Synthesis, binding affinity, radiolabeling, and microPET evaluation of 4-(2-substituted-4-substituted)-8-(dialkylamino)-6-methyl-1-substituted-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-ones as ligands for brain corticotropin-releasing factor type-1 (CRF1

Article abstract of DOI:10.1016/j.bmcl.2015.10.010

Compounds 1-14 were synthesized in a search for high-affinity CRF₁ receptor ligands that could be radiolabeled with ¹¹C or ¹⁸F for use as positron emission tomography (PET) radiotracers. Derivatives of 2 were developed whi

Full text of DOI:10.1016/j.bmcl.2015.10.010

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5111–5114
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, binding affinity, radiolabeling, and microPET evaluation
of 4-(2-substituted-4-substituted)-8-(dialkylamino)-6-methyl-1-
substituted-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-ones as ligands
for brain corticotropin-releasing factor type-1 (CRF1) receptors
b
b
a
c
Jeffrey S. Stehouwer a, , Chase H. Bourke , Michael J. Owens , Ronald J. Voll , Clinton D. Kilts ,
⇑
Mark M. Goodman a,b
a Center for Systems Imaging, Department of Radiology and Imaging Sciences, Emory University, WWHC 209, 1841 Clifton Rd NE, Atlanta, GA 30329, USA
b Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA, USA
c Psychiatric Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds 1–14 were synthesized in a search for high-affinity CRF1 receptor ligands that could be radi-
olabeled with 11C or 18F for use as positron emission tomography (PET) radiotracers. Derivatives of 2 were
developed which contained amide N-fluoroalkyl substituents. Compounds [18F]24 and [18F]25 were
found to have appropriate lipophilicities of logP7.4 = 2.2 but microPET imaging with [18F]25 demonstrated
limited brain uptake.
Received 13 August 2015
Revised 1 October 2015
Accepted 5 October 2015
Available online 8 October 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Corticotropin-releasing factor
CRF-1 receptor
Fluorine-18
PET imaging
Positron emission tomography
The neuropeptide corticotropin-releasing factor (CRF)1,3 coordi-
nates the neuroendocrine response to stress through the hypotha-
lamic–pituitary–adrenal axis by acting on the CRF Type-1 (CRF1)
receptor.4 Dysregulation of brain CRF signaling has been proposed
to be involved in stress, anxiety, depression, and addiction; and
brain regional alterations in CRF1 density have been detected in
depressed patients and victims of suicide.5–8 This evidence has
resulted in a more than 20-year search for small molecule, brain
bioavailable CRF1 antagonists that could be used as therapeutics
for treating CRF1-related disorders.9–11 Therapeutic medication
development can be uniquely aided by the availability of validated
positron emission tomography (PET) and single-photon emission
computed tomography (SPECT) radiotracers that can be used to
both support target occupancy measurements of candidate
therapeutics and to explore mechanisms of pathophysiology and
treatment response by measurement of receptor density and
expression levels.12–15 Numerous attempts to develop a viable
brain CRF1 receptor PET radiotracer have been reported over the
years but no candidates to date have been successful due to
problems such as high lipophilicity and inability to cross the
blood–brain barrier (BBB), rapid metabolism, or insufficient speci-
fic receptor binding.16–19 As part of an effort to develop a viable
brain CRF1 receptor PET tracer we have been exploring various
structural motifs20 and report here our results with compounds
1–15 (Scheme 1), and derivatives thereof, which are based on a
previous report that compounds 2, 6, and 9 displayed high binding
affinity (IC50 = 0.70, 0.49, and 0.92 nM, respectively) at the rat brain
CRF1 receptor.21 We anticipated that the reported high binding
affinity of 2, 6, and 9 would enable radiotracer derivatives of this
structural class to achieve specific binding at CRF1 and that the
presence of the carbonyl group would enhance water solubility,
thereby reducing logP and increasing BBB passage.22–25
Furthermore, the synthetic route for this class of compounds21
(Scheme S1, Supplementary material) allows the aromatic
core and the N,N-dialkyl group to be held constant while the
Abbreviations: CRF, corticotropin-releasing factor; CRF1, CRF type-1 receptor;
PET, positron emission tomography; SPECT, single-photon emission computed
tomography; BBB, blood–brain barrier; DMA, dimethylacetamide; SUV, standard
uptake value; TACs, time–activity curves; CPCU, chemical processing control unit;
rcy, radiochemical yield; EOB, end-of-bombardment.
⇑
Corresponding author.
E-mail address: jstehou@emory.edu (J.S. Stehouwer).
http://dx.doi.org/10.1016/j.bmcl.2015.10.010
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

5112
J. S. Stehouwer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5111–5114
Scheme 2.
Table 2
Results of in vitro competition binding assays at 23 °C using transfected human CRF1
receptor in HEK293T cells
Scheme 1.
Compd Ro Rp
R1
R2
Et
R3
Ki (nM) SEMa n=
16
17
18
20
21
22
23
24
25
Br Br
Br i-Pr Me Me
Br i-Pr Me Et
Bu
Me
H
H
2.4 0.6
180
2
2
3
3
3
3
3
3
3
2,4-substituted pendant aryl ring is varied to produce a library of
high-affinity candidates that are amenable to radiolabeling with
11C or 18F.
5
16.4 1.6
3.1 0.3
Br i-Pr Pr
Pr
H
Br i-Pr Me Me CH2CH2F
Br i-Pr Et Et CH2CH2F
Br i-Pr Me Me (E)-CH2CH = CHCH2F 14.2 2.9
Br i-Pr Me Me CH2CH2CH2CH2F
Br i-Pr Me Et CH2CH2CH2CH2F
59
9
The binding affinities of compounds 1–14 at the CRF1 and CRF2
receptors were determined by in vitro competition binding assays
at 23 °C using HEK293T cells transfected with the human CRF1 or
CRF2 receptor (Table 1). Compounds 1–14 all bound to the CRF1
receptor with high affinity (Ki = ꢀ1.3–5.4 nM). Thus, although
these compounds have various combinations of substituents on
the pendant aryl ring, they all have binding affinities within a nar-
row range that differs by ꢀ4 nM. The difference in affinities of 2, 6,
and 9 between our results and the previously reported results21 is
presumably due to the difference between human and rat CRF1
receptors as well as the fact that we are reporting Ki values and
the previous work reported IC50 values. Sauvagine and R12191926
were used as positive controls in our assays: R121919 was found
to have an affinity of Ki = 2.6 0.4 nM for the CRF1 receptor which
is comparable to the previously reported values of Ki = 3.5 nM26
and Ki = 3.0 0.16 nM;27 and sauvagine was found to have an affin-
ity of Ki = 0.8 0.1 nM which is in agreement with the previously
reported value of Ki = 0.8–1 nM.28 The competing radioligand for
these studies, [125I]-Tyr0-sauvagine, has a reported affinity of
0.2–0.4 nM for the CRF1 receptor.28 Compounds 1–3, 6, and 8–12
7.3 0.7
21.6 2.8
13.9 1.9
a
Versus [125I]-Tyr0-sauvagine.28
did not display binding affinity at the CRF2 receptor (they were
unable to significantly displace [125I]antisauvagine-30),29 thus
demonstrating selectivity for the CRF1 receptor and so further
screening of compounds 4, 5, 7, 13, and 14 at the CRF2 receptor
was not performed.
Compound 1 was found to have the highest binding affinity for
the CRF1 receptor followed by compounds 2–5, but these com-
pounds do not have a position available for radiolabeling with 11C
or 18F. Thus, we shifted our focus to derivatives that could poten-
tially be radiolabeled on the amide nitrogen atom. Compound 16
(Scheme 2) was prepared by amide N-methylation of 1 with CH3I.
This resulted in only a small decrease in CRF1 binding affinity
(Table 2) relative to 1 (Table 1). Compounds 17–20 (Scheme 3) were
synthesized (Scheme S2, Supplementary material) to be used as
intermediates in the synthesis of amide N-fluoroalkyl target com-
pounds. The shorter N,N-dialkyl chains of 17–19 were expected to
compensate for the increased lipophilicity that would result from
adding alkyl groups to the amide nitrogen atom, while 20 was
included to evaluate the effect on binding affinity of rearranging
Table 1
Results of in vitro competition binding assays at 23 °C using transfected human CRF1
and CRF2 receptors in HEK293T cells
Compd
Ro
Rp
CRF1
CRF2
Ki
n=
%
n=
(nM) SEMa
Displacementb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Br
Br
Et
Br
Cl
Cl
Me
Me
Me
Br
Me
Br
Br
1.3 0.3
3
3
6
3
3
3
3
3
3
3
3
3
3
3
9
9
9.4 2.3
0.7 3.3
2.1 2.5
N/D
N/D
0.9 3.4
N/D
6.8 1.4
À2.3 3.2
À3.8 0.5
5.9 0.7
5.5 4.9
N/D
N/D
23.8 0.3
100.0 0.0
2
2
2
N/A
N/A
2
N/A
2
2
2
2
2
N/A
N/A
2
2
2
i-Pr 2.4 0.2
Br
Cl
Br
Cl
I
2.5 0.7
2.5 0.2
2.7 0.4
3.4 0.4
3.4 0.6
3.9 1.2
Me
OMe 4.0 0.6
t-Bu 4.2 2.2
Br
Me
Me
Cl
N/A 2.6 0.4
N/A 0.8 0.1
N/A N/D
4.4 0.9
4.4 1.1
4.4 0.4
5.4 0.9
Cl
Me
N/A
N/A
N/A
R121919
Sauvagine
Antisauvagine-
30
N/A 99.0 0.6
N/D = not determined. N/A = not applicable.
a
Versus [125I]-Tyr0-sauvagine.28
b
Mean% displacement of radiotracer ([125I]antisauvagine-30)29 specific binding
at CRF2 receptors by 1 lM of competing ligand.
Scheme 3.

J. S. Stehouwer et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5111–5114
5113
Table 3
LogP7.4 values
the methylene groups of the N,N-dialkyl substituent of 2 into a
symmetrical substitution pattern. Furthermore, derivatives of 2
were used, rather than 1, because 2-bromo-4-iso-propylaniline
can be reacted neat without the need for solvent or NaH which
simplified the synthetic procedure. Compounds 21–25 (Scheme 3)
were synthesized (Scheme S2, Supplementary material) by
N-alkylation of the amide nitrogen atom.
Compd
LogP7.4 SD
n=
[
[
18F]24
18F]25
2.19 0.01
2.22 0.02
4
4
Replacing the N-butyl-N-ethyl substituent of
2 with N,N-
dimethyl to give 17 resulted in a ꢀ75-fold loss of binding affinity
at CRF1 (Table 2) compared to 2 (Table 1) due to the inability to fill
the requisite lipophilic binding pocket on the receptor.21,30,31
When one of the N-methyl groups of 17 was replaced with N-ethyl
to give 18 an ꢀ11-fold gain in binding affinity was obtained rela-
tive to 17 and which corresponds to a ꢀ6.8-fold loss in binding
affinity relative to 2. Compound 20, which contains the same
number of N,N-dialkyl methylene groups as 2 but which are
arranged in a symmetrical fashion, had only a slight reduction in
binding affinity relative to 2 which indicates that symmetrical N,
N-dialkyl substitution can be tolerated by the CRF1 receptor for this
class of compounds as long as the lipophilic binding pocket on the
receptor is filled. Amide N-alkylation of 17 to give 21 resulted in a
ꢀ3-fold increase in binding affinity relative to 17, and when the N,
N-dimethyl group of 21 was replaced with N,N-diethyl to give 22
this resulted in a ꢀ8-fold increase in binding affinity relative to
21. Replacing the N-fluoroethyl group of 21 with an N-(E)-fluoro-
2-butenyl group32 to give 23 resulted in a ꢀ4-fold increase in
binding affinity while incorporation of an N-fluorobutyl group to
give 24 resulted in a ꢀ2.7-fold increase relative to 21. Amide
N-fluorobutylation of 18 to give 25 resulted in a negligible change
in binding affinity relative to 18 but the presence of the N-ethyl
group on 25 resulted in a slight increase in binding affinity relative
to 24. Thus, the significant loss of binding affinity that resulted
from using an N,N-dimethyl group on 17 can be minimized
through amide N-fluoroalkylation (which also provides a position
for radiolabeling), and by replacing the N,N-dimethyl group with
an N-ethyl-N-methyl or N,N-diethyl group.
The radiolabeling precursors 26 and 27 (Scheme 3) were
synthesized (Scheme S2, Supplementary material) by N-alkylation
of the amide nitrogen atoms of 17 and 18, respectively, with
1,4-ditosyloxybutane.20 Radiolabeling of [18F]24 and [18F]25 was
performed in one-step with K18F/K2.2.2 (Scheme 4) from 26 and
27, respectively. The logP7.4 values of [18F]24 and [18F]25 (Table 3)
were measured by the octanol/aqueous buffer shake-flask
method33,34 and were both found to be logP7.4 = 2.2 which is in
the range for passive diffusion across the BBB.22,23,25
MicroPET imaging was performed in an anesthetized male
cynomolgus monkey using a Siemens MicroPET Focus 220 as pre-
viously described.20 The whole-brain time-activity curves (TACs)
for [18F]25 (Fig. 1) show that the radiotracer did not significantly
enter the brain (SUV = <1)2 even though the logP7.4 value of [18F]
25 (Table 3) indicates that it should be able to diffuse across the
Figure 1. MicroPET whole-brain TACs of
cynomolgus monkey.
[
18F]25 in an anesthetized male
BBB.22,23,25 The binding affinity of 25 (Table 2) may not be as strong
as would be desired for a CRF1 PET tracer, but this would not affect
entry of the radiotracer into the brain, only retention of the radio-
tracer once in the brain. Thus, it would appear that the radiotracer
was metabolized in the blood.
In summary, compounds 1–14 were found to have high binding
affinities at the CRF1 receptor with a range of Ki = ꢀ1.3–5.4 nM, but
the highest affinity compounds, 1–5, do not have positions avail-
able for radiolabeling with 11C or 18F (although 76Br derivatives
are a possibility). Using shorter N,N-dialkyl chains allows for amide
N-alkylation but these changes reduce the binding affinities at the
CRF1 receptor. One-step radiolabeling using amide N-butyltosylate
precursors 26 and 27 was successful and yielded [18F]24 and [18F]
25, respectively. Compounds [18F]24 and [18F]25 had appropriate
lipophilicities (logP7.4 = 2.2) to support BBB permeability but
microPET imaging with
[
18F]25 demonstrated minimal brain
uptake. This program of synthesis, radiochemistry, in vitro charac-
terization, and PET imaging, while failing to yield a valid brain CRF1
receptor PET radioligand, further describes the theoretical and
methodological complexities of developing such an in vivo molec-
ular imaging probe.
Acknowledgments
This research was sponsored by NIH/NIMH (2U19 MH069056).
We acknowledge the use of shared instrumentation provided by
Grants from the NIH and the NSF.
Supplementary data
Supplementary data (experimental details, and spectroscopic
and analytical data) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
010.
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