Tritiation of SR141716 by metallation-iodination-reduction: Tritium-proton nOe study

Article abstract of DOI:10.1002/jlcr.529

The central cannabinoid receptor antagonist SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide, was synthesized from commercially available starting materials. Condensation of an aryl hydrazine with a diketone followed by base promoted isomerization/cyclization of the intermediate anti-imine gave the pyrazole acid which was converted to the title hydrazide via its acid chloride. Facile iodination via metallation followed by in situ trapping with an iodine source gave a modest yield of the iodinated SR 141716. The iodine was selectively reduced with tritium gas and catalyst while retaining the three aryl chlorine atoms in the structure. The tritiated SR 141716 exhibited a tritium-proton nOe both definitively identifying the position of the tritium as well as the sought isomer of the diarylpyrazole. This work provides a direct method for the preparation of preferred iodinated aryl substrates that offer advantages where selectivity and high incorporation in catalytic reduction is sought. Copyright

Full text of DOI:10.1002/jlcr.529

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2002; 45: 59–70.
DOI: 10.1002/jlcr.529
Research Article
Tritiation of SR141716 by metallation–
iodination–reduction:
tritium-proton nOe study
Herbert H. Seltzman*, F. Ivy Carroll, Jason P. Burgess,
Christopher D. Wyrick and David F. Burch
Chemistry and Life Sciences, Research Triangle Institute, 3040 Cornwallis Road,
POBox 12194, Research Trian gle Park, NC 27709-2194, USA
Summary
The central cannabinoid receptor antagonist SR141716, N-(piperidin-1-yl)-5-
(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide,
was synthesized from commercially available starting materials. Condensation
of an aryl hydrazine with a diketone followed by base promoted isomerization/
cyclization of the intermediate anti-imine gave the pyrazole acid which was
converted to the title hydrazide via its acid chloride. Facile iodination via
metallation followed by in situ trapping with an iodine source gave a modest
yield of the iodinated SR141716. The iodine was selectively reduced with
tritium gas and catalyst while retaining the three aryl chlorine atoms in the
structure. The tritiated SR141716 exhibited a tritium-proton nOe both
definitively identifying the position of the tritium as well as the sought isomer
of the diarylpyrazole. This work provides a direct method for the preparation
of preferred iodinated aryl substrates that offer advantages where selectivity
and high incorporation in catalytic reduction is sought. Copyright # 2002
John Wiley & Sons, Ltd.
Key Words: metallation; iodination; tritiation; tritium-proton nOe; SR141716
*Correspondence to: H. H. Seltzman, Research Triangle Institute, Chemistry and Life Sciences,
3040 Cornwallis Road, P.O. Box 12194, Research Triangle Park, NC 27709-2190, USA.
E-mail: hss@rti.org
Contract/grant sponsor: National Institute on Drug Abuse; contract/grant number: N01DA-1-
7311
Copyright # 2002 John Wiley & Sons, Ltd.
Received 4 May 2001
Revised 24 September 2001
Accepted 24 September 2001

60
H. H. SELTZMAN ET AL.
Introduction
Tritium labelling of a compound in the preferred final step of its
preparation often involves the synthesis of a derivative that can be
reduced back to the parent compound. Halogenation of the target
compound on an aromatic ring can preclude a separate, complete
synthesis of the halo-analog. However, if the target compound contains
chlorine atoms that are to be maintained in the final product, the
introduction of the significantly more reactive iodine would be required
to achieve good selectivity in the reduction step. The rapid reduction of
an aryl iodide offers both greater selectivity and higher specific activities
than the more commonly used bromides. Iodine incorporation,
however, typically by an iodonium reagent, is often problematic. In
this paper, an approach of metallation–iodination followed by selective
catalytic reduction is described for the tritiation of the cannabinoid
receptor antagonist SR141716.
