Synthesis and Biological Evaluation of Endocannabinoid Uptake Inhibitors Derived from WOBE437

Article abstract of DOI:10.1002/cmdc.202000153

WOBE437 ((2E,4E)-N-(3,4-dimethoxyphenethyl)dodeca-2,4-dienamide, 1) is a natural product-derived, highly potent inhibitor of endocannabinoid reuptake. In this study, we synthesized almost 80 analogues of 1 with different types of modifications in the dodecadienoyl domain as well as the dimethoxyphenylethyl head group, and we investigated their effects on anandamide uptake into U937 cells. Intriguingly, none of these analogues was a more potent inhibitor of anandamide uptake than WOBE437 (1). At the same time, a number of WOBE437 variants exhibited potencies in the sub-100 nM range, with high selectivity over inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase; two compounds were virtually equipotent with 1. Interestingly, profound activity differences were observed between analogues in which either of the two methoxy substituents in the head group had been replaced by the same bulkier alkoxy group. Some of the compounds described here could be interesting departure points for the development of potent endocannabinoid uptake inhibitors with more drug-like properties.

Full text of DOI:10.1002/cmdc.202000153

Accepted Article
Title: Synthesis and Biological Evaluation of Endocannabinoid Uptake
Inhibitors Derived from WOBE437
Authors: Karl-Heinz Altmann, Patrick Mäder, Ruben Bartholomäus,
Simon Nicolussi, Alice Baumann, Melanie Weis, Andrea
Chicca, Mark Rau, Ana Catarina Simão, and Jürg Gertsch
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000153
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01/2020

10.1002/cmdc.202000153
ChemMedChem
FULL PAPER
Synthesis and Biological Evaluation of Endocannabinoid Uptake
Inhibitors Derived from WOBE437
Patrick Mäder,[a][c] Ruben Bartholomäus,[a][c] Simon Nicolussi,[b] Alice Baumann,[a] Melanie Weis,[a]
Andrea Chicca,[b] Mark Rau,[b] Ana Catarina Simão,[a] Jürg Gertsch,[b] and Karl-Heinz Altmann*[a]
[a]
Dr. P. Mäder, Dr. R. Bartholomäus (ORCID ID 0000-0003-1696-5045), Msc. ETH A. Baumann, MSc. ETH M. Weiss, Dr. A. C. Simão, Prof. Dr. K.-H.
Altmann (ORCID ID 0000-0002-0747-9734)
Department of Chemistry and Applied Biosciences
ETH Zürich
HCI H405
Vladimir-Prelog-Weg 4
CH-8093 Zürich, Switzerland
E-mail: karl-heinz.altmann@pharma.ethz.ch
[b]
[c]
Dr. S. Nicolussi (ORCID ID 0000-0003-1260-3282), Dr. M. Rau, Dr. A. Chicca, Prof. Dr. J. Gertsch (ORCID ID 0000-0003-0978-1555)
Institute of Biochemistry and Molecular Medicine
University of Bern, Switzerland
Bühlstrasse 28
CH-3012 Bern, Switzerland
PM and RB contributed equally to this work
Supporting information for this article is given via a link at the end of the document.
Abstract: WOBE437 ((2E,4E)-N-(3,4-dimethoxyphenethyl)dodeca-
2,4-dienamide, 1) is a natural product-derived, highly potent inhibitor
of endocannabinoid reuptake. In this study, we have synthesized
almost 80 analogs of 1 with different types of modifications in the
dodecadienoyl domain as well as the dimethoxyphenylethyl head
group and we have investigated their effects on anandamide uptake
into U937 cells. Intriguingly, none of these analogs was a more potent
inhibitor of anandamide uptake than WOBE437 (1). At the same time,
a number of WOBE437 variants exhibited potencies in the sub-100
nM range with high selectivity over inhibition of the endocannabinoid-
degrading enzyme fatty acid amide hydrolase; two compounds were
virtually equipotent with 1. Interestingly, profound activity differences
were observed between analogs where either of the two methoxy
substituents in the head group had been replaced by the same bulkier
alkoxy group. Some of the compounds described here could be
interesting departure points for the development of potent
endocannabinoid uptake inhibitors with more drug-like properties.
neurological disorders,[1a] inflammatory conditions, and metabolic
diseases.[1c]
Given its involvement in many pathophysiological processes, it is
not surprising that the ECS has emerged as an important target
system for drug development in different indication areas.[1a-c] In
particular, a number of CB1 or CB2 agonists and antagonists have
been evaluated in clinical trials in humans, although the success
of these efforts so far has been limited.[1c] The most advanced
example of a synthetic CB receptor ligand is the CB1 antagonist
rimonabant, which was approved in 2006 in the European Union
for the treatment of obese or overweight patients with additional
risk factors such as diabetes or dyslipidemia as adjunct to diet and
exercise.[5] However, the compound was never approved in the
US[6] and it was withdrawn from the European market in 2008 due
to frequent psychiatric side effects.[5] Likewise, CB1 receptor
agonists are fraught with significant central side effects.[1c]
Compared to direct CB activation, promoting EC signalling
through inhibition of the major EC-catabolic enzymes fatty acid
amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) may
offer a safer alternative towards ECS-directed therapeutics.[1a][6]
Based on this premise, a number of FAAH inhibitors have been
advanced into clinical trials, mostly against neurological
disorders.[1a][1c] Unfortunately, this approach suffered at least a
temporary setback in 2016 due to very serious adverse events in
a Phase I clinical trial with BIA 10-2474.[7] However, subsequent
work has shown that these events were most likely caused by off-
targets effects in the brain, rather than inhibition of FAAH.[8]
Apart from inhibition of FAAH or MAGL, EC signaling can also be
enhanced through inhibition of AEA or 2-AG uptake,[1a][9] as this
prolongs the availability of these transmitters for interaction with
CB receptors. In this context, we have recently reported the
discovery of the dodeca-2,4-dienamide WOBE437 (1) as the first
selective, potent AEA uptake inhibitor.[10]
Introduction
The endocannabinoid system (ECS) is a ubiquitous lipid signaling
system that comprises two G protein-coupled receptors (GPCRs),
the cannabinoid receptors 1 and 2 (CB1 and CB2, respectively),
their endogenous ligands, 2-arachidonoyl glycerol (2-AG) and
N-arachidonoyl ethanolamine (anandamide, AEA), and five major
enzymes that are responsible for ligand biosynthesis and
degradation.[1] While CB1 is primarily found in the central nervous
system[2] (and has been suggested to be the most abundant
GPCR in the mammalian brain),[1c] CB2 is mostly found on
immune cells;[3] however, the latter has also been detected in
microglial cells, in astrocytes and in certain neurons.[4] The ECS
plays a key role in the regulation of cell and tissue homeostasis in
virtually all organ systems and its dysregulation, consequently, is
associated with a variety pathological conditions, including
1
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WOBE437 (1) blocks uptake of AEA into U937 cells with an IC50
of 10 nM, while neither affecting the uptake of 2-AG nor the
hydrolysis of AEA by FAAH (selectivity ratios of 28 and 977,
respectively). The compound has been demonstrated to exert
cannabimimetic effects in vivo and, more recently, has also been
shown to be orally active.[11] At a more general level, the specific
effect of WOBE437 (1) on AEA uptake, together with other,
indirect evidence points to the existence of a dedicated AEA
membrane transport system.[9][12] However, the exact
component(s) of this system (protein(s), lipids) and its working
mechanism still need to be elucidated.
mediated amide bond formation, while 15 was obtained through
catalytic hydrogenation of 1. (See the SI for details).
Here, we describe the synthesis of a series of more than 70
analogs of WOBE437 (1) with modifications either in the C12 acyl
chain or in the (dimethoxyphenyl)ethyl head group and the
assessment of their effects on AEA uptake in U937 cells. In
addition, we have also determined the effect of most compounds
on FAAH activity, in order to ensure that potent AEA uptake
inhibition did not result from inhibition of AEA degradation; the
latter would reduce the inward-directed concentration gradient
that provides the major driving force for AEA uptake into cells.[13]
While some of these compounds were already included in the SI
of our previous WOBE437 study,[10] their synthesis was not
described there and the corresponding biological data were not
commented on in detail. Thus, the current study represents the
first comprehensive SAR analysis of WOBE437-derived
structures. An SAR study around the natural product guineensine,
which one of our groups had disclosed as a potent inhibitor of AEA
uptake prior to publication of our work on WOBE437 (1),[14] has
recently appeared.[15] While our investigations of WOBE437
analogs so far have not yielded any compounds with an improved
inhibitory profile, our SAR data provide important insights into the
relevance of individual structural features of WOBE437 (1) for
potent and selective inhibition of AEA uptake.
Figure 1. WOBE437 analogs with modified acyl chains I.
Results and Discussion
Scheme 1. Reagents and conditions: a) Chloroacetyl chloride, Et3N, CH2Cl2,
0 °C to rt, 15 min, 93%; b) P(OEt)3, 150 °C, 2 h, 99%; c) R-CHO, KOtBu, THF,
rt, 15 min, 52-88%; d) pent-4-enoic acid, (COCl)2, CH2Cl2, rt, 20 min, then 81,
Et3N, CH2Cl2, rt, 10 min, 44%; e) non-1-ene, Grubbs II catalyst, CH2Cl2, 40 °C,
6 h, 29% (13/14 = 5:1).
Acyl chain modifications. The initial questions to be addressed
in our SAR work were related to the significance of the length of
the acyl chain in WOBE437 (1), the number and positions of
double bonds, and the tolerance to modifications in the saturated
C7 segment following the dienoyl moiety. The corresponding
target structures 2 - 21 are depicted in Fig. 1.
As for the variations in acyl chain length, changes in the number
of double bonds led to a clear loss in potency relative to 1, with
the incorporation of an additional double bond between C6 and
C7 being better tolerated than the removal of either of the double
bonds present in WOBE437 (1). Surprisingly, the fully reduced
analog 15 was more potent than any of the analogs containing a
single double bond. As we have shown previously, the
replacement of the dodecadienoyl moiety by an arachidonoyl
residue is reasonably well tolerated[10] and the corresponding
analog 16 is only 6-fold less potent than 1, without affecting FAAH
activity (selectivity index (SI) = 106; for definition of SI see Table
1).
Overall, the SAR around WOBE437 (1) with regard to acyl chain
length as well as the number and position of double bonds is
intriguingly steep. Thus, with the exception of arachidonoyl
derivative 16, which incorporates the acyl chain of the natural
substrate, all modifications investigated here led to a decrease in
potency of at least 10-fold. This finding re-enforces our previous
conclusion that WOBE437 (1) is a highly specific inhibitor of AEA
uptake, whose activity is not based on simple, non-specific
As shown in Scheme 1, analogs 2 - 11 and 17 - 21 were obtained
by HWE olefination of phosphonate 82 with the appropriate
aldehydes. The latter were either commercially available or
prepared according to standard procedures (see the SI).
Phosphonate 82 was synthesized from O,O-dimethyl dopamine
(81) by acylation with chloroacetyl chloride and reaction of the
ensuing chloroacetamide with triethyl phosphite at 150 °C.
An alternative approach for acyl chain assembly was followed in
the synthesis of analogs 13 and 14, which was based on cross-
metathesis between terminally unsaturated amide 83 and non-1-
ene in the presence of Grubbs II catalyst[16] (Scheme 1). The
reaction gave a 5:1 mixture of 13 and 14 in a total yield of 29%;
13 and 14 could be separated by preparative RP-HPLC to yield
stereochemically pure samples of both isomers. Amide 83 was
obtained by EDC/HOBt-mediated coupling[17] of 4-pentenoic acid
with amine 81. Likewise, analogs 12 and 16 were prepared from
amine 81 and the requisite carboxylic acids by EDC/HOBt-
2
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10.1002/cmdc.202000153
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lipophilic interactions. We assume that the specificity of 1 arises
from both defined interactions with a hitherto uncharacterized
protein target but also from specific interactions with membrane
components. Perhaps unsurprisingly then, the incorporation of
heteroatoms into the saturated segment of the acyl chain (analog
17) proved to be detrimental for AEA uptake inhibitory activity.
