Note: Descriptions are shown in the official language in which they were submitted.
CA 02316392 2000-06-27
. ~ , , ' ~ , ~ . . ; , . . , ~ , ~ . , , < <
a , . ~ ~ ~ a , , , . , , , , ,
' ~ < < < , , , . ,
CHELATORS THAT PREDOMINATELY FORM A SINGLE STEREOISOMERIC ,
SPECIES UPON COORDINATION TO A METAL CENTER
Technical Field
This invention relates to chelators that form a mixture enriched for a single
stereoisomeric species upon coordination to a metal center.
Background of the Invention
The current interest in radiolabeling biologically important molecules
(proteins,
antibodies, and peptides) with 99mTc stems from the desire to develop a target
specific
diagnostic- radiopharmaceutical.l-lo_ ~e advantages- of using 99'"Tc in
diagnostic
nuclear medicine are well knownl i-is and a number of techniques have been
developed for the 99mTc labeling of biologically important molecules.l6.zo
One obvious approach is to coordinate a 99mTc metal directly with the
targeting
molecule. This approach is known as the direct labeling method and it involves
the
use of a reducing agent to convert disulfide linkages into free thiolates,
which then
bind to the 99mTc metal. A major disadvantage of this method is the lack of
control
over the coordination of the 99mTc metal and the stability of the resulting
metal
complex. In addition, the lack of suitable or accessible coordination sites in
some
proteins and peptides exclude direct labeling as a viable technique.
Two common alternatives to direct labeling are the final step labeling method
and the
pre-formed chelate method. Both techniques involve the use of a bifunctional
chelator, which provides the site of 99mTc coordination. The difference
between the
two methods lies in the order in which the 99mTc complex is formed. In the
final step
labeling method, complexation occurs after the chelator has been attached onto
the
targeting molecule. With the pre-formed chelate method, the 99mTc complex is
initially prepared and purified before being attached to the targeting
molecule. In both
methods, the bifunctional chelator must coordinate t0 99mTc to form a complex
that is
stable in vivo and the chelator must have an active moiety that can react with
a
functional group on the targeting molecule.
A number of bifunctional chelators have been used in the labeling of proteins,
peptides and monoclonal antibodies.2° 9~ io, m, 2i-2s Depending on the
chelator, the
labeling of biologically important molecules with bifunctional chelators often
results
AMENDED SHEET
CA 02316392 2000-06-27
<, " " ~< . "
. , < < . , , , , , ,
, ~ , , ~ , , , , , . . .
< < . ~ , , , . , . , < ,
, ~ , , , ,
, ,< , _, ~ . ..
in the formation of multiple species or isomeric complexes. An example is the
99"'Tc
labeling of molecules using the hydrazinonicotinamide (HYNIC) system. Since
the
HYNIC group can only occupy one or two sites of Tc coordination, co-ligands
are
required to complete the coordination sites. Glucoheptonate29-so
tris(hydroxymethyl)methylglycine (tricine)25, ethylenediamine-N, N'-diacetic
acid
(EDDA)9, water soluble phosphines25 [trisodium triphenylphosphine-3,3',3"-
trisulfonate (TPPTS), disodium triphenylphosphine-3,3'disulfonate (TPPDS), and
sodium triphenylphosphine-3-monosulfonate (TPPMS)] and polyamino
polycarboxylates9 have all been used as co-ligand in the HYNIC system.
It has been clearly shown that the Tc-99m labeling of molecules via the
HYNIC/co-
ligand system produces multiple species, which is due to the different
coordination
modalities of the hydrazine moiety and the co-ligands. The number of species,
the
type, the stability and the properties of the species vary greatly from one co-
ligand to
another. In the labeling of chemotactic peptides using the HYNIC system, the
nature
of the co-ligand also greatly affects the biodistribution of the labeled
peptide.31
Another example of a bifunctional chelator producing multiple species is
dithiosemicarbazone (DTS) system. It has been shown that the DTS bifunctional
chelator produces at least four complexes with technetium.32 Two of the
complexes
are known to be charged; hence they have different biodistribution from the
uncharged
species.
As in the development of a pharmaceutical based on organic molecules, the
stereochemistry or isomerism of a metal complex is very important in the
development of a radiopharmaceutical or metallodrug. It is well known that
isomers
can have different lipophilicities, biodistribution patterns, and biological
activities.
An example of this is the 99"'Tc complex of 3,6,6,9-tetramethyl-4,8-
diazaundecane-
2,10-dione dioxime (99mTc-d,l-HMPAO or Ceretec), which is a cerebral perfusion
imaging agent.14,33-3s Though 99"'Tc-d,l-HMPAO is active, it has been shown
that the
meso analogs of the 99°'Tc HM-PA0~4~3s complex and the 99'"Tc complex
of 3,3,9,9-
tetramethyl-4,8-diazaundecane-2,10-dione dioximela,37 (pnAO) do not possess
the
properties necessary for use as a cerebral perfusion imaging agent.
A type of Tc and Re coordination modality common in Tc and Re
radiopharmaceuticals is the coordination of a tetradentate N4_XSX chelator to
a metal
AMENDEt~ SHEET
CA 02316392 2000-06-27
' , ; . ~ , , . . ' , , , , , ,
a , , , , , ,
" ' , " < "
oxo moiety to form a square pyramidal or octahedral metal oxo complex. A host
of
bifunctional chelators have been developed based on the tetradentate N~XSX
coordination motif. Examples include N4 propylene amine oxime38, N3S triamide
thiols9~ 39-43' N2S2 ~~de dithiols9° 4a-as, N2S2 monoamide
monoaminedithiols4T~9 and
N2S2 diamine dithiolsso.ss, Fictionalization of the chelator backbone enables
these
chelators to be attached to biologically interesting molecules. The labeling
of these
bifunctional chelators with Tc03+ or Re03+ often produces isomers or
epimers.39-a3, as.
ss ,I,he isomers or epimers (syn and anti) arise from the configuration of the
metal oxo
group relative to the functional group on the chelator backbone. It has been
clearly
shown that the biodistribution and biological activity of the syn and anti
isomers are
often different.39~3, 46, s6 The Tc complex of
mercaptoacetylglycylglycylglycine
(MAG3), a renal imaging agent, exists in the syn and anti isomers. The
biological
activities of the syn and anti isomers are known to be different.3s,ao .tee
syn and anti
isomers of the Tc complex of 2,3-bis(mercaptoacetamide)propanoate (map) wwere
also shown to have different biological activities.46 It was reported that, in
humans,
58% of the syn isomer was excreted at 30 minutes as compared to only 19 % of
the
anti isomer. Another example of the isomers exhibiting a difference in
biological
behaviour is the 99mTc labeled diamino dithiol piperidine conjugates, which
were
investigated as brain perfusion imaging agents. It was shown that the two
isomeric
complexes exhibit widely disparate brain uptake.ss At 2 minute post-
administration in
rats, uptake of the anti isomer in the brain was 1.08 % dose/g, while the
uptake of the
syn isomer was 2.34 % dose/g. The brain/blood ratio at 2 minute post-
administration
was 2.09 for the anti isomer and 5.91 for the syn isomer.
