Radiosynthesis of the 11C-methyl derivative of LBQ657 for PET investigation of the neprilysin inhibitor sacubitril

Article abstract of DOI:10.1002/jlcr.3817

Neprilysin, also known as neutral endopeptidase, is a cell surface membrane metalo-endopeptidase that cleaves various peptides. Altered neprilysin expression has been correlated with various cancers and cardiovascular diseases. In this work, we present the radiosynthesis of the novel O-¹¹C-methylated derivative of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [¹¹C]methyl ester ([¹¹C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a ¹¹C-methyl instead of an ethyl. [¹¹C]MeOLBQ was produced in a one-pot two-step synthesis. The O-¹¹C-methylation of the pentanoic acid part was done with [¹¹C]methyl triflate followed by the deprotection of the tert-butyl ester precursor in acidic conditions. [¹¹C]MeOLBQ ([¹¹C]7) was produced in 9.5 ± 2.5% RCY (25 ± 6% decay-corrected from [¹¹C]CO₂, n = 3) high molar activity 348 ± 100 GBq/μmol (9425 ± 2720 mCi/μmol) at EOS, in high chemical (>95%) and radiochemical (>99%) purities. The total synthesis time including HPLC purification and reformulation was 29 minutes. To our knowledge, this is the first PET-labeled analog of the clinically used NEP inhibitor sacubitril.

Full text of DOI:10.1002/jlcr.3817

Received: 4 October 2019
DOI: 10.1002/jlcr.3817
Revised: 19 November 2019
Accepted: 21 November 2019
R E S E A R C H A R T I C L E
Radiosynthesis of the 11C-methyl derivative of LBQ657 for
PET investigation of the neprilysin inhibitor sacubitril
Valentin R. Teyssier1,2 | José-Mathieu Simard1 | Mark H. Dornan1,3
François Tournoux4,5 | Jean N. DaSilva1,2,3
|
1Laboratoire de Radiochimie et Cyclotron,
Neprilysin, also known as neutral endopeptidase, is a cell surface membrane
metalo-endopeptidase that cleaves various peptides. Altered neprilysin expres-
sion has been correlated with various cancers and cardiovascular diseases. In
this work, we present the radiosynthesis of the novel O-11C-methylated deriva-
tive of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-
4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [11C]methyl ester
([11C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a 11C-methyl
instead of an ethyl. [11C]MeOLBQ was produced in a one-pot two-step synthe-
sis. The O-11C-methylation of the pentanoic acid part was done with [11C]
methyl triflate followed by the deprotection of the tert-butyl ester precursor in
acidic conditions. [11C]MeOLBQ ([11C]7) was produced in 9.5 2.5% RCY (25
Centre de Recherche du Centre
Hospitalier de l'Université de Montréal,
Montréal, Québec, Canada
2Institut de Génie Biomédical, Faculté de
Médecine, Université de Montréal,
Québec, Canada
3Département de Radiologie, radio-
oncologie et médecine nucléaire, Faculté
de médecine, Université de Montréal,
Québec, Canada
4Département de Médecine, Centre de
Recherche du Centre Hospitalier de
l'Université de Montréal, Montréal,
Québec, Canada
6% decay-corrected from [11C]CO2, n = 3) high molar activity 348
100
5Centre cardiovasculaire, Centre
Hospitalier de l'Université de Montréal,
Montréal, Québec, Canada
GBq/μmol (9425 2720 mCi/μmol) at EOS, in high chemical (>95%) and
radiochemical (>99%) purities. The total synthesis time including HPLC purifi-
cation and reformulation was 29 minutes. To our knowledge, this is the first
PET-labeled analog of the clinically used NEP inhibitor sacubitril.
Correspondence
Jean N. DaSilva, Centre de Recherche du
Centre Hospitalier de l'Université de
Montréal, Laroratoire de, Radiochimie et
Cyclotron, 900 rue Saint-Denis, Montréal,
Québec, H2X 0A9, Canada.
K E Y W O R D S
carbon-11, 11C-esterification, heart failure, natriuretic peptides, radiosynthesis, sacubitril
Email: jean.dasilva@umontreal.ca
Funding information
RBIQ, Grant/Award Number: 14.04;
SQIC-Roche
1 | INTRODUCTION
by reducing their availability at the cell surface.1 NEP is
widely expressed in mammalian tissues, such as kidneys,
Neprilysin (NEP), also called neutral endopeptidase,
enkephalinase, or CD10, is a zinc dependent type II inte-
gral membrane peptidase that cleaves peptides such as
substance P, endothelin-1, natriuretic peptides, and
encephalin and thus regulates their physiological action
brain, heart, and lung.2 In cancer, by regulating the bio-
logical activities of peptide substrates, NEP plays an
important role in maintenance of homeostasis, neoplastic
transformation, and tumor progression.3,4 Elevated NEP
expression in several solid tumors and carcinomas has
been reported to be a marker of cancer progression.5,6
NEP is also a target of choice for therapeutic intervention
in heart failure. NEP plays a key role into cardiorenal dis-
orders. By breaking down natriuretic peptides, NEP
The authors declare the following patent application
(CAN_DMS_126825411 USPR Radiolabeled Sacubitril Derivatives to V.
R.T., F.T., and J.N.D.).
J Label Compd Radiopharm. 2020;63:65–71.
wileyonlinelibrary.com/journal/jlcr
© 2020 John Wiley & Sons, Ltd.
65

66
TEYSSIER ET AL.
participates in the regulation of the sodium level, water
balance, and blood pressure homeostasis.7-9 Blocking this
enzyme with NEP inhibitors (NEPi) increases the con-
centration of the natriuretic peptides and thus has thera-
peutic potential.10,11 It has been demonstrated that a
combined therapy consisting of the NEPi sacubitril and
the angiotensin II blocker valsartan (ie, Entresto,
Novartis) improved the outcome in the treatment of heart
failure and patient survival.12 Sacubitril is metabolized
into the desethylated active NEPi sacubitrilat (LBQ657).13
instrument equipped with a UV detector (254 nm) and
Raytest Gabi Star radioactivity detector.
2.2 | Chemistry
2.2.1 | (3R,5S)-5-Biphenyl-4-ylmethyl-3-
methylpyrrolidin-2-one (2)
(2R,4S)-4-Amino-5-(biphenyl-4-yl)-2-methylpentanoic
acid ethyl ester (1) (500 mg, 1.43 mmol) was dissolved in
1:1 THF/MeOH (12 mL) and 2M NaOH (3.6 mL) was
added dropwise. The reaction mixture was quenched
with water and extracted with EtOAc (3 × 30 mL). The
organic fractions were combined, washed with brine,
dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification by silica column chroma-
tography (30% EtOAc in hexanes) afforded compound 2
Taking into account the terminal half-life of 0.7
0.3
hours in humans14 and interpatient variability of
Sacubitril,15 the novel 11C-methylated derivative of the
potent NEPi LBQ657 (as opposed to the ethyl ester deriv-
ative of LBQ657, sacubitril) was developed with the
potential to investigate in vivo the hydrolysis and metab-
olism of an alkylated ester analog allowing for a better
understanding of the distribution and pharmacokinetics
of the prodrug. This paper describes the radiosynthesis of
the 11C-methylated derivative of LBQ657 ([11C]
MeOLBQ).
1
(360 mg, 1.35 mmol, 95%) as a white powder. H NMR
(300 MHz, CDCl3) δ 7.56 (t, J = 8.2 Hz, 4H), 7.44 (t, J =
7.4 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.25 (d, J = 7.8 Hz,
2H), 6.00 (s, 1H), 3.94-3.75 (m, 1H), 2.91-2.70 (m, 2H),
2.56-2.39 (m, 1H), 2.21-2.07 (m, 1H), 1.99-1.83 (m, 1H),
1.20 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
180.5, 140.8, 139.9, 136.8, 129.6, 128.9, 127.6, 127.4, 127.1,
53.5, 42.5, 35.2, 35.0, 16.3. HRMS (ESI): Exact mass calcd
for (C18H19NO) [M + H]+: 266.1539. Found: 266.1537.
2 | EXPERIMENTAL
2.1 | Materials
Commercially available chemicals were used without
further purification unless otherwise noted. The
syntheses of 1-O-tert-butyl 4-O-(2,5-dioxopyrrolidin-1-yl)
2.2.2 | (2R,4S)-4-Amino-5-biphenyl-4-yl-2-
methylpentanoic acid hydrochloride (3)
butanedioate
(8)
and
1-O-benzyl
4-O-(2,-
5-dioxopyrrolidin-1-yl) butanedioate (4) were completed
Compound 2 (300 mg, 1.13 mmol) was dissolved in 1:1
acetic acid/HCl (20 mL). The reaction mixture was stirred
under reflux for 20 hours and then concentrated under
reduced pressure. Recrystallization from 9:1 EtOAc/acetic
acid gave the product 3 (290 mg, 0.90 mmol, 80%) as a
yellow powder. 1H NMR (300 MHz, DMSO) δ 7.65 (t, J =
7.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 7.7 Hz,
1H), 3.64-3.23 (m, 1H), 3.13-2.97 (m, 1H), 2.95-2.78 (m,
1H), 2.74-2.57 (m, 1H), 1.93-1.77 (m, 1H), 1.67-1.49 (m, J
= 13.7 Hz, 1H), 1.06 (d, J = 6.9 Hz, 1H). 13C NMR (75
MHz, DMSO) δ 177.0, 140.2, 139.1, 136.7, 130.5, 129.4,
127.9, 127.7, 127.0, 50.8, 38.6, 36.2, 35.4, 18.0. HRMS
(ESI): Exact mass calcd for (C18H22NO2+) [M − HCl +
H]+: 284.1645. Found: 284.1644.
as reported previously.16,17 Flash chromatography was
1
carried out with silica gel (40-60 μm). H NMR and 13C
NMR spectra were acquired with a Bruker 300- MHz
spectrometer at ambient temperature. Spectral data are
reported in parts per million (ppm) using residual solvent
as a reference. High resolution and accurate mass mea-
surements were done in positive mode by flow injection
analysis into a Thermo Scientific Q-Exactive Plus
Orbitrap Mass Spectrometer (San Jose, CA, USA) inter-
faced with a heated electrospray ion source. A synthesis
platform (Synthra MeIplus Research) was utilized for
automated radiotracer synthesis, purification, and
reformulation. Sep-Pack Vac C18 plus short (100 mg,
Waters) solid-phase extraction cartridges were
preconditioned by passing through ethanol (10 mL)
followed by deionized water (20 Ml). Semipreparative
HPLC was performed with a Phenomenex Luna C18 (2)
HPLC column (250 × 10 mm, 10 μm) at a flow rate of 7
mL/min (4.5:5 acetonitrile/ammonium formate 0.1M, pH
4.8). Analytical HPLC was carried out on a Luna C18 (2)
column (250 × 4.6 mm, 10 μm) at a flow rate of 3
mL/min (1:1 CH3CN/H2O + 0.1% TFA) with a Waters
2.2.3 | (2R,4S)-5-(Biphenyl-4-yl)-4-(4-
(benzyloxy)-4-oxobutanamido)-2-
methylpentanoic acid (5)
Compounds 3 (250 mg, 0.78 mmol), 4 (358 mg, 1.17
mmol, 1.5 eq.), and DMAP (955 mg, 7.8 mmol, 10 eq.)
were dissolved in 1:1 DMF/PBS (20 mL), and the reaction

TEYSSIER ET AL.
67
was stirred at room temperature for 18 hours. The mix-
ture was quenched with water (20 mL) and extracted
with EtOAc (3 × 30 mL). The organic fractions were com-
bined and washed with 2N HCl (30 mL), water (20 mL),
and brine (30 mL). The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pres-
sure. Compound 5 (245 mg, 0.51 mmol, 65%) was purified
by silica column chromatography (40% EtOAc in hex-
temperature for 5 minutes under nitrogen atmosphere.