The hydrochloride of the diarylpyrazole hydrazide SR141716, N-
(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- 4-methyl-1H-
pyrazole-3-carboxamide, is the first antagonist for the cannabinoid
receptor.1,2 This ligand has become an important tool in studies leading
to a better understanding of the neurochemical system represented by
the receptor.3,4 Essential in pharmacological studies of the receptor is
the availability of the corresponding tritiated radioligand. The details of
the synthesis of both the unlabelled free base SR141716 and of the
tritium-labelled ligand from a metallation–iodination–tritiation se-
quence are reported. The nuclear Overhauser enhancement (nOe)
spectroscopy that was used to unambiguously identify the structure of
SR141716 versus its 1,3-isomer is described as well as its extension to a
tritium-proton nOe.
Results and discussion
The synthesis of the unlabelled SR141716 is outlined in Figure 1. A
preliminary account of this synthesis has been published5 as have
syntheses from other laboratories.6–8 The synthesis begins with the
lithium hexamethyldisilazide promoted condensation9 of 4-chloropro-
piophenone 1 with diethyl oxylate 2 to give the readily isolated solid
lithium enolate 3 in 35% yield. Use of the stronger base LDA modestly
increased the yield to 40%. The further enhancement of the kinetic
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TRITIATION OF SR141716 BY METALLATION
61
Figure 1.
basicity of LDA by the addition of 10 mol% of potassium t-butoxide10
raised the yield of the salt 3 to 65%. Acidification of 3 gave 4 in 52%
overall yield from 1.
Condensation of 4 with the hydrazine free base 5 in ethanol at reflux11
gave the pyrazole ester 6 (40%) and the imine 7 (60%) which were
readily separated due to solubility differences between the two
compounds in the reaction mixture. Further heating of 7 failed to
generate more 6, indicative of a non-reactive anti configuration between
the anilino nitrogen and the 4-keto group. However, when 7 or the
reaction mixture containing 6 and 7 were heated with ethanolicsodium
hydroxide,5 the pyrazole acid 8 was obtained directly in 89% yield. The
cyclization of 7 proceeded via base promoted isomerization at the imine
double bond to afford a reactive syn relationship between the anilino
nitrogen and the 4-keto group. In situ hydrolysis of the ester 6 then
provided the acid 8.
The acid 8 was converted to the corresponding acid chloride with
thionyl chloride12,13 in 94% yield. Treatment of the acid chloride with 1-
aminopiperdine and triethylamine readily afforded SR141716 (free base)
in 73% yield after chromatography and recrystallization, for an overall
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H. H. SELTZMAN ET AL.
Figure 2.
yield of 32%. A trace amount of the 1,3-isomer 10 was obtained from
the mother liquor (see Experimental).
Two isomers are possible from the condensation of the diketone 4
with the hydrazine 5 affording the desired 1,5-diarylpyrazole acid 8 and
the isomeric1,3-diarylpyrazole. The former is usually preferred due to
initial attack of the unsubstituted hydrazine nitrogen at the more
electrophilic 2-carbonyl of 2,4-diketo esters.9,14 An nOe spectral
determination was used to show the regiochemistry of the product
SR141716 and by inference the structure of 8. The proximity of the two
aryl rings in the 1,5-isomer suggests that an nOe interaction would exist
between the ortho protons Ha and Hb on the respective aryl rings, thus
identifying SR141716. No such effect from the similar protons would be
anticipated for the 1,3-isomer (10). The five aromaticresonances were
completely resolved in MeOD allowing a definitive experiment (in
contrast to the overlap in CDCl3 or C6D6). The NOESY spectrum
clearly demonstrated an interaction between the 6-H and the 20,60–H2 of
the respective rings and thereby rigorously identified the final product as
the 1,5-diarylpyrazole SR141716.
