Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer

Article abstract of DOI:10.1021/jm3002372

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K_D) and PDE10A target density (B_max) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [ ³H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [³H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

Full text of DOI:10.1021/jm3002372

Article
pubs.acs.org/jmc
Rapid Identification of a Novel Small Molecule Phosphodiesterase
10A (PDE10A) Tracer
Essa Hu,*,† Ji Ma,# Christopher Biorn,⊥ Dianna Lester-Zeiner,⊥ Robert Cho,# Shannon Rumfelt,†
Roxanne K. Kunz,† Thomas Nixey,† Klaus Michelsen,§ Silke Miller,‡ Jianxia Shi,# Jamie Wong,#
Geraldine Hill Della Puppa,‡ Jessica Able,⊥ Santosh Talreja,⊥ Dah-Ren Hwang,∥ Stephen A. Hitchcock,†,∞
Amy Porter,⊥ David Immke,‡ Jennifer R. Allen,† James Treanor,‡ and Hang Chen*,⊥
†Department of Small Molecule Chemistry, ‡Department of Neuroscience, §Department of Molecular Structures, and ∥Department of
Medical Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States
#
⊥Department of Neuroscience and Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard,
South San Francisco, California 94080, United States
ABSTRACT: A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead
optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid
identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC−MS/MS
technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake
in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in
vivo. The PDE10A affinity (KD) and PDE10A target density (Bmax) were determined to be 0.94 nM and 2.3
pmol/mg protein, respectively, using [3H]5 on rat striatum homogenate. Autoradiography on rat brain
sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific
binding of [3H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-
10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats
using LC−MS/MS technology.
cleaves the cyclic 3′,5′-phosphodiester bonds of cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP) to generate adenosine monophosphate (AMP) and
guanosine monophosphate (GMP).2 PDE10A is most highly
expressed in the GABAergic medium spiny neurons of the
striatum which is the main relay for glutamatergic cortical input
and dopaminergic input within the basal ganglia.3 Phenotypic
analyses of PDE10A knockout mice (PDE10A−/−)4 have shown
that these mice have dampened responses in models of
psychosis and disrupted basal ganglia function. Furthermore,
TP-104 and MP-105 (Figure 1), both selective inhibitors of
INTRODUCTION
■
Radiotracers have been used in clinical studies as biomarkers to
determine target coverage in the human brain. Molecular
imaging approaches using target-specific, well-behaved PET
(positron emission tomography) or SPECT (single-photon
emission computed tomography) radiotracers have generated
valuable data, providing researchers with a noninvasive method
to correlate CNS target coverage with drug pharmacokinetics
(PK) in the plasma, to facilitate clinical design and to help
interpret clinical outcomes.1 Despite their demonstrated utility
in the clinic, use of radiotracers in CNS preclinical drug
discovery efforts has been limited because of challenges of
developing a novel target specific radiotracer. In addition to
high intrinsic potency and brain permeability, radiotracers must
also exhibit high specificity, high brain uptake, and suitable
target binding kinetics. Nevertheless, a target-specific radio-
tracer can be a very powerful translational tool in preclinical
research, allowing the measurement of binding characteristics of
the target (KD and Bmax) in tissues across species as well as
enabling assessment of target occupancy during the lead
optimization. Furthermore, such a tool can be used to correlate
target occupancy of a drug candidate with preclinical efficacy
and target engagement experiments to support dose selection
in clinical studies.
Figure 1.
PDE10A, have been reported to be efficacious in preclinical
rodent behavioral models of schizophrenia such as phencycli-
dine (PCP) stimulated hyperlocomotor activity model,
prepulse inhibition model (PPI), and conditioned avoidance
The impetus to develop a radiotracer for preclinical research
stemmed from our interest in developing an inhibitor for
phosphodiesterase 10A (PDE10A) for the treatment of
schizophrenia. Phosphodiesterase 10A is a hydrolase that
Received: February 22, 2012
© XXXX American Chemical Society
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a
Scheme 1. Synthesis of Tracer Candidates 5, 6, and 7
a
Reagents and conditions: (a) NaNO2, HCl, water, 75 °C, 71% yield; (b) POBr3, MeCN, 70 °C, 77% yield; (c) Cs2CO3, Pd(PPh3)4, DME, water;
75% yield; (d) for 5, propan-2-amine, K2CO3, DMSO, 90 °C, 71% yield; (i) for 6, 2,2-difluoroethanamine, K2CO3, DMSO, 90 °C, 45% yield; (ii)
NaH, EtI, DMF, 48% yield; for 7, 1-(pyridin-2-yl)propan-2-amine, K2CO3, DMSO, 90 °C, then chiral separation by SFC.
a
Scheme 2. Synthesis of Precursor 15 and Subsequent Radiosynthesis of [3H]5
a
Reagents and conditions: (a) acetic anhydride, nitric acid, 82% yield; (b) AcOH, iron, 89% yield; (c) NaNO2, HCl, water, 65 °C, 91% yield; (d)
POBr3, 41% yield; (e) LHMDS, BnOH, 50 °C, 47% yield; (f) Na2CO3, Pd(PPh3)2Cl2, DME, water; (g) H2, Pd/C; (h) K2CO3, CT3I; (i) propan-2-
amine, K2CO3, DMSO, 90 °C.
response (CAR).6 These results support the concept that
inhibition of PDE10A could have therapeutic benefit in
neurological disorders, including schizophrenia, Parkinson’s
disease, and Huntington’s disease.7
RESULTS AND DISCUSSION
■
Chemistry and Radiochemistry. Syntheses of the three
tracer candidates discussed in this report are described in
Scheme 1. All three compounds shared the same core scaffold.
The commercially available 1-(2-amino-4,5-dimethoxyphenyl)-
ethanone (1) was heated with sodium nitrite to produce 6,7-
dimethoxycinnolin-4-ol (2) in 71% yield. The resulting phenol
was subjected to phosphorus oxybromide to generate 4-bromo-
6,7-dimethoxycinnoline (3). Suzuki coupling with (6-fluoro-5-
methylpyridin-3-yl)boronic acid afforded the 4-(6-fluoro-5-
methylpyridin-3-yl)-6,7-dimethoxycinnoline scaffold (4). Trac-
er candidates 5 and 7 were synthesized by SNAr displacement
of pyridyl fluoride in DMSO with propan-2-amine and 1-
(pyridin-2-yl)propan-2-amine, respectively. Enantiopure tracer
7 was isolated after chiral separation of racemic mixture 5-(6,7-
dimethoxycinnolin-4-yl)-3-methyl-N-(1-(pyridin-2-yl)propan-
2-yl)pyridin-2-amine using supercritical fluid chromatography
(SFC). Candidate 6 was obtained in two steps starting with a
similar SNAr displacement of intermediate 4 with 2,2-
difluoroethanamine followed by alkylation with iodoethane.
Traditionally, a tracer candidate has to be radiolabeled prior
to any in vivo kinetics profiling. The need to radiolabel multiple
candidates and the need to develop different synthetic routes to
enable radiolabeling prior to tracer selection make this
approach both time and cost prohibitive. Instead, we directly
evaluated the kinetics of unlabeled compounds in vivo by
utilizing a method based upon a liquid chromatography
coupled to mass spectrometry (LC−MS) technology, first
reported by Phebus et al.8 to evaluate profiles of known tracers
in rats. Using both a set of in vitro profile criterion to identify
brain penetrant compounds and this LC−MS technology to
measure brain distribution, we have rapidly identified 5 (AMG-
7980)9 as the optimal tracer. Further in vivo studies confirmed
suitable kinetic properties of the tracer.
