JP2012207995A - Radioactive bromine indicator pet molecule imaging probe targetting norepinephrine transporter in brain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide chemical compounds usable as a PET radioactive tracer and a pharmaceutical composition including the compounds.SOLUTION: A chemical compound of positron emitting -2-(α-(2-bromophenoxy)benzyl)morpholines expressed by the following formula (1) (in the formula, Br is radioactive bromine selected from a group consisting ofBr,Br,Br, andBr; and Ris hydrogen or alkyl), a chemical compound of positron emitting -3-(2-bromophenoxy)-3-phenylpropane amines, optical isomers of the compounds, and medically permissible salts including the compounds are provided.

Description

  The present invention relates to a molecular imaging probe labeled with radioactive bromine and the like used for nuclear medicine diagnosis.

In recent years, it is said that 1 in 15 people will suffer from depression due to increased stress in social life. According to the 2005 vital statistics in Japan, suicide is the first cause of death from the 20s to 30s, and suicide is the second cause of death even in the 40s. It is unclear whether all of these causes of death are depression, but depression is thought to account for a significant portion.
Also, psychosomatic diseases are particularly costly. General medical care expenses in Japan in FY2005 were 24,967.7 billion yen, of which the treatment expenses for mental illness (mental and behavioral disorders) accounted for 1,886.3 billion yen (7.6%) ( (From the Ministry of Health, Labor and Welfare's “Summary of National Health Expenditure 2005”) However, it does not include indirect costs related to unemployment due to mental disorders, leave of absence, etc., and these indirect costs are said to be several times the direct costs. Considering the suffering of families with patients with neuropsychiatric disorders and the impact at work, it goes without saying that the medical costs required for neuropsychiatric disorders are only the tip of the iceberg of their social costs.

  On the other hand, in nuclear medicine diagnostic methods using positron emission computed tomography (PET) and single photon emission computed tomography (SPECT), functional changes in vivo are non-invasive. It is expected to be clinically developed by elucidating the pathophysiology of functional psychiatric disorders and developing diagnostic and therapeutic methods. To that end, it will be necessary to develop radiopharmaceuticals for PET and SPECT that target functional psychiatric disorders.

  The monoamine hypothesis has been proposed as one of the causes of functional mental illness. This is a hypothesis that mental disorders occur when the released amounts and transmission levels of monoamines serotonin and norepinephrine (hereinafter also referred to as NE) are not balanced. In order to prove this monoamine hypothesis, probes related to a number of serotonergic nerves have been developed. In addition, several groups have started to develop molecular imaging probes for in vivo imaging of the function of norepinephrine-operating nerves (hereinafter also referred to as NE-operating nerves or NE nerves). However, development research targeting the norepinephrine transporter (hereinafter also referred to as NET) is conducted only by the group of the present inventors in Japan, and only by a few groups in the West. It is.

  NET is a membrane protein that exists in the presynaptic part of the NE nerve. NET consists of 617 amino acids, has a molecular weight of about 69 kDa, and has 12 transmembrane sites. The main role of NET is to re-use NE, which is a neurotransmitter released into the synaptic cleft, to re-use NE and terminate neurotransmission. This NET has been reported to change its expression level in the autopsy brain of patients with functional psychiatric disorders (Non-Patent Documents 1 to 6), and is considered to be an important protein that leads to the elucidation of the disease state. In addition, since NET is a target site for therapeutic drugs for functional psychiatric disorders, analysis of the occupancy ratio of therapeutic drugs for NET, etc. by imaging can be used to determine and predict therapeutic effects.

  As a molecular imaging probe targeting NET, a probe labeled with C-11 is known (Non-patent Document 7). Although this probe has achieved high affinity and selectivity for NET, it has been reported that detailed analysis is difficult due to the short half-life of C-11 of about 20 minutes (Non-patent Document 8). ). A probe that overcomes this problem by introducing F-18 is also known (Non-Patent Document 9). However, this probe has a problem in the introduction portion of F-18, and it has been reported that F-18 is detached from the probe after in vivo administration and the image quality is deteriorated (Non-patent Document 10).

V Klimek et al., The Journal of Neuroscience, Vol. 17, pp. 8451-8458 (1997) Ryu et al., Neuropsychobiology, Vol. 49, pp. 174-177 (2004) Biederman et al., Biological Psychiatry, Vol. 46, pp. 1234-1242 (1999) Viggiano et al., Neural Plast, Vol. 11, pp. 133-149 (2004) Tejani-Butt et al., Brain Research, Vol. 631, pp. 147-50 (1993) Marien et al., Brain Research Reviews, Vol. 45, pp. 38-78 (2004) Wilson et al., Nuclear Medical Biology, Vol. 30, pp. 85-92 (2003) Logan et al., Nuclear Medical Biology, Vol. 34, pp. 667-679 (2007) Schou et al., Synapse, Vol. 53, pp. 57-67 (2004) Takano et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 36, pp. 1885-1891 (2009)

  As described above, many of the causes of death of active generations are related to functional psychiatric disorders such as depression. The development of is of high social significance and is also important from the viewpoint of medical economic effect. Therefore, the development of a new probe that overcomes the problems of existing probes as described above has great value. The present invention provides compounds that can be used as such probes.

