Fully automated high yield synthesis of (R)- and (S)-[11C]verapamil for measuring P-glycoprotein function with positron emission tomography

Article abstract of DOI:10.1002/jlcr.632

Racemic (±) verapamil is a well characterized substrate for P-glycoprotein (P-gp). However, the in vivo pharmacokinetics and pharmacodynamics of both enantiomers are reported to be different. In the preparation of evaluation studies of both enantiomers in animals and humans, the purpose of the present study was to optimize and automate the synthesis of (R)- and (S)- [¹¹C]verapamil. (R)- and (S)-[¹¹C]verapamil were prepared from (R)- and (S)-desmethyl-verapamil, respectively, by methylation with no-carrier added [¹¹C]methyliodide or [¹¹C]methyltriflate. Different conditions of the methylation reaction were studied: reaction time, temperature, base and solvent, and chemical form of the precursor using either the hydrochloric acid salt or the free base of the starting material. After optimization, the synthesis was fully automated using home-made modules and performed according to GMP guidelines. Optimal yields of 60-70% for the methylation reaction were obtained using 1.5 mg of the free base of (R)- or (S)-desmethyl-verapamil in 0.5ml of acetonitrile at 50°C for 5min with [¹¹C]methyltriflate as methylating agent. Under the same reaction conditions, but with a reaction temperature of 100°C, the radiochemical yield starting with [¹¹C]methyliodide as methylation reagent was 40%. The specific activity of (R)- and (S)-[¹¹C]verapamil was > 20 GBq/μmol and the radiochemical purity was > 99% for both methods. The total synthesis time was 45 min. The automated high yield synthesis of (R)- and (S)-[¹¹C]verapamil provides the means for evaluating both enantiomers as in vivo tracers of P-gp function. Copyright

Full text of DOI:10.1002/jlcr.632

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 1199–1207.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.632
Research Article
Fully automated high yield synthesis of
(R)- and (S)-[11C]verapamil for measuring
P-glycoprotein function with positron
emission tomography
Gert Luurtsema1*, Albert D. Windhorst2, Martien P.J. Mooijer2,
Jacobus D.M. Herscheid2, Adriaan A. Lammertsma2 and
Eric J.F. Franssen1,3
1 PET Centre, VU University Medical Centre, P.O. Box 7057,
1007 MB Amsterdam, The Netherlands
2 Radionuclide Centre, Vrije Universiteit, Amsterdam, The Netherlands
3 Department of Pharmacy, VU University Medical Centre, Vrije Universiteit,
Amsterdam, The Netherlands
Summary
Racemic ( ꢀ ) verapamil is a well characterized substrate for P-glycoprotein
(P-gp). However, the in vivo pharmacokinetics and pharmacodynamics of both
enantiomers are reported to be different. In the preparation of evaluation
studies of both enantiomers in animals and humans, the purpose of the present
study was to optimize and automate the synthesis of (R)- and (S)-
[11C]verapamil.
(R)- and (S)-[11C]verapamil were prepared from (R)- and (S)-desmethyl-
verapamil, respectively, by methylation with no-carrier added [11C]methylio-
dide or [11C]methyltriflate. Different conditions of the methylation reaction
were studied: reaction time, temperature, base and solvent, and chemical
form of the precursor using either the hydrochloric acid salt or the free
base of the starting material. After optimization, the synthesis was fully
automated using home-made modules and performed according to GMP
*Correspondence to: G. Luurtsema, PET Centre, VU University Medical Centre, P.O. Box 7057,
1007 MB Amsterdam, The Netherlands. E-mail: g.luurtseme@vumc.nl
Copyright # 2002 John Wiley & Sons, Ltd.
Received 4 February 2002
Revised 6 May 2002
Accepted 31 May 2002

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G. LUURTSEMA ET AL.
guidelines. Optimal yields of 60–70% for the methylation reaction
were obtained using 1.5 mg of the free base of (R)- or (S)-desmethyl-verapamil
in 0.5 ml of acetonitrile at 508C for 5 min with [11C]methyltriflate as
methylating agent. Under the same reaction conditions, but with a reaction
temperature of 1008C, the radiochemical yield starting with [11C]methyliodide
as methylation reagent was 40%. The specific activity of (R)- and (S)-
[11C]verapamil was >20 GBq/mmol and the radiochemical purity was >99%
for both methods. The total synthesis time was 45 min. The automated high
yield synthesis of (R)- and (S)-[11C]verapamil provides the means for
evaluating both enantiomers as in vivo tracers of P-gp function. Copyright
# 2002 John Wiley & Sons, Ltd.
Key Words: [11C]methyliodide; [11C]methyltriflate; enantiomers; P-gp;
verapamil
Introduction
Positron emission tomography (PET) is a powerful technique for the
quantification of biochemical processes in vivo. Several PET-radio-
pharmaceuticals have been developed for P-glycoprotein (P-gp)
imaging. Amongst others these are [11C]colchicine, [94mTc]-complexes,
[64Cu]–complexes, [68Ga]-complexes, [11C]carvedilol and [11C]verapa-
mil.1–6 P-gp is a multi-drug transporter and is expressed at high levels in
a variety of tissues such as the endothelial cells of the blood–brain
barrier (BBB) capillaries. P-gp acts as an ATP dependent efflux pump
for a large range of hydrophobic drugs and natural compounds such as
neurotransmitters and peptides. The P-gp pump plays an important role
in multi-drug resistance of tumours7 and in drug transport8 and there is
evidence that P-gp is an important factor in several neurological
diseases such as Alzheimer’s disease.9
Verapamil is a specific high affinity substrate for the P-gp pump. The
compound is well characterized both pharmacologically and with regard
to its metabolic pathway. Therefore, verapamil seems to be a suitable
PET tracer for measuring P-gp function in vivo. Recently, a method was
developed using racemic [11C]verapamil.10 For the quantification of
P-gp function in vivo, a mathematical model to describe the tracer
kinetic data is required. As the kinetics of the two isomers might be
different, for accurate quantification, an optically pure compound is
needed. This is also true for verapamil as it has been shown that the two
enantiomers have different pharmacokinetic profiles.11–13
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AUTOMATED SYNTHESIS OF C-VERAPAMIL
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For the evaluation of these enantiomers and to select the most
appropriate candidate for clinical studies, a reliable high yield, fully
automated, GMP compliant synthesis of (R) or (S)-[11C]verapamil is
needed.
Results and discussion
Automated radiosynthesis
The automated synthesis was performed using home-made units, which
have been described in detail elsewhere.14 (R)-[11C]verapamil and (S)-
[11C]verapamil 2 were prepared from the corresponding des-methyl-
starting materials 1 (free base or hydrochloric acid salt) by methylation
with no-carrier added [11C]methyliodide or [11C]methyltriflate according
to the following scheme (Figure 1):
The [11C]methyliodide production was performed in a small volume
trapping vessel. Therefore, less LiAlH4 could be used for the reaction
with [11C]CO2. This resulted in a higher specific activity, because of the
reduced amount of atmospheric 12C carbon in the LiAlH4 solution. In
7 min after trapping [11C]CO2 in the liquid nitrogen cooled coil, 70–80%
(corrected for decay) was converted into [11C]methyliodide. The
[11C]methyltriflate production was performed according to literature
procedures.15,16 As previously reported, for optimal performance of the
home-packed silvertriflate columns, it proved to be very important to
impregnate the GraphpacTM material with silvertriflate using a
silvertriflate solution (1 g/10 ml) in acetonitrile instead of diethy-
lether.16,17 In addition, just prior to use, the packed silvertriflate
column had to be pre-conditioned at 2008C with a helium flow of
100 ml/min for at least 10 min. The conversion of [11C]methyliodide to
[11C]methyltriflate was higher than 80%. The conversion was tested on
(nitrobenzyl)pyridine according to the method of Jewett.15
Figure 1. Synthesis of the enantiomers of [11C]verapamil (experimental details
mentioned in Table 1)
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G. LUURTSEMA ET AL.
The reactivity of the nucleophilic amine was studied by changing
reaction parameters like solvent (DMF/ acetonitrile), base, reaction
time, temperature and by using the hydrochloric acid salt or the free
base as the starting material (Table 1). The overall radiochemical yields,
including synthesis, purification and formulation, were calculated from
the trapped amount of [11C]methyliodide or [11C]methyltriflate in the
starting reaction mixture. The reaction conditions as previously
reported1 were 0.5 mg 1 (HCl), 25 mg Al2O3/KF as a base, 1008C and
10 min in 0.5 ml of acetonitrile. The radiochemical yield of the synthesis
of the racemic [11C]verapamil described in the literature was 16%. In the
present study, these reaction conditions (except for the use of 1.5 mg
rather than 0.5 mg (R/S) 1) resulted in a comparable radiochemical yield
of 17.2 ꢀ 7.1% (n=6). Initially, the methylation reaction was optimized
by shortening the reaction time to 5 min and reducing the reaction
volume to 250 ml. No significant effects were observed.18 An overall
radiochemical yield of 13.3 ꢀ 3.4% (n=3) was obtained. For practical
reasons 1.5 mg (R/S) 1 in a volume of 500 ml of dry acetonitrile was
maintained. The rationale of using solid Al2O3/KF was to absorb
radiochemical impurities, which otherwise were difficult to remove from
the final product. In the HPLC-system used, these radiochemical
impurities did not disturb purification and therefore Al2O3/KF was
omitted from the reaction mixture.
In common polar–aprotic solvents such as dimethylformamide
(DMF), the nucleophile is less solvated and is more susceptible to
nucleophilic reactions in comparison to protic solvents. The methyla-
tion with [11C]methyliodide in DMF with sodium bis(trimethylsilyl)
amide (TMSA) as a base resulted in a very low radiochemical yield of
2%. It can be assumed that methylation of the TMSA has occurred
Table 1. Optimizing the methylation reaction conditions of [11C]-verapamila
Nor-
verapamil
form
Radio-
precursor
Temperature
(8C)
Reaction
Time
(min)
Base
% radiochemical
yielda
N
HCl
HCl
11CH3I
100
100
50
10
5
Al2O3/KF
Al2O3/KF
Al2O3/KF
}
17.2 ꢀ 7.1
13.3 ꢀ 3.4
14.2 ꢀ 2.8
45.8 ꢀ 10.1
70.5 ꢀ 10.6
6
3
5
6
2
11CH3I
HCl
Free amine
Free amine
11CH3I
5
11CH3I
100
50
5
11CH3triflate
5
}
aThe following standard reaction conditions were used: 1.5 mg starting material in 0.5 ml of dry
acetonitrile; closed reaction vessel. The overall radiochemical yields were calculated from the
starting amount of [11C]methyliodide or [11C]methyltriflate (corrected for decay).
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under these conditions. Significantly higher yields were obtained with
the free base of (R/S) 1 in acetonitrile. A radiochemical yield of
45.8 ꢀ 10.1% (n=6) of 2 was achieved under optimal conditions.
About 55% of the [11C]methyliodide activity, however, still had not
reacted. For this reason, the more reactive [11C]methyltriflate was used
as an alternative methylation reagent. Under the same reaction
conditions, a radiochemical yield of 70.5 ꢀ 10.6% was achieved with
[11C]methyltriflate, independent of the reaction time. Increasing the
reaction temperature to 1008C did not increase the radiochemical yield.
Taking into account the 80% conversion yield of [11C]methyltriflate
from [11C]methyliodide, the overall yields for both methods are not
Figure 2. The purification of the reaction mixture. Semipreperative HPLC
chromatograms purification of the reaction mixture
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significantly different. From a chemical point of view there is no
preference for one method or the other. For practical reasons, therefore,
the methylation with [11C]methyliodide instead of [11C]methyltriflate is
preferred.
Using the optimal reaction conditions, 2 was synthesized by
methylation of the secondary amine with [11C]methyltriflate in a
solution of 1.5 mg of 1 in 500 ml of dry acetonitrile. The reaction was
carried out at 508C for 5 min in a closed reaction vial. Subsequently,
2 ml of HPLC eluent was added to the reaction mixture. The complete
solution was transferred to the HPLC unit and subjected to a
preparative HPLC purification equipped with a UV detector (230 nm)
and a radioactivity detector (Figure 2). The collected fraction contain-
ing the product was diluted with 50 ml of sterile water in the formulation
unit. The diluted product was passed through a Sep Pak cartridge,
which was subsequently washed with sterile H2O. The product was
eluted from the Sep Pak with ethanol and saline and filtered. To check
for possible occurrence of racemization during this procedure, a sample
of the tracer was analysed using a Chiracel HPLC column. For
enantiomeric identification a sample of the tracer was spiked with an
optically pure reference of verapamil. The product was analysed by
HPLC on a Symmetryshield C18 Column. A stereo and (radio)chemical
purity of 99.5% was obtained. Characterization of 2 was performed
using HPLC-MS/MS.
Conclusion
After optimization and automation of the [11C]-synthesis of (R)- and
(S)-[11C]verapamil, a reproducible and high yield synthesis was
developed. (R)- and (S)-[11C]verapamil, synthesized according to this
procedure, are now available for in vivo PET-studies to select the most
appropriate candidate for quantitative PET-studies.
Experimental
Materials
The starting materials (R)- and (S)-desmethyl-verapamil as well as the
reference compounds (R)- and (S)-verapamil were a gift from Knoll
Pharmaceuticals, The Netherlands. DIPA, Al2O3/KF were obtained
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AUTOMATED SYNTHESIS OF C-VERAPAMIL
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from Aldrich; methanol, acetonitrile, NaClO4 and 57% HI from Merck.
All chemicals were of analytical grade.
Other laboratory supplies were obtained from Alltech; the HPLC-
MS/MS from Perkin- Elmer API3000.
Production of [11C]methyliodide
[11C]CO2 was produced by the 14N(p,a)11C nuclear reaction with an IBA
18/9 cyclotron. An irradiation for 10 min with 30 mA of 18 MeV protons
yielded 25–30 GBq of [11C]CO2 at EOB. After irradiation the [11C]CO2
was transferred through 190 m of 1/800 stainless steel tubing with a
helium flow of 1000 ml/min to the hot cell. The [11C]CO2 was trapped in
a liquid nitrogen trap, subsequently transferred to the synthesis modules
with a helium flow of 10 ml/min and reacted with 100 ml of 0.1 M LiAlH4
in THF. After evaporation of the solvent, 200 ml of 57% HI was added
and the [11C]methyliodide obtained was distilled into a second reaction
vessel containing the methylation reaction mixture. Following this
procedure, a yield of 15–20 GBq of [11C]methyliodide was produced in
12 min synthesis time after EOB (not decay corrected). The conversion
yield was 70–80% (decay corrected).
Production of [11C]methyltriflate
For the conversion to [11C]methyltriflate, [11C]methyliodide was passed
through a pre-conditioned column (7 ꢁ 0.5 cm) containing silvertriflate
and GraphpacTM (80–100 mesh) at
a
temperature of 2008C.
The [11C]methyltriflate was trapped at room temperature in the reaction
vessel containing the mixture for the methylation reaction.
The conversion yield was >80% (decay corrected).
Automated radiosynthesis
Compound 2 was synthesized by methylation of the secondary amine
with [11C]methyltriflate in a solution of 1.5 mg of (R/S)1 (free base) in
500 ml of dry acetonitrile. The reaction was carried out at 508C for 5 min
in a closed reaction vial. The reaction mixture was diluted with 2 ml of
HPLC eluent and subsequently transferred to the HPLC unit using a
remote controlled operated HPLC injection system and subjected to a
semi-preparative HPLC purification using
a
Varian Kromasil
250 ꢁ 22 mm (10 mm) with MeOH:H2O:DIPA 82:18:0.2 (%v/v/v) as
eluent and a flow of 10 ml/min. [11C]verapamil had a retention time of
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15 min, as detected by UV (230 nm) and radioactivity (Figure 2). The
collected fraction containing the product was diluted with 50 ml of
sterile water in the formulation unit.14 The diluted product was passed
through an activated tC18 Sep Pak cartridge using a helium over-
pressure, which was subsequently washed with 20 ml of sterile H2O.
Compound 2 was eluted from the Sep Pak with 1 ml of ethanol followed
by 9 ml saline and filtered through a sterile 0.22 mm filter using a helium
overpressure yielding a sterile and pyrogen free-ready for injection
solution. The total synthesis time was 45 min including the purification
and the formulation.