The peptide dimethylglycine-L-serine-L-cysteine-glycine is a bifunctional
chelator
that can be used to label biologically important molecules.6i,6z It has been
shown that
dimethylglycine-L-serine-L-cysteine-glycine coordinates to Tc03+ and Re03+ via
a
monoamine diamide monothiol coordination modality.61 The resulting Tc and Re
complexes exist as two isomers; the serine CH20H side chain is in the syn and
anti
conformations with respect to the metal oxo bond. The presence of the syn and
anti
isomers is very evident from the NMR spectral data. In the'H NMR spectrum of
the
Re complex, there were two pairs of singlets associated with the nonequivalent
methyl
groups in the dimethylglycine residue . Each pair of singlets corresponded to
either
AMEN~FD ~~!E~i
CA 02316392 2000-06-27
a ~ ; ' ~ , ; , , y ~ ; ~, ; ,
the syn or anti isomers. The presence ~of the two isomers is clearly evident
from the ~ '
NMR data. In the coordination of dimethylglycine-L-isoleucine-L-cysteine-
glycine
(RP349) to Re03+, two isomers (syn and anti) were also observed. The 99"'Tc
labeling
of RP294 and RP349 produced syn and anti isomers; two peaks were observed in
the
HPLC using the radiometric detector. ~e 99mTc labeling of biotin with
dimethylglycine-L-lysine-L-cysteine-NH2 (RP332) also produced syn and anti
isomers; two peaks were observed in the HPLC. These results are consistent
with the
coordination of other tetradentate N4_xSX chelators to Tc03+ and Re03+.9, 39-
Ss
The labeling of biologically important molecules via a bifunctional chelator
can result
in the formation of isomers or multiple species, which can have significant
impact on
the biological properties of the radiopharmaceutical. For receptor-based
radiopharmaceuticals, the target uptake is largely dependent on the receptor
binding
affinity of the targeting molecule and the blood clearance of the labeled
molecule,
which is determined by the physical properties of both the targeting molecule
and the
metal chelate. Hence, the presence of isomers for the metal chelate can have
significant impact on the radiopharmaceutical. Therefore, in the development
of a
radiopharmaceutical or metallodrug, it is necessary to separate the isomers
and
evaluate the biological activities of each individual isomer. It would
therefore be
desirable to develop chelators that predominately form a single stereoisomeric
species
upon coordination to a metal center.
An article entitled Imaging a Model of Colitis with RP128, A Tc-99m Chelated
Tuftsin Antagonist by S.H. Peers et al (The Journal of Nuclear Medicine,
Proceedings
of 42nd Annual Meeting, vol. 36., 15 June 1995, page 114, XP002102963)
discloses
the ability of Tc99M-RP 128 to image rat model inflammatory disease. The
article
does not discuss isomerization of the targeting agent.
An article entitled Rhenium (~ and Technetium (~ Oxo Complexes of an N2N'S
Peptidic Chelator: Evidence of Interconversion Between the Syn and Anti
Conformations by Wong et al (Inorg Chem. (1997), 36(25), 5799-5808) shows the
radiolabelling of oxo complexes of the peptide RP294. Although interconversion
between syn and anti isomers is shown, a compound that predominately forms a
single
isomer upon coordination to a metal center is not disclosed.
4
ANILP~DED S~IcET
CA 02316392 2000-06-27
;, ,, ,,~, ,,, ',,, ,
' ' ~ , , , ; , ~ , , , . a , , ,
, , , , . , , , , ,
:, , ~, , " "
European Patent Application 0284071 discloses chelated radionuclide
compositions
that are provided for conjugation to polypeptides and carbohydrates.
International Publication Number WO 95/33497 describes radiopharmaceuticals
for
targeting sites within a mammalian body. In particular, the
radiopharmaceuticals
comprise targeting molecules that are covalently linked to monoamine, diamine,
thio-
containing metal chelators.
International Publication Number WO 96/40293 describes metallo-constructs that
can
be used for diagnostic imaging and therapy.
International Publication Number WO 95/22996 describes peptide-chelator
conjugates
that are useful for diagnostic imaging of sites of inflammation.
International Publication Number WO 96/03427 describes peptide derived
radionuclide chelators for use in imaging sites of disgnostic interest within
the body.
AM~r~~~~ s~-~~~T
CA 02316392 2000-06-27
. , . . y , , : ,
. . ~ . . ~ ~ ~ ,
' ; , ~ , . ~ ; ~ ; ; , . , ~ ; a : , , .
Summary of the Invention
Chelators and chelator-targeting molecule conjugates are provided that form a
mixture
with a predominant stereoisomeric species upon coordination to a metal center.
According to an aspect of the invention, there is provided a chirally pure
compound of
the formula I:
R8
R7 S R9
R3 R4 O
R' ~ R ~ o
wN ~ w
O R5 R6 O
R2
wherein
Rl is a linear or branched, saturated or unsaturated Cl~allcyl chain that is
optionally
interrupted by one or two heteroatoms selected from N, O and S; and is
optionally substituted by one or more substituents selected from halogen,
hydroxyl, amino, carboxyl, Cl.~alkyl, aryl and C(O)Rlo;
R2 is H or a substituent defined by R';
Rl and R2 may together form a 5- to 8-membered saturated or unsaturated
heterocyclic
ring optionally substituted by one or more substituents selected from halogen,
hydroxyl, amino, carboxyl, oxo, CI.~aIkyl, aryl and C(O)Z;
R3, R4 and RS are selected independently from H; carboxyl; Cl~alkyl;
Cl.~allcyl
substituted with a substituent selected from hydroxyl, amino, sulfllydryl,
halogen, carboxyl, Cl.~allcoxycarbonyl and aminocarbonyl; an alpha carbon side
chain of a D- or L-amino acid other than proline; and C(O)Rlo;
R6 is selected from a group consisting of
i) an optionally subsituted 3- to 6-membered heterocylic or carbocylic ring,
ii) a compound of the following formula:
rr~rr~~~~ s~EE-r
CA 02316392 2000-06-27
' ; ~ , '' ' , ~ , . ; ' , . ' . .
R~~ ' ; ',.' ~ '<. '
I
C R~2
R~3
wherein Rll, Ri2 _ and R13 are independently selected from H, linear or
branched, saturated or unsaturated Cl.~alkyl chain that is optionally
interrupted
by one or two heteroatoms selected from N, O and S; and is optionally
substituted by one or more substituents; alkoxycarbonyl, alninocarbonyl,
allcoxy,
an optionally subsituted 3- to 6-membered heterocylic or carbocylic ring; with
the proviso that a least one of RI1, Ri2 and R13 is not H; .
iii) a compound of the following formula:
R ~4
R~s
wherein Ri4 and Rls are independently selected from H, linear or branched,
saturated or unsaturated CL~alkyl chain that is optionally interrupted by one
or
two heteroatoms selected from N, O and S; and is optionally substituted by one
or more substituents (; alkoxycarbonyl, aminocarbonyl, alkoxy, an optionally
subsituted 3- to 6-membered heterocylic or carbocylic ring; with the proviso
that
a least one of R14 and Rls is not H;
and iv) a compound of the following formula:
R~s
X
wherein X is selected from O or S and R16 is selected from linear or branched,
saturated or unsaturated CI_6alkyl chain that is optionally interrupted by one
or
two heteroatoms selected from N, O and S; and is optionally substituted by one
or more substituents; alkoxycarbonyl, aminocarbonyl, alkoxy, and an optionally
subsituted 3- to 6-membered heterocylic or carbocylic ring;
R' and R8 are selected independently from H; carboxyl; amino; Cl.~alkyl;
C~.~allcyl
substituted by a substituent selected from hydroxyl, carboxyl and amino; and
C(O)Rlo;
AMENDcp SWEET
CA 02316392 2000-06-27
a , , , , , , , ,
R9 is selected from H and a sulfur protecting~group; and 1
R1° is selected from hydroxyl, allcoxy, an amino acid residue, a
linking group and a
targeting molecule.
