Synthesis and Preliminary Evaluation of 11C-Labeled VU0467485/AZ13713945 and Its Analogues for Imaging Muscarinic Acetylcholine Receptor Subtype 4

Article abstract of DOI:10.1002/cmdc.201800710

Muscarinic acetylcholine receptors (mAChRs) have five distinct subunits (M₁–M₅) and are involved in the action of the neurotransmitter acetylcholine in the central and peripheral nervous system. Attributed to the promising clinical efficacy of xanomeline, an M₁/M₄-preferring agonist, in patients of schizophrenia and Alzheimer's disease, M₁- or M₄-selective mAChR modulators have been developed that target the topographically distinct allosteric sites. Herein we report the synthesis and preliminary evaluation of ¹¹C-labeled positron emission tomography (PET) ligands based on a validated M₄R positive allosteric modulator VU0467485 (AZ13713945) to facilitate drug discovery. [¹¹C]VU0467485 and two other ligands were prepared in high radiochemical yields (>30 %, decay-corrected) with high radiochemical purity (>99 %) and high molar activity (>74 GBq μmol^?1). In vitro autoradiography studies indicated that these three ligands possess moderate-to-high in vitro specific binding to M₄R. Nevertheless, further physiochemical property optimization is necessary to overcome the challenges associated with limited brain permeability.

Full text of DOI:10.1002/cmdc.201800710

DOI: 10.1002/cmdc.201800710
Communications
11
Synthesis and Preliminary Evaluation of C-Labeled
VU0467485/AZ13713945 and Its Analogues for Imaging
Muscarinic Acetylcholine Receptor Subtype 4
Xiaoyun Deng+,[a] Akiko Hatori+,[b] Zhen Chen,[a] Katsushi Kumata,[b] Tuo Shao,[a]
Xiaofei Zhang,[a] Tomoteru Yamasaki,[b] Kuan Hu,[b] Qingzhen Yu,[a] Longle Ma,[a]
Gangqiang Wang,[c] Lu Wang,[a, d] Yihan Shao,[e] Lee Josephson,[a] Shaofa Sun,*[c] Ming-
Rong Zhang,*[b] and Steven Liang*[a]
Muscarinic acetylcholine receptors (mAChRs) have five distinct
subunits (M1–M5) and are involved in the action of the neuro-
transmitter acetylcholine in the central and peripheral nervous
system. Attributed to the promising clinical efficacy of xanome-
line, an M1/M4-preferring agonist, in patients of schizophrenia
and Alzheimer’s disease, M1- or M4-selective mAChR modula-
tors have been developed that target the topographically dis-
tinct allosteric sites. Herein we report the synthesis and pre-
liminary evaluation of 11C-labeled positron emission tomogra-
phy (PET) ligands based on a validated M4R positive allosteric
modulator VU0467485 (AZ13713945) to facilitate drug discov-
ery. [11C]VU0467485 and two other ligands were prepared in
high radiochemical yields (>30%, decay-corrected) with high
radiochemical purity (>99%) and high molar activity (>
74 GBqmmolÀ1). In vitro autoradiography studies indicated that
these three ligands possess moderate-to-high in vitro specific
binding to M4R. Nevertheless, further physiochemical property
optimization is necessary to overcome the challenges associat-
ed with limited brain permeability.
