NOVEL PRECURSOR
Field of the Invention
The present invention describes a novel radiotracer(s) for Positron Emission
Tomography (PET) or Single Photon Emission Computed Tomography (SPECT)
imaging of disease states related to altered choline metabolism (e.g., tumor imaging of
prostate, breast, brain, esophageal, ovarian, endometrial, lung and prostate cancer -
primary tumor, nodal disease or metastases). The present invention also describes
intermediate(s), precursor(s), pharmaceutical composition(s), methods of making, and
methods of use of the novel radiotracer(s).
Description of Related Art
The biosynthetic product of choline kinase (EC 2.7.1.32) activity, phosphocholine, is
elevated in several cancers and is a precursor for membrane phosphatidylcholine
(Aboagye, E.O., et al., Cancer Res 1999; 59:80-4; Exton, J.H., Biochim Biophys Acta
1994; 1212:26-42; George, T.P., et al., Biochim Biophys Acta 1989; 104:283-91; and
Teegarden, D., et al, J Biol Chem 1990; 265(1 1):6042-7). Over-expression of
choline kinase and increased enzyme activity have been reported in prostate, breast,
lung, ovarian and colon cancers (Aoyama, C , et al, Prog Lipid Res 2004; 43(3):266-
81; Glunde, K., et al. , Cancer Res 2004; 64(12):4270-6; Glunde, K., et al. , Cancer
Res 2005; 65(23): 11034-43; Iorio, E., et al, Cancer Res 2005; 65(20): 9369-76;
Ramirez de Molina, A., et al., Biochem Biophys Res Commun 2002; 296(3): 580-3;
and Ramirez de Molina, A., et al, Lancet Oncol 2007; 8(10): 889-97) and are largely
responsible for the increased phosphocholine levels with malignant transformation
and progression; the increased phosphocholine levels in cancer cells are also due to
increased breakdown via phospholipase C (Glunde, K., et al, Cancer Res 2004;
64(12):4270-6).
Because of this phenotype, together with reduced urinary excretion, [ C]choline has
become a prominent radiotracer for positron emission tomography (PET) and PETComputed
Tomography (PET-CT) imaging of prostate cancer, and to a lesser extent
imaging of brain, esophageal, and lung cancer (Hara, T., et al, J Nucl Med 2000;
41:1507-13; Hara, T., etal, J Nucl Med 1998; 39:990-5; Hara, T., etal, J Nucl Med
1997; 38:842-7; Kobori, O., et al, Cancer Cell 1999; 86:1638-48; Pieterman, R.M.,
et al. , Nucl Med 2002; 43(2): 167-72; and Reske, S.N. Eur J Nucl Med Mol Imaging
2008; 35:1741). The specific PET signal is due to transport and phosphorylation of
the radiotracer to [ C]phosphocholine by choline kinase.
Of interest, however, is that [ Cjcholine (as well as the fluoro-analog) is oxidized to
[ C]betaine by choline oxidase (see Figure 1 below)(EC 1.1.3.17) mainly in kidney
and liver tissues, with metabolites detectable in plasma soon after injection of the
radiotracer (Roivainen, A., et al. , European Journal of Nuclear Medicine 2000;
27:25-32). This makes discrimination of the relative contributions of parent
radiotracer and catabolites difficult when a late imaging protocol is used.
Lipid
,OH Phosphorylation ,oro ,2- Incorporation
H3
1 1 H3
1 1
O-Choline ne
Excretion
betaine
Figure 1. Chemical structures of major choline metabolites and their pathways.
[ F]Fluoromethylcholine ([ F]FCH):
FCH
was developed to overcome the short physical half-life of carbon- 11 (20.4 min)
(DeGrado, T.R., et al. , Cancer Res 2001 ; 61(1): 110-7) and a number of PET and
PET-CT studies with this relatively new radiotracer have been published (Beheshti,
M., et al., Eur J Nucl Med Mol Imaging 2008;35(10):1766-74; Cimitan, M., et al,
Eur J Nucl Med Mol Imaging 2006; 33(12): 1387-98; de Jong, I.J., et al, Eur J Nucl
Med Mol Imaging 2002; 29:1283-8; and Price, D.T., et al., J Urol 2002; 168(1):273-
80). The longer half-life of fluorine-18 (109.8 min) was deemed potentially
advantageous in permitting late imaging of tumors when sufficient clearance of parent
tracer in systemic circulation had occurred (DeGrado, T.R., et al., J Nucl Med 2002;
43(l):92-6).
WO2001/82864 describes 18F-labeled choline analogs, including
[18F]Fluoromethylcholine ([18FJ-FCH) and their use as imaging agents {e.g., PET)
for the non-invasive detection and localization of neoplasms and pathophysiologies
influencing choline processing in the body (Abstract). WO200 1/82864 also describes
18F-labeled di-deuterated choline analogs such as [18F]fluoromethyl-[l- 2H2]choline
([ F]FDC)(hereinafter referred to as "[ F]D2-FCH"):
FDC
The oxidation of choline under various conditions; including the relative oxidative
stability of choline and [l,2- H 4]choline has been studied (Fan, F., et al, Biochemistry
2007, 46, 6402-6408; Fan, F., et a , Journal of the American Chemical Society 2005,
127, 2067-2074; Fan, F., et al., Journal of the American Chemical Society 2005, 127,
17954-17961; Gadda, G. Biochimica et Biophysica Acta 2003, 1646, 112-118; Gadda,
G., Biochimica et Biophysica Acta 2003, 1650, 4-9). Theoretically the effect of the
extra deuterium substitution was found to be neglible in the context of a primary
isotope effect of 8-10 since the b-secondary isotope effect is -1.05 (Fan, F., et al.,
Journal of the American Chemical Society 2005, 127, 17954-17961).
[ F]Fluoromethylcholine is now used extensively in the clinic to image tumour status
(Beheshti, M., et al, Radiology 2008, 249, 389-90; Beheshti, M., et al, Eur J Nucl
Med Mol Imaging 2008, 35, 1766-74).
The present invention, as described below, provides a novel precursor compound.
These novel precursor compounds can be used in the synthesis of, for example, Fradiolabeled
radiotracers which in turn that can be used for PET imaging of choline
metabolism.
Brief Description of the Drawings
Figure 1 depicts the chemical structures of major choline metabolites and their
pathways.
Figure 3 shows NMR analysis of tetradeuterated choline precursor. Top, H NMR
spectrum; bottom, C NMR spectrum. Both spectra were acquired in CDCI 3.
Figure 4 depicts the HPLC profiles for the synthesis of [ F]fluoromethyl tosylate (9)
and [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) showing (A) radio-HPLC profile
for synthesis of (9) after 15 mins; (B) UV (254 nm) profile for synthesis of (9) after
15 mins; (C) radio-HPLC profile for synthesis of (9) after 10 mins; (D) radio-HPLC
profile for crude (9); (E) radio-HPLC profile of formulated (9) for injection; (F)
refractive index profile post formulation (cation detection mode).
Figure 5a is a picture of a fully assembled cassette of the present invention for the
production of [18F]fluoromethyl-[l,2- 2H4]choline (D4-FCH) via an unprotected
precursor.
Figure 5b is a picture of a fully assembled cassette of the present invention for the
production of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) via a PMB-protected
precursor.
Figure 6 depicts representative radio-HPLC analysis of potassium permanganate
oxidation study. Top row are control samples for [18FJfluoromethylcholine
([ F]FCH) and [ F]fluoromethyl-[l,2- H4]choline ([ F]D4-FCH), extracts from the
reaction mixture at time zero (0 min). Bottom row are extracts after treatment for 20
mins. Left hand side are for [ F]fluoromethylcholine ([ F]FCH), right are for [ F]
fluoromethyl- [1,2- H4]choline ([ F]D4-FCH).
Figure 7 shows chemical oxidation potential of [18FJfluoromethylcholine and
[18F]fluoromethyl-[l,2- 2H4]choline in the presence of potassium permanganate.
Figure 8 shows time-course stability assay of [ FJfluoromethylcholine and
[18F]fluoromethyl-[l,2- 2H4]choline in the presence of choline oxidase demonstrating
conversion of parent compounds to their respective betaine analogues.
Figure 9 shows representative radio-HPLC analysis of choline oxidase study. Top
row are control samples for [ F]fluoromethylcholine and [ F]fluoromethyl-[l,2-
H4]choline, extracts from the reaction mixture at time zero (0 min). Bottom row are
extracts after treatment for 40 mins. Left hand side are of [18FJfluoromethylcholine,
right are of [ F]fluoromethyl- [1,2- H4]choline.
Figure 10. Top: Analysis of the metabolism of [ F]fluoromethylcholine (FCH) to
[ F]FCH-betaine and [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) to [ F]D4-FCHbetaine
by radio-HPLC in mouse plasma samples obtained 15 min after injecting the
tracers i.v. into mice. Bottom: summary of the conversion of parent tracers,
[ F]fluoromethylcholine (FCH) and [ F]fluoromethyl-[l,2- H4]choline (D4-FCH), to
metabolites, [ F]FCH-betaine (FCHB) and [ F]D4-FCH betaine (D4-FCHB), in
plasma.
Figure 11. Biodistribution time course of [ F]fluoromethylcholine (FCH),
[ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2- H4]choline
(D4-FCH) in HCT-116 tumor bearing mice. Inset: the time points selected for
evaluation. A) Biodistribution of [ F]fluoromethylcholine; B) biodistribution of
[ F]fluoromethyl-[l- H 2]choline; C) biodistribution of [ F]fluoromethyl-[l,2-
H4]choline; D) time course of tumor uptake for [ F]fluoromethylcholine (FCH),
[ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2- H4]choline
(D4-FCH) from charts A-C. Approximately 3.7 MBq of [18FJfluoromethylcholine
(FCH), [ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2-
H4]choline (D4-FCH) injected into awake male C3H-Hej mice which were sacrificed
under isofluorane anesthesia at the indicated time points.
Figure 12 shows radio-HPLC chromatograms to show distribution of choline
radiotracer metabolites in tissue harvested from normal white mice at 30 min p.i. Top
row, radiotracer standards; middle row, kidney extracts; bottom row, liver extracts.
On the left is [ F]FCH, on the right [ F]D4-FCH.
Figure 13 show radio-HPLC chromatograms to show metabolite distribution of
choline radiotracers in HCT1 16 tumors 30 min post-injection. Top-row, neat
radiotracer standards; bottom row, 30 min tumor extracts. Left side, [18F]FCH;
middle, [ F]D4-FCH; right, [ C]choline.