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Table 1. Summary of Tracer Candidates Properties
Figure 2. LC−MS/MS tracer kinetics distribution of 5 in vivo in rats. (A) Peak intensity in striatal tissue. LC−MS/MS quantification of 5 was done
by measuring MRM transition of m/z 391.1−281.0. (B) Peak intensity of internal control. (C) Time course and tissue distribution of 5 in vivo in
rats. The concentrations of 5 in striatum (blue circle), thalamus (purple triangle), and plasma (red dot) were measured by LC−MS/MS. Green
squares represent specific uptake (striatum minus thalamus). (D) Percentage of injected dose per gram of tissue (% ID/g) in the target tissue
(striatum). Error bars represent standard error of mean, and N = 4 animals per group.
Rodent metabolite identification (ID) studies suggested that
the 7-methoxy group on the cinnoline was a suitable position
for tritium radiolabeling. In the original synthetic route
(Scheme 1), this 7-methoxy group was introduced in the first
step. To maximize the radiochemical yield of [3H]5, we
designed a different synthetic route in order to reduce the
number of steps after tritium labeling (Scheme 2). Our
redesigned synthetic route began with nitration of commercially
available 1-(4-fluoro-3-methoxyphenyl)ethanone (8) to pro-
duce 1-(4-fluoro-5-methoxy-2-nitrophenyl)ethanone (9). Iron
reduction afforded 1-(2-amino-4-fluoro-5-methoxyphenyl)-
ethanone (10). Cyclization with sodium nitrite formed 7-
fluoro-6-methoxycinnolin-4-ol (11). Reflux with phosphorus
oxybromide generated 4-bromo-7-fluoro-6-methoxycinnoline
(12). Treatment of sodium benzylate with 4-bromo-7-fluoro-
6-methoxycinnoline produced the protected 7-(benzyloxy)-4-
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bromo-6-methoxycinnoline (13). After Suzuki coupling to
make 7-(benzyloxy)-4-(6-fluoro-5-methylpyridin-3-yl)-6-me-
thoxycinnoline (14), radiolabel precursor cinnolin-7-ol (15)
was unmasked by palladium hydrogenation. Tritiation with
[3H]methyl iodide followed by SNAr displacement with propan-
2-amine produced the tritiated radiotracer [3H]5.
Pharmacology. Three tracer candidates 5, 6, and 7, were
selected from our collection of PDE10A inhibitors for
advancement into in vivo studies. These compounds were
chosen based on a set of in vitro and physiochemical criteria:10
single-digit nanomolar IC50 potency on PDE10A, greater than
100-fold selectivity over other PDEs, moderate lipophilicity
(log P of 1−4), and good permeability devoid of P-gp substrate
recognition (P-gp efflux ratio of <3) (Table 1). These
parameters were selected to ensure that the tracer candidates
would be specific to PDE10A and CNS permeable.
Figure 3. Biacore assay for KD determination. Unlabeled 5 was tested
at 1000, 200, 40, and 8 nM. Kinetic titration experiments were
performed by injecting the low to high inhibitor solutions at 2 min
intervals onto the PDE10A chip surface (black trace, measured data;
red trace, best fit to 1:1 binding model).
LC−MS/MS Tracer Kinetics. We employed the LC−MS/
MS based technology platform developed by Phebus et al.7 to
evaluate the binding kinetics of these tracer candidates in rats
using unlabeled compounds. Unlabeled compound was
administered as a microdose (3−10 μg/kg) in rats by bolus
intravenous injection. The concentrations of the tracer
candidate in striatum (as the target tissue), thalamus (as the
reference tissue), and plasma samples were measured through
quantitative LC−MS/MS analysis with the MRM11 (multiple
reaction monitoring) transition as m/z 391.1→ 281.0 (e.g., 5,
Figure 2A and Figure 2B). The lowest level of quantitation
(LOQ) was 0.1 ng/g for brain tissues and 0.01 ng/mL for
plasma samples.
Whereas all three candidates exhibited comparable in vitro
profiles, their tracer candidacies were readily differentiated in
vivo through our LC−MS/MS platform. In terms of target
tissue specificity, both compounds 5 and 6 exhibited good
striatum to thalamus ratios of 9.4 and 11.8, respectively, based
on the AUC (area under the curve). Candidate 7 demonstrated
a low ratio of 2.6. High striatal uptake was observed with
compounds 5 and 7, both achieving greater than 1% injected
dose per gram of tissue (% ID/g) at 10 min after injection,
whereas the value was lower with compound 6 at 0.3% ID/g. As
a result, 5 clearly emerged as the compound for advancement
because it showed both high target tissue specificity and high
target tissue uptake (Figure 2C and Figure 2D).
Surface Plasmon Resonance (SPR) Binding Assay. The
LC−MS/MS results showed a steep drop of 5 concentration
over time, suggesting fast in vivo kinetics. To better characterize
the PDE10A binding properties of this molecule, we utilized a
surface plasmon resonance (SPR) spectroscopy binding assay
with purified truncated human PDE10A enzyme (amino acids
442−779) covalently attached to a CM5 (carboxymethyl 5)
chip to measure affinity (KD), on-rate (ka), and off-rate (kd).
Consistent with its PDE10A inhibition potency (IC50 = 1.9
nM), 5 showed high affinity to PDE10A with a KD of 1.2 nM
(Figure 3). This molecule exhibited fast on-rate (ka = 1.4 × 108
M−1 s−1) and fast off-rate (kd = 1.7 × 10−1 s−1). The derived
T1/2 was only 0.1 min. This fast off-rate in vitro supports the
fast washout observed in Figure 2 above.
In Vivo Saturable Binding and PDE10A Bmax Determi-
nation. High saturable specific binding in target tissue is one
requisite tracer characteristic for measurement of target−ligand
interaction. To demonstrate saturable striatal uptake of the
tracer candidate in vivo, 5 was administered at a range of
dosages from 1 to 1000 μg/kg in rats by bolus intravenous
injection. Striatal tissues, thalamic tissues, and blood samples
were collected at 10 min after dosing. Compound concen-
trations in the striatum and the thalamus were measured using
LC−MS/MS. Specific striatum uptake was derived by
subtraction of the tracer levels in the thalamus (as the
reference tissue for nonspecific binding due to its low
PDE10A expression level) from the levels in the striatum (as
total binding due to its high PDE10A expression level) (Figure
4A). The resulting curve shape indicated that specific striatal
binding of 5 was able to reach saturation in vivo. This study
also enabled measurement of target density of PDE10A in the
striatum showing a Bmax of 1.15 0.11 pmol/mg (388.4 38.4
ng/g) of wet tissue. The ED50 (dosage to reach 50% of plateau)
was determined by nonlinear regression to arrive at a value of
219.6 μg/kg. To derive target occupancy of 5, 100% target
coverage was set as the maximum (plateau) value of striatal
specific uptake. Plotting percent of target occupancy against the
tracer concentration in the plasma (Figure 4B) revealed a
plasma EC50 of 101.8 10.3 nM (53.2 3 ng/mL).
Characterization of in Vitro [3H]5 Binding to PDE10A
Using Rat Striatal Homogenate. Radiolabeled tracer, [3H]5,
was utilized to perform KD and Bmax assessments on rat striatal
homogenate. For comparison, nonspecific binding was
calculated in two different ways: one using an excess of
nonradiolabeled compound (Figure 5A), the other using [3H]5
bound to thalamus tissue (Figure 5B). Both approaches showed
[3H]5 bound to striatal homogenates in a saturable manner.
The binding affinity was calculated using nonlinear regression
to an equilibrium dissociation constant (KD) of 0.94 nM. This
is consistent with the value measured by the Biacore assay (KD
= 1.2 nM). The Bmax of rat striatum tissue for PDE10A binding
by [3H]5 was 2.1 pmol/mg of protein. The PDE10A target
density in the striatum derived from in vitro homogenate
binding data was consistent with target density derived from in
vivo striatal binding using our LC−MS/MS technology. This
comparison of nonspecific binding calculations further
confirmed that thalamus was sufficient as the reference tissue
to compare against target tissue binding in the striatum.