(analogs 22 and 23) or separated from the carbonyl carbon by
one CH2 group (analogs 24 - 27) (Fig. 2). For the meta-substituted
analogs the number of consecutive carbons in the acyl chain (12)
corresponds with that in WOBE437 (1) (for the shorter path
through the aromatic ring).
Table 1. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
modified acyl chains I.a
FAAH IC50 [M][d]
Max. Inh.
EC50 [M]
Cpd.[a]
SI[e]
(%)[c]
(95% CI)[b]
(95% CI)[b]
2
3
4
5
6
7
1
>100 (ND)
43
>10 (79)[f]
>10 (83)[f]
>10 (89)[f]
>10 (72)[f]
>10 (74)[f]
>10 (67)[f]
9.8
ND
ND
ND
ND
ND
ND
980
87
(ND)
54
35
(ND)
67
10
(ND)
73
2.1
0.6
(1.332-3.366)
(0.3-1)
69
73
0.01 (0.007-0.015)
0.14 (0.11-0.19)
0.17 (0.12-0.25)
0.097 (0.062-0.218)
0.11 (0.06-0.19)
0.55 (0.44-0.70)
79
Figure 2. WOBE437 analogs with modified acyl chains II.
8
74
73
69
96
74
74
86
74
73
ND
65
54
61
65
16.5 (10.3-21.9) 117
9
9.8 (5.6-15.1)
7.7 (4.9-11.0)
12.3 (7.5-16.2)
5.1 (3.4-6.3)
>1
101
79
Analogs 22 and 23 were prepared by EDC/HOBt-mediated amide
bond formation[17][18] from amine 81 (cf. Scheme 1) and p- or m-
octylbenzoic acid, respectively. (See the SI for details). The
synthesis of WOBE analogs 24 - 27 is summarized in Scheme 2
and in the key step involved the attachment of the distal part of
the acyl side chain to aromatic bromides 86 and 87 by Suzuki
coupling;[19] the latter, in turn, had been obtained by EDC/HOBt-
mediated couplings[17][18] between amine 81 and p- and m-bromo
phenylacetic acids 84 and 85, respectively. The Suzuki couplings
proceeded in good yields (59% and 76%) and the ensuing
unsaturated analogs 24 and 26 were then transformed into
saturated derivatives 25 and 27, respectively, by Pd/C-catalyzed
reduction with Et3SiH.[20] Sulfonamide-based analog 28 was
obtained by the reaction of p-octylphenylsulfonyl chloride with
amine 81 (see the SI for details).
10
11
12
13
14
15
16
17
18
19
20
21
112
9
1.1
2.0
(1.0-1.3)
(1.6-2.7)
ND
ND
>45
106
ND
ND
ND
ND
ND
>1
0.22 (0.140-0.360)
0.064 (0.041-0.099)
> 10
>10
6.8 (4.9-8.2)
>10
3.02 (1.411-6.455)
49.0 (ND)
>1 (69)[f]
>1 (67)[f]
>1 (74)[f]
>1 (78)[f]
61.7 (ND)
69.0 (ND)
[a] For structures, cf. Fig. 1. The data for cpds. 2 - 16 are included in the SI for
ref[10], but they are not discussed in this reference in any detail. ND = Not
determined. [b] Concentration required for half-maximal inhibition of uptake of
AEA into U937 cells. EC50 values represent the mean of at least three
independent experiments. 95% CI, 95% confidence interval. [c] Maximal
inhibition of uptake of AEA into U937 cells. Maximum inhibition data in our
uptake assay data varied significantly; thus, at this point, we consider numbers
>60% as essentially indistinguishable. [d] IC50 for the inhibition of FAAH in U937
cell homogenates. [e] Selectivity index = EC50 (AEA uptake inhibition)/IC50
(FAAH inhibition). [f] Number in parentheses indicates the remaining FAAH
activity at 1 M cpd. concentration. [3H]-AEA was used to determine uptake
inhibition by WOBE437 analogs; FAAH inhibition was assessed in U937 cell
Scheme 2. Reagents and conditions: a) EDC•HCl, HOBt, Et3N, CH2Cl2, rt, 8-16
h, 60-90%; b) Pd(OAc)2, K2CO3, DME/H2O 2:3, 90 °C, 10-14 h, 24: 59%, 26:
76%; c) Et3SiH, Pd/C, EtOH, 1-2 h, 25: 83%, 27: 96%.
homogenates. For experimental details cf. ref[14]
.
With the impact of acyl chain length variations on the activity of
WOBE437 analogs established, we next wanted to investigate the
effects of modifications leading to additional rigidization of the acyl
chain at the site of the diene moiety. To this end, we prepared
analogs 22 - 27 that incorporate a para- or meta-substituted
phenylene moiety either directly attached to the carbonyl carbon
All analogs 22 - 27 were less potent inhibitors of AEA uptake into
U937 cells than WOBE437 (1), albeit to different extents (Table
2). While 22 was only ca. 10-fold less active than 1, the IC50 for
24 was increased by more than 100-fold. Para-substituted analog
3
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22 appeared to be slightly more potent than the meta-substituted
variant 23, although the meta-substitution pattern provides the
better match for a dodeca-2,4-dienoyl chain in a fully extended
conformation; however, the difference between 22 and 23 is very
small and may not be significant. For analog pairs 24/26 and
25/27, there is no clear preference for one of the substitution
patterns; likewise, saturation of the double bond of the styryl
moiety of 25 and 26 led to opposite effects on inhibitory activity.
The replacement of the amide bond in 22 by a sulfonamide moiety
resulted in a ca. 20-fold reduction in potency.
Table 2. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
modified acyl chains II.a
FAAH
IC50 [M][d]
Max. Inh.
(%)[c]
EC50 [M]
Cpd.[a]
SI[e]
(95% CI)[b]
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
0.11 (0.08-0.15)
0.23 (0.17-0.32)
1.2 (0.3-4.2)
65
71
5.3 (4.5-5.9)
12.3 (10.6-14.1)
>5
49
52
Figure 3. WOBE437 analogs with modified acyl chains III.
65
ND
20
Analogs 33, 34, and 36 were obtained through EDC/HOBt-
mediated coupling[17][18] of the corresponding (commercially
available) carboxylic acids and amine 81 (see the SI for details).
0.69 (0.47-1.00)
0.33 (0.047-2.33)
0.72 (0.367-1.41)
2.3 (0.846-6.31)
0.28 (0.18-0.44)
1.0 (0.47-2.4)
1.3 (0.74-2.5)
0.73 (0.38-1.4)
0.96 (0.67-1.36)
2.8 (1.2-6.6)
64
13.6 (12.3-15.4)
68
21.8 (18.5.-22.9) 66
63
>5
ND
ND
49
81
ND
103
128
74
13.7 (11.6-17.0)
>10 (85)[f]
ND
11
14.1 (11.1-16.8)
16.7 (13.0-21.1)
2.1 (1.4-3.0)
4.6 (3.3-6.3)
12.6 (8.6-18.1)
ND (15)[g]
64
23
56
2.2
1.6
9.5
ND
ND
63
1.3 (0.6-2.9)
58
Scheme 3. Reagents and conditions: a) EDC•HCl, HOBt, Et3N, CH2Cl2, rt, 8-16
h, 91: 90%, 92: 98%; b) 35: 93, K2CO3, DMF, rt, 20 h, 90%; 37: C7H15NH2,
K2CO3, DMSO, 120 °C, 12 h, 33%; c) Pd(OAc)2, K2CO3, DME/H2O 2:3, 90 °C,
14 h, 84%; d) Et3SiH, Pd/C, EtOH, 1 h, 99%.
Ca. 20% Inh. @1uM
Ca. 41% Inh. @1uM
ND
ND
ND (30)[g]
[a] For structures, cf. Figs. 2 and 3. [b] Concentration required for half-maximal
inhibition of uptake of AEA into U937 cells. EC50 values represent the mean of
at least three independent experiments. 95% CI, 95% confidence interval. [c]
Maximal inhibition of uptake of AEA into U937 cells. Maximum inhibition data in
our uptake assay data varied significantly; thus, at this point, we consider
numbers >60% as essentially indistinguishable. [d] IC50 for the inhibition of
FAAH in U937 cell homogenates. [e] Selectivity index = EC50 (AEA uptake
inhibition)/IC50 (FAAH inhibition). [f] Number in parentheses indicates the
remaining FAAH activity at 1 M cpd. concentration. [g] Number in parentheses
indicates the remaining FAAH activity at 5 M cpd. concentration. [3H]-AEA was
used to determine uptake inhibition by WOBE437 analogs; FAAH inhibition was
As illustrated by the data in the lower part of Table 2, all
modifications of the substituent in the para-position of the
phenylacetamide moiety in 22 led to a reduction in potency by ca.
3- (29) to ca. 30-fold (34 and perhaps 36). Thus, shortening of the
octyl substituent in 22 is not tolerated, even if the nominal length
of the acyl chain in 29 corresponds with that of WOBE437 (1) (12
carbons), while it is one carbon longer in 22. Of note, the simple
replacement of the CH2 group directly attached to the aromatic
ring by a heteroatom led to less potent analogs (36 and 37),
compared to the parent compound 29.
assessed in U937 cell homogenates. For experimental details cf. ref[14]
.
Based on the observations with WOBE437 analogs 22 - 27, we
subsequently prepared a series of congeners of 22, with
variations in the nature of the substituent para to the amide moiety
(Fig. 3). The synthesis of analog 31 involved Suzuki coupling[17]
between aromatic bromide 92 and boronic acid 94, which
proceeded in high yield (84%) (Scheme 3); reduction of the
double bond in 31 with Et3SiH in the presence of Pd/C gave 32.
Hetero-substituted analogs 35 and 37 were derived from aromatic
fluoride 90 by nucleophilic aromatic substitution with 4-
heptylphenol and heptylamine, respectively (33% and 90% yield).
Head group modifications. In a first set of head group
modifications (i. e. modifications of the N-phenylethyl moiety), we
investigated the importance of each of the methoxy groups on the
phenyl ring (analogs 38 - 47) and of the length of the linker
between the phenyl group and the amide nitrogen (analogs 48
and 49) (Fig. 4).
4
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Replacement of either of the methoxy groups in 1 by a larger
allyloxy group (analogs 46 and 47) was reasonably well tolerated,
with the corresponding analogs both exhibiting IC50 values for
AEA transport inhibition below 100 nM.
Table 3. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
modified head groups I.a
Max. Inh.
EC50 [M]
FAAH IC50 [M][d]
Cpd.[a]
SI[e]
12
(%)[c]
64
56
69
77
67
98
72
79
66
78
51
73
(95% CI)[b]
(95% CI)[b]
38
39
40
41
42
43
44
45
46
47
48
49
0.22 (0.16-0.35)
0.27 (0.10-0.72)
0.36 (0.18-0.73)
0.31 (0.21-0.46)
0.067 (0.045-0.098)
1.1 (0.4-2.9)
2.61 (0.8-3.0)
Figure 4. WOBE437 analogs with modified head groups I.
12.4 (10.4-14.1) 46
16.5 (12.2-19.3) 46
18.2 (15.2-23.4) 59
The majority of these analogs were prepared by direct
EDC/HOBt-mediated coupling[17][18] of acid 95 with the respective
(commercially available) amines; 46 and 47 were obtained by
alkylation of 42 and 41, respectively with allyl bromide in excellent
yields (Scheme 4).
5.9 (2.6-7.4)
>10 (100)[f]
88
ND
7.6
4.5
161
0.29 (0.20-0.44)
0.22 (0.11-0.44)
0.08 (0.016-0.480)
0.057 (0.038-0.085)
0.41 (0.29-0.58)
0.32 (0.063-1.6)
2.2 (1.0-2.6)
1.0 (0.7-1.6)
12.9 (9.7-15.2)
20.5 (18.3-22.7) 44
>100
ND
>100
ND
[a] For structures, cf. Fig. 4. Data for cpds. 38 - 40, 43, and 49 were already
reported in the SI of ref[10]. Data for 38, 39, and 43 are directly taken from ref[10]
,
those for 40 and 49 were (re)determined in this study. ND = Not determined. [b]
Concentration required for half-maximal inhibition of uptake of AEA into U937
cells. EC50 values represent the mean of at least three independent
experiments. 95% CI, 95% confidence interval. [c] Maximal inhibition of uptake
of AEA into U937 cells. Maximum inhibition data in our uptake assay varied
significantly; thus, at this point, we consider numbers >60% as essentially
indistinguishable. [d] IC50 for the inhibition of FAAH in U937 cell homogenates.
