APOPTOSIS IMAGING AGENTS BASED ON LANTIBIOTIC PEPTIDES
Field of the Invention.
The present invention relates to radiopharmaceutical imaging in vivo of apoptosis and
other forms of cell death. The invention provides imaging agents which target
apoptotic cells via selective binding to the aminophospholipid
phosphatidylethanolamine (PE), which is exposed on the surface of apoptotic cells.
Also provided are pharmaceutical compositions, kits and methods of in vivo imaging.
Background to the Invention.
Apoptosis or programmed cell death (PCD) is the most prevalent cell death pathway
and proceeds via a highly regulated, energy-conserved mechanism. In the healthy
state, apoptosis plays a pivotal role in controlling cell growth, regulating cell number,
facilitating morphogenesis, and removing harmful or abnormal cells. Dysregulation
of the PCD process has been implicated in a number of disease states, including those
associated with the inhibition of apoptosis, such as cancer and autoimmune disorders,
and those associated with hyperactive apoptosis, including neurodegenerative
diseases, haematologic diseases, AIDS, ischaemia and allograft rejection. The
visualization and quantitation of apoptosis is therefore useful in the diagnosis of such
apoptosis-related pathophysiology.
Therapeutic treatments for these diseases aim to restore balanced apoptosis, either by
stimulating or inhibiting the PCD process as appropriate. Non-invasive imaging of
apoptosis in cells and tissue in vivo is therefore of immense value for early assessment
of a response to therapeutic intervention, and can provide new insight into devastating
pathological processes. Of particular interest is early monitoring of the efficacy of
cancer therapy to ensure that malignant growth is controlled before the condition
becomes terminal.
There has consequently been great interest in developing imaging agents for apoptosis
[see eg. Zeng etal, Anti-cancer Agent Med.Chem., 9(9), 986-995 (2009); Zhao, ibid,
9(9), 1018-1023 (2009) and M. De Saint-Hubert etal, Methods, 48, 178-187 (2009)].
Of the probes available for imaging cell death, radiolabelled Annexin V has received
the most attention. Annexin V binds only to negatively charged phospholipids, which
renders it unable to distinguish between apoptosis and necrosis.
The lanthionine-containing antibiotic peptides ("lantibiotics") duramycin and
cinnamycin are two closely related 19-mer peptides with a compact tetracyclic
structure [Zhao, Amino Acids, DOI 10.1007/s00726-009-0386-9, Springer-Verlag
(2009), and references cited therein]. They are crosslinked via four covalent,
intramolecular bridges, and differ by only a single amino acid residue at position 2 .
The structures of duramycin and cinnamycin are shown schematically below, where
the numbering refers to the position of the linked amino acid residues in the 19-mer
sequence:
Duramycin X = Lys,
Cinnamycin X2 = Arg.
Programmed cell death or apoptosis is an intracellular, energy-dependent selfdestruction
of the cell. The redistribution of phospholipids across the bilayer of the
cell plasma membrane is an important marker for apoptosis. Thus, in viable cells, the
aminophospholipids phosphatidylethanolamine (PE or PtdE) and phosphatidyl serine
(PS) are predominantly constituents of the inner leaflet of the cell plasma membrane.
In apoptopic cells, there is a synchronised externalization of PE and PS.
Both duramycin and cinnamycin bind to the neutral aminophospholipid PE with
similar specificity and high affinity, by forming a hydrophobic pocket that fits around
the PE head-group. The binding is stabilised by ionic interaction between the b-
hydroxyaspartic acid residue (HO-Asp15) and the ethanolamine group. Modifications
to this residue are known to inactivate duramycin [Zhao et al, J.Nucl.Med, 49, 1345-
1352 (2008)]. Zhao [Amino Acids, DOI 10.1007/s00726-009-0386-9, Springer-
Verlag (2009)] cites earlier work by Wakamatsu et al [Biochemistry, 29, 113-188
(1990)], where MR studies show that none of the H MR resonances of the 5
terminal amino acids of cinnamycin are shifted on binding to PE - suggesting that
they are not involved in interactions with PE.
US 2004/0147440 Al (University of Texas System) describes labelled antiaminophospholipid
antibodies, which can be used to detect pre-apoptopic or
apoptopic cells, or in cancer imaging. Also provided are conjugates of duramycin
with biotin, proteins or anti-viral drugs for cancer therapy.
WO 2006/055855 discloses methods of imaging apoptosis using a radiolabelled
compound which comprises a phosphatidylserine-binding C2 domain of a protein.
WO 2009/1 14549 discloses a radiopharmaceutical made by a process comprising:
(i) providing a polypeptide having at least 70% sequence similarity with
CKQSCSFGPFTFVCDGNTK,
wherein the polypeptide comprises a thioether bond between amino acids
residues 1-18, 4-14, and 5-1 1, and an amide bond between amino acids
residues 6-19, and, wherein one or more distal moieties of structure
are covalently bound to the amino acid at position 1, position 2, or,
positions 1 and 2 of the polypeptide, and wherein R and R2 are each
independently a straight or branched, saturated or unsaturated C
alkyl; and
chelating one or more of the distal moieties with Tc , ( mTc=0) +,
( TcºN) +, (0= mTc=0) + or [ mTc(CO)3]+, wherein x is a redox or
oxidation state selected from the group consisting of +7, +6, +5, +4, +3, +2,
+1, 0 and -1, or, a salt, solvate or hydrate thereof.
The 'distal moiety' of WO 2009/1 14549 is a complexing agent for the radioisotope
9 mTc, which is based on hydrazinonicotinamide (commonly abbreviated "HYNIC").
HY IC is well known in the literature [see e.g. Banerjee et al, Nucl.Med.Biol, 32, 1-
20 (2005)], and is a preferred method of labelling peptides and proteins with mTc
[R.Alberto, Chapter 2, pages 19-40 in IAEA Radioisotopes and Radiopharmaceuticals
Series 1 : "Technetium-99m Radiopharmaceuticals Status and Trends" (2009)].
WO 2009/1 14549 discloses specifically mTc-HYNIC-duramycin, and suggests that
the radiopharmaceuticals taught therein are useful for imaging apoptosis and/or
necrosis, atherosclerotic plaque or acute myocardial infarct.
Zhao et al [J.Nucl.Med, 49, 1345-1352 (2008)] disclose the preparation of mTc-
HYNIC-duramycin. Zhao et al note that duramycin has 2 amine groups available for
conjugation to HYNIC: at the N-terminus (Cys1 residue), and the epsilon e side
chain of the Lys2 residue. They purified the HYNIC-duramycin conjugate by HPLC
to remove the te-HYNIC-functionalised duramycin, prior to radiolabelling with
mTc. Zhao et al acknowledge that the mTc-labelled mofto-HYNIC-duramycin
conjugates studied are probably in the form of a mixture of isomers.
Whilst HYNIC forms stable Tc complexes, it requires additional co-ligands to
complete the coordination sphere of the technetium metal complex. The HYNIC may
function as a monodentate ligand or as a bidentate chelator depending on the nature of
the amino acid side chain functional groups in the vicinity [King et al, Dalton Trans.,
4998-5007 (2007); Meszaros et al [Inorg.Chim.Acta, 363, 1059-1069 (2010)]. Thus,
depending on the environment, HYNIC forms metal complexes having 1- or 2- metal
donor atoms. Meszaros et al note that the nature of the co-ligands used with HYNIC
can have a significant effect on the behaviour of the system, and state that none of the
co-ligands is ideal.
The Present Invention.
The present invention provides radiopharmaceutical imaging agents, particularly for
imaging disease states of the mammalian body where abnormal apoptosis is involved.
The imaging agents comprise radiometal chelator conjugates of a lantibiotic peptide.
The invention provides radiometal complexes which form reproducibly, in high
radiochemical purity (RCP), without the need for co-ligands. The present inventors
have also established that attachment of the radiometal complex at the N-terminus
(Cys residue) of the lantibiotic peptide of Formula II herein is strongly preferred,
since attachment of the uncomplexed chelator at even the amino acid adjacent to the
N-terminus (Xaa of Formula II) has a deleterious effect on binding to
phosphatidylethanolamine. This effect was not recognized previously in the prior art,
and hence the degree of impact on binding affinity is believed novel.
Detailed Description of the Invention.
In a first aspect, the present invention provides an imaging agent which comprises a
compound of Formula I :
(I)
wherein:
LBP is a lantibiotic peptide of Formula II:
'-Xaa-Gln-Serb-Cysc-Serd-Phe-Gly-Pro-Phe-Thr -Phe-Val-Cys
(HO-Asp)-Gly-Asn-Thr -Lysd
(P)
Xaa is Arg or Lys;
Cys -Thr , Serb-Cysb and Cys -Thr are covalently linked via thioether
bonds;
Serd-Lysd are covalently linked via a lysinoalanine bond;
HO-Asp is b-hydroxyaspartic acid;
Z1- - is attached to Cys and optionally also to Xaa of LBP, wherein Z1
comprises a radiometal complex of a chelating agent having
at least 4 metal donor atoms;
attached to the C-terminus of LBP and is OH, OB , or M ,
where B is a biocompatible cation; and
MIG is a metabolism inhibiting group which is a biocompatible group
which inhibits or suppresses in vivo metabolism of the LBP peptide;
L is a synthetic linker group of formula -(A)m- wherein each A is
independently -CR2- , -CR=CR- , -CºC- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO - , - (C=0 ) -, -NR(C=S)NR-, -S0 2NR- , -NRS0 2- , -CR2OCR2- ,
-CR SCR2- , -CR2 RCR2- , a C 4-8 cycloheteroalkylene group, a C 4-8
cycloalkylene group, a Cs.i2 arylene group, or a C3-12 heteroarylene group, an
amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building
block;
each R is independently chosen from H, C 1 alkyl, C2-4 alkenyl, C -4 alkynyl,
Ci-4 alkoxyalkyl or C hydroxyalkyl;
m is an integer of value 1 to 20;
n is an integer of value 0 or 1.
By the term "imaging agent" is meant a compound suitable for imaging the
mammalian body. Preferably, the mammal is an intact mammalian body in vivo, and
is more preferably a human subject. Preferably, the imaging agent can be
administered to the mammalian body in a minimally invasive manner, i.e. without a
substantial health risk to the mammalian subject when carried out under professional
medical expertise. Such minimally invasive administration is preferably intravenous
administration into a peripheral vein of said subject, without the need for local or
general anaesthetic. The imaging agents of the first aspect are particularly suitable for
imaging apoptosis and other forms of cell death, as is described in the sixth aspect
(below).
The term "in vivo imaging" as used herein refers to those techniques that noninvasively
produce images of all or part of an internal aspect of a mammalian subject.
By the term "amino acid" is meant an L- or D- amino acid, amino acid analogue (eg.
naphthylalanine) or amino acid mimetic which may be naturally occurring or of
purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence
chiral, or a mixture of enantiomers. Conventional 3-letter or single letter
abbreviations for amino acids are used herein. Preferably the amino acids of the
present invention are optically pure.
By the term "peptide" is meant a compound comprising two or more amino acids, as
defined above, linked by a peptide bond (i.e. an amide bond linking the amine of one
amino acid to the carboxyl of another).
