
1975, 59, 96-99,
which is incorporated herein by reference in its entirety. Fragments of L27
can be from about 10
amino acids to about 90 amino acids, more preferably from about 15 amino acids
to about 75
amino acids, more preferably from about 20 amino acids to about 50 amino
acids, more
preferably from about 25 amino acids to about 40 amino acids, and more
preferably from about
amino acids to about 35 amino acids in length. Preferably, the fragments
comprise contiguous
25 amino acids from within the amino acid sequence for L27. L27 can be
isolated by the methods
described above, or can be isolated or produced by standard methodology, and
can be isolated
from any of the organisms recited°above.
In some embodiments of the invention, a probe is contacted with a plurality of
different
biological targets including any combination of the biological targets
described above.
30 The biological target is also contacted with a test compound. The amount of
binding
between the probe and the biological target in the presence and absence of the
test compound is
compared. Binding can be measured either quantitatively or qualitatively by
numerous methods
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known to those skilled in the art. A decrease in the amount of binding between
the probe and the
biological target in the presence of the test compound indicates that the test
compound inhibits
binding of the probe to the biological target. A plurality of test compounds
can also be screened
at the same time.
In some embodiments of the invention, the test compounds can be further tested
using
mammalian cells. Those test compounds that are able to inhibit binding of a
probe to a biological
target in non-mammalian cells (e.g., bacterial, fungi, etc.) but which are not
able to inhibit or
insignificantly binding of the probe to a biological target in a mammalian
cell can be ideal
candidates for treatment of mammals. In these circumstances, the test compound
would have
activity against a microbe or bacteria while having very little, if any,
effect on host mammalian
cells. The methods described herein are useful for, inter alia, the molecular
description of the
nature of the toxicity of antibiotic compounds in eukaryotic cells. In this
particular case, the
sensitive eukaryotic cells or organelles are incubated and treated as defined
above for bacterial
cells and the relevant targets are identified. This allows the definition of
screens, assays, and
structure-based design as discussed below for the discovery of active
antibiotics and when used
as a negative selection technique allows for optimization of new antibacterial
or other therapeutic
agents.
The photoaffinity probes that can be used in any of the methods described
herein are
shown below and include, but are not limited to, photoaffinity probes
comprising Formula I,
Formula II, Formula III, Formula IV, Formula V, or Formula VI, or any mixture
thereof. The
preferred configuration at C-5 is (S). It will be appreciated by those skilled
in the art that
compounds of the present can have additional chiral centers and be isolated in
optically active or
racemic form. The present invention encompasses any racemic, optically-active
(such as
enantiomers, diastereomers), tautomeric, or stereoisomeric form, or mixture
thereof, of a
compound of the invention. Preferred compounds of this invention have one
radioactive element
which is either 3H (T3), 3s5, or lasl. It is understood, however, that the
Formulas include all
isotopic forms of the compounds depicted.
In some embodiments of the invention, photoaffinity probes comprise Formula I,
shown
below.
10
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R2
16
R1 / I R R3
L
N Y
N3 ~ 1~
R O ~N
R
X Q
Formula I
wherein X and Y are, independently, F, H or CH3 in a variety of substitution
patterns. Preferred
compounds have one fluorine and one H. R1 is H, F or I. Rz is H, F or OH. R16
is H or F. Rl~ is H
or F. R3 is H or Cl-C8 alkyl. L is a bond or -OCHZC(=O). Q is
O O
NCO O
, N 4 or ~N N 4
Z 2
Z
wherein R4 is H, CH3, CH2CH3 or cyclopropyl. Z is O or S. Compounds comprising
Formula I
also include pharmaceutically acceptable salts thereof.
Preferred compounds comprising Formula I have the following substituents: X is
F, Y is
H, R3 is H, and R4 is CH3. More preferably, compounds of Formula I include,
but are not limited
to, 2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-
fluorophenyl]-1-piperazinyl]-
2-oxoethyl-4-azido-2-hydroxy-5-iodo-lzsl-benzoate, N-[[(5S)-3-[4-[4-(4-Azido-2-
hydroxy-5-
iodo-lzsl-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]
acetamide, 2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-
fluorophenyl]-1-
piperazinyl]-2-oxoethyl 4-azido-3-iodo-lzsl-benzoate, and N-[[(5S)-3-[4-[4-(4-
Azido-3-iodo-lzsl-
benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]
acetamide.
In other embodiments of the invention, photoaffinity probes comprise Formula
II,
shown below.
11
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R1
Formula II
wherein X and Y are, independently, F, H or CH3 in a variety of substitution
patterns. Preferred
compounds have one fluorine and one H. R1 is H, F or I. R2 is H, F or OH. R16
is H or F. R1' is H
or F. Q is
O O
N O
H 4 N\O H a. \~ /O
N R , N or ~N 1H 4
z
wherein R4 is H, CH3, CHZCH3 or cyclopropyl. Z is O or S. Compounds comprising
Formula II
also include pharmaceutically acceptable salts thereof..
Preferred compounds comprising Formula II have the following substituents: X
is F, Y
is H, and R4 is CH3. More preferably, compounds of Formula II include, but are
not limited to,
N-[[(5S)-3-(4,'-Azido-2-fluoro [ 1,1'-biphenyl]-4.-yl)-2-oxo-5-
oxazolidinyl]methyl]-T3-acetamide,
N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo [ 1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]-T3-
acetamide, N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo[1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]ethane-35S-thioamide, and N-[[(5S)-3-(4'-Azido-2-fluoro-3'-
iodo-lzsl-[1,1'-
biphenyl]-4-yl)-2-oxo-S-oxazolidinyl]methyl]acetamide.
In other embodiments of the invention, photoaffinity probes comprise Formula
III,
shown below.
Y
Rs
X ~ P
Formula III
12
R2
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wherein X and Y are, independently, F, H or CH3 in a variety of substitution
patterns. Preferred
compounds have one fluorine and one H. RS is
Ni ~ N
or
' r ~ ~ .,~'v
R6 R6
wherein R6 is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, C1-C4
dialkylamino, C1-C4 alkyl,
nitrile, carboxamide, Cl-C~. alkoxy, Cl-C4 alkylthio, or C1-C4 alkoxycarbonyl.
P is
O O
N
N ~ r ~~ ~O
N R~ ~ N R or ' N N R~
Z Z Z
wherein Z is O or S. R' is
16
' 1
R16 Rl R2 N Ns R16 R
or R2
\ I Rl~ I I \ \
~ \N3 R1 R2 I Ns
R1~ Rl~
wherein Rl is H, F or I. R2 is H, F or OH. R16 is H or F. Rl~ is H or F.
Compounds comprising
Formula DI also include pharmaceutically acceptable salts thereof.
Preferred compounds comprising Formula III have the following substituents: X
is F, Y
is H, and R6 is H. More preferably, compounds of Formula IZI include, but are
not limited to,
(2E)-3-(4-azido-3-iodo-lasl-phenyl)-N-[ [(5S)-3-[3-fluoro-4-(4-
pyridinyl)phenyl]-2-oxo-5-
oxazolidinyl]methyl]-2-propenamide, 4-azido-N-[[(5S)-3-[3-fluoro-4-(4-
pyridinyl)phenyl]-2-
oxo-5-oxazolidinyl]methyl]-2-hydroxy-5-iodo-lzsl-benzamide, and N-(4-
azidophenyl)-N'-[[(5S)-
3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] 35S-thiourea.
In other embodiments of the invention, the photoaffinity probes comprise
Formula IV
shown below.
13
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R9
Rls
Rg / Rlo
L
N Y
N3 ~ 19
R O
R1
Formula IV
wherein X and Y are, independently, F, H or CH3; R8 is H, F or I; R9 is H, F
or OH; Rl$ is H or
F; R19 is H or F; Rl° is H or C1-C8 alkyl; L is a bond or -OCH2C(=O);
and Q is
O O
N
N O ~ 'O 'O
~ N Rll N Rll or ~ N N Rll
Z Z 2
wherein Rll is H, CH3, CHZCH3 or cyclopropyl; and Z is O or S; or a
pharmaceutically
acceptable salt thereof.
In other embodiments of the invention, the photoaffinity probes comprise
Formula V
shown below.
Y
R12
X Q
Formula V
wherein X and Y are, independently, F, H or CH3; R12 is N3 or
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Rs R19
R9
V3 Ris
wherein R8 is H, F or I; R~ is H, F or OH; Rl$ is H or F; R19 is H or F; and Q
is
O O
N
N o ~ 'o 'o
H Rn H Rm
N~ a N or ~ N N R11
Z ~ Z 2
wherein R11 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S; or a
pharmaceutically
acceptable salt thereof.
