Synthesis of fluorine-containing phosphodiesterase 10A (PDE10A) inhibitors and the in vivo evaluation of F-18 labeled PDE10A PET tracers in rodent and nonhuman primate

Article abstract of DOI:10.1021/acs.jmedchem.5b01205

A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [¹¹C]MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: 18a-j, 19d-j, 20a-b, and 21b had IC₅₀ values <5 nM for PDE10A. Seven F-18 labeled compounds [¹⁸F]18a-e, [¹⁸F]18g, and [¹⁸F]20a were radiosynthesized by ¹⁸F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ～2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [¹⁸F]18a-d and [¹⁸F]20a. MicroPET studies of [¹⁸F]18d and [¹⁸F]20a in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a ¹⁸F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington's disease and schizophrenia.

Full text of DOI:10.1021/acs.jmedchem.5b01205

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Article
Synthesis of fluorine-containing phosphodiesterase 10A
(PDE10A) inhibitors and the in vivo evaluation of F-18 labeled
PDE10A PET tracers in rodent and nonhuman primate
Junfeng Li, Xiang Zhang, Hongjun Jin, Jinda Fan, Hubert Flores, Joel S. Perlmutter, and Zhude Tu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01205 • Publication Date (Web): 02 Oct 2015
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Synthesis of fluorineꢀcontaining phosphodiesterase 10A (PDE10A) inhibitors and
the in vivo evaluation of Fꢀ18 labeled PDE10A PET tracers in rodent and
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nonhuman primate
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Junfeng Li,a Xiang Zhang,a Hongjun Jin,a Jinda Fan,a Hubert Flores,b Joel S. Perlmuttera,b and Zhude Tua*
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aDepartment of Radiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
bDepartment of Neurology, Washington University School of Medicine, St. Louis, MO, 63110, USA.
*Corresponding author
Keywords: Phosphodiesterase 10A (PDE10A) inhibitor, central nervous system (CNS), schizophrenia,
Huntington’s disease, MPꢀ10, positron emission tomography (PET), Fluorineꢀ18.
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ABSTRACT
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A series of fluorineꢀcontaining PDE10A inhibitors were designed and synthesized to improve the metabolic
stability of [11C]MPꢀ10. 20 of the 22 new analogues had high potency and selectivity for PDE10A: 18a-j, 19d-j,
20a-b, and 21b had IC50 values <5 nM for PDE10A. Seven Fꢀ18 labeled compounds [18F]18a-e, [18F]18g, and
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[18F]20a were radiosynthesized by Fꢀintroduction onto the quinoline rather than the pyrazole moiety of the
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MPꢀ10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ~2ꢀfold higher
activity in the PDE10Aꢀenriched striatum than nonꢀtarget brain regions; this ratio increased from 5 to 30 min
postꢀinjection, particularly for [18F]18a-d and [18F]20a. MicroPET studies of [18F]18d and [18F]20a in
nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable
metabolic stability. These results suggest this strategy may identify a 18Fꢀlabeled PET tracer for quantifying the
levels of PDE10A in patients with CNS disorders including Huntington’s disease and schizophrenia.
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INTRODUCTION
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Phosphodiesterase 10A (PDE10A) is a dualꢀspecificity phosphodiesterase enzyme that is able to hydrolyze both
cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) to AMP and GMP,
respectively.1ꢀ3 PDE10A mRNA is highly expressed in the brain; expression in peripheral tissues, with the
exception of the testis, is low.4 Both PDE10A mRNA and protein are specifically enriched in the medium spiny
neurons (MSNs) of the striatum.5 Abnormal striatal levels of PDE10A play a major role in schizophrenia,
Huntington’s disease, addiction and other neuropsychiatric disorders.6ꢀ8
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Positron emission tomography (PET) is a preclinical molecular imaging technique which permits
quantitative noninvasive measurement of drug disposition and localization in animal models of human disease
and an important tool for clinical studies. PDE10A inhibitors have shown promise as therapeutics in preclinical
models of human disease, several have progressed to clinical trials.7, 9ꢀ16 The highly potent (IC50 = 0.37 nM) and
highly
selective
(>100ꢀfold)
PDE10A
inhibitor
2ꢀ[4ꢀ(1ꢀmethylꢀ4ꢀpyridinꢀ4ꢀylꢀ1Hꢀpyrazolꢀ3ꢀyl)ꢀ
phenoxymethyl]ꢀquinoline (MPꢀ10) 17 has been extensively evaluated as a therapeutic inhibitor of PDE10A and
has completed several clinical trials in human subjects.18 [11C]MPꢀ10 (Figure 1) was independently investigated
by several groups as a PET tracer for quantifying PDE10A in vivo. Although variable success and significant
species differences have been reported in preclinical studies, 19ꢀ21 [11C]MPꢀ10 remains a potentially viable PET
tracer for clinical studies. Numerous other 11Cꢀ and 18Fꢀlabeled PDE10A inhibitors have been evaluated in
preclinical rodent models and although several candidate tracers have proceeded to studies in nonhuman
primates and human subjects, the search for a PET tracer with ideal pharmacological properties is ongoing.22ꢀ31
Although [11C]MPꢀ10 provides a clear anatomic image of the brain with high accumulation in the striatum
of nonhuman primates (NHP), tissue timeꢀactivity curves in macaques revealed an increasing trend of
radioactivity accumulation in both the target region (striatum) and the reference region (cerebellum) which may
limit its utility.19 Our metabolite analysis of [11C]MPꢀ10 suggested that a brain penetrant radiometabolite was
formed by Oꢀdealkylation of the pyrazole moiety of [11C]MPꢀ10.19 An additional consideration in the search for
an improved PET tracer is that the longer halfꢀlife of 18F places fewer time constraints on production and
permits longer scan sessions that usually result in improved target: nonꢀtarget ratios and higher quality images.
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Although a potent and selective quinoline analogue of MPꢀ10 was formed by introducing a [18F]fluoroethyl
group on the Nꢀatom of the pyrazole moiety ([18F]JNJ41510417, Figure 1), 32, 33 slow equilibrium kinetics were
reported in rodents and NHPs, possibly due to brainꢀpenetrant radioactive metabolites that accumulate nonꢀ
specifically in the brain. To overcome this potential liability, we recently 11Cꢀlabeled the quinoline moiety
instead of the pyrazole moiety of several new PDE10A inhibitors ([11C]1ꢀ4, Figure 1).34 We hypothesized that
this strategy might lead to the identification of a new analogue with (i) increased in vivo metabolic stability,
and/or; (ii) a metabolic pathway which generates radioactive metabolites that do not cross the bloodꢀbrain
barrier (BBB). Our earlier structureꢀactivity relationship (SAR) study explored introduction of a methoxy group
(ꢀOCH3) in the 3, 4 or 6ꢀposition of the 2ꢀmethylquinoline moiety of the MPꢀ10 pharmacophore and the
regioisomers of the new analogues. The most potent and selective compounds were the 3ꢀ and 4ꢀmethoxy
substituted quinolines with an Oꢀatom bridge linkage; little difference was seen in the in vitro biological activity
of regioisomers.35 Subsequent evaluation of the 11Cꢀlabeled lead tracers showed prolonged favorable target:
background ratios in rats for the 3ꢀ and 4ꢀmethoxy substituted quinolines with high selectivity in the target
region and nonꢀtarget region and promising kinetic behavior in both the target region (striatum) and the
reference regions of NHPs.34
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We have continued our exploration of 18Fꢀlabeled PET radiotracers for PDE10A by introducing fluoroꢀ,
fluoromethyl, fluoropropyl or triazole fluoroethyl groups into the quinoline moiety of MPꢀ10. We also explored
the introduction of a short fluoroPEGylated (FPEG) unit. Incorporation of a short FPEG unit has been shown to:
(i) enable facile 18Fꢀlabeling of the target compound; (ii) improve the in vivo pharmacokinetics of molecules
interacting with biologically interesting proteins or peptides;36, 37 (iii) decrease the lipophilicity of molecules,
which enhances their ability to cross the BBB.38 Radiolabeling of [11C]MPꢀ10 also yielded the regioisomer
([11C]isoMPꢀ10 Figure 1),19 a slightly less potent PDE10A inhibitor reported in the discovery of MPꢀ10;17
[11C]isoMPꢀ10 also showed selective binding in the rat striatum, though initial brain uptake was significantly
lower than that of [11C]MPꢀ10.28 We further expanded our synthetic efforts to include modification of the
pyrazole moiety to generate regioisomers of the new fluorineꢀcontaining MPꢀ10 quinoline analogues; this
permitted expanded SAR studies. Here we report (i) the synthesis and screening of new analogues which allow
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18Fꢀlabeling in the quinoline moiety rather than the pyrazole moiety; (ii) 18Fꢀlabeling of the potent and selective
candidates and the in vivo evaluation of their suitability as PDE10A PET imaging agents using rats and
macaques.
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RESULTS AND DISCUSSION
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Chemistry. The syntheses of target new PDE10A analogues was accomplished by a Mitsunobu reaction39 of
benzyl alcohols on quinoline moiety coupling with substituted phenols on pyrazole moiety 17a-b, or a Oꢀ
alkylation of substituted methyl bromide on quinoline moiety with substituted phenols 17a-b.