In Vitro Binding Displacement. To determine whether
the tracer could be displaced, [3H]5 was studied in binding
displacement experiments first with unlabeled 5 and then with a
known selective and structurally distinct PDE10A inhibitor 17
(MP-10) (Figure 6). By use of the Cheng−Prusoff equation,12
Ki values were calculated for 5 (IC50 = 3.47 nM and Ki = 1.18
nM) and 17 (IC50 = 0.50 nM and Ki = 0.17 nM). The
displacement of [3H]5 by the unlabeled 5 showed that the
binding to PDE10A was competitive. Meanwhile, the displace-
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Figure 4. In vivo saturation binding of PDE10 by 5. Rats were intravenously administered 5 doses ranging from 1 to 1000 μg/kg. At 10 min after
injection, animals were euthanized and tissues were collected. The tracer concentration levels were determined using LC−MS/MS. (A) Saturable
striatum specific uptake of 5. Blue circles represent striatum concentration as total binding. Red circles represent the thalamus concentration as
nonspecific binding. Purple squares represent (STR-THA) specific binding. GraphPad Prism, version 5, software was used for nonlinear regression of
the curve fit. Results are expressed as the mean SEM (standard error of the mean), N = 4 animals per group. (B) Correlation of the tracer plasma
concentration with calculated target coverage (RO%). The plateau value of the striatum specific binding (STR-THA) was used as 100% of target
occupancy.
Figure 5. Saturation binding of [3H]5 in rat striatum homogenates for PDE10A target density (Bmax) and the apparent affinity (KD) determination
using two nonspecific binding calculations. Saturation binding was determined by incubating rat striatum homogenates with concentrations of [3H]5
ranging from 0.01 to 50 nM in a final volume of 200 μL. (A) Nonspecific binding was determined using an excess (1 μM) of unlabeled compound.
Blue circles represent total binding. Red circles represent nonspecific binding, and purple circles represent specific binding. (B) Nonspecific binding
was defined as [3H]5 binding to thalamic tissue homogenate. Blue squares indicate total binding. Red squares indicate nonspecific binding ([3H]5
binding to thalamic tissue homogenate), and purple squares indicate specific binding. GraphPad Prism, version 5, software was used for the nonlinear
regression of the curve fit. Data are presented as the mean SEM. Error bars in the graph represent N = 4 data points.
specific and can be used to measure target occupancy of other
PDE10A inhibitors binding to the same active site.
Ex Vivo Autoradiography of [3H]5 Distribution in Rat
Brain. To visualize the distribution of 5 in the brain in vivo,
rats (n = 3−4 per group) were dosed with 3, 10, 30, or 100 μCi
[3H]5 via a jugular vein catheter and brains were collected 10
min after dosing. The best signal-to-noise ratio of >3 was
obtained with 30 μCi (not shown). Therefore, a subsequent set
of rats (n = 3−4 per group) was dosed with vehicle or 10 mg/
kg 17 and 30 μCi [3H]5 was administered via a jugular vein
catheter after 50 min. Brains were collected 10 min after tracer
Figure 6. [3H]5 binding displacement with a selective PDE10A
inhibitor. The dose−response curves of various PDE10A inhibitors
injection, and sagittal brain sections (n = 4 per brain) were
analyzed using real-time autoradiography. Radioactive uptake
was the highest in the striatum, and very low binding was
detectable in other brain regions including the thalamus (Figure
7A). The binding pattern of [3H]5 in vivo was consistent with
the published results.3 Striatum and thalamus were selected as
regions of interest, and binding was measured as counts/mm2
using M3vision (Biospace, Paris, France). Specific striatal
binding was determined by subtracting thalamus binding from
striatum and blocked by predosing with 17 by an average of
74% (Figure 7B).
were determined in competition binding studies using native striatum
tissue homogenates with the concentration of [3H]5 at approximately
the KD in the presence of increasing concentrations of displacer
compound from 5 pM to 100 nM. Open circles represent 17, and filled
circles represent 5. Curves were analyzed using nonlinear regression
on GraphPad Prism. Results are expressed as the mean
SEM
(standard error of mean), and error bars in the graph represent N = 2
per data point. The Ki values were calculated for 17 (IC50 = 0.50 nM
and Ki = 0.17 nM) and the 5 (IC50 = 3.47 nM and Ki = 1.18 nM).
ment of [3H]5 by 17, a structurally distinct and potent
PDE10A inhibitor, indicated that the tracer is not scaffold
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CONCLUSION
■
Using a combination of stringent selection criterion and the
LC−MS/MS technology to screen unlabeled tracer candidates
in rodents, we rapidly identified a promising PDE10A tracer 5
based on in vivo evaluation of three nonradiolabeled PDE10A
tracer candidates with similarly promising in vitro character-
istics, bypassing the traditionally cost and time prohibitive
requirement of radiolabeling tracer candidates prior to in vivo
tracer profiling. Tracer 5 exhibited the superior in vivo profile,
with high affinity, high specificity (good striatum to thalamus or
target to reference ratio), and good striatum uptake. The in
vitro and in vivo features of this molecule were extensively
characterized using both the unlabeled 5 and the [3H]5. By use
of ex vivo autoradiography, [3H]5 was used to assess relative
expression levels of PDE10A in rat brain tissues and target
occupancy of 17. Using LC−MS/MS technology, we
demonstrated the utility of 5 by measuring the in vivo
PDE10A target occupancy of 17. These results further
suggested that 5 could possess suitable characteristics to be a
PET imaging tracer similar to other PDE10A PET tracers
reported to date.13 Our findings on advancing 5 as a PET tracer
candidate will be reported in a separate publication.
EXPERIMENTAL SECTION
■
Chemistry, Materials, and General Methods. Unless otherwise
noted, all reagents and solvents were obtained from commercial
suppliers such as Aldrich, Sigma, Fluka, Acros, EDM Sciences, etc. and
used without further purification. Dry organic solvents (dichloro-
methane, acetonitrile, DMF, etc.) were purchased from Aldrich
packaged under nitrogen in Sure/Seal bottles. All reactions involving
air or moisture sensitive reagents were performed under a nitrogen or
argon atmosphere. Silica gel chromatography was performed using
prepacked silica gel cartridges (Biotage or RediSep). Microwave
assisted reactions were performed in Biotage Initiator Sixty microwave
Figure 7. Visualization of in vivo PDE10A binding and occupancy by
17 by autoradiography with [3H]5. (A) Diagram of sagittal section of
rat brain which indicates the areas of autoradiography results: Ctx,
cortex; Th, thalamus; Str, striatum; Bs, brain stem; Cer, cerebellum.
(B) Representative autoradiograph of a sagittal section from a rat
dosed with vehicle and 30 μCi [3H]5 showing high binding in the
striatum and only low binding in other brain areas. (C) Representative
autoradiograph of a sagittal section from a rat dosed with 17 (10 mg/
kg po, 60 min) and 30 μCi [3H]5, demonstrating a strong blockade of
uptake in the striatum. Color bar represents counts, and scale bar
respresents 5 mm.
1
reactor. H NMR spectra were recorded on a Bruker DRX 300 MHz,
Bruker DRX 400 MHz, Bruker AV 400 MHz, Varian 300 MHz, or a
Varian 400 MHz spectrometer at ambient temperature. Chemical
shifts are reported in parts per million (ppm, δ units). Data are
reported as follows: chemical shift, number of protons, multiplicity (s
= singlet, d = doublet, t = triplet, q = quartet, br = broad, m =
multiplet), coupling constants, and number of protons. Reactions were
monitored using Agilent 1100 series LC/MSD SL high performance
liquid chromatography (HPLC) systems with UV detection at 254 nm
and a low resonance electrospray mode (ESI). All final compounds
were purified to >95% purity, as determined by high performance
liquid chromatography (HPLC). HPLC methods used the followung:
Agilent 1100 spectrometer, Zorbax SB-C18 column (50 mm × 3.0
mm, 3.5 μm) at 40 °C with a 1.5 mL/min flow rate; solvent A of 0.1%
TFA in water, solvent B of 0.1% TFA in MeCN; 0.0−3.0 min, 5−95%
B; 3.0−3.5 min, 95% B; 3.5−3.51 min, 5% B. Flow from UV detector
was split (50:50) to the MS detector, which was configured with API-
ES as ionizable source. All high resolution mass spectrometry (HRMS)
data were acquired on a Synapt G2 HDMS instrument (Waters
Corporation, Manchester, U.K.) operated in positive electrospray
ionization mode. The sample was diluted to 10 μM in 50% acetonitrile
(v/v), 0.1% formic acid (v/v) and infused into the mass spectrometer
at a flow rate of 5 μL/min through an electrospray ionization source
operated with a capillary voltage of 3 kV. The sample cone was
operated at 30 V. The time-of-flight analyzer was operated at a
resolution (fwhm) of 30 000 at m/z 785 and was calibrated over the
m/z range 50−1200 using a 1 μM sodium iodide (NaI) solution (50%
v/v acetonitrile solution). To obtain accurate mass measurements, an
internal lock-mass correction was applied using leucine enkephalin
(m/z 556.2771). Collision induced fragmentation (CID) was
performed using an injection voltage of 28 V. Instrument control
was performed through the software suite MassLynx, version 4.1.
In Vivo Measurement of Target Occupancy. To
demonstrate the full utility of 5, we performed PDE10A
occupancy studies in vivo in rats with 17 using the LC−MS/
MS technology. Blocker 17 was administered orally with
dosages 1, 3, 10, and 30 mg/kg. Tracer 5 (6 μg/kg) was
injected intravenously at 50 min after 17 dosing. The dosage of
the tracer was chosen to occupy less than 5% of the target,
sufficient to generate reliable signal values for the binding
potential (BP) calculation.8 Ten minutes later, samples from
blood, striatum, and thalamus were collected. Using LC−MS/
MS analysis, we obtained blocker 17 exposure levels in the
plasma and striatum (Figure 8A) and obtained tracer 5
exposure levels in the target tissue (striatum), reference tissue
(thalamus), and plasma (Figure 8B). The maximum striatal
PDE10A occupancy by 17 at 30 mg/kg was 91.2%. Also
noteworthy was that target occupancy by 17 at 10 mg/kg was
83.8%, comparable to the occupancy of 74% measured by [3H]
5 with autoradiography. The ED50 of 17 to occupy PDE10A in
the striatum was measured with a value of 2.3
0.3 mg/kg
(Figure 8C). The target coverage of 17 was also plotted against
plasma and striatum exposure levels to obtain the EC50 values
of 241.5 36.1 nM and 639.7 69.1 nM, respectively. This
experiment confirmed that 5 is a useful tracer for measurement
of target occupancy of PDE10A inhibitors in vivo.
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Figure 8. Determination of in vivo PDE10A occupancy of 17 using tracer 5. Both the blocker 17 concentration and tracer 5 concentrations in the
plasma, striatum, and thalamus tissues were measured by LC−MS/MS from the same sample preparation. (A) 17 exposure levels in the plasma (red
asterisks), striatum (blue circles), and thalamus (purple squares) at 1 h after oral dosing. (B) 5 concentrations in the plasma (red asterisks), striatum
(blue circles), and thalamus (purple squares) were plotted against the 17 dosages. (C−E) 17 target occupancy in the striatum was plotted against 17
dosages, exposure levels in the plasma, and exposure levels in the striatum, respectively, to calculate ED50 and EC50 in plasma and striatum. Curves
were analyzed using nonlinear regression on GraphPad Prism. Results in parts A and B are expressed as the mean SEM (standard error of the
mean), and error bars in graph represent N = 4 animals per dosage group.
Compounds. Tritium-labeled compound [3H]5 was synthesized
by Moravek Biochemicals, Inc., with a specificity of 45 Ci/mmol (in
ethanol solution) and 99.5% purity as analyzed by HPLC.
with 40 mL of chloroform. The reaction mixture was allowed to stir at
65 °C for 3.5 h. The mixture was allowed to cool to room temperature,
and the volatile solvent was removed by vacuum. The solid residue was
then poured onto ice, and to it was added saturated NaHCO3. The
aqueous mixture was filtered to collect the solid. The solid was placed
in a rbf and triturated with 4 mL of DCM and 20 mL of hexane. The
solid was filtered and dried by vacuum. The solid was recrystallized in
hot MeOH to afford desired intermediate 3 (1.63 g, 62.3% yield). H
NMR (300 MHz, DMSO): δ ppm 4.05 (s, 6H), 7.22 (s, 1H), 7.79 (s,
1H), 9.40 (s, 1H). LC−MS: (m + 1 = 269.0).
4-(6-Fluoro-5-methylpyridin-3-yl)-6,7-dimethoxycinnoline
(4). To a round-bottomed flask were added 3 (0.6372 g, 2.4 mmol), 6-
fluoro-5-methylpyridin-3-ylboronic acid (0.4094 g, 2.6 mmol),
palladium tetrakistriphenylphosphine (0.1525 g, 0.12 mmol), and
cesium carbonate (2.1 g, 6.4 mmol) in DME (250 mL) and water (10
mL). The mixture was stirred at 80 °C overnight. The reaction mixture
was diluted with water and extracted with EtOAc. The organic extract
was washed with water, saturated NaCl, dried over MgSO4, filtered,
and concentrated in vacuo. The solid was allowed to stir in ether for 15
min and was filtered. The solid was dried by vacuum to afford the
desired intermediate 4 (0.46 g, 65% yield). LC−MS shows product
peak at 4.852 min (m + 1 = 300.1). 1H NMR (300 MHz, chloroform-
6,7-Dimethoxycinnolin-4-ol (2). 2′-Amino-4′,5′-dimethoxyaceto-
phenone (15.82 g, 81.0 mmol) was solubilized in concentrated HCl
(460 mL) and water (80 mL). The temperature was cooled to −5 °C
using ice/brine bath, and the resulting mixture was stirred vigorously.
A solution of sodium nitrite (5.67 g, 81.8 mmol) and water (29.2 mL,
1621 mmol) was added to the reaction mixture over 1 h. The mixture
was allowed to continue to stir for 1 h. Then the temperature was
raised to 60 °C. After heating for 4 h, the solution was allowed to cool
to room temperature. The solid was filtered and collected. The solid
was transferred to a rbf and suspended with deionized water. The pH
was adjusted to ∼12 by adding NaOH. The solution was then
neutralized with concentrated HCl. A precipitate was formed and
1
1
filtered to produce desired intermediate 2 (13.78 g, 82.5% yield). H
NMR (300 MHz, DMSO): δ ppm 3.88 (s, 6H), 6.94 (s, 1H), 7.31 (s,
1H), 7.62 (s, 1H), 13.35 (s, 1H). LC−MS: (m + 1 = 207.1). Material
was carried to the next step without further purification.
4-Bromo-6,7-dimethoxycinnoline (3). To a 150 mL round-
bottomed flask were added 6,7-dimethoxycinnolin-4-ol (2.0049 g,
9.723 mmol) and phosphorus oxybromide (1.387 mL, 13.61 mmol)
G
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d): δ ppm 2.44 (s, 3 H), 3.95 (s, 3 H), 4.14 (s, 3 H), 6.99 (s, 1 H),
7.80 (dd, J = 9.21, 1.61 Hz, 1 H), 7.84 (s, 1 H), 8.26 (s, 1 H), 9.02 (s,
1 H).
Hz, 3 H), 3.64 (q, J = 7.02 Hz, 2 H), 3.89−4.07 (m, 5 H), 4.13 (s, 3
H), 6.78 (d, J = 8.48 Hz, 1 H), 7.18 (s, 1 H), 7.72 (dd, J = 8.77, 2.48
Hz, 1 H), 7.80 (s, 1 H), 8.39 (d, J = 1.90 Hz, 1 H), 9.02 (s, 1 H).
HRMS (ES+) calculated for [C19H20F2N4O2]+: 374.385. Found:
374.155.
5-(6,7-Dimethoxycinnolin-4-yl)-N-isopropyl-3-methylpyri-
din-2-amine (5). In a glass microwave reaction vessel were placed 4
(0.0545 g, 0.18 mmol) and isopropylamine (0.156 mL, 1.8 mmol) in
DMSO (2 mL), and the mixture was stirred at 90 °C overnight. The
crude product was adsorbed onto a plug of silica gel and
chromatographed through a Biotage prepacked silica gel column
(40S), eluting with a gradient of 1−5% MeOH in CH2Cl2 over 20
column volumes to provide 5 (0.0438 g, 71% yield, 99% purity). LC−
1-(4-Fluoro-5-methoxy-2-nitrophenyl)ethanone (9). A 2 L
three-neck round-bottom flask equipped with an overhead stirrer,
addition funnel with nitrogen inlet, and thermocouple was charged
with 8 (54.06 g, 321 mmol) and acetic anhydride (220 mL). The
resulting solution was cooled with an ice/water bath. Nitric acid (70%
a.c.s, 43.4 mL, 964 mmol) was added dropwise over 30 min. At the
end of the addition, the cooling bath was removed and stirring was
continued at room temperature. After ∼18 h of stirring, the reaction
mixture was heated to an internal temperature of 45 °C for 1 h and 20
min. Heating was stopped, and the reaction mixture was cooled to
internal temperature of 3 °C. The solid that formed was isolated by
filtration, washed with ice cold water, and dried. The filtrates were
carefully combined and allowed to stand at an internal temperature of
7 °C for 30 min. A second batch of solids were isolated by filtration
and dried. The filtrate was partitioned between water and ethyl acetate.
The water layer was extracted with EtOAc, and the organic layer was
washed with an aqueous saturated NaHCO3 solution, dried over
MgSO4, filtered, and concentrated in vacuum. Purification was by
MPLC (hexanes/EtOAc, 100/0 to 90/10, then 80/20). The isolated
product plus the solids collected by filtration combined gave the
desired intermdiate 9 (68.5 g, 82% yield). 1H NMR (400 MHz,
DMSO-d6): δ ppm 8.15 (d, J = 10.96 Hz, 1 H), 7.46 (d, J = 8.22 Hz, 1
H), 4.01 (s, 3 H), 2.55 (s, 3 H).
1
MS product peak was found at 2.8 min (m + 1 = 339.2). H NMR
(300 MHz, chloroform-d): δ ppm 1.38 (d, J = 6.28 Hz, 6 H), 2.24 (s, 3
H), 4.00 (s, 3 H), 4.16 (s, 3 H), 4.31 (d, J = 7.16 Hz, 1 H), 4.38−4.53
(m, 1 H), 7.30 (s, 1 H), 7.49 (s, 1 H), 7.83 (s, 1 H), 8.32 (s, 1 H).
HRMS (ES+) calculated for [C19H22N4O2]+: 338.404. Found:
338.174.
(S or R)-5-(6,7-Dimethoxycinnolin-4-yl)-3-methyl-N-(1-(pyr-
idin-2-yl)propan-2-yl)pyridin-2-amine (6). A mixture of 4 (200
mg, 0.67 mmol), 1-(pyridin-2-yl)propan-2-amine (140 mg, 1.0 mmol),
and DMSO (2 mL) was heated at 120 °C for 18 h. The resulting
solution was purified by preparative HPLC (10−60% CH3CN/H2O/
0.1%TFA) to give a mixture of isomers as the TFA salts (235 mg).
Chiral separation of the isomers was performed on Berger Multigram
II SFC (supercritical fluid chromatography) using a Chiralcel AS-H
(250 mm × 21 mm, 5 μm) column with 80% liquid CO2 and 20%
methanol with 0.2% DEA (diethylamine) as the mobile phase and flow
rate of 65 mL/min at 40 °C. The desired compound 6 was the second
eluting compound, and it was isolated in 33 mg yield (0.077 mmol)
with >99% enantiopurity. Enantiopurity of 6 was determined on an
analytical SFC/MS instrument using a Chiralpak AS-H (150 mm × 4.6
mm, 5 μm) with 80% liquid CO2 and 20% methanol with 0.2% DEA
(diethylamine) as the mobile phase and flow rate of 4.0 mL/min at 40
°C. Absolute stereochemistry was not determined. 1H NMR (400
MHz, MeOH-d): δ ppm 9.20 (1 H, s), 8.79 (1 H, dd, J = 5.8, 0.9 Hz),
8.49 (1 H, td, J = 7.9, 1.6 Hz), 8.35 (1 H, d, J = 2.0 Hz), 8.07 (1 H, d, J
= 8.0 Hz), 7.95−8.01 (1 H, m), 7.87−7.94 (1 H, m), 7.79 (1 H, s),
7.44 (1 H, s), 4.76−4.88 (1 H, m), 4.17 (3 H, s), 4.10 (3 H, s), 3.43−
3.64 (2 H, m), 2.37 (3 H, s), 1.49 (3 H, d, J = 6.7 Hz). HRMS (ES+)
calcd for [C24H26N5O2]+: 415.488. Found: 415.201.
N-(2,2-Difluoroethyl)-5-(6,7-dimethoxycinnolin-4-yl)-N-
ethyl-3-methylpyridin-2-amine (7). In a glass microwave reaction
vessel were placed 4 (0.0555 g, 0.195 mmol) and 2,2-difluoroethan-
amine (0.1609 g, 1.95 mmol) in DMSO (2 mL), and the mixture was
stirred at 90 °C. The solution was allowed to stir for 2 days. The
reaction mixture was diluted with water and extracted with EtOAc.
The organic extract was washed with water, saturated NaCl, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product was
adsorbed onto a plug of silica gel and chromatographed through a
Biotage prepacked silica gel column (40S), eluting with a gradient of
1−5% MeOH in CH2Cl2, to provide N-(2,2-difluoroethyl)-5-(6,7-
dimethoxycinnolin-4-yl)pyridin-2-amine (0.0308 g, 45.7% yield, 97.9%
purity). LC−MS product peak was found at 4.174 min (m + 1 =
347.1). 1H NMR (300 MHz, DMSO-d6): δ ppm 3.30 (d, J = 7.16 Hz,
3 H), 3.80 (td, J = 15.60, 4.02 Hz, 1 H), 3.99−4.07 (m, 6 H), 6.81 (d, J
= 8.62 Hz, 1 H), 7.19 (s, 1 H), 7.76 (s, 1 H), 7.82 (dd, J = 8.70, 2.41
Hz, 1 H), 8.35 (d, J = 1.90 Hz, 1 H), 9.03 (s, 1 H).
1-(2-Amino-4-fluoro-5-methoxyphenyl)ethanone (10). A 1 L
three-neck round-bottom flask with mechanical stirrer, nitrogen inlet,
and thermocouple was charged with 9 (13.8 g, 64.7 mmol), acetic acid
(280 mL), and iron powder, where <10 μm (18.08 g, 324 mmol) was
added in one portion at room temperature. The resulting reaction
mixture was heated at 100 °C internal temperature. Stirring was
continued for 1.5 h. The reaction mixture was cooled to room
temperature, filtered through a plug of Celite, and rinsed with EtOAc.
The filtrate was concentrated in vacuo. Purification by MPLC
(hexanes/EtOAc, 100/0 to 80/20) afforded the desired intermediate
1
10 (10.5 g, 89% yield). H NMR (400 MHz, DMSO-d6): δ ppm 7.37
(d, J = 9.68 Hz, 1 H), 7.10 (br s, 2 H), 6.59 (d, J = 13.79 Hz, 1 H),
3.78 (s, 3 H), 2.50 (s, 3 H)
7-Fluoro-6-methoxycinnolin-4-ol (11). Hydrochloric acid, 37%
(1820 μL, 21.84 mmol), was added to a cooled (ice/water) suspension
of 10 (200 mg, 1.092 mmol) in water (4 mL). A solution of sodium
nitrite (181 mg, 2.62 mmol) in water (1 mL) was then added dropwise
at 0 °C. Stirring was continued for ∼1 h at 0 °C. The cooling bath was
removed, and the reaction mixture was stirred at 65 °C for 4 h. The
reaction mixture was cooled with an ice bath. The yellow solid that
formed upon cooling was isolated by filtration, washed with ice-cold
water, and dried under high vacuum overnight to produce the desired
1
intermediate 11 (192 mg, 91% yield). H NMR (400 MHz, DMSO-
d6): δ ppm 13.57 (br s, 1 H), 7.71 (s, 1 H), 7.55 (d, J = 9.10 Hz, 1 H),
7.45 (d, J = 11.35 Hz, 1 H), 3.95 (s, 3 H).
4-Bromo-7-fluoro-6-methoxycinnoline (12). A suspension of
11 (2.6 g, 0.013 mol) in chloroform (30 mL) under nitrogen
atmosphere was treated with phosphorus oxybromide (14.5 g, 0.0493
mol) and stirred at room temperature for 12 h. The mixture was then
heated to reflux for 4 h. Reaction mixture was poured on crushed ice
and extracted with ethyl acetate (3 × 100 mL). The combined organic
extracts were washed with aqueous saturated sodium bicarbonate
solution, dried over anhydrous sodium sulfate, and concentrated in
vacuum. The crude mixture was then purified by silica gel column
chromatography to afford the desired intermediate 12 (1.4 g, 41%
To a round-bottomed flask were added N-(2,2-difluoroethyl)-5-
(6,7-dimethoxycinnolin-4-yl)pyridin-2-amine (0.0625 g, 0.18 mmol)
and sodium hydride (0.0052 mL, 0.20 mmol) in DMF (10 mL). The
mixture was allowed to stir for 10 min, and iodoethane (0.030 mL,
0.36 mmol) was added. The mixture was stirred overnight. The
reaction mixture was diluted with water and extracted with EtOAc.
The organic extract was washed with water, saturated NaCl, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product was
adsorbed onto a plug of silica gel and chromatographed through a
Biotage prepacked silica gel column (40S), eluting with a gradient of
1−5% MeOH in CH2Cl2 to provide 7 (0.0327 g, 48% yield, 99%
purity). LC−MS product peak was found at 5.223 min (m + 1 =
1
yield) as a white solid. H NMR (DMSO): δ 4.13 (s, 3H, OCH3),
7.43−7.45 (d, 1H, 2-Ar-H), 8.36−8.39 (d, 1H, 5-Ar-H), 9.58 (s, 1H, 3-
Ar-H). TOF MS (ES+) 257.11/259.11.
7-(Benzyloxy)-4-bromo-6-methoxycinnoline (13). To a micro-
wave vial was added 12 (1.0804 g, 4.2 mmol), benzyl alcohol (0.440
mL, 4.2 mmol), and LHMDS (1 M solution in THF, 6.3 mL, 6.3
mmol) in DMF. The resulting mixture was heated to 50 °C for 3 h.
1
375.2). H NMR (300 MHz, chloroform-d): δ ppm 1.30 (t, J = 7.09
H
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The reaction mixture was allowed to cool to room temperature and
then diluted with water (10 mL) and extracted with EtOAc (3× 10
mL). The organic extract was washed with water (3 × 10 mL),
saturated NaCl solution, dried with magnesium sulfate, filtered, and
concentrated. The crude product was adsorbed onto a plug of silica gel
and chromatographed through a Biotage prepacked silica gel column
(40M), eluting with a gradient of 0.5−2.5% MeOH in dichloro-
methane to provide desired intermediate 13 (0.6807 g, 47% yield).
LC−MS: [m + 1] = 345.2 with purity of 96.7%. 1H NMR (300 MHz,
CDCl3): δ ppm 4.11 (s, 3 H), 5.37 (s, 2 H), 7.23 (s, 1 H), 7.30−7.47
(m, 3 H), 7.48−7.57 (m, 2 H), 7.78 (s, 1 H), 9.25 (s, 1 H).
7-(Benzyloxy)-4-(6-fluoro-5-methylpyridin-3-yl)-6-methoxy-
cinnoline (14). In a microwave vessel were added 13 (0.6807 g, 1.972
mmol), 6-fluoro-5-methylpyridin-3-ylboronic acid (0.3095 g, 1.998
mmol), trans-dichlorobis(triphenylphosphine)palladium(II) (0.1087 g,
0.1578 mmol), and an aqueous solution of sodium carbonate (0.1937
mL, 3.944 mmol) in DME at 80 °C. The mixture was stirred
overnight. The mixture was allowed to cool to room temperature,
diluted with water (10 mL), and extracted with DCM (3 × 10 mL).
The organic extract was washed with water (3 × 10 mL), saturated
NaCl solution, dried with magnesium sulfate, filtered, and concen-
trated. The residue was filtered through a plug of silica to produce
intermediate 14 which was directly advanced to the next step.
4-(6-Fluoro-5-methylpyridin-3-yl)-6-methoxycinnolin-7-ol
(15). To a suspension of 14 (355 mg, 946 μmol) in EtOH was added
palladium on carbon (403 mg, 378 μmol). Hydrogen was bubbled
through solution. Then the flask was flushed with hydrogen (3×), and
the mixture was stirred overnight at room temperature. The resulting
mixture was filtered through Celite, rinsing with DCM. Reverse phase
purification was performed to produce the desired intermediate 15
which was directly taken to the next step. LC−MS: 286.0 [M + H],
>99% purity.
Pharmacology. PDE10 Biochemical Assay. Functional inhib-
ition of human recombinant PDE10A was measured as described in
the IMAP TR-FRET (time-resolved fluorescence energy transfer)
assay kit protocol (Molecular Devices, Sunnyvale, CA, catalog no.
R8160, R8176, or R8159). Compound was serially diluted in 100%
DMSO from a 10 mM stock and further diluted in complete reaction
buffer (10 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3, 0.01%
Tween 20, and 1 mM freshly added DTT) to a 4× concentration.
Recombinant PDE10A enzyme and cAMP substrate were diluted in
complete reaction buffer. The optimized binding buffer was 70%
binding buffer A and 30% binding buffer B. Binding reagent (1:800)
and terbium donor (1:400) were added to the binding buffer. All
incubations were carried out at room temperature. Testing or control
compounds (5 μL of each 4× concentration per well, 12-point dose−
response curve ranging from 5.1 pM to 10 μM, tested in
quadruplicate) were incubated with 5 μL of recombinant human
PDE10A (0.06 units per well) in a 384-well microplate. After 30 min,
10 μL of cAMP substrate was added to each well for a final substrate
concentration of 100 nM. After 1 h, 60 μL of binding buffer was added
to each well. The plate was then incubated from 3 h to overnight
before reading on an EnVision plate reader. The IMAP binding
reagent binds to the nucleotide monophosphate generated from cyclic
nucleotides (cAMP/cGMP) through phosphodiesterases and enables
measurement of substrate turnover.
Permeability and Transcellular Transport. Materials. Digoxin
and mannitol were purchased from Sigma-Aldrich (St. Louis, MO).
[3H]Digoxin and [14C]mannitol were purchased from PerkinElmer
Life and Analytical Sciences (Boston, MA). Transport buffer was
prepared using Hank’s balanced salt solution (HBSS) supplemented
with 10 mM Hepes, pH 7.4, and 0.1% BSA (HBSS, Invitrogen, Grand
Island, NY; BSA, bovine serum albumin, Calbiochem, La Jolla, CA).
Cell Lines and Cultures. Cultures were incubated at 37 °C in a
humidified (95% relative humidity) atmosphere of 5% CO2/95% air.
The parental cell line LLC-PK1 (porcine renal epithelial cells) was
purchased from American Type Culture Collection (ATCC, Manassas,
VA). Human MDR1 and Sprague-Dawley rat mdra1 transfectants in
LLC-PK1 were generated at Amgen (Thousand Oaks, CA). Cells were
cultured in medium 199 supplemented with 2 mM L-glutamine,
penicillin (50 units/mL), streptomycin (50 μg/mL), and 10% (v/v)
fetal bovine serum (all from Invitrogen).14
Permeability and Transcellular Transport of Test Compounds.
LLC-PK1, MDR1-LLC-PK1, and mdr1a-LLC-PK1 cell monolayers
were seeded onto porous (1.0 μm) polycarbonate 96-well transwell
membrane filters (Millipore Corp., Billerica, MA) and cultured for 6
days with one medium replacement on day 4 prior to transwell
experiments. Cells were washed once with warmed HHBSS prior to
transwell experiments. Experiments were initiated by replacing the
buffer in each compartment with 0.15 mL of HBSS containing 0.1%
BSA with and without 5 μM of test compound in triplicate wells. The
plates were incubated for 2 h at 37 °C in an EVO incubator with
shaking. Aliquots (100 μL) from both donor and receiver chambers
were transferred to 96-well plates or scintillation vials. Protein was
precipitated by addition of 200 μL of acetonitrile containing 0.1%
formic acid and prazosin (25 ng/mL) as internal standard. After
vortexing and centrifugation at 3000 rpm for 20 min, 150 μL
supernatant samples were transferred to a new plate containing 50 μL
water for LC−MS/MS analysis. Transcellular transport of [3H]digoxin
was used as a positive control for Pgp. Paracellular permeability of
[14C]mannitol was used to measure the integrity of the monolayer.
Sample radioactivity was measured using a liquid scintillation counter
(Packard Tri-Carb 2910TR, PerkinElmer).
4 - ( 6 - F l u o r o - 5 - m e t h y l p y r i d i n - 3 - y l ) - 6 , 7 - [ 3 H ] -
dimethoxycinnoline (16). To a solution of 15 (8 mg, 28 μmol) in
dimethylformamide, 1.1 mL, was added potassium carbonate (12 mg,
90 μmol). The resulting mixture was stirred for 10 min. Tritiated
methyl iodide (CT3I, 50 μmol, 2500 mCi) was distilled over the
vacuum line. The mixture was stirred at room temperature overnight.
The reaction mixture was evaporated to dryness and purified by Gilson
HPLC (Supelco SDC-18 column, 250 mm × 21.2 mm, 5 μm), eluting
with a mobile phase of 40% acetonitrile in water with 0.1% TFA and a
flow rate of 6 mL/min. UV detection was performed at 260 nm. The
radiolabeled product 16 was collected after 22 min. Analytical HPLC
to determine radiochemical purity was performed with a SpectraSYS-
TEM UV 1000 (SDC18 4.0 mm × 250 mm), with a flow rate of 1
mL/min. Mobile phases used were solvent A of water with 0.1% TFA
and solvent B of acetonitrile with 0.1% TFA. Elution began at 0−10
min at 5% solvent B, 10−35 min at 5−100% B, then 35−40 min hold
at 100% B. UV detection was performed at 254 nm. Compound 16
was synthesized with a decay corrected radiochemical yield of 4% and
with a radiochemical purity of 99%.
5-(6,7-[3H]Dimethoxycinnolin-4-yl)-N-isopropyl-3-methyl-
pyridin-2-amine ([3H]5). To a solution of 16 (100 mCi) in dimethyl
sulfoxide (200 μL) was added excess of isopropylamine (100 μL). The
reaction mixture was heated to 90 °C overnight. The mixture was
evaporated to dryness and purified by Discovery HPLC instrument
(Supelco C-18 column, 250 mm × 21.2 mm, 5 μm), eluting with a
mobile phase of 18% acetonitrile in water with 0.1% TFA and a flow
rate of 5 mL/min. UV detection was performed at 254 nm. Analytical
HPLC to determine radiochemical purity was performed with a
SpectraSYSTEM UV 1000 (SDC18 4.0 mm × 250 mm), with a flow
rate of 1 mL/min. Mobile phases used were solvent A of water with
0.1% TFA and solvent B of acetonitrile with 0.1% TFA. Elution began
at 0−10 min at 5% solvent B, 10−35 min at 5−100% B, then 35−40
min hold at 100% B. UV detection was performed at 254 nm. The final
40 mCi of radiolabeled compound isolated was then formulated in
ethanol at 1 mL/mCi. Compound [3H]5 was synthesized with decay
corrected radiochemical yield of 1.8% and with a radiochemical purity
of 99%. The average specific radioactivity was found to be 46.7 Ci/
mmol.
The apparent permeability coefficient (Papp) of all tested agents was
estimated from the slope of cumulative amount (dQ) of the agent vs
time (dt) and the following equation:
P
= (dQ /dt)/(AC0)
app
where dQ/dt is the penetration rate of the agent (μm/s), A is the
surface area of the cell layer on the transwell (0.11 cm2), and C0 is the
initial concentration of the test compound (μM).
I
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Administration of Tracer Candidates in Rats and LC−MS/MS
Detection. Animals. All experiments were conducted under approved
research protocols by Amgen’s Animal Care and Use Committee
(IACUC) and in accordance with National Institutes of Health Guide
for Care and Use of Laboratory Animals in facilities accredited by the
Association for the Assessment and Accreditation of Laboratory
Animal Care (AALAC). Adult male Sprague-Dawley (SD) rats (250−
280 g) were purchased from Harlan (Harlan, Indianapolis, IN). For
the autoradiography experiments jugular vein catheters were inserted
by the vendor. Rats were group-housed on a filtered, forced air
isolation rack and maintained on sterile wood chip bedding in a quiet
room on a 12 h light−dark cycle, with food and water available ad
libitum. Animals were allowed a minimum of 3 days of adaptation to
the laboratory conditions prior to being utilized in the experiments.
Methods. The tracer candidates were formulated in 20% Captisol,
pH 4, with methanesulfonic acid (MSA). The tracer was administered
by bolus intravenous injection via lateral tail vein. Animals were
euthanized by decapitation under anesthetized conditions at different
postdosing time points. Blood was collected and discrete brain areas
(striatum, and thalamus) were rapidly dissected. The brain tissues were
collected with preweighed flat-bottom 2 mL tubes, and the amount of
tissue collected in each tube was weighed and calculated. The samples
were snap frozen on dry ice and stored at −80 °C until use. Then 20%
w/v of HPLC graded water was added to the sample tubes and
samples were homogenized with an acoustic homogenizer (E210
Covaris, Inc.). Covaris settings were as follows: 4 °C, duty cycle 20%,
intensity 8, cycles per burst 500, treatment time 6 × 10 s. Then 50 μL
of homogenate was transferred to a new tube and 10 μL of methanol
and 150 μL of acetonitrile were added followed by extraction and
centrifugation. The supernatant was transferred to well autosampler
plate, and 150 μL of HPLC grade water was added and mixed. The
compound concentrations in the brain and plasma samples were
analyzed employing high performance liquid chromatography (HPLC)
coupled with triple quad mass spectrometry (API 4000, Applied
Biosystem). The HPLC system employed a Waters Atlantis column.
Surface Plasmon Resonance (SPR) Spectroscopy Binding Assay.
SPR measurements were performed on a Biacore T100 (GE
Healthcare). His-PDE10A (442-779) was generated in-house; all
other reagents were purchased from GE Healthcare or Sigma-Aldrich.
PDE10 (∼3000 RU) was coupled to the CM5 chip using standard
amine coupling. The immobilization running buffer consisted of 10
mM Hepes, pH 7.4, with 150 mM NaCl. Immobilization steps
consisted of a 7 min EDC/NHS activation step [200 mM 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, 50 mM N-hydrox-
ysuccinimide], 0.2 min of 20 μg/mL PDE10A in 10 mM MES, pH 6.0,
and 7 min of 1 M ethanolamine hydrochloride, pH 8.5.
Binding kinetics were measured with a 20 mM Tris-HCl buffer (pH
7.5) containing 50 mM NaCl, 5 mM MgCl2, and 2% (v/v) DMSO.
Stock solution of 5 was prepared at 10 mM in DMSO and diluted with
running buffer to 1 μM, 0.2 μM, 40 nM, and 8 nM. Kinetic titration
experiments15 were performed by injecting the low to high compound
solutions in 2 min intervals onto the PDE10A surface. All experiments
were performed at 25 °C.
The data were processed and analyzed using the T100 evaluation
software and the CLAMP analysis software package (Center for
Biomolecular Interactions, University of Utah, Salt Lake City, UT).
The sample response was baseline corrected by subtracting reference
flow cell data to correct for systematic noise and baseline drift. The
response from blank injections was used to double-reference the
binding data. The kinetic parameters were established by fitting the
data to a 1:1 binding model which included a mass transfer limitation
term.
KD and Bmax Measurement Using Rat Striatal Homogenate. Brain
homogenates were prepared from SD rat striatum and thalamus
regions. Brain samples were weighed and 20% w/v of PDE10
homogenization buffer (50 mM Tris-HCl, pH 7.5, 1.2 mM MgCl2, 0.1
mM DTT, 10% sucrose, 1× Roche complete protease inhibitor mix)
was added, followed by homogenization using an acoustic homoge-
nizer (E210 Covaris, Inc.). The brain homogenate was then
centrifuged at 4200g to pellet unbroken cells and nuclei. The
supernatant was collected and protein concentration of the total
brain homogenate was determined using Biorad DC protein
concentration assay. An amount of 20 μg of homogenate protein of
rat striatum and thalamus was added per well.
Brain homogenates were incubated with tritium labeled compound
(with concentrations from 0.01 to 50 nM in a final volume of 200 μL.)
Nonspecific binding was determined using excess nonlabeled
compound (1 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 5
mM MgCl2, 1× Roche complete protease inhibitor mix) for 1 h at
room temperature. The binding reaction was terminated by rapid
filtration through GF/C filter, and the mixture was presoaked in 0.3%
PEI for 30 min and immediately rinsed 6 times with 4 °C wash buffer
(50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). The filter plates were
dried in a vacuum oven at 56 °C for approximately 40 min, and 50 μL
of Microscint-20 was added per well. Plates were sealed and read on a
Packard TopCount NXT plate reader for 2 min/well. Specific binding
was calculated as total (cpm) minus nonspecific (cpm) binding.
Data were analyzed by nonlinear regression analysis using
GraphPad Prism software, version 5 (GraphPad Inc., San Diego,
CA), which fitted binding curves to the following equation for
saturation binding experiments:
B = BmaxL/(Kd + L)
where B is the concentration of bound ligand, Bmax is the maximal
number of binding sites, Kd is the ligand dissociation constant, and L is
the ligand concentration.
Competition Binding Studies: Ki Determination. Competition
binding was determined by incubating homogenates with a
concentration of [3H]5 at approximately KD in the presence of
increasing concentrations of 17 or nonlabeled 5 ranging from 5 pM to
100 nM. The actual [3H]5 concentration added to the mixture was
calculated by taking control counts of the experimental sample of [3H]
5 to accurately calculate cpm/fmol per well. The concentration of cold
compound that inhibited 50% of the specific binding of [3H]5 was
determined (IC50). By use of the Kd and IC50 values obtained, Ki
values were calculated according to Cheng and Prusoff:12 Ki = (IC50/
(1 + (L/Kd)). IC50 is the inhibitor concentration for half-maximal
binding inhibition, and L is ligand concentration. The Y-axis in
competition binding experiments is defined as the percent of control
(POC).
Ex Vivo Autoradiography. Jugular cannulated SD rats were either
dosed with [3H]5 (3, 10, 30, or 100 μCi iv) or pretreated with vehicle
or 17 (10 mg/kg, po), and 50 min afterward, [3H]5 (30 μCi) was
administered by bolus intravenous injection via the jugular vein. Ten
minutes after injection of the tracer, animals were euthanized by CO2
inhalation and brains were collected, frozen in ice-cold methylbutane
(Sigma-Aldrich, St. Louis, MO), and stored at −80 °C until usage.
Sagittal brain sections (20 μm) were cut on a cryostat (Leica,
Nussloch, Germany) and air-dried, and autoradiographic images were
acquired with a BetaIMAGER D (Biospace, Paris, France) and
analyzed with M3vision (Biospace, Paris, France).
Target Occupancy Analysis of 17. A target occupancy dose−
response study was conducted with 17. Sprague-Dawley (SD) rats
were pretreated with vehicle (2% HPMC, 1% Tween 80, pH 2.2, with
methanesulfonic acid) or 17 (0.1, 0.3, 1, 3, 10, and 30 mg/kg, po).
Fifty minutes after dosing 17, 5 (6 μg/kg) was administered by bolus
intravenous injection via lateral tail vein. Ten minutes after injection of
5, animals were euthanized and blood and brain samples (striatum as
target tissue and thalamus as reference tissue) were collected. Brain
samples were weighed and HPLC-grade water was added (20%
weight/volume), followed by homogenization using a Covaris E210
acoustic homogenizer, as described previously. Homogenized samples
were stored at −20 °C before analysis using API 4000 LC−MS/MS
(Applied Biosystems, Carlsbad, CA).
PDE10A occupancy based on the reference tissue model was
determined using the following equations:2,16
BP = (STR − THA)/THA = (STR/THA) − 1
RO% = 100 × [1 − (BPdrug/BPveh)]
J
dx.doi.org/10.1021/jm3002372 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
where BP refers to binding potential, RO refers to receptor occupancy,
BPdrug refers to the binding potential of the test article dosed (17), and
BPveh refers to the binding potential of the vehicle.
Results were expressed as the mean STD (standard deviation) or
the mean SEM (standard error of mean). Curve fit was assessed
using a four-parameter nonlinear regression in GraphPad Prism
software, version 5 (GraphPad Inc., San Diego, CA).
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Immunohistochemical localization of PDE10A in the rat brain. Brain
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Quantitative comparison of phosphodiesterase mRNA distribution in
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James, L. C.; Williams, R. D.; Stock, J. L.; McNeish, J. D.; Strick, C. A.;
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analysis: log(inhibitor) vs response − variable slope
equation:
x)(Hill slope)
−
Y = bottom + (top − bottom)/[1 + 10(log EC
]
50
Data outliers were determined using Grubb’s test outlined in
GraphPad Prism software (GraphPad Inc., San Diego, CA).
AUTHOR INFORMATION
Corresponding Author
*For E.H.: phone (805) 313-5300; e-mail, ehu@amgen.com.
For H.C.: phone, (650) 244-2490; e-mail, hangc@amgen.com.
■
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Present Address
∞Envoy Therapeutics, 555 Heritage Drive, Jupiter, Florida
33458, United States.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express our gratitude to Lee Phebus for his valuable insights
and to David Bauer for his contributions to the experimental
conditions.
DEDICATION
■
We dedicate this paper to our dear friend and colleague Robert
Cho who passed away in 2011. We will always be inspired by
his dedication, talents, and kindness.
ABBREVIATIONS USED
■
BP, binding potential; CM5, carboxymethyl 5; CNS, central
nervous system; HCl, hydrogen chloride; HHBSS, Hepes
buffered Hank’s balanced salt solution; ID/g, injected dose per
gram; LC−MS/MS, liquid chromatography coupled to mass
spectrometry; PDE, phosphodiesterase; rbf, round-bottom
flask; RO, receptor occupancy; SEM, standard error of the
mean; SFC, supercritical fluid chromatography; SNAr,
nucleophilic aromatic substitution; STR, striatum; THA,
thalamus
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