Synthesis of 14C- and 14C3-labelled Org 37462

Article abstract of DOI:10.1002/jlcr.1499

Org 37462 (1) is the active ingredient in Orgalutran, an innovative product that reduces the time of treatment in in vitro fertilization from four to less than two weeks. Org 37462 is a synthetic decapeptide containing several amino acids that are unnatur

Full text of DOI:10.1002/jlcr.1499

Research Article
Received 17 October 2007,
Revised 10 December 2007,
Accepted 10 January 2008
Published online 18 February 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1499
Synthesis of 14C- and 14C3-labelled Org 37462
Ã
P. H. G. Wiegerinck, L. W. H. Hofstede, F. M. G. M. Sperling,
and J. F. Vader
Org 37462 (1) is the active ingredient in Orgalutrans, an innovative product that reduces the time of treatment in in vitro
fertilization from four to less than two weeks. Org 37462 is a synthetic decapeptide containing several amino acids that are
unnatural in stereochemistry and/or in structure. The synthesis, starting with a ProtectingGroup-D-Ala-resin, is a typical
solid state synthesis. For the conduction of several metabolism studies, Org 37462 had to be labelled with carbon 14. It was
decided to label D-3-(2-naphthyl)alanine, the last amino acid to be coupled to the resin. We report the synthesis of [14C]-
and [14C3]-Org 37462, starting from 2-bromo-[14C-methyl]-naphthalene and [14C2]-tert-butyl glycinate.
Introduction
Results and Discussion
Org 37462 (1) (Figure 1) is the active ingredient in Orgalutrans, For the mono-labelled Org 37462 it was decided to synthesize
an innovative product that reduces the time of treatment in in N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine (8) (Scheme 1) to be
vitro fertilization (IVF) from four to less than two weeks. Patients coupled to the peptide moiety. Because the N-acetyl group is
given Orgalutrans have a much simpler IVF procedure. Their already present, the use of 8 would minimize the number of
exposure to drugs as well as the physical and emotional reaction steps with radiolabelled material. In the case of the
burdens, associated with IVF treatment, are reduced. Orgalu- triple-labelled product it was decided to synthesize and use
trans prevents the premature rise in levels of the luteinizing N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine (10) in order to
hormone (LH) during specific infertility treatments such as IVF follow the same coupling procedure as for unlabelled Org
and intracytoplasmic sperm injection. When the growth of 37462 to increase the chemical yield.
multiple follicles is stimulated with gonadotropins such as The syntheses of these two compounds are outlined in
follicle-stimulating hormone, an early increase in LH may cause Scheme 1. (1R)-(1)-Camphor (2) was used as an auxiliary group
unfavorable effects on the eggs and the possibility of getting to introduce the correct stereochemistry in the alkylating step,
pregnant.1–4
as described by McIntosh and Mishra4 and McIntosh and
Org 37462 is a synthetic decapeptide containing several Leavitt5 (1R)-(1)-Camphor (2) was converted to (1R)-(1)-
amino acids that are unnatural in stereochemistry and/or in thiocamphor (3) using phosphorus pentasulfide.6 Condensation
structure. The synthesis of unlabelled Org 37462 is a linear of 3 with tert-butyl glycinate and [14C2]-tert-butyl glycinate,
process that starts with a protected-D-Ala-resin to which leading to imines 4a and 4b, respectively, followed by alkylation
subsequently the amino acids are coupled in the right order. with 2-bromo-[14C-methyl]-naphthalene gave the corresponding
This is a typical solid state synthesis: removal of the protecting imines 5a and 5b and their C-2 epimers. In test experiments
group, coupling of the protected amino acid, washing away the with unlabelled materials imine 5 and its C-2 epimer were
1
reagents and repetition of this sequence. After nine cycles, formed in a 9/1 ratio, according to H NMR. The imines 5a and
subsequently followed by removal of the remaining protecting 5b and their C-2 epimers were converted to the corresponding
groups, cleavage from the resin and purification by preparative final products 12 and 14. In both cases the undesired
high-performance liquid chromatography (HPLC), the final diastereoisomer, Org 14735, could easily be separated by
product, Org 37462, is isolated.
preparative HPLC from Org 37462 (Figure 2). This figure shows
For the conduction of several metabolism studies, among that the acid Org 14734, which was formed during the split-off
others a human ADME study, the availability of Org 37462 of the resin, could also be removed in the same HPLC
labelled with carbon-14 was required. It was decided to label 3- purification system.
