New structure–activity relationships of N-acetamide substituted pyrazolopyrimidines as pharmacological ligands of TSPO

Article abstract of DOI:10.1016/j.bmcl.2016.06.041

Translocator protein (TSPO) represents an attractive target for molecular imaging and therapy due to its prevalence and critical roles played in oncology and other pathologies. Based upon our previously optimized pyrazolopyrimidine scaffold, we elucidated new structure activity relationships related to N,N-disubstitutions of the terminal acetamide on pyrazolopyrimidines and further explored the impacts of these substituents on lipophilicity and plasma protein binding. Several novel chemical probes reported here exhibited significantly increased binding affinity, suitable lipophilicity and protein binding compared with contemporary TSPO ligands. We illustrate that N,N-acetamide disubstitution affords opportunities to introduce diverse chemical moieties distal to the central pyrazolopyrimidine core, without sacrificing TSPO affinity. We anticipate that further exploration of N-acetamide substitutions may yield additional TSPO ligands capable of furthering the field of precision medicine.

Full text of DOI:10.1016/j.bmcl.2016.06.041

Accepted Manuscript
New structure-activity relationships of N-acetamide substituted pyrazolopyri-
midines as pharmacological ligands of TSPO
Jun Li, Michael L. Schulte, Michael L. Nickels, H. Charles Manning
PII:
S0960-894X(16)30652-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.041
BMCL 23994
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
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5 May 2016
13 June 2016
15 June 2016
Please cite this article as: Li, J., Schulte, M.L., Nickels, M.L., Charles Manning, H., New structure-activity
relationships of N-acetamide substituted pyrazolopyrimidines as pharmacological ligands of TSPO, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.041
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New structure-activity relationships of N-
acetamide substituted pyrazolopyrimidines
as pharmacological ligands of TSPO
Jun Li, Michael L. Schulte, Michael L. Nickels and H. Charles Manning

Bioorganic & Medicinal Chemistry Letters
New structure-activity relationships of N-acetamide substituted
pyrazolopyrimidines as pharmacological ligands of TSPO
Jun Lia,b,c , Michael L. Schulteb,c,d , Michael L. Nickelsb,c,d and H. Charles Manninga-i, 
a Interdisciplinary Materials Science Program, Vanderbilt University, Nashville, TN 37232, United States
b Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University Medical Center, Nashville, TN 37232, United States
c Center for Molecular Probes, Vanderbilt University Medical Center, Nashville, TN 37232, United States
d Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, United States
e Program in Chemical and Physical Biology, Vanderbilt University Medical Center, Nashville, TN 37232, United States
f Vanderbilt-Ingram Cancer Center (VICC), Vanderbilt University Medical Center, Nashville, TN 37232, United States
g Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, United States
h Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States
i Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, TN 37232, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Translocator protein (TSPO) represents an attractive target for molecular imaging and therapy
due to its prevalence and critical roles played in oncology and other pathologies. Based upon our
previously optimized pyrazolopyrimidine scaffold, we elucidated new structure activity
relationships related to N,N-disubstitutions of the terminal acetamide on pyrazolopyrimidines
and further explored the impacts of these substituents on lipophilicity and plasma protein
binding. Several novel chemical probes reported here exhibited significantly increased binding
affinity, suitable lipophilicity and protein binding compared with contemporary TSPO ligands.
We illustrate that N,N-acetamide disubstitution affords opportunities to introduce diverse
chemical moieties distal to the central pyrazolopyrimidine core, without sacrificing TSPO
affinity. We anticipate that further exploration of N-acetamide substitutions may yield additional
TSPO ligands capable of furthering the field of precision medicine.
Keywords:
TSPO
Pyrazolopyrimidine
SAR
Cancer
2009 Elsevier Ltd. All rights reserved.
Precision medicine
Imaging now plays a critical role in cancer diagnosis and the
molecular characterization of actionable lesions. Thus, imaging is
central in the successful deployment of precision cancer
medicine. Among clinical modalities available for cancer
imaging, the sensitivity and quantitative nature of positron
emission tomography (PET), coupled with the ability to produce
biologically active tracers bearing positron-emitting isotopes,
renders PET imaging uniquely capable of detecting tumors and
profiling their molecular features. Despite this potential, a lack of
specific and biologically validated tracers that are capable of
providing detailed molecular information about individual tumors
limits the breadth of biological questions addressable with PET.
By far, the most widely used PET tracer in oncology is 2-deoxy-
2-(18F)fluoro-D-glucose (FDG), which accumulates in tissues as a
function of glucose uptake. Many tumors utilize glycolysis to
partially fuel their growth, which has made FDG PET a
mainstream tool for cancer detection and assessment.
Importantly, however, FDG has several limitations. FDG uptake
in tumors can be confounded by accumulation in tissues
possessing increased glycolytic metabolism such as the brain.
Conversely, not all tumors exhibit high metabolic rates such as
prostate cancer.1 These issues highlight a currently unmet need to
explore and validate additional molecular targets for cancer
imaging.
Previously termed as peripheral benzodiazepine receptor
(PBR), translocator protein (TSPO) is an 18 kDa protein typically
localized to the outer mitochondrial membrane whose primary
role is to facilitate cholesterol metabolism. TSPO expression and
function is shown to be linked with steroid biosynthesis, cellular
proliferation, and apoptosis.2 In oncology, TSPO overexpression
has been reported in many human cancers, including glioma,
colon cancer, and breast cancer.3 As a promising target for cancer
imaging, TSPO ligands have been synthesized based upon a
diversity of core scaffolds.4-6 Many TSPO ligands were
previously developed to image CNS disorders and thus, have
sub-optimal pharmacokinetic properties for peripheral organ
———
 Corresponding author. Tel.: +01-615-322-3793; fax: +01-615-322-0734; e-mail: henry.c.manning@vanderbilt.edu

sites. Our lab has focused on the development and
characterization of high-affinity TSPO ligands applicable to a
wide variety of imaging modalities, especially in tumors that
arise outside of the central nervous system.1, 7-13 We previously
we report a variety of novel pyrazolopyrimidine TSPO candidate
ligands (n = 16), bearing alkyl, alicyclic, aryl, and heterocyclic
pendant acetamides. In doing so, we also report the development
of a novel synthetic route to these compounds that allows for a
rapid library expansion approach.
Our prior synthetic route to pyrazolopyrimidine-based
libraries required a divergent synthesis at the first step,1 which
reduced the efficiency of producing products with various
substituents. To achieve a more efficient pathway, we developed
a new route which allows diversification in the final step,
providing a facile approach to a diverse library. To further
accelerate the library generation, we utilized microwave-assisted
organic synthesis (MAOS) as shown previously.7,16 As shown in
Scheme 1, starting from commercially available compounds 3
Figure
1.
Structure
of
DPA-713
(1),
2-(5,7-diethyl-2-(4-
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide,
previously reported by our group (2) and indicated substituents of new
pyrazolopyrimidine-based ligands.
and 4, compound
5 was synthesized in a single step.
Subsequently, the pyrazolo ring of compound 6 was formed by
the reaction of compound 5 with hydrazine in ethanol, in the
presence of acetic acid. Preparation of compound 7 was
accomplished in a one-pot reaction procedure, combining
compound 6 with 3,5-heptanedione to close pyrimidine ring,
followed by deprotection to reveal the free carboxylic acid.
Finally, a variety of secondary amines were subsequently reacted
with carboxylic acid 7 to generate the final chemical probes
evaluated here. In total, 16 probes were synthesized with a
diversity of N-acetamide substituents.
optimized the 5, 6, and 7 positions of a pyrazolopyrimidine,
DPA-7136,14 and reported a novel fluorine-18-labeled PET tracer
(VUIIS 1008) with improved affinity and pharmacokinetic
properties for cancer imaging.1,11 Based upon our prior work and
motivated by the work of Selleri et al.,15 we further elaborated on
the pyrazolopyrimidine structure activity relationships related to
N,N-disubstitutions of the terminal acetamide (Figure 1). Herein,
Scheme 1. Synthetic route to N-acetamide substituted pyrazolopyrimidines.
Table 1. Structure, yield, affinity, and lipophilicity of pyrazolopyrimidines.
c,d
e
Yield
Compound
Structure
Ki (nM)
log P7.5
a
8
---
4.7
2.40
(DPA-713)
97%
37.59±5.96
2.47
9

Table 1 (continued)
c,d
e
Yield
Compound
Structure
Ki (nM)
log P7.5
b
86%
0.18
2.84
10
82%
82%
5.57±2.98
7.34±0.89
3.35
3.81
11
12
80%
79%
2.17±0.74
4.29
4.78
13
14
18.57±10.27
64%
70%
94.79±43.05
59.12±16.12
3.13
3.32
15
16
67%
85%
20.03±1.56
13.25±1.76
3.26
3.77
17
18
76%
51%
14.40±8.07
6.44±1.44
3.75
3.11
19
20

Table 1 (continued)
c,d
e
Yield
Compound
21
Structure
Ki (nM)
log P7.5
49%
0.28±0.14
3.27
76%
397.29±69.39
3.73
22
96%
83%
87.75±37.96
2.58
2.72
23
24
127.20±13.13
84%
49.22±9.24
2.96
25
a
b
c
d
DPA-713, see ref 6. Probe 5b, see ref 1. Ki versus 3H-flunitrazepam > 10000 nM. All Ki values were averaged from triple runs with corresponding standard
e
derivation. log P7.5 were averaged from triple runs with 1% or less standard deviation for each sample.
Upon synthesis and analytical characterization, TSPO
affinities of the chemical probes were determined using a C6
glioma cell lysate binding assay previously described.1,9,17
Lipophilicity was determined using analytical reverse phase
HPLC as previously reported.1 Plasma protein binding was
performed using the equilibrium dialysis method. After
extraction, the buffer and plasma fractions were analyzed using
standard LC-MS methods.18
over 300-fold, from 0.18 nM (compound 10) to 59.12 nM
(compound 16).
Interestingly, the introduction of a single phenyl ring rescued
the binding affinity. We observed the heterozygous phenyl-ethyl
substitution pattern to be particularity favorable, resulting in a
novel ligand with pico-molar activity (Ki = 0.28nM), which is
among the highest binding affinity for TSPO ligands reported to
date. In contrast, when both N-acetamide substitutions were
homologated to benzyl groups, affinity was decreased
significantly (Ki = 397.29nM). It was also found that acetamide
substitution with ethyl group was generally well-tolerated in
combination with many other N-substitutions on the acetamide.
For example, mixed ethyl acetamides fared better than other
substituents of similar structure, e.g. 15 vs 16, 20 vs 21. In
contrast to acyclic derivates, alicyclic constrained derivatives (23,
24 and 25) exhibited very poor binding affinity.
Previous investigation of 5/7 substitutions illuminated that
TSPO affinity was highly sensitive to alkyl substitutions and
intolerant of steric bulk.1 However, we found the acetamide
position particularly tolerant to N-alkyl substitution, with entries
of differentially substituted alkyl groups exhibiting affinities in
the nano molar to sub-nano molar range with the chain length
ranging from one to six carbons. Among them, N-alkyl
substitutions of up to 5 carbon length exhibited affinities
analogous or better than DPA-713.
Recognizing acetamide substitution as an opportunity to
diversify the binding affinity of novel TSPO ligands, we further
evaluated the lipophilicity and plasma protein binding.
Lipophilicity is an important factor that dictates biodistribution
and clearance. Chain length was a critical determinant of
lipophilicity within the N-alkyl series. Long carbon chain
substitutions, such as 12 (n=4), 13 (n=5) and 14 (n=6), were
extremely lipophilic as expected. Compared to straight chain
Chemical probes possessing branched alkane substituents
typically maintained reasonably high affinity, yet branching
generally diminished affinities relative to straight chain alkyl
groups of similar carbon number. It was noted that the bulky t-
butyl group significantly decreased binding affinity. When
substituting ethyl groups with t-butyls, the affinity decreased by

alkyl groups of similar carbon number, branched alkyl
substituents tended to exhibit more moderate lower lipophilicity.
The lipophilicity of phenyl substitution compounds (20 and 21,
3.11 and 3.27 respectively) appeared to represent a compromise
between longer alkyl chains and bulky branched substituents.
Lustgarten Foundation, and the Vanderbilt Center for Molecular
Probes.
References and notes
1.
2.
Tang, D.; McKinley, E. T.; Hight, M. R.; Uddin, M. I.; Harp, J.
M.; Fu, A.; Nickels, M. L.; Buck, J. R.; Manning, H. C., J. Med.
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C., Life Sci. 1983, 32, 1839.
The majority of the compounds reported here exhibited
unbound fractions of approximately 1%, including phenyl
substituents (Table 2). It was noted that chemical probes
exhibited higher lipophilicity tended to also exhibit lower
unbound fraction. For example, the unbound fraction of
compound 12 (n=4, logP7.5=3.81) was 0.3%, the lowest among
the examined library members.
3.
4.
Table 2. Protein binding of selected compounds.
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6.
Marangos, P. J.; Patel, J.; Boulenger, J.; Clark-Rosenberg, R.,
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Compound
Structure
Fraction Unbound
James, M. L.; Fulton, R. R.; Henderson, D. J.; Eberl, S.; Meikle,
S. R.; Thomson, S.; Allan, R. D.; Dolle, F.; Fulham, M. J.;
Kassiou, M., Bioorg. Med. Chem. 2005, 13, 6188.
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Lett. 2010, 51, 4595.
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Smith, R. A.; Tantawy, M. N.; Peterson, T. E.; Colvin, D. C.;
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1.1%
10
7.
8.
9.
Buck, J. R.; McKinley, E. T.; Hight, M. R.; Fu, A.; Tang, D.;
Smith, R. A.; Tantawy, M. N.; Peterson, T. E.; Colvin, D.; Ansari,
M. S., J. Nucl. Med. 2011, 52, 107.
0.7%
0.3%
11
12
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H. C., Mol. Imag. Biol. 2014, 16, 813.
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H.; Bornhop, D. J., Bioconjugate Chem. 2004, 15, 1488.
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Cederquist, K.; Stella, N.; Bornhop, D. J., Bioconjugate Chem.
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1.3%
1.1%
15
20
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0.6%
21
In summary, we illustrate here that the pendant acetamide
represents an opportunity to diversify the chemical and physical
properties of pyrazolopyrimidine-based TSPO ligands. Several
new chemical probes exhibited suitable binding properties,
lipophilicity and protein binding to potentially serve as TSPO
imaging tracers when functionalized with an appropriate positron
emitter such as Carbon-11. We anticipate that these results may
expand opportunities to deploy TSPO ligands to image and treat
diseases especially relevant to peripheral tissues.
Acknowledgments
The authors acknowledge funding from the National Institutes
of Health (K25 CA127349, P30
CA068485,
P30
DK058404, 1R01 CA163806), The Kleberg Foundation, The