receptor (mAChR) have been identified throughout the body
in mammals.[5–8] In particular, immunoprecipitation studies in
rodent brain revealed that M4 receptor (M4R) is abundant in
the cerebral cortex, hippocampus, striatum and thalamus, with
most density in striatum and lowest in hindbrain, respective-
ly.[9,10] A similar distribution of M4R in the human brain was
also observed.[10,11] By autoradiography studies in neurological
disorders and neurodegenerative diseases, researchers found
that brain sections from Alzheimer’s disease (AD) patients
showed a significant decrease in M4R density in the dentate
gyrus, CA4 regions, mediodorsal nucleus, and temporal
cortex,[12–14] increased levels in striatum and frontal cortex,[15,16]
but no changes in visual and frontal cortex.[17] Postmortem
studies with schizophrenia patients showed that significantly
decreased expression levels of M4R were observed in different
brain regions, including anterior cingulate cortex, superior tem-
poral gyrus, prefrontal cortex, hippocampus, caudate, and pu-
tamen.[18–24] The findings in patients of dementia with Lewy
bodies (DLB) revealed that the expression of M4R was in-
creased in cortex and claustrum.[16] In addition, the M1/M4-pre-
ferring agonist xanomeline showed clinical efficacy in schizo-
phrenic and AD patients. By a large-scale, placebo-controlled
clinical trial, the observed improvements in Alzheimer’s Disease
Assessment Scale (ADAS-Cog) and the Clinician’s Interview-
Based Impression of Change (CIBIC+) following treatment
with xanomeline showed that the agonist can be effective for
the AD patients in cognitive function improvement.[25] Cogni-
Muscarinic acetylcholine receptors (mAChRs), together with
nicotinic acetylcholine receptors (nAChRs) constitute the ace-
tylcholine receptor (AChR) families. mAChRs are members of
the G protein-coupled receptor superfamily and involved in
cognitive, behavior, sensory, motor and autonomic func-
tions.[1–4] Five subtypes (M1–M5) of the muscarinic acetylcholine
[a] Dr. X. Deng,+ Dr. Z. Chen, Dr. T. Shao, Dr. X. Zhang, Dr. Q. Yu, Dr. L. Ma,
Dr. L. Wang, Prof. Dr. L. Josephson, Prof. Dr. S. Liang
[d] Dr. L. Wang
Department of Nuclear Medicine and PET/CT-MRI Center, the First Affiliated
Hospital of Jinan University & Institute of Molecular and Functional Imag-
ing, Jinan University, Guangzhou 510630 (China)
Department of Radiology, Division of Nuclear Medicine and Molecular
Imaging, Massachusetts General Hospital and Harvard Medical School, 55
Fruit Street, Boston, MA 02114 (USA)
[e] Prof. Dr. Y. Shao
E-mail: liang.steven@mgh.harvard.edu
Department of Chemistry and Biochemistry, University of Oklahoma,
Norman, OK 73019 (USA)
[b] Dr. A. Hatori,+ Dr. K. Kumata, Dr. T. Yamasaki, Dr. K. Hu,
[+] These authors contributed equally to this work.
Prof. Dr. M.-R. Zhang
Department of Radiopharmaceuticals Development, National Institute of
Radiological Sciences, National Institutes for Quantum and Radiological
Science and Technology, Chiba 263-8555 (Japan)
E-mail: zhang.ming-rong@qst.go.jp
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201800710.
[c] Dr. G. Wang, Prof. Dr. S. Sun
Hubei Collaborative Innovation Centre for Non-power Nuclear Technology,
College of Nuclear Technology & Chemistry and Biology, Hubei University of
Science and Technology, Xianning (China)
E-mail: sunshaofa@hbust.edu.cn
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tive improvement for schizophrenia patients provided the evi-
dence that further investigation of xanomeline is worth pursu-
ing as an innovative approach.[26] These convincing data result-
ing from postmortem studies of patients and clinical trials
have stimulated the development of therapeutics targeting
M4R. In particular, the development of M4-selective positive al-
losteric modulators (PAMs) is a novel strategy to overcome the
challenges associated with subtype-selective orthosteric mus-
carinic agonists.[27,28] However, at early stage the development
of M4-selective PAMs was challenging and progressed slowly
due to species differences in M4 PAM potency, challenges in M4
over M2 selectivity, P-glycoprotein-mediated efflux, and the
lack of chemical diversity.[27,29–36] Several M4 PAMs such as
LY2033298, VU10010, ML253, VU0152099, and VU0467154
have been reported in recent years (Figure 1).[27,29,30,33–35,37–39]
Among these, VU0467485/AZ13713945, a potent and selective
Scheme 1. Reagents and conditions: (i) 2 equiv 2, DMF, 1008C, 2 h, 37%;
(ii) 33 equiv POCl3, 1,4-dioxane, reflux, 5 h, 67%; (iii) 1.05 equiv 5, 1.6 equiv
K2CO3, tBuOH/MeOH, room temperature to 458C, 15 h, 98%; (iv) 8 equiv
KOH, THF/H2O, 608C, 4 h; (v) HCl (2m), 87%; (vi) 1.2 equiv HATU, 3 equiv
DIPEA, DMF, room temperature, 10 min; (vii) 2 equiv 9 or 10, room tempera-
ture, 2 h, 85% for 11 a, 81% for 11 b, 66% for 11 c, 41% for 12a, 56% for
12b, 46% for 12c; (viii) 2.0 equiv LiAlH4, THF, room temperature, 6 h, 52%
for 9b; (ix) BH3-THF, THF, reflux, 8 h, 53% for 9c; (x) HBr (48% aqueous solu-
tion), reflux, 5 h, 85% for 10a, 50% for 10b, 53% for 10c; DMF=N,N-dime-
thylformamide; HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’N’-tetramethyluroni-
um hexafluorophosphate; DIPEA=N,N-diisopropylethylamine; THF=tetrahy-
drofuran.
