Potential CRF1R PET imaging agents: 1-Fluoroalkylsubstituted 5-halo-3-(arylamino)pyrazin-2(1H)-ones

Article abstract of DOI:10.1016/j.bmcl.2013.02.009

A series of pyrazinones were prepared and evaluated as potential CRF ₁R PET imaging agents. Optimization of their CRF₁R binding potencies and octanol-phosphate buffer phase distribution coefficients are discussed herein.

Full text of DOI:10.1016/j.bmcl.2013.02.009

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2052–2055
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potential CRF1R PET imaging agents: 1-Fluoroalkylsubstituted
5-halo-3-(arylamino)pyrazin-2(1H)-ones
a,
a
a
a
Derek J. Denhart a, , Dmitry Zuev , Jonathan L. Ditta , Richard A. Hartz , Vijay T. Ahuja ,
Ronald J. Mattson a, Hong Huang a, Gail K. Mattson a, Larisa Zueva b, Julia M. Nielsen b,
Edward S. Kozlowski a, Nicholas J. Lodge a, Joanne J. Bronson a, John E. Macor a
⇑
⇑
a Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA
b Spectrix Analytical Services, LLC, PO Box 234, Middletown, CT 06457, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazinones were prepared and evaluated as potential CRF1R PET imaging agents. Optimiza-
tion of their CRF1R binding potencies and octanol–phosphate buffer phase distribution coefficients are
discussed herein.
Received 13 November 2012
Revised 25 January 2013
Accepted 1 February 2013
Available online 13 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
CRF
PET
Depression
Pyrazinones
Fluoroalkyl
Corticotropin-releasing factor, a 41-amino acid neuropeptide
produced in the hypothalamus, plays a central role in the coordina-
tion of neuroendocrine, autonomic, behavioral and immune re-
sponses to stress.1,2 Secreted from the hypothalamus in response
to acute physical or psychological stress, CRF activates the tran-
scription of a gene which results in the secretion of adrenocortico-
tropin hormone (ACTH) from the pituitary gland. In turn, ACTH
stimulates the release of the steroid hormone cortisol from the
adrenal gland. To restore the balance of the hypothalamic–pitui-
tary–adrenal (HPA) axis, cortisol exerts negative feedback control
on the secretion of CRF in the hypothalamus.3 One hypothesis is
that severe or prolonged stress results in an excessive secretion
of CRF and a long-term activation of the HPA axis, which may lead
to a variety of stress-related illnesses, such as anxiety, depression,
obsessive–compulsive and posttraumatic stress disorders.4a–c
CRF mediates its function through the CRF1 and CRF2 receptor
subtypes,5 of which the CRF1 receptor appears to play a significant
role in the stress-related responses. It has been hypothesized that
selective CRF1R antagonists may be useful for the treatment of the
psychiatric disorders. In the past decade and a half, a number of
potent non-peptide CRF1R antagonists have been reported, reflect-
ing significant efforts of many research groups in this area.6,7
In light of this progress, the development of a selective CRF1R
Positron Emission Tomography (PET) radioligand could provide
scientists with a powerful tool to assess receptor occupancy in clin-
ical trials of CRF1R receptor antagonists in normal subjects and pa-
tients. Development of PET radioligands, however comes with its
own set of unique challenges, requiring compounds with potent
and selective target affinity, and low non-specific binding.8
In 2000, Rice and co-workers9 published the synthesis of unla-
belled fluorinated pyrrolo[2,3-d]pyrimidines as high-affinity po-
tential CRF1R PET ligands 1 ( Fig. 1). A year later, Martarello
et al.10 described the radiosynthesis, in vitro binding studies and
in vivo rat tissue distribution of [18F] FBPPA 2, the first reported
CRF1R 18F ligand. The initial level of accumulation of radioactivity
of 2 in the rat pituitary was fairly high (5.59%) but the ligand exhib-
ited fast washout (79.2% after 60 min). Very poor levels of radioac-
tivity and retention were displayed in the hypothalamus, amygdala
and cerebellum: the brain areas rich in CRF1 receptor sites. The
authors speculated that the high lipophilicity of 2 (clogP >6)
and/or the insolubility of the radiopharmaceutical in blood could
account for its exceedingly low blood–brain barrier penetration.
In 2003, Eckelman et al.11 disclosed the synthesis of [76Br]-MJL-
1-109-2 3, a high-affinity CRF1R potential PET radioligand (Ki
ꢀ2 nM) with appropriate lipophilicity (clogP = 3). In rat biodistri-
bution studies, this compound penetrated the blood–brain barrier
with cerebellum and cortex uptakes of 0.29 0.01% ID/g and
0.32 0.03% ID/g after 30 min. More recently, Kumar and Sullivan
⇑
Corresponding authors. Fax: +1 203 677 7702.
E-mail addresses: derek.denhart@bms.com (D.J. Denhart), dmitry.zuev@
bms.com (D. Zuev).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.009

D. J. Denhart et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2052–2055
2053
18F
O
O
R
N
F
N
N
N
N
N
N
F
O
N
HO
N
N
N
HN
HN
a
b
N
N
Cl
76Br
10
18F] FBPPA
[
76Br]-MJL-1-109-2
9
[
R = Et, Pr, Bu
2
1
3
F
F
c
d
O
O
11
CH3
O
O
N
N
HN
N
NH2
O
N
N
N
N
N
CN
N
N
11
O
12
N
N
N
F
F
N
O
11CH3
N 11
N
CH
CH3
N
N
O
X
O
N
N
O
e, f
3
[
11C] SN003
[
11C] R121920
[
11C] DMP696
X
X
NH
Ar
4
5
6
Figure 1.
13 X = Cl, Br
7 X = Cl, Br, CN
Scheme 1. Reagents and conditions: (a) 1-Bromo-2-fluoroethane, NaH, DMF, rt, 6 h,
20–84%; (b) H2, 10% Pd–C, EtOH, 5 psi, rt, 30 min, 100%; (c) ClCH2CN, KI, K2CO3,
CH3CN, 550 °C, 18 h, 52–82%; (d) (COX)2, toluene, À78 °C, 15 min, then 55 °C, 15 h,
12–55%; (e) NaHMDS, ArNH2, THF, rt, 1.5 h, 8–97%; (f) for 13 (X = Br) to 7 (X = CN)
Zn(CN)2, Pd(PPh3)4, DMF, 175 °C, 18 h, 49%.
evaluated [11C]SN003 4,12 11C]R121920 5 and [11C]DMP696 613 in
[
baboons. Metabolism of all three radioligands was rapid in baboon,
with compounds 5 and 6 showing just 50% of the parent molecule
remaining after 9 and 13 min, respectively. There was a lack of
detectable specific binding with all three ligands, and this was
attributed to the lower density of CRF1R receptors in primate brain
as compared to rat or human brain.
The goal of the present work was to identify an effective CRF1R
antagonist PET ligand with appropriate physico-chemical proper-
ties that would be useful in assessing the receptor occupancy of
clinical candidates in vivo. In particular, we turned to our pyrazi-
none series7c of potent CRF1R antagonists to find a receptor ligand
having an IC50 65 nM with lipophilicity as measured by logD 64.
Our initial efforts focused on the incorporation of a fluorine la-
bel in the side chain of 1-substituted 5-halo-3-(arylamino)pyrazin-
2(1H)-ones7d with compounds 7 and 8 as the PET ligand targets (
Fig. 2). One hypothesis was that the monocyclic 5-halo-3-(arylami-
no)pyrazin-2(1H)-one core would be advantageous in producing
target compounds with reduced lipophilicity compared to the liter-
ature bicyclic core CRF1R antagonist chemotypes. In addition, an
optimal balance between potency and polarity might be achieved
by a systematic modulation of the steric and electronic properties
of aryl rings, as well as by a variation in substituent X.
n
n
a - d
e
BnO
HO
HN
NH2
CN
14
15
n
n
N
N
BnO
X
BnO
h, i
f
N
N
O
X
O
NHAr
X
17 X = Cl
18 X =Br
19 X = Me
16
The synthetic pathway to derivatives 7 is illustrated in Scheme
1. Previously described (R)-2-cyclopropyl-2-((R)-1-phenylethyl-
amino)ethanol7d 9 was treated with NaH and bromofluoroethane
to give fluoroethyl ether 10. Mild hydrogenolysis followed by
g
n
n
MsO
F
X
j
N
O
N
O
n
F
F
X
O
X
X
N
NHAr
N
NHAr
N
N
O
N
N
O
8 X = Cl, Br, Me
20
NH
Ar
NH
Ar
Scheme 2. Reagents and conditions: (a) Boc2O, K2CO3, CH2Cl2, H2O, rt, 91–96%; (b)
NaH, BnBr, DMF, rt, 63–75%; (c) TFA, rt, 90–95%; (d) ClCH2CN, KI, K2CO3, CH3CN,
55 °C, 18 h, 88–95%; (e) (COX)2, toluene, À78 °C, 15 min, then 55 °C, 15 h, 53–81%;
(f) NaHMDS, ArNH2, THF, rt, 1.5 h, 40–91%; (g) SnMe4, Pd(PPh3)4, DMF, 175 °C, 18 h,
25%; (h) BBr3, CH2Cl2, À78 °C, 81–85%; (i) MsCl, Et3N, CH2Cl2, rt, 77–88%; (j) KF,
DMSO, 90 °C, 3–10%.
7
8
Figure 2.

2054
D. J. Denhart et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2052–2055
Table 1
Table 1 (continued)
CRF1R binding affinities and phase distribution coefficients of amines 7a–k
Compd
Ar-NH2
X
IC50 (nM)
LogD
F
HN
O
N
N
O
7j
Cl
3.2
4.0
Cl
X
NH
Ar
OMe
F
NH2
7
Compd
Ar-NH2
NH2
X
IC50 (nM)
0.46
LogD
7k
Cl
>1000
4.6
F
CN
7a
Cl
Cl
4.6
OMe
NH2
Table 2
CRF1R binding affinities and phase distribution coefficients of amines 8a–d
Cl
Cl
Cl
Cl
Cl
F
F
7b
7c
7d
7e
7f
0.60
0.46
7.7
19
4.0
4.1
3.4
3.3
3.4
N
N
O
N
N
O
OMe
NH2
X
NH
Cl
NH
OMe
Br
CN
Cl
Y
N
N
(+/-)
OMe
OMe
OMe
NH2
8a-c
8d
Cl
Compd
X
Y
IC50 (nM)
LogD
8a
8b
8c
8d
Cl
CH
CH
N
1.1
0.61
4.7
4.5
4.2
4.1
2.9
Me
Me
—
OMe
—
53
NH2
OMe
alkylation of the resulting amine 11 with chloroacetonitrile pro-
duced cyanomethylamine 12. Upon treatment with either oxalyl
chloride or oxalyl bromide, 12 underwent cyclization to pyrazinon-
es 13, which were coupled with a number of anilines to provide
target compounds 7 (X = Cl or Br). For target compound 7 with
X = CN, the corresponding bromide was substituted with cyanide
using zinc cynanide and a palladium catalyst.
N
N
N
Cl
NH2
OMe
Br
14
N
Fluoroalkyl pyrazinones
8 were synthesized as shown in
Cl
Scheme 2. Hydroxyalkylamines 14 were converted to cyanometh-
ylamines 15 by a sequence of standard transformations.7c Upon
treatment with either oxalyl chloride or oxalyl bromide, deriva-
tives 15 were cyclized to the corresponding pyrazinones 16. Cou-
NH2
Cl
7g
7h
7i
Cl
Cl
Cl
4.5
7.4
5.0
3.7
3.4
3.6
pling of the latter compounds with
a variety of anilines
conveniently provided pyrazinones 17 and 18 in moderate to high
yields. 5-Bromo derivatives 18 were converted to 5-methylpyrazi-
nones 19 by a palladium-catalyzed coupling with tetramethylst-
annane. The benzyl groups in 17, 18 and 19 were carefully
removed by the action of boron tribromide in dichloromethane
at low temperature, and the resulting alcohols were converted to
the corresponding mesylates 20. Consecutive displacement with
potassium fluoride provided target compounds 8, albeit in low
yields.
CN
NH2
CN
NH2
Cl
Cl
The CRF1R binding affinities of analogs 7a–k and 8a–d were
determined by measuring the inhibition of specific binding of
[
125I]-o-CRF in a CRF1 receptor binding assay using rat frontal cor-
tex homogenate.14 The logD values of the compounds were deter-
mined by
classical octanol–phosphate buffer (pH ꢀ7.4)
CN
a

D. J. Denhart et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2052–2055
2055
partitioning of the compounds.15 Both sets of data are presented in
Tables 1 and 2.
7. (a) Gilligan, P. J.; Robertson, D. W.; Zaczek, R. J. Med. Chem. 2000, 43, 1641; (b)
Hartz, R. A.; Ahuja, V. T.; Schmitz, W. D.; Molski, T. F.; Mattson, G. K.; Lodge, N.
J.; Bronson, J. J.; Macor, J. E. Bioorg. Med. Chem. Lett. 1890, 2010, 20; (c) 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; (d) 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.
8. Zhang, L.; Villalobos, A. Annu. Rep. Med. Chem. 2012, 47, 105.
9. Hsin, L.-W.; Webster, E. L.; Chrousos, G. P.; Gold, P. W.; Eckelman, W. C.;
Contoreggi, C.; Rice, K. C. Bioorg. Med. Chem. Lett. 2000, 10, 707.
10. Martarello, L.; Kilts, C. D.; Ely, T.; Owens, M. J.; Nemeroff, C. H.; Camp, M.;
Goodman, M. M. Nucl. Med. Biol. 2001, 28, 187.
11. 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.
The most potent compounds in the fluoroethoxy series—7a, 7b,
and 7c—all had subnanomolar CRF1R binding affinities. Unfortu-
nately, the logD values of these compounds were among the high-
est in the series. Variations in a trisubstitution pattern of 7a and 7b
as well as changes in the size of a halogen atom X in 7b and 7c gave
compounds with similar binding affinity. The introduction of a po-
lar cyano-group to the pyrazinone core of 7d and to the aromatic
rings of 7g, 7h and 7i resulted in some loss of potency, however,
these compounds were still near the appropriate potency and lipo-
philicity range. A further increase in polarity led to a significant
reduction in potency, as evidenced by the data for pyrimidinyl ana-
logs 7e and 7f, the compounds with the lowest logD values in this
subseries. This inverse relationship between CRF1R IC50 and logD
values has been also observed in a related series.16 Conformational
constraint of the aniline sector (as in 7j) did not provide a signifi-
cant improvement of the potency-lipophilicity balance. The total
loss of activity observed for 4-cyano-2,5-difluoro-analog 7k sug-
gested the importance of a larger substituent (i.e., a chlorine atom
or a methyl group) at the 2-position of the phenyl ring.
12. Kumar, J. S. D.; 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.
13. Sullivan, G. M.; Parsey, R. V.; Kumar, J. S. D.; Arango, V.; Kassir, S. A.; Huang, Y.;
Simpson, N. R.; Van Heertum, R. L.; Mann, J. J. Nucl. Med. Biol. 2007, 34, 353.
14. CRF1R binding assay. Frozen rat frontal cortex (source of CRF1 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
The data for the fluoroalkyl pyrazinones are shown in Table 2.
The highest logD compound, 8a, has a potent CRF1R binding affin-
ity with an IC50 of 1.1 nM. Replacement of Cl by Me gives 8b which
has similar potency and a trend to a lower logD. Replacement of
the lower aryl group with a pyridine residue (8c) did not provide
a significant improvement in logD, but resulted in some loss of
activity. Compounds with much lower logD such as 8d showed a
large loss of potency, a trend which had also been seen in the flu-
oroethoxy-series (vide supra).
In summary, the present research produced a number of potent
fluorinated pyrazinones as CRF1R antagonists. While the most po-
tent compounds in both series consistently had higher logD values
then desired for potential PET ligands, a reasonable compromise
between potency and polarity was achieved with cyano-deriva-
tives 7g, 7h and 7i. The data displayed by these three compounds
met our established selection criteria, thus making them potential
candidates for radiosynthesis and biodistribution studies. The con-
tinuation of these efforts will be presented in due course.17
pepstatin A, 0.005% Triton X-100, 10 U/mL bacitracin and 0.1% ovalbumin and
homogenized. The suspension was centrifuged at 32,000Âg for 30 min. The
resulting supernatant was discarded and the pellet resuspended by
homogenization in assay buffer and centrifuged again. The supernatant was
discarded and the pellet resuspended by homogenization in assay buffer and
frozen at À70 °C. On the day of the experiment aliquots of the homogenate
were thawed quickly and homogenate (25
lg/well rat frontal cortex) added to
ligand (150 pM 125I-o-CRF) and drugs in a total volume of 100
l
L of assay
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
using 1
lM DMP696. Filters were then counted in a Wallac Wizard gamma
counter.
15. 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
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 the shake-
flask logD.
16. Zuev, D.; Mattson, R. J.; Huang, H.; Mattson, G. K.; Zueva, L.; Nielsen, J. M.;
Kozlowski, E. S.; Huang, X. S.; Wu, D.; Gao, Q.; Lodge, N. J.; Bronson, J. J.; Macor,
J. E. Bioorg. Med. Chem. Lett. 2011, 21, 2484.
17. For a recently published related series see: Deskus, J. A.; Dischino, D. D.;
Mattson, R. J.; Ditta, J. L.; Parker, M. F.; Denhart, D. J.; Zuev, D.; Huang, H.; Hartz,
R. A.; Ahuja, V. T.; Wong, H.; Mattson, G. K.; Molski, T. F.; Grace, J. E.; Zueva, L.;
Nielsen, J. M.; Dulac, H.; Li, Y.-W.; Guaraldi, M.; Azure, M.; Onthank, D.; Hayes,
M.; Wexler, E.; McDonald, J.; Lodge, N. J.; Bronson, J. J.; Macor, J. E. Bioorg. Med.
Chem. Lett. 2012, 22, 6651.
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