Synthesis and pharmacological evaluation of sulfamide-based analogues of anandamide

Article abstract of DOI:10.1016/j.ejmech.2009.08.003

Arachidonyl and linoleyl sulfamide derivatives have been synthesized and their potential cannabimimetic properties evaluated in in vitro functional and binding assays. Replacement of the ethanolamide moiety of anandamide by -CH₂NHSO₂NH-R considerably reduces the CB1 receptor activity and only some of the compounds showed modest cannabinoid properties in binding assays. The new compounds were also tested as inhibitors of the FAAH enzyme but were inactive.

Full text of DOI:10.1016/j.ejmech.2009.08.003

European Journal of Medicinal Chemistry 44 (2009) 4889–4895
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of sulfamide-based analogues
of anandamide
a
a
a
,
b
b
*
´
´
Carolina Cano , Juan Antonio Paez , Pilar Goya , Antonia Serrano , Javier Pavon ,
b
c
c
´
´
´
´
Fernando Rodrıguez de Fonseca , Margarita Suardıaz , Marıa Isabel Martın
a Instituto de Qu´ımica Me´dica, CSIC, Juan de la Cierva 3, Madrid 28006, Spain
b
´
Fundacio´n Hospital Carlos Haya, Avda. Carlos Haya 82, Malaga 29010, Spain
c Unidad de Farmacolog´ıa, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Avda. Atenas s/n 28922, Alcorco´n, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Arachidonyl and linoleyl sulfamide derivatives have been synthesized and their potential cannabimi-
metic properties evaluated in in vitro functional and binding assays. Replacement of the ethanolamide
moiety of anandamide by –CH2NHSO2NH–R considerably reduces the CB1 receptor activity and only
some of the compounds showed modest cannabinoid properties in binding assays. The new compounds
were also tested as inhibitors of the FAAH enzyme but were inactive.
Received 10 March 2009
Received in revised form
29 July 2009
Accepted 3 August 2009
Available online 12 August 2009
Ó 2009 Published by Elsevier Masson SAS.
Keywords:
Sulfamide
Anandamide
Arachidonyl
Cannabinoid
FAAH
1. Introduction
Oleylethanolamide (OEA) (Fig. 1) is another important acyle-
thanolamide involved in metabolism pathways which acts through
The therapeutic properties of cannabinoids including plant
derived compounds as well as synthetic derivatives are due to their
interaction with the cannabinoid receptors CB1 and CB2 [1,2]. The
identification of these G-protein coupled receptors [3] prompted
the discovery of the endogenous ligands, of which the most
important are anandamide (AEA) [4] (Fig. 1) and 2-arachidonoyl
glycerol [5]. Recently new evidences are appearing to prove the
existence of a third cannabinoid-like receptor called GPR55 [6].
The endocannabinoids together with the two proteins involved
in their regulation mechanisms, the anandamide transporter (ANT)
and the fatty acid amide hydrolase (FAAH), and the cannabinoid
receptors responsible for many biochemical processes, integrate
the endocannabinoid system and represent important therapeutic
targets. Thus, many analogues of anandamide have been synthe-
sized and their structure activity relationships are well established.
Besides, improved metabolically stable derivatives and inhibitors of
the transporter and of the FAAH have also been prepared and
evaluated [7].
PPAR
a receptors [8]. We have reported the synthesis and biological
evaluation of a series of novel sulfamide analogs of OEA that shows
in vivo satiety inducing actions and PPAR
a activation, the most
interesting compounds being N-oleyl-N0-propylsulfamide and
N-propyl-N0-stearylsulfamide [9] (Fig. 2).
The fact that incorporation of the sulfamide moiety in OEA
derivatives led to compounds with potent in vivo activity encour-
aged us to evaluate this replacement in the structurally related
anandamide derivatives.
So, in the present study, we wish to report the synthesis and
pharmacological evaluation of novel anandamide analogues with
the particular feature that they incorporate an aminosulfonylamino
group. Besides, the hydroxyethyl of the ethanolamide has been
replaced by a propyl and, as a different approach to include highly
lipophilic chains, adamantyl sulfamide derivatives have also been
prepared. These compounds together with the OEA sulfamide
analogs, previously described [9], have been tested for cannabinoid
activity both in isolated tissue assays and in binding experiments.
On the other hand, alkane sulfonyl derivatives of anandamide
are also of interest since fatty acid sulfonyl fluorides, such as pal-
mitylsulfonylfluoride (AM374) (Fig. 2), are potent inhibitors of
FAAH and thus they can enhance the action of endogenous
* Corresponding author. Tel.: þ34915622900; fax: þ34915644853.
E-mail address: iqmg310@iqm.csic.es (P. Goya).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.08.003

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C. Cano et al. / European Journal of Medicinal Chemistry 44 (2009) 4889–4895
Table 1
Structures and biological assays data of compounds 3–19 R1–NH–SO2–NH–R2.
R1
R2
Compound
Ki (nM)b
FAAH activity (% of control)c [Compound] (M)
10ꢀ6
10ꢀ7
H
3
4010 ꢁ 825
NDd
H3C
H
4
6406 ꢁ 267
NDd
H C
3
Pr
Pr
Pr
8a
>10,000
96.5
NDd
NDd
101.1
H C
3
9
2339 ꢁ 81
7190 ꢁ 865
H C
3
10
H C
3
H
H
11a
1890 ꢁ 360
>10,000
101.7
99.4
100.2
105.9
H C
3
12a
H3C
13a
14a
15a
ND
ND
ND
102.9
108.8
99.3
113.5
108.6
107.5
CH3
H
H
H
CH3
CH3
Pr
16a
9102 ꢁ 140
109.8
108.8
H3C
Pr
Pr
17a
18a
6670 ꢁ 1320
>10,000
NDd
NDd
CH3

C. Cano et al. / European Journal of Medicinal Chemistry 44 (2009) 4889–4895
4891
Table 1 (continued)
R1
R2
Pr
Compound
Ki (nM)b
FAAH activity (% of control)c [Compound] (M)
10ꢀ6
10ꢀ7
19a
8580 ꢁ 669
132 ꢁ 23
92.6
NDd
90.4
–
AEA
a
Described in Ref. [9].
b
Affinity of compounds for the CB1 receptor was evaluated by displacing experiments using mouse cerebellum membrane and [3H]SR141716A as CB1 receptor ligand.
Membrane-bound FAAH activity was assayed using arachidonyl-[1-3H]-ethanolamide as a substrate, and measuring metabolized [3H]anandamide (as [3H]ethanolamine)
c
in the aqueous phase after chloroform extraction.
d
ND: not determined.
the major compound, together with the mono oleyl propyl sulfa-
mide 8 (Scheme 3) [9]. Therefore, another synthetic approach was
envisaged which involved the use of N-propylsulfamoyl chloride
prepared from propylamine and chlorosulfonic acid [17]. This was
treated in situ with the fatty acid amines 1 and 2 in toluene with
triethylamine as base and the corresponding derivative N-arach-
idonyl-N0-propylsulfamide (9) and derivative N-linoleyl-N0-pro-
pylsulfamide (10) were obtained (Scheme 2).
O
H
N
OH
OH
N
H
O
H3C
H3C
OEA
AEA
Fig. 1. Structures of anandamide and oleylethanolamide.
The structures of the novel compounds 3–6, 9 and 10 were
established according to their analytical and spectroscopic data.
cannabinoids [10]. Due to the structural similarity of some of our
compounds with these FAAH inhibitors, and to the fact that the
sulphonamide group should, in principle, not be prone to the action
of the amidohydrolase, the potential inhibition of FAAH by our
compounds has also been evaluated.
2.2. Biological assays
Radioligand displacement assays were used to evaluate the
affinity of the compounds to the CB1 receptor in rat cerebellar
membranes using either 3[H]WIN-55212-2 or 3[H]SR141716A [18].
The cannabinoid properties of the compounds were also examined
using the mouse vas deferens, a preparation supposed to contain
cannabinoid receptors that can mediate an inhibitory effect of
cannabinoid receptor agonists on electrically evoked contractions
[19].
Binding affinity for the cannabinoid CB1 receptor of the new
compounds here described, together with those of other fatty acid
sulfamides previously synthesized in our group [9] are gathered in
Table 1.
2. Results and discussion
2.1. Chemistry
The synthesis of the long-chain sulfamoyl derivatives (Table 1)
can be achieved starting from the amines of the corresponding fatty
acids either by a transamination [11,12] with a sulfamide or by
reaction with a sulfamoyl chloride [13].
First, N-arachidonylamine 1 was prepared from the parent acid
following a described procedure [14] which involves mesylation,
formation of the azide and subsequent reduction to the amine.
N-Linoleylamine 2, previously obtained by a different way [15], was
prepared by following the procedure mentioned above (Scheme 1).
Then, reaction of 1 and 2 with sulfamide in water/ethanol
afforded the corresponding N-monosubstituted derivatives 3 and 4,
as can be seen in Scheme 2. In a similar manner, N-2-adamantyl-
amine and N-1(1-adamantylethyl)amine provided the ada-
mantylsulfamides 5 and 6 (Scheme 2). (Compound 5 has been
reported by a completely different synthetic procedure from the
primary amine and N-(tert-butoxycarbonyl)-N-[4-(dimethylaza-
niumylidene)-1,4-dihydropyridin-1-ylsulfonyl]azanide [16]).
In a similar manner, it would be possible to obtain the desired
N-propylsulfamide derivatives using propylsulfamide. However,
when commercially available N-oleylamine reacted with N-pro-
pylsulfamide [11,12] the disubstitution product 7 was obtained, as
Examination of these data indicates that, in general, these sul-
famoyl derivatives do not bind to the receptor or show only modest
affinity. It is interesting to mention that contrary to what occurs
with the sulfamide analogs of OEA, in which PPARa affinity and in
vivo activity is kept, in the case of anandamide, the inclusion of
sulfamide results in a loss of activity. In line with this, a structurally
related compound, N-(2-hydroxyethyl)arachidonyl sulfonamide
[20] turned out to be 2500-fold less potent than anandamide at the
CB1 agonist site (Fig. 2).
We also performed binding assays using tritiated WIN 55,212-2.
Ki values were usually higher using this agonist, as previously
reported [21]. Thus, Ki values for compounds 16, 17, 18 and 19 were
>10,000; > 10,000; >100,000 and 4229 ꢁ 830 respectively. Since
WIN 55,22-2 also labeled CB2 receptors and we did not observe any
displacing curve fitting a two binding sites model, these results also
H
O
O
O
N
S
O
S
F
O
S
O
HN
HN
CH3
CH3
H3C
H3C
OH
AM374
N -(2-hydroxyethyl)arachidonylsu lfonamid e N -propyl-N '-stearylsulfamide
Fig. 2. Long chain derivatives as FAAH inhibitors and PPARa ligands.

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C. Cano et al. / European Journal of Medicinal Chemistry 44 (2009) 4889–4895
O
NaN , DMF
3
LiAlH , Et O
MsCl,Py
0 °C, 5 h
4
2
1
1
1
1
R
N
3
R
OMs
R
OH
R
OH
1)10 °C, 30 min
2) reflux 30 min
90 °C, 24 h
LiAlH , Et O
4
2
reflux 3 h
1
1
R = linoleyl
R = arachidonyl
NH
2
NH
2
1
2
Scheme 1. Synthesis of arachidonyl and linoleyl amines 1 and 2.
activity (Table 1). Therefore, the lack of inhibition of fatty acid
amidohydrolase by these compounds discards a potential indirect
action on the endocannabinoid/oleylethanolamide system through
interferences on their enzymatic degradation.
O
O
O
O
R1
S
EtOH/H2O
S
N
NH2
H2
N
NH2
H
3-6
R1-NH2
+
O
O
1) TEA, toluene, 0°C
2) r. t.
R1
S
3. Conclusion
ClO2SHN
N
N
H
H
9,10
Compounds
Novel sulfamoyl and propyl sulfamoyl derivatives with fatty acid
and adamantyl chains have been synthesized and their cannabi-
mimetic properties evaluated in functional and binding assays.
Replacement of ethanolamide moiety of anandamide with
–CH2NHSO2NH–R considerably reduces the activity at the CB1
receptor and only some compounds showed modest cannabinoid
properties in the binding assay. None of these compounds were
inhibitors of FAAH.
1
R
3, 9
H3C
4,10
H3C
5
6
4. Experimental
1H and 13C NMR spectra were recorded on Varian Unity 300 and
400 Varian Gemini and Bruker Avance 300 spectrometers. Chem-
CH3
ical shifts are reported in ppm on the
d scale. The signal of the
solvent was used as reference. Mass spectra (electrospray ioniza-
tion) were determined on an MSD-Serie 1100 Hewlett Packard
instrument. Melting points were determined on a Reichert Jung
Thermovar melting point apparatus and are uncorrected. Elemental
analyses were performed on a Heraeus CHN–O Rapid Analysis in
Scheme 2. Synthesis of sulfamides 3–6, 9 and 10.
O
O
O
O
O O
EtOH/H2O
R1-NH2
S
R1
S
+
S
+
R1
R1
´
´
our Analytical Services at Centro de Quımica Organica ‘‘Manuel Lora
Tamayo’’ (CSIC). Flash column chromatography was carried out
using Merck silica gel 60 (0.040–0.063). All starting materials were
commercially available in Aldrich or Fluka and used without further
purification. N-arachidonylamine and N-linoleylamine were
obtained from their carboxylic acids purchased in Fluka, as
described in the literature [14]. N-propylsulfamide 5 was prepared
from the procedure reported by Paquin [11] and N-propylsulfamoyl
chloride 7 was synthesized following the procedure of Kloek and
Leschinsky [17].
N
N
H2N
N
N
N
H
H
H
H
H
8
7
1
R
=
H3C
Scheme 3. Reaction of N-oleylamine with N-propylsulfamide to give 7 and 8.
confirm the lack of binding affinities of these compounds to either
CB1 or CB2 receptor.
The functional assays performed in compounds 3–6, 8–12 and
16–19 confirmed also this lack of cannabinoid activity and so, none
of the compounds were able to inhibit the electrically-evoked
contractions on mouse isolated vas deferens (Fig. 3).
The negative results obtained in the binding and functional
assays prompted us not to evaluate CB2 activity although an
‘‘inversion’’ for affinity in anandamide derivatives has been repor-
ted by Appendino et al. [22] who found CB2 selectivity for fatty acid
derived compounds lacking polyunsaturation.
4.1. General procedure for the preparation of monosubstituted
sulfamides
The amine dissolved in EtOH was added dropwise to a solution
of sulfamide in H2O. The reaction mixture was refluxed and evap-
orated to dryness; the crude was purified by column chromatog-
raphy on silica gel with CH2Cl2:MeOH/NH3 9:1, giving the expected
product.
4.1.1. N-(cis-5-cis-8-cis-11-cis-14-eicosatetraenyl)sulfamide (3)
Due to the structural similarity with some FAAH inhibitors, the
saturated long-chain derivatives (12–16), the oleyl derivatives 8
and 11 and the N-adamantyl-N0-propylsulfamide 19 were evaluated
as FAAH inhibitors. None of the compounds showed significant
From
N-(cis-5-cis-8-cis-11-cis-14-eicosatetraenyl)amine
1
(0.100 g, 0.3 mmol), sulfamide (0.033 g, 0.3 mmol), H2O (20 mL) and
EtOH (10 mL); reaction time: 24 h; yield (0.020 g of yellow oil, 14%).
Mp ¼ oil.1H NMR (400 MHz, CDCl3)
d: 5.37 (m, 8H, CH]CH); 4.60 (br

C. Cano et al. / European Journal of Medicinal Chemistry 44 (2009) 4889–4895
4893
J ¼ 12.1 Hz, H-4eq); 1.85 (d, 6H, J ¼ 12.1 Hz, H-2ec and H-4ax); 1.71
(d, 3H, J ¼ 12.1 Hz, H-2ax); 1.35 (d, 3H, J ¼ 7.0 Hz, CH3). 13C NMR
3
4
9
10
12
16
17
18
19
WIN
% of inhibition
70
60
50
40
30
20
10
0
(100 MHz, CDCl3) d: 59.6 (C-1); 39.5 (C-4); 38.2 (C-2); 29.9 (C-3);
14.9 (CH3). MS (ESþ) [M-NHSO2NH2]þ 163 (100%). Anal. calcd. for
C12H22N2SO2: C 55.77, H 8.58, N 10.84, S 12.41; found: C 55.56, H
8.36, N 10.61, S 12.62.
4.2. Preparation of N,N0-dioleylsulfamide (7) and N-oleyl-N0-
propylsulfamide (8) from N-propylsulfamide
0.68 mL of N-oleylamine (1.4 mmol) dissolved in EtOH was
added dropwise to a solution of 0.200 g of N-propylsulfamide
(1.4 mmol) in 2 mL of H2O. The reaction mixture was refluxed for
48 h and evaporated to dryness; the crude was purified by column
chromatography on silica gel with CH2Cl2, giving 0.260 g of white
solid (7) and 0.120 g of desired white solid (8); Yield (7) 31%;
-10
10-6M
8,1x10-8M
2,4x10-7M
7,2x10-7M
Fig. 3. Effect of the addition of increasing concentrations of WIN or of the new
compounds in the contractile response induced by electrical stimulation of mouse vas
deferens. Each point is the mean ꢁ ESM of at least 6 data.
Mp ¼ 85–88 ꢂC. 1H NMR (400 MHz, CDCl3)
d: 5.32 (m, 4H, CH]CH);
4.25 (br s, 2H, NH); 3.00 (m, 4H, CH2NHSO2); 1.99 (m, 8H, CH2–
CH]CH–CH2); 1.53 (sextet, J ¼ 7.3 Hz, 4H, CH2CH3); 1.26 (bm, 44H);
0.87 (t, 6H, J ¼ 7.3 Hz, CH3). 13C NMR (100 MHz, CDCl3)
d: 129.9,
129.7 (CH]CH); 43.2 (CH2NHSO2); 31.9 (CH2–CH2CH3); 29.7, 29.6,
29.5, 29.4, 29.3, 29.2, 27.2, 26.7 (–CH2); 22.6 (CH2–CH3); 14.0 (CH3).
MS (ESþ) [M þ H]þ 598 (100%). Anal. calcd. for C36H72N2SO2: C
72.42, H 12.16, N 4.69, S 5.37; found: C 72.65, H 13.41, N 4.92, S 4.32.
s, 2H, NH2); 4.37 (br s,1H, NH); 3.13 (m, 2H, CH2NHSO2); 2.81 (m, 6H,
C]CH–CH2–CH]C); 2.07 (m, 4H, CH]CH–CH2CH2); 1.60 (m, 2H,
CH2–CH2NHSO2); 1.43 (m, 2H, CH2–CH2CH2NHSO2); 1.30 (m, 6H,
–CH2–); 0.89 (t, 3H, J ¼ 6.8 Hz, CH2–CH3). 13C NMR (100 MHz, CDCl3)
Yield (8) 22%; Mp ¼ 82–83 ꢂC; 1H NMR (400 MHz, CDCl3)
d: 5.30 (m,
d
: 130.7, 129.4, 128.6, 128.5, 128.2, 128.0, 127.8, 127.7 (CH]CH); 43.7
2H, CH]CH); 4.25 (br s, 2H, NH); 2.97 (m, 4H, CH2NHSO2); 1.97 (m,
4H, CH2–CH]CH–CH2); 1.54 (sextet, J ¼ 7.3 Hz, 2H, CH2CH3 (Pr));
1.51 (m, 2H, CH2CH2NHSO2); 1.21 (bm, 22H); 0.91 (t, 3H, J ¼ 7.3 Hz,
CH3 (propyl)); 0.84 (t, 3H, J ¼ 6.6 Hz, CH3 (oleyl)). 13C NMR
(CH2NH); 31.8 (CH2–CH2CH3); 29.8, 29.3 (CH2–CH2CH2CH3, CH2–
CH2NHSO2); 27.4 (CH]CH–CH2CH2); 26.8 (CH2–CH2CH2NHSO2);
25.6 (C]CH–CH2–CH]C); 22.8 (CH2–CH3); 14.3 (CH2–CH3). MS
(ESþ) [M þ H]þ 369 (100%). Anal. calcd. for C20H36N2SO2: C 65.17, H
9.84, N 7.60, S 8.70; found: C 64.85, H 9.75, N 7.54, S 8.50.
(100 MHz, CDCl3) d: 129.9, 129.7 (CH]CH); 44.9 (NHSO2NHCH2
(Pr)); 43.2 (oleyl–CH2NHSO2); 31.8 (CH2–CH2CH3); 29.8–28.9, 26.7
(–CH2); 27.1 (CH2–CH]CH–CH2); 22.8 (CH2–CH3 (Pr)); 22.6 (CH2–
CH3 (oleyl)); 14.1 (CH3 (oleyl)); 11.2 (CH3(Pr)). MS (ESþ) [M þ H]þ
389 (100%); Anal. calcd. for C21H44N2SO2: C 64.86, H 11.32, N 7.20,
S 8.23; found: C 65.06, H 11.60, N 7.34, S 8.33.
4.1.2. N-(cis-9-cis-12-octadecadienyl)sulfamide (4)
From N-(cis-9-cis-12-octadecadienyl)amine 2 (0.044 g, 0.16 mmol),
sulfamide (0.016 g, 0.16 mmol), H2O (20 mL) and EtOH (10 mL); reac-
tion time: 35 h; yield (0.007 g of white solid, 12%). Mp ¼ 55–57 ꢂC. 1H
NMR (400 MHz, CDCl3) d: 5.33 (m, 4H, CH]CH); 4.59 (br s, 2H, NH2);
4.36 (br s, 1H, NH); 3.10 (m, 2H, CH2NHSO2); 2.76 (m, 2H, C]CH–CH2–
CH]C); 2.03 (m, 4H, CH]CH–CH2CH2); 1.56 (m, 2H, CH2–CH2NHSO2);
1.29 (m, 16H, –CH2–); 0.87 (t, 3H, J ¼ 7.0 Hz, CH2–CH3). 13C NMR
4.3. General procedure for the preparation of disubstituted
sulfamides
(100 MHz, CDCl3,)
d
: 130.2,130.0,128.1,127.9 (CH]CH); 43.6 (CH2NH);
Freshly distilled N-propylsulfamoyl chloride was added drop-
wise to a solution of the amine and TEA in dry toluene at 0 ꢂC under
nitrogen atmosphere. The reaction mixture was stirred at room
temperature, the white solid TEA$HCl was filtered, the solvent was
evaporated to dryness and the crude was purified by column
chromatography on silica gel with CH2Cl2:MeOH/NH3 9:1, giving
the expected product.
31.5 (CH2–CH2CH3); 29.1 (CH2–CH2NHSO2); 27.2 (CH]CH–CH2CH2);
29.6–26.6 (–CH2–); 25.6 (C]CH–CH2–CH]C); 22.5 (CH2–CH3); 14.0
(CH2–CH3). MS (ESþ) [M þ H]þ 345 (100%). Anal. calcd. for
C18H36N2SO2: C 62.79, H 10.46, N 8.14, S 9.30; found: C 62.90, H 10.80,
N 8.03, S 9.51.
4.1.3. N-(2-adamantyl)sulfamide (5)
From N-(2-adamantyl)amine hydrochloride (0.190 g, 1.0 mmol),
sulfamide (0.080 g, 0.8 mmol), H2O (20 mL) and EtOH (10 mL),
TEA 0.200 g (2.0 mmol), reaction time: 10 h; yield (0.059 g of
white solid, 31%). Mp ¼ 197–199 ꢂC. 1H NMR (400 MHz, CDCl3)
4.3.1. N-(cis-5-cis-8-cis-11-cis-14-eicosatetraenyl)-N0-
propylsulfamide (9)
From
N-(cis-5-cis-8-cis-11-cis-14-eicosatetraenyl)amine
1
(0.353 g, 1.2 mmol)), N-propylsulfamoyl chloride (0.192 g,
d: 3.71 (m, 1H, H-2); 2.25 (m, 2H, H-1 and H-3); 2.21 (m, 2H,
1.2 mmol), TEA (0.36 g, 3.6 mmol), toluene (30 mL); reaction time:
H-4ax, H-10ax); 1.97–2.12 (bm, 8H, H-5, H-6, H-7, H-8, H-9); 1.77
51 h; yield (0.090 g of yellow oil, 18%). 1H NMR (400 MHz, CDCl3)
d:
(bm, 2H, H-4ec and H-10eq). 13C NMR (100 MHz, CDCl3)
d
: 59.3
5.34 (m, 8H, CH]CH); 4.25 (br s, 1H, NH ((Pr)); 4.22 (br s, 1H, NH
(arachidonyl)); 3.02 (m, 4H, CH2NHSO2); 2.81 (m, 6H, C]CH–CH2–
CH]C); 2.07 (m, 4H, CH]CH–CH2CH2); 1.54 (m, 2H,
CH2CH2NHSO2); 1.57 (sextet, 2H, J ¼ 7.3 Hz, CH2CH3 (Pr)); 1.42 (m,
2H, CH2–CH2CH2NHSO2); 1.29 (bm, 6H, –CH2–); 0.95 (t, 3H,
(C-2); 38.7 (C-6); 38.5 (C-8 and C-9); 34.0 (C-3 and C-1); 32.2 (C-4
and C-10); 28.6 (C-5 and C-7). Anal. calcd. for C10H18N2SO2:
C 52.15, H 7.88, N 12.16, S 13.92; found: C 51.97, H 7.71, N 11.98,
S 13.40.
J ¼ 7.3 Hz, CH3 (Pr)); 0.89 (t, 3H, J ¼ 7.0 Hz, CH3 (arachidonyl)). 13
C
4.1.4. N-(1-(1-adamantyl)ethyl)sulfamide (6)
NMR (100 MHz, CDCl3) d: 130.5, 129.4, 128.6, 128.5, 128.3, 128.1,
From N-(1-(1-adamantyl)ethyl)amine hydrochloride (0.172 g,
0.8 mmol), sulfamide (0.064 g, 0.6 mmol), H2O (20 mL) and EtOH
(10 mL); TEA 0.161 g (1.6 mmol), reaction time: 14 h; yield (0.085 g
127.8, 127.5 (CH]CH); 44.9 (CH2NH (Pr)); 43.1 (CH2NHSO2
(arachidonyl)); 31.5 (CH2–CH2CH3); 27.1 (CH]CH–CH2CH2); 29.3
(CH2–CH2CH2CH3); 26.8 (CH2–CH2CH2NH SO2); 25.6 (C]CH–CH2–
CH]C); 22.8 (CH2–CH3 (Pr)); 22.5 (CH2–CH3 (arachidonyl)); 14.3
(CH2–CH3 (arachidonyl)); 11.2 (CH2–CH3 (Pr)). MS (ESþ) [M þ H]þ
of white solid, 55%). Mp ¼ 156–158 ꢂC. 1H NMR (400 MHz, CDCl3)
d:
3.09 (q, 1H, H-1, J ¼ 7.0 Hz); 2.16 (m, 3H, H-3); 1.93 (d, 3H,

4894
C. Cano et al. / European Journal of Medicinal Chemistry 44 (2009) 4889–4895
411 (100%). Anal. calcd. for C23H42N2SO2: C 67.31, H 10.24, N 6.82,
S 7.80; found: C 67.24, H 10.35, N 6.88, S 8.02.
Dupont, Boston, MA, 40–60 Ci/mmol) as ligands. For competition
analysis, drugs were dissolved in 100% DMSO to a final concen-
tration of 10 nM. Further dilutions were made in assay buffer to
reach concentrations spanning between 10ꢀ5 and 10ꢀ12 M (final
concentration in tubes). Radioligand binding was initiated by the
4.3.2. N-(cis-9-cis-12-octadecadienyl)-N0-propylsulfamide (10)
From
N-(cis-9-cis-12-octadecadienyl)amine
2
(0.093 g,
0.35 mmol), N-propylsulfamoyl chloride (0.055 g, 0.35 mmol), TEA
(0.050 g, 0.5 mmol), toluene (30 mL); reaction time: 24 h; yield
(0.023 g of white solid, 17%). Mp ¼ 70–75 ꢂC; 1H NMR (400 MHz,
addition of 25 m
g of protein to tubes in triplicates containing [3H]-
SR141716A (0.4 nM) or [3H]-WIN-55212-2 (1 nM) and increasing
concentrations of tested compounds in 0.5 mL of buffer HEPES
20 mM pH 7.4 and MgCl2 1 mM plus free fatty acids BSA 0.5%. Tubes
were incubated at 30 ꢂC during 90 min. The reaction was stopped
by the addition of 2.5 mL of ice chilled buffer C, rapidly filtered in
a Millipore vacuum manifold through Whatman GF/C glass-fiber
filters previously soaked in assay buffer containing 5% BSA. The
tubes were washed twice with 2.5 mL of ice-cold buffer C and the
filters rinsed with 5 mL of the same buffer. Non-specific binding
CDCl3) d: 5.34 (m, 4H, CH]CH); 4.27 (br s, 1H, NH, (Pr)); 4.25 (br s,
1H, NH (linoleyl)); 3.01 (m, 4H, CH2NHSO2); 2.77 (m, 2H, C]CH–
CH2–CH]C); 2.05 (m, 4H, CH]CH–CH2CH2); 1.60 (m, 4H, CH2–
CH2NH); 1.29 (m, 16H, –CH2–); 0.95 (t, 3H, J ¼ 7.3 Hz, CH3 (Pr)); 0.87
(t, 3H, J ¼ 6.7 Hz, CH3 (linoleyl)). 13C NMR (100 MHz, CDCl3)
d: 130.2,
130.0, 128.0, 127.9 (CH]CH); 44.9 (SO2NHCH2 (Pr)); 43.2 (linoleyl–
CH2NH); 31.5 (CH2–CH2CH3); 29.1 (CH2–CH2NHSO2); 27.2
(CH]CH–CH2CH2); 29.6–26.6 (–CH2–); 25.6 (C]CH–CH2–CH]C);
22.8 (CH2–CH3 (Pr)); 22.5 (CH2–CH3 (linoleyl)); 14.0 (CH3 (lino-
leyl)); 11.2 (CH3 (Pr)). MS (ESþ) [M þ H]þ 387 (100%). Anal. calcd.
for C21H42N2SO2: C 65.28, H 10.88, N 7.25, S 8.29; found: C 65.30,
H 11.12, N 7.22, S 8.40.
was assessed in the presence of 10 mM cold SR141716A. The filters
were placed in vials containing liquid scintillation cocktail Ecolite
(ICN Biomedicals), incubated at room temperature overnight and
counted in a scintillation counter.
A single population of high-affinity binding sites was demon-
strated using LIGAND analysis for both radioligands. Ki for the
different drugs assayed were calculated from the equation of Cheng
and Prusoff [24], using fixed Kd values for either 3[H]WIN-55212-2
or 3[H]SR141716A obtained from independent experimental assays.
The different results (see Table 1) in terms of compound Ki for
displacing the different CB1 tritiated ligands are based on: (1) the
lower affinity of the cannabinoid agonist WIN-55212-2 with
respect to 3[H]SR141716A for the CB1 binding sites in rat cerebellar
membranes, (2) the potential different recognition sites for dis-
placing drugs.
4.4. In vitro assays
4.4.1. Agonistic activity
1) Contractile responses in isolated tissues
The functional activity of the new compounds was tested on
isolated tissues commonly used to study and characterize
cannabinoid effects: mouse vas deferens Male CD-1mice weighing
25–30 g were used for this study. The mouse vas deferens was
isolated as described by Hughes [23]. Tissues were suspended in
a 10 mL organ bath containing 5 mL of Krebs solution (NaCl 118;
KCl 4.75; CaCl2 2.54; KH2PO4 1.19; MgSO4 1.2; NaHCO3 25; glucose
11 mM). This solution was continuously gassed with 95% O2 and
CO2. Tissues were kept under 0.5 g of resting tension at 32 ꢂC and
were electrically stimulated through two platinum ring elec-
trodes. Mouse vas deferens were subjected to alternate periods of
stimulation (5 min) and rest (10 min), during the stimulation
period strains of 15 rectangular pulses of 70 V, 15 Hz and 2 ms
duration for each min were applied. The isometric force was
monitored by computer using a MacLab data recording and
analysis systems.
4.5. FAAH assay
Brain tissues were homogenized in 50 mM Tris buffer, pH 8,
containing 0.32 M sucrose. Homogenates were centrifuged first at
1000g (5 min), the pellet discarded and the supernatant centri-
fuged at 45,000g (30 min). The pellets obtained were solubilized at
0–4 ꢂC in Tris buffer. Protein content in the membrane fraction was
measured with the Bradford method [25]. All tissue samples and
membrane fractions were stored at ꢀ70 ꢂC until used. Both enzy-
matic assays were run under conditions that were linear with time
and protein concentrations. We assayed membrane-bound FAAH
activity using arachidonyl-[1-3H]-ethanolamide as a substrate, and
measuring metabolized [3H]anandamide (as [3H]ethanolamine) in
the aqueous phase after chloroform extraction, as described [26].
Concentration–response curves for reference cannabinoid
receptor agonists or the new compounds were constructed in
a
step-by-step manner. The interval between application of
increasing concentrations was 15 min curves were constructed for
the synthetic agonist WIN 55,212-2 (10ꢀ10 to 5 ꢃ10ꢀ8 M), the
natural agonist anandamide and the new compounds (10ꢀ8 to
10ꢀ6 mM) in mouse vas deferens.
The effect of the drugs was evaluated 15 min after the addition
of each dose, as % of inhibition, taking the amplitude of the last
contraction before the first addition of agonist as 100%. The agonists
were added to the organ bath 15 min after the beginning of elec-
trical stimulation. Each tissue was employed to construct only one
concentration–response curve. In mice vas deferens the electrical
stimulation was stopped, Krebs solution was replaced before the
addition of each dose of the agonist and then, 10 min later, tissues
were again stimulated during 5 min.
Acknowledgments
Financial support from Spanish MCYT (SAF 2003-08003, SAF
2006-13391) FIS and Fundacion Eugenio Rodrıguez Pascual is
gratefully acknowledged.
´
´
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