According to another aspect of the invention, there is provided a chirally
pure compound
of the formula II:
Ra O Rb R~ O Rf Rg
S~ b Rn
N '
i
d a
H R R C)
wherein
R8 is selected from H and a sulfur protecting group;
Rb, R° Rd, Rf and Rg are selected independently from H; carboxyl;
C»allcyl; Cl.~alkyl
substituted with a substituent selected from hydroxyl, amino, sulfhydryl,
halogen, carboxyl, Cl~alkoxycarbonyl and aminocarbonyl; an alpha carbon side
chain of a D- or L-amino acid other than proline; and C(O)Rh;
R' is selected from a group consisting of
an optionally subsituted 3- to 6-membered heterocylic or carbocylic ring;
and
R~
C R~
Rk
wherein R', R~ and R~' are independently selected from H, linear or branched,
saturated or unsaturated C»allcyl chain that is optionally interrupted by one
or
two heteroatoms selected from N, O and ~S; and is optionally substituted by
one
or more substituents; alkoxycarbonyl, aminocarbonyl, alkoxy, an optionally
s AMENDED SHEET,
CA 02316392 2000-06-27
;' ', ~ ~ ,' ; ; , , , . ,
' ; , , ' , , ~ , ,
~. , " ,.
subsituted 3- to 6-membered heterocylic or carbocylic ring; with the proviso
that
a least one of R', R' and Rk is not H;
and
R~
Rm
wherein R~ and R"' are independently selected from H, linear or branched,
saturated or unsaturated Cl.~allcyl chain that is optionally interrupted by
one or
two heteroatoms selected from N, O and S; and is optionally substituted by one
or more substituents; alkoxycarbonyl, aminocarbonyl, alkoxy, an optionally
subsituted 3- to 6-membered heterocylic or carbocylic ring; with the proviso
that
a least one of R~ and Rm is not H;
and
Rn
X
wherein X is selected from O or S and Rn is selected from linear or branched,
saturated
or unsaturated Cl~alkyl chain that is optionally interrupted by one or two
heteroatoms
selected from N, O and S; and is optionally substituted by one or more
substituents;
allcoxycarbonyl, aminocarbonyl, alkoxy, and an optionally subsituted 3- to 6-
membered
heterocylic or carbocylic ring; and
Rh is selected from hydroxyl, alkoxy, an amino acid residue, a linking group
and a
targeting molecule.
According to another aspect of the invention, the chelator-targeting molecule
conjugates
are provided in combination with a diagnostically useful metal or an oxide or
nitride
thereof.
According to another aspect of the present invention, there is provided a
method of
imaging a site of diagnostic interest, comprising the step of administering a
diagnostically effective amount of a composition comprising a chelator-
targeting
molecule conjugate which is complexed to a diagnostically useful metal or an
oxide or
nitride thereof.
~MEPdDED SHEET
CA 02316392 2000-06-27
Detailed Description of the Invention
In the coordination of dimethylglycine-t-butylglycine-cysteine-glycine to
Tc03+ and
Re03+, a single isomer was observed. A single pair of singlets associated with
the
methyl groups in the dimethylglycine residue was observed. The 99"'Tc labeling
of
dimethylglycine-L-t-butylglycine-L-cysteine-glycine (RP455) and of
dimethylglycine-
D-t-butylglycine-L-cysteine-glycine (RPSOS) produced a single peak as observed
in
the HPLC using the radiometric detector. This was an unexpected result and
contrasted with what was observed in the Tc and Re oxo complexes of other
tetradentate N4-xSx chelators,9~ 39-ss which existed as the syn and anti
isomers.
The presence of a sterically bulky group in the side chain of the peptidic
chelator
caused the formation of a single isomeric metal complex. In the cases of
dimethylglycine-L-lysine-L-cysteine and dimethylglycine-L-serine-L-cysteine-
glycine,
there was insufficient bulk to cause one isomer to be preferred over another;
hence
the ratio of the syn and anti isomers was approximately 1:1.
In the case of dimethylglycine-L-isoleucine-L-cysteine, a more sterically
bulky
CH(CH3)-CH2-CH3 group was introduced into the peptidic backbone. This
additional
bulk caused the ratio of the syn and anti isomers to be 3:1; hence, one isomer
was
more favored over the other. In the case of dimethylglycine-t-butylglycine-
cysteine-
glycine, the incorporation of the C(CH3)3 group introduced su~cient bulk into
the
peptide to cause one of the isomers to be completely favored over the other;
hence, a
single isomeric metal complex was observed.
Molecular modeling with Quanta Charm indicated that the syn isomer was
favoured
because in the anti isomer there was steric interaction between the bulky side
group
and the oxygen atoms of the adjacent amide groups. For example, the dihedral
angles
of the beta carbon of serine with the backbone of the chelate in the anti
isomer of the
Re complex of dimethylglycine-L-serine-L-cysteine-glycine (ReORP414) were -
27.39° (O-C-C-C) and 8.35° (C-C-N-C). The corresponding dihedral
angles for the
anti isomer of the Re complex of dimethylglycine-L-t-butylglycine-L-cysteine-
glycine
(ReORP455) were -11.95° and -6.87°. The difference of about
15° for each angle
was a result of the shift of the amide oxygen atoms and the side group atoms
of
ReORP455 to a position of least contact. The shift of atomic positions induced
some
strain on the chelate system and therefore lessened its stability.
to AMEP~DED SF~EET
CA 02316392 2000-06-27
.. .~. ., r ., ,.
. . ~ . ~ a . ~ . . . ,
. ; ~ . ~ . . , . ,
. ~ . , , . , . , , ,
' " ' , ' , a , . . . ,
Molecular modeling of each of the Re complexes of the peptides was in
agreement
with experimental results. Molecular modeling of the Re complex of
dimethyglycine-L-serine-L-cysteine-glycine showed the two isomers possessing
thermodynamic potential energies of -67.02 and -68.37 kcal/mole. There was
only a
small difference in the energy of the two isomers. There was no preferred
isomer for
the Re complex and both the syn and anti isomers were observed at an
approximate
ratio of 1:1. Molecular modeling of the Re complex of dimethylglycine-L-lysine-
L-
cysteine showed a difference between the thermodynamic potential energies of
the
two isomers to be approximately 1 kcal/mole. There was again only a small
difference in the energy of the two isomers; hence, both the syn and anti
isomers
would be observed.
In the case of dimethylglycine-L-isoleucine-L-cysteine-glycine, a more bulky
side
chain was incorporated into the peptidic backbone. Molecular modeling of the
Re
complex of the dimethylglycine-L-isoleucine-L-cysteine-glycine showed one of
the
isomers having a potential energy that was approximately 3 kcaUmole lower than
the
energy of the other isomer. There was now a greater difference in the energies
and
there was a slight preference for one isomer over the ather. Accordingly, the
observed
experimental ratio of the two isomers was 3:1.
In the case of dimethylglycine-L-t-butylglycine-L-cysteine-glycine, molecular
modeling of the Re complex showed the difference in the potential energies of
the two
isomers to be approximately 6.5 kcal/mole. With the Re complex of
dimethylglycine-
D-t-butylglycine-L-cysteine-glycine, the difference in the energies of the two
isomers
was about 8.5 kcaI/mole. One isomer was significantly preferred over the
other;
hence, only a single isomer was observed for the Re and Tc complexes.
Molecular modeling of the Re complex of mercaptoacetyl-L-t-butylglycine-
glycine-
glycine showed that the syn and a»ti isomers of the complex with a energy
difference
of 7.4. The metal complexes of mercaptoacetyl-L-t-butylglycine-glycine-glycine
preferred one isomer over the other and would exist as a single isomer.
Artificial amino acids with bulky side chains can be prepared according to
known
literature methods.63'~~ For example, both L- and D- amino acid derivatives
can be
prepared starting directly from the commercially available L- or D-serine,
respectively.6~ Using this method, alkyl, phenyl and other bulky groups can be
11
AMENDED SHEET
CA 02316392 2000-06-27
, : a . , , , , , , .
