Synthesis and in vitro evaluation of e - And Z-geometrical isomers of PSS232 as potential metabotropic glutamate receptors subtype 5 (mGlu 5) binders

Article abstract of DOI:10.1055/s-0033-1338843

Based on the core structure of [¹¹C]-ABP688, our group developed a novel fluorine-18 labeled PET radiotracer, (E)-[¹⁸F]-PSS232, with significantly improved in vivo properties compared to the earlier fluorine-18 derivative [¹⁸F]-FDEGPECO. The synthetic approach used to obtain PSS232 and the radiolabeling precursor mesylate is disclosed as well as the evaluation of the two geometrical isomers of PSS232. In vitro displacement assays showed higher binding affinity of the E-geometrical isomer (1 nM vs 15 nM, for Z-isomer), which was, for this reason, selected for radiolabeling. One-step radiolabeling conditions (K₂₂₂/¹⁸F^-, DMSO, 95 °C, 10 min) to synthesize (E)-[¹⁸F]-PSS232 from the mesylate via nucleophilic substitution are described. At ambient temperature, (E)-[¹⁸F]-PSS232, with log D_7.4 value of 2.0, was chemically stable over six hours in the presence of sodium ascorbate as a radical scavenger. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0033-1338843

PAPER
▌1877
paper
Synthesis and In Vitro Evaluation of E- and Z-Geometrical Isomers of PSS232
as Potential Metabotropic Glutamate Receptors Subtype 5 (mGlu5) Binders
Synthesis and Evaluation of E- and Z-Isomers of PSS232
Selena Milicevic Sephton,* Linjing Mu, Martina Dragic, Stefanie D. Krämer, Roger Schibli, Simon M. Ametamey*
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of Chemical and Applied Biosciences,
Swiss Federal Institute of Technology (ETH), Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland
Fax +41(44)6331367; E-mail: selena.milicevic@pharma.ethz.ch; E-mail: simon.ametamey@pharma.ethz.ch
Received: 31.03.2013; Accepted after revision: 01.05.2013
Dedicated to Prof. Scott E. Denmark on the occasion of his 60th birthday
[(E)-[18F]-PSS223, (E)-[18F]-3],18,19 was evaluated and al-
Abstract: Based on the core structure of [11C]-ABP688, our group
though (E)-[18F]-3 was selective and specific for mGlu5, it
developed a novel fluorine-18 labeled PET radiotracer, (E)-[18F]-
underwent rapid defluorination in vivo. In comparison to
PSS232, with significantly improved in vivo properties compared to
(E)-[18F]-2, (E)-[18F]-3 has a PEGylated side-chain ex-
the earlier fluorine-18 derivative [18F]-FDEGPECO. The synthetic
approach used to obtain PSS232 and the radiolabeling precursor tended by one methylene unit, which presumably was
mesylate is disclosed as well as the evaluation of the two geometri-
cal isomers of PSS232. In vitro displacement assays showed higher
prone to enzymatic oxygenation by cytochrome P450 and
subsequent defluorination.20 In order to prevent the
binding affinity of the E-geometrical isomer (1 nM vs 15 nM, for Z-
observed defluorination, an alternative derivative 3-
isomer), which was, for this reason, selected for radiolabeling. One-
(pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-[18F]fluo-
step radiolabeling conditions (K222/18F–, DMSO, 95 °C, 10 min) to
roethoxy)propyl] oxime ([18F]-PSS232, [18F]-4, Figure 1)
was hypothesized, such that a methylene unit was added
next to oxime functionality; in essence reducing the num-
ber of methylene units at the end of PEGylated side-chain
by one, to maintain the length of the side-chain, which re-
sulted in (E)-[18F]-4 exhibiting excellent in vivo proper-
ties and subsequently being selected as a PET radiotracer
for further evaluation in human subjects.21 In another re-
cent report, we explored a series of α-fluoro derivatives of
ABP688 and found a significant difference in the binding
affinity of E- and Z-isomers,22 which prompted us to eval-
uate this effect between E- and Z-isomers of 4. Herein, we
report on the syntheses of these two geometrical isomers
(E)-4 and (Z)-4, their in vitro binding affinity determina-
tion, and the optimization of radiolabeling method to ob-
tain (E)-[18F]-4 as well as the in vitro radiochemical
stability of our new mGlu5 PET radiotracer.
synthesize (E)-[18F]-PSS232 from the mesylate via nucleophilic
substitution are described. At ambient temperature, (E)-[18F]-
PSS232, with log D7.4 value of 2.0, was chemically stable over six
hours in the presence of sodium ascorbate as a radical scavenger.
Key words: mGlu5, PET imaging, PSS223, PSS232, ABP688
To date, the most widely clinically applied positron emis-
sion tomography (PET)1 radiotracer for imaging metabo-
tropic glutamate receptor subtype 5 (mGlu5) is (E)-3-[(6-
methylpyridin-2-yl)ethynyl]cyclohex-2-enone O-[11C]meth-
yl oxime ([11C]-ABP688, [11C]-1, Figure 1).2–7 Despite its
excellent in vitro and in vivo properties,8–10 the clinical
application of [11C]-1 is limited to centers with on-site cy-
clotron facilities due to the short physical half-life of car-
bon-11 (20 min). With respect to physical half-life,
fluorine-18 (110 min) is a preferred radionuclide and ef-
forts have been made to develop the fluorine-containing
analogue of [11C]-1. Among others,11–15 our group report-
ed on one such analogue, (E)-3-(pyridin-2-ylethynyl)cy-
clohex-2-enone O-[2-(2-[18F]fluoroethoxy)ethyl] oxime
([18F]-FDEGPECO, (E)-[18F]-2),16,17 which demonstrated
desirable in vitro properties (e.g., binding affinity, speci-
ficity, and selectivity for mGlu5), but a poor signal-to-
noise ratio in in vivo PET studies. Due to its in vitro pro-
file, (E)-[18F]-2 presented a strong basis for further opti-
mization and we rationalized that increased lipophilicity
as reflected through log D7.4 values (1.7 ± 0.1 vs 1.9 ± 0.1
for (E)-[18F]-2 and (E)-[18F]-3, respectively)16,18 would
benefit permeation through the blood-brain barrier (BBB)
and potentially lead to an improved signal-to-noise ratio.
The (E)-[18F]-2 analogue, (E)-3-(pyridin-2-ylethynyl)cy-
clohex-2-enone O-[2-(3-[18F]fluoropropoxy)ethyl] oxime
(E)-[18F]-2
n = m = 1, [18F]-FDEGPECO
Ki = 3.8 nM, log D7.4 = 1.7
N
N
[
11C]-1
(E)-[18F]-3
[
11C]-ABP688
n = 1, m = 2, [18F]-PSS223
Ki = 4.4 nM
18F
m
Ki = 6.0 nM, log D7.4 = 1.9
log D7.4 = 2.4
N
N
O
O
[
18F]-4
11CH3
n
n = 2, m = 1, [18F]-PSS232
O
this work
Figure 1 Structure of [11C]-ABP688 and its fluorine-containing an-
alogues. A crossed double bond is used to indicate oxime ether double
bond isomers (E and Z); for a list of abbreviations, see the experimen-
tal section
SYNTHESIS 2013, 45, 1877–1885
Advanced online publication: 28.05.2013
DOI: 10.1055/s-0033-1338843; Art ID: SS-2013-C0257-OP
© Georg Thieme Verlag Stuttgart · New York
The synthesis of geometrical isomers of 4 was envisioned
via the hydroxy oximes (E)-10 and (Z)-10 (Scheme 1),
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X

1878
S. Milicevic Sephton et al.
PAPER
which were prepared using the Sonogashira cross-cou- Further conformation was compounded by invoking the γ-
pling reaction conditions as previously reported.16,19 Ox- effect24,25 and a 7.8 ppm upfield chemical shift was deter-
ime (E)-10 was reacted with the bromoethers 8a/8b to mined in the 13C NMR for the sterically compressed car-
afford substituted oxime (E)-11 after cleavage of protect- bon atom (Callylic) in (Z)-4. This analysis was in complete
ing groups (TBS and TES) with tetrabutylammonium agreement with our observation made previously for α-
fluoride. Similar to the synthesis of 3, which we have de- fluorinated ABP688 analogues.22 One should note, how-
scribed previously,18 alcohol (E)-11 was treated with me- ever, the inversed nomenclature of geometrical isomers in
syl chloride to give the mesylate (E)-12 (Scheme 1; for the two series of analogues (Z/E α-fluorinated analogues
structure see Table 2), a common precursor for the synthe- vs E/Z-4).
sis of reference compounds (E)-4 and (E)-[18F]-4. The
synthesis of fluoride (E)-4 was accomplished in 62% yield
in the substitution reaction with KF/K222. The synthesis of
(Z)-4 was accomplished in an analogous manner starting
from (Z)-10 and bromoether 8. After the removal of silyl
protecting groups, alcohol (Z)-11, was obtained as a 1:9
(NMR ratio) E/Z mixture and mesylated to give (Z)-12 as
a 1:7 E/Z mixture. Finally, compound (Z)-4 was obtained
as a mixture with an E/Z ratio of 1:5.
Br
Br
InBr3
Et3SiH
OTBS
OR2
conditions
OR1
HO
see Table 1
CHCl3, 80 °C
6 h, 54%
O
O
O
5, R1 = H
7
8a, R2 = TBS
8b, R2 = TES
TBSCl, i-Pr2NEt
DMF, r.t., 34%
6, R1 = TBS
Figure 2 1H and 13C NMR analysis of (E)- and (Z)-4 based on the
anisotropic deshielding by the oxime oxygen on the equatorial pro-
tons and the γ-effect; for the carbon spectra, refer to the Supporting
Information
10, NaH, DMF, r.t.
R3O
N
N
N
9a, R2 = TBS
9b, R2 = TES
For the synthesis of bromoether 8, which was employed in
the syntheses of both isomers, an approach similar to that
employed for the synthesis of 3 was used.18,19 Ethylene
glycol was monoprotected with TBS and reacted with an
acid chloride to yield ester 7, which was further reduced
N
OR2
(E)- or (Z)-10, R3 = H
(E)-13, R3 = (CH2)3OH
Br(CH2)3OH
NaH, DMF
O
O
TBAF⋅THF, THF
(E): 64%, (Z): 75%
(two steps)
26
Br(CH2)2F
NaH, DMF
to bromoether 8 using Et3SiH and InBr3 in moderate
yield (54%). In agreement with our earlier observation, 8
was isolated as a mixture of TBS and TES silyl enol
ethers. The ratio of TBS/TES silyl enol ethers varied, but
this did not affect the subsequent coupling.
a) MsCl, Et3N, THF
0 °C to r.t.
N
N
(E): 97%, (Z): 58%
Reaction of the acid chloride with alcohol 6 required op-
timization. Under the phase-transfer reaction conditions
(Table 1, entries 1 and 2) only traces of the product were
formed. Similarly, the reaction of ethylene glycol and bro-
mopropionic acid using a Dean–Stark apparatus gave only
trace amounts of the product. In a further attempt (entry 4)
nucleophilic substitution was done using Et3N as a base
and a catalytic amount of DMAP. This reaction yielded
b) KF, CHCl3
K222, 80 °C
(E): 62%, (Z): 62%
4
11
N
F
N
OH
O
O
O
O
Scheme 1 Synthesis of PSS232 geometrical isomers via oxime 10
Structural assignment of the oxime ether double bond was only 6% of ester 7. Employing Hünig’s base (entry 5) in-
stead of Et3N afforded 11% of 7. Ester 7 was produced in
22% when bromopropionic acid was used under Mitsunobu
conditions (entry 6); however, the reaction was not repro-
ducible. The use of NaH as a base afforded 7 in 32% and
50% yield (entries 7 and 8).
done by analogy with other oxime ether derivatives (Fig-
ure 2).22 In particular, strong anisotropic deshielding (de-
scribed in the comprehensive study by Denmark and co-
workers for the series of α-chloroketoximes)23 by the ox-
ime oxygen on the equatorial protons results in a down-
field shift of Ha (these protons are closer through space to
the oxime oxygen) and upfield shift of Hcy (these protons
are closer through space to the lone pairs on the oxime ni-
trogen) in the E-isomer and vice versa in the Z-isomer.
Interestingly, the reaction of alcohol (E)-13, obtained
from the reaction of oxime (E)-10 with bromopropanol in
Synthesis 2013, 45, 1877–1885
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis and Evaluation of E- and Z-Isomers of PSS232
1879
Table 1 Optimization of Reaction Conditions for the Preparation of Ester 7
O
Br
Br
O
OTBS
R
OTBS
HO
O
7
6
Entry
R
6 (g)
Base
Additive/Activator Solvent
Time (h)
Yield (%)a
1
2
3
4
5
6
7
8
Cl
0.1
0.1
1.5
0.4
3.4
0.3
0.1
3.3
NaOH
NaOH
–
TBAF·THF
TBAC
TsOH
DMAP
DMAP
DIAD/PPh3
–
CH2Cl2
CH2Cl2
toluene
CH2Cl2
CH2Cl2
THF
1
2
nd
nd
nd
6
Cl
OH
Cl
21
15
17
70
2
Et3N
i-Pr2NEt
–
Cl
11
22
32
50
OH
Cl
NaH
NaH
THF
Cl
–
THF
16
a nd: Not determined.
73% yield, with bromofluoroethane failed to afford the Ametamey group reported a similar effect for the E- and
desired (E)-4 directly (Scheme 1) or in reaction with bro- Z-isomers when the substituent on the oxime ether was 2-
moacetyl bromide.
fluoropyridine16 with a 4.8-fold higher binding affinity for
the E-isomer. In the same report, only a slight preference
for Z-isomer was observed (1.1-fold) when 3-fluoro-4-cy-
anobenzene constituted the oxime ether.
While (E)-4 was completely stable upon storage at –20 °C
a neat (Z)-4 mixture with E-isomer (E/Z ratio of 1:5) un-
derwent isomerization. The E/Z ratio changed to 2:1 under
the same conditions, over a period of two months and an
additional unidentified by-product was observed. Prepar-
ative HPLC was performed to isolate (Z)-4 in E/Z NMR
ratio of 1:11 and this material was used further for IC50 de-
termination. The observed isomerization suggests that the
E-isomer is likely the thermodynamically more stable iso-
mer. Next an experiment was performed in which CDCl3
solutions at 4 mg/mL of (E)-4 and (Z)-4 were monitored
by 1H NMR over 25 days and observed no change in the
former case; however, in the latter the E/Z NMR ratio
changed from 1:5 to 1:3. The two solutions were next
heated to 60 °C over five hours; once again isomerization
was observed for the Z-isomer and the E/Z ratio decreased
to 1:2. In this case, no other by-products were detected.
The binding affinity toward mGlu5 of the two analogues
was determined in a displacement assay with [3H]-1 using
rat brain homogenates in triplicate.16,18 For the determina-
tion of nonspecific binding, 2-[(3-methoxyphenyl)ethy-
nyl]-6-methylpyridine (MMPEP) was applied.
Figure 3 Displacement curves for (E)-4 and (Z)-4 showing clear dif-
ference in IC50 values of the two geometrical isomers. For each iso-
mer three experiments were performed in triplicate and the average
values with standard deviations are depicted. Solid curves represent
fitted values assuming one binding site. Dotted lines represent esti-
mated IC50 values.
The IC50 values estimated from the plotted curves were
1.3 ± 0.1 nM and 15.3 ± 1.4 nM for (E)-4 and (Z)-4, re-
spectively (Figure 3). While the difference in binding af-
finity of E- and Z-isomers, in the case of α-fluorinated
analogues was 5.5-fold,22 a 11.8-fold higher binding affin-
ity was determined for (E)-4 when compared to the Z-iso-
mer. This suggests that the configuration of the oxime
ether double bond bearing the long lipophilic side-chain
plays a significant role in binding to the target mGlu5. The
Due to its favorable IC50 value (comparable to that of
ABP6888,16), our attention was focused on the further de-
velopment of (E)-4. Conditions to establish a successful
radiolabeling protocol were next sought involving the nu-
cleophilic substitution with 18F–. Mesylate (E)-12, which
was stable for months both in solution and neat, was em-
ployed as a precursor for this purpose. Radiosynthesis was
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1877–1885

1880
S. Milicevic Sephton et al.
PAPER
Table 2 Optimization of the Reaction Conditions for the Radiolabeling of Mesylate (E)-12 To Obtain (E)-[18F]-4a,b
K222, K2CO3
18F–
N
N
18F
N
OMs
N
O
O
O
O
(E)-[18F]-4
(E)-12
Entry
(E)-12
(mg)
Solvent
Temp
(°C)
RCC at 5 min
(%)
RCC at 10 min
(%)
RCC at 15 min RCC at 20 min
(%)
(%)
1
2
3
4
5
6
2.0
2.0
1.8
1.8
1.8
1.8
MeCN
MeCN
DMSO
DMSO
DMF
95
80
17
42
44
76
31
51
34
–
46
51
58
71
51
60
48
66
55
73
49
62
95
54
76
45
60
120
95
DMF
110
a Manual approach.
b RCC: Radiochemical conversion.
initially accomplished at 90 °C over 10 minutes in DMF parent compound as an E/Z mixture was intact. Also, the
to give (E)-[18F]-4. The decay corrected radiochemical activity concentration of investigated samples was of im-
yields of 13–37% (n = 40) were not significantly im- portance, and radiolysis was typically observed when con-
proved when the labeling was performed at 95 °C. In a centration exceeded 500 MBq/mL.
typical experiment 3–12 GBq of the formed product was
As a measure of lipophilicity log D7.4 was determined to
obtained starting from 30–70 GBq of 18F-fluoride. Specif-
give a value of 2.0 ± 0.1 for (E)-[18F]-4 using the shake-
ic activity was 80–400 GBq/μmol at the end of the synthe-
sis. Total synthesis time was 70 minutes from the end of
flask method, which was comparable to (E)-[18F]-3 and
slightly higher than that measured for (E)-[18F]-2. The
bombardment and the E/Z HPLC ratio was 99:1. Subse-
measured log D7.4 value was within the range established
quently, it was shown that the quality of images obtained
for successful passage through the BBB.16
with (E)-[18F]-4 was not reduced due to 1% of Z-isomer in
In conclusion, we have prepared two geometrical isomers
(E and Z) of novel mGlu5 PET radiotracer 4, and measured
11.8-fold difference in the binding affinity toward mGlu5
in favor of the E-isomer. Not only did (Z)-4 have lower
binding affinity for mGlu5, but also underwent isomeriza-
tion upon storage. The syntheses of the two isomers were
accomplished via substitution reactions of bromoether 8
with oximes (E)-10 and (Z)-10, respectively, in six syn-
thetic steps, and 7% and 5% overall yields. Mesylate (E)-
12 was used as a precursor for radiolabeling to obtain (E)-
[18F]-4. Compound (E)-[18F]-4 exhibited desirable log
D7.4 value. We have established practical synthetic ap-
proach to prepare radiolabeling precursor mesylate (E)-12
and optimized the radiolabeling protocol to accommodate
the product mixture.21
For the purposes of translating the manually performed ra-
diosynthesis to a module-based radiolabeling procedure,
several different reaction conditions were evaluated (Ta-
ble 2), considering solvent, reaction time, and tempera-
ture. Generally, it was established that higher reaction
temperature resulted in higher conversions to radiolabeled
product (Table 2, entries 3 and 5 vs 4 and 6) and reaction
times had very little effect (entries 1–6; 20 vs 15 vs 10
min). The highest conversion was accomplished with
DMSO as the solvent (entries 3 and 4), which was select-
ed for further application in translating manual synthesis
to the module based approach.
Compound (E)-[18F]-4 was found to be prone to radiolysis translation to the module-based synthesis for the purposes
when produced in amounts larger than 5 GBq for which of preparing (E)-[18F]-4 under GMP conditions with the
reason, sodium ascorbate (25 mg/mL) was employed as a ultimate aim of applying (E)-[18F]-4 in human clinical
radical scavenger in the final formulation solution. After practice. The results of the in vivo studies (in rodents and
six hours, in the radiotracer solution, more than 95% of humans) will be reported in due course.
Synthesis 2013, 45, 1877–1885
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis and Evaluation of E- and Z-Isomers of PSS232
1881
All reactions requiring anhydrous conditions were conducted in
flame-dried glass apparatus under an atmosphere of inert gas. All
chemicals and anhydrous solvents were purchased from Aldrich or
ABCR and used as received unless otherwise noted. Reported den-
sity values are for ambient temperature. [3H]-ABP688 (2.405
GBq/μmol, 37 MBq/mL solution in EtOH) was obtained from
AstraZeneca. Oximes (E)-10 and (Z)-10 were prepared as described
previously.16,19 MMPEP was prepared based on the published pro-
cedure.27
with 20% EtOAc–pentane) to afford 6 (2.93 g, 16.6 mmol, 34%) as
a colorless oil.
1H NMR (400 MHz, CDCl3): δ = 3.73–3.70 (m, 2 H), 3.66–3.62 (m,
2 H), 2.05 (t, J = 6.2 Hz, 1 H), 0.91 (s, 9 H), 0.08 (s, 6 H).
13C NMR (100 MHz, CDCl3): δ = 64.3 (2), 63.9 (2), 26.1 (3, 3 C),
18.5 (0), –5.1 (3, 2 C).
MS (ES+): m/z = 177 (M + H)+.
HRMS (EI+): m/z calcd for C8H21O2Si: 177.1305; found: 177.1308.
Preparative chromatographic separations were performed on Al-
drich Science silica gel 60 (35–75 μm) and reactions were followed
by TLC analysis using Sigma-Aldrich silica gel 60 plates (2–25 μm)
with fluorescent indicator (254 nm) and visualized with UV or
KMnO4.
2-[(tert-Butyldimethylsilyl)oxy]ethyl 3-Bromopropanoate (7)
A stirred solution of 6 (3.27 g, 18.6 mmol) in anhyd THF (160 mL)
was treated with NaH (60% suspension in oil, 1.1 g, 27.9 mmol) and
the resulting heterogeneous pale yellow mixture was allowed to stir
for 60 min at r.t. under N2 atmosphere. After this time, the mixture
was further treated dropwise with 3-bromopropionyl chloride (1.9
mL, 3.2 g, 18.6 mmol, d = 1.7) over 3 min and the resulting almost
colorless mixture was allowed to stir for 16 h. After this time, the
mixture was poured over sat. aq NH4Cl (120 mL) and ice (150 g) in
a separatory funnel and the mixture was diluted with EtOAc (280
mL) and the two layers were well shaken and separated. The aque-
ous phase was washed with EtOAc (2 × 280 mL). The combined or-
ganic extracts were washed with brine (200 mL), dried (Na2SO4),
and concentrated in vacuo. The crude product was purified by chro-
matography on a silica gel column (eluting with a gradient of 5%
Et2O–pentane to 10% Et2O–pentane) to afford 7 (2.87 g, 9.22 mmol,
50%) as a colorless oil.
IR spectra were recorded on a JASCO FT/IR 6200 (OmniLab) spec-
trometer using a CHCl3 solution of the compound. 1H and 13C NMR
spectra were recorded in Fourier transform mode at the field
strength specified on Bruker Avance FT-NMR spectrometers.
NMR spectra were obtained from the specified deuterated solvents
in 5 mm diameter tubes. Chemical shift in ppm is quoted relative to
residual solvent signals calibrated as follows: CDCl3 δH
(CHCl3) = 7.26, δC = 77.2; DMSO-d6 δH (CD3SOCHD2) = 2.50,
δC = 39.5. Standard abbreviations were used for denoting the multi-
plicities in the 1H NMR spectra ; coupling constants are reported in
Hz. Numbers in parentheses following carbon atom chemical shifts
refer to the number of attached hydrogen atoms as revealed by the
DEPT spectral editing technique. Electrospray (ES) mass spectra
(LRMS) were obtained with a Micromass Quattro micro API LC
electrospray ionization and ES mass spectra (HRMS) were obtained
with a Bruker FTMS 4.7 T BioAPEXII spectrometer. Electron im-
pact (EI) and chemical ionization (CI) mass spectra (LRMS and
HRMS) were obtained with a Waters Micromass AutoSpec Ultima
MassLynx 4.0 spectrometer. Ion mass/charge (m/z) ratios are re-
ported as values in atomic mass units. Semi-preparative purification
of radiolabeled material was performed on a Merck-Hitachi
L6200A system equipped with Knauer variable wavelength detec-
tor and an Eberline radiation detector or Smartline system with
Pump 1000, Manager 5000, Knauer UV detector, and GabiStar
(Raytest) radiodetector using a reverse phase column (C18 Phe-
nomenex Gemini, 5 μm, 250 × 10 mm) and eluting with gradient:
0–5 min 5% aq MeCN, 5–15 min 5–50% aq MeCN, 15–30 min 50%
aq MeCN, 30–50 min, 50–90% aq MeCN, 50–65 min, 65% MeCN
at flow rate 5 mL/min. Analytical HPLC samples were analyzed by
Agilent HPLC 1100 system equipped with UV multi-wavelength
detector and Raytest Gabi star radiation detector using reverse
phase column (ACE 111-0546, C18, 3 μm, 50 × 4.6 mm) and elut-
ing with 45% aq MeCN at flow rate 1 mL/min.
IR (neat): 2954, 2929, 2857, 1740, 1472, 1254, 1130, 1108, 834,
777 cm–1.
1H NMR (400 MHz, CDCl3): δ = 4.20 (ddm, J = 6.2, 4.0 Hz, 2 H),
3.82 (ddm, J = 6.1, 3.8 Hz, 2 H), 3.58 (t, J = 6.9 Hz, 2 H), 2.94 (t,
J = 6.9 Hz, 2 H), 0.89 (s, 9 H), 0.07 (s, 6 H).
13C NMR (100 MHz, CDCl3): δ = 66.4 (2), 61.3 (2), 37.9 (2), 26.0
(3, 3 C), 25.9 (2), 18.5 (0), –5.1 (3, 2 C).
MS (ES+): m/z = 312 (M + H)+.
[2-(3-Bromopropoxy)ethoxy](tert-butyl)dimethylsilane (8a)
A stirred solution of 7 (418 mg, 1.34 mmol) in CHCl3 (1.3 mL) was
treated with InBr3 (24.0 mg, 0.07 mmol) in one portion under N2 at-
mosphere. Et3SiH (0.86 mL, 623.4 mg, 5.36 mmol, d = 0.728) was
then added dropwise over 1 min and the resulting heterogeneous
mixture was allowed to heat at 60 °C (oil bath temperature) for 6 h
in a flask equipped with a condenser. Immediately upon heating, the
mixture turned creamy and yellow. After this time, the mixture was
allowed to cool to r.t. and then diluted with H2O (5 mL) and CH2Cl2
(10 mL), and the two layers were well shaken and separated. The
aqueous phase was further extracted with CH2Cl2 (2 × 10 mL). The
combined organic extracts were washed with brine (1 × 15 mL),
dried (Na2SO4), and concentrated in vacuo to give a brown oily res-
idue (1.5 g). The crude product was purified by chromatography on
a silica gel column (eluting with 5% EtOAc–pentane) to afford 8a
(219 mg, 0.74 mmol, 54%) as a mixture with [2-(3-bromopro-
poxy)ethoxy]triethylsilane (8b) in a 2:1 ratio.
Specific radioactivity of (E)-[18F]-4 was determined from a calibra-
tion curve prepared with decreasing concentrations of (E)-4.
2-[(tert-Butyldimethylsilyl)oxy]ethanol (6)
To a flame-dried flask charged with anhyd DMF (87 mL) was added
1,2-ethyleneglycol (5; 2.7 mL, 3.0 g, 48.3 mmol, d = 1.113) at r.t.
under N2 atmosphere. The resulting colorless solution was treated
with i-Pr2NEt (78.0 mL, 59.0 g, 459 mmol, d = 0.755) in one por-
tion and the pale yellow biphasic mixture was vigorously stirred.
This mixture was further treated with a solution of tert-butyldimeth-
ylchlorosilane (7.3 g, 48.3 mmol) in DMF (52 mL) dropwise over
68 min during which time the mixture turned cloudy and it was al-
lowed to stir for 19 h. After this time, the mixture was partitioned
between H2O (100 mL) and Et2O (180 mL) and the two layers were
well shaken and separated. The aqueous phase was extracted with
Et2O (2 × 180 mL). The combined organic extracts were washed
with 2 M aq HCl (2 × 180 mL; Caution! vigorous bubbling), sat. aq
NaHCO3 (1 × 180 mL), brine (1 × 180 mL), dried (Na2SO4), and
concentrated in vacuo to give a pale yellow oil. The crude product
was purified by chromatography on a silica gel column (eluting
1H NMR (400 MHz, CDCl3): δ = 3.76 (tm, J = 5.0 Hz, 4 H, 8a and
8b), 3.60 (tm, J = 5.9 Hz, 4 H, 8a and 8b), 3.54–3.50 (m, 8 H, 8a
and 8b), 2.13–2.07 (m, 4 H, 8a and 8b), 0.96 (t, J = 8.0 Hz, 9 H, 8b),
0.90 (s, 9 H, 8a) 0.61 (q, J = 7.6 Hz, 6 H, 8b), 0.07 (s, 6 H, 8a).
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(tert-Bu-
tyldimethylsilyl)oxy]ethoxy}propyl) Oxime [(E)-9a] and (E)-3-
(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(Triethylsi-
lyl)oxy]ethoxy}propyl) Oxime [(E)-9b]
To a flame-dried flask charged with (E)-3-(pyridin-2-ylethynyl)cy-
clohex-2-enone oxime [(E)-10; 141 mg, 0.66 mmol] was added an-
hyd DMF (5 mL). The clear pale yellow mixture was treated with
NaH (63 mg of 60% suspension in oil, 1.58 mmol) and the resulting
bright yellow heterogeneous mixture was stirred for 47 min at r.t.
under N2 atmosphere. The resulting orange mixture of the deproton-
© Georg Thieme Verlag Stuttgart · New York
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1882
S. Milicevic Sephton et al.
PAPER
ated oxime was then treated with a solution of 8a/8b (2:1 ratio, re-
spectively, 217 mg, 0.73 mmol) in DMF (5 mL) dropwise over 20
min during which time the mixture turned brown and was allowed
to stir further for 2.5 h. After this time, the crude mixture was
quenched with sat. aq NaHCO3 (15 mL) and diluted with H2O (10
mL) and Et2O (45 mL). The two layers were well shaken and sepa-
rated. The aqueous phase was extracted with Et2O (2 × 45 mL). The
combined organic extracts were washed with H2O (3 × 35 mL),
brine (1 × 40 mL), dried (Na2SO4), and concentrated in vacuo to
give the crude mixture as a brown oily residue (699 mg). Low-
resolution mass spectrometry confirmed the presence of the desired
product. The crude mixture was used in the next step without puri-
fication.
7.25 (ddd, J = 7.6, 4.9, 1.1 Hz, 1 H), 7.20 (br t, J = 1.8 Hz, 1 H), 4.19
(t, J = 6.1 Hz, 2 H), 3.74 (br t, J = 4.3 Hz, 2 H), 3.62–3.57 (m, 4 H),
2.45 (td, J = 6.0, 1.8 Hz, 2 H), 2.42–2.39 (m, 2 H), 1.96 (quint,
J = 6.2 Hz, 2 H), 1.89 (quint, J = 6.1 Hz, 2 H).
(E)-2-[3-({[3-(Pyridin-2-ylethynyl)cyclohex-2-en-1-yli-
dene]amino}oxy)propoxy]ethyl Methanesulfonate [(E)-12]
A stirred solution of (E)-11 (134 mg, 0.43 mmol) in anhyd THF (4.3
mL) was treated with Et3N (120 μL, 87.0 mg, 0.86 mmol, d = 0.726)
in one portion and MsCl (40 μL, 58.0 mg, 0.51 mmol, d = 1.477)
was then added dropwise (addition time <1 min) and the resulting
pale yellow mixture was allowed to stir at r.t. under N2 atmosphere.
After 21 min, another portion of MsCl (20 μL, 29.0 mg, 0.26 mmol)
was added and the mixture was stirred for an additional 15 min. Af-
ter this time, the mixture was partitioned between H2O (20 mL) and
EtOAc (30 mL) and the two layers were well shaken and separated.
The aqueous phase was extracted with EtOAc (2 × 35 mL). The
combined organic extracts were washed with H2O (3 × 30 mL),
brine (1 × 35 mL), dried (Na2SO4), and concentrated in vacuo to
give a pale yellow oil. The crude product was purified by chroma-
tography on a silica gel column (eluting with 80% EtOAc–pentane)
to afford (E)-12 (161.4 mg, 0.41 mmol, 97%) as a pale yellow oil.
MS (ES+): m/z = 430 (M + H)+.
(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(tert-Bu-
tyldimethylsilyl)oxy]ethoxy}propyl) Oxime [(Z)-9a] and (Z)-3-
(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(Triethylsi-
lyl)oxy]ethoxy}propyl) Oxime [(Z)-9b)
An analogous approach to that described for (E)-9a and (E)-9b was
employed to prepare the crude mixture as a brown oily residue (327
mg). Without purification the crude mixture was used in the next
step.
IR (neat): 2935, 2872, 1580, 1561, 1352, 1175, 1127, 779, 742 cm–1.
1H NMR (400 MHz, CDCl3): δ = 8.58 (ddd, J = 4.8, 1.5, 0.9 Hz, 1
H), 7.65 (td, J = 7.8, 1.8 Hz, 1 H), 7.44 (dt, J = 7.8, 1.0 Hz, 1 H),
7.22 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 6.55 (br t, J = 1.6 Hz, 1 H),
4.37–4.35 (m, 2 H), 4.18 (t, J = 6.3 Hz, 2 H), 3.71–3.69 (m, 2 H),
3.58 (t, J = 6.4 Hz, 2 H), 3.05 (s, 3 H), 2.53 (br m, J = 6.4 Hz, 2 H),
2.39 (td, J = 6.1, 1.6 Hz, 2 H), 1.95 (quint, J = 6.3 Hz, 2 H), 1.79
(quint, J = 6.4 Hz, 2 H).
13C NMR (100 MHz, CDCl3): δ = 155.5 (0), 150.3 (1), 143.4 (0),
136.3 (1), 131.2 (1), 127.4 (1), 127.3 (0), 123.0 (1), 92.0 (0), 90.0
(0), 71.0 (2), 69.3 (2), 68.7 (2), 68.3 (2), 37.8 (3), 29.6 (2), 29.5 (2),
22.3 (2), 20.9 (2).
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Hydroxy-
ethoxy)propyl] Oxime [(E)-11]
To the crude mixture of (E)-9a and (E)-9b (285 mg, 0.66 mmol) in
a round-bottomed flask was added anhyd THF (13 mL) at r.t. under
N2 atmosphere. The resulting clear orange mixture was further
treated with TBAF solution in THF (1.33 mL, 1.33 mmol, c = 1 M)
dropwise over 4 min and the resulting brown mixture was allowed
to stir at r.t. under N2 for 61 min. After this time, the mixture was
partitioned between H2O (30 mL) and EtOAc (40 mL) and the two
layers were well shaken and separated. The aqueous phase was ex-
tracted with EtOAc (2 × 40 mL, slow separation of phases). The
combined organic extracts were washed with brine (1 × 40 mL),
dried (Na2SO4), and concentrated in vacuo to give a brown oil (779
mg). The crude product was purified by chromatography on a silica
gel column (eluting with gradient 90% EtOAc–pentane to 100%
EtOAc) to give (E)-11 (134.7 mg, 0.43 mmol, 64%) as a pale yellow
oil.
MS (ES+): m/z = 393 (M + H)+.
HRMS (ESI): m/z calcd for C19H25N2O5S: 393.1479; found:
393.1477.
(Z)-2-[3-({[3-(Pyridin-2-ylethynyl)cyclohex-2-en-1-yli-
dene]amino}oxy)propoxy]ethyl Methanesulfonate [(Z)-12]
An analogous approach to that described for (E)-12 was employed
to prepare the title compound (129.6 mg, 0.33 mmol, 58%) as a mix-
ture with the E-isomer in NMR E/Z ratio of 1:7. The mixture was
identified by 1H NMR spectrum (only peaks corresponding to Z-iso-
mer are listed; several peaks are overlapped) and used in the next
step.
1H NMR (400 MHz, CDCl3): δ = 8.61 (ddd, J = 4.8, 1.7, 0.9 Hz, 1
H), 7.68 (td, J = 7.7, 1.8 Hz, 1 H), 7.47 (br t, J = 7.8, 1.0 Hz, 1 H),
7.25 (ddd, J = 7.7, 4.9, 1.2 Hz, 1 H), 7.17 (br t, J = 1.8 Hz, 1 H),
4.39–4.37 (m, 2 H), 4.15 (t, J = 6.2 Hz, 2 H), 3.73–3.71 (m, 2 H),
3.61 (t, J = 6.4 Hz, 2 H), 3.07 (s, 3 H), 2.45 (td, J = 6.1, 1.8 Hz, 2
H), 2.41–2.38 (m, 2 H), 1.96 (quint, J = 6.3 Hz, 2 H), 1.89 (quint,
J = 6.2 Hz, 2 H).
IR (neat): 3401, 2934, 2869, 2205, 1581, 1463, 1429, 1121, 1059,
866, 779 cm–1.
1H NMR (400 MHz, CDCl3): δ = 8.58 (ddd, J = 4.8, 1.7, 0.9 Hz, 1
H), 7.65 (td, J = 7.7, 1.8 Hz, 1 H), 7.44 (dt, J = 7.8, 1.0 Hz, 1 H),
7.22 (ddd, J = 7.6, 4.9, 1.2 Hz, 1 H), 6.57 (t, J = 1.6 Hz, 1 H), 4.21
(t, J = 6.2 Hz, 2 H), 3.72 (br t, J = 4.0 Hz, 2 H), 3.57 (t, J = 6.4 Hz,
2 H), 3.56–3.54 (m, 2 H), 2.54 (br m, J = 6.4 Hz, 2 H), 2.39 (td,
J = 5.9, 1.6 Hz, 2 H), 2.26 (br s, 1 H), 1.96 (quint, J = 6.3 Hz, 2 H),
1.79 (quint, J = 6.4 Hz, 2 H).
13C NMR (100 MHz, CDCl3): δ = 155.5 (0), 150.2 (1), 143.4 (0),
136.3 (1), 131.2 (1), 127.4 (1), 127.3 (0), 123.0 (1), 92.0 (0), 90.0
(0), 72.0 (2), 71.2 (0), 67.9 (2), 61.9 (2), 29.8 (2), 29.5 (2), 22.4 (2),
20.9 (2).
MS (ES+): m/z = 315 (M + H)+.
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Fluoro-
ethoxy)propyl) Oxime [(E)-4]
HRMS (ESI): m/z calcd for C18H23N2O3: 315.1703; found:
A stirred solution of Kryptofix-222® (K222, 251 mg, 0.67 mmol) and
KF (38.7 mg, 0.67 mmol) in anhyd MeCN (5.5 mL) was treated
with a solution of (E)-12 (130.7 mg, 0.33 mmol) in anhyd MeCN
(5.5 mL) dropwise over 7 min at r.t. under N2 atmosphere during
which time the heterogeneous mixture turned brown. The flask was
equipped with a condenser and the mixture was placed in an oil bath
and heated to 80 °C over 65 min. After this time, the mixture was
allowed to cool to r.t. and quenched with sat. aq NH4Cl (20 mL).
The mixture was diluted further with H2O (10 mL) and EtOAc (50
mL) and the two layers were well shaken and separated. The aque-
ous phase was extracted with EtOAc (2 × 50 mL). The combined
organic extracts were washed with H2O (3 × 30 mL), brine (40 mL),
315.1707.
(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Hydroxy-
ethoxy)propyl] Oxime [(Z)-11]
An analogous approach to that described for (E)-11 was employed
to prepare the title compound (180.8 mg, 0.57 mmol, 75%) as a mix-
ture with the E-isomer in NMR E/Z ratio of 1:9. The mixture was
identified by 1H NMR spectrum (only peaks corresponding to Z-iso-
mer are listed; several peaks are overlapped) and used in the next
step.
1H NMR (400 MHz, CDCl3): δ = 8.61 (ddd, J = 4.9, 1.7, 0.9 Hz, 1
H), 7.67 (td, J = 7.8, 1.8 Hz, 1 H), 7.47 (dt, J = 7.8, 1.0 Hz, 1 H),
Synthesis 2013, 45, 1877–1885
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis and Evaluation of E- and Z-Isomers of PSS232
1883
dried (Na2SO4), and concentrated in vacuo. The crude product was
purified by chromatography on a silica gel column (eluting with
50% EtOAc–pentane) to afford (E)-4 (65.4 mg, 0.21 mmol, 62%) as
a pale yellow oil.
1H NMR (400 MHz, CDCl3): δ = 5.73 (dm, J = 4.8 Hz, 1 H), 7.66
(td, J = 7.7, 1.8 Hz, 1 H), 7.45 (dt, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd,
J = 7.6, 4.9, 1.1 Hz, 1 H), 6.57 (tm, J = 1.6 Hz, 1 H), 4.27 (t, J = 5.9
Hz, 2 H), 3.76 (dd, J = 10.9, 5.5 Hz, 2 H), 2.54 (dd, J = 6.4 Hz, 2
H), 2.40 (td, J = 6.2, 1.6 Hz, 2 H), 1.93 (quint, J = 6.0 Hz, 2 H), 1.80
(quint, J = 6.4 Hz, 2 H).
IR (neat): 2948, 2872, 2201, 1580, 1462, 1428, 1130, 1048, 778,
739 cm–1.
13C NMR (100 MHz, CDCl3): δ = 155.7 (0), 150.3 (1), 143.3 (0),
136.3 (1), 131.0 (1), 127.7 (0), 127.4 (1), 123.0 (1), 92.2 (0), 89.9
(0), 72.0 (2), 60.5 (2), 32.5 (2), 29.5 (2), 22.4 (2), 20.9 (2).
1H NMR (400 MHz, CDCl3): δ = 8.59 (br m, J = 4.4 Hz, 1 H), 7.66
(td, J = 7.7, 1.8 Hz, 1 H), 7.44 (br t, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd,
J = 7.6, 4.9, 1.1 Hz, 1 H), 6.57 (t, J = 1.6 Hz, 1 H), 4.56 (dm,
J = 47.7 Hz, 2 H), 4.21 (t, J = 6.3 Hz, 2 H), 3.69 (dm, J = 29.7 Hz,
2 H), 3.60 (t, J = 6.4 Hz, 2 H), 2.54 (br d, J = 6.4 Hz, 2 H), 2.40 (td,
J = 6.2, 1.5 Hz, 2 H), 1.98 (quint, J = 6.4 Hz, 2 H), 1.80 (quint,
J = 6.4 Hz, 2 H).
13C NMR (100 MHz, CDCl3): δ = 155.5 (0), 150.3 (1), 143.4 (0),
136.3 (1), 131.4 (1), 127.4 (1), 127.1 (0), 123.0 (1), 91.9 (0), 90.1
(0), 84.2 (2), 83.3 (d, J = 168 Hz, 2), 71.2 (2), 70.2 (d, J = 19.8 Hz,
2), 68.5 (2), 29.7 (2), 29.5 (2), 22.4 (2), 21.0 (2).
MS (ES+): m/z = 271 (M + H)+.
HRMS (ESI): m/z calcd for C16H19N2O2: 271.1441; found:
271.1430.
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-18F-Fluo-
roethoxy)propyl] Oxime [(E)-[18F]-4]
[18F]-Fluoride was produced via nuclear 18O(p, n)18F reaction in cy-
clotron, which involved proton bombardment of 18O-enriched water
(940 μL in a medium volume liquid target) for 70 min at a target
current of 15–30 μAh. [18F]-Fluoride was obtained as an aqueous
solution, which was immediately passed through QMA cartridge
[preconditioned with 0.5 M aq K2CO3 (1 × 5 mL), then H2O (1 × 5
mL) and dried in air] and the trapped [18F]-fluoride was eluted from
the cartridge with 0.25% wt Kryptofix-222® solution (1 mL) in ba-
sic (0.05% wt K2CO3) aq MeCN (75% vv) into a tightly closed re-
action vial to give 30–70 GBq. The solvents were evaporated in
vacuo (130 mbar) with a gentle stream of N2 gas at 110 °C over 5
min. To the resulting solid residue was added anhyd MeCN (1 mL)
and the mixture was azeotropically dried in vacuo (130 mbar) with
a gentle stream of N2 at 110 °C over 3 min and this was repeated
twice more. After this time, the solid residue was additionally dried
in vacuo (100 mbar) at r.t. over 1 min to afford 20–60 GBq of activ-
ity. The resulting solid Kryptofix-222®/[18F–] complex was treated
with a solution of (E)-12 (2.80 mg, 7.13 μmol) in anhyd DMF (0.3
mL) and the resulting dark brown mixture was allowed to heat at 90
or 95 °C (in a metal heating block) over 10 min during which time
the reaction mixture turned light brown. The crude mixture was di-
luted with 50% aq (v/v) MeCN (2 mL) and purified by semi-prepar-
ative HPLC (Phenomenex C18 Gemini 5 μm, 10 × 250 mm column,
eluting with 5% aq MeCN for 10 min, then a gradient of 5% to 50%
aq MeCN for 10 min, and finally 50% aq MeCN for 30 min at 5
mL/min) and the desired product was collected over 1 min (reten-
tion time: 31.0 min) and immediately diluted with H2O (10 mL) to
give 4–15 GBq of activity. The aqueous solution was passed
through a C18 cartridge [preconditioned with EtOH (1 × 5 mL),
then H2O (1 × 5 mL) and dried in air] and the cartridge was washed
with H2O (2 × 1 mL). The product was eluted from the C18 car-
tridge with EtOH (1 × 0.3 mL) in a sterile vial containing 50% aq
PEG 200 (5 mL) to afford 3–12 GBq of the radiolabeled title com-
pound (13–37% decay corrected yield: 95–99% HPLC purity, 80–
400 GBq/μmol specific activity).
19F NMR (376 MHz, CDCl3): δ = –222.7 to –223.1 (m).
MS (ES+): m/z = 318 (M + H)+.
HRMS (ESI): m/z calcd for C18H22FN2O2: 317.1660; found:
317.1660.
(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Fluoro-
ethoxy)propyl] Oxime [(Z)-4]
An analogous approach to that described for the synthesis of (E)-4
was employed to prepare the title compound (64.3 mg, 0.20 mmol,
62%); pale yellow oil.
IR (neat): 2950, 2927, 2873, 1582, 1462, 1428, 1131, 1048, 885,
778 cm–1.
1H NMR (400 MHz, CDCl3): δ = 8.61 (br s, 1 H), 7.67 (td, J = 7.8,
1.5 Hz, 1 H), 7.47 (br d, J = 7.7 Hz, 1 H), 7.25–7.23 (m, 1 H), 7.19
(br t, J = 1.6 Hz, 1 H), 4.56 (dm, J = 47 Hz, 2 H), 4.17 (t, J = 6.2 Hz,
2 H), 3.70 (dm, J = 29 Hz, 2 H), 3.62 (t, J = 6.5 Hz, 2 H), 2.45 (td,
J = 6.1, 1.7 Hz, 2 H), 2.41–2.38 (m, 2 H), 1.98 (quint, J = 6.3 Hz, 2
H), 1.89 (quint, J = 6.3 Hz, 2 H).
13C NMR (100 MHz, CDCl3): δ = 151.9 (0), 150.4 (1), 143.2 (0),
136.4 (1), 130.6 (0), 127.6 (1), 123.6 (1), 123.3 (1), 89.9 (0, 2 C),28
83.3 (d, J = 168 Hz, 2), 70.9 (2), 70.2 (d, J = 19 Hz, 2), 68.5 (2),
30.6 (2), 29.6 (2), 27.7 (2), 22.4 (2).
19F NMR (376 MHz, CDCl3): δ = –223.2 to –223.7 (m).
MS (ES+): m/z = 318 (M + H)+.
HRMS (ESI): m/z calcd for C18H22FN2O2: 317.1660; found:
317.1660.
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-Hydroxy-
propyl) Oxime [(E)-13]
To a flame-dried flask charged with (E)-10 (200 mg, 0.94 mmol)
was added DMF (12 mL) at r.t. under N2 atmosphere. The resulting
pale yellow clear solution was treated with NaH (60% suspension
in oil, 45 mg, 1.13 mmol) and the heterogeneous mixture was al-
lowed to stir for 50 min during which time it turned orange. The
mixture was then further treated with a solution of 3-bromopropanol
(90 μL, 144 mg, 1.03 mmol, d = 1.6) in DMF (7 mL) dropwise over
12 min and the resulting brown mixture was allowed to stir for 2 h.
After this time, the crude mixture was partitioned between H2O (30
mL) and EtOAc (60 mL) and the two layers were well shaken and
separated. The aqueous phase was extracted with EtOAc (2 × 60
mL). The combined organic extracts were washed with H2O (3 × 30
mL), brine (40 mL), dried (Na2SO4), and concentrated to give the
crude product, which was purified by chromatography on a silica
gel column (eluting with gradient of 50% to 80% EtOAc–pentane)
to give (E)-13 (187.2 mg, 0.69 mmol, 73%) as a pale yellow oil.
Pharmacological Evaluation
Determination of log D7.4
The determination of log D value was performed using the shake-
flask method as previously reported.16,29 Formulated solution of
(E)-[18F]-4 (ca. 1 MBq) was partitioned between phosphate buffer
(pH 7.4) saturated with octan-1-ol (700 μL) and octan-1-ol saturated
with phosphate (pH 7.4) buffer (700 μL). Washed octan-1-ol phase
was divided in five individual aliquots (100 μL) and each was dilut-
ed with phosphate (pH 7.4) buffer saturated with octan-1-ol (100
μL) and the two phases were shaken and radioactivity in each phase
was measured in a γ-counter.
Competition Binding Assay
Brain membranes were prepared from Sprague-Dawley rat brains as
described previously.16,22 Frozen membranes were thawed on ice
and pelleted at 45000 g at 4 °C for 5 min. The membranes were
washed twice with HEPES buffer (30 mM HEPES, 110 mM NaCl,
5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, pH 8 at 4 °C) and resus-
IR (neat): 3372, 2940, 2874, 2197, 1581, 1464, 1355, 1059, 865,
779 cm–1.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1877–1885

1884
S. Milicevic Sephton et al.
PAPER
pended in HEPES buffer at a protein concentration of 1.3 mg/mL.
The binding assay was performed as previously described.16 In
brief, brain membranes (0.1 mg protein) were incubated in tripli-
cates at ambient tempearture with 2 nM [3H]-1 and (E)-4 or (Z)-4 at
concentrations between 10 pM and 100 μM in a total volume of 0.2
mL HEPES. Compound (E)-4 or (Z)-4 was diluted from a 1 mM
ethanolic (50%) solution. The corresponding EtOH concentrations
did not affect [3H]-1 binding (data not shown). Unspecific binding
of [3H]-1 was estimated with 100 μM MMPEP. After 45 min, the
samples were filtered and the filters containing the membranes with
bound [3H]-1 were measured in a β-counter (Beckman LS6500). To
estimate IC50, bound [3H]-1 (B, pmol per mg protein) was fitted
with Excel solver to
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Y. P.; Sovago, J.; Stockmeier, C.; Buck, A.; Hasler, G. Am.
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B = Bmin + {(Bmax – Bmin)/[1 + (C/IC50)]}
where C is the total (E)-4 or (Z)-4 concentration; Bmax is the maxi-
mal B, that is, the plateau in the B/C plot at low log C; and Bmin is
the minimal B, that is, the plateau at high log C. The inhibition con-
stant Ki of (E)-4 or (Z)-4 was estimated from IC50 and Kd of 1 (1.7
± 0.2 nM) with the Cheng–Prusoff equation. For each compound
three independent experiments were performed in triplicate.
Abbreviations
mGlu5: Metabotropic glutamate receptor subtype 5; PET: positron
emission tomography; [11C]-ABP688: (E)-3-[(6-methylpyridin-2-
yl)ethynyl]cyclohex-2-enone O-[11C]methyl oxime; [3T]-ABP688:
(10) Hintermann, S.; Vranesic, I.; Allgeier, H.; Bruelisauer, A.;
Hoyer, D.; Lemaire, M.; Moenius, T.; Urwyler, S.;
Whitebread, S.; Gasparini, F.; Auberson, Y. P. Bioorg. Med.
Chem. 2007, 15, 903.
(E)-3-[(6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone
O-
[3T]methyl oxime; [18F]-FDEGPECO: (E)-3-(pyridin-2-ylethy-
nyl)cyclohex-2-enone O-[2-(2-[18F]fluoroethoxy)ethyl] oxime;
[18F]-PSS223: (E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[2-
(3-[18F]fluoropropoxy)ethyl] oxime; [18F]-PSS232: (E)-3-(pyridin-
2-ylethynyl)cyclohex-2-enone O-[3-(2-[18F]fluoroethoxy)propyl]
oxime; MMPEP: 2-[(3-methoxyphenyl)ethynyl]-6-methylpyridine;
PBS: phosphate buffer in saline; IC50: half maximal inhibitory con-
centration; Ki: inhibition constant; TBS: tert-butyldimethylsilyl;
TES: triethylsilyl, TBAF: tetrabutylammonium fluoride; TBAC:
triethylbenzylammonium chloride; DIAD: diisopropylazodicarbox-
ylate.
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Acknowledgment
The authors would like to acknowledge Dr. Thomas Nauser and Mr.
Mathias Nobst for technical assistance in the radiochemistry lab.
Mr. Patrick Dennler (PSI) is acknowledged for suggesting applica-
tion of InBr3/Et3SiH method for the ester reduction. Dr. Mark A.
Sephton (ZHAW) is acknowledged for proofreading the manuscript
and discussions on NMR assignments. Prof. P. A. Schubiger, Drs.
Cindy A. Wanger-Baumann, Thomas Betzel, Adrienne Müller, and
Miss Cindy R. Fischer are acknowledged for many useful discus-
sions.
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M. F.; Schubiger, P. A.; Kramer, S. D.; Ametamey, S. M.
NeuroImage 2011, 56, 984.
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Baumann, C. A.; Schibli, R.; Krämer, S. D.; Ametamey, S.
M. Am. J. Nucl. Med. Mol. Imaging 2012, 2, 14.
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257.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis. Included
are full experimental procedures and characterization of all com-
pounds, NMR data, HPLC traces, and IC50 binding curves in tripli-
cate.SungIifo
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(21) Patent for this work was filed. Application No. 12170914.1-
2101, 05.06.2012.
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Krämer, S. D.; Ametamey, S. M. J. Med. Chem. 2012, 55,
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