2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.12.007

Calcium-independent phospholipase A₂(GVIA iPLA₂) has recently attracted interest as a medicinal target. The number of known GVIA iPLA₂inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyflu

Full text of DOI:10.1016/j.bmc.2016.12.007

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Oxoamides based on dipeptides as selective calcium-independent
phospholipase A2 inhibitors
Anneta Smyrniotou a, Maroula G. Kokotou b,c, Varnavas D. Mouchlis c, Efrosini Barbayianni b,
George Kokotos b, Edward A. Dennis c, , Violetta Constantinou-Kokotou a,
⇑
⇑
a Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
b Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Athens 15771, Greece
c Department of Pharmacology and Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0601, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target.
The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol
lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on
dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on
human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted
GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which
presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectiv-
ity over GIVA cPLA2 and GV sPLA2.
Received 9 September 2016
Revised 4 December 2016
Accepted 5 December 2016
Available online xxxx
Keywords:
Dipeptides
Inhibitors
Oxoamides
Ó 2016 Elsevier Ltd. All rights reserved.
Phospholipase A2
Pseudodipeptides
1. Introduction
As summarized in a recent review,4 increased or decreased
expression of iPLA2s may affect the metabolic state, CNS function,
Phospholipases A2 (PLA2s) are a superfamily of enzymes charac-
terized by their ability to hydrolyze the ester bond at the sn-2 posi-
tion of glycerophospholipids.1 Historically, much attention has
been paid to two types of PLA2s: the cytosolic enzymes (cPLA2)2
and the secreted enzymes (sPLA2).3 More recently in comparison
to them, another group of enzymes, the Ca2+-independent phos-
pholipases A2, designated as GVI iPLA2s, has attracted growing
interest and attention.4–6 The iPLA2s are intracellular enzymes that
do not require Ca2+ either for their activity or for translocation to
membranes. The GVIA iPLA2 is the most widely described of the
iPLA2s and expression of its activity was described in P388D1
macrophage-like cells in 1994.7 In contrast to cPLA2, which exhi-
bits a marked preference for hydrolysis of arachidonic acid from
the sn-2 position, this enzyme does not demonstrate this substrate
specificity. It is a 85 kDa protein (752 amino acids) with a serine
lipase consensus sequence (GTSGT) for an Asp Ser dyad catalytic
mechanism for its catalytic domain which is preceded by eight
N-terminal ankyrin (Ank) repeats.8,9
cardiovascular performance and cell survival and therefore, dysreg-
ulation of iPLA2s may play a critical role in the development of
many diseases. Due to the involvement of PLA2s in chronic inflam-
matory conditions, a variety of synthetic inhibitors have been
developed.1,10–12 Bromoenol lactone (1, BEL, Fig. 1) is the first selec-
tive GVIA iPLA2 inhibitor reported in literature.13 It is an irreversible
and covalent inhibitor and its (R)- and (S)-enantiomers present dif-
ferent inhibitory properties.14 Although BEL has been widely used
to inhibit iPLA2 in cellular systems and in vivo, it has to keep in
mind that it is able to inhibit other enzymes, like magnesium-
dependent phosphatidate phosphohydrolase-1.15 Fluoroketones
FKGK11 (2),16 FKGK18 (3)17 and GK187 (4)18 (Fig. 1) constitute a
very important class of selective and potent GVIA iPLA2 inhibitors.
These inhibitors have been used to study the role of the enzyme
ex vivo and in vivo and in particular in autoimmune diseases.19–22
FKGK11 showed strong reduction in the clinical severity and
progression of experimental autoimmune encephalomyelitis.19
Administration of FKGK18 to non-obese diabetic (NOD) mice signif-
icantly reduced diabetes incidence in association with reduced
insulitis, improved glucose homeostasis, and b-cell preservation.21
Computational studies as well as deuterium exchange mass spec-
trometry, shed light on the binding mode of fluoroketone inhibitors
⇑
Corresponding authors.
E-mail
addresses:
edennis@ucsd.edu
(E.A.
Dennis),
vikon@aua.gr
(V. Constantinou-Kokotou).
http://dx.doi.org/10.1016/j.bmc.2016.12.007
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

2
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Br
O
2.2. Synthesis of inhibitors
C2F5
O
2-Hydroxy acids 8a and 8b were synthesized as described ear-
lier33 and the synthesis of 8c is depicted in Scheme 1. The key-
intermediate was the cyanohydrin 6, which was converted to car-
boxylic acid under hydrolytic conditions.
O
2, FKGK11
1, BEL
O
O
The synthesis of 2-oxoamides 12a–c based on the dipeptide
C2F5
Nle-Gly started by the coupling of carbobenzoxy-L-norleucine (9)
with tert-butyl glycinate (Scheme 2).
CF3
H3CO
After hydrogenation and coupling with 2-hydroxy acids 8a–c,
oxidation of 2-hydroxy amides 11a–c gave the target 2-oxoamides
12a–c. The corresponding acids 13a,b were prepared by treatment
with trifluoroacetic acid. Derivatives 12d–f containing beta-ala-
nine, gamma-aminobutyric acid or 5-aminovaleric acid instead of
glycine were synthesized by similar procedures (Scheme 2). In
addition, the ethyl ester derivative 16 was synthesized in a similar
manner (Scheme 3).
The pseudodipeptides 17a,b were used as starting materials for
the synthesis of the 2-oxoamide ether analogs 19a–d (Scheme 4),
after removal of the N-protecting group and coupling with 2-
hydroxy acids 8a–c.
3, FKGK18
4, GK187
Fig. 1. Structures of known GVIA iPLA2 inhibitors.
in the active site of GVIA iPLA2 and the role that membranes play in
the binding and hydrolysis of the phospholipid substrates.23,24
The aim of this work was to develop new potent and selective
inhibitors of GVIA iPLA2. Based on our observation that some of
the 2-oxoamides we have developed as inhibitors of GIVA cPLA2
presented some GVIA iPLA2 inhibition, we synthesized several
new 2-oxoamides based on dipeptides and pseudodipeptides and
we studied their in vitro inhibitory activity on human GVIA iPLA2
as well as their selectivity over GIVA cPLA2 and GV sPLA2.
2-Oxoamides 25a–g based on a variety of dipeptide tert-butyl
esters were synthesized as shown in Scheme 5. The synthesis of
additional analogs required for the structure-activity relationship
studies is depicted in Schemes 6 and 7.
2. Results and discussion
2.3. In vitro inhibition of GIVA cPLA2, GVIA iPLA2 and GV sPLA2
2.1. Design of inhibitors
All synthesized inhibitors were tested for inhibition of human
GVIA iPLA2, GIVA cPLA2, and GV sPLA2 using previously described
mixed micelle-based assays.26,28,30 The inhibition results are pre-
sented in Tables 1 and 2, either as percent inhibition or as XI(50)
values. At first, the percent of inhibition for each PLA2 enzyme at
0.091 mol fraction of each inhibitor was determined. Then, the
XI(50) values were measured for compounds that displayed greater
than 95% inhibition. The XI(50) is the mole fraction of the inhibitor
in the total substrate interface required to inhibit the enzyme by
50%.
Initially, we studied the role of the dipeptide or dipeptide ana-
log using as aromatic rings phenyl, p-methoxy-phenyl and naph-
thyl (Table 1). First of all, it is obvious that a free carboxyl group
led to totally inactive compounds for GVIA iPLA2 (13a, 13b, 20d).
Thus, an ester group (ethyl or tert-butyl) was employed for the
other derivatives. Comparing compounds 12a with 19b and 19d
with 12c, it seems that the dipeptide unit gave better inhibitory
results for GVIA iPLA2 than the ether pseudodipeptide, irrespec-
tively of which aromatic group was used. Comparing compounds
Both GIVA cPLA2 and GVIA iPLA2 share the same catalytic mech-
anism utilizing serine for their catalytic action. We have developed
a novel class of 2-oxoamides as inhibitors of GIVA cPLA2.25–31 2-
Oxoamides were initially designed to target the active site serine
of GIVA cPLA2. However, it became clear that some 2-oxoamides
could also inhibit GVIA iPLA2. Long chain 2-oxoamides based on
c
- or d-amino acids containing a free carboxyl group were found
to be selective inhibitors of GIVA cPLA2.26,28,30,32 However, the cor-
responding esters inhibit both GIVA cPLA2 and GVIA iPLA2.28 In
particular, we have observed that some 2-oxoamides based on
dipeptides or ether dipeptide analogs presented a slight preference
to inhibit GVIA iPLA2.31
To develop 2-oxoamides as selective GVIA iPLA2 inhibitors, we
designed compounds, where the 2-oxoamide functionality, that
ensures the interaction with the active site serine, was accompa-
nied by an aromatic group at a medium distance from the activated
carbonyl and a dipeptide or a pseudodipeptide unit (Fig. 2). We
have shown by extended structure-activity relationship studies
on polyfluoroketone derivatives that the optimum distance
between the activated carbonyl and the aromatic group corre-
sponds to four carbon atoms.16,17 In addition, the favorable aro-
matic groups were either a phenyl ring, alone or with a para-
methoxy substitution, or a naphthalene ring.17,18 We envisaged
that the presence of a small peptide unit could create favorable
interactions with the amino acid residues of the enzyme. Our pre-
vious studies have shown that polar groups create unfavorable
interactions with the residues of the GVIA iPLA2 active site. Thus,
we employed mainly non-polar amino acids in combination with
an ester C-terminal group. The amino component of the inhibitor
was initially designed to be the dipeptide Nle-Gly.
OH
OH
OH
OH
OH
O
O
8b
8a
5
OH
CN
c
a, b
MeO
MeO
MeO
6
OH
OH
OH
OMe
d
O
O
MeO
7
8c
Scheme 1. Reagents and conditions: (a) NaBr, NaOCl, H2O, NaHCO3, toluene, AcNH-
Tempo, AcOEt; (b) (i) NaHSO3, CH2Cl2 (ii) KCN, H2O; (c) 3 N HCl/CH3OH; (d) NaOH
1 N, CH3OH.
Fig. 2. Design of 2-oxoamides based on dipeptides.
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A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
O
OH
O
O
H
N
OBut
OBut
CbzHN
CbzHN
b, c
a
OH
Ar
N
H
2
N
H
n
O
n
O
O
2
2
10a-d
9
11a-f
O
O
O
O
H
N
H
N
e
OBut
d
OH
Ar
Ar
N
H
N
H
n
n
O
O
O
O
2
2
12a-f
13a,b
11-13
n
Ar
10
n
a
b
c
d
1
a
b
1
2
3
4
1
c
1
2
MeO
MeO
d
e
3
4
MeO
MeO
f
Scheme 2. Reagents and conditions: (a) HClÁH2N(CH2)nCOOC(CH3)3, WSCIÁHCl, HOBt, Et3N, CH2Cl2, (b) H2, 10% Pd/C, THF; (c) 8a–c, WSCIÁHCl, HOBt, Et3N, CH2Cl2; (d) Dess-
Martin periodinane, CH2Cl2; (e) 50% TFA/CH2Cl2.
O
OH
the activity. The next step was the elongation of the carbon chain
of Gly keeping Nle as the first amino acid of the dipeptide.
Thus, analogs of 12c containing beta-alanine (compound 12d),
or GABA (compound 12e) or aminovaleric acid (compound 12f)
were tested. It was gratifying that compound 12e (GK317) exhib-
ited higher activity (XI(50) 0.007) in comparison to 12c. Finally,
the tert-butyl ester of 12e was replaced by a benzyl one (com-
pound 31b) and Nle of 12e by Leu (compound 22g). However, both
derivatives presented reduced inhibitory activity of GVIA iPLA2 in
comparison to 12e.
O
H
N
OEt
BocHN
OEt
a,b
N
H
2
N
H
O
O
O
2
14
15
O
O
H
N
OEt
c
N
H
O
O
2
16
Taken together, we have identified a 2-oxoamide based on Nle-
GABA-OBut, compound 12e (GK317), which presents potent inhibi-
tion of GVIA iPLA2 (XI(50) value of 0.007). This compound was
found to present weak inhibitory activity over the other major
intracellular GIVA cPLA2 (52.6% at 0.091 mol fraction) and the
secreted GV sPLA2 (44.8% at 0.091 mol fraction). The length of
the chain of the C-terminal amino acid seems critical, because
either increase or decrease results in reduction of the inhibitory
activity on GVIA iPLA2. Two commercially available inhibitors of
GVIA iPLA2 and GIVA cPLA2 have been included in Table 2 for com-
parison purposes. FKGK11 selectively inhibits GVIA iPLA2 [XI(50)
0.014]17, while AACOCF3 inhibits both GVIA iPLA2 [XI(50) 0.028]13
and GIVA cPLA2 [XI(50) 0.036].1
Phospholipases A2 are water-soluble enzymes acting on water-
insoluble substrates that exist in aggregated form in aqueous solu-
tion. Mixed micelles and the surface dilution kinetics model were
successfully used by our laboratory for assaying the enzymatic
activity of these enzymes and conducting inhibitory response
curves for small molecule inhibitors. As part of surface-dilution
considerations, the enzyme may undergo surface binding to mem-
branes, whereby the enzyme either associates nonspecifically with
the surface of the lipid aggregate or associates specifically with a
phospholipid in the aggregate’s surface. Thus, a two dimensional
unit such as mole fraction is more relevant for expressing the inhi-
bitory activity of PLA2 inhibitors since they will first incorporate in
the aggregated substrate in order to access the enzyme active site
Scheme 3. Reagents and conditions: (a) HCl/Et2O; (b) 8b, WSCIÁHCl, HOBt, Et3N,
CH2Cl2; (c) Dess-Martin periodinane.
16 with 12b and 19a with 19d, tert-butyl ester provided better
results than the ethyl ester. From the results summarized in
Table 1, compound 12c stands out as for the inhibition of GVIA
iPLA2. It inhibited GVIA iPLA2 with an XI(50) value of 0.012, while
it presented only weak inhibition of GIVA cPLA2 and GV sPLA2.
Thus, further modifications were accomplished on compound
12c, which is based on the dipeptide Nle-Gly-OBut.
The results of the in vitro potency and selectivity for analogs of
12c are summarized in Table 2. Replacement of the tert-butyl
group of 12c by the benzyl one (compound 31a) reduced the inhi-
bitory potency (from XI(50) 0.012 to XI(50) 0.026). Increase or
decrease of the peptide size destroyed the activity. The 2-oxoa-
mides based on either a single amino acid derivative, Nle-OBut
(compound 29), Nle-NH2 (compound 27), or a tripeptide, Nle-
Gly-Gly-OBut (compound 33), presented weak activity on GIVA
iPLA2. Replacement of Nle by other amino acids containing small
aliphatic chains, such as Leu, Ile, and Val (compounds 22a–c) was
examined. Only Leu analog (22a) produced an interesting activity,
however half potency (XI(50) 0.024) in comparison with the Nle
derivative 12c. Then, Gly was replaced by
22c and 22d), but again a decrease of the activity was observed.
The replacement of -Nle by -Nle (compound 22f) also decreased
L- and D-Ala (compounds
L
D
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4
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
OH
H
PHN
OR
N
OR
d
a or b, c
O
O
Ar
O
O
O
2
2
18a-d
17a,b
O
O
H
N
H
OR
N
OH
O
O
e
O
Ar
Ar
O
O
O
2
2
20d
Ar
19a-d
17
a
P
R
18-20
a
R
Boc
Et
Et
MeO
Cbz But
b
But
But
b
c
MeO
But
d
Scheme 4. Reagents and conditions: (a) TFA/CH2Cl2; (b) H2, 10% Pd/C, THF; (c) 8a–c, WSCIÁHCl, HOBt, Et3N, CH2Cl2; (d) Dess-Martin periodinane, CH2Cl2; (e) 50% TFA/CH2Cl2.
and compete with phospholipid substrate. With this caveat, we
hereby provide the effective concentrations translated to solution
for the most potent iPLA2 inhibitors of this series and the reference
a
b, c
+
H-AA2-OBut
Z-AA1-AA2-OBut
Z-AA1-OH
21a-e
22a-d
23a-g
inhibitors: compound 12c IC50 6.6
compound 22a IC50 13 M, compound 12e IC50 3.8
IC50 0.77 M, AACOCF3 IC50 15 M.
l
M, compound 31a IC50 14
lM,
OH
l
lM, FKGK11
AA1-AA2-OBut
d
l
l
O
MeO
MeO
3. Conclusion
24a-g
O
AA1-AA2-OBut
In conclusion, a variety of 2-oxoamides based on dipeptides and
ether dipeptides were synthesized. Structure-activity relationship
studies revealed the first iPLA2 selective 2-oxoamide, compound
12e (GK317), which presents around 13 times more potent inhibi-
tion of GVIA iPLA2 than of GIVA cPLA2. This inhibitor has the same
order of magnitude of GVIA iPLA2 inhibition with our previously
described fluoroketone inhibitor FKGK11 (XI(50) 0.0014). Impor-
tantly, FKGK11 has been studied in vivo in various animal models
and significant effects have been reported for its action in animal
models of autoimmune diseases. Thus, we propose that the new
oxoamide inhibitor GK317 may be a useful tool for studies in cells
and in vivo and may serve as a lead for the development of more
potent and selective oxoamide inhibitors of GVIA iPLA2. The dipep-
tidic component of the inhibitor seems that contributes to the
selectivity for GVIA iPLA2.
O
25a-g
21
23-25
AA1 AA2
L-Leu Gly
AA1
22
AA2
a
b
c
d
e
f
a
b
c
d
e
L-Leu
L-Ile
L-Val
L-Nle
D-Nle
a
b
c
d
Gly
L-Ile
Gly
Gly
L-Ala
L-Val
D-Ala
GABA
L-Nle L-Ala
L-Nle D-Ala
D-Nle Gly
L-Leu GABA
g
Scheme 5. Reagents and conditions: (a) WSCIÁHCl, HOBt, Et3N, CH2Cl2; (b) H2, 10%
Pd/C, THF; (c) 8c, WSCIÁHCl, HOBt, Et3N, CH2Cl2; (d) Dess-Martin periodinane,
CH2Cl2.
OH
O
O
O
H
N
H
N
b
NH2
NH2
O
O
MeO
MeO
2
2
26
8c
27
a
a
O
O
OH
O
H
N
H
N
OBut
OBut
b
O
O
MeO
MeO
2
2
29
28
Scheme 6. Reagents and conditions: (a) Nle-NH2 (for 26) or Nle-OBut (for 28), WSCIÁHCl, HOBt, Et3N, CH2Cl2; (b) Dess-Martin periodinane, CH2Cl2.
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A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
O
OH
Nle-AA-OBn
Nle-AA-OBn
b
O
O
MeO
MeO
31a,b
30a,b
a
30, 31
n
8c
a
b
Gly
GABA
a
O
OH
Nle-Gly-Gly-OBut
Nle-Gly-Gly-OBut
b
O
O
MeO
MeO
33
32
Scheme 7. Reagents and conditions: (a) Nle-AA-OBn (for 30a,b) or Nle-Gly-Gly-OBut (for 32), WSCIÁHCl, HOBt, Et3N, CH2Cl2; (b) Dess-Martin periodinane, CH2Cl2.
mixture was stirred for 18 h at room temperature. Then, the aque-
ous layer was washed with CH2Cl2 (10 mL) and the organic layer
was washed with brine and dried over Na2SO4. The solvent was
evaporated under reduced pressure and the residue was purified
by column chromatography using EtOAc/petroleum ether (bp 40–
60 °C) 2:8 as eluent. Yield 67%; Yellowish oil; 1H NMR (CDCl3): d
7.11 (d, J = 8.2 Hz, 2H, arom), 6.85 (d, J = 8.6 Hz, 2H, arom), 4.43
(q, J = 6.6 Hz, 1H, CH), 3.80 (s, 3H, OCH3), 3.58 (d, J = 6.6 Hz, 1H,
OH), 2.60 (t, J = 7.0 Hz, 2H, PhCH2), 1.95–1.80 (m, 2H, CH2), 1.75–
1.45 (m, 4H, 2ÂCH2); 13C NMR (CDCl3): d 157.5, 134.0, 129.2,
120.0, 113.7, 61.0, 55.2, 34.8, 34.5, 30.8, 24.0; MS (ESI) m/z (%):
237 ([M+NH4]+, 100); Anal. Calcd for C13H17NO2: C, 71.21; H,
7.81; N, 6.39. Found: C, 71.01; H, 7.94; N, 6.33.
4. Experimental section
4.1. General procedures
Merck Silica Gel 60 (70–230 or 230–400 mesh) was used for the
chromatographic purification of products and Silica Gel 60,254 alu-
minum plates for the thin-layer chromatography (TLC). UV light
and/or phosphomolybdic acid and/or ninhydrin in EtOH was
employed for visualizing spots. A Büchi 530 apparatus was used
to estimate melting points and they were uncorrected. 1H and
13C NMR spectra were recorded on Varian Mercury at 200 MHz
and 50 MHz respectively. Samples were diluted in CDCl3. Chemical
shifts are given in ppm, and coupling constants (J) in Hz. Peak mul-
tiplicities are typified as: s, singlet, d, doublet, dd, doublet of dou-
plets, t, triplet, q, quartet 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 °C on a Perkin-Elmer 343 polarimeter using a
10 cm cell. Dichloromethane was dried by standard procedures
and stored over molecular sieves. No further purification of other
solvents and chemicals needed as they were reagent grade. HRMS
4.2.2. Methyl 2-hydroxy-6-(4-methoxyphenyl)hexanoate (7)
A solution of cyanhydrin 6 (1.0 mmol) in dry HCl/CH3OH 6 M
(0.33 mL) was stirred for 18 h at room temperature. The organic
solvent was evaporated under reduced pressure and H2O was
added (1 mL) as well as K2CO3 for neutralizing pH. The aqueous
layer was washed with EtOAc (3 Â 15 mL) and the combined
organic layers were dried over Na2SO4. The solvent was evaporated
under reduced pressure and the residue was purified by column
chromatography using EtOAc/petroleum ether (bp 40–60 °C) 2:8
as eluent. Yield 70%; Colorless oil; 1H NMR (CDCl3): d 7.09 (d,
J = 8.4 Hz, 1H, arom), 6.83 (d, J = 8.8 Hz, 1H, arom), 4.19 (q,
J = 5.2 Hz, 1H, CH), 3.79 (s, 3H, PhOCH3), 3.77 (s, 3H, COOCH3),
2.77 (d, J = 5.6 Hz, 1H, OH), 2.57 (t, J = 7.2 Hz, 2H, PhCH2), 1.90–
1.40 (m, 6H, 3ÂCH2); 13C NMR (CDCl3): d 175.7, 157.6, 134.4,
129.2, 113.6, 70.3, 55.2, 52.5, 34.8, 34.2, 31.4, 24.4; MS (ESI) m/z
(%): 270 ([M+NH4]+, 100); Anal. Calcd for C14H20O4: C, 66.65; H,
7.99. Found: C, 66.42; H, 8.05.
spectra were recorded on
Spectrometer.
a Bruker Maxis Impact QTOF
Compounds 8a, 8b have been described elsewhere and their
analytical data are in accordance with literature.33
4.2. Chemistry
4.2.1. 2-Hydroxy-6-(4-methoxyphenyl)hexanenitrile (6)
To a stirred solution of alcohol 5 (1.0 mmol) in toluene and
EtOAc (1:1, 6.0 mL), H2O (0.5 mL) and NaBr (0.11 g, 1.1 mmol) were
added. The mixture was cooled at À5 °C and under vigorous stir-
ring AcNH-Tempo (0.21 mg, 0.01 mmol) was added, followed by
the addition of an aqueous solution of NaOCl 0.5 M (2.2 mL,
1.1 mmol) and NaHCO3 (0.24 g, 3.0 mmol) within 1 h. The aqueous
layer was separated and washed with EtOAc (20 mL). The com-
bined organic layers were washed consecutively with 5% aqueous
citric acid (10 mL) containing KI (0.04 g), 10% aqueous Na2S2O3
(10 mL), and brine and dried over Na2SO4. The solvents were evap-
orated under reduced pressure and the residue was used directly to
the next reaction.
To a solution of the resulting aldehyde (1.0 mmol) in CH2Cl2
(1.3 mL) an aqueous solution of NaHSO3 (0.25 mL, 6 M) was added
and a white solid was formed. The mixture was stirred for 30 min
at room temperature, the organic solvent was evaporated under
reduced pressure, water (1.0 mL) was added and the mixture was
cooled at 0 °C. Under vigorous stirring an aqueous solution of
KCN 6 M (0.25 mL, 15.0 mmol) was added dropwise. The reaction
4.2.3. 2-Hydroxy-6-(4-methoxyphenyl)hexanoic acid (8c)
To a stirred solution of a methyl ester 7 (1.0 mmol) in methanol
(10 mL), 1 M NaOH (1.5 mmol) was added and the mixture was left
overnight at room temperature. After the completion of the reac-
tion, methanol was evaporated under reduced pressure, water
(10 mL) was added and the mixture was acidified with 1 M HCl
to pH 1. The aqueous layer was extracted with EtOAc
(3 Â 15 mL). The combined organic layers were washed with brine,
dried over Na2SO4 and evaporated under reduced pressure. Yield
96%; White solid; mp 94–96 °C; 1H NMR (CDCl3): d 7.10 (d,
J = 8.4 Hz, 2H, arom), 6.83 (d, J = 8.8 Hz, 2H, arom), 4.35–4.20 (m,
1H, CH), 3.79 (s, 3H, OCH3), 5.65–2.50 (m, 2H, CH2CH), 2.00–1.40
(m, 7H, 3ÂCH2, OH);); 13C NMR (CDCl3): d 179.8, 157.6, 134.4,
129.2, 113.7, 70.1, 55.2, 34.7, 33.9, 31.3, 24.4; MS (ESI) m/z (%):
237 ([MÀH]À, 100); Anal. Calcd for C13H18O4: C, 65.53; H, 7.61.
Found: C, 65.32; H, 7.78.
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6
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 1
2-Oxoamides based on the dipeptide Nle-Gly and its ether analog containing various aromatic rings.a
Number
Structure
% Inhibition
GVIA iPLA2
91.1 0.6
GIVA cPLA2
46.9 3.8
GV sPLA2
43.4 5.5
12a
13a
N.D.
N.D.
N.D.
19b
12b
13b
76.0 3.5
89.1 1.0
N.D.
82.3 1.4
57.5 4.3
43.1 5.1
57.5 4.6
44.8 5.7
N.D.
16
77.1 1.1
88.4 2.1
93.6
70.5 3.8
85.5 1.0
–
54.4 4.6
63.9 4.3
–
19c
19d
20d
19a
12c
N.D.
–
–
75.2
–
–
>95 XI(50) 0.012 0.002
40.2 2.5
45.3 3.3
a
Average percent inhibition and standard error (n = 3) are reported for each compound at 0.091 mol fraction. XI(50) values were determined for inhibitors with greater
than 95% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).
4.2.4. Coupling method
To a stirred solution of the amino component (1.0 mmol) in
CH2Cl2 (10.0 mL) cooled at 0 °C, Et3N (0.3 mL, 2.2 mmol) and sub-
4.2.4.1. tert-Butyl (S)-(2-(((benzyloxy)carbonyl)amino)hexanoyl)gly-
cinate (10a). Yield 62%; Yellow oil; 1H NMR (CDCl3): d 7.39–7.28
(m, 5H, arom), 6.70 (br s, 1H, NHCO), 5.47 (d, J = 7.4 Hz, 1H,
OCONH), 5.09 (s, 2H, PhCH2), 4.20–4.03 (m, 1H, CH), 3.97–3.92
(m, 2H, CH2), 1.80–1.50 (m, 2H, CHCH2), 1.44 [s, 9H, C(CH3)3],
1.35–1.22 (m, 4H, 2ÂCH2), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR
(CDCl3): d 172.3, 169.8, 156.2, 136.3, 128.6, 128.1, 128.0, 81.2,
66.9, 56.8, 41.3, 34.9, 31.9, 28.3, 25.7, 22.4, 13.9; MS (ESI) m/z
(%): 377 ([MÀH]À, 100); Anal. Calcd for C20H30N2O5: C, 63.47; H,
7.99; N, 7.40. Found: C, 63.12; H, 8.13; N, 7.35.
sequently
1-(3-dimethyl-aminopropyl)-3-ethyl
carbodiimide
hydrochloride (WSCIÁHCl) (0.21 g, 1.1 mmol) and 1-hydroxybenzo-
triazole (HOBt) (0.14 g, 1.0 mmol) were added. The acid compo-
nent (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, 5% citric acid, brine, 5%
NaHCO3 and brine, dried over Na2SO4 and evaporated under
reduced pressure. Purification by column chromatography eluting
with the appropriate mixture of EtOAc/petroleum ether (bp 40–
60 °C) afforded the product.
4.2.4.2. (S)-tert-Butyl 3-(2(((benzyloxy)carbonyl)amino) hexanamido)
20
propanoate (10b). Yield 46%; Yellow oil; [
a]
À7.2 (c 1.00, CHCl3);
D
1H NMR (CDCl3): d 7.41–7.28 (m, 5H, arom), 6.65–6.45 (m, 1H,
NHCO), 5.39 (d, J = 7.4 Hz, 1H, OCONH), 5.10 (s, 2H, PhCH2),
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A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
Table 2
2-Oxoamide analogs of inhibitor 12c.a
Number
Structure
% Inhibition
GVIA iPLA2
GIVA cPLA2
40.2 2.5
GV sPLA2
45.3 3.3
12c
>95 XI(50) 0.012 0.002
O
O
H
N
O
N
H
O
O
O
O
2
31a
29
>95 XI(50) 0.026 0.008
52.3 1.2
85.7 1.0
N.D.
O
O
H
O
N
N
H
O
O
2
60.0 3.0
66.2 4.0
60.6 3.2
O
O
H
N
O
O
O
2
27
29.3 2.6
31.1 3.1
O
O
H
N
NH2
O
O
O
2
33
66.5 2.5
27.1 3.6
51.6 4.2
N.D.
O
O
O
H
N
H
N
N
O
H
O
O
O
2
22a
>95 XI(50) 0.024 0.06
40.8 5.2
O
H
N
O
N
H
O
O
O
22b
81.8 4.6
57.3 3.3
48.2 9.3
O
O
H
O
N
N
H
O
O
O
22c
22d
85.7 1.7
87.1 1.0
35.4 4.4
73.4 1.3
47.1 4.0
49.9 0.8
O
O
H
O
N
N
H
O
O
O
O
O
O
O
H
N
O
N
H
O
2
22c
87.5 1.9
57.4 3.1
46.6 6.6
O
O
H
N
O
N
H
O
O
O
O
O
2
22f
O
O
>95 XI(50) 0.04 0.04
68.0 0.4
37.8 2.3
39.3 4.6
32.5 6.2
H
N
N
H
O
O
2
12d
83.1 1.4
O
O
O
H
N
O
N
H
O
O
2
12e
GK317
O
O
>95 XI(50) 0.007 0.001
52.6 2.5
64.5 5.8
44.8 4.5
56.3 4.5
H
N
O
N
H
O
O
O
O
O
( )2
O
O
12f
90.1 2.5
H
N
N
H
( )2
O
O
(continued on next page)
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8
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 2 (continued)
Number
Structure
% Inhibition
GVIA iPLA2
GIVA cPLA2
47.9 8.6
GV sPLA2
27.7 4.8
31b
22g
78.3 9.3
O
O
H
N
O
N
H
O
O
O
( )2
92.8 1.0
58.0 3.7
N.D.
O
O
H
N
O
N
H
O
O
O
FKGK11
XI(50) 0.01417
XI(50) 0.02813
N.D.17
2817
O
C2F5
O
AACOCF3
XI(50) 0.03613
–
CF3
a
Average percent inhibition and standard error (n = 3) are reported for each compound at 0.091 mol fraction. XI(50) values were determined for inhibitors with greater
than 95% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).
4.20–4.00 (m, 1H, CH), 3.57–3.38 (m, 2H, NHCH2), 2.43 (t,
J = 6.0 Hz, 2H, CH2COOBut), 1.80–1.50 (m, 2H, CHCH2), 1.45 [s,
9H, C(CH3)3], 1.35–1.22 (m, 4H, 2ÂCH2), 0.88 (t, J = 6.6 Hz, 3H,
CH3); 13C NMR (CDCl3): d 171.7, 171.6, 156.0, 136.2, 128.5, 128.1,
128.0, 81.1, 66.9, 55.0, 35.0, 34.9, 32.6, 28.1, 27.4, 22.4, 13.9; MS
(ESI) m/z (%): 391 ([MÀH]À, 100); Anal. Calcd for C21H32N2O5: C,
64.26; H, 8.22; N, 7.14. Found: C, 64.08; H, 8.44; N, 7.01.
4.2.4.6. tert-Butyl 2-((2S)-2-(2-hydroxy-6-(naphthalen-2-yl)hex-
anamido) hexanamido)acetate (mixture of diastereomers) (11b).
Yield 47%; Colorless oil; 1H NMR (CDCl3): d 7.80–7.23 (m, 8H, arom,
NH), 7.15–6.99 (m, 1H, NH), 4.63–4.40 (m, 1H, CH), 4.23–4.06 (m,
1H, CH), 4.02–3.75 (m, 2H, CH2NH), 2.76 (t, J = 7.6 Hz, 2H, PhCH2),
2.02–1.18 [m, 21H, 6ÂCH2, C(CH3)3], 0.88 (t, J = 6.4 Hz, 3H, CH3);
13C NMR (CDCl3): d 174.5, 174.8, 172.0, 172.3, 168.7, 168.6,
139.9, 133.5, 131.8, 127.7, 127.5, 127.3, 127.2, 126.2, 125.8,
125.0, 82.3, 72.0, 71.9, 52.7, 52.5, 41.9, 35.8, 34.6, 32.0, 31.1,
27.9, 27.5, 24.7, 22.3, 22.2, 13.9; MS (ESI) m/z (%): 485 ([M+H]+,
100); Anal Calcd for C28H40N2O5: C, 69.39; H, 8.32; N, 5.78. Found:
C, 69.24; H, 8.44; N, 5.60.
4.2.4.3. (S)-tert-Butyl 4-(2-(((benzyloxy)carbonyl)amino)hexa-namido)
butanoate (10c). Yield 70%; Pink-brown solid; mp 57–59 °C; [a D20
]
À2.9 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.35–7.28 (m, 5H, arom),
6.75 (br s, 1H, NHCO), 5.69 (d, J = 8.4 Hz, 1H, OCONH), 5.07 (s, 2H,
PhCH2), 4.20–4.10 (m, 1H, CH), 3.32–3.15 (m, 2H, NHCH2), 2.23 (t,
J = 7.2 Hz, 2H, CH2COOBut), 1.83–1.55 (m, 4H, CH2CH2COOBut,
CHCH2), 1.41 [s, 9H, C(CH3)3], 1.35–1.24 (m, 4H, 2ÂCH2), 0.85 (t,
J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3): d 172.9, 172.2, 156.4, 136.5,
128.7, 128.3, 128.2, 80.8, 67.1, 55.3, 39.1, 33.0, 32.9, 28.3, 27.8,
24.8, 22.6, 14.1; MS (ESI) m/z (%): 405 ([MÀH]À, 100); Anal. Calcd
for C22H34N2O5: C, 65.00; H, 8.43; N, 6.89. Found: C, 64.87; H,
8.56; N, 6.78.
4.2.4.7. tert-Butyl 2-((2S)-2-(2-hydroxy-6-(4-methoxy-phenyl)hex-
anamido)hexanamido)acetate (mixture of diastereomers) (11c). Yield
81%; Colorless oil; 1H NMR (CDCl3): d 7.26–7.13 (m, 1H, NH), 7.07
(d, J = 8.4 Hz, 2H, arom), 6.95–6.85 (m, 1H, NH), 6.81 (d, J = 8.6 Hz,
2H, arom), 4.57–4.41 (m, 1H, CH), 4.19–4.05 (m, 1H, CH), 4.00–3.93
(m, 1H, OH), 3.89 (d, J = 5.2 Hz, 2H, CH2COOBut), 3.78 (s, 3H, CH3O),
2.55 (t, J = 7.6 Hz, 2H, PhCH2), 1.95–1.49 (m, 6H, 3ÂCH2), 1.46 [s,
9H, C(CH3)3], 1.39–1.21 (m, 6H, 3ÂCH2), 0.89 (t, J = 6.2 Hz, 3H,
CH3); 13C NMR (CDCl3): d 174.6, 174.3, 172.2, 171.9, 168.7, 168.6,
157.6, 134.5, 129.2, 113.6, 82.4, 72.1, 55.2, 52.7, 52.6, 42.0, 34.7,
34.6, 32.0, 31.4, 28.0, 27.6, 24.7, 22.3, 13.9; MS (ESI) m/z (%): 463
([MÀH]À, 100); Anal Calcd for C25H40N2O6: C, 64.63; H, 8.68; N,
6.03. Found: C, 64.45; H, 8.79; N, 5.91.
4.2.4.4. (S)-tert-Butyl 5-(2-(benzyloxycarbonylamino)hexa-namido)
pentanoate (10d). Yield 60%; Yellow oil; 1H NMR (CDCl3): d 7.44–
7.15 (m, 5H, arom), 6.66–6.41 (m, 1H, NHCO), 5.61 (d, J = 8.2 Hz,
1H, OCONH), 5.05 (s, 2H, CH2), 4.24–3.95 (m, 1H, CH), 3.34–3.00
(m, 2H, CH2), 2.32–2.03 (m, 2H, CH2), 1.90–1.11 [m, 19H, 5ÂCH2,
C(CH3)3], 0.95–0.71 (m, 3H, CH3); 13C NMR (CDCl3): d 172.8,
171.8, 156.1, 136.1, 128.4, 128.0, 127.9, 80.2, 66.8, 55.0, 38.9,
34.8, 32.5, 28.7, 28.0, 27.5, 23.7, 22.3, 13.8; MS (ESI) m/z (%): 419
([MÀH]À, 100); Anal. Calcd for C23H36N2O5: C, 65.69; H, 8.63; N,
6.66. Found: C, 65.43; H, 8.79; N, 6.54.
4.2.4.8. tert-Butyl 3-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)hexanamido)propanoate (mixture of diastereomers) (11d).
Yield 57%; Yellow oil; 1H NMR (CDCl3): d 7.21–7.12 (m, 1H, NH),
7.07 (d, J = 8.6 Hz, 2H, arom), 6.81 (d, J = 8.6 Hz, 2H, arom), 4.45–
4.28 (m, 1H, CH), 4.17–4.04 (m, 1H, CH), 3.78 (s, 3H, CH3O),
3.52–3.35 (m, 2H, NHCH2), 2.55 (t, J = 7.4 Hz, 2H, PhCH2), 2.43 (t,
J = 6.4 Hz, 2H, CH2COOBut), 1.91–1.48 (m, 6H, 3ÂCH2), 1.45 [s,
9H, C(CH3)3], 1.36–1.16 (m, 6H, 3ÂCH2), 0.88 (t, J = 6.2 Hz, 3H,
CH3); 13C NMR (CDCl3): d 174.3, 174.1, 171.7, 171.5, 157.6, 134.5,
129.2, 113.6, 81.3, 71.9, 55.2, 52.7, 35.1, 34.8, 34.5, 32.3, 31.4,
29.7, 28.1, 27.5, 24.7, 22.3, 13.9; MS (ESI) m/z (%): 477 ([MÀH]À,
100); Anal Calcd for C26H42N2O6: C, 65.25; H, 8.84; N, 5.85. Found:
C, 65.01; H, 8.98; N, 5.72.
4.2.4.5. tert-Butyl 2-((2S)-2-(2-hydroxy-6-phenylhexa-namido)hex-
anamido)acetate (mixture of diastereomers) (11a). Yield 66%; Color-
less oil; 1H NMR (CDCl3): d 7.53–7.03 (m, 6H, arom, NH), 6.95–6.78
(m, 1H, NH), 4.58–4.41 (m, 1H, CH), 4.19–4.06 (m, 1H, CH), 4.02–
3.76 (m, 2H, CH2), 3.48 (br s, 1H, OH), 2.61 (t, J = 7.2 Hz, 2H, PhCH2),
1.99–1.19 [m, 21H, 6ÂCH2, C(CH3)3], 0.89 (t, J = 6.4 Hz, 3H, CH3);
13C NMR (CDCl3): d 174.4, 174.1, 171.9, 171.6, 168.8, 168.6,
142.4, 128.3, 128.2, 125.7, 82.5, 82.0, 71.9, 52.6, 52.8, 42.0, 35.8,
35.7, 34.5, 34.7, 31.8, 31.9, 31.2, 28.0, 27.6, 24.7, 24.6, 22.3, 22.4,
13.9; MS (ESI) m/z (%): 435 ([M+H]+, 75); Anal Calcd for
4.2.4.9. tert-Butyl 4-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)hexanamido)butanoate (mixture of diastereomers) (11e).
Yield 37%; Yellow oil; 1H NMR (CDCl3): d 7.26–7.16 (m, 1H, NH),
C24H38N2O5: C, 66.33; H, 8.81; N, 6.45. Found: C, 66.09; H, 8.92;
N, 6.32.
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A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
7.07 (d, J = 8.2 Hz, 2H, arom), 6.99–6.87 (m, 1H, NH), 6.81 (d,
J = 8.4 Hz, 2H, arom), 4.49–4.29 (m, 1H, CH), 4.17–4.03 (m, 1H,
CH), 3.78 (s, 3H, CH3O), 3.36–3.11 (m, 2H, NHCH2), 2.55 (t,
J = 7.6 Hz, 2H, PhCH2), 2.25 (t, J = 7.2 Hz, 2H, CH2COOBut), 1.90–
1.47 (m, 8H, 4ÂCH2), 1.43 [s, 9H, C(CH3)3], 1.35–1.18 (m, 6H,
3ÂCH2), 0.88 (t, J = 6.2 Hz, 3H, CH3); 13C NMR (CDCl3): d 174.6,
174.3, 173.0, 172.7, 172.0, 171.7, 157.6, 134.4, 129.2, 113.6, 80.7,
71.9, 55.2, 52.9, 39.0, 34.8, 32.9, 32.1, 31.5, 29.7, 28.0, 27.7, 25.9,
24.7, 22.4, 13.9; MS (ESI) m/z (%): 493 ([M+H]+, 100); Anal Calcd
for C27H44N2O6: C, 65.83; H, 9.00; N, 5.69. Found: C, 65.68; H,
9.22; N, 5.56.
67%; Yellowish oil; 1H NMR (CDCl3): d 7.15–7.04 (m, 1H, NH),
7.06 (d, J = 8.6 Hz, 2H, arom), 6.79 (d, J = 8.6 Hz, 2H, arom), 4.20–
3.95 (m, 2H, 2ÂCH), 3.93 (s, 2H, OCH2CO), 3.76 (s, 3H, CH3O),
3.60 (dd, J1 = 9.6 Hz, J2 = 3.6 Hz, 1H, CHCHHO), 3.50 (dd,
J1 = 10.8 Hz, J2 = 4.0 Hz, 1H, CHCHHO), 2.54 (t, J = 7.2 Hz, 2H,
PhCH2), 2.44 (br s, 1H, OH), 1.95–1.50 (m, 6H, 3ÂCH2), 1.45 [s,
9H, C(CH3)3], 1.40–1.20 (m, 6H, 3ÂCH2), 0.88 (t, J = 6.2 Hz, 3H,
CH3); 13C NMR (CDCl3): d 174.1, 173.9, 170.1, 170.0, 157.4, 134.6,
134.5, 129.1, 125.4, 113.5, 82.1, 82.0, 73.0, 72.0, 71.6, 68.5, 68.3,
55.1, 48.8, 48.5, 34.8, 34.5, 30.1, 29.6, 31.5, 30.9, 29.5, 28.1, 28.0,
24.6, 24.4, 22.4, 13.9; MS (ESI) m/z (%): 452 ([M+H]+, 100); Anal
Calcd for C25H41NO6: C, 66.49; H, 9.15; N, 3.10. Found: C, 66.21;
H, 9.31; N, 3.02.
4.2.4.10. tert-Butyl 5-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)hexanamido)pentanoate (mixture of diastereomers) (11f).
Yield 37%; Yellow oil; 1H NMR (CDCl3): d 7.70–7.29 (m, 2H,
2ÂNHCO), 7.06 (d, J = 8.2 Hz, 2H, arom), 6.79 (d, J = 8.2 Hz, 2H,
arom), 4.49–4.27 (m, 1H, CH), 4.19–3.98 (m, 1H, CH), 3.76 (s, 3H,
CH3O), 3.31–3.01 (m, 2H, NHCH2), 2.63–2.37 (m, 2H, PhCH2),
2.29–2.06 (m, 2H, CH2), 1.93–1.71 (m, 2H, CH2), 1.70–1.06 [m,
23H, C(CH3)3, 7ÂCH2], 0.87 (t, J = 6.6 Hz, 3H, CH3); 13C NMR
(CDCl3): d 174.9, 173.2, 172.0, 157.5, 134.5, 129.1, 113.6, 80.4,
71.9, 55.1, 52.6, 39.0, 34.8, 34.5, 31.8, 31.4, 28.6, 28.0, 27.6, 24.7,
22.3, 21.9, 13.8; MS (ESI) m/z (%): 507 ([M+H]+, 100); Anal Calcd
for C28H46N2O6: C, 66.37; H, 9.15; N, 5.53. Found: C, 66.04; H,
9.34; N, 5.42.
4.2.4.15. tert-Butyl 2-((2S)-2-(2-hydroxy-6-(naphthalen-2-yl)hex-
anamido)hexyl)oxy)acetate (mixture of diastereomers) (18d). Yield
69%; Yellow oil; 1H NMR (CDCl3): d 7.91–7.18 (m, 7H, arom),
7.01 (br s, 1H, NH), 4.23–3.77 (m, 4H, 2ÂCH, CH2COO), 3.70–3.32
(m, 2H, CH2O), 2.79 (t, J = 6.6 Hz, 2H, PhCH2), 2.01–1.04 [m, 21H,
6ÂCH2, C(CH3)3], 0.89 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3): d
179.6, 170.4, 140.0, 133.6, 131.9, 127.8, 127.7, 127.6, 127.4,
126.3, 125.8, 125.0, 82.1, 73.1, 72.1, 68.3, 49.1, 48.9, 35.9, 31.1,
31.0, 28.2, 28.0, 24.7, 24.6, 22.5, 14.0; MS (ESI) m/z (%): 472 ([M
+H]+, 100); Anal Calcd for C28H41NO5: C, 71.31; H, 8.76; N, 2.97.
Found: C, 71.12; H, 8.84; N, 2.93.
4.2.4.11. Ethyl 2-((2S)-2-(2-hydroxy-6-(naphthalen-2-yl)hexanamido)
hexanamido)acetate (mixture of diastereomers) (15). Yield 42%; Whi-
tish oil; 1H NMR (CDCl3): d 7.83–6.77 (m, 9H, 2ÂNH, arom), 4.57–
4.34 (m, 1H, CH), 4.26–4.06 (m, 3H, COOCH2, CH), 3.97 (d,
J = 5.0 Hz, 2H, NHCH2), 2.78 (t, J = 7.4 Hz, 2H, PhCH2), 2.02–1.41
(m, 8H, 4ÂCH2), 1.39–1.17 (m, 7H, 2ÂCH2, CH3), 0.89 (t, J = 6.6 Hz,
3H, CH3); 13C NMR (CDCl): d 174.9, 172.3, 169.7, 139.9, 133.5,
131.8, 127.7, 127.5, 127.3, 127.2, 126.2, 125.8, 125.0, 72.0, 61.5,
41.2, 35.8, 34.4, 33.7, 31.0, 27.5, 24.7, 22.3, 14.0, 13.9; MS (ESI) m/
z (%): 457 ([M+H]+, 100); Anal Calcd for C26H36N2O5: C, 68.40; H,
7.95; N, 6.14. Found: C, 68.29; H, 8.06; N, 6.01.
4.2.4.16. (S)-tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)-4-methylpen-
tanamido)acetate (23a). Yield 80%; Yellowish oil; [a D20
]
À16.1 (c 1.00,
CHCl3); 1H NMR (CDCl3): d 7.32–7.18 (m, 5H, arom), 7.13 (t, J = 5.0 Hz,
1H, NHCO), 5.97 (d, J = 8.6 Hz, 1H, OCONH), 5.01 (q, J = 8.4 Hz, 2H,
PhCH2), 4.39–4.22 (m, 1H, CH), 3.82 (dd, J1 = 2.6 Hz, J2 = 5.2 Hz, 2H,
CH2COOBut), 1.70–1.47 (m, 3H, CHCH2), 0.86 (d, J = 5.8 Hz, 6H,
2ÂCH3); 13C NMR (CDCl3): d 172.7, 168.7, 156.2, 136.0, 128.2, 127.8,
127.7, 81.8, 66.6, 41.6, 41.2, 27.7, 24.3, 22.8, 21.5; MS (ESI) m/z (%):
379 ([M+H]+, 100); Anal. Calcd for C20H30N2O5: C, 63.47; H, 7.99; N,
7.40. Found: C, 63.29; H, 8.17; N, 7.32.
4.2.4.17. tert-Butyl 2-((2S,3R)-2-(((benzyloxy)carbonyl)amino) -3-
4.2.4.12. Ethyl 2-(((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hexanamido)
hexyl)oxy)acetate (mixture of diastereomers) (18a). Yield 19%; Color-
less oil; 1H NMR (CDCl3): d 7.09 (d, J = 8.4 Hz, 2H, arom), 6.91 (d,
J = 8.6 Hz, 1H, NH), 6.82 (d, J = 8.0 Hz, 2H, arom), 4.20 (q, J = 6.6 Hz,
2H, OCH2CH3), 4.15–3.95 (m, 4H, 2ÂCH, OCH2CO), 3.78 (s, 3H,
CH3O), 3.65 (dd, J1 = 9.2 Hz, J2 = 3.4 Hz, 1H, CHHO), 3.46 (dd,
J1 = 10.4 Hz, J2 = 3.4 Hz, CHHO), 2.56 (t, J = 7.2 Hz, 2H, PhCH2),
1.82–1.75 (br s, 1H, OH), 1.70–1.48 (m, 6H, 3ÂCH2), 1.21 (t, 3H,
OCH2CH3), 1.48–1.10 (m, 6H, 3ÂCH2); 13C NMR (CDCl3): d 173.6,
170.9, 157.6, 134.5, 129.2, 113.6, 73.1, 71.7, 68.1, 61.0, 55.2, 49.0,
34.9, 34.8, 31.5, 31.1, 30.3, 29.7, 28.2, 24.5, 22.5, 14.1, 14.0; MS
(ESI) m/z (%): 424 ([M+H]+, 100); Anal Calcd for C23H37NO6: C,
65.22; H, 8.81; N, 3.31. Found: C, 65.02; H, 8.99; N, 3.23.
methylpentanamido)acetate (23b). Yield 78%; White solid; mp
À2.5 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.41–
20
133–135 °C; [
a]
D
7.23 (m, 5H, arom), 6.76 (t, J = 4.6 Hz, 1H, NHCO), 5.65 (d,
J = 9.0 Hz, 1H, OCONH), 5.16–4.98 (m, 2H, PhCH2), 4.19–4.08 (m,
1H, CH), 3.91 (dd, J1 = 5.0 Hz, J2 = 8.6 Hz, 2H, CH2COOBut), 1.97–
1.76 (m, 1H, CH), 1.45 [s, 9H, C(CH3)3], 1.30–0.99 (m, 2H, CH2),
0.95–0.79 (m, 6H, 2ÂCH3); 13C NMR (CDCl3): d 171.4, 168.6,
156.3, 136.1, 128.4, 128.0, 127.9, 82.2, 66.9, 59.5, 41.9, 37.3, 27.9,
24.6, 15.4, 11.3; MS (ESI) m/z (%): 379 ([M+H]+, 100); Anal. Calcd
for C20H30N2O5: C, 63.47; H, 7.99; N, 7.40. Found: C, 63.29; H,
8.12; N, 7.31.
4.2.4.18. (S)-tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)-3-methylbu-
tanamido)acetate (23c). Yield 57%; White solid; mp 145–148 °C;
4.2.4.13. tert-Butyl 2-(((2S)-2-(2-hydroxy-6-phenylhexanamido)hexyl)
oxy)acetate (mixture of diastereomers) (18b). Yield 53%; Colorless
oil; 1H NMR (CDCl3): d 7.37–6.76 (m, 6H, NH, arom), 4.15–3.78
(m, 4H, OCH2CO, 2ÂCH), 3.61 (dd, J1 = 9.4 Hz, J2 = 3.6 Hz, 1H,
CHCHHO), 3.41 (dd, J1 = 9.4 Hz, J2 = 3.4 Hz, 1H, CHCHHO), 2.60 (t,
J = 7.2 Hz, 2H, PhCH2), 1.98–1.04 (m, 21H, 6ÂCH2, C(CH3)3), 0.87
(t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3): d 174.1, 170.2, 142.5,
128.3, 128.2, 125.5, 82.8, 73.1, 72.0, 68.4, 48.7, 35.8, 34.6, 31.3,
30.9, 28.2, 28.0, 24.7, 22.5, 14.0; MS (ESI) m/z (%): 422 ([M+H]+,
100); Anal Calcd for C24H39NO5: C, 68.38; H, 9.32; N, 3.32. Found:
C, 68.15; H, 9.47; N, 3.28.
[
a 2D0
]
À5.3 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.44–7.20 (m, 5H,
arom), 6.82 (br s, 1H, NHCO), 5.69 (d, J = 9.0 Hz, 1H, OCONH), 5.17–
5.00 (m, 2H, PhCH2), 4.19–4.05 (m, 1H, CH), 3.91 (dd, J1 = 5.4 Hz,
J2 = 13.2 Hz, 2H, CH2COOBut), 2.21–2.03 (m, 1H, CH), 1.45 [s, 9H, C
(CH3)3], 0.97 (d, J = 6.8 Hz, 3H, CH3), 0.93 (d, J = 6.8 Hz, 3H, CH3);
13C NMR (CDCl3): d 171.4, 169.6, 156.4, 136.1, 128.4, 128.0, 127.9,
82.2, 66.9, 60.1, 41.8, 31.0, 27.9, 19.2, 17.7; MS (ESI) m/z (%): 365
([M+H]+, 100); Anal. Calcd for C19H28N2O5: C, 62.62; H, 7.74; N,
7.69. Found: C, 62.44; H, 7.81; N, 7.59.
4.2.4.19. (S)-tert-Butyl 2-((S)-2-(((benzyloxy)carbonyl)amino) hex-
anamido)propanoate (23d). Yield 72%; White solid; mp 91–93 °C;
1H NMR (CDCl3): d 7.42–7.28 (m, 5H, arom), 6.50 (d, J = 7.2 Hz,
1H, NHCO), 5.36 (d, J = 7.8 Hz, 1H, OCONH), 5.12 (s, 2H, PhCH2),
4.2.4.14. tert-Butyl 2-(((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)hexyl)oxy)acetate (mixture of diastereomers) (18c). Yield
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

10
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
4.55–4.35 (m, 1H, CHCOOBut), 4.25–4.06 (m, 1H, CH), 1.95–1.22
[m, 18H, 3ÂCH2, C(CH3)3, CHCH3], 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C
NMR (CDCl3): d 171.8, 171.1, 156.4, 136.01, 128.5, 128.1, 128.0,
82.1, 66.9, 54.9, 48.6, 32.6, 27.9, 27.4, 22.4, 18.5, 13.9; MS (ESI)
m/z (%): 393 ([M+H]+, 80); Anal. Calcd for C21H32N2O5: C, 64.26;
H, 8.22; N, 7.14. Found: C, 64.01; H, 8.37; N, 7.06.
0.79 (m, 6H, 2ÂCH3); 13C NMR (CDCl3): d 174.7, 174.5, 171.8,
171.5, 168.5, 157.4, 134.5, 129.1, 113.5, 82.2, 72.0, 57.2, 55.1,
41.9, 36.9, 34.8, 34.6, 31.5, 27.9, 24.6, 15.4, 15.3, 11.1; MS (ESI)
m/z (%): 465 ([M+H]+, 100); Anal Calcd for C25H40N2O6: C, 64.63;
H, 8.68; N, 6.03. Found: C, 64.47; H, 8.81; N, 5.92.
4.2.4.25. tert-Butyl 2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl) hex-
anamido)-3-methylbutanamido)acetate (mixture of diastereomers)
(24c). Yield 68%; Colorless oil; 1H NMR (CDCl3): d 7.52–7.28 (m,
2H, 2ÂNHCO), 7.05 (d, J = 8.6 Hz, 2H, arom), 6.79 (d, J = 8.4 Hz,
2H, arom), 4.45–4.32 (m, 1H, CH), 4.17–4.06 (m, 1H, CH), 4.06–
3.78 (m, 2H, CH2COOBut), 3.76 (s, 3H, CH3O), 2.84 (br s, 1H, OH),
2.52 (t, J = 7.0 Hz, 2H, PhCH2), 1.96–1.26 (m, 6H, 3ÂCH2), 1.44 [s,
9H, C(CH3)3], 1.04–0.80 (m, 6H, 2ÂCH3); 13C NMR (CDCl3): d
174.8, 174.7, 171.8, 171.6, 168.5, 157.4, 134.5, 129.1, 113.5, 82.1,
71.8, 57.9, 57.7, 55.1, 41.9, 34.8, 31.5, 30.9, 27.9, 24.6, 19.2, 18.1;
MS (ESI) m/z (%): 451 ([M+H]+, 100); Anal Calcd for C24H38N2O6:
C, 63.98; H, 8.50; N, 6.22. Found: C, 63.79; H, 8.72; N, 6.09.
4.2.4.20. (R)-tert-Butyl 2-((S)-2-(((benzyloxy)carbonyl)amino) hex-
anamido)propanoate (23e). Yield 36%; White solid; mp 80–82 °C;
1H NMR (CDCl3): d 7.40–7.22 (m, 5H, arom), 6.76 (d, J = 5.2 Hz,
1H, NHCO), 5.56 (d, J = 6.6 Hz, 1H, OCONH), 5.09 (s, 2H, PhCH2),
4.53–4.34 (m, 1H, CHCOOBut), 4.30–4.09 (m, 1H, CH), 1.95–1.52
(m, 2H, CH2), 1.44 [s, 9H, C(CH3)3], 1.32–1.20 (m, 4H, 2ÂCH2),
0.86 (t, J = 6.2 Hz, 3H, CH3); 13C NMR (CDCl3): d 171.9, 171.1,
156.1, 136.1, 128.4, 128.0, 127.9, 82.0, 66.8, 54.8, 48.5, 32.4, 27.8,
27.4, 22.3, 18.4, 13.8; MS (ESI) m/z (%): 393 ([M+H]+, 75); Anal.
Calcd for C21H32N2O5: C, 64.26; H, 8.22; N, 7.14. Found: C, 64.03;
H, 8.39; N, 7.05.
4.2.4.21. (R)-tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)he-xana-
mido)acetate (23f). Yield 66%; White solid; mp 84–85 °C; [a]
D
4.2.4.26. (2S)-tert-Butyl 2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)
hexanamido)hexanamido)propanoate (mixture of diastereomers)
(24d). Yield 68%; Colorless oil; 1H NMR (CDCl3): d 7.18 (d,
J = 8.4 Hz, 1H, NHCO), 7.06 (d, J = 8.6 Hz, 2H, arom), 6.85–6.70
[(m, 3H, 2Âarom, NHCO)], 4.49–4.29 (m, 2H, 2ÂCH), 4.16–4.04
(m, 1H, CH), 3.83 (br s, 1H, OH), 3.76 (s, 3H, CH3O), 2.53 (t,
J = 7.6 Hz, 2H, PhCH2), 1.90–1.48 (m, 6H, 3ÂCH2), 1.44 [s, 9H, C
(CH3)3], 1.39–1.19 (m, 6H, 3ÂCH2), 1.33 [d, J = 7.2 Hz, 3H, CH
(CH3)], 0.87 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (CDCl3): d 174.0,
171.7, 171.1, 157.6, 134.4, 129.2, 113.6, 82.1, 71.9, 55.2, 52.8,
48.7, 34.8, 32.3, 31.5, 27.9, 27.5, 24.6, 22.4, 18.4, 13.9; MS (ESI)
m/z (%): 479 ([M+H]+, 100); Anal Calcd for C26H42N2O6: C, 65.25;
H, 8.85; N, 5.85. Found: C, 65.01; H, 8.99; N, 5.72.
20
9.2 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.40–7.20 (m, 5H, arom),
6.75 (br s, 1H, NHCO), 5.60 (d, J = 8.2 Hz, 1H, OCONH), 5.17–5.00
(m, 2H, PhCH2), 4.30–4.13 (m, 1H, CH), 3.89 (d, J = 5.0 Hz, 2H, CH2-
COOBut), 1.93–1.47 (m, 2H, CHCH2), 1.44 [s, 9H, C(CH3)3], 1.35–
1.17 (m, 4H, 2ÂCH2), 0.86 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3):
d 172.0, 168.7, 156.1, 136.1, 128.4, 128.1, 128.0, 82.2, 66.9, 54.8,
41.8, 32.4, 27.9, 27.5, 22.3, 13.8; MS (ESI) m/z (%): 379 ([M+H]+,
78); Anal calcd for C20H30N2O5: C, 63.47; H, 7.99; N, 7.40. Found:
C, 63.32; H, 8.05; N, 7.30.
4.2.4.22. (S)-tert-Butyl 4-(2-(benzyloxycarbonylamino)-4-methylpen-
tanamido)butanoate (23g). Yield 60%; Colorless oil; 1H NMR
(CDCl3): d 7.34–7.18 (m, 5H, arom), 6.66–6.41 (t, J = 4.6 Hz, 1H,
NHCO), 5.83 (d, J = 8.4 Hz, 1H, OCONH), 5.13–4.88 (m, 2H, PhCH2),
4.26–4.05 (m, 1H, CH), 3.33–3.00 (m, 2H, CH2), 2.17 (t, J = 7.2 Hz,
2H, CH2), 1.81–1.37 [m, 14H, C(CH3)3, 2ÂCH2, CH), 0.85 (d,
J = 6.8 Hz, 6H, 2ÂCH3); 13C NMR (CDCl3): d 172.5, 169.0, 156.2,
136.1, 128.3, 128.2, 127.9, 80.3, 66.7, 53.4, 41.4, 38.7, 32.6, 27.9,
24.5, 22.7, 21.8; MS (ESI) m/z (%): 407 ([M+H]+, 85); Anal calcd
for C22H34N2O5: C, 65.00; H, 8.43; N, 6.89. Found: C, C, 64.75; H,
8.61; N, 6.78.
4.2.4.27. (2R)-tert-Butyl 2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)
hexanamido)hexanamido)propanoate (mixture of diastereomers)
(24e). Yield 38%; Colorless oil; 1H NMR (CDCl3):
d 7.09 (d,
J = 8.6 Hz, 2H, arom), 7.03–6.88 (m, 1H, NHCO), 6.82 (d, J = 8.8 Hz,
2H, arom), 6.63 (t, J = 6.8 Hz, 1H, NHCO), 4.52–4.33 (m, 2H,
2ÂCH), 4.26–4.19 (m, 1H, CH), 3.79 (s, 3H, CH3O), 3.20 (br s,
1/2H, OH), 2.83 (br s, 1/2H, OH), 2.56 (t, J = 7.6 Hz, 2H, PhCH2),
1.98–1.52 (m, 6H, 3ÂCH2), 1.46 [s, 9H, C(CH3)3], 1.40–1.25 (m,
6H, 3ÂCH2), 1.37 [d, J = 7.2 Hz, 3H, CH(CH3)], 0.89 (t, J = 6.6 Hz,
3H, CH3); 13C NMR (CDCl3): d 174.5, 174.1, 172.1, 171.8, 171.3,
171.0, 157.5, 134.5, 129.2, 113.6, 82.3, 82.2, 72.1, 71.9, 55.2, 52.7,
52.4, 48.7, 34.8, 34.4, 31.9, 31.5, 27.9, 26.0, 24.7, 22.3, 18.4, 18.3,
13.9; MS (ESI) m/z (%): 479 ([M+H]+, 100); Anal Calcd for
4.2.4.23. tert-Butyl 2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)-4-methylpentanamido)acetate (mixture of diastereomers)
(24a). Yield 66%; Yellow oil; 1H NMR (CDCl3): d 7.33 (d, J = 8.8 Hz,
1H, NHCO), 7.18 (t, J = 5.2 Hz, 1H, NHCO), 7.06 (d, J = 8.6 Hz, 2H,
arom), 6.80 (d, J = 8.4 Hz, 2H, arom), 4.64–4.49 (m, 1H, CH), 4.16–
4.05 (m, 1H, CH), 3.87 (t, J = 4.8 Hz, 2H, CH2COOBut), 3.77 (s, 3H,
CH3O), 2.77 (br s, 1H, OH), 2.53 (t, J = 7.6 Hz, 2H, PhCH2), 1.98–
1.35 (m, 9H, 4ÂCH2, CH), 1.44 [s, 9H, C(CH3)3], 0.91 (t, J = 5.6 Hz,
6H, 2ÂCH3); 13C NMR (CDCl3): d 174.8, 174.6, 172.7, 172.4, 168.8,
168.6, 157.4, 134.4, 129.1, 113.5, 82.3, 55.1, 51.1, 41.9, 40.9, 34.8,
31.5, 27.9, 24.6, 22.9, 21.8; MS (ESI) m/z (%): 465 ([M+H]+, 100);
Anal Calcd for C25H40N2O6: C, 64.63; H, 8.68; N, 6.03. Found: C,
64.49; H, 8.79; N, 5.95.
C26H42N2O6: C, 65.25; H, 8.85; N, 5.85. Found: C, 65.02; H, 8.99;
N, 5.74.
4.2.4.28. tert-Butyl 2-((2R)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)hexanamido)acetate (mixture of diastereomers) (24f). Yield
79%; Yellow oil; 1H NMR (CDCl3): d 7.38 (d, J = 8.4 Hz, 1H, NHCO),
7.25–7.12 (m, 1H, NHCO), 7.06 (d, J = 8.4 Hz, 2H, arom), 6.79 (d,
J = 8.4 Hz, 2H, arom), 4.60–4.45 (m, 1H, CH), 4.14–4.06 (m, 1H,
CH), 3.94–3.80 (m, 2H, CH2COOBut), 3.76 (s, 3H, CH3O), 2.53 (t,
J = 7.0 Hz, 2H, PhCH2), 1.90–1.48 (m, 6H, 3ÂCH2), 1.44 [s, 9H, C
(CH3)3], 1.36–1.21 (m, 6H, 3ÂCH2), 0.95–0.77 (m, 3H, CH3); 13C
NMR (CDCl3): d 174.8, 174.5, 172.4, 172.1, 168.7, 168.6, 157.4,
134.4, 129.1, 113.5, 82.2, 71.8, 55.1, 52.6, 41.9, 34.8, 32.1, 31.5,
27.9, 27.6, 24.6, 22.3, 13.9; MS (ESI) m/z (%): 465 ([M+H]+, 100);
Anal Calcd for C25H40N2O6: C, 64.63; H, 8.68; N, 6.03. Found: C,
64.42; H, 8.76; N, 5.96.
4.2.4.24. tert-Butyl 2-((2S,3R)-2-(2-hydroxy-6-(4-methoxyphenyl)
hexanamido)-3-methylpentanamido)acetate (mixture of diastere-
omers) (24b). Yield 90%; Yellow oil; 1H NMR (CDCl3): d 7.43 (dd,
J1 = 3.4 Hz, J2 = 9.2 Hz, 1H, NHCO), 7.19 (t, J = 5.0 Hz, 1H, NHCO),
7.06 (d, J = 8.6 Hz, 2H, arom), 6.79 (d, J = 8.2 Hz, 2H, arom), 4.67
(br s, 1/2H, OH), 4.47–4.35 (m, 1H, CH), 4.27 (br s, 1/2H, OH),
4.17–4.07 (m, 1H, CH), 4.05–3.80 (m, J = 4.8 Hz, 2H, CH2COOBut),
3.76 (s, 3H, CH3O), 2.53 (t, J = 7.4 Hz, 2H, PhCH2), 1.98–1.46 (m,
6H, 3ÂCH2), 1.44 [s, 9H, C(CH3)3], 1.28–1.01 (m, 2H, CH2), 0.98–
4.2.4.29. tert-Butyl 4-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hex-
anamido)-4-methylpentanamido)butanoate (mixture of diastere-
omers) (24g). Yield 71%; Yellow oil; 1H NMR (CDCl3): d 7.18 (d,
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
11
J = 8.4 Hz, 1H, NHCO), 7.12–6.92 (m, 3H, NHCO, arom), 6.80 (d,
4.2.5. Oxidation of 2-hydroxyamides
J = 7.8 Hz, 2H, arom), 4.53–4.33 (m, 1H, CH), 4.17–4.01 (m, 1H,
CH), 3.77 (s, 3H, CH3), 3.32–3.09 (m, 2H, CH2), 2.54 (t, J = 7.2 Hz,
2H, CH2), 2.24 (t, J = 7.0 Hz, 2H, CH2), 1.97–1.31 [m, 20H, 5ÂCH2,
CH, C(CH3)3], 0.97–0.86 (m, 6H, 2ÂCH3); 13C NMR (CDCl3): d
174.7, 173.0, 172.1, 157.5, 134.5, 129.2, 113.6, 80.7, 72.1, 55.2,
52.6, 41.0, 38.9, 34.3, 34.8, 32.9, 31.5, 28.0, 24.7, 22.9, 22.1; MS
(ESI) m/z (%): 493 ([M+H]+, 100); Anal Calcd for C27H44N2O6: C,
65.83; H, 9.00; N, 5.69. Found: C, 65.62; H, 9.14; N, 5.56.
To a stirred solution of 2-hydroxyamide (1.0 mmol) in dry
CH2Cl2 (10 mL), Dess–Martin periodinane was added (0.64 g,
1.5 mmol) and the mixture was stirred for 1 h at room tempera-
ture. The organic phase was washed with 5% NaHCO3, dried over
Na2SO4 and the organic solvent was evaporated under reduced
pressure. The residue was purified by column chromatography
with the appropriate mixture of solvents as eluent.
4.2.5.1. (S)-tert-Butyl 2-(2-(2-oxo-6-phenylhexanamido)hexanamido)
20
4.2.4.30. N-((S)-1-Amino-1-oxohexan-2-yl)-2-hydroxy-6-(4-methox-
yphenyl)hexanamide (mixture of diastereomers) (26). Yield 52%; Yel-
low syrup; 1H NMR (CDCl3): d 7.26–7.16 (m, 1H, NH), 7.07 (d,
J = 8.6 Hz, 2H, arom), 6.81 (d, J = 8.8 Hz, 2H, arom), 6.76 (br s,
1/2H, NH2), 6.54 (br s, 1/2H, NH2), 6.19 (br s, 1/2H, NH2), 5.91 (br
s, 1/2H, NH2), 4.47–4.28 (m, 1H, CH), 4.17–4.03 (m, 1H, CH), 3.78
(s, 3H, CH3O), 2.55 (t, J = 7.0 Hz, 2H, PhCH2), 1.94–1.20 (m, 13H,
6ÂCH2, OH), 0.89 (t, J = 6.2 Hz, 3H, CH3); 13C NMR (CDCl3): d
175.2, 174.4, 157.6, 134.5, 129.2, 113.7, 72.0, 55.2, 52.6, 34.9,
33.9, 31.7, 31.5, 29.8, 27.6, 24.7, 22.3, 13.9; MS (ESI) m/z (%): 351
([M+H]+, 100); Anal Calcd for C19H30N2O4: C, 65.12; H, 8.63; N,
7.99. Found: C, 64.97; H, 8.79; N, 7.94.
acetate (12a). Yield 80%; Colorless oil; [
a
]
D
À17.2 (c 1.00, CHCl3);
1H NMR (CDCl3): d 7.40 (d, J = 8.6 Hz, 1H, COCONH), 7.34–7.09 (m,
5H, arom), 6.39 (t, J = 4.8 Hz, 1H, NH), 4.46–4.29 (m, 1H, CH), 3.92
(dd, J1 = 1.8 Hz, J2 = 5.2 Hz, 2H, NHCH2), 2.95 (t, J = 6.6 Hz, 2H,
COCOCH2), 2.62 (t, J = 7.0 Hz, 2H, CH2Ph), 2.03–1.58 (m, 6H,
3ÂCH2), 1.45 [s, 9H, C(CH3)3], 1.40–1.22 (m, 4H, 2ÂCH2), 0.88 (t,
J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.1, 170.5, 168.5,
160.0, 142.0, 128.4, 128.3, 125.8, 82.6, 53.2, 42.0, 36.6, 35.6, 32.0,
30.8, 28.0, 27.5, 22.7, 22.3, 13.8; MS (ESI) m/z (%): 450 ([M
+NH4]+, 100); Anal. Calcd for C24H36N2O5: C, 66.64; H, 8.39; N,
6.48. Found: C, 66.51; H, 8.52; N, 6.39.
4.2.5.2. (S)-tert-Butyl 2-(2-(6-(naphthalen-2-yl)-2-oxohexanamido)
20
4.2.4.31. (2S)-tert-Butyl 2-(2-hydroxy-6-(4-methoxyphenyl)hexanamido)
hexanoate (mixture of diastereomers) (28). Yield 46%; Yellow oil; 1H
NMR (CDCl3): d 7.08 (d, J = 8.8 Hz, 2H, arom), 6.98 (d, J = 8.2 Hz, 1H,
NH), 6.82 (d, J = 8.6 Hz, 2H, arom), 4.57–4.40 (m, 1H, CH), 4.18–4.07
(m, 1H, CH), 3.79 (s, 3H, CH3O), 3.09 (br s, 1/2H, OH), 2.90 (br s,
1/2H, OH), 2.56 (t, J = 7.2 Hz, 2H, PhCH2), 1.93–1.53 (m, 6H, 3ÂCH2),
1.47 [s, 9H, C(CH3)3], 1.41–1.16 (m, 6H, 3ÂCH2), 0.90 (t, J = 6.6 Hz,
3H, CH3); 13C NMR (CDCl3): d (173.5, 171.8), 157.6, 134.5, 129.2,
113.7, 82.1, 72.0, 55.2, 52.3, 34.8, 34.7, 32.2, 31.4, 28.0, 27.1, 24.6,
22.3, 13.9; MS (ESI) m/z (%): 408 ([M+H]+, 100); Anal Calcd for
hexanamido)acetate (12b). Yield 99%; Colorless oil; [
a]
À16.0 (c
1.00, CHCl3); 1H NMR (CDCl3): d 7.88–7.21 (m, 8H, 7ÂDarom, NH),
6.61 (br s, 1H, NH), 4.53–4.33 (m, 1H, CH), 4.02–3.82 (m, 2H, CH2-
COO), 2.97 (t, J = 6.6 Hz, 2H, PhCH2), 2.79 (t, J = 7.0 Hz, 2H, CH2-
COCO), 2.12–1.56 (m, 6H, 3ÂCH2), 1.45 [s, 9H, C(CH3)3], 1.37–
1.16 (m, 4H, 2ÂCH2), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3):
d 198.0, 170.7, 168.5, 160.0, 139.5, 133.5, 131.9, 127.8, 127.5,
127.3, 127.2, 126.3, 125.8, 125.0, 82.4, 53.1, 41.9, 36.6, 35.7, 32.0,
30.5, 27.9, 27.5, 22.6, 22.3, 13.8; MS (ESI) m/z (%): 483 ([M+H]+,
45); Anal. Calcd for C28H38N2O5: C, 69.68; H, 7.94; N, 5.80. Found:
C, 69.45; H, 8.03; N, 5.66.
C23H37NO5: C, 67.78; H, 9.15; N, 3.44. Found: C, 67.61; H, 9.39; N,
3.35.
4.2.5.3. (S)-tert-Butyl 2-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
20
4.2.4.32. Benzyl 2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)hexanamido)
hexanamido)acetate (mixture of diastereomers) (30a). Yield 40%; White
oil; 1H NMR (CDCl3): d 7.42–7.28 (m, 5H, arom), 7.21–7.02 [(m, 3H,
NH, 2Âarom), 7.01–6.87 (m, 1H, NH), 6.81 (d, J = 8.2 Hz, 2H, arom),
5.15 (s, 2H, OCH2Ph), 4.56–4.36 (m, 1H, CH), 4.19–3.95 (m, 3H, CH,
NHCH2), 3.77 (s, 3H, CH3O), 3.55 (br s, 1/2H, OH), 3.15 (br s, 1/2H,
OH), 2.55 (t, J = 7.6 Hz, 2H, PhCH2), 2.06–1.14 (m, 12H, 6ÂCH2), 0.89
(t, J = 5.8 Hz, 3H, CH3); 13C NMR (CDCl3): d 174.6, 174.3, 172.3,
172.0, 169.4, 157.6, 135.0, 134.4, 129.2, 128.6, 128.5, 128.4, 113.6,
71.9, 67.2, 55.2, 52.7, 41.3, 34.8, 31.8, 31.4, 29.7, 27.6, 24.6, 22.4,
13.9; MS (ESI) m/z (%): 497 ([MÀH]À, 100); Anal Calcd for
hexanamido)acetate (12c). Yield 72%; Colorless oil; [
a]
À16.6 (c
D
1.00, CHCl3); 1H NMR (CDCl3): d 7.49 (d, J = 8.6 Hz, 1H, NH), 7.08
(d, J = 8.6 Hz, 2H, arom), 6.81 (d, J = 8.8 Hz, 2H, arom), 6.60 (t,
J = 5.0 Hz, 1H, NH), 4.48–4.34 (m, 1H, CH), 3.91 (dd, J1 = 2.2 Hz,
J2 = 5.2 Hz, 2H, NHCH2), 3.78 (s, 3H, CH3O), 2.93 (t, J = 6.4 Hz, 2H,
PhCH2), 2.57 (t, J = 7.0 Hz, 2H, CH2COCO), 1.99–1.75 (m, 2H, CH2),
1.69–1.55 (m, 4H, 2ÂCH2), 1.46 [s, 9H, C(CH3)3], 1.40–1.27 (m, 4H,
2ÂCH2), 0.88 (t, J = 7.6 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.0,
170.7, 168.6, 160.0, 157.6, 134.1, 129.2, 113.6, 82.5, 55.2, 53.1,
42.0, 36.6, 34.6, 32.0, 31.0, 27.9, 27.5, 22.6, 22.3, 13.8; MS (ESI) m/
z (%): 461 ([MÀH]À, 100); HRMS (ESI) calcd for C25H37N2OÀ6 [MÀH]À:
461.2657. Found: 461.2654; Anal. Calcd for C25H38N2O6: C, 64.91;
H, 8.28; N, 6.06. Found: C, 64.79; H, 8.43; N, 5.94.
C
28H38N2O6: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.21; H, 7.84; N,
5.52.
4.2.4.33. tert-Butyl 2-(2-((2S)-2-(2-hydroxy-6-(4-methoxyphenyl)
hexanamido)hexanamido)acetamido)acetate (mixture of diastere-
omers) (32). Yield 46%; Colorless oil; 1H NMR (CDCl3): d 7.61–
7.40 (m, 2H, 2ÂNHCO), 7.20 (t, J = 5.2 Hz, 1H, NHCO), 7.07 (d,
J = 8.4 Hz, 2H, arom), 6.81 (d, J = 8.8 Hz, 2H, arom), 4.51–4.31 (m,
1H, CH), 4.13–3.83 (m, 5H, CH, 2ÂNHCH2], 3.78 (s, 3H, CH3O),
3.16–3.01 (m, 1H, OH), 2.55 (t, J = 7.6 Hz, 2H, PhCH2), 1.98–1.48
(m, 6H, 3ÂCH2), 1.45 [s, 9H, C(CH3)3], 1.38–1.20 (m, 6H, 3ÂCH2),
0.89 (t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3): d 175.8, 175.2,
172.5, 169.4, 169.2, 157.5, 134.4, 129.2, 113.6, 82.7, 82.5, 72.0,
70.4, 55.2, 53.6, 42.9, 41.9, 34.8, 34.4, 31.4, 28.8, 28.0, 24.9, 22.6,
22.3, 13.9; MS (ESI) m/z (%): 520 ([MÀH]À, 100); Anal Calcd for
4.2.5.4. (S)-tert-Butyl 3-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
20
hexanamido)propanoate (12d). Yield 78%; Yellowish oil; [
a
]
D
À8.6
(c 1.00, CHCl3); 1H NMR (CDCl3): d 7.45 (d, J = 8.6 Hz, 1H, NH), 7.09
(d, J = 8.6 Hz, 2H, arom), 6.82 (d, J = 8.6 Hz, 2H, arom), 6.53 (br t, 1H,
NH), 4.33–4.20 (m, 1H, CH), 3.78 (s, 3H, CH3O), 3.55–3.37 (m, 2H,
NHCH2), 2.93 (t, J = 6.8 Hz, 2H, PhCH2), 2.57 (t, J = 6.8 Hz, 2H,
CH2COCO), 2.44 (t, J = 6.2 Hz, 2H, CH2COOBut), 1.96–1.69 (m, 2H,
CH2), 1.69–1.54 (m, 4H, 2ÂCH2), 1.44 [s, 9H, C(CH3)3], 1.37–1.22
(m, 4H, 2ÂCH2), 0.88 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (CDCl3): d
198.1, 171.8, 170.4, 159.9, 157.7, 134.1, 129.2, 113.7, 81.3, 55.2,
53.3, 36.6, 35.1, 34.6, 32.2, 31.0, 29.7, 28.0, 27.5, 22.6, 22.3, 13.8;
MS (ESI) m/z (%): 475 ([MÀH]À, 100); HRMS (ESI) calcd for C26H39N2-
OÀ6 [MÀH]À: 475.2814. Found 475.2820; Anal. Calcd for C26H40N2O6:
C, 65.52; H, 8.46; N, 5.88. Found: C, 65.37; H, 8.61; N, 5.79.
C
27H43N3O7: C, 62.17; H, 8.31; N, 8.06. Found: C, 61.90; H, 8.45;
N, 7.96.
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

12
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
4.2.5.5. (S)-tert-Butyl 4-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
3ÂCH2), 1.45 [s, 9H C(CH3)3], 1.36–1.14 (m, 4H, 2ÂCH2), 0.87
(t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.9, 169.5, 159.9,
142.1, 128.3, 128.3, 125.7, 81.8, 72.4, 68.8, 49.4, 36.6, 35.6, 31.0,
30.8, 28.1, 22.8, 22.5, 13.9; MS (ESI) m/z (%): 420 ([M+H]+, 40);
Anal. Calcd for C24H37NO5: C, 68.71; H, 8.89; N, 3.34. Found: C,
68.45; H, 9.05; N, 3.21.
hexanamido)butanoate (12e). Yield 70%; Yellow solid; mp 50–
20
52 °C; [
a]
À9.3 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.42 (d,
D
J = 8.2 Hz, 1H, NH), 7.09 (d, J = 8.8 Hz, 2H, arom), 6.82 (d,
J = 8.6 Hz, 2H, arom), 6.39 (br t, 1H, NHCH2), 4.36–4.17 (m, 1H,
CH), 3.78 (s, 3H, CH3O), 3.36–3.17 (m, 2H, NHCH2), 3.00–2.83 (m,
2H, PhCH2), 2.64–2.50 (m, 2H, CH2COCO), 2.27 (t, J = 7.2 Hz, 2H,
CH2COOBut), 1.89–1.68 (m, 4H, 2ÂCH2), 1.68–1.56 (m, 4H,
2ÂCH2), 1.44 [s, 9H, C(CH3)3], 1.38–1.20 (m, 4H, 2ÂCH2), 0.89 (t,
J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.1, 172.8, 170.5,
159.9, 157.7, 134.1, 129.2, 113.7, 80.8, 55.2, 53.4, 39.2, 36.6, 34.6,
33.0, 32.1, 31.0, 28.0, 27.6, 24.4, 22.6, 22.3, 13.8; MS (ESI) m/z
(%): 489 ([MÀH]À, 100); HRMS (ESI) calcd for C27H41N2OÀ6 [MÀH]À:
489.2970. Found 489.2970; Anal. Calcd for C27H42N2O6: C, 66.10; H,
8.63; N, 5.71. Found: C, 65.92; H, 8.87; N, 5.57.
4.2.5.10. (S)-tert-Butyl 2-((2-(6-(4-methoxyphenyl)-2-oxohexanamido)
hexyl)oxy)acetate (19c). Yield 86%; Yellow oil; [a D20
]
À7.0 (c 1.00,
CHCl3); 1H NMR (CDCl3): d 7.29 (d, J = 9.2 Hz, 1H, NH), 7.09 (d,
J = 8.8 Hz, 2H, arom), 6.81 (d, J = 8.6 Hz, 2H, arom), 4.05–3.97 (m,
1H, CH), 3.95 (s, 2H, OCH2CO), 3.78 (s, 3H, CH3O), 3.63 (dd,
J1 = 9.4 Hz, J2 = 4.2 Hz, 1H, CHCHHO), 3.50 (dd, J1 = 9.4 Hz,
J2 = 3.8 Hz, 1H, CHCHHO), 2.94 (t, J = 6.4 Hz, 2H, PhCH2), 2.57 (t,
J = 7.0 Hz, 2H, CH2COCO), 1.75–1.55 (m, 6H, 3ÂCH2), 1.47 [s, 9H, C
(CH3)3], 1.40–1.20 (m, 4H, 2ÂCH2), 0.88 (t, J = 6.4 Hz, 3H, CH3); 13C
NMR (CDCl3): d 198.9, 169.4, 159.9, 157.6, 134.1, 129.2, 113.6,
81.7, 72.4, 68.7, 55.2, 49.4, 36.6, 34.6, 31.0, 30.9, 28.0, 22.6, 22.4,
4.2.5.6. (S)-tert-Butyl 5-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
hexanamido)pentanoate (12f). Yield 65%; White solid; 1H NMR
(CDCl3): d 7.49 (d, J = 8.4 Hz, 1H, NH), 7.08 (d, J = 8.4 Hz, 2H, arom),
6.81 (d, J = 8.4 Hz, 2H, arom), 6.36 (t, J = 5.6 Hz, 1H, NH), 4.39–4.20
(m, 1H, CH), 3.77 (s, 3H, CH3O), 3.37–3.11 (m, 2H, CH2), 3.00–2.77
(m, 2H, CH2), 2.65–2.43 (m, 2H, CH2), 2.23 (t, J = 6.6 Hz, 2H, CH2),
1.95–1.48 (m, 10H, 5ÂCH2), 1.43 [s, 9H, C(CH3)3], 1.36–1.18 (m,
4H, 2ÂCH2), 0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): d
198.0, 172.9, 170.5, 159.9, 157.6, 134.0, 129.2, 113.6, 80.3, 55.2,
53.3, 39.0, 36.6, 34.7, 34.6, 32.1, 30.9, 28.6, 28.0, 27.5, 22.6, 22.3,
13.9; MS (ESI) m/z (%): 448 ([MÀH]À, 100); HRMS (ESI) calcd for C25
-
H
38NOÀ6 [MÀH]À: 448.2705. Found 448.2699; Anal. Calcd for
C25H39NO6: C, 66.79; H, 8.74; N, 3.12. Found: C, 66.55; H, 8.87; N,
3.09.
4.2.5.11. (S)-tert-Butyl 2-((2-(6-(naphthalen-2-yl)-2-oxohexanamido)
20
hexyl)oxy)acetate (19d). Yield 84%; Yellowish oil; [
a]
À7.2 (c
D
1.00, CHCl3); 1H NMR (CDCl3): d 7.88–7.19 (m, 8H, NH, arom),
4.10–3.84 (m, 3H, CH, CH2COO), 3.64 (dd, J1 = 4.0 Hz, J2 = 9.4 Hz,
1H, CHHO), 3.52 (dd, J1 = 3.8 Hz, J2 = 9.4 Hz, 1H, CHHO), 2.99 (t,
J = 6.6 Hz, 2H, CH2COCO), 2.82 (t, J = 6.6 Hz, 2H, PhCH2), 1.89–
1.53 (m, 6H, 3ÂCH2), 1.47 [s, 9H, C(CH3)3], 1.37–1.15 (m, 4H,
2ÂCH2), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.9,
169.5, 159.9, 139.5, 133.5, 131.9, 127.8, 127.5, 127.4, 127.3,
126.3, 125.8, 125.0, 81.8, 72.4, 68.7, 49.4, 36.6, 35.7, 31.0, 30.6,
28.0, 22.7, 22.4, 13.9; MS (ESI) m/z (%): 470 ([M+H]+, 33); Anal.
Calcd for C28H39NO5: C, 71.61; H, 8.37; N, 2.98. Found: C, 71.39;
H, 8.61; N, 2.85.
21.9, 13.8; HRMS (ESI) calcd for
C
28H44N2NaO+6 [M+Na]+:
527.3092. Found: 527.3096; Anal. Calcd for C28H44N2O6: C, 66.64;
H, 8.79; N, 5.55. Found: C, 66.41; H, 8.99; N, 5.41.
4.2.5.7. (S)-Ethyl 2-(2-(6-(naphthalen-2-yl)-2-oxohexanamido)hex-
20
anamido)acetate (16). Yield 80%; Yellowish oil; [
a]
D
À21.4 (c
1.00, CHCl3); 1H NMR (CDCl3): d 7.85–7.28 (m, 8H, NH, arom),
6.67 (t, J = 5.0 Hz, 1H, NH), 4.52–4.34 (m, 1H, NHCH), 4.20 (q,
J = 7.2 Hz, 2H, OCH2), 4.02 (dd, J1 = 2.6 Hz, J2 = 5.0 Hz, 2H, NHCH2),
2.97 (t, J = 6.8 Hz, 2H, CH2COCO), 2.80 (t, J = 6.6 Hz, 2H, CH2Ph),
2.06–1.59 (m, 6H, 3ÂCH2) 1.45–1.18 (m, 7H, 2ÂCH2, CH3), 0.89
(t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.0, 170.9, 169.5,
160.0, 139.4, 133.5, 131.9, 127.8, 127.5, 127.3, 127.2, 126.3,
125.8, 125.0, 61.6, 53.1, 41.3, 36.6, 35.7, 31.9, 30.6, 27.5, 22.6,
22.3, 14.0, 13.8; MS (ESI) m/z (%): 455 ([M+H]+, 100); Anal. Calcd
for C26H34N2O5: C, 68.70; H, 7.54; N, 6.16. Found: C, 68.54; H,
7.68; N, 6.02.
4.2.5.12. (S)-tert-Butyl 2-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)-
4-methylpentanamido)acetate (25a). Yield 82%; Colorless oil; [a D20
]
À21.4 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.51 (d, J = 8.0 Hz, 1H,
NH), 7.07 (d, J = 8.8 Hz, 2H, arom), 6.80 (d, J = 8.6 Hz, 2H, arom),
6.78–6.67 (m, 1H, NH), 4.57–4.44 (m, 1H, CH), 3.90 (dd, J1 = 2.4 Hz,
J2 = 5.2 Hz, 2H, NHCH2), 3.76 (s, 3H, CH3O), 2.92 (t, J = 6.4 Hz, 2H,
PhCH2), 2.55 (t, J = 7.0 Hz, 2H, CH2COCO), 1.74–1.55 (m, 6H,
3ÂCH2), 1.44 [s, 9H, C(CH3)3], 0.93 (d, J = 4.2 Hz, 3H, CH3), 0.90 (d,
J = 4.2 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.1,171.2, 168.5, 160.0,
157.5, 133.9, 129.0, 113.5, 82.1, 55.0, 51.3, 41.8, 40.9, 36.5, 34.5,
30.8, 27.8, 24.5, 22.8, 22.4, 21.6; MS (ESI) m/z (%): 461 ([MÀH]À,
100); HRMS (ESI) calcd for C25H37N2OÀ6 [MÀH]À: 461.2657. Found
461.2658; Anal. Calcd for C25H38N2O6: C, 64.91; H, 8.28; N, 6.06.
Found: C, 64.73; H, 8.50; N, 5.93.
4.2.5.8. (S)-Ethyl 2-((2-(6-(4-methoxyphenyl)-2-oxohexanamido)
20
hexyl)oxy)acetate (19a). Yield 61%; Yellow oil; [
a]
À4.9 (c 1.00,
D
CHCl3); 1H NMR (CDCl3): d 7.32 (d, J = 9.0 Hz, 1H, NH), 7.09 (d,
J = 8.6 Hz, 2H, arom), 6.82 (d, J = 8.6 Hz, 2H, arom), 4.22 (q,
J = 7.2 Hz, 2H, OCH2CH3), 4.07 (s, 2H, OCH2CO), 4.05–3.90 (m, 1H,
CH), 3.79 (s, 3H, CH3O), 3.65 (dd, J1 = 9.4 Hz, J2 = 4.2 Hz, 1H,
CHCHHO), 3.53 (dd, J1 = 9.4 Hz, J2 = 3.8 Hz, 1H, CHCHHO), 2.95 (t,
J = 6.8 Hz, 2H, PhCH2), 2.58 (t, J = 6.8 Hz, 2H, CH2COCO), 1.70–
1.55 (m, 6H, 3ÂCH2), 1.35–1.20 (m, 7H, 2ÂCH2, OCH2CH3), 0.89
(t, J = 6.4 Hz, 3H, CH3); 13C NMR (CDCl3): d 199.0, 170.4, 159.9,
134.1, 129.2, 113.6, 72.5, 68.2, 61.0, 55.2, 49.4, 36.6, 34.6, 31.0,
30.9, 28.0, 22.7, 22.5, 14.2, 13.9; MS (ESI) m/z (%): 422 ([M+H]+,
100); HRMS (ESI) calcd for C23H34NOÀ6 [MÀH]À: 420.2392. Found
420.2389; Anal. Calcd for C23H35NO6: C, 65.54; H, 8.37; N, 3.32.
Found: C, 65.39; H, 8.49; N, 3.27.
4.2.5.13. tert-Butyl 2-((2S,3R)-2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)-3-methylpentanamido)acetate (25b). Yield 69%; Colorless
20
oil; [a
]
À17.0 (c 0.50, CHCl3); 1H NMR (CDCl3): d 7.56 (d,
D
J = 9.0 Hz, 1H, NH), 7.07 (d, J = 8.2 Hz, 2H, arom), 6.80 (d,
J = 8.4 Hz, 2H, arom), 6.77–6.67 (m, 1H, NH), 4.38–4.27 (m, 1H,
CH), 3.92 (dd, J1 = 5.2 Hz, J2 = 14.4 Hz, 2H, NHCH2), 3.76 (s, 3H,
CH3O), 2.92 (t, J = 6.4 Hz, 2H, PhCH2), 2.55 (t, J = 6.6 Hz, 2H,
CH2COCO), 2.04–1.85 (m, 1H, CH), 1.65–1.53 (m, 4H, 2ÂCH2),
1.44 [s, 9H, C(CH3)3], 1.32–1.03 (m, 2H, CH2), 0.99–0.81 (m, 6H,
2ÂCH3); 13C NMR (CDCl3): d 197.9, 170.3, 168.4, 159.9, 157.5,
133.9, 129.0, 113.5, 82.1, 57.5, 55.0, 41.8, 37.1, 36.5, 34.5, 30.8,
27.8, 24.7, 22.4, 15.2, 11.0; MS (ESI) m/z (%): 461 ([MÀH]À, 100);
4.2.5.9. (S)-tert-Butyl 2-((2-(2-oxo-6-phenylhexanamido)hexyl)oxy)
20
acetate (19b). Yield 95%; Colorless oil; [
a]
À10.2 (c 1.00, CHCl3);
D
1H NMR (CDCl3): d 7.44–7.02 (m, 6H, NH, arom), 4.05–3.85 (m, 3H,
OCH2COO, CH), 3.61 (dd, J1 = 9.4 Hz, J2 = 4.2 Hz, 1H, CHHO), 3.49
(dd, J1 = 9.4 Hz, J2 = 4.0 Hz, 1H, CHHO), 2.94 (t, J = 6.6 Hz, 2H,
CH2COCO), 2.62 (t, J = 6.8 Hz, 2H, CH2Ph), 1.77–1.53 (m, 6H,
HRMS (ESI) calcd for
C
25H37N2OÀ6 [MÀH]À: 461.2657. Found
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13
461.2662; Anal. Calcd for C25H38N2O6: C, 64.91; H, 8.28; N, 6.06.
Found: C, 64.79; H, 8.39; N, 6.01.
solid; 1H NMR (CDCl3): d 7.58 (d, J = 8.4 Hz, 1H, NH), 7.06 (d,
J = 8.4 Hz, 2H, arom), 6.80 (d, J = 8.4 Hz, 2H, arom), 6.36 (t,
J = 5.6 Hz, 1H, NH), 4.50–4.42 (m, 1H, CH), 3.90 (m, 2H, CH2), 3.72
(s, 3H, CH3O), 2.92 (t, J = 6.5 Hz, 2H, PhCH2), 2.54 (t, J = 6.7 Hz,
2H, CH2), 1.99–1.31 [m, 20H, 5ÂCH2, CH, C(CH3)3], 0.98–0.85 (m,
6H, 2ÂCH3); 13C NMR (CDCl3): d 198.1, 170.5, 168.5, 159.8, 157.5,
134.5, 129.2, 113.6, 82.2, 55.1, 53.4, 41.7, 38.9, 36.6, 34.3, 32.7,
31.0, 27.9, 27.5, 24.7, 22.9, 22.1; HRMS (ESI) calcd for C27H42N2-
NaO+6 [M+Na]+: 513.2935. Found: 513.2949; Anal. Calcd for
4.2.5.14.
(S)-tert-Butyl
2-(2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)-3-methylbutanamido)acetate (25c). Yield 80%; Colorless
20
oil; [a]
À13.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 200 MHz): d 7.60
D
(d, J = 9.2 Hz, 1H, NH), 7.06 (d, J = 8.6 Hz, 2H, arom), 6.97–6.85
(m, 1H, NH), 6.79 (d, J = 8.6 Hz, 2H, arom), 4.32 (dd, J1 = 7.0 Hz,
J2 = 9.0 Hz, 1H, CHCO), 3.92 (dd, J1 = 5.4 Hz, J2 = 18.8 Hz, 2H,
NHCH2), 3.76 (s, 3H, CH3O), 2.92 (t, J = 5.0 Hz, 2H, PhCH2), 2.55 (t,
J = 6.8 Hz, 2H, CH2COCO), 2.29–2.08 (m, 1H, CH), 1.68–1.54 (m,
4H, 2ÂCH2), 1.44 [s, 9H, C(CH3)3], 1.03–0.87 (m, 6H, 2ÂCH3); 13C
NMR (CDCl3, 50 MHz): d 197.9, 170.2, 168.5, 160.0, 157.5, 133.9,
129.1, 113.5, 82.2, 58.3, 55.0, 41.8, 36.5, 34.5, 31.1, 30.9, 27.8,
22.4, 19.1, 18.0; MS (ESI) m/z (%): 447 ([MÀH]À, 100); HRMS
(ESI) calcd for C24H35N2O6À [MÀH]À: 447.2501. Found 447.2509;
Anal. Calcd for C24H36N2O6: C, 64.26; H, 8.09; N, 6.25. Found: C,
64.02; H, 8.03; N, 6.09.
C27H42N2O6: C, 66.10; H, 8.63; N, 5.71. Found: C, 65.89; H, 8.78;
N, 5.64.
4.2.5.19. (S)-N-(1-Amino-1-oxohexan-2-yl)-6-(4-methoxyphenyl)-2-
20
oxohexanamide (27). Yield 71%; Yellow oil; [
a]
À16.6 (c 0.05,
D
CHCl3); 1H NMR (CDCl3): d 7.44 (d, J = 7.8 Hz, 1H, NH), 7.09 (d,
J = 8.6 Hz, 2H, arom), 6.82 (d, J = 8.6 Hz, 2H, arom), 6.09, 5.71
(2Âbr s, 2H, NH2), 4.46–4.31 (m, 1H, CH), 3.79 (s, 3H, CH3O), 2.93
(t, J = 6.4 Hz, 2H, PhCH2), 2.58 (t, J = 6.8 Hz, 2H, CH2COCO), 1.99–
1.76 (m, 2H, CH2), 1.70–1.57 (m, 4H, 2ÂCH2), 1.39–1.30 (m, 4H,
2ÂCH2), 0.89 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.1,
172.8, 160.0, 157.7, 134.0, 129.2, 113.7, 55.2, 52.8, 36.6, 34.6,
31.8, 31.0, 27.5, 22.6, 22.3, 13.8; MS (ESI) m/z (%): 347 ([MÀH]À,
100); HRMS (ESI) calcd for C19H27N2OÀ4 [MÀH]À: 347.1976. Found
347.1976; Anal. Calcd for C19H28N2O4: C, 65.49; H, 8.10; N, 8.04.
Found: C, 65.23; H, 8.27; N, 7.93.
4.2.5.15. (S)-tert-Butyl 2-((S)-2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)hexanamido)propanoate (25d). Yield 94%; Colorless oil;
1H NMR (CDCl3): d 7.47 (d, J = 8.6 Hz, 1H, NH), 7.08 (d, J = 8.6 Hz,
2H, arom), 6.82 (d, J = 8.6 Hz, 2H, arom), 6.51 (d, J = 7.4 Hz, 1H,
NH), 4.53–4.30 (m, 2H, 2ÂCH), 3.78 (s, 3H, CH3O), 2.93 (t,
J = 7.0 Hz, 2H, PhCH2), 2.57 (t, J = 7.0 Hz, 2H, CH2COCO), 1.97–
1.72 (m, 2H, CH2), 1.68–1.56 (m, 4H, 2ÂCH2), 1.46 [s, 9H, C
(CH3)3], 1.36 (d, J = 7.2 Hz, 3H, CH3), 1.34–1.21 (m, 4H, 2ÂCH2),
0.88 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.1, 171.7,
170.0, 159.9, 157.7, 134.1, 129.2, 113.7, 82.2, 55.2, 53.2, 48.7,
36.6, 34.6, 32.1, 31.0, 27.9, 27.5, 22.6, 22.3, 18.5, 13.8; MS (ESI)
m/z (%): 475 ([MÀH]À, 100); HRMS (ESI) calcd for C26H39N2O6À
[MÀH]À: 475.2814. Found 475.2819; Anal. Calcd for C26H40N2O6:
C, 65.52; H, 8.46; N, 5.88. Found: C, 65.27; H, 8.62; N, 5.73.
4.2.5.20. (S)-tert-Butyl 2-(6-(4-methoxyphenyl)-2-oxohexanamido)
20
hexanoate (29). Yield 88%; Yellow oil; [
a]
À8.7 (c 1.00, CHCl3);
D
1H NMR (CDCl3): d 7.38 (d, J = 8.2 Hz, 1H, NH), 7.09 (d, J = 8.6 Hz,
2H, arom), 6.83 (d, J = 8.8 Hz, 2H, arom), 4.53–4.34 (m, 1H, CH),
3.79 (s, 3H, CH3O), 2.94 (t, J = 7.0 Hz, 2H, PhCH2), 2.58 (t,
J = 6.8 Hz, 2H, CH2COCO), 1.95–1.72 (m, 1H, CHCHH), 1.72–1.55
(m, 5H, 2ÂCH2, CHCHH), 1.48 [s, 9H, C(CH3)3], 1.39–1.19 (m, 4H,
2ÂCH2), 0.90 (t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.4,
170.6, 159.6, 157.7, 134.1, 129.2, 113.7, 82.3, 55.2, 52.7, 36.5,
34.6, 32.1, 31.0, 28.0, 27.1, 22.7, 22.3, 13.8; MS (ESI) m/z (%): 404
([MÀH]À, 100); HRMS (ESI) calcd for C23H34NOÀ5 [MÀH]À:
404.2442. Found 404.2442; Anal. Calcd for C23H35NO5: C, 68.12;
H, 8.70; N, 3.45. Found: C, 67.94; H, 8.88; N, 3.31.
4.2.5.16. (R)-tert-Butyl 2-((S)-2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)hexanamido)propanoate (25e). Yield 85%; Colorless oil;
1H NMR (CDCl3): d 7.42 (d, J = 8.4 Hz, 1H, NH), 7.08 (d, J = 8.4 Hz,
2H, arom), 6.80 (d, J = 8.6 Hz, 2H, arom), 6.49 (d, J = 6.8 Hz, 1H,
NH), 4.54–4.23 (m, 2H, 2ÂCH), 3.77 (s, 3H, CH3O), 2.93 (t,
J = 6.6 Hz, 2H, PhCH2), 2.56 (t, J = 6.8 Hz, 2H, CH2COCO), 1.98–
1.53 (m, 6H, 3ÂCH2), 1.44 [s, 9H, C(CH3)3], 1.37 (d, J = 7.0 Hz, 3H,
CH3), 1.33–1.19 (m, 4H, 2ÂCH2), 0.87 (t, J = 7.0 Hz, 3H, CH3); 13C
NMR (CDCl3): d 198.1, 171.8,169.8, 159.9, 157.6, 134.1, 128.2,
113.7, 82.3, 55.2, 53.1, 48.7, 36.6, 34.6, 32.1, 31.0, 27.9, 27.5,
22.6, 22.3, 18.5, 13.9; MS (ESI) m/z (%): 475 ([MÀH]À, 100); HRMS
(ESI) calcd for C26H39N2O6À [MÀH]À: 475.2814. Found 475.2820;
Anal. Calcd for C26H40N2O6: C, 65.52; H, 8.46; N, 5.88. Found: C,
65.33; H, 8.69; N, 5.73.
4.2.5.21. (S)-Benzyl 2-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
hexanamido)acetate (31a). Yield 75%; Yellowish solid; mp 69–
20
73 °C; [
a]
À17.5 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.48–7.28
D
(m, 6H, NH, arom), 7.08 (d, J = 8.4 Hz, 2H, arom), 6.82 (d,
J = 8.6 Hz, 2H, arom), 6.52 (t, J = 5.2 Hz, 1H, NHCH2), 5.17 (s, 2H,
OCH2Ph), 4.47–4.27 (m, 1H, CH), 4.08 (d, J = 5.2 Hz, 2H, NHCH2),
3.78 (s, 3H, CH3O), 2.92 (t, J = 6.6 Hz, 2H, PhCH2), 2.57 (t,
J = 7.0 Hz, 2H, CH2COCO), 2.01–1.77 (m, 1H, CHCHH), 1.77–1.54
(m, 5H, 2ÂCH2, CHCHH), 1.38–1.20 (m, 4H, 2ÂCH2), 0.88 (t,
J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.0, 170.8, 169.3,
160.0, 157.7, 134.9, 134.1, 129.2, 113.7, 67.3, 55.2, 53.1, 41.3,
36.6, 34.6, 31.8, 31.0, 27.5, 22.6, 22.3, 13.8; MS (ESI) m/z (%): 495
([MÀH]À, 100); HRMS (ESI) calcd for C28H35N2OÀ6 [MÀH]À:
495.2501. Found 495.2497; Anal. Calcd for C28H36N2O6: C, 67.72;
H, 7.31; N, 5.64. Found: C, 67.52; H, 7.47; N, 5.52.
4.2.5.17. (R)-tert-Butyl 2-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
hexanamido)acetate (25f). Yield 84%; Colorless oil; [a]D
20 16.0 (c 0.05,
CHCl3); 1H NMR (CDCl3): d 7.54 (d, J = 8.4 Hz, 1H, NH), 7.05 (d,
J = 8.4 Hz, 2H, arom), 6.79 (d, J = 8.4 Hz, 2H, arom), 6.75–6.66 (m,
1H, NH), 4.53–4.30 (m, 1H, CH), 3.90 (dd, J1 = 1.6 Hz, J2 = 5.0 Hz,
2H, NHCH2), 3.75 (s, 3H, CH3O), 2.91 (t, J = 6.4 Hz, 2H, PhCH2), 2.54
(t, J = 6.8 Hz, 2H, CH2COCO), 1.94–1.51 (m, 6H, 3ÂCH2), 1.43 [s, 9H,
C(CH3)3], 1.32–1.19 (m, 4H, 2ÂCH2), 0.85 (t, J = 6.4 Hz, 3H, CH3);
13C NMR (CDCl3): d 198.0, 170.7, 168.5, 159.9, 157.6, 134.0, 129.1,
113.6, 82.3, 55.1, 53.0, 41.9, 36.5, 34.5, 32.0, 30.9, 27.9, 27.5, 22.5,
22.2, 13.8; MS (ESI) m/z (%): 461 ([MÀH]À, 100); HRMS (ESI) calcd
for C25H37N2OÀ6 [MÀH]À: 461.2657. Found 461.2660; Anal. Calcd
for C25H38N2O6: C, 64.91; H, 8.28; N, 6.06. Found: C, 64.73; H,
8.46; N, 6.01.
4.2.5.22. Benzyl (S)-4-(2-(6-(4-methoxyphenyl)-2-oxohexanamido)
hexanamido)butanoate (31b). Yield 70%; Colorless oil; 1H NMR
(CDCl3): d 7.48–7.20 (m, 6H, NH, arom), 7.08 (d, J = 8.4 Hz, 2H,
arom), 6.80 (d, J = 8.4 Hz, 2H, arom), 6.50 (t, J = 5.3 Hz, 1H, NH),
5.10 (s, 2H, OCH2Ph), 4.40–4.25 (m, 1H, CH), 3.98 (d, J = 5.3 Hz,
2H, CH2), 3.77 (s, 3H, CH3O), 2.92 (t, J = 6.6 Hz, 2H, PhCH2), 2.56
(t, J = 6.9 Hz, 2H, CH2), 2.01–1.20 (m, 14H, 7ÂCH2), 0.89 (t,
J = 6.9 Hz, 3H, CH3); 13C NMR (CDCl3): d 198.0, 170.7, 169.0,
159.9, 157.6, 134.8, 134.1, 129.2, 128.2, 127.6, 126.2, 113.7, 67.2,
55.3, 53.2, 41.3, 36.6, 34.6, 32.1, 31.0, 29.8, 27.5, 23.4, 22.6, 22.3,
4.2.5.18.
(S)-tert-Butyl
4-(2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)-4-methylpentanamido)butanoate (25g). Yield 79%; White
Please cite this article in press as: Smyrniotou A., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.007

14
A. Smyrniotou et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13.8; MS (ESI) m/z (%): 523 ([MÀH]À, 100); HRMS (ESI) calcd for
[MÀH]À: 392.2079. Found 392.2084; Anal. Calcd for C21H31NO6: C,
C
C
30H40N2OÀ6 [MÀH]À: 523.2814. Found 523.2811; Anal. Calcd for
64.10; H, 7.94; N, 3.56. Found: C, 63.92; H, 8.09; N, 3.45.
30H40N2O6: C, 68.81; H, 7.51; N, 5.35. Found: C, 68.62; H, 7.72;
N, 5.22.
4.3. In vitro PLA2 activity assays
The activities of human GVIA iPLA2, GIV cPLA2 and GV sPLA2
were determined using a group-specific mixed micelle modified
Dole assay.26,28,30 The substrate was prepared using slightly differ-
ent conditions for each enzyme to achieve optimum activity: (i)
4.2.5.23. (S)-tert-Butyl 2-(2-(2-(6-(4-methoxyphenyl)-2-oxohex-
anamido)hexanamido)acetamido)acetate (33). Yield 72%; Brown
20
syrup; [
a]
À10.8 (c 1.00, CHCl3); 1H NMR (CDCl3): d 7.53 (d,
D
J = 8.0 Hz, 1H, NH), 7.17 (br s, 1H, NH), 7.08 (d, J = 8.4 Hz, 2H, arom),
6.90 (br s, 1H, NH), 6.81 (d, J = 8.2 Hz, 2H, arom), 4.40–4.20 (m, 1H,
CH), 4.04–3.97 (m, 2H, NHCH2CO), 3.94 (d, J = 5.2 Hz, 2H, NHCH2-
CO), 3.78 (s, 3H, CH3O), 2.99–2.83 (m, 2H, PhCH2), 2.61–2.50 (m,
2H, CH2COCO), 1.97–1.52 (m, 4H, 2ÂCH2), 1.44 [s, 9H, C(CH3)3],
1.36–1.28 (m, 4H, 2ÂCH2), 0.88 (t, J = 6.2 Hz, 3H, CH3); 13C NMR
(CDCl3): d 197.9, 171.3, 168.9, 168.8, 160.3, 157.6, 134.0, 129.2,
113.6, 82.5, 55.2, 53.6, 41.9, 36.6, 34.6, 31.7, 31.0, 29.7, 27.6,
27.9, 22.5, 22.3, 13.8; MS (ESI) m/z (%): 518 ([MÀH]À, 100); HRMS
(ESI) calcd for C27H40N3O7À [MÀH]À: 518.2872. Found 518.2869;
Anal. Calcd for C27H41N3O7: C, 62.41; H, 7.95; N, 8.09. Found: C,
62.24; H, 8.07; N, 8.00.
GIVA cPLA2 mixed micelle substrate consisted of 400
X-100, 95.3 M PAPC, 1.7
M arachidonyl-1-14C PAPC, and 3
PIP2 in a buffer containing 100 mM HEPES pH 7.5, 90 M CaCl2,
2 mM DTT, and 0.1 mg/ml BSA; (ii) GVIA iPLA2 mixed micelle sub-
strate consisted of 400 M Triton X-100, 98.3 M PAPC, and
1.7
M arachidonyl-1-14C PAPC in a buffer containing 100 mM
HEPES pH 7.5, 2 mM ATP, and 4 mM DTT; and (iii) GV sPLA2 mixed
micelles substrate consisted of 400 M Triton X-100, 98.3
PAPC, and 1.7
M arachidonyl-1-14C PAPC in a buffer containing
50 mM Tris-HCl pH 8.0, and 5 mM CaCl2. The compounds were ini-
tially screened at 0.091 mol fraction (5 L of 5 mM inhibitor in
lM Triton
l
l
lM
l
l
l
l
l
lM
l
l
DMSO) in substrate (495 uL). XI(50) was determined for com-
pounds exhibiting greater than 95% inhibition. Inhibition curves
were generated using GraphPad Prism 5.0 and the non-linear
regression by plotting percentage of inhibition vs log (mole frac-
tion) to calculate the reported XI(50) and its associated error.
4.2.6. Cleavage of tert-butyl protecting group
A solution of the tert-butyl ester derivative (1.0 mmol) in 50%
TFA/CH2Cl2 (0.5 M) was stirred for 1–3 h at room temperature.
After the completion of the reaction, the organic solvent was evap-
orated under reduced pressure and the residue was purified by
recrystallization using diethyl ether/petroleum ether (bp 40–
60 °C).
Acknowledgments
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 (NSRF) – Research
Funding Program: Heracleitus II. Investing in knowledge society
through the European Social Fund (AS and VK), and NIH grant
RO1 GM20501-40 (EAD).
4.2.6.1. (S)-2-(2-(2-Oxo-6-phenylhexanamido)hexanamido)acetic acid
(13a). Yield 88%; Colorless oil; [a D20
]
À14.2 (c 1.00, CHCl3); 1H NMR
(CDCl3): d 8.52 (br s, 1H, OH), 7.78 (d, J = 8.8 Hz, 1H, NH), 7.47–7.06
(m, 6H, 5Âarom, NH), 4.70–4.45 (m, 1H, CH), 4.15–3.90 (m, 2H, CH2-
COOH), 2.92 (t, J = 6.4 Hz, 2H, COCOCH2), 2.62 (t, J = 6.6 Hz, 2H,
PhCH2), 2.05–1.50 (m, 6H, 3ÂCH2), 1.48–1.12 (m, 4H, 2ÂCH2), 0.88
(t, J = 6.2 Hz, 3H, CH3); 13C NMR (CDCl3): d 197.8, 172.4, 171.8,
160.2, 141.9, 128.29, 128.27, 125.7, 53.0, 41.3, 36.6, 35.5, 31.9,
30.7, 27.5, 22.6, 22.2, 13.8; MS (ESI) m/z (%): 375 ([MÀH]À, 100);
Anal. Calcd for C28H28N2O5: C, 63.81; H, 7.50; N, 7.44. Found: C,
63.64; H, 7.69; N, 7.35.
References
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acetic acid (13b). Yield 98%; Yellowish syrup; [a D20
]
À10.9 (c 1.00,
CHCl3); 1H NMR (CDCl3): d 7.77–7.22 (m, 8H, arom, NH), 7.01 (br s,
1H, NH), 4.65–4.40 (m, 1H, CH), 4.02 (br s, 2H, CH2COO), 2.93 (t,
J = 6.6 Hz, 2H, CH2COCO), 2.78 (t, J = 6.6 Hz, 2H, PhCH2), 1.98–1.49
(m, 6H, 3ÂCH2), 1.39–1.02 (m, 4H, 2ÂCH2), 0.87 (t, J = 5.6 Hz, 3H,
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131.8, 127.8, 127.5, 127.3, 127.1, 126.3, 125.8, 125.0, 53.0, 36.6,
35.6, 31.9, 30.5, 30.4, 27.5, 22.6, 22.2, 13.8; MS (ESI) m/z (%): 425
([MÀH]À, 100); Anal. Calcd for C24H30N2O5: C, 67.59; H, 7.09; N,
6.57. Found: C, 67.24; H, 7.23; N, 6.44.
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4.2.6.3. (S)-2-((2-(6-(4-Methoxyphenyl)-2-oxohexanamido)he-xyl)oxy)
acetic acid (20d). Yield 93%; Brown solid; mp 52–55 °C; [a D20
]
À10.9
(c 1.00, CHCl3); 1H NMR (CDCl3): d 7.63 (br s, 1H, NH), 7.12 (d,
J = 8.4 Hz, 2H, arom), 6.82 (d, J = 8.6 Hz, 2H, arom), 4.21–3.91 (m,
3H, CH, OCH2COOH), 3.75 (s, 3H, CH3O), 3.70–3.50 (m, 2H, CH2O),
2.97–2.81 (m, 2H, PhCH2), 2.65–2.48 (m, 2H, CH2COCO), 1.74–1.48
(m, 6H, 3ÂCH2), 1.40–1.20 (m, 4H, 2ÂCH2), 0.93–0.80 (m, 3H,
CH3); 13C NMR (CDCl3): d 198.9, 160.1, 157.6, 134.1, 129.2, 113.6,
55.2, 49.5, 36.6, 34.6, 30.9, 30.7, 29.6, 28.0, 22.6, 22.4, 13.9; MS
(ESI) m/z (%): 392 ([MÀH]À, 100); HRMS (ESI) calcd for C21H30NOÀ6
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