. . , , , ~ ; ; a
,~ .,
incorporated into serine to produce (3-hydroxy-a-amino acids.67 Hence,
artificial
amino acids with bulky side chains can be incorporated into peptidic
chelators, which
would produce a single species and a single isomeric metal complex.
The advantage of having a bifunctional chelator that forms a single isomeric
metal
complex is that in the labeling of biologically important molecules, there is
only a
single radiolabeled species. Hence, there is no need to isolate and evaluate
the
biological activity and toxicity of multiple compounds. It is also easier to
formulate a
radiopharmaceutical kit that consistently produces a single radiolabeled
compound
than one that produces a series of radiolabeled compounds. In the labeling of
a
biologically important molecule with a chelator that results in multiple
species, there
is a necessity to formulate the kit such that the labeling consistently
produces the same
set of compounds in the same ratio. This is eliminated with the use of a
chelator that
produces a single metal complex. Quality control of a radiopharmaceutical is
also
simplified by the use of a chelator that results in a single species as it is
much easier to
develop a quality control protocol that identifies a single well characterized
compound
than one that has to identify the presence and quantity of multiple compounds.
An additional benefit of the incorporation of different side chain groups into
the
peptidic chelator backbone to cause a single isomer is that the lipophilicity
of the
resulting metal complexes is altered by the addition of the different groups.
The log D
of the 99'"Tc complex of dimethylglycine-L-t-butylglycine-L-cysteine-glycine
is -1.3,
compared to -2.3 for the 99mTc complex of dimethylglycine-L-serine-L-cysteine-
glycine.
The terms defining the variables Rl - Rl° , Ra - R° and X as
used hereinabove in formula
(1] have the following meanings:
"alkyl" refers to a straight or branched C~-Cg chain and includes lower C~-C4
alkyl;
"alkoxy" refers to straight or branched C1-Cg allcoxy and includes lower C1-C4
allcoxy;
"thiol" refers to a sulfhydryl group that may be substituted with an alkyl
group to form a
thioether;
"sulfur protecting group" refers to a chemical group that is bonded to a
sulfur atom and
inhibits oxidation of sulfur and includes groups that are cleaved upon
chelation of the
12
~~~CN~~~ J~~L'T
CA 02316392 2000-06-27
, a ~ , . . ~ . ; , a .
. < , ~ ~ ~ ,
, , . ~ ~ , , , . < .
' , . , . , . , .
,, " "
.
metal. Suitable sulfur protecting groups include ~ known alkyl, aryl, acyl,
alkauoyl,
aryloyl, mercaptoacyl and organothio groups.
"Linlting group" refers to a chemical group that serves to couple the
targeting molecule
to the chelator while not adversely affecting either the targeting function of
the peptide
or the metal binding function of the chelator. Suitable linking groups include
alkyl
chains; alkyl chains optionally substituted with one or more substituents and
in which
one or more carbon atoms are optionally replaced with nitrogen, oxygen or
sulfur atoms.
Other suitable linking groups include those having the formula AI-A2-A3
wherein A'
and A3 are independently selected from N, O and S; and A2 includes allcyl
optionally
substituted with one or more substituents and in which one or more carbon
atoms are
optionally replaced with nitrogen, oxygen or sulfur atoms; aryl optionally
substituted
with one or more substituents; and heteroaryl optionally substituted with one
or more
substituents. Still other suitable linking groups include amino acids and
amino acid
chains functionalized with one or more reactive groups for coupling to the
glycopeptide
and/or chelator. In one embodiment, the linking group is a peptide of 1 to 5
amino acids
and includes, for example, chains of 1 or more synthetic amino acid residues
such as 13-
Alanine residues. In another embodiment, the linking group is NH-alkyl-NH.
"Targeting molecule" refers to a molecule that can selectively deliver a
chelated
radionuclide or MRI contrasting agent to a desired location in a mammal.
Preferred
targeting molecules selectively target cellular receptors, transport systems,
enzymes,
glycoproteins and processes such as fluid pooling. Examples of targeting
molecules
suitable for coupling to the chelator include, but are not limited to,
steroids, proteins,
peptides, antibodies, nucleotides and saccharides. Preferred targeting
molecules include
proteins and peptides, particularly those capable of binding with specificity
to cell
surface receptors characteristic of a particular pathology. For instance,
disease states
associated with over-expression of particular protein receptors can be imaged
by
labeling that protein or a receptor binding fragment thereof coupled to a
chelator of
invention. Most preferably targeting molecules are peptides capable of
specifically
binding to target sites and have three or more amino acid residues. The
targeting
moiety can be synthesised either on a solid support or in solution and is
coupled to the
next portion of the chelator-targeting moiety conjugates using known
chemistry.
13
AMENDE~J ~~-itET
CA 02316392 2000-06-27
a . . , , , . ~ ; ; ;
' , ' ~ ~ ; , , , ,
,.
Chelator conjugates of the invention may be prepared by various methods
depending
upon the chelator chosen. The peptide portion of the conjugate if present is
most
conveniently prepared by techniques generally established in the art of
peptide synthesis,
such as the solid-phase approach. Solid-phase synthesis involves the stepwise
addition
of amino acid residues to a growing peptide chain that is linked to an
insoluble support
or matrix, such as polystyrene. The C-terminus residue of the peptide is first
anchored
to a commercially available support with its amino group protected with an N-
protecting
agent such as a t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl
(FMOC) group. The amino protecting group is removed with suitable deprotecting
agents such as TFA in the case of tBOC or piperidine for FMOC and the next
amino
acid residue (in N-protected form) is added with a coupling agent such as
dicyclocarbodiimide (DCC). Upon formation of a peptide bond, the reagents are
washed from the support. After addition of the final residue, the peptide is
cleaved from
the support with a suitable reagent such as trifluoroacetic acid (TFA) or
hydrogen
fluoride (I-~).
Conjugates may further incorporate a linking group component that serves to
couple the
peptide to the chelator while not adversely affecting either the targeting
function of the
peptide or the metal binding function of the chelator.
In accordance with one aspect of the invention, chelator conjugates
incorporate a
diagnostically useful metal capable of forming a complex. Suitable metals
include
radionuclides such as technetium and rhenium in their various forms such
as99'"Tc03+,
~mTc02+, Re03+ and Re02+. Incorporation of the metal within the conjugate can
be
achieved by various methods common in the art of coordination chemistry. When
the
metal is technetium-99m, the following general procedure may be used to form a
technetium complex. A peptide-chelator conjugate solution is formed initially
by
dissolving the conjugate in aqueous alcohol such as ethanol. The solution is
then
degassed to remove oxygen then thiol protecting groups are removed with a
suitable
reagent, for example with sodium hydroxide and then neutralized with an
organic acid
such as acetic acid (pH 6.0-6.5). In the labelling step, a stoichiometric
excess of sodium
pertechnetate, obtained from a molybdenum generator, is added to a solution of
the
conjugate with an amount of a reducing agent such as stannous chloride
sufficient to
reduce technetium and heated. The labelled conjugate may be separated from
H.MEND~D ~~-!~rT
CA 02316392 2000-06-27
- . , , ~ (, , ~
( ' ; , ' ' ~ , , , ,
. , . , , , , . , , ,
, ~ . ~ , a v , , ,
t ( . , f f i ( n .
contaminants 99"'Tc04 and colloidal 99mTc02 chromatographically, for example
with a
C-18 Sep Pak carhidge.
In an alternative method, labelling can be accomplished by a transchelation
reaction.
The technetium source is a solution of technetium complexed with labile
ligands
facilitating ligand exchange with the selected chelator. Suitable ligands for
transchelation include tartarate, citrate and heptagluconate. In this instance
the preferred
reducing reagent is sodium dithionite. It will be appreciated that the
conjugate may be
labelled using the techniques described above, or alternatively the chelator
itself may be
labelled and subsequently coupled to the peptide to form the conjugate; a
process
referred to as the "prelabelled ligand" method.
