Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

Article abstract of DOI:10.1016/j.bmc.2016.02.040

Inhibition of group IIA secreted phospholipase A₂ (GIIA sPLA₂) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA₂s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC₅₀ = 143 nM and 68 nM against human and mouse GIIA sPLA₂, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity.

Full text of DOI:10.1016/j.bmc.2016.02.040

Accepted Manuscript
Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2
guided by molecular docking calculations and molecular dynamics simulations
Sofia Vasilakaki, Efrosini Barbayianni, Georgios Leonis, Manthos G.
Papadopoulos, Thomas Mavromoustakos, Michael H. Gelb, George Kokotos
PII:
S0968-0896(16)30130-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.040
BMC 12842
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 January 2016
24 February 2016
27 February 2016
Please cite this article as: Vasilakaki, S., Barbayianni, E., Leonis, G., Papadopoulos, M.G., Mavromoustakos, T.,
Gelb, M.H., Kokotos, G., Development of a potent 2-oxoamide inhibitor of secreted phospholipase A guided by
2
molecular docking calculations and molecular dynamics simulations, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.02.040
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Bioorganic & Medicinal Chemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m
Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2
guided by molecular docking calculations and molecular dynamics simulations
a
a
b
b
Sofia Vasilakaki , Efrosini Barbayianni , Georgios Leonis , Manthos G. Papadopoulos , Thomas
a
c
a,
Mavromoustakos , Michael H. Gelb and George Kokotos ∗
a
Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 15771, Greece
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece
b
c
Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
2 2
Inhibition of group IIA secreted phospholipase A (GIIA sPLA ) has been an important
objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-
oxoamide functionality may inhibit human and mouse GIIA sPLA s. Herein, the development of
2
new potent inhibitors by molecular docking calculations using the structure of the known
inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds
revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity
Available online
Keywords:
Docking
Inhibitors
Molecular dynamics
(
2
IC50 = 143 nM and 68 nM against human and mouse GIIA sPLA , respectively). In addition,
molecular dynamics simulations were employed to shed light on GK241 potent and selective
inhibitory activity.
2
016 Elsevier Ltd. All rights reserved.
2
-Oxoamides
Secreted phospholipase A
2
1
. Introduction
Phospholipases A (PLA ) are a superfamily of enzymes
The up-regulation or down-regulation of the expression of
these enzymes is related to pathological conditions, such as
atherosclerosis, cardiovascular disease and cancer, as it has
2
2
whose main role is the hydrolysis of the ester bond of the
membrane glycerophospholipids at the sn-2 position. The
5,6
been summarized in a number of recent reviews. As a
1
consequence, a variety of synthetic inhibitors of sPLA have
2
products of this enzymatic activity are mainly unsaturated fatty
acids which can act as bioactive mediators, correlating with
numerous pathological conditions. PLA2 enzymes are
categorized into six types according to their dependency on
calcium, their amino acid sequence domain, their molecular
been developed by pharmaceutical companies and research
1,7-9
institutes.
In particular, GIIA sPLA has been related to
2
10
inflammation since it was identified in 1989. GIIA sPLA is
2
highly expressed in synovial cells, while its concentration
increases in the plasma of patients who suffer from coronary
artery disease, making it a useful tool for prognostic purposes.
1
weight and their evolutionary relationship.
Approximately, one third of PLA enzymes belong to the
2
Many attempts for selective inhibition of GIIA sPLA have
2
secreted phospholipase (sPLA ) type, whose members are
11
2
been made with many positive outcomes. Varespladib (1a,
2
-4
characterized by a low-molecular weight. They can act either
by catalyzing reactions as enzymes or by binding to receptors
and hence, they are involved in the activation of several
Fig. 1), which is a potent but not selective GIIA sPLA2
inhibitor, was advanced into clinical trials as an intravenously-
administered
therapy
for
sepsis-induced
systemic
biological pathways. To date, 11 gene products of sPLA s (IB,
2
inflammatory response syndrome. Although it was found to
have an acceptable safety profile in patients with severe sepsis,
its development was terminated because Phase II studies
resulted in little efficacy. Also, Varespladib methyl (1b, Fig. 1)
which functions as a prodrug and is rapidly converted in vivo
to Varespladib, was synthesized as an attempt to improve
Varespladib efficacy. Both inhibitors were claimed by Anthera
Pharmaceuticals as new agents for the treatment of
cardiovascular diseases and underwent clinical trials from
IIA, IIC, IID, IIE, IIF, III, V, X, XIIA and XIIB) have been
identified in mammals. Their domains are highly stabilized by
six common disulfide bonds and additionally one or two that
are typical of each enzyme. Their active site contains the
His/Asp dyad that is utilized in the catalytic mechanism. Their
activation may require concentration of Ca in the mM range
and thus these proteins are localized in the extracellular space,
targeting both cellular glycerophospholipids and soluble lipids.
2
+
—
——
∗
Corresponding author. Tel.: +30-210-727-4462; fax: +30-210-727-4761; e-mail: gkokotos@chem.uoa.gr

various sPLA s are presented. Moreover, we report our results
2
from docking, molecular dynamics simulations and free energy
calculations of the complexes, in an attempt to understand their
structure-activity relationships.
2
2
. Results and discussion
.1. Design and docking of new 2-oxoamides
New 2-oxoamides were designed based on the structure of
the lead inhibitor 7 (Fig. 2). Proteinogenic • -amino acids of
both S and R configuration were used to replace the (S)-leucine
part of 7 and the resulted derivatives were docked in the active
2
3
site of GIIA sPLA using GOLD v5.2 program by CCDC.
2
The evaluation of the generated poses was mainly based on the
number of interactions they formed with the residues of the
active site upon binding. The • EClash
,
• EInternal
,
Chemscore.Internal.Correction.Weighted
and
Chemscore.Rot.Weighted penalty terms of the ChemScore
scoring function were also considered. The highly scored
penalty terms usually indicate poor geometry of the bound
inhibitors.
2
Figure 1. Common inhibitors of secreted PLA .
2
006 to 2012. However, on 2012 phase III clinical studies were
terminated due to lack of efficacy.
The expression of the full set of human and mouse groups I,
II, V, X, and XII sPLA s in Escherichia coli and insect cells
2
provided pure recombinant enzymes for detailed comparative
1
2
interfacial kinetic and binding studies. Among the class of
inhibitors of sPLA s based on substituted indoles, 6,7-
2
1
3,14
benzoindoles and indolizines,
(Fig. 1) was a selectively potent inhibitor against hGX over
all other human and mouse sPLA enzymes, while compound 3
it was found that compound
2
2
(
Fig. 1) inhibited nearly all human and mouse sPLA s in the
2
low nanomolar range. Another important class of sPLA2
inhibitors is amides based on non-natural amino acids.
Compound FPL67047XX (4, Fig. 1) was found to be a potent
Figure 2. The workflow of the development of new potential inhibitors
based on the inhibitor 7 guided by molecular docking calculations.
1
5
inhibitor of human platelet sPLA , and its precise binding
2
According to the docking results, the derivatives containing
-amino acids with R configuration did not generate favorable
interactions with the human nonpancreatic sPLA2 were
determined by high-resolution X-ray crystallography. The
structurally related compound 5 (Fig. 1) was co-crystallized
•
conformations. This, in combination with the low inhibitory
20
activity of similar derivatives previously reported led us to
with GIIA hnpsPLA and the crystal structure revealed a
2
discard these structures.
2
+
chelation to a Ca ion through its carboxylate and amide
oxygen atoms, H-bonding through an amide NH group to
His48, multiple hydrophobic contacts and a T-shaped aromatic
The derivatives containing non-polar • -amino acids with S
configuration frequently generated favorable binding modes
with comparatively good scoring values. Therefore, in order to
better understand this behavior, simulated annealing was
employed to generate 30 possible conformations for each of
these molecules, which subsequently were docked in GOLD.
By docking multiple conformations of each ligand, improved
statistical analysis of the docking results was achieved.
Indeed, the recurrence statistics of the desirable binding
motif of (S)-valine, (S)-alanine and (S)-proline derivatives was
high, according to their docking solutions. More specifically,
the carboxyl group of the derivatives chelates the calcium ion
and also forms a hydrogen bond with either Gly31 or Lys62
residues. In addition, the 2-carbonyl group or the oxygen atom
1
6
17
group - His6 interaction. In addition, compound GK115 (6,
Fig. 1) was found to inhibit GV sPLA (X (50) = 0.003 ±
2
I
0
.0004) without affecting the activities of intracellular GIVA
cPLA and GVIA iPLA . In a continuation of our studies on a
2
2
1
8,19
novel class of cPLA inhibitors,
the 2-oxoamide based on
20
2
the natural • -amino acid (S)-leucine GK126, (7, Fig. 1),
demonstrated a promising IC value of 0.30 µM against GIIA
5
0
2
0
sPLA2.
Computer-aided drug design has recently offered powerful
tools for the rational design of PLA inhibitors, raising the
2
potential for the development of new compounds with
improved inhibitor properties.
A
recent review article
2+
of the amide group point to Ca ion. A hydrogen bond is
demonstrates various applications of rational design on PLA
2
formed between Gly29 and the 2-carbonyl group in most cases
Supporting info). Similar results were generated when
derivatives of two non-natural amino acids ((S)-tert-leucine
and 2-aminoisobutyric acid) were docked. Moreover, some 2-
hydroxy amides were also docked and no significant
differences between the generated poses and the corresponding
2
1
inhibitors. For example, docking calculations have led to the
(
2
2
design of new indole inhibitors of sPLA2. The aim of this
work was to develop improved inhibitors of GIIA sPLA by
2
keeping the 2-oxoamide functionality of the lead 2-oxoamide
inhibitor 7 and altering its other structural features. Using
molecular docking simulations, we designed new 2-oxoamides
based on • -amino acids. Herein, the synthesis of new 2-
oxoamides derivatives and their in vitro activities against
2
-oxoamides ones were observed. The two (S)-Glu derivatives
were docked for comparison.

Synthesis and biological evaluation of the docked ligands
were accomplished. According to the experimental data, which
are discussed later, the (S)-valine derivative 16a (GK241)
demonstrated inhibitory activity against GIIA sPLA2.
Therefore, an effort to modify its structure, in order to succeed
a higher potency, was made. It is well established that when a
ligand has multiple conformations while unbound, then
narrowing down this number to the only favorable
conformations in the binding pocket, is entropically expensive.
Thus, the replacement of 16a long aliphatic chain by a shorter,
carrying an aromatic system (phenyl, biphenyl or naphthyl),
was a reasonable concept. New analogues were designed and
some of the most promising structures, according to the
docking simulations, were synthesized and tested.
Scheme 3. Reagents and conditions: (a) CH (CH ) CHOHCOOH (8),
3
2 13
WSCI·HCl, HOBt, Et N, CH Cl ; (b) Dess-Martin periodinane, CH Cl ;
3
2
2
2
2
3 2 2
(c) CF COOH, CH Cl .
2
.2. Synthesis of inhibitors
esters were then converted to alcohols 25a,b using the di-
isobutyl aluminum hydride (DIBAL-H) reducing agent,
followed by the NaOCl/AcNH-TEMPO oxidation method, to
form the corresponding aldehydes 26a,b. The synthesis of 31c
started from 5-phenyl-1-pentanol 25c, which was converted to
the corresponding aldehyde 26c, as it described above.
Cyanohydrin reaction followed by acid and then alkaline
conditions afforded the desirable • -hydroxy acids 28a-c. These
acids were coupled with (S)-valine, followed by oxidation and
The synthesis of 2-oxoamides and 2-hydroxyamides is
presented in Schemes 1-3. The protected (S)-• -amino acids 9a-
d and 13a-c were coupled with 2-hydroxyhexadecanoic acid
using
1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide
(WSCI·HCl) as a condensing agent in the presence of 1-
hydroxybenzotriazole (HOBt). The • -hydroxyamide methyl
esters 10a-d were saponified to produce the acids 11a-d.
Compounds 11b-d were then oxidized to the corresponding 2-
oxoamide acids 12b-d using Dess-Martin periodinane. In
addition, • -hydroxyamide tert-butyl esters 14a-c were first
oxidized using Dess-Martin reagent to afford 15a-c and
subsequently, the protecting group was removed by
trifluoroacetic acid to afford the desired 2-oxoamides 16a,c,d.
The synthesis of compound 21 is described in Scheme 3.
Scheme 1. Reagents and conditions: (a) WSCI·HCl, HOBt, Et
b) 1N aq. NaOH, MeOH; (c) Dess-Martin periodinane, CH Cl
3
N, CH
2
Cl ;
2
Scheme
OOCH=CHCH
THF; (c) DIBAL-H, Et
TEMPO, 0.5• NaOCl/NaHCO
M KCN; (f) (i) HCl conc. (ii) EtOH/H
WSCI·HCl, HOBt, Et N, CH Cl ; (i) Dess-Martin periodinane, CH
CF COOH, CH Cl
4.
Reagents
and
conditions:
(a)
(
2
2
.
C
2
H
5
2
P(=O)(OC
H
2 5
)
2
, LiOH·H
2
O, dry THF; (b) H
2
/Pd,
2
O; (d) NaBr/H
/• • ; (e) (i) NaHSO
O, KOH; (g) (S)-Val-OtBu,
Cl ; (j)
2
O, toluene/EtOAc, AcNH-
3
2
3
, CHCl (ii) H O,
3
2
6
2
3
2
2
2
2
3
2
2
.
Scheme 2. Reagents and conditions: (a) WSCI·HCl, HOBt, Et
b) Dess-Martin periodinane, CH Cl ; (c) CF COOH, CH Cl
3 2 2
N, CH Cl ;
(
2
2
3
2
2
.
The synthesis of 2-oxoamide (S)-valine analogues, where
the long chain has been replaced by a medium one carrying an
aromatic system, is depicted in Scheme 4. Horner-Wadsworth-
Emmons olefination of 1,1'-biphenyl-4-carbaldehyde and 2-
naphthaldehyde 22a,b led to esters 23a,b and subsequently
catalytic hydrogenation to saturated products 24a,b. The ethyl
Scheme 5. Reagents and conditions: (a) EtOAc/THF/80% CH
KCN; (b) (i) HCl conc. (ii) EtOH/H O, KOH; (c) (S)-Val-OtBu,
WSCI·HCl, HOBt, Et N, CH Cl ; (d) Dess-Martin periodinane, CH Cl
(e) CF COOH, CH Cl
3
COOH, 6M
2
3
2
2
2
2
;
3
2
2
.