As a solution to the problems of the prior art as described above, the present inventors have conceived the use of radioactive bromine that emits β ⁺ rays, particularly Br-76. Br-76 is a nuclide that emits β ⁺ rays and has a relatively long half-life of 16 hours. By using this nuclide, the present inventors can overcome the difficulty of analysis due to the short half-life, and further, by introducing this nuclide directly into the benzene ring, it is possible to construct a structure that is difficult to desorb in vivo. I thought. Based on this idea, we designed a drug based on reboxetine, which has been reported to bind selectively to NET, as a parent compound. The present inventors have proposed a molecular imaging probe for PET, such as a probe designed in such a way that has a long half-life, a high affinity and selectivity for NET, and excellent biodistribution dynamics. It was confirmed that it has excellent characteristics as a probe for diagnosis of functional psychiatric disorders, and the present invention was completed.

That is, the present invention provides the following.
[1] The following formula (I):

(Wherein Br represents a radioactive bromine selected from the group consisting of ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, and ⁸⁰ Br; R ₁ represents hydrogen or alkyl) and optical isomers thereof And the following formula (II):

Wherein Br and R ₁ are as defined above, and R ₂ is hydrogen or alkyl, or a compound selected from the group consisting of optical isomers thereof, or a pharmaceutically acceptable Its salt.
[2] The compound of the above-mentioned [1], which is a compound represented by the above formula (I), wherein Br is ⁷⁶ Br and R ₁ is hydrogen, or a pharmaceutically acceptable compound Its salt to be.
[3] The compound represented by the formula (II), wherein Br is ⁷⁶ Br, R ₁ is hydrogen, and R ₂ is methyl in the formula (II) A compound, or a pharmaceutically acceptable salt thereof.
[4] A pharmaceutical composition for PET, comprising the compound according to any one of [1] to [3] above or a pharmaceutically acceptable salt thereof as a radioactive tracer.
[5] The pharmaceutical composition according to the above [4], which is used for diagnosis of a functional psychiatric disorder.

  The compound according to the above [1] or a salt thereof (hereinafter collectively referred to as the compound of the present invention) has a property of being easily taken into the brain, and has a high binding ability and selectivity to NET. have. In addition, because the radionuclide used has a long half-life, long-term imaging is possible with PET. Therefore, the pharmaceutical composition containing the compound of the present invention is useful as a NET imaging agent for PET.

(S, S) - ⁷⁷ is a diagram showing a measurement result of the compound by HPLC after labeling synthesis of Br-BPBM. It is a figure which shows the measurement result of non-radioactive (S, S) -BPBM in HPLC. (S, S) - after ⁷⁷ Br-BPBM labeling synthesis is a diagram showing a chart of the reaction solution in preparative using HPLC. And purified (S, S) - ⁷⁷ is a graph showing the analysis results of HPLC of Br-BPBM. And purified (S, S) - ⁷⁷ is a graph showing the analysis results of TLC of Br-BPBM. FIG. ³ shows a binding inhibition curve of ³ H-Nisoxetine. The horizontal axis represents the ligand concentration (Log [M]), and the vertical axis represents the% binding of ³ H-Nisoxetine. (S, S) - ⁷⁷ shows the binding inhibition curve of the Br-BPBM. The horizontal axis ligand concentration (Log [M]), the vertical axis (S, S) - shows the percent binding of ⁷⁷ Br-BPBM. Of each organ (S, S) - ⁷⁷ is a diagram showing the time variation of the Br-BPBM uptake. The horizontal axis represents time (min) after administration, and the vertical axis represents% ID / g. (S, S) in each brain tissue - ⁷⁷ is a diagram showing the time variation of the Br-BPBM uptake. The horizontal axis represents time (min) after administration, and the vertical axis represents% ID / g. (S, S) of the striatum - ⁷⁷ Br-BPBM for uptake at each brain tissue (S, S) - ⁷⁷ is a diagram showing the ratio of Br-BPBM uptake. The horizontal axis represents the time (min) after administration, and the vertical axis represents the ratio. (S, S) of each brain tissue - is a view showing a captured image of the ⁷⁷ Br-BPBM. In the figure, areas with high% ID / g are displayed in black, and areas with low ID / g are displayed in white. It is a figure which shows the correlation with% ID / g and NET expression level in each brain tissue. The horizontal axis represents NET expression level (fmol / mg protein), and the vertical axis represents% ID / g. ^{(S, S) - 77 Br} -BPBM only control, and, Nisoxetine, Fluoxetine, or each inhibitor that GBR12909 (S, S) - A diagram illustrating ⁷⁷ the captured image when administered Br-BPBM simultaneously . In the figure, areas with high% ID / g are displayed in black, and areas with low ID / g are displayed in white. ^{(S, S) - 77 Br} -BPBM only control, and, Nisoxetine, Fluoxetine or each inhibitor that GBR12909 (S, S), - 77 Br-BPBM% in co-administered each brain tissue if the ID / It is a figure which shows g.

