U.S. patent application number 13/362658 was filed with the patent office on 2013-08-01 for 99mtc imaging agents and methods of use.
This patent application is currently assigned to GENERAL ELECTRIC COMPANY. The applicant listed for this patent is Randall Lee Carter, Mark Christopher Patrick Darey, Bruce Fletcher Johnson, Michael James Rishel, Karin Ann Stephenson, John Fitzmaurice Valliant, Tao Wu, Yang Yang. Invention is credited to Randall Lee Carter, Mark Christopher Patrick Darey, Bruce Fletcher Johnson, Michael James Rishel, Karin Ann Stephenson, John Fitzmaurice Valliant, Tao Wu, Yang Yang.
Application Number | 20130195756 13/362658 |
Document ID | / |
Family ID | 47628179 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130195756 |
Kind Code |
A1 |
Johnson; Bruce Fletcher ; et
al. |
August 1, 2013 |
99mTc IMAGING AGENTS AND METHODS OF USE
Abstract
An embodiment of the invention comprises a ligand of Formula I
##STR00001## wherein R.sup.1 and R.sup.2 are independently an alkyl
or cycloalkyl; R.sup.3 is and alkyl; X is CO or SO.sub.2; Y is
(CH2).sub.n, C.sub.6H.sub.4,
(OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n and
(OCH.sub.2CH.sub.2CH.sub.2).sub.n, or a combination thereof; Z is
linker group capable of conjugating to a vector; and n is an
integer between 0 and 10. Also included are an imaging agent
comprising a compound of Formula I complexed to .sup.99mTc and
their method of use to image a subject having a target site using
single photon emission computed tomography (SPECT).
Inventors: |
Johnson; Bruce Fletcher;
(Scotia, NY) ; Carter; Randall Lee; (Clifton Park,
NY) ; Rishel; Michael James; (Saratoga Springs,
NY) ; Darey; Mark Christopher Patrick; (Charleston,
WV) ; Wu; Tao; (Ancaster, CA) ; Yang;
Yang; (Edmonton, CA) ; Valliant; John
Fitzmaurice; (Ancaster, CA) ; Stephenson; Karin
Ann; (Burlington, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Johnson; Bruce Fletcher
Carter; Randall Lee
Rishel; Michael James
Darey; Mark Christopher Patrick
Wu; Tao
Yang; Yang
Valliant; John Fitzmaurice
Stephenson; Karin Ann |
Scotia
Clifton Park
Saratoga Springs
Charleston
Ancaster
Edmonton
Ancaster
Burlington |
NY
NY
NY
WV |
US
US
US
US
CA
CA
CA
CA |
|
|
Assignee: |
GENERAL ELECTRIC COMPANY
Schenectady
NY
|
Family ID: |
47628179 |
Appl. No.: |
13/362658 |
Filed: |
January 31, 2012 |
Current U.S.
Class: |
424/1.49 ;
424/1.65; 424/1.69; 424/1.73; 530/300; 530/391.3; 534/14; 564/169;
564/197; 564/88; 564/95 |
Current CPC
Class: |
A61K 51/08 20130101;
A61K 51/1093 20130101; A61K 51/04 20130101; A61K 51/088 20130101;
C07C 311/18 20130101; A61K 51/0474 20130101; A61K 51/0491
20130101 |
Class at
Publication: |
424/1.49 ;
564/95; 564/88; 564/197; 564/169; 534/14; 530/300; 530/391.3;
424/1.65; 424/1.69; 424/1.73 |
International
Class: |
A61K 51/04 20060101
A61K051/04; C07C 311/18 20060101 C07C311/18; C07C 237/10 20060101
C07C237/10; C07C 235/84 20060101 C07C235/84; A61K 51/06 20060101
A61K051/06; C07K 4/00 20060101 C07K004/00; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00; A61K 51/08 20060101
A61K051/08; A61K 51/10 20060101 A61K051/10; C07C 311/32 20060101
C07C311/32; C07F 13/00 20060101 C07F013/00 |
Claims
1. A ligand of Formula I ##STR00012## wherein R.sup.1 and R.sup.2
are independently an alkyl or cycloalkyl; R.sup.3 is and alkyl; X
is CO or SO.sub.2, Y is (CH.sub.2).sub.n, C.sub.6H.sub.4,
(OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n and
(OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; Z is
linker group capable of conjugating to a vector; and n is an
integer between 0 and 10.
2. The ligand of claim 1 wherein R.sup.1, R.sup.2, and R.sup.3 are
independently lower alkyl groups.
3. The ligand of claim 1 wherein R.sup.1, R.sup.2, and R.sup.3 are
CH.sub.3, X is SO.sub.2, Y is CH.sub.2, and n is 1.
4. The ligand of claim 1 wherein Z comprises carboxylic acid, an
activated ester, a phosphoramidite, isocyanate, isothiocyanate,
aldehyde, acid chloride, sulfonyl chloride, alkyl halide, amine,
phosphine, phosphate, alcohol, thiol, or a combination thereof.