The term "lantibiotic peptide" refers to a peptide containing at least one lanthionine
bond. "Lanthionine" has its conventional meaning, and refers to the sulfide analogue
of cystine, having the chemical structure shown:
By the term "covalently linked via thioether bonds" is meant that the thiol functional
group of the relevant Cys residue is linked as a thioether bond to the Ser or Thr
residue shown via dehydration of the hydroxyl functional group of the Ser or Thr
residue, to give lanthionine or methyllanthionine linkages. Such linkages are
described by Willey et al [Ann.Rev.Microbiol., 61, 477-501 (2007)].
By the term "lysinoalanine bond" is meant that the epsilon amine group of the Lys
residue is linked as an amine bond to the Ser residue shown via dehydration of the
hydroxyl functional group of the Ser giving a -(CH 2)-NH-(CH2)4- linkage joining the
two -carbon atoms of the amino acid residues.
By the term "radiometal complex" is meant a coordination metal complex of the
radiometal with the chelator, wherein said chelator is covalently bonded to the LBP
peptide via the linker group (L) of Formula I . The coordination complex does not
comprise hydrazinonicotinamide (HYNIC) ligands bound to the radiometal. Hence,
the chelator is the principal species binding to the radiometal it is not simply a coligand
for HYNIC.
The term "chelating agent" has its conventional meaning and refers to 2 or more metal
donor atoms arranged such that chelate rings, preferably 5- to 7-membered chelate
rings, result upon metal coordination, more preferably 5- or 6- membered chelate
rings. The metal donor atoms are covalently linked by a non-coordinating backbone
of either carbon atoms or non-coordinating heteroatoms. The chelating agent can be
macrocyclic or open chain. The chelating agents of the present invention comprise at
least 4 metal donor atoms, suitably 4 to 8 metal donor atoms, in which at least 4 such
metal donor atoms are bound to the radiometal in the radiometal complex.
Suitable radiometals of the present invention include: Tc, 4 Tc, 1 Re, 1 Re,
4Cu, Cu, Ga, Ga, 105Rh, 10 Rh, ln, Zr or 4 Ti.
When Z1 is attached to Cysa, it is attached to the N-terminus of the LBP peptide.
When Z1 is also attached to Xaa, that means that Xaa is Lys, and Z is attached to the
epsilon amino group of the Lys residue.
The Z2 group substitutes the carbonyl group of the last amino acid residue of the LBP
- i.e. the carboxy terminus. Thus, when Z2 is OH, the carboxy terminus of the LBP
terminates in the free CO2H group of the last amino acid residue, and when Z2 is OBc
that terminal carboxy group is ionised as a C0 2B group.
By the term "biocompatible cation" (Bc) is meant a positively charged counterion
which forms a salt with an ionised, negatively charged group, where said positively
charged counterion is also non-toxic and hence suitable for administration to the
mammalian body, especially the human body. Examples of suitable biocompatible
cations include: the alkali metals sodium or potassium; the alkaline earth metals
calcium and magnesium; and the ammonium ion. Preferred biocompatible cations are
sodium and potassium, most preferably sodium.
By the term "metabolism inhibiting group" (M G) is meant a biocompatible group
which inhibits or suppresses in vivo metabolism of the LBP peptide at the carboxy
terminus (Z2) . Such groups are well known to those skilled in the art and are suitably
chosen from: carboxamide, tert-buty\ ester, benzyl ester, cyclohexyl ester, amino
alcohol or a polyethyleneglycol (PEG) building block. The LBP peptides of the
invention are known to exhibit high in vivo metabolic stability (95% at 60 min), hence
Z2 is preferably OH or OB .
Preferred embodiments.
The chelating agent is preferably designed such that the chelate rings formed on
complexation with the radiometal comprise at least one 5- or 6- membered ring, more
preferably 2 to 4 such rings, most preferably 3 or 4 such rings.
The chelating agent is preferably chosen from: an aminocarboxylate ligand having at
least 6 donor atoms; or a tetradentate chelator having an N3S, N2S2 or N4 donor set.
The chelating agent is more preferably either an aminocarboxylate ligand having at
least 6 donor atoms, or a tetradentate chelator having an N4 donor set, and most
preferably a tetradentate chelator having an N4 donor set.
The term "aminocarboxylate ligand" has its conventional meaning, and refers to a
chelating agent of the EDTA, DTPA type. The donor atoms of such chelators are a
mixture of amine (N) donors and carboxylic acid (O) donors. Such chelators may be
open chain (e.g. EDTA, DTPA or HBED), or macrocyclic (eg. DOTA or NOTA).
Suitable such chelators include DOTA, HBED and NOTA, which are well known in
the art and are preferred for radiometals such as Ga or 6 Ga, In, radioisotopes of
copper, Zr and 4 Ti.
The term "tetradentate chelator" has its conventional meaning and refers to a
chelating agent in which the radiometal is coordinated by the four metal donor atoms
of the tetradentate chelating agent.
By the term "N3S donor set" is meant that the four metal donor atoms of the
tetradentate chelator are made up of 3 nitrogen donor atoms and one sulfur donor
atom. Examples of suitable such N donor atom types are: amines (especially primary
or secondary amines); amides or oximes, or combinations thereof. Examples of
suitable such S donor atom types are: thiol and thioether. Preferred such N3S
chelators have a thioltriamide donor set, and are preferably open chain chelators such
as MAG3 (mercaptoacetyltri glycine).
By the term "N2S2 donor set" is meant that the four metal donor atoms of the
tetradentate chelator are made up of 2 nitrogen donor atoms and 2 sulfur donor atoms.
Suitable N and S donor atoms are as described for N3S (above). Preferred such N2S2
chelators have a diaminedithiol or amideaminedithiol donor set, and are preferably
open chain chelators such as BAT or N,N -ethylenedi-Z-cysteine [Inorg Chem.,
35(2):404-414 (1996)].
By the term "N4 donor set" is meant that the four metal donor atoms of the
tetradentate chelator are all based on nitrogen. Examples of suitable such N donor
atom types are: amines (especially primary or secondary amines); amides or oximes,
or combinations thereof.
The N4 donor set is preferably chosen from: diaminedioxime; tetra-amine;
amidetriamine, or diamidediamine. The N4 chelator can be open-chain or
macrocyclic (eg. cyclam, cyclen, monoxocyclam or dioxocyclam). Preferred N4
tetradentate chelating agents of the present invention have a diaminedioxime or a
tetra-amine donor set, and are more preferably open-chain diaminedioximes or openchain
tetra-amines.
Preferred diaminedioxime chelators are of formula:
where E^E 6 are each independently an R' group;
each R' is independently H or C Oalkyl, C3.10 alkylaryl, C2-10 alkoxyalkyl, C1-10
hydroxyalkyl, C1.10 fluoroalkyl, C2-10 carboxyalkyl or Ci-ioaminoalkyl, or two or more
R' groups together with the atoms to which they are attached form a carbocyclic,
heterocyclic, saturated or unsaturated ring;
and Q is a bridging group of formula -(J)f ;
where f is 3, 4 or 5 and each J is independently -0-, -NR' - or -C(R ' )2- provided that
-(J)f may contain a maximum of one J group which is -O- or -NR ' - .
Preferred Q groups are as follows:
Q = -(CH )(CHR ' )(CH2)- i.e. propyleneamine oxime or PnAO derivatives;
Q = -(CH 2)2(CHR ' )(CH 2)2- i.e. pentyleneamine oxime orPentAO derivatives;
Q = -(CH 2) N '(CH2) - .
E1 to E6 are preferably chosen from: C1-3 alkyl, C2 -4 alkoxyalkyl, C1-3 hydroxyalkyl,
Ci-3 fluoroalkyl, C2 -6 carboxyalkyl or C1- aminoalkyl. Most preferably, each E1 to E6
group is CH .
Q is preferably -(CH 2)(CHR ' )(CH 2)- , -(CH )2(CHR ')(CH 2) - or -(CH ) ' (CH ) -,
most preferably -(CH 2)2(CHR ')(CH 2)2- . An especially preferred diaminedioxime
chel ator has the Formul a:
(Chelator 1)
wherein the bridgehead primary amine group is conjugated to (L) (i.e. the linker
group) and/or LBP peptide.
Preferred tetra-amine chelators are of formula:
(Chelator 2)
wherein the bridgehead carboxyl group is conjugated to the linker group and/or
LBP peptide.
In Chelator 2, the [linker] is preferably a group of formula (A') mi, where ml is an
integer of value 0 to 6, and each A' is independently CH2 or p-phenylene, where no
more than one of the A groups is or ^-phenylene. Preferably, each of the A' groups is
CH2 and ml is 1 to 6 . A preferred such chelator is Chelator 2A, where the [linker] is
-(CH )-.
The radiometal of the imaging agent is preferably 4 Tc or mTc, and is more
preferably Tc. For these technetium radioisotopes, the chelator is preferably a
tetradentate with an N4 donor set as defined above.
Z2 is preferably OH or OBc.
In the imaging agent of the first aspect, Z1 is preferably attached only to Cys of LBP.
When Xaa is Arg, that means that Z1 is attached to the LBP N-terminus, at the free
amino group of the Cys residue. When Xaa is Lys, that means that steps are taken to
either:
(i) selectively functionalise the LBP peptide at the Cysa residue in
preference to the epsilon amine group of the Xaa residue; or
(ii) a composition comprising LBP functionalized with Z1 at both Cys and
Xaa is prepared, then the Xaa-functionalised species is removed.
In the imaging agent of the first aspect, Xaa is preferably Arg.
The imaging agent of the first aspect preferably comprises a Linker Group (L), .i.e. n
in Formula (I) is preferably 1. L preferably comprises a PEG group of formula
-(OCH 2CH2)x- where x is an integer of value 6 to 18, preferably 8 to 14, more
preferably 10 to 12. Such linker groups are advantageous in reducing liver
background retention and increasing urinary excretion of the imaging agent in vivo
Preferably, L comprises a biomodifier group of Formula IA or IB:
(IA)
17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula
wherein p is an integer from 1 to 10. In Formula IA, p is preferably 1, 2 or 3 .
Alternatively, a PEG-like structure based on a propionic acid derivative of Formula IB
can be used:
(IB)
where p is as defined for Formula IA and
q is an integer from 3 to 15.
In Formula IB, p is preferably 1 or 2, more preferably 1, and q is preferably 5 to 12,
more preferably 12.
By the term "biomodifier" is meant a group which has an effect on the biodistribution
of the agent in vivo.
The imaging agents of the first aspect can be obtained as described in the third aspect.
In a second aspect, the present invention provides a chelator conjugate of Formula III:
Z -(L) -[LBP]-Z2
(III)
wherein:
Z3 is a chelating agent having at least 4 metal donor atoms; and
L, n, LBP and Z2 are as defined in the first aspect.
Preferred aspects of L, n, LBP and Z2 and the chelating agent (Z ) in the second
aspect are as defined in the first aspect (above).
Certain LBP peptides are commercially available. Thus, cinnamycin and duramycin
are available from Sigma-Aldrich. Duramycin is produced by the strain: D3168
Duramycin from Streptoverticillium cinnamoneus. Cinnamycin can be biochemically
produced by several strains, eg. from Strptomyces cinnamoneus or from
Streptoverticillium griseoverticillatum. See the review by C. Chatterjee et al [Chem.
Rev., 105, 633-683 (2005)]. Other peptides can be obtained by solid phase peptide
synthesis as described in P. Lloyd-Williams, F. Albericio and E. Girald; Chemical
Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997.
