Carbon-isotope monoxide labeling of DAA1106 and its analogues to be used as tracers for a peripheral type benzodiazepine binding site

Field of the Invention

The present invention relates to analogues of DAAl 106 and a carbonylation method of labeling DAAl 106 and its analogues by carbon-isotope monoxide in a palladium mediated carbonylation reaction for the study of the function of peripheral benzodiazepine receptors in the neuroinflammation of a patient. The synthetic routes for the preparation of precursor amines and reference compounds were also developed.

Background of the Invention

Positron emission tomography ("PET") is a non-invasive imaging technique based on the detection of positron annihilation radiation and has become a tool of choice for many diagnostic medical applications. Moreover, this technique allows the investigation of biochemical transformations of drugs or other molecules in living systems and thereby offers possibilities to study physiology, molecular biology, energy metabolism, drug-receptor or drug-enzyme interactions etc. and can play an important role in the discovery of new drugs. The micro dosing concept of PET can be a useful strategy for speeding up the drug development process. ¹

Any investigation using PET requires the target molecules to label with short lived positron emitting radionuclides like ¹¹ C, ¹⁸ F, ⁷⁶ Br, ⁶⁸ Ga etc. Among these, ¹¹ C is very important to PET radiochemists because of its half life (t> _/2 = 20.4 min) which is almost optimal from the synthetic point of view and also a wide rage of compounds can be labelled with ¹¹ C. The ¹¹ C-labelled biologically interesting molecules are used as PET tracer for various biological investigations in vitro and in vivo. The demand for new method for the synthesis of labelled molecules is continuously increasing with increasing use of PET in medical and biomedical research as well as in various clinical applications. Although the methods most commonly used for the synthesis of ^π C-labelled compounds are S-, O- and iV-methylations using [ ^u C]methyl iodide or [ ⁿ C]methyl triflate, the application of carbonylation using [ ^u C]carbon monoxide has recently become an increasingly employed ^l ^-labelling strategy.

During the last two decades carbonylation chemistry using carbon monoxide has developed significantly. The recent development of methods such as palladium- catalyzed carbonylative coupling reactions has provided a mild and efficient tool for the transformation of carbon monoxide into different carbonyl compounds.

Carbonylation reactions using [ ¹¹ C] carbon monoxide have a primary value for

PET -tracer synthesis since biologically active substances often contain carbonyl groups or functionalities that can be derived from a carbonyl group. The syntheses are tolerant to most functional groups, which means that complex building blocks can be assembled in the carbonylation step to yield the target compound. This is particularly valuable in PET-tracer synthesis where the unlabeled substrates should be

combined with the labeled precursor as late as possible in the reaction sequence, in order to decrease synthesis-time and thus optimize the uncorrected radiochemical yield.

When compounds are labeled with ¹¹ C, it is usually important to maximize specific radioactivity. In order to achieve this, the isotopic dilution and the synthesis time must be minimized. Isotopic dilution from atmospheric carbon dioxide may be

substantial when [ ¹¹ C] carbon dioxide is used in a labeling reaction. Due to the low

reactivity and atmospheric concentration of carbon monoxide (0.1 ppm vs. 3.4 x I O ⁴

ppm for CO ₂ ), this problem is reduced with reactions using [ ¹¹ C] carbon monoxide.

The synthesis of [ ¹¹ C] carbon monoxide from [ ¹¹ C] carbon dioxide using a heated column containing reducing agents such as zinc, charcoal or molybdenum has

been described previously in several publications. Although [ ¹¹ C] carbon monoxide

was one of the first ^π C-labeled compounds to be applied in tracer experiments in

! human, it has until recently not found any practical use in the production of PET- tracers. One reason for this is the low solubility and relative slow reaction rate of

[ ¹¹ C] carbon monoxide which causes low trapping efficiency in reaction media. The

general procedure using precursors such as [ ¹¹ C] methyl iodide, [ ¹¹ C] hydrogen cyanide or f ¹¹ C] carbon dioxide is to transfer the radioactivity in a gas-phase, and trap the radioactivity by leading the gas stream through a reaction medium. Until recently this has been the only accessible procedure to handle [ ¹¹ C] carbon monoxide in labeling synthesis. With this approach, the main part of the labeling syntheses with

[ ¹¹ C] carbon monoxide can be expected to give a very low yield or fail completely.

