Design, synthesis and biological evaluation of novel desmuramyldipeptide analogs

Article abstract of DOI:10.1016/j.ejmech.2011.05.042

A series of novel desmuramyldipeptides have been designed and synthesized as part of our search for therapeutically useful muramyldipeptide (MDP) analogs. Their immunomodulatory properties were initially assessed in vitro, evaluating their effect on lipop

Full text of DOI:10.1016/j.ejmech.2011.05.042

European Journal of Medicinal Chemistry 46 (2011) 3762e3777
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel desmuramyldipeptide
analogs
a
b
a
a
a,
*
ꢀ
ꢀ
ꢀꢀ
Ziga Jakopin , Emanuela Corsini , Martina Gobec , Irena Mlinaric-Rascan , Marija Sollner Dolenc
a
ꢀ
ꢀ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000 Ljubljana, Slovenia
b Laboratory of Toxicology, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel desmuramyldipeptides have been designed and synthesized as part of our search for
therapeutically useful muramyldipeptide (MDP) analogs. Their immunomodulatory properties were
initially assessed in vitro, evaluating their effect on lipopolysaccharide (LPS)-induced cytokine release in
THP-1 cells. Following the initial screening, selected compounds were further investigated for immu-
nomodulatory properties using LPS and phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated
human peripheral blood mononuclear cells. The results confirmed the immunomodulatory properties of
some of the synthesized desmuramyldipeptide analogs. Taken together, presented data confirmed the
immunostimulatory effect of compound 44, MDP derivative incorporating a pyrido-fused [1,2]-benzi-
sothiazole moiety, while for compounds 32 and 39, indole scaffold-based derivatives of MDP, an
immunosuppressive effect was observed. Further studies will be necessary to address their potential
therapeutic use as immunomodulatory drugs, both as immunostimulants or anti-inflammatory agents.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 7 December 2010
Received in revised form
14 May 2011
Accepted 17 May 2011
Available online 27 May 2011
Keywords:
MDP
Immunomodulator
Desmuramyldipeptide
Immunotoxicity
Immunostimulant
Anti-inflammatory agent
1. Introduction
confer non-specific resistance against bacterial, viral or parasitic
infections and protection against tumors by engagement of host
Protective immunity against pathogen exposure is achieved in
vertebrates by the integration of two distinct arms of the immune
response, the innate and antigen-specific (adaptive) responses. The
innate immune response constitutes the first line of defense against
microorganisms. It is activated to resist infection by molecular
recognition of common bacterial components such as peptido-
glycan, LPS, lipoproteins and bacterial DNA. This resistance is non-
specific, and extends to unrelated species of bacteria, fungi, and
viruses. The adaptive immune response takes time to become
effective and comprises cellular or humoral elements that are most
appropriate for protection against the particular infectious agent.
A component of Gram-negative and Gram-positive bacterial cell
defense mechanisms by stimulating immunocompetent cells, such
as monocytes and macrophages [2]. Macrophage activation is
essential for resistance to infections caused by many species of
pathogens, its enhanced production of oxygen radicals being the
pivotal feature required for killing the pathogens and also for its
tumoricidal activity. Activated macrophages also increase their
production of proinflammatory cytokines, such as interleukin-1
b
(IL-1 and tumor necrosis factor- (TNF- ), thus activating
b
)
a
a
neutrophils, B lymphocytes and T lymphocytes resulting in path-
ogen clearance [2]. The immunomodulatory activity of MDP is thus
based mainly on stimulation of monocytes/macrophages.
MDP is a far weaker stimulator (on a molar basis) of immuno-
competent cells than most toll-like receptor (TLR) ligands, and
induces the release of modest amounts of proinflammatory cyto-
kines [3e5]. However, it was found to synergize with LPS and
a plethora of other stimuli ranging from TLR ligands to PMA,
resulting in increased proinflammatory cytokine releasing capacity
[6]. In pathological conditions such as bacterial infection, MDP and
LPS are likely to be found simultaneously in the same tissues, which
further reinforces the notion that such synergy could be of great
physiological importance [7]. Thus MDP is an immune-amplifier
and modulator of immune reaction, rather than an immunosti-
mulant per se [8].
wall, muramyldipeptide MurNAc-L-Ala-D-isoGln (MDP) was found
to be the minimal structural unit of peptidoglycan possessing
immunoadjuvant and immunoenhancing activity [1]. MDP can
Abbreviations: DAG, diacylglycerol; GMDP, glucosaminyl muramyl dipeptide;
H4R, H4 receptor; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysac-
charide; LTA, lipoteichoic acid; MB, murabutide; MDP, muramyl dipeptide; MTP-PE,
muramyl tripeptide phosphatidylethanolamine; PBMC, peripheral blood mono-
nuclear cells; PMA, phorbol 12-myristate 13-acetate; TLR, toll-like receptor; TNF-
a,
tumor necrosis factor-
a
.
* Corresponding author. Tel.: þ386 1 4769572; fax: þ386 1 4258031.
E-mail address: marija.sollner@ffa.uni-lj.si (M.S. Dolenc).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.042

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3763
Fig. 1. Design of novel desmuramyldipeptide analogs.
With traditional immunomodulatory drugs exhibiting limited
efficacy and adverse side effects, the development of new, improved,
and specifically targeted immunotherapeutics is extremely impor-
tant. However, immunomodulation, as a therapeutic need for the
third millenium, remains in its infancy [9]. MDP would be a useful
immunomodulator if certain drawbacks, such as its poor cellular
penetration and rapid elimination could be overcome [10]. Further-
more, despite extensive research on MDP, the molecule is too pyro-
genic and arthritogenic to be used as an adjuvant in humans [11].
Numerous derivatives of MDP have therefore been synthe-
sized, with the intention of improving the pharmacological
properties and lowering the toxicological profile of the parent
molecule. Of the hydrophilic derivatives, murabutide (MB),
temurtide, nor-MDP and glucosaminyl-MDP (GMDP) eventually
reached the clinical stage of development [12]. Furthermore,
romurtide and muramyl tripeptide phosphatidylethanolamine
(MTP-PE) were the two major lipophilic derivatives that have
been developed by pharmaceutical industries [12].
forthcoming to decisively demonstrate Nod2-MDP binding to, and
subsequent activation of, immunocompetent cells [3].
Some desmuramylpeptides are able to enhance the host defense
against microbial infections as well as exhibiting strong antiviral
activity and remarkable antitumor potency [21e23]. LK-409 and
LK-410 stimulated the macrophages in a dose-dependent manner
to produce superoxide radicals and significantly accelerated the
differentiation of mouse B lymphocytes in plaque forming cells
[24]. A phthalimido desmuramyldipeptide compound, LK-423,
proved to be the most promising analog and has been selected
for further study to develop it as an anti-inflammatory agent
[25e27]. The absence of structural data for the binding site of MDP
makes the design of novel immunomodulators difficult, the struc-
ture/activity relationship of well known immunomodulatory
compounds being the only route for rational drug design. Looking
for another appropriate surrogate for the N-acetylmuramyl group
we focused our attention on the saccharin and indole heterocycles
and their derivatives as potential immunomodulatory compounds.
The receptor and signaling system for MDP are still a matter of
debate. Various receptors have been discussed as possible candi-
dates, such as 5-HT receptor [13], CD14 [14,15] and TLR 2 and 4
receptors [16]. Recently, an intracellular protein, Nod2, was
discovered as a putative receptor for MDP [17,18]. In peripheral
blood cells, the highest levels of Nod2 were observed in monocytes,
granulocytes, dendritic cells, Paneth cells and, to a lesser extent, in T
lymphocytes [19,20]. The binding site of MDP however, remains
elusive since no biochemical or structural data have been
1.1. Design
The aromatic part of the saccharin ring system [28] may be
considered as a good
residue of the parent molecule. Thus, acyclic substituted carboxylic
acid derivatives 1e3 of different lengths based on saccharin were
envisaged (shown in Fig. 1) in which we tried to retain the specific
position of the carbonyl group of the N-acetyl muramic part of MDP,
p-electronic substitute for the glucosamine

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
O
O
CH3
O
O
CH3
O
O
O
CH3
O
O
O
H
N
H
N
H
N
O
( )
i
O
O
N
H
O
Ph
Ph
H2N
CF3COOH
O
Ph
Ph
R
R
N
H
O
O
Ph
Ph
O
R
OH
O
8
NH2
O
NH2
O
NH2
1-7
10
12-18
(ii
)
CH3
O
O
CH3
O
CH3
H
N
H
N
H
N
(
i
)
N
H
O
H2N
CF3COOH
O
N
H
O
O
O
O
Ph
O
O
Ph
O
19-25
O
Ph
9
11
(iii)
O
O
CH3
O
O
H
N
R
R
N
H
OH
No.
No.
R
R
O
NH2
OH
O
S
O
O
O
26-32
S
N
N
29, 36
CH3
O
1, 12, 19,
26, 33
H
N
H
O
N
H
OH
O
O
O
O
33-39
O
S
N
S
N
O
30, 37
2, 13, 20,
27, 34
H
O
O
O
H
O
O
N
S
N
H
N
3, 14, 21,
28, 35
6, 17, 24,
31, 38
O
O
S
O
N
NH
4, 15, 22
7, 18, 25,
32, 39
H
N
O
O
O
S
N
5, 16, 23
Scheme 1. Reagents and conditions: (i) TFA/dichloromethane (5:1), 0 ꢀC to rt, 3 h; (ii) TBTU, Et3N, acetonitrile, 0 ꢀC to rt, 3 h; (iii) H2, Pd/C, acetic acid, rt, 2 h.
O
O
CH3
O
O
O
CH3
O
O
O
O
CH3
O
O
O
H
N
H
N
H
N
O
(i)
(i)
O
O
N
H
O
O
H2N
CF3COOH
O
O
R
R
N
H
O
O
O
R
OH
O
NH2
O
42
NH2
O
NH2
4, 5
40
44, 45
(ii)
CH3
CH3
CH3
O
H
N
H
N
H
N
N
H
H2N
CF3COOH
N
H
O
O
O
O
O
43
O
O
46, 47
O
41
(iii)
O
O
CH3
O
O
O
No.
R
H
N
R
R
N
H
OH
OH
O
S
O
O
48, 49
NH2
OH
N
4, 44, 46, 48, 50
CH3
O
H
N
O
N
H
O
S
N
O
5, 45, 47, 49, 51
50, 51
Scheme 2. Reagents and conditions: (i) TFA/dichloromethane (5:1), 0 ꢀC to rt, 3 h; (ii) TBTU, Et3N, acetonitrile, 0 ꢀC to rt, 3 h; (iii) 1M LiOH/EtOH, rt, 3 h.

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3765
O
CH3
O
CH3
O
No.
R
H
N
N
O
N
H
O
O
N
H
O
Ph
O
N
52
O
O
OH
O
S
N
O
NH2
1, 54, 59
(i)
64 (L, L)
65 (D, L)
66 (D, D)
O
CH3
O
S
N
N
2, 55, 60
O
(i)
HCl H2N
O
O
N
53
(ii)
O
O
CH3
O
O
H
N
R OH
1, 2, 3,
O
S
O
H2N
CF3COOH
O
Ph
O
N
3, 56, 61
4, 6
O
O
NH2
O
O
R
CH3
N
67 (L, L)
68 (D, L)
69 (D, D)
O
N
O
N
H
O
S
N
O
4, 57, 62
O
54-58
(ii)
(iii) or (iv)
O
CH3
O
O
O
H
H
O
N
N
S
O
R
CH3
N
H
N
N
H
O
Ph
6, 58, 63
N
N
H
O
O
O
NH2
O
N
O
70 (L, L)
71 (D, L)
OH
59-63
72 (D, D)
Scheme 3. Reagents and conditions: (i) SOCl2, EtOH, 0 ꢀC, 30 min, then 0 ꢀC to rt for
2 h; (ii) TBTU, Et3N, acetonitrile, 0 ꢀC to rt, 3 h; (iii) 3M HCl, 80 ꢀC, 30 min; (iv) for 57
and 58; 1M LiOH/EtOH, rt, 3 h.
(iii)
O
CH3
O
O
H
N
O
which had been proposed as essential for recognition and binding
S
[29]. nor-MDP analogs were synthesized in which the
D-lactyl
N
N
H
OH
residue is modified, with the aim of deleting the toxicity and
pyrogenicity of MDP. Different lengths of acyclic side chain were
used to explore the structure/activity relationship. A conforma-
tional restriction of the parent saccharin analogs was achieved by
incorporating an alkyl side chain into a pyrido- or pyrrolo-fused
[1,2]benzisothiazole ring systems 4 and 5 [30]. We applied the
approach of combining two pharmacophores in a single synthetic
compound by introducing indole-2-ylcarboxamido moiety as
a replacement for N-acetylmuramyl fragment (mimetics 6 and 7).
Indole-2-ylcarboxamido moiety is present in one of the first
reported selective H4 receptor (H4R) antagonists, JNJ 7777120, and
we postulated that connecting this moiety to the essential dipep-
tide part of MDP could produce new immunomodulators with
improved pharmacological properties. Recent research indicates
a crucial role for H4R, expressed mainly on hematopoietic cells in
allergy and inflammation [31,32].
O
O
NH2
O
73 (L, L)
74 (D, L)
75 (D, D)
Scheme 4. Reagents and conditions: (i) TFA/dichloromethane (5:1), 0 ꢀC to rt, 3 h; (ii)
TBTU, Et3N, acetonitrile, 0 ꢀC to rt, 3 h; (iii) H2, Pd/C, acetic acid, rt, 2 h.
THP-1. They were then assessed for their ability to modulate proin-
flammatory cytokine production, evoked by LPS or PMA/ionomycin in
human peripheral blood mononuclear cells (PBMC), relative to that of
murabutide, a well known immunomodulator.
2. Chemistry
The mimetics of N-acetyl muramic part of MDP thus obtained
were coupled with both parent dipeptide moieties,
L
-Ala-
D
-iGln and
The protected dipeptides Boc-L-alanyl-D-isoglutamine benzyl
L
-Ala- -Glu, since most of the known MDP analogs possess an intact
D
ester 8 [35] and Boc- -alanyl- -glutamic acid dibenzyl ester 9 [36]
L
D
dipeptide moiety, thus affording novel desmuramyldipeptide
analogs. Moreover, we also incorporated the 1,2,4-oxadiazole-
containing dipeptidomimetic [33,34] into a series of novel des-
muramyldipeptide analogs to gain a new perspective and further
understanding of the structure/activity relationship (shown in
Fig. 1). Taken together, 26 novel desmuramyldipeptide analogs
were synthesized and screened for their immunomodulatory
properties (their structures depicted in Schemes 1e4).
were prepared by known procedures. Removal of the Boc-
protecting group, using trifluoroacetic acid in dichloromethane,
gave the corresponding trifluoroacetate salts of the dipeptides 10
and 11, which were immediately used in the following reaction.
Synthesized acids 1e7 were coupled with benzyl L-alanyl-D-iso-
glutaminate trifluoroacetate 10 using TBTU as a coupling reagent to
give benzyl esters 12e18. Hydrogenolysis of 12e18 over palladium
on charcoal in glacial acetic acid afforded the target derivatives of
alanyl- -isoglutamine, 26e32.
The same strategy was applied to the synthesis of derivatives of
-alanyl- -glutamic acid. Coupling of dibenzyl -alanyl- -gluta-
L-
The THP-1 cell line, like primary human monocytes, expresses
mRNA coding for Nod2, CD14, TLR2 and TLR 4 receptors [7]. The
synthesized desmuramyldipeptides, MDPand murabutide (MB), were
D
L
D
L
D
screened for their ability to induce TNF-
a
release from naive THP-1
mate trifluoroacetate 11 with acids 1e7 afforded dibenzyl esters
19e25 which were subsequently deprotected by hydrogenolysis to
cells and to modulate the TNF- inducing activity of LPS-stimulated
a

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
250
200
150
100
50
160
***
**
140
187,4
119,3
*
**
**
115,3
182,6
114
120
100
80
60
40
20
0
100
91
**
126,2
**
120,2
**
11*8*,5
*
** 1*14*,3
11*3*,4
117
113,3
**
114
110,5
100
0
0
LPS
26 (L,D)
73 (L,L)
74 (D,L)
75 (D,D)
Contr LPS 26
28
32
35
36
39
48
50
63 MDP MB
Stimulus
Stimulus
Fig. 2. Effect of MDP, MB and the novel desmuramyldipeptide analogs (in form of free
acid) on LPS-induced TNF- release in THP-1 cells. Cells were incubated for 24 h with
the selected compounds (20.2 M) in the presence of LPS (1 ng/ml). Values are
expressed as % of LPS-induced TNF-
samples. Statistical analysis was performed with ManneWhitney U-Rank test; with
*p < 0.05; **p < 0.01 and ***p < 0.001 vs LPS treated cells.
Fig. 4. Influence of stereochemistry on TNF-
with the selected compounds (20.2 M) in the presence of LPS (1 ng/ml). Values are
expressed as % of LPS-induced TNF-
samples. Statistical analysis was performed with ManneWhitney U-Rank test; with
*p < 0.05; **p < 0.01 vs LPS treated cells.
a release. Cells were incubated for 24 h
a
m
m
a
release. Each value represents the mean ꢁ SD of 6
a
release. Each value represents the mean ꢁ SD of 6
give the desired derivatives of
(Scheme 1).
L-alanyl-D-glutamic acid, 33e39
[33], was treated with thionyl chloride in EtOH affording 1,2,4-
oxadiazole derivative 53.
To prepare pyrido[1,2-b] [1,2]benzisothiazole derivatives 48 and
50 and pyrrolo[1,2-b] [1,2]benzisothiazole derivatives 49 and 51,
a different synthetic strategy was employed (Scheme 2).
Carboxylic acid derivatives 1e4 and 6 were then coupled with
crude 1,2,4-oxadiazole derivative 53 to give corresponding amides
54e58. Acid hydrolysis of 54e56 in refluxing 3M HCl afforded the
Removal of the Boc-protecting group from Boc-L-alanyl-D-iso-
corresponding
1,2,4-oxadiazole
dipeptidomimetics
59e61,
glutamine ethyl ester 40 [35] was achieved using trifluoroacetic
acid in dichloromethane to give the trifluoroacetate 42, which was
immediately coupled with acids 4 and 5, affording the corre-
sponding ethyl esters 44 and 45. Alkaline hydrolysis of the latter in
1M LiOH/EtOH furnished the desired compounds 48 and 49. The
whereas compounds 62 and 63 were obtained by alkaline hydro-
lysis of 57 and 58, respectively (Scheme 3). To determine the
influence of configuration at the chiral centers within the peptide
part of the molecule on its immunomodulatory activity, all
stereoisomers of 26 (derivative of
The protected dipeptides Boc-
ester 64, Boc- -alanyl- -isoglutamine benzyl ester 65 and Boc-
alanyl- -isoglutamine benzyl ester 66 were prepared according to
L
-Ala-
D
-iGln) were synthesized.
TFA-induced cleavage of Boc group from Boc-L-alanyl-D-glutamate
L-alanyl-
L-isoglutamine benzyl
dimethyl ester 41 [36,37] gave the trifluoroacetate 43, which was
immediately coupled with acids 4 and 5, affording the corre-
sponding dimethyl esters 46 and 47. Conventional alkaline hydro-
lysis of the latter yielded the target compounds 50 and 51.
3-(3-((S)-1-tertbutyloxycarbonylaminoethyl)-1,2,4-oxadiazol-
5-yl)propanoate 52 synthesized according to known procedure
D
L
D-
D
known procedures [35,36]. Subsequent acidolytic removal of the
Boc-protecting group afforded the corresponding trifluoroacetate
salts 67e69, which were immediately coupled with 5-(1,1,3-
trioxo-1,3-dihydro-2H-1,2-benzisothiazol-2-yl)pentanoic acid (1),
160
**
12*0*,2
**
306,8
140
120
100
80
111,2
122
300
**
204,3
100
250
**
1*87*,4
171,8
200
150
100
50
LPS
MDP
48
120,2
60
108
90,3
100
92,5
40
20
0
0
LPS
15
44
48
LPS
0.2 µM
2 µM
20 µM
200 µM
Stimulus
Concentration of stimulus
Fig. 3. Influence of concentration on TNF-
increasing concentrations of compound 48 and MDP (0.2e200
LPS (1 ng/ml). Values are expressed as % of LPS-induced TNF-
a
release. Cells were incubated for 24 h with
M) in the presence of
release. Each value
Fig. 5. Influence of lipophilicity on TNF-
the selected compounds (20.2 M) in the presence of LPS (1 ng/ml). Values are
expressed as % of LPS-induced TNF-
samples. Statistical analysis was performed with ManneWhitney U-Rank test; with
*p < 0.05; **p < 0.01 vs LPS treated cells.
a release. Cells were incubated for 24 h with
m
a
m
a
release. Each value represents the mean ꢁ SD of 6
represents the mean ꢁ SD of 6 samples. Statistical analysis was performed with
Dunnet test; with *p < 0.05; **p < 0.01 vs LPS treated cells.

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3767
O
CH3
O
O
O
S
O
CH3
O
O
O
S
H
N
O
O
H
N
O
NH
O
CH3
O
O
O
H
N
H
N
N
N
N
H
OH
N
N
H
OH
N
H
OH
O
NH2
O
O
O
NH2
O
O
O
NH2
26
28
32
O
CH3
O
O
O
S
H
N
O
NH
O
CH3
O
O
H
N
H
N
N
H
OH
N
H
OH
O
OH
O
O
OH
OH
35
39
O
HO
O
OH
O
S
NH
O
O
S
O
O
N
O
N
CH3
O
NH
O
O
H
N
CH3
O
O
H
N
O
O
O
N
OH
HN
H
O
N
H
O
O
O
NH2
O
NH2
NH2
48
Murabutid
44
Fig. 6. Selection of desmuramyldipeptides for further evaluation.
yielding benzyl esters 70e72. Hydrogenolytic deprotection gave
the target compounds 73e75 (Scheme 4).
26.2%. The saccharin-based compounds 26, 28 (
L
-Ala-
D
-iGln series)
and 35 ( -Ala-D-Glu series), incorporating a spacer with 5 C-atoms
L
separating the saccharin fragment from the dipeptide moiety, were
confirmed as weak up-modulators. Both 32 and 39, in which the
3. Results and discussion
indole-2-carboxamide and the dipeptide moiety (either
iGln or -Ala- -Glu) are linked by a spacer with 2 C-atoms, were also
effective in enhancing the LPS-induced TNF- release. Moreover, of
L-Ala-D-
L
D
3.1. Biological activity
a
the conformationally restrained analogs, only the pyrido-fused
[1,2]-benzisothiazoles 36, 48 and 50 proved as active. In contrast,
of the series of compounds incorporating a 1,2,4-oxadiazole based
The synthesized desmuramyldipeptide analogs, MDP and MB as
reference compounds, were screened for their ability to induce
TNF-
a
release from naive THP-1 cells and to modulate LPS-induced
release.
L
-Ala-
D
-iGln mimetic, only the indole-based compound 63 showed
release.
TNF-a
significant up-modulation of TNF-
a
Preliminary experiments were conducted with MDP to define
the optimal experimental conditions. The exposure time was
chosen based on preliminary time-course experiments of TNF-
A dose-dependent response was also investigated and the
observed results confirmed the influence of concentration of des-
muramyldipeptide 48 and MDP on the modulation of TNF-a release
from LPS-stimulated THP-1 cells. As shown in Fig. 3 both compound
a
release induced by LPS alone or in combination with MDP. The
synergistic effects of MDP/synthesized analogs and LPS were
evident at all chosen time points (3 h, 8 h, and 24 h) (data not
shown). No difference in TNF-a secretion was observed between
48 and MDP induced a dose-dependent stimulatory effect on LPS-
induced TNF-
It has been demonstrated that the replacement of
-isoGln by -isoGln eliminated the ability of MDP to stimulate
Nod2, indicating stereoselective recognition [17]. To shed more
light on the influence of stereochemistry on TNF- release we
a release, with MDP being more active.
L-Ala by D-Ala
preincubation and co-incubation of cells with MDP and LPS, sug-
gesting that pretreatment was not necessary for the effect of MDP.
From these preliminary data, the 24 h exposure time and co-
incubation conditions were chosen for all further experiments.
Lactate dehydrogenase (LDH) leakage was also initially assessed
to evaluate cytotoxicity of the tested compounds. Results (data not
shown) confirmed none of the compounds was cytotoxic at the
maximum concentration tested.
or
D
L
a
prepared and tested three stereoisomers of 26 (derivative with L,D
configuration), 73 (L,L), 74 (D,L) and 75 (D,D). Compound 73,
carrying the
L
-Ala-L-iGln moiety, proved even more active than the
parent compound 26, whereas the
D
-Ala- -iGln containing analog
L
74 was inactive. The obtained results are presented in Fig. 4. The
MDP (L,D) and MDP (L,L) isomers both induced pronounced TNF-
THP-1 cells were treated with desmuramyldipeptide analogs,
MDP and MB, alone or in combination with 1 ng/ml LPS. TNF-
a
secretion in an in vitro experiment using PBMC [38]. An analogous
a
release was assessed 24 h later. Neither desmuramyldipeptide
analogs nor the positive controls MDP nor MB induced a significant
TNF- release on their own (data not shown). However, a synergistic
effect on LPS-induced TNF- secretion was observed by some of the
finding was described by Kengatharan, who found that, in the lip-
oteichoic acid (LTA) co-stimulated J774.2 murine macrophage cell
line, MDP (L,D) proved to be the most active isomer with regard to
nitrite production. MDP (L,L) was also active whereas MDP (D,D)
proved to be inactive [39]. Another report revealed that replace-
a
a
synthesized desmuramyldipeptide analogs and the positive controls
MDP and MB (shown in Fig. 2). None of the synthesized compounds
was as effective as MDP and MB, suggesting that either the absence of
the N-acetylmuramyl moiety affected recognition and binding
capacity to its receptor or decreased cell permeability of those
compounds caused the reduction of immunostimulatory property.
The positive controls, MDP and MB, enhanced the LPS-induced
TNF-a release by 87.4 and 82.6%, respectively. In this experiment
our compounds exerted only a weak up-modulating activity,
the most effective being compound 32, an indole-based desmur-
amyldipeptide, that enhanced the aforementioned TNF-a release by
ment of D-isoGln by L-isoGln in O-acylated derivatives of MDP did
not abrogate their immunomodulatory effect [16]. The results
obtained from our experiment are in accordance with previous
research [38,39]. Taking all this into consideration, it can be
concluded that MDP analogs exert their effects not only via Nod2
but also other receptors.
The plasma membrane transporter hPepT1, has been shown to
enable MDP, but no other molecule, to enter the host cell [40,41].
The fact that MDP and MB, our positive controls, can serve as

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
Table 1
Effect of selected desmuramyldipeptide analogs on LPS-induced cytokine production in human PBMC.
Cytokine
TNF-
a
IL-10
IL-6
IL-1
b
IL-8
(% release vs. LPS) ꢁ SD
(% release vs. LPS) ꢁ SD
(% release vs. LPS) ꢁ SD
(% release vs. LPS) ꢁ SD
(% release vs. LPS) ꢁ SD
Control
LPS
2.0
100 ꢁ 6.9
2.2
100 ꢁ 3.5
0.6
100 ꢁ 4.6
64.3
100 ꢁ 4.1
8.8
100 ꢁ 1.6
26 þ LPS
28 þ LPS
32 þ LPS
35 þ LPS
39 þ LPS
48 þ LPS
44 þ LPS
MB þ LPS
108.5 ꢁ 2.9
104.5 ꢁ 0.6
101 ꢁ 5.8
98 ꢁ 2.3
84.5 ꢁ 1.7*
71.5 ꢁ 0.6*
65.5 ꢁ 0.6*
68.5 ꢁ 0.6*
94 ꢁ 4.6
109 ꢁ 1.2*
95.5 ꢁ 0.6
74 ꢁ 2.3*
106 ꢁ 1.2*
95.5 ꢁ 0.6
90.5 ꢁ 2.9*
85 ꢁ 1.2*
78 ꢁ 1.2*
94 ꢁ 3.5
97.5 ꢁ 2.9
96.5 ꢁ 0.6*
98.5 ꢁ 1.7
89.5 ꢁ 0.6*
95.5 ꢁ 1.7*
98.5 ꢁ 0.6
94.5 ꢁ 2.9*
107 ꢁ 1.2*
104 ꢁ 1.2
86 ꢁ 2.3*
103.5 ꢁ 0.6
115 ꢁ 3.5*
122.5 ꢁ 2.9*
696.5 ꢁ 7.5*
76.8 ꢁ 0.5*
103.5 ꢁ 4.0
120 ꢁ 1.2*
594.5 ꢁ 9.8*
105.5 ꢁ 2.9
95 ꢁ 4.6
559 ꢁ 2.7*
416.5 ꢁ 7.5*
PBMC were treated with the selected compounds (20.2
m
M) in the presence of LPS (10 ng/ml) for 18 h. Each value represents the mean ꢁ SD of 4 samples, statistical analysis was
performed by ManneWhitney U-rank test, with *p < 0.05 vs LPS treated cells. Data is representative of two independent donors.
substrates for hPepT1 could explain their superiour efficacy over
our synthesized compounds. The synthesized desmuramyldipep-
tides, which do not act as substrates for the hPepT1 transporter, are
fully ionized under the experimental pH conditions (7.2), which
makes it extremely difficult for them to penetrate the cell
membrane. The pharmacokinetics of desmuramyldipeptide LK-423
were reported recently and show that LK-423 is absorbed passively,
most probably by the paracellular route [42].
TNF-
significantly stimulated TNF-
not be confirmed for compounds 26, 28, 32, 35 and 39.
In addition to TNF- , the release of IL-1 , IL-6, IL-8 and IL-10 was
also assessed. A complex cytokine profile was observed. Regarding
IL-1 release, it was increased by compound 26, and significantly
reduced by 32, 35 and 39. Similarly, IL-6 release was increased by
compounds 26 and 44, and reduced by 32, 35 and 39. IL-8 release
was modestly affected by all compounds, including the positive
control MB. Finally, IL-10 was significantly reduced by 28, 32, 35
and 39.
a
release, whereas in PBMC, only compounds 44 and 48
a
release. The stimulatory effect could
a
b
b
To address the influence of lipophilicity, desmuramyldipeptide
48 and its corresponding ethyl and benzyl esters, compounds 44
and 15, were tested for modulation of LPS-induced TNF-a release
from THP-1 cells. We tested the hypothesis that the formation of
ester prodrugs of desmuramyldipeptides could facilitate the
passive transport through the cell membrane. No correlation
As for LPS, cytokine production induced by PMA/ionomycin was
also considerably affected by the selected compounds (Table 2). For
TNF-
a
release, an increase was observed for compounds 35 and 44,
, an
between lipophilicity and TNF-
highly lipophilic benzyl ester 15 did not increase TNF-
a
release was observed since the
release
while a reduction was found for compounds 28 and 39. For IL-1b
a
increase was found only for compounds 35 and 44. Regarding IL-6,
an increase was observed only for compound 44, while a reduction
was found for compounds 26, 28, 32, 39 and 48. As for LPS also for
PMA/ionomycin, the release of IL-8 was modestly affected with the
exception of compound 44 which increased its release. Finally, IL-
10 release was significantly reduced by compounds 26, 39 and 44.
Taken together, data presented confirmed the immunostimu-
latory effect of compound 44, while for compounds 32 and 39 an
immunosuppressive effect was observed. The selected desmur-
amyldipeptide analogs definitely have a different behavior if
compared to MB, which significantly up-regulated the release of all
cytokines tested induced by both LPS and PMA/ionomycin.
from THP-1 cells if compared to the value of parent compound
48 (Fig. 5).
To further characterize the immunomodulatory capacity of
selected synthesized desmuramyldipeptide analogs (Fig. 6), their
ability to modulate cytokine synthesis was assessed in human
PBMC [43]. LPS (10 ng/ml) and ionomycin (500 nM) plus PMA
(3.33 ng/ml) were used as stimuli. LPS mainly stimulates mono-
cytes and B cells, whereas the combination of diacylglycerol (DAG)
analog such as PMA and ionomycin was used to mimic antigen
receptor stimulation [44,45]. The results shown in Tables 1 and 2
demonstrate the immunomodulatory capacity of the selected
desmuramyldipeptide analogs compared with that of MB on cyto-
kine production.
The majority of the deleterious effects of bacteremia are caused
by inflammatory responses to specific bacterial components [46].
Clinical studies have demonstrated that high concentrations of
The selected compounds differently affect LPS-induced TNF-
a
release modulation if compared with data obtained in THP-1 cells.
proinflammatory cytokines in the circulation, such as TNF-
a and IL-
In the latter, all the selected compounds increased LPS-induced
1b, correlate with the severity of the disease and poor prognosis
Table 2
Effect of selected desmuramyldipeptide analogs on PMA/ionomycin-induced cytokine production in human PBMC.
Cytokine
TNF-
a
IL-10
IL-6
IL-1
b
IL-8
(% release vs.
PMA/iono) ꢁ SD
(% release vs.
PMA/iono) ꢁ SD
(% release vs.
PMA/iono) ꢁ SD
(% release vs.
PMA/iono) ꢁ SD
(% release vs.
PMA/iono) ꢁ SD
Control
<0.1
52
5.7
21.8
12.8
PMA/iono
100 ꢁ 9.2
100 ꢁ 11.5
100 ꢁ 8.1
100 ꢁ 17.3
100 ꢁ 5.8
26 þ PMA/iono
28 þ PMA/iono
32 þ PMA/iono
35 þ PMA/iono
39 þ PMA/iono
48 þ PMA/iono
44 þ PMA/iono
MB þ PMA/iono
96 ꢁ 1.2
83.5 ꢁ 4.0*
93.5 ꢁ 14.4
124 ꢁ 10.4*
63.5 ꢁ 1.7*
105.5 ꢁ 1.7
142 ꢁ 8.1*
332.5 ꢁ 29.4*
84.5 ꢁ 1.7*
89.5 ꢁ 4.0
81 ꢁ 2.3*
63.5 ꢁ 6.4*
76 ꢁ 4.6*
91.5 ꢁ 9.8
122 ꢁ 40.4
91.5 ꢁ 5.2
91 ꢁ 4.6
103 ꢁ 1.2
110.5 ꢁ 14.4
98.5 ꢁ 9.8
104 ꢁ 1.2
90 ꢁ 9.2
102 ꢁ 12.7
116 ꢁ 9.2
111.5 ꢁ 2.9*
42.5 ꢁ 0.6*
74 ꢁ 2.3*
75.8 ꢁ 8.6*
88.5 ꢁ 11.0
82.5 ꢁ 4.0*
158.5 ꢁ 50.2*
96.5 ꢁ 0.6
88 ꢁ 10.4
103 ꢁ 9.2
134 ꢁ 15.0*
152 ꢁ 2.3*
139.5 ꢁ 8.7*
439.5 ꢁ 16.7*
406 ꢁ 52.0*
2964.5 ꢁ 444*
PBMC were treated with the selected compounds (20.2
m
M) in the presence of PMA (3.33 ng/ml) and ionomycin (500 nM) for 18 h. Each value represents the mean ꢁ SD of 4
samples, statistical analysis was performed by ManneWhitney U-rank test, with *p < 0.05 vs PMA/ionomycin treated cells. Data is representative of two independent donors.

