RADIOACTIVE IODINE- OR BROMINE LABELLED PIPERIDINE COMPOUND AS WELL AS METHOD OF PREPARING SAME AND COMPOSITION COMPRISING SAID COMPOUND

Abstract: The invention relates to a piperidine compound labelled with radioactive iodine or bromine, having general formula (I), wherein Y* is a radioactive iodine atom or a radioactive bromine atom; and Q is a group derived from a heterocyclic ring system having two anellated rings which collectively comprise at least two hetero atoms selected from the group consisting of N, O and S, which ring system is substituted with at least one oxo- and/or thioxo function and may be substituted with one or more substitutents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, halogen, C1-C4 alkylamino and di (C1-C4) alkylamino. The invention also relates to a method of preparing the said compound, to a radiopharmaceutical composition which comprises said compound, and to a kit for preparing said composition.

Description: RADIOACTIVE IODINE- OR BROMINE LABELLED PIPERIDINE COMPOUND AS WELL AS METHOD OF PREPARING SAME AND COMPOSITION COMPRISING SAID COMPOUND Radioac t ive iodine - or bromine - labe l led p iperidine compound as we l l as me thod of preparing same and compos i t ion compris ing s aid compound . The invention relates to a piperidine compound which is labelled with radioactive iodine or bromine. Recent investigations have demonstrated that certain piperidine compounds, for example, ketanserine, ritanseri- ne , pirenperone and altanserine behave like serotonine-So- receptor antagonists.- Meanwhile it has also become known that serotonine-S2-receptors are involved in certain neurological and mental pathological processes and in particular play an important part in disorders which influence mood, for example, agression and depression, and in migraine and related disorders. In order to enable a therapeutic treatment of the said disorders, the identification thereof, i.e. a correct diagnosis, is a prerequisite. It is therefore of the utmost importance to dispose of means which can selectively demonstrate serotonine-S2- ece tor binding sites in vivo, hence in the brains themselves. As a result of this a better recognition of these disorders could be obtained, offering the possibility of arriving at a specific therapy. Janssen et al (J.Labelled Compounds and Radiopharma- ceuticals, Vol. " XXV, NO. 7, 1988, ~~ £__ 783-788) have - 14 radiolabelled ketanserine with J H and C for in vitro investigation. Due to their radionuclidic properties, such isotopes cannot be used for in vivo investigation. Crouzel et al have radiolabelled ketanserine and ritanserine with 11 18 C and F, respectively, (J. Labelled Compounds and Radiopharmaceuticals , Vo . XXV, No. 8, jjp_. 827-831) so as to be able to demonstrate serotine-S2-binding sites in vivo by means of "positron emission tomography" (PET) . By means of this technique, a computer tomogram of the brains can be made, as a result of which certain brain regions can be visualised. PET is an imaging technique in which very short-living radioisotopes are used which emit positrons. 11 18 Such- a short half-life as C and F, namely 20 and 110 minutes, respectively, has for its practical consequen¬ ce that compounds labelled with these isotopes can be prepared only in situ; moreover, a cyclotron is necessary for this purpose. All this hence means an enormous investment in a comparatively complicated infrastructure in the hospital or clinic. Another suitable technique, in particular for identifying abnormal brain functions is the "single photon emission computer tomography" (SPECT) . In this technique gamma rays are used which are detected by sensitive detectors. SPECT is a less expensive technique than PET. Moreover, in this technique compounds may be used which are labelled with suitable gamma emittors, for example, with radioactive iodine of a high specific activity, for 123 example I . As a result of this, tissues with a compara- tively small number of receptor binding sites can be studied by means of this technique. SPECT therefore is a more or less "established" technique in nuclear medicine, for which the apparatus is already present in most of the better equipped clinics and hospitals. In 1986, Wouters at al (Biochem. Pharmacology, 1986, Vol. 35, no. 19, pp. 3199-3202) synthetised 7-amino-8- 125 I- ketanserine, a compound which in principle should be suitable for functional brain study by means of the above SPECT technique. The chemical structure of said compound is presented in the attached table sub "A" . As appears from the publication, said compound in in vitro experiments, performed on frontal cortex tissue of a rat, indeed has a high affinity to serotonine-S2-receptors . However, the spcificity is less pronounced than in ketanserine, because a considerable binding to histamine-H2 receptors was also found. As a result of this a less clear image could be obtained-of the specific serotonine-S2 - receptor binding sites, so that a diagnosis of the involved disorders is impeded. A much more serious disadvantage is that, when using the said compound synthesised by Wouters et al in vivo, no radioactivity useful for practical purposes is detected in the brains, as will become apparent from the examples. This compound, therefore, is only of scientific significance. Obviously, the properties of the molecules have changed by the introduction of the substituents in the selected sites in the molecule to such an extent that the compound can no longer reach the serotonine-S2- receptors in the brains. In fact, it is