U.S. patent application number 13/685414 was filed with the patent office on 2013-06-13 for 5-pyrrolidinylsulfonyl-isatin derivatives.
This patent application is currently assigned to Universitatsklinikum Munster. The applicant listed for this patent is Universitatsklinikum Munster. Invention is credited to Klaus Kopka, Bodo Levkau, Michael Schafers.
Application Number | 20130149239 13/685414 |
Document ID | / |
Family ID | 36588499 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149239 |
Kind Code |
A1 |
Kopka; Klaus ; et
al. |
June 13, 2013 |
5-PYRROLIDINYLSULFONYL-ISATIN DERIVATIVES
Abstract
The present invention relates to novel 5-pyrrolidinylsulfonyl
isatin derivatives, non-peptidyl Caspase binding Radioligands
(CbRs) and CbR-transporter conjugates derived from said isatin
derivatives, diagnostic compositions comprising said compounds of
the invention and their use for non-invasive diagnostic
imaging.
Inventors: |
Kopka; Klaus; (Munster,
DE) ; Levkau; Bodo; (Munster, DE) ; Schafers;
Michael; (Havixbeck, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universitatsklinikum Munster; |
Munster |
|
DE |
|
|
Assignee: |
Universitatsklinikum
Munster
Munster
DE
|
Family ID: |
36588499 |
Appl. No.: |
13/685414 |
Filed: |
November 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11794878 |
Feb 15, 2008 |
|
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PCT/EP2005/013908 |
Dec 22, 2005 |
|
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13685414 |
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Current U.S.
Class: |
424/1.69 ;
424/1.81; 424/1.89 |
Current CPC
Class: |
C07D 403/12 20130101;
C07D 401/14 20130101; A61K 51/0446 20130101; A61K 51/087 20130101;
A61K 51/08 20130101 |
Class at
Publication: |
424/1.69 ;
424/1.81; 424/1.89 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 51/04 20060101 A61K051/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2005 |
EP |
05000828.3 |
Claims
1. A method of preparing a diagnostic composition for non invasive
imaging of caspase activity in vivo by Single Photon Emission
Computed Tomography (SPECT) or Positron Emission Tomography (PET),
comprising a) providing a non-peptidyl CbR or CbR-transporter
conjugate according to Formula 1, ##STR00079## wherein
R.sub.1--X--Y is methoxymethyl; wherein R.sub.2 is an optionally
substituted alkyl, heteroalkyl, aralkyl, heteroarylalkyl,
carboxymethyl or methyloxycarbonylmethyl group, wherein the
substituents are selected from F, I, Br, OH, NH.sub.2, methylamino,
methoxy, fluoroethyloxy, fluoropropyloxy, trimethylamino, nitro,
tosylate, triflate, mesylate, diazonium N.sub.2.sup.+,
3-fluorobenzoyl, 4-fluorobenzoyl, 4-fluorophenyl, tributylstannyl,
trimethylstannyl, trimethylsilyl, 2-hydrazino-pyridin-5-carbonyl;
or a metal-chelator or a metal-chelator bound to an aralkyl,
aminoalkyl, hydroxyalkyl or piperazin-1-carbonylmethyl group; and
optionally additionally comprises a spacer, linker or molecular
transporter selected from Annexin V, PEG.sub.1-200, an
oligopeptide, polyamide, polysaccharide,
--NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200; wherein R.sub.2
can also contain an amino acid selected from histidine, lysine,
tyrosine, cysteine, arginine and aspartic acid; and wherein R.sub.2
is labelled with a positron-emitting non-metal radionuclide
selected from C-11, N-13, and F-18; and b) formulating the
non-peptidyl CbR or CbR-transporter conjugate in an isotonic or
isohydric solution in an amount effective for use in non invasive
imaging of caspase activity in vivo by SPECT or PET.
2. A method for the diagnosis of disorders connected with
apoptosis, comprising a) administering a 5-Pyrrolidinylsulfonyl
isatin derivative of Formula 1 in vivo to a subject in need of
diagnosis, ##STR00080## wherein, R.sub.1--X--Y is methoxymethyl;
R.sub.2 is an optionally substituted alkyl, heteroalkyl, aralkyl,
heteroarylalkyl, carboxymethyl or methyloxycarbonylmethyl group,
wherein the substituents are selected from F, I, Br, OH, NH.sub.2,
methylamino, methoxy, fluoroethyloxy, fluoropropyloxy,
trimethylamino, nitro, tosylate, triflate, mesylate, diazonium
N.sub.2.sup.+, 3-fluorobenzoyl, 4-fluorobenzoyl, 4-fluorophenyl,
tributylstannyl, trimethylstannyl, trimethylsilyl,
2-hydrazino-pyridin-5-carbonyl; or a metal-chelator or a
metal-chelator bound to an aralkyl, aminoalkyl, hydroxyalkyl or
piperazin-1-carbonylmethyl group; and optionally additionally
comprises a spacer, linker or molecular transporter selected from
Annexin V, PEG.sub.1-200, an oligopeptide, polyamide,
polysaccharide, --NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200 and wherein R.sub.2
can also contain an amino acid selected from histidine, lysine,
tyrosine, cysteine, arginine and aspartic acid; b) detecting formed
enzyme-inhibitor complexes via a nuclear medicinal technique; and
c) determining in a clinical environment whether the apoptosis is
physiological or pathological apoptosis.
3. The method of claim 2, wherein the disorder to be diagnosed is
selected from the group consisting of atherosclerosis, acute
myocardial infarction, chronic heart failure, allograft rejection,
stroke or neurodegenerative disorders.
4-5. (canceled)
6. A method for monitoring therapeutic responses connected with
apoptosis, comprising a) administering a 5-Pyrrolidinylsulfonyl
isatin derivative of Formula 1 in vivo to a subject in need of
monitoring of therapeutic responses in treatment of an already
diagnosed disorder, ##STR00081## wherein, R.sub.1--X--Y is
methoxymethyl; R.sub.2 is an optionally substituted alkyl,
heteroalkyl, aralkyl, heteroarylalkyl, carboxymethyl or
methyloxycarbonylmethyl group, wherein the substituents are
selected from F, I, Br, OH, NH.sub.2, methylamino, methoxy,
fluoroethyloxy, fluoropropyloxy, trimethylamino, nitro, tosylate,
triflate, mesylate, diazonium N.sub.2.sup.+, 3-fluorobenzoyl,
4-fluorobenzoyl, 4-fluorophenyl, tributylstannyl, trimethylstannyl,
trimethylsilyl, 2-hydrazino-pyridin-5-carbonyl; or a metal-chelator
or a metal-chelator bound to an aralkyl, aminoalkyl, hydroxyalkyl
or piperazin-1-carbonylmethyl group; and optionally additionally
comprises a spacer, linker or molecular transporter selected from
Annexin V, PEG.sub.1-200, an oligopeptide, polyamide,
polysaccharide, --NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200 and wherein R.sub.2
can also contain an amino acid selected from histidine, lysine,
tyrosine, cysteine, arginine and aspartic acid; b) detecting formed
enzyme-inhibitor complexes via a nuclear medicinal technique; and
c) monitoring therapeutic response to treatment of an already
diagnosed disorder by determining apoptosis in the subject during a
treatment regime in a clinical environment.
7. The method of claim 6, wherein the method comprises the
monitoring of induction of apoptosis in tumors.
8. The method of claim 6, wherein the apoptosis is
chemotherapy-induced or ionizing radiation-induced apoptosis.
Description
[0001] This application is a continuation application of U.S.
application Ser. No. 11/794,878 filed Feb. 15, 2008, which is a 371
application of PCT application PCT/EP05/13908 filed Dec. 22, 2005,
which claims priority to EP application serial no. 05000828.3 filed
Jan. 17, 2005, the disclosures of which are incorporated herein by
reference.
[0002] The present invention relates to novel
5-pyrrolidinylsulfonyl isatin derivatives, non-peptidyl caspase
binding radioligands (CbR) and CbR-transporter conjugates derived
from said isatin derivatives, diagnostic compositions comprising
said non-peptidyl CbR and CbR-transporter conjugates of the
invention and their use for non invasive diagnostic imaging.
[0003] The present invention relates to the establishment of a
non-invasive molecular imaging technique for the molecular imaging
of caspase activity in vivo. More particularly the inventions
pertain to targeting intracellularly the apoptotic process with
non-peptidyl imaging agents (namely radiolabeled non-peptidyl
caspase inhibitors) that specifically bind to activated caspases
(cysteinyl aspartate-specific proteases). In the following these
new imaging agents are called CbRs which stands for Caspase binding
Radioligands. In addition, to actively translocate the CbRs into
cells the principle of molecular transporter conjugates is applied
[1-5].
[0004] The caspases belong to an enzyme class that play a critical
role in the execution of the programmed cell death (apoptosis).
Thus, this in vivo target offers the feasibility to diagnose
directly diseases (e.g. atherosclerosis, acute myocardial
infarction, chronic heart failure, allograft rejection, stroke,
neurodegenerative disorders etc.) and/or therapeutic responses
(induction of apoptosis in tumors etc.) that correlate immediately
with the apoptotic process. The here presented invention can be
directly applied in non-invasive nuclear medicinal diagnosis with
high clinical impact to differentiate between balanced
(physiological) and unbalanced (pathological) apoptosis using
Single Photon Emission Computed Tomography (SPECT) or Positron
Emission Tomography (PET). In contrast to the known radiolabeled
AnnexinV radiotracers that bind to negatively charged phospholipids
(especially to phosphatidylserine residues) and therefore are not
exclusive markers for apoptosis [6-15], the here described CbRs and
CbR-transporter conjugates should be capable to directly target
apoptosis in vivo in human beings as imaging agents thereby
excluding the imaging of necrotic processes. Consequently, the CbRs
and CbR-transporter conjugates could enhance the effectiveness and
accuracy of therapeutic interventions in the clinics and offer
improved perspectives for the disease management in a variety of
clinical disciplines.
BACKGROUND ART
[0005] Known potent peptide caspase inhibitors (e.g. the
irreversible pan-caspase inhibitor Z-VAD-fmk) [16] are only
moderately selective and possess only poor cell permeabilities
hindering the intracellular targeting of activated caspases
[17].
[0006] In contrast, the 5-pyrrolidinylsulfonyl isatins represent a
rare class of non-peptidyl caspase inhibitors which bind
selectively to the downstream caspases, preferably to the effector
caspases 3 and 7 [19]. The dicarbonyl functionality of the isatins
bind in a tetrahedral manner to the caspase active site. A
thiohemiketal is formed via the electrophilic C-3 carbonyl of the
isatin and the nucleophilic thiolate function of the Cys163 residue
of the enzyme. Consequently, the ability of the caspases to cleave
substrates possessing a P1 Asp residue that reaches into the
primary S1 pocket is blocked (reversible inhibitory effect) [20].
In contrast to the peptidomimetic caspase inhibitors, the
5-pyrrolidinylsulfonyl isatins do not possess an acidic
functionality which may bind in the primary Asp binding pocket.
Various N-substituted 5-pyrrolidinylsulfonyl isatins with the
general formula 1 have been synthesized and disclosed so far
[20-22]. Compounds bearing an allyl-, cyclohexylalkyl- or arylalkyl
substituent at the N-1 nitrogen of the isatin are highly affine
caspase 3 and 7 inhibiting agents. Their potency was proved by in
vitro inhibition of recombinant human caspase 3 and 7 using
standard fluorometric assays [21]. As recently described a
non-peptidyl 5-pyrrolidinylsulfonyl isatin derivative was shown to
possess cardioprotective potential in isolated rabbit hearts after
ischemic injury as well as in cardiomyocytes [17].
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a western blot analysis of active caspase-3 in
apoptotically dying human endothelial cells.
[0008] FIG. 2 is another western blot analysis of active caspase-3
in apoptotically dying human endothelial cells.
[0009] FIG. 3 is a PET Scan of the in vivo biodistribution
behaviour of [.sup.18F]VI in NMRI athymic nude mice.
DESCRIPTION OF THE INVENTION
[0010] The present invention deals with the in vivo imaging of
caspases using the synthetic biomarkers CbR as imaging probes. The
caspases represent a family of intracellularly activated enzymes
that could be targeted by 5-pyrrolidinylsulfonyl isatins, a class
of non-peptidyl caspase inhibitors with high caspase affinity and
moderate lipophilicity which implies a potent cell permeability. In
addition, CbR-transporter conjugates are intended to improve the
translocation of the CbRs into the cells and to advance their
target specificity [1-5]. Within the scope of the invention
chemically modified and radiolabeled 5-pyrrolidinylsulfonyl isatins
should result in potential non-peptidyl CbR tracers as well as
CbR-transporter conjugates that form--after non-invasive
application (preferably i.v.)--intracellular enzyme-inhibitor
complexes by binding of the directly administered CbR or by binding
of CbR released from the administered CbR-transporter conjugate at
the enzyme active site. The specifically formed enzyme-CbR complex
should be detectable in vivo via the nuclear medicinal techniques
Positron Emission Tomography (PET) or Single Photon Emission
Computed Tomography (SPECT), respectively [23-24]. For this purpose
positron-emitting radioactive metals (e.g. Cu-62, Cu-64, Ga-68,
Tc-94m) or non-metals (e.g. C-11, N-13, F-18, Br-76, I-124) for PET
application as well as gamma-emitting radioactive metals (e.g.
Tc-99m, In-111, In-113m, Ga-67) or halogens (e.g. I-123, I-131,
Br-77) for SPECT application have to be introduced into the CbRs.
The radiochemical modification of the 5-pyrrolidinylsulfonyl
isatins should result in similar or even improved pharmacokinetic
characteristics of the CbRs or CbR-transporter conjugates to
achieve intracellular caspase targeting. Suitable
radionuclides/radiosynthons to be used for the radiolabeling of the
isatins are preferably C-11-methyliodide [25] or F-18-fluoride
[26-29] for PET and I-123-iodide [30-31] or Tc-99m-chelators for
SPECT [32-34] that could be coupled each to the biological tracer
resulting in the CbR radiotracers. For first in vitro (e.g.
cellular assays) and ex vivo (e.g. autoradiography) pharmacological
evaluation studies the relevant radioisotopes C-14 and 1-125 can
also be used to establish the CbR ligands in vitro. In summary, the
development of the here presented CbR tracers and CbR-transporter
conjugates offer the realization of the non-invasive in vivo
monitoring of the rate and extent of apoptosis.