Three M4R PAMs 11 a–c and their corresponding labeling
precursors were synthesized. As summarized in Scheme 1, the
cyclization of biacetyl 1 and 2-cyanoacetohydrazide 2 led to in-
termediate 3 in 37% yield. Phosphorus oxychloride was used
to chlorinate compound 3 to obtain the pyridazine chloride 4
in 67% yield. Methyl carboxylate 6 was prepared by cyclization
of compound 4 and methyl 2-mercaptoacetate 5 in 98% yield.
After hydrolysis, intermediate 6 was converted into the corre-
sponding carboxylic acid 7 in 87% yield. By condensation reac-
tions using HATU and DIPEA, a series of amides 11 and 12
were produced with corresponding amines 8 or 9 in 41–85%
yields. It is worth mentioning that sodium carboxylate deriva-
tive, obtained by microwave reaction of pyridazine chloride 4
and methyl 2-mercaptoacetate 5 under sodium hydroxide,
could also be used as an alternative carboxylate to obtain 11
and 12. Amine 8b was generated by reduction of benzonitrile
compound with LiAlH4 in 52% yield. Because it was not appli-
cable for the preparation of amine 8c as one of the fluorine
atoms on the arene ring was replaced by hydrogen under
LiAlH4 reduction, BH3-THF complex was used to obtain 8c in
53% yield. By demethylating the methoxy compound 9 with
hydrobromic acid under reflux, we obtained phenolic precur-
sors 10 in 50–85% yields. In all, M4R PAMs 11 a–c and their cor-
responding precursors 12a–c were synthesized in 14–18% and
9–12% overall yields, respectively. The potency of three PAM li-
gands 11 a–c was reported,[40] which showed human M4 PAM
Figure 1. Structures of reported M4 PAMs.
M4 PAM, represents a major advance in the field with robust ef-
ficacy in cross species and behavioral models.[40]
Based on the favorable pharmacological and physiochemical
characteristics of VU0467485/AZ13713945,[40] we hypothesize
that this scaffold and its analogues (11 a–11 c) may enable the
development of a positron emission tomography (PET) ligand
targeting M4R, and provide a quantitative imaging biomarker
for monitoring biochemical and pharmacological process of
M4R in vivo between normal and disease conditions.[41–44] At
present, no M4R-specific PET ligand is available for human use,
which represents an urgent and unmet clinical need. Herein
we describe the synthesis of three potent and selective M4R
PAMs that are amenable for 11C-labeling and their preliminary
evaluation in rodents. In vitro autoradiography studies confirm
moderate-to-high binding specificity of these analogues based
on VU0467485/AZ13713945 scaffold (with highest levels of
specific binding found in [11C]11c; also called [11C]M4R-1023),
which may provide an entry point for future M4R PET ligand
development.
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pEC50 values of 7.10, 7.38 and 7.36, respectively. No off-target
subtype binding (M1, M2, M3 and M5) was observed. Although
the potency values of candidate compounds may be not ideal
for PET ligand development, as proof of concept, we perform
the radiochemistry and preliminary evaluation to study in vitro
binding properties and pharmacokinetic profiles, which may
provide us a roadmap to guide further SAR studies based on
these tool compounds.
As shown in Scheme 2, the methyl ether of M4R PAMs was
identified as the most accessible labeling site with carbon-
Figure 2. In vitro autoradiography of [11C]11 a binding in rat brain sections.
(A) Brain sections were treated with [11C]11 a in the absence (baseline) or
presence of VU0467485/AZ13713945 (11 a; 10 mm). Cr, cerebellum; Cx,
cortex; St, striatum; Hi, hippocampus; Th, thalamus. (B) The radioactivity dis-
tribution was quantified in regional rat brain. The data are expressed as radi-
oactivity per mm2 (n=4). (C) Blocking studies. The data are normalized to
percent of radioactivity vs. control (n=4). Asterisks indicate statistical signifi-
cance: **p<0.01 and ****p<0.0001 vs. control.
4. In the baseline study, the distribution of bound radioactivity
was heterogeneous with signal levels in decreasing order of
thalamus, striatum, cerebellum, cerebral cortex and hippocam-
pus (Figure 2B, Figure 3B and Figure 4B, respectively). In par-
ticular, regional distribution of [11C]11 c resembled the most
similar expression pattern of M4R in the brain as prior re-
ports.[9,10] As shown in Figure 2C, quantitative analysis of radio-
activity binding in the striatum with VU0467485/AZ13713945
(11 a; 10 mm) showed ~20% reduced binding compared with
that of baseline while other regions showed less reduction. In
Figure 3C for [11C]11 b, self-blocking study in the five tested re-
Scheme 2. Radiosynthesis of M4R PAM ligands 11 a–c: [11C]CH3I, NaOH, DMF;
808C, 5 min.
11.[45] The synthon [11C]CH3I was transferred into a pre-cooled
(À15 to À208C) reaction vessel containing precursor 12a–c,
NaOH and anhydrous DMF. After the radioactivity was deliv-
ered, the reaction vessel was warmed to 808C and maintained
for 5 min. The reaction mixture was purified by semi-prep
radio-HPLC, and reformulated in a saline solution (3 mL) con-
taining 100 mL of 25% ascorbic acid in sterile water and 100 mL
of 20% Tweenꢁ 80 in ethanol. The M4R PAM ligands [11C]11 a–
[11C]11 c were synthesized in 31–42% radiochemical yield
based on [11C]CO2 (decay-corrected). Specifically, starting from
420–600 mCi (15.5–22.2 GBq) of [11C]CO2, tracers were obtained
in 48–63 mCi (1.8–2.3 GBq) at end-of-synthesis (35–40 min syn-
thesis time) with high radiochemical purity (>99%) and high
molar activity (>2 CimmolÀ1; 74 GBqmmolÀ1). No radiolysis was
observed within 90 min.
Figure 3. In vitro autoradiography of [11C]11 b binding in rat brain sections.
(A) Brain sections were treated with [11C]11 b in the absence (baseline) or
presence of 11 b (10 mm). Cr, cerebellum; Cx, cortex; St, striatum; Hi, hippo-
campus; Th, thalamus. (B) The radioactivity distribution was quantified in re-
gional rat brain. The data are expressed as radioactivity per mm2 (n=4).
(C) Blocking studies. The data are normalized to percent of radioactivity vs.
control (n=4). Asterisks indicate statistical significance: *p<0.05, **p<0.01,
***p<0.001 and ****p<0.0001 vs. control.
The binding specificity of [11C]VU0467485/[11C]AZ13713945
([11C]11 a), [11C]11 b and [11C]11 c was evaluated by in vitro auto-
radiography. Representative in vitro autoradiograms of three li-
gands on sagittal sections of rat brains are shown in Figures 2–
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Information). A possible explanation was as follows: While the
ligand of this type showed reasonable CNS penetration in vitro
and in vivo (exemplified in 11 a with efflux ratio of 1.4 in
MDCK/MDR1 assay; total brain/total plasma, Kp of 0.31 and un-
bound brain/unbound plasma, Kp,uu of 0.37 in Sprague–Dawley
rats),[40] the PAM can be functionally potent, but is a possible
weak binder due to cooperativity, leading to greater chance of
nonspecific binding.
We have efficiently synthesized three PAMs targeting M4R
and their corresponding labeling precursors. The 11C-labeling
experiments were carried out in excellent radiochemical yield,
high radiochemical purity and high molar activity. In vitro auto-
radiography studies confirmed moderate-to-high specific bind-
ing of all these M4R ligands. While [11C]11c (also called [11C]M4R-
1023) is not likely advanced for in vivo mapping of M4R due to
limited brain permeability, in vitro high specific binding
showed a promising chemotype for further M4R PET ligand de-
velopment. Further in-depth pharmacokinetic studies are nec-
essary to test PgP/Bcrp brain efflux accountability in cross spe-
cies studies and to facilitate new tier design with improved
brain permeability.
Figure 4. In vitro autoradiography of [11C]11 c binding in rat brain sections.
