Cannabinoid CB1 receptor chemical affinity probes: Methods suitable for preparation of isopropyl [11,12-3H]dodecylfluorophosphonate and [11,12-3H]dodecanesulfonyl fluoride

Article abstract of DOI:10.1081/SCC-120021043

Preparations of isopropyl dodec-11-enylfluorophosphonate and dodec-11-enesulfonyl fluoride are described along with conditions for their reduction with hydrogen (or potentially tritium) gas to the 1-dodecane derivatives as candidate high-potency (IC₅₀ 0.5-7 nM) chemical affinity probes for the mouse brain cannabinoid CB1 receptor and fatty acid amide hydrolase.

Full text of DOI:10.1081/SCC-120021043

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Cannabinoid CB1 Receptor Chemical Affinity
Probes: Methods Suitable for Preparation of
Isopropyl [11,12-3H]Dodecylfluorophosphonate and
[11,12-3H]Dodecanesulfonyl Fluoride
Yoffi Segall a b , Gary B. Quistad a & John E. Casida a
a Environmental Chemistry and Toxicology Laboratory, Department of Environmental
Science, Policy and Management, University of California, Berkeley, California, USA
b Israel Institute for Biological Research, Ness-Ziona, Israel
Published online: 17 Aug 2006.
To cite this article: Yoffi Segall , Gary B. Quistad & John E. Casida (2003): Cannabinoid CB1 Receptor Chemical Affinity
Probes: Methods Suitable for Preparation of Isopropyl [11,12-3H]Dodecylfluorophosphonate and [11,12-3H]Dodecanesulfonyl
Fluoride, Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
33:12, 2151-2159
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SYNTHETIC COMMUNICATIONSÕ
Vol. 33, No. 12, pp. 2151–2159, 2003
Cannabinoid CB1 Receptor Chemical Affinity
Probes: Methods Suitable for Preparation of
Isopropyl [11,12-3H]Dodecylfluorophosphonate
and [11,12-3H]Dodecanesulfonyl Fluoride
#
Yoffi Segall, Gary B. Quistad, and John E. Casida
*
Environmental Chemistry and Toxicology Laboratory,
Department of Environmental Science, Policy and
Management, University of California, Berkeley,
California, USA
ABSTRACT
Preparations of isopropyl dodec-11-enylfluorophosphonate and
dodec-11-enesulfonyl fluoride are described along with conditions
for their reduction with hydrogen (or potentially tritium) gas to the
1-dodecane derivatives as candidate high-potency (IC50 0.5–7 nM)
chemical affinity probes for the mouse brain cannabinoid CB1
receptor and fatty acid amide hydrolase.
#Current address: Israel Institute for Biological Research, Ness-Ziona, Israel.
*Correspondence: John E. Casida, Environmental Chemistry and Toxicology
Laboratory, Department of Environmental Science, Policy, and Management,
114 Wellman Hall, University of California, Berkeley, CA 94720-3112, USA;
Fax: (510) 642-6497; E-mail: ectl@nature.berkeley.edu.
2151
DOI: 10.1081/SCC-120021043
Copyright & 2003 by Marcel Dekker, Inc.
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com