[0011] The skeletal structure in formula 1
##STR00001##
[0012] Formula 1: R.sub.1--X--Y=e.g. methoxymethyl, phenoxymethyl;
R.sub.2=e.g. allyl, benzyl, cyclohexylmethyl gives the basis to
modify this putative class of non-peptidyl caspase inhibitors by
inserting imaging moieties (preferably radionuclides for PET or
SPECT) into the residues R.sub.1--X--Y and/or R.sub.2. In such a
way a diagnostic imaging agent for the non-invasive in vivo imaging
of apoptosis can be designed.
[0013] Preferred synthetic 5-pyrrolidinylsulfonyl isatin caspase
inhibitors of the present invention contain substituents as
follows:
[0014] R.sub.1--X--Y=alkyl, heteroalkyl-, alkyloxyalkyl-,
aryloxyalkyl-, alkyloxycarbonyl-, alkylaminoalkyl-,
alkylaminocarbonyl-, aryl-, aryloxyalkyl-, arylthioalkyl-,
heteroaryl-, arylaminoalkyl-, arylaminocarbonyl- (all of the
substituents R.sub.1--X--Y can be radiolabeled with PET or SPECT
radionuclides and can contain spacers or linkers like PEG,
oligopeptides, polyamides, polysaccharides,
--NH--(CH.sub.2).sub.n--NH--, --O--(CH.sub.2).sub.n--O-- or
succinidyl units etc.)
[0015] R.sub.2=alkyl-, heteroalkyl-, allyl- (e.g. fluoroallyl-),
aryl-, arylalkyl- (e.g. benzyl-), heteroarylalkyl- (e.g.
pyridylmethyl-, picolyl-), alkyloxycarbonylmethyl-,
aryloxycarbonylmethyl, Tc-chelators, Ga-chelators (all of the
substituents R.sub.2 can be radiolabeled with PET or SPECT
radionuclides and can contain spacers or linkers like PEG,
oligopeptides, polyamides, polysaccharides,
--NH--(CH.sub.2).sub.n--NH--, --O--(CH.sub.2).sub.n--O--,
succinidyl or 1,4-disubstituted 1,2,3-triazole units etc.)
[0016] In particular the present invention relates to
5-pyrrolidinylsulfonyl isatin derivatives of the formula 1:
##STR00002## [0017] wherein, [0018] X.dbd.--O--, --S--, --NH-- and
Y.dbd.--CH.sub.2--, --C(O)-- [0019] R.sub.1 is an alkyl group such
as methyl, ethyl, or propyl; a substituted alkyl group such as
trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl; an aryl group such
as phenyl, 4-fluorophenyl or 4-iodophenyl; a heteroarylalkyl group
such as 4-picolyl-, 3-picolyl, 2-picolyl-;
6-fluoro-2-picolyl-(=6-fluoropyridyl-2-methyl), 2- or
6-fluoro-3-picolyl (=2- or 6-fluoropyridyl-3-methyl),
2-fluoro-4-picolyl (=2-fluoropyridyl-4-methyl), and optionally
additionally comprises a spacer or linker selected from
PEG.sub.1-200, oligopeptide, polyamide, polysaccharide,
--NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200; [0020] R.sub.2 is
an optionally substituted alkyl, heteroalkyl, aralkyl,
heteroarylalkyl carboxymethyl or methyloxycarbonylmethyl group,
wherein the substituents are selected from F, I, Br, OH, NH.sub.2,
methylamino, isopropylamino, methoxy, fluoroethyloxy,
fluoropropyloxy, trimethylamino, nitro, tosylate, triflate,
mesylate, diazonium --N.sub.2.sup.+, 3-fluorobenzoyl,
4-fluorobenzoyl, 4-fluorophenyl, tributylstannyl, trimethylstannyl,
trimethylsilyl and 2-hydrazino-pyridin-5-carbonyl, such as methyl,
ethyl, propyl, allyl, cyclohexylmethyl, 2-aminoethyl,
3-aminopropyl, 2-methylaminoethyl, 3-methylaminopropyl,
2-hydroxyethyl, 3-hydroxypropyl, 2-fluoroethyl, 3-fluoropropyl, 2-
or 3-fluoroallyl, benzyl, 4-benzyloxybenzyl, 4-fluorobenzyl,
4-(2-fluoroethyloxy)benzyl, 4-(3-fluoropropyloxy)benzyl,
4-hydroxybenzyl, 4-iodobenzyl, 4-methoxybenzyl,
piperazin-1-carbonylmethyl, 4-methyl-piperazin-1-carbonylmethyl,
4-isopropyl-piperazin-1-carbonylmethyl,
4-(3-fluoropropyl)piperazin-1-carbonylmethyl, 4-picolyl-,
3-picolyl, 2-picolyl-;
6-fluoro-2-picolyl-(=6-fluoropyridyl-2-methyl), 2- or
6-fluoro-3-picolyl (=2- or 6-fluoropyridyl-3-methyl),
2-fluoro-4-picolyl (=2-fluoropyridyl-4-methyl); [0021] or a
metal-chelator (e.g. hydrazinonicotinamide HYNIC, histidine, DOTA
and DOTA derivatives, MAG.sub.3, BAT, DTPA, EDTA, DAD, Pn216,
carbaPn216, Pn44 etc.) or a metall-chelator bound to an aralkyl,
aminoalkyl, hydroxyalkyl or a piperazin-1-carbonylmethyl group;
[0022] and optionally additionally comprises a spacer, linker or
molecular transporter selected from AnnexinV, PEG.sub.1-200,
oligopeptide, polyamide, polysaccharide,
--NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200 and wherein R.sub.2
can also contain an amino acid selected from histidine, lysine,
tyrosine, cysteine, arginine, aspartic acid (e.g. cysteine as
linker or spacer bound to octaarginine (see Scheme 6) or Annexin V
(see Scheme 7) in CbR-transporter conjugates; or histidine as
chelator in .sup.99mTc-labeled CbR (Table 4, 2.sup.nd
example)).
[0023] In a preferred embodiment the group R.sub.1--X--Y is an
alkoxyalkyl, aryloxyalkyl, arylthioalkyl, alkyloxycarbonyl,
aryloxycarbonyl or arylaminocarbonyl group.
[0024] Furthermore it is preferred that R.sub.2 is an aralkyl group
or a Tc-, Cu-, Ga- or In-chelator or a Tc-, Cu-, Ga- or In-chelator
bound to an aralkyl, aminoalkyl, hydroxyalkyl or a
piperazin-1-carbonyl group.
[0025] Moreover, compounds are preferred, wherein R.sub.1--X--Y
and/or R.sub.2 additionally comprises a spacer, linker or molecular
transporter selected from AnnexinV, polyethylene glycol
PEG.sub.1-200, from an oligopeptide (e.g. heptaarginine,
octaarginine, homopolyarginine, heteropolyarginine), from a
polyamide, from a polysaccharide,
--NHC(O)--((CH.sub.2).sub.n--NH--C(O)).sub.m--,
--O--((CH.sub.2).sub.n--O).sub.m--, succinyl and 1,4-disubstituted
1,2,3-triazole units, wherein n=0-6 and m=1-200.
[0026] In a further embodiment the present invention provides
non-peptidyl CbRs (Caspase binding Radioligands) having the formula
as defined in any one of claims 1 to 4, wherein at least one of the
substituents R.sub.1--X--Y or R.sub.2 is labelled with a
positron-emitting metal radionuclide selected from Cu-62, Cu-64,
Ga-68 and Tc-94m, a positron-emitting non-metal radionuclide
selected from C-11, N-13, F-18, Br-76 and 1-124, gamma- and/or
beta-emitting metal radionuclide selected from Tc-99m, In-111,
In-113m, Ga-67 and Cu-67 and gamma- and or/beta-emitting non-metal
radionuclide selected from C-14, I-123, I-125, I-131 and Br-77.
[0027] In a preferred embodiment of the CbR the group R.sub.1--X--Y
is 4-[.sup.123I]iodophenoxymethyl-,
4-[.sup.18F]fluorophenoxymethyl-,
[.sup.18F]trifluoromethyloxymethyl-,
2-[.sup.18F]fluoroethyloxymethyl,
3-[.sup.18F]fluoropropyloxymethyl,
2-[.sup.18F]fluoroethyloxycarbonyl,
4-[.sup.11C]methyloxyphenoxymethyl, or [.sup.11C]methyloxycarbonyl,
and/or R.sub.2 is AnnexinV-S-Cys-acyloxybenzyl-, thus forming a
phosphatidyl serinopathy-dependent CbR-transporter conjugate (see
Scheme 7); Arg.sub.8-S-Cys-acyloxybenzyl-(see Scheme 6), thus
forming a phosphatidyl serinopathy-independent CbR-transporter
conjugate; 3-[.sup.123I]iodo-4-hydroxybenzyl-,
4-[.sup.123I]iodobenzyl-, [.sup.11C]methyl,
3-[.sup.11C]methylaminopropyl,
3-(2'-[[.sup.11C]isopropyl)aminopropyl, [.sup.11C]methyloxycarbonyl
methyl, 4-[.sup.11C]methyloxybenzyl,
4-(2-[.sup.18F]fluoroethyloxy)benzyl,
4-(3-[.sup.18F]fluoropropyloxy)benzyl,
4-[.sup.11C]methyl-piperazin-1-carbonylmethyl,
4-(2'-[.sup.11C]isopropyl)piperazin-1-carbonylmethyl,
4-(3-[.sup.18F]fluoropropyl)piperazin-1-carbonylmethyl),
6-[.sup.18F]fluoro-2-picolyl-(=6-[.sup.18F]fluoropyridyl-2-methyl),
2- or 6-[.sup.18F]fluoro-3-picolyl (=2- or
6-[.sup.18F]fluoropyridyl-3-methyl), 2-[.sup.18F]fluoro-4-picolyl
(=2-[.sup.18F]fluoropyridyl-4-methyl);
[.sup.11C]methyloxycarbonylmethyl, a .sup.99mTc-chelator-group, or
a .sup.68Ga-chelator-group.
[0028] Moreover the present invention provides a diagnostic
composition comprising a non-peptidyl CbR (Caspase binding
Radioligand) and/or a CbR-transporter conjugate as described
above.
[0029] In a further embodiment the present invention provides the
use of a non-peptidyl CbR and/or a CbR-transporter conjugate as
described above for the preparation of a diagnostic composition for
non-invasive imaging of caspase activity in vivo by Single Photon
Emission Computed Tomography (SPECT) or Positron Emission
Tomography (PET) [23-24].
[0030] The diagnostic compositions according the present invention
in particular be used for the diagnosis of disorders connected with
apoptosis and/or monitoring therapeutic responses connected with
apoptosis, thus in the diagnosis of atherosclerosis, acute
myocardial infarction, chronic heart failure, allograft rejection,
stroke or neurodegenerative disorders.
[0031] In a further preferred embodiment the diagnostic
compositions according the present invention may be used in the
monitoring of induction of apoptosis in tumors, in particular for
monitoring chemotherapy-induced or ionizing radiation-induced
apoptosis.
[0032] It will be appreciated by the person of ordinary skill in
the art that the present invention also comprises all stereoisomers
of the compounds according to the invention, including its
enantiomers and diastereomers. Individual stereoisomers of the
compounds according to the invention can be substantially present
pure of other isomers, in admixture thereof or as racemates or as
selected stereoisomers.
[0033] The nomenclature of the compound numbering used herein is as
follows:
[0034] I, II, III, IV, V etc.=non-radioactive reference compounds
of PET-compatible CbR tracers or CbR-transporter conjugates
[.sup.11C]II, [.sup.11C]III, [.sup.18F]IV etc. .dbd.PET-compatible
CbR tracers or CbR-transporter conjugates Ia, Ib, Ic, IIa, IIa,
IIb, IIc etc.=precursors of PET-compatible CbR tracers or
CbR-transporter conjugates for radiolabeling 1, 2, 3, 4, 5
etc.=non-radioactive reference compounds of SPECT-compatible CbR
tracers or CbR-transporter conjugates
[0035] [.sup.123I]1, [.sup.123I]2, [.sup.99mTc]3
etc.=SPECT-compatible CbR tracers or CbR-transporter conjugates
[0036] 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c etc.=precursors of
SPECT-compatible CbR tracers or CbR-transporter conjugates for
radiolabeling
[0037] especially: Iaa, 1bb, IIcc etc=intermediates of precursor
compounds Ia, 1b, IIc etc.
[0038] The caspase 3 and 7 selective isatin sulfonamides
(S)-1-methyl-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)isatin I
(K.sub.i(Caspase 3)=15 nM) and
(S)-5-(1-[2-(methoxymethyl)pyrrolidinyl]-sulfonyl)isatin IIa
(K.sub.i (Caspase 3)=60 nM) were chosen as lead structures to
develop CbRs (Scheme 1) [20].
##STR00003##
[0039] Concerning the here disclosed invention compound 11a is an
example of a CbR precursor that could be radiolabeled by
C-11-methylation of the N-1 isatin nitrogen resulting in the
potential PET-compatible CbR
(S)-5-(1-[2-(methoxymethyl)pyrrolidinyl]sulfonyl)-1-[.sup.11C]methyl-isat-
in [.sup.11C]II. Compound I represents the non-radioactive
counterpart of a PET-compatible CbR which should be available by
authentic radiolabeling (here: again N--[.sup.11C]methylation) of
the desmethyl precursor
(S)-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)isatin la
resulting in the feasible PET-compatible
CbR(S)-1-[.sup.11C]methyl-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)is-
atin [.sup.11C]I (Scheme 2).
##STR00004##
[0040] The F-18-fluoroalkylation of the isatin N-1 nitrogen should
be also possible (e.g. nucleophilic substitution reaction of a
corresponding 3-tosylpropyl precursor with
[.sup.18F]K(Kryptofix222)F). In table 1 further PET-compatible CbRs
are summarised that are achievable using the radiosynthons
[.sup.18F]F.sub.2, [.sup.18F]K(Kryptofix222)F,
[.sup.18F]F--(CH.sub.2).sub.n-LG (n=1-3, LG=Tos, Hal, Tf, Ms),
[.sup.11C]CH.sub.3X (X.dbd.I, Tf) or [.sup.11C]acetone.
TABLE-US-00001 TABLE 1 Selection of CbRs for PET, radiolabeled in
R.sub.2 (LG = Tos, Tf, Ms) Precursor PET-Tracer Lead structure
R.sub.2 = R.sub.2 = ##STR00005## H ##STR00006## ##STR00007##
.sup.11CH.sub.3 ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031##
##STR00032## ##STR00033## wherein Tos = tosylate Tf = triflate Ms =
mesylate
TABLE-US-00002 TABLE 2 Selection of CbRs for PET, rediolabeled in
R.sub.1 (LG = Tos, Tf, Ms) Precursor PET-Tracer Lead structure
R.sub.1 = R.sub.1 = ##STR00034## ##STR00035## ##STR00036##
##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048##
[0041] In addition to the PET-compatible CbR tracers to be
developed especially SPECT-compatible CbR tracers are attractive
for commercialisation purposes owing to the somewhat longer lived
SPECT nuclides I-123 (T.sub.1/2=13.2 h) and Tc-99m (T.sub.1/2=6 h).
This circumstance allows professional shipment and distribution of
the corresponding CbR tracers as radiopharmaceuticals after
realisation of the necessary clinical phase studies regarding the
pharmaceutical as well as the radiation protection guidelines. In
contrast to C-11-labeled CbR tracers (T.sub.1/2=20 min), the
commercialisation of F-18-labeled (T.sub.1/2=110 min) and
Ga-68-labeled (T.sub.1/2=67.6 min) CbR ligands would be also
possible but is limited to a so called satellite distribution
system.
[0042] In scheme 3 the I-123-labeled SPECT-compatible CbR tracer
(S)-1-(4-[.sup.123I]iodobenzyl)-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfo-
nyl)isatin [.sup.123I]1 is displayed which is available by
iododemetalation reaction [30] of the precursor
(S)-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)-1-(4-(tributylstannyl)b-
enzyl)isatin 1a (For the synthesis of non-radioactive SPECT CbR
references and radiolabeled SPECT CbR model tracers please see
below).
##STR00049##
##STR00050##
TABLE-US-00003 TABLE 3 Selection of I-123-labeled CbRs for SPECT
Precursor SPECT-Tracer Lead structure R.sub.2 = R.sub.2 =
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
Precursor SPECT-Tracer Lead structure R.sub.1 = R.sub.1 =
##STR00056## ##STR00057## ##STR00058## ##STR00059##
##STR00060##
##STR00061##
[0043] Furthermore, the isatin N-1 nitrogen provides a promising
position for the coupling with Tc-99m-chelators to yield potential
Tc-99m-technetium CbR tracers. A modified isatin lead structure is
suggested in scheme 5 which offers the opportunity to link a
variety of Tc-99m-technetium chelates with N.sub.4, N.sub.2O.sub.2,
N.sub.2S.sub.2, N.sub.3S, N.sub.3O.sub.3, N.sub.2O(CO).sub.3 etc.
coordination sphere. Examples are given as follows:
[0044] All the compounds derived by the chelator modifications (see
items 1.-5., below) represent precursors for the radiosyntheses of
Tc-99m-SPECT-compatible CbRs which are available via ordinary kit
preparation procedures.
[0045] Tc-chelators according to the present invention are e.g. the
compounds as listed below, however, they are not limited to them
[34]:
1.
##STR00062## [0046] Further chelators according to the present
invention are e.g. derivatives of MAG3 (mercapto acetyl triglycine)
or tripodand ligands with N.sub.3S--, N.sub.2S.sub.2-etc.
coordination sphere, which could be also linked to the isatin N-1
nitrogen using similar spacers (alkyl, polyethylenglycol (PEG),
oligopeptide, polyamide, oligosaccharide spacers etc.) in the
manner presented in scheme 5. [0047] Moreover, also the chelators
Pn44, Pn216, carbaPn216 or BAT or any other suitable chelator with
N.sub.4--, N.sub.2O.sub.2--, N.sub.2S.sub.2--, N.sub.3S-- etc.
coordination sphere may be attached to the lead structure by the
substitution of the corresponding halogeno isatin derivative via
the NH.sub.2 residues of the chelators (scheme 5: coupling moiety
X.ident.NH--). [0048] Additional chelators that may be used in the
present invention are the chelators DAD, MAG3 or any other suitable
chelator with N.sub.4--, N.sub.2O.sub.2--, N.sub.2S.sub.2--,
N.sub.3S--etc. coordination sphere and may be attached to an amino
group of a suitable precursor by an amidation reaction (see Scheme
5: coupling moiety X.ident.N--C(.dbd.O)--). [0049] Moreover the
chelators Pn44, Pn216, carbaPn216 or BAT may be attached to the
carboxy group of a suitable precursor by an amidation reaction
(scheme 5: coupling moiety X.ident.C(.dbd.O)--N--).
[0050] In addition, further SPECT-compatible Tc-99m-labeled CbRs
are summarised in table 4 that are achievable by histidine [35]
and/or HYNIC chelators [36]attached to the isatin N-1 position via
alkyl, polyethylenglycol (PEG), oligopeptide, polyamide and/or
oligosaccharide spacers or via the amino group of a suitable
precursor by an amidation reaction (see Scheme 5: coupling moiety
X.ident.N--C(.dbd.O)--).
TABLE-US-00004 TABLE 4 Tc-99m-labeled CbRs for SPECT (Lead
structure see Table 1) Precursor SPECT-Tracer R.sub.2 = R.sub.2 =
##STR00063## ##STR00064## ##STR00065## ##STR00066##
[0051] Furthermore, the isatin N-1 nitrogen provides a promising
position for the coupling with Ga-68-chelators to yield potential
Ga-88-gallium CbR tracers for PET (Table 5).
TABLE-US-00005 TABLE 5 Example of a Ga-68-labeled CbR for PET (Lead
structure see Table 1) Precursor PET-Tracer R.sub.2 = R.sub.2 =
##STR00067## ##STR00068##
[0052] Furthermore, the isatin N-1 nitrogen provides a position for
the coupling with molecular transporters like hepta- or
octaarginine [1-4] or Annexin V [5] to yield potential
CbR-transporter conjugates for SPECT and/or PET.
[0053] In a further aspect the present invention provides
CbR-transporter conjugates which may be used for an active caspase
targeting. Hereby the substituent R.sub.1--X--Y of the isatin
structure is radiolabeled in contrast to the labeling for the
unconjugated CbR wherein particularly the R.sub.2 substituent is
radiolabeled.
[0054] The following SPECT- and PET-compatible R.sub.1--X--Y groups
are preferred: [0055] 4-[.sup.123I]iodophenoxymethyl-, [0056]
4-[.sup.18F]fluorophenoxymethyl-, [0057]
2-[.sup.18F]fluoroethyloxymethyl, [0058]
3-[.sup.18F]fluoropropyloxymethyl, [0059]
2-[.sup.18F]fluoroethyloxycarbonyl, [0060]
[.sup.11C]methyloxycarbonyl
[0061] The CbR-transporter conjugates according to the present
invention, i.e. the linking of suitably radiolabeled CbRs with
so-called molecular transporters may be used to introduce the CbR
actively into the cells.
[0062] The CbR is linked via the N-1 nitrogen atom of the isatin
structure. The CbR-transporter conjugates according the present
invention will release the CbR after intracellular intake via
cleavage or due to lysosomal degradation of the molecular
transporter and thus finally binds to the caspases.
[0063] By this implementation of the releasable drug-transporter
conjugate approach as described by Wender et al. [1-4], a profound
enhancement of sensitivity of caspase detection can be obtained due
to the active transport via the membrane into the cells thus also
providing an improved apoptosis imaging.
TABLE-US-00006 TABLE 6 Selection of radiolabeled CbR-transporter
conjugates for SPECT or PET Lead structure R.sub.1--X--Y = R.sub.2
= PET ##STR00069## [.sup.18F]F(CH.sub.2).sub.2OCH.sub.2
[.sup.18F]F(CH.sub.2).sub.3OCH.sub.2
4-[.sup.18F]F--C.sub.6H.sub.4--OCH.sub.2
[.sup.18F]F(CH.sub.2).sub.2OCO [.sup.11C]CH.sub.3OCO
[.sup.18F]F(CH.sub.2).sub.2OCH.sub.2
[.sup.18F(CH.sub.2).sub.3OCH.sub.2
4-[.sup.18F]F--C.sub.6H.sub.4--OCH.sub.2
[.sup.18F]F(CH.sub.2).sub.2OCO [.sup.11C]CH.sub.3OCO
Arg.sub.8-S-Cys-acyloxybenzyl '' '' '' ''
AnnexinV-S-Cys-acyloxybenzyl '' '' '' '' CH.sub.3OCH.sub.2
.sup.58Ga-AnnexinV-S-Cys-acyloxybenzyl C.sub.6H.sub.5OCH.sub.2 ''
CH.sub.3OCH.sub.2 .sup.18F-AnnexinV-S-Cys-acyloxybenzyl
C.sub.6H.sub.5OCH.sub.2 '' SPECT
4-[.sup.123I]I--C.sub.6H.sub.4--OCH.sub.2
Arg.sub.8-S-Cys-acyloxybenzyl
4-[.sup.123I]I--C.sub.6H.sub.4--OCH.sub.2
AnnexinV-S-Cys-acyloxybenzyl CH.sub.3OCH.sub.2
.sup.99mTc-AnnexinV-S-Cys-acyloxybenzyl C.sub.6H.sub.5OCH.sub.2 ''
CH.sub.3OCH.sub.2 .sup.123I-AnnexinV-S-Cys-acyloxybenzyl
C.sub.6H.sub.5OCH.sub.2 ''
[0064] As molecular transporters according to the present invention
the following may be used:
Annexin V
Heptaarginine
Oktaarginine
Heteropolyarginines
Homopolyarginines
[0065] Targeted drug delivery using the CbRs according to the
present invention can be distinguished between: [0066] A
phosphatidyl serinopathy-independent transport of the
CbR-polyarginine conjugate, [0067] B phosphatidyl
serinopathy-dependent transport of the CbR-AnnexinV conjugate.
[0068] C dual specificity probes for the detection of
apoptosis.
Synthesis of Phosphatidyl Serinopathy-Independent CbR-Polyarginine
Conjugates.
[0069] Two different species of CbR-transporter-conjugates are
synthesized according to the present invention.
[0070] A CbR-octaarginine conjugate which may optionally be labeled
at the R.sub.1--X--Y group with [.sup.11C] or
[.sup.18F](PET-Tracer) or with [.sup.123I](SPECT-Tracer).
[0071] This is exemplified in scheme 6 for a [.sup.18F]-labeled
target conjugate, using a modified Balz-Schiemann-Reaktion for the
[.sup.18F]-fluorination labeling [28-29]. A [.sup.123I]-labeling
can be achieved via a tributylstannyl intermediate [31].
[0072] Moreover a nitro moiety can be introduced into the
phenoxyprolinol of the group R.sub.1--X--Y to prepare a subsequent
reduction, diazotisation and subsequent [.sup.18F]-fluorination. In
a further embodiment a R.sub.1--X--Y tosylate intermediate (e.g.
R.sub.1--X--Y=3-tosylpropyloxymethyl) can be [.sup.18F]fluorinated
with [.sup.18F]K(Kryptofix222)F (see Table 6,
R.sub.1--X--Y=3-[.sup.18F]fluoropropyloxymethyl). The modification
of the isatin-nitrogen substituent can be achieved via a protected
p-hydroxybenzylfunction and the molecular transporter such as
octaarginine, can be bound to the CbR via a modified
cysteine-bridge.
[0073] Various substituents R (see Scheme 6) can be used to obtain
different in vitro und in vivo-stabilities of the conjugate.
[0074] Depending on the nature of R CbR is relased by an
intramolecular substitution within minutes to several hours,
whereby the active ingredient acts as a leaving group [1-2]. The
last 5 steps of the synthesis beginning with the
[.sup.18F]-labeling are carried out with an automated synthesis
module.
##STR00070##
Synthesis of Phosphatidyl Serinopathy-Dependent CbR-AnnexinV
Conjugates.
[0075] Similarly also a [.sup.11C], [.sup.18F]- or
[.sup.123I]-CbR-AnnexinV conjugate is synthesized.
[0076] Scheme 7 shows the synthesis in accordance with the
synthesis of 4-[.sup.18F]fluorobenzoyl-annexinV ([.sup.18]FBA)
[37]which is congruent to the above synthesis of the corresponding
octaarginine conjugate.
##STR00071##
[0077] An accordingly modifiable isatin which can be prepared for
radioactive labeling may be radiolabeled via automated synthesis
and coupled with the protein AnnexinV.
Synthesis of Dual Specificity Probes for Apoptosis.
[0078] Phosphatidyl Serinopathy-Dependent CbR-AnnexinV Conjugates
May be Labeled with Different Radioisotopes.
[0079] AnnexinV may be first labeled with [.sup.99mTc] or
[.sup.123I] and the thus obtained product may be used as a
radiosynthon for conjugation with non-radiolabeled CbR. In a
double-nuclide study first the SPECT-compatible conjugate
--[.sup.99mTc]- or [.sup.123I]-labeled at the Annexin V site--and
subsequently the same but analogous PET-compatible conjugate
--[.sup.18-F]- or [.sup.11C]-labeled at the CbR site--can be
applied. The result is an image depicting phosphatidyl serinopathy
using SPECT (e.g. CbR-[.sup.99mTc]Tc-HYNIC-AnnexinV) and depicting
intracellular caspase-CbR interaction using PET (e.g.
[.sup.18F]fluoroCbR-AnnexinV). Of course AnnexinV can also be
labeled as a PET-compatible phosphatidyl serinopathy
CbR-transporter conjugate with a .sup.68Ga-chelator and
subsequently the CbR portion may be labeled with
[.sup.123I]reflecting the Caspase-CbR interaction using SPECT.
[0080] This provides a meaningful tool for the detection of
individual cell reactions to a potentially deadly stimulus which
can be used to differentiate between potentially reversible
PS-exposition in myocardial ischemia (determining the area at risk)
and apoptotic tissue (moribund by caspases) (see Table 6 for
preferable variations).
Synthesis of the Compounds of the Invention
1. Synthesis of 5-pyrrolidinylsulfonyl Isatins
##STR00072##
[0082] The compounds of structures I and IIa were synthesised
according to Lee et al. [20-21]. In scheme 8 the general synthesis
route for the preparation of the potential PET-compatible CbR
radiotracer
(S)-1-[.sup.11C]methyl-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)isati-
n [.sup.11C]I is exemplified.
[0083] It will be apparent for the person of ordinary skill in the
art how to vary the above scheme 8 to arrive at the other compounds
with various R.sub.1--X--Y as well as R.sub.2 substituents.
[0084] A General Procedure for the Synthesis of New Isatin
Derivatives is as Follows:
[0085] 5-[1-(2-phenoxymethylpyrrolidinyl)-sulfonyl]isatin Ia or
5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa (Scheme 9)
were placed in a round bottom flask and dissolved in 50 mL of dry
dimethylformamide. Under argon-atmosphere 1 equivalent of sodium
hydride was added. During stirring for 30 minutes at room
temperature the solution became dark red. Afterwards an access of
the benzylbromide was added and the reaction mixture was stirred
for another 3 hours at room temperature. In the case of
benzylchlorides the reaction mixture was warmed up to 80.degree. C.
Removal of the solvent in vacuo afforded the crude product, which
was purified by silica gel chromatography.
##STR00073##
EXAMPLES
1.1. PET-Compatible References (I, II, III, IV, V etc.)
1.1.1 Synthesis of
(S)-(+)-1-(methyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin
I
(Compound I was Synthesised in Accordance to ref. [21].)
[0086] (S)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia
(500 mg, 1.3 mmol) was reacted with sodium hydride (52 mg, 1.3
mmol, 60% in mineral oil) and methyl iodide (553 mg, 3.9 mmol, 0.24
mL) as described in the general procedure and stirred 5 h at room
temperature. The crude orange product was purified by silica gel
chromatography (diisopropyl ether:acetone 6:1) and yielded I as an
orange solid.
[0087] Yield: 280 mg (0.7 mmol, 54%). .sup.1H-NMR (300 MHz,
d.sub.6-DMSO): .delta. [ppm]: 1.58-1.67, 1.83-1.93, 3.20-3.37,
3.39-3.43, 3.89-4.11 (m, 9H, pyrrolidine-CH/H.sub.2, OCH.sub.2),
3.17 (s, 3H, NCH.sub.3), 6.90-6.93 (m, 3H, Ar--H), 7.28 (d, 1H,
.sup.3J.sub.H,H=8.1 Hz, isatin-H), 7.25-7.31 (m, 2H, ArH), 7.81 (d,
1H, .sup.4J.sub.H,H=1.8 Hz, isatin-H), 8.12 (dd, 1H,
.sup.3J.sub.H,H=8.1 Hz, .sup.4J.sub.H,H=1.8 Hz, isatin-H).
.sup.13C-NMR (75 MHz, d.sub.6-DMSO): .delta. [ppm]: 24.0, 28.8
(pyrrolidine-CH.sub.2), 26.7 (NCH.sub.3), 49.6, 58.7
(pyrrolidine-NCH.sub.2), 69.9 (OCH.sub.2), 111.6, 114.8 (ArCH),
118.1 (q-ArCCO), 121.2, 122.8, 129.9, 131.8 (ArCH), 137.1
(q-ArCSO.sub.2), 154.6 (q-ArCNH), 158.5 (q-ArC), 158.8 (COCONH),
187.5 (COCONH).
[0088] MS (EI): m/e (intensity %): 400 (M.sup.+, 28), 293 (100),
224 (76), 160 (48).
[0089] Anal (C.sub.20H.sub.20N.sub.2O.sub.5S) C, H, N; calcd: C,
59.99; H, 5.03; N, 7.00. found: C, 59.90; H, 4.95; N, 6.99.
1.1.2 Synthesis of
(S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-1-methyl-isatin
II
[0090] 485 mg (1.25 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 50 mg (1 mmol) sodium hydride (60% in mineral oil)
and 248 mg (1.5 mmol; 0.1 mL) methyliodide as described in the
general procedure and stirred 2 hours at room temperature. The
crude dark orange product was purified by silica gel chromatography
(diisopropylether/acetone 4:1) and yielded 175 mg of II (0.58 mmol;
46%) as an orange solid.
[0091] mp.: 143-144.degree. C.
[0092] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.61, 1.82,
3.18, 3.51, 3.52 and 3.68 (bs, 9H, pyrrolidine-CH.sub.2 and CH);
3.24 (s, 3H, OCH.sub.3); 3.28 (s, 3H, OCH.sub.3); 6.94-6.98 (m, 1H,
isatin-H); 7.96 (bs, 1H, isatin-H); 8.02-8.04 (m, 1H,
isatin-H).
[0093] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=26.1, 28.6,
30.8, 32.3, 51.3, 61.0, 61.2, 76.8, 112.2, 119.2, 126.3, 136.0,
139.4, 156.0, 159.8, 183.8.
[0094] MS (MALDI-TOF) m/e: 361
(C.sub.15H.sub.18N.sub.2O.sub.5S+Na).sup.+.
[0095] Anal. Calc. for C.sub.15H.sub.18N.sub.2O.sub.5S: C, 53.24;
H, 5.36; N, 8.28. found: C, 53.54; H, 5.34; N, 8.49.
1.1.3 Synthesis
of(S)-1-(4-methoxybenzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isat-
in II
[0096] 386 mg (1 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 40 mg (1 mmol) sodium hydride (60% in mineral oil)
and 670 mg (3 mmol) 4-methoxybenzylchloride as described in the
general procedure. The crude dark orange product was purified by
silica gel chromatography (diisopropylether/acetone 8:1) and
yielded 310 mg of III (0.61 mmol; 61%) as an orange solid.
[0097] mp.: 152.degree. C.
[0098] .sup.1H-NMR (300 MHz, CDCl.sub.3): 6 (ppm)=1.77-1.81,
2.00-2.04, 3.22-3.26, 3.47-3.51 and 4.15-4.19 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.80 (s, 3H, OCH.sub.3); 3.88-3.98
(m, 2H, PhOCH.sub.2); 4.86 (s, 2H, NCH.sub.2Ph); 6.81-6.98 (m, 6H,
PhH, isatin-H); 7.21-7.28 (m, 4H, PhH); 7.95 (dd, 1H, J=1.5 Hz, 8.4
Hz, isatin-H); 8.01 (d, 1H, J=1.8 Hz, isatin-H).
[0099] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=26.8, 28.7,
30.0, 43.6, 49.2, 55.0, 58.3, 110.9, 114.0, 114.3, 117.1, 120.7,
123.9, 125.1, 128.4, 128.7, 129.2, 133.8, 136.7, 153.0, 157.4,
157.9, 159.4, 181.4.
[0100] MS (EI-directly intake): m/e (intensity %): 506 (M.sup.+,
17); 399 (M-CH.sub.2OPh.sup.+, 100).
[0101] Anal. Calc. for C.sub.27H.sub.26N.sub.2O.sub.6S: C, 64.02;
H, 5.17; N, 5.53. found: C, 63.89; H, 5.34; N, 5.51.
1.1.4 Synthesis of
(S)-1-(4-methoxybenzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
IV
[0102] 500 mg (1.54 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 61 mg (1.54 mmol) sodium hydride (60% in mineral
oil) and 723 mg (0.65 mL, 4.62 mmol) 4-methoxybenzylchloride as
described in the general procedure. The crude dark orange product
was purified by silica gel chromatography (petrolether/ethyl
acetate 3:1-1:1) and yielded 462 mg of IV (1.04 mmol; 68%) as an
orange foam.
[0103] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.65-1.69,
1.85-1.89, 3.10-3.13, 3.35-3.41 (m, 7H, pyrrolidine-CH.sub.2 and
CH); 3.33 (s, 3H, OCH.sub.3); 3.52-3.54 and 3.72-3.75 (m, 2H,
PhOCH.sub.2); 3.79 (s, 3H, PhOCH.sub.3); 4.90 (s, 2H, NCH.sub.2Ph);
6.87 (d, 1H, J=8.1 Hz, isatin-H); 6.94 (d, 2H, J=8.4 Hz, PhH); 7.25
(d, 2H, J=8.4 Hz, PhH); 7.98 (dd, 1H, J=1.8 Hz, 8.1 Hz, isatin-H);
8.02 (d, 1H, J=1.8 Hz, isatin-H).
[0104] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=24.1, 28.2,
28.9, 44.0, 49.3, 55.4, 59.1, 74.9, 111.2, 114.6, 117.6, 124.4,
125.8, 129.1, 134.1, 137.3, 153.4, 157.8, 159.8, 181.9.
[0105] MS (EI-directly intake): m/e (intensity %): 444 (M*, 90);
399 (M-CH.sub.2OCH.sub.3, 100).
[0106] Anal. Calc. for C.sub.22H.sub.24N.sub.2O.sub.6S: C, 59.45;
H, 5.44;
[0107] N, 6.30. found: C, 59.36; H, 5.46; N, 6.05.
1.1.5 Synthesis of
(S)-1-(4-(2-fluoroethoxy)benzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfon-
yl]isatin V
[0108] 374 mg (1 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 60 mg (1.5 mmol) sodium hydride (60% in mineral oil)
and 1.18 g (5 mmol) 4-(2-fluoroethoxy)benzylbromide as described in
the general procedure. The crude dark orange product was purified
by silica gel chromatography (cyclohexane/ethyl acetate 1:1) and
yielded 170 mg of V (0.32 mmol; 32%) as an orange foam.
[0109] mp.: 145-146.degree. C.
[0110] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.73-1.84,
1.94-2.06, 3.18-3.26, 3.45-3.51 and 4.13-4.14 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.94-3.97 (m, 2H, PhOCH.sub.2);
4.14-4.16, 4.22-4.25, 4.63-4.66, 4.79-4.82 (each m, each 1H,
PhCH.sub.2CH.sub.2F); 4.85 (s, 2H, NCH.sub.2Ph); 6.79-6.92 (m, 6H,
PhH and isatin-H); 7.15-7.27 (m, 4H, PhH); 7.93 (dd, 1H, J=1.5 Hz,
8.4 Hz, isatin-H); 7.98 (d, 1H, J=1.5 Hz, isatin-H).
[0111] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=24.2, 27.9,
29.1, 43.9, 49.5, 58.7, 67.4, 69.2, 80.7, 82.9, 111.2, 114.4,
115.4, 117.5, 121.1, 124.3, 126.5, 128.2, 129.2, 129.5, 134.2,
137.1, 153.3, 157.8, 158.3, 158.6, 181.8.
[0112] .sup.19F-NMR (282 MHz, CDCl.sub.3): .delta.
(ppm)=-224.0.
[0113] MS (EI-directly intake): m/e (intensity %): 538 (M.sup.+,
8); 431 (M-CH.sub.2OPh, 100).
[0114] Anal. Calc. for C.sub.28H.sub.27N.sub.2FO.sub.6S: C, 62.44;
H, 5.05; N, 5.20. found: C, 62.70; H, 5.02; N, 4.91.
1.1.6 Synthesis of
(S)-1-(4-(2-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfon-
yl]isatin VI
[0115] 324 mg (1.00 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 60 mg (1.5 mmol) sodium hydride (60% in mineral oil)
and 1.18 g (5.16 mmol) 4-(2-fluoroethoxy)benzylbromide as described
in the general procedure. The crude orange product was purified by
silica gel chromatography (petrolether/ethyl acetate 3:1) and
yielded 313 mg of VI (0.66 mmol; 66%) as a yellow powder.
[0116] mp.: 68-69.degree. C.
[0117] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.54-1.64,
1.78-1.84, 3.00-3.07, 3.45-3.61 and 3.60-3.65 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.25 (s, 3H, OCH.sub.3); 3.25-3.35
(m, 2H, CH.sub.3OCH.sub.2); 4.08, 4.17, 4.58, 4.74 (each dd, each
1H, J=5.2 Hz, PhCH.sub.2CH.sub.2F); 4.83 (s, 2H, NCH.sub.2Ph);
6.81-6.87 (m, 3H, PhH and isatin-H); 7.19-7.23 (m, 2H, PhH); 7.89
(dd, 1H, J=1.5 Hz, 8.1 Hz, isatin-H); 7.95 (d, 1H, J=1.5 Hz,
isatin-H).
[0118] .sup.13C-NMR (75 MHz, CDCl.sub.3): S (ppm)=24.5, 29.2, 44.3,
49.7, 59.4, 67.8, 75.2, 81.0, 83.3, 111.6, 115.7, 117.9, 124.8,
126.8, 129.5, 134.6, 137.7, 153.7, 158.2, 159.0, 182.3.
[0119] .sup.19F-NMR (282 MHz, CDCl.sub.3): .delta.
(ppm)=-224.0.
[0120] MS (EI-directly intake): m/e (intensity %): 476 (M.sup.+,
8); 431 (M-CH.sub.2OCH.sub.3.sup.+, 100).
[0121] Anal. Calc. for C.sub.23H.sub.25N.sub.2FO.sub.6S: C, 57.97;
H, 5.27; N, 5.88. found: C, 57.61; H, 5.18; N, 5.51.
1.2 Precursors for PET Chemistry (Ia, IIa, IIIa, IVa, Va etc.)
1.2.1 Synthesis of
(S)-(+)-1-(4-benzyloxybenzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)-sulfonyl-
]isatin IIIaa
[0122] 500 mg (1.3 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 52 mg (1.3 mmol) sodium hydride (60% in mineral oil)
and 605 mg (2.6 mmol) 4-benzyloxybenzylchloride as described in the
general procedure. The crude dark orange product was purified by
silica gel chromatography (petroletherlethyl acetate 3:1) and
yielded 675 mg of Illaa (1.16 mmol; 89%) as an orange foam.
[0123] mp.: 69-70.degree. C.
[0124] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.75-1.85,
1.93-2.05, 3.17-3.25, 3.44-3.50 and 3.90-3.97 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.86-3.91 (m, 2H, PhOCH.sub.2); 4.83
(s, 2H, NCH.sub.2Ph); 5.03 (s, 2H, NCH.sub.2Ph); 6.78-6.96 (m, 7H,
isatin-H and PhH); 7.18-7.40 (m, 8H, PhH); 7.93 (dd, 1H, J=1.5 Hz,
7.8 Hz, isatin-H); 7.98 (d, 1H, J=1.5 Hz, isatin-H).
[0125] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=26.7, 31.6,
46.5, 52.1, 61.2, 71.7, 72.7, 113.7, 116.9, 118.1, 120.0, 123.6,
126.8, 128.5, 130.0, 131.2, 131.7, 132.1, 136.7, 139.2, 139.6,
155.9, 160.3, 161.5, 182.8.
[0126] MS (MALDI-TOF) m/e: 606
(C.sub.33H.sub.30N.sub.2O.sub.6S+Na).sup.+. Anal. Calc. for
C.sub.33H.sub.30N.sub.2O.sub.6S: C, 68.03; H, 5.19; N, 4.81. found:
C, 68.38; H, 5.34; N, 4.51.
1.2.2 Synthesis of
(S)-1-(p-tert-butyldimethylsilyloxybenzyl)-5-[1-(2-phenoxymethylpyrrolidi-
nyl)sulfonyl]-isatin IIab
[0127] (S)-5-[1-(2-Phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia
(750 mg, 2 mmol) was reacted with sodium hydride (88 mg, 2.2 mmol,
60% in mineral oil) and
p-[(tert-butyldimethylsilyl)oxy]benzylbromide (1.81 g, 6 mmol) as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (cyclohexane:ethyl acetate
9:1 to 4:1) to yield a yellow sticky oil.
[0128] Yield: 630 mg (1.01 mmol, 52%).
[0129] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. [ppm]: 0.18 (s,
6H, SiCH.sub.3); 0.97 (s, 9H, SitBu); 1.77-1.80, 1.99-2.05,
3.22-3.24, 3.48-3.51, 3.89-3.97, 4.14-4.17 (m, 9H,
pyrrolidine-CH/H.sub.2, CH.sub.2O); 4.84 (s, 2H, NCH.sub.2Ar);
6.80-6.94 (m, 6H, Ar--H, isatin-H), 7.17-7.24 (m, 4H, Ar--H); 7.94
(dd, 1H, .sup.3J.sub.H,H=8.4 Hz, .sup.4J.sub.H,H=1.6 Hz, isatin-H);
8.07 (d, 1H, .sup.4J.sub.H,H=1.6 Hz, isatin-H). .sup.13C-NMR (100
MHz, CDCl.sub.3): .delta. [ppm]: -4.6 (SiCH.sub.3), 18.1
(SiCCH.sub.3), 26.8 (C(CH.sub.3).sub.3), 24.0, 28.9, 49.4, 58.5
(pyrrolidine-C), 43.9 (CCH.sub.2Ar), 69.0 (OCH.sub.2), 117.0
(q-ArC(CO)), 114.3, 120.2, 122.7, 122.8, 124.1, 126.2, 128.9 (ArC),
129.4 (q-CCH.sub.2N), 134.1 (isatin-CH), 136.9 (q-CSO.sub.2), 153.3
(q-CN(CO)), 155.9 (q-COSi), 157.6 (isatin-N(CO)), 158.1
(q-COCH.sub.2), 181.6 (N(CO)CO).
[0130] MS (MALDI-TOF) m/e: 629 (M+Na); 607 (M+H).sup.+.
[0131] Anal. Calc. for C.sub.32H.sub.38N.sub.2O.sub.6SSi+EtOAc: C,
62.22; H, 6.67; N, 4.03. found: C, 62.01; H, 6.57; N, 4.04.
1.2.3 Synthesis of
(S)-(+)-1-(4-benzyloxybenzyl)-5-[1-(2-methoxymethylpyrrolidinyl)-sulfonyl-
]isatin IVaa
[0132] 560 mg (1.72 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 69 mg (1.72 mmol) sodium hydride (60% in mineral
oil) and 1.2 g (5.16 mmol) 4-benzyloxybenzylchloride as described
in the general procedure. The crude orange product was purified by
silica gel chromatography (petrolether/ethyl acetate 3:1) and
yielded 700 mg of IVaa (1.34 mmol; 78%) as a yellow powder.
[0133] mp.: 73-74.degree. C.
[0134] .sup.1H-NMR (300 MHz, CDC3): .delta. (ppm)=1.65-1.69,
1.85-1.89, 3.10-3.12, 3.52-3.57 and 3.70-3.73 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.32 (s, 3H, OCH.sub.3); 3.34-3.39
(m, 2H, CH.sub.3OCH.sub.2); 4.89 (s, 2H, NCH.sub.2Ph); 5.04 (s, 2H,
OCH.sub.2Ph); 6.92-6.97 (m, 4H, PhH); 7.25-7.41 (m, 6H, PhH and
isatin-H); 7.96 (dd, 1H, J=1.5 Hz, 8.4 Hz, isatin-H); 8.02 (d, 1H,
J=1.5 Hz, isatin-H).
[0135] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=24.1, 28.7,
44.0, 49.3, 59.1, 70.2, 74.9, 111.3, 115.6, 117.5, 124.4, 126.1,
127.5, 128.1, 128.6, 129.1, 134.1, 136.6, 137.3, 153.4, 157.9,
159.0, 181.9.
[0136] MS (EI-directly intake): m/e (intensity %): 520 (M.sup.+,
15); 475 (M-CH.sub.2OCH.sub.3.sup.+, 100).
1.2.4 Synthesis of
(S)-1-(p-tert-butyldimethylsilyloxybenzyl)-5-[1-(2-methoxymethylpyrrolidi-
nyl)sulfonyl]-isatin IVab
[0137] (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin IIa
(648 mg, 2 mmol) was reacted with sodium hydride (88 mg, 2.2 mmol,
60% in mineral oil) and
p-[(tert-butyldimethylsilyl)oxy]benzylbromide (1.81 g, 6 mmol) as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (cyclohexane:ethyl acetate
9:1 to 3:2) and yielded IVab as a yellow sticky oil.
[0138] Yield: 510 mg (0.94 mmol, 47%).
[0139] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. [ppm]: 0.18 (s,
6H, SiCH.sub.3); 0.97 (s, 9H, SitBu); 1.65-1.67, 1.88-1.90,
3.09-3.12, 3.40-3.42, 3.54-3.57, 3.71-3.73 (m, 9H,
pyrrolidine-CHIH.sub.2, CH.sub.2O); 3.33 (s, 3H, OCH.sub.3); 4.89
(s, 2H, NCH.sub.2Ar); 6.83 (d, 2H, .sup.3J.sub.H,H=8.4 Hz, Ar--H),
6.93 (d, 1H, .sup.3J.sub.H,H=8.4 Hz, isatin-H); 7.20 (d, 2H,
.sup.3J.sub.H,H=8.4 Hz, Ar--H); 7.97 (dd, 1H, .sup.3J.sub.H,H=8.4
Hz, .sup.4J.sub.H,H=1.6 Hz, isatin-H); 8.04 (d, 1H,
.sup.4J.sub.H,H=1.6 Hz, isatin-H). .sup.13C-NMR (100 MHz, CDC31):
.delta. [ppm]: -4.6 (SiCH.sub.3), 18.1 (SiCCH.sub.3), 25.5
(C(CH.sub.3).sub.3), 24.0, 28.7, 49.2, 58.9 (pyrrolidine-C), 43.8
(NCH.sub.2Ar), 59.1 (OCH.sub.3), 74.7 (OCH.sub.2), 117.2
(q-ArC(CO)), 120.2, 122.7, 122.8, 124.3 (Ar--C), 129.5
(q-CCH.sub.2N), 134.1 (isatin-CH), 137.1 (q-CSO.sub.2), 153.3
(q-CN(CO)), 155.8 (q-COSi), 157.7 (isatin-N(CO)), 181.8
(N(CO)CO).
[0140] MS (MALDI-TOF) m/e: 567 (M+Na).sup.+, 545 (M+H).sub.t.
[0141] Anal. Calc. for C.sub.27H.sub.36N.sub.2O.sub.6SSi: C, 59.53;
H, 6.66; N, 5.14. found: C, 59.87; H, 6.38; N, 4.89.
1.2.5 Synthesis of
(S)-1-(p-hydroxybenzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin
IIa
[0142]
(S)-1-(p-tert-Butyldimethylsilyloxybenzyl)-5-[1-(2-phenoxymethyl-py-
rrolidinyl)sulfonyl]isatin Illab (400 mg, 0.66 mmol) was dissolved
in methanol (15 mL) and conc. HCl (1 mL) was added. The resulting
mixture was stirred for 2 h at ambient temperature and then diluted
with ethyl acetate (100 mL). The organic layer was washed With
NaHCO.sub.3, water and brine and dried with magnesium sulphate.
After removal of the solvent the yellow residue was purified by
silica gel chromatography (cyclohexane:ethyl acetate 2:1 to 3:2) to
yield a yellow sticky oil.
[0143] Yield: 210 mg (0.43 mmol, 65%).
[0144] .sup.1H-NMR (300 MHz, CDC3): .delta. [ppm]: 1.71-1.82,
1.91-2.05, 3.19-3.26, 3.43-3.51, 3.60-3.71, 4.12-4.16 (m, 9H,
pyrrolidine-CH/H.sub.2, CH.sub.2O); 4.82 (s, 2H, NCH.sub.2Ar); 5.58
(m, 1H, ArOH); 6.79-6.94 (m, 6H, Ar--H, isatin-H), 7.17-7.31 (m,
4H, Ar--H); 7.95 (dd, 1H, .sup.3J.sub.H,H=8.4 Hz,
.sup.4J.sub.H,H=1.6 Hz, isatin-H); 7.99 (d, 1H, .sup.4J.sub.H,H=1.6
Hz, isatin-H). .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. [ppm]:
24.5, 29.4, 49.9, 59.1 (pyrrolidine-C), 44.4 (NCH.sub.2Ar), 59.2
(OCH.sub.3), 72.7 (OCH.sub.2), 117.9 (q-ArC(CO)), 116.5, 124.7,
126.1, 127.6, 127.7, 129.1, (Ar--C), 129.6 (q-CCH.sub.2N), 134.6
(isatin-CH), 137.5 (q-CSO.sub.2), 153.7 (q-CN(CO)), 156.5 (q-COH),
158.2, 158.6 (isatin-N(CO), q-COCH.sub.2), 182.2 (N(CO)CO)).
[0145] MS (MALDI-TOF) m/e: 516 (M+Na).sup.+, 494 (M+H).sup.+.
[0146] Anal. Calc. for C.sub.26H.sub.25N.sub.2O.sub.6S: C, 63.40;
H, 4.91; N, 5.69. found: C, 63.25; H, 4.76; N, 5.98.
1.2.6 Synthesis of
(S)-1-(p-hydroxybenzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
IVa
[0147]
(S)-1-(p-tert-Butyldimethylsilyloxybenzyl)-5-[1-(2-methoxymethyl-py-
rrolidinyl)sulfonyl]isatin IVab (500 mg, 0.92 mmol) was dissolved
in methanol (15 mL) and conc. HCl (1 mL) was added. The resulting
mixture was stirred for 2 h at ambient temperature and then diluted
with ethyl acetate (100 mL). The organic layer was washed with
NaHCO.sub.3, water and brine and dried with magnesium sulphate.
After removal of the solvent the residue was purified by silica gel
chromatography (cyclohexane:ethyl acetate 3:2 to 1:1) to yield a
yellow sticky oil.
[0148] Yield: 350 mg (0.81 mmol, 88%).
[0149] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. [ppm]: 1.65-1.71,
1.85-1.92, 3.10-3.13, 3.41-3.44, 3.53-3.57, 3.71-3.73 (m, 9H,
pyrrolidine-CH/H.sub.2, CH.sub.2O); 3.33 (s, 3H, OCH.sub.3); 4.61
(m, 1H, ArOH), 4.87 (s, 2H, NCH.sub.2Ar); 6.83 (d, 2H,
.sup.3J.sub.H,H=8.4 Hz, Ar--H), 6.94 (d, 1H, .sup.3J.sub.H,H=8.4
Hz, isatin-H); 7.18 (d, 2H, .sup.3J.sub.H,H=8.4 Hz, Ar--H); 7.96
(dd, 1H, .sup.3J.sub.H,H=8.4 Hz, .sup.4J.sub.H,H=1.8 Hz, isatin-H);
8.02 (d, 1H, .sup.4J.sub.H,H=1.8 Hz, isatin-H). .sup.13C-NMR (75
MHz, CDCl.sub.3): .delta. [ppm]: 24.1, 28.8, 49.3, 59.1
(pyrrolidine-C), 44.1 (CCH.sub.2), 59.2 (OCH.sub.3), 74.8
(OCH.sub.2), 117.5 (q-ArC(CO)), 116.2, 122.8, 124.4, 124.9 (ArC),
129.5 (q-CCH.sub.2N), 134.1 (isatin-CH), 137.3 (q-CSO.sub.2), 153.5
(q-CN(CO)), 156.9 (q-COH), 157.9 (isatin-N(CO)), 182.0
(N(CO)CO)).
[0150] MS (MALDI-TOF) m/e: 453 (M+Na)*, 431 (M+H).
[0151] Anal. Calc. for C.sub.21H.sub.22N.sub.2O.sub.6S: C, 58.59;
H, 5.15; N, 6.51. found: C, 58.72; H, 4.98; N, 6.21.
1.2.7 Synthesis of
(S)-1-(4-(2-bromoethoxy)benzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfony-
l]isatin Vaa
[0152] 730 mg (1.90 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 80 mg (1.90 mmol) sodium hydride (60% in mineral
oil) and 882 mg (3 mmol) 4-(2-bromoethoxy)benzylbromide as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (petrolether/ethyl acetate
3:1-1:1) and yielded 910 mg of Vaa (1.52 mmol; 80%) as a yellow
solid.
[0153] mp.: 162-163.degree. C.
[0154] .sup.1H-NMR (300 MHz, CDC.sub.3): .delta. (ppm)=1.68-1.75,
1.91-1.97, 3.13-3.17, 3.39-3.42 (m, 6H, pyrrolidine-CH.sub.2 and
CH); 3.54 (t, 2H, J=6.0 Hz, PhCH.sub.2CH.sub.2Br); 3.80-3.90 (m,
2H, PhOCH.sub.2); 4.05-4.09 (m, 1H, pyrrolidine-CH); 4.19 (t, 2H,
J=6.0 Hz, PhCH.sub.2CH.sub.2Br); 4.77 (s, 2H, NCH.sub.2Ph);
6.71-6.87 (m, 6H, PhH and isatin-H); 7.11-7.20 (m, 4H, PhH); 7.86
(dd, 1H, J=1.8 Hz, 8.4 Hz, isatin-H); 7.91 (d, 1H, J=1.5 Hz,
isatin-H).
[0155] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=24.5, 29.3,
29.4, 44.3, 49.9, 59.1, 68.4, 69.6, 11.6, 114.8, 115.8, 117.9,
121.5, 124.7, 127.0, 129.6, 129.9, 134.6, 137.5, 153.7, 158.2,
158.6, 182.1.
[0156] MS (EI-directly intake): m/e (intensity %): 600 (3), 598
(M*, 3); 493 (100), 491 (M-CH.sub.2OPh, 100).
[0157] Anal. Calc. for C.sub.28H.sub.27BrN.sub.2O.sub.6S: C, 56.10;
H, 4.54; N, 4.67. found: C, 56.10; H, 4.40; N, 4.56.
1.2.8 Synthesis of
(S)-1-(4-(2-(p-methylphenylsulfonyloxy)ethoxy)benzyl)-5-[1-(2-phenoxymeth-
ylpyrrolidinyl)sulfonyl]isatin Va
[0158] 500 mg (0.83 mmol) of
(S)-(+)-1-(4-(2-bromoethoxy)benzyl)-5-[1-(2-phenoxymethyl-pyrrolidinyl)su-
lfonyl]isatin Vaa was solved in 20 mL dry acetonitrile under argon
atmosphere. After adding 1.26 g (4 mmol) silver tosylate the
reaction mixture was heated to reflux for 24 h. During the reaction
grey precipitation was formed. The solvent was removed in vacuo and
the crude orange product was purified by silica gel chromatography
(toluene/ethyl acetate 2:1). It yielded 510 mg of Va (0.75 mmol;
90%) as an orange solid.
[0159] mp.: 83-83.degree. C.
[0160] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.74-1.85,
1.95-2.05, 3.22-3.27, 3.46-3.53 (m, 6H, pyrrolidine-CH.sub.2 and
CH); 2.45 (s, 3H, PhCH.sub.3); 3.96-3.99 (m, 2H, PhOCH.sub.2);
4.12-4.18 (m, 3H, PhCH.sub.2CH.sub.2OTos and pyrrolidine-CH);
4.34-4.37 (m, 2H, PhCH.sub.2C.sub.2HOTos); 4.85 (s, 2H,
NCH.sub.2Ph); 6.79-6.94 (m, 6H, PhH and isatin-H); 7.21-7.36 (m,
6H, PhH); 7.79-7.82 (m, 2H, PhH), 7.96 (dd, 1H, J=1.8 Hz, 8.4 Hz,
isatin-H); 8.00 (d, 1H, J=1.8 Hz, isatin-H).
[0161] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=22.0, 24.5,
29.4, 44.3, 49.9, 59.1, 66.1, 68.3, 69.6, 111.6, 114.8, 115.7,
117.9, 121.4, 124.6, 126.9, 128.4, 128.6, 129.5, 129.9, 130.3,
133.3, 134.6, 137.5, 145.4, 153.7, 158.2, 158.6, 182.1.
[0162] MS (EI-directly intake): m/e (intensity %): 583
(M-PhOCH.sub.2.sup.+, 10); 385 (Ia.sup.+, 39); 91 (100)
(PhCH.sub.2.sup.+, 100).
[0163] Anal. Calc. for C.sub.35H.sub.34N.sub.2O.sub.9S.sub.2: C,
60.85; H, 4.96; N, 4.06. found: C, 61.04; H, 4.87; N, 3.88.
1.2.9 Synthesis of
(S)-1-(4-(2-bromoethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)-sulfon-
yl]isatin Vlaa
[0164] 800 mg (2.46 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 98 mg (2.46 mmol) sodium hydride (60% in mineral
oil) and 1.4 g (4.92 mmol) 4-(2-bromoethoxy)benzylbromide as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (petrolether/ethyl acetate
3:1.fwdarw.1:2) and yielded 1.02 g of VIaa (1.90 mmol; 77%) as a
yellow foam.
[0165] mp.: 61-62.degree. C.
[0166] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.66-1.70,
1.86-1.90, 3.10-3.13, 3.53-3.57 and 3.71-3.73 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.33 (s, 3H, OCHa); 3.35-3.39 (m, 2H,
CH.sub.3OCH.sub.2); 3.63 (t, 2H, J=5.7 Hz, PhCH.sub.2CH.sub.2Br);
4.28 (t, 2H, J=5.7 Hz, PhCH.sub.2CH.sub.2Br); 4.91 (s, 2H,
NCH.sub.2Ph); 6.89-6.97 (m, 3H, PhH and isatin-H); 7.27-7.30 (m,
2H, PhH); 7.97 (dd, 1H, J=1.8 Hz, 8.4 Hz, isatin-H); 8.02 (d, 1H,
J=1.5 Hz, isatin-H).
[0167] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=24.5, 29.2,
29.4, 44.3, 49.7, 59.4, 68.4, 75.2, 111.6, 115.8, 117.9, 124.8,
127.0, 129.6, 134.7, 137.7, 153.7, 158.2, 158.6, 182.3.
[0168] MS (EI-directly intake): m/e (intensity %): 538 (42), 536
(42) (M.sup.+, 42); 493 (100), 491 (100)
(M-CH.sub.2OCH.sub.3.sup.+, 100).
[0169] Anal. Calc. for C.sub.23H.sub.25BrN.sub.2O.sub.6S: C, 51.40;
H, 4.69; N, 5.21. found: C, 51.08; H, 4.48; N, 5.00.
1.2.10 Synthesis of
(S)-1-(4-(2-(p-methylphenylsulfonyloxy)ethoxy)benzyl)-5-[1-(2-methoxymeth-
ylpyrrolidinyl)sulfonyl]isatin Via
[0170] 500 mg (0.93 mmol) of
(S)-(+)-1-(4-(2-bromoethoxy)benzyl)-5-[1-(2-methoxymethyl-pyrrolidinyl)su-
lfonyl]isatin VIaa was solved in 20 mL dry acetonitrile under argon
atmosphere. After adding 1.26 g (4 mmol) silver tosylate the
reaction mixture was heated to reflux for 24 h. During the reaction
grey precipitation was formed. The solvent was removed in vacuo and
the crude orange product was purified by silica gel chromatography
(toluene/ethyl acetate 2:1). It yielded 540 mg of Via (0.88 mmol;
94%) as an orange solid.
[0171] mp.: 61-62.degree. C.
[0172] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.63-1.68,
1.85-1.89, 3.09-3.13, 3.52-3.57 and 3.70-3.74 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 2.44 (s, 3H, PhCH.sub.3); 3.33 (s,
3H, OCH.sub.3); 3.33-3.39 (m, 2H, CH.sub.3OCH.sub.2); 4.12-4.15 (m,
2H, PhCH.sub.2CH.sub.2OTos); 4.33-4.36 (m, 2H,
PhCH.sub.2C.sub.2HOTos); 4.89 (s, 2H, NCH.sub.2Ph); 6.78-6.81 (m,
2H, PhH) 6.91 (d, 1H, J=8.4 Hz, isatin-H); 7.15-7.35 (m, 6H, PhH);
7.78-7.82 (m, 2H, PhH), 7.97 (dd, 1H, J=1.8 Hz, 8.4 Hz, isatin-H);
8.03 (d, 1H, J=1.8 Hz, isatin-H).
[0173] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=22.0, 24.5,
29.2, 44.3, 49.7, 59.6, 60.7, 66.0, 68.3, 75.2, 111.5, 115.7,
117.9, 124.8, 126.9, 128.4, 129.4, 130.3, 133.3, 134.7, 137.7,
145.4, 153.7, 158.2, 158.6, 182.3.
[0174] MS (EI-directly intake): m/e (intensity %): 628 (M.sup.+,
1.5); 583 (100) (M-CH.sub.2OCH.sub.3, 100).
[0175] Anal. Calc. for C.sub.30H.sub.32N.sub.2O.sub.9S.sub.2: C,
57.31; H, 5.13; N, 4.36. found: C, 57.36; H, 5.25; N, 3.99.
1.3 SPECT-compatible References (1, 2, 3, 4, 5 etc.)
1.3.1 Synthesis of
(S)-1-(4-iodobenzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin
1
[0176] 385 mg (1 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 40 mg (1 mmol) sodium hydride (60% in mineral oil)
and 445 mg (1.5 mmol) 4-iodobenzylbromide as described in the
general procedure. The crude dark orange product was purified by
silica gel chromatography (diisopropylether/acetone 4:1) and
yielded 400 mg of 1 (0.66 mmol; 66%) as an orange solid.
[0177] mp.: 88-90.degree. C.
[0178] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.71-1.74,
1.91-1.98, 3.13-3.18, 3.39-3.43 and 4.05-4.09 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.81-3.91 (m, 2H, PhOCH.sub.2); 4.78
(s, 2H, NCH.sub.2Ph); 6.72-6.76 (m, 3H, isatin-H and PhH);
6.83-6.87 (m, 1H, PhH); 6.98-7.01 (m, 2H, PhH); 7.13-7.19 (m, 2H,
PhH); 7.61-7.63 (m, 2H, PhH); 7.86 (dd, 1H, J=1.5 Hz, 7.8 Hz,
isatin-H); 7.93 (d, 1H, J=1.5 Hz, isatin-H).
[0179] .sup.13C-NMR (100 MHz, CDC3): .delta. (ppm)=24.1, 28.9,
43.8, 49.4, 58.6, 69.1, 94.0, 110.9, 114.3, 117.4, 120.9, 124.3,
129.2, 129.3, 129.4, 133.4, 134.4, 137.0, 138.3, 152.8, 157.7,
158.1, 181.3.
[0180] MS (ES): m/e (intensity %): 657 (100) (M+MeOH+Na).sup.+; 625
(25) (M+Na).sup.+; 603 (10) (M+H)*.
[0181] Anal. Calc. for C.sub.26H.sub.23IN.sub.2O.sub.5S: C, 51.84;
H, 3.85; N, 4.65. found: 52.29; H, 4.11; N, 4.57.
1.3.2 Synthesis of
(S)-1-(4-iodobenzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin
2
[0182] 750 mg (2.3 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 92 mg (2.3 mmol) sodium hydride (60% in mineral oil)
and 1.02 g (3.45 mmol) 4-iodobenzylbromide as described in the
general procedure. The crude dark orange product was purified by
silica gel chromatography (diisopropyl ether/acetone 8:1) and
yielded 820 mg of 2 (1.52 mmol; 66%) as an orange solid.
[0183] mp.: 129-130.degree. C.
[0184] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=1.64-1.69,
1.86-1.91, 3.11-3.13, 3.35-3.41 (m, 7H, pyrrolidine-CH.sub.2 and
CH); 3.30 (s, 3H, OCH.sub.3); 3.52-3.57 and 3.72-3.74 (m, 2H,
PhOCH.sub.2); 4.91 (s, 2H, NCH.sub.2Ph); 6.87 (d, 1H, J=8.4 Hz,
isatin-H); 7.08 (d, 2H, J=8.7 Hz, PhH); 7.69 (d, 2H, J=8.7 Hz,
PhH); 7.97 (dd, 1H, J=1.8 Hz, 8.4 Hz, isatin-H); 8.04 (d, 1H, J=1.8
Hz, isatin-H).
[0185] .sup.13C-NMR (75 MHz, CDCt.sub.3): S (ppm)=24.1, 27.2, 28.9,
44.0, 49.2, 59.1, 59.2, 74.8, 94.1, 111.0, 117.6, 124.6, 129.4,
133.5, 134.5, 137.4, 138.4, 153.0, 157.8, 181.5.
[0186] MS (EI-directly intake): m/e (intensity %): 540 (M.sup.+,
2); 495 (100) (M-CH.sub.2OCH.sub.3.sup.+, 100).
[0187] Anal. Calc. for C.sub.21H.sub.21N.sub.2IO.sub.5S: C, 46.68;
H, 3.92; N, 5.18. found: 47.00; H, 3.91; N, 5.01.
1.4 Precursors for SPECT Chemistry (1a, 2a, 3a, 4a, 5a etc.)
1.4.1 Synthesis of
(S)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]-1-(4-tributylstannylbenzy-
l)-isatin 1a
[0188] 385 mg (1 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 60 mg (1.5 mmol) sodium hydride (60% in mineral oil)
and 1.42 g (3 mmol) 4-Tributylstannylbenzylmethansulfonate as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (petrolether/ethyl acetate
6:1) and yielded 410 mg of I (0.53 mmol; 53%) as an orange oil.
[0189] .sup.1H-NMR (300 MHz, CDClt): .delta. (ppm)=0.92 (t, 12H,
J=7.5 Hz, SnBu-C H.sub.3); 1.07-1.13, 1.31-1.43, 1.53-1.60 (m, 18H,
SnCH.sub.2) 1.81-1.89, 2.04-2.11, 3.28-3.34, 3.50-3.56 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.94-4.04 and 4.19-4.23 (m, 2H,
PhOCH.sub.2); 4.94 (s, 2H, NCH.sub.2Ph); 6.86 (d, 2H, J=8.1 Hz,
4-SnBu.sub.3PhH); 6.91-6.99 (m, 2H, PhH); 7.23-7.34 (m, 4H, PhH);
7.51 (d, 2H, J=8.1 Hz, 4-SnBu.sub.3PhH); 8.00 (dd, 1H, J=1.8 Hz,
8.4 Hz, isatin-H); 8.07 (d, 1H, J=1.8 Hz, isatin-H).
[0190] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=10.0, 14.0,
24.6, 27.7, 28.2, 29.4, 44.8, 49.9, 59.1, 69.6, 111.6, 114.8,
117.9, 121.5, 124.6, 127.4, 129.9, 133.7, 134.7, 137.5, 143.4,
153.8, 158.2, 158.6, 180.1, 182.1.
[0191] MS (MALDI-TOF) m/e: 709
(C.sub.3-8HsoN.sub.2O.sub.5SSn--C.sub.4H.sub.9).sup.+.
1.4.2 Synthesis of
(S)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]-1-(4-trimethylsilylbenzyl-
)-isatin 1b
[0192] 500 mg (1.29 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 64 mg (1.61 mmol) sodium hydride (60% in mineral
oil) and 314 mg (1.29 mmol) 4-trimethylsilylbenzylbromide as
described in the general procedure and stirred 21 hours at room
temperature. The reaction mixture was diluted with 50 mL water and
extracted with 100 mL chloroform three times. The combined organic
extracts were washed with brine and dried (Na.sub.2SO.sub.4). After
evaporation the product was purified by silica gel chromatography
(petrolether/ethyl acetate 2:1) and yielded 217 mg of 1b (0.4 mmol;
31%) as an orange solid.
[0193] mp.: 128-130.degree. C. (decomposition)
[0194] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=0.25 (s,
9H, Si(CH.sub.3).sub.3); 1.77-1.81, 1.99-2.05, 3.23-3.26, 3.47-3.49
and 3.88-4.17 (m, 9H, pyrrolidine-CH.sub.2 and CH); 4.90 (s, 2H,
NCH.sub.2Ph); 6.79-6.94 (m, 4H, isatin-H and PhH); 7.19-7.30 (m,
4H, PhH); 7.50-7.53 (m, 2H, PhH); 7.95 (dd, 1H, J=1.8 Hz, 8.4 Hz,
isatin-H); 8.01 (d, 1H, J=1.8 Hz, isatin-H).
[0195] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (ppm)=-1.21,
14.2, 24.2, 29.1, 44.4, 49.5, 58.7, 69.2, 111.2, 114.4, 117.5,
121.1, 124.3, 126.9, 129.5, 134.2, 137.1, 141.3, 153.7, 158.0,
158.3, 182.2.
[0196] MS (EI-directly intake): m/e (intensity %): 548 (M.sup.+,
5); 441 (M-CH.sub.2OPh.sup.+, 100).
[0197] Anal. Calc. for C.sub.29H.sub.32N.sub.2O.sub.5SSi: C, 63.48;
H, 5.88; N, 5.11. found: C, 62.67; H, 6.02; N, 4.86.
1.4.3 Synthesis of
(S)-(+)-1-(4-bromobenzyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isat-
in 1c
[0198] 500 mg (1.3 mmol) of
(S)-(+)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]isatin Ia was
converted with 60 mg (1.5 mmol) sodium hydride (60% in mineral oil)
and 647 mg (2.6 mmol) 4-bromobenzylbromide as described in the
general procedure. The crude dark orange product was purified by
silica gel chromatography (petroletherlethyl acetate 2:1) and
yielded 480 mg of 1c (0.87 mmol; 67%) as an orange solid.
[0199] mp.: 74-76.degree. C.
[0200] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. (ppm)=1.78-1.85,
1.98-2.05, 3.23-3.27, 3.47-3.52 and 3.97-3.99 (m, 7H,
pyrrolidine-CH.sub.2 and CH); 3.89-3.93 (m, 2H, PhOCH.sub.2); 4.87
(s, 2H, NCH.sub.2Ph); 6.79-6.81 (m, 3H, isatin-H and PhH);
6.91-6.95 (m, 1H, PhH); 7.19-7.26 (m, 4H, PhH); 7.50-7.51 (m, 2H,
PhH); 7.96 (dd, 1H, J=1.6 Hz, 8.4 Hz, isatin-H); 8.02 (d, 1H, J=1.6
Hz, isatin-H).
[0201] .sup.13C-NMR (100 MHz, CDCl.sub.3): .delta. (ppm)=24.2,
29.1, 43.9, 49.5, 58.7, 69.1, 75.7, 111.0, 114.4, 117.5, 121.1,
122.7, 124.5, 129.3, 129.5, 132.5, 132.8, 134.7, 137.2, 152.9,
157.8, 158.2, 181.2.
[0202] MS (EI-directly intake): m/e (intensity %): 555 (5), 553
(M.sup.+, 5); 449 (100), 447 (M-CH.sub.2OPh.sup.+, 95).
[0203] Anal. Calc. for C.sub.26H.sub.23BrN.sub.2O.sub.5S: C, 56.18;
H, 4.17; N, 5.04. found: C, 56.50; H, 4.28; N, 4.68.
1.4.4 Synthesis of
(S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]-1-(4-tributylstannylbenzy-
l)isatin 2a
[0204] 324 mg (1 mmol) of
(S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin IIa was
converted with 60 mg (1.5 mmol) sodium hydride (60% in mineral oil)
and 680 mg (1.4 mmol) 4-tributylstannylbenzylmethansulfonate as
described in the general procedure. The crude orange product was
purified by silica gel chromatography (petrolether/ethyl acetate
4:1) and yielded 378 mg of 2a (0.54 mmol; 54%) as an orange
oil.
[0205] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (ppm)=0.80 (t,
12H, J=7.5 Hz, SnBu--CH.sub.3); 0.94-1.00, 1.16-1.28, 1.39-1.48 (m,
18H, SnCH.sub.2) 1.58-1.61, 1.78-1.83, 3.02-3.09, 3.45-3.50,
3.64-3.69 (m, 7H, pyrrolidine-CH.sub.2 and CH); 3.25 (s, 3H,
CH.sub.3OCH.sub.2); 3.27-3.35 (m, 2H, CH.sub.3OCH.sub.2); 4.87 (s,
2H, NCH.sub.2Ph); 6.86 (d, 2H, J=8.4 Hz, isatin-H); 7.20 (d, 2H,
J=7.2 Hz, 4-SnBu.sub.3PhH); 7.38 (d, 2H, J=7.2 Hz,
4-SnBu.sub.3PhH); 7.91 (dd, 1H, J=1.8 Hz, 8.4 Hz, isatin-H); 7.97
(d, 1H, J=1.8 Hz, isatin-H).
[0206] .sup.13C-NMR (75 MHz, CDCl.sub.3): S (ppm)=9.6, 13.6, 24.1,
27.3, 28.8, 29.0, 44.4, 49.3, 59.2, 60.3, 74.8, 111.2, 117.5,
124.4, 126.9, 133.2, 137.3, 143.0, 153.4, 157.8, 180.0.
[0207] MS (MALDI-TOF) m/e: 647
(C.sub.33H.sub.48N.sub.2O.sub.5SSn--C.sub.4H.sub.9).sup.+.
##STR00074##
##STR00075## ##STR00076##
2. Radiosynthesis of PET- and SPECT-Compatible CbRs
2.1 PET-compatible CbRs (eg. [.sup.11C]II, [.sup.11C]III,
[.sup.11CIV, [.sup.18F]V etc.)
[0208] 2.1.1 Radiosynthesis.sup.a of
(S)-(+)-1-([.sup.11C]methyl)-5-[1-(2-phenoxymethylpyrrolidinyl)sulfonyl]i-
satin ([.sup.11C]I
[0209] [.sup.11]C]CO.sub.2 was produced by the
.sup.14N(p,.alpha.).sup.11C nuclear reaction of research grade
nitrogen gas target mixture containing 2.5% oxygen with a
CTI-RDS-111 cyclotron using 11 MeV proton beams at currents of 40
.mu.A and trapped in a stainless steel loop cooled with liquid
nitrogen to -150.degree. C. [.sup.11C]CH.sub.3I was prepared from
[.sup.11C]CO.sub.2, 50 .mu.l 1 M LiAlH.sub.4 (ABX advanced
biochemical compounds), 100 .mu.l 0.5 M H.sub.3PO.sub.4, a column
filled with PPh.sub.3I.sub.2 adsorbed at Al.sub.2O.sub.3
(180.degree. C.) and a column filled with P.sub.2O.sub.5 using a
procedure similar to that previously described [38]. 1.0 mg (2.6
.mu.mol) desmethyl-precursor Ia and 0.2 mg (60% mineral oil, 5.0
.mu.mol) NaH in 200 .mu.l DMF was reacted with [.sup.11C]CH.sub.3I
at 80.degree. C. for 5 min. After cooling to 50.degree. C., 200
.mu.l water for injection were added and the crude mixture was
loaded onto a semi-preparative HPLC-column and the product
[.sup.11C]I was eluated with H.sub.2O/CH.sub.3CN 65/35 at a flow of
4 ml/min at 43.6-50 min in 150 ml water for injection. The mixture
was passed through a C18 SepPak.RTM.-cartridge (Waters). The
cartridge was washed with 5 ml water for injection and [.sup.11C]I
was eluated with 2 ml EtOH in 10 ml saline. Finally the solution
was filtered through a sterile filter (0.2 .mu.m). The time of
synthesis and purification was 91 min from the EOB. The absolute
radiochemical yield was 290 MBq. The radiochemical purity,
determined via radio-HPLC (eluent: 500 mM NH.sub.4COO/CH.sub.3CN
6/4, flow: 0.3 ml/min, retention time: 23.8 min), was >99% with
a specific activity of 1.0 GBq/.mu.mol at the EOS (n=1). Chemical
identity of [.sup.11C]I was proved by HPLC coinjection of
[.sup.11C]I and non-radioactive reference I.
.sup.a Radiosynthesis was Carried Out Using an Automated PET Tracer
Synthesizer TRACERLab Fxc (GE Functional Imaging GmbH).
[0210] Separation of the radiosynthesized compounds, and analyses
of the radiochemical yields were performed by radio-HPLC using a
Syknm S1021 pump, a Knauer K-2001 UV-detector (wavelength 254 nm),
a Raytest Ramona-90/92 .gamma.-detector, a Nucleosil 100-10 C18
precolumn (20.times.8 mm.sup.2) and a Nucleosil 100-7 C18 column
(250.times.16 mm.sup.2). Sample injection was carried out using a
VICI injector block (type C6W incl. 1000 .mu.l loop). The recorded
data were processed by the TRACERLab C software (GE Functional
Imaging GmbH).
[0211] The radiochemical purities and the specific activities were
acquired with a radio-HPLC system composed of a Syknm S1021 pump, a
Knauer K-2501 UV-detector (wavelength 254 nm), a Crismatec Na(TI)
Scintibloc 51 SP51 .gamma.-detector, a Nucleosil 100-3 C18 column
(200.times.3 mm.sup.2), a VICI injector block (type C1 incl. 20
.mu.l loop) and the NINA version 4.8, Rev. 4 software (GE
Functional Imaging GmbH).
2.1.2 Radiosynthesis.sup.b of
(S)-1-(4-(2-[.sup.18F]Fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidi-
nyl)-sulfonyl]isatin[.sup.18F]VI
[0212] No-carrier-added aqueous [18F]fluoride was produced on a
CTI-RDS-111 cyclotron by irradiation of a 1.2 ml water target using
10 MeV proton beams on 97.0% enriched [.sup.18O]water by the
.sup.18O(p,n).sup.18F nuclear reaction. A typical ion batch was 5.9
GBq of [.sup.18F]fluoride at the end of bombardment for currents of
20 .mu.A and irradiation times of 5 min. To recover the
[.sup.18O]water the ion batch of aqueous [.sup.18F]fluoride was
passed through an anion exchange resin (Sep-Pak.RTM. Light Waters
Accell.TM. Plus QMA cartridge, preconditioned with 5 ml 1 M
K.sub.2CO.sub.3 and 10 ml water for injection). [.sup.18F]fluoride
was eluted from the resin with a mixture of 40 .mu.l 1 M
K.sub.2CO.sub.3, 200 .mu.l water for injection, and 800 .mu.l
DNA-grade CH.sub.3CN containing 10 mg Kryptofix.RTM.222.
Subsequently, the aqueous [.sup.18F]K(Kryptofix222)F solution was
carefully evaporated to dryness in vacuo.
[0213] [.sup.18F]VI was prepared by treating the tosylate precursor
(1.3 mg, 2.1 .mu.mol) VIa with the carefully dried
[.sup.18F]K(Kryptofix222)F residue in DNA-grade CH.sub.3CN (1 ml)
at 84.degree. C. for 5 min. Then CH.sub.3CN was evaporated in vacuo
at 50.degree. C. After cooling to rt the crude reaction mixture was
passed through a Waters Sep-Pak.RTM. Light C18 cartridge with water
for injection (10 mL). The cartridge was washed with additional
water for injection (10 ml), followed by elution of the [18F]VI raw
product with ethanol (1.5 ml). The ethanolic solution was
fractionised using a semiautomatical radio-RP-HPLC procedure
(conditions: flow 2 ml/min, .lamda.=254 nm; eluents:
A=CH.sub.3CN/H.sub.2O/TFA, 950150/1, B.dbd.CH.sub.3CN/H.sub.2O/TFA,
50/950/1; Nucleosil 100 C18 5.mu. column (250.times.4.6 mm.sup.2)
with corresponding precolumn (20.times.4.6 mm.sup.2); eluent B from
70% to 10% in 35 min, from 10% to 70% in 5 min) resulting in
[.sup.18F]VI with radiochemical yields of 32% (decay-corrected) and
radiochemical purities >90% (Retention time R.sub.t=26 min). The
determined specific radioactivity was 48 GBq/.mu.mol at the end of
synthesis (EOS). The time of synthesis and purification was 82 min
from the end of bombardement (EOB). The absolute radiochemical
yield was 1109 MBq at the EOS. Chemical identity of [.sup.18F]VI
was proved by RP-HPLC and coinjection of [18F]VI and nonradioactive
counterpart VI.
[0214] For in vivo experiments, the [.sup.18F]VI fraction was
collected in 0.5 ml 8.4% sodium bicarbonate solution and dried in
vacuo. Finally, [18F]VI was diluted in saline to reconstitute
injectable doses with radioactivity concentrations of 70
MBq/ml.
[0215] .sup.b Radiosynthesis was carried out using a modified
automated PET Tracer Synthesizer TRACERLab FX.sub.FDG (GE
Functional imaging GmbH). The recorded data were processed by the
TRACERLab FDG software (GE Functional Imaging GmbH).
[0216] Separation of the radiosynthesised and unlabelled compounds,
analyses of the radiochemical yields and radiochemical purities as
well as specific activities were performed by a gradient radio-HPLC
system composed of a RP-HPLC Nucleosil column 100 C-18 5.mu.
250.times.4.6 mm.sup.2, a corresponding 20.times.4.6 mm.sup.2
precolumn, a Knauer K-500 and a Latek P 402 pump, a Knauer K-2000
UV-detector (wavelength 254 nm) and a Crismatec Na(TI) Scintibloc
51 SP51 gamma detector. Sample injection was carried out using a
Rheodyne injector block (type 7125 incl. 200 .mu.l loop). The
recorded data were processed by the NINA radio-HPLC software,
version 4.9 (GE Functional Imaging GmbH, Germany).
2.2 SPECT-Compatible CbRs (eg. [.sup.123]1, [.sup.123I]2,
[.sup.99mTc]3 etc.) 2.2.1 Radiosynthesis of
(S)-1-(4-[.sup.125I]iodobenzyl)-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfo-
nyl)isatin [.sup.125I]1
[0217] In a conical glas vial 0.56 mg (0.725 .mu.mol)
(S)-5-(1-[2-(phenoxymethyl)pyrrolidinyl]sulfonyl)-1-(4-(tributylstannyl)b-
enzyl)-isatin Ia in 100 .mu.l ethanol were added to a solution of 4
.mu.l [.sup.125I]NaI (approx. 14 MBq) in 0.05; N NaOH and 4 .mu.l
0.05 M H.sub.3PO.sub.4. The radiosynthesis was started by adding
0.25 mg (1.095 .mu.mol) chloramine-T hydrate (CAT) in 25 .mu.l 0.1
M K.sub.2HPO.sub.4 (pH 7.36). The reaction mixture was vortexed and
allowed to stand 5 min at RT. The resulting reaction suspension was
diluted with 50 .mu.l ethanol and was injected onto a gradient
radio-RP-HPLC-chromatograph with a Nucleosil 100 column (C-18 5.mu.
250.times.4.6 mm) with a corresponding precolumn (20.times.4.6 mm)
and combined .gamma.-/UV-detectors to isolate the radiolabeled
product [.sup.125]I. Radiochemical yield: 90%. Radiochemical
purity: >95%. Calculated specific activity: 0.134 GBq/.mu.g.
HPLC-conditions: eluent A: CH.sub.3CN/H.sub.2O/TFA 950/50/1, eluent
B: CH.sub.3CN/H.sub.2O/TFA 50/950/1; time-program: isocratic run
with 37% of eluent B; flow: 2.5 ml/min, .lamda.: 254 nm,
R.sub.t(product): 17.7 min.
Quality Control
[0218] 200 .mu.l of the product fraction was re-injected onto the
HPLC column. The quality control did not show any impurities within
the .gamma.-range. Only the injection peak was detectable within
the UV-range. HPLC-conditions: eluent A: CH.sub.3CN/H.sub.2O/TFA
950/50/1, eluent B: CH.sub.3CN/H.sub.2O/TFA 50/950/1; time-program:
eluent B from 50% to 20% within 20 min, eluent B 20% for 10 min,
eluent B from 20% to 50% within 10 min; flow: 2.5 ml/min; .lamda.:
254 nm; R.sub.t (product): 17.2 min.
Reference Control
[0219] The radioiodinated product [.sup.125]I was verified by
concentrating 150 .mu.l of the isolated .gamma.-fraction with 50
.mu.l of a solution of the non-radioactive reference compound 1 in
methanol (c=1 mg/ml methanol). The concentrated 200 .mu.l mixture
was again injected onto the HPLC column. Both the radiolabeled
product and the non-radioactive reference standard corresponded to
each other. HPLC-conditions: see Quality control; R.sub.t
(product): 16.9 min.
##STR00077##
3. Caspase Inhibition Assay
[0220] The inhibition of recombinant human caspase 3 by twentythree
isatin sulfonamides (twenty of those representing new isatin
derivatives) has been assessed by using standard fluorometric
assays [21a].
[0221] Recombinant full-length human caspase-3 was purified as
described previously [21b]. The caspase-3 substrate Ac-DEVD-AMC
(Ac-Asp-Glu-Val-Asp-AMC, K.sub.M=9.7 mM.+-.1 mM) was purchased from
Alexis Biochemicals (Switzerland) and dissolved in a buffer
consisting of 140 mM NaCl, 2.7 mM KCl, and 10 mM KH.sub.2PO.sub.4.
Enzyme assays were performed in a 200 .mu.l volume at 37.degree. C.
in reaction buffer containing 0.1% CHAPS, 50 mM KCl, 5 mM
P-mercaptoethanol, 25 mM HEPES (pH 7.5) and nonradioactive isatins
in DMSO each in single doses (end concentrations 500 M, 50 .mu.M, 5
.mu.M, 500 nM, 50 nM, 5 nM, 500 .mu.M, 50 .mu.M or 5 .mu.M).
Recombinant caspase-3 was diluted into the appropriate buffer to a
concentration of 1 unit per assay (=0.5 .mu.M, i.e. 100 .mu.M
substrate conversion after 10 min). After 10 min incubation time
Ac-DEVD-AMC (end concentration 10 .mu.M) was added and reacted for
further 10 min. Reaction rates showing inhibitory activity of the
nonradioactive model inhibitor were measured with a Fusion.TM.
universal microplate analyzer (PerkinElmer) at excitation and
emission wavelengths of 360 and 460 nm, respectively. The
IC.sub.50-values were determined by non-linear regression analysis
using the XMGRACE program (Linux software) and converted into the
corresponding Ki-values by the equation
K.sub.i=IC.sub.50/(1+[S]/K.sub.M) assuming competitive inhibition
by the isatin derivatives, where [S] is the concentration and
K.sub.M is the Michaelis constant of substrate Ac-DEVD-AMC.
[0222] The resulting Kir.sub.app) values in table 7 show that the
in vitro affinities of the modified and new isatin sulfonamides
have been significantly improved compared with the compounds of
structures I, Ia and IIa (Schemel).
TABLE-US-00007 TABLE 7 Inhibition constants of N-1-alkylated isatin
derivatives ##STR00078## Inhibitor Inhibitor K.sub.i(app)/nM .sup.a
log D R.sub.1 = R.sub.2 = Caspase 3 values .sup.b Ph H-- (Ia) 89
([21]: 2.23 IC.sub.50 = 44 nM) Ph .sup.c CH.sub.3-- (I) 124 ([20]:
15 nM) 2.27 Ph .sup.c 4-CH.sub.3O--C.sub.6H.sub.4--CH.sub.2-- (III)
n.d. 3.96 Ph .sup.d 4-I--C.sub.6H.sub.4--CH.sub.2-- (1) 3 5.08 Ph
4-(CH.sub.3).sub.3Si--C.sub.6H.sub.4--CH.sub.2-- (1b) 0.7 6.55 Ph
4-Br--C.sub.6H.sub.4--CH.sub.2-- (1c) 3 4.82 Ph
4-HO--C.sub.6H.sub.4--CH.sub.2 (IIIa) 3 3.31 Ph
4-BnO--C.sub.6H.sub.4--CH.sub.2-- (IIIaa) 6.9 5.62 Ph
4-TBDMSO--C.sub.6H.sub.4--CH.sub.2 (IIIab) 20 2.44 Ph
4-TosO(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (Va) 17 5.05 Ph
4-Br(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (Vaa) 25 4.73 Ph
.sup.c 4-F(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (V) 0.4
4.19 CH.sub.3 H-- (IIa) 77 ([20]: 60 nM) 0.26 CH.sub.3 .sup.c
CH.sub.3-- (II) 2 0.28 CH.sub.3 .sup.c
4-CH.sub.3O--C.sub.6H.sub.4--CH.sub.2-- (IV) 9 1.97 CH.sub.3 .sup.d
4-I--C.sub.6H.sub.4--CH.sub.2-- (2) 11 3.09 CH.sub.3
4-Bu.sub.3Sn--C.sub.6H.sub.4--CH.sub.2-- (2a) 22 9.86 CH.sub.3
4-HO--C.sub.6H.sub.4--CH.sub.2 (IVa) 45 1.32 CH.sub.3
4-BnO-C.sub.6H.sub.4--CH.sub.2-- (IVaa) 5 3.62 CH.sub.3
4-TBDMSO--C.sub.6H.sub.4--CH.sub.2 (IVab) 4 0.45 CH.sub.3
4-TosO(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (VIa) 2 3.05
CH.sub.3 4-Br(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (VIaa)
13 2.73 CH.sub.3 .sup.c
4-F(CH.sub.2).sub.2O--C.sub.6H.sub.4--CH.sub.2-- (VI) 36 2.20
.sup.a K.sub.i(app) = IC.sub.50/(1 + [S]/K.sub.M) with [S] = 10
.mu.M, K.sub.M = 9.7 mM .+-. 1.0 mM; S = Ac-DEVD-AMC .sup.b logD
values calculated with ACD/Chemsketch Labs 6.00 (log D = log P at
physiological pH (pH 7.4) .sup.c Non-radioactive target compounds
of potential PET-compatible CbRs. .sup.d Non-radioactive target
compounds of potential SPECT-compatible CbRs.
4. Cellular Caspase Assays
[0223] In the context of cellular apoptosis assays concentration-
and time-dependent kinetics of mentioned CbRs and CbR-transporter
conjugates are performed to evaluate the pharmacological inhibition
of apoptosis in viable apoptotic cells (e.g. growth factor
withdrawal-induced, drug-induced, or ionizing radiation-induced
apoptosis in endothelial cells).
[0224] HUVEC (Human umbilical vein endothelial cells) were
cultivated on gelatine (2%)-coated dishes in RPMI-1640 containing
15% bovine calf serum, 1% Pen/Strep/Amph, 1% Heparin and 0.05 mg/ml
bovine pituitary extract (BPE) at 37.degree. C. in 5% CO.sub.2.
Apoptosis was induced by growth factor withdrawal as previously
described [39]. For caspase inhibition experiments, cells were
pre-incubated for 30 min with the compounds in the indicated
concentrations. The medium was then removed and replaced with
RPMI-1640 without serum or BPE, and the cells were incubated in the
presence or absence of the various inhibitor concentrations for 8
hours. All cells were then harvested in lysis buffer, incubated for
10 min on ice, and cell debris was removed by centrifugation at
14000 rpm at 4.degree. C. for 10 min. Protein concentration was
determined by the Pierce protein assay, and 30 .mu.g cell lysate
were loaded on 15% SDS-Page gels and transferred to Immobilon PVDF
membranes. Western blots were performed with antibodies to active
caspase-3 (Cell Signaling) and developed using ECL (Amersham).
[0225] A quantitative (pharmacological) inhibition of before
mentioned target caspases with the non-radioactive PET- or
SPECT-compatible CbRs of presented invention needs macroscopic
amounts of the inhibitor, i.e. concentrations of caspase inhibitor
that are definitely more than necessarily needed for molecular
imaging purposes. Therefore, concentrations of inhibitor in the
micromolar range (see FIGS. 1 and 2: Western blots of specific
non-radioactive CbRs of the present invention) clearly demonstrate
an incisive non-invasive imaging compatibility. The inhibition of
caspase progression in the presence of compounds 2, II, IV, and VI,
is already recognizable at 10 .mu.M (FIGS. 1 and 2).
[0226] FIG. 1 is a Western blot analysis of active caspase-3 in
apoptotically dying human endothelial cells in the presence of
different concentrations (c=1 .mu.M i c=10 .mu.M) of PET--(cpds.
II, I, IV, III) and SPECT-compatible (cpds. 2, 1) nonradioactive
conterparts of the CbRs. The methoxymethyl compounds II, IV, and 2
inhibit caspase processing to its p12 subunit with compensatory
accumulation of the p17 subunit at 10 .mu.M. Z-VAD-fmk is used as a
control for full inhibition of caspase processing.
[0227] FIG. 2 is a Western blot analysis of active caspase-3 in
apoptotically dying human endothelial cells in the presence of
different concentrations (c=1-300 .mu.M) of fluorinated
PET--compatible nonradioactive conterparts of the CbRs VI and V.
Inhibition of caspase processing by compound VI occurs at 10
.mu.M.
[0228] As can be seen from above the compounds of the present
invention lead to PET- and SPECT-compatible CbR tracers with a
5-pyrrolidinylsulfonyl isatin skeletal structure that are able to
target intracellular caspases, preferably the effector caspases 3
and 7. The potency of several new CbR reference substances
(nonradioactive) has been proved in vitro using caspase inhibition
assays. The new compounds comprise even higher affinities to
caspase 3 compared with the compounds of structures I and IIa.
[0229] Thus the above CbRs enable a specific imaging of apoptosis
leading to a enhanced efficacy and precision of therapeutic
interventions (disease monitoring) and open new perspectives in
many areas of disease management (therapy control).
5. In Vivo Experiments
Data Acquisition--PET.
[0230] PET was performed using a high-resolution dedicated
small-animal PET system (32-module quadHIDAC; Oxford Positron
Systems) which uses multiwire chamber detectors with
submillimeter-resolution potency. For each data acquisition, up to
two mice were placed on a heating pad to maintain a normal body
temperature. The animals were anesthetised by inhalation of
isoflurane (1.5%) and intravenously injected with approximately 7
MBq of each radiotracer in 100 .mu.L isotonic as well as isohydric
solution.
[0231] FIG. 3 is an examination of the in vivo biodistribution
behavior of [.sup.18F]VI in NMRI athymic nude mice (nu/nu) using
the quadHIDAC small-animal PET scanner (scanning time: 180 min
after i.v. injection of 7 MBq [.sup.18F]VI). All organs are cleared
from radioactivity after 3 h except the bowels and the gall
bladder.
Acquisition Protocol--Biodistribution in WT Mouse (nu/nu)
[0232] A small-animal PET scan was performed with the quadHIDAC
device to trace the in vivo biodistribution behavior of the
PET-compatible
CbR(S)-1-(4-(2-[.sup.18F]fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrol-
idinyl)-sulfonyl]isatin [.sup.18F]VI. Immediately after i.v.
injection of [.sup.18F]VI (A=7 MBq, pH=8, A.sub.s=48 GBq/.mu.mol,
V=100 .mu.l in sodium bicarbonate buffered saline solution) data
acquisition was started. List-mode data were acquired for 180 min
and subsequently reconstructed into an image volume of
90.times.90.times.120 mm.sup.3, voxel size 0.4.times.0.4.times.0.4
mm.sup.3, using an iterative reconstruction algorithm (OPL-EM). As
shown in FIG. 3, [.sup.18F]VI was cleared 180 min p.i. from all
peripheral organs. Radioactivity only remains in the bowels and in
a hot spot nearby the liver which putatively can be assigned to the
gall bladder. Mentioned hot spot remains even 6 h p.i. (data not
shown). According to the here described invention [.sup.18F]VI is a
PET-compatible CbR with corresponding pharmacokinetics, plasma
clearance characteristics as well as imaging potency for the
detection of locally upregulated caspase activity that is
associated with induced apoptosis.
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