BLZ945 | Micro rna2

BLZ945 derivatives for PET imaging of colony stimulating factor-1 receptors in the brain☆
Berend van der Wildt a,b, Zheng Miao a, Samantha T. Reyes a, Jun H. Park a, Jessica L. Klockow a, Ning Zhao a,
Alex Romero a, Scarlett G. Guo a, Bin Shen a, Albert D. Windhorst b, Frederick T. Chin a,⁎
a Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University, School of Medicine, Stanford, CA, USA
b Amsterdam UMC, Vrije Universiteit Amsterdam, Radiology & Nuclear Medicine, de Boelelaan 1117, Amsterdam, Netherlands

a r t i c l e i n f o

Article history:
Received 16 March 2021
Received in revised form 4 May 2021
Accepted 17 June 2021

Keywords:
Positron emission tomography Colony stimulating Factor-1 receptor Tumor associated macrophages Glioblastoma
Companion diagnostic BLZ945
a b s t r a c t

Background: The kinase colony stimulating factor-1 receptor (CSF-1R) has recently been identified as a novel ther- apeutic target for decreasing tumor associated macrophages and microglia load in cancer treatment. In glioblas- toma multiforme (GBM), a high-grade cancer in the brain with extremely poor prognosis, macrophages and microglia can make up to 50% of the total tumor mass. Currently, no non-invasive methods are available for mea- suring CSF-1R expression in vivo. The aim of this work is to develop a PET tracer for imaging of CSF-1R receptor expression in the brain for future GBM patient selection and treatment monitoring.
Methods: BLZ945 and a derivative that potentially allows for ﬂuorine-18 labeling were synthesized and evaluated in vitro to determine their affinity towards CSF-1R. BLZ945 was radiolabeled with carbon-11 by N-methylation of des-methyl-BLZ945 using [11C]CH3I. Following administration to healthy mice, metabolic stability of [11C]BLZ945 in blood and brain and activity distribution were determined ex vivo. PET scanning was performed at baseline, ef- ﬂux transporter blocking, and CSF-1R blocking conditions. Finally, [11C]BLZ945 binding was evaluated in vitro by autoradiography on mouse brain sections.
Results: BLZ945 was the most potent compound in our series with an IC50 value of 6.9 ± 1.4 nM. BLZ945 was radiolabeled with carbon-11 in 20.7 ± 1.1% decay corrected radiochemical yield in a 60 min synthesis procedure with a radiochemical purity of >95% and a molar activity of 153 ± 34 GBq·μmol−1. Ex vivo biodistribution showed moderate brain uptake and slow wash-out, in addition to slow blood clearance. The stability of BLZ945 in blood plasma and brain was >99% at 60 min post injection. PET scanning demonstrated BLZ945 to be a sub- strate for efﬂux transporters. High brain uptake was observed, which was shown to be mostly non-specific. In ac- cordance, in vitro autoradiography on brain sections revealed high non-specific binding.
Conclusions: [11C]BLZ945, a CSF-1R PET tracer, was synthesized in high yield and purity. The tracer has high po- tency for the target, however, future studies are warranted to address non-specific binding and tracer efﬂux be- fore BLZ945 or derivatives could be translated into humans for brain imaging.

⦁ Introduction

Glioblastoma multiforme (GBM) is a grade IV brain cancer with ex- tremely poor prognosis; chemotherapy alone or combined with radia- tion therapy and surgery only prolongs the median survival time of GBM patients from 10 months to 14 months [1–3]. Newer therapies, such as bevacizumab, approved as a second line treatment for patients with recurrent GBM, and local drug-release implants introduced after surgical resection, only provide limited effects [4–6]. Therefore, novel treatment strategies are essential for combatting this devastating

☆ Given his role as Editor in Chief of this journal, Albert D. Windhorst had no involve- ment in the peer-review of this article.
⁎ Corresponding author at: 3165 Porter Drive, CA, 94304 Palo Alto, USA.
E-mail address: [email protected] (F.T. Chin).
disease.
A new strategic target for GBM treatment is the colony stimulating factor 1 receptor (CSF-1R), which is expressed on tumor-associated mi- croglia and macrophages (TAMs). Together TAMs can comprise up to 50% of the tumor mass [7,8]. Microglia and macrophages depend on the activation of CSF-1R by their endogenous ligand Colony Stimulating Factor-1 (CSF-1) for their survival and proliferation. In the absence of CSF-1 or the presence of CSF-1R inhibitors macrophage and microglia are unable to differentiate and will eventually be depleted from the tis- sue. This strategy of CSF-1R inhibition has resulted in reduced GBM tumor mass and a corresponding prolonged survival in GBM mouse models and thus provides a promising new approach to combatting brain cancers [9]. Currently, many small molecule and antibody inhibi- tors of CSF-1R are in (pre)clinical development [10,11]. In addition to cancer, CSF-1R has been implicated as a pharmacological target for neuroinﬂammatory and neurodegenerative diseases [12].

Positron emission tomography (PET) is a molecular imaging tech- nique that allows for the quantitative and non-invasive assessment of biological processes such as transporter and enzyme activity and recep- tor density [13]. A validated CSF-1R selective PET tracer would have enormous value in facilitating study of brain diseases, selecting appro- priate patient therapies, monitoring treatment responses, and develop- ing new drugs. Three CSF-1R PET tracers have been reported to date (Fig. 1) [14–16]. However, these tracers suffer from various limitations, which could prevent their clinical translation. E.g. [18F]10 has poor affin- ity and low selectivity [14], whereas [11C]AZD6495 displayed insuffi- cient brain uptake [15]. The most promising CSF-1R tracer reported to date is [11C]CPPC, which has a high affinity for CSF-1R and displays good brain penetration in mice and baboon [16]. However, this com- pound is derived from a rather non-selective compound [17] and in ad- dition, shows high non-specific binding in the brain.
Among the multiple CSF-1R small molecule inhibitors currently in clinical development [17–22] is BLZ945, a highly potent and selective CSF-1R inhibitor (Ki = 1 nM, > 3200 fold selectivity over other kinases) [9]. This CSF-1R inhibitor demonstrated great efficacy in GBM mice, im- plicating good brain uptake. Taken together, BLZ945 has high potential for translation into the first highly potent and selective CSF-1R PET tracer, either by direct labeling with carbon-11 or by development of an- alogues that allow for ﬂuorine-18 labeling. The aim of the current study is to develop a new CSF-1R PET tracer based on BLZ945 as a companion diagnostic for imaging CSF-1R expression in the brain.

⦁ Material and methods

⦁ General

All chemical reagents and solvents were obtained from commercial suppliers and used as received. Microwave reactions were performed on a CEM Discover Legacy (Matthews, NC, USA). Reaction monitoring was performed by thin layer chromatography on pre-coated silica 60 F254 aluminum plates (Merck, Darmstadt, Germany). Spots were visu- alized with UV light (254 nm), KMnO4, or ninhydrin staining. NMR spec- troscopy was performed using an Agilent 400 MR (Agilent Technologies, Santa Clara, CA, USA) with chemical shifts (δ) reported in parts per mil- lion (ppm) relative to the solvent (CDCl3 1H: 7.26 ppm, 13C: 77.16 ppm; DMSO‑d6 1H: 2.50 ppm, 13C 39.52 ppm; CD3OD 1H: 3.31 ppm, 13C: 49.00
ppm). High resolution mass spectrometry was performed on a Bruker Daltonics – apex-Qe (Bruker, Billerica, MA, USA) in either positive or negative ion mode. Enzyme inhibition assay kits (Z-LYTE™ assay) and human recombinant enzymes were obtained from ThermoFisher Scien- tific (Waltham, MA, USA). Fluorescent readout of 384-well plates was performed on a ﬂuorimeter (Safire, Tecan, XFluo). Carbon-11 was pre- pared by the 14N(p,α)11C nuclear reaction on a GE PETtrace 880 cyclo- tron and was delivered as [11C]CO2 using nitrogen as a carrier gas to the experimental set up (GE TRACERlab FX-C Pro, Boston, MA, USA). Preparative HPLC was performed on a Dionex P680 HPLC pump equipped with a Phenomenex Gemini semi preparative HPLC column (250 × 10 mm, 5u) using a mixture of A) H2O and B) MeCN according

to the following scheme (Method A): 0 min, 35% B; 2 min, 35% B; 20 min, 60% B. UV detection was performed using a Knauer k-2001 UV de- tector. Radioactivity levels were determined using a dose calibrator (Capintec CRC-15 PET). Analytical HPLC was performed on an Agilent Technologies 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) connected to a Raytest GABI* radiodetector (Elisia Raytest GmbH, Straubenhardt, Germany, sensitivity 370 Bq (10 nCi)) using a Luna 5u C18 column (250 × 4.6 mm, 5 μm particle size) using a mixture of A) H2O and B) MeCN as a mobile phase according to the following scheme (Method B): 0 min, 50% B; 15 min, 70% B; 15.1 min, 95% B; 17
min, 95% B; 17.1 min, 50% B; 17.1–20.0 min 50% B. Mice were housed
in 12 h light/dark cycles and were provided food and water ad libitum. Blood was obtained by arterial puncture and centrifuged using an ultra- centrifuge system (Eppendorf Minispin Plus). Gamma counting was performed on a Hidex Automatic Gamma Counter (Turku, Finland). TLC plates were exposed to a phosphorimager screen (Fujifilm, Valhalla, NY, USA) for 30 min in a cassette (Hypercasette, Amersham Biosciences, Little Chalfont, United Kingdom) and subsequently developed using a Typhoon Trio Imager (GE Healthcare, Waukesha, WI, USA). Images were analyzed using ImageJ version 1.49v (National Institute of Health, USA). PET scanning was performed on a Siemens Inveon dPET and Sie- mens Inveon Hybrid MicroPET/CT (Munich, Germany) with identical PET components. Scans were acquired in list mode and rebinned into the following frame sequence: 7.5 s, 4 × 15 s, 37.5 s, 3 × 60 s, 180 s, 9
× 300 s and 435 s. Following a transmission scan for attenuating correc- tion, reconstruction was performed (3D OSEM). Images were analyzed using PMOD (PMOD Technologies LLC, Zurich, Switzerland) by drawing regions of interest over selected organs and tissues. PLX3397 was pur- chased from Selleckchem. CPPC and ‘compound 22’ were synthesized using reported literature procedures [16,23]. Spectroscopic and spectro- metric data (1H NMR, 13C NMR and ESI-HRMS) was in accordance with reported values (data not shown).

⦁ Chemistry

Compound 1 ((1R,2R)-2-((6-methoxybenzo[d]thiazol-2-yl)amino) cyclohexan-1-ol):
A solution of 2-chloro-6-methoxybenzo[d]thiazole (1.0 g, 5.0 mmol), (1R,2R)-2-aminocyclohexan-1-ol (0.76 g, 5.0 mmol), TEA (1.7 mL, 12.5 mmol) in NMP (2 mL) was heated at 120 °C for 48 h. The reaction mix- ture was cooled to rt. and the precipitates were removed by filtration. The filtrate was concentrated in vacuo and purified by ﬂash column chromatography (EtOAc/hexanes 1:1) to afford the product as a white solid (0.82 g, 59%). 1H NMR (400 MHz, CDCl3): δ = 7.39 (d, 1H, J = 9.1 Hz), 7.05 (d, 1H, J = 2.5 Hz), 6.86 (dd, 1H, J = 2.6, 8.8 Hz), 5.81
(bs, 1H), 3.79 (s, 3H), 3.44 (m, 2H), 2.11 (m, 2H), 1.70 (m, 2H), 1.41–1.16 (m, 4H), 0.27 (s, 1H). 13C NMR (101 MHz, CDCl3): δ =
166.81, 155.35, 145.93, 131.43, 119.21, 113.58, 105.47, 75.28, 61.81,
55.99, 34.27, 32.07, 24.86, 24.15. ESI-HRMS: m/z calculated for C14H18N2O2S: 278.1089; found: 301.0980 [M + Na]+.
Compound 2 (2-(((1S,2R)-2-hydroxycyclohexyl)amino)benzo[d] thiazol-6-ol).

Fig. 1. Previously reported CSF-1R PET tracers [14–16] and BLZ945 as a potential PET tracer with improved characteristics (this work). The positions of the carbon-11 labels are depicted with *.

To a solution of 1 (0.56 g, 2.0 mmol) and TEA (0.56 mL, 4.0 mmol) in DCM (5 mL) at 0 °C was added boron tribromide (1.0 M in DCM, 4.0 mL, 4.0 mmol) and the resulting solution was stirred for 3 h while allowing gradual warming to room temperature. After concentration in vacuo the residue was purified by ﬂash column chromatography (EtOAc/hexanes 2:1) to afford the product as a white solid (0.48 g, 90%). 1H NMR (400 MHz, CD3OD): δ = 7.22 (d, 1H, J = 8.7 Hz), 6.99
(d, 1H, J = 2.1 Hz), 6.73 (dd, 1H, J = 2.2, 8.6 Hz), 3.52 (m, 1H), 3.42
(m, 1H), 2.17 (m, 1H), 2.01 (m, 1H), 1.73 (m, 2H), 1.41–1.21 (m, 4H).
13C NMR (101 MHz, CD3OD): δ = 167.59, 153.70, 146.27, 132.03,
119.09, 114.91, 107.85, 74.50, 61.49, 35.38, 32.59, 25.60, 25.27. ESI-
HRMS: m/z calculated for C13H16N2O2S: 264.0932; found: 265.1006 [M
+ H]+.
Compound 3 (4-((2-(((1S,2R)-2-hydroxycyclohexyl)amino)benzo [d]thiazol-6-yl)oxy)picolinamide):
A mixture of 2 (66 mg, 0.25 mmol), methyl 4-ﬂuoropicolinate (39 mg, 0.25 mmol) and K2CO3 (69 mg, 0.50 mmol) in DMF (4 mL) was heated at 100 °C for 16 h. The mixture was diluted with EtOAc, filtered and concentrated in vacuo. After ﬂash column chromatography (EA -
> 2% MeOH in EtOAc) the product was obtained as a yellow solid (51 mg, 51%). 1H NMR (400 MHz, CDCl3): δ = 8.54 (d, 1H, J = 5.6 Hz), 7.62 (d, 1H, J = 2.5 Hz), 7.52 (d, 1H, J = 8.6 Hz), 7.28 (d, 1H, J = 2.4
Hz), 7.00 (dd, 1H, J = 2.5, 8.7 Hz), 6.97 (dd, 1H, J = 2.5, 5.6 Hz), 5.9
(m, 2H), 3.96 (s, 3H), 3.51 (m, 2H), 2.18 (m, 2H), 1.74 (m, 2H), 1.33
(m, 4H). 13C NMR (101 MHz, CDCl3): δ = 168.37, 166.55, 165.53,
151.41, 149.97, 148.21, 131.94, 119.87, 119.37, 114.73, 113.51, 113.39,
113.38, 75.24, 61.88, 53.13, 34.35, 32.02, 24.81, 24.15. ESI-HRMS: m/z
calculated for C20H21N3O4S: 399.1253; found: 422.1145 [M + Na]+.
Compound 4 4-((2-(((1S,2R)-2-hydroxycyclohexyl)amino)benzo [d]thiazol-6-yl)oxy)picolinamide.
A mixture of 2 (66 mg, 0.25 mmol), 4-ﬂuoropicolinamide (35 mg,
0.25 mmol) and K2CO3 (69 mg, 0.50 mmol) in DMF (4 mL) was heated at 100 °C for 16 h. The mixture was diluted with EtOAc, filtered and con- centrated in vacuo. After ﬂash column chromatography (EA – > 2% MeOH in EA) the product was obtained as a yellow solid (80 mg, 80%). 1H NMR (400 MHz, CDCl3): δ = 8.38 (d, 1H, J = 5.6 Hz), 7.86 (d, 1H, J
= 4.2 Hz), 7.67 (d, 1H, J = 2.6 Hz), 7.48 (d, 1H, J = 8.7 Hz), 7.27 (m,
1H), 6.97 (m, 2H), 6.24 (bs, 1H), 6.07 (d, 1H, J = 4.1 Hz), 4.32 (bs,
2H), 3.49 (m, 2H), 2.17 (m, 2H), 1.73 (m, 2H), 1.43–1.23 (m, 4H). 13C NMR (101 MHz, CDCl3): δ = 168.42, 166.92, 166.62, 151.81, 150.02,
149.98, 148.17, 131.86, 119.67, 119.32, 114.43, 113.52, 110.41, 74.93,
61.90, 34.28, 31.92, 24.79, 24.19. ESI-HRMS: m/z calculated for C19H20N4O3S: 384.1256; found: 385.1331 [M + H]+.
Compound 5 N-(2-ﬂuoroethyl)-4-((2-(((1S,2R)-2-hydroxycy- clohexyl)amino)benzo[d]thiazol-6-yl)oxy)picolinamide.
A solution of compound 3 (40 mg, 0.10 mmol) in MeOH/THF/KOH (4 M in H2O) (1 mL, 4:9:2, v/v/v) was stirred at room temperature for 1 h. The solution was concentrated in vacuo, diluted with water (10 mL) and then acidified to pH 3 with 1 M HCl. The mixture was extracted with DCM (3 × 10 mL). The combined organic fractions were concentrated to dryness and the residue was resuspended in DMF (2 mL). Then, DiPEA (39 mg, 0.30 mmol), 2-ﬂuoroethylamine·HCl (10 mg, 0.10 mmol) and HATU (45 mg, 0.10 mmol) were added and the solution was left for 16 h at room temperature. After concentration in vacuo, the residue was purified by ﬂash column chromatography (2% MeOH in DCM) to afford the product as a white solid (25 mg, 58%). Intermedi- ate: 1H NMR (400 MHz, CD3OD): δ = 8.62 (d, 1H, J = 6.1 Hz), 7.75 (m, 2H), 7.64 (d, 1H, J = 8.7 Hz), 7.39 (m, 2H), 3.55 (m, 2H), 2.14 (m, 2H),
1.82 (m, 2H), 1.43 (m, 4H); 13C NMR (101 MHz, CD3OD): δ = 171.25,
170.23, 163.84, 150.59, 149.69, 147.97, 140.89, 127.96, 121.88, 117.38,
116.50, 116.42, 114.62, 74.55, 63.97, 35.40, 31.83, 25.51, 25.10. Title compound 5: 1H NMR (400 MHz, CD3OD): 8.46 (d, 1H, J = 5.6 Hz), 7.54 (s, 1H), 7.47 (d, 1H, J = 8.6 Hz), 7.41 (s, 1H), 7.04 (m, 2H), 4.61
(t, 1H, J = 5.1 Hz), 4.49 (t, 1H, J = 5.1 Hz), 3.66 (m, 3H), 3.46 (m, 1H),
2.20 (m, 1H), 2.06 (m, 1H), 1.78 (m, 2H), 1.37 (m, 4H); 13C NMR (101
MHz, CD3OD): 169.77, 168.46, 166.52, 153.01, 151.54, 151.45, 149.31,

132.83, 120.08, 119.70, 115.19, 114.69, 110.77, 83.08 (d, J = 167.7 Hz),
74.35, 61.66, 41.06 (d, J = 21.3 Hz), 35.44, 32.53, 25.62, 25.29.
4-((2-(((1S,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl) oxy)-N-methylpicolinamide, BLZ945:
To a solution of 3 (25 mg, 63 μmol) in THF was added methylamine·HCl (21 mg, 0.30 mmol) and K2CO3 (43 mg, 0.30 mmol). The mixture was reacted for 16 h at 50 °C. After filtration and concentration in vacuo, the product was purified by ﬂash column chro- matography (EA – > 2% MeOH in EA) to obtain the product as an off- white solid (20 mg, 80%). 1H NMR (400 MHz, CDCl3): δ = 8.35 (d, 1H, J = 5.6 Hz), 8.02 (m, 1H), 7.68 (d, 1H, J = 2.5 Hz), 7.49 (d, 1H, J = 8.7
Hz), 7.27 (s, 1H), 6.99 (dd, 1H, J = 2.5, 8.7 Hz), 6.92 (dd, 1H, J = 2.6,
5.6 Hz), 6.18 (bs, 1H), 3.72 (bs, 1H), 3.50 (m, 2H), 3.00 (d, 3H, J = 5.2
Hz), 2.11 (m, 2H), 1.74 (m, 2H), 1.44–1.24 (m, 4H). 13C NMR (101
MHz, CDCl3): δ = 168.43, 166.94, 164.75, 152.26, 149.82, 149.77,
148.27, 131.81, 119.65, 119.33, 114.02, 113.52, 110.15, 75.06, 61.88,
34.28, 31.93, 26.29, 24.79, 24.17. ESI-HRMS: m/z calculated for C20H22N4O3S: 398.1413; found: 421.1308 [M + Na]+.

⦁ In vitro IC50 determination

Affinities for designed compounds were determined using a com- mercially available Z’-LYTE™ assay according to the manufacturer’s in- structions. Brieﬂy, to a 384 well plate were added the respective kinase (either CSF-1R, PDGFR-β or c-KIT in a final concentration of 0.5,
1.0 and 1.0 ng/μL, respectively) in supplied kinase buffer, ATP (in a final concentration of 50 μM for CSF-1R and PDGFR-β, 100 μM for c- KIT), the respective inhibitor (2-fold dilution series in a concentration range from 1 μM to 0.5 nM), and the assay FRET-peptide. The resulting solutions (total volume of 10 μL per well) were incubated at room temperature for 1 h, followed by the addition of protease solution (5 μL per well). After careful mixing, the solutions were incubated for 1 h followed by the addition of stop buffer (5 μL per well). Readout of the plate was performed using excitation wavelength of 400 nm and emission wavelengths of 445 nm (coumarin) and 520 nm (ﬂuorescein) with a 12 nm bandwidth. Negative control and positive control reac- tions were performed by omission of ATP and inhibitor, respectively. The obtained values are the result of three independent experiments and are expressed as average IC50 value ± standard deviation.

⦁ Radiosynthesis

After proton bombardment, [11C]CO2 was delivered to a TRACERlab FXC-pro module and trapped on a mixture of molecular sieves, pre- charged with hydrogen gas, and nickel. After completing delivery, the molecular sieve trap was heated to 350 °C and the formed [11C]CH4 was released to a silica trap, cooled to −80 °C using liquid nitrogen. This trap was heated to release [11C]CH4 into a closed circuit, where it was mixed with iodine vapor and converted to [11C]CH3I at 720 °C. The [11C]CH3I that was formed was accumulated on a cooled Porapak trap. When the radioactivity levels on this Porapak trap reached a pla- teau, it was heated to 220 °C to release [11C]CH3I, which was transferred to a reaction vial using a gentle stream of helium (20 mL/min). The reac- tion vial was previously charged with precursor 4 (1.0 mg, 2.6 μmol) and TBAOH (10 μL, 1.0 M in MeOH) in DMSO (400 μL) and was loaded with [11C]CH3I until radioactivity reached a maximum. The reactor was sealed and the reaction solution was heated at 100 °C for 4 min and subsequently cooled to 20 °C. Next, the reaction solution was diluted with H20 (1.0 mL) and purified using HPLC using method A. The collected HPLC product fraction was diluted with H2O (40 mL) and passed over a preconditioned solid phase extraction cartridge (C18 Sep-Pak Light, Waters). After washing the cartridge with H2O (10 mL), the product was obtained by sequential elution with ethanol (1.0 mL) and saline (9.0 mL). A sample was analyzed by analytical HPLC to deter- mine the (radio)chemical purity and molar activity (Method B).

⦁ Partition coefﬁcient LogD

The partitioning of [11C]BLZ945 between 1-octanol and 0.2 M phos- phate buffer (pH = 7.4) was determined by vigorously mixing [11C] BLZ945 (100 μL, 20 MBq) with a solution of 0.2 M phosphate buffer (2 mL, pH 7.4) and 1-octanol (2 mL) for 1 min using a vortex apparatus. After a settling period of 1 h, three samples of 100 μL were taken from both layers. Samples were counted for radioactivity and the Log D values were calculated according to the following formula: Log Doct,7.4 = Log (Aoct/Aphosphate buffer), where Aoct and Aphosphate buffer represent average radioactivities of three 1-octanol and three phosphate buffer samples, respectively. The result is expressed as mean ± standard deviation (n = 3).

⦁ Plasma stability

[11C]BLZ945 (50 μL, 10 MBq) was added to freshly thawed (500 μL) mouse plasma (Sigma Aldrich, catalogue number P9275) and incubated at 37 °C for 1 h. Ice-cold acetonitrile (1.0 mL) was added and the mixture was centrifuged for 5 min at 15,000 RPM. An aliquot of the supernatant was analyzed by analytical HPLC using method B (retention time of [11C] BLZ945 is 8.4 min).

⦁ Radiometabolite analysis

Mice (10–16 weeks old, 25–30 g, n = 3 per time point) were injected with [11C]BLZ945 (100 μL, approximately 10 MBq) under isoﬂurane an- esthesia (2% in O2 at 1 L/min). At the indicated time-points, blood was collected by arterial puncture, approximately 0.5 mL) and transferred to a Heparin coated Eppendorf tube. Mice were perfused gently (25 mL of phosphate buffer) and brains (left hemisphere) were collected. Blood samples were centrifuged for 5 min at 4600 RPM to separate blood plasma from cells. The plasma (100 μL) was diluted in acetonitrile (200 μL at 0 °C) and centrifuged for 5 min at 15,000 RPM for removal of proteins. Brains were homogenized in acetonitrile (200 μL at0 °C) and centrifuged for 5 min at 15,000 RPM. An aliquot of each supernatant (10 μL) was transferred to a TLC plate, which was subsequently dried at room temperature for 5 min and ran in a solution of DCM/MeOH/ TEA (90:10:1, v/v/v). The radioTLC plate was transferred to a phosphorimager storage screen and left for 1 h. Readout was performed on a Typhoon phosphorimager and subsequent analysis was performed using ImageJ.

⦁ Biodistribution of [11C]BLZ945

Mice (10–16 weeks old, 25–30 g, n = 3 per time point) were injected with [11C]BLZ945 (100 μL, approximately 10 MBq) under isoﬂurane

anesthesia (2% in O2 at 1 L/min). At the indicated time-points blood was collected by arterial puncture, approximately 0.5 mL) and trans- ferred to a Heparin coated Eppendorf tube. Mice were perfused gently (25 mL of phosphate buffer) to remove the blood component from or- gans. Organs were collected, weighed, and counted using a gamma counter. Results are expressed as percent of injected dose per gram (% ID/g) ± standard deviation.

⦁ PET and PET/CT scanning

Mice were anesthetized with 2% isoﬂuorane gas in O2 at 1 L/min and catheterized in their tail vein. When indicated, cyclosporin A was ad- ministered by tail vein injection 30 min prior to the start of PET scanning at 30 mg/kg, formulated at 20 mg/mL in DMSO, EtOH, polyethylene gly- col 400, and H2O (1/15/50/34 v/v/v/v). Unlabeled BLZ945 was formu- lated in 10% EtOH in saline and administered by co-injection with [11C]BLZ945. PLX3397 (Selleckchem) was dosed at 5 mg/kg, formulated by dissolving in the cyclosporin A solution and administered by tail vein injection 30 min prior to tracer administration. [11C]BLZ945 was admin- istered (10–15 MBq in 100 μL) by tail vein injection. After PET scanning, mice were sacrificed by cervical dislocation and brains were collected, weighed, and activity levels were determined using a gamma counter. Time-activity curves were obtained by drawing regions of interest using PMOD. Results are expressed as the average %ID/g ± standard de- viation (n =4 per experimental group).

⦁ Autoradiography

Autoradiography was performed in ﬂash frozen mouse brain sec- tions (10 μm thickness). Sections were washed with 50 mM Tris-HCl buffer (pH 7.4) for 15 min. After drying under a gentle air ﬂow the sec- tions were incubated with [11C]BLZ945 (0.5 MBq·mL−1) in 50 mM Tris- HCl, pH 7.4 with 3% BSA in the absence or presence of a CSF-1R inhibitor at 1 μM concentration for 30 min. Washing was performed with cold Tris-HCl (5 mM, 4 °C, two times) followed by dipping in ice cold water. After drying in an air stream, mouse brain sections were exposed to a phosphorimaging screen (GE Healthcare, Buckinghamshire, UK) for 30 min and developed on a Typhoon FLA 7000 phosphor imager (GE Healthcare, Buckinghamshire, UK). Visualisation of binding was per- formed using ImageQuantTL v8.1.0.0 (GE Healthcare, Buckinghamshire, UK). Data are expressed as % of binding relative to [11C]BLZ945 binding in the absence of CSF-1R inhibitor (n = 3).

⦁ Statistical analysis

Statistical analysis was performed using a one-sided, unpaired Stu- dent’s t-test.

Scheme 1. Synthesis of BLZ945, analogue 5 and precursor 4 for carbon-11 radiolabeling. Reagents and conditions: a) (1R,2R)-2-aminocyclohexan-1-ol, TEA, NMP, 120 °C, 48 h, 59%; b) BBr3, TEA, DCM, 0 °C to rt., 3 h, 90%; c) methyl 4-ﬂuoropicolinate, K2CO3, DMF, 100 °C, 16 h, 51%; d) methylamine·HCl, K2CO3, THF, 50 °C, 16 h, 80%; e) 4-ﬂuoropicolinamide, K2CO3, DMF, 100 °C, 16 h, 80%; f) i) KOH, MeOH/THF/H2O, rt., 1 h; ii) 2-ﬂuoroethylamine·HCl, HATU, DiPEA, rt., 16 h, 58%.

Fig. 2. Inhibition of compounds towards CSF-1R.

⦁ Results and discussion

⦁ Chemistry

Based on the chemical structure, direct radiolabeling of BLZ945 at the N-methyl functionality with carbon-11 (Fig. 1) is feasible by reacting [11C]CH3I with the des-methyl precursor molecule [24]. In addition, efforts were made to design a derivative that allows for ﬂuorine-18 labeling and thus has improved properties for PET imaging (Scheme 1) [25]. A substitu- tion of the methyl functionality for a ﬂuoroethyl group was envisioned to allow for potential ﬂuorine-18 labeling [26]. The synthesis of the target compounds and their respective precursor molecules for radiolabeling was based on reported literature procedures [9,27] and is depicted in Scheme 1. Brieﬂy, 2-chloro-6-methoxybenzothiazole was reacted with (1R,2R)-2-aminocyclohexanol to obtain benzothiazole 1, which was then subjected to O-demethylation using BBr3, affording compound 2. Subse- quent nucleophilic aromatic substitution using 4-ﬂuoropicolinamide re- sulted in BLZ945 precursor 4. Methyl ester 3 was obtained by reaction of compound 2 with methyl 4-ﬂuoropiconilate. Compound 3 was then con- verted to BLZ945 by treatment with methylammonium chloride and to ﬂuoro-ethyl analogue 5 by alkaline deprotection followed by HATU- mediated coupling with ﬂuoroethylamine.

⦁ In vitro evaluation

Both BLZ945 and analogue 5 were tested for their inhibitory potency in vitro against human recombinant CSF-1R (Fig. 2) using a commercially available Z-LYTE™ assay kit. In addition, selectivity over c-Kit and PDGFR- β, close family members of CSF-1R with high structural homology [28], was determined. BLZ945 was found to be most potent, with IC50 value of 6.9 ± 1.4 nM. The ﬂuoroethyl analogue 5 resulted in an increase in IC50 to 40.5 ± 3.7 nM, demonstrating the preference for a methylamide

at this position. In accordance with the general selectivity for this scaffold, no inhibition of c-KIT and PDGFR-β was observed at concentration up to 4 μM [27]. As a result of this affinity and selectivity screening, BLZ945 was selected as the optimal candidate for radiolabeling.

⦁ Radiolabeling, QC and in vitro evaluation

Carbon-11 synthesis of BLZ945 was pursued by direct methylation of precursor amide 4. Although this precursor has an aminocyclohexanol functionality that might benefit from protection to avoid the formation of side products, such strategy would require multi-step syntheses and the resulting elongated synthesis time might not outweigh the improved radiochemical conversion to [11C]BLZ945. Following a one-step strategy, [11C]BLZ945 was efficiently synthesized by reacting precursor 4 with [11C]CH3I in the presence of tetrabutylammonium hydroxide (1.0 M solu- tion in MeOH) in DMSO (Fig. 3). The main radiochemical product was [11C]BLZ945, the identity of which was confirmed by coinjection of an au- thentic reference sample on HPLC using two distinct HPLC conditions (Supporting Information Fig. S1). The full synthetic procedure, from end of bombardment to formulation in 10% EtOH in saline, was performed in a fully automated fashion in 60 min and gave [11C]BLZ945 in 20.7 ± 1.1% decay corrected yield (2.58 ± 0.14 GBq at end of synthesis), a molar activity of 153 ± 34 GBq·μmol−1 and a radiochemical purity
>95% (n = 5, Supporting Information Fig. S1).
[11C]BLZ945 remained stable in solution as well as in mouse plasma up to 1 h (Supporting information Fig. S1). The partition coefficient LogD was empirically determined as 2.26 ± 0.01 by the shake ﬂask method. This value is comparable with the calculated LogP of 3.09 (ChemDraw Professional, v16.0.1.4) and well within the range for successful brain penetration [29].

⦁ Ex vivo evaluation

Potential metabolism of [11C]BLZ945 was determined ex vivo by radioTLC analysis of blood plasma and brain homogenates at 15, 30, and 60 min post injection in healthy mice (n = 3 per time point). Both in plasma and brain only, intact tracer was detected, demonstrating the ex- cellent metabolic stability of BLZ945 up to 1 h post injection (Supporting information Fig. S2). These findings correspond well with the reported high stability of [14C]BLZ945 in human hepatocytes and microsomes [30]. Ex vivo tracer distribution was determined at 15, 30, and 60 min post injection in healthy mice (Fig. 4 and Supporting Information Table S1). In accordance with its pharmaceutical activity in mouse models of glioblastoma [9], [11C]BLZ945 displayed good brain penetration of ap- proximately 1%ID/g. Moderate washout was observed, which could po- tentially be attributed to the basal expression of CSF-1R on microglia in the healthy brain [31]. Blood radioactivity concentrations were relatively high, potentially due to the high plasma protein binding (96%) and low free fraction (4%) of BLZ945 [30]. Excretion mainly occurs via the hepatic system, as evidenced by high radioactivity concentrations in liver and in- testines and low values for kidney and urine.

Fig. 3. Optimization of radiolabeling conditions towards [11C]BLZ945. Reagents and conditions: a) TBAOH (1.0 M in MeOH), DMSO, 100 °C, 5 min.

Fig. 4. Ex vivo tissue distribution of [11C]BLZ945 following administration to healthy mice (n = 3). Error bars indicate standard deviations (n = 3 per time point).

⦁ In vivo evaluation

Dynamic distribution of [11C]BLZ945 in mice brain was evaluated by means of PET scanning. Brain uptake was determined in different exper- imental conditions; besides administration to non-treated mice, [11C] BLZ945 was administered to mice that were pretreated with cyclospor- ine A (30 mg/kg, 30 min prior to PET tracer administration). Blocking ex- periments were performed using excess unlabeled BLZ945 (1 mg·kg−1, co-injection with radiotracer) and PLX3397 (5 mg/kg, 30 min prior to PET tracer administration). BLZ945 is a substrate for efﬂux transporters, as evidenced by the approximate 2-fold increase of brain activity levels when comparing baseline with efﬂux transporter inhibition at 60 min

post injection (p = 0.0003). Specific binding of [11C]BLZ945 was dem- onstrated by co-administration of unlabeled BLZ945, which resulted in a 20% decrease in brain activity concentrations at 60 min post injection (p = 0.05). At time-points up to 8 min post injection, brain radioactivity levels were higher for the BLZ945 blocking conditions, potentially due to displacement of [11C]BLZ945 in peripheral organs, resulting in in- creased blood concentrations. Indeed, higher blood concentrations (de- termined as heart TACs) were found in this blocking condition at earlier time points (Supporting information Fig. S3). The suggestion that this initial increase in brain activity level is due to increased tracer availability and not a result of CSF-1R binding is further supported by the accelerated washout of [11C]BLZ945 when compared to unblocked conditions. PLX3397 pretreatment (i.v. administration, 5 mg/kg, 30 min prior to tracer injection), did not result in a blocking effect. PLX3397 is designed to bind at the juxtamembrane domain of CSF-1R in the auto-inhibited state [32], which is only partially overlapping with the ATP-binding do- main. The binding domain of BLZ945 has not been reported, but based on the ATP competitive binding, it is likely that BLZ945 directly occupies the ATP binding site. The potentially differing binding domains could ex- plain the lack of a blocking effect of [11C]BLZ945 binding upon PLX3397 pretreatment. Similar lack of blocking has previously been reported using CSF-1R PET tracer [11C]CPPC. Here, no blocking effect of PLX3397 was observed by in vitro autoradiography studies on human Alzheimer’s disease brain sections [16], whereas BLZ945 co-incubation did result in anticipated blocking. The brain radioactivity concentrations determined by PET imaging were confirmed by post-scanning biodistribution (Fig. 6), which overlapped very well with PET scanning results (Fig. 5B). The efﬂux transporter substrate behavior of BLZ945, which is evidenced by the increased brain uptake upon Cyclosporin A pretreatment, could po- tentially hamper clinical translation, as efﬂux transporter inhibition in human is not recommended.

⦁ Autoradiography

Finally, in vitro autoradiography on mouse brain sections using [11C]
BLZ945 was performed. Various CSF-1R inhibitors were used for

Fig. 5. In vivo brain uptake of [11C]BLZ945 in healthy mice. A) Time-activity-curves (TACs) of whole mouse brain at different experimental conditions; B) Brain uptake at 60 min post injection and representative coronal sections of mice from the corresponding experimental groups. Brain regions are indicated with white dotted circles. Results are expressed as average ± standard deviation (n = 4).

Fig. 6. Post-mortem brain activity concentrations. Results are expressed as the average ± standard deviation (n = 4 per group).

blocking of [11C]BLZ945 binding (Fig. 7) [9,16,23,32].
The most pronounced blocking of [11C]BLZ945 binding was ob- served when using an excess of BLZ945 or when using CPPC as a CSF- 1R inhibitor. The blocking effect was about 30%, indicating specific and selective binding of [11C]BLZ945 to the brain tissue. However, this result also demonstrates that about 70% of binding is non-specific. This modest drop in binding corresponds well with PET imaging results, where a modest decrease of [11C]BLZ945 binding of approximately 20% was ob- served. PLX3397 blocking showed a less pronounced blocking effect, again in close accordance with PET imaging results. Similar results were obtained using the bisamide CSF-1R inhibitor ‘compound 22’. Overall, the autoradiography results confirm the high non-specific bind- ing observed in PET imaging experiments. This non-specific binding was observed with [11C]CPPC as well and drastically hampers CSF-1R PET imaging [16]. Given that [11C]BLZ945 is also a strong substrate for efﬂux transporters, no further efforts were undertaken to evaluate the com- pound in animal models of CSF-1R overexpression, such as a glioblas- toma model. Instead, future work will focus on identifying CSF-1R PET tracers that do not suffer from efﬂux transport and less from non- specific binding compared to the currently reported CSF-1R PET tracers, which is especially relevant when considering the modest increases in CSF-1R expression in various animal models [33–36]. To assist in the compound selection, the use of both in silico (e.g. CNS-MPO-PET scoring) and in vitro screening tools (e.g. lipid binding assay [37]) are recom- mended to optimize the tracer development process.

⦁ Conclusion

[11C]BLZ945, a highly potent and selective CSF-1R inhibitor, was suc- cessfully obtained in high yield and purity. Unfortunately, because of the strong efﬂux transporter substrate behavior and high non-specific bind- ing in the brain, [11C]BLZ945 seems unsuitable as a PET tracer for imag- ing of CSF-1R expression levels in the brain. Therefore, future work will

Fig. 7. In vitro autoradiography with [11C]BLZ945 on mouse brain sections. Representative brain slices are depicted together with quantification of binding relative to baseline conditions ([11C]BLZ945 only). CSF-1R inhibitors were used at 1 μM. (* indicates p < 0.05, ** indicates p < 0.005, n.s. not significant, n = 3).

focus on radiolabeling and evaluation of structurally distinct CSF-1R in- hibitors. Further research on [11C]BLZ945 or ﬂuorine-18 labeled deriva- tives could focus on imaging of accumulation of macrophages in the periphery, where efﬂux transport at the blood-brain-barrier is irrelevant and non-specific binding in the relevant tissue might be lower (e.g. cancers, inﬂammation or atherosclerotic plaques) [38].

Funding

The project was supported, in part, by The Ben and Catherine Ivy Foundation (FTC), the National Cancer Institute: R21 CA205564 (FTC) & T32 CA118681 (JLK), and the National Institutes of Health: S10 OD018130 (FTC).

Availability of data and materials

Please contact author for data requests.

Ethics approval and consent to participate

Not applicable.

Declaration of competing interest

There are no competing interests to report.

Acknowledgments

We thank Tim Doyle and the Stanford Small Animal Imaging Facility for their technical assistance.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.nucmedbio.2021.06.005.

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