removal of the protecting group to afford the final products
1a-c. The synthesis of 37 was accomplished by similar
methods (Scheme 5).
The lowest energy docking poses of 16a and 31c were
3
subjected to MD simulations in AMBER v12 molecular
2
5
dynamics package. Poses were selected so that the key
interactions between the ligands and the binding site to be
present. The total simulation time was 200 ns in explicit
solvent and RMSD calculations were performed with respect
to the starting coordinates of each complex.
2
.3. In vitro inhibition of GIIA sPLA2
The new 2-oxoamide derivatives were tested for inhibitory
activity against a panel of human and mouse sPLA enzymes,
2
In the initial poses, the inhibitor interacts with the calcium
ion via its carboxyl group and the 2-carbonyl group of the
amide (Fig. 3 & 4). During the first 20 ns of the simulation,
both molecules were reoriented within the active site in such a
way that the carboxylate oxygen atoms chelated the metal in
bidentate fashion (Fig. 3 & 4). This interaction lasted until the
end of the simulation of both complexes.
2
4
using a previously described continuous fluorometric assay.
None of the compounds 12b-d, 16c,d and 21 presented
inhibition of GIIA sPLA2 higher than 25% at 1 µM
concentration and thus we did not further study them.
However, the (S)-valine derivative 16a, exhibited high and
selective potency for inhibition of GIIA sPLA and the IC
2
50
values are summarized in Table 1. Compound 16a
demonstrates a two times higher potency for GIIA sPLA than
2
the previously reported inhibitor 7 and it is ten times more
selective for GIIA than GV sPLA . The IC values for
2
50
varespladib (1a, Fig. 1) using the same assay conditions, are
included in Table 1 for comparison.
Table 1. IC50 values of 1a and 16a on hGIIA, hGV, and mGIIA.
IC50 value (nM)
Compound
hGIIA
hGV
mGIIA
1
3,14
13,14
13,14
1
a
125 ± 20
500 ± 50
70 ± 20
68 ± 16
1
6a
143 ± 50
1200 ± 50
In addition, 16a does not exhibit any appreciable inhibition
against various other human and mouse sPLA (Table 2).
2
Table 2. Inhibitory activity of 16a on other sPLA
remaining activity
of the enzymes at
2
enzymes.
%
% remaining activity
Isoform
Isoform
of the enzymes at
1
000 nM
95
1000 nM
hGX
hGIB
hGIIE
hGIIF
mGX
mIID
mGV
98
99
90
98
93
94
Moreover, the lack of inhibitory activity of 2-hydroxyamide
derivatives (data not shown) indicates that indeed the 2-
oxoamide functionality has a crucial contribution to the
binding affinity of these compounds.
2
Table 3. Inhibitory activity of analogues of 16a on sPLA enzymes.
%
hGIB
20
Inhibition*
hGV
No
hGIIA
27
hGX
3
1a
26
50
40
53
51
54
40
56
3
1b
21
30
2
Figure 3. Conformational changes of the complex 16a-GIIA sPLA at the
3
1c
11
56
beginning (up) and at the end (down) of the MD simulation.
3
7
50
50
*
10 µM final concentration of the inhibitor.
During the MD simulation of 16a complex, two hydrogen
bonds are formed between the amide group and the residues
His47 and Gly29 (Table 4), resulting in the stable binding of
16a to the enzyme. The RMSD distribution further supports
the low mobility of the 16a functional groups, which allows
the formation of this H-bonds network (Fig. 5). However, the
2-oxoamide group of 31c does not form any H-bond with the
residues of the catalytic centre, during the simulation time
The analogues of 16a were also tested against several
sPLA s and the results are summarized in Table 3. The in vitro
activities of 31a-c and 37 failed to meet our docking
predictions. Molecular dynamics simulations were applied on
1
2
6a and on one of its analogues in an attempt to understand the
in vitro data.
(
Table 4). Due to conformational changes of 31c, the 2-
2
.4. Molecular dynamics simulations
oxoamide group moves away from the catalytic centre and
periodically interacts only with the solvent molecules.

The fluctuations of the RMSD values manifest the high
mobility of the 31c in the active site (Fig. 5). The RMSD
values were calculated for the part of the molecules which
form the key interactions and consists of the C• atom of (S)-
valine, the two carbon atoms of the 2-oxoamide moiety and the
nitrogen atom of the amide.
Using the MD trajectories, free energy calculations were
performed with the Molecular Mechanics / Poisson-Boltzmann
Surface Area method (MM-PBSA) (Table 5). Frames from the
second half of the simulation were used for the calculations.
Analysis of the energetic terms for each complex, revealed a
high contribution from van der Waals forces.
Table 5. Energetic analysis for 16a-GIIA sPLA
2
and 31c-GIIA sPLA
2
complexes.
*
Energy
1
6a
31c
-
1
(
kcal·mol )
∆
E
vdW
elec
-36.86 ± 4.34
-515.69 ± 12.53
519.78 ± 11.93
-4.90 ± 0.19
-30.12 ± 3.85
∆
∆
∆
E
-501.60 ± 10.45
506.27 ± 9.42
-3.76 ± 0.10
G
PB
G
NP
∆
E
G
solv
514.88 ± 11.88
-552.55 ± 12.51
-37.67 ± 5.16
20.09 ± 9.03
502.51 ± 9.407
-531.73 ± 8.69
-29.21 ± 4.62
22.59 ± 11.10
-6.62 ± 1.81
∆
∆
-
MM, gas
H
(MM+solv)
T·∆Stot
∆
G
MM-PBSA
-17.58 ± 3.37
*
Errors represent the standard deviation error out of the total number of
trajectory snapshots used in MM-PBSA calculations.
1
6a creates two additional H-bonds within the cavity of the
enzyme compared to 31c, which is reflected on the difference
between the • E electrostatic term of the two complexes (Table
5
). Moreover, the entropic term does not greatly differ between
the two complexes. Partly, this could be due to the fact that
both molecules are similarly flexible. Interestingly, in the case
of 16a the overall flexibility of the compound does not
increase the mobility of the 2-oxoamide group, which creates
the main interactions with the residues. The difference in
binding energies between 16a and 31c is 10.96 kcal·mol . This
indicates that 16a complex formation is more favored over 31c
complex formation, in agreement with the in vitro results.
2
Figure 4. Conformational changes of the complex 31c-GIIA sPLA at the
beginning (up) and at the end (down) of the MD simulation.
-
1
Table 4. Main H-bond interactions between the ligands and the residues of
the GIIA sPLA active site. The numbers describe the H-bond distance and
2
the occurrence as the percentage of simulation time that the interaction
exists.
3
. Conclusion
Structural modifications were applied on the GIIA sPLA2
1
6a
31c
-
-
inhibitor 7 and the new compounds were docked using GOLD.
The results suggested that the non-polar • -amino acids
derivatives with S configuration could generate favorable
conformations. Their in vitro evaluation revealed that the 2-
oxoamide inhibitor based on (S)-valine 16a is a potent and
COO group
.05 Å
2%
NHCO group
.9 Å
1%
COO group
3
2.94 Å
75.8%
5
GLY29
HIS47
2
selective inhibitor of GIIA sPLA . Further attempts to optimize
2
5
the 16a activity by altering its structure, did not lead to any
desirable outcomes.
Thus, molecular dynamics simulations were used in order to
understand how the replacement of the long aliphatic chain of
NHCOCO group
3.18 Å
3
0.47%
1
6a resulted in the decrease of its potency. It was suggested
that the aliphatic chain, which creates van der Waals
interactions, adopts a conformation that keeps the 2-oxoamide
moiety close to the key residues of the catalytic centre.
Subsequently, the 2-oxoamide moiety forms a permanent H-
bond network which further stabilize the 16a binding. The
replacement of the long aliphatic chain by a four C atoms
chain carrying a phenyl ring led to an increase of 2-oxoamide
group’s mobility and the loss of the H-bonding interactions it
crated with the active side. Although its carboxylic group
Figure 5. RMSD for the 16a and 31c.
2
+
chelates the Ca ion and creates an H-bond with Gly29, these

interactions seems to be not enough to keep the derivative
bound to the enzyme.
4.1.3. Molecular dynamics simulations
Further work will be needed to test the in vivo potency of
6a. Molecular dynamics simulations are a useful tool for
explaining and evaluating the experimental data and should be
used towards this direction.
MD simulations in explicit solvent were performed using
2
5
1
AMBER. The Antechamber tool was used to generate the
33
partial atomic charges of each ligand with the AM1-BCC
method. Modification of the force field parameters of each
structure was made by Parmchk program. The AMBER force
4
4
4
. Experimental section
34
35
field ff12SB and the general AMBER force field (GAFF)
were loaded in tLEaP module to create the parameters and
topologies of the protein and the organic molecule,
respectively. The complex was loaded in tLEaP along with the
parameters file of calcium ions, which were created by a
.1. Computational methods
.1.1. Preparation of ligands’ structures
The preparation of the structures was accomplished using
1
6
combination of crystallographic data, GAFF force field
3
5
36
parameters and the Aqvist et al. method. Disulfide bonds
between Cys83-Cys59, Cys124-Cys49, Cys117-Cys26, Cys50-
Cys90, Cys28-Cys44, Cys43-Cys97 and Cys88-Cys77 were
assigned, as well as the coordination between the metal ion of
the catalytic centre and the residues His27, Gly29 and Gly31.
The complex was then immersed in a truncated octahedral
water box using the TIP3P water model with 12.0 Å periodic
boundary conditions. Finally, the system was neutralized by
2
6
SYBYL v8.0. The charges were defined at physiological pH
and the or configuration was assigned. Several
S
R
minimization steps were performed to accomplish optimization
of the structures, using initially the steepest descent algorithm
for 300 iteration steps followed by the conjugate gradients
method for another 300 iteration steps and finally the Powell
2
7
28
algorithm for 700 iteration steps. The Tripos force field was
applied during optimization and each method was terminated
-
adding 18 Cl counter ions using the Joung and Cheatham ion
-
1
-1
3
7
when a convergence of 0.05 kc ·aml ol ·Å was reached. For
parameters and its coordinate and parameter topology files
were saved.
2
9
the Powell algorithm, the simplex method was used for its
initial optimization.
Prior to the MD simulation, an initial minimization was
performed using 1500 iteration steps of steepest descent and
3
0
The simulated annealing method was used in order to
sample 30 relevant conformations for each structure.
Molecules were heated at 600 K for 1500 fs and annealed at
3
8
1
500 iteration steps of conjugate gradient in SANDER,
keeping the complex almost fixed with a restraint set to 400
-
1
-1
2
50 K for 2000 fs. The Tripos force field was used and the
kcal·mol ·Å . In addition, four sets of steepest descent and
conjugate gradient steps were applied to the system, with the
non-bonded cutoff was set to 8.0 Å. The structures produced
were optimized using 200 iteration steps with the Powell
-
strength of the restraint being gradually reduced to 8 kca· lm ol
-
1
-1
1
-1
algorithm with a gradient of 0.05 kc a· ml ol ·Å . The simplex
method was used as well.
·
Å . The system was then smoothly heated from 0 to 300 K in
three steps under constant volume. During these steps of total
-
1
-1
time 100 ps, the restraint was retained to 8 k ·cma ol l
·Å ,
4
.1.2. Docking
hydrogen atoms were constrained using the SHAKE algorithm
and the Langevin thermostat was used for temperature control.
Two steps of equilibration were applied keeping the restraint to
The atomic coordinates of the human secreted
-
1
-1
phospholipase group IIA protein were downloaded from
8 kcal·mol ·Å for the first 50 ps and then removed it for the
next 50 ps, under constant pressure. MD simulations were
3
1
16
Protein Data Bank (PDB code: 1KQU). Missing loops were
32
39
added with the Prime module of Schrödinger Suite.
performed with the pmemd.cuda implementation for 200 ns
Hydrogen atoms were added in GOLD and atoms types were
automatically assigned by the program.
and a 8 Å nonbonded cutoff was applied. SHAKE was kept
during the production runs to allow the use of 2 fs time step,
and the long-range electrostatic interactions were accounted for
Docking simulations were performed in GOLD. The cavity
4
0
of the catalytic centre of GIIA sPLA was oriented within 5 Å
using the particle mesh Ewald (PME) method. Analysis on
the produced trajectories (RMSD, distances, H-bond
interactions) was performed with the ptraj analysis tool
2
around the crystallographic ligand including Leu2, Phe5, His6,
Gly29, Gly31, His47, Asp48, Lys52, Lys62 and Asp91 amino
acids. Eight crystal water molecules in the spatial vicinity to
the catalytic center were kept and assigned as toggle and spin,
while the rest were deleted. His6 was assigned to have flexible
side chain (1 rotamer). H-bond donors / acceptors were treated
as solvent accessible. The options ‘flip amide bonds and ring
corners’ were set on for ligand flexibility. The ChemScore
scoring function (1) along with chemscore.p450_csd.params
parameters file were used to rank the generated binding modes.
4
1
available in the AmberTools13.
The relative binding energies of each system (Table 5) were
4
2
estimated using the MM-PBSA. The • Eelect term is the
electrostatic interaction energy and the • EvdW term is the van
der Waals interaction energy.
4
3
The Poisson-Boltzmann (PB) model was used for the
estimation of the electrostatic contribution to solvation
energies (• G ) and the solvent-accessible surface area
PB
(SASA) was used for the estimation of the non electrostatic
ChemScore = - (• G + • E(clash) + • E(int)) (1)
contributions (• GNP). The SASA is calculated by the following
equation:
where • G is the free energy change upon ligand binding, • E
(
clash) is the protein-ligand clash penalty and • E (int) is the
•
GNP = γSASA + β
(2)
ligand internal torsion strain penalty. It was established via
additional computational experiments that this combination of
scoring function and parameters were suitable for the docking.
Internal ligand energy offset was enabled and the preset
genetic algorithm parameter settings were used and set to
where the SASA term is the surface area of the solute, the γ
-
1
-2
-
term is 0.00542 kca ·l mol ·Å while β is -1.008000 kcal·mol
1
44
.
The γ and β are constants related with the surface tension
coefficient and the offset, respectively.
1
00,000 operations.

1
3
The entropic term T·• S was calculated using the nmode
program available in AMBER. The metal ions were removed
during the calculation (strip_mask) to avoid misleading results.
(t, J = 6.4 Hz, 3H, CH ); C NMR (CDCl , 50 MHz): • 173.6,
3 3
172.4, 81.9, 69.5, 59.4, 59.1, 56.7, 52.5, 46.7, 45.6, 34.4, 32.7,
32.1, 29.9, 29.7, 29.6, 29.2, 28.9, 25.3, 24.9, 22.9, 14.3; MS
ESI) m/z (%): 384.4 (100) [M + H] ; Anal. Calcd for
+
(
C H NO : C, 68.89; H, 10.77; N, 3.65. Found: C, 68.63; H,
4
.2. Chemistry
22 41
4
1
0.84; N, 3.56.
Merck Silica Gel 60 (70-230 or 230-400 mesh) z d v#x vh g #i r u#
wk h#fkur p d wr j udsklf#sxulilfd wl r q#r i#sur gxf wv #dqg#Vlolfd#J ho#93#
I 587# doxp lqxp # sod wh v# ir u# wk h# wk lq-od| hu# f k ur p d wr j uds k | #
4
.2.1.3. (2S)-Methyl
2-(2-hydroxyhexadecanamido)-3,3-
dimethylbutanoate (mixture of diastereomers) (10c). Yield
o
20
8
4%; White solid; mp 73-75 C; [• ] 2.30 (c 1.00, CHCl3);
D
+
WO F , 1# XY#olj k w# dqg2r u#s kr vs kr p r o| eglf #df lg#lq#H wR K#z dv#
1
H NMR (CDCl , 200 MHz): • 7.29-7.07 (m, 1H, NH), 4.47-
3
hp s or | hg#ir u#ylvxdol}lqj #vs r wv 1# D#E˙ f kl#530 apparatus was
1
4.37 (m, 1H, NHCH), 4.21-4.04 (m, 1H, CHOH), 3.70 (s, 3H,
CH ), 1.93-1.70 (m, 2H, CH CHOH), 1.52-1.16 (m, 24H,
CH ), 0.96 (s, 9H, CH ), 0.85 (t, J = 6.8 Hz, 3H, CH );
used to estimate melting points and they were uncorrected. H
1
3
3
2
and C NMR spectra were recorded on Varian Mercury at 200
MHz or 300 MHz and 50 MHz respectively. Samples were
diluted in CDCl , CD OD or DMSO. Chemical shifts are given
13
C
2
3
3
NMR (CDCl , 50 MHz): • 174.0, 172.1, 171.7, 72.2, 71.9,
3
3
3
5
2
9.5, 59.4, 51.7, 34.7, 31.8, 29.6, 29.3, 29.2, 26.4, 25.0, 24.9,
2.6, 14.0; MS (ESI) m/z (%): 400.2 (100) [M + • ] ; Anal.
+
in ppm, and coupling constants (J) in Hz. Peak multiplicities
are typified as: s, singlet, d, doublet, t, triplet and m, multiplet.
Electron spray ionization (ESI) mass spectra were recorded on
a Finnigan Surveyor MSQ Plus spectrometer. Specific
rotations of the compounds were measured at 25 ºC on a
Calcd for C H NO : C, 69.13; H, 11.35; N, 3.51. Found: C,
2
3
45
4
6
9.05; H, 11.51; N, 3.32.
4
.2.1.4. Methyl
2-(2-hydroxyhexadecanamido)-2-
methylpropanoate (racemic mixture) (10d). Yield 54%;
Perkin-Elmer 343 polarimeter using
a
10 cm cell.
o
20
1
White solid; mp 53-56 C; [• ]
NMR (CDCl , 200 MHz): • 7.13 (s, 1H, NH), 4.09-3.93 (m,
2.35 (c 1.02, CHCl ); H
Dichloromethane, diethylether and toluene were dried by
standard procedures and stored over molecular sieves. No
further purification of other solvents and chemicals needed as
they were reagent grade. HRMS spectra were recorded on a
Bruker Maxis Impact QTOF Spectrometer.
D
3
3
1
1
2
H, CH• • ), 3.72 (s, 3H, COOCH ), 3.53 (br, 1H, OH), 1.84-
3
.58 (m, 2H, CH CHOH), 1.53 (s, 6H, CH ), 1.34-1.10 (m,
2
3
1
3
4H, CH ), 0.86 (t, J = 6.8 Hz, 3H, CH ); C NMR (CDCl , 50
2
3
3
4
5
45
46
46
47
48
MHz): • 174.9, 173.7, 71.8, 55.9, 52.5, 34.5, 31.8, 29.6, 29.4,
9.3, 24.8, 24.6, 22.6, 14.0; MS (ESI) m/z (%): 372.2 (100) [M
Compounds 23b, 24b, 25a, 26a, 25b, 26b have
been described elsewhere and their analytical data are in
accordance with literature.
2
+
+
• ] ; Anal. Calcd for C H NO : C, 67.88; H, 11.12; N, 3.77.
21 41 4
Found: C, 67.66; H, 11.29; N, 3.72.
4
.2.1. Coupling method. To
a
stirred solution of
4.2.1.5. (2S)-tert-Butyl
2-(2-hydroxyhexadecanamido)-3-
hydrochloride amino component (1.0 mmol) in CH Cl (10
methylbutanoate (mixture of diastereomers) (14a). Yield
2
2
o
20
mL), Et N (0.3 mL, 2.2 mmol) and subsequently 1-(3-
62%; White solid; mp 49-53 C; [• ] 9.80 (c 1.01, CHCl3);
3
D
1
dimethyl-aminopropyl)-3-ethyl carbodiimide hydrochloride
H NMR (CDCl , 200 MHz): • 7.17 (d, J = 9.0 Hz, ½H, NH),
3
(
WSCI·HCl) (0.21 g, 1.1 mmol) and 1-hydroxybenzotriazole
7.05 (d, J = 9.0 Hz, ½H, NH), 4.46-4.31 (m, 1H, CH), 4.18-
4.03 (m, 1H, CHOH), 4.00 (d, J = 4.8 Hz, ½H, OH), 3.83 (d, J
= 4.8 Hz, ½H, OH), 2.24-2.03 (m, 1H, NHCHCH), 1.90-1.52
(m, 2H, CH CHOH), 1.44 (s, 9H, CH ), 1.34-1.13 (m, 24H,
(
HOBt) (0.14 g, 1.0 mmol) were added at 0˚C. The acid
reactant (1.0 mmol) was added and the reaction mixture was
stirred for 1 h at 0˚C and then overnight at room temperature.
After the completion of the reaction, the solvent was
evaporated under reduced pressure and EtOAc (20 mL) was
added. The organic layer was washed consecutively with brine,
2
3
1
3
CH ), 0.97-0.75 (m, 9H, CH ); C NMR (CDCl , 50 MHz): •
2
3
3
174.1, 174.0, 171.3, 170.9, 82.0, 81.9, 72.2, 71.9, 57.0, 56.8,
34.8, 31.8, 31.3, 31.2, 29.6, 29.5, 29.3, 27.9, 25.0, 24.8, 22.6,
18.9, 18.8, 17.5, 17.4, 14.0; MS (ESI) m/z (%): 428.3 (95) [M
1
N HCl, brine, 5% NaHCO and brine, dried over Na SO and
3
2
4
+
evaporated under reduced pressure. Purification by flash
column chromatography eluting with the appropriate mixture
of EtOAc/petroleum ether (bp 40-60 °C) afforded the product.
+ • ] ; Anal. Calcd for C H NO : C, 70.21; H, 11.55; N, 3.28.
2
5
49
4
Found: C, 70.03; H, 11.70; N, 3.19.
4
.2.1.6. (2S)-di-tert-Butyl 2-(2-hydroxyhexadecanamido)
pentanedioate (mixture of diastereomers) (14b). Yield 76%;
4
.2.1.1. (2S)-Methyl 2-(2-hydroxyhexadecanamido)-3-(1H-
2
0
1
Yellowish oil; [• ] 8.1 (c 1.00, CHCl ); H NMR (CDCl ,
D
3
3
indol-3-yl)propanoate (mixture of diastereomers) (10a).
Yield 52%; White solid; mp 78-85 C; [• ]
CHCl ); H NMR (CDCl , 200 MHz): • 8.22 (s, 1H, NH),
7
5
3
1
o
20
200 MHz): • 7.24-7.06 (m, 1H, NH), 4.61-4.38 (m, 1H,
NHCH), 4.22-4.02 (m, 1H, CHOH), 3.61 (br, 1H, OH), 2.44-
41.1 (c 1.00,
D
1
3
3
1
2
.71 (m, 6H, CH ), 1.53-1.38 (m, 18H, CH ), 1.36-1.17 (m,
2 3
13
.61-7.50 (m, 1H, arom), 7.43-6.88 (m, 5H, arom & NH),
.08-4.88 (m, 1H, NHCH), 4.14-3.97 (m, 1H, CHOH), 3.81-
.67 (m, 3H, COOCH ), 3.34 (d, J = 5.6 Hz, 2H, NHCHCH ),
4H, CH ), 0.97-0.78 (m, 3H, CH ); C NMR (CDCl , 50
2
3
3
MHz): • 174.2, 174.0, 172.0, 170.8, 82.3, 82.2, 80.7, 71.9,
1.7, 34.7, 31.8, 31.5, 29.6, 29.3, 29.2, 27.9, 27.6, 24.9, 22.6,
3
2
5
.87-1.64 (m, 2H, CH CHOH), 1.62-1.15 (m, 24H, CH ), 0.89
2
2
+
1
3
14.0; MS (ESI) m/z (%): 514.6 (90) [M + H] ; Anal. Calcd for
C H NO : C, 67.80; H, 10.79; N, 2.73. Found: C, 67.62; H,
(t, J = 6.6 Hz, 3H, CH3); C NMR (CDCl , 50 MHz): • 173.6,
3
2
9
55
6
1
1
2
72.3, 169.0, 168.4, 135.9, 122.7, 122.2, 119.6, 119.5, 118.5,
18.3, 111.3, 109.8, 80.6, 72.0, 52.4, 34.6, 31.9, 29.6, 29.5,
1
0.88; N, 2.67.
9.4, 29.3, 27.6, 24.9, 22.6, 14.1; MS (ESI) m/z (%): 473.3
4
.2.1.7. (2S)-tert-Butyl
2-(2-hydroxyhexadecanamido)
+
(100) [M + H] ; Anal. Calcd for C H N O : C, 71.15; H,
2
8
44
2
4
propanoate (mixture of diastereomers) (14c). Yield 40%;
9
.38; N, 5.93. Found: C, 71.03; H, 9.45; N, 5.87.
20
1
Colorless oil; [• ] 7.7 (c 0.99, CHCl ); H NMR (CDCl , 200
D
3
3
MHz): • 7.22-7.04 (m, 1H, NH), 4.56-4.35 (m, 1H, NHCH),
.17-4.02 (m, 1H, CHOH), 3.45 (br, 1H, OH), 1.93-1.55 (m,
2H, CH CHOH), 1.45 (s, 9H, CH ), 1.36 (d, J = 7.0 Hz, 3H,
4
.2.1.2. (2S)-Methyl 1-(2-hydroxyhexadecanoyl) pyrrolidine
4
-2-carboxylate (mixture of diastereomers) (10b). Yield 64%;
o
20
1
2
3
White solid; mp 42-55 C; [• ]
NMR (CDCl , 200 MHz): • 4.75-4.43 (m, 1H, NCH), 4.29-
4
NCH ), 3.06 (br, 1H, OH), 2.57-1.74 (m, 6H, NCH CH CH &
CH CHOH), 1.73-1.42 (m, 4H, CH ), 1.23 (s, 20H, CH ), 0.85
-50.0 (c 1.00, CHCl ); H
D
3
NHCHCH ), 1.32-1.18 (s, 24H, CH ), 0.86 (t, J = 6.4 Hz, 3H,
3
2
3
13
CH3); C NMR (CDCl , 50 MHz): • 173.9, 173.7, 172.3,
3
.14 (m, 1H, CHOH), 3.71 (s, 3H, CH ), 3.62-3.44 (m, 2H,
3
1
2
72.0, 81.9, 71.9, 48.1, 34.7, 34.6, 31.8, 29.6, 29.5, 29.4, 29.3,
9.2, 27.8, 24.9, 22.6, 18.4, 14.0; MS (ESI) m/z (%): 400.4
2
2
2
2
2
2
2

+
(
100) [M + H] ; Anal. Calcd for C H NO : C, 69.13; H,
136.7, 133.1, 133.0, 128.5, 128.3, 128.0, 127.5, 126.2, 126.1,
2
3
45
4
1
1.35; N, 3.51. Found: C, 68.91; H, 11.46; N, 3.43.
126.0, 125.9, 124.0, 123.9, 82.1, 74.3, 74.1, 57.3, 57.1, 31.3,
-
2
;
7.8, 18.7, 17.4, 17.2; MS (ESI) m/z (%): 356.2 (100) [M - H]
Anal. Calcd for C H NO : C, 70.56; H, 7.61; N, 3.92.
4
.2.1.8. (2S)-5-tert-Butyl 1-ethyl-2-(2-
2
1
27
4
hydroxyhexadecanamido)pentanedioate
diastereomers) (19). Yield 53%; Yellow oil; [• ]
1
1
&
(mixture
of
5.80 (c
Found: C, 70.38; H, 7.70; N, 3.86.
2
0
D
1
.00, CHCl ); H NMR (CDCl , 200 MHz): • 7.33-7.19 (m,
4.2.2. Saponification of methyl esters. To a stirred solution of
a methyl ester (1.0 mmol) in water, 1N NaOH (1 mL, 1.0
mmol) was added and the mixture was left overnight at room
temperature. After the completion of the reaction, the mixture
was washed with EtOAc, acidified with 1N HCl to pH 1 and
extracted with EtOAc (3 x 10 mL). The combined organic
layers were washed with brine, dried over Na SO and
3
3
H, NH), 4.55-4.38 (m, 1H, NHCH), 4.18-3.95 (m, 3H, CHOH
COOCH ), 2.33-1.74 (m, 6H, CH & CH CHOH), 1.34 (s,
2
2
2
9
3
1
2
H, CH ), 1.29-1.07 (m, 27H, CH & CH ), 0.76 (t, J = 6.8 Hz,
3
2
3
1
3
H, CH3); C NMR (CDCl , 50 MHz): • 174.6, 174.4, 171.8,
3
71.5, 80.6, 71.8, 61.4, 51.1, 34.5, 34.5, 34.4, 31.7, 31.2, 29.4,
9.2, 29.1, 27.8, 27.1, 24.8, 24.7, 22.4, 13.9; MS (ESI) m/z
2
4
+
(%): 486.4 (100) [M + H] ; Anal. Calcd for C H NO : C,
evaporated under reduced pressure.
2
7
51
6
6
6.77; H, 10.58; N, 2.88. Found: C, 66.48; H, 10.72; N, 2.78.
4
.2.2.1. (2S)-2-(2-Hydroxyhexadecanamido)-3-(1H-indol-3-
4
.2.1.9. (2S)-tert-Butyl 2-(2-hydroxy-6-(naphthalen-2-yl)
yl) propanoic acid (mixture of diastereomers) (11a). Yield
hexanamido)-3-methylbutanoate
diastereomers) (29a). Yield 73%; Colorless oil; [• ] 10.1 (c
(mixture of
o
20
8
0%; White solid; mp 144-146 C; [• ] 18.7 (c 1.00, MeOH);
D
2
0
1
D
H NMR (CD OD, 200 MHz): • 7.63-7.51 (m, 1H, NH), 7.36-
1
3
1
3
7
4
.00, CHCl ); H NMR (CDCl , 200 MHz): • 7.86-7.68 (m,
3 3
7
1
.26 (m, 1H, arom), 7.15-6.92 (m, 3H, arom), 4.82-4.67 (m,
H, NHCH), 4.01-3.88 (m, 1H, CHOH), 3.44-3.19 (m, 2H,
H, arom), 7.63-7.54 (m, 1H, arom), 7.51-7.24 (m, 3H, arom),
.08 (d, J = 8.8 Hz, ½H, NH), 6.91 (d, J = 8.8 Hz, ½H, NH),
.51-4.39 (m, 1H, NHCH), 4.21-4.06 (m, 1H, CHOH), 3.02
NHCHCH ), 1.77-1.50 (m, 2H, CH CHOH), 1.48-1.12 (m,
2
2
13
2
5
1
2
4H, CH ), 0.89 (t, J = 6.6 Hz, 3H, CH ); C NMR (CD OD,
2
3
3
(br, 1H, OH), 2.78 (t, J = 7.4 Hz, 2H, CH ), 2.27-2.06 (m, 1H,
2
0 MHz): • 177.0, 175.0, 142.0, 128.9, 124.5, 122.4, 119.9,
19.4, 112.2, 110.4, 72.5, 53.3, 35.5, 33.1, 30.8, 30.5, 28.5,
5.9, 23.8, 14.5; HRMS (ESI) calcd for C H N NaO [M +
NHCHCH), 2.01-1.64 (m, 4H, CH ), 1.62-1.40 (m, 11H, CH
&
•
1
5
1
2
2
1
3
CH ), 0.98-0.80 (m, 6H, CH ); C NMR (CDCl , 50 MHz):
3
3
3
2
7
42
2
4
173.9, 173.8, 171.3, 170.9, 139.8, 133.5, 131.8, 127.7, 127.5,
27.3, 127.2, 126.2, 125.7, 124.9, 82.1, 82.0, 72.0, 71.8, 57.0,
6.8, 35.8, 35.8, 34.7, 34.6, 31.3, 31.2, 31.0, 27.9, 24.6, 18.9,
+
Na] : 481.3037. Found: 481.3057; Anal. Calcd for
C H N O : C, 70.71; H, 9.23; N, 6.11. Found: C, 70.59; H,
9
2
7
42
2
4
.35; N, 6.01.
-
8.8, 17.5, 17.3; MS (ESI) m/z (%): 412.2 (100) [M - H] ;
Anal. Calcd for C H NO : C, 72.61; H, 8.53; N, 3.39. Found:
C, 72.38; H, 8.69; N, 3.20.
4.2.2.2. (2S)-1-(2-Hydroxyhexadecanoyl)pyrrolidine-2-
carboxylic acid (mixture of diastereomers) (11b). Yield
2
5
35
4
o
20
1
8
0%; White solid; mp 71-74 C; [• ] -5.2 (c 1.02, CHCl ); H
D
3
4
.2.1.10. (2S)-tert-Butyl
2-(6-([1,1'-biphenyl]-4-yl)-2-
(mixture of
NMR (CDCl , 200 MHz): • 4.82-4.38 (m, 1H, NCH), 4.37-
3
hydroxyhexanamido)-3-methylbutanoate
diastereomers) (29b). Yield 68%; Colorless oil; [• ] 10.8 (c
1
1
4
4
.08 (m, 1H, CHOH), 3.82-3.30 (m, 2H, NCH ), 2.56-1.76 (m,
2
0
2
D
H, NCHCH CH ), 1.73-1.00 (m, 26H, CH ), 0.86 (t, J = 6.8
1
2
2
2
.00, CHCl ); H NMR (CDCl , 200 MHz): • 7.64-6.90 (m,
13
3
3
Hz, 3H, CH3); C NMR (CDCl , 50 MHz): • 174.6, 167.8,
3
0H, arom & NH), 4.47 (dd, J = 9.0 Hz, J = 4.6 Hz, 1H,
1
2
8
2
1.5, 69.5, 59.5, 56.4, 46.7, 45.4, 33.9, 32.3, 31.8, 29.5, 29.4,
NHCH), 4.25-4.05 (m, 1H, CHOH), 3.72-3.33 (m, 1H, OH),
9.3, 28.9, 28.4, 25.0, 24.5, 22.6, 22.1, 14.0; HRMS (ESI)
2
1
.67 (t, J = 7.4 Hz, 2H, CH ), 2.31-2.08 (m, 1H, NHCHCH),
+
2
calcd for C H NNaO [M + Na] : 392.2771. Found:
1
3
21 39
4
.98-1.34 (m, 15H, CH & CH ), 1.00-0.81 (m, 6H, CH );
C
2
3
3
3
3
92.2785; Anal. Calcd for C H NO : C, 68.25; H, 10.64; N,
.79. Found: C, 68.14; H, 10.73; N, 3.65.
21 39 4
NMR (CDCl , 50 MHz): • 174.0, 171.3, 170.9, 141.4, 141.0,
3
1
3
38.5, 128.8, 128.6, 126.8, 82.1, 82.0, 72.0, 71.8, 57.0, 56.8,
5.3, 34.6, 31.3, 31.2, 31.1, 27.9, 24.6, 18.9, 18.8, 17.5, 17.4;
4.2.2.3. (2S)-2-(2-Hydroxyhexadecanamido)-3,3-
-
MS (ESI) m/z (%): 438.4 (100) [M - H] ; Anal. Calcd for
C H NO : C, 73.77; H, 8.48; N, 3.19. Found: C, 73.55; H,
dimethylbutanoic acid (mixture of diastereomers) (11c).
o
20
2
7
37
4
Yield 80%; White solid; mp 99-101 C; [• ]
D
1.8 (c 1.00,
1
8
.65; N, 3.14.
CHCl ); H NMR (CDCl , 200 MHz): • 7.52-7.01 (m, 1H,
3
3
NH), 4.51-4.25 (m, 1H, NHCH), 4.24-4.03 (m, 1H, CHOH),
.92-1.11 (m, 26H, CH ), 0.96 (s, 9H, CH ), 0.89-0.72 (m, 3H,
4
.2.1.11. (2S)-tert-Butyl 2-(2-hydroxy-6-phenylhexanamido)
1
2
3
-3-methylbutanoate (mixture of diastereomers) (29c). Yield
13
CH3); C NMR (CDCl , 50 MHz): • 175.5, 174.6, 174.4, 74.5,
2
0
1
3
9
2%; Colorless oil; [• ]
8.38 (c 0.99, CHCl ); H NMR
D
3
5
2
9.8, 34.6, 34.5, 31.9, 29.7, 29.6, 29.5, 29.3, 26.5, 25.1, 24.5,
2.6, 14.1; MS (ESI) m/z (%): 386.2 (100) [M + H] ; Anal.
(
CDCl , 200 MHz): • 7.33-6.96 (m, 6H, arom & NH), 4.42
+
3
(dd, J = 9.0 Hz, J = 4.6 Hz, 1H, NHCH), 4.21-4.03 (m, 1H,
1
2
Calcd for C H NO : C, 68.53; H, 11.24; N, 3.63. Found: C,
2
2
43
4
CHOH), 3.72 (br, 1H, OH), 2.61 (t, J = 7.4 Hz, 2H, CH2),
2
9
MHz): • 174.1, 174.0, 171.3, 170.8, 142.3, 128.2, 128.1, 125.5,
8
1
6
8.44; H, 11.35; N, 3.52.
.30-2.06 (m, 1H, NHCHCH), 1.96-1.52 (m, 6H, CH ), 1.47 (s,
2
13
H, CH ), 0.97-0.82 (m, 6H, CH ); C NMR (CDCl , 50
4.2.2.4. 2-(2-Hydroxyhexadecanoylamino)-2-
3
3
3
methylpropanoic acid (racemic mixture) (11d). Yield 72%;
•
20
D
1
2.0, 81.9, 71.9, 71.8, 56.9, 56.7, 35.7, 34.5, 31.1, 27.9, 24.6,
White solid; mp 81-83 C; [• ] 1.7 (c 1.02, MeOH); H NMR
-
8.8, 17.5, 17.4; MS (ESI) m/z (%): 362.4 (100) [M - H] ;
(
CDCl , 200 MHz): • 7.20 (br s, 1H, NH), 4.21-3.92 (m, 1H,
3
Anal. Calcd for C H NO : C, 69.39; H, 9.15; N, 3.85. Found:
C, 69.27; H, 9.27; N, 3.75.
CHOH), 1.94-1.03 (m, 32H, CH & CH ), 0.88 (t, J = 6.8 Hz,
2
1
33
4
2
3
1
3
3
7
1
H, CH3); C NMR (CDCl , 50 MHz): • 173.1, 96.9, 96.4,
3
2.0, 34.3, 31.9, 29.8, 29.6, 29.5, 29.3, 25.1, 24.7, 24.5, 22.7,
4.1; MS (ESI) m/z (%): 358.3 (100) [M + H] ; Anal. Calcd
4
.2.1.12. (2S)-tert-Butyl 2-(2-hydroxy-2-(naphthalen-2-yl)
+
acetamido)-3-methylbutanoate (mixture of diastereomers)
for C H NO : C, 67.19; H, 10.99; N, 3.92. Found: C, 67.02;
2
0
1
20 39
4
(35). Yield 66%; Yellow oil; [• ]
7.9 (c 1.00, CHCl ); H
D
3
H, 11.08; N, 3.86.
NMR (CDCl , 200 MHz): • 7.88-7.68 (m, 4H, arom), 7.55-
3
7
.38 (m, 3H, arom), 7.29-6.77 (m, 1H, NH), 5.20 (d, J = 3.0
Hz, 1H, CHOH), 4.47-4.33 (m, 1H, NHCH), 2.20-1.97 (m, 1H,
NHCHCH), 1.46-1.33 (m, 9H, CH ), 0.93-0.66 (m, 6H, CH );
4
.2.3. Oxidation of 2-hydroxyamides. To a solution of 2-
hydroxyamide (1.0 mmol) in dry CH Cl (10 mL) Dess-Martin
periodinane was added (0.64 g, 1.5 mmol) and the mixture was
stirred for 1-3 h at room temperature. The organic solvent was
2
2
3
3
1
3
C NMR (CDCl , 50 MHz): • 172.1, 172.0, 170.7, 170.6,
3

evaporated under reduce pressure and Et O (30 mL) was
4.2.3.6. (S)-tert-Butyl 2-(2-oxohexadecanamido)propanoate
2
o
20
added. The organic phase was washed with saturated aqueous
NaHCO (20 mL) containing Na S O (1.5 g, 9.5 mmol), H O
(15c). Yield 95%; White solid; mp 35-36 C; [• ] 5.0 (c 1.02,
D
1
CHCl ); H NMR (CDCl , 200 MHz): • 7.44 (d, J = 7.0 Hz,
3
2
2
3
2
3
3
(
20 mL), dried over Na SO and the organic solvent was
1H, NH), 4.52-4.33 (m, 1H, NHCH), 2.90 (t, J = 7.4 Hz, 2H,
CH CO), 1.76-1.38 (m, 14H, CH & CH CH CO), 1.38-1.17
2
4
evaporated under reduced pressure. The residue was purified
by flash column chromatography [EtOAc/petroleum ether (bp
2
3
2
2
1
3
(m, 22H, CH ), 0.86 (t, J = 6.4 Hz, 3H, CH ); C NMR
2
3
4
0-60 °C), 2:8 or CHCl /MeOH, 9:1].
(CDCl , 50 MHz): • 198.5, 171.1, 159.4, 82.4, 48.3, 36.6, 31.9,
3
3
2
9.6, 29.0, 27.9, 23.1, 22.6, 18.2, 14.1; MS (ESI) m/z (%):
+
4
.2.3.1. (S)-1-(2-Oxohexadecanoyl)pyrrolidine-2-carboxylic
415.3 (100) [M + NH
] ; Anal. Calcd for C23H43NO : C, 69.48;
4 4
2
0
D
H, 10.90; N, 3.52. Found: C, 69.34; H, 10.96; N, 3.43.
acid (rotamers) (12b). Yield 80%; Colorless semi solid; [• ]
1
-3.2 (c 1.01, CHCl ); H NMR (CDCl , 200 MHz): • 7.40 (br,
3
3
4
.2.3.7. (S)-5-tert-Butyl 1-ethyl 2-(2-oxohexadecanamido)
20
1
H, COOH), 4.86-4.31 (m, 1H, NCH), 3.95-3.41 (m, 2H,
pentanedioate (20). Yield 85%; Colorless oil; [• ]
1.00, CHCl ); H NMR (CDCl , 300 MHz): • 7.52 (d, J = 8.2
11.6 (c
D
NCH ), 3.06-2.60 (m, 2H, CH CO), 2.46-1.12 (m, 28H,
NCHCH CH & CH ), 0.88 (t, J = 6.6 Hz, 3H, CH ); C NMR
2
2
1
1
3
3
3
2
2
2
3
Hz, 1H, NH), 4.60-4.49 (m, 1H, NHCH), 4.21 (q, J = 7.2 Hz,
H, COOCH ), 2.89 (t, J = 7.2 Hz, 2H, CH COCO), 2.41-2.17
(CDCl , 50 MHz): • 200.0, 177.4, 163.0, 61.3, 48.3, 47.4, 39.1,
3
2
2
2
3
8.5, 31.9, 29.7, 29.5, 29.3, 29.1, 25.1, 23.1, 22.7, 22.6, 14.0;
+
(m, 3H, CH CHH), 2.08-1.92 (m, 1H, CHH), 1.66-1.52 (m,
2
HRMS (ESI) calcd for C H NO [M + H] : 368.2795. Found:
2
1
38
4
2
&
H, CH CH CO), 1.43 (s, 9H, CH ), 1.37-1.17 (m, 25H, CH
2
2
2
3
3
3
68.2794; Anal. Calcd for C H NO : C, 68.63; H, 10.15; N,
21 37 4
13
CH ), 0.85 (t, J = 6.6 Hz, 3H, CH ); C NMR (CDCl , 50
3
3
3
.81. Found: C, 68.51; H, 10.22; N, 3.74.
MHz): • 198.2, 171.6, 170.8, 159.9, 80.9, 61.7, 51.7, 36.7,
4
.2.3.2. (S)-3,3-Dimethyl 2-(2-oxohexadecanamido)butanoic
31.8, 31.2, 29.6, 29.4, 29.3, 29.0, 28.0, 27.2, 23.0, 22.6, 14.0;
MS (ESI) m/z (%): 428.2 (100) [M - tBu + H] ; Anal. Calcd
+
2
0
acid (12c). Yield 62%; Colorless oil; [• ]
CHCl ); H NMR (CDCl , 200 MHz): • 7.50 (d, J = 9.2 Hz,
1.8 (c 1.00,
D
1
for C27
H49NO
: C, 67.05; H, 10.21; N, 2.90. Found: C, 66.96;
3
3
6
H, 10.29; N, 2.81.
1
2
2
H, NH), 4.31 (d, J = 9.2 Hz, 1H, NHCH), 2.91 (t, J = 6.8 Hz,
H, CH CO), 1.74-1.49 (m, 2H, CH CH CO), 1.43-1.18 (m,
2
2
2
4
.2.3.8. (S)-tert-Butyl
3-methyl-2-(6-(naphthalen-2-yl)-2-
2H, CH ), 1.01 (s, 9H, CH ), 0.88 (t, J = 6.4 Hz, 3H, CH );
2
3
3
1
3
oxohexanamido)butanoate (30a). Yield 91%; Colorless oil;
C NMR (CDCl , 50 MHz): • 198.5, 175.7, 159.8, 60.8, 36.8,
3
20
1
[
• ]
11.1 (c 1.00, CHCl ); H NMR (CDCl , 200 MHz): •
D 3 3
3
4.7, 31.9, 29.6, 29.4, 29.3, 29.0, 26.5, 23.0, 22.6, 14.1; HRMS
+
7.86-7.68 (m, 3H, arom), 7.65-7.53 (m, 1H, arom), 7.52-7.18
m, 4H, arom & NH), 4.39 (dd, J = 9.2 Hz, J = 4.6 Hz, 1H,
(ESI) calcd for C H NNaO [M + Na] : 406.2928. Found:
2
2
41
4
(
1
2
4
3
06.2945; Anal. Calcd for C H NO : C, 68.89; H, 10.77; N,
22 41 4
NHCH), 2.99 (t, J = 7.2 Hz, 2H, CH ), 2.80 (t, J = 7.0 Hz, 2H,
2
.65. Found: C, 69.11; H, 10.88; N, 3.59.
CH CO), 2.32-2.08 (m, 1H, NHCHCH), 1.86-1.58 (m, 4H,
2
1
3
4
.2.3.3. 2-Methyl 2-(2-oxo-hexadecanoylamino)propanoic
CH
2
), 1.48 (s, 9H, CH ), 1.00-0.82 (m, 6H, CH ); C NMR
3
3
1
(CDCl , 50 MHz): • 198.3, 169.9, 159.8, 139.4, 133.5, 131.8,
acid (12d). Yield 95%; Colorless oil; H NMR (CDCl , 200
MHz): • 7.77 (br s, 1H, COOH), 2.88 (t, J = 6.8 Hz, 2H,
CH CO), 1.66-1.40 (m, 8H, CH & CH CH CO), 1.39-1.15 (m,
3
3
127.8, 127.5, 127.3, 127.2, 126.3, 125.8, 125.0, 82.3, 57.4,
6.5, 35.6, 31.4, 30.6, 27.9, 22.7, 18.9, 17.4; MS (ESI) m/z
3
2
3
2
2
1
3
+
2
2H, CH ), 0.88 (t, J = 6.6 Hz, 3H, CH ); C NMR (CDCl , 50
(%): 356.2 (100) [M - tBu + H] ; Anal. Calcd for C25
H33NO :
4
2
3
3
C, 72.96; H, 8.08; N, 3.40. Found: C, 72.85; H, 8.17; N, 3.26.
MHz): • 199.0, 198.8, 180.6, 160.0, 57.2, 36.4, 31.9, 29.6,
9.5, 29.4, 29.3, 29.1, 24.2, 23.0, 22.6, 14.1; HRMS (ESI)
2
+
4.2.3.9. (S)-tert-Butyl 2-(6-([1,1'-biphenyl]-4-yl)-2-
calcd for C H NNaO [M + Na] : 378.2615. Found:
3
3
2
0
37
4
oxohexanamido)-3-methylbutanoate (30b). Yield 82%;
78.2627; Anal. Calcd for C H NO : C, 67.57; H, 10.49; N,
20 37 4
20
1
Yellow oil; [• ] 12.2 (c 1.00, CHCl ); H NMR (CDCl , 200
D
3
3
.94. Found: C, 67.36; H, 10.61; N, 3.87.
MHz): • 7.75-7.16 (m, 10H, arom & NH), 4.48-4.29 (m, 1H,
4
.2.3.4. (S)-tert-Butyl 3-methyl-2-(2-oxohexadecanamido)
NHCH), 3.11-2.87 (m, 2H, CH
2
), 2.80-2.55 (m, 2H, CH
2
CO),
2
0
2
.34-2.09 (m, 1H, NHCHCH), 1.85-1.60 (m, 4H, CH ), 1.48 (s,
2
13
butanoate (15a). Yield 92%; Yellow oil; [• ] 12.7 (c 1.00,
CHCl ); H NMR (CDCl , 200 MHz): • 7.40 (d, J = 9.0 Hz,
D
1
9H, CH
) 1.04-0.82 (m, 6H, CH ); C NMR (CDCl , 50
3 3
3
3
3
MHz): • 198.3, 169.9, 159.8, 141.1, 141.0, 138.6, 128.7, 128.6,
127.0, 126.9, 82.3, 57.4, 36.5, 35.1, 31.4, 30.7, 27.9, 22.7,
1
H, NH), 4.40 (dd, J = 9.2 Hz, J = 4.4 Hz, 1H, NHCH), 2.91
1 2
(t, J = 7.2 Hz, 2H, CH COCO), 2.33-2.10 (m, 1H, NHCHCH),
2
-
1
8.9, 17.4; MS (ESI) m/z (%): 436.4 (100) [M - H] ; Anal.
1
.71-1.53 (m, 2H, CH CH CO), 1.48 (s, 9H, CH ) 1.40-1.16
2 2 3
13
(m, 22H, CH ), 1.01-0.81 (m, 9H, CH ); C NMR (CDCl , 50
Calcd for C27
H35NO : C, 74.11; H, 8.06; N, 3.20. Found: C,
4
2
3
3
7
4.02; H, 8.17; N, 3.09.
MHz): • 198.2, 169.6, 159.5, 81.9, 57.1, 36.4, 31.5, 31.1, 29.2,
9.0, 28.7, 27.6, 22.8, 22.3, 18.6, 17.1, 13.7; MS (ESI) m/z
2
+
4.2.3.10. (S)-tert-Butyl
3-methyl-2-(2-oxo-6-
(%): 387.2 (100) [M - tBu + NH ] ; Anal. Calcd for
4
phenylhexanamido)butanoate (30c). Yield 74%; Colorless
C H NO : C, 70.54; H, 11.13; N, 3.29. Found: C, 70.43; H,
1
2
5
47
4
20
1
oil; [• ] 11.3 (c 1.00, CHCl ); H NMR (CDCl , 200 MHz): •
D
3
3
1.24; N, 3.21.
7
.50-6.94 (m, 6H, arom & NH), 4.36 (dd, J = 9.2 Hz, J = 4.6
1 2
Hz, 1H, NHCH), 2.93 (t, J = 6.8 Hz, 2H, CH ), 2.62 (t, J = 6.8
4
.2.3.5. (S)-di-tert-Butyl
2-(2-oxohexadecanamido)
2
2
0
pentanedioate (15b). Yield 84%; Yellow oil; [• ]
9.18 (c
Hz, 2H, CH
CO), 2.31-2.06 (m, 1H, NHCHCH), 1.73-1.55 (m,
2
13
D
1
4
H, CH ), 1.46 (s, 9H, CH ), 1.00-0.74 (m, 6H, CH );
C
1
.02, CHCl ); H NMR (CDCl , 300 MHz): • 7.47 (d, J = 8.4
2
3
3
3
3
Hz, 1H, NH), 4.49-4.37 (m, 1H, NHCH), 2.88 (t, J = 7.2 Hz,
NMR (CDCl
3
, 50 MHz): • 197.9, 169.7, 159.8, 142.0, 128.3,
1
25.6, 82.3, 57.1, 36.5, 35.5, 31.4, 30.7, 27.9, 22.6, 18.9, 17.4;
2
1
1
H, CH COCO), 2.38-2.08 (m, 3H, CH CHH), 2.05-1.87 (m,
2
2
-
H, CHH), 1.66-1.51 (m, 2H, CH CH CO), 1.46 (s, 9H, CH ),
MS (ESI) m/z (%): 360.4 (100) [M - H] ; Anal. Calcd for
C H NO : C, 69.78; H, 8.64; N, 3.87. Found: C, 69.61; H,
2
2
3
.42 (s, 9H, CH ), 1.35-1.16 (m, 22H, CH ), 0.86 (t, J = 6.6
21 31
4
3
2
1
3
Hz, 3H, CH3); C NMR (CDCl , 50 MHz): • 198.3, 171.7,
8.73; N, 3.81.
3
1
2
70.0, 159.8, 82.7, 80.8, 52.1, 36.7, 31.8, 31.3, 29.6, 29.5,
4
.2.3.11. (S)-tert-Butyl 3-methyl-2-(2-(naphthalen-2-yl)-2-
20
9.4, 29.3, 29.2, 29.0, 28.0, 27.9, 27.4, 23.0, 22.6, 14.0; MS
+
oxoacetamido)butanoate (36). Yield 95%; Yellow oil; [• ]
D
(ESI) m/z (%): 529.5 (100) [M + NH ] ; Anal. Calcd for
4
1
7
.03 (c 1.01, CHCl ); H NMR (CDCl , 200 MHz): • 9.21-8.96
3
3
C H NO : C, 68.06; H, 10.44; N, 2.74. Found: C, 67.90; H,
1
2
9
53
6
(m, 1H, arom), 8.23-7.05 (m, 7H, arom & NH), 4.55 (dd, J =
1
0.55; N, 2.68.
9
.2 Hz, J2 = 4.6 Hz, 1H, NHCH), 2.43-2.08 (m, 1H,

1
3
NHCHCH), 1.50 (s, 9H, CH ), 1.09-0.79 (m, 6H, CH );
C
(m, 1H, NHCH), 4.22 (q, J = 7.0 Hz, 2H, COOCH ), 2.89 (t, J
3
3
2
NMR (CDCl , 50 MHz): • 186.6, 170.0, 161.6, 136.0, 134.8,
= 7.2 Hz, 2H, CH CO), 2.36-1.94 (m, 2H, NHCHCH CH ),
3
2
2
2
1
8
3
7
34.7, 132.2, 130.4, 130.2, 129.2, 128.2, 127.6, 126.7, 125.1,
2.3, 57.6, 31.5, 27.9, 19.0, 18.9, 17.6; MS (ESI) m/z (%):
1.70-1.48 (m, 2H, NHCHCH ), 1.46-1.08 (m, 24H, CH ), 0.87
2
2
1
3
(t, J = 6.8 Hz, 3H, CH3); C NMR (CDCl , 50 MHz): • 198.1,
3
-
54.1 (100) [M - H] ; Anal. Calcd for C H NO : C, 70.96; H,
177.8, 170.7, 160.0, 61.9, 51.5, 36.7, 31.8, 29.9, 29.6, 29.3,
29.2, 29.0, 26.9, 23.0, 22.6, 14.0; HRMS (ESI) calcd for
2
1
25
4
.09; N, 3.94. Found: C, 70.74; H, 7.20; N, 3.86.
+
C H NNaO [M + Na] : 450.2826. Found: 450.2826; Anal.
2
3
41
6
4
.2.4. Cleavage of tert-butyl protecting group. A solution of
Calcd for C H NO : C, 64.61; H, 9.67; N, 3.28. Found: C,
2
3
41
6
the tert-butyl ester derivative (1.0 mmol) in 50% TFA/CH Cl2
2
64.49; H, 9.74; N, 3.22.
(0.5M) was stirred for 1-3 h at room temperature. After the
completion of the reaction, the organic solvent was evaporated
under reduced pressure and the residue was purified by
recrystallization.
4.2.4.6. (S)-3-Methyl
2-(6-(naphthalen-2-yl)-2-
oxohexanamido)butanoic acid (31a). Yield 83%; Colorless
2
0
1
solid; [• ] -2.8 (c 0.5, CHCl ); H NMR (CDCl , 200 MHz):
D
3
3
•
7.85-7.69 (m, 3H, arom), 7.64-7.58 (m, 1H, arom), 7.51-7.28
4
.2.4.1. (S)-3-Methyl
2-(2-oxohexadecanamido)butanoic
(m, 4H, arom & NH), 5.94 (br, 1H, COOH), 4.52 (dd, J = 9.2
1
o
Hz, J = 4.6 Hz, 1H, NHCH), 2.98 (t, J = 6.8 Hz, 2H, CH ),
acid (16a, GK241). Yield 72%; White solid; mp 55-58 C;
2
2
2
0
1
[
• ]
5.04 (c 1.03, CHCl ); H NMR (CDCl , 200 MHz): •
2.81 (t, J = 7.0 Hz, 2H, CH
2
CO), 2.41-2.21 (m, 1H,
D
3
3
NHCHCH), 1.88-1.59 (m, 4H, CH ), 1.08-0.89 (m, 6H, CH );
7
.43 (d, J = 8.0 Hz, 1H, NH), 4.59-4.35 (m, 1H, NHCH), 2.91
2
3
1
3
(t, J = 6.8 Hz, 2H, CH CO), 2.42-2.16 (m, 1H, NHCHCH),
C NMR (CDCl
33.5, 131.9, 127.8, 127.5, 127.3, 127.2, 126.3, 125.8, 125.0,
56.9, 36.5, 35.6, 31.0, 30.5, 22.7, 19.0, 17.4; HRMS (ESI)
3
, 50 MHz): • 198.1, 175.4, 159.9, 139.4,
2
1
1
1
1
2
.74-1.50 (m, 2H, CH CH CO), 1.47-1.15 (m, 22H, CH ),
.08-0.79 (m, 9H, CH3); C NMR (CDCl , 50 MHz): • 198.4,
2
2
2
1
3
3
+
calcd for C H NO [M + H] : 356.1856. Found: 356.1841;
76.0, 160.2, 57.4, 36.8, 31.9, 31.0, 29.7, 29.4, 29.3, 29.1,
3.1, 22.7, 19.1, 17.5, 14.1; HRMS (ESI) calcd for C H NO
4
21 26
4
Anal. Calcd for C21
H25NO
: C, 70.96; H, 7.09; N, 3.94. Found:
2
1
40
4
+
C, 70.84; H, 7.16; N, 3.87.
[
M + H] : 370.2952. Found: 370.2938; Anal. Calcd for
C H NO : C, 68.25; H, 10.64; N, 3.79. Found: C, 68.09; H,
2
1
39
4
4
.2.4.7. (S)-2-(6-([1,1'-Biphenyl]-4-yl)-2-oxohexanamido)-3-
1
0.76; N, 3.70.
methylbutanoic acid (31b). Yield 92%; White solid; mp 123-
1
MHz): • 7.70-7.08 (m, 10H, arom & NH), 4.56-4.26 (m, 1H,
•
2.39-2.08 (m, 1H, NHCHCH), 1.83-1.44 (m, 4H, CH
0
o
20
1
24 C; [• ]
-6.1 (c 1.00, CHCl ); H NMR (CDCl , 200
4
.2.4.2. (S)-2-(2-Oxohexadecanamido)propanoic acid (16c).
D 3 3
o
20
Yield 90%; White solid; mp 98-101 C; [• ]
CHCl ); H NMR (CDCl , 200 MHz): • 8.92 (br, 1H, COOH),
5.8 (c 1.03,
D
1
• CH), 3.12-2.80 (m, 2H, CH ), 2.76-2.46 (m, 2H, CH CO),
2
2
3
3
7
.43 (d, J = 7.6 Hz, 1H, NH), 4.69-4.50 (m, 1H, NHCH), 2.91
2
), 1.10-
1
3
.68 (m, 6H, CH3); C NMR (CDCl , 50 MHz): • 198.1,
(
t, J = 7.2 Hz, 2H, CH CO), 1.70-1.48 (m, 5H, CH CH CO &
3
2
2
2
CH ), 1.44-1.19 (s, 22H, CH ), 0.88 (t, J = 6.6 Hz, 3H, CH );
176.2, 160.1, 141.0, 140.9, 138.6, 128.7, 128.6, 127.0, 126.9,
7.5, 36.6, 35.2, 30.7, 27.3, 22.6, 19.1, 17.5; HRMS (ESI)
3
2
3
1
3
5
C NMR (CDCl , 50 MHz): • 198.3, 176.8, 159.6, 47.8, 36.7,
3
+
3
1.9, 29.6, 29.5, 29.4, 29.3, 29.0, 23.0, 22.6, 17.7, 14.1; HRMS
calcd for C23
H28NO [M + H] : 382.2013. Found: 382.1995;
4
+
Anal. Calcd for C H NO : C, 72.42; H, 7.13; N, 3.67. Found:
C, 72.64; H, 7.24; N, 3.25.
(ESI) calcd for C H NO [M + H] : 342.2639. Found:
23 27
4
1
9
36
4
3
4
42.2650; Anal. Calcd for C H NO : C, 66.83; H, 10.33; N,
.10. Found: C, 66.61; H, 10.45; N, 4.01.
1
9
35
4
4
.2.4.8. (S)-3-Methyl
2-(2-oxo-6-
4
.2.4.3. (S)-2-(2-Oxohexadecanamido)pentanedioic
acid
phenylhexanamido)butanoic acid (31c). Yield 50%;
2
0
1
o
20
Colorless oil; [• ] 5.8 (c 1.00, CHCl ); H NMR (CDCl , 200
(16d). Yield 90%; White solid; mp 100-103 C; [• ] 30.5 (c
D
3
3
D
1
1
.02, CHCl ); H NMR (CDCl , 200 MHz): • 9.67 (br, 1H,
MHz): • 9.43 (br, 1H, COOH), 7.37 (d, J = 9.0 Hz, 1H, NH),
3
3
7
.32-7.03 (m, 5H, arom), 4.50 (dd, J = 9.0 Hz, J = 4.6 Hz,
COOH), 7.63 (d, J = 8.0 Hz, 1H, NH), 4.72-4.56 (m, 1H,
NHCH), 2.91 (t, J = 7.0 Hz, 2H, CH CO), 2.64-2.43 (m, 2H,
NHCHCH CH ), 2.38-2.20 (m, 2H, NHCHCH ), 1.72-1.51 (m,
2
3
1
1
4
6
1
2
1H, NHCH), 3.04-2.86 (m, 2H, CH
), 2.72-2.50 (m, 2H,
2
2
CH CO), 2.41-2.16 (m, 1H, NHCHCH), 1.79-1.50 (m, 4H,
2
2
2
2
1
3
H, CH CH CO), 1.44-1.18 (m, 22H, CH ), 0.88 (t, J = 6.4 Hz,
CH
98.0, 175.8, 159.9, 141.9, 128.2, 125.7, 57.1, 36.5, 35.5, 30.9,
30.6, 22.5, 19.0, 17.4; HRMS (ESI) calcd for C17 [M +
2
), 1.11-0.81 (m, 6H, CH ); C NMR (CDCl , 50 MHz): •
3
3
2
2
2
1
3
1
H, CH3); C NMR (CDCl , 50 MHz): • 198.1, 178.5, 176.2,
3
59.8, 51.2, 36.7, 31.9, 29.6, 29.5, 29.4, 29.3, 29.0, 23.0, 22.6,
H24NO
4
+
+
H] : 306.1700. Found: 306.1695; Anal. Calcd for C H NO :
4.1; HRMS (ESI) calcd for C H NNaO [M + Na] :
17 23
4
2
1
37
6
22.2513. Found: 422.2513; Anal. Calcd for C H NO : C,
C, 66.86; H, 7.59; N, 4.59. Found: C, 66.65; H, 7.71; N, 4.52.
2
1
37
6
3.13; H, 9.33; N, 3.51. Found: C, 62.99; H, 9.44; N, 3.43.
4
.2.4.9. (S)-3-Methyl
2-(2-(naphthalen-2-yl)-2-
oxoacetamido) butanoic acid (37). Yield 80%; Yellow solid;
4
.2.4.4. (2S)-2-(2-Hydroxyhexadecanamido)-3-
o
20
1
methylbutanoic acid (mixture of diastereomers) (17). Yield
mp 122-124 C; [• ]
00 MHz): • 9.58 (br, 1H, COOH), 9.15 (s, 1H, arom), 8.17 (d,
J = 8.6 Hz, 1H, NH), 8.00 (d, J = 7.8 Hz, 1H, arom), 7.94-7.76
m, 2H, arom), 7.75-7.44 (m, 3H, arom), 4.71 (dd, J = 9.0 Hz,
-25.3 (c 1.03, CHCl ); H NMR (CDCl ,
D 3 3
o
20
2
9
3%; White solid; mp 95-99 C; [• ] 17.1 (c 1.00, MeOH);
D
1
H NMR (CDCl , 200 MHz): • 7.01 (d, J = 9.2 Hz, 1H, NH),
3
(
4
.57-4.42 (m, 1H, NHCH), 4.24-4.10 (m, 1H, CHOH), 3.68
1
(br, 1H, OH), 2.39-2.15 (m, 1H, NHCHCH), 1.95-1.53 (m, 2H,
J = 4.6 Hz, 1H, NHCH), 2.54-2.26 (m, 1H, NHCHCH), 1.18-
2
13
0
.95 (m, 6H, CH3); C NMR (CDCl , 50 MHz): • 186.4,
3
CH CHOH), 1.51-1.12 (s, 24H, CH ), 1.10-0.76 (m, 9H,
2
2
1
3
3
7
2
+
xCH3); C NMR (CDCl , 50 MHz): • 176.2, 175.3, 174.4,
176.4, 161.9, 136.0, 135.0, 132.2, 130.3, 130.2, 129.4, 128.4,
27.6, 126.8, 125.0, 57.1, 31.1, 19.1, 17.5; HRMS (ESI) calcd
3
1
2.2, 57.0, 56.7, 34.5, 34.4, 31.8, 30.6, 30.5, 29.6, 29.3, 25.0,
2.6, 19.0, 17.4, 14.1; HRMS (ESI) calcd for C H NNaO [M
+
for C17
H18NO
[M + H] : 300.1230. Found: 300.1216; Anal.
2
1
41
4
4
+
Calcd for C H NO : C, 68.21; H, 5.72; N, 4.68. Found: C,
Na] : 394.2928. Found: 394.2947; Anal. Calcd for
17 17
4
C H NO : C, 67.88; H, 11.12; N, 3.77. Found: C, 67.73; H,
68.12; H, 5.80; N, 4.59.
2
1
41
4
1
1.23; N, 3.69.
4
.2.5. (2E,4E)-Ethyl 5-(naphthalen-2-yl)penta-2,4-dienoate
(
23a). To a stirred solution of aldehyde (1.0 mmol) in dry THF
(10 mL) and molecular sieves (1.5 g/mmol aldehyde) under
Ar, C H OOCH=CHCH P(=O)(OC H ) (1.5 mmol) was
4
.2.4.5. (S)-5-Ethoxy-5-oxo-4-(2-oxohexadecanamido)
o
pentanoic acid (21). Yield 93%; White solid; mp 64-66 C;
2
0
1
[
• ]
13.4 (c 1.03, CHCl ); H NMR (CDCl , 200 MHz): •
2
5
2
2
5 2
D
3
3
o
9
.75 (br, 1H, COOH), 7.57 (d, J = 8.2 Hz, 1H, NH), 4.67-4.49
added at 0 C. Then, LiOH·H O (1.5 mmol) was added
2

+
dropwise and the reaction mixture heated at reflux for 24 h.
The reaction was filtered over celite and the organic solvent
was evaporated under reduced pressure. The residue was
purified by flash column chromatography [EtOAc/petroleum
ether (bp 40-60 ˚C), 5:95]. Yield 50%; White solid; mp 100-
24.1; MS (ESI) m/z (%): 257.1 (100) [M + NH ] ; Anal. Calcd
4
for C H NO: C, 80.30; H, 7.16; N, 5.85. Found: C, 80.07; H,
1
6
17
7.32; N, 5.66.
4.2.9. 6-([1,1'-Biphenyl]-4-yl)-2-hydroxyhexanenitrile (27b).
Yield 80%; Pink solid; mp 78-82 °C; H NMR (CDCl , 200
MHz): • 7.69-7.25 (m, 9H, arom), 4.50-4.46 (m, 1H, CHOH),
3.64 (br, 1H, OH), 2.83-2.70 (m, 2H, CH ), 1.97-1.90 (m, 2H,
o
1
1
1
02 C; H NMR (CDCl , 200 MHz): • 7.90-7.60 (m, 5H,
3
3
arom), 7.55-7.45 (m, 3H, arom & CH), 7.10-6.95 (m, 2H, CH),
.04 (s, 1H, CH), 4.26 (m, 2H, CH ), 1.28 (t, J = 7.0 Hz, 3H,
6
2
2
1
3
13
CH3); C NMR (CDCl , 50 MHz): • 167.1, 144.6, 140.4,
CH ), 1.80-1.61 (m, 4H, CH ); C NMR (CDCl , 50 MHz): •
3
2
2
3
1
1
33.5, 133.4, 128.5, 128.2, 128.1, 127.7, 126.6, 126.5, 126.4,
1
40.9, 140.7, 138.5, 128.6, 126.9, 126.7, 120.0, 60.8, 35.0,
34.7, 30.5, 24.1; MS (ESI) m/z (%): 282.9 (100) [M + NH ] ;
Anal. Calcd for C H NO: C, 81.47; H, 7.22; N, 5.28. Found:
+
23.3, 121.3, 60.4, 14.3; MS (ESI) m/z (%): 253 (100) [M +
4
+
H] ; Anal. Calcd for C H O : C, 80.93; H, 6.39. Found: C,
1
7
16
2
1
8
19
8
0.85; H, 6.55.
C, 81.25; H, 7.39; N, 5.23.
4
.2.6. Ethyl 5-(naphthalen-2-yl)pentanoate (24a). To
a
4.2.9.1. 2-Hydroxy-6-phenylhexanenitrile (27c). Yield 78%;
1
stirred solution of the alkene (1.0 mmol) in absolute EtOH (10
mL), Pd/C 10% was added. The reaction left overnight under
H at room temperature. The reaction was filtered over celite
and the organic solvent was evaporated under reduced
pressure. The residue was purified by flash column
chromatography [EtOAc/petroleum ether (bp 40-60 ˚C), 5:95].
Colorless oil; H NMR (CDCl , 200 MHz): • 7.44-7.15 (m,
3
5
H, arom), 4.56-4.32 (m, 1H, CHOH), 4.03 (br, 1H, OH),
13
2
2.81-2.59 (m, 2H, CH ), 2.01-1.48 (m, 6H, CH ); C NMR
2
2
(
CDCl , 50 MHz): • 141.7, 128.1, 125.5, 119.9, 60.7, 35.3,
3
+
34.6, 30.4, 23.9; MS (ESI) m/z (%): 207.3 (90) [M + NH ] ;
4
Anal. Calcd for C H NO: C, 76.16; H, 7.99; N, 7.40. Found:
1
2
15
1
Yield 80%; Colorless oil; H NMR (CDCl , 200 MHz): • 8.03-
C, 76.04; H, 8.11; N, 7.30.
3
7
2
.18 (m, 7H, arom), 4.34-4.06 (m, 2H, COOCH CH ), 3.03-
2 3
.69 (m, 2H, CH ), 2.51-2.31 (m, 2H, CH ), 1.91-1.69 (m, 4H,
4.2.10. 2-Hydroxy-2-(naphthalen-2-yl)acetonitrile (33). To a
stirring solution of naphthalene-2-carbaldehyde (1.0 mmol) in
EtOAc/THF/AcOH 4:4:2, aqueous solution of NaCN 6M (0.25
mL, 1.5 mmol) was added. After the completion of the
2
2
1
3
CH ), 1.41-1.22 (m, 3H, CH ); C NMR (CDCl , 50 MHz): •
2
3
3
1
1
73.4, 139.4, 133.4, 131.8, 127.6, 127.4, 127.2, 127.1, 126.2,
25.7, 124.9, 60.0, 35.5, 34.0, 30.5, 24.4, 14.1; MS (ESI) m/z
+
(%): 274.1 (100) [M + NH ] ; Anal. Calcd for C H O : C,
reaction, the organic layer was extracted with CH
mL), washed with brine and dried over Na SO . The organic
2 2
Cl (2 x 20
4
17 20
2
7
9.65; H, 7.86. Found: C, 79.39; H, 8.00.
2
4
solvent was evaporated under reduced pressure and the
compound was purified with flash column chromatography
4
.2.7. General method of oxidation of primary alcohols to
[
EtOAc/petroleum ether (bp 40-60 °C), 2:8]. Yield 95%; White
aldehydes and then to cyanohydrins. To a solution of
primary alcohol (1.0 mmol) in a mixture of toluene (3 mL) and
EtOAc (3 mL), a solution of NaBr (0.11 g, 1.1 mmol) in water
• 1
solid; mp 110-113 C; H NMR (CDCl , 200 MHz): • 7.99-
3
7
.24 (m, 7H, arom), 5.78-5.59 (m, 1H, CHOH), 2.08 (d, J = 2.4
13
Hz, 1H, OH); C NMR (CDCl , 50 MHz): • 133.6, 132.8,
(
0.5 mL) was added and the mixture was cooled down to -5 ºC.
3
1
32.3, 129.4, 128.3, 127.7, 127.2, 123.6, 63.8; MS (ESI) m/z
To the resulting biphasic system, AcNH-TEMPO (2.2 mg, 0.01
mmol) was added, followed by the drop-wise addition of an
aqueous solution of 0.5 M NaOCl (2.2 mL, 1.1 mmol) and
+
(%): 157.1 (73) [M - CN] ; Anal. Calcd for C H NO: C,
12 9
7
8.67; H, 4.95; N, 7.65. Found: C, 78.49; H, 5.10; N, 7.56.
NaHCO (0.24 g, 3.0 mmol) over a period of 1 h under
vigorous stirring. After the mixture was stirred for a further 15
3
4.2.11. Converting the nitriles into corresponding
carboxylic acids. The nitrile (1.0 mmol) was dissolved in HCl
min at 0 ºC, EtOAc (10 mL) and H O (10 mL) were added.
2
The aqueous layer was separated and washed with EtOAc (2 x
conc. (2.5 mL) and stirred for 18 h at room temperature. H
(5 mL) was then added and the organic layer was extracted
with CH Cl , washed with brine and dried over Na SO . The
2
O
1
0 mL). The combined organic layers were washed
consecutively with 5 % aqueous citric acid (10 mL) containing
KI (0.04 g), 10 % aqueous Na S O (10 mL), and brine and
2
2
2
4
organic solvent was evaporated under reduced pressure and the
amide derivative was crystallized over cold petroleum ether.
To a solution of the amide derivative (0.8 mmol) in
2
2
3
dried over Na SO . The solvents were evaporated under
2
4
reduced pressure and the aldehyde was used straight to the next
reaction without further purification.
MeOH/H O (2:1, 6 mL), KOH (8.0 mmol) was added and the
2
To a solution of the aldehyde (1.0 mmol) in CH Cl (1.3
reaction mixture was heated under reflux for 2 h. Then,
2
2
mL), an aqueous solution of NaHSO (0.25 mL, 1.5 mmol) was
methanol was evaporated under reduced pressure, H O was
3
2
added and the mixture was stirred for 30 min at room
temperature. The organic solvent was evaporated under
added and the aqueous layer was acidified with 1N HCl to pH
1. The organic layer was extracted with EtOAc (3 x 15 mL),
dried over Na SO and the solvent was evaporated under
reduced pressure and H O (5 mL) was added. The mixture was
2
2
4
cooled down to 0 ºC and KCN 6 M (0.25 mL, 1.5 mmol) was
added drop-wise under vigorous stirring. The reaction was left
stirring for 18 h at room temperature. After the completion of
the reaction, the organic layer was extracted with CH Cl (2 x
reduced pressure. The compound was purified by
recrystallization [CH Cl /petroleum ether (bp 40-60 °C)].
2
2
4
.2.11.1. 2-Hydroxy-6-(naphthalen-2-yl)hexanoic
acid
2
2
1
(28a). Yield 98%; White solid; mp 115-118 °C; H NMR
2
0 mL), washed with brine and dried over Na SO . The
2 4
(
CDCl , 200 MHz): • 7.81-7.25 (m, 7H, arom), 4.35-4.10 (m,
3
organic solvent was evaporated under reduced pressure and the
compound was purified with flash column chromatography
1
H, CHOH), 2.78 (t, J = 7.4 Hz, 2H, CH ), 2.07-1.31 (m, 6H,
2
13
CH2); C NMR (CDCl , 50 MHz): • 177.1, 139.8, 133.4,
3
[
EtOAc/petroleum ether (bp 40-60 °C), 2:8].
1
4
2
31.7, 127.6, 127.4, 127.2, 127.2, 126.1, 125.7, 124.9, 69.9,
9.7, 49.2, 48.8, 35.7, 33.9, 30.9, 24.5; MS (ESI) m/z (%):
-
4
.2.8. 2-Hydroxy-6-(naphthalen-2-yl)hexanenitrile
(27a).
57.2 (100) [M - H] ; Anal. Calcd for C H O : C, 74.39; H,
1
6
18
3
1
Yield 56%; Yellow oil; H NMR (CDCl , 200 MHz): • 7.95-
7.02. Found: C, 74.31; H, 7.18.
3
7
1
.19 (m, 7H, arom), 4.41 (t, J = 6.6 Hz, 1H, CHOH), 2.99 (br,
13
H, OH), 2.88-2.66 (m, 2H, CH ), 1.98-1.42 (m, 6H, CH );
C
4.2.11.2. 6-([1,1'-Biphenyl]-4-yl)-2-hydroxyhexanoic
(28b). Yield 93%; White solid; H NMR (DMSO, 200 MHz): •
7.48-7.11 (m, 9H, arom), 4.06-3.94 (m, 1H, CHOH), 2.58-2.50
acid
2
2
1
NMR (CDCl , 50 MHz): • 139.3, 133.4, 131.8, 127.8, 127.5,
3
1
27.3, 127.1, 126.3, 125.9, 125.1, 119.9, 61.0, 35.6, 34.9, 30.5,

1
3
(
m, 2H, CH ), 1.74-1.37 (m, 6H, CH ); C NMR (CDCl , 50
11. Draheim, S. E.; Bach, N. J.; Dillard, R. D.; Berry, D. R.;
Carlson, D. G.; Chirgadze, N. Y.; Clawson, D. K.; Hartley, L.
W.; Johnson, L. M.; Jones, N. D.; McKinney, E. R.; Mihelich,
E. D.; Olkowski, J. L.; Schevitz, R. W.; Smith, A. C.; Snyder,
D. W.; Sommers, C. D.; Wery, J.-P. J. Med. Chem. 1996, 39,
2
2
3
MHz): • 177.1, 141.4, 140.9, 138.4, 128.6, 128.5, 126.8, 126.8,
9.9, 35.2, 33.9, 31.0, 24.5; MS (ESI) m/z (%): 282.9 (100) [M
H] ; Anal. Calcd for C H O : C, 76.03; H, 7.09. Found: C,
6
-
7
-
1
8
20
3
5.77; H, 7.23.
5
159-5175.
1
2. Singer, A. G.; Ghomashchi, F.; Le Calvez, C.; Bollinger, J.;
Bezzine, S.; Rouault, M.; Sadilek, M.; Nguyen, E.; Lazdunski,
M.; Lambeau, G.; Gelb, M. H. J. Biol. Chem. 2002, 277,
48535-48549.
4
6
.2.11.3. 2-Hydroxy-6-phenylhexanoic acid (28c). Yield
1
3%; White solid; mp 100-102 °C; H NMR (CDCl , 200
3
MHz): • 7.41-7.10 (m, 5H, arom), 4.36-4.20 (m, 1H, CHOH),
2
NMR (CDCl , 50 MHz): • 179.5, 142.3, 128.4, 128.3, 125.7,
7
1
3
.64 (t, J = 7.2 Hz, 2H, CH ), 2.02-1.37 (m, 6H, CH );
C
13. Smart, B. P.; Oslund, R. C.; Walsh, L. A.; Gelb, M. H. J. Med.
Chem. 2006, 49, 2858-2860.
1
2
2
3
4. Oslund, R. C.; Cermak, N.; Gelb, M. H. J. Med. Chem. 2008,
51, 4708-4714.
0.1, 35.7, 34.0, 31.1, 24.4; MS (ESI) m/z (%): 208.1 (100)
-
[
M] ; Anal. Calcd for C H O : C, 69.21; H, 7.74. Found: C,
12 16 3
1
5. Beaton, H. G.; Bennion, C.; Connolly, S.; Cook, A. R.;
Gensmantel, N. P.; Hallam, C.; Hardy, K.; Hitchin, B.; Jackson,
C. G.; Robinson, D. H. J. Med. Chem. 1994, 37, 557-559.
6. Hansford, K. A.; Reid, R. C.; Clark, C. I.; Tyndall, J. D. A.;
Whitehouse, M. W.; Guthrie, T.; McGeary, R. P.; Schafer, K.;
Martin, J. L.; Fairlie, D. P. Chembiochem. 2003, 4, 181-185.
6
9.04; H, 7.89.
.2.11.4. 2-Hydroxy-2-(naphthalen-2-yl)acetic acid (34).
4
1
1
Yield 90%; White solid; mp 57-60 °C; H NMR (CDCl , 200
MHz): • 7.89-7.24 (m, 7H, arom), 5.28 (s, 1H, CHOH), 3.32-
3
1
3
3
1
7
.08 (m, 1H, OH); C NMR (CDCl , 50 MHz): • 175.1, 135.8,
17. Antonopoulou, G.; Barbayianni, E.; Magrioti, V.; Cotton, N.;
Stephens, D.; Constantinou-Kokotou, V.; Dennis, E. A.;
Kokotos, G. Bioorg. Med. Chem. 2008, 16, 10257-10269.
3
33.1, 128.2, 127.9, 127.5, 126.2, 126.1, 126.1, 125.8, 124.1,
+
2.6; MS (ESI) m/z (%): 202.21 (100) [M + H] ; Anal. Calcd
1
8. Kokotos, G.; Six, D. A.; Loukas, V.; Smith, T.; Constantinou-
Kokotos, V.; Hadjipavlou-Litina, D.; Kotsovolou, S.; Chiou,
A.; Beltzner, C. C.; Dennis, E. A. J. Med. Chem. 2004, 47,
3615-3628.
9. Six, D. A.; Barbayianni, E.; Loukas, V.; Constantinou-
Kokotou, V.; Hadjipavlou-Litina, D.; Stephens, D.; Wong, A.
C.; Magrioti, V.; Moutevelis-Minakakis, P.; Baker, S. F.;
Dennis, E. A.; Kokotos, G. J. Med. Chem. 2007, 50, 4222-
for C H O : C, 71.28; H, 4.98. Found: C, 71.11; H, 5.13.
1
2
10
3
Acknowledgments
1
This research has been co-financed by the European Union
(European Social Fund – ESF) and Greek national funds
through the Operational Program "Education and Lifelong
Learning" of the National Strategic Reference Framework
4
235.
20. Mouchlis, V. D.; Magrioti, V.; Barbayianni, E.; Cermak, N.;
Oslund, R. C.; Mavromoustakos, T. M.; Gelb, M. H.; Kokotos,
G. Bioorg. Med. Chem. 2011, 19, 735-743.
(NSRF) - Research Funding Program: Heracleitus II. Investing
in knowledge society through the European Social Fund.
2
1. Mouchlis, V. D.; Barbayianni, E.; Mavromoustakos, T. M.;
Kokotos, G. Curr. Med. Chem. 2011, 18, 2566-2582.
22. Mouchlis, V. D.; Mavromoustakos, T. M.; Kokotos, G. J.
Comput.-Aided Mol. Des. 2010, 24, 107-115.
3. GOLD 5.2, CCDC, Cambridge, UK, 2013.
4. Smart, B. P.; Pan, Y. H.; Weeks, A. K.; Bollinger, J. G.;
Bahnson, B. J.; Gelb, M. H. Bioorg. Med. Chem. 2004, 12,
This work was supported in part by Grant HL36235 from the
National Institutes of Health (to M.•H.•G.).
2
2
Partial support was offered by LinkSCEEM-2 project, funded
by the European Commission under the 7th Framework
Program through Capacities Research Infrastructure, INFRA-
1
737-1749.
25. Case, D. A.; Darden, T.; Cheatham, T. E., III; Simmerllling, C.
L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.;
Merz, K. M.; Roberts, B. P.; Hayik, S.; Roitberg, A.; Seabra,
G.; Swails, J.; Goetz, A. W.; Kolossvai, I.; Wong, K. F.;
Paesani, F.; Vanicek, J.; Wolf, R. M.; Liu, J.; Wu, X.; Brozell,
S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, B.;
Hsieh, M. J.; Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M.
G.; Salomon-Ferrer, R.; Sagui, C.; Babin, V.; Luchko, T.;
Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 12;
University of California: San Francisco, 2012.
6. SYBYL, molecular modeling software packages, version 8.0,
2007, Tripos Inc., 1699 South Hanley Rd., St. Louis, MO
63144-2917.
7. Powell, D. J. M. Math. Program. 1977, 12, 241-254.
8. Clark, M.; Crammer, D. R., III; Van Opdenbosch, N. J.
Comput. Chem. 1989, 10, 982-1012.
2
010-1.2.3 Virtual Research Communities, Combination of
Collaborative Project and Coordination and Support Actions
CP-CSA) under grant agreement no. RI-261600.
(
Supplementary Material
1
13
Code numbers of tested compounds, H, C NMR and
HRMS spectra of inhibitor 16a are given. Docking results of
the synthesized compounds and MD simulations of 16a and
2
3
1c are provided. Supplementary data associated with this
2
2
article can be found, in the online version, at http://dx.doi.org/
References and notes
2
9. Murty, K. G. Linear programming. New York: John Wiley &
Sons Inc. 1983.
1
.
Dennis, E. A.; Cao, J.; Hsu, Y. H.; Magrioti, V.; Kokotos, G.
Chem. Rev. 2011, 111, 6130-6185.
30. Kirkpatrick, S.; Gelatt, C. D., Jr.; Vecchi, M. P. Science 1983,
220, 671-680.
2
3
.
.
Murakami, M.; Lambeau, G. Biochimie 2013, 95, 43-50.
Murakami, M.; Taketomi, Y.; Miki, Y.; Sato, H.; Yamamoto,
K.; Lambeau, G. Biochimie. 2014, 107Pt A, 105-113.
Murakami, M.; Sato, H.; Miki, Y.; Yamamoto, K.; Taketomi,
Y. J. Lipid Res. 2015, 56, 1248-1261.
31. RCSB
Protein
Data
Bank.
http://www.rcsb.org/pdb/home/home.do (accessed November
2015).
32. Schrödinger Suite 2009 Prime version 2.1, Schrödinger, LLC,
New York, NY, 2009.
4
.
5
6
.
.
Hui, D. Y. Curr. Opin. Lipidol. 2012, 23, 235-240.
Quach, N. D.; Arnold, R. D.; Cummings, B. S. Biochem.
Pharmacol. 2014, 90, 338-348.
Magrioti, V.; Kokotos, G. Exp. Opin. Ther. Pat. 2010, 20, 1-18.
Magrioti, V.; Kokotos, G. Exp. Opin. Ther. Pat. 2013, 3, 333-
33. Jakalian, A.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2002,
23, 1623-1641.
34. Cheatham, T. E., III; Cieplak, P.; Kollman, P. A. J. Biomol.
Struct. Dyn. 1999, 16, 845-862.
35. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case,
D. A. J. Comput. Chem. 2004, 25, 1157-1174.
36. Aqvist, J. J. Phys. Chem. 1990, 94, 8021-8024.
37. Joung, I. S.; Cheatham, T. E., III J. Phys. Chem. B 2008, 112,
9020-9041.
7
8
.
.
3
44.
9
1
.
Ong, W. Y.; Farooqui, T.; Kokotos, G.; Farooqui, A. A. ACS
Chem. Neurosci. 2015, 6, 814-831.
0. Seilhamer, J. J.; Pruzanski, W.; Vadas, P.; Plant, S.; Miller, J.
A.; Kloss, J.; Johnson, L. K. J. Biol. Chem. 1989, 264, 5335-
5
338.

3
8. Salomon-Ferrer, R.; Case, D. A.; Walker, R. C. Wiley
Interdiscip. Rev. Comput. Mol. Sci. 2012, DOI:
1
0.1002/wcms.1121.
3
4
4
9. Salomon-Ferrer, R.; Goetz, A. W.; Poole, D.; Le Grand, S.;
Walker, R. C. J. Chem. Theory Comput. 2013, 9, 3878-3888.
0. Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98,
1
0089-10092.
1. Case, D. A.; Cheatham, T. E., III; Darden, T.; Gohlke, H.; Luo,
R.; Merz, K. M., Jr; Onufriev, A.; Simmerling, C.; Wang, B.;
Woods, R. J. J. Comput. Chem. 2005, 26, 1668-1688.
4
2. Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.;
Chong, L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.; Donini, O.;
Cieplak, P.; Srinivasan, J.; Case, D. A.; Cheatham, T. E., III
Acc. Chem. Res. 2000, 33, 889-897.
4
4
3. Honig, B.; Nicholls, A. Science 1995, 268, 1144-1149.
4. Sitkoff, D.; Sharp, K. A.; Honig, B. J. Phys. Chem. 1994, 98,
1
978-1988.
4
5. Magrioti, V.; Nikolaou, A.; Smyrniotou, A.; Shah, I.;
Constantinou-Kokotou, V.; Dennis, A. E.; Kokotos, G. Bioorg.
Med. Chem. 2013, 21, 5823-5829.
4
4
6. Nicolaou, K. C.; Reingruber, R.; Sarlah, D.; Bräse, S. J. Am.
Chem. Soc. 2009, 131, 2086-2087.
7. Gribble, A. D.; Dolle, R. E.; Shaw, A.; McNair, D.; Novelli, R.;
Novelli, C. E.; Slingsby, B. P.; Shah, V. P.; Tew, D.; Saxty, B.
A.; Allen, M.; Groot, P. H.; Pearce, N.; Yates, J. J. Med. Chem.
1
996, 39, 3569-3584.
8. Bethesda Pharmaceuticals INC. Patent: WO 2005/009437 A1,
005.
4
2

Graphical Abstract
Development of a Potent 2-Oxoamide
Inhibitor of Secreted Phospholipase A2
Guided by Molecular Docking Calculations
and Molecular Dynamics Simulations
a
a
b
b
Sofia Vasilakaki , Efrosini Barbayianni , Georgios Leonis , Manthos G. Papadopoulos , Thomas
a
c
a
Mavromoustakos , Michael H. Gelb and George Kokotos ,*
a
Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian University of Athens,
Panepistimiopolis, Athens 15771, Greece
b
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas.
Constantinou Avenue, Athens 11635, Greece
c
Department of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA