Design, synthesis and immunological evaluation of novel amphiphilic desmuramyl peptides

Article abstract of DOI:10.1016/j.ejmech.2017.09.070

Muramyl dipeptide (MDP) – an essential bacterial cell wall component – is recognized by our immune system as pathogen-associated molecular pattern (PAMP) which results in immune responses with adverse toxic effects. In order to harness the beneficial prop

Full text of DOI:10.1016/j.ejmech.2017.09.070

European Journal of Medicinal Chemistry 141 (2017) 26e36
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and immunological evaluation of novel amphiphilic
desmuramyl peptides
,
Farooq-Ahmad Khan a b, Marina Ulanova b, Bing Bai c, Damayanthi Yalamati c,
,
*
Zi-Hua Jiang a
a Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada
b Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada
c Alberta Research Chemicals Inc., 11421 Saskatchewan Drive, Edmonton, Alberta, T6G 2M9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2017
Received in revised form
15 September 2017
Accepted 29 September 2017
Available online 30 September 2017
Muramyl dipeptide (MDP) e an essential bacterial cell wall component e is recognized by our immune
system as pathogen-associated molecular pattern (PAMP) which results in immune responses with
adverse toxic effects. In order to harness the beneficial properties from the pro-inflammatory charac-
teristics of the bacterial cell wall motif, MDP was strategically re-designed while conserving the L-D
configurations of the dipeptide moiety. The muramic acid was replaced with a hydrophilic arene and
lipophilic chain was introduced at peptide end to give the amphiphilic desmuramyl peptides (DMPs). The
novel DMPs were found to modulate the immune response by amplifying the LPS-induced surface
glycoprotein (ICAM-1) expression in THP-1 cells without showing significant toxicity. Furthermore, these
Keywords:
Amphiphilic desmuramyl peptides
Immunostimulants
THP-1 cells
compounds were able to trigger the secretion of higher levels of pro-inflammatory cytokine (TNF-a) than
the well-studied NOD2 agonist, Murabutide.
© 2017 Elsevier Masson SAS. All rights reserved.
Immunomodulation
TNF-
a
ICAM-1
1. Introduction
the production of proinflammatory cytokines such as interlukin-1
beta (IL-1b) and tumor necrosis factor-alpha (TNF-a) [1,3]. How-
Innate immune system is the first line of defense against
invading pathogens [1]. The system is comprised of different
pattern recognition receptors (PRRs) such as Toll-like receptor
(TLR), Nod-like receptor (NLR) and retinoic acid inducible gene
(RIG)-like receptor [2]. PRRs are capable of detecting the invading
microorganisms by recognizing the pathogen-associated molecular
patterns (PAMPs) and triggering a cascade of responses against
them. Common PAMPs usually encompass bacterial cell wall
components like peptidoglycan (PGN), lipopolysaccharides (LPS)
and bacterial DNA [3]. The innate immune response to PGN is
largely mediated by NLRs such as NOD2, which recognizes mur-
amyl dipeptide (MDP) [2]. MDP is the smallest bioactive fragment
of bacterial PGN consisting of one carbohydrate and two amino
acids (Fig. 1) [1]. This molecule is found both in Gram-positive and
Gram-negative bacteria [1]. When mammalian cells expressing
NOD2 are treated with MDP, an inflammatory response is activated
triggering the expression of adhesion molecules (e.g. ICAM-1) and
ever, MDP possesses serious drawbacks such as poor penetration
through cell membrane, and rapid elimination [3]. Furthermore, it
induces severe local reactions similar to some bacterial infections in
humans and is considered too toxic to be used in clinical applica-
tions [4]. Numerous structural modifications in MDP have been
done with the intention of improving the pharmacological prop-
erties and lowering the toxicological profile [2]. These efforts led to
the discovery of many useful hydrophilic derivatives such as mur-
abutide (MB), temurtide, nor-MDP, glucosaminyl-MDP and
paclitaxel-MDP [3,5e7]. Lipophilic derivatives of MDP containing
an additional amino acid residue at C-terminus including mafi-
murtide, romurtide and muramyl tripeptide phospatidylethanol-
amine also reached the clinical stage of development [3].
Furthermore, a variety of carbohydrate analogs of MDP containing
manno-, galacto-, xylo-, allo- and
synthesized [8]. -manno- and -galacto-type muramyl dipeptides
were found as active as the -gluco-type muramyl dipeptide on the
induction of type IV hypersensitivity in guinea pigs [9]. Some fur-
anoid MDP analogs containing -glucofuranose had better immu-
noadjuvant activity compared to the parent MDP molecule [10],
L-idomuramic acid have been
D
D
D
D
* Corresponding author.
E-mail address: zjiang@lakeheadu.ca (Z.-H. Jiang).
https://doi.org/10.1016/j.ejmech.2017.09.070
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
27
Fig. 1. Structural components of novel amphiphilic desmuramyl peptides (right).
whereas the 2-deoxy-
D
-arabinohexose analogue was completely
is lost, when
enantiomer [23]. Although
amino acid such as -serine or
D
-isoglutamine is replaced by the
L
-isoglutamine
inactive [11]. Synthetic PGN fragments containing di-, tetra- and
octasaccharides coupled to dipeptide moiety also had definite
immunostimulating activities similar to that of MDP but their tu-
mor necrosis factor-alpha (TNF-a) inducing potencies decreased as
the glycan chain became longer from di-, tetra-to octasaccharides
[12].
Carbohydrate moiety is apparently not essential for the pyro-
genic and immunoadjuvant activity of peptidoglycan fragments [8].
Desmuramyl peptides (DMPs), which are MDP derivatives lacking
the carbohydrate moiety, have also been reported. For example, O-
L-alanine can be replaced by a similar
L
L
-valine, -isoglutamine cannot be
D
replaced by another amino acid [24]. However, peptide chain can be
prolonged at carboxy-terminus [25]. It's therefore imperative to
conserve L-D configuration of the dipeptide in the design of DMPs.
Several studies have delineated that the intact muramyl moiety
containing N-acetylglucosamine and lactic acid is not essential for
biological activity [18,19]. However, previously reported DMPs lack
hydrophilic character e an important variable towards the activa-
tion of NLRs found in cytosol [2]. A further literature search
revealed that existing methods did not describe the preparation of
DMPs presenting both hydrophilic and lipophilic characters in the
same molecule. In our attempt to rationally modulate the hydro-
philicity in DMPs, aryl amine containing hydroxylated N-alkyl
groups was introduced (Fig. 1). The hydrophilic aromatic amines
(L-alanyl-D-isoglutamine-L-alanyl)-glycerol-3-mycolate was found
as active as MDP in stimulating mouse resistance to infection [13].
Several DMPs have shown significant immunomodulatory activity
and remarkable antitumor potency [2,14,15]. For example, Gang Liu
and co-workers replaced the muramic acid moiety in MDP with
hydrophobic arenes. The novel DMP e paclitaxel conjugates
combine chemotherapy and immunotherapy in the treatment of
cancer and had dual antitumor growth with metastasis activities
[14,15]. Sollner et al. synthesized 7-oxo-octanoyl group containing
DMP. The new molecule was found to be an apyrogenic immuno-
modulator and had tumor growth delay effect [16]. Moriguchi and
were then linked by glycolic acid linker to the N-terminus of
alanine- -isoglutamine dipeptide. The glycolic acid linker was
employed to replace the -lactic acid in MDP, thus further simpli-
L-
D
D
fying the structure by removing the chirality present in the lactic
acid moiety. Furthermore, an aliphatic chain was added at the
peptide end to facilitate their penetration through cell membrane.
