Pergamon

N&. Med. Biol. Vol. 21. No. 2. DD. 211-217. 1994 Copyright c 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0969-805 l/94 $6.00 + 0.00

Synthesis and Preliminary Evaluation of (R,S )- 1-[2-((Carbamoyl-4-hydroxy)phenoxy)- ethylamino]-3-[4-( 1 -[11C]-methyl-4-trifluoro- methyl-2-imidazolyl)phenoxy]-2-propanol

([“C]CGP 20712A) as a Selective P,-Adrenoceptor Ligand for PET

PHILIP H. ELSINGA*, AREN VAN WAARDE, GERBEN M. VISSER and WILLEM VAALBURG

PET Center and Groningen Center for Catalysis and Synthesis, University Hospital, P.O. Box 30.001. NL-9700 RB Groningen, The Netherlands

(Accepted 25 August 1993)

The most selective P,-adrenoceptor ligand known at this moment is (S)-l-[2-((carbamoyI-C hydroxy)phenoxy)ethylamino]-3-[4-(1-methyI-4-trifluoromethyl-2-imidazoIyl)phenoxy]-2-propanol (CGP 26505), the S-isomer of CGP 20712A. We prepared the racemic “C analogue by methylation with [“C]CH,I of the corresponding desmethyl compound using a microwave oven to accelerate the reaction. Several radioactive by-products (about 70% of the non-volatile radioactive products) were formed. After HPLC purification [“CJCGP 20712A with a specific activity of 35 TBq/mmol was dissolved in a propylene glycol-ethanol-saline mixture to prepare it for injection. The total preparation time was 35 min. The radiochemical yield was 5% (calculated from [“C]CH,I, not corrected for decay). The identity of [“CICGP 20712A was proved by liquid chromatography-mass spectrometry (LC-MS).

Tissue distribution studies in male Wistar rats have been performed. At 20 min after injection of the radioligand (0.1 nmol) the DAR [differential absorption ratio = (counts per minute recovered/g tissue)/(couits per min injected/g body weight)] in heart tissue decreased significantly (P < 0.005) from 1.84 + 0.11 to 1.21 + 0.12 after blocking of a-adrenoceptors with 500 tig (R,S)-propranolol.

A preliminary PI!?? study in a Wista; rat ‘showed maximal uptake in the time- frame IO-20 min after injection. The ratio of specific/non-specific binding at this interval was 2.6.

Introduction

p-Adrenergic receptors play an important role in the regulation of the heart rate and of myocardial con- tractility. In various forms of heart failure, the fl- adrenoceptor density is reduced (Bristow et al., 1987;

Brodde et al., 1989a,b; Brodde, 1991; Michel et al., 1988). The loss of receptors can be global (the /3, and p2 subtypes being equally affected) (Brodde et a/., 1989a,b; Brodde, 1991) or subtype-selective (the number of /?,-adrenoceptors being lowered whereas that of /I,-adrenoceptors is relatively un- changed) (Bristow et al., 1987; Michel et al., 1988). Little information is available aboui the development in time of these alterations, their spatial distribution within the heart and the influence of therapy. A

*Author for correspondence.

suitable procedure for visualization and quantitation of myocardial /$-receptors by PET would therefore

be of great clinical interest. Some positron emitting /?-ligands have been pre-

pared and evaluated for imaging purposes, such as

carbon-l 1 labeled CGP 12177 (Boullais et al., 1985), (S)-CGP 12177 (Aigbirhio et al., 1992; Brady et al., 1990; Hammadi and Crouzel, 1990, 1991), meto- prolol, propranolol, atenolol (Antoni et al., 1989), carazolol (Berridge et al., 1992), fluorine-18-labeled metoprolol (de Groot et al., 1993) and carazolol (Zheng and Berridge, 1992; Kinsey et al., 1992). However none of these have both a high selectivity for the p,-adrenoceptor and high affinity for p- adrenoceptors in general. Because the /3,-adrenocep- tor density is predominantly affected in heart failure (Bristow et al., 1987; Michel et al., 1988) we would like to develop PI-selective ligands with high affinity.

211

212 PHILIP H. ELSINGA et al.

Fig. 1. Reaction scheme for the synthesis of [“C]CGP 20712A.

The most selective /I,-ligand known at this moment is CGP 26505, the biologically active S-isomer of racemic CGP 20712A. Its /I,/& selectivity is lOOO-lO,OOO-fold, while the Kd at /3’-receptors = 0.3 nM (Dooley et al., 1986).

In our laboratories pharmacological studies with [3H]CGP 26505 (3H-label on the 5-phenoxy ring position) on male Wistar rats have been performed (van Waarde et al., 1992a,b). The ligand shows a high uptake in the heart (l-Z% injected dose/g) and a significant fraction of this uptake (47%) can be blocked by previous injection of propranolol. CGP 26505 is rapidly metabolized, but the metabolites are not taken up by the heart, which indicates that they have negligible /?-blocking potency. The initial clear- ance from plasma is very rapid (t,,* = 0.6min). Association to receptors is also rapid, maximal myo- cardial binding being reached within 2-20 min. Sixty percent of the initially bound CGP 26505 dissociates from the receptor during the first hour after injection. Myocardial uptake of [‘H]CGP 26505 is also reduced after treatment of rats with the subtype-selective CI- and b-agonists or antagonists: CGP 12177 (/I ), iso- prenaline (/I), atenolol (/I,), ICI 118,551 (&), prazosin (CQ) and yohimbine (~1~). From these results it was concluded, that the uptake of CGP 26505 does not reflect binding to &-adrenoceptors. This conclusion was unexpected because of the excellent selectivity of CGP 20712A in vitro (Dooley et al., 1986; Pauwels et al., 1988). Since the tritiated CGP 26505 had a low specific activity of 740 GBq/mmol, we wanted to continue the CGP 26505-experiments with a “C analogue which can be obtained with a much higher specific activity (35 TBq/mmol). In the experiments with tritiated CGP 26505 a dose

of 4nmol was used, while with [“C]CGP 26505 dosages of < 1 nmol can be applied. Moreover, after injection of [“CICGP 26505 the kinetics of myo- cardial uptake in the presence or absence of receptor agonists or antagonists can be monitored using a PET camera.

The racemic “C analogue can be prepared by methylation with [“Clmethyliodide of the corre- sponding desmethyl compound (Fig. 1) analogous to the synthesis of unlabeled CGP 20712A described by Fuhrer et al. (1981).

Here we report the successful synthesis of [“C]CGP 20712A and the results of preliminary animal stadies.

Materials and Methods

Materials

[“C]CH31 was produced from [“C]CO_, ( “N(p,a)“C nuclear reaction with 17 MeV protons) using an Anatech robotic system, yielding 30 GBq carbon-l l-labeled methyliodide with a specific activity of 150 TBq/mmol. The synthesis time was 11 min and the radiochemical yield was 70-90% (corrected for decay). Unlabeled CGP 20712A and the desmethyl analogue were kind gifts of Dr K. Scheibli and Dr H. Schiiter, Ciba-Geigy AG, Basel, Switzerland. All chemicals were analytical grade and purchased from Merck, Darmstadt, Germany. (R,S)-Propranolol was from Sigma, St Louis, MO., U.S.A.

Chemistry

A solution of 1 mg (2.1 pmol) desmethyl precursor and 0.5 mg (2.1 pmol) KO-tBu in 0.5 mL aceto-

Synthesis and preliminary evaluation of [“CICGP 20712A 213

nitrile was prepared at least 30min before adding [“Clmethyliodide. The methyliodide was trapped in 0.5 mL acetonitrile at -40°C. After addition of [“Clmethyliodide, the reaction mixture was heated in a Miele M 686 microwave oven. In the Miele- microwave experiments the reaction mixture (1 mL) was transferred into a borosilicate glass tube (11 mL), sealed tightly by a Schott GL 14 cap. The tube was placed in a Parr microwave acid digestion bomb. The digestion bomb was placed on a fixed position in the microwave oven. After 90 s of microwave treat- ment (600 W), the bomb was cooled for 2 min.

After evaporation of the solvent at reduced press- ure at 50°C the residue was dissolved in 0.5 mL HPLC-eluent. The eluent was dichloromethane/ methanol/ammonia 240/25/O. 1 (v/v/v). The reaction mixture was applied on a Zorbax-NH, column. The Zorbax-NH, column (250 x 4.6 mm, 7 pm) was obtained from Chrompack, Middelburg, The Nether- lands. Using a flow rate of 1 mL/min, [“CICGP 20712A was eluted after 14 min (Fig. 2: peak 2). After evaporation of the eluent under reduced pressure at 50°C [“C]CGP 20712A was dissolved in a propylene glycol: ethanol 96% : 0.9% NaCl mixture (2: 1: 2, v/v/v) to prepare it for injection.

The by-products were identified by LC-MS (liquid chromatography-mass spectrometry) by using an ion spray interface (Bruins et al., 1987). The HPLC eluate

was treated with a nebulizing gas to form a charged aerosol. The positive ions are attracted into the mass spectrometer. Every 2 s a mass spectrum is collected and is stored in the computer. The sensitivity of this technique is in the order of magnitude of 1 nmol/mL. Besides determination of the molecular mass, fragmentation experiments were performed in order to determine the methylation position. By application of a voltage of 170 V, CGP 20712A is fragmented on two positions (Fig. 3).

Safety precautions during microwave reactions

Increasing the volume of solvent and (or) the power level or decreasing the size of the reaction vessel can cause an increase in pressure and tempera- ture leading to deformation of the reaction vessels or even to an explosion. To ensure a safe experiment solvent volumes should not exceed 10% of the volume of the reaction vessel. Heating times of more than 2.5 min at a power of 600 W should be avoided and the reaction vessel must be placed in a microwave digestion bomb.

Tissue distribution studies

All experiments were carried out in compliance with the Law on Animal Experimentation of The Netherlands. Male Wistar rats (200g) were anes- thetized by i.p. injection of pentobarbital (60 mg/kg

20

time (min)

radioactivity

Fig. 2. HPLC chromatogram obtained from the purification of [“CICGP 20712A. Peaks denoted as 1 are the dimethylated products, peak 2 is the phenolic methylated compound, peak 3 is the imidazol isomer

of CGP 20712A, peak 4 is authentic CGP 20712A and finally peak 5 is the N-methyl compound.

214 PHILIP H. ELSINGA et al.

Fig. 3. Fragmentation positions in CGP 20712A after application of 170 V in LC-MS.

body weight). Before administration of the radio- ligand, the rats were treated either with saline (con- trol group, n = 3) or with 500 pg (R,S)-propranolol (blocking experiments, n = 3) by iv. injection in a tail vein. Subsequently 1 MBq [“C]CGP 20712A (0.1 nmol) was injected in a volume of 0.3 mL. After 20 min the rats were killed by extirpation of the heart and several tissues were dissected. Uptake of “C was measured in heart, lung, kidney, liver, skeletal muscle (m. vastus medialis), subcutaneous fat and plasma. Plasma was obtained from blood by centrifugation (3 min, 1OOOg). Tissue uptakes were expressed as differential absorption ratios [DAR = (counts per min recovered/g tissue)/(counts per minute injected/g body weight)]. Differences between the propranolol treated group and the control group were analyzed using Student’s t-test.

PET study

A PET study was carried out in a Siemens ECAT 951/31 positron camera. Data acquisition was per- formed using a dynamic protocol and in stationary mode. In this mode the in-plane spatial resolution amounts to 6 mm FWHM. During the reconstruction a zoom factor of 1.5 was applied and the matrix size was 128 x 128. The rat was anesthetized as described in the tissue distribution studies and was positioned in the PET camera parallel to the transaxial plane of the tomograph, so sagittal sections were obtained. Subsequently 2 MBq [“CICGP 20712A (0.15 nmol) was administered by injection in a tail vein. The following frames were defined: &2, 2-5, 5-10, lo-20 and 20-40 min after injection. Ninety minutes after the first injection the animals were treated with 500 pg (R,S)-propranolol, followed after 5 min by a second injection with 2 MBq [“CICGP 20712A and a second data acquisition sequence as described above. Data analysis was performed using the Siemens ECAT software (V6.3 D) on a Sun work- station.