Figure 14 shows radio-HPLC chromatograms for phosphocholine HPLC validation
using HCT1 16 cells. Left, neat [18F]FCH standard; middle, phosphatase enzyme
incubation; right, control incubation.
Figure 15 shows distribution of radiometabolites for [ F]fluoromethylcholine
analogs: 18FJfluoromethylcholine, [18F]fluoromethyl-[l- 2H2]choline and
[ F]fluoromethyl-[l,2- H 4]choline at selected time points.
Figure 16 shows tissue profile of [ F]FCH and [ F]D4-FCH. (a) Time versus
radioactivity curve for the uptake of [18F]FCH in liver, kidney, urine (bladder) and
muscle derived from PET data, and (b) corresponding data for [18FJD4-FCH. Results
are the mean + SE; n = 4 mice. For clarity upper and lower error bars (SE) have been
used. (Leyton, et al, Cancer Res 2009: 69:(19), pp 7721-7727).
Figure 17 shows tumor profile of [ F]FCH and [ F]D4-FCH in SKMEL28 tumor
xenograft (a) Typical [ F]FCH-PET and [ F]D4-FCH-PET images of SKMEL28
tumor-bearing mice showing 0.5 mm transverse sections through the tumor and
coronal sections through the bladder. For visualization, 30 to 60 min summed image
data are displayed. Arrows point to the tumors (T), liver (L) and bladder (B). (b).
Comparison of time versus radioactivity curves for [18F]FCH and [18FJD4-FCH in
tumors. For each tumor, radioactivity at each of 19 time frames was determined. Data
are mean %ID/vox6omean + SE (n = 4 mice per group) (c) Summary of imaging
variables. Data are mean + SE, n = 4; *P = 0.04. For clarity upper and lower error bars
(SE) have been used.
Figure 18 shows the effect of PD0325901, a mitogenic extracellular kinase inhibitor,
18 on uptake of [ FJD4-FCH in HCTl 16 tumors and cells (a) Normalized time versus
radioactivity curves in HCTl 16 tumors following daily treatment for 10 days with
vehicle or 25mg/kg PD0325901. Data are the mean + SE; n = 3 mice (b) Summary of
imaging variables %ID/vox 6o, %ID/vox60max, and AUC. Data are mean + SE; * P =
0.05.(c) Intrinsic cellular effect of PD0325901 (ImM ) on [ F]D4-FCH
phosphocholine metabolism after treating HCTl 16 cells for 1 hr with [ F]D4-FCH in
culture. Data are mean + SE; n=3 ; * P = 0.03.
Figure 19 shows expression of choline kinase A in HCTl 16 tumors (a) A typical
Western blot demonstrating the effect of PD0325901 on tumor choline kinase A
(CHKA) protein expression. HCTl 16 tumors from mice that were injected with
PD0325901 (25mg/kg daily for 10 days, orally) or vehicle were analyzed for CHKA
expression by western blotting b-actin was used as the loading control (b) Summary
densitometer measurements for CHKA expression expressed as a ratio to b-actin. The
results are the mean ratios + SE; n = 3, * P = 0.05.
Summary of the invention
The present invention further provides a precursor compound of Formula (II):
(P)
wherein:
Ri, R2, , and R4 are each independently hydrogen or deuterium (D);
Rs, R6, and R 7 are each independently hydrogen, Rs, -(CH2)mR , -(CD2)mR ,
(CF2)mR , -CH(R )2, or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
The present invention further provides a method of making a precursor
compound of Formula (II).
The present invention further provides a pharmaceutical composition
comprising a precursor compound of Formula (II) and a pharmaceutically acceptable
carrier or excipient.
Detailed Description of the Invention
The present invention provides a novel radiolabeled choline analog compound
of formula (I):
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF2)mR , -CH(R )2, or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope; and
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine,
fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline,
fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutylcholine,
fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyldiethanol-
choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, 1,1-
dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-
dideuterofluoromethyl-propyl-choline, or an [ F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
Ri, R2, R , and R4 are each independently hydrogen;
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF2)mR , -CH(R )2, or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine,
fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline,
fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutylcholine,
fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyldiethanol-
choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, or
an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
R and R2 are each hydrogen;
R and R 4 are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not 1,1-
dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-
dideuterofluoromethyl-propyl-choline, or an [ F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion.
According to the present invention, when Z of a compound of Formula (I) as
described herein is a halogen, it can be a halogen selected from F, CI, Br, and I ;
preferably, F.
According to the present invention, when Z of a compound of Formula (I) as
described herein is a radioisotope (hereinafter referred to as a "radiolabeled
compound of Formula (I)"), it can be any radioisotope known in the art. Preferably, Z
is a radioisotope suitable for imaging (e.g., PET, SPECT). More preferably Z is a
radioisotope suitable for PET imaging. Even more preferably, Z is F, 6Br, I, 4I,
or 5l . Even more preferably, Z is F .
According to the present invention, Q of a compound of Formula (I) as
described herein can be any anionic counterion known in the art suitable for cationic
ammonium compounds. Suitable examples of Q include anionic: bromide (Br ),
chloride (CI ), acetate (CH CH2C(0)0 ), or tosylate (OTos). In a preferred
embodiment of the invention, Q is bromide (Br ) or tosylate (OTos). In a preferred
embodiment of the invention, Q is chloride (CI ) or acetate (CH CH2C(0)0 ) . In a
preferred embodiment of the invention, Q is chloride (CI ) .
According the invention, a preferred embodiment of a compound of Formula
(I) is the following comp
,Q Q'
(la)
wherein:
Ri, R2, R3, and R4 are each independently deuterium (D);
R5, R6, and R 7 are each hydrogen;
X and Y are each independently hydrog
Z is F;
Q is c .
According to the invention, a preferred compound of Formula (la) is
[ F]fluoromethyl-[l,2- H4]-choline ([ F]-D4-FCH). [ F]-D4-FCH is a more
metabolically stable fluorocholine (FCH) analog. [ F]-D4-FCH offers numerous
advantages over the corresponding 18F-non-deuterated and/or 18F-di-deuterated
18 analog. For example, [ FJ-D4-FCH exhibits increased chemical and enzymatic
oxidative stability relative to [ F]fluoromethylcholine. [ F]-D4-FCH has an
improved in vivo profile (i. e. , exhibits better availability for in vivo imaging) relative
to dideuterofluorocholine, [18F]fluoromethyl-[l- 2H2]choline, that is over and above
18 what could be predicted by literature precedence and is, thus, unexpected. [ F]-D4-
FCH exhibits improved stability and consequently will better enable late imaging of
tumors after sufficient clearance of the radiotracer from systemic circulation. [ F]-
D4-FCH also enhances the sensitivity of tumor imaging through increased availability
of substrate. These advantages are discussed in further detail below.
The present invention provides a compound of formula (III):
(III)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen
selected from F, CI, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl
group; and
Q is an anionic counterion; with the proviso the compound of Formula (III) is
not C-choline.
According to the invention, C* of the compound of formula (III) can be any
radioisotope of carbon. Suitable examples of C* include, but are not limited to, C,
C, and 4C. Q is a described for the compound of Formula (I).
In a preferred embodiment of the invention, a compound of Formula (III) is
provided wherein C* is C; X and Y are each hydrogen; and Z is F.
Pharmaceutical or Radiopharmaceutical Composition
The present invention provides a pharmaceutical or radiopharmaceutical
composition comprising a compound for Formula (I), including a compound of
Formula (la), each as defined herein together with a pharmaceutically acceptable
carrier, excipient, or biocompatible carrier. According to the invention when Z of a
compound of Formula (I) or (la) is a radioisotope, the pharmaceutical composition is
a radiopharmaceutical composition.
The present invention further provides a pharmaceutical or
radiopharmaceutical composition comprising a compound for Formula (I), including a
compound of Formula (la), each as defined herein together with a pharmaceutically
acceptable carrier, excipient, or biocompatible carrier suitable for mammalian
administration.
The present invention provides a pharmaceutical or radiopharmaceutical
composition comprising a compound for Formula (III), as defined herein together
with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier.
The present invention further provides a pharmaceutical or
radiopharmaceutical composition comprising a compound for Formula (III), as
defined herein together with a pharmaceutically acceptable carrier, excipient, or
biocompatible carrier suitable for mammalian administration.
As would be understood by one of skill in the art, the pharmaceutically
acceptable carrier or excipient can be any pharmaceutically acceptable carrier or
excipient known in the art.
The "biocompatible carrier" can be any fluid, especially a liquid, in which a
compound of Formula (I), (la), or (III) can be suspended or dissolved, such that the
pharmaceutical composition is physiologically tolerable, e.g., can be administered to
the mammalian body without toxicity or undue discomfort. The biocompatible carrier
is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection;
an aqueous solution such as saline (which may advantageously be balanced so that the
final product for injection is either isotonic or not hypotonic); an aqueous solution of
one or more tonicity-adjusting substances (e.g., salts of plasma cations with
biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g.,
sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol materials (e.g.,
polyethyleneglycols, propylene glycols and the like). The biocompatible carrier may
also comprise biocompatible organic solvents such as ethanol. Such organic solvents
are useful to solubilise more lipophilic compounds or formulations. Preferably the
biocompatible carrier is pyrogen-free water for injection, isotonic saline or an aqueous
ethanol solution. The pH of the biocompatible carrier for intravenous injection is
suitably in the range 4.0 to 10.5.
The pharmaceutical or radiopharmaceutical composition may be administered
parenterally, i. e., by injection, and is most preferably an aqueous solution. Such a
composition may optionally contain further ingredients such as buffers;
pharmaceutically acceptable solubilisers (e.g., cyclodextrins or surfactants such as
Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or
antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid). Where
a compound of Formula (I), (la), or (III) is provided as a radiopharmaceutical
composition, the method for preparation of said compound may further comprise the
steps required to obtain a radiopharmaceutical composition, e.g., removal of organic
solvent, addition of a biocompatible buffer and any optional further ingredients. For
parenteral administration, steps to ensure that the radiopharmaceutical composition is
sterile and apyrogenic also need to be taken. Such steps are well-known to those of
skill in the art.
