(R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2, 5-dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3- phenylpyrazinones as potential CRF-R1 PET imaging agents

Article abstract of DOI:10.1016/j.bmcl.2012.08.112

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [¹⁸F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.

Full text of DOI:10.1016/j.bmcl.2012.08.112

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6651–6655
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-
dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3-phenylpyrazi-
nones as potential CRF-R1 PET imaging agents
Jeffrey A. Deskus a, , Douglas D. Dischino a, Ronald J. Mattson a, Jonathan L. Ditta a, Michael F. Parker a,
⇑
Derek J. Denhart a, Dmitry Zuev a, Hong Huang a, Richard A. Hartz a, Vijay T. Ahuja a, Henry Wong a,
Gail K. Mattson a, Thaddeus F. Molski a, James E. Grace Jr. a, Larisa Zueva b, Julia M. Nielsen b, Heidi Dulac a,
Yu-Wen Li a, Mary Guaraldi c, Michael Azure c, David Onthank c, Megan Hayes c, Eric Wexler c,
Jennifer McDonald c, Nicholas J. Lodge a, Joanne J. Bronson a, John E. Macor a
a Bristol-Myers Squibb Co., Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
b Spectrix Analytical Services, LLC, PO Box 234, Middletown, CT 06457, USA
c Bristol-Myers Squibb Medical Imaging, North Billerica, MA 01862, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10
was evaluated for potential development as PET radioligand. Compound [18F]10 was prepared with high
radiochemical purity and showed promising binding properties in rat brain imaging experiments.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 June 2012
Revised 13 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
Corticotropin-releasing factor
Positron Emission Tomography
Antidepressants
Anxiolytics
Radioligand
Corticotropin-releasing factor (CRF), a 41 amino acid-containing
neuropeptide, mediates the endocrine, autonomic, and behavioral
responses to stress.1 CRF regulates the hypothalamic-pituitary-
adrenal (HPA) axis through binding to the CRF receptor, a G-pro-
tein coupled receptor for which two subtypes have been identified
(CRF-R1 and CRF-R2). In response to physical or psychological
stress, CRF is released from the hypothalamus and binds to CRF
receptors in the anterior pituitary, resulting in the secretion of
adrenocorticotropin hormone (ACTH). Increased ACTH stimulates
the release of cortisol from the adrenal gland.2 In order to stabilize
the HPA axis, cortisol exerts negative feedback control on the
secretion of CRF in the hypothalamus.3 Severe stress, resulting in
a hyper-secretion of CRF and extended activation of the HPA axis,
may lead to disorders such as anxiety or depression.4–6 CRF recep-
tor antagonists, in particular antagonists of the CRF-R1 subtype,
may have potential as novel antidepressants and anxiolytics.
In support of the evaluation of potential CRF-R1 antagonist
clinical candidates, efforts have been undertaken to develop an
appropriate radioligand for a Positron Emission Tomography
(PET) study that would provide a useful clinical tool to monitor lev-
els of receptor occupancy. The earliest reported effort to develop
CRF PET radioligands was in 2001, but the 18F-labeled compound
described suffered from high lipophilicity and poor brain penetra-
tion.7 Later, Eckelman8 disclosed [76Br]-MJL-1-109-2, 1a (Fig. 1), a
high affinity CRF-R1 PET radioligand (Ki ꢀ2 nM) with a reasonable
lipophilicity (clogP = 3). In rat biodistribution studies, this com-
pound showed limited retention of activity in brain with cerebel-
lum and cortex uptake of 0.29 0.01% injected dose per gram
(ID/g) and 0.32 0.03% ID/g, respectively, after 30 min. However,
no further experiments have been reported. Recently, Lang re-
ported a radiosynthesis of a similar analog, [76Br]-BMK-I-152, 1b,
with lower lipophilicity and improved brain penetration, but has
yet to publish any in vivo results.9 Kumar10 evaluated [11C]SN003
(2), and Sullivan11 studied [11C]R121920 (3) and [11C]DMP696 (4)
in baboons (Fig. 1). Each of these studies concluded that the lack
of detectable specific binding may be due to lower density CRF-
R1 receptors in primate brain as compared to rat or human brain.
In addition, while these latter PET ligands had high affinities for the
CFR-R1 receptor (Ki 1.7–4 nM) and reasonable lipophilicities (logD
⇑
Corresponding author.
E-mail address: jeffrey.deskus@bms.com (J.A. Deskus).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.112

6652
J. A. Deskus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6651–6655
O
R
N
R
N
NH2
O
O
O
O
O
X
N
N
N
N
N
a
X
N
NH
+
R'
N
N
N
X
N
R'
N
NH
Y
R"
5
X
6a, R"= CH3
6b, R"= H
R'= H, Cl, CH3
O
X= Br, Cl
R"
Br76
7a, R"= CH3
7b, R"= H
O 11
b
CH3
R
N
76Br]-MJL-1-109-2; X=H, Y=Cl
1a:
1b:
[
11C]SN003
O
[
[
76Br]-BMK-I-152; X=Y=OMe
N
2
c
7b
X
N
NH
11CH3
O
R'
N
N
O
N
N
O
N
R"
N
7a, R"= CH3
7c, R"= CH2F
7d, R"= CH2CH2F
N
Cl
N
N 11CH3
Scheme 1. Reagents and conditions: (a) NaHMDS, THF; (b) BBr3, CH2Cl2, -78 °C; (c)
CH3I or FCH2Br or FCH2CH2Br, KOH, DMSO, 65 °C.
Cl
11C]R121920
[
11C]DMP696
[
4
3
7a with boron tribromide at low temperature (Scheme 1). Alterna-
tively, the pyrazinone core 5 could be coupled directly to phenol 6b
to give 7b directly, but the yields of this reaction were lower com-
pared to the two step process. From phenol 7b, 7a (for a 11C label),
7c and 7d were prepared by alkylation with methyl iodide, bromo
fluoromethane, or 1-bromo-2-fluoroethane respectively, in the
presence of potassium hydroxide in dimethyl sulfoxide with heat-
ing. This synthetic route (Scheme 1) provided a rapid, general
method to use for the radiolabeling and evaluation of candidate
compounds 8–17 as shown in Tables 1 and 2.
Key parameters for analogs 8–11 are shown in Table 1 and for
analogs 12–17 in Table 2. The CRF-R1 binding affinities of these
analogs were determined by measuring the inhibition of specific
binding of [125I]-o-CRF in a CRF-R1 receptor binding assay using
rat frontal cortex homogenate.15 The partition coefficients (logD)
of these and previous analogs were determined by classical octa-
nol-phosphate buffer (pHꢀ7.4) partitioning methodology.16
Figure 1. Previously reported potential CRF PET ligands.
3.1–4), the studies found that all three 11C radioligands underwent
rapid metabolism in baboon, with compounds 3 and 4 showing
only 50% of parent remaining after only
9 and 13 min
respectively.11
To identify compounds as potential CRF PET radioligands, we
hoped to achieve high CRF-R1 affinity (Ki < 5 nM) and provide ana-
logs with reasonable protein binding (free fractions >1%). Suitable
compounds would have high brain uptake and be stable with no
radiolabeled metabolites in brain. Ideal compounds would also
not be subject to efflux mechanisms. Each candidate molecule
would make use of a facile synthesis of 11C or 18F -labeled deriva-
tives. Lipophilicity, measured as an experimental octanol-water
partition coefficient (logD),12 was also an important consideration.
It is well appreciated in the CRF antagonist field that the most po-
tent compounds tend to be highly lipophilic (logD > 4).2 For a suit-
able PET ligand, we set a minimum criteria of logD < 4.5, although
a logD in the 1–2.5 range was preferred.13
Our previously reported series of N3-phenylpyrazinone CRF-R1
antagonists14 was chosen to explore the potential of identifying a
suitable PET ligand. An extensive series of pyrazinone analogs
(i.e., 7, Scheme 1) had been prepared with several compounds ana-
lyzed to determine their lipophilicities. While CRF-R1 affinity in
this series of compounds typically increased with higher lipophil-
icity, we focused our efforts on analogues with an N-1 side-chain
bearing a methoxy group (in R, Scheme 1), since these derivatives
had generally lower lipophilicity. The site chosen for introduction
of a 11C or 18F label was the hydroxyl group on the 3-arylamino
substituent. Here in we describe the properties and SAR of several
analogs appropriate for labeling, and the radiosynthesis and initial
evaluation of [18F]10 as a potential CRF-R1 PET imaging agent.
The preparation of these N3-phenylpyrazinone analogs has been
previously published.14 Initially, 3-(4-methoxy)arylamino analogs
(6, Scheme 1) were prepared from anilines obtained through com-
mercial sources, then coupled to the pyrazinone cores (Scheme 1).
The preparation of phenol 7b was achieved by coupling pyraz-
inone core 5 to a 4-methoxy aniline 6a, followed by treatment of
Table 1
CRF-R1 binding affinities, phase distribution coefficients and the metabolic stabilities
of selected initial analogs
R1
O
N
N
O
Cl
NH
O
R2
Compounds
R1
R2
IC50,nM
logD
hLM/rLMa
8
9
10
11
c-Pr
c-Pr
c-Pr
Me
Me
CH2F
CH2CH2F
Me
0.26
4.1
2.4
4.4
3.1
3.7
3.4
0.17/0.23
À/À
0.07/0.12
0.06/0.19
0.88
a
Rate of compound depletion (pmol/min/mg protein); see Ref. 19.

J. A. Deskus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6651–6655
6653
Table 2
CRF-R1 binding affinities, phase distribution coefficients and the metabolic stabilities
of later synthesized, more polar analogs
O
N
N
O
18
F
O
O
O
a
Br
Br
+
OTf
N
NH
Cl
19
18
X
N
NH
R1
R4
O
OH
N
N
O
R2
20
b
O
R3
NH
Cl
Compounds
X
R1/R2/R3/R4
IC50,nM
logD
hLM/rLMa
12
13
14
15
16
17
Cl
Cl
Cl
Br
Br
Br
Cl/H/Me/Cl
5.0
5.4
210
7.8
4.3
3.4
3.1
3.1
2.8
2.9
3.8
0.03/0.17
À/À
Cl/H/CH2F/Cl
Cl/H/CH2CH2F/Cl
Me/H/Me/Me
Me/H/CH2F/Me
H/Me/Me/Me
0.07/0.12
À/À
18
O
F
0.22/0.26
0.07/0.14
[
18F]10
0.68
a
Scheme 2. Reagents and conditions: (a) freshly distilled 18, K18F, Kryptofix 222,
1,2-dichlorobenzene, 125 °C, under N2, 10 min.; (b) solid KOH, DMSO, 60–70 °C,
12 min, water, C-18 Sep-Pak/acetonitrile, preparative HPLC.
Rate of compound depletion (pmol/min/mg protein); see Ref. 19.
Initial pyrazinone analogs 8–11 had good CRF-R1 binding affin-
ities due to a less polar top amine side chain. Compound 8 was
found to have a rat plasma protein free fraction of 2.1%.17 Com-
pound 1018 displayed good binding affinity to the CRF-R1 receptor,
but was 10-fold less potent than its methoxy counterpart 8. The
presence of the fluoroethoxy group in 10, however, lowered the
logD by nearly 0.7 of a unit relative to 8, and improved metabolic
stability19 in both human and rat liver microsomes when com-
pared to 8. Compound 11 was more potent at CRF-R1 and slightly
more polar than 10, but was less metabolically stable in rat. Thus,
analog 10 offered a balance between binding potency, lipophilicity
and metabolic stability when compared with 8 or 11.
Biodistribution of F-18 Labeled 10 in Rats
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Ave % ID/G 15 min
Ave % ID/G 60 min
During the development of 10 as a first candidate for radio-
labeling, several newer analogs were synthesized in an effort to
further lower logD values while retaining CRF-R1 potency. These
compounds, which bear two methoxy groups on N-1, are shown
in Table 2. By the incorporation of an additional oxygen on the
top amine side chain, replacement of the core chlorine with bro-
mine, plus the choice and movement of the substituents on the
lower aniline ring moiety, logD values of <3 were obtained for
compounds 15 and 16, with single digit nanomolar CRF-R1 binding
being retained. In addition, these compounds had free fractions
ranging between 3-6%.17 By replacement of the 4-methoxy group
with fluoromethoxy as in compound 16, affinity increases without
a substantial increase of lipophilicity as compared to 15. Com-
pound 17 showed excellent affinity and metabolic stability, but
the compromise was slightly higher lipophilicity than 12 and 13.
In all, compounds like 12, 13, and 15–17 were of interest due to
their combined properties, particularly with their logD values
being in a more desirable range for a potential PET ligand.
Figure 2. Multi-tissue biodistribution of [18F]10 in rats.
Metabolism studies were conducted 10 min post injection with
[
18F]10 (353
lCi per rat) to determine the nature of activity in
brain and blood. Collected blood and brain samples were analyzed
by HPLC (Fig. 3) and compared to the initial radioligand with a
radiochemical purity (RCP) of 100 percent. After 10 min, 50 percent
of the radioactivity found in the blood was due to a radiolabeled
metabolite. Analysis of the brain sample at 10 min showed less
than 10 percent of a similar Rt radioactive material, which was de-
rived from blood present in the brain. The appearance of the sub-
stantial metabolite peak seen within the blood sample was not
seen in the brain sample. This study demonstrated that only one la-
beled compound was the major component present in the brain
after 10 min.
Based on its overall properties, the 18F -labeled version of 10
was first chosen for further study in rats. The radiosynthesis of
[
18F]1020 is shown in Scheme 2. [18F]10 was routinely obtained
in yields of 5–12 mCi, with specific activities of 0.25–0.50 Ci/
and radiochemical purities >98%.
lmol,
Compound [18F]10 was then characterized in a series of studies
which looked at organ/tissue distribution, the possible formation
of radiolabeled metabolites, and brain uptake imaging. Compound
[
18F]10 was first evaluated in a whole body biodistribution study
(Fig. 2), with samples analyzed at 15 min and 60 min intervals.
Compound levels were uniform across ten tissue types at both time
points. Of particular interest was the relatively low distribution to
the tibia, which demonstrated that a loss of fluorine locating to
bone was not a significant issue with this compound, and thus sug-
gested good stability of the radiolabel.
Figure 3. Nature of radioactivity in blood and brain for [18F] 10 in rats.

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J. A. Deskus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6651–6655
concluded that this particular compound was likely not an opti-
mum tool, and that compounds with either higher CRF-R1 poten-
cies and/or lower logD values may be needed.
In conclusion, we identified compound 10 with a balance be-
tween CRF-R1 affinity, low lipophilicity and metabolic stability
for potential development as a PET radioligand. 18F radiolabeled
compound 10 was then prepared with high RCP, and was then
studied in a series of rat experiments. Based on its promising prop-
erties, [18F]10 was evaluated in rat brain slice imaging experiments
to determine if this compound could be advanced into further
studies in primates. While [18F]10 did not prove to be the best tool
to advance into primate studies, we were encouraged to continue
to develop newer analogs from this series of compounds (see
Table 2) with similar CRF-R1 affinity and even lower lipophilicity.
These improved compounds, their radiosynthesis, and biological
studies will be the subject of future publications.
Figure 4. MicroPET sagittal images of [18F] 10 in rat.
References and notes
1. (a) Owens, M.; Nemeroff, C. B. Pharmacol. Rev. 1991, 43, 425; (b) Grigoriadis, D.
E.; Haddach, M.; Ling, N.; Saunders, J. Curr. Med. Chem. CNS Agents 2001, 1, 63.
2. Dzierba, C. D.; Hartz, R. A.; Bronson, J. J. Annu. Rep. Med. Chem. 2008, 43, 3.
3. Plotsky, P. M. J. Neuroendocrinol. 1991, 3, 1.
4. Banki, C. M.; Karmasci, L.; Bissette, G.; Nemeroff, C. B. Eur.
Neuropsychopharmacol. 1992, 2, 107.
5. Holsboer, F. J. Psychiatric Res. 1999, 33, 181.
6. Kaskow, J. W.; Baker, D.; Geracioti, T. D. Peptides 2001, 22, 845.
7. Martarello, L.; Kilts, C. D.; Ely, T.; Owens, M. J.; Nemeroff, C. H.; Camp, M.;
Goodman, M. M. Nucl. Med. Biol. 2001, 28, 187.
8. Jagoda, E.; Contoreggi, C.; Lee, M.-J.; Kao, C.-H. K.; Szajek, L. P.; Listwak, S.; Gold,
P.; Chrousos, G.; Greiner, E.; Kim, B. M.; Jacobson, A. E.; Rice, K. C.; Eckelman, W.
J. Med. Chem. 2003, 46, 3559.
9. Lang, L.; Ma, Y.; Kim, B. M.; Jagoda, E. M.; Rice, K. C.; Szajek, L. P.; Contoreggi, C.;
Gold, P. W.; Chrousos, G. P.; Eckelman, W. C.; Kiesewetter, D. O. J. Label. Compd.
Radiopharm. 2009, 52, 394.
10. Dileep Kumar, J. S.; Majo, V. J.; Sullivan, G. M.; Prabhakaran, J.; Simpson, N. R.;
Van Heertum, R. L.; Mann, J. J.; Parsey, R. V. Bioorg. Med. Chem. 2006, 14, 4029.
11. Sullivan, G. M.; Parsey, R. V.; Dileep Kumar, J. S.; Arango, V.; Kassir, S. A.; Huang,
Y.; Simpson, N. R.; Van Heertum, R. L.; Mann, J. J. Nucl. Med. Biol. 2007, 34, 353.
12. Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 6, 525.
13. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E. J. Nucl. Med. 1983,
24, 1030.
14. (a) Hartz, R. A.; Ahuja, V. T.; Zhuo, X.; Mattson, R. J.; Denhart, D. J.; Deskus, J. A.;
Vrudhula, V. M.; Pan, S.; Ditta, J. L.; Shu, Y-Z.; Grace, J. E.; Lentz, K. A.; Lelas, S.;
Li, Y-W.; Molski, T. F.; Krishnananthan, S.; Wong, H.; Qian-Cutrone, J.;
Schartman, R.; Denton, R.; Lodge, N. J.; Zaczek, R.; Macor, J. E.; Bronson, J. J. J.
Med. Chem. 2009, 52, 7653; (b) Hartz, R. A.; Ahuja, V. T.; Arvanitis, A. G.;
Rafalski, M.; Yue, E. W.; Denhart, D. J.; Schmitz, W. D.; Ditta, J. L.; Deskus, J. A.;
Brenner, A. B.; Hobbs, F. W.; Payne, J.; Lelas, S.; Li, Y-W.; Molski, T. F.; Mattson,
G. K.; Peng, Y.; Wong, H.; Grace, J. E.; Lentz, K. A.; Qian-Cutrone, J.; Zhuo, X.;
Shu, Y-Z.; Lodge, N. J.; Zaczek, R.; Combs, A. P.; Olson, R. E.; Bronson, J. J.;
Mattson, R. J.; Macor, J. E. J. Med. Chem. 2009, 52, 4173; (c) Hartz, R. A.; Ahuja, V.
T.; Rafalski, M.; Schmitz, W. D.; Brenner, A. B.; Denhart, D. J.; Ditta, J. L.; Deskus,
J. A.; Yue, E. W.; Arvanitis, A. G.; Lelas, S.; Li, Y-W.; Molski, T. F.; Wong, H.;
Grace, J. E.; Lentz, K. A.; Li, J.; Lodge, N. J.; Zaczek, R.; Combs, A. P.; Olson, R. E.;
Mattson, R. J.; Bronson, J. J.; Macor, J. E. J. Med. Chem. 2009, 52, 4161.
15. CRF-R1 binding assay: Frozen rat frontal cortex (source of CRF-R1 receptor) was
thawed rapidly in assay buffer containing 50 mM HEPES (pH 7.0 at 23 °C)
10 mM MgCl2, 2 mM EGTA, 1 lg/mL aprotinin, 1 lg/mL leupeptin, 1 lg/mL
pepstatin A, 0.005% Triton X-100, 10U/mL bacitracin and 0.1% ovalbumin and
homogenized. The suspension was centrifuged at 32,000g for 30 min. The
resulting supernatant was discarded and the pellet re-suspended by
homogenization in assay buffer and centrifuged again. The supernatant was
discarded and the pellet re-suspended by homogenization in assay buffer and
frozen at À70 °C. On the day of the experiment aliquots of the homogenate
Figure 5. In vitro autoradiographic images of [18F]10 in rat brain slices.
Brain uptake was evaluated using in vivo MicroPET studies in
rats. The sagittal images shown in Figure 4 are ear side views of
rat head and neck. These studies showed rapid brain uptake within
10 min of dosing with subsequent rapid washout by 30 min. The
MicroPET images also demonstrate very little defluorination of
were thawed quickly and homogenate (25
l
g/well rat frontal cortex) added to
L of assay
ligand (150 pM 125I-o-CRF) and drugs in a total volume of 100
l
buffer. The assay mixture was incubated for 2 h at 21 °C. Bound and free
radioligand were then separated by rapid filtration, using glass fiber filters
(Whatman GF/B, pretreated with 0.3% PEI) on a Brandel Cell Harvester. Filters
were then washed multiple times with ice cold wash buffer (PBS w/o Ca2+ and
Mg2+, 0.01% Triton X-100 (pH 7.0 at 23 °C)). Nonspecific binding was defined
[
18F]10 as evidenced by a lack of skull bone uptake of radioactivity.
Finally, an in vitro autoradiography imaging study21 was carried
out to determine the localization of the radioligand [18F]10 in rat
brain slices. This study evaluated the uptake of the compound at
the receptors and showed the extent of non-specific (NS) binding.
Analysis of the sagittal images (Fig. 5) showed 18% specific binding
for the neocortex region of the brain, and 19% specific binding for
the cerebellum. Since [18F]10 showed low specific binding, we
using 1 lM DMP696. Filters were then counted in a Wallac Wizard gamma
counter. Affinity is reported as an average from at least two test occasions,
where each sample was evaluated at five concentrations. Materials: Rat frontal
cortex was obtained from Analytical Biological Services, Inc. (Wilmington, DE).
125I-o-CRF (2200 Ci/mmol) was obtained from PerkinElmer Life Sciences, Inc.
(Boston, MA).
16. Shake-flask logD determination assay for lipophilicity: In a 250 mL separatory
funnel were added 25 mM phosphate buffer (200 mL) and octanol (10 mL). The

J. A. Deskus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6651–6655
6655
two-phase system was mixed well and let stand overnight to allow complete
saturation and separation of both layers. A sample (1.0 mg) was dissolved in
octanol (1 mL) and transferred to a volumetric flask, containing 50 mL of the
phosphate buffer, saturated with octanol, as described above. The resulting
mixture was shaken intensely for 30–40 min and was allowed to stand until
two layers separated completely. A sample of each layer was analyzed by an
HPLC method twice. The sample area counts were used to calculate shake-flask
logD.
165 mg of K222, 14 mL of CH3CN) followed by 1 mL of CH3CN. The solution was
then evaporated by placing the vial into a 90 °C oil bath, and applying a gentle
stream of N2 and a partial vacuum. When the volume of the liquid was reduced
to less than 0.3 mL, a 0.5 mL aliquot of CH3CN was added and the solution
reduced by azeotropic distillation. This process was repeated 3 times in an
effort to remove all traces of water. During the final procedure, the vial was
removed from the oil bath before the solution had gone to dryness and
the residue in the vial was placed under full vacuum at room temperature
for 6 min. After 6 min, the vacuum was removed and 1,2-dichlorobenzene
17. Protein binding studies: Unbound fraction of test compounds in rats was
determined in vitro by equilibrium dialysis using the Dianorm dialysis system.
Rat plasma was spiked with the test compound and equilibrated against
isotonic phosphate buffer for 3 h at 37 °C. Following the incubation period,
plasma and buffer samples were analyzed for compound concentrations using
LC/MS/MS. Unbound fraction was calculated based on the ratio between buffer
concentration and the plasma concentration.
(300 lL) containing freshly distilled bromoethyl triflate (20 lL) was added
to the vial. Formation and distillation of the F-18 bromofluoroethane
was achieved by placing the reaction vial in a 130 °C oil bath and having a
slow stream of N2 gas, flow through the vial and into
a receiving flask
containing the corresponding phenol (2.3 mg), KOH (3.8 mg), DMSO (0.3 mL)
and a small stirring bar. After 10 min, the N2 gas flow was discontinued, the
receiving flask sealed and the reaction mixture allowed to stir at 60–70 °C for
15 min. The vial was then cool to RT and diluted with 7 mL of water and passed
through a C-18 SEP-PAKÒ cartridge. The cartridge rinsed with H2O (10 mL) and
the activity eluted from the cartridge with CH3CN (2.5 mL). The sample was
then diluted with H2O (2.5 mL) and purified by semi-preparative HPLC (Zorbax
18. Analytical data for (R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-
fluoroethoxy)-2,5-dimethylphenylamino)pyrazin-2(1H)-one:
1H
NMR
(400 MHz, CDCl3): d 0.35–0.32 (m, 1H), 0.50–0.48 (m, 1H), 5.88 (m, 1H), 0.76
(m, 1H), 0.86 (m, 1H), 2.24 (s, 3H), 2.28 (s, 3H), 3.33 (s, 3H), 3.68–3.64 (m, 1H),
3.75–3.71 (m, 1H), 4.16–4.13 (m, 1H), 4.22–4.20 (m, 2H), 4.68–4.66 (m, 1H),
4.80–4.78 (m, 1H), 6.65 (s, 1H), 6.68 (s, 1H), 7.24 (s, 1H), 8.01–7.92 (m, 1H).
HRMS: 410.03 (MH+); Rt 2.78 min.; analytical LC/MS: Phenomenex LUNA
Rx-C18,
5
micron column, 9.4 Â 250 mm, 4.6 mL/min, 260 nm) with an
isocratic mobile phase of 62% CH3CN and 38% 0.1% TFA water. Under these
conditions the radioactive product eluted from the column ꢀ14 min. The
radioactive product was collected and formulated by diluting the sample with
H2O (20 mL), concentrating the product on a C-18 SEP-PAKÒ cartridge, rinsing
the cartridge with H2O (5 mL) and eluting with EtOH (1 mL). The EtOH sample
was then diluted with saline to yield a 10% EtOH/saline solution. In this
procedure a sample of 9.2 mCi of HPLC purified product was available in
132 min.
4.6 Â 50 mm; S5; inj. 5
l
L; solvent A, CH3OH(10%)/H2O(90%) with TFA(0.1%);
solvent B, CH3OH(90%)/H2O(10%)/with
TFA(0.1%); starting%B = 0;
final%B = 100; flow rate 4 mL/min; gradient time, 2 min; end time, 3 min; UV
at 220 nm; purity 95%.
19. Metabolic stability assay: Estimates of rates of compound turnover (pmol/min/
mg protein) were generated using human and rat liver microsomes (BD
Gentest, Woburn, MA). The stability in liver microsomes was determined by a
high-throughput in-house assay in which 3
l
M of compound was incubated
21. [ lm
18F]10 in vitro autoradiographic studies: Fresh sagittal brain sections (20
with 1.3 mM NADPH and 1 mg/ml of microsomal protein at 37 °C. Incubations
were performed in sodium phosphate buffer (100 mM), at pH 7.4, and were
terminated at 0 and 10 min following the start of the incubations. The final
organic concentration of solvent was 0.015% DMSO and 0.985% acetonitrile.
Compound concentrations were determined by LC/MS/MS.
thick) were obtained from adult male Sprague–Dawley rats. Sets of the brain
sections were preincubated in buffer containing 50 mM HEPES, 10 mM MgCl2,
0.2 M EGTA, pH 7.4) for 10 min. The sections were then incubated with 1.3 nM
[
18F]10 (1.3 mCi/mL) in the same buffer at 22–24 °C for 40 min. Nonspecific
binding was defined by incubation of adjacent sections with [18F]10 in the
presence of DMP696, selective CRF-R1 receptor antagonist. After
20. F-18 labeled 10: A 250 mCi sample of aqueous F-18 fluoride was obtained
from P.E.T. NET Pharmaceutical Services, Milstein Hospital, New York, NY
and applied to a previously activated QMA cartridge contained within our
remote control radiosynthesis system. After passing N2 through the cartridge,
the activity was eluted from the cartridge and introduced into a 5 mL custom
crimp seal vial by the addition of 0.1 mL of K2CO3 (6 mg/mL deionized water),
1 mL of the following stock solution (30 mg of K2CO3, 1 mL of deionized water,
1
l
M
a
incubation, slides were washed in ice-cold PBS (pH 7.4) for 2 min. The slides
were then air dried and exposed to multi-purpose phosphor screens overnight
and scanned by the Cyclone Storage Phosphor Imaging System (Packard
Instruments Co.). The imaging of [18F]10 in the brain sections was analyzed
using the OptiQuant acquisition and analysis program (Packard Instruments
Co.).