The chelator conjugates of the second aspect can be obtained as follows. When the
chelator is a diaminedioxime, by reaction of the appropriate diamine with either:
(i) the appropriate chloronitroso derivative Cl-C(R 1) 2-CH(NO)R 1;
(ii) an alpha-chloro oxime of formula Cl-C(R )2-C(=NOH)R 1;
(iii) an alpha-bromoketone of formula Br-C(R 1) 2-C(=0)R 1 followed by
conversion of the diaminediketone product to the diaminedioxime with
hydroxylamine.
Route (i) is described by S. Jurisson et al [Inorg. Chem., 26, 3576-82 (1987)].
Chloronitroso compounds can be obtained by treatment of the appropriate alkene with
nitrosyl chloride (NOC1) as is known in the art. Further synthetic details of
chloronitroso compounds are given by: Ramalingam [Synth. Commun., 25(5), 743-
752 (1995)]; Glaser [J. Org. Chem., 61(3), 1047-48 (1996)]; Clapp [J. Org Chem.,
36(8) 1169-70 (1971)]; Saito [Shizen Kagaku, 47, 41-49 (1995)] and Schulz [Z.
Chem., 21(1 1), 404-405 (1981)] Route (iii) is described in broad terms by Nowotnik
et al [Tetrahedron, 50(29), p .8617-8632 (1994)]. Alpha-chloro-oximes can be
obtained by oximation of the corresponding alpha-chloro-ketone or aldehyde, which
are commercially available. Alpha-bromoketones are commercially available.
More preferred tetra-amine chelators are of formula:
2
(Chelator 3)
where:
L, LBP, n and Z2 are as defined in the first aspect;
Q1 to Q6 are independently Q groups, where Q is H or an amine
protecting group.
By the term "protecting group" is meant a group which inhibits or suppresses
undesirable chemical reactions, but which is designed to be sufficiently reactive that it
may be cleaved from the functional group in question under mild enough conditions
that do not modify the rest of the molecule. After deprotection the desired product is
obtained. Amine protecting groups are well known to those skilled in the art and are
suitably chosen from: Boc (where Boc is ferZ-butyloxycarbonyl), Fmoc (where Fmoc
is fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde [i.e. l-(4,4-
dimethyl-2,6-dioxocyclohexylidene)ethyl] or pys (i.e. 3-nitro-2-pyridine sulfenyl).
In some instances, the nature of the protecting group may be such that both the Q Q
or Q5/Q6 groups, i.e. there is no NH bond on the associated amine nitrogen atom. The
use of further protecting groups are described in 'Protective Groups in Organic
Synthesis', 4th Edition, Theorodora W. Greene and Peter G.M. Wuts, [Wiley
Blackwell, (2006)]. Preferred amine protecting groups are Boc and Fmoc, most
preferably Boc. When Boc is used, Q1 and Q6 are both H, and Q2, Q3, Q4 and Q5 are
each teri-butoxycarbonyl.
Preferred aspects of L, LBP, n and Z2 in Chelator 3 are as defined in the first aspect
(above). Preferred Chelator 3 chelators have (L) = (A')ml , where A' and ml and
preferred aspects thereof are as described for Chelator 2 (above).
Tetra-amine chelators can be obtained as described in Scheme 1 (below). Further
synthetic information on amino- and carboxy- functionalised tetra-amine chelators is
provided by Abiraj et al [Chem.Eur ., 16, 2 115-2124 (2010)]. The synthesis of the
Boc-protected tetra-amine analogue with a -(CH2)sOH bridgehead substituent has
been described by Turpin et al [I.Lab.Comp.Radiopharm., 45, 379-393 (2002)]. The
conjugation of tetra-amine chelators to biological targeting peptides is described by
Nock et al [EurJ.Nucl.Med., 30(2), 247-258 (2003)], and Maina et al
[EurJ.Nucl.Med., 30(9), 1211-1219 (2003)]. A bifunctional HBED derivative having
a pendant active ester group is taught by Eder et al [EurJ.Nucl. Med.Mol.Imaging, 35,
1878-1886 (2008)].
cheme 1: Synthesis of Boc-protected Chelator 2A.
N3S bifunctional chelators can be prepared by the method of Sudhaker et al [Bioconj .
Chem., Vol. 9, 108-1 17(1998)]. N2S2 Diamidedithiol compounds can be prepared by
the method of Kung et al [Tetr. Lett., Vol 30, 4069-4072 (1989].
Monoamidemonoaminebisthiol compounds can be prepared by the method of Hansen
etal [Inorg. Chem., Vol 38, 5351-5358 (1999)].
In a third aspect, the present invention provides a method of preparation of the
imaging agent of the first aspect, which comprises reaction of the chelator conjugate
of the second aspect with a supply of the desired radiometal in a suitable solvent.
Preferred aspects of the chelator conjugate and the radiometal in the third aspect are
as described in the first and second aspects of the present invention (above).
The suitable solvent is typically aqueous in nature, and is preferably a biocompatible
carrier solvent as defined in the fourth aspect (below).
In a fourth aspect, the present invention provides a radiopharmaceutical composition
which comprises the imaging agent of the first aspect, together with a biocompatible
carrier, in a form suitable for mammalian administration.
Preferred aspects of the imaging agent in the fourth aspect are as described in the first
aspect of the present invention (above).
The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent
can be suspended or preferably dissolved, such that the composition is physiologically
tolerable, i.e. can be administered to the mammalian body without toxicity or undue
discomfort. The biocompatible carrier is suitably an inj ectable carrier liquid such as
sterile, pyrogen-free water for injection; an aqueous solution such as saline (which
may advantageously be balanced so that the final product for inj ection is isotonic); an
aqueous buffer solution comprising a biocompatible buffering agent (e.g. phosphate
buffer); an aqueous solution of one or more tonicity-adjusting substances (eg. salts of
plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (eg. sorbitol or mannitol), glycols (eg. glycerol), or other non-ionic
polyol materials (eg. polyethyleneglycols, propylene glycols and the like). Preferably
the biocompatible carrier is pyrogen-free water for injection, isotonic saline or
phosphate buffer.
By the phrase "in a form suitable for mammalian administration" is meant a
composition which is sterile, pyrogen-free, lacks compounds which produce toxic or
adverse effects, and is formulated at a biocompatible pH (approximately pH 4.0 to
10.5). Such compositions lack particulates which could risk causing emboli in vivo,
and are formulated so that precipitation does not occur on contact with biological
fluids (eg. blood). Such compositions also contain only biologically compatible
excipients, and are preferably isotonic.
The imaging agents and biocompatible carrier are each supplied in suitable vials or
vessels which comprise a sealed container which permits maintenance of sterile
integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen
or argon), whilst permitting addition and withdrawal of solutions by syringe or
cannula. A preferred such container is a septum-sealed vial, wherein the gas-tight
closure is crimped on with an overseal (typically of aluminium). The closure is
suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on
septum seal closure) whilst maintaining sterile integrity. Such containers have the
additional advantage that the closure can withstand vacuum if desired (eg. to change
the headspace gas or degas solutions), and withstand pressure changes such as
reductions in pressure without permitting ingress of external atmospheric gases, such
as oxygen or water vapour.
Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm3
volume) which contains multiple patient doses, whereby single patient doses can thus
be withdrawn into clinical grade syringes at various time intervals during the viable
lifetime of the preparation to suit the clinical situation. Pre-filled syringes are
designed to contain a single human dose, or "unit dose" and are therefore preferably a
disposable or other syringe suitable for clinical use. The pharmaceutical compositions
of the present invention preferably have a dosage suitable for a single patient and are
provided in a suitable syringe or container, as described above.
The pharmaceutical composition may contain additional optional excipients such as:
an antimicrobial preservative, pH-adjusting agent, filler, radioprotectant, solubiliser or
osmolality adjusting agent. By the term "radioprotectant" is meant a compound which
inhibits degradation reactions, such as redox processes, by trapping highly-reactive
free radicals, such as oxygen-containing free radicals arising from the radiolysis of
water. The radioprotectants of the present invention are suitably chosen from:
ascorbic acid, ¾ -aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e.
2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cation as described
above. By the term "solubiliser" is meant an additive present in the composition
which increases the solubility of the imaging agent in the solvent. A preferred such
solvent is aqueous media, and hence the solubiliser preferably improves solubility in
water. Suitable such solubilisers include: C1-4 alcohols; glycerine; polyethylene
glycol (PEG); propylene glycol; polyoxyethylene sorbitan monooleate; sorbitan
monooloeate; polysorbates; poly(oxyethylene)poly(oxypropylene)poly(oxyethylene)
block copolymers (Pluronics™); cyclodextrins (e.g. alpha, beta or gamma
cyclodextrin, hydroxypropyl -P-cyclodextrin or hydroxypropyl-y-cyclodextrin) and
lecithin.
By the term "antimicrobial preservative" is meant an agent which inhibits the growth
of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The
antimicrobial preservative may also exhibit some bactericidal properties, depending on
the dosage employed. The main role of the antimicrobial preservative(s) of the present
invention is to inhibit the growth of any such micro-organism in the pharmaceutical
composition. The antimicrobial preservative may, however, also optionally be used to
inhibit the growth of potentially harmful micro-organisms in one or more components
of kits used to prepare said composition prior to administration. Suitable antimicrobial
preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or
mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred
antimicrobial preservative(s) are the parabens.
The term "pH-adjusting agent" means a compound or mixture of compounds useful to
ensure that the pH of the composition is within acceptable limits (approximately pH
4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting
agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS
[i.e. im(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such
as sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition
is employed in kit form, the pH adjusting agent may optionally be provided in a
separate vial or container, so that the user of the kit can adjust the pH as part of a
multi-step procedure.
By the term "filler" is meant a pharmaceutically acceptable bulking agent which may
facilitate material handling during production and lyophilisation. Suitable fillers
include inorganic salts such as sodium chloride, and water soluble sugars or sugar
alcohols such as sucrose, maltose, mannitol or trehalose.
The pharmaceutical compositions of the second aspect may be prepared under aseptic
manufacture (i.e. clean room) conditions to give the desired sterile, non-pyrogenic
product. It is preferred that the key components, especially the associated reagents
plus those parts of the apparatus which come into contact with the imaging agent (eg.
vials) are sterile. The components and reagents can be sterilised by methods known in
the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation,
autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred
to sterilise some components in advance, so that the minimum number of
manipulations needs to be carried out. As a precaution, however, it is preferred to
include at least a sterile filtration step as the final step in the preparation of the
pharmaceutical composition.
As noted above, the pharmaceutical compositions of the present invention preferably
comprise a solubiliser, so that a sterile filtration step may be used without undue loss
of radioactivity adsorbed to the filter material. Similar considerations apply to
manipulations of the pharmaceutical compositions in clinical grade syringes, or using
plastic tubing, where adsorption may cause loss of radioactivity without the use of a
solubiliser.
The radiopharmaceutical compositions of the present invention may be prepared by
various methods:
(i) aseptic manufacture techniques in which the radiometal complex
formation is carried out in a clean room environment;
(ii) terminal sterilisation, in which the radiometal complex formation is carried
out without using aseptic manufacture and then sterilised at the last step
[eg. by gamma irradiation, autoclaving dry heat or chemical treatment (e.g.
with ethylene oxide)];
(iii) kit methodology in which a sterile, non-radioactive kit formulation
comprising the chelator conjugate of Formula III and optional excipients
reacted with a supply of the desired radiometal.
Method (iii) is preferred, and kits for use in this method are described in the fifth
embodiment (below).