In other embodiments of the invention, the photoaffinity probes comprise
Formula VI
shown below.
Y
R13
X ~ P
Formula VI
wherein X and Y are, independently, F, H or CH3; R13 is
Ni ~ N
or
R14 Rm ° .
wherein~Rl4 is H, N3, halogen, NH2, OH, SH, Cl-C4 alkylamino, Cl-C4
dialkylamino, Cl-C4
alkyl, nitrile, carboxamide, C1-C4 alkoxy, Ci-C4 alkylthio, or C1-C~
alkoxycarbonyl; and P is
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O O
N
~O
R15 N R15 or ~ N H Rls
~ ~ ~ ~N
Z Z
Z
wherein: Z is O or S; and Rl$ is
18
H
R18 R R N ' N3 Rls R
or ~ R~
R19
Rl9Ns Rg R9 N3.
R19
wherein Rg is H, F or I; and R9 is H, F or OH; Rl$ is H or F; and Rl~ is H or
F; or a
pharmaceutically acceptable salt thereof.
Methods for preparing the photoaffinity probes described in Formulas I, II,
III, IV, V,
and VI are depicted in the following synthesis schemes. It will be apparent to
those skilled in the
art that the described synthetic procedures are merely representative in
nature and that alternative
procedures are feasible and may be preferred in some cases.
~ Non-radioactive compounds of Formulas I and IV are prepared by the methods
described in Schemes A and B. As shown in Scheme A, coupling of a benzoic acid
moiety (Al)
with an appropriate hydroxyacetyl piperazine fragment (A2) leads to compounds
A3 of Formula I
where L is -OCH2C(=O). Coupling can be accomplished with 1-[3-
(dirnethylamino)propyl]-3-
ethylcarbodiimide hydrochloride or any other reagents familiar to ones skilled
in the art.
Appropriate benzoic acid fragments can be made by procedures known in the
literature. (Dupuis,
Can. J. Claem., 1987, 65, 2450-2453; Shu, J. Labelled Compounds afzd
Radiopharmaceuticals,
1996, 3~, 227-237, each of which is incorporated herein by reference in its
entirety). Appropriate
hydroxyacetyl piperazine fragments can also be made by methods known in the
literature
(Barbachyn, U.S. Patent No. 5,547,950; Barbachyn, U.S. Patent No. 5,990,136;
and Snyder,
International Publication WO 00/10566-A1, each of which is incorporated herein
by reference in
its entirety). Methods for incorporation of lasI into compounds A3 are shown
in Schemes C and
D. Scheme A can also be used where the compounds of A1 and A3 have R16 and Rl'
substituents
16
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WO 02/056013 PCT/USO1/48455
ortho to the acid substituent (as iri Formulas I and IV).
Scheme A:
R1 O O R1 R2
OH HO~N~ Y ~O
+ ~N ~ O~N~ Y
R2 N \ I Na O ~N /
3 X a I
\ O
X
A1 A2 A3
Non-radioactive compounds of Formulas I and IV where L is a bond are prepared
by the
synthetic sequence shown in Scheme B. An appropriate benzoic acid fragment (Al
of Scheme A)
is coupled with an appropriate piperazine (B2) using 1,1-carbonyldiimidazole
in tetrahydrofuran
to give the desired compound (B3). Other coupling methods known to those
skilled in the art are
also possible. The piperazine fragment is made by methods known in the
literature (Hutchinson,
U.S. Patent No. 5,700,799, which is incorporated herein by reference in its
entirety; Barbachyn,
U.S. Patent No. 5,990,136; and Snyder, International Publication WO 00/10566-
A1). Methods
for incorporation of lasl into compounds B3 are shown in Schemes C and D.
Scheme B can also
be used where the compound of B3 has R16 and Rl~ substituents ortho to the
amide substituent (as
in Formulas I and IV).
Scheme B:
R2 O
HN~ Y
~N R1 / I ~ Y
A~ + X \ I Q ~ \ s N /
N
X \ Q
B
Radioactive iodine is introduced into the compounds of Formulas I and IV by
the
methods shown in Schemes C and D. Compounds CZ of Formula I (where Rl is OH
and R2 is
iasl) are prepared by reaction of compounds Cl (prepared according to the
methods of Schemes A
and B) with Na125I and chloramine-T.
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WO 02/056013 PCT/USO1/48455
Scheme C:
0 0
HO (~).N~ Y HO U).N~ Y
~ ~IN , ~ / ~ ~N
N3 X \ I O N3 1251 X ~ I Q
Ci C2
Alternatively, compounds D3 of Formulas I and IV (where R1 is H and RZ is 12s~
are
prepared as shown in Scheme D. Reaction of compounds D1 (prepared by the
methods shown in
Schemes A and B) with hexamethylditin affords the stannanes D2. Reaction of D2
with Nalzsl
and chloramine-T leads to D3.
Scheme D:
0 0
H (~).N~ Y H t~)'N~ Y
/ ~ ~IN / ~ / ~ ~N
N3 I X ~ O N3 SnMe3 X ~ O
p ~2
O
H ~~)'N~ Y
vN
N3 1251 p3 X ' Q
Non-radioactive compounds of Formulas II and V are prepared by the method
shown in
Scheme E. The appropriate biphenyl nitro fragment (El) is reduced in the
presence of hydrogen
gas and a palladium catalyst to give the appropriate biphenyl aniline fragment
(E2). Other
reduction methods familiar to those skilled in the art may also be used.
Conversion to the azido
moiety (E3) can be accomplished via displacement of the appropriate diazonium
salt with sodium
azide using conditions familiar to those skilled in the art. The appropriate
nitro fragments (El)
can be prepared by methods known in the literature (Barbachyn, U.S. Patent No.
5,654,435,
which is incorporated herein by reference in its entirety; Barbachyn U.S.
Patent No. 5,990,136;
and Synder, International Publication WO 00/10566-A1) or by other methods
familiar to those
skilled in the art. Introduction of radioactive elements into compounds of
Formulas II and V are
depicted in Schemes F, G, and H. Scheme E can also be used where the compounds
of El, EZ,
and E3 have R16 and Rl~ or Rl8 and R19 substituents in the ortho position (as
in Formulas II and
18
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WO 02/056013 PCT/USO1/48455
V).
Scheme E:
Rz Rz Rz
02N / I Y HzN / I Y N3 / I Y
R~ / I ~ R1 / I ~ R1 / I
X ~ Q X ~ Q X ~ O
E1 Ez Es
Scheme F shows the procedure for incorporation of tritium into compounds of
Formulas
II and V where Q is oxazolidinone, Z is O, and R4 is CH3. Reaction of Fl
(prepared according to
Scheme E) with 6N HCl and methanol affords the free amine F2. Reaction of F2
with tritiated
sodium acetate and a coupling reagent affords the tritiated acetamide F3.
Suitable coupling
reagents include O-benzotriazol-1-yl-N,N,N',N',tetramethyluronium
hexafluorophosphate and
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
Other
acceptable coupling reagents are known by those skilled in the art.
Alternatively, tritiated acetic
anhydride and a suitable base can be used in place of tritiated sodium acetate
and a coupling
reagent. Incorporation of tritium into compounds of Formulas II and V where Q
is isoxazoline is
carried out in similar fashion. Scheme F can also be used where the compounds
of Fl, F2, and F3
have R16 and Rl~ or Rl8 and Rl9 substituents in the ortho position (as in
Formulas II and V).
Scheme F:
Rz Rz
N3 / I Y N / I Y
3
Ri / I ~ Ri / I OII
X ~ N O H X ~ N~O
F~ ~N~CHs F2 ~NH2
I IO
R2
N / I Y
3
/I o
R1 II
X ~ N~O
F3 ~N~CT3
I IO
Scheme G shows the method for incorporation of 35S into compounds of Formulas
II
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WO 02/056013 PCT/USO1/48455
and V where Q is oxazolidinone, Z is S, and R4 is CH3. Reaction of F2 (from
Scheme F) with
ethyl 3sS-dithioacetate affords the 3sS-thioacetamide, Gz, Incorporation of
3sS into compounds of
Formulas II and V where Q is isoxazoline is carried out in similar fashion.
Scheme G can also be
used where the compound of G2 has R16 and Rl' or Rl$ and Rl~ substituents in
the ortho position
(as in Formulas II and V).
