Synthesis of [11C]MK-1064 as a new PET radioligand for imaging of orexin-2 receptor

Article abstract of DOI:10.1016/j.bmcl.2016.05.083

The reference standard MK-1064 {5″-chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,2′:5′,3″-terpyridine]-3′-carboxamide} was synthesized from methyl 2-chloro-5-iodonicotinate and 5-(chloropyridin-3-yl)boronic acid in 4 steps with 33% overall chemical yield. The precursor desmethyl-MK-1064 {5″-chloro-N-((5-hydroxy-6-methoxypyridin-2-yl)methyl)-[2,2′:5′,3″-terpyridine]-3′-carboxamide} for radiolabeling was synthesized from 2-bromopyridin-3-ol and 5″-chloro-[2,2′:5′,3″-terpyridine]-3′-carboxylic acid in 6 steps with 17% overall chemical yield. The target tracer [¹¹C]MK-1064 {5″-chloro-N-((5-[¹¹C]methoxy-6-methoxypyridin-2-yl)methyl)-[2,2′:5′,3″-terpyridine]-3′-carboxamide} was prepared by O-[¹¹C]methylation of its corresponding precursor desmethyl-MK-1064 with [¹¹C]CH₃OTf under basic condition and isolated by a simplified solid-phase extraction (SPE) method in 50–60% decay corrected radiochemical yields based on [¹¹C]CO₂at end of bombardment (EOB). The overall synthesis time from EOB was 23?min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555?GBq/μmol.

Full text of DOI:10.1016/j.bmcl.2016.05.083

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3694–3699
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [11C]MK-1064 as a new PET radioligand for imaging of
orexin-2 receptor
⇑
Mingzhang Gao, Min Wang, Qi-Huang Zheng
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
The reference standard MK-1064 {500-chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,20:50,300-ter-
pyridine]-30-carboxamide} was synthesized from methyl 2-chloro-5-iodonicotinate and 5-(chloropy-
ridin-3-yl)boronic acid in 4 steps with 33% overall chemical yield. The precursor desmethyl-MK-1064
{500-chloro-N-((5-hydroxy-6-methoxypyridin-2-yl)methyl)-[2,20:50,300-terpyridine]-30-carboxamide} for
radiolabeling was synthesized from 2-bromopyridin-3-ol and 500-chloro-[2,20:50,300-terpyridine]-30-
carboxylic acid in 6 steps with 17% overall chemical yield. The target tracer [11C]MK-1064 {500-
chloro-N-((5-[11C]methoxy-6-methoxypyridin-2-yl)methyl)-[2,20:50,300-terpyridine]-30-carboxamide} was
prepared by O-[11C]methylation of its corresponding precursor desmethyl-MK-1064 with [11C]CH3OTf
under basic condition and isolated by a simplified solid-phase extraction (SPE) method in 50–60% decay
corrected radiochemical yields based on [11C]CO2 at end of bombardment (EOB). The overall synthesis
time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of
Article history:
Received 10 May 2016
Revised 23 May 2016
Accepted 27 May 2016
Available online 28 May 2016
Keywords:
[
11C]MK-1064
Orexin-2 receptor (OX2R)
Positron emission tomography (PET)
Sleep disorders
synthesis (EOS) was 185–555 GBq/lmol.
Ó 2016 Elsevier Ltd. All rights reserved.
The orexin system consists of two neuropeptides: orexin-A (a
33 amino-acid peptide) and orexin-B (a 28 amino-acid peptide);
two G-protein coupled receptors: orexin-1 receptor (OX1R) and
orexin-2 receptor (OX2R).1–4 Orexin-A activates both OX1R and
OX2R with near equal affinity whereas orexin-B preferentially
binds to OX2R. OX1R is selectively expressed in the locus coeru-
leus. Conversely, OX2R is expressed in the tuberomammillary
nucleus.5 The available experimental evidences strongly support
that OX2R plays a key role in motivation, arousal and sleep-wake
regulation, and is associated with sleep disorders, irregularities in
central vestibular motor control, feeding and gastrointestinal dis-
orders and drug abuse.6 Although OX1R is thought to regulate
sleep-wakefulness and energy homeostasis, particularly food
intake, the therapeutic potential of selective antagonism of OX1R
is still under evaluation.6 Orexin receptor is an interesting thera-
peutic target, and many selective orexin receptor antagonists have
been developed.3,4 However, the in vivo selectivity, distribution
and involvements of OX1R and OX2R in the pathophysiology of
orexin-related disorders are not fully understood.7 Biomedical
imaging technique positron emission tomography (PET) is a useful
tool for in vivo quantification of various biological processes.7
OX2R has also become a promising target for molecular imaging
of OX2R-mediated diseases and image-guided therapy using PET
modality, and several PET radioligands including [11C]BBAC, [11C]
BBPC, [11C]EMPA, [11C]CW4, and (1R,2S)-2-(3-fluorophenyl)-N-(5-
fluoropyridin-2-yl)-2-(((4-((methoxy-11C)methyl)-2-methylpyrimi-
din-5-yl)oxy)methyl)cyclopropane-1-carboxamide ([11C]FFMMCC)
have been evaluated for imaging of OX2R, as indicated in
Figure 1.7–10 PET scans with [11C]BBAC and [11C]BBPC in a rhesus
monkey showed no tracer retention and appropriate brain uptake.7
[
11C]EMPA-PET also showed poor brain uptake in both rats and
baboon.8 [11C]CW4-PET in a baboon showed appropriate early brain
uptake, but fast kinetics and high nonspecific binding.9 PET scan
with [11C]FFMMCC showed moderate uptake in rat and monkey
brains under deficiency or blockade of P-glycoprotein, but this radi-
oligand is not selective, and it binds to OX1R and OX2R with the
equal affinity (Ki 1 nM for OX1R and OX2R).10 In view of these radi-
oligands recently developed for PET imaging of OX2R, only a limited
success is achieved. Therefore, to investigate the therapeutic effect
and receptor occupancy of the OX2R antagonists and to guide the
therapy, a new suitable radioligand for OX2R is still needed.
