U.S. patent application number 13/123329 was filed with the patent office on 2011-11-24 for 18f-labelled folates as pet radiotracers.
This patent application is currently assigned to MERCK & CIE. Invention is credited to Simon Mensah Ametamey, Viola Groehn, Rudolf Moser, Cristina Magdalena Muller, Tobias Ludwig Ross, Roger Schibli.
Application Number | 20110286921 13/123329 |
Document ID | / |
Family ID | 41397772 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110286921 |
Kind Code |
A1 |
Schibli; Roger ; et
al. |
November 24, 2011 |
18F-LABELLED FOLATES AS PET RADIOTRACERS
Abstract
The present invention is directed towards new .sup.18F-folate
radiopharmaceuticals, wherein fluorine-18 is covalently linked to
the glutamate portion of a folate or derivative thereof, a method
of their preparation, as well as their use in diagnosis and
monitoring of therapy of cancer and inflammatory and autoimmune
diseases.
Inventors: |
Schibli; Roger; (Baden,
CH) ; Moser; Rudolf; (Schaffhausen, CH) ;
Muller; Cristina Magdalena; (Nussbaumen, CH) ;
Ametamey; Simon Mensah; (Zurich, CH) ; Ross; Tobias
Ludwig; (Eschborn, DE) ; Groehn; Viola;
(Dachsen, CH) |
Assignee: |
MERCK & CIE
Schaffhausen
CH
|
Family ID: |
41397772 |
Appl. No.: |
13/123329 |
Filed: |
October 12, 2009 |
PCT Filed: |
October 12, 2009 |
PCT NO: |
PCT/EP2009/063258 |
371 Date: |
August 1, 2011 |
Current U.S.
Class: |
424/1.89 ;
435/7.21; 544/261 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 475/04 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/1.89 ;
544/261; 435/7.21 |
International
Class: |
A61K 51/04 20060101
A61K051/04; G01N 33/566 20060101 G01N033/566; C07D 475/04 20060101
C07D475/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2008 |
EP |
08166356.9 |
Claims
1. A compound of formula I, ##STR00009## wherein P is a pteroyl
group or derivative thereof, X.sub.a, X.sub.b are independently of
each other C, N, O, S, R.sub.a, R.sub.b are independently of each
other H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; and Z.sub.1, Z.sub.2 are
independently of each other H or .sup.18F, with the proviso that
one of Z.sub.1 and Z.sub.2 is .sup.18F.
2. A compound according to claim 1 having formula II ##STR00010##
wherein X.sub.1 to X.sub.5 are independently of each other C or N,
R.sub.1 and R.sub.2 are independently of each other H, Hal, --OR',
--NR''R''', C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino)carbonyl, wherein R' is H or
C.sub.1-C.sub.6 alkyl, and wherein R'' and R''' are independently
of each other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, R.sub.3,
R.sub.4 are independently of each other H, formyl, trifluoroacetyl,
iminomethyl, nitroso, straight chain or branched C.sub.1-C.sub.12
alkyl, which is unsubstituted or substituted by at least one CN,
Hal, or NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 form together a
C.sub.1 or C.sub.2-bridge between X.sub.3 and X.sub.5, R.sub.5 is
H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino)carbonyl, m is 0 or 1, p is 0, 1 or 2,
q has a value of 1 to 7 X.sub.a, X.sub.b are independently of each
other C, N, O, S, R.sub.a, R.sub.b are independently of each other
H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; and Z.sub.1, Z.sub.2 are
independently of each other H or .sup.18F, with the proviso that
one of Z.sub.1 and Z.sub.2 is .sup.18F.
3. A compound according to claim 1 having formulae IIIa, IIIb, IVa,
or IVb, ##STR00011## wherein X.sub.1 to X.sub.5 are independently
of each other C or N, R.sub.1 and R.sub.2 are independently of each
other H, Hal, --OR', --NR''R''', C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl,
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl, wherein R' is H or C.sub.1-C.sub.6 alkyl, and
wherein R'' and R''' are independently of each other selected from
H, formyl, straight chain or branched C.sub.1-C.sub.12 alkyl, which
is unsubstituted or substituted by at least one CN, Hal, or
NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, R.sub.3, R.sub.4 are independently of
each other H, formyl, trifluoroacetyl, iminomethyl, nitroso,
straight chain or branched C.sub.1-C.sub.12 alkyl, which is
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is H or
C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 form together a
C.sub.1 or C.sub.2-bridge between X.sub.3 and X.sub.5, R.sub.5 is
H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, or
(C.sub.1-C.sub.12 alkylamino)carbonyl, m is 0 or 1, p is 0, 1 or 2,
q has a value of 1 to 7, X.sub.a, X.sub.b are independently of each
other C, N, O, S, R.sub.a, R.sub.b are independently of each other
H or straight-chain or branched C.sub.1-C.sub.12 alkyl, C3-C6
cycloalkyl, C5-C14 aryl or C5-C14 heteroaryl, which independently
of each other are unsubstituted or substituted by at least one CN,
Hal, or NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--,
wherein R' is H or C.sub.1-C.sub.6 alkyl.
4. A compound according to claim 1, wherein R.sub.1 and R.sub.2 are
independently of each other H, alkyl, --OR', --NHR', wherein R'
represents H or C.sub.1-C.sub.6 alkyl.
5. A compound according to claim 1, wherein R.sub.3 is H, formyl,
C1-C12 alkyl or C1-C12 alkanoyl.
6. A compound according to claim 1, wherein R.sub.3 is H, formyl,
or methyl.
7. A compound according to claim 1, wherein R.sub.4 is H, formyl,
nitroso, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, or
C.sub.1-C.sub.6 alkanoyl.
8. A compound according to claim 1, wherein R.sub.4 is H, formyl,
or methyl.
9. A compound according to claim 1, wherein R.sub.5 is H,
C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12
alkanoyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, or (C.sub.1-C.sub.12
alkylamino)carbonyl.
10. A compound according to claim 1, wherein R.sub.5 is H.
11. A compound according to claim 1 having formulae Va, Vb, VIa or
VIb, ##STR00012## wherein, X.sub.a, X.sub.b are independently of
each other C, N, O, S, R.sub.a, R.sub.b are independently of each
other H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; Y.sub.1, Y.sub.2 are independently
of each other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, Y.sub.3 is
selected from H, formyl, trifluoroacetyl, nitroso, straight chain
or branched C.sub.1-C.sub.12 alkyl, which is unsubstituted or
substituted by at least one CN, Hal, or NO.sub.2, and wherein one
or more of embedded, non-adjacent CH.sub.2 groups may independently
be replaced by --O--, --CO--, --CO--O--, --CO--NR'--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl.
12. A compound according to claim 1 having formulae VIIa, VIIb,
VIIIa or VIIIb, ##STR00013## wherein, X.sub.a, X.sub.b are
independently of each other C, N, O, S, R.sub.a, R.sub.b are
independently of each other H or straight-chain or branched
C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.5-C.sub.14 aryl or C.sub.5-C.sub.14 heteroaryl, which
independently of each other are unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --SO.sub.2--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl; p is 0, 1 or 2, Y.sub.1, Y.sub.2 are independently of each
other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, R.sub.3 is
H, formyl, iminomethyl, nitroso, C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl, halosubstituted
C.sub.1-C.sub.12 alkanoyl, and Y.sub.3 is selected from H, formyl,
trifluoroacetyl, nitroso, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, or R.sub.3
and Y.sub.3 form together a C.sub.1 or C.sub.2-bridge between the
two N-atoms to which they are attached to.
13. (canceled)
14. (canceled)
15. Method for diagnostic imaging of a cell or population of cells
expressing a folate-receptor, said method comprising the steps of
administering at least one compound according to claim 1 in a
diagnostic imaging amount, and obtaining a diagnostic image of said
cell or population of cells.
16. Method according to claim 15, wherein the diagnostic imaging is
performed of a cell or population of cells expressing a
folate-receptor in vitro or in vivo.
17. Method for in vitro detection of a cell expressing the folate
receptor in a tissue sample which includes contacting said tissue
sample with a compound according to claim 1 in effective amounts
and for sufficient time and conditions to allow binding to occur
and detecting such binding by PET imaging.
18. Method of diagnostic imaging or monitoring a subject comprising
the steps of (i) administering at least one compound according to
claim 1 in a diagnostic imaging amount, and (ii) performing
diagnostic imaging using PET by detecting a signal from said at
least one compound.