HPLC}analysis and identification
To check for racemization during this procedure, a sample of 20 ml of 2
was analysed using a Chiracel OD-R column 250 ꢁ 4.6 mm, with 1 M
NaClO4:CH3CN 60:40 (%v/v) as eluent and a flow of 0.5 ml/min. The
eluent was monitored using UV absorbance at a wavelength of 232 nm
and the radioactivity using a sodium iodide scintillation detector. For
identification, a sample of 2 was spiked with an optically pure reference
of verapamil. Under these conditions the retention times of (R)- and (S)-
verapamil were 15.5 and 18.5 min.
For the routine check on chemical and radiochemical purity a Waters
Symmetryshield RP18, 5 mm, 3.9 ꢁ 150 mm, with acetonitrile:methanol:
50 nM potassium phosphate pH=7.0 33:18:49 (%v/v/v) as eluent and a
flow of 1 ml/min, was used with detection as described above. The
retention time for both enantiomers of 2 was 15 min with this HPLC-
system. The characterization was performed using HPLC-MS/MS with
a Varian Kromasil 250 ꢁ 4.6 mm with MeOH:H2O:DIPA 72:28:0.05
(%v/v/v) as eluent and a flow of 1 ml/min. The batch was recorded in a
range of m/z 200–600. The spectra showed a principal peak at m/z 455,
with a retention time of 6 min. The mass spectrum corresponds with a
reference of verapamil. MS: 455 (M+1), 303, 260 (C16H3NO+2 ), 165
(C10H13O+2 ), 150 (C9H10O2+).
Acknowledgements
The authors thank Knoll Pharmaceuticals (Almere, the Netherlands)
for their gift of (R)- and (S)-(desmethyl)-verapamil and Eduard Struys
(VU Medical centre, Amsterdam, the Netherlands) for his expertise of
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AUTOMATED SYNTHESIS OF C-VERAPAMIL
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the LC-MS/MS. Peter van Leuffen and the personnel of the BV
Cyclotron VU are gratefully acknowledged for the production of
[11C]CO2.
References
1. Metha BM, Rosa E, Biedler JL, et al. J Nucl Med 1994; 35: 1179–1184.
.
2. Bigott HM, McCarthy DW, Wust FR, et al. J Label Compd Radiopharm
2001; 44: S119–S121.
3. Lewis JS, Dearling JLJ, Sosabowski JK, et al. Eur J Nucl Med 2000; 27:
638–646.
4. Sharma V, Beatty A, Wey SP, et al. Chem Biol 2000; 7: 335–343.
5. Elsinga PH, Franssen EJ, Hendrikse NH, et al. J Nucl Med 1996; 37:
1571–1575.
6. Hendrikse NH, Dijkers ECF, Wegman TD, et al. J Nucl Med 2001; 42:
279.
7. Endicott JA, Ling V. Annu Rev Biochem 1989; 58: 137–171.
8. King M, Wendy S, Chang A, et al. Nature Neurosci 2001; 4: 268–274.
9. Lam FC, Liu R, Lu P, et al. J Neurochem 2001; 76: 1121–1128.
10. Hendrikse NH, de Vries EG, Franssen EJ, Vaalburg W, van der Graaf
WT. Eur J Clin Pharmacol 2001; 56: 827–829.
11. Eichelbaum M, Mikus G, Vogelgesang B. Br J Clin Pharmacol 1984; 17:
453–458.
12. Vogelgesang B, Echizen, Smidt H, et al. Br J Clin Pharmacol 1984; 18:
733–740.
13. Echizen H, Vogelgesang B, Eichelbaum M. Clin Pharmacol Ther 1985; 38:
71–76.
14. Windhorst AD, Linden ter T, Nooij de A, et al. J Label Compd
Radiopharm 2001; 44: S1052–S1055.
15. Jewett DM. Appl Radiat Isot 1992; 43: 1383–1385.
16. Mock HM, Mulholland GK, Vavrek MT. Nucl Med Biol 1999; 26:
467–471.
17. Iwata R, Pascali C, Bogni A, et al. Appl Radiat Isot 2001; 55: 17–22.
18. Luurtsema G, Windhorst AD, Herscheid JDM, et al. J Label Compd
Radiopharm 2001; 44: s313–s315.
Copyright # 2002 John Wiley & Sons, Ltd.
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