Synthesis and initial in vivo evaluation of [11C]AZ683-A Novel PET Radiotracer for Colony Stimulating Factor 1 Receptor (CSF1R)

Article abstract of DOI:10.3390/PH11040136

Positron emission tomography (PET) imaging of Colony Stimulating Factor 1 Receptor (CSF1R) is a new strategy for quantifying both neuroinflammation and inflammation in the periphery since CSF1R is expressed on microglia and macrophages. AZ683 has high aff

Full text of DOI:10.3390/PH11040136

pharmaceuticals
Article
Synthesis and Initial In Vivo Evaluation of
[11C]AZ683—A Novel PET Radiotracer for Colony
Stimulating Factor 1 Receptor (CSF1R)
Sean S. Tanzey 1 , Xia Shao 2, Jenelle Stauff 2, Janna Arteaga 2, Phillip Sherman 2,
Peter J. H. Scott 1,2,
*
and Andrew V. Mossine 2,
*
1
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA;
tanzeys@umich.edu
Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA; xshao@umich.edu (X.S.);
jrstauff@umich.edu (J.S.); jannaa@umich.edu (J.A.); psherman@umich.edu (P.S.)
Correspondence: pjhscott@umich.edu (P.J.H.S.); avmossine@gmail.com (A.V.M.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 October 2018; Accepted: 11 December 2018; Published: 13 December 2018
Abstract: Positron emission tomography (PET) imaging of Colony Stimulating Factor 1 Receptor
(CSF1R) is a new strategy for quantifying both neuroinflammation and inflammation in the
periphery since CSF1R is expressed on microglia and macrophages. AZ683 has high affinity
for CSF1R (Ki = 8 nM; IC50 = 6 nM) and >250-fold selectivity over 95 other kinases. In this
paper, we report the radiosynthesis of [11C]AZ683 and initial evaluation of its use in CSF1R PET.
[11C]AZ683 was synthesized by 11C-methylation of the desmethyl precursor with [11C]MeOTf in
3.0% non-corrected activity yield (based upon [11C]MeOTf), >99% radiochemical purity and high
molar activity. Preliminary PET imaging with [11C]AZ683 revealed low brain uptake in rodents and
nonhuman primates, suggesting that imaging neuroinflammation could be challenging but that the
radiopharmaceutical could still be useful for peripheral imaging of inflammation.
Keywords: neuroinflammation; microglia; carbon-11; radiochemistry; positron emission tomography
1. Introduction
Colony Stimulating Factor 1 Receptor (CSF1R, M-CSF, or cFMS) is a class III receptor tyrosine
kinase [
and macrophage-like cells [
IL-34), plays a role in many disorders that have an immune/inflammatory component [
1
] that regulates immune response by controlling the survival and activity of macrophages
]. Aberrant activity of CSF1R, or its endogenous ligands (CSF1 and
]. Specifically,
2
3
chronic inflammation caused by increased activity of macrophages due to increased CSF1R response is
present in many autoimmune disorders such as rheumatoid arthritis, inflammatory bowel disease, and
autoimmune nephritis, among others [
Disease (AD) is also well known, due in part to its proliferative effects on microglia, which are
associated with neuroinflammation, a hallmark clinical symptom of AD [ ]. A mechanism for CSF1R
involvement in inter-neuronal transmission of pathogenic tau protein by microglia was also recently
elucidated [ ]. Involvement of CSF1R in certain types of cancers, such as gliomas, also correlates with
4,5]. The contribution of CSF1R to symptomatic Alzheimer’s
6,7
8
poor disease prognosis, as proliferation of CSF1R-controlled tumor-associated macrophages (TAMs)
correlates with tumor angiogenesis and metastasis [4,9–11].
Consequently, CSF1R inhibitors (both small molecules and biologics) have been proposed as
a means of controlling inflammation in this multitude of diseases and disorders via macrophage
depletion/regulation [12]. Many CSF1R inhibitors, including some that are kinase-specific, can be
found in both academic and patent literature [
4
,5,13], and several have proceeded to clinical trials for
Pharmaceuticals 2018, 11, 136; doi:10.3390/ph11040136
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the treatment of RA [14] and various types of cancer [11]. However, not all macrophage populations
are CSF1R-sensitive, necessitating that CSF1R involvement must be positively identified prior to the
start of treatment. As CSF1R is a cell surface receptor, its upregulation is only present at the site of
inflammation. Although blood biomarkers can be used to directly measure CSF1R involvement in
certain diseases, such as lymphoma [11], methods of determining CSF1R involvement and quantifying
CSF1R levels at loci not directly connected to the central circulatory system is difficult, particularly in
the CNS, and employs either indirect means (i.e., measurement of CSF1 levels as a proxy for CSF1R [14])
or invasive procedures (i.e., immunohistochemistry using a biopsy sample or surgically excised
tissue [15,16]). In fact, despite the implication of irregular CSF1R levels in numerous diseases [4],
quantitative information on expression levels in disease is generally lacking from the literature. In
part this is because CSF1R levels are constantly changing as, for example, macrophages are produced
and/or cleared, but also because there is currently an unmet need for a non-invasive method that
can positively identify and quantify CSF1R involvement in disease. This goal can be readily achieved
with positron emission tomography (PET) imaging, wherein a CSF1R-selective radiolabeled ligand
(radiopharmaceutical) would be used to detect changes in activity, expression levels, and localization of
CSF1R in a minimally-invasive manner. Furthermore, a brain-penetrant CSF1R-selective PET imaging
agent could be used to selectively image microglia, as they are the only cells in the brain that express
CSF1R under normal conditions [17]. Microglia associate with amyloid beta plaques in the brain [18],
and are implicated in pathological tau transmission [8] and neuroinflammation [6,7], both of which are
important in the progression of neurodegenerative disorders.
The current method of choice for imaging macrophages/microglia is by targeting the translocator
protein 18 kDa (TSPO). However, TSPO is not an ideal imaging target since it is expressed in various
tissue types (in addition to immune cells). Moreover, a single nucleotide polymorphism (SNP) in the
TSPO gene has been identified that leads to considerable variability in its expression levels between
patients and, consequently, variability in the corresponding PET data [19]. Therefore, a CSF1R imaging
agent selective for microglia is of considerable interest for using PET both to quantify CSF1R and as a
surrogate biomarker for neuroinflammation (and peripheral inflammation).
Radiopharmaceuticals used in PET imaging are often structural analogs of existing pharmaceutical
agents that have been labeled with a positron-emitting radionuclide such as 11C, 18F or 124I. As
such, the radiopharmaceutical can be expected to possess the same pharmacokinetic properties as
its nonradioactive counterpart and behave accordingly in vivo. Fortunately, lead identification for
CSF1R PET radiopharmaceutical development is relatively straightforward because recent interest in
developing CSF1R inhibitors has led to hundreds of active compounds, several of which have also been
translated into clinical trials (see Figure 1 for several leads) [4,5,13]. PET imaging agents for CSF1R
have been reported [20 21], but none have seen widespread use to date. One is a mixed inhibitor of both
,
CSF1R and tropomyosin receptor kinases B and C (Trk B/C) [20], while the second ([11C]JHU11744)
has shown promise in preliminary evaluation in rodent models of AD and neuroinflammation [21].
PET imaging of CSF1R therefore remains underdeveloped and herein we attempt to address this
issue through development of [11C]AZ683, a new radiopharmaceutical for CSF1R. AZ683 (Figure 1)
was selected because it has >250-fold selectivity for CSF1R over 95 other kinases, low plasma protein
binding, a good pharmacokinetic (PK) profile, and both fluorine and N-methyl moieties which are
potential sites for radiolabeling with 18F or 11C, respectively [22
–24]. Moreover, AZ683 has low
nanomolar affinity for CSF1R (Ki = 8 nM; IC50 = 6 nM), making it ideal for PET studies which typically
utilize nanomoles-picomoles of radiotracer, and the cLogP of the neutral (uncharged) compound is
3.1 which suggests that it should cross the BBB. Since N-methylation of the desmethyl precursor with
[11C]MeI (or [11C]MeOTf) was envisioned to be simpler than 18F-labeling of this scaffold, the synthesis
and carbon-11 radiolabeling of [11C]AZ683 was undertaken for initial evaluation and is described
herein. We also report preliminary evaluation of the radiotracer as a CSF1R imaging agent in rodent
and non-human primate PET imaging studies.

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Figure 1. Potential lead compounds for CSF1R radiopharmaceutical development (proposed radiolabeling
sites are shown in red).
2. Results and Discussion
2.1. Synthesis of Reference Standard and Precursor
AZ683 reference standard 6a and N-desmethyl precursor
literature procedures in five and six steps, respectively (Scheme 1) [23]. Both syntheses diverged
from a common intermediate . This common intermediate was synthesized via condensation of
4-bromo-3-ethoxyaniline ( ) with diethylethoxymethylenemalonate to yield . This was followed
by cyclization/chlorination with POCl3 and tetrabutylammonium chloride to form chloroquinoline
. A subsequent SNAr reaction with 2,4-difluoroaniline yielded intermediate . Buchwald–Hartwig
cross-coupling was then used to couple either N-Boc piperazine or N-methylpiperazine with , yielding
intermediates 5a and 5b for the reference standard and precursor, respectively. Amidation of the ethyl
ester of was performed using formamide/NaOEt to generate reference standard 6a and N-Boc
protected precursor 6b. Final deprotection of the Boc group of 6b with trimethylsilyl chloride in
methanol furnished precursor . Precursor and reference standard 6a were readily separable on
7 were synthesized via modified
4
1
2
3
4
4
5
7
7
analytical and semipreparative Phenomenex Luna C18 columns using a 30% ethanolic eluent buffered
with sodium phosphate at a pH of 6.6 (see Materials and Methods for details).

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Scheme 1. Synthesis of precursor and reference standar◦d for [11C]AZ683. Reagents and conditio◦ns:
(i) diethylethoxymethylenemalonate, K2CO3, MeCN, 70 C (71%); (ii) POCl3, TBACl, toluene, 130 C
(17%); (iii) 2,4-difluoroaniline, 20% AcOH, EtOH, 80 ◦C (66%); (iv) 1–5 mol % Pd2(dba)3, BINAP,
Cs2CO3, toluene, 100 ◦C (5a: 54%; 5b: 45%); (v) formamide, NaOEt, EtOH/THF, reflux (6a: 32%; 6b
30%); (vi) TMSCl, MeOH, room temp (100% from 6b).
:
2.2. Radiosynthesis of [11C]AZ683
Radiolabeling of [11C]AZ683 was accomplished by treating precursor
7
with [11C]MeOTf
(Scheme 2). The labeling reaction was automated using a TRACERLab FXC-pro synthesis module
and our standard carbon-11 procedures [25]. Following radiolabeling, [11C]AZ683 was purified within
the synthesis module via semipreparative HPLC and formulated for injection (0.9% saline solution
containing 10% ethanol) using a Waters C18 1cc vac cartridge to trap/release the product. This resulted
in an overall non-decay corrected activity yield of 1125
±
229 MBq (3.0% based upon 37 GBq of
38 GBq/ mol (n = 4), confirming
[11C]MeOTf), radiochemical purity >99%, and molar activity of 153
doses were suitable for preclinical evaluation.
±
µ
Scheme 2. Radiosynthesis of [11C]AZ683. Reagents and conditions: (i) [11C]MeOTF, DMF, rt, 3 min
(3.0% activity yield).
2.3. Preclinical PET Imaging
Initial evaluation of the imaging properties of [11C]AZ683 was undertaken in female
Sprague–Dawley rat. [11C]AZ683 was administered via intravenous tail vein injection and rodent
brain imaging was conducted for 60 min. To our surprise, [11C]AZ683 showed very little brain uptake
but did show high uptake and retention in the pituitary and thyroid glands (Figure 2, left). Although
both glands are known for expression of CSF1R protein (thyroid) and CSF1R RNA (thyroid and
pituitary) [26], we assume the very high uptake is more likely indicative of non-specific binding
associated with the lipophilic nature of the compound (Table 1). Since inter-species differences are
sometimes apparent between rodents and non-human primates due to the higher metabolic rate in
rodents and differing BBB efflux systems, imaging in rhesus macaque brain was also performed. The
primate imaging results largely mirrored the rat data, with fairly poor brain influx during the early
frames, followed by almost complete washout and little brain retention in a normal brain (Figure 2,
right). There was some retention in the central region of the brain that was likely ventricular uptake
and, as before, the pituitary gland could be observed in frame and showed a much greater degree

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of uptake than brain. Overall though, brain uptake in monkey was higher than in rat and there was
perhaps some focal uptake in the monkey cerebellum (standardized uptake value (SUV) ~0.3–0.4 at
late time points). Given that the cerebellum is an area of known CSF1R expression in humans [26], and
CSF1R function is thought to be conserved between vertebrates [27], this signal could correspond to
CSF1R, presumably associated with microglia found in the monkey cerebellum [28]. However, this will
need to be confirmed in future in vitro experiments with primate brain sections. Target receptor density
of CSF1R could ostensibly be low in a non-diseased control animal and would explain poor brain
retention, but again normal CSF1R levels are challenging to quantify in vivo as they are transient and
expected to fluctuate with the turnover of macrophages and microglia. However, low receptor density
would not limit first pass brain influx and efflux which was also quite low. Overall these PET imaging
data suggest imaging CSF1R associated with neuroinflammation using [11C]AZ683 may be challenging,
but that uptake in monkey could be sufficient to observe accumulation in a brain inflammation model.
There is literature precedent for TSPO radiotracers with low brain uptake being used to successfully
image inflammation in rat models [29,30]. Moreover, the present studies do not rule out labeling the
scaffold with a longer-lived PET radionuclide (e.g., 18F or 124I) and using a prolonged infusion protocol
so that sufficient radiotracer accumulates at sites of inflammation. [11C]AZ683 could also possibly be
used for imaging of peripheral CSF1R to evaluate its role in inflammation outside of the brain.
Given that [11C]AZ683 possesses properties mostly consistent with BBB permeability
(Table 1) [23,31,32], the lack of brain uptake was unexpected and the reasons for it are unclear. It is
possible that [11C]AZ683 is a substrate for an efflux transporter on the BBB and since, for example,
P-glycoprotein transporter (P-gp) expression is higher in rodents than monkeys (and humans) [33],
this could explain the 2–3-fold higher uptake of the radiotracer observed in monkey brain. Given the
differences in type and expression levels of efflux transporters between species, monkeys are better for
predicting the role of P-gp in limiting brain penetration of drugs in humans [33]. However, as we take a
conservative view towards primate safety, methods to determine whether efflux activity is responsible
for the low brain uptake of [11C]AZ683 (e.g., cyclosporin A blockade of the P-gp transporter [34])
have not been pursued at this time. Alternatively, in this case, cLogP estimates (Table 1) may not be a
good indicator of BBB permeability. [11C]AZ683 has multiple groups containing nitrogen and oxygen
atoms which are ionizable, corresponding to multiple pKa values (Figure 3 [32]). We do not expect
the primary amide to limit BBB permeability since we conduct brain imaging with other primary
amide-containing radiopharmaceuticals such as [11C]LY2795050 [35]. Understanding the relationship
between cLogP of charged species as a function of pH is complicated [31], but it is likely that AZ683
is charged at physiological pH and this could be the reason for poor brain uptake. The oxygen and
nitrogen atoms in question also participate in hydrogen bonding, and cLogP—the total number of
oxygen and nitrogen atoms in a drug molecule (N + O) offers information about logBBB. If cLogP—(N
+ O) >0, logBBB is likely to be positive and the drug has a good probability of entering the CNS [31].
In the case of AZ863, cLogP—(N + O) =
may be too high for good CNS penetration. All of these issues should be considered in the design of
next generation CSF1R radiopharmaceuticals going forward.
−
4, suggesting the number of oxygen and nitrogen atoms
Table 1. Properties of [11C]AZ683 compared to a typical CNS drug.
Property
Activity
cLogP
Preferred Value for Successful CNS Drugs [31]
[
11C]AZ683 [23,32]
Low nM
<5 (Lipinski’s Ro5 [29])
Ki = 8 nM; IC50 = 6 nM
3.1
<2.7 (optimized [29])
60–70 Å2
≤450 g/mol
≤3
83 Å2
441 g/mol
tPSA
Molecular weight
H-bond donors
H-bond acceptors
Rotatable bonds
Metabolic stability
Solubility
2
6
8
≤7
<8
T1/2 > 3.1 h
>60 µg/mL
7.5–10.5
>0
2.1 h
128 µg/mL
6.5–7.5
−4
pKa
cLogP—(N + O)

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4
3
2
Ventricle
P ituitary
1
0
Thyroid
P ituitary
C erebellum
R at
M onkey
Figure 2. Summed rodent (left) and primate (right) PET images of [11C]AZ683 (0–60 min after injection
of the radiotracer) and associated time–radioactivity curves (SUV = standardized uptake value).
Figure 3. Multiple pKa values for AZ683 [32].
3. Materials and Methods
3.1. Synthesis
3.1.1. General Considerations
All the chemicals were purchased from commercially available suppliers and used without
purification. Automated flash chromatography was performed with Biotage Isolera Prime system.

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High-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT
1
system. H NMR spectra were acquired using a Varian 400 apparatus (400 MHz) in CDCl3 or CD3OD.
δ
are reported in ppm relative to tetramethylsilane (δ = 0), J are given in Hz. Mass spectra were measured
on an Agilent Technologies (Santa Clara CA, USA) Q-TOF HPLC-MS or Micromass (Manchester, UK)
VG 70-250-S Magnetic sector mass spectrometer employing the electrospray ionization (ESI) method.
3.1.2. Compounds Synthesized
Diethyl 2-(((4-bromo-3-ethoxyphenyl)amino)methylene)malonate (
4-bromo-3-ethoxyaniline hydrochloride ( ) (0.66 g, 3 mmol) and K2CO3 (1.68 g, 12.2 mmol) in MeCN
(30 mL) was added diethyl-ethoxymethylene malonate (620 L, 3 mmol). The reaction was heated to
2). To a solution mixture of
1
µ
reflux and allowed to stir for 36 h, at which time it was cooled and vacuum filtrated through celite to
remove potassium carbonate. The filtrate was purified by flash chromatography using a hexane-EtOAc
gradient to yield
2 δ 11.00 (d, J = 13.5 Hz, 1H), 8.45 (d,
(0.84 g, 71%). 1H-NMR (400 MHz, CDCl3)
J = 13.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 6.63 (d, J = 8.5 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 4.33–4.22 (m,
4H), 4.09 (q, J = 7.0 Hz, 2H), 1.49 (t, J = 7.0 Hz, 3H), 1.38–1.31 (m, 6H). [M + H]+: Expected 386.0598,
Found 386.0604.
Ethyl 6-bromo-4-chloro-7-ethoxyquinoline-3-carboxylate (3). Compound 2 (0.84 g, 2.2 mmol) was dissolved
in dry Toluene (2.5 mL). Tert-Butyl ammonium chloride (TBACl: 1.94 g, 7 mmol) was added, followed
by POCl3 (2 mL, 22 mmol) while stirring at room temperature for 5 min. The reaction mixture was
then heated to reflux and stirred for 68 h. After this time the reaction was cooled, diluted with DCM
(30 mL) and quenched with water (30 mL). The aq. layer was extracted with further DCM (30 mL) and
the combined organic fractions were washed with brine (60 mL), dried (Na2SO4) and concentrated.
The crude material was purified by flash chromatography using a hexane/EtOAc gradient to yield
3
1
(0.15 g, 17%). H-NMR (400 MHz, CDCl3)
δ
9.16 (s, 1H), 8.61 (s, 1H), 7.42 (s, 1H), 4.48 (q, J = 7.2 Hz,
2H), 4.28 (dd, J = 14.1, 7.0 Hz, 2H), 1.58 (t, J = 7.0 Hz, 3H), 1.45 (t, J = 7.2 Hz, 3H). [M + H]+: Expected
357.9840, Found 359.9820. Cl-37 accounts for difference in expected value.
Ethyl 6-bromo-4-((2,4-difluorophenyl)amino)-7-ethoxyquinoline-3-carboxylate (
0.41 mmol) was dissolved in ethanol (10 mL). 20 mol % acetic acid (4.7 L, 0.082 mmol) was added
followed by 2,4-difluoroaniline (46 L, 0.45 mmol, 1.1 eq.). The reaction was heated to reflux and
stirred for 24 h. After this time the reaction was cooled and Et3N (100 L) was added to neutralize
acetic acid. The crude reaction mixture was purified by flash chromatography to yield title compound
(0.11 g, 66%). 1H-NMR (400 MHz, CDCl3)
10.30 (s, 1H), 9.19 (s, 1H), 7.73 (s, 1H), 7.03 (s, 1H),
4). Compound 3 (0.15 g,
µ
µ
µ
4
δ
6.97–6.94 (m, 1H), 6.94–6.92 (m, 1H), 6.84 (t, J = 8.3 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 4.23 (q, J = 7.0 Hz,
2H), 1.53 (t, J = 7.0 Hz, 3H), 1.45 (t, J = 7.1 Hz, 3H). [M + H]+: Expected 451.0463, Found 451.0463.
Ethyl 4-((2,4-difluorophenyl)amino)-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxylate
Compound (113 mg, 0.251 mmol) was dissolved in dry toluene (10 mL). To this solution,
1-methylpiperizine (33.4 L, 0.301 mmol, 1.2 eq.) was added. This solution was aspirated with a
(5a).
3
µ
syringe and added to a mixture of 2.5 mol % Pd2(dba)3 (5.75 mg, 0.007 mmol), 2.5 mol % BINAP
(3.9 mg, 0.007 mmol) and 1.6 eq. of Cs2CO3 (0.13 g, 0.402 mmol) under Ar. The reaction was heated to
100◦C and stirred for 60 h. After this time the reaction was cooled and quenched with satd. KHCO3
(20 mL). The organic layer was washed with water (50 mL) and the water was extracted twice with
EtOAc. The organic layers were combined, washed with brine (60 mL), concentrated and dried
(Na2SO4). The residue was purified by flash chromatography using an EtOAc/MeOH gradient to
1
yield compound 5a (64 mg, 54%). H-NMR (400 MHz, CDCl3)
δ 10.14 (s, 1H), 9.11 (s, 1H), 7.32 (s,
1H), 7.26 (s, 1H), 7.00–6.90 (m, 1H), 6.89 (s, 1H), 6.75 (t, J = 8.4 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 4.21 (q,
J = 6.9 Hz, 2H), 2.83 (m, 4H), 2.55 (m, 4H), 2.30 (s, 3H), 1.51 (t, J = 6.9 Hz, 3H), 1.48–1.44 (m, 3H). [M +
H]+: Expected 471.2202, Found 471.2202.

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Ethyl 6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-4-((2,4-difluorophenyl)amino)-7-ethoxyquinoline -3-carboxylate
5b). The same procedure described for the synthesis of 5a was also used to prepare 5b (61 mg, 44.5%).
1H-NMR (400 MHz, CDCl3)
10.08 (s, 1H), 9.12 (s, 1H), 7.30 (s, 1H), 6.96–6.88 (m, 2H), 6.86 (s, 1H),
(
δ
6.75 (t, J = 8.2 Hz, 1H), 4.43 (q, J = 7.1 Hz, 3H), 4.21 (q, J = 7.0 Hz, 2H), 3.53–3.45 (m, 4H), 2.75–2.67 (m,
4H), 1.52 (t, J = 7.0 Hz, 3H), 1.47 (s, 9H), 1.44 (d, J = 7.1 Hz, 3H). [M + H]+: Expected 557.2571, Found
557.2571.
4-((2.,4-Difluorophenyl)amino)-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide
Reference Standard 6a). Compound 5a (53 mg, 0.113 mmol) was dissolved in THF (0.1 mL) and
formamide (22.4 L, 0.563 mmol, 5 eq.) was added. To this solution, a 21% wt solution of NaOEt in
EtOH (231 L, 0.563 mmol, 5 eq.) was added. The reaction was heated to reflux and stirred for 16 h,
(AZ683
µ
µ
after which time it was cooled and quenched with NH4Cl (53 mg, 1 mmol). The reaction mixture was
concentrated onto silica and purified by flash chromatography using an EtOAc/MeOH gradient to
1
yield compound 6a (16 mg, 32%). H-NMR (400 MHz, CD3OD)
δ
8.78 (s, 1H), 7.27 (s, 1H), 7.16–7.06
(m, 3H), 6.95 (t, J = 8.6 Hz, 1H), 4.25 (q, J = 6.9 Hz, 3H), 3.10 (m, 4H), 2.73 (m, 4H), 1.96 (s, 3H), 1.51 (t,
J = 6.9 Hz, 3H). [M + H]+: Expected 442.2049, Found 442.2048.
tert-Butyl 4-(3-carbamoyl-4-((2,4-difluorophenyl)amino)-7-ethoxyquinolin-6-yl)piperazine-1-carboxylate (6b).
1
The same procedure described for the synthesis of 6a was used to prepare 6b (18 mg, 30%). H-NMR
(400 MHz, CDCl3)
δ 10.44 (s, 1H), 8.76 (s, 1H), 7.27 (s, 1H), 6.94–6.88 (m, 2H), 6.87 (s, 1H), 6.74 (t,
J = 8.3 Hz, 1H), 6.21 (br. s, 1H), 4.20 (q, J = 6.9 Hz, 2H), 3.48–3.47 (m, 4H), 2.73–2.71 (m, 4H), 1.51 (t,
J = 6.9 Hz, 3H), 1.47 (s, 9H). [M + H]+: Expected 528.2417, Found 528.2419.
4-((2,4-Difluorophenyl)amino)-7-ethoxy-6-(piperazin-1-yl)quinoline-3-carboxa◦mide (
7). Compound 6b
(18 mg, 0.034 mmol) was dissolved in dry MeOH (5 mL) and cooled to -78 C for 5 min. Trimethylsilyl
chloride (TMS-Cl, 43.3 µL, 0.341 mmol, 10 eq.) was added and the reaction was allowed to warm
up to room temperature and was stirred until deprotection was complete as determined by TLC
(~25 h). The reaction was quenched with water and concentrated to remove solvent and excess TMS-Cl.
The concentrate was re-dissolved in MeOH and re-concentrated two more times to ensure complete
removal of TMS-Cl. The product was further dried in a vacuum desiccator to yield compound
7
(15 mg,
1
100%). H-NMR (400 MHz, CD3OD)
δ
8.80 (s, 1H), 7.56–7.50 (m, 1H), 7.38 (s, 1H), 7.34 (s, 1H), 7.26–7.18
(m, 1H), 7.14 (t, J = 8.4 Hz, 1H), 4.33 (q, J = 7.0 Hz, 2H), 3.39–3.33 (m, 4H), 3.22–3.20 (m, 4H), 1.55 (t,
J = 7.0 Hz, 3H). [M + H]+: Expected 428.1893, Found 428.1892.
3.2. Radiochemistry
3.2.1. General Considerations
All the chemicals (except for reference standard 6a and precursor
7 noted above) were purchased
from commercially available suppliers and used without purification: sodium chloride, 0.9% USP
and Sterile Water for Injection, USP were purchased from Hospira; Dehydrated Alcohol for Injection,
USP was obtained from Akorn Inc. (Lake Forest IL, USA) HPLC was performed using a Shimadzu
(Kyoto, Japan) LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector, and HPLC
columns were acquired from Phenomenex (Torrance CA, USA). Other synthesis components were
obtained as follows: sterile filters were acquired from MilliporeSigma (Burlington MA, USA); C18 Vac
1cc Sep-Paks were purchased from Waters Corporation (Milford MA, USA); Sep-Paks were flushed
with 5 mL of ethanol followed by 10 mL of sterile water prior to use.
3.2.2. Radiosynthesis of [11C]AZ683
[11C]CO2 was produced with a General Electric Healthcare (GE, Uppsala, Sweden) PETTrace
cyclotron via the 14N(p,
40
synthesis module and converted to [11C]MeOTf (~37 GBq) as previously described [25]. [11C]MeOTf
α
)11C reaction. High purity N2 (g) containing 0.5% O2 was irradiated at
µ
A for 30 min to generate [11C]CO2 (~111 GBq), which was delivered to a GE TRACERLab FXC-Pro

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was bubbled at 15 mL/min through a solution of precursor
7
(1 mg) in DMF (100
µL) at room
temperature for 3 min. Following radiolabeling, the reaction mixture was diluted with HPLC mobile
phase and purified by semipreparative HPLC (column: Phenomenex Luna C18, 10 , 10 250 mm;
µ
×
mobile phase: 27% ethanol, 10 mM Na2HPO4, pH = 5.75; flow rate: 5 mL/min; see Figure 4 for a
representative semipreparative HPLC trace). The peak corresponding to [11C]AZ683 (tR ~12–14 min)
was collected, diluted in water (50 mL), and the resulting solution was passed through a Waters C18 1cc
vac cartridge to trap the product. [11C]AZ683 was eluted from the cartridge with ethanol (1 mL) and
diluted with 0.9% saline solution (9 mL) to provide the formulated product in 10% EtOH. The dose was
passed through a 0.22
µ
m sterile filter into a sterile dose vial. The overall non-decay corrected activity
yield of [11C]AZ683 was 1125
±
229 MBq (3.0% based upon 37 GBq of [11C]MeOTf) and quality control
testing (see below) confirmed radiochemical purity >99%, and molar activity of 153
(n = 4), confirming doses were suitable for preclinical evaluation.
±
38 GBq/µmol
Figure 4. Typical semi-preparative HPLC trace for [11C]AZ683.
3.2.3. Quality Control Testing of [11C]AZ683
Visual inspection
Doses were visually examined and required to be clear, colorless, and free of particulate matter.
Dose pH
The pH of the doses was determined by applying a small amount of the dose to pH-indicator
strips and determined by visual comparison to the scale provided. pH needs to be between 4.5 and 7.5,
and the pH of each [11C]AZ683 dose synthesized in this study was 5.0.
Analytical HPLC
Analytical HPLC was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan
B-FC-1000 radiation detector (column: Phenomenex Luna C18, 5
µ, 4.6
×
150 mm; mobile phase: 27%

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ethanol, 10 mM Na2HPO4, pH: 5.75; flow rate: 0.75 mL/min). Analysis confirmed radiochemical
purity >99% (tR of [11C]AZ683 ~6 min; see Figure 5 for a typical analytical HPLC trace) and coinjection
with unlabeled reference standard 6a confirmed radiochemical identity (see Figure 6 for a coinjection
HPLC trace).
Figure 5. Analytical HPLC trace for formulated [11C]AZ683 dose.
Figure 6. Analytical HPLC trace for formulated [11C]AZ683 dose co-injected with AZ683 reference
standard 6a.
3.3. Preclinical PET Imaging
3.3.1. General Considerations
Rodent and primate imaging studies were performed at the University of Michigan (UM) using
a Concorde (CTI-Concorde, Knoxville TN, USA) MicroPET P4 scanner. The University of Michigan

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is accredited by the Council on Accreditation of the Association for Assessment and Accreditation
of Laboratory Animal Care (AAALAC International, Frederick MD, USA) and imaging studies were
conducted in accordance with the standards set by the Institutional Animal Care and Use Committee
(IACUC) at the University of Michigan (PRO00008103: Biodistribution and Pharmacokinetics of
Radiolabeled Compounds; Approval date: 1/16/2018).
3.3.2. Animal Husbandry and Housing
Husbandry and housing for rodents and primates is provided by the University Laboratory for
Animal Medicine (ULAM) at UM, and animal facilities are in compliance with the regulations defined
by the US Department of Agriculture (USDA).
Monkeys: The University of Michigan PET Center has maintained 2 rhesus macaques for
~15 years and the monkeys are individually housed in adjacent steel cages (83.3 cm high
×
152.4 cm
wide × 78.8 cm deep) equipped with foraging boxes. They are currently housed in adjacent cages as
repeated attempts to socially house them in the same cage have been unsuccessful due to aggressive
incompatibility. Cages are metal and do contain gridded floors for radiation safety reasons (radioactive
waste is contained to the gridded floor and is easier to clean). Temperature and humidity are carefully
controlled, and the monkeys are kept on a 12 h light/12 h dark schedule. Monkeys are fed Lab Fiber
Plus Monkey Diet (PMI Nutrition Intl. LLC, Shoreview MN, USA) that is supplemented with fresh fruit
and vegetables daily. Water and enrichment toys (manipulanda and food-based treats) are available
continuously in the home cage.
Rodents: Rats are housed in Allentown #3 micro ventilated cages (27 cm wide
27 cm high, floor area 923 Sq cm) with animal housing densities set by ULAM and the Guide for
×
49 cm deep
×
the Care and Use of Laboratory Animals. Housing is located on ventilated racks with continuous
water and air supply exchange. All animals are provided with LabDiet 5LOD as well as enrichment
materials, and are on a light schedule of 12 h light/12 h dark.
3.3.3. Rodent Imaging Protocol
Rodent imaging studies were done using a female Sprague–Dawley rat (weight = 237 g, n = 1).
The rat was anesthetized (isoflurane), intubated, and positioned in the PET scanner. Following a
transmission scan, the animal was injected (via intravenous (i.v.) tail vein injection) with [11C]AZ683
(14.8 MBq) as a bolus over 1 min, and the brain imaged for 60 min (5
frames-2 × 5 min frames-4 × 10 min frames).
×
1 min frames-2
×
2.5 min
3.3.4. Primate Imaging Protocol
Primate imaging studies were done using a mature female rhesus monkey (weight = 9.4 kg, n = 1).
The animal was anesthetized in the home cage with ketamine and transported to the PET imaging
suite. The monkey was intubated for mechanical ventilation, and anesthesia was continued with
isoflurane. Anesthesia was maintained throughout the duration of the PET scan. A venous catheter
was inserted into one hind limb and the monkey was placed on the PET gantry with its head secured to
prevent motion artifacts. Following a transmission scan, the animal was injected i.v. with [11C]AZ683
(145.0 MBq) as a bolus over 1 min, and the brain imaged for 60 min (5
frames-3 × 10 min frames).
×
2 min frames-4
×
5 min
3.3.5. PET Image Analysis
Emission data were corrected for attenuation and scatter, and reconstructed using the 3D
maximum a priori (3D MAP) method. By using a summed image, regions of interest (ROI) were drawn
on multiple planes, and the volumetric ROIs were then applied to the full dynamic data set to generate
time-radioactivity curves.

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4. Conclusions
In conclusion, we have developed a radiosynthesis of [11C]AZ683 for PET imaging of CSF1R and
neuroinflammation that provides doses suitable for preclinical use. However, preliminary preclinical
PET imaging in rodents and nonhuman primates revealed low brain uptake of [11C]AZ683. Overall,
these PET imaging data suggest imaging CSF1R associated with neuroinflammation using [11C]AZ683
could be challenging and emphasize that high affinity, good selectivity, and appropriate drug-like
properties do not guarantee that a compound will make a good radiopharmaceutical for in vivo
brain PET. Nevertheless, uptake in monkey could be sufficient to observe accumulation in a brain
inflammation model. These studies also do not rule out labeling the scaffold with a longer-lived
PET radionuclide (e.g., 18F or 124I) and using a prolonged infusion protocol to ensure that sufficient
radiotracer accumulates at sites of inflammation for imaging and quantitation of CSF1R. [11C]AZ683
could potentially also be used for imaging of peripheral CSF1R to evaluate its role in inflammation
outside the brain. Future evaluation in animal models of inflammation appears warranted.
Author Contributions: Conceptualization: P.J.H.S. and A.V.M.; Investigation: S.S.T., X.S., J.S., J.A., P.S. and
A.V.M.; Methodology: S.S.T., X.S. and A.V.M; Writing: S.S.T., P.J.H.S. and A.V.M.; Supervision: P.J.H.S.; Funding
acquisition: P.J.H.S.
Funding: Financial support from the US Department of Energy/National Institute of Biomedical Imaging and
Bioengineering (DE-SC0012484) and the University of Michigan (College of Pharmacy) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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