Biological evaluation of pyridone alkaloids on the endocannabinoid system

Article abstract of DOI:10.1016/j.bmc.2017.02.031

Naturally occurring pyridone alkaloids as well as synthetic derivatives were previously shown to induce neurite outgrowth. However, the molecular basis for this biological effect remains poorly understood. In this work, we have prepared new pyridones, and tested the effect of thirteen 4-hydroxy-2-pyridone derivatives on the components of the endocannabinoid system. Investigation of binding affinities towards CB₁ and CB₂ receptors led to the identification of a compound binding selectively to CB₁ (12). Compound 12 and a closely related derivative (11) also inhibited anandamide (AEA) hydrolysis by fatty acid amide hydrolase. Interestingly, none of the compounds tested showed any effect on 2-AG hydrolysis by monoacylglycerol lipase at 10 μM. Assessment of AEA uptake did, however, lead to the identification of four inhibitors with IC₅₀ values in the submicromolar range and high selectivity over the other components of the endocannabinoid system.

Full text of DOI:10.1016/j.bmc.2017.02.031

Accepted Manuscript
Biological Evaluation of Pyridone Alkaloids on the Endocannabinoid System
Andrea Chicca, Regina Berg, Henning J. Jessen, Nicolas Marck, Fabian Schmid,
Patrick Burch, Jürg Gertsch, Karl Gademann
PII:
S0968-0896(16)31251-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.031
BMC 13561
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 December 2016
10 February 2017
14 February 2017
Please cite this article as: Chicca, A., Berg, R., Jessen, H.J., Marck, N., Schmid, F., Burch, P., Gertsch, J., Gademann,
K., Biological Evaluation of Pyridone Alkaloids on the Endocannabinoid System, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biological Evaluation of Pyridone Alkaloids on the Endocannabinoid System
Andrea Chiccaa, Regina Bergb, Henning J. Jessenc, Nicolas Marckb, Fabian Schmidc,
Patrick Burchc, Jürg Gertscha*, Karl Gademannb*
a
NCCR-TransCure, Institute of Biochemistry and Molecular Medicine, University of
Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland.
b
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland.
c
Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel,
Switzerland.
* Corresponding authors. E-mail addresses: juerg.gertsch@ibmm.unibe.ch (Prof. Dr. J.
Gertsch); karl.gademann@uzh.ch (Prof. Dr. K. Gademann).

Abstract
Naturally occurring pyridone alkaloids as well as synthetic derivatives were previously
shown to induce neurite outgrowth. However, the molecular basis for this biological
effect remains poorly understood. In this work, we have prepared new pyridones, and
tested the effect of thirteen 4-hydroxy-2-pyridone derivatives on the components of the
endocannabinoid system. Investigation of binding affinities towards CB1 and CB2
receptors led to the identification of a compound binding selectively to CB1 (12).
Compound 12 and a closely related derivative (11) also inhibited anandamide (AEA)
hydrolysis by fatty acid amide hydrolase. Interestingly, none of the compounds tested
showed any effect on 2-AG hydrolysis by monoacylglycerol lipase at 10 µM.
Assessment of AEA uptake did, however, lead to the identification of four inhibitors with
IC50 values in the submicromolar range and high selectivity over the other components
of the endocannabinoid system.
1. Introduction
Hydroxy-2-pyridones constitute a compelling class of secondary metabolites,[1] which
have been suggested to act as chemical mediators in interactions between
entomopathogenic fungi and their insect hosts. Over the past years, a number of
biological activities have been reported for these compounds, mostly focusing on their in
vitro properties, such as cytotoxicity.[2] For example, ricinine, a 2-pyridone alkaloid first
isolated from castor oil plants in the 19th century,[3] has long been known as an
insecticide[4] and lately been reported to have stimulatory effects on the central nervous
system.[5] Pronounced antifungal activity has been observed with the structurally more
complex compounds ilicicolin H,[2d, 6] apiosporamide,[7] and YM-215343.[2e] However, the
molecular targets of 4-hydroxy-2-pyridones have generally remained elusive.[8] A recent
study by the group of Waldmann, however, demonstrated an inhibitory effect on kinase
activity, and a crystal structure of 3-acyl-4-hydroxy-2-pyridone complexed with wild-type
mitogen-activated protein kinase 4 (MAP4K4) with the inhibitor residing in the ATP
binding site supports this hypothesis.[9]
There have been numerous efforts directed towards the total synthesis of 4-hydroxy-2-
pyridones, as it is difficult to obtain these alkaloids in pure form from natural sources.[10]
The work of one of our groups has focused on a family of 3-acyl-4-hydroxy-2-pyridone

alkaloids isolated from entomopathogenic fungi (Table 1).[2b, 11] Chemical synthesis
yielded the congeners pretenellin B, prebassianin B, farinosone A, militarinone D and
another homologue of the militarinone family (HJJ-510). All of these substances show
neuritogenic activity in the PC12 cell assay.[12] Based on these phenotypic observations,
we became interested in the potential mechanism of action of these compounds.
Table 1: Family of pyridone alkaloids from entomopathogenic fungi.
pretenellin B
tenellin
R = H, n = 1
R = OH, n = 1
R = H, n = 2
R = OH, n = 2
R = H, n = 3
R = OH, n = 3
prebassianin B
bassianin
farinosone A
farinosone B
R = H, n = 1
R = OH, n = 1
R = H, n = 2
R = OH, n = 2
unknown (HJJ-510)
unknown
militarinone D
unknown
X = CH3, n = 1
X = H, n = 2
pyridovericin
torrubiellone C
The structurally related compound farinosone C triggers neurite outgrowth, and we have
shown that structurally simplified analogues retain this activity.[13] In order to investigate
the underlying biochemical pathways responsible for neurite outgrowth, these
substances were tested for their ability to bind to cannabinoid receptors (CB1 and CB2).
It had been previously reported that CB1 receptor activation promotes neuritogenesis in
Neuro-2A cells via a complex signalling pathway[14] and to restore neuritogenic activity in
hyperglycemic PC12 cells.[15] One of the farinosone C analogues (BSL-34; Scheme 1)
very effectively inhibits anandamide (AEA) uptake – most likely by interacting with the

putative endocannabinoid membrane transporter, which is supposed to regulate the
bidirectional shuttle of endocannabinoids across the plasma membrane or via interaction
with cytoplasmic shuttle proteins.[16]
Scheme 1: Neuritogenic compounds farinosone C and BSL-34.
Based on these observations, we reasoned that AEA uptake inhibition might be the
cause for the neuritogenic effects exhibited by BSL-34 because such inhibition would
lead to an increase in the extracellular concentrations of AEA, which can in turn activate
cannabinoid receptors. In addition, increasing levels of AEA might also affect TRPV1
activity, which plays a role in neuronal differentiation of SH-SY5Y human neuroblastoma
cells.[17] Therefore, we investigated whether the neuritogenic effects of the pyridone
alkaloids are related to activation of CB1 and CB2 and/or indirect modulation of AEA
levels by inhibition of the degrading enzymes and/or specifically AEA uptake. All
compounds tested – eight natural products, two truncated ester derivatives and three
derivatives with ketone side chains of various length – are shown in Table 2.
Table 2. Chemical structure of the pyridone alkaloids tested on the different components
of the ECS and known inhibitors for positive controls.
entry
name
structure
1
farinosone A

2
3
4
5
militarinone D
torrubiellone C
HJJ-510
prebassianin B
6
7
8
9
SF-182
pretenellin B
pyridovericin
NM33

10
11
12
13
NM34/36
NM56
NM57
NM58
14
CP55940
UCM707[18]
JZL184[19]
15
16

WWL70[20]
17
THL[20]
18
URB597[18, 21]
19
20
DuP697[22]
OMDM-2[23]
21
22
WIN55,212-2[24]
2. Chemistry

The natural products tested in this study for their activity towards components of the
endocannabinoid system were obtained by total synthesis as reported in literature.[10i, 12,
25]
As we aimed to produce 4-hydroxy-2-pyridone derivatives with diverse side chains at
position C-3, we first envisioned a late-stage transacylation of the methyl ester analogue
(27) of the known ethyl ester intermediate from the total synthesis of natural pyridone
alkaloids.[12] We thus prepared methyl (Z)-2-cyano-3-methoxybut-2-enoate by conden-
sation of methyl cyanoacetate (22) with trimethylorthoacetate according to a procedure
first reported by Arndt in 1936 with concomitant distillation of methanol.[26] Upon cooling,
the product solidified. It was re-crystallized from an aqueous methanol solution (62 %
yield) and used without further purification in the subsequent condensation with N,N-
dimethylformamide dimethyl acetal to give the bright yellow push-pull substituted olefin
23 with 74 % yield. Heating this enamine in acetic acid triggers nucleophilic attack of the
amino group at the cyano group, finally leading to formation of the pyridine ring, as
reported by the group of Prager.[27] However, due to the harsh reaction conditions this
procedure is prone to side product formation and the isolated yield of the desired
product was only 40 %. Subsequently, the pyridone ring was site-selectively brominated
in 5-position with an excellent yield of 96 % to give methyl 5-bromo-4-methoxy-2-oxo-
1,2-dihydropyridine-3-carboxylate 24. As in the total synthesis of pyridone alkaloid
natural products,[12] the pyridone ring was protected by reaction with 2-(trimethylsilyl)-
ethoxymethyl chloride giving the N-SEM (25a) and the O-SEM product (25b) in a ratio of
4:3. Both derivatives undergo facile Suzuki coupling with {4-[(4-methoxybenzyl)oxy]-
phenyl}boronic acid, but in this study the N-SEM isomer was further utilized. With the
PMB-protected 4-hydroxy aryl group installed (26), we now tested the attempted
transesterification under mild conditions as reported by Seebach and co-workers.[28]
Using molecular sieves as a scavenger for methanol, we were able to obtain the
pentenyl (27) and decanyl ester (28) in moderate yields of 28 % and 22 % with the
desired concomitant deprotection of the hydroxyl group on position C-4 of the
heterocycle, which is much more critical to carry out at a later stage of the synthesis.
Deprotection of the pyridone ring and the phenol with TBAF and TFA, respectively, gave
the desired products 9 and 10.

1) HOAc/H2O (4/1)
reflux
2) NBS, NH4NO3
MeCN, reflux
1) trimethyl orthoacetate, neat, 110 °C, 7 h;
then 135 °C
2) N,N-dimethylformamide dimethylacetale
MeOH, reflux, 45 min
MeO
COOMe
CN
O
NC
OMe
Me2N
22
23
46 %
(over two steps)
OMe
OMe
OMe
Br
COOMe
Br
COOMe
OSEM
Br
COOMe
O
SEM-Cl
Et3N, DCM, 0 °C
N
H
O
N
N
SEM
25a
24
25b
38 %
(over two steps)
40 %
:
33 %
PMBO
PMBO
OMe
OMe
B(OH)2
[Pd(PPh3)4], K2CO3
COOMe
O
Br
COOMe
N
N
O
DME/DMF/H2O
SEM
SEM
25a
16 h, 60 °C
26
92 %
PMBO
HO
OH
O
O
OH
O
O
1) TBAF, THF, 65 °C
2) TFA, DCM, rt
OR
OR
ROH, LiBr, DBU
THF/DCM 3/1, reflux
N
H
N
SEM
27
9
, R = (CH2)3CH=CH2: 39 %
, R = (CH2)3CH=CH2: 22 %
28, R = C10H21: 28 %
10, R = C10H21: 22 %
(over two steps)
Scheme 2: The transesterification route to pyridine alkaloid derivatives.
Due to the limitations of this route regarding general applicability and yield, we prepared
derivatives with ketone side chains of different length following synthetic procedures
established by Baldwin and Waldmann.[9, 10h] One of the key advantages of this route is
the possibility to start from commercially available 2,4-dihydroxy pyridine (29) without
having to form the heterocyclic ring. Following procedures reported by Baldwin et al.,[10h]

a double bromination led to 3,5-dibromopyridine-2,4-diol in 71 % yield after recrystalli-
zation from ethanol/water. Protection of the free hydroxyl groups with methyl iodide in
the presence of silver carbonate proceeded sluggishly to give the desired 3,5-dibromo-
2,4-dimethoxypyridine (30) in 74 % yield after stirring the reaction mixture for five days at
room temperature. The step conferring structural diversity is the subsequent
regioselective lithiation and alkylation. With octanal, dodecanal and octadecanal this
reaction proceeded with good to excellent yields to give the racemic mixtures of alcohols
31–33. The protected 4-hydroxy aryl group was installed by Suzuki coupling with [(tert-
butyldimethylsilyl)oxy]phenyl boronic acid following procedures of Waldmann and
coworkers.[9] Subsequent Dess-Martin oxidation of 34–36 (82–94 % yield) was followed
by deprotection of both the 4-hydroxy group and the phenol to give the desired pyridone
compounds 11–13 in good yield. Although the diversifying key step is carried out early
on, this strategy proved to be much more effective as it comprises only six synthetic
steps, most of them with high to excellent yields.
Scheme 3: Alkylation-Suzuki route for the preparation of pyridone alkaloid derivatives.

We were thus able to assess the biological activity of eight natural products, two
truncated ester derivatives and three derivatives with ketone side chains of various
length (Table 2).
3. Results and discussion
Thirteen 4-hydroxy-2-pyridone compounds were tested for their activity on the different
components of the endocannabinoid system (ECS). The molecular structure of the
compounds is given in Table 2. Among them are natural products such as farinosone A,
militarinone D, torrubiellone C, prebassianin B and pyridovericin, which have been
produced by total synthesis.[10h-j, 12, 25] Entries 4 and 6 represent compounds that are
structurally very similar, but have not been isolated from natural sources. As the side
chain of 4-hydroxy-2-pyridones was shown to hardly have any influence on their
neuritogenic activity in PC12 cell assays,[29] truncated structurally simplified derivatives
with an ester and a ketone side-chain of different lengths were included as well.
First, we investigated the binding properties to CB1 and CB2 receptors.[30] As reported in
Fig. 1A, HJJ-510 (4) and pretenellin B (7) showed a very weak binding to CB1 defined by
approximately 40 % displacement of [3H]CP55,940 at a concentration of 1 µM.
Compound 12 reached 50 % displacement of the radioligand and was assessed in a full
concentration-dependent curve showing a Ki value of 1.0 µM (Fig. 1B). In contrast, the
other structurally simplified pyridones did not show any significant binding to CB1 and
CB2 receptors (< 20 %) at a concentration of 1 µM (Fig 1A). Compound 12 did not bind
to CB2 receptors, thus showing a selective interaction with CB1 receptors.
A
B
CB2
CB1
Ki= 1.0 (0.7 -1.5) µM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9 9 10 10 11 11 12 12 13 13
-9 -8 -7 -6 -5 -4
log [12] (M)

Fig. 1. A: CB1 and CB2 receptors binding properties of pyridone alkaloids tested at the
screening concentration of 1 µM. B: Concentration-dependent binding curve for
compound 12 to CB1 receptors.
We then investigated the effects of these compounds on 2-OG hydrolysis by monoacyl-
glycerol lipase (MAGL), α,β-hydrolase domain-6 (ABHD6) and -12 (ABHD12), as well as
AEA hydrolysis by fatty acid amide hydrolase (FAAH). As shown in Fig. 2A, none of the
tested compounds showed significant inhibition of MAGL activity at the concentration of
10 µM. Similarly, our compound library did not inhibit ABHD6 and ABDH12, as
compared to the positive control compounds WWL70 (17) and THL (18) used for the
ABHD6 and ABHD12 assay, respectively (data not shown).[20] In contrast, militarinone D
(2), 10 and 12 inhibited FAAH activity by 40–50 % at 10 µM, while compound 11
inhibited AEA hydrolysis by 80 % compared to vehicle. In further concentration-
dependent experiments, 11 showed an IC50 value of 4.6 µM (Fig. 2B). Interestingly, this
compound did not show any significant inhibition of MAGL, thus indicating a selectivity
towards FAAH inhibition.
A
B
FAAH
MAGL
120
100
80
60
40
20
0
120
100
80
60
40
20
0
IC50= 4.6 (3.8-5.6) µM
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 19 16
-7
-6
-5
-4
log [11] (M)
Fig. 2. A: FAAH and MAGL activity upon treatment with the seven natural pyridone
alkaloids and five simplified analogs tested at the screening concentration of 10 µM.
URB597 (19) (1 µM)[21] and JZL184 (16)[19] (1 µM) were used as positive control for
FAAH and MAGL inhibition, respectively. B: Concentration-dependent inhibition of FAAH
activity induced by 11 in U937 cell homogenate.

Alternatively, AEA and 2-AG can undergo oxidative metabolism mediated by COX-2
activity.[31] This degradation pathway generates prostaglandin-ethanolamines and
prostaglandin-glycerol esters, which were shown to possess bioactivity unrelated to the
interaction with cannabinoid receptors and the classic prostaglandin receptors. In the
last years, it has been observed that several compounds can act as specific inhibitors by
blocking the COX-2-mediated oxygenation of AEA and 2-AG without affecting
arachidonic acid oxidation.[32] Here, we tested the pyridone alkaloids for the inhibition of
COX-2-mediated oxidation of the classic substrate arachidonic acid or the
endocannabinoid 2-AG. As shown in Fig. 3, some of the compounds showed partial
inhibition of COX-2 (ranging from 20 % to 50 %) at a screening concentration of 10 µM,
which was not selective for either of the substrates.
Arachidonic Acid
2-AG
120
100
80
60
40
20
0
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 20 20
Fig. 3. COX-2 activity upon treatment with the natural pyridone alkaloids and derivatives
at a screening concentration of 10 µM using either arachidonic acid or 2-AG as
substrate. DuP697 (1 µM)[22] (20) was used as a positive control.
Finally, all compounds were screened on AEA uptake using U937 cells as previously
described. At a concentration of 10 µM, farinosone A (1) induced the most potent
inhibition of AEA uptake (Fig. 4A) and in further concentration-dependent experiments, it
showed an IC50 value of 1.2 µM (Fig. 4B), being in range with the potency reported for
the positive controls UCM707[18] and OMDM-2.[23, 33] Militarinone D (2), SF-182 (6) and
pretenellin B (7) only partially inhibited AEA uptake at 10 µM, while torrubiellone C (3),