Following the above strategy, quinoline fragments of molecules including fluorineꢀcontaining intermediates
6a-j and 15a were first synthesized as shown in Scheme 1. Different approaches were used for the synthesis of
compounds 6a-j and 15a: (i) aniline 5 was converted to the aryl fluoride 6a via diazonium fluoroborates under
the BalzꢀSchiemann conditions40; (ii) the alcohol 7 was converted directly into 6b by treatment with
diethylaminosulfur trifluoride (DAST); (iii) treatment of compound 8 with 1,1,1ꢀtrifluoroꢀNꢀphenylꢀNꢀ
((trifluoromethyl)sulfonyl)methaneꢀsulfonamide afforded the triflate. Stille reaction41ꢀ43 of the triflate with
allyltributyltin in the presence of tetrakis triphenylphosphine palladium and lithium bromide generated
compound 9. Hydroborationꢀoxidation of 9 to afford the alcohol, followed by fluorination generated compound
6c; (iv) alkylation of commercially available 10 (ethyl 4ꢀhydroxy quinolineꢀ2ꢀcarboxylate) with 2ꢀfluoroethyl
bromide afforded ethyl 4ꢀ(2ꢀfluoroethoxy)quinolineꢀ2ꢀcarboxylate (6d) in 60% yield; (v) alkylation of the
phenol group of 10 with either a –Cl atom or –OTs group afforded the corresponding alcohols which were
converted using DAST to the corresponding fluoroPEGylated intermediates 6e-g in modest yields (60ꢀ71%);
(vi) alkylation of (2ꢀmethylquinolinꢀ4ꢀyl)methanol 7 with 2ꢀfluoroethyl bromide under basic conditions
afforded 4ꢀ((2ꢀfluoroethoxy)methyl)ꢀ2ꢀmethylquinoline 6h; (vii) the aniline group of 5 was protected using diꢀ
tertꢀbutyl dicarbonate to form the Bocꢀprotected compound 11, subsequent Nꢀalkylation using 2ꢀfluoroethyl
bromide afforded 6i; (viii): 10 was brominated using POBr3 to afford ethyl 4ꢀbromoquinolineꢀ2ꢀcarboxylate 12,
which was converted to the azide compound and subjected to click reaction conditions,44 followed by DAST
fluorination to afford compound 6j; (ix) using Friedlander synthesis45, 2ꢀaminobenzaldehyde 13 reacted with 1ꢀ
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methoxypropanꢀ2ꢀone afforded the 3ꢀmethoxyꢀ2ꢀmethylquinoline46, subsequent demethylation utilizing BBr3
followed by Oꢀalkylation afforded compound 15a.
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The synthesis of intermediate compounds 16a-n was accomplished using strategies shown in Scheme 2. (i)
bromination of 6a or 15d using NBS afforded compound 16a and 16n respectively (Scheme 2, Method 1); (ii)
the methylquinoline derivatives 6b-c, 6h-i, 15a-c were oxidized using selenium dioxide (SeO2), followed by
NaBH4 reduction, while the ethyl quinolineꢀ2ꢀcarboxylate 6d-g, 6j were reduced using NaBH4 to afford the
corresponding alcohols 16b-m (Scheme 2, Methods 2ꢀ3). The detailed methods for synthesis of intermediates
are provided in the Supporting Information.
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The synthesis of final compounds 18a-j, 19d-j, 20a-d, and 21b was accomplished as shown in Scheme 3.
Substituted phenols 17a-b were prepared as previously published (Scheme 3).35 16b-m were coupled with the
phenol 17a or 17b with a Mitsunobu reaction39 to afford 18b-j, 20a-c, 19d-j and 21b. Compounds 18a, 20d
were made by Oꢀalkylation of the substituted phenol 17a with substituted methyl bromide 16a or 16n.
In vitro inhibition assays identified candidate potent and selective analogues for radiolabeling: 18a-e, 18g
and 20a. Schemes 4 and 5 outline the synthesis of the precursors for radiolabeling; the detailed methods for
their synthesis are provided in the Supporting Information. Scheme 4 outlines the synthesis of precursors 23a,
23d and 24a. The method described above for synthesis of 16d from 6d was used for the synthesis of 23a. A
methoxymethyl ether (MOM) group was used to protect phenols 10 and 24b under basic conditions, the
protected intermediates were reacted with the substituted phenol 17a under Mitsunobu reaction conditions to
afford 27a-b. The MOM ether functional group was easily deprotected by TFA to afford precursors 23d and
24a.
The precursors 23b-c, 23e, 23g were obtained by following Scheme 5. Commercially available tertꢀ
butyldimethylsilyl (TBS) chloride was used as a silylation agent to protect alcohols 28a-d. The use of 1.7 eq.
imidazole as base with 1.7 eq. of TBSCl and DMF as solvent proved effective and resulted in TBSꢀethers 29a-d
in high yield. Compounds 29a-d were converted to the respective alcohols 30a-d, which were coupled with the
phenol 17a or 17b in the presence of Ph3P and diꢀtertꢀbutyl azodicarboxylate (DBAD) via a Mitsunobu Oꢀ
alkylation39 to afford compounds 31a-d. The TBSꢀethers 31a-d were easily deprotected with a catalytic amount
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of tetrabutyl ammonium fluoride (TBAF), the intermediate alcohol was then chlorinated or sulfonated as
indicated in Scheme 5 to afford precursors 23b-c, 23e, and 23g.
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Radiochemistry. An overview of the radiolabeling strategies for [18F]18a-e, [18F]18g, and [18F]20a is shown in
Scheme 6. These tracers were labeled by the following strategies: (i) a oneꢀstep direct nucleophilic halogen
exchange was used for labeling [18F]18a and [18F]18b; (ii) a oneꢀstep direct nucleophilic aliphatic substitution
with a tosylate (Ts) or mesylate (Ms) leaving group was used for labeling [18F]18c, [18F]18e, and [18F]18g; (iii)
a twoꢀstep strategy was used for labeling [18F]18d and [18F]20a using the 2ꢀ[18F]fluoroethyl tosylate 34 as a
secondary labeling agent. The structures of the new fluorineꢀcontaining PDE10A inhibitors [18F]18a-e,
[18F]18g, and [18F]20a are shown in Figure 2.
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Scheme 6 shows the conditions used for the successful radiolabeling of [18F]18a-e, [18F]18g, and [18F]20a.
Labeling of [18F]18b was initially planned by a direct nucleophilic aliphatic substitution using a tosylate (Ts) or
mesylate (Ms) leaving group. However, because heating the precursor with 18Fꢀ at 85 °C for 15 min yielded only
a trace of [18F]18b, an alternative strategy of halogen exchange reaction was pursued, this was successfully
performed using the chloride precursor 23b. As shown in Scheme 6, a oneꢀstep displacement reaction using
mesylate and tosylate leaving groups was employed for radiolabeling [18F]18b, [18F]18c, [18F]18e, and [18F]18g.
A twoꢀstep strategy was used for the radiosynthesis of [18F]18d and [18F]20a using the tosylate [18F]34 as a
secondary labeling agent. First, [18F]KF/4,7,13,16,21,24ꢀHexaoxaꢀ1,10ꢀdiazabicyclo[8.8.8]hexacosane
(Kryptofix[222]) was substituted into the diꢀtosylate 33. Second, the resulting 2ꢀ[18F]fluoroethyl tosylate,
[18F]34, was directly used in a nucleophilic substitution by reaction with the precursors to afford [18F]18d and
[18F]20a. Details are provided in the Experimental section. All tracers were obtained with high specific activity
(SA), and with a radiochemical yield sufficient for in vivo studies. The radiochemical purity of each tracer was
> 99% and the chemical purity was >95%. Details of the radiosynthesis for each 18Fꢀlabeled compound are
provided in the Experimental section.
In Vitro Assays. We previously reported that the introduction of a 3ꢀ or 4ꢀ methoxy group on the quinoline
fragment of MPꢀ10 and isoMPꢀ10 yielded potent and PDE10A selective analogues.35 Here we report our
continued exploration of fluorineꢀcontaining quinoline analogues as PDE10A inhibitors. In vitro potency and
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selectivity of the new substituted quinoline analogues as PDE10A inhibitors are shown in Table 1; the top
section of the table contains analogs of MPꢀ10 while the lower section contains select corresponding
regioisomers which are analogues of isoMPꢀ10. Comparison of the in vitro assay results for MPꢀ10 analogues
18d-j and 20b, with the regioisomers 19d-j and 21b showed no significant difference for paired regioisomers,
although the slightly lower IC50 values for the analogues based on MPꢀ10 suggest they may be modestly more
potent. New analogues with a fluorineꢀcontaining group into 4ꢀposition of the quinoline moiety of the MPꢀ10
pharmacophore (18a-j, 19d-j), had high potency for PDE10A with IC50 values <3 nM, particularly 18a-b, 18d-i
and 19f-i with IC50 values <1 nM. These results are consistent with the SAR we reported for the methoxyꢀ
substituted quinolone analogues.35
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We also explored substitution at the 3ꢀ and 6ꢀ positions. Compound
(3ꢀfluoroethoxy substitution)
20a
demonstrated high PDE10A inhibition (IC50 = 0.26 nM). Replacement of the hydrogen atom at 6ꢀposition of the
quinoline moiety with fluoride(ꢀF) yielded regioisomers and which demonstrated high potency with
20b
21b
IC50 values of 0.29 and 0.76 nM respectively, however, replacement of the hydrogen at 6ꢀposition using larger
fluorineꢀcontaining groups resulted in analogues with low potency ( , ꢀCH F, IC > 76 nM;
20c
, –
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OCH2CH2F, IC50 = 33.5 ± 0.08 nM).
Selectivity of a PET ligand for PDE10A over the other subtypes is also an important consideration.
Inhibition of PDE3A/B has been associated with arrhythmia and increased mortality,47, 48 while PDE4 is widely
expressed in the central nervous system.49, 50 The potent new PDE10A inhibitors (20 compounds, PDE10A IC50
<5 nM) were also evaluated for inhibition of PDE3A/B and PDE4A/B. The results for final compounds 18a-j,
19d-j, 20a-d, and 21b (Table 1) revealed almost all compounds had very low potency for inhibiting PDE3A/B
and PDE4A/B with the IC50 value > 750 nM, except 18c which had slight potency for PDE4A/B (IC50 460 nM
for PDE4A and 380 nM for PDE4B). All of the new compounds, including 18c, had >210ꢀfold selectivity ratios
for PDE10A vs PDE3A/B and PDE4A/B. This suggests that 18a-j, 19d-j, 20a-b and 21b are both potent and
selective inhibitors of PDE10A.
Based on these screening results and our previous experience with the 11Cꢀlabeled regioisomers, seven
analogues of MPꢀ10 were identified as candidate ligands for 18Fꢀlabeleing: –F (18a), ꢀ(CH2)nF (18b, 18c), ꢀ
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OCH2CH2F (18d), ꢀCH2OCH2CH2F (18e), and ꢀFPEG (18g, 20a). The precursors were synthesized (details are
provided in the Supporting Information) for the subsequent radiolabeling and the in vivo evaluation as PET
tracers for quantification of PDE10A in rodents and nonhuman primates.
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Biodistribution. The biodistribution of [18F]18a-e, [18F]18g, and [18F]20a was evaluated in normal adult male
SpragueꢀDawley rats at 5, 30 and 60 min postꢀinjection (p.i.) Table 2 shows the uptake and washout in
peripheral organs and whole brain for each of the seven tracers. The initial brain uptake values were 0.410 ±
0.063, 0.128 ± 0.012, 0.114 ± 0.015, 0.118 ± 0.012, 0.213 ± 0.039, 0.353 ± 0.052, and 0.092 ± 0.011 %ID/g for
[18F]18a, [18F]18b, [18F]18c, [18F]18d, [18F]18e, [18F]18g, and [18F]20a, respectively. At 5 min, 30 min, and 60
min p.i., brain uptake of each of the tracers was comparable to or higher than [11C]MPꢀ10 (0.162 ± 0.025, 0.066
± 0.008, and 0.056 ± 0.006 %ID/g 5 min, 30 min and 60 min p.i.).19 Modest but progressive accumulation of
radioactivity in bone was observed for [18F]18a, [18F]18b, and [18F]18c: bone activity increased from 0.522 ±
0.116, 0.421 ± 0.063, and 0.222 ± 0.014 %ID/g at 5 min to 6.300 ± 0.314, 5.620 ± 1.132, and 2.037 ± 0.303
%ID/g at 60 min p.i. respectively. This progressive increase in bone activity was most likely the result of
metabolic defluorination. These results suggest that functional groups including –F (18a), ꢀCH2F (18b), and ꢀ
CH2CH2CH2F (18c) on the quinoline fragment of MPꢀ10 undergo in vivo metabolic defluorination in rodents.
In contrast, [18F]18d, [18F]18e, [18F]18g and [18F]20a appeared more stable to in vivo defluorination.
The cerebellum and cortex are accepted reference (nonꢀtarget) regions in kinetic modeling of PDE10A PET
tracers.29 Therefore, in addition to high striatal uptake and retention, a candidate tracer for clinical imaging
should show low uptake and rapid washout in the cerebellum and cortex; this will facilitate high target: nonꢀ
target ratios and yield high quality images. The new radiotracers displayed generally heterogeneous regional
brain distribution with the highest uptake observed in the striatum; by 30 min p.i., the ratio of activity in the
striatum versus nonꢀtarget brain regions was > 2ꢀfold for [18F]18a, [18F]18b, [18F]18c, [18F]18d, and [18F]20a
(Figure 3). Between 5 and 30 min p.i., the striatum: cerebellum and striatum: cortex ratios increased for the five
tracers, which suggests that they were rapidly cleared from nonꢀtarget regions and displayed specific binding
within the PDE10Aꢀenrichedꢀstriatum.
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Taken together, the rodent biodistribution and regional brain uptake and washout data identified 4ꢀ
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fluoroethoxy [18F]18d and 3ꢀfluoroethoxy [18F]20a as the more promising candidates for further evaluation in
nonhuman primates. Both tracers appeared stable to metabolic defluorination and showed favourable ratios in
the striatum versus nonꢀtarget brain regions.
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MicroPET Imaging Studies of Cynomolgus Macaques. The two fluoroethoxy lead PET radioligands for
imaging PDE10A, [18F]18d and [18F]20a, underwent PET imaging studies (n = 3 for each tracer) in male
cynomolgus monkeys (Figure 4) using a microPET Focus 220 scanner. The representative summed images
from 0 to 180 min were coꢀregistered with MRI images to accurately identify the anatomical regions of interest
(ROI’s) are shown in Figure 4. MicroPET studies of both [18F]18d and [18F]20a gave clear visualization of the
PDE10Aꢀenriched striatum. Both the caudate and putamen striatal regions had high uptake of [18F]18d and
[18F]20a ; the ratios of uptake in the striatum vs. nonꢀtarget brain regions were > 3ꢀfold by 40 min p.i. In
particular, for [18F]20a, target: nonꢀtarget ratios reached > 5ꢀfold by 70 min p.i. More importantly, although the
two tracers showed differences in striatal clearance, tissue timeꢀactivity curves of both [18F]18d and [18F]20a
demonstrated improved washout kinetics from both target and nonꢀtarget regions compared with [11C]MPꢀ10.
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The improved washout kinetics suggest that the strategy of Fꢀlabeling the quinoline rather than the pyrazole
moiety reduced the formation of radioactive metabolites that cross the BBB.
Metabolite Studies in Cynomolgus Macaques. Initial NHP brain imaging of [18F]18d and [18F]20a showed
improved kinetics over previously published PET tracers for PDE10A. To further investigate the in vivo
metabolic stability of these tracers, HPLC analysis was performed to identify the percentage of parent
compound versus radiometabolites in solvent extracts of NHP plasma obtained during microPET imaging
studies of [18F]18d and [18F]20a. NHP arterial blood was obtained from cynomolgus macaques 5, 15, 30, 60 and
90 min p.i for [18F]18d and [18F]20a. Plasma was separated from packed red cells; ~ 65ꢀ75% of the radioactivity
was in the plasma. Following solvent extraction and deproteination, ~90% of the plasma activity was in the
supernatant and only 10% of the activity was in the protein pellet. The solvent extract was injected onto an
HPLC to quantify the percentage of the radioactivity for the parent compound versus radiolabeled metabolites
based on the different retention times. As shown in Table 3, HPLC analysis of NHP plasma revealed good
metabolic stability for both [18F]18d and [18F]20a. Particularly, at 30 min p.i., solvent extracts of arterial blood
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contained ~90% parent compound for [18F]18d. The major radioactive metabolite of [18F]18d was more polar
(retention time ～3.5 min) than the parent compound (retention time ～10.5 min), negligible amounts of a
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minor radioactive metabolite (retention time ~7.5 min) were also observed. For [18F]20a, solvent extracts of
arterial blood contained > 90% parent compound 15 min p.i., 73% parent remained 30 min p.i. The major
radioactive metabolite was more polar (retention time ～3.5 min) than the parent compound (retention time ～
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10.5 min). The minor radioactive metabolite was < 14% of the recovered activity 90 min p.i. (retention time
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~7.5 min). The results of the metabolite analysis confirmed that our new strategy of Fꢀlabeling the quinoline
rather than the pyrazole moiety resulted in significant metabolic stability in NHP blood.
CONCLUSION
In the present study, we optimized the structure of MPꢀ10 to identify new compounds that can be 18Fꢀlabeled in
the quinoline moiety instead of the pyrazole moiety. As a result, 20 highly potent compounds 18a-j, 19d-j, 20a-
b and 21b (IC50 < 5 nM) were identified which also displayed high selectivity for PDE10A against PDE3A/B,
PDE4A/B. Among these potent compounds, 18a-e, 18g and 20a were evaluated as potential PET radioligands
for quantifying the level of PDE10A. [18F]18a, [18F]18b, [18F]18c, [18F]18d, [18F]18e, [18F]18g, and [18F]20a
are, to our knowledge, the first MPꢀ10 analogues successfully 18Fꢀlabeled in the quinoline instead of the
pyrazole moiety. Rat biodistribution indicated [18F]18a, [18F]18b, [18F]18c, [18F]18d, [18F]18e, [18F]18g, and
[18F]20a can cross the bloodꢀbrainꢀbarrier and accumulate in the PDE10Aꢀenriched striatum. [18F]18d and
[18F]20a proceeded to further evaluation in NHPs. The preliminary microPET imaging studies in NHPs
demonstrated that [18F]18d and [18F]20a provided very clear visualization of striatum. Although different
patterns of striatal clearance were observed, both tracers showed rapid clearance from nonꢀtarget brain regions.
Compared with [11C]MPꢀ10, [18F]18d and [18F]20a displayed more suitable washout kinetics in NHP brain. In
addition, both tracers displayed favorable in vivo metabolic stability based on HPLC analysis of NHP plasma.
These promising preliminary results suggest that our strategy has a high probability of identifying a clinical
PET tracer for quantifying PDE10A in the human brain. Future NHP microPET imaging studies with [18F]18d
and [18F]20a will explore the specificity of the tracer uptake.
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In summary, the strategy of introducing fluorineꢀcontaining group in a suitable position of the 2ꢀ
methylquinoline moiety of MPꢀ10 identified new potent and selective PD10A inhibitors; this approach will help
guide the design of potent and selective ligands with suitable lipophilicity as candidates for treating
schizophrenia and other CNS diseases using PDE10A enzyme inhibition strategies. Further validation of these
new analogues is ongoing to determine if either is a suitable PET tracer to quantify the level of PDE10A in the
human brain.
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EXPERIMENTAL SECTION
General. All reagents and chemicals were purchased from commercial suppliers and used without further
purification unless otherwise stated. All anhydrous reactions were carried out in ovenꢀdried glassware under an
inert nitrogen atmosphere unless otherwise stated. When reactions involved extraction with DCM (CH2Cl2),
chloroform (CHCl3), or ethyl acetate (EtOAc), the organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Free bases were converted into the corresponding oxalate salt by addition
of oxalic acid for all final compounds. Melting points of salts were determined on the MELꢀTEMP 3.0
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apparatus and left uncorrected. H NMR spectra were recorded at 400 MHz on a Varian MercuryꢀVX
spectrometer with CDCl3 as solvents and tetramethylsilane (TMS) was used as the internal standard. Elemental
analyses (C, H, N) were determined by Atlantic Microlab, Inc. Synthesis of starting materials and intermediates
are shown in Supporting Online Information.
General Method for Synthesis of 18a-j, 19d-j, 20a-d, and 21b.
4-Fluoro-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)quinoline
(18a).
Sodium
hydride (31 mg, 1.3 mmol) was added to a solution of 16a (251 mg, 1.00 mmol) in anhydrous DMF (20 mL) at
0 °C. After 30 min, 17a (238 mg, 1.00 mmol) was added to the reaction mixture which was stirred overnight at
rt. The reaction mixture was then diluted with water (30 mL), extracted with DCM (3 × 60 mL), washed with 1
N NaOH (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was
purified by silica gel chromatography using MeOH: DCM (1:30, v/v) to afford 18a as a light yellow solid (205
mg, 50%). 1H NMR (CDCl3) δ 8.48 – 8.45 (m, 2H), 8.09 – 8.07 (m, 2H), 7.80 – 7.76 (m, 1H), 7.61 – 7.57 (m,
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2H), 7.41 – 7.38 (m, 3H), 7.18 – 7.16 (m, 2H), 7.01 – 6.99 (m, 2H), 5.37 (s, 2H), 3.96 (s, 3H). mp: 176.2 –
177.2 °C. Anal. (C25H19FN4O • 2H2C2O4) C, H, N: Calcd. C: 58.98, H: 3.93, N: 9.49. Found C: 58.78, H: 3.84,
N: 9.26.
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4-(Fluoromethyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl) quinoline (18b).
Triphenylphosphine (84 mg, 0.32 mmol) and DBAD (73 mg, 0.32 mmol) were added to a solution of 17a (50
mg, 0.26 mmol) and 16b (50 mg, 0.28 mmol) in 1,4ꢀdioxane (2 mL) and heated at 60 °C for 18 h. The reaction
mixture was quenched with 1 N NaOH (50 mL), extracted DCM (3 × 50 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure. The crude product was purified by silica gel column chromatography
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using MeOH: DCM (1:30, v/v) to afford 18b as a yellow solid (55 mg, 65%). H NMR (CDCl3) δ 8.55 – 8.37
(m, 2H), 8.22 – 8.00 (m, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.80 – 7.70 (m, 2H), 7.63 – 7.53 (m, 2H), 7.43 – 7.33
(m, 2H), 7.20 – 7.11 (m, 2H), 7.08 – 6.93 (m, 2H), 5.94 (s, 1H), 5.83 (s, 1H), 5.39 (s, 2H), 3.95 (s, 3H). mp:
174.4 – 174.5 °C. Anal. (C26H21FN4O • 2H2C2O4) C, H, N: Calcd. C: 59.60, H: 4.17, N: 9.27. Found C: 59.89,
H: 4.36, N: 9.15.
4-(3-Fluoropropyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl) quinoline (18c).
Compound 16c was used as the starting material following the procedure described above for the preparation of
18b to synthesize 18c as a yellow solid (40 mg, 49%). 1H NMR (CDCl3) δ 8.45 (d, J = 5.0 Hz, 2H), 8.05 (m,
2H), 7.71 (t, J = 7.5 Hz, 1H), 7.55 (dd, J = 14.5, 7.1 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.24 (s, 1H), 7.14 (d, J =
5.0 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 5.35 (s, 2H), 4.56 (t, J = 5.6 Hz, 1H), 4.44 (t, J = 5.5 Hz, 1H), 3.95 (s,
3H), 3.22 (t, J = 7.7 Hz, 2H), 2.25 – 2.05 (m, 2H). mp: 125.3 – 125.5 °C. Anal. (C28H25FN4O • 2H2C2O4) C, H,
N: Calcd. C: 60.76, H: 4.62, N: 8.86. Found C: 60.86, H: 4.51, N: 8.63.
4-(2-Fluoroethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl) quinoline (18d).
Compound 16d was used as the starting material following the procedure described above for the preparation of
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18b to synthesize 18d as a white solid (82 mg, 67%). HꢀNMR (CDCl3): δ 8.46 (s, 2H), 8.24 (d, J = 7.5 Hz,
1H), 7.99 (d, J = 6.7 Hz, 1H), 7.71 (s, 1H), 7.64 – 7.46 (m, J = 22.3 Hz, 2H), 7.38 (d, J = 6.6 Hz, 2H), 7.15 (s,
2H), 7.00 (s, 3H), 5.33 (s, 2H), 4.89 (d, J = 47.9 Hz, 2H), 4.43 (d, J = 26.9 Hz, 2H), 3.95 (s, 3H). mp: 126.2ꢀ
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126.3 °C. Anal. (C27H23FN4O2 • 2H2C2O4) C, H, N: Calcd. C: 58.68, H: 4.29, N: 8.83. Found C: 58.98, H: 4.19,
N: 8.91.
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4-((2-Fluoroethoxy)methyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl)quinoline
(18e). Compound 16h was used as the starting material following the procedure described above for the
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preparation of 18b to synthesize 18e as a white solid (69 mg, 69%). HꢀNMR (CDCl3):δ 8.45 (dd, J = 4.6, 1.5
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Hz, 2H), 8.09 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.78 – 7.67 (m, 2H), 7.61 – 7.50 (m, 2H), 7.38 (d, J
= 8.8 Hz, 2H), 7.14 (dd, J = 4.6, 1.6 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 5.37 (s, 2H), 5.06 (s, 2H), 4.71 – 4.61
(m, 1H), 4.58 – 4.49 (m, 1H), 3.94 (s, 3H), 3.87 – 3.81 (m, 1H), 3.80 – 3.74 (m, 1H). mp: 126.8ꢀ127.4 °C. Anal.
(C28H25FN4O2 • 2H2C2O4) C, H, N. Calcd. C: 59.26, H: 4.51, N: 8.64. Found C: 58.09, H: 4.77, N: 8.88.
4-(2-(2-Fluoroethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)
methyl)
quinoline (18f). Compound 16e was used as the starting material following the procedure described above for
the preparation of 18b to synthesize 18f as a white solid (64 mg, 68%). 1H NMR (CDCl3) δ 8.50 – 8.42 (m, 2H),
8.20 (d, J = 8.3 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.49 (t, J = 7.5 Hz, 1H),
7.38 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 5.8 Hz, 2H), 7.04 – 6.95 (m, 3H), 5.33 (s, 2H), 4.68 – 4.61 (m, 1H), 4.58 –
4.50 (m, 1H), 4.40 – 4.32 (m, 2H), 4.06 – 3.99 (m, 2H), 3.95 (s, 3H), 3.92 – 3.86 (m, 1H), 3.85 – 3.79 (m, 1H).
mp: 170.1ꢀ170.3 °C. Anal. (C29H27FN4O3 • 2H2C2O4) C, H, N: C: 58.41, H: 4.60, N: 8.26. Found C: 58.70, H:
4.81, N: 8.45.
4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)-
methyl) quinoline (18g). Compound 16f was used as the starting material following the procedure described
above for the preparation of 18b to synthesize 18g as a white solid (55 mg, 63%). 1HꢀNMR (CDCl3): δ 8.47 (d,
J = 5.8 Hz, 2H), 8.22 (d, J = 8.3 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.57 (s, 1H), 7.49 (t,
J = 7.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 5.9 Hz, 2H), 7.07 – 6.96 (m, 3H), 5.33 (s, 2H), 4.63 –
4.56 (m, 1H), 4.51 – 4.44 (m, 1H), 4.41 – 4.31 (m, 2H), 4.01 (t, J = 4.6 Hz, 2H), 3.96 (s, 3H), 3.83 – 3.71 (m,
6H). mp: 166.0ꢀ166.2 °C. Anal. (C31H31FN4O4 • 2H2C2O4) C, H, N: Calcd. C: 58.17, H: 4.88, N: 7.75. Found C:
58.33, H: 4.98, N: 7.94.
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4-(2-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-
yl)phenoxy) methyl)quinoline (18h). Compound 16g was used as the starting material following the procedure
described above for the preparation of 18b to synthesize 18h as a white solid (48 mg, 59%). 1HꢀNMR (CDCl3):
δ 8.46 (d, J = 5.9 Hz, 2H), 8.21 (d, J = 8.3 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.56 (s,
1H), 7.48 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 5.9 Hz, 2H), 7.05 – 6.95 (m, 3H), 5.32 (s,
2H), 4.61 – 4.55 (m, 1H), 4.49 – 4.42 (m, 1H), 4.35 (t, J = 4.6 Hz, 2H), 4.00 (t, J = 4.6 Hz, 2H), 3.95 (s, 3H),
3.82 – 3.62 (m, 10H). mp: 128.4ꢀ129.0 °C. Anal. (C33H35FN4O5 • 2H2C2O4) C, H, N: Calcd. C: 57.96, H: 5.13,
N: 7.31. Found C: 58.06, H: 5.31, N: 7.50.
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N-(2-Fluoroethyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl) quinolin-4-amine
(18i). Compound 16i was used as the starting material following the procedure described above for the
preparation of 18b, then used TFA for deprotection to afford 18i as a light yellow solid (42 mg, 63%). 1HꢀNMR
(CDCl3): δ 8.47 (d, J = 4.0 Hz, 2H), 7.99 (d, J = 4.4 Hz, 1H), 7.86 – 7.83 (m, 1H), 7.64ꢀ7.61 (m, 1H), 7.57 (s,
1H), 7.42ꢀ7.37 (m, 3H), 7.17 – 7.15 (m, 2H), 7.03ꢀ7.01 (m, 2H), 6.63 (s, 1H), 5.33 (s, 2H), 4.82 – 4.79 (m, 1H),
4.70 – 4.67 (m, 1H), 3.96 (s, 3H), 3.76 – 3.69 (m, 2H). mp: 210.6 – 210.7 °C. Anal. (C27H24FN5O • 2H2C2O4)
C, H, N: Calcd. C: 58.77, H: 4.45, N: 11.05. Found C: 58.98, H: 4.34, N: 11.23.
4-(4-(2-Fluoroethyl)-1H-1,2,3-triazol-1-yl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-
yl)phenoxy)methyl)quinoline (18j). Compound 16j was used as the starting material following the procedure
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described above for the preparation of 18b to synthesize 18j as a light yellow solid (59 mg, 64%). H NMR
(CDCl3) δ 8.52 – 8.44 (m, 2H), 8.23 – 8.17 (m, 1H), 8.02 – 7.96 (m, 1H), 7.96 – 7.93 (m, 1H), 7.88 – 7.80 (m,
2H), 7.65ꢀ7.63 (m, 1H), 7.57 (s, 1H), 7.44 – 7.37 (m, 2H), 7.19 – 7.13 (m, 2H), 7.07 – 6.99 (m, 2H), 5.45 (s,
2H), 4.89 (t, J = 5.9 Hz, 1H), 4.77 (t, J = 5.9 Hz, 1H), 3.97 (s, 3H), 3.35 (t, J = 5.9 Hz, 1H), 3.28 (t, J = 5.9 Hz,
1H). mp: 214.2ꢀ214.6°C. Anal. (C29H24FN7O • 2H2C2O4) C, H, N: Calcd. C: 57.81, H: 4.12, N: 14.30. Found C:
57.97, H: 4.01, N: 14.42.
4-(2-Fluoroethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy) methyl) quinoline (19d).
Compound 16d with 17b (in place of 17a) were used as the starting materials following the procedure described
above for the preparation of 18b to synthesize 19d as a white solid (86 mg, 70%). 1HꢀNMR (CDCl3): δ 8.38 (d,
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J = 2.4 Hz, 2H), 8.29 – 8.27 (m, 1H), 8.03ꢀ8.01 (m, 1H), 7.82 (s, 1H), 7.75 (m, 1H), 7.57ꢀ7.53 (m, 1H), 7.26 –
7.22 (m, 3H), 7.16 – 7.14 (m, 2H), 7.06 – 7.03 (m, 3H), 5.38 (s, 2H), 5.00 (s, 1H), 4.86 (s, 1H), 4.55 (s, 1H),
4.45 (s, 1H), 3.75 (s, 3H). mp: 189.1 – 189.3 °C. Anal. (C27H23FN4O2 • 2H2C2O4) C, H, N: Calcd. C: 58.68, H:
4.29, N: 8.83. Found C: 58.58, H: 4.49, N: 9.01.
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4-((2-Fluoroethoxy)methyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy) methyl)quinoline
(19e). Compound 16h with 17b (in place of 17a) were used as the starting materials following the procedure
described above for the preparation of 18b to synthesize 19e as a white solid (62 mg, 69%). 1H NMR (CDCl3) δ
8.39 – 8.38 (m, 2H), 8.14 – 8.12 (m, 1H), 8.02 – 8.00 (m, 1H), 7.82 (s, 2H), 7.77 – 7.74 (m, 1H), 7.62 – 7.58
(m, 1H), 7.26 – 7.23 (m, 2H), 7.18 – 7.16 (m, 2H), 7.05 – 7.04 (m, 2H), 5.43 (s, 2H), 5.12 (s, 2H), 4.73 – 4.71
(m, 1H), 4.61 – 4.59 (m, 1H), 3.94 – 3.92 (m, 1H), 3.87 – 3.85 (m, 1H), 3.75 (s, 3H). mp: 121.3 – 121.8 °C.
Anal. (C28H25FN4O2 • 2H2C2O4) C, H, N: Calcd. C: 59.26, H: 4.51, N: 8.64. Found C: 59.45, H: 4.31, N: 8.51.
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4-(2-(2-Fluoroethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy)
methyl)
quinoline (19f). Compound 16e with 17b (in place of 17a) were used as the starting materials following the
procedure described above for the preparation of 18b to synthesize 19f as a white solid (58 mg, 62%). 1HꢀNMR
(CDCl3): δ 8.39ꢀ8.38 (m, 2H), 8.24 (d, J = 4.0 Hz, 1H), 8.01 (d, J = 4.3 Hz,1H), 7.81 (s, 1H), 7.73 (m, 1H), 7.52
(m, 1H), 7.26 – 7.04 (m, 7H), 5.38 (s, 2H), 4.69 (m, 1H), 4.53 (m, 1H), 4.41 (m, 2H), 4.06 (m, 2H), 3.94 – 3.93
(m, 1H), 3.85ꢀ3.83 (m, 1H), 3.75 (m, 3H). mp: 109.6 – 109.7 °C. Anal. (C29H27FN4O3 • 2H2C2O4) C, H, N:
Calcd. C: 58.41, H: 4.60, N: 8.26. Found C: 58.63, H: 4.79, N: 8.45.
4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy)
methyl)quinoline (19g). Compound 16f with 17b (in place of 17a) were used as the starting materials
following the procedure described above for the preparation of 18b to synthesize 19g as a white solid (52 mg,
60%). 1HꢀNMR (CDCl3): δ 8.39 – 8.38 (m, 2H), 8.25 (d, J = 4.0 Hz, 1H), 8.01 (d, J = 4.3 Hz,1H), 7.82 (s, 1H),
7.75 – 7.70 (m, 1H), 7.54 – 7.49 (m, 1H), 7.26ꢀ7.03 (m, 7H), 5.38 (s, 2H), 4.63 (m, 1H), 4.47 – 4.40 (m, 3H),
4.06 – 4.04 (m, 2H), 3.82ꢀ3.72 (m, 9H). mp: 82.0 – 82.1 °C. Anal. (C31H31FN4O4 • 2H2C2O4) C, H, N: Calcd. C:
58.17, H: 4.88, N: 7.75. Found C: 58.35, H: 4.99, N: 7.62.
(2-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-
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Journal of Medicinal Chemistry
yl)phenoxy) methyl)quinoline (19h). Compound 16g with 17b (in place of 17a) were used as the starting
materials following the procedure described above for the preparation of 18b to synthesize 19h as a white solid
(46 mg, 57%). 1HꢀNMR (CDCl3): δ 8.39 – 8.37 (m, 2H), 8.24 (d, J = 4.0 Hz, 1H), 8.00 (d, J = 4.3 Hz,1H), 7.81
(s, 1H), 7.74 – 7.70 (m, 1H), 7.53 – 7.49 (m, 1H), 7.24 – 7.22 (m, 2H), 7.16 – 7.13 (m, 2H), 7.05 – 7.03 (m,
3H), 5.37 (s, 2H), 4.50 – 4.58 (m, 1H), 4.48ꢀ4.46 (m, 1H), 4.40 – 4.38 (m, 2H), 4.40 – 4.38 (m, 2H), 3.81 – 3.67
(m, 13H). mp: 108.8 – 108.9 °C. Anal. (C33H35FN4O5 • 2H2C2O4) C, H, N: Calcd. C: 57.96, H: 5.13, N: 7.31.
Found C: 57.71, H: 5.33, N: 7.47.
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N-(2-Fluoroethyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy)methyl)
quinolin-4-amine
(19i). Compound 16i with 17b (in place of 17a) were used as the starting materials following the procedure
described above for the preparation of 18b, then deprotection with TFA afforded 19i as a yellow solid (40 mg,
60%). 1HꢀNMR (CDCl3): δ 8.37 (d, J = 3.0 Hz, 2H), 7.99 (d, J = 4.2 Hz, 1H), 7.81 – 7.77 (m, 2H), 7.71 – 7.67
(m, 1H), 7.50ꢀ7.46 (m, 1H), 7.23 – 7.21 (m, 2H), 7.15 – 7.13 (m, 2H), 7.05 – 7.03 (m, 2H), 6.75 (s, 1H), 5.33 (s,
2H), 4.82 – 4.79 (m, 1H), 4.70 – 4.67 (m, 1H), 3.76ꢀ3.69 (m, 5H). mp: 206.5 – 206.7 °C. Anal. (C27H24FN5O •
2H2C2O4) C, H, N: Calcd. C: 58.77, H: 4.45, N: 11.05. Found C: 58.53, H: 4.71, N: 11.22.
4-(4-(2-Fluoroethyl)-1H-1,2,3-triazol-1-yl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-
yl)phenoxy)methyl)quinoline (19j). Compound 16j with 17b (in place of 17a) were used as the starting
materials following the procedure described above for the preparation of 18b to synthesize 19j as a light yellow
solid (62 mg, 67%). 1H NMR (CDCl3) δ 8.38 – 8.36 (m, 2H), 8.22 – 8.20 (m, 1H), 7.99 – 7.97 (m, 2H), 7.87 –
7.84 (m, 2H), 7.80 (s, 1H), 7.68 – 7.64 (m, 1H), 7.26 – 7.23 (m, 2H), 7.16 – 7.14 (m, 2H), 7.04 – 7.02 (m, 2H),
5.48 (s, 2H), 4.89 (t, J = 5.8 Hz, 1H), 4.77 (t, J = 5.8 Hz, 1H), 3.74 (s, 3H), 3.35 (t, J = 5.8 Hz, 1H), 3.28 (t, J =
5.8 Hz, 1H). mp: 172.3ꢀ172.7°C. Anal. (C29H24FN7O • 2H2C2O4) C, H, N: Calcd. C: 57.81, H: 4.12, N: 14.30.
Found C: 58.01, H: 4.02, N: 14.52.
3-(2-Fluoroethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl) quinoline (20a).
Compound 16k was used as the starting material following the procedure described above for the preparation of
18b to synthesize 20a as a light yellow solid (60 mg, 67%). 1HꢀNMR (CDCl3): δ 8.44 (d, J = 4.9 Hz, 2H), 8.07
(d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.60 – 7.46 (m, 3H), 7.43 (s, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.19 –
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7.11 (m, 2H), 7.05 (d, J = 8.5 Hz, 2H), 5.43 (s, 2H), 4.85 (d, J = 3.8 Hz, 1H), 4.73 (d, J = 3.9 Hz, 1H), 4.40 (d, J
= 3.7 Hz, 1H), 4.33 (d, J = 3.6 Hz, 1H), 3.96 (s, 3H). mp: 191.4 ꢀ 191.7 °C. Anal. (C27H23FN4O2 • 2H2C2O4) C,
H, N: Calcd. C: 58.68, H: 4.29, N: 8.83. Found C: 58.73, H: 4.48, N: 8.99.
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6-Fluoro-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl) phenoxy) methyl) quinoline (20b). Compound
16l was used as the starting material following the procedure described above for the preparation of 18b to
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synthesize 20b as a white solid (56 mg, 69%). H NMR (CDCl3) δ 8.52 – 8.42 (m, 2H), 8.14 (d, J = 8.6 Hz,
1H), 8.07 (dd, J = 9.3, 5.3 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.57 (s, 1H), 7.55 – 7.42 (m, 2H), 7.43 – 7.35 (m,
2H), 7.20 – 7.12 (m, 2H), 7.04 – 6.97 (m, 2H), 5.38 (s, 2H), 3.97 (s, 3H). mp: 213.3 – 213.5 °C. Anal.
(C25H19FN4O • 2H2C2O4) C, H, N: Calcd. C: 58.98, H: 3.93, N: 9.49. Found C: 59.09, H: 3.71, N: 9.57.
6-(Fluoromethyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl) quinoline (20c).
Compound 16m was used as the starting material following the procedure described above for the preparation
of 18b to synthesize 20c as a light solid (50 mg, 61%). 1H NMR (CDCl3) δ 8.51 – 8.34 (m, 2H), 8.16 (d, J = 8.5
Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.79 (t, J = 2.2 Hz, 1H), 7.74 – 7.64 (m, 2H), 7.53 (s, 1H), 7.43 – 7.32 (m,
2H), 7.22 – 7.09 (m, 2H), 7.02 – 6.90 (m, 2H), 5.59 (s, 1H), 5.47 (s, 1H), 5.37 (s, 2H), 3.96 (s, 3H). mp: 220.7 –
220.8 °C. Anal. (C26H21FN4O • 2H2C2O4) C, H, N: Calcd. C: 59.60, H: 4.17, N: 9.27. Found C: 59.78, H: 4.29,
N: 9.15.
6-(2-Fluoroethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl) quinoline (20d).
Compound 16n was used as the starting material following the procedure described above for the preparation of
18a to synthesize 20d as a light yellow solid (290 mg, 61%). 1HꢀNMR (CDCl3): δ 8.48 – 8.46 (m, 2H), 8.07 (d,
J = 8.5 Hz,1H), 7.99 (d, J = 9.2 Hz,1H), 7.64ꢀ7.62 (m, 1H), 7.57 (s, 1H), 7.43ꢀ7.37 (m, 3H), 7.16 – 7.14 (m,
2H), 7.09 – 7.08 (m, 1H), 7.01 – 6.99 (m, 2H), 5.35 (s, 2H), 4.90 – 4.88 (m, 1H), 4.78 – 4.76 (m, 1H), 4.38 –
4.36 (m, 1H), 4.31 – 4.29 (m, 1H), 3.96 (s, 3H). mp: 228.8 – 228.9 °C. Anal. (C27H23FN4O2 • 2H2C2O4) C, H,
N: Calcd. C: 58.68, H: 4.29, N: 8.83. Found C: 58.83, H: 4.56, N: 8.73.
6-Fluoro-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-5-yl)phenoxy)methyl)quinoline
(21b).
6ꢀ
Fluoroquinolinꢀ2ꢀyl)methanol (16l) with 17b (in place of 17a) were used as the starting materials following the
procedure described above for the preparation of 18b to synthesize 21b as a white solid (53 mg, 65%) 1H NMR
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Journal of Medicinal Chemistry
(CDCl3) δ 8.39 (dd, J = 4.7, 1.4 Hz, 2H), 8.19 (d, J = 8.5 Hz, 1H), 8.09 (dd, J = 9.2, 5.3 Hz, 1H), 7.82 (s, 1H),
7.74 (d, J = 8.5 Hz, 1H), 7.58 – 7.42 (m, 2H), 7.30 – 7.21 (m, 2H), 7.15 (d, J = 8.8 Hz, 2H), 7.07 – 7.00 (m,
2H), 5.42 (s, 2H), 3.75 (s, 3H). mp: 220.8 – 220.9 °C. Anal. (C25H19FN4O • 2H2C2O4) C, H, N: Calcd. C: 58.98,
H: 3.93, N: 9.49. Found C: 58.85, H: 4.11, N: 9.68.
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General Radiochemistry. We used both oneꢀ and twoꢀstep approaches for radiolabeling; methods for each are
described below. [18F]Fluoride is produced by a 18O(p, n)18F reaction through proton irradiation of enriched 18O
water (95%) using Washington University’s RDSꢀ111 cyclotron (Siemens/CTI Molecular Imaging, Knoxville,
TN, USA). [18F]Fluoride is first passed through an ionꢀexchange resin then eluted with 0.02 M potassium
carbonate (K2CO3) solution.
Following the oneꢀ or twoꢀstep incorporation of [18F]Fluoride, the reaction mixtures were purified using a
semiꢀpreparative HPLC system which contains a 5ꢀmL injection loop, a semiꢀpreparative column, UV detector
at 254 nm, and an inꢀline radioactivity detector which is used to identify the desired product for collection in a
vial containing 50 mL sterile water. The diluted eluent was trapped on a Cꢀ18 Plus SepꢀPak cartridge, which
was then rinsed with 10 mL sterile water; the product was eluted with 0.6 mL ethanol followed by 5.4 mL saline.
After the diluted radiotracer was sterileꢀfiltered through a 0.22 µm syringe filter into a sterile glass vial, and an
aliquot was removed for QC testing. The total synthesis time was ~ 2 h for oneꢀstep labeling and ~ 3 h for twoꢀ
step labeling. Quality control was conducted on an analytical HPLC system with UV detection at 254 nm. The
radioactive dose was authenticated by coꢀinjection with the nonradioactive reference compound.
Synthesis of [18F]18a: a one-step general procedure for radiosynthesis of [18F]18a, [18F]18b, [18F]18c,
[18F]18e, and [18F]18g
4-(Fluoro-18F)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)quinoline ([18F]18a). A
sample of ~7.40 GBq [18F]/fluoride/K2CO3 was added to a reaction vessel containing Kryptofix [222] (6.5–7.0
mg), and dried by azeotropic distillation at 110 °C using MeCN (3 × 1 mL) under a gentle flow of nitrogen. 1.0
– 3.0 mg of the bromide precursor 23a dissolved in DMSO (200 ꢀ 300 ꢁL) was then transferred to the reaction
vessel which was capped, vortexed, and heated in an oil bath at 170 ꢀ 175 °C for 20 min. After the heat source
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was removed, the reaction mixture was diluted with 3.0 mL of HPLC mobile phase and purified using an
Agilent SBꢀC18 column with MeCN/0.1 M ammonium formate buffer pH 4.5 (48/52, v/v) as the mobile phase
at a flow rate of 4.0 mL/min; the retention time for the desired product was 19 ꢀ 21 min. QC was conducted on a
Grace Alltima Cꢀ18 column (250 mm × 4.6 mm), with a mobile phase of MeCN/0.1 M ammonium formate
buffer pH 4.5 (62/38, v/v) at 1.8 mL/min. The retention time of [18F]18a was 5.1 min. The radiochemical purity
was >99%, the yield was 4.2ꢀ4.8% (decayꢀcorrected) and ꢀ SA was > 74 GBq/µmol (decayꢀcorrected to EOS).
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4-((Fluoro-18F)methyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy) methyl)
quinoline
([18F]18b). The chloride precursor 23b was used for the oneꢀstep radiolabeling of [18F]18b as described above
for [18F]18a. The reaction temperature was 105 ꢀ 110 °C for 20 min. The reaction mixture was purified using an
Agilent SBꢀC18 column with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5 (40/60, v/v) at
4.0 mL/min; the retention time for the desired product was 24 ꢀ 26 min. QC was performed using a Phenomenex
Prodigy Cꢀ18 column (250 mm × 4.6 mm) with a mobile phase of MeCN/0.1 M ammonium formate buffer pH
4.5 (62/38, v/v) at 1.5 mL/min. The retention time of [18F]18b was 4.9 min. The radiochemical purity was >
99%, the chemical purity was > 95%, the yield was 1.1ꢀ1.5% (decayꢀcorrected) and the SA was > 74 GBq/µmol
(decayꢀcorrected to EOS).
4-(3-(Fluoro-18F)propyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)
quinoline
([18F]18c). The mesylate precursor 23c was used for the oneꢀstep radiolabeling of [18F]18c as described above
for [18F]18a. The reaction temperature was 75 ꢀ 80 °C for 20 min. The reaction mixture was purified using an
Agilent SBꢀC18 column with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5 (50/50, v/v) at
4.0 mL/min; the retention time for the desired product was 17 ꢀ 18 min. QC was performed using an Agilent
SBꢀ C18 column (250 mm × 4.6 mm) with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5
(65/35, v/v) at 1.5 mL/min. The retention time of [18F]18c was 4.9 min. The radiochemical purity was > 99%,
the yield was 10.1ꢀ12.2% (decayꢀcorrected) and the SA was > 74 GBq/µmol (decayꢀcorrected to EOS).
4-((2-(Fluoro-18F)ethoxy)methyl)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl) phenoxy) methyl)-
quinoline ([18F]18e). The mesylate precursor 23e was used for the oneꢀstep radiolabeling of [18F]18e as
described above for [18F]18a. The reaction temperature was 85 ꢀ 90 °C for 15 min. The reaction mixture was
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Journal of Medicinal Chemistry
purified using an Agilent SBꢀC18 column with a mobile phase of MeCN/0.1 M ammonium formate buffer pH
4.5 (40/60, v/v) at 4.0 mL/min; the retention time was 21.8 min. QC was performed using a Grace Alltimaꢀ C18
column (250 mm × 4.6 mm) with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5 (62/38,
v/v) at 1.8 mL/min. The retention time of [18F]18e was 4.4 min. The radiochemical purity was >99%, the
chemical purity was > 95%, the yield was 15ꢀ18% (decayꢀcorrected) and the SA was > 74 GBq/µmol (decay
corrected to EOS).
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2-(2-(2-((2-((4-(1-Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)quinolin-4-yl)oxy)ethoxy)-
ethoxy)ethyl hypofluorite-18F ([18F]18g). The tosylate precursor 23g was used for the oneꢀstep radiolabeling of
[18F]18g as described above for [18F]18a. The reaction temperature was 95 ꢀ 100 °C for 15 min. The reaction
mixture was purified using an Agilent SBꢀC18 column with a mobile phase of MeCN/0.1 M ammonium
formate buffer pH 6.5 (50/50, v/v) at 3.5 mL/min; the retention time for the desired product was 13 min. QC
was performed using an Agilent SBꢀC18 column (250 mm × 4.6 mm) with a mobile phase of MeCN/0.1 M
ammonium formate buffer pH 6.5 (56/44, v/v) at 1.5 mL/min. The retention time of [18F]18g was 4.9 min. The
radiochemical purity was > 99%, the chemical purity was > 95%, the yield was 27ꢀ30% (decayꢀcorrected) and
the SA was > 74 GBq/µmol (decayꢀcorrected to EOS).
Synthesis of [18F]20a: a two–step general procedure for radiosynthesis [18F]20a and [18F]18d
3-((2-Fluoro-18F)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)
quinoline
([18F]20a). A sample of ~7.40 GBq [18F]/fluoride/K2CO3 was added to a reaction vessel containing Kryptofix
[222] (7.0 – 8.0 mg). The syringe was washed with 2 × 0.4 mL ethanol, evaporated and dried by azeotropic
distillation at 110 °C using MeCN (3 × 1 mL) under a gentle flow of N2 gas. After all water was removed, 5.0ꢀ
5.5 mg of the diꢀtosylate 33 was dissolved in MeCN (200 ꢁL) mixed, then transferred into the reaction vessel
containing [18F]fluoride/Kryptofix[222]/K2CO3 which was capped, mixed, and heated in an oil bath to 100 °C
for 15 min. After the heat source was removed, the reaction mixture was diluted with 3.0 mL of HPLC mobile
phase and passed through an alumina Neutral SepꢀPak Plus cartridge. The reaction mixture was then purified
with an Agilent SBꢀC18 column (250 mm × 10 mm) with a mobile phase of MeCN/0.1 M ammonium formate
buffer pH 6.5 (50/50, v/v) at 4.0 mL/min; the retention time of the [18F]fluoroethyl tosylate product was 9.5ꢀ10
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min. The retention time of the precursor was 23ꢀ24 min. The product was diluted with 50 mL sterile water then
trapped on a Cꢀ18 SepꢀPak Plus cartridge. The trapped product was eluted with diethyl ether (2.5 mL).
The eluted solution formed two phases, the top organic phase was removed and reserved, and the bottom
aqueous phase was extracted with 1 mL of ether. The ether extracts were combined and passed through two
SepꢀPak Plus dry cartridges in series into a reaction vessel; the ether was evaporated under a stream of nitrogen.
1 ꢀ 2 mg of phenol precursor 24a was then dissolved in 200 µL DMSO and transferred to a vial containing 2.0
mg Cs2CO3 and vortexed for 1 min; this saturated Cs2CO3 solution was added to the reaction vessel which was
capped, mixed, and heated in an oil bath at 85ꢀ90 °C for 15 min. The reaction mixture was diluted with 3.0 mL
HPLC mobile phase and purified using an Agilent SB Cꢀ18 column with a mobile phase of MeCN/0.1 M
ammonium formate buffer pH 4.5 (40/60, v/v) at 4.0 mL/min; the retention time was 21.4 min. QC was
performed using an Agilent Zorbax, SBꢀC18 column (250 mm × 4.6 mm) with a mobile phase of MeCN/0.1 M
ammonium formate buffer pH 4.5 (56/44, v/v) at 1.5 mL/min. The retention time of [18F]20a was 4.8 min. The
radiochemical purity was > 99%, the chemical purity was > 95%, the yield was 65ꢀ72% (decayꢀcorrected) and
the SA was > 74 GBq/µmol (decayꢀcorrected to EOS).
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4-((2-Fluoro-18F)ethoxy)-2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)
quinoline
([18F]18d). The phenol precursor 23d was used for the twoꢀstep labeling of [18F]18d as described above for
[18F]20a. The reaction temperature was 95ꢀ100 °C for 15 min. The reaction mixture was purified on an Agilent
SBꢀC18 column with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5 (45/55, v/v) at 4.0
mL/min; the retention time was 16.2 min. QC was performed using an Agilent Zorbax, SBꢀC18 column (250
mm × 4.6 mm) with a mobile phase of MeCN/0.1 M ammonium formate buffer pH 4.5 (50/50, v/v) at 1.4
mL/min. The retention time of [18F]18d was 7.5 min. The radiochemical purity was >99%, the chemical purity
was 95%, the yield was 30ꢀ35% (decayꢀcorrected) and the SA was > 74 GBq/µmol (decayꢀcorrected to EOS).
In vitro assay. The PDE10A in vitro screening assay followed our previously published procedures.35 PDE
activity was measured using the Phosphodiesterase [3H]cAMP Scintillation Proximity Assay (SPA) (Cat.
#TRKQ7090, Perkin Elmer, Waltham, MA) with minor modifications. Recombinant human PDE10A
containing catalytic domain of human PDE10A (SwissꢀProt accession number Q9Y233) was purchased from
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Enzo Life Sciences Inc., Farmingdale, NY. Recombinant human PDE3A and 3B, PDE4A and PDE4B were
purchased from EMD Chemicals, Inc., San Diego, CA. Optimal concentrations of enzymes were tested in the
linear range of the enzyme activation curves with substrate. Inhibitor compounds were tested along with
compound whose IC50 values are known (e.g.: MPꢀ10) as an internal control for each PDE assay. All inhibitors
were dissolved in DMSO, and serial dilutions were performed to obtain desired concentrations. Diluted
compounds (10 µL/well) were added to a 96ꢀwell plate, after addition of 50 µL diluted PDE enzyme in buffer
the plate was incubated on ice for 5 min. 50 µL of radiolabeled substrate ([3H]cAMP) at a fixed (1/3 of the Km)
concentration of 1.48 kBq/mL was then added to each well and plates were incubated on ice for an additional 20
min to give ~30% substrate turnover before termination of the reaction with 50 µL yttrium silicate SPA beads (8
mg/mL). Plates were allowed to settle for 1.5 h at rt, then counted on a Trilux MicroꢀBeta Counter
(PerkinElmer, Waltham, MA). CPM values for each well were plotted against inhibitor concentration in
GraphPad Prism (GraphPad Software, Inc., La Jolla, CA) and inhibitor IC50 values were calculated by nonꢀ
linear regression with Prism’s oneꢀsite receptor competitive binding model. All compounds were independently
assayed at least twice and fitting R2 values were ≥ 90%; results are reported as fitted IC50 ± SD.
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Biodistribution and regional brain uptake in rats. All animal experiments were conducted in compliance
with the Guidelines for the Care and Use of Research Animals under protocols approved by the Washington
University Animal Studies Committee. For the biodistribution studies, 1.48 – 2.22 MBq of the radiotracer was
injected in the tail vein of mature male SpragueꢀDawley rats (250 − 400 g) under anesthesia (2.5% isoflurane in
oxygen). At 5, 30, and 60 min post ꢀinjection (p.i.), rats (n=4 per study group) were anesthetized and
euthanized. The whole brain was quickly removed and the hippocampus, striatum, cortex, thalamus, brain stem,
and cerebellum were separated by gross dissection. The remainder of the brain was collected to determine total
brain uptake. Peripheral tissues including blood, lung, heart, liver, kidney, muscle, fat, and bone were also
collected. Samples were weighed and counted in an automated gamma counter (Beckman Gamma 8000 well
counter) with a standard of diluted injectate, and the %ID/g was calculated.
MicroPET brain imaging studies in male cynomolgus monkeys. Three independent PET studies were done
on adult male cynomolgus macaques (~4−6 kg) for each tracer with a microPET Focus 220 scanner
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(Concorde/CTI/Siemens Microsystems, Knoxville, TN). The animal was fasted for 12 h before PET study and
initially anesthetized with ketamine (10 mg/kg) and glycopyrrolate (0.13 mg/kg) intramuscularly. Upon arrival
at the scanner, the animal was intubated and percutaneous catheters were placed for venous tracer injection and
arterial blood sampling. The head was positioned supine in the adjustable head holder with the brain in the
center of the field of view. Anesthesia was maintained at 0.75−2.0% isoflurane in oxygen and core temperature
maintained at ~37 °C. A 10 min transmission scan was performed to confirm positioning; this was followed by
a 45 min transmission scan for attenuation correction. Subsequently, a 3 h dynamic emission scan was acquired
after venous injection of 185 ꢀ 259 MBq of [18F]18d or [18F]20a.
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MicroPET image processing and analysis. Acquired list mode data were histogrammed into a 3D set of
sinograms and binned to the following time frames: 3 × 1 min, 4 × 2 min, 3 × 3 min, and 20 × 5 min. Sinogram
data were corrected for attenuation and scatter. Maximum a posteriori (MAP) reconstructions were done with
18 iterations and a β value of 0.004. A 1.5 mm Gaussian filter was applied to smooth each MAP reconstructed
image. These images were then coꢀregistered with MRI images to accurately identify the anatomical regions of
interest with Amira software (Visage Imaging, Inc., Carlsbad, CA). The 3D regions of interest were manually
drawn through all planes of coꢀregistered MRI images for the caudate, putamen, and cerebellum. The regions of
interest were then overlaid on all reconstructed PET images to obtain timeꢀactivity curves (TACs). Activity
measures were standardized to body weight and dose of radioactivity injected to yield standardized uptake value
(SUV).
HPLC metabolite analysis of NHP plasma. Arterial blood samples (~1.5 mL) were collected 2, 15, 30, 60 and
90 min p.i. from a percutaneous catheter in a heparinized syringe and 1 mL aliquots were counted in a well
counter then centrifuged to separate red blood cells from plasma. Plasma was deproteinated with 2 parts iceꢀ
cold methanol and plasma proteins were separated by centrifugation. The solvent extract (200 µL) was injected
on an Agilent SB Cꢀ18 analytic HPLC column (250 mm × 4.6 mm, 5 ꢁÅ), with a mobile phase of MeCN/0.1 M
ammonium formate buffer, pH 4.5 ([18F]18d, 50/50, v/v; ([18F]20a, 52/48, v/v), flow rate 0.9 mL/min, mass
peaks were detected by UV at 254 nm. HPLC fractions were collected at 1 min intervals for 16 min and counted
on a well counter and decayꢀcorrected. The percent parent and percent radiolabeled metabolites for [18F]18d and
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[18F]20a was calculated by dividing the recovered activity in the respective peaks by the sum of the total
recovered off the column and multiplying by 100. The parent compound peaks were authenticated by coꢀ
injection with the respective nonradioactive standard.
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Associated Content
9
Molecular formula strings of final products and IC50 values for PDEs in a csv format.
Supporting Information Table of Contents
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General chemistry methods
Synthesis of intermediate compounds: 6a-j, 9, 11, 12, 14, 15a-d, and 16a-n
Synthesis of radiolabeling precursors 23a-e, 23g and 24a and intermediate compounds
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Author Information
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Zhude Tu, Ph.D.
Department of Radiology
Washington University School of Medicine
Campus Box 8225
510 S. Kingshighway Blvd.
St. Louis, MO 63110, USA
Tel: 1ꢀ314ꢀ362ꢀ8487
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Fax: 1ꢀ314ꢀ362ꢀ8555
eꢀmail: tuz@mir.wustl.edu
Acknowledgement
Financial support for these studies was provided by the National Institute of Health (NIH/NINDS, NS061025,
NS075527, NIH/NIMH MH092797). We would like to acknowledge the Department of Chemistry staff and the
Washington University High Resolution NMR Facility for assistance with NMR spectra. Purchase of the 400
MHz NMR instrument was partially supported by S10 RR027207 from the NIH Shared Instrument Grant
program. We would like to thank personnel of the Washington University Cyclotron Facility for [18F]fluoride
production. We would also like to thank personnel in the East Building Imaging Research Center for technical
assistance with the nonhuman primate imaging studies, and Lynne Jones, Adam Rosenberg and Xuyi Yue for
assistance in editing the manuscript.
Abbreviations Used
cGMP, cyclic guanosine monophosphate; DAST, diethylaminosulfur trifluoride; DBAD, diꢀtertꢀbutyl
azodicarboxylate; DIEA, N,Nꢀdiisopropylethylamine; DMC, dimethyl carbonate; EOS, end of synthesis; EtOH,
ethanol; EtOAc, ethyl acetate; FPEG, fluoroPEGylated; MeCN, acetonitrile; MSNs, medium spiny neurons;
MOMꢀBr, bromo(methoxy)methane; MPꢀ10, 2ꢀ((4ꢀ(1ꢀmethylꢀ4ꢀ(pyridinꢀ4ꢀyl)ꢀ1Hꢀpyrazolꢀ3ꢀyl)phenoxy)ꢀ
methyl)quinoline; NHP, nonhuman primate; phenyl triflimide, Nꢀphenylꢀbis(trifluoromethane sulfonamide);
PDE3A, phosphodiesterase 3A; PDE3B, phosphodiesterase 3B; PDE4A, phosphodiesterase 4A; PDE4B,
phosphodiesterase 4B; PDE10A, phosphodiesterase 10A; p.i., postꢀinjection; QC, quality control; RCY,
radiochemical yield; ROI, region of interest; SA, specific activity; TBSCl, tertꢀbutyldimethylsilyl chloride;
TEA, triethylamine.
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