19. Method of monitoring therapy of cancer and inflammatory and
autoimmune diseases in a subject comprising the steps of (i)
administering to a subject in need thereof at least one compound
according to claim 1 in a diagnostic imaging amount in combination
with a therapeutically active, and (ii) performing diagnostic
imaging using PET by detecting a signal from said at least one
compound to follow the course of therapy of cancer and inflammatory
and autoimmune diseases.
20. Method of claim 15 used in combination with any other methods
of diagnosis or therapy of cancer and inflammatory and autoimmune
diseases.
Description
FIELD OF INVENTION
[0001] The present invention is directed towards new
.sup.18F-folate radiopharmaceuticals, wherein fluorine-18 is
covalently linked to the glutamate portion of a folate or
derivative thereof, a method of their preparation, as well as their
use in diagnosis and monitoring of cancer and inflammatory and
autoimmune diseases and therapy thereof.
BACKGROUND
[0002] Cell-specific targeting for delivery of effector moieties
such as diagnostic or therapeutic agents is a widely researched
field and has led to the development of non-invasive diagnostic
and/or therapeutic medical applications. In particular in the field
of nuclear medicine procedures and treatments, which employ
radioactive materials emitting electromagnetic radiations as
.gamma.-rays or photons or particle emitting radiation, selective
localization of these radioactive materials in targeted cells or
tissues is required to achieve either high signal intensity for
visualization of specific tissues, assessing a disease and/or
monitoring effects of therapeutic treatments, or high radiation
dose, for delivering adequate doses of ionizing radiation to a
specified diseased site, without the risk of radiation injury in
other e.g. healthy tissues. It is thus of crucial interest to
determine and assess cell-specific structures and in particular
structures that are present in case of tumors (i.e. cancer) or
inflammatory and autoimmune diseases, such as receptors, antigens,
haptens and the like which can be specifically targeted by the
respective biological vehicles.
[0003] The folate receptor (FR) has been identified as one of these
structures. The FR is a high-affinity (K.sub.D<10.sup.-9 M)
membrane-associated protein. In normal tissues and organs
FR-expression is highly restricted to only a few organs (e.g.
kidney, lungs, choroids plexus, and placenta), where it largely
occurs at the luminal surface of epithelial cells and is therefore
not supplied with folate in the circulation. The FR-alpha is
frequently overexpressed on a wide variety of specific cell types,
such as epithelial tumours (e.g. ovarian, cervical, endometrial,
breast, colorectal, kidney, lung, nasopharyngeal), whereas the
FR-beta is frequently overexpressed in leukaemia cells (approx. 70%
of acute myelogenous leukaemia (AML) are FR-beta positive). Both
may therefore be used as a valuable tumour marker for selective
tumour-targeting (Elnakat and Ratnam, Adv. Drug Deliv. Rev. 2004;
56:1067-84). In addition, the FR-beta isoform has been found on
activated (but not resting) macrophages. Activated macrophages are
involved in inflammatory pathologies such as e.g. rheumatoid
arthritis, psoriasis, Crohn's disease, ulcerative colitis, systemic
lupus erythematosus, atherosclerosis, diabetes, osteoarthritis,
glomerulonephritis, infections, etc.
[0004] The literature reports several preclinical studies of
folate-based imaging agents for detection/localization of sites of
inflammation as well as folate receptor targeted therapy of theses
diseases. Recently, a clinical study has been published that
reports the results of imaging studies in patients with rheumatoid
arthritis using the FolateScan (Turk et al., Arthritis and
Rheumatism 2002, 45, 1947-1955; Paulos et al., Adv. Drug Deliv.
Rev. 2004, 56, 1205-1217; Chen et al., Arthritis Research &
Therapy 2005, 7, 310-317; Hattori et al., Biol. & Pharm. Bull.
2006, 29, 1516-1520; Chandraseka et al., J. Biomed. Mat. Res. Part
A 2007, 82, 92-103; Varghese et al., Mol. Pharmaceutics 2007, 4,
679-685; Low et al. Discovery and development of folic-acid-based
receptor targeting for imaging and therapy of cancer and
inflammatory diseases 2008, 41, 120-129; Matteson et al., Clinical
and Experimental Rheumatology 2009, 27, 253-259).
[0005] Folic acid, which is based on a pteridine skeleton
conjugated through a benzoylamino moiety to a glutamate, and its
derivatives have thus been intensively studied over the past 15
years as targeting agents for the delivery of therapeutic and/or
diagnostic agents to cell populations bearing folate receptors in
order to achieve a selective concentration of therapeutic and/or
diagnostic agents in such cells relative to normal cells.
[0006] Various folic acid derivatives and conjugates are known and
have been (pre)clinically evaluated, including folate
radiopharmaceuticals (Leamon and Low, Drug Discov. Today 2001;
6:44-51; U.S. Pat. No. 4,276,280), fluorinated folate
chemotherapeutics (U.S. Pat. No. 4,628,090), folate-conjugates with
chemotherapeutic agents (Leamon and Reddy, Adv. Drug Deliv. Rev.
2004; 56:1127-41; Leamon et al, Bioconjugate Chem. 2005;
16:803-11), with proteins and protein toxins (Ward et al., J. Drug
Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem. 1993;
268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12), with
antisense oliconucleotides (Li et al, Pharm. Res. 1998; 15:1540-45;
Zhao and Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), with
liposomes (Lee and Low, Biochim. Biophys. Acta-Biomembr. 1995;
1233:134-44; Gabizon et al, Adv. Drug Deliv. Rev. 2004;
56:1177-92), with hapten molecules (Paulos et al, Adv. Drug Deliv.
Rev. 2004; 56:1205-17), with MRI contrast agents (Konda et al,
Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc.
[0007] Folate radiopharmaceuticals can be in particular very useful
for an improved diagnosis and evaluation of the effectiveness of
cancer and inflammatory and autoimmune disease therapy. This may
include assessment and/or prediction of a treatment response and
consequently improvement of radiation dosimetry. Typical
visualization techniques suitable for radioimaging are known in the
art and include positron emission tomography (PET), planar or
single photon emission computerized tomography (SPECT) imaging,
gamma cameras, scintillation, and the like.
[0008] Both PET and SPECT use radiotracers to image, map and
measure activities of target sites of choice. Yet while PET uses
positron emitting nuclides which require a nearby cyclotron, SPECT
uses single photon emitting nuclides which are available by
generator systems, which may make its use more convenient. However
SPECT provides less sensitivity than PET and beside a few
approaches quantification methods are lacking. In case of PET, the
positron annihilation results in two gamma rays of 511 keV which
provide the basis for well developed quantification methods. Thus
PET is one of the most sophisticated functional imaging
technologies to assess regional uptake and affinity of ligands or
metabolic substrates in brain and other organs and thus provides
measures of imaging based on metabolic activity. This is for
example achieved by administering a positron emitting isotope to a
subject, and as it undergoes radioactive decay the gamma rays
resulting from the positron/electron annihilation are detected by
the PET scanner.
[0009] Factors that need to be considered in the selection of a
suitable isotope useful for PET include sufficient half-life of the
positron-emitting isotope to permit preparation of a diagnostic
composition optionally in a pharmaceutically acceptable carrier
prior to administration to the patent, and sufficient remaining
half-life to yield sufficient activity to permit extra-corporeal
measurement by a PET scan. Furthermore, a suitable isotope should
have a sufficiently short half-life to limit patient exposure to
unnecessary radiation. Typically, a suitable radiopharmaceutical
for PET may be based on a metal isotope, such as gallium or copper.
These two require however a chelator for entrapment of the metal,
which may have an effect on steric and chemical properties.
Alternatively a radiopharmaceutical may be based on a covalently
linked isotope which provides minimal structural alteration.
Radionuclides used for covalent attachment and which could be
suitable for PET scanning are typically isotopes with short half
lives such as .sup.11C (ca. 20 min), .sup.13N (ca. 10 min),
.sup.15O (ca. 2 min), .sup.18F (ca. 110 min).
[0010] To date, a number of chelate-based folate
radiopharmaceuticals have been synthesized and successfully
evaluated as diagnostic agents for imaging folate receptor-positive
tumors. The most widely studied derivatives were labeled either
with .sup.111In and .sup.99mTc (Siegel et al., J. Nucl. Med. 2003,
44:700; Muller et al., J. Organomet. Chem. 2004, 689:4712) for
SPECT or with .sup.68Ga for PET (Mathias et al., Nucl. Med. Biol.
2003, 30(7):725). However, all of the above need a suitable
chelating agent, which is typically linked to folic acid through
its glutamate portion.