Results and Discussion

Microwave heating is a useful tool to accelerate chemical reactions, especially when short-lived radio- nuclides such as “C and “F are involved (Gedye et al., 1988, 1991; Hwang, 1987; Stone-Elander and Elander, 1991; Thorell et al., 1992). The methylation

reaction as shown in Fig. 1 could be performed conveniently in a microwave oven. The total incor- poration of “C, including the formation of [“CICGP 20712A (peak 4) and of by-products with peak numbers 1, 2, 3 and 5 as depicted in Fig. 2 was 5@70% after heating for 90 s. In cold experiments a comparison was made between microwave and ther- mal heating. Comparable yields of [“CICGP 20712A were achieved with thermal heating at 110°C for 20 min and for microwave treatment for 90 s at 600 W as described above. When the reaction was carried out at ambient temperature for 30min as described for the synthesis of unlabeled CGP 207 12A (Fuhrer et af., 1981) the “C incorporation was only 30%. Consequently, microwave treatment increased the radiochemical yield (corrected for decay), while the total synthesis time was significantly reduced with one half life. The addition of NaI to increase energy absorption in the microwave tube (Zijlstra et al., 1993) did not affect the radiochemical yield, but changed the relative yields of the by-products. By adding 2 mg of NaI to the reaction mixture, the yield of the product with the methyl group on the N-alkyl chain (product 5 in Fig. 2) increased from 2 to 1 1 %, while methylation on the phenolic moiety decreased from 26 to 16% (product 2 in Fig. 2). The regioselec- tivity as observed under microwave conditions could also be achieved with thermal heating at 110°C.

We changed the solvent from DMF (Fuhrer et al., 1981) to acetonitrile, because of the lower boiling point of the latter solvent, which facilitates its removal by evaporation. The yield of methylated product decreased rapidly when the amount of pre- cursor was diminished. When the reaction was carried out at ambient temperature with 0.5 mg desmethyl compound, the radiochemical yield was only 3%. The presence of the base KO-tBu to make the desmethyl compound more nucleophilic, seemed to be essential, since without KO-tBu the reaction had a yield of less than 1%.

HPLC purification on a Zorbax-NH, column indicated that several radioactive by-products were formed (Fig. 2), representing 70% of the non-volatile radioactivity. This is conflicting with the results of Fuhrer et al. (1981) who reported the formation of only one N-imidazole isomer besides CGP 20712A. In order to have a good HPLC separation between the radioactive products an injection volume of

Synthesis and preliminary evaluation of [“CICGP 20712A 215

Table 1. Spectral data from LC-MS exper- iments. The data for the dimethylated com- pounds are not displayed. These data are not relevant and are dificult to interpret, be- cause a mixture of compounds is analyzed.

The M +-value was 509

Peak No. m/r fragments Base ueak

2 151,328,477 495 3 243,343,477 495 4 243,343,477 495 5 228,343,477 495

maximal 0.5mL is applied. A disadvantage is the concommitant loss of about 50% of radioactivity.

The by-products were identified by LC-MS. The spectral data are presented in Table 1. Fragment A without a methyl group has a MW of 228 (with methyl group 243). CGP 20712A (MW = 494) with- out fragment C has a MW of 343 (without fragment C + methyl MW of 328). The products, denoted as 1, were shown to be dimethylated compounds. The number and radiochemical yield of dimethylated compounds (O-4) varied in each synthesis. In Table 1 no m/z fragments are given for the dimethylated compounds. The m +-peak was 509. No correlation could be found between the specific activity of [“C]CH31 and the percentage of dimethylated products. Product 2 proved to be a compound with the methyl group in the carbamoyl phenolic moiety. It is probably formed by methylation on the phenolic group. Product 3 was shown to be the N-imidazole isomer of CGP 20712A. Product 4 is authentic CGP 20712A. Finally product 5 is a compound with the methyl group on the N-alkyl chain.

The [“C]CGP 20712A peak was also identified by spiking with authentic material. Using dichloro- methane/methanol/ammonia 240/25/O. 1 as eluent, the retention time was 14min. After evaporation of the eluent, [“CJCGP 20712A with a specific activity of 35 TBq/mmol was dissolved in a propylene glycol- ethanol-saline mixture and was suitable for injection. The total synthesis time (including purification) was 35 min (EOB). Practical yields are 744185 MBq [“C]CGP 20712A.

Tissue distribution studies on male Wistar rats were performed (Table 2). Tissue uptake was evalu- ated at 20 min after injection, because experiments

Table 2. Tissue distribution studies in rats with [“C]CGP 20712A, 20 min post-injection. The uptakes are expressed as DAR. n = 3 in each group. Errors are

Tissue

SD. NS is not significant

Untreated Propranolol P*

Heart 1.84~0.11 1.21 f0.12 < 0.005 Lung 2.16+0.31 1.03 + 0.23 <O.Ol Kidney 1.98 f 0.58 1.28 f 0.74 NS Liver 1.92 + 0.44 1.74 0.62 f NS Muscle 0.21 * 0.04 0.22 _+ 0.09 NS Fat 0.17 * 0.03 0.19+0.19 NS Plasma 0.12 * 0.01 0.10 + 0.02 NS

*Dual-tail probability, Student’s I -test.

with [3H]CGP 26505 (van Waarde et al., 1992a) and the PET study described in this paper have shown that the ratio of specific/non-specific binding is maximal at this time point. The DAR of “C in heart tissue decreased significantly to 66% of the control value, when the rats were treated with 500 ,ug (R,S)-propranolol. Compared to the studies with the tritiated S-isomer, where this ratio showed a decrease to 53% compared to the control value by propranolol treatment, a higher non-specific binding was observed. This may be due to the use of a racemate rather than the pure S-isomer in the “C experiments. The biologically inactive R-isomer does not bind to /I-adrenoceptors, when it is administered at tracer dosages, but it does show non-specific binding.

In general, [“C]CGP 20712A-binding was only moderately blocked by (R,S)-propranolol, in con- trast to a more hydrophilic tracer like [“C]CGP 12177, of which >80% of tissue uptake can be blocked by pretreatment of animals with P-adrenoceptor antagonists (van Waarde et af., 1992a).

In lung tissue, which has relatively few &- adrenoceptors, but many /?,-adrenoceptors (Barnett et al., 1978; Minneman et al., 1979; Vanscheeuwijck et al., 1989) a decrease of the DAR is observed after treatment of the animals with propranolol. Despite the relatively small proportion of jY,- receptors in lung compared to heart, 25 and 85% of total ,!?-adrenoceptors, respectively, the absolute &- receptor density is higher in the lung than in the heart. It is therefore not surprising that in the lung the decrease in DAR after blocking with propranolol is larger compared to heart. A similar pattern of total/non-specific binding in the lung was found after administration of [‘H]CGP 26505 (van Waarde et al., 1992a).

In a preliminary PET study in a Wistar rat the heart could clearly be visualized (Fig. 4). The Siemens ECAT 951/31 positron camera seems to be suitable for dynamic PET studies in rats. A marked difference between the unblocked and the propranolol-blocked study was observed (Figs 4 and 5). In the unblocked situation the uptake reaches a maximum in the 10-20min time frame, whereafter a washout is observed (Fig. 5). After treatment with propranolol the uptake is maximal in the 5-10 min time frame, followed by a much faster washout. In the 20-40 min time frame the ratio of specific/non-specific binding reached a maximum of 2.7.

The results of the experiments with [“C]CGP 20712A are qualitatively similar to those obtained with [3HJCGP 26505 (van Waarde et a/., 1992a). Work is in progress to synthesize the enantiomerically pure S-isomer in order to evaluate [“C]CGP 26505 as a new potent fl,-ligand for PET studies. Block- ing experiments with subtype-selective a- and p- antagonists in combination with dynamic PET studies have to be performed to study the myocardial uptake of [‘QCGP 26505.

216 PHILIP H. ELSINGA et al.

Fig. 4. PET study with [“CICGP 20712A in a Wistar rat. Integrated activity from 10 to 20min after injection in a plane 5 mm to the left of the median axis is displayed. The left-hand image is the control study, the right-hand image the propranolol-blocked study of the same animal. “Hot spots” are from left

to right: bladder, liver and heart. .The heart is barely visible after propranolol treatment.

0.20

0.15

v1

.Z

5 + 0.10

2 0.05

0

Heart

- Control

- - - Propranolol

I I I U 10 20 30

Time (min)

Fig. 5. Time-activity uptake curves as measured by the Siemens-ECAT system. The myocardial counts are plotted

against the median time of each frame.

Acknowledgements-The authors gratefully acknowledge A. P. Bruins and M. Jeronimus (Department of Pharmacy) for performing the LC-MS experiments.

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