Synthesis and μ-Opioid Activity of the Primary Metabolites of Carfentanil

Article abstract of DOI:10.1021/acsmedchemlett.9b00404

Carfentanil is a synthetic opioid significantly more potent than clinically prescribed fentanyl. The primary metabolites of carfentanil, generated from human liver microsomes, were structurally confirmed through chemical synthesis. The synthesized compounds were evaluated for μ-opioid receptor (MOR) functional activity. Of the six metabolites assayed, a major metabolite showed comparable activity to the parent opioid. Three other metabolites showed significant MOR functional activity. The availability of the metabolites could aid improvements in the analysis of biomedical samples obtained from suspected human exposures to carfentanil and development of treatment protocols.

Full text of DOI:10.1021/acsmedchemlett.9b00404

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Synthesis and μ‑Opioid Activity of the Primary Metabolites of
Carfentanil
Fu-Lian Hsu,† Andrew J. Walz,*,† James M. Myslinski,† Li Kong,† Michael G. Feasel,†
Tyler D. P. Goralski,† Tim Rose,‡ Nicholas J. Cooper,‡ Neil Roughley,‡ and Christopher M. Timperley‡
†United States Army CCDC Chemical Biological Center, Aberdeen Proving Ground, Maryland, United States
‡Defence Science and Technology Laboratory (Dstl), Porton Down, Salisbury, Wiltshire, U.K.
S
* Supporting Information
ABSTRACT: Carfentanil is a synthetic opioid significantly
more potent than clinically prescribed fentanyl. The primary
metabolites of carfentanil, generated from human liver
microsomes, were structurally confirmed through chemical
synthesis. The synthesized compounds were evaluated for μ-
opioid receptor (MOR) functional activity. Of the six
metabolites assayed, a major metabolite showed comparable
activity to the parent opioid. Three other metabolites showed
significant MOR functional activity. The availability of the metabolites could aid improvements in the analysis of biomedical
samples obtained from suspected human exposures to carfentanil and development of treatment protocols.
KEYWORDS: Carfentanil, μ-opioid receptor, metabolite, chemical synthesis
arfentanil is a potent synthetic opioid developed by
Janssen Pharmaceuticals.1,2 It is approved for veterinarian
M3−M6 were considered both structurally and synthetically
viable, whereas M7 and M8 were deemed problematic.
For M7, it was reasoned that the product resulting from
hydroxylation alpha to the nitrogen atom was likely to be
unstable due to the presence of a hemiaminal. The alternative
hydroxylation site, alpha to the quaternary amino-ester carbon
atom, would be sterically congested. Given these factors, M2
was targeted for synthesis instead of M7. M2 would result from
a sterically more favorable hydroxylation, avoid hemiaminal
formation, and would be consistent with the reported mass
spectral data, except for a single fragment assigned as an
unmodified phenylethyl group. M2 is synthetically more
accessible than M7 and its synthesis has been reported.2
Three structural isomers of M6 were targeted for synthesis
to determine which isomer was generated by the liver
microsomes.
M8 contains an oxidized amide nitrogen atom. Metabol-
ically, this transformation is unlikely. Synthetically, selective
amide oxidation would be very difficult in the presence of the
piperidine nitrogen; therefore, M8 was not targeted for
synthesis.
Synthesis of M4 and M2 metabolites began with M1
(Scheme 1). This intermediate was employed in Janssen’s
original synthesis of carfentanil.2 Reported improvements to
the Janssen methodology were used herein to produce M1 as
an intermediate in metabolite production.15
C
use as a large animal sedative but is severely restricted for
human sedation due to its extremely high potency and low
therapeutic index.3 Limited human in vivo pharmacological
data has been reported.4 Illicit carfentanil use has been
increasing worldwide and many unintentional overdose deaths
have been documented.5 Furthermore, British researchers
confirmed the fatal use of aerosolized carfentanil and
remifentanil by Russian Defense Forces to resolve a 2002
terrorist siege.6
Carfentanil selectively binds to the μ-opioid receptor
(MOR).7 Treatment for exposure utilizes a nonselective,
competitive opioid receptor antagonist such as naloxone.8,9
The serum half-life of naloxone is generally less than that of
known opioids. Postnaloxone renarcotization has been
documented in carfentanil sedated large animals.10−12 Non-
linear serum accumulation of the drug, documented in rats,13
combined with the reported 5.7 h human serum half-life4 could
contribute to carfentanil’s prolonged toxidromic effects along
with any MOR-active metabolites.
