Novel Pyrazolo[1,5-a]pyrimidines as Translocator Protein 18 kDa (TSPO) Ligands: Synthesis, in Vitro Biological Evaluation, [18F]-Labeling, and in Vivo Neuroinflammation PET Images

Article abstract of DOI:10.1021/acs.jmedchem.5b00932

A series of novel pyrazolo[1,5-a]pyrimidines, closely related to N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (2, DPA-714), were synthesized and biologically in vitro evaluated for their potential to bind

Full text of DOI:10.1021/acs.jmedchem.5b00932

Subscriber access provided by Stockholm University Library
Article
Novel pyrazolo[1,5-a]pyrimidines as translocator protein 18
kDa (TSPO) ligands: Synthesis, in vitro biological evaluation,
18
[
F]-labelling and in vivo neuroinflammation PET images.
Annelaure Damont, Vincent Medran-Navarrete, Fanny Cacheux, Bertrand Kuhnast, Géraldine
Pottier, Nicholas Bernards, Frank Marguet, Frédéric Puech, Raphael Boisgard, and Frédéric Dollé
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00932 • Publication Date (Web): 17 Aug 2015
Downloaded from http://pubs.acs.org on August 24, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 58
Journal of Medicinal Chemistry
1
2
3
Novel pyrazolo[1,5-a]pyrimidines as translocator protein 18 kDa (TSPO)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
ligands: Synthesis, in vitro biological evaluation, [ F]-labelling and in
vivo neuroinflammation PET images.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
,
a,b
a,b
a,b
a,b
a,b
Annelaure Damont,*
Vincent MedranꢀNavarrete,
Fanny Cacheux,
Bertrand Kuhnast,
a,b
a,b
c
c
Géraldine Pottier, Nicholas Bernards, Frank Marguet, Frédéric Puech, Raphaël Boisgard,
a,b
Frédéric Dollé.
a
CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France.
b
Inserm / CEA / Université Paris Sud, UMR 1023 – ERL 9218 CNRS, IMIV, Orsay, France.
c
LGCR, Sanofi, ChillyꢀMazarin, France.
ꢀ
ABSTRACT
A series of novel pyrazolo[1,5ꢀa]pyrimidines, closely related to N,Nꢀdiethylꢀ2ꢀ(2ꢀ(4ꢀ(2ꢀ
fluoroethoxy)phenyl)ꢀ5,7ꢀdimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀyl)acetamide (2, DPAꢀ714), were
synthesised and biologically in vitro evaluated for their potential to bind the translocator protein 18
kDa (TSPO), a protein today recognized as an early biomarker of neuroinflammatory processes. This
series is composed of fluoroalkylꢀ and fluoroalkynylꢀ analogues, prepared from a common iodinated
intermediate via Sonogashira coupling reactions. All derivatives displayed subnanomolar affinity for
the TSPO (0.37 to 0.86 nM), comparable to that of 2 (0.91 nM). Two of them were radiolabelled with
fluorineꢀ18 and their biodistribution was investigated by in vitro autoradiography and positron
emission tomography (PET) imaging on a rodent model of neuroinflammation. Brain uptake and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
local accumulation of both compounds in the AMPAꢀmediated lesion confirm their potential as in
1
8
vivo PETꢀradiotracers. In particular, [ F]23 exhibited a significantly higher ipsiꢀ to contralateral ratio
1
8
at 60 minutes than the parent molecule [ F]2 in vivo.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ INTRODUCTION
Nearly all brain diseases reveal pronounced changes in the functional states of glial cells and most
prominent is the presence of activated microglia in areas of progressive disease or tissue destruction.
Microglia activation is characterized by the overexpression of the translocator protein 18 kDa
(TSPO) on the outer mitochondrial membranes, supporting for over two decades considerable efforts
in the design of radioligands for the in vivo imaging of this pharmacological target by Positron
1
Emission Tomography (PET).
11
11
1
Today, the isoquinoline carboxamide [ C]1 ([ C]PK11195 ) is still the most widely used PETꢀ
radioligand to localise TSPO and visualise its expression level changes in vivo. Nevertheless the nonꢀ
optimal profile of this radioligand, especially with regard to the high level of nonꢀspecific binding
resulting in a poor signal to noise ratio obtained in vivo during PET studies, encourage the search for
more adequate radioligands. Few analogues of 1 labelled with a shortꢀlived isotope have been
11
2
11
2
18
2
described so far, among which [ C]PK11211 , [ C]PK14105 and [ F]PK14105 or more recently
11
3
11
3
11
3
[
C]PK13015 , [ C]PK12090 and [ C]PK14045 (Figure 1). SAR studies are still onꢀgoing to
4
,5
identify new lead TSPO ligands derived from 1 such as 4ꢀphenylquinazolineꢀ2ꢀcarboxamides or
6
derivatives featuring a 2ꢀarylquinoline scaffold. Besides these analogues of 1, a number of new
compounds, belonging to various chemical families, have also been prepared and investigated as
TSPO ligands. Among them, some have been labelled (usually on the basis of their promising in vitro
profiles) either with carbonꢀ11 (T = 20.4 min.) or with the longer halfꢀlife isotope fluorineꢀ18 (T
1
/2
1/2
ACS Paragon Plus Environment

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
7,8
=
109.8 min.), and in vivo evaluated. The early discovery of the benzodiazepine Ro5ꢀ4864,
a
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
chlorinated analogue of diazepam, as high affinity TSPO ligand and its carbonꢀ11ꢀlabelling in 1984,
have encouraged the development of a series of compounds of which structures result from the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
10−12
cleavage of the diazepine ring, giving rise to the phenoxyarylacetamides family. [ C]DAA1106
was the first carbonꢀ11ꢀlabelled representative of this series to emerge because of its subnanomolar
binding affinity and selectivity for the TSPO. Its initial development was concomitant with the
1
8
13
18
13
synthesis of two fluorineꢀ18ꢀanalogues, [ F]FMDAA1106 and [ F]FEDAA1106, which was
1
8
14,15
18
16
rapidly followed by the preparation of [ F]PBR06
in 2005 and [ F]DAA1106 in 2007. So far,
other closely related compounds have appeared in the literature for their high potential to in vivo
1
1
17,18
PETꢀimage the TSPO. For examples, [ C]PBR28,
a carbonꢀ11ꢀlabelled phenoxypyridinyl
18
19
acetamide, was proposed by Briard et al., followed by two radiofluorinated analogues: [ F]FEPPA ,
1
8
20
and more recently 6ꢀ[ F]FꢀPBR28.
Figure 1. Representative examples of TSPO ligands belonging to the following families: (a)
1
1
isoquinolines, including [ C]1, (b) other analogues of 1, (c) a benzodiazepine, (d)
phenoxyphenylacetamides and (e) phenoxypyridinylacetamides.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Besides these derivatives inspired from 1 or benzodiazepines, TSPO PET imaging has encouraged
the design of many other radioligands that share a number of common structural characteristics and
can be grouped together around the schematic architectural representation proposed in Figure 2 (a).
Indeed, they exhibit a multicyclic heteroaromatic planar skeleton as central molecular core and have
the particularity to commonly feature an acetamide function, linked to the heteroaromatic backbone,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
whose presence seems to be a requirement for efficient binding with the protein. It is also
interesting to note that most of these molecules display an aromatic ring directly attached to their
planar scaffold. The first radiolabelled molecules of this kind were inspired from the structure of the
imidazopyridine alpidem that binds both peripheral (TSPO) and central (CBR) benzodiazepine
1
1
22 11
23−26
11
27
receptors. Within this series, [ C]CLINME , [ C]PBR170
and [ C]ꢀA are carbonꢀ11ꢀlabelled
1
8
28,29
18
28,29
representative examples while [ F]PBR111
and [ F]PBR102
are fluorineꢀ18ꢀlabelled
specimens. Beside imidazopyridine acetamides, a number of bioisosteric structures have also been
explored as potential scaffolds allowing specific and efficient binding to the TSPO, such as
11
pyrazolopyrimidine or imidazopyridazine acetamides. The carbonꢀ11ꢀlabelled compound [ C]DPAꢀ
3
0ꢀ35
18
18
36ꢀ39
18
40,41
713
and the fluorineꢀ18 close analogues [ F]2 ([ F]DPAꢀ714)
and [ F]VUIIS1008
are
illustrations of TSPO imaging agents belonging to the pyrazolopyrimidine acetamides family,
18
42
whereas [ F]PBR132 is an example of imidazopyridazine acetamide radioligand. A number of 2ꢀ
4
3
arylꢀindoleꢀ3ꢀacetamide derivatives, also known as FGINꢀ1 class compounds , were screened
against TSPO and were shown to display high affinity and selectivity. Recently, conformationally
constrained versions of this series of molecules have been explored as novel TSPO ligands and one
4
4
of them has been labelled at its methylindole moiety (Figure 2). In this series, compounds feature a
glyoxylamide moiety in replacement of the acetamide motif rendering the structures less flexible in
hopes of increased affinity and selectivity toward the TSPO. Other closely related structures,
ACS Paragon Plus Environment

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
resulting from the fusion of the indole moiety with a pyridazine ring, have been proposed as potential
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
45
probes to image TSPO expression. [ C]SSR180575 is a carbonꢀ11ꢀlabelled illustration of this
18
46
phenylꢀpyridazinoindole acetamide family. Another indoleacetamide, [ F]GEꢀ180 , bearing a
labelling at the Nꢀindole position has recently been reported. Unlike the other previously mentioned
radioligands, it does not share the particularity of featuring a freely rotating aryl group linked to the
main heteroaromatic core of the molecule and was conformationally constrained by cyclisation via an
αꢀbranching of the acetamide part. Finally, a number of 2ꢀarylꢀ8ꢀoxodihydropurine acetamides have
also been radiolabelled and evaluated for their potential to selectively image the TSPO expression.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11
47 18
48
18
48
[
C]ACꢀ5216, [ F]FEDAC and [ F]FAC are representative examples belonging to the latter
series.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. (a) Schematic representation of a number of TSPO ligands and some representative
examples belonging to the following families: (b) 2ꢀarylꢀimidazo[1,2ꢀa]pyridineꢀ3ꢀacetamides, (c) 2ꢀ
arylꢀpyrazolo[1,5ꢀa]pyrimidineꢀ3ꢀacetamides, (d) 2ꢀarylꢀimidazo[1,2ꢀb]pyridazineꢀ3ꢀacetamides, (e)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2ꢀarylꢀindoleꢀ3ꢀacetamides, (f) 2ꢀarylꢀindoleꢀ3ꢀglyoxylamides, (g) 3ꢀarylꢀpyridazino[4,5ꢀb]indoleꢀ5ꢀ
acetamides, (h) αꢀbranchedꢀindole acetamides and (i) 2ꢀ(8ꢀoxoꢀ2ꢀarylꢀdihydropurine) acetamides.
Among all these series of compounds, we have been particularly interested in the design of novel 3,5ꢀ
dimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀylacetamide (DPA) specimens, leading to the discovery of the
18
38−39
11
fluoroethoxy derivative [ F]2,
today considered as a serious challenger of [ C]1. However,
recent studies clearly demonstrate that this compound is rapidly and extensively in vivo metabolised
4
9
in both rodents (rats) and nonꢀhuman primates (baboons). The major radiometabolites generated
have been identified: they mainly result from oxidation at the sensitive methyl groups of the DPA
scaffold or oxidation at the alphaꢀposition of the nitrogen atom of the acetamide moiety leading to Nꢀ
deethylated metabolites. These metabolites have been proved not to cross the human BBB.
1
8
Nevertheless and in a lesser extent, the brainꢀpenetrant radiometabolite [ F]fluoroacetate has also
4
9
18
been shown to be liberated. Indeed, [ F]2 is also subjected to oxidation at the alphaꢀposition of the
oxygen bridging the fluorineꢀ18 atom to the DPA scaffold (ether linkage). The presence of
18
18
[
F]fluoroacetate as well as its further in vivo conversion to [ F]fluoride may reduce the PET image
quality. Thus, within the present work, novel DPA analogues were designed by replacing the ether
linkage of 2 with fragments preventing metabolic fluoroacetate release. Two subꢀseries of
compounds were prepared: four fluoroalkylꢀ and four fluoroalkynylꢀ derivatives of 2. Their TSPO
binding and selectivity versus the CBR were evaluated in vitro as well as their lipophilicity. In vitro
metabolism studies were also performed, using human, rat and mouse microsomes. Based on the in
ACS Paragon Plus Environment

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
vitro data obtained, two representatives, one in each subꢀclass of analogues, were selected for
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
radiolabelling with fluorineꢀ18 to evaluate their potency as TSPO radiotracers in animal models.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
Figure 3. Structures of [ F]2 and the investigated new fluoroalkylꢀ and fluoroakynylꢀ analogues.
ꢀ
RESULTS AND DISCUSSION
Chemistry. Compound 12, the structurally closest analogue of 2 in the fluoroalkyl series, featuring a
methylene group in replacement of the oxygen atom, was the first candidate of the new series to be
prepared. The initial synthetic route employed for its preparation is depicted on scheme 1. From
commercially available methyl 4ꢀhydroxybenzoate, the iodopropoxy or bromopropoxy derivatives 3
and 4 were obtained in good yields (72 and 83 % respectively) by Oꢀalkylation with the appropriate
1,3ꢀdihalopropane in presence of a base in acetone. They were subsequently converted to methyl 4ꢀ
(3ꢀhydroxypropyl)benzoate (5) via aryl translocation from oxygen to carbon using standard radical
50
generating conditions (13ꢀ18 %). The resulting alcohol 5 was subsequently protected with tertꢀ
butyldiphenylsilyl chloride (TBDPSCl) to give compound 6 (80 % yield). Reaction of 6 with sodium
methoxide in refluxing acetonitrile yielded nitrile 7 which was alkylated with N,Nꢀ
diethylchloroacetamide to afford amide 8 in sufficient amount (28 % over 2 steps). A first
condensation reaction was conducted between 8 and hydrazine hydrochloride to afford the
aminopyrazole 9 in excellent yield (95 %). The latter compound was subsequently submitted to a
second condensation step using pentanꢀ2,4ꢀdione to achieve the construction of the DPA scaffold and
generate the pyrazolo[1,5ꢀa]pyrimidine 10 (75 % yield) to which hydroxyl group was subsequently
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
unprotected with tetrabutylammonium fluoride in THF to afford 11 (66 % yield). Finally, alcohol 11
was treated with the deoxofluorinating agent bis(2ꢀmethoxyethyl)aminosulfur trifluoride in toluene at
room temperature to afford compound 12 in 52 % yield.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 1. First synthetic pathway explored for the preparation of compound 12 .
a
Reaction conditions: (a) 1,3ꢀdiiodopropane or 1,3ꢀdibromopropane, K CO , acetone, 50 °C, 16 h; (b)
2
3
Bu SnH, AIBN, toluene, reflux, 12 h; (c) TBDPSCl, pyridine, r.t., 4 h; (d) NaOMe, CH CN, reflux, 4
3
3
h; (e) N,Nꢀdiethylꢀchloroacetamide, NaOH, EtOH, H O, 75 °C, 12 h; (f) NH NH .H O, AcOH,
2
2
2
2
EtOH, 60 °C, 3 h; (g) pentanꢀ2,4ꢀdione, EtOH, 60 °C, 16 h; (h) TBAF, THF, r.t., 8 h; (i) bis(2ꢀ
methoxyethyl)aminosulfur trifluoride, toluene, r.t., 6 h.
Afterwards, a more general synthetic method was developed for the preparation of the fluoroalkylꢀ
and fluoroalkynylꢀ analogues of 2, via a common iodophenyl intermediate (compound 16), as
depicted in scheme 2. This alternative pathway was also reused for the preparation of 12 at a higher
scale stimulated by the need to increase its global yield of production due to the very low yield of the
translocation reaction (13 to 18 %). Thus, obtainment of cyanoketone 13 was achieved via formation
of the lithium salt of acetonitrile by treatment with nꢀBuLi in THF at ꢀ60 °C followed by reaction
ACS Paragon Plus Environment

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
with commercial methyl 4ꢀiodobenzoate at ꢀ45 °C (70 % yield). Subsequent alkylation of 13 with
N,Nꢀdiethylchloroacetamide in presence of sodium hydroxide and sodium iodide in ethanol yielded
amide 14 (54 % yield) which, upon reaction with hydrazine monohydrate in refluxing ethanol with a
catalytic amount of acetic acid, afforded aminopyrazole 15 in 77 % yield. Pyrazolo[1,5ꢀa]pyrimidine
16 was efficiently obtained (78 % yield) by reacting 15 with pentanꢀ2,4ꢀdione in refluxing ethanol.
Sonogashira coupling of 16 with the appropriate alkynol using Pd(PPh ) Cl and CuI in triethylamine
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
2
afforded the expected hydroxyalkynylphenyl compounds 17, 18, 19 and 20 in good yields (64 %, 78
%, 69 % and 80 % respectively). Alcohols 17, 18, 19 and 20 were then reacted with bis(2ꢀ
methoxyethyl)aminosulfur trifluoride in dichloromethane to generate the corresponding fluorine
derivatives 21, 22, 23 and 24 (8 to 62 % yields). They were also hydrogenated in methanol in
presence of Pd/C for reduction of the triple bond to lead to their hydroxyalkylphenyl counterparts 11,
25, 26 and 27 respectively (40 to 92% yields). It is worth noting that compound 21 was not easily
accessible using this strategy since reaction of bis(2ꢀmethoxyethyl)aminosulfur trifluoride with the
propargylic alcohol 17 led to side products difficult to separate from the expected fluoropropynyl
analogue 21. Finally, in the alkylꢀ series, alcohols 11, 25, 26 and 27 were converted to their
fluorinated counterparts by deoxofluorination in dichloromethane to give fluoropropylꢀcompound 12,
fluorobutylꢀcompound 28, fluoropentylꢀcompound 29 and fluorohexylꢀcompound 30 in moderate
yields (43 to 58 %).
Scheme 2. Synthetic preparation of fluoroalkynyl- (21-24) and fluoroalkyl- (12, 28-30)
a
analogues .
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reaction conditions: (a) nꢀBuLi, CH CN, THF, ꢀ65 °C, 30 min then methyl 4ꢀiodobenzoate, ꢀ65 °C
3
to ꢀ45 °C, 2 h; (b) NaOH, EtOH, r.t., 15 min then N,Nꢀdiethylꢀchloroacetamide, NaI, r.t., 4 d.; (c)
N H .H O, AcOH, EtOH, 80 °C, 5ꢀ8 h; (d) acetylacetone, EtOH, 80 °C, 5 h; (e) alkynyl alcohol, CuI,
2
4
2
Pd(PPh ) Cl , Et NH, r.t., 24 h; (f) bis(2ꢀmethoxyethyl)aminosulfur trifluoride, CH Cl , r.t., 2ꢀ4 d.;
3 2
2
2
2
2
(g) H , Pd/C 10 %, CH Cl , r.t., 48 h.
2 2 2
In vitro binding assays. The binding affinity for the TSPO of these novel analogues of 2 were
3
measured by competition experiments against [ H]1 in membrane homogenates of rat heart. The
binding affinity of the newly synthesised compounds were also evaluated for the CBR by competitive
3
binding assays against [ H]flunitrazepam using membrane homogenates of rat cerebral cortex. The
inhibition constants (KiTSPO) and percentage of inhibition at 1 ꢀM (CBR) were determined for each
ligand and are reported in Table 1. The lipophilicity parameter LogD7.4 was also evaluated for this
ACS Paragon Plus Environment

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
new series of compounds on the basis of their HPLC retention times and the corresponding lipophilic
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
1
efficiencies (LipE) were calculated. Data are summarized in Table 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. In vitro competitive binding assays, lipophilicity determination and LipE calculation.
CBR % inhib.
d
TSPO
pIC50
e
TSPO Ki
(nM)
LogD7.4
LipE
Series
Ligand
R
a,b
c
at 1ꢀM
2
Oꢀ(CH
2
)
2
ꢀF
0.91 ± 0.08
0.37 ± 0.02
0.86 ± 0.06
0.54 ± 0.03
0.45 ± 0.03
0.54 ± 0.04
0.74 ± 0.06
0.35 ± 0.04
0.79 ± 0.09
0 %
0 %
0 %
3 %
12 %
0 %
0 %
0 %
0 %
2.89
3.51
3.90
4.31
4.70
3.61
3.67
4.06
4.35
9.00
9.39
9.02
9.22
9.30
9.22
9.09
9.41
9.06
6.11
5.88
5.12
4.91
4.60
5.61
5.42
5.35
4.71
1
2
(CH ) ꢀF
2
2
2
2
3
4
5
6
2
8
(CH
(CH
(CH
)
)
)
ꢀF
ꢀF
ꢀF
Alkyl-
29
3
2
0
1
C≡CꢀCH ꢀF
2
2
2
C≡Cꢀ(CH
C≡Cꢀ(CH
C≡Cꢀ(CH
2
2
2
)
)
)
2
3
4
ꢀF
ꢀF
ꢀF
Alkynyl-
23
2
4
a
TSPO Ki values were determined using membrane homogenates of rat heart and screened against
3
b
[ H]1 (K = 1.8 nM, C = 0.2 nM). In vitro binding affinity values are the mean ± SE of duplicate
d
c
measurements. CBR binding affinity was evaluated using homogenates of rat cerebral cortex and
3
d
expressed as % of inhibition at 1 ꢀM against [ H]flunitrazepam. Retention time recorded for the
tested compounds were converted into their logD7.4 values using a validated, standardized HPLC
e
method. LipE values were calculated as follows : LipE = pIC – LogD .
5
0
7.4
All analogues displayed subnanomolar affinity for the TSPO (0.37 to 0.86 nM), comparable to 2
0.91 nM). In the fluoroalkyl series, the lowest Ki value was obtained for compound 12 (0.37 nM)
(
whereas in the alkynyl series, it was found for compound 23 (0.35 nM). Additionally, all compounds
showed excellent selectivity towards TSPO, except compounds 29 and 30 that both exhibited a slight
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
binding affinity for the CBR (3 and 12 % at 1 ꢀM, respectively). The LogD7.4 values were all higher
(3.51 – 4.70) than that of 2 (2.89), most likely due to the replacement of the oxygen atom attached to
the phenyl ring, with alkylꢀ or alkynylꢀ fragments leading to less hydrophilic structures. These
LogD7.4 values followed the expected trend of increase with the length of the carbonated sideꢀchain
in both series. Nevertheless, most of the LogD7.4 values keep in the range for expected good passive
cerebral penetration of the ligands. Calculated LipE scores ranged from 4.60 (compound 30) to 6.11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
compound 2). The highest LipE score was obtained with 2 and is due to the combination of a
subnanomolar affinity for the TSPO and a rather low logD7.4 value slightly inferior to 3. Compound
2 also displays a high LipE score of 5.88, while the lengthening of the side chain, especially in the
1
fluoroalkylꢀ series, resulted in a drop of LipE values below 5 for compounds 29, 30 and 24. In the
fluoroalkynylꢀ series, except for compound 24, LipE values were all comparable (5.35 to 5.61). In
particular, despite a LogD7.4 value above 4 (4.06), compound 23 exhibited a rather good LipE score
(5.35) due to a high TSPO binding affinity. A LipE plot of the binding and lipophilicity data of the
compounds is presented in Figure 4.
Figure 4. LipE plot of the newly synthesised compounds (12, 21−24, 28−30) and 2: pIC vs
5
0
LogD .
7
.4
ACS Paragon Plus Environment

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
In vitro metabolism. Oxidative metabolism of 2 and the newly synthesised compounds (12, 21ꢀ24
and 28ꢀ30) was investigated using hepatic microsomes from human, rat and mouse. The results,
expressed as the percentage of biotransformation obtained after 20 minutes of microsomal
incubation, are summarised in Table 2. All compounds are rapidly and extensively metabolised in
presence of rat and mouse microsomes, more than 90 % of biotransformation is generally observed
after 20 minutes. Biotransformation using human microsomes is more variable, with percentage
ranging from 31 % for 2 to 91 % for compound 29. It can be noted that alkynyl derivatives are less
metabolised compared to their alkyl counterparts. These rather pronounced rates of biotransformation
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
49
are in line with what has already been observed with the parent molecule [ F]2: indeed, it has been
shown that it is rapidly and extensively metabolised in vitro but also in vivo in both rodents (rats) and
18
49
nonꢀhuman primates (baboons). Identification of the metabolites of 2 and [ F]2 notably revealed
that oxidation reactions occurred mainly (i) at the sensitive methyl positions of the pyrazolo[1,5ꢀ
a]pyrimidine core, (ii) at the alphaꢀposition of the nitrogen atom of the acetamide function leading to
Nꢀdealkylation and, in a lesser extent, (iii) at the alpha position of the oxygen atom of the
fluoroethoxy moiety of 2 leading to the release of fluoroacetate. These oxidative pathways generated
1
8
hydrophilic species, mainly nonꢀbrain penetrant except for [ F]fluoroacetate that cross the BBB and
might alter the PET image quality. Contrary to 2, the newly synthesised compounds (12, 21−24 and
28−30) don’t feature the sensitive ether linkage bridging the fluorine atom to the DPA scaffold and
therefore their metabolism may not lead to the unwanted liberation of fluoroacetate. Nevertheless,
they still feature the same DPA scaffold and may thus be subjected to a similar oxidative metabolic
pathway with regard to oxidation at the methyl positions and Nꢀdeethylation (or a combination of
both). Overall, this in vitro study suggests that 2, 23 and 24 are the less sensitive compounds to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
microsomal metabolism, especially with the human subcellular fractions (biotransformation at 20
min < 50 %). And, more specifically, in the fluoroalkyl series, 12 is the less metabolised compound
using human microsomes with 79 % biotransformation at 20 minutes compared to 90 ± 1 % for 28,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29 and 30.
Table 2. In vitro oxidative metabolism.
a
%
biotransformation at 20 min
Series
Ligand
Human
Rat
Mouse
2
31 %
79 %
89 %
91 %
89 %
55 %
60 %
47 %
39 %
100 %
90 %
1
2
100 %
99 %
99 %
97 %
99 %
98 %
91 %
96 %
99 %
100 %
100 %
96 %
28
Alkyl-
2
3
2
2
9
0
1
2
100 %
100 %
98 %
Alkynyl-
23
2
4
78 %
a
hepatic microsomal (male CD1 mouse, male SpragueꢀDawley rat or humans (BD pool)) incubation,
followed by analysis of the supernatant using HPLC/ESIꢀMS/MS.
On the basis of the in vitro preliminary results (affinity, lipophilicity and metabolism) of the newly
synthesised compounds: 12, in the fluoroalkyl series, and 23, in the fluoroalkynyl series, were chosen
for radiolabelling with fluorineꢀ18 to further investigate their in vivo PET properties as TSPO
radiotracers.
Radiochemistry. Fluorineꢀ18ꢀlabelling of compounds 12 and 23 was performed using a
TRACERLab FXꢀFN synthesiser (GEMS) by nucleophilic aliphatic substitution on their
18
corresponding tosylates 31 and 32 (precursors for labelling) with [ F]fluoride (Scheme 3).
ACS Paragon Plus Environment

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
Compounds 31 and 32 were synthesised from the corresponding alcohols 11 and 19 by reaction of pꢀ
toluenesulfonylchloride or pꢀtoluenesulfonylanhydride in dichloromethane in presence of
triethylamine as depicted in Scheme 3 (85 and 45 % yield, respectively). The efficiency of
incorporation of fluorineꢀ18 was first investigated with compound 12 in various reaction conditions
and the optimized conditions were then used for the labelling of compound 23 (Table 2). Briefly, the
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18
noꢀcarrierꢀadded dried K[ F]Fꢀ[2.2.2]ꢀcryptand complex was first prepared from cyclotronꢀproduced
18
[
F]fluoride, potassium carbonate and [2.2.2]ꢀcryptand. An acetonitrile or DMSO solution of the
1
8
tosylate 31 (or 32) was then added to the reaction vessel containing K[ F]Fꢀ[2.2.2]ꢀcryptand and the
resulting mixture was heated at various temperature and for variable duration. The resulting mixture
®
TM
was then diluted with the HPLC mobile phase, preꢀpurified on a SepPak Alumina N cartridge and
TM
purified on an HPLC semiꢀpreparative XꢀTerra RP18 column. Identity of the collected radioactive
product was confirmed by coꢀelution with reference compound 12 (or 23, respectively) on an
18
18
analytical HPLC system. Formulated solutions of [ F]12 (or [ F]23), ready for use, were typically
obtained after a synthesis time of 50 ± 5 min from end of bombardment (EOB). Average batch
product activities, radiochemical yields (RCY) and specific radioactivities (SRA) obtained from the
18
various [ F]12 radiosynthesis trials are compiled in Table 3.
18
18
a
Scheme 3. Preparation of tosylates 31 and 32, and labelling of [ F]12 and [ F]23 .
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
18
Reaction conditions: (a) TsCl or Ts O, TEA, CH Cl , 0°C to r.t., 5 to 16 h; (b) K[ F]Fꢀ[2.2.2]ꢀ
2
2
2
cryptand, K CO , solvent, T (°C), time (min) [see Table 3 for details].
2
3
1
8
18
Table 3. Labelling conditions and results of radiosynthesis trials for [ F]12 and [ F]23
preparation.
Temperature Reaction time
a
a
Precursor
Solvent
DMSO
RCY (d.c.)
SRA (GBq/ꢀmol)
Product
(°C)
(min)
18
3
3
1
1
165
100
100
100
5
10
5
38 ± 6
50 ± 11
37 ± 10
24 ± 7
110 ± 5
75 ± 15
78 ± 14
83 ± 9
[ F]12
1
8
CH CN
[ F]12
3
1
8
31
CH CN
[ F]12
3
18
3
2
CH CN
10
[ F]23
3
a
Values are the mean ± SD of at least 3 independent radiosyntheses
1
8
18
Typically, starting from 33–52 GBq of [ F]fluoride, 9−17 GBq of [ F]12 were obtained, within 50 ±
5
min, with moderate SRA ranging from 60 to 115 GBq/ꢀmol at end of synthesis (EOS).
18
[ F]Fluorination of 12 proved to be the most efficient in acetonitrile when heating the reaction
mixture at 100°C for 10 minutes with an average decayꢀcorrected (d.c.) RCY of 50 ± 11%. Attempts
to reduce reaction time to 5 min or increase reaction temperature to 165°C in DMSO, led to a lower
18
labelling efficacy with d.c. RCY below 40%. [ F]Fluoride incorporation for the preparation of
18
[
F]23 was satisfactory using acetonitrile as solvent at 100°C for 10 min and these labelling
ACS Paragon Plus Environment

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
18
conditions were used for the preparation of readyꢀtoꢀinject [ F]23 batches for in vivo studies. Quality
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
18
controls were performed on aliquots of formulated radioligands [ F]12 and [ F]23. The radiotracers
preparations were clear solutions with a measured pH between 5 and 7. As demonstrated by
analytical HPLC analysis, radiochemical and chemical purities were greater than 95 % and the
preparations were chemically and radiochemically stable for four hours at ambient temperature on
shelve.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Autoradiography. The high binding affinity and selectivity for TSPO of 12 and 23 were confirmed
by in vitro autoradiography studies of their corresponding radiofluorinatedꢀversions in sections of rat
brain with acute local neuroinflammation. Rat brain slices were generated from our inꢀhouse model,
2
3,34,52
seven days after an excitotoxic AMPA injection in the right striatum of Wistar rats.
In a first set
of experiments, the radioligand was incubated alone and accumulation of the tracer was observed in
the lesioned area (rightꢀhand side) when compared to the control side (leftꢀhand side) as seen on
1
8
Figure 5A for [ F]12. In a second and third set of experiments the radioligand was incubated
together with an excess of either its nonꢀlabelled version (12 or 23) or the TSPO ligand of reference
1
8
18
1. Images obtained either with [ F]12 (Figure 5B and 5C) or [ F]23 (not shown here) proved the
absence of nonꢀspecific binding of the ligands and the specificity for the TSPO target, the binding
being fully inhibited in the lesioned area in both cases. Finally, in a fourth set of experiments, the
evaluated radioligand was incubated with an excess of flumazenil, a CBRꢀspecific ligand, to confirm
its absence of affinity for this target. As shown on Figure 5D and as expected from the previous in
3
vitro competition assays with [ H]flunitrazepam (another CBRꢀspecific ligand), the binding of
1
8
[
F]12 was not affected by the presence of an excess of flumazenil, proving again its selectivity for
the TSPO.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
Figure 5. Autoradiography in vitro of [ F]12 (5ꢀ10 nM) on rat AMPAꢀlesioned brain sections : (A)
alone, (B) with nonꢀlabelled 12 (20 ꢀM), (C) with 1 (20 ꢀM) and (D) with flumazenil (20 ꢀM).
18
Autoradiographic images, obtained with [ F]23 as radioligand, were comparable to the one
1
8
18
presented for [ F]12: [ F]23 accumulates in the lesioned area, and the use of 1 or nonꢀlabelled 23
18
fully inhibited the binding of [ F]23 while flumazenil did not. The target to background ratio (TBR),
calculated as the bound radiotracer in the lesion versus the bound radiotracer in the contralateral side,
18
18
was determined after incubation of the radiotracer alone and values obtained for [ F]12 and [ F]23
1
8
18
18
are presented in Table 4 for comparison to [ F]2. The TBR of [ F]12 (2.4 ± 0.3) and [ F]23 (1.9 ±
1
8
0
.3) were surprisingly more than 50 % lower than the one of [ F]2 (5.4 ± 2.0).
18
18
18
Table 4. TBR calculated from in vitro autoradiographies of [ F]2, [ F]12 and [ F]23.
1
8
a
[
F]ꢀtracer
TBR
18
[
F]12
F]23
2.4 ± 0.3
1.9 ± 0.3
5.4 ± 2.0
18
[
1
8
[ F]2
a
TBR values are the mean ± SD of at least 3 independent experiments
18
18
Small animal PET studies. PET imaging was performed with [ F]12 and [ F]23 on anaesthetised
23,34,52
Wistar rats seven days after AMPAꢀinduced brain lesion in the right striatum.
As representative
ACS Paragon Plus Environment

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
examples, Figure 6 shows summed coronal, sagittal and axial images of rat brain from 5 to 60
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
18
minutes after injection of [ F]12. Similar images were obtained after injection of [ F]23.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. microPET images acquired as summed data from 5 to 60 minutes after intravenous
18
injection of [ F]12 to AMPAꢀinduced brain lesioned rat.
1
8
18
Data reported in Table 5 show that both [ F]12 and [ F]23 rapidly enter the brain with initial uptake
values, 2 minutes p.i., in the lesioned striatum of 0.43 and 0.39 percentages of injected dose per
milliliter (% ID/mL) respectively. These uptakes were lower 60 minutes p.i. but still relatively high
1
8
18
(0.33% for [ F]12 and 0.24% for [ F]23), suggesting that the radiotracers still bound their target. In
contrast, uptake in the contralateral side decreased along the study and was rather low 60 minutes p.i.
1
8
18
with values of 0.09 and 0.05 % ID/mL for [ F]12 and [ F]23, respectively. A marked contrast
between the lesioned area and the corresponding area in the intact contralateral hemisphere of the rat
18
brain was thus obtained for [ F]12 after 60 minutes, with a calculated ipsilateralꢀtoꢀcontralateral
ratio of 3.57 ± 0.48 (n=4) that reflects a high in vivo specific binding for the TSPO and is comparable
1
8
18
to that measured for [ F]2 (3.71 ± 0.39). Analogously, the calculated ratio for [ F]23 reached a
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
18
value of 4.62 ± 0.44 (n=4) which is greater than that of [ F]2. Noteworthy, these ratios increased
along the study and may even be higher using extended imaging protocols. The better contrast
18
obtained with [ F]23 mainly results from the quicker wash out of the radiotracer in the nonꢀlesioned
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
area. Table 5 compiles the uptake values obtained in the lesioned and nonꢀlesioned area over a period
18
of 60 minutes, as well as the corresponding calculated ipsi/contra ratios for radioligands [ F]12,
18
18
[
F]23 and [ F]2.
1
8
18
18
Table 5. [ F]12, [ F]23 and [ F]2 uptake values in the lesioned and non-lesioned rat brain
areas over a period of 60 minutes post-injection, and the corresponding calculated ipsi/contra
ratios.
Time
(min)
Ipsi uptake (% ID/mL)
Contra uptake (% ID/mL)
Ipsi/contra ratio
1
8
18
18
18
18
18
18
18
18
[ F]12
[ F]23
[ F]2
[ F]12
[ F]23
[ F]2
[ F]12
[ F]23
[ F]2
2
5
0.43 ± 0.20 0.39 ± 0.07
0.40 ± 0.17 0.38 ± 0.05
0.37 ± 0.16 0.30 ± 0.05
0.32 ± 0.14 0.25 ± 0.06
0.33 ± 0.15 0.24 ± 0.04
0.40 ± 0.02
0.36 ± 0.01
0.34 ± 0.03
0.31 ± 0.05
0.30 ± 0.05
0.28 ± 0.13 0.23 ± 0.04
0.23 ± 0.11 0.16 ± 0.03
0.16 ± 0.08 0.09 ± 0.02
0.10 ± 0.05 0.06 ± 0.01
0.09 ± 0.04 0.05 ± 0.01
0.24 ± 0.02
0.19 ± 0.01
0.12 ± 0.02
0.08 ± 0.02
0.08 ± 0.01
1.53 ± 0.16 1.70 ± 0.12
1.72 ± 0.35 2.43 ± 0.32
2.15 ± 0.33 3.38 ± 0.31
3.05 ± 0.51 4.01 ± 1.05
3.57 ± 0.48 4.62 ± 0.44
1.63 ± 0.12
1.91 ± 0.20
2.76 ± 0.14
3.71 ± 0.49
3.71 ± 0.39
15
4
6
0
0
ꢀ
CONCLUSION
A novel series of 5,7ꢀdimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀylacetamides modified at the 4ꢀpositon of
the phenyl ring were synthesised and in vitro evaluated for their potential to bind the TSPO. All
newly synthesised compounds showed comparable or higher affinity for the TSPO than the parent
molecule 2, with Ki values in the subnanomolar range. The TSPO/CBR selectivity was also assessed
for all compounds and was proved to be conserved. Determination of their LogD7.4 demonstrated
moderately increased lipophilicity compared to 2, however most of the values were in the range for
ACS Paragon Plus Environment

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
suitable blood brain barrier passive penetration. Preliminary metabolism studies were also conducted
for the title compounds. Among them, two candidates were radiolabelled with fluorineꢀ18 and
evaluated in vitro and in vivo for their specific binding to the TSPO and their potential as PET probes
on a rat model of acute neuroinflammation. The data obtained revealed that the newly developed
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
18
radioligands, [ F]12 and [ F]23, are well suited for imaging neuroinflammation in vivo. The
potential of these promising PET radiotracers will be further investigated, in particular with regard to
their metabolic profiling in vivo.
ꢀ
EXPERIMENTAL SECTION
General. Chemicals were purchased from Aldrich France and were used without further purification.
Flash chromatographies were conducted on silica gel or alumina gel (0.63ꢀ0.200 mm, VWR)
columns. TLCs were run on preꢀcoated plates of silica gel 60F254 (VWR, France). The compounds
were localized at 254 nm using a UVꢀlamp and by dipping the TLCꢀplates in a 1% ethanolic
ninhydrin solution or a basic potassium permanganate aqueous solution and heating on a hot plate.
1
13
H and C NMR spectra were recorded on a Bruker (Wissembourg, France) Avance 400 MHz
apparatus and chemical shifts were referenced to the hydrogenated residue of the deutereted solvent
δ[CD HCN] = 1.94 ppm, δ[CD HOD] = 3.31 ppm, δ[CDHCl ] = 5.32 ppm, δ[CHCl ] = 7.24 ppm)
(
2
2
2
3
1
for H NMR and to the deuterated solvent (δ[CD CN] = 118.7 ppm, δ[CD OD] = 49.2 ppm,
3
3
1
3
δ[CD Cl ] = 54.0 ppm and δ[CDCl ] = 77.2 ppm) for C NMR experiments. The standard
2
2
3
concentration of the analysed samples was 20 mg/mL. The chemical shifts are reported in ppm,
downfield from TMS (s, d, t, q, m and b for singlet, doublet, triplet, quadruplet, multiplet and broad,
respectively). The lowꢀresolution mass spectra (MS) were measured on a Thermo Electron (Les Ulis,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
France) Ion Trap LCQ Deca XP1 spectrometer (positive electrospray ionization (ESI)). The highꢀ
resolution mass spectrometry (HRMS) analyses were performed by Imagif (ICSNꢀCNRS, Gifꢀsurꢀ
Yvette, France) by electrospray with positive (ESI+) or negative (ESIꢀ) ionization mode. Purity of the
synthesised compounds was determined using analytical HPLC (HPLC A) and was found to be more
than 95 %. HPLC A: UPLC/SQD Acquity Waters, Acquity BEH C18 (2.1 x 50 mm) column, 1.7 ꢁm,
mobile phase: H O (A), CH CN + 0.1 % formic acid (B), linear gradient from 2 to 100 % (B) in 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
min with a flow rate of 1.0 mL/min. LogD7.4 values were determined based on a validated and
standardized HPLC method (HPLC B): Alliance 2695 ꢀ PDA Waters, XꢀTerra MS C18 (4.6 x 20
mm, 3.5 µm) column, mobile phase: 5 mM MOPS/(CH ) NOH pH 7.4 (A), 5 % MOPS/(CH ) NOH
3
4
3 4
(
100 mM, pH 7.4) / 95 % CH CN (B), gradient (A / B): 98:2 (0.5 min), 0:100 (4.8 min), 98:2 (1.6
3
min), with a flow rate of 1.2 mL/min, 25 °C, detection at 254 nm.
Chemistry. Methyl 4-(3-iodopropoxy)benzoate (3). To a suspension of 18.7 g of potassium
carbonate (136 mmol) in acetone (1 L) was added diiodopropane (22.4 mL, 195 mmol). The mixture
was stirred at ambient temperature under argon before addition of a solution of 10.0 g of methyl 4ꢀ
hydroxybenzoate (65.8 mmol) in acetone (100 mL). The reaction mixture was then heated to 50 °C
and stirring was prolonged for 16 h. The resulting mixture was filtered to remove insoluble potassium
carbonate and the filtrate was evaporated under reduced pressure. The crude product was purified by
silica gel column chromatography using heptane/EtOAc (5/1, v/v) as eluent to yield compound 3
1
(15.2 g, 47.6 mmol, 72 % yield) as light yellow crystals. R (heptane/EtOAc: 2/1 v/v): 0.48. H NMR
f
(
CDCl ) δ 7.97 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 4.07 (t, J = 6.2 Hz, 2H), 3.87 (s, 3H),
3
1
3
3
.36 (t, J = 6.2 Hz, 2H), 2.28 (q, J = 6.2 Hz, 2H). C NMR (CDCl ) δ 166.9 [C], 162.4 [C], 131.7
3
[
2×CH], 122.8 [C], 114.1 [2×CH], 67.4 [CH ], 51.9 [CH ], 32.8 [CH ], 2.2 [CH I]. Mp: 47ꢀ48°C.
2 3 2 2
+
ESI(+)ꢀMS: m/z 321 [M+H] .
ACS Paragon Plus Environment

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
Methyl 4-(3-bromopropoxy)benzoate (4). For the preparation of methyl 4ꢀ(3ꢀ
bromopropoxy)benzoate 4, the procedure described above with methyl 4ꢀhydroxybenzoate 2 was
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reproduced with dibromopropane instead of diiodopropane as alkylating agent. Starting from 10.0 g
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of 2 (65.8 mmol), 14.9 g of compound 4 (54.7 mmol, 83 % yield) could be obtained as a white solid
1
after silica gel column chromatography using pure toluene as eluent. R (toluene 100 %): 0.32. H
f
NMR (CDCl ) δ 7.98 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 3.88 (s,
3
13
3
H), 3.60 (t, J = 6.4 Hz, 2H), 2.34 (m, 2H). C NMR (CDCl ) δ 166.9 [C], 162.5 [C], 131.7 [2×CH],
3
122.9 [C], 114.2 [2×CH], 65.6 [CH ], 51.9 [CH ], 32.2 [CH ], 29.7 [CH ]. ESI(+)ꢀMS: m/z, not seen.
2
3
2
2
Methyl 4-(3-hydroxypropyl)benzoate (5). Starting from methyl 4ꢀ(3ꢀbromopropoxy)benzoate
4): 12.73 g of 4 (46.6 mmol) were dissolved in dry toluene (1 L) and the solution was stirred under
(
argon and heated to 100 °C. A solution containing azobisisobutyronitrile (AIBN, 1.54 g, 9.4 mmol)
and tributyltin hydride (15 mL, 55.9 mmol) in dry toluene (100 mL) was slowly added to the solution
of 4 over a period of 1 h and stirring was prolonged at 100 °C overnight. Full conversion of the
starting material was confirmed by TLC and the reaction mixture was then concentrated under
reduced pressure. The residue was purified by silica gel column chromatography using
heptane/EtOAc (2/1, v/v) as eluent to yield compound 5 (1.61 g, 8.30 mmol, 18 % yield) as a light
yellow oil.
Starting from methyl 4ꢀ(3ꢀiodopropoxy)benzoate (3):15.2 g of 3 were subjected to the procedure
described above. Purification of the crude product afforded compound 5 (1.13 g, 5.82 mmol, 13 %
1
yield). R (heptane/EtOAc: 2/1 v/v): 0.14. H NMR (CDCl ) δ 7.95 (d, J = 8.4 Hz, 2H), 7.26 (d, J =
f
3
1
3
8
.4 Hz, 2H), 3.90 (s, 3H), 3.67 (t, J = 6.4 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H), 1.90 (m, 2H). C NMR
(
CDCl ) δ 167.4 [C], 147.6 [C], 129.9 [2×CH], 128.7 [2×CH], 128.1 [C], 62.2 [CH ], 52.2 [CH ],
3
2
3
3
4.0 [CH ], 32.3 [CH ]. ESI(+)ꢀMS: m/z, not seen.
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Methyl 4-(3-((tert-butyldiphenylsilyl)oxy)propyl)benzoate (6). To 1.31 g of 5 (6.75 mmol)
dissolved in dry pyridine (25 mL) was added tertꢀbutylꢀdiphenylsilyl chloride (2.11 mL, 8.10 mmol)
and the reaction mixture was stirred for 4 h at ambient temperature. After addition of ethyl acetate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(200 mL) the solution was washed twice with a 1.0 M aqueous hydrochloric acid solution (2 x 300
mL) and once with brine (250 mL). The organic layer was then dried over sodium sulfate before
being filtered and evaporated to dryness. The resulting residue was purified by silica gel column
chromatography using toluene/heptane (2/1, v/v) as eluent to yield compound 6 (2.34 g, 5.40 mmol,
1
80% yield) as a colorless oil. R (toluene 100 %): 0.36. H NMR (CDCl ) δ 7.93 (d, J = 8.0 Hz, 2H),
f
3
7.66 (d, J = 6.4 Hz, 4H), 7.43ꢀ7.38 (m, 6H), 7.22 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3.68 (t, J = 6.4 Hz,
1
3
2
H), 2.79 (t, J = 7.6 Hz, 2H), 1.88 (m, 2H), 1.07 (s, 9H). C NMR (CDCl ) δ 167.2 [C], 147.8 [C],
3
135.6 [4×CH], 133.9 [2×C], 129.7 [2×CH], 129.6 [2×CH], 128.5 [2×CH], 127.7 [C], 127.6 [4×CH],
2.9 [CH ], 52.0 [CH ], 33.8 [CH ], 32.2 [CH ], 26.9 [3×CH ], 19.3 [C]. ESI(+)ꢀMS: m/z 433
6
2
3
2
2
3
+
[
M+H] .
-(4-(3-((tert-Butyldiphenylsilyl)oxy)propyl)phenyl)-3-oxopropanenitrile
3
(7).
Sodium
methoxide (250 mg, 4.62 mmol), 6 (2.00 g, 4.62 mmol) and dry acetonitrile (25 mL) were mixed at
ambient temperature and the resulting reaction mixture was stirred and heated to reflux until the
reaction was complete (1ꢀ2 days, TLC monitoring). Addition of water (5 mL) and diethyl ether (5
mL) to the mixture allowed extraction in the organic layer of unreacted starting material. The
aqueous phase was collected and acidified to pH 6 with 20% aqueous sulfuric acid solution to
precipitate the desired compound 7 that was filtered off and dried under vacuum. Compound 7 (695
mg, 1.57 mmol, 34% yield) was obtained as a white powder in a sufficient pure form to be used in
1
the next step without further purification. R (toluene/AcOEt 95/5, v/v): 0.33. H NMR (CDCl ) δ
f
3
7.81 (d, J = 8.0 Hz, 2H), 7.65 (m, 4H), 7.46ꢀ7.36 (m, 6H), 7.30 (d, J = 8.0 Hz, 2H), 4.05 (s, 2H), 3.68
ACS Paragon Plus Environment

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
13
(
t, J = 6.0 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 1.88 (m, 2H), 1.07 (s, 9H). C NMR (CDCl ) δ 186.5
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
C], 150.2 [C], 135.5 [4×CH], 133.7 [2×C], 132.0 [C], 129.6 [2×CH], 129.2 [2×CH], 128.5 [2×CH],
1
27.6 [4×CH], 113.8 [C], 62.6 [CH ], 33.5 [CH ], 32.2 [CH ], 29.2 [CH ], 26.8 [3×CH ], 19.2 [C].
2 2 2 2 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
+
ESI(ꢀ)ꢀMS: m/z 440 [MꢀH] . HRꢀESI(+)ꢀMS m/z calcd for C H NO Si: 440.2046 [M+H] , found
2
8
30
2
440.2042.
-(4-(3-((tert-Butyldiphenylsilyl)oxy)propyl)phenyl)-3-cyano-N,N-diethyl-4-oxobutanamide
8). To 16.3 mL of a 0.125 M solution (2.04 mmol) of sodium hydroxide in a 8/2 mixture of ethanol
4
(
and water, were successively added 7 (750 mg, 1.70 mmol), sodium iodide (765 mg, 5.10 mmol) and
N,Nꢀdiethylchloroacetamide (256 ꢀL, 1.87 mmol). The resulting mixture was stirred at 75 °C for 12
h, diluted then with a 0.1 M aqueous hydrochloric acid solution (100 mL) and extracted with ethyl
acetate (100 mL). The organic layer was separated, washed with brine, dried over sodium sulfate
before being filtered and evaporated to dryness. The resulting residue was purified by silica gel
column chromatography using toluene/EtOAc (95/5, v/v) as eluent to yield compound 8 (764 mg,
1
1
.38 mmol, 81 % yield) as an orange oil. R (toluene/AcOEt 9/1, v/v): 0.29. H NMR (CD Cl ) δ 7.97
f
2
2
(d, J = 8.0 Hz, 2H), 7.70 (m, 4H), 7.45ꢀ7.35 (m, 8H), 7.25 (m, 1H), 7.19 (m, 1H), 5.00 (dd, J = 8.8, J
= 4.8 Hz, 1H), 3.73 (t, J = 6.0 Hz, 2H), 3.40ꢀ3.23 (m, 5H), 2.93 (dd, J = 16.0, J = 4.8 Hz, 1H), 2.86 (t,
13
J = 7.6 Hz, 2H), 1.92 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H), 1.08 (m, 12H). C NMR (CD Cl ) δ 189.5
2
2
[
C], 166.8 [C], 149.9 [C], 135.4 [4×CH], 133.8 [2×C], 132.0 [C], 129.5 [2×CH], 129.1 [2×CH],
1
3
28.9 [2×CH], 127.6 [4×CH], 117.4 [C], 62.7 [CH ], 41.8 [CH ], 40.4 [CH ], 34.0 [CH], 33.6 [CH ],
2 2 2 2
2.9 [CH ], 32.1 [CH ], 26.5 [3×CH ], 19.0 [C], 13.8 [CH ], 12.6 [CH ]. ESI(+)ꢀMS: m/z 555
2
2
3
3
3
+
+
+
[M+H] , 577 [M+Na] , 593 [M+K] . HRꢀESI(+)ꢀMS m/z calcd for C H N O Si: 555.3043
3
4
43
2
3
+
[
M+H] , found 555.3036.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
-(3-Amino-5-(4-(3-((tert-butyldiphenylsilyl)oxy)propyl)phenyl)-1H-pyrazol-4-yl)-N,N-
diethylacetamide (9). To 750 mg of 8 (1.35 mmol) dissolved in ethanol (6.8 mL) were added glacial
acetic acid (140 ꢀL) and hydrazine monohydrate (100 µL, 2.03 mmol). The resulting reaction
mixture was stirred at 60 °C for 3 h, cooled down to room temperature and diluted with ethyl acetate
(100 mL). The organic layer was successively washed with a saturated aqueous ammonium chloride
solution, brine and dried over sodium sulfate before being filtered and evaporated to dryness. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resulting residue was purified by silica gel column chromatography using CH Cl /MeOH (9/1, v/v)
2
2
as eluent to yield compound 9 (734 mg, 1.29 mmol, 95 % yield) as a light pink oil. Rf
1
(
CH Cl /MeOH 95/5, v/v): 0.15. H NMR (CD Cl ) δ 7.68 (m, 4H), 7.46ꢀ7.38 (m, 6H), 7.32ꢀ7.26 (m,
2
2
2
2
4H), 3.72 (t, J = 6.0 Hz, 2H), 3.49 (s, 2H), 3.32 (q, J = 7.2 Hz, 2H), 3.07 (q, J = 7.2 Hz, 2H), 2.80 (t, J
1
3
=
7.6 Hz, 2H), 1.91 (m, 2H), 1.09 (m, 12H), 0.90 (t, J = 7.2 Hz, 3H). C NMR (CD Cl ) δ 170.0 [C],
2 2
1
53.5 [C], 142.9 [C], 142.8 [C], 135.4 [4×CH], 134.0 [C], 133.9 [2×C], 129.5 [2×CH], 129.0
[2×CH], 127.6 [4×CH], 127.5 [2×CH], 98.0 [C], 62.9 [CH ], 42.2 [CH ], 40.3 [CH ], 34.0 [CH ],
2
2
2
2
3
1.7 [CH ], 28.2 [CH ], 26.5 [3×CH ], 19.0 [C], 13.8 [CH ], 12.7 [CH ]. ESI(+)ꢀMS: m/z 569
2 2 3 3 3
+
+
+
[
M+H] , 591 [M+Na] , 607 [M+K] .
2-(2-(4-(3-((tert-Butyldiphenylsilyl)oxy)propyl)phenyl)-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)-N,N-diethylacetamide (10). To 720 mg of 9 (1.27 mmol) dissolved in ethanol
(16 mL) was added pentanꢀ2,4ꢀdione (143 ꢀL, 1.39 mmol) and the resulting mixture stirred overnight
at 60 °C. Evaporation of the reaction solvent afforded a crude product that was purified by silica gel
column chromatography using pure ethyl acetate as eluent to yield compound 10 (600 mg, 0.95
1
mmol, 75% yield) as a beige powder. R (EtOAc 100 %): 0.18. H NMR (CD Cl ) δ 7.70 (m, 6H),
f
2
2
7
.46ꢀ7.38 (m, 6H), 7.29 (d, J = 8.4 Hz, 2H), 6.58 (s, 1H), 3.91 (s, 2H), 3.75 (t, J = 6.0 Hz, 2H), 3.50
(
q, J = 7.2 Hz, 2H), 3.40 (q, J = 7.2 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.76 (s, 3H), 2.57 (s, 3H), 1.94
ACS Paragon Plus Environment

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
13
(
m, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 7.2 Hz, 3H), 1.09 (s, 9H). C NMR (CD Cl ) δ 169.6
2 2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
C], 157.6 [C], 154.5 [C], 147.6 [C], 144.8 [C], 142.5 [C], 135.5 [4×CH], 133.9 [2×C], 131.3 [C],
29.5 [2×CH], 128.5 [2×CH], 128.2 [2×CH], 127.5 [4×CH], 108.2 [CH], 100.9 [C], 63.0 [CH ], 42.1
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
CH ], 40.4 [CH ], 34.1 [CH ], 31.7 [CH ], 27.9 [CH ], 26.6 [3×CH ], 24.3 [CH ], 19.0 [C], 16.6
2 2 2 2 2 3 3
+
+
+
[CH ], 13.9 [CH ], 12.8 [CH ]. ESI(+)ꢀMS: m/z 633 [M+H] , 655 [M+Na] , 671 [M+K] . HRꢀ
3
3
3
+
ESI(+)ꢀMS m/z calcd for C H N O Si: 633.3625 [M+H] , found 633.3607.
39
49
4
2
N,N-Diethyl-2-(2-(4-(3-hydroxypropyl)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)acetamide (11). From 10: To 585 mg of 10 (0.93 mmol) dissolved in tetrahydrofuran (5 mL), was
added tetrabutylammonium fluoride (TBAF, 1.0 M in tetrahydrofuran, 1.85 mL, 1.85 mmol) and the
resulting reaction mixture stirred at ambient temperature overnight. The mixture was then diluted
with ethyl acetate and successively washed with a saturated aqueous ammonium chloride solution
and brine. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The
resulting residue was purified by silica gel column chromatography using CH Cl /MeOH (97/3, v/v)
2
2
as eluent to yield compound 11 (240 mg, 0.61 mmol, 66 % yield) as a white powder.
From 17: To a solution of 209 mg of N,Nꢀdiethylꢀ2ꢀ(2ꢀ(4ꢀ(4ꢀhydroxypropꢀ1ꢀynꢀ1ꢀyl)phenyl)ꢀ5,7ꢀ
dimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀyl)acetamide 17 (0.536 mmol) in dichloromethane (4ꢀ5 mL) was
added 10 % palladium on charcoal (5 mol %). The reaction flask was degassed under vacuum and
filled with hydrogen at 1 atm. and the reaction mixture was stirred at ambient temperature for 24 h.
The mixture was then filtered on a silica pad and washed with a mixture of CH Cl /MeOH (8/2, v/v).
2
2
The filtrate was concentrated to dryness and the residue was purified by silica gel column
chromatography using CH Cl /MeOH (99/1 to 95/5, v/v) as eluent to afford compound 11 (194 mg,
2
2
1
0
.492 mmol, 92 % yield) as a white solid. R (CH Cl /MeOH 95/5, v/v): 0.17. H NMR (CD Cl ) δ
f 2 2 2 2
7
.70 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.56 (s, 1H), 3.89 (s, 2H), 3.63 (t, J = 6.4 Hz, 2H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
1
1
.49 (q, J = 7.2 Hz, 2H), 3.39 (q, J = 7.2 Hz, 2H), 2.73 (m, 5H), 2.53 (s, 3H), 1.87 (m, 2H), 1.70 (bs,
1
3
H), 1.22 (t, J = 7.2 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H). C NMR (CD Cl ) δ 169.6 [C], 157.6 [C],
2
2
54.5 [C], 147.6 [C], 144.8 [C], 142.3 [C], 131.4 [C], 128.5 [2×CH], 128.3 [2×CH], 108.2 [CH],
00.9 [C], 61.9 [CH ], 42.1 [CH ], 40.4 [CH ], 34.3 [CH ], 31.8 [CH ], 27.9 [CH ], 24.3 [CH ], 16.6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
2
2
3
+
+
+
[CH ], 14.0 [CH ], 12.8 [CH ]. ESI(+)ꢀMS: m/z 395 [M+H] , 417 [M+Na] , 433 [M+K] . HRꢀ
3
3
3
+
ESI(+)ꢀMS m/z calcd for C H N O : 395.2447 [M+H] , found 395.2447.
23
31
4
2
N,N-Diethyl-2-(2-(4-(3-fluoropropyl)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)acetamide (12). 168 mg (0.43 mmol) of N,Nꢀdiethylꢀ2ꢀ(2ꢀ(4ꢀ(3ꢀhydroxypropyl)phenyl)ꢀ5,7ꢀ
dimethylpyrazolo[1,5ꢀa]pyrimidinꢀ3ꢀyl)acetamide 11, were reacted with bis(2ꢀmethoxyethyl)
®
aminosulfur trifluoride (DeoxoꢀFluor , 50% solution in toluene, 0.28 mL, 1.07 mmol) in toluene (2.5
mL) at room temperature for 6 h. The reaction mixture was then evaporated to dryness and the
residue purified by silica gel column chromatography using CH Cl /MeOH (99/1, v/v) as eluent to
2
2
give compound 1 (88 mg, 0.22 mmol, 52 % yield) as a beige solid. R (CH Cl /MeOH 98/2 v/v):
f
2
2
1
0
6
.50. t (HPLC A) = 1.18 min. H NMR (CDCl ) δ 7.74 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H),
R
3
2
3
.52 (s, 1H), 4.46 (dt, J
= 46.8 Hz, J
= 6.0 Hz, 2H), 3.93 (s, 2H), 3.49 (q, J = 7.2 Hz, 2H), 3.40
HꢀH
HꢀF
(q, J = 7.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.74 (s, 3H), 2.54 (s, 3H), 2.10ꢀ1.96 (m, 2H), 1.20 (t, J =
13
7.2 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H). C NMR (CD Cl ) δ 169.6 [C], 157.6 [C], 154.4 [C], 147.6
2 2
[
C], 144.8 [C], 141.4 [C], 131.7 [C], 128.5 [2×CH], 128.4 [2×CH], 108.2 [CH], 101.0 [C], 83.2 [d,
1
2
CꢀF
3
J
= 163.0 Hz, CH F], 42.1 [CH ], 40.4 [CH ], 31.9 [d, J
= 20.0 Hz, CH ], 31.0 [d, J = 6.0
CꢀF
CꢀF
2
2
2
2
+
Hz, CH ], 27.9 [CH ], 24.3 [CH ], 16.5 [CH ], 14.0 [CH ], 12.8 [CH ]. ESI(+)ꢀMS: m/z 397 [M+H] ,
2
2
3
3
3
3
+
+
419 [M+Na] . HRꢀESI(+)ꢀMS m/z calcd for C H FN O: 397.2404 [M+H] , found 397.2411.
23 30 4
3
-(4-Iodophenyl)-3-oxopropanenitrile (13). To 50 mL of anhydrous tetrahydrofuran cooled at ꢀ
6
0 °C was added first a 1.6 M nꢀbutyllithium solution in hexanes (62.5 mL, 100 mmol) and then,
ACS Paragon Plus Environment

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
cautiously, a solution of acetonitrile (5.2 mL, 100 mmol) in anhydrous tetrahydrofuran (50 mL) over
a period of 15ꢀ20 min while maintaining the temperature below ꢀ50 °C. The mixture was then stirred
for 30 min at ꢀ60 °C. 12.0 g of methyl 4ꢀiodobenzoate (45.8 mmol) dissolved in anhydrous
tetrahydrofuran (70 mL) was then cautiously channeled to the mixture over 20 min while maintaining
the temperature below ꢀ50 °C. Once the addition finished, the reaction mixture was stirred for 1 h at ꢀ
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
60 °C and 2 h at ꢀ45 °C. Once completed (TLCꢀmonitoring), the reaction was quenched at ꢀ40 °C
with addition of water (200 mL) under vigorous stirring. Then, a 37 % aqueous hydrochloric acid
solution was added to acidify the aqueous layer to pH 2. A white inorganic precipitate was filtered
off and the filtrate was extracted twice with ethyl acetate (2 x 200 mL). The combined organic layers
were washed with water (2 x 100 mL) and brine (100 mL), dried over sodium sulfate, filtered and
concentrated to dryness to afford pure compound 13 (8.64g, 31.9 mmol, 70 % yield) as a beige solid.
1
R (heptane/acetone 3/1, v/v): 0.28. H NMR (CD CN) δ 7.95 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz,
f
3
13
2
H), 4.29 (s, 2H). C NMR (CD CN) δ 188.5 [C], 138.2 [2xCH], 134.1 [C], 129.7 [2xCH], 114.6
3
ꢀ
[
C], 101.8 [C], 29.7 [CH ]. ESI(ꢀ)ꢀMS: m/z 270 [MꢀH] . HRꢀESI(ꢀ)ꢀMS m/z calcd for C H NOI:
2
9
5
ꢀ
2
69.9416 [MꢀH] , found 269.9421.
3-Cyano-N,N-diethyl-4-(4-iodophenyl)-4-oxobutanamide (14). To a solution containing 8.64 g
of 13 (31.9 mmol) in a mixture of ethanol (170 mL) and water (30 mL) was added portionwise and
under vigorous stirring sodium hydroxide (1.40 g, 35.0 mmol). The reaction mixture was stirred for
15 min at ambient temperature and then sodium iodide (9.60 g, 64.0 mmol) was added in one portion,
followed by the dropwise addition of N,Nꢀdiethylchloroacetamide (4.40 mL, 32.1 mmol). The
reaction mixture was stirred for 4 days at ambient temperature. Once the reaction completed (TLCꢀ
monitoring), the suspension was filtered to remove inorganic salt and the filtrate was concentrated to
dryness. The residue was was purified by silica gel column chromatography using CH Cl /MeOH
2
2
(100/0 to 98/2, v/v) as eluent to yield compound 14 (6.61 g, 17.2 mmol, 54 % yield) as a beige
ACS Paragon Plus Environment