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3769
[47]. In addition, Crohn’s disease, a common inflammatory disease
B in 30 min. The purity of all pharmacologically investigated
of the intestinal tract, is associated with high concentrations of IL-
compounds was >95% as determined by RP-HPLC.
1
b
, IL-6, IL-12 and TNF-
a
. Antibodies to TNF-
a, IL-6 and IL-12, as
well as direct inhibition of NF-
k
B, are effective therapies [48].
5.1. Chemistry
Down-regulation of the production of proinflammatory mediators
may also overcome these problems and provide an alternative
strategy in the treatment of these inflammatory responses.
5.1.1. General procedure for TBTU-mediated coupling of carboxylic
acids with amines
Conversely, TNF-
a
and IL-1
b
are important for non-specific innate
To a stirred solution of corresponding carboxylic acid deriva-
immunity [49]. Immunomodulators capable of influencing cytokine
production can contribute to achieving homeostasis.
tive (1 mmol) and benzyl L-alanyl-
roacetate (1 mmol) or dibenzyl L-alanyl-
D
-isoglutaminate trifluo-
-glutamate trifluo
D
-roacetate (1 mmol) or ethyl 3-(3-((S)-1-aminoethyl)-1,2,4-
oxadiazol-5-yl)propanoate hydrochloride (1 mmol) in dry aceto-
nitrile, triethylamine (2.5 mmol) was added at 0 ꢀC. After stirring
for 15 min TBTU was added, and the mixture was allowed to
warm to room temperature and stirring then continued for 3 h.
The precipitate was filtered off and washed with 10% citric acid
(2 ꢂ 10 ml), water (20 ml), saturated NaHCO3 solution (2 ꢂ 10 ml)
and water (20 ml). Drying the precipitate at elevated tempera-
tures (50 ꢀC) afforded solvent-free products.
4. Conclusion
A series of novel desmuramyldipeptides have been designed
and synthesized as part of our search for therapeutically useful
muramyldipeptide (MDP) analogs. Following the initial screening,
selected compounds were further investigated for immunomodu-
latory properties using LPS and phorbol 12-myristate 13-acetate
(PMA)/ionomycin-stimulated human peripheral blood mono-
nuclear cells. The results confirmed the immunomodulatory
properties of some of the synthesized desmuramyldipeptide
analogs. On the one hand, indole scaffold-based compounds 32 and
39 proved to be effective down-modulators of proinflammatory
cytokine synthesis and provide a promising basis for further
improvement of their pharmacological profile.
5.1.1.1. Benzyl (4R)-5-amino-5-oxo-4-[((2S)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoate (12). Synthesized from 10 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 372 mg (65%); m.p. 170e174 ꢀC 1H NMR (CDCl3):
d
¼ 1.37 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.71e1.81 (m, 2H, CH2CH2CH2),
1.83e1.93 (m, 2H, CH2CH2CH2), 1.99e2.11 and 2.17e2.24 (2m, 1H
each, CH2- -iGln), 2.40e2.66 (m,
On the other hand, the desmuramyldipeptide 44, incorporating
a pyrido-fused [1,2]-benzisothiazole moiety and ethyl ester func-
tionality, significantly enhanced the production of multiple proin-
b
-iGln), 2.28 (t, 2H, J ¼ 7.5 Hz, CH2-
g
2H, CH2CO) 3.79 (t, 2H, J ¼ 6.9 Hz, NCH2), 4.20e4.25 (m, 1H, CH-
iGln), 4.40e4.47 (m, 1H, CH-Ala), 5.10 (5.15) (AB-system, 2H,
J ¼ 12.3 Hz, CH2-benzyl), 5.46 (s,1H, NH-Ala), 6.09 (s, 1H, NH2-iGln),
6.72 (s, 1H, NH2-iGln), 7.08 (s, 1H, NH-iGln), 7.32e7.37 (m, 5H,
phenyl), 7.80e7.93 (m, 3H, 3 saccharinyl-H), 8.02e8.07 (m, 1H,
saccharinyl-H4). MS (ESI): m/z (%) ¼ 573.2 (M þ H)þ. IR (KBr):
flammatory cytokines, including TNF-a, IL-6, IL-1b and IL-8.
Although MB was superior to the selected compounds in every
aspect, compound 44 also proved to be an immunostimulant
capable of increasing proinflammatory cytokine production.
Further in vitro as well as in vivo biological tests are needed to
investigate the true potential of these compounds and their
mechanism of action which remains to be elucidated. However,
their potency needs to be balanced against their potential to
overactivate or deactivate the immune system, with damaging
consequences.
n
¼ 3433, 1612, 1322 cmꢃ1. HRMS Calcd for C27H33N4O8S m/z:
573.2019 (M þ H)þ, found 573.2023.
5.1.1.2. Benzyl (4R)-5-amino-5-oxo-4-[((2S)-2-{[4-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)butanoyl]amino}propanoyl)
amino]pentanoate (13). Synthesized from 10 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 419 mg (75%); m.p. 165e168 ꢀC 1H NMR (CDCl3):
5. Experimental protocols
Chemicals were obtained from Acros, Aldrich Chemical Co. and
Fluka, and used without further purification. Murabutide was from
Invivogen, and MDP and LPS (from Escherichia coli, 0127:B8) from
Sigma. Analytical TLC was performed on Merck 60 F254 silica gel
plates (0.25 mm), using visualization with ultraviolet light and
ninhydrin. Column chromatography was carried out on silica gel 60
(particle size 240e400 mesh). Microwave assisted reactions were
performed using a CEM Discover microwave reactor (CEM Corpo-
ration, USA). Melting points were determined on a Reichert hot
stage microscope and are uncorrected. 1H NMR and 13C NMR
spectra were recorded at 300 MHz and 75 MHz, respectively on
a Bruker AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6
solution with TMS as the internal standard. Spectra were assigned
using gradient COSY and HSQC experiments. IR spectra were
recorded on a PerkineElmer 1600 FT-IR spectrometer. Microanal-
yses were performed on a PerkineElmer C, H, N analyzer 240 C.
Analyses indicated by the symbols of the elements were within
ꢁ0.4% of the theoretical values. Mass spectra were obtained using
a VG-Analytical Autospec Q mass spectrometer. HPLC analyses were
performed on an Agilent Technologies HP 1100 instrument with
G1365B UVeVIS detector (220 nm), using a Luna C18 column
(4.6 ꢂ 150 mm) at flow rate 1 ml/min. The eluant was a mixture of
0.1% TFA in water (A) and acetonitrile (B). Gradient was 10% B to 80%
d
¼ 1.37 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 2.00e2.29 (m, 4H, CH2-
b-iGln
and CH2CH2CH2), 2.34 (t, 2H, J ¼ 7.5 Hz, CH2- -iGln), 2.45e2.66 (m,
g
2H, CH2CO) 3.75e3.94 (m, 2H, NCH2), 4.24e4.35 (m, 1H, CH-iGln),
4.42e4.49 (m, 1H, CH-Ala), 5.10 (5.15) (AB-system, 2H,
J ¼ 12.3 Hz, CH2-benzyl), 5.46 (s, 1H, NH-Ala), 6.35 (s, 1H, NH2-iGln),
6.77 (s, 1H, NH2-iGln), 7.21 (s, 1H, NH-iGln), 7.31e7.39 (m, 5H,
phenyl), 7.81e7.94 (m, 3H, 3 saccharinyl-H), 8.06 (d, 1H, J ¼ 6.6 Hz,
saccharinyl-H4). MS (ESI): m/z (%) ¼ 559.2 (M þ H)þ. IR (KBr):
n
¼ 3412, 3285, 2935, 1736, 1720, 1671, 1628, 1560, 1457, 1394, 1326,
1260, 1177, 1060, 987, 753, 702, 676, 589, 510 cmꢃ1. HRMS Calcd for
C26H31N4O8S m/z: 559.1863 (M þ H)þ, found 559.1852.
5.1.1.3. Benzyl (4R)-5-amino-5-oxo-4-{[(2S)-2-({2-[2-(1,1,3-trioxo-
1,3-dihydro-2H-1,2-benzisothiazol-2-yl)ethoxy]acetyl}amino)prop-
anoyl]amino}pentanoate (14). Synthesized from 10 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 505 mg (88%); m.p. 140e142 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.18 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.69e1.82 and 1.96e2.07 (2m,
1H each, CH2- -iGln), 3.78 (t, 2H,
b
-iGln), 2.34 (t, 2H, J ¼ 7.5 Hz, CH2-
g
J ¼ 5.4 Hz, NCH2CH2O), 3.93e3.96 (m, 4H, NCH2 and OCH2CO),
4.11e4.18 (m, 1H, CH-iGln), 4.23e4.32 (m, 1H, CH-Ala), 5.08 (s, 2H,
CH2-benzyl), 7.08 (s,1H, NH2-iGln), 7.27 (s,1H, NH2-iGln), 7.32e7.40
(m, 5H, phenyl), 7.68 (d, 1H, J ¼ 6.9 Hz, NH-Ala), 7.96e8.14 (m, 4H, 3

ꢀ
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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
saccharinyl-H and NH-iGln), 8.30 (d, 1H, J ¼ 6.9 Hz, saccharinyl-H4).
(%) ¼ 530.2 (M þ Na)þ. IR (KBr):
n
¼ 3290, 1724, 1646, 1542, 1449,
MS (ESI): m/z (%) ¼ 575.2 (M þ H)þ. IR (KBr):
n
¼ 3374, 1727, 1659,
1420, 1317, 1270, 1200, 959, 744, 696 cmꢃ1. HRMS Calcd for
1331, 1262, 1190, 1130 cmꢃ1. HRMS Calcd for C26H31N4O9S m/z:
C26H29N5O6Na m/z: 530.2016 (M þ Na)þ, found 530.2019.
575.1812 (M þ H)þ, found 575.1812.
5.1.1.8. Dibenzyl
(2R)-2-[((2S)-2-{[5-(1,1,3-trioxo-1,3-dihydro-2H-
5.1.1.4. Benzyl (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-
1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)amino]pentane-
dioate (19). Synthesized from 11 according to the General procedure
for coupling with TBTU. White amorphous powder, yield: 477 mg
5l
6-pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}prop-
anoyl)amino]-5-oxopentanoate (15). Synthesized from 10 according
to the General procedure for coupling with TBTU. White amorphous
powder, yield: 244 mg (44%); m.p. 136e140 ꢀC 1H NMR (DMSO-d6):
(72%); m.p. 113e116 ꢀC 1H NMR (CDCl3):
d
¼ 1.34 (d, 3H, J ¼ 7.2 Hz,
CH3-Ala), 1.70e1.79 (m, 2H, CH2CH2CH2), 1.83e1.93 (m, 2H,
CH2CH2CH2), 1.96e2.09 (m, 1H, H- -Glu), 2.17e2.27 (m, 3H, H-b-
d
¼ 1.30 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.81e1.93 (m, 1H, H-CH2-
b
b
-
-
b
iGln), 2.01e2.07 (m, 2H, NCH2CH2), 2.11e2.20 (m, 1H, H-CH2-
Glu and CH2CO), 2.39 (t, 2H, J ¼ 7.2 Hz, CH2CO), 3.78 (t, 2H,
J ¼ 6.9 Hz, NCH2), 4.45e4.55 (m,1H, CH-Glu), 4.56e4.63 (m,1H, CH-
Ala), 5.09 (5.15) (AB-system, 2H, J ¼ 12.3 Hz, CH2-benzyl), 5.09 (s,
2H, CH2-benzyl), 6.01 (d, 1H, J ¼ 7.2 Hz, NH-Ala), 6.95 (d, 1H,
J ¼ 7.8 Hz, NH-Glu), 6.95 (d, 1H, J ¼ 7.2 Hz, AreH), 7.30e7.38 (m,
10H, 2ꢂphenyl), 7.78e7.92 (m, 3H, saccharinyl-H), 8.02e8.05 (m,
1H, saccharinyl-H4). MS (ESI): m/z (%) ¼ 664.2 (M þ H)þ. IR (KBr):
iGln), 2.47 (t, 2H, J ¼ 7.8 Hz,
g-iGln), 3.52e3.56 (m, 2H, NCH2CH2),
4.26e4.35 (m, 1H, CH-iGln), 4.38e4.47 (m, 1H, CH-Ala), 5.15 (s, 2H,
CH2-benzyl), 7.22 (s, 1H, NH2-iGln), 7.38e7.44 (m, 6H, 5 phenyl-H
and H-NH2-iGln), 7.71e7.75 (m, 2H, AreH), 7.99e8.06 (m, 2H,
AreH), 8.34 (d, 1H, J ¼ 8.1 Hz, NH-iGln), 8.87 (d, 1H, J ¼ 6.0 Hz, NH-
Ala). * signal for NCH2CH2CH2 is hidden under broad peak of DMSO
at 2.5 ppm MS (ESI): m/z (%) ¼ 555.2 (M þ H)þ. IR (KBr):
n
¼ 3282,
n
¼ 3340, 1723, 1630, 1529, 1331, 1275, 1180, 958, 753 cmꢃ1. HRMS
1734, 1654, 1618, 1540, 1457, 1306, 1178, 1138, 1061, 756, 698, 601,
579 cmꢃ1. HPLC: 97%, tr ¼ 16.47 min. HRMS Calcd for C27H31N4O7S
m/z: 555.1913 (M þ H)þ, found 555.1932.
Calcd for C34H37N3O9SNa m/z: 686.2148 (M þ Na)þ, found 686.2140.
5.1.1.9. Dibenzyl
(2R)-2-[((2S)-2-{[4-(1,1,3-trioxo-1,3-dihydro-2H-
1,2-benzisothiazol-2-yl)butanoyl]amino}propanoyl)amino]pentane-
dioate (20). Synthesized from 11 according to the General procedure
for coupling with TBTU. White amorphous powder, yield: 604 mg
5.1.1.5. Benzyl
(4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-
3H-5
l
6-pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}
propanoyl)amino]-5-oxopentanoate (16). Synthesized from 10
according to the General procedure for coupling with TBTU. White
amorphous powder, yield: 275 mg (51%); m.p. 218e220 ꢀC 1H NMR
(93%); m.p. 90e93 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.18 (d, 3H, J ¼ 7.2 Hz,
-Glu and CH2CH2CH2), 2.26 (t, 3H,
-Glu), 3.73 (t, 2H,
CH3-Ala), 1.83e2.12 (m, 4H, CH2-
b
J ¼ 7.2 Hz, CH2CO), 2.42 (t, 3H, J ¼ 7.5 Hz, CH2-
g
(DMSO-d6):
2.03e2.14 (2m, 2H, CH2-
d
¼ 1.31 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.73e1.86 and
J ¼ 7.2 Hz, NCH2), 4.30e4.39 (m, 2H, CH-Glu and CH-Ala), 5.02e5.16
(m, 4H, 2ꢂCH2-benzyl), 7.34e7.36 (m, 10H, 2ꢂphenyl), 7.97e8.11
(m, 3H, saccharinyl-H and NH-Ala), 8.27e8.31 (m, 2H, AreH and
b
-iGln), 2.38 (t, 2H, J ¼ 7.65 Hz, CH2-
g-
iGln), 3.38 (t, 2H, J ¼ 7.65 Hz, CH2Cq), 3.74 (t, 2H, J ¼ 8.7 Hz,
NCH2CH2), 4.18e4.25 (m, 1H, CH-iGln), 4.33e4.42 (m, 1H, CH-Ala),
5.07 (s, 2H, CH2-benzyl), 7.23 (s, 1H, NH2-iGln), 7.27e7.44 (m, 6H, 5
phenyl-H and H-NH2-iGln), 7.74e7.86 (m, 2H, AreH), 8.01e8.04 (m,
2H, AreH), 8.17 (d, 1H, J ¼ 8.1 Hz, NH-iGln), 8.86 (d, 1H, J ¼ 7.8 Hz,
NH-Glu). MS (ESI): m/z (%) ¼ 650.2 (M þ H)þ. IR (KBr):
n
¼ 3301,
3032, 2944, 1737, 1719, 1643, 1525, 1456, 1418, 1392, 1362, 1336,
1263, 1205, 1183, 1060, 1021, 999, 964, 908, 786, 757, 694, 676, 587,
542, 510 cmꢃ1. HRMS Calcd for C33H36N3O9S m/z: 650.2177
(M þ H)þ, found 650.2172.
NH-Ala). MS (ESI): m/z (%) ¼ 541.2 (M þ H)þ. IR (NaCl):
n
¼ 3410,
3297, 1726, 1642, 1613, 1528, 1319, 1227, 1180, 1084 cmꢃ1. HRMS
Calcd for C26H29N4O7S m/z: 541.1757 (M þ H)þ, found 541.1754.
5.1.1.10. Dibenzyl (2R)-2-{[(2S)-2-({2-[2-(1,1,3-trioxo-1,3-dihydro-
2H-1,2-benzisothiazol-2-yl)ethoxy]acetyl}amino)propanoyl]amino}
pentanedioate (21). Synthesized from 11 according to the General
procedure for coupling with TBTU. White amorphous powder, yield:
5.1.1.6. Benzyl (4R)-5-amino-4-{[(2S)-2-({3-[(1H-indol-2-ylcarbonyl)
amino]propanoyl}amino)propanoyl]amino}-5-oxopentanoate (17).
Synthesized from 10 according to the General procedure for coupling
with TBTU. White amorphous powder, yield: 396 mg (76%); m.p.
505 mg (88%); m.p. 70e73 ꢀC 1H NMR (CDCl3):
d
¼ 1.38 (d, 3H,
-Glu), 2.16e2.51 (m, 3H,
g-Glu), 3.80e3.85 (m, 2H, NCH2CH2O), 3.96e4.08
J ¼ 7.2 Hz, CH3-Ala), 1.90e2.03 (m, 1H, H-
b
233e236 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.19 (d, 3H, J ¼ 6.9 Hz, CH3-
Ala), 1.72e1.85 and 1.96e2.11 (2m, 1H each, CH2- -iGln), 2.34e2.49
(m, 4H, CH2- -iGln and NHCH2CH2CO), 3.45e3.52 (m, 2H,
H- -Glu and CH2-
b
b
(m, 4H, NCH2CH2O and OCH2CO), 4.45e4.63 (m, 2H, CH-Glu and
CH-Ala), 5.08e5.19 (m, 4H, 2ꢂCH2-benzyl), 7.06 (d, 1H, J ¼ 7.8 Hz,
NH-Ala), 7.30e7.40 (m, 11H, 2ꢂphenyl-H and NH-Glu), 7.83e7.86
(m, 2H, saccharinyl-H), 7.93e7.96 (m, 1H, saccharinyl-H), 8.06e8.09
(m,1H, saccharinyl-H). MS (ESI): m/z (%) ¼ 666.2 (M þ H)þ. IR (KBr):
g
NHCH2CH2CO), 4.15e4.31 (m, 2H, CH-Ala and CH-iGln), 5.08 (s, 2H,
CH2-benzyl), 6.99e7.43 (m, 11H, 9 AreH and NH2-iGln), 7.59 (d, 1H,
J ¼ 7.8 Hz, indole-H4), 8.12 (d, 1H, J ¼ 8.1 Hz, NH-Ala), 8.21 (d, 1H,
J ¼ 6.3 Hz, NH-iGln), 8.44 (t, 1H, J ¼ 5.7 Hz, CONHCH2), 11.52 (s, 1H,
n
¼ 3386, 3300, 3066, 2938, 1734, 1654, 1595, 1541, 1456, 1389, 1327,
indole-NH). MS (ESI): m/z (%) ¼ 544.2 (M þ Na)þ. IR (KBr):
n
¼ 3339,
1266, 1190, 1126, 1063, 1029, 953, 910, 831, 784, 750, 696, 677, 606,
589, 509 cmꢃ1. HRMS Calcd for C33H36N3O10S m/z: 666.2121
(M þ H)þ, found 666.2116.
1620, 1309, 1170 cmꢃ1
. HRMS Calcd for C27H31N5O6Na m/z:
544.2172 (M þ Na)þ, found 544.2186.
5.1.1.7. Benzyl (4R)-5-amino-4-{[(2S)-2-({2-[(1H-indol-2-ylcarbonyl)
amino]acetyl}amino)propanoyl]amino}-5-oxopentanoate (18). Synth-
esized from 10 according to the General procedure for coupling with
TBTU. Orange amorphous powder, yield: 365 mg (72%); m.p.
5.1.1.11. Dibenzyl
(2R)-2-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-
5l
6-pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}prop-
anoyl)amino]pentanedioate (22). Synthesized from 11 according to
the General procedure for coupling with TBTU. White amorphous
powder, yield: 555 mg (86%); m.p. 142e145 ꢀC 1H NMR (CDCl3):
161e164 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.23 (d, 3H, J ¼ 7.2 Hz, CH3-
Ala),1.73e1.86 and 2.01e2.10 (2m,1H each, CH2- -iGln), 2.36 (t, 2H,
-iGln), 3.86e3.99 (m, 2H, NHCH2CO), 4.16e4.23 (m,
b
d
¼ 1.41 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 2.01e2.11(m, 3H, H-CH2-
and NCH2CH2CH2), 2.16e2.27 (m, 1H, H-CH2- -Glu), 2.41 (t, 2H,
-Glu),
b-Glu
J ¼ 7.5 Hz, CH2-
g
b
1H, CH-iGln), 4.26e4.36 (m, 1H, CH-Ala), 5.06 (s, 2H, CH2-benzyl),
7.01e7.45 (m, 11H, 9 AreH and NH2-iGln), 7.62 (d, 1H, J ¼ 7.8 Hz,
indole-H4), 8.09e8.17 (m, 2H, NH-Ala and NH-iGln), 8,74 (t, 1H,
J ¼ 5.7 Hz, NHCH2), 11.53 (s, 1H, indole-NH). MS (ESI): m/z
J ¼ 6.9 Hz, NCH2CH2CH2), 2.53 (t, 2H, J ¼ 6.3 Hz, CH2-
g
3.53e3.66 (m, 2H, NCH2CH2), 4.57e4.71 (m, 2H, CH-Glu and CH-
Ala), 5.10 (s, 2H, CH2-benzyl), 5.15 (s, 2H, CH2-benzyl), 6.36 (d, 1H,
J ¼ 7.2 Hz, NH-Glu), 6.95 (d, 1H, J ¼ 7.5 Hz, NH-Ala), 7.29e7.39 (m,