There are only a few examples of practically valuable ^{1 !} C-labeling syntheses using high pressure techniques (> 300 bar). In principal, high pressures can be utilized for increasing reaction rates and minimizing the amounts of reagents. One problem with this approach is how to confine the labeled precursor in a small high- pressure reactor. Another problem is the construction of the reactor. If a common column type of reactor is used (i.e. a cylinder with tubing attached to each end), the gas-phase will actually become efficiently excluded from the liquid phase at pressurization. The reason is that the gas-phase, in contracted form, will escape into the attached tubing and away from the bulk amount of the liquid reagent.

The cold-trap technique is widely used in the handling of ^π C-labeled precursors, particularly in the case of [ ¹¹ C] carbon dioxide. The procedure has, however, only been performed in one single step and the labeled compound was always released in a continuous gas-stream simultaneous with the heating of the cold- trap. Furthermore, the volume of the material used to trap the labeled compound has been relative large in relation to the system to which the labeled compound has been transferred. Thus, the option of using this technique for radical concentration of the labeled compound and miniaturization of synthesis systems has not been explored. This is especially noteworthy in view of the fact that the amount of a ^l ^-labeled compound usually is in the range of 20-60 nmol.

Recent technical development for the production and use of [ ^l C] carbon monoxide has made this compound useful in labeling synthesis. WO 02/102711

describes a system and a method for the production and use of a carbon-isotope monoxide enriched gas-mixture from an initial carbon-isotope dioxide gas mixture. [ C] carbon monoxide may be obtained in high radiochemical yield from cyclotron produced [ ¹¹ C] carbon dioxide and can be used to yield target compounds with high specific radioactivity. This reactor overcomes the difficulties listed above and is useful in synthesis of ^l ^-labeled compounds using [ ¹¹ C] carbon monoxide in palladium or selenium mediated reaction. With such method, a broad array of carbonyl compounds can be labeled (Kilhlberg, T.; Langstrom, B. J., Org. Chem., vol. 64, 1999, 9201-9205; Kihlberg, T., Karimi, F., Langstrom, B., J. Org. Chem., vol. 67, 2002, 3687-3692). Additionally, [ ¹³ C] and [ ¹⁴ C]-labeled compounds using [ ¹³ C] and [ ¹⁴ C] carbon monoxide in palladium or selenium mediated reactions are presented as part of this carbon monoxide carbon monoxide carbon monoxide

In the present invention, carbonylation methods for labeling [ ¹¹ C ] compounds was studied in order to obtain the best tracer for visualization and characterization of a peripheral type benzodiazepine binding site, peripheral benzodiazepine receptors (PBR). A PBR is an 18 IeDa protein that is found in outer mitochondrial membranes in almost all tissues and is abundant in steroid-producing cells. PBRs are also found on glial cells of central and peripheral nervous systems and its density increases with the inflammatory reaction of the nervous tissue. J. Maeda et. al, SYNAPSE, 2004, vol. 52, 283-291.

Furthermore, PBR is expressed in most organs and its expression is reported to be increased in activated microglia in the brain. PBR ligands such as [ ¹¹ C]PKl 1195

have been widely used for the in vivo imaging of PBRs, Id. The prototype PBR ligands Ro5-4864 and the isoquinoline carboxamide, PKl 1195 have been widely used to determine the cellular expression and function of PBR in various tissues wherein a ligand is defined as a group, ion, or molecule coordinated to a central atom or molecule in a complex.

Accordingly, there is a need for creating new synthesis methods, expanding such methods to generate new compounds for the labeling of DAAl 106 and making a library of analogues of these compounds for finding PET tracers for the study of the function of peripheral benzodiazepine receptor (PBR) in neuroinflammation within a patient wherein PBR is a key element of the steroidogenic pathway in peripheral tissues.

The term "analogue" used throughout this invention is defined as a chemical compound that is structurally similar to DAAl 106 but differs in composition i.e. elements, functional groups.

Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

Summary of the Invention

In view of the needs of the prior art, the present invention provides a method that relates to a carbonylation method for the labeling of DAAl 106 and making a library of analogues of this compound for finding Positron Emission Tomography (PET) tracers for the study of the function of peripheral benzodiazepine receptors (PBR) in the neuroinflammation of a patient.

Additionally, the present invention depicts a method for labeling synthesis of a ligand and its analogues for a peripheral type bezodiazepine receptor, comprising: providing a carbon-isotope using a carbon-isotope monoxide in a palladium mediated carbonylation reaction; introducing a micro autoclave technique into said carbonylation reaction whereby converting the carbon-isotope monoxide to a crude product; flashing the crude product with nitrogen to remove furan residues and adding acetonitile whereby forming a resulting solution; and injecting the resulting soultion into a liquid chromatograph.

In a further embodiment, a method for labeling synthesis of precursor amines for iV-(2,5-dimethoxybenzyl)-iV-(5-fluoro-2-phenoxyphenyl) acetamide analogues is claimed as Scheme (2).

R1 = OMe, Cl, H

X = F, Cl, H Y = Br, Cl R2 = H, OMe R3 = H, OMe wherein Rl is OMe, F, or hydrogen; R2 is hydrogen or OMe; and R3 is hydrogen or OMe.

Scheme (2)

In yet another embodiment of the present invention presents another method for labeling synthesis of precursors forN-(2,5-dimethoxybenzyl)-iV-(5-fluoro-2- phenoxyphenyl) acetamide analogues, which comprises the reaction performed by Scheme (3):

Scheme (3)

In another embodiment, the present invention shows a method for labeling synthesis of precursors for N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (4):

R1 = OMe or H R2 = OMe or H R3 = Ome or H wherein Rl is OMe or hydorgen; R2 is OMe or hydrogen; and R3 is OMe or hydrogen.

Scheme (4)

Additionally, another embodiment shows a method for labeling synthesis of reference compounds for iV-(2,5-dimethoxybenzyl)-iV-(5-fluoro-2-phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (5):

10 11 X = F, Cl wherein X is F or Cl

Scheme (5)

Brief Description of Figures

Fig. 1 shows the different structures and composition make up of three peripheral benzodiazepine receptor (PBR) ligands, Ro5-4864, PKl 1195, and DAAl 106 that have been labeled with [ ¹¹ C].

Fig. 2 depicts twenty-five (25) analogues of [carbonyl- ⁿ C\ DAAl 106 which have been selected as target molecules from where compounds will be selected according to their affinity and specificity for PBR. Autoradiography and organ distribution using frozen tissues and rats are and have been used as a searching

technique for the selection of these compounds. The selected compounds will be used for further biological experiments.

Fig. 3 shows nine (9) precursor amines used in the synthesis of [ ¹¹ C] DAA analogues.

Detailed Description of the Invention

A ligand for peripheral type benzodiazepine receptor DAAl 106 and analogues were labeled with ¹¹ C using [ ¹¹ C] carbon monoxide in a palladium mediated carbonylation reaction. The reaction was performed in one step in a home made completely automated micro autoclave device. The ^l ^-labeled products were obtained with 15-45% decay-corrected radiochemical yields. The synthetic routes for the preparation of precursor amines and reference compounds were also developed.

The compound N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide or DAAl 106 is a potent and selective ligand for peripheral benzodiazepine receptors (PBR). The PBR is an 18 kDa protein and found in outer mitochondrial membrane in almost all patient tissues and is abundant in steroid-producing cells. These receptors are also found on glial cells of central and peripheral nervous systems and its density increases with the inflammatory reaction of the nervous tissue. The ligands for this receptor in combination with PET might be used as markers for neuroinflamation since the inflammation increases the concentration of the receptors for these ligands. Three different PBR ligands such as Ro5-4864, PKl 1195 and DAAl 106 (Fig. 1) have so far been labeled with ¹¹ C and used in PET studies, and in

all cases a methylation method for [ ¹¹ C] methyl iodide was used for labeling syntheses. The present invention contains the development of a carbonylatiόn method for carbon-isotope labeling of DAAl 106 and making a library of analogues of this compound for finding PET tracers for the study of the function of PBR in neuroinflamation within a patient.

A useful method for ^π C-labeling of DAAl 106 using [ ¹¹ C] carbon monoxide and a micro autoclave technique has been developed and a small library of 25 compounds (Fig 2) have been selected as target molecules from where the most useful compounds will be selected according to their affinity and specificity for PBR. Autoradiography and organ distribution using frozen tissues and rats were used for the searching technique for selection of useful compounds. The selected compounds will be used for further biological experiments.

The aforementioned micro auto clave technique is a 200 μL volume autoclave that is connected to a carbon-isotope monoxide system through a five points valve. The valve is also connected to a High Performance Liquid Chromotography (HPLC) pump, a product outlet capillary, reagent injection loop and a waste outlet capillary. The autoclave is first charged with carbon-isotope monoxide and then the reagent is loaded into the loop that was forced through the autoclave using a HPLC pump.

Furthermore, the carbonylation method of labeling DAAl 106 and its analogues by a carbon-isotope monoxide have suggested a clearly favorable contrast in animals when compared to other selective PBR ligands and methods; One

advantage with the use of the carbonylatioii method is the development of new analogues for carbon-isotope labeling of DAAl 106 analogues. There are several additional advantages with using the present method over previous methods. Since labeled metabolites might be a confounding factor, the option of labeling DAAl 106 in other positions may be valuable and, accordingly, the carbonyl labeling will allow the formation of analogues of the compound. For example, by using [ ¹¹ C] carbon monoxide for labeling [ ¹¹ C] DAAl 106 as opposed to labeling [ ¹¹ C] DAAl 006 with [ ¹¹ C] methyl iodide may enable a clinician to see a greater contrast between the binding and non-binding site expressing areas of PBR.

Below a detailed description is given of a method for labeling DAAl 106 and making a library of analogues of this compound for finding PET tracers for the study of the function of PBR in neoroinfmammation.

In one embodiment of the present invention comprises a method for labeling synthesis of a ligand and its analogues for a peripheral type bezodiazepine receptor by: providing a carbon-isotope using a carbon-isotope monoxide in a palladium mediated carbonylation reaction; introducing a micro autoclave technique into said carbonylation reaction whereby converting the carbon-isotope monoxide to a crude product; flashing the crude product with nitrogen to remove furan residues and adding acetonitile whereby forming a resulting solution; and then injecting the" resulting soultion into a liquid chromatograph.

Wherein, the ligand for the peripheral type bezodiazepine receptor is 7V-(2,5- dimethoxybenzyl)-AT-(5-fluoro-2-phenoxyphenyi) acetamide or also know as DAAl 106 and the furan residues comprise tetrahydrofαran or similar furans.

In a further embodiment, the analogues of the ligand for the peripheral type bezodiazepine receptor is represented by any one of the labeled compounds below or a combination thereof:

DORP DORQ DORR

DORV DORW DORX

DORY

An additional embodiment of the present invention is that carbon-isotope can be ¹¹ C, ¹³ C, or ¹⁴ C.

Another embodiment of the present invention is where the palladium mediated carbonylation reaction is represented by Scheme (1):

Scheme (1)

wherein the concentration of the carbon-isotope monoxide is about ca 10 ^"2 M to about ca 10 ^"6 M. The concentration of the carbon-isotope monoxide is preferably about ca 10 ^"4 M. Additionally, the resulting solution is a purified

product and the liquid chromatograph used to evaluate the resulting solution is semi-preparitive.

Another embodiment of the present invention is a method for labeling synthesis of precursor amines for N-(2,5-dimethoxybenzyl)-iV-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (2):

4 1

R1 = OMe, Cl, H

X = F, Cl, H Y = Br, Cl R2 = H, OMe R3 = H, OMe wherein Rl is OMe, F, or hydrogen; R2 is hydrogen or OMe; and R3 is hydrogen or OMe.

Scheme (2)

In yet another embodiment of the present invention depicts a labeling synthesis of precursors for N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (3):

Scheme (3)

A further embodiment of the present invention involves a labeling synthesis of precursors for N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (4):

R1 = OMe or H R2 = OMe or H R3 = Ome or H wherein Rl is OMe or hydorgen; R2 is OMe or hydrogen; and R3 is OMe or hydrogen.

Scheme (4)

Additionally, in a preferred embodiment, the present invention also defines a method for labeling synthesis of reference compounds forN-(2,5- dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (5):

wherein X is F or Cl

Scheme (5)

Additionally, in another embodiment, the present invention shows a method for labeling synthesis of precursor amines wherein the precursor amines are represented by one or more of the following compounds:

The present invention also provides a kit for a PET study comprising an effective amount of carbon-isotope labeled compound of fig. (3), and pharmaceutically acceptable salts and solvates thereof.

The present invention further provides a method of use for a PET study

comprising an effective amount of carbon-isotope labeled compound of fig. (3), and

pharmaceutically acceptable salts and solvates thereof, wherein the compound can be

one or more of the following structures:

DORM DORN DORO

DORP DORQ DORR

DORS DORT DORU

DORV DORW DORX

DORY

wherein * is the Labeling position.

A further embodiment of the present invention is wherein the method of use for labeling synthesis of a ligand and its analogues for a peripheral type bezodiazepine receptor is used according to the follow method:

(i) providing a carbon-isotope using a carbon-isotope monoxide in a palladium mediated carbonylation reaction; (ii) introducing a micro autoclave technique into said carbonylation reaction whereby converting the carbon- isotope monoxide to a crude product; (iii) flashing the crude product with nitrogen to remove furan residues and adding acetonitile whereby forming a resulting solution; and

(iv) injecting the resulting soultion into a liquid chromatograph.

wherein the ligand for the peripheral type bezodiazepine receptor is N-(2,5- dimethoxybenzyl)-iV-(5-fluoro-2-phenoxyphenyl) acetamide and wherein the analogues of the ligand for the peripheral type bezodiazepine receptor is represented by any one of the compounds below or a combination thereof:

DORJ DORL

DORK

DORP DORQ DORR

DORV DORW DORX

DORY wherein * is the Labeling position.

Furthermore, the carbon-isotope is ¹¹ C, ¹³ C, or ¹⁴ C, wherein the carbon-isotope is preferably ¹¹ C.

Yet another embodiment of the present invention presents that the palladium mediated carbonylation reaction is represented by scheme (1):

Scheme (1) wherein the concentration of the carbon-isotope monoxide is about ca 10 v ^"z 2 M- to about ca 10 ^"6 M.

A further embodiment of the present invention presents a method of use wherein the concentration of the carbon isotope monoxide is about ca 10 ^~4 M. and wherein the resulting solution is a purified product.

Still another embodiment of the present invention presents the liquid chromatograph as being semi-preparative.

Yet another embodiment of the present invention presents a method of use for labeling synthesis of precursor amines foriV-(2,5-dimethoxybenzyl)-iV-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme

(2):

R1 = OMe, Cl, H

X = F, Cl, H Y = Br, Cl R2 = H, OMe R3 = H, OMe

wherein Rl is OMe, F, or hydrogen; R2 is hydrogen or OMe; and R3 is hydrogen or OMe

Scheme (2).

A further embodiment of the present invention presents a method of use for labeling synthesis of precursors for N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (3):

Scheme (3).

Another embodiment of the present invention depicts a method of use for labeling synthesis of precursors for N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2- phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme

(4):

9

R1 = OMe or H R2 = OMe or H R3 = Ome or H wherein Rl is OMe or hydorgen; R2 is OMe or hydrogen; and R3 is OMe or hydrogen

Scheme (4).

Still another embodiment of the present invention depicts a method of use for labeling synthesis of reference compounds forN-(2,5-dimethoxybenzyl)-N-(5-fluoro- 2-phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (5):

10 11

X = F, Cl wherein X is F or Cl

Scheme (5).

The present invention also depicts the method for labeling synthesis of precursor amines according to a method of use for labeling synthesis of precursor amines for N- (2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide analogues, comprising the reaction performed by Scheme (2):

4 1

R1 = OMe, Cl, H

X = F, Cl, H Y = Br, Cl R2 =, H, OMe R3 = H, OMe

Scheme (2) wherein Rl is OMe, F, or hydrogen; R2 is hydrogen or OMe; and R3 is hydrogen or OMe, and further wherein the precursor amines are represented by one or more of the following compounds:

8

Examples

The invention is further described in the following examples which are in no way intended to limit the scope of the invention.