(2-naphthyl)-D-alanine since this is the final amino acid to be
Hydrolysis7 of the imine 5a and acetylation of the amino
coupled to the resin and thus the number of actions with group of 6a resulted in the fully protected amino acid 7. The
carbon-14-labelled material is minimized. Another reason for
this position of the label is that it is an unnatural amino acid and
aDepartment of Process Chemistry (DPC), N.V. Organon, P.O. Box 20, 5340 BH
Oss, The Netherlands
it might be interesting to follow this amino acid in the metabolic
pathway. In this paper we report the synthesis of the mono- and
the triple-[14C]-labelled versions of 3-(2-naphthyl)-D-alanine and
*Correspondence to: P. H. G. Wiegerinck, Department of Process Chemistry
(DPC), N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands.
E-mail: peter.wiegerinck@organon.com
its conversion to the corresponding mono- and triple-[14C]-
labelled versions of Org 37462.
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P. H. G. Wiegerinck et al.
Figure 1. Structure of Org 37462.
Scheme 1
amino acid was purified by column chromatography. Subse- with reverse-phase HPLC resulted in the desired [14C]-Org 37462
quently, the tert-butyl ester of 7 was cleaved yielding N-acetyl- (12) in a yield of 1.74 mCi (3.1% from 5a) with a specific activity
[3-14C]-3-(2-naphthyl)-D-alanine (8).
In a similar way, hydrolysis of the imine 5b, Fmoc-protection
of 58 mCi/mmol.
In a later stage, Org 37462 was developed starting with a
of the amino group of 6b, purification by column chromato- ‘base-sensitive’ resin, which can be split off using a mixture of
graphy and cleavage of the tert-butyl ester 9, gave the N-Fmoc- ammonia (10%) in methanol. Since the synthesis of [14C3]-Org
[1,2,3-14C3]-3-(2-naphthyl)-D-alanine (10).
The N-protected amino acids 8 and 10 were used without nonapeptide moiety 13, obtained from the production depart-
37462 (14) was performed in a later stage of development,
further purification in the coupling reaction with the peptide– ment, was used as the starting material (Scheme 3). This peptide
was coupled to N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine
resin moiety.
In the early stage, an ‘acid-sensitive’ resin was used to build (10) under the same conditions as mentioned above. After
up the decapeptide, Org 37462, in nine steps. From this route 24 hr the resin was split off upon treatment with a solution of
nonapeptide moiety 11 was built up and coupled to N-acetyl- 10% ammonia in methanol, the protecting groups were
[3-14C]-3-(2-naphthyl)-D-alanine (8) in the presence of removed and the crude peptide was purified by reverse-phase
1-hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N0,N0- HPLC, yielding 7.2 mCi (2.9% from 5b) [14C3]-Org 37462 (14) with
tetramethyluronium tetrafluoroborate (TBTU) and N-methylmor- a specific activity of 161 mCi/mmol.
pholine (Scheme 2). After 24 hr the protected peptide was In the coupling reaction the isolated yield of 12 (5.0%) and 14
separated from the resin upon treatment with a mixture of (4.0%), respectively, was comparable. Whereas the yield in the
trifluoroacetic acid, anisole and 1,2-ethanedithiol. Purification unlabelled procedure was significantly higher (450%). The
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P. H. G. Wiegerinck et al.
Figure 2. HPLC chromatogram Org 37462, Org 14734 and Org 14735 (HPLC: Symmetry C18 (5 mm 250 ꢀ 4.6 mm) with a gradient of water/acetonitrile/trifluoroacetic acid
(90/10/0.1–10/90/0.1, V/V/V), 1 ml/min, 220 nm).
Scheme 2
lower yield can be ascribed to the fact that in both labelled [14C]-methylnaphthalene were purchased from PerkinElmer Life
coupling reactions two to three equivalents of the labelled and Analytical Sciences. Resin-bound peptides 11 and 13 were
amino acid were added to the resin, whereas in the unlabelled supplied by Organon, the Department of Development Chem-
procedure 10 equivalents of the amino acid were added and not istry Riom. The remaining solvents and reagents were purchased
to the difference in N-protection group.
from Sigma-Aldrich or Acros Organics.
[14C]-Org 37462 (12) was further processed and formulated
and successfully applied in a human ADME study. [14C3]-Org
37462 (14) was successfully used in ADME studies, which
required a higher specific activity.
Instrumentation and equipment
The HPLC purification and purity controls were performed on a
Waters 510 HPLC system equipped with an Applied Biosystems
785A UV-detector and a Lablogic b-RAM radioactivity detector
and on a Waters 600 HPLC system equipped with an Applied
Biosystems 757 UV-detector and a Lablogic b-RAM radioactivity
detector Thin layer chromatography (TLC) plates, Merck silica gel
60 F-254, were scanned with a TLC scanner from Raytest, type
RITA 92 and type RITA Star.
Experimental
Materials
2-Bromo-[14C]-methylnaphthalene was purchased from BlyChem
Ltd. [14C2]-Glycine tert-butyl ester hydrochloride and 2-bromo-
J. Label Compd. Radiopharm 2008, 51 195–201
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P. H. G. Wiegerinck et al.