Another approach for labelling conjugates of the present invention involves
techniques
described in International Publication Number WO 95/13832, incorporated herein
by
reference. Briefly, the chelator conjugates are immobilized on a solid-phase
support
through a linkage that is cleaved upon metal chelation. This is achieved when
the
chelator is coupled to a functional group of the support by one of the
complexing atoms.
Preferably, a complexing sulfur atom is coupled to the support which is
functionalized
with a sulfur protecting group such as maleimide.
A conjugate labelled with a radionuclide metal such as technetium-99m may be
administered to a mammal by intravenous injection in a pharmaceutically
acceptable
solution such as isotonic saline. The amount of labelled conjugate appropriate
for
administration is dependent upon the distribution profile of the chosen
conjugate in the
sense that a rapidly cleared conjugate may be administered in higher doses
than one that
clears less rapidly. Unit doses acceptable for imaging inflammation are in the
range of
about 5-40 mCi for a 70kg individual. In vivo distribution and localization is
tracked by
standard scintigraphic techniques at an appropriate time subsequent to
administration;
typically between 30 minutes and 180 minutes depending upon the rate of
accumulation
at the target site with respect to the rate of clearance at non-target tissue.
AMENDED ~ ~ v~ i
CA 02316392 2000-06-27
. , , , , . . ,
a ~ ~ , ; , , , , < < ; . . ;
, , ~ , , , , , , ,
. ,
List of Abbreviations
Abbreviation Description
Acm acetoamidomethyl
Ar argon
Arg arginine
Boc tent-butyloxycarbonyl
Cys cysteine
DIEA diisopropylethylamine
Dimethylgly N,N-dimethylglycine
DMF N,N-dimethylformamide
ES-MS Electron Spray Mass Spectrometry
Fmoc 9-fluorenylmethyloxycarbonyl
Gly glycine
HBTU 2-(1H-benzotriazol-1-yl}-1,1,3,3-tetramethyl-uronium
hexafluorophosphate
HOBT 1-hydroxybenzotriazole
HPLC high performance liquid chromatography
Ile isoleucine
Leu leucine
Lys lysine
mL millilitre(s)
mmol millimole(s)
mol moles}
Mott 4-methoxytrityl
NaOH sodium hydroxide
NMP N-methylpyrrolidone
Phe phenylalanine
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
R~ retention time
sasrin 2-methoxy-4-alkoxybenzyl alcohol (super
acid sensitive
resin)
Ser serine
t-Bu tert-butyl
A~IEf~DE~ E~-~~==v
CA 02316392 2000-06-27
. . ~ ; ;
" , " , " , .
TFA trifluoroacetic acid
Thr threonine
Trt trityl
Tyr tyrosine
YE-R protection group R is attached to the peptide chain via the atom,
Y, ~ on the amino acid side chain (Y is N, O or S and R is
Acm, Boc, Molt, t-Bu or Trt)
Ezamules
Materials. N-methylpyrrolidone, N,N-dimethylformamide, 100 mmol 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate/ O.SM 1-
hydroxybenzotriazole DMF, 2.OM diisopropylethylamine/ NMP, dichloromethane and
trifluoroacetic acid were purchased from Applied Biosystems Inc. Triethylamine
and
tert-butyl methyl ether were purchased from Aldrich Chemical Inc. Fmoc amino
acid
derivatives and Fmoc-Gly sasrin resin was purchased from Bachem Bioscience
Inc.
All chemicals were used as received. [Re02(en)2)Cl was prepared according to
literature methods.s~°ss
Instrumentation. NMR spectra were recorded on a Broker AC-300 and on a Broker
DRX-500 NMR spectrometer and are reported as S in ppm from external TMS. Mass
spectra (electrospray) were obtained on a Sciex API#3 mass spectrometer in the
positive ion detection mode. HPLC analyses and purifications were made on a
Beckman System Nouveau Gold chromatographic system with a Waters 4 mrn radial
pak C-18 column. During analytical HPLC analysis, the mobile phase was changed
from 100% 0.1% aqueous trifluoroacetic acid to 100% acetonitrile containing
0.1%
trifluoroacetic acid over 20 minutes at a flow rate of 2 mL/min. All HPLC
analyses
were monitored with a UV detector set at 214 and 254 nm. Solid phase peptide
syntheses were performed on an ABI Peptide Synthesizer model 433A using
FastMoc
chemistry and preloaded Fmoc amino acid sasrin resin.59>6o Molecular modeling
of
the Re complexes was performed using Quanta Charm version 3.3.63 HPLC analyses
of the 99mTc samples were made on a Beckman System Gold chromatographic system
with a Vydac 4.6 mm radial pak C-18 column. The mobile phase was changed from
AMENDED Si-~rtT
CA 02316392 2000-06-27
a ; ; ~ , , , , , , ~ ; , ,
< , ~ , "
100% water containing 0.1 % trifluoroacetic acid to~ 70% acetonitrile
containing 0.1
trifluoroacetic acid over 25 minutes at a flow rate of 1 mL/min. The HPLC
analyses
of the 99'"Tc samples were monitored with a W detector set at 215 nm and a
radiometric gamma detector.
Example 1
Synthesis of Peptides. Peptides of various amino acid sequences were prepared
via a
solid phase peptide synthesis method on an automated peptide synthesizer using
FastMoc 1.0 mmole chemistry.s9°6o preloaded Fmoc amino acid sasrin
resin and Fmoc
amino acid derivatives were used. Prior to the addition of each amino acid
residue to
the N-terminus of the peptide chain, the FMOC group was removed with 20%
piperidine in NMP. Each Fmoc amino acid residue was activated with 0.50 M
HBTU/
HOBt/ DMF, in the presence of 2.OM DIEA/ NMP. The C-terminus of the completed
peptide was attached to the resin via the sasrin linker. The peptidyl resin
was washed
with dichloromethane and dried under vacuum for 20-24 hours. The peptide was
cleaved off the resin by stirring the peptidyl resin in 95 % aqueous
trifluoroacetic acid
for 3-4 hours. The sasrin resin was filtered and the filtrate was added
dropwise to tert-
butyl methyl ether at 0 °C. The peptide precipitate out of the ether.
The precipitate
was collected by centrifugation and dissolved in minimal amount of water. The
aqueous peptide solution was lyophilized to yield the product. The product was
analyzed by mass spectrometry and by HPLC. The product was purified by HPLC.
This method was used to prepare the following peptides
1)RP349: Dimethylgly-L-Ile-L-Cys(SE-Acm)-Gly
2)RP332: Dimethylgly-L-lysine(N~-Biotin)-L- Cys(S~-Acm)
3)1ZP455: Dimethylgly-L-t-Butylgly-L-Cys(Se-Acm)-Gly
4)RP505: Dimethylgly-D-t-Butylgly-L-Cys(S~-Acm)-Gly
5)RP502: Dimethylgly-L-t-Butylgly-L-Cys(SE-Acm)-Gly-Thr-Lys-Pro-Pro-Arg
6)RP573: Dimethylgly-L-t-Butylgly-L-Cys(SE-Acm)-Gly-Arg-Ile-Lys-Pro-His
Example 2
Synthesis of Re Oxo Complex of Dimethylglycine-L-t-butylgly-L-Cys-Gly: To
remove the acm protecting group, dimethylgly-L-t-butylgly-L-Cys-(SE-Acm)-Gly
APJIENG'G SH~~T
CA 02316392 2000-06-27
, ~ . ~ , , ~ , < , , ,
,~ , " , <, ,.