General method for preparing "C-Iabeling synthesis

Tetrakis (triphenylphosphine) palladium (3.0 mg, 2.7 micromol) was placed in a vial (1 ml), flashed with nitrogen gas in order to make the tetrakis palladium inert inside the vial be removing air and moisture from the vial and anhydrous tetrahydofuran (THF) (200 microL) was added thereafter. The mixture was shaken until the solution was homogeneous and methyl iodide (13.6 mg , 6 microL, 96.4 micromol) was added. The mixture was heated for 5 minutes at 6O ⁰ C and the solvent was removed by flashing with nitrogen. The residue was dissolved in fresh anhydrous THF (250 microL).

An amine (as shown in fig. 3) (14.1 micromol) was placed in another vial (1 niL), flashed with nitrogen and dissolved in anhydrous THF (200 microL). The solution was treated with BuLi (10 microL of 1.6 M solution, 16.0 micromol), heated for 5 minutes at 60°C (no heating was needed for DORA) and the solvent was removed by flashing with nitrogen. The residue was dissolved in fresh anhydrous THF (250 microL).

The two reagent solutions were mixed together, filtered and injected into the loop of a Liquid Chromatography (LC) from where the mixture was transferred with pressure (35 Mpa) into the micro autoclave (200 microl), pre-charged with [ ¹¹ C] carbon monoxide in helium. The micro-autoclave was heated for 5 minutes at 150 ⁰ C. The crude product was transferred to a vial (1 niL), THF was removed by flashing with nitrogen and acetonitrile (1 mL) was added. The resulting solution was injected in a semi-preparative LC or LC.

A AMF(40)-MeCN/H ₂ O (50:7)(60) solvent was used in the semi preparative LC system. A linear gradient to 0:90 within 5 minutes of the LC reading and a flow of 5 niL/min. were achieved. A reverse-phase column such as the Jones Chromatography Genesis C 18, 4 micrometers, 250 mm. by 10 mm. was used. Data collection and LC control were performed using a Beckman System Gold chromatography software package.

A AMF(50)-MeCN/H ₂ O (50:7)(50) solvent was used in the analytical LC system with a linear gradient to 0:100 within 6 minutes of the LC reading and a flow of 2 mL/min. A reverse-phase column such as the Jones Chromatography Genesis C 18, 4 micrometers, 250 mm. by 4.6 mm. was used. Data collection and LC control were performed using a Beckman System Gold chromatography software package.

The biologically suitable solution to be injected into the LC was prepared as follows. The purified fraction of the product is placed inside an evaporator and the solvent was removed under reduced pressure at 95°C. The residue was dissolved in a

mixture of sterile propyleneglycol (1 niL), ethanol (99.5%, 1 mL) and a sterile phosphate buffer (4 mL) and was transferred into an injection vial. The automated synthesis system, Synthia, was used for LC injection and fraction collection transfer of the sample and operation of the evaporator.

Synthia is a synthetic robot developed at the Uppsala PET center for the handling of radioactive substance and performing some labeling synthesis. A description of this device can be found in P. Bjurling, R. Reineck, G. Westerberg, A. D. Gee, J. Sutcliffe, B. Langstrδm, Proceedings of the VUh workshop on targetry and target chemistry; TRIUMF, Vancouver, Canada, 1995, pp 282-284.

Example 1 - Experiemental Studies

I. Labeling synthesis of DAA1106 and analogues

The peripheral type benzodiazepine receptor ligand DAAl 106 and its analogues were labeled with ¹¹ C using [ ¹¹ C] carbon monoxide. Palladium mediated carbonylation using tetrakis (triphenylphosphine) palladium, methyl iodide, a concentration of about ca 10 ^"2 M to about ca 10 ^"6 M, or a concentration more preferably ca 10 ^"4 M, of [ ¹¹ C] carbon monoxide and nine different amines (fig. 3) were used in the labeling synthesis (Scheme 1).

Scheme 1. Synthesis of [carbonyl- 11/ C]DAA analogues.

The reaction was performed in a micro autoclave of 200 μL. The conversion of [ rl l C] carbon monoxide to crude products (trapping efficiency) was over 95% for all cases (Table 1) and the decay corrected radiochemical yields of LC-purified products, calculated from [ ¹¹ C] carbon monoxide, were 10 to 40% (Table 1).

Table 1. Trapping efficiency and radiochemical yields for the * ^-labelled DAAl 106 and analogues shown in Fig 2.

TE = Trapping Efficiency, decay-corrected, the fraction of radioactivity left in the crude product after purge with nitrogen, ⁸ RCY= radiochemical yield; decay-corrected and determined by analytical LC on the basis of radioactivity in the crude product before nitrogen purging and the purity of crude product, the value is the mean value of at least 3 experiments. Values in parenthesis are the isolated yields.

The radiochemical purity was over 97% for all compounds. The identification of the labeled products was performed using analytical LC with co-injection of non- radioactive materials. The final identification of ^l ^-labeled compounds will be confirmed by Liquid Chromatography-Mass Spectometry (LC-MS). Some of the selected compounds will be labeled with ¹³ C followed by Nucleur Magnetic Resonance (NMR) analysis for the confirmation of labeling position.

Compound DAAl 106 was previously labeled with ¹¹ C using [ ¹¹ C] methyl iodide and in that strategy only DAAl 106 could be labeled with ¹¹ C. No other ¹¹ C- labeled analogues of that compound could be labeled using [ ¹¹ C] methyl iodide. On the other hand, carbonylation using [ ¹¹ C] carbon monoxide gave a possibility to prepare a series of ^x ^-labeled analogues of DAAl 106 and 25 compounds have been selected as the initial step of this study.

In initial experiments, said reaction was performed with non activated amines i.e. amines without any additional base and the reaction worked very poorly. The radiochemical yield was only 5%. The activation of amines is usually essential for improving the radiochemical yields of ' ^-labeled amides prepared by carbonylation reactions using [ ¹¹ C] carbon monoxide. Different bases for the activation of amines

were used. Furthermore, the activation of amines with BuLi gave considerable improvement to the radiochemical yield. Wherein the radioactive yield of activated amines was 40% when the reaction was performed after activation of amine using BuLi, i.e. the improved radiochemical yield difference between the activated and non activated amines was 35%.

The procedure described above can be used for ¹³ C and ¹⁴ C-labeling of DAAl 006 and its analogues by [ ¹³ C] and [ ¹⁴ C] monoxide in a palladium mediated carbonylation reaction.

II. Synthesis of the precursors of the DAA analogues

The precursors for DAAl 106 analogues were prepared by iV-alkylation of 3 different 2-phenoxy anilines (3) with different benzyl halides (4). The reaction was performed in presence of CsOH.H ₂ O and molecular sieves (MS) in dry DMF.

X=F, R1 =R3=OMe, R2=H (1a); X=F, RI =CI, R2=R3=H (1b); X=H, R1 =R3=OMe, R2=H (1c); X=H, R1=H, R2=R3=OMe (1d); X=H, RI=CI, R2=R3=H (1e); X=H, R1=R2=OMe, R3=H (1f); X=CI, R1=H, R2=R3=OMe (1g); X=CI, R1=R3=OMe, R2=H (1 h).

The yields for the target amines varied from 65 to 90% (Table 2). The yields were lower, 45 and 40% for compounds Id and Ig respectively (entries 4 and 7 in Table 2) when benzyl chloride instead of benzyl bromide was used as substrate, since chloride is worse leaving group than bromide in substitution reactions. Only one (Ia) of eight prepared amines (Ia-Ih) was known previously. All other seven amines (Ib- Ih) are novel compounds, The problem of over alkylation, which is the main problem in N-alkylation reactions, could be avoided using this method. Almost no di- or tri- alkylated product was formed.

Table 2. Yields of the precursor amines (Ia-Ih).

41

The compound 5-fluoro-2-phenoxyaniline (6) used in the JV-alkylation reaction was not commercially available and prepared by reduction of 4-fluoro-2-nitro-l- phenoxybenzene (5). The reduction was carried out using hydrazine hydrate in presence of ferric chloride and activated carbon (M. Protiva, J. Jilek, I. Cervena, J. Pomykacek, V. Bartl, A. Dlabak, M. Valchar, J. Metysova, J. Holubak and E. Svatek, Collec. Czech. Chem. Commun. 1986, vol. 51, 2598-2616) and the yield was 96%. Scheme 2. Synthesis of precursor amines for DAA analogues.

One of three phenoxy anilines i.e. 5-fluoro-2-phenoxyaniline (7) used in the iV-alkylation reaction was prepared in three steps starting with 2,5-difluoro- nitrobenzene (5) (Scheme 3).

Scheme 3. Synthesis of 5-fluoro-2-phenoxyaniline.

The nucleophilic aromatic substitution of 2,5-difluoro-nitrobenzene with phenoxide ion gave 5-fluoro-2-phenoxy-nitrobenzene (6). The yield for this step was 97%. The nitrobenzene 5 was reduced to aniline 7 using hydrazine hydrate in presence of ferric chloride and activated coal. The yield for this step was 96%.

Benzyl bromides such as 2,5-Dimethoxybenzyl bromide and 2,3- dimethoxybenzyl bromide used in JV-alkylation reactions were optionally prepared from the corresponding benzyl alcohols by treating with carbon tetrabromide and triphenyl phosphine in dry dichloromethane (Scheme 4). The reaction was performed at ambient temperature for 30 minutes and the yield was 85 to 90%.

R1 = OMe or H R2 = OMe or H R3 = Ome or H

Scheme 4. Synthesis of benzyl bromides from benzyl alcohols.

The procedure described above can be used for ¹³ C and ¹⁴ C-labeling of

DAA1006 and its analogues by [ ¹³ C] and [ ¹⁴ C] monoxide in a palladium mediated carbonylation reaction.

Synthesis of precursor amines Ia-Ih. The procedure is exemplified by the preparation of 2V-(2,5- dimethoxybenzyl)-iV-(5- fluoro-2-phenoxyphenyl)amine (Ia).

A suspension of activated powdered 4 A molecular sieves (5.00 g) and cesium hydroxide monohydrate (3.12 g, 18.93 mmol) in anhydrous DMF (100 mL) was stirred vigorously for 10 min at ambient temperature. A solution of 5-fluoro-2- phenoxyaniline (3.30 g, 16.25 mmol) in anhydrous DMF (10 mL) was added slowly and the mixture was stirred for an additional 30 min. at the same temperature. Finally a solution of 2-(bromomethyl)-l,4- dimethoxybenzene (3.61 g, 16.24 mmol) in dry DMF (10 mL) was added to the white suspension, and the reaction was allowed to proceed at 70 ⁰ C for 4 h. The reaction mixture was filtered to remove the undissolved

materials and washed ethyl acetate (50 niL). The filtrate was concentrated under reduced pressure and the residue was taken up in 1 N NaOH (100 ml), and extracted with ethyl acetate (4 x 100 ml). The combined organic layers were washed with brine (2 x 100 ml), dried over anhydrous magnesium sulfate, filtered, and the solvent was removbd under reduced pressure to give the crude product. All of the products were purified by flash column chromatography. Two times flashing, first using pentane/dichloromethane/di ethyl ether (8:1 :1 v/v) and then pentane/diethyl ether (8:2 v/v) as the eluents were needed to get the pure product.

III. Synthesis of reference compounds for DAA analogues

The reference compounds for two of the labeled target analogues (fig. 2) were prepared by treating the amines with acetyl chloride in the presence of pyridine (Scheme 5). These compounds yielded over 90% in both cases.

10 11 X = F ₁ CI

Scheme 5. Synthesis of reference compounds for DAA analogues.

The procedure described above can be used for ¹³ C and ¹⁴ C-labeling of DAAl 006 and its analogues by [ ¹³ C] and [ ¹⁴ C] monoxide in a palladium mediated carbonylation reaction.

IV. Results

The compounds and methods of use thereof described above will allow the clinician to use carbon-isotope labeling of DAAl 106 and make a library of analogues of DAAl 106 for finding useful Positron Emission Tomography tracers for the study of the function of peripheral benzodiazepine receptors in neuroinflamation within a patient.

Specific Embodiments, Citation of References

The present invention is not to be limited in scope by specific embodiments described herein. Indeed, various modifications of the inventions in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.