Optimization of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors

Article abstract of DOI:10.1002/cmdc.201402453

At present, inhibitors of α/β-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430) potently and irreversibly inhibited hABHD6 (IC₅₀ = 44 nM) and showed ～ 230-fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling indicated that JZP-430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP-430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.

Full text of DOI:10.1002/cmdc.201402453

DOI: 10.1002/cmdc.201402453
Full Papers
Optimization of 1,2,5-Thiadiazole Carbamates as Potent
and Selective ABHD6 Inhibitors
Jayendra Z. Patel,*[a] Tapio J. Nevalainen,[a] Juha R. Savinainen,[b] Yahaya Adams,[a]
Tuomo Laitinen,[a] Robert S. Runyon,[c] Miia Vaara,[b] Stephen Ahenkorah,[a]
Agnieszka A. Kaczor,[a, d] Dina Navia-Paldanius,[b] Mikko Gynther,[a] Niina Aaltonen,[a]
Amit A. Joharapurkar,[e] Mukul R. Jain,[e] Abigail S. Haka,[c] Frederick R. Maxfield,[c]
Jarmo T. Laitinen,[b] and Teija Parkkari*[a]
Dedicated to Dr. Saurin Raval, principal scientist at Zydus Research Centre, India.
At present, inhibitors of a/b-hydrolase domain 6 (ABHD6) are
viewed as a promising approach to treat inflammation and
metabolic disorders. This article describes the development of
1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether,
34 compounds were synthesized, and their inhibitory activity
was tested using lysates of HEK293 cells transiently expressing
human ABHD6 (hABHD6). Among the compound series, 4-mor-
pholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-
430) potently and irreversibly inhibited hABHD6 (IC50 =44 nm)
and showed ~230-fold selectivity over fatty acid amide hydro-
lase (FAAH) and lysosomal acid lipase (LAL), the main off-tar-
gets of related compounds. Additionally, activity-based protein
profiling indicated that JZP-430 displays good selectivity
among the serine hydrolases of the mouse brain membrane
proteome. JZP-430 has been identified as a highly selective,
irreversible inhibitor of hABHD6, which may provide a novel
approach in the treatment of obesity and type II diabetes.
Introduction
In the central nervous system (CNS), the a/b hydrolase domain
containing 6 (ABHD6), an integral membrane serine hydrolase,
contributes to a small portion of the in vivo degradation of 2-
arachidonoylglycerol (2-AG), an endogenous lipid signaling
molecule activating the cannabinoid receptors.[1] In the brain,
ABHD6 along with the serine hydrolases monoacylglycerol
lipase (MAGL) and a/b hydrolase domain containing 12
(ABHD12) account for ~98% of 2-AG degradation;[2] 85% of
2-AG is metabolized by MAGL and 9% by ABHD12 while only
4% is attributed to ABHD6.[2] The remaining ~2% is hydrolyzed
by additional enzymes, including fatty acid amide hydrolase
(FAAH). MAGL, ABHD12 and ABHD6 have different tissue distri-
bution and subcellular localization, suggesting that they may
have distinct roles in controlling the lifetime of 2-AG.[1] To dis-
tinguish between these roles and to gain in-depth understand-
ing of their physiological significance, selective ABHD6 inhibi-
tors are needed.
Recent reports have suggested ABHD6 as an emerging ther-
apeutic target for the treatment of inflammation, metabolic
disorders (obesity and type II diabetes mellitus) and epilep-
sy.[3–6] ABHD6 inhibitors may have certain advantages over in-
hibitors of MAGL and ABHD12. First, genetic inactivation of
MAGL causes a massive increase in brain 2-AG levels, leading
to psychotropic side effects and cannabinoid receptor desensi-
tization.[7–9] Second, even though ABHD12 is still poorly charac-
terized, studies with genetically ABHD12 deficient mice sug-
gest that inactivation of this serine hydrolase leads to age-de-
pendent symptoms that resemble the human neurodegenera-
tive disorder PHARC (polyneuropathy, hearing loss, ataxia, reti-
nosis pigmentosa, cataract).[10] Inhibition of ABHD6, on the
other hand, is expected to induce only a slight increase in
2-AG levels suggesting that ABHD6 inhibitors may have less
CNS-related side effects.[2,4,11]
[a] J. Z. Patel, Dr. T. J. Nevalainen, Y. Adams, Dr. T. Laitinen, S. Ahenkorah,
Dr. A. A. Kaczor, Dr. M. Gynther, N. Aaltonen, Dr. T. Parkkari
School of Pharmacy, University of Eastern Finland
P.O. Box 1627, 70211 Kuopio (Finland)
E-mail: jayendra.patel@uef.fi
teija.parkkari@uef.fi
[b] Dr. J. R. Savinainen, M. Vaara, D. Navia-Paldanius, Dr. J. T. Laitinen
School of Medicine, Institute of Biomedicine/Physiology
University of Eastern Finland
P.O. Box 1627, 70211 Kuopio (Finland)
[c] R. S. Runyon, Dr. A. S. Haka, Prof. F. R. Maxfield
Department of Biochemistry, Weill Cornell Medical College
1300 York Avenue, New York, NY 10065 (USA)
[d] Dr. A. A. Kaczor
Department of Synthesis & Chemical Technology of Pharmaceutical
Substances, Faculty of Pharmacy with Division of Medical Analytics
Medical University of Lublin, 4a Chodzki St., 20093 Lublin (Poland)
[e] Dr. A. A. Joharapurkar, Dr. M. R. Jain
Department of Pharmacology & Toxicology, Zydus Research Centre
Sarkhej Bavla NH8A, Moraiya, Ahmedabad 382210, Gujarat (India)
To date, only a few ABHD6 inhibitors have been reported
(Figure 1). In 2007, Cravatt and co-workers reported the iden-
tification of WWL70 (1), a potent and selective carbamate-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402453.
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its LAL. So far, numerous carba-
mate compounds have been re-
ported as inhibitors of endocan-
nabinoid
metabolizing
en-
zymes,[12,15,20–23] (for recent re-
views, see References [24–27]).
We therefore thought to use
1,2,5-thiadiazole carbamate (I,
Figure 2) scaffold for the devel-
opment of inhibitors of the en-
docannabinoid metabolizing en-
zymes. A limited structure-activi-
ty relationship (SAR) study based
on this scaffold has been report-
ed,[19] thus leaving room for fur-
ther optimization of the 1,2,5-
Figure 1. Selective and non-selective ABHD6 inhibitors (1–7).
thiadiazole carbamate scaffold
(II, Figure 2). The mechanism for
based inhibitor whose selectivity among the serine hydrolases
was evaluated using activity-based protein profiling (ABPP).[12]
Marrs et al. described UCM710 (2), a dual inhibitor of ABHD6
and FAAH.[13] Examples of non-selective ABHD6 inhibitors in-
clude methylarachidonoyl fluorophosphonate (MAFP), orlistat
(tetrahydrolipstatin, THL, 3), RHC-80267, and the triterpene
pristimerin.[14] Recently, Cravatt and colleagues disclosed sever-
al other ABHD6 inhibitors such as carbamate based compound
WWL123 (4), an isostere analogue of WWL70, and triazole urea
analogues (e.g., KT195 (5) and KT182 (6)) as potent and selec-
tive ABHD6 inhibitors.[15–17] Very recently, Janssen et al. reported
glycine sulfonamide analogue LEI-106 (7) as a potent and se-
lective dual inhibitor of sn-1-diacylglycerol lipase a (DAGL-a)
and ABHD6.[18]
LAL inhibition via 1,2,5-thiadiazole carbamates is suggested to
occur by carbamoylation of the active site serine with the
1,2,5-thiadiazole alcohol group serving as the leaving group (I,
Figure 2). In our compound series (Figure 2 and 3), we used
1,2,5-thiadiazole scaffold by introducing different cyclic and
non-cyclic secondary amines at the main core while a small set
of different cyclic amines were introduced as potential leaving
groups.
Herein we report the optimization of 1,2,5-thiadiazole carba-
mates as novel ABHD6 inhibitors. The selectivity against other
endocannabinoid targets, serine hydrolases of the mouse
membrane proteome as well as LAL has been evaluated, and
the inhibitory activity data have been used to explore the SAR.
Finally, homology modeling and molecular docking were used
in attempts to provide insight into how the best compounds
interacted optimally with the active site of ABHD6.
In 2010, Helquist and co-workers reported 1,2,5-thiadiazole
carbamates (I, Figure 2) as potent inhibitors of lysosomal acid
lipase (LAL, also known as LIPA).[19] LAL was recently identified
as a potential therapeutic target for Niemann–Pick disease
type C (NPC), a condition characterized by a gradual lysosomal Results and Discussion
accumulation of lipids such as cholesteryl esters and triglycer-
Chemistry
ides. Additionally, Helquist and colleagues reported that orlistat
(3), which acts as a broad-spectrum lipase inhibitor, also inhib-
The synthesis of 1,2,5-thiadiazole carbamates (22–55) is shown
in Scheme 1. Commercially avail-
able 3,4-dichloro-1,2,5-thiadia-
zole was coupled with the ap-
propriate secondary amine to
afford a corresponding mono-
chloro 1,2,5-thiadiazole deriva-
tive (8–14), which was then con-
verted in to 1,2,5-thiadiazole al-
cohol (15–21) via treatment with
aqueous alkali. Finally, coupling
with appropriate carbamoyl
chloride gave the desired 1,2,5-
thiadiazole carbamates (22–55).
The synthesis of monochloro
1,2,5-thiadiazole derivatives (8–
14), 1,2,5-thiadiazole alcohol de-
rivatives (15–21) and carbamoyl
Figure 2. Optimization of 1,2,5-thiadiazole carbamates.
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Cyclic “N” containing thiadi-
azole carbamates (structural
modifications of main core and
leaving group)
As an initial step, we synthesized
two previously reported LAL in-
hibitors having piperidine and
morpholine rings at opposite
sides of the thiadiazole core,
that is, compounds 22 and 23
(Table 1). Both 22 and 23
showed excellent ABHD6 inhibi-
tory activities with potencies in
the low nanomolar range (IC50
52 nm and 85 nm, respectively)
but these compounds inhibited
also FAAH with moderate poten-
cies (IC50 0.40 and 0.30 mm, re-
Figure 3. Variations around the 1,2,5-thiadiazole scaffold.
spectively). As compound 22
was more potent of these two
we retained the thiadiazole pi-
peridine core in the newly synthesized analogues 24 and 25.
We found a similar inhibitory activity trend for pyrrolidine ana-
logue 24 and 1,2,3,4-tetrahydroisoquinoline analogue 25, al-
though decreased inhibitory potencies toward ABHD6 and
FAAH were observed. Because none of the analogues showed
significant improvement in selectivity, we clarified the effect of
the leaving group by synthesizing different thiadiazole carba-
mates (26–30) in which the piperidine carbamate scaffold was
kept intact. Substituted piperidine analogues 26 and 28 as
well as piperazine analogue of 26 (compound 27) showed sim-
ilar FAAH inhibition, while only weak inhibition of ABHD6 was
observed. However, fused bicyclic analogues (compounds 29
and 30) showed improved FAAH inhibition (IC50 17 nm and
31 nm, respectively) while moderate inhibitory activities were
observed against ABHD6 (IC50 0.46 and 0.56 mm, respectively).
Compounds 22–30 did not show any appreciable inhibition of
hMAGL or hABHD12 (Table S3, Supporting Information).
To reveal additional off-targets, we screened selected ana-
logues (22, 23, 29 and 30) at 1 mm concentration against the
serine hydrolases of the mouse brain membrane proteome
using competitive ABPP, essentially as previously described[14,28]
(Figure S1, Supporting Information). We found that all the
tested compounds showed complete inhibition of FAAH, and
inhibition of ABHD6 was also evident. Moreover, an unidenti-
fied serine hydrolase (a protein band migrating at ~30 kDa)
was found as an off-target site of the four analogues.
Scheme 1. Synthesis of 1,2,5-thiadiazole carbamate derivatives 22–55. Re-
agents and conditions: a) 110–1208C, 2–6 h or K2CO3, DMF, 100–1108C, 6–
10 h; b) aq. NaOH or KOH, DMSO, reflux, 1–6 h; c) pyridine, CH2Cl2, triphos-
gene, 0–58C or À788C, 3–4 h; d) dry THF, 15–21, KOtBu, 0–258C, 16–24 h.
chloride compounds was performed as per literature proce-
dures with minor modifications (see Supporting Information).
SAR of ABHD6 inhibitors
The inhibitory activities of the synthesized compounds were
initially screened at 1 mm concentration against hABHD6 and
hABHD12, and at 10 mm concentration against hFAAH and
hMAGL. As FAAH was found to be the main off-target site, in-
hibitory activity data concerning hABHD6 and hFAAH are pre-
sented in Tables 1–4, while results of the hABHD12 and hMAGL
inhibition experiments are presented in Tables S3 and S4 (see
Supporting Information).
Non-cyclic “N” containing thiadiazole carbamates (structural
modifications of the main core)
As no satisfactory selectivity for ABHD6 over FAAH was ach-
ieved with the analogues 22–30 (selectivity ratio <30-fold), we
explored the thiadiazole carbamates further by opening the
“N” containing ring system in the main core (see Figure 2).
N,N-dimethyl analogue 31 showed weak FAAH inhibition (IC50
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6.45 mm) while no inhibition was
observed against the other
tested enzymes (Table 2 and Ta-
bles S3,S4 in Supporting Infor-
mation). Replacing one methyl
Table 1. Inhibitory activities of 1,2,5-thiadiazole carbamates 22–30 against hABHD6 and hFAAH.
Compd
Structure
pIC50 (range)[a][b]
hABHD6
hFAAH
group of 31 with
a phenyl
22
7.28 (7.23–7.32) [0.052]
7.07 (7.03–7.10) [0.085]
6.39 (6.29–6.49) [0.40]
group (compound 32) resulted
in excellent ABHD6 inhibitory ac-
tivity (IC50 22 nm), and also im-
proved ABHD6 selectivity (404-
fold) over FAAH (IC50 8.9 mm).
However, adding another phenyl
group in the compound 32
(compound 33) resulted in com-
plete loss of activity toward all
the tested enzymes. Additionally,
the N,N-diisopropyl analogue
(compound 34) showed loss of
activity, which may be due to
shielding of the carbonyl group
from attack by the serine hy-
droxy group at the active site of
the enzyme. As compound 32
turned out to be the best
ABHD6 inhibitor, we investigated
further the optimal structural re-
quirement needed for inhibitory
activity and selectivity. Changing
the methyl group of compound
32 into an ethyl (compound 35)
resulted in a ~20-fold drop in
potency, while changing the
phenyl (32) into benzyl (36) re-
sulted in a 2-fold increase in
ABHD6 inhibitory activity (IC50
10 nm). Compound 35 showed
no noticeable inhibition of the
other tested enzymes (Table 2
and Table S3 in Supporting Infor-
mation), while loss of selectivity
was observed for compound 36
as it also showed improved
FAAH inhibition (IC50 67 nm) as
well as weak MAGL inhibition
(IC50 5.6 mm; Table S3, Support-
ing Information).
23
6.48 (6.41–6.55) [0.30]
24
25
6.58 (6.43–6.73) [0.26]
6.88 (6.80–6.95) [0.13]
6.09 (6.01–6.18) [0.81]
6.25 (6.23–6.27) [0.56]
26
27
28
29
30
41%[b]
5.83 (5.34–6.31) [1.47]
6.68 (6.51–6.84) [0.21]
6.49 (6.30–6.67) [0.32]
7.77 (7.71–7.83) [0.017]
7.51 (7.48–7.53) [0.031]
40%[b]
15%[b]
6.34 (6.22–6.45) [0.46]
6.25 (6.19–6.31) [0.56]
WWL70 (1)
THL (3)
JZP-327A[e]
–
–
–
7.07Æ0.05 [0.085][c]
7.32Æ0.11 [0.048][c]
NI[d]
30%[b]
NA[d]
In competitive ABPP of the
mouse brain membrane pro-
teome, compounds 32 and 36
were found to inhibit ABHD6
completely (Figure S2, Support-
ing Information) at 1 mm concen-
tration. As expected, 36 also tar-
geted FAAH. In addition, an un-
identified serine hydrolase (a
protein band migrating at
~30 kDa) was inhibited by 32.
7.94 (7.91–7.97) [0.011]
[a] pIC50 values (Àlog10 [IC50, mm]) represent the mean (range) of two independent experiments performed in
duplicate. IC50 values were calculated for those compounds exhibiting ꢀ50% inhibition at 1 mm for hABHD6
and at 10 mm for hFAAH; where <50% inhibition was observed, the percent inhibition at the aforementioned
concentration is given. IC50 values are derived from the mean pIC50 values as shown in parentheses. [b] Percent
inhibition values represent the mean of two independent experiments performed in duplicate. [c] pIC50 values
are the mean ÆSEM of three independent experiments performed in duplicate; data taken from Ref. [14].
[d] NA: not analyzed; NI: no inhibition. [e] S-3-(1-(4-isobutylphenyl)ethyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one
(JZP-327A)[29] was used as reference FAAH inhibitor.
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ABHD6 inhibition, while methoxy analogues (com-
pounds 47 and 48) showed only marginal differences
in their ABHD6 inhibitory activities. However, their
ortho analogues (46 and 49) showed complete loss
of ABHD6 inhibition. Finally, substitution of the
phenyl ring with the meta-phenyl resulted in almost
a 40-fold loss (compound 50) of ABHD6 inhibitory
potency, and the bulky trimethyl substitution (com-
pound 51) lead to complete loss of activity. None of
the analogues 37–51 showed appreciable inhibition
of hFAAH, hMAGL or hABHD12 (Table 3 and Table S4
in Supporting Information).
Table 2. Inhibitory activities of novel 1,2,5-thiadiazole carbamates 31–36 against
ABHD6 and FAAH.
[a]
Compd
Structure
pIC50
hABHD6
10%[b]
hFAAH
31
5.19 (5.17–5.20) [6.45]
32
33
34
35
36
7.66Æ0.07 [0.022]
5.06 (5.05–5.07) [8.91]
To screen inhibitor selectivity among the serine hy-
drolases in mouse brain membrane proteome, we
performed competitive ABPP for selected analogues
(42 and 45) and found complete inhibition of ABHD6
at 1 mm concentration (Figure S3, Supporting Infor-
mation). In addition, an unidentified serine hydrolase
migrating at ~30 kDa was targeted by the com-
pounds 42 and 45.
NI[c]
NI[c]
NI[c]
NI[c]
N-Methyl-N-cycloalkyl thiadiazole carbamates
6.33Æ0.13 [0.47]
8.01Æ0.03 [0.010]
24%[b]
As no further improvement in ABHD6 inhibitory ac-
tivity or selectivity was obtained with the analogues
37–51, we replaced the phenyl ring of compound 32
by different cycloalkyl rings (compounds 52–55,
Table 4). Increasing the size of the cycloalkyl ring
from a six- to eight-membered ring (52–54) resulted
in an approximate 2–4-fold loss of ABHD6 inhibition,
while no inhibition of FAAH was observed at 10 mm.
As increased ring size also causes increased lipophi-
licity (i.e., clogP for 52 is 4.4 while for 54 it is 5.5;
Table S5, Supporting Information), we replaced the
piperidine ring of compound 54 with a morpholine
ring (compound 55). Consequently, compound 55
had ABHD6 inhibitory activity similar to that of com-
pounds 52 and 53, while being less lipophilic
7.20 (7.17–7.23) [0.063]
[a] pIC50 values (Àlog10 [IC50, mm]) represent the mean ÆSEM of three independent ex-
periments performed in duplicate (for hABHD6) or the mean (range) of two independ-
ent experiments performed in duplicate (for hFAAH). IC50 values were calculated for
those compounds exhibiting ꢀ50% inhibition at 1 mm for hABHD6 and at 10 mm for
hFAAH; where <50% inhibition was observed, the percent inhibition at the afore-
mentioned concentration is given. IC50 values are derived from the mean pIC50 values
as shown in parentheses. [b] Percent inhibition values represent the mean of two
independent experiments performed in duplicate. [c] NI: no inhibition.
N-Methyl-N-substituted phenyl thiadiazole carbamates
(clogP=4.1). None of these compounds 52–55 showed any in-
hibition of the other enzymes tested (Table S4, Supporting In-
formation). Finally, when these analogues (52–55) were tested
using competitive ABPP, all the compounds except compound
52 selectively targeted ABHD6 when tested at 1 mm concentra-
tion (Figure S4, Supporting Information). Compound 52 addi-
tionally targeted the ~30 kDa serine hydrolase with unknown
identity.
Next, we investigated the effect of different substituents on
the phenyl ring of compound 32 by synthesizing the ana-
logues 37–51 (Table 3). Among these, compounds having an
electron withdrawing group (EWG) at the para position of the
phenyl ring (compounds 37, 41 and 42) showed a 4- to 55-
fold loss of ABHD6 inhibitory activity, and the cyano analogue
40 showed complete loss of activity. Switching the para-nitro
substituent (compound 37) to the meta position (compound
38) retained activity, while in the ortho position (compound
39) ABHD6 inhibitory activity was completely lost. Further-
more, both para- and meta-fluoro analogues (compounds 42
and 43) were almost equipotent in inhibiting ABHD6. In a simi-
lar fashion, electron donating groups (EDG) at the para-posi-
tion resulted in a 6- to 12-fold loss of ABHD6 inhibitory activity,
depending on the nature of EDG (44 and 47). However, switch-
ing back the methyl substituent from the para (44) to the
meta position (45) showed almost a threefold improvement in
ABHD6 selectivity
LAL inhibitory activity
As our compound series was developed from the compounds
that were originally designed as LAL inhibitors, we tested the
activity of these compounds toward LAL, essentially as previ-
ously described.[19] We selected several potent analogues (22,
23, 29, 30, 32, 36, 42, 45 and 52–55) from our compound
series containing both known LAL inhibitors as well as novel
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Table 3. Inhibitory activities of novel 1,2,5-thiadiazole carbamates 37–51
Table 3. (Continued)
against ABHD6 and FAAH.
[a]
Compd Structure
pIC50
Inhib. [%][b]
hFAAH
[a]
Compd Structure
pIC50
Inhib. [%][b]
hFAAH
hABHD6
hABHD6
50
51
6.04Æ0.10 [0.91]
13
7
37
5.90Æ0.08 [1.25]
19
NI[c]
38
39
40
5.92Æ0.05 [1.20]
11
46
19
[a] pIC50 values (Àlog10 [IC50, mm]) represent the mean ÆSEM of three in-
dependent experiments performed in duplicate. IC50 values were calculat-
ed for those compounds exhibiting ꢀ50% inhibition at 1 mm and are de-
rived from the mean pIC50 values as shown in parentheses. [b] Percent in-
hibition at 1 mm for hABHD6 and at 10 mm for hFAAH is reported; values
represent the mean of two independent experiments performed in dupli-
cate. [c] NI: no inhibition.
NI[c]
15%[b]
Table 4. Inhibitory activities of novel 1,2,5-thiadiazole carbamates 52–55
against ABHD6 and FAAH.
41
6.39Æ0.03 [0.41]
16
[a]
Compd
Structure
pIC50
Inhib. [%][b]
hFAAH
hABHD6
42
43
44
45
46
47
48
49
7.11Æ0.07 [0.078]
7.22Æ0.05 [0.060]
6.83Æ0.04 [0.15]
7.27Æ0.07 [0.054]
17%[b]
22
48
21
9
52
7.36Æ0.05 [0.044]
7.37Æ0.05 [0.043]
16
21
53
54
7.14Æ0.06 [0.072]
7.36Æ0.05 [0.044]
13
18
JZP-430
(55)
40
17
17
18
[a] pIC50 values (Àlog10 [IC50, mm]) represent the mean ÆSEM of three in-
dependent experiments performed in duplicate. IC50 values were calculat-
ed for those compounds exhibiting ꢀ50% inhibition at 1 mm and are de-
rived from the mean pIC50 values as shown in parentheses. [b] Percent in-
hibition at 10 mm; values represent the mean of two independent experi-
ments performed in duplicate.
6.58Æ0.04 [0.26]
6.71Æ0.07 [0.19]
11 %[b]
ABHD6 inhibitors, and tested them at 10 mm concentration.
(Figure 4). Among the cyclic analogues (22, 23, 29 and 30) the
previously reported LAL inhibitors 22 and 23 were found to in-
hibit LAL activity almost completely. A similar trend was ob-
served for our compounds 29 and 30, both having bulky cyclic
rings as potential leaving groups. Among the non-cyclic ana-
logues (32, 36, 42, 45 and 52–55), N-methyl-N-aryl analogues
32, 42 and 45 were found to inhibit LAL activity by 25–35%,
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Figure 4. Lysosomal acid lipase (phLAL) activity in the presence of selected
thiadiazole carbamates, each at 10 mm. Enzymatic activity at 378C was quan-
tified as background-corrected 4-methylumbelliferone fluorescence, normal-
ized to the DMSO control average value. Data are averages ÆSEM from two
independent experiments (n=5 wells used for quantification per experi-
ment).
and interestingly, N-methyl-N-benzyl analogue 36 showed
>99% inhibition. N-methyl-N-cycloalkyl analogues 52–55 were
also weak LAL inhibitors showing <33% inhibition at 10 mm
concentration. Notably, the ABHD6 inhibitor 55 (JZP-430) was
found to have only a slight inhibition (<20%) of LAL at 10 mm
concentration. We determined the dose–response curves and
calculated the IC50 values for those compounds that in the
initial screen showed >50% inhibition (Table S6, Supporting
Information).
Activity-based protein profiling (ABPP)
Next, we tested in more detail the selectivity of our carba-
mate-based analogue JZP-430 (55) using competitive ABPP of
the mouse brain membrane proteome (Figure 5). We used ear-
lier reported inhibitors WWL70 (1)[12] and JZP-327A[29] at the in-
dicated concentrations to locate the bands of ABHD6 and
FAAH, respectively. We found that JZP-430 (55) inhibited
ABHD6 dose-dependently, being effective already at 0.25 mm
concentration. Selective inhibition of ABHD6 was detected
even at 1 mm concentration while negligible inhibition of FAAH
was observed at 2.5 mm concentration. At 20-fold (5 mm) con-
centration partial inhibition of FAAH was detected. In short,
when tested at below 2.5 mm concentration, JZP-430 (55) ap-
peared to be selective for ABHD6 over the other detectable
brain serine hydrolases, including FAAH, MAGL and ABHD12.
Figure 5. Competitive ABPP of compound 55 (JZP-430) among the serine
hydrolases in mouse whole-brain membrane proteome. Molecular weight
markers are indicated at left. Reference inhibitors WWL70 (1) and JZP-327A
were used at the indicated concentrations to identify the following serine
hydrolases from the gel: ABHD6, inhibited by WWL70 (1)[12] and FAAH, inhib-
ited by JZP-327A.[29] In addition, protein bands corresponding to MAGL (dou-
blet) and ABHD12 are indicated. Note that JZP-430 (55) inhibits only probe
labeling of ABHD6 at 0.25 mm. Selective inhibition of ABHD6 was evident at
concentrations <2.5 mm, whereas partial inhibition of FAAH was observed at
5 mm (20-fold). The gel is representative of two ABPP experiments with simi-
lar outcomes.
Reversibility of ABHD6 inhibition
To get deeper insight into ABHD6 binding mode of JZP-430
(55), we tested its potency to inhibit ABHD6 using a 96-well
format dilution method.[28] As a result, both the established ir-
reversible ABHD6 inhibitor WWL70 (1) and JZP-430 (55) fully
retained their potencies during the 90 min incubation period
following a fast 40-fold dilution of the enzyme-inhibitor com-
plex (Figure 6), a finding suggesting that compound 55 inacti-
vated hABHD6 in an irreversible manner.
Selectivity over the other endocannabinoid targets
Finally, JZP-430 (55) was tested against the cannabinoid CB1
and CB2 receptors but it did not show any appreciable agonist
or antagonist activity when tested at 10 mm concentration
(Table S7, Supporting Information).
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the main core, have a comple-
mentary shape with the active
site cavity of our model
(Figure 7). In addition, the piperi-
dine/morpholine rings dock well
to the other end of the l-shaped
site. However, the N-containing
bicyclic rings of compounds 29
and 30 seem to be too rigid and
thus failed to dock at this posi-
tion. As a consequence, model-
ing studies suggest that good
inhibitory activity is gained
when proper shape complemen-
tarity meets easy access for the
carbonyl to oxyanion hole prior
to nucleophilic attack. Com-
pounds 54 and 55 (JZP-430)
have more spacious aliphatic
ring structures located in this
narrower region of the FAAH
active site, so no converged
docking poses were found.
When examining the interaction
of compounds 29 and 30 with
FAAH, the bulkiest N-containing
bicyclic ring system was found
Figure 6. Potencies (pIC50) of the irreversible ABHD6 inhibitor WWL70 (1) and compound 55 (JZP-430) are not
time-dependently changed following a rapid 40-fold dilution of inhibitor-treated hABHD6 preparation, indicating
that within the timeframe studied, compound 55 acts as an irreversible ABHD6 inhibitor. Note that due to meth-
odological limitations, the IC50 values obtained by the dilution method are not directly comparable to those ob-
tained using the routine assay protocol (Table 4).[28,30] Data are mean ÆSEM from three independent experiments
(each with duplicate wells).
Molecular modeling
to dock to the entrances of the acyl binding site and mem-
brane access channel, while the piperidine/morpholine rings fit
well in the mouth of the cytoplasm exit (Figure S5, Supporting
Information).
We assumed in our homology modeling studies that the cata-
lytic triad of ABHD6 comprises Ser148–His306–Asp278 and
the oxyanion hole is formed by Met149 and Phe80.[14] A ho-
mology model of ABHD6 has been successfully used in dock-
ing studies.[31] Our comparative modeling studies suggested
that among the current template structures available, template
PDB ID: 2XMZ[32] resulted in optimal active site geometry for
docking studies.
Conclusions
In this study, we identified 1,2,5-thiadiazole carbamates as
novel ABHD6 inhibitors and used molecular modeling to
define their interactions with the catalytic site of the enzyme.
The best compound of the series, in terms of both potency
and selectivity, was 4-morpholino-1,2,5-thiadiazol-3-yl cyclooc-
tyl(methyl)carbamate (JZP-430, 55), as this compound inhibited
human a/b hydrolase domain 6
The docking poses of highest affinity support the idea that
bulkiness at the main core and leaving group modulate the se-
lectivity for ABHD6 over FAAH. In the case of ABHD6, com-
pounds 54 and 55 (JZP-430), which have larger cyclic rings at
with low-nanomolar potency
(IC50 44 nm) and was >200-fold
selective for ABHD6 over FAAH
and LAL enzymes. Moreover,
compound 55 showed good se-
lectivity for ABHD6 over the
other serine hydrolases detected
in the mouse brain membrane
proteome using ABPP. Com-
pound 55 (JZP-430) showed irre-
versible binding in our reversibil-
ity assays and in molecular mod-
eling studies, it was docked well
into the active site of hABHD6
Figure 7. Most favorable Glide docking poses of high-affinity compounds 54 (left) and 55 (JZP-430) (right) to the
ABHD6 active site in a homology model. Catalytic residues are colored using yellow carbon atoms, and the surface
of the active site is presented.
and was shown to have favora-
ble interactions, including impor-
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4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl pyrrolidine-1-carboxylate
(24): White solid product (55 mg, 12%); H NMR (CDCl3): d=3.58–
tant hydrogen bonding of the carbonyl oxygen atom, to the
oxyanion hole.
1
3.55 (m, 2H), 3.53–3.50 (m, 2H), 3.43–3.40 (m, 4H), 2.0–1.93 (m,
4H), 1.67–1.62 ppm (m, 6H); 13C NMR (CDCl3): d=153.7, 150.3,
146.6, 49 (2C), 46.8, 46.7, 29.7, 25.8, 25.4, 24.9, 24.2 ppm; MS
(ESI+): m/z=283.22 [M+H]+.
Experimental Section
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl 3,4-dihydroisoquinoline-
2(1H)-carboxylate (25): Brown oil (90 mg, 62%); 1H NMR (CDCl3):
d=7.24–7.10 (m, 4H), 4.79 (s, 1H), 4.71 (s, 1H), 3.87 (t, J=5.6 Hz,
1H), 3.81 (t, J=5.6 Hz, 1H), 3.38–3.35 (m, 4H), 2.95 (t, J=5.9 Hz,
2H), 1.65–1.59 ppm (m, 6H); 13C NMR (CDCl3): d=153.7, 151.2,
146.4, 134.6, 132.5, 128.9, 127, 126.7, 126.4, 49, 46.5, 42.6, 38.7, 30,
25.9, 25.4, 24.4 ppm; MS (ESI+): m/z=345.64 [M+H]+.
Chemistry
Materials and methods: Reagents and solvents were purchased
from commercial suppliers and were used without further purifica-
tion. Reactions were monitored by thin-layer chromatography
using aluminum sheets coated with silica gel F245 (60 ꢁ, 40–63 mm,
230–400 mesh) with suitable UV visualization. Purification was car-
ried out by flash chromatography (FC) on J. T. Baker’s silica gel for
chromatography (pore size 60 ꢁ, particle size 50 nm). Petroleum
4-(4-Phenylpiperidin-1-yl)-1,2,5-thiadiazol-3-yl piperidine-1-car-
1
boxylate (26): White solid product (542 mg, 42%); H NMR (CDCl3):
1
ether (PE) used for chromatography is of fraction 40–608C. H NMR
d=7.30 (t, J=7.5 Hz, 2H), 7.21 (t, J=8.3, 3H), 4.13–4.07 (m, 2H),
3.57–3.55 (brs, 2H), 3.54–3.50 (brs, 2H), 3.03–2.97 (m, 2H), 2.72–
2.68 (m, 1H), 1.92–1.80 (m, 4H), 1.65–1.61 (m, 4H), 1.56–1.52 ppm
(m, 2H); 13C NMR (CDCl3): d=153.5, 150.8, 146.7, 145.6, 128.5 (2C),
126.7 (2C), 126.4, 48.7 (2C), 45.9, 45.5, 42.4, 32.9, 32.8, 26, 25.4,
24 ppm; MS (ESI+): m/z=373.25 [M+H]+.
and 13C NMR were recorded on a Bruker Avance AV 500 (Bruker
Biospin, Switzerland) spectrometer operating on 500.1 and
125.8 MHz, respectively. Tetramethylsilane (TMS) was used as an in-
1
ternal standard for H NMR. Chemical shifts are reported in ppm on
the d scale from an internal standard of solvent (CDCl3 7.26 and
77.0 ppm, DMSO 2.50). The spectra were processed from the re-
corded FID files with TOPSPIN 2.1 software. The following abbrevia-
tions are used: s, singlet; brs, broad singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. Coupling constants are reported in Hz.
ESI-MS spectra were acquired using a LCQ quadrupole ion trap
mass spectrometer equipped with an electrospray ionization
source (Thermo LTQ, San Jose, CA, USA). Elemental analyses were
performed on a ThermoQuest CE instrument (EA 1110 CHNS-O) or
a PerkinElmer PE 2400 Series II CHNS-O Analyzer.
4-(4-Phenylpiperazin-1-yl)-1,2,5-thiadiazol-3-yl piperidine-1-car-
1
boxylate (27): White solid (63 mg, 8%); H NMR (CDCl3): d=7.26 (t,
J=8.0 Hz, 2H), 6.94 (d, J=7.9 Hz, 2H), 6.88 (t, J=7.3 Hz, 1H), 3.60–
3.58 (m, 6H), 3.53–3.49 (brs, 2H), 3.27–3.25 (m, 4H), 1.67–1.63 ppm
(brs, 6H); 13C NMR (CDCl3): d=153.1, 151.1, 150.8, 147.2, 129.2
(2C), 120.4, 116.5 (2C), 49 (2C), 47.9 (2C), 46, 45.6, 26, 25.4,
24.1 ppm; MS (ESI+): m/z=374.21 [M+H]+.
4-(4-Benzylpiperidin-1-yl)-1,2,5-thiadiazol-3-yl piperidine-1-car-
boxylate (28): White solid (134 mg, 31%); 1H NMR (DMSO): d=
7.27 (t, J=7.5 Hz, 2H), 7.17 (t, J=7.0 Hz, 3H), 4.02 (s, 1H), 3.86 (d,
J=12.8 Hz, 2H), 3.56–3.51 (brs, 2H), 3.43–3.38 (brs, 2H), 3.32–3.28
(m, 1H), 2.83 (t, J=11.9 Hz, 2H), 2.53–2.51 (m, 2H), 1.75–1.71 (m,
1H), 1.63–1.52 (m, 6H), 1.27–1.19 ppm (m, 2H); 13C NMR (CDCl3):
d=153.5, 150.8, 146.6, 140.2, 129.1 (2C), 128.3 (2C), 126, 48.3, 46,
45.5, 43.1, 37.8, 31.6 (2C), 29.7, 26, 25.4, 24.1 ppm; MS (ESI+):
m/z=387.23 [M+H]+.
General procedures for preparation of 1,2,5-thiadiazole carba-
mates (22–55):[19] KOtBu (1.3 equiv) was added to a solution of
1,2,5-thiadiazole alcohol (1.0 equiv) in dry THF (0.2m) at 08C. The
mixture was stirred at the same temperature for 10–30 min. Carba-
moyl chloride (1.0 equiv) was added slowly under inert atmos-
phere. The reaction mixture was allowed to warm and stirred at
20–258C for another 16–24 h. The progress of the reaction was
monitored by TLC using 20% EtOAc in PE as a mobile phase. Reac-
tion mixture was diluted with EtOAc. It was washed with H2O and
brine. The organic layer was dried over sodium sulfate, filtered and
concentrated under vacuum to afford crude 1,2,5-thiadiazole car-
bamates which were purified by flash column chromatography
using PE/EtOAc (9:1) as an eluent. The desired fractions were col-
lected and solvents were evaporated on a rotatory evaporator to
afford 1,2,5-thiadiazole carbamates. The obtained solid 1,2,5-thia-
diazole carbamate was stirred in minimum amount of solvent (n-
hexane or di-isopropyl ether (DIPE)) for 10–12 min and, filtered and
dried. The purity of the synthesized 1,2,5-thiadiazole carbamates
(22–55) were determined through combustion analyses and are
ꢀ95% (see Table S1 and S2, Supporting Information).
4-(3,4-Dihydroisoquinoline-2-(1H)-yl)-1,2,5-thiadiazole-3-yl piper-
idine-1-Carboxylate (29): Off white solid (230 mg, 42%); 1H NMR
(CDCl3): d=7.18–7.09 (m, 4H), 4.67 (s, 2H), 3.75 (t, J=5.7 Hz, 2H),
3.65–3.61 (brs, 2H) 3.55–3.51 (brs, 2H), 2.96 (t, J=5.9 Hz, 2H),
1.68–1.64 ppm (m, 6H); 13C NMR (CDCl3): d=152.8, 150.9, 146.2,
134, 133.4, 128.8, 126.5, 126.3 (2C), 49.7, 46, 45.6, 45.3, 28.7, 26,
25.4, 24.1 ppm; MS (ESI+): m/z=345.19 [M+H]+.
4-(Octahydroisoquinoline-2-(1H)-yl)-1,2,5-thiadiazole-3-yl piperi-
dine-1-carboxylate (30): White solid (200 mg, 68%); 1H NMR
(DMSO): d=3.93–3.90 (brs, 1H), 3.75–3.72 (brs, 1H), 3.54 (d, J=
4.8 Hz, 2H), 3.40 (d, J=5.3 Hz, 2H), 2.87 (t, J=12.4 Hz, 1H), 2.54–
2.51 (m, 1H), 1.69–1.65 (m, 2H), 1.59–1.48 (m, 9H), 1.25–1.06 (m,
5H), 0.97–0.90 ppm (m, 2H); 13C NMR (CDCl3): d=153.5, 150.8,
146.5, 54.3, 48.8, 45.9, 45.5, 41.8, 41.5, 32.9, 32.4, 30.1, 26.3, 26,
25.9, 25.4, 24.1 ppm; MS (ESI+): m/z=351.23 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl morpholine-4-carboxylate
(22): White solid (270 mg, 56%); 1H NMR (CDCl3): d=3.74–3.70
(brs, 4H), 3.66–3.62 (brs, 2H), 3.55–3.51 (brs, 2H), 3.37–3.35 (m,
4H), 1.64–1.60 ppm (m, 6H); 13C NMR (CDCl3): d=153.7, 150.9,
146.2, 66.6, 66.4, 49 (2C), 45.2, 44.5, 25.4 (2C), 24.2 ppm; MS
(ESI+): m/z=299.05 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl dimethylcarbamate (31):
1
White solid (220 mg, 79%); H NMR (CDCl3): d=3.41–3.39 (m, 4H),
3.11 (s, 3H), 3.04 (s, 3H), 1.66–1.62 ppm (m, 6H); 13C NMR (CDCl3):
d=153.6, 152.1, 146.5, 48.9 (2C), 37, 36.6, 25.3, 25.1, 24.1 ppm; MS
(ESI+): m/z=257.04 [M+H]+.
4-Morpholino-1,2,5-thiadiazol-3-yl piperidine-1-carboxylate (23):
1
White solid (190 mg, 42%); H NMR (CDCl3): d=3.81–3.79 (m, 4H),
3.59–3.57 (m, 2H), 3.54–3.53 (m, 2H), 3.45–3.44 (m, 4H), 1.69–
1.57 ppm (m, 6H); 13C NMR (CDCl3): d=153.1, 150.8, 146.7, 66.3
(2C), 48.1 (2C), 46, 45.6, 26, 25.4, 24 ppm; MS (ESI+): m/z=299.02
[M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(phenyl)carbamate
1
(32): White solid (132 mg, 38%); H NMR (CDCl3): d=7.42–7.39 (m,
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2H), 7.32–7.25 (m, 3H), 3.43–3.38 (brs, 4H), 3.17 (s, 3H), 1.57–
1.55 ppm (brs, 6H); 13C NMR (CDCl3): d=153.4, 151, 146.2, 142,
129.3 (2C), 127.6 (2C), 126.4, 48.8 (2C), 38.7, 25.4, 25.1, 24.2 ppm;
MS (ESI+): m/z=319.04 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl (4-fluorophenyl)(methyl)-
carbamate (42): White solid (250 mg, 46%); 1H NMR (CDCl3): d=
7.30–7.01 (m, 4H), 3.42–3.36 (m, 4H), 3.24–3.17 (m, 3H), 1.63–
1.55 ppm (m, 6H); 13C NMR (CDCl3): d=162.6, 160.6, 153.4, 151,
146.1, 138, 128.3, 127, 116.4, 48.9, 38.9, 29.7, 25.4, 25.3, 24.1 ppm;
MS (ESI+): m/z=337.12 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl diphenylcarbamate (33):
1
White solid (273 mg, 88%); H NMR (CDCl3): d=7.38–7.36 (m, 8H),
7.27–7.25 (m, 2H), 3.24–3.20 (brs, 4H), 1.61–1.56 ppm (brs, 6H);
13C NMR (CDCl3): d=153.6, 150.1, 145.9, 141.4 (2C), 129.2 (8C), 127
(2C), 48.9, 48.7, 25.5, 25.3, 24.1 ppm; MS (ESI+): m/z=381.03
[M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl (3-fluorophenyl)(methyl)-
carbamate (43): White solid (500 mg, 50%); 1H NMR (CDCl3): d=
7.39–7.36 (m, 1H), 7.15–7.02 (m, 3H), 3.42 (s, 3H), 3.26–3.22 (m,
4H), 1.59–1.55 ppm (m, 6H); 13C NMR (CDCl3): d=163.7, 161.8,
153.5, 150.8, 147, 143.5, 130.5 (3C), 49 (2C), 29.7, 25.4 (2C),
24.2 ppm; MS (ESI+): m/z=337.12 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl diisopropylcarbamate (34):
1
Brown oil (627 mg, 36%); H NMR (CDCl3): d=4.63–4.12 (brs, 1H),
3.93–3.91 (brs, 1H), 3.40 (t, J=5.3 Hz 4H), 1.68–1.59 (m, 6H), 1.33–
1.29 ppm (m, 12H); 13C NMR (CDCl3): d=153.9, 150.4, 146.5, 48.7
(2C), 47.2, 46.8, 25.1 (4C), 24, 21.1, 20.1 ppm; MS (ESI+): m/z=
313.63 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(p-tolyl)carbamate
(44): White solid (110 mg, 20%); H NMR (CDCl3): d=7.28–7.19 (m,
4H), 3.39–3.35 (m, 4H), 3.19 (s, 3H), 2.38 (s, 3H), 1.62–1.55 ppm (m,
6H); 13C NMR (CDCl3): d=153.5, 151.1, 146.3, 139.4, 137.6, 130,
126.2, 49.2, 48.8 (2C), 38.8, 25.6, 25.4 (2C), 24.1, 21 ppm; MS
(ESI+): m/z=333.06 [M+H]+.
1
4-(Piperidin-1-yl)-1,2,5-thiadiazole-3-yl ethyl(phenyl)carbamate
1
(35): White solid (160 mg, 18%); H NMR (CDCl3): d=7.43–7.27 (m,
5H), 3.83–3.78 (brs, 2H), 3.18–3.15 (brs, 4H), 1.56–1.52 (brs, 6H),
1.25–1.16 ppm (m, 3H); 13C NMR (CDCl3): d=153.4, 150.5, 146.2,
140.3, 129.3, 129.1, 127.8, 127.6, 48.8, 46.2, 25.5, 25.4 (2C), 24.8,
24.1, 13 ppm; MS (ESI+): m/z=333.06 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(m-tolyl)carbamate
(45): Colorless oil (290 mg, 40%); H NMR (CDCl3): d=7.27 (d, J=
1
7.17 Hz, 1H), 7.15–7.10 (brs, 3H), 3.40–3.35 (brs, 3H), 3.23–3.17
(brs, 4H), 2.39–2.34 (s, 3H), 1.58–1.53 ppm (brs, 6H); 13C NMR
(CDCl3): d=153.6, 151.1, 146.3, 142, 139.5, 129.2, 128.5, 127.2,
123.6, 48.9 (2C), 38.8, 25.5 (2C), 24.2, 21.3 ppm; MS (ESI+): m/z=
333.06 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl benzyl(methyl)carbamate
1
(36): White solid (450 mg, 35%); H NMR (CDCl3): d=7.38–7.26 (m,
5H), 4.61 (d, J=2.7 Hz, 1H), 4.56 (d, J=2.9 Hz, 1H) 3.40–3.32 (m,
4H), 3.30 (s, 3H), 1.58–1.54 ppm (m, 6H); 13C NMR (CDCl3): d=
153.8, 152.2, 146.5, 136.2, 128.8, 127.9, 127.8, 127.1, 53.2, 49, 35.2,
34.2, 29.7, 25.3 (2C), 24.2 ppm; MS (ESI+): m/z=333.08 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(o-tolyl)carbamate
1
(46): White solid (233 mg, 32%); H NMR (CDCl3): d=7.28–7.20 (m,
4H), 3.31 (s, 3H), 3.17–3.14 (brs, 4H), 2.35 (s, 3H), 1.55–1.49 ppm
(brs, 6H); 13C NMR (CDCl3): d=153.4, 151.2, 146.2, 140.6, 135.4,
131.2 (2C), 128.4, 127.1, 49.1, 48.8, 37.7, 25.4 (2C), 24.2, 17.5 ppm;
MS (ESI+): m/z=333.08 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(4-nitrophenyl)car-
1
bamate (37): White solid (410 g, 70%); H NMR (CDCl3): d=8.28 (d,
J=8.95 Hz, 2H), 7.56 (d, J=8.81 Hz, 2H), 3.52 (s, 3H), 3.31–3.26
(brs, 4H), 1.62–1.57 ppm (brs, 6H); 13C NMR (CDCl3): d=153.5,
150.5, 147.6, 145.6, 144.5, 126.4, 125.9 (2C), 124.6 (2C), 49 (2C),
38.1, 25.5, 25.3, 24 ppm; MS (ESI+): m/z=364.03 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl
(4-methoxyphenyl)(me-
thyl)carbamate (47): White solid (290 mg, 52%); 1H NMR (CDCl3):
d=7.22 (d, J=8.05 Hz, 2H), 6.90 (d, J=8.81 Hz, 2H), 3.81 (s, 3H),
3.35 (s, 3H), 3.22–3.17 (brs, 4H), 1.61–1.58 ppm (brs, 6H); 13C NMR
(CDCl3): d=158.7, 153.4, 151.2, 146.2, 134.7, 127.6, 126.3, 114.4
(2C), 55.4, 48.8 (2C), 39, 25.5, 25.3, 24.1 ppm; MS (ESI+): m/z=
349.04 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(3-nitrophenyl)car-
bamate (38): Yellow solid (406 mg, 52%); 1H NMR (CDCl3): d=
8.25–8.19 (m, 2H), 7.77–7.62 (m, 2H), 3.54–3.50 (brs, 4H), 3.28 (s,
3H), 1.62–1.57 ppm (brs, 6H); 13C NMR (CDCl3): d=153.5, 150.7,
148.7, 145.7, 143.1, 132.3, 130.1, 122.2, 121.3, 49.3, 49, 38.4, 25.5,
25.3, 24.1 ppm; MS (ESI+): m/z=364.04 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl
(3-methoxyphenyl)(me-
thyl)carbamate (48): White solid (600 mg, 80%); 1H NMR (CDCl3):
d=7.34–7.28 (m, 1H), 6.94–6.87 (m, 3H), 3.83 (s, 3H), 3.42 (d, J=
8.9 Hz, 7H), 1.71–1.65 ppm (brs, 6H); 13C NMR (CDCl3): d=160.2,
153.4, 151, 146.1, 143.1, 130 (2C), 118.6, 113, 112.5, 55.4, 48.8, 38.6,
29.6, 25.1, 24.1 ppm; MS (ESI+): m/z=349.07 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl methyl(2-nitrophenyl)car-
1
bamate (39): Brown oil (1.3 g, 66%); H NMR (CDCl3): d=8.11–8.04
(m, 1H), 7.73–7.68 (m, 1H), 7.57–7.49 (m, 2H), 3.38 (s, 3H), 3.19–
3.15 (brs, 4H), 1.55–1.50 ppm (brs, 6H); 13C NMR (CDCl3): d=153.4,
150.5, 145.6, 134.6, 130.5, 129.4, 128.9, 125.8, 125.7, 48.8 (2C), 38.4,
25.3 (2C), 24.1 ppm; MS (ESI+): m/z=364.05 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl
(2-methoxyphenyl)(me-
thyl)carbamate (49): White solid (102 mg, 15%); 1H NMR (CDCl3):
d=7.35–7.27 (m, 2H), 6.99–6.97 (m, 2H), 3.88 (s, 3H), 3.31 (s, 3H),
3.26–3.21 (brs, 4H), 1.58–1.52 ppm (brs, 6H); 13C NMR (CDCl3): d=
154.9, 153.2, 151.5, 146.2, 130.5, 129.4, 128.6, 120.8, 112.1, 55.6, 49,
48.6, 37.7, 25.1 (2C), 24.1 ppm; MS (ESI+): m/z=349.01 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl (4-cyanophenyl)(methyl)-
carbamate (40): White solid (333 mg, 60%); 1H NMR (CDCl3): d=
7.71 (d, J=8.65 Hz, 2H), 7.50 (d, J=8.95 Hz, 2H), 3.48 (s, 3H), 3.29–
3.24 (brs, 4H), 1.62–1.57 ppm (brs, 6H); 13C NMR (CDCl3): d=153.5,
150.5, 145.9, 145.6, 133.2, 126.2, 118, 110.8, 49 (2C), 38, 25.3, 25.1,
24 ppm; MS (ESI+): m/z=344.05 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl
[1,1’-biphenyl]-3-yl(me-
thyl)carbamate (50): White solid (280 mg, 60%); 1H NMR (CDCl3):
d=7.57–7.52 (m, 4H), 7.48–7.43 (m, 3H), 7.37 (t, J=7.25 Hz, 1H),
7.32–7.28 (m, 1H), 3.45 (s, 3H), 3.22–3.17 (brs, 4H), 1.51–1.48 ppm
(brs, 6H); 13C NMR (CDCl3): d=153.5, 151, 146.2, 142.8, 142.6, 140,
129.7, 129 (3C), 127.9, 127.1 (2C), 126.4, 125.2, 53.4 (2C), 38, 25.3,
25.1, 24 ppm; MS (ESI+): m/z=395.03 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl (4-chlorophenyl)(methyl)-
carbamate (41): White solid (290 mg, 51%); 1H NMR (CDCl3): d=
7.38–7.26 (m, 4H), 3.41–3.37 (m, 4H), 3.20 (s, 3H), 1.60–1.57 ppm
(m, 6H); 13C NMR (CDCl3): d=153.5, 150.8, 146, 140.5, 133.4, 129.5
(2C), 127.8, 126.3, 49.7, 48.9, 38.6, 25.5 (2C), 24.1 ppm; MS (ESI+):
m/z=353.03 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl mesityl(methyl)carbamate
1
(51): White solid (233 mg, 30%); H NMR (CDCl3): d=6.91 (s, 2H),
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3.24 (s, 3H), 3.19–3.14 (brs, 4H), 2.29 (d, J=5.15 Hz, 9H), 1.58–
1.53 ppm (brs, 6H); 13C NMR (CDCl3): d=153.5, 151.6, 146.4, 137.9,
136.9, 136, 135 (2C), 129.4, 129.3, 49.1, 48.7, 36.3, 25.4 (2C), 24.1,
20.9, 17.5 ppm (2C); MS (ESI+): m/z=361.06 [M+H]+.
previously described.[33] The assay buffer was 50 mm Tris-HCl
(pH 7.4); 1 mm EDTA and the test compounds were dissolved in
DMSO (the final DMSO concentration was max 5% v/v). The incu-
bations were performed in the presence of 0.5% (w/v) BSA (essen-
tially fatty acid free). Protein (55 mL, 1 mg protein per well) was pre-
incubated with solvent (DMSO) or the inhibitor (5 mL, five to six dif-
ferent concentrations spanning the range 10À9 m to 10À4 m) for
10 min at 378C (60 mL). At the 10 min time point, 20 mm AEA was
added so that its final concentration was 2 mm in 100 mL (contain-
ing 10 nm of 3H-AEA having specific activity of 60 CimmolÀ1 and
concentration of 1 mCimLÀ1). The incubations proceeded for
10 min at 378C. Ethyl acetate (400 mL) was added at the 20 min
time point to stop the enzymatic reaction. Additionally, 100 mL of
50 mm Tris-HCl, pH 7.4; 1 mm EDTA was added. Samples were cen-
trifuged for 4 min at RT 13000 rpm, and aliquots (100 mL) from the
aqueous phase containing 1-[3H]ethanolamine were measured for
radioactivity by liquid scintillation counting (Wallac 1450 Micro-
Beta; Wallac Oy, Finland).
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl cyclohexyl(methyl)carba-
mate (52): White solid (118 mg, 19%); 1H NMR (CDCl3): d=4.02–
3.97 (m, 1H), 3.42–3.37 (brs, 4H), 2.95–2.91 (d, 3H, two conforma-
tions), 1.87–1.77 (m, 4H), 1.75–1.58 (m, 8H), 1.53–1.33 ppm (m,
4H); 13C NMR (CDCl3): d=153.9, 151.8, 146.8, 56.3, 49 (2C), 30.7,
29.9, 29.7, 25.7, 25.5, 25.4 (2C), 25.3, 24.2 ppm; MS (ESI+): m/z=
325.06 [M+H]+.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl cycloheptyl(methyl)carba-
mate (53): White solid (175 mg, 22%); 1H NMR (CDCl3): d=4.16–
4.14 (m, 1H), 3.43–3.39 (brs, 4H), 2.96–2.90 (d, 3H, two conforma-
tions), 1.91–1.86 (brs, 2H), 1.74–1.66 (m, 12H), 1.57–1.51 ppm (m,
4H); 13C NMR (CDCl3): d=153.9, 151.5, 146.8, 58.3, 49 (2C), 32.9,
32.3, 31, 29.7, 29.4, 27.5, 25.4, 25.2, 25.1, 24.2 ppm; MS (ESI+):
m/z=339.08 [M+H]+.
Determination of LAL activity using 4-methylumbelliferone oleate as
a substrate: LAL activity was determined using a previously de-
scribed method.[19] Briefly, purified human LAL overexpressed in
Pichia pastoris (phLAL, 0.01 UmLÀ1, 105 UmgÀ1) was mixed with
compounds at 10 mm and pre-incubated for 20 min at 378C. The
reaction was started by addition of 4-methylumbelliferone oleate,
which was cleaved by enzymatic activity to 4-methylumbelliferone.
The reaction was allowed to proceed for 1 h at 378C, and enzymat-
ic activity was quantified by subtracting background fluorescence
from all the values, and results were normalized to the DMSO con-
trol value.
4-(Piperidin-1-yl)-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carba-
mate (54): White solid (42 mg, 11%); H NMR (CDCl3): d=4.30–4.26
(m, 1H), 3.44–3.39 (brs, 4H), 2.95–2.89 (d, 3H, two conformations),
1.90–1.72 (m, 6H), 1.69–1.51 ppm (m, 14H); 13C NMR (CDCl3): d=
153.9, 151.6, 146.8, 56.9, 49 (2C), 32.1, 31.4, 29.7, 26.3, 26.1, 25.5,
25.4 (2C), 24.9 (2C), 24.2 ppm; MS (ESI+): m/z=353.09 [M+H]+.
1
4-Morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate
1
(55): White solid (71 mg, 17%); H NMR (CDCl3): d=4.27–4.22 (m,
1H), 3.81–3.79 (m, 4H), 3.45–3.44 (m, 4H), 2.94–2.89 (d, 3H, two
conformations), 1.78–1.70 (m, 6H), 1.67–1.51 ppm (m, 8H);
13C NMR (CDCl3): d=153.2, 151.5, 146.8, 66.4 (2C), 57.1, 48.2, 48.1,
32.2, 31.4, 29.8, 26.3 (2C), 26, 25, 24.9 ppm; MS (ESI+): m/z=
355.02 [M+H]+.
Activity-based protein profiling (ABPP) of serine hydrolases: Competi-
tive ABPP using mouse whole-brain membranes was conducted to
visualize the selectivity of inhibitors toward ABHD6 against other
serine hydrolases in brain membrane proteome. We used the
active site serine-targeting fluorescent fluorophosphonate probe
TAMRA-FP as previously described.[14,28] Briefly, brain membranes
(100 mg) were treated for 1 h with DMSO or the selected inhibitors,
after which TAMRA-FP labeling was conducted for 1 h at RT (final
probe concentration 2 mm). The reaction was quenched by addi-
tion of 2ꢃ gel loading buffer, after which 10 mg protein was loaded
per lane and the proteins were resolved in 10% SDS-PAGE togeth-
er with molecular weight standards. TAMRA-FP labeling was visual-
ized (lex 552; lem 575 nm) using a fluorescent scanner (FLA-3000
laser fluorescence scanner, Fujifilm, Tokyo, Japan).
In vitro assays
Determination of ABHD6 activity and reversibility using a sensitive
fluorescent glycerol assay: Glycerol liberated from 1-AG hydrolysis
was determined with a sensitive fluorescent glycerol assay using ly-
sates of HEK293 cells expressing hABHD6 as previously de-
scribed.[14,28] In this approach, glycerol production was coupled via
a three-step enzymatic cascade to hydrogen peroxide (H2O2) de-
pendent generation of resorufin whose fluorescence (lex 530;
lem 590 nm) was kinetically monitored using a Tecan Infinite M200
plate reader (Tecan Group Ltd., Mꢂnnedorf, Switzerland). Briefly,
hABHD6-HEK lysates (99 mL, 0.3 mg protein/well) were pretreated
for 30 min with the solvent (DMSO) or the inhibitor (1 mL, four to
five different concentrations spanning the range 10À9 m to 10À5 m),
after which 1-AG (100 mL, 12.5 mm final concentration) was added
and the reaction kinetically monitored for 90 min. The assays rou-
tinely contained 0.5% (w/v) BSA (essentially fatty acid free) as a car-
rier; 1-AG was used instead of 2-AG, as this is the preferred endo-
cannabinoid isomer for hABHD6.[14] The IC50 values at time-point
90 min were calculated after nonlinear fitting of the inhibitor
dose–response curves. Assay blanks without enzyme were included
in each experiment and fluorescence of the assay blank was sub-
tracted before calculation of the final results. Reversibility of com-
pounds to inhibit hABHD6 were tested in 96-well plate format
using a 40-fold dilution method previously described for testing
reversibility of MAGL inhibitors.[28]
Ethics statement: For the ABPP experiments in vitro with native
mouse brain membrane proteome, membranes prepared from
brain tissue of four-week-old male mice were used. The animals
were obtained from the National Laboratory Animal Centre, Univer-
sity of Eastern Finland. The animals were sacrificed using decapita-
tion. Approval for the harvesting of animal tissue was applied, reg-
istered and obtained from the local welfare officer of the University
of Eastern Finland.
Data analyses: The inhibitor dose–response curves and IC50 values
derived thereof were calculated from nonlinear regressions using
GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego,
CA (USA): www.graphpad.com) and Matlab.
Molecular modeling
Determination of FAAH activity using anandamide as a substrate: In-
hibitory activities of the synthesized compounds were determined
using membranes of COS-7 cells expressing hFAAH, essentially as
Molecular modeling was performed using Schrçdinger Maestro
software package[34] and comparative modeling was done using
Accelrys Discovery Studio Client. Structures of small molecules
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were prepared using the LigPrep module of Schrçdinger suite.
X-ray crystal structure for the FAAH (PDB ID: 3QK5)[35] and homolo-
gy model for ABHD6 were used for docking studies. The homology
model of ABHD6 is based on 2XMZ template and the model is
based on sequence alignment derived from the default blast
search (2XMZ:[32] identity 25%, alignment length 269, E-value
1.59373e-12, positive 44%, resolution 1.94 ꢁ). The model was con-
structed using standard settings of Discovery Studio homology
modeling protocol. Side chains of the active site residues were fur-
ther refined using Prime module of Schrçdinger. X-ray structure of
the FAAH was pre-processed using the protein preparation wizard
of Schrçdinger suite in order to optimize the hydrogen bonding
network and to remove any possible crystallographic artefacts.[36]
Prior to Glide docking studies the grid box was centered using cor-
responding X-ray ligand as template in the case of FAAH and clos-
est active site residues in the case of ABHD6 model. The Ligand
docking was performed using default SP settings of Schrçdinger
Glide using hydrogen bond constraints to oxyanion hole residues
(at least one contact required). Graphical illustrations were generat-
ed using Molecular Operating Environment software (MOE,
2013.8).[37]
Keywords: ABHD6 · 2-arachidonoylglycerol · cannabinoids ·
homology modeling · receptors · 1,2,5-thiadiazole carbamates
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The Supporting Information contains synthesis and spectroscopic
characterization of all intermediates 8–21; elemental analyses for
all final compounds 22–55; determination of MAGL activity using
2-AG as a substrate; determination of ABHD12 activity using a pre-
viously validated sensitive fluorescent glycerol assay; determination
of lipophilicity values for compounds 52–55; determination of LAL
inhibitory activity (IC50); TAMRA-FP labeling in mouse brain pro-
teome through competitive ABPP assay; cannabinoid receptor
activity; molecular modeling studies, and related references.
Author contributions
All authors contributed equally and gave approval to the final ver-
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Acknowledgements
We thank Ms. Minna Glad, Ms. Tiina Koivunen, Ms. Helly Rissa-
nen, Ms. Taija Hukkanen, and Ms. Satu Marttila for their skillful
technical assistance. We are grateful to Dr. Hong Du (Indiana
University School of Medicine) and Dr. Gregory Grabowski (Syna-
geva BioPharma) for the gift of the LAL enzyme. CSC-Scientific
Computing, Ltd. is gratefully acknowledged for software licenses
and computational resources. Financial support for this study
was provided by the Graduate School of Drug Design, University
of East Finland (to J.Z.P.), The Academy of Finland (grants 139140
to T.J.N., 127653 to T.P., and 139620 to J.T.L.), Drug Discovery and
Chemical Biology (DDCB) Consortium, Biocenter Finland and the
US National Institutes of Health (NIH) (grant R37-DK27083). Part
of the research was performed under a Marie Curie Intra-Europe-
an Fellowship awarded to A.A.K. Some of the calculations were
performed under a computational grant from the Interdisciplina-
ry Center for Mathematical and Computational Modeling (ICM),
Warsaw, Poland (grant G30-18). For collaborative purposes, we
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Received: October 21, 2014
Published online on && &&, 0000
ChemMedChem 0000, 00, 0 – 0
www.chemmedchem.org
13
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
J. Z. Patel,* T. J. Nevalainen,
Irreversible and selective: a/b-Hydro-
lase domain 6 (ABHD6) is an emerging
target to treat inflammation, metabolic
disorders, and epilepsy. Herein we de-
scribe the potent and irreversible inhibi-
tion of hABHD6 with 1,2,5-thiadiazole
carbamate based compound JZP-430.
This compound showed good selectivity
over its main supposed off-targets, fatty
acid amide hydrolase and lysosomal
acid lipase.
J. R. Savinainen, Y. Adams, T. Laitinen,
R. S. Runyon, M. Vaara, S. Ahenkorah,
A. A. Kaczor, D. Navia-Paldanius,
M. Gynther, N. Aaltonen,
A. A. Joharapurkar, M. R. Jain, A. S. Haka,
F. R. Maxfield, J. T. Laitinen, T. Parkkari*
&& – &&
Optimization of 1,2,5-Thiadiazole
Carbamates as Potent and Selective
ABHD6 Inhibitors
&
ChemMedChem 0000, 00, 0 – 0
www.chemmedchem.org
14
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!