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Segall, Quistad, and Casida
Key Words: Cannabinoid CB1 receptor; Chemical affinity probes;
Fatty acid amide hydrolase; Dodecanesulfonyl fluoride; Isopropyl
decylfluorophosphonate.
RESULTS AND DISCUSSION
The cannabinoid system of the brain consists of the CB1 receptor with
theagonist binding sitefor anandamideand Á9-tetrahydrocannabinol[1]
and fatty acid amide hydrolase (FAAH) that hydrolyzes anandamide.[2]
Both components are inhibited by alkyl alkylfluorophosphonates and
alkanesulfonyl fluorides presumably by phosphonylation and sulfonyla-
tion, respectively, of a nucleophilic site in the receptor and the catalytic
serine of the hydrolase.[3–5] Characterization of these targets as to structure
and function would be greatly facilitated by a potent and selective chemical
affinity probe radiolabeled at high specific activity, properties met in part
by ethyl oleylfluorophosphonate used for FAAH but prepared with a
specific activity of only 1.0 mCi/mmol.[5] An ideal probe would have at
least one tritium atom (about 30 Ci/mmol) and be potent on both CB1 and
FAAH but not other brain targets such as neuropathy target esterase
(NTE), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).
Chlorpyrifos oxon (CPO) (diethyl 3,5,6-trichloro-2-pyridinyl phosphate)
3
has been labeled with H at high specific activity[6] but lacks the desired
potency for FAAH and CB1 and selectivity relative to other esterases
(Table 1). Probe optimization has been achieved with isopropyl 1-dodecyl-
fluorophosphonate( 1) and 1-dodecanesulfonyl fluoride (2) with IC50
values of 0.5–7 nM for FAAH and CB1[12] and lower potency on other
esterases (except for 1 and NTE) (Table 1). The preferred chemical affinity
radioprobe for the cannabinoid system components is 1 based on potency,
but theoutstanding specificity of 2 would minimize interference due to
labeling AChE, BChE, NTE, and possibly other brain components. This
article reports methods suitable for one-step radiolabeling of 1 and 2 by
reduction of the double bond in isopropyl dodec-11-enylfluorophospho-
nate( 7) and dodec-11-enesulfonyl fluoride (9), respectively, with tritium
gas (or hydrogen in method development) (Sch. 1).
EXPERIMENTAL
Chemicals and Spectroscopy
Sources for the chemicals were: [3H]CP 55,940 (120 Ci/
mmol) from New England Nuclear (Boston, MA); methyl

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Cannabinoid CB1 Receptor Chemical Affinity Probes
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Table 1. Potency and specificity of candidate chemical affinity probes for mouse
brain CB1 receptor, FAAH, and esterases.
Compound and IC50 (nM)
Receptor or esterase
1
2
CPO
Cannabinoid system
CB1 receptora
FAAHc
2
2
7
2
14b
40d
Esterasesc
AChE
BChE
700
55
>100,000
>100,000
250
19d
<1d
150e
NTE
ca. 0.2
aMethyl arachidonylfluorophosphonate-sensitive portion of cannabinoid agonist
[3H]CP 55,940 binding.[7]
bRef.[7]
cHydrolysis assays with specific substrates: [14C]oleamide for FAAH[8]
acetylthiocholine and butyrylthiocholine for AChE and BChE, respectively[9,10]
;
;
paraoxon-resistant and mipafox-sensitive phenyl valerate hydrolytic activity for
NTE.[11]
dRef.[10] (mouseplasma for BChE).
eRef.[11] (hen brain).
arachidonylfluorophosphonatefrom Cayman Chme ical (Ann Arbor,
MI); 1,12-dibromododecane and 1-dodecanesulfonyl chloride from
Lancaster (Windham, OH); others from Aldrich (Milwaukee, WI).
NMR spectra were recorded for CDCl3 solutions (unless indicated other-
wise) with a Bruker AM-300 spectrometer. Chemical shifts (ꢀ) are
1
reported for H at 300 MHz relative to internal CHCl3 (at 7.27 ppm),
for 31P at 121.5 MHz relative to external trimethyl phosphate and for
13C at 75.5 MHz relative to CDCl3 (at 77.23 ppm).
Synthesis of Isopropyl 1-Dodecylfluorophosphonate (1) and
1-Dodecanesulfonyl Fluoride (2)
Isopropyl 1-dodecylfluorophosphonate (1). Thestandard compound
was prepared from diethyl 1-dodecylphosphonate via 1-dodecylphos-
phonic acid and 1-dodecyldifluorophosphonate utilizing the same
procedures as for the analogous dodec-11-enyl derivatives (see below)
(87%, purified with silica, initially eluted with CHCl3 followed by
1
EtOAc) (TLC/silica/EtOAc Rf 0.8). H NMR: 4.82 (septet, 1H), 1.77

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Segall, Quistad, and Casida
BrCH2CH2(CH2)10Br
CH2=CH(CH2)10P(O)(OC2H5)2
4
5
6
7
b
a
c
CH2=CH(CH2)10Br
3
8
9
CH2=CH(CH2)10P(O)(OH)2
g
d
CH2=CH(CH2)10SO3Na
CH2=CH(CH2)10P(O)F2
h
e
CH2=CH(CH2)10SO2F
CH2=CH(CH2)10P(O)(Oi-C3H7)F
f
f
*H
CH(CH2)10SO2F
*H
*H
CH2
*H
CH2
2
CH(CH2)10P(O)(Oi-C3H7)F
1
a) (i-C3H7)2NLi, THF, -20 0C. b) (C2H5O)2P(O)H, NaH, THF, reflux. c) 1. (CH3)3SiBr,
CH2Cl2; 2. (CH3)2CO, H2O. d) 1. (CH3)2NSF3, CHCl3; 2. 0.5N HCl, 0 0C. e) i-C3H7OH,
+
(C2H5)3N, (C2H5)2O. f) H2, 10% Pd/C, EtOAc. g) Na2SO3, C6H5CH2N(C2H5)3 Br-, CH3OH,
H2O, reflux. h) (CH3)2NSF3, CHCl3.
Scheme 1. Preparation of isopropyl dodec-11-enylfluorophosphonate (7)
and dodec-11-enesulfonyl fluoride (9) and conditions for their reduction with
hydrogen (or potentially tritium) gas to the 1-dodecane derivatives (1 and 2,
respectively).
(m, 2H), 1.55 (m, 2H), 1.27 (dd, 6H, JP–H ¼ 3.1 Hz), 1.28 (br s, 18H), 0.88
(t, 3H). 13C NMR: 72.33 (d, JP–C ¼ 6.8 Hz), 31.94, 30.32 (d,
JP–C ¼ 18.1 Hz), 29.63, 29.57, 29.37, 29.01, 24.71 (dd, JP–C ¼ 142.5 Hz,
JF–C ¼ 22.6 Hz), 23.88, 23.85, 22.72, 22.03, 21.97, 14.09. 31P NMR:
23.15 (d, JF–P ¼ 1068.1 Hz).
1-Dodecanesulfonyl fluoride (2). 1-Dodecanesulfonyl chloride (2.68 g,
10 mmol), (n-C4H9)4NþBrꢀ (48.2 mg, 0.15 mmol), KFꢁ2H2O (2.35 g,
25 mmol), CHCl3 (5 mL), and H2O (1 mL) were vigorously stirred for
72 h at rt. Water (40 mL) was added to dissolve the precipitated NaCl
and the solution was extracted with hexane (2 ꢂ 20 mL), dried (MgSO4),
and evaporated to leave 2.2 g pure 2 (87%). Alternatively, 2 was
synthesized from 1-dodecanesulfonyl chloride on reaction with 2 equiv.
of (n-C4H9)4NþFꢀ (1 M solution in THF) for 2 h at rt. Thereaction was
quenched with water, and extracted with hexane, which was then dried

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Cannabinoid CB1 Receptor Chemical Affinity Probes
2155
and evaporated to give 100% yield of the fluoride with the same NMR
characteristics. An additional synthesis comprised reduction of 9 (Sch. 1).
1H NMR: 3.36 (m, 2H), 1.95 (m, 2H), 1.49 (m, 2H), 1.27 (br s, 16H), 0.89
(t, 3H). 13C NMR: 51.5 (d, JF–C ¼ 15.8 Hz), 32.09, 29.75–29.00 (5C),
28.08, 23.61, 22.87, 14.27.
Synthesis of Isopropyl
Dodec-11-enylfluorophosphonate (7) (Sch. 1)
Dodec-11-ene bromide (3). A THF solution of (i-C3H7)2NLi (70 mL,
2 M, 0.14 mol) was added dropwise (3 h) to a cooled (ꢀ20ꢃC) solution of
1,12-dibromododecane (32.6 g, 0.1 mol) in dry THF (400 mL). The
mixture was stirred 3 h at rt, the THF distilled, hexane (100 mL) was
added, and extracted with water (pH 1) (2 ꢂ 50 mL) and theorganic
phase was dried. The product was distilled at 71ꢃC/0.1 mm Hg (33%
1
yield). H and 13C NMR data were similar to those reported.[13]
Diethyl dodec-11-enylphosphonate (4). Diethyl phosphite (1.4 g,
10 mmol) in THF (10 mL) was added dropwise, under N2, to a slurry
of NaH in THF (20 mL) maintaining the temperature between 0–5ꢃC and
then stirring for 2 h at rt. Bromide 3 (2.46 g, 10 mmol) was added and the
mixture was refluxed overnight. The THF was distilled and then ether
was added and extracted with water (2 ꢂ 30 mL). Theproduct was pur-
ified on column chromatography with ether (TLC/silica/ether, Rf 0.35) to
1
give2.42 g pure 4 (80%). H NMR: 5.68 (m, 1H), 4.84 (m, 2H), 3.98
(m, 4H), 1.92 (q, 2H), 1.61 (2H), 1.49 (m, 2H), 1.21 (t, 6H), 1.16
(m, 14H). 13C NMR: 139.08, 114.10, 61.37 (d, JP–C ¼ 6.7 Hz), 33.77,
30.56 (d, JP–C ¼ 17.9 Hz), 29.49–28.92 (6C), 25.68 (d, JP–C ¼ 138.3 Hz),
22.37 (d, JP–C ¼ 6.7 Hz), 16.44 (d, JP–H ¼ 6.7 Hz). 31P NMR: 25.20.
Dodec-11-enylphosphonic acid (5). (CH3)3SiBr was used to convert
diethyl ester 4 to thecorrseponding diacid
would havechlorinatde across thedoublebond. Thus, (CH
5, rather than PCl5 that
3)3SiBr
(6.7 g, 39.6 mmol) in dry CH2Cl2 (10 mL) was added dropwise to a
cooled (0ꢃC) and stirred solution of 4 (2 g, 6.6 mmol) in CH2Cl2
(20 mL).[14] Thesolution was stirred for 30 min at 0 ꢃC and 60 min at rt.
Evaporating the solvent and excess reagent gave crude bis(trimethylsilyl)-
dodec-11-enylphosphonate (ꢀ 31P NMR: 10.26 ppm), which was dissolved
in acetone (20 mL) without further purification. Water (20 mL, distilled)
was added and the mixture was stirred for 60 min. at rt. The product,
after solvent evaporation, was recrystallized from hexane to give >98%
5, m.p. 98–99ꢃC. 1H NMR: 5.81 (m, 1H), 4.95 (m, 2H), 2.03 (q, 2H), 1.72

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Segall, Quistad, and Casida
(m, 2H), 1.55 (m, 2H), 1.37 (m, 2H), 1.27 (br s, 12H). 31P NMR (10%
C6D6/acetone): 23.03.
Dodec-11-enyldifluorophosphonate (6). (CH3)2NSF3 (DAST) (650 mg,
4.9 mmol, in 10 mL CHCl3) was added to a cooled (0ꢃC) and stirred
solution of 5 (430 mg, 1.73 mmol) in CHCl3 (20 mL). Themixturewas
stirred for 10 min at 0ꢃC followed by 10 min at rt and treated at 0ꢃC with
0.5 N HCl (30 mL) followed by H2O (30 mL). Theorganic layre was
quickly dried and the CHCl3 removed to give 432 mg pure 6 (99%).
1H NMR: 5.74 (m, 1H), 4.95 (m, 2H), 1.99 (m, 4H), 1.97 (q, 2H), 1.67
(m, 2H), 1.39 (m, 2H), 1.22 (br s, 10H). 13C NMR: 139.23, 114.22, 33.88,
30.13 (d, JP–C ¼ 15.6 Hz), 29.52, 29.28, 29.19, 29.04, 28.92, 22.92
(dt, JP–C ¼ 138.3 Hz, JF–C ¼ 17.8 Hz), 21.61, 21.55. 31P NMR: 19.05
(t, JF–P ¼ 1134.3 Hz).
Isopropyl dodec-11-enylfluorophosphonate (7).
A
solution of
i-C3H7OH (108 mg, 1.71 mmol) and (C2H5)3N (181 mg, 1.71 mmol) in dry
ether (15 mL) was added dropwise to a cooled (0ꢃC) and stirred solution of
6 (432 mg, 1.71 mmol) in ether (5 mL). After stirring for 60 min, the pro-
duct was purified by silica column chromatography, initially eluted with
CHCl3 to remove impurities and further with 1:1 EtOAc:CHCl3 to give
482 mg pure 7 (96%). 1H NMR: 5.74 (m, 1H), 4.88 (m, 3H), 1.98 (q, 2H),
1.75 (m, 2H), 1.59 (m, 2H), 1.31 (dd, 6H, JP–H ¼ 3.1 Hz), 1.28 (br s, 14H).
13C NMR: 139.17, 114.19, 72.41 (d, JP–C ¼ 9.0 Hz), 33.86, 31.85 (d,
JP–C ¼ 17.9 Hz), 29.54, 29.49, 29.37, 29.19, 29.04, 25.74 (d,
JP–C ¼ 22.3 Hz), 23.94, 22.81 (dd, JP–C ¼ 122.7 Hz, JF–C ¼ 4.5 Hz), 22.09.
31P NMR: 23.13 (d, JP–F ¼ 1063.1 Hz).
Synthesis of Dodec-11-enesulfonyl Fluoride (9) (Sch. 1)
Sodium dodec-11-enesulfonate (8). Bromide 3 (1.23 g, 5 mmol),
Na2SO3 (1.26 g, 10 mmol), C6H5CH2N(C2H5)3þBrꢀ (13.6 mg), CH3OH
(8 mL), and H2O (18 mL) were refluxed for 15 h. The mixture was
extracted with ether (2 ꢂ 20 mL). The white crystals, precipitated on cool-
ing theaqueous solution to 0 ꢃC, were filtered and dried to give 0.9 g pure
8 (80%). 1H NMR (D2O): 5.78 (m, 1H), 4.94 (m, 2H), 2.85 (m, 2H), 2.02
(q, 2H), 1.74 (m, 2H), 1.30 (br s, 14H). 13C NMR (D2O, DSS): 141.10,
116.64, 53.86, 36.13, 32.02–31.09 (7C), 26.93.
Dodec-11-enesulfonyl fluoride (9). A solution of DAST (1.08 g,
8.2 mmol) in CHCl3 (5 mL, ethanol free) was added to a stirred slurry
of 8 (730 mg, 2.7 mmol) in CHCl3 (10 mL) at 0ꢃC under dry N2
atmosphere. After 2 h stirring at rt, the CHCl3 was evaporated and

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then ether (30 mL) was added and extracted with 0.5 N HCl (30 mL)
followed by H2O (30 mL). Theproduct was purified by silica chromatog-
raphy eluting with CHCl3:hexane (1:1) to give 130 mg of pure 9 (27%).
1H NMR: 5.81 (m, 1H), 4.97 (m, 2H), 3.36 (m, 2H), 2.04 (q, 2H), 1.95 (m,
2H), 1.48 (m, 2H), 1.29 (br s, 12H). 13C NMR: 139.35, 114.34, 51.1 (d,
JF–C ¼ 15.8 Hz), 33.98, 29.57–28.04 (7C), 23.61.
Methods Suitable for Preparation of [3H]1 and [3H]2by
Reduction of Phosphonofluoridate 7 and Sulfonyl Fluoride 9
with Tritium at Atmospheric Pressure
The radiolabeling procedure should ideally introduce tritium at
the last step such as on regioselective reduction of the dodecenyl
precursors (7 and 9) with a terminal double bond vs. potential
competing sites of reaction such as the P-F or S-F linkages. Earlier
studies showed that analogous reductions of oct-7-enyl-benzodioxa-
phosphorin 2-oxide[15] and S-(but-3-butenyl), S,S-dibutylphosphoro-
trithioate[16] are specific for the terminal double bond with no effect
at other molecular sites. The reduction of 7 and 9 was carried out
cleanly and quantitatively at rt and atmospheric pressure on a
5–10 mg scalewith 10% Pd/C catalyst in EtOAc. Thus, a solution
of 7 or 9 (10 mg in 1 mL EtOAc) was stirred for 2 h at rt in the
presence of 10 mg of 10% Pd/C catalyst under a slight positive H2
pressure (from a 2 L balloon directly attached to the reaction flask,
initially flushed with H2). After filtration through a short column of
Celite layered on silica, the solvent was evaporated at reduced pres-
sureand pumpde at 0.01 mm Hg for 1 h (100%). Phosphonofluoridate
7 gavesaturatde 1 and sulfonyl fluoride 9 gavesaturatde 2 identified
by comparison with the corresponding independently synthesized pro-
ducts (1H, 13C and 31P NMR spectra and MS). The biological activity
(IC50, CB1) of the reduced products were the same as those obtained
by independent synthesis.
ACKNOWLEDGMENT
This work was supported by Grant R01 ES08762 from the National
Institute of Environmental Health Sciences (NIEHS), NIH, and its con-
tents are solely the responsibility of the authors and do not necessarily
represent the official views of the NIEHS, NIH.

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Received in the USA October 28, 2002

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