Synthesis of PNU-243922 labelled with 14C

Article abstract of DOI:10.1002/jlcr.646

The arylpiperazine PNU-243922 (1) has been labelled with ¹⁴C. A two-step sequence starting from the ¹⁴C labelled alcohol 6a led to radiochemically pure (> 98%) [¹⁴C]PNU-243922 with a specific activity of 546 MBq/mmol. Copyright

Full text of DOI:10.1002/jlcr.646

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 85–92.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.646
Research Article
14
Synthesis of PNU-243922 labelled with C
1,
1
1
2
3
.
C. Felicini *, E. Fontana , D. Giribone , B. M. Nilsson , K. Osterlund
and A. Paptchikhine4
1 Global Drug Metabolism Department, Pharmacia, viale Pasteur 10,
20014 Nerviano (MI), Italy
2 Department of Medicinal Chemistry, Biovitrum AB, 6443, S751 37 Uppsala,
Sweden
3 Department of Medicinal Chemistry, Biovitrum AB, Lindhagensgatan 133,
SE 112 76 Stockholm, Sweden
4 Department of Chemical Technologies, Biovitrum AB, Lindhagensgatan 133,
SE 112 76 Stockholm, Sweden
Summary
The arylpiperazine PNU-243922 (1) has been labelled with 14C. A two-step
sequence starting from the 14C labelled alcohol 6a led to radiochemically pure
(> 98%) [14C]PNU-243922 with a specific activity of 546 MBq=mmol.
Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: PNU-243922; arylpiperazine; carbon-14; anti-obesity agent
Introduction
PNU-243922 (8-[2-({3-[(2R)-2-methylpiperazinyl}-2-pyrazinyl}oxy)ethoxy]-
1, 2
2-quinolinylamine (1)) is a novel agonist at the 5HT2C
receptor
belonging to the arylpiperazine chemical class.
The compound was evaluated for its potential application as control
calorie intake and nutrient balance, which should result in weight loss
without inducing psychotropic side effects. The preparation of a
*Correspondence to: C. Felicini, Global Drug Metabolism Department, Pharmacia, viale Pasteur
10, 20014 Nerviano (MI), Italy. E-mail:chiara.felicini@pharmacia.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 25 June 2002
Accepted 29 July 2002

86
C. FELICINI ET AL.
N
N
O
O
N
N
NH2
NH
1
specifically labelled 14C form of PNU-243922 was required at an early
stage of the research program in order to help the selection of a
compound with an adequate ADME profile. The piperazine ring seemed
to be at first glance the most convenient group to introduce a radiolabel
in the molecule. However, previous metabolism studies carried out with
compounds belonging to the same chemical, class showed that this
labelling position was not suitable. On the other hand, preliminary
metabolism studies indicated that the –O–CH2–CH2–O– chain was
unlikely to be lost through metabolic activity. In this paper the synthesis
of the 14C labelled form of the title compound as well as its precursor is
reported.
Discussion and results
During the preparation of a promising compound of the same chemical
class, useful information was obtained about the Mitsunobu re-
action.325 Moreover, the radiolabelled intermediate 6a was identified
as a common precursor to prepare a series of 14C compounds of
potential interest for the 5HT2C project. Compound 6 was synthesized
according to Scheme 1, using trityl (Tr) as protecting group. Attempts
Tr
Tr
N
N
H
N
H
N
N
O
N
N
b
N
c
N
N
a
Cl
Cl
N
N
H
N
Cl
Tr
N
6
5
3
4
OH
Scheme 1. Reagents and conditions: (a) TrCl=CH2Cl2; (b) K2CO3=DMF and
(c) ethylene glycol, NaH/DMF
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SYNTHESIS OF PNU-243922
87
to use Boc protection was unsuccessful due to its instability under the
reaction conditions. Compound 3 was treated with TrCl in CH2Cl2 to
form only the isomer 4,6; 7 the second nitrogen acting as base to accept
the proton. Condensation of 4 with excess 2,3-dichloropyrazine in DMF
gave quantitative conversion to 5. Finally, coupling with ethylene glycol
in the presence of NaH in DMF gave 6.
With the synthetic method to 6 in hand, it seemed most convenient to
introduce 14C in the –O–CH2–CH2–O– moiety of the title compound as
shown in Scheme 2. However the Mitsunobu reaction conditions
successfully applied during the preparation of similar compounds5; 6 did
not work in case of PNU-243922 and only unreacted 6a was recovered.
Several exploratory trials were carried out to optimize the reaction
conditions such as temperature, solvents, reagents addition order, etc.
The Mitsunobu reaction turned out to be extremely sensitive to the
concentration gradient of the reactants while reaction temperature and
reaction time did not substantially affect the yield. The best results were
obtained when a 4:5 equivalents of diisopropyl azodicarboxylate
(DIAD) and a 4:5 equivalents of triphenylphosphine ðPh3PÞ were added
in portions to the 14C labelled alcohol 6a and 2-amino-8-hydroxyquino-
OH
14C
14C
14C
14C
O
N
N
Tr
O
N
NH2
O
N
N
OH
N
N
N
NH2
N
N
d
Tr
7
6a
e
14C
14C
O
N
O
N
N
N
NH2
NH
[14C]PNU-243922
Scheme 2. Reagents and conditions: (d) Ph3P, DIAD, THF and (e) CH3CN,
TFA, then MeOH
Copyright # 2002 John Wiley & Sons, Ltd.
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88
C. FELICINI ET AL.
line 2:5 equivalents in dry tetrahydrofuran (THF) under nitrogen. The
obtained diether 7 was purified by flash-chromatography followed by
preparative thin layer chromatography (TLC). The removal of the trityl
group was achieved by treating 7 with trifluoroacetic acid (TFA) in
acetonitrile. After purification by preparative high-performance liquid
chromatography (HPLC), [14C]PNU-243922 was obtained in > 98%
radiochemically pure with a specific activity of 546 MBq=mmol. The
overall radiochemical yield was about 2% from 6a.
Experimental
General methods
2-(f3-[(2R)-2-methyl-4-tritylpiperazinyl]-2-pyranizylgoxy)-1-[U-14C] etha-
nol 6a was purchased from Amersham, synthesized according to
our method for the unlabelled equivalent 6. All solvents and reagents
were of analytical grade and were used without purification
unless otherwise indicated. Radioactivity measurements were performed
on a Tri-Carb 2100 TR liquid scintillation analyzer (Packard)
using Rialuma (Lumac System) as liquid scintillation cocktail. TLC
and radio-TLC measurements were made using a Packard Bioscan
System 200 imaging scanner and silica gel Merck F254 plates
(20 ꢀ 5 cm, 0:25 mm thick): toluene:ethyl acetate 7:3 (v:v) (system I),
methylene chloride:methanol:triethylamine 2:8:0.1 (v:v) (system II).
Preparative TLC were performed using silica gel Merck F254
plates (20 ꢀ 20 cm, 0:5 mm thick). Chemical purities were determined
by HPLC performed at 258C using a series-200 pump (Perkin-Elmer)
equipped with a LC-295 UV/VIS detector (Perkin-Elmer) and
PE-Nelson Turbochrom 4.0 software. Radiochemical purities
were determined using an A-515TR radio-HPLC analyzer (Packard)
equipped with a 0:5 ml homogeneous cell (liquid scintillation cocktail:
Ultima Flo-M (Packard) ratio to HPLC effluent: 3:1), under the
following conditions: YMC C8 column (100 ꢀ 4:6 mm ID; particle
size 5 mm, supplied by YMC Europe-GmbH) eluting with
H2O:CH3CN:TFA 94:6:0.1 (v:v) (A) and H2O:CH3CN:TFA 10:90:0.1
(v:v) (B) mixtures (isocratic at 100% A for 2 min, linear gradient
from 100% A to 0% A over 8 min, isocratic at 100% B for 5 min,
linear gradient from 0% A to 100% A over 0:5 min, isocratic at 100%
A for 2:5 min); flow rate 1:5 ml=min; wavelength 254 nm. The
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SYNTHESIS OF PNU-243922
89
purification of the final compound was performed by preparative HPLC
under the following conditions: YMC J’sphere ODS-H80 column
(150 ꢀ 100 mm ID; particle size 4 mm, supplied by YMC Europe-
GmbH) eluting with H2O:CH3CN:TFA 94:6:0.1 (v:v) (A) and
H2O:CH3CN:TFA 10:90:0.1 (v:v) (B) mixtures (isocratic at 93% A
for 10 min, linear gradient from 93% A to 40% A over 3 min, isocratic
at 40% A for 10 min, linear gradient from 40% A to 0% A over 1 min,
isocratic at 100% B for 5 min, linear gradient from 0% A to 93% A
over 1 min, isocratic at 93% A for 3 min); flow rate 8 ml=min;
wavelength 254 nm.
3-(R)-Methyl-1-tritylpiperazine (4)
Solid TrCl (260 g, 0:935 mol) was slowly added to a well-stirred and
cooled solution of 3 ð95 g; 0:95 molÞ in 1:2 l of CH2Cl2 (EXOTHER-
MIC!). After 45 min, the mixture was poured into an aqueous solution
of K2CO3 (140 g in 0:5 l of water), the organic phase was separated and
dried by K2CO3, and the solvent was evaporated. This gave 370 g oily, 4
which was used without purification. 4 can, however, be crystallized
1
from heptane. H-NMR ðCDCl3Þ: 7.57–7.10 (m, 15H) Tr; 3.23–2.85
(m, 7H) piperazine; 0.95 (d, 3H, J ¼ 6:0 Hz) Me.
1-(3-Chloro-2-pyrazinyl)-2-(R)-methyl-4-trityl-piperazine (5)
A mixture of 4 from above, 2,3-dichloropyrazine (154 g, 1:0 mol)
and K2CO3 ð160 g, 0:6 molÞ in 1 l DMF was heated (100–1108C)
for 20 h with intensive stirring (monitored by TLC in CHCl3 : EtOH
20:1). The mixture was then cooled and poured slowly into 6 l of water
with stirring. Compound 5 was filtered, washed with water and dried
under vacuum (3 mm=508C, 2 days) to remove DMF completely. Yield
299 g (70.8%). For analysis, 3 g of 5 was dissolved in 10 ml of
hot ethanol, and HCl (10% aqueous solution, 3 ml) was added
slowly (to keep the compound in solution). TrOH begins to crystallize
after 10–15 min. After 30 min, TrOH was filtered and ethanol
was evaporated. The aqueous solution was extracted by ether ð2 ꢀ
200 mlÞ and made basic by K2CO3 to pH 12. The compound
was extracted by CHCl3 ð3 ꢀ 10 mlÞ dried with K2CO3, and then the
solvent was evaporated to give 1:4 g of the detritylated product
1-(3-chloro-2-pyrazinyl)-2-(R)-methylpiperazine. 1H-NMR ðCDCl3Þ:
8.08 (d, 1H, J ¼ 2:7 Hz) Ar; 7.83 (d, 1H) Ar; 4.11(m, 1H)
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C. FELICINI ET AL.
CH–Me; 3.32(m, 2H) + 3.13–2.72 (m, 5H) piperazine; 1.17 (d, 3H,
%
J ¼ 6:6 Hz) Me.
2-(f3-[(2R)-2-Methyl-4-tritylpiperazinyl]-2-pyrazinylgoxy)-1-
ethanol (6)
The title compound was prepared using 5 ð0:227 g, 0:5 mmolÞ which
was added to glycol (0:310 g, 5:0 mmol) and NaH (0:097 g, 60% in
mineral oil, 2:5 mmol) in DMF. The reaction was stirred at 658C
overnight. Purification by silica gel chromatography (20% ethylacetate
in n-hexane) gave 6 as a yellow solid (0:120 g, 50% yield); mp 86–888C;
13H-NMR (DMSO) d 7.67–7.65 (m, 1H), 7.50–7.45 (m, 5H), 7.45–7.42
(m, 1H), 7.32–7.27 (m, 5H), 7.20–7.15 (m, 3H), 4.67–4.61, (m, 1H),
4.38–4.28 (m, 2H), 4.16–4.10 (m, 1H), 3.95–3.69 (m, 2H), 3.65–3.58 (m,
1H), 2.95–2.89 (m, 1H), 2.85–2.80 (m, 1H), 2.04–1.97 (m, 1H), 1.73–1.65
(m, 1H), 1.50–1.46 (m, 3H).
8-[2-(f3-[(2R)-2-methyl-4-tritylpiperazinyl]-2-pyrazinylgoxy)[U-14C]
ethoxy]-2-quinolinylamine (7)
To a cooled ð08CÞ solution of 6a (195:36 MBq, 0:264 mmol, with
a radiochemical purity of about 75%) and 2-amino-8-hydroxyquino-
line ð105:9 mg; 0:66 mmolÞ in dry THF ð1 mlÞ a first portion of
Ph3P ð43:3 mg; 0:172 mmolÞ was added. When the mixture
of reaction became clear, a freshly prepared solution of DIAD in dry
THF ð330 mg=ml; 100 ml; 33:4 mg; 0:172 mmolÞ was added. After
removing the cold bath and stirring at room temperature for
about 30 min, 90% of the unreacted precursor 6a was still present
in the reaction mixture (determined by radio-TLC, systems I and II).
At a distance of about 30 min, the Mitsunobu reagents were added
again at 08C as follows: Ph3P ð129:8 mg; 0:488 mmolÞ and DIAD in
dry THF (330 mg=ml, 300 ml, 100 mg, 0:488 mmol) and twice Ph3P
ð43:3 mg; 0:172 mmolÞ and DIAD in THF (330 mg=ml, 100 ml,
33:4 mg, 0:172 mmol). The reaction mixture was then stirred at room
temperature for 24 h. As the conversion of 6a was still not complete
(29% of unreacted precursor, checked by radio-TLC, systems I and II)
further Ph3P ð43:3 mg; 0:172 mmolÞ and a solution of DIAD in THF
(330 mg=ml, 100 ml, 33:4 mg, 0:172 mmol) were added. At the end of the
reaction (determined by radio-TLC, systems I and II), the solution
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SYNTHESIS OF PNU-243922
91
was evaporated to dryness and the non-radioactive yellow impurities
were eliminated by a flash chromatography on SiO2 eluting
with toluene:ethyl acetate 7:3. The fractions containing the
product (determined by radio-TLC, system I and by radio-HPLC)
were combined and evaporated to dryness giving the crude intermediate
3 (77:6 MBq, 0:1422 mmol, about 20% radiochemically pure by
radio-HPLC). The above material was then purified twice
by preparative TLC eluting with a mixture methylene chloride:me-
tha-nol:triethylamine 2:8:0.1 (v:v) and the chromatographic
bands corresponding to 7 were extracted with a mixture ethyl
acetate:toluene 1:1 (v:v). After organic phases evaporation to dryness,
the compound 3 was obtained (5:66 MBq, 0:0104 mmol, 88% radio-
chemically pure by radio-HPLC). The overall radiochemical yield was
4% from 6a.
8-[2-(f3-[(2R)-2-methylpiperazinyl]-2-pyrazinylgoxy)[U-14C]
ethoxy]-2-quinolinylamine([14C]PNU-243922)
A solution of TFA (10 ml, 0:13 mmol) in dry acetonitrile ð1:1 mlÞ
was added dropwise into a cold ð48CÞ solution of 7 (7:43 MBq,
0:0136 mmol) in dry acetonitrile ð1:1 mlÞ in about 5 min under
nitrogen. The cold bath was removed and the reaction mixture was
stirred under nitrogen at room temperature for about 1 h. After the
disappearance of the precursor (determined by radio-HPLC),
the solution was evaporated to dryness, methanol ð1:5 mlÞ was added
and the reaction mixture stirred at room temperature for three days.
At the end of the reaction (determined by radio-HPLC) the solution
was evaporated to dryness and the crude product was dissolved in
the mobile phase A ð2 mlÞ and purified by preparative HPLC. The
fractions containing the pure product (checked by radio-HPLC)
were combined and evaporated to small volume until the organic phase
was removed. The aqueous phase was adjusted to pH 8.5 with
1 N NaOH and extracted with methylene chloride ð3 ꢀ 20 mlÞ. The
organic phases were collected and washed with brine ð2 ꢀ 10 mlÞ. After
drying over anhydrous sodium sulfate and solvent evaporation to
dryness, [14C]PNU-243922 ð4:37 MBq) was obtained in 98% radio-
chemically pure form (by radio-HPLC, Rt ¼ 6:70 min), with a specific
activity of 546 MBq=mmol. The radiochemical yield of this step was
about 60%.
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Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 85–92