  Hereinafter, the compound of the present invention and the pharmaceutical composition for PET containing the compound will be described in detail.

  The compound of the present invention has the following formula (I):

(Wherein Br represents a radioactive bromine selected from the group consisting of ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, and ⁸⁰ Br; R ₁ represents hydrogen or alkyl) (S, S) -2- (α- (2-bromophenoxy) benzyl) morpholines, and the following formula (II):

(Wherein Br and R ₁ are as defined above, and R ₂ is hydrogen or alkyl.) Positron-releasing (R) -3- (2-bromophenoxy) -3-phenylpropanamine And their optical isomers, as well as their pharmaceutically acceptable salts. The compound of the present invention has a high affinity and selectivity for NET, as shown in Examples and the like to be described later, and has a particularly high functional spirit such as high accumulation in tissues expressing NET in the body after administration. It has advantageous properties for use in diagnosis and treatment of disease and brain function. Therefore, the compound of the present invention can be suitably used as a radioactive tracer for PET.

In this specification, PET (positron emission tomography) refers to a computer tomography technique using positron detection. In PET diagnosis, a drug containing a compound (radiotracer) labeled with a nuclide that positron-anti-β decays is administered to a subject. A β ⁺ ray emitted from a radioactive tracer administered into a living body (eg, human body) annihilates with an electron in the vicinity of the emitted position, and at this time, two photons (γ rays having diametrically opposite momentum) ) Is released. In PET, information on the position of pair annihilation is accumulated by detecting a number of gamma ray pairs thus emitted, and a tomographic image is generated by processing the information with a computer. Therefore, in this specification, a radioactive tracer for PET is a compound labeled with a positron anti-β ⁺ decaying nuclide, and when it is administered to a living body at the time of PET diagnosis, it decays in the living body. By emitting positrons is meant a compound that can be utilized by a PET device to determine the distribution of the compound in vivo. In this specification, a radioactive tracer is used synonymously with an imaging probe or simply a probe.

The radioactive bromine may be any one selected from the group consisting of ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸⁰ Br, ^80m Br, and ⁸² Br, but it is halved when used as a radioactive tracer for PET. From the viewpoint of the length of the period, the energy released, etc., ⁷⁶ Br is particularly preferable. Production of radioactive bromine can be performed by a known method using a cyclotron. Although the radioactive bromine used in the present invention has a longer half-life than the radioactive substances (eg C-11, F-18) used in conventional PET radioactive tracers, the length is limited (eg: ⁷⁵ Br in 96.7 min; ⁷⁶ in Br 16.2hr; ⁷⁷ Br in 57.0 hr; ⁸⁰ Br in 17.7 min; the ^80m Br 4.4 hr; ⁸² Br in 35.3 hr). Therefore, the radioactive bromine is preferably produced using a small medical cyclotron installed in a hospital.

R ₁ and R ₂ in formula (I) or formula (II) are the same or different and each represents hydrogen or alkyl. More specifically, R ₁ and R ₂ are preferably hydrogen or alkyl having 1 to 6 carbon atoms (eg, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl). More preferably hydrogen or alkyl having 1 to 3 carbon atoms (for example, methyl or ethyl), and even more preferably hydrogen or methyl. R ₁ is particularly preferably hydrogen, while R ₂ is particularly preferably methyl. That is, the affinity of the viewpoints such as for NET, as particularly preferred compounds of the invention include, but are not limited to, compounds of formula (I) R ₁ is hydrogen, and, R ₁ is hydrogen and R Mention may be made of the compounds of the formula (II) in which ₂ is methyl, as well as their salts.

  The pharmaceutically acceptable salt is not particularly limited as long as it has the desired pharmacological activity of the present invention. Specific examples of pharmaceutically acceptable salts include addition salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid and maleic acid And addition salts of organic acids such as fumaric acid, methanesulfonic acid and p-toluenesulfonic acid.

  The compounds of the present invention can be synthesized using conventional synthesis methods. Materials useful for the synthesis of the compounds of the invention are either commercially available or can be prepared by well-known synthetic methods.

Specifically, the compound of the present invention is, for example, a compound of the above formula (I) wherein R ₁ is hydrogen, for example, (S, S) -2- (α- (2-iodophenoxy) benzyl) morpholine (hereinafter referred to as “S”) (Also referred to as (S, S) -IPBM) can be synthesized by substituting iodine with an appropriate radioactive bromine. Here, (S, S) -IPBM is represented by the following formula (III):

It is a compound which has the chemical formula. (S, S) -IPBM can be synthesized, for example, according to the teaching of N. Kanegawa et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 33, pp. 639-647 (2006).
The substitution of iodine with bromine in (S, S) -IPBM should be performed based on the method described in Loc'h et al., Nuclear Medicine and Biology Vol. 21, pp. 49-55 (1994), for example. Can do. Specifically, it can be carried out by a method described in Examples and the like described later, but an example of the procedure is briefly described as follows. That is, ultrapure water containing the desired radioactive bromine is placed in a vial, heated to 110 ° C. with the inside of the vial filled with nitrogen gas, and the ultrapure water is evaporated. In the vial, an ethanol solution containing (S, S) -IPBM, CuSO ₄ .5H ₂ O and (NH ₄ ) ₂ SO ₄ are added. The vial is filled with nitrogen gas and heated to 140 ° C., and the vial is evacuated to completely evaporate the water. React at 140 ° C for 1 hour.
Further, regarding the compound of the above formula (I) in which R ₁ is alkyl, those skilled in the art will follow the above-mentioned method when R ₁ is hydrogen, based on the above-mentioned teaching of N. Kanegawa et al. A compound into which an alkyl group is introduced can be synthesized.
On the other hand, the compound of the above formula (II) can also be synthesized by synthesizing an iodine precursor and carrying out an iodine-radioactive bromine exchange reaction in the same manner as (S, S) -BPBM. The synthesis of the iodine precursor can be performed, for example, according to the method described in Y. Kiyono et al., Nuclear Medicine and Biology, Vol. 30, pp. 697-706 (2003). For example, according to the method described in Loc'h et al., Nuclear Medicine and Biology, Vol. 21, pp. 49-55 (1994).

  The compound of the present invention obtained as described above is preferably purified. Purification can be performed by known means such as high performance liquid column chromatography.

  The present invention also provides a pharmaceutical composition for PET containing the compound of the present invention as a radioactive tracer (hereinafter also referred to as the pharmaceutical composition of the present invention). In addition to the compound of the present invention, the pharmaceutical composition of the present invention contains carriers (eg, solution, inorganic salt, pH adjuster, excipient, stabilizer, isotonicity) that are usually used in pharmaceutical preparations as necessary. Etc.) may be included. The pharmaceutical composition of the present invention is preferably used as an injection. When used as an injection, the compound of the present invention is usually administered after being dissolved in an appropriate solvent (eg, physiological saline).

  The content of the compound of the present invention in the injection is not particularly limited as long as it is an amount sufficient to enable imaging with PET when administered to an administration subject. The specific dose may vary depending on various factors such as the body weight of the administration subject and the target site, but is, for example, 37 MBq to 740 MBq, preferably 111 MBq to 370 MBq.

  The pharmaceutical composition of the present invention is suitably used for diagnosis of a biological function disorder using PET or a tumor expressing NET. The biological function may be any biological function possessed by any organ that expresses NET (eg, lung, heart, adrenal gland, small intestine, large intestine, kidney, brain parts), etc. is there. Examples of neuropsychiatric dysfunction that changes in NET include mood disorders, anxiety disorders, and developmental disorders.

  Hereinafter, the present invention will be described more specifically with reference to examples and the like. However, the scope of the present invention is not limited to the following examples.

Synthesis Example 1: (S, S) - 77 Synthesis and purification of Br-BPBM
(1) Production and extraction of Br-77
The half-life of Br-76 is about 16 hours, which is short for basic experiments, and has the same chemical properties and half-life because there is a risk of exposure under consideration to emit radiation with higher energy. The bromine radioisotope Br-77, which has a long and low energy, was used in this study. The properties of Br-76 and Br-77 are shown in Table 1 and Table 2 below, respectively.

(2) (S, S) - 77 Br-BPBM labeled synthetic
Reboxetine, a selective NET inhibitor, was selected as the parent compound for molecular imaging probes for PET, and radioactive bromine-76 was introduced at the 2-position of the phenoxy group (S, S) -2- (α- It was designed ^{(2- [76 Br] bromophenoxy)} benzyl) morpholine and (BPBM). Our previous report that introduction of a substituent at the 2-position of the phenoxy group of the parent compound does not affect the binding to NET (N Kanegawa et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 33, pp. 639-647 (2006)), Br-76 was introduced at the 2-position of the phenoxy group. The chemical formulas of Reboxetine and (S, S) ^-76 Br-IPBM are as follows.

Using Br-77, which was extracted with ultrapure water in the above (S, S) - labeled synthetic experiment was carried out to obtain a ⁷⁷ Br-BPBM. The label synthesis route is as follows.

The synthesis was performed according to the following procedure. Br-77 ultrapure water was placed in a vial, and the vial was filled with nitrogen gas and heated to 110 ° C. to evaporate the ultrapure water. In a vial obtained by evaporating ultrapure water, solution A (precursor (S, S) -IPBM as a solute, ethanol as a solvent (100 μl)), catalyst A (copper sulfate pentahydrate 36.25 mg / ml, 200 μl) and catalyst B (ammonium sulfate 50 mg / ml, 200 μl). The vial was filled with nitrogen gas and heated to 140 ° C. to evaporate water. The vial was evacuated to completely evaporate the water. The mixture was reacted at 140 ° C. for 1 hour and dissolved in 400 μl of acetonitrile solution (MeCN: H ₂ O = 1: 3).
The results are HPLC (T-Flow: 1, P-Max: 200, UV: 254, Oven: 40 ° C, column: Cosmosil 5C18-ARII 4.6 × 150, mobile phase-acetonitrile (trifluoroacetic acid 0.1%): water (tri Fluoroacetic acid 0.1%) = 3: 7), TLC (solvent-chloroform: methanol: triethylamine = 10: 1: 0.5), and compare the results with non-radioactive (S, S) -BPBM results It was evaluated. FIG. 1 shows the measurement result of the synthesized product by HPLC, and FIG. 2 shows the measurement result of non-radioactive (S, S) -BPBM by HPLC. Table 3 shows the measurement results of the synthesized product by TLC and the measurement results of non-radioactive (S, S) -BPBM.

The elution times in Fig. 1 and Fig. 2 are the same, and TLC results are also significantly different between the Rf values of the synthesized compounds shown in Table 3 and the Rf values of non-radioactive (S, S) -BPBM. Was not recognized. Accordingly, the present labeling synthesis (S, S) - could be achieved in ⁷⁷ Br-BPBM labeled synthetic.
(3) (S, S) - performed for ⁷⁷ Br-BPBM fractionation - from the product of ⁷⁷ Br-BPBM Purification above (2), using HPLC with radioactivity detector (S, S) It was. At this time, the elution time of non-radioactive (S, S) -BPBM detected by HPLC is confirmed in advance.
Purification was performed as follows. The solution containing the target compound obtained in (2) above is HPLC with a radioactivity detector (T-Flow: 4, P-Max: 200, UV: 254, Oven: 40 ° C, column: Cosmosil 5C18- ARII 10 × 250, mobile phase-acetonitrile (trifluoroacetic acid 0.1%): water (trifluoroacetic acid 0.1%) = 35:65) was injected. The radioactive fraction detected at the same time as the elution time of non-radioactive (S, S) -BPBM examined in advance was fractionated. Preparative radioactive fraction ^{(S, S) - 77 Br} -BPBM to confirm that it is, radioactivity detector with a HPLC (T-Flow: 1, P-Max: 200, UV: 254, Oven : 40 ° C., column: Cosmosil 5C18-ARII 4.6 × 150, mobile phase-acetonitrile (trifluoroacetic acid 0.1%): water (trifluoroacetic acid 0.1%) = 3: 7). In order to improve the analysis accuracy, further analysis was performed by TLC (solvent-chloroform: methanol: triethylamine = 10: 1: 0.5). After analysis, if confirmed that can be purified, ethanol added after evaporation to dryness, in aluminum foil (S, S) - ⁷⁷ wrapped container that contains the Br-BPBM, and stored at 4 ° C..
Figure 3 charts the reaction solution in prep using HPLC, fractionated (S, S) - ⁷⁷ analysis by HPLC of Br-BPBM 4, 5 the analysis results by TLC Show. A portion surrounded by a circle in FIG. 3 was collected. Than 4, since the peak detected was one, (S, S) - it was proved to be able to purify ⁷⁷ Br-BPBM other impurities without. Furthermore, since it was confirmed that a peak is one, a high purity (S, S) in FIG. 5 - ⁷⁷ Br-BPBM is confirmed that could be purified.
Obtained by purification (S, S) - ⁷⁷ radiochemical purity of Br-BPBM is 44.9 ± 5.0% (n = 6 ), obtained as a result of 99.0% is radiochemical yield (n = 6) is It was.

Test Example 1: (S, S) - 77 Br-BPBM measured synthesis of octanol / water partition coefficient (S, S) - ⁷⁷ whether Br-BPBM is easily taken into the brain, to obtain the fat-soluble Can be judged. Generally, an octanol / water partition coefficient of 1.5 to 2.5 is regarded as an index of fat solubility that is easily taken into the brain. Therefore, referring to the experimental method described in the literature (Y Kiyono et al., Nuclear Medicine and Biology, Vol. 31, pp. 147-153 (2004)), the following formula:

It is calculated from (S, S) - ⁷⁷ was performed Br-BPBM experiments to determine the octanol / water partition coefficient.
The experimental procedure is as follows. 20 ml each of 1-octanol and 0.1 M phosphate buffer (pH 7.4) were added to the tube, stirred, and allowed to stand overnight at room temperature. 3 ml of 1-octanol in a separate tube in the upper layer of the tube, phosphate buffer new 0.1 M (pH 7.4) and 3 ml, (S, S) - 77 Br-BPBM (74 KBq) and 2 [mu] l I put it in. The mixture was stirred for 1 minute × 3 sets and allowed to stand at room temperature for 20 minutes. Furthermore, the treatment of stirring for 1 minute × 3 sets and allowing to stand at room temperature for 20 minutes was repeated three times, and centrifuged at 1,000 × g for 5 minutes. The 1-octanol in the upper layer was collected in another tube and separated from the phosphate buffer in the lower layer. 500 μl of each was collected, and the radioactivity was measured using a γ counter.
As a result, the synthesized (S, S) - octanol / water partition coefficient of ⁷⁷ Br-BPBM is 2.28 ± 0.03 (n = 5) becomes, (S, S) - and ⁷⁷ Br-BPBM likely are incorporated into the brain properties Was found to have.

Test Example 2: (S, S) - 77 affinity experiment for NET of Br-BPBM
(S, S) - ⁷⁷ affinity NET of Br-BPBM are described in the literature (N Kanegawa et al, European Journal of Nuclear Medicine and Molecular Imaging, Vol 33, pp 639-647 (2006)...) The degree of inhibition of ³ H-Nisoxetine binding to the cerebral cortex crude synaptic membrane fraction was evaluated with reference to the experimental methods described above. ³ H-Nisoxetine is a compound known to have a specific binding ability to NET.
(1) Preparation of rat cerebral cortex crude synapse fraction The brain of male Sprague-Dawley Rat (10 weeks old) was excised, and a crude synapse membrane fraction of the cerebral cortex was prepared. The method is shown below.
Add 30 ml of ice-cold 50 mM Tris-HCl buffer A (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) to the cerebral cortex, crush it using Polytron Homogenizer, and centrifuge 4 ml at a time. The tube was placed in a tube and centrifuged at 28,000 × g and 4 ° C. for 10 minutes. 2 ml of ice-cold 50 mM Tris-HCl buffer A was added to the residue and resuspended. The two tubes were combined and centrifuged again at 28,000 × g and 4 ° C. for 10 minutes. Add 2 ml of ice-cold 50 mM Tris-HCl buffer B (50 mM Tris, 300 mM NaCl, 5 mM KCl, pH 7.4), resuspend the residue, and centrifuge at 28,000 × g for 10 minutes at 4 ° C. did. 1 ml of ice-cooled 50 mM Tris-HCl buffer B was added to the residue and resuspended. The suspension was stored at -80 ° C until use.
(2) Protein quantification The protein concentration of the suspension was measured by the BCA method.
BSA (bovine serum albumin) was diluted with 50 mM Tris-HCl buffer B to prepare BCA protein standard. Concentrations are 1000 μg / ml, 500 μg / ml, 250 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 10 μg / ml, 0 μg / ml (50 mM Tris-HCl buffer B Only). The suspension was diluted 1/10 and 1/50 with 50 mM Tris-HCl buffer B to prepare a sample. BCA working reagent (WR) was prepared by mixing 5000 μl of BCA Protein Assay Reagent A and 100 μl of BCA Protein Assay Reagent B. 10 μl each of BCA protein standard and sample was placed in an assay plate, and 200 μl of WR was added thereto. Incubated at 37 ° C for 30 minutes. Absorbance at a wavelength of 562 nm was measured with a spectrophotometer, and protein concentration was measured from a standard curve.
(3) ³ H-Nisoxetine binding inhibition experiment Next, ³ H-Nisoxetine binding inhibition experiment was performed using this crude synaptic membrane fraction. The procedure is as follows.
Prepare the suspension to 1.0 mg protein / ml with 50 mM Tris-HCl buffer B and add 400 μl of suspension (0.4 mg protein), ³ H-Nisoxetine (final concentration 1.0 nM, 50 μl), and A test drug having a concentration of 10 ⁻¹² M to 10 ⁻³ M (dissolved in 50 mM Tris-HCl buffer B and prepared to 50 μl) was added. In addition to non-radioactive (S, S) -BPBM and its optical isomer (R, R) -BPBM, the test drugs include selective NET inhibitors Nisoxetine, Desipramine, and Five types of Reboxetine were used.
Incubated at 4 ° C for 240 minutes. Stop the reaction by adding 5 ml of ice-cold 50 mM Tris-HCl buffer B to the sample, suction filter with a glass filter soaked in 0.1% polyethyleneimine solution, and filter the filter with 5 ml of ice-cold 50 mM Tris-HCl. Washed 3 times with buffer B. 8 ml of ACS II (Amersham) was added, and the radioactivity was measured with a liquid scintillation counter, which was defined as the total amount of binding.
Nonspecific binding was measured with protein free of 450 μl of 50 mM Tris-HCl buffer B only. Inhibition curves were obtained using the difference between the total binding amount and the non-specific binding amount as the specific binding amount, and the IC ₅₀ value was determined using GraphPad Prism. The IC ₅₀ value is the concentration at which the amount of binding reaches 50%. The smaller the IC ₅₀ value, the higher the affinity. Also, the following formula:

K _i values were calculated according to The K _i value is an index of binding ability, and like the IC ₅₀ value, the smaller the value, the higher the affinity.
The resulting ³ H-Nisoxetine binding inhibition curve is shown in FIG. 6, and the Ki values calculated from the graph are shown in Table 4.

Non-radioactive (S, S) and Ki values -BPBM, Nisoxetine, because significant differences in Ki values of Desipramine was ^{observed, (S, S) - 77} Br-BPBM for NET, existing NET inhibitor It became clear that it has the strong binding ability of the same level.

Test Example 3: (S, S) - 77 binding selectivity experiments on NET of Br-BPBM
(S, S) - binding selectivity for NET for ⁷⁷ Br-BPBM are described in the literature (N Kanegawa et al, European Journal of Nuclear Medicine and Molecular Imaging, Vol 33, pp 639-647 (2006)...) Using the inhibitors that bind to SERT (Serotonin Tansporter) and DAT (Dopamine Transporter), which are similar in protein structure to NET, with reference to the experimental methods described above, rough cerebral cortex synaptic membrane used in Test Example 2 to min (S, S) - it was assessed by comparing the degree of inhibition on the binding of ⁷⁷ Br-BPBM.
The experimental procedure is as follows. The suspension 50 mM Tris - prepared 1.0 mg protein / ml HCl buffer B, the suspension in the tube 400 μl (0.4 mg protein), (S, S) - 77 Br-BPBM ( final concentration 1.9 nM , 50 μl), and a test drug having a concentration of 10 ⁻¹² M to 10 ⁻³ M (prepared in 50 μl dissolved in 50 mM Tris-HCl buffer B). As test drugs, selective NET inhibitors Nisoxetine, Desipramine, Reboxetine, selective SERT inhibitor Fluoxetine, and selective DAT inhibitor GBR12909 were used. Incubated at 25 ° C for 1 hour. Stop the reaction by adding 5 ml of ice-cold 50 mM Tris-HCl buffer B to the sample, suction filter with a glass filter soaked in 0.1% polyethyleneimine solution, and filter the filter with 5 ml of ice-cold 50 mM Tris-HCl. Washed 3 times with buffer B. Radioactivity was measured with a γ counter, and this was defined as the total amount of binding.
The resulting (S, S) - binding inhibition curves for ⁷⁷ Br-BPBM 7 shows an IC ₅₀ value calculated from the graph shown in Table 5.

From Table 5, the IC ₅₀ value of Fluoxetine was about 200 times that of Nisoxetine and the IC ₅₀ value of GBR12909 was about 750 times that of Nisoxetine, indicating a significant difference.
This ^{result, (S, S) - 77} Br-BPBM are considered to be specifically binding to NET.

Test Example 4: (S, S) in normal rats - ⁷⁷ Br-BPBM biodistribution experiments
(S, S) - ⁷⁷ in order to study the distribution in the body of Br-BPBM, was biodistribution experiments with rats.
(1) (S, S) - 77 Procedure for the distribution kinetics experiments for each organ Br-BPBM is as follows. Normal rats (SD: 10 weeks old, male) to and dissolved in physiological saline (5% ethanol) (S, S) - and ⁷⁷ Br-BPBM (approximately 74 kBq / 200 μl) was administered through the tail vein. After a certain period of time (5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours), the tissue was dissected and each organ was removed. Each organ was weighed and packed in a tube. The amount of radioactivity in each organ was measured using a γ counter. % ID / g was used for comparison of the amount of uptake.
(S, S) in each organ - a time variation of ⁷⁷ Br-BPBM uptake shown in FIG. ^{(S, S) - 77 Br} -BPBM is raised accumulation in organs that express NET, such as after administration, the heart, lungs, adrenal glands, then showed a rapid loss. In addition, accumulation in the stomach, which is an indicator of in vivo debromination, decreased with time, suggesting that it is stable in vivo.
(2) (S, S) - 77 Distribution kinetics of the brain of Br-BPBM
(S, S) - About ⁷⁷ the rats receiving Br-BPBM, brains removed, and the radioactivity was measured quantity in the same manner for each brain tissue.
(S, S) in each brain tissue - the time variation of ⁷⁷ Br-BPBM uptake shown in FIG. ^{(S, S) - 77 Br} -BPBM promptly goes to the brain after the administration, the brain-blood ratio as an index of brain imaging increases with time, peaking at 60 minutes of administration. In the brain tissue, compared to the striatum with a small distribution of NET, the accumulation of sites with high expression of NET such as cerebral cortex, thalamus and hypothalamus was high and localization was recognized.
In order to examine the localization in the brain, the value obtained by dividing the radioactivity accumulation in each tissue by the radioactivity accumulation in the striatum was used as an indicator of specific binding. As a result, it was considered that the specific binding was maximum at 180 minutes after administration (FIG. 10). From the above, subsequent experiments are (S, S) - was carried out at ⁷⁷ time points 180 minutes after administration of Br-BPBM.

Test Example 5: Ex vivo autoradiography experiment in normal rats
(S, S) - in order to study the detailed accumulation sites within the rat brain ⁷⁷ Br-BPBM, were ex vivo autoradiography experiments.
The experimental procedure is as follows. In normal rats, dissolved in saline (5% ^{ethanol) (S, S) - 77} Br-BPBM (about 2.2 MBq / 400 μl) was administered from the tail vein, brains were removed after 180 minutes, dry Frozen with ice. (S, S) as a calibration curve ^{- 77 Br-BPBM (1.85 -} 370 kBq) was dissolved in 10% gelatin, and weighed to calculate the% ID / g, and cooled at -37 ° C.. After obtaining a 20 mm thick brain section using a cryostat, it was placed on an imaging plate and exposed for 2 days. Images were acquired using FLA-7000, images were analyzed using MultiGauge, and% ID / g was calculated.
A part of the obtained image is shown in FIG. 11, calculated% ID / g, and NET expression level (Tejani-Butt SM et al., Journal of Pharmacology and Experimental Therapeutics, Vol. 260, pp. 427) -436 (1992)) is shown in FIG. High locus coeruleus of the expression of NET, the thalamus before nuclear group ^{(S, S) - 77 Br} -BPBM high accumulation was observed. In addition, accumulation in the thalamus, hypothalamus, and cerebral cortex with moderate expression was low compared with the locus coeruleus and prethalamic nucleus groups, but higher than that in the striatum without NET expression. Comparison with the expression level of a reported NET, (S, S) - 77 very good correlation between the Br-BPBM and NET expression level was observed.

Test Example 6: Examination of brain selectivity in normal rats
(S, S) - or ⁷⁷ integrated in the rat brain Br-BPBM is NET selective, it was examined by ex vivo autoradiography experiments with inhibitors used in the study of the selectivity of the in vitro .
The experiment was performed according to the following procedure. Dissolved in saline (5% ^{ethanol) (S, S) - 77} Br-BPBM (about 2.2 MBq / 400 μl), Nisoxetine that binds to NET (10 mg / kg), bind to SERT Flioxetine ( 10 mg / kg) or each mixture of GBR12909 (1 mg / kg) binding to DAT was administered from the tail vein of SD male rats (9 weeks old). (S, S) as a calibration curve ^{- 77 Br-BPBM (1.85 -} 370 kBq) was dissolved in 10% gelatin, and weighed to calculate the% ID / g, and cooled at -37 ° C.. After 180 minutes, the brain was removed, frozen with dry ice, and a 20 mm thick brain section was obtained using a cryostat, and then placed on an imaging plate and exposed for 2 days. The obtained image was analyzed and% ID / g was calculated.
^{(S, S) - 77 Br} -BPBM only 13 control and image of each inhibitor coadministration of administration, indicating each inhibitor was calculated from the image and a graph of the% ID / g of each brain tissue 14 . When Nisoxetine was coadministered, substitution with Nisoxetine occurred, and accumulation of (S, S) -77Br-BPBM was not observed. When Flioxetine or GBR12909 was administered simultaneously,% ID / g at each site was not significantly different from the control in which only (S, S) -77Br-BPBM was administered. Therefore, even in the brain ^{(S, S) - 77 Br} -BPBM is to be specifically bound to NET indicated.

The study of the ^{above, (S, S) - 77} Br-BPBM revealed to have selectivity with high binding capacity for NET. In the rat brain, it was shown that accumulation according to the amount of NET expression and that the accumulation was NET-specific.
Or from that ^{(S, S) - 77 Br} -BPBM the (S, S) - be a NET imaging agent using PET by conversion to ⁷⁶ Br-BPBM was suggested.

Claims (5)

The following formula (I):
(Wherein Br represents a radioactive bromine selected from the group consisting of ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, and ⁸⁰ Br; R ₁ represents hydrogen or alkyl) and optical isomers thereof And the following formula (II):
Wherein Br and R ₁ are as defined above, and R ₂ is hydrogen or alkyl, or a compound selected from the group consisting of optical isomers thereof, or a pharmaceutically acceptable Its salt. The compound represented by the formula (I), wherein Br is ⁷⁶ Br and R ₁ is hydrogen in the formula (I), or a pharmaceutically acceptable salt thereof. . The compound represented by the formula (II), wherein Br is ⁷⁶ Br, R ₁ is hydrogen, and R ₂ is methyl in the formula (II), or a pharmaceutical Acceptable salts thereof.   A pharmaceutical composition for PET, comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as a radioactive tracer.   The pharmaceutical composition according to claim 4, which is used for diagnosis of a functional psychiatric disorder.