(84.0 mg, 0.187 mmoles) was dissolved in 2 mL of 30% acetic acid. Mercury(In
acetate (91.6 mg, 0.287 mmoles) was added to the solution and the solution was
stirred under Ar at room temperature for 18 hours. H2S gas was then bubbled
through
the solution for 5 minutes, causing black HgS to precipitate. The precipitate
was
removed by centrifugation, and the filtrate was frozen and lyophilized
overnight.
[Re02(en)2]Cl (88.6 mg, 0.237 mmoles) was dissolved in 3 mL of distilled water
and
added to the lyophilized deprotected peptide. The solutions was a light green
colour.
The pH of the solution was adjusted to 6 using 1 M NaOH. The solution was
refluxed
under Ar for 2 hours, during which time the solution changed from green to
red. The
solution was frozen and lyophilized overnight, yielding a red solid.
Purification of the
product was done by HPLC. Mass spectrum (electrospray): m/z = 577 ([MH]~,
[C15H27N4O6Re1S1]. HPLC retention time: 9.52 min. 1H NMR and 13C NMR (500
MHz, D20) spectral data are shown in Table 3 and 4. Log D (pH: 7.4): -1.3.
Example 3
Synthesis of Re Ogo Complex of Dimethylgly-D-t butylgly-L-Cys-Gly: The
procedure for the synthesis of the Re oxo complex of dimethylgly-D-t-butylgly-
L-Cys-
Gly was the same as the one described for the synthesis of the Re complex of
Dimethylgly-L-t-butylgly-L-Cys-Gly. Mass spectrum (electrospray): m/z = 577
([~]~~ [CisH26NaOsRelS1]. HPLC retention time: 9.62 min. 'H NMR (300 MHz,
D20): 2.89 (s, methyl 1H in the dimethylglycine residue), 3.65 (s, methyl 1H
in the
dimethylglycine residue).
Exan~le 4
Synthesis of Re Ozo Complez of Dimethylgly-L-t-Butylgly-L-Cys-Gly-Thr-Lys-
Pro-Pro-Arg: The procedure for the synthesis of the Re oxo complex Dimethylgly-
L-
t-Butylgly-L-Cys-Gly-Thr-Lys-Pro-Pro-Arg was the same as the one described for
the
synthesis of the Re complex of dimethylgly-L-t-butylgly-L-Cys-Gly. Mass
spectrum
(electrospray): mlz = 1155 ([MH]~, [C41H71Ni3O~zReiSi]+). HPLC retention time:
8.82 min. 'H NMR (500 MHz, D20): 2.63 (s, methyl IH in the dimethylglycine
residue), 3.56 (s, methyl 1H in the dimethylglycine residue).
19
Ai~JELjs~J~~ ~~'~_~~-
CA 02316392 2000-06-27
. " , ~ . . . .
, , , . , . , ~, : . . ; ~ ,
. , , , , ~ , . . , , , ,
. ~ . , , . , : ,
,. . :< . " ..
Example 5
Synthesis of Re Ogo Comple$ of DimethylgIy-L-lle-L-Cys-Gly: The procedure for
the synthesis of the Re oxo complex Dimethylgly-L-ile-L-cys-gly was the same
as the
one described for the synthesis of the Re complex of dimethylgly-L-t-butylgly-
L-cys-
gly. Mass spectrum (electrospray): mlz = 577 ([MH]~, [C41H~INi30~2ReiS1]~, mlz
=
598 ([MH]+, [C41H~IN13012ReiS1]~. HPLC retention time: 9.50 min. 1H NMR (300
MHz, D20): 2.60 (s, methyl 1H in the dimethylglycine residue of isomer A),
2.76 (s,
methyl 1H in the dimethylglycine residue of isomer B), 3.68 (s, methyl IH in
the
dimethylglycine residue of isomer A), 3.72 (s, methyl iH in the
dimethylglycine
residue of isomer B).
Example 6
Synthesis of the Re Ozo Complex of Dimethylgly-L-t-Butylgly-L-Cys-GIy-Arg-
lle-Lys-Pro-His: The procedure for the synthesis of the Re oxo complex of
dimethylgly-L-t-Butylgly-L-Cys-Gly-Arg-Ile-Lys-Pro-His was the same as the one
described for the synthesis of the Re complex of dimethylgly-L-t-butylgly-L-
Cys-Gly.
Mass spectrum (electrospray): m/z= 1207 ([MH]+), [C43H~1NisOioRe~S1]. HPLC
retention time: 8.78 min. iH NMR (300 MHz, D20): 2.71 (s, methyl 1H in the
dimethylglycine residue), 3.65 (s, methyl'H in the dimethylglycine residue).
Example 7
Synthesis of the 99'°Tc complex. The peptide (0.2-0.5 ,moles) was
dissolved in 200
~.L, of saline. Na[99mTc04] (10 mCi) was added to the solution , followed by
tin(I~
chloride (7.5 x 103 fig, 39 pmoles), sodium gluconate (1.3 x 103 ~.g, 5.8
N,rnoles), and
20 ~,L of 0.1 M NaOH. The solution was left at room temperature for 1 hour or
heated at 100 °C for 15 minutes. In the synthesis of the ~mTc complex,
the
acetoamidomethyl protection group was displaced from the cysteine residue in
RP414.
The 99"'Tc complex was analyzed by HPLC. The 99"'Tc complexes of RP455, RP505
and RP502 was co-injected with the corresponding Re complexes. The HPLC
retention times of the 99mTc peptidic complexes are as follows:
1 )99mTc complex of RP349 (Dimethylgly-L-Ile-L-Cys-Gly): HPLC retention time:
99mTc(RP349) RL = 19.41, 21.53 min (radiometric gamma detector).
2o f,f,prp~~il~~: ~~'~i-~-
~IY:L~~L ~.
CA 02316392 2000-06-27
. ' , , , , , , , ,
' ' : : : , ' ~ : ~ . , ' , , : , .
, < , , , : , , , ~ ,
2)99'"Tc complex of RP332 (Dimethylgly-L-lysine(Ne-Biotin)-L- Cys): HPLC
retention time: 99mTc(RP332) Rt = 11.54, 11.97 min (radiometric gamma
detector).
3)99'"Tc complex of RP455 (Dimethylgly-L-t-Butylgly-L-Cys-Gly): HPLC
retention time: Re0(RP455) Rt = 21.18 min (UV detector set at 215 nm);
99mTc(RP445) Rt = 21..49 min (radiometric gamma detector).
4) 99mTc complex of RP505 (Dimethylgly-D-t-Butylgly-L-Cys-Gly): HPLC
retention time: Re0(RP505) RL = 18.16 min (UV detector set at 215 nm);
99mTc(RP505) Rt =18.89 min (radiometric gamma detector).
5)99mTc complex of RP502 (Dimethylgly-L-t-Butylgly-L-Cys(SE-Acm)-Gly-Thr-
Lys-Pro-Pro-Arg): HPLC retention time: Re0(RP502) RL = 19.76 min (LTV
detector set at 215 nm); 99'°Tc(RP502) Rt = 20.10 min (radiometric
gamma
detector).
6) 99"'Tc complex of RP573 (Dimethylgly-L-t-Butylgly-L-Cys(SE-Acm)-Gly-Arg-
Ile-Lys-Pro-His): HPLC retention time: (ReORP573) Rt = 16.43 min (UV
detector set at 215 nm); ~'"Tc(RP573) Rt = 20.75 min (radiometric gamma
detector).
Example 8
Synthesis of Dimethylgly-L-Beta-hydrozyvaline-L-Cys-Gly. The beta-
hydroxyvaline is synthesized according to the method of Shao, H., and Goodman,
M.,
J. O~g. Chem. 1996, 61, 2582-2583 or Beloken, Yu. N.; Bulychev, A. G.; Vitt,
S. V.;
Struchkov, Yu. T.; Batsanov, A. S.; Timofeeva, T. V.; Tsyryapkin, V. A.;
Ryzhov,
M. G.; Lysova, L. A.; Et al. J. Am. Soc. Chem.,1985,107(14), 4252-9.. The
FMOC group is added to the amino terminus according to the method of Carpino,
L.