Metabolism of carfentanil by human liver microsomes was
reported in 2016.14 The authors proposed chemical structures
for the metabolites using mass spectral data and computational
methods. The proposed primary metabolite structures were
M1 and M3−M8 (Figure 1). The present work reports the
confirmation of the primary metabolite structures through
chemical synthesis in conjunction with metabolic screening.
The synthesized primary metabolites were then evaluated for
MOR functional activity.
Received: September 3, 2019
Accepted: October 9, 2019
Prior to the initiation of synthesis, analysis of the proposed
structures and their mass spectral data was conducted. M1 and
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00404
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Letter
a
Scheme 2
a
Reagents and conditions: (a) K2CO3, H2O2, acetonitrile, H2O.
in carfentanil metabolism to yield M1 and phenylacetaldehyde
cannot be discounted at the present time.
M5 was synthesized by acylation of the secondary amine
with acrylolyl chloride followed by a Michael-type hydroxide
addition (Scheme 3). The starting amine was prepared as
reported.17
a
Scheme 3
a
Reagents and conditions: (a) acryolyl chloride, 1,2-dichloroethane;
(b) 2 M NaOH, t-butanol; (c) oxalic acid, isopropanol.
Synthesis of three M6 structural isomers is shown in Scheme
4. 2-(4-Hydroxyphenyl)ethyl bromide was commercially
Figure 1. Structures of carfentanil and proposed primary metabolites.
a
Scheme 4
a
Scheme 1
a
(a) Reagents and conditions: 2-bromoacetophenone, K2CO3,
methylethyl ketone; (b) oxalic acid, isopropanol; (c) NaBH4,
methanol. (d) oxalic acid, isopropanol.
a
Reagents and conditions: (a) 2-(4-hydroxyphenyl)ethyl bromide,
K2CO3, 4-methyl-2-pentanone; (b) oxalic acid, methanol; (c) 2-(3-
hydroxyphenyl)ethyl bromide, K2CO3, 4-methyl-2-pentanone; (d)
oxalic acid, isopropanol; (e) 2-(2-THPO-phenyl)ethyl bromide,
K2CO3, 4-methyl-2-pentanone; (f) p-toluenesulfonic acid(H2O),
methanol; (g) oxalic acid, isopropanol.
M1 was alkylated with 2-bromoacetophenone to provide
M4. The ketone was reduced using sodium borohydride to
generate M2 as a racemic mixture. M3 cis/trans was
synthesized by oxidation of carfentanil using basic hydrogen
peroxide (Scheme 2). The starting carfentanil oxalate was
synthesized as reported.15 The isomers were chromato-
graphically separated and characterized. Unfortunately, the
chromatographically separated neat isomers proved to be
unstable. It is known that some amine oxides decompose on
standing at room temperature.16 Although decomposition of
the M3 isomers was not studied, usual decomposition products
of such amine oxides are a secondary amine and an aldehyde.
The possibility that this decomposition pathway might occur
available. 2-(3-Hydroxy-phenyl)ethyl bromide was synthesized
as previously reported.18 Both were used to alkylate M1
without protection of the phenolic hydroxyl group. Generation
of M6-ortho required the use of a phenolic protecting group
during amine alkylation. Tetrahydropyranyl (THP) protected
2-(2-hydroxyphenyl)ethyl) bromide was used as the alkylating
B
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Letter
reagent and was synthesized from commercially available 2-(2-
methoxyphenyl)ethanol.19
groups in M2 and M6-ortho might result in a favorable
hydrogen bonding interaction with the receptor that could
account for their higher binding affinities.
In the current study, the metabolism of carfentanil was
accomplished and the structures of the primary metabolites
M1−M6 were confirmed by comparison with the synthesized
compounds (metabolic methods and data are available in the
Supporting Information). M6-para was the only phenolic
isomer detected.
The primary metabolites of carfentanil have been synthe-
sized and evaluated for MOR functional activity. The
synthesized metabolites could aid in the analysis of human
biomedical samples from subjects with suspected carfentanil
exposure and in the development of treatment protocols.
While there exist a wide range of parameters which can
influence biological effects of psychotropic drug metabolism,20
the MOR data presented indicate that four of the synthesized
metabolites could prolong the already potent analgesic effects
of carfentanil, particularly the major active metabolite M2.
