Synthesis of novel thiourethane analogs of neurolipins

Article abstract of DOI:10.1007/s10600-013-0566-4

Thiourethane analogs of anandamide and arachidonic acid amides with cyclopropylamine, dopamine, and serotonin that were capable of inhibiting hydrolysis of fatty-acid amides and interacting with cannabinoid receptor type 1 were synthesized for the first t

Full text of DOI:10.1007/s10600-013-0566-4

Chemistry of Natural Compounds, Vol. 49, No. 2, May, 2013 [Russian original No. 2, March–April, 2013]
SYNTHESIS OF NOVEL THIOURETHANE
ANALOGS OF NEUROLIPINS
N. M. Gretskaya, A. A. Druzhinina,
M. Yu. Bobrov, and V. V. Bezuglov
UDC 547.392.52.057
*
Thiourethane analogs of anandamide and arachidonic acid amides with cyclopropylamine, dopamine, and
serotonin that were capable of inhibiting hydrolysis of fatty-acid amides and interacting with cannabinoid
receptor type 1 were synthesized for the first time.
Keywords: anandamide, arachidonic acid, thiourethanes, fatty-acid amide hydrolase, CB1-receptor.
Several nor-arachidonoylurethane analogs of anandamide (an endogenous ligand of cannabinoid receptors) were
shown previously to be stable to the action of fatty-acid amide hydrolase, an enzyme that inactivates endocannabinoid amides
(anandamide etc.) in vivo in animals. Several of these compounds exhibited high affinity for cannabinoid receptor type 1
(CB1-receptor) and a pharmacologic profile similar to anandamide. The researchers hypothesized that the additional electron-
donating nitrogen atom in the structure of the urethane derivatives was responsible for these properties [1]. In order to refine
concepts about the structure–activity relationship of neurolipins with respect to endocannabinoid system components, we
synthesized novel analogs of anandamide (1a); arachidonoylcyclopropylamine (1b), a selective ligand of the CB1-receptor;
arachidonoyldopamine (1c), an endogenous ligand of the cannabinoid and vanilloid receptors; and arachidonoylserotonin
(1d), an endogenous neurolipin and inhibitor of fatty-acid amide hydrolase. The amide group was replaced by thiourethane in
the corresponding analogs 2a–d.
S
O
X
X
N
H
N
H
N
H
1a - d
2a - d
d:
7'
H
N
OH
OH
4'
7'
3'
1'
1'
1'
1'
OH
X = a:
b:
c:
2'
4'
10'
OH
The starting compound for the synthesis of neurolipin thiourethane analogs 2a–d was arachidonic acid (3). It was
converted quantitatively in the first synthetic step (Scheme 1) into the mixed anhydride with arachidonic anhydride 4 by the
literature method [2]. Reduction of anhydride 4 by sodium borohydride according to the published procedure [3] gave the
arachidyl alcohol (5) in 70% yield. The resulting arachidyl alcohol (5) was transformed into mesylate 6. The reaction with
mesylchloride gave a better yield (85%) but was slower if pyridine was used as the base rather than triethylamine [4]. Next,
the mesylate was transformed into azide 7 by sodium azide in the dark for 20 h. We chose a tandem Staudinger/azo-Wittig
reaction to transform azide 7 into isothiocyanate 8 [5]. This enabled the use of thiophosgene to be avoided. Carbon disulfide
was used instead in the synthesis.
M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997,
Moscow, Ul. Miklukho-Maklaya, 16/10, fax: (095) 335 71 03, e-mail: vvbez@ibch.ru. Translated from Khimiya Prirodnykh
Soedinenii, No. 2, March–April, 2013, pp. 193–195. Original article submitted October 26, 2012.
©
222
0009-3130/13/4902-0222 2013 Springer Science+Business Media New York

O
O
O
Ms
iii
i
ii
88%
R
OH
O
R
R
O
O
R
OH
75%
90%
3
4
5
6
iv
v
vi
R
N
R
N
C
S
3
2a - d
71%
62%
7
8
S
C
N
S
CS
2
PPh
3
PPh
3
R
N
8
C S
R
N
PPh
R
R
N
N
3
3
N
N
N
N
+ SPPh
3
9
10
7
2
R =
i. iso-butyl chloroformate, Et N, CH CN, 0–4ꢁC, 20 min, Ar; ii. NaBH , THF–H O, –8ꢁC,
3
3
4
2
40 min, Ar; iii. MsCl, Et N, CH Cl , 2 h, room t; iv. NaN , DMF–H O, 2 h, 66ꢁC and 18 h,
3
2
2
3
2
room t, in the dark; v. PPh , CS , THF, room t, 48 h, in the dark; vi. 11a-d, THF or DMF, 4ꢁC,
3
2
30 min, 1 h
Scheme 1
Thiourethane analogs 2a–d were obtained by condensation of isothiocyanate 8 with ethanolamine (11a),
cyclopropylamine (11b), dopamine (11c), and serotonin (11d) in yields of 99, 87, 76, and 50%, respectively. The condensation
with isothiocyanate 8 was carried out in the presence of an equimolar amount of Et N if dopamine and serotonin hydrochlorides
3
were used in order to convert the amines into the free bases and bind the released HCl. The differences in yields of thiourethanes
2a,b and 2c,d were apparently due to the ability of the dopamine and serotonin derivatives to be oxidized by oxygen. This led
to losses of the compounds during their isolation.
The synthesized compounds were moderate inhibitors of arachidonoylglycine hydrolysis by fatty-acid amide hydrolase
from the membrane fraction of rat-brain homogenate. Thus, thiourethane derivatives 2b and 2c at concentration 50 ꢀM
inhibited arachidonoylglycine (2 ꢀM) hydrolysis by 20.4 1.2 and 33.7 8.0%. The affinity of the synthesized derivatives for
3
the CB1-receptor was estimated in a test for displacement of the labeled CB1-receptor agonist [ H]-CP55940 in the membrane
fraction of rat-brain homogenate. Arachidonoyldopamine analog 2c, which inhibited agonist binding with K = 0.5 ꢀM
i
(compared with K of anandamide of 10 nM), displayed the lowest activity. The thiourethane analog of anandamide 2a and
i
cyclopropyl derivative 2b (K ~ 40 nM) had activities comparable with that of anandamide.
i
Thus, we synthesized novel biologically active thiourethane analogs of neurolipins that were capable of interacting
with key proteins of the cannabinoid system.
EXPERIMENTAL
We used arachidonic acid (95% pure, Russia); isobutylchloroformate, serotonin, dopamine, sodium borohydride
(Fluka, Switzerland); triethylamine (Merck, Germany); methanesulfonylchloride, ethanolamine, triphenylphosphine, carbon
disulfide (Acros, Germany); cyclopropylamine (Aldrich, USA); and sodium azide (Sigma, USA). Solvents were purified by
standard methods [6]. Tetrahydrofuran (THF) was purified by distillation from Na in the presence of benzophenone and was
stored under Ar.
Thin-layer chromatography (TLC) was carried out on Silufol UV-254 (Kavalier, Czech Rep.) and DC-Alufolien
Kieselgel 60 F
plates (Merck, Germany). The detectors were phosphomolybdic acid solution (10%) in EtOH, H SO
254
2 4
solution (5%) in MeOH, and ninhydrin solution (5%) in EtOH. TLC used solvent systems CHCl :MeOH (19:1, A) and
3
hexane:Et O (2:1, B).
2
Column chromatography was performed over Kieselgel 60 silica gel (Merck, Germany). Ionol was added to and Ar
was bubbled through a suspension of the sorbent before filling the column. Fractions containing the desired product (TLC
analysis) were evaporated in a rotary evaporator at bath temperature <37–40°C using a water-aspirator with subsequent removal
of traces of solvent by an oil vacuum pump.
223

Mass spectra were recorded in an MX-5303 TOF mass spectrometer with electrospray ionization (St. Petersburg,
Russia). PMR spectra were recorded from CDCl solutions on a Bruker CXP-200 pulsed NMR spectrometer at operating
3
frequency 200 MHz. Chemical shifts were given on the ꢂ-scale in ppm relative to Me Si standard.
4
Standard Work-up of Reaction Mixtures. The reaction mixture was treated with H O and extracted with EtOAc.
2
The extract was dried over Na SO , filtered, and evaporated in vacuo (water aspirator).
2
4
Preparation of Eicosa-5,8,11,14-tetraenylisothiocyanate (8). Arachidonoyl(isobutyl)carbonate (4). A solution
of arachidonic acid (3, 427 mg, 1.4 mmol) in CH CN (8 mL) was stirred at 0°C under Ar, treated with Et N (214 ꢀL,
3
3
1.54 mmol) and isobutylchloroformate (200 ꢀL, 1.54 mmol), stirred at this temperature for 20 min, and worked up as above.
The product was used without further purification. Yield 508 mg (90%), colorless oil, R 0.88 (A).
f
Arachidyl Alcohol (5). A solution of mixed anhydride 4 (508 mg, 1.26 mmol) in THF (15 mL) was treated with a
freshly prepared solution of NaBH (80 mg, 2.1 mmol) in H O (400 ꢀL, 200 mg/mL). The reaction was carried out under Ar
4
2
with constant stirring at –8°C (saltwater bath). After 1 min, the mixture was treated with H O (23 mL) and allowed to warm
2
slowly to room temperature. After 40 min, THF was removed in a rotary evaporator. Arachidyl alcohol (5) was extracted from
the aqueous solution by EtOAc. Yield 357 mg (88%), light-yellow oil, R 0.5 (A).
f
Eicosa-5,8,11,14-tetraenylmethanesulfonate (6). A solution of 5 (357 mg, 1.23 mmol) and Et N (510 ꢀL,
3
3.7 mmol) in freshly distilled CH Cl (10 mL) was treated with methanesulfonylchloride (143 ꢀL, 1.8 mmol) with constant
2
2
stirring under Ar at 0°C. The reaction mixture was allowed to warm to room temperature, stored for another 2 h, worked up as
above, and purified by column chromatography over silica gel using a hexane:Et O gradient (0–20%). Yield 335 mg (75%),
2
colorless oil, R 0.3 (B).
f
Eicosa-5,8,11,14-tetraenylazide (7). A solution of methanesulfonate 6 (335 mg, 0.9 mmol) in DMF:H O (1500 ꢀL,
2
5:1) was stirred, treated with sodium azide (200 mg, 3.1 mmol), stirred for 18 h at 23°C and 2 h at 66°C in the dark, cooled to
room temperature, and worked up as above. The azide was isolated by column chromatography over silica gel using CHCl .
3
Yield 180 mg (62%), light-yellow oil, R 0.9 (A).
f
Eicosa-5,8,11,14-tetraenylisothiocyanate (8). A solution of azide 7 (180 mg, 0.57 mmol) in THF (5 mL) was stirred
under Ar, treated with PPh (223 mg, 0.85 mmol) and CS (341 ꢀL, 5.67 mmol), held for 48 h in the dark, and evaporated. The
3
2
solid was dissolved in cold hexane and filtered. The product was isolated over a column of silica gel using hexane:Et O
2
(0–5%). Yield 134 mg (71%), colorless oil, R 0.9 (B).
f
Preparation of Thiourethane Derivatives (2a–d) (2a). A solution of isothiocyanate 8 (30 mg, 0.1 mmol) in CH CN
3
(3 mL, 10 mg/mL) was treated with distilled ethanolamine (11 ꢀL, 0.2 mmol), stirred at 4°C for 30 min, and worked up as
above. The product was purified by chromatography over a column of silica gel (hexane:Et O, 0–15%). Yield 35 mg (99%),
2
+
yellow oil, R 0.55 (A). Mass spectrum m/z 393.5255 [M + H] . C H N OS. PMR spectrum (ꢂ, ppm, J/Hz): 0.89 (3H, t,
f
23 40 2
J = 6.7, H-20), 1.57–1.24 (10H, m, H-2, 3, 17, 18, 19), 2.07–2.01 (4H, m, H-4, 16), 2.79–2.77 (6H, m, H-7, 10, 13), 3.72–3.38
(6H, m, H-1, 1ꢃ, 2ꢃ), 5.4–5.2 (8H, m, H-5, 6, 8, 9, 11, 12, 14, 15).
(2b). Asolution of isothiocyanate 8 (30 mg, 0.09 mmol) in THF (3 mL, 10 mg/mL) was treated with cyclopropylamine
(13 ꢀL, 0.121 mmol), stirred at 4°C for 30 min, and worked up as above. The product was purified by column chromatography
over silica gel (hexane:Et O, 0–15%). Yield 30 mg (87%), light-yellow oil, R 0.7 (A). Mass spectrum m/z 393.5255
2
f
+
[M + H] . C H N S. PMR spectrum (ꢂ, ppm, J/Hz): 0.84–0.69 (4H, m, H-2ꢃ, 3ꢃ), 0.877 (3H, t, J = 6.5, H-20), 1.32–1.24
24 40
2
(11H, m, H-2, 3, 17, 18, 19, 1ꢃ), 2.08–2.00 (4H, m, H-4, 16), 2.78–2.76 (6H, m, H-7, 10, 13), 3.62–3.59 (2H, q, H-1), 5.4–5.2
(8H, m, H-5, 6, 8, 9, 11, 12, 14, 15).
(2c). A solution of isothiocyanate 8 (30 mg, 0.09 mmol) in DMF (600 ꢀL, 50 mg/mL) was stirred at 4°C, treated with
dopamine hydrochloride (21 mg, 0.11 mmol) and Et N (16.5 ꢀL, 0.11 mmol), stirred for 1 h, and evaporated using an oil
3
vacuum pump. The product was purified by chromatography over a column of silica gel (CHCl :EtOAc, 0–20%). Yield 33.5 mg
3
+
(76%), light-yellow oil, R 0.53 (A). Mass spectrum m/z 484.3062 [M] . C H N O S. PMR spectrum (ꢂ, ppm, J/Hz): 0.88
f
29 44 2 2
(3H, t, J = 6.6, H-20), 1.38–1.24 (6H, m, H-17, 18, 19), 1.49–1.46 (4H, m, H-2, 3), 2.05–1.99 (4H, m, H-4, 16), 2.61 (2H, m,
H-2ꢃ), 2.78–2.77 (6H, m, H-7, 10, 13), 3.3 (2H, m, H-1), 3.48 (2H, m, H-1ꢃ), 5.4–5.2 (8H, m, H-5, 6, 8, 9, 11, 12, 14, 15), 6.44
(1H, m, H-8ꢃ), 6.62 (1H, m, H-7ꢃ), 6.74 (1H, m, H-4ꢃ).
(2d). Asolution of isothiocyanate 8 (30 mg, 0.09 mmol) in DMF (600 ꢀL, 50 mg/mL) was stirred at 4°C, treated with
serotonin hydrochloride (23 mg, 0.11 mmol) and Et N (15 ꢀL, 0.11 mmol), stirred for 1 h, and evaporated using an oil vacuum
3
pump. The product was purified by chromatography over a column of silica gel (CHCl :EtOAc, 0–20%). Yield 23 mg (50%),
3
+
light-yellow oil, R 0.50 (A). Mass spectrum m/z 507.4196 [M] . C H N OS. PMR spectrum (ꢂ, ppm, J/Hz): 0.85 (3H, t,
f
31 45 3
224

J = 6.4, H-20), 1.57–1.07 (10H, m, H-2, 2, 17, 18, 19), 2.16–1.80 (4H, m, H-4, 16), 2.59–2.34 (2H, s, H-2ꢃ), 2.94–2.59 (6H, m,
H-7, 10, 13), 3.26–2.76 (4H, d, H-1, 1ꢃ), 5.43–5.06 (8H, m, H-5, 6, 8, 9, 11, 12, 14, 15), 6.93–6.47 (3H, m, H-7ꢃ, 8ꢃ, 10ꢃ), 7.50
(1H, m, H-4ꢃ).
ACKNOWLEDGMENT
The work was supported partially by the RFBR (Projects 11-04-01469a and 12-04-00608a). We thankA. V. Blazhenova
for performing the experiments on inhibition of arachidonoylglycine in the presence of the synthesized compounds.
REFERENCES
1.
2.
3.
4.
E. W. Ng, M. M. Aung, M. E. Abood, B. R. Martin, and R. K. Razdan, J. Med. Chem., 42, 1975 (1999).
D. V. Kuklev and V. V. Bezuglov, Bioorg. Khim., 20, 67 (1994).
M. Rodriguez, M. Linares, S. Doulut, A. Heitz, and J. Martinez, Tetrahedron Lett., 32, 923 (1991).
S. Lin, A. Khanolkar, P. Fan, A. Goutopoulos, CeQin, D. Papahadjis, and A. Makryannis, J. Med. Chem., 41,
5353 (1998).
5.
6.
T. Isoda, K. Hayashi, S. Tamai, T. Kumagai, and Y. Nagao, Chem. Pharm. Bull. (Tokyo), 54, 1616 (2006).
A. J. Gordon and R. A. Ford, A Chemist’s Companion, Wiley-Interscience, New York, 1972 [Russian translation, p. 437].
225