Scheme G
R2
/I Y
F2 ~ / O
R1
X N~O
~N~CH3
2
35S
Radioactive iodine can be introduced into compounds of Formulas II and V by
the
method shown in Scheme H. Reaction of Hl (prepared according to the route
shown in Scheme
E) with hexamethylditin affords the organostannane Hz. Reaction of HZ with
Nalzsl and
chloramine-T affords the radioiodinated compound H3.
Scheme H:
t Me3Sn ~125~
Y I Y N \ I Y
N3 \ ~ ---~ N3 \ -~ 3 /
X \ Q X \ Q X Q
H1 H2 H3
Compounds of Formula V where Rlz is N3 are made according to the procedures
I5 described in U.S. Patent No. 5,910,504, Example I7, which is incorporated
herein by~reference
in its entirety.
Scheme I illustrates a synthetic method for the preparation of non-radioactive
compounds of Formulas III and VI where P is oxazolidinone, Z is O, and R~ is
optionally
substituted azidophenyl or azidocinnamoyl. Refluxing an appropriate acetamide
fragment (h) in
methanolic hydrochloric acid affords the free amine Iz. The acetamide
fragments (h) are prepared
by methods known in the literature (Barbachyn, U.S. Patent No. 5,565,571,
which is incorporated
herein by reference in its entirety; Barbachyn, U.S. Patent No. 5,990,136; and
Synder,
International Publication WO 00/10566-A1). Coupling of I2 with an appropriate
benzoic acid
fragment (I3, n = 0) or cinnamic acid fragment (I3, n = 1) leads to the amide
I4.. Coupling can be
CA 02432162 2003-06-13
WO 02/056013 PCT/USO1/48455
accomplished with EDC or other reagents familiar to ones skilled in the art.
Appropriate benzoic
acid fragments (I3, n = 0) are prepared by the same method used to prepare A1
of Scheme A.
Appropriate cinnamic acid fragments (I3, n =1) can be prepared by coupling of
an appropriate
benzaldehyde with Wittig-Horner reagents. Benzaldehyde fragments can be
prepared by
procedures known in the literature (Shu, J. Labelled Compouyzds and
Radiophanyzaceuticals,
1996, 3~, 227-237) or by other methods familiar to those skilled in the art.
Compounds of
Formulas III and VI where P is isoxazoline or isoxazolinone are made by
similar methods.
Scheme I can also be used where the compounds of I3 and Iq. have R16 and Rl~
or Rl8 and Rl~
substituents in the ortho position (as in Formulas III and VI).
Scheme I:
Y R5 Y O Ri
5 ~~ '
R / I O ~ ~ ~ ~ + HO~
X N~O X \ N O n ~~R2
/\
~N ~NH2 N3
11 O 12 13
Y
R5
O
I' R
X w N~O H N3
L--~ N
n R2
14
Introduction of lasI into compounds of Formulas III and VI where P is
oxazolidinone, Z
is O, and R' is optionally substituted azidophenyl or azidocinnamoyl is
accomplished by the
method shown in Scheme J. Reaction of I4. (from Scheme I, where Rl is H and R2
is OH) with
Nalasl and chloramine-T affords the radioiodinated compound JZ. Introduction
of 12$I into
compounds of Formulas III and VI where P is isoxazoline or isoxazolinone is
carried out by
similar methods.
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WO 02/056013 PCT/USO1/48455
Scheme J:
Y
R'
1251
/
H N3
N \
OH
J2 O
Alternatively, introduction of lzsl into compounds of Formulas III and VI
where P is
oxazolidinone, Z is O, and R' is optionally substituted azidophenyl or
azidocinnamoyl is carried
out by the method shown in Scheme K. Reaction of I4 (from Scheme I, where Rl
is I and Rz is H)
with hexamethylditin affords the organostannane Kz. Reaction of Kz with Nalzsl
and
chloramine-T affords the radioiodinated compound K3. Radioiodination of
compounds of
Formulas DI and VI where P is isoxazoline or isoxazolinone is carried out in
similar fashion.
Scheme K:
Y
Y
R5
R5
O
SnMe~ / ~ ~ lzSI
X N~/\O / X ~ /
~N \ n~ I ~N
K ~ N3 v/a ~~Ns
K3 O
Scheme L illustrates a synthetic method for the preparation of radioactive
compounds of
Formulas III and VI where P is oxazolidinone, Z is 3sS, and R~ is optionally
substituted
azidoaniline. An appropriate 3sS-isothiocyanate Lz is reacted with the
appropriate aminornethyl
fragment Iz (Scheme I) in refluxing THF to give the desired 3sS-thiourea,
(L3). The required 3sS
isothiocyanate L2 is prepared by reaction of an appropriate aniline with 3sS-
thiophosgene.
Introduction of 3sS into compounds of Formulas III and VI where P is
isoxazoline or
isoxazolinone is carried out in similar fashion. Scheme L can also be used
where the compounds
of L2 and L3 have R16 and Rl' or Rl$ and Rl~ substituents in the ortho
position (as in Formulas Ill
and VI).
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Scheme L:
Y
R5
1
R N=C=35S / I O
~2 + ~ ~ I ~ X N O H N R2
R2 vN /
N3 g S I
1
L2 Ls R Ns
The invention is further illustrated by way of the following examples which
are intended
to elucidate the invention. These examples are not intended, nor are they to
be construed, as
limiting the scope of the disclosure.
EXAMPLES
Example 1: Synthesis
2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-
piperazinyl]-2-oxoethyl 4-azido-2-hydroxy-5-iodo-lzsI-benzoate (Compound C2 of
Scheme C
where L is -CH2C(=O), X is F, Y is H, Q is oxazolidinone, Z is O and R4 is
CH3) is prepared as
follows.
Ns / OH O
125 \ I ~~N~
O vN /
F' v 'N O
~N~CH3
I IO
Step 1. To a stirred solution of (S)-N-[[3-[3-fluoro-4[4-(hydroxyacetyl)-1-
piperazinyl]
phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide (515.8 mg, 1.31 mmol) in
dimethylformamide
(10 ml) and pyridine (1 ml) is added 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide
hydrochloride (509.9 mg, 2.66 mmol) followed by 4-azidosalicylic acid (Dupuis,
Caya. J. Chem.,
1987, 65, 2450-2453) and a catalytic amount of 4-dimethylaminopyridine. The
reaction mixture
is stirred at room temperature for 72 hours then concentrated. The residue is
diluted with CHZCl2
(100 ml) and is washed successively with HZO (2 x 30 ml), 1 N HCl (2 x 30 ml),
saturated
NaHC03 (1 x 30 ml), dried (MgS04), filtered and concentrated. The residue is
dissolved in
CH30H/CHZC12, absorbed onto silica gel and is purified on a Biotage 40S column
with a SIM
using 2.5 % CH30H in CHZC12 as the eluent to give 186.5 mg (0.33 mmol, 25 %)
of the benzoate
ester. mp 177-178 °C (dec). 1H-NMR (DMSO) 8: 10.4, 8.24, 7.87, 7.53,
7.17, 7.09, 6.76, 6.70,
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WO 02/056013 PCT/USO1/48455
5.17, 4.71, 4.09, 3.71, 3.60, 3.40, 3.02, 2.96, 1.83.
Step 2. All reagents are prepared in 0.1 N NaP04 buffer, pH 7.4 unless
otherwise
specified. Buffer (70 ~1), chloramine-T (70 ~1 of a 1 mM stock solution), and
the azido phenol of
Step 1 (10 ~.l of a 50 ~,M stock solution in DMSO) are added to a 1.5 ml glass
reaction vial. A
rubber septum cap is crimped onto the reaction vial and a solution of l2sIz in
sodium hydroxide
(10 ~l containing 1 mCi (Amersham #IMS 30) is added. The reaction is gently
vortexed in the
dark for 2 hours at room temperature then quenched with 10% solution of sodium
bisulfite (100
~.I). The quenched reaction is diluted with buffer (800 ~,1) and transferred
from the reaction vial
with a 1 ml tuberculin syringe fitted with an 18 gauge needle. The reaction
volume (1 ml) is
loaded onto a preconditioned C18 sep-pak cartridge (Millipore Corporation) and
the
unincorporated lzs I2 is washed from the C 18 resin with HPLC grade water
containing 0.1 %
trifluoroacetic acid (20 ml). Product is eluted using of 80% CH3CN/0.1 TFA (3
ml). The typical
yield of iodinated product is approximately 30% of the total lzs IZ added to
the reaction.
Example 2: Synthesis
N-[[(5S)-3-[4-[4-(4-Azido-2-hydroxy-5-iodo-lasI-benzoyl)-1-piperazinyl]-3-
fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide (Compound C2 of Scheme C
where L is a
bond, X is F, Y is H, Q is oxazolidinone, Z is O and R4 is CH3) is prepared as
follows.
HO O
~ \ ~N
N3125~ ~
F' v \
H
'--~ N ' /
~O
Step 1. To a stirred suspension of (S)-N-[[3-[4-[3-fluoro-4-(1-
piperazinyl)]phenyl]-2-
oxo-5-oxazolidinyl]methyl]-acetamide (498.0 mg, 1.3 mmol) in CH2Cl2 (10 ml) is
added
diisopropylethylamine (0.70 ml, 4.0 mmol) followed by 4-azidosalicoyl chloride
(342.6 mg, 1.7
mmol) in CH2C12 (7 ml). The reaction mixture is stirred at room temperature
for 18 hours and
then is partitioned between CH2C12 (50 ml) and H20 (10 ml). The phases are
separated. The
organic layer is washed with H20 (10 ml), dried (MgSO~), filtered and
concentrated. The residue
is dissolved in CH30H/CH2C12, absorbed onto silica gel and is purified on a
Biotage 40S column
with a SIM using 3% CH30H in CH2C12 as the eluent to afford 412.3 mg (0.83
mmol, 62%) of
the desired benzamide as a tan solid. mp 188-189 °C (dec). 1H-NMR
(DMSO) 8: 10.2, 8.24,
7.51, 7.22, 7.16, 7.07, 6.64, 6.58, 4.70, 4.07, 3.70, 3.36, 2.96, 1.83.
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Step 2. Starting with the phenol prepared in Step 1, lasI is introduced
according to the
procedure described in Step 2 of example 1.
Example 3: Synthesis
2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-
piperazinyl]-2-oxoethyl 4-azido-3-iodo-lasl-benzoate (Compound D3 of Scheme D
where L is -
CH2C(=O), X is F, Y is H, Q is oxazolidinone, Z is O and R4 is CH3)
N3 / O
125 ~ ~ ~~N~
O ~N / I O
F~N
~H
'~N' /
~(O
Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid (103.8 ring, 0.36
mmol, (Shu,
J. of Labelled Co~zpoufzds ayad Radiopharmaceuticals, 1996, 38, 227-237)) in
dry THF (2.0 ml)
is added 1,1-carbonyldiimidazole (58.2 mg, 0.36 mmol). The reaction mixture is
stirred at room
temperature for 1 hour, then (S)-N-[[3-[3-fluoro-4[4-(hydroxyacetyl)-1-
piperazinyl]phenyl]-2-
oxo-5-oxazolidinyl]methyl]acetamide (141.9 mg, 0.36 mmol) is added followed by
a catalytic
amount of DMAP. The reaction mixture is heated at reflux for 72 hours. The
reaction mixture
was cooled to room temperature, and poured into CHZC12 (30 ml) and washed
successively with
H20 (15m1), 1 N HCl (15 ml), saturated aqueous NaHC03 (15 ml), brine (15 ml),
dried
(MgS04), filtered and concentrated. The residue is purified on a Biotage 12M
column using 2 %
CH30H in CHZCl2 as the eluent to afford 119.6 mg (0.18 mmol, 50%) of the
benzoate ester. mp
137-139°C. 1H-NMR (CDC13) 8: 8.52, 8.14, 7.49, 7.19, 7.07, 6.95, 6.01,
5.00, 4.72, 4.02, 3.81,
3.75, 3.62, 3.05, 1.58.
Step 2. To a stirred solution of the iodobenzoate prepared in Step 1 (62.7 mg,
0.094
mmol) and hexamethylditin (46.3 mg, 0.14 mmol) in dry THF (3 ml) is added
dichlorobis(triphenylphosphine)palladium (II) (2.0 mg, 0.003 mmol). The
reaction mixture is
degassed and is heated at reflux for 3 hours. The reaction mixture is cooled
and filtered through a
pad of celite. The filtrate is absorbed onto silica gel and purified on a
Biotage 12M column with
SIM using 2 % CH30H in CH2Cl2 as the eluent to afford 28.6 mg (0.04 mmol, 43
%) of the
stannane.lH-NMR (DMSO) b: 8.25, 8.04, 8.00, 7.48, 7.42, 7.15, 7.10, 5.10,
4.73, 4.09, 3.71,
3.60, 3.40, 3.02, 2.96, 1.83, 0.33.
Step 3. To a stirred solution of the stannane prepared in Step 2 in dry
acetonitrile is
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added a solution of 1M aqueous Nalz$I followed by chloramine-T hydrate. After
stirring at room
temperature for 30 minutes, the reaction mixture is quenched with saturated
aqueous NazS203
and purified to give the radioiodinated material.
Example 4: Synthesis
N-[[(5S)-3-[4-[4-(4-Azido-3-iodo-lzsl-benzoyl)-1-piperazinyl]-3-fluorophenyl]-
2-oxo-5-
oxazolidinyl]methyl]acetamide (Compound D3 of Scheme D where L is a bond, X is
F, Y is H, Q
is oxazolidinone, Z is O and R4 is CH3)
0
125
/ I N~
Ns \ ~N / I
F~N O
~H
'--~N' /
~(O
Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid (272.0 mg, 0.94
mmol) in
dry THF (4 ml) is added 1,1-carbonyldiimidazole (152.6 mg, 0.94 mmol). The
reaction mixture
is stirred at room temperature for 1 hour, then (S)-N-[[3-[4-[3-fluoro-4-(1-
piperazinyl)]phenyl]-
2-oxo-5-oxazolidinyl]methyl]-acetamide (315.9 mg, 0.94 mmol) is added followed
by DMF (2
ml). The reaction mixture is heated at reflux for 18 hours. The reaction
mixture is cooled and
poured into CHzClz (40 ml) and successively washed with H20 (20 ml), 1 N HCl
(20 ml),
saturated aqueous NaHC03 (20 ml), brine (20 ml), dried (MgS04), filtered and
concentrated. The
residue is dissolved in CH2Clz, absorbed onto silica gel and is purified on a
Biotage 40S column
with S1M using 2.5 % CH30H in CHZCIz as the eluent to afford 376.7 mg (0.62
mmol) of the
benzamide as a yellow solid. 1H-NMR (DMSO) b: 8.24, 7.88, 7.53, 7.47, 7.39,
7.19, 7.08, 4.71,
4.08, 3.70, 3.51, 3.40, 2.99, 1.83.
Step 2. To a stirred solution of the iodobenzamide prepared in Step 1 (82.4
mg, 0.13
mmol) and hexamethylditin (71.1 mg 0.22 mmol) in dry THF (6 ml) is added
tetrakis(triphenylphosphine)palladium(0). The reaction mixture is degassed and
heated at reflux
for 12 hours. The cooled reaction mixture is filtered through a plug of celite
and the filtrate is
absorbed onto silica gel and purified on a Biotage 12M column with S1M using 2
% CH30H in
49% CHZCIz and 49% EtOAC as the eluent to afford 28.2 mg (0.044 mmol, 34 %) of
the
stannane. 1H-NMR (DMSO) 8: 8.24, 7.50, 7.43, 7.35, 7.18, 7.17, 4.71, 4.08,
4.01, 3.70, 3.40,
2.99, 1.83, 0.32.
Step 3. To a stirred solution of the stannane prepared in Step 2 in dry
acetonitrile is
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added a solution of 1M aqueous NalzsI followed by chloramine -T hydrate. After
stirring at room
temperature for 30 minutes, the reaction mixture is quenched with saturated
aqueous Na2S203
and purified to give the desired radioiodinated material.
Example 5: Synthesis
N-[[(5S)-3-(4'-Azido-2-fluoro[1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]-T3-
acetamide (Compound F3 of Scheme F where Rlis H, RZ is H, X is F, and Y is H)
F O
CT3
O
Step 1. To a stirred solution of 4-iodonitrobenzene (6.86 g, 27.5 mmol) in dry
DMF
(230 ml) is added bis(pinacolato)diboron (8.24 g, 32.4 mmol) followed by
potassium acetate
(8.68 g, 88.5 mmol) and [1,1'-
bis(diphenylphosphino)ferrocene]dichloropalladium(lI) (624.6 mg,
0.76 mmol). The reaction mixture is degassed and heated at 85 °C for 2
hours. To the cooled
dark reaction mixture is added (S)-N-[[3-(3-fluoro-4-iodophenyl)-2-oxo-5-
oxazolidinyl]methyl]
acetamide (5.8 g, 15.3 mmol) followed by 2 N aqueous Na2C03 (143 ml) and [1,1'-
bis
(diphenylphosphino)ferrocene]dichloropalladium(II) (312.0 mg, 0.38 mmol). The
reaction
mixture is degassed and heated at 85 °C for 3 hours. The cooled
reaction mixture is partitioned
between EtOAC (500 ml) and H20 (300 ml). The phases are separated. The aqueous
layer is
extracted with EtOAC (300 ml). The organic layers are combined and
successively washed with
H20 (500 ml), brine (500 ml), dried (MgS04), filtered and concentrated. The
residue is dissolved
in CH30H/CH2C12, absorbed onto silica gel and is purified on a Biotage 40 M
column (2 lots)
with S1M using 75 % EtOAC in CH2Clz to 100 % EtOAC as the eluent to afford
3.74 g (10.0
mmol, 65%) of the desired nitrobiphenyl compound. 1H-NMR (DMSO) &: 8.30, 7.83,
7.68, 7.50,
4.78, 4.18, 3.80, 3.44, 1.84.
Step 2. A mixture of the nitrobiphenyl compound prepared in Step 1 (3.74 g,
10.0
mmol), 10% palladium on carbon in THF (100 ml), CH3OH (100 ml) and CH2C12 (100
ml) is
hydrogenated under a balloon of hydrogen for 18 hours. The reaction mixture is
filtered through
a pad of celite and the filtrate is concentrated to afford 2.50 g (7.3 mmol,
73%) of the desired
aminobiphenyl.1H-NMR (DMSO) 8: 8.26, 7.45, 7.34, 7.23, 6.64, 5.28, 4.73, 4.14,
3.76, 3.42,
1.84.
Step 3. To a stirred solution of the aminobiphenyl prepared in Step 2 (508.93
mg, 1.48
mmol) in CH30H (40 ml) and 1 M HCl (40 ml), cooled to 0 °C is added a
1.2 M aqueous NaN02
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solution (1.48 ml, 1.78 mmol). The reaction mixture is stirred at 0 °C
for 90 minutes, then
sulfamic acid (143.5 mg, 1.48 mmol) is added followed by sodium azide (115.4
mg, 1.78 mmol)
in H20 (1.5 ml). The reaction mixture is stirred at 0 °C for 45
minutes, then diluted with CHZC12
(200 ml). The phases are separated. The aqueous phase is extracted with CHZC12
(75 ml). The
combine organic phases are dried (MgS04), filtered and concentrated. The
residue is dissolved in
CH30H/CH2C12, absorbed onto silica gel and is purified on a Biotage 40S column
with SIM
using 10% CH3OH in CH2Cl2 as the eluent to afford 262.9 mg (0.71 mmol, 48%) of
the desired
azidobiphenyl as a pale yellow solid. 1H-NMR (DMSO) 8: 8.27, 7.58, 7.42, 7.24,
4.76, 4.16,
3.78, 3.43, 1.84.
Step 4. The azidobiphenyl prepared in Step 3 (102.4 mg, 0.27 mmol) in 6 N HCl
(2 ml)
and CH30H (6 ml) is heated at reflux for 18 hours. The CH3OH is removed in
vacuo and the
solid precipitate is isolated by filtration and is washed successively with
H20 (10 ml), ether (2 x
ml) then dried to afford 82.1 mg (0.23 mmol, 82%) of the desired amine
hydrochloride.1H-
NMR (DMSO) b: 8.30, 7.62, 7.42, 7.25, 4.98, 4.25, 3.91.
15 Step 5. To a stirring solution of 0.57 mg (6.94 pmol, 250 mCi) of tritiated
acetic acid
sodium salt (American Radiolabeled Chemicals, lot no ARC 990519) in 1 ml of
dry DMF and
2.71 mg (21 ~.mol) of diisopropylethylamine at room temperature, is added 6.94
pmol of 0.45M
O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in
dry DMF.
The solution instantly turned pale yellow and is stirred at room temperature
for 10 minutes. The
activated [3H]acetic acid sodium salt was then added to a stirring solution of
2.73 mg (7.5 p,mol)
of the amine hydrochloride prepared in Step 4 in 2 ml of dry DMF. The reaction
is stirred at
room temperature for 4.5 hours, then all solvents are removed by vacuum
distillation at room
temperature. The crude reaction mixture is purified on a preparative TLC plate
(Analtech Silica
gel GF, 500 micron, 20 cm x 20 cm plate), eluted with 8% methanol in
dichloromethane. The
desired band is scrapped. The product is eluted from the silica gel with 20%
methanol in
dichloromethane and filtered. The filtrate is concentrated under vacuum, and
the residue is
dissolved in 65.5 ml of methanol to afford 94.4 mCi of the desired tritiated
material (1.44
mCi/ml methanol, specific activity 57.37 mCi/mg (57.37 Ci/mmol), radiochemical
purity 99.5%
by HPLC).
Example 6: Synthesis
N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo[1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]-T3-acetamide (Compound F3 of Scheme F where Rlis H, R2 is
I, X is F, and
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YisH)
O
N3 ~ ~ ~ ~ ~N CT3
O
Step 1. To a stirred solution of the aniline prepared 'in Step 2 of Example 5
(284.9 mg,
0.83 mmol) in acetic acid (3 ml) is added iodine monochloride (134.5 mg, 0.83
mmol) in acetic
acid (0.25 ml). The reaction mixture is stirred at room temperature for 1.5
hours. The reaction
mixture is partitioned between EtOAc and aqueous Na2S203. The phases are
separated. The
aqueous phase is extracted with EtOAc (20 ml). The combined organic phases
were dried
(MgS04), filtered and concentrated. The residue is dissolved in CH30H,
absorbed onto silica gel
and is purified on a Biotage 40S column with SIM using 10-25 % acetone in
CHZC12 as the
eluent to afford 48.4 mg (0.10 mmol, 12%) of the desired iodoaniline as a
yellow oil. 1H-NMR
(CH30D) b: 7.73, 7.52, 7.29, 6.85, 4.81, 4.10, 3.79, 3.53, 3.32, 1.98.
Step 2. To a stirred solution of the iodoaniline prepared in Step 1 (47. 2 mg,
0.10 mmol)
in CH3OH (2 ml) and 1N HCl (2 ml) cooled to 0 °C, is added a solution
of NaN02 (8.5 mg, 0.12
mmol) in HZO (1 ml). The yellow reaction mixture is stirred at 0 °C for
30 minutes, then a
solution of NaN3 (8.0 mg, 0.12 mmol) in H20 (1 ml) is added. The reaction
mixture is stirred at 0
°C for 1 hour, during which time a yellow precipitate formed. The solid
is isolated by filtration
and washed with H20 and dried to afford 41.0 mg (0.083 mmol, 83%) of the
desired
iodoazidobiphenyl as a yellow solid. mp 173-175 °C (dec). 1H-NMR (DMSO)
8: 8.27, 7.98,
7.60, 7.42, 4.76, 4.16, 3.78, 3.43, 1.84.
Step 3. A mixture of 129 mg (0.26 mmol) of the iodoazidobiphenyl prepared in
Step 2
(129.0 mg, 0.26 mmol), CH30H (6 ml) and 1 N HCl (2 ml) are heated at reflux
for 48 hours. The
cooled reaction mixture is concentrated to afford quantitative yield the
desired amine
hydrochloride as a tan solid. 1H-NMR (CH30D) b: 7.96, 7.63, 7.46, 7.38, 7.29,
5.04, 4.34, 3.93,
3.38, 1.30.
Step 4. To a solution of 5.1 mg (0.05 mmol, 25 mCi) of tritiated acetic
anhydride
(Amersham Batch B77, isotope # 00-0316) is added 2 N PC13 in CHaCl2 (25 ~,1).
The reaction
mixture is left at room temperature for 5 hours with occasional mixing. To
this mixture is added
a solution of the amine hydrochloride prepared in Step 3 (47.7 mg, 0.104 mmol)
in pyridine (0.25
ml) followed by DMAP (4.6 mg). After 30 minutes, the reaction mixture is
partitioned between
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H20 and CHZC12. The phases are separated. The aqueous phase is extracted
exhaustively with
CH2C12 and then concentrated. The residue is purified on silica gel (4 g)
using 20 % acetone in
toluene as the eluent to afford 38.2 mg (0.077 mmol, 74 %) of desired
tritiated acetamide.
Example 7: Synthesis
N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo[ 1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]ethane-3sS-thioamide (Compound G2 of Scheme G where Rlis
H, R2 is I, X
is F,andYisH)
O
N3 ~ ~ ~ ~ N~O N
35~
Step 1. Methylmagnesium chloride in tetrahydrofuran (THF) is treated with 3sS
labeled
carbon disulfide at 40 °C, followed by treatment with ethyl iodide. The
reaction is stirred at 60 °C
for 1.5 hours. After workup with water and ethyl ether, the desired ethyl 3sS-
dithioacetate is
obtained.
Step 2. The amine hydrochloride salt prepared in Step 3 of Example 6 and the
ethyl
[ssS]dithioacetate prepared in Step 1 are stirred in methylene chloride,
methanol, and
triethylamine to give the desired 3sS labeled thioamide.
Example 8: Synthesis
N-[ [(5S)-3-(4'-Azido-2-fluoro-3'-iodo-lzsl-[ 1,1'-biphenyl]-4-yl)-2-oxo-5-
oxazolidinyl]methyl]acetamide (Compound H3 of Scheme H where X is F, Y is H, Q
is
oxazolidinone, Z is O, and R4 is CH3)
125
N3 ~ ~ ~ ~ ~N
Step 1. To a stirred solution of the iodobiphenyl prepared in Step 2 of
Example 6 (56.2
mg, 0.11 mmol) and hexamethylditin (73.9 mg, 0.22 mmol) in toluene (5 ml) is
added palladium
(II) acetate (2.6 mg, 0.011 mmol) followed by triphenylphosphine (6.5 mg,
0.022 mmol). The
reaction mixture is degassed and heated at 80 °C for 20 hours. The
cooled reaction mixture is
concentrated to one half the volume, then purified on a Biotage 12S column
using 10-20 %
acetone in CH2C12 as the eluent to afford 53.5 mg (0.10 mmol, 89%) of the
desired stannane. 1H-
NMR (CDC13) ~: 7.53, 7.43, 7.30, 7.21, 6.07, 4.83, 4.11, 3.83, 3.73, 2.05,
0.35.
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Step 2. To a stirred solution of the stannane prepared in Step 1 in dry CH3CN
and pH 7
phosphate buffer is added chloramine-T followed by a solution of 1M aqueous
Nalasl. After 30
minutes, the reaction mixture is quenched with saturated aqueous Na2S203 and
purified to give
the title compound.
Example 9: Synthesis
(2E)-3-(4-Azido-3-iodo-lasl-phenyl)-N-[ [(5S)-3-[3-fluoro-4-(4-
pyridinyl)phenyl]-2-oxo-
5-oxazolidinyl]methyl]-2-propenamide (Compound I4. of Scheme I where Rs is 4-
pyridyl, X is F,
Y is H, n is 1, R1 is l2sl, and R2 is H)
0
Ns
N\ ~ ~ ~ ~N
~/\ X125
O
, Step 1. To a stirred solution of oxalyl chloride (0.10 ml, 1.2 mmol) in
CH2C12 (1.5 ml),
cooled to -78 °C, is added dry DMSO (0.14 ml, 1.97 mmol). After 10
minutes, a solution of 4-
azido-3-iodobenzyl alcohol (217.0 mg, 0.79 mmol (Shu, J. of Labeled Compounds
afad
Radiophannaceuticals, 1996, 38, 227-237)) in CH2C12 (2.5 ml) is added. After
l5.minutes,
triethylamine (0.33 ml, 2.37 mmol) is added and the reaction mixture is
allowed to warm to room
temperature. The reaction mixture is poured into CHZCIz (30 ml) and washed
successively with
H20 (20 ml), brine (20 ml), dried (MgSO~.), filtered and concentrated. The
residue is purified on
a Biotage 12M column using 10% EtOAC in hexane to afford 195.5 mg (0.72 mmol,
91%) of the
desired aldehyde.1H-NMR (CDC13) 8: 9.9, 8.31, 7.93, 7.28.
Step 2. To a stirred solution of the aldehyde prepared in Step 1 (190.0 mg,
0.69 mmol)
in dry THF (1 ml) is added triethylphosphonoacetate (0.15 ml, 0.76 mmol)
followed by lithium
hydroxide monohydrate (32.1 mg, 0.76 mmol). The reaction mixture is stirred at
room
temperature for 48 hours. The reaction mixture is poured into CHZC12 (40 ml)
and successively
washed with H20 (20 ml), brine (20 ml), dried (MgS04), filtered and
concentrated. The residue
is dissolved in CHZC12, absorbed onto silica gel and purified on a Biotage 40S
column with a
. SIM using 5% EtOAC in hexane as the eluent to afford 165.1 mg (0.48 mmol, 70
%) of the
desired ester. mp 93-94 °C. 1H-NMR (CDC13) b: 7.97, 7.56, 7.16, 6.40,
4.29, 1.35.
Step 3. To a stirred solution of the ester prepared in Step 2 (66.7 mg, 0.19
mmol) in
CH30H (2 ml) is added 1 N LiOH (0.19 ml, 0.19 mmol). The reaction mixture is
heated at reflux
for 12 hours. The cooled reaction mixture is concentrated and used
immediately.
Step 4. (S)-N-[[3-[3-fluoro-4-(4-pyridyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]
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acetamide (1.40 g, 4.25 mmol) in CH30H (62 ml) and 6 N HCl (31 ml) is heated
at reflux for 18
hours. The reaction mixture is concentrated to afford 1.48 g of the amine bis-
hydrochloride salt.
1H-NMR (DMSO) 8: 8.98, 8.62, 8.26, 7.75, 7.56, 7.35, 5.76, 5.07, 4.28, 4.30,
3.27.
Step 5. The amine bis-hydrochloride (from Step 4) (68.2 mg, 0.19 mmol), the
lithium
carboxylate prepared in Step 3, 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride
(72.8 mg, 0.38 mmol) and 1-hydroxybenzotriazole hydrate (30.8 mg, 0.23 mmol)
are dissolved in
pyridine (2 ml) and stirred at room temperature for 72 hours. The reaction
mixture is
concentrated. The residue is dissolved in CH2C12 (40 ml) and washed with HZO
(20 ml), brine
(20 ml), dried (MgS04), filtered and concentrated. The residue is dissolved in
CH30H/CHZC12,
absorbed onto silica gel and is purified on a Biotage 12M column with SIM
using 2 % CH30H
(saturated with NH3) in CH2C12 as the eluent to afford 56.2 mg (0.096 mmol,
51%) of the desired
cinnamide. 1H-NMR (DMSO) &: 8.66, 8.48, 8.02, 7.64, 7.49, 7.40, 7.35, 6.70,
4.86, 4.22, 3.84,
3.60.
Step 6. To a stirred solution of the iodocinnamide prepared in Step 4 and
hexamethylditin in dry THF is added tetrakis(triphenylphosphine)palladium(0).
The reaction
mixture is degassed and heated at reflux for 12 hours. The cooled reaction
mixture is filtered
through a plug of celite and the filtrate is absorbed onto silica gel and
purified on a Biotage 12M
column with SIM to afford the stannane.
Step 7. To a stirred solution of the stannane prepared in Step 5 in dry
acetonitrile is
added a solution of 1M aqueous NalasI followed by chloramine -T hydrate. After
stirring at room
temperature for 30 minutes, the reaction mixture is quenched with saturated
aqueous Na2S203
and purified to give the desired radioiodinated material.
Example 10: Synthesis
4-Azido-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-
oxazolidinyl]methyl]-2-
hydroxy-5-iodo-lasI -benzamide (Compound JZ of Scheme J where RS is 4-pyridyl,
X is F, and Y
is H, and n is 0)
X125
Ns
~N
I I
O OH
Step 1. To a stirred suspension of the amine bis-hydrochloride salt prepared
in Step 4 of
Example 9 (172.4 mg, 0.48 mmol) in pyridine (4 ml) and CH2C12 (1 ml) is added
4-azidosalicylic
acid (128.9 mg 0.72 mrnol) followed by added 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide
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hydrochloride (184.0 mg, 0.96 mmol) and 1-hydroxybenzotriazole hydrate (77.8
mg, 0.58
mmol). The reaction mixture is stirred at room temperature for 72 hours then
concentrated. The
residue is dissolved in CH30H/CH2C12, absorbed onto silica gel and purified on
a Biotage 40S
column with SIM using EtOAC as the eluent to afford 44.8 mg (0.10 mmol, 21 %)
of the
benzamide as a tan solid. mp 200-202 °C (dec). 1H-NMR (DMSO) 8: 12.5,
9.1, 8.66, 7.92, 7.70,
7.67, 7.61, 7.50, 7.46, 6.70, 6.60, 4.93, 4.25, 3.92, 3.70.
Step 2. Starting with the phenol prepared in Step 1, lzsI is introduced
according to the
procedure described in Step 2 of Example 1.
Example 11: Synthesis
N-(4-Azidophenyl)-N'-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-
oxazolidinyl]
methyl] 35S-thiourea (Compound L3 of Scheme L where RS is 4-pyridyl, X is F, Y
is H, Rl is H
and RZ is H)
0
Nv ~ ~ ~ ~N N
F s S \ ~ N
3
To a stirred solution of the amine bis-hydrochloride (from Step 4 of Example
9) in dry
THF is added Hunig's base followed by 35S-4-azidophenylisothiocyanate in THF.
The reaction
mixture is heated at reflux for 1 hour. The cooled reaction mixture is cooled
and purified to give
the desired thiourea.
Example 12: Identification Of Biological Targets
Bacteria with sensitivity to parent antibiotic (for example S. aureus or other
sensitive
gram positive or gram negative bacteria) are grown in complete Mueller-Hinton
medium to mid-
exponential phase. Representative aliquots of the culture are briefly
sedimented in RNase-free
tubes (1.5 ml capacity) and then resuspended in fresh Mueller-Hinton medium.
Competitor
compounds (active or inactive as antibiotics) are added from a stock
containing DMSO (total
final concentration = 0.2%). The active photoaffinity probe (for example,
compounds such as
those described in Formulas within; mixture of unlabeled and lasI-labeled, 3H-
labeled, or any
other suitable radiolabeled or non-radiolabeled, detectable compound) is then
added to a final
concentration near the minimum inhibitory concentration (MIC) for
antibacterial action. This
exposure is continued in the dark for 30 minutes at 37°C.
The bacteria are then exposed directly to ultraviolet light or briefly
sedimented,
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resuspended in phosphate buffered saline (PBS), and then exposed to
ultraviolet light to activate
the photoprobe (Stratalinker at energy setting 180,000 microjoules). The
labeled cells are then
washed with PBS, resuspended in Buffer A (10 mM Tris-HCL (pH 7.6), 30 mM
NH4C1, 30 mM
MgCl2) and lysed with lysostaphin (5 ~,g/ml final concentration of lysostaphin
for 15 minutes at
37°C ). Cell wall and large membrane fragments are then removed by
centrifugation at 18,000 x
g for 15 minutes. The resulting supernatant is sedimented at 450,000 x g for
60 minutes, yielding
a pellet containing cell membrane and ribosomes. The pellet is resuspended in
Buffer B (10 mM
Tris-HCl (pH 7.6), 0.5 % SDS and 6 mM EDTA), and the RNA is extracted by a
standard phenol
extraction procedure. RNA extracts are precipitated with ethanol and
resuspended in the
appropriate buffer for electrophoresis of RNA. Crude pellets are subjected to
electrophoresis for
proteins. Crosslinking of the labeled photoprobe to, for example, ribosomal
RNA (ribosomal
RNA of the 235, 165, 5S size) or to transfer RNA is detected by RNase H
digestion and
subsequent primer extension assays, yielding the precise base at which
crosslinking occurs.
Specific crosslinking events can be validated through the use of biologically
active antibacterial
compounds, resulting in a reduction in the amount of crosslinking. Additional
validation of the
crosslinking events) is obtained by observing that competitor antibiotics,
which are not
biologically active (devoid of antibacterial activity), fail to decrease the
amount of specific
crosslinking to the RNA and/or protein target(s).
Using this approach, portions of RNA (23S RNA peptidyltransferase region
including
A2602, U2506, A2451), tRNA together with the 64 kDa LepA and 11 kDa L27
proteins have
been identified as biological targets and can be used in the discovery of
unique inhibitors of
protein translation based on this mechanism.
Example 13: Identification of Antibiotic Compounds
The proteins (e.g., the 64 kDa and the 11 kDa proteins, LepA and L27,
respectively)
and/or RNA components are used either alone or in combination in recognition
assays to detect
the binding of a representative photoaffinity probe, such as an oxazolidinone
photoaffinity probe
or equivalent compound, that is labeled by either radioactive or non-
radioactive (e.g., enzymatic,
chemiluminescent or fluorescent) means. The ability of test compounds to
interfere with the
interaction is measured. Increased interaction with these sites is/are used as
positive selection
criteria, whereas interaction with eukaryotic sites is used as a negative
selection criterion to
optimize the potential therapeutic.
Ribosomal RNA is bound to a solid surface such as, for example, scintillation
proximity
34
CA 02432162 2003-06-13
WO 02/056013 PCT/USO1/48455
bead (SPA) by charge interaction. Separate additions to the assay can include
tRNA and the
associated 64kDa LepA and L27 proteins, as well as other biological targets
identified in the
methods described above. Biological targets and/or test compounds can be
tested alone or in any
combination. A representative oxazolidinone or equivalent compound (e.g.,
eperezolid) is added
in a labeled form as described (e.g., 3H-eperezolid) and the ability of test
compounds to compete
or reduce the binding of the labeled compound is measured. For the tritiated
SPA example, ,
measurement can be by liquid scintillation. Alternate probes with alternate
methods of
measurement (e.g., coupled enzymatic activity, chemical shifts/measurement,
fluorescence),
however, can also be used. The effective concentrations of compounds that
specifically reduce
binding to the targets identified as described above are taken as a positive
selection for potential
therapeutics. Attractive compounds so identified are then tested for
selectivity with respect to the
toxicity targets identified in the eukaryotic target cells by similar
techniques but using eukaryotic
target molecules. Proteins can also be bound to selection matrixes by
antibodies or by known
chemical tags attached to the identified targets (e.g. histidine-metal,
streptavidin-biotin, etc.) for
these purposes.
In particular, iodinated probe with competition of cross-linking by both
active and
inactive enantiomers of eperezolid can be used to illustrate this particular
embodiment. A strain
of S. aureus is grown at exponential rate. Aliquots of cells (1 m1/1.5 ml
tube) are gently pelleted
and resuspended in fresh medium with or without 40 ~,M active eperezolid (S)
or inactive
enantiomer (R) and 8 ~,M lasl-probe (2 ~,Ci/tube) for 30 minutes. The samples
were then treated
as described above in Example 12. Examination of the RNA from such procedures
shows that
the 23S RNA is cross-linked I a particular fashion, e.g. cross-linking is
prevented by the active
enantiomer (S) but not the inactive enantiomer (R). Separation of RNA on a 1%
agarose gel
demonstrates that the 23S RNA is specifically cross-linked (data not shown).
Separation on 10%
TBE urea electrophoresis gels demonstrates that tRNA is also cross-linked
(data not shown). An
autoradiogram of 10-20% Tris Tricine polyacrylamide analysis of the ribosomal
pellets
demonstrates cross-linking of a 64 kDa protein (LepA) and an 11 kDa protein
(L27) (data not
shown).
The information taught by cross-linking studies can also be used for structure-
based
design. Briefly, co-crystals are prepared with an oxazolidinone or equivalent
molecule together
with one or more of the key components of the interaction as identified and
described above.
The description of the direct interaction with the relevant binding site is
used as a positive guide
CA 02432162 2003-06-13
WO 02/056013 PCT/USO1/48455
in the construction of new potential therapeutics. The co-crystal of the same
test compounds with
the eukaryotic target can be used as a negative selection.
By the same rationale, NMR can be used to study the interaction of a suitable
test
compound (oxazolidinone or equivalent molecule) with any or all of the
respective target
molecules that have been identified by the cross-linking studies. In this
case, either the test
compound and/or the biological targets are suitably modified to allow
measurements of the site
of interaction by standard techniques.
As those skilled in the art will appreciate, numerous changes and
modifications may be
made to the preferred embodiments of the invention without departing from the
spirit of the
invention. It is intended that all such variations fall within the scope of
the invention. The entire
disclosure of each publication cited herein is hereby incorporated by
reference.
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SEQUENCE LISTING
<110> Pharmacia & Upjohn Company
Colca, Jerry R.
McDonald, William Gerald
Shinabarger, Dean L.
<120> Oxazolidinone Photoaffinity Probes, Uses And Compounds
<130> 00172.PCT1
<150> 60/256,053
<151> 2000-12-15
<160> 5
<170> PatentIn version 3.1
<210> 1
<211> 566
<212> PRT
<213> Lep A Protein
<220>
<223> B. subtilis
<400> 1
Asn Lys Arg Tyr Ala Arg Ser Val Thr Arg Phe Asn Gly Phe Arg Lys
1 . 5 10 15
Arg Thr Trp Tyr Tyr Asn Gln Ile Lys Arg Val Arg Leu Lys Tyr Glu
20 25 30
Ala Lys Asp Gly Asn Thr Tyr Thr Phe His Leu Ile Asp Thr Pro Gly
35 40 45
His Val Asp Phe Thr Tyr Glu Val Ser Arg Ser Leu Ala Ala Cys Glu
50 55 60
Gly Ala Ile Leu Val Val Asp Ala Ala Gln Gly Ile Glu Ala Gln Thr
65 70 75 80
Leu Ala Asn Val Tyr Leu Ala Leu Asp Asn Glu Leu Glu Leu Leu Pro
85 90 95
Val Ile Asn Lys Ile Asp Leu Pro Ala Ala Glu Pro Glu Arg Val Lys
100 105 110
Gln Glu Ile Glu Asp Met Ile Gly Leu Asp Gln Asp Asp Val Val Leu
115 120 125
Ala Ser Ala Lys Ser Asn Ile Gly Ile Glu Glu Ile Leu Glu Asp Ile
130 135 140
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Val Glu Val Val Pro Ala Pro Asp Gly Asp Pro Glu Ala Pro Leu Lys
145 150 155 160
Ala Leu Ile Phe Asp Ser Glu Tyr Asp Pro Tyr Arg Gly Val Ile Ser
165 170 175
Ser Ile Arg Ile Val Asp Gly Val Val Lys Ala Gly Asp Lys Ile Arg
180 185 190
Met Met Ala Thr Gly Lys Glu Phe Glu Val Thr Glu Val Gly Ile Asn
195 200 205
Thr Pro Lys Gln Leu Pro Val Asp Glu Leu Thr Val Gly Asp Val Gly
210 215 220
Tyr Ile Ile Ala Ser Ile Lys Asn Val Asp Asp Ser Arg Val Gly Asp
225 230 235 240
Thr Ile Thr Leu Ala Ser Arg Pro Ala Ser Glu Pro Leu Gln Gly Tyr
245 250 255
Lys Lys Met Asn Pro Met Val Tyr Cys Gly Leu Phe Pro Ile Asp Asn
260 265 270
Lys Asn Tyr Asn Asp Leu Arg Glu Ala Leu Glu Lys Leu Gln Leu Asn
275 280 285
Asp Ala Ser Leu Glu Phe Glu Pro Glu Ser Ser Gln Ala Leu Gly Phe
290 295 300
Gly Tyr Arg Thr Gly Phe Leu Gly Met Leu His Met Glu Ile Ile Gln
305 310 315 320
Glu Arg Tle Glu Arg Glu Phe Gly Ile Glu Leu Ile Ala Thr Ala Pro
325 330 335
Ser Val Ile Tyr Gln Cys Val Leu Arg Asp Gly Ser Glu Val Thr Val
340 345 350
Asp Asn Pro Ala Gln Met Pro Asp Arg Asp Lys Ile Asp Lys Ile Phe
355 360 365
Glu Pro Tyr Val Arg Ala Thr Met Met Val Pro Asn Asp Tyr Val Gly
370 375 380
Ala Val Met Glu Leu Cys Gln Arg Lys Arg Gly Gln Phe Ile Asn Met
385 390 395 400
2
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WO 02/056013 PCT/USO1/48455
Asp Tyr Leu Asp Asp Ile Arg Val Asn Ile Val Tyr Glu Leu Pro Leu
405 410 415
Ala Glu Val Val Phe Asp Phe Phe Asp Gln Leu Lys Ser Asn Thr Lys
420 425 430
Gly Tyr Ala Ser Phe Asp Tyr Glu Phe Ile Glu Asn Lys Glu Ser Asn
435 440 445
Leu Val Lys Met Asp Ile Leu Leu Asn Gly Asp Lys Val Asp Ala Leu
450 455 460
Ser Phe Ile Val His Arg Asp Phe Ala Tyr Glu Arg Gly Lys Ala Leu
465 470 475 480
Val Glu Lys Leu Lys Thr Leu Ile Pro Arg G1n Gln Phe Glu Val Pro
485 490 495
Val Gln Ala Ala Ile Gly Gln Lys Ile Val Ala Arg Thr Asn Ile Lys
500 505 510
Ser Met Gln Lys Asn Val Leu Ala Lys Cys Tyr Gly Gly Asp Ile Ser
515 520 525
Arg Lys Arg Lys Leu Leu Glu Lys Gln Lys Ala Gly Lys Arg Lys Met
530 535 540
Lys Ala Val Gly Asn Val Glu Ile Pro Gln Asp Ala Phe Leu Ala Val
545 550 555 560
Leu Lys Met Asp Asp Glu
565
<210> 2
<211> 98
<212> PRT
<213> L27 Protein
<220>
<223> S. aureus
<400> 2
Val Arg Cys Ile Pro Met Leu Lys Leu Asn Leu Gln Phe Phe Ala Ser
1 5 10 15
Lys Lys Gly Val Ser Ser Thr Lys Asn Gly Arg Asp Ser Glu Ser Lys
20 25 30
3
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WO 02/056013 PCT/USO1/48455
Arg Leu Gly Ala Lys Arg Ala Asp Gly Gln Phe Val Thr Gly Gly Ser
35 40 45
Ile Leu Tyr Arg Gln Arg Gly Thr Lys Ile Tyr Pro Gly Glu Asn Val
50 55 60
Gly Arg Gly Gly Asp Asp Thr Leu Phe Ala Lys Ile Asp Gly Val Lys
65 70 75 80
Phe Glu Arg Lys Gly Arg Asp Lys Lys Gln Val Ser Val Tyr Ala Val
85 90 95
Ala Glu
<210> 3
<211> 94
<212> PRT
<213> L27 Protein
<220>
<223> B. subtilis
<400> 3 ,
Met Leu Arg Leu Asp Leu Gln Phe Phe Ala Ser Lys Lys Gly Val Gly
1 5 10 1'5
Ser Thr Lys Asn Gly Arg Asp Ser Glu Ala Lys Arg Leu Gly Ala Lys
20 25 30
Arg Ala Asp Gly Gln Phe Val Thr Gly Gly Ser Ile Leu Tyr Arg Gln
35 40 45
Arg Gly Thr Lys Ile Tyr Pro Gly Glu Asn Val Gly Arg Gly Gly Asp
50 55 60
Asp Thr Leu Phe Ala Lys Ile Asp Gly Thr Val Lys Phe Glu Arg Phe
65 70 75 80
Gly Arg Asp Arg Lys Lys Val Ser Val Tyr Pro Val Ala Gln
85 90
<210> 4
<211> 85
<212> PRT
<213> L27 Protein
<220>
<223> E. coli
<400> 4
4
CA 02432162 2003-06-13
WO 02/056013 PCT/USO1/48455
Met Ala His Lys Lys Ala Gly Gly Ser Thr Arg Asn Gly Arg Asp Ser
1 5 ' 10 15
Glu Ala Lys Arg Leu Gly Val Lys Arg Phe Gly Gly Glu Ser Val Leu
20 25 30
Ala Gly Ser Ile Ile Val Arg Gln Arg Gly Thr Lys Phe His Ala Gly
35 40 45
Ala Asn Val Gly Cys Gly Arg Asp His Thr Leu Phe Ala Lys Ala Asp
50 55 60
Gly Lys Val Lys Phe Glu Val Lys Gly Pro Lys Asn Arg Lys Phe Ile
65 70 75 80
Ser Ile Glu Ala Glu
<210> 5
<211> 85
<212> PRT
<213> L27 Protein .
<220>
<223> H. influenzae
<400> 5
Met Ala Thr Lys Lys Ala Gly Gly Ser Thr Arg Asn Gly Arg Asp Ser
1 5 10 15
Glu Ala Lys Arg Leu Gly Val Lys Arg Phe Gly Gly Glu Ser Val Leu
20 25 30
Ala Gly Ser Ile Ile Val Arg Gln Arg Gly Thr Lys Phe His Ala Gly
35 40 45
Asn Asn Val Gly Met Gly Arg Asp His Thr Leu Phe Ala Thr Ala Asp
50 55 60
Gly Lys Val Lys Phe Glu Val Lys Gly Glu Lys Ser Arg Lys Tyr Val
65 70 75 80
Val Ile Val Thr Glu
S