5. The ligand of claim 4 wherein the activated ester comprises
N-hydroxysuccinimide ester, pentafluorophenyl ester, or a
combination thereof
6. The ligand of claim 1 wherein Formula I is ##STR00013##
7. An imaging agent comprising a compound of Formula I complexed to
.sup.99mTc wherein Formula I comprises: ##STR00014## wherein
R.sup.1 and R.sup.2 are independently an alkyl or cycloalkyl;
R.sup.3 is and alkyl; X is CO or SO.sub.2, Y is (CH.sub.2).sub.n,
C.sub.6H.sub.4, (OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n
and (OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; Z
is linker group capable of conjugating to a vector; and n is an
integer between 0 and 10.
8. The imaging agent of claim 7 wherein R.sup.1, R.sup.2, and
R.sup.3 are independently lower alkyl groups.
9. The imaging agent of claim 8 wherein R.sup.1, R.sup.2, and
R.sup.3 are CH.sub.3, X is SO.sub.2, Y is CH.sub.2, and n is 1
10. The imaging agent of claim 7 wherein Z comprises carboxylic
acid, an activated ester a phosphoramidite, isocyanate,
isothiocyanate, aldehyde, acid chloride, sulfonyl chloride, e,
alkyl halide, amine, phosphine, phosphate, alcohol, thiol, or a
combination thereof.
11. The imaging agent of claim 10 wherein the activated ester
comprises N-hydroxysuccinimide ester, pentafluorophenyl ester, or a
combination thereof
12. The imaging agent of claim 7 wherein Formula I is
##STR00015##
13. The imaging agent of claim 7 further comprising a vector
covalently bound to the ligand through a linker moiety.
14. The imaging agent of claim 13 wherein the vector comprises
3-100 mer peptides or peptide analogues, monoclonal anitbodies or
fragments thereof; enzyme substrates, enzyme inhibitors; synthetic
receptor-binding compounds; oligonucleotides, oligo-DNA fragments,
oligo-RNA fragments, or a combination thereof.
15. The imaging agent of claim 14 wherein the vector comprises a
3-100 mer peptide, peptide analogues, or combinations thereof.
16. A method of imaging a target site comprising: administering to
a subject having a target site, an imaging agent comprising a
ligand, complexed with .sup.99mTc, and chemically bound to a vector
through a linker moiety, and wherein said ligand comprises a
compound of Formula I ##STR00016## wherein R.sup.1 and R.sup.2 are
independently an alkyl or cycloalkyl; R.sup.3 is and alkyl; X is CO
or SO.sub.2, Y is (CH.sub.2).sub.n, C.sub.6H.sub.4,
(OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n and
(OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; Z is
linker group capable of conjugating to a vector; and n is an
integer between 0 and 10; allowing the imaging agent to localize to
the target site;and detecting the imaging agent at the target using
single photon emission computed tomography (SPECT).
17. The method of claim 16 wherein R.sup.1, R.sup.2, and R.sup.3
are independently lower alkyl groups.
18. The method of claim 17 wherein R.sup.1, R.sup.2, and R.sup.3
are CH.sub.3, X is SO.sub.2, Y is CH.sub.2, and n is 1.
19. The method of claim 17 wherein Z comprises carboxylic acid, an
activated ester a phosphoramidite, isocyanate, isothiocyanate,
aldehyde, acid chloride, sulfonyl chloride, alkyl halide, amine,
phosphine, phosphate, alcohol, thiol, or a combination thereof.
20. The method of claim 19 wherein the activated ester comprises
N-hydroxysuccinimide ester, pentafluorophenyl ester, or a
combination thereof
21. The method of claim 17 wherein Formula I is ##STR00017##
22. The method of claim 16 wherein the vector comprises 3-100 mer
peptides or peptide analogues, monoclonal antibodies or fragments
thereof; enzyme substrates, enzyme inhibitors; synthetic
receptor-binding compounds, oligonucleotides, oligo-DNA fragments,
oligo-RNA fragments, or a combination thereof.
23. The method of claim 22 wherein the vector comprises a 3-100 mer
peptide, peptide analogues, or combinations thereof.
Description
BACKGROUND
[0001] The invention relates to imaging agents and methods for
SPECT, and more particularly to imaging agents radiolabeled with
.sup.99mTc.
[0002] The most widely used radionuclide in nuclear medicine is
technetium-99m (.sup.99mTc; T.sub.1/2=6.0 h, 140 KeV .gamma.
emission). While the majority of clinically approved
.sup.99Tc-radiopharmaceuticals are perfusion-type agents
(diagnostic images of blood flow), there is a growing interest to
develop and commercialize single photon emission computed
tomography (SPECT) imaging agents that target specific
biomarkers.
[0003] Exploitation of this opportunity requires the creation of
ligand which acts as a technetium chelator to incorporate
.sup.99mTc and is capable of regioselectively conjugating to a
variety of vectors, including biomolecules. Vectors refer to a
vehicle used to transfer material to a target or target site.
Ideally, the ligand should be capable of incorporating .sup.99mTc
without impairing the biological properties of the vector.
[0004] While ligands capable of chelating with .sup.99mTc are well
known, few ligands meet the criteria needed to develop an effective
agent. For example in certain synthetic methodologies .sup.99mTc
incorporation is achieved under basic conditions, which can be
deleterious to certain peptides/proteins.
[0005] For example European Patent EP0738158 and U.S. Pat. No.
7,597,875 disclose a ligand with an all-carbon bridge and shown in
FIG. 1 (structure A). The ligand (A) is capable of conjugation a
broad array of vectors via the primary nitrogen. However, like
diaminodioximes in general it requires a pH of approximately 9-10
to label optimally. This pH is incompatible with many sensitive
biomolecules. Furthermore the synthesis of the structure A yields
mono, bi, and tri functionalized products from which the desired
bi-functionalized chelate must be isolated by preparative HPLC.
[0006] U.S. Pat. No. 7,049,289 discloses a ligand, (FIG. 1,
structure B), that may also be appropriate for conjugation a broad
array of vectors via the primary nitrogen; it is also synthetically
more accessible than the all carbon backbone (structure A). However
as taught in U.S. Pat. No. 7,597,875, this ligand does not form a
single radiolabeled species with .sup.99mTc under mild
conditions.
[0007] Similarly, U.S. Pat. No. 5,688,487 and U.S. Pat. No.
5,997,843 disclose a ligand, (FIG. 1, structure C) with an all
carbon bridge and nitroimidazole vector (X) attached at the C1
position. This construct is limited in that it is not easy for a
facile and broad conjugation to vectors as the vector is
incorporated early in a multistep synthesis.
[0008] As such, the development of an alternative approach
involving chelation that is effective at slightly basic to acidic
pH and is readily synthesized would provide a technology to enable
technetium radiolabeling of vectors without pH limitations.
Furthermore it would be desirable to have .sup.99mTc incorporation
in a single step in a manner suitable for clinical production of
agents with high effective specific activity. Accordingly, there is
a need for imaging systems and methods that can provide a high
resolution, high sensitivity image in a shorter period of time and
which may be produced under mild aqueous conditions.
BRIEF DESCRIPTION
[0009] An embodiment of the invention comprises a ligand of Formula
I
##STR00002##
wherein R.sup.1 and R2 are independently an alkyl or cycloalkyl;
R.sup.3 is an alkyl; X is CO or SO.sub.2, Y is (CH.sub.2).sub.n,
C.sub.6H.sub.4, (OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n
and (OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; Z
is linker group capable of conjugating to a vector; and n is an
integer between 0 and 10.
[0010] In one embodiment, an imaging agent comprising a compound of
Formula I complexed to .sup.99mTc.
[0011] Another embodiment of the invention comprises administering
to a subject having a target site, an imaging agent comprising a
ligand, complexed with .sup.99mTc, and chemically bound to a vector
through a linker moiety, and wherein the ligand comprises a
compound of Formula I. An example of the method generally
comprises, allowing the imaging agent to localize to the target
site; and detecting the imaging agent at the target using single
photon emission computed tomography (SPECT).
DRAWINGS
[0012] These and other features, aspects, and advantages of the
present invention will become better understood when the following
detailed description is read with reference to the accompanying
drawings in which like characters represent like parts throughout
the drawings, wherein:
[0013] FIG. 1 are structures of ligands capable of chelating with
.sup.99mTc.
[0014] FIG. 2 is a schematic representation of the
.sup.99mTc-ligand complex.
[0015] FIG. 3 is a schematic representation of examples of the
"N--X--(Y).sub.n--Z" structure of Formula I.
[0016] FIG. 4 is graph representing HPLC analysis of Formula 2.
[0017] FIG. 5 are graphical representations of competition
experiments comparing Structure C( X.dbd.H) (FIG. 1) and Formula 2
at different pH values.
DEFINITIONS
[0018] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the examples included therein. In
the following specification and the claims which follow, reference
will be made to a number of terms which shall be defined to have
the following meanings:
[0019] In the context of the present invention, alkyl is intended
to include linear, branched, or cyclic hydrocarbon structures and
combinations thereof, including lower alkyl and higher alkyl.
Preferred alkyl groups are those of C20 or below. Lower alkyl
refers to alkyl groups of from 1 to 6 carbon atoms, preferably from
1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl,
isopropyl, and n-, s- and t-butyl. Higher alkyl refers to alkyl
groups having seven or more carbon atoms, preferably 7-20 carbon
atoms, and includes n-, s- and t-heptyl, octyl, and dodecyl.
Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon
groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups
include cyclopropyl, cyclobutyl, cyclopentyl, and norbomyl. Alkenyl
and alkynyl refer to alkyl groups wherein two or more hydrogen
atoms are replaced by a double or triple bond, respectively.
[0020] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon
atoms of a straight, branched, cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, and cyclohexyloxy. Lower alkoxy refers to groups
containing one to four carbons.