The order of abundance for the primary metabolites in the
previous report was as follows: M7 > M1 > M4 > M5 > M6 >
M3 > M8 as determined by mass spectral peak intensity
utilizing human hepatocytes. Proposed structure M7 was the
most abundant metabolite after 1, 4, and 6 h. In our hands,
employing human liver microsomes, the order of abundance
was M1 (40.0%), M2 (12.0%), M3 (2.5%), M4 (1.9%), M5
(<1.0%), and M6 (<1.0%) after 2 h. The order of HPLC
elution, in both studies, was M1, M5, M6, (M7/M2), M4, and
(M3/M8). On the basis of mass spectral fragmentation data,
relative abundance, and elution order, it is reasonable to
conclude that proposed structures M7 and M8 correspond to
structures M2 and an isomer of M3, respectively. In the
present work, an unconfirmed primary metabolite was
observed which did not correspond to any synthetic
compound. This metabolite had a relative abundance below
1%, a MW = 411.22773, and may correspond to proposed
structure M7.
The pharmacological assay evaluated the potency of
reference compound Ala2-MePhe4-Glyol5-Enkephalin
(DAMGO), carfentanil, M1−M5, and all M6 isomers with
respect to their ability to elicit a second messenger cyclic
adenosine monophosphate (cAMP) signal via agonism of the
MOR. (MOR assay methods and concentration−response
curves are shown in Supporting Information.) Median effective
concentration (EC50) values and relative potency are shown in
Table 1. The confirmed metabolites are listed in decreasing
order of metabolic abundance.
EXPERIMENTAL PROCEDURES
■
Synthetic starting materials were purchased from Aldrich Chemical
Co. (Milwaukee, WI). Flash chromatography was performed on a
Grace Reveleris X2 semiautomatic purification system. NMR data
were obtained on a JEOL 400 MHz spectrometer, and chemical shifts
(δ) are reported in parts per million (ppm) downfield from
tetramethylsilane. UHPLC-HRMS was performed on a Thermo-
Fisher Scientific Ultimate 3000 HPLC system coupled to an Orbitrap
Fusion Tribrid mass spectrometer.
Chemical Synthesis. M4 Oxalate. M1 (1.23 g, 4.24 mmol), 2-
bromoacetophenone (0.93 g, 4.66 mmol), and potassium carbonate
(5.86 g, 42.40 mmol) were stirred with 20 mL of methylethyl ketone.
The mixture was heated at reflux for 16 h, allowed to cool to room
temperature, and filtered. The filtrate was concentrated under reduced
pressure. The residue was flash chromatographed eluting with 0% to
5% methanol in dichloromethane. This provided 1.90 g of the free
base as a tan oil. Oxalic acid dihydrate (0.64 g. 5.08 mmol) was
dissolved in a minimum of hot isopropanol and added to the free base
dissolved in minimal hot isopropanol. The mixture was allowed to
cool and refrigerated for 16 h. The precipitated salt was filtered and
dried to provide 1.30 g of M4 oxalate in a 65% yield. Further
purification was accomplished by recrystallization from methanol. mp
1
206−209 °C; H-NMR (400 MHz, methanol-d4) δ 7.98−7.96 (m,
2H), 7.69 (m, 1H), 7.58−7.50 (m, 5H), 7.45−7.43 (m, 2H), 4.86−
4.84 (m, 2H), 3.82 (s, 3H), 3.48−3.42 (m, 4H), 2.53 (br d, J = 14.2
Hz, 2H), 2.09−2.01 (m, 2H), 1.94 (q, J = 7.5 Hz, 2H), 0.94 (t, J = 7.6
Hz, 3H); 13C-NMR (101 MHz, methanol-d4) δ 190.9, 175.5, 172.6,
165.1, 138.2, 134.6, 133.7, 130.3, 129.8, 129.4, 128.8, 128.0, 60.3,
51.9, 50.5, 50.4, 30.2, 28.5, 8.1; HRMS (ESI-Orbitrap), m/z
calculated for C24H29N2O4 [M + H]+ 409.21288, found 409.21218.
