Synthesis and evaluation of novel potent TSPO PET ligands with 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide

Article abstract of DOI:10.1016/j.bmc.2019.07.036

Translocator protein (TSPO) expression is closely related with neuroinflammation and neuronal damage which might cause several central nervous system diseases. Herein, a series of TSPO ligands (11a–c and 13a–d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714. Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity for in vivo brain studies. After radiolabeling with fluorine-18, [¹⁸F]11a was used for a dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation model; the inflammatory lesion was clearly visualized with a superior target-to-background ratio compared to [¹⁸F]DPA-714. An immunohistochemical examination of the dissected brains confirmed that the uptake location of [¹⁸F]11a in the PET study was consistent with a positively activated microglia region. This study proved that [¹⁸F]11a could be employed as a potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis of other diseases, such as cancers related with TSPO expression.

Full text of DOI:10.1016/j.bmc.2019.07.036

Accepted Manuscript
Synthesis and Evaluation of Novel Potent TSPO PET Ligands with 2-Phenyl-
pyrazolo[1,5-a]pyrimidin-3-yl Acetamide
Van Hieu Tran, Hyunjun Park, Jaekyung Park, Young-Do Kwon, Shinwoo
Kang, Jae Ho Jung, Keun-A Chang, Byung Chul Lee, Sang-Yoon Lee, Soosung
Kang, Hee-Kwon Kim
PII:
S0968-0896(19)30637-6
https://doi.org/10.1016/j.bmc.2019.07.036
BMC 15020
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 April 2019
9 July 2019
19 July 2019
Please cite this article as: Hieu Tran, V., Park, H., Park, J., Kwon, Y-D., Kang, S., Ho Jung, J., Chang, K-A., Chul
Lee, B., Lee, S-Y., Kang, S., Kim, H-K., Synthesis and Evaluation of Novel Potent TSPO PET Ligands with 2-
Phenylpyrazolo[1,5-a]pyrimidin-3-yl Acetamide, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.07.036
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Synthesis and Evaluation of Novel Potent TSPO PET Ligands with 2-
Phenylpyrazolo[1,5-a]pyrimidin-3-yl Acetamide
Van Hieu Tran a,‡, Hyunjun Park b,c,d‡, Jaekyung Park e, Young-Do Kwon f, Shinwoo Kang b,c, Jae Ho Jung g,h
,
Keun-A Chang b,c,d, Byung Chul Lee g,h, Sang-Yoon Lee c,e,i, Soosung Kangj, Hee-Kwon Kim a,k
*
[a]
Department of Nuclear Medicine, Molecular Imaging & Therapeutic Medicine Research Center, Chonbuk National
University Medical School and Hospital, Jeonju 54907, Republic of Korea
[b] Department of Pharmacology, College of Medicine, Gachon University, Incheon 21936, Republic of Korea
[c] Neuroscience Research Institute, Gachon University, Incheon 21565, Republic of Korea
[d] Department of Health Sciences and Technology, Gachon Advanced Institute for Health Sciences and Technology
(GAIHST), Gachon University, Incheon 21999, Republic of Korea
[e] Gachon Advanced Institute for Health Sciences and Technology, Graduate School, Gachon University, Incheon 21936,
Republic of Korea
[f] Department of Nuclear Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
[g] Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul National University Bundang
Hospital, Seongnam 13620, Republic of Korea
[h] Center for Nanomolecular Imaging and Innovative Drug Development, Advanced Institutes of Convergence Technology,
Suwon 16229, Republic of Korea
[i] Department of Neuroscience, College of Medicine, Gachon University, Incheon 21936, Republic of Korea
[j] College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic
of Korea
[k]
Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk
National University Hospital, Jeonju 54907, Republic of Korea
‡ These authors contributed equally.
* Corresponding author.
Tel: +82 63 250 2768; Fax: +82 63 255 1172.
E-mail address: hkkim717@jbnu.ac.kr (H-K Kim).
Postal address: Department of Nuclear Medicine, Chonbuk National University Medical School, Geonji-ro 20,
Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

ABSTRACT
Translocator protein (TSPO) expression is closely related with neuroinflammation and
neuronal damage which might cause several central nervous system diseases. Herein, a series
of TSPO ligands (11a–c and 13a–d) with a 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide
structure were prepared and evaluated via an in vitro binding assay. Most of the novel ligands
exhibited a nano-molar affinity for TSPO, which was better than that of DPA-714.
Particularly, 11a exhibited a subnano-molar TSPO binding affinity with suitable lipophilicity
for in vivo brain studies. After radiolabeling with fluorine-18, [18F]11a was used for a
dynamic positron emission tomography (PET) study in a rat LPS-induced neuroinflammation
model; the inflammatory lesion was clearly visualized with a superior target-to-background
ratio compared to [18F]DPA-714. An immunohistochemical examination of the dissected
brains confirmed that the uptake location of [18F]11a in the PET study was consistent with a
positively activated microglia region. This study proved that [18F]11a could be employed as a
potential PET tracer for detecting neuroinflammation and could give possibility for diagnosis
of other diseases, such as cancers related with TSPO expression.
Keywords: Translocator protein; Positron emission tomography probe; Structure activity
relationships; Neuroinflammation.

1. Introduction
Positron emission tomography (PET) is a noninvasive molecular imaging technique
that can provide crucial information including disease progress and biochemical events. In a
PET image study, radiopharmaceuticals containing a positron emitter were employed.1–4
Widely used positron-emitting radionuclides for PET include 11C (t1/2 = 20.4 min), 13N (t1/2
=
9.97 min), 15O (t1/2 = 2.04 min), and 18F (t1/2 = 110 min), which are produced from cyclotrons.
Specially, fluorine-18 offers several advantages such as a long half-life and low-energy
positron (0.635 MeV) compared to the other cyclotron-produced radionuclides; fluorine-18 is
adequate to high resolution PET imaging, multistep synthesis, and transportation to research
18
centers and hospitals. In this sense, various F-labeled radiotracers have been developed to
study biologically important targets of interest using PET modality.4,5
One biological target in medical studies is the 18-kDa translocator protein (TSPO),
which was previously named as a peripheral type benzodiazepine receptor.6 Although TSPO
is highly expressed in many cell types related to steroidogenesis, it has been reported that
TSPO expression in the brain is restricted to microglia and astrocytes.7,8 Microglial cells are
resident macrophages in the central nervous system (CNS); their activation resulting from
endogenous or exogenous signals such as bacterial lipopolysaccharide (LPS) is involved in
CNS inflammation.8,9 Additionally, there are several reports that astrocytes are activated and
related to neuroinflammation.8–10 Because TSPO overexpression in the CNS is caused by
microglial activation, TSPO has been considered as an important biomarker for CNS diseases
such as Parkinson’s disease and Alzheimer’s disease.8–11
Since [11C]PK11195 was first reported to detect diseases associated with TSPO
expression,12,13 it has been widely studied and used as a TSPO PET ligand; however, several
studies have demonstrated critical limitations, such as a low signal to noise ratio and poor
brain uptake.14–16 Moreover, [11C]PK11195 used carbon-11 which possesses a short half-life

for PET imaging studies. Such hindrances and the advantages of fluorine-18 have led
research groups to develop new 18F-labeled TSPO PET imaging agents such as
[18F]FEDAA1106,17 [18F]DPA-714,18 [18F]AB5186,19 [18F]GE-180,20 and [18F]FEPPA.21
Among them, [18F]DPA-714 is a promising TSPO PET ligand with a high binding affinity
and has also been most extensively investigated and employed in preclinical to clinical
studies.22–25 Various derivatives from structure activity relationship (SAR) studies with 2-
phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide scaffold, the core chemical structure of DPA-
714, have been reported to establish improved radiotracers for TSPO.26–31 However, very few
2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide based ligands have exhibited both high
TSPO binding affinity and proper lipophilicity for brain studies. Recently, we also reported
several TSPO PET probes with the scaffold; one of our previously reported ligands exhibited
comparable TSPO binding affinity and an improved relative uptake of ipsilateral areas
administrated with LPS compared to the contralateral areas than those of [18F]DPA-714.32
However, it also possesses a high lipophilicity even though PET images in animal brains
were successfully obtained using a micro PET study.
To overcome this limitation, we tried to design novel ligands with a much higher
TSPO binding affinity and more suitable lipophilicity for brain PET studies through the
modification of substituents of the 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide
scaffold. Here, new TSPO ligands bearing branched aliphatic groups and cyclic groups based
on 2-phenylpyrazolo[1,5-a]pyrimidin-3-yl acetamide were prepared, and in vitro experiments
were performed to measure their binding affinity with TSPO. Newly synthesized ligands
exhibiting a higher in vitro binding affinity to TSPO and suitable lipophilicity from the SAR
study were employed to perform in vivo animal PET studies. Through in vivo brain studies,
the novel ligand was proven to be a promising PET imaging probe for TSPO.

2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 2-(5,7-dialkyl-2-(4-hydroxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-
N,N-dialkylacetamides (5a–d) were prepared via modifications in our previously reported
method.32
A starting material, commercially obtained ethyl 4-hydroxybenzoate 1 was protected with
an isopropyl ether and treated with acetonitrile in the presence of with potassium tert-
butoxide to yield a beta-ketonitrile derivative (2). Alpha carbon deprotonation of the ketone
of 2 to the enolate followed by treatment with hydrazine monohydrate afforded
aminopyrazoles (3a–c). Condensation of 3a–c with acetylacetone or 3,5-heptanedione
yielded the pyrazolopyrimidine motif (4a–d); subsequent removal of the isopropyl of 4a–d in
the presence of aluminum chloride generated 5a–d. After dialcohols (6 and 8) were treated
with p-toluenesulfonyl chloride to yield the corresponding ditosylates (7 and 9), each
ditosylate (7 or 9) was reacted with the phenol group of 5a–d in the presence of potassium
carbonate to yield precursors (10a–c and 12a–d) with a 64–80% yield. The reaction of 10a–c
or 12a–d with tetrabutylammonium fluoride hydrate yielded the corresponding new fluoro
based TSPO ligand compounds (11a–c and 13a–d) with an 81–96% yield.

Scheme 1. Synthetic route toward novel TSPO ligands.
N
N
HN
H2N
(1) a
(2) b
(1) c
(2) d
O
O
O
O
OH
O
O
N
C
3a: R1 = ethyl
3b: R1 = propyl
3c: R1 = isopropyl
R1
1
2
R1
R2
R2
N
N
N
N
N
O
OH
e
HO
R2
f
N
N
R2
R2
O
O
N
R1
R1
R1
R1
5a: R1 = ethyl, R2 = methyl
5b: R1 = propyl, R2 = methyl
5c: R1 = isopropyl, R2 = methyl
5d: R1 = ethyl, R2 = ethyl
4a: R1 = ethyl, R2 = methyl
4b: R1 = propyl, R2 = methyl
4c: R1 = isopropyl, R2 = methyl
4d: R1 = ethyl, R2 = ethyl
g
g
OTs
TsO
TsO
OTs
OH
OH
HO
7
9
6
8
5a-5c
5a-5d
h
i
R2
N
R2
N
N
N
N
OTs
N
N
O
O
OTs
R2
N
O
12a: R1 = ethyl, R2 = methyl
12b: R1 = propyl, R2 = methyl
12c: R1 = isopropyl, R2 = methyl
12d: R1 = ethyl, R2 = ethyl
O
10a: R1 = ethyl, R2 = methyl
10b: R1 = propyl, R2 = methyl
10c: R1 = isopropyl, R2 = methyl
R1
R1
R1
R1
j
j
R2
N
R2
N
N
N
N
F
N
O
O
F
R2
N
R2
O
O
13a: R1 = ethyl, R2 = methyl
13b: R1 = propyl, R2 = methyl
13c: R1 = isopropyl, R2 = methyl
13d: R1 = ethyl, R2 = ethyl
11a: R1 = ethyl, R2 = methyl
11b: R1 = propyl, R2 = methyl
11c: R1 = isopropyl, R2 = methyl
N
R1
R1
R1
R1
Reagents and conditions. a) 2-Bromopropane, potassium carbonate, acetone, reflux, 3 days. b) Acetonitrile,
potassium tert-butoxide, tetrahydrofuran, rt, 2 h. c) Sodium hydride, 2-bromo-N,N-diethylacetamide or 2-
bromo-N,N-dipropylacetamide or 2-bromo-N,N-diisopropylacetamide, tetrahydrofuran, rt, 21–22 h. d)
Hydrazine monohydrate, acetic acid, ethanol, reflux, 13–14 h. e) 2,4-Pentanedione or 3,5-heptanedione, ethanol,
reflux, 14–16 h. f) Aluminum chloride, dichloromethane, rt, 14–16 h. g) p-Toluenesulfonyl chloride,
triethylamine, dichloromethane, rt, 15–18 h. h) Compound 7, potassium carbonate, acetonitrile, 60 oC, 19–23 h.
o
i) Compound 9, potassium carbonate, acetonitrile, 60 C, 12–14 h. j) Tetrabutylammonium fluoride hydrate, 2-
methyl-2-butanol, 80 oC, 12–15 h.

2.2 In vitro binding assay and lipophilicity
Newly prepared ligands (11a–c and 13a–d) were used to evaluate the in vitro TSPO binding
affinities (Ki) via a competition study with [3H]PK11195. All new ligands exhibited a better
binding affinity to TSPO compared with DPA-714 (Table 1). TSPO ligands 11a with a
branched aliphatic structure and 13a with a cyclic hexyl group at R3 position, each bearing an
ethyl group at R1, yielded 3.5- and 2.4- fold higher affinity to TSPO compared with DPA-
714, respectively: The Ki values of 11a and 13a were 0.94 nM and 1.32 nM, respectively.
These results indicated that a branched aliphatic structure at the R3 position provided a more
positive binding affinity to TSPO compared with a cyclic structure at the R3 position. In
addition, affinities were found to increase as smaller moieties were introduced at the R1
position: ethyl- (11a and 13a) > propyl- (11b and 13b) > isopropyl- (11c and 13c). This result
indicated that the binding affinity to TSPO was also influenced by a substituent at the R1
position. It was noted that, when an isopropyl group (branched) was introduced to the R1
position, the binding affinity to TSPO was improved compared with an n-propyl group
(linear) at the R1 position.
The lipophilicity (log P7.5) of compounds is an important factor in brain studies because it
can indicate penetration of the brain-blood barrier in the brain and thus can be used as an
indirect indicator. The log P7.5 of the newly prepared ligands (11a–c and 13a–d) was
examined using a previously reported method.32 As shown in Table 1, log P7.5 values were
affected by ligand substituents and ranged between 2.44 and 3.92. It was found that a (4-
(fluoromethyl)cyclohexyl)methoxy group at the R3 position yielded a higher lipophilicity
than a 3-fluoro-2-mehtylpropoxy group at the R3 position; propyl and isopropyl groups at the
R1 position yielded higher lipophilicity values than ethyl groups at the R1 position. Improved
lipophilicity results were observed for ethyl groups at the R2 position compared with methyl
groups at the R2 position. Among the new ligands, 11a and 11c exhibited lower lipophilicity

(log P7.5 for 11a = 2.44, and log P7.5 for 11c = 2.93) compared with the other new ligands,
suggesting that 11a and 11c possessed more suitable log P7.5 values for in vivo brain studies.
Table 1. Chemical characteristic and binding affinity values of the new TSPO ligands and
reference compound
R2
N
N
OR3
N
R2
O
N
R1
R1
Ki
Compound
R1
R2
R3
LogP7.5
1.99
(nM ± SD)a
3.26 ± 0.390
DPA-714
-Et
-Me
-CH2CH2F
11a
11b
11c
11db
13a
13b
13c
13d
-Et
-Pr
-iPr
-Et
-Et
-Pr
-iPr
-Et
-Me
-Me
-Me
-Et
-CH2CH(CH3)CH2F
-CH2CH(CH3)CH2F
-CH2CH(CH3)CH2F
-CH2CH(CH3)CH2F
-CH2C6H10CH2F
0.94± 0.045
1.11 ± 0.074
1.04 ± 0.065
3.12 ± 0.065
1.32 ± 0.067
1.88 ± 0.072
1.59 ± 0.093
2.39 ± 0.054
2.44
3.17
2.93
3.21
3.34
3.92
3.73
3.91
-Me
-Me
-Me
-Et
-CH2C6H10CH2F
-CH2C6H10CH2F
-CH2C6H10CH2F
a The mean ± SD of three experiments.
b See reference 32.
2.3. Modeling docking study
Molecular docking is a study used to predict the mode of action of novel compounds
when binding with target proteins. TSPO is comprised of five transmembrane helices
(TM1–5) with a short extra-membranous helix (α1,2) between TM1 and TM2. The 3D

structures of the TSPO-PK11195 complex (PDB: 4RYI, 2MGY) showed the drug binding
site is in the upper cytosolic part of the five transmembrane helical bundles.33-34 Newly
prepared compounds were docked into the PK11195 binding site (PDB: 4RYI) using the
Glide module of the Schrödinger software with the Maestro interface. As shown in Fig. 1a-b,
the pyrazolopyrimidine of 11a and 11d was located in a PK11195 binding pocket formed by
Trp51, Phe55, Asn87, Phe90, Ser91, Cys107 and Val110. Interestingly, the aromatic residues
of Trp51 and Phe90 in the pocket were involved in π - π stacking interaction with the
pyrazolopyrimidine of the inhibitors. This pocket appears to only fit with methyl- or ethyl-
substituted pyrazolopyrimidines, docking analysis with a propyl-substituted dummy
derivative (white molecule in Fig. 1b) showed altered positioning at the binding pocket with
worse docking score. The N,N-dialkyl amide chain of the synthesized molecules contacts
Pro42, Ile47, Phe90, Trp138, Phe 141, Ala142, Leu145, and Ser146. Apparently, steric
repulsion between N-alkyl group of the amide and hydrophobic residues on the binding site
can destabilize the binding. Docking simulations with the newly prepared compounds as well
as with more bulky dummy amides indicated that the particular N-diethyl or N-diisopropyl
substitution on the amide is subjected to minimal repulsive interactions in the TSPO amide
binding site. The repulsive interaction between bulky amides and TSPO slightly altered the
position of the pyrazolopyrimidine and middle aromatic ring to limit retention of the
fluoroalkyl ether group to the originally occupied binding site. This negative modulation was
more severe when a bulky fluoroalkyl group was utilized as an ether moiety (13a–d). In
additions, the 4-(fluoromethyl)cyclohexyl group on 13a–d was partially outside the drug
binding site, and probably should pay solvation penalty during exposure to a biological
solvent (docking score: -9.220 kcal/mol for 13a, -7.425 kcal/mol for 13b, -7.624 kcal/mol for
13c, and -6.896 kcal/mol for 13d). The favorable binding orientation of N-alkyl amide and
pyrazolopyrimidine, coupled with fluorinated alkyl chains on the para position of the middle

phenyl ring of 11a, resulted in a significantly enhanced inhibitory activity of 11a (docking
score: -9.451 kcal/mol for 11a).
Fig. 1. Predicted binding modes of compound 11a (green), 11d (deepblue), and 13a (blue)
with TSPO (PDB:4RYI).
(a) Top and side views of the 11a-TSPO docking model; (b) Detailed side view of 11a and 11d in the binding
pocket with protein surface; (c) Detailed view of 13a in the binding cavity. Key residues interacting with amide
(yellow), pyrazolopyrimidine (sky blue), and fluoroalkyl (orange) of the compounds are highlighted. The
ligands are drawn as stick-ball figures with atom coloring of nitrogen (blue), oxygen (red), and fluorine (light
sky blue). The protein surface is colored blue (positive potential) and red (negative potential). Images were
generated with Pymol

2.4. Radiosynthesis
Among the newly prepared ligands, [18F]11a was chosen for an animal study because of its
high binding affinity and proper lipophilicity. The radiosynthesis of [18F]11a was performed
via the reaction of 10a with fluorine-18, which was produced from a cyclotron. To obtain
optimal radiosynthesis conditions for [18F]11a, several reaction factors that influenced the
radiofluorination of precursor 10a were screened. Examination of base and solvent effects on
the radiofluorination showed a reaction using K2CO3 and K222 in dimethyl sulfoxide (DMSO)
that produced a higher radiochemical yield (RCY) as shown in Table 2. Different quantities
of base reagent were also investigated to obtain better radiofluorination results, indicating
that a 1:1.5:2.8 molar ratio of precursor-base-K222 could produce a higher RCY. In addition,
radiofluorination for 10 min afforded a higher RCY than other reaction periods.
Using the optimal radiofluorination reaction conditions, the radiosynthesis of [18F]11a was
o
conducted at 100 C for 10 min, and HPLC purification was performed using ammonium
acetate (NH4OAc) and MeCN (30/70 (v/v); 0.1 M ammonium acetate/MeCN) to yield
[18F]11a, the radiolabeled target TSPO ligand, with an isolated RCY of 28.7  3.5% (n=5,
decay-corrected). In the HPLC analysis study, [18F]11a was compared to the control ligand
11a to validate the target radiolabeled product: the peak of the final product ([18F]11a) was
consistent with that of 11a (Figures S1 and S2), and the HPLC peak of [18F]11a was observed
approximately 14 min after injection. In addition, the radiochemical purity of the product at
the end of synthesis was greater than 99%, and specific activity was 60 GBq/μmol (n = 5).
The in vitro stability of [18F]11a was also investigated, and no radioactive metabolites were
observed over 4 hours (Figure S3), suggesting that [18F]11a was stable enough for in vivo
animal study.

Table 2. Screening of reaction conditions for the radiosynthesis of precursor 10a.a
18F, Base/K222
Solvent
Temp.
N
N
N
N
18F
O
OTs
O
N
N
O
O
N
N
18F]11a
10a
[
Entry Base (equiv.)/K222 (equiv.) Solvent Temp. (oC) RCY (%)b
1
2
KHCO3(1.5)/K222 (2.8)
Cs2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.0)/K222 (1.5)
K2CO3(1.0)/K222 (2.5)
K2CO3(1.5)/K222 (2.0)
K2CO3(1.5)/K222 (2.5)
K2CO3(1.5)/K222 (3.5)
K2CO3(1.5)/K222 (2.8)
K2CO3(1.5)/K222 (2.8)
DMSO
DMSO
DMSO
MeCN
DMF
100
100
100
100
100
80
53.4 ± 3.1
37.4 ± 2.8
70.2 ± 3.4
41.4 ± 2.7
48.6 ± 8.5
40.5 ± 1.7
64.1 ± 5.2
35.3 ± 2.5
43.7 ± 3.7
53.3 ± 6.4
63.2 ± 4.5
51.3 ± 3.2
60.8 ± 4.8
69.2 ± 5.3
3
4
5
6
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
7
120
100
100
100
100
100
100
100
8
9
10
11
12
13c
14d
a Reaction conditions: precursor 10a (3.0 mg, 5.2 μmol), solvent (250 µL), 10 min, radioactivity of [18F]fluoride:
185 MBq.
b Radiochemical yield was determined by radio-TLC (n=3).
c Reaction was conducted for 5 min.
d Reaction was conducted for 20 min.
2.5. PET imaging study in an LPS-induced neuroinflammation rat model.
A micro-PET imaging study was performed in LPS-induced neuroinflammatory rat
models to observe the potential of [18F]11a as a PET ligand for neuroinflammation under
neuropathological conditions. Micro-PET imaging was obtained 4 days after the injection of
LPS into the brain of a rat. [18F]11a was reconstituted in less than 5% ethanol-saline solution;
the new ligand was injected through the tail vein so that it could act on the brain. A dynamic

PET scan was performed at 24 frames for 90 min to assess the behavior of [18F]11a. Micro-
PET images and time-activity curves (TACs) for the radioactivity of [18F]11a were obtained
at the contralateral and ipsilateral regions of the rat brains as shown in Fig. 2. The binding of
[18F]11a in the ipsilateral region was significantly enhanced compared to that of [18F]11a in
the contralateral region (Fig. 2a). TAC curve analysis showed that the amount of [18F]11a
was higher in the ipsilateral region compared with that of the contralateral region (Fig. 2b).
[18F]11a exhibited a 2.0-fold higher uptake ratio between the ipsilateral region and
contralateral region compared with [18F]DPA-714, a clinically used TSPO PET tracer (Figure
S4), suggesting that the area of neuroinflammation in the brain was more clearly identified
using [18F]11a compared with [18F]DPA-714 ([18F]11a = 3.65 ± 0.28; [18F]DPA-714 = 1.82 ±
0.19). The PET study indicated that the newly prepared TSPO ligand [18F]11a could be a
more effective PET probe to identify neuroinflammation in the brain than DPA-714.
In vivo selectivity and specificity of [18F]11a was examined via blockade
experiments using PK11195, a previously reported TSPO PET ligand used for CNS diseases.
In this experiment, PK11195 (3 mg/kg) was administered into the tail vein 60 min after the
injection of [18F]11a. The uptake of [18F]11a was significantly reduced in the ipsilateral
region of the brain by PK11195 (Fig. 3), confirming that the value of SUV decreased to a
similar extent in the contralateral area after 30 min (Figure S5). These results confirmed that
[18F]11a exhibited a high specific binding capacity for TSPO in the brain.

Fig. 2. Micro-PET images and time-activity curves for the reactivity of [18F]11a in a
neuroinflammatory rat model injected with LPS
(a)
(b)
Representative micro-PET brain images and time-activity curves of radiotracers in a rat LPS model of
neuroinflammation (n=3). The above image shows PET images (a) and time-activity curves (b) taken for 90 min
after [18F]11a injection. Ipsilateral (orange dot) and contralateral (blue dot) indicate ipsilateral and contralateral
sides of the LPS-injected brain in the striatum. Data are expressed as SUV (mean and SE of three animals).

Fig. 3. Blocking study of [18F]11a with PK11195.
Representative coronal brain PET images (a) before and (b) after PK11195 injection.
2.6. Immunohistochemical study
After PET scanning, LPS-injected rat brains were collected, and the
neuroinflammatory response was investigated using GFAP antibody for a protein marker of
reactive astrocytes, Iba-1 antibody for a protein marker of microglia, CD68 antibody for a
protein marker of microglial activation, and PBR antibody for a protein marker of TSPO. The
anatomical area of neuroinflammation in the brains of rats injected with LPS was
successfully visualized using immunohistochemical staining (Fig. 4). Fluorescence confirmed
that GFAP-positive astrocytes (Fig. 4a), Iba-1-positive microglia (Fig. 4b), CD68-positive
activated microglia (Fig. 4c), and PBR-positive TSPO proteins (Fig. 4d) were increased in the
ipsilateral region compared with the contralateral region of LPS-injected rat brains. The
location of neuroinflammation identified using immunohistochemical staining was closely
associated with [18F]11a binding in the PET images. This result showed that [18F]11a can
potentially identify neuroinflammation in the brain using PET imaging.

Fig. 4. The immunohistochemical staining of rat brains in a neuroinflammatory rat model
injected with LPS.
(a) GFAP
(b) Iba-1
(c) CD68
(d) PBR/TSPO
Representative images of immunohistochemistry in LPS induced neuroinflammatory animal model.
Immunohistochemistry revealed that astrocytes were stained with GFAP antibody (a; green), activated microglia
were stained with Iba-1 or CD68 antibody (b & c; green), or TSPO proteins were stained with PBR antibody (d;
green). Nucleus was counterstained with DAPI (blue). Immunohistochemical analysis showed the significantly

increased number of GFAP-, Iba-1-, CD68- and PBR/TSPO-positive cells in the ipsilateral region compared
with the contralateral region. Ipsilateral and contralateral indicate ipsilateral and contralateral region of the LPS-
injected brain in the striatum.
2.7. Metabolite analysis in mouse blood and brain
The metabolites from blood and brain tissue of normal rats were analyzed at 30 min and 90
min after administration of [18F]11a via tail vein (Figure S6). At 30 min after injection,
approximately 35% of the two major polar metabolites were found in blood samples. At 90
min after injection, total metabolites increased to approximately 41%, with the majority
consisting of less polar metabolites. During metabolism, polar metabolites were produced and
quickly excreted from the blood vessels but did not penetrate the brain tissue. In brain tissues,
approximately 96% of unchanged [18F]11a was observed at both time points and the
metabolite portions were less than 4% in brain tissue at both time points. The polar
metabolites were not found in brain samples at 30 and 90 min, indicating polar metabolites
did not penetrate the blood-brain barrier.
2.8. In vitro Cytotoxicity study
A standard WST-1 assay was used to investigate the cytotoxicity of 11a and 13a.
SH-SY5Y cells (a human neuroblastoma cell line) were treated with various concentrations
(0.1, 1, 10, 100, or 1000 nM) of 11a or 13a for 24 h and then the cell viability was evaluated
using a WST-1 assay. The results indicated the treatment of 11a or 13a did not induce
cytotoxic events (Figure S7).
3. Conclusions
In this study, several novel fluorinated TSPO ligands containing 2-phenylpyrazolo[1,5-
a]pyrimidin-3-yl acetamide scaffolds were prepared. In vitro assays suggest that branch and

cyclic substitution could be important factors yielding high TSPO affinity; all novel ligands
exhibited nano-molar TSPO affinities that were better than that of DPA-714.
Among the novel ligands, ligand 11a was proven to be a potential ligand due to its high
TSPO affinity and suitable lipophilicity. After radiosynthesis via labeling procedure with
fluorine-18, [18F]11a was used in an animal PET imaging study to demonstrate that
inflammatory lesions were visualized with a high target-to-background ratio and to confirm
the high accumulation of [18F]11a in microglia, an enhanced TSPO expression location.
Immunohistochemical study of the dissected brains validated that a high [18F]11a uptake
region in the PET study was in agreement with the positively activated microglia location.
Overall, these results illuminated [18F]11a as a promising TSPO PET ligand for the
evaluation of neuroinflammation and other diseases related to TSPO expression.
4. Experimental section
General information was placed in Supporting Information.
4.1. Chemistry
4.1.1.
General
procedure
for
the
synthesis
of
2-(5,7-diethyl-2-(4-
isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-dialkylacetamide (4a–d)
A mixture of the appropriate compound 3a, 3b, or 3c (1.0 equiv.) and 2,4-pentanedione or
3,5-heptanedione (1.3 equiv.) in ethanol (HPLC grade, 40 mL) was stirred at reflux for 14–16
h and then evaporated. The residue was purified by flash column chromatography (ethyl
acetate/hexane, 1.5:1) on silica gel to yield the desired product.
4.1.1.1.
N,N-Diethyl-2-(2-(4-isopropoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)acetamide (4a)
Treating compound 3a (3.85 g, 11.6 mmol) with 2,4-pentanedione (1.52 g, 15.2 mmol)
according to the general procedure gave compound 4a (4.15 g, 90.6%) as a yellowish solid.

o
m.p. 163–165 C; 1H NMR (400 MHz, CDCl3) δ 7.74 – 7.72 (m, 2H), 6.96 – 6.94 (m, 2H),
6.49 (s, 1H), 4.62 – 4.56 (m, 1H), 3.92 (s, 2H), 3.52 – 3.47 (q, J = 6.8Hz, 2H), 3.43–3.38 (q, J
= 6.8 Hz, 2H), 2.73 (s, 3H), 2.53 (s, 3H), 1.36 (d, J = 5.6 Hz, 6H), 1.19 (t, J = 7.2 Hz, 3H),
13
1.10 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl3) δ 170.08, 158.13, 157.38, 155.20,
147.45, 144.87, 129.96, 126.03, 115.92, 108.06, 100.78, 69.88, 42.30, 40.60, 28.17, 24.54,
22.05, 16.96, 14.34, 13.08; HRMS (ESI) m/z (M+H)+ calcd for C23H31N4O2: 395.2447; found
395.2449.
4.1.1.2.
2-(2-(4-Isopropoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
dipropylacetamide (4b)
Treating compound 3b (3.20 g, 8.93 mmol) with 2,4-pentanedione (1.16 g, 11.6 mmol)
according to the general procedure gave compound 4b (3.12 g, 83.6%) as a yellowish solid.
o
m.p. 110–112 C; 1H NMR (400 MHz, CDCl3) δ 7.76 – 7.72 (m, 2H), 6.96 – 6.94 (m, 2H),
6.49 (s, 1H), 4.62 – 4.58 (m, 1H), 3.92 (s, 2H), 3.37 – 3.32 (t, J = 8.0 Hz, 2H), 3.31 – 3.28 (t,
J = 7.6 Hz, 2H), 2.73 (s, 3H), 2.53 (s, 3H), 1.64 – 1.62 (m, 2H), 1.55–1.52 (m, 2H), 1.38 (d, J
= 6.0 Hz, 6H), 0.91 – 0.83 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 170.57, 158.13, 157.35,
155.15, 147.51, 144.81, 129.95, 126.06, 115.93, 108.06, 100.87, 69.88, 50.00, 48.11, 28.29,
24.55, 22.28, 22.04, 16.93, 11.38, 11.26; HRMS (ESI) m/z (M+H)+ calcd for C25H35N4O2:
423.2760; found 423.2764.
4.1.1.3.
2-(2-(4-Isopropoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
diisopropylacetamide (4c)
Treating compound 3c (2.70 g, 7.50 mmol) with 2,4-pentanedione (0.98 g, 9.80 mmol)
according to the general procedure gave compound 4c (2.62 g, 82.5%) as a yellowish solid.
o
m.p. 170–172 C; 1H NMR (400 MHz, CDCl3) δ 7.73 – 7.69 (m, 2H), 6.96 – 6.94 (m, 2H),
6.48 (d, J = 0.8 Hz, 1H), 4.61–4.58 (m, 1H), 4.24 – 4.22 (m, 1H), 3.89 (s, 2H), 3.42 – 3.40
(m, 1H), 2.72 (s, 3H), 2.54 (s, 3H), 1.37 – 1.34 (dd, J = 6.0, 2.8 Hz, 12H), 1.16 (d, J = 6.4 Hz,

6H); 13C NMR (100 MHz, DMSO-d6) δ 169.11, 158.01, 157.69, 153.85, 147.62, 144.96,
129.71, 126.02, 115.81, 108.68, 101.92, 69.57, 48.58, 45.33, 29.88, 24.61, 22.23, 20.99,
20.93, 16.76; HRMS (ESI) m/z (M+H)+ calcd for C25H35N4O2: 423.2760; found 423.2761.
4.1.1.4.
2-(5,7-Diethyl-2-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
diethylacetamide (4d)
Treating compound 3d (4.14 g, 12.5 mmol) with 3,5-heptanedione according to the general
o
procedure gave compound 4d (4.37 g, 82.6%) as a yellowish solid. m.p. 84–86 C; 1H NMR
(600 MHz, CDCl3) δ 7.81 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.49 (s, 1H), 4.62 –
4.58 (m, 1H), 3.92 (s, 2H), 3.56 (q, J = 7.2 Hz, 2H), 3.41 (q, J = 7.2 Hz, 2H), 3.19 (q, J = 7.4
Hz, 2H), 2.82 (q, J = 7.6 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H), 1.36 – 1.32 (m, 9H), 1.21 (t, J =
7.2 Hz, 3H), 1.11 (t, J = 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 170.53, 162.29, 158.19,
155.08, 149.79, 147.74, 130.16, 126.36, 116.00, 104.91, 100.91, 69.97, 42.57, 40.80, 31.55,
28.21, 22.18, 23.43, 14.53, 13.24, 13.02, 10.45; HRMS (ESI) m/z (M+H)+ calcd for
C25H35N4O2: 423.2760; found 423.2763.
4.1.2.
General
procedure
for
the
synthesis
of
2-(5,7-diethyl-2-(4-
hydroxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-dialkylacetamide (5a–d)
Aluminum chloride (3.3 equiv.) was added to a solution of the appropriate compound 4a, 4b,
4c, or 4d (1.0 equiv.) in anhydrous dichloromethane (50 mL). The mixture was stirred at rt
o
for 14–16 h and treated slowly with half sat. aq. ammonium chloride (100 mL) at 0 C. The
mixture was stirred at rt for 2 h and then separated. The aqueous phase was extracted with
dichloromethane (2 x 60 mL). The combined organic phase was dried over with Na2SO4 and
evaporated. The residue was purified by flash column chromatography
(dichloromethane/methanol, 20:1) on silica gel to yield the desired product.
4.1.2.1.
N,N-Diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-

yl)acetamide (5a)
Treating compound 4a (3.93 g, 10.0 mmol) with aluminum chloride (3.99 g, 30.0 mmol)
according to the general procedure gave compound 5a (2.39 g, 67.9%) as a white solid. m.p.
247–249 oC; 1H NMR (400 MHz, CDCl3) δ 7.58 - 7.55 (m, 2H), 6.76 – 6.74 (m, 2H), 6.46 (d,
J = 0.8 Hz, 1H), 3.96 (s, 2H), 3.49 – 3.47 (q, J = 7.2 Hz, 2H), 3.39 – 3.37 (q, J = 7.2 Hz, 2H),
13
2.71 (s, 3H), 2.54 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H), 1.08 (t, J = 6.8 Hz, 3H); C NMR (100
MHz, CDCl3) δ 170.65, 157.57, 157.36, 155.65, 147.22, 145.18, 129.82, 124.73, 115.68,
108.15, 100.21, 42.54, 40.90, 28.35, 24.34, 16.99, 14.13, 12.97; HRMS (ESI) m/z (M+H)+
calcd for C20H25N4O2 : 353.1978; found 353.1975.
4.1.2.2.
2-(2-(4-Hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
dipropylacetamide (5b)
Treating compound 4b (2.11 g, 5.00 mmol) with aluminum chloride (2.00 g, 15.0 mmol)
according to the general procedure gave compound 5b (1.73 g, 90.8%) as a yellowish solid.
m.p. 214–216 oC; 1H NMR (400 MHz, CDCl3) δ 7.60-7.57 (m, 2H), 6.78 – 6.75 (m, 2H), 6.47
(s, 1H), 3.98 (s, 2H), 3.36 – 3.27 (q, J = 8.0 Hz, 2H), 3.27 – 3.26 (t, J = 7.6 Hz, 2H), 2.72 (s,
3H), 2.54 (s, 3H), 1.59 – 1.51 (m, 4H), 0.86 – 0.81 (q, J = 7.6 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 171.07, 157.53, 157.37, 155.69, 129.82, 124.73, 115.68, 108.12, 100.28, 50.15,
48.36, 28.56, 24.25, 22.11, 20.84, 16.99, 11.35, 11.21; HRMS (ESI) m/z (M+H)+ calcd for
C22H29N4O2: 381.2291; found 381.2295.
4.1.2.3.
2-(2-(4-Hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
diisopropylacetamide (5c)
Treating compound 4c (2.03 g, 4.50 mmol) with aluminum chloride (1.98 g, 14.9 mmol)
according to the general procedure gave compound 5c (1.20 g, 70.0%) as a yellowish solid.
o
m.p. 267 – 268 C; 1H NMR (400 MHz, DMSO-d6) δ 7.58 – 7.56 (m, 2H), 6.84 – 6.81 (m,
3H), 4.35–4.29 (m, 1H), 3.77 (s, 2H), 3.50 – 3.48 (m, 1H), 3.33 (s, 1H) 2.68 (s, 3 H), 2.51 (s,

13
3H), 1.28 – 1.26 (d, J = 6.8 Hz, 6H), 1.16 - 1.15 (d, J = 6.0 Hz, 6H); C NMR (100 MHz,
DMSO-d6) δ 169.19, 158.16, 157.55, 154.19, 147.66, 144.89, 129.70, 124.70, 115.60, 108.53,
101.05, 29.95, 24.59, 20.96, 16.76; HRMS (ESI) m/z (M+H)+ calcd for C22H29N4O2:
381.2291; found 381.2292.
4.1.2.4.
2-(5,7-Diethyl-2-(4-hydroxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-
diethylacetamide (5d)
Treating compound 4d (3.57 g, 8.45 mmol) with aluminum chloride (3.48 g, 26.2 mmol)
according to the general procedure gave compound 5d (2.87 g, 89.4%) as a yellowish solid.
o
m.p. 207–208 C; 1H NMR (400 MHz, CDCl3) δ 7.69 - 7.66 (m, 2H), 6.82 - 6.80 (m, 2H),
6.49 (s, 1H), 3.98 (s, 2H), 3.56 (q, J = 7.2 Hz, 2H), 3.38 (q, J = 7.0 Hz, 2H), 3.18 (q, J = 7.6
Hz, 2H), 2.83 (q, J = 7.6 Hz, 2H), 1.42 (t, J = 7.5 Hz, 3H), 1.33 (t, J = 7.5 Hz, 3H), 1.14 (t, J
= 6.9 Hz, 3H), 1.07 (t, J = 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 171.16, 162.47,
157.56, 155.57, 150.00, 147.53, 130.03, 124.99, 115.83, 104.93, 100.30, 42.83, 41.12, 31.46,
28.57, 23.42, 14.28, 13.11, 13.06, 10.40; HRMS (ESI) m/z (M+H)+ calcd for C22H29N4O2:
381.2291; found 381.2292.
4.1.3. 2-Methylpropane-1,3-diyl bis(4-methylbenzenesulfonate) (7)
p-Toluenesulfonyl chloride (10.2 g, 53.2 mmol) was added to a mixture of 2-methylpropane-
1,3-diol 6 (1.20 g, 13.3 mmol) and triethylamine (5.40 g, 54.3 mmol) in anhydrous
dichloromethane (50 mL). The mixture was stirred at rt for 18 h. The mixture was washed
with brine (80 mL) and water (80 mL), dried over with Na2SO4, and evaporated. The residue
was purified by flash column chromatography (ethyl acetate/hexane, 1:3) on silica gel to
o
yield compound 7 (4.94 g, 93.1%) as a white solid. m.p. 73 – 75 C; 1H NMR (400 MHz,
CDCl3) δ 7.75 - 7.73 (m, 4H), 7.36 - 7.34 (d, J = 8.4 Hz, 4H), 3.93–3.86 (m, 4H), 2.46 (s, 6H),
2.20 – 2.12 (m, 1H), 0.92 (d, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 145.17, 132.62,

130.07, 127.99, 70.44, 33.13, 21.78, 13.08; HRMS (ESI) m/z (M+H)+ calcd for C18H23O6S2:
399.0936; found 399.0939.
4.1.4. Cyclohexane-1,4-diylbis(methylene) bis(4-methylbenzenesulfonate) (9)
p-Toluenesulfonyl chloride (7.93 g, 41.6 mmol) was added to a mixture of 1,4-
cyclohexanedimethanol (2.39 g, 16.6 mmol) and triethylamine (5.61 g, 55.4 mmol) in
o
anhydrous dichloromethane (50 mL) at 0 C. The mixture was stirred at rt for 15 h. The
mixture was filtered and washed with H2O (40 mL) and methanol (40 mL). The filtrate was
evaporated, filtered and washed with methanol (40 mL). The combined filtered solid was
washed with methanol (40 mL) and dichloromethane (2 x 15 mL) and dried to yield
compound 9 (3.17 g, 42.3%) as a white solid. m.p. 160–162 oC; 1H NMR (400 MHz, CDCl3)
δ 7.77 – 7.75 (dt, J = 8.4 Hz, 1.3 Hz, 4H), 7.34 – 7.32 (d, J = 8.0 Hz, 4H), 3.80 – 3.78 (d, J =
6.4 Hz, 4H), 2.44 (s, 6H), 1.74 (d, J = 7.2 Hz, 4H) 1.59 – 1.57 (m, 2H), 0.92 – 0.87 (m, 4H);
13C NMR (100 MHz, CDCl3) δ 144.72, 133.00, 129.82, 127.86, 74.82, 36.96, 28.45, 28.03,
21.65; HRMS (ESI) m/z (M+H)+ calcd for C22H29O6S2: 453.1406; found 453.1408.
4.1.5. General procedure for the synthesis of 3-(4-(3-(2-(dialkylamino)-2-oxoethyl)-5,7-
dimethylpyrazolo[1,5-a]pyrimidin-2-yl)phenoxy)-2-methylpropyl 4-methylbenzenesulfonate
(10a–c)
The appropriate compound 5a, 5b or 5c (1.0 equiv.), compound 7 (2.0 equiv.), and
potassium carbonate (4.0 equiv.) were dissolved in anhydrous acetonitrile (8 mL). The
mixture was stirred at 60 oC for 19–23 h. The mixture was diluted with ethyl acetate (20 mL)
and washed with H2O (20 mL). The organic phase was dried over with Na2SO4 and
evaporated. The residue was purified by flash column chromatography (ethyl acetate/hexane,
1.5:1) on silica gel to yield the desired product.

4.1.5.1.
3-(4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)-2-methylpropyl 4-methylbenzenesulfonate (10a)
Treating compound 5a (0.526 g, 1.50 mmol) with compound 7 (1.23 g, 3.00 mmol) according
to the general procedure gave compound 10a (0.690 g, 78.0%) as a white solid. m.p. 97–98
oC; 1H NMR (400 MHz, CDCl3) δ 7.76 - 7.74 (m, 4H), 7.28 - 7.27 (m, 2H), 6.86 - 6.84 (m,
2H), 6.55 (s, 1H), 4.16 – 4.12 (m, 2H), 4.01 (s, 2H), 3.90 – 3.82 (m, 2H), 3.57 – 3.51 (q, J =
6.8 Hz, 2H), 3.46 – 3.41 (q, J = 7.2 Hz, 2H), 2.79 (s, 3H), 2.62 (s, 3H), 2.42 (s, 2H), 2.36 –
2.31 (m, 1H), 1.25 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 6.8 Hz, 3H), 1.10 (d, J = 6.8 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ 169.71, 158.97, 157.41, 144.81, 132.72, 129.92, 129.83, 127.84,
114.47, 107.91, 100.88, 42.33, 40.66, 33.30, 28.27, 21.64, 17.13, 14.37, 13.47, 13.10; HRMS
(ESI) m/z (M+H)+ calcd for C31H39N4O5S: 579.2641; found 579.2644.
4.1.5.2.
3-(4-(3-(2-(Dipropylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)-2-methylpropyl 4-methylbenzenesulfonate (10b)
Treating compound 5b (0.400 g, 1.05 mmol) with compound 7 (0.840 g, 2.10 mmol)
according to the general procedure gave compound 10b (0.480 g, 80.6%) as a colorless oil;
1H NMR (400 MHz, CDCl3) δ 7.78 – 7.74 (m, 4H), 7.29-7.28 (m, 2H), 6.85 – 6.83 (m, 2H),
6.52 (s, 1H), 4.14 – 4.12 (m, 2H), 3.95 (s, 2H), 3.90 – 3.81 (m, 2H), 3.42 (t, J = 8.0 Hz, 2H),
3.36 – 3.31 (m, 2H), 2.76 (s, 3H), 2.56 (s, 3H), 2.41(s, 3H), 2.37 – 2.29 (m, 1H), 1.71 – 1.62
13
(m, 2H), 1.62 – 1.52 (m, 2H) 1.09 (d, J = 7.2 Hz, 3H), 0.94 – 0.86 (m, 6H); C NMR (100
MHz, CDCl3) δ 170.53, 158.76, 157.42, 154.93, 144.82, 132.71, 129.86, 129.84, 127.84,
126.54, 114.39, 108.13, 100.84, 49.99, 48.11, 33.30, 28.31, 24.54, 22.30, 21.64, 20.93, 16.92,
13.48, 11.40, 11.30; HRMS (ESI) m/z (M+H)+ calcd for C33H43N4O5S: 607.2954; found
607.2955.
4.1.5.3. 3-(4-(3-(2-(Diisopropylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)-2-methylpropyl 4-methylbenzenesulfonate (10c)

Treating compound 5c (0.400 g, 1.05 mmol) with compound 7 (0.840 g, 2.10 mmol)
according to the general procedure gave compound 10c (0.298 g, 76.7%) as a colorless oil; 1H
NMR (400 MHz, CDCl3) δ 7.77 – 7.72 (m, 4H), 7.29 – 7.28 (m, 2H), 6.85 – 6.82 (m, 2H),
6.52 (s, 1H), 4.32 – 4.25 (m, 1H), 4.14 – 4.11 (m, 2H), 3.93 (s, 2H), 3.89 – 3.81(m, 2H), 3.48
– 3.44 (m, 1H), 2.76 (s, 3H), 2.58 (s, 3H), 2.41 (s, 3H), 2.36 – 2.29 (m, 1H), 1.39 (d, J = 6.8
Hz, 6H), 1.20 (d, J = 6.8 Hz, 6H), 1.09 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
169.47, 158.76, 157.40, 154.88, 144.82, 132.69, 129.84, 127.83, 126.51, 114.33, 108.07,
101.01, 71.51, 68.26, 48.67, 45.90, 33.29, 30.25, 24.45, 21.65, 20.91, 20.64, 16.97, 13.08;
HRMS (ESI) m/z (M+H)+ calcd for C33H43N4O5S: 607.2954; found 607.2958.
4.1.6. General procedure for the synthesis of 2-(5,7-diethyl-2-(4-(3-fluoro-2-
methylpropoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-dialkylacetamide (11a–c)
The appropriate compound 11a, 11b or 11c (1.0 equiv.) and tetrabutylammonium fluoride
hydrate (2.0 equiv.) were dissolved in 2-methyl-2-butanol (10 mL). The mixture was stirred
o
at 80 C for 12–15 h. The mixture was diluted with ethyl acetate (20 mL) and washed with
H2O (20 mL). The organic phase was dried over with Na2SO4 and evaporated. The residue
was purified by flash column chromatography (ethyl acetate/hexane, 1:5) on silica gel to
yield the desired product
4.1.6.1. N,N-Diethyl-2-(2-(4-(3-fluoro-2-methylpropoxy)phenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)acetamide (11a)
Treating compound 10a (0.210 g, 0.350 mmol) with tetrabutylammonium fluoride hydrate
(0.180 g, 0.700 mmol) according to the general procedure gave compound 11a (0.144 g,
o
1
96.5%) as a white solid. m.p 117 – 118 C; H NMR (400 MHz, CDCl3) δ 7.80 – 7.74 (m,
2H), 6.98-6.96 (m, 2H), 6.50 (s, 1H), 4.57 – 4.55 (m, 1H), 4.45 – 4.43 (m, 1H), 3.99 – 3.94
(m, 2H), 3.92 (s, 2H), 3.52 – 3.47 (q, J = 7.2 Hz, 2H), 3.43 – 3.38 (q, J = 7.2 Hz, 2H), 2.74 (s,

3H), 2.57(s, 3H), 2.43 – 2.30 (m, 1H), 1.22-1.20 (t, J = 6.8 Hz, 3H), 1.13 – 1.09 (m, 6H); 13C
NMR (100 MHz, CDCl3) δ 170.03, 159.15, 157.42, 155.12, 129.97, 126.41, 114.55, 108.09,
100.80, 85.94, 84.26, 68.71, 68.66, 42.30, 40.60, 34.64, 34.45, 28.19, 24.48, 16.95, 14.34,
13.08, 12.92, 12.86; HRMS (ESI) m/z (M+H)+ calcd for C24H32FN4O2: 427.2509; found
427.2505.
4.1.6.2. 2-(2-(4-(3-Fluoro-2-methylpropoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)-N,N-dipropylacetamide (11b)
Treating compound 10b (0.200 g, 0.330 mmol) with tetrabutylammonium fluoride hydrate
(0.172 g, 0.660 mmol) according to the general procedure gave compound 11b (1.34 g,
89.4%) as a white solid. m.p 154–155 oC; 1H NMR (400 MHz, CDCl3) δ 7.78 – 7.74 (m, 2H),
7.98 – 6.96 (m, 2H), 6.49 (s, 1H), 4.56 – 4.45 (m, 1H), 4.45 – 4.44 (m, 1H), 3.98 – 3.96 (m,
2H), 3.92 (s, 2H), 3.38 – 3.32 (t, J = 8.0 Hz, 2H), 3.30 – 3.28 (m, 2H), 2.73 (s, 3H), 2.53 (s,
3H), 2.37 – 2.32 (m, 1H), 1.66 – 1.57 (m, 2H), 1.57 – 1.51 (m, 2H), 1.12 - 1.11(dd, J = 7.2,
13
1.2 Hz, 3H), 0.91 – 0.84 (m, 6H); C NMR (100 MHz, CDCl3) δ 170.52, 159.13, 157.40,
155.05, 147.46, 144.88, 129.96, 126.42, 114.55, 108.10, 100.89, 85.94, 84.27, 68.71, 68.65,
49.99, 48.11, 34.64, 34.45, 28.30, 24.51, 22.28, 20.93, 16.95, 12.93, 12.86, 11.39, 11.30;
HRMS (ESI) m/z (M+H)+ calcd for C26H36FN4O2: 455.2822; found 455.2824.
4.1.6.3. 2-(2-(4-(3-Fluoro-2-methylpropoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)-N,N-diisopropylacetamide (11c)
Treating compound 10c (0.200 g, 0.330 mmol) with tetrabutylammonium fluoride hydrate
(0.172 g, 0.660 mmol) according to the general procedure gave compound 11c (0.138 g,
o
1
92.2%) as a white solid. m.p. 178 – 180 C; H NMR (400 MHz, CDCl3) δ 7.75 – 7.71 (m,
2H), 6.97 – 6.95 (m, 2H), 6.49 (s, 1H), 4.57 – 4.54 (m, 1H), 4.47 – 4.44 (m, 1H), 4.28 – 4.2
(m, 1H), 4.01 – 3.92 (m, 2H), 3.91 (s, 2H), 3.43 – 3.41 (m, 1H), 2.73 (s, 3H), 2.55 (s, 3H),
2.37–2.30 (m, 1H), 1.36 (d, J = 6.8 Hz, 6H), 1.17 (d, J = 6.8 Hz, 6H), 1.10 (d, J = 6.8 Hz,

3H); 13C NMR (100 MHz, CDCl3) δ 169.48, 159.12, 157.37, 154.97, 129.93, 126.43, 114.48,
108.04, 101.05, 85.94, 84.27, 68.73, 68.68, 48.64, 45.89, 34.68, 34.45, 30.23, 24.48, 20.89,
20.62, 16.97, 12.92, 12.86; HRMS (ESI) m/z (M+H)+ calcd for C26H36FN4O2: 455.2822;
found 455.2823.
4.1.7. General procedure for the synthesis of (4-((4-(3-(2-(dialkylamino)-2-oxoethyl)-5,7-
dimethylpyrazolo[1,5-a]pyrimidin-2-yl)phenoxy)methyl)cyclohexyl)methyl
4-
methylbenzenesulfonate (12a–d)
The appropriate compound 5a, 5b, 5c, or 5d (1.0 equiv.), compound 9 (2.0 equiv.), and
potassium carbonate (4.0 equiv.) were dissolved in anhydrous acetonitrile (4 mL). The
mixture was stirred at 60 oC for 12–14 h. The mixture was diluted with ethyl acetate (10 mL)
and washed with H2O (10 mL). The organic phase was dried over with Na2SO4 and
evaporated. The residue was purified by flash column chromatography (ethyl acetate/hexane,
1.5:1) on silica gel to yield the desired product.
4.1.7.1.
(4-((4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)methyl)cyclohexyl)methyl 4-methylbenzenesulfonate (12a)
Treating compound 5a (0.410 g, 1.14 mmol) with compound 9 (1.03 g, 2.28 mmol) according
to the general procedure gave compound 12a (0.469 g, 64.9%) as a white solid. m.p. 75 oC;
1H NMR (400 MHz, CDCl3) δ 7.80 – 7.72 (m, 4H), 7.35 – 7.26 (m, 2H), 6.94 – 6.92 (m, 2H),
6.50 (s, 1H), 3.92 (s, 2H), 3.85 (d, J = 6.4 Hz, 2H), 3.79 (d, J = 6.0 Hz, 2H), 3.52 – 3.47 (q, J
= 7.2 Hz, 2H), 3.43 – 3.37 (q, J = 7.2 Hz, 2H), 2.73 (s, 3H), 2.55 (s, 3H), 2.45 (s, 3H), 1.95-
1.91 (d, J = 13.2 Hz, 2H), 1.81 - 1.78 (d, J = 12.8 Hz, 2H), 1.75–1.67 (m, 2H), 1.22 (t, J = 7.2
Hz, 3H), 1.13 (t, J = 7.2 Hz, 4H), 1.10 – 0.85 (m, 6H); 13C NMR (100 MHz, CDCl3) δ
169.98, 159.41, 157.39, 155.38, 144.64, 145.18, 133.13, 129.93, 129.81, 127.88, 126.04,
114.54, 108.02, 100.78, 75.15, 73.05, 42.30, 40.60, 37.44, 37.32, 28.79, 28.44, 28.21, 25.28,

24.32, 22.32, 21.65, 16.98, 14.34, 13.08; HRMS (ESI) m/z (M+H)+ calcd for C35H45N4O5S:
633.3111; found 633.3112.
4.1.7.2. (4-((4-(3-(2-(Dipropylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)methyl)cyclohexyl)methyl 4-methylbenzenesulfonate (12b)
Treating compound 5b (0.400 g, 1.05 mmol) with compound 9 (0.95 g, 2.10 mmol) according
to the general procedure gave compound 12b (0.500 g, 72.0%) as a white solid. m.p. 54–56
oC; 1H NMR (400 MHz, CDCl3) δ 7.80 – 7.72 (m, 4H), 7.36 – 7.34 (m, 2H), 6.94 – 6.91 (m,
2H), 6.50 (s, 1H), 3.94 (s, 2H), 3.86 (d, J = 8.0 Hz, 2H), 3.79 (d, J = 6.4 Hz, 2H), 3.40 – 3.36
(t, J = 7.6 Hz, 2H), 3.32 – 3.28 (t, J = 7.6 Hz, 2H), 2.74 (s, 3H), 2.55 (s, 3H), 2.45 (s, 3H),
1.91 – 1.81 (d, J = 13.2 Hz, 2H), 1.78 (d, J = 12.4 Hz, 2H), 1.77 – 1.52 (m, 6H), 1.04 – 0.98
(m, 4H), 0.91 – 0.83 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 170.39, 159.46, 157.37, 144.64,
133.18, 129.93, 129.81, 127.88, 125.92, 114.55, 107.96, 100.89, 75.15, 73.05, 49.98, 48.12,
37.45, 37.33, 28.80, 28.44, 28.36, 24.08, 22.25, 21.65, 20.95, 17.02, 11.40, 11.30; HRMS
(ESI) m/z (M+H)+ calcd for C37H49N4O5S: 661.3424; found 661.3421.
4.1.7.3. (4-((4-(3-(2-(Diisopropylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-
2-yl)phenoxy)methyl)cyclohexyl)methyl 4-methylbenzenesulfonate (12c)
Treating compound 5c (0.400 g, 1.05 mmol) with compound 9 (0.950 g, 2.10 mmol)
according to the general procedure gave compound 12c (0.520 g, 74.9%) as a yellow solid.
o
m.p. 158–160 C; 1H NMR (400 MHz, CDCl3) δ 7.80 – 7.70 (m, 4H), 7.36 – 7.34 (m, 2H),
6.92 – 6.91 (m, 2H), 6.49 (d, J = 0.8 Hz, 1H), 4.28 – 4.21 (m, 1H), 3.89 (s, 2H), 3.85 (d, J =
8.0 Hz, 2H), 3.77 (t, J = 5.2 Hz, 2H), 3.44 – 3.40 (m, 1H), 2.73 (s, 3H), 2.55 (s, 3H), 2.45 (s,
3H), 1.94 (d, J = 13.2 Hz, 2H), 1.81 (d, J = 12.8 Hz, 2H), 1.76 – 1.52 (m, 2H), 1.36 – 1.34 (d,
J = 6.8 Hz, 6H), 1.16 (d, J = 6.4 Hz, 6H), 1.10 – 0.95 (m, 4H); 13C NMR (100 MHz, CDCl3)
δ 169.51, 159.34, 157.37, 144.66, 133.08, 129.89, 129.82, 127.89, 126.08, 114.46, 108.04,
101.01, 75.16, 73.06, 48.64, 45.88, 37.44, 37.31, 30.24, 28.78, 28.43, 24.50, 21.67, 20.89,

20.62, 16.97; HRMS (ESI) m/z (M+H)+ calcd for C37H49N4O5S: 661.3424; found 661.3422.
4.1.7.4. (4-((4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-diethylpyrazolo[1,5-a]pyrimidin-2-
yl)phenoxy)methyl)cyclohexyl)methyl 4-methylbenzenesulfonate (12d)
Treating compound 5d (0.100 g, 0.263 mmol) with compound 9 (0.234 g, 517 mmol)
according to the general procedure gave compound 12d (0.131 g, 75.1%) as a white solid.
m.p. 104 – 107 oC; 1H NMR (400 MHz, CDCl3) δ 7.76 – 7.75 (m, 4H), 7.36 – 7.34 (m, 2H),
6.95 – 6.92 (m, 2H), 6.50 (s, 1H), 3.91 (s, 2H), 3.85 (d, J = 6.4 Hz, 2H), 3.78 (d, J = 6.4 Hz,
2H), 3.58 – 3.52 (q, J = 7.2 Hz, 2H), 3.44 – 3.37 (q, J = 7.2 Hz, 2H), 3.21 – 3.15 (q, J = 7.6
Hz, 2H), 2.84 – 2.78 (q, J = 7.6 Hz, 2H), 2.45 (s, 3H), 1.95 (d, J = 13.2 Hz, 2H), 1.81 (d, J =
12.0, 2H), 1.75 – 1.64 (m, 2H), 1.43 (t, J = 7.6 Hz, 3H), 1.32 (t, J = 7.6 Hz, 3H), 1.20 (t, J =
13
6.8 Hz, 3H), 1.00 (t, J = 6.8 Hz, 3H), 1.04 – 0.87 (m, 4H); C NMR (100 MHz, CDCl3) δ
170.37, 162.20, 159.27, 154.88, 149.72, 144.74, 144.67, 133.05, 132.96, 129.98, 129.82,
127.91, 126.38, 114.48, 104.82, 100.74, 75.19, 74.82, 73.01, 42.44, 40.66, 37.42, 37.31,
36.95, 31.40, 28.79, 28.44, 28.02, 23.32, 21.68, 14.42, 13.13, 12.92, 10.32; HRMS (ESI) m/z
(M+H)+ calcd for C37H49N4O5S : 661.3424; found 661.3425.
4.1.8.
General
procedure
for
the
synthesis
of
N,N-dialkyl-2-(2-(4-((4-
(fluoromethyl)cyclohexyl)methoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)acetamide (13a–d)
The appropriate compound 12a, 12b, 12c, or 12d (1.0 equiv.) and tetrabutylammonium
fluoride hydrate (2.0 equiv.) were dissolved in 2-methyl-2-butanol (10 mL). The mixture was
stirred at 80 oC for 14–15 h. The mixture was diluted with ethyl acetate (20 mL) and washed
with H2O (20 mL). The organic phase was dried over with Na2SO4 and evaporated. The
residue was purified by flash column chromatography (ethyl acetate/hexane, 1:1) on silica gel
to yield the desired product.