DETAILED DESCRIPTION
[0021] .sup.99mTc labeled vectors are of great interest for
molecular imaging via SPECT and their basic configuration as
illustrated in FIG. 1. As shown, a ligand acts as a technetium
chelator to incorporate .sup.99mTc while binding to a vector
through a linker moiety. The vector is a biologically active
molecule which affects the biodistribution of the .sup.99mTc by
binding to targets, or target sites, such as receptors or enzymes
associated with specific tissues, lesions or pathological
processes. This enables the presence or absence of these receptors
to be imaged noninvasively via single-photon emission computed
tomography (SPECT). The vector can be a small molecule, peptide or
biomacromolecule such as an antibody. The ligand is critical for
linking the .sup.99mTc to vector. A schematic representation of the
.sup.99mTc-ligand complex is shown in FIG. 2
[0022] Imaging agents for use in the compositions and methods of
the present invention include structural units derived from a
ligand represented by Formula I
##STR00003## [0023] wherein R.sup.1 and R.sup.2 are independently
an alkyl or cycloalkyl; [0024] R.sup.3 is an alkyl; [0025] X is CO
or SO.sub.2, [0026] Y is (CH.sub.2).sub.n, C.sub.6H.sub.4,
(OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n and
(OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; [0027]
Z is a group capable of conjugating to a vector; and n is an
integer between 0 and 10.
[0028] Ligands of Formula I have a tertiary nitrogen which makes
this class of ligands more synthetically accessible than their
counterparts lacking the nitrogen. Furthermore the electron
withdrawing properties of X may facilitate the complexation of
Formula I with .sup.99mTc by making the tertiary nitrogen less able
to participate in binding to the Tc which allows only one species
to form. As such, using the ligands of Formula I as part of a
radiolabeled molecular imaging agents, provide a means of
incorporating a broad array of vectors useful in a wide variety of
diagnostic and therapeutic monitoring applications
[0029] In certain embodiments, R.sup.1, R.sup.2, and R.sup.3 are
independently lower alkyl groups. In certain other embodiments,
R.sup.1, R.sup.2, and R.sup.3 are CH.sub.3, X is SO.sub.2, Y is a
direct bond such that n is 0, and Z is linker group capable of
conjugating to a vector.
[0030] Exemplary structures of the "N--X--Y--Z moiety of Formula I
are shown in FIG. 3.
[0031] In certain embodiments the Z group, includes a moiety that
attaches or conjugates the ligand to the vector. Example of linkers
include, but are not limited to, carboxylic acids, activated
esters, such as N-hydroxysuccinimide ester or pentafluorophenyl
ester, phosphoramidite, isocyanate, isothiocyanate, aldehyde, acid
chloride, sulfonyl chloride, alkyl halide, amine, phosphine,
phosphate, alcohol, thiol, or a combination thereof.
[0032] Exemplary ligands include, but are not limited to,
##STR00004##
[0033] In certain embodiment the ligand of Formula I is chemically
bound to a vector through a linker moiety
[0034] The vector is a compound that differentially accumulates in
a target site. A site may be a cell, group of cells, organ, tumor
or lesion relative to nearby sites. The accumulation of the vector
in the target site may be due to the vector binding to and/or
interaction with a biomarker that is differentially expressed at
the target site relative to nearby sites. Representative examples
of biomarkers include, but is not limited to human epidermal growth
factor receptor 2 (HER-2) brain thymidine kinase 1 (TK-1), and
peripheral benzodiazepine receptors (PBRs).
[0035] Non-limiting examples of vectors includes small molecules,
proteins, peptides, polypeptides, glycoproteins, lipoproteins,
phospholipids, oligonucleotides, steroids, alkaloids or the like,
e.g., hormones, lymphokines, growth factors, albumin, cytokines,
enzymes, immune modulators, receptor proteins, oligonucleotides or
mimics thereof, and antibodies and antibody fragments, individually
or in any combination thereof as well as derivatives thereof. In
certain embodiments the vectors may be classified as 3-100 mer
peptides or peptide analogues which may be linear peptides or
cyclic peptides or combinations thereof; monoclonal anitbodies or
fragments thereof; or enzyme substrates or inhibitors; synthetic
receptor-binding compounds; oligonucleotides, or oligo-DNA or
oligo-RNA fragments. The vectors may be of synthetic or natural
origin. Examples of particular vectors include aptamers and
thioaptamers. Preferred vectors are 3-20 mer peptides. Examples of
vectors, which may also be referred to as a "biological targeting
moiety" may be found in U.S. Pat. No. 7,597,875 entitled "Chelator
Conjugates" and issued Oct. 6, 2009. The patent is hereby
incorporated by reference.
[0036] In certain embodiments, R.sup.1, R.sup.2, and R.sup.3 are
independently lower alkyl groups. In certain other embodiments,
R.sup.1, R.sup.2, and R.sup.3 are CH.sub.3, X is SO.sub.2, Y is a
direct bond wherein n is 0, and Z is linker.
[0037] In certain embodiments the Z group, includes a moiety that
attaches or conjugates the ligand to the vector. Example of linkers
include, but are not limited to, carboxylic acids, activated
esters, such as N-hydroxysuccinimide ester or pentafluorophenyl
ester, phosphoramidite, isocyanate, isothiocyanate, aldehyde, acid
chloride, sulfonyl chloride, alkyl halide, amine, phosphine,
phosphate, alcohol, thiol, or a combination thereof.
[0038] In certain embodiments, the ligand of Formula I is complexed
with .sup.99mTc to form a radiolabel. The complexation chemistry of
.sup.99mTc may be produced by using different methods. In certain
methods it is produced using pertechnetate
.sup.99mTcO.sub.4-requiring reduction and complexation using the
reduced state. The trans dioxo .sup.99mTcO.sub.2.sup.+ core may
also be used and has the advantage of being symmetrical around the
Tc core when complexed.
[0039] In certain embodiments the complexation of the ligand of
Formula I with .sup.99mTc may be depicted as Formula II. Wherein
the .sup.99mTc complexes of the chelators are neutral, Tc(V) dioxo
complexes:
##STR00005##
wherein R.sup.1 and R.sup.2 are independently an alkyl or
cycloalkyl; [0040] R.sup.3 is and alkyl; [0041] X is CO or
SO.sub.2, [0042] Y is (CH.sub.2).sub.n, C.sub.6H.sub.4,
(OCH.sub.2CH.sub.2).sub.n(NHCH.sub.2CH.sub.2).sub.n and
(OCH.sub.2CH.sub.2CH.sub.2).sub.n or a combination thereof; [0043]
Z is linker group capable of conjugating to a vector; and [0044] n
is an integer between 0 and 10.
[0045] In certain embodiments, a target may be detected by
administering to a subject an imaging agent comprising the ligand
of Formula I, complexed with .sup.99mTc and chemically bound to a
vector through a linker moiety, allowing the imaging agent to
travel to the target site via intracellular diffusion and to
subsequently bind to the target of interest thorough noncovalent or
covalent (rare) association. The pattern of accumulated agent is
detected in the subject using SPECT. For example, for some
applications the labeled may be detected in cancerous cells wherein
the ligand binds to a biomarker such as Human Epidermal growth
factor Receptor 2 (HER2) which is overexpressed in certain breast
cancers.
[0046] In certain embodiments, the imaging agent may be dissolved
or suspended in a pharmaceutical carrier to allow for administering
the imaging agent to a subject. Pharmaceutical carrier refers to a
composition which allows the application of the agent material to
the site of the application, surrounding tissues, or prepared
tissue section to allow the agent to have an effective residence
time for specific binding to the target or to provide a convenient
manner of release. Formulation strategies may include but are not
limited to: pH adjustments, salt formation, formation of ionizable
compounds, use of co-solvents, complexation, surfactants and
micelles, emulsions and micro-emulsions. The pharmaceutical carrier
may include, but is not limited to, a cosolvent, detergent, buffer
solution, stabilizers, and preservatives. Examples of these include
but are not limited to, HCl, citric acid, DMSO, propylene glycol,
ethanol PEG 300, cyclodextrans, citrate, acetate, phosphate,
carbonate or tris(hydroxymethyl)aminomethane. Particularly, the
pharmaceutical carrier is suitable for intravenous, intramuscular,
subcutaneous, or parenteral administration (e.g., by injection).
These pharmaceuticals may also be administered orally under
appropriate circumstances
EXPERIMENTAL
[0047] Complexation with .sup.99mTc may be accomplished while
maintaining a solution pH of about 6 to about 10 and more
preferable a pH of about 7 to about 9. This is depicted in the
experimental results shown in FIG. 4, whereby the radiolabeling of
Formula 2
##STR00006##
was achieved at pH 9 and 7.about.7.5 in 15 min in 1/1(v/v)
DMSO/H.sub.2O as evidenced by HPLC analysis of retention time
(minutes) vs. response (mV).
[0048] In each case, with or without the use of a common co-ligand
used in .sup.99mTc radiolabelling, good radiochemical purity (RCP)
was achieved and colloid formation was minimal (Table 1).
Noteworthy is the high RCP that occurred at pH 7-7.5 which
addresses the need for a mild pH conditions. The co-ligand used was
methylenediphosphonic acid (MDP).
TABLE-US-00001 TABLE 1 .sup.99mTc (V) Labeling of 2 at pH 9 and
7-7.5. pH MDP % RCP Colloid Activity Rxn Time 9 yes 96% 0.33% 0.24
GBq (6.6 mCi) 15 min 9 no 95% 1.06% 0.27 GBq (7.2 mCi) 15 min 7~7.5
yes 94% 1.19% 0.28 GBq (7.6 mCi) 15 min
[0049] Competition experiments between structure C (X.dbd.H) (FIG.
1) and Formula 2, were carried out at pH 9, 7.about.7.5 and 6.
Results are presented in FIG. 5 which shows radiochemical purity
(RCP) as a percentage vs. time (minutes) for a range of pH values.
At pH 9, the complexation of C was still dominating regardless the
presence of MDP (C/2 product ratio.about.3:1). However, at lower pH
(7.about.7.5), the complex of 2 became the major product (product
ratio.about.1:1.5). At even lower pH (6), 2 showed a higher
complexation amount than C (product ratio.about.1:3). The results
demonstrate improved performance of 2 relative to C as pH is
reduced.
[0050] Reaction amounts are given in tables. Often a reagent will
list a volume and weight; this means that the reagent was dispensed
by volume but the amount determined by weight. All calculations are
based on weight.
Synthesis of Diaminedioxime C(X.dbd.H)
[0051] Diaminedioxime C(X.dbd.H) has been shown to cleanly form a
complex with .sup.99mTc that forms a dioxo TcO.sub.2.sup.+
core.
3-chloro-3-methyl-2-butanone oxime 20
[0052] The chloroxime was synthesized following the procedure from
European Patent Application EP404377 filed Apr. 6, 2009.
##STR00007##
TABLE-US-00002 TABLE 2 purity MW amt amt d vol w/w g/mol g mmol eq
g/ml ml CA # 21 100% 70.1 33.15 470 1.24 0.66 50.0 513-35-9 isoamyl
96% 117.2 46.22 379 1.00 0.87 50.0 110-46-3 nitrite HCl 37% 36.5
56.5 574 1.51 1.19 47.5 conc EtOH 34 ml 20 95% 135.6 33.4 234 62%
yield 3238-16-2
[0053] 2-methyl-2-butene 21 was mixed with isoamyl nitrite and
cooled to -70.degree. C. in a MeOH/dry ice bath. HCl.sub.conc was
added over 50 min., keeping the temperature between -30 and
-20.degree. C. EtOH (34 mL) was added towards the end of the
addition, but the mixture still solidified to a gel after the
addition was complete. The batch was stirred for a further 2 h. at
-20 to -10.degree. C., then filtered (filtration and washes took
about 40 min.). The filter cake was washed with chilled EtOH (40
mL) followed by ice-cold water (50 mL) and was left to dry for 90
min. The wet weight was 66.4 g. After drying under vacuum, the
weight fell to 33.4 g (0.246 moles, 62% theory). Proton NMR
indicated that the product (now a mixture of viscous liquid and
solid) was a mixture of the trans-oxime 20 (49%), nitroso-tautomer
22 (40%) and 3-nitroso-2-chlorobutane 23 (11%). Upon storing in the
refrigerator, the material solidified yielding the trans-oxime
20.
3,3'-(1,5-pentanediyldiimino)bis[3-methyl-2-butanone]dioxime C
(X.dbd.H)
##STR00008##
TABLE-US-00003 [0054] TABLE 3 purity MW amt amt d vol w/w g/mol g
mmol eq g/ml ml CA # 22 95% 135.6 9.40 66 2.29 3238-16-2 24 95%
102.2 3.10 29 1.00 0.87 3.55 462-94-2 MeOH 40 ml C 95% 300.4 0.638
2 7.0% yield 109929-73-9
[0055] Chloroxime 20 was dissolved in MeOH (40 mL) to give a pale
green solution. The solution was cooled to below 0.degree. C. in an
ice/acetone bath, and a white solid precipitated.
1,5-diaminopentane 24 was added over about 30 m, keeping the
temperature below 0.degree. C. (initial exotherm to 10.degree. C.).
On commencing the addition, the mixture became an orange brown
color, turning to a thin dark violet slurry by the end of the
addition. The mixture was stirred at room temperature overnight.
TLC in CH.sub.2Cl.sub.2/MeOH/NH.sub.3 and visualization with
ninhydrin stain showed that the mixture consisted of two major
components: one very polar (presumably monoalkylated) and one less
polar (presumably dialkylated). Heating to reflux for 2 h. did not
appear to change the composition of the mixture. Water (100 mL) was
added to the cooled mixture, and 0.9 g of a yellow solid was
collected by filtration. The pH of the filtrate was adjusted to pH
12, whereupon a viscous oil partitioned from the aqueous layer. The
oil was extracted into CH.sub.2Cl.sub.2, and the organic layer
concentrated to give 8 g of a purple gum. The gum was further dried
under vacuum over night at room temperature, to give 6 g of a
sticky solid. The solid was triturated with water (75 mL) and
filtered to give 4 g of dirty white solid, which was still slightly
sticky. The solid was dissolved in refluxing MeOH (20 mL) and
cooled in an ice/water bath over 90 min. The white solid was
collected by filteration, and dried under vacuum to give 638 mg
(2.1 mmol, 7% theory) of the desired ligand as a white solid. MS,
.sup.1H and .sup.13C NMRs confirmed the structure. In this
procedure a Hunig's base was not used but may be added
Synthesis of Formula 2
N,N-bis[2-[(phenylsulfonyl)oxy]ethyl]-benzenesulfonamide 18
##STR00009##
TABLE-US-00004 [0056] TABLE 4 purity MW amt amt d vol w/w g/mol g
mmol eq g/ml ml CA # di- 98% 105.1 4.0 37.3 1.00 111-42-2 ethanol-
amine TsCl 98% 190.7 23.5 120.8 3.24 616-47-7 Et3N 100% 101.2 13.2
130.4 3.50 0.726 18 121-44-8 Et2O 120 ml CH3CN 100 ml 261 reaction
volume 18 95% 567.7 13.8 23.1 62% yield 22185-13-3
[0057] Et.sub.3N in 20 mL of Et.sub.2O was added to tosyl chloride
in 100 mL Et.sub.2O in a 500 mL flask. This was stirred and cooled
in an ice bath. Diethanolamine was melted in an oven and
transferred to a 100 ml flask, 80 mL of CH.sub.3CN was added to the
amine to dissolve it, and the mixture was added via cannula to the
mixture of tosylchloride and Et.sub.3N to form a white suspension.