[e] Selectivity index = EC50 (AEA uptake inhibition)/IC50 (FAAH inhibition). [f]
Value in parentheses indicates the remaining FAAH activity at 1 M cpd.
concentration. [3H]-AEA was used to determine uptake inhibition by WOBE437
analogs; FAAH inhibition was assessed in U937 cell homogenates. For
Scheme 4. Reagents and conditions: a) For all compounds, except for 51 and
56: EDC•HCl, HOBt, Et3N, CH2Cl2, rt, 8-16 h, 27-91%. For 51: (COCl)2, CH2Cl2,
rt, 20 min, then 2-(2-fluoro-4,5-dimethoxyphenyl)ethylamine (105), CH2Cl2, rt,
10 min, 27%; for 56: (COCl)2, CH2Cl2, rt, 15 min, then 2-(3,4,5-
trimethoxyphenyl)ethylamine (110), CH2Cl2, rt, 15 min, 27%; b) allyl bromide,
K2CO3, DMF, 50 °C, 5.5 h, 46: 84%, 47: 78%; c) 4M HCl in 1,4-dioxane, rt, 1 h,
87%.
experimental details cf. ref[14]
.
Shortening the N-ethyl linker in WOBE437 (1) to a CH2 group
produced an analog (48) with ca. 40-fold reduced potency;
interestingly, and in contrast to what is observed for the parent
compound 1, the activity of 48 was not reduced upon removal of
the methoxy groups (Table 3).
As an extension of the above studies on demethoxylated and
demethylated derivatives of WOBE437 (1), we have also
investigated head group-modified analogs incorporating an
additional substituent at position 2 or 3 of the dimethoxyphenyl
moiety (Fig. 5).
We have previously reported that the removal of each or both of
the methoxy groups in WOBE437 (1) (analogs 38 - 40) leads to a
significant loss in AEA uptake inhibitory activity;[10] these results
have been confirmed in the current study (within the bounds of
the reproducibility of our transport assay) (Table 3).
A similar loss in potency was incurred by demethylation of the
methoxy group in position meta to the ethyl linker to the amide
nitrogen (analog 41), while demethylation of the p-methoxy group
appeared to be less critical (analog 42). Thus, 42 was only 7-fold
less potent than WOBE437 (1) (Table 3). Removal of both methyl
groups produced an analog (43) with an activity that was >100-
fold lower than that of WOBE437 (1). Interestingly, the
demethylation products of mono-methoxy derivatives 38 and 39,
i. e. 44 and 45, respectively, both exhibited comparable activity as
the corresponding parent compound. Thus, once one of the
methoxy substituents in WOBE437 (1) is removed, the
demethylation of the remaining methoxy group is of no further
consequence, in contrast to the effects observed upon
demethylation of 1, 41, and 42. In fact, mono-hydroxylated
analogs 44 and 45 were more potent than bis-phenol 43.
Figure 5. WOBE437 analogs with modified head groups II.
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000153
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FULL PAPER
Analogs 52 - 55 were prepared by EDC/HOBt-mediated
coupling[17][18] of acid 95 with the requisite amines; 51 and 56 were
obtained via the acid chloride (Scheme 4). For the synthesis of 50
see the SI.
Table 4. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
modified head groups II.a
EC50 [M]
FAAH IC50 [M][d]
Max. Inh.
Cpd.[a]
SI[e]
(%)[c]
(95% CI)[b]
(95% CI)[b]
As depicted in Scheme 5, chlorinated and iodinated amine
precursors 106 and 108, respectively, were obtained by
halogenation of N-BOC-protected 2-(4,5-dimethoxyphenyl)ethyl
amine (114) with N-chlorosuccinimide or elementary iodine
followed by BOC-removal with HCl/dioxane in good overall yields.
Brominated amine 107 and nitro-substituted derivative 109 were
prepared by direct bromination and nitration, respectively, of
unprotected amine 81. The syntheses of 2-(2-methyl-4,5-
dimethoxyphenyl)ethyl amine, 2-(2-fluoro-4,5-dimethoxyphenyl)-
ethyl amine, and 2-(3,4,5-trimethoxyphenyl)ethyl amine are
described in the SI.
50
51
52
53
54
55
56
0.14 (0.078-0.27)
0.146 (0.123-0.173)
0.185 (0.078-0.435)
0.081 (0.049-0.134)
0.043 (0.017-0.111)
0.12 (0.017-0.18)
72
1.4 (1.0-2.0)
>1 (70)[f]
10
81
ND
63
15.0 (11.1-17.4) 81
14.7 (12.4-16.9) 181
13.9 (10.5-15.1) 323
62
62
74
0.8 (0.5-1.2)
>10
6.8
ND
3.4
(1.1-9.9)
71
[a] For structures, cf. Fig. 5. ND = Not determined. [b] Concentration required
for half-maximal inhibition of uptake of AEA into U937 cells. EC50 values
represent the mean of at least three independent experiments. 95% CI, 95%
confidence interval. [c] Maximal inhibition of uptake of [3H]-AEA into U937 cells.
Maximum inhibition data in our uptake assay varied significantly; thus, at this
point, we consider numbers >60% as essentially indistinguishable. [d] IC50 for
the inhibition of FAAH in U937 cell homogenates. [e] Selectivity index = EC50
(AEA uptake inhibition)/IC50 (FAAH inhibition). [f] The value in parentheses
indicates the remaining FAAH activity at 1 M cpd. concentration. [3H]-AEA was
used to determine uptake inhibition by WOBE437 analogs; FAAH inhibition was
assessed in U937 cell homogenates. For experimental details cf. ref[14]
.
Scheme 5. Reagents and conditions: a) Boc2O, THF, 0 °C to rt, 12 h, 99%; b)
NCS, Si(CH3)3Cl, MeCN, rt, 2 h, 81%; c) I2, CF3COOAg, CHCl3, rt, 2 h, 97%; d)
4M HCl in 1,4-dioxane, 1,4-dioxane, rt, 2 h, 106: quant, 108: 99%; e) Br2, AcOH,
rt, 16 h, 98%; f) HNO3, H2O, rt, 3 h, 77%.
Substitution of WOBE437 (1) at the 2-position of the aromatic ring
was associated with a decrease in potency for all substituent
groups investigated, although IC50 values for AEA uptake
inhibition still remained below 200 nM or even 100 nM (53 and 54)
(Table 4). No difference was observed between the effect of a
slightly electropositive methyl group and (strongly) electron-
withdrawing substituents, such as fluoro, chloro, or nitro. For
analogs 51 - 54 there was a trend of increasing potency with
decreasing electronegativity/increasing size of the halogen
substituent. It may thus be speculated that the potent activity of
iodo derivative 56 is related to halogen bonding[21] with the
hitherto elusive protein target(s) of WOBE437-type AEA uptake
inhibitors.
Compared to all other analogs in Fig. 5, 56, which incorporates
an additional methoxy group at the 3-position of the phenyl ring,
showed substantially lower activity. The IC50 value for 56 was >3
M, which makes the compound ca. 300-fold less potent than
Figure 6. WOBE437 analogs with modified head groups III.
These studies were carried out in the context of our work on the
identification of fluorescently labelled and/or reactive probe
molecules for the identification of proteins involved in EC
transport. This work has led to the discovery of the previously
reported, highly versatile probe RX-55 (66 in Fig. 6),[10] but it has
also revealed some intriguing features of the WOBE437 SAR
(vide infra).
The synthesis of analogs 57 - 70 proceeded through analogs 41
and 42 as intermediates, which were alkylated with mesylate 116
in yields of 66% and 84%, respectively (Scheme 6). Subsequent
removal of the BOC-protecting group with TFA gave the
WOBE437 (1). Whether this effect reflects
a
general
incompatibility of transport inhibition with the presence of
substituents at the 3-position of WOBE437 (1) or if it may be
caused by the increased electron density in the aromatic ring
remains to be determined.
Based on the results obtained with allyloxy analogs 46 and 47,
which had indicated that alkoxy substituents larger than a simple
methoxy group are not necessarily detrimental for AEA uptake
inhibitory activity, we have subsequently investigated WOBE437
analogs with significantly extended alkoxy substituents and bulky
end groups (Fig. 6).
6
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10.1002/cmdc.202000153
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corresponding free amines, which were further elaborated into the
desired target structures by acylation (61 - 66, 69), alkylation (67,
68), or sulfonylation (70) in very good to excellent yields (Scheme
6). (The synthesis of 65 and 66 has also been described in ref[10]).
Table 5. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
modified head groups II.a
Max. Inh.
EC50 [M]
FAAH IC50 [M][d]
Cpd.[a]
SI[e]
(%)[c]
43
85
69
88
78
72
76
80
65
82
94
86
82
85
(95% CI)[b]
(95% CI)[b]
57
58
59
60
61
62
63
64
65
66
67
68
69
70
0.16
0.04
0.16
(0.056-0.50)
2.1 (1.2-2.9)
2.24 (1.0-4.3)
1.0 (0.63-1.7)
13
56
6
(0.03-0.06)
(0.07-0.37)
0.036 (0.026-0.051)
0.19 (0.108-0.34)
0.62 (0.35-0.90) 17
>10 >50
0.013 (0.010-0.018)
0.028 (0.018-0.043)
0.069 (0.058-0.828)
13.2 (10.1-17.9) 1015
16.8 (10.1-20.0) 600
>10
ND
>145
ND
2.0
(0.60-6.5)
0.016 (0.011-0.023)
14.8 (12.4-18.0) 925
Scheme 6. Reagents and conditions: a) Boc2O, CH2Cl2, rt, 16 h, quant.; b)
MesCl, Et3N, CH2Cl2, 0 °C, 20 min, quant.; c) 42, K2CO3, KI, DMF, 90 °C, 12 h,
84%; d) 41, K2CO3, KI, DMF, 90 °C, 12 h, 66%; e) TFA, CH2Cl2, rt, 2 h, 87%; f)
TFA, CH2Cl2, rt, 2 h, 86%; g) 61: PhCOCl, Et3N, CH2Cl2, rt, 5 min, 85%; 65:
RCOOH, EDC•HCl, HOBt, Et3N, CH2Cl2, rt, 12 h, 88%; 67: 4-chloro-7-
nitrobenzoxadiazole, NaHCO3, CHCl3/ MeOH 1:1, rt, 18 h, 98%; h) 62: PhCOCl,
Et3N, CH2Cl2, rt, 5 min, 96%; 63, 64, and 66: RCOOH, EDC•HCl, HOBt, Et3N,
CH2Cl2, rt, 8-16 h, 85-87%; 68: 4-chloro-7-nitrobenzoxadiazole, NaHCO3,
CHCl3/ MeOH 1:1, rt, 18 h, 78%; 69: Ac2O, Et3N, DMAP, CH2Cl2, rt, 30 min,
87%; 70: PhSO2Cl, Et3N, CH2Cl2, rt, 30 min, 84%.
1.9
(0.55-6.4)
ND
ND
28
0.21
0.12
(0.14-0.31)
(0.079-0.18)
6.0 (2.3-14.7)
9.2 (6.7-11.2)
12.9 (5.6-15.5)
77
0.063 (0.054-0.071)
204
[a] For structures, cf. Fig. 6. Data for cpds. 65 and 66 have already been
reported in ref[10]. ND = Not determined. [b] Concentration required for half-
maximal inhibition of uptake of AEA into U937 cells. EC50 values represent the
mean of at least three independent experiments. 950% CI, 95% confidence
interval. [c] Maximal inhibition of uptake of AEA into U937 cells. Maximum
inhibition data in our uptake assay data varied significantly; thus, at this point,
we consider numbers >60% as essentially indistinguishable. [d] IC50 for the
inhibition of FAAH in U937 cell homogenates. [e] Selectivity index = EC50 (AEA
uptake inhibition)/IC50 (FAAH inhibition). [3H]-AEA was used to determine
uptake inhibition by WOBE437 analogs; FAAH inhibition was assessed in U937
As illustrated by the data summarized in Table 5, several of the
analogs 57 - 70 were highly potent inhibitors of AEA uptake, with
63 and 66 closely approaching the activity of WOBE437 (1). It is
noteworthy that these analogs retained the outstanding selectivity
over FAAH, thus clearly indicating that inhibition of AEA uptake
was not caused by compromised FAAH activity; as the only
example, selectivity was somewhat reduced for analog 59,
although the compound was still selective. Compared to their
BOC-protected precursors 57 and 59, amines 58 and 60,
respectively, were clearly more potent; with no differences in
activity being observed between the respective regioisomers. In
contrast, and very intriguingly, a profound dependence of AEA
uptake inhibitory potency on the position of the extension on the
aromatic ring is observed for 65 and 66 and, albeit to a lesser
extent, for the pairs of regioisomers 61/62 and 67/68. In all three
cases the isomer with the extension attached to the meta oxygen
is significantly more potent. It is somewhat puzzling, why such a
difference is not observed for amines 58 and 60 or, in particular,
their BOC-derivatives 57 and 59, as the latter also carry a bulky
N-substituent at the chain terminus. Comparison of 62, 63, and
64 reveals that both electron-donating as well as electron-
withdrawing substituents are reasonably well tolerated at the para
position of the benzamide moiety. Likewise, sulfonamide-based
analog 70 almost retains the activity of the amide-based parent
compound 61. Our previous report on WOBE437 (1)[10] also
included analogs with an N-iso-butyl or an N-(2-hydroxyethyl)
amide head group, which are part of the natural product (2E,4E)-
N-iso-butyl dodeca-2,4-dienamide from different Echinacea
species[22] and of the natural transport substrate AEA
(anandamide), respectively. Both compounds were found to be
poor inhibitors of AEA uptake into U937 cells.
cell homogenates. For experimental details cf. ref[14]
.
Notwithstanding these negative findings, we have also
investigated a very small set of WOBE437 analogs with other non-
aromatic head groups; in addition, we have prepared one analog
incorporating an alternative (non-phenylethyl/benzyl) aromatic
amide substituent (Fig. 7).
Figure 7. WOBE437 analogs with non-phenylethyl/benzyl amide head groups
Compounds 71, 73, and 74 were prepared by EDC/HOBt-
mediated coupling[17][18] of acid 95 with the (commercially
available) amines 111-113, respectively, in good yields (Scheme
4; for structures of 111-113 see the SI); 72 was obtained by BOC-
removal from 71 with HCl/dioxane.
Analogs 71 and 72 were >10-fold and >30-fold less active,
respectively, than the corresponding parent compound 49 (and,
thus, several hundred-fold less active than WOBE437 (1);
7
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10.1002/cmdc.202000153
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similarly, indole derivative 74 was >100-fold less potent than
WOBE437 (1) (Table 6).
Table 6. Inhibition of AEA uptake into U937 cells by WOBE437 analogs with
non-phenylethyl/benzyl amide head groups.a
FAAH IC50 [M][d]
Max. Inh.
EC50 [M]
Cpd.[a]
SI[e]
(%)[c]
(95% CI)[b]
(95% CI)[b]
Scheme 7. Reagents and conditions: a) 81 (cf. Scheme 1), EDC•HCl, HOBt,
Et3N, CH2Cl2, rt, 12 h, 100%; b) For 75: LDA, THF, -78 °C, 30 min, then 119, -
78 °C to -20 °C, 20 min, 32%; for 76: LDA, THF, -78 °C, 30 min, then 120, -78
°C to -20 °C, 20 min, 47%.
71
72
73
74
9.5
(3.5-15.6)
49
>10
>10
ND
ND
ND
ND
ND
ND
4.19 (1.37-12.5)
0.18 (0.069-0.49)
1.11 (0.68-1.83)
79
49
68
Analogs 75 and 76 both were less potent than WOBE (1) by ca.
25- to 30-fold, with no significant difference being detectable
between the two enantiomers (Table 7). Likewise, the
incorporation of a methyl or a hydroxymethyl substituent at C1 of
the ethylene linker between the amide nitrogen and the phenyl
ring resulted in a reduction in AEA uptake inhibitory activity;
however, measurable differences were now observed between
enantiomers. Thus, methyl-substituted analog 78, which is only 7-
fold less active than WOBE437 (1), is ca. 3-fold more potent than
its R-enantiomer 77. The stereochemical preference is reversed
for hydroxymethyl substitution, with 79 now being ca. 4.5-fold
more potent than 80 (note that due to a change in priority for a
hydroxymethyl vs. a methyl substituent, the more potent isomer
79 is still S-configured). Overall, there is an almost 8-fold
preference for a C1-methyl over a C1-hydroxymethyl group for
one spatial orientation of the substituent (78 vs. 80), while the
difference is only 2-fold for the other (77 vs. 90). However, it
needs to be kept in mind that all differences between analogs
75 - 80 are relatively small and should not be overinterpreted.
Nevertheless, the fact that chirality can be incorporated into
WOBE437 (1) with tolerable effects on potency could be important
for future optimization efforts on the WOBE437 scaffold, as chiral
molecules have been suggested to be less prone to promiscuous
effects than those without chiral centers.[23][24]
[a] For structures, cf. Fig. 7. ND = Not determined. [b] Concentration required
for half-maximal inhibition of uptake of AEA into U937 cells. EC50 values
represent the mean of at least three independent experiments. 950% CI, 95%
confidence interval. [c] Maximal inhibition of uptake of AEA into U937 cells.
Maximum inhibition data in our uptake assay data varied significantly; thus, at
this point, we consider numbers >60% as essentially indistinguishable. [d] IC50
for the inhibition of FAAH in U937 cell homogenates. [e] Selectivity index = EC50
(AEA uptake inhibition)/IC50 (FAAH inhibition). [3H]-AEA was used to determine
uptake inhibition by WOBE437 analogs; FAAH inhibition was assessed in U937
cell homogenates. For experimental details cf. ref[14]
.
In contrast, cyclohexyl derivative 73, even if being ca. 20-fold less
active than WOBE437 (1), retained substantial potency; in fact,
the compound was somewhat more potent than the
corresponding phenyl derivative 49 (Fig. 4, Table 3) and it is
orders of magnitude more potent than its iso-butyl amide
congener.
In order to assess the effects of substitution of either the acyl
chain or the ethyl linker on AEA uptake inhibition, we finally
investigated the chiral WOBE437 analogs depicted in Fig. 8.
Table 7. Inhibition of AEA uptake into U937 cells by chiral WOBE437 analogs.a
Max. Inh.
EC50 [M]
FAAH IC50 [M][d]
Cpd.[a]
SI[e]
(%)[c]
103
121
60
(95% CI)[b]
(95% CI)[b]
Figure 8. Chiral WOBE437 analogs.
75
76
77
78
79
80
0.279 (0.18-0.44)
0.247 (0.18-0.34)
0.23 (0.16-0.33)
0.071 (0.040-0.12)
0.12 (0.05-0.2)
0.54 (0.38-0.77)
>1
ND
ND
9.6
ND
37
We were well aware that analogs 75 and 76 incorporate multiple
modifications, which would prevent an unambiguous structural
interpretation of any activity differences to WOBE437 (1);
however, these compounds were attractive, due to their ready
accessibility from citronellal, which is commercially available in
both enantiomeric forms. Thus, HWE reaction of either S-
citronellal (119) or R-citronellal (120) with phosphonate 118
directly led to 75 and 76, respectively (Scheme 7). (For the
synthesis of 77 - 80 see the SI).
>1
2.2 (1.4-3.5)
ND
61
71
4.4 (2.9-5.7)
7.9 (4.5-13.0)
72
15
[a] For structures, cf. Fig. 7. ND = Not determined. [b] Concentration required
for half-maximal inhibition of uptake of AEA into U937 cells. EC50 values
represent the mean of at least three independent experiments. 95% CI, 95%
confidence interval. [c] Maximal inhibition of uptake of AEA into U937 cells.
Maximum inhibition data in our uptake assay data varied significantly; thus, at
this point, we consider numbers >60% as essentially indistinguishable. [d] IC50
for the inhibition of FAAH in U937 cell homogenates. [e] Selectivity index = EC50
(AEA uptake inhibition)/IC50 (FAAH inhibition). [3H]-AEA was used to determine
uptake inhibition by WOBE437 analogs; FAAH inhibition was assessed in U937
cell homogenates. For experimental details cf. ref[14]
.
8
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10.1002/cmdc.202000153
ChemMedChem
FULL PAPER
Conclusions
Acknowledgements
We have assessed the inhibition of AEA uptake into U937 cells
by almost 80 analogs of the potent AEA uptake inhibitor
This research was supported by the Swiss National Science
Foundation through the National Centre of Competence in
Research (NCCR) TransCure. We are indebted to Dr. B. Pfeiffer.
Dr. L. Betschart, and Philipp Waser for NMR support, to Dr. X.
Zhang, L. Bertschi, R. Häfliger, and O. Greter for HRMS spectra,
and to Kurt Hauenstein for general technical support. We thank
Patricia Schenker und Tatiana Hofer for excellent support with
compound testing. We thank Dr. Lea Radtke for providing
intermediates 141 and 143.
WOBE437
((2E,4E)-N-(3,4-dimethoxyphenethyl)dodeca-2,4-
dienamide, 1), incorporating a diverse set of modifications both in
the acyl chain as well as the dimethoxyphenylethyl head group.
This has included variations in acyl chain length and its degree of
unsaturation, the introduction of rigidifying elements, the complete
removal, demethylation or extension of methoxy groups, the full
replacement of the phenyl moiety by other entities, and the
incorporation of chirality in both the fatty acid moiety as well as
the head group. As the bottom line finding, none of these
compounds was more active than WOBE437 (1), with 23 out of
79 analogs exhibiting IC50 values for AEA uptake of >1 M (vs. 10
nM for WOBE (1)). At the same time, several analogs exhibited
IC50 values close to or below 100 nM; importantly, these
compounds proved to be highly selective AEA uptake inhibitors
(with selectivity indices vs. FAAH inhibition between 17 and
>1000), thus attesting to the exquisite selectivity potential
associated with the WOBE437 scaffold. The most potent analogs
identified were 62 and 66, whose activity is essentially identical
with that of WOBE437 (1). Perhaps the most intriguing finding of
this SAR study is the >100-fold activity difference between
regioisomers 65 and 66; similarly, 62 is more potent than 61 and
68 is more active than 67, although the differences are less
pronounced (ca. 13-fold and 9-fold, respectively). While these
differences cannot be rationalized in the absence of structural
information on the interacting protein(s) (which are still elusive at
this point), our findings provide important guidance for future
optimization studies. Likewise, analog 22, which does not contain
any olefinic double bonds and exhibits an IC50 for AEA uptake of
107 nM and chiral analog 78 (IC50 of 78 nM) are interesting
departure points for the development of potent AEA inhibitors with
more drug-like properties. In particular, 22, with a selectivity index
of 49, offers significant room for variations of and around the
newly introduced aromatic site and efforts in this direction have
been initiated in our laboratories.
Conflict of interest
This authors declare no conflict of interest.
Keywords: endocannabinoid system • endocannabinoid
membrane transport • anandamide transport inhibitor • SAR •
WOBE437
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Experimental Section
Detailed protocols for the synthesis of all final products and
intermediates, analytical data for all new compounds, and copies
1
of the relevant H- and 13C-NMR spectra can be found in the SI.
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Entry for the Table of Contents
The dodeca-2,4-dienamide WOBE437 (1) is a potent inhibitor of endocannabinoid uptake. To elucidate the key structural features
underlying the specific inhibitory activity of 1, we have conducted a comprehensive SAR study on almost 80 WOBE437 analogs with
modifications in the dodecadienoyl domain as well as the dimethoxyphenylethyl head group. Several of these variants exhibited
potencies in the sub-100 nM range and were highly selective over inhibition of the endocannabinoid-degrading enzyme fatty acid
amide hydrolase. However, none of the analogs surpassed the potency of WOBE437 (1).
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