Scheme 3
Radioactivity was counted with a Packard Tri-CARB 2200 CA/4 by column chromatography (silica gel, heptane/ethyl acetate:
liquid scintillation analyzer, using Ultima Gold mv6013159 75/25, v/v), yielding tert-butyl-N-(1R)-bornylideneglycinate 4a in
scintillation fluid.
1H NMR spectra were recorded in CD3OD on a Bruker DRX400 2H), d2.3 (dt, Jd = 18 Hz, Jt = 4.0 Hz, 1H), d1.95 (t, J = 4.0 Hz, 1H),
spectrometer and on a Bruker AVANCE 600 spectrometer. d1.87 (m, 1H), d1.83 (t, J = 18 Hz, 1H), d1.67 (m, 1H), d1.47 (s, 9H),
78% yield (730 mg, 2.8 mmol). 1H NMR, CDCl3, d4.00 (q, J = 16 Hz,
Mass spectra (ESI) were recorded on a PerkinElmer Sciex d1.44 (m, 1H), d1.22 (m, 1H), d1.02 (s, 3H), d0.94 (s, 3H), d0.80
API 100 spectrometer and on a Thermo Fisher Scientific LTQ (s, 3H). MS (ESI) m/z 266 (M11).
Orbitrap spectrometer.
tert-Butyl N-[(1R)-bornylidene]-[1,2-14C2]-glycinate (4b)
Synthesis of [14C]-Org 37462 (12) and [14C3]-
Org 37462 (14)
Thiocamphor 3 (1.08 g, 6.4 mmol) was dissolved in toluene
(10 ml). Subsequently, [14C2]-glycine tert-butyl ester hydrochlor-
Thiocamphor (3)
ide (300 mCi) and triethylamine (0.65 ml, 0.65 mmol) were
added. The formed orange suspension was slowly heated to
1201C and stirred for 5 hr. The reaction mixture was then cooled
to room temperature and concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel,
heptane/ethyl acetate: 8/2, v/v), giving the tert-butyl-N-(1R)-
bornylidene-[1,2-14C2]-glycinate 4b (225 mCi, 75% yield). Radio-
chemical purity 98% (TLC: silica gel, toluene/ethanol 9/1, v/v and
heptane/ethyl acetate 8/2, v/v).
(1R)-(1)-Camphor (2) (5.05 g, 33.2 mmol) was dissolved in
ethylene glycol dimethyl ether (50 ml) to give a colorless
solution.
A mixture of phosphorous pentasulfide (14 g,
63.0 mmol) and sodium hydrogen carbonate (14 g, 167 mmol)
was prepared. Part of this mixture (3 g) was added to the
solution mentioned above, which became
a
greenish
suspension. After stirring at room temperature for 30 min,
this suspension was slowly heated to 901C. At 601C gas
formation was visible. The remaining amount of the mixture
(25 g), mentioned above, was added in small portions
within 30 min at 901C. The suspension was stirred for an
additional 60 min at 901C, and subsequently cooled to room
temperature. The reaction mixture, an orange solution contain-
ing a sticky precipitation, was poured into ice–water (200 ml),
leading to the formation of an orange suspension.
The suspension was stirred for 30 min and the formed crystals
were filtered off. The crystals were dissolved in toluene
(50 ml) and evaporated to dryness. This was repeated twice to
remove residual water. In this way, thiocamphor 3 (3.7 g,
22.2 mmol) was isolated in 67% yield, which was used as such
in the next step.
tert-Butyl N-[(1R)-bornylidene]-[3-14C]-3-(2-naphthyl)-D-
alanate (5a)
Diisopropylamine (125 ml) was dissolved in dry tetrahydrofuran
(2.5 ml) at 01C under nitrogen atmosphere. n-Butyllithium
(560 ml, 1.6 M in hexane) was added and the mixture was
cooled to ꢁ781C. A solution of tert-butyl N-[(1R)-bornylidene]-
glycinate (4a) (212 mg, 0.80 mmol) in tetrahydrofuran (1 ml) and
hexamethylphosphoramide (155 ml) was added and the mixture
was stirred for 15 min at ꢁ781C. Next a solution of 2-bromo-
[14C]-methylnaphtalene (45 mCi) in tetrahydrofuran (1 ml) was
added and the reaction mixture was stirred for 3 hr at ꢁ781C.