DMPs possessing both hydrophilic and lipophilic properties
have not yet been explored. In this report, we describe the synthesis
of novel amphiphilic DMPs (1e2), in which the carbohydrate
moiety of MDP was replaced by hydrophilic arene e an entity
suitable in cytosol to target the intracellular NLRs for the activation
of innate immune system. Complementary lipophilicity was ach-
ieved by introducing a modest aliphatic chain to assist cell mem-
brane penetration. We also examined whether these novel
structures, alone or in combination, can modulate the expression of
cell surface glycoprotein viz. intercellular adhesion molecule-1
(ICAM-1) which plays an important role in immune and inflam-
co-workers prepared
a phthalimido-desmuramyldipeptide e
another immunomodulating agent capable of restoring the
interleukin-10 capacity in rodents [17]. Jakopin et al. synthesized
DMPs incorporating either a pyrido-fused [1,2]-benzisothiazole
moiety or an indole scaffold and studied their immunomodulatory
properties [2,3]. DMPs containing carbocyclic ring have also been
reported to be immunologically active [18]. Although a good
number of DMPs have been reported in the literature, they lack
hydrophilicity due to the elimination of muramic acid e the car-
bohydrate moiety of MDP. A balanced approach towards lip-
ophilicity and hydrophilicity is important for the exhibition of
interesting biological activity [19]. Murabutide (MB) is a prime
example of such approach in which MDP was strategically modified
by introducing a modest lipophilic chain at peptide end. The new
molecule proved to be a safe immunomodulator, capable of
enhancing the host's resistance against bacterial and viral in-
fections without significant toxicity [2,16]. With traditional
immunomodulatory agents exhibiting limited efficacy, the devel-
opment of new multifunctional and non-toxic drugs capable of
altering the immune response safely is extremely important [3].
However, immunomodulation e a therapeutic need of the new
millennium e is still in its infancy [3,20].
matory processes. Tumour necrosis factor-alpha (TNF-a) is one of
the major cytokines and important mediator of immunologic and
inflammatory reactions [26]. Investigation using enzyme linked
immunosorbent assay (ELISA) also demonstrated the production of
pro-inflammatory cytokine (TNF-
amylpeptides (1e2).
a) by these amphiphilic desmur-
2. Results and discussion
2.1. Synthesis
MDP is composed of N-acetyl-
D-glucosamine linked with two
The convergent synthesis of amphiphilic DMPs began with the
alkylation of 3-nitrophenol to give tert-butyl 2-(3-nitrophenoxy)
acetate 3. Reduction of nitro group in 3 via Pd/C with molecular
hydrogen afforded tert-butyl 2-(3-aminophenoxy)acetate 4.
Refluxing 4 with 3-bromopropanol in the presence of N,N-diiso-
propylethylamine resulted in mono-alkylation of free amine to give
amino acids ( -alanine and -isoglutamine) via lactic acid moiety
L
D
(Fig. 1) [1]. Recognition of MDP by innate immune system is highly
stereospecific of the L-D isomer. D-L diastereomer (N-acetylmur-
amyl-D-alanyl-L-isoglutamine) was found completely inactive [21]
just like D-D [22] and L-L isomers [1]. Biological activity of MDP

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F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
tert-butyl 2-(3-((3-hydroxypropyl)amino)phenoxy) acetate 5 along
with the di-alkylated product 7, which was isolated in <10% yield.
Treating 5 with trifluoroacetic acid in dichloromethane gave 6
containing free acid group. Alternatively, a prolonged reflux of 5 in
acetonitrile with the excess of 3-bromopropanol also gave di-
alkylated product 7 in high yields, which was converted into free
acid group containing 8 by treating with trifluoroacetic acid in
dichloromethane (see Scheme 1).
values ranging from 32 Hz for 100ꢀ angle to 0 Hz for 125ꢀ angle
[27,28]. In amphiphilic desmuramyl peptides 1 and 2, the scalar
coupling of the two diastereotopic protons in OCH2 appear as a pair
of doublets at ~4.41 ppm with 2J constant being 15 Hz in both
compounds (Fig. 2, see also Fig. 5s in Supporting Information for
DMP 2).
2.2. Biological studies
The amine moiety in hexylamine was acylated with N-(tert-
butoxycarbonyl)-D-glutamic acid a-benzyl ester 9 under the pro-
motion of peptide coupling reagent O-benzotriazole-N,N,N0,N0-tet-
ramethyl-uronium-hexafluorophosphate (HBTU) to give benzyl N2-
Nucleotide binding oligomerization domain 2 (NOD2) and Toll-
like receptor 4 (TLR4) are two major pattern recognition receptors
(PRR) capable of sensing pathogen-associated molecular patterns
(PAMPs) and initiating a cascade of response leading to the release
of pro-inflammatory cytokines and an increased expression of
cellular adhesion molecules [29]. Although synergism of NOD2
agonist muramyl dipeptide (MDP) or its derivatives (e.g. MDP-C) in
combination with LPS is well documented, yet a cross-talk among
LPS and DMPs in the regulation of intercellular adhesion molecule 1
(ICAM-1) has never been explored [5,30]. Therefore, amphiphilic
DMPs were initially assessed with LPS in their capacity of inducing
ICAM-1 expression in human monocytic THP-1 cells. Following the
initial screening, differentiated THP-1 cells were employed to
further investigate the immunomodulatory potential of the
selected compound and the results are presented in comparison
with a NOD2 agonist murabutide.
THP-1 monocytes were chosen due to high expression of NOD2
receptor in this cell line [31]. NOD2 confers responsiveness to MDP
type molecules by activating NF-kB transcriptional factor pathway
that plays a central role in innate immunity [31]. In our initial ex-
periments, when THP-1 monocytes were incubated for 21 h with
20 mM concentrations of each DMP or murabutide, very low level of
ICAM-1 expression was detected (Fig. 1s in Supporting Informa-
tion). Previously synergistic effect of MDP in combination with LPS
has been reported [32]. DMPs 1 and 2 were also found to amplify
the LPS-induced expression of ICAM-1 by monocytic THP-1 cells
(Fig. 2s in Supporting Information). Up to 46% increase of ICAM-1
(tert-butoxycarbonyl)-N5-hexyl-
-glutaminate 10 in 82% yield.
D
Aminolysis of benzyl ester in 10 was achieved via freshly prepared
7N methanolic ammonia solution. 1H NMR spectral data of 11
indicated that the two amide protons were not identical. Two
distinct singlets were observed at
d 5.75 ppm and at d 6.78 ppm e
each signal corresponding to one proton e a consequence of the
prohibited amide bond rotation. Removal of the N-Boc protecting
group in 11 via treatment with trifluoroacetic acid in dichloro-
methane (1:1) provided free amine 12 (Scheme 2), which was
reacted with N-(tert-butoxycarbonyl)-L-alanine using the peptide
coupling reagent HBTU to give lipophilic dipeptide 13. Free amine
14, obtained by removing N-Boc in 13, was directly acylated with
the acid group containing 6 under the promotion of peptide
coupling reagent HBTU to generate mono-alkylated DMP 1 which
was isolated in low yield (28%). A similar poor yield (29%) was
observed for di-alkylated DMP 2, when the amine 14 was reacted
with 8 under the similar conditions. The cause of this poor yield can
be attributed to the presence of reactive hydroxyl group(s) in
building block 6 or 8. Given the poor yield encountered for 1 and 2,
an alternative coupling condition was employed. An equimolar
mixture of acid 6 or 8, free amine 14 and N-hydroxysuccinimide
(NHS) was stirred in DMF-THF followed by the addition of dicy-
clohexylcarbodiimide (DCC). The new coupling procedure pre-
sumably produced the NHS ester intermediate of 6 or 8, which
reacted more preferably with an amine group over a hydroxyl
group, resulting in a satisfactory yield of 1 and 2 (53% and 48%,
respectively).
expression was observed for LPS in combination with 2 at 16 mM.