The reaction was then placed under an atmosphere of H2
(balloon) which was bubbled through the mixture for 20
minutes. The reaction was stirred for an additional 20
minutes, filtered over celite, and concentrated under
reduced pressure. The product 7 (18 mg, 0.04 mmol, 88%)
was purified by silica column chromatography (50%
EtOAc in hexanes + 0.1% acetic acid). 1H NMR (300
MHz, CDCl3) δ 7.56 (d, J = 7.1 Hz, 2H), 7.51 (d, J = 8.1
Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H),
7.22 (d, J = 8.1 Hz, 2H), 6.02 (d, J = 8.8 Hz, 1H), 4.32-
4.15 (m, 1H), 3.65 (s, 3H), 2.90-2.76 (m, 2H), 2.66-2.51 (m,
3H), 2.41 (t, J = 6.5 Hz, 2H), 2.00-1.85 (m, 1H), 1.63-1.47
(m, 1H), 1.15 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 176.9, 176.6, 172.0, 140.8, 139.4, 136.6, 129.8,
128.8, 127.2, 127.1, 127.0, 51.9, 48.8, 40.5, 37.4, 36.4, 30.8,
29.7, 17.6. HRMS (ESI): Exact mass calcd for C23H28NO5
[M + H]+: 398.1962. Found: 398.1961.
1
anes). H NMR (300 MHz, CDCl3) δ 7.59-7.48 (4H, m),
7.46-7.38 (2H, m), 7.37-7.28 (6H, m), 7.21 (2H, d, J = 8.2),
6.03 (1H, d, J = 8.8), 5.09 (2H, s, J = 2.6), 4.40-4.24 (1H,
m), 2.92-2.39 (7H, m), 1.99-1.87 (1H, m), 1.60-1.48 (1H,
m), 1.16 (3H, d, J = 6.9). 13C NMR (75 MHz, CDCl3) δ
178.7, 173.1, 172.8, 140.7, 139.7, 136.1, 135.8, 129.6, 128.8,
128.6, 128.4, 128.2, 127.3, 127.3, 127.0, 66.7, 49.0, 40.7,
39.7, 36.5, 31.0, 29.5, 17.8. HRMS (ESI): Exact mass calcd
for (C29H31NO5) [M + H]+: 474.2275. Found: 474.2275.
2.2.4 | Methyl (2R,4S)-5-(biphenyl-4-yl)-4-
(4-(benzyloxy)-4-oxobutanamido)-2-
methylpentanoate (6)
2.2.6 | (2R,4S)-5-(Biphenyl-4-yl)-4-(4-(tert-
butoxy)-4-oxobutanamido)-2-
methylpentanoic acid (9)
Compound 5 (35 mg, 0.07 mmol) and Na2CO3 (12 mg,
0.11 mmol, 1.6 eq.) were mixed in DMF (5 mL). Methyl
iodide (15 mg, 0.11 mmol, 1.6 eq.) was added, and the
solution was stirred at room temperature for 4 hours. The
reaction was quenched with water (20 mL) and extracted
with EtOAc (3 × 15 mL). The organic fractions were com-
bined and washed with 2N HCl (15 mL), water (20 mL),
and brine (15 mL). The solution was then dried over
MgSO4, filtered, and concentrated under reduced pres-
sure. The product 6 (32 mg, 0.06 mmol, 88%) was purified
by silica column chromatography (50% EtOAc in hex-
Compounds 3 (50 mg, 0.15 mmol), 8 (85 mg, 0.31 mmol),
and DMAP (191 mg, 1.5 mmol) were dissolved in 1:1
DMF/PBS (20 mL), and the reaction was stirred at room
temperature for 18 hours. The mixture was quenched
with 10% citric acid (20 mL) and extracted with EtOAc (3
× 35 mL). The organic fractions were combined and
washed with 10% citric acid (2 × 50 mL), water (50 mL),
and brine (30 mL). The solution was dried over MgSO4,
filtered, and concentrated under reduced pressure. Com-
pound 9 (23 mg, 0.05 mmol, 34%) was purified by silica
column chromatography (40% EtOAc in hexanes + 0.1%
acetic acid). 1H NMR (300 MHz, CDCl3) δ 7.57 (d, J = 7.1
Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H),
7.33 (t, J = 7.3 Hz, 1H), 7.23 (d, J = 8.2 Hz, 2H), 6.16 (d, J
= 8.8 Hz, 1H), 4.40-4.22 (m, 1H), 2.96-2.75 (m, 2H), 2.69-
2.32 (m, 5H), 1.99-1.86 (m, 1H), 1.60-1.47 (m, 1H), 1.42 (s,
9H), 1.17 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 176.8, 172.9, 170.9, 140.8, 139.4, 136.7, 135.8, 129.9,
128.8, 128.6, 128.3, 128.2, 127.2, 127.1, 127.0, 66.5, 51.8,
48.4, 40.5, 37.6, 36.4, 31.2, 29.6, 17.7. HRMS (ESI): Exact
mass calcd for (C26H33NO5) [M + H]+: 440.2431. Found:
440.2431.