M2 Oxalate. M4 oxalate (0.82 g, 1.64 mmol) was stirred with 50
mL of saturated aqueous sodium bicarbonate and 25 mL of
chloroform for 1 h at room temperature. The organic layer was
separated, and the aqueous solution was extracted two times with
chloroform. The combined organic extracts were dried with sodium
sulfate and filtered, and the filtrate was concentrated under reduced
pressure. The residue was dissolved in 50 mL of methanol and cooled
in an ice water bath. Sodium borohydride (0.13 g, 3.28 mmol) was
added in small portions. After the addition, the ice bath was removed,
and the reaction mixture was stirred for 6 h. Acetic acid (0.50 mL)
was added, and the mixture was stirred for 16 h, and then
concentrated under reduced pressure. The residue was taken up in
chloroform and saturated aqueous sodium bicarbonate solution. The
organic layer was separated, and the aqueous solution was extracted
two times with chloroform. The combined organic extracts were dried
with sodium sulfate and filtered. The filtrate was concentrated under
reduced pressure. The residue was purified using flash chromatog-
raphy eluting with 0% to 4% methanol in dichloromethane. This
provided 0.63 g of the free base as a tan oil. The oxalate salt was
prepared as described for M4 oxalate. The precipitate was filtered and
dried to provide 0.65 g of a white solid in a 79% yield from M1. mp
Table 1. Calculated EC50 Values Observed Using the Lance
Ultra cAMP Assay
a
compound
EC50 (nM)
SEM
0.041
0.0016
4.1
0.0011
0.73
0.052
0.026
0.00064
0.0027
0.0061
rel. potency
fentanyl
carfentanil
M1
M2
M3 cis/trans
M4
0.51
0.0049
17
0.0051
4.4
0.20
1/100
1
1/3400
1
1/900
1/42
1/56
2
M5
0.28
M6-ortho
M6-meta
M6-para
0.0024
0.014
0.028
1/3
1/6
a
Relative potency to carfentanil is provided for reference. EC50 and
standard error of mean (SEM) values expressed as nM.
The most abundant metabolite in this work, M1, showed
very little MOR agonism, while M2 showed comparable
activity to carfentanil. All confirmed metabolites, except M1
and M3, are more active than the clinically prescribed MOR
binding fentanyl. Interestingly, M2 was found to have a median
effective dose 1.67 times less potent than carfentanil in a rat tail
withdrawal test.2 M6-ortho was the most active compound
tested but was not a confirmed liver microsomal metabolite. It
is speculated that the relatively similar location of the hydroxyl
1
189−191 °C; H-NMR (400 MHz, methanol-d4) δ 7.55−7.27 (m,
10H), 5.04 (t, J = 6.9 Hz, 1H), 3.81 (s, 3H), 3.62−3.54 (m, 2H),
3.44−3.38 (m, 2H), 3.20 (d, J = 6.9 Hz, 2H), 2.50 (td, J = 17.6, 2.9
C
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Letter
Hz, 2H), 2.08−1.91 (m, 4H), 0.93 (t, J = 7.3 Hz, 3H); 13C-NMR
(101 MHz, methanol-d4) δ 175.5, 172.6, 165.4, 140.8, 138.2, 130.3,
130.2, 129.7, 129.4, 128.4, 128.1, 125.8, 67.4, 62.3, 60.4, 51.9, 48.7,
30.2, 30.0, 28.5, 8.1; HRMS (ESI-Orbitrap), m/z calculated for
C24H31N2O4 [M + H]+ 411.22783, found 411.22826.
138.1, 136.3, 130.3, 129.8, 129.4, 128.6, 128.5, 126.9, 60.4, 57.5, 57.4,
51.9, 49.5, 38.0, 30.3, 30.1; HRMS (ESI-Orbitrap), m/z calculated for
C24H31N2O4 [M + H] 411.22783, found 411.22837.
M6-para Oxalate. M1 (1.23 g, 4.25 mmol), 2-(4-hydroxyphenyl)-
ethyl bromide (0.95 g, 4.70 mmol), and potassium carbonate (1.76 g,
12.75 mmol) were mixed with 40 mL of 4-methyl-2-pentanone and
heated at reflux for 20 h. The mixture was allowed to cool to room
temperature. The solids were filtered, and the filtrate was
concentrated under reduced pressure. The residue was taken up in
chloroform and water. The organic layer was separated, and the
aqueous solution was washed two times with chloroform. The
combined organic layers were washed with brine. The organic
solution was dried with sodium sulfate, filtered, and the volatiles were
evaporated to provide a brown oil. The residue was purified using
flash chromatography eluting with 0−6% methanol in dichloro-
methane to provide 1.18 g of the free base as a tan oil. Oxalic acid
dihydrate (0.17 g. 1.36 mmol) was dissolved in a minimum of hot
methanol and added to the free base (0.51 g, 1.24 mmol) dissolved in
minimal hot methanol. The mixture was allowed to cool, and the
solvent removed under reduced pressure to a volume of
approximately 1.5 mL. The mixture was allowed to cool and
refrigerated for 16 h. The precipitate was filtered and dried. The
collected salt was recrystallized from methanol/isopropanol followed
by heated vacuum drying to provide 0.56 g of a white solid in a 61%
yield from M1. mp 194−196 °C; 1H-NMR (400 MHz, methanol-d4)
δ 7.51−7.35 (m, 5H), 7.03 (d, J = 8.7 Hz, 2H), 6.70 (d, J = 8.7 Hz,
1H), 3.80 (s, 3H), 3.46−3.17 (m, 6H), 2.89−2.85 (m, 2H), 2.47 (br
d, J = 14.2 Hz, 2H), 1.98−1.89 (m, 4H), 0.92 (t, J = 7.6 Hz, 3H); 13C-
NMR (101 MHz, methanol-d4) δ 175.4, 172.6, 165.3, 156.4, 138.2,
130.2, 129.8, 129.5, 129.4, 126.7, 115.3, 60.4, 57.9, 51.9, 49.4, 30.3,
29.3, 28.5, 8.1; HRMS (ESI-Orbitrap), m/z calculated for
C24H31N2O4 [M + H] 411.22783, found 411.22836.
M3 cis/trans. Carfentanil oxalate (0.27 g, 0.55 mmol) and
potassium carbonate (0.08 g, 0.60 mmol) were dissolved in 2 mL
of 1:1 v/v water/acetonitrile and stirred at room temperature for 30
min. Acetonitrile (4.5 mL) and hydrogen peroxide (1 mL, 30% aq.)
were added, and the solution was heated to 60 °C for 3 h. Upon
consumption of the starting material, the solution was allowed to cool
to room temperature and mixed with brine and dichloromethane. The
organic layer was separated, and the aqueous solution was extracted
two times with dichloromethane. The combined organic extracts were
dried with sodium sulfate and filtered. The filtrate was concentrated
under reduced pressure. The oil was triturated with diethyl ether to
afford 0.28 g of a pale yellow solid. The isomers were separated using
flash chromatography eluting with 0% to 40% methanol in ethyl
acetate. M3 trans was eluted first as the major product, followed by
M3 cis. (The isolated isomers were not stable, which necessitated the
use of the crude M3 cis/trans mixture for receptor assays whose purity
1
was determined to be 80% by H NMR using p-xylene as an internal
standard.)
1
M3 trans H-NMR (400 MHz, chloroform-d) δ 7.34−7.39 (m,
3H), 7.20−7.28 (m, 4H), 7.13−7.16 (m, 3H), 3.73 (s, 3H), 3.47 (t, J
= 11.4 Hz, 2H), 3.28−3.32 (m, 3H), 3.10−3.17 (m, 4H), 2.50 (td, J =
13.6, 3.9 Hz, 2H), 2.14 (d, J = 13.6 Hz, 2H), 1.86 (q, J = 7.4 Hz, 2H),
0.91 (t, J = 7.4 Hz, 3H); 13C-NMR (101 MHz, chloroform-d) δ
174.36, 173.45, 138.60, 137.43, 130.12, 129.74, 129.09, 128.90,
128.79, 126.75, 72.56, 61.83, 60.66, 52.57, 49.92, 28.85, 27.88, 9.10;
HRMS (ESI-Orbitrap), m/z calculated for C24H31N2O4 [M + H]+
411.22783, found 411.22840.
1
M3 cis H-NMR (400 MHz, chloroform-d) δ 7.49 (s, 5H), 7.28−
M6-meta Oxalate. M1 (1.19 g, 4.10 mmol), 2-(3-hydroxyphenyl)-
ethyl bromide (0.95 g, 4.51 mmol), and potassium carbonate (1.70 g,
12.30 mmol) were mixed with 40 mL of 4-methyl-2-pentanone and
heated at reflux for 20 h. The mixture was allowed to cool to room
temperature. The solids were filtered, and the filtrate was
concentrated under reduced pressure. The residue was taken up in
chloroform and water. The organic layer was separated, and the
aqueous solution was washed two times with chloroform. The
combined organic layers were washed with brine. The organic
solution was dried with sodium sulfate and filtered, and the volatiles
were evaporated to provide a brown oil. The residue was flash
chromatographed eluting with 0% to 6% methanol in dichloro-
methane. The process was repeated to improve purity, and 0.91 g of
the free base was obtained as a tan oil. The oxalate salt was prepared
as described for M4 oxalate. The precipitate was filtered and dried to
provide 0.48 g of a white solid in a 47% yield from M1. Further
purification was accomplished by recrystallization from water/
7.31 (m, 2H), 7.21−7.25 (m, 1H), 7.18 (d, J = 7.1 Hz, 2H), 3.84 (s,
3H), 3.29−3.33 (m, 2H), 3.15−3.25 (m, 4H), 2.97−3.09 (m, 4H),
2.13 (d, J = 11.0 Hz, 2H), 1.92 (q, J = 7.4 Hz, 2H), 0.95 (t, J = 7.4 Hz,
3H); 13C-NMR (101 MHz, chloroform-d) δ 175.25, 173.70, 139.62,
137.34, 130.52, 129.93, 129.40, 128.95, 128.92, 126.94, 61.69, 61.54,
53.02, 29.20, 28.82, 27.85, 9.06; HRMS (ESI-Orbitrap m/z calculated
for C24H31N2O4 [M + H] 411.22783, found 411.22830.
M5 Oxalate. Synthesized amine (0.77 g, 2.29 mmol) and acryloyl
chloride (1.03 g, 11.43 mmol) were dissolved in 15 mL of 1,2-
dichloroethane in a pressure tube. The vessel was heated in an oil bath
at 85 °C for 16 h, then allowed to cool. The mixture was concentrated
under reduced pressure. The residue was taken up in saturated
aqueous sodium bicarbonate and chloroform. The organic layer was
separated, and the aqueous solution was extracted two times with
chloroform. The combined organic extracts were dried with sodium
sulfate. The mixture was filtered through Celite with a layer of silica
gel underneath. The filtrate was concentrated under reduced pressure.
In a pressure tube, the residue was dissolved in 27 mL of t-butanol,
and 13.50 mL of 2 M aqueous sodium hydroxide was added. The
reaction vessel was heated in an oil bath at 105 °C for 48 h then
allowed to cool to room temperature. The solution was filtered
through Celite, which was then rinsed with ethanol. The filtrate was
concentrated under reduced pressure. The residue was taken up in
saturated brine and chloroform. The organic layer was separated, and
the aqueous solution was extracted two times with chloroform. The
combined organic extracts were dried with sodium sulfate. The
mixture was filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by flash chromatography, eluting
with 50% to 0% hexanes in dichloromethane then 0% to 75% acetone
in dichloromethane to provide 0.33 g of the free base as a tan oil. The
oxalate salt was prepared as described for M4 oxalate. The precipitate
was filtered, and 0.37 g of a white solid were obtained in a 32% yield
1
methanol followed by heated vacuum drying. mp 99−101 °C; H-
NMR (400 MHz, methanol-d4) δ 7.52−7.35 (m, 5H), 7.11−7.07 (m,
1H), 6.68−6.63 (m, 3H), 3.80 (s, 3H), 3.48−3.22 (m, 6H), 2.91−
2.87 (m, 2H), 2.48 (d, J = 14.4 Hz, 2H), 1.94−1.89 (m, 4H), 0.92 (t,
J = 7.4 Hz, 3H); 13C-NMR (101 MHz, methanol-d4) δ 175.4, 172.5,
165.1, 157.7, 138.2, 137.6, 130.2, 129.8, 129.6, 129.4, 119.4, 115.3,
113.8, 60.4, 57.5, 51.9, 49.5, 30.3, 30.0, 28.5, 8.1; HRMS (ESI-
Orbitrap), m/z calculated for C24H31N2O4 [M + H] 411.22783, found
411.22849.
M6-ortho Oxalate. M1 (0.99 g, 3.40 mmol), 2-(2-THPO-
phenyl)ethyl bromide (0.97 g, 3.40 mmol), and potassium carbonate
(1.70 g, 12.30 mmol) were mixed with 40 mL of 4-methyl-2-
pentanone and heated at reflux for 48 h. The mixture was allowed to
cool to room temperature. The solids were filtered, and the filtrate
was concentrated under reduced pressure. The residue was taken up
in chloroform and water. The organic layer was separated, and the
aqueous solution was washed two times with chloroform. The
combined organic layers were washed with brine. The organic
solution was dried with sodium sulfate and filtered, and the volatiles
were evaporated to provide a brown oil. The oil was dissolved in 40
mL of methanol, and p-toluenesulfonic acid monohydrate (1.00 g,
1
from amine. mp 168−171 °C; H-NMR (400 MHz, methanol-d4) δ
7.53−7.37 (m, 5H), 7.30−7.19 (m, 5H), 3.80 (s, 3H), 3.65 (t, J = 6.4
Hz, 2H), 3.49−3.48 (m, 2H), 3.35−3.24 (m, 4H), 2.99−2.95 (m,
2H), 2.49 (d, J = 14.4 Hz, 2H), 2.13 (t, J = 6.3 Hz, 2H), 1.98−1.96
(m, 2H); 13C-NMR (101 MHz, methanol-d4) δ 172.7, 172.6, 165.1,
D
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5.10 mmol) was added. The solution was heated for 16 h. The
solution was allowed to cool and the solvents removed under reduced
pressure. The residue was taken up in chloroform and saturated
aqueous sodium bicarbonate solution. The organic layer was
separated, and the aqueous solution was extracted two times with
chloroform. The combined organic extracts were dried with sodium
sulfate and filtered. The filtrate was concentrated under reduced
pressure. The residue purified using flash chromatography eluting
with 0% to 6% methanol in dichloromethane and 0.84 g of the free
base obtained as a tan oil. The oxalate salt was prepared as described
for M4 oxalate. The precipitate was filtered and recrystallized from
ethanol. After heated vacuum drying, 0.54 g of a white solid was
obtained in a 54% yield from M1. Further purification was
accomplished by recrystallization from methanol/ethanol followed
(3) Leen, J. L. S.; Juurlink, D. N. Carfentanil: a narrative review of its
pharmacology and public health concerns. Can. J. Anaesth. 2019, 66,
414−421.
(4) Uddayasankar, U.; Lee, C.; Oleschuk, C.; Eschun, G.; Ariano, R.
E. The Pharmacokinetics and Pharmacodynamics of Carfentanil After
Recreational Exposure: A Case Report. Pharmacotherapy 2018, 38,
e41−e45.
(5) O’Donnell, J.; Gladden, R. M.; Mattson, C. L.; Kariisa, M.
Overdose Deaths with Carfentanil and Other Fentanyl Analogs
Detected-10 States, July 2016-June 2017. MMWR-Morb. Mortal. Wkly.
Rep. 2018, 67, 767−768.
(6) Riches, J. R.; Read, R. W.; Black, R. M.; Cooper, N. J.;
Timperley, C. M. Analysis of Clothing and Urine from Moscow
Theatre Siege Casualties Reveals Carfentanil and Remifentanil Use. J.
Anal. Toxicol. 2012, 36, 647−656.
(7) Eriksson, O.; Antoni, G. C-11 Carfentanil Binds Preferentially to
mu-Opioid Receptor Subtype 1 Compared to Subtype 2. Mol. Imaging
2015, 14, 476−483.
(8) Gourlay, G. K.; Coulthard, K. The Role of Naloxone Infusions in
the Treatment of Overdoses of Long Half-Life Narcotic Agonists -
Application to Nor-Methadone. Br. J. Clin. Pharmacol. 1983, 15, 269−
272.
(9) Clarke, S. F. J.; Dargan, P. I.; Jones, A. L. Naloxone in opioid
poisoning: walking the tightrope. Emerg. Med. J. 2005, 22, 612−616.
(10) Jacobson, E. R.; Kollias, G. V.; Heard, D. J.; Caligiuri, R.
Immobilization of African Elephants with Carfentanil and Antagonism
with Nalmefene and Diprenorphine. Journal of Zoo Animal Medicine
1988, 19, 1−7.
(11) Miller, M. W.; Wild, M. A.; Lance, W. R. Efficacy and safety of
naltrexone hydrochloride for antagonizing carfentanil citrate immobi-
lization in captive Rocky Mountain elk (Cervus elaphus nelsoni). J.
Wildl. Dis. 1996, 32, 234−239.
(12) Cole, A.; Mutlow, A.; Isaza, R.; Carpenter, J. W.; Koch, D. E.;
Hunter, R. P.; Dresser, B. L. Pharmacokinetics and pharmacody-
namics of carfentanil and naltrexone in female common eland
(Taurotragus oryx). J. Zoo Wildl Med. 2006, 37, 318−326.
(13) Bergh, M. S.-S.; Bogen, I. L.; Garibay, N.; Baumann, M. H.
Evidence for nonlinear accumulation of the ultrapotent fentanyl
analog, carfentanil, after systemic administration to male rats.
Neuropharmacology 2019, 158, 107596.
1
by heated vacuum drying. mp 210−212 °C; H-NMR (400 MHz,
methanol-d4) δ 7.50−7.35 (m, 5H), 7.07−7.02 (m, 2H), 6.75−6.71
(m, 2H), 3.79 (s, 3H), 3.47−3.44 (m, 2H), 3.29−3.16 (m, 4H),
2.96−2.92 (m, 2H), 2.46 (br d, J = 14.4 Hz, 2H), 1.99−1.88 (m, 4H),
0.91 (t, J = 7.4 Hz, 3H); 13C-NMR (101 MHz, methanol-d4) δ 175.4,
172.7, 166.3, 155.3, 138.3, 130.3, 130.2, 129.7, 129.3, 128.2, 122.7,
119.5, 114.8, 60.6, 56.3, 51.9, 49.3, 30.4, 28.5, 25.6, 8.1; HRMS (ESI-
Orbitrap), m/z calculated for C24H31N2O4 [M + H] 411.22783, found
411.22853.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00404.
Spectral data on the synthesized compounds, metabo-
lism methods, UHPLC conditions, metabolite mass
spectral data, and MOR assay methods and data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: andrew.j.walz.civ@mail.mil.
■
ORCID
Andrew J. Walz: 0000-0001-6306-6320
Michael G. Feasel: 0000-0001-7029-2764
Author Contributions
All authors contributed to this manuscript.
Funding
US Defense Threat Reduction Agency, project #3662.
Notes
The authors declare no competing financial interest.
(14) Feasel, M. G.; Wohlfarth, A.; Nilles, J. M.; Pang, S.; Kristovich,
R. L.; Huestis, M. A. Metabolism of Carfentanil, an Ultra-Potent
Opioid, in Human Liver Microsomes and Human Hepatocytes by
High-Resolution Mass Spectrometry. AAPS J. 2016, 18, 1489−1499.
(15) Walz, A. J.; Hsu, F.-L. Scaled-Up Synthesis: Salts of Carfentanil
and Remifentanil. ECBC-TR-1473; U.S. Army Combat Capabilities
Development Command Chemical Biological Center: Aberdeen
Proving Ground, MD, 2017; AD1039337. https://apps.dtic.mil/
dtic/tr/fulltext/u2/1039337.pdf.
(16) Sidgwick, N. V. Amine oxides. In The Organic Chemistry of
Nitrogen; Millar, I. T., Springhall, H. D., Eds.; Clarendon Press:
Oxford, UK, 1966; pp 134−138.
(17) Walz, A. J.; Hsu, F.-L. Synthesis of 4-anilinopiperidine methyl
esters, intermediates in the production of carfentanil, sufentanil, and
remifentanil. Tetrahedron Lett. 2014, 55, 501−502.
DEDICATION
■
This work is dedicated to Dr. Fu-Lian Hsu on the occasion of
his happy retirement after 36 years of US government service.
(18) Butini, S.; Gemma, S.; Brindisi, M.; Borrelli, G.; Lossani, A.;
Ponte, A. M.; Torti, A.; Maga, G.; Marinelli, L.; La Pietra, V.; Fiorini,
I.; Lamponi, S.; Campiani, G.; Zisterer, D. M.; Nathwani, S.-M.;
Sartini, S.; La Motta, C.; Da Settimo, F.; Novellino, E.; Focher, F.
Non-nucleoside inhibitors of human adenosine kinase: synthesis,
molecular modeling, and biological studies. J. Med. Chem. 2011, 54,
1401−1420.
ABBREVIATIONS
■
MOR μ-opioid receptor; THP tetrahydropyranyl; DAMGO
Ala2-MePhe4-Glyol5-Enkephalin; cAMP cyclic adenosine
monophosphate; EC50 median effective concentration; SEM
standard error of mean
(19) Baker, R. K.; Kayser, F.; Bao, J.; Miao, S.; Parsons, W. H.;
Rupprecht, K. M. Preparation of tetracyclic triterpenes with
immunosuppressant activity. Patent WO9920274A1, 1999.
(20) Hendset, M.; Haslemo, T.; Rudberg, I.; Refsum, H.; Molden, E.
The complexity of active metabolites in therapeutic drug monitoring
of psychotropic drugs. Pharmacopsychiatry 2006, 39, 121−127.
REFERENCES
■
(1) Van Daele, P. G. H.; De Bruyn, M. F. L.; Boey, J. M.; Sanczuk,
S.; Agten, J. T. M.; Janssen, P. A. J. Synthetic analgesics: N-(1-[2-
arylethyl]-4-substituted 4-piperidinyl)-N-arylalkanamides. Arzneim.-
Forsch. 1976, 26, 1521−1531.
(2) Janssen, P. A. J.; Van Daele, G. H. P. 4-Anilinopiperidines. US
Patent US4179569A, 1979.
E
DOI: 10.1021/acsmedchemlett.9b00404
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX