Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family

Article abstract of DOI:10.1021/acs.jmedchem.0c00522

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the α-ketoamide series using activity-based protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family members. LEI-301 reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, LEI-301 may help to dissect the physiological role of the PLAATs.

Full text of DOI:10.1021/acs.jmedchem.0c00522

Subscriber access provided by CARLETON UNIVERSITY
Article
Structure-activity relationship studies of #-ketoamides as inhibitors
of the phospholipase A and acyltransferase (PLAAT) enzyme family
Juan Zhou, Elliot D. Mock, Karol Al Ayed, Xinyu Di, Vasudev Kantae, Lindsey Burggraaff, Anna
Stevens, Andrea Martella, Florian Mohr, Ming Jiang, Tom van der Wel, Tiemen Wendel, Tim Ofman,
Yvonne Tran, Nicky de Koster, Gerard J.P. van Westen, Thomas Hankemeier, and Mario van der Stelt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00522 • Publication Date (Web): 02 Aug 2020
Downloaded from pubs.acs.org on August 2, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
Structure-activity relationship studies of -ketoamides as inhibitors
6
7
8
of the phospholipase A and acyltransferase (PLAAT) enzyme family
9
Juan Zhou1,‡, Elliot D. Mock1,‡, Karol Al Ayed1, Xinyu Di2, Vasudev Kantae2, Lindsey Burggraaff3, Anna F. Stevens1,
Andrea Martella1, Florian Mohr1, Ming Jiang1, Tom van der Wel1, Tiemen J. Wendel1, Tim P. Ofman1, Yvonne
Tran1, Nicky de Koster1, Gerard J.P. van Westen3, Thomas Hankemeier2 & Mario van der Stelt1,*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University & Oncode Institute, 2300 RA Leiden,
The Netherlands.
2
Department of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University,
2300 RA Leiden, The Netherlands.
3 Department of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, 2300 RA Leiden,
The Netherlands.
E-mail: m.van.der.stelt@chem.leidenuniv.nl
‡
These authors contributed equally.
Abstract
The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which
are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid
precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such
as food intake, pain, inflammation, stress and anxiety. Recently, we identified -ketoamides as the first pan-
active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in
HepG2 cells. Here, we report the structure-activity relationships of the -ketoamide series using activity-based
protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family
members. LEI-301 reduced NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5.
Collectively, LEI-301 may help to dissect the physiological role of the PLAATs.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 42
1
2
3
4
Introduction
5
6
7
8
9
The subfamily of phospholipase A and acyltransferases (PLAATs) consists of five members with reported roles
in tumor suppression and phospholipid metabolism.1,2 They belong to the lecithin retinol acyltransferase (LRAT)
protein family that is part of the NlpC/P60 superfamily of thiol hydrolases. The LRAT family has a conserved
catalytic motif of six amino acids (NCEHFV) containing a cysteine residue that acts as the active site
nucleophile.3 A C-terminal hydrophobic tail is shared by PLAAT1-4, serving as a single-pass transmembrane
anchoring domain.4 The physiological functions of the PLAAT family members are only partly understood.5
PLAATs are multifunctional enzymes that display varying degrees of N- and O-acyltransferase or phospholipase
A1/2 activity in vitro.5-11 In addition, PLAAT4 (also known as RARRES3, HRASLS4, TIG3 or RIG1) is involved in
protein deacylation of WNT and H-RAS proteins in breast cancer cells, thereby controlling cell growth.12
Biological functions have been described for PLAAT3 (also known as PLA2G16, HRASLS3, AdPLA or HREV107),
which primarily acts as a phospholipase A1/2 and regulates lipolysis in adipose tissue.4,10,13 Notably, Plaat3
knockout mice were protected against diet-induced obesity.12 Recently, PLAAT3 was reported to be a host
factor for picornaviridae by facilitating the entry of viral RNA into the cytosol from virus-containing endosomes.
14,15 As such, inhibitors of PLAAT3 hold promise as anti-obesity or anti-viral agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Less is known about the other PLAAT enzymes. Ueda and co-workers reported that in vitro, PLAAT2 (also
known as HRASLS2) displays the highest N-acyltransferase activity of all PLAATs, followed at some distance by
PLAAT5 (also known as iNAT or HRASLS5).16 This reaction involves the transfer of an acyl group from
phosphatidylcholine (PC) to phosphatidylethanolamine (PE), generating N-acylphosphatidylethanolamine
(NAPE) and lyso-phosphatidylcholine (LPC) (Scheme 1). Firstly, the PLAAT enzyme forms an acyl thioester
intermediate using its Cys-His-His catalytic triad, thereby expelling LPC. This is followed by nucleophilic attack
of the PE-amino group, producing the NAPE product and liberating the catalytic cysteine. NAPEs are an
underexplored class of triacylated phospholipids that serve as precursors for N-acylethanolamines (NAEs), an
important family of signaling molecules that includes the endocannabinoid anandamide (N-
arachidonoylethanolamine, AEA).17 Through activation of the cannabinoid CB1 receptor, anandamide is
involved in physiological processes such as appetite, pain sensation, memory formation, stress and anxiety.18-20
The canonical enzyme responsible for NAPE biosynthesis in the brain is a Ca2+-dependent N-acyltransferase (Ca-
NAT), recently identified as PLA2G4E.21 NAPEs are in turn converted to NAEs in one step by NAPE-
2
ACS Paragon Plus Environment

Page 3 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
phospholipase D (NAPE-PLD) as well as other multistep pathways.5 In contrast, the PLAAT family members
operate via a calcium-independent mechanism, providing an alternative pathway through which NAPEs and
NAEs are biosynthesized.16 PLAAT2 was reported to preferably transfer the sn-1 over the sn-2 acyl group of PC,
which suggests that it mostly generates saturated or mono-unsaturated NAEs.16 Furthermore, HEK293 cells
stably overexpressing PLAAT2 exhibited highly increased NAPE and NAE levels. Gene expression of PLAAT2 was
found in the lung, liver, kidney, small intestine, colon, testis and trachea.9,22 NAEs have well-established
signaling roles in the gastrointestinal system.23 For instance, N-oleoylethanolamide (OEA) was found to act as a
satiety factor via activation of peroxisome proliferator-activated receptor (PPAR)-.24 This raises the possibility
that PLAAT2 is involved in NAE biosynthesis in the gut. Notably, rodents lack the gene that encodes PLAAT2,
thereby hindering the development of genetic models.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As of yet, no physiological functions are known for PLAAT5. Of all PLAAT family members, PLAAT5 does not
have a reported tumor suppressing role.5 High gene expression levels were found in testis of mice, rats and
humans as well as in human pancreas.8,25 PLAAT5 activity was mainly localized to the cytosol fraction, while an
inactive form of the enzyme was found to be membrane associated.8 In vitro, PLAAT5 displayed higher
N-acyltransferase than phospholipase A1/2 activity.16 Importantly, compared to PLAAT2, PLAAT5 showed no
preference for the sn-1 or sn-2 acyl group of PC, suggesting that it could be involved in N-arachidonoyl-PE and
thus anandamide biosynthesis.6,8 PLAAT inhibitors would be valuable pharmacological tools to study the
biological role of PLAAT2 and PLAAT5.
Recently, we described the discovery of -ketoamide LEI-110 as the first pan-active PLAAT inhibitor using a
gel-based competitive activity-based protein profiling (ABPP) assay.26 LEI-110 was able to reduce arachidonic
acid levels in PLAAT3-overexpressing cells and in HepG2 cells. Here, we report the structure-activity
relationship of a library of -ketoamides on the PLAAT family members. Next to LEI-110, we also identified LEI-
301 as a nanomolar potent PLAAT2 inhibitor with similar potency for other members of the PLAAT family,
which was selective over the proteins of the endocannabinoid system (ECS). LEI-301 reduced NAE levels in
PLAAT2- and PLAAT5-overexpressing cells, but not in control cells. These findings show that LEI-301 is a new
pharmacological tool to study the biological role of PLAATs in cellular systems.
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 42
1
2
3
O
O
4
5
R2
O
O
R4
O
O
O-
P
O-
P
R1
O
O
R3
O
O
+
NH2
N+
6
7
O
O
O
O
PE
PC
8
PLA2G4E
9
or
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PLAAT1-5
O
O
R2
O
O
O
R4
O
O-
P
O-
P
R1
O
O
O
O
+
N
H
R3
N+
O
O
OH
O
NAPE
LPC
H2O
Alternative
pathways*
NAPE-PLD
O
R2
O
O
O
O-
R1
O
OH
+
HO
P
N
H
R3
O
O
PA
NAE
Scheme 1. Biosynthesis of N-acylphosphatidylethanolamines (NAPEs) and N-acylethanolamines (NAEs). The sn-1 acyl
group of phosphatidylcholine (PC) is transferred to the amine of phosphatidylethanolamine (PE) by the acyltransferase
activity of PLA2G4E or PLAAT1-5 forming N-acyl-PE (NAPE) and lyso-PC (LPC). NAPE-PLD hydrolyzes the phosphodiester
bond of NAPE to form NAE and phosphatidic acid (PA). R1, R2 and R3 denote saturated, mono- or polyunsaturated fatty
acids. * For the alternative pathways see ref. 5.
Results & Discussion
Screening for PLAAT inhibitors using competitive ABPP.
A focused in-house library of lipase inhibitors was screened for PLAAT inhibition using gel-based competitive
ABPP.26 This method uses an activity-based probe (ABP) containing an electrophilic group that forms a covalent
bond with the catalytic nucleophile of an enzyme.27 ABPs are also equipped with a reporter group such as a
fluorophore or biotin, which allows visualization of enzymatic activity in a native biological setting. In a
competitive ABPP experiment, potential inhibitors are pre-treated with a cell lysate that contains the protein of
interest followed by incubation with an ABP. After resolving the proteins by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluorescence scanning, the residual enzymatic activity
can be determined by measuring the probe labeling intensity. Previously, MB06428, which incorporates a -
4
ACS Paragon Plus Environment

Page 5 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
lactone electrophilic group, was validated as an effective ABP for the PLAAT enzyme family.26 Here, cytosol
fractions of human embryonic kidney HEK293T cells that overexpressed PLAAT2-5 were treated with potential
inhibitors (10 M, 30 min), followed by incubation with MB064 (250 nM, 20 min) (Figure 1A). The proteins
were then resolved by SDS-PAGE and PLAAT activity was quantified by in-gel fluorescence scanning. A
compound that showed residual protein labeling of ≤ 50% was considered to be active. For these active
compounds an IC50 curve was generated using a dose-response ABPP experiment (Figure 1B). Data for PLAAT2,
PLAAT3, PLAAT4 and PLAAT5 is reported in Table 1 as pIC50 ± SEM (n = 3). Unfortunately, PLAAT1 could not be
tested due to a lack of protein expression in HEK293T cells. -Ketoamides 1 and 2 were identified as
submicromolar hits, showing similar potency for all tested PLAAT members. An early structure-activity
relationship emerged from the structurally similar keto- and hydroxyamides (3-22) present in this focused
screening library. The -position of the ketone next to the amide was essential for binding (compare -
ketoamides 1 and 2 with -ketoamides 5-8). -Hydroxyamides (3 and 4) were inactive. Removing the alkyl
spacer (11) was also detrimental for activity. Furthermore, the phenethylamine of 1 was preferred over
benzylamine (9) and ethylamine (10). N-methylation resulted in complete loss of activity (12), which suggested
that the N-H is potentially involved in hydrogen bond formation or that the methyl group has a steric clash with
the protein. Similarly, secondary amides incorporating either (hetero)cyclic (13, 15, 16, 18-21) or acyclic (14, 17,
22) motifs, did not show any activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 42
1
2
3
4
A
B
PLAAT2 activity
5
6
7
8
100
50
0
O
OH
H
N
O
9
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
F
N+
O
F
B-
N
O
-10
-9
-8
-7
-6
-5
-4
-3
N
N
log[compound 1] (M)
N
[1] (µM)
Cy3
0
0.001 0.01 0.1
1
10
100
MB064
Coomassie
Figure 1. Evaluating PLAAT activity using competitive activity-based protein profiling (ABPP). A) Structure of broad-
spectrum lipase probe MB064. B) Representative gel and apparent IC50 curve of a competitive ABPP experiment for PLAAT2.
Labeling by MB064 and dose-dependent inhibition by 1 (apparent pIC50 = 6.2 ± 0.1, dotted lines show 95% confidence
interval). Data represent mean values ± SEM (n = 3). Coomassie staining was used as a protein loading control.
Table 1. Structure-activity relationship (SAR)-analysis of keto- and hydroxyamides 1-22.
apparent pIC50 ± SEM
ID
1
Structure
PLAAT2
PLAAT3
PLAAT4
PLAAT5
O
H
N
6.2 ± 0.1
6.0 ± 0.1
5.9 ± 0.1
6.4 ± 0.1
O
Cl
Cl
O
H
N
2
6.2 ± 0.1
< 5
6.0 ± 0.1
< 5
5.8 ± 0.1
< 5
6.7 ± 0.1
< 5
O
OH
O
N
H
3
4
5
F
F
OH
O
O
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
F
F
O
O
N
H
F
F
O
O
6
< 5
< 5
< 5
< 5
N
H
F
F
O
O
N
H
7
8
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
F
O
O
N
H
O
H
N
9
5.6 ± 0.1
5.8 ± 0.1
5.6 ± 0.1
6.6 ± 0.1
O
Cl
6
ACS Paragon Plus Environment

Page 7 of 42
Journal of Medicinal Chemistry
1
2
3
O
H
N
10
11
12
13
14
15
5.6 ± 0.1
< 5
5.1 ± 0.1
5.4 ± 0.1
< 5
6.0 ± 0.1
< 5
4
5
6
7
8
9
O
Cl
Cl
Cl
O
H
N
< 5
< 5
< 5
< 5
< 5
O
O
N
< 5
< 5
< 5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
N
N
< 5
< 5
< 5
O
O
Cl
Cl
< 5
< 5
< 5
O
O
O
N
< 5
< 5
< 5
O
Cl
Cl
N
N
16
17
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
O
O
O
O
O
N
N
O
O
O
Cl
18
19
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
Cl
Cl
N
O
O
N
20
< 5
< 5
< 5
< 5
O
Cl
O
O
N
N
21
22
< 5
< 5
< 5
< 5
< 5
< 5
< 5
< 5
O
O
Cl
Cl
Evaluation of an -ketoamide inhibitor library delivers nanomolar hit LEI-301.
-Ketoamide 1 exhibited the smallest molecular weight (MW = 316) and highest overall potency for the
PLAAT enzymes, therefore this compound was resynthesized and its activity was confirmed on all PLAAT
members with a pIC50 ranging from 6.0-6.4 (Table 2). It was envisioned that the electrophilic ketone of 1 could
bind with the PLAAT active site cysteine through a reversible covalent mechanism forming a hemithioketal
adduct, similar to other reported -ketoamide inhibitors.26,29 To test this hypothesis, compound 23 was
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 42
1
2
3
4
prepared in which the ketone was replaced by an alcohol. This compound showed no activity at 10 M
5
6
(Table 2), which is in line with the hypothesis.
7
8
9
To systematically investigate the SAR and improve the potency of 1, R1-ketone and R2-phenethyl analogues
were synthesized (compounds 24-56) (Table 2 and 3). First, the effect of various substitutions on the R1-group
of 1 was evaluated with derivatives 24-36 (Table 2). Removal of the chlorine (24) was detrimental for the
activity for PLAAT2-4, but not PLAAT5. The length of the alkyl chain was studied in analogues 24-27, showing
that propylene derivative 25 was optimal, which had increased potency for PLAAT2, PLAAT3 and PLAAT5. The
4-chloro substituent on the phenyl ring seemed to be optimal based on the inhibitory activity of compounds
29-33. Electron-donating groups such as 4-methyl (29) and 4-methoxy (32) substituents decreased potency, but
a lipophilic electron withdrawing group (e.g. 4-trifluoromethyl, 30) was tolerated for PLAAT5. A small (4-fluoro,
31) substituent lowered the activity, while a large group (4-phenoxy, 33) provided a selectivity window for
PLAAT5 over the other PLAATs of 10- to 30-fold. Furthermore, substitution of the 4-chloro to the ortho or meta
position, did not result in improved potency (compounds 34 and 35). The 3,4-dichloro derivative 36, however,
presented increased activity for PLAAT3 and PLAAT5. Taken together, these compound series point towards the
presence of a small lipophilic pocket, restricted in size, which is occupied by the alkylphenyl group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, ,-unsaturated -ketoamides 37-42 were evaluated to test whether conformational restriction of
the alkyl linker would lead to a gain in activity (Table 2). Although unsaturation was tolerated in the alkyl chain,
no or little improvement in potency was observed for these derivatives (compare compounds 1, 24, 32 and 37-
39). Also 4-bromo, 3-bromo or 3-phenyl substitutions (40-42), did not provide the desired inhibitory activity
increase. Overall, this suggests that the R1 group is positioned towards a shallow pocket.
Table 2. Structure-activity relationship (SAR)-analysis of α-ketoamide analogues 1, 23-42.
O
H
N
R1
O
apparent pIC50 ± SEM
ID
1
R1:
cLogPa
PLAAT2
PLAAT3
PLAAT4
PLAAT5
4.37
6.2 ± 0.1
6.0 ± 0.1
6.2 ± 0.1
6.4 ± 0.1
Cl
8
ACS Paragon Plus Environment

Page 9 of 42
Journal of Medicinal Chemistry
1
2
3
OH
H
N
23
3.83
< 5
< 5
< 5
< 5
4
5
O
Cl
6
7
8
9
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
3.66
4.04
4.57
3.04
1.56
4.16
4.54
3.80
3.58
5.76
4.37
4.37
4.97
4.37
3.66
3.58
4.52
4.52
< 5
< 5
5.5 ± 0.1
6.2 ± 0.1
5.8 ± 0.1
< 5
6.0 ± 0.1
7.0 ± 0.1
6.8 ± 0.1
< 5
6.3 ± 0.1
5.5 ± 0.1
< 5
6.4 ± 0.1
6.0 ± 0.1
< 5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
< 5
< 5
< 5
< 5
5.3 ± 0.1
5.7 ± 0.1
5.1 ± 0.1
5.5 ± 0.1
5.4 ± 0.1
5.9 ± 0.1
5.6 ± 0.1
5.7 ± 0.2
5.8 ± 0.1
5.1 ± 0.1
5.8 ± 0.1
5.3 ± 0.1
< 5
5.7 ± 0.1
5.1 ± 0.1
5.3 ± 0.1
5.5 ± 0.1
5.4 ± 0.1
5.7 ± 0.1
5.8 ± 0.1
7.0 ± 0.2
5.6 ± 0.1
5.8 ± 0.1
5.3 ± 0.1
5.6 ± 0.1
< 5
5.8 ± 0.1
5.6 ± 0.1
5.0 ± 0.1
5.9 ± 0.1
5.9 ± 0.1
5.6 ± 0.1
5.9 ± 0.1
6.0 ± 0.1
5.6 ± 0.1
5.0 ± 0.1
5.6 ± 0.1
5.2 ± 0.1
5.1 ± 0.1
5.6 ± 0.1
6.5 ± 0.1
5.8 ± 0.1
5.9 ± 0.1
6.9 ± 0.1
5.8 ± 0.1
5.9 ± 0.1
7.0 ± 0.1
6.2 ± 0.1
5.2 ± 0.2
6.0 ± 0.1
6.2 ± 0.1
5.5 ± 0.1
F3C
F
O
O
Cl
Cl
Cl
Cl
Cl
O
Br
Br
42
5.55
5.6 ± 0.1
5.5 ± 0.1
5.6 ± 0.1
6.0 ± 0.1
a cLogP was calculated using Chemdraw 15.
Next, the focus was shifted towards the optimization of the R2-phenethyl moiety. Analogues 43-56
incorporating substituted phenethylamines were prepared in combination with the 2-oxo-5-phenylpentanoyl
motif of compound 25, which demonstrated the highest PLAAT2 activity (Table 3). The compounds showed a
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 42
1
2
3
4
5
6
7
8
9
comparable SAR on PLAAT2 and PLAAT4, as substitutions on the para position were unfavorable for methyl
(43), methoxy (44) and hydroxyl (46). Substitutions on the meta and ortho position (48, 49) or 2,4-disubstituted
methoxy (45) or chloro (50) also did not afford an improvement in potency. Increasing the lipophilicity gave a 2-
to 3-fold increase in activity for 4-bromo analog 47. Further expansion with a 4-phenoxy moiety (51) improved
the potency both for PLAAT2 and PLAAT4 with 10-fold compared to 25. Introduction of a 4-methyl group in
compound 43 enhanced the inhibitory activity for PLAAT3 and PLAAT5. Other lipophilic groups (4-bromo (47)
and 4-phenoxy (51)) gave even higher activities for PLAAT5 especially.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Addition of the phenoxy group raised the cLogP of 51 with two log units to 6.14 (calculated with Chemdraw
15), therefore more polar heteroaryl rings were investigated to lower the lipophilicity (52-56) (Table 3).
Compound 55 (LEI-110) was identified as the most potent inhibitor of PLAAT3 and its biological characterization
has previously been described in detail.26 With regard to PLAAT2, a decrease in activity was observed for
compounds 52-56 compared to 51. Therefore, being the most potent inhibitor of PLAAT2, 51 (termed LEI-301)
was selected for further characterization.
10
ACS Paragon Plus Environment

Page 11 of 42
Journal of Medicinal Chemistry
1
2
3
Table 3. SAR-analysis of phenethyl analogues 25, 43-56.
4
5
O
H
N
R2
6
O
7
8
9
apparent pIC50 ± SEM
ID
R2:
cLogPa
PLAAT2
PLAAT3
PLAAT4
PLAAT5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
43
44
45
46
47
48
49
4.04
4.54
3.96
3.70
3.37
4.90
4.75
4.75
6.3 ± 0.1
6.4 ± 0.1
6.2 ± 0.1
7.0 ± 0.1
5.7 ± 0.1
5.8 ± 0.1
5.3 ± 0.1
5.9 ± 0.1
6.6 ± 0.1
6.1 ± 0.1
5.2 ± 0.1
6.9 ± 0.1
6.1 ± 0.1
6.0 ± 0.2
5.8 ± 0.1
6.8 ± 0.1
6.7 ± 0.1
5.9 ± 0.1
5.9 ± 0.1
5.8 ± 0.1
5.2 ± 0.1
5.6 ± 0.1
6.7 ± 0.1
6.0 ± 0.1
5.6 ± 0.1
7.4 ± 0.1
7.1 ± 0.1
6.5 ± 0.1
6.8 ± 0.1
8.0 ± 0.1
7.3 ± 0.1
6.1 ± 0.2
O
O
O
OH
Br
Cl
Cl
Cl
50
5.47
5.9 ± 0.1
6.6 ± 0.1
6.1 ± 0.1
7.2 ± 0.1
Cl
O
51
(LEI-301)
6.14
3.68
3.68
5.67
5.67
4.44
7.3 ± 0.1
6.3 ± 0.1
5.7 ± 0.1
6.7 ± 0.1
6.8 ± 0.1
6.2 ± 0.1
6.6 ± 0.1
6.6 ± 0.1
5.7 ± 0.1
6.8 ± 0.1
7.0 ± 0.1
6.6 ± 0.1
7.3 ± 0.2
6.2 ± 0.1
5.9 ± 0.1
7.1 ± 0.1
6.2 ± 0.1
6.7 ± 0.1
7.4 ± 0.1
7.2 ± 0.1
6.6 ± 0.1
7.1 ± 0.1
7.5 ± 0.1
7.1 ± 0.1
N
N
52
53
54
O
O
O
O
O
N
N
N
CF3
CF3
55
(LEI-110)
N
N
N
56
Cl
a cLogP was calculated using Chemdraw 15.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 42
1
2
3
4
In silico modeling of -ketoamides inhibitors.
5
6
7
8
To explain the binding mode of the -ketoamides inhibitors in PLAAT2, LEI-301 and 1 were docked in a PLAAT2
crystal structure (PDB: 4DPZ).4 Residues 39-52 and 105-111 were absent from this structure, therefore a
homology model was prepared using the closely related PLAAT3 crystal structure (PDB: 4DOT)4 from which the
shape of the loop for residues 105-111 could be adopted. A second loop comprising residues 39-52 was
modeled based on sequence, as it is not present in both crystal structures. Since our data suggested that the
electrophilic ketone of -ketoamides could engage with the active site cysteine through a reversible covalent
mechanism29, LEI-301 and 1 were covalently docked to Cys113 in the enzyme (Figure 2). Both compounds
revealed a hydrogen bonding network between the oxy-anion and amide carbonyl with His23 and the Trp24
backbone amide N-H, while the backbone carbonyl of Leu108 formed a H-bond with the amide of the
inhibitors. Introduction of the 4-phenoxy group in LEI-301 suggested that an additional - stacking interaction
with Tyr21 would be possible. This offered a potential reason for the observed activity increase of LEI-301.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Docking pose of LEI-301 and 1 with PLAAT2. Compounds 1 (blue) and LEI-301 (orange) in complex with PLAAT2,
covalently bound to Cys113. Yellow dotted lines represent a hydrogen bond, cyan represent -interactions.
12
ACS Paragon Plus Environment

Page 13 of 42
Journal of Medicinal Chemistry
1
2
3
4
Selectivity profile of LEI-301 for the endocannabinoid system.
5
6
7
8
The affinity or activity of LEI-301 for the receptors and metabolic enzymes of the endocannabinoid system
(ECS) was determined to assess its selectivity profile. Minimal affinity (< 50 %) was observed at 10 M for the
cannabinoid receptors type 1 and 2 (CB1/CB2) (Table 4). The enzymes involved in NAE biosynthesis (PLA2G4E,
NAPE-PLD) and degradation (FAAH) were also not inhibited at this concentration (Table 5). Enzymes involved in
the metabolism of the other endocannabinoid 2-arachidonoylglycerol (2-AG), such as diacylglycerol lipase
 and  (DAGL/), monoacylglycerol lipase (MAGL) and ,-hydrolase domain containing 6 (ABHD6) were not
inhibited.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Affinity of LEI-301 for cannabinoid receptors CB1 and CB2.
Radioligand displacement at
10 M LEI-301 (% ± SD; N = 2, n = 2)
hCB1
hCB2
49 ± 8
32 ± 4
Table 5. Inhibitory activities of LEI-301 for metabolic enzymes of the endocannabinoid system (ECS).
Remaining enzyme activity at 10 M LEI-301 (% ± SD; n = 3)
hNAPE-PLD
92 ± 8
hPLA2G4E
95 ± 5
hMAGL
mFAAH
108 ± 4
mABHD6
92 ± 5
mDAGL
mDAGL
97 ± 10
83 ± 1
105 ± 19
Activities were obtained from surrogate (hNAPE-PLD) or natural (hMAGL) substrate assays. hPLA2G4E, mDAGL/, mFAAH
and mABHD6 were determined by gel-based ABBP.
Targeted lipidomics shows that LEI-301 reduces NAEs in PLAAT2-overexpressing cells.
Having established that LEI-301 is a potent inhibitor of PLAAT2 and selective over the other enzymes of the
ECS, it was investigated whether LEI-301 is active in a cellular setting. To this end, NAE levels, which are
downstream metabolites of NAPEs generated by NAPE-PLD, were measured in living cells. Human U2OS
osteosarcoma cells were therefore transiently transfected with a pcDNA3.1 plasmid containing the gene for
PLAAT2, PLAAT5 or an empty (mock) vector. Of note, mRNA of NAPE-PLD was detected by quantitative PCR
(qPCR) in this cell line, suggesting that NAPEs can be converted to NAEs (NAPEPLD: quantification cycle Cq ±
SEM = 27.3 ± 0.05, RPS18 (housekeeping gene): Cq ± SEM = 17.8 ± 0.01, n = 3; the presence of reference gene
mRNA in combination with a Cq ≤ 29 for the targeted mRNA is considered sufficient30). Targeted lipidomics on
the lipid extracts of the transfected cells allowed the quantification of eight different NAEs and ten free fatty
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 42
1
2
3
4
5
6
7
8
9
acids (FFAs) by liquid chromatography-mass spectrometry (LC-MS). A striking increase of 9- to 99-fold for all
NAE species was observed for the PLAAT2-overexpressing cells compared to control, including anandamide
(AEA, 54-fold) (Figure 3A, Table 6). This result confirms previous findings.16 Notably, PLAAT2 overexpression did
not elevate most fatty acid species except for arachidonic acid (fold change ± SD = 1.81 ± 0.45, P = 0.04), while
levels of -linolenic (18:3-6) and linoleic acid (18:2-6) were significantly decreased (Figure 3A, Table S1). In
PLAAT5-transfected cells also a significant increase of NAE content was observed, although smaller in
magnitude compared to PLAAT2 (Figure 3B, Table 6). Interestingly, N-docosahexaenoylethanolamine (DHEA)
levels were unaffected by overexpression of PLAAT5. Furthermore, fatty acid levels including arachidonic acid,
were not elevated for this enzyme (Figure 3B, Table S1). Next, the PLAAT2/PLAAT5 or mock-transfected cells
were incubated with 10 M LEI-301 for 4 h. A significant 2-fold reduction of anandamide was apparent in the
PLAAT2 and PLAAT5 cells, which was absent in the control samples (for PLAAT2: P = 0.006; for PLAAT5: P <
0.0001) (Figure 3C,F). Other saturated, mono- and polyunsaturated NAEs also showed significant reductions
upon treatment with LEI-301 in the PLAAT2 and PLAAT5 overexpressing cells but not in the mock cells (Figure
3F, Figure S1). LEI-301 did reduce arachidonic acid levels in PLAAT2-transfected cells, however this did not meet
significance (P = 0.06) (Figure 3E). In the case of PLAAT5, DHEA as well as arachidonic acid levels were not
affected by treatment with LEI-301 (Figure 3E,F). Notably, LEI-301 did not alter levels of the other
endocannabinoid 2-AG in all tested conditions (Figure 3D). Previously, -ketoamides (also termed 2-oxo-
amides) were reported as inhibitors of various PLA2 enzymes.29,31-34 To test the selectivity of LEI-301 in U2OS
cells, we performed competitive ABPP experiments in membrane and cytosol fractions with broad-spectrum
lipase probes FP-TAMRA and MB064. No inhibitory activity was observed for LEI-301 at 10 M for any of the
probe-labelled enzymes (Figure S2A). In addition, members of the PLA2G4 family were also not inhibited by LEI-
301 (Figure S2B-C). Taken together, these results indicate that LEI-301 can be used to study the biological role
of PLAAT2 and PLAAT5 in cellular systems.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14
ACS Paragon Plus Environment

Page 15 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
6
Table 6. PLAAT2 and PLAAT5 overexpression greatly increases NAE content in U2OS cells. Data represent mean values ± SD
for 4 biological replicates. P-values were determined by one-way ANOVA.
Absolute NAE levels (pmol/106 cells ± SD)
Fold change ± SD
P-value
7
8
9
NAE
PLAAT2/
mock
PLAAT5/
mock
mock
PLAAT2
PLAAT5
PLAAT2
PLAAT5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PEA (16:0)
POEA (16:1)
SEA (18:0)
OEA (18:1)
0.196 ± 0.05
0.031 ± 0.01
0.516 ± 0.12
0.190 ± 0.05
18.62 ± 5.97
2.247 ± 0.78
47.39 ± 13.1
18.83 ± 5.02
0.569 ± 0.10
0.093 ± 0.01
2.916 ± 0.42
1.027 ± 0.17
95 ± 30
73 ± 25
92 ± 25
99 ± 26
2.9 ± 0.5
3.0 ± 0.4
5.7 ± 0.8
5.4 ± 0.9
0.0008
0.0013
0.0004
0.0003
0.0005
0.0001
< 0.0001
0.0001
LEA (18:2)
0.047 ± 0.01
1.569 ± 0.50
0.121 ± 0.01
33 ± 10
2.6 ± 0.3
0.0009
0.0002
AEA (20:4)
EPEA (20:5)
DHEA (22:6)
0.040 ± 0.01
0.010 ± 0.01
0.032 ± 0.01
2.194 ± 0.79
0.555 ± 0.15
0.286 ± 0.11
0.337 ± 0.09
0.059 ± 0.01
0.027 ± 0.01
54 ± 20
53 ± 14
8.9 ± 3.4
8.4 ± 2.2
5.7 ± 0.8
0.84 ± 0.1
0.0016
0.0004
0.0034
0.0005
< 0.0001
0.1886
Abbreviations: PEA = N-palmitoylethanolamine, POEA = N-palmitoleoylethanolamine, SEA = N-stearoylethanolamine, OEA =
N-oleoylethanolamine, LEA = N-linoleoylethanolamine, AEA = N-arachidonoylethanolamine, EPEA = N-eicosapentaenoylethanolamine,
DHEA = N-docosahexaenoylethanolamine.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 42
1
2
3
4
5
A
B
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
D
E
F
mock
PLAAT2
PLAAT5
Figure 3. U2OS cells transfected with PLAAT2 or PLAAT5 exhibit highly increased NAE levels and LEI-301 can inhibit NAE
formation. A,B) Volcano plots depicting the log2(fold change) vs. –log10(P-value) of NAEs (green diamonds) and free fatty
acids (FFAs, red circles) in (A) PLAAT2- or (B) PLAAT5- vs. mock-transfected U2OS cells. C-E) Absolute levels of
(C) anandamide (AEA), (D) 2-AG and (E) arachidonic acid in mock-, PLAAT2- or PLAAT5-transfected cells treated with vehicle
(DMSO) or LEI-301 (10 M, 4 h). F) Normalized NAE levels of mock-, PLAAT2- or PLAAT5-transfected cells treated with LEI-
301 (10 M, 4 h) represented as effect %. Data were normalized against mock-, PLAAT2- or PLAAT5-transfected cells
treated with vehicle (DMSO). Absolute values are depicted in Figure S1. Data represent mean values ± SD for 4 biological
replicates. *, P < 0.05, **, P < 0.01, ***, P < 0.001 by one-way ANOVA.
16
ACS Paragon Plus Environment

Page 17 of 42
Journal of Medicinal Chemistry
1
2
3
4
Conclusion
5
6
7
8
9
In summary, we have described the discovery and optimization of an -ketoamide inhibitor library for the
PLAAT enzyme subfamily. The SAR of the R1-ketone moiety proved to be narrow with little room for expansion
(Figure 4). The R2-phenethylamine side did allow introduction of a large hydrophobic para-phenoxy group,
which led to the identification of LEI-301 as a potent inhibitor for PLAAT2-5, having a 10-fold higher potency for
PLAAT2 and PLAAT5 than our initial hit. Covalent docking in the PLAAT2 crystal structure provided a possible
binding mode of LEI-301. Overexpression of PLAAT2 in U2OS cells resulted in a large increase of all measured
NAE species, including the endocannabinoid anandamide, while no significant elevations of fatty acids were
observed except for arachidonic acid. Also PLAAT5 was able to increase NAE content upon transient
transfection, although this was smaller in magnitude compared to PLAAT2. These findings support the notion
that PLAAT2 and PLAAT5 are involved in the biosynthetic pathways of the NAEs. Furthermore, treatment of
overexpressing PLAAT2 and PLAAT5 cells with LEI-301 gave a 2-fold reduction of anandamide levels, which was
absent in control cells. This validates LEI-301 as a promising tool compound to study PLAAT2 and PLAAT5
function in biological systems. LEI-301 allows acute blockade of these enzymes, which can be beneficial
compared to genetic knockout models, where long-term compensatory effects can occur. In addition, Plaat2 is
not present in the rodent genome, which hampers the study of its biological function. Currently, it is unknown
if the Ca2+-independent PLAAT enzymes contribute to physiological NAPE and thus NAE biosynthesis. In
contrast to the Ca2+-dependent NAPE production by PLA2G4E, PLAATs may continuously produce NAPEs from
their abundant PE and PC substrates. So far, PLA2G4E activity has been reported in heart, brain and skeletal
muscle.21 Peripheral organs such as kidney, small intestine and testis have well-established NAE signaling roles
and reported PLAAT2 or PLAAT5 expression.5,18 Furthermore, these tissues show low Ca2+-dependent PLA2G4E
activity.21 Therefore, these organs are prime candidates to assess the contribution of PLAAT enzymes with
regard to NAPE and NAE formation using the inhibitors here disclosed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
- Shallow lipophilic pocket
- Lipophilic pocket:
- Propyl chain optimal
- Cl can be removed
- o, m and p-position available,
but no increase in potency
Cl
O
- Ethyl chain optimal
- Large hydrophobic groups
favorable on p-position
O
N
H
O
- Amide NH essential
-
-ketoamide > -ketoamide
Figure 4. Structure-activity map for the PLAAT -ketoamide inhibitor library.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 42
1
2
3
4
Chemistry
5
6
7
A
O
O
O
O
O
OH
H
N
H
N
a
b
c
d
e
8
9
Cl
Cl
O
O
O
OH
N
R1
R1
R1
R2
R1
R2
O
O
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
57
58
59a-k
60a-k
1, 24-36,
43-51
23
B
O
O
H
N
O-K+
f
g
O
R
R
R
O
O
61a-f
62a-f
37-42
C
O
RHN
RHN
H
N
i
k
Het
R1
Het
OH
O
O
O
63: R = H
65a-e: R = Boc
66a-e: R = H * HCl
52-56
h
j
64: R = Boc
Scheme 2. General synthetic routes for A) -ketoamide 1 analogues, B) ,-unsaturated -ketoamides and C) O-heteroaryl
phenethylamine derivatives. Reagents and conditions: a) i. t-BuOH, THF, 0 oC; ii. N,O-dimethylhydroxylamine·HCl, Et3N, 0 oC,
75%; b) i. Mg, alkylbromide, Et2O, reflux; ii. Weinreb amide, -78 oC, 21% – 83%; c) TFA, DCM, rt, 99%; d) HATU or HCTU,
o
DiPEA, amine, DMF, rt, 22% – 80%; e) NaBH4, THF, rt, 72%. f) pyruvic acid or sodium pyruvate, KOH, MeOH, 0 C to rt; g) i.
oxalyl chloride, DCM, 0 oC to rt; ii. phenethylamine, DCM, 0 oC to rt, 14% – 35% over two steps. h) Boc2O, NaHCO3, THF, H2O,
o
rt, 85%; i) heteroaryl halide, K2CO3, DMSO or DMF, rt or 85 C, 63% - 92%; j) HCl, dioxane, rt, 99%; k) EDC·HCl, HOBt,
ketoacid, NMM, DCM, 0 oC to rt, 15% – 30%.
Oxalyl chloride (57) was reacted with tert-butanol and N,O-dimethylhydroxylamine·HCl giving Weinreb amide
58. Treatment with an in situ formed Grignard reagent from 4-chlorophenethyl bromide followed by tert-butyl
deprotection gave ketoacid 60a. Finally, amide coupling using HCTU afforded -ketoamide 1.
R1-derivatives 24-36 and R2-analogues 43-51 were synthesized via the general route (Scheme 2A). ,-
Unsaturated -ketoamides (37-42) were prepared using a two-step procedure (Scheme 2B): condensation of a
benzaldehyde (61a-f) with pyruvic acid, which afforded the ,-unsaturated -ketoacid as the potassium salt
(62a-f), followed by acid chloride formation and coupling with phenethylamine. O-Arylated 4-
hydroxyphenethylamine derivatives 52-56 were synthesized via Scheme 2C. Tyramine (63) was Boc-protected,
18
ACS Paragon Plus Environment

Page 19 of 42
Journal of Medicinal Chemistry
1
2
3
4
followed by nucleophilic aromatic substitution (SNAr) with a heteroaryl halide. Boc deprotection and
5
6
subsequent amide coupling provided the -ketoamides 52-56.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental section
Biological procedures
Plasmids
Full-length cDNA of human PLAAT1-5 (obtained from Natsuo Ueda8) was cloned into mammalian expression
vector pcDNA3.1 with a C-terminal FLAG-tag and containing genes for ampicillin and neomycin resistance.
Plasmids were isolated from transformed XL10-Gold competent cells (prepared using E. coli transformation
buffer set; Zymo Research) using plasmid isolation kits following the supplier’s protocol (Qiagen). All sequences
were analyzed by Sanger sequencing (Macrogen) and verified (CLC Main Workbench).
Cell culture
o
HEK293T and U2OS cells (ATCC) were cultured at 37 C and 7% CO2 in DMEM (Sigma Aldrich, D6546) with
GlutaMax (2 mM), penicillin (100 g/ml, Duchefa), streptomycin (100 g/ml, Duchefa) and 10% (v/v) newborn
calf serum (Seradigm). Medium was refreshed every 2-3 days and cells were passaged twice a week at 80-90%
confluence. Cells were passaged twice a week by thorough pipetting (HEK293T) or trypsinization (U2OS) to
appropriate confluence.
Transient transfection
Transient transfection was performed as described previously.1 In brief, 107 HEK293T cells were seeded in 15
cm petri dishes one day before transfection. Two hours before transfection the medium was refreshed with 13
mL medium. Transfection was performed with polyethyleneimine (PEI, 60 g per dish) in a ratio of 3:1 with
plasmid DNA (20 g per dish). PEI and plasmid DNA were incubated in serum-free medium (2 mL per dish) at rt
for 15 min, followed by dropwise addition to the cells. Transfection with the empty pcDNA3.1 vector was used
to generate control (mock) samples. The medium was refreshed after 24 hours and cells were harvested after
48 or 72 hours in cold PBS. Cells were pelleted by centrifugation (5 min, 1,000 g) and the pellet was washed
with PBS. The supernatant was removed and cell pellets were flash frozen in liquid N2 and stored at -80 oC.
Cell lysate preparation
Cell pellets were thawed on ice, and resuspended in cold lysis buffer (50 mM Tris-HCl pH 8, 2 mM DTT, 1 mM
MgCl2, 2.5 U/mL benzonase) and incubated on ice for 30 minutes. The cytosolic fraction (supernatant) was
separated from the membranes by ultra-centrifugation (100,000 g, 45 min, 4 C, Beckman Coulter, Ti 70.1
rotor). The pellet (membrane fraction) was resuspended in cold storage buffer (50 mM Tris-HCl pH 8, 2 mM
DTT) and homogenized by thorough pipetting and passage through an insulin needle (29G). Protein
concentrations were determined by a Quick Start™ Bradford protein assay (Bio-Rad) or QubitTM protein assay
(Invitrogen). Samples were flash frozen in liquid N2 and stored at -80 oC.
o
Mouse brain lysate preparation
Mouse brains were thawed on ice, dounce homogenized in cold lysis buffer (20 mM HEPES pH 7.2, 2 mM DTT, 1
mM MgCl2, 2.5 U/mL benzonase) and incubated on ice for 30 minutes. The membrane and cytosolic fractions
of cell or tissue lysates were separated by ultra-centrifugation (100,000 g, 45 min, 4 C). The supernatant was
collected (cytosolic fraction) and the membrane pellet was resuspended in cold storage buffer (20 mM HEPES
pH 7.2, 2 mM DTT) and homogenized by thorough pipetting and passage through an insulin needle (29G).
o
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 42
1
2
3
4
5
Protein concentrations were determined using a Bradford assay (Bio-Rad). Samples were flash frozen in liquid
N2 and stored at -80 oC.
6
7
Activity-based protein profiling on PLAAT2-5 transfected HEK293T cell lysate.
Gel-based activity based protein profiling (ABPP) was performed with minor changes as described previously.28
For ABPP assays on HEK293T cells overexpressing PLAAT2, the cytosol proteome (0.25 g/L, 20 L) was pre-
incubated with vehicle (DMSO) or inhibitor (0.5 L in DMSO, 30 min, rt) followed by incubation with MB064
(final concentration: 250 nM, 20 min, rt). For PLAAT3, PLAAT4 and PLAAT5 the protocols differed for the
protein concentrations (0.5 g/L, 1 g/L and 1 g/L, respectively) and MB064 concentration (250 nM, 500
nM and 500 nM, respectively). Final concentrations for the inhibitors are indicated in the main text and figure
legends. For the dose-response experiments only cytosol proteome was used. Proteins were denatured with 4x
Laemmli buffer (5 L, stock concentration: 240 mM Tris pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 5% (v/v) -
mercaptoethanol, 0.04% (v/v) bromophenol blue). 10 l sample per reaction was resolved on a 10% acrylamide
SDS-PAGE gel (180 V, 70 min). Gels were scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55
filters, respectively) on a ChemiDoc™ Imaging System (Bio-Rad). Fluorescence was normalized to Coomassie
staining and quantified with Image Lab (Bio-Rad). Experiments were performed in triplicate. Dose-response IC50
curves were generated with Graphpad Prism 6.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
qPCR
For primer sequences used see Table S2. RNA isolation and cDNA synthesis: Total RNA from U2OS cells was
extracted using a NucleoSpin® RNA kit (Macherey-Nagel) according to the manufacturer’s instructions.
Subsequently, cDNA synthesis was carried out with a SuperScript™ First-Strand Synthesis System (Invitrogen)
according to the manufacturer’s instructions. qPCR analysis: 2.5 ng of input cDNA was analyzed using
SYBRGreen qPCR master mix (Thermo-Fisher) on a CFX96 optical thermal cycler (Bio-Rad). Data analysis was
performed using CFX Manager software (Bio-Rad). The housekeeping gene 40S ribosomal protein S18 (RPS18)
was used as a control. Data are expressed in quantitation cycles (Cq) ± SEM of three technical replicates.
Radioligand displacement assays CB1 and CB2 receptor
[3H]CP55940 displacement assays to determine the affinity for the cannabinoid CB1 and CB2 receptors were
performed as previously described.35
NAPE-PLD surrogate substrate (PED6) activity assay
The human NAPE-PLD activity assay was performed as previously described.35
Natural substrate-based fluorescence assay MAGL
The natural substrate assay for human MAGL was performed as reported previously.36
Activity-based protein profiling for determining mDAGL/, mFAAH, mABHD6 and hPLA2G4E activities
Gel-based activity based protein profiling (ABPP) was performed as previously described.28 In brief, mouse
brain membrane proteome or hPLA2G4E-overexpressing membrane lysate (9.5 L and 2 g/L or 19.5 L and 1
g/L, respectively) was pre-incubated with vehicle or inhibitor (0.5 L 20x or 0.5 L 40x inhibitor stock in
DMSO, respectively, 30 min, rt) followed by incubation with the activity based probe MB064 (250 nM, 0.5 L
20x stock in DMSO) or FP-TAMRA (500 nM, 0.5 L 20x stock in DMSO) for mouse brain lysate (15 min, rt) or FP-
TAMRA (50 nM, 0.5 L 40x stock in DMSO) for PLA2G4E overexpressing lysate (5 min, rt). Final concentrations
for the inhibitors are indicated in the main text and figure legends. Proteins were denatured with 4x Laemmli
buffer (3.5 L, stock concentrations: 240 mM Tris-HCl pH 6.8, 8% w/v SDS, 40% v/v glycerol, 5% v/v -
mercaptoethanol, 0.04% v/v bromophenol blue, 30 min, rt). The samples (10 L per slot) were resolved by SDS-
PAGE (respectively, 10% or 8% acrylamide for mouse brain or PLA2G4E lysate, 180 V, 75 min). Gels were
scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55 filters, respectively) on a ChemiDoc™
20
ACS Paragon Plus Environment

Page 21 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
Imaging System (Bio-Rad). Fluorescence was normalized to Coomassie staining and quantified with Image Lab
(Bio-Rad).
6
7
8
9
Activity-based protein profiling for determining the selectivity profile of LEI-301 in U2OS cell lysate
U2OS cytosol or membrane lysate (19 L, 2.5 g/L) was pre-incubated with vehicle or inhibitor (0.5 L 40x
stock in DMSO, 30 min, rt) followed by incubation with the activity based probe MB064 (2 M, 0.5 L 40x stock
in DMSO, 15 min, rt) or FP-TAMRA (500 nM, 0.5 L 40x stock in DMSO, 15 min, rt). Final concentrations for the
inhibitors are indicated in the figure legends. Proteins were denatured with 3x Laemmli buffer (10 L, stock
concentrations: 240 mM Tris-HCl pH 6.8, 8% w/v SDS, 40% v/v glycerol, 5% v/v -mercaptoethanol, 0.04% v/v
bromophenol blue, 15 min, rt). The samples (12.5 L per slot) were resolved by SDS-PAGE (180 V, 75 min). Gels
were scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55 filters, respectively) on a
ChemiDoc™ Imaging System (Bio-Rad). Fluorescence was normalized to Coomassie staining and quantified with
Image Lab (Bio-Rad).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Activity-based protein profiling for determining PLA2G4B, PLA2G4C and PLA2G4D activities
hPLA2G4B, hPLA2G4C or hPLA2G4D-overexpressing membrane lysate (19 L, 1 g/L) was pre-incubated with
vehicle or inhibitor (0.5 L 40x inhibitor stock in DMSO, 30 min, rt) followed by incubation with the activity
based probe FP-TAMRA (500 nM, 0.5 L 20x stock in DMSO, 20 min, rt). Final concentrations for the inhibitors
are indicated in the figure legends. Proteins were denatured with 3x Laemmli buffer (10 L, stock
concentrations: 240 mM Tris-HCl pH 6.8, 8% w/v SDS, 40% v/v glycerol, 5% v/v -mercaptoethanol, 0.04% v/v
bromophenol blue, 30 min, rt). The samples (12.5 L per slot) were resolved by SDS-PAGE (180 V, 75 min). Gels
were scanned using Cy3 and Cy5 multichannel settings (605/50 and 695/55 filters, respectively) on a
ChemiDoc™ Imaging System (Bio-Rad). Fluorescence was normalized to Coomassie staining and quantified with
Image Lab (Bio-Rad).
Activity-based protein profiling for determining PLA2G4A activity
Recombinant full length human PLA2G4A (0.73 L, 440 ng/L, R&D systems, 6659-PL) was diluted with assay
buffer (13.3 L, 50 mM Tris HCl pH 8.0, NaCl 500 mM, CaCl2 20 mM in MilliQ) and pre-incubated with vehicle or
o
inhibitor (0.5 L 29x inhibitor stock in DMSO, 30 min, 37 C). FP-alkyne was used as a positive control.37 This
was followed by incubation with FP-TAMRA (500 nM, 0.5 L 30x inhibitor stock in DMSO, 20 min, rt). Final
concentrations for the inhibitors are indicated in the figure legends. Proteins were denatured with 4x Laemmli
buffer (5 L, stock concentrations: 240 mM Tris-HCl pH 6.8, 8% w/v SDS, 40% v/v glycerol, 5% v/v -
o
mercaptoethanol, 0.04% v/v bromophenol blue, 30 min, 95 C). The samples (12.5 L, ~200 ng per slot) were
resolved by SDS-PAGE (180 V, 75 min). Gels were scanned using Cy3 and Cy5 multichannel settings (605/50 and
695/55 filters, respectively) on a ChemiDoc™ Imaging System (Bio-Rad). Fluorescence was normalized to
Coomassie staining and quantified with Image Lab (Bio-Rad).
Targeted lipidomics in U2OS cells
The targeted lipidomics experiments are based on previously reported methods with small alterations as
specified below.38
Sample preparation
o
2 · 106 U2OS cells (grown at 37 C, 7% CO2) were seeded 1 day before transfection in 6 cm dishes. After 24 h,
PLAAT2, PLAAT5 or mock plasmid DNA (2.7 g/dish) and polyethyleneimine (PEI, 1 g/L, 8 g/dish) were
incubated in serum-free culture medium (15 min, rt), and then added dropwise to the cells in a ratio of 1:5
(plasmid/PEI). After 24 h, medium was aspirated and cells were washed once with serum-free medium. New
serum-free medium was added with LEI-301 (final concentration: 10 M, 0.1% DMSO) or DMSO as a control.
After incubating for 4 hours (37 oC, 7% CO2) the medium was removed and the cells were washed with cold PBS
(3x). The cells were harvested in 1.5 mL Eppendorf tubes by trypsinization, followed by centrifugation (10 min,
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 42
1
2
3
4
5
1,500 rpm). PBS was removed and the cell pellets were flash frozen with liquid N2 and stored at -80 oC. Live cell
count with trypan blue was performed after compound treatment to test for cell viability and for sample
normalization after lipid measurements.
6
7
8
Lipid extraction
Lipid extraction was performed on ice. In brief, cell pellets with 2 · 106 cells were spiked with 10 L each of
deuterium labeled internal standard mix for endocannabinoids (N-arachidonoylethanolamine (AEA)-d8, N-
docosahexaenoylethanolamine (DHEA)-d4, 2-arachidonoylglycerol (2-AG)-d8, N-stearoylethanolamine (SEA)-
d3, N-palmitoylethanolamine (PEA)-d4, N-linoleoylethanolamine (LEA)-d3 and N-oleoylethanolamine (OEA)-d4),
and negative polar lipids (fatty acid (FA)17:0-d33), followed by the addition of ammonium acetate buffer (100
L, 0.1 M, pH 4). After extraction with methyl tert-butyl ether (MTBE, 1 mL), the tubes were thoroughly mixed
for 4 min using a bullet blender at medium speed (Next Advance Inc., Averill park, NY, USA), followed by a
centrifugation step (5,000 g, 12 min, 4 oC). Then 925 L of the upper MTBE layer was transferred into clean 1.5
mL Eppendorf tubes. Samples were dried in a speedvac followed by reconstitution in acetonitrile/water (50 L,
90 : 10, v/v). The samples were centrifuged (14,000 g, 3 min, 4 oC) before transferring into LC-MS vials. Each
sample was injected on two different lipidomics platforms: endocannabinoids (5 L) and negative polar lipids (8
L).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC-MS/MS analysis for endocannabinoids
A targeted analysis of endocannabinoids and related NAEs (N-acylethanolamines) was measured using an
Acquity UPLC I class binary solvent manager pump (Waters, Milford, USA) in conjugation with AB SCIEX 6500
quadrupole ion trap (QTRAP) (AB Sciex, Massachusetts, USA). Separation was performed with an Acquity HSS
o
T3 column (1.2 x 100 mm, 1.8 m) maintained at 40 C. The aqueous mobile phase A consisted of 2 mM
ammonium formate and 10 mM formic acid, and the organic mobile phase B was acetonitrile. The flow rate
was set to 0.4 ml/min; initial gradient conditions were 55% B held for 2 min and linearly ramped to 100% B over
6 minutes and held for 2 min; after 10 s the system returned to initial conditions and held 2 min before next
injection. Electrospray ionization-MS was operated in positive mode for measurement of endocannabinoids
and NAEs, and a selective Multiple Reaction Mode (sMRM) was used for quantification.
LC-MS/MS analysis for negative polar lipids
This method is measured on an Acquity UPLC binary solvent manager 8 pump (Waters) coupled to an Agilent
6530 electrospray ionization quadrupole time-of-flight (ESI-Q-TOF, Agilent, Jose, CA, USA) high resolution mass
spectrometer using reference mass correction. The chromatographic separation was achieved on an Acquity
HSS T3 column (1.2 x 100 mm, 1.8 m) maintained at 40 oC. The negative apolar lipids that constitute free fatty
acids were separated with a flow of 0.4 mL/min over 15 min gradient. In negative mode, the aqueous mobile
phase A consisted of 5:95 (v/v) acetonitrile:H2O with 10 mM ammonium formate, and the organic mobile phase
B consisted of 99% (v/v) methanol with 10 mM ammonium formate.
Statistical analysis
Absolute values of lipid levels were corrected using the measured live cell count numbers (cell viability was >
90%). Data were tested for significance with GraphPad v6 using one-way ANOVA with Tukey correction for
multiple comparisons. P-values < 0.05 were considered significant.
Computational Chemistry
Ligand preparation
Molecular structures of LEI-301 and 1 were drawn with specified chirality and prepared for docking using
Ligprep from Schrödinger.39 Default Ligprep settings were applied: states of heteroatoms were generated using
Epik at a pH 7.40 No tautomers were created by the program, which resulted in one standardized structure per
ligand.
22
ACS Paragon Plus Environment

Page 23 of 42
Journal of Medicinal Chemistry
1
2
3
4
Protein preparation
The x-ray structure of PLAAT2 was extracted from the PDB (PDB ID: 4DPZ).4 The apo protein structure was
prepared for docking with the Protein Preparation tool from the Schrödinger 2017-4 suite. Waters were
removed and explicit hydrogens were added. Missing side chains and loops were added with homology
modeling using Prime41: loop 39-53 was modeled based on the protein sequence and loop 105-111 was based
on the structure of PLAAT3 (PDB ID: 4DOT).4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Docking
The PLAAT2 binding pocket was induced using the binding pose from 1 in PLAAT3 as previously reported.26,42
The complex of superposed 1 covalently bound to PLAAT2, was optimized using molecular dynamic simulations
(10 ns). Compound 1 was removed and the cysteine was restored to its non-bonded state. Subsequently, LEI-
301 and 1 were covalently docked to Cys113 using the Schrödinger 2017-4 suite.42 The poses with the lowest
docking scores were manually examined and one pose per ligand was selected. Selection was based on docking
score, frequency of recurring poses, and interactions made between the ligand and the protein.
Synthetic procedures
General
All chemicals (Sigma-Aldrich, Fluka, Acros, Merck) were used as received. All solvents used for reactions were of
analytical grade. THF, Et2O, DMF, CH3CN and DCM were dried over activated 4 Å molecular sieves, MeOH over 3
Å molecular sieves. Flash chromatography was performed on silica gel (Screening Devices BV, 40-63 μm, 60 Å).
The eluent EtOAc was of technical grade and distilled before use. Reactions were monitored by thin layer
chromatography (TLC) analysis using Merck aluminium sheets (Silica gel 60, F254). Compounds were visualized
by UV-absorption (254 nm) and spraying for general compounds: KMnO4 (20 g/L) and K2CO3 (10 g/L) in water,
o
1
or for amines: ninhydrin (0.75 g/L) and acetic acid (12.5 mL/L) in ethanol, followed by charring at ~150 C. H
and 13C NMR experiments were recorded on a Bruker AV-300 (300/75 MHz), Bruker AV-400 (400/100 MHz) or
Bruker DMX-400 (400/101 MHz). Chemical shifts are given in ppm (δ) relative to tetramethylsilane or CDCl3 as
internal standards. Multiplicity: s = singlet, br s = broad singlet, d = doublet, dd = doublet of doublet, t = triplet,
q = quartet, p = pentet, m = multiplet. Coupling constants (J) are given in Hz. LC-MS measurements were
performed on a Thermo Finnigan LCQ Advantage MAX ion-trap mass spectrometer (ESI+) coupled to a Surveyor
HPLC system (Thermo Finnigan) equipped with a standard C18 (Gemini, 4.6 mmD x 50 mmL, 5 μm particle size,
Phenomenex) analytical column and buffers A: H2O, B: CH3CN, C: 0.1% aq. TFA. High resolution mass spectra
were recorded on a LTQ Orbitrap (Thermo Finnigan) mass spectrometer or a Synapt G2-Si high definition mass
spectrometer (Waters) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV,
sheath gas flow 10 mL min-1, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass range
m/z = 150-2000) and dioctylphtalate (m/z = 391.28428) as a lock mass. Preparative HPLC was performed on a
Waters Acquity Ultra Performance LC with a C18 column (Gemini, 150 x 21.2 mm, Phenomenex). All final
compounds were determined to be >95% pure by integrating UV intensity recorded via HPLC.
General procedure A: -ketoester synthesis. Magnesium turnings were activated by stirring in a 3 M solution
of HCl for 5 min. The magnesium was then washed with water and acetone and dried under reduced pressure.
A round-bottom flask connected to a reflux condenser was flame dried before addition of activated magnesium
turnings (2 eq) under an argon atmosphere. Dry Et2O (2 mL) and a small piece of iodine were added followed by
dropwise addition of a solution of alkyl bromide (1 - 1.5 eq) in dry Et2O (1 M). The reaction was initiated with a
heat gun and refluxed for 1 h. In a separate flask, a solution of the Weinreb amide 58 (1 eq) in dry Et2O (1 M)
was prepared and cooled to -78 oC. The Grignard solution was taken up by syringe and added dropwise to the
o
Weinreb amide solution. After stirring for 2 h at -78 C the reaction was quenched with sat. aq. NH4Cl and
extracted with Et2O (2x). The combined organic layers were dried (MgSO4), filtered and concentrated under
reduced pressure. The crude residue was purified using silica gel column chromatography (EtOAc/pentane)
affording the α-ketoester.
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 42
1
2
3
4
5
6
7
8
General procedure B: -ketoester deprotection. A round bottom flask was charged with -ketoester (1 eq),
DCM (0.3 M) and TFA (5-10 eq) and stirred for 1-24 h at rt. The reaction mixture was concentrated under
reduced pressure after TLC analysis showed complete consumption of the starting material, followed by
coevaporation with toluene (3x). The obtained -ketoacid was used in the next step without further
purification.
9
General procedure C: -ketoamide synthesis. A round bottom flask was charged with -ketoacid (1 eq) and
DMF (0.2 M). HATU or HCTU (1-1.2 eq), DiPEA (1-2 eq) or Et3N (1-2 eq), and amine (1-1.1 eq) were added and
the mixture was stirred for 2-24 h at rt. Water was added and the mixture was extracted with DCM (2x). The
combined organic layers were washed with 1 M HCl, sat. aq. NaHCO3, brine, dried (MgSO4), filtered and
concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography
(EtOAc/pentane) affording the -ketoamide.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure D: -ketoamide synthesis. A round bottom flask was charged with -ketoacid (1 eq) and
THF or DCM (0.2 M) at 0 oC. EDC·HCl (1-1.5 eq) and HOBt (1-1.5 eq) were added and the mixture was stirred for
30 min, followed by addition of NMM (optional, 4 eq) and the amine (1.2 eq). The mixture was stirred for 1-4
days warming to rt. Work-up involved addition of sat. aq. NaHCO3 and extraction with EtOAc (2 x 25 mL). The
combined organic layers were washed with brine (1x), dried (MgSO4), filtered and concentrated under reduced
pressure. The crude residue was purified by silica gel column chromatography (EtOAc/pentane) or preparative
HPLC affording the -ketoamide.
General procedure E: N-Boc-tyramine O-arylation via SNAr. A microwave vial was charged with N-Boc-
tyramine 64 (1 eq), heteroaryl halide (1 eq), K2CO3 (2 eq) in DMSO or DMF (0.2 – 1 M) and capped. The mixture
was stirred for 24 - 42 h at 85°C in an oil bath until TLC showed complete conversion. The mixture was diluted
with H2O and extracted with EtOAc (3x). The combined organic layers were washed with brine (1x), dried
(MgSO4), filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column
chromatography (EtOAc/pentane) affording the product.
General procedure F: ,-unsaturated -ketoamide synthesis. A round bottom flask was charged with pyruvic
o
acid or sodium pyruvate (1 eq), aldehyde (1 eq) and MeOH (1 M) and cooled to 0 C. A solution of KOH (2 M,
1.5 eq) in MeOH was added dropwise while keeping the temperature below 25 oC. The reaction was stirred at rt
overnight, forming a yellow precipitate. The reaction mixture was filtered, the precipitate was washed with
cold MeOH (2x), Et2O (2x) and dried affording the -ketoacid as the potassium salt. A new round bottom flask
was charged with the potassium salt and DCM (0.5 M) and the suspension was sonicated for 20 min. This was
followed by cooling to 0 oC and addition of oxalyl chloride (2 eq). After consumption of the potassium salt, the
reaction mixture was concentrated under reduced pressure and coevaporated with toluene (2x). The α-
ketoacid chloride was then dissolved in DCM (0.5 M) and cooled to 0 oC, followed by addition of
phenethylamine (1 eq) and Et3N (2 eq). The reaction was stirred for 2 h. Work-up involved addition of H2O and
extraction with EtOAc. The organic layer was then washed with 1 M HCl (2x), sat. aq. NaHCO3 (2x) and brine
(1x), dried (MgSO4), filtered and concentrated under reduced pressure. The crude residue was purified by silica
gel column chromatography (EtOAc/pentane) affording the -ketoamide.
4-(4-Chlorophenyl)-2-oxo-N-phenethylbutanamide (1). t-Butyl deprotection 59a: the -ketoacid was prepared
according to general procedure B using -ketoester 59a (0.85 g, 3.2 mmol, 1 eq) and TFA (2.5 mL, 32 mmol, 10
eq) affording the α-ketoacid 60a (0.68 g, 3.2 mmol, quant.). Amide coupling: the title compound was prepared
according to general procedure C using the -ketoacid 60a (0.68 g, 3.2 mmol, 1 eq), phenethylamine (0.15 mL,
1.2 mmol, 1.1 eq), HATU (1.2 g, 3.2 mmol, 1 eq) and DiPEA (0.61 mL, 3.5 mmol, 1.1 eq) in DMF, affording the
product (0.70 g, 2.2 mmol, 70%). 1H NMR (300 MHz, CDCl3) δ 7.54 – 6.79 (m, 10H), 3.52 (q, J = 6.9 Hz, 2H), 3.21
(t, J = 7.4 Hz, 2H), 2.91 – 2.73 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 197.80, 159.82, 138.78, 138.13, 131.88,
24
ACS Paragon Plus Environment

Page 25 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
129.71, 128.67, 128.60, 128.51, 126.67, 40.44, 38.06, 35.28, 28.35. HRMS [C18H18NClO2 + H]+: 316.1099
calculated, 316.1099 foun.
6
7
8
9
4-(4-Chlorophenyl)-2-hydroxy-N-phenethylbutanamide (23). A round bottom flask was charged with -
ketoamide 1 (70 mg, 0.22 mmol, 1 eq) and THF (1 mL). NaBH4 (12 mg, 0.33 mmol, 1.5 eq) was added and the
mixture was stirred for 15 min. The reaction was quenched with water (10 mL) and extracted with EtOAc (1 x
10 mL). The organic layer was washed with 1 M aq. HCl (2 x 10 mL), brine (1 x 10 mL), dried (MgSO4), filtered
and concentrated under reduced pressure. Purification by silica gel column chromatography afforded the
product (50 mg, 0.16 mmol, 72%). 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.25 (m, 2H), 7.25 – 7.14 (m, 5H), 7.08 (d,
J = 8.4 Hz, 2H), 6.61 (t, J = 5.4 Hz, 1H), 4.03 (dd, J = 7.9, 3.7 Hz, 1H), 3.66 – 3.40 (m, 2H), 3.24 (br s, 1H), 2.80 (t, J
= 7.0 Hz, 2H), 2.65 (t, J = 7.9 Hz, 2H), 2.11 – 1.97 (m, 1H), 1.90 – 1.80 (m, 1H). 13C NMR (101 MHz, CDCl3) δ
173.79, 139.74, 138.63, 131.87, 129.93, 128.81, 128.78, 128.65, 126.74, 71.33, 40.33, 36.38, 35.76, 30.57.
HRMS [C18H20NClO2 + H]+: 318.1255 calculated, 318.1252 found.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Oxo-N-phenethyl-4-phenylbutanamide (24). t-Butyl deprotection 59b: the -ketoacid was prepared
according to general procedure B using -ketoester 59b (0.50 g, 2.1 mmol, 1 eq) and TFA (0.80 mL, 32 mmol, 5
eq) affording the -ketoacid 60b (0.40 g, 2.1 mmol, quant.). Amide coupling: the title compound was prepared
according to general procedure C using the -ketoacid 60b (0.20 g, 1.2 mmol, 1 eq), phenethylamine (0.15 mL,
1.2 mmol, 1.1 eq), HCTU (0.48 g, 1.15 mmol, 1 eq) and DiPEA (0.22 mL, 1.3 mmol, 1.1 eq) in DMF, affording the
1
product (80 mg, 0.28 mmol, 24%). H NMR (300 MHz, CDCl3) δ 7.40 – 7.07 (m, 10H), 7.07 – 6.88 (m, 1H), 3.55
(q, J = 6.9 Hz, 2H), 3.26 (t, J = 7.5 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H). 13C NMR (75 MHz, CDCl3)
δ 198.25, 160.05, 140.46, 138.27, 128.85, 128.77, 128.60, 128.46, 126.85, 126.36, 40.58, 38.40, 35.51, 29.22.
HRMS [C18H19NO2 + H]+: 282.1489 calculated, 282.1487 found.
2-Oxo-N-phenethyl-5-phenylpentanamide (25). The title compound was prepared according to general
procedure C using the -ketoacid 60c (0.12 g, 0.63 mmol, 1 eq), phenethylamine (86 L, 0.69 mmol, 1.1 eq),
HCTU (0.26 g, 0.63 mmol, 1 eq) and DiPEA (0.12 mL, 0.70 mmol, 1.1 eq) in DCM, affording the product (70 mg,
0.24 mmol, 38%). 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.20 (m, 5H), 7.20 – 7.13 (m, 5H), 6.98 (br s, 1H), 3.53 (q, J
= 7.0 Hz, 2H), 2.92 (t, J = 7.3 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.92 (p, J = 7.4 Hz, 2H). 13C
NMR (101 MHz, CDCl3) δ 198.97, 160.15, 141.42, 138.30, 128.83, 128.75, 128.57, 128.51, 126.83, 126.12, 40.56,
36.20, 35.52, 35.12, 24.92. HRMS [C19H21NO2 + H]+: 296.1645 calculated, 296.1646 found.
2-Oxo-N-phenethyl-6-phenylhexanamide (26). t-Butyl deprotection 59d: the -ketoacid was prepared
according to general procedure B using -ketoester 59d (0.33 g, 1.3 mmol, 1 eq) and TFA (1.9 mL, 25 mmol, 19
eq) affording the α-ketoacid 60d (0.26 g, 1.3 mmol, quant.). Amide coupling: the title compound was prepared
according to general procedure C using the -ketoacid 60d (0.26 g, 1.3 mmol, 1 eq), phenethylamine (0.22 mL,
1.72 mmol, 1.3 eq), HATU (0.59 g, 1.56 mmol, 1.2 eq) and DiPEA (0.30 mL, 1.72 mmol, 1.3 eq), affording the
1
product (0.34 g, 1.11 mmol, 71%). H NMR (300 MHz, CDCl3) δ 7.43 – 7.08 (m, 10H), 7.09 – 6.89 (m, 1H), 3.52
(q, J = 6.9 Hz, 2H), 3.01 – 2.86 (m, 2H), 2.81 (t, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.62 (p, J = 3.5 Hz, 4H). 13C
NMR (75 MHz, CDCl3) δ 198.99, 160.11, 142.05, 138.25, 128.74, 128.68, 128.40, 128.34, 126.73, 125.81, 40.50,
36.55, 35.62, 35.42, 30.79, 22.81. HRMS [C20H23NO2 + H]+: 310.1802 calculated, 310.1801 found.
2-Oxo-N-phenethyl-2-phenylacetamide (27). t-Butyl deprotection 59e: the -ketoacid was prepared according
to general procedure B using -ketoester 59e (0.68 g, 3.3 mmol, 1 eq) and TFA (2.4 mL, 32 mmol, 10 eq)
affording the -ketoacid 60e (0.58 g, 3.2 mmol, quant.). Amide coupling: the title compound was prepared
according to general procedure C using the -ketoacid 60e (0.25 g, 1.7 mmol, 1 eq), phenethylamine (0.23 mL,
1.8 mmol, 1.1 eq), HCTU (0.69 g, 1.7 mmol, 1 eq) and DiPEA (0.32 mL, 1.8 mmol, 1.1 eq) in DCM, affording the
product (0.22 g, 0.86 mmol, 52%). 1H NMR (400 MHz, CDCl3) δ 8.33 – 8.19 (m, 2H), 7.62 – 7.52 (m, 1H), 7.47 –
7.38 (m, 2H), 7.34 – 7.26 (m, 2H), 7.26 – 7.14 (m, 4H), 3.62 (q, J = 7.0 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 187.86, 161.98, 138.36, 134.36, 133.30, 131.11, 128.75, 128.72, 128.47, 126.68, 40.60,
35.46. HRMS [C16H15NO2 + H]+: 254.1176 calculated, 254.1175 found.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 42
1
2
3
4
5
6
2-Oxo-N-phenethylpropanamide (28). A round bottom flask was charged with pyruvic acid (0.79 mL, 12 mmol,
1 eq) and cooled to 0 oC. Thionyl chloride (0.93 mL, 13 mmol, 1.1 eq) was added and the mixture was stirred for
3 h at rt. The reaction mixture was concentrated under reduced pressure and coevaporated with toluene (3x).
o
The acid chloride was dissolved in DCM (50 mL) and cooled to 0 C. Phenethylamine (1.5 mL, 12 mmol, 1 eq)
7
and Et3N (1.8 mL, 13 mmol, 1.1 eq) were added and the reaction was stirred for 2 h. Work-up involved addition
of H2O and extraction with EtOAc. The organic layer was then washed with 1 M HCl (2x), sat. aq. NaHCO3 (2x)
and brine (1x), dried (MgSO4), filtered and concentrated under reduced pressure. The crude residue was
purified by silica gel column chromatography (EtOAc/pentane) affording the product (100 mg, 0.52 mmol, 4%).
1H NMR (300 MHz, CDCl3) δ 7.43 – 7.15 (m, 5H), 7.13 – 6.82 (m, 1H), 3.57 (q, J = 7.0 Hz, 2H), 2.87 (t, J = 7.1 Hz,
2H), 2.47 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 197.16, 160.18, 138.27, 128.88, 128.78, 126.87, 40.65, 35.54,
24.58. HRMS [C11H13NO2 + H]+: 192.1019 calculated, 192.1019 found.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Oxo-N-phenethyl-4-(p-tolyl)butanamide (29). t-Butyl deprotection 59f: the -ketoacid was prepared
according to general procedure B using -ketoester 59f (0.54 g, 2.2 mmol, 1 eq) and TFA (1.6 mL, 22 mmol, 10
eq) affording the α-ketoacid 60f (0.42 g, 2.2 mmol, quant.). Amide coupling: the title compound was prepared
according to general procedure C using the α-ketoacid 60f (0.42 g, 2.2 mmol, 1 eq), phenethylamine (0.30 mL,
2.4 mmol, 1.1 eq), HATU (0.83 g, 2.2 mmol, 1 eq) and DiPEA (0.42 mL, 2.4 mmol, 1.1 eq) in DCM, affording the
1
product (0.48 g, 1.6 mmol, 74%). H NMR (300 MHz, CDCl3) δ 7.32 – 7.23 (m, 2H), 7.23 – 7.18 (m, 1H), 7.18 –
7.11 (m, 2H), 7.11 – 6.97 (m, 5H), 3.51 (q, J = 6.9 Hz, 2H), 3.21 (t, J = 7.5 Hz, 2H), 2.97 – 2.69 (m, 4H), 2.28 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 198.18, 159.96, 138.21, 137.24, 135.61, 129.12, 128.66, 128.62, 128.18,
126.64, 40.44, 38.37, 35.33, 28.64, 20.97. HRMS [C19H21NO2 + H]+: 296.1645 calculated, 296.1643 found.
2-Oxo-N-phenethyl-4-(4-(trifluoromethyl)phenyl)butanamide (30). t-Butyl deprotection 59g: the -ketoacid
was prepared according to general procedure B using α-ketoester 59g (0.25 g, 0.83 mmol, 1 eq) and TFA (0.62
mL, 8.3 mmol, 10 eq) affording the -ketoacid 60g (0.20 g, 0.83 mmol, quant.). Amide coupling: The title
compound was prepared according to general procedure C using the -ketoacid 60g (0.20 g, 0.80 mmol, 1 eq),
phenethylamine (0.11 mL, 0.88 mmol, 1.1 eq), HATU (0.38 g, 0.80 mmol, 1 eq) and DiPEA (0.15 mL, 0.80 mmol,
1.1 eq) in DMF. Column chromatography (20% -> 60% EtOAc in pentane) afforded the product (0.22 g, 0.64
mmol, 80%). 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.1 Hz, 2H), 7.39 – 7.31 (m, 4H), 7.31 – 7.25 (m, 1H), 7.25 –
7.19 (m, 2H), 7.12 – 7.00 (m, 1H), 3.60 (q, J = 7.0 Hz, 2H), 3.32 (t, J = 7.4 Hz, 2H), 3.01 (t, J = 7.4 Hz, 2H), 2.88 (t, J
= 7.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 197.70, 159.88, 144.56 (q, J = 1.3 Hz), 138.19, 128.78, 128.70,
128.48, 126.80, 125.46 (q, J = 3.8 Hz), 124.31 (q, J = 271.8 Hz), 40.55, 37.89, 35.39, 28.89. HRMS [C19H18F3NO2 +
H]+: 350.1362 calculated, 350.1362 found.
4-(4-Fluorophenyl)-2-oxo-N-phenethylbutanamide (31). t-Butyl deprotection 59h: the -ketoacid was
prepared according to general procedure B using α-ketoester 59h (0.15 g, 0.59 mmol, 1 eq) and TFA (0.44 mL,
5.9 mmol, 10 eq) affording the -ketoacid 60h (0.12 g, 0.59 mmol, quant.). Amide coupling: the title compound
was prepared according to general procedure C using the -ketoacid 60h (0.12 g, 0.63 mmol, 1 eq),
phenethylamine (80 μL, 0.63 mmol, 1 eq), HCTU (0.26 g, 0.63 mmol, 1 eq) and DiPEA (0.12 mL, 0.69 mmol, 1.1
1
eq) in DMF, affording the product (0.12 g, 0.39 mmol, 62%). H NMR (300 MHz, CDCl3) δ 7.35 – 7.21 (m, 3H),
7.21 – 7.08 (m, 4H), 7.08 – 6.99 (m, 1H), 6.99 – 6.88 (m, 2H), 3.54 (q, J = 6.9 Hz, 2H), 3.22 (t, J = 7.4 Hz, 2H), 2.99
– 2.64 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 198.05, 163.13, 159.97, 138.22, 136.05 (d, J = 3.2 Hz), 129.85 (d, J =
7.9 Hz), 128.77 (d, J = 6.8 Hz), 126.82, 115.32 (d, J = 21.2 Hz), 40.55, 38.44, 35.44, 28.39. HRMS [C18H18FNO2 +
H]+: 300.1394 calculated, 300.1393 found.
4-(4-Methoxyphenyl)-2-oxo-N-phenethylbutanamide (32). A round bottom flask was charged with
unsaturated -ketoamide 39 (0.10 g, 0.32 mmol, 1 eq) and MeOH (1 mL) and flushed with N2. Pd/C (10 wt. %,
10 mg, 9.4 mol, 3 mol%) was added and the flask was purged again with N2, followed by H2 and the reaction
was stirred overnight under a H2 atmosphere (balloon). The reaction was filtered over Celite, which was
washed with MeOH and the filtrate was concentrated under reduced pressure. The crude residue was purified
by silica gel column chromatography (EtOAc/pentane) affording the product (50 mg, 0.16 mmol, 50%). 1H NMR
26
ACS Paragon Plus Environment

Page 27 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
6
(400 MHz, CDCl3) δ 7.36 – 7.27 (m, 2H), 7.27 – 7.21 (m, 1H), 7.18 (d, J = 7.0 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 7.05
– 6.90 (m, 1H), 6.88 – 6.71 (m, 2H), 3.77 (s, 3H), 3.55 (q, J = 7.0 Hz, 2H), 3.22 (t, J = 7.5 Hz, 2H), 2.95 – 2.73 (m,
4H). 13C NMR (101 MHz, CDCl3) δ 198.37, 160.05, 158.13, 138.26, 132.48, 129.41, 128.86, 128.77, 126.85,
113.99, 55.35, 40.57, 38.67, 35.51, 28.38. HRMS [C19H21NO3 + H]+: 312.1594 calculated, 312.1593 found.
7
8
9
2-Oxo-N-phenethyl-4-(4-phenoxyphenyl)butanamide (33). A round bottom flask was charged with
unsaturated -ketoamide 67 (0.12 g, 0.33 mmol, 1 eq) and MeOH (1 mL) and flushed with N2. Pd/C (10 wt. %,
10 mg, 9.4 mol, 3 mol%) was added and the flask was purged again with N2, followed by H2 and the reaction
was stirred overnight under a H2 atmosphere (balloon). The reaction was filtered over Celite, which was
washed with MeOH and the filtrate was concentrated under reduced pressure. The ketone was overreduced to
the alcohol according to NMR analysis, therefore it was reoxidized. A round bottom flask was charged with the
alcohol, Dess-Martin periodinane (0.21 g, 0.49 mmol, 1.5 eq) and DCM (5 mL) and stirred at rt. Work-up
involved addition of H2O and extraction with EtOAc. The organic layer was washed with 1 M HCl (2x), sat. aq.
NaHCO3 (2x) and brine (1x), dried (MgSO4), filtered and concentrated under reduced pressure. The crude
residue was purified by silica gel column chromatography (EtOAc/pentane) affording the product (80 mg, 0.23
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
mmol, 65%). H NMR (400 MHz, CDCl3) δ 7.36 – 7.27 (m, 4H), 7.27 – 7.20 (m, 1H), 7.20 – 7.11 (m, 4H), 7.11 –
7.04 (m, 1H), 7.04 – 6.95 (m, 3H), 6.95 – 6.88 (m, 2H), 3.55 (q, J = 7.0 Hz, 2H), 3.25 (t, J = 7.5 Hz, 2H), 2.90 (t, J =
7.5 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 198.20, 160.01, 157.51, 155.62, 138.23, 135.36,
129.79, 129.71, 128.85, 128.76, 126.85, 123.16, 119.14, 118.74, 40.58, 38.55, 35.49, 28.49. HRMS [C24H23NO3 +
H]+: 374.1751 calculated, 374.1748 found.
4-(2-Chlorophenyl)-2-oxo-N-phenethylbutanamide (34). t-Butyl deprotection 59i: the -ketoacid was prepared
according to general procedure B using -ketoester 59i (0.23 g, 0.87 mmol, 1 eq) and TFA (0.94 mL, 13 mmol,
15 eq) affording the -ketoacid 60i (0.18 g, 0.87 mmol, quant.). Amide coupling: the title compound was
prepared according to general procedure C using the -ketoacid 60i (0.18 g, 0.87 mmol, 1 eq), phenethylamine
(0.12 mL, 0.95 mmol, 1.1 eq), HATU (0.33 g, 0.87 mmol, 1 eq) and DiPEA (0.16 mL, 0.95 mmol, 1.1 eq) in DCM,
affording the product (0.13 g, 0.42 mmol, 48%). 1H NMR (300 MHz, CDCl3) δ 7.50 – 7.08 (m, 9H), 7.08 – 6.89 (m,
1H), 3.55 (q, J = 6.9 Hz, 2H), 3.27 (t, J = 7.5 Hz, 2H), 3.01 (t, J = 7.4 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H). 13C NMR (75
MHz, CDCl3) δ 197.96, 159.93, 138.24, 138.01, 134.04, 130.52, 129.65, 128.82, 128.74, 127.92, 126.96, 126.82,
40.56, 36.78, 35.48, 27.23. HRMS [C18H18ClNO2 + H]+: 316.1099 calculated, 316.1100 found.
4-(3-Chlorophenyl)-2-oxo-N-phenethylbutanamide (35). t-Butyl deprotection 59j: the -ketoacid was prepared
according to general procedure B using -ketoester 59j (0.55 g, 2.0 mmol, 1 eq) and TFA (2 mL, 26 mmol, 13
eq) affording the -ketoacid 60j (0.47 g, 2.0 mmol, quant.). Amide coupling: The title compound was prepared
according to general procedure C using the -ketoacid 60j (0.47 g, 2.0 mmol, 1 eq), phenethylamine (0.31 mL,
2.4 mmol, 1.2 eq), HATU (0.84 mg, 2.2 mmol, 1.1 eq) and DiPEA (0.42 mL, 2.4 mmol, 1.2 eq), affording the
product (0.37 g, 1.2 mmol, 53%). 1H NMR (300 MHz, CDCl3) δ 7.73 – 6.73 (m, 10H), 3.58 (q, J = 6.9 Hz, 2H), 3.28
(t, J = 7.4 Hz, 2H), 3.07 – 2.74 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 197.71, 159.83, 142.40, 138.16, 134.13,
129.72, 128.69, 128.62, 128.53, 126.69, 126.55, 126.42, 40.47, 37.95, 35.32, 28.65. HRMS [C18H18ClNO2 + H]+:
316.1099 calculated, 316.1098 found.
4-(3,4-Dichlorophenyl)-2-oxo-N-phenethylbutanamide (36). t-Butyl deprotection 59k: the -ketoacid was
prepared according to general procedure B using -ketoester 59k (90 mg, 0.30 mmol, 1 eq) and TFA (0.25 mL,
32 mmol, 10 eq) affording the -ketoacid 60k (74 mg, 0.30 mmol, quant.). Amide coupling: the title compound
was prepared according to general procedure C using -ketoacid 60k (66 mg, 0.27 mmol, 1 eq),
phenethylamine (36 μL, 0.29 mmol, 1.1 eq), HATU (110 mg, 0.29 mmol, 1.1 eq) and DiPEA (92 μL, 0.53 mmol, 2
1
eq) in DCM, affording the product (42 mg, 0.12 mmol, 44%). H NMR (400 MHz, CDCl3) δ 7.41 – 7.32 (m, 4H),
7.32 – 7.26 (m, 1H), 7.22 (d, J = 7.0 Hz, 2H), 7.07 (dd, J = 8.2, 2.0 Hz, 1H), 7.05 – 6.97 (m, 1H), 3.60 (q, J = 7.0 Hz,
2H), 3.28 (t, J = 7.4 Hz, 2H), 3.03 – 2.79 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 197.63, 159.85, 140.68, 138.17,
132.46, 130.53, 130.50, 130.39, 128.88, 128.76, 127.98, 126.90, 40.61, 37.94, 35.49, 28.29. HRMS [C18H17Cl2NO2
+ H]+: 350.0709 calculated, 350.0708 found.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 42
1
2
3
4
5
6
7
8
9
(E)-4-(4-Chlorophenyl)-2-oxo-N-phenethylbut-3-enamide (37). -Ketoacid formation: the -ketoacid salt was
prepared according to general procedure F using pyruvic acid (1.7 mL, 18 mmol, 1 eq), 4-chlorobenzaldehyde
(2.2 mL, 19 mmol, 1 eq), KOH (2.1 g, 38 mmol, 2 eq) in MeOH affording potassium 4-(4-chlorophenyl)-2-oxobut-
3-enoate 62a (2.0 g, 8.0 mmol, 42%). Amide coupling: the title compound was prepared according to general
procedure F using potassium salt 62a (2.0 g, 8.0 mmol, 1 eq), oxalyl chloride (1.4 mL, 16 mmol, 2 eq),
phenethylamine (1.1 mL, 8.8 mmol, 1.1 eq) and Et3N (2.2 mL, 16 mmol, 2 eq) in DCM, affording the product
(0.30 g, 0.96 mmol, 12%). 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 16.2 Hz, 1H), 7.73 (d, J = 16.1 Hz, 1H), 7.59 (d,
J = 8.5 Hz, 2H), 7.43 – 7.36 (m, 2H), 7.36 – 7.28 (m, 2H), 7.28 – 7.17 (m, 4H), 3.63 (q, J = 7.0 Hz, 2H), 2.89 (t, J =
7.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 185.32, 161.24, 146.51, 138.34, 137.61, 132.94, 130.37, 129.48, 128.87,
128.81, 126.86, 119.09, 40.77, 35.58. HRMS [C18H16ClNO2 + H]+: 314.0942 calculated, 314.0939 found.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(E)-2-Oxo-N-phenethyl-4-phenylbut-3-enamide (38). -Ketoacid formation: the -ketoacid salt was prepared
according to general procedure F using pyruvic acid (0.79 mL, 11 mmol, 1 eq), benzaldehyde (1.2 g, 11 mmol, 1
eq), KOH (0.98 g, 17 mmol, 1.5 eq) in MeOH affording potassium 2-oxo-4-phenylbut-3-enoate 62b (0.85 g, 3.9
mmol, 36%). Amide coupling: the title compound was prepared according to general procedure C using
potassium salt 62b (0.20 g, 0.93 mmol, 1 eq), phenethylamine (0.12 mL, 0.93 mmol, 1 eq), HCTU (0.39 g, 0.93
mmol, 1 eq) and DiPEA (0.32 mL, 1.86 mmol, 2 eq) in DMF (5 mL) affording the product (70 mg, 0.25 mmol,
27%). 1H NMR (300 MHz, CDCl3) δ 7.93 (d, J = 16.2 Hz, 1H), 7.77 (d, J = 16.1 Hz, 1H), 7.71 – 7.62 (m, 2H), 7.49 –
7.37 (m, 3H), 7.36 – 7.16 (m, 6H), 3.63 (q, J = 7.0 Hz, 2H), 2.89 (t, J = 7.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
185.49, 161.38, 148.12, 138.40, 134.48, 131.60, 129.28, 129.15, 128.85, 128.82, 126.83, 118.67, 40.76, 35.61.
HRMS [C18H17NO2 + H]+: 280.1332 calculated, 280.1331 found.
(E)-4-(4-Methoxyphenyl)-2-oxo-N-phenethylbut-3-enamide (39). -Ketoacid formation: the -ketoacid salt
was prepared according to general procedure F using sodium pyruvate (3.0 g, 27 mmol, 1 eq), 4-
methoxybenzaldehyde (3.3 mL, 27 mmol, 1 eq), KOH (2.3 g, 41 mmol, 1.5 eq) in MeOH affording potassium 4-
(4-methoxyphenyl)-2-oxobut-3-enoate 62c (6.5 g, 27 mmol, 98%). Amide coupling: the title compound was
prepared according to general procedure F using potassium salt 62c (1.0 g, 4.1 mmol, 1 eq), oxalyl chloride
(0.87 mL, 8.2 mmol, 2 eq), phenethylamine (0.52 mL, 4.1 mmol, 1 eq) and Et3N (1.1 mL, 8.2 mmol, 2 eq) in
DCM, affording the product (1.2 g, 3.8 mmol, 93%). 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 16.0 Hz, 1H), 7.68 –
7.57 (m, 3H), 7.37 – 7.27 (m, 3H), 7.26 – 7.18 (m, 3H), 6.95 – 6.88 (m, 2H), 3.83 (s, 3H), 3.61 (q, J = 7.0 Hz, 2H),
2.88 (t, J = 7.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 185.15, 162.54, 161.66, 147.93, 138.44, 131.23, 128.76,
127.27, 126.72, 116.20, 114.59, 55.50, 40.69, 35.56. HRMS [C19H19NO3 + H]+: 310.1438 calculated, 310.1435
found.
(E)-4-(4-Bromophenyl)-2-oxo-N-phenethylbut-3-enamide (40). -Ketoacid formation: the -ketoacid salt was
prepared according to general procedure F using pyruvic acid (1.4 mL, 16 mmol, 1 eq), 4-bromobenzaldehyde
(1.6 mL, 16 mmol, 1 eq), KOH (1.8 g, 32 mmol, 2 eq) in MeOH affording potassium 4-(4-bromophenyl)-2-
oxobut-3-enoate 62d (2.0 g, 6.8 mmol, 42%). Amide coupling: the title compound was prepared according to
general procedure F using potassium salt 62d (2.0 g, 6.8 mmol, 1 eq), oxalyl chloride (1.2 mL, 14 mmol, 2 eq),
phenethylamine (0.94 mL, 7.5 mmol, 1.1 eq) and Et3N (1.90 mL, 14 mmol, 2 eq) in DCM, affording the product
1
(0.84 g, 2.3 mmol, 34%). H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 16.2 Hz, 1H), 7.74 (d, J = 16.1 Hz, 1H), 7.64 –
7.42 (m, 4H), 7.38 – 7.15 (m, 6H), 3.62 (q, J = 7.0 Hz, 2H), 2.89 (t, J = 7.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
185.37, 161.22, 146.55, 138.35, 133.37, 132.45, 130.52, 128.87, 128.81, 126.86, 126.08, 119.23, 40.77, 35.60.
HRMS [C18H16BrNO2 + H]+: 358.0437 calculated, 358.0437 found.
(E)-4-(3-Bromophenyl)-2-oxo-N-phenethylbut-3-enamide (41). -Ketoacid formation: the -ketoacid salt was
prepared according to general procedure F using sodium pyruvate (1.0 g, 11 mmol, 1 eq), 3-
bromobenzaldehyde (1.3 mL, 11 mmol, 1 eq), KOH (0.96 g, 17 mmol, 1.5 eq) in MeOH affording potassium 4-(3-
bromophenyl)-2-oxobut-3-enoate 62e (0.40 g, 1.4 mmol, 12%). Amide coupling: the title compound was
prepared according to general procedure F using potassium salt 62e (0.40 g, 1.4 mmol, 1 eq), oxalyl chloride
(0.24 mL, 2.7 mmol, 2 eq), phenethylamine (0.19 mL, 1.5 mmol, 1.1 eq) and Et3N (0.38 mL, 2.7 mmol, 2 eq) in
28
ACS Paragon Plus Environment

Page 29 of 42
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
DCM, affording the product (0.31 g, 0.87 mmol, 62%). 1H NMR (400 MHz, CDCl3) δ 7.86 – 7.78 (m, 2H), 7.74 (d, J
= 16.2 Hz, 1H), 7.62 – 7.51 (m, 2H), 7.37 – 7.28 (m, 3H), 7.28 – 7.19 (m, 4H), 3.63 (q, J = 7.0 Hz, 2H), 2.89 (t, J =
7.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 185.33, 161.12, 146.13, 138.34, 136.52, 134.25, 131.84, 130.63,
128.88, 128.82, 127.72, 126.88, 123.27, 119.96, 40.78, 35.59. HRMS [C18H16BrNO2 + H]+: 358.0437 calculated,
358.0437 found.
9
(E)-4-([1,1'-Biphenyl]-3-yl)-2-oxo-N-phenethylbut-3-enamide (42). A round bottom flask was charged with aryl
bromide 41 (0.20 g, 0.56 mmol, 1 eq) and toluene/EtOH (4:1, 3 mL) and degassed for 20 min with sonication.
Pd(PPh3)4 (13 mg, 0.01 mmol, 2 mol%), phenylboronic acid (0.10 g, 0.84 mmol, 1.5 eq) and K2CO3 (0.46 g, 3.4
mmol, 6 eq) were added and the reaction was stirred for 16 h at 80 oC. The reaction mixture was filtered over a
pad of Celite and concentrated under reduced pressure. The crude residue was purified by silica gel column
chromatography (EtOAc/pentane) affording the product (0.15 g, 0.42 mmol, 75%). 1H NMR (300 MHz, CDCl3) δ
8.03 (d, J = 16.2 Hz, 1H), 7.94 – 7.78 (m, 2H), 7.73 – 7.18 (m, 14H), 3.67 (q, J = 6.9 Hz, 2H), 2.93 (t, J = 7.1 Hz,
2H). 13C NMR (75 MHz, CDCl3) δ 185.46, 161.38, 148.00, 142.21, 140.23, 138.39, 134.95, 130.35, 129.57,
129.02, 128.85, 128.81, 128.04, 127.94, 127.91, 127.24, 126.82, 118.96, 40.76, 35.59. HRMS [C24H21NO2 + H]+:
356.1645 calculated, 356.1641 found.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4-Methylphenethyl)-2-oxo-5-phenylpentanamide (43). The title compound was prepared according to
general procedure D using -ketoacid 60c (57 mg, 0.29 mmol, 1 eq), 2-(p-tolyl)ethan-1-amine (47 μL, 0.32
mmol, 1.1 eq), EDC·HCl (85 mg, 0.44 mmol, 1.5 eq), HOBt (60 mg, 0.44 mmol, 1.5 eq) in DCM. Column
1
chromatography (2.5% -> 20% EtOAc in pentane) afforded the product (18 mg, 58 mol, 20%). H NMR (400
MHz, CDCl3) δ 7.39 – 7.22 (m, 3H), 7.22 – 7.14 (m, 3H), 7.12 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 7.01 –
6.86 (m, 1H), 3.52 (q, J = 7.0 Hz, 2H), 2.93 (t, J = 7.3 Hz, 2H), 2.80 (t, J = 7.1 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.32
(s, 3H), 1.93 (p, J = 7.5 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 199.02, 160.11, 141.47, 136.44, 135.16, 129.56,
128.65, 128.61, 128.56, 126.21, 126.16, 40.68, 36.24, 35.13, 24.94, 21.18. HRMS [C20H23NO2 + H]+: 310.1802
calculated, 310.1803 found.
N-(4-Methoxyphenethyl)-2-oxo-5-phenylpentanamide (44). The title compound was prepared according to
general procedure D using -ketoacid 60c (89 mg, 0.46 mmol, 1 eq), 2-(4-methoxyphenyl)ethan-1-amine (75 l,
0.51 mmol, 1.1 eq), EDC·HCl (0.13 g, 0.69 mmol, 1.5 eq), HOBt (94 mg, 0.69 mmol, 1.5 eq) in DCM. Column
1
chromatography (2.5% -> 40% EtOAc in pentane) afforded the product (13 mg, 40 mol, 9%). H NMR (300
MHz, CDCl3) δ 7.32 – 7.24 (m, 2H), 7.23 – 7.14 (m, 3H), 7.10 (d, J = 8.6 Hz, 2H), 7.04 – 6.90 (m, 1H), 6.85 (d, J =
8.6 Hz, 2H), 3.79 (s, 3H), 3.50 (q, J = 6.9 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H), 2.65 (t, J = 7.6 Hz,
2H), 1.93 (p, J = 7.5 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 199.03, 160.10, 158.52, 141.45, 130.25, 129.75, 128.60,
128.54, 126.15, 114.27, 55.39, 40.77, 36.23, 35.14, 34.65, 24.94. HRMS [C20H23NO3 + H]+: 326.1751 calculated,
326.1752 found.
N-(3,4-Dimethoxyphenethyl)-2-oxo-5-phenylpentanamide (45). The title compound was prepared according
to general procedure D using -ketoacid 60c (57 mg, 0.30 mmol, 1 eq), 2-(3,4-dimethoxyphenyl)ethan-1-amine
(56 L, 0.33 mmol, 1.1 eq), EDC·HCl (86 mg, 0.45 mmol, 1.5 eq), HOBt (61 mg, 0.45 mmol, 1.5 eq) in DCM.
1
Column chromatography (2.5% -> 40% EtOAc in pentane) afforded the product (6 mg, 17 mol, 6%). H NMR
(400 MHz, CDCl3) δ 7.31 – 7.26 (m, 2H), 7.22 – 7.11 (m, 3H), 7.03 – 6.91 (m, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.77 –
6.66 (m, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 3.52 (q, J = 7.0 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H),
2.65 (t, J = 7.6 Hz, 2H), 1.93 (p, J = 7.5 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 199.04, 160.13, 149.01, 147.96,
141.44, 130.77, 128.56, 126.17, 120.73, 111.86, 111.53, 56.02, 40.69, 36.25, 35.16, 24.95. HRMS [C21H25NO4 +
H]+: 356.1856 calculated, 356.1858 found.
N-(4-Hydroxyphenethyl)-2-oxo-5-phenylpentanamide (46). The title compound was prepared according to
general procedure D using -ketoacid 60c (98 mg, 0.51 mmol, 1 eq), 4-(2-aminoethyl)phenol (77 mg, 0.56
mmol, 1.1 eq), EDC·HCl (0.15 g, 0.77 mmol, 1.5 eq), HOBt (0.10 g, 0.77 mmol, 1.5 eq) in DCM. Column
chromatography (10% -> 60% EtOAc in pentane) afforded the product (18 mg, 58 mol, 20%). 1H NMR (400
29
ACS Paragon Plus Environment