Preparation of a Compound of the Invention
The present invention provides a method to prepare a compound for Formula
(I), including a compound of Formula (la), wherein said method comprises reaction of
the precursor compound of Formula (II) with a compound of Formula (Ilia) to form a
compound of Formula (I) (Scheme A):
Scheme A
wherein the compounds of Formulae (I) and (II) are each as described herein and the
compound of Formula (Ilia) is as follows:
ZXYC-Lg ( I lia)
wherein X, Y and Z are each as defined herein for a compound of Formula (I) and
"Lg" is a leaving group. Suitable examples of "Lg" include, but are not limited to,
bromine (Br) and tosylate (OTos). A compound of Formula (Ilia) can be prepared by
any means known in the art including those described herein.
Synthesis of a compound of Formula (Ilia) wherein Z is F; X and Y are both H
and the Lg is OTos (i.e., fluoromethyltosylate) can be achieved as set forth in Scheme
3 below:
CH2I2 * CH2OTos2 * FCH2OTos
SCHEME 3
wherein: i : Silver p-toluenesulfonate, MeCN, reflux, 20 h;
ii: KF, MeCN, reflux, 1 h.
According to Scheme 3 above:
(a) Synthesis of methylene ditosylate
Commercially available diiodomethane can be reacted with silver tosylate,
using the method of Emmons and Ferris, to give methylene ditosylate (Emmons,
W.D., et a , "Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of
Alkyl Sulfonates", Journal of the American Chemical Society, 1953; 75:225).
(b) Synthesis of cold Fluoromethyltosylate
Fluoromethyltosylate can be prepared by nucleophilic substitution of
Methylene ditosylate from step (a) using potassium fluoride/Kryptofix K222 in
acetonitrile at 80°C under standard conditions.
When Z is a radioisotope, the radioisotope can be introduced by any means
known by one of skill in the art. For example, the radioisotope [18F]-fluoride ion
(18F ) is normally obtained as an aqueous solution from the nuclear reaction
0(p,n) F and is made reactive by the addition of a cationic counterion and the
subsequent removal of water. Suitable cationic counterions should possess sufficient
solubility within the anhydrous reaction solvent to maintain the solubility of 18F .
Therefore, counterions that have been used include large but soft metal ions such as
rubidium or caesium, potassium complexed with a cryptand such as Kryptofix™, or
tetraalkylammonium salts. A preferred counterion is potassium complexed with a
cryptand such as Kryptofix™ because of its good solubility in anhydrous solvents and
enhanced 18F- reactivity. 18F can also be introduced by nucleophilic displacement of a
suitable leaving group such as a halogen or tosylate group. A more detailed
discussion of well-known F labelling techniques can be found in Chapter 6 of the
"Handbook of Radiopharmaceuticals" (2003; John Wiley and Sons: M.J. Welch and
C.S. Redvanly, Eds.). For example, [18F]Fluoromethyltosylate can be prepared by
nucleophilic substitution of Methylene ditosylate with [18F]-fluoride ion in acetonitrile
containing 2-10%water (see Neal, T.R., et a , Journal of Labelled Compounds and
Radiopharmaceuticals 2005; 48:557-68).
Automated Synthesis
In a preferred embodiment, the method to prepare a compound for Formula (I),
including a compound of Formula (la), is automated. For example, [ F]-radiotracers
may be conveniently prepared in an automated fashion by means of an automated
radiosynthesis apparatus. There are several commercially-available examples of such
platform apparatus, including TRACERlab™ {e.g., TRACERlab™ MX) and
FASTlab™ (both from GE Healthcare Ltd.). Such apparatus commonly comprises a
"cassette", often disposable, in which the radiochemistry is performed, which is fitted
to the apparatus in order to perform a radiosynthesis. The cassette normally includes
fluid pathways, a reaction vessel, and ports for receiving reagent vials as well as any
solid-phase extraction cartridges used in post-radiosynthetic clean up steps.
Optionally, in a further embodiment of the invention, the automated radiosynthesis
apparatus can be linked to a high performance liquid chromatograph (HPLC).
The present invention therefore provides a cassette for the automated synthesis
of a compound of Formula (I), including a compound of Formula (la), each as defined
herein comprising:
i) a vessel containing the precursor compound of Formula (II) as defined
herein; and
ii) means for eluting the contents of the vessel of step (i) with a compound of
Formula (Ilia) as defined herein.
For the cassette of the invention, the suitable and preferred embodiments of the
precursor compound of Formulae (II) and (Ilia) are each as defined herein.
In one embodiment of the invention, a method of making a compound of
Formula (I), including a compound of Formula (la), each as described herein, that is
compatible with FASTlab™ from a protected ethanolamine precursor that requires no
HPLC purification step is provided.
The radiosynthesis of [ F]fluoromethyl-[l,2- H4]choline ( F-D4-FCH) can
be performed according to the methods and examples described herein. The
radiosynthesis of 18F-D4-FCH can also be performed using commercially available
synthesis platforms including, but not limited to, GE FASTlab™ (commercially
available from GE Healthcare Inc.).
An example of a FASTlab™ radiosynthetic process for the preparation of
[ F]fluoromethyl-[l,2- H4]choline from a protected precursor is shown in Scheme 5 :
F-, FCH2OTs / Ts-[ F]F
[ F]FCH 2OTs /Ts-[ F]F
Scheme 5
wherein:
a. Preparation of [ F]KF/K222/K2CC>3 complex as described in more detail
below;
b. Preparation of [18F]FCH 20Ts as described in more detail below;
c . SPE purification of [18F FC OTs as described in more detail below;
d. Radiosynthesis of O-PMB -[ F]-D4-Choline (0-PMB-[ F]-D4-FCH) as
described in more detail below; and
e. Purification & formulation of [ F]-D4-Choline ( F-D4-FCH) as the
hydrochloric salt as described in more detail below.
The automation of [18F]fluoro-[l,2- 2H4]choline or [18FJfluorocholine (from the
protected precursor) involves an identical automated process (and are prepared from
the fluoromethylation of 0-PMB-N,N-dimethyl-[l,2- ¾]ethanolamine and O-PMB-
N,N-dimethylethanolamine respectively) .
According to one embodiment of the present invention, FASTlab™ syntheses
of [18F]fluoromethyl-[l,2- 2H4]choline or [18FJfluoromethylcholine comprises the
following sequential steps :
(i) Trapping of [ F]fluoride onto QMA;
(ii) Elution of [ F]fluoride from a QMA;
(iii) Radiosynthesis of [ F]FCH 2OTs;
(iv) SPE clean up of [ F]FCH 2OTs;
(v) Reaction vessel clean up;
(vi) Drying reaction vessel and [18FJfluoromethyl tosylate retained on SPE t-C18
plus simultaneously;
(vii) Alkylation reaction;
(viii) Removal of unreacted O-PMB-precursor; and
(ix) Deprotection & formulation.
Each of steps (i)-(ix) are described in more detail below.
In one embodiment of the present invention, steps (i)-(ix) above are performed
on a cassette as described herein. One embodiment of the present invention is a
cassette capable of performing steps (i)-(ix) for use in an automated synthesis
platform. One embodiment of the present invention is a cassette for the
radiosynthesis of [ F]fluoromethyl-[l,2- H4]choline ([ F]-D4-FCH) or
[18F]fluoromethylcholine from a protected precursor. An example of a cassette of the
present invention is shown in Figure 5b.
(i) Trapping of [ F]fluoride onto QMA
[18F]fluoride (typically in 0.5 to 5mL H2
18O) is passed through a pre
conditioned Waters QMA cartridge.
(ii) Elution of [ 8F]fluoride from a QMA
The eluent, as described in Table 1 is withdrawn into a syringe from the eluent
vial and passed over the Waters QMA into the reaction vessel. This procedure elutes
[ F]fluoride into the reaction vessel. Water and acetonitrile are removed using a
well-designed drying cycle of "nitrogen/vacuum/heating/cooling".
(iii) Radiosynthesis of [ 8F]FCH2OTs
Once the K[ F]Fluoride/K222/K 2C0 complex of (ii) is dry, CH2(OTs) 2
methylene ditosylate in a solution containing acetonitrile and water is added to the
reaction vessel containing the K[ F]fluoride/K222/K 2CC>3 complex. The resulting
reaction mixture will be heated (typically to 110°C for 10 min), then cooled down
(typically to 70°C).
(iv) SPE clean up of [ 8F]FCH2OTs
Once radiosynthesis of [ F]FCH 2OTs is completed and the reaction vessel is
cooled, water is added into the reaction vessel to reduce the organic solvent content in
the reaction vessel to approximately 25%. This diluted solution is transferred from the
reaction vessel and through the t-C18-light and t-C18 plus cartridges - these
cartridges are then rinsed with 12 to 15mL of a 25% acetonitrile / 75% water solution.
At the end of this process:
the methylene ditosylate remains trapped on the t-C18-light and
- the [ F]FCH 2OTs, tosyl-[ F]fluoride remains trapped on the t-C18
plus.
(v) Reaction vessel clean up
The reaction vessel was cleaned (using ethanol) prior to the alkylation of
[ FJfluoroethyl tosylate and O-PMB-DMEA precursor.
(vi) Drying reaction vessel and [ 8F]fluoromethyl tosylate retained on SPE t-
C18 plus simultaneously
Once clean up (v) was completed, the reaction vessel and the
[18FJfluoromethyl tosylate retained on SPE t-C18 plus was dried simultaneously.
(vii) Alkylation reaction
Following step (vi), the [18F]FCH 2OTs (along with tosyl-[ 18FJfluoride)
retained on the t-C18 plus was eluted into the reaction vessel using a mixture of OPMB-
N,N-dimethyl-[l,2- H4]ethanolamine (or O-RMB-N,N-dimethylethanolamine)
in acetonitrile.
The alkylation of [18F]FCH2OTs with O-PMB-precursor was achieved by
heating the reaction vessel (typically 110°C for 15min) to afford [ F]fluoro-[l,2-
H4]choline (or 0-PMB-[ F]fluorocholine).
(viii) Removal of unreacted O-PMB-precursor
Water (3 to 4mL) was added to the reaction and this solution was then passed
through a pre-treated CM cartridge, followed by an ethanol wash - typically 2 x 5mL
(this removes unreacted O-PMB-DMEA) leaving "purified" [ F]fluoro-[l,2-
H4]choline (or 0-PMB-[ FJfluorocholine) trapped onto the CM cartridge.
(ix) Deprotection & formulation
Hydrochloric acid was passed through the CM cartridge into a syringe: this
resulted in the deprotection of 0-PMB-[ 18FJfluorocholine (the syringe contains
[18FJfluorocholine in a HC1 solution). Sodium acetate was then added to this syringe
to buffer to pH 5 to 8 affording [ F]-D4-choline (or [ F]choline) in an acetate
buffer. This buffered solution is then transferred to a product vial containing a
suitable buffer.