In a fifth aspect, the present invention provides a kit for the preparation of the
radiopharmaceutical composition of the fourth aspect, which comprises the chelator
conjugate of the second aspect in sterile, solid form such that upon reconstitution with
a sterile supply of the radiometal in a biocompatible carrier, dissolution occurs to give
the desired radiopharmaceutical composition.
Preferred aspects of the chelator conjugate in the fifth aspect are as described in the
second aspect of the present invention (above).
By the term "kit" is meant one or more non-radioactive pharmaceutical grade
containers, comprising the necessary chemicals to prepare the desired
radiopharmaceutical composition, together with operating instructions. The kit is
designed to be reconstituted with the desired radiometal to give a solution suitable for
human administration with the minimum of manipulation.
The sterile, solid form is preferably a lyophilised solid.
For mTc, the kit is preferably lyophilised and is designed to be reconstituted with
sterile Tc-pertechnetate (TcCV) from a Tc radioisotope generator to give a
solution suitable for human administration without further manipulation. Suitable kits
comprise a container (eg. a septum-sealed vial) containing the chelator conjugate in
either free base or acid salt form, together with a biocompatible reductant such as
sodium dithionite, sodium bisulfite, ascorbic acid, formamidine sulfinic acid, stannous
ion, Fe(II) or Cu(I). The biocompatible reductant is preferably a stannous salt such as
stannous chloride or stannous tartrate. Alternatively, the kit may optionally contain a
non-radioactive metal complex which, upon addition of the technetium, undergoes
transmetallation (i.e. metal exchange) giving the desired product. The non-radioactive
kits may optionally further comprise additional components such as a transchelator,
radioprotectant, antimicrobial preservative, pH-adjusting agent or filler - as defined
above.
In a sixth aspect, the present invention provides a method of imaging the human or
animal body which comprises generating an image of at least a part of said body to
which the imaging agent of the first aspect, or the composition of the fourth aspect has
distributed using PET or SPECT, wherein said imaging agent or composition has been
previously administered to said body.
Preferred aspects of the imaging agent or composition in the sixth aspect are as
described in the first and fourth aspects respectively of the present invention (above).
The method of the sixth aspect is preferably carried out where the part of the body is
disease state where abnormal apoptosis is involved. By the term "abnormal
apoptosis" is meant dysregulation of the programmed cell death (PCD) process. Such
dysregulation has been implicated in a number of disease states, including those
associated with the inhibition of apoptosis, such as cancer and autoimmune disorders,
and those associated with hyperactive apoptosis, including: neurodegenerative
diseases; haematologic diseases; AIDS; ischaemia; allograft rejection and cardiology
(myocardial infarction, atherosclerosis and/or cardiotoxicity follow drug therapy).
The visualization and quantitation of apoptosis is therefore useful in the diagnosis of
such apoptosis-related pathophysiology.
The imaging method of the sixth aspect may optionally be carried out repeatedly to
monitor the effect of treatment of a human or animal body with a drug, said imaging
being effected before and after treatment with said drug, and optionally also during
treatment with said drug. Therapeutic treatments for these diseases aim to restore
balanced apoptosis, either by stimulating or inhibiting the PCD process as appropriate.
Of particular interest is early monitoring of the efficacy of cancer therapy to ensure
that malignant growth is controlled before the condition becomes terminal.
In a seventh aspect, the present invention provides the use of the imaging agent of the
first aspect, the composition of the fourth aspect, or the kit of the fifth aspect in a
method of diagnosis of the human or animal body.
In an eighth aspect, the present invention provides a method of diagnosis of the
human or animal body which comprises the method of imaging of the sixth aspect.
Preferred aspects of the imaging agent or composition in the seventh and eighth
aspects are as described in the first and fourth aspects respectively of the present
invention (above). The diagnosis of the human or animal body of both aspects is
preferably of a disease state where abnormal apoptosis is involved. Such "abnormal
apoptosis" is as described in the sixth aspect (above).
The invention is illustrated by the non-limiting Examples detailed below. Examples 1
to 3 provide the synthesis of Chelator 1 (a diaminedioxime) of the invention, and
Example 4 the synthesis of Chelator 1A (a diaminedioxime functionalised with
glutaric acid) and synthesis of the corresponding active ester Chelator 1A-TFTP ester.
Example 5 the synthesis of Chelator IB (a diaminedioxime functionalised with
glutaryl-amino-PEG12 propionic acid). Example 6 provides the synthesis of a Bocprotected
tetra-amine chelator of the invention (Chelator 2A). Example 7 provides
the synthesis of a HYNIC-duramycin conjugate (prior art) for comparative purposes.
Example 8 provides the synthesis of duramycin functionalised with Chelator 1A
(Conjugate 3A and Conjugate 3B). Example 9 provides the synthesis of cinnamycin
with Chelator 1A (Conjugate 5). Example 10 provides the synthesis of duramycin
with Chelator IB (Conjugate 6). Example 11 provides the synthesis of cinnamycin
with Chelator IB (Conjugate 6). Example 12 provides the synthesis of duramycin
functionalised with Chelator 2A (Conjugate 2A and Conjugate 2B) and Example 13
of cinnamycin with Chelator 2A (Conjugate 4). Example 14 provides the
radiolabelling of the chelator conjugates of the invention with the radiometal Tc.
The mTc complexes form as a single species with high RCP. That is an advantage
over HY IC, where multiple species form when HYNIC/phosphine/tricine labelling
is used. The procedure is simple, with efficient labeling at room temperature. The
RCP is very good, even at high radioactive concentration (>90% RCP at >
500MBq/mL).
Example 15 provides determination of the site of conjugation of a chelator and
Example 16 demonstrates that the site of conjugation of a chelator has a significant
effect on the binding affinity for phosphatidylethanolamine, with a factor of 18
difference (K< 5 nM vs 90 nM). This provides evidence that attachment of the
radiometal complex at the N-terminus (Cys of Formula II) is preferred over
attachment at Xaa of Formula II. The EL4 lymphoma mouse xenograft tumour model
of Example 17 has been used as a model to mimic the apoptotic response following
chemotherapy. Therapy-treated mice (etoposide/cyclophosphamide) showed a 4 fold
increase in tumour apoptosis compared to vehicle control treated animals. The
biodistribution results of Example 17 show a higher uptake of each agent in
chemotherapy-treated tumours, while correlation analysis suggests a trend of higher
binder uptake in tumours with higher levels of apoptosis. Tc-[Conjugate 5] had
similar tumour and improved liver performance mTc-[Conjugate 3A] mTc-
[Conjugate 2A] shows similar tumour but inferior lung performance vs Tc-
[Conjugate 5]. Repeat imaging studies with mTc-[Conjugate 5] showed a consistent
increase in tumour: muscle ratios following therapy. Example 18 shows that a PEG
linker group is advantageous in reducing liver background and increasing urinary
excretion in vivo.
Abbreviations.
Conventional single letter or 3-letter amino acid abbreviations are used.
% id: Percentage injected dose
Ac: Acetyl.
Acm: Acetamidomethyl .
ACN: Acetonitrile.
Boc: ter/-Butyloxycarbonyl .
Bz: Benzyl.
DCM: Dichloromethane.
DIPEA: N-Diisopropylethylamine.
DMF: N,N -Dimethylformamide.
DMSO: Dimethyl sulfoxide.
Fmoc: 9-Fluorenylmethoxycarbonyl
Glut: Glutaric acid.
HATU: (9-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
HOAt: 7-Aza- 1-hydroxybenzotriazole.
HPLC: High performance liquid chromatography.
IBX: l-Hydroxy-l,2-benziodoxole-3(lH)-one-l-oxide.
MDP: Methylenediphosphonic acid.
NaPABA: Sodium ^ara-aminobenzoate.
NHS: N-Hydroxy-succinimide.
NMM: N-Methylmo holine.
NMP: l-Methyl-2-pyrrolidinone.
PBS: Phosphate-buffered saline.
PEG12: -(OCH 2CH2)12 - .
PyAOP: (7-Azabenzotriazole- 1-yl-oxy-irw-pyrrolidino-phosphonium
hexafluorophosphate
PyBOP: Benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate.
RAC: Radioactive concentration.
RCP: Radiochemical purity.
tBu: rt-Butyl.
TFA: Trifluoroacetic acid.
TFTP: Tetrafluorothiophenol.
THF: Tetrahydrofuran .
TIS: Triisopropylsilane.
Trt: Trityl.
Table 1: Compounds of the Invention.
Formula II (with bridges as specified in the first aspect):
Cysa-Xaa-Gln-Ser b-Cys -Ser -Phe-Gly-Pro-Phe-Thr -Phe-Val-Cys b-
(HO-Asp)-Gly-Asn-Thr -Lysd
Name Structure
LBP1 Duramycin Formula II, where Xaa = Lys.
LBP2 Cinnamycin Formula II, where Xaa = Arg.
Conjugate 1 [HYNIC]-LBP1, with HYNIC attached at either of Cys & Xaa.
(prior art) (A mixture of mono- and bis- functionalised species).
Conjugate 1A [HYNIC]-LBP1, with HYNIC attached at Cys
(^owo-functionalised species).
Conjugate IB [HYNIC]-LBP1, with HYNIC attached at Xaa
(mowofunctionalised species).
Conjugate 2A [Chelator 2A]-LBP1, with Chelator 2A attached at Cys
-funct ionalised species).
Conjugate 2B [Chelator 2A]-LBP1, with Chelator 2A attached at Xaa
species).
Conjugate 3A [Chelator 1A]-LBP1, with Chelator 1A attached at either Cys
and Xaa (mixture of -funct ionalised species).
Conjugate 3B [Chelator 1A]-LBP1, with Chelator 1A attached at both Cys
and Xaa (fos-functionalised species).
Conjugate 4 [Chelator 2A]-LBP2, with Chelator 2A attached at Cys .
( -funct ionalised species).
Conjugate 5 [Chelator 1A]-LBP2, with Chelator 1A attached at Cysa.
species).
Conjugate 6 [Chelator 1B]-LBP1, with Chelator IB attached at either Cys
and Xaa (mixture of -funct ionalised species).
Conjugate 7 [Chelator 1BJ-LBP2, with Chelator IB attached at Cys (monofunctionalised
species).
Example 1; Synthesis of l,l,l-fm(2-aminoethvQmethane,
Step 1(a): 3(methoxycarbonylmethylene)glutaric acid dimethylester.
Carbomethoxymethylenetriphenylphosphorane (167g, 0.5mol) in toluene (600ml) was
treated with dimethyl 3-oxoglutarate (87g, 0.5mol) and the reaction heated to 100°C
on an oil bath at 120°C under an atmosphere of nitrogen for 36h. The reaction was
then concentrated in vacuo and the oily residue triturated with 40/60 petrol
ether/diethylether 1:1, 600ml. Triphenylphosphine oxide precipitated out and the
supernatant liquid was decanted/filtered off. The residue on evaporation in vacuo was
Kugelrohr distilled under high vacuum Bpt (oven temperature 180-200°C at 0.2torr)
to give 3-(methoxycarbonylmethylene)glutaric acid dimethylester (89.08g, 53%).
NMR CDCl ): d 3.31 (2H, s, CH2), 3.7(9H, s, 3xOCH3), 3.87 (2H, s, CH2), 5.79 (1H, s,
=CH, ) ppm.