1
anes). H NMR (300 MHz, CDCl3) δ 7.58 (d, J = 7.1 Hz,
2H), 7.52 (d, J = 8.2 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H),
7.37-7.29 (m, 6H), 7.24 (d, J = 8.2 Hz, 2H), 5.62 (d, J =
8.8 Hz, 1H), 5.11 (s, 2H), 4.36-4.17 (m, 1H), 3.67 (s, J =
2.9 Hz, 3H), 2.83 (d, J = 6.4 Hz, 2H), 2.71-2.63 (m, 2H),
2.62-2.51 (m, 1H), 2.42 (t, J = 6.8 Hz, 2H), 2.00-1.88 (m,
1H), 1.59-1.45 (m, 1H), 1.16 (d, J = 7.1 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ 176.8, 172.9, 170.9, 140.8, 139.4, 136.7,
135.8, 129.9, 128.8, 128.6, 128.3, 128.2, 127.2, 127.1, 127.0,
66.5, 51.8, 48.4, 40.5, 37.6, 36.4, 31.2, 29.6, 17.7. HRMS
(ESI): Exact mass calcd for (C30H33NO5) [M + H]+:
488.2428. Found: 488.2428.
2.2.5 | (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-
carboxypropionyl)amino]-2-
methylpentanoic (7), MeOLBQ standard
2.3 | Radiochemistry
2.3.1 | Production of [11C]CH3I and [11C]
CH3OTf
Compound 6 (25 mg, 0.05 mmol) was dissolved in metha-
nol (6 mL); 10 wt% Pd/C (3 mg, 0.002 mmol, 0.04 eq.)
was added, and the reaction was stirred at room
[11C]CO2 was produced using our IBA Cyclone 18/9
cyclotron via the 14N(p,α)11C nuclear reaction by

68
TEYSSIER ET AL.
irradiating a gas target of 0.2% O2 in N2 with a 40 to 45
μA beam at 15-16 MeV for 20 to 25 minutes. Following
transfer with the target gas, [11C]CO2 was trapped at
−180ꢀC and reduced to [11C]CH4 using a nickel catalyst
in the presence of H2 gas at 425ꢀC. The [11C]CH4 was
trapped on a Carbosphere column at −120ꢀC. Rec-
irculation using high purity He (grade 6) following
release of [11C]CH4 by temperature increase allowed
reaction with elemental iodine at 750ꢀC to produce [11C]
CH3I that was trapped on a Porapak column at room
temperature. When maximum activity level was reached,
the Porapak column was heated at 200ꢀC to permit the
transfer with He (grade 6) of [11C]CH3I through an
online silver triflate column heated at 200ꢀC, allowing
the generation of [11C]CH3OTf which was then bubbled
into the reactor. Alternatively, [11C]CH3I was transferred
directly from the Porapak column into the reactor for
11C-methylation.
potassium carbonate (4 mg, 28.9 μmol, 26 eq.), and
Kryptofix 222 (5.1 mg, 13.5 μmol, 12 eq.) dissolved in
DMF (200 μL) in various conditions (see Table 1). [11C]
CH3I or [11C]CH3OTf was bubbled (flow 35 mL/min) and
trapped at −25ꢀC in the reaction mixture before heating
the vial at 50ꢀC or 120ꢀC for 1 or 3 minutes to conduct
the alkylation reaction. The deprotection reaction was
then completed at 50ꢀC to 90ꢀC for 0.5 to 3 minutes using
200 μL of 18% HCl, 6% HCl or TFA. The reaction mixture
was cooled to 20ꢀC, quenched with the HPLC buffer (0.8
mL), and then injected onto semipreparative HPLC.
2.3.3 | Semipreparative HPLC
purification and reformulation
The semipreparative HPLC fraction containing [11C]7 (tR
= 5.8 minutes, Figure 1) was collected into a vessel con-
taining Milli-Q water (40 mL). A flow of He was used to
transfer the solution through a preconditioned Sep-Pack
Vac C18 1 cc cartridge. The cartridge was washed with
sterile water (10 mL) to remove residual HPLC solvent.
[11C]7 was eluted with USP ethanol (0.6 mL) followed by
saline (5.4 mL) into a sterile vial to provide the final
product. (Figure 1A).
2.3.2 | Automated radiosynthesis of [11C]
(2R,4S)-5-(biphenyl-4-yl)-4-[(3-
carboxypropionyl)amino]-2-
methylpentanoic acid methyl ester ([11C]
MeOLBQ, [11C]7)
Radiolabeling optimization reactions were performed
with approximately 2 Ci (74 GBq) of [11C]CO2 at end of
beam. 11C-Methylation was carried out with either [11C]
methyl iodide or [11C]methyl triflate with a mixture of
the tert-butyl-protected precursor 9 (0.5 mg, 1.13 μmol),
2.3.4 | Analytical HPLC analyses
An aliquot of the final product was removed for analyti-
cal HPLC to determine the chemical and radiochemical
TABLE 1 Optimization of reaction conditions
Reaction Time
Temperature of of Deprotection,
Reagent
Reaction Time
Temperature of
Deprotection, ꢀC
Acid for
Deprotection
RCY,a
%
Entry MeI/MeOTf of Alkylation, s
Alkylation, ꢀC
s
1
MeI
180
180
180
60
120
120
50
50
50
50
50
50
50
50
50
50
60
60
60
60
90
120
180
30
60
30
30
60
60
60
60
60
90
60
60
60
60
60
90
60
HCl 18%
HCl 18%
HCl 18%
HCl 18%
HCl 18%
HCl 18%
HCl 18%
HCl 18%
HCl 6%
HCl 6%
HCl 6%
TFA
12
10
22
9
2
MeI
3
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
MeOTf
4
5
180
180
180
180
180
180
180
180
5
6
10
8
7
8
19
10
6
9
10
11
12
2
0
aRCY (decay-corrected from CO2) corresponds to the radioactivity values of [11C]7 from the recovered semipreparative HPLC peak without reformulation.

TEYSSIER ET AL.
69
FIGURE 1 A, Representative semipreparative HPLC chromatogram of [11C]7, radioactivity in blue ([11C]7 tR = 5.8 min) and UV in
black (λ = 254 nm). B, Representative analytical HPLC chromatogram of [11C]7, radioactivity in blue ([11C]7 tR = 3.7 min), UV in black (λ =
254 nm, tR = 3.8 minutes) and the nonradioactive standard 7 in red (UV, λ = 254 nm, tR = 3.8 min). The difference in the retention times
between radiation and UV detection is due to detectors being in series
purities. The chemical identity of [11C]MeOLBQ was con-
firmed by a coelution with the nonradioactive standard 7.
The molar activity was measured by comparing the UV
mass of [11C]MeOLBQ sample at 254 nm (tR = 3.7
minutes) with the UV response of a single injection of
the nonradioactive standard 7 (Figure 1B).
give the ammonium salt 3 in high yield (80%). The
ammonium salt 3 was then coupled with the NHS-
activated acid 4 in DMF/PBS with DMAP to form the
oxobutanamido benzylic protected ester 5 which was
then methylated in DMF in the presence of K2CO3
(88%). The benzyl group was cleaved using H2 with
Pd/C in methanol at room temperature to afford the
standard, MeOLBQ (7), in an overall yield of 38%. The
tert-butyl protected carboxylic acid 9 was synthesized by
coupling the ammonium salt 3 with the NHS-activated
acid 8 in DMF with DMAP to give the precursor 9 in 34
% yield. An overall yield of 26% was obtained for the
three-step synthesis of the precursor 9 as shown in
Scheme 1.
3 | RESULTS AND DISCUSSION
The production of [11C]MeOLBQ by a simple one-step
11C-methylation of LBQ657 cannot be realized due to the
presence of two carboxylic acids which would require the
synthesis of both methyl ester derivatives as standards
and precise analytical HPLC process in order to confirm
selective methylation. Direct methylation of the free car-
boxylic acid precursor with the protected oxobutanoic
acid moiety has thus been developed (Scheme 1), which
allows the selective methylation of the 2-methylbutanoic
acid moiety. The benzyl ester precursor 6 was synthesized
to produce the standard compound 7. However, the use
of Pd/C and H2 for deprotection of the [11C]MeOLBQ
requires a filtration of the catalyst before semipreparative
HPLC. The filtration reduced significantly the amount of
the radiolabeled product in the final formulation and was
not optimal. Therefore, we decided to use another
approach to produce [11C]C-7 via 11C-methylation of the
tert-butyl ester precursor 9 (instead of 6) allowing rapid
deprotection in acidic conditions without the need of
solid catalyst before HPLC purification.
[11C]MeOLBQ was synthesized by 11C-methylation of
the tert-butyl ester precursor 9 followed by deprotection
under acid conditions. 11C-Methylation was carried out
in the presence of Kryptofix 222 and potassium carbon-
ate. Utilization of this cryptant was previously reported
to enhance the reactivity of the base increasing the for-
mation of the carboxylate ion for methylation.18,19 In
order to improve the RCY of the reaction, both methylat-
ing agents were tested, [11C]methyl iodide and [11C]
methyl triflate. As expected, [11C]methyl triflate resulted
in a higher RCY (Table 1, entries 1-3). Higher RCYs were
obtained at lower temperature and less time. Reducing
the time of the methylation from 3 to 1 minute was not
beneficial (Table 1, entry 4), indicating that a longer reac-
tion time is needed to complete the alkylation reaction in
high RCY. The duration of the deprotection reaction was
then studied. Prolonged deprotection reaction times
(Table 1, entries 5-7) led to diminished RCYs. Reducing
the time of deprotection from 60 to 30 seconds did not
offer a significant improvement in RCY (Table 1, entries
3 and 8). It was thus concluded that 1 minute was suffi-
cient for the deprotection reaction. Given that longer
The nonradioactive standard 7 was synthesized in a
five-step route. The first step of the synthesis involved
the hydrolysis of the amino pentanoate 1 with sodium
hydroxide to generate product 2 in 95 % yield. The ring
of the formed lactam 2 was then opened after treatment
with acetic acid and hydrochloric acid under reflux to

70
TEYSSIER ET AL.
SCHEME 1 Synthetic route of [11C]MeOLBQ
deprotection reaction times resulted in lower RCY, vari-
ous concentrations of HCl were tested for the
deprotection reaction, while minimizing the potential
degradation of the 11C-methyl ester derivative. Reducing
the HCl concentration from 18% to 6% did not increase
the final yield (Table 1, entries 3 and 9-11). Deprotection
with TFA was not successful (Table 1, entry 12). The best
results were therefore obtained with 3 minutes of [11C]
MeOTf alkylation at 50ꢀC, followed by deprotection with
18% HCl for 1 minute at 60ꢀC. These conditions were
tested in validation runs including reformulation to
obtain [11C]MeOLBQ ([11C]7) in 9.5 2.5% RCY (25
6% decay-corrected from [11C]CO2, n = 3) and high molar
activity 348 100 GBq/μmol (9425 2720 mCi/μmol) at
EOS, in high chemical (>95%) and radiochemical (>99%)
purities. The total synthesis time including HPLC purifi-
cation and reformulation was 29 minutes.
ester precursor with acidic condition. To our knowledge
[11C]MeOLBQ is the first PET-labeled analog of the clini-
cally used NEPi sacubitril.
ACKNOWLEDGEMENTS
The authors thank the staff from the Radiochemistry and
Cyclotron Platform at the CRCHUM for the radioisotope
production and Fleur Gaudette of the CRCHUM Pharma-
cokinetics core facility for high-resolution and accurate
mass measurements. This work was supported by Société
Québécoise d'Insuffisance Cardiaque (SQIC-Roche) and
RBIQ (grant #14.04).
ORCID
Jean N. DaSilva https://orcid.org/0000-0001-6631-2320
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