Synthesis and biological activity of cyclolinopeptide A analogues modified with γ3-bis(homophenylalanine

Article abstract of DOI:10.1016/j.ejmech.2014.09.014

Cyclolinopeptide A, naturally occurring immunomodulatory nonapeptide, was modified with S or R-γ³- bis(homophenylalanine) in positions 3 or 4, or both 3 and 4. The replacement of one or both Phe residues by γ³-hhPhe led to decrease of their conformational flexibility in the analogues in comparison to CLA. All cyclic peptides, except 11, exist as isomers with the cis ProePro peptide bond. Cyclic peptide 11 with single modification S-γ³-hhPhe4 exists as a mixture of two isomers and the major isomer (89%) contains all peptide bonds of the trans geometry. The peptides were subjected to several immunological tests in vitro and in vivo. Linear peptides 1e8, precursors of CLA analogues 9e16, were not toxic against human peripheral blood mononuclear cells (PBMC) but cyclic analogues showed dose-dependent toxicity with exception of peptide 11. Linear peptides did not inhibit mitogen-induced PBMC proliferation whereas cyclic ones inhibited the proliferation in a dose-dependent manner. The actions of linear and cyclic peptides with regard to lipopolysaccharide (LPS) -induced tumour necrosis factor alpha (TNF a) production in whole human blood cultures were differential but particularly suppressive in the case of linear compound 6. Therefore, for in vivo tests compounds 6 and 11 were selected. The compounds showed comparable, suppressive actions in induction and effector phases of delayed type hypersensitivity as well as in the carrageenaninduced foot pad edema in mouse models. In summary, linear peptide 6 and cyclic peptide 11 are attractive as potential immune suppressor drugs.

Full text of DOI:10.1016/j.ejmech.2014.09.014

European Journal of Medicinal Chemistry 86 (2014) 515e527
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of cyclolinopeptide A analogues
modified with
3-bis(homophenylalanine)
g
Karol Je˛drzejczak a, Paweł Hrynczyszyn a, Jolanta Artym b, Maja Kocie˛ba b,
,
, *
Michał Zimecki b, Janusz Zabrocki a c, Stefan Jankowski a
a
_
ꢀ ꢀ
Institute of Organic Chemistry, Faculty of Chemistry, Technical University of Łodz, Zeromskiego 116, 90-924, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Science, R. Weigla 12, 53-114 Wroclaw, Poland
c Peptaderm Ltd., Rydygiera 8, 01-793 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Cyclolinopeptide A, naturally occurring immunomodulatory nonapeptide, was modified with S or R-g3
-
Article history:
Received 18 May 2014
Received in revised form
1 September 2014
Accepted 4 September 2014
Available online 6 September 2014
bis(homophenylalanine) in positions 3 or 4, or both 3 and 4. The replacement of one or both Phe residues
by
3-hhPhe led to decrease of their conformational flexibility in the analogues in comparison to CLA. All
cyclic peptides, except 11, exist as isomers with the cis ProePro peptide bond. Cyclic peptide 11 with
single modification S-
3-hhPhe4 exists as a mixture of two isomers and the major isomer (89%) contains
g
g
all peptide bonds of the trans geometry.
The peptides were subjected to several immunological tests in vitro and in vivo. Linear peptides 1e8,
precursors of CLA analogues 9e16, were not toxic against human peripheral blood mononuclear cells
(PBMC) but cyclic analogues showed dose-dependent toxicity with exception of peptide 11. Linear
peptides did not inhibit mitogen-induced PBMC proliferation whereas cyclic ones inhibited the prolif-
eration in a dose-dependent manner. The actions of linear and cyclic peptides with regard to lipopoly-
Keywords:
Cyclolinopeptide A
Immune response
Gamma amino acids
TNF-
a
Proliferation
Immunosuppression
saccharide (LPS) -induced tumour necrosis factor alpha (TNF
a) production in whole human blood
cultures were differential but particularly suppressive in the case of linear compound 6. Therefore, for
in vivo tests compounds 6 and 11 were selected. The compounds showed comparable, suppressive ac-
tions in induction and effector phases of delayed type hypersensitivity as well as in the carrageenan-
induced foot pad edema in mouse models. In summary, linear peptide 6 and cyclic peptide 11 are
attractive as potential immune suppressor drugs.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
[10]. Cyclic immunosuppressive peptides derived from natural
sources are regarded as a new class of pharmaceuticals [11].
Naturally occurring and synthetic cyclopeptides exhibit
different activities: anti-cancer [1,2], anti-HIV [3], antinoceptive
[4,5], cytotoxic [6,7], anti-bacterial [8], antifungal [9] or insecticidal
Cyclolinopeptide
A
(cyclo-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-
Val), CLA), discovered in linseed oil in 1959 [12], possesses a range
of biological activities including the ability to inhibit hepatocyte cell
transport [13] and their immunosuppressive activity at low doses is
equal to that of cyclosporine A, that has been confirmed in a
number of assays [14,15]. All aspects of the biological activity of CLA
and its analogues based on the structureeactivity relationship have
been discussed in detail in reviewed papers [16,17]. According to
the well-known hypothesis the Pro-Pro-Phe-Phe sequence and
conformational flexibility are the most important factors respon-
sible for its biological activity. We applied several modifications of
Abbreviations: ANOVA, analysis of variance; Boc, tert-Butoxycarbonyl; C8, octyl
stationary phase; C18, octadecyl stationary phase; CLA, cyclolinopeptide A; COSY-
DQF, double quantum filtered proton spin correlation; DEX, dexamethasone; DIPEA,
N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
hhPhe, bis(homophenylalanine); HOBt, 1-hydroxybezotriazole hydrate; Ile, isoleu-
cine; Leu, leucine; LPS, lipopolysaccharide; MTT, 93-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide; PBMC, peripheral blood mononucleated cell; PHA,
Phytohemagglutinin A; Phe, phenylalanie; Pro, proline; TBTU, O-(Benzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid;
CLA using (S)-
proline [18], or replacement of phenylalanine by
[19], or homophenylalanine [20]. The introduction of ethylene
bridge between phenylalanine nitrogens resulted in
b
2-isoproline or (S)-
b
3-homo-proline instead of
3-phenylalanine
b
TFMSA, trifluoromethanesulfonic acid; TNF-
valine.
a, tumour necrosis factor alpha; Val,
* Corresponding author.
E-mail address: stefan.jankowski@p.lodz.pl (S. Jankowski).
http://dx.doi.org/10.1016/j.ejmech.2014.09.014
0223-5234/© 2014 Published by Elsevier Masson SAS.

516
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
conformational flexibility of CLA molecule being probably a cause
of its antagonistic action towards methotrexate [21].
chromatograms were visualized by KMnO4 or ninhydrin. HPLC was
performed on a Thermo Spectra System, a Vydac C8 column
(0.46 ꢀ 25 cm): flow 1.5 mL/min or 1 mL/min, detection at 220 nm
and eluents (A) 0.05% trifluoroacetic acid in water and (B) 0.038%
trifluoroacetic acid in acetonitrile/water 90:10 with a gradient
application. Purification of peptides was performed by the pre-
parative reversed-phase HPLC on Gilson 322 pump and UV/Vis-152
detector on a Vydac C8 column (2.21 ꢀ 25 cm): flow rate 20 mL/
min, UV detection at 220 nm. The identities of the pure peptides
were confirmed by Maldi-TOF mass spectrometry on Voyager Elite,
g-Amino acids are homologues of a-amino acids with two
additional carbon atoms separating amino and carboxyl groups.
Such a modification leads to the resistance against enzymatic
degradation [22].
[23] as well as antiviral [24] and anticancer properties [25,26].
Amino acids are also important elements of foldamers [27] and self-
assembling nanotubes [28]. Despite their significant flexibility,
amino acids form well-defined secondary structures in solutions. In
particular, homogenous oligomers containing monosubstituted
a,g-Peptide hybrids display antibiotic activity
g-
g-
g-
Perceptive Biosystems using a-cyano-4-hydroxy-cinnamic acid as a
amino acids can form stable helical conformations in solution [29].
matrix. The one- and two-dimensional 1H NMR spectra were
recorded on a Bruker Avance II Plus spectrometer at 700.4 MHz in
DMSO-d6, CDCl3, MeOD or CD3CN using as an internal shift
standard solvent signals of 2.50 ppm, 7.26 ppm, 4.87 ppm, CD3CN
1.94 ppm, respectively. Spin-lock time in TOCSY experiments was
80 ms and NOESY experiments were recorded with 300 ms mixing
time. 2D NMR spectra were processed with TopSpin 2.1 (Bruker)
and analyzed by Sparky software [30]. All amino acid derivatives
and peptide bond forming reagents were purchased from IRIS
Biotech (Germany). All reagents were purchased from Sigma-
eAldrich and solvent were purchased from POCh.
In order to evaluate the role and significance of the presence of
g-amino acid residue on the biological activity we have synthetized
8 linear and 8 cyclic analogues of CLA, in which one or both phe-
nylalanines were replaced by one or two S or R
nylalanine) residues (Fig. 1).
g
3-bis(homophe-
2. Materials and methods
2.1. General remarks
All solvents were purified by conventional methods. Evapora-
tions were carried out under reduced pressure. Melting points were
determined in a capillary melting point apparatus Büchi SMP-20.
The optical rotation was measured in a 1 dm cell (1 mL) on a
PolAAr 3001 automatic polarimeter at 589 nm. For thin layer
chromatography 254 nm silica gel on TLC plates (Fluka) was used
with ethyl acetate: hexane (4:1) solvent system. The
2.2. Peptide synthesis and purification
The linear peptides 1e8 were synthesized by the manual solid-
phase method using chloromethylated Merrifield resin as a solid
support. Attachment of the first Boc-AA-OH (Boc-Leu) to the resin
was performed according to cesium salt procedure [31] and the
substitution level was determined by weight gain measurements
(Boc-Leu-Ⓟ, 0.62 mmol for peptides 1e3 or 0.72 mmol for peptides
4e8). Synthesis of peptides was achieved by stepwise coupling of
Boc-amino acids to the growing peptide chain on the resin. Starting
with 0.2 mmol of Boc protected amino acid attached to the resin,
standard single TBTU/HOBt/DIPEA coupling protocol (with depro-
tonation steps omitted) was used for all amino acids and was
repeated if Kaiser test [32] or Chloranil test (proline residue) [33]
was found positive. In all cases, when second coupling test was
slightly positive, remaining free amino groups were acetylated with
the aid of acetic anhydride (190
ml) in the presence of diisopropy-
lethylamine (350 l). After coupling step the Boc protecting group
m
was removed with 50% TFA in methylene chloride. The peptide
resin was treated with TFMSA (1 mL), TFA (10 mL) and anisole
(0.5 mL) at 0 ꢁC and stirred for 60 min at room temperature. The
resin had been filtered off and washed with TFA, and the crude
peptide was precipitated upon concentration of solvents and
addition of diethyl ether. The analytical samples were purified by
the preparative HPLC. The physicochemical properties of the syn-
thesized linear peptide are summarized in Table 1.
2.3. 1H NMR analysis of cyclolinopeptides 9e16
2.3.1. c(Pro1-Pro2-S-
Val9) (9)
g
3-hhPhe3-S-
g
3-hhPhe4-Leu5-Ile6-Ile7-Leu8-
(DMSO-d6, 50 ꢁC)
d
: 0.75 (3H, Hg, Ile7), 0.77 (3H, Hd, Leu8), 0.80
(3H, Hd, Ile7), 0.81 (3H, Hd, Ile6), 0.82 (3H, Hg, Ile6), 0.85 (3H, Hg,
Val9), 0.86 (3H, Hd, Leu8), 0.89 (6H, Hd, Leu5), 0.94 (3H, Hg, Val9),1.00
(1H, Hg, Ile7), 1.06 (1H, Hg, Ile6), 1.37 (1H, Hg, Ile7), 1.40 (1H, Hg, Ile6),
1.47 (1H, Hb, Leu5), 1.49 (2H, Hb, Leu8), 1.53 (1H, Hb, Leu5), 1.55 (1H,
Hg, Leu8), 1.62 (1H, Hg, Leu5), 1.66 (1H, Hg, Pro2), 1.70 (1H, Hb, Pro1),
1.71 (1H, Hb, Ile7), 1.80 (1H, Hb, Ile6), 1.80 (1H, Hg, Pro2), 1.80 (1H, Hg,
Pro1), 1.97 (1H, Hg, Pro1), 1.99 (1H, Hb, Val9), 2.03 (1H, Ha, hhPhe4),
2.03 (1H, Hb, Pro2), 2.06 (2H, Ha, hhPhe3), 2.07 (1H, Hb, hhPhe4),
2.21 (1H, Hb,Pro2), 2.24 (1H, Hb, Pro1), 2.34 (1H, Ha, hhPhe4), 2.45
Fig. 1. The sequences of linear (1e8) and cyclic (9e16) analogues of CLA containing g3
hhPhe 17.
-
00
(1H, Hg”, hhPhe3), 2.53 (1H, Hb, hhPhe3), 2.57 (1H, Hg , hhPhe4),

K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
517
Table 1
Physicochemical properties of peptides 1e16.
Peptide
Yield [%]
HPLC
Molecular formula
Formula mass
MALDI MS, m/z
[MþH]þ
tR (min)
Purity [%]
Crude
[MþNa]þ
Purified
1
2
3
4
5
6
7
8
74a
96a
88a
91a
90a
90a
90a
89a
12b
12b
18b
25b
40b
25b
36b
46b
13.233
12.690
13.177
13.085
13.938
14.073
13.383
14.168
10.110
9.890
11.630
9.605
10.543
9.242
10.033
11.870
86.57
90.58
85.26
91.24
92.25
94.16
90.20
89.73
98.43
99.08
95.97
97.23
95.74
96.52
97.98
100.00
98.39
100.00
95.32
98.09
100.00
96.82
98.43
97.07
C61H95N9O10
C59H91N9O10
C59H91N9O10
C61H95N9O10
C61H95N9O10
C61H95N9O10
C59H91N9O10
C59H91N9O10
C61H93N9O9
C59H89N9O9
C59H89N9O9
C61H93N9O9
C61H93N9O9
C61H93N9O9
C59H89N9O9
C59H89N9O9
1113.72
1085.69
1085.69
1113.72
1113.72
1113.72
1085.69
1085.69
1095.71
1067.68
1067.68
1095.71
1095.71
1095.71
1067.68
1067.68
1114.35
1086.51
1086.50
1114.64
1114.66
1114.61
1086.48
1086.55
1096.38
1068.50
1068.53
1096.60
1096.62
1096.67
1068.69
1068.52
1136.29
1108.41
1108.48
1136.61
1136.61
1136.54
1108.47
1108.51
1118.34
1090.47
1090.50
1118.57
1118.56
1118.62
1090.63
1090.49
9
10
11
12
13
14
15
16
a
Before HPLC purification.
Yield of cyclization.
b
00
00
2.62 (1H, Hg , hhPhe3), 2.65 (1H, Hg , hhPhe3), 3.04 (1H, Hg,
hhPhe3), 3.15 (1H, Hg, hhPhe4), 3.21 (1H, Hg, hhPhe3), 3.31 (1H, Hg,
hhPhe4), 3.39 (1H, Hd, Pro2), 3.41 (1H, Hd, Pro2), 3.58 (1H, Hd, Pro1),
3.67 (1H, Hd, Pro1), 4.13 (1H, Ha, Ile6), 4.19 (1H, Ha, Leu5), 4.20 (1H,
Ha, Ile7), 4.23 (1H, Ha, Pro1), 4.34 (1H, Ha, leu8), 4.42 (1H, Ha, Val9),
4.44 (1H Ha, Pro2), 7.11 (2H, H2,6, hhPhe4), 7.18 (1H, H4, hhPhe3), 7.20
(2H, H2,6, hhPhe3), 7.22 (2H, H3,5, hhPhe4),7.24 (1H, H4, hhPhe4),
7.22 (2H, H3,5, hhPhe4), 7.27 (2H, H3,5, hhPhe3), 7.46 (1H, HN, Ile7),
7.53 (1H, HN, Val9), 7.64 (1H, HN, Ile7), 7.78 (1H, HN, Leu5), 7.81 (1H,
HN, hhPhe4), 8.05 (1H, HN, Leu8), 8.07 (1H, HN, hhPhe3).
(1H, Hg, Pro1), 2.02 (1H, Hb, Val9), 2.10 (1H, Ha, hhPhe4), 2.13 (1H, Hb,
Pro1), 2.16 (1H, Hb, Pro2), 2.23 (1H, Ha, hhPhe4), 2.26 (1H, Hb,
hhPhe4), 2.56 (1H, Hg”, hhPhe4), 2.61 (1H, Hg”, hhPhe4), 2.97 (1H, Hd,
Pro2), 3.08 (1H, Hg, hhPhe4), 3.23 (1H, Hg, hhPhe4), 3.22 (1H, Hb,
Phe3), 3.26 (1H, Hd, Pro2), 3.28 (1H, Hb, Phe3), 3.35 (1H, Hd, Pro1),
3.61 (1H, Hd, Pro1), 4.02 (1H, Ha, Ile7), 4.14 (1H, Ha, Leu5), 4.19 (1H,
Ha, Ile6), 4.20 (1H, Ha, Leu8), 4.36 (1H, Ha, Pro1), 4.36 (1H, Ha, Pro2),
4.41 (1H, Ha, Phe3), 4.44 (1H, Ha, Val9), 7.18 (1H, HN, Val9), 7.18 (1H,
H4, Phe3), 7.19 (2H, H2,6, hhPhe4), 7.23 (2H, H3,5, Phe3), 7.26 (2H, H2,6
,
Phe3), 7.26 (1H, H4, hhPhe4), 7.26 (2H, H3,5, hhPhe4), 7.67 (1H, HN,
Ile6), 7.79 (1H, HN, Ile7), 7.81 (1H, HN, Leu5), 7.81 (1H, HN, hhPhe4),
8.07 (1H, HN, Leu8), 8.47 (1H, HN, Phe3)
2.3.2. c(Pro1-Pro2-S-
(DMSO-d6, 50 ꢁC)
g
3-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) (10)
: 0.70 (6H, Hd, Leu5), 0.73 (3H, Hd, Ile6), 0.80
d
(3H, Hd, Ile7), 0.81 (3H, Hg, Ile6),0.81 (6H, Hd, Leu5), 0.86 (3H, Hg,
Ile7), 0.86 (3H, Hg, Val9), 0.96 (3H, Hg, Val9), 0.99 (1H, Hg, Ile7), 1.04
(1H, Hg, Ile6), 1.34 (1H, Hg, Ile6), 1.37 (1H, Hg, Leu5), 1.40 (1H, Hb,
Leu8), 1.40 (1H, Hg, Ile7), 1.47 (1H, Hg, Leu5), 1.56 (2H, Hb, Leu5), 1.58
(1H, Hb, Leu5), 1.58 (1H, Hg, Pro2), 1.69 (1H, Hb, Pro1), 1.81 (1H, Hg,
Pro1), 1.82 (1H, Hg, Pro2), 1.88 (1H, Hb, Ile7), 1.88 (1H, Hb, Ile6), 2.01
2.3.4. c(Pro1-Pro2-Phe3-S-
(11b) (10.7%)
g
3-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9)
(DMSO-d6, 50 ꢁC) : 0.71 (3H, Hd, Ile6), 0.76 (3H, Hd, Ile7), 0.77
d
(3H, Hd, Leu8), 0.85 (3H, Hg, Ile6), 0.85 (3H, Hg, Ile7), 0.86 (3H, Hd,
Leu8), 0.87 (3H, Hg, Val9), 0.87 (6H, Hd, Leu5), 0.93 (3H, Hg, Val9),
1.02 (1H, Hg, Ile6), 1.06 (1H, Hg, Ile7), 1.16 (1H, Hg, Pro2), 1.54 (2H, Hb,
Leu5), 1.55 (1H, Hg, Ile7), 1.56 (1H, Hg, Leu8), 1.61 (1H, Hg, Ile6), 1.65
(2H, Hb, Leu8), 1.65 (1H, Hg, Pro1), 1.66 (1H, Hg, Pro2), 1.67 (1H, Hb,
Pro1), 1.68 (1H, Hg, Leu5), 1.69 (1H, Hg, Pro1), 1.87 (1H, Hb, Ile6), 1.90
(1H, Hb, Pro2), 1.94 (1H, Hb, Val9), 1.96 (1H, Ha, hhPhe4),1.96 (1H, Hb,
Ile7), 2.11 (1H, Ha, hhPhe4), 2.17 (1H, Hb, Pro2), 2.17 (1H, Hb, hhPhe4),
2.23 (1H, Hb, Pro1), 2.53 (2H, Hg”, hhPhe4), 2.71 (1H, Hg, hhPhe4),
3.06 (1H, Hd, Pro2), 3.19 (1H, Hb, Phe3), 3.25 (1H, Hg, hhPhe4), 3.27
(1H, Hd,Pro2), 3.29 (1H, Hb, Phe3), 3.33 (1H, Hd, Pro1), 4.40 (1H, Hd,
Pro1), 3.96 (1H, Ha, Leu8), 3.97 (1H, Ha, Ile7), 4.13 (1H, Ha, Ile6) 4.15
(1H, Ha, Val9), 4.30 (1H, Ha, Pro2), 4.41 (1H, Ha, Phe3), 4.43 (1H, Ha,
Leu5), 4.77 (1H, Ha, Pro1), 7.04e7.35 (10H, H2,6, Phe3, H3,5, Phe3, H4,
Phe3, H2,6, hhPhe4, H3,5, hhPhe4, H4, hhPhe4), 7.55 (1H, HN, Val9),
7.73 (1H, HN, hhPhe4), 7.73 (1H, HN, Leu8), 8.06 (1H, HN, Ile7), 8.14
(1H, HN, Leu5), 8.17 (1H, HN, Ile6), 8.43 (1H, HN, Phe3).
(1H, Hb, Pro2), 2.02 (1H, Hg, Pro1), 2.06 (2H, Ha, hhPhe3), 2.07 (1H,
00
Hb, Val9), 2.29 (1H, Hb, Pro2), 2.29 (1H, Hg00 , hhPhe3), 2.33 (1H, Hb,
Pro1), 2.39 (1H, Hb, hhPhe3), 2.57 (1H, Hg , hhPhe3), 2.76 (1H, Hg,
hhPhe3), 2.81 (1H, Hb, Phe4), 3.04 (1H, Hg, hhPhe3), 3.05 (1H, Hb,
Phe4), 3.31 (1H, Hd, Pro2), 3.42 (1H, Hd, Pro2), 3.58 (1H, Hd, Pro1),
3.75 (1H, Hd, Pro1), 3.75 (1H, Ha, Ile6), 4.15 (1H, Ha, Leu5), 4.20 (1H,
Ha, Pro1), 4.26 (1H, Ha, Leu8), 4.35 (1H, Ha, Ile7), 4.48 (1H, Ha, Pro2),
4.48 (1H,Ha, Val9), 4.68 (1H, Ha, Phe4), 7.11 (1H, H2,6, hhPhe3), 7.12
(1H, H4, Phe4), 7.14 (2H, H2,6, Phe4), 7.16 (2H, H3,5, Phe4), 7.18 (1H,
H4, hhPhe3), 7.25 (2H, H3,5,hhPhe3), 7.54 (1H, HN, Ile7), 7.55 (1H, HN,
Val9), 7.78 (1H, HN, Ile6), 7.82 (1H, HN, Leu5), 7.95 (1H, HN, Leu8), 8.06
(1H, HN, hhPhe3), 8.34 (1H, HN, Phe4).
2.3.3. c(Pro1-Pro2-Phe3-S-
g
3-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9)
(11a) (89.3%)
(DMSO-d6, 50 ꢁC)
d
:0.75 (3H, Hd, Leu8), 0.80 (3H, Hd, Ile7), 0.82
2.3.5. c(Pro1-Pro2-R-
Val9) (12)
g
3-hhPhe3-S-
g
3-hhPhe4-Leu5-Ile6-Ile7-Leu8-
(3H, Hg, Ile6), 0.84 (3H, Hg, Ile7), 0.85 (3H, Hd, Leu8), 0.86 (3H, Hg,
Val9), 0.86 (3H, Hd, Leu5), 0.90 (3H, Hd, Leu5), 0.96 (3H, Hg, Val9),1.00
(1H, Hg, Ile6), 1.02 (1H, Hg, Pro2),1.09 (1H, Hg, Ile6), 1.11 (1H, Hg, Ile7),
1.44 (3H, Hd, Ile6), 1.47 (1H, Hg, Ile7), 1.52 (1H, Hb, Leu8), 1.55 (1H, Hb,
Leu8), 1.56 (1H, Hg, Leu8), 1.57 (1H, Hb, Leu5), 1.59 (1H, Hg, Leu5), 1.61
(1H, Hg, Pro2), 1.62 (1H, Hb, Pro1), 1.64 (1H, Hb, Leu5), 1.78 (1H, Hg,
Pro1), 1.81 (1H, Hb, Ile6), 1.85 (1H, Hb, Pro2), 1.92 (1H, Hb, Ile7), 1.95
(DMSO-d6, 50 ꢁC)
d
: 0.72 (3H, Hd, Leu8), 0.79 (3H, Hg, Ile7), 0.80
(3H, Hd, Ile7), 0.81 (3H, Hg, Val9), 0.81 (3H, Hd, Leu8), 0.82 (3H, Hd,
Ile6), 0.83 (3H, Hg, Ile6), 0.88 (3H, Hd, Leu5), 0.91 (3H, Hd, Leu5), 0.92
(3H, Hg, Val9), 1.01 (1H, Hg, Ile7), 1.06 (1H, Hg, Ile6), 1.36 (1H, Hb,
Leu8), 1.40 (1H, Hg, Ile7), 1.41 (1H, Hg, Ile6), 1.47 (1H, Hb, Leu5), 1.50
(1H, Hb, Leu8), 1.54 (1H, Hb, Leu5), 1.55 (1H, Hg, Leu8), 1.62 (1H, Hg,

518
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
Leu5), 1.64 (1H, Hg, Pro2), 1.68 (1H, Hb, Pro1), 1.72 (1H, Hb, Ile7), 1.80
(1H, Hg, Pro2), 1.81 (1H, Hg, Pro1), 1.82 (1H, Hb, Ile6), 1.90 (1H, Hb,
Val9), 1.98 (1H, Hg, Pro1), 1.99 (1H, Hb, Pro2), 2.03 (1H, Ha, hhPhe3),
2.06 (1H, Hb, hhPhe4), 2.17 (1H, Ha, hhPhe4), 2.21 (1H,Ha, hhPhe3),
2.3.8. c(Pro1-Pro2-R-
(DMSO-d6, 50 ꢁC)
g
3hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) (15)
: 0.76 (3H, Hd, Leu5), 0.78 (3H, Hd, Ile7), 0.82
d
(3H, Hd, Ile6), 0.83 (3H, Hg, Ile6), 0.83 (3H, Hd, Leu5),0.84 (3H, Hd,
Leu8), 0.86 (3H, Hg, Ile7), 0.87 (3H, Hg, Val9), 0.88 (3H, Hd, Leu8), 0.97
(3H, Hg, Val9), 1.04 (1H, Hg, Ile7), 1.10 (1H, Hg,Ile6), 1.41 (1H, Hg, Ile7),
1.43 (1H, Hb, Leu5), 1.45 (1H, Hg, Ile6), 1.52 (1H, Hb, Leu5), 1.53 (1H,
Hg, Leu8), 1.54 (1H, Hb, Leu8), 1.55 (1H, Hg, Leu5), 1.63 (1H, Hb, Leu8),
1.66 (1H, Hg, Pro2), 1.70 (1H, Hb, Pro1), 1.81 (1H, Hb, Ile7), 1.84 (1H,
Hg, Pro2), 1.86 (1H, Hg, Pro1), 1.97 (1H, Hb, Ile6), 1.97 (1H, Hb, Val9),
1.99 (1H, Hg, Pro1), 2.06 (1H, Ha, hhPhe3), 2.06 (1H, Hb, Pro2), 2.16
(1H, Ha, hhPhe3), 2.22 (1H, Hb, hhPhe3), 2.24 (1H, Hb, Pro2), 2.28
2.23 (1H,Hb, Pro2), 2.24 (1H,Hb00, Pro1), 2.30 (1H,Ha, hhPhe4), 2.36
00
(1H, Hb, hhPhe3), 2.50 (1H, Hg , hhPhe300 ), 2.58 (1H, Hg , hhPhe3),
00
2.66 (1H, Hg , hhPhe4), 2.95 (1H, Hg , hhPhe4), 2.98 (1H, Hg,
hhPhe3), 3.17 (1H, Hg, hhPhe3), 3.17 (1H, Hg, hhPhe4), 3.30 (1H, Hd,
Pro2), 3.34 (1H, Hg, hhPhe4), 3.40 (1H, Hd, Pro2), 3.59 (1H, Hd, Pro1),
3.67 (1H, Hd, Pro1), 4.10 (1H, Ha, Ile6), 4.22 (1H, Ha, Ile7), 4.23 (1H,
Ha, Leu5), 4.39 (1H, Ha, Leu8), 4.43 (1H, Ha, Val9), 4.44 (1H, Ha, Pro2),
4.29 (1H, Ha, Pro1), 7.14 (2H, H3,5, hhPhe3), 7.16 (2H, H3,5, hhPhe4),
7.17 (1H, H4, hhPhe4), 7.18 (1H, H2,6, hhPhe3), 7.19 (2H, H2,6, hhPhe4),
7.19 (1H, H4, hhPhe3), 7.51 (1H, HN, Ile7), 7.58 (1H, HN, Ile6), 7.66 (1H,
HN, Val9), 7.77 (1H, HN, Leu5), 7.88 (1H, HN, hhPhe4), 8.10 (1H, HN,
Leu8), 8.23 (1H, HN, hhPhe3).
00
00
(1H, Hb, Pro1), 2.37 (1H, Hg , hhPhe3), 2.45 (1H, Hg , hhPhe3), 2.85
(1H, Hb, Phe4), 2.93 (2H, Hg, hhPhe3), 3.05 (1H, Hb, Phe4), 3.37 (1H,
Hd, Pro2), 3.44 (1H, Hd, Pro2), 3.59 (1H, Hd, Pro1), 3.72 (1H, Hd, Pro1),
3.39 (1H, Ha, Ile6), 4.18 (1H, Ha, Leu8), 4.25 (1H, Ha, Ile7), 4.25 (1H,
Ha, Pro1), 4.26 (1H, Ha, Leu5), 4.40 (1H, Ha, Pro2), 4.48 (1H, Ha, Val9),
4.61 (1H, Ha, Phe4), 7.09 (1H, H4, Phe4), 7.10 (2H, H2,6, hhPhe3), 7.16
(1H, H4, hhPhe3), 7.16 (2H, H3,5, Phe4), 7.23 (2H, H2,6, Phe4), 7.23 (2H,
H3,5, hhPhe3), 7.38 (1H, HN, Val9), 7.64 (1H, HN, Ile7), 7.85 (1H,HN,
Ile6), 7.86 (1H, HN, Leu8), 7.87 (1H, HN, Leu5), 7.96 (1H, HN, Phe3),
8.03 (1H, HN, hhPhe3).
2.3.6. c(Pro1-Pro2-S-
Val9) (13)
g
3-hhPhe3-R-
g
3-hhPhe4-Leu5-Ile6-Ile7-Leu8-
(DMSO-d6, 50 ꢁC)
d
: 0.67 (3H, Hg, Ile6), 0.78 (3H, Hg, Ile7), 0.79
(3H, Hd, Ile6), 0.80 (3H, Hd, Leu5), 0.81 (3H, Hd, Ile7), 0.84 (3H, Hd,
Leu8), 0.84 (3H, Hd, Leu8), 0.86 (3H, Hd, Leu5), 0.88 (3H, Hg, Val9),
0.95 (1H, Hg, Ile6), 0.96 (3H, Hg, Val9), 0.98 (1H, Hg, Ile7), 1.36 (1H,
Hg, Ile7), 1.39 (1H, Hg, Ile6), 1.44 (2H, Hb, Leu5), 1.44 (1H, Hb, Leu8),
1.56 (1H, Hg, Leu5), 1.53 (1H, Hb, Leu8), 1.59 (1H, Hg, Leu8), 1.64 (1H,
Hb, Ile6), 1.68 (1H, Hg, Pro2), 1.70 (1H, Hb, Ile7), 1.73 (1H, Hb, Pro1),
1.81 (1H, Hg, Pro1), 1.81 (1H, Hg, Pro2), 1.91 (1H, Hg, Pro1), 1.97 (1H,
Ha, hhPhe3), 2.00 (1H, Hb, Val9), 2.01 (1H, Ha, hhPhe4), 2.06 (1H, Hb,
2.3.9. c(Pro1-Pro2-Phe3-R-
g
3hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9) (16)
(DMSO-d6, 50 ꢁC) : 0.76 (3H, Hg, Ile7), 0.79 (3H, Hd, Leu8), 0.80
d
(3H, Hd, Ile7), 0.82 (3H, Hd, Ile6), 0.84 (3H, Hg, Ile6), 0.84 (3H, Hd,
Leu8), 0.84 (3H, Hd, Leu5), 0.86 (1H, Hg, Pro2), 0.89 (3H, Hd, Leu5),
0.92 (3H, Hg, Val9), 0.99 (3H, Hg, Val9), 1.05 (1H, Hg, Ile7), 1.08 (1H,
Hg, Ile6),1.41 (1H, Hg, Ile7),1.42 (1H, Hg, Ile6),1.50 (2H, Hb, Leu5),1.52
(2H, Hb, Leu8), 1.55 (1H, Hg, Pro2), 1.60 (1H, Hg, Leu5), 1.62 (1H, Hg,
Leu5), 1.67 (1H, Hb, Pro1), 1.79 (1H, Hb, Pro2), 1.80 (1H, Hb, Ile7), 1.82
(1H, Hg, Pro1), 1.91 (1H, Hb, Ile6), 1.99 (1H, Hg, Pro1), 2.05 (1H, Hb,
Val9), 2.10 (1H, Ha, hhPhe4), 2.13 (1H, Hb, Pro2), 2.18 (1H, Ha00,
Pro2), 2.07 (1H, Ha, hhPhe4), 2.10 (1H, Ha, hhPhe3), 2.20 (1H, Hb,
”
Pro2), 2.22 (1H, Hb, Pro1), 2.26 (1H, Hb, hhPhe4), 2.26 (1H, Hg
,
hhPhe4), 2.28 (1H, Hg”, hhPhe3), 2.56 (1H, Hb, hhPhe3), 2.64 (1H,
Hg”, hhPhe4), 2.69 (1H, Hg”, hhPhe3), 2.86 (1H, Hg, hhPhe4), 3.08
(1H, Hg, hhPhe3), 3.19 (1H, Hg, hhPhe4), 3.22 (1H, Hg, hhPhe3), 3.40
(1H, Hd, Pro2), 3.44 (1H, Hd, Pro2), 3.60 (1H, Hd, Pro1), 3.63 (1H, Hd,
Pro1), 4.01 (1H, Ha, Ile6), 4.09 (1H, Ha, Leu5), 4.15 (1H, Ha, Ile7), 4.20
(1H, Ha, Pro1), 4.33 (1H, Ha, Leu8), 4.43 (1H, Ha, Val9), 4.43 (1H, Ha,
Pro2), 7.14 (2H, Ha, hhPhe4), 7.17 (2H, H2,6, hhPhe3),7.19 (1H, H4,
hhPhe4), 7.21 (1H, Ha, hhPhe3), 7.24 (2H, H3,5, hhPhe4),7.25 (2H, Ha,
hhPhe3), 7.25 (1H, HN, Ile7),7.53 (1H, HN, Val9),7.85 (1H, HN, hhPhe4),
7.90 (1H, HN, Ile6), 7.96 (1H, HN, Leu5), 8.06 (1H, HN, hhPhe3), 8.15
(1H, HN, Leu8).
hhPhe4), 2.19 (1H, Hb, Pro1), 2.30 (1H, Hb, hhPhe4), 2.39 (1H, Hg
,
hhPhe4), 2.69 (1H, Hg”, hhPhe4), 2.85 (1H, Hd, Pro2), 2.99 (1H, Hg,
hhPhe4), 3.09 (1H, Hb, Phe3), 3.18 (1H, Hb, Phe3), 3.24 (1H, Hd, Pro2),
3.30 (1H, Hg, hhPhe4), 3.58 (1H, Hd, Pro1), 3.69 (1H, Hd, Pro1), 3.99
(1H, Ha, Ile7), 4.00 (1H, Ha, Ile6), 4.19 (1H, Ha, Leu5), 4.28 (1H, Ha,
Leu8), 4.3 (1H, Ha, Pro1), 4.40 (1H, Ha, Pro2), 4.40 (1H, Ha, Val9), 4.49
(1H, Ha, Phe3), 7.16 (1H, H4, Phe3), 7.19 (2H, H2,6, hhPhe4), 7.21 (2H,
H3,5, Phe3), 7.25 (2H, Ha, Phe3), 7.27 (2H, H3,5, hhPhe4), 7.28 (1H, H4,
hhPhe4), 7.33 (1H, HN, Val9), 7.63 (1H, HN, Ile6), 7.65 (1H, HN, Ile7),
7.75 (1H, HN, hhPhe4), 7.92 (1H, HN, Leu5), 7.93 (1H, HN, Leu8), 8.52
(1H, HN, Phe3).
2.3.7. c(Pro1-Pro2-R-
Val9) (14)
g
3hhPhe3-R-
g
3hhPhe4-Leu5-Ile6-Ile7-Leu8-
(DMSO-d6, 50 ꢁC)
d
: 0. 75 (3H, Hd, Leu5), 0.76 (3H, Hg, Ile7), 0.80
2.4. Synthetic procedures
(3H, Hd, Ile6), 0.81 (3H, Hd, Ile7), 0.81 (3H, Hg, Ile6), 0.82 (3H, Hd,
Leu8), 0.83 (3H, Hd, Leu5), 0.86 (3H, Hg, Val9), 0.89 (3H, Hd, Leu5),
0.93 (3H, Hg, Val9), 1.02 (1H, Hg, Ile7), 1.04 (1H, Hg, Ile6), 1.37 (1H, Hb,
Leu8), 1.40 (1H, Hg, Ile7), 1.42 (1H, Hg, Ile6), 1.46 (2H, Hb, Leu5) 1.50
(1H, Hb, Leu8), 1.57 (1H, Hg, Leu8), 1.59 (1H, Hg, Leu5), 1.65 (1H, Ha
Pro2), 1.68 (1H, Hb, Pro1), 1.69 (1H, Hb, Ile7), 1.79 (1H, Hg, Pro1), 1.80
(1H, Hb, Ile6), 1.81 (1H, Hg, Pro1), 1.85 (1H, Hg, Pro2), 1.94 (1H, Hb,
Val9), 1.98 (1H, Hb, Pro2), 2.01 (1H, Ha, hhPhe3), 2.01 (1H, Ha,
hhPhe4), 2.22 (1H, Ha, hhPhe3), 2.22 (1H, Hb, Pro2), 2.23 (1H, Hb,
Pro1), 2.24 (1H, Ha, hhPhe4), 2.28 (1H, Hb, hhPhe3), 2.33 (1H, Hb,
hhPhe4), 2.42 (1H, Hg”, hhPhe4), 2.59 (2H, Hg, hhPhe3), 2.67 (1H,
Hg”, hhPhe4), 2.98 (1H, Hg, hhPhe4), 3.02 (1H, Hg, hhPhe3), 3.16 (1H,
Hg, hhPhe4), 3.20 (1H, Hg, hhPhe3), 3.31 (1H, Hd, Pro2), 3.41 (1H,Ha
Pro1), 3.49 (1H, Hd, Pro2), 3.61 (1H, Hd, Pro1), 4.13 (1H, Ha, Ile6), 4.17
(1H, Ha, Ile7), 4.20 (1H, Ha,Leu5), 4.27 (1H,Ha, Pro1), 4.42 (1H, Ha,
Pro2), 4.44 (1H, Ha, Val9), 4.44 (1H, Ha, Leu8), 7.14 (2H, H2,6, hhPhe3),
7.17 (1H, H4, hhPhe3), 7.17 (1H, H4, hhPhe4), 7.18 (2H, H2,6, hhPhe4),
7.24 (2H, H3,5, hhPhe3), 7.26 (2H, H3,5, hhPhe4), 7.40 (1H, HN, Ile6),
7.54 (1H, HN, Ile7), 7.71 (1H, HN, Val9), 7.76 (1H, HN, Leu5), 7.82 (1H,
HN, hhPhe4), 8.08 (1H, HN, Leu8), 8.13 (1H, HN, hhPhe3).
(3S)- and (3R)-4-((tert-Butoxycarbonyl)amino-)-3-benzyl-buta-
noic acid 17 was obtained from racemic ( )-3-aminomethyl-4-
phenylbutanoic acid hydrochloride, which was synthesized ac-
cording to the earlier published procedure [34] with some modi-
fications.
Ethyl
( )-3-nitromethyl-4-phenylbutanoate
was
hydrolysed and then hydrogenated using 10% Pd/C to get acid,
which was transformed into Boc-derivative. The products were
purified by crystallization from ethyl acetate/hexane to yield crys-
talline solids. The enantiomeric purity was determined according to
the known procedure using Na-(2,4-dinitro-5-fluorophenyl)-L-
valinamide as a derivatizing reagent [35]. Diastereomeric de-
rivatives were detected at different retention times (min): 12.67
and 13.62 for 17-3R and 17-3S, respectively.
2.4.1. Synthesis and enantiomeric resolution of 17
2.4.1.1. (3RS)-4-((tert-Butoxycarbonyl)amino-)-3-benzyl-butanoic
acid, 17. ( )-3-Aminomethyl-4-phenylbutanoic acid hydrochloride
(6.66 g, 29 mmol) and Boc2O (7 g, 32.1 mmol) in 1,4-dioxane
(30 mL) and 2 N NaOH (30 mL) were stirred at RT for 24 h. After

K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
519
the solvents removal the residue was dissolved in water (40 mL),
pH was adjusted to 9e10 with 1 N NaHSO4 and solution was
washed with EtOAc (3 ꢀ 50 mL). The solution was then washed
with dichloromethane (3 ꢀ 50 mL) at pH 3e4 and (2 ꢀ 50 mL) at pH
1. The organic phases were combined, washed with brine
(2 ꢀ 50 mL) and dried over MgSO4. After evaporation of the solvent
the racemic acid 17 was obtained in 63.6% yield (5.41 g, 95.3%
purity).
Enantiomeric resolution of 17 was achieved by crystallization in
the presence of (R)-(þ)-methylbenzylamine or (S)-(ꢂ)-methyl-
benzylamine starting from 5409 g of 4-((tert-butoxycarbonyl)
amino-)-3-phenyl-pentanoic acid 17-3R,S. For crystallization of 1 g
of racemic acid 17 ethyl acetate (110 mL) was applied. (S)-
(ꢂ)-Methylbenzylamine was used to obtain (3S)-(þ)-4-((tert-
butoxycarbonyl)amino-)-3-phenyl-pentanoic acid 17-3R, then the
solid was recrystallized three times to obtain final product with
ee ¼ 97.4%. (R)-(þ)-Methylbenzylamine was applied to the mother
liquor after the first crystallization of 17-3S and (3R)-(ꢂ)-4-((tert-
butoxycarbonyl)amino-)-3-phenyl-pentanoic acid 17-3R and
ee ¼ 98.1% was obtained. Each time the solutions were left for 24 h
at þ5 ꢁC for crystallization. After each crystallization, 3e5 mg were
used for enantiomeric excess examination using Marfey's reagent.
Acids 17-3R and 17-3S were generated from the ethyl acetate
solution by treatment with 1 M NaHSO4.
3.04e3.09 (m, 1H, NHeCHH), (minor 5.00 (bs, 1H, NH), major 5.36
(bs,1H, NH)) 7.19e7.22 (m, 3H, Ar), 7.28e7.31 (m, 2H, Ar) 9.52 (s,1H,
OH); 13C NMR (175 MHz, CD3CN)
d
29.0, 36.9, 39.1, 39.3, 44.5, 79.8,
127.5, 129.7, 130.6, 141.5, 157.8, 174.9. 1H NMR (700 MHz, DMSO)
major rotamer (90%) 1.38 (s, 9H, (CH3)3C), 1.99e2.04 (m, 1H,
d
HOOCeCH2), 2.11e2.20 (m, 2H, HOOCeCH2, CH), 2.47e2.51 (m, 1H,
Ph-CHH), 2.59 (dd, J ¼ 6.7, 14.0 Hz, 1H, Ph-CHH), 2.82e2.87 (m, 1H,
NHeCHH), 2.91e2.97 (m, 1H, NHeCHH), 6.87 (t, J ¼ 5.4 Hz, 1H, NH),
7.16e7.20 (m, 3H, Ph2,6;4), 7.26e7.29 (m, 2H, Ph3,5), 12.06 (bs, 1H,
OH); 1H NMR (700 MHz, DMSO-d6) minor rotamer (10%)
d 1.33 (s,
9H, (CH3)3C), 1.99e2.04 (m, 1H,HOOCeCH2), 2.11e2.20 (m, 2H,
HOOCeCH2, CH), 2.47e2.51 (m, 1H, Ph-CHH), 2.53e2.58 (m, 1H, Ph-
CHH), 2.75e2.83 (m, 1H, NHeCHH), 2.91e2.97 (m, 1H, NHeCHH),
6.55 (t, J ¼ 5.4 Hz, 1H, NH), 7.16e7.20 (m, 3H, Ph2,6;4), 7.26e7.29 (m,
2H, Ph3,5), 12.06 (bs, 1H, OH); 13C NMR (175 MHz, DMSO)
d 28.2,
35.7, 37.0, 37.2, 42.8, 77.5, 125.9, 128.2, 129.1, 139.8, 155.8, 173.7; CI
MS m/z (%) 294.3 [Mþ 1]þ, 294.2 (100), 238.1 (46), 194.1 (26), 176.1
(3). For C16H23NO4 calculated 293.162.
2.4.1.4. (3S)-(þ)-4-((tert-Butoxycarbonyl)amino-)-3-benzyl-buta-
25
noic acid, 17-3S. Mp 84e86 ꢁC; [
a
]
D
¼ 14.0ꢁ; HPLC (40e80% B,
15 min), tR ¼ 10.64 min (97.4%), ee ¼ 97.36%. All spectroscopic data
(NMR, IR, MS) were identical as recorded for 17-3R.
2.5. Biological methods
2.4.1.2. (3RS)-4-((tert-Butoxycarbonyl)amino-)-3-benzyl-butanoic
acid, 17. Mp 64e66 ꢁC; HPLC (50e90% B, 15 min), tR ¼ 7.65 min
2.5.1. Reagents
(95.3%); IR
n
3332, 2977, 2930, 1701, 1656, 1514, 1453, 1406, 1366,
DMSO, RPMI-1640, PHA, MTT, LPS, OVA, Freund's complete
adjuvant, dexamethasone, carrageenan and all other reagents were
supplied from SigmaeAldrich (USA). LSM 1077 was obtained from
1249, 1161, 909, 731, 699, 504, 461 cmꢂ1; 1H NMR (700 MHz, CD3Cl)
(78%) 1.44 (s, 9H, (CH3)3C), 2.29e2.39 (m, 3H, CH, HOOCeCH2),
d
2.65 (d, 2H, J ¼ 6.3 Hz, Ph-CH2), 3.06e3.14 (m, 1H, NHeCHH),
3.20e3.27 (m, 1H, NHeCHH), 4.71 (bs, 1H, NH), 7.17 (d, 2H,
J ¼ 7.5 Hz, Ph), 7.21 (t, 1H, J ¼ 7.5 Hz, Ph), 7.29 (t, 2H, J ¼ 7.5 Hz, Ph);
CytoGen, Germany. Human TNF-
vided by eBioscience, USA.
a ELISA Ready-SET-Go was pro-
1H NMR (700 MHz, CD3Cl) minor rotamer (22%)
d
1.44 (s, 9H,
2.5.2. Preparation of the compounds for biological assays
(CH3)3C), 2.21 (bs, 1H, CH, HOOCeCHH), 2.30e2.40 (m, 1H, CH,
HOOCeCHH), 2.45 (bs, 1H, CH), 2.57 (bs, 1H, Ph-CHH), 2.59e2.68
(m, 1H, Ph-CHH), 2.95 (bs, 1H, NHeCHH), 3.21e3.27 (m, 1H,
NHeCHH), 5.89 (bs, 1H, NH), 7.17 (d, 2H, J ¼ 7.5 Hz, Ph), 7.21 (t, 1H,
J ¼ 7.5 Hz, Ph), 7.29 (t, 2H, J ¼ 7.5 Hz, Ph); 13C NMR (175 MHz, CD3Cl)
28.5, 36.4, 38.2, 38.5, 43.6, 79.9, 126.6, 128.7, 129.3, 139.4, 156.7,
177.6.
The compounds were dissolved in DMSO (5 mg/200 ml) and
subsequently diluted to 5 mL of RPMI-1640 medium for in vitro
studies or in 0.9% NaCl for injections. As a control appropriate di-
lutions of DMSO in RMPI-1640 medium or in 0.9% NaCl were used.
2.5.3. Mice
CBA female and male mice, 8e12 weeks old were supplied by
the Animal Breeding Center in Ilkowice, Poland. The mice were kept
in 12 h light and dark cycles with free access to water and granu-
lated food. The local ethics committee approved the study.
2.4.1.3. (3R)-(ꢂ)-4-((tert-Butoxycarbonyl)amino-)-3-benzyl-buta-
25
noic acid, 17-3R. Mp 84e86 ꢁC; [
a]
¼ ꢂ14.3ꢁ; HPLC (40e70% B,
D
15 min), tR ¼ 10.67 min (100%), ee ¼ 98.10%; IR
n 3372, 2977, 2966,
2931, 1705, 1656, 1455, 1441, 1398, 1365, 1200, 1169, 1138, 1106,
2.5.4. Isolation of the peripheral blood mononuclear cells (PBMC)
Venous blood from a single donor was withdrawn into hepa-
rinized syringes and diluted twice with PBS. PBMC were isolated by
centrifugation on ficoll-uropoline gradient (density 1.077 g/mL)
and centrifuged at 800 ꢀ g for 20 min at 4 ꢁC. The interphase cells,
consisting of lymphocytes (20%) and monocytes (80%), were then
washed three times with Hanks’ medium and re-suspended in a
culture medium, referred to below as the culture medium, con-
1083.47, 991, 863, 774, 738, 698, 658, 596, 496, 437 cmꢂ1; 1H NMR
(700 MHz, CDCl3 300 K) major rotamer (76%) d 1.43 (s, 9H, (CH3)3C),
2.29e2.39 (m, 3H, CH, HOOCeCH2), 2.65 (d, 2H, J ¼ 4.2 Hz, Ph-CH2),
3.06e3.14 (m, 1H, NHeCHH), 3.20e3.27 (m, 1H, NHeCHH), 4.71 (bs,
1H, NH), 7.17 (d, 2H, J ¼ 7.5 Hz, Ph), 7.21 (t, 1H, J ¼ 7.5 Hz, Ph), 7.29 (t,
2H, J ¼ 7.5 Hz, Ph), 10.81 (bs, 1H, OH); 1H NMR (700 MHz, CDCl3
300 K) minor rotamer (24%) d 1.44 (s, 9H, (CH3)3C), 2.20 (bs, 1H, CH,
HOOCeCHH), 2.30e2.40 (m, 1H, CH, HOOCeCHH), 2.48 (bs, 1H, CH),
2.57 (bs, 1H, Ph-CHH), 2.59e2.68 (m, 1H, Ph-CHH), 2.91 (bs, 1H,
NHeCHH), 3.21e3.27 (m, 1H, NHeCHH), 6.07 (bs, 1H, NH), 7.17 (d,
2H, J ¼ 7.5 Hz, Ph), 7.21 (t, 1H, J ¼ 7.5 Hz, Ph), 7.29 (t, 2H, J ¼ 7.5 Hz,
Ph) 10.81 (bs, 1H, OH); 13C NMR (175 MHz, CDCl3) major rotamer
sisting of RPMI-1640, supplemented with 10% foetal calf serum, L-
glutamine, sodium pyruvate, 2-mercaptoethanol and antibiotics, at
density of 2 ꢀ 106 cells/mL.
2.5.5. Phytohemagglutinin A (PHA)-induced proliferation of human
blood mononuclear cells
d
28.5, 36.3, 38.1, 38.5, 43.7, 79.8, 126.5, 128.7, 129.3, 139.3, 156.6,
178.0; 13C NMR (175 MHz, CDCl3) minor rotamer
28.4, 37.0, 37.7,
38.7, 45.3, 81.0, 126.5, 128.7, 129.3, 139.3, 157.9, 178.0; 1H NMR
(700 MHz, CD3CN)) major/minor rotamer (88/(12) 1.40 (s, 9H,
d
PBMC were distributed into 96-well flat-bottom plates in 100 ml
aliquots (2 ꢀ 105 cells/well). PHA was added at a concentration of
d
5 mg/mL. The compounds were tested at doses of 1, 10 and 100 mg/
C(CH3)3), 2.16 (dd, 1H, J ¼ 5.00, 14.4 Hz, HOOCeCHH), 2.19e2.26 (m,
2H, HOOCeCHH, CH), 2.56 (dd, 1H, J ¼ 6.9, 13.7 Hz, Phe-CHH), 2.61
(dd, 1H, J ¼ 6.6, 13.7 Hz, Phe-CHH), 2.94e3.00 (m, 1H, NHeCHH),
mL. DMSO at appropriate dilutions served as control. After a four-
day incubation in cell culture incubator, the proliferative
response of the cells was determined by the colorimetric MTT
a

520
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
Table 2
method [36]. The data are presented as a mean OD value from
quadruplicate wells standard error (SE).
3
Vicinal coupling constants JNHC [Hz] of peptides 9e16 in DMSO-d6 at 323 K.
aH
Peptide Phe3 or
g
3-hhPhe3 Phe4 or
g
3-hhPhe4 Leu5 Ile6
Ile7 Leu8 Val9
2.5.6. Cytotoxicity of the compounds against human blood
mononuclear cells
9
5.8
5.9
7.4
7.5a
5.4
7.4
5.4
7.4
7.6
5.8
6.7
8.1
8.0
8.9 8.4 8.6 9.2
7.3 8.5 6.2 8.8
10
11a
11b
12
13
14
15
16
PBMC at density of 3 ꢀ 105/100
mL/well, re-suspended in the
8.5a
10.2a
6.0
8.1a 10.9a 9.9a 8.8a 9.3a
8.0a
8.3
8.1
8.3
8.2
8.0
8.7a 7.5a 7.0a 8.7a
8.9 8.6 8.1 9.2
culture medium, were cultured for 24 h in a cell culture incubator
with the preparations at 1, 10 and 100 mg/mL concentrations. Cell
survival was determined by MTT colorimetric method. The results
are given in percentage of viable cells as compared with appro-
priate DMSO controls.
7.8
5.5
5.6
e
8.1
e
8.8 9.2
9.1 8.5 8.8 9.2
8.2 8.2 8.2 8.9
8.5 7.8 7.8 8.3
a
Vicinal coupling constants 3JNHC
aH [Hz] of peptides determined from COSY-
DQF spectra.
2.5.7. Lipopolysaccharide (LPS)-induced tumour necrosis factor
alpha (TNF-a) production in whole blood cell culture
The test was performed as described elsewhere [37]. In brief,
venous blood from a single donor was diluted 10 ꢀ with RPMI-1640
medium and distributed in 1 mL aliquots in 24-well culture plates
3. Results and discussion
3.1. Chemistry
(Nunc). The cultures were stimulated by addition of 1
from Escherichia coli, serotype O:111:B4. The compounds were
added to the cultures at concentrations of 5 and 25 g/mL. Higher
concentrations of the compounds could not be used because of
inhibitory effects on TNF- production by corresponding DMSO
mg/mL of LPS
All analogues were modified with g
3-hhPhe residue in position
m
3 or 4 and both 3 and 4. All analogues were synthetized according
to manual SPPS procedure based on Boc procedure. The scale of
synthesis was 0.2 mmol for each analogue. The deprotection took
place with 50% solution TFA in DCM through 10 min. TBTU was used
as a coupling reagent, HOBt as an antiracemic additive and DIPEA as
an amin. Resin was washed with DCM, methanol and DCM, then the
synthetic procedure (coupling, deprotection, acylation) was
continued. Standard Boc-protected amino acids were used in 3 eq
a
(the solvent) dilutions. Appropriate dilutions of DMSO served as
controls. After overnight incubation in a cell culture incubator, the
supernatants were harvested and frozen at ꢂ20 ꢁC until cytokine
determination by immunoassay.
2.5.8. Delayed type hypersensitivity to ovalbumin (OVA)
and Boc-g
3-hhPhe-OH was used in 1.5 eq in coupling to the resin.
The test was performed as described previously [38]. Mice were
The cleavage took place in 30 min using TFMSA, TFA and anisol. The
solution was evaporated under vacuum and the resulting peptide
was achieved by precipitation with diethyl ether. The cyclisation
was achieved with syringe pump and commonly occurred through
24 h with 12e46% yield. For biological assays all peptides were
purified by HPLC.
sensitized subcutaneously into tail base with 5
Freund's adjuvant. After 4 days the mice received 50
incomplete Freund's adjuvant in a volume of 50 l in the hind foot
pads. After 24 h the foot pad edema was measured using a spring
caliper. Control (background group) mice were not sensitized but
received the eliciting dose of antigen. The compounds were given
m
g OVA in complete
m
g OVA in
m
to mice intraperitoneally, in 100 mg doses, 2 h before sensitization
or 2 h before elicitation of the DTH reaction with the second dose of
antigen. Control mice received appropriate doses of the solvent
(DMSO). The results were presented as mean values of antigen-
specific increase of foot pad swelling measured in 5 mice per
group (10 determinations from 10 feet) and expressed in DTH units
standard error (SE). One DTH unit ¼ 10ꢂ2 cm.
3.2. NMR measurements
1H NMR spectra of cyclic peptides 9e16 in DMSO-d6 at 300 K are
well resolved, much better than those recorded for the unmodified
CLA [40]. The NH resonances are spread within the narrow range of
0.5e0.8 ppm, except range of 1.2 ppm for peptides 11a and 16. Most
of the NH signals are well resolved, enabling determination of NH-
2.5.9. Carrageenan-induced paw inflammation
We followed the procedure as recently published [39]. Mice
were administered 0.1 mL of 2% carrageenan in 0.9% NaCl in hind
CHa coupling constants.
For CLA analogues 9e16 the geometries of peptide bonds were
established from ROESY spectra. The ROE between Ca eH and Cd-H
proline atoms is present for trans ProePro peptide bond and be-
tween Ca eH and Ca-H for cis ProePro bonds. In addition the dif-
ference in chemical shifts between Cb and Cg in prolines was
examined. The differences of about 9 ppm and about 5 ppm are
foot pads. The studied compounds (100 mg doses) were injected
intraperitoneally at 24 h or 24 h and 30 min before administration
of carrageenan. The foot pad edema was measured using a spring
caliper after: 2, 3 and 4 h after carrageenan administration. Dexa-
methasone (50 mg/ml), administered 30 min before carrageenan,
was served as a reference drug. The results were presented as a
mean increase in foot pad swelling from 5 mice per group (10 de-
terminations from 10 feet) standard error.
Table 3
Temperature dependence of the NH chemical shifts (ꢂDd
/DT, ppb/K) of peptides
9e16 in DMSO-d6, in the range of 300e340 K.
Peptide Phe3 or
g
3-hhPhe3 Phe4 or 3-hhPhe4 Leu5 Ile6 Ile7 Leu8 Val9
g
2.5.10. Statistics
The results are presented as mean values standard error (SE).
Brown-Forsyth’s test was used to determine the homogeneity of
variance between groups. When the variance was homogenous,
analysis of variance (One-way ANOVA) was applied, followed by
post hoc comparisons with the Tukey's test to estimate the signif-
icance of the difference between groups. Nonparametric data were
evaluated with the KruskaleWallis’ analysis of variance, as indi-
cated in the text. Significance was determined at p < 0.05. Statistical
analysis was performed using STATISTICA 6.1 for Windows.
9
0.1
1.3
3.8
3.7
0.8
2.6
3.4
5.5
5.1
0.7
3.5
5.5
5.0
5.1
5.4
5.3
3.5
2.6
3.5
2.5
5.2
3.8
3.8
2.1
0.1 5.6 5.7
4.2 2.1 2.9
4.2 3.5 3.9
1.8 3.6 3.6
4.9
3.3
0.1
5.3
3.6
4.3
1.8
4.3
10
11aa
12
13
14
15
16
8.9
?
5.6
1.2 5.3 4.3
3.8 4.9 3.8
2.5 3.4 4.4
a
The temperature dependence of the NH chemical shifts for cyclopeptide 11b
was unable to determine and is omitted.

K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
521
Fig. 2. The toxicity of linear peptides 1e8 (A) and cyclic peptides 9e16 (B) with regard to human peripheral blood mononuclear cells. The peptides were used at concentrations of
1e100 g/mL. The cell toxicity was compared with toxicity of DMSO used as solvent.
m
characteristic for trans and cis Xxx-Pro geometries, respectively
[41]. All cyclic peptides, except major isomer of peptide 11, exist as
isomers with the cis ProePro peptide bond, similarly as in CLA [40].
mixture of two isomers in the ratio 89:11. The major isomer con-
tains all peptide bonds of the trans geometry.
Chemical shifts of the
cyclopeptides except 11 and 16 containing fragment of Phe3-R-g3
hhPhe4. For these peptides one of
protons experiences a high-
g
protons of Pro2 are very similar for all
The CLA analogue 11 with single modification S-
g
3-hhPhe4 is a
-
g

522
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
Fig. 3. Effects of linear peptides 1e8 (A) and cyclic peptides 9e16 (B) on PHA-induced PBMC proliferation. The peptides were used at concentrations of 1e100
mg/mL and
cyclosporine A was applied as reference drug.
field shift up to 1.02 ppm (11a), 1.16 (11b) and 0.86 ppm (16). The
observed up-field shift is not as large as observed for CLA (Pro2
modified with g-hhPhe do not manifest the face-to-edge interac-
tion of phenyl rings as in native CLA [40,42,43] or its analogues
g
proton observed at 0.33 ppm), but evidences much closer proximity
of the Phe3 aromatic rings and Pro2 side chains in peptides 11 and
16, than in other cyclopeptides.
For all studied cyclopeptides' signals of both Phe aromatic rings
are placed in narrow and similar range. This suggests that peptides
modified with tyrosine [44].
Vicinal coupling constants 3JNHC H were measured directly from
a
1H NMR spectra or from COSY-DQF spectrum (peptide 11) and are
summarized in Table 2. Most of 3JNHC H vicinal couplings constants
a
3
are about 8 Hz. For Phe or hhPhe residues JNHC are about 6 Hz,
a
H

K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
523
Fig. 4. Effects of linear peptides 1e8 (A) and cyclic peptides 9e16 (B) on LPS-induced TNF-
and 25 g/mL.
a
production in human whole blood cultures. The peptides were used at concentrations 5
m
except peptides 11, 13 and 16. Vicinal couplings indicates the sim-
The solvent exposure of NH protons was probed by measuring
ilarity of the NH-C
a
H moieties with 4 angles in the range ꢂ80ꢁ/
the temperature effect coefficients (Dd
temperature coefficients (Dd T < 3 ppb/K) indicate the presence of
intramolecular hydrogen bonds [30]. For CLA the NH's of Phe4, Ile7,
/DT) (Table 3). The low
ꢂ160ꢁ [45].
/D

524
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
Fig. 5. Inhibitory effects of peptides 6 and 11 on delayed type hypersensitivity to ovalbumin. A: the compound administered before sensitization, B: the compound administered
before elicitation of DTH.
Leu8, and Val9 were found engaged in intramolecular hydrogen
two isomers and the major isomer (89%) contains all peptide bonds
bonds in chloroform in the range of 204e264 K. The analogues of
of the trans geometry.
CLA modified with
different numbers of hydrogen bonds: five hydrogen bonds (10 e S-
3-hhPhe3, Phe4, Leu5, Ile7, Leu8), two hydrogen bonds (9 e S-g3
hhPhe3, Ile6,12 e Leu5, Ile6,14 e R- 3-hhPhe3, Ile6,16 e Leu5, Ile6) or
one hydrogen bond (11a e Val9, 13 e S- 3-hhPhe3, 15 e Val9).
In comparison to native CLA the replacement of one or both Phe
g
3-hhPhe are characterized in DMSO by
g
-
3.3. Biology
g
g
3.3.1. Effects of the compounds on viability of PBMC
The cytotoxic effects of the compounds, at the concentration
range of 1e100 mg/mL, were evaluated in the cultures of PBMC. For
linear peptides 1e8 we have not found any effects on the viability of
cells in the studied concentration range (Fig. 2A). All cyclic ana-
logues 9e16, except for 11, appeared to be toxic in a dose-
dependent manner. Typically, a significant toxicity occurred at
residues by g
3-hhPhe increased the number of stabilizing hydrogen
bonds for all analyzed analogues, except peptide 10. 1H NMR
spectra are better resolved, probably due to lower exchange be-
tween conformers in the NMR scale. All cyclic peptides, except 11,
exist as isomers with the cis ProePro peptide bond. The CLA
analogue 11 with single modification S-g
3-hhPhe3 is a mixture of
10
mg/mL (Fig. 2B. Interestingly, peptide 11 was virtually devoid of
toxicity.

K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
525
Fig. 6. The anti-inflammatory actions of peptide 6 (A) and 11 (B) on carrageenan-induced foot pad inflammation in mice. Dexamethasone (DEX) was applied as reference drug.
Peptides 1e8 at the concentration range of 1e100
mg/mL did not
two remaining linear compounds e 6 and 7 were strongly inhibi-
affect proliferation of PBMC to PHA (Fig. 3A). Cyclosporine A, used
as a reference drug, showed a typical, strong anti-proliferative ac-
tivity already at 1 mg/mL. On the other hand, the cyclic peptides
exhibited strong anti-proliferative activities in the model of PHA-
induced PMBC proliferation (Fig. 3B). Although that strong inhibi-
tion of cell proliferation could be attributed to high cell toxicity of
majority of the peptides, the anti-proliferative action of peptide 11
(the non-toxic peptide) was probably associated with a different
mechanism.
tory at both concentrations.
The actions of the cyclic CLA analogues on LPS-induced TNF-
production (Fig. 4B) were differentiated but predominantly inhib-
itory, depending on a dose. Providing that at 25 g/mL concentra-
tion and in part at 5 g/mL, the compounds, except for peptide 11,
were cytotoxic, in these cases the suppressive effects on TNF-
production could be due to toxicity. Interestingly, the non-toxic 11
displayed about 55% inhibition of TNF- production at 5 g/mL and
42% inhibition at 25 g/mL.
a
m
m
a
a
m
m
The effects of the linear peptides on LPS-induced TNF-
duction are shown in Fig. 4A. The peptides were used at 5 and
25 g/mL. Among them the peptides 1e4 showed some stimulatory
activities and peptide 8 was strongly inhibitory at 25 g/mL. The
a
pro-
For the in vivo experiments peptides 6 and 11 were chosen
considering their strong antiproliferative and anti-inflammatory
actions in vitro. Peptide 6 and 11 were administered at doses of
m
m
100
mg to mice 2 h before sensitization of animals with OVA
(Fig. 5A) or 2 h before elicitation of the antigen-specific cellular

526
K. Je˛drzejczak et al. / European Journal of Medicinal Chemistry 86 (2014) 515e527
response with a second dose of antigen (Fig. 5B). The results
revealed a substantial (about 50%) inhibition of the DTH reaction
upon administration of compound 6 before sensitization and even
deeper inhibition of the immune response when 6 was given before
elicitation of the reaction. CLA analogue 11 exerted even stronger
suppressive actions on DTH reaction to OVA, both on the sensiti-
zation (Fig. 5A) and on elicitation phase of the response (Fig. 5B). In
the latter case it virtually blocked the response.
hypersensitivity as well as in the carrageenan-induced foot pad
edema. In conclusion, linear peptide 6 and cyclic peptide 11 are
attractive as potential drugs, although the cyclic structure of com-
pound 11 predisposes the peptide as a better candidate as an anti-
inflammatory and immunosuppressive therapeutic.
Acknowledgements
Next, peptides
carrageenan-induced foot pad inflammation in mice. The com-
pounds were administered intraperitoneally at 100 g dose, 24 h
and 2 h before carrageenan or only 2 h before carrageenan injec-
tion. Dexamethasone, a control drug (50 g), was given i.p. 2 h
6 and 11 were tested in the model of
This work was supported by Technical University of Lodz Grant
DS I18/10/2014 and IIET Grant 501-4/2014.
m
Appendix A. Supplementary data
m
before application of carrageenan. Fig. 6 shows the results of
measurements of foot pad edema after 3 h following carrageenan
administration. It appeared that both compounds were strongly
inhibitory in this model, particularly when given at two doses.
In summary, among the studied compounds two of them (6 and
11) showed distinct anti-inflammatory actions. The ability of 6 to
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.014.
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The replacement of one or two Phe residues in the CLA molecule
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