To obtain the iodinated analog for reduction, direct iodination of the
unlabelled ligand of interest would be preferable to conducting a
separate, multistep synthesis from an iodinated fragment, especially if
the latter was not readily available. The availability of a site on
SR141716 that can be metallated15–17 by directed ortho metallation and
followed by trapping with an iodine source made such direct iodination
possible. Thus, metallation by n-butyllithium at the 6-position of the
2,4-dichlorophenyl ring, directed by the N-linked pyrazole ortho
directing metallation group, followed by quenching with 1-chloro-2-
iodoethane afforded the 6-iodophenyl derivative 11 in 17% yield
(Figure 2). Also obtained from this process was the 3-iodophenyl
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TRITIATION OF SR141716 BY METALLATION
63
derivative 12, in 14% yield, which derived from the combined weak ortho
metallation directing effects of the two chlorine atoms. The compounds
were isolated chromatographically and characterized by 1H NMR
(MeOD, only two meta coupled doublets for the trihalogenated ring of
11, d 7.94 and 7.66, J ¼ 2:2 Hz) and MS (DIP, m/z 588 M+, 84 base).
Reduction of 11 under a deuterium atmosphere in the presence of 10%
Pd/C (10% w/w) and Et3N (258C, Et2O) afforded deuterium-labelled
2
1
SR141716 (93% H) that was identical (HPLC, H NMR, MS) in all
other respects with SR141716. No over-reduced product was detected.
Tritiation under similar conditions with carrier-free tritium gas gave
[3H]SR141716 as pre-dominantly a single component (HPLC, bRAM).
Purification by HPLC afforded >99% pure ligand at a specific activity
of 22.4 Ci/mmol as determined by UV quantitation and scintillation
counting. While effective, the tritiation in ether proceeded sluggishly
and with modest conversion to the radioligand. A change of solvents to
ethanol gave faster and more complete reduction in both deuterium and
tritium experiments. A specific activity of 23 Ci/mmol was obtained. A
3
proton-decoupled H NMR spectrum (MeOD) exhibited one singlet
(d 7.50) for the 6- 3H on the 2,4-dichlorophenyl ring, thus identifying the
single site of label incorporation. A proton observed tritium-proton nOe
difference spectrum of [6-3H]SR141716 exhibited an enhancement of the
2,6-protons of the 4-chlorophenyl ring and an enhancement of the 5-
proton of the 2,4-dihlorophenyl ring upon irradiation of the tritium in a
1:3 ratio, respectively.y This demonstrates the position of the tritium
atom and its proximity to the 4-chlorophenyl ring with the same
rigorousness as for the unlabelled SR141716. In addition to its
application to structure determination, tritium-proton nOe can con-
tribute significant understanding to pivotal interactions between
proteins and tritiated substrates, in suitable studies. For example, a
more complex protein counterpart to this small molecule tritium-proton
nOe has been demonstrated in an nOe study of a tritiated substrate’s
covalent adduct to an enzyme’s binding site.18
Conclusion
This paper provides the details of a metallation–iodination–tritiation
sequence that offers a facile entry to tritiated ligands from numerous
y The use of a direct difference nOe experiments for observation of {3H}1H nOe makes
determination of percent enhancements difficult. However, the superior spectra obtained without
subtraction artifacts makes this method preferable for qualitative detection of nOe interactions.
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H. H. SELTZMAN ET AL.
aryl ligands. The chemistry suggests its wider application to other
structures that are amenable to metallation and trapping with the
preferred halogen iodine. It is applied here to a cannabinoid receptor
ligand of considerable current interest that is proving to be a valuable
tool in pharmacological studies of this emerging neurochemical system.
Our synthesis of the unlabelled SR141716 from commercially available
fragments without the need for purification of intermediates recommend
this route for the preparation of this important compound. The
demonstration of an intramolecular proton-tritium nOe has implica-
tions for the study of the interactions of ligands with receptors.
Experimental
NMR spectra were obtained on a Bruker AMX-500 spectrometer
1
operating at 500.13 MHz for H or on a Bruker AM-250 spectrometer
1
operating at 250.13 MHz for H. Proton spectra were referenced to
internal tetramethylsilane at 0.0 ppm and tritium spectra were
referenced to internal tetramethylsilane by the relationship
dtritium ¼ ftritium-( fTMS*gT/gH) ꢀ 106 where ftritium and fTMS are the
observational frequencies of the tritium of interest and tetramethylsi-
3
lane, respectively, and gT/gH is the ratio of the gyromagneticratios of H
and H or 1.06663975. Two-dimensional (2D) NOESY spectroscopy at
1
500 MHz was obtained on a sample of SR141716 that was dissolved in
d4-methanol and then degassed using several freeze–pump–thaw cycles
and sealed under vacuum. NOESY spectra were acquired with a 800 ms
mixing time as a 2K ꢀ 1K data matrix and then zero-filled to 2K ꢀ 2K
prior to apodization with a phase shifted squared sine function and 2D
Fourier transformation. {3H}1H nOe difference spectra were obtained
on a 10 mCi sample of tritium-labelled SR141716 that was dissolved in
d4-methanol and placed in a Teflon NMR tube insert that had been cut
to a length of 500 and placed in an 800 NMR tube. The sample was then
degassed using several freeze–pump–thaw cycles and sealed under
vacuum. Difference spectra were obtained by a proton observation
direct difference. In this method, a transient is obtained after low power
(ꢁ10 mW) continuous wave irradiation at the tritium resonance
frequency during the relaxation delay of 2 s. The irradiation frequency
is gated off during acquisition of the proton signal during which time the
irradiation frequency is shifted off resonance by 5000 Hz and the next
transient is obtained with the detector phase shifted by 1808. This results
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TRITIATION OF SR141716 BY METALLATION
65
in the accumulation of a difference spectrum after each even number of
transients.
Mass spectra were obtained on a model HP 5989A mass spectrometer
or a VG ZAB-E high resolution, double focusing mass spectrometer.
Ethyl 2,4-dioxo-3-methyl-4-(4-chlorophenyl)-butanoate (4)
Under anhydrous conditions, potassium t-butoxide (70 mg, 0.59 mmol)
was cooled to ꢂ788C under dry argon and treated with anhydrous ethyl
ether (20 ml) and stirred for 10 min. Commercial LDA in heptane/THF/
ethyl benzene (2.77 ml, 5.93 mmol) was added by syringe and the
mixture stirred for 15 min. 4-Chloropropiophenone (1.00 g, 5.93 mmol)
in ether (7 ml) was added drop-wise over 30 min with stirring. After a
further 30 min, diethyl oxylate (0.81 ml, 5.93 mmol) was added in one
portion. The cold bath was removed and the reaction stirred 2.5 h after
reaching ambient temperature. After standing 3 days, the precipitated
yellow solid was filtered to afford 1.06 g (65%) of the lithium enolate of
the title compound. On larger scale (723 mmol), a second crop of
lithium enolate was also obtained for a total yield of 54%. The solid was
sequentially partitioned between 1 N HCl and CH2Cl2 (3 times)