The preliminary data also indicated that 2 was more effective than
1 in enhancing LPS-induced ICAM-1 expression (Fig. 2s). Since the
monocytic THP-1 cell line is weakly responsive to immunomodu-
latory signals [33], we then carried our experiments with the more
sensitive differentiated THP-1 cells [34].
1H NMR analysis of amphiphilic DMPs 1 and 2 show geminal
interaction in methylenoxy protons (OCH2) of the linker. Geminal
coupling usually show up as pair of doublets if the system has
asymmetric centres. The coupling constant of geminal interaction is
strongly dependent on the angle between HeCeH bonds with
THP-1 cells can be differentiated into macrophages by various
compounds such as phorbol 12-myristate 13-acetate (PMA),
Scheme 1. a) tert-Butyl bromoacetate, K2CO3, acetone, reflux, 81%; b) Pd/C, H2, 94%; c) Br(CH2)3OH, iPr2NEt, C2H5OH, reflux, 41%; d) TFA/CH2Cl2 (1:1), quant.; e) Br(CH2)3OH, iPr2NEt,
CH3CN, reflux, 66%; f) TFA/CH2Cl2 (1:1), quant.

F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
29
Scheme 2. a) 1-Aminohexane, HBTU, iPr2NEt, DMF, 82%; b) 7N NH3 in CH3OH, 78%; c) TFA/CH2Cl2 (1:1), 72%; d) N-(tert-butoxycarbonyl)-L-alanine, HBTU, iPr2NEt, DMF, 71%; e) TFA/
CH2Cl2 (1:1), 81%; f) HBTU, iPr2NEt, DMF, 28%; g) NHS, DCC, DMFeTHF (2:3), 0 ꢀC to rt, 53%; h) HBTU, iPr2NEt, DMF, 29%; i) NHS, DCC, DMFeTHF (2:3), 0 ꢀC to rt, 48%.
retinoic acid and 1,2,5-dihydroxyvitamin D3 (VD3) [33,34]. How-
ever, elevated concentrations of these compounds are also capable
of stimulating various genes in THP-1 cells, thus overwhelming the
effect of stimuli on differentiated macrophages [33]. Therefore,
optimisation of THP-1 differentiation is necessary to avoid unde-
sired results. For the present study, PMA was employed for cell
differentiation into macrophages. THP-1 cells were treated with
different PMA concentrations (5-30 ng/mL), which results in im-
mediate loss of cell proliferation [35]. THP-1 cells treated with
20 ng/mL or higher concentration of PMA for 48 h showed >90%
adherence to dishes indicating that they had been sufficiently
differentiated to macrophages (Fig. 3). PMA concentration of
20 ng mLꢁ1 was chosen to ensure that THP-1 cells were sufficiently
differentiated to macrophages (Fig. 4) so that responses to weak
stimuli by these cells would be measurable.
increase in overall ICAM-1 expression was observed when
compared to cells treated with LPS alone (Fig. 6). This level of in-
crease is greater than that induced by compound 2 at 20 mM alone
(see Fig. 5). This might be the result of synergistic effect between
LPS and DMP 2; however, additional experiments are needed to
confirm that. Murabutide also shows similar effect on enhancing
LPS-induced ICAM-1 expression, but to a lesser extent than DMP
2 at 8
significantly when cells were pre-treated with 2 at a lower con-
centration (ꢂ4 M). In the presence of 2 at a higher concentration
(ꢃ16 M), the overall ICAM-1 expression level decreased. ICAM-1
expression is NF- B-dependent and induction of high levels of
mM. The overall ICAM-1 expression level did not change
m
m
k
ICAM-1 occurs in response to various inflammatory mediators,
including bacterial LPS and pro-inflammatory cytokines such as
tumor necrosis factor-a (TNF-a), interleukin-1b, and g-interferon
When differentiated THP-1 macrophages were incubated for
(g-IFN) [36e38]. Therefore, the effect of 2 on LPS-induced ICAM-1
21 h with 20
mM concentrations of each DMP, FACS analysis
expression can be attributed towards the intracellular presence of 2
revealed the mean fluorescence intensity of ICAM-1 expression
being 885.76 and 777.29 under the effect of 1 and 2 respectively,
compared to 602.95 of unstimulated cells (Fig. 5). Average ICAM-1
expression of murabutide treated cells was found to be 1436.05
thus implying that the amphiphilic DMPs (1 and 2) are agonistically
less active in inducing ICAM-1 expression.
due to its lipophilic chain, which potentiates the action of LPS via
NF-kB mediated pro-inflammatory cytokines [39]. It is imperative
to measure the release of cytokines induced by DMPs (1 and 2)
potentially mediated by intracellular NOD2.
The binding of agonistic ligands to their receptors leads to the
release of various pro-inflammatory cytokines, immunomodulating
The effect of 2 on LPS-induced ICAM-1 expression in PMA
differentiated THP-1 cells was measured and the results were
compared to murabutide. In a typical procedure, THP-1 cells were
differentiated for 48 h employing PMA (20 ng mLꢁ1), followed by
the treatment with a different concentration of 2 for 1 h prior to
mediators and nitric oxide [40]. Tumour necrosis factor-a (TNF-a), a
pro-inflammatory cytokine, has been suggested to be the mediator
of anti-infectious activity [41]. In vitro studies with various mur-
amyl peptides have been shown to induce the production of TNF-
a
in human monocytes and myeloid-derived cells [26,42]. Boons and
stimulation by LPS (at 0.1
mg/mL). When macrophages were pre-
co-workers also showed the pro-inflammatory properties of mur-
treated with compound 2 at 8
mM, up to a 33% (3583.43 307.3)
amyl tripeptides capable of inducing TNF-a gene expression

30
F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
Fig. 2. 1H NMR spectrum of DMP 1 showing a pair of doublets at around
d
4.41 ppm arising due to H-C-H coupling in the constrained linker.
without TNF-
a
protein production in a human monocytic cell line
responses. At 15
peptides, average TNF-
and 3606
m
M concentration of amphiphilic desmuramyl
released by 1 and 2 was 3622 371 pg/mL
231 pg/mL, respectively, which were significantly
[43]. Desmuramyl peptides [2] and nor-MDP with tuftsin or retro-
tuftsin derivatives [44] have been found to stimulate TNF- secre-
a
a
tion in differentiated human peripheral blood mononuclear cells
(PBMCs) and lymphocytes.
higher than that by Murabutide (2694 271 pg/mL). It's interesting
to note that further increase in the concentration of amphiphilic
To further characterize the immunostimulatory properties of
desmuramyl peptides dampens the TNF-a release. In earlier ex-
amphiphilic DMPs (1e2), the production of TNF-
THP-1 cells in the presence of such stimuli was measured and the
results are presented in Fig. 7. Significant levels of TNF- were
induced by amphiphilic DMPs (1e2). A maximum response level
was achieved at a stimulus concentration of 15 M, after which a
further increase in stimulus concentration resulted in decreased
a
by differentiated
periments, higher concentrations of 2 were also found to lower the
LPS-induced ICAM-1 expression (Fig. 6), although no sign of cell
deaths was observed (Fig. 3s, Supporting Information). A similar
effect was previously reported for high doses of MDP [45]. Further
studies are needed to understand the mechanistic details of these
amphiphilic desmuramyl peptides immunostimulatory activity and
a
m
Fig. 3. THP-1 cells without PMA treatment (left) and the cells treated with 20 ng/mL PMA for 48 h (right).

F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
31
Fig. 4. THP-1 cell differentiation dependence on phorbol 12-myristate 13-acetate
(PMA). Cell adherence results are expressed as the average of three separate
Fig. 6. ICAM-1 expression in PMA-differentiated THP-1 cells induced by DMP (2) plus
LPS and Murabutide plus LPS. ICAM-1 expression results are expressed as the average
experiments
standard error mean (SEM). Statistical significance comparison was
of three separate experiments
standard error mean (SEM). Statistical significance
determined by one way ANOVA, where (*) p < 0.05.
their therapeutic potential.
3. Conclusion
comparison was determined by one way ANOVA, where (*) p < 0.05, (***) p < 0.001 vs
LPS treated cells.
molecules needs to be further evaluated by using different immune
cells and profiling other cytokines. In vivo studies may be followed
to explore their therapeutic potential as immunostimulatory/
immunomodulatory agents.
In summary, we have designed a class of new desmuramyl
peptides containing a lipophilic chain and hydrophilic arene moi-
ety. An efficient synthetic route was adopted to provide the target
compounds in good yield. The amphiphilic DMPs were primarily
evaluated in vitro through the investigation of their synergism on
LPS-induced surface glycoprotein (ICAM-1) expression followed by
measuring the release of pro-inflammatory cytokine (TNF-
novel compounds could amplify LPS-induced ICAM-1 expression
and were capable of stimulating the release of TNF- at levels
higher than murabutide. It is remarkable to observe the overall
similarity in the immunomodulatory activity displayed by these
amphiphilic desmuramyl peptides and murabutide when consid-
ering their huge structural difference. Further studies are war-
ranted to show whether these amphiphilic desmuramyl peptides
exhibit their immunostimulatory effect through the activation of
NOD2 receptor. As well, the immunomodulating potential of these
4. Experimental section
4.1. General procedure for the Boc removal
The Boc containing compound was dissolved in dichloro-
methane and cooled to 0 ꢀC. An equal amount of TFA was added
drop wise. The solution was stirred at room temperature until all
the starting material consumed (TLC monitoring). The solution was
a). The
a
Fig. 7. Cytokine (TNF-
(1e2). THP-1 cells were differentiated for 48 h with phorbol 12-myristate 13-acetate
(PMA, 20 ng mLꢁ1) followed by stimulation with 1, 15 and 30
M of each agonist for
the next 24 h. The cytokine (TNF- ) expression in the supernatant was measured via
a) expression by THP-1 cells after exposure to amphiphilic DMPs
Fig. 5. ICAM-1 expression in differentiated THP-1 cells induced by 20
1 and 2. ICAM-1 expression was measured via immunostaining and flow cytometry
m
M murabutide,
m
a
analysis and the results expressed as the average of three separate
enzyme-linked immunosorbent assay (ELISA) and the results are expressed as the
average of two separate experiments standard error mean (SEM). Statistical signif-
icance comparison was determined by one way ANOVA, where (*) p < 0.05, (**)
experiments
standard error mean (SEM). Statistical significance comparison was
determined by one way ANOVA, where (*) p < 0.05, (**) p < 0.01 vs murabutide treated
cells.
p < 0.01 vs 15 mM murabutide treated cells.

32
F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
concentrated in vacuo, neutralized with saturated aqueous NaHCO3,
washed with water. After brine treatment, the organic layers were
dried over MgSO4, filtered, and evaporated in vacuo to yield the
crude product. When the amine salt was the desired product, the
reaction mixture was evaporated in vacuo. The residual TFA was
removed by co-evaporating with methanol several times to give the
product in quantitative yield.
(0.12 g, 100%) as brown salt. 1H NMR (500 MHz, (CD3)2SO):
d 1.85
(quint, 2H, J ¼ 7 Hz, CH2), 3.09 (m, 2H, CH2NH), 4.20 (m, 2H,
CH2OH), 4.70 (s, 2H, OCH2), 6.20e6.30 (m, 3H, CH), 7.01 (t, 1H,
J ¼ 7 Hz, CH). 13C NMR (125 MHz, (CD3)2SO):
d 32.41 (CH2), 44.10
(CH2NH), 59.13 (CH2OH), 65.24 (OCH2), 98.10 (CH), 102.15 (CH),
106.30 (CH), 129.95 (CH), 150.10 (CCH), 159.27 (CCH), 168.56 (C¼O).
MALDI-TOF (m/z) Calcd for C11H15NO4 [MþH]þ: 226.1079, found:
226.1020.
4.1.1. tert-butyl 2-(3-nitrophenoxy)acetate (3)
To a refluxing solution of 3-nitrophenol (1.00 g, 7.2 mmol) and
potassium carbonate (1.99 g, 14.4 mmol) in dry acetone (50 mL),
tert-butyl 2-(bromomethoxy)acetate (1.27 mL, 8.6 mmol) was
added drop wise in about 15 min. After refluxing overnight, the
mixture was cooled and the solids were filtered off. The filtrate was
evaported in vacuo and the residue was purified via flash column
chromatography (hexane/EtOAc, 7:1) which yielded 3 (1.53 g, 84%)
as light brown syrup: Rf ¼ 0.47 (hexane/EtOAc, 7:1); 1H NMR
4.1.5. tert-butyl 2-(3-(bis(3-hydroxypropyl)amino)phenyl)acetate
(7)
A mixture of 5 (0.1 g, 0.35 mmol), N,N-Diisopropylethylamine
(0.05 g, 0.43 mmol) and 3-bromopropanol (0.06 g, 0.43 mmol) was
refluxed overnight in acetonitrile (10 mL). Then reaction mixture
was cooled and the solvent removed in vacuo. The residue was
purified via flash column chromatography employing EtOAc as
eluent, which yielded 7 (0.08 g, 66%) as brown syrup: Rf ¼ 0.24
(500 MHz, CDCl3):
d
1.50 (s, 9H, C(CH3)3), 4.61 (s, 2H, CH2), 7.26 (dd,
(EtOAc, 100%); 1H NMR (500 MHz, CDCl3):
d 1.49 (s, 9H, C(CH3)3),
1H, J ¼ 2, 8 Hz, CH), 7.45 (t, 1H, J ¼ 8 Hz, CH), 7.70 (t, 1H, J ¼ 2 Hz,
1.83 (quint. 4H, J ¼ 7 Hz, CH2), 3.42 (t, 4H, J ¼ 7 Hz, NCH2), 3.72 (t,
4H, J ¼ 6 Hz, OCH2), 4.85 (s, 2H, CH2), 6.19 (dd, 1H, J ¼ 2, 8 Hz, CH),
6.38 (s, 1H, CH), 6.41 (dd, 1H, J ¼ 2, 8 Hz, CH), 7.10 (t, 1H, J ¼ 8 Hz,
CCHC), 7.86 (dd, 1H, J ¼ 2, 8 Hz, CH); 13C NMR (125 MHz, CDCl3):
d
28.03 (C(CH3)3), 65.83 (CH2), 83.10 (C(CH3)3), 108.85 (CH), 116.60
(CH), 121.87 (CH), 130.13 (CH), 149.10 (CCH), 158.41 (CCH), 167.08
(C¼O); MALDI-TOF (m/z) Calcd for C12H15NO5 [MþNa]þ: 276.0848,
found: 276.3185.
CH). 13C NMR (125 MHz, CDCl3):
d 28.05 (C(CH3)3), 29.99 (CH2)2,
48.16 (NCH2), 60.63 (CH2OH)2, 65.72 (OCH2), 82.25 (C(CH3)3),
100.31 (CH), 101.59 (CH), 106.82 (CH), 129.90 (CH), 149.62 (CCH),
159.21 (CCH), 168.51 (C¼O); MALDI-TOF (m/z) Calcd for C18H29NO5
[MþNa]þ: 362.1943, found: 362.0771.
4.1.2. tert-butyl 2-(3-aminophenoxy)acetate (4)
To a solution of 9 (1.53 g, 6.0 mmol) in ethanol (45 mL), palla-
dium on charcoal (10%, 0.15 g) was added and the mixture was
4.1.6. 2-(3-(bis(3-hydroxypropyl)amino)phenyl)acetic acid (8)
The Boc containing 7 (0.1 g, 0.29 mmol) was dissolved in
dichloromethane (3 mL) and cooled to 0 ꢀC. An equal amount of TFA
was added drop wise. The solution was stirred at room temperature
until all the starting material consumed (TLC monitoring). The re-
action mixture was evaporated in vacuo. The residual TFA was
removed by co-evaporating with methanol several times to give 8
stirred at room temperature under
a hydrogen atmosphere
(balloon) for 24 h. The mixture was filtered, and the filtrate
concentrated. The residue was purified by flash column chroma-
tography (Hexane/EtOAc, 1:1) to afford 10 (1.27 g, 94%) as brown
syrup: Rf ¼ 0.55 (hexane/EtOAc, 1:1); 1H NMR (500 MHz, CDCl3):
d
1.47 (s, 9H, C(CH3)3), 3.70 (br s, 2H, NH2), 4.43 (s, 2H, CH2), 6.25 (m,
3H, CH), 7.01 (t,1H, J ¼ 8 Hz, CH). 13C NMR (125 MHz, CDCl3):
d
28.06
(0.12 g, 100%) as brown salt. 1H NMR (500 MHz, (CD3)2SO):
d 1.93
(C(CH3)3), 65.60 (CH2), 82.18 (C(CH3)3), 101.93 (CH), 104.03 (CH),
108.76 (CH), 130.08 (CH), 148.10 (CCH), 159.06 (CCH), 168.25 (C¼O);
MALDI-TOF (m/z) Calcd for C12H17NO3 [MþNa]þ: 246.1106, found:
246.2956.
(quint. 4H, J ¼ 6 Hz, CH2), 3.40 (m, 4H, NCH2), 4.42 (q, 4H, J ¼ 6 Hz,
CH2OH), 4.60 (s, 2H, OCH2), 6.19 (d, 1H, J ¼ 7 Hz, CH), 6.25 (s, 1H,
CH), 6.36 (d, 1H, J ¼ 7 Hz, CH), 7.06 (t, 1H, J ¼ 7 Hz, CH). 13C NMR
(125 MHz, (CD3)2SO):
d 25.75 (CH2)2, 31.72 N(CH2)2, 58.52
(CH2OH)2, 64.90 (OCH2), 113.60 (CH), 114.54 (CH), 115.88 (CH),
130.42 (CH), 158.70 (CCH), 159.00 (CCH), 170.80 (C¼O); MALDI-TOF
(m/z) Calcd for C14H21NO5 [MþH]þ: 284.1498, found: 284.1316.
4.1.3. tert-butyl 2-(3-((3-hydroxypropyl)amino)phenoxy)acetate
(5)
To a refluxing solution of 4 (0.2 g, 0.9 mmol) and N,N-Diiso-
propylethylamine (0.14 g, 1.08 mmol) in ethanol (10 mL), 3-
bromopropanol (0.15 g, 1.08 mmol) was added drop wise in about
45 min. After refluxing 6 h, the mixture was cooled and the solvent
was evaported in vacuo. The residue was purified via flash column
chromatography (hexane/EtOAc, 1:1) which yielded 5(0.10 g, 41%)
as brown syrup: Rf ¼ 0.42 (hexane/EtOAc, 1:1); 1H NMR (500 MHz,
4.1.7. benzyl N2-(tert-butoxycarbonyl)-N5-hexyl-D-glutaminate
(10)
A solution of hexylamine (0.15 g, 1.48 mmol) in DMF (2 mL) was
added to a mixture of N-(tert-butoxycarbonyl)- -glutamic acid 1-
D
benzyl ester 9 (0.5 g, 1.48 mmol), HBTU (0.56 g, 1.48 mmol) and
iPr2NEt (0.38 g, 2.96 mmol) in DMF (8 mL). The resulting mixture
was stirred at room temperature for 18 h. The solvent was removed
in vacuo and the residue poured into a separatory funnel with water
(25 mL), and then extracted with CH2Cl2 (3x, 40 mL). The combined
organic phase was washed with a cold saturated sodium chloride
solution (8 mL), dried over Na2SO4, and concentrated. Flash column
chromatography of the residue (hexane/EtOAc, 1:1) afforded 10
(0.51 g, 82%) as white powder: Rf ¼ 0.42 (hexane/EtOAc, 1:1); 1H
CDCl3):
d
1.49 (s, 9H, C(CH3)3), 1.87 (quint, 2H, J ¼ 6 Hz, CH2), 3.26 (t,
2H, J ¼ 6 Hz, CH2), 3.80 (t, 2H, J ¼ 6 Hz, CH2), 4.47 (s, 2H, CH2), 6.22
(dd, 1H, J ¼ 2, 12 Hz, CH), 6.24 (s, 1H, CH), 6.28 (dd, 1H, J ¼ 2, 8 Hz,
CH),7.06 (t, 1H, J ¼ 8 Hz, CH). 13C NMR (125 MHz, CDCl3):
d 28.17
(C(CH3)3), 32.41 (CH2), 44.10 (CH2NH), 59.13 (CH2OH), 65.24
(OCH2), 81.64 (C(CH3)3), 98.10 (CH), 102.15 (CH), 106.30 (CH), 129.95
(CH), 150.10 (CCH), 159.27 (CCH), 168.56 (C¼O); MALDI-TOF (m/z)
Calcd for C15H23NO4 [MþNa]þ: 304.1524, found: 304.2307.
NMR (500 MHz, CDCl3):
d
0.88 (t, 3H, J ¼ 7 Hz, CH3), 1.30 (m, 6H,
(CH2)3), 1.40e1.55 (m, 11H, C(CH3)3, CH2), 1.91e1.95 (m, 1H, CHCH2),
2.17e2.23 (m, 3H, CHCH2, COCH2), 3.21 (d, 2H, J ¼ 13 Hz, CH2NH),
4.32 (m, 1H, CH), 5.17 (dd, 2H, J ¼ 12, 32 Hz, OCH2), 5.34 (s, 1H,
OCONH), 5.96 (s, 1H, NHCO), 7.30e7.40 (m, 5H, CH). 13C NMR
4.1.4. 2-(3-((3-hydroxypropyl)amino)phenoxy)acetic acid (6)
The Boc containing 5 (0.1 g, 0.35 mmol) was dissolved in
dichloromethane (3 mL) and cooled to 0 ꢀC. An equal amount of TFA
was added drop wise. The solution was stirred at room temperature
until all the starting material consumed (TLC monitoring). The re-
action mixture was evaporated in vacuo. The residual TFA was
removed by co-evaporating with methanol several times to give 6
(125 MHz, CDCl3):
(CH3CH2CH2), 28.30
d
14.05 (CH3), 22.57 (CH3CH2), 26.60
(C(CH3)3), 29.14(CHCH2), 29.46
(CH2CH2CH2NH), 31.48 (CH2CH2NH), 32.70 (CH2CO), 39.66
(CH2NH), 53.10 (NCH), 67.23 (OCH2), 80.13 (C(CH3)3), 128.41 (CH-

F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
33
Ar), 128.51 (CH-Ar), 128.64 (CH-Ar), 135.27 (C-Ar), 155.85
(OC¼ONH), 171.70 (OC¼O), 172.18 (HNC¼O). MALDI-TOF (m/z)
Calcd for C23H36N2O5 [MþNa]þ: 443.2519, found: 443.1882.
(CH2CH2CH3), 38.92 (CH2CH2NH), 50.43 (CHCH3), 52.46 (CHCH2),
78.69 (C(CH3)3), 155.81 (OC¼O), 171.70 (NHC¼O), 173.16
(CH2C¼O), 173.72 (NH2C¼O). MALDITOF (m/z) Calcd for
C
19H36N4O5 [MþNa]þ: 423.2583, found: 423.3919.
4.1.8. tert-butyl (R)-(1-amino-5-(hexylamino)-1,5-dioxopentan-2-
yl)carbamate (11)
4.1.11. (R)-4-((S)-2-aminopropanamido)-N1-hexylpentanediamide
(14)
The benzyl ester containing 10 (0.5 g, 1.19 mmol) was added to
7 N methanolic ammonia (15 mL) at 0 ꢀC. The mixture was stirred
at room temperature until all the starting material consumed
(48 h). The solution was concentrated in vacuo and the residue
purified via recrystallization in EtOAc to afford 11 (0.3 g, 78%) as
white precipitates: Rf ¼ 0.30 (EtOAc/MeOH, 5:0.3); 1H NMR
The Boc containing 13 (0.4 g, 1 mmol) was dissolved in
dichloromethane (3 mL) and cooled to 0 ꢀC. An equal amount of TFA
was added drop wise. The solution was stirred at room temperature
until all the starting material consumed (TLC monitoring). The so-
lution was concentrated in vacuo, neutralized with saturated
aqueous NaHCO3 and washed with water. After brine treatment,
the organic layers were dried over Na2SO4, filtered and evaporated
in vacuo to yield 14 (0.24 g, 81%) as off-white powder. 1H NMR
(500 MHz, CDCl3):
d
0.89 (t, 3H, J ¼ 7 Hz, CH3), 1.30 (m, 6H, (CH2)3),
1.40e1.55 (m, 11H, C(CH3)3, CH2), 1.90e2.00 (m, 1H, CHCH2),
2.08e2.18 (m, 1H, COCH2), 2.28e2.36 (m, 1H, CHCH2), 2.38e2.46
(m, 1H, COCH2), 3.25 (m, 2H, CH2NH), 4.16 (m, 1H, CH), 5.46 (s, 1H,
OCONH), 5.75 (s, 1H, NH2), 5.94 (NHCO), 6.78 (s, 1H, NH2). 13C NMR
(500 MHz, (CD3)2SO):
d
0.86 (t, 3H, J ¼ 7 Hz, CH3CH2),1.20e21.4 (m,
11H, CHCH3 (CH2)4), 1.72 (td, 1H, J ¼ 8, 15 Hz, CHCHH), 1.93 (td, 1H,
8, 14 Hz, CHCHH), 2.07 (t, 2H, J ¼ 8 Hz, COCH2), 3.00 (q, 2H, J ¼ 6 Hz,
NHCH2), 3.89 (quint, 1H, J ¼ 6 Hz, CHCH3), 4.24 (m, 1H, CHCH2), 7.17
(s, 1H, NH2), 7.47 (s, 1H, NH2), 7.82 (t, 1H, J ¼ 5 Hz, NHCH2), 8.05 (s,
2H, CHNH2), 8.55 (d, 1H, J ¼ 8 Hz, CHNH). 13C NMR (125 MHz,
(125 MHz, CDCl3):
(CH3CH2CH2), 28.34
d
14.04 (CH3), 22.57 (CH3CH2), 26.63
(C(CH3)3), 28.34 (CHCH2), 29.44
(CH2CH2CH2NH), 31.49 (CH2CH2NH), 32.82 (CH2CO), 39.77
(CH2NH), 53.45 (NHCH)s6.05 (OC¼ONH), 172.64 (NH2C¼O), 174.24
(HNC¼O); MALDI-TOF (m/z) Calcd for C16H31N3O4 [MþNa]þ:
352.2212, found: 352.1974.
(CD3)2SO):
d 14.34 (CH3CH2), 17.76 (CH3CH), 22.50 (CH2CH3), 26.53
(CH2), 28.55 (CH2), 29.51 (CH2), 31.44 (CH2), 32.08 (CH2), 38.93
(CH2NH), 48.71 (CHCH3), 52.65 (CHCH2), 169.89 (C¼O), 171.49
(C¼O), 173.25 (C¼O). MALDI-TOF (m/z) Calcd for C14H28N4O3
[MþNa]þ: 323.2059, found: 323.2036.
4.1.9. (R)-4-amino-N1-hexylpentanediamide (12)
The Boc containing 11 (1 g, 3 mmol) was dissolved in
dichloromethane (5 mL) and cooled to 0 ꢀC. An equal amount of
TFA was added drop wise. The solution was stirred at room
temperature until all the starting material consumed (TLC
monitoring). The solution was concentrated in vacuo, neutralized
with saturated aqueous NaHCO3 and washed with water. After
brine treatment, the organic layers were dried over Na2SO4,
filtered and evaporated in vacuo to yield 12 (0.5 g, 72%) as off-
4.1.12. (R)-N1-hexyl-4-((S)-2-(2-(3-((3-hydroxypropyl)amino)
phenoxy)acetamido)propanamido) pentanediamide (1)
A solution of 14 (0.075 g, 0.25 mmol) in DMF (2 mL) was added
to a mixture of 6 (0.056 g, 0.25 mmol), HBTU (0.095 g, 0.25 mmol)
and iPr2NEt (0.064 g, 0.50 mmol) in DMF (5 mL). The resulting
mixture was stirred at room temperature for 18 h. The solvent was
removed in vacuo and the residue purified via flash column
chromatography (CHCl3/MeOH, 5:1) to afford air-sensitive 1
(0.035 mg, 28%) as white powder. Alternatively, a solution of 6
(0.05 g, 0.22 mmol) in DMF (2 mL) was added a mixture of 14
(0.067 g, 0.22 mmol) and 1-hydroxy-2,5-pyrrolidinedione (N-
hydroxysuccinimide, NHS) (0.025 g, 0.22 mmol) in tetrahydro-
furan (3 mL). With ice-cooling and stirring, dicyclohex-
ylcarbodiimide (0.046 g, 0.22 mmol) was added thereto. The
mixture was stirred overnight at room temperature. The precipi-
tated dicyclohexyl urea was filtered off, the filtrate was concen-
trated in vacuo and the residue was purified via flash column
chromatography (CHCl3/MeOH, 5:1) which yielded air sensitive 1
(0.06 g, 53%) as white precipitate: Rf ¼ 0.41 (CHCl3/MeOH, 5:1); 1H
white product. 1H NMR (500 MHz, CD3OD):
d 0.92 (t, 3H,
J ¼ 7 Hz, CH3), 1.22e1.40 (m, 6H, CH3(CH2)3), 1.51 (quint, 2H,
J ¼ 7 Hz, NHCH2CH2), 1.92 (m, 1H, COCH2CHH), 2.04 (m, 2H,
COCH2), 2.35 (m, 1H, COCH2CHH), 3.18 (t, 2H, CH2NH), 4.12 (q, 1H,
J ¼ 7 Hz, CH). 13C NMR (125 MHz, CD3OD):
d 12.95 (CH3), 19.47
(CH3CH2), 22.23 (CH3CH2CH2), 26.29 (CHCH2), 28.91 (CH2), 31.26
(CH2), 31.56 (CH2), 39.10 (NHCH2), 60.16 (NHCH), 171.68 (C¼O),
173.43 (C¼O). MALDI-TOF (m/z) Calcd for C11H23N3O2 [MþNa]þ:
252.1688, found: 252.1732.
4.1.10. tert-butyl ((S)-1-(((R)-1-amino-5-(hexylamino)-1,5-
dioxopentan-2-yl)amino)-1-oxopropan-2yl)carbamate (13)
A solution of 12 (0.21 g, 0.92 mmol) in DMF (2 mL) was added
to
a
mixture of N-(tert-butoxycarbonyl)-
L
-alanine (0.17 g,
NMR (500 MHz, (CD3)2SO):
d
0.87 (t, 3H, J ¼ 7 Hz, CH3CH2), 1.24
0.92 mmol), HBTU (0.0.35 g, 0.92 mmol) and iPr2NEt (0.35 g,
2.76 mmol) in DMF (5 mL). The resulting mixture was stirred at
room temperature for 18 h. The solvent was removed in vacuo and
the residue poured into a separatory funnel with water (15 mL),
and then extracted with CH2Cl2 (3x, 35 mL). The combined organic
phase was washed with a cold saturated sodium chloride solution
(10 mL), dried over Na2SO4, and concentrated. The residue was
further purified via recrystallization in CH2Cl2 to afford 13 (0.37 g,
71%) as white precipitates: Rf ¼ 0.70 (CHCl3/MeOH, 4:1); 1H NMR
(m, 6H, CH3(CH2)3), 1.27 (d, 3H, J ¼ 7 Hz, CHCH3), 1.37 (quint., 2H,
J ¼ 7 Hz, CH3(CH2)3CH2), 1.69 (quint., 2H, J ¼ 6 Hz, CH2CH2OH), 1.72
(m, 1H, CHCHH), 1.94 (m, 1H, CHCHH), 2.08 (t, 2H, J ¼ 8 Hz,
CHCH2CH2), 3.02 (m, 4H, CH2OH, CONHCH2), 3.56 (q, 2H, J ¼ 6 Hz,
NHCH2CH2CH2OH), 4.13 (td, 1H, J ¼ 5, 8 Hz, CHCH2), 4.34 (quint.,
1H, J ¼ 7 Hz, CHC H3), 4.43 (dd, 2H, J ¼ 15 Hz, OCH2), 4.50 (t, 1H,
J ¼ 5 Hz, OH), 5.61 (t, 1H, J ¼ 5 Hz, NHCH2CH2CH2OH), 6.12 (dd, 1H,
J ¼ 2, 8 Hz, OCCHCHCH), 6.15 (t, 1H, J ¼ 2 Hz, CCHC), 6.20 (dd, 1H,
J ¼ 2, 8 Hz, OCCHCHCH), 6.96 (t, 1H, J ¼ 8 Hz, OCCHCHCH), 7.11 (s,
1H, NH2), 7.33 (s, 1H, NH2), 7.79 (t, 1H, J ¼ 5 Hz, CONHCH2), 8.10 (d,
1H, J ¼ 6 Hz, CH3CHNH), 8.25 (d, 1H, J ¼ 8 Hz, CHNHCO). 13C NMR
(500 MHz, (CD3)2SO):
d
0.86 (t, 3H, J ¼ 7 Hz, CH3CH2), 1.16 (d, 3H,
J ¼ 7 Hz, CHCH3), 1.23 (m, 6H, (CH2)3), 1.32e1.40 (m, 11H, C(CH3)3,
CH2), 1.70 (m, 1H, CHCH2), 1.93 (m, 1H, CHCH2), 2.05 (t, 2H,
J ¼ 7 Hz, COCH2), 2.99 (q, 2H, J ¼ 6 Hz, NHCH2), 3.94 (quint, 1H,
J ¼ 7 Hz, CHCH2), 4.10 (m, 1H, CHCH3), 7.08 (d, 1H, J ¼ 7 Hz,
CONHCH), 7.13 (s, 1H, NH2), 7.27 (s, 1H, NHCH2), 7.60 (s, 1H, NH2),
7.96 (d, 1H, J ¼ 8 Hz, OCONH). 13C NMR (125 MHz, (CD3)2SO):
(125 MHz, (CD3)2SO):
d 14.40 (CH3CH2), 18.83 (CH3CH), 22.52
(CH2CH3), 26.55 (CH2), 28.13 (CH2), 29.52 (CH2), 31.46 (CH2), 32.22
(CH2), 32.42 (CH2), 38.95 (NCH2), 45.00 (NHCH2), 48.56 (CHCH3),
52.65 (CHCH2), 59.15 (CH2OH), 67.07 (OCH2), 98.63 (CHCHCH),
101.78 (CHCHCH), 106.40 (CCHC), 130.00 (CHCHCH), 150.91
(NCCH), 159.23 (NCCHC), 168.12 (CH3CHC¼O), 171.71 (CH2C¼O),
172.34 (OCH2C¼O), 173.67 (NH2C¼O); MALDI-TOF (m/z) Calcd for
d
14.38 (CH3CH2), 18.19 (CH3CH), 22.51 (CH2CH3), 26.53 (C(CH3)3),
28.33 (CH2), 28.65 (CH2CO), 29.53 (CH2), 31.45 (CH2CH), 32.19

34
F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
C25H41N5O6 [MþNa]þ: 530.2954, found: 530.2084.
Serum Albumin/PBS to proceed with staining and flow cytometry
analysis.
4.1.13. (R)-4-((S)-2-(2-(3-(bis(3-hydroxypropyl)amino)phenoxy)
acetamido)propanamido)-N1-hexylpentanediamide (2)
4.2.3. Optimization of PMA concentration for cell differentiation
THP-1 monocytes were plated at 0.5 ꢄ 106 cells wellꢁ1 in 6-well
tissue culture plates containing the RPMI medium further supple-
mented with different PMA concentrations (5e30 ng/mL). After
48 h, unattached THP-1 cells were collected and counted to
determine cell adherence. Cell counting was performed using he-
mocytometer, with viability results being determined through the
trypan blue cellular exclusion method.
A solution of 14 (0.075 g, 0.25 mmol) in DMF (2 mL) was added
to a mixture of 8 (0.071 g, 0.25 mmol), HBTU (0.094 g, 0.25 mmol)
and iPr2NEt (0.065 g, 0.50 mmol) in DMF (5 mL). The resulting
mixture was stirred at room temperature for 18 h. The solvent was
removed in vacuo and the residue purified via flash column
chromatography (CHCl3/MeOH, 5:1) to afford air-sensitive 2
(0.051 g, 29%) as white powder. Alternatively, a solution of 14
(0.05 g, 0.17 mmol) in DMF (2 mL) was added to a mixture of 8
(0.05 g, 0.17 mmol) and 1-hydroxy-2,5-pyrrolidinedione (N-
hydroxysuccinimide, NHS) (0.02 g, 0.17 mmol) in tetrahydrofuran
(3 mL). With ice-cooling and stirring, dicyclohexylcarbodiimide
(0.03 g, 0.17 mmol) was added thereto. The mixture was stirred
overnight at room temperature. The precipitated dicyclohexyl
urea was filtered off, the filtrate was concentrated in vacuo and the
residue was purified via flash column chromatography (CHCl3/
MeOH, 5:1) which yielded air sensitive 2 (45 mg, 48%) as white
precipitate: Rf ¼ 0.37 (CHCl3/MeOH, 5:1); 1H NMR (500 MHz,
4.2.4. ICAM-1 induction in differentiated THP-1 cells
THP-1 monocytes were plated at 0.5 ꢄ 106 cells wellꢁ1 in 6-
well tissue culture plates containing the RPMI medium further
supplemented with 20 ng mLꢁ1 of PMA. After 48 h, the medium
was removed and now adhered THP-1 cells were washed with
PBS. The wells were then refilled with serum-free RPMI medium,
cells were stimulated with amphiphilic DMP 1 or 2 (20
the positive control (Murabutide, 20 M). For synergistic ICAM-1
expression, PMA-differentiated THP-1 cells were first incubated
with different concentrations (0.1e64 M) of amphiphilic DMP 2
for 1 h and then with LPS (0.1 g/mL) for a further 20 h. Cells were
mM) and
m
(CD3)2SO):
d
0.86 (t, 3H, J ¼ 7 Hz, CH3CH2), 1.23 (m, 6H, CH3(CH2)3),
1.27 (d, 3H, J ¼ 7 Hz, HCH3), 1.65 (m, 3H, CH2CH2N, CHCHH), 1.93
(m, 1H, CHCHH), 2.07 (t, 2H, J ¼ 8 Hz, CHCH2CH2), 3.00 (m, 2H,
NHCH2), 3.31 (m, 4H, CH2OH), 3.45 (q, 4H, J ¼ 6 Hz, NCH2), 4.12 (td,
1H, J ¼ 5, 9 Hz, CHCH2), 4.37 (quint., 1H, J ¼ 7 Hz, CHC H3), 4.44 (d,
2H, J ¼ 15 Hz, OCH2), 4.53 (t, 2H, J ¼ 5 Hz, OH), 6.15 (dd, 1H, J ¼ 2,
8 Hz, OCCHCHCH), 6.24 (t, 1H, J ¼ 2 Hz, CCHC), 6.32 (dd, 1H, J ¼ 2,
8 Hz, OCCHCHCH), 7.02 (t, 1H, J ¼ 8 Hz, OCCHCHCH), 7.10 (s, 1H,
NH2), 7.32 (s, 1H, NH2), 7.79 (t, 1H, J ¼ 5 Hz, CONHCH2), 8.12 (d, 1H,
J ¼ 7 Hz, CHNH), 8.25 (d, 1H, J ¼ 8 Hz, CHNH). 13C NMR (125 MHz,
m
m
washed with PBS and the washings were centrifuged at 400 ꢄ g
for 4 min. The cells in the washings were stained to determine
Trypan blue-positive cells and calculate the overall cell viability.
For immunostaining and flow cytometry analysis, the differenti-
ated THP-1 cells were detached via gentle cold shocks and then
suspended in 100
mL 0.1% Bovine Serum Albumin/PBS. The
expression of ICAM-1 was determined via immunostaining with
phycoerythrin-conjugated mAb to ICAM-1 (CD54) from BD Bio-
sciences, San Jose, CA. The antibody was added to the cells and the
mixture incubated at 4 ꢀC for 1 h in the dark. After incubation,
(CD3)2SO):
d 14.40 (CH3CH2), 18.77 (CH3CH), 22.52 (CH2CH3),
26.55 (CH2), 28.09 (CH2), 29.51 (CH2), 30.48 (CH2), 31.46 (CH2),
32.22 (CH2), 38.95 (NCH2), 47.66 (NHCH2), 48.63 (CHCH3), 52.65
(CHCH2), 58.88 (CH2OH), 67.15 (OCH2), 98.76 (CHCHCH), 101.27
(CHCHCH), 105.69 (CCHC), 130.20 (CHCHCH), 149.66 (NCCH),
159.41 (NCCHC), 168.23 (CH3CHC¼O), 171.76 (CH2C¼O), 172.38
(OCH2C¼O), 173.71 (NH2C¼O); MALDI-TOF (m/z) Calcd for
cells were washed twice with PBS, resuspended in 500 mL PBS, and
subjected to flow cytometry analysis on FACS Calibur with CELL-
QUEST PRO software (BD Biosciences), acquiring 20,000 events.
The results were presented as the mean fluorescence intensity
(MFI) on the FL2 channel.
C
28H47N5O7 [MþNa]þ: 588.3373, found: 588.2733.
4.2. Reagents for biological experiments
4.2.5. Cytokine induction and measurement
THP-1 monocytes were plated at 0.5 ꢄ 106 cells per well in 6-
well tissue culture plates containing the RPMI media further
supplemented with 20 ng mLꢁ1 of PMA. After 48 h, the media was
removed and the adhered THP-1 cells were washed with PBS. The
wells were then refilled with serum-free RPMI media, incubated
for 3 h, and then exposed to stimuli. After 24 h stimulation, culture
supernatants were collected and stored frozen (ꢁ80 ꢀC) until
tested. Enzyme-linked immunosorbent assays (ELISA) were per-
formed in 96-well MaxiSorp plates. Ready-Set-Go! ELISA kits
(eBioscience) were used for cytokine quantification of human
E. coli LPS 0111:B4 was obtained from InvivoGen, San Diego, CA.
Murabutide was purchased from Sigma-Aldrich. Synthetic DMPs 1
and 2 were dissolved in DMSO, reconstituted in water and stored
at ꢁ20 ꢀC.
4.2.1. Cell maintenance
THP-1 cells were obtained from American Type Culture Collec-
tion (ATCC). The cells were maintained at 37 ꢀC and 5% CO2 atmo-
sphere in RPMI-1640 medium supplemented with 10% heat
inactivated fetal bovine serum and 1% antibioticeantimycotic 100x
(all from Gibco BRL, Grand Island, NY, US). Cell counting was per-
formed using hemocytometer, with viability being determined
through the trypan blue cellular exclusion method.
TNF-
a according to the manufacturer's instructions. The absor-
bance was measured at 450 nm with wavelength correction set to
540 nm using a micro-plate reader (BMG Labtech). A standard
curve was generated using polynomial second order regression
analysis and the cytokine values were presented as the
mean SEM of two separate experiments.
4.2.2. ICAM-1 induction in THP-1 monocytes
THP-1 monocytes were plated at 0.5 ꢄ 106 cells/well in 6-well
tissue culture plates and incubated for 24 h. Cells were then incu-
bated for 21 h with 20
mM concentrations of each compound.
Alternatively for combinatory synergistic effect, cells were first
4.2.6. Statistical analyses
incubated with different concentration of amphiphilic DMPs
Statistical analyses were done using one-way ANOVA, followed
by multiple comparisons with Dunnett's test in GraphPad Prism
(version 6) software. A value of p < 0.05 was considered statistically
significant. All the data are presented as the mean SEM.
(0.1e32
Cells were centrifuged at 1000 g for 5 min, washed with phosphate
buffered saline (PBS), and then re-suspended in 100 L 0.1% Bovine
mM) for 1 h and then with LPS (0.1 mg/mL) for a further 20 h.
m

F.-A. Khan et al. / European Journal of Medicinal Chemistry 141 (2017) 26e36
35
-alanyl- D -glutamic Acid: a novel immunologically active carbocyclic mur-
amyl dipeptide analogue, J. Med. Chem. 41 (1998) 530e539.
Acknowledgement
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This work was supported by Natural Sciences and Engineering
Research Council of Canada (NSERC, Grant EGP 469917-14) and
Lakehead University.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.09.070.
^
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