known from the literature menntioned hereinbefore that ketanserine, if labelled with tritium, has very selective serotonine-S2- receptor affinity in vivo. Otherwise it is generally known that the introduction of certain atoms or groups, and certainly of voluminous atoms such as iodine, may have a disastrous influence on the pharmacokinetic behaviour, as a result of which the compounds either can no longer reach their target, or can no longer bind to the target recep¬ tors . It is the object of the present invention to provide a radiolabelled compound for functional brain study, in particular for neurological and mental pathological processes which are associated with the serotonine-S2" receptor binding sites in the brains, while using the ECT (Emission Computerised Tomography) techniques, in which the compound has a very selective serotonine-S2-receptor affinity and may moreover be used in vivo. This object can be achieved with a piperidine compound which is labelled whit radioactive iodine or bromine and which is characterised according to the invention by the general formula wherein Y is a radioactive iodine atom or a radioactive bromine atom; and Q is a group derived from a heterocyclic ring system having two anellated rings which collectively comprise at least two hetero atoms selected from the group consisting of N, 0 and S, which ring system is substituted with at least one oxo- and/or thioxo function and may be substituted with one or more substitutents selected from the group consisting of C 1 -C alkyl, C1-C4 alkoxy, halogen, C1-C4 alkylamino and di (C^-C )alk lamino . As a result of the specific position of the radioactive iodine atom or bromine atom, namely meta with respect to the fluorine atom with which the phenyl group is substituted, a compound is obtained which in vivo does not behave essentially differently from the corresponding compound not substituted with radioactive iodine or bromine. 123 76 Radioactive halogen isotopes, for example I, Br or Br are excellently suitable for scintigraphic imaging. A compound of the general formula (ID where in Y has the meaning given hereinbefore; and Q 1 is a group derived form a heterocyclic ring system having two anellated rings of which the ring bonded to the ethyl group is an at least partially hydrated pyrimidine ring which is substituted with one or two functions selected from the group consisting of oxo and thioxo and which may be substituted with a methyl group, and in which the other ring, in addition to a nitrogen atom optionally joined with the first ring, may comprise a hetero atom selected from N, 0 and S. Examples of the above preferred compounds are recorded in the attached table under nos . 1, 2 and 3. As a particular aspect of the present invention it was found that the new piperidine compounds labelled with radioactive iodine or bromine can be excellently prepared from the corresponding non-radioactive halogen compounds, provided the preparation method is used as described in European Patent Application 165630. For that purpose, the starting compound in question is reacted with a water- soluble radioactive iodide or bromide, for example, radioactive sodium iodide, sodium bromide, potassium iodide and the like, in the presence of a water-soluble acid, copper ions as a catalyst and at lest one reducing agent which is stable in acid medium. The reaction is preferably carried out in an aqueous medium to which optionally one or more water- iscible solvents, for example, ethanol, methanol, acetone, and the like, have been added. The reaction is preferably carried out at a slightly elevated temperature and then occurs quantitatively or sustantially quantitatively. Various copper salts are suitable for use as a catalyst in the above-described reaction, provided they are soluble in the medium used in the reaction, for example, copper nitrate, copper sulphate, and the like. As water-soluble acids are preferably chosen organic acids, for example, acetic acid, ascorbic acid, gentisic acid, and the like. The quantity of reducing agent which is necessary in this reaction must be larger than the quantity of catalyst. Suitable reducing agents are Sn(II) salts, ascorbic acid, gentisic acid, citric acid, a monosaccharide or a sulphite. Several reducing agents may optionally be used in combination with each other. If desired, antioxi- dants may be used in this reaction, for example, metallic tin, ascorbic acid, citric acid, a monosaccharide or gentisic acid. The invention also relates to a radiopharmaceutical composition which comprises, in addition to a pharmaceuti¬ cally acceptable liquid carrier material, e.g. a physiolo¬ gical saline solution, the piperidine compound labelled with radioactive iodine or bromine, as defined hereinbefo¬ re. For performing a radiodiagnostic examination, in particular of certain brain functions as described hereinbefore, the composition, if so desired after dilution with a pharmaceutically acceptable liquid, may be adminis¬ tered to a warm-blooded living being in a quantity from 1 to 1,000 MBq, preferably from 5 to 500 MBq, per 70 kg of body weight, after which the emitted radioactive radiation is recorded. The ECT tehniques described hereinbefore are preferably used in this examination. As stated hereinbefore, the preparation reaction runs off quantitatively or substantially quantitatively at a slightly elevated temperature, for example, on a boiling water bath. The preparation reaction is therefore excel¬ lently suitable for being performed in a clinic or a clinical laboratory. The desired piperidine compound labelled with radioactive iodine or bromine, which has a comparatively short shelf life in connection with the natural decay of the radioisotope , may then be prepared by the user in situ and be used immediately thereafter. It may be advantageous .to perform the reaction in a solution which is entirely or substantially entirely free from organic solvent and at a pH suitable for physiological application, the desired product being formed quantitativily or substantially quantitatively. The resulting reaction mixture is then immediately ready for pharmaceutical use without any laborious purification and may then be administered to a living being intravenously or subcutane- ously. However, it may also be desired to prepare a radioactive composition having the desired high specific radioactivity by a non-isotopic exchange, succeeded by a simple separation, for example, a column chromatographic separation. Since the radiopharmaceutical composition can be prepared so easily and so simply, this preparation is particularly suitable starting from a so-called "kit". This means that a so-called "cold-kit", in which all the ingre¬ dients required for the preparation reaction with the exception of the radioactive iodide or bromide are present, can be placed at the user's disposal. The radioactive material may be supplied to the user separately. The user can now perform the above-mentioned reaction himself by mixing the ingredients of the "cold kit" with the radioac¬ tive iodide or bromide in the presence of a suitable solvent, and by then treating the reaction mixture as prescribed, for example, by heating it for a given period of time on a boiling water bath. If desired, a simple separation may be carried out and a formulation liquid may be added after termination of the reaction. The ingredients of the "cold kit" may be present, for example, in a dry form, e.g. in lyophilized form; this may favour the stability. It will be obvious from the above that the present invention also relates to such a "cold kit" for preparing a radiopharmaceutical composition. In addition to the starting substance, i.e. the corresponding non-radioactive halogen compound, said kit will comprise a water-soluble acid, a copper salt, at least one reducing agent which is stable in acid medium, and optionally a pharmaceutically acceptable formulation liquid and/or auxiliary substances, e.g. stabilisers and the like. The kit further comprises instructions for use including a prescription for carrying out the reaction described hereinbefore and optionally for carrying out a simple separation. The invention will now be described in greater detail with reference to the ensuing specific examples. EXAMPLE I Prepartion of 3- \2- f - ( 2-radioiodo-4-fluorobenzovDpjperi- dinyl) -ethyl1 -2. (1H .3H)quinazolinedione (compound 1 of the table) . A solution of 1 mg of SnSθ4, 25 mg of gentisic acid, 35 mg of citric acid, 500 /ul of 50% acetic acid in 2 ml of water is filtered. 500 /ul of this solution are added to 0.5 mg of 3- [2- {4- (2-bromo-4-fluorobenzoyl)piperidinyl) - ethyl] -2,4(lH,3H)quinazolinedione and 30 /ul of a Cu 2+ - solution, obtained by dissolving 32.5 mg of CUSO .5H2O in 10 ml of water. The mixture is shaken in a closed tube; thereupon the mixture is flushed with nitrogen for 15 minutes. 30 ul of a iodine-123-labelled sodium iodide with an activity of 370 MBq are added to this mixture. The reaction mixture is heated in a closed vial in a boiling water bath for 60 minutes. The resulting labelling efficiency of the radioactive iodine-labelled product is determined by means of radio-HPLC and is > 90%. In order to obtain a high specific activity the resulting radioactive product is further separated by means of HPLC , in which a semi-preparative "reversed phase" column is used [column packing: silica gel RP18 , eluent: methanol/acetonitri- le/water/trimethylamine/acetic acid mixture; pH 4.8] . The labelled product can be concentrated on a so-called "mini reversed phase" column [column packing: silica gel RP18^; eluent: ethanol acidified with HC1] . The radiopharma¬ ceutical is finally diluted with a isotonic saline solution and sterilised by a 0.22 u bacteria filter. The compositi¬ on is now ready for use. The desired 2'-radioiodo compound can also be obtained by starting from the non-radioactive 2'iodo compound. The corresponding iodine-131- and iodine-125-labelled compounds can be prepared in the same manner by using sodium iodide solutions labelled with these radioisotopes . In a corresponding manner the bromine-76- and bromine-77- labelled compounds have been prepared by means of sodium bromide labelled with bromine-76 and bromine-77, respecti¬ vely. The compounds 2 and 3 recorded in the table are prepared in a corresponding manner from the corresponding non-radioactive 2'-bromo or 2'-iodo compounds. EXAMPLE II In vitro binding of the compound 1 (I - 1-125) obtained according to example I The in vitro binding is examined in a membrane composition of the frontal cortex of rats. The binding is assayed on 0.5 ml of this tissue fraction to which 25 ul of the radiopharmaceutical composition obtained according to Example I and 25 /ul of 10% ethanol have been added. After incubation at 37°C in Tris-HCl buffer of pH 7.7 and filtering (plus rinsing), the radioactivity on the filter is measured. From a Scatchard and Hill analysis of the results appears a linear saturation curve with a dissocia- tion constant KQ of 0.82 nM and a B max of 37 fmol per mg of tissue. The linearity of the curve points to the fact that the examined compound 1 binds to a uniform single receptor population. From inhibition studies it can be determined what type of receptor this is. For that purpose the inhibition of the binding of compound 1 by the notoriously selective serotonine-S2-receptor antagonists ketanserine, ritanserine and methysergide is determined by adding in the above-described experiment 25/ul of antagonist. It is found that the binding is strongly prevented by the said antagonists: K.r_ values of 2.3 x 10 "9 M, 0.53 x 10 " 9 M and 0.64 x 10 "9 M, respectively. It appears from the above inhibition experiments that compound 1 binds selectively to serotonine-S2-receptors . In comparative experiments with tritium-labelled ketanserine no essential differences are found in binding capacity between the latter compound and compound 1. EXPERIMENT III In vivo study in rats The radiopharmaceutical composition obtained according to example I is administered intravenously to male Whistar rats of 200-500 g. Both the compounds labelled with bromine-76 and with iodine-131 and with iodine-125 are used as radioactive compounds. After a given period of time the experimental animals are sacrificed, after which the radioactivity of the organs and the relevant brain parts is measured by means of a "multichannel" spectrometer and an automated ^-scintillation counter. The results obtained show a high uptake of the compounds labelled with radioac¬ tive iodine and with radioactive bromine in the brains of the rats, so that the compounds obviously easily cross the blood-brain barrier. This in contrast with the known compound, namely 7-amino-8- -~ - > l -ketanserine (compound A in the table), which in a comparative experiment cannot or can hardly be detected in the brains. When using compound 1 the brain activity increases linearly with the plasma activity and with the quantity of injected compound. This means that the brain activity is directly related to blood flow. The activity in the frontal cortex also increases linearly with the injected quantity intil the beginning of saturation of the receptors; this indicates a saturable reversible binding. In this connection the frontal cortex is the most relevant part of the brains because this is a brain region with a high concentration of serotonine-S2-receptors (see also example IV) . The radioactive ratio between the frontal cortex and the cerebellum still increases between 3 and 15 hours after injection, which indicates a strong binding. EXAMPLE IV In vivo study in primates A male baboon weighing about 15 kg is initially anaesthesised by injecting 250 mg of ketamine, succeeded by 15 mg/kg pentobarbital . 200 MBq of a radiopharmaceutical composition of compound 1, labelled with 1-123, is then administered intravenously to the animal; the composition has a specific activity of 7.10' MBq/mmol. Between 0 and 60 minutes after injection a series of 60 images per minute is obtained with a large field of view gamma camera positioned over the head and body. One and two hours after injection SPECT studies of the head are performed using a rotating gamma camera (40 sec/view) , 64 angles, 360° rotation). Total volume reconstructions are obtained by "backfiltering" , in which a Hamming Hann filter is used. The reconstructed volume is reoriented so as to obtain transverse slices parallel to the orbito-meatal plane. The resulting SPECT images are shown in Figures 1 and 2, 1 and 2 hours respectively after injection. The planes of the tranverse (from top) slices are shown. A highly selective uptake in the frontal cortex is observed in both studies. Two hours after injection the activity ratio between frontal cortex and the cerebellum has increased to >10. The frontal cortex is a brain region in which serotonine-S2-receptors are present in a high density. The selective uptake of the tested compound in the frontal cortex hence indicates a selective binding of the tested compound to serotonine-S2-receptors . This is confirmed once again by the experiments described in Example V. The above experiments have been repeated three times, each time with the same results . EXAMPLE V In vivo study in primates: displacement experiments Displacement experiments are carried out so as to confirm the selective binding of the compounds according to the invention to serotonine-S2-receptors . The experiments are carried out in the same manner as described in Example IV, with the proviso that one hour prior to the injection of the radiopharmaceutical composi¬ tion 5 mg of ketanserine are injected intravenously. As noted hereinbefore, ketanserine is a notoriously selective serotonine-S2-receptor antagonist which will be going to occupy all the serotonine-S2-receptor binding sites in the frontal cortex. The iodine-123-labelled compound is administered in a very small quantity, namely 203.5 MBq; specific activity 7.10' MBq/mmol; this means an administra- tion of only a few nanogrammes of active substance, equal to the quantity used in Example IV. The SPECT-studies as described in Example IV indicate only an extremely small uptake of radioactivity in the frontal cortex, namely approximately 1% of the radioactivity found in Example IV. This means that consequently there is a significant competition between the labelled compound according to the invention and ketanserine in selective binding to the serotonine-S2-receptors in the frontal cortex, or, in other words, that the labelled compound according to the invention binds to the same type of receptors as ketanse¬ rine, namely to serotonine-S2-receptors .

Claims: CLAIMS: 1. A piperidine compound labelled with radioactive iodine or bromine, having the general formula wherein * is a radioactive iodine atom or a radioactive bromine atom; and is a group derived from a heterocyclic ring system having two anellated rings which collectively comprise at least two hetero atoms selected from the group consisting of N, 0 and S, which ring system is substituted with at least one oxo- and/or thioxo function and may be substituted with one or more substitutents selected from the group consisting of C1-C4 alkyl, C -C4 alkoxy, halogen, C1L-C4 alkylamino and di(C^-C4)alkylamino. A compound as claimed in Claim 1 of the general formula wherein Y* has the meaning given in Claim 1; and Q' is a group derived from a heterocyclic ring system having two anellated rings of which the ring bonded to the ethyl group is an at least partially hydrated pyrimidine ring which is substituted with one or two functions selected from the group consisting of oxo and thioxo and which may be substituted with a methyl group, and in which the other ring, in addition to a nitrogen atom optionally joined with the first ring, may comprise a hetero atom selected from N, 0 and S. 3. A method of preparing a compound of th ° general formula I shown in Claim 1, wherein the symbols have the meanings given in claim 1, by reacting a compound of the general formula wherein Q has the meaning given in Claim 1, and X is a non-radioactive halogen atom, with a water-soluble radioactive iodide or bromide in the presence of a water-soluble acid, copper ions as a catalyst, and at least one reducing agent which is stable in acid medium. 4. A method of preparing a compound of the general formula II shown in claim 2, wherein the symbols have the meanings given in Claim 2, by reacting a compound of the general formula (IV) wherein Q' has the meaning given in Claim 2, and X has the meaning given in Claim 3 , with a water-soluble radioactive iodide or bromide in the presence of a water-soluble acid, copper ions as a catalyst, and at least one reducing agent which is stable in acid medium. 5. A radiopharmaceutical composition which comprises, in addition to a pharmaceutically acceptable liquid carrier material, a radioactive labelled compound, characterised in that the composition comprises a piperidine compound labelled with radioactive iodine or bromine according to Claim 1. 6. A composition as claimed in Claim 5, characterised in that the composition comprises a piperidine compound labelled with radioactive iodine or bromine according to Claim 2. 7. A method of performing a radiodiagnostic examination, in particular of certain brain functions, characterised in that a composition as claimed in Claim 5 or 6 , optionally after dilution with a pharmaceutically acceptable liquid, is administered to a warm-blooded living being in a quantity from 1 to 1,000 MBq, preferably from 5 to 500 MBq, per 70 kg of body weight, and the emitted radioactive radiation is then recorded. 8. A kit for preparing a radiopharmaceutical composition as claimed in Claim 5, comprising a compound of the general formula III shown in Claim 3, wherein the symbols have the meanings given in the preceding Claims, a water-soluble acid, a copper salt, at least one reducing agent which is stable in acid medium, optionally a pharmaceutically acceptable formulation liquid and/or auxiliary substances, and instruction for use including a prescription for reacting the above ingredients with an aqueous solution of a radioactive iodide or bromide as claimed in Claim 3. 9. A kit for preparing a radiopharmaceutical composition as claimed in Claim 6, comprising a compound of the general formula IV shown in Claim 4, wherein the symbols have the meanings given in the preceding Claims, a water-soluble acid, a copper salt, at least one reducing agent which is stable in acid medium, optionally a pharmaceutically acceptable formulation liquid and/or auxiliary substances, and instructions for use including a prescription for reacting the above ingredients with an aqueous solution of a radioactive iodide or bromide as claimed in Claim 4.

Inventors: MERTENS JOHN, MERTENS, JOHN
Applicants: MALLINCKRODT INC, MALLINCKRODT, INC.
Application year: 1990
Main classification: A61K49
IPC codes: A61K51, C07D401