The mixture was quenched with water (1 ml) and allowed to
warm up to room temperature. Brine (1 ml) was added and the
mixture was extracted three times with diethyl ether. The
combined organic layers were dried over sodium sulfate and
tert-Butyl N-[(1R)-bornylidene]-glycinate (4a)
tert-Butyl glycinate (470 mg, 3.6 mmol) was dissolved in toluene concentrated under reduced pressure to give the crude tert-
(10 ml). Thiocamphor 3 (600 mg, 3.6 mmol) was added. The butyl N-[(1R)-bornylidene]-[3-14C]-(2-naphthyl)-D-alanate (5a)
reaction mixture was heated to reflux temperature and stirred (34 mCi). In a similar experiment, starting from tert-butyl N-
for 16 hr. An additional amount of tert-butyl glycinate (280 mg, [(1R)-bornylidene]-glycinate (4a) (240 mg, 0.91 mmol) and 2-
2.1 mmol) was added and the mixture was again stirred for 16 hr bromo-[14C]-methylnaphtalene (55 mCi), a second amount of
at reflux temperature. The reaction mixture was cooled and crude tert-butyl N-[(1R)-bornylidene]-[3-14C]-(2-naphthyl)-D-ala-
concentrated under reduced pressure. The residue was purified nate (5a) (54 mCi) was obtained. The two portions were
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P. H. G. Wiegerinck et al.
combined and purified by column chromatography (silica gel, methanol 95/5 (v/v)10.1% triethylamine). This compound was
toluene/ethyl acetate 95/5, v/v), yielding tert-butyl N-[(1R)- used directly in the following step.
bornylidene]-[3-14C]-(2-naphthyl)-D-alanate (5a) (57 mCi, 57%
yield). Radiochemical purity 97% (TLC: silica gel, toluene/ethyl tert-Butyl N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine (7)
acetate 95/5, v/v).
tert-Butyl [3-14C]-3-(2-naphthyl)-D-alanate (6a) (46 mCi) was dis-
solved in acetic acid anhydride (0.5 ml). After the addition of
pyridine (2.5 ml), the reaction mixture was stirred for 16 hr at room
temperature. The reaction mixture was concentrated under
reduced pressure. The residue was purified by column chromato-
tert-Butyl N-[(1R)-bornylidene]-[1,2,3-14C3]-3-(2-naphthyl)-
D-alanate (5b)
tert-butyl-N-(1R)-bornylidene-[1,2-14C]-glycinate 4b (225 mCi)
was dissolved in tetrahydrofuran (1 ml), under nitrogen
atmosphere, and cooled to –781C. Lithium diisopropylamide
(1.19 g, 3.00 mmol) and hexamethylphosphoramide (0.5 ml)
were added. The reaction mixture was stirred for 10 min at
–781C. Next a solution of 2-bromo-[14C]-methylnaphtalene
(100 mCi) in tetrahydrofuran (1 ml) was added. The reaction
mixture was stirred for 3.5 hr at ꢁ781C. The mixture was
quenched with water (2.5 ml) and allowed to warm up to
room temperature. Brine (2.5 ml) was added and the mixture
was extracted three times with methyl tert-butyl ether.
The combined organic layers were dried over sodium
sulfate and concentrated under reduced pressure to give
graphy (silica gel, dichloromethane/acetone 9/1, v/v) to give tert-
Butyl N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine) (7) (39 mCi, 85%
yield). Radiochemical purity 96% (TLC: silica gel, dichloromethane/
acetone 9/1, v/v and heptane/ethyl acetate 6/4, v/v).
N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine (8)
tert-Butyl N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine) (7) (39 mCi)
was dissolved in a mixture of trifluoroacetic acid (9 ml) and
dichloromethane (1 ml). The reaction mixture was stirred for 3 hr
at room temperature. The reaction mixture was concentrated
under reduced pressure. The residue was dissolved in dichlor-
omethane and again concentrated under reduced pressure to
yield the crude N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine) (8).
This compound was used directly in the following step.
the
crude
tert-butyl
N-[(1R)-bornylidene]-[1,2,3-14C3]-
(2-naphthyl)-D-alanate (309 mCi). Purification by column
chromatography (silica gel, toluene/ethyl acetate 9/1, v/v)
resulted in tert-butyl N-[(1R)-bornylidene]-[1,2,3-14C3]-(2-
naphthyl)-D-alanate (5b) (260 mCi, 80% yield). Radiochemical
purity 98% (TLC: silica gel, toluene/ethanol 9/1, v/v and
heptane/ethyl acetate 8/2, v/v).
tert-Butyl N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine (9)
tert-Butyl [1,2,3-14C]-3-(2-naphthyl)-D-alanate (6b) (213mCi) was
dissolved in tetrahydrofuran (3.5 ml) and an aqueous solution of
sodium carbonate (0.315 g in 3.5 ml) was added. The two-layer
suspension was cooled to 01C under thorough stirring. Next a
suspension of N-(9-fluorenylmethoxycarbonyloxy)succinimide
(Fmoc-OSu) (511 mg, 1.5 mmol) in tetrahydrofuran (2 ml) was
added. The reaction mixture was allowed to warm up to room
temperature and stirred for 15 min. The organic layer was
separated from the water layer. The latter was extracted twice
with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by column chromatography
(silica gel, toluene/ethyl acetate 9/1, v/v) obtaining tert-butyl N-
Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine) (9) (188 mCi, 88% yield).