(A) Brain sections were treated with [11C]11 c in the absence (baseline) or
presence of 11 c (10 mm). Cr, cerebellum; Cx, cortex; St, striatum; Hi, hippo-
campus; Th, thalamus. (B) The radioactivity distribution was quantified in re-
gional rat brain. The data are expressed as radioactivity per mm2 (n=4).
(C) Blocking studies. The data are normalized to percent of radioactivity vs.
control (n=4). Asterisks indicate statistical significance: **p<0.01,
***p<0.001, and ****p<0.0001 vs. control.
Experimental Section
gions showed ~30–40% reduced binding compared with that
of baseline. In Figure 4C, [11C]11 c showed highest level of spe-
cific binding among three ligands, as ~90% reduced binding
was observed in the five tested regions. These encouraging re-
sults prompted us to perform in vivo evaluation of [11C]11 c in
PET studies.
The general experimental section was described previously[46] with
minor modification as follows: All the chemicals employed in the
syntheses were purchased from commercial vendors and used
without further purification. Thin-layer chromatography (TLC) was
conducted with 0.25 mm silica gel plates (60F254) and visualized by
exposure to UV light (254 nm) or stained with potassium perman-
ganate. Flash column chromatography was performed using silica
gel (particle size 0.040–0.063 mm). Nuclear magnetic resonance
(NMR) spectra were obtained on a Bruker spectrometer 300 MHz.
Chemical shifts (d) are reported in ppm and coupling constants are
reported in Hertz. The multiplicities are abbreviated as follows: s=
singlet, d=doublet, t=triplet, q=quartet, quint=quintet, sext=
sextet, sept=septet, m=multiplet, br=broad signal, dd=doublet
of doublets. For LC–MS/MS measurements, the ionization method
is ESI using Agilent 6430 Triple Quad LC–MS. The animal experi-
ments were approved by the Institutional Animal Care and Use
Committee of Massachusetts General Hospital or the Animal Ethics
Committee at the National Institutes for Quantum and Radiological
Science and Technology, National Institute of Radiological Sciences.
Sprague–Dawley rats (male, 7 weeks old) were used for PET imag-
ing studies.
Dynamic PET acquisitions (0–60 min) were conducted with
[11C]11 c in Sprague–Dawley rats. Representative PET images of
[11C]11 c (sagittal and coronal, summed 0–60 min) and time-ac-
tivity curves of five brain regions at baseline conditions are
shown in Figure 5. The potential M4R ligand [11C]11 c showed
limited brain uptake (~0.4 SUV peak uptake) with a heteroge-
neous distribution in decreasing order of cerebellum, cerebral
cortex, hippocampus and striatum. Pretreatment with unradio-
labeled M4R PAM VU0467485/AZ13713945 (11 a; 1 mgkgÀ1)
failed to show the substantial difference of [11C]11 c uptake in
the brain relative to baseline conditions (Figure S1, Supporting
General procedure for the condensation reaction: A mixture of
compound
7 (0.2 mmol), N,N-diisopropylethylamine (0.6 mmol,
3 equiv) and O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU, 0.24 mmol, 1.2 equiv) in DMF (1 mL)
was allowed to stir at ambient temperature for 10 min; then 9a–
10c (0.4 mmol, 2 equiv) in DMF (1 mL) was added. After stirring for
an additional 2 h, the mixture was purified by chromatography on
silica gel, elution being carried out with ethyl acetate. Compounds
11 a–12c were prepared in 41–85% yields.
5-amino-N-(3-fluoro-4-methoxybenzyl)-3,4-dimethylthieno[2,3-
c]pyridazine-6-carboxamide (VU0467485/AZ13713945; 11a): Ac-
cording to the general procedure for the condensation reaction,
compound 11 a was prepared in 85% yield as a yellow solid.
1H NMR (300 MHz, DMSO): d=8.60 (t, J=5.8 Hz, 1H), 7.15–6.96 (m,
5H), 4.35 (d, J=5.9 Hz, 2H), 3.80 (s, 3H), 2.70 (s, 3H), 2.69 ppm (s,
Figure 5. PET/MRI fused images of [11C]11 c in rat brain: (A) baseline and
(B) time-activity curves in different brain regions at baseline.
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3H); 13C NMR (75 MHz, DMSO): d=165.15 (s), 160.21 (s), 154.97 (s),
151.69 (d, J=243.0 Hz), 146.79 (s), 146.40 (d, J=10.3 Hz), 133.06 (s),
132.98 (s), 127.45 (s), 124.00 (s), 115.44 (d, J=18.0 Hz), 114.15 (s),
102.90 (s), 56.45 (s), 42.10 (s), 20.19 (s), 15.01 ppm (s).
J=3.1, 1.5 Hz), 102.76 (s), 36.17 (s), 20.18 (s), 15.01 ppm (s); MS: m/
z [M+1]+ =365.
General procedure for 11C-methylation: The general procedure
for 11C-labeling was described previously[46] with minor modifica-
tion in this work. Briefly, [11C]methyl iodide ([11C]CH3I) was synthe-
sized from cyclotron-produced [11C]CO2, which was produced by
14N(p,a)11C nuclear reaction. Briefly, [11C]CO2 was bubbled into a so-
lution of LiAlH4 (0.4m in THF, 300 mL). After evaporation, the re-
maining reaction mixture was treated with hydroiodic acid (57%
aqueous solution, 300 mL). The resulting [11C]CH3I was transferred
under helium gas with heating into a pre-cooled (À15 to À208C)
reaction vessel containing precursor (1.0 mg), NaOH (6.0 mL, 0.5m)
and anhydrous DMF (300 mL). After the radioactivity reached a pla-
teau during transfer, the reaction vessel was warmed to 808C and
maintained for 5 min. CH3CN/H2O+0.1% Et3N (0.5 mL) was added
to the reaction mixture, which was then injected to a semi-prepa-
rative HPLC system. HPLC purification was completed on a Capcell
Pak UG80 C18 column (10 mm IDꢂ250 mm) using a mobile phase
of CH3CN/H2O+0.1% Et3N. The radioactive fraction corresponding
to the desired product was collected in a sterile flask, evaporated
to dryness in vacuo, and reformulated in a saline solution (3 mL)
containing 100 mL of 25% ascorbic acid in sterile water and 100 mL
of 20% Tweenꢁ 80 in ethanol. Radiochemical and chemical purity
were measured by analytical HPLC (Capcell Pak UG80 C18, 4.6 mm
ID ꢂ 250 mm, UV at 254 nm; CH3CN/H2O+0.1% Et3N at a flow rate
of 1.0 mLminÀ1). The identity of [11C]11 a–c was confirmed by the
co-injection with unlabeled 11 a–c.
5-amino-N-(2,5-difluoro-4-methoxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide (11 b): According to the gener-
al procedure for the condensation reaction, compound 11 b was
prepared in 81% yield as a yellow solid. Melting point: 235–2378C;
1H NMR (300 MHz, DMSO): d=8.57 (t, J=5.6 Hz, 1H), 7.14 (ddd, J=
23.0, 11.6, 7.1 Hz, 2H), 6.94 (s, 2H), 4.38 (d, J=5.6 Hz, 2H), 3.82 (s,
3H), 2.70 (s, 3H), 2.68 ppm (s, 3H); 13C NMR (75 MHz, DMSO): d=
165.27 (s), 160.23 (s), 156.21 (dd, J=241.0, 2.2 Hz), 154.99 (s),
148.03 (dd, J=239.7, 2.8 Hz), 147.33 (dd, J=12.3, 10.7 Hz), 146.92
(s), 133.07 (s), 127.35 (s), 117.94 (dd, J=17.7, 5.9 Hz), 116.32 (dd, J=
20.7, 6.3 Hz), 102.62 (s), 102.27 (d, J=1.6 Hz), 56.93 (s), 36.08 (s),
20.19 (s), 15.01 ppm (s).
5-amino-N-(2,3-difluoro-4-methoxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide (11 c): According to the gener-
al procedure for the condensation reaction, compound 11 c was
prepared in 66% yield as a yellow solid. Melting point: 217–2198C;
1H NMR (300 MHz, DMSO): d=8.62 (t, J=5.6 Hz, 1H), 7.16–6.95 (m,
4H), 4.42 (d, J=5.6 Hz, 2H), 3.84 (s, 3H), 2.71 (s, 3H), 2.69 ppm (s,
3H); 13C NMR (75 MHz, DMSO): d=165.20 (s), 160.23 (s), 154.94 (s),
148.85 (dd, J=245.0, 10.4 Hz), 147.91 (dd, J=7.7, 3.0 Hz), 146.89
(s), 140.32 (dd, J=245.0, 14.3 Hz), 133.29 (s), 127.49 (s), 123.89 (dd,
J=5.1, 4.4 Hz), 120.08 (dd, J=12.3, 1.3 Hz), 109.27 (d, J=3.2 Hz),
102.76 (s), 56.96 (s), 36.24 (s), 20.12 (s), 15.03 ppm (s).
5-amino-N-(3-fluoro-4-[11C]methoxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide
([11C]VU0467485/
5-amino-N-(3-fluoro-4-hydroxybenzyl)-3,4-dimethylthieno[2,3-
c]pyridazine-6-carboxamide (12a): According to the general pro-
cedure for the condensation reaction, compound 12a was pre-
pared in 41% yield as a yellow solid. Melting point: 188–1908C;
1H NMR (300 MHz, DMSO): d=9.70 (s, 1H), 8.56 (t, J=5.9 Hz, 1H),
7.07 (dd, J=12.3, 1.3 Hz, 1H), 6.98–6.84 (m, 4H), 4.31 (d, J=5.9 Hz,
2H), 2.70 (s, 3H), 2.68 ppm (s, 3H); 13C NMR (75 MHz, DMSO): d=
165.10 (s), 160.20 (s), 154.95 (s), 151.18 (d, J=240.5 Hz), 146.74 (s),
144.05 (d, J=12.2 Hz), 132.99 (s), 131.42 (d, J=5.8 Hz), 127.41 (s),
124.00 (d, J=2.9 Hz), 117.95 (d, J=3.0 Hz), 115.63 (d, J=18.5 Hz),
[11C]AZ13713945/[11C]11a): According to the general procedure[46]
for the 11C-methylation, the retention time for [11C]11 a was
12.0 min. The synthesis time was ~40 min from end-of-bombard-
ment. HPLC purification was completed on a Capcell Pak UG80 C18
column (10 mm IDꢂ250 mm) using a mobile phase of CH3CN/H2O
(v/v, 4:6)+0.1% Et3N at a flow rate of 4.0 mLminÀ1. Radiochemical
and chemical purity were measured by analytical HPLC (Capcell
Pak UG80 C18, 4.6 mm IDꢂ250 mm, UV at 254 nm; CH3CN/H2O (v/v,
4:6)+0.1% Et3N at a flow rate of 1.0 mLminÀ1). Radiochemical
yield was 31.3% decay-corrected based on [11C]CO2 with >99% ra-
diochemical purity and greater than 2 CimmolÀ1 molar activity.
102.98 (s), 42.09 (s), 20.19 (s), 15.00 ppm (s); MS: m/z [M+1]+
=
347.
5-amino-N-(2,5-difluoro-4-[11C]methoxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide ([11C]11b): According to the
general procedure[46] for the 11C-methylation, the retention time for
[11C]11 b was 8.1 min. The synthesis time was ~35 min from end-
of-bombardment. HPLC purification was completed on a Capcell
Pak UG80 C18 column (10 mm ID ꢂ 250 mm) using a mobile phase
5-amino-N-(2,5-difluoro-4-hydroxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide (12b): According to the gen-
eral procedure for the condensation reaction, compound 12b was
1
prepared in 56% yield as a yellow solid. H NMR (300 MHz, DMSO):
d=10.25 (s, 1H), 8.53 (t, J=5.6 Hz, 1H), 7.15–6.70 (m, 4H), 4.35 (d,
J=5.6 Hz, 2H), 2.69 (s, 3H), 2.68 ppm (s, 3H); 13C NMR (75 MHz,
DMSO): d=165.23 (s), 160.22 (s), 155.98 (dd, J=240.5, 1.9 Hz),
154.94 (s), 147.77 (dd, J=236.6, 2.5 Hz), 146.86 (s), 145.16 (dd, J=
14.3, 11.8 Hz), 133.05 (s), 127.35 (s), 116.79 (dd, J=6.0, 4.0 Hz),
116.53 (dd, J=7.3, 6.1 Hz), 105.06 (dd, J=26.4, 3.1 Hz), 102.71 (s),
36.09 (s), 20.16 (s), 15.00 ppm (s); MS: m/z [M+1]+ =365.
of CH3CN/H2O (v/v, 45:55)+0.1% Et3N at
a flow rate of
5.0 mLminÀ1. Radiochemical and chemical purity were measured
by analytical HPLC (Capcell Pak UG80 C18, 4.6 mm IDꢂ250 mm, UV
at 254 nm; CH3CN/H2O (v/v, 45:55)+0.1% Et3N at a flow rate of
1.0 mLminÀ1). Radiochemical yield was 38.8% decay-corrected
based on [11C]CO2 with >99% radiochemical purity and greater
than 2 CimmolÀ1 molar activity.
5-amino-N-(2,3-difluoro-4-hydroxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide (12c): According to the gener-
al procedure for the condensation reaction, compound 12c was
prepared in 81% yield as an orange red solid. Melting point: 245–
2478C; 1H NMR (300 MHz, DMSO): d=10.26 (s, 1H), 8.56 (t, J=
5.5 Hz, 1H), 7.02–6.70 (m, 4H), 4.39 (d, J=5.5 Hz, 2H), 2.70 (s, 3H),
2.69 ppm (s, 3H); 13C NMR (75 MHz, DMSO): d=165.18 (s), 160.23
(s), 154.97 (s), 149.24 (dd, J=244.5, 10.6 Hz), 146.85 (s), 145.91 (dd,
J=9.0, 2.8 Hz), 139.97 (dd, J=242.1, 14.2 Hz), 133.08 (s), 127.37 (s),
123.81 (dd, J=5.3, 4.2 Hz), 118.12 (dd, J=12.2, 1.4 Hz), 113.00 (dd,
5-amino-N-(2,3-difluoro-4-[11C]methoxybenzyl)-3,4-dimethylthie-
no[2,3-c]pyridazine-6-carboxamide ([11C]11c): According to the
general procedure[46] for the 11C-methylation, the retention time for
[11C]11 c was 8.5 min. The synthesis time was ~35 min from end-of-
bombardment. HPLC purification was completed on a Capcell Pak
UG80 C18 column (10 mm IDꢂ250 mm) using a mobile phase of
CH3CN/H2O (v/v, 45:55)+0.1% Et3N at a flow rate of 5.0 mLminÀ1
.
Radiochemical and chemical purity were measured by analytical
ChemMedChem 2018, 13, 1 – 8
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Communications
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kept under anesthesia with 1–2% (v/v) isoflurane during the scan.
The [11C]11 c (~1 mCi/100 mL) was injected via a preinstalled cathe-
ter via the tail vein. A dynamic scan was acquired for 60 min in
three-dimensional list mode. For pretreatment studies, unlabeled
VU0467485/AZ13713945 (11 a; 1 mgkgÀ1
) dissolved in 300 mL
saline containing 10% ethanol and 5% Tweenꢁ 80 was injected at
5 min via the pre-embedded tail vein catheter before the injection
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Acknowledgements
We thank Professor Thomas J. Brady (Nuclear Medicine and Mo-
lecular Imaging, Radiology, MGH and Harvard Medical School)
for helpful discussions.
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Conflict of interest
[32] N. E. Byun, M. Grannan, M. Bubser, R. L. Barry, A. Thompson, J. Rosanelli,
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The authors declare no conflict of interest.
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Keywords: carbon-11
subtype 4 · positive allosteric modulator · positron emission
tomography · VU0467485/AZ13713945
· muscarinic acetylcholine receptor
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Communications
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Manuscript received: November 5, 2018
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
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ChemMedChem 2018, 13, 1 – 8
www.chemmedchem.org
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
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COMMUNICATIONS
X. Deng, A. Hatori, Z. Chen, K. Kumata,
T. Shao, X. Zhang, T. Yamasaki, K. Hu,
Q. Yu, L. Ma, G. Wang, L. Wang, Y. Shao,
L. Josephson, S. Sun,* M.-R. Zhang,*
S. Liang*
Carbon-11 footprint: A validated M4R
positive allosteric modulator VU0467485
(AZ13713945) was evaluated as a pre-
clinical candidate. A series of 11C-labeled
positron emission tomography (PET) li-
gands based on VU0467485 were syn-
thesized and preliminarily evaluated. In
vitro autoradiography studies indicated
that these PET ligands possess moder-
ate-to-high in vitro specific binding to
M4R. Nevertheless, further optimization
is necessary to improve brain permea-
bility.
&& – &&
Synthesis and Preliminary Evaluation
of 11C-Labeled VU0467485/
AZ13713945 and Its Analogues for
Imaging Muscarinic Acetylcholine
Receptor Subtype 4
&
ChemMedChem 2018, 13, 1 – 8
www.chemmedchem.org
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!