Recently, a new selective OX2R antagonist (2-SORA) MK-1064
{500-chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,20:50,300-ter-
pyridine]-30-carboxamide} has been discovered by Merck, it
exhibited Ki (nM) and IC50 (nM) for OX2R and OX1R, 0.5 and 18,
1584 and 1789, respectively, with selectivity index (SI) Ki/IC50
3168/99, and it is one of the most potent and selective 2-SORA
compounds reported to date.11 Here we report the design, synthe-
⇑
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
sis and radiolabeling of
[
11C]MK-1064 {500-chloro-N-((5-[11C]
http://dx.doi.org/10.1016/j.bmcl.2016.05.083
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

M. Gao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3694–3699
3695
11CH3
O
11CH3
N
N
O
HN
N
N
N
O
X
N
O
Cl
S
N
11
N
N
O CH3
N
S
O
O
O
[
[
11C]BBAC, X=S
11C]BBPC, X=CH2
11C]EMPA
[
11C]CW4
[
F
H311CO
O
O
HN
N
N
N
F
(1R,2S)-2-(3-Fluorophenyl)- N-(5-fluoropyridin-2-yl)-2-(((4-((methoxy- 11C)methyl)-2-methylpyrimidin-5-
yl)oxy)methyl)cyclopropane-1-carboxamide ([11C]FFMMCC)
N
N
O
HN
O11CH3
Cl
O
N
N
[
11C]MK-1064
Figure 1. PET tracers for orexin-2 receptor.
N
N
I
CO2Me
Cl
CO2Me
N
ii
Cl
CO2Me
Cl
i
Cl
N
N
N
1
2
N
N
O
O
N
O
HN
N
H2N
Cl
O
N
4
O
CO2H
iii
Cl
N
iv
N
N
3
5
MK-1064 ( )
Scheme 1. Synthesis of the reference standard MK-1064 (5). Reagents, conditions and yields: (i) 5-(chloropyridin-3-yl)boronic acid, PdCl2(dppf), Cs2CO3, DMF/water, room
temperature (RT), 20 h; 90%. (ii) 2-(tri-n-butylstannyl)pyridine, Pd(PPh3)4, CsF, CuI, DMF, 80 °C, 2 h; 53%. (iii) KOH, MeOH, RT, 2 d; 98%. (iv) (5,6-dimethoxypyridin-2-yl)
methanamine (4), EDC, HOAt, DMF, DIPEA, RT, 15 h; 70%.
methoxy-6-methoxypyridin-2-yl)methyl)-[2,20:50,300-terpyridine]-
30-carboxamide} (Fig. 1).
with N,N-diisopropylethylamine (DIPEA) to give MK-1064 in 70%
yield.
The reference standard MK-1064 (5) was synthesized according
to the reported procedures11 with modifications, as shown in
Scheme 1. Commercially available starting material methyl 2-
chloro-5-iodonicotinate was undergone a Suzuki reaction with 5-
(chloropyridin-3-yl)boronic acid to obtain compound 1 in 90%
yield. Compound 1 was then converted to the intermediate 2
through a Stilling reaction with 2-(tri-n-butylstannyl)pyridine in
53% yield. Compound 2 was hydrolyzed in methanol/water solu-
tion of KOH to yield the acid 3 in 98% yield, which subsequently
reacted with (5,6-dimethoxypyridin-2-yl)methanamine (4) using
catalysts 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
and 1-hydroxy-7-azabenzotriazole (HOAt) under basic condition
Attempt to desmethylation of MK-1064 to synthesize the pre-
cursor desmethyl-MK-1064 {500-chloro-N-((5-hydroxy-6-methoxy-
pyridin-2-yl)methyl)-[2,20:50,300-terpyridine]-30-carboxamide, 11}
employing several methods was unsuccessful. Thus a new route
for the synthesis of the precursor 11 was designed as depicted in
Scheme 2. 2-Bromo-3-hydroxy pyridine was reacted with I2 and
K2CO3 in water to afford compound 6 in 96% yield. Compound 6
was then converted to compound 7 protecting phenol hydroxyl
group via benzyl bromide in 81% yield. The bromide 7 was alco-
holized in DMF by sodium methoxide to afford compound 8 in
85% yield. Compound 8 was reacted with CuCN at 150 °C for
1.5 h to obtain compound 9 in 73% yield. Compound 9 was

3696
M. Gao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3694–3699
Br
Br
Br
O
HO
HO
i
ii
O
N
N
N
N
I
I
6
7
O
iii
iv
O
O
N
CN
9
I
8
N
N
O
HN
O
3
Cl
O
N
OH
v
HO
N
vi
N
NH2
10
11
Desmethyl-MK-1064 (
)
Scheme 2. Synthesis of the precursor desmethyl-MK-1064 (11). Reagents, conditions and yields: (i) K2CO3, H2O, I2, 5 h, RT; 96%. (ii) BnBr, K2CO3, CH3CN, 60 °C, 12 h; 81%.
(iii) MeONa, MeOH, DMF, 100 °C, 1.5 h; 85%. (iv) CuCN, DMF, 150 °C, 2 h; 73%. (v) H2 (50 psi), Pd(OH)2/C, MeOH, 8 h. (vi) EDC, HOAt, DMF, DIPEA, RT, 15 h; 35%.
undergone a 2-step reaction: deprotection of hydroxyl group and
reduction of nitrile group, in one-pot under hydrogen atmosphere
using Pd(OH)2/C as catalyst to give the key intermediate 10, which
was converted to the precursor desmethyl-MK-1064 using similar
procedure as synthesis of MK-1064 in 35% yield.
Sep-Pak, we need to increase the volume of the eluent ethanol.
Thus, the formulation was completed by Sep-Pak trap/release
and rotatory evaporation methods. For the radiotracer produced
for animal study, we used ethanol (2 ꢀ 2 mL) for elution, and
rotatory evaporation was required before reformulation. For the
radiotracer produced for human study, we used ethanol
(2 ꢀ 1 mL), no evaporation required, and a C-18 Sep-Pak was used
for direct reformulation.22–25 We have tried to use a C-18 Light
Sep-Pak cartridge instead of a C-18 Plus Sep-Pak cartridge to allow
smaller volume (1 mL) of ethanol and to avoid laborious rotary
evaporation before formulation. However, there is more serious
back pressure in the Light Sep-Pak than in the Plus Sep-Pak, in
addition, dead volume in the transfer tubing also affects the elu-
tion, and thus it is more difficult to efficiently elute the labeled pro-
duct from a Light Sep-Pak, which resulted in the low radiochemical
yield. The reason is that our home-built fully automated module
used many PTFE(polytetrafluoroethylene)/Silicone liners (septa)
and Teflon tubing, and these materials cannot afford too high pres-
sure gas (N2) push. The pressure of N2 gas introduced in our mod-
ule is set at 207 kPa (30 Psi). C-18 Light Sep-Pak cartridge works
well in manual or semi-automated radiosynthesis in our lab,
because we can easily introduce high pressure gas push during
the purification and reformulation process. Overall synthesis time
was 23 min from EOB, including approximately 11 min for [11C]
CH3OTf production, 5 min for O-[11C]methylation reaction, and
7 min for SPE purification, evaporation and reformulation. SPE
technique is fast, efficient and convenient and works very well
for the O-methylated ether tracer purification using the phenolic
hydroxyl precursor for radiolabeling.15,26,27
Synthesis of [11C]MK-1064 ([11C]5) is indicated in Scheme 3.
The desmethyl-MK-1064 was labeled by a reactive [11C]methylat-
ing agent, [11C]methyl triflate ([11C]CH3OTf)12,13 prepared from
[
11C]CO2, through the O-[11C]methylation. The labeling reaction
was under basic conditions in acetonitrile, and the labeling mix-
ture was isolated by a simplified solid-phase extraction (SPE)
method14–17 to provide target tracers [11C]5. The radiochemical
yields were 50–60%, decay corrected to end of bombardment
(EOB), based on [11C]CO2.
[
11C]Methyl iodide ([11C]CH3I) is a most commonly used [11C]
methylating agent,18 but [11C]CH3OTf is more reactive than [11C]
CH3I, and thus, the radiochemical yield of 11C]5 was relatively high.
The radiosynthesis including reaction, purification and formulation
was performed in an automated self-designed multi-purpose 11C-
radiosynthesis module, allowing measurement of specific activity
at EOB during synthesis.19–21 The labeling reaction was conducted
using a V-vial method. The purification was performed by a SPE
method, because the large polarity difference between the sodium
salt of the phenolic hydroxyl precursor (2 N NaOH) and the corre-
sponding labeled O-methylated ether product permitted the use of
SPE technique for purification of the labeled product from the radi-
olabeling reaction mixture. A C-18 Plus Sep-Pak cartridge was used
in SPE purification technique. It is difficult to directly elute the
labeled product from a C-18 Plus Sep-Pak to a vial using small vol-
ume of ethanol (1 ꢀ 1 mL or 2 ꢀ 0.5 mL), due to the back pressure
in the C-18 Sep-Pak and dead volume in the transfer tubing. In
order to elute most of the labeled product from the C-18
Specific activity is an important parameter for a brain radio-
tracer. If a reverse-phase (RP) high performance liquid chromatog-
raphy (HPLC) is used as purification method, then on line
N
N
O
N
N
O
HN
HN
i
O11CH3
Cl
O
N
Cl
O
N
OH
N
N
11C]MK-1064 ([11C] )
5
11
Desmethyl-MK-1064 (
)
[
Scheme 3. Synthesis of target tracer [11C]MK-1064 ([11C]5). Reagents, conditions and yields: (i) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 °C, 3 min; SPE; 50–60%.

M. Gao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3694–3699
3697
determination of specific activity at EOB is accurate. If a SPE car-
tridge is used as purification method, then the on-the-fly technique
to determine specific activity at EOB is not applied. The specific
activity for the 11C-tracers produced in our PET chemistry facility
[
11C]MK-1064 has been developed. An automated self-designed
multi-purpose [11C]-radiosynthesis module for the synthesis of
11C]MK-1064 has been built. The target tracer was easily prepared
[
by O-[11C]methylation of its corresponding desmethylated
precursor using a reactive [11C]methylating agent, [11C]CH3OTf,
and isolated by a simplified SPE purification procedure in high
radiochemical yields, short overall synthesis time, and high specific
radioactivities. These methods are efficient and convenient. It is
anticipated that the approaches for the design, synthesis and
automation of new tracer, authentic standard and radiolabeling
precursor, and improvements to increase radiochemical yield and
specific activity of the tracer described here can be applied with
advantages to the synthesis of other 11C-radiotracers for PET imag-
ing. These chemistry results combined with the reported in vitro
and in vivo biological data11 of MK-1064 encourage further
in vivo biological evaluation of [11C]MK-1064 as a new candidate
PET radioligand for imaging of OX2R in brain diseases.
usually ranges from 370 to 1110 GBq/
our previous works. The specific activity of [11C]MK-1064 was
185–555 GBq/ mol at the end of synthesis (EOS) determined by
lmol at EOB according to
l
analytical HPLC28,29 and calculated, which are in agreement with
the ‘on line’ determined values. To achieve the highest specific
activity, several procedures were performed.30,31 The 11C gas target
we used is the Siemens RDS-111 Eclipse cyclotron 11C gas target.
The technical trick to produce high specific activity [11C]CO2 is
we will usually do 2–3 times pre-burn with the same beam current
and short time like 10 min before production run. This pre-burn
will warm up the cyclotron and eliminate significant amount of
12C carrier-added in the cyclotron 11C gas target. We use an Eckert
& Ziegler Modular Lab C-11 Methyl Iodide/Triflate module to pro-
duce [11C]CH3OTf, convenient gas phase bromination of [11C]
methane, and production of [11C]CH3OTf. This is a ‘dry’ method
using Br2 to generate a [11C]CH3Br intermediate. Comparing with
other ‘dry’ methods using I2 and ‘wet’ methods using LiAlH4 and
HI, our ‘dry’ method seems to help minimize introduction of addi-
tional 12C carrier after [11C]CO2 production.13 To further help pro-
duce high specific activity [11C]CH3OTf, we usually do 2 ‘test
loop’ procedures when we set up the module for the actual [11C]
CH3OTf production run, and 1 actual [11C]CH3OTf production run
before we do [11C]methylation labeling reaction. These procedures
avoid any leak in the module to introduce additional 12C carrier
and eliminate significant amount of original 12C carrier accumu-
lated in the [11C]CH3OTf production system. Therefore, the specific
activity of our 11C-tracers is significantly improved.
Acknowledgments
This work was partially supported by the United States Indiana
State Department of Health (ISDH) Indiana Spinal Cord & Brain
Injury Fund (ISDH EDS-A70-2-079612). 1H NMR spectra were
recorded at 500 MHz on a Bruker Avance II 500 MHz NMR spec-
trometer in the Department of Chemistry and Chemical Biology
at Indiana University-Purdue University Indianapolis (IUPUI),
which is supported by the United States National Science Founda-
tion (NSF) Major Research Instrumentation Program (MRI) grant
CHE-0619254.
References and notes
Chemical purity and radiochemical purity were determined by
analytical HPLC.28,29 The chemical purity of the precursor and ref-
erence standard was >95%. The radiochemical purity of the target
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diode) flow detector, and the chemical purity of the target tracer
was >90% determined by reversed-phase HPLC through UV flow
detector.
The octanol–water partition coefficient (commonly expressed
as LogP) is an important physical parameter directly correlated
with the biological activities of a wide variety of organic com-
pounds.32–35 LogP provides an assessment of lipophilicity that
often correlates with a compound’s ability to penetrate the blood
brain barrier (BBB). We obtained LogP and calculated LogP (CLogP)
values of PET tracers for OX2R (Fig. 1) from ChemBioDraw Ultra
14.0 (ChemOffice) as listed in Table 1. LogP and CLogP data of
[
11C]MK-1064 are similar to those of [11C]FFMMCC, and in the opti-
mal range of LogD7.4 (2.0–3.5) reported for an optimum central
nervous system (CNS) penetration of drug molecules.36 These data
suggest the [11C]MK-1064 has appropriate lipophilicity for brain
uptake.
The experimental details and characterization data for com-
pounds 1–3, 5–11 and for the tracer [11C]5 are given.37
In summary, a simple and moderate-to-high-yield synthetic
route to synthesize MK-1064, desmethyl-MK-1064 and
Table 1
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LogP and CLogP values of PET tracers for OX2R
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Compound
LogP
CLogP
[
[
[
[
[
[
11C]BBAC
4.21
3.79
2.24
4.98
3.65
3.2
4.40
3.06
3.43
4.11
3.29
3.15
11C]BBPC
11C]EMPA
11C]CW4
11C]FFMMCC
11C]MK-1064

3698
M. Gao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3694–3699
26. Gao, M.; Gao, A.; Wang, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2014, 91, 71.
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Radiat. Isot. 2013, 74, 61.
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residue was purified by column chromatography on silica gel with eluent
(0:100 to 3:97 MeOH/CH2Cl2) to give a white solid product 5 (226 mg, 70%).
Rf = 0.56 (1:9 MeOH/CH2Cl2), mp 170–172 °C. 1H NMR (DMSO-d6): d 3.79 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 4.37 (d, J = 6.0 Hz, 2H, CH2), 7.16 (d, J = 8.0 Hz,
1H, Ar-H), 7.33 (d, J = 8.0 Hz, 1H, Ar-H), 7.42 (ddd, J = 1.0, 4.5, 7.5 Hz, 1H, Ar-H),
7.94 (dt, J = 1.5, 7.5 Hz, 1H, Ar-H), 8.07 (d, J = 7.5 Hz, 1H, Ar-H), 8.36 (d,
J = 2.0 Hz, 1H, Ar-H), 8.50 (t, J = 2.0 Hz, 1H, Ar-H), 8.53 (d, J = 4.0 Hz, 1H, Ar-H),
8.72 (d, J = 2.0 Hz, 1H, Ar-H), 8.88 (t, J = 6.0 Hz, 1H, NH), 9.06 (d, J = 2.0 Hz, 1H,
Ar-H), 9.16 (d, J = 2.0 Hz, 1H, Ar-H). MS (ESI): 462 ([M+H]+, 100%).
(f) 2-Bromo-6-iodopyridin-3-ol (6): To
a solution of 2-bromo-3-hydroxy
pyridine (20.0 g, 115 mmol) in water (250 mL) was added K2CO3 (31.8 g,
230 mmol) and I2 (29.2 g, 115 mmol). The mixture was stirred at RT for 5 h,
then cooled to 0 °C and treated with concentrated HCl until solids precipitated
from solution (pH ꢂ 6). The solids were isolated by filtration and dried to give a
36. Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376.
37. (a) General: All commercial reagents and solvents were purchased from Sigma-
brown solid 6 (33.1 g, 96%). Rf = 0.46 (1:49 MeOH/CH2Cl2), mp 152–154 °C. 1
H
Aldrich and Fisher Scientific, and used without further purification.
CH3OTf was prepared according to a literature procedure.13 Melting points
were determined on MEL-TEMP II capillary tube apparatus and were
[
11C]
NMR (DMSO-d6): d 7.07 (d, J = 8.0 Hz, 1H, Ar-H), 7.59 (d, J = 8.0 Hz, 1H, Ar-H),
11.31 (br s, 1H, OH). MS (ESI): 298 ([MꢁH]ꢁ, 100%).
a
(g) 3-(Benzyloxy)-2-bromo-6-iodopyridine (7): To a solution of compound 6
(12.0 g, 40.0 mmol) in CH3CN (150 mL) was added K2CO3 (11.6 g, 80.0 mmol)
and benzyl bromide (8.21 g, 48.0 mmol). The mixture was stirred at 60 °C for
12 h, then cooled to RT, filtered, washed with CH3CN, and concentrated. The
residue was recrystallized from EtOAc/hexanes to give a pale brown solid 7
(12.6 g, 81%). Rf = 0.82 (1:2 EtOAc/hexanes), mp 82–84 °C. 1H NMR (CDCl3): d
5.16 (s, 2H, CH2), 6.82 (d, J = 8.5 Hz, 1H, Ar-H), 7.32–7.36 (m, 1H, Ar-H), 7.37–
7.43 (m, 4H, Ar-H), 7.51 (d, J = 8.5 Hz, 1H, Ar-H). MS (ESI): 390 ([M+H]+, 100%).
(h) 3-(Benzyloxy)-6-iodo-2-methoxypyridine (8): To a solution of compound 7
(6.50 g, 16.6 mmol) in DMF (60 mL) was added MeONa (5.76 g, 26.6 mmol, 25%
in methanol). The mixture was heated to 100 °C and stirred for 1.5 h, then
cooled to RT, and partitioned between EtOAc and water. The organic phase was
washed with water and brine, dried over MgSO4, filtered, and concentrated.
The crude product was purified by column chromatography on silica gel with
eluent (5:95 to 20:80 EtOAc/hexanes) to give a white solid product 8 (4.81 g,
85%). Rf = 0.70 (1:5 EtOAc/hexanes), mp 48–50 °C. 1H NMR (CDCl3): d 3.99 (s,
3H, OCH3), 5.10 (s, 2H, CH2), 6.71 (d, J = 8.0 Hz, 1H, Ar-H), 7.13 (d, J = 8.0 Hz, 1H,
Ar-H), 7.31–7.33 (m, 1H, Ar-H), 7.34–7.39 (m, 4H, Ar-H). MS (ESI): 342 ([M+H]+,
100%).
uncorrected. 1H NMR spectra were recorded at 500 MHz on a Bruker Avance
II 500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal
standard. Chemical shift data for the proton resonances were reported in parts
per million (ppm, d scale) relative to internal standard TMS (d 0.0), and
coupling constants (J) were reported in hertz (Hz). Liquid chromatography–
mass spectra (LC–MS) analysis was performed on an Agilent system, consisting
of an 1100 series HPLC connected to a diode array detector and a 1946D mass
spectrometer configured for positive-ion/negative-ion electrospray ionization.
The high resolution mass spectra (HRMS) were obtained using a Waters/
Micromass LCT Classic spectrometer. Chromatographic solvent proportions are
indicated as volume: volume ratio. Thin-layer chromatography (TLC) was run
using Analtech silica gel GF uniplates (5 ꢀ 10 cm2). Plates were visualized
under UV light. Normal phase flash column chromatography was carried out
on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated
solvent system in the proportions described below. All moisture- and air-
sensitive reactions were performed under a positive pressure of nitrogen
maintained by a direct line from a nitrogen source. Analytical RP HPLC was
performed using a Prodigy (Phenomenex) 5
mobile phase 43% CH3CN/57% H2O; flow rate 1.5 mL/min; and UV (254 nm)
and -ray (PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were
lm C-18 column, 4.6 ꢀ 250 mm;
(i) 5-(Benzyloxy)-6-methoxypicolinonitrile (9): To a solution of compound 8
(4.09 g, 12.0 mmol) in DMF (120 mL) was added copper cyanide (2.15 g,
24.0 mmol). The mixture was heated to 150 °C for 2 h, then cool to RT, diluted
with EtOAc (250 mL), and filtered through a pad of Celite. The reaction mixture
was washed with water (2 ꢀ 50 mL) and brine (50 mL), dried over MgSO4,
filtered, and concentrated. The residue was purified by column chromatogra-
phy on silica gel with eluent (5:95 to 30:70 EtOAc/hexanes) to give a white
c
obtained from Waters Corporation (Milford, MA). Sterile Millex-GS 0.22
filter units were obtained from Millipore Corporation (Bedford, MA).
lm
(b) Methyl 50,6-dichloro-[3,30-bipyridine]-5-carboxylate (1): To
a solution of
methyl 2-chloro-5-iodonicotinate (5.95 g, 20.0 mmol) in N,N-dimethylfor-
mamide (DMF, 82 mL)/water (4.0 mL) was added 5-(chloropyridin-3-yl)
boronic acid (3.20 g, 20.3 mmol), [1,10-bis(diphenylphosphino)ferrocene]
dichloropalladium(II) (Pd(dppf)Cl2, 1.46 g, 2.0 mmol) and Cs2CO3 (19.6 g,
60.0 mmol). The reaction was stirred at RT for 24 h. Then the reaction mixture
was partitioned between EtOAc and water, the organic layer was washed with
water and brine, dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by trituration with MeOH followed by EtOAc/Et2O to give
a yellow solid 1 (5.10 g, 90%). Rf = 0.70 (1:19 MeOH/CH2Cl2), mp 135–137 °C.
1H NMR (CDCl3): d 4.01 (s, 3H, OCH3), 7.89 (t, J = 2.5 Hz, 1H, Ar-H), 8.34 (d,
J = 2.5 Hz, 1H, Ar-H), 8.66 (d, J = 2.5 Hz, 1H, Ar-H), 8.72–8.74 (m, 2H, Ar-H). MS
(ESI): 283 ([M+H]+, 100%).
solid product 9 (2.10 g, 73%). Rf = 0.28 (1:5 EtOAc/hexanes), mp 106–108 °C. 1
H
NMR (DMSO-d6): d 3.90 (s, 3H, OCH3), 5.22 (s, 2H, CH2), 7.35–7.38 (m, 1H, Ar-
H), 7.40–7.43 (m, 2H, Ar-H), 7.45–7.46 (m, 2H, Ar-H), 7.50 (d, J = 8.0 Hz, 1H, Ar-
H), 7.64 (d, J = 8.0 Hz, 1H, Ar-H). MS (ESI): 242 ([M+H]+, 100%); MS (ESI): 239
([MꢁH]ꢁ, 1%).
(j) 6-(Aminomethyl)-2-methoxypyridin-3-ol (10): To a solution of compound 9
(360 mg, 1.5 mmol) in methanol (70 mL) was added Pearlman’s catalyst (Pd
(OH)2 on carbon, 120 mg). The mixture was reacted under an atmosphere of
hydrogen (50 psi) for 8 h. The reaction contents were filtered through a pad of
Celite and the filtrate was collected. The solvent was removed in vacuo to give
a bone semi-solid crude product. Because of instability of the product on silica
gel column, the product was directly used in next step reaction without further
purification. Rf = 0.22 (1:1 MeOH/CH2Cl2). MS (ESI): 155 ([M+H]+, 100%); MS
(ESI): 153 ([MꢁH]ꢁ, 5%).
(c) Methyl 500-chloro-[2,20:50,300-terpyridine]-30-carboxylate (2): To a solution of
compound 1 (1.66 g, 5.86 mmol) in DMF (30 mL) was added 2-(tri-n-butyl-
stannyl)pyridine (3.02 g, 8.20 mmol), CsF (2.67 g, 17.6 mmol), CuI (223 mg,
1.17 mmol) and Pd(PPh3)4 (677 mg, 0.586 mmol). The mixture was heated to
80 °C for 2 h. The reaction mixture was cooled, diluted with EtOAc (120 mL),
and filtered through a pad of Celite. The filtrate was washed with water
(2 ꢀ 50 mL) and brine (50 mL), dried over MgSO4, filtered and concentrated.
The residue was purified by column chromatography on silica gel with eluent
(5:95 to 50:50 EtOAc/hexanes) to give a white solid product 2 (1.01 g, 53%).
Rf = 0.26 (1:2 EtOAc/hexanes), mp 111–113 °C. 1H NMR (CDCl3): d 3.84 (s, 3H,
OCH3), 7.34 (ddd, J = 1.0, 5.0, 7.5 Hz, 1H, Ar-H), 7.85 (dt, J = 2.0, 8.0 Hz, 1H, Ar-
H), 7.94 (t, J = 2.0 Hz, 1H, Ar-H), 8.12 (d, J = 2.0 Hz, 1H, Ar-H), 8.22 (d, J = 8.0 Hz,
1H, Ar-H), 8.63 (d, J = 4.0 Hz, 1H, Ar-H), 8.65 (d, J = 2.0 Hz, 1H, Ar-H), 8.79 (d,
J = 2.0 Hz, 1H, Ar-H), 8.94 (d, J = 2.0 Hz, 1H, Ar-H). MS (ESI): 326 ([M+H]+,
100%).
(k)
500-Chloro-N-((5-hydroxy-6-methoxypyridin-2-yl)methyl)-[2,20:50,300-ter-
pyridine]-30-carboxamide (desmethyl-MK-1064, 11): Compound 10 was reacted
with compound 3 using the similar procedure as synthesis of compound 5 to
give a white solid 11, yield 35%. Rf = 0.45 (1:19 MeOH/CH2Cl2), mp > 251 °C
(decomposed). 1H NMR (DMSO-d6): d 3.30 (s, 3H, OCH3), 4.33 (d, J = 6.0 Hz, 2H,
CH2), 7.00 (d, J = 7.5 Hz, 1H, Ar-H), 7.09 (d, J = 7.5 Hz, 1H, Ar-H), 7.41 (dd, J = 5.0,
7.5 Hz, 1H, Ar-H), 7.92 (dt, J = 1.5, 8.0 Hz, 1H, Ar-H), 8.06 (d, J = 8.0 Hz, 1H, Ar-
H), 8.34 (d, J = 2.0 Hz, 1H, Ar-H), 8.49 (d, J = 4.0 Hz, 1H, Ar-H), 8.50 (t, J = 2.0 Hz,
1H, Ar-H), 8.72 (d, J = 2.0 Hz, 1H, Ar-H), 8.82 (t, J = 6.0 Hz, 1H, NH), 9.06 (d,
J = 2.0 Hz, 1H, Ar-H), 9.14 (d, J = 2.0 Hz, 1H,Ar-H), 9.26 (s, 1H, OH). MS (ESI):
448 ([M+H]+, 100%); MS (ESI): 446 ([MꢁH]ꢁ, 18%). HRMS (ESI): calcd for
(d) 500-Chloro-[2,20:50,300-terpyridine]-30-carboxylic acid (3): To
a
solution of
C
23H18N5O3NaCl 470.0996 ([M+Na]+), found 470.0986.
compound 2 (0.33 g, 1.0 mmol) in methanol (30 mL)/water (3 mL) was added
KOH (0.56 g, 10 mmol). The mixture was stirred at RT for 48 h. The reaction
mixture was concentrated in vacuo. The residue was added water and HCl
(1 N), and pH value of the mixture was adjusted to 6. The resulting precipitate
was filtered, washed with cold water, and dried to afford a white solid product
3 (0.306 g, 98%). Rf = 0.38 (1:1 MeOH/CH2Cl2), mp 185–187 °C. 1H NMR (DMSO-
d6): d 7.47 (t, J = 6.0 Hz, 1H, Ar-H), 7.98 (t, J = 7.5 Hz, 1H, Ar-H), 8.11 (d,
J = 7.5 Hz, 1H, Ar-H), 8.43 (s, 1H, Ar-H), 8.50 (s, 1H, Ar-H), 8.60 (d, J = 4.0 Hz, 1H,
Ar-H), 8.72 (s, 1H, Ar-H), 9.05 (s, 1H, Ar-H), 9.16 (s, 1H, Ar-H), 13.18 (br s, 1H,
OH). MS (ESI): 312 ([M+H]+, 56%); MS (ESI): 310 ([MꢁH]ꢁ, 14%).
(l) 500-Chloro-N-((5-[11C]methoxy-6-methoxypyridin-2-yl)methyl)-[2,20:50,300-ter-
pyridine]-30-carboxamide ([11C]MK-1064, [11C]5): [11C]CO2 was produced by the
14N(p, 11C nuclear reaction in the small volume (9.5 cm3) aluminum gas
a)
target provided with the Siemens RDS-111 Eclipse cyclotron. The target gas
consisted of 1% oxygen in nitrogen purchased as a specialty gas from Praxair,
Indianapolis, IN. Typical irradiations used for the development were 50
lA
beam current for 15 min on target. The production run produced approx-
imately 25.9 GBq of [11C]CO2 at EOB. Desmethyl-MK-1064 (11) (0.1–0.3 mg)
was dissolved in CH3CN (300 lL). To this solution was added 2 N NaOH (2 lL).
The mixture was transferred to a 5-mL small reaction vial. No-carrier-added
(high specific activity) [11C]CH3OTf (13.9 GBq) that was produced by the gas-
phase production method13 within 11 min from [11C]CO2 (25.9 GBq) through
(e) 500-Chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,20:50,300-terpyridine]-30-
carboxamide (MK-1064, 5): To a solution of compound 3 (218 mg, 0.70 mmol)
in DMF (30 mL) was added (5,6-dimethoxypyridin-2-yl)methanamine (4,
124 mg, 0.735 mmol), EDC (268 mg, 1.4 mmol) HOAt (237 mg, 1.4 mmol) and
DIPEA (361 mg, 2.8 mmol). The mixture was stirred at RT for 15 h. The reaction
mixture was diluted with EtOAc (120 mL), washed with water (2 ꢀ 30 mL) and
brine (30 mL), dried over MgSO4, filtered, and concentrated in vacuo. The
[
11C]CH4 (21.8 GBq) and [11C]CH3Br (13.9 GBq) with silver triflate (AgOTf)
column was passed into the reaction vial at RT until radioactivity reached a
maximum (2 min), and then the reaction vial was isolated and heated at 80 °C
for 3 min. The contents of the reaction vial were diluted with NaHCO3 (0.1 M,
1 mL). The reaction vial was connected to a C-18 Plus Sep-Pak cartridge. The

M. Gao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3694–3699
3699
labeled product mixture solution was passed onto the cartridge for SPE
purification by gas pressure. The cartridge was washed with H2O (2 ꢀ 3 mL),
and the aqueous washing was discarded. The product was eluted from the
cartridge with EtOH (2 ꢀ 2 mL), and then passed onto a rotatory evaporator.
The solvent was removed by evaporation (3 min) under vacuum. The final
volume of ethanol after evaporation was ꢂ1 mL. The labeled product [11C]5
was reformulated with saline (10 mL), sterile-filtered through a sterile vented
Millex-GS 0.22 m cellulose acetate membrane and collected into a sterile vial.
l
Total radioactivity (4.7–7.1 GBq) was assayed and the total volume (10–11 mL)
was noted for tracer dose dispensing. Retention times in the analytical HPLC
system were: tR 11 = 4.13 min, tR 5 = 6.56 min, and tR [
11C]5 = 6.64 min.