A., Han, G. Y. J. Org. Chem. 1972, 37, 3404. The FMOC-beta-hydroxyvaline is
purified by column chromatography. The peptide dimethylgly-L-beta-
hydroxyvaline-
L-cys-gly is synthesized on the peptide synthesizer in the same manner as set
out in
Example 1. The Re and Tc-99m complexes are synthesized by the same method as
the Re and Tc-99m complexes of dimethylgly-L-t-butylgly-L-cys-gly as shown in
examples 2 and 7 respectively.
2~
AMENDED SliEET
CA 02316392 2000-06-27
. ; , ~ ~ , ~ ' , , ~ , : , ~ , ,
. . . , . , . , , .
' ~ ~ < < < , . ~ ; : ,
, , .. ~ " , ,. ,.
These Re and Tc-99m chelates form the syn isomer predominantly but are more
hydrophilic than the Re and Tc-99m complexes already mentioned. This is an
advantage when the chelates are attached to hydrophilic targeting molecules.
Although the invention has been described with preferred embodiments, it is to
be
understood that modifications may be resorted to as will be apparent to those
skilled
in the art. Such modifications and variations are to be considered within the
purview
and scope of the present invention.
References
(1) Baidoo, K. E.; Lever, S. Z. Bioconjugate Chem.1990, l, 132.
(2) Eisenhut. M.; Missfeldt, M.; Lehmann, W. D.; Karas, M. J. Label Compound
Radiopharm. 1991, 29, 1283.
(3) Fritzberg, A. R.; Beaumier, P. L. J. Nucl. Med. 1992, 33, 394.
(4) Fischman, A. J.; Babich, J. W.; Strauss, H. W. J. Nucl. Med. 1993, 34,
2253.
(5) Thakur, M. L. Nucl. Med. Commun. 1995,16, 724.
(6) Malin, R.; Steinbrecher, R.; Jannsen, J.; Semmler, W.; Noll, B.;
Johannsen, B.;
Frommel, C.; Hohne, W.; Schneider-mergener, J. J. Am. Chem. Soc. 1995,
117, 11821.
(7) Pearson, D. A.; Lister-James, J.; McBride, W. J.; Wilson, D. M.; Martel,
L. J.;
Civitello, E. R.; Taylor, J. E.; Moyer, B. R.; Dean, R. T. J. Med. Chem. 1996,
39, 1361.
(8) Lister-James, J.; Knight, L. C.; Maurer, A. H.; Bush, L. R. J. Nucl. Med.
1996,
37, 775.
(9) Liu, S.; Edwards, D. S.; Looby, R. J.; Hams, A. R.; Poirier, M. J.;
Barrett, J.
A.; Heminway, S. J.; Carroll, T. R. Bioconjugate Chem. 1996, 7, 63.
(10) Liu, S.; Edwards, D. S.; Looby, R. J.; Poirier, M. J.; Rajopadhye, M.;
Bourque,
J. P.; Carroll, T. R. Bioconjugate Chem. 1996, 7, 196.
(11) Deutsch, E.; Libson, K.; Jurisson, S.; Lindoy, L. F. Prog. Ino~g. Chem.
1983,
30, 75.
22
pt~ENDED SHEtT
CA 02316392 2000-06-27
. , , ~ ; , , ,
(12) Melnik, M.; Van Lier, J. E. Coord. Chem. Rev. 1987, 77, 275.
(13) Mazzi, U. Polyhedron, 1989, 8, 1633.
(14) Jurisson, S.; Berning, D.; Jia, W.; Ma, D. Chem. Rev. 1993, 93, 1137.
(15) Tisato, F.; Refosco, F.; Bandoli,G. Coord Chem. Rev. 1994, 135, 325.
(I6) Otsuka, F. L.; Welch, M. J. Nucl. Med. Biol. 1987,14, 243.
(17) Fritzberg, A. R.; Berninger, R W.; Hardey, S. W.; Wester, D. W. Pharm.
Res.
1988, 5, 325.
(18) Eckelman, W. C.; Paik, C. H.; Steigman, J. Nucl. Med. Biol. 1989,16, 171.
(19) Hnatowich, D. J. Semin. Nucl. Med. 1990, 20, 80.
(20) Srivastava, s. C.; Mease, R. C. Nucl. Med. Biol. 1991, 18, 589.
(21) Liang, F. H.; Virzi, F.; Hnatowich, D. J. Nucl. Med. Biol. 1987, 14, 63.
(22) Chianelli, M.; Signore, A.; Fritzberg, A. R.; Mather, S. J. Eur. J. Nucl.
Med.
1992, 19, 625.
(23) Baidoo, K. E.; Lever, S. Z.; Scheffel, U. Bioconjugate Chem. 1994, S,
114.
(24) Eisenhut, M.; Lehmann, W. D.; Becker, W.; Behr, T. J. Nucl. Med. 1996,
37,
362.
(25) Edwards, D. S.; Liu, S.; Barrett, J. A.; Hams, A. R; Looby, R. J.;
Ziegler, M.
C.; Heminway, S. J.; Carroll, T. R. Bioconjugate Chem. 1997, 8, 146.
(26) Barrett, J. A.; Crocker, A. C.; Damphousse, D. J.; Heminway, S. J.; Liu,
S.;
Edwards, D. S.; Lazewatsky, J. L.; Kagun, M.; Mazaika, T. J.; Carroll, T. R.
Bioconjugate Chem. 1997, 8, 155.
(27) Thakur, M. L.; Kolan, H.; Li, J.; Wiaderkiewicz, R.; Pallela, V. R.;
Duggaraju,
R.; Schally, A. V. Nucl. Med. Biol. 1997, 24, 105.
(28) Childs, R. L.; Hnatowich, D. J. J. Nucl. Med. 1985, 26, 293.
(29) Fischman, A. J.; Babich, J. W.; Rubin, H. R. Semin. Nucl. Med. 1993, 24,
1954.
23 AMENDED S~~ET
CA 02316392 2000-06-27
' . " ,.., " . " ,.
' : ' ; ~ , , , ~ , , , , , ; ; , ,
. , ' " ; ~ , . ' " '
(30) Babich, J. W.; Solomon, H.; Pike, M. C.; Kroon, D.; Graham, W.; Abrams,
M.
J.; Tompkins, R. G.; Rubin, R. H.; Fischman, A. J. J. Nucl. Med. 1993, 34,
1967.
(31) Babich, J. W.; Fichman, A. J. Nucl. Med. Biol. 1995, 22, 25.
(32) Hosotani, T.; Yolcoyama, A.; Arano, Y.; Horiuchi, K.; Wasaki, H.; Saji,
H.;
Torizuka, K. Nucl. Med. Biol. 1986,12, 431.
(33) Leonard, J. P.; Nowotnik, D. P.; Neirinckx, R. D. J. Nucl. Med. 1986, 27,
1819.
(34) Neirinckx, R. D.; Canning, L. R.; Piper, I. M.; Nowotnik, d. P.; Pickett,
R. D.;
Holmes, R. A.; Volkert, W. A.; Forster, A. M.; Weisner, P. S.; Marriott, J.
A.;
Chaplin, S. B. J. Nucl. Med. 1987, 28, 191.
(35) Neirinckx, R. D.; Burke, J. F.; Harrison, R. C.; Forster, A. M.;
Andersen, A.
R.; Lassen, N. A. J. Cereb. Blood Flow Metab. 1988, 8, S4.
(36) Holm, S.; Anderson, A. R.; Vorstrup, S.; Lassen, N. A.; Paulson, O. B.;
Holmes, R. A.; J. Nucl. Med. 1985, 26, 1129.
(37) Sharp, P. F.; Smith, F. W.; Gemmell, H. G.; Lyall, D.; Evans, N. T. S.;
Gvozdanovic, D.; Davidson, J.; Tyrrell, D. A.; Pickett, R. D.; Neirinckx, R.
D.
J. Nucl. Med. 1986, 27, 171
(38) Linder, K. E.; Wen, M. D.; Nowotnik, D. P.; Malley, M. F.; Gougoutas, J.
Z.;
Nunn, A. D.; Eckelman, W. C. Bioconjugate Chem . 1991, 2, 160
(39) Rao, T. N.; Adhikesavalu, D.; Camerman, A.; Fritzberg, A. R. J. Am. Chem.
Soc. 1990,112, 5798
(40) Eshima, D.; Taylor Jr., A.; Fritzberg, A. R.; Kasina, S.; Hansen, L.;
Sorenson,
J. F. J. Nucl. Med. 1987, 28, 1180
(41) Subhani, M.; Cleynhens, B.; Bormans, G.; Hoogmartens, M.; De Roo, M.;
Verbruggen, A. M. In Technetium and Rhenium in Chemistry and Nuclear
Medicine-3; Nicotine M.; Banoli, G.; Mazzi, U., Eds.; Cortina International,
Verona, Italy, 1990, p. 453.
24 AMENDED Jr~i=~_-:
CA 02316392 2000-06-27
,, ,.<, " , , ..
a . , , , ~ , , , ; ; ,
< ' ~ , . . , , ~ , , . ,
, , , < ~ , , , . ,
,. ,
(42) Bormans, G.; Cleynhens, B.; Hoogniartens,' IvL; De Roo, M.;
Verbruggeri,'A. '
M. In Technetium and Rhenium in Chemistry and Nuclear Medicine-3;
Nicoline M.; Banoli, G.; Mazzi, U., Eds.; Cortina International, Verona,
Italy,
1989, p. 661.
(43) Bormans, G.; Cleynhens, B.; Adriaens, P.; De Roo, M.; Verbruggen, A. M.
J.
Labelled Compounds and Radiopharmaceuticals, 1993, 33, 1065
(44) Lister-James, J.; Knight, L. C.; Mauer, A. H.; Bush, L. R.; Moyer, B. R.;
Dean,
R. T. J. Nucl. Med. 1996, 37, 775
(45) Muto, P.; Lastoria, S.; Varrella, E.; Salvatore, M.; Morgano, G.; Lister-
James,
J.; Bernardy, J. D.; Dean, R. T. Wencker, D.; Boer, J. S. J. Nucl. Med. 1995,
36, 1384
(46) Klingensnuth III, W. C.; Fritzberg, A. R.; Spitzer, V. M.; Johnson, D.
L.;
Kuni, C. C.; Williamson, M. R.; Washer, G.; Weil III, R. J. Nucl. Med. 1984,
25, 42.
(47) Marchi, A.; Marvelli, L.; Rossi, R.; Magon, L.; Bertolasi, V.; Ferretti,
V.;
Gilli, P.; J. Chem. Soc., Dalton Trans. 1992, 1485
(48) Kung, H. F.; Bradshaw, J. E.; Chumpradit, S.; Zhang, Z. P.; Kung, M. P.;
Mu,
M.; Frederick, D. In Technetium and Rhenium in Chemistry and Nuclear
Medicine-4; Nicoline M.; Banoli, G.; Mazzi, U., Eds.; Cortina International,
Verona, Italy, 1995, p. 293.
(49) Meegalla, S.; Plossl, K.; Dung, M.-P.; Chumpradt, S.; Stevenson, D. A.;
Kushner, S. A.; McElgin, W. T.; Mozley, P. D.; Kung, H. F. J. Med: Chem.
1997, 40, 9
(50) Edwards, D. S.; Cheesman, E. H.;; Watson, M. W.; Maheu, L. J.; Nguyen, S.
A.; Dimitre, L.; Nason, T.; Watson, A. D.; Walovitch, R. In Technetium and
Rhenium in Chemistry and Nuclear Medicine-3; Nicoline M.; Banoli, G.;
Mazzi, U., Eds.; Corona International, Verona, Italy, 1990, p. 431.
(51) Oya, S.; Kung, M.-P.; Frederick, D.; Kung, H. F. Nucl. Med. Biol. 1995,
22,
749.
AMENDED ~'L~~r"
.:
CA 02316392 2000-06-27
a a , ~ , , ; , ; ; ~ ,
, , , . , ~ , . . , , . ~ ,
.
(52) Kung, H. F.; Guo, Y. Z.; Yu, C. C.; Billings, J.; Subramanyam, B.;
Calabrese,
J. C. J. Med. Chem. 1989, 32, 433.
(53) Mach, R. H.; Kung, H. F.; Guo, Y. Z.; Yu, C. C.; Subramanyam, V.;
Calabrese, J. C. Nucl. Med Biol. 1989,16, 829.
(54) Francesconi, L. C.Graczyk, G.; Wehrli, S.; Shaikh, S. N.; McClinton, D.;
Liu,
S.; Zubieta, J.; Kung, H. F. Inorg. Chem. 1993, 32, 3114.
(55) Efange, S. M. N.; Kung, H. F.; Billings, S. S.; Blau, M. J. Med. Chem.
1988,
31, 1043.
(56) Walovitch, R. C.; Cheesman, E. H.; Maheu, L. J.; Hall, K. M. J. Cereb.
Blood
Flow Metab. 1988, 8, S4.
(57) Rouschias, G. Chem. Rev. 1974, 74, 531.
(58) Fergusson, J. E. Coord. Chem. Rev. 1966, l, 459.
(59) User' Manual of Peptide Synthesizer Model 433A, Applied BioSystems,
Philadelphia, 1993.
(60) Introduction to Cleavage Techniques, Applied BioSystems, Philadelphia,
1990.
(61) Wong, E.; Fauconnier, T.; Bennett, S.; Valliant J.; Nguyen, T.; Lau, F.;
Lu, L. F.
L.; Pollak,; Bell, R. A.; Thornback, J. R. Inorg. Chem. 1997, in press.
(62) Peers, S. H.; Tran, L. L.; Eriksson, S. J.; Ballinger, J.; Goodbody, A.
E. J. Nucl.
Med. 1995, 36, 114P.
(63) Williams, R. M. Synthesis of Optically Active a-Amino Acids; Pergamon:
Toronto, Canca, 1987.
(64) Arnold, L. D.; May, r. G.; Vederas, J. C. J. Am. Chem. Soc. 1987, 109,
4649.
(65) Arnold, L. D.; May, R. G.; Vederas, J. C. J. Am. Chem. Soc. 1988, ll D,
2237.
(66) Reetz, M. T. Angew. Chem., Int. Ed. Engl. 1991, 30, 1531.
(67) Blaskovich, M. A.; Lajoie, G. A. J. Am. Chem. Soc. 1993, 115, 5021.
(68) Shao, H., and Goodman, M., J. O~g. Chem. 1996, 61, 2582-2583.
(69) Beloken, Yu. N.; Bulychev, A. G.; Vitt, S. V.; Struchkov, Yu. T.;
Batsanov,
26
vl iL
AMEND~~ C~~r i
CA 02316392 2000-06-27
. : ~ ~ ; , , ;
S.; Timofeeva, T. V.; Tsyryapkin, V. A.; Ryzhov, ~IvI. G.; Lysova, L. A.;~ Et
al. J. Am. Soc. Chem., 1985, 107(14), 4252-9.
(70) Carpino, L. A., Han, G. Y. J. Org. Chem. 1972, 37, 3404.
27
AMENDEQ SH L