[0011] Thus a folate radiopharmaceutical having a covalently linked
isotope would be of great interest. In particular a
.sup.18F-labeled folate radiopharmaceutical would be most suitable
for PET Imaging because of its excellent imaging characteristics
which would fulfil all of the above considerations. Compared with
other suitable radionuclides (.sup.11C, .sup.13N, .sup.15O),
.sup.18F is very useful because of its long half-life of
approximately 110 minutes and because it decays by emitting
positrons having the lowest positron energy, which allows for the
sharpest images with a high-resolution PET. Furthermore, the longer
half-life of .sup.18F also allows for syntheses that are more
complex and satellite distribution to PET centers with no
radiochemistry facilities.
[0012] Yet, to date, there have been only few .sup.18F-labeled
folic acid derivatives reported in the literature (Bettio et al.,
J. Nucl. Med., 2006, 47(7), 1153; Ross et al., Bioconjugate Chem.,
2008, 19, 2402; WO 2006/071754; WO 2008/098112; WO 2008/125613; WO
2008/125615; WO 2008/125617), in addition to few reports on folate
derivatives labeled with isotopes having much longer half-lives,
such as .sup.131I (60 days) and .sup.125I (8 days: U.S. Pat. No.
4,276,280, U.S. Pat. No. 4,298,735) and .sup.75Se (120 days: GB
1501119, U.S. Pat. No. 4,202,976). Moreover, some of the
methodologies suffer from drawbacks including time-consuming
radiosyntheses giving only low radiochemical yields of less than 5%
(Bettio et al., J. Nucl. Med., 2006, 47(7), 1153) or unfavoruable
pharmacokinetics for molecular imaging purposes (Ross et al.,
Bioconjugate Chem., 2008, 19, 2402). Thus, there is still a need
for specific radiopharmaceuticals suitable for metabolic imaging of
tumors to improve diagnosis and treatment of cancer and
inflammatory and autoimmune diseases.
[0013] Applicants have now found efficient and versatile methods
for production of new .sup.18F-labeled folate radiopharmaceuticals
wherein fluorine-18 is linked to the glutamate portion of a folate
or derivative thereof. The present method is highly effective
giving the desired .sup.18F-folate in good yields to meet the
expectations for a clinical application in humans. In addition, the
new radiosynthesis is applicable in an automated synthesis module
which allows a fast and convenient labeling procedure which meets
the requirements of GMP guidelines. Preliminary in-vitro and
in-vivo studies suggested their suitability as powerful diagnostic
agents for FR-positive tumours.
SUMMARY OF THE INVENTION
[0014] The present invention is in a first aspect directed to new
.sup.18F-folate radiopharmaceuticals (hereinafter also called
compounds of the invention), wherein fluorine-18 is linked to the
glutamate portion of a folate or derivative thereof.
[0015] In one specific embodiment, the new folate
radiopharmaceuticals are compounds of formula I,
##STR00001##
wherein P is a pteroyl group or derivative thereof, X.sub.a,
X.sub.b are independently of each other C, N, O, S, R.sub.a,
R.sub.b are independently of each other H or straight-chain or
branched C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.5-C.sub.14 aryl or C.sub.5-C.sub.14 heteroaryl, which
independently of each other are unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --SO.sub.2--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl; and Z.sub.1, Z.sub.2 are independently of each other H or
.sup.18F, with the proviso that one of Z.sub.1 and Z.sub.2 is
.sup.18F.
[0016] In another specific embodiment, the new folate
radiopharmaceuticals are compounds of formula II
##STR00002##
wherein X.sub.1 to X.sub.5 are independently of each other C or N,
R.sub.1 and R.sub.2 are independently of each other H, Hal, --OR',
--NR''R''', C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino)carbonyl, wherein R' is H or
C.sub.1-C.sub.6 alkyl, and wherein R'' and R''' are independently
of each other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, R.sub.3,
R.sub.4 are independently of each other H, formyl, trifluoroacetyl,
iminomethyl, nitroso, straight chain or branched C.sub.1-C.sub.12
alkyl, which is unsubstituted or substituted by at least one CN,
Hal, or NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 form together a
C.sub.1 or C.sub.2-bridge between X.sub.3 and X.sub.5, R.sub.5 is
H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino) carbonyl, m is 0 or 1, p is 0, 1 or
2, q has a value of 1 to 7 X.sub.a, X.sub.b are independently of
each other C, N, O, S, R.sub.a, R.sub.b are independently of each
other H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; and Z.sub.1, Z.sub.2 are
independently of each other H or .sup.18F, with the proviso that
one of Z.sub.1 and Z.sub.2 is .sup.18F.
[0017] In a further aspect the present invention is directed to a
method of their preparation.
[0018] In another aspect the present invention is directed to
pharmaceutical compositions of the compounds of the invention.
[0019] In yet another aspect the present invention is directed to
the use in diagnosis and monitoring of therapy of cancer and
inflammatory and autoimmune diseases in vitro or in vivo.
[0020] In one embodiment, the present invention is directed towards
uses of the compounds of the invention for diagnostic imaging of a
cell or population of cells expressing a folate-receptor.
[0021] More specifically the present invention includes methods for
diagnostic imaging of a cell or population of cells expressing a
folate-receptor, which includes for example methods for in vitro
detection of a cell expressing the folate receptor, for example a
tumor cell or an activated macrophage, in a tissue sample. Such
methods may also be performed in vivo.
[0022] Thus, in a further embodiment the present invention is
directed towards uses of the compounds of the invention for
convenient and effective administration to a subject in need for
diagnostic imaging and/or monitoring of therapy of cancer and
inflammatory and autoimmune diseases. The subject of the methods of
the present invention is preferably a mammal, such as an animal or
a human, preferably a human.
[0023] Such methods of the invention may be performed in
combination with any other methods of diagnosis or therapy of
cancer and inflammatory and autoimmune diseases including methods
using other already developed diagnostic and/or therapeutic agents
and utilizing x-ray computed tomography (CT), magnetic resonance
imaging (MRI), functional magnetic resonance imaging (fMRI), single
photon emission computed tomography (SPECT), optical imaging, and
ultrasound.
[0024] Other features and advantages of the invention will be
apparent from the following detailed description thereof and from
the claims.
BRIEF DESCRIPTION OF FIGURES
[0025] FIG. 1: Synthesis pathway of
.gamma.-[.sup.18F]-compounds.
[0026] FIG. 2: Specific uptake of the
.gamma.-[.sup.18F]fluoro-folic acid in folate receptor-positive
tissues.
[0027] FIG. 3: Representative series of normalized horizontal
slices of whole body PET scans using .gamma.-[.sup.18F]fluoro-folic
acid under control and blockade conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention is in a first aspect directed to new
.sup.18F-folate radiopharmaceuticals (hereinafter also called
compounds of the invention), wherein fluorine-18 is linked to the
glutamate portion of a folate or derivative thereof.
[0029] The term "folate" as used herein, comprises compounds based
on a pteroyl group, which is coupled through a peptide bond to a
glutamic acid (or derivative thereof). The term "pteroyl" as used
herein represents a condensed pyrimidine heterocycle, which is
linked to an aminobenzoyl moiety. As used herein a "condensed
pyrimidine heterocycle" includes a pyrimidine fused with a further
5- or 6-membered heterocycle, resulting in a pteridine (i.e. a
fused 6-6 heterocycle) or a pyrrolopyrimidine bicycle (i.e. a fused
6-5 heterocycle). Derivatives of a condensed pyrimidine heterocycle
include carbocyclic derivatives such as indoles, and isoindoles,
quinolines and isoquinolines, and the like. As used herein a
"condensed pyrimidine heterocycle, which is linked to an
aminobenzoyl moiety" also includes three fused ring systems, i.e.
wherein the amino group of the aminobenzoyl moiety forms a further
fused ring with the condensed pyrimidine heterocycle, resulting in
a fused 6-6-6, 6-6-5, 6-5-6, or 6-5-5 heterocycle. Preferred
representatives of folates as used herein are based on a folate
skeleton, i.e. pteroyl-glutamic acid resp.
N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl-
]-L-(or D-)glutamic acid, and derivatives thereof and includes
optionally substituted folic acid, folinic acid, pteropolyglutamic
acid, 5,10-methenyl-5,6,7,8-tetrahydrofolate and folate
receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, tetrahydrofolates, and their deaza and dideaza
analogs. Folic acid, 5-methyl-(6S)-tetrahydrofolic acid and
5-formyl-(6S)-tetrahydrofolic acid are the preferred basic
structures used for the compounds of this invention. The terms
"deaza" and "dideaza" analogs refers to the art recognized analogs
having a carbon atom substituted for one or two nitrogen atoms in
the naturally occurring folic acid structure. For example, the
deaza analogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and
10-deaza analogs. The dideaza analogs include, for example,
1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs.
Preferred deaza analogs compounds include
N-[4-[2-[(6R)-2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-
-yl]ethyl]benzoyl]-L-glutamic acid (Lometrexol) and
N-[4-[1-[(2,4-diamino-6-pteridinyl)methyl]propyl]benzoyl]-L-glutamic
acid (Edatrexate).
[0030] More specifically, the new folate radiopharmaceuticals are
compounds of formula I,
##STR00003##
wherein P is a pteroyl group or derivative thereof, X.sub.a,
X.sub.b are independently of each other C, N, O, S, R.sub.a,
R.sub.b are independently of each other H or straight-chain or
branched C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.5-C.sub.14 aryl or C.sub.5-C.sub.14 heteroaryl, which
independently of each other are unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --SO.sub.2--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl; and Z.sub.1, Z.sub.2 are independently of each other H or
.sup.18F, with the proviso that one of Z.sub.1 and Z.sub.2 is
.sup.18F.
[0031] In another specific embodiment, the new folate
radiopharmaceuticals are compounds of formula II
##STR00004##
wherein X.sub.1 to X.sub.5 are independently of each other C or N,
R.sub.1 and R.sub.2 are independently of each other H, Hal, --OR',
--NR''R''', C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino)carbonyl, wherein R' is H or
C.sub.1-C.sub.6 alkyl, and wherein R'' and R''' are independently
of each other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, R.sub.3,
R.sub.4 are independently of each other H, formyl, trifluoroacetyl,
iminomethyl, nitroso, straight chain or branched C.sub.1-C.sub.12
alkyl, which is unsubstituted or substituted by at least one CN,
Hal, or NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 form together a
C.sub.1 or C.sub.2-bridge between X.sub.3 and X.sub.5, R.sub.5 is
H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino) carbonyl, m is 0 or 1, p is 0, 1 or
2, q has a value of 1 to 7 X.sub.a, X.sub.b are independently of
each other C, N, O, S, R.sub.a, R.sub.b are independently of each
other H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; and Z.sub.1, Z.sub.2 are
independently of each other H or .sup.18F, with the proviso that
one of Z.sub.1 and Z.sub.2 is .sup.18F.
[0032] It is understood, that the abbreviations "N" and "C" are
representative for all possible degrees of saturation, i.e. N
includes --NH-- and --N.dbd. linkages and C includes --CH.sub.2--
and --CH.dbd. linkages.
[0033] It is further understood, that (H).sub.q represents all H
substituents on the indicated ring (i.e. on X.sub.3, C6, C7 and
X.sub.4). For example q=5 for a fully saturated unsubstituted
analog (X.sub.3=X.sub.4=N, p=j) or q=7 for a fully saturated
unsubstituted 5,8-dideaza analog (X.sub.3=X.sub.4=C, p=0) and q=1
for a fully unsaturated analog with X.sub.3=X.sub.4=N, p=0.
[0034] It is further understood that all isomers, including
enantiomers, diastereoisomers, rotamers, tautomers and racemates of
the compounds of formula I are contemplated as being part of this
invention. The invention includes stereoisomers in optically pure
form and in admixture, including racemic mixtures. Isomers can be
prepared using conventional techniques, either by reacting
optically pure or optically enriched starting materials or by
separating isomers of a compound of formula I. This applies
specifically to any amino acid groups present in a compound of
formula I (and subsequent formulas), which may be present in the
natural L- or non-natural D-form. In a specific embodiment, unless
specified otherwise the term "glutamic acid" or "glutamate portion"
always refers to both the natural L- and non-natural D-isomer.
[0035] In another specific embodiment, the new folate
radiopharmaceuticals are compounds of formulae IIIa, IIIb, IVa, or
IVb,
##STR00005##
wherein X.sub.1 to X.sub.5 are independently of each other C or N,
R.sub.1 and R.sub.2 are independently of each other H, Hal, --OR',
--NR''R''', C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy,
C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, and
(C.sub.1-C.sub.12 alkylamino)carbonyl, wherein R' is H or
C.sub.1-C.sub.6 alkyl, and wherein R'' and R''' are independently
of each other selected from H, formyl, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl, R.sub.3,
R.sub.4 are independently of each other H, formyl, trifluoroacetyl,
iminomethyl, nitroso, straight chain or branched C.sub.1-C.sub.12
alkyl, which is unsubstituted or substituted by at least one CN,
Hal, or NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 form together a
C.sub.1 or C.sub.2-bridge between X.sub.3 and X.sub.5, R.sub.5 is
H, CN, Hal, NO.sub.2, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12
alkoxy, C.sub.1-C.sub.12 alkanoyl, C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, (C.sub.1-C.sub.12 alkoxy)carbonyl, or
(C.sub.1-C.sub.12 alkylamino)carbonyl, m is 0 or 1, p is 0, 1 or 2,
q has a value of 1 to 7, X.sub.a, X.sub.b are independently of each
other C, N, O, S, and R.sub.a, R.sub.b are independently of each
other H or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl.
[0036] Preferably, R.sub.1 and R.sub.2 may independently of each
other H, alkyl, --OR.sub.5, --NR''R''', more preferably --OR',
--NR''R''', wherein R' represents H or C.sub.1-C.sub.6 alkyl, and
wherein R'' and R''' are independently of each other selected from
H, formyl, straight chain or branched C.sub.1-C.sub.12 alkyl, which
is unsubstituted or substituted by at least one CN, Hal, or
NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl.
[0037] Preferably, R.sub.3 is H, formyl, C.sub.1-C.sub.12 alkyl or
C.sub.1-C.sub.12 alkanoyl.
[0038] Preferably, R.sub.4 is H, formyl, nitroso, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, or C.sub.1-C.sub.6 alkanoyl.
[0039] Preferably, R.sub.3 and R.sub.4 form together a C.sub.1 or
C.sub.2-bridge between X.sub.3 and X.sub.5.
[0040] Preferably, R.sub.5 is H, CN, Hal, NO.sub.2,
C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12
alkanoyl, or (C.sub.1-C.sub.12 alkoxy)carbonyl, more preferably H,
CN, Hal, NO.sub.2, or C.sub.1-C.sub.8 alkyl.
[0041] Preferably, X.sub.a and X.sub.b are C, N, O most preferably
O.
[0042] Preferably R.sub.a and R.sub.b are H or straight-chain or
branched C.sub.1-C.sub.12 alkyl or C.sub.5-C.sub.14 aryl, which
independently of each other are, which is unsubstituted or
substituted by at least one Hal, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --SO.sub.2--, --CH.dbd.CH--.
In a specific embodiment Ra and Rb may independently represent a
natural or non-natural amino acid.
[0043] The term "natural amino acid" indicates one of the natural
L-amino acids found in the natural proteins (Gly, Ala, Val, Leu,
Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr,
Pro, Trp and His) as well as polymeric forms thereof. Preferred
natural amino acids include glutamic acid and polymeric forms
thereof such as polyglutamate. The term "non-natural amino acid"
refers to both optical isomers of natural .alpha.-amino acids, such
as D-glutamic acid, as well as modified natural .alpha.-amino acids
such as chemical derivatives or polymeric forms thereof. Examples
of such modifications include, but are not limited to: (i)
functional group transformation caused by introduction of a
functional group (such as alkylation, esterification, halogenation
or amination), oxidation, reduction, addition or dissociation, (ii)
introduction of a sugar compound (monosaccharide, disaccharide,
oligosaccharide or polysaccharide) or a lipid compound, (iii)
phosphorylation, (iv) biotinylation, and the like. Specific
examples include e.g., hydroxyproline, .alpha.-carboxyglutamate,
methionine sulfoxide, methionine methyl sulfonium and
O-phosphoserine; N-alkyl, preferably N-methyl amino acids, e.g.,
N-methyl-valine, N-methyl-isoleucine, N-methyl-leucine,
N-methyl-alanine; compounds in which a methylene residue was added
to the amino acid backbone, e.g., homoserine, homoleucine,
homoisoleucine, homolysine, or in which a methylene residue was
deleted from the amino acid backbone, e.g., norvaline (Nva),
norleucine (NIe); ornithine (2,5-diamino pentanoic acid),
citrulline (2-amino-5-(carbamoylamino)pentanoic acid),
diaminobutyric acid (DAB), 2-methyl-alanine.
[0044] Those skilled in the art recognize that numerous other
unnatural amino acids can be synthesized and utilized in the
context of the present invention.
[0045] One specific embodiment of the compounds of the invention
includes for example compounds wherein
(a) X.sub.1 to X.sub.5 are N, R.sub.1 is NY.sub.1Y.sub.2, R.sub.2
is O, R.sub.4 is Y.sub.3, m is 1, p is 0 or 1 and q is 1 or 3,
or
(b) X.sub.1 to X.sub.5 are N, R.sub.1 is NY.sub.1Y.sub.2, R.sub.2
is NH.sub.2, R.sub.4 is Y.sub.3, m is 1, p is 0 and q is 1.
[0046] Thus, in a further specific embodiment the present invention
is for example directed to compounds of formulae Va, Vb, VIa or
VIb,
##STR00006##
wherein, X.sub.a, X.sub.b are independently of each other C, N, O,
S, R.sub.a, R.sub.b are independently of each other H or
straight-chain or branched C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.5-C.sub.14 aryl or C.sub.5-C.sub.14 heteroaryl,
which independently of each other are unsubstituted or substituted
by at least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --SO.sub.2--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl; Y.sub.1, Y.sub.2 are independently of each other selected
from H, formyl, straight chain or branched C.sub.1-C.sub.12 alkyl,
which is unsubstituted or substituted by at least one CN, Hal, or
NO.sub.2, and wherein one or more of embedded, non-adjacent
CH.sub.2 groups may independently be replaced by --O--, --CO--,
--CO--O--, --CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is
H or C.sub.1-C.sub.6 alkyl, and Y.sub.3 is selected from H, formyl,
trifluoroacetyl, nitroso, straight chain or branched
C.sub.1-C.sub.12 alkyl, which is unsubstituted or substituted by at
least one CN, Hal, or NO.sub.2, and wherein one or more of
embedded, non-adjacent CH.sub.2 groups may independently be
replaced by --O--, --CO--, --CO--O--, --CO--NR'--, --CH.dbd.CH--,
--C.ident.C--, wherein R' is H or C.sub.1-C.sub.6 alkyl.
[0047] Preferably, Y.sub.3 is H, formyl, nitroso, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, or C.sub.1-C.sub.6 alkanoyl.
[0048] In another specific embodiment, the present invention is
directed towards a compound according to formula 1 having formulae
VIIa, VIIb, VIIIa or VIIIb,
##STR00007##
wherein, [0049] X.sub.a, X.sub.b are independently of each other C,
N, O, S, [0050] R.sub.a, R.sub.b are independently of each other H
or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; [0051] Y.sub.1, Y.sub.2 are
independently of each other selected from H, formyl, straight chain
or branched C.sub.1-C.sub.12 alkyl, which is unsubstituted or
substituted by at least one CN, Hal, or NO.sub.2, and wherein one
or more of embedded, non-adjacent CH.sub.2 groups may independently
be replaced by --O--, --CO--, --CO--O--, --CO--NR'--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl, [0052] R.sub.3 is H, formyl, iminomethyl, nitroso,
C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12
alkanoyl, halosubstituted C.sub.1-C.sub.12 alkanoyl, and [0053]
Y.sub.3 is selected from H, formyl, trifluoroacetyl, nitroso,
straight chain or branched C.sub.1-C.sub.12 alkyl, which is
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is H or
C.sub.1-C.sub.6 alkyl, or [0054] R.sub.3 and Y.sub.3 form together
a C.sub.1 or C.sub.2-bridge between the two N-atoms to which they
are attached to.
[0055] Preferably, Y.sub.3 is H, formyl, nitroso, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, or C.sub.1-C.sub.6 alkanoyl.
[0056] In another specific embodiment, the present invention is
directed towards a compound according to formula 1 having formulae
IXa, IXb, Xa or Xb,
##STR00008##
wherein, [0057] X.sub.a, X.sub.b are independently of each other C,
N, O, S, [0058] R.sub.a, R.sub.b are independently of each other H
or straight-chain or branched C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.5-C.sub.14 heteroaryl, which independently of each other are
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --SO.sub.2--, --CH.dbd.CH--, --C.ident.C--, wherein R'
is H or C.sub.1-C.sub.6 alkyl; [0059] Y.sub.1, Y.sub.2 are
independently of each other selected from H, formyl, straight chain
or branched C.sub.1-C.sub.12 alkyl, which is unsubstituted or
substituted by at least one CN, Hal, or NO.sub.2, and wherein one
or more of embedded, non-adjacent CH.sub.2 groups may independently
be replaced by --O--, --CO--, --CO--O--, --CO--NR'--,
--CH.dbd.CH--, --C.ident.C--, wherein R' is H or C.sub.1-C.sub.6
alkyl, [0060] R.sub.3 is H, formyl, iminomethyl, nitroso,
C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12
alkanoyl, halosubstituted C.sub.1-C.sub.12 alkanoyl, and [0061]
Y.sub.3 is selected from H, formyl, trifluoroacetyl, nitroso,
straight chain or branched C.sub.1-C.sub.12 alkyl, which is
unsubstituted or substituted by at least one CN, Hal, or NO.sub.2,
and wherein one or more of embedded, non-adjacent CH.sub.2 groups
may independently be replaced by --O--, --CO--, --CO--O--,
--CO--NR'--, --CH.dbd.CH--, --C.ident.C--, wherein R' is H or
C.sub.1-C.sub.6 alkyl, or [0062] R.sub.3 and Y.sub.3 form together
a C.sub.1 or C.sub.2-bridge between the two N-atoms to which they
are attached to.
[0063] Preferably, Y.sub.3 is H, formyl, nitroso, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, or C.sub.1-C.sub.6 alkanoyl.
[0064] The term "alkyl", when used singly or in combination, refers
preferably to straight chain or branched alkyl groups containing 1
to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl,
butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,
hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.
More preferred alkyl groups contain 1 to 8, more preferably 1 to 4
carbon atoms.
[0065] As used herein, the term "alkenyl", singly or in combination
with other groups, refers to straight chain or branched alkyl
groups containing 2 to 12 carbon atoms, such as methylene,
ethylene, propylene, isopropylene, butylene, t-butylene,
sec-butylene, isobutylene, amylene, isoamylene, pentylene,
isopentylene, hexylene and the like. The preferred alkenyl groups
contain 2 to 6 carbon atoms.
[0066] The term "alkynyl" as used herein refers to a linear or
branched chain of carbon atoms with one or more carbon-carbon
triple bonds. The preferred alkynyl groups contain 2 to 12, more
preferably 2 to 6 carbon atoms.
[0067] The term "alkoxy" as used herein refers to alkyl, as defined
above, substituted with oxygen, such as methoxy, ethoxy, propoxy,
isopropoxy, butoxy, tert-butoxy and the like.
[0068] The term "alkanoyl" as used herein refers to formyl, or
alkyl, as defined above, terminally-substituted with a carbonyl
such as acetyl, propanoyl, butanoyl, pentanoyl and the like.
[0069] The term "alkylamino" as used herein refers to alkyl, as
defined above, substituted with nitrogen, including both
monoalkylamino such as methylamino, ethylamino, propylamino,
tert-butylamino, and the like, and dialkylamino such as
dimethylamino, diethylamino, methylpropylamino, and the like.
[0070] The term "halo" as used herein refers to any Group 17
element and includes fluoro, chloro, bromo, iodo, and
astatine(o).
[0071] The term "(C.sub.3-C.sub.6)cycloalkyl" as used herein alone
or in combination with other groups includes saturated or partially
unsaturated cyclic hydrocarbon groups having 1 ring of a total of 3
to 6 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl.
[0072] The term "(C.sub.5-C.sub.14)aryl" as used herein alone or in
combination with other groups refers to mono- and bicyclic aromatic
groups, such as phenyl, naphthyl, anthracenyl.
[0073] The term "(C.sub.5-C.sub.14)heteroaryl" as used herein alone
or in combination with other groups, means a mono- or bicyclic
radical having at least one aromatic ring containing one, two, or
three ring heteroatoms selected from N, O and S, the remaining ring
atoms being C, such as pyridyl, furyl, imidazolyl, benzimidazolyl,
pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl.
[0074] In a further aspect the present invention also provides a
method of synthesizing a compound of the invention. Clearly, the
introduction of a .sup.18F moiety should occur as late in the
synthesis as possible due to its decaying nature. Thus, applicants
have found that the compounds of the invention may be obtained in
an effective manner by a process which comprises (i) activating the
position to be labelled in the suitably protected glutamic acid
portion, (ii) coupling the activated glutamic acid with pteroic
acid or a derivative thereof and (iii) finally substituting the
activating group with .sup.18F. If desired, steps (i) and (ii) may
be reversed.
[0075] A synthetic pathway using fluorinated glutamate residues as
starting material and subsequent coupling with a pteroyl group may
be used to obtain .sup.19F-labeled compounds as reference
compounds, while an analogous synthesis using a .sup.18F-labeled
glutamic acid (see e.g. WO 2008/052788) and subsequent coupling
with a pteroyl group is disadvantageous with respect to the half
life of .sup.18F isotope due to time consuming reaction steps (e.g.
laborious work up and purification procedures).
[0076] Activation of the glutamic acid portion may be achieved by
introducing any known activating group A that is susceptible to
nucleophilic displacement at the .gamma.-position. These include
but are not limited to tosylate, mesylate, brosylate,
fluorosulfonate, triflate, nonaflate, alkoxy having 1 to 10 carbon
atoms, aryloxy having 6 to 12 carbon atoms, trifluoracetate, nitro,
bromo, chloro, iodo, and the like, preferably mesylate, tosylate,
nosylate and other sulfonates, sulfonium salts and iodonium
salts.
[0077] In one embodiment the displacement reaction of the
activating group by fluorine-18 and subsequent deprotection may be
performed in solution in a one-pot procedure.
[0078] In another embodiment the activated precursor is bound
directly or via a linker to a solid support and reaction with
fluorine-18 will yield the labeled compound in solution.
[0079] A suitable solid support may be any suitable solid-phase
support which is insoluble in any solvents to be used in the
process but to which the linker and/or activated precursor can be
covalently bound. Examples of a suitable solid support include
polymers such as polystyrene (which may be block grafted, for
example with polyethylene glycol), polyacrylamide, or polypropylene
or glass or silicon coated with such a polymer. The solid support
may be in the form of small discrete particles such as beads or
pins, or as a coating on the inner surface of a cartridge or on a
microfabricated vessel. If necessary a linker is used, which may be
any suitable organic group which serves to space the reactive site
sufficiently from the solid support structure so as to maximise
reactivity. Suitably, a linker comprises up to four aryl groups
(suitably phenyl) and/or a C(1-16)alkyl (preferably C(1-6)alkyl) or
C(1-16)haloalkyl (preferably C(1-6)haloalkyl), typically
C(1-16)fluoroalkyl (preferably C(1-6)fluoroalkyl), or C(1-16)alkoxy
or C(1-16)haloalkoxy (preferably C(1-6)alkoxy or C(1-6)haloalkoxy)
typically C(1-16)fluoroalkoxy (preferably C(1-6)fluoroalkoxy), and
optionally one to four additional functional groups such as amide
or sulphonamide groups.
[0080] Treatment of the support-bound activated precursor with
fluorine-18 may be effected by treatment with any suitable source
of .sup.18F, such as Na.sup.18F, K.sup.18F, Cs.sup.18F,
tetraalkylammonium .sup.18F fluoride, tetraalkylphosphonium
.sup.18F fluoride or electrochemically bound .sup.18F fluoride. To
increase the reactivity of the fluoride, a phase transfer catalyst
such as 4,7,13,16,21,24 hexaoxa-1,10-diazabicyclo[8,8,8] hexacosane
may be added and the reaction performed in a non protic solvent or
in a combination of a non protic solvent and sterically hindered
alcohols, such as tert.-butanol, tert.-amyl-alcohol and the like.
The treatment with fluorine-18 is suitably effected in the presence
of a suitable organic solvent such as acetonitrile,
dimethylformamide, dimethylsulphoxide, tetrahydrofuran, dioxan, 1,2
dimethoxyethane, sulpholane, N-methylpyrolidinineone, at a
temperature from 15.degree. C. to 180.degree. C., preferably at
elevated temperature. On completion of the reaction, the
.sup.18F-labeled compound dissolved in the solvent is conveniently
separated from the solid-phase by filtration.
[0081] In a further aspect the present invention provides uses of
folate radiopharmaceuticals of the invention for convenient and
effective administration to a subject in need for diagnostic
imaging.
[0082] Thus the present invention provides a method for diagnostic
imaging of a cell or population of cells expressing a
folate-receptor, said method comprising the steps of administering
at least one folate radiopharmaceutical of the invention in a
diagnostic imaging amount, and obtaining a diagnostic image of said
cell or population of cells.
[0083] Such imaging may be performed on a cell or population of
cells expressing a folate-receptor in vitro or in vivo.
[0084] Thus, the present invention provides a method for in vitro
detection of a cell expressing the folate receptor in a tissue
sample which includes contacting said tissue sample with at least
one folate radiopharmaceutical of the invention in effective
amounts and for sufficient time and conditions to allow binding to
occur and detecting such binding by PET imaging.
[0085] In a further aspect the present invention provides uses of
folate radiopharmaceuticals of the present invention for convenient
and effective administration to a subject in need for diagnostic
imaging or monitoring of therapy of cancer and inflammatory and
autoimmune diseases.
[0086] In another aspect the present invention provides a method
for simultaneous diagnosis and therapy, comprising the steps of
administering to a subject in need thereof at least one folate
radiopharmaceutical of the present invention in a diagnostically
effective amount in combination with a therapeutically active, and
obtaining a diagnostic image of said tissues to follow the course
of treatment.
[0087] The subject of the methods of the present invention is
preferably a mammal, such as an animal or a human, preferably a
human.
[0088] The dosage depends on the nature of the effect desired, such
as the form of diagnosis or therapy, on the kind and frequency of
treatment, on the diagnostic instrumentation, on the form of
application of the preparation, and on the age, weight, nutrition
and condition of the recipient, kind of concurrent treatment, if
any.
[0089] However, the most preferred dosage can be tailored to the
individual subject, as is understood and determinable by one of
skill in the art, without undue experimentation. This typically
involves adjustment of a standard dose, e.g., reduction of the dose
if the patient has a low body weight.
[0090] Treatment can commence with a smaller amount, below the
optimum amount, which can be increased in order to achieve the
optimum effect.
[0091] The imaging procedure in the PET scanner takes place from
within minutes to 2-4 hours after administration of the
radiotracer. The schedule depends on the imaging target and
kinetics of the radiotracer as well as the desired information.
[0092] The preferred route of administration of the folate
radiopharmaceuticals of the present invention is by intraveneous
injection.
[0093] The suitable forms for injection include sterile aqueous
solutions or dispersions of the above mentioned folate
radiopharmaceuticals of the present invention. Typically the
radiopharmaceutical will be formulated in physiological buffer
solutions.
[0094] The folate radiopharmaceuticals can undergo sterilization by
any art recognized technique, including but not limited to,
addition of antibacterial of antifungal agents, for example,
paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. Preferably they undergo a sterile filtration before
administration eliminating the need of additional sterilisation
agents.
[0095] For a solution to be injected a preferred unit dosage is
from about 0.01 mL to about 10 mL. After intravenous
administration, imaging of the organ or tumor in vivo can take
place, if desired, from within minutes to 2-4 hours after the
radiolabeled reagent has been administered to a subject to allow a
sufficient amount of the administered dose to accumulate in the
targeted area of choice.
[0096] The folate radiopharmaceuticals of the invention may also be
used for in vitro detection of a cell expressing the folate
receptor in a tissue biopsy taken from a subject. Thus in a further
embodiment the present invention provides a method for in vitro
detection of a cell expressing the folate receptor, e.g. a tumor
cell, in a tissue sample which includes contacting said tissue
sample with a folate radiopharmaceutical of the present invention
in effective amounts and for sufficient time and conditions to
allow binding to occur and detecting such binding by imaging
techniques.
[0097] Samples can be collected by procedures known to the skilled
person, e.g., by collecting a tissue biopsy or a body fluid, by
aspirating for tracheal or pulmonary samples and the like.
[0098] Tissue samples to be tested include any tissue suspected to
contain a cell expressing a folate receptor, such as tumour cells,
epithelial cells, kidneys, gastrointestinal or the hepatobiliary
system, and others. Samples can be sectioned, e.g., with a
microtome, to facilitate microscopic examination and observation.
Samples can also be fixed with an appropriate fixative either
before or after incubation with one of the folate
radiopharmaceuticals of the present invention to improve the
histological quality of sample tissues.
[0099] Time and conditions sufficient for binding of a folate
radiopharmaceutical of the present invention to a folate receptor
on the cell include standard tissue culture conditions, i.e.
samples can be cultured in vitro and incubated with one of the
complexes or compositions of the present invention in physiological
media. Such conditions are well known to the skilled person.
Alternatively, samples can be fixed and then incubated with a
folate radiopharmaceutical of the present invention in an isotonic
or physiological buffer.
[0100] For all applications it is convenient to prepare the
compounds of the present invention at, or near, the site where they
are to be used.
[0101] All of the compounds and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. It will be apparent to those of
skill in the art that variations may be applied to the present
invention without departing from the scope of the invention. The
Examples provided herein are intended to be illustrative and are
not exhaustive; therefore the illustrated Examples should not be
viewed as limiting the invention in any way.
EXAMPLES
Materials and Methods
[0102] Nuclear magnetic resonance spectra were recorded with a with
a Varian Mercury Plus 200 (200 MHz) spectrometer. Chemical shifts
are reported in parts per million (ppm) relative to
tetramethylsilane (0.00 ppm). The following abbreviations are used
in the experimental section for the description of .sup.1H-NMR
spectra: singlet (s), doublet (d), triplet (t), multiplet (m),
doublet of doublets (dd). The chemical shifts of complex multiplets
are given as the range of their occurrence. HR-ESI-MS were recorded
with a Bruker FTMS 4.7 T BioAPEXII (ESI) spectrometer.
[0103] Water sensitive reactions were run under argon in
flame-dried glass ware. Reactions were monitored by thin layer
chromatography (TLC, performed on EM Science 0.25 mm thick,
precoated silica gel 60 F-254 glass supported plates) or HPLC. HPLC
was performed on a Merck-Hitachi L-7000 system equipped with a
L-7400 tunable absorption detector. Analytical HPLC was performed
with a Nucleosil column (C18, 5 .mu.m, 4.times.250 mm, Macherey
Nagel) using the following solvent system (1 mL/min): Solvent A:
0.05M aq. NaH2PO4, adjusted to pH 7.0 with 32% NaOH, Solvent B: 800
ml MeOH/200 ml 0.05M NaH.sub.2PO.sub.4, 1 mL/min; 0 min, 100% A;
0-30 min, 100-0% A. UV detection at 230 nm. 20 mg of the sample
were dissolved in a buffer consisting of 20 g NaHCO.sub.3 and 20 g
KHCO.sub.3 in 1000 ml of water. All chemicals were used as supplied
unlike stated otherwise.
[0104] Semi-preparative HPLC purification of the
.gamma.-[.sup.18F]fluorofolic acid was carried out on a RP 18
column, Gemini 5.mu. C18, 250.times.10 mm, using a gradient as
follows. Solvent A=0.05M phosphate buffer solution (5% methanol),
B=methanol, 0-35 min: A: 100%.fwdarw.40%, 35-40 min:
A:40%.fwdarw.20%, 50-60 min: A: 20%.fwdarw.100%. Flow: 4
ml/min.
[0105] Production of n.c.a. [.sup.18F]fluoride. N.c.a.
[.sup.18F]fluoride was produced via the .sup.18O(p,n).sup.18F
nuclear reaction at a Cyclone 18/9 cyclotron (IBA, Belgium).
Isotopically 97% enriched [.sup.18O]water was irradiated by a 16
MeV proton beam using a 2.1 ml liquid target. The
[.sup.18F]fluoride/[.sup.18O]water solution was transferred from
the target to a manipulator equipped syntheses hotcell using a
helium stream.
[0106] The [.sup.18F]fluoride which was trapped on an anion
exchange cartridge, was directly eluted into a 10 ml sealed
reaction vessel using a solution of tetrabutylammonium hydroxide in
methanol (0.7 ml). At 85-90.degree. C. the solvents were removed by
vacuum and a stream of nitrogen. Subsequently, 0.8-1.0 ml of dry
acetonitrile was added three times and evaporated to dryness.
[0107] A .gamma.-Fluoro-folic acid reference standard can be
synthesized e.g. according to B. Hart et al, J. Med. Chem., 39,
1996, 56-65. A .beta.-Fluoro-folic acid reference standard can be
synthesized in analogy to the same literature using the analogous
.beta.-fluoro-glutamic acid which can be synthesized according to
e.g. A. Vidal-Cros et al, J. Org. Chem., 54, 498, 1989.
EXPERIMENTAL
Synthesis of .gamma.-[.sup.18F]Fluorofolic Acid (According to FIG.
1)
Example 1
Synthesis of 4-methanesulfonyloxy-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester (step a)
[0108] To 10.0 g of 4-hydroxy-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester (purchased from Bachem) in 200
ml dry dichloromethane were added 4 ml methanesulfonyl chloride.
The mixture was cooled to 0.degree. C. and 17 ml triethylamine were
added. The mixture was allowed to warm to room temperature. After 2
hours 500 ml dichloromethane were added. The mixture was washed
with 500 ml cold 1 M HCl and two times with 500 ml cold water. The
organic layer was evaporated to dryness to give an oil, which was
crystallized at 4.degree. C. by addition of 100 ml
methyl-tert-butyl ether. The crystals were sucked off, washed two
times with 25 ml methyl-tert-butyl ether and dried at 35.degree. C.
under vacuum to give 9.27 g of
4-methanesulfonyloxy-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester. .sup.1H-NMR (CDCl.sub.3, TMS
as internal standard, 200 MHz): .delta.=5.27 (m, 1H, C(.gamma.)-H),
4.44 (m, 1H, C(.alpha.)-H), 3.78 (m, 2H, C(.delta.)-H.sub.2), 3.75
(s, 3H, OCH.sub.3), 3.05 (s, 3H, SO.sub.2CH.sub.3), 2.62 (m, 1H,
C(.beta.)-H), 2.19-2.33 (m, 1H, C(.beta.)-H'), 1.46, 1.42 (two s,
9H, OtBu). .sup.13C-NMR (CDCl.sub.3, TMS as internal standard, 200
MHz): .delta.=172.7, 153.3, 80.9, 77.9, 57.4, 52.5, 52.2, 38.7,
37.5, 28.2. HR-MS: m/z [M+Na]+ calcd. for
C.sub.12H.sub.21NNaO.sub.7S: 346.0931; found: 346.0933.
Example 2
Synthesis of
4-methanesulfonyloxy-5-oxo-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester (step b)
[0109] To 99.8 g of
4-methanesulfonyloxy-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester in 4000 ml ethyl acetate were
added 264 sodium perjodate and 3.2 g ruthenium (III) chloride in
4000 ml water. The mixture was stirred vigorously for 96 hours at
room temperature. After filtration the aqueous layer was separated
and extracted two times with 1400 ml ethyl acetate. The ethyl
acetate layers were combined and 863 ml iso-propanol were added.
After stirring for 30 min., magnesium sulphate was added, filtered
off and the residue was evaporated to dryness. The residue was
purified by flash chromatography using 1 kg silica gel 60 and ethyl
acetate/n-hexane 4.5:5.5. 32.8 g of colourless crystals were
obtained which were treated with 100 ml of methyl-tert-butylester
at r.t. After cooling to 4.degree. C. the crystals were sucked off,
washed with methyl-tert-butylester and dried overnight at
40.degree. C. in vacuum to give 28.9 g of
4-methanesulfonyloxy-5-oxo-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester. .sup.1H-NMR (CDCl.sub.2, TMS
as internal standard, 200 MHz): .delta.=5.28-5.38 (m, 1H,
C(.gamma.)-H), 4.65-4.70 (m, 1H, C(.alpha.)-H), 3.82 (s, 3H,
OCH.sub.3), 3.30 (s, 3H, SO.sub.2CH.sub.3), 2.38-2.70 (m, 2H,
C(.beta.)-H2), 1.52 (s, 9H, OtBu). .sup.13C-NMR (CDCl.sub.3, TMS as
internal standard, 200 MHz): .delta.=170.7, 167.6, 148.8, 85.1,
75.2, 55.3, 53.2, 40.0, 29.3, 27.9. HR-MS: m/z [M+Na]+ calcd. for
C.sub.12H.sub.19NNaO.sub.8S: 360.0724; found: 360.0728.
Example 3
Synthesis of
2-tert-butoxycarbonylamino-4-methanesulfonyloxy-pentanedicarboxylic
acid dimethylester (step c)
[0110] To 28.3 g of
4-methanesulfonyloxy-5-oxo-pyrrolidine-1,2-dicarboxylic
acid-1-tert-butylester-2-methylester in 354 ml dichloromethane were
added 71 ml methanol and 0.58 g potassium carbonate. The mixture
was stirred for three hours at room temperature. After filtration
the filtrate was evaporated in vacuum and the oily residue was
purified by chromatography (90 g silica gel 60, solvent
CH.sub.2Cl.sub.2:MeOH, 99:1 to 95:5) to give 9.8 g of
2-tert-butoxycarbonylamino-4-methanesulfonyloxy-pentanedicarboxy-
lic acid dimethylester as a colourless oil. .sup.1H-NMR
(CDCl.sub.3, TMS as internal standard, 200 MHz): .delta.=5.3 (s,
1H, NH), 5.14-5.20 (m, 1H, C(.gamma.)-H), 4.50 (m, 1H,
C(.alpha.)-H), 3.81 (s, 3H, OCH.sub.3), 3.78 (s, 3H, OCH.sub.3),
3.15 (s, 3H, SO.sub.2CH.sub.3), 2.32-2.64 (m, 2H,
C(.beta.)-H.sub.2), 1.45 (s, 9H, OtBu). .sup.13C-NMR (CDCl.sub.3,
TMS as internal standard, 200 MHz): .delta.=171.5, 169.1, 74.4,
74.1, 52.9, 52.7, 50.0, 39.2, 34.2, 28.5. HR-MS: m/z [M+Na]+ calcd.
for C.sub.13H.sub.23NNaO.sub.9S: 392.0986; found: 392.0981.
Example 4
Deprotection of
2-tert-butoxycarbonylamino-4-methanesulfonyloxy-pentanedicarboxylic
acid dimethylester (step d)
[0111] To a solution of 1 g of
2-tert-butoxycarbonylamino-4-methanesulfonyloxy-pentanedicarboxylic
acid dimethylester in 10 ml dichloromethane were added 10 ml of
trifluoroacetic acid and stirred for 30 min. at room temperature.
The mixture was evaporated to dryness, the residue was dried in
vacuum to give 1.8 g of an oily residue which was directly used for
synthesis of
N.sup.2-N,N-dimethylaminomethylen-10-formyl-.gamma.-methanesulfonyloxy-fo-
lic acid dimethylester in example 5 without further purification.
DC (CH.sub.2Cl.sub.2/MeOH, 9:1, Ninhydrin), Rf=0.29.
Example 5
Synthesis of
N.sup.2-N,N-dimethylaminomethylene-10-formyl-.gamma.-methanesulfonyloxy-f-
olic acid dimethylester (step e)
[0112] To 1.07 g of
N.sup.2,N,N-dimethylaminomethylene-10-formyl-pteroic acid in 100 ml
N,N-dimethylformamide were added 1.03 g of
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate
(=HBTU) and 0.93 ml of N,N-diisopropylethylamine. After 5 min. a
solution of 1.8 g of crude
2-amino-4-methanesulfonyloxy-pentanedicarboxylic acid dimethylester
trifluoroacetate (crude product from example 4) in 10 ml absolute
N,N-dimethylformamide were added. The mixture was stirred for 18
hours at room temperature and evaporated to dryness in vacuum. The
residue was dissolved in 50 ml dichloromethane and washed three
times with 10 ml of aqueous 5% sodium bicarbonate solution, three
times with 10 ml of 5% citric acid and three times with 10 ml of
water. After drying the dichloromethane layer over magnesium
sulphate it was evaporated to dryness to give 1.2 g of a light
yellow foam which was purified by chromatography (120 g silica gel
60, eluent dichloromethane/methanol 95:5) to give 0.3 g of a yellow
residue which was further purified by chromatography (30 g silica
gel 60, eluent dichloromethane/methanol 95:5) to give 0.16 g of
N.sup.2-N,N-dimethylaminomethylen-10-formyl-.gamma.-methanesulfonyloxy-fo-
lic acid dimethylester (Ms=SO.sub.2CHT3). HPLC: 94.1% area (UV
detection at 230 nm); DC (dichloromethane/methanol 85:15) Rf=0.49.
.sup.1H-NMR (DMSO-d.sub.6, TMS as internal standard, 200 MHz):
.delta.=11.99 (bs, 1H, N.sup.3--H), 8.82, 8.78 8.71 (three s, 3H,
CHNMe.sub.2, CHO, C(7)-H), 7.86 (d, 2H, C(1', 5'), Pte), 7.60 (d,
2H, C(2',4'), Pte), 5.24-5.33 (m, s, 3H, C(6)-H.sub.2,
C(.gamma.)-H), 4.53-4.67 (m, 1H, C(.alpha.)-H), 3.64 (s, 3H, OMe),
3.60 (s, 3H, OMe), 3.24 (s, 3H, NCH.sub.3), 3.21 (s, 3H,
NCH.sub.3), 3.08 (s, 3H, SO.sub.2CH.sub.3), 2.58-2.29 (m, 2H,
C(.beta.)-H.sub.2). HR-MS: m/z [M+Na]+ calcd. for
C.sub.26H.sub.30N.sub.8NaO.sub.10S: 669.1698; found: 669.1706.
Example 6
Synthesis of .gamma.-[.sup.18F]fluorofolic acid by
.sup.18F-fluorination of N.sup.2,
N,N-dimethylaminomethylene-10-formyl-.gamma.-methanesulfonyloxy-folic
acid dimethylester and subsequent deprotection (step f and g)
[0113] To the dry tetrabutylammonium [.sup.18F]fluoride the
precursor
N.sup.2,N,N-dimethylaminomethylene-10-formyl-.gamma.-methanesulfonyloxy-f-
olic acid dimethylester (5.2 mg) in 0.25 ml acetonitrile were
added. The mixture was heated to 80-85.degree. C. for 35 min. After
cooling, 9 ml water were added and the mixture was passed though a
reversed phase cartridge (Sep-Pak.RTM. .sup.tC18 plus, Waters AG).
The cartridge was washed three times with 10 ml of water and dried
2 min by a stream of nitrogen. The .sup.18F-labeled protected
compound was eluted with 2.5 ml of acetonitrile into another 10 ml
sealed reaction vessel. The volume of acetonitrile was reduced to
0.1 ml under reduced pressure, nitrogen stream and slight warming
of 80-90.degree. C.
[0114] For hydrolysis (step g), 0.5 ml of 1M NaOH solution was
added and the mixture was heated to 50.degree. C. for 20 min. After
cooling, the mixture was neutralized by 0.5 ml 1M HCl solution. 2.0
ml of HPLC solvent A was added to give the final injection volume
for the semi-preparative HPLC purification. The HPLC solvent of the
product fraction was evaporated under reduced pressure and a stream
of nitrogen at 90.degree. C. For formulation, water for injection
and 0.15 M phosphate buffer solution were added to the dry product
and the mixture was passed through a sterile filter into a sterile
and pyrogen-free vial.
Example 7
In Vivo and Ex Vivo Studies Using .gamma.-[.sup.18F]Fluoro-Folic
Acid
[0115] .gamma.-[.sup.18F]fluoro-folic acid was applied in ex vivo
biodistribution studies using male NMRI nu/nu mice bearing KB tumor
xenografts. .about.2 MBq of the radiotracer were injected into each
animal. In a blockade group, 200 .mu.g natural folic acid was
injected 10 min prior to the radiotracer. The animals were
sacrificed 105 min post injection. The folate receptor-positive KB
tumors show a high specific uptake of the radiotracer with a ratio
of 90.0% specific blockade. Furthermore, a high specific uptake of
86.0% specific blockade was also found in the kidneys, which are
known to express the folate receptor.
[0116] FIG. 2 shows the high specific uptake of the
.gamma.-[.sup.18F]fluoro-folic acid in folate receptor-positive
tissues, such as tumor and kidney (two bars are shown for each
tissue, wherein the left bar represents the control group uptake
and the right bar represents the blockade group uptake).
[0117] In vivo PET imaging using the .gamma.-[.sup.18F]fluoro-folic
acid was performed in male NMRI nu/nu mice bearing KB tumor
xenografts. Ca. 10 MBq of the radiotracer were injected into each
animal. In the blockade group, 200 .mu.g natural folic acid was
injected 10 min prior to the radiotracer. The PET scans were
acquired from 75 min to 105 min post injection.
[0118] PET studies using .gamma.-[.sup.18F]fluoro-folic acid
provided excellent images of KB tumor xenografts. Furthermore, the
uptake is highly specific and blocked by natural folic acid. A high
specific uptake of the radiotracer was also found in the kidney
cortex, while no uptake was found in the kidney medulla. This
pattern is consistent with the physiological distribution of the
folate receptor and points out the high specificity of
.gamma.-[.sup.18F]fluoro-folic acid. Besides the specific tumor and
kidney uptake, the .gamma.-[.sup.18F]fluoro-folic acid also shows
radioactivity accumulation in liver cells.
[0119] FIG. 3 show representative series of normalized horizontal
slices of whole body PET scans using .gamma.-[.sup.18F]fluoro-folic
acid under control and blockade conditions. Arrows with circles
indicate the tumor site and arrows with squares indicate the
kidneys.
* * * * *