The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.01.007

A series of para-substituted phenolic N-alkyl carbamates were evaluated for their FAAH and MGL inhibitory activities. The compounds were generally selective for FAAH, with IC₅₀ values in the nM range, whereas inhibition of MGL required concentrations three orders of magnitude higher. The most potent compounds, dodecylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl (12) and 4-(1,2,3-thiadiazol-4-yl)phenyl (26) esters, inhibited FAAH and MGL with IC₅₀ values at the low-nanomolar (IC₅₀s; 0.0063 and 0.012 μM) and the low-micromolar ranges (IC₅₀s; 2.1 and 1.0 μM), respectively. Compound 26 also inhibited both FAAH-dependent AEA uptake and AEA hydrolysis (IC₅₀; 0.082 μM) by intact RBL2H3 cells, and could also reduce 2-AG hydrolysis by these cells at concentrations ≥0.030 μM.

Full text of DOI:10.1016/j.ejmech.2009.01.007

European Journal of Medicinal Chemistry 44 (2009) 2994–3008
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis and biological evaluation of para-substituted phenolic N-alkyl
carbamates as endocannabinoid hydrolyzing enzyme inhibitors
,
b
a
b
Anna Minkkila¨ a , Mikko J. Myllyma¨ki , Susanna M. Saario , Joel A. Castillo-Melendez ,
*
Ari M.P. Koskinen b, Christopher J. Fowlerc, Jukka Leppa¨nen a, Tapio Nevalainen a
a University of Kuopio, Department of Pharmaceutical Chemistry, P.O. Box 1627, FI-70211 Kuopio, Finland
b Department of Chemistry, Helsinki University of Technology, P.O. Box 6100, FI-02015 TKK, Finland
c Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-90187 Umeå, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of para-substituted phenolic N-alkyl carbamates were evaluated for their FAAH and MGL
inhibitory activities. The compounds were generally selective for FAAH, with IC50 values in the nM range,
whereas inhibition of MGL required concentrations three orders of magnitude higher. The most potent
compounds, dodecylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl (12) and 4-(1,2,3-thiadiazol-4-
yl)phenyl (26) esters, inhibited FAAH and MGL with IC50 values at the low-nanomolar (IC50s; 0.0063 and
Received 21 August 2008
Received in revised form
23 December 2008
Accepted 9 January 2009
Available online 20 January 2009
0.012
m
M) and the low-micromolar ranges (IC50s; 2.1 and 1.0
m
M), respectively. Compound 26 also
M) by intact RBL2H3 cells,
M.
inhibited both FAAH-dependent AEA uptake and AEA hydrolysis (IC50; 0.082
and could also reduce 2-AG hydrolysis by these cells at concentrations ꢀ0.030
m
Keywords:
Endocannabinoid
N-Arachidonoylethanolamine
2-Arachidonoylglycerol
Fatty acid amide hydrolase
Monoglyceride lipase
m
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
selectivity, and it has been well characterized both pharmacologi-
cally and biochemically. Therefore, several FAAH inhibitors have
been developed (see for review Ref. [12]), including various fatty
The endocannabinoids constitute an important class of
endogenous lipids that serve as chemical messengers both in the
brain and the periphery. The two main endocannabinoids,
N-arachidonoylethanolamine (anandamide, AEA, 1) [1] and
2-arachidonoylglycerol (2-AG, 2) [2,3], bind to and activate both
cannabinoid receptors, CB1 [4,5] and CB2 [6], that bind also exog-
acid derivatives [13–17], and non-lipid inhibitors such as
a-keto
heterocycles [18–20], carbamate derivatives [21–25], (thio)-
hydantoins [26] and most recently, selective piperidine/piperazine
ureas [27,28]. Probably, the most well-studied FAAH inhibitors are
the carbamate-based 30-carbamoylbiphenyl-3-yl ester (4, URB597)
enous cannabinoids like
D
9-tetrahydrocannabinol
(D
9-THC, 3)
(Fig. 2) [21,29] and the a-keto heterocycle-based 7-phenyl-1-(5-
(Fig. 1). The activation of CB1 and CB2 receptors by cannabinoids
induces several beneficial therapeutic effects such as pain relief and
reduction of inflammation [7]. However, the cannabinergic effects
of AEA and 2-AG remain weak in vivo owing to their rapid inacti-
vation by the hydrolytic enzymes fatty acid amide hydrolase (FAAH,
FAAH-1) [8] and monoglyceride lipase (MGL) [9], respectively.
FAAH is the main metabolizing enzyme responsible for the AEA
degradation. AEA is also hydrolyzed by other enzymes such as
FAAH-2, a second FAAH isoform [10], and N-acylethanolamine-
hydrolyzing acid amidase (NAAA) [11]. FAAH has a broad substrate
(pyridin-2-yl)oxazol-2-yl)heptan-1-one (OL-135) [19], which have
gained considerable promise for treating anxiety, inflammation,
and pain [7,19,29–31].
Besides being the main metabolizing enzyme for AEA, FAAH is
also able to hydrolyze 2-AG [32]. However, the main enzyme
responsible for the inactivation of 2-AG in the brain has been
suggested to be MGL (EC 3.1.1.23) [9,33,37], although other hydro-
lytic activities may also contribute to the metabolism [34–37]. MGL
is a serine hydrolase, which hydrolyzes medium- and long-chain
fatty acid esters such as 2-AG and its analogue 2-oleoylglycerol
(2-OG) [9,38].
In contrast to FAAH, there is neither a 3D structure available for
MGL nor the exact information of amino acid residues in the
vicinity of the active site of the enzyme. Therefore, the design of
potent and selective MGL inhibitors has remained a challenging
Abbreviations: AEA, N-arachidonoylethanolamine, anandamide; 2-AG, 2-arach-
idonoylglycerol; FAAH, fatty acid amide hydrolase; MGL, monoglyceride lipase.
* Corresponding author. Tel.: þ358 40 355 3715; fax: þ358 017 162 252.
E-mail address: anna.minkkila@uku.fi (A. Minkkila¨).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.007

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
2995
Fig. 1. Structures of N-arachidonoylethanolamine (AEA, 1), 2-arachidonoylglycerol (2-
AG, 2), and
9-tetrahydrocannabinol ( 9-THC, 3).
D
D
Fig. 3. The lead structures SPB01403 (7) and SEW01169 (8).
task so far. However, very recently, during revision of this manu-
script, Long et al. published a potent and selective MGL inhibitor
called JZL184, which was shown to raise brain 2-AG in mice by
eight-fold without altering AEA [39]. The other known potent
inhibitors of this enzyme are compounds like hexadecylsulfonyl-
fluoride (HDSF), methyl arachidonylfluorophosphonate (MAFP),
and diisopropyl fluorophosphate [9,34,38] that are highly
unselective and not useful for in vivo studies. The same is true
for bis(dimethylthio)mercury, the contaminant responsible for
the MGL inhibitory effects originally ascribed to 6-methyl-2-
FAAH (IC50; 0.52 mM) and MGL (IC50; 30 mM) [50]. This observation
raises the possibility that O-phenyl carbamate-based compounds
can be optimized to give compounds with potent effects toward
both FAAH and MGL, and that these compounds can be used in vivo
to investigate the therapeutic potential of a concomitant blockade
of hydrolysis of both AEA and 2-AG. Consequently, the structurally
similar compound, SEW01169 (8) (Fig. 3), was purchased and tested
for its FAAH and MGL inhibitory activity, and it was found to be
slightly more potent FAAH inhibitor (IC50; 0.16
less potent inhibitor against 2-AG hydrolysis with remaining MGL
activity 54% at 100 M compound concentration in rat cerebellar
membranes. Compound was also tested against human
recombinant MGL (hrMGL), and it was found to inhibit 2-OG
hydrolysis with an IC50 value of 43 M. Herein, we present the
mM) than 7, albeit
[(4-methylphenyl)amino]-4H-3,1-benzoxazin-4-one
(URB754)
[40]. Additionally, the N-biphenyl-3-cyclohexylcarbamic acid
cyclohexyl ester (5, URB602) (Fig. 2) was originally suggested to be
a selective MGL inhibitor [41], although its selectivity data has
recently been questioned [42,43]. Sulfhydryl-reactive compounds
such as p-chloromercuribenzoic acid, N-ethylmaleimide (NEM)
[9,44,45] and disulfiram [46] inhibit MGL activity, indicating the
presence of an essential sulfhydryl group at the active site of the
enzyme. In a series of sulfhydryl-specific maleimide derivatives,
N-arachidonylmaleimide (NAM, 6) (Fig. 2) was found to be a potent
inhibitor of MGL, inhibiting 2-AG hydrolysis in rat cerebellar
membranes with an IC50 value of 140 nM [45], a result confirmed
using human recombinant MGL (hrMGL), and 2-OG as substrate
[46]. Despite containing a highly reactive maleimide group, NAM
has been shown to exhibit rather high selectivity for MGL relative to
other brain serine hydrolases [37]. Other approaches have also
yielded compounds that inhibit MGL, but with potencies in the
micromolar range [47]. For current knowledge of MGL and the
inhibitors developed so far, see reviews [12,48,49].
m
8
m
synthesis and biological evaluation of a series of phenolic N-alkyl
carbamates derived from 7 and 8, and their structure–activity
relationships (SAR) for the inhibition of endocannabinoid
hydrolyzing enzymes, FAAH and MGL.
2. Chemistry
The synthesis of compounds is outlined in Schemes 1 and 2. The
majority of the carbamates were obtained by treating various
phenols with suitable alkyl isocyanates in the presence of trie-
thylamine; the O-phenyl carbamates 9–29, 30 [51], 31–33, 35
[53,54], 37–39 [55], 40 [52,55], 41, 42 [53,56], 43 [57], 44, 46, and
48–51 were prepared from the corresponding phenols, and in the
case of N-phenyl carbamate 52, from cyclohexanol by a route
described in Schemes 1 and 2. On the contrary, the O-phenyl
carbamates 34 [52,55,58–60] and 36 [61] were prepared from
4-nitrophenyl chloroformate and the appropriate amine, yielding
the desired products in 32–99% yield, with shorter reaction time
than in the isocyanate method. The O-phenyl N-n-hexylcarbamates
45 and 47 were prepared from the corresponding methyl benzoates
(Scheme 2). 4-(4,5-Dihydrothiazol-2-yl)phenol 54 [62] for the
preparation of the carbamates 9–20 was prepared by simply
heating the mixture of cysteamine and 4-cyanophenol (Scheme 1).
Unfortunately this method was not suitable for the preparation of
4-(4,5-dihydrooxazol-2-yl)phenol 56 for the synthesis of the
carbamates 21–24, thus it was obtained by the reaction described
by Witte and Seeliger [63] (Scheme 1). For this purpose the
hydroxyl group of 4-cyanophenol was protected as the benzyl ether
(55) [64]. (1,2,3-Thiadiazol-4-yl)phenols 58a–c [65,66] for the
preparation of the carbamates 25–29 and 48–51 [67] were obtained
from phenyl ketones by the Hurd–Mori reaction via hydrazones
57a–c [65,66] (Scheme 1). The same method was applied in the
preparation of 4-(1,2,3-thiadiazol-4-yl)aniline hydrochloride (58d)
for the synthesis of the urea compound 53 (Scheme 1).
The carbamate compound SPB01403 (7) (Fig. 3), found in virtual
screening as reported in our previous study, could inhibit both
Fig. 2. Structures of URB597 (4), URB602 (5), and N-arachidonylmaleimide (NAM, 6).

2996
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
Scheme 1. Synthesis of compounds 9–29, 48–51 and 53–58a–d. Reagents and conditions: (a) 2-aminoethanethiol, 100 ꢁC, 88%; (b) i) BnBr, K2CO3, KI, acetone, reflux, 98%, ii) 2-
aminoethanol, PhCl, reflux, 50%; (c) H2, Pd/C, ethanol, rt, 75%; (d) ethyl carbazate, p-TsOH, toluene/ethanol, Dean–Stark, 69–100%; (e) SOCl2, rt, 43–85%; (f) RNCO, Et3N, toluene, rt-
reflux, 20–100%; wherein R ¼ Et, n-Pr, n-Hex, dodecyl, t-Bu, c-pentyl, c-Hex, Bn, 2-MeBn, 4-MeBn, 4-MeOBn, phenethyl.
3. Biological testing
selective MGL inhibitor URB602 (5), were evaluated. The results of
reference compounds showed good correlation with the ones
reported in the literature [29,42,43].
The inhibitory potencies against AEA hydrolysis (by FAAH) of all
the prepared compounds 9–53, and the leads 7 and 8 were deter-
mined in rat brain homogenate [50]. The compounds 7–10, 13–26,
28, 31–33, 36, 42, 43, 49, 51, 52, and 53 were tested for their ability
to inhibit 2-AG hydrolysis (by MGL) in rat cerebellar membranes
[34], while the MGL inhibitory potencies of the compounds 8, 11, 12,
26, 27, 29–31, 34, 35, 37–41, 44–48, 50, and 51 were evaluated in
hrMGL (Cayman Chemical), using tritium-labelled oleoylglycerol as
substrate. In order to demonstrate the comparability of the two
MGL methods used, the compounds 8, 26, 31 and 51 were tested in
both assays. Moreover, inhibition of FAAH and MGL by reference
compounds, FAAH inhibitor URB597 (4) and previously reported
4. Results and discussion
4.1. Structural optimization of the lead SPB01403 (7)
A series of N-alkyl derivatives of the lead SPB01403 (7) with
dihydrothiazoline (9–20, Table 1) or dihydrooxazoline moiety (21–
24, Table 2) at para-position in the phenyl ring were prepared and
tested for their FAAH and MGL inhibitory activities.
Among the dihydrothiazoline moiety containing open chain
N-alkyl carbamates (7, 9–12) the chain length was the main factor
Scheme 2. Synthesis of compounds 30–47 and 52. Reagents and conditions: (a) RNCO, Et3N, toluene, rt-reflux, 50–100%; wherein R ¼ n-Bu, n-Hex, c-Hex, dodecyl; (b) RNH2,
pyridine, CH2Cl2, reflux, 32–99%; wherein R ¼ n-Bu, c-Hex; (c) methanol, H2SO4, reflux, 100%.

