U.S. patent application number 12/098537 was filed with the patent office on 2008-11-06 for radioflorination methods.
This patent application is currently assigned to GE HEALTHCARE LIMITED. Invention is credited to Joseph Maduabuchi Arukwe, Alan Cuthbertson, Hege Karlsen, Magne Solbakken.
Application Number | 20080274052 12/098537 |
Document ID | / |
Family ID | 9933482 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274052 |
Kind Code |
A1 |
Cuthbertson; Alan ; et
al. |
November 6, 2008 |
RADIOFLORINATION METHODS
Abstract
The present invention relates to methods and reagents for
[.sup.18F]-fluorination, particularly of peptides. The resultant
.sup.18F-labelled compounds are useful as radiopharmaceuticals,
specifically for use in Positron Emission Tomography (PET). Thus, a
compound of formula .sup.18F-(Linker)-SH, such as a compound of
formula (IV), (V), or (VI): ##STR00001## may be reacted with an
activated peptide as a means for .sup.18F-labelling.
Inventors: |
Cuthbertson; Alan; (Oslo,
NO) ; Solbakken; Magne; (Skien, NO) ; Arukwe;
Joseph Maduabuchi; (Oslo, NO) ; Karlsen; Hege;
(Oslo, NO) |
Correspondence
Address: |
GE HEALTHCARE, INC.
IP DEPARTMENT, 101 CARNEGIE CENTER
PRINCETON
NJ
08540-6231
US
|
Assignee: |
GE HEALTHCARE LIMITED
Buckinghamshire
GB
|
Family ID: |
9933482 |
Appl. No.: |
12/098537 |
Filed: |
April 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10508682 |
Sep 20, 2004 |
7368474 |
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PCT/GB03/01332 |
Mar 20, 2003 |
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12098537 |
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Current U.S.
Class: |
424/1.69 ;
530/300; 564/182; 568/62; 568/65 |
Current CPC
Class: |
C07B 59/008
20130101 |
Class at
Publication: |
424/1.69 ;
568/62; 568/65; 564/182; 530/300 |
International
Class: |
A61K 51/08 20060101
A61K051/08; C07C 321/04 20060101 C07C321/04; C07C 233/64 20060101
C07C233/64; C07K 2/00 20060101 C07K002/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 22, 2002 |
GB |
0206750.2 |
Claims
1.-3. (canceled)
4. A compound of formula (IV):
.sup.18F--(CH.sub.2CH.sub.2O).sub.n--(CH.sub.2).sub.m--SH (IV) or a
thiol protected precursor thereof, wherein: n is an integer of 1 to
20; m is an integer of 1 to 10.
5. A compound of formula (V): .sup.18F--(CH.sub.2).sub.p--SH (V) or
a thiol protected precursor thereof, wherein p is an integer of 1
to 20.
6. A compound of formula (VI): ##STR00041## or a thiol protected
precursor thereof, wherein: q is an integer of 0 to 4; and r is an
integer of 1 to 10.
7. A compound of formula (VII), (VIII), or (IX): ##STR00042##
wherein: n is an integer of 1 to 20; and m is an integer of 1 to
10. p is an integer of 1 to 20. q is an integer of 0 to 4; and r is
an integer of 1 to 10.
8. A compound of formula (X), (XI), or (XII): ##STR00043## wherein:
n is an integer of 1 to 20; m is an integer of 1 to 10; p is an
integer of 1 to 20; q is an integer of 0 to 4; r is an integer of 1
to 10; and Y is a C.sub.1-10 hydrocarbyl group optionally including
1 to 6 heteroatoms.
9. A compound of formula (XII) according to claim 8 which is:
##STR00044##
10. A radiofluorination kit comprising: (i) a compound of formula
(IIa) L-(Linker)-SR (IIa) wherein L is a leaving group such as
p-toluenesulphonate, trifluoromethanesulphonate, or
methanesulphonate, the Linker is a C.sub.1-30 hydrocarbyl group
optionally including 1 to 10 heteroatoms; R is hydrogen or a thiol
protecting group; and (ii) an activated peptide of formula (I) or
(Ia). ##STR00045##
11. A radiofluorination kit according to claim 10, comprising: (i)
a compound of formula (IVa), (Va), or (VIa): ##STR00046## n is an
integer of 1 to 20; m is an integer of 1 to 10; p is an integer of
1 to 20; q is an integer of 0 to 4; r is an integer of 1 to 10; L
is a leaving group such as p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate; L' is a leaving
group such as iodo, p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate and when q is 0,
L' can be nitro or an iodonium or ammonium salt, R is hydrogen or a
thiol protecting group; and (ii) an activated peptide of formula
(I) or (Ia). ##STR00047##
Description
[0001] The present invention relates to methods and reagents for
[.sup.18F]-fluorination, particularly of peptides. The resultant
18F-labelled compounds are useful as radiopharmaceuticals,
specifically for use in Positron Emission Tomography (PET).
[0002] The application of radiolabelled bioactive peptides for
diagnostic imaging is gaining importance in nuclear medicine.
Biologically active molecules which selectively interact with
specific cell types are useful for the delivery of radioactivity to
target tissues. For example, radiolabelled peptides have
significant potential for the delivery of radionuclides to tumours,
infarcts, and infected tissues for diagnostic imaging and
radiotherapy. .sup.18F, with its half-life of 110 minutes, is the
positron-emitting nuclide of choice for many receptor imaging
studies. Therefore, .sup.18F-labelled bioactive peptides have great
clinical potential because of their utility in PET to
quantitatively detect and characterise a wide variety of
diseases.
[0003] WO 99/11590 describes methods for [.sup.18F]fluorination of
thiol containing peptides and proteins.
[0004] One difficulty with .sup.18F-labelled peptides is that the
existing .sup.18F-labelling agents are time-consuming to prepare.
Efficient labelling of peptides and proteins with .sup.18F is only
achieved by using suitable prosthetic groups. Several such
prosthetic groups have been proposed in the literature, including
N-succinimidyl-4-[.sup.18F]fluorobenzoate,
m-maleimido-N-(p-[18F]fluorobenzyl)-benzamide,
N-(p-[.sup.18F]fluorophenyl)maleimide, and
4-[.sup.18F]fluorophenacylbromide. Almost all of the methodologies
currently used today for the labelling of peptides and proteins
with .sup.18F utilise active esters of the fluorine labelled
synthon. As peptides and proteins may contain a multitude of
functional groups capable of reaction with active esters these
current methods are not site-specific. For example a peptide
containing 3 lysine residues has three amine functions all equally
reactive towards the labelled synthon. Therefore, there still
exists a need for .sup.18F-labelled prosthetic groups and
methodologies which allow rapid, chemoselective introduction of
.sup.18F, particularly into peptides, under mild conditions to give
.sup.18F-labelled products in high radiochemical yield and purity.
Additionally, there is a need for such methodologies which are
amenable to automation to facilitate preparation of
radiopharmaceuticals in the clinical setting.
[0005] Accordingly, the present invention provides a method for
radiofluorination comprising reaction of a compound of formula (I)
or (la):
##STR00002##
with a compound of formula (II):
.sup.18F-(Linker)-SH (II)
wherein: X is a leaving group selected from chloro, bromo, and
iodo, and is preferably chloro; Y is a C.sub.1-10 hydrocarbyl group
optionally including 1 to 6 heteroatoms; and the Linker in formula
(II) is a C.sub.1-30 hydrocarbyl group optionally including 1 to 10
heteroatoms; to give a compound of formula (III) or (IIIa)
respectively:
##STR00003##
wherein the Linker group is as defined in the compound of formula
(II), Y is as defined in the compound of formula (Ia), and the
peptide is as defined in the compound of formula (I) or (Ia)
respectively.
[0006] The present invention provides a more chemoselective
approach to radiolabelling where the exact site of introduction of
the label is pre-selected during the synthesis of the peptide
precursor. The thiol function of the synthon reacting at a
pre-determined site in the peptide giving only one possible
product. This methodology is therefore chemoselective and its
application is considered generic for a wide range of peptides and
biomolecules.
[0007] In a preferred aspect, the present invention provides a
method for radiofluorination comprising reaction of a compound of
formula (I):
##STR00004##
with a compound of formula (IV), (V), or (VI):
##STR00005##
wherein: X is a leaving group selected from chloro, bromo, and
iodo, and is preferably chloro; n is an integer of 1 to 20; m is an
integer of 1 to 10; p is an integer of 1 to 20; q is an integer of
0 to 4; r is an integer of 1 to 10; to give a compound of formula
(VII), (VIII), or (IX) respectively:
##STR00006##
wherein m, n, p, q, and r are as defined for the compound of
formula (IV), (V), or (VI).
[0008] This reaction may be effected in a suitable solvent, for
example, in an aqueous buffer in the pH range 5 to 11, and at a
non-extreme temperature of from 5 to 60.degree. C., preferably at
ambient temperature.
[0009] In a further preferred aspect, the present invention
provides a method for radiofluorination comprising reaction of a
compound of formula (Ia):
##STR00007##
with a compound of formula (IV), (V), or (VI):
##STR00008##
wherein: Y is a C.sub.1-10 hydrocarbyl group optionally including 1
to 6 heteroatoms; n is an integer of 1 to 20; m is an integer of 1
to 10; p is an integer of 1 to 20; q is an integer of 0 to 4; r is
an integer of 1 to 10; to give a compound of formula (X), (XI), or
(XII) respectively:
##STR00009##
wherein m, n, p, q, and r are as defined for the compound of
formula (IV), (V), or (VI), Y and the peptide are defined for the
compound of formula (Ia).
[0010] This reaction may be effected in a suitable solvent, for
example, in an aqueous buffer in the pH range 5 to 11, and at a
non-extreme temperature of from 5 to 60.degree. C., preferably at
ambient temperature.
[0011] In formula (I) and (Ia), suitable peptides for labelling may
include somatostatin analogues, such as octreotide, bombesin,
vasoactive intestinal peptide, chemotactic peptide analogues,
.alpha.-melanocyte stimulating hormone, neurotensin, Arg-Gly-Asp
peptide and its analogues, human pro-insulin connecting peptide,
endothelin, angiotensin and
formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine.
Preferred peptides for labelling are Arg-Gly-Asp peptide and its
analogues, such as those described in WO 01/77415 and WO 03/006491.
In one particular aspect, the peptide in formula (I) or (Ia) is of
formula (A):
##STR00010##
wherein X.sup.7 is either --NH.sub.2 or
##STR00011##
wherein a is an integer of from 1 to 10, preferably a is 1.
[0012] As will be appreciated by the skilled person, the methods of
the invention may also be used for radiofluorination of other
biomolecules such as proteins, hormones, oligonucleotides, and
antibody fragments, as well as small molecules to provide a variety
of PET tracers.
[0013] In formula (Ia), Y is a C.sub.1-10 hydrocarbyl group
optionally including 1 to 6 heteroatoms such as oxygen or nitrogen,
suitably Y is a C.sub.1-10 alkyl group optionally including a
C.sub.4-7cycloalkyl group, for example Y may be
methylcyclohexyl.
[0014] In formula (II) the Linker is a C.sub.1-30 hydrocarbyl group
optionally including 1 to 10 heteroatoms such as oxygen or
nitrogen, and may be chosen to provide good in vivo
pharmacokinetics, such as favourable excretion characteristics.
Suitable Linker groups include alkyl, alkenyl, alkynyl chains,
aromatic, polyaromatic, and heteroaromatic rings, and polymers
comprising ethyleneglycol, amino acid, or carbohydrate
subunits.
[0015] In the compounds of formulae (IV) and (VII), n is typically
2 to 6, suitably 3, and m is typically 1 to 4, suitably 2.
[0016] In the compounds of formulae (V) and (VIII), p is typically
1 to 6, suitably 3.
[0017] In the compounds of formulae (VI) and (IX), the group
.sup.18F(CH.sub.2).sub.q-- is suitably attached in the para
position relative to the amide group, q is typically 0 to 4,
suitably 1, and r is typically 1 to 4, suitably 2.
[0018] As a further aspect of the invention, there is provided a
compound of formula (IV), (V), or (VI) as defined above. These
compounds having general utility as prosthetic groups for
radio-fluorination. Thiol protected derivatives of the compounds of
formulae (IV), (V), and (VI) also have utility as synthetic
precursors for such prosthetic groups, and those wherein the thiol
is protected by a trityl group are particularly preferred. Other
thiol protecting groups in precursors of formula (IV), (V), or (VI)
are well known to the person skilled in the art and are described,
for example, in Protecting Groups in Organic Synthesis, Theodora W.
Greene and Peter G. M. Wuts, published by John Wiley & Sons
Inc.
[0019] As a further aspect of the invention, there is provided a
compound of formula (VII), (VIII), (IX), (X), (XI), or (XII) as
defined above. These compounds having utility as PET tracers. Of
these compounds, those in which the peptide is Arg-Gly-Asp peptide
or its analogues are preferred, such as the peptides described in
WO 01/77145 and WO 03/006491. Particularly preferred peptides in
this aspect of the invention are those of formula (A) as defined
above for the compounds of formula (I) and (Ia).
[0020] Compounds of formula (I) may be prepared by standard methods
of peptide synthesis, for example, solid-phase peptide synthesis,
for example, as described in Atherton, E. and Sheppard, R. C.;
"Solid Phase Synthesis"; IRL Press: Oxford, 1989. Incorporation of
the group "X--CH.sub.2C(O)-" in a compound of formula (I) may be
achieved by reaction of the N-terminus of the peptide with the
reagent of formula (XIII):
X--CH.sub.2C(O)Z (XIII)
under standard conditions for peptide bond formation; wherein X is
as defined for the compound of formula (I), and Z is --OH or a
suitable activating group such as, chloro, bromo, fluoro,
--OC(O)CH.sub.2--X wherein X is as defined for the compound of
formula (I), or when Z is --OH the acid may be activated using in
situ agents such as
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) or
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylen-
e]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU).
[0021] Compounds of formula (Ia) may be prepared by standard
methods of peptide synthesis, for example, solid-phase peptide
synthesis, for example, as described in Atherton, E. and Sheppard,
R. C.; "Solid Phase Synthesis"; IRL Press: Oxford, 1989.
Incorporation of the group "maleimide-Y-" in a compound of formula
(Ia) may be achieved by reaction of the N-terminus of the peptide
with the reagent of formula (XIV):
##STR00012##
under standard conditions for peptide bond formation; wherein Y is
as defined for the compound of formula (Ia), and Z is --OH or a
suitable activating group such as, chloro, bromo, fluoro, or active
esters such as pentafluorophenol or N-hydroxysuccinimide ester, or
when Z is --OH then the acid may be activated by use of in situ
activating agents such as HBTU or HATU as described above for the
compound of formula (XIII).
[0022] Compounds of formula (II) may be prepared from the
corresponding compound of formula (IIa):
L-(Linker)-SR (IIa)
wherein L is a leaving group such as p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate, and the Linker is
as defined for the compound of formula (II) and R is hydrogen or a
thiol protecting group; by reaction with cyclotron produced aqueous
[.sup.18F]-fluoride, suitably pre-activated by evaporation from a
base (for example, from tetrabutylammonium or
K.sub.2CO.sub.3/Kryptofix-222), in a suitable solvent such as
acetonitrile, N,N-dimethylformamide, or dimethyl sulphoxide,
typically at elevated temperature, for example 60 to 120.degree.
C., followed by removal of any thiol protecting group using
standard methods.
[0023] Compounds of formula (IV) may be prepared from the
corresponding compound of formula (IVa):
L-(CH.sub.2CH.sub.2O).sub.n--(CH.sub.2).sub.m--SR (IVa)
wherein L is a leaving group such as p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate, and n and m are
as defined for the compound of formula (IV) and R is hydrogen or a
thiol protecting group; by reaction with cyclotron produced aqueous
[.sup.18F]-fluoride, suitably pre-activated by evaporation from a
base (for example, from tetrabutylammonium or
K.sub.2CO.sub.3/Kryptofix-222), in a suitable solvent such as
acetonitrile, N,N-dimethylformamide, or dimethyl sulphoxide,
typically at elevated temperature, for example 60 to 120.degree.
C., followed by removal of any thiol protecting group using
standard methods.
[0024] Compounds of formula (V) may be prepared from the
corresponding compound of formula (Va):
L-(CH.sub.2).sub.p--SR (Va)
wherein L is a leaving group such as p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate, and p is as
defined for the compound of formula (V) and R is hydrogen or a
thiol protecting group; by reaction with cyclotron produced aqueous
[.sup.18F]-fluoride, suitably pre-activated by evaporation from a
base (for example, from tetrabutylammonium or
K.sub.2CO.sub.3/Kryptofix-222), in a suitable solvent such as
acetonitrile, N,N-dimethylformamide, or dimethyl sulphoxide,
typically at elevated temperature, for example 60 to 120.degree.
C., followed by removal of any thiol protecting group using
standard methods.
[0025] Compounds of formula (VI) may be prepared from the
corresponding compound of formula (VIa):
##STR00013##
wherein L' is a leaving group such as iodo, p-toluenesulphonate,
trifluoromethanesulphonate, or methanesulphonate and when q is 0,
L' can be nitro or an iodonium or ammonium salt, and q and r are as
defined for the compound of formula (VI) and R is hydrogen or a
thiol protecting group; by reaction with cyclotron produced aqueous
[.sup.18F]-fluoride, suitably pre-activated by evaporation from a
base (for example, from tetrabutylammonium or
K.sub.2CO.sub.3/Kryptofix-222), in a suitable solvent such as
acetonitrile, N,N-dimethylformamide, or dimethyl sulphoxide,
typically at elevated temperature, for example 60 to 120.degree.
C., followed by removal of any thiol protecting group using
standard methods.
[0026] In formulae (IIa), (IVa), (Va), and (VIa), suitable thiol
protecting groups include (Phenyl).sub.3C-(trityl) and others as
may be found described in Protecting Groups in Organic Synthesis,
Theodora W. Greene and Peter G. M. Wuts, published by John Wiley
& Sons Inc. Removal of such thiol protecting groups may be
effected by standard methods, such as those described in Greene.
For example, where R is trityl, the free thiol may be formed by
treatment with dilute acid, for example trifluoroacetic acid in a
chlorinated solvent, such as dichloromethane.
[0027] In one preferred aspect, the compounds of formulae (IIa),
(IVa), (Va), and (VIa) may be bound to a solid support, such as
polymer beads or coatings, for example, a trityl or chlorotrityl
resin. In this aspect, the excess reagents and by-products of the
radio-fluorination reaction may be separated from the polymer-bound
product by washing. Using the deprotection methods as described
above, effects cleavage of the compound of formula (II), (IV), (V),
or (VI) from the solid support. This approach may be particularly
suitable for automated production of the compounds of formulae
(II), (IV), (V), and (VI). Alternatively, the by-products of thiol
deprotection, where insoluble in the reaction mixture, may be
removed by filtration.
[0028] According to a further aspect of the invention there is
provided a kit for the preparation of a radiofluorinated tracer
comprising a prosthetic group of formula (IIa), (IVa), (Va), or
(VIa) and an activated peptide of formula (I) or (Ia).
[0029] In use of the kit, the compound of formula (IIa), (IVa),
(Va), or (VIa) would be converted to the corresponding compound of
formula (II), (IV), (V), or (VI) using methods described above.
Preferably, the compound of formula (II), (IV), (V), (VI) or a
thiol protected precursor of any thereof, may be separated from
waste reactants by passing the reaction mixture through a Solid
Phase Extraction (SPE) cartridge. The SPE cartridge may comprise a
graphite pad or C.sub.18 stationary phase. Any thiol protecting
group may be removed, for example, by addition of an acid such as
trifluoroacetic acid. Where the thiol group in the compound of
formula (II), (IV), (V), or (VI) is protected with a hydrophobic
group, such as a trityl group, the deprotection may conveniently be
effected on the SPE cartridge, whereby the hydrophobic thiol
protecting group (such as trityl) remains bound on the stationary
phase while the labelled prosthetic group of formula (II), (IV),
(V), or (VI) is eluted in high purity and yield. The compound of
formula (II), (IV), (V), or (VI) would then be added to the
compound of formula (I) or (Ia) which may suitably be dissolved in
aqueous buffer (pH 7-11). After reaction at a non-extreme
temperature for 1 to 60 minutes, the labelled peptide may be
purified, for example, by SPE and collected.
[0030] The invention is illustrated by way of the following
examples.
EXAMPLES
Production of .sup.18F
[0031] Fluorine-18 was produced by a cyclotron using the
.sup.18O(p,n).sup.18F reaction. Enriched [.sup.18O]H.sub.2O (95%
.sup.18O) was irradiated by protons (19 MeV) with an integrated
beam current of 5-10 .mu.Ah.
Synthesis of ClCH.sub.2CO-LVs-Gly-Phe-Gly-Lys-OH peptide
##STR00014##
[0033] Assembly of the amino acid sequence using fully automated
synthesis (ABI 433A), Lys-sasrin resin (0.10 mmol) and 10 mmol
cartridges of aminoacids. After the peptide synthesis the peptide
on resin was placed in a manual bubbler apparatus and washed with
N,N dimethylformamide (DMF) and freshly made chloroacetic anhydride
(0.5 mmol) (chloroacetic acid (94.5 mg, 1.0 mmol) and
dicyclohexyl-carbodiimide (DCCl) (102.6 mg, 0.5 mmol) in
dichloromethane (DCM) for 10 minutes. Dicyclohexylurea (DCU)
filtered off and the solution was evaporated under reduced pressure
affording the anhydride) in DMF was added. After 1 hour the Kaiser
test was negative (yellow, red resin (due to the anhydride)), the
solvent removed, and the resin washed with DMF 8 times then DCM and
diethyl ether (DEE) and N.sub.2-dried. triisopropylsilane (TIS) and
a drop of water were added to the resin before trifluoroacetic acid
(TFA) was added. The resin was filtered off after 50 minutes and
the solution evaporated under reduced pressure and washed with DEE
3 times. The crude product was purified by reverse phase
preparative chromatography affording 64.3 mg of pure product (Vydac
218TP1022 column; solvents A=water/0.1% TFA and B=CH.sub.3CN/0.1%
TFA; gradient 00-30% B over 40 min; flow 10 ml/minute; detection at
254 nm). (Analytical HPLC: Phenomenex Luna 00B-4251-E0 column;
solvents: A=water/0.1% TFA and B=CH.sub.3CN/0.1% TFA; gradient
00-30% B over 10 min; flow 2.0 ml/minute; retention time 5.1
minutes detected at 214 and 254 nm). Further characterisation was
carried out using mass spectrometry, giving m/z value
612.8-[MH.sup.+].
Example 1
Preparation of
4-Fluoromethyl-N-[2-(tritylsulphanyl)ethyl]benzamide, deprotection
and site-specific conjugation to a chloroacetyl modified
peptide
1. a) S-Trityl cysteamine
##STR00015##
[0035] To a stirred solution of cysteamine (Fluka, 3.85 g, 50.0
mmol) in trifluoroacetic acid (TFA) (50 ml) was added
triphenylmethanol (13.0 g, 50.0 mmol). The mixture was stirred at
room temperature for 30 min and concentrated. To the residue was
added ether (250 ml). Precipitated material was filtered off and
washed with ether. The TFA-salt was partitioned between 1 M aqueous
KOH solution (150 ml) and ether (150 ml). The phases were separated
and the ether phase was dried (MgSO.sub.4). The solution was
filtered and concentrated and the product was crystallised from
ether/n-hexane, giving 9.20 g (58%) of white solid.
1. b) 4-Hydroxymethyl-N-[2-(tritylsulphanyl)ethyl]benzamide
##STR00016##
[0037] To a solution of S-trityl cysteamine (1.60 g, 5.00 mmol) and
4-hydroxymethylbenzoic acid pentafluorophenyl ester (MilliGen, 1.51
g, 5.00 mmol) in dichloromethane (40 ml) was added
N-methylmorpholine (0.55 ml, 5.0 mmol). The mixture was stirred at
room temperature for 36 hrs. Precipitated material was filtered
off, washed with dichloromethane and dried to give 1.30 g (57%) of
white solid. NMR analysis was in accordance with the structure.
1. c) Methanesulphonic acid
4-[2-(tritylsulphanyl)ethylcarbamoyl]benzyl ester
##STR00017##
[0039] To a solution of
4-hydroxymethyl-N-[2-(tritylsulphanyl)ethyl]benzamide (226 mg,
0.500 mmol) in dry THF (8 ml) was added N-methylpyrrolidinone (NMP)
(61 .mu.l, 0.55 mmol) and mesyl chloride (85 .mu.l, 1.1 mmol). The
reaction mixture was stirred at room temperature for 48 hours,
filtered through silica and concentrated. The product was purified
by column chromatography (silica, 1% methanol in chloroform) to
give 213 mg of an oil that solidified slowly. NMR analysis was in
accordance with the structure.
1. d) 4-Fluoromethyl-N-[2-(tritylsulphanyl)ethyl]benzamide
##STR00018##
[0041] A solution of Kryptofix (Fluka, 15 mg, 40 .mu.mol) in dry
acetonitrile (200 .mu.l) was added to solid potassium fluoride (2.3
mg, 80 .mu.mol). The mixture was shaken for 5 min. The
Kryptofix/KF-solution was added to a solution of methanesulphonic
acid 4-[2-(tritylsulphanyl)ethylcarbamoyl]benzyl ester (21 mg, 40
.mu.mol) in acetonitrile (400 .mu.l). The mixture was heated at
65.degree. C. for 10 min. An aliquot was analysed by HPLC (column
Phenomenex Luna 3 .mu.m C18(2) 50.times.4.60 mm; solvents:
A=water/0.1% TFA and B=acetonitrile/0.1% TFA; gradient 40-80% B
over 10 min; flow 2.0 ml/min, UV detection at 214 and 254 nm),
showing complete conversion of starting material (t.sub.R 5.6 min)
to a new product (t.sub.R 6.3 min). Purification by reverse phase
preparative HPLC (column: Phenomenex Luna C18(2) 5 .mu.m
250.times.21.2 mm, solvents: A=water/0.1% TFA and
B=acetonitrile/0.1% TFA; gradient 40-80% B over 60 min; flow 10.0
ml/min, UV detection at 214 nm) gave 4.5 mg of white solid. The
structure was confirmed by NMR spectroscopy.
1. e) 4-Fluoromethyl-N-(2-mercaptoethyl)benzamide
##STR00019##
[0043] To 1.4 mg (3.0 .mu.mol) of
4-fluoromethyl-N-[2-(tritylsulphanyl)ethyl]benzamide was added 50
.mu.l of TFA/TIS/H.sub.2O (95:2.5:2.5) mixture. The flask was
swirled gently for 5 minutes and then concentrated in vacuo. LC-MS
analysis (column Phenomenex Luna 3 .mu.m C18(2) 50.times.2.00 mm;
solvents: A=water/0.1% HCOOH and B=acetonitrile/0.1% HCOOH;
gradient 5-60% B over 10 min; flow 0.3 ml/min, UV detection at 214
and 254 nm, ESI-MS) gave a peak (t.sub.R 6.8 min) with m/z at 214
corresponding to MH.sup.+.
1. f) Site-Specific Conjugation to the Chloroacetyl Modified
Peptide
##STR00020##
[0045] The residue from above was taken up in 100 .mu.l 0.1 M
sodium hydrogencarbonate/disodium carbonate buffer at pH 9.1. To
the mixture was added a solution of the peptide
chloroacetyl-KGFGK-OH (3.7 mg, 6.0 .mu.mol) in 150 .mu.l buffer.
The mixture was heated at 45.degree. C. for 20 min. LC-MS analysis
(column Phenomenex Luna 3 .mu.m C18(2) 50.times.2.00 mm; solvents:
A=water/0.1% HCOOH and B=acetonitrile/0.1% HCOOH; gradient 5-60% B
over 10 min; flow 0.3 ml/min, UV detection at 214 and 254 nm,
ESI-MS) showed complete conversion of the fluoro compound to a new
product (t.sub.R 4.3 min) giving m/z at 789.4 corresponding to
MH.sup.+ for the conjugate. The conjugate product was purified by
reverse phase HPLC (column: Phenomenex Luna C18(2) 5 .mu.m
250.times.21.2 mm, solvents: A=water/0.1% TFA and
B=acetonitrile/0.1% TFA; gradient 5-60% B over 60 min; flow 10.0
ml/min, UV detection at 214 nm) to give 2 mg of white solid.
Example 2
Preparation of (3-fluoro-propylsulfanyl)triphenylmethane,
deprotection and site-specific conjugation to a chloroacetyl
peptide
2. a) Synthesis of 3-tritylsulfanyl-propan-1-ol
##STR00021##
[0047] Trityl chloride (27.9 mg, 0.1 mmol) and triethyl amine (49
.mu.l, 0.5 mmol) were dissolved in DCM (2 ml) before
3-mercapto-1-propanol (9 .mu.l, 0.1 mmol) was added. DCM was
evaporated under reduced pressure after 6 hours and the crude
product purified by reverse phase preparative chromatography (Vydac
218TP1022 column; solvents A=water/0.1% TFA and B=CH.sub.3CN/0.1%
TFA; gradient 30-70% B over 40 min; flow 10 ml/minute; detection at
254 nm). A yield of 6 mg of purified material was obtained
(analytical HPLC: column phenomenex Luna C18,00B-4251-E0: solvents:
A=water/0.1% TFA and B=CH.sub.3CN/0.1% TFA; gradient 30-70% B over
10 min; flow 1.0 ml/minute; retention time 7.73 minutes detected at
214 and 254 nm). Structure verified by NMR.
2. b) Synthesis of methanesulfonic acid 3-tritylsulfanyl-propyl
ester
##STR00022##
[0049] Mesyl chloride (6 .mu.l, 0.075 mmol) was added to a solution
of 3-tritylsulfanyl-propan-1-ol (5 mg, 0.015 mmol) and triethyl
amine (32 .mu.l, 0.23 mmol) in THF (1 ml). After 30 minutes THF was
evaporated under reduced pressure and the crude product dissolved
in DCM, washed with a saturated solution of sodium
hydrogencarbonate in water, a saturated solution of sodium chloride
and dried with MgSO.sub.4. A yield of 10 mg was obtained after
evaporation under reduced pressure (analytical HPLC: column Luna
C18, 00B-4251-E0: solvents: A=water/0.1% TFA and B=CH.sub.3CN/0.1%
TFA; gradient 40-80% B over 10 min; flow 1.0 ml/minute; retention
time 7.12 minutes detected at 214 and 254 nm). Structure verified
by NMR.
2. c) Synthesis of (3-fluoro-propylsulfanyl)triphenylmethane
##STR00023##
[0051] KF (1.4 mg, 0.024 mmol) and Kryptofix 222 (9.0 mg, 0.024
mmol) were dissolved in acetonitrile (0.2 ml) (heating).
Methanesulfonic acid 3-tritylsulfanyl-propyl ester (5 mg, 0.012
mmol) in acetonitrile (0.2 ml) was added. The reaction mixture was
heated to 80 degrees for 90 minutes. The crude product was purified
by reverse phase preparative chromatography (Vydac 218TP1022
column; solvents A=water/0.1% TFA and B=CH.sub.3CN/0.1% TFA;
gradient 40-90% B over 40 min; flow 10 ml/minute; detection at 254
nm). A yield of 2 mg of purified material was obtained (analytical
HPLC: column Phenomenex Luna C18, 00B-4251-E0: solvents:
A=water/0.1% TFA and B=CH.sub.3CN/0.1% TFA; gradient 40-80% B over
10 min; flow 1.0 ml/minute; retention time 8.2 minutes detected at
214 and 254 nm). Structure verified by NMR.
2. d) Conjugation of 3-fluoro-propane-1-thiol with
Cl-Ac-Lys-Gly-Phe-Gly-Lys-OH
##STR00024##
[0053] The trityl group from
(3-fluoro-propylsulfanyl)triphenylmethane (1.0 mg, 0.003 mmol) was
cleaved with TFA (25 .mu.l) in the presence of TIS (5 .mu.l) and
water (5 .mu.l) (5 minutes). The TFA solution was cooled on ice
during addition of ammonia (25%) until pH was 9. A solution of
ClCH.sub.2CO-KGFGK-OH 3.6 mg, 0.006 mmol) in 50 .mu.l of water was
added and the pH adjusted to 9 with ammonia. The reaction mixture
was heated to 60 degrees for 30 minutes. The reaction mixture was
quenched with water+0.1% TFA and purified using reverse phase
preparative chromatography (Phenomenex, C18, 00G-4253-N0 column;
solvents A=water/0.1% TFA and B=CH.sub.3CN/0.1% TFA; gradient
00-30% B over 30 min; flow 5 ml/minute; detection at 254 nm). A
yield of 0.4 mg of purified material was obtained (analytical HPLC:
column Luna 00B-4251-E0: solvents: A=water/0.1% TFA and
B=CH.sub.3CN/0.1% TFA; gradient 00-30% B over 10 min; flow 1.0
ml/minute; retention time 6.88 minutes detected at 214 and 254 nm).
Further characterisation was carried out using mass spectrometry,
giving m/z value 670.35. [M-H.sup.+].
Example 3
Preparation of
(2-{2-[2-(2-Fluoro-ethoxy)-ethoxy]-ethoxy}-ethyl)-trityl sulfide,
deprotection and site-specific conjugation to a chloroacetyl
modified peptide
3. a) Toluene-4-Sulfonic acid
2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester
##STR00025##
[0055] To a stirred suspension of p-toluenesulfonyl chloride (9.82
g, 51.5 mmol) in
2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethanol, (20 g, 103
mmol) at 0.degree. C. was added dropwise triethylamine (13 ml, 93
mmol). The suspension was stirred at 0.degree. C. for 1 hour and
then at room temperature for another 16 hours. The resulting
mixture was dissolved in dichloromethane (350 ml). The clear and
colourless solution that was obtained was extracted with 1 M
hydrochloric acid (2.times.100 ml) and once with water (200 ml).
The organic phase was dried (MgSO.sub.4), filtered and evaporated
to afford a colourless oil. The crude product was purified by flash
chromatography using ethyl acetate. Tetraethylene glycol
ditosylate, eluted first followed by toluene-4-Sulfonic acid
2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester, as a
colourless oil; 8.89 g (49%).
3. b) 2-{2-[2-(2-Mercapto-ethoxy)-ethoxy]-ethoxy}-ethanol
##STR00026##
[0057] A solution of thiourea (0.94 g, 12.35 mmol) and
toluene-4-sulfonic acid
2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester (4.09 g,
11.75 mmol) in absolute alcohol (32 ml) was heated to reflux under
argon atmosphere for 24 h. The mixture was cooled and a solution of
NaOH (1.23 g, 30.75 mmol) in 15 ml of ethanol/water (9:1, v/v) was
added. Refluxing was continued for another 2.5 h under argon. The
reaction mixture was then cooled to ambient temperature and
acidified to pH 2 using concentrated hydrochloric acid and then
concentrated under reduced pressure. The residue was purified by
flash chromatography using ethyl acetate/absolute ethanol (10:1) to
afford the product as a colourless oil. Yield 1.55 g (66%).
3. c) 2-{2-[2-(2-Tritylsulfanyl-ethoxy)-ethoxy]-ethoxy}-ethanol
##STR00027##
[0059] To 2-{2-[2-(2-mercapto-ethoxy)-ethoxy]-ethoxy}-ethanol (310
mg, 1.47 mmol) and trityl chloride (452 mg, 1.62 mmol, 1.1 eq.) in
a round bottomed flask was added simultaneously triethylamine (2.1
ml, 10 eq.) and THF (25 ml) under argon. The initial red colour
faded upon stirring at ambient temperature for 1 hour. The reaction
was monitored by TLC, ethyl acetate/ethanol (1:1) and was completed
after 6 hours. Thereafter methanol (6 ml) was added to consume
excess trityl chloride and the mixture stirred for 5 minutes before
evaporation of the solvents. The residue was purified by flash
chromatography using 100% ethyl acetate to afford the product (550
mg, 83%) as a colourless oil.
3 d) Methanesulfonic acid
2-{2-[2-(2-tritylsulfanyl-ethoxy)-ethoxy]-ethoxy}-ethyl ester
##STR00028##
[0061] To a stirring THF (12 ml) solution of
2-{2-[2-(2-Tritylsulfanyl-ethoxy)-ethoxy]-ethoxy}-ethanol (227 mg,
0.50 mmol) and triethylamine (139 .mu.l, 1.0 mmol) was added methyl
sulfonyl chloride (47 .mu.l, 0.60 mmol, 1.2 eq.). After stirring
for 2 hours at ambient temperature, the mixture was filtered to
eliminate precipitated triethylamine hydrochloride salt and the
solvents were evaporated. The residue was purified by flash
chromatography to afford the product (238 mg, 90%) as a colourless
oil.
3. e) Synthesis of
(2-{2-[2-(2-Fluoro-ethoxy)-ethoxy]-ethoxy}-ethyl)-trityl
sulfide
##STR00029##
[0063] A stirring solution of methanesulfonic acid
2-{2-[2-(2-tritylsulfanyl-ethoxy)-ethoxy]-ethoxy}-ethyl ester
(71.67 mg, 0.14 mmol) and tetrabutylammonium fluoride (1.1 M in
THF, 127 .mu.l, 0.14 mmol) was heated at 90.degree. C. for 30
minutes. The mixture was cooled to ambient temperature and
evaporated to dryness. Purification of product was by flash
chromatography using ethyl acetate/hexane, (1:1) to give the
product (71%) as a colourless oil.
Example 3
f) Deprotection of the Trityl Group and Site-Specific Conjugation
to the Chloroacetyl Peptide
##STR00030##
[0065] Deprotection of the trityl group and site-specific
conjugation to the chloroacetyl functionalised peptide was carried
out as described in example 1. e) and f) above.
HPLC Method for Examples 4 to 6.
[0066] Beckman System Gold.RTM., column: Luna (Phenomenex), C18, 3
.mu.m, 50.times.4.6 mm i.d.; flow rate 1 ml/min; solvent A: water
(0.1%) TFA, solvent B: acetonitrile (0.1% TFA).
gradient system 1: 1 min 40% B, 15 min 40.fwdarw.80% B, 5 min 80% B
gradient system 2: 1 min 0% B, 10 min 0.fwdarw.30% B, 5 min 30% B,
5 min 30.fwdarw.80% B
Example 4
Preparation of
4-[.sup.18F]-Fluoromethyl-N-[2-(tritylsulphanyl)ethyl]-benzamide,
deprotection and site-specific conjugation to a chloroacetyl
modified peptide
4.a) Preparation of
3-[.sup.18F]Fluoromethyl-N-(2-mercaptotrityl-ethyl)-benzamide
##STR00031##
[0068] The preparation was carried out by methods analogous to
those described in example 5a). The mesylate precursor prepared as
described in Example 1c) was stirred at room temperature for 15
minutes to give desired product in a 22% radiochemical yield
(HPLC).
4.b) Chemoselective ligation to peptide
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH
##STR00032##
[0070] The deprotection and ligation of .sup.18F-synthon from
Example 4a) with peptide precursor
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH was carried out following the
methods described in example 5. HPLC analysis revealed formation of
the desired product with a radiochemical purity of 80%.
Example 5
Preparation of
(3-[.sup.18F]-fluoro-propylsulfanyl)triphenylmethane, deprotection
and site-specific conjugation to a chloroacetyl peptide
5.a) Preparation of .sup.18F synthon:
3-[.sup.18F]fluoro-1-mercaptotrityl-propane
##STR00033##
[0072] To a Wheaton vial (2 ml) charged with Kryptofix.RTM. 222 (10
mg), potassium carbonate (1 mg dissolved in 50 .mu.l water), and
acetonitrile (0.8 ml) the fluorine-18 containing water (10 mCi, 1
ml) was added. The solvent was removed by heating at 110.degree. C.
for one hour under a stream of nitrogen. Anhydrous acetonitrile
(0.5 ml) was added and again evaporated as before. This step was
repeated twice. The vial was cooled to room temperature followed by
injecting a solution of mesylate prepared as described in Example
2b) (1 mg) in anhydrous DMSO (0.2 ml). The reaction mixture was
stirred at 80.degree. C. for 5 min and analysed by HPLC (gradient
1, radiochemical yield 90%).
[0073] The reaction mixture was diluted with DMSO/water (1:1 v/v,
0.15 ml) and loaded onto a SepPak-Plus cartridge (.sup.tC18,
Waters) that had been conditioned (10 ml acetonitrile, 20 ml
water). The cartridge was washed with water (10 ml) and the product
eluted using acetonitrile. The radiochemical purity was 99%.
5.b) Chemoselective ligation of a) to
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH
##STR00034##
[0075] A solution of product from 5 a) in acetonitrile (0.5 ml, 1.1
mCi) was evaporated to dryness using a stream of nitrogen and
heating at 100.degree. C. A mixture of TFA (0.05 ml),
triisopropylsilane (0.01 ml), and water (0.01 ml) was added
followed by heating for 10 min at 80.degree. C. After the vial was
cooled to 0.degree. C., ammonia (27% in water, 0.1 ml) and
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH
[0076] (1 mg) in water (0.05 ml) were added. The mixture was
stirred for 30 min at 80.degree. C. Analysis by HPLC (gradient 2)
revealed formation of desired product with a radiochemical purity
of 93%.
Example 6
Preparation of
(2-{2-[2-(2-[.sup.18F]-Fluoro-ethoxy)-ethoxy]-ethoxy}-ethyl)-trityl
sulfide, deprotection and site-specific conjugation to a
chloroacetyl modified peptide
6.a) Preparation of .sup.18F synthon:
2-{2-[2-(2-[.sup.18F]Fluoro-ethoxy)-ethoxy]-ethoxy}-mercaptotrityl-ethane
##STR00035##
[0078] This preparation was carried out similarly as described in
Example 5a). After reacting the mesylate precursor prepared as
described in Example 3d) for 15 min in DMSO at 80.degree. C. a
radiochemical yield of 76% of product was obtained (HPLC).
Extraction by Sep-Pak gave the desired product in a radiochemical
purity of 97%.
6.b) Chemoselective ligation of to peptide
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH
##STR00036##
[0080] The deprotection and ligation reaction of 18F-synthon from
Example 6a) with peptide precursor
Cl--CH.sub.2CO-Lys-Gly-Phe-Gly-Lys-OH was carried out following the
methods described in Example 5. HPLC analysis revealed formation of
the desired product in a radiochemical purity of 41%.
Example 7
Synthesis of
[Cys.sup.2-6]cyclo[CH.sub.2CO-Lys(4-{3-[2-(4-fluoromethyl-benzoylamino)-e-
thylsulfanyl]-2,5-dioxo-pyrrolidin-1-ylmethyl}-cyclohexane-1-carbonyl)-Cys-
.sup.2-Arg-Gly-Asp-Cys.sup.6-Phe-Cys]-NH.sub.2
##STR00037##
[0081] 7 a) Synthesis of
[Cys.sup.2-6]cyclo[CH.sub.2CO-Lys-Cys.sup.2-Arg-Gly-Asp-Cys.sup.6-Phe-Cys-
]-NH.sub.2
##STR00038##
[0083] The title compound was synthesised as described in WO
03/006491.
7 b) Synthesis of
[Cys.sup.2-6]cyclo[CH.sub.2CO-Lys(4-[N-maleimidomethyl]-cyclohexane-1-car-
bonyl)-Cys.sup.2-Arg-Gly-Asp-Cys.sup.6-Phe-Cys]-NH.sub.2
##STR00039##
[0085] To 5 mg of the peptide from 4 a) above in DMF 1 mL was added
3 mg of sulfosuccinimidyl
4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (Pierce) and 0.05
mL NMM. The reaction was left stirring at room temperature for 2
hours then diluted with 5 mL of water and charged onto a
semi-preparative Luna C18 column. The product fraction was
collected and freeze-dried yielding 1 mg of desired product. The
structure was confirmed by MALDI-MS: Expected M+H+, 1189; Found
1190.
7. c) Site-Specific Conjugation to the Maleimide Modified
Peptide
##STR00040##
[0087] To 0.16 mg (0.34 .mu.mol) of
4-fluoromethyl-N-[2-(tritylsulphanyl)ethyl]benzamide (from example
1d above) was added 50 .mu.l of TFA/TIS/H.sub.2O (95:2.5:2.5)
mixture. The flask was swirled gently for 5 minutes and then
concentrated in vacuo. The residue was taken up in 50 .mu.l water.
To the mixture was added a solution of 0.4 mg (0.34 .mu.mol)
maleimide modified peptide in 50 .mu.l water and pH adjusted to 6.5
with 0.001 M sodium hydroxide. After 50 minutes LC-MS analysis
(column Phenomenex Luna 3 .mu.m C18(2) 50.times.2.00 mm; solvents:
A=water/0.1% HCOOH and B=acetonitrile/0.1% HCOOH; gradient 0-50% B
over 10 min; flow 0.3 ml/min, UV detection at 214 and 254 nm,
ESI-MS) showed almost complete conversion of the maleimide modified
peptide to a new product(t.sub.R 7.6 min) giving m/z at 1400.7
corresponding to the M+H+ for the conjugate.
Example 8
Synthesis of [Cys.sup.2-6]cyclo[CH.sub.2CO-Lys(4-{3-[2-(4-[.sup.18
F]-fluoromethyl-benzoylamino)-ethylsulfanyl]-2,5-dioxo-pyrrolidin-1-ylmet-
hyl}-cyclohexane-1-carbonyl)-Cys.sup.2-Arg-Gly-Asp-Cys.sup.6-Phe-Cys]-NH.s-
ub.2
[0088] The title compound is prepared from the modified peptide of
Example 7b by reaction with
4-[.sup.18F]-fluoromethyl-N-[2-(tritylsulphanyl)ethyl]benzamide
(Example 4) using methods analogous to those described in Example
7c.
* * * * *