The progress of the reaction was monitored via HPLC.
[0058] The reaction was worked up after 5 days by evaporating the
solvent under an active stream on nitrogen gas. Diethylether was
added to the residue to produce a suspension and the precipitate
was collected by filtration. The precipitate was purified on a
gravity silica gel column starting with 60/40
hexanes/CH.sub.2Cl.sub.2 progressing to 100% CH.sub.2Cl.sub.2.
N,N-bis(2-aminoethyl)-4-methyl-benzenesulfonamide 19
##STR00010##
TABLE-US-00005 [0059] TABLE 5 purity MW amt amt w/w g/mol g mmol eq
CA # 18 95% 567.7 6.25 10.5 1.00 22185-13-3 NaN3 99% 65.0 2.12 32.2
3.08 26628-22-8 DMF 102 ml 10% Pd/C 10% 106.4 0.36 0.3 0.03 EtOH
102 ml 19 95% 257.4 0.87 3.2 31% yield 23539-15-3
[0060] Compound 18 (6.25 g) and NaN.sub.3 (2.115 g) in 102 mL DMF
was heated at 100.degree. C. for 2 h. After this time, HPLC
indicated that the reaction was complete. The reaction mixture was
allowed to cool, and 10% K.sub.2CO.sub.3 aq solution (100 mL) was
added. The mixture was extracted 3 times with hexanes (60 mL) but
most of the azide separated out as an interphase solid, which was
dissolved in CH.sub.2Cl.sub.2. EtOH (100 mL) was added and the
solution concentrated to 100 mL. Pd/C catalyst (0.36 g) was added,
the flask flushed with N.sub.2, then evacuated and flushed with
H.sub.2 (from a balloon). The reaction was stirred under H.sub.2
for 3 hr, after which HPLC indicated that no starting material
remained. The reaction mixture was filtered and concentrated to
give 1.7 g of pale yellow oil (60% theory). The oil was dissolved
in water and then was purified by chromatography on C-18 (10 g, 60
mL). The column was first flushed with CH.sub.3CN, then with
water/0.05% TFA. The crude solution was loaded onto the column, and
was eluted with water (35 mL), followed by water/CH.sub.3CN 98:2
(20 mL), then 20 mL fractions in which the percentage of CH.sub.3CN
was increased by 2% each time, up to 16%. Fractions 2-11 contained
pure diamine by HPLC and were concentrated, to give 1.3 g of a
sticky white solid. After drying under vacuum, the weight fell to
0.87 g (31% theory).
N,N-bis(2-((E)-3-(hydroxyimino)-2-methylbutan-2-ylamino)ethyl)-4-methylben-
zenesulfonamide 2
##STR00011##
TABLE-US-00006 [0061] TABLE 6 purity MW amt amt d w/w g/mol g mmol
eq g/ml ml CA # 19 95% 257.4 0.85 3.1 1.0 22185-13-3 20 95% 135.6
3.60 25.2 8.0 3238-16-2 DIPEA 99% 129.2 3.40 26.0 8.3 0.742 4.60
7087-68-5 EtOH 20 ml 2 95% 455.6 0.535 1.1 36% yield
[0062] Tosyl diamine 19 (850 mg) was slurried with EtOH and cooled
in an ice water bath to 0-5.degree. C. A total of 3.4 g DIPEA
(diisopropylethylamine) and 3.6 g of 20 were added in 3 portions
over about 3 hours after which time HPLC showed that no free
diamine remained. The reaction mixture was concentrated, water (40
mL) was added, and the solution acidified with 1.5 mL HCl.sub.conc.
The aqueous layer was extracted with 2.times.50 mL
CH.sub.2Cl.sub.2. The pH of the aqueous layer was then adjusted to
10 with K.sub.2CO.sub.3 (a precipitate formed). The aqueous layer
was extracted with CH.sub.2Cl.sub.2 which was concentrated and
slurried with MeOH. The dialkylated product was collected by
filtration as a white solid. Dry weight was 535 mg (36%
theory).
Radiolabeling
[0063] Ligand stability studies: A stock solution (prepared by
dissolving .about.300 .mu.g of ligand in 4 mL of water; in the
cases of Compound 2, 4 mL of DMSO/H.sub.2O [1:1 v/v] was used) was
stored on bench top at room temperature without extra precautions.
LC-MS was taken every 24 h and no decomposition was observed after
3 days.
[0064] General labeling procedure (pH 9): To a 10 mL vial
containing 0.21 mL of cpn ligand (3) stock solution (16 .mu.g, 75
.mu.g/mL, aq.), 200 .mu.L of NaOAc (4 mg, 20 mg/mL, aq.), 0.5 mL of
pH 9 bicarbonate buffer and 13.2 .mu.L of MDP (13.2 .mu.g, 1.0
mg/mL, aq.), were added 1 mL of Na.sup.99mTcO.sub.4.sup.- solution
and 14.3 .mu.L of SnCl.sub.2.2H.sub.2O (36 .mu.g, 2.51 mg/mL, aq.),
sequentially. The pH value of the reaction solution was measured by
a pH strip and verified to be pH 9. The reaction mixture was
allowed to sit at room temperature for 15 min At the end of the
experiment, the pH value was measured again with a pH strip, and
the reaction solution was filtered through a filter (Acrodisc.RTM.
13 mm Syringe filter with 0.2 .mu.m Nylon Membrane, HPLC Certified
filter, Pall corporation, NY) on the tip of a 5 mL syringe. 2 mL of
water was pushed through the filter followed by the measurement of
radioactivities of combined filtrate, filter, original vial,
syringe and needle. An aliquot of the filtrate was subjected to
HPLC analysis.
[0065] General labeling procedure (pH 7.about.7.5, NaHCO.sub.3
buffer): To a 10 mL vial containing 0.21 mL of cpn ligand (3) stock
solution (16 .mu.g, 75 .mu.g/mL, aq.), 0.15 mL of 100 mM
NaHCO.sub.3 solution (pH 8.0-8.5) and 13.2 .mu.L of MDP (13.2
.mu.g, 1.0 mg/mL, aq.), were added 1 mL of
Na.sup.99mTcO.sub.4.sup.- solution and 14.3 .mu.L of
SnCl.sub.2.2H.sub.2O (36 .mu.g, 2.51 mg/mL, aq.), sequentially. The
pH value of the reaction solution was measured to be 7.about.7.5
(by a pH strip) and the reaction mixture allowed to sit at rt for
15 min. At the end of the experiment, the pH value was measured
again with a pH strip, and the reaction solution was filtered
through a filter (Acrodisc.RTM. 13 mm Syringe filter with 0.2 .mu.m
Nylon Membrane, HPLC Certified filter) on the tip of a 5 mL
syringe. 2 mL of water was pushed through the filter followed by
the measurement of radioactivities of combined filtrate, filter,
original vial, syringe and needle. An aliquot of the filtrate was
subjected to HPLC analysis.
[0066] General labeling procedure (pH 7-7.5): The above general
procedure was adopted with the following changes: pH value of the
solution was adjusted to 7 to 7.5 by the addition of a 0.1 N NaOH
solution after the addition of SnCl.sub.2.
[0067] General labeling procedure (pH 6): To a 10 mL vial
containing 0.42 mL of cpn ligand (3) stock solution (32 .mu.g, 75
.mu.g/mL, aq.), 0.025 mL of 100 mM NaHCO.sub.3 solution (pH
8.0-8.5) and 13.2 .mu.L of MDP (13.2 .mu.g, 1.0 mg/mL, aq.), were
added 1.21 mL of Na.sup.99mTcO.sub.4.sup.- solution and 14.3 .mu.L
of SnCl.sub.2.2H.sub.2O (36 .mu.g, 2.51 mg/mL, aq.), sequentially.
The pH value of the reaction solution was measured to be .about.6
(by a pH strip) and the reaction mixture was allowed to sit at rt
for 15 min At the end of the experiment, the pH value was measured
again with a pH strip, and the reaction solution was filtered
through a filter (Acrodisc.RTM. 13 mm Syringe filter with 0.2 .mu.m
Nylon Membrane, HPLC Certified filter) on the tip of a 5 mL
syringe. 2 mL of water was pushed through the filter followed by
the measurement of radioactivities of combined filtrate, filter,
original vial, syringe and needle. An aliquot of the filtrate was
subjected to HPLC analysis.
[0068] Analytical Methods HPLC conditions: All analytical studies
were performed on Waters Acquity UPLC system. Column: Waters
Acquity Analytical UPLC column (100.times.2.1 mm, C18, 1.7 .mu.m
BEH. Mobile Phase: solvent A is 0.4% ammonium formate in H.sub.2O
and solvent B is acetonitrile.
TABLE-US-00007 TABLE 7 Flow rate: 0.3 ml/min Time (mins) 0 3 10 15
16 20 % B 10 10 75 75 10 10
[0069] While only certain features of the invention have been
illustrated and described herein, many modifications and changes
will occur to those skilled in the art. It is, therefore, to be
understood that the appended claims are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
* * * * *