Practical (14C)-synthesis of molecules containing an acetic acid moiety: Application to (14C)-labeled DP1 antagonists

Article abstract of DOI:10.1002/jlcr.1146

Efficient carbon-14 labeling of four potent and selective DP1 antagonists is reported. The synthetic sequence began with α-hydroxylation, reduction of an ester, followed by oxidative diol cleavage and aldehyde reduction. The resulting alcohol 4 was conver

Full text of DOI:10.1002/jlcr.1146

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 1–5.
JLCR
Published online 7 December 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1146
Research Article
Practical [14C]-synthesis of molecules containing an acetic
acid moiety: application to [14C]-labeled DP1 antagonists
CARL BERTHELETTE* and ZHAOYIN WANG
´
Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire/Dorval, Quebec, Canada H9R 4P8
Received 19 September 2006; Accepted 29 September 2006
Abstract: Efficient carbon-14 labeling of four potent and selective DP1 antagonists is reported. The synthetic
sequence began with a-hydroxylation, reduction of an ester, followed by oxidative diol cleavage and aldehyde
reduction. The resulting alcohol 4 was converted to a mesylate then nucleophilic substitution with [14C]-sodium
cyanide was performed to yield a nitrile, which upon basic hydrolysis provided the carbon-14 labeled acid 1.
Compound 2 was obtained from the same alcohol intermediate 4 and two diastereomeric compounds 6 and 7 were
easily prepared from compound 2. Carbon-14 synthesis of compounds 1, 2, 6 and 7 were achieved in good
yields, high radiochemical purity (>99%) and with high specific activity (45 mCi/mmol). Copyright # 2006 John
Wiley & Sons, Ltd.
Keywords: [14C]-synthesis; labeled compounds; tetrahydrocyclopenta[b]indol-3-yl acetic acid; DP1 antagonists
Introduction
tetrahydrocyclopenta[b]indol-3-yl acetic acid, and we
wish to report herein a novel, short and efficient
radiosynthesis for such compounds.
The importance of labeled compounds has increased
dramatically over recent years. It is worth mentioning
that every preclinical candidate will have received
special attention regarding its own radiosynthesis. In
this regard, the specific site of incorporation of the
isotope in a molecule is crucial in order to quantita-
tively trace the parent molecule as well as possible
metabolites. Despite the fact that tritium labeled
compounds are easier to synthesize and provide higher
specific activity, carbon-14 labeled compounds are the
most frequently used in drug pharmacokinetics and
metabolic studies. The development of short and
efficient procedures for the incorporation of carbon-14
in a metabolically stable position is therefore desirable.
The introduction of the marker should be done
preferably at the latest stage in the synthesis. During
the course of our work, compound 1, 2, 6, 7 proved to
be potent prostaglandin D1 (DP1) receptor antagonists
with Ki values of 1.5, 1.1, 0.9 and 1.0 nM, respectively.1
Toward this end, we became interested in developing
synthetic routes to prepare carbon-14 labeled 1,2,3,4-
Results and discussion
The procedure described herein involves the removal of
one carbon atom, located at the acetic acid moiety end,
and its replacement with a carbon-14 atom originating
from radiolabeled sodium cyanide. To this end, synth-
esis of the desired precursor 4 for the introduction of
the carbon-14 labeled has been accomplished in few
steps and in good yield (Scheme 1). First, the acid 1 is
converted to a methyl ester using diazomethane in
quantitative yield. An a-hydroxylation is then carried
out using KHMDS as a base and Davis reagent2 as the
oxygen source followed by the reduction of the ester
moiety with DIBAL in the same reaction flask to afford
diol 3 in 70% yield. The next reaction involved an
oxidative diol cleavage using sodium periodate to afford
the corresponding terminal aldehyde which was then
immediately reduced to alcohol 4 using DIBAL in 69%
yield. No epimerization at the C3 center has been
observed during this transformation as confirmed by
chiral HPLC analysis. This intermediate was used for
the synthesis of our desired radiolabeled compounds.
*Correspondence to: Carl Berthelette, Department of Medicinal Chem-
istry, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005,
Pointe-Claire/Dorval, Que´bec, Canada, H9R 4P8.
E-mail: carl berthelette@merck.com
Copyright # 2006 John Wiley & Sons, Ltd.

2
C. BERTHELETTE AND Z. WANG
Scheme 1
At this point, the synthesis of radiolabeled compound
The synthesis of compound 6 and 7 was straightfor-
ward since only a ketone reduction and an alcohol
methylation were required in order to access both
diastereomers (Scheme 2). Carbon-14 labeled 2 was
treated first with diazomethane, and then the ketone
moiety was reduced with sodium borohydride in
methanol to cleanly provide a mixture of the corre-
sponding alcohols (1.5:1), which were directly methy-
lated using sodium hydride and methyl iodide. The
ester was hydrolyzed to the acid and finally a separa-
tion was necessary to unveil the diastereomeric carbon-
14 labeled 6 and 7. All radiolabeled compounds 1, 2, 6,
7 were obtained in high radiochemical purity (>99%)
and with a specific activity of 45 mCi/mmol as deter-
mined by analytical HPLC and a radiometric detector.
Analysis of the enantiomeric purity at the acetic acid
side chain showed that no epimerization had taken
place during the synthesis, neither in the SN2 displace-
ment with [14C]-sodium cyanide nor in the hydrolysis
step.
1
was accomplished directly in three steps by
converting the alcohol 4 to a mesylate, followed by a
displacement of the mesylate with [14C]-sodium cya-
nide in DMSO at 608C to afford the corresponding
nitrile, which was directly hydrolyzed to the acid using
sodium hydroxide (5 N) in refluxing ethanol to afford
the carbon-14 labeled compound 1 in 70% overall
yield.
The synthesis of radiolabeled compound 2 started
from the same precursor 4 which was first protected as
an acetate under standard conditions in quantitative
yield. Secondly, a Stille (1986) coupling was used to
introduce the methyl ketone at the 5-position of the
core structure and finally the acetate was removed to
obtain compound 5 in 67% overall yield for the three
steps. Identical procedures as described above (mesy-
late formation, SN2 displacement with [14C]-sodium
cyanide, hydrolysis) proceeded smoothly and provided
carbon-14 labeled compound 2 in 75% yield.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1–5
DOI: 10.1002.jlcr

[
14C]-SYNTHESIS OF MOLECULES CONTAINING AN ACETIC ACID MOIETY
3
Scheme 2
Conclusion
1-[(3R)-5-bromo-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl]ethane-1,2-diol (3)
In summary, we have developed an efficient synthetic
pathway for the synthesis of carbon-14 labeled 1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl acetic acids 1, 2, 6
and 7. This mild procedure can be utilized to introduce
a carbon-14 label at the acid position of a molecule
possessing an acetic acid moiety. The carbon-14
marker is introduced at a late stage and only two steps
were required in order to successfully synthesize
compounds 1 and 2 in good yields with high radio-
chemical purity (>99%) and high specific activity
Compound 1 (1.35 g, 3 mmol) was treated with diazo-
methane (1M/Et2O) then evaporated to dryness. The
resulting methyl ester was dissolved in 10 ml of THF
and was slowly added to a solution of KHMDS (7.8 ml,
3.9 mmol, 0.5M/tol) in 30 ml of THF at ꢁ788C under an
inert atmosphere of nitrogen. The reaction mixture was
stirred for 15 min then Davis reagent (1.57 g, 6 mmol)
was added and stirred for 1h. DIBAL (15 ml, 15 mmol,
1M/Hex) was introduced and the reaction mixture was
placed at ꢁ408C for 1 h. Quenching of the reaction was
done by adding 50 ml of HCl 10%, 100 ml of EtOAc and
stirred for 30 min. The reaction was extracted twice
with 100 ml of EtOAc, washed with 50 ml of Rochelle’s
salt, brine, dried over Na2SO4 and evaporated. The
compound 3 was obtained in 70% yield and used
directly for the next step.
(45 mCi/mmol) from
[
14C]-sodium cyanide. Com-
pounds 6 and 7 were easily obtained from carbon-14
labeled compound 2 using standard chemistry.
Experimental
General
All reagents and solvents are commercially available
and used directly without any purification. Davis
reagent can be easily obtained according to literature
procedure.3,y Reactions were performed in a well-
ventilated fume-hood. Evaporations were carried out
on a Buchi evaporator in vacuo at bath temperatures
less than 358C. TLC was performed on Merck kGaA
F254 precoated silica plates. Analytical HPLC was
performed on a Waters instrument equipped with a
Zorbax C18 column (4.6 ꢀ 150 mm) and peak detection
was done simultaneously by UV (254 nm, Waters 996
Photodiode Array detector) and a liquid scintillation
flow monitor. Radioactivity measurements were per-
[(3R)-5-bromo-4-(4-chlorobenzyl)-7-fluoro-1,2,3,
4-tetrahydrocyclopenta[b]indol-3-yl]methanol (4)
Compound 3 (910 mg, 2.1 mmol) was dissolved in 20 ml
of a mixture of THF/H2O (3:1) at room temperature and
sodium periodate (890 mg, 4.2 mmol) was slowly
added. After 15 min, the reaction mixture was
quenched with 20 ml of H2O, extracted twice with
50 ml Et2O, washed with brine, dried over Na2SO4
and evaporated. The crude was redissolved in 10 ml of a
mixture of THF/toluene (1:1) and cooled to ꢁ788C prior
to the addition of DIBAL (4.2 ml, 4.2 mmol, 1M/Hex).
The mixture was stirred at ꢁ408C for 1 h. Quenching of
the reaction was done by adding 30 ml of HCl 10%,
50 ml of EtOAc and stirred for 10 min. The reaction was
extracted twice with 50 ml of EtOAc, washed with 50 ml
of Rochelle’s salt, brine, dried over Na2SO4 and
evaporated. Purification by flash chromatography over
silica gel using 40% EtOAc/hexanes afforded the
desired compound 4 in 72% yield as a colorless oil.
1H NMR (acetone-d6): d 7.29 (d, J=8.3 Hz, 2H), 7.17 (d,
J=8.9 Hz, 1H), 7.06 (d, J=8.9 Hz, 1H), 6.90 (d, J=8.2 Hz,
formed on
a Beckman Coulter LS6000SC liquid
scintillation counter using Ultima Gold XR. A Bruker
500 MHz instrument was used for NMR characteriza-
tion and chemical shifts are reported in ppm downfield
from TMS.
y The oxaziridine (Davis reagent) is readily prepared by
literature procedures from benzaldehyde via an N-sulfonyli-
mine.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1–5
DOI: 10.1002.jlcr

4
C. BERTHELETTE AND Z. WANG
2H), 5.98 (d, JAB=17.8 Hz, 1H), 5.82 (d, JAB=17.8 Hz,
1H), 4.02 (bs, 1H), 3.69–3.65 (m, 2H), 3.31–3.28 (m,
1H), 2.89–2.83 (m, 1H), 2.79–2.74 (m, 1H), 2.67–2.63
(m, 1H), 2.37–2.33 (m, 1H).
1-[(3S)-4-(4-chlorobenzyl)-7-fluoro-3-(hydroxy-
methyl)-1,2,3,4-tetrahydrocyclo-penta[b]indol-5-
yl]ethanone (5)
Step 1: The common intermediate
4
(170 mg,
[(3R)-5-bromo-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl]acetic acid (1)
0.41 mmol) was dissolved in 5 ml of CH2Cl2 at 08C
under an inert atmosphere of nitrogen. Triethylamine
(115 ml, 0.82 mmol) and DMAP (1 mg, cat.) were added
followed by acetyl chloride (31 ml, 0.42 mmol). The
reaction was stirred for 30 min then the cooling bath
was removed for another 30 min of stirring. The
reaction mixture was quenched by adding 5 ml of a
saturated solution of NH4Cl, extracted three times with
10 ml of CH2Cl2, washed with brine, dried over Na2SO4
and evaporated. The desired acetate was obtained as a
colorless oil in quantitative yield and was used directly
for the next step.
Step 1: Mesylate formation. Compound
4 (45 mg,
0.11 mmol) was dissolved in 2 ml of CH2Cl2 at ꢁ308C
under an inert atmosphere of nitrogen. Triethylamine
(25 ml, 0.18 mmol) was added followed by methanesul-
fonyl chloride (11 ml, 0.14 mmol) and the reaction was
stirred for 30 min. The reaction was allowed to warm-
up to room temperature and stirred for 30 min.
Quenching of the reaction was performed using 1 ml
of a saturated solution of NaHCO3, extraction with 5 ml
of CH2Cl2 twice, washing with brine, drying over
Na2SO4 and evaporation. The crude mesylate was
directly used for the next step.
Step 2: To
a solution of the acetate (180 mg,
0.4 mmol) in 3 ml of DMF under an inert atmosphere
of nitrogen was added (1-ethoxyvinyl)tributylstannane
(270 ml, 0.8 mmol). In a separate flask, triphenylarsine
(50 mg, 0.16 mmol) and Pd2(dba)3 (36 mg, 0.04 mmol)
were mixed in 2 ml of DMF and sonicated for 1 min.
This solution was then added to the previous solution
containing the acetate, purged three times with va-
cuum/N2 then heated to 908C for 18 h.
Step 3: The reaction mixture was cooled to 358C prior
to the addition of 5 ml of HCl 1 N and stirring was
continued for 15 min. Filtration through a pad of celite
removed palladium residues and two washes with
25 ml of EtOAc were necessary. The crude reaction
mixture was extracted with three portions of 25 ml of
EtOAc, washed with brine, dried over Na2SO4 and
evaporated. Purification by flash chromatography
using silica gel and 5% EtOAc/Hexanes afforded the
methyl ketone in 67% yield.
Step 2: Cyanide displacement. The mesylate (40 mg,
0.08 mmol) was dissolved in 1 ml of DMSO at room
temperature before addition of [14C]-sodium cyanide
(3.5 mg, 0.07 mmol). The reaction mixture was heated
to 608C and stirred for 18 h. The crude reaction mixture
was cooled to room temperature and directly purified
on silica gel using 20% EtOAc/hexanes to afford the
desired nitrile as a white solid. The compound was
obtained in quantitative yield and used directly for the
next step.
Step 3: Hydrolysis. To a solution of the nitrile (17 mg,
0.04 mmol) in 1.2 ml of EtOH at room temperature was
added sodium hydroxide 5 N (600 ml). The reaction was
heated to reflux and stirred for 18 h. Quenching of the
reaction was done using HCl 3 N until acidic by pH
paper, dilution with 2 ml of H2O, extracted twice with
5 ml EtOAc, washed with brine, dried over Na2SO4 and
evaporated. Purification by prep HPLC using a Novapak
C18 (7.8 ꢀ 300 mm) using 65% CH3CN/H2O + 0.1%
AcOH at 3.5 ml/min at 270 nm afforded the desired
Step 4: Finally, the methyl ketone (110 mg,
0.26 mmol) was dissolved in 4 ml of a mixture of THF/
MeOH (1:1) at room temperature. A 2 N solution of
NaOH (0.5 mL, 1 mmol) was added and the mixture was
heated to reflux for 15 min. The reaction was cooled to
room temperature and neutralized with 1 ml of HCl 1 N,
extracted twice with 10 ml of EtOAc, washed with brine,
dried over Na2SO4 and evaporated. Purification by
flash chromatography using silica gel and a gradient
of 0–30% EtOAc/Hexanes afforded the alcohol 5 in
quantitative yield.
[
14C]-acid 1 as a white solid in 70% overall yield for the
three steps. Chemical and radiochemical purity were
greater than 99% and the specific activity calculated to
be 45 mCi/mmol. 1H NMR (acetone-d6): d 10.69 (br s,
1H), 7.33 (d, J=8.3 Hz, 2H), 7.20 (dd, J=8.9 and 2.1 Hz,
1H), 7.09 (dd, J=9.0 and 2.2 Hz, 1H), 6.94 (d, J=8.2 Hz,
2H), 5.92 (d, JAB=17.8 Hz, 1H), 5.77 (d, JAB=17.8 Hz,
1H), 3.65–3.62 (m, 1H), 2.95–2.65 (m, 4H), 2.42 (dd,
J=16.1 and 10.1 Hz, 1H), 2.32–2.27 (m, 1H); 13C NMR
(acetone-d6): d 172.2, 156.9 (d, JCF = 237 Hz), 151.9,
138.7, 134.1, 132.2, 128.6, 127.5 (d, JCF = 10.2 Hz),
127.2, 119.7 (d, JCF = 4.8 Hz), 113.6 (d, JCF = 28.7 Hz),
103.4 (d, JCF = 22.7 Hz), 102.9 (d, JCF = 12.1 Hz), 48.4,
38.1, 35.6, 35.2, 22.4.
[(3R)-5-acetyl-4-(4-chlorobenzyl)-7-fluoro-1,2,3,4-
tetrahydrocyclopenta[b]indol-3-yl]acetic acid (2)
Compound 2 was obtained as a white powder in 70%
overall yield from the previous alcohol 5 according to
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1–5
DOI: 10.1002.jlcr

[
14C]-SYNTHESIS OF MOLECULES CONTAINING AN ACETIC ACID MOIETY
5
the three step procedure cited above (mesylate forma-
tion, cyanide displacement and hydrolysis). Chemical
and radiochemical purity were greater than 99% and
the specific activity calculated to be 45 mCi/mmol. 1H
NMR (acetone-d6) d 10.9 (br s, 1H), 7.38 (dd, J=8.9 and
2.5 Hz, 1H), 7.26 (d, J=8.4 Hz, 2H), 7.21 (dd, J=9.8 and
2.5 Hz, 1H), 6.71 (d, J=8.3 Hz, 2H), 5.42 (s, 3H), 3.77–
3.73 (m, 1H), 3.01–2.95 (m, 1H), 2.91–2.78 (m, 3H),
2,53 (dd, J=16.1 and 10.0 Hz, 1H), 2.39–2.27 (m, 1H),
extracted twice with 2 ml of EtOAc, washed with brine,
dried over Na2SO4 and evaporated. Purification by
HPLC using a ChiralPak AD column (4.6 ꢀ 250 mm)
using 7% i PrOH / Hexanes + 0.2% AcOH at 254 nM
at a flow rate of 1 ml/min afforded cleanly carbon-14
labeled 6 and 7 with a retention time of 12 and 17 min,
respectively. Radiochemical purity was greater than
99% and the specific activity calculated to be 45 mCi/
mmol for both compounds.
2.14 (s, 3H). 13C NMR (acetone-d6) d 199.8 (d, JCF
=
1.5 Hz), 172.9, 156.8 (d, JCF=235 Hz), 153.0, 137.6,
133.2, 133.0, 129.2, 128.9, 128.4 (d, JCF=9.6 Hz),
128.2 (d, JCF=7.0 Hz), 119.6 (d, JCF=4.4 Hz), 109.6
(d, JCF=26.5 Hz), 108.1 (d, JCF=23.0 Hz), 50.1, 38.9,
36.2, 35.9, 28.7, 23.2.
Acknowledgement
We would like to thank John Colucci for proof-reading
this manuscript.
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Step 1: Diazomethane (1 ml, 1M/Et2O) was added to
radiolabeled compound 2 (3 mg), stirred 5 min then
evaporated to dryness.
Step 2: The corresponding methyl ester (ꢂ3 mg) was
dissolved in 1 ml of MeOH at 08C under an inert
atmosphere of nitrogen. Sodium borohydride (10 mg)
was added and stirred for 15 min. The reaction was
quenched with 300 ml of AcOH and evaporated to
dryness. The crude compound was quickly purified
through a short pad of silica gel using 70% EtOAc/
Hexanes.
Step 3: The alcohol (ꢂ3 mg) was dissolved in 1 ml of
DMF and sodium hydride (5 mg) was added, stirred
15 min then methyl iodide (20 ml) was introduced. The
reaction was stirred for 30 min, and then quenched by
adding 200 mL of HCl 1 N, extracted twice with 2 ml of
EtOAc, washed with brine, dried over Na2SO4 and
evaporated.
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reflux for 15 min. The reaction was cooled down to
room temperature and neutralized with 1 ml of HCl 1 N,
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1–5
DOI: 10.1002.jlcr