NMR 1 C(CDC13), d 36.56,CH3, 48.7, 2xCH3, 52.09 and 52.5 (2xCH ); 122.3 and 146.16
C=CH; 165.9, 170 0 and 170.5 3xCOO ppm.
Step Kb): Hydrogenation of 3-(methoxycarbonylmethylene)glutaric acid
dimethylester.
3-(methoxycarbonylmethylene)glutaric acid dimethylester (89g, 267mmol) in
methanol (200ml) was shaken with ( 10% palladium on charcoal: 50% water) (9 g)
under an atmosphere of hydrogen gas (3.5 bar) for (3Oh). The solution was filtered
through kieselguhr and concentrated in vacuo to give 3-
(methoxycarbonylmethyl)glutaric acid dimethylester as an oil, yield (84. 9g, 94 %).
NMR ( C d 2.48 (6H, d, J=8Hz, 3xCH2), 2.78 (1H, hextet, J=8Hz CH, ) 3.7 (9H, s,
3xCH3) .
NMR 1 C(CDC13), d 28.6, CH; 37.50, 3xCH3; 51.6, 3xCH2; 172.28,3xCOO.
Step 1(c): Reduction and esterification of trimethyl ester to the triacetate.
Under an atmosphere of nitrogen in a 3 necked 2L round bottomed flask lithium
aluminium hydride (20g, 588mmol) in THF (400ml) was treated cautiously with
ira(methyloxycarbonylmethyl)methane (40g, 12mmol) in THF (200ml) over lh. A
strongly exothermic reaction occurred, causing the solvent to reflux strongly. The
reaction was heated on an oil bath at 90°C at reflux for 3 days. The reaction was
quenched by the cautious dropwise addition of acetic acid ( 100ml) until the evolution
of hydrogen ceased. The stirred reaction mixture was cautiously treated with acetic
anhydride solution (500ml) at such a rate as to cause gentle reflux. The flask was
equipped for distillation and stirred and then heating at 90°C (oil bath temperature) to
distil out the THF. A further portion of acetic anhydride (300ml) was added, the
reaction returned to reflux configuration and stirred and heated in an oil bath at 140°C
for 5h. The reaction was allowed to cool and filtered. The aluminium oxide precipitate
was washed with ethyl acetate and the combined filtrates concentrated on a rotary
evaporator at a water bath temperature of 50°C in vacuo (5 mraHg) to afford an oil.
The oil was taken up in ethyl acetate (500ml) and washed with saturated aqueous
potassium carbonate solution. The ethyl acetate solution was separated, dried over
sodium sulfate, and concentrated in vacuo to afford an oil. The oil was Kugelrohr
distilled in high vacuum to give fr /X2-acetoxyethyl)methane (45 .3g, 95.9%) as an oil.
Bp. 220 °C at 0 .1 mmHg.
NMR ^CDCls), d 1.66(7H, m, 3xCH¾CH), 2.08( 1H, s, 3xCH3); 4.1(6H, t, 3xCH 0)
NMR 1 C(CDC13), d 20.9, CH3; 29.34, CH; 32. 17, CH2; 62. 15, CH 0 ; 17 1, CO.
Step 1(d): Removal of Acetate groups from the triacetate.
7ra(2-acetoxyethyl)methane (45. 3g, 165mM) in methanol (200ml) and 880 ammonia
(100ml) was heated on an oil bath at 80°C for 2 days. The reaction was treated with a
further portion of 880 ammonia (50ml) and heated at 80°C in an oil bath for 24h. A
further portion of 880 ammonia (50ml) was added and the reaction heated at 80°C for
24h. The reaction was then concentrated in vacuo to remove all solvents to give an
oil. This was taken up into 880 ammonia (150ml) and heated at 80°C for 24h. The
reaction was then concentrated in vacuo to remove all solvents to give an oil.
Kugelrohr distillation gave acetamide bp 170-180 0.2mm. The bulbs containing the
acetamide were washed clean and the distillation continued. Tris(2-
hydroxyethyl)methane (22.53g, 92%) distilled at bp 220 °C 0.2mm.
NMR ^(CDCls), d 1.45(6H, q, 3xCH2), 2.2(1H, quintet, CH); 3.7(6H, 3xCH2OH); 5.5(3H,
brs, 3xOH).
NMR 1 C(CDC13), d 22.13, CH; 33.95, 3xCH2; 57 8, 3xCH2OH.
Step 1(e): Conversion of the triol to the ?rzs(methanesulphonate).
To an stirred ice-cooled solution of fr/s(2-hydroxyethyl)methane (10g, 0.0676mol) in
dichloromethane (50ml) was slowly dripped a solution of methanesulfonyl chloride
(40g, 0.349mol) in dichloromethane (50ml) under nitrogen at such a rate that the
temperature did not rise above 15°C. Pyridine (21.4g, 0.27mol, 4eq) dissolved in
dichloromethane (50ml) was then added drop-wise at such a rate that the temperature
did not rise above 15°C, exothermic reaction. The reaction was left to stir at room
temperature for 24h and then treated with 5N hydrochloric acid solution (80ml) and
the layers separated. The aqueous layer was extracted with further dichloromethane
(50ml) and the organic extracts combined, dried over sodium sulfate, filtered and
concentrated in vacuo to give frzs[2-(methylsulphonyloxy)ethyl]methane
contaminated with excess methanesulfonyl chloride. The theoretical yield was 25. 8g.
NMR ^(CDCy, d 4.3 (6H, t, 2xCH ), 3.0 (9H, s, 3xCH3), 2 (1H, hextet, CH), 1.85 (6H, q,
3xCH ) .
Step 1(f): Preparation of 1,1,1 -fr?s(2-azidoethyl)methane.
A stirred solution of ira[2-(methylsulfonyloxy)ethyl]methane [from Step 1(e),
contaminated with excess methylsulfonyl chloride] (25. 8g, 67mmol, theoretical) in dry
DMF (250ml) under nitrogen was treated with sodium azide (30. 7g, 0.47mol) portionwise
over 15 minutes. An exotherm was observed and the reaction was cooled on an
ice bath. After 30 minutes, the reaction mixture was heated on an oil bath at 50°C for
24h. The reaction became brown in colour. The reaction was allowed to cool, treated
with dilute potassium carbonate solution (200ml) and extracted three times with 40/60
petrol ether/diethyl ether 10:1 (3x1 50ml). The organic extracts were washed with
water (2x1 50ml), dried over sodium sulfate and filtered. Ethanol (200ml) was added
to the petrol/ether solution to keep the triazide in solution and the volume reduced in
vacuo to no less than 200ml. Ethanol (200ml) was added and reconcentrated in vacuo
to remove the last traces of petrol leaving no less than 200ml of ethanolic solution.
The ethanol solution of triazide was used directly in Step 1(g).
CARE DO NOT REMOVE ALL THE SOLVENT AS THE AZIDE IS
POTENTIALLY EXPLOSIVE AND SHOULD BE KEPT IN DILUTE SOLUTION
AT ALL TIMES
Less than 0.2ml of the solution was evaporated in vacuum to remove the ethanol and
an NMR run on this small sample:
NMR ^(CDCy, d 3.35 (6H, t, 3xCH ), 1.8 (1H, septet, CH, ), 1.6 (6H, q, 3xCH2) .
Step Kg): Preparation of l - (2-aminoethyl)methane.
ra (2-azidoethyl)methane (15.06g, 0.0676 mol), (assuming 100% yield from previous
reaction) in ethanol (200ml) was treated with 10% palladium on charcoal (2g, 50%
water) and hydrogenated for 12h. The reaction vessel was evacuated every 2 hours to
remove nitrogen evolved from the reaction and refilled with hydrogen. A sample was
taken for NMR analysis to confirm complete conversion of the triazide to the
triamine.
Caution: unreduced azide could explode on distillation. The reaction was filtered
through a celite pad to remove the catalyst and concentrated in vacuo to give tris(2-
aminoethyl)methane as an oil. This was further purified by Kugelrohr distillation
bp.l80-200°C at 0.4mm/Hg to give a colourless oil (8. lg, 82.7% overall yield from
the triol).
NMR ^(CDCls), d 2.72 (6H, t, 3xCH N), 1.41 (H, septet, CH), 1.39 (6H, q, 3xCH ) .
NMR 1 C(CDC13), d 39.8 (CH2NH2), 38.2 (CH .), 31.0 (CH).
Example 2: Preparation of 3-chloro-3-methyl-2-nitrosobutane.
A mixture of 2-methylbut-2-ene (147ml, 1.4mol) and isoamyl nitrite (156ml, 1.16mol)
was cooled to -30 °C in a bath of cardice and methanol and vigorously stirred with an
overhead air stirrer and treated dropwise with concentrated hydrochloric acid (140ml,
1.68mol ) at such a rate that the temperature was maintained below -20°C. This
requires about l h as there is a significant exotherm and care must be taken to prevent
overheating. Ethanol (100ml) was added to reduce the viscosity of the slurry that had
formed at the end of the addition and the reaction stirred at -20 to -10°C for a further
2h to complete the reaction. The precipitate was collected by filtration under vacuum
and washed with 4x30ml of cold (-20°C) ethanol and 100ml of ice cold water, and
dried in vacuo to give 3-chloro-3-methyl-2-nitrosobutane as a white solid. The ethanol
filtrate and washings were combined and diluted with water (200ml) and cooled and
allowed to stand for l h at -10°C when a further crop of 3-chloro-3-methyl-2-
nitrosobutane crystallised out. The precipitate was collected by filtration and washed
with the minimum of water and dried in vacuo to give a total yield of 3-chloro-3-
methyl-2-nitrosobutane (115g 0.85mol, 73%) >98% pure by MR.
NMR T^CDCy, As a mixture of isomers (isomerl, 90%) 1.5 d, (2H, CH ), 1.65 d, (4H, 2
xCH3), 5.85,q, and 5.95,q, together 1H. (isomer2, 10%), 1.76 s, (6H, 2x CH3), 2.07(3H,
CH3) .
Example 3: Synthesis of Ms[N-(14-dimethyl-2-N-hydroxyimine propyl)2-
aminoethyl1-(2-aminoethyl)methane (Chelator 1).
To a solution of ir?X2-aminoethyl)methane (4.047g, 27.9mmol) in dry ethanol (30ml)
was added potassium carbonate anhydrous (7.7g, 55.8mmol, 2eq) at room temperature
with vigorous stirring under a nitrogen atmosphere. A solution of 3-chloro-3-methyl-
2-nitrosobutane (7.56g, 55.8mol, 2eq) was dissolved in dry ethanol (100ml) and 75ml
of this solution was dripped slowly into the reaction mixture. The reaction was
followed by TLC on silica [plates run in dichloromethane, methanol, concentrated
(0.88sg) ammonia; 100/30/5 and the TLC plate developed by spraying with ninhydrin
and heating]. The mono-, di- and tri-alkylated products were seen with RF's
increasing in that order. Analytical HPLC was run using RPR reverse phase column in
a gradient of 7.5-75% acetonitrile in 3% aqueous ammonia. The reaction was
concentrated in vacuo to remove the ethanol and resuspended in water ( 110ml). The
aqueous slurry was extracted with ether (100ml) to remove some of the trialkylated
compound and lipophilic impurities leaving the mono and desired dialkylated product
in the water layer. The aqueous solution was buffered with ammonium acetate (2eq,
4.3g, 55.8mmol) to ensure good chromatography. The aqueous solution was stored at
4°C overnight before purifying by automated preparative HPLC.
Yield (2.2g, 6.4mmol, 23%).
Mass spec; Positive ion 10 V cone voltage. Found: 344; calculated M+H= 344.
NMR T^CDCl,), d 1.24(6H, s, 2xCH ), 1.3(6H, s, 2xCH3), 1.25-1 .75(7H, m, 3xCH CH),
(3H, s, 2xCH2), 2.58 (4H, m, CH2N), 2.88(2H, t CH2N2), 5.0 (6H, s, NH2 , 2xNH, 2xOH).
NMR ¾ ((CD3) SO) 1.1 4xCH; 1.29, 3xCH ; 2 .1 (4H, t, 2xCH );
NMR 1 C((CD3) SO), d 9.0 (4xCH3), 25.8 (2xCH3), 31.0 2xCH2, 34.6 CH2, 56.8 2xCH2N;
160.3, C=N.
HPLC conditions: flow rate 8ml/min using a 25mm PRP column [A=3% ammonia
solution (sp.gr = 0.88) /water; B=Acetonitrile].
Time %B
0 7 .5
15 75.0
20 75.0
22 7 .5
30 7 .5
Load 3ml of aqueous solution per run, and collect in a time window of 12.5- 13.5 min.
Example 4: Synthesis of Tetrafluorothiophenyl ester of Chelator 1-glutaric acid
rChelator 1A-TFTP Ester).
a) Synthesis of [Chelator l |-glutaric acid (Chelator 1A .
Chelator 1A
Chelator 1 ( 100 mg, 0.29 mmol) was dissolved in DMF (10 ml) and glutaric
anhydride (33 mg, 0.29 mmol) added by portions with stirring. The reaction was
stirred for 23 hours to afford complete conversion to the desired product. The pure
acid was obtained following RP-HPLC in good yield.
b) Synthesis of Chelator 1A-TFTP Ester.
Chelator 1A-TFTP ester
To Chelator 1A (from Step a; 300 mg, 0.66 mmol) in DMF (2 ml) was added HATU
(249 mg, 0.66 mmol) and NMM (132 , 1.32 mmol). The mixture was stirred for 5
minutes then tetrafluorothiophenol (0.66 mmol, 119 mg) was added. The solution
was stirred for 10 minutes then the reaction mixture was diluted with 20 %
acetonitrile/water (8 ml) and the product purified by RP-FIPLC yielding 110 mg of the
desired product following freeze-drying.
Example 5; Synthesis of Chelator l-glutaryl-amino-PEG12 propionic acid
(Chelator IB).
Boc-amino-PEG12 propionic acid (Polypure; 45 mg, 0.060 mmol) was treated with
TFA/water (19: 1) ( 1 mL) for 30 min. The TFA was then evaporated in vacuo and the
residue dried in vacuo overnight affording 52 mg crude amino-PEG12 propionic acid.
Chelator 1A (46 mg, 0.10 mmol) and PyAOP (31 mg, 0,060 mmol) were dissolved in
NMP ( 1 mL). DIPEA (42 m , 0.24 mmol) was added and the solution shaken for 5
min and added to amino-PEG12 propionic acid (0.06 mmol). The reaction mixture
was shaken overnight. Additional Chelator 1A (0.03 mmol) was added to the reaction
mixture and after 1 h the mixture was diluted with water/0. 1% TFA (7 mL) and the
product purified by preparative RP-FIPLC.
Purification and characterization
Purification by RP-HPLC (gradient: 10-30% B over 40 min, t : 34.5 min) afforded 44
mg (67% yield) of Chelator IB after lyophilisation. Chelator IB was characterized by
LC-MS (gradient: 10-40% B over 5 min, t : 2.2 min; calcd. m/z 1057.7 [MH] +, found
m/z 1058.0).
Example 6; Synthesis of Chelator 2A.
Step (a): Diethyl [2-(benzyloxy)ethyl]malonate.
The compound was prepared by a modification of the method of Ramalingam et al
Tetrahedron, 51, 2875-2894 (1995)]. Thus, sodium (1.20g) was dissolved in absolute
ethanol (25 ml) under argon. Diethyl malonate (14.00g) was added and the mixture
was refiuxed for 30 min. Benzyl bromoethyl ether (lOg) was added and the mixture
was stirred at reflux for 16 hours. The ethanol was removed by rotary evaporation
and the residue was partitioned between ether (100ml) and water (50 ml). The
ethereal layer was washed with water (3x50 ml) and dried over sodium sulfate. The
ether was removed by rotary evaporation and the residue was distilled in vacuoo. The
fraction distilling at 40-55 °C was discarded (unreacted diethyl malonate). The
product distilled at 140-150 °C (1mm), [lit. bp 138-140 C (1mm)]. The yield was
12.60g of colourless oil.
¾ NMR (270 MHz, CDC13, 25 °C, TMS) d = 7.28 (m, 5H C H ), 4.47 (s, 2H, CH2-Ph), 4.16
(m, 4H, COOCH ), 3.58 (t, 1H, CH), 3.50 (t, 2H, 0 -G¾-CH ), 2.21 (t, 2H, 0 -CH -(¾), 1.20
(t, 6H, COOCH2- ). 1 CNMR (67.5 MHz, CDC13, 25 °C, TMS) d = 169.20 (CO), 138.10,
128.60, 127.80 (aromatic), 73.00 (CH Ph), 67.30 (0 - H -CH2), 61.70 (COO H ), 49.10
(CH), 28.90 (0 -CH - H2), 14.10 (COOCH2 ¾ ).
Step (b): N.N'-i?/.s,(2-aminoethyl)-2-(2-benzyloxy-ethyl)malonamide.
Diethyl [2-(benzyloxy)ethyl]malonate (4.00g) was added to ethylene diamine (30 ml)
and the solution was stirred at room temperature for two days. The excess ethylene
diamine was removed by rotary evaporation and the residue was dried under high
vacuum for 2 days to give a yellow oil (4.28g) that crystallized on standing. The
product still contained traces of ethyl enedi amine, as detected in the NMR spectra.
H NMR (270 MHz, CDC13, 25 °C, TMS) d = 7.74 (br t, 2H, CO-NH), 7.32 (m, 5H,
C6H5), 4.46 (s, 2H, CH2-Ph), 3.50 (t, 2H, OCH -CH2- ), 3.33 (t 1H, CH), 3.23 (m, 4H,
CO-NH-C H2), 2.74 (t, 4H, CH -NH2) 2.18 (q, 2H, 0 -CH 2-G¾-) 1.55 (br s 4H, NH2) .
1 C NMR (67.5 MHz, CDC13, 25 °C, TMS) d = 171.10 (CO), 138.20, 128.30, 127.70
(aromatic), 73.00 (CH 2-Ph), 67.80 (0 - H2-CH 2), 51.40 (CH), 42.40 (CO-NH-CH 2),
41.20 (CH 2-NH 2), 31.90 (0 -CH 2-CH 2-).
Step (c): N,N'-i¾s(2-amino-ethyl)-2-(2-benzyloxy ethyl)- 1,3 -diaminopropane.
N,N'-i?/.y-(2-aminoethyl)-2-(2-benzyloxy-ethyl)malonamide (3.80g) was dissolved in
THF (20 ml) and the flask was immersed in an ice bath. The flask was flushed with
argon and THF borane complex (80 ml, 1M in THF) was added through a syringe.
The reaction mixture was allowed to warm up to room t emp and then stirred at 4 0 °C
for 2 days and refluxed for lh. Methanol (50ml) was added dropwise and the solution
was stirred at 40 °C overnight. The solvents were removed by rotary evaporator and
the residue was dissolved in methanol (20 ml). Sodium hydroxide (lOg in 15ml of
water) was added and the methanol was boiled away. A colourless oil separated that
was extracted into CH2CI2 (3x50ml). The solution was dried over N a2SC>4. Removal
of the solvent gave 3.40g of colourless oil.
NMR (270 MHz, CDC13, 2 5 °C, TMS) d = 7.34 (m, 5H, C6H5), 4.49 (s, 2H, CH2-
Ph), 3.55 (t, 2H, OCH2-CH 2-), 2.76 (t, 4H, N-CH 2), 2.63 (m, 8H, N-CH 2), 1.84 (m,
1H, CH), 1.58 (m, 2H, CH-G¾-CH 2-0), 1.41 (br s, 6H, H ) . 1 C NMR (67.5 MHz,
CDCI 3, 25 °C, TMS) d = 138.60, 128.30, 127.60 (aromatic), 72.80 (CH 2-Ph), 68.70
(O-CH2-CH2), 53.50 (N-CH 2) , 52.80 (N-CH ), 41.60 (N-CH 2) 36.40 (CH), 31.30
(CH-CH2-CH2-O). MS-EI: 295 [M+H] +, (calcd.: 295.2).
Step (d): N.N' -Bis(2-tert-butoxycarbonylamino-ethyn-2-(2-benzyloxyethyl)- 1.3-
di(tert-butoxycarb ony 1amino)propane .
N,N'-i?/X2-aminoethyl)-2-(2-benzyloxy-ethyl)- 1,3 -diaminopropane (3.30g) was
dissolved in CH2CI2 (100ml) and triethylamine (5.40g) and tert-b dicarbonate
(10.30g) were added. The reaction mixture was stirred at room t emp for 2 days. The
mixture was washed with water (100 ml), citric acid solution (100 ml, 10% in water)
and with water (2x100ml). The organic layer was dried over N a2SC>4, and the solvent
was removed by rotary evaporation giving a yellow oil which was dried to a constant
mass under high vacuum. The crude product (7.70g) was purified on a silica gel
column (250g, 230-400 mesh, CH2C 12, CH2C 12-Et 20 1:1) to give 6.10g (78.3%) of a
clear oil.
¾ NMR (270 MHz, CDC13, 25 °C, TMS) d = 7.32 (m, 5H, C H ), 5.12 (br d, 2H, NH), 4.47
(s, 2H, CH2-Ph), 3.49 (t, 2H, OC -CH2-), 3.24 (br, 12H, N-CH 2), 2.14 (br, 1H, CH), 1.59
(m, 2H, CH-CH2-CH -0) 1.45 (s, 18H, t-Bu), 1.42 (s, 18H, t-Bu). 1 C NMR (67.5 MHz,
CDCI 3, 25 °C, TMS) d = 155.90 (NH-CO), 138.20, 128.30 127.60, 127.50 (aromatic), 79.90,
78.90 (CMe3), 72.80 (CH2-Ph), 68.00 (0-CH 2-CH2), 50.00 (br, N-CH ), 46.90 (br, N-CH 2),
39.20 (N-CH 2), 34.40 (br, CH), 29.80 (CH-CH 2-CH2-0), 28.30 (t-Bu). MS-EI: 695 [M+H]+,
(calcd.: 695.5)
Step (e): N,N'-j?M2-tert-butoxycarbonylamino-ethyl)-2-(2-hydroxy ethyl)- 1,3-di(tertbutoxycarbony
1amino)propane .
N,N'- S/.y(2-tert-butoxycarbonylamino-ethyl)-2-(2-benzyloxy-ethyl)-l,3-di(tertbutoxycarbonylamino)
propane (3.16g) was dissolved in absolute ethanol (100ml) and
Pd on activated carbon (l.OOg, dry, 10%) was added. The mixture was hydrogenated
in a Parr hydrogenation apparatus at 35 psi for two days. The catalyst was filtered off,
washed with ethanol (3x20ml). The ethanol was removed by rotary evaporation to
give a colourless oil that was dried to a constant mass (2.67g, 97.1%) under high
vacuum.
1H NMR (270 MHz, CDC1 , 25 °C, TMS) d = 5.25 (br d, 2H, NH), 3.69 (t, 2H, OCH -
CH2-), 3.28 (br, 12H, N-CH2), 2.71 (br, OH), 2.23 (br, 1H, CH), 1.56 (shoulder,m,
2H, CH-CH2-CH2 -O) 1.48 (s, 18H, t-Bu), 1.44 (s, 18H, t-Bu). 13C NMR (67.5 MHz,
CDCI 3, 25 °C, TMS) d = 156.10 (NHCO), 80.00, 79.20 (CMe3), 59.60 (0-CH 2-CH2),
49.90 (br, N-CH 2), 47.00 (br, N-CH 2), 39.34 (N-CH2), 33.80 (CH), 32.30 (CH- 2-
CH2-0), 28.30 (t-Bu). MS-EI: 605 [M+H]+, (calcd.: 605.4).
Step (f): N,N'-i?^(2-tert-butoxycarbonylamino-ethyl)-2-(2-carboxymethyl)-l,3-
di(tert-butoxycarbonylamino)propane (Boc-protected Chelator 2A).
The method of Mazitschek et al [Ang. Chem. Int. Ed., 41, 4059-4061 (2002)] was
used. Thus, N,N -i¾X2-tert-butoxycarbonylamino-ethyl)-2-(2-hydroxy ethyl)- 1,3 -
di(tert-butoxycarbonylamino)propane (2.60g) was dissolved in DMSO (15 ml) and 1-
hydroxy-l,2-benziodoxole-3(lH)-one-l-oxide (IBX, 3.50g) was added. The mixture
was stirred at room temp for 1 hour then N-hydroxysuccinimide (2.50g) was added.
The reaction mixture was stirred at room temp for 2 days. Sodium hydroxide
solution (2M, 40ml) was added and the mixture was stirred at room temp for 4 hours.
The solution was immersed in an ice bath and was acidified with 2M hydrochloric
acid to pH 2 . The aqueous layer was extracted with ether (4x1 00ml) and the
combined ether extracts were washed with water (3x50ml). The ethereal layer was
dried over a2S0 4 and the solvent was removed by rotary evaporation to give a
yellow solid residue that contained the product and 2-iodosobenzoic acid. Most of the
iodosobenzoic acid (2.1g) was removed by crystallization from chloroform-hexanes
(1:3) (80ml). Evaporation of the chloroform-hexanes mother liquor gave a yellow oil
(3g) that was loaded on a silica column (300g, CH2Cl2 -Et20 , 1:1). The remaining
iodosobenzoic acid was eluted with ether. The product was eluted with ethermethanol
(9:1). The fractions containing the product were combined and removal of
the solvent gave 1.5g of pale yellow oil. This was re-chromatographed on a silica
column (50g, Et20). The product was eluted with ether-acetic acid (95:5). The
fractions containing the product were combined and the solvent was removed by
rotary evaporation to give an oil that was dried under high vacuum. The yield was
1.1 Og (41.3%).
¾ NMR (270 MHz, CDC13, 25 °C, TMS) d = 7.61 (br s, 1H, COOH), 5.19 (br d, 2H, NH),
3.22 (br, 12H, N-CH ), 2.47 (br m, 1H, CH), 2.26 (br, 2H, CH-(¾-COOH), 1.41 (s, 18H, t-
Bu), 1.37 (s, 18H, t-Bu). 1 C NMR (67.5 MHz, CDC13, 25 °C, TMS) d = 175.90 (COOH),
156.10 (NHCO), 80.40, 79.10 (CMe3), 49.50 (N-CH ), 46.80 (N-CH ), 39.00 (N-CH2), 34.70
(CH-CH -COOH), 34.20 (CH-CH2-COOH), 28.30 , 28.20(t-Bu). MS-EI: 619 [M+H]+,
(calcd.: 619.4).
Example 7; Synthesis of HYNIC-Duramycin (Conjugate 1A and Conjugate IB).
Duramycin (Sigma-Aldrich; 5.0 mg, 2.5 mihoΐ) and N-Boc-HYNIC succinimidyl ester
(ABX Advanced Biochemical Compounds; 1.0 mg, 2.8 mihoΐ) were dissolved in DMF
( 1 ml) and DIPEA (2.0 m , 13 mihoΐ ) was added to the mixture. The reaction progress
was monitored by LC-MS analysis. Addition of HOAt (1.1 eq) after 3 hrs, in order to
drive the sluggish reaction, afforded -60% product formation overnight. Additional
N-Boc-HYNIC succinimidyl ester ( 1 eq) and HOAt (2 eq) were needed to obtain
80% HYNIC-conjugate formation after one subsequent day at room temperature and
3 days at 4 °C. Two baseline separated peaks corresponding to mono-conjugates were
observed by LC-MS analysis in addition to the bis-conjugate (-35%).
Purification and characterisation.
Water/0.1% TFA (4 ml) was added to the reaction mixture and the two monoconjugated
products were purified by preparative RP-HPLC (gradient: 0% B over 15
min; 0-45% B over 10 min; 45-60%. B over 40 min, tR: 47.9 and 49.3 min) and then
Boc-deprotected in TFA affording two mono-conjugated Duramycin isomers, in 1.7
mg and 1.1 mg yield, respectively.
The two isomers were characterised by LC-MS (gradient: 20-40%> B over 5 min, :
2.8 min (Conjugate 1A), found m/z 1074.8, expected MH2
+ : 1074.4, t : 2.9 min
(Conjugate IB), found m/z: 1074.8, expected MH2
2+: 1074.4.
Example 8; Synthesis of [Chelator lAI-Duramycin -conjugate fConjugate
3A and [Chelator lAI-Duramycin to-Conjugate (Coniugate 3B).
Chelator 1A (Example 4; 3.0 mg, 6.6 mpioΐ), PyBOP (2.6 mg, 5.0 mpioΐ) and DIPEA
(1.7 m , 9.7 mhioΐ) were dissolved in MP (0.7 ml). The mixture was shaken for 5
min and added to a solution of Duramycin (Sigma- Aldrich; 5.0 mg, 2.5 mihoΐ) in
NMP (0.5 ml). The reaction mixture was shaken for 40 min, and then diluted with
water/0.1% TFA (6 ml) and the product purified using preparative HPLC.
Purification by preparative HPLC (gradient: 5-35% B over 40 min where A =
H2O/0.1% HCOOH and B = ACN/0.1% HCOOH) afforded 2.5 mg pure Conjugate
3A (yield 41%) and 1.7 mg pure Conjugate 3B (yield 28%).
The purified Conjugate 3A was analysed by analytical LC-MS (gradient: 25-3 5% B
over 5 min, /R: 1.93 min, found m/z: 1227.0, expected MH2
+ : 1226.6).
The purified Conjugate 3B was analysed by analytical LC-MS (gradient: 25-35% B
over 5 min, t : 2.35 min, found m/z: 1446.7, expected MH2
2+ : 1446.3).
Separation of the two mono-conjugates (Conjugate 3A) could not be achieved using
either analytical or preparative HPLC. In each case the two regioisomers eluted as
one single peak.
Example 9; Synthesis of [Chelator lAI-Cinnamycin Coniugate (Conjugate S).
Cinnamycin (Sigma- Aldrich; 2.0 mg, 1.0 mhioΐ ), Chelator 1A (Example 4; 0.9 mg, 1.5
mhioΐ) and DIPEA (0.5 m , 2.9 mhioΐ) were dissolved in a solution of NMP (0.2 ml),
DMF (0.2 ml) and DMSO (0.6 ml). The reaction mixture was shaken overnight. The
mixture was then diluted with 10% ACN/water/0. 1% TFA (7 ml) and the product
purified using preparative HPLC.
Purification and characterisation
Purification by preparative HPLC (gradient: 20-40% B over 40 min) afforded 1.9 mg
pure Conjugate 5 (yield 78%). The purified material was analysed by analytical LCMS
(gradient: 20-40% B over 5 min, tR: 2.86 min, found m/z 1241.0, expected
MH2
+ : 1240.6).
Example 10; Synthesis of [Chelator lBl-Duramycin Conjugate (Conjugate 6).
Chelator IB (Example 5; 1.6 mg, 1.5 mhioΐ), PyBOP (0.4 mg, 0.8 m hoΐ) and DIPEA
( 1 m , 6 mihoΐ) were dissolved in MP (0.5 mL). The mixture was shaken for 5 min
and added to a solution of duramycin (3.0 mg, 1.5 mihoΐ) in NMP (0.5 mL). The
reaction mixture was shaken for 30 min. Two additional aliquots of activated
Chelator IB (2 x 1.6 mg) were added at 30 min intervals. The mixture was diluted
with water/0. 1% TFA (6 mL) and the product purified using preparative RP-HPLC.
Purification and characterisation
Purification by preparative HPLC (gradient: 20-50% B over 40 min where A =
water/0.1% ammonium acetate and B = ACN) afforded 3.9 mg pure Conjugate 6
(yield 87%). The purified material was analysed by LC-MS (gradient: 20-40% B over
5 min, t : 2.89 min, found m/z 1526.5, expected MH2
+ : 1526.2).
Example 11; Synthesis of [Chelator lBl-Cinnamycin Conjugate (Conjugate 7).
Chelator IB (Example 5; 4.8 mg, 4.4 m hoΐ), PyBOP (2.1 mg, 4.0 mihoΐ) and DIPEA
(2.3 m , 13.2 m o ) were dissolved in DMF (0.5 mL). The mixture was shaken for 5
min and added to a solid cinnamycin (4.5 mg, 2.2 mihoΐ) . Additional pre-activated
Chelator IB was added after 2 h and after 3.5 h in order to drive the reaction close to
completion within 4h. The mixture was diluted with 20% ACN/water/0. 1% TFA (8
mL) and the product purified using preparative RP-HPLC.
Purification and characterization
Purification by preparative RP-HPLC (gradient: 25-35% B over 40 min; ¾ 38.6 min)
afforded 3.9 mg purified Conjugate 7 (yield 58%).
The purified material was analysed by LC-MS (gradient: 20-40% B over 5 min: 2.9
min, found m/z 1028.0, expected MH2
+ : 1027.5 (purity -93.5%, -3% unreacted
starting material).
Example 12; Synthesis of [Chelator 2A1-Duramycin Coniugate (Coniugate 2A
and Coniugate 2B).
Duramycin (Sigma-Aldrich; 7.5 mg, 3.8 mhioΐ), Boc-protected Chelator 2A (Example
6; 5.0 mg, 6.9 m oΐ), HOAt ( 1.9 mg, 8.8 m oΐ) and DIPEA (4.1 i , 20.0 mihoΐ ) were
dissolved in NMP (1.5 ml). The reaction mixture was shaken overnight. The mixture
was then diluted with 20% ACN/water/0. 1% TFA (6 ml) and the product purified
using
preparative HPLC.
Purification and characterisation.
Purification by preparative HPLC (gradient: 0% B over 10 min; 0-30% B over 5 min;
30-70% B over 40 min, tR: 42.4 and 45.0 min), followed by Boc-deprotection in TFA
affordedg two mono-conjugated Duramycin isomers, in 2.0 mg and 0.4 mg yield,
respectively.
The two isomers were characterized by LC-MS (gradient: 20-60% B over 5 min, t :
1.7 min (Conjugate 2A), found m/z: 1107.5, expected MH2
+ : 1107.0, t : 1.6 min
(Conjugate 2B), found m/z 1107.5, expected MH2
2+: 1107.0).
Example 13; Synthesis of [Chelator 2A1-Cinnamycin Coniugate (Coniugate 4).
Cinnamycin (Sigma-Aldrich; 2.0 mg, 1.0 m oΐ ), Boc-protected Chelator 2A
(Example 6; 1. 1 mg, 1.5 m o ) and DIPEA (0.5 m , 2.9 mhioΐ) were dissolved in
DMF (1.0 ml). The reaction mixture was shaken overnight. The mixture was then
diluted with 20% ACN/water/0. 1% TFA (6 ml) and the product purified using
preparative HPLC.
Purification and characterization.
Purification by preparative HPLC (gradient: 30-70% B over 40 min) afforded 1.8 mg
pure Boc-protected Conjugate 4 . The purified material was Boc-deprotected in
TFA/4% water (2 ml) for 45 min and lyophilized from 50% ACN/water affording 1.6
mg Conjugate 4 (yield 73%). The material was analysed by analytical LC-MS
(gradient: 10-40% B over 5 min, t : 3.7 min, found m/z 1120.9, expected MH2
+ :
1121.0).
Example 14; Preparation of mTc-labelled Chelator-conjugates.
The radiolabelled preparations were used either (i) without purification (high RCP at
high RAC ); or (ii) with purification to remove unlabelled LBP peptide.
Conjugate 3A (0. 1 mg, 40 nmol) was dissolved in a mixture of ethanol (100 ) and
water (100 ΐ ) and placed in a sonic bath for ~ 20 min to aid solubility. The solution
was added to a lyophilised kit [formulation: SnCl2.2H20 (0.0 16 mg, 0.07 m oΐ),
MDP(H 4) (0.025 mg, 0 .14 m oΐ ), NaHC0 3 (4.5 mg, 53.6 mihoΐ ), Na2C0 (0.6 mg,
5.66 m hoΐ) and NaPABA (0.2 mg, 1.26 m hoΐ)].
[ mTc0 4] eluate (~ 1 ml) from a Mo/ Tc generator was then added and the
mixture was left to stand for ~ 10 min at room temperature. A portion of crude
product (~ 400 m ) was inj ected onto the HPLC column (see HPLC conditions
below). The radioactive peak with a retention time of ca. 18 min was "cut" into a vial
containing PBS (various volume depending on desired RAC) and then dried in vacuo
to remove excess mobile phase.
Crude RCP = 93 + 6% (n = 13). Formulated RCP (t = 0) = 99 + 1% (n = 13).
Formulated RCP (t = 120 min) = 97 + 3% (n = 13).
Specific Activity = 4.2 + 0.5 GBq/nmol (n = 13).
RT ( mTc-Conjugate 3A) = 18 min.
Tc-Conjugate 5 was prepared following the same procedure as for Conjugate 3A:
Crude RCP = > 85% (n = 12). Formulated RCP (t = 0) = 93 ± 7% (n = 12).
Formulated RCP (t = 120 min) = 9 1 ± 7% (n = 6).
Specific Activity = 3.5 + 0.5 GBq/nmol (n = 13).
RT ( mTc-Conjugate 5) = 18 min.
HPLC Conditions
The RCP of the prior art HY IC counterpart yymTc-[Conjugate 1] was 78-89%
(crude).
The conjugates of the tetra-amine chelator (Conjugates 2A and 4) were prepared
similarly, except that 0 .1% TFA was used as mobile phase A in place of 50 mM
ammonium acetate. The retention time of Tc-[Conjugate 2A] was 12.2 min, and of
mTc-[Conjugate 4] was 12.4 min.
Example 15; Ednian Degradation of Conjugates 2A and 2B.
The use of MS-analysis techniques alone were found not to be feasible for
determination of the site of conjugation of the chelator, manual Edman degradation
chemistry combined with LC-MS analysis was applied.
A modified literature method was used [Xu e t al; PNAS, 106, p . 193 10-193 15 (2009);
Onisko e t al, J . Am. Soc. Mass Spectrom., 18, p . 1070-1079 (2007) and Hayashi e t al,
J Antibiotics, 43, 142 1-1430 (1990)] .
The data obtained demonstrated that Conjugate 2A corresponds to the N amino
conjugated isomer, whereas Conjugate 2B corresponds to the Lys2 N -amino
conjugate. The data did not fit with degradation products expected for a secondary
amino conjugate (Lysd of Formula II), proving that this site is not reactive under the
conditions used for chelate conjugation. It was noted, however, that the secondary
amino group does react with phenylisothiocyanate under the more forcing coupling
conditions used during the Edman degradation cycles.
Example 16; Affinity for Phosphatidyl Ethanolamine.
A Biacore 3000 (GE Healthcare, Uppsala) was equipped with an L I chip. Liposomes
made of POPE/POPC (20% PE) were applied for the affinity study using the capture
technique recommended by the manufacturer. Each run consisted of activation of the
chip surface, immobilization of liposomes, binding of peptide and wash off of both
liposomes and peptide (regeneration). Similar applications can be found in Frostell-
Karlsson et al [Pharm. Sciences, V.94 (1), (2005)]. Thorough washing of needle,
tubing and liquid handling system with running buffer was performed after each
cycle.
BIACORE software: The BIACORE control software including all method
instructions was applied. A method with commands was also written in the
BIACORE Method Definition Language (MDL) to have full control over pre
programmed instructions. BIACORE evaluation software was applied for analysing
the sensorgrams. All substances were found to be good binders to phosphatidyl
ethanolamine. The KD for all substances was less than 100 nM. The results are given
in Table 2 :
Table 2 :
Example 17: Tumour Uptake Studies.
mTc- [Conjugate 2A], Tc-[Conjugate 3A] and mTc- [Conjugate 5] were assessed
by biodistribution in the EL4 mouse lymphoma xenograft model. Briefly, following
establishment of tumour growth in C57/B16 mice, the animals were treated with
either:
(i) a saline/DMSO solution; or
(ii) with chemotherapy (67 mg/kg etoposide and 100 mg/kg
cyclophosphamide in 50% saline 50% DMSO).
Twenty four hours after therapy or vehicle treatment, the animals were assessed for
the biodistribution of the appropriate radiolabelled compound. In addition, the
tumours were extracted and assessed for levels of apoptosis by measuring caspase
activity (caspase-Glo assay). The correlation of binder uptake and caspase activity
was then plotted for various time points. The results are shown in Table 3 (below) at
120 minutes post-injection, and in Figures 1 and 2 for Tc-[Conjugate 5]:
Table 3 :
Example 18; of Tc-labelled Chelator-coniugates.
mTc-Conjugates were assessed by biodistribution in naive rats to determine the
pharmacokinetic profiles of the different compounds. The correlation of binder
retention in different organs/tissues was then plotted for various time points. Data
generated demonstrated that the inclusion of PEG in LBP1 and LBP2 conjugates
improved pharmacokinetics by reducing the liver retention (see Table 4 below):

CLAIMS.
An imaging agent which comprises a compound of Formula
Z L LB -Z
(I)
wherein:
LBP is a lantibiotic peptide of Formula II:
Cysa-Xaa-Gln-Serb-Cysc-Serd-Phe-Gly-Pro-Phe-Thr -Phe-Val-Cysb-
(HO-Asp)-Gly-Asn-Thr -Lysd
(P)
Xaa is Arg or Lys;
Cys -Thra, Ser -Cysb and Cys -Thrc are covalently linked via thioether
bonds;
Serd-Lysd are covalently linked via a lysinoalanine bond;
HO-Asp is b-hydroxyaspartic acid;
Z - ) - is attached to Cysa and optionally also Xaa of LBP, wherein Z1
comprises a radiometal complex of a chelating agent having
at least 4 metal donor atoms;
Z2 is attached to the C-terminus of LBP and is OH, OB , or MI ,
where B is a biocompatible cation; and
MIG is a metabolism inhibiting group which is a biocompatible group
which inhibits or suppresses in vivo metabolism of the LBP peptide;
L is a synthetic linker group of formula -(A)m- wherein each A is
independently -CR2- , -CR=CR- , -CºC- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO R- , - R(C=0 ) R-, -NR(C=S)NR-, -S0 2NR- , -NRS0 2- , -CR2OCR2- ,
-CR2SCR2- , -CR2 RCR2- , a C4- cycloheteroalkylene group, a C4-
cycloalkylene group, a C - arylene group, or a C -12 heteroarylene group, an
amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building
block;
each R is independently chosen from H, C alkyl, C2-4 alkenyl, C2-4 alkynyl,
Ci-4 alkoxyalkyl or C1-4 hydroxyalkyl;
m is an integer of value 1to 20;
n is an integer of value 0 or 1.
2 . The imaging agent of claim 1, where the chelating agent is either an
aminocarboxylate ligand having at least 6 donor atoms or a tetradentate chelator
having an N4 donor set.
3 . The imaging agent of claim 2, where the N4 donor set is a diaminedioxime
chelator or a tetra-amine chelator.
4 . The imaging agent of any one of claims 1 to 3, where Z1 is attached only to
Cys ofLBP.
5. The imaging agent of any one of claims 1 to 4, where Xaa is Arg.
6 . The imaging agent of any one of claims 1 to 5, where Z2 is OH or OBc.
7 . The imaging agent of any one of claims 1 to 6, where n is i and L comprises a
PEG group of formula (OCH2CH2)x- where x is an integer of value 6 to 18.
8. The imaging agent of any one of claims 1 to 7, where the radiometal is mTc.
9 . A chelator conjugate of Formula III:
Z -(L) -[LBP]-Z2
(III)
wherein:
Z3 is a chelating agent having at least 4 metal donor atoms; and
L, n, LBP and Z2 are as defined in any one of claims 1 to 7 .
10. A method of preparation of the imaging agent of any one of claims 1 to 8,
which comprises reaction of the chelator conjugate of claim 9 with a supply of the
desired radiometal in a suitable solvent.
11. A radiopharmaceutical composition which comprises the imaging agent of any
one of claims 1 to 8, together with a biocompatible carrier, in a form suitable for
mammalian administration.
12. A kit for the preparation of the radiopharmaceutical composition of claim 11,
which comprises the chelator conjugate of claim 9 in sterile, solid form such that upon
reconstitution with a sterile supply of the radiometal in a biocompatible carrier,
dissolution occurs to give the desired radiopharmaceutical composition.
13. The kit of claim 12, where the sterile, solid form is a lyophilised solid.
14. A method of imaging the human or animal body which comprises generating an
image of at least a part of said body to which the imaging agent of any one of claims 1
to 8, or the composition of claim 11 has distributed using PET or SPECT, wherein
said imaging agent or composition has been previously administered to said body.
15. The method of claim 14, where the part of the body is a disease state where
abnormal apoptosis is involved.
16. The method of claim 14 or claim 15, which is carried out repeatedly to monitor
the effect of treatment of a human or animal body with a drug, said imaging being
effected before and after treatment with said drug, and optionally also during
treatment with said drug.
17. The use of the imaging agent of any one of claims 1 to 8, the composition of
claim 11, or the kit of claim 12 in a method of diagnosis of the human or animal body.
18. A method of diagnosis of the human or animal body which comprises the method
of imaging of claim 14 or claim 15.