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
2997
Table 1
The differences in the FAAH and MGL inhibition potencies among
the other dihydrothiazoline derivatives with different N-alkyl
groups (14–20) were mainly not notable. However, with cycloalkyl
and benzylic groups (14–19) complete MGL inhibition was not
observed, even at the highest concentrations tested (1 mM). This
remaining enzyme activity can be explained by an additional
enzyme activity distinct from MGL or by low aqueous solubility of
these compounds. An interesting observation concerning the FAAH
IC50 values for the inhibition of MGL and FAAH by dihydrothiazoline derivatives 7,
and 9–20.
H
O
N
R
O
S
N
inhibition was that the N-2-methylbenzyl derivative (17) (IC50
0.062 M) was more potent FAAH inhibitor than the other deriva-
tives with cyclic or benzylic N-alkyl moiety (14–16, 18, 19) (IC50s;
0.10–0.32 M).
;
Compound
R
IC50 (95% CI)a
m
FAAH [ M]
m
MGL [mM]
7
9
n-Bu
Et
n-Pr
0.52 (0.39–0.70)b
15 (10–22)
31 (25–39)b
102 (80–131)c
46 (39–56)d
8.4 (6.8–10)e
2.1 (1.7–2.6)e
No inhibition at 100
11 (9–12)d
m
With respect to dihydrooxazoline derivatives 21–24 (Table 2),
no clear difference in the inhibition potencies toward either FAAH
or MGL was seen when comparing the results to those of the cor-
responding dihydrothiazoline derivatives 7, 14, 17, and 18. However,
like in the series of dihydrothiazoline substituted N-cycloalkyl or
10
11
12
13
14
15f
16
17
18
19
20
1.7 (1.3–2.3)
n-Hex
Dodecyl
t-Bu
c-Pentyl
c-Hex
Bn
2-MeBn
4-MeBn
4-MeOBn
CH2Ph
0.0082 (0.0068–0.0099)
0.0063 (0.0045–0.0087)
7.0 (5.0–10.0)
0.13 (0.11–0.15)
0.10 (0.095–0.10)
0.29 (0.25–0.33)
0.062 (0.056–0.060)
0.28 (0.12–0.40)
0.32 (0.25–0.34)
m
M
27 (21–35)c
42 (35–49)c
46 (30–52)c
94 (85–105)c
46 (38–55)g
28 (25–31)
N-benzylic derivatives, the N-2-methylbenzyl derivative (23) (IC50
0.034 M) displayed better inhibitory potency against FAAH than
the other N-alkyl derivatives in the dihydrooxazoline series (IC50s;
0.10–0.18 M).
;
m
m
0.038 (0.035–0.042)
a
IC50 values represent the mean from three independent experiments performed
in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity
was completely abolished at the highest tested concentration unless otherwise
stated.
4.2. Structural optimization of the lead SEW01169 (8)
After establishing the optimal N-alkyl groups for the inhibition
of FAAH and MGL within the dihydrothiazol-2-yl and dihydroox-
azol-2-yl substituted O-phenyl carbamates, a series of SEW01169
(8) derivatives with various and selected N-alkyl groups were
prepared (Table 3).
Not surprisingly, in this series of 1,2,3-thiadiazol-4-yl
substituted derivatives (8, 25–29), the length and the shape of the
N-alkyl group had a similar effect on the inhibitory potencies
against both enzymes, FAAH and MGL, as it had in the previous
series. Thus, by replacing the N-n-butyl chain of 8 by N-n-hexyl (25)
the inhibitory potencies toward FAAH and MGL were improved
b
See Ref. [50].
Remaining enzyme activity was 12–19% at 1 mM.
Remaining enzyme activity was 6–7% at 1 mM.
c
d
e
Inhibition of hrMGL.
f
Compound contained 5% of unidentified impurity by 1H NMR.
g
Remaining enzyme activity was 21–26% at 1 mM.
affecting the inhibition potencies toward both enzymes; with short
N-ethyl group (9) the IC50 values against FAAH was 15
with longer N-n-hexyl (11) and N-dodecyl (12) groups the FAAH
IC50 values were 0.0082 M and 0.0063 M, respectively. Moreover,
compounds 11 and 12 inhibited hrMGL mediated 2-OG hydrolysis
with IC50 values of 8.4 M and 2.1 M, respectively.
mM, whereas
m
m
(IC50s; 0.019 and 11
potent inhibitor in this series with N-dodecyl moiety (26) inhibited
FAAH with an IC50 value of 0.012 M. Moreover, hydrolysis of 2-AG
mM, respectively). Furthermore, the most
m
m
m
Replacement of the open chain N-alkyl groups with bulky
N-tert-butyl group (13) reduced the inhibitory potencies toward
in rat brain membranes as well as hrMGL mediated 2-OG hydrolysis
FAAH and MGL (IC50s; 7.0 mM and >100 mM, respectively). This is
probably due to steric hindrance near the carbamate group, thus
reducing crucial interactions within the active sites of the enzymes.
Table 3
IC50 values for the inhibition of MGL and FAAH by 1,2,3-thiadiazoline derivatives 8,
and 25–29.
H
O
N
R
Table 2
IC50 values for the inhibition of MGL and FAAH by dihydrooxazoline derivatives 21–
24.
O
N
N
H
O
N
S
R
Compound
R
IC50 (95% CI)a or percentage of inhibitionb
FAAH [ M] MGL [ M]
O
O
m
m
8
n-Bu
0.16 (0.13–0.20)
46% at 100 mM
43 (27–66)c,d
11 (9.8–13)
N
Compound
R
IC50 (95% CI)a
FAAH [ M]
25
26
n-Hex
Dodecyl
0.019 (0.015–0.024)
0.012 (0.011–0.014)
1.9 (1.7–2.1)
1.0 (0.8–1.4)c
m
MGL [mM]
21
22
23
24
n-Bu
0.12 (0.11–0.13)
0.10 (0.082–0.11)
0.034 (0.031–0.038)
0.18 (0.14–0.24)
34 (27–42)
11 (9–14)b
27
28
29
c-Pentyl
c-Hex
Bn
0.020 (0.014–0.029)
0.021 (0.018–0.025)
0.044 (0.034–0.056)
44% at 100
28% at 100
m
m
Mc
M
c-Pentyl
2-MeBn
4-MeBn
35 (32–38)c
125 (111–141)d
24 (15–39)c,d
a
IC50 values represent the mean from three independent experiments performed
in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity
was completely abolished at the highest tested concentration unless otherwise
stated.
a
IC50 values represent the mean from three independent experiments performed
in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity
was completely abolished at the highest tested concentration unless otherwise
stated.
b
The percentage of inhibition is represented as the mean from two independent
b
Remaining enzyme activity was 21–26% at 1 mM.
Remaining enzyme activity at 1 mM was 12–19%.
Remaining enzyme activity was 6–7% at 1 mM.
experiments performed in duplicates.
c
c
Inhibition of hrMGL.
Remaining enzyme activity 22–26% at 1 mM.
d
d

2998
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
were inhibited by 26 with IC50 values of 1.9
m
M and 1.0
m
M,
inhibitory potencies against FAAH were hardly notable when
comparing the results of EWG-substituted N-n-butyl derivatives 30,
34, and 37 with that of corresponding methoxy-substituted 40. On
the contrary, within N-n-hexyl derivatives EWG-substituted 31, 35,
38, and 39 were more potent than methyl-substituted 41.
As observed in the series of the derivatives of 7 and 8, the FAAH
and MGL inhibitory potencies were enhanced by increasing the
length of the N-alkyl group. The same was true in this series; for
example, the N-dodecyl derivative with electron-withdrawing
methoxy carbonyl group (33) was found to be as potent toward
respectively. When comparing these results to those observed with
the corresponding dihydrothiazoline derivative 12, we can
conclude that the heterocyclic moiety in these two compounds has
a minor effect on their FAAH and MGL inhibitory capacities.
However, the compounds with N-cyclopentyl (27), N-cyclohexyl
(28) and N-benzyl (29) moieties were more potent FAAH inhibitors
than the corresponding dihydrothiazoline derivatives 14–16, indi-
cating that FAAH inhibitory activity could be affected by the type of
heterocycle. This effect was not seen for MGL.
FAAH and MGL (IC50s; 0.025
26. Moreover, to our surprise, the nonsubstituted compounds 42
and 43 were quite potent FAAH inhibitors (IC50s; 0.091–0.092 M)
mM and 2.8 mM, respectively) as 12 and
4.3. The effect of para-substituents with different electronic
properties
m
in comparison with the EWG-substituted 31, 33, 35, 38 and 39,
while Tarzia et al. have reported that phenylcarbamic acid cyclo-
hexyl ester lacks inhibitory activity against FAAH [21]. This
observed increase in FAAH inhibition potencies might be due to
more flexible and lipophilic nature of N-n-hexyl and N-dodecyl
groups. The similar observation can be seen when comparing the
results of N-cyclohexyl (32, 36) and/or N-n-butyl derivatives (30,
34, 37, and 40) to those of other N-n-hexyl (31, 35, 38, 39, and 41)
and N-dodecyl derivatives (33).
Altogether, these results indicate that FAAH inhibition potencies
might be more dependent on the N-alkyl moiety than the electronic
properties of the para-substituent, while MGL inhibition
potencies can be enhanced by EWG at para-position in the phenyl
ring as well.
The catalytic site serine –OH of FAAH [68] and MGL is expected to
have a strong interaction with the reactive carbonyls of the ligands.
Therefore, we decided to study whether the electronic properties of
the para-substituent in O-phenyl carbamates have an effect on the
FAAH and MGL inhibitory activities. The presumption that electron-
withdrawing groups in para-position on the phenyl ring would
increase the electrophilicity of the carbonyl carbon prompted us to
prepare a series of N-alkyl carbamates with various electron-with-
drawing and electron-donating substituents (Table 4). Methoxy
carbonyl (30–33), nitro (34–36), cyano (37 and 38), and bromine
(39) were selected as electron-withdrawing (EWG), and methoxy
(40) and methyl (41) as electron-donating groups (EDG). The neutral
effect was evaluated with the unsubstituted O-phenyl carbamates
42 and 43.
As evident from the results presented in Table 4, the EWG and
EDG had opposite effects on the inhibitory potencies against MGL.
EWG (methoxycarbonyl, nitro, and cyano) substituted compounds
30–39 maintained the potencies against MGL similar to those of the
corresponding N-alkyl derivatives presented in Tables 1–3, whereas
EDG (methoxy and methyl) substituted compounds 40 and 41 had
clearly diminished inhibition potencies. However, differences in
Table 5
Effect of the ortho-substituent in the phenyl ring on MGL and FAAH inhibition; IC50
values for the inhibition of MGL and FAAH by compounds 44–47.
Compound Structure
IC50 (95% CI)a or percentage of
inhibitionb
FAAH [
mM]
MGL [mM]
O
H
N
Table 4
O
O
n-Hex
n-Hex
IC50 values for the inhibition of MGL and FAAH by various EWG- and EDG-
substituted O-phenyl carbamates 30–43.
0.0055
(0.0049–0.0062)
44
4.2 (2.7–6.2)c
O
O
O
H
O
O
N
R
O
H
N
X
Compound
X
R
IC50 (95% CI)a or percentage of inhibitionb
FAAH [ M] MGL [ M]
35% at 0.1
0.055
(0.038–0.079)
22 (18–27)c
O
45
m
m
30
31
CO2Me n-Bu
CO2Me n-Hex
mM
19 (12–29)c
35 (30–41.5)
19 (16–24)c
14 (12–18)
O
O
0.031 (0.023–0.041)
32
33
34
35
36
37
38
39
40
41
42
43
CO2Me c-Hex
CO2Me Dodecyl 0.025 (0.022–0.030) 2.8 (2.5–3.2)
41% at 0.1
mM
I
H
N
O
NO2
NO2
NO2
CN
CN
Br
OMe
Me
H
n-Bu
16% at 0.1
0.027 (0.021–0.035)
m
M
24 (18–33)c
n-Hex
n-Hex
c-Hex
n-Bu
n-Hex
n-Hex
n-Bu
18 (12–27)c
0.024
(0.021–0.025)
14 (11–16)c
O
O
O
46
47
25% at 0.1
25% at 0.1
0.013 (0.012–0.014)
0.018 (0.016–0.020)
m
m
M
M
19 (17–21)
30 (20–45)c
12 (9.0–17)c
17 (11–26)c,d
No inhibition at 100
No inhibition at 100
5% at 0.1
27% at 0.1
0.091 (0.075–0.110)
m
M
mM
m
m
Mc
Mc
t-Bu
n-Hex
n-Hex
H
41% at 100
mM
18% at 100 m
Mc
O
N
4% at 0.1
m
M
H
Dodecyl 0.092 (0.076–0.112)
36% at 100 mM
n-Hex
a
O
IC50 values represent the mean from three independent experiments performed
in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity
was completely abolished at the highest tested concentration unless otherwise
stated.
t-Bu
IC50 values represent the mean from three independent experiments performed
O
a
in duplicatewith 95% confidence intervals shown inparenthesis. Enzyme activity was
completely abolished at the highest tested concentration unless otherwise stated.
The percentage of inhibition is represented as the mean from two independent
experiments performed in duplicates.
b
The percentage of inhibition is represented as the mean from two independent
b
experiments performed in duplicates.
c
Inhibition of hrMGL.
Remaining enzyme activity was 16% at 1 mM.
d
c
Inhibition of hrMGL.

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
2999
In addition, a few derivatives of 31 with EDG (methoxy, 44;
methyl, 45; tert-butyl, 47) or EWG (iodine, 46) at the ortho-position
in the phenyl ring were prepared to study the effects of steric and
electronic properties of ortho-substituents to MGL and FAAH
inhibitory activity. As evident from Table 5, the ortho-methoxy
substituent (44) improved the inhibitory potency toward both
FAAH and hrMGL when compared to the results obtained with the
compound 31 without any ortho-substituent. This might be due to
favourable interactions between the oxygen atom in the methoxy
group and the active site residues of the enzymes. Me- or
I-substitution (45 and 46) did not have a significant effect on the
affinity toward either FAAH or MGL. However, a bulky tert-butyl
substituent (47) diminished the affinity toward these enzymes
notably, which could be due to steric hindrance.
MGL inhibitory activities) [22], meta- and ortho-analogues of 8 and
its most potent derivative with N-dodecyl group (26) were
prepared and tested (Table 6).
In our previous studies, meta-substituted carbamate URB597 (4)
was found to inhibit AEA hydrolysis with an IC50 value of 0.0038
[50], which is comparable to the previously reported IC50 value of
0.0046 M [29], and only 30% inhibition of 2-AG hydrolysis in rat
mM
m
brain membranes was evident at 1 mM [34].
Compound 48, the ortho-substituted analogue of 8, did not
inhibit either of the enzymes; only 27% inhibition of MGL was
observed at 100
0.1 M. On the contrary, the meta-substituted analogue 49 inhibi-
ted FAAH with an IC50 value of 0.0069 M. However, only 13%
inhibition of MGL by 49 was observed at 100 M concentration,
mM, and no inhibition of FAAH could be detected at
m
m
m
which is similar to the results obtained with the ortho-substituted
48 and the para-substituted 8.
4.4. Effect of the meta- and ortho-substitution in the phenyl ring
The meta-substituted compound 51 with a more lipophilic
N-dodecyl group inhibited MGL with an IC50 value of 8.0
slightly less potent than the para-substituted analogue 26 (IC50
1.0 M). Moreover, 51 inhibited FAAH with an IC50 value of
mM, being
To support our previous results of the influence of the
substituent’s position in the phenyl ring on the FAAH and MGL
inhibitory activities (i.e. para-substitution is more favourable to the
;
m
Table 6
Comparison of the meta- and ortho-substitution in the phenyl ring on MGL and FAAH inhibition; IC50 values for the inhibition of MGL and FAAH by 4 (URB597), and compounds
48–51.
Compound
Structure
IC50 (95% CI)a or percentage of inhibitionb
FAAH [ M]
m
MGL [mM]
O
NH2
H
N
4
0.0038 (0.0029–0.0050)c
30% at 1 mMd
O
c-Hex
O
S
N
N
N
H
O
N
48
No inhibition at 0.1
m
M
27% at 100 m
Me
n-Bu
O
N
H
N
S
O
n-Bu
49
50
0.0069 (0.0054–0.0088)
13% at 100 mM
O
S
N
N
H
N
O
0.050 (0.042–0.058)
18 (12–26)e,f
dodecyl
O
57% at 30
8.0 (5.9–11.0)e,h
m
Mg
N
N
H
N
S
O
51
0.00024 (0.00020–0.00030)
dodecyl
O
a
IC50 values represent the mean from three independent experiments performed in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity was
completely abolished at the highest tested concentration unless otherwise stated.
b
The percentage of inhibition is represented as the mean from two independent experiments performed in duplicate.
See Ref. [50].
See Ref. [34].
Inhibition of hrMGL.
c
d
e
f
Remaining enzyme activity was 26% at 1 mM.
g
Compound started to precipitate at concentrations over 30
Remaining enzyme activity was 13% at 1 mM.
mM.
h

3000
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
Table 7
Effect of the O-phenyl carbamate moiety on MGL and FAAH inhibition; IC50 values
for the inhibition of MGL and FAAH by the reference compound 5 (URB602), and
compounds 52 and 53.
Compound Structure
IC50 (95% CI)a or percentage of
inhibitionb
FAAH [
m
M]
MGL [mM]
H
N
O
O
5
108 (86–136) 30% at 100 m
Mc
Fig. 5. Effect of 26 upon the uptake of 0.10 m
M [3H]AEA by either RBL2H3 cells in the
absence or presence of URB597 (4) or by wells alone. Data are means and s.e.m., n ¼ 4.
*p < 0.05 vs corresponding control, Dunnett’s multiple comparison test following
significant one-way ANOVA for repeated measures.
H
N
O
No inhibition
at 100
52
9% at 0.1
9% at 0.1
m
m
M
M
O
m
M
O2N
has been reported to be a selective inhibitor of MGL [41], although
in our previous study it was shown to be totally inactive against
2-AG hydrolysis in rat brain membranes at 1 mM [42]. Moreover, in
H
H
N
N
n-Hex
No inhibition
at 100
53
O
mM
N
the present study 30% inhibition of hrMGL was seen at 100
URB602 has also been reported to inhibit AEA hydrolysis by rat
brain membranes (IC50; 17 M) [43]. In the present study URB602
inhibited FAAH with an IC50 value of 108 M. Consequently,
mM.
N
S
m
a
IC50 values represent the mean from three independent experiments performed
in duplicate with 95% confidence intervals shown in parenthesis. Enzyme activity
was completely abolished at the highest tested concentration unless otherwise
stated.
The percentage of inhibition is represented as the mean from two independent
experiments performed in duplicate.
m
compounds 52 and 53 lacking O-phenyl carbamate structure
showed no inhibition activity toward MGL, and only 9% inhibition
of FAAH at 0.1 mM was observed with these compounds. These
results establish that the O-phenyl carbamate structure in the
compounds investigated in the present study is essential for the
inhibitory activities toward both enzymes, FAAH and MGL. The
results are presented in Table 7.
b
c
Inhibition of hrMGL.
0.00024 mM, being more potent against FAAH than 26. The ortho-
substituted compound 50 with N-dodecyl group inhibited both
FAAH and MGL, although with slightly higher IC50 values than
para- and meta-substituted analogues 26 and 51.
4.6. Reversibility
These data support the previous findings that para-substitution
is more favourable to MGL inhibition, whereas meta-substitution is
favourable for FAAH [22].
Compound 26 was investigated in more detail with respect to
reversibility of FAAH and MGL inhibition. Reversibility was deter-
mined by measuring the recovery of FAAH and MGL enzymatic
activity after a rapid and large dilution (500-fold) of the enzyme–
inhibitor complexes.
4.5. The effectiveness of O-phenyl carbamate structure
on MGL and FAAH inhibition
In the reversibility studies the compound 26 was found to
inhibit both FAAH and hrMGL in an irreversible manner (Fig. 4a and
b). Inhibition of FAAH by URB597 (4) was shown to be irreversible
and by 1-(oxazolo[4,5-b]pyridin-2-yl)-1-oxo-dodecane (CAY10435)
[18] inhibition was reversible (Fig. 4a), as has been described in the
To investigate the effectiveness of the O-phenyl carbamate
structure on MGL and FAAH inhibition, two N-phenyl carbamates
(URB602 (5) and 52) and a urea compound 53 were tested. URB602
Fig. 4. (A) Reversibility of FAAH inactivation by 26 (240 nM), URB597 (76 nM) and CAY10435 (2.8 nM); (B) reversibility of hrMGL inactivation by 26 (36 mM) and MAFP (0.3 mM).
Enzyme activity was measured at the indicated time-points after 500-fold dilution of the enzyme-inhibitor complex. For a fully reversible compound, the dilution should result in
d.p.m. values approaching those seen with vehicle (DMSO) alone. The data represent the means and s.e.m. from three independent experiments performed in duplicate.

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
3001
Fig. 6. Effect of 26 upon the hydrolysis of 0.10
m
M [3H]AEA and [3H]2-AG by RBL2H3 cells. Data are means and s.e.m., n ¼ 5 (AEA) or n ¼ 4 (2-AG). *p < 0.05 vs corresponding control,
Dunnett’s multiple comparison test following significant one-way ANOVA for repeated measures. Expressing the data as % of control for AEA gave values of 92 ꢂ 7, 70 ꢂ 8, 55 ꢂ 6 and
15 ꢂ 7 for 0.001, 0.01, 0.1 and 1
mM 26, respectively (means ꢂ s.e.m.). These values give an IC50 value of 0.082
mM (constraining ‘‘top’’ and ‘‘bottom’’ values of the analysis to 100 and
0%, respectively).
literature [18,68]. Inhibition of hrMGL by MAFP was also shown to
be irreversible (Fig. 4b).
further design of compounds with more potent inhibitory activities
against MGL.
6. Experimental protocols
4.7. The effects in intact cells
6.1. Chemistry
Compound 26 was investigated to its effects in intact cells. The
effects of a compound upon FAAH activity in intact cells can be
determined by measuring either the URB597 sensitive uptake of
AEA or by following its hydrolysis [69]. In these assays 26 was found
to be efficacious, and the IC50 value for the inhibition of the
Commercially available starting materials were used without
further purification. SEW01169 and SPB01403 were purchased from
Maybridge Chemicals LTD. URB597, URB602, CAY10435, and MAFP
were purchased from Cayman Chemical. Solvents were distilled or
dried with 4 Å molecular sieves prior to use. All dry reactions were
performed under argon in flame- or oven-dried glassware.
Analytical thin-layer chromatography was carried out on Merck
hydrolysis by intact RBL2H3 cells was 0.082
Compound 26 also reduced the hydrolysis of 2-AG by these cells at
(Fig. 6). However, this presumably
mM (Figs. 5 and 6).
concentrations ꢀ0.03
mM
silica gel F254 (60 Å, 40–63
mm, 230–400 mesh) precoated
reflects a FAAH component of 2-AG hydrolysis in these cells, since
URB597 (4) also produced a partial inhibition of the 2-AG hydro-
lysis (data not shown).
aluminium sheets and detected under UV-light. Purification of
reaction products was carried out by flash chromatography (FC) on
J.T. Bakers silica gel for chromatography (pore size 60 Å, particle
size 50 nM). The 1H NMR and 13C NMR spectra were recorded on
a Bruker Avance 500 or 400 spectrometers operating at 500.1/
400 MHz for 1H and 125.1/100 MHz for 13C. Chemical shifts are
5. Conclusions
In the present paper, we have developed a novel series of para-
substituted phenolic N-alkyl carbamates as inhibitors of FAAH and
MGL. The compounds inhibited FAAH with IC50 values in the nM
range, whereas inhibition of MGL required concentrations three
orders of magnitude higher. The FAAH inhibition potencies of the
prepared para-substituted carbamates were highly affected by the
shape and lipophilicity of the N-alkyl group; compounds with
flexible and lipophilic N-n-hexyl and N-dodecyl groups were most
potent inhibitors of FAAH.
reported in ppm on the d scale from an internal standard of solvent
(CDCl3 7.26 and 77.0 ppm; DMSO-d6 2.50 and 39.51 ppm).
Elemental analyses (CHN or CHNS) were recorded using a Thermo
Quest CE Instrument EA 1110 CHNSO or Perkin Elmer 2400 CHN-
elemental analyzers (Table 8). HRMS(ESIþ)-spectra were recorded
on Waters LCT Premier spectrometer (Table 8). Melting points were
¨
determined in open capillaries using BUCHI Melting Point B-545 or
Stuart SMP and are uncorrected.
Due the importance of FAAH as
a
potential target for
6.1.1. 4-(4,5-Dihydrothiazol-2-yl)phenol (54) [62]
the development of new therapeutical agents, these novel
para-substituted carbamates present a useful extension to the
previously reported meta-substituted carbamates as FAAH inhibi-
tors [21–25], and may be useful for the development of new classes
of FAAH inhibitors. Furthermore, this data may be valuable,
together with that of the compounds reported earlier by us [22], in
the derivation of 3D-QSAR models for the FAAH inhibition by
O-phenyl carbamates.
Moreover, O-phenyl carbamates have been previously described
extensively as potent FAAH inhibitors, but less is known about their
ability to inhibit MGL. The present results indicate that in the
compounds investigated in the present study the O-aryl carbamate
structure, with an acyclic or a cyclic electron-withdrawing moiety
at the para-position in the phenyl ring, and a lipophilic N-alkyl
group are essential for MGL inhibitory activity. These novel
compounds, albeit not being potent and selective, may be helpful in
A mixture of 4-cyanophenol (1.20 g, 10 mmol, 100 mol%) and
2-aminoethanethiol (1.20 g, 15 mmol, 150 mol%) was heated at
100 ꢁC for 10 min and cooled to rt. The resulting yellow solid was
suspended in water (8 mL) and the mixture was acidified with 1 M
HCl (8 mL). Filtration and drying gave 54 (1.58 g, 88%) as a white
solid: mp 201.8–203.2 ꢁC; 1H NMR (DMSO-d6 400 MHz)
d 9.95 (s,
1H), 7.65–7.60 (m, 2H), 6.86–6.81 (m, 2H), 4.32 (t, J ¼ 8.2 Hz, 2H),
3.38 (t, J ¼ 8.2 Hz, 2H); 13C NMR (DMSO-d6)
d 165.6, 160.2, 129.9,
124.0, 115.4, 64.8, 33.0; HRMS calcd. for C9H10NOS [M þ H]:
180.0483, found: 180.0505.
6.1.2. 2-(4-Benzyloxyphenyl)-4,5-dihydrooxazole (55) [64]
To a refluxing solution of 4-cyanophenol (6.0 g, 50 mmol,
100 mol%), K2CO3 (13.8 g, 100 mmol, 200 mol%), and KI (4.15 g,
25 mmol, 50 mol%) in acetone (100 mL) was added BnBr (9.4 g,
55 mmol, 110 mol%) and the mixture was refluxed for 2 h. The

3002
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
Table 8
Elemental and HRMS analysis data.
Compound
Formula
Calculated C, H, N
Found C, H, N
9
C12H14N2O2S
C13H16N2O2S
C16H22N2O2S
C22H34N2O2S
C14H18N2O2S
C, 57.58; H, 5.64; N, 11.19
C, 59.07; H, 6.10; N, 10.60
C, 62.72; H, 7.24; N, 9.14
C, 67.65; H, 8.77; N, 7.17
C, 60.41; H, 6.52; N, 10.06
C, 62.04; H, 6.25; N, 9.65; S, 11.04
HRMS for C15H19N2O2S: 291.1167
HRMS for C16H20N2O2SNa: 327.1143
C, 65.36; H, 5.16; N, 8.97
C, 66.23; H, 5.56; N, 8.58
C, 66.23; H, 5.56; N, 8.58
C, 63.14; H, 5.30; N, 8.18
C, 66.23; H, 5.56; N, 8.58; S, 9.82
C, 64.10; H, 6.92; N, 10.68
C, 61.63; H, 6.90; N, 9.58
C, 69.66; H, 5.85; N, 9.03
C, 69.66; H, 5.85; N, 9.03
C, 59.62; H, 6.54; N, 13.36
C, 64.75; H, 8.02; N, 10.79
C, 58.11; H, 5.23; N, 14.52
C, 59.39; H, 5.65; N, 13.85
C, 61.72; H, 4.21; N, 13.50
C, 62.14; H, 6.82; N, 5.57
C, 64.50; H, 7.58; N, 5.01
C, 64.97; H, 6.91; N, 5.05
C, 69.39; H, 9.15; N, 3.85
C, 55.46; H, 5.92; N, 11.76
C, 58.64; H, 6.81; N, 10.52
C, 59.08; H, 6.10; N, 10.60
C, 66.04; H, 6.47; N, 12.83
C, 68.27; H, 7.37; N, 11.37
C, 52.01; H, 6.04; N, 4.67
C, 64.55; H, 7.67; N, 6.27
C, 71.46; H, 9.00; N, 5.95
C, 70.56; H, 8.65; N, 6.33
C, 74.71; H, 10.23; N, 4.59
C, 62.12; H, 7.49; N, 4.53
C, 65.51; H, 7.90; N, 4.77
C, 44.46; H, 4.97; N, 3.46
C, 70.55; H, 9.53; N, 3.58
C, 56.30; H, 5.45; N, 15.15
C, 56.30; H, 5.45; N, 15.15
C, 64.75; H, 8.02; N, 10.79
C, 64.75; H, 8.02; N, 10.79
C, 59.08; H, 6.10; N, 10.60
C, 57.85; H, 5.48; N, 11.11
C, 59.27; H, 6.00; N, 10.54
C, 62.79; H, 7.25; N, 8.99
C, 67.97; H, 9.05; N, 7.07
C, 60.54; H, 6.39; N, 10.14
C, 62.88; H, 6.42; N, 9.91; S, 10.78
Found: 291.1176
10
11
12
13
14
C15H18N2O2S
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
C16H20N2O2S
C17H16N2O2S
C18H18N2O2S
C18H18N2O2S
C18H18N2O3S
C18H18N2O2S
Found: 327.1126
C, 65.51; H, 5.42; N, 9.09
C, 66.15; H, 5.63; N, 8.36
C, 66.09; H, 5.65; N, 8.55
C, 63.33; H, 5.14; N, 8.23
C, 65.93; H, 5.49; N, 8.70; S, 9.60
C, 64.01; H, 6.94; N, 10.71
C, 61.53; H, 6.72; N, 9.75
C, 69.67; H, 5.71; N, 9.11
C, 69.67; H, 5.71; N, 9.11
C, 60.02; H, 6.37; N, 13.74
C, 64.36; H, 8.37; N, 10.58
C, 57.86; H, 5.28; N, 14.23
C, 59.00; H, 5.66; N, 13.86
C, 61.41; H, 4.20; N, 13,24
C, 62.41; H, 6.93; N, 5.45
C, 64.39; H, 7.75; N, 5.06
C, 64.83; H, 7.10; H, 5.11
C, 69.66; H, 9.35; N, 3.90
C, 55.31; H, 6.00; N, 11.65
C, 59.05; H, 7.21; N, 10.10
C, 58.90; H, 6.21; N, 10.49
C, 65.99; H, 6.54; N, 12.70
C, 68.22; H, 7.47; N, 11.18
C, 52.08; H, 6.04; N, 5.04
C, 64.57; H, 7.79; N, 6.18
C, 71.35; H, 9,18; N, 6.02
C, 70.60; H, 8.78; N, 6.45
C, 74.81; H, 10.41; N, 4.70
C, 62.35; H, 7.65; N, 4.45
C, 65,23; H, 8.14; N, 5.17
C, 44,89; H, 5.12; N, 3.47
C, 70.37; H, 9.56; N, 3.61
C, 56.06; H, 5.38; N, 14.89
C, 55.87; H, 5.46; N, 15.14
C, 64.48; H, 8.02; N, 10.73
C, 64.67; H, 8.26; N, 10.75
C, 59.07; H, 6.51; N, 10.81
C, 60.50; H, 7.42; N, 17.45
C14H18N2O3
C15H18N2O3$H2O
C18H18N2O3
C18H18N2O3
C15H19N3O2S
C21H31N3O2S
C14H15N3O2S
C15H17N3O2S
C16H13N3O2S
C13H17NO4
C15H21NO4
C15H19NO4
C21H33NO4
C11H14N2O4
C13H18N2O4
C13H16N2O4
C12H14N2O2
C14H18N2O2
C13H18BrNO2
C12H17NO3
C14H21NO2
C13H19NO2
C19H31NO2
C16H23NO5
C16H23NO4
C15H20INO4
C23H37NO4
C13H15N3O2S
C13H15N3O2S
C21H31N3O2S
C21H31N3O2S
C13H16N2O4
C15H20N4OS$1/14 n-hexylamine
C, 60.07; H, 7.24; N, 17.40
mixture was cooled to rt, poured into water (250 mL) and extracted
with EtOAc (200 mL þ 100 mL). The organic phase was washed
with brine (150 mL), dried (Na2SO4), evaporated to dryness and the
resulting residue crystallized from EtOAc/hexane giving in 4-ben-
zyloxybenzonitrile as colourless needles (10.2 g, 97%).
6.1.3. 4-(4,5-Dihydrooxazol-2-yl)phenol (56) [70]
Compound 55 (2.0 g, 7.9 mmol,100 mol%) was dissolved in EtOH
(50 mL), and the mixture was degassed with argon. 5% Pd/C
(330 mg, 0.16 mmol, 2 mol%) was added and the mixture was
degassed with hydrogen. The mixture was stirred under H2 at rt for
90 min and filtered through a pad of celite and evaporated to
dryness. Crystallization of the residue from EtOH gave 56 (0.96 g,
75%) as a white solid: mp 209.1–210.1 ꢁC; 1H NMR (CDCl3,
ZnCl2 (0.65 g, 4.8 mmol, 10 mol%) was melted by flame heating
under high vacuum and cooled to rt under argon atmosphere. 4-Ben-
zyloxy-benzonitrile (10.2 g, 48.7 mmol, 100 mol%) and chlorobenzene
(180 mL) were added and the mixture was heated to reflux. Ethanol-
amine (14.5 mL, 245 mmol, 500 mol%) was added and the mixture was
refluxed for 24 h. Another portion of ethanolamine (29 mL,1000 mol%)
was added and the mixture refluxed foradditional 24 h and partitioned
between water (500 mL) and EtOAc (500 mL). The organic phase was
washed with brine (200 mL), dried (Na2SO4), filtered and evaporated to
dryness. The residue was purified by FC (eluent 1–5% MeOH in CH2Cl2)
and recrystallized (EtOAc:hexane) to give 55 (6.16 g, 50%) as white
400 MHz)
d 7.75–7.71 (m, 2H), 6.81–6.77 (m, 2H), 4.45–4.41 (t,
J ¼ 9.5 Hz, 2H), 3.98–3.93 (t, J ¼ 9.5 Hz, 2H); 13C NMR (CDCl3,
100 MHz)
d 165.8, 160.8, 129.7, 118.0, 114.8, 67.4, 53.4; HRMS calcd.
for C9H10NO2 [M þ H]: 164.0712, found: 164.0738.
6.1.4. General procedure for the preparation of compounds 57a,
57b, 57d; N0-[1-(4-hydroxy-phenyl)-ethylidene]-
hydrazinecarboxylic acid ethyl ester (57a) [65]
crystals: mp 136.7–137.5 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
7.90–7.80 (m,
A
mixture of 4-hydroxyacetophenone (4.1 g, 30 mmol,
2H), 7.43–7.30 (m, 5H) 7.00–6.98 (m, 2H), 5.11 (s, 2H), 4.46 (t, J ¼ 9.5 Hz,
100 mol%), ethyl carbazate (3.3 g, 31.5 mmol,105 mol%) and p-TsOH
(280 mg,1.5 mmol, 5 mol%) in dry toluene (100 mL) was refluxed for
2 h with a Dean–Stark trap. The solvent was removed under reduced
pressure, and the crude residue was washed with diethyl ether to
2H), 4.04 (t, J ¼ 9.5 Hz, 2H); 13C NMR (CDCl3, 100 MHz)
d 164.3, 161.1,
136.4, 129.8, 128.6, 128.1, 127.5, 120.5, 114.5, 70.0, 67.5, 54.8; HRMS
calcd. for C16H16NO2 [M þ H]: 254.1181, found: 254.1169.

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
3003
give 57a (6.85 g, 100%) as a pale yellow solid: 1H NMR (DMSO-d6,
500.1 MHz)
2H), 4.19 (q, J ¼ 7.1 Hz, 2H), 2.18 (s, 3H), 1.28 (t, J ¼ 7.1 Hz, 3H).
Purification by FC (eluent EtOAc/PE, 1:2) and recrystallization
d
9.93 (s, 1H), 7.61 (d, J ¼ 8.6 Hz, 2H), 6.80 (d, J ¼ 8.6 Hz,
(EtOAc/hexane, 1:1) gave 26 (196 mg, 82%) as a white solid: mp
149.5–150.0 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
d
8.60 (s, 1H), 8.04 (d,
J ¼ 8.6 Hz, 2H), 7.29 (d, J ¼ 8.6 Hz, 2H), 5.06 (s, 1H), 3.29 (m, 2H),
1.68–0.83 (m, 23H); 13C NMR (CDCl3, 125.1 MHz)
162.2, 154.2,
6.1.4.1. N0-[1-(3-Hydroxy-phenyl)-ethylidene]-hydrazinecarboxylic
acid ethyl ester (57b). Pale brown solid (4.6 g, 69%): 1H NMR
d
152.0, 129.7, 128.4, 127.8, 122.3, 41.3, 31.9, 29.8, 29.64, 29.62, 29.58,
29.54, 29.34, 29.26, 26.8, 22.7, 14.1; Anal. (C21H31N3O2S) C, H, N.
(DMSO-d6, 500.1 MHz)
d 10.08 (br s, 1H), 7.26–7.14 (m, 3H), 6.80
(ddd, J ¼ 7.9, 2.4, 0.9 Hz,1H), 4.20 (q, J ¼ 7.1 Hz, 2H), 2.19 (s, 3H),1.29
(t, J ¼ 7.1 Hz, 3H); 13C NMR (DMSO-d6, 125.1 MHz) 158.1, 155.1,
140.6, 130.1, 126.4, 118.0, 116.9, 113.5, 61.4, 15.5, 14.8.
6.1.8.1. Ethylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl ester
(9). White solid (480 mg, 74%): mp 130.5–131.9 ꢁC; 1H NMR (CDCl3,
400 MHz) d 7.83–7.81 (m, 2H), 7.27–7.16 (m, 2H), 5.18 (s,1H), 4.44 (t,
6.1.4.2. N0-[1-(4-Amino-phenyl)-ethylidene]-hydrazinecarboxylic
acid ethyl ester (57d). Pale yellow solid (2.2 g, 100%). 1H NMR
J ¼ 8.2 Hz, 2H), 3.39 (t, J ¼ 8.2 Hz, 2H), 3.29 (q, J ¼ 7.1 Hz, 2H), 1.21 (t,
J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 167.5, 153.8, 153.2,
130.1, 129.5, 121.4, 65.2, 36.1, 33.8, 15.0; Anal. (C12H14N2O2S) C, H, N.
(DMSO-d6, 500.1 MHz)
d
9.82 (s, 1H), 7.48 (d, J ¼ 8.6 Hz, 2H), 6.58 (d,
J ¼ 8.6 Hz, 2H), 5.55 (br s, 2H), 4.17 (q, J ¼ 7.1 Hz, 2H), 2.13 (s, 3H),
1.28 (t, J ¼ 7.1 Hz, 3H).
6.1.8.2. 2 n-Propylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
ester (10). White solid (520 mg, 80%): mp 121.0–121.2 ꢁC; 1H NMR
6.1.5. General procedure for the preparation of compounds 58a,
58b, 58d; 4-(1,2,3-thiadiazol-4-yl)phenol (58a) [65]
(CDCl3, 400 MHz) d 7.85–7.82 (m, 2H), 7.20–7.17 (m, 2H), 5.05
(s, 1H), 4.45 (t, J ¼ 8.4 Hz, 2H), 3.42 (t, J ¼ 8.4 Hz, 2H), 3.27
Compound 57a (4.5 g, 20.2 mmol, 100 mol%) was stirred over-
night in thionyl chloride (50 mL, 3000 mol%) at rt. Excess thionyl
chloride was removed under reduced pressure giving a crude
product which was washed with hot CHCl3 to afford 58a (3.0 g,
(q, J ¼ 6.8 Hz, 2H), 1.62 (sext, J ¼ 7.3 Hz, 2H), 0.99 (t, J ¼ 7.5 Hz, 3H);
13C NMR (CDCl3, 100 MHz)
d 167.4, 153.9, 153.3, 130.2, 129.5, 121.3,
65.2, 42.9, 33.8, 22.9, 11.1; Anal. (C13H16N2O2S) C, H, N.
85%) as a pale yellow solid: 1H NMR (DMSO-d6, 500.1 MHz)
(s, 1H), 7.99 (d, J ¼ 8.6 Hz, 2H), 6.96 (d, J ¼ 8.6 Hz, 2H).
d
9.42
6.1.8.3. n-Hexylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
ester (11). Colourless crystals (290 mg, 88%): mp 87.6–88.7 ꢁC; 1H
NMR (CDCl3, 400 MHz)
d 7.86–7.80 (m, 2H), 7.21–7.15 (m, 2H), 5.11
6.1.5.1. 3-(1,2,3-Thiadiazol-4-yl)phenol (58b). Pale yellow solid
(app t, 1H), 4.44 (t, J ¼ 8.3 Hz, 2H), 3.41 (t, J ¼ 8.3 Hz, 2H), 3.25
(2.0 g, 71%): 1H NMR (DMSO-d6, 500.1 MHz)
d 9.76 (br s,1H), 9.59 (s,
(q, J ¼ 6.8 Hz, 2H), 1.56 (qui, J ¼ 7.2 Hz, 2H), 1.40–1.25 (m, 6H), 0.90
1H), 7.60–7.57 (m, 2H), 7.38 (dd, J ¼ 7.9, 7.9 Hz,1H); 13C NMR (DMSO-
(t, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 400 MHz)
d 167.5, 153.9, 153.3,
d6, 125.1 MHz)
d
162.9, 158.9, 134.0, 132.8, 131.3, 118.9, 117.3, 114.8.
130.1, 129.5, 121.4, 65.2, 41.3, 33.8, 31.4, 29.7, 26.4, 22.5, 14.0; Anal.
(C16H22N2O2S) C, H, N.
6.1.5.2. 4-(1,2,3-Thiadiazol-4-yl)phenylamine
hydrochloride (58d). Yellow solid (805 mg, 75%): 1H NMR (DMSO-
6.1.8.4. Dodecylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
d6, 500.1 MHz)
d
9.59 (s, 1H), 8.17 (d, J ¼ 7.0, 2 H), 7.35 (d, J ¼ 7.0,
ester (12). Colourless crystals (290 mg, 88%): mp 87.6–88.7 ꢁC; 1H
2H); Anal. calcd. for (C8H8N3SCl): C, 44.97; H, 3.77; N, 19.66; found:
C, 44.84; H, 3.69; N, 19.41.
NMR (CDCl3, 400 MHz) d 7.86–7.80 (m, 2H), 7.21–7.15 (m, 2H), 5.11
(app t, 1H), 4.44 (t, J ¼ 8.3 Hz, 2H), 3.41 (t, J ¼ 8.3 Hz, 2H), 3.25
(q, J ¼ 6.8 Hz, 2H), 1.56 (qui, J ¼ 7.2 Hz, 2H), 1.40–1.25 (m, 6H), 0.90
6.1.6. N0-[1-(2-Hydroxy-phenyl)-ethylidene]-hydrazinecarboxylic
acid ethyl ester (57c) [66]
(t, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 400 MHz)
d 167.5, 153.9, 153.3,
130.1, 129.5, 121.4, 65.2, 41.3, 33.8, 31.4, 29.7, 26.4, 22.5, 14.0; Anal.
(C22H34N2O2S) C, H, N.
A mixture of 2-hydroxyacetophenone (3.6 ml, 4.1 g, 30 mmol,
100 mol%), ethyl carbazate (3.3 g, 31.5 mmol, 105 mol%) and p-TsOH
(280 mg, 1.5 mmol, 5 mol%) in ethanol (100 mL) was refluxed for
22 h and kept overnight in the refrigerator. The precipitate was
filtered off with suction and washed with diethyl ether to give 57c
(5.24 g, 78%) as light yellow needles: 1H NMR (DMSO-d6,
6.1.8.5. tert-Butylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
ester (13). White solid (580 mg, 83%); mp 148.9–151.1 ꢁC; 1H NMR
(CDCl3, 400 MHz) d 7.82–7.79 (m, 2H), 7.17–7.15 (m, 2H), 5.19 (s, 1H),
4.42 (t, J ¼ 8.4 Hz, 2H), 3.42 (t, J ¼ 8.3 Hz, 2H), 1.35 (s, 9H); 13C NMR
500.1 MHz)
d
12.94 (br s, 1H), 10.80 (br s, 1H), 7.57 (dd, J ¼ 8.3,
(CDCl3, 100 MHz) d 167.4, 153.2, 152.0, 130.0, 129.5, 121.9, 65.2, 50.8,
1.7 Hz, 1H), 7.30–7.27 (m, 1H), 6.91–6.88 (m, 1H), 4.25 (q, J ¼ 7.1 H,
33.7, 28.7; Anal. (C14H18N2O2S) C, H, N.
2H), 1.31 (t, J ¼ 7.1 Hz, 3H).
6.1.8.6. Cyclopentylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
6.1.7. 2-(1,2,3-Thiadiazol-4-yl)phenol (58c) [66]
ester (14). Pale yellow solid (670 mg, 92%): mp 170.5–173.0 ꢁC; 1H
Compound 57c (3.5 g, 15.7 mmol, 100 mol%) was stirred over-
night in thionyl chloride (35 mL, 3000 mol%) at rt. Excess thionyl
chloride was removed under reduced pressure. The crude product
was filtered through a plug of silica (CH2Cl2) and then purified by FC
(eluent CH2Cl2) to give 58c (1.2 g, 43%) as a yellow solid:1H NMR
NMR (CDCl3, 400 MHz) d 7.87–7.79 (m, 2H), 7.21–7.16 (m, 2H), 5.06
(br d, J ¼ 6.8 Hz, 1H), 4.44 (t, J ¼ 8.3 Hz, 2H), 4.05 (sext, J ¼ 6.9 Hz,
1H), 3.41 (t, J ¼ 8.3 Hz, 2H), 2.02 (sext, J ¼ 6.1 Hz, 2H), 1.77–1.56
(m, 4H),1.54–1.43 (m, 2H); 13C NMR (CDCl3,100 MHz)
d 167.9,153.8,
153.7, 130.5, 129.9, 121.9, 65.6, 53.4, 34.2, 33.5, 23.9; Anal.
(C15H18N2O2S) C, H, N.
(CDCl3, 500.1 MHz)
1.7 Hz, 1H), 7.35 (ddd, J ¼ 8.4, 7.1, 1.5 Hz, 1H).
d
10.53 (br s, 1H), 8.80 (s, 1H), 7.65 (dd, J ¼ 7.9,
6.1.8.7. Cyclohexylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
6.1.8. General procedure for the preparation of the compounds 9–
33, 35, 37–44, 46, and 48–51; dodecylcarbamic acid 4-(1,2,3-
thiadiazol-4-yl)phenyl ester (26)
To a mixture of 58a (89 mg, 0.5 mmol, 100 mol%) and a catalytic
amount of Et3N in dry toluene (2 mL) was added dodecyl isocyanate
ester (15). White solid (560 mg, 73%): mp 167.1–168.8 ꢁC; 1H NMR
(CDCl3, 400 MHz)
d 7.83–7.81 (m, 2H), 7.19–7.17 (m, 2H), 5.06
(s, 1H), 4.44 (t, J ¼ 8.2 Hz, 2H), 3.41 (t, J ¼ 8.4 Hz, 2H), 2.04–2.00
(m, 3H), 1.76–1.71 (m, 3H), 1.64–1.60 (m, 1H), 1.21–1.18 (m, 3H); 13C
NMR (CDCl3, 100 MHz) d 168.8, 163.0, 156.7, 128.8, 125.5, 114.7, 65.6,
(130
mL, 0.55 mmol, 100 mol%). The reaction mixture was gently
49.8, 39.1, 32.6, 25.2, 24.6; HRMS calcd. for C16H20N2O2SNa
refluxed for 2.5 h. After cooling, the solvent was evaporated.
[M þ Na]: 327.1143, found: 327.1126.

3004
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
6.1.8.8. Benzylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl ester
6.1.8.16. (4-Methyl)-benzylcarbamic acid 4-(4,5-dihydrooxazol-2-
(16). White solid (400 mg, 79%): mp 129.8–131.4 ꢁC; 1H NMR
yl)phenyl ester (24). White solid (100 mg, 30%): mp 141–142.5 ꢁC;
(CDCl3, 400 MHz)
(m, 2H), 5.88 (app t, 1H), 4.45–4.34 (m, 4H), 3.35 (t, J ¼ 8.3 Hz, 2H);
13C NMR (CDCl3, 100 MHz)
167.6, 154.0, 153.1, 137.8, 130.0,
d
7.82–7.77 (m, 2H), 7.35–7.20 (m, 5H), 7.17–7.08
1H NMR (CDCl3, 400 MHz)
d
7.92–7.89 (m, 2H), 7.24–7.12 (m, 6H),
5.74 (br s, 1H), 4.45–4.38 (m, 4H), 4.04 (t, J ¼ 9.5 Hz, 2H), 2.32
(s, 3H); 13C NMR (CDCl3, 100 MHz)
164.0, 154.0, 153.4, 137.4, 134.8,
d
d
129.4, 128.6, 127.5, 127.5, 121.3, 64.9, 45.0, 33.6; Anal. (C17H16N2O2S)
C, H, N.
129.4, 127.7, 124.7, 121.3, 67.6, 54.9, 45.0, 21.7; Anal. (C18H18N2O3) C,
H, N.
6.1.8.9. (2-Methyl)-benzylcarbamic acid 4-(4,5-dihydrothiazol-2-
6.1.8.17. n-Hexylcarbamic acid 4-(1,2,3-thiadiazol-4-yl)phenyl ester
(25). White solid (44 mg, 28%): mp 146.4–148.2 ꢁC; 1H NMR
yl)phenyl ester (17). White solid (300 mg, 53%): mp 117.1–118.7 ꢁC;
1H NMR (CDCl3, 400 MHz)
d
7.84–7.79 (m, 2H), 7.30–7.26 (m, 1H),
(CDCl3, 500.1 MHz)
d
8.61 (s, 1H), 8.04 (d, J ¼ 8.6 Hz, 2H), 7.29 (d,
7.25–7.15 (m, 5H), 5.47–5.41 (m, 1H), 4.50–4.38 (m, 4H), 3.38 (t,
J ¼ 8.6 Hz, 2H), 5.06 (s, 1H), 3.31–3.27 (m, 2H), 1.62–1.58 (m, 2H),
J ¼ 8.3 Hz, 2H), 2.35 (s, 3H); 13C NMR (CDCl3, 100 MHz)
d 167.4,
1.41–1.33 (m, 6H), 0.91 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3,
153.8, 153.2, 136.2, 135.4, 130.5, 130.2, 129.5, 128.3, 127.9, 126.2,
121.3, 65.1, 43.3, 33.8, 19.0; Anal. (C18H18N2O2S) C, H, N.
125.1 MHz) d 162.2, 154.2, 152.0, 129.7, 128.4, 127.8, 122.3, 41.3, 31.4,
29.8, 26.4, 22.5, 14.0; Anal. (C15H19N3O2S) C, H, N.
6.1.8.10. (4-Methyl)-benzylcarbamic acid 4-(4,5-dihydrothiazol-2-
6.1.8.18. Cyclopentylcarbamic acid 4-(1,2,3-thiadiazol-4-yl)phenyl
yl)phenyl ester (18). White solid (460 mg, 85%): mp 144.0–144.6 ꢁC;
ester (27). White solid (262 mg, 90%): mp 189.7–190.3 ꢁC; 1H NMR
1H NMR (CDCl3, 400 MHz)
d
7.87–7.76 (m, 2H), 7.27–7.06 (m, 6H),
5.56 (br s, 1H), 4.48–4.33 (m, 4H), 3.39 (t, J ¼ 8.1 Hz, 2H), 2.34
(s, 3H); 13C NMR (CDCl3, 100 MHz)
167.4, 154.0, 153.2, 137.4, 134.7,
(CDCl3, 500.1 MHz)
J ¼ 8.6 Hz, 2H), 5.03 (d, J ¼ 6.7 Hz, 1H), 4.11–4.05 (m, 1H), 2.08–2.01
(m, 2H), 1.77–1.48 (m, 6H); 13C NMR (CDCl3, 125.1 MHz)
162.2,
d
8.60 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.29 (d,
d
d
130.2, 129.4, 129.3, 127.6, 121.4, 65.1, 45.0, 33.7, 21.0; Anal.
(C18H18N2O2S) C, H, N.
153.6, 152.0, 129.7, 128.4, 127.8, 122.3, 53.1, 33.2, 23.5; Anal.
(C14H15N3O2S) C, H, N.
6.1.8.11. (4-Methoxy)-benzylcarbamic acid 4-(4,5-dihydrothiazol-2-
6.1.8.19. Cyclohexylcarbamic acid 4-(1,2,3-thiadiazol-4-yl)phenyl
yl)phenyl ester (19). White solid (640 mg, 88%): mp 148.2–148.8 ꢁC;
ester (28). Light yellow solid (250 mg, 55%): mp 175.0–177.1 ꢁC; 1H
1H NMR (CDCl3, 400 MHz)
d
7.84–7.79 (m, 2H), 7.26–7.21 (m, 2H),
NMR (CDCl3, 500.1 MHz)
d
8.64 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.29
7.19–7.14 (m, 2H), 6.89–6.84 (m, 2H), 5.69–5.62 (m, 1H), 4.41
(d, J ¼ 8.6 Hz, 2H), 4.96 (d, J ¼ 7.5 Hz, 1H), 3.63–3.55 (m, 1H), 2.05–
(t, J ¼ 8.3 Hz, 2H), 4.34 (d, J ¼ 5.8 Hz, 2H), 3.78 (s, 3H), 3.38
2.02 (m, 2H), 1.78–1.74 (m, 2H), 1.44–1.35 (m, 2H), 1.65–1.17
(t, J ¼ 8.3 Hz, 2H); 13C NMR (CDCl3, 100 MHz)
d
167.4, 159.0, 153.9,
(m, 6H); 13C NMR (CDCl3, 125.1 MHz)
d 162.2, 153.2, 152.0, 129.7,
153.1, 130.1, 129.9, 129.4, 129.0, 121.4, 114.0, 65.0, 55.2, 44.6, 33.7;
Anal. (C18H18N2O3S) C, H, N.
128.3,127.7,122.3, 50.2, 33.2, 25.4, 24.7; Anal. (C15H17N3O2S) C, H, N.
6.1.8.20. Benzylcarbamic acid 4-(1,2,3-thiadiazol-4-yl)phenyl ester
6.1.8.12. Phenethylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl
(29). White solid (300 mg, 96%): mp 164.5–165.2 ꢁC; 1H NMR
ester (20). White solid (200 mg, 37%): mp 138.1–138.8 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
d
8.61 (s, 1H), 8.05 (d, J ¼ 8.5 Hz, 2H), 7.40–7.31
(CDCl3, 400 MHz)
d
7.85–7.80 (m, 2H), 7.36–7.31 (m, 2H), 7.28–7.21
(m, 7 H), 5.40 (s, 1H), 4.49 (d, J ¼ 6.0 Hz, 2H); 13C NMR (CDCl3,
(m, 3H), 7.18–7.13 (m, 2H), 5.09 (app t, 1H), 4.44 (t, J ¼ 8.3 Hz, 2H),
125.1 MHz) d 162.1, 154.3, 151.9, 137.8, 129.8, 128.8, 128.4, 128.0,
3.54 (dd, J ¼ 13.1, 6.8 Hz, 2H), 3.41 (t, J ¼ 8.3 Hz, 2H), 2.89 (t,
127.8, 127.7, 122.3, 45.4; Anal. (C16H13N3O2S) C, H, N.
J ¼ 7.0 Hz, 2H); (CDCl3, 100 MHz)
d 167.5, 153.9, 153.2, 138.4, 130.2,
129.5, 128.8, 128.7, 126.7, 121.4, 65.2, 42.3, 35.9, 33.8; Anal.
(C18H18N2O2S) C, H, N, S.
6.1.8.21. 4-(Butylcarbamoyloxy)benzoic acid methyl ester (30)
[51]. White crystals (88 mg, 70%): mp 90.3–91.0 ꢁC; 1H NMR (CDCl3,
500.1 MHz)
d
8.03 (d, J ¼ 8.5 Hz, 2H), 7.20 (d, J ¼ 8.5 Hz, 2H), 5.12
6.1.8.13. n-Butylcarbamic acid 4-(4,5-dihydrooxazol-2-yl)phenyl
ester (21). White solid (260 mg, 87%): mp 116–117 ꢁC; 1H NMR
(s, 1H), 3.90 (s, 3H), 3.27–3.25 (m, 2H), 1.58–1.53 (m, 2H), 1.43–1.35
(m, 2H), 0.95 (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
d 166.4,
(CDCl3, 400 MHz)
d
7.98–7.91 (m, 2H), 7.21–7.16 (m, 2H), 5.04 (br s,
154.8, 153.7, 131.0, 126.8, 121.3, 52.1, 41.0, 31.8, 19.9, 13.7; Anal.
(C13H17NO4) C, H, N.
1H), 4.43 (t, J ¼ 8.2 Hz, 2H), 4.06 (t, J ¼ 8.2 Hz, 2H), 3.28–3.26
(m, 2H),1.60–1.52 (m, 2H),1.45–1.35 (m, 2H), 0.96 (t, J ¼ 7.3 Hz, 3H);
13C NMR (CDCl3, 100 MHz)
d
164.0, 154.0, 153.5, 129.3, 124.5, 121.3,
6.1.8.22. 4-(Hexylcarbamoyloxy)benzoic
(31). White crystals (30 mg, 22%): mp 87.0–87.7 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
acid
methyl
ester
67.6, 54.8, 40.9, 31.8, 19.8, 13.6; Anal. (C14H18N2O3) C, H, N.
d
8.04 (d, J ¼ 8.7 Hz, 2H), 7.21 (d, J ¼ 8.7 Hz, 2H),
6.1.8.14. Cyclopentylcarbamic acid 4-(4,5-dihydrooxazol-2-yl)phenyl
5.04 (s, 1H), 3.91 (s, 3H), 3.29–3.25 (m, 2H), 1.60–1.56 (m, 2H),
ester (22). Pale yellow solid (300 mg, 90%): mp 81–82 ꢁC; 1H NMR
1.38–1.33 (m, 6H), 0.90 (t, J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3,
(CDCl3, 400 MHz)
(m, 1H), 4.43 (t, J ¼ 9.5 Hz, 2H), 4.09–4.02 (m, 3H), 2.08–1.98
(m, 2H), 1.87–1.44 (m, 6H); 13C NMR (CDCl3, 100 MHz)
164.0,
d
8.00–7.89 (m, 2H), 7.23–7.14 (m, 2H), 5.10–5.01
125.1 MHz) d 166.4, 154.8, 153.7, 131.0, 126.8, 121.3, 52.1, 41.3, 31.4,
29.7, 26.4, 22.5, 14.0; Anal. (C15H21NO4) C, H, N.
d
153.5, 153.4, 129.4, 124.5, 121.3, 67.6, 54.9, 53.0, 33.1, 23.5; Anal.
(C15H18N2O3$H2O) C, H, N.
6.1.8.23. 4-(Cyclohexylcarbamoyloxy)benzoic acid methyl ester
(32). White solid (491 mg, 59%): mp 135.3–136.2 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
d
8.04 (d, J ¼ 8.7 Hz, 2H), 7.21 (d, J ¼ 8.7 Hz, 2H),
6.1.8.15. (2-Methyl)-benzylcarbamic acid 4-(4,5-dihydrooxazol-2-
4.95 (d, J ¼ 7.4 Hz,1H), 3.90 (s, 3H), 3.61–3.53 (m,1H), 2.03–2.00 (m,
yl)phenyl ester (23). White solid (190 mg, 50%): mp 125–126 ꢁC; 1H
2H), 1.77–1.73 (m, 2H), 1.65–1.16 (m, 6H); 13C NMR (CDCl3,
NMR (CDCl3, 400 MHz)
d
7.94–7.90 (m, 2H), 7.30–7.25 (m,1H), 7.23–
125.1 MHz) d 166.4, 154.8, 152.8, 130.9, 126.7, 121.2, 52.0, 50.2, 33.1,
7.09 (m, 5H), 5.62 (app t, J ¼ 5.2 Hz, 1H), 4.44–4.36 (m, 4H), 4.00
25.4, 24.7; Anal. (C15H19NO4) C, H, N.
(t, J ¼ 9.5 Hz, 2H), 2.35 (s, 3H); 13C NMR (CDCl3, 100 MHz)
d 164.0,
153.9, 153.4, 136.2, 135.4, 130.5, 129.4, 128.2, 127.8, 126.2, 124.5,
121.3, 67.6, 54.7, 43.2, 18.9; Anal. (C18H18N2O3) C, H, N.
6.1.8.24. 4-(Dodecylcarbamoyloxy)benzoic
acid
methyl
ester
(33). White solid (168 mg, 92%): mp 93.4–94.4 ꢁC; 1H NMR

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
3005
(CDCl3, 500.1 MHz)
d
8.04 (d, J ¼ 8.7 Hz, 2H), 7.21 (d, J ¼ 8.7 Hz,
151.1, 129.2, 125.2, 121.6, 41.3, 31.9, 29.8, 29.63, 29.61, 29.6, 29.5,
29.3, 29.3, 26.7, 22.7, 14.1; Anal. (C19H31NO2) C, H, N.
2H), 5.03 (s, 1 H), 3.91 (s, 3H), 3.29–3.25 (m, 2H), 1.61–1.55 (m,
2H), 1.38–1.27 (m, 18H), 0.88 (t, J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3,
125.1 MHz)
d
154.6, 151.1, 131.0, 129.2, 125.1, 121.6, 41.3, 31.9, 29.8,
6.1.8.33. 3-Methoxy-4-(hexylcarbamoyloxy)benzoic acid methyl
29.62, 29.61, 29.6, 29.5, 29.32, 29.25, 26.7, 22.7, 14.1; Anal.
(C21H33NO4) C, H, N.
ester (44). White solid (91 mg, 59%): mp 86.9–88.6 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
d
7.66–7.63 (m, 2H), 7.16 (d, J ¼ 8.2 Hz, 1H), 5.11
(s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.28–3.24 (m, 2H), 1.60–1.54 (m,
6.1.8.25. n-Hexylcarbamic acid 4-nitrophenyl ester (35) [54]. White
2H), 1.38–1.32 (m, 6H), 0.90 (t, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3,
solid (108 mg, 81%): mp 84.8–85.9 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
125.1 MHz) d 166.5, 153.6, 151.5, 143.9, 128.1, 123.1, 122.6, 113.3, 56.1,
d
8.24 (d, J ¼ 9.2 Hz, 2H), 7.31 (d, J ¼ 9.2 Hz, 2H), 5.12 (s, 1H), 3.30–
3.26 (m, 2H),1.61–1.56 (m, 2H),1.40–1.32 (m, 6H), 0.90 (t, J ¼ 6.8 Hz,
3H); 13C NMR (CDCl3, 125.1 MHz)
156.0, 153.0, 144.7, 125.1, 121.9,
52.2, 41.4, 31.4, 29.7, 26.3, 22.5, 14.0; Anal. (C16H23NO5) C, H, N.
d
6.1.8.34. 3-Iodo-4-(hexylcarbamoyloxy)benzoic acid methyl ester
41.4, 31.4, 29.4, 26.4, 22.5, 14.0; Anal. (C13H18N2O4) C, H, N.
(46). White crystals (89 mg, 44%): mp 83.1–83.5 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
d
8.48 (d, J ¼ 2.6 Hz,1H), 8.01 (dd, J ¼ 8.4, 2.6 Hz,
6.1.8.26. n-Butylcarbamic acid 4-cyanophenyl ester (37). White
1H), 7.26 (d, J ¼ 8.4 Hz, 1H), 5.19 (s, 1H), 3.91 (s, 3H), 3.31–3.27 (m,
solid (286 mg, 66%): mp 105.2–106.9 ꢁC; 1H NMR (CDCl3, 400 MHz)
2H), 1.63–1.57 (m, 2H), 1.40–1.31 (m, 6H), 0.90 (t, J ¼ 6.8 Hz, 3H); 13C
d
7.64 (d, J ¼ 8.5 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H), 5.26 (s, 1H), 3.36–
NMR (CDCl3, 125.1 MHz) d 165.1, 154.7, 152.4, 140.7, 130.8, 128.8,
3.22 (m, 2H), 1.45–1.33 (m, 2H), 0.95 (t, J ¼ 7.3 Hz, 3H); 13C NMR
122.9, 90.3, 52.4, 41.5, 31.4, 29.7, 26.4, 22.6, 14.0; Anal. (C15H20INO4)
C, H, N.
(CDCl3, 100 MHz) d 154.5, 153.2, 133.4, 122.3, 118.4, 108.6, 40.9, 31.6,
19.8, 13.6; Anal. (C12H14N2O2) C, H, N.
6.1.8.35. n-Butylcarbamic acid 2-(1,2,3-thiadiazol-4-yl)phenyl ester
(48). White solid (100 mg, 73%): mp 102.8–104.7 ꢁC; 1H NMR
6.1.8.27. n-Hexylcarbamic acid 4-cyanophenyl ester (38). White
solid (393 mg, 80%): mp 94.4–95.2 ꢁC; 1H NMR (CDCl3, 400 MHz)
(CDCl3, 500.1 MHz)
d
8.70 (s, 1H), 8.16 (d, J ¼ 6.8 Hz, 1H), 7.46
d
7.69–7.61 (m, 2H), 7.31–7.23 (m, 2H), 5.24 (app. t, 1H), 3.35–3.21
(m, 2H),1.62–1.52 (m, 2H),1.41–1.26 (m, 6H), 0.90 (t, 3H, J ¼ 6.8 Hz);
13C NMR (CDCl3, 100 MHz)
154.5, 153.2, 133.4, 122.3, 118.4, 108.6,
41.3, 31.3, 29.6, 26.3, 22.4, 13.9; Anal. (C14H18N2O2) C, H, N.
(pt, J ¼ 8.1, 6.8 Hz,1H), 7.37 (pt, J ¼ 8.1, 6.8 Hz,1H), 7.28 (d, J ¼ 8.1 Hz,
1H), 5.12 (s, 1H), 3.25–3.21 (m, 2H), 1.54–1.48 (m, 2H), 1.38–1.31 (m,
d
2H), 0.93 (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
d 158.2,
153.9, 148.2, 133.1, 130.6, 130.3, 126.2, 124.2, 123.6, 41.1, 31.8, 19.8,
13.7; Anal. (C13H15N3O2S) C, H, N.
6.1.8.28. n-Hexylcarbamic
[53]. White solid (444 mg, 74%): mp 98.8–99.8 ꢁC; 1H NMR (CDCl3,
400 MHz) 7.47–7.43 (m, 2H), 7.04–6.99 (m, 2H), 5.04 (br s, 1H),
acid
4-bromophenyl
ester
(39)
6.1.8.36. n-Butylcarbamic acid 3-(1,2,3-thiadiazol-4-yl)phenyl ester
(49). Pale yellow solid (244 mg, 81%): mp 101.6–102.9 ꢁC; 1H NMR
d
3.28–3.21 (m, 2H), 1.60–1.51 (m, 2H), 1.40–1.25 (m, 6H), 0.90 (t,
(CDCl3, 500.1 MHz)
d
8.65 (s, 1H), 7.89 (d, J ¼ 7.9 Hz, 1H), 7.83 (s, 1H),
J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
154.1, 150.1, 132.2,
7.49 (pt, J ¼ 8.1, 7.9 Hz,1H), 7.22 (d, J ¼ 8.1 Hz,1H), 5.07 (s, 1H), 3.32–
123.3, 118.1, 41.3, 31.4, 29.7, 26.4, 22.5, 14.0; Anal. (C13H18BrNO2) C,
H, N.
3.28 (m, 2H), 1.61–1.55 (m, 2H), 1.45–1.38 (m, 2H), 0.97 (t, J ¼ 7.4 Hz,
3H); 13C NMR (CDCl3, 125.1 MHz)
d 162.0, 154.3, 151.6, 131.9, 130.6,
130.0, 124.1, 122.6, 120.6, 41.0, 31.8, 19.8, 13.7; Anal. (C13H15N3O2S)
C, H, N.
6.1.8.29. n-Butylcarbamic acid 4-methoxyphenyl ester (40)
[52,55]. White crystals (122 mg, 100%): mp 75.6–76.7 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
d
7.03 (d, J ¼ 8.9 Hz, 2H), 6.86 (d, J ¼ 8.9 Hz, 2H),
6.1.8.37. Dodecylcarbamic acid 2-(1,2,3-thiadiazol-4-yl)phenyl ester
5.00 (s, 1H), 3.78 (s, 3H), 3.24–3.23 (m, 2H), 1.57–1.51 (m, 2H), 1.42–
(50). White solid (187 mg, 96%): mp 107.1–108.4 ꢁC; 1H NMR
1.35 (m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
(CDCl3, 500.1 MHz)
d
8.70 (s, 1H), 8.17 (d, J ¼ 7.4 Hz, 1H), 7.47 (pt,
d
156.8, 155.0, 144.6, 122.4, 114.3, 55.6, 40.9, 31.9, 19.9, 13.7; Anal.
J ¼ 8.1, 7.4 Hz, 1H), 7.38 (pt, J ¼ 8.1, 7.4 Hz, 1H), 7.29 (d, J ¼ 8.1 Hz,
1H), 5.08 (s, 1H), 3.25–3.21 (m, 2H), 1.55–1.52 (m, 2H), 1.30–1.26
(m, 18H), 0.88 (t, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
(C12H17NO3) C, H, N.
6.1.8.30. n-Hexylcarbamic acid p-tolyl ester (41). White solid
d 158.3, 153.9, 148.2, 133.0, 130.6, 130.3, 126.2, 124.2, 123.6, 41.4,
(375 mg, 80%): mp 72.7–73.7 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
7.17–
31.9, 29.8, 29.61, 29.57, 29.5, 29.3, 29.2, 26.7, 22.7, 14.1; Anal.
(C21H31N3O2S) C, H, N.
7.11 (m, 2H), 7.02–6.97 (m, 2H), 5.00 (br s, 1H), 3.24 (q, J ¼ 6.7 Hz,
2H), 2.32 (s, 3H), 1.60–1.50 (m, 2H), 1.40–1.27 (m, 6H), 0.90 (t,
J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) 154.8, 148.8, 134.7, 129.7,
121.3, 41.2, 31.4, 29.8, 26.4, 22.5, 20.8, 14.0; Anal. (C14H21NO2)
C, H, N.
6.1.8.38. Dodecylcarbamic acid 3-(1,2,3-thiadiazol-4-yl)phenyl ester
(51). White solid (99 mg, 90%): mp 106.4–107.4 ꢁC; 1H NMR (CDCl3,
500.1 MHz)
d
8.65 (s, 1H), 7.89 (d, J ¼ 7.8 Hz, 1H), 7.83 (s, 1H), 7.50
(pt, J ¼ 8.1, 7.8 Hz, 1H), 7.23 (d, J ¼ 8.1 Hz, 1H), 5.06 (s, 1H), 3.29
(m, 2H), 1.62–1.59 (m, 2H), 1.37–1.27 (m, 18H), 0.88 (t, J ¼ 6.5 Hz,
6.1.8.31. n-Hexylcarbamic acid phenyl ester (42) [56]. White solid
(95 mg, 86%): mp 37.6–38.8 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
d
7.35
3H); 13C NMR (CDCl3, 125.1 MHz)
d 162.1, 154.3, 151.7, 132.0, 130.4,
(pt, J ¼ 7.8 Hz, 2H), 7.18 (t, J ¼ 7.4 Hz,1H), 7.13 (d, J ¼ 7.8 Hz, 2H), 4.98
130.1, 124.2, 122.7, 120.7, 41.4, 31.9, 29.8, 29.62, 29.58, 29.5, 29.3,
29.3, 26.8, 14.1; Anal. (C21H31N3O2S) C, H, N.
(br s, 1H), 3.29–3.24 (m, 2H), 1.60–1.54 (m, 2H), 1.38 (m, 6H), 0.90 (t,
J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
d 154.6, 151.1, 129.2,
125.1, 121.6, 41.2, 31.4, 29.7, 26.4, 22.5, 14.0; Anal. (C13H19NO2)
C, H, N.
6.1.9. General procedure for the preparation of the alkyl carbamic
acid 4-nitrophenyl esters 34 and 36; n-butylcarbamic acid 4-
nitrophenyl ester (34) [52,55]
6.1.8.32. Dodecylcarbamic acid phenyl ester (43) [57]. White solid
4-Nitrophenyl chloroformate (670 mg, 3.3 mmol, 110 mol%) in
CH2Cl2 (10 mL) was added to a mixture of butylamine (0.3 mL,
3.0 mmol, 100 mol%) and pyridine (0.27 mL, 3.3 mmol, 110 mol%).
The mixture was gently refluxed for 1 h, cooled and evaporated to
dryness. The residue was purified by FC (eluent EtOAc/PE, 1:4) to
(153 mg, 100%): mp 56.9–57.7 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
d
7.35 (pt, J ¼ 7.8 Hz, 2H), 7.18 (t, J ¼ 7.4 Hz, 1H), 7.13 (d, J ¼ 7.7 Hz,
2H), 4.97 (s, 1H), 3.28–3.24 (m, 2H), 1.58–1.56 (m, 2H), 1.33–1.27 (m,
18H), 0.89 (t, J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
d 154.6,

3006
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
give 34 (232 mg, 32%) as a white solid: mp 96.7–97.4 ꢁC; 1H NMR
(CDCl3, 500.1 MHz)
8.24 (d, J ¼ 9.1 Hz, 2H), 7.32 (d, J ¼ 9.1 Hz, 2H),
d
9.47 (s, 1H), 8.68 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.59 (d, J ¼ 8.6 Hz,
d
2H), 6.25 (t, J ¼ 5.6 Hz, 1H), 3.15–3.11 (m, 2H), 1.49–1.45 (m, 2H),
5.09 (s, 1H), 3.32–3.28 (m, 2H), 1.61–1.55 (m, 2H), 1.45–1.37 (m, 2H),
1.37–1.27 (m, 6H), 0.93–0.88 (m, 3H); 13C NMR (DMSO-d6,
0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (CDCl3, 125.1 MHz)
d
156.0, 153.1,
125.1 MHz) d 163.0, 156.0, 142.6, 132.0, 128.6, 124.6, 118.7, 32.0, 31.0,
30.6, 27.0, 23.0,14.9; Anal. (C15H20N4OS$1/14 n-hexylamine) C, H, N.
144.6, 125.0, 121.9, 41.0, 31.7, 19.8, 13.6; Anal. (C11H14N2O4) C, H, N.
6.1.9.1. Cyclohexylcarbamic acid 4-nitrophenyl ester (36) [61]. Pale
yellow solid (261 mg, 99%): mp 146.3–147.1 ꢁC; 1H NMR (CDCl3,
6.2. Assay of MGL activity
500.1 MHz)
J ¼ 6.1 Hz, 1H), 3.61–3.54 (m, 1H), 2.04–2.01 (m, 2H), 1.79–1.73 (m,
2H), 1.67–1.17 (m, 6H); 13C NMR (CDCl3, 125.1 MHz)
156.1, 152.1,
d
8.24 (d, J ¼ 9.1 Hz, 2H), 7.32 (d, J ¼ 9.1 Hz, 2H), 4.99 (d,
6.2.1. Animals and preparation of rat cerebellar membranes for
MGL assay
d
144.6, 125.1, 121.9, 50.4, 33.1, 25.3, 24.7; Anal. (C13H16N2O4) C, H, N.
Four-week-old male Wistar rats were used in these studies. All
animal experiments were approved by the local ethics committee.
The animals lived in a 12-h light/12-h dark cycle (lights on at
0700 h), with water and food available ad libitum. The rats were
decapitated eight hours after lights on (1500 h), the whole brains
were removed, dipped in isopentane on dry ice and stored at
ꢃ80 ꢁC.
6.1.10. General procedure for the preparation of the carbamic acid
esters 45, and 47 via esterification; 3-methyl-4-
(hexylcarbamoyloxy)benzoic acid methyl ester (45)
4-Hydroxy-3-methyl benzoic acid (152 mg, 100 mol%) in
methanol (10 mL) and a catalytic amount of conc. H2SO4 was
refluxed overnight and the remaining methanol was evaporated
giving 177 mg (100%) of the corresponding methyl ester. This was
used directly without further purification to the preparation of the
title compound.
The membranes were prepared as previously described [71–73].
Briefly, cerebella were dissected and homogenized in nine volumes
of ice-cold 0.32 M sucrose with a glass Teflon homogenizer. The
crude homogenate was centrifuged at low speed (1000 ꢄ g for
10 min at 4 ꢁC) and the pellet was discharged. The supernatant was
centrifuged at high speed (100,000 ꢄ g for 10 min at 4 ꢁC). The
pellet was resuspended in ice-cold deionized water and washed
twice, repeating the high-speed centrifugation. Finally, the
membranes were resuspended in 50 mM Tris–HCl, pH 7.4 with
1 mM EDTA and aliquoted for storage at ꢃ80 ꢁC until use. The
protein concentration of the final preparation was measured by the
Bradford method [74].
To a mixture of 4-hydroxy-3-methyl benzoic acid methyl ester
(83 mg, 0.5 mmol, 100 mol%) and a catalytic amount of Et3N in dry
toluene (2 mL) was added hexyl isocyanate (80 mL, 110 mol%). After
gently refluxing overnight the reaction mixture was cooled and the
solvent evaporated. Purification by FC (eluent EtOAc/PE, 1:1) and
crystallization (EtOAc/hexane, 1:2) gave 45 (81 mg, 55%) as white
crystals: mp 52.4–53.5 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
d 7.91 (s,
1H), 7.87 (d, J ¼ 8.4 Hz, 1H), 7.15 (d, J ¼ 8.4 Hz, 1H), 5.08 (s, 1H), 3.89
(s, 3H), 3.29–3.24 (m, 2H), 2.25 (s, 3H), 1.61 (m, 2H), 1.38 (m, 6H),
0.90 (t, J ¼ 6.8 Hz, 2H); 13C NMR (CDCl3, 125.1 MHz)
d
166.6, 153.7,
6.2.2. In vitro assay for MGL activity in rat cerebellar membranes
153.2, 132.5, 130.7, 128.5, 127.2, 122.2, 52.1, 41.3, 31.4, 29.8, 26.4,
22.5, 16.1, 14.0; Anal. (C16H23NO4) C, H, N.
The assay for MGL has been described previously [34]. Briefly,
experiments were carried out with preincubations (80
mL, 30 min
at 25 ꢁC) containing 10
mg membrane protein, 44 mM Tris–HCl (pH
6.1.10.1. 3,5-Di-tert-butyl-4-(hexylcarbamoyloxy)benzoic
methyl ester (47). White crystals (50 mg, 26%): mp 104.1–104.9 ꢁC;
1H NMR (CDCl3, 500.1 MHz)
8.00 (s, 2H), 5.09 (s, 1H), 3.90 (s, 3H),
3.29–3.25 (m, 2H), 1.67–1.31 (m, 29H); 13C NMR (CDCl3, 125.1 MHz)
167.2, 154.7, 152.3, 143.8, 127.9, 126.7, 52.0, 41.3, 35.7, 31.4, 31.2,
acid
7.4), 0.9 mM EDTA, 0.5% (wt/vol) BSA and 1.25% (vol/vol) DMSO as
a solvent for inhibitors. The preincubated membranes were kept at
0 ꢁC just prior to the experiments. The incubations (90 min at
d
25 ꢁC) were initiated by adding 40
mL
of the preincubated
membrane cocktail, giving a final volume of 400 L. The final
volume contained 5 g membrane protein, 54 mM Tris–HCl (pH
7.4), 1.1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 0.5% (wt/vol) BSA
and 50 M of 2-AG. At time-points of 0 and 90 min, 100 l aliquots
were removed from the incubation, acetonitrile (200 L) was added
d
m
29.8, 26.4, 22.6, 14.0; Anal. (C23H37NO4) C, H, N.
m
6.1.11. 4-Nitrophenylcarbamic acid cyclohexyl ester (52)
m
m
To a mixture of cyclohexanol (47
mL, 0.5 mmol, 100 mol%) and
m
a catalytic amount of Et3N in dry toluene (2 ml) was added 4-
nitrophenyl isocyanate (90 mg, 0.55 mmol, 110 mol%). The reaction
mixture was gently refluxed and monitored by TLC until cyclo-
hexanol could not be detected on TLC. The reaction mixture was
cooled and solvent evaporated. Purification by FC (eluent EtOAc/PE,
1:1) gave compound 52 (145 mg, 100%) as white solid: mp 115.2–
to stop the enzymatic reaction, and the pH of the samples was
simultaneously decreased with phosphoric acid (added to aceto-
nitrile) to 3.0, in order to stabilize 2-AG against chemical acyl
migration reaction to 1(3)-AG. The samples were centrifuged at
23,700g for 4 min at rt prior to HPLC analysis of the supernatant.
116.4 ꢁC; 1H NMR (CDCl3, 500.1 MHz)
d
8.18 (d, J ¼ 8.9, 2H), 7.55
6.2.3. HPLC method for in vitro assay for MGL
(d, J ¼ 8.9, 2H), 7.03 (s, 1H), 4.81–4.76 (m, 1H), 1.94–1.91 (m, 2H),
The analytical HPLC was performed as previously described [34].
Briefly, the analytical HPLC system consisted of a Merck Hitachi
(Hitachi Ltd., Tokyo, Japan) L-7100 pump, D-7000 interface module,
L-7455 diode-array UV detector (190–800 nm, set at 211 nm) and L-
7250 programmable autosampler. The separations were accom-
plished on a Zorbax SB-C18 endcapped reversed-phase precolumn
1.76–1.73 (m, 2H), 1.57–1.23 (m, 6H); 13C NMR (CDCl3, 125.1 MHz)
d
152.4, 144.2, 142.8, 125.2, 117.6, 74.6, 31.7, 25.2, 23.7; Anal.
(C13H16N2O4) C, H, N.
6.1.12. 1-Hexyl-3-(4-(1,2,3-thiadiazol-4-yl)phenyl) urea (53)
To a mixture of 4-(1,2,3-thiadiazol-4-yl)phenylamine hydro-
(4.6 ꢄ 12.5 mm, 5
m
m) and column (4.6 ꢄ 150 mm, 5
mm) (Agilent,
l. A mobile phase mixture of
chloride (58d, 89 mg, 0.41 mmol, 100 mol%) and Et3N (63
110 mol%) in dry toluene (2 mL) was added hexyl isocyanate (65
m
m
L,
L,
U.S.A). The injection volume was 50 m
28% phosphate buffer (30 mM, pH 3.0) in acetonitrile was used at
a flow rate of 2.0 ml minꢃ1. Retention times were 5.8 min for 2-AG,
6.3 min for 1(3)-AG and 10.2 min for arachidonic acid. The relative
concentrations of 2-AG, 1(3)-AG and arachidonic acid were deter-
mined by the corresponding peak areas. This was justified by the
equivalence of response factors for the studied compounds, and
0.451 mmol, 110 mol%). The reaction mixture was gently refluxed
for 5 h and then stirred overnight at rt. Solvent was evaporated and
the resulting crude product was purified by FC (eluent EtOAc/PE,
1:1) and crystallization (EtOAc) to give compound 53 (85 mg, 68%)
as yellow solid: mp 159.5–160.7 ꢁC; 1H NMR (DMSO-d6, 500.1 MHz)

A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
3007
was supported by the observation that the sum of the peak areas
6.5. Reversibility studies
was constant throughout the experiments.
The reversibility of FAAH inhibition by compounds 26,
CAY10435 and 4 was determined by measuring the recovery of
enzymatic activity after a rapid and large dilution of enzyme–
6.3. Human recombinant MGL (hrMGL) assay
inhibitor complex. In the preincubation mixture (70 mL) the amount
The endpoint enzymatic assay was developed to quantify
human recombinant MGL (Cayman Chemical, cat# 10008354)
activity with tritium-labelled 2-oleoylglycerol [glycerol-1,2,3-3H]
(American Radiolabeled Chemicals Inc., St Louis, MO, USA). The
assay buffer was 50 mM Tris–HCl, pH 7.4; 1 mM EDTA and test
compounds were dissolved in DMSO (the final DMSO concentration
was not more than 5% v/v). The incubations were performed in the
presence of 0.5% (w/v) BSA (essentially fatty acid-free). hrMGL
of rat brain homogenate was 20-fold the amount required for the
activity assay, and the inhibitor concentration was approximately
20-fold the IC50 value. After the preincubation, 1.8 mL of enzyme–
inhibitor mixture was taken and immediately diluted 500-fold into
the assay buffer containing AEA. The final incubation concentration
of AEA was 2
100 L were taken at 0, 5, 10, 15, 20, 30, 45 and 60 min time-points
and added to tubes containing 400 L of cold ethyl acetate. Addi-
tionally,100 l of assay buffer was added. Samples were centrifuged
at 16,000g for 4 min at rt, and aliquots (100
m
M (including w20 nM [3H]AEA). The samples of
m
m
(0.015
m
L) was preincubated with test compounds for 10 min at
m
37 ꢁC (60
mL). At the 10 min time point, 2-oleoylglycerol was added
mL) were taken from the
to achieve the final concentration of 50
m
M
(containing
aqueous phase, which contained ethanolamine 1-3H, and measured
for radioactivity by liquid scintillation counting (Wallac 1450
MicroBeta; Wallac Oy, Finland).
112 ꢄ10ꢃ3 mCi of 40 Ci/mmol [3H]2-OG) with the final incubation
volume of 100 m
L. The incubations proceeded for 10 min at 37 ꢁC.
Ethyl acetate (400
the enzymatic reaction. Additionally, 100
HCl, pH 7.4; 1 mM EDTA) was added. The samples were centrifuged
m
L) was added at the 20 min time point to stop
The reversibility of hrMGL inhibition by compound 26 and
MAFP was determined analogously to the method described for the
reversibility of FAAH. Briefly, in the preincubation mixture (30 mL)
m
L of buffer (50 mM Tris–
at 16,000g for 4 min at rt, and aliquots (100
m
L) were taken from the
the hrMGL concentration was 50-fold the concentration required
for the activity assay, and the inhibitor concentration was
approximately 30-fold the IC50 value. After the preincubation,
aqueous phase, which contained glycerol-1,2,3-3H, and measured
for radioactivity by liquid scintillation counting (Wallac 1450
MicroBeta; Wallac Oy, Finland).
1.8
diluted 500-fold into the assay buffer containing 2-OG. The final
incubation concentration of 2-OG was 50 M (containing 1.2 Ci of
40 Ci/mmol [3H]2-OG). The samples of 100
L were taken at 0, 5,10,
15, 20, 30, 45 and 60 min time-points and added to tubes con-
taining 400 L of cold ethyl acetate. Additionally, 100 l of assay
buffer was added. Samples were centrifuged at 16,000g for 4 min at
mL of enzyme–inhibitor mixture was taken and immediately
m
m
6.4. Assay of FAAH activity
m
6.4.1. Animals and preparation of rat brain homogenate for FAAH
assay
m
m
Eight-week-old male Wistar rats were used in these studies. All
animal experiments were approved by the local ethics committee.
The animals lived in a 12-h light/12-h dark cycle (lights on at
0700 h) with water and food available ad libitum.
rt, and aliquots (100 mL) were taken from the aqueous phase, which
contained glycerol-1,2,3-3H, and measured for radioactivity by
liquid scintillation counting (Wallac 1450 MicroBeta; Wallac Oy,
Finland).
The rats were decapitated, forebrains were dissected and
homogenized in one volume (v/w) of ice-cold 0.1 M potassium
phosphate buffer (pH 7.4) with a Potter–Elvehjem homogenizer
(Heidolph). The homogenate was centrifuged at 10,000g for 20 min
(at 4 ꢁC). The protein concentration of the supernatant was deter-
mined by the method of Bradford with BSA as a standard [74].
Aliquots of the supernatant were stored at ꢃ80 ꢁC until use.
6.6. Uptake of AEA by intact RBL2H3 cells
The uptake assay using RBL2H3 basophilic leukaemia cells was
undertaken as described previously [75] using tritium-labelled
arachidonoylethanolamide [arachidonoyl 5,6,8,9,11,12,14,15-3H]
(American Radiolabeled Chemicals Inc., St Louis, MO, USA). The
cells were preincubated with compound 26 and/or URB597 for
10 min at 37 ꢁC prior to addition of [3H]AEA (assay concentration
100 nM) and incubation for a further 10 min at 37 ꢁC. In these cells,
the uptake of AEA is driven by its subsequent FAAH-catalysed
hydrolysis to arachidonic acid [76], and the difference between the
observed activity in the absence and presence of an FAAH inhibitor
like URB597 represents the component of uptake due to FAAH.
6.4.2. In vitro assay for FAAH activity
The assay for FAAH has been described previously [50]. The
endpoint enzymatic assay was developed to quantify FAAH activity
with tritium-labelled arachidonoylethanolamide [ethanolamine
1-3H] (American Radiolabeled Chemicals Inc., St Louis, MO, USA).
The assay buffer was 0.1 M potassium phosphate (pH 7.4) and test
compounds were dissolved in DMSO (the final DMSO concentration
was not more than 5% v/v). The incubations were performed in the
presence of 0.5% (w/v) BSA (essentially fatty acid-free). Test
compounds were preincubated with rat brain homogenate
6.7. Hydrolysis of AEA and 2-AG by intact RBL2H3 cells
The assay used was that of Paylor et al. [76] using tritium-
labelled arachidonoylethanolamide [ethanolamine 1-3H] and
2-arachidonoylglycerol [glycerol-1,2,3-3H] (American Radiolabeled
Chemicals Inc., St Louis, MO, USA). The cells were preincubated
with compound 25 and/or URB597 for 10 min at 37 ꢁC prior to
addition of [3H]AEA or [3H]2-AG, as appropriate, (assay concen-
tration 100 nM) and incubation for a further 20 min at 37 ꢁC.
protein (18 m mL). At the 10 min time point,
g) for 10 min at 37 ꢁC (60
N-arachidonoylethanolamine was added to achieve the final
concentration of 2
m
M (containing 50 ꢄ 10ꢃ3 mCi of 60 Ci/mmol
[3H]AEA) with the final incubation volume of 100
mL. The incuba-
tions proceeded for 10 min at 37 ꢁC. Ethyl acetate (400
added at the 20 min time point to stop the enzymatic reaction.
Additionally, 100 L of unlabeled ethanolamine (1 mM) was added.
Samples were centrifuged at 16,000g for 4 min at rt, and aliquots
mL) was
m
6.8. Data analyses
(100 mL) were taken from the aqueous phase, which contained
ethanolamine 1-3H, and measured for radioactivity by liquid scin-
tillation counting (Wallac 1450 MicroBeta; Wallac Oy, Finland).
The results from the enzyme inhibition experiments are
presented as mean ꢂ 95% confidence intervals of at least three

3008
A. Minkkila¨ et al. / European Journal of Medicinal Chemistry 44 (2009) 2994–3008
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Chemistry and Chemical Biology (GSOCCB), The National Tech-
nology Agency of Finland, The Academy of Finland (grant 110277),
and The Research and Science Foundation of Farmos have provided
financial support for this study. In addition, CJF thanks Mrs. Eva
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Council (Grant no. 12158, medicine) for financial support.
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