affording 0.84 g (52% overall) of the title compound as an orange oil
after drying the organiclayers (Na SO4), filtering and evaporation of
2
the volatiles in vacuo. 1H NMR (CDCl3), d 7.93 (d, 2H, 8.8 Hz, ArH2),
7.50 (d, 2H, 8.8 Hz, ArH20 ), 5.00 (q, 1H, 7.1 Hz, HCMe), 4.28 (q, 2H,
7.2 Hz, OCH2), 1.45 (d, 3H, 7.1 Hz, HCCH3), 1.30 (t, 3H, 7.1 Hz).
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
carboxylic acid (8)
The diketone 4 (36.0 g, 134 mmol) dissolved in 500 ml of absolute
ethanol was treated with solid 2,4-dichlorophenylhydrazine free-base
(23.7 g, 134 mmol) in one portion with mechanical stirring under dry
nitrogen. The reaction was heated at reflux for 2.5 h. An aliquot that
was evaporated in vacuo, dissolved in CH2Cl2 and analyzed by TLC
(SiO2 F254, EtOAc:hex 1:2, UV detection) showed the diketone to be
essentially consumed and two higher Rf products to be present. The
ambient temperature reaction was treated with solid NaOH (8.0 g,
200 mmol) and heated under nitrogen at reflux for 2 h. TLC (SiO2,
ace:CH2Cl2:HOAc, 1:1: trace, UV detection) of an aliquot quenched in
aqueous HCl and extracted with ether showed the intermediate imine
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H. H. SELTZMAN ET AL.
and pyrazole ester to be consumed and a lower Rf product to be
formed. Water was added (ꢁ1000 ml) and the ethanol removed by
rotary evaporation in vacuo. The residual basic solution was extracted
with ether (2 ꢀ ) to remove neutrals. The aqueous phase was acidified to
pH 1 with HCl and extracted with ether (3 ꢀ ). The combined organic
layers of the acid extract was dried (Na2SO4) and evaporated in vacuo
and high vacuum dried to afford 45.9 g (89%) of the title acid as a beige
1
solid. This acid was suitable for use in the next reaction. H NMR
(MeOD) d 7.58 (d, 1H, J=2.2 Hz, 3-H), 7.53 (d, 1H, J=8.5 Hz, 6-H),
7.44 (dd, 1H, J=2.2 Hz, 8.5 Hz, 5-H), 7.37 (d, 2H, J=8.5 Hz, 30, 50-H2),
7.21 (d, 2H, J=8.5 Hz, 20, 60-H2), 2.30 (s, 3H, CH3).
When the reaction is run in more concentrated solution (10 ml
ethanol per gram of diketone), the intermediate anti imine precipitates
(60%) leaving the pyrazole ester (40%), derived from the syn-imine, in
solution, thus enabling their easy separation. In situ processing of the
mixture often requires further dilution with ethanol to maintain fluidity
in the subsequent base-promoted step of the reaction.
5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
carboxylic acid chloride
The unpurified acid 8 (43.7 g, 114 mmol) dissolved in dry toluene
(2500 ml) was treated with SOCl2 (16.7 ml, 229 mmol) under N2 and
heated at reflux for 3–6 h. Rotary evaporation in vacuo and high
vacuum drying afforded 44.7 g (94%) of the acid chloride as a golden
foam. The completeness of the reaction can be determined by the
observation of SR141716, and the absence of the precursor acid 8, after
treatment of a high vacuum dried reaction aliquot residue with
triethylamine and 1-aminopiperidine, as in the next step, and analyzing
by TLC. HRMS: calculated for C17H10N2OCl4 397.9547, found
397.9549.
N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxamide, SR141716
The acid chloride (44.7 g, 107 mmol) dissolved in dry CH2Cl2 (2000 ml)
was treated with 1-aminopiperidine (17.4 ml, 161 mmol) followed by
triethylamine (46.0 ml, 321 mmol) under N2 with stirring at ambient
temperature. After 2 h, TLC (SiO2, EtOAc:hex, 4:1, phosphomolybdic
acid, Ce+4) on a worked up aliquot showed the reaction to be complete.
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67
The reaction solution was washed with water (3 ꢀ ), aqueous NaHCO3
(3 ꢀ ), and 1 N HCl (3 ꢀ ) then dried over Na2SO4 and rotary
evaporated to afford 52.4 g (105%) of a foam. The crude product was
dissolved in EtOAcand diluted with three volumes of hexane. The
resulting precipitate of the title compound (13.3 g) was filtered and the
mother liquor was loaded on a silica gel column (2500 g) and eluted with
25% EtOAc–hexane (14 l) followed by 50% EtOAc–hexane (22 l). The
product containing fractions were combined, rotary evaporated and the
residue (36 g) recrystallized from in methanol/water. Filtration afforded
17.7 g (36%) of 98% pure (HPLC: Reverse Phase C-18 Waters Radial
Nova Pak; 10 mml 8 mm ꢀ 10 cm, CH3CN–H2O, 75:25 UV, 280 nm) title
compound. A second crop of 7.45 g (14%) of 95% pure SR141716 was
similarly obtained from the mother liquor residue. The above 13.3 g of
precipitate was recrystallized from methanol/water to afford 11.5 g
(23%) of 98% pure SR141716. Analytically pure product was obtained
from subsequent recrystallization from ether/hexane; m.p. 157–1598C.
1H NMR (MeOD), d 7.56 (d, 1H, J=2.2 Hz, 3-H), 7.53 (d, 1H,
J=8.5 Hz, 6-H), 7.45 (dd, 1H, J=2.3, 8.5 Hz, 5-H), 7.36 (d, 2H,
J=8.3 Hz, 30,50-H2), 7.19 (d, 2H, J=8.3 Hz, 20,60-H2), 2.82 (br, 4H, CH2-
N-CH2), 2.30 (s, 3H, CH3), 1.73 (p, 4H, J=5.7 Hz, 2 CH2), 1.44 (br, 2H,
CH2). HRMS: calculated for C22H21N4OCl3 462.0783, found 462.0781.
CHNCl within 0.06%. NOESY (MeOD), interaction between 6-H and
20,60-H2.
Recrystallization, from ether, of the mother liquor residue from the
above second crop afforded 645 mg of the isomeric N-(piperidin-1-yl)-3-
(4-chlorophenyl) - 1-(2, 4 - dichlorophenyl) -4-methyl-1H-pyrazole-5-car-
1
boxamide (10). H NMR (MeOD), d 7.61 (d, 2H, J=8.5 Hz, 30,50-H2),
7.51 (d, 1H, J=2.2 Hz, 3-H), 7.48 (d, 1H, J=8.5 Hz, 6H), 7.42 (d, 2H,
J=8.5 Hz, 20,60-H2), 7.38 (dd, 1H, J=2.2, 8.5 Hz, 5-H), 2.76 (br, 4H,
CH2-N-CH2), 2.40 (s, 3H, CH3), 1.70 (p, 4H, J=5.5 Hz, 2CH2), 1.61
(br, 2H, CH2), MS, m/z; 462 (M+), 344 (M-84-35+1), 99 (base).
N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-6-iodophenyl)-
4-methyl-1H-pyrazole-3-carboxamide (11) and N-(Piperidin-1-yl)-
5-(4-chlorophenyl)-1-(2,4-dichloro-3-iodophenyl)-4-methyl-1H-pyrazole-
3-carboxamide (12)
N - (Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3- carboxamide (SR141716) (300 mg, 0.65 mmol) and a few
crystals of 1,10-phenanthroline indicator were dissolved in 12 ml dry
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H. H. SELTZMAN ET AL.
ether (distilled from Na/benzophenone) in a 50 ml flask fitted with a
septum, a gas inlet and a Teflon stir bar under a dry argon atmosphere.
The flask was cooled to ꢂ308C and treated with n-BuLi (0.416 ml,
1.56 M, 0.65 mmol) with stirring until a dark red end-point. This was
followed by a further addition of n-BuLi (1.25 ml, 1.95 mmol, 3 eq).
After stirring for 2.5 h at ꢂ308C, the reaction was cooled to ꢂ788C and
1-chloro-2-iodoethane (0.94 ml, 10.4 mmol) was added. The reaction
was stirred at ꢂ788C for 0.5 h and ꢂ308C to ꢂ188C for 0.75 h after
which the reaction was allowed to warm slowly over the next 0.5 h to
ambient temperature. The opaque brown mixture was quenched with
MeOH (10 ml) and the volatiles evaporated in vacuo. The residue was
partitioned between water (50 ml) and ether (3 ꢀ 50 ml). The combined
organics were dried (Na2SO4) and evaporated in vacuo to yield 0.41 g of
a yellow resin which was chromatographed on a Merck size B
prepacked silica gel column eluting with methyl t-butylether: toluene
(1:9) to afford 64 mg (17%) of 11 and 53 mg (14%) of 12. 11: 1H NMR
(250 MHz, MeOD), d 7.94 (d, 1H, J=2.2 Hz, 5-H), 7.66 (d, 1H,
J=2.2 Hz, 3-H), 7.39 (d, 1H, J=8.5 Hz, 30,50-H2), 7.30 (d, 1H,
J=8.5 Hz, 20,60-H2), 2.84 (br m, 4H, N–(CH2)2), 2.31 (s, 3H, CH3),
1.73 (br p, 4H, J=5.5 Hz, N–(CH2-CH2)2), 1.45 (br m, 2H, N-CH2-
CH2-CH2); MS (DIP) (m/z 588 M+, 84 base).
12: 1H NMR (250 MHz, MeOD), d 7.60 (d, 1H, J=8.6 Hz, 6-H), 7.54
(d, 1H, J=8.3 Hz, 5-H), 7.38 (d, 2H, J=8.5 Hz, 30,50-H2), 7.20 (d, 2H,
J=8.5 H,z, 20,60–H2), 2.82 (t, 4H, J=5.2 Hz, N–(CH2)2), 2.30 (s, 3H,
CH3), 1.73 (br p, 4H, J=5.4 Hz, N-(CH2-CH2)2), 1.44 (br m, 2H, N-
CH2-CH2-CH2).
N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-([6-3H]-2,4-dichlorophenyl)-
4-methyl-1H-pyrazole-3-carboxamide, tritium-labelled SR141716
N-(Piperidin - 1-yl)-5 -(4-chlorophenyl)-1-(2,4-dichloro-6-iodophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (10 mg, 0.017 mmol) in 1 ml abso-
lute EtOH was shaken with 10% (w/w) Pd/C (4 mg) and filtered,
evaporated under a stream of nitrogen and high vacuum dried. The
residue was redissolved in absolute EtOH (0.8 ml) and transferred to a
2 ml tritiation flask containing 10% (w/w) Pd/C (4 mg) and then treated
with anhydrous Et3N (from basicAl 2O3) (7 ml, 0.0507 mmol) and stirred
under carrier-free tritium gas (4.9 Ci) for 3.5 h. The catalyst was
removed by filtration through celite and diluted with 25 ml absolute
EtOH for temporary storage. Evaporation of EtOH (3 ꢀ ) to remove
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69
exchangeable tritium afforded 317 mCi as a single component by TLC-
radioscan (SiO2, 10% MeOH/CHCl3). The product was eluted from a
Waters RP-C18 25 mm ꢀ 10 cm, 10 cm column with 75% CH3CN/H2O
monitoring by UV at 280 nm. The radioactivity containing fractions
were identified by non-eluted TLC-radioscan and analyzed by HPLC in
the above system. The product was identified by HPLC retention time
versus an unlabelled standard. Fractions were combined affording
139 mCi at a specific activity of 23 Ci/mmol determined by counting and
1
UV determination of yield. H NMR (MeOD), d 7.56 (s, 3-H), 7.53 (d,
J=7.0 Hz, residual 6-H), 7.45 (d, J=7.0 Hz, 5-H), 7.36 (d, J=8.0 Hz,
30,50-H2), 7.19 (d, J=8.0 Hz, 20,60-H2), 2.82 (br s, CH2-N-CH2), 2.33 (s,
CH3), 1.74 (br s, 2 CH2), 1.47 (br, 2H, CH2). 3H NMR (MeOD) 7.51 (d,
J=7.4 Hz). Difference nOe (MeOD), interaction between 6-3H and 5-H,
and 6-3H and 20,60-H2.
Acknowledgement
This work was supported, in part, by the National Institute on Drug
Abuse on Contract No. NO1DA-1-7311.
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