Journal of Medicinal Chemistry
Article
acetate/hexane, 5:5 (v/v), as eluent afforded the pure product (30−
50%).
levels (data not shown), thus ruling out the involvement of
FAAH in the effect of 37 on 2-AG levels.
2-(4-Benzhydrylpiperazin-1-yl)-1-phenyl-2-thioxoethanone
1
(03). Mp 125−127 °C. H NMR (CDCl3): δ (ppm) 7.89−7.22 (m,
CONCLUSIONS
■
15H), 4.22 (s, 1H) 3.51 (t, 2H, J = 4.84 Hz), 3.49 (t, 2H, J = 4.76 Hz),
2.56 (t, 2H, J = 4.84 Hz), 2.35 (t, 2H, J = 4.76 Hz). 13C NMR
(CDCl3): δ (ppm) 195.05, 188.00, 141.60, 134.27, 133.34, 129.83,
128.89, 128.78, 127.75, 127.44, 75.58, 51.75, 51.68, 51.12, 46.99.
HRMS: [M + H]+ = 401.166 81.
Starting from a modest arylthioamide hit,25 we have developed
a novel series of aryldithiocarbamate inhibitors of MAGL. The
activity of these inhibitors is highly dependent on the presence
of 2,4-dinitrophenolate as a leaving group, suggesting that the
inhibitors react with their enzymatic target. Indeed, we
demonstrate here that these inhibitors irreversibly inhibit
MAGL via formation of a DTT-sensitive covalent bond with
either Cys208 or Cys242, two noncatalytic cysteine residues,
and/or the catalytic Ser122 of the MAGL. By structure−activity
relationships studies we have identified 2,4-dinitrophenyl 4-(4-
tert-butylbenzyl)piperazine-1-carbodithioate (37) as a potent
and quite selective MAGL inhibitor. We have also identified
2,4-dinitrophenyl 4-benzhydrylpiperazine-1-carbodithioate (16)
as a submicromolar and highly selective MAGL inhibitor. The
efficacy in inhibiting pure hMAGL in vitro, coupled to its ability
to increase cellular levels of 2-AG in intact cells, makes 37 a
promising MAGL inhibitor, which should prove to be useful for
future investigation of endocannabinoid degradation pathways.
The details for compound 04 can be found in the Supporting
Information (p S3).
Dithiocarbamate Derivatives Synthesis. Dithiocarbamates
derivatives 05−15 were obtained by a Ullman-type coupling reaction.
To a solution of N,N-dimethylglycine (30 mol %), dithiocarbamic
acid sodium salt (1.2 mmol), obtained as reported by Sattigeri et al.,30
and substituted aryl iodide (1 mmol) in anhydrous DMF (2 mL) was
added CuI (15 mol %). Under nitrogen atmosphere, the mixture was
stirred at 110 °C for 22 h. The reaction mixture was then cooled to
room temperature, poured in water and extracted with ethyl acetate.
The combined organic layer was dried over magnesium sulfate. After
evaporation of ethyl acetate, the product was purified by column
chromatography (petroleum ether/ethyl ether, v/v = 4/1) (55−90%).
Phenyl 4-Benzhydrylpiperazine-1-carbodithioate (05). Mp
1
157−159 °C. H NMR (CDCl3): δ (ppm) 7.44−7.19 (m, 15H), 4.29
(s, 1H), 4.29(t, 2H, J = 3.28 Hz), 4.04(t, 2H, J = 3.92 Hz), 2.54 (m,
4H). 13C NMR (CDCl3) δ (ppm) 197.09, 141.87, 137.10, 131.28,
130.07, 129.83, 129.10, 128.73, 127.86, 127.34, 75.71, 51.40, 48.8.
HRMS: [M + H]+ = 405.144 17.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. All reagents and solvents of
analytical grade purchased from commercial sources (Sigma-Aldrich
and Acros Organics) were used without further purification. The
structures of all compounds synthesized were consistent with their
NMR spectra and high resolution mass spectra. Melting points were
determined in open capillaries using the Electrothermal 9100
apparatus and are reported uncorrected. Nuclear magnetic resonance
(1H and 13C) spectra were recorded on a Bruker Avance 400 MHz
Ultrashield instrument. Chemical shifts (δ) are reported relative to the
tetramethylsilane peak set at 0 ppm. In the case of multiplets, the
signals are reported as intervals. Signals are abbreviated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; qt, quintet; m, multiplet.
Coupling constants are expressed in hertz. HRMS data for all final
compounds were obtained using a LTQ-Orbitrap mass spectrometer
(Thermo-Fisher Scientific) with the analysis performed using an ESI
source in both positive and negative modes. All tested compounds
were at least 95% pure as determined using an Accela HPLC system
(Thermo-Fisher Scientific). Separation was performed using a RP-18
column (3 μM, 4 mm × 150 mm; Sigma-Aldrich).
The details for compounds 06−15 can be found in the Supporting
Information (pp S3−S5).
Nucleophilic Aromatic Substitution (16−37). The target
aryldithiocarbamates 16−37 were obtained by a nucleophilic aromatic
substitution using the following protocol. To a solution of
dithiocarbamic acid triethylammonium salt (see Supporting Informa-
tion, p S2) (1.2 mmol) in N,N-dimethylformamide (5 mL) was added
2,4-dinitrofluorobenzene (1 mmol). The mixture was stirred overnight
at room temperature, dissolved in water, and extracted with
dichloromethane. The organic phase was dried over sodium sulfate
and evaporated under reduced pressure. Crystallization from ethanol
afforded the pure product. In some cases further purification using
column chromatography (silica gel, ethyl acetate/hexane, 3:7 v/v) was
needed (80−95%).
2,4-Dinitrophenyl 4-Benzhydrylpiperazine-1-carbodithioate
(16). Mp 157−159 °C. 1H NMR (CDCl3): 8.75 (d, 1H, J = 2.32 Hz),
8.32 (dd, 1H, J = 6.16 Hz), 7.78 (d, 1H, J = 8.64 Hz), 7.36−7.11 (m,
10H), 4.23 (s, 1H), 4.01 (t, 2H, J = 4.84 Hz), 3.99 (t, 2H, J = 4.84
Hz), 2.48 (t, 4H, J = 4.84 Hz). 13C NMR (CDCl3) δ (ppm) 189.90,
151.20, 147.90, 141.67, 139.35, 135.14, 128.81, 127.82, 127.47, 126.19,
Thiourea Derivatives Synthesis (01 and 02). To a solution of
aryl isothiocyanate (see Supporting Information, p S2) in methanol
(0.01 mol) was added 1-benzhydrylpiperazine (0.012 mol). The
mixture was stirred for 3 h at room temperature and the reaction
monitored by TLC. After completion of the reaction, the precipitate
was filtered off and purified by column chromatography (ethyl acetate/
hexane, 2:8) to yield the target product (80−90%).
4-Benzhydryl-N-phenylpiperazine-1-carbothioamide (01).
Mp 215−217 °C. 1H NMR (DMSO-d6): δ (ppm) 9.29 (s, 1H),
7.48−7.10 (m, 15H), 4.38 (s, 1H), 3.93 (t, 4H, J = 4.88 Hz), 2.38 (t,
4H, J = 4.88 Hz). 13C NMR (DMSO-d6): δ (ppm) 181.29, 142.34,
140.97, 133.05, 128.58, 127.98, 127.65, 126.99, 125.17, 124.24, 74.47,
51.20, 47.97. HRMS: [M + H]+ = 388.183 24.
120.44, 75.56, 52.16, 51.94, 51.64, 51.23 . HRMS: [M + H]+
495.113 34.
=
2,4-Dinitrophenyl 4-(4-tert-Butylbenzyl) piperazine-1-carbo-
1
dithioate (37). Mp 128−130 °C. H NMR (CDCl3): δ (ppm) 8.85
(d, 1H, J = 2.32 Hz), 8.43 (dd, 1H, J = 6.32 Hz), 7.89 (d, 1H, J = 8.60
Hz), 7.37−7.23 (m, 4H), 4.34 (t, 2H, J = 4.36 Hz), 4.06 (t, 2H, J =
4.52 Hz), 3.52 (s, 1H), 2.61 (t, 2H, J = 4.36 Hz), 2.57 (t, 2H, J = 4.52
Hz), 1.35 (s, 9H). 13C NMR (CDCl3): δ 190.01, 151.31, 150.53,
147.95, 139.46, 135.07, 134.05, 128.87, 126.45, 125.38, 120.43, 62.01,
52.59, 52.28, 51.90, 34.54, 31.39. HRMS: [M + H]+ = 475.144 97.
The details for compounds 17−36 can be found in the Supporting
Information (pp S6−S10).
The details for compound 02 can be found in the Supporting
Information (p S3).
Pharmacological Evaluation. MAGL Esterase Activity Assay.
Measurement of radiolabeled 2-oleoylglycerol (2-OG) hydrolysis by
MAGL was performed as previously described.21 Briefly, 2-OG (10
μM, [3H]-2-OG, 50 000 dpm, American Radiolabeled Chemicals) was
incubated at 37 °C for 10 min in the presence of purified recombinant
hMGL (5 ng in Tris buffer, pH 8.0, 50 mM, 0.1% BSA, 200 μL of total
volume assay) and 10 μL of DMSO (controls) or inhibitors (dissolved
in DMSO). The incubation was stopped by adding 400 μL of an ice-
cold 1:1 methanol−chloroform mixture to each tube and thorough
mixing. After centrifugation at 700g for 5 min, radioactivity in the
Aryloxothioamide Derivatives Synthesis (03 and 04). The
used protocol was adapted from Asinger et al.29 To a suspension of 2-
bromo-1-phenylethanone (see Supporting Information, p S2) (0.1
mol) and sulfur (19.2 g) in DMF (50 mL) was added 1-
benzhydrylpiperazine (0.35 mol). The reaction mixture was stirred
overnight at room temperature. The mixture was then poured into
water, and the crude product was extracted with dichloromethane. The
organic phase was dried on sodium sulfate and then, concentrated
under reduced pressure. Purification on silica gel column using ethyl
5781
dx.doi.org/10.1021/jm3006004 | J. Med. Chem. 2012, 55, 5774−5783