tert-Butyl [3-14C]-3-(2-naphthyl)-D-alanate (6a)
Citric acid (5 ml of a 15% aqueous solution) was added to a
solution of tert-butyl N-[(1R)-bornylidene]-[3-14C]-(2-naphthyl)-D-
alanate (5a) (57 mCi) in tetrahydrofuran (10 ml) and the reaction
mixture was stirred for 4 hr at 801C. The mixture was cooled to
room temperature diluted with water (5 ml) and extracted once
with methyl tert-butyl ether. Subsequently, the water layer was
adjusted to pH 8 with a saturated aqueous solution of sodium
carbonate and extracted twice with ethyl acetate. The combined
organic layers were dried over sodium sulfate and concentrated
under reduced pressure. The residue was purified by column
chromatography (silica gel, dichloromethane/methanol/triethyl
amine 80/20/1, v/v/v) to give tert-butyl [3-14C]-3-(2-naphthyl)-D-
alanate (6a) (46 mCi, 81% yield). Radiochemical purity 97% (TLC:
silica gel, toluene/ethanol 85/15, v/v and dichloromethane/
methanol 95/5, v/v).
N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine (10)
tert-Butyl
N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine
(9)
(188 mCi) was dissolved in a mixture of dichloromethane
(1.5 ml) and trifluoroacetic acid (10 ml). The reaction mixture
was stirred for 2 hr at room temperature. The reaction mixture
was concentrated under reduced pressure. The residue was
dissolved in dichloromethane (1 ml) and again concentrated
under reduced pressure, this procedure was repeated twice.
The residue, N-Fmoc-[1,2,3-14C3]-3-(2-naphthyl)-D-alanine)
(10), was used directly in the following reaction step.
tert-Butyl [1,2,3-14C3]-3-(2-naphthyl)-D-alanate (6b)
Citric acid (45ml of a 15% aqueous solution) was added to a
solution of tert-butyl N-[(1R)-bornylidene]-[1,2,3-14C3]-(2-naphthyl)-
D-alanate (5b) (260 mCi) in tetrahydrofuran (20 ml) and the
reaction mixture was stirred for 3.5 hr at 801C. The mixture was
cooled to room temperature. The tetrahydrofuran was removed
via evaporation under reduced pressure. The residual water was
extracted twice with methyl tert-butyl ether. Subsequently, the
water layer was brought to pH 8 with a saturated aqueous
solution of sodium carbonate and extracted twice with ethyl
acetate. The combined organic layers were dried over sodium To
N-Acetyl-[14C]-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylala-
nyl-3-(3-pyridyl)-D-alanyl-L- seryl-L-tyrosyl-N9,N10-diethyl-D-
homoarginyl-L-leucyl-N9,N10-diethyl-L-homoarginyl-L-prolyl-
D-alanylamide acetate, [14C]-Org 37462 (12)
a
solution of N-acetyl-[3-14C]-3-(2-naphthyl)-D-alanine (8)
sulfate and concentrated under reduced pressure, giving crude (39 mCi) in 1-methyl-2-pyrrolidinone (NMP, 18 ml), subsequently
tert-butyl [1,2,3-14C3]-3-(2-naphthyl)-D-alanate (6b) (213 mCi, 82% 1-hydroxybenzotriazole hydrate (HOBt, 180mg, 1.33 mmol) and O-
yield). Radiochemical purity 98% (TLC: silica gel, dichloromethane/ (benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate
J. Label Compd. Radiopharm 2008, 51 195–201
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P. H. G. Wiegerinck et al.
(TBTU, 360 mg, 1.12 mmol) were added. The resulting mixture was
Next the resin was suspended in NMP (50 ml) and piperidine
added to the ‘acid-sensitive’ resin 11 (1.7 g, 0.22 mmol/g) and (10 ml) was added. After 10 min stirring this reaction mixture was
thoroughly shaken manually. After 10min, N-methylmorpholine filtered. This procedure was repeated twice. Next the resin was
(90 ml) was added to the reaction mixture. After 4 h, additional subsequently washed twice with a mixture of HOBt (20 ml) in
amounts of HOBt (150 mg, 1.11 mmol) and TBTU (150 mg, NMP (20 ml), once with ethanol (20 ml), twice with a mixture of
0.46 mmol) were added. After 16h, the solvent was removed HOBt (20 ml) in NMP (20 ml), once with ethanol (20 ml) and again
and the resin was washed, three times alternating with NMP twice with a mixture of HOBt (20 ml) in NMP (20 ml).
(10 ml containing 0.1% HOBt) and with ethanol (10 ml), once with
The deprotected resin was added to a solution of N-Fmoc-
ethanol (45 ml) and once with dichloromethane (30 ml). The resin [1,2,3-14C3]-3-(2-naphthyl)-D-alanine) (10) (188 mCi), HOBt
was dried under reduced pressure. The residue was suspended in (120 mg, 0.89 mmol) and TBTU (275 mg, 0.84 mmol) in NMP
a mixture of trifluoroacetic acid/anisole/1,2-ethanedithiol (15 ml, (10 ml). The reaction mixture was stirred for 2 min, before a
95/2.5/2.5, v/v/v). The mixture was set aside for 2hr and shaken mixture of N-methylmorpholine (130 ml) in NMP (660 ml) was
occasionally. Next the solvent was removed, the residue was added. The reaction mixture was stirred for 2 h, additional
washed with trifluoroacetic acid and the soluble phases were amounts of HOBt (70 mg, 0.52 mmol) and TBTU (152 mg,
lyophilized. The residue was dissolved in trifluoroacetic acid (6 ml) 0.46 mmol) and a mixture of N-methylmorpholine (40 ml) in
and precipitated with diethyl ether (15 ml) to give the crude [14C]- NMP (200 ml) was added. The reaction mixture was stirred at
Org 37462 (5.57 mCi). This crude product was purified by HPLC on room temperature. After 16 hr the mixture was filtered. The
Symmetry Prep C18 (7 mm, 19 ꢀ 150 mm) with a gradient of water/ residual resin was subsequently washed with a mixture of HOBt
acetonitrile/trifluoroacetic acid (90/10/0.1–10/90/0.1, v/v/v), 10ml/ (20 ml) in NMP (20 ml), with ethanol (10 ml), with a mixture of
min, 300 nm, yielding the trifluoroacetic acid salt of 12. This was HOBt (20 ml) in NMP (20 ml), with ethanol (10 ml) and with a
converted into the acetic acid salt by dissolving it in water (15 ml), mixture of HOBt (20 ml) in NMP (20 ml).
next an ion exchange resin (BioRad AG 1-X2, 1.0 g) was added and
Next the resin was suspended in NMP (50 ml) and piperidine
this suspension was stirred for 10min before being filtered. To the (10 ml) was added. After stirring this reaction mixture for 10 min,
filtrate a second amount of ion exchange resin (1.0 g) was added. it was filtered. This procedure was repeated twice. Next the resin
After stirring for 10min the suspension was filtered again. The was subsequently washed twice with a mixture of HOBt (10 ml) in
filtrate was lyophilized to give N-acetyl-[14C]-3-(2-naphthyl)-D- NMP (10 ml), once with ethanol (10 ml), twice with a mixture of
alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L- seryl-L-tyro- HOBt (10 ml) in NMP (10 ml), once with ethanol (10 ml) and again
syl-N9,N10-diethyl-D-homoarginyl-L-leucyl-N9,N10-diethyl-L-homoar- twice with a mixture of HOBt (10 ml) in NMP (10 ml). To the
ginyl-L-prolyl-D-alanylamide acetate, [14C]-Org 37462 (12) deprotected resin, a mixture of nitrophenyl acetate (160 mg,
(1.74 mCi, 5.0% yield) with a specific activity of 34 mCi/mg, 0.88 mmol) and HOBt (125 mg, 0.93 mmol) in NMP (10 ml) was
58 mCi/mmol. Radiochemical purity 95% (HPLC: Symmetry C18 added and the reaction mixture was stirred for 5 min. Next a
(5 mm 250 ꢀ 4.6 mm) with
a gradient of water/acetonitrile/ solution of N-methylmorpholine (40 ml) in NMP (200 ml) was
trifluoroacetic acid (90/10/0.1–10/90/0.1, v/v/v), 1 ml/min, UV added. This mixture (pH 6) was stirred for 90 min. After filtration,
220 nm and Supelcosil LC 18-DB (250 ꢀ 4.6mm) with an aqueous the residue was washed with dichloromethane (10 ml). This
0.5 M NaH2PO4 (pH 2.1)/acetonitrile gradient, 1 ml/min, UV acetylation step with nitrophenyl acetate was repeated. The
220 nm. 1H NMR spectrum is in agreement with the corresponding resin was dried on air for 64 hr.
spectrum of the unlabelled Org 37462: 1H NMR, CD3OD, d
Next a solution of 10% ammonia in methanol (v/v) (3.5 ml)
Naphthylalanine 1.83 (s, 3H), 2.92 (m, 1H), 3.14 (m, 1H), 4.68 (dd, was added to the resin. The reaction mixture was set aside for
1H), 7.26 (dd, 1H), 7.39–7.44 (m, 2H), 7.58 (s, 1H), 7.71 (m, 1H), 7.74 16 h, before being filtered. The residue was washed twice with
(m, 1H), 7.78 (m, 1H). p-Cl-Phenylalanine, 2.86 (dd, 1H), 3.18 (dd, methanol (5 ml). The filtrate was dissolved in a solution of
1H), 4.59 (dd, 1H), 7.15 (d, 2H), 7.19 (d, 2H). Pyridylalanine, 3.05 (m, trifluoroacetic acid (3.5 ml), triisopropylsilane (100 ml) and water
1H), 3.14 (m, 1H), 4.48 (t, 1H), 7.35 (dd, 1H), 7.72 (m, 1H), 8.39 (m, (100 ml). This mixture was stirred at room temperature for 1 hr.
1H), 8.40 (s, 1H). Serine, 3.52 (dd, 1H), 3.73 (dd, 1H), 4.27 (m, 1H). Next acetonitrile (15 ml) was added and the mixture was
Tyrosine, 3.01 (m, 1H), 3.07 (m, 1H), 4.38 (dd, 1H), 6.65 (d, 2H), 7.01 concentrated under reduced pressure. This was repeated twice,
(d, 2H). Homo-arginine, 1.19 (t, 6H), 1.19–1.29 (m, 2H), 1.48–1.58 yielding the crude [14C3]-Org 37462 (29.1 mCi). The crude
(m, 2H), 1.68 (m, 1H), 1.79 (m, 1H), 3.12–3.18 (m, 2H), 3.24 (q, 4H), product was purified twice by HPLC on Symmetry Prep C18
4.34 (t, 1H). Leucine, 0.85 (d, 3H), 0.91 (d, 3H), 1.61 (m, 1H), (7 mm, 19 ꢀ 150 mm) with a gradient of water/acetonitrile/
1.64–1.65 (m, 2H), 4.50 (m, 1H). Homo-arginine, 1.20 (m, 1H), 1.30 trifluoroacetic acid (95/5/0.1–5/95/0.1, v/v/v), 10 ml/min, UV
(m, 1H), 1.35 (m, 1H), 1.42–1.50 (m, 2H), 1.67 (m, 1H), 3.08–3.16 (m, 280 nm, yielding the trifluoroacetic acid salt of 14. This was
2H), 4.28 (m, 1H). Proline, 1.88 (m, 2H), 2.05 (m, 1H), 2.15 (m, 1H), converted into the acetic acid salt by dissolving it in water
3.50 (m, 1H), 3.87 (m, 1H), 4.30 (m, 1H). Alanine, 1.35 (d, 3H), 4.22 (15 ml), next an ion exchange resin (BioRad AG 1-X2, 1.75 g) was
(q, 1H). Acetic acid, 1.90 (s, 6H). MS (ESI) unlabelled Org 27462 (1): added and this suspension was stirred for 10 min before being
m/z 1570; MS (ESI) [14C]-Org 37462 m/z 1572 (8% 14C0, 92% 14C1). filtered. To the filtrate a second amount of ion exchange resin
(1.75 g) was added. After stirring for 1.5 hr the suspension was
filtered again. The filtrate was lyophilized to give pure N-acetyl-
N-Acetyl-[1,2,3-14C3]-3-(2-naphthyl)-D-alanyl-4-chloro-D-
[1,2,3-14C3]-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-
phenylalanyl-3-(3-pyridyl)-D-alanyl-L- seryl-L-tyrosyl-N9,N10
diethyl-D-homoarginyl-L-leucyl-N9,N10-diethyl-L-homoargi-
nyl-L-prolyl-D-alanylamide acetate, [14C3]-Org 37462 (14)
-
pyridyl)-D-alanyl-L- seryl-L-tyrosyl-N9,N10-diethyl-D-homoarginyl-
L-leucyl-N9,N10-diethyl-L-homoarginyl-L-prolyl-D-alanylamide
acetate, [14C3]-Org 37462 (14) (7.6 mCi, 4.0% yield), with a
specific activity of 94.8 mCi/mg, 161 mCi/mmol. Radiochemical
purity 96% (HPLC: Symmetry C18 (5 mm 250 ꢀ 4.6 mm) with
a gradient of water/acetonitrile/trifluoroacetic acid (90/10/
‘Base-sensitive’ resin 13 (5.0 g, 0.079 mmol/g) was swollen in
dichloromethane (20 ml). After 1 hr the suspension was filtered
and the resin was washed with NMP (50 ml).
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 195–201

P. H. G. Wiegerinck et al.
0.1–10/90/0.1, v/v/v), 1 ml/min, 220 nm). 1H NMR- and the 13C (d, CH), 30.3 (t, CH2), 26.2 (t, CH2), 49.5 (t, CH2), 174.2 (s, C==O
NMR-spectra are in agreement with the corresponding spectra amide)). Alanine, 50.4 (d, CH), 17.7 (q, CH3), 177.7 (s, C==O
of the unlabelled Org 37462, with that respect that in the 13C (amide)). Acetic acid, 24.0 (q, CH3), 179.7 (s, C==O). MS (ESI)
NMR spectrum of the labelled Org 37462 the signals of unlabelled Org 27462 (1): m/z 1576; MS (ESI) [14C3]-Org 37462 m/
naphthylalanine, the carbonyl is 30%, the CH is 25% and the z 1576 (0.4% 14C0, 13.5% 14C1, 13.8% 14C2, 72.3% 14C3).
CH2 is 5% meaning that the carbonyl is 70%, the CH is 75% and
the CH2 is 95% 14C-labelled. 13C NMR, CD3OD: d Napthylalanine, Acknowledgements
22.6 (q, CH3 (C(O)CH3)), 173.3 (s, C==O (C(O)CH3)), 56.3 (d, CH
(14C)), 38.9 (t, CH2 (14C)), 135.9 (s, 1-naphthyl), 128.9 (d, 2-
The authors thank Christine Flouzat, Serge Wilmouth and
naphthyl), 134.8 (s, 3-naphthyl), 128.6 (d, 4-naphthyl), 126.7 (d, 5-
naphthyl), 127.1 (d, 6-naphthyl), 128.8 (d, 7-naphthyl), 133.8 (s, 8-
naphthyl), 129.0 (d, 9-naphthyl), 128.5 (d, 10-naphthyl), 174.0 (s,
C==O (14C, amide)). P-Cl-Phenylalanine, 55.9 (d, CH), 37.8 (t, CH2),
137.5 (s, 1-pCl-phenyl), 132.1 (d, 216-pCl-phenyl), 129.5 (d, 315-
pCl-phenyl), 133.5 (s, 4-pCl-phenyl), 173.5 (s, C==O (amide)).
Pyridylalanine, 56.5 (d, CH), 35.2 (t, CH2), 134.9 (s, 1-pyridyl),
150.9 (d, 2-pyridyl), 148.5 (d, 3-pyridyl), 125.2 (d, 4-pyridyl), 139.2
(d, 5-pyridyl), 173.3 (s, C==O (amide)). Serine, 57.6 (d, CH), 62.5 (t,
CH2), 172.7 (s, C==O (amide)). Tyrosine, 57.7 (d, CH), 37.3 (t, CH2),
129.0 (s, 1-phenol), 131.2 (d, 216-phenol), 116.4 (d, 315-
phenol), 157.3 (d, 4-phenol), 174.0 (s, C==O (amide)). Homo-
arginine, 52.3 (d, CH), 32.2 (t, CH2), 24.2 (t, CH2), 29.3 (t, CH2), 42.4
(t, CH2), 155.5 (s, C==N), 37.5 (t, CH2), 14.6 (t, CH3), 172.5 (s, C==O
(amide)). Leucine, 52.8 (d, CH), 41.9 (t, CH2), 25.9 (d, CH), 22.0
(q, CH3), 174.1 (C==O (amide)). Homo-arginine, 54.6 (d, CH), 32.2
(t, CH2), 23.3 (t, CH2), 29.7 (t, CH2), 42.5 (t, CH2), 155.5 (s, C==N),
37.5 (t, CH2), 14.6 (t, CH3), 174.4 (s, C==O (amide)). Proline, 61.9
Christiane Ray from the DDCR of Organon in Riom (France) for
their support and for the supply of the resin-bound peptide
moieties and Frans Kaspersen, R&D Fellow at Organon, for the
fruitful discussions.
References
[1] J. Itskov-Eldor, S. Kol, B. Mannaerts, H. Coelingh Bennink, Hum.
Reprod. 1998, 13, 294–295.
[2] P. Devroey, K. Abyholm, K. De Jong, T. Hillensjo, B. Hedon,
J. Itskovitz-Eldor, J. Kahn, O. Naether, F. Olivennes, S. Pavlou,
B. Tarlatzis, L. Westergaard, B. Mannaerts, B. Van der Heijden,
H. Coelingh Bennink, Hum. Reprod. 1998, 13, 3023–3031.
[3] H. J. Out, B. M. J. L. Mannaerts, Hum. Fertil. 2002, 5, G5–G12.
[4] J. M. McIntosh, P. Mishra, Can. J. Chem. 1985, 64, 726–731.
[5] J. M. McIntosh, R. K. Leavitt, Tetrahedron Lett. 1986, 27, 3839–3842.
[6] B. S. Pedersen, S. Scheibe, N. H. Nilsson, S. O. Lawesson, Bull. Soc.
Chim. Belg. 1978, 87, 223–228.
[7] S. Hoarau, J. L. Fauchere, L. Pappalardo, M. L. Roumestant, P.
Viallefont, Tetrahedron Asymmetry 1996, 7, 2585–2593.
J. Label Compd. Radiopharm 2008, 51 195–201
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org