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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3771
10H, 2ꢂphenyl), 7.49e7.57 (m, 2H, AreH), 7.79e7.88 (m, 2H, AreH).
(m, 2H, AreH), 7.93e8.00 (m, 2H, AreH), 8.27 (d, 1H, J ¼ 8.1 Hz, NH-
iGln), 8.82 (d, 1H, J ¼ 6.0 Hz, NH-Ala). MS (ESI): m/z (%) ¼ 491.2
MS (ESI): m/z (%) ¼ 646.2 (M þ H)þ. IR (KBr):
n
¼ 3315, 1645, 1179,
696 cmꢃ1. HRMS Calcd for C34H36N3O8S m/z: 646.2223 (M þ H)þ,
(M ꢃ H)ꢃ. IR (KBr):
n
¼ 3788, 3422, 3299, 3258, 3056, 2969, 2940,
found 646.2207.
2886, 2679,1737,1672,1641,1620,1543,1472,1451,1429,1377,1316,
1276, 1262, 1184, 1138, 1100, 1064, 1032, 1020, 980, 949, 919, 893,
863, 846, 817, 799, 781, 758, 748, 737, 721, 705, 684, 626, 605, 581,
543, 520 cmꢃ1. HPLC: 98%, tr ¼ 11.67 min. HRMS Calcd for
C22H27N4O7S m/z: 491.1600 (M ꢃ H)ꢃ, found 491.1602.
5.1.1.12. Dibenzyl (2R)-2-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-3H-5l6
-
pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}propanoyl)
amino]pentanedioate (23). Synthesized from 11 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 517 mg (82%); m.p. 138e140 ꢀC 1H NMR (CDCl3):
5.1.1.16. Ethyl (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-3H-
d
¼ 1.44 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 2.01e2.13 and 2.19e2.29 (2m,
5l
6-pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}prop-
1H each, CH2- -Glu), 3.24 (t, 2H,
b
-Glu), 2.41 (t, 2H, J ¼ 7.5 Hz, CH2-
g
anoyl)amino]-5-oxopentanoate (45). Synthesized from 42 accord-
ing to the General procedure for coupling with TBTU. Orange
amorphous powder, yield: 411 mg (65%); m.p. 135e138 ꢀC 1H NMR
J ¼ 8.7 Hz, NCH2CH2), 3.84 (t, 2H, J ¼ 9.0 Hz, NCH2CH2), 4.61e4.65
(m, 2H, CH-Glu and CH-Ala), 5.08e5.19 (m, 4H, 2ꢂCH2-benzyl), 6.14
(d, 1H, J ¼ 6.9 Hz, NH-Glu), 6.97 (d, 1H, J ¼ 7.5 Hz, NH-Ala),
7.28e7.39 (m, 10H, 2ꢂphenyl), 7.58e7.70 (m, 2H, 2AreH),
7.79e7.82 (m, 1H, AreH), 9.04e9.06 (m, 1H, AreH). MS (ESI): m/z
(DMSO-d6):
J ¼ 7.2 Hz, CH3-Ala), 1.70e1.82 and 1.99e2.10 (2m, 1H each, CH2-
-iGln), 3.31e3.46 (m, 2H,
d
¼ 1.17 (t, 3H, J ¼ 7.2 Hz, CH3CH2), 1.32 (d, 3H,
b
-
iGln), 2.30 (t, 2H, J ¼ 7.65 Hz, CH2-
g
(%) ¼ 632.2 (M þ H)þ. IR (NaCl):
n
¼ 3315, 1724, 1654, 1623, 1543,
NCH2CH2), 3.74 (t, 2H, J ¼ 8.85 Hz, NCH2CH2), 4.02 (q, 2H, J ¼ 7.2 Hz,
CH3CH2), 4.15e4.23 (m, 1H, CH-iGln), 4.33e4.42 (m, 1H, CH-Ala),
7.22 (s, 1H, CONH2), 7.31 (s, 1H, CONH2), 7.76e7.88 (m, 2H, AreH),
8.00e8.05 (m, 2H, AreH), 8.15 (d, 1H, J ¼ 8.1 Hz, NH-Ala), 8.86 (d,
1H, J ¼ 7.8 Hz, NH-iGln). MS (ESI): m/z (%) ¼ 477.1 (M ꢃ H)ꢃ. IR
1518, 1456, 1315, 1224,1162, 1128, 1061, 963, 837, 756, 736, 696, 670,
594 cmꢃ1. HRMS Calcd for C33H34N3O8S m/z: 632.2067 (M þ H)þ,
found 632.2072.
5.1.1.13. Dibenzyl (2R)-2-{[(2S)-2-({3-[(1H-indol-2-ylcarbonyl)amino]
propanoyl}amino)propanoyl]amino}pentanedioate (24). Synthesized
from 11 according to the General procedure for coupling with TBTU.
Beige amorphous powder, yield: 532 mg (87%); m.p. 127e130 ꢀC 1H
(KBr):
n
¼ 3393, 3279, 3213, 2989, 2940, 2880, 2679, 1711, 1657,
1624, 1577, 1540, 1531, 1464, 1418, 1368, 1356, 1315, 1289, 1259,
1229, 1178, 1156, 1130, 1076, 1026, 973, 954, 844, 771, 753, 730, 693,
668, 637, 610, 594, 534, 500 cmꢃ1. HRMS Calcd for C21H25N4O7S m/
z: 477.1444 (M ꢃ H)ꢃ, found 477.1447.
NMR (CDCl3):
2.20e2.31 (2m, 1H each, CH2-
d
¼ 1.36 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 2.01e2.14 and
b
-Glu), 2.35e2.56 (m, 4H, CH2- -Glu
g
and CH2CONH), 3.53e3.64 (m, 1H, CH-Glu), 3.83e3.93 (m, 1H, CH-
Ala), 4.57e4.71 (m, 2H, NHCH2CH2), 4.92 (5.07) (AB-system, 2H,
J ¼ 12.3 Hz, CH2-benzyl), 5.09 (s, 2H, CH2-benzyl), 6.79 (d, 1H,
J ¼ 1.2 Hz, AreH), 6.97 (t,1H, J ¼ 7.5 Hz, AreH), 7.13e7.42 (m,15H,13
AreH and NH-Ala and NH-Glu), 7.53 (t, 1H, J ¼ 6.6 Hz, CONHCH2),
9.85 (s,1H, indole-NH). MS (ESI): m/z (%) ¼ 613.2 (M þ H)þ. IR (KBr):
5.1.1.17. Dimethyl (2R)-2-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-5l6
-
pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}propanoyl)
amino]pentanedioate (46). Synthesized from 43 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 320 mg (65%); m.p. 170e172 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.26 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.85e2.08 (m, 4H, CH2-
b-Glu
n
¼ 3289, 1727, 1646, 1534, 1419, 1364, 1312, 1264, 1187, 812, 776,
and NCH2CH2CH2), 2.37e2.47 (m, 4H, NCH2CH2CH2 and CH2-g-
750, 697 cmꢃ1
.
HRMS Calcd for C34H37N4O7 m/z: 613.2662
Glu), 3.46e3.50 (m, 2H, NCH2CH2), 3.59 (s, 3H, CH3O), 3.64 (s, 3H,
CH3O), 4.27e4.35 (m, 1H, CH-Glu), 4.43e4.53 (m, 1H, CH-Ala),
7.62e7.72 (m, 2H, AreH), 7.82e7.85 (m, 1H, AreH), 7.97e8.00 (m,
1H, AreH), 8.46 (d, 1H, J ¼ 7.8 Hz, NH-Glu), 8.70 (d, 1H, J ¼ 7.5 Hz,
(M þ H)þ, found 613.2667.
5.1.1.14. Dibenzyl (2R)-2-{[(2S)-2-({2-[(1H-indol-2-ylcarbonyl)amino]
acetyl}amino)propanoyl]amino}pentanedioate (25). Synthesized from
11 according to the General procedure for coupling with TBTU. Orange
amorphous powder, yield: 437 mg (73%); m.p. 96e99 ꢀC 1H NMR
NH-Ala). MS (ESI): m/z (%) ¼ 492.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3305,
1754, 1727, 1658, 1629, 1529, 1442, 1305, 1262, 1209, 1181, 1062,
1020 cmꢃ1. HRMS Calcd for C22H26N3O8S m/z: 492.1441 (M ꢃ H)ꢃ,
found 492.1455.
(DMSO-d6):
d
¼ 1.23 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.83e2.13 (m, 2H,
CH2-
b-Glu), 2.42 (t, 2H, J ¼ 7.35 Hz, CH2-
g-Glu), 3.94 (d, 2H,
J ¼ 5.7 Hz, NHCH2CO), 4.32e4.50 (m, 1H, CH-Glu and CH-Ala),
5.04e5.17 (m, 4H, 2ꢂCH2-benzyl), 7.01e7.21 (m, 2H, 2 AreH),
7.28e7.35 (m, 10H, AreH), 7.44 (d, 1H, J ¼ 8.1 Hz, AreH), 7.62 (d, 1H,
J ¼ 8.1 Hz, AreH), 8.13 (d, 1H, J ¼ 8.1 Hz, NH-Ala), 8.35 (d,
1H, J ¼ 7.5 Hz, NH-Glu), 8.73 (t, 1H, J ¼ 7.5 Hz, NHCH2), 11.54 (s, 1H,
5.1.1.18. Dimethyl (2R)-2-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-3H-5l6
-
pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}propanoyl)
amino]pentanedioate (47). Synthesized from 43 according to the
General procedure for coupling with TBTU. Orange amorphous
powder, yield: 340 mg (71%); m.p. 142e146 ꢀC 1H NMR (DMSO-d6):
indole-NH). MS (ESI): m/z (%) ¼ 599.3 (M þ H)þ. IR (KBr):
n
¼ 3274,
d
¼ 1.32 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.79e1.89 and 1.92e2.08 (2m,
3061, 2936, 1734, 1638, 1553, 1455, 1419, 1379, 1312, 1264, 1165,
1026, 961, 822, 794, 745, 697, 579 cmꢃ1. HRMS Calcd for C33H35N4O7
m/z: 599.2506 (M þ H)þ, found 599.2494.
1H each, CH2- -Glu), 3.34e3.41
b
-Glu), 2.36 (t, 2H, J ¼ 7.5 Hz, CH2-
g
(m, 2H, NCH2CH2), 3.55 (s, 3H, CH3O), 3.63 (s, 3H, CH3O), 3.71e3.79
(m, 2H, NCH2CH2), 4.27e4.34 (m, 1H, CH-Glu), 4.39e4.50 (m, 1H,
CH-Ala), 7.75e7.94 (m, 3H, AreH), 8.02e8.05 (m, 1H, AreH), 8.32
(d, 1H, J ¼ 8.1 Hz, NH-Ala), 8.92 (d, 1H, J ¼ 6.9 Hz, NH-Glu). MS (ESI):
5.1.1.15. Ethyl (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-
5
l
6-pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}propanoyl)
m/z (%) ¼ 478.1 (M ꢃ H)ꢃ. IR (KBr):
¼ 3322, 2990, 2949, 1748,1661,
n
amino]-5-oxopentanoate (44). Synthesized from 42 according to
the General procedure for coupling with TBTU. White amorphous
powder, yield: 320 mg (65%); m.p. 163e166 ꢀC 1H NMR (DMSO-d6):
1626, 1563, 1524, 1468, 1438, 1414, 1310, 1230, 1163, 1130, 1077, 990,
915, 843, 820, 756, 730, 593 cmꢃ1. HRMS Calcd for C21H24N3O8S m/
z: 478.1284 (M ꢃ H)ꢃ, found 478.1281.
d
¼ 1.18 (t, 3H, J ¼ 7.05 Hz, CH3CH2), 1.26 (d, 3H, J ¼ 7.2 Hz, CH3-Ala),
1.71e1.84 (m, 1H, H-CH2- -iGln), 1.98e2.11 (m, 3H, H-CH2- -iGln
and NCH2CH2CH2), 2.32 (t, 2H, J ¼ 7.65 Hz, NCH2CH2CH2), 2.45
b
b
5.1.1.19. Ethyl
3-[3-((1S)-1-{[5-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)pentanoyl]amino}ethyl)-1,2,4-oxadiazol-5-yl]
propanoate (54). Synthesized from 53 according to the General
procedure for coupling with TBTU. White amorphous powder, yield:
(t, 2H, J ¼ 6.0 Hz, CH2- -iGln), 3.48 (t, 2H, J ¼ 4.8 Hz, NCH2CH2), 4.02
g
(q, 2H, J ¼ 7.2 Hz, CH3CH2), 4.19e4.26 (m, 1H, CH-iGln), 4.33e4.42
(m, 1H, CH-Ala), 7.16 (s, 1H, CONH2), 7.33 (s, 1H, CONH2), 7.66e7.70
397 mg (83%); m.p. 82e86 ꢀC 1H NMR (CDCl3):
d
¼ 1.26 (t, 3H,

ꢀ
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Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
J ¼ 7.05 Hz, CH3CH2), 1.51 (d, 3H, J ¼ 6.9 Hz, CH3CH), 1.73e1.83 (m,
2H, CH2CH2CH2), 1.87e1.96 (m, 2H, CH2CH2CH2), 2.31 (t, 2H,
J ¼ 7.05 Hz, CH2CH2CH2N), 2.85 (t, 2H, J ¼ 7.05 Hz, CH2COOEt), 3.17
(t, 2H, J ¼ 7.20 Hz, Ox-CH2), 3.81 (t, 2H, J ¼ 7.05 Hz, CH2N), 4.17 (q,
2H, J ¼ 7.2 Hz, CH3CH2), 5.26e5.36 (m, 1H, CH), 6.09 (d, 1H,
J ¼ 8.1 Hz, NH), 7.80e7.94 (m, 3H, AreH), 8.04e8.07 (m, 1H, AreH).
1449, 1420, 1379, 1312, 1266, 1199, 1136, 1072, 1044, 1018, 884, 858,
832, 780, 751, 678, 610, 592, 547, 525 cmꢃ1. HRMS Calcd for
C21H26N5O5 m/z: 428.1934 (M þ H)þ, found 428.1940.
5.1.1.24. Benzyl (4S)-5-amino-5-oxo-4-[((2S)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoate (70). Synthesized from 67 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 418 mg (73%); m.p. 172e176 ꢀC 1H NMR (DMSO-d6):
MS (ESI): m/z (%) ¼ 479.2 (M þ H)þ. IR (KBr):
¼ 3282, 2986, 1734,
n
1639, 1578, 1542, 1458, 1338, 1262, 1186, 992, 878, 785, 751, 676,
590, 551, 510 cmꢃ1. HRMS Calcd for C21H26N4O7SNa m/z: 501.1420
(M þ Na)þ, found 501.1424.
d
¼ 1.18 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.51e1.61 (m, 2H, CH2CH2CH2),
1.67e1.85 (m, 3H, CH2CH2CH2 and H-CH2- -iGln), 1.85e1.93 (m, 1H,
H-CH2- -iGln), 2.35 (t, 2H,
-iGln), 2.17 (t, 2H, J ¼ 6.6 Hz, CH2-
b
5.1.1.20. Ethyl 3-[3-((1S)-1-{[4-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)butanoyl]amino}ethyl)-1,2,4-oxadiazol-5-yl]
propanoate (55). Synthesized from 53 according to the General
procedure for coupling with TBTU. Colorless oil, yield: 362 mg (78%);
b
g
J ¼ 7.2 Hz, CH2CO), 3.70 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.15e4.25 (m, 2H,
CH-iGln and CH-Ala), 5.07 (s, 2H, CH2-benzyl), 7.07 (s, 1H, NH2-
iGln), 7.28e7.37 (m, 6H, phenyl and NH2-iGln), 7.86 (d, 1H,
J ¼ 8.4 Hz, NH-Ala), 7.97e8.07 (m, 3H, AreH and NH-iGln),
8.29e8.31 (m, 1H, AreH). MS (ESI): m/z (%) ¼ 573.2 (M þ H)þ. IR
1H NMR (CDCl3):
d
¼ 1.25 (t, 3H, J ¼ 7.05 Hz, CH3CH2), 1.54 (d, 3H,
J ¼ 7.2 Hz, CH3CH), 2.16e2.26 (m, 2H, CH2CH2CH2N), 2.36 (t, 2H,
J ¼ 6.6 Hz, CH2CH2CH2N), 2.85 (t, 2H, J ¼ 7.35 Hz, CH2COOEt), 3.19 (t,
2H, J ¼ 7.35 Hz, Ox-CH2), 3.80e3.97 (m, 2H, CH2N), 4.16 (q, 2H,
J ¼ 7.2 Hz, CH3CH2), 5.28e5.38 (m, 1H, CH), 6.40 (d, 1H, J ¼ 8.4 Hz,
NH), 7.81e7.95 (m, 3H, AreH), 8.05e8.10 (m, 1H, AreH). MS (ESI):
(KBr):
n
¼ 3433, 1612, 1322 cmꢃ1. HRMS Calcd for C27H33N4O8S m/z:
573.2019 (M þ H)þ, found 573.2023.
5.1.1.25. Benzyl (4S)-5-amino-5-oxo-4-[((2R)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoate (71). Synthesized from 68 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 441 mg (77%); m.p. 171e174 ꢀC 1H NMR (DMSO-d6):
m/z (%) ¼ 465.1 (M þ H)þ. IR (NaCl):
n
¼ 3379,1735,1334,1182 cmꢃ1
.
HRMS Calcd for C20H24N4O7SNa m/z: 487.1263 (M þ Na)þ, found
487.1252.
5.1.1.21. Ethyl 3-{3-[(1S)-1-({2-[2-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)ethoxy]acetyl}amino)ethyl]-1,2,4-oxadiazol-5-yl}
propanoate (56). Synthesized from 53 according to the General
procedure for coupling with TBTU. Colorless oil, yield: 380 mg
d
¼ 1.17 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.50e1.61 (m, 2H, CH2CH2CH2),
1.67e1.83 (m, 3H, CH2CH2CH2 and H-CH2- -iGln), 1.85e1.93 (m, 1H,
H-CH2- -iGln), 2.35 (t, 2H,
b
b
-iGln), 2.17 (t, 2H, J ¼ 6.6 Hz, CH2-
g
J ¼ 7.8 Hz, CH2CO), 3.70 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.12e4.24 (m, 2H,
CH-iGln and CH-Ala), 5.07 (s, 2H, CH2-benzyl), 7.10 (s, 1H, NH2-
iGln), 7.28e7.37 (m, 6H, phenyl and NH2-iGln), 7.97e8.15 (m, 5H,
AreH and NH-Ala and NH-iGln), 8.29e8.31 (m,1H, AreH). MS (ESI):
(79%); 1H NMR (CDCl3):
d
¼ 1.26 (t, 3H, J ¼ 7.05 Hz, CH3CH2), 1.51
(d, 3H, J ¼ 6.9 Hz, CH3), 2.82 (t, 2H, J ¼ 7.35 Hz, CH2COOEt), 3.13
(t, 2H, J ¼ 7.35 Hz, Ox-CH2), 3.82e3.86 (m, 2H, CH2N), 3.91e4.05
(m, 4H, CH2CH2O and OCH2CO), 4.17 (q, 2H, J ¼ 7.2 Hz, CH3CH2),
5.22e5.32 (m, 1H, CH), 7.50 (d, 1H, J ¼ 8.4 Hz, NH), 7.83e7.97
(m, 4H, 3 AreH), 8.02e8.08 (m, 1H, AreH). MS (ESI): m/z
m/z (%) ¼ 573.2 (M þ H)þ. IR (KBr):
n
¼ 3433, 1612, 1322 cmꢃ1
.
HRMS Calcd for C27H33N4O8S m/z: 573.2019 (M þ H)þ, found
573.2023.
(%) ¼ 481.1 (M þ H)þ. IR (KBr):
n
¼ 3368, 1734, 1684, 1540, 1337,
1262, 1184, 612 cmꢃ1. HRMS Calcd for C20H24N4O8SNa m/z:
5.1.1.26. Benzyl (4R)-5-amino-5-oxo-4-[((2R)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoate (72). Synthesized from 69 according to the
General procedure for coupling with TBTU. White amorphous
powder, yield: 384 mg (67%); m.p. 169e172 ꢀC 1H NMR (DMSO-d6):
503.1213 (M þ Na)þ, found 503.1212.
5.1.1.22. Ethyl 3-[3-((1S)-1-{[(5,5-dioxo-5,7,8,9-tetrahydro-5l
6-pyr-
ido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}ethyl)-1,2,4-
oxadiazol-5-yl]propanoate (57). Synthesized from 53 according to
the General procedure for coupling with TBTU. Green oil, yield:
d
¼ 1.17 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.50e1.61 (m, 2H, CH2CH2CH2),
1.66e1.85 (m, 3H, CH2CH2CH2 and H-CH2- -iGln), 1.85e1.93 (m, 1H,
H-CH2- -iGln), 2.35 (t, 2H,
-iGln), 2.17 (t, 2H, J ¼ 6.75 Hz, CH2-
b
382 mg (83%); 1H NMR (CDCl3):
d
¼ 1.25 (t, 3H, J ¼ 7.2 Hz, CH3CH2),
b
g
1.60 (d, 3H, J ¼ 6.9 Hz, CH3), 2.04e2.15 (m, 2H, CH2CH2N),
2.48e2.66 (m, 2H, CH2CH2CH2N), 2.84 (t, 2H, J ¼ 7.35 Hz,
CH2COOEt), 3.18 (t, 2H, J ¼ 7.05 Hz, Ox-CH2), 3.54e3.65 (m, 2H,
CH2N), 4.15 (q, 2H, J ¼ 7.2 Hz, CH3CH2), 5.41e5.51 (m, 1H, CH), 6.38
(d, 1H, J ¼ 8.1 Hz, NH), 7.51e7.67 (m, 2H, AreH), 7.80e7.95 (m, 2H,
J ¼ 7.95 Hz, CH2CO), 3.70 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.15e4.27 (m,
2H, CH-iGln and CH-Ala), 5.07 (s, 2H, CH2-benzyl), 7.07 (s, 1H, NH2-
iGln), 7.27e7.39 (m, 6H, phenyl and NH2-iGln), 7.85 (d, 1H,
J ¼ 8.1 Hz, NH-Ala), 7.97e8.12 (m, 4H, AreH and NH-iGln),
8.29e8.31 (m, 1H, AreH). MS (ESI): m/z (%) ¼ 573.2 (M þ H)þ. IR
AreH). MS (ESI): m/z (%) ¼ 483.1 (M þ Na)þ. IR (NaCl):
n
¼ 3374,
(KBr):
n
¼ 3433, 1612, 1322 cmꢃ1. HRMS Calcd for C27H33N4O8S m/z:
1734, 1655, 1534, 1468, 1305, 1176, 1061, 766 cmꢃ1. HRMS Calcd for
573.2019 (M þ H)þ, found 573.2023.
C21H24N4O6SNa m/z: 483.1314 (M þ Na)þ, found 483.1295.
5.1.2. General procedure for hydrogenolytic cleavage of benzyl
protecting groups
Compounds were dissolved in glacial acetic acid and hydroge-
nated for 2 h over 10% Pd/C at room temperature and normal
pressure. The catalyst was removed by filtration, and the filtrate
evaporated in vacuo to give deprotected acid.
5.1.1.23. Ethyl 3-{-[(1S)-1-({3-[(1H-indol-2-ylcarbonyl)amino]prop-
anoyl}amino)ethyl]-1,2,4-oxadiazol-5-yl}propanoate
(58). Synthesized from 53 according to the General procedure for
coupling with TBTU. Red oil, yield: 320 mg (75%); 1H NMR (CDCl3):
d
¼ 1.24 (t, 3H, J ¼ 7.2 Hz, CH3CH2), 1.51 (d, 3H, J ¼ 6.9 Hz, CH3), 2.57
(t, 2H, J ¼ 5.7 Hz, CH2CONH), 2.79 (t, 2H, J ¼ 7.2 Hz, CH2COOEt), 3.11
(t, 2H, J ¼ 7.2 Hz, Ox-CH2), 3.66e3.90 (m, 2H, CH2N), 4.14 (q, 2H,
J ¼ 7.2 Hz, CH3CH2), 5.26e5.37 (m, 1H, CH), 6.51 (d, 1H, J ¼ 8.1 Hz,
NH), 7.09 (d, 1H, J ¼ 0.9 Hz, AreH), 7.14 (dt, 1H, J ¼ 7.2 Hz, J ¼ 1.2 Hz,
AreH), 7.26e7.41 (m, 2H, AreH), 7.63 (d, 1H, J ¼ 7.8 Hz, NH), 8.01 (s,
1H, AreH), 9.45 (s, 1H, indole-NH). MS (ESI): m/z (%) ¼ 428.2
5.1.2.1. (4R)-5-amino-5-oxo-4-[((2S)-2-{[5-(1,1,3-trioxo-1,3-dihydro-
2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)amino]pen-
tanoic acid (26). Synthesized from 12 according to the General
procedure for hydrogenolytic cleavage of benzyl protecting groups.
White amorphous powder, yield: 185 mg (91%); m.p.194e198 ꢀC 1H
(M þ H)þ. IR (KBr):
n
¼ 3361, 3268, 2986, 2939, 1709, 1639, 1557,
NMR (DMSO-d6):
d
¼ 1.19 (d, 3H, J ¼ 6.9 Hz, CH3-Ala), 1.52e1.63 (m,

ꢀ
Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3773
2H, CH2CH2CH2), 1.66e1.78 (m, 3H, CH2CH2CH2 and H-CH2-
b
-iGln),
according to the General procedure for hydrogenolytic cleavage of
benzyl protecting groups. White amorphous powder, yield: 106 mg
1.91e2.03 (m, 1H, H-CH2- -iGln), 2.12e2.31 (m, 4H, CH2- -iGln and
b
g
CH2CO) 3.71 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.06e4.27 (m, 2H, CH-iGln
and CH-Ala), 7.06 (s, 1H, NH2-iGln), 7.26 (s, 1H, NH2-iGln),
7.97e8.12 (m, 5H, 4 AreH and NH-Ala), 8.30 (d, 1H, J ¼ 7.5 Hz, NH-
iGln), 12.01 (s, 1H, COOH). MS (ESI): m/z (%) ¼ 483.2 (M þ H)þ. IR
(91%); m.p. 124e128 ꢀC 1H NMR (D2O):
d
¼ 1.12e1.27 (m, 3H, CH3-
Ala), 1.81e1.93 and 2.05e2.16 (2m, 1H each, CH2- -iGln), 2.26e2.51
(m, 4H, CH2- -iGln and NeCH2eCH2), 3.40e3.47 (m, 1H,
b
g
HeCH2eN), 3.49e3.56 (m, 1H, NeCH2eCH2eCH), 3.75e3.90 (m,
3H, HeCH2eN and CH-Ala), 4.04e4.08 and 4.21e4.26 (m, 1H, CH-
iGln), 5.33 and 5.37 (d, 1H, J ¼ 7.2 Hz, CHeN), 7.41e7.80 (m, 4H,
(KBr):
n
¼ 3410, 1636, 1541, 1457, 1340, 1261, 1183, 1061, 993, 789,
752, 676, 589, 526 cmꢃ1. HPLC: 100%, tr ¼ 10.97 min. Anal. Calcd for
C20H26N4O8S ꢂ H2O: C, 47,99; H, 5,64; N, 11,19. Found: C, 48,24; H,
AreH). MS (ESI): m/z (%) ¼ 453.2 (M þ H)þ. IR (KBr):
¼ 3369, 1663,
n
20
5,50; N, 10,93. [
a]
¼ ꢃ13.6ꢀ (c 0.2, MeOH).
1540, 1399, 1287, 1168, 1135, 1067, 1014, 763, 588 cmꢃ1. HRMS Calcd
D
for C19H24N4O7SNa m/z: 475.1263 (M þ Na)þ, found 475.1250. Anal.
5.1.2.2. (4R)-5-amino-5-oxo-4-[((2S)-2-{[4-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)butanoyl]amino}propanoyl)
amino]pentanoic acid (27). Synthesized from 13 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. Green amorphous powder, yield: 232 mg (87%); m.p.
Calcd for C19H24N4O7S ꢂ AcOH ꢂ H2O: C, 47,21; H, 5,88; N, 10,80.
20
Found: C, 47,54; H, 5,70; N, 10,56. [
a
]
D
¼ ꢃ17.7ꢀ (c 0.2, MeOH).
5.1.2.6. (4R)-5-amino-4-{[(2S)-2-({3-[(1H-indol-2-ylcarbonyl)amino]
propanoyl}amino)propanoyl]amino}-5-oxopentanoic acid (31). Syn-
thesized from 17 according to the General procedure for hydro-
genolytic cleavage of benzyl protecting groups. Green amorphous
powder, yield: 75 mg (83%); m.p. 186e189 ꢀC 1H NMR (D2O):
122e126 ꢀC 1H NMR (D2O):
1.87e1.99 (m, 1H, H-CH2-
and CH2CH2CH2), 2.39e2.44 (m, 4H, CH2-
d
¼ 1.33 (d, 3H, J ¼ 7.2 Hz, CH3-Ala),
-iGln), 2.06e2.20 (m, 3H, H-CH2- -iGln
-iGln and CH2CO) 3.80 (t,
b
b
g
2H, J ¼ 6.9 Hz, NCH2), 4.22 (q, 1H, J ¼ 7.2 Hz, CH-Ala), 4.29 (q, 1H,
d
¼ 1.27 (d, 3H, J ¼ 6.6 Hz, CH3-Ala), 1.66e1.80 and 1.88e2.08 (2m,
J ¼ 4.8 Hz, CH-iGln), 7.91e8.09 (m, 4H, AreH). MS (ESI): m/z
1H each, CH2- -iGln), 2.28e2.57 (m, 4H, CH2- -iGln and
b
g
(%) ¼ 469.1 (M þ H)þ. IR (KBr):
n
¼ 3291, 2931, 1720, 1631, 1543,
NHCH2CH2CO), 3.56e3.66 (m, 2H, NHCH2CH2CO), 4.13e4.32 (m,
2H, CH-Ala and CH-iGln), 7.03 (s, 1H, AreH), 7.14 (t, 1H, J ¼ 6.3 Hz,
AreH), 7.30 (t, 1H, J ¼ 6.3 Hz, AreH), 7.49 (d, 1H, J ¼ 7.5 Hz, AreH),
7.68 (d, 1H, J ¼ 7.8 Hz, AreH). MS (ESI): m/z (%) ¼ 432.2 (M þ H)þ. IR
1451, 1327, 1260, 1181, 1127, 1060, 987, 788, 752, 676 cmꢃ1. HPLC:
95%, tr ¼ 10.00 min. Anal. Calcd for C19H24N4O8S ꢂ AcOH ꢂ ½ H2O:
C, 47,07; H, 6,06; N, 10,72. Found: C, 46,92; H, 5,44; N, 10,42.
20
[
a]
¼ ꢃ1.7ꢀ (c 0.2, MeOH).
(KBr):
n
¼ 3284, 2981, 1654, 1624,1558, 1541, 1420, 1386, 1342, 1312,
D
1261, 934, 809, 769, 746 cmꢃ1. HPLC: 97%, tr ¼ 10.63 min. Anal.
5.1.2.3. (4R)-5-amino-5-oxo-4-{[(2S)-2-({2-[2-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)ethoxy]acetyl}amino)propanoyl]
amino}pentanoic acid (28). Synthesized from 14 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. Beige amorphous powder, yield: 137 mg (93%); m.p.
Calcd for C20H25N5O6 ꢂ 2/3 AcOH ꢂ 2/3H2O: C, 53,11; H, 5,97; N,
20
14,77. Found: C, 53,00; H, 6,05; N, 14,49. [
MeOH).
a
]
D
¼ þ11.4ꢀ (c 0.2,
5.1.2.7. (4R)-5-amino-4-{[(2S)-2-({2-[(1H-indol-2-ylcarbonyl)amino]
acetyl}amino)propanoyl]amino}-5-oxopentanoic acid (32). Syn-
thesized from 18 according to the General procedure for hydro-
genolytic cleavage of benzyl protecting groups. Green amorphous
powder, yield: 65 mg (79%); m.p. 155e158 ꢀC 1H NMR (D2O):
203e205 ꢀC 1H NMR (D2O):
1.63e1.76 and 1.89e2.00 (2m, 1H each, CH2-
d
¼ 1.16 (d, 3H, J ¼ 7.2 Hz, CH3-Ala),
b
-iGln), 2.22 (t, 2H,
J ¼ 7.35 Hz, CH2- -iGln), 3.75 (t, 2H, J ¼ 4.8 Hz, NCH2CH2O),
g
3.89e3.92 (m, 2H, NCH2), 3.95 (s, 2H, OCH2CO), 4.05 (q, 1H,
J ¼ 4.5 Hz, CH-iGln), 4.16 (q, 1H, J ¼ 7.2 Hz, CH-Ala), 7.81e8.02 (m,
d
¼ 1.38 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.90e1.99 and 2.08e2.19 (2m,
4H, AreH). MS (ESI): m/z (%) ¼ 485.1 (M þ H)þ. IR (KBr):
n
¼ 3457,
1H each, CH2- -iGln), 4.10 (s, 2H,
b
-iGln), 2.39 (t, 2H, J ¼ 7.5 Hz, CH2-
g
3376, 3307, 2927, 1719, 1676, 1647, 1552, 1447, 1333, 1299, 1269,
1192, 1131, 1067, 954, 788, 754, 679, 606, 590, 531, 508 cmꢃ1. HPLC:
100%, tr ¼ 10.41 min. Anal. Calcd for C19H24N4O9S ꢂ 1/3H2O: C,
NHCH2CO), 4.25e4.36 (m, 2H, CH-iGln and CH-Ala), 7.15e7.20 (m,
2H, AreH), 7.34 (t, 1H, J ¼ 7.65 Hz, AreH), 7.53 (d, 1H, J ¼ 8.4 Hz,
AreH), 7.73 (d, 1H, J ¼ 8.1 Hz, AreH). MS (ESI): m/z (%) ¼ 418.2
46,68; H, 5,01; N, 11,36. Found: C, 46,53; H, 5,07; N, 11,42.
(M þ H)þ. IR (KBr):
n
¼ 3299, 1654, 1542, 1419, 1341, 1312, 1248,
20
[
a]
¼ þ8.2ꢀ (c 0.25, MeOH).
1135, 1020, 937, 818, 750, 658 cmꢃ1. HPLC: 95%, tr ¼ 10.38 min. Anal.
D
Calcd for C19H23N5O6 ꢂ AcOH ꢂ H2O: C, 50,60; H, 5,98; N, 14,50.
20
5.1.2.4. (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-5,7,8,9,10,10a-hexahy-
dro-5
6-pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}
Found: C, 50,90; H, 5,90; N, 14,13. [
a
]
D
¼ ꢃ16.9ꢀ (c 0.2, MeOH).
l
propanoyl)amino]-5-oxopentanoic acid (29). Synthesized from 15
according to the General procedure for hydrogenolytic cleavage of
benzyl protecting groups. Gray amorphous powder, yield: 87 mg
5.1.2.8. (2R)-2-[((2S)-2-{[5-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)pentanoyl]amino}propanoyl)amino]pentanedioic
acid (33). Synthesizedfrom 19 accordingtotheGeneral procedurefor
hydrogenolytic cleavage of benzyl protecting groups. Beige amorphous
(86%); m.p. 135e138 ꢀC 1H NMR (D2O):
d
¼ 1.18 (d, 3H, J ¼ 7.2 Hz,
-iGln and NeCH2eCH2eCH2),
-iGln and NeCH2eCH2), 2.40 (t, 2H,
-iGln), 3.02e3.12 (m, 1H, H-CH2-N), 3.39e3.42 (m,
CH3-Ala), 1.71e1.91 (m, 3H, H-CH2-
2.02e2.23 (m, 3H, H-CH2-
J ¼ 7.5 Hz, CH2-
b
powder, yield: 160 mg (92%); m.p. 92e96 ꢀC 1H NMR (D2O):
d
¼ 1.34
(d, 3H, J ¼ 7.5 Hz, CH3-Ala),1.64e1.75 (m, 2H, CH2CH2CH2),1.77e1.83
(m, 2H, CH2CH2CH2), 1.86e1.96 and 2.09e2.17 (2m, 1H each, CH2-
Glu), 2.21e2.40 (m, 4H, CH2-
b
g
b-
1H, NeCHeCH), 3.78e3.82 (m, 1H, HeCH2eN), 3.96e4.05 (m, 1H,
CH-Ala), 4.07e4.14 and 4.16e4.21 (m, 1H, J ¼ 4.8 Hz, CH-iGln),
7.51e7.87 (m, 4H, AreH). * signal for NeCHeCH is hidden under
the signal of water. MS (ESI): m/z (%) ¼ 467.2 (M þ H)þ. IR (KBr):
g
-Glu and CH2CO), 3.78 (t, 2H, J ¼ 6.9 Hz,
NCH2), 4.26 (q,1H, J ¼ 7.2 Hz, CH-Ala), 4.36 (q,1H, J ¼ 4.2 Hz, CH-Glu),
7.89e8.07 (m, 4H, AreH). MS (ESI): m/z (%) ¼ 482.1 (M ꢃ H)ꢃ. IR
(KBr):
n
¼ 3384, 1734, 1653, 1540, 1457, 1337, 1260, 1182, 1060, 985,
n
¼ 3421, 2078, 1663, 1540, 1259, 1169, 1131, 1060, 744 cmꢃ1. HRMS
790, 753, 677, 587, 511 cmꢃ1. HPLC: 98%, tr ¼ 11.67 min. Anal. Calcd for
Calcd for C20H26N4O7SNa m/z: 489.1420 (M þ Na)þ, found 489.1407.
C20H25N3O9S ꢂ 6/5H2O: C, 47,56; H, 5,47; N, 8,32. Found: C, 47,93; H,
20
Anal. Calcd for C20H26N4O7S ꢂ 3/2AcOH ꢂ H2O: C, 48,08; H, 5,96; N,
5,84; N, 8,30. [
a]
D
¼ ꢃ16.7ꢀ (c 0.2, MeOH).
20
9,75. Found: C, 48,01; H, 6,12; N, 9,75. [
a
]
¼ ꢃ30.8ꢀ (c 0.2, MeOH).
D
5.1.2.9. (2R)-2-[((2S)-2-{[4-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)butanoyl]amino}propanoyl)amino]pentanedioic
acid (34). Synthesized from 20 according to the General procedure
for hydrogenolytic cleavage of benzyl protecting groups. White
5.1.2.5. (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-2,3,5,9b-tetrahydro-
1H-5
6-pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}
propanoyl)amino]-5-oxopentanoic acid (30). Synthesized from 16
l

ꢀ
3774
Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
amorphous powder, yield: 134 mg (90%); m.p. 112e114 ꢀC 1H NMR
(D2O):
¼ 1.33 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.92e2.04 (m, 1H, H-
CH2- -Glu), 2.05e2.24 (m, 3H, H-CH2- -Glu and CH2CH2CH2),
2.39e2.47 (m, 4H, CH2-
yield: 67 mg (87%); m.p. 220e224 ꢀC 1H NMR (D2O):
J ¼ 7.2 Hz, CH3-Ala), 1.67e1.77 and 1.91e2.02 (2m, 1H each, CH2-
d
¼ 1.17 (d, 3H,
d
b-
b
b
Glu), 2.18 (t, 2H, J ¼ 7.2 Hz, CH2- -Glu), 2.46 (t, 2H, J ¼ 6.6 Hz,
g
g
-Glu and CH2CO) 3.80 (t, 2H, J ¼ 6.75 Hz,
NHCH2CH2CO), 3.51 (t, 2H, J ¼ 6.9 Hz, NHCH2CH2CO), 4.15e4.20 (m,
2H, CH-Ala and CH-Glu), 6.97 (d, 1H, J ¼ 0.9 Hz), 7.00e7.05 (m, 1H,
AreH), 7.16e7.21 (m, 1H, AreH), 7.37e7.40 (m, 1H, AreH), 7.57 (d,
1H, J ¼ 7.8 Hz, AreH). MS (ESI): m/z (%) ¼ 431.2 (M ꢃ H)ꢃ. IR (KBr):
NCH2), 4.22 (q, 1H, J ¼ 7.2 Hz, CH-Ala), 4.29 (q, 1H, J ¼ 4.8 Hz, CH-
Glu), 7.89e8.08 (m, 4H, AreH). MS (ESI): m/z (%) ¼ 468.1
(M ꢃ H)ꢃ. IR (KBr):
n
¼ 3469, 2923, 1642, 1182 cmꢃ1. HPLC: 100%,
tr ¼ 10.75 min. Anal. Calcd for C19H23N3O9S ꢂ ½ AcOH ꢂ H2O: C,
n
¼ 3317, 3061, 2939, 1744, 1654, 1613, 1560, 1540, 1430, 1388, 1342,
46,56; H, 5,36; N, 8,08. Found: C, 46,56; H, 5,77; N, 9,16.
1312, 1266, 1158, 1049, 968, 931, 806, 765, 739, 636, 543 cmꢃ1
.
20
[
a]
¼ ꢃ4.3ꢀ (c 0.2, MeOH).
HPLC: 96%, tr ¼ 11.27 min. Anal. Calcd for C20H24N4O7 ꢂ H2O: C,
D
53,33; H, 5,82; N, 12,44. Found: C, 53,20; H, 5,69; N, 12,30.
20
5.1.2.10. (2R)-2-{[(2S)-2-({2-[2-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)ethoxy]acetyl}amino)propanoyl]amino}pentane-
dioic acid (35). Synthesized from 21 according to the General
procedure for hydrogenolytic cleavage of benzyl protecting groups.
White amorphous powder, yield: 154 mg (87%); m.p. 128e130 ꢀC
[a
]
¼ þ5.2ꢀ (c 0.2, MeOH).
D
5.1.2.14. (2R)-2-{[(2S)-2-({2-[(1H-indol-2-ylcarbonyl)amino]acetyl}
amino)propanoyl]amino}pentanedioic acid (39). Synthesized from
25 according to the General procedure for hydrogenolytic cleavage of
benzyl protecting groups. Yellow-green amorphous powder, yield:
1H NMR (D2O):
2.05e2.19 (2m, 1H each, CH2-
d
¼ 1.29 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.75e1.88 and
b
-Glu), 2.35 (t, 2H, J ¼ 7.2 Hz, CH2-
g-
87 mg (82%); m.p. 175e177 ꢀC 1H NMR (D2O):
d
¼ 1.24 (d, 3H,
Glu), 3.87e3.93 (m, 2H, NCH2CH2O), 4.02e4.06 (m, 2H, NCH2), 4.11
(s, 2H, OCH2CO), 4.25 (q, 1H, J ¼ 4.8 Hz, CH-Glu), 4.33 (q, 1H,
J ¼ 7.2 Hz, CH-Ala), 7.92e8.11 (m, 4H, AreH). MS (ESI): m/z
J ¼ 7.2 Hz, CH3-Ala), 1.78e1.88 and 1.98e2.08 (2m, 1H each, CH2-
b-
Glu), 2.26 (t, 2H, J ¼ 7.2 Hz, CH2- -Glu), 3.94 (d, 2H, J ¼ 2.7 Hz,
g
NHCH2CO), 4.17e4.34 (m, 2H, CH-Glu and CH-Ala), 7.02e7.07 (m,
2H, AreH), 7.18e7.24 (m, 1H, AreH), 7.39e7.42 (m, 1H, AreH), 7.60
(d, 1H, J ¼ 8.1 Hz, AreH). MS (ESI): m/z (%) ¼ 417,2 (M ꢃ H)ꢃ. IR
(%) ¼ 484.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3374, 3333, 1726, 1663, 1636,
1597, 1561, 1540, 1458, 1418, 1388, 1332, 1320, 1299, 1267, 1191, 1169,
1132, 1062, 1032, 1012, 953, 899, 788, 752, 742, 680, 656, 605, 590,
532, 503 cmꢃ1. HPLC: 97%, tr ¼ 11.05 min. Anal. Calcd for
(KBr):
n
¼ 3379, 1653, 1559, 1419, 1317, 1256, 820, 748, 654 cmꢃ1
.
HPLC: 96%, tr ¼ 11.12 min. Anal. Calcd for C19H22N4O7 ꢂ 1/3
C19H23N3O10S: C, 45,17; H, 5,05; N, 8,10. Found: C, 45,18; H, 4,94; N,
AcOH ꢂ 8/3H2O: C, 48,56; H, 5,94; N, 11,52. Found: C, 48,33; H, 5,57;
20
20
8,90. [
a
]
¼ þ14.9ꢀ (c 0.2, MeOH).
N, 11,51. [
a
]
D
¼ ꢃ9.4ꢀ (c 0.2, MeOH).
D
5.1.2.11. (2R)-2-[((2S)-2-{[(5,5-dioxo-5,7,8,9,10,10a-hexahydro-5l6
-
5.1.2.15. (4S)-5-amino-5-oxo-4-[((2S)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoic acid (73). Synthesized from 70 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. White amorphous powder, yield: 165 mg (87%); m.p.
pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}propanoyl)
aminopentanedioic acid (36). Synthesized from 22 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. Beige amorphous powder, yield: 138 mg (90%); m.p.
118e122 ꢀC 1H NMR (D2O):
1.77e1.94 (m, 3H, H-CH2-
(m, 3H, H-CH2-
-Glu and NeCH2eCH2), 2.36 (t, 2H, J ¼ 7.5 Hz, CH2-
-Glu), 3.03e3.12 (m, 1H, HeCH2eN), 3.38e3.45 (m, 1H,
d
¼ 1.12e1.17 (m, 3H, CH3-Ala),
195e198 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.19 (d, 3H, J ¼ 7.2 Hz, CH3-
Ala), 1.53e1.62 (m, 2H, CH2CH2CH2), 1.68e1.77 (m, 3H, CH2CH2CH2
and H-CH2- -iGln), 1.85e1.96 (m, 1H, H-CH2- -iGln), 2.15e2.21 (m,
4H, CH2-
b
-Glu and NeCH2eCH2eCH2), 2.02e2.25
b
b
b
g
g
-iGln and CH2CO), 3.71 (t, 2H, J ¼ 7.05 Hz, NCH2),
NeCHeCH), 3.78e3.84 (m, 1H, HeCH2eN), 3.99e4.06 and
4.08e4.15 (m, 1H, CH-Ala), 4.20e4.28 (m, 1H, CH-Glu), 7.51e7.87
(m, 4H, AreH). * signal for NeCHeCH is hidden under the signal of
4.09e4.26 (m, 2H, CH-iGln and CH-Ala), 7.04 (s, 1H, NH2-iGln), 7.28
(s, 1H, NH2-iGln), 7.97e8.13 (m, 5H, 4 AreH and NH-Ala), 8.30 (d,
1H, J ¼ 6.9 Hz, NH-iGln), 12.01 (s, 1H, COOH). MS (ESI): m/z
water. MS (ESI): m/z (%) ¼ 466.2 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3350, 2370,
(%) ¼ 483.2 (M þ H)þ. IR (KBr):
n
¼ 3410, 1636, 1541, 1457, 1340,
2052, 1688, 1540, 1388, 1257, 1167, 1132, 1061, 765, 744 cmꢃ1. HRMS
1261, 1183, 1061, 993, 789, 752, 676, 589, 526 cmꢃ1. HPLC: 99%,
20
Calcd for C20H25N3O8SNa m/z: 490.1260 (M þ Na)þ, found 490.1255.
tr ¼ 10.93 min [
a]
¼ ꢃ24.5ꢀ (c 0.2, MeOH).
D
20
[
a]
¼ ꢃ25.3ꢀ (c 0.2, MeOH).
D
5.1.2.16. (4S)-5-amino-5-oxo-4-[((2R)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoic acid (74). Synthesized from 71 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. White amorphous powder, yield: 187 mg (89%); m.p.
5.1.2.12. (2R)-2-[((2S)-2-{[(5,5-dioxo-2,3,5,9b-tetrahydro-1H-5l6
-
pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}propanoyl)
amino]pentanedioic acid (37). Synthesized from 23 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting
groups. Yellow amorphous powder, yield: 59 mg (86%); m.p.
195e198 ꢀC 1H NMR (DMSO-d6):
d
¼ 1.18 (d, 3H, J ¼ 7.2 Hz, CH3-Ala),
1.52e1.61 (m, 2H, CH2CH2CH2), 1.67e1.77 (m, 3H, CH2CH2CH2 and
H-CH2- -iGln), 1.81e1.94 (m, 1H, H-CH2- -iGln), 2.09 (t, 2H,
J ¼ 7.2 Hz, CH2- -iGln
102e105 ꢀC 1H NMR (D2O):
d
¼ 1.15 and 1.22 and 1.37 and 1.44* (d,
3H, J ¼ 7.2 Hz, CH3-Ala),1.87e1.97 and 2.11e2.20 (2m,1H each, CH2-
-Glu), 2.27e2.51 (m, 4H, CH2- -Glu and NeCH2eCH2), 3.39e3.56
b
b
b
g
g-iGln or CH2CO), 2.17 (t, 2H, J ¼ 6.75 Hz, CH2-
g
(m, 2H, HBeCH2eN and CHeCHeCH2), 3.79e3.92 (m, 2H, CH-Ala
and HAeCH2eN), 4.30e4.35 and 4.40e4.45 (m, 1H, CH-Glu), 5.33
and 5.37 (d,1H, J ¼ 7.2 Hz, CHeCHeCH2), 7.48e7.80 (m, 4H, AreH). *
mixture of diastereomers. MS (ESI): m/z (%) ¼ 454.1 (M þ H)þ. IR
or CH2CO), 3.71 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.01e4.08 (m,1H, CH-iGln
or CH-Ala), 4.14e4.23 (m, 1H, CH-iGln or CH-Ala), 7.00 (s, 1H, NH2-
iGln), 7.32 (s, 1H, NH2-iGln), 7.97e8.16 (m, 4H, 4 AreH and NH-Ala),
8.31 (d,1H, J ¼ 7.8 Hz, NH-iGln), 8.61 (d,1H, J ¼ 8.1 Hz, NH-Ala),12.02
(KBr):
n
¼ 3368, 2362, 1718, 1654, 1540, 1456, 1400, 1288, 1168, 1135,
(s,1H, COOH). MS (ESI): m/z (%) ¼ 483.2 (M þ H)þ. IR (KBr):
¼ 3410,
n
1067,1013, 760, 590, 536 cmꢃ1. HRMS Calcd for C19H23N3O8SNa m/z:
1636, 1541, 1457, 1340, 1261, 1183, 1061, 993, 789, 752, 676, 589,
476.1104 (M þ Na)þ, found 476.1093. [
a
]
¼ ꢃ32.2ꢀ (c 0.25, MeOH).
526 cmꢃ1. HPLC: 96%, tr ¼ 10.97 min [
a]
D
¼ þ13.6ꢀ (c 0.2, MeOH).
20
20
D
5.1.2.13. (2R)-2-{[(2S)-2-({3-[(1H-indol-2-ylcarbonyl)amino]prop-
anoyl}amino)propanoyl]amino}pentanedioic acid (38). Synthesized
from 24 according to the General procedure for hydrogenolytic
cleavage of benzyl protecting groups. Beige amorphous powder,
5.1.2.17. (4R)-5-amino-5-oxo-4-[((2R)-2-{[5-(1,1,3-trioxo-1,3-
dihydro-2H-1,2-benzisothiazol-2-yl)pentanoyl]amino}propanoyl)
amino]pentanoic acid (75). Synthesized from 72 according to the
General procedure for hydrogenolytic cleavage of benzyl protecting

ꢀ
Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3775
groups. White amorphous powder, yield: 148 mg (89%); m.p.
196e198 ꢀC 1H NMR (DMSO-d6):
¼ 1.19 (d, 3H, J ¼ 7.2 Hz, CH3-
Ala), 1.53e1.62 (m, 2H, CH2CH2CH2), 1.68e1.85 (m, 3H, CH2CH2CH2
and H-CH2- -iGln), 1.81e1.94 (m, 1H, H-CH2- -iGln), 2.09 (t, 2H,
J ¼ 7.2 Hz, CH2- -iGln
(m, 1H, CH-Ala), 7.63e7.70 (m, 2H, AreH), 7.82e7.84 (m, 1H, AreH),
7.97e8.04 (m, 1H, AreH), 8.31 (d, 1H, J ¼ 7.8 Hz, NH-Glu), 8.71 (d,
1H, J ¼ 8.1 Hz, NH-Ala), 12.40 (br s, 2H, 2 COOH). MS (ESI): m/z
d
b
b
(%) ¼ 464.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3788, 33777, 1734, 1653, 1540,
g
-iGln or CH2CO), 2.20 (t, 2H, J ¼ 7.2 Hz, CH2-
g
1472,1458,1305,1176,1135,1061,1016, 978, 891, 821, 764, 576 cmꢃ1
.
or CH2CO), 3.71 (t, 2H, J ¼ 7.05 Hz, NCH2), 4.01e4.08 (m,1H, CH-iGln
or CH-Ala), 4.13e4.21 (m, 1H, CH-iGln or CH-Ala), 6.97 (s, 1H, NH2-
iGln), 7.25 (s, 1H, NH2-iGln), 7.97e8.15 (m, 4H, 4 AreH and NH-Ala),
8.29e8.32 (m, 1H, AreH), 8.56 (d, 1H, J ¼ 7.2 Hz, NH-Ala), 12.02 (s,
HPLC: 98%, tr ¼ 10.24 min. HRMS Calcd for C20H22N3O8S m/z:
20
464.1128 (M ꢃ H)ꢃ, found 464.1132. [
a
]
¼ þ6.7ꢀ (c 0.15, MeOH).
D
5.1.3.4. (2R)-2-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-3H-5
[1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}propanoyl)amino]
l
6-pyrrolo
1H, COOH). MS (ESI): m/z (%) ¼ 483.2 (M þ H)þ. IR (KBr):
¼ 3410,
n
1636, 1541, 1457, 1340, 1261, 1183, 1061, 993, 789, 752, 676, 589,
pentanedioic acid (51). Synthesized from 47 according to the
General procedure for alkaline ester hydrolysis. Yellow amorphous
solid, yield: 72 mg (63%); m.p. 124e126 ꢀC; 1H NMR (DMSO-d6):
20
526 cmꢃ1. HPLC: 99%, tr ¼ 10.93 min [
a
]
D
¼ þ 25.8ꢀ (c 0.2, MeOH).
5.1.3. General procedure for alkaline hydrolysis of alkyl esters
To a solution of the corresponding ester in ethanol/water (1:1)
mixture (6 ml), LiOH (3 mmol) was added and the reaction mixture
stirred at room temperature for 3 h. Ethanol was evaporated under
vacuum and the resulting aqueous solution acidified with 1M HCl
to pH 3. The water phase was then extracted with ethyl acetate
(3 ꢂ 10 ml) which was subsequently washed with water (10 ml),
brine (10 ml) and dried over Na2SO4 to afford crude products. The
products were purified, if necessary, by column chromatography
using chloroform/methanol/acetic acid (6:1:0.1) as eluant.
d
¼ 1.32 (d, 3H, J ¼ 7.2 Hz, CH3-Ala), 1.73e1.86 and 1.94e2.06 (2m,
1H each, CH2- -Glu), 3.36 (t, 2H,
b
-Glu), 2.26 (t, 2H, J ¼ 7.5 Hz, CH2-
g
J ¼ 9.3 Hz, NCH2CH2), 3.73 (t, 2H, J ¼ 8.7 Hz, NCH2CH2), 4.21e4.29
(m, 1H, CH-Glu), 4.43e4.53 (m, 1H, CH-Ala), 7.75e7.88 (m, 3H,
2AreH and NH-Ala), 8.03 (d, 1H, J ¼ 8.1 Hz, AreH), 8.19 (d, 1H,
J ¼ 6.9 Hz, NH-Glu), 8.91 (d, 1H, J ¼ 8.1 Hz, AreH), 12.40 (br s, 2H, 2
COOH). MS (ESI): m/z (%) ¼ 452.1 (M þ H)þ. IR (KBr):
n
¼ 3374, 1718,
1654, 1522, 1458, 1308, 1177, 763, 595 cmꢃ1
.
HPLC: 95%,
20
tr ¼ 11.79 min [
a
]
¼ ꢃ28.8ꢀ (c 0.25, MeOH).
D
5.1.3.5. 3-[3-((1S)-1-{[(5,5-dioxo-5,7,8,9-tetrahydro-5
l
6-pyrido [1,2-
5.1.3.1. (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-
b] [1,2]benzisothiazol-10-yl)carbonyl]amino}ethyl)-1,2,4-oxadiazol-
5-yl]propanoic acid (62). Synthesized from 57 according to the
General procedure for alkaline ester hydrolysis. Orange oil, yield:
5l
6-pyrido [1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}prop-
anoyl)amino]-5-oxopentanoic acid (48). Synthesized from 44
according to the General procedure for alkaline ester hydrolysis.
79 mg (63%); 1H NMR (DMSO-d6):
d
¼ 1.46 (d, 3H, J ¼ 7.2 Hz, CH3),
Green oil, yield: 48 mg (47%); 1H NMR (DMSO-d6):
d
¼ 1.26 (d, 3H,
1.96e2.03 (m, 2H, CH2CH2N), 2.46 (t, 2H, J ¼ 6.45 Hz, CH2CH2CH2N),
2.76 (t, 2H, J ¼ 6.75 Hz, CH2COOH), 3.14 (t, 2H, J ¼ 6.9 Hz, Ox-CH2),
3.45e3.52 (m, 2H, CH2N), 5.15e5.25 (m, 1H, CH), 7.67e7.72 (m, 2H,
AreH), 7.82e7.85 (m, 1H, AreH), 7.99e8.02 (m, 1H, AreH), 9.11 (d,
1H, J ¼ 7.8 Hz, NH), 12.40 (br s, 1H, COOH). MS (ESI): m/z (%) ¼ 431.1
J ¼ 7.2 Hz, CH3-Ala), 1.66e2.14 (m, 4H, CH2-
b-iGln and NCH2CH2),
3.46e3.49 (m, 2H, NCH2CH2), 3.90e3.93 (m, 1H, CH-iGln),
4.37e4.49 (m, 1H, CH-Ala), 6.65 (s, 1H, NH2-iGln), 7.36 (s, 1H,
NH2-iGln), 7.63e7.90 (m, 4H, AreH), 7.98 (d, 1H, J ¼ 7.8 Hz, NH-
iGln), 8.91 (d, 1H, J ¼ 7.2 Hz, NH-Ala). * signals for NCH2CH2 and
(M ꢃ H)ꢃ. IR (KBr):
n
¼ 3336, 2938, 1734, 1636, 1579, 1541, 1471,
CH2-
g-iGln are hidden under broad peak of DMSO at 2.5 ppm and
1451, 1362, 1306, 1266, 1218, 1176, 1137, 1103, 1061, 1017, 978, 891,
the signal for COOH under peak of water at 3.3 ppm. MS (ESI): m/z
864, 822, 765, 746, 635, 600, 578, 513 cmꢃ1
. HPLC: 96%,
20
(%) ¼ 463.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3412, 2068, 1648,1177, 497 cmꢃ1
.
tr ¼ 13.58 min; [
a
]
D
¼ ꢃ19.0ꢀ (c 0.2, MeOH).
HPLC: 99%, tr ¼ 9.04 min. HRMS Calcd for C20H23N4O7S m/z:
20
463.1104 (M ꢃ H)ꢃ, found 463.1093. [
a
]
D
¼ þ0.9ꢀ (c 0.25, MeOH).
5.1.3.6. 3-{3-[(1S)-1-({3-[(1H-indol-2-ylcarbonyl)amino]propanoyl}
amino)ethyl]-1,2,4-oxadiazol-5-yl}propanoic acid (63). Synthesized
from 58 according to the General procedure for alkaline ester
hydrolysis. Orange oil, yield: 60 mg (72%); 1H NMR (DMSO-d6):
5.1.3.2. (4R)-5-amino-4-[((2S)-2-{[(5,5-dioxo-2,5-dihydro-3H-5l6
pyrrolo [1,2-b] [1,2]benzisothiazol-1-yl)carbonyl]amino}propanoyl)
amino]-5-oxopentanoic acid (49). Synthesized from 45 according to
the General procedure for alkaline ester hydrolysis. Green oil, yield:
-
d
¼ 1.38 (d, 3H, J ¼ 6.9 Hz, CH3), 2.41e2.46 (m, 2H, CH2CONH), 2.73
(t, 2H, J ¼ 6.9 Hz, CH2COOH), 3.08 (t, 2H, J ¼ 6.9 Hz, Ox-CH2),
3.45e3.52 (m, 2H, CH2N), 5.02e5.11 (m, 1H, CH), 7.00e7.19 (m, 3H,
AreH), 7.41e7.43 (m, 1H, AreH), 7.60 (d, 1H, J ¼ 7.8 Hz, NH), 8.45 (t,
1H, J ¼ 5.4 Hz, NH), 8.54 (d, 1H, J ¼ 8.1 Hz, indole-NH), 12.36 (br s,
45 mg (41%); 1H NMR (DMSO-d6):
Ala), 1.68e2.13 (m, 2H, CH2-
d
¼ 1.31 (d, 3H, J ¼ 6.9 Hz, CH3-
b
-iGln), 2.48 (t, 2H, J ¼ 7.5 Hz, CH2-
g-
iGln), 3.74 (t, 2H, J ¼ 9.0 Hz, NCH2CH2), 3.81e3.87 (m, 1H, CH-iGln),
4.41e4.51 (m, 1H, CH-Ala), 6.61 (s, 1H, NH2-iGln), 7.33 (s, 1H, NH2-
iGln), 7.57e7.59 (m, 1H, AreH), 7.74e7.86 (m, 2H, 2AreH),
8.01e8.06 (m, 2H, NH-iGln and AreH), 8.91 (d, 1H, J ¼ 7.5 Hz, NH-
Ala). * signals for CH2Cq and COOH are hidden under peak of water
1H, COOH). MS (ESI): m/z (%) ¼ 398.2 (M ꢃ H)ꢃ. IR (KBr):
¼ 3348,
n
2106, 1647, 1559, 1341, 1310, 1260, 815, 749 cmꢃ1. HPLC: 95%,
20
tr ¼ 13.21 min; [
a]
¼ ꢃ7.7ꢀ (c 0.2, MeOH).
D
at 3.3 ppm. MS (ESI): m/z (%) ¼ 449.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3412,
5.1.4. General procedure for acid hydrolysis of alkyl esters
2078, 1653 cmꢃ1. HPLC: 96%, tr ¼ 10.68 min. HRMS Calcd for
A solution of ester in 3M HCl (5 ml) was stirred at 80 ꢀC for
30 min. Water was added (5 ml) and the solution extracted with
ethyl acetate (3 ꢂ 10 ml) which was subsequently washed with
brine (10 ml) and dried over Na2SO4 to afford crude products. The
products were purified, if necessary, by column chromatography
using hexane/ethyl acetate/acetic acid (1:3:0.1) as eluant.
C19H21N4O7S m/z: 449.1118 (M
ꢃ
H)ꢃ, found 449.1122.
20
[
a]
¼ ꢃ9.5ꢀ (c 0.25, MeOH).
D
5.1.3.3. (2R)-2-[((2S)-2-{[(5,5-dioxo-5,7,8,9-tetrahydro-5l
6-pyrido
[1,2-b] [1,2]benzisothiazol-10-yl)carbonyl]amino}propanoyl)amino]
pentanedioic acid (50). Synthesized from 46 according to the
General procedure for alkaline ester hydrolysis. Beige amorphous
powder, yield: 86 mg (57%); m.p. 128e132 ꢀC; 1H NMR (DMSO-d6):
5.1.4.1. 3-[3-((1S)-1-{[5-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)pentanoyl]amino}ethyl)-1,2,4-oxadiazol-5-yl]
propanoic acid (59). Synthesized from 53 according to the General
procedure for acid ester hydrolysis. Colorless oil, yield: 64 mg (53%);
d
¼ 1.26 (d, 3H, J ¼ 6.9 Hz, CH3-Ala),1.75e1.89 (m,1H, H-CH2-
1.93e2.09 (m, 3H, H-CH2- -Glu and NCH2CH2), 2.29 (t, 2H,
J ¼ 7.2 Hz, CH2- -Glu), 2.45 (t, 2H, J ¼ 6.0 Hz, NCH2CH2CH2),
3.46e3.50 (m, 2H, NCH2CH2), 4.22e4.29 (m,1H, CH-Glu), 4.46e4.56
b-Glu),
b
g
1H NMR (DMSO-d6):
2H, CH2CH2CONH), 1.63e1.78 (m, 2H, CH2CH2N), 2.16 (t, 2H,
d
¼ 1.37 (d, 3H, J ¼ 7.2 Hz, CH3), 1.53e1.60 (m,

ꢀ
3776
Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
J ¼ 6.9 Hz, CH2CONH), 2.67 (t, 2H, J ¼ 7.2 Hz, CH2COOH), 3.06 (t, 2H,
J ¼ 6.9 Hz, Ox-CH2), 3.71 (t, 2H, J ¼ 7.05 Hz, CH2N), 4.97e5.07 (m,
1H, CH), 7.97e8.12 (m, 3H, AreH), 8.28e8.31 (m, 1H, AreH), 8.41 (d,
1H, J ¼ 8.1 Hz, NH), 12.16 (br s, 1H, COOH). MS (ESI): m/z (%) ¼ 449.1
and PMA 3.33 ng/ml final concentration in the cell culture medium.
0.5 ꢂ 106 cells (final culture volume 0.75 ml) were plated in 24-well
culture plates (Nunc, Denmark) with each of desmuramyldipeptide
compounds separately, a combination of a desmuramyldipeptide
compound, 500 nM ionomycin and 3.33 ng/ml PMA, a combination
of a desmuramyldipeptide compound with 10 ng/ml LPS, or
medium alone, at 37 ꢀC in a humidified atmosphere of 5% CO2 in air.
The cell-free supernatants were collected at 18 h and stored
at ꢃ78 ꢀC before being evaluated for cytokines by the commercially
available kit.
(M ꢃ H)ꢃ. IR (KBr):
n
¼ 3401, 2432, 2302, 1728, 1658, 1582, 1458,
HPLC: 96%, tr 14.16 min;
1334, 1260, 1183, 752 cmꢃ1
.
¼
20
[
a]
¼ ꢃ32.8ꢀ (c 0.2, MeOH).
D
5.1.4.2. 3-[3-((1S)-1-{[4-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)butanoyl]amino}ethyl)-1,2,4-oxadiazol-5-yl]
propanoic acid (60). Synthesized from 55 according to the General
procedure for acid ester hydrolysis. Colorless oil, yield: 47 mg (49%);
1H NMR (DMSO-d6):
d
¼ 1.38 (d, 3H, J ¼ 6.9 Hz, CH3), 1.90e2.00 (m,
5.3. Cytokine assays
2H, CH2CH2CH2N), 2.22e2.28 (m, 2H, CH2CH2CH2N), 2.69 (t, 2H,
J ¼ 7.2 Hz, CH2COOH), 3.07 (t, 2H, J ¼ 7.05 Hz, Ox-CH2), 3.71e3.77
(m, 2H, CH2N), 4.98e5.08 (m, 1H, CH), 7.95e8.12 (m, 3H, AreH),
8.29e8.31 (m, 1H, AreH), 8.45 (d, 1H, J ¼ 7.8 Hz, NH), 12.17 (br s, 1H,
5.3.1. Cytokine assay employing ELISA
TNF-
a release from THP-1 cells was measured in cell-free
supernatants obtained by centrifugation at 3000 rpm for 5 min
and stored at ꢃ78 ꢀC until measurement. TNF-
a production was
COOH). MS (ESI): m/z (%) ¼ 435.1 (M ꢃ H)ꢃ. IR (KBr):
¼ 3436, 2090,
n
1730, 1650, 1582, 1458, 1333, 1261, 1181, 1061, 752, 677, 513 cmꢃ1
.
assessed by specific sandwich ELISA (Immunotools, Friesoythe,
Germany). Results are expressed in pg/ml. Limit of detection
15.6 pg/ml.
20
HPLC: 94%, tr ¼ 13.38 min; [
a]
¼ ꢃ26.2ꢀ (c 0.2, MeOH).
D
5.1.4.3. 3-{3-[(1S)-1-({2-[2-(1,1,3-trioxo-1,3-dihydro-2H-1,2-
benzisothiazol-2-yl)ethoxy]acetyl}amino)ethyl]-1,2,4-oxadiazol-5-yl}
propanoic acid (61). Synthesized from 56 according to the General
procedure for acid ester hydrolysis. Colorless oil, yield: 89 mg (57%);
5.3.2. Cytokine assay employing flow cytometry
Cytokine release from PBMCs was measured in cell-free super-
natants obtained by centrifugation at 1200 rpm for 5 min and
stored at ꢃ78 ꢀC until measurement. Flow cytometric analyses
were performed using a FACSCalibur flow cytometer with Sorting
Option: 4-Color and CELLQuest software (BD Biosciences; San
Diego, CA, USA). Cytokine production was assessed by BD Cyto-
metric Bead Array (CBA) Human Inflammatory Cytokine Kit
1H NMR (DMSO-d6):
d
¼ 1.38 (d, 3H, J ¼ 6.9 Hz, CH3), 2.68 (t, 2H,
J ¼ 6.9 Hz, CH2COOH), 3.05 (t, 2H, J ¼ 7.05 Hz, Ox-CH2), 3.78
(t, 2H,J ¼ 5.4 Hz, CH2N), 3.95 (t, 2H, J ¼ 5.4 Hz, CH2CH2O), 3.97 (s,
2H, OCH2CO), 5.00e5.11 (m, 1H, CH), 7.98e8.12 (m, 4H, 3 AreH and
NH), 8.30e8.32 (m, 1H, AreH), 12.17 (br s, 1H, COOH). MS (ESI): m/z
(%) ¼ 451.1 (M ꢃ H)ꢃ. IR (KBr):
n
¼ 3361, 2938, 1733, 1670, 1581,
(Content: IL-8, IL-1
b
, IL-6, IL-10, TNF-
a, IL-12p70). Results are
expressed in pg/ml.
1536, 1459, 1334, 1263, 1184, 1129, 1061, 1028, 950, 790, 753, 677,
20
586 cmꢃ1. HPLC: 95%, tr ¼ 13.79 min; [
a
]
D
¼ ꢃ3.9ꢀ (c 0.2, MeOH).
Cytokine
IL-8
Standard Curve Range [pg/ml]
Limit of detection [pg/ml]
5.2. Cell culture procedures
20e5000
20e5000
20e5000
20e5000
20e5000
3.6
7.2
2.5
3.3
3.7
IL-1
IL-6
b
5.2.1. Cell culture procedure for THP-1 cells
For all experiments, THP-1 cells (Istituto Zooprofilattico di
IL-10
TNF-
Brescia, Brescia, Italy) were diluted to 106 cells/ml in RPMI 1640
a
containing 2 mM
penicillin, 50 M 2-mercaptoethanol, supplemented with 10% heat-
inactivated fetal calf serum (media) and cultured at 37 ꢀC in 5% CO2.
To evaluate TNF- production, cultures were set up in 24-well
culture plates containing 500 L of cells and treated with selected
desmuramyldipeptides, MDP and MB at a final concentration of
20.2 M and lipopolysaccharide (LPS, from E. coli serotype 0127:B8,
Sigma) at a final concentration of 1 ng/ml. DMSO was the vehicle
control (0.2% final concentration was not exceeded). Cells were
incubated for different times with increasing concentrations of the
selected chemicals or with DMSO as vehicle control as indicated in
the figure legends.
L-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml
m
5.4. Statistical analysis
a
m
All experiments were performed at least twice, with average
results shown. The data are expressed as means ꢁ SD. Statistical
analyses were performed using GraphPad InStat (GraphPad Soft-
ware, San Diego, California, USA). Statistical significance was
determined by ManneWhitney U-rank or Dunnet multiple
comparison test as reported in the figure/table legends. Differences
were considered significant for P < 0.05, very significant for
P < 0.01 and extremely significant for P < 0.001.
m
5.2.2. Cell culture procedure for PBMC cells
Acknowledgments
Human PBMC from the buffy coat of healthy blood donors were
isolated by density gradient centrifugation with FicollePaque
(Pharmacia, Sweden). The cells were cultured in RPMI 1640 (Sigma,
This work was supported financially by the Slovenian Research
Agency (Grant No. P1-0208). The authors thank Matevz Prijatelj for
providing the PBMC cells and for his help with evaluation of the
cytokine production by flow cytometry. The authors also thank
Roger Pain for critically reading the manuscript.
ꢀ
Germany) supplemented with 100 U/ml penicillin (Sigma), 100
mg/
ml streptomycin (Sigma), 2 mM -glutamine (Sigma), 50 M 2-
L
m
mercaptoethanol (Sigma) and 10% heat-inactivated fetal calf
serum (Gibco, USA). The desmuramyldipeptide compounds, MDP
and MB, required for in vitro stimulation of human PBMC in molar
concentration equivalent to 10
mg/ml MDP, i.e. 20.2
mM concen-
Appendix. Supplementary material
tration, were dissolved in DMSO and further diluted in RPMI 1640
supplemented as described below, so that the final concentration of
DMSO did not exceed 0.1%. LPS was 10 ng/ml, ionomycin 500 nM
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.05.042

ꢀ
Z. Jakopin et al. / European Journal of Medicinal Chemistry 46 (2011) 3762e3777
3777
LK-409, with immunomodulatory properties, Agents Actions 38 (1993)
References
273e280;
ꢀ
ꢀ
ꢀ ꢀ
ꢀ ꢀ
(b) D. Kikelj, S. Pecar, V. Kotnik, A. Stalc, B. Wraber-Herzog, S. Simcic, A. Ihan,
L. Klamfer, L. Povsic, R. Grahek, E. Suhadolc, M. Hocevar, H. Hönig, R. Rogi-
Kohlenprath, N-{trans-[[2’(acetylamino)cyclohexyl]oxy]acetyl}-L-alanyl-D-
[1] F. Ellouz, A. Adam, R. Ciorbaru, E. Lederer, Minimal requirements for adjuvant
activity of bacterial peptidoglycan derivatives, Biochem. Biophys. Res. Com-
mun. 59 (1974) 1317e1325.
[2] M.J. Pabst, S. Beranova-Giorgianni, J.M. Krueger, Effects of muramyl peptides
on macrophages, monokines, and sleep, Neuroimmunomodulation 6 (1999)
261e283.
[3] P.J. Murray, Beyond peptidoglycan for Nod2, Nat. Immunol. 10 (2009)
1053e1054.
[4] A. Todate, T. Suda, H. Kuwata, K. Chida, H. Nakamura, Muramyl dipeptide-Lys
stimulates the function of human dendritic cells, J. Leukoc. Biol. 70 (2001)
723e729.
[5] A.-L. Pauleau, P.J. Murray, Role of Nod2 in the response of macrophages to toll-
like receptor agonists, Mol. Cell. Biol. 23 (2003) 7531e7539.
[6] S. Traub, S. von Aulock, T. Hartung, C. Hermann, MDP and other muropeptides
e direct and synergistic effects on the immune system, J. Endotoxin Res. 12
(2006) 69e85.
[7] J.H. Fritz, S.E. Girardin, C. Fitting, C. Werts, D. Mengin-Lecreulx, M. Caroff, J.-
M. Cavaillon, D.J. Philpott, M. Adib-Conquy, Synergistic stimulation of human
monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-
activating molecules, Eur. J. Immunol. 35 (2005) 2459e2470.
[8] S. Traub, N. Kubasch, S. Morath, M. Kresse, T. Hartung, R.R. Schmidt,
C. Hermann, Structural requirements of synthetic muropeptides to synergize
with lipopolysaccharide in cytokine induction, J. Biol. Chem. 279 (2004)
8694e8700.
[9] M.-T. Labro, Interference of antibacterial agents with phagocyte functions:
immunomodulation or “immuno-fairy tales”? Clin. Microbiol. Rev. 13 (2000)
615e650.
[10] G. Merhi, A.W. Coleman, J.-P. Devissaguet, G.M. Barratt, Synthesis and
immunostimulating properties of lipophilic ester and ether muramyl peptide
derivatives, J. Med. Chem. 39 (1996) 4483e4488.
[11] K. Geddes, J.G. Magalhães, S.E. Girardin, Unleashing the therapeutic potential
of NOD-like receptors, Nat. Rev. Drug Disc. 8 (2009) 465e479.
[12] G.M. Bahr, Immune and antiviral effects of the synthetic immunomodulator
murabutide. in: C.J. Hackett, D.A. HarnJr. (Eds.), Vaccine Adjuvants: Immu-
nological and Clinical Principles. Humana Press, Totowa, New Jersey, 2006, pp.
193e219.
[13] D.H. Silverman, H. Wu, M.L. Karnovsky, Muramyl peptides and serotonin
interact at specific binding sites on macrophages and enhance superoxide
release, Biochem. Biophys. Res. Commun. 131 (1985) 1160e1167.
[14] B. Weidemann, J. Schletter, R. Dziarski, S. Kusumoto, F. Stelter, E.T. Rietschel,
H.-D. Flad, A.J. Ulmer, Specific binding of soluble peptidoglycan and mur-
amyldipeptide to CD14 on human monocytes, Infect. Immun. 65 (1997)
858e864.
[15] S. Albright, B. Chen, K. Holbrook, N.U. Jain, Solution NMR studies provide
structural basis for endotoxin pattern recognition by the innate immune
receptor CD14, Biochem. Biophys. Res. Commun. 368 (2008) 231e237.
[16] J. Uehori, K. Fukase, T. Akazawa, S. Uematsu, S. Akira, K. Funami, M. Shingai,
M. Matsumoto, I. Azuma, K. Toyoshima, S. Kusumoto, T. Seya, Dendritic cell
maturation induced by muramy dipeptide (MDP) derivatives: monoacylated
MDP confers TLR2/TLR4 activation, J. Immunol. 174 (2005) 7096e7103.
[17] N. Inohara, Y. Ogura, A. Fontalba, O. Gutierrez, F. Pons, J. Crespo, K. Fukase,
S. Inamura, S. Kusumoto, M. Hashimoto, S.J. Foster, A.P. Moran, J.L. Fernandez-
Luna, G. Nuñez, Host recognition of bacterial muramyl dipeptide mediated
through NOD2, J. Biol. Chem. 278 (2003) 5509e5512.
[18] S.E. Girardin, I.G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas,
D.J. Philpott, P.J. Sansonetti, Nod2 is a general sensor of peptidoglycan through
muramyl dipeptide (MDP) detection, J. Biol. Chem. 278 (2003) 8869e8872.
[19] O. Gutierrez, C. Pipaon, N. Inohara, A. Fontalba, Y. Ogura, F. Prosper, G. Nuñez,
J.L. Fernandez-Luna, Induction of Nod2 in myelomonocytic and intestinal
ꢀ
glutamic acid: a novel immunologically active carbocyclic muramyl dipeptide
analogue, J. Med. Chem. 41 (1998) 530e539.
ꢀ ꢀ
ꢀ
[25] U. Urleb, A. Krbavcic, M. Sollner, D. Kikelj, S. Pecar, Synthesis of phthalimido-
desmuramylpeptide analogues as potential immunomodulating agents, Arch.
Pharm. (Weinheim) 328 (1995) 113e117.
ꢀ
[26] C. Ochi, N. Norisada, M. Moriguchi, A. Stalc, U. Urleb, S. Muraoka, Interleukin-
10 inducing activity of LK423,
a
phthalimido-desmuramyldipeptide
compound, Drug Res. 49 (1999) 72e79.
ꢀ
[27] M. Moriguchi, N. Norisada, C. Ochi, A. Stalc, U. Urleb, S. Muraoka, Therapeutic
effects of LK423, a phthalimido-desmuramyldipeptide compound, on dextran
sulfate sodium induced colitis in rodents through restoring their interleukin-
10 capacity, Drug Res. 49 (1999) 184e192.
ꢀ
[28]
Z
Jakopin, M.S. Dolenc, Advances in the Chemistry of saccharins: from
synthetic novelties towards biologically active compounds, Curr. Med. Chem.
17 (2010) 651e671.
ꢀ
ꢀ
[29] M. Sollner, S. Pecar, A. Stalc, The influence of the lipophilicity of 7-oxoacyl-L-
alanyl-D-isoglutamines on their immunorestoration activity in vivo, Eur. J.
Med. Chem. 31 (1996) 927e933.
ꢀ
[30]
Z
Jakopin, M.S. Dolenc, Microwave-assisted preparation of N-alkylated
saccharins and their reactions with potassium t-butoxide, Synth. Comm. 40
(2010) 2464e2474.
[31] P. Igel, E. Schneider, D. Schnell, S. Elz, R. Seifert, A. Buschauer, NG-acylated
imidazolylpropylguanidines as potent histamine H4 receptor agonists: selec-
tivity by variation of the NG-substitutent, J. Med. Chem. 52 (2009) 2623e2627.
[32] R. Kiss, B. Kiss, Á Könczöl, F. Szalai, I. Jelinek, V. László, B. Noszál, A. Falus,
}
G.M. Keseru, Discovery of novel human histamine H4 receptor ligands by large-
scale structure-based virtual screening, J. Med. Chem. 51 (2008) 3145e3153.
ꢀ
ꢀ
[33] Z Jakopin, R. Roskar, M.S. Dolenc, Synthesis of 3,5-disubstituted 1,2,4-oxadiazoles
as peptidomimetic building blocks, Tetrahedron Lett. 48 (2007) 1465e1468.
ꢀ
[34] Z Jakopin, M.S. Dolenc, Recent advances in the synthesis of 1,2,4- and 1,3,4-
oxadiazoles, Curr. Org. Chem 12 (2008) 850e898.
[35] S. Kusumoto, Y. Tarumi, K. Ikenaka, T. Shiba, Synthesis of N-acetylmuramyl
peptides with partial structures of bacterial cell wallandtheiranalogs in relation
to immunoadjuvant activities, Bull. Chem. Soc. Jpn. 49 (1976) 533e539.
[36] P. Lefrancier, J. Choay, M. Derrien, I. Lederman, Synthesis of N-acetyl-mur-
amyl-L-alanyl-D-isoglutamine, an adjuvant of the immune response and of
some N-acetyl-muramyl-peptide analogs, Int. J. Pept. Protein Res. 9 (1977)
249e257.
[37] K. Kamisongo, I. Saiki, T. Yoshiro, S. Kobayashi, T. Fukuda, I. Sekikawa,
I. Azuma, Y. Yamamura, Chemical synthesis and adjuvant activity of N-ace-
tylmuramyl-L-alanyl-D-isoglutamine (MDP) analogs, Chem. Pharm. Bull. 29
(1981) 1644e1654.
[38] P. Cottagnoud, C.M. Gerber, P.A. Majcherczyk, F. Acosta, M. Cottagnoud,
K. Neftel, P. Moreillon, M.G. Täuber, The stereochemistry of the amino acid
side chain influences the inflammatory potential of muramyl dipeptide in
experimental meningitis, Infect. Immun. 71 (2003) 3663e3666.
[39] K.M. Kengatharan, S. De Kimpe, C. Robson, S.J. Foster, C. Thiemermann,
Mechanism of Gram-positive shock: identification of peptidoglycan and lip-
oteichoic acid moieties essential in the induction of nitric oxide synthase,
shock, and multiple organ failure, J. Exp. Med. 188 (1998) 305e315.
[40] M.G. Ismair, S.R. Vavricka, G.A. Kullak-Ublick, M. Fried, D. Mengin-Lecreulx,
S.E. Girardin, hPepT1 selectively transports muramyl dipeptide but not Nod1-
activating muramyl peptides, Can. J. Physiol. Pharmacol. 84 (2006) 1313e1319.
[41] L. Charrier, D. Merlin, The oligopeptide transporter hPepT1: gateway to the
innate immune response, Lab. Invest. 86 (2006) 538e546.
ꢀ
ꢀ
ꢀ ꢀ
[42] P. Smrdel, I. Grabnar, I. Locatelli, M. Cerne, S. Andrensek, N. Kovacic, A. Kristl,
M. Bogataj, U. Urleb, A. Mrhar, Physicochemical and preclinical pharmacoki-
netic and toxicological evaluation of LK-423, a new phthalimido-desmuramyl-
dipeptide derivative with immunomodulating activity, Drug Dev. Ind. Pharm.
35 (2009) 1293e1304.
epithelial cells via nuclear factor-
kB activation, J. Biol. Chem. 277 (2002)
41701e41705.
[20] D.A. Van Heel, S. Ghash, M. Butler, K.A. Hunt, A.M.C. Lundberg, T. Ahmad,
D.P.B. McGovern, C. Onnie, K. Negoro, S. Goldthorpe, B.M.J. Foxwell,
C.G. Mathew, A. Forbes, D.P. Jewell, R.J. Playford, Muramyl dipeptide and toll-
like receptor sensitivity in NOD2-associated Crohn’s disease, Lancet 365
(2005) 1794e1796.
[43] A. Goodchild, N. Nopper, A. Craddock, T. Law, A. King, G. Fanning, L. Rivory,
T. Passioura, Primary leukocyte screens for innate immune agonists, J. Biomol.
Screen. 14 (2009) 723e730.
[44] M. Spitaler, D.A. Cantrell, Protein kinase C and beyond, Nat. Immunol. 5 (2004)
785e790.
[21] (a) K. Dzierzbicka, A.M. Ko1odziejczyk, Muramyl peptides e synthesis and
biological activity, Polish J. Chem. 77 (2003) 373e395;
ꢀ ꢀ
[45] S. Gobec, M. Sollner-Dolenc, U. Urleb, B. Wraber, S. Simcic, M. Filipi&ccaron,
(b) K. Dzierzbicka, A.M. Ko1odziejczyk, Synthesis and antitumor activity of
conjugates of muramyldipeptide or normuramyldipeptide with hydrox-
yacridine/acridone derivatives, J. Med. Chem. 46 (2003) 183e189;
Modulation of cytokine production by some phthalimido-desmuramyl
dipeptides and their cytotoxicity, Il Farmaco 59 (2004) 345e352.
[46] M.A. Wolfert, T.F. Murray, G.-J. Boons, J.N. Moore, The origin of the synergistic
effect of muramyl dipeptide with endotoxin and peptidoglycan, J. Biol. Chem.
277 (2002) 39179e39186.
[47] J.G. Cannon, R.G. Tompkins, J.A. Gelfand, H.R. Michie, G.G. Stanford, J.W. van
der Meer, S. Endres, G. Lonnemann, J. Corsetti, B. Chernow, Circulating
interleukin-1 and tumor necrosis factor in septic shock and experimental
endotoxin fever, J. Infect. Dis. 161 (1990) 79e84.
[48] J.H. Fritz, R.L. Ferrero, D.J. Philpott, S.E. Girardin, Nod-like proteins in immu-
nity, inflammation and disease, Nat. Immunol. 7 (2006) 1250e1257.
[49] G.J. Nau, J.F.L. Richmond, A. Schlesinger, E.G. Jennings, E.S. Lander, R.A. Young,
Human macrophage activation programs induced by bacterial pathogens,
Proc. Natl. Acad. Sci. USA 99 (2002) 1503e1508.
ꢁ
(c) K. Dzierzbicka, A.M. Ko1odziejczyk, B. Wysocka-Skrzela, A. Mysliwski,
D. Sosnowska, Synthesis and antitumor activity of conjugates of mur-
amyldipeptide, normuramyldipeptide, and desmuramylpeptides with acri-
dine/acridone derivatives, J. Med. Chem. 44 (2001) 3606e3615.
[22] C.D. Leclerc, F.M. Audibert, L.A. Chedid, E.J. Deriaud, N.K. Masihi, E. Lederer,
Comparison of immunomodulatory activities in mice and guinea pigs of
a synthetic desmuramylpeptidolipid triglymc, Infect. Immun. 43 (1984) 870e875.
[23] Z. Zidek, Immune-related edemagemic activity of glutamines and glutamic acid,
components of immunomodulatory agents, Agents Actions 42 (1994) 163e166.
ꢀ
ꢀ ꢀ
ꢀ ꢀ
[24] (a) M. Sollner, V. Kotnik, A. Stalc, S. Simcic, L. Povsic, B. Herzog-Wraber,
L. Klampfer, A. Ihan, P. Grosman, Apyrogenic synthetic desmuramyldipeptide,