CA2432739A1 - Oxazolidinone photoaffinity probes - Google Patents
Oxazolidinone photoaffinity probes Download PDFInfo
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- CA2432739A1 CA2432739A1 CA002432739A CA2432739A CA2432739A1 CA 2432739 A1 CA2432739 A1 CA 2432739A1 CA 002432739 A CA002432739 A CA 002432739A CA 2432739 A CA2432739 A CA 2432739A CA 2432739 A1 CA2432739 A1 CA 2432739A1
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- compound
- oxo
- oxazolidinyl
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
- C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
- C07D263/08—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
- C07D263/16—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D263/18—Oxygen atoms
- C07D263/20—Oxygen atoms attached in position 2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Abstract
Disclosed are novel compounds that are useful and effective as photoaffinity probes and methods of using oxazolidinone photoaffinity probes.
Description
OXAZOLIDINONE PHOTOAFFINITY PROBES
FIELD OF THE INVENTION
The present invention is directed, in part, to novel photolabile oxazolidinones and preparation thereof. The novel compounds can be used as photoaffinity probes within sensitive bacteria including both gram-positive and gram-negative bacteria and mammalian cells.
BACKGROUND OF THE INVENTION
A number of antibiotic compounds have been developed and shown to be effective in inhibiting translation. The antibiotics neomycin, thiostrepton, and hygromycin appear to inhibit translocation and occupation of the A site within a ribosome but have no effect on formation of the fMet-tRNA Met/ribosome translation complex nor on the peptide-bond synthesis which occurs in the absence of elongation factor P. Streptomycin, which causes misreading and also inhibits A-site binding, interacts with two sites on the 16S rRNA of the 30S subunit.
Lincomycin inhibits peptidyltransferase and occupation of the A-site. Erythromycin inhibits peptidyltransferase and destabilizes the peptidyl-tRNA/ribosome/mRNA complex.
A number of additional 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., Proc. Nat!. 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 andlor antibacterial agents and methods of using the same are highly desired.
The present invention is directed, inter alia, to novel oxazolidinone compounds and use of oxazolidinone compounds as photoaffinity probes. Compounds suspected of having antimicrobial and/or antibacterial activity can be used in intact cells and can be evaluated for a mechanism of action by using active and inactive enantiomers of oxazolidinone compounds as competitors for crosslinking to components within the cell. The compounds and methods of the present invention allow one skilled in the art to elucidate the mechanism of action of antibacterial andlor antimicrobial agents that are suspected of inhibiting protein translation. These and other aspects of the invention are described below.
SUMMARY OF THE INVENTION
The present invention is directed to novel compounds that are useful and effective as photoaffinity probes useful for, iyiter alia, identification of the oxazolidinone binding site within gram-positive and gram-negative bacteria. These compounds can also be used for aiding in the determination of oxazolidinone binding sites within mammalian cells.
The compounds of the present invention comprise Formula I shown below.
Ri / ~ R3 L
N
N3 ~ i7 ~
R O ~.~1 R
Formula I
wherein X and Y are, independently, F, H or CH3; Rl is H, F or I; R2 is H, F
or OH; Rl6 is H or F; R17 is H or F; R3 is H or C1-C8 alkyl; L is a bond or -OCHZC(=O); and Q is O . O
N
~O
~.~/ N ~ ~ \ O H a. ~ O H a.
' ~N or N N
Z Z Z
wherein R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S; or a pharmaceutically acceptable salt thereof.
Other compounds of the present invention comprise Formula II shown below.
Formula II
wherein X and Y are, independently, F, H or CH3; Rl is H, I or F; RZ is H, OH
or F; R16 is H or F;Rl7isHorF; andQis O O
N O
H 4. N\0 H 4 ~ /O
N , N or ~N N
Z Z Z
wherein R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S; or a pharmaceutically acceptable salt thereof.
Other compounds of the present invention comprise Formula III shown below.
Y
Rs X
FIELD OF THE INVENTION
The present invention is directed, in part, to novel photolabile oxazolidinones and preparation thereof. The novel compounds can be used as photoaffinity probes within sensitive bacteria including both gram-positive and gram-negative bacteria and mammalian cells.
BACKGROUND OF THE INVENTION
A number of antibiotic compounds have been developed and shown to be effective in inhibiting translation. The antibiotics neomycin, thiostrepton, and hygromycin appear to inhibit translocation and occupation of the A site within a ribosome but have no effect on formation of the fMet-tRNA Met/ribosome translation complex nor on the peptide-bond synthesis which occurs in the absence of elongation factor P. Streptomycin, which causes misreading and also inhibits A-site binding, interacts with two sites on the 16S rRNA of the 30S subunit.
Lincomycin inhibits peptidyltransferase and occupation of the A-site. Erythromycin inhibits peptidyltransferase and destabilizes the peptidyl-tRNA/ribosome/mRNA complex.
A number of additional 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., Proc. Nat!. 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 andlor antibacterial agents and methods of using the same are highly desired.
The present invention is directed, inter alia, to novel oxazolidinone compounds and use of oxazolidinone compounds as photoaffinity probes. Compounds suspected of having antimicrobial and/or antibacterial activity can be used in intact cells and can be evaluated for a mechanism of action by using active and inactive enantiomers of oxazolidinone compounds as competitors for crosslinking to components within the cell. The compounds and methods of the present invention allow one skilled in the art to elucidate the mechanism of action of antibacterial andlor antimicrobial agents that are suspected of inhibiting protein translation. These and other aspects of the invention are described below.
SUMMARY OF THE INVENTION
The present invention is directed to novel compounds that are useful and effective as photoaffinity probes useful for, iyiter alia, identification of the oxazolidinone binding site within gram-positive and gram-negative bacteria. These compounds can also be used for aiding in the determination of oxazolidinone binding sites within mammalian cells.
The compounds of the present invention comprise Formula I shown below.
Ri / ~ R3 L
N
N3 ~ i7 ~
R O ~.~1 R
Formula I
wherein X and Y are, independently, F, H or CH3; Rl is H, F or I; R2 is H, F
or OH; Rl6 is H or F; R17 is H or F; R3 is H or C1-C8 alkyl; L is a bond or -OCHZC(=O); and Q is O . O
N
~O
~.~/ N ~ ~ \ O H a. ~ O H a.
' ~N or N N
Z Z Z
wherein R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S; or a pharmaceutically acceptable salt thereof.
Other compounds of the present invention comprise Formula II shown below.
Formula II
wherein X and Y are, independently, F, H or CH3; Rl is H, I or F; RZ is H, OH
or F; R16 is H or F;Rl7isHorF; andQis O O
N O
H 4. N\0 H 4 ~ /O
N , N or ~N N
Z Z Z
wherein R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S; or a pharmaceutically acceptable salt thereof.
Other compounds of the present invention comprise Formula III shown below.
Y
Rs X
wherein X and Y are, independently, F; H or CH3; R5 is N~ I N
or I
R6 , R6 _ .
wherein RG is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, Ci-C4 dialkylamino, C1-C4 alkyl, nitrile, carboxamide, Cl-C4 alkoxy, Cl-C4 allcylthio, or Cl-Cø alkoxycarbonyl;
and P is O O
N
to N o s ~o 'o N R~ , N R7 or \ N ~ R~
Z Z Z
wherein Z is O or S; and R7 is R
I ' 1 . I or R ~ I ~N3 ~N3 R1 R2 17 wherein R1 is H, F or I; and R2 is H, F or OH; R16 is H or F; R17 is H or F;
or a pharmaceutically acceptable salt thereof.
In other embodiments of the invention, methods of using a compound comprising Formula IV as a photoaffinity probe are provided, wherein Formula IV is Rg / Rls Rlo L~ Y
N3 ~ 19 ~ ~ NI
R O ~T
R o~ ~1 Formula IV
wherein X and Y are; independently, F, H or CH3; Rg is H, F or I; R~ is H, F
or OH; Rl8 is H or F; Rl~ is H or F; Rl° is H or C1-Cg alkyl; L is a bond or -OCH2C(=O);
and Q is O O
N o r 'o 'o H Rll H R11 ~ N N Rll ~N~ ' ~N~ or Z Z Z
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, methods of using a compound comprising Formula V as a photoaffinity probe are provided, wherein Formula V is Y
. X
Q
Formula V
wherein X and Y are, independently, F, H or CH3; R12 is N3 or ~3 \
wherein Rg is H, I or F; R~ is H, OH or F; Rlg is H or F; R19 is H or F; and Q
is O O
N
N o ~ 'o 'o ~ N Rll' N R11 or \ N N R11 Z Z Z
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, methods of using a compound comprising Formula VI as a photoaffinity probe are provided, wherein Formula VI is Y
X ~ P
Formula VI
wherein X and Y are, independently, F, H or CH3; R13 is N ,. I N
or wherein Rlø is H, N3, halogen, NHZ, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, Cl-C4 alkyl, nitrite, carboxamide, Cl-C4 alkoxy, Cl-C4 alkylthio, ox Cl-C4 alkoxycarbonyl; and P is O O
N I
N o ~ 'o 'o R1s' N R1s or ~ N H R1s ~ ~ ~ ~ \ 'N
Z Z Z
wherein: Z is O or S; and R15 is R1g s 18 R8 R9 N N3 R1g R
' 19 . ~ or R ~ I ~N
~N3 Rg R9 R19 wherein R$ is H, F or I; and R~ is H, F or OH; Rl$ is H or F; Rl~ is H or F;
or a pharmaceutically acceptable salt thereof.
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.
As used herein, the term "cross-linking" ox "binding" means the physical interaction between the photoaffinity probe and at least one component 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, indirect being 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 "component" within a cell means any protein, nucleic acid, lipid, etc. within a cell. Components include, but are not limited to, the contents of the cytoplasm, nucleus, cell membrane, cell wall, and the like.
or I
R6 , R6 _ .
wherein RG is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, Ci-C4 dialkylamino, C1-C4 alkyl, nitrile, carboxamide, Cl-C4 alkoxy, Cl-C4 allcylthio, or Cl-Cø alkoxycarbonyl;
and P is O O
N
to N o s ~o 'o N R~ , N R7 or \ N ~ R~
Z Z Z
wherein Z is O or S; and R7 is R
I ' 1 . I or R ~ I ~N3 ~N3 R1 R2 17 wherein R1 is H, F or I; and R2 is H, F or OH; R16 is H or F; R17 is H or F;
or a pharmaceutically acceptable salt thereof.
In other embodiments of the invention, methods of using a compound comprising Formula IV as a photoaffinity probe are provided, wherein Formula IV is Rg / Rls Rlo L~ Y
N3 ~ 19 ~ ~ NI
R O ~T
R o~ ~1 Formula IV
wherein X and Y are; independently, F, H or CH3; Rg is H, F or I; R~ is H, F
or OH; Rl8 is H or F; Rl~ is H or F; Rl° is H or C1-Cg alkyl; L is a bond or -OCH2C(=O);
and Q is O O
N o r 'o 'o H Rll H R11 ~ N N Rll ~N~ ' ~N~ or Z Z Z
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, methods of using a compound comprising Formula V as a photoaffinity probe are provided, wherein Formula V is Y
. X
Q
Formula V
wherein X and Y are, independently, F, H or CH3; R12 is N3 or ~3 \
wherein Rg is H, I or F; R~ is H, OH or F; Rlg is H or F; R19 is H or F; and Q
is O O
N
N o ~ 'o 'o ~ N Rll' N R11 or \ N N R11 Z Z Z
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, methods of using a compound comprising Formula VI as a photoaffinity probe are provided, wherein Formula VI is Y
X ~ P
Formula VI
wherein X and Y are, independently, F, H or CH3; R13 is N ,. I N
or wherein Rlø is H, N3, halogen, NHZ, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, Cl-C4 alkyl, nitrite, carboxamide, Cl-C4 alkoxy, Cl-C4 alkylthio, ox Cl-C4 alkoxycarbonyl; and P is O O
N I
N o ~ 'o 'o R1s' N R1s or ~ N H R1s ~ ~ ~ ~ \ 'N
Z Z Z
wherein: Z is O or S; and R15 is R1g s 18 R8 R9 N N3 R1g R
' 19 . ~ or R ~ I ~N
~N3 Rg R9 R19 wherein R$ is H, F or I; and R~ is H, F or OH; Rl$ is H or F; Rl~ is H or F;
or a pharmaceutically acceptable salt thereof.
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.
As used herein, the term "cross-linking" ox "binding" means the physical interaction between the photoaffinity probe and at least one component 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, indirect being 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 "component" within a cell means any protein, nucleic acid, lipid, etc. within a cell. Components include, but are not limited to, the contents of the cytoplasm, nucleus, cell membrane, cell wall, and the like.
As used herein, the term "competitor compound" means any identifiable chemical or molecule, small molecule, peptide, protein, sugar, natural or synthetic, that is suspected (such as a test compound) to potentially interact with or compete with the photoaffinity probe for cross-linking to a component within a cell or from a cell.
As used herein, the term "contacting" means either direct or indirect, application of a photoaffinity compound or competitor compound within a cell, on or to a cell, or to components from a cell. The competitor compound or photoaffinity compound can be present within a buffer, salt, solution, etc.
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/US90/06220, PCT/US94/08904, PCT/US94/10582, PCT/LTS95/02972, PCT/LTS95/10992, PCT/US93/04850, PCT/LJS95/12751, PCT/LTS96/00718, PCT/US93103570, PCT/US93/09589, PCT/US96/05202, PCT/US97/03458, PCT/LTS96/12766, PCT/LTS97/01970, PCT/LTS96/14135, PCT/LTS96/19149, PCT/US96/17120, PCT/US98/09889, PCT/US98/13437;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.
The present invention is directed to novel photoaffinity probes comprising Formula I, Formula II, or Formula III. 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. The present invention is also directed to compositions comprising photoaffinity probes which comprise Formula I, Formula II, or Formula Ill, or a mixture thereof.
Preferred compounds of this invention have one radioactive element which is either 3H
(T3)a 355, or lzsl. It is understood, however, that the Formulas include all isotopic forms of the compounds depicted.
In some embodiments of the invention, compounds comprise Formula I, shown below.
L
N
N I
R O ~l R/ ~3 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. R2 is H, F or OH. R16 is H or F. R17 is H
or F. R3 is H or Cl-C8 alkyl. L is a bond or -OCHZC(=O). Q is O O
N O NCO
- , N 4 N 4 or Z 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 T 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-IZSI-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, compounds comprise Formula II, shown below.
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, I or F. Rz is H, OH or F. R16 is H or F. R17 is H
or F. Q is O O
O H a. ~ /O
\~N or ~N
Z
Z
wherein R4 is H, CH3, CHZCH3 or cyclopropyl. Z is O or S. Compounds comprising Formula II
also include pharmaceutically acceptable salts thereof.
Rz ,~
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-5-oxazolidinyl]methyl]acetamide.
In other embodiments of the invention, compounds comprise Formula III, shown below.
Y
Rs X ~ P
Formula nI
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 N .~ I N
or R6 6 _ R
wherein R~ is H, N3, halogen, NHz, OH, SH, Cl-C4 alkylamino, Cl-C4 dialkylamino, C1-C4 alkyl, nitrile, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl.
P is O O
N
N O ~ ~ ~O H 7 H
~ N R ~ N R or R~
Z Z Z
wherein Z is O or S. R7 is R
R16 Rl R2 N N3 R16 or \ I Rm . ~ \ \
~ ~ \ .. \ N3 R1~N3 Rl R2 Ri7 .
wherein Rl is H, F or I. R2 is H, F or OH. Rl~ is H or F. R17 is H or F.
Compounds comprising Formula III 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 III include, but are not limited to, (2E)-3-(4-azido-3-iodo-lzsl-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-lasl-benzamide, and N-(4-azidophenyl)-N'-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] 35S-thiourea.
The present invention is also directed to methods of using a compound having Formula I, II, III, IV, V, or VI (shown above) as a photoaffinity probe. Briefly, a cell, or components) thereof, is contacted with a photoaffinity probe comprising Formula I, II, III, IV, V, or VI.
Preferably, the photoaffinity probe is radiolabled. The photoaffinity probe is then exposed to light, preferably ultraviolet, in order to activate the photoaffinity probe.
Cross-linking of the photoaffinity probe is determined by methods well known to those skilled in the art such as, for example, by detecting the radiolabel. Radiolabel, such as 3H, lzsh or 355, for example, can be detected by a variety of autoradiography techniques well known to the slcilled artisan. In other embodiments of the invention, the method further comprises contacting the cell or components) thereof with a competitor compound. The ability of the competitor compound to interfere with cross-linking of the photoaffinity probe indicates the bioactivity of the competitor compound.
Cells of the present invention include, but are not limited to, gram positive bacterial pathogens, including, for example, Staphylococcus aureus; Staphylococcus epiden~aidis (A, B, C
biotypes); Staphylococcus caseolyticus; Staphylococcus gallinarum;
Staphylococcus laaemolyticus; Staphylococcus horninzs; Staphylococcus saprophyticus;
Streptococcus agalactiae (group B); Streptococcus mutafaslrattus; Streptococcus pneumof2iae;
Streptococcus pyogerzes (group A); Streptococcus salivarius; Streptococcus sanguis; Streptococcus sobrinus;
Actirzomyces spps.; Arthrobacter histidinolovorans; Corynebacterium diptheriae; Clostridium difficle; Clostridiunz spps.; Ezzterococcus casseliflavus; Enterococcus durans; Enterococcus faecalis; Enterococcus faecium; Enterococcus gallinarunz; Erysipelothrix rhusiopathiae;
Fusobacteriurn spps.; Listeria monocytogenes; Prevotella spps.;
Propionibacteriunz aches; and Porphyromonas gingivalis.
Cells also include, but are not limited to, gram negative bacterial pathogens, including, for example, Acinetobacter calcoaceticus; Acinetobacter haemolyticus;
Aeromonas hydroplzila;
Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica;
Bacteroides fragilis;
Bartonella bacilliforrrzis; Brucella abortus; Brucella melitensis;
Cafnpylobacter fetus;
Campylobacter jejuni; Clzlamydia pneunzoniae; Chlamydia psittaci; Clzlanzydia trachonzatis;
Citrobacter freundii; Coxiella burnetti; Edwardsiella tarda; Edwardsiella hoslzinae;
Enterobacter aerogenes, Enterobacter cloacae (groups A and B); Escherichia coli (to include all pathogenic subtypes) Elarlicia spps.; Francisella tularejzsis; Haemophilus actinomycetenzconzitaTZS; Haemophilus ducreyi; Haemoplzilus haemolyticus;
Haemophilus infZuenzae; Haenzophilus paralzaenzolyticus; Haemophilus parainfluenzae;
Hafnia alvei;
Helicobacter pylori; Kingella kingae; Klebsiella oxytoca; Klebsiella pneum.oniae; Legionella pneurnophila; Legionella spps.; Morgarzella spps.; Moraxella cattarhalis;
Neisseria gonorrlzoeae; Neisseria meningitidis; Plesiomonas shigelloides; Proteus tnirabilis; Proteus penneri; Providencia spps.; Pseudomofzas aeruginosa; Pseudomonas species;
Rickettsia prowazekii; Rickettsia rickettsii; Rickettsia tsutsugamuslzi; Rochalimaea spps.; Salmonella subgroup 1 serotypes (to include S. paratyphi and S. typhi); Salmonella subgroups 2, 3a, 3b, 4, and 5; Serratia marcesans; Serratia spps.; Slzigella boydii; Slzigella flexneri; Shigella dysenteriae; Slzigella son fzei; Yersinia enterocolitica; Yersinia pestis;
Yersinia pseudotuberculosis; Vibrio cholerae; Vibrio vulnificus; and Vibrio parahaenzolyticus.
Cells also include, but are not limited to, Mycobacterial species, including, for example, Mycobacterium tuberculosis; Mycobacterium aviunz; and other Mycobacterium spps.
Cells also include, but are not limited to, Mycoplasmas (or pleuropneumonia-like organisms), including, for example, Mycoplasnza genitalium; Mycoplasnza przeumoniae; and other Mycoplasma spps.
Cells also include, but are not limited to, Treponemataceae (spiral organisms) including, for example, Borrelia burgdozferi; other Borrelia species; Leptospira spps.;
Trepozzezzza pallidu>72.
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 (A1) with an appropriate hydroxyacetyl piperazine fragment (Az) leads to compounds A3 of Formula I
where L is -OCH2C(=O). Coupling can be accomplished with 1-[3-(dimethylamino)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. Chem., 1987, 65, 2450-2453; Shu, ,l. Labelled CompouzZds and Radiopharnzaceuticals, 1996, 38, 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 Synder, Int'1 Publication WO 00/10566-A1, each of which is incorporated herein by reference in its entirety).
Methods for incorporation of lzsl into compounds A3 are shown in Schemes C and D. Scheme A
can also be used where the compounds of Al and A3 have R16 and RI7 substituents ortho to the acid substituent (as in Formulas I and IV).
Scheme A:
OH HO~N~ Y O
~N
N~ Y
R2 N \ I N3 O vN /
s X Q I
\ O
X
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 (Bz) using l,l-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 lasI into compounds B3 are shown in Schemes C and D.
Scheme B can also be used where the compound of B3 has R~6 and R17 substituents ortho to the amide substituent (as in Formulas I and IV).
Scheme B:
HN~ Y
~N R1 / I N~ Y
A1 -i- / I --~ W ~N /
X ~ Q . Ns X ~ I Q
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 CI (prepared according to the methods of Schemes A
and B) with Nalasl and chloramine-T.
Scheme C:
HO O)~N~ Y HO U)~N~ Y
~ ~N ' I -~ / ~ ~N /
N3 X Q N3 125 X \ Q
Alternatively, compounds D3 of Formulas I and IV (where R1 is H and R2 is lasI) 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 DZ
with Nalasl and chloramine-T leads to D3.
Scheme D:
H (~).N~ Y H U).N~ Y
~ ~ IN / ~ / ' ~ IN ~~~ ~
N I X ~ I Q N3 SnMe3 X~O
p ~2 O
H ~~)~N~ Y
IN /
N3 1251 p3 X
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, E2, and E3 have R16 and R17 or Rl$ and Rl~ substituents in the ortho position (as in Formulas II and V).
Scheme E:
~2N / I Y H2N / I Y N3 / I Y
R, ~ ~ '--~ R1 _ ~ I -~ R' X Q X Q X Q
E~ E2 E3 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, FZ, and F3 have R16 and R17 or Rl$ and R19 substituents in the ortho position (as in Formulas II and V).
Scheme F:
N3 ~ I Y N ~ I Y
Ri \ I ~ Ri X N O H X N O
F1 ~N~CH3 F2 ~NH2 O
Y
Ns O
R1 Ir X \ N~O
Fa ~N~CT3 O
Schema G shows the method for incorporation of 35S into compounds of Formulas II
and V where Q is oxazolidinone, Z is S, and R4 is CH3. Reaction of F2 (from Scheme F) with ethyl 35S-dithioacetate affords the 35S-thioacetamide, GZ, Incorporation of 35S 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 R17 or Rl8 and Rl~ substituents in the ortho position (as in Formulas II and V).
Scheme G
Y
F2 --~ N3 I
/ OII
R1 \
X N~O
~N~CH3 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 H2. Reaction of H2 with Nalzsl and chloramine-T affords the radioiodinated compound H3.
Scheme H:
Me3Sn Y
Y Y N
N3 \ ~ -~ N3 \ I --.~ 3 \ /
/I /I \I
X \ Q X \ (~ X Q
Compounds of Formula V where R12 is N3 are made according to the procedures described in U.S. Patent No. 5,910,504, Example 17, 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 R7 is optionally substituted azidophenyl or azidocinnamoyl. Refluxing an appropriate acetamide fragment (h) in methanolic hydrochloric acid affords the free amine I2. 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 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 Compounds and Radioplvarmaceuticals, 1996, 38, 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 I4. have R16 and R17 or Rl8 and Rl~
substituents in the ortho position (as in Formulas III and VI).
Scheme I:
R / I O -~ ~ I ~ + HO~ _ X N~O X \ N O n I_ i-H-R2 ~N ~NH2 Ns 11 O 12 ~3 Y
O
II R
X ~ N~O H Ns ~--~N
O n R2 ~4 Introduction of lasl into compounds of Formulas III and VI where P is oxazolidinone, Z
is O, and R7 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 Nalzsl and chloramine-T affords the radioiodinated compound J2. Introduction of laslinto compounds of Formulas III and VI where P is isoxazoline or isoxazolinone is carried out by similar methods.
Y
R' Scheme J:
izsI
X /
Ns v n OH
Jz O
Alternatively, introduction of lzsl into compounds of Formulas III and VI
where P is oxazolidinone, Z is O, and R7 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 I~2. Reaction of I~2 with Nalasl and chloramine-T affords the radioiodinated compound K3. Radioiodination of compounds of Formulas III and VI where P is isoxazoline or isoxazolinone is carried out in similar fashion.
Scheme I~:
Rs I4 --~ nMe3 / I 0 i2sl X N o /
~N \ \ .
v v in Scheme L illustrates a synthetic method for the preparation of radioactive compounds of Formulas DI and VI where P is oxazolidinone, Z is 355, and R7 is optionally substituted azidoaniline. An appropriate 35S-isothiocyanate I~ is reacted with the appropriate aminomethyl fragment IZ (Scheme I) in refluxing THF to give the desired 35S-thiourea, (L3). The required 355-isothiocyanate Lz, is prepared by reaction of an appropriate aniline with 355-thiophosgene.
Introduction of 35S into compounds of Formulas DI and VI where P is isoxazoline or isoxazolinone is can-ied out in similar fashion. Scheme L can also be used where the compounds of Lz, and L3 have Ri6 and R17 or Rl8 and R19 substituents in the ortho position (as in Formulas III
and VI).
Scheme L:
Y
R N=C_35S / I O
I ~ X N O H N R2 N R2 ~N~ /
3 355 i I
L2 Ls R1 N3 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-lasI-benzoate (Compound CZ 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.
i25~ ~ ~ ~~N~
O ~IN
/I O
F ~ N~O H
~N~CN3 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 CH2C12 (100 ml) and is washed successively with H20 (2 x 30 ml), 1 N HCl (2 x 30 ml), saturated NaHC03 (1 x 30 ml), dried (MgSO4), 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 % CH3OH in CH2C12 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, 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 ~l of a 1 mM stock solution), and the azido phenol of Step 1 (10 ~1 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 lasI2 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 ~1). 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 1zs Iz 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 I2 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~ ~~ I O
F' v \
~H
'--~N~
I IO
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 CH2C12 (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 (MgS04), filtered and concentrated. The residue is dissolved in CH30H/CH2Cl2, 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.
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-lzsI-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) Ns 125 ~ ~ N
O ~N
O
N~O
~H
'~N~
j(0 Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid (103.8 mg, 0.36 mmol, (Shu, J. of Labelled Cos~apoufzds ayzd Radiopha~naceuticals, 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 CH2Cl2 (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 (CDCl3) 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) 8: 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, I.83, 0.33.
Step 3. To a stirred solution of the stannane prepared in Step 2 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 radioiodinated material.
Example 4: Synthesis N-[[(5S)-3-[4-[4-(4-Azido-3-iodo-l2sI-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) / I N'1 Ns w ~N / I
F~N O
~--~N~
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 CHZC12 (40 ml) and successively washed with H20 (20 ml), 1 N HCl (20 ml), saturated aqueous NaHCO3 (20 ml), brine (20 ml), dried (MgS04), filtered and concentrated. The residue is dissolved in CHZCl2, absorbed onto silica gel and is purified on a Biotage 40S column with SIM using 2.5 % CH30H in CHZCl2 as the eluent to afford 376.7 mg (0.62 mmol) of the benzamide as a yellow solid. 1H-NMR (DMSO) 8: 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 SIM using 2 % CH30H in 49% CH2C12 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 added a solution of 1M aqueous NalasI followed by chloramine -T hydrate. After stirnng at room temperature for 30 minutes, the reaction mixture is quenched with saturated aqueous NaZS203 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
N3 ~ ~ ~ ~ N /O N CT3 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(ll) (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 HZO (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 CH2C12 to 100 % EtOAC as the eluent to afford 3.74 g (10.0 mmol, 65%) of the desired nitrobiphenyl compound. 1H-NMR (DMSO) 8: 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), CH30H (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 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 HZO (1.5 ml). The reaction mixture is stirred at 0 °C for 45 minutes, then diluted with CH2C12 (200 ml). The phases are separated. The aqueous phase is extracted with CH2C12 (75 ml). The combine organic phases are dried (MgSOø), filtered and concentrated. The residue is dissolved in CH30H/CHZCl2, absorbed onto silica gel and is purified on a Biotage 40S column with SIM
using 10% CH30H in CH2C12 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 ~,mol, 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 wmol of 0.45M
O-Benzotriazol-I-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 ~.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-[ [ (5 S )-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, RZ is I, X is F, and 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 CHZCl2 as the eluent to afford 4~.4 mg (0.10 mmol, 12%) of the desired iodoaniline as a yellow oil. iH-NMR
(CH30D) &: 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 CH30H (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: 8.2T, 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) 8: 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 CHZCl2 (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 H20 and CH2C12. 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 °Io) of desired tritiated acetamide.
Example 7: Synthesis N-[ [ (5 S )-3-(4' -Azido-2-fluoro-3' -iodo [ 1,1' -biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]
ethane-35S-thioamide (Compound GZ of Scheme G where Rlis H, RZ is I, X is F, and Y is H) O
N3 ~ ~ ~ ~ N~O N
35~
Step 1. Methylmagnesium chloride in tetrahydrofuran (THF) is treated with 35S
labeled carbon disulfide at 40 °C, followed by treatment with ethyl iodide. The reaction is stirred at 60 °C
for 1.5 hours. After worlcup with water and ethyl ether, the desired ethyl 35S-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 35S labeled thioamide.
Example 8: Synthesis N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo-lasl-[ 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) _ _ N3 ~ ~ ~ ~ ~N
O
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 (CDCl3) 8: 7.53, 7.43, 7.30, 7.21, 6.07, 4.83, 4.11, 3.83, 3.73, 2.05, 0.35.
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-lzsl-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-propenamide (Compound Iø of Scheme I where Rs is 4-pyridyl, X is F, Y is H, n is l, Rl is lash and R2 is H) Ns Nv / ~ / ~N W
~/~/\ X125 O
Step 1. To a stirred solution of oxalyl chloride (0.10 mL, 1.2 mmol) in CH2Cl2 (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 and Radiopharzrzaceuticals, 1996, 3~, 227-237)) in CH2C12 (2.5 ml) is added. After 15 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 CH2C12 (30 ml) and washed successively with H20 (20 ml), brine (20 ml), dried (MgSOø), 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 CH2C12 (40 ml) and successively washed with H20 (20 ml), brine (20 ml), dried (MgSO4), filtered and concentrated. The residue is dissolved in CH2C12, 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) 8: 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]
acetamide (1.40 g, 4.25 mrnol) 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 CHZC12 (40 ml) and washed with H20 (20 ml), brine (20 ml), dried (MgS04), filtered and concentrated. The residue is dissolved in CH30H/CH2C12, absorbed onto silica gel and is purified on a Biotage 12M column with S1M
using 2 % CH30H
(saturated with NH3) in CHZC12 as the eluent to afford 56.2 mg (0.096 mmol, 51%) of the desired cinnamide. 1H-NMR (DMSO) 8: 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 NalzsI 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 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 J2 of Scheme J where RS is 4-pyridyl, X is F, and Y
is H, and n is 0) Nv l ~ ~ ~N \
F 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 CHZC12 (1 ml) is added 4-azidosalicylic acid (128.9 mg 0.72 mmol) followed by added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide 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/CHZC12, 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) b: 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, lzsl 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-oxazolidiriyl]
methyl] 35S-thiourea (Compound L3 of Scheme L where RS is 4-pyridyl, X is F, Y
is H, Rl is H
and R2 is H) N~ ~ ~ ~ N O H H
~--~N N
F 35S \ I N
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.
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.
As used herein, the term "contacting" means either direct or indirect, application of a photoaffinity compound or competitor compound within a cell, on or to a cell, or to components from a cell. The competitor compound or photoaffinity compound can be present within a buffer, salt, solution, etc.
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/US90/06220, PCT/US94/08904, PCT/US94/10582, PCT/LTS95/02972, PCT/LTS95/10992, PCT/US93/04850, PCT/LJS95/12751, PCT/LTS96/00718, PCT/US93103570, PCT/US93/09589, PCT/US96/05202, PCT/US97/03458, PCT/LTS96/12766, PCT/LTS97/01970, PCT/LTS96/14135, PCT/LTS96/19149, PCT/US96/17120, PCT/US98/09889, PCT/US98/13437;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.
The present invention is directed to novel photoaffinity probes comprising Formula I, Formula II, or Formula III. 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. The present invention is also directed to compositions comprising photoaffinity probes which comprise Formula I, Formula II, or Formula Ill, or a mixture thereof.
Preferred compounds of this invention have one radioactive element which is either 3H
(T3)a 355, or lzsl. It is understood, however, that the Formulas include all isotopic forms of the compounds depicted.
In some embodiments of the invention, compounds comprise Formula I, shown below.
L
N
N I
R O ~l R/ ~3 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. R2 is H, F or OH. R16 is H or F. R17 is H
or F. R3 is H or Cl-C8 alkyl. L is a bond or -OCHZC(=O). Q is O O
N O NCO
- , N 4 N 4 or Z 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 T 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-IZSI-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, compounds comprise Formula II, shown below.
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, I or F. Rz is H, OH or F. R16 is H or F. R17 is H
or F. Q is O O
O H a. ~ /O
\~N or ~N
Z
Z
wherein R4 is H, CH3, CHZCH3 or cyclopropyl. Z is O or S. Compounds comprising Formula II
also include pharmaceutically acceptable salts thereof.
Rz ,~
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-5-oxazolidinyl]methyl]acetamide.
In other embodiments of the invention, compounds comprise Formula III, shown below.
Y
Rs X ~ P
Formula nI
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 N .~ I N
or R6 6 _ R
wherein R~ is H, N3, halogen, NHz, OH, SH, Cl-C4 alkylamino, Cl-C4 dialkylamino, C1-C4 alkyl, nitrile, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl.
P is O O
N
N O ~ ~ ~O H 7 H
~ N R ~ N R or R~
Z Z Z
wherein Z is O or S. R7 is R
R16 Rl R2 N N3 R16 or \ I Rm . ~ \ \
~ ~ \ .. \ N3 R1~N3 Rl R2 Ri7 .
wherein Rl is H, F or I. R2 is H, F or OH. Rl~ is H or F. R17 is H or F.
Compounds comprising Formula III 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 III include, but are not limited to, (2E)-3-(4-azido-3-iodo-lzsl-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-lasl-benzamide, and N-(4-azidophenyl)-N'-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] 35S-thiourea.
The present invention is also directed to methods of using a compound having Formula I, II, III, IV, V, or VI (shown above) as a photoaffinity probe. Briefly, a cell, or components) thereof, is contacted with a photoaffinity probe comprising Formula I, II, III, IV, V, or VI.
Preferably, the photoaffinity probe is radiolabled. The photoaffinity probe is then exposed to light, preferably ultraviolet, in order to activate the photoaffinity probe.
Cross-linking of the photoaffinity probe is determined by methods well known to those skilled in the art such as, for example, by detecting the radiolabel. Radiolabel, such as 3H, lzsh or 355, for example, can be detected by a variety of autoradiography techniques well known to the slcilled artisan. In other embodiments of the invention, the method further comprises contacting the cell or components) thereof with a competitor compound. The ability of the competitor compound to interfere with cross-linking of the photoaffinity probe indicates the bioactivity of the competitor compound.
Cells of the present invention include, but are not limited to, gram positive bacterial pathogens, including, for example, Staphylococcus aureus; Staphylococcus epiden~aidis (A, B, C
biotypes); Staphylococcus caseolyticus; Staphylococcus gallinarum;
Staphylococcus laaemolyticus; Staphylococcus horninzs; Staphylococcus saprophyticus;
Streptococcus agalactiae (group B); Streptococcus mutafaslrattus; Streptococcus pneumof2iae;
Streptococcus pyogerzes (group A); Streptococcus salivarius; Streptococcus sanguis; Streptococcus sobrinus;
Actirzomyces spps.; Arthrobacter histidinolovorans; Corynebacterium diptheriae; Clostridium difficle; Clostridiunz spps.; Ezzterococcus casseliflavus; Enterococcus durans; Enterococcus faecalis; Enterococcus faecium; Enterococcus gallinarunz; Erysipelothrix rhusiopathiae;
Fusobacteriurn spps.; Listeria monocytogenes; Prevotella spps.;
Propionibacteriunz aches; and Porphyromonas gingivalis.
Cells also include, but are not limited to, gram negative bacterial pathogens, including, for example, Acinetobacter calcoaceticus; Acinetobacter haemolyticus;
Aeromonas hydroplzila;
Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica;
Bacteroides fragilis;
Bartonella bacilliforrrzis; Brucella abortus; Brucella melitensis;
Cafnpylobacter fetus;
Campylobacter jejuni; Clzlamydia pneunzoniae; Chlamydia psittaci; Clzlanzydia trachonzatis;
Citrobacter freundii; Coxiella burnetti; Edwardsiella tarda; Edwardsiella hoslzinae;
Enterobacter aerogenes, Enterobacter cloacae (groups A and B); Escherichia coli (to include all pathogenic subtypes) Elarlicia spps.; Francisella tularejzsis; Haemophilus actinomycetenzconzitaTZS; Haemophilus ducreyi; Haemoplzilus haemolyticus;
Haemophilus infZuenzae; Haenzophilus paralzaenzolyticus; Haemophilus parainfluenzae;
Hafnia alvei;
Helicobacter pylori; Kingella kingae; Klebsiella oxytoca; Klebsiella pneum.oniae; Legionella pneurnophila; Legionella spps.; Morgarzella spps.; Moraxella cattarhalis;
Neisseria gonorrlzoeae; Neisseria meningitidis; Plesiomonas shigelloides; Proteus tnirabilis; Proteus penneri; Providencia spps.; Pseudomofzas aeruginosa; Pseudomonas species;
Rickettsia prowazekii; Rickettsia rickettsii; Rickettsia tsutsugamuslzi; Rochalimaea spps.; Salmonella subgroup 1 serotypes (to include S. paratyphi and S. typhi); Salmonella subgroups 2, 3a, 3b, 4, and 5; Serratia marcesans; Serratia spps.; Slzigella boydii; Slzigella flexneri; Shigella dysenteriae; Slzigella son fzei; Yersinia enterocolitica; Yersinia pestis;
Yersinia pseudotuberculosis; Vibrio cholerae; Vibrio vulnificus; and Vibrio parahaenzolyticus.
Cells also include, but are not limited to, Mycobacterial species, including, for example, Mycobacterium tuberculosis; Mycobacterium aviunz; and other Mycobacterium spps.
Cells also include, but are not limited to, Mycoplasmas (or pleuropneumonia-like organisms), including, for example, Mycoplasnza genitalium; Mycoplasnza przeumoniae; and other Mycoplasma spps.
Cells also include, but are not limited to, Treponemataceae (spiral organisms) including, for example, Borrelia burgdozferi; other Borrelia species; Leptospira spps.;
Trepozzezzza pallidu>72.
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 (A1) with an appropriate hydroxyacetyl piperazine fragment (Az) leads to compounds A3 of Formula I
where L is -OCH2C(=O). Coupling can be accomplished with 1-[3-(dimethylamino)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. Chem., 1987, 65, 2450-2453; Shu, ,l. Labelled CompouzZds and Radiopharnzaceuticals, 1996, 38, 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 Synder, Int'1 Publication WO 00/10566-A1, each of which is incorporated herein by reference in its entirety).
Methods for incorporation of lzsl into compounds A3 are shown in Schemes C and D. Scheme A
can also be used where the compounds of Al and A3 have R16 and RI7 substituents ortho to the acid substituent (as in Formulas I and IV).
Scheme A:
OH HO~N~ Y O
~N
N~ Y
R2 N \ I N3 O vN /
s X Q I
\ O
X
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 (Bz) using l,l-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 lasI into compounds B3 are shown in Schemes C and D.
Scheme B can also be used where the compound of B3 has R~6 and R17 substituents ortho to the amide substituent (as in Formulas I and IV).
Scheme B:
HN~ Y
~N R1 / I N~ Y
A1 -i- / I --~ W ~N /
X ~ Q . Ns X ~ I Q
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 CI (prepared according to the methods of Schemes A
and B) with Nalasl and chloramine-T.
Scheme C:
HO O)~N~ Y HO U)~N~ Y
~ ~N ' I -~ / ~ ~N /
N3 X Q N3 125 X \ Q
Alternatively, compounds D3 of Formulas I and IV (where R1 is H and R2 is lasI) 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 DZ
with Nalasl and chloramine-T leads to D3.
Scheme D:
H (~).N~ Y H U).N~ Y
~ ~ IN / ~ / ' ~ IN ~~~ ~
N I X ~ I Q N3 SnMe3 X~O
p ~2 O
H ~~)~N~ Y
IN /
N3 1251 p3 X
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, E2, and E3 have R16 and R17 or Rl$ and Rl~ substituents in the ortho position (as in Formulas II and V).
Scheme E:
~2N / I Y H2N / I Y N3 / I Y
R, ~ ~ '--~ R1 _ ~ I -~ R' X Q X Q X Q
E~ E2 E3 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, FZ, and F3 have R16 and R17 or Rl$ and R19 substituents in the ortho position (as in Formulas II and V).
Scheme F:
N3 ~ I Y N ~ I Y
Ri \ I ~ Ri X N O H X N O
F1 ~N~CH3 F2 ~NH2 O
Y
Ns O
R1 Ir X \ N~O
Fa ~N~CT3 O
Schema G shows the method for incorporation of 35S into compounds of Formulas II
and V where Q is oxazolidinone, Z is S, and R4 is CH3. Reaction of F2 (from Scheme F) with ethyl 35S-dithioacetate affords the 35S-thioacetamide, GZ, Incorporation of 35S 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 R17 or Rl8 and Rl~ substituents in the ortho position (as in Formulas II and V).
Scheme G
Y
F2 --~ N3 I
/ OII
R1 \
X N~O
~N~CH3 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 H2. Reaction of H2 with Nalzsl and chloramine-T affords the radioiodinated compound H3.
Scheme H:
Me3Sn Y
Y Y N
N3 \ ~ -~ N3 \ I --.~ 3 \ /
/I /I \I
X \ Q X \ (~ X Q
Compounds of Formula V where R12 is N3 are made according to the procedures described in U.S. Patent No. 5,910,504, Example 17, 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 R7 is optionally substituted azidophenyl or azidocinnamoyl. Refluxing an appropriate acetamide fragment (h) in methanolic hydrochloric acid affords the free amine I2. 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 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 Compounds and Radioplvarmaceuticals, 1996, 38, 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 I4. have R16 and R17 or Rl8 and Rl~
substituents in the ortho position (as in Formulas III and VI).
Scheme I:
R / I O -~ ~ I ~ + HO~ _ X N~O X \ N O n I_ i-H-R2 ~N ~NH2 Ns 11 O 12 ~3 Y
O
II R
X ~ N~O H Ns ~--~N
O n R2 ~4 Introduction of lasl into compounds of Formulas III and VI where P is oxazolidinone, Z
is O, and R7 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 Nalzsl and chloramine-T affords the radioiodinated compound J2. Introduction of laslinto compounds of Formulas III and VI where P is isoxazoline or isoxazolinone is carried out by similar methods.
Y
R' Scheme J:
izsI
X /
Ns v n OH
Jz O
Alternatively, introduction of lzsl into compounds of Formulas III and VI
where P is oxazolidinone, Z is O, and R7 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 I~2. Reaction of I~2 with Nalasl and chloramine-T affords the radioiodinated compound K3. Radioiodination of compounds of Formulas III and VI where P is isoxazoline or isoxazolinone is carried out in similar fashion.
Scheme I~:
Rs I4 --~ nMe3 / I 0 i2sl X N o /
~N \ \ .
v v in Scheme L illustrates a synthetic method for the preparation of radioactive compounds of Formulas DI and VI where P is oxazolidinone, Z is 355, and R7 is optionally substituted azidoaniline. An appropriate 35S-isothiocyanate I~ is reacted with the appropriate aminomethyl fragment IZ (Scheme I) in refluxing THF to give the desired 35S-thiourea, (L3). The required 355-isothiocyanate Lz, is prepared by reaction of an appropriate aniline with 355-thiophosgene.
Introduction of 35S into compounds of Formulas DI and VI where P is isoxazoline or isoxazolinone is can-ied out in similar fashion. Scheme L can also be used where the compounds of Lz, and L3 have Ri6 and R17 or Rl8 and R19 substituents in the ortho position (as in Formulas III
and VI).
Scheme L:
Y
R N=C_35S / I O
I ~ X N O H N R2 N R2 ~N~ /
3 355 i I
L2 Ls R1 N3 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-lasI-benzoate (Compound CZ 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.
i25~ ~ ~ ~~N~
O ~IN
/I O
F ~ N~O H
~N~CN3 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 CH2C12 (100 ml) and is washed successively with H20 (2 x 30 ml), 1 N HCl (2 x 30 ml), saturated NaHC03 (1 x 30 ml), dried (MgSO4), 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 % CH3OH in CH2C12 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, 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 ~l of a 1 mM stock solution), and the azido phenol of Step 1 (10 ~1 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 lasI2 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 ~1). 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 1zs Iz 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 I2 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~ ~~ I O
F' v \
~H
'--~N~
I IO
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 CH2C12 (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 (MgS04), filtered and concentrated. The residue is dissolved in CH30H/CH2Cl2, 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.
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-lzsI-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) Ns 125 ~ ~ N
O ~N
O
N~O
~H
'~N~
j(0 Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid (103.8 mg, 0.36 mmol, (Shu, J. of Labelled Cos~apoufzds ayzd Radiopha~naceuticals, 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 CH2Cl2 (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 (CDCl3) 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) 8: 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, I.83, 0.33.
Step 3. To a stirred solution of the stannane prepared in Step 2 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 radioiodinated material.
Example 4: Synthesis N-[[(5S)-3-[4-[4-(4-Azido-3-iodo-l2sI-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) / I N'1 Ns w ~N / I
F~N O
~--~N~
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 CHZC12 (40 ml) and successively washed with H20 (20 ml), 1 N HCl (20 ml), saturated aqueous NaHCO3 (20 ml), brine (20 ml), dried (MgS04), filtered and concentrated. The residue is dissolved in CHZCl2, absorbed onto silica gel and is purified on a Biotage 40S column with SIM using 2.5 % CH30H in CHZCl2 as the eluent to afford 376.7 mg (0.62 mmol) of the benzamide as a yellow solid. 1H-NMR (DMSO) 8: 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 SIM using 2 % CH30H in 49% CH2C12 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 added a solution of 1M aqueous NalasI followed by chloramine -T hydrate. After stirnng at room temperature for 30 minutes, the reaction mixture is quenched with saturated aqueous NaZS203 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
N3 ~ ~ ~ ~ N /O N CT3 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(ll) (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 HZO (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 CH2C12 to 100 % EtOAC as the eluent to afford 3.74 g (10.0 mmol, 65%) of the desired nitrobiphenyl compound. 1H-NMR (DMSO) 8: 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), CH30H (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 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 HZO (1.5 ml). The reaction mixture is stirred at 0 °C for 45 minutes, then diluted with CH2C12 (200 ml). The phases are separated. The aqueous phase is extracted with CH2C12 (75 ml). The combine organic phases are dried (MgSOø), filtered and concentrated. The residue is dissolved in CH30H/CHZCl2, absorbed onto silica gel and is purified on a Biotage 40S column with SIM
using 10% CH30H in CH2C12 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 ~,mol, 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 wmol of 0.45M
O-Benzotriazol-I-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 ~.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-[ [ (5 S )-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, RZ is I, X is F, and 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 CHZCl2 as the eluent to afford 4~.4 mg (0.10 mmol, 12%) of the desired iodoaniline as a yellow oil. iH-NMR
(CH30D) &: 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 CH30H (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: 8.2T, 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) 8: 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 CHZCl2 (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 H20 and CH2C12. 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 °Io) of desired tritiated acetamide.
Example 7: Synthesis N-[ [ (5 S )-3-(4' -Azido-2-fluoro-3' -iodo [ 1,1' -biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]
ethane-35S-thioamide (Compound GZ of Scheme G where Rlis H, RZ is I, X is F, and Y is H) O
N3 ~ ~ ~ ~ N~O N
35~
Step 1. Methylmagnesium chloride in tetrahydrofuran (THF) is treated with 35S
labeled carbon disulfide at 40 °C, followed by treatment with ethyl iodide. The reaction is stirred at 60 °C
for 1.5 hours. After worlcup with water and ethyl ether, the desired ethyl 35S-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 35S labeled thioamide.
Example 8: Synthesis N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo-lasl-[ 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) _ _ N3 ~ ~ ~ ~ ~N
O
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 (CDCl3) 8: 7.53, 7.43, 7.30, 7.21, 6.07, 4.83, 4.11, 3.83, 3.73, 2.05, 0.35.
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-lzsl-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-propenamide (Compound Iø of Scheme I where Rs is 4-pyridyl, X is F, Y is H, n is l, Rl is lash and R2 is H) Ns Nv / ~ / ~N W
~/~/\ X125 O
Step 1. To a stirred solution of oxalyl chloride (0.10 mL, 1.2 mmol) in CH2Cl2 (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 and Radiopharzrzaceuticals, 1996, 3~, 227-237)) in CH2C12 (2.5 ml) is added. After 15 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 CH2C12 (30 ml) and washed successively with H20 (20 ml), brine (20 ml), dried (MgSOø), 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 CH2C12 (40 ml) and successively washed with H20 (20 ml), brine (20 ml), dried (MgSO4), filtered and concentrated. The residue is dissolved in CH2C12, 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) 8: 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]
acetamide (1.40 g, 4.25 mrnol) 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 CHZC12 (40 ml) and washed with H20 (20 ml), brine (20 ml), dried (MgS04), filtered and concentrated. The residue is dissolved in CH30H/CH2C12, absorbed onto silica gel and is purified on a Biotage 12M column with S1M
using 2 % CH30H
(saturated with NH3) in CHZC12 as the eluent to afford 56.2 mg (0.096 mmol, 51%) of the desired cinnamide. 1H-NMR (DMSO) 8: 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 NalzsI 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 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 J2 of Scheme J where RS is 4-pyridyl, X is F, and Y
is H, and n is 0) Nv l ~ ~ ~N \
F 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 CHZC12 (1 ml) is added 4-azidosalicylic acid (128.9 mg 0.72 mmol) followed by added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide 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/CHZC12, 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) b: 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, lzsl 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-oxazolidiriyl]
methyl] 35S-thiourea (Compound L3 of Scheme L where RS is 4-pyridyl, X is F, Y
is H, Rl is H
and R2 is H) N~ ~ ~ ~ N O H H
~--~N N
F 35S \ I N
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.
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.
Claims (26)
1. A compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R1 is H, F or I;
R2 is H, F or OH;
R16 is H or F;
R17 is H or F;
R3 is H or C1-C8 alkyl;
L is a bond or -OCH2C(=O); and Q is wherein:
R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof.
X and Y are, independently, F, H or CH3;
R1 is H, F or I;
R2 is H, F or OH;
R16 is H or F;
R17 is H or F;
R3 is H or C1-C8 alkyl;
L is a bond or -OCH2C(=O); and Q is wherein:
R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1 wherein X is F, Y is H, R3 is H, and R4 is CH3.
3. A compound of claim 1 wherein said compound is 2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl-4-azido-2-hydroxy-5-iodo-125I-benzoate.
4. A compound of claim 1 wherein said compound is N-[[(5S)-3-[4-[4-(4-Azido-2-hydroxy-5-iodo-125I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide.
5. A compound of claim 1 wherein said compound is 2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl 4-azido-3-iodo-125I-benzoate.
6. A compound of claim 1 wherein said compound is N-[[(5S)-3-[4-[4-(4-Azido-3-iodo-125I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide.
7. A compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R1 is H, F or I;
R2 is H,F or OH;
R16 is H or F;
R17 is H or F; and Q is wherein:
R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof.
X and Y are, independently, F, H or CH3;
R1 is H, F or I;
R2 is H,F or OH;
R16 is H or F;
R17 is H or F; and Q is wherein:
R4 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof.
8. A compound of claim 7 wherein X is F, Y is H, and R4 is CH3.
9. A compound of claim 7 wherein said compound is N-[[(5S)-3-(4'-Azido-2-fluoro[1,1'-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T3-acetamide.
10. A compound of claim 7 wherein said compound is N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo[1,1'-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T3-acetamide.
11. A compound of claim 7 wherein said compound is N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo[1,1'-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]ethane-35S-thioamide.
12. A compound of claim 7 wherein said compound, is N-[[(5S)-3-(4'-Azido-2-fluoro-3'-iodo-125I-[1,1'-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]acetamide.
13. A compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R5 is wherein:
R6 is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4 alkyl, nitrite, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl; and P is wherein:
Z is O or S; and R7 is wherein:
R1 is H, F or I;
R2 is H, F or OH;
R16 is H or F; and R17 is H or F;
or a pharmaceutically acceptable salt thereof.
X and Y are, independently, F, H or CH3;
R5 is wherein:
R6 is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4 alkyl, nitrite, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl; and P is wherein:
Z is O or S; and R7 is wherein:
R1 is H, F or I;
R2 is H, F or OH;
R16 is H or F; and R17 is H or F;
or a pharmaceutically acceptable salt thereof.
14. A compound of claim 13 wherein X is F, Y is H, and R6 is H.
15. A compound of claim 13 wherein said compound is (2E)-3-(4-azido-3-iodo-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-propenamide.
16. A compound of claim 13 wherein said compound is 4-azido-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-hydroxy-5-iodo-125I-benzamide.
17. A compound of claim 13 wherein said compound is N-(4-azidophenyl)-N'-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-35S-thiourea.
18. A method of using a compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F;
R19 is H or F;
R10 is H or C1-C8 alkyl;
L is a bond or -OCH2C(=O); and Q is wherein:
R11 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
X and Y are, independently, F, H or CH3;
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F;
R19 is H or F;
R10 is H or C1-C8 alkyl;
L is a bond or -OCH2C(=O); and Q is wherein:
R11 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
19. The method of claim 18 comprising the steps:
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
20. The method of claim 19 further comprising contacting said cell or components thereof with a competitor compound.
21. A method of using a compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R12 is N3 or wherein:
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F; and R19 is H or F; and Q is wherein:
R11 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
X and Y are, independently, F, H or CH3;
R12 is N3 or wherein:
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F; and R19 is H or F; and Q is wherein:
R11 is H, CH3, CH2CH3 or cyclopropyl; and Z is O or S;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
22. The method of claim 21 comprising the steps:
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
23. The method of claim 22 further comprising contacting said cell or components thereof with a competitor compound.
24. A method of using a compound comprising the formula wherein:
X and Y are, independently, F, H or CH3;
R13 is wherein:
R14 is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4alkyl, nitrite, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl; and P is wherein:
Z is O or S; and R15 is wherein:
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F; and R19 is H or F;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
X and Y are, independently, F, H or CH3;
R13 is wherein:
R14 is H, N3, halogen, NH2, OH, SH, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4alkyl, nitrite, carboxamide, C1-C4 alkoxy, C1-C4 alkylthio, or C1-C4 alkoxycarbonyl; and P is wherein:
Z is O or S; and R15 is wherein:
R8 is H, F or I;
R9 is H, F or OH;
R18 is H or F; and R19 is H or F;
or a pharmaceutically acceptable salt thereof, as a photoaffinity probe.
25. The method of claim 24 comprising the steps:
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
contacting a cell or component thereof with said compound, wherein said compound is radiolabeled;
exposing said radiolabeled compound to light; and detecting said radiolabel.
26. The method of claim 25 further comprising contacting said cell or components thereof with a competitor compound.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/738,022 US6861433B2 (en) | 2000-12-15 | 2000-12-15 | Oxazolidinone photoaffinity probes |
| US09/738,022 | 2000-12-15 | ||
| PCT/US2001/048063 WO2002048139A2 (en) | 2000-12-15 | 2001-12-14 | Oxazolidinone photoaffinity probes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2432739A1 true CA2432739A1 (en) | 2002-06-20 |
Family
ID=24966243
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|---|---|---|---|
| CA002432739A Abandoned CA2432739A1 (en) | 2000-12-15 | 2001-12-14 | Oxazolidinone photoaffinity probes |
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|---|---|
| US (3) | US6861433B2 (en) |
| EP (1) | EP1368326A2 (en) |
| JP (1) | JP2004520298A (en) |
| AU (1) | AU2002234016A1 (en) |
| CA (1) | CA2432739A1 (en) |
| WO (1) | WO2002048139A2 (en) |
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| EP1386153A2 (en) * | 2000-12-15 | 2004-02-04 | PHARMACIA & UPJOHN COMPANY | Oxazolidinone photoaffinity probes, uses and compounds |
| US7022705B2 (en) | 2001-10-25 | 2006-04-04 | Astrazeneca Ab | Isoxazoline derivatives useful as antimicrobials |
| WO2005051933A1 (en) * | 2003-11-28 | 2005-06-09 | Ranbaxy Laboratories Limited | An improved process for the synthesis of 4-(4-benzyloxy-carbonylamino-2-fluorophenyl)-piperazine-1-carboxylic acid tert-butyl ester, a key intermediate for oxazolidinone antimicrobials and compounds prepared thereby |
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| KR100257418B1 (en) | 1991-11-01 | 2000-05-15 | 로렌스 티. 마이젠헬더 | Substituted aryl-and heteroaryl-phenyloxazolidinone |
| JP3698724B2 (en) | 1993-11-22 | 2005-09-21 | ファルマシア・アンド・アップジョン・カンパニー | Substituted-esters of hydroxyacetylpiperazine phenyloxazolidinone |
| BR9607017A (en) | 1995-02-03 | 1997-10-28 | Upjohn Co | Compound and use thereof |
| PT1054874E (en) | 1998-02-13 | 2003-01-31 | Upjohn Co | UTILIZED AMINOFENYL-ISOXAZOLINE DERIVATIVES AS ANTI-MICROBIALS |
| TW572757B (en) | 1998-08-24 | 2004-01-21 | Bristol Myers Squibb Co | Novel isoxazolinone antibacterial agents |
| US6689779B2 (en) | 2000-06-05 | 2004-02-10 | Dong A Pharm. Co., Ltd. | Oxazolidinone derivatives and a process for the preparation thereof |
-
2000
- 2000-12-15 US US09/738,022 patent/US6861433B2/en not_active Expired - Fee Related
-
2001
- 2001-12-14 JP JP2002549670A patent/JP2004520298A/en not_active Withdrawn
- 2001-12-14 WO PCT/US2001/048063 patent/WO2002048139A2/en not_active Application Discontinuation
- 2001-12-14 CA CA002432739A patent/CA2432739A1/en not_active Abandoned
- 2001-12-14 AU AU2002234016A patent/AU2002234016A1/en not_active Abandoned
- 2001-12-14 EP EP01985023A patent/EP1368326A2/en not_active Withdrawn
-
2003
- 2003-02-06 US US10/359,767 patent/US6875871B2/en not_active Expired - Fee Related
- 2003-02-06 US US10/359,766 patent/US6858635B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US6875871B2 (en) | 2005-04-05 |
| US6861433B2 (en) | 2005-03-01 |
| US20030073696A1 (en) | 2003-04-17 |
| WO2002048139A2 (en) | 2002-06-20 |
| JP2004520298A (en) | 2004-07-08 |
| AU2002234016A1 (en) | 2002-06-24 |
| US6858635B2 (en) | 2005-02-22 |
| WO2002048139A3 (en) | 2003-10-02 |
| US20030232840A1 (en) | 2003-12-18 |
| US20030232008A1 (en) | 2003-12-18 |
| EP1368326A2 (en) | 2003-12-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |