Discovery of a High-Affinity Fluoromethyl Analog of [¹¹C]5-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([¹¹C]CPPC) and Their Comparison in Mouse and Monkey as Colony-Stimulating Factor 1 Receptor Positron Emission Tomography Radioligands

Abstract

[¹¹C]CPPC has been advocated as a radioligand for colony-stimulating factor 1 receptor (CSF1R) with the potential for imaging neuroinflammation in human subjects with positron emission tomography (PET). This study sought to prepare fluoro analogs of CPPC with higher affinity to provide the potential for labeling with longer-lived fluorine-18 (t_1/2 = 109.8 min) and for delivery of higher CSF1R-specific PET signal in vivo. Seven fluorine-containing analogs of CPPC were prepared and four were found to have high inhibitory potency (IC₅₀ in low to sub-nM range) and selectivity at CSF1R comparable with CPPC itself. One of these, a 4-fluoromethyl analog (Psa374), was investigated more deeply by labeling with carbon-11 (t_1/2 = 20.4 min) for PET studies in mouse and monkey. [¹¹C]Psa374 showed high peak uptake in monkey brain but not in mouse brain. Pharmacological challenges revealed no CSF1R-specific binding in either species at baseline. [¹¹C]CPPC also failed to show specific binding at baseline. Moreover, both [¹¹C]Psa374 and [¹¹C]CPPC showed brain efflux transporter substrate behavior in both species in vivo, although Psa374 did not show liability toward human efflux transporters in vitro. Further development of [¹¹C]Psa374 in non-human primate models of neuroinflammation with demonstration of CSF1R-specific binding would be required to warrant the fluorine-18 labeling of Psa374 with a view to possible application in human subjects.

Microglia are brain-resident macrophages that are pivotal to the development and homeostasis of the central nervous system (CNS). They originate in the yolk sac¹ and constitute about 10% of all cells in the CNS.² The colony-stimulating factor 1 receptor (CSF1R) is mainly expressed in microglia and, to a lower extent, in other cells, such as neurons.³ Cell-surface CSF1R, also known as macrophage colony-stimulating factor 1 receptor, belongs to class III of the tyrosine kinases. The signals arising from the interaction of CSF1R with its two endogenous ligands, macrophage colony-stimulating factor 1 and interleukin-34, are essential for the regulation, differentiation, proliferation, and maintenance of microglia.⁴

Various findings from post-mortem patient tissues and animal models closely associate CSF1R with neuroinflammation. For example, upregulated levels of CSF1R have been observed in Alzheimer’s disease, multiple sclerosis, and glioblastoma.⁵ Moreover, neuroinflammation is diminished after attenuation of the CSF1R signal.⁶ These findings indicate the potential of CSF1R to act as a biomarker in the imaging of neuroinflammation with positron emission tomography (PET). Notably, an effective PET radioligand for imaging brain CSF1R could aid in obtaining a better understanding of neuroinflammation and in the development of new anti-inflammatory CSF1R-targeted treatments.

Only a few CSF1R PET radioligands have been reported (Figure 1).⁵ [¹⁸F]5-(4-((4-Fluorobenzyl)oxy)-3-methoxybenzyl)pyrimidine-2,4-diamine was the first to be reported but without imaging data.⁷ A few CSF1R inhibitors have been labeled with carbon-11 (t_1/2 = 20.4 min) to produce candidate PET radioligands. One example is 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (BLZ945), a potent (IC₅₀ = 1 nM) and selective CSF1R inhibitor.⁸ Racemic BLZ945(9) and its eutomer, (+)-BLZ945,¹⁰ have each been labeled with carbon-11. Despite BLZ945 being known as a brain-penetrant drug, [¹¹C]BLZ945 showed low brain uptake (<0.7 standardized uptake value; SUV) in healthy mice and non-human primates. One reason for the low uptake is that [¹¹C]BLZ945 acts as a substrate for the P-gp efflux transporter at the blood–brain barrier (BBB).

Figure 1.

Some previously reported candidate radioligands for PET imaging of brain CSF1R.

Among the most recent CSF1R radioligand candidates, [¹¹C]5-cyano-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([¹¹C]CPPC) is the most investigated.¹¹⁻¹⁴CPPC is a potent CSF1R inhibitor with reported IC₅₀ values of 0.8 nM¹⁵ and 4.1 nM.¹⁶ PET imaging in healthy mice showed [¹¹C]CPPC to have low brain uptake (∼0.6 SUV). This uptake increased by 53% and 55% following intracranial or systemic lipopolysaccharide (LPS) injection, respectively. LPS injection is intended to promote a neuroinflammatory response but may compromise the BBB by, for example, inhibiting efflux transporter function at the BBB.¹⁷ Moreover, LPS may indirectly decrease CSF1R activity.¹⁸ At best, [¹¹C]CPPC gave very low CSF1R-specific signal in microglial-depleted and CSF1R-knockout mice. [¹¹C]CPPC gave a much higher brain radioactivity uptake (∼3.5 SUV) in healthy baboons. The total volume of distribution (V_T) increased by 120% after LPS pretreatment, and this increase could be fully blocked by a high intravenous dose of CPPC.¹¹ Furthermore, the binding of [³H]CPPC to post-mortem human brain was partially blocked by the known CSF1R inhibitors, PLX3397, PLX5622, and BLZ945 (<25%).¹² Results from a recent study of [¹¹C]CPPC in healthy human subjects extended support for the use of [¹¹C]CPPC to study microglia in the human brain.¹⁹V_T values were somewhat higher in thalamus, striatum, and non-cerebellar cortical regions than in hippocampus, cerebellar cortex, and total white matter. Nonetheless, direct evidence for the specific binding of [¹¹C]CPPC to human brain CSF1R in vivo has not been demonstrated through rigorous pharmacological challenge experiments, there being no CSF1R inhibitors available for safe use in humans.

Another candidate CSF1R PET radioligand is [¹¹C]GW2580 ([methoxy-¹¹C]5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine),^13,14 which is closely related in structure to the aforementioned ¹⁸F-labeled radioligand (Figure 1). This radioligand has been compared to [¹¹C]CPPC in mouse models of acute and chronic neuroinflammation and in rhesus monkey. Despite reportedly much lower CSF1R affinity (IC₅₀ = 10 nM²⁰ and IC₅₀ = 30 nM²¹) [¹¹C]GW2580 appeared to be more sensitive than [¹¹C]CPPC for detecting CSF1R in mouse models of acute and chronic neuroinflammation, based on LPS injection and genetic modification, respectively. The uptake of [¹¹C]GW2580 in the rhesus monkey brain at baseline showed susceptibility to homologous preblock (∼30%), unlike that of the higher uptake seen for [¹¹C]CPPC.

In the study reported here, we sought to modify the structure of CPPC to allow labeling not only with carbon-11 but potentially with longer-lived fluorine-18 (t_1/2 = 109.8 min) and to increase inhibitory potency for potentially better CSF1R-specific signal-to-noise ratio in PET imaging. Fluorine-18 is attractive as a radiolabel because (i) the physical half-life allows more extended kinetics to be monitored in PET experiments than with carbon-11, (ii) [¹⁸F]fluoride ion can be produced in very high activities and molar activities by diverse radiolabeling methods, and (iii) ¹⁸F-labeled ligands can be distributed from central pharmacies to multiple imaging centers who might then feasibly engage in powerful multicenter studies. From the radioligand design perspective, a fluorine-18 atom can replace a hydrogen atom or a hydroxy group, sometimes conferring increased affinity on a ligand for the PET imaging target.^22,23

Results and Discussion

Ligand Design

Structure–activity relationship studies and binding models for the interaction of 8g, a methylated analog of CPPC, with CSF1R have been reported.¹⁵ According to that study, a CSF1R inhibitor should give certain intra- and extra-molecular interactions to be effective. First, an internal hydrogen bond between the amide NH with the oxygen atom of the 5-membered ring in 8g is required to confer the necessary planarity for fitting the inhibitor into the binding pocket of CSF1R. An extra-molecular hydrogen bond between the amide carbonyl group of the inhibitor and the backbone NH of the Cys666 residue plus hydrophobic interactions for both the 2- and 4-substituents of the aryl ring also favor inhibitor–protein binding.

While considering these structural requirements, we designed ligands 8a–8f and Psa374 (Scheme 1) as analogs of the known 8g(15) and CPPC that would not only preserve the possibility of labeling with carbon-11 but also eventually allow alternate and more desirable labeling with fluorine-18.

Scheme 1. Synthesis of CSF1R Ligands 8a–8g, Psa374, and CPPC.

Reagents and conditions: (a) EtOH, 0 °C for 1 h, then rt for 16 h, (b) 140 °C, 16 h, (c) H₂ (70 psi), Pd/C 10%, MeOH, rt, 16 h, (d) HATU, DIPEA, DMF, rt, 16 h.

Our ligand design also considered other properties that are desirable in PET radioligands,²⁴ such as low molecular weight (<500 Da), low tPSA, and moderate computed lipophilicity from ChemDraw software (Table S1).

Chemical Synthesis

Ligand Synthesis

Ligands CPPC, Psa374, and 8a–8g were prepared by multistep pathways according to generally high-yielding literature methods for group transformations, with some minor technical modifications (Scheme 1).¹¹ Thus, treatment of 4-chloro-2-fluoro-1-nitrobenzene with the corresponding piperidines (1a–1c) at room temperature gave 3a–3c, which upon treatment with 1-methylpiperazine at 140 °C gave the nitro derivatives 5a–5c. These derivatives were readily reduced to the corresponding anilines 6a–6c with palladium catalyst over carbon (10%) under hydrogen (70 psi). These conditions gave cleaner reactions than the reported use of hydrogen generated in situ from zinc metal in the presence of ammonium chloride.¹¹ Treatment of 6a–6c with various carboxylic acids 7a–7d in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) finally gave the desired ligands, 8a–8g, Psa374, and CPPC.

Precursor Synthesis

The N-desmethyl precursors 12a–12d for radiolabeling were synthesized by similar routes to the ligands (Scheme 2). Thus, the chloro-intermediates 3a–3c were treated with tert-butyl piperazine-1-carboxylate in the presence of potassium carbonate to give the nitro derivatives 9a–9c in moderate yields. These nitro compounds were reduced to the respective anilines 10a–10c and then coupled with a carboxylic acid, either 7a or 7b, to give the amides 11a–11d in high overall yields. The Boc protecting group was removed efficiently in the last step with trifluoroacetic acid (TFA). Finally, the resulting ammonium salt was neutralized with saturated aqueous sodium bicarbonate to give the free amine precursors 12a–12d.

Scheme 2. Synthesis of Secondary Amine Precursors for Radiolabeling.

Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, K₂CO₃, 130 °C, 16 h, (b) H₂ (70 psi), Pd/C 10%, MeOH, rt, 16 h, (c) HATU, DIPEA, DMF, rt, 16 h, (d) 1. TFA, DCM, rt; 2. aq. NaHCO₃.

Ligand Inhibitory Potencies at Human CSF1R

Inhibitors 8a–8f and Psa374, along with previously known 8g and CPPC, were initially assayed in vitro toward human CSF1R at a concentration of 50 nM to give single-point percentages of inhibition (Table S2). All the ligands, except 8a and 8b, showed greater than 90% inhibition of human CSF1R at this concentration. The half-maximal inhibitory concentrations (IC₅₀s) were then accurately determined for these high-affinity compounds, as reported in Table 1.

Table 1. In Vitro Determination of Half-Maximal Inhibitory Concentrations (IC₅₀s) of Inhibitors at Human CSF1R and Closely Related Tyrosine Kinases^a,b.

	IC50 (nM)
inhibitor	at CSF1R	at FLT3	at PDGFRβ	at RET
CPPC	0.37 (0.32–0.42)	>39.7	>100	>91.5
Psa374	0.22 (0.20–0.25)	>24.1	>58.4	>47.5
8c	0.33 (0.28–0.37)	>24.7	>100	21.2 (11.6–30.7)
8d	1.08 (0.88–1.28)	n/a	n/a	n/a
8e	0.70 (0.61–0.80)	>54.6	>100	>100
8f	3.30 (2.70–3.89)	n/a	n/a	n/a
8g	0.16 (0.14–0.18)	13.2 (7.6–18.7)	>100	>37.1

^aWith 95% confidence limits in parentheses for n = 2. IC₅₀ values are reported as “greater than” (>) when the dose–response curve did not reach a plateau (Table S3).

^bAll listed inhibitors, except 8d and 8f, were also tested against FGFR1, FGFR2, FGFR3, FGFR4, FLT1, FLT4, KDR, KIT, and PDGFRα, giving IC₅₀ values of >100 nM. n/a = not assessed.

Our measurements of the IC₅₀s for CPPC (0.37 nM) and 8g (0.16 nM) were in line with literature values of 0.8 nM and 1 nM, respectively.¹⁵ Replacement of the nitrile group in 8g with fluorine, as in 8e, slightly decreased the affinity for CSF1R (IC₅₀ = 0.16 nM vs. IC₅₀ = 0.70 nM). This small loss in affinity could be due to the lower availability of the oxygen lone electron pairs for hydrogen bonding with the neighboring nitrogen because of the high electronegativity of the nearby 5-fluoro substituent.^25,26

Replacement of the 5-cyano-furanyl substituent with a 4-fluoro-pyrrolyl substituent, as in 8c, slightly decreased affinity (IC₅₀ = 0.16 nM vs. IC₅₀ = 0.33 nM). This result aligns with the requirement for a hydrogen bond between the amido NH group and the ring heteroatom to maintain ligand planarity.

Finally, substituting the nitrogen atom with a bulkier sulfur atom, as in 8a, dramatically reduced affinity (Table S2).

The corresponding C-desmethyl-CPPC analogs (8f, 8d, and 8b) showed a similar rank-order trend in IC₅₀ values, although affinities for CSF1R were slightly lower than for C-methyl compounds. Therefore, we decided to retain a C-methyl substituent on the piperazinyl group but with one hydrogen atom replaced with a fluorine atom as a potential site for future ¹⁸F-labeling. This ligand, Psa374, proved to be the most potent fluorine-containing CSF1R inhibitor among the new ligands (IC₅₀ = 0.22 nM) (Table 1) and therefore became the main focus of this study in comparison with CPPC.

Selectivity of Ligands for CSF1R In Vitro

Knight et al.¹² found that CPPC binds to numerous kinases (204 out of 403 tested) and other CNS targets at 10 μM concentration in vitro. In particular, three kinases, i.e., an insulin receptor, a vascular endothelial growth factor receptor 2, and a tyrosine-protein kinase LCK, generated submicromolar IC₅₀ values of 70 nM, 74 nM, and 22 nM, respectively. It remains unclear whether CPPC has a sufficiently high affinity at any off-target site to cause possible concerns for PET imaging. In general, binding affinities to off-target sites in the micromolar range are very unlikely to be problematic.

To gain further understanding of possible off-target binding for CPPC, 8g, and other inhibitors that have CSF1R IC₅₀ values of less than 1 nM (i.e., 8c, 8e, and Psa374), we determined their IC₅₀s for 12 human tyrosine kinases that are closely related to CSF1R (Table 1).²⁷ At 100 nM inhibitor concentration, none of the dose–concentration curves reached saturation (Table S3). Therefore, these compounds showed more than 13-fold greater selectivity for binding to CSF1R than the dozen tested kinases.

Radiochemistry

The synthesis of [¹¹C]8e from the desmethyl precursor 12c was selected to optimize labeling conditions that could be applied generically. Conditions reported for the radiosynthesis and purification of [¹¹C]CPPC²⁸ did not give us adequate yield for in vivo imaging. Indeed, treatment of 12c with [¹¹C]methyl triflate for 3 min in acetonitrile, either at room temperature (n = 1) or at 50 °C (n = 2), gave [¹¹C]8e in less than 4% decay-corrected formulated yield from cyclotron-produced [¹¹C]carbon dioxide.

Higher yields were achieved when more accessible [¹¹C]iodomethane was used as a labeling agent. Different conditions, including solvents, bases, and reaction temperatures, were tested to optimize radiosynthesis. The reaction of 12c with [¹¹C]iodomethane in DMSO for 5 min at 80 °C produced the formulated radioligand in a 6% decay-corrected yield (n = 1). Yields were further improved up to 13% when DMF was used as solvent at 80 °C (Scheme 3, n = 7). Reactions did not proceed in the presence of tetra-butylammonium hydroxide (1 M) as a base.

Scheme 3. Radiosyntheses of Radioligands [¹¹C]CPPC, [¹¹C]Psa374, [¹¹C]8e, and [¹¹C]8g.

[¹¹C]CPPC, [¹¹C]Psa374, [¹¹C]8e, and [¹¹C]8g were amenable to separation with reverse-phase high-performance liquid chromatography (HPLC) using a mobile phase comprising a slightly basic aqueous phase plus an organic solvent (MeCN). With an initial mobile phase composed of a mixture of aq. NH₄OH (10 mM, pH ∼ 10.6) and acetonitrile, each radioligand eluted close to an unidentified minor radioactive byproduct. The subsequent use of a mixture of water and a binary combination of water (99%) and 1% TEA/H₃PO₄ (8:2 v/v) at a final pH of 9.8 gave better separations. However, the phosphoric acid could not be removed efficiently from the collected mobile phase during radioligand formulation. Replacement of this inorganic acid with more volatile formic acid (pH ∼ 9.9) resolved this issue and provided good separations (Figure S1).

All four radioligands were prone to autoradiolysis²⁹ during formulation for intravenous injection. This radiolysis was prevented by the addition of aqueous ascorbic acid (10 mg/mL, 100 μL) to the isolated HPLC fraction before it was dried and taken up into 10 mL of saline (0.9%, w/v)–ethanol (9:1, v/v).³⁰

Formulated [¹¹C]CPPC, [¹¹C]Psa374, [¹¹C]8e, and [¹¹C]8g were obtained in useful and reproducible decay-corrected yields of 12.0–14.1% from [¹¹C]carbon dioxide (Table S4) when the non-radioactive precursor (selected from 12a–d: 1.9–2.5 μmol) was treated with [¹¹C]iodomethane in DMF at 80 °C for 5 min (Scheme 3). All four CSF1R radioligands were obtained in 40–45 min with high radiochemical purities (>97.5%) and with molar activities (A_m) in the range of 1316–1936 GBq/μmol, decay-corrected to the end of bombardment (Table S4). Radioligand identities were verified by co-injection with the corresponding reference compound on HPLC (Figure S2) and LC-MS analysis of the related carrier (Table S5). All the formulated radioligands were stable for at least 1 h at room temperature.

Radioligand Lipophilicity

The log of the distribution coefficient (D) of a compound between sodium phosphate buffer (pH 7.4) and 1-octanol at room temperature, denoted as logD_7.4, is an index of lipophilicity and an important property influencing brain radioligand uptake, plasma free fraction (f_P), metabolism, and non-specific binding. The logD_7.4 measured for [¹¹C]Psa374 was 2.96 ± 0.01 (n = 6), a value that falls within the range that is considered desirable for brain-penetrant PET radioligands (1.5 < logD < 4).²⁴ Computations with ChemDraw software imply that Psa374 has a slightly lower logP value than CPPC (2.37 vs. 2.58), and this outcome accords with the corresponding reverse-phase HPLC retention times (t_R = 4.4 vs. t_R = 5.6, Figure S2).

[¹¹C]Psa374 Biostability Measured In Vitro and Ex Vivo

The biostability of [¹¹C]Psa374 when incubated in vitro at 37 °C for 60 min in whole blood, plasma, or brain homogenate from wild-type rats was assessed with radio-HPLC. At the end of incubations, parent radioligand accounted for 74% of radioactivity in plasma, 95% of radioactivity in whole blood, and 98% of radioactivity in brain homogenates.

Lower plasma [¹¹C]Psa374 stability was seen ex vivo. Following an intravenous bolus injection of [¹¹C]Psa374 into a rat, parent radioligand represented 42% and 13.5% of radioactivity in plasma at 30 and 120 min after injection, respectively (Figure 2, panel A, and Figure S3, panel A). Radiometabolites eluted earlier than unchanged radioligand. The respective whole brain radioactivity concentrations were 5.9 and 2.3 SUV and were comprised mainly of parent radioligand (>96.3% after 30 or 120 min postinjection; Figure 2, panel B and Figure S3, panel B). Thus, the in vitro and ex vivo results imply that [¹¹C]Psa374 is not subject to extensive metabolism within rat brain. The ex vivo results also suggest that radiometabolites do not readily enter rat brain from plasma. These findings are comparable to those that have been found ex vivo for [¹¹C]CPPC in mouse plasma and brain.^11,13,14

Figure 2.

Ex vivo reverse-phase HPLC analysis of radioactivity in plasma (panel A) and brain (panel B) at 30 min after intravenous injection of [¹¹C]Psa374 into a rat. See Experimental Section for HPLC conditions.

PET Imaging of [¹¹C]CPPC and [¹¹C]Psa374 in Wild-Type and Dual P-gp and Breast Cancer Resistance Protein (BCRP) Knockout Mice

We imaged wild-type mice and mice with dual P-gp (abcb1a/b gene deletion) and BCRP (abcg2 gene deletion) knockout after intravenous injection of [¹¹C]CPPC or [¹¹C]Psa374 to evaluate whether these radioligands are substrates for brain efflux transporters. Brain radioactivity uptakes were measured at baseline and after treatment with the homologous non-radioactive compound to test for specific binding (Figure 3).

Figure 3.

Whole brain time–activity curves from PET in wild-type and dual P-gp and BCRP knockout mice after intravenous injection of [¹¹C]CPPC (panel A) or [¹¹C]Psa374 (panel B) at baseline and after homologous treatments. Error bars are mean ± SD and within symbol size if not shown.

At baseline, both [¹¹C]CPPC and [¹¹C]Psa374 quickly accumulated in wild-type mouse brain to give peak values of 1.8 and 1.5 SUV, respectively. The low radioligand uptake is consistent with a previously reported finding for [¹¹C]CPPC in mice.¹¹ Following pretreatment with the homologous non-radioactive compound (4 mg/kg, i.v.), as much as a 2-fold higher radioactivity uptake was seen in the whole brain. Thus, these experiments did not detect any specific binding of the radioligands to CSF1R at baseline. Broadly these findings agree with those earlier found for [¹¹C]CPPC.^11,13,14 One possibility is that the CSF1R inhibitory potencies of the radioligands are too low for successful imaging. In this regard, inhibitory potency against mouse CSF1R is unknown and could be much lower than for human CSF1R. Human and mouse CSF1R share 75% amino acid homology,³¹ suggesting a significant possibility for a species difference in inhibitor potency. Another possible explanation is that mouse CSF1R density (B_max) is too low at baseline for imaging with either radioligand. We also considered the possibility of brain CSF1R occupancy by carrier in the baseline experiments at the administered radioligand doses. However, based on the CSF1R IC₅₀s measured for CPPC and Psa374 (Table S3), the injected doses of the carrier, peak radioactivity uptakes in the mouse brain, and an assumption that free fractions in the brain would be close to that seen in monkey plasma, we estimated that peak occupancy of CSF1R would have been lower than 40%.

The increased peak brain radioactivity uptakes for [¹¹C]CPPC and [¹¹C]Psa374 following homologous ligand treatment are not readily explained. Interaction of the radioligands and pretreatment agents with efflux transporters was suspected and therefore investigated. Both [¹¹C]CPPC and [¹¹C]Psa374 gave substantially higher peak radioactivity uptakes at baseline in dual P-gp and BCRP knockout mice of 2.7 SUV at 4.5 min and 2.9 SUV at 2.5 min, respectively (Figure 3). These results indicate that both [¹¹C]Psa374 and [¹¹C]CPPC are substrates for mouse brain efflux transporters. In this regard, and consistent with our findings, a prior study on [¹¹C]CPPC in normal mice found that brain radioactivity uptake increased with intravenous dose of CPPC preblocking agent over the range 0.3 to 20 mg/kg (Figure S4 in ref (11)).

Pretreatment with homologous ligands did not appreciably affect the time–activity curves for [¹¹C]CPPC or [¹¹C]Psa374 in efflux transporter knockout mice. Therefore, these experiments again show no evidence for CSF1R-specific binding by either of these two radioligands in healthy mouse brain.

PET Imaging in Monkey Brain

Each of the four prepared radioligands was imaged in rhesus macaque (Macaca mulatta) under baseline conditions and following a pharmacological challenge. Radioligands were injected intravenously at similarly high molar activities and radiochemical purities. Figure 4 shows the time–activity curves for the monkey whole brain at baseline and after attempted CSF1R blockade with CPPC (1 mg/kg, i.v.) for all four radioligands and with homologous inhibitor (4 mg/kg, i.v.) for [¹¹C]CPPC and [¹¹C]Psa374.

Figure 4.

Time–activity curves in the brain after intravenous injection of [¹¹C]8e (panel A), [¹¹C]8g (panel B), [¹¹C]CPPC (panel C), and [¹¹C]Psa374 (panel D) into monkey at baseline and after treatment with CPPC or Psa374. Solid lines show fits of the data to a 2-tissue compartmental model. Error bars are SD values for n ≥ 3 or half range for n = 2 and are within symbol size if not shown.

At baseline, [¹¹C]8e, [¹¹C]8g, and [¹¹C]Psa374 entered the brain to give peak radioactivity uptakes of about 2.28, 2.24, and 1.97 SUV at 65, 35, and 17 min, respectively (Figure 4). [¹¹C]Psa374 showed the fastest decline in radioactivity from peak level, whereas [¹¹C]8e exhibited a much slower washout. The kinetics for [¹¹C]CPPC were similar in giving peak radioactivity uptake of 2.59 SUV at 35 min and slow subsequent radioactivity decline in accordance with a prior rhesus monkey study.^13,14 This finding is also similar to that reported for baseline uptake of [¹¹C]CPPC in the baboon brain (2.5 to 4.0 SUV at 20 min).¹¹

Pretreatment of monkeys with CPPC at a dose of 1 mg/kg (i.v.) influenced imaging results in an unexpected direction (Figure 4). The time–activity curves obtained under the attempted CSF1R blocking conditions showed substantially higher peak brain radioactivity uptakes for both [¹¹C]CPPC and [¹¹C]Psa374. Except for [¹¹C]Psa374, the radioligands showed a slow decline from peak radioactivity levels up to the end of scanning at 85 min, as in the corresponding baseline curves. Therefore, these experiments failed to reveal any CSF1R specific binding. The possibility for occupancy of brain CSF1R by the administered carrier in the monkey baseline experiments is much less than in mouse. We estimated in the same manner as we did for the mouse experiments that the maximal occupancy would be less than 20%.

In experiments with [¹¹C]Psa374 and [¹¹C]CPPC in which blockade of CSF1R was attempted at a higher dose (4 mg/kg, i.v.) of homologous inhibitor, peak brain radioactivity levels were little altered from those in the experiments with the lower dose of CPPC (1 mg/kg, i.v.) (Figure 4). However, for [¹¹C]Psa374, the decline of radioactivity from a peak level of 3.1 SUV at 27 min was slower under the Psa374 dose condition (4 mg/kg, i.v.) than under the lower CPPC dose condition (1 mg/kg, i.v.) (Figure 4, panel D). These time–activity curves alone do not evidence the presence of CSF1R-specific binding for any of the four radioligands in monkey at baseline.

A reason to be considered for the increase in brain radioactivity concentration after attempted CSF1R blockade in monkey is a displacement of radioligand that may be bound to peripheral CSF1R. However, for most of the imaged radioligands, radiometabolite-corrected arterial input functions did not change appreciably due to intravenous administration of the CSF1R blocking agent (Figure S4). This shows that peripheral displacement of the radioligands by the non-radioactive blocking agent did not occur. In the case of [¹¹C]Psa374, a slight increase in the concentration of the parent radioligand in blood plasma was recorded, but this variation did not seem to be blocker concentration-dependent.

Another possible explanation for the unexpectedly higher brain radioligand accumulations could be that the blocking agents CPPC and Psa374, and corresponding radioligands, are weak substrates of the ATP-binding cassette (ABC) family. ATP-dependent efflux transporters regulate the influx and efflux of various xenobiotic and endogenous substances at the BBB and thereby protect the body from potential toxins. At the BBB, the three most predominant ABC transporters are P-glycoprotein (P-gp), multidrug resistance protein (MRP), and BCRP.³²

Because we suspected that Psa374 and the other CSF1R tracers sharing the same phenylpiperazine core could be weak substrates for ABC transporters, as already demonstrated in mice, we selected [¹¹C]CPPC and [¹¹C]Psa374 for PET study in monkeys at baseline and after administration of elacridar, a dual P-gp and BCRP inhibitor.³³Figure 5 shows time–activity curves for [¹¹C]CPPC and [¹¹C]Psa374 at baseline and after intravenous injection of elacridar at 3 mg/kg. [¹¹C]Psa374 injection after elacridar pretreatment resulted in peak brain radioactivity uptake of 2.5 SUV after 22 min, a 25% increase over that in the baseline experiment. Subsequent radioactivity decline resembled that seen in the baseline experiment. An even more marked effect of elacridar was seen on [¹¹C]CPPC kinetics. Indeed, for this radioligand, peak brain radioactivity concentration increased almost 2-fold, reaching 4.5 SUV after 12.5 min. Subsequent radioactivity washout was quicker than under baseline conditions. Radioligand arterial input functions did not vary between baseline and elacridar pretreatment experiments (Figure S5). These results therefore suggest that at high concentrations CPPC and Psa374 act as partial and competitive substrates of the ABC transporters in non-human primates. An interpretation of our results in mice and monkey and those of Horti et al. in mice (Figure S4 in ref (11)) is that at low no-carrier-added radioligand dose the efflux transporters limit brain radioligand entry substantially. However, when these compounds are administered as non-radioactive blocking agents at high dose, they overwhelm efflux pump function and consequently radioligand uptake is markedly increased.

Figure 5.

Time–activity data fitted (solid lines) using the two-tissue compartmental model in monkey brain for [¹¹C]CPPC (panel A) and [¹¹C]Psa374 (panel B) at baseline and after partial blocking of efflux transporters with elacridar (3 mg/kg, i.v.). For each radioligand, the same monkey was used in the baseline and blocking experiment in 1 day (n = 1).

Emergence of Radiometabolites in Monkey Plasma

Following the intravenous injection of [¹¹C]CPPC into monkey, parent radioligand represented 47.4% of radioactivity in plasma at 30 min (Figure 6, panel A). Corresponding values for [¹¹C]Psa374 were 54.7% at 30 min (Figure 6, panel B). Radiometabolites eluted much earlier than unchanged radioligand in each case, implying they are much less lipophilic and less likely to enter brain than parent radioligand. In this regard, radiometabolites with similar retention times and lipophilicities were not found to a large extent in rat brain after intravenous administration of [¹¹C]Psa374 (Figure 2).

Figure 6.

Reverse-phase HPLC analysis of radioactivity in monkey plasma at 30 min after intravenous injection of [¹¹C]CPPC (panel A) and [¹¹C]Psa374 (panel B). See Experimental Section for HPLC conditions.

Monkey Radioligand Plasma Free Fractions

Only the fraction of radioligand in plasma that is free (f_p) (i.e., not bound to blood protein or sequestered into blood cells) is considered to be able to enter the brain. In general, brain radioactivity will be proportional to f_p. Therefore, f_p values should be known and accurately measurable if these values are to be considered for biomathematical determination of output measures, such as radioligand-specific binding. For [¹¹C]Psa374, we obtained an f_p value of 5.62 ± 0.23% (n = 3) in rat plasma in vitro. For this and other measurements, radioligand degradation had to be inhibited with sodium fluoride. Higher f_p values were measured in monkey plasma for both [¹¹C]CPPC and [¹¹C]Psa374 (7.0 ± 0.4%, n = 2 and 7.8 ± 1.2%, n = 3, respectively; Table S6). These values changed slightly in experiments where monkeys were pretreated with various agents. Remarkably, the plasma free fractions for [¹¹C]8g and [¹¹C]8e were much lower than for [¹¹C]CPPC and [¹¹C]Psa374 (Table S6).

Biomathematical Analyses of PET Scans

All the time–activity curves in the monkey PET experiments were well-fitted according to a two-tissue compartment model (2TCM) (Figures 4 and 5). Quantitative PET analysis of volume of distribution (V_T) (Table S7) and volume of distribution normalized by free fraction (V_T/f_P) (Table S8) revealed that [¹¹C]CPPC and [¹¹C]Psa374 gave quite similar regional distributions of radioactivity uptake in brain, although V_T and V_T/f_P values were markedly lower for [¹¹C]Psa374 than for [¹¹C]CPPC. Radioactivity predominantly accumulated in the frontal cortex, insula, and thalamus, whereas the cerebellum and the occipital cortex saw the lowest concentrations. The distribution expected for an effective CSF1R radioligand is unclear, because of lack of information in the literature. Plots of regional [¹¹C]CPPC and [¹¹C]Psa374V_T values against reported human brain mRNA distributions showed no correlations for two probes, CUST_15663_PI416261804 (Figure S6) and A_23_P110791 (Figure S7), in the Allen Brain Atlas,³⁴ nor data in the Human Protein Atlas (Figure S8).^35,36 Each radioligand showed good time stability of whole brain V_T, consistent with an absence of radiometabolite accumulation in the brain (Figure S9).

The values V_T and V_T/f_P for [¹¹C]CPPC showed some decrease by pretreatment with CPPC at the high dose of 4 mg/kg (i.v.) in different brain regions, amounting to about 41% across whole brain in one monkey in comparison to average baseline values (n = 2), but not at the lower dose of 1 mg/kg (i.v.), which actually increased these parameter values (Figures 7 and 8; Tables S7 and S8). The values for V_T and V_T/f_P [¹¹C]Psa374 were also increased by pharmacological challenges with CPPC (1 mg/kg; i.v.) or Psa374 (4 mg/kg; i.v.). These results therefore show no clear evidence of any specific binding of these radioligands to CSF1R in monkey brain. Moreover, Lassen plots of brain regional V_T in the baseline experiment versus the regional reductions in V_T under blocking conditions showed poor goodness of fit (R² < 0.4, Figure S10), consistent with an absence of CSF1R specific binding. Another cause of non-linearity might be regional variation in V_ND, which violates a requirement for the Lassen plot.

Figure 7.

Whole brain V_T (panel A) and V_T/f_P (panel B) values for [¹¹C]CPPC and [¹¹C]Psa374 in monkeys at baseline (n = 3 for [¹¹C]Psa374 and n = 2 for [¹¹C]CPPC) and after intravenous pharmacological challenge with either CPPC (n = 2 for [¹¹C]Psa374 and n = 1 for [¹¹C]CPPC) or Psa374 (n = 1). Error bars are mean ± SD for n > 2, and mean ± half of the range for n = 2. See Tables S7 and S8 for respective V_T and V_T/f_P values for individual brain regions.

Figure 8.

Parametric images of one monkey brain derived from Logan plots at baseline and after self-block at 4 mg/kg (i.v.) for [¹¹C]CPPC (panel A) and [¹¹C]Psa374 (panel B). V_T values for these images were derived from Logan plots. Left images are coronal, middle are sagittal, and right are horizontal.

As expected from the time–activity curves shown in Figure 5, biomathematical analysis with 2TCM (Figure 9) showed that whole brain V_T and V_T/f_P values increased by 60% and 33% for [¹¹C]CPPC and 54% and 38% for [¹¹C]Psa374, respectively, following pretreatment with elacridar (3 mg/kg, i.v.). Brain regional values are shown in Tables S9 and S10. Figure 10 shows the regional V_T and V_T/f_P values derived from Logan plots. These data verify the weak substrate behavior of the radioligand for brain efflux transporters in monkey.

Figure 9.

Monkey whole brain V_T (panel A, n = 1) and V_T/f_P values (panel B, n = 1) for [¹¹C]CPPC and [¹¹C]Psa374 in monkeys at baseline and after challenge with elacridar. See Tables S9 and S10 for respective V_T and V_T/f_P values for individual brain regions.

Figure 10.

Parametric images of monkey brain at baseline and after intravenous administration of elacridar at 3 mg/kg for [¹¹C]CPPC (panel A) and [¹¹C]Psa374 (panel B). V_T values for these images were derived from Logan plots. Left images are coronal, middle are sagittal, and right are horizontal.

Evaluation of Psa374 as a Substrate for Human P-gp and BCRP

Species differences in the transport of PET radioligands from blood to the brain have been reported, despite, for example, high amino acid sequence homology across species for P-gp protein.^37,38 Because [¹¹C]Psa374 showed interactions with mouse and monkey brain efflux transporters, we decided to evaluate the behavior of Psa374 toward human P-gp and BCRP efflux pumps.

Bidirectional assays are recognized models for evaluating novel drugs as potential substrates for efflux transporters.³⁹ We used a bidirectional assay performed by Eurofins Panlabs to assess the interaction of Psa374 with human efflux transporters. This assay uses a polarized monolayer of Caco-2 cells on a semiporous filter at the interface of a dual-chamber apparatus, partitioning the apical and basolateral chambers.⁴⁰ Caco-2 cell lines express BCRP and multidrug resistance-associated protein 2 in addition to P-gp.⁴¹

The test compound is added to the apical compartment, and the basolateral chamber’s measured appearance rate defines its apical-to-basolateral apparent permeability (P_app-AB), which is proportional to the absorptive transport. Adding the test compound to the basolateral chamber gives the basolateral-to-apical apparent permeability (P_app-BA), an indicator for secretive transport.⁴² An efflux ratio index (ER) for the tested compound can be defined as the ratio of P_app-BA to P_app-AB. A compound is commonly considered to be a substrate for efflux transporters if its ER is greater than 2.⁴³

Psa374 was assayed at 10 μM concentration and gave an ER value of 0.24. Psa374P_app was further investigated in the presence of the P-gp inhibitor verapamil and the BCRP inhibitor KO143 (Table 2). In both cases, the basal-to-apical P_app did not change appreciably, indicating that Psa374 is not a substrate for human efflux transporters under these conditions. However, as discussed earlier for PET experiments in mouse and monkey, it may be that the high concentration of Psa374 overwhelms the action of the efflux transporters by acting as a weak substrate inhibitor.

Table 2. Apparent Permeabilities and Calculated Efflux Ratios for Psa374.

Compound(s)	Papp (Caco-2)	Permeabilitya (10–6 cm/s)	ERb
Psa374	A-B	12.2 ± 0.97	0.24
Psa374	B-A	2.9 ± 0.18
Psa374 + verapamil	A-B	7.8 ± 0.04	0.49
Psa374 + verapamil	B-A	3.8 ± 0.04
Psa374 + KO143	A-B	13.7 ± 0.19	0.18
Psa374 + KO143	B-A	2.5 ± 0.28

^aValues are mean ± half of the range (n = 2).

^bER is reported as P_app-BA/P_app-AB

Conclusions

Seven new fluorinated analogs of CPPC were prepared, and four were found to have high (sub-nanomolar) potencies and selectivities to inhibit CSF1R. In particular, Psa374, a simple 4-fluoromethyl derivative of CPPC, showed high CSF1R inhibitory potency and selectivity comparable to that of CPPC itself. [¹¹C]Psa374 behaved similarly to [¹¹C]CPPC in PET imaging of brain in mouse and monkey. Neither radioligand showed definite specific binding to brain CSF1R in either species. Moreover, our results show that [¹¹C]Psa374 and [¹¹C]CPPC interact weakly with mouse and monkey P-gp efflux pumps in vivo, which alone would preclude any utility for robust PET imaging of CSF1R in these species. Although Psa374 was found not to be a substrate for human ABC transporters in vitro, the high concentration used in this assay (10 μM) may be masking a weak substrate behavior at low concentration. This leaves open the possibility that [¹¹C]Psa374 and [¹¹C]CPPC would be subject to the actions of brain efflux transporters in living human subjects. Further development of [¹¹C]Psa374 in non-human primate models of neuroinflammation showing CSF1R-specific binding would be needed to warrant the fluorine-18 labeling of Psa374 with a view to possible application in human subjects. Further studies are required to verify the efficacy of [¹¹C]CPPC for measuring CSF1R in human brain. Probably, the field needs a radioligand that is better than [¹¹C]Psa374 and [¹¹C]CPPC in having higher affinity to allow detection of CSF1R in healthy brain without efflux transporter liability.

Experimental Section

Materials

All reagents were obtained commercially and were used without further purification. Commercially available chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO) and Alfa Aesar (Ward Hill, MA). Elacridar was purchased from Sigma-Aldrich.

General Methods

All reactions requiring an inert atmosphere were performed under argon. Reactions were monitored using reverse-phase LC-MS on an LC-MS-2020 instrument (Shimadzu, Long Beach, CA) equipped with a Luna C18(2) column (4.6 × 250 mm, 10 μm; Phenomenex, Torrance, CA) and a UV absorbance detector or by TLC (thin layer chromatography) on silica plates (type 60 F254; 400–630 mesh; Sigma-Aldrich) that were visualized under ultraviolet light (λ = 254 nm). Compounds were purified with flash chromatography on a semiautomated apparatus (CombiFlash Rf + UV; Teledyne ISCO Inc., Lincoln, NE).

¹H (400 MHz), ¹³C (100 MHz), and ¹⁹F NMR (376.49 MHz) spectra were acquired at room temperature (rt) with an Avance III HD NMR spectrometer (Bruker BioSpin Corp., Billerica, MA) and reported using the chemical shift of residual deuterated solvent as the internal standard. ¹H and ¹³C chemical shifts are reported in δ units (ppm) downfield relative to the chemical shift for tetramethylsilane (δ = 0) and ¹⁹F chemical shifts relative to that for CFCl₃. Abbreviations bs, d, dd, ddd, dt, m, q, s, t, and td denote broad singlet, doublet, doublet of doublet, doublet of doublet of doublet, doublet of triplet, multiplet, quartet, singlet, triplet, and triplet of doublet, respectively. Coupling constants (J) are in Hz. NMR spectra are shown in the Supporting Information.

Melting points were measured on a digital melting point apparatus (Stuart SMP 20). High-resolution mass spectra (HRMS) were recorded on an ESI-TOF MS spectrometer (DART ion source) at the Bioorganic Chemistry Laboratory of NIDDK (NIH).

The purities of CPPC, 8g, and the new compounds submitted to the biological assay were >97.7%, as determined with HPLC on a Luna C18 column (5 μm, 250 mm × 4.6 mm; Phenomenex) eluted isocratically with aq. TEA/formic acid (8:2, 1% v/v, pH = 9.9):MeCN, (40:60 v/v, 1.5 mL/min) and with eluate monitored for absorbance at 254 nm (Figure S11).

All radiochemistry was performed in lead-shielded hot-cells to ensure radiation protection for personnel. The main apparatus for radiochemistry consisted of a PETtrace MeI Process Module (GE Medical Systems, Severna Park, MD) for [¹¹C]iodomethane production and a semirobotic Synthia instrument⁴⁴ (Synthia AB, Uppsala, Sweden) upgraded⁴⁵ with PLC-control for labeling reactions. Dedicated recipes were created in the Autorad software and followed step by step for each radiosynthesis. The HPLC apparatus for radioactive compound separation comprises a pump (P4.1S; Knauer, Berlin, Germany), a UV absorbance detector (UVD2.1S; Knauer), and a radioactivity detector (flow-count; Eckert & Ziegler, Berlin, Germany). The Clarity Chromatography Station software (DataApex, Prague, Czech Republic) was used to record the chromatograms. The equipment for radio-HPLC analyses was comprised of two pumps (LC-20AD; Shimadzu, Columbia, MD), a UV absorbance detector (SPD-M20A; Shimadzu), and a radioactivity detector (flow-count; Eckert & Ziegler).

All animals used in this study were handled in accordance with “Animal Research: Reporting of In Vivo Experiments” guidelines as well as “Guidelines for the Care and Use of Laboratory Animals” ⁴⁶ and the requirements of the National Institute of Mental Health Animal Care and Use Committee.

The formulation of pharmacological agents for intravenous administration to animals in PET experiments is detailed in Supporting Information.

All statistical analyses were performed in GraphPad Prism (version 5.02; GraphPad Software, Inc., San Diego, CA). Results with p-values <0.05 were considered statistically significant. Data are presented as mean ± standard deviation (S.D.) or mean with a range.

Chemistry

Syntheses of CSF1R Ligands

1-(5-Chloro-2-nitrophenyl)piperidine (3a)

Piperidine (1a, 1.7 mL; 16.75 mmol) was added dropwise over 5 min to a cooled solution of 4-chloro-2-fluoronitrobenzene (2, 1 g, 5.58 mmol) in EtOH (18 mL). The resultant orange solution was stirred at 0 °C for 1 h, left to warm to rt, and stirred overnight. The EtOH was removed almost entirely under vacuum. The obtained slurry was diluted with H₂O and extracted with EtOAc. The organic phase was washed with sat. aq. NaHCO₃ and brine, dried over Na₂SO₄, and evaporated in vacuo to dryness. The crude product was purified by automated reverse-phase CombiFlash chromatography (H₂O: MeCN), giving 3a as an orange solid (93%). Mp: 66–66.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.88 (dd, J = 8.7, 2.1 Hz, 1H), 3.06–2.99 (m, 4H), 1.77–1.66 (m, 4H), 1.65–1.55 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 147.89, 140.00, 139.58, 127.65, 120.59, 120.02, 52.64, 25.73, 24.01. HRMS (ESI) m/z: 241.1 [M + H]⁺. Calcd for C₁₁H₁₄N₂O₂³⁵Cl: 241.0744; found 241.0745.

1-(5-Chloro-2-nitrophenyl)-4-methylpiperidine (3b)

Similarly, 4-methylpiperidine (1b, 6.2 mL, 50.26 mmol) and 4-chloro-2-fluoronitrobenzene (2, 3 g, 16.75 mmol) in EtOH (55 mL) gave 3b as an orange solid (98%). Mp: 38.5–39.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.06 (dd, J = 8.8, 2.1 Hz, 1H), 3.43 (dt, J = 12.1, 2.7 Hz, 2H), 3.03 (td, J = 12.1, 2.4 Hz, 2H), 1.89 (dd, J = 12.7, 3.6 Hz, 2H), 1.73 (ddtt, J = 12.9, 9.6, 6.2, 3.1 Hz, 1H), 1.58 (qd, J = 11.9, 3.8 Hz, 2H), 1.19 (d, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 147.54, 139.89, 139.36, 127.56, 120.48, 119.84, 51.83, 33.94, 30.36, 21.72. HRMS (ESI) m/z: 255.1 [M + H]⁺. Calcd for C₁₂H₁₆N₂O₂³⁵Cl: 255.0900; found 255.0898.

1-(5-Chloro-2-nitrophenyl)-4-(fluoromethyl)piperidine (3c)

Similarly, 4-(fluoromethyl)piperidinium chloride (1c, 5g, 30.71 mmol), 4-chloro-2-fluoronitrobenzene (2, 5.0 g, 27.92 mmol), and TEA (8.3 mL, 58.63 mmol) in EtOH (93 mL) gave 3c as an orange solid (81%). Mp: 55.4–57 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 2.1 Hz, 1H), 6.93 (dd, J = 8.7, 2.1 Hz, 1H), 4.39 (d, J = 6.2 Hz, 1H), 4.27 (d, J = 6.0 Hz, 1H), 3.31 (dt, J = 12.2, 2.9 Hz, 2H), 2.86 (td, J = 12.2, 2.4 Hz, 2H), 1.96–1.85 (m, 1H), 1.81 (dd, J = 13.8, 3.2 Hz, 2H), 1.60–1.47 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 147.51, 140.34, 139.64, 127.63, 120.88, 120.69, 87.34 (d, J = 168.9 Hz), 51.44, 36.34 (d, J = 18.9 Hz), 27.65 (d, J = 6.2 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −223.12. HRMS (ESI) m/z: 273.1 [M + H]⁺. Calcd for C₁₂H₁₅N₂O₂F³⁵Cl: 273.0806; found 273.0806.

1-Methyl-4-(4-nitro-3-(piperidin-1-yl)phenyl)piperazine (5a)

A mixture of 3a (4.56 g, 18.03 mmol) and 1-methylpiperazine (4, 6.1 mL, 54.08 mmol) was heated with stirring at 140 °C for 16 h. The mixture was cooled to rt and then quenched with H₂O. The crude product was extracted portion-wise with EtOAc to avoid the formation of an emulsion. The organic phase extracts were combined, washed with H₂O and brine, dried over Na₂SO₄, and evaporated in vacuo to dryness. The crude product was purified by automated reverse-phase CombiFlash chromatography (H₂O:MeCN), giving a brown oil. The oil was stripped a few times with Et₂O, giving 5a as a brown solid (91%). Mp: 70–71.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 9.3 Hz, 1H), 6.39 (dd, J = 9.4, 2.6 Hz, 1H), 6.31 (d, J = 2.6 Hz, 1H), 3.41–3.33 (m, 4H), 3.05–2.97 (m, 4H), 2.57–2.50 (m, 4H), 2.35 (s, 3H), 1.75 (p, J = 5.7 Hz, 4H), 1.61 (ddd, J = 11.9, 7.7, 4.6 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.94, 150.62, 132.05, 129.61, 106.32, 103.46, 54.64, 53.21, 47.12, 46.25, 25.95, 24.21. HRMS (ESI) m/z: 305.2 [M + H]⁺. Calcd for C₁₆H₂₅N₄O₂: 305.1978; found 305.1974.

1-Methyl-4-(3-(4-methylpiperidin-1-yl)-4-nitrophenyl)piperazine (5b)

Similarly, 3b (1.8 g, 6.71 mmol) and 1-methylpiperazine (4, 2.3 mL, 20.14 mmol) gave 5b as an orange solid (86%). Mp: 87–89 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 9.3 Hz, 1H), 6.35 (dd, J = 9.4, 2.6 Hz, 1H), 6.28 (d, J = 2.6 Hz, 1H), 3.34 (t, J = 5.1 Hz, 4H), 3.24 (d, J = 11.9 Hz, 2H), 2.74 (t, J = 11.7 Hz, 2H), 2.50 (t, J = 5.1 Hz, 4H), 2.31 (s, 3H), 1.73–1.60 (m, 2H), 1.52–1.38 (m, 3H), 0.96 (d, J = 5.6 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.92, 150.38, 132.03, 129.63, 106.29, 103.50, 54.64, 52.49, 47.11, 46.10, 34.21, 30.71, 21.86. HRMS (ESI) m/z: 319.2 [M + H]⁺. Calcd for C₁₇H₂₇N₄O₂: 19.2134; found 319.2138.

1-(3-(4-(Fluoromethyl)piperidin-1-yl)-4-nitrophenyl)-4-methylpiperazine (5c)

Similarly, 3c (6 g, 20.9 mmol) and 1-methylpiperazine (4, 7 mL, 62.71 mmol) gave 5c as a dark yellow solid (50%). Mp: 93.1–95.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 9.4 Hz, 1H), 6.42 (dd, J = 9.4, 2.6 Hz, 1H), 6.32 (d, J = 2.6 Hz, 1H), 4.33 (dd, J = 47.4, 6.2 Hz, 2H), 3.45–3.28 (m, 6H), 2.80 (td, J = 11.9, 2.4 Hz, 2H), 2.54 (t, J = 5.1 Hz, 4H), 2.35 (s, 3H), 1.95–1.85 (m, 1H), 1.81 (d, J = 13.3 Hz, 2H), 1.59 (qd, J = 12.1, 3.9 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.96, 150.21, 132.24, 129.68, 106.71, 103.69, 87.66 (d, J = 168.5 Hz), 54.63, 51.95, 47.11, 46.09, 36.65 (d, J = 18.7 Hz), 27.88 (d, J = 6.2 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −222.56. HRMS (ESI) m/z: 337.2 [M + H]⁺. Calcd for C₁₇H₂₆N₄O₂F: 337.2040; found 337.2043.

4-(4-Methylpiperazin-1-yl)-2-(piperidin-1-yl)aniline (6a)

10% Pd/C (1.3 g, 1.24 mmol) was added to a mixture of 5a (3.9 g, 12.44 mmol) in MeOH (6 mL) under nitrogen. The nitrogen was evacuated, and the reaction vessel was refilled with hydrogen (70 psi). The reaction mixture was then stirred overnight at rt. The catalyst was removed by filtration over a bed of Celite and the solution evaporated in vacuo to dryness. The crude product was purified by automated reverse-phase CombiFlash chromatography (H₂O: MeCN), giving 6a as a brown solid (88%). Mp: 73–75.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.69 (d, J = 2.6 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.55 (dd, J = 8.5, 2.6 Hz, 1H), 3.71 (s, 2H), 3.10–3.04 (m, 4H), 2.83 (t, J = 5.2 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H), 1.69 (p, J = 5.6 Hz, 4H), 1.61–1.51 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 144.63, 141.54, 135.47, 115.43, 112.75, 110.88, 55.43, 52.65, 51.16, 46.19, 26.89, 24.42. HRMS (ESI) m/z: 275.2 [M + H]⁺. Calcd for C₁₆H₂₇N₄: 275.2236; found 275.2235.

4-(4-Methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)aniline (6b)

Similarly, 5b (1.7 g 5 mmol), 10% Pd/C (0.53 g, 0.5 mmol), and H₂ (70 psi) in MeOH (2.5 mL) gave 6b as a gray solid (90%). Mp: 106.5–107 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.69 (d, J = 2.6 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.55 (dd, J = 8.5, 2.6 Hz, 1H), 3.51 (bs, 2H), 3.15–3.05 (m, 6H), 2.68 (t, J = 4.9 Hz, 4H), 2.57 (td, J = 11.6, 2.3 Hz, 2H), 2.41 (s, 3H), 1.73 (dd, J = 12.7, 3.4 Hz, 2H), 1.49 (ddt, J = 14.9, 9.3, 5.0 Hz, 1H), 1.33 (qd, J = 11.8, 3.8 Hz, 2H), 0.98 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 144.23, 141.20, 135.70, 115.48, 113.10, 110.65, 55.15, 51.92, 50.76, 45.75, 35.23, 30.76, 22.09. HRMS (ESI) m/z: 289.2 [M + H]⁺. Calcd for C₁₇H₂₉N₄: 289.2392; found 289.2387.

2-(4-(Fluoromethyl)piperidin-1-yl)-4-(4-methylpiperazin-1-yl)aniline (6c)

Similarly, 5c (3.47 g, 10.13 mmol), 10% Pd/C (1.08 g, 1.01 mmol), and H₂ (70 psi) in MeOH (10 mL) gave 6c as a white solid (84%). Mp: 62.5–63 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.70 (d, J = 2.6 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.57 (dd, J = 8.4, 2.6 Hz, 1H), 4.34 (dd, J = 47.5, 5.6 Hz, 2H), 3.70 (s, 2H), 3.19 (d, J = 11.5 Hz, 2H), 3.07 (t, J = 5.0 Hz, 4H), 2.67–2.53 (m, 6H), 2.35 (s, 3H), 1.83 (d, J = 12.0 Hz, 3H), 1.46 (qt, J = 12.7, 6.3 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 144.68, 140.84, 135.42, 115.57, 113.07, 110.56, 87.89 (d, J = 168.4 Hz), 55.40, 51.28, 51.14, 46.17, 36.72 (d, J = 18.7 Hz), 28.73 (d, J = 5.9 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −223.13. HRMS (ESI) m/z: 307.2 [M + H]⁺. Calcd for C₁₇H₂₈N₄F: 307.2298; found 307.2299.

4-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)thiophene-2-carboxamide (8a)

DIPEA (0.22 mL, 1.25 mmol) was added to 6b (0.20 g, 0.62 mmol), 4-fluorothiophene-2-carboxylic acid (7d, 0.11 g, 0.75 mmol), and HATU (0.29 g, 0.75 mmol) in DMF (3.1 mL) under argon. The reaction mixture was stirred at rt overnight. The following day, the reaction was quenched with H₂O and the organic phase was extracted with DCM. The organic phases were combined, washed with H₂O and brine, dried over Na₂SO₄, and evaporated in vacuo to dryness. The crude product was purified with automated reverse-phase Combiflash chromatography (H₂O:MeCN), giving 8a as a white solid (62%). Mp: 146–149.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.03 (s, 1H), 8.26 (d, J = 8.9 Hz, 1H), 7.29 (s, 1H), 6.88 (s, 1H), 6.76 (d, J = 2.7 Hz, 1H), 6.68 (dd, J = 8.9, 2.7 Hz, 1H), 3.13 (t, J = 4.9 Hz, 4H), 2.94 (dt, J = 12.3, 3.3 Hz, 2H), 2.67 (td, J = 11.6, 2.3 Hz, 2H), 2.53 (t, J = 4.9 Hz, 4H), 2.30 (s, 3H), 1.78 (dd, J = 13.2, 3.6 Hz, 2H), 1.58–1.45 (m, 1H), 1.35 (qd, J = 11.8, 3.8 Hz, 2H), 1.00 (d, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.78 (d, J = 2.2 Hz), 157.03 (d, J = 261.2 Hz), 148.21, 143.38, 138.41 (d, J = 5.9 Hz), 125.73, 119.99, 117.23 (d, J = 25.7 Hz), 112.44, 109.22, 108.27 (d, J = 20.5 Hz), 55.09, 53.15, 49.54, 46.11, 35.49, 30.53, 21.96. ¹⁹F NMR (376 MHz, CDCl₃) δ −124.73. HRMS (ESI) m/z: 417.2 [M + H]⁺. Calcd for C₂₂H₃₀N₄OF³²S: 417.2124; found 417.2120.

4-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)thiophene-2-carboxamide (8b)

Similarly, 6a (0.14 g, 0.48 mmol), 4-fluorothiophene-2-carboxylic acid (7d, 0.09 g, 0.58 mmol), HATU (0.22 g, 0.58 mmol) and DIPEA (0.17 mL, 0.97 mmol) in DMF (3 mL) gave 8b as a yellow solid (44%). Mp: 162–162.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.06 (s, 1H), 8.24 (d, J = 8.9 Hz, 1H), 7.26 (s, 1H), 6.85 (s, 1H), 6.72 (d, J = 2.7 Hz, 1H), 6.66 (dd, J = 8.9, 2.7 Hz, 1H), 3.11 (t, J = 4.9 Hz, 4H), 2.76 (t, J = 5.1 Hz, 4H), 2.51 (t, J = 5.0 Hz, 4H), 2.29 (s, 3H), 1.70 (p, J = 5.7 Hz, 4H), 1.56 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 157.91 (d, J = 2.2 Hz), 157.15 (d, J = 261.5 Hz), 148.30, 143.71, 138.58 (d, J = 5.5 Hz), 125.90, 120.01, 117.26 (d, J = 25.7 Hz), 112.64, 109.40, 108.38 (d, J = 20.5 Hz), 55.20, 53.93, 49.67, 46.21, 27.23, 24.12. ¹⁹F NMR (376 MHz, CDCl₃) δ −124.66. HRMS (ESI) m/z: 403.2 [M + H]⁺. Calcd for C₂₁H₂₈N₄OF³²S: 403.1968; found 403.1961.

4-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (8c)

Similarly, 6b (0.20 g, 0.62 mmol), 4-fluoro-1H-pyrrole-2-carboxylic acid (7c, 0.10 g, 0.75 mmol), HATU (0.29 g, 0.75 mmol), and DIPEA (0.22 mL, 1.25 mmol) in DMF (4 mL) gave 8c as a light brown solid (33%). Mp: 211–212.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.72 (s, 1H), 8.82 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 2.6 Hz, 1H), 6.77–6.69 (m, 2H), 6.38 (s, 1H), 3.17 (t, J = 5.0 Hz, 4H), 2.98 (d, J = 11.2 Hz, 2H), 2.75–2.65 (m, 2H), 2.59 (t, J = 4.9 Hz, 4H), 2.36 (s, 3H), 1.83 (d, J = 11.9 Hz, 2H), 1.64–1.51 (m, 1H), 1.39 (qd, J = 11.8, 3.7 Hz, 2H), 1.06 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.93 (d, J = 2.9 Hz), 151.93 (d, J = 242.1 Hz), 148.11, 143.48, 126.22, 122.93 (d, J = 4.8 Hz), 120.06, 112.61, 109.55, 106.09 (d, J = 27.9 Hz), 96.00 (d, J = 15.8 Hz), 55.31, 53.29, 49.90, 46.29, 35.69, 30.78, 22.12. ¹⁹F NMR (376 MHz, CDCl₃) δ −162.65. HRMS (ESI) m/z: 400.3 [M + H]⁺. Calcd for C₂₂H₃₁N₅OF: 400.2513; found 400.2509.

4-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (8d)

Similarly, 6a (0.20 g, 0.71 mmol), 4-fluoro-1H-pyrrole-2-carboxylic acid (7c, 0.12 g, 0.86 mmol), HATU (0.33 g, 0.86 mmol), and DIPEA (0.25 mL,0.86 mmol) in DMF (4 mL) gave compound 8d as a light-yellow solid (33%). Mp: 235–236 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.05 (s, 1H), 8.86 (s, 1H), 8.26 (d, J = 8.9 Hz, 1H), 6.79 (d, J = 2.7 Hz, 1H), 6.76–6.69 (m, 2H), 6.40 (t, J = 2.3 Hz, 1H), 3.17 (t, J = 5.0 Hz, 4H), 2.83 (t, J = 5.2 Hz, 4H), 2.59 (t, J = 4.9 Hz, 4H), 2.36 (s, 3H), 1.77 (p, J = 5.9 Hz, 4H), 1.63 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 157.84 (d, J = 2.6 Hz), 151.95 (d, J = 242.1 Hz), 148.09, 143.72, 126.26, 123.00 (d, J = 4.4 Hz), 119.98, 112.68, 109.60, 105.98 (d, J = 27.9 Hz), 96.03 (d, J = 15.8 Hz), 55.30, 53.95, 49.89, 46.28, 27.32, 24.22. ¹⁹F NMR (376 MHz, CDCl₃) δ −162.54. HRMS (ESI) m/z: 386.2 [M + H]⁺. Calcd for C₂₁H₂₉N₅OF: 386.2356; found 386.2351.

5-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)furan-2-carboxamide (8e)

Similarly, 6b (0.13 g, 0.40 mmol), 5-fluorofuran-2-carboxylic acid (7a, 0.06 g, 0.48 mmol), HATU (0.19 g, 0.48 mmol), and DIPEA (0.14 mL, 0.79 mmol) in DMF (2 mL) gave 8e as a light brown solid (62%). Mp: 126–128 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.14 (s, 1H), 8.28 (d, J = 8.9 Hz, 1H), 7.08 (t, J = 3.5 Hz, 1H), 6.75 (d, J = 2.7 Hz, 1H), 6.68 (dd, J = 8.9, 2.7 Hz, 1H), 5.63 (dd, J = 7.2, 3.6 Hz, 1H), 3.15 (t, J = 5.0 Hz, 4H), 2.96 (dt, J = 12.2, 3.2 Hz, 2H), 2.67 (td, J = 11.5, 2.3 Hz, 2H), 2.55 (t, J = 5.0 Hz, 4H), 2.32 (s, 3H), 1.81–1.72 (m, 2H), 1.58–1.48 (m, 1H), 1.42 (qd, J = 11.6, 3.7 Hz, 2H), 1.01 (d, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.22 (d, J = 284.0 Hz), 154.53, 148.13, 143.55, 138.84 (d, J = 2.2 Hz), 125.84, 119.94, 116.24, 112.26, 109.27, 85.11 (d, J = 12.8 Hz), 55.19, 53.09, 49.70, 46.17, 35.30, 30.59, 22.05. ¹⁹F NMR (376 MHz, CDCl₃) δ −110.09. HRMS (ESI) m/z: 401.2 [M + H]⁺. Calcd for C₂₂H₃₀N₄O₂F: 401.2353; found 401.2346.

5-Fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide (8f)

Similarly, 6a (0.20 g, 0.71 mmol), 5-fluorofuran-2-carboxylic acid (7a, 0.12 g, 0.85 mmol), HATU (0.33 g, 0.86 mmol), and DIPEA (0.25 mL, 1.43 mmol) in DMF (4 mL) gave 8f as a black solid (36%). Mp: 145.5–147 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.11 (t, J = 3.5 Hz, 1H), 6.77 (d, J = 2.7 Hz, 1H), 6.70 (dd, J = 8.9, 2.7 Hz, 1H), 5.65 (dd, J = 7.2, 3.6 Hz, 1H), 3.20–3.13 (m, 4H), 2.83 (t, J = 5.2 Hz, 4H), 2.61–2.54 (m, 4H), 2.35 (s, 3H), 1.77 (p, J = 5.7 Hz, 4H), 1.62 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 157.33 (d, J = 283.9 Hz), 154.63 (d, J = 8.4 Hz), 148.24, 143.91 (d, J = 2.9 Hz), 138.88, 125.86 (d, J = 9.5 Hz), 120.02, 116.38, 112.37, 109.36, 85.20 (d, J = 12.5 Hz), 55.29, 53.88, 49.81, 46.28, 26.99, 24.30. ¹⁹F NMR (376 MHz, CDCl₃) δ −109.87. HRMS (ESI) m/z: 387.2 [M + H]⁺. Calcd for C₂₁H₂₈N₄O₂F: 387.2196; found 387.2191.

5-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)furan-2-carboxamide (8g)

Similarly, 6b (0.2 g, 0.62 mmol), 5-cyanofuran-2-carboxylic acid (7b, 0.11 g, 0.75 mmol), HATU (0.29 g, 0.75 mmol), and DIPEA (0.22 mL, 1.25 mmol) in DMF (3 mL) gave 8g as a yellow solid (46%). Mp: 151–153 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.55 (s, 1H), 8.25 (d, J = 8.8 Hz, 1H), 7.18 (q, J = 3.8 Hz, 2H), 6.76 (d, J = 2.7 Hz, 1H), 6.68 (dd, J = 9.0, 2.6 Hz, 1H), 3.15 (t, J = 4.8 Hz, 4H), 2.95 (d, J = 11.4 Hz, 2H), 2.69 (t, J = 11.4 Hz, 2H), 2.54 (t, J = 4.9 Hz, 4H), 2.31 (s, 3H), 1.79 (d, J = 12.2 Hz, 2H), 1.58–1.37 (m, 3H), 1.03 (d, J = 6.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 153.08, 152.29, 148.56, 143.78, 126.44, 125.17, 123.47, 119.85, 114.43, 112.13, 110.55, 109.19, 55.08, 53.13, 49.41, 46.12, 35.36, 30.53, 22.01. HRMS (ESI) m/z: 408.2 [M + H]⁺. Calcd for C₂₃H₃₀N₅O₂: 408.2400; found 408.2393.

5-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide (CPPC)

Similarly, 6a (2.84 g, 10.14 mmol), 5-cyanofuran-2-carboxylic acid (7b, 1.78 g, 12.17 mmol), HATU (4.68 g, 12.17 mmol), and DIPEA (3.6 mL, 20.29 mmol) in DMF (51 mL) gave CPPC as a yellow solid (72%). Mp: 164.8–166.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.25–7.18 (m, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 9.1, 2.7 Hz, 1H), 3.19 (t, J = 4.8 Hz, 4H), 2.84 (t, J = 5.1 Hz, 4H), 2.58 (t, J = 4.8 Hz, 4H), 2.36 (s, 3H), 1.80 (p, J = 5.5 Hz, 4H), 1.65 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.20, 152.37, 148.63, 144.13, 126.50, 125.12, 123.59, 120.04, 114.58, 112.16, 110.63, 109.20, 55.13, 53.88, 49.46, 46.16, 26.96, 24.11. HRMS (ESI) m/z: 394.2 [M + H]⁺. Calcd for C₂₂H₂₈N₅O₂: 394.2243; found 394.2239.

5-Cyano-N-(2-(4-(fluoromethyl)piperidin-1-yl)-4-(4-methylpiperazin-1-yl)phenyl)furan-2-carboxamide (Psa374)

Similarly, 6c, (2.43 g, 7.54 mmol), 5-cyanofuran-2-carboxylic acid (7b, 1.31 g, 9.05 mmol), HATU (3.48 g, 9.05 mmol), and DIPEA (2.6 mL, 15.08 mmol) in DMF (38 mL) gave Psa374 as a yellow solid (87%). Mp: 170.9–173 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 8.29 (d, J = 8.9 Hz, 1H), 7.23 (d, J = 3.7 Hz, 1H), 7.20 (d, J = 3.8 Hz, 1H), 6.79 (d, J = 2.6 Hz, 1H), 6.73 (dd, J = 8.9, 2.7 Hz, 1H), 4.39 (dd, J = 47.2, 5.8 Hz, 2H), 3.18 (t, J = 5.0 Hz, 4H), 3.06 (d, J = 11.4 Hz, 2H), 2.77 (td, J = 11.9, 2.2 Hz, 2H), 2.57 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H), 1.99–1.85 (m, 3H), 1.57 (qd, J = 13.1, 3.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.12, 152.27, 148.65, 143.40, 126.51, 125.11, 123.46, 120.05, 114.55, 112.49, 110.54, 109.21, 87.61 (d, J = 169.1 Hz), 55.11, 52.48, 49.45, 46.16, 36.41 (d, J = 18.7 Hz), 28.91 (d, J = 6.2 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −222.58. HRMS (ESI) m/z: 426.2 [M + H]⁺. Calcd for C₂₃H₂₉N₅O₂F: 426.2305; found 426.2303.

Syntheses of Precursors for Radiolabeling

tert-Butyl 4-(4-nitro-3-(piperidin-1-yl)phenyl)piperazine-1-carboxylate (9a)

tert-Butyl piperazine-1-carboxylate (2 g, 10.59 mmol) and K₂CO₃ (2.2 g, 15.88 mmol) were added to a suspension of 3a (1.3 g, 5.29 mmol) in DMSO (18 mL). The reaction mixture was stirred at 130 °C for 16 h. After cooling the reaction mixture to rt, H₂O was added, and the organic phase was extracted portion-wise (to avoid emulsion) with EtOAc. The organic phases were combined, washed with H₂O and brine, dried over Na₂SO₄, and evaporated in vacuo to dryness. The crude product was purified with automated reverse-phase Combiflash chromatography (H₂O: MeCN), giving 9a as a yellow solid (35%). Mp: 162.3–165 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 9.3 Hz, 1H), 6.38 (dd, J = 9.3, 2.6 Hz, 1H), 6.31 (d, J = 2.6 Hz, 1H), 3.58 (t, J = 5.0 Hz, 4H), 3.34 (t, J = 5.3 Hz, 4H), 3.02 (t, J = 5.2 Hz, 4H), 1.76 (p, J = 5.5 Hz, 4H), 1.65–1.58 (m, 2H), 1.49 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.90, 154.70, 150.59, 132.64, 129.75, 106.58, 103.98, 80.40, 53.32, 47.30, 43.15, 28.52, 26.05, 24.31. HRMS (ESI) m/z: 391.3 [M + H]⁺. Calcd for C₂₀H₃₁N₄O₄: 391.2345; found 391.2349.

tert-Butyl 4-(3-(4-methylpiperidin-1-yl)-4-nitrophenyl)piperazine-1-carboxylate (9b)

Similarly, 3b (2 g, 7.46 mmol), tert-butyl piperazine-1-carboxylate (2.8 g, 14.92 mmol), and K₂CO₃ (3.1 g, 22.38 mmol) in DMSO (25 mL) gave 9b as a yellow solid (60%). Mp: 117.5–119 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 9.3 Hz, 1H), 6.37 (dd, J = 9.3, 2.6 Hz, 1H), 6.31 (d, J = 2.6 Hz, 1H), 3.58 (t, J = 5.3 Hz, 4H), 3.34 (t, J = 5.3 Hz, 4H), 3.28 (d, J = 12.2 Hz, 2H), 2.78 (t, J = 11.6 Hz, 2H), 1.74–1.66 (m, 2H), 1.53–1.43 (m, 12H), 1.00 (d, J = 5.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.84, 154.66, 150.32, 132.49, 129.71, 106.50, 103.91, 80.34, 52.54, 47.21, 43.04, 34.25, 30.77, 28.47, 21.90. HRMS (ESI) m/z: 405.2 [M + H]⁺. Calcd for C₂₁H₃₃N₄O₄: 405.2502; found 405.2498.

tert-Butyl 4-(3-(4-(fluoromethyl)piperidin-1-yl)-4-nitrophenyl)piperazine-1-carboxylate (9c)

Similarly, 3c (0.8 g, 2.79 mmol), tert-butyl piperazine-1-carboxylate (1.1 g, 5.57 mmol), and K₂CO₃ (1.2 g, 8.36 mmol) in DMSO (14 mL) gave 9c as a yellow solid (59%). Mp: 132.5–133 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 9.3 Hz, 1H), 6.41 (dd, J = 9.4, 2.6 Hz, 1H), 6.32 (d, J = 2.6 Hz, 1H), 4.34 (dd, J = 47.4, 6.2 Hz, 2H), 3.59 (t, J = 5.3 Hz, 4H), 3.35 (t, J = 5.6 Hz, 6H), 2.81 (td, J = 11.8, 2.0 Hz, 2H), 1.95–1.85 (m, 1H), 1.82 (d, J = 13.4 Hz, 2H), 1.61 (td, J = 12.2, 3.9 Hz, 2H), 1.49 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.80, 154.60, 150.11, 132.58, 129.67, 106.83, 104.00, 87.61 (d, J = 168.7 Hz), 80.34, 51.93, 47.10, 43.30, 36.62 (d, J = 18.7 Hz), 28.40, 27.85 (d, J = 6.2 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −222.69.HRMS (ESI) m/z: 423.2 [M + H]⁺. Calcd for C₂₁H₃₂N₄O₄F: 423.2408; found 423.2404.

tert-Butyl 4-(4-amino-3-(piperidin-1-yl)phenyl)piperazine-1-carboxylate (10a)

The synthesis was performed as for 6a. Thus, 9a (0.64 g, 1.61 mmol), 10% Pd/C (10%, 0.171 g, 0.16 mmol), and H₂ (70 psi) in MeOH (5 mL) gave 10a as a gray solid (79%). Mp: 96.5–97.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.71–6.63 (m, 2H), 6.55 (dd, J = 8.4, 2.6 Hz, 1H), 3.74 (bs, 2H), 3.57 (t, J = 5.1 Hz, 4H), 2.96 (t, J = 5.1 Hz, 4H), 2.83 (t, J = 4.4 Hz, 4H), 1.70 (p, J = 5.6 Hz, 4H), 1.61–1.56 (m, 2H), 1.48 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.83, 144.63, 141.60, 136.08, 115.46, 113.52, 111.20, 79.78, 52.71, 51.59, 43.70, 28.53, 26.93, 24.46. HRMS (ESI) m/z: 361.3 [M + H]⁺. Calcd for C₂₀H₃₃N₄O₂: 361.2604; found 361.2607.

tert-Butyl 4-(4-amino-3-(4-methylpiperidin-1-yl)phenyl)piperazine-1-carboxylate (10b)

Similarly, 9b (1.6 g, 3.76 mmol), 10% Pd/C (0.40 g, 0.38 mmol) and H₂ (70 psi) in MeOH (12 mL) gave 10b as a white solid (83%). Mp: 106–106.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.71–6.63 (m, 2H), 6.54 (dd, J = 8.5, 2.5 Hz, 1H), 3.83–3.65 (m, 2H), 3.56 (t, J = 5.1 Hz, 4H), 3.16–3.06 (m, 2H), 2.96 (t, J = 5.1 Hz, 4H), 2.57 (td, J = 11.6, 2.3 Hz, 2H), 1.73 (dd, J = 12.7, 3.5 Hz, 2H), 1.52–1.44 (m, 10H), 1.34 (qd, J = 11.7, 3.8 Hz, 2H), 0.99 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.72, 144.51, 141.19, 136.02, 115.37, 113.45, 111.10, 79.66, 51.91, 51.52, 44.27, 35.24, 30.78, 28.45, 22.08. HRMS (ESI) m/z: 375.3 [M + H]⁺. Calcd for C₂₁H₃₅N₄O₂: 375.2760; found 375.2759.

tert-Butyl 4-(4-amino-3-(4-(fluoromethyl)piperidin-1-yl)phenyl)piperazine-1-carboxylate (10c)

Similarly, 9c (0.66 g, 1.48 mmol), 10% Pd/C (0.16 g, 0.15 mmol), and H₂ (70 psi) in MeOH (5 mL) gave 10c as a white solid (91%). Mp: 104–105 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.71–6.64 (m, 2H), 6.56 (dd, J = 8.4, 2.6 Hz, 1H), 4.34 (dd, J = 47.4, 5.6 Hz, 2H), 3.73 (s, 2H), 3.57 (t, J = 5.1 Hz, 4H), 3.19 (d, J = 11.5 Hz, 2H), 2.96 (t, J = 5.1 Hz, 4H), 2.66–2.56 (m, 2H), 1.83 (d, J = 12.1 Hz, 3H), 1.60–1.55 (m, 2H), 1.48 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.85, 144.70, 140.91, 136.05, 115.62, 113.86, 111.22, 87.94 (d, J = 168.4 Hz), 79.85, 51.60, 51.37, 44.23, 36.79 (d, J = 18.3 Hz), 28.79 (d, J = 5.9 Hz), 28.54. ¹⁹F NMR (376 MHz, CDCl₃) δ −223.19.HRMS (ESI) m/z: 393.3 [M + H]⁺. Calcd for C₂₁H₃₄N₄O₂F: 393.2666; found 393.2661.

tert-Butyl 4-(4-(5-cyanofuran-2-carboxamido)-3-(piperidin-1-yl)phenyl)piperazine-1-carboxylate (11a)

The synthesis was performed as for 8a. Thus, 10a (0.38 g, 1.01 mmol), 5-cyanofuran-2-carboxylic acid (7b, 0.17 g, 1.21 mmol), HATU (0.47 g, 1.21 mmol), and DIPEA (0.35 mL, 2.02 mmol) in DMF (4 mL) gave 11a as a yellow solid (90%). Mp: 205–207 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 8.32 (d, J = 8.9 Hz, 1H), 7.25–7.20 (m, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 8.9, 2.7 Hz, 1H), 3.59 (t, J = 5.1 Hz, 4H), 3.11 (t, J = 5.1 Hz, 4H), 2.84 (t, J = 5.3 Hz, 4H), 1.81 (p, J = 5.7 Hz, 4H), 1.69–1.61 (m, 2H), 1.49 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.68, 153.26, 152.27, 148.62, 144.17, 126.53, 125.64, 123.61, 120.05, 114.66, 112.82, 110.60, 109.87, 79.92, 53.87, 49.83, 43.45, 28.43, 26.94, 24.09. HRMS (ESI) m/z: 480.3 [M + H]⁺. Calcd for C₂₆H₃₄N₅O₄: 480.2611; found 480.2615.

tert-Butyl 4-(4-(5-cyanofuran-2-carboxamido)-3-(4-(fluoromethyl)piperidin-1-yl)phenyl)piperazine-1-carboxylate (11b)

Similarly, 10c (0.49 g, 1.20 mmol), 5-cyanofuran-2-carboxylic acid (7b, 0.21 g, 1.44 mmol), HATU (0.55 g, 1.44 mmol), and DIPEA (0.4 mL, 2.40 mmol) in DMF (6 mL) gave 11b as a yellow solid (81%). Mp: 243–245 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.54 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.23 (dd, J = 17.5, 3.8 Hz, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.74 (dd, J = 8.9, 2.7 Hz, 1H), 4.40 (dd, J = 47.2, 5.8 Hz, 2H), 3.59 (t, J = 5.1 Hz, 4H), 3.14–3.03 (m, 6H), 2.83–2.72 (m, 2H), 1.95 (d, J = 12.3 Hz, 3H), 1.67–1.57 (m, 2H), 1.49 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 154.74, 153.25, 152.23, 148.71, 143.51, 126.61, 125.71, 123.54, 120.12, 114.70, 113.22, 110.58, 109.95, 87.63 (d, J = 169.1 Hz), 80.01, 52.54, 49.88, 43.42, 36.45 (d, J = 19.1 Hz), 28.95 (d, J = 5.9 Hz), 28.49. ¹⁹F NMR (376 MHz, CDCl₃) δ −222.66. HRMS (ESI) m/z: 512.3 [M + H]⁺. Calcd for C₂₇H₃₅N₅O₄F: 512.2668; found 512.2673.

tert-Butyl 4-(4-(5-fluorofuran-2-carboxamido)-3-(4-methylpiperidin-1-yl)phenyl)piperazine-1-carboxylate (11c)

Similarly, 10b 0.3 g, 0.79 mmol), 5-fluorofuran-2-carboxylic acid (7a, 0.13 g, 0.94 mmol), HATU (0.36 g, 094 mmol), and DIPEA (0.27 mL, 1.57 mmol) in DMF (3 mL) gave 11c as a white solid (83%). Mp: 171–172.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.17 (s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.12 (t, J = 3.5 Hz, 1H), 6.77 (d, J = 2.7 Hz, 1H), 6.70 (dd, J = 8.9, 2.7 Hz, 1H), 5.66 (dd, J = 7.1, 3.6 Hz, 1H), 3.58 (t, J = 5.1 Hz, 4H), 3.08 (t, J = 5.1 Hz, 4H), 3.04–2.94 (m, 2H), 2.69 (td, J = 11.5, 2.3 Hz, 2H), 1.80 (dd, J = 12.6, 2.4 Hz, 2H), 1.56–1.51 (m, 1H), 1.48 (s, 9H), 1.43 (td, J = 11.7, 3.7 Hz, 2H), 1.04 (d, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.21 (d, J = 283.9 Hz), 154.68, 154.54, 148.10, 143.57, 138.74, 126.34, 119.93, 116.32, 112.91, 109.91, 85.13 (d, J = 12.5 Hz), 79.83, 53.06, 50.07, 43.54, 35.24, 30.54, 28.43, 22.01. ¹⁹F NMR (376 MHz, CDCl₃) δ −109.94. HRMS (ESI) m/z: 487.3 [M + H]⁺. Calcd for C₂₆H₃₆N₄O₄F: 487.2721; found 487.2721.

tert-Butyl 4-(4-(5-cyanofuran-2-carboxamido)-3-(4-methylpiperidin-1-yl)phenyl)piperazine-1-carboxylate (11d)

Similarly, 10b (0.36 g, 0.95 mmol), 5-cyanofuran-2-carboxylic acid (7b, 0.16 g, 1.14 mmol), HATU (0.44 g, 1.14 mmol), and DIPEA (0.33 mL, 1.91 mmol) in DMF (3 mL) gave 11d as a yellow solid (94%). Mp: 235–235.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.59 (s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.22 (dd, J = 16.1, 4.0 Hz, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 8.8, 2.7 Hz, 1H), 3.59 (t, J = 5.2 Hz, 4H), 3.10 (t, J = 5.1 Hz, 4H), 3.03–2.95 (m, 2H), 2.72 (t, J = 11.1 Hz, 2H), 1.88–1.80 (m, 2H), 1.54–1.42 (m, 12H), 1.07 (d, J = 6.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.78, 153.31, 152.34, 148.69, 143.97, 126.63, 125.83, 123.56, 120.02, 114.64, 112.95, 110.62, 110.01, 80.03, 53.26, 49.95, 43.67, 35.44, 30.64, 28.52, 22.08. HRMS (ESI) m/z: 494.3 [M + H]⁺. Calcd for C₂₇H₃₆N₅O₄: 494.2767; found 494.2762.

5-Cyano-N-(4-(piperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide (12a)

TFA (1.8 mL, 22.68 mmol) was added dropwise over 5 min to a stirred solution of 11a (0.37 g, 0.76 mmol) in DCM (1.5 mL). The reaction mixture was then stirred for 90 min until complete. Most of the TFA was removed azeotropically with DCM. The residue was diluted with DCM and washed with saturated aq. NaHCO_3. Removal of DCM gave 12a as a yellow solid (85%). Mp: 153–155 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.53 (s, 1H), 8.32 (d, J = 8.9 Hz, 1H), 7.23 (dd, J = 15.0, 3.8 Hz, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 8.9, 2.7 Hz, 1H), 3.18–3.11 (m, 4H), 3.11–3.03 (m, 4H), 2.84 (t, J = 5.2 Hz, 4H), 1.98 (bs, 1H), 1.80 (p, J = 5.7 Hz, 4H), 1.65 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.32, 152.47, 149.25, 144.23, 126.61, 125.32, 123.69, 120.13, 114.69, 112.39, 110.73, 109.45, 53.99, 50.83, 46.21, 27.07, 24.21. HRMS (ESI) m/z: 380.1 [M + H]⁺. Calcd for C₂₁H₂₆N₅O₂: 380.2087; found 380.2093.

5-Cyano-N-(2-(4-(fluoromethyl)piperidin-1-yl)-4-(piperazin-1-yl)phenyl)furan-2-carboxamide (12b)

Similarly, 11b (0.2 g, 0.38 mmol) and TFA (0.9 mL, 11.49 mmol) in DCM (2 mL) gave 12b as a yellow solid (91%). Mp: 171.3–173 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.54 (s, 1H), 8.30 (d, J = 8.9 Hz, 1H), 7.25–7.19 (m, 2H), 6.83–6.69 (m, 2H), 4.39 (dd, J = 47.2, 5.7 Hz, 2H), 3.25–3.13 (m, 4H), 3.13–3.01 (m, 7H), 2.77 (td, J = 11.9, 2.2 Hz, 2H), 2.01–1.84 (m, 3H), 1.59 (td, J = 12.2, 3.9 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.24, 152.36, 149.31, 143.51, 126.62, 125.27, 123.56, 120.13, 114.65, 112.67, 110.64, 109.42, 87.71 (d, J = 168.4 Hz), 52.59, 50.93 (d, J = 11.7 Hz), 46.25, 36.51 (d, J = 18.7 Hz), 29.01 (d, J = 6.2 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −222.60. HRMS (ESI) m/z: 412.2 [M + H]⁺. Calcd for C₂₂H₂₇N₅O₂F: 412.2149; found 412.2157.

5-Fluoro-N-(2-(4-methylpiperidin-1-yl)-4-(piperazin-1-yl)phenyl)furan-2-carboxamide (12c)

Similarly, 11c (0.15 g, 0.30 mmol) and TFA (0.7 mL, 9.06 mmol) in DCM (2 mL) gave 12c as a gray solid (86%). Mp: decomposition (>160 °C). ¹H NMR (400 MHz, CDCl₃) δ 9.18 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.11 (t, J = 3.5 Hz, 1H), 6.78 (d, J = 2.7 Hz, 1H), 6.71 (dd, J = 8.9, 2.7 Hz, 1H), 5.66 (dd, J = 7.1, 3.6 Hz, 1H), 3.14–3.08 (m, 4H), 3.07–3.02 (m, 4H), 2.99 (d, J = 11.7 Hz, 2H), 2.70 (td, J = 11.7, 2.3 Hz, 2H), 1.80 (d, J = 12.1 Hz, 2H), 1.59–1.37 (m, 4H), 1.04 (d, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.32 (d, J = 283.9 Hz), 154.66, 148.79, 143.64, 138.92, 126.00, 120.01, 116.34, 112.44, 109.50, 85.20 (d, J = 12.8 Hz), 53.18, 51.18, 46.31, 35.37, 30.69, 22.13. ¹⁹F NMR (376 MHz, CDCl₃) δ −110.04. HRMS (ESI) m/z: 387.2 [M + H]⁺. Calcd for C₂₁H₂₈N₄O₂F: 387.2196; found 387.2198.

5-Cyano-N-(2-(4-methylpiperidin-1-yl)-4-(piperazin-1-yl)phenyl)furan-2-carboxamide (12d)

Similarly, 11d (0.4 g, 0.79 mmol) and TFA (1.8 mL, 23.82 mmol) in DCM (2 mL) gave 12d as a yellow solid (88%). Mp: 155–157.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.60 (s, 1H), 8.30 (d, J = 8.9 Hz, 1H), 7.21 (s, 2H), 6.79 (d, J = 2.7 Hz, 1H), 6.72 (dd, J = 8.9, 2.7 Hz, 1H), 3.12 (dd, J = 6.4, 3.3 Hz, 4H), 3.04 (dd, J = 6.3, 3.3 Hz, 4H), 2.99 (d, J = 11.4 Hz, 2H), 2.73 (t, J = 11.2 Hz, 2H), 1.84 (d, J = 12.6 Hz, 2H), 1.55–1.41 (m, 4H), 1.07 (d, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 153.28, 152.48, 149.30, 143.96, 126.62, 125.39, 123.58, 120.02, 114.58, 112.40, 110.69, 109.50, 53.31, 50.97, 46.31, 35.51, 30.70, 22.13. HRMS (ESI) m/z: 394.2 [M + H]⁺. Calcd for C₂₂H₂₈N₅O₂: 394.2243; found 394.2242.

Computation and Measurement of LogD_7.4

cLogP and clogD_7.4 values were computed with ChemDraw software (version 20.1; PerkinElmer; Shelton, CT). LogD_7.4 was measured on [¹¹C]Psa374 by partition between n-octanol and the sodium phosphate buffer (pH 7.4) at rt, as described previously.^47,48

Determination of Tyrosine Kinase Inhibitory Potency

The half-maximal inhibitory concentrations of the compounds 8c–8g, Psa374, and CPPC for CSF1R and the 12 related tyrosine kinases were measured by Thermofisher Scientific (Waltham, MA) following the Z′-LYTE binding assay screening protocol.⁴⁹

Evaluation of Human P-Glycoprotein–Substrate Behavior

The interaction of Psa374 with human P-gp and BCRP transporters was assessed with a bidirectional assay (Cat. ref. G206 and G330) performed by Eurofins Panlabs (St. Charles, MO).

Radiosyntheses of ¹¹C-Labeled CSF1R Radioligands

No-carrier-added [¹¹C]carbon dioxide was prepared by the ¹⁴N(p,α)¹¹C nuclear reaction by irradiation of nitrogen (164 psi) containing oxygen (1%) for 20 min with a proton beam (16.5 MeV, 45 μA) generated from a cyclotron (PETtrace 200; GE Healthcare, Milwaukee, WI). The [¹¹C]carbon dioxide (∼74 GBq) was converted into [¹¹C]iodomethane by reduction to [¹¹C]methane, followed by a gas-phase reaction with molecular iodine in an automated apparatus (PETrace MeI MicroLab; GE).⁵⁰

A crimp-sealed pulled-point vial (1.1 mL; Thermo Scientific) containing a solution of a precursor (selected from 12a–12d; 0.7–1.0 mg, 1.9–2.5 μmol) in anhydrous DMF (400 μL) was placed in a [¹¹C]iodomethane trapping station. [¹¹C]Iodomethane was bubbled into the reaction mixture at rt, and then the mixture was heated at 80 °C for 5 min. The reaction was quenched with H₂O (500 μL) and loaded into the HPLC injection loop. The radioligand product was isolated with HPLC on an XBridge BEH C18 OBD column (5 μm; 10 × 250 mm; Waters) eluted at 7 mL/min with aq. TEA/formic acid (8:2, 1% v/v): MeCN, (55:45 v/v for [¹¹C]Psa374; t_R 13.3 ± 1.4 min, 50:50 v/v for [¹¹C]CPPC, t_R 10.7 ± 0.4 min, and 45:55 v/v for [¹¹C]8g, t_R 11.7 ± 0.1, and [¹¹C]8e, t_R 15.1 ± 0.2 min). The eluate was monitored for absorbance at 254 nm and radioactivity. The mobile phase containing the ¹¹C-labeled products was collected into a rotary evaporator flask preloaded with aqueous ascorbic acid (10 mg/mL; 100 μL) and then evaporated under vacuum at 80 °C for 1 min. The dried products were then reconstituted in saline (10 mL) containing EtOH (9% v/v) and filtered through a sterile filter (25 mm Millex-LG, 0.20 μm; Millipore) for analysis and intravenous injection into the animals.

Determination of Radiochemical Purity and Molar Radioactivity

Samples of formulated [¹¹C]Psa374, [¹¹C]8g, [¹¹C]8e, and [¹¹C]CPPC of known radioactivity (∼6 MBq), were analyzed with HPLC on an XBridge C18 column (5 μm; 4.6 × 250 mm; Waters) eluted with aq. TEA/formic acid (8:2, 1% v/v):MeCN, (40:60 v/v) at 1.5 mL/min. The eluate was monitored for absorbance at 254 nm and radioactivity. The mass of carriers Psa374, 8g, 8e, and CPPC in the injectates was determined from precalibrated mass response curves obtained under identical HPLC conditions. Molar activities (GBq/μmol) are reported as the radioactivity of the [¹¹C]product in the injected sample (GBq), divided by the mass of the corresponding carrier (μmol) decay-corrected to end of radionuclide production.

Determination of the Radiochemical Stability of Formulated ¹¹C-Labeled CSF1R Radioligands

The radiochemical purity of formulated [¹¹C]Psa374, [¹¹C]8g, [¹¹C]8e, and [¹¹C]CPPC kept at rt for 1 h was analyzed with radio-HPLC. All the radioligands maintained greater than 97.2% radiochemical purity.

PET Imaging in Mouse

Imaging experiments were performed on a microPET Focus 120 camera (Siemens Medical Solution; Knoxville, TN). P-gp-1a/b and BCRP knockout mice (FVB.129P2-Abcb1a^tm1Bor Abcb1b^tm1BorAbcg2^tm1Ahs; 37.3 ± 1.6 g; n = 6) and wild-type FVB mice (39.9 ± 1.3 g; n = 6) were anesthetized with 1.5% isoflurane in oxygen at a flow rate of 1 L/min. Formulated radioligand (∼150 μL) was injected intravenously through the tail vein of each mouse. At baseline, the administered [¹¹C]CPPC doses (n = 6) were 5.8 ± 1.1 MBq with an A_m value of 101 GBq/μmol, corresponding to 0.0015 ± 0.0003 nmol carrier per g weight. For [¹¹C]Psa374, the administered doses (n = 6) were 13.7 ± 2.8 MBq with an A_m value of 213 GBq/μmol, corresponding to 0.0017 ± 0.0003 nmol per g weight. Similar radioligand doses and molar activities were used in preblocking experiments. Preblock scans were imaged after intravenous administration of 4 mg/kg of Psa374 or CPPC at 10 min before the radioligand. All the scans were acquired for 100 min. Data were histogrammed into 23 time frames (6 × 20, 5 × 60, 4 × 120, 3 × 300, 3 × 600 and 2 × 1200 s) and reconstructed using Fourier rebinning plus a two-dimensional filtered back-projection. No scatter, or attenuation corrections were applied. PET images were analyzed using PMOD with a single region-of-interest of the whole brain.

PET Imaging in Monkeys

Six healthy rhesus monkeys (body weight, 9.3 ± 0.7 kg) were initially immobilized with ketamine hydrochloride (10 mg/kg, i.m.). Anesthesia was maintained with 1–3% isoflurane and 98% oxygen. Body temperature was maintained between 37.0 and 37.5 °C. Furthermore, electrocardiogram, body temperature, heart rate, and respiratory rate measures were monitored throughout the scan.

Brain PET imaging was performed using a microPET Focus 220 camera (Siemens Medical Solution, Knoxville, TN). Following a transmission scan using a ⁵⁷Co point source, 120 min baseline and 90 min preblock dynamic PET scans were acquired after the i.v. injection of [¹¹C]8e, [¹¹C]8g, [¹¹C]CPPC, or [¹¹C]Psa374. At baseline, the administered [¹¹C]CPPC doses (n = 3) were 266 ± 21 MBq with A_m values of 129 ± 32 GBq/μmol, corresponding to 0.25 ± 0.10 nmol carrier per kg weight. For [¹¹C]Psa374, the administered doses (n = 3) were 242 ± 10 MBq with A_m values of 137 ± 19 GBq/μmol, corresponding to 0.18 ± 0.02 nmol per kg weight. Similar radioligand doses and molar activities were used for [¹¹C]8e, [¹¹C]8g, and all the preblocking experiments. 20 mL of the vehicle to be used for the pharmacological agent in the following PET experiment, to be done on the same day, was injected intravenously 10 min before radioligand administration. For the second PET experiment, CPPC (1 or 4 mg/kg), Psa374 (4 mg/kg), or elacridar (3 mg/kg) was dissolved in the vehicle and then injected intravenously 10 min before the radioligand. PET images were reconstructed using Fourier rebinning plus a two-dimensional filtered back-projection with scatter and attenuation correction.

Emergence of Radiometabolites in Rat Plasma and Stability of Radioligand Ex Vivo and In Vitro

Arterial blood was sampled from the rat at set time-points (30 and 120 min) after radioligand injection and stabilized with sodium fluoride. The percentage of radioactivity in each plasma sample that was represented by parent radioligand was measured with radio-HPLC on an X-Terra C18 column (10 μm, 7.8 × 300 mm; Waters Corp.) eluted isocratically with MeOH: H₂O: Et₃N (75:25:0.1 by vol) at 5.0 mL/min. The same HPLC method was used to assess the stability of [¹¹C]Psa374 in brain tissue and plasma in vitro.

Emergence of Radiometabolites in Monkey Plasma

Arterial blood was sampled during all monkey scans to determine the time course of the plasma concentration of the parent radioligand. During a PET scan, 15 blood samples were drawn from an implanted port in the femoral artery and stabilized with sodium fluoride at 15-s intervals for the first 2 min, followed by samples at 3, 5, 10, 30, 60, 90, and 120 min (volumes varying from 1.0 to 5.0 mL). The percentage of radioactivity in the plasma of each sample represented by parent radioligand was then measured with HPLC, as for rat plasma, and plotted against time after injection. Furthermore, the total plasma radioactivity was corrected for the radiometabolites by its mathematical product with the parent plasma fraction determined by radio-HPLC.

Analysis of Plasma Free Fractions

Radioligand plasma free fractions (f_P) in rat and monkey plasma were measured by ultrafiltration, as previously described.^51,52

Analysis of Monkey PET Scans

PET images were coregistered to a standardized monkey MRI template using PMOD software (version 4.1, PMOD Technologies Ltd.; Zurich, Switzerland). Thirty-five predefined regions of interest from a monkey brain template were applied to the coregistered PET images to obtain regional time–activity curves. Brain uptake was expressed as a SUV, which normalizes for injected radioactivity and body weight. Using the brain time–activity curves and the radiometabolite-corrected arterial input function, the total V_T was calculated with a two-tissue compartment model (2TCM). The Lassen plot was used to determine target occupancy by blocker and non-displaceable distribution volume (V_ND), which was used to calculate binding potential (BP_ND). Logan plots were used to create images of brain V_T and V_T/f_P.

To determine the minimum scan duration needed to reliably measure V_T as well as to indirectly assess whether radiometabolites accumulate in the brain, the time stability of V_T was examined by truncating the scan duration from 120 min down to 20 min in 20 min increments.

Glossary

Abbreviations

ABC

ATP-binding cassette

A_m

molar activity

BBB

blood–brain barrier

BCRP

breast cancer resistance protein

BP_ND

binding potential

CNS

central nervous system

CPPC

5-cyano-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide

CSF1R

colony-stimulating factor-1 receptor

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DMF

N,N-dimethylformamide

DMSO

dimethyl sulfoxide

ER

efflux ratio

EtOH

ethanol alcohol

FGFR1

fibroblast growth factor receptor 1

FGFR2

fibroblast growth factor receptor 2

FGFR3

fibroblast growth factor receptor 3

FGFR4

fibroblast growth factor receptor 4

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectra

i.m.

intramuscular

i.v.

intravenous

KDR

kinase insert domain receptor

KIT

KIT proto-oncogene

KO

knockout

LPS

lipopolysaccharide

MeI

methyl iodide

MRP

multidrug resistance protein

NIDDK

National Institute of Diabetes and Digestive and Kidney Diseases

NMR

nuclear magnetic resonance

PDGFRα

platelet-derived growth factor receptor alpha

PDGFRβ

platelet-derived growth factor receptor beta

PET

positron emission tomography

P-gp

P-glycoprotein

PMOD

peripheral module interface

RET

RET proto-oncogene

rt

room temperature

SD

standard deviation

SUV

standardized uptake value

TAC

time–activity curve

TEA

triethylamine

TFA

trifluoroacetic acid

TLC

thin-layer chromatography

tPSA

topological polar surface area

UV

ultraviolet

V_ND

non-displaceable volume of distribution

V_T

total volume of distribution

WT

wild-type

2TCM

two-tissue compartmental model

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00003.

• Compound structures, Smiles, and characterization; biological data; quantitative PET imaging results (PDF)

Author Contributions

S.A. contributed to project design and performed synthesis and radiochemistry. C.L.M. performed radiochemistry. X.Y. and J.-S.L. performed PET studies and their analyses. S.S.Z., J.A.M.S., and M.D.J. performed radiometabolite analyses and other in vitro analyses. R.B.I. and V.W.P. contributed to project design and provided overall supervision. The manuscript was written through contributions of all authors.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue “Diagnostic and Therapeutic Radiopharmaceuticals”.