HJJ-510 (4), prebassianin B (5) and pyridovericin (8) were inactive (Fig. 4A). This data
indicates that, in comparison to farinosone A (1), shorter side chains in combination with
a lower degree of unsaturation (4 and 5) and in the presence of a hydroxyl group (3 and
8) lead to impaired binding capacity.
The synthetic analogs of the natural pyridone alkaloids (9–13) were also tested under
the same conditions. At a concentration of 10 µM, 10, 11and 12 induced maximal
inhibition of AEA uptake in the same range as the positive controls (Fig. 4A). In further
concentration-dependent experiments, these compounds showed IC50 values in the
submicromolar range (0.67–0.78 µM) (Fig 4B). In contrast, compounds 9 and 13 were
inactive (Fig. 4A). This indicates that fully saturated unbranched side chains in the range
of C8–C12 (10–12) represent the optimal length for inhibition of AEA uptake , as
compared to longer (C20, 13) and shorter (C4, 9) chains. When comparing natural
pyridone alkaloids with their synthetic analogs, the data indicates that branched side
chains (1–8) are less favourable for AEA uptake inhibition as compared to linear alkyl
side chains (10–12).
A
120
100
80
60
40
20
0
B
120
100
80
60
40
20
0
1
10
11
12
1
2
3
4
5
6
7
8
9
10 11 12 13 15 21
-8 -7 -6 -5 -4
Log [compound] (M)
Fig. 4. A: Inhibition of AEA uptake into U937 cells induced by natural pyridone alkaloids
and their analogs at a screening concentration of 10 µM. B: The most potent inhibitors
were tested in concentration-dependent experiments. UCM707 (15)[18] and OMDM-2
(21)[23] (both at 10 µM) were used as positive controls. Data in graph B have been
normalized.

AEA hydrolysis and uptake are coupled processes as the FAAH-dependent AEA
concentration gradient needs to be maintained. This gradient is the driving force for
34]
AEA’s energy-independent uptake.[16b,
In cellular systems, FAAH inhibitors can
indirectly inhibit AEA uptake by affecting this concentration gradient,[34-35] and a
correlation between IC50 values for FAAH and AEA uptake inhibition was reported.[36]
Importantly, farinosone A (1) and the synthetic analogs 10–12 showed IC50 values >10
µM for FAAH. These compounds also showed IC50 values >10 µM for MAGL and COX-
2. Thus, 1 and 10–12 are selective inhibitors of AEA uptake (Table 3). Among them, only
12 showed weak binding to the CB1 receptor (Ki = 1.0 µM), while the other compounds
did not significantly bind to CB1 or CB2 (0–30% binding at 1 µM).
Table 3. Summary of the Ki and IC50 values of natural pyridone alkaloids 1 and their
analogs 10-13 on the different components of the ECS.
Ki value (CB1 and CB2) or IC50 values (mean SD, µM)
AEA
Compound
CB1
CB2
FAAH
MAGL
COX-2
uptake
1.2 0.6
0.6 0.4
0.7 0.4
0.8 0.3
>10
1
>1
>1
>1
>1
>1
>1
>1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10
11
12
13
>1
>10
1.0 0.5
>1
4.6 0.7
>10
4. Conclusion
In conclusion, we describe that the natural products 1, 2, 6 and 8 as well as synthetic
pyridone alkaloids 10–12 act as AEA uptake inhibitors. Their IC50 values lie in the
micromolar (1, 2, 6, 8) and in submicromolar (10–12) range and a selectivity >10 fold
over the main components of the ECS, including FAAH. In agreement with other studies
on different scaffolds, our results indicate that small modifications of the pyridone
alkaloid side chain in terms of length, branching, degree of unsaturation and the
presence of hydrophilic substituents can lead to a significant loss of potency for AEA
uptake inhibition.[34-36]

5. Experimental section
5.1 Chemistry
The total syntheses of natural products 1–8 have been published by one of our groups
and the substance samples used in this work have been produced by these
procedures.[10i, 12, 25] Derivatives 9–13 are novel compounds that have not been reported
elsewhere. Esters 9 and 10 are produced by transesterification of the respective methyl
ester derivatives. The preparation of ketones 11–13 has been carried out in close
analogy to previously reported synthetic work by the groups of Baldwin and
Waldmann.[9, 10h] All synthetic procedures as well as analytical data of all intermediates
can be found in the Supporting Information. Chemicals and solvents used for reactions
were supplied by Sigma-Aldrich, Alfa Aesar, Fluka, Fluorochem, Maybridge, Acros, and
Merck, and were used without further purification. Distilled technical grade solvents were
used for column chromatography and work-up. Solvents were removed in vacuo on a
rotary evaporator with 40 °C water bath temperature. Unless otherwise stated, reactions
were carried out under argon atmosphere and reactions involving air-sensitive reagents
were carried out using solvents degassed by freeze-pump-thaw cycling. Deuterated
solvents used for NMR spectroscopy were supplied by Cambridge Isotope Laboratories.
All reported yields are isolated yields, unless otherwise indicated. Flash chromatography
was performed by dry-pack method using silica gel with particles size between 0.040
and 0.063 mm as stationary phase according to the procedure developed by Mitra and
coworkers.[37] Thin layer chromatography was performed on silica gel-coated glass
plates (Merck 60 F254, thickness: 210–270 µm), spots were observed under UV light (λ =
254 nm) and plates were stained with potassium permanganate solution. Preparative
HPLC purifications were carried out on a Dionex Chromatography System (Interface
Chromeleon, ASI 100 automatic sample injector, PDA 100 (USB) PD detector, pump
P680); flow rate: 20 mL min-1; column: Phenomenex Synergi Hydro-RP, 10 µm, 80 Å,
250 mm x 21.2 mm); solvents: A – H2O, B – MeCN.
Method 1: from 80 % B to 100 % B over 2 minutes and maintaining 100 % B during 40
minutes. Method 2: from 50 % B to 100 % B over 25 minutes and maintained 100 % B
during 15 minutes. Reversed-phase LC-MS analyses were performed on a Shimadzu
LC system with a Reprospher column (Dr. Maisch, 100 Å C18-Aqua, 5 µm, 125 mm x 2

mm) and an amaZon X Bruker mass spectrometer. The solvents used were
demineralized water (milli-Q, 0.01 % formic acid, solvent A) and acetonitrile (HPLC
grade, 0.01% formic acid, solvent B) at a flow rate of 0.5 mL min-1. The gradient applied
was 0.0–2.0 minutes 10 % B; 2.0–12.0 minutes 10 % B to 95 % B; 12.0–15.0 minutes
95 % B; 15.0–16.0 minutes 95 % B to 10 % B; 16.0–18.0 minutes 10 % B. NMR
experiments were performed on Bruker Avance III NMR spectrometers operating at
250 MHz, 400 MHz or 500 MHz proton frequency. Chemical shifts δ are reported in ppm
1
and were determined by reference to the residual H solvent peaks (chloroform: 7.26
ppm (1H) and 77.16 (13C); DMSO: 2.50 ppm (1H) and 39.52 ppm (13C); methanol: 3.31
ppm (1H), 49.00 ppm(13C).[38] The assignment of signals in the 1H and 13C NMR spectra
was achieved by interpretation of DEPT and two-dimensional NMR spectra (COSY,
NOESY, HMBC and HSQC). Processing, analyses and interpretations of NMR spectra
were performed with MestReNova (version: 10.0.1-14719). Infrared spectra were
recorded using a Varian 800 FT-IR ATR Spectrometer. The absorptions were reported in
cm–1, both the intensity and profile of signals were described as weak, medium, strong
or broad. ESI-HRMS spectra. Melting points (m.p.) were determined in open capillaries
using a Büchi B-545 apparatus.
Methyl (Z)-2-cyano-3-methoxybut-2-enoate.[26] In a one-necked 100 ml round-
bottomed flask, methyl cyanoacetate (18.0 mL, 20.1 g, 0.203 mol, 1.0 equiv) and
trimethyl orthoacetate (28.86 g, 0.240 mol, 1.2 equiv) were stirred at 110 °C and the
methanol formed during this reaction was removed via a short distillation bridge. After
three hours, a second portion of trimethyl orthoacetate was added (30.2 g, 0.251 mmol,
1.2 equiv) and the reaction mixture stirred at 110 °C for another four hours. The
temperature was then raised to 135 °C and excess reagents distilled off the reaction
mixture. Upon cooling, the mixture solidified. A mixture of water and methanol (75 mL,
water/methanol 2/1) was added and the flask heated until the solid had completely
dissolved. The hot solution was allowed to cool to room temperature and was stored at 8
°C to allow for crystallization of the product. Colorless crystals, yield: 62 %; m.p. = 97
°C; 1H NMR (400 MHz, CDCl3) δ 3.99 (s, 3H), 3.78 (s, 3H), 2.62 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 184.6, 164.5, 115.1, 85.8, 56.9, 52.3, 15.3; HRMS (ESI+) exact mass
calculated for [C7H10NO3]+ m/z 156.0655 [M+H]+, found m/z 156.0656; FT-IR ??
cm–1): 2957, 2222, 1715, 1572, 1470, 1438, 1429, 1410, 1381, 1315, 1288, 1194, 1129,
̃ (neat,

1088, 1022, 969, 873, 788, 768, 641. Methyl (2Z,4E)-2-cyano-5-(dimethylamino)-3-
methoxypenta-2,4-dienoate (23).[27] Methyl (Z)-2-cyano-3-methoxybut-2-enoate (16.9
g, 0.109 mol, 1.0 equiv) was suspended in dry methanol (8 mL) under an inert
atmosphere and N,N-dimethylformamide dimethylacetale (23.2 mL, 20.7 g, 0.174 mmol,
1.6 equiv) was added. The reaction mixture was stirred under reflux conditions for 45
min and turned dark red. Excess solvents and reagents were removed at the rotary
evaporator. The red solid was dissolved in DCM and purified by filtration over a silica gel
column. After evaporation of the solvent, the orange product was recrystallized from
ethyl acetate/petroleum ether to give a bright yellow solid. Yellow crystals, yield: 74 %;
m.p. = 129 °C; Rf = 0.27 (50 % EtOAc in pentane); 1H NMR (400 MHz, CDCl3) δ 7.45 (d,
J = 12.4 Hz, 1H), 6.09 (s, br, 1H), 3.93 (s, 3H), 3.65 (s, 3H), 3.13 (s, 3H), 2.90 (s, 3H,
NCH3)2); 13C NMR (101 MHz, CDCl3) δ 181.8 ((H3CO)C=C), 166.4 (COOCH3), 153.3
(C=C(NMe2)), 119.3 (CN), 91.4 ((Me2N)C=C), 74.9 (C=C(CN)), 62.5 (OCH3), 51.3
(COOCH3), 45.7 (N(CH3)2), 37.5 (N(CH3)2); HRMS (ESI+) exact mass calculated for
[C10H15N2O3]+ m/z 211.1077 [M+H]+, found m/z 211.1074; FT-IR ?? (neat, cm–1): 2927,
̃
2917, 2187 (CN), 1690, 1612, 1525, 1459, 1432, 1389, 1359, 1315, 1265, 1225, 1205,
1185, 1133, 1052, 1000, 982, 891, 871, 823, 794, 769, 726, 643. Methyl 4-methoxy-2-
oxo-1,2-dihydropyridine-3-carboxylate.
Methyl
2-cyano-5-(dimethylamino)-3-
methoxypenta-2,4-dienoate (15.1 g, 71.9 mmol) was dissolved in an aqueous solution of
acetic acid (80 vol.-%, 50 ml) in a 250 ml round-bottomed flask and stirred under reflux
conditions under inert gas for one hour. The dark red reaction mixture was diluted with
water (25 ml) and solid sodium carbonate was carefully added until pH 7 was reached.
The product was extracted from the aqueous phase with ethyl acetate (8 x 50 mL) and
with DCM (4 x 50 mL). The combined organic phases (red) were dried over sodium
sulfate. After filtration of the drying agent the solvent was removed under vacuum. Flash
chromatography yielded the product as an orange solid (7.7 % MeOH in DCM). Orange
crystals, yield: 40 %; m.p. = 202–204 °C (decomposition); Rf = 0.29 (9.1 % MeOH in
DCM); 1H NMR (400 MHz, CDCl3) δ 13.47 (s, 1H), 7.50 (d, J = 7.4 Hz, 1H), 6.15 (d, J =
7.5 Hz, 1H), 3.89 (s, 3H), 3.88 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 167.5, 165.5,
163.8, 138.3, 108.4, 95.0, 56.7, 52.6; HRMS (ESI+) exact mass calculated for
̃
[C8H9NO4Na]+ m/z 206.0424 [M+Na]+, found m/z 206.0425; FT-IR ?? (neat, cm–1): 2952,
2839, 2360, 1733, 1632, 1610, 1552, 1477, 1429, 1327, 1272, 1241, 1181, 1124, 1094,

1073, 966, 952, 919, 836, 786, 770, 674, 629. Methyl 5-bromo-4-methoxy-2-oxo-1,2-
dihydropyridine-3-carboxylate (24). In a 100 ml round-bottomed flask, methyl 4-
methoxy-2-oxo-1,2-dihydropyridine-3-carboxylate (5.73 g, 31.3 mmol, 1.0 equiv) was
suspended in acetonitrile (50 ml). NBS (5.57 g, 13.1 mmol, 1.0 equiv; freshly crystallized
from water and dried under high vacuum) and ammonium nitrate (0.25 mg, 1.37 mmol,
0.1 equiv) were added. The reaction mixture was heated to reflux for 45 min. The
solvent was removed under vacuum. Ethyl acetate was added (80 ml), but the orange
solid did not completely dissolve. Thus, the solid was filtered and dissolved in DCM.
After evaporation of DCM, the pure product was obtained. The red solution of EtOAc
was washed with brine (4 x 100 ml) and the combined organic phases were dried over
sodium sulfate. After filtration of the drying agent, the solvent was removed under
vacuum. The red solid residue partly dissolved in EtOAc and the solution overlain by
toluene for recrystallization to give the product as slightly yellow crystals. The part of the
red solid that was completely insoluble in EtOAc was collected by filtration and subjected
to column chromatography (7.7 % MeOH in DCM). Slightly yellow crystals, yield: 96 %;
1
m.p. = 154 °C (decomposition); Rf = 0.39 (7.7 % MeOH in DCM); H NMR (400 MHz,
13
CDCl3) δ 13.27 (s, 1H), 7.59 (s, 1H), 3.99 (s, 3H), 3.93 (s, 3H); C NMR (101 MHz,
CDCl3) δ 165.6, 163.9, 163.2, 137.2, 111.2, 96.5, 59.9, 53.2; HRMS (ESI+) exact mass
calculated for [C8H8(79Br)NO4Na]+ m/z 283.9529 [M+Na]+, found m/z 283.9528; FT-IR ??
̃
(neat, cm–1): 3005, 2951, 2900, 2809, 2774, 2686, 1728, 1618, 1475, 1464, 1448, 1432,
1391, 1317, 1266, 1246, 1212, 1099, 1070, 984, 959, 877, 860, 778, 766, 746, 692,
674. Methyl 5-bromo-4-methoxy-2-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1,2-
dihydropyridine-3-carboxylate (25a).[12] To an ice-cooled solution of methyl 5-bromo-
4-methoxy-2-oxo-1,2-dihydropyridine-3-carboxylate (2.989 g, 11.40 mmol, 1.0 equiv) in
dry DCM (24 mL), 2-(trimethylsilyl)ethoxymethyl chloride (2.4 mL, 14 mmol, 1.2 equiv)
and freshly distilled triethylamine (2.1 mL, 15 mmol, 1.3 equiv) were added. The reaction
mixture was stirred at 0 °C for ten minutes and then further stirred at room temperature
for four hours. The reaction mixture was diluted with DCM and washed with brine (200
mL). The aqueous phase was extracted with DCM (2 x 200 mL). The combined organic
phases were dried over sodium sulfate, filtered and dried in vacuo. The product mixture
was subjected to column chromatography (gradient: Et2O in pentane 25 % to 33 %).
Colorless waxy solid, yield: 40 % (O-SEM product: 33 %); m.p. = 60–62 °C; Rf: 0.39

(6.3 % MeOH in DCM); 1H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H), 5.26 (s, 2H), 3.99 (s,
13
3H), 3.92 (s, 3H), 3.62 (m, 2H,), 0.98–0.88 (m, 2H), 0.00 (s, 9H); C NMR (101 MHz,
CDCl3) δ 165.7, 161.5, 160.9, 137.1, 111.0, 95.9, 76.4, 67.8, 59.6, 53.2, 18.1, –1.3;
HRMS (ESI+) exact mass calculated for [C14H22BrNNaO5Si]+ m/z 414.0343
̃
[M(79Br)+Na]+, found m/z 414.0340, [M(79Br)+Na]+; FT-IR ?? (neat, cm–1): 2952, 2895,
1733, 1645, 1605, 1544, 1462, 1388, 1323, 1249, 1220, 1141, 1082, 992, 916, 835,
757, 689, 623. Methyl 4-methoxy-5-{4-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1-{[2-
(trimethylsilyl)ethoxy]methyl}-1,2-dihydropyridine-3-carboxylate (26).[12] A 25 mL
Schlenk tube was loaded with ethyl 5-bromo-4-methoxy-2-oxo-1-{[2-(trimethylsilyl)-
ethoxy]methyl}-1,2-dihydropyridine-3-carboxylate (1.764 g, 4.496 mmol, 1.0 equiv),
K2CO3 (1.865 g, 13.494 mmol, 3.0 equiv), [Pd(PPh3)4] (520 mg, 0.450 mmol, 0.1 equiv)
and {4-[(4-methoxybenzyl)oxy]phenyl}boronic acid (1.743 g, 6.754 mmol, 1.5 equiv). The
previously degassed solvent mixture (DME/water/DMF 9/1/0.5; 9 mL) was added. The
suspension was frozen in liquid nitrogen and was degassed in three freeze-pump-thaw-
cycles. The reaction was then stirred for 16 hours at 60 °C. The reaction mixture was
diluted with EtOAc (100 mL), washed with a saturated aqueous solution of ammonium
chloride (1 x 100 mL) and with brine (2 x 100 mL). The organic phase was dried over
sodium sulfate, filtered and the solvents were removed in vacuo. The crude product was
purified by column chromatography (25 % pentane in diethyl ether + 0.1 % Et3N).
1
Colorless solid, yield: 92 %; Rf = 0.20 (25 % pentane in diethyl ether + 0.1 % Et3N); H
NMR (500 MHz, CDCl3) δ 7.37 (m, 2H, 7.34 (s, 1H), 7.33 (m, 2H), 6.99 (m, 2H), 6.93 (m,
2H), 5.33 (s, 2H), 5.01 (s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.73 (s, 3H), 3.67 (m, 2H), 0.95
13
(m, 2H), 0.00 (s, 9H); C NMR (126 MHz, CDCl3) δ 166.6, 164.0, 161.2, 159.7, 158.7,
136.1, 130.3, 129.4, 128.8, 126.2, 116.7, 115.0, 114.2, 111.6, 76.3, 70.0, 67.6, 59.4,
55.4, 53.0, 18.2, –1.2; HRMS (ESI+) exact mass calculated for [C28H36NO7Si]+ m/z
̃
526.2256 [M+H]+, found m/z 526.2256, [M+H]+; FT-IR ?? (neat, cm–1): 2955, 1733, 1612,
1587, 1561, 1518, 1462, 1448, 1401, 1389, 1366, 1330, 1308, 1266, 1247, 1208, 1166,
1079, 1056, 1036, 1007, 96, 976, 958, 904, 856, 832, 815, 753, 692, 668, 637.
General Procedure for the Pyridone Methyl Ester Transesterification[28]
A two-necked flask equipped with an adapter piece featuring a Por2 frit was filled with
molecular sieves (4 Å), thoroughly flame-dried and flushed with argon. The flask was
charged with 4-methoxy-5-{4-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1-{[2-(trimethylsilyl)-

ethoxy]methyl}-1,2-dihydro pyridine-3-carboxylate (1.0 equiv) and LiBr (5–6 equiv).
Then, the flask was evacuated under high vacuum and flushed with argon. The solvent
mixture (25 % DCM in THF, 0.1 M with respect to the pyridone methyl ester derivative)
was added and the solution was stirred for 15 min. The alcohol (1.8–2.5 equiv) and DBU
(0.5 equiv) were added over a septum via a syringe and the reaction mixture was heated
to reflux under an atmosphere of argon for eight to nine hours. DCM and a saturated
aqueous solution of ammonium chloride were added to the reaction mixture. The phases
were separated and the aqueous phase was extracted with DCM (3 x 50 mL). The
combined organic phases were dried over sodium sulfate. After filtration of the drying
agent, the solvents were removed in vacuo. Preparative HPLC (method 1) was carried
out to obtain the corresponding pyridone ester derivative.
Pent-4-en-1-yl 4-hydroxy-5-{4-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1-{[2-(tri-
methyl silyl)ethoxy]methyl}-1,2-dihydropyridine-3-carboxylate (27). Colorless solid,
yield: 22%, m.p. = 83 °C; Rf = 0.52 (25 % pentane in Et2O); 1H NMR (500 MHz, CDCl3)
δ 14.12, 7.54 (s, 1H), 7.39 (m, 2H), 7.36 (m, 2H), 7.01 (m, 2H), 6.92 (m, 2H), 5.88 (ddt,
J = 17.0, 10.2, 6.6 Hz, 1H), 5.29 (s, 2H), 5.08 (dd, J = 17.0, 1.6 Hz, 1H), 5.02 (s, 2H),
5.00 (m, 1H), 4.38 (t, J = 6.7 Hz, 2H), 3.81 (s, 3H), 3.60 (m, 2H), 2.20 (m, 2H), 1.89 (m,
2H), 0.93 (m, 2H), 0.00 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 173.8, 173.0, 159.8,
159.7, 158.8, 140.1, 137.5, 130.4, 129.4, 128.9, 125.4, 115.5, 115.0, 114.2, 114.1, 98.6,
76.4, 70.0, 67.5, 65.6, 55.5, 30.1, 27.7, 18.2, –1.2; HRMS (ESI+) exact mass calculated
for [C31H40NO7Si]+ m/z 566.2569 [M+H]+, found m/z 566.2563, [M+H]+; FT-IR ??
̃ (neat,
cm–1): 3075, 2998, 2952, 2901, 2880, 1731, 1665, 1612, 1515, 1468, 1446, 1406, 1347,
1323, 1300, 1283, 1241, 1218, 1178, 1140, 1080, 1032, 1007, 974, 944, 920, 833, 823,
762,
744,
708,
693,
650,
641,
611.
Decyl
4-hydroxy-5-{4-[(4-
methyl}-1,2-
methoxybenzyl)oxy]phenyl}-2-oxo-1-{[2-(trimethylsilyl)ethoxy]
dihydropyridine-3-carboxylate (28). Colorless solid, yield: 28 %; Rf = 0.70 (4.8 %
MeOH in DCM); 1H NMR (500 MHz, CDCl3) δ 14.18 (s, 1H), 7.54 (s, 1H), 7.37 (m, 2H),
7.36 (dd, J = 2.2, 1.5 Hz, 2H), 7.08–6.96 (m, 2H), 6.91 (m, 2H), 5.34 (s, 2H), 5.02 (s,
2H), 4.40 (t, J = 7.1 Hz, 2H), 3.82 (s, 3H), 3.69–3.64 (m, 2H), 1.85 (m, 2H), 1.47–1.40
(m, 2H), 1.38–1.23 (m, 12H), 1.00–0.93 (m, 2H), 0.88 (t, J = 7.0 Hz, 3H), 0.01 (s, 9H);
13C NMR (126 MHz, CDCl3) δ 173.8, 173.0, 159.7, 159.6, 158.7, 140.0, 130.3, 129.3,
128.9 , 125.4, 114.9, 114.1, 114.0, 98.5, 76.3, 70.0, 67.4, 66.3, 55.4, 32.0, 29.7, 29.6,

29.4, 29.4, 28.7, 25.9, 22.8, 18.2, 14.2, –1.3; HRMS (ESI+) exact mass calculated for
[C36H52NO7Si+] m/z 638.3508 [M+H]+, found m/z 638.3507, [M+H]+.
General Procedure for N-SEM Deprotection of Pyridone Derivatives[12]
To a solution of the N-SEM protected pyridone ester in THF (dry, 0.1 M) was added a
solution of TBAF in THF (dry, 1 M, 2.2 equiv) via syringe. The reaction mixture was
heated to 65 °C for 48 hours. The reaction mixture was concentrated in vacuo, diluted
with DCM and poured on brine/DCM. The phases were separated and the aqueous
phase was extracted with DCM. The combined organic phases were dried over sodium
sulfate. After filtration of the drying agent, the solvent was evaporated in vacuo. The
crude product was suspended in cold MeOH (–20 °C). The solvent was removed via a
teflon cannula with a filter paper attached to it by teflon tape, and rinsed with cold MeOH
to obtain the pure compound.
Pent-4-en-1-yl 4-hydroxy-5-{4-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,2-dihydropy-
ridine-3-carboxylate. Colorless solid, yield: 81%; m.p. = 192 °C; Rf = 0.18 (25 %
1
pentane in Et2O); H NMR (400 MHz, CDCl3): δ 14.01 (s, 1H), 12.64 (s, 1H), 7.47 (s,
1H), 7.41–7.32 (m, 4H), 7.04 (m, 2H), 6.92 (m, 2H), 5.85 (ddt, J = 16.9, 10.2, 6.6 Hz,
1H), 5.16–4.96 (m, 4H), 4.43 (t, J = 6.9 Hz, 2H), 3.82 (s, 3H), 2.15 (m, 2H), 1.93 (m, 2H);
13C NMR (101 MHz, CDCl3): δ 174.3, 172.5, 162.8, 159.6, 158.7, 138.8, 137.5, 130.3,
13
129.4, 129.0, 125.3, 115.6, 115.0, 114.4, 114.2, 98.8, 65.6, 55.4, 30.1, 27.8; the C
signal of C12H2 was not observed; HRMS (ESI+) exact mass calculated for [C25H26NO6]+
̃
m/z 436.1755 [M+H]+, found m/z 436.1748 [M+H]+; FT-IR ?? (neat, cm–1): 2975, 2911,
2845, 2758, 1647, 1549, 1514, 1487, 1451, 1412, 1382, 1351, 1307, 1277, 1236, 1206,
1178, 1080, 1022, 971, 889, 826, 764, 729, 610. Decyl 4-hydroxy-5-{4-[(4-
methoxybenzyl)oxy]phenyl}-2-oxo-1,2-dihydropyridine-3-carboxylate.
Colorless
solid, yield: 53 %; Rf = 0.17 (4.8 % MeOH in DCM); 1H NMR (500 MHz, CDCl3) δ 14.07
(s, 1H), 12.21 (s, 1H), 7.45 (s, 1H), 7.41–7.37 (m, 4H), 7.35 (m, 2H), 5.02 (s, 2H), 4.41
(t, J = 7.1 Hz, 2H), 3.83 (s, 3H), 1.84 (m, 2H), 1.45 (m, 2H), 1.37–1.13 (m, 12H), 0.86 (t,
J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 174.3, 172.6, 162.6, 159.7, 158.8, 138.6,
130.4, 129.4, 129.0, 125.3, 115.0, 114.4, 114.2, 98.9, 70.0, 66.4, 55.5, 32.0, 29.7, 29.7,
29.4, 29.4, 28.7, 26.0, 22.8, 14.3; FT-IR ??̃
(neat, cm-1): 2921, 2834, 1725, 1680, 1634,
1552, 1515, 1466, 1441, 1410, 1346, 1278, 1243, 1178, 1078, 1037, 956, 873, 829,
742, 652, 612.

General Procedure for PMB Deprotection of Pyridone Ester Derivatives[12]
To an ice-cooled solution of PMB-protected pyridone ester derivative in DCM (0.2 M)
was added a solution of TFA in DCM (2.5 weight-%, 15 equiv). The reaction mixture was
stirred with cooling for 30 minutes and then allowed to warm to room temperature (45–
60 minutes). The reaction mixture was diluted with DCM and poured on brine. The
phases were separated and the aqueous phase was extracted with DCM. The organic
phases were dried over sodium sulfate. After filtration of the drying agent, the solvent
was removed in vacuo. The resulting colorless solid was purified via flash
chromatography (6.3 % MeOH in DCM).
Pent-4-en-1-yl 4-hydroxy-5-(4-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-carboxy-
late (9). Colorless solid, yield: 48 %; m.p. = 225–230 °C (decomposition); Rf = 0.15 (6.3 %
1
MeOH in DCM); H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 7.32–7.23 (m, 2H), 6.81 (m,
2H), 5.87 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 5.03 (m, 2H), 4.39 (t, J = 6.7 Hz, 2H), 2.25 (m,
2H), 1.89 (m, 2H); 13C NMR (101 MHz, CD3OD) δ 175.4, 173.5, 162.8, 158.3, 139.8, 138.9,
131.4, 125.3, 116.1, 115.7, 101.4, 66.3, 31.1, 28.8; HRMS (ESI+) exact mass calculated
for [C17H18NO5]+ m/z 316.1179 [M+H]+, found m/z 316.1176, [M+H]+; FT-IR ?? (neat, cm–1):
̃
3214, 2987–2757, 1650, 1540, 1513, 1484, 1443, 1418, 1343, 1303, 1276, 1249, 1202,
1175, 1110, 1081, 994, 950, 910, 870, 836, 724, 678. Decyl 4-hydroxy-5-(4-
hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-carboxylate (10). Colorless solid, yield:
62 %; m.p. = 190 °C; Rf = 0.10 (6.3 % MeOH in DCM); 1H NMR (500 MHz, CD3OD) δ 7.45
(s, 1H), 7.28 (m, 2H), 6.81 (m, 2H), 4.37 (t, J = 6.8 Hz, 2H), 1.79 (m, 2H), 1.55 (m, 2H),
13
1.37 (m, 12H), 0.90 (m, 3H); C NMR (101 MHz, CD3OD) δ 175.5, 173.5, 162.8, 158.3,
139.7, 131.4, 125.4, 116.1, 99.9, 66.9, 33.1, 30.7, 30.7, 30.5, 30.4, 29.6, 27.0, 23.7, 14.4;
HRMS (ESI+) exact mass calculated for [C22H30NO5]+ m/z 388.2118 [M+H]+, found m/z
̃
388.2119, [M+H]+; FT-IR ?? (neat, cm–1): 3271 , 2923, 2852, 2439 , 2287, 1640, 1543,
1513, 1470, 1423, 1343, 1310, 1244, 1192, 1106, 1076, 1002, 896, 837, 720, 653.
General Procedure for Regioselective Lithiation and Alkylation of 3,5-Dibromo-2,4-
dimethoxypyridine[9]
In a flame-dried two-necked flask, 3,5-dibromo-2,4-dimethoxypyridine (1.0 equiv) was
dissolved in dry THF (0.1 M with respect to the 3,5-dibromo-2,4-dimethoxypyridine). This
solution was then cooled down to –78 °C and a previously titrated s-BuLi solution (1.35

M in cyclohexane, 1.0–1.2 equiv) was added dropwise over 10–15 minutes. The mixture
was stirred for one hour at –78 °C. A solution of the aldehyde in dry THF (1.2 M, 1.0–2.5
equiv) was slowly added. The mixture was stirred for another hour at -78 °C. The
mixture was then allowed to warm to room temperature with continued stirring (14–16
hours). The mixture was quenched with water (1/3 of the volume of THF). A saturated
aqueous ammonium chloride solution (1/3 of the volume of THF) and ethyl acetate (two
times the volume of THF) were added. The phases were separated and the aqueous
phase was extracted with ethyl acetate. The combined organic phases were washed
with brine and dried over magnesium sulfate. After filtration of the drying agent, the
solvents were removed in vacuo. The crude product was purified by flash chromatogra-
phy (7 % ethyl acetate in pentane).
1-(5-Bromo-2,4-dimethoxypyridin-3-yl)octan-1-ol (31). Colorless liquid, yield: 69%; Rf
= 0.15 (7% ethyl acetate in pentane); 1H NMR (250 MHz, CDCl3) δ 8.12 (s, 1H), 4.93 (m,
1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.21 (d, J = 11.5 Hz, 1H), 1.94–1.81 (m, 1H), 1.69–1.63
13
(m, 1H), 1.26 (m, 10H), 0.86 (t, J = 6.7 Hz, 3H); C NMR (101 MHz, CDCl3) δ 162.2,
162.0, 147.8, 121.3, 108.1, 67.9, 61.8, 54.1, 37.3, 32.0, 29.5, 29.3, 26.3, 22.8, 14.2;
HRMS (ESI+) exact mass calculated for [C15H25O3NBr]+ m/z 346.10123 [M(79Br)+H]+,
̃
found m/z 346.1012 [M(79Br)+H]+; FT-IR ?? (neat, cm-1): 3568, 3446, 2925, 2855, 1729,
1564 1458, 1416, 1381, 1297, 1250, 1160, 1086, 1008, 914, 832, 794, 723, 663, 624. 1-
(5-Bromo-2,4-dimethoxypyridin-3-yl)dodecan-1-ol (32). Colorless oil, yield: 93 %; Rf
1
= 0.17 (7 % ethyl acetate in pentane); H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 4.95–
4.92 (m, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.20 (bs, 1H), 1.89 (m, 1H), 1.70 (m, 1H), 1.48
(m, 1H), 1.32–1.21 (m, 17H), 0.87 (t, J = 6.8 Hz, 3H); HRMS (ESI+) exact mass
calculated for [C19H33BrNO3]+ m/z 402.16383 [M(79Br)+H]+, found m/z 402.1639
[M(79Br)+H]+; FT-IR ?? (neat, cm–1): 3572, 3436, 2923, 2853, 1729, 1564 1459, 1416,
̃
1381, 1298, 1250, 1162, 1089, 1009, 914, 826, 794, 664, 630. 1-(5-Bromo-2,4-
dimethoxypyridin-3-yl)octadecan-1-ol (33). Colorless solid, yield: 77 %; m.p.= 52 °C;
1
Rf = 0.24 (7 % ethyl acetate in pentane); H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H),
4.995–4.89 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.19 (d, J = 11.2 Hz, 1H), 1.94–1.81 (m,
13
1H), 1.70–1.61 (m, 1H), 1.33–1.17 (m, 30H), 0.85 (t, J = 6.8 Hz, 3H); C NMR (101
MHz, CDCl3) δ 162.1, 162.0, 147.7, 121.3, 108.1, 67.8, 61.7, 54.1, 37.3, 32.0, 29.8,
29.8, 29.8, 29.7, 29.7, 29.5, 29.5, 26.3, 22.8, 14.2; HRMS (ESI+) exact mass calculated

for [C25H45BrNO3]+ m/z 486.25773 [M(79Br)+H]+, found m/z 486.2576 [M(79Br)+H]+; FT-IR
??
(neat, cm–1): 3440, 3297, 2919, 2851, 1565 1461, 1416, 1381, 1297, 1251, 1119,
̃
1089, 1012, 936, 910, 844, 794, 665, 628.
General Procedure for Suzuki Cross-Coupling Reactions[9]
A flame-dried Schlenk tube was charged with 5-bromo-2,4-dimethoxypyridine (1.0
equiv), 4-[(tert-butyldimethylsilyl)oxy]phenyl boronic acid (1.8–2.0 equiv) and [Pd(PPh3)4]
(8 mol%). The flask was evacuated and flushed with argon. A degassed mixture of
toluene/ethanol (20 % ethanol in toluene, 0.05 M with respect to the 5-bromo-2,4-
dimethoxypyridine) and a degassed aqueous solution of sodium carbonate (2 M, 70 Vol-
% of the organic solvent) were added. The reaction mixture was heated to reflux (85 °C)
for 14–20 hours. Ethyl acetate was added and the phases were separated. The organic
phase was washed with water and brine, and dried over anhydrous magnesium sulfate.
After filtration of the drying agent, the solvent was removed in vacuo. The crude product
was purified by column chromatography (7 % ethyl acetate in pentane).
1-{5-[4-((tert-Butyldimethylsilyl)oxy)phenyl]-2,4-dimethoxypyridin-3-yl}octan-1-ol
1
(34). Colorless liquid, yield: 94 %; Rf = 0.07 (7 % ethyl acetate in pentane); H NMR
(400 MHz, CDCl3) δ 7.96 (s, 1H), 7.38–7.29 (m, 2H), 6.94–6.86 (m, 2H), 5.03 (ddd, J =
11.6, 8.4, 5.8 Hz, 1H), 4.01 (s, 3H), 3.42 (d, J = 11.6 Hz, 1H), 3.39 (s, 3H), 1.97–1.84
(m, 1H), 1.78–1.68 (m, 1H), 1.59–1.46 (m, 1H), 1.35–1.24 (m, 8H), 1.00 (s, 9H), 0.88 (t,
J = 6.8 Hz, 3H), 0.23 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 163.2, 161.7, 155.4, 146.8,
130.0, 128.5, 124.9, 120.4, 118.6, 67.8, 60.8, 53.8, 37.8, 32.0, 29.6, 29.4, 26.4, 25.8,
22.8, 18.4, 14.3, -4.2; HRMS (ESI+) exact mass calculated for C27H44NO4Si m/z
474.30341 [M+H]+, found m/z 474.3040 [M+H]+; FT-IR ??
2929, 2857, 1589, 1513, 1467, 1413, 1377, 1255, 1171, 1136 1072, 1012, 911, 840,
806, 781, 657, 624. 1-{5-[4-((tert-Butyldimethylsilyl)oxy)phenyl]-2,4-
̃
(neat, cm–1): 3568, 3462,
dimethoxypyridin-3-yl}dodecan-1-ol (35). Colorless liquid, yield: 94 %; Rf = 0.09 (7 %
ethyl acetate in pentane); 1H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.38–7.30 (m, 2H),
6.96–6.84 (m, 2H), 5.03 (ddd, J = 11.6, 8.3, 5.8 Hz, 1H), 4.01 (s, 3H), 3.42 (d, J = 11.6
Hz, 1H), 3.39 (s, 3H), 1.96–1.85 (m, 1H), 1.77–1.66 (m, 1H), 1.53 (m, 1H), 1.38–1.20
(m, 17H), 1.00 (s, 9H), 0.87 (t, J = 6.7 Hz, 3H), 0.23 (s, 6H); 13C NMR (101 MHz, CDCl3)
δ 163.2, 161.7, 155.4, 146.8, 130.0, 128.5, 124.9, 120.4, 118.6, 67.8, 60.8, 53.8, 37.8,
32.1, 29.8, 29.8, 29.8 29.7, 29.5, 26.4, 25.8, 22.8, 18.4, 14.3, -4.2; HRMS (ESI+) exact

mass calculated for [C31H52NO4Si]+ m/z 530.36601 [M+H]+, found m/z 530.3666 [M+H]+;
FT-IR ??
(neat, cm-1): 3566, 2927, 2852, 1590, 1514, 1468, 1423, 1377, 1258, 1171,
1136 1073, 1013, 912, 842, 782, 735, 688, 632. 1-{5-[4-((tert-
Butyldimethylsilyl)oxy)phenyl]-2,4-dimethoxypyridin-3-yl}octadecan-1-ol
̃
(36).
1
Colorless solid, yield: 93 %, m.p. = 48 °C; Rf = 0.10 (7 % ethyl acetate in pentane); H
NMR (500 MHz, CDCl3) δ 7.96 (s, 1H), 7.38–7.29 (m, 2H), 6.94–6.85 (m, 2H), 5.03 (ddd,
J = 11.6, 8.3, 5.8 Hz, 1H), 4.01 (s, 3H), 3.41 (d, J = 11.6 Hz, 1H), 3.39 (s, 3H), 1.95–1.86
(m, 1H), 1.75 -1.68 (m, 1H), 1.56–1.47 (m, 1H), 1.35–1.23 (m, 29H), 1.00 (s, 9H), 0.88
13
(t, J = 6.9 Hz, 3H), 0.23 (s, 6H); C NMR (126 MHz, CDCl3) δ 163.2, 161.7, 155.4,
146.8, 130.0, 128.5, 124.9, 120.4, 118.6, 67.8, 60.8, 53.8, 37.9, 32.1, 29.9, 29.9, 29.8,
29.8, 29.8, 29.7, 29.5, 26.4, 25.8, 22.9, 18.4, 14.3, –4.2; HRMS (ESI+) exact mass
calculated for C37H64NO4Si m/z 614.45991 [M+H]+, found m/z 614.4609 [M+H]+; FT-IR ??
̃
(neat, cm–1): 3574, 2924, 2854, 1590, 1514, 1468, 1423, 1377, 1258, 1171, 1135 1073,
1014, 913, 842, 806 782, 719, 632.
General Procedure for Dess-Martin Oxidation[9]
The racemic mixture of secondary pyridine alcohol (1.0 equiv) was dissolved in dry DCM
(0.02 M) and Dess-Martin periodinane (1.5–1.8 equiv) was added portionwise. The
colorless suspension was stirred at room temperature until completion of the reaction
(1–2 hours). The reaction mixture was quenched with a saturated aqueous solution of
sodium hydrogen carbonate and ethyl acetate. The mixture was treated with solid
sodium thiosulfate (300 mg per 100 mg of Dess-Martin periodinane) and stirred until the
precipitate was dissolved. The phases were separated and the aqueous phase was
extracted with ethyl acetate. The combined organic phases were dried over magnesium
sulfate and the solvent was removed in vacuo. The product was purified by elution
through a short silica gel column (7 % ethyl acetate in petroleum ether).
1-{5-[4-((tert-Butyldimethylsilyl)oxy)phenyl]-2,4-dimethoxypyridin-3-yl}octan-1-
one. Colorless oil, yield: 92 %; Rf = 0.46 (7 % ethyl acetate in petroleum ether);1H NMR
(400 MHz, CDCl3) δ 8.06 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 3.96
(s, 3H), 3.44 (s, 3H), 2.81 (t, J = 7.4 Hz, 2H), 1.70 (p, J = 7.4 Hz, 2H), 1.39–1.24 (m,
8H), 1.00 (s, 9H), 0.92–0.86 (m, 3H), 0.23 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 203.9,
162.7, 160.6, 155.6, 148.7, 130.1, 127.8, 124.6, 120.4, 118.2, 61.4, 54.1, 44.9, 31.9,
29.3, 29.2, 25.8, 23.7, 22.8, 18.4, 14.2, –4.2; HRMS (ESI+) exact mass calculated for

[C27H42NO4Si]+ m/z 472.28776 [M+H]+, found m/z 472.2879 [M+H]+; FT-IR ?? (neat, cm–
̃
1): 2930, 2858, 1708, 1588, 1513, 1467, 1409, 1378, 1259, 1172, 1142, 1110 1085,
1009, 913, 841, 782, 668, 632. 1-{5-[4-((tert-Butyldimethylsilyl)oxy)phenyl]-2,4-
dimethoxypyridin-3-yl}dodecan-1-one. Colorless oil, yield: 82 % ; Rf = 0.48 (7 % ethyl
acetate in pentane); 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.37–7.29 (m, 2H), 6.94–
6.85 (m, 2H), 3.96 (s, 3H), 3.44 (s, 3H), 2.81 (t, J = 7.4 Hz, 2H), 1.75–1.64 (m, 2H), 1.28
(m, 16H), 1.04–0.94 (m, 9H), 0.87 (t, J = 6.8 Hz, 3H), 0.23 (s, 6H); 13C NMR (101 MHz,
CDCl3) δ 203.9, 162.7, 160.5, 155.6, 148.7, 130.1,127.8, 124.6, 120.4,118.2, 61.4, 54.0,
44.9, 32.1, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 25.8, 23.7, 22.8, 18.4, 14.3, –4.2; HRMS
(ESI+) exact mass calculated for [C31H50NO4Si]+ m/z 528.35036 [M+H]+, found m/z
528.3510 [M+H]+; FT-IR ?? (neat, cm–1): 2928, 2856, 1709, 1588, 1516, 1468, 1409,
̃
1378, 1317, 1260, 1172, 1090 1010, 913, 841, 782, 688, 632. 1-{5-[4-((tert-
Butyldimethylsilyl)oxy)phenyl]-2,4-dimethoxypyridin-3-yl}octadecan-1-one.
1
Colorless solid, yield: 94 %, m.p.= 42 °C; Rf = 0.51 (7 % ethyl acetate in pentane); H
NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.40–7.30 (m, 2H), 6.94–6.84 (m, 2H), 3.96 (s,
3H), 3.44 (s, 3H), 2.81 (t, J = 7.4 Hz, 2H), 1.70 (p, J = 7.4 Hz, 2H), 1.36–1.22 (m, 28H),
1.00 (s, 9H), 0.88 (t, J = 6.9 Hz, 3H), 0.23 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 203.9,
162.7, 160.5, 155.6, 148.7, 130.1, 127.8, 124.6, 120.4, 118.2, 61.4, 54.0, 44.9, 32.1,
29.9, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 25.8, 23.7, 22.8, 18.4, 14.3, –4.2; HRMS (ESI+)
exact mass calculated for [C37H62NO4Si]+ m/z 612.4443 [M+H]+, found m/z 612.4433
[M+H]+; FT-IR ?? (neat, cm–1): 2919, 2851, 2361, 1703, 1585, 1559, 1515, 1462, 1407,
̃
1364, 1328, 1258, 1173, 1135 1107, 1076, 1005, 914, 839, 806 780, 750, 719, 722,
704, 681, 654.
General Procedure for Pyridine Deprotection[9]
The protected pyridine (1.0 equiv) was dissolved in MeCN (dry, 0.01–0.02 M). Dry
aluminium chloride (25 equiv) and dry sodium iodide (20 equiv) were added. The mixture
was stirred at room temperature or 45 °C for one to two hours. The reaction mixture was
quenched with a few milliliters of hydrochloric acid (1 M, aq) and extracted four times
with ethyl acetate. The combined organic phases were washed with an aqueous solution
of sodium thiosulfate (5 weight-%), brine and dried over magnesium sulfate. The solvent
was removed in vacuo and the crude product was purified by preparative HPLC
(MeCN/water).

4-Hydroxy-5-(4-hydroxyphenyl)-3-octanoylpyridin-2(1H)-one (11). Colorless solid,
1
yield: 68 %, m.p. = 154 °C; Rf = 0.29 (6.3 % methanol in DCM); H NMR (400 MHz,
DMSO-d6) δ 11.67 (d, J = 6.5 Hz, 1H), 9.48 (s, 1H), 7.56 (d, J = 6.5 Hz, 1H), 7.30–7.19
(m, 2H), 6.82–6.71 (m, 2H), 3.12 (t, J = 7.4 Hz, 2H), 1.58 (p, J = 7.0 Hz, 2H), 1.37–1.19
(m, 8H), 0.90–0.81 (m, 3H); 13C NMR (101 MHz, DMSO-d6) δ 208.1, 175.3, 161.4,
156.7, 140.7, 130.1, 123.3, 115.0, 112.2, 106.0, 41.9, 31.2, 28.7, 28.6, 23.9, 22.1, 13.9;
HRMS (ESI+) exact mass calculated for [C19H24NO4]+ m/z 330.1700 [M+H]+, found m/z
330.1695 [M+H]+; FT-IR ?? (neat, cm-1): 3295, 2925, 2854, 1659, 1607, 1542, 1512,
̃
1453, 1427, 1379, 1231, 1175, 1109, 988, 901, 833, 705, 635. 3-Dodecanoyl-4-
hydroxy-5-(4-hydroxyphenyl)pyridin-2(1H)-one (12). Colorless solid, yield: 78 %,
1
m.p. = 162 °C; Rf = 0.30 (6.3 % methanol in DCM); H NMR (400 MHz, DMSO-d6) δ
11.67 (d, J = 5.7 Hz, 1H), 9.48 (s, 1H), 7.56 (d, J = 5.7 Hz, 1H), 7.32–7.15 (m, 2H),
6.87–6.68 (m, 2H), 3.12 (t, J = 7.4 Hz, 2H), 1.58 (p, J = 7.0 Hz, 2H), 1.33–1.23 (m, 16H),
0.97–0.78 (m, 3H); 13C NMR (101 MHz, DMSO-d6) δ 208.1, 175.3, 161.4, 156.7, 140.7,
130.1, 123.3, 115.0, 112.2 , 106.0, 41.9, 31.3, 29.0, 29.0, 28.9, 28.8, 28.7, 23.8, 22.1,
14.0; HRMS (ESI+) exact mass calculated for [C23H32NO4]+ m/z 386.2326 [M+H]+, found
m/z 386.2330 [M+H]+; FT-IR ?? (neat, cm–1): 3540, 2923, 2851, 2339, 2210, 1652, 1605,
̃
1537, 1512, 1457, 1420, 1381, 1317, 1251, 1209, 1173, 1106, 1043, 980, 896, 832,
769, 703, 650. 4-Hydroxy-5-(4-hydroxyphenyl)-3-stearoylpyridin-2(1H)-one (13).
1
Colorless solid, yield: 78 %, m.p.= 154 °C ; Rf = 0.32 (6.3 % methanol in DCM); H
NMR (400 MHz, DMSO-d6) δ 11.67 (d, J = 6.5 Hz, 1H), 9.47 (s, 1H), 7.56 (d, J = 6.5 Hz,
1H), 7.33–7.14 (m, 2H), 6.84–6.72 (m, 2H), 3.12 (t, J = 7.4 Hz, 2H), 1.58 (p, J = 7.0 Hz,
13
2H), 1.23 (m, 28H), 0.87–0.80 (t, J = 6.5 Hz, 3H); C NMR (101 MHz, DMSO-d6) δ
208.1, 175.3, 161.4, 156.7, 140.7, 130.1, 123.3, 115.0, 112.2, 106.0, 41.9, 31.3, 29.0,
28.9, 28.8, 28.7, 23.8, 22.1, 13.9; HRMS (ESI+) exact mass calculated for [C29H44NO4]+
̃
m/z 470.3265 [M+H]+, found m/z 470.3267 [M+H]+; FT-IR ?? (neat, cm–1): 3534, 2919,
2850, 2224, 1654, 1608, 1512, 1460, 1420, 1235, 1174, 1106, 988, 895, 832, 722, 638.
5.2 Biological Testing
AEA, N-(2-hydroxyethyl-1,1,2,2-d4)-(5Z,8Z,11Z,14Z)-eicosatetraenamide, 2-AG, 2-
[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)
cyclohexyl]-5-(2-methyloctan-2-yl)phenol
(CP55,940, 14), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide

(UCM707, 15),[18] 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-
carboxylate (JZL184, 16),[19] N-methyl-N-{[3-(4-pyridinyl)phenyl]methyl}-carbamic acid
4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester (WWL70, 17),[20] (S)-1-[(2S,3S)-3-hexyl-4-
oxooxetan-2-yl]tridecan-2-yl formyl-L-leucinate (tetrahydrolipstatin, THL, 18),[20]
(5Z,8Z,11Z,14Z)-eicosatetraenoic acid, [3-(aminocarbonyl)(1,1-biphenyl)-3-yl]-cyclo-
hexylcarbamate (URB597, 19),[21] 5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phe-
nyl]-thiophene (DuP697, 20),[22] (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-
octadecenamide (OMDM-2, 21),[23] (11R)-2-methyl-11-[(morpholin-4-yl)methyl]-3-
(naphthalene-1-carbonyl)-9-oxa-1-azatricyclo[6.3.1.04,12]dodeca-2,4(12),5,7-tetraene
(WIN55,212-2)[24] and methylarachidonylfluorophosphonate (MAFP)[39] were purchased
from Cayman Chemicals Europe.[40] [Ethanolamine-1-3H]-AEA (60 Ci/mmol) and [1,2,3-
3H]2-AG (20–40 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc..
5.2.1 Cell Culture
Human monocytic leukemia U937 cells were purchased from American Type Culture
Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with
10% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, 100
g/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen).[41] Cells were grown in a
humidified incubator at 37 °C and in humidified atmosphere with 5% CO2.
5.2.2 CB1 and CB2 binding
CB1 and CB2 receptor affinities were determined using [3H]CP55,940 (0.5 nM) as
displacing ligand and membrane preparations of stably transfected hCB1 and hCB2
CHO-K1 cells as previously described.[42] (R)-WIN55,212-2 was used as positive control
(10 µM)[24] to determine total specific binding. Compounds were screened in triplicates
(10 µM). Ki values were calculated using the Cheng-Prusoff equation (CP55,940 hCB1
Kd=0.5 nM, hCB2 Kd=0.59 nM).
5.2.3 FAAH, MAGL and ABHD activity assays
FAAH and MAGL activity assays were performed using U937 cell homogenate and pig
brain homogenate, respectively, as previously described.[36] ABHDs assay was
performed using BV-2 cell homogenate as previously described.[43] Briefly, FAAH activity
was assessed using 100 µg of U937 cell homogenate diluted in 200 µL of Tris–HCl 10
mM, EDTA 1 mM, pH 8 containing 0.1% fatty acid-free BSA. Compounds were added (2