Table 1 provides a listing of reagents and other components required for preparation
of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) (or [ F]fluoromethylcholine)
radiocassette of the present invention:
Table 1
According to one embodiment of the present invention, FASTlabTM
synthesis of [18F]fluoromethyl-[l,2- 2H4]choline via an unprotected precursor
comprises the following sequential steps as depicted in Scheme 6 below:
Scheme 6
18 1. Recovery of [ F]fluoride from QMA;
2 Preparation of K[ F]F/K2 22/K2C0 3 complex;
3 Radiosynthesis of FCH2OTs;
4 SPE cleanup of FCH2OTs;
5 Clean up of reaction vessel cassette and syringe;
6 Drying of reaction vessel and C18 SepPak;
7 Elution off and coupling of FCH2OTs with D4-DMEA;
8 Transfer of reaction mixture onto CM cartridge;
9 Clean up of cassette and syringe;
10 Washing of CM cartridge with dilute aq ammonia solution, Ethanol
and water;
11 Elution of [ F]fluoromethyl-[l,2- H4]choline from CM cartridge with
0.09% sodium chloride (5 ml), followed by water (5ml).
In one embodiment of the present invention, steps (l)-(ll) above are
performed on a cassette as described herein. One embodiment of the present
invention is a cassette capable of performing steps (l)-(l 1) for use in an automated
synthesis platform. One embodiment of the present invention is a cassette for the
radiosynthesis of [ F]fluoromethyl-[l,2- H4]choline ([ F]-D4-FCH) from an
unprotected precursor. An example of a cassette of the present invention is shown in
Figure 5a.
Table 2 provides a listing of reagents and other components required for preparation
of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) (or [ F]fluoromethylcholine) via an
unprotected precursor radiocassette of the present invention:
Table 2
Imaging Method
The radiolabeled compound of the invention, as described herein, will be
taken up into cells via cellular transporters or by diffusion. In cells where choline
kinase is overexpressed or activated the radiolabeled compound of the invention, as
described herein, will be phosphorylated and trapped within that cell. This will form
the primary mechanism of detecting neoplastic tissue.
The present invention further provides a method of imaging comprising the
step of administering a radiolabeled compound of the invention or a pharmaceutical
composition comprising a radiolabeled compound of the invention, each as described
herein, to a subject and detecting said radiolabeled compound of the invention in said
subject. The present invention further provides a method of detecting neoplastic
tissue in vivo using a radiolabeled compound of the invention or a pharmaceutical
composition comprising a radiolabeled compound of the invention, each as described
herein. Hence the present invention provides better tools for early detection and
diagnosis, as well as improved prognostic strategies and methods to easily identify
patients that will respond or not to available therapeutic treatments. As a result of the
ability of a compound of the invention to detect neoplastic tissue, the present
invention further provides a method of monitoring therapeutic response to treatment
of a disease state associated with the neoplastic tissue.
In a preferred embodiment of the invention, the radiolabeled compound of the
invention for use in a method of imaging of the invention, as described herein, is a
radiolabeled compound of Formula (I).
In a preferred embodiment of the invention, the radiolabeled compound of the
invention for use in a method of imaging of the invention, as described herein, is a
radiolabeled compound of Formula (III).
As would be understood by one of skill in the art the type of imaging (e.g.,
PET, SPECT) will be determined by the nature of the radioisotope. For example, if
the radiolabeled compound of Formula (I) contains 18F it will be suitable for PET
imaging.
Thus the invention provides a method of detecting neoplastic tissue in vivo
comprising the steps of:
i) administering to a subject a radiolabeled compound of the
invention or a pharmaceutical composition comprising a
radiolabeled compound of the invention, each as defined herein;
ii) allowing said a radiolabeled compound of the invention to bind
neoplastic tissue in said subject;
iii) detecting signals emitted by said radioisotope in said bound
radiolabeled compound of the invention;
iv) generating an image representative of the location and/or amount
of said signals; and,
v) determining the distribution and extent of said neoplastic tissue in
said subject.
The step of "administering" a radiolabeled compound of the invention is
preferably carried out parenterally, and most preferably intravenously. The
intravenous route represents the most efficient way to deliver the compound
throughout the body of the subject. Intravenous administration neither represents a
substantial physical intervention nor a substantial health risk to the subject. The
radiolabeled compound of the invention is preferably administered as the
radiopharmaceutical composition of the invention, as defined herein. The
administration step is not required for a complete definition of the imaging method of
the invention. As such, the imaging method of the invention can also be understood
as comprising the above-defined steps (ii)-(v) carried out on a subject to whom a
radiolabeled compound of the invention has been pre-administered.
Following the administering step and preceding the detecting step, the
radiolabeled compound of the invention is allowed to bind to the neoplastic tissue.
For example, when the subject is an intact mammal, the radiolabeled compound of the
invention will dynamically move through the mammal's body, coming into contact
with various tissues therein. Once the radiolabeled compound of the invention comes
into contact with the neoplastic tissue it will bind to the neoplastic tissue.
The "detecting" step of the method of the invention involves detection of
signals emitted by the radioisotope comprised in the radiolabeled compound of the
invention by means of a detector sensitive to said signals, e.g., a PET camera. This
detection step can also be understood as the acquisition of signal data.
The "generating" step of the method of the invention is carried out by a
computer which applies a reconstruction algorithm to the acquired signal data to yield
a dataset. This dataset is then manipulated to generate images showing the location
and/or amount of signals emitted by the radioisotope. The signals emitted directly
correlate with the amount of enzyme or neoplastic tissue such that the "determining"
step can be made by evaluating the generated image.
The "subject" of the invention can be any human or animal subject.
Preferably the subject of the invention is a mammal. Most preferably, said subject is
an intact mammalian body in vivo. In an especially preferred embodiment, the subject
of the invention is a human.
The "disease state associated with the neoplastic tissue" can be any disease
state that results from the presence of neoplastic tissue. Examples of such disease
states include, but are not limited to, tumors, cancer (e.g., prostate, breast, lung,
ovarian, pancreatic, brain and colon). In a preferred embodiment of the invention the
disease state associated with the neoplastic tissue is brain, breast, lung, espophageal,
prostate, or pancreatic cancer.
As would be understood by one of skill in the art, the "treatment" will be
depend on the disease state associated with the neoplastic tissue. For example, when
the disease state associated with the neoplastic tissue is cancer, treatment can include,
but is not limited to, surgery, chemotherapy and radiotherapy. Thus a method of the
invention can be used to monitor the effectiveness of the treatment against the disease
state associated with the neoplastic tissue.
Other than neoplasms, a radiolabeled compound of the invention may also be
useful in liver disease, brain disorders, kidney disease and various diseases associated
with proliferation of normal cells. A radiolabeled compound of the invention may
also be useful for imaging inflammation; imaging of inflammatory processes
including rheumatoid arthritis and knee synovitis, and imaging of cardiovascular
disease including artherosclerotic plaque.
Precursor Compound
The present invention provides a precursor compound of Formula (II):
(P)
as described above.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
Ri, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
R and R2 are each hydrogen;
R and R are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
According to the invention, compound of Formula (II) is a compound of
Formula (Ila):
(Ha).
embodiment of the invention, a compound of Formula (lib) is provided:
(lib)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, -(CH2 ) m R8, -(CD2)mR ,
(CF2)mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; and
Pg is a hydroxyl protecting group.
In a preferred embodiment of the invention, a compound of Formula (lib) is
provided wherein Pg is a p-methoxybenyzl (PMB), trimethylsilyl (TMS), or a
dimethoxytrityl (DMTr) group.
In a preferred embodiment of the invention, a compound of Formula (lib) is
provided wherein Pg is a p-methoxybenyzl (PMB) group.
In one embodiment of the invention, a compound of Formula (lie) is provided:
(lie)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4;
with the proviso that when Ri, R2, R3, and R4 are each hydrogen, R5, R6, and
R7 are each not hydrogen; and with the proviso that when Ri, R2, R3, and R 4 are each
deuterium, R5, R6, and R7are each not hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
Ri, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; with the proviso that R5, R , and R7 are each not
hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; with the proviso that R5, R6, and R 7 are each not
hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
R and R2 are each hydrogen; and
R and R 4 are each deuterium (D).
A precursor compound of Formula (II), including a compound of Formula
(Ha), (lib) and (lie), can be prepared by any means known in the art including those
described herein. For example, the compound of Formula (Ila) can be synthesized by
alkylation of dimethylamine in THF with 2-bromoethanol-l,l,2,2-d4 in the presence
of potassium
SCHEME 1
wherein i = K2CO3, THF, 50°C, 19 h. The desired tetra-deuterated product can be
purified by distillation. The H NMR spectrum of the compound of Formula (Ila)
(Figure 3) in deuteriochloroform showed only the peaks associated with the N,Ndimethyl
groups and the hydroxyl of the alcohol; no peaks associated with the
hydrogens of the methylene groups of the ethyl alcohol chain were observed.
Consistent with this, the C NMR spectrum (Figure 3) showed the large singlet
associated with the N,N -dimethyl carbons; however, the peaks for the ethyl alcohol
methylene carbons at 60.4 ppm and 62.5 ppm were substantially reduced in
magnitude, suggesting the absence of the signal enhancement associated with the
presence of a covalent carbon-hydrogen bond. In addition, the methylene peaks are
both split into multiplets, indicating spin-spin coupling. Since C NMR is typically
run with H decoupling, the observed multiplicity must be the result of carbondeuterium
bonding. On the basis of the above observations the isotopic purity of the
desired product is considered to be > 98% in favour of the H isotope (relative to the
H isotope).
A di-deuterated analog of a precursor compound of Formula (II) can be
synthesized from N,N-dimethylglycine via lithium aluminium hydride reduction as
shown in Scheme 2 below:
SCHEME 2
wherein i = LiAlD4, THF, 65°C, 24 h. C NMR analysis indicated that isotopic
purity of greater than 95% in favor of the H isomer (relative to the H isotope) can be
achieved.
According to the invention, the hydroxyl group of a compound of Formula
(II), including a compound of Formula (Ila) can be further protected with a protecting
group to give a comp
(lib)
wherein Pg is any hydroxyl protecting group known in the art. Preferably, Pg is any
acid labile hydroxyl protecting group including, for example, those described in
""Protective Groups in Organic Synthesis", 3rd Edition, A Wiley Interscience
Publication, John Wiley & Sons Inc., Theodora W. Greene and Peter G. M. Wuts, pp
17-200. Preferably, Pg is a p-methoxybenzyl (PMB), trimethylsilyl (TMS), or a
dimethoxytrityl (DMTr) group. More preferably, Pg is a p-methoxybenyzl (PMB)
group.
Validation of r Flfluoromethyl-ri,2- 2H4lcholine (D4-FCH)
Stability to oxidation resulting from isotopic substitution was evaluated in in
vitro chemical and enzymatic models using [ F]fluoromethylcholine as standard.
[ F]Fluoromethyl-[l,2- H 4]choline was then evaluated in in vivo models and
11 18 18 compared to [ CJcholine, [ FJfluoromethylcholine and [ F]Fluoromethyl-[l-
H 2]choline:
OH
C-Choline
[ 1 8F]fluo ro methylcholine [ 1 8F] Fluo ro methyl-[1 -2H2]choline
[1 8F]Fluoromethyl-[1 ,2-2H4]choline
Potassium Permanganate oxidation study
The effect of deuterium substitution on bond strength was initially tested by
evaluation of the chemical oxidation pattern of [18FJfluoromethylcholine and
[18F]Fluoromethyl-[l,2- 2H4]choline using potassium permanganate. Scheme 6 below
details the base catalyzed potassium permanganate oxidation of
[18FJfluoromethylcholine and [18F]Fluoromethyl-[l,2- 2H4]choline at room
temperature, with aliquots removed and analyzed by radio-HPLC at pre-selected time
oints:
c) R -| ,R , R3, R4
Scheme 6
Reagents and Conditions: i) KMn0 4, Na2C0 , H20 , rt.
The results are summarized in Figures 6 and 7. The radio-HPLC
chromatogram (Figure 6) showed a greater proportion of the parent compound
remaining at 20 min for [18F]Fluoromethyl-[l,2- 2FLJcholine. The graph in Figure 7
further showed a significant isotope effect for the deuterated analogue,
[ F]Fluoromethyl-[l,2- H4]choline, with nearly 80% of parent compound still present
1 hour post-treatment with potassium permanganate, compared to less than 40% of
parent compound [18FJFluoromethylcholine still present at the same time point.
Choline oxidase model
[18FJfluoromethylcholine and [18F]fluoromethyl-[l,2- 2H4]choline were
evaluated in a choline oxidase model (Roivainen, A., et al. , European Journal of
Nuclear Medicine 2000; 27:25-32). The graphical representation in Figure 8 clearly
shows that, in the enzymatic oxidative model, the deuterated compound is
significantly more stable than the corresponding non-deuterated compound. At the 60
minute time point the radio-HPLC distribution of choline species revealed that for
[18FJfluoromethylcholine the parent radiotracer was present at the level of 11+8%; at
60 minutes the corresponding parent deuterated radiotracer [ F]fluoromethyl-[l,2-
H4]choline was present at 29+4%. Relevant radio-HPLC chromatograms are shown
in Figure 9 and further exemplify the increased oxidative stability of
[18F]fluoromethyl-[l,2- 2H4]-choline relative to [18FJfluoromethylcholine. These radio-
HPLC chromatograms contain a third peak, marked as 'unknown' , that is speculated
to be the intermediate oxidation product, betaine aldehyde.
In vivo stability analysis
[18F]fluoromethyl-[l,2- 2H4]-choline is more resistant to oxidation in vivo. The
relative rates of oxidation of the two isotopically radiolabeled choline species,
[18FJfluoromethylcholine and [18F]fluoromethyl-[l,2- 2H4]-choline to their respective
metabolites, [18FJfluoromethylcholine -betaine ( [18F]-FCH-betaine) and
[ F]fluoromethyl-[l,2- H4]-choline-betaine ([ F]-D4-FCH-betaine) was evaluated by
high performance liquid chromatography (HPLC) in mouse plasma after intravenous
(i.v.) administration of the radiotracers. [18F]fluoromethyl-[l,2- 2H4]-choline was
found to be markedly more stable to oxidation than [ F]fluoromethylcholine. As
shown in Figure 10, [ F]fluoromethyl-[l,2- H4]-choline was markedly more stable
than [18FJfluoromethylcholine with -40% conversion of r1F8]fluoromethyl-[l,2-2H4]-
choline to [18F]-D4-FCH-betaine at 15 min after i.v. injection into mice compared to ~
80% conversion of [ F]fluoromethylcholine to [ F]-FCH-betaine. The time course
for in vivo oxidation is shown in Figure 10 showing overall improved stability of
[18F]fluoromethyl- [1,2- 2Ή 4]-choline over [18FJfluoromethylcholine.
Biodistribution
Time course biodistribution
Time course biodistribution was carried out for [ FJfluoromethylcholine,
18 2 [ F]fluoromethyl-[l- H 18 2 2]choline and [ F]fluoromethyl-[l,2- H4]choline in nude
mice bearing HCT116 human colon xenografts. Tissues were collected at 2, 30 and 60
minutes post-injection and the data summarized in Figure 11A-C. The uptake values
for [18FJfluoromethylcholine were in broad agreement with earlier studies (DeGrado,
T.R., et al. , "Synthesis and Evaluation of 18F-labeled Choline as an Oncologic Tracer
for Positron Emisson Tomography: Initial Findings in Prostate Cancer", Cancer
Research 2000; 61:110-7). Comparison of the uptake profiles revealed a reduced
uptake of radiotracer in the heart, lung and liver for the deuterated compounds
[18F]fluoromethyl-[l- 2H 18 2 2]-choline and [ F]fluoromethyl-[l,2- H4]-choline. The tumor
uptake profile for the three radiotracers is shown in Figure 11D and shows increased
localization of radiotracer for the deuterated compounds relative to
[18FJfluoromethylcholine at all time points. A pronounced increase in tumor uptake of
[18F]fluoromethyl-[l,2- 2H4]choline at the later time points is evident.
Distribution of choline metabolites
Metabolite analysis of tissues including liver, kidney and tumor by HPLC was
also accomplished. Typical HPLC chromatograms of [18F]FCH and [18F]D4-FCH and
their respective metabolites in tissues are shown in Figure 12. Tumor distribution of
metabolites was analyzed in a similar fashion (Figure 13). Choline and its metabolites
lack any UV chromophore to permit presentation of chromatograms of the cold
unlabelled compound simultaneously with the radioactivity chromatograms. Thus, the
presence of metabolites was validated by other chemical and biological means. Of
note the same chromatographic conditions were used for characterization of the
metabolites and retention times were similar. The identity of the phosphocholine peak
was confirmed biochemically by incubation of the putative phosphocholine formed in
untreated HCT116 tumor cells with alkaline phosphatase (Figure 14).
A high proportion of liver radioactivity was present as phosphocholine at 30 min post
injection for both [ F]FCH and [ F]D4-FCH (Figure 12). An unknown metabolite
(possibly the aldehyde intermediate) was observed in both the liver (7.4 + 2.3%) and
kidney (8.8 + 0.2%) samples of [ F]D4-FCH treated mice. In contrast, this unknown
metabolite was not found in liver samples of [18F]FCH treated mice and only to a
smaller extent (3.3 + 0.6%) in kidney samples. Notably 60.6 + 3.7% of [ F]D4-FCH
derived kidney radioactivity was phosphocholine compared to 31.8 + 9.8% from
[ F]FCH (P = 0.03). Conversely, most of the [ F]FCH-derived radioactivity in the
kidney was in the form of [ F]FCH-betaine; 53.5 + 5.3% compared to 20.6 + 6.2%
for [18F]D4-FCH (Figure 12). It could be argued that levels of betaine in plasma
reflected levels in tissues such as liver and kidneys. Tumors showed a different HPLC
profile compared to liver and kidneys; typical radio-HPLC chromatograms obtained
from the analysis of tumor samples (30 min after intravenous injection of [18F]FCH,
[18FJD4-FCH and [11dcholine) are shown in Figure 12. In tumors, radioactivity was
mainly in the form of phosphocholine in the case of [ F]D4-FCH (Figure 13). In
contrast [ F]FCH showed significant levels of [ F]FCH-betaine. In the context of
late imaging, these results indicate that [18FJD4-FCH will be the superior radiotracer
for PET imaging with an uptake profile that is easier to interpret.
The suitable and preferred aspects of any feature present in multiple aspects of the
present invention are as defined for said features in the first aspect in which they are
described herein. The invention is now illustrated by a series of non-limiting
examples.
Isotopic Carbon Choline Analogs
The present invention provides a compound of Formula (III) as described
herein. Such compounds are useful as PET imaging agents for tumor imaging, as
described herein. In particular, a compound of Formula (III), as described herein,
may not be excreted in the urine and hence provide more specific imaging of pelvic
malignancies such as prostate cancer.
The present invention provides a method to prepare a compound for Formula
(III), wherein said method comprises reaction of the precursor compound of Formula
(II) with a compound of Formula (IV) to form a compound of Formula (III) (Scheme
Scheme A
wherein the compounds of Formulae (I) and (III) are each as described herein and the
compound of Formula (IV) is as follows:
ZXYC*- Lg (IV)
wherein C*, X, Y and Z are each as defined herein for a compound of Formula (III)
and "Lg" is a leaving group. Suitable examples of "Lg" include, but are not limited
to, bromine (Br) and tosylate (OTos) . A compound of Formula (IV) can be prepared
by any means known in the art including those described herein (e.g. , analogous to
Examples 5 and 7).
Examples
Reagents and solvents were purchased from Sigma-Aldrich (Gillingham, UK) and
used without further purification. Fluoromethylcholine chloride (reference standard)
was purchased from ABCR Gmbh & Co. (Karlsruhe, Germany). Isotonic saline (0.9
% w/v) was purchased from Hameln Pharmaceuticals (Gloucester, UK). NMR
Spectra were obtained using either a Bruker Avance NMR machine operating at 400
MHz ( H NMR) and 100 MHz ( C NMR) or 600 MHz ( H NMR) and 150 MHz ( C
NMR). Accurate mass spectroscopy was carried out on a Waters Micromass LCT
Premier machine in positive electron ionisation (EI) or chemical ionisation (CI) mode.
Distillation was carried out using a Biichi B-585 glass oven (Biichi, Switzerland).
Example 1. Preparation of N,N-dimethyl-[l,2- 2H ]-ethanolamine (3)
1 2
To a suspension of K2CO (10.50 g, 76 mmol) in dry THF (10 mL) was added
dimethylamine (2.0 M in THF) (38 mL, 76 mmol) followed by 2-bromoethanoll
,l,2,2-d 4 (4.90 g, 38 mmol) and the suspension heated to 50°C under argon. After 19
h, thin layer chromatography (TLC) (ethyl acetate/alumina/I 2) indicated complete
conversion of (2) and the reaction mixture was allowed to cool to ambient
temperature and filtered. Bulk solvent was then removed under reduced pressure.
Distillation gave the desired product (3) as a colorless liquid, b.p. 78°C/88 mbar (1.93
g, 55%). H NMR (CDCI3, 400 MHz) d 3.40 (s, 1H, OH), 2.24 (s, 6H , N(CH )2) . C
NMR (CDCI3, 75 MHz) d 62.6 (NCD2 D2OH), 60.4 (N D2CD2OH), 47.7 (N(CH )2) .
HRMS (EI) = 93. 1093 (M+) . C4H7 H4NO requires 93. 1092.
Example 2. Preparation of N,N-dimethyl-[l- 2H2]-ethanolamine (5)
\ OH
C0 2H
/ D D
To a suspension of N,N-dimethylglycine (0.52 g, 5 mmol) in dry THF(10 mL) was
added lithium aluminium deuteride (0.53 g, 12.5 mmol) and the resulting suspension
refluxed under argon. After 24 h the suspension was allowed to cool to ambient
temperature and poured onto sat. aq. Na2S0 4 (15 mL) and adjusted to pH 8 with 1 M
Na2CC>3, then washed with ether (3 x 10 mL) and dried (Na 2S0 4) . Distillation gave
the desired product (5) as a colorless liquid, b.p. 65°C/26 mbar (0.06 g, 13%). H
NMR (CDC1 , 400 MHz) d 2.43 (s, 2H, NCH2CD2) , 2.25 (s, 6H, N(C H )2), 1.43 (s,
1H, OH) . C NMR (CDCI 3, 150 MHz) d 63.7 (N H2CD2OH), 57.8 (NCH 2 D2OH),
45.7 (N(CH3)2) .
Example 3. Preparation of Fluoromethyltosylate (8)
CH2OTos2 FCH2OTos
7 8
Methylene ditosylate (7) was prepared according to an established literature procedure
and analytical data was consistent with reported values (Emmons, W.D., et al. ,
Journal of the American Chemical Society, 1953; 75:2257; and Neal, T.R., et al.,
Journal of Labelled Compounds and Radiopharmaceuticals 2005; 48:557-68).
To a solution of methylene ditosylate (7) (0.67 g, 1.89 mmol) in dry acetonitrile (10
mL) was added Kryptofix K222 [4,7,13,16,21,24-hexaoxa-l,10-
diazabicyclo[8.8.8]hexacosane] (1.00 g, 2.65 mmol) followed by potassium fluoride
(0.16 g, 2.83 mmol). The suspension was then heated to 110°C under nitrogen. After
1 h TLC (7:3 hexane/ethyl acetate/silica/UV 254 ) indicated complete conversion of (7).
The reaction mixture was diluted with ethyl acetate (25 mL), washed with water (2 x
15 mL) and dried over MgS0 4. Chromatography (5 ® 10% ethyl acetate/hexane)
gave the desired product (8) as a colorless oil (40 mg, 11%). H NMR (CDC1 , 400
MHz) d 7.86 (d, 2H, = 8 Hz, aryl CH), 7.39 (d, 2 H, = 8 Hz, aryl CH), 5.77 (d, 1
H, = 52 Hz, CH2F), 2.49 (s, 3H, tolyl CH ) . C NMR (CDC1 ) d 145.6 (aryl), 133.8
(aryl), 129.9 (aryl), 127.9 (aryl), 98.1 (d, = 229 Hz, CH2F), 21.7 (tolyl CH ) . HRMS
(CI) = 222.0604 (M + NH4)+. Calcd. for C H FN0 S 222.0600.
Example 4. Preparation of N,N-Dimethylethanolamine(0-4-methoxybenzyl)
ether (O-PMB-DM
N,N-Dimethylethanolamine(0-4-methoxybenzyl) ether
To a dry flask was added dimethylethanolamine (4.46 g, 50 mmol) and dry DMF (50
mL). The solution was stirred under argon and cooled in an ice bath. Sodium hydride
(2.0 g, 50 mmol) was then added portionwise over 10 min and the reaction mixture
then allowed to warm to room temperature. After 30 min 4-methoxybenzyl chloride
(3.92 g, 25 mmol) was added dropwise over 10 min and the resulting mixture left to
stir under argon. After 60 h GC-MS indicated reaction completion (disappearance of
4-methoxybenzyl chloride) and the reaction mixture was poured onto 1M sodium
hydroxide (100 mL) and extracted with dichloromethane (DCM)(3 x 30 mL) then
dried (Na2S0 4) . Column chromatography (0®10 methanol/DCM; neutral silica)
gave the desired product (O-PMB-DMEA) as a yellow oil (1.46 g, 28 ). H NMR
(CDC1 , 400 MHz) d 7.28 (d, 2H, J = 8.6 Hz, aryl CH), 6.89 (d, 2H, J = 8.6 Hz, aryl
CH), 4.49 (s, 2H, -CH2-), 3.81 (s, 3H, OCH ), 3.54 (t, 2H, = 5.8, NCH2CH20), 2.54
(t, 2H, = 5.8, NCH2CH20), 2.28 (s, 6H, N(CH )2) . HRMS (ES) = 210.1497 (M+H+) .
C 2H20NO2 requires 210.1494.
Example 4a. Preparation of Dueterated Analogues of N,NDimethylethanolamine(
0-4-methoxybenzyl) ether (O-PMB-DMEA)
The di- and tetra-deuterated analogs of N,N-Dimethylethanolamine(0-4-
methoxybenzyl) ether can be prepared according to Example 4 from the appropriate
di- or tetra-deuterated dimethylethanolamine.
Example 5. Preparation of Synthesis of [ 8F]fluoromethyl tosylate (9)
CH2OTos2
1 FCH2OTos
7 9
To a Wheaton vial containing a mixture of K2CO (0.5 mg, 3.6 mihoΐ , dissolved in 100
water), 18-crown-6 (10.3 mg, 39 mihoΐ ) and acetonitrile (500 ) was added
[18F]fluoride (-20 mCi in 100 water). The solvent was then removed at 110°C
under a stream of nitrogen (100 mL/min). Afterwards, acetonitrile (500 ) was
added and distillation to dryness continued. This procedure was repeated twice. A
solution of methylene ditosylate (7) (6.4 mg, 18 mihoΐ ) in acetonitrile (250 )
containing 3 % water was then added at ambient temperature followed by heating at
100°C for 10-15 min., with monitoring by analytical radio-HPLC. The reaction was
quenched by addition of 1:1 acetonitrile/water (1.3 mL) and purified by semi-
preparative radio-HPLC. The fraction of eluent containing [18FJfluoromethyl tosylate
(9) was collected and diluted to a final volume of 20 mL with water, then immobilized
on a Sep Pak C18 light cartridge (Waters, Milford, MA, USA) (pre-conditioned with
DMF (5 mL) and water (10 mL)). The cartridge was washed with further water (5
mL) and then the cartridge, with [ F]fluoromethyl tosylate (9) retained, was dried in
a stream of nitrogen for 20 min. A typical HPLC reaction profile for synthesis of
[ F](13) is shown in Figure 4A/4B below.
Example 6. Radiosynthesis of [ F]fluoromethylcholine derivatives by reaction
with [ 8F]fluorobromomethane
lla-c
11a: R,, R2, R3, R4 = H
lib: R , R2= H; R3, R4 = D
11c: R,, R2, R3, R4 = D
[ FJFluorobromomethane (prepared according to Bergman et al (Appl Radiat Isot
2001;54(6):927-33)) was added to a Wheaton vial containing the amine precursor
N,N-dimethylethanolamine (150 ) or N,N-dimethyl-[l,2- H 4]ethanolamine
i ) in dry acetonitrile (1 mL), pre-cooled to 0°C. The vial was sealed and then heated
to 100°C for 10 min. Bulk solvent was then removed under a stream of nitrogen, then
the sample remaining was redissolved in 5% ethanol in water (10 mL) and
immobilized on a Sep-Pak CM light cartridge (Waters, Milford, MA, USA) (preconditioned
with 2 M HC1 (5 mL) and water (10 mL)) to effect the chloride anion
exchange. The cartridge was then washed with ethanol (10 mL) and water (10 mL)
followed by elution of the radiotracer (11a) or (11c) using saline (0.5-2.0 mL) and
passing through a sterile filter (0.2 mih) (Sartorius, Goettingen, Germany).
Example 7. Radiosynthesis of [ F]Fluoromethylcholine, [ F]fluoromethyl-[l
2H2]choline and [ 8F]fluoromethyl-[l,2- 2H ]choline by reaction with
[ 8F]fluoromethylmethyl tosylate
12a-c
[ FJFluoromethyl tosylate (9)(prepared according to Example 5) and eluted from the
Sep-Pak cartridge using dry DMF (300 ), was added in to a Wheaton vial
containing one of the following precursors: N,N-dimethylethanolamine (150 )
N,N-dimethyl-[l,2- FL]ethanolamine (3) (150 [iL) (prepared according to Example
1); or N,N-dimethyl-[l- H 2]ethanolamine (5)(150 (prepared according to
Example 2 ), and heated to 100°C with stirring. After 20 min the reaction was
quenched with water (10 mL) and immobilized on a Sep Pak CM light cartridge
(Waters) (pre-conditioned with 2M HC1 (5 mL) and water (10 mL)) in order to effect
the chloride anion exchange and then washed with ethanol (5 mL) and water (10 mL)
followed by elution of the radiotracer [ F]Fluoromethylcholine (12a),
[ F]fluoromethyl-[l- H2]choline (12b) or [ F]fluoromethyl-[l,2- H4]choline [ F]
(12c) with isotonic saline (0.5-1.0 rriL).
Example 8. Synthesis of cold Fluoromethyltosylate (15)
i ii
CH2I2 * CH2OTos2 FCH2OTos
13 14 5
Scheme 3
i : Silver p-toluenesulfonate, MeCN, reflux, 20 h;
ii: KF, MeCN, reflux, 1 h.
According to Scheme 3 above:
(a) Synthesis of methylene ditosylate (14)
Commercially available diiodomethane (13) (2.67 g, 10 mmol) was reacted
with silver tosylate (6.14 g, 22 mmol), using the method of Emmons and Ferris, to
give methylene ditosylate (10) (0.99g) in 28% yield (Emmons, W.D., et a ,
"Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of Alkyl
Sulfonates", Journal of the American Chemical Society, 1953; 75:225).
(b) Synthesis of cold Fluoromethyltosylate (15)
Fluoromethyltosylate (11) (0.04g) was prepared by nucleophilic substitution of
Methylene ditosylate (10) (0.67 g, 1.89 mmol) of Example 3(a) using potassium
fluoride (0.16 g, 2.83 mmol)/Kryptofix K222 (1-0 g, 2.65 mmol) in acetonitrile (10
rriL) at 80°C to give the desired product in 11% yield .
Example 9. Synthesis of [ F]fluorobromomethane (17)
r18 F]KF
FCH2Br
16 17
Adapting the method of Bergman et al (Appl Radiat Isot 2001;54(6):927-33),
commercially available dibromomethane (16) is reacted with [ F]potassium
fluoride/Kryptofix K222 in acetonitrile at 110°C to give the desired
[ F]fluorobromomethane (17), which is purified by gas-chromatography and trapped
by elution into a pre-cooled vial containing acetonitrile and the relevant choline
precursor.
Example 10. Analysis of radiochemical purity
Radiochemical purity for [ F]Fluoromethylcholine, [ F]fluoromethyl-[l- H2]choline
and [ F]fluoromethyl-[l,2- H4]choline [ F] was confirmed by co-elution with a
commercially available fluorocholine chloride standard. An Agilent 1100 series
HPLC system equipped with an Agilent G1362A refractive index detector (RID) and
a Bioscan Flowcount FC-3400 PIN diode detector was used. Chromatographic
separation was performed on a Phenomenex Luna C reverse phase column (150 mm
x 4.6 mm) and a mobile phase comprising of 5 mM heptanesulfonic acid and
acetonitrile (90:10 v/v) delivered at a flow rate of 1.0 mL/min.
Example 11. Enzymatic oxidation study using choline oxidase
This method was adapted from that of Roivannen et al (Roivainen, A., et al. ,
European Journal of Nuclear Medicine 2000; 27:25-32). An aliquot of either
[ F]Fluoromethylcholine or [ F]fluoromethyl-[l,2- H4]choline [ F] (100 uL, -3.7
MBq) was added to a vial containing water (1.9 mL) to give a stock solution. Sodium
phosphate buffer (0.1 M, pH 7) (10 uL) containing choline oxidase (0.05 units/uL)
was added to an aliquot of stock solution (190 uL) and the vial was then left to stand
at room temperature, with occasional agitation. At selected time-points (5, 20, 40 and
60 minutes) the sample was diluted with HPLC mobile phase (buffer A, 1.1 mL),
filtered (0.22 mih filter) and then ~ 1 mL injected via a 1mL sample loop onto the
HPLC for analysis. Chromatographic separation was performed on a Waters C
Bondapak (7.8 x 300 mm) column (Waters, Milford, Massachusetts, USA) at
3mL/min with a mobile phase of buffer A, which contained acetonitrile, ethanol,
acetic acid, 1.0 mol/L ammonium acetate, water, and 0.1 mol/L sodium phosphate
(800:68:2:3:127:10 [v/v]) and buffer B, which contained the same constituents but in
different proportions (400:68:44:88:400:10 [v/v]). The gradient program comprised
100% buffer A for 6 minutes, 0-100% buffer B for 10 minutes, 100-0% B in 2
minutes then 0% B for 2 minutes.
Example 12. Biodistribution
Human colon (HCT116) tumors were grown in male C3H-Hej mice (Harlan, Bicester,
United Kingdom) as previously reported (Leyton, J., et a , Cancer Research 2005;
65(10):4202-10). Tumor dimensions were measured continuously using a caliper and
tumor volumes were calculated by the equation: volume = (p/6) x a x b x c, where a,
b, and c represent three orthogonal axes of the tumor. Mice were used when their
tumors reached approximately 100 mm3. [18FJFluoromethylcholine,
[ F]fluoromethyl-[l- H2]choline and [ F]fluoromethyl-[l,2- H4]choline (-3.7 MBq)
were each injected via the tail vein into awake untreated tumor bearing mice. The
mice were sacrificed at pre-determined time points (2, 30 and 60 min) after
radiotracer injection under terminal anesthesia to obtain blood, plasma, tumor, heart,
lung, liver, kidney and muscle. Tissue radioactivity was determined on a gamma
counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK)
and decay corrected. Data were expressed as percent injected dose per gram of tissue.
Example 13. Oxidation potential of [ 8F]Fluoromethylcholine ([ 8F]FCH) and
[ 8F]fluoromethyl-[l,2- 2H ]choline ([ 8F]D4-FCH) in vivo
[ F]FCH or [ F](D4-FCH) (80-100 m ) was injected via the tail vein into
anesthetized non-tumor bearing C3H-Hej mice; isofluorane/0 2/N20 anesthesia was
used. Plasma samples obtained at 2, 15, 30 and 60 minutes after injection were snap
frozen in liquid nitrogen and stored at -80°C. For analysis, samples were thawed and
kept at 4°C. To approximately 0.2 mL of plasma was added ice-cold acetonitrile (1.5
mL). The mixture was then centrifuged (3 minutes, 15,493 x g; 4°C). The supernatant
was evaporated to dryness using a rotary evaporator (Heidoloph Instruments GMBH
& CO, Schwabach, Germany) at a bath temperature of 45°C. The residue was
suspended in mobile phase (1.1 mL), clarified (0.2 mih filter) and analyzed by HPLC.
Liver samples were homogenized in ice-cold acetonitrile (1.5 mL) and then
subsequently treated as per plasma samples. All samples were analyzed on an Agilent
1100 series HPLC system equipped with a g -RAM Model 3 radio-detector (IN/US
Systems inc., FL, USA). The analysis was based on the method of Roivannen
(Roivainen, A., et ah, European Journal of Nuclear Medicine 2000; 27:25-32) using a
Phenomenex Luna SCX column (10m, 250 x 4.6 mm) and a mobile phase comprising
of 0.25 M sodium dihydrogen phosphate (pH 4.8) and acetonitrile (90:10 v/v)
delivered at a flow rate of 2 ml/min.
Example 14. Distribution of choline metabolites
Liver, kidney, and tumor samples were obtained at 30 min. All samples were snapfrozen
in liquid nitrogen. For analysis, samples were thawed and kept at 4°C
immediately before use. To -0.2 mL plasma was added ice-cold methanol (1.5 mL).
The mixture was then centrifuged (3 min, 15,493 x g , 4jC). The supernatant was
evaporated to dryness using a rotary evaporator (Heidoloph Instruments) at a bath
temperature of 40°C. The residue was suspended in mobile phase (1.1 mL), clarified
(0.2 Am filter), and analyzed by HPLC. Liver, kidney, and tumor samples were
homogenized in ice-cold methanol (1.5 mL) using an IKA Ultra-Turrax T-25
homogenizer and subsequently treated as per plasma samples (above). All samples
were analyzed by radio-HPLC on an Agilent 1100 series HPLC system (Agilent
Technologies) equipped with a g -RAM Model 3 g -detector (IN/US Systems) and
Laura 3 software (Lablogic). The stationary phase comprised a Waters Bondapak
C18 reverse-phase column (300 x 7.8 mm)(Waters, Milford, MA, USA). Samples
were analyzed using a mobile phase comprising solvent A
(acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1mol/L sodium
phosphate; 800/127/68/2/3/10) and solvent B (acetonitrile/water/ethanol/acetic
acid/1.0 mol/L ammonium acetate/0.1 mol/L sodiumphosphate; 400/400/68/44/88/10)
with a gradient of 0% B for 6 min, then 0 ® 100% B in 10 min, 100% B for 0.5 min,
100®0% B in 1.5 min then 0% B for 2 min, delivered at a flow rate of 3 mL/min.
Example 15. Metabolism of [ 8F]D4-FCH and [ 8F]FCH by HCT116 tumor cells.
HCT116 cells were grown in T150 flasks in triplicate until they were 70% confluent
and then treated with vehicle (1% DMSO in growth medium) or 1 mihoI/L
PD0325901 in vehicle for 24 h. Cells were pulsed for 1 h with 1.1 MBq of either
18 [ F]D4- FCH or [18F]FCH. The cells were washed three times in ice-cold phosphate
buffered saline (PBS), scraped into 5 mL PBS, and centrifuged at 500 x g for 3 min
and then resuspended in 2 mL ice-cold methanol for HPLC analysis as described
above for tissue samples. To provide biochemical evidence that the 5'-phosphate was
the peak identified on the HPLC chromatogram, cultured cells were treated with
alkaline phosphatase as described previously (Barthel, H., et a , Cancer Res 2003;
63(13):3791-8). Briefly, HCT116 cells were grown in 100 mm dishes in triplicate and
incubated with 5.0 MBq [ F]FCH for 60 min at 37°C to form the putative [ F]FCHphosphate.
The cells were washed with 5 mL ice-cold PBS twice and then scraped
and centrifuged at 750 x g (4°C, 3 min) in 5 mL PBS. Cells were homogenized in 1
mL of 5 mmol/L Tris- H (pH 7.4) containing 50% (v/v) glycerol, 0.5mmol/L
MgCl2, and 0.5mmol/L ZnCl2 and incubated with 10 units bacterial (type III) alkaline
phosphatase (Sigma) at 37°C in a shaking water bath for 30 min to dephosphorylate
the [18F]FCH-phosphate. The reaction was terminated by adding ice-cold methanol.
Samples were processed as per plasma above and analyzed by radio-HPLC.
Control experiments were done without alkaline phosphatase.
Example 16. Small animal PET imaging
PET imaging studies. Dynamic [ F]FCH and [ F]D4-FCH imaging scans were
carried out on a dedicated small animal PET scanner, quad-HIDAC (Oxford Positron
Systems). The features of this instrument have been described previously (Barthel, H.,
et a , Cancer Res 2003; 63(13):3791-8). For scanning the tail veins, vehicle- or drugtreated
mice were cannulated after induction of anesthesia (isofluorane/0 2/N20). The
animals were placed within a thermostatically controlled jig (calibrated to provide a
rectal temperature of ~37°C) and positioned prone in the scanner. [18F]FCH or
[18FJD4-FCH (2.96-3.7 MBq) was injected via the tail vein cannula and scanning
commenced. Dynamic scans were acquired in list mode format over a 60 min period
as reported previously (Leyton, J., et a , Cancer Research 2006; 66(15):7621-9). The
acquired data were sorted into 0.5 mm sinogram bins and 19 time frames (0.5 x 0.5 x
0.5 mm voxels; 4 x 15, 4 x 60, and 11 x 300 s) for image reconstruction, which was
done by filtered back-projection using a two-dimensional Hamming filter (cutoff 0.6).
The image data sets were visualized using the Analyze software (version 6.0;
Biomedical Imaging Resource, Mayo Clinic). Cumulative images of 30 to 60 min
dynamic data were used for visualization of radiotracer uptake and to draw regions of
interest. Regions of interest were defined manually on five adjacent tumor regions
(each 0.5 mm thickness). Dynamic data from these slices were averaged for each
tissue (liver, kidney, muscle, urine, and tumor) and at each of the 19 time points to
obtain time versus radioactivity curves. Corresponding whole body time versus
radioactivity curves representing injected radioactivity were obtained by adding
together radioactivity in all 200 x 160 x 160 reconstructed voxels. Tumor
radioactivity was normalized to whole-body radioactivity and expressed as percent
injected dose per voxel (%ID/vox). The normalized uptake of radiotracer at 60 min
(%ID/vox60) was used for subsequent comparisons. The average of the normalized
maximum voxel intensity across five slices of tumor %IDvox60max was also use for
comparison to account for tumor heterogeneity and existence of necrotic regions in
tumor. The area under the curve was calculated as the integral of %ID/vox from 0 to
60 min.
Examplel7. Effect of PD0325901 treatment in mice. Size-matched HCT116
tumor bearing mice were randomized to receive daily treatment by oral gavage of
vehicle (0.5% hydroxypropyl methylcellulose + 0.2% Tween 80) or 25 mg/kg (0.005
m g mouse) of the mitogenic extracellular kinase inhibitor, PD0325901, prepared in
vehicle. [ F]D4-FCH-PET scanning was done after 10 daily treatments with the last
dose administered 1 h before scanning. After imaging, tumors were snap-frozen in
liquid nitrogen and stored at ~80°C for analysis of choline kinase A expression. The
results are illustrated in Fig. 18 and 19.
This exemplifies use of [ F]D4-FCH-PET as an early biomarker of drug response.
Most of the current drugs in development for cancer target key kinases involved in
cell proliferation or survival. This example shows that in a xenograft model for which
tumor shrinkage is not significant, growth factor receptor-Ras-MAP kinase pathway
inhibition by the MEK inhibitor PD0325901 leads to a significant reduction in tumor
[18FJD4-FCH uptake signifying inhibition of the pathway. The figure also shows that
inhibition of [ F]D4-FCH uptake was due at least in part to the inhibition of choline
kinase activity.
Example 18. Comparison of [ 8F]FCH and [ 8F]D4-FCH for Imaging
As illustrated in Figure 16, [ F]FCH and [ F]D4-FCH were both rapidly
taken up into tissues and retained. Tissue radioactivity increased in the following
order: muscle < urine < kidney < liver. Given the predominance of phosphorylation
over oxidation in the liver (Figure 12), little differences were found in overall liver
radioactivity levels between the two radiotracers. Liver radioactivity at levels 60 min
after [ F]D4-FCH or [ F]FCH injection, %ID/vox60, was 20.92 + 4.24 and 18.75 +
4.28, respectively (Figure 16). This is also in keeping with the lower levels betaine
with [ F]D4-FCH injection than with [ F]FCH injection (Figure 12). Thus,
pharmacokinetics of the two radiotracers in liver determined by PET (which lacks
chemical resolution) were similar. The lower kidney radioactivity levels for [ F]D4-
FCH compared to [18F]FCH (Figure 16), on the other hand, reflect the lower oxidation
potential of [ F]D4-FCH in kidneys. The %ID/vox60 for [ F]FCH and [ F]D4-FCH
were 15.97 + 4.65 and 7.59 + 3.91, respectively in kidneys (Figure 16). Urinary
excretion was similar between the radiotracers. Regions of interest (ROIs) that were
drawn over the bladder showed %ID/vox6ovalues of 5.20 + 1.71 and 6.70 + 0.71 for
[ F]D4-FCH and [ F]FCH, respectively. Urinary metabolites comprised mainly of
the unmetabolized radiotracers. Muscle showed the lowest radiotracer levels of any
tissue.
Despite the relatively high systemic stability of [18FJD4-FCH and high
proportion of phosphocholine metabolites, higher tumor radiotracer uptake by PET in
mice that were injected with [ F]D4-FCH compared to the [ F]FCH group was
observed. Figure 17 shows typical (0.5 mm) transverse PET image slices
demonstrating accumulation of [18F]FCH and [18FJD4-FCH in human melanoma
SKMEL-28 xenografts. In this mouse model, the tumor signal-to-background contrast
was qualitatively superior in the [ F]D4-FCH PET images compared to [ F]FCH
images. Both radiotracers had similar tumor kinetic profiles detected by PET (Figure
17). The kinetics were characterized by rapid tumor influx with peak radioactivity at
~ 1 min (Figure 17). Tumor levels then equilibrated until ~5 min followed by a
plateau. The delivery and retention of [18FJD4-FCH were quantitatively higher than
those for FCH (Figure 17). The %ID/vox60 for [ F]D4-FCH and [ F]FCH were 7.43
+ 0.47 and 5.50 + 0.49, respectively (P=0.04). Because tumors often present with
heterogeneous population of cells, another imaging variable that is probably less
sensitive to experimental noise was exploited - an average of the maximum pixel
%ID/vox oacross 5 slices (%IDvox6om a x)- This variable was also significantly higher
for [ F]D4-FCH (P=0.05; Figure 17). Furthermore, tumor area under the time versus
radioactivity curve (AUC) was higher for D4-FCH mice than FCH (P =0.02).
Although the 30 min time point was selected for a more detailed analysis of tissue
samples, the percentage of parent compound in plasma was consistently higher for
[ F]D4-FCH compared to [ F]FCH at earlier time points. Regarding imaging, tumor
uptake for both radiotracers was similar at the early (15 min) and late (60 min) time
points (Supplementary Tablel). The earlier time points may be appropriate for pelvic
imaging.
Example 19. Imaging response to treatment
Having demonstrated that [18FJD4-FCH was a more stable fluorinated-choline
analog for in vivo studies, the use of this radiotracer to measure response to therapy
was investigated. These studies were performed in a reproducible tumor model
system in which treatment outcomes had been previously characterized, i.e., , the
human colon carcinoma xenograft HCT116 treated with PD0325901 daily for 10 days
(Leyton, J., et al., "Noninvasive imaging of cell proliferation following mitogenic
extracellular kinase inhibition by PD0325901", Mol Cancer Ther 2008; 7(9):3112-
21). Drug treatment led to tumor stasis (reduction in tumor size by only 12.2% at day
10 compared to the pretreatment group); tumors of vehicle-treated mice increased by
375%. Tumor [ F]D4-FCH levels in PD0325901 -treated mice peaked at
approximately the same time as those of vehicle-treated ones, however, there was a
marked reduction in radiotracer retention in the treated tumors (Figure 18). All
imaging variables decreased after 10 days of drug treatment (P=0.05, Figure 18). This
indicates that [ F]D4-FCH can be used to detect treatment response even under
conditions where large changes in tumor size reduction are not seen (Leyton, J., et al.,
"Noninvasive imaging of cell proliferation following mitogenic extracellular kinase
inhibition by PD0325901", Mol Cancer Ther 2008; 7(9):3 112-21). To understand the
biomarker changes, the intrinsic cellular effect of PD0325901 on D4-FCHphosphocholine
formation was examined by treating exponentially growing HCT1 16
cells in culture with PD0325901 for 24 h and measuring the 60-min uptake of
[ F]D4-FCH in vitro. As shown in Figure 18, PD0325901 significantly inhibited
[18F]D4-FCH-phosphocholine formation in drug-treated cells demonstrating that the
effect of the drug in tumors is likely due to cellular effects on choline metabolism
rather than hemodynamic effects.
To understand further the mechanisms regulating [ F]D4-FCH uptake with
drug treatment, changes in CHKA expression in PD0325901 and vehicle-treated
tumors excised after PET scanning were assessed. A significant reduction in CHKA
protein expression was seen in vivo at day 10 (P=0.03) following PD0325901
treatment (Figure 19) indicating that reduced CHKA expression contributed to the
lower D[18FJ4-FCH uptake in drug-treated tumors. The drug-induced reduction of
CHKA expression also occurred in vitro in exponentially growing cells treated with
PD0325901.
Example 20. Statistics.
Statistical analyses were done using the software GraphPad Prism version 4
(GraphPad). Between-group comparisons were made using the nonparametric Mann-
Whitney test. Two-tailed P < 0.05 was considered significant.
All patents, journal articles, publications and other documents discussed and/or cited
above are hereby incorporated by reference.

Claims:
1. A compound of Formula
(P)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2 ) m R8, -(CD2 ) m R8,
,R , -CH(R )2, or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
2. A compound according to Claim 1 wherein:
Ri, R2, R , and R4 are each independently hydrogen;
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
3. A compound according to Claim 1 wherein:
R and R2 are each hydrogen;
R3 and R 4 are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
A compound according to Claim 1 wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD ,
-CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
5. A compound according to Claim 1 of Formula (Ila):
6. A compound of Formula (lib) is provided:
(lib)
wherein:
Ri, R2, R , and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R8, -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; and
Pg is a hydroxyl protecting group.
7. A compound according to Claim 6 wherein Pg is a p-methoxybenyzl (PMB),
trimethylsilyl (TMS), or a dimethoxytrityl (DMTr) group.
A compound according to Claim 6 wherein Pg is a p-methoxybenyzl (PMB)
group.
9. A compound of Formula lie):
(lie)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R8, -(CH2)mR , -(CD2)mRs, -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4;
with the proviso that when Ri, R2, R3, and R4 are each hydrogen, R5, R6, and
R7 are each not hydrogen; and with the proviso that when Ri, R2, R3, and R4 are each
deuterium, R5, R6, and R 7 are each not hydrogen.
A compound according to Claim 9 wherein:
Ri, R2, R3, and R4 are each independently hydrogen;
with the proviso that R5, R6, and R 7 are each not hydro
A compound according to Claim 9 wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R 7 are each not hydrogen.
A compound according to Claim 9, wherein:
R and R2 are each hydrogen; and
R3 and R4 are each deuterium (D).