Synthesis and antiproliferative activity of conjugates of adenosine with muramyl dipeptide and nor-muramyl dipeptide derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.05.043

We synthesized a series of MDP(D,D) and nor-MDP(D,D) derivatives conjugated with adenosine through a spacer as potential immunosuppressants. New conjugates 8a-k were evaluated on two leukemia cell lines (Jurkat and L1210) and PBMC from healthy donors. The conjugates 8a-k and MDP(D,D)/nor-MDP(D,D) derivatives 7e, f, i, j were active against L1210 cell line. Unconjugated nor-MDP(D,D) had better antiproliferative properties, but the conjugates 8b, f, g had the highest values of selectivity index. Both cell lines as well as PBMC were resistant to analogs 11a, b with the 6-aminohexanoic linker.

Full text of DOI:10.1016/j.bmcl.2014.05.043

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of conjugates of adenosine
with muramyl dipeptide and nor-muramyl dipeptide derivatives
b
Monika Samsel a, Krystyna Dzierzbicka a, , Piotr Trzonkowski
⇑
a Department of Organic Chemistry, Gdansk University of Technology, ul. Narutowicza 11/12, 80-233 Gdansk, Poland
b Department of Clinical Immunology and Transplantology, Medical University of Gdansk, ul. Debinki 7, 80-211 Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a series of MDP(D,D) and nor-MDP(D,D) derivatives conjugated with adenosine through a
spacer as potential immunosuppressants. New conjugates 8a–k were evaluated on two leukemia cell
lines (Jurkat and L1210) and PBMC from healthy donors. The conjugates 8a–k and MDP(D,D)/nor-
MDP(D,D) derivatives 7e, f, i, j were active against L1210 cell line. Unconjugated nor-MDP(D,D) had better
antiproliferative properties, but the conjugates 8b, f, g had the highest values of selectivity index. Both
cell lines as well as PBMC were resistant to analogs 11a, b with the 6-aminohexanoic linker.
Ó 2014 Published by Elsevier Ltd.
Received 19 March 2014
Revised 12 May 2014
Accepted 13 May 2014
Available online xxxx
Keywords:
Muramyl dipeptide conjugates
MDP(D,D)
Nor-MDP(D,D)
Adenosine
Synthesis
Biological activity
Immunosuppression is well-established strategy of treatment
in medicine. Depression of immune activities with pharmacologi-
cal compounds is able to cure or alleviate symptoms of autoim-
mune and allergic diseases as well as maintain transplanted
organs.
modifications have led to construction of compounds not only with
nonpyrogenic adjuvants13 but also derivatives and conjugates with
antitumor,14 antiviral,15 antibacterial or hepatoprotective
actions.16 However, in 1976 it was observed that a change of
Ala to
D-Ala in a peptide part of MDP entirely alternates its
Many naturally derived substances have an impact on the
human immune system. They can either promote or suppress the
immune response. Compounds with recognized immunosuppres-
sive action are, that is, cyclosporine1 isolated from the soil fungus
Tolypocladium inflatum, mycophenolic acid produced by Penicillium
spp.2,3 or tacrolimus that was first isolated from the fermentation
broth of Streptomyces tsukubaensis.4 The bacterial cell wall peptido-
glycan (PGN) is a component that has an opposite effect – it
activates the immune system. In 1974 it was demonstrated that
properties. The data showed depression of the humoral response
by N-AcMur-D-Ala-D-Glu-NH2.17 Later, researchers revealed that
the replacement of
L-alanine for D-alanine causes the loss of
activity towards NOD2 receptor.18
In this Letter we would like to report the synthesis of new series
of MDP(D,D) and nor-MDP(D,D) derivatives linked through an
2-aminoethyl (as reported previously)19 or 6-aminohexanoic acid
spacer to the adenosine, whose cytotoxic and antiproliferative
activities were evaluated. In order to achieve compounds that
decrease the humoral immune response we modified the peptide
muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine,
MDP 1) (Fig. 1) is the minimal structure of bacterial PGN required
for immunoadjuvant activity.5 MDP acts through intracellular
NOD2 receptor expressed in immune cells, including monocytes,
macrophages, T lymphocytes, granulocytes, dendritic cells and also
in intestinal epithelial cells.6 Injection of MDP is pyrogenic7 and
can induce uveitis,8 arthritis9 or meningitis.10 Therefore, many
derivatives of MDP have been synthesized in order to gain less
toxic agents with better pharmacological properties.11,12 Structure
part of MDP by replacing L-alanine with D-amino acids. To amplify
the immunosuppressive action an adenosine fragment was
attached. Adenosine 2 (Fig. 1) is a purine nucleoside that is consti-
tutively present at low levels outside cells.20,21 In case of hypoxia
or ischemia its concentration raises and protects cells and tissues
mediating its effects via four types of adenosine receptors: A1,
A
2A, A2B and A3, belonging to the G-protein-coupled receptor
family.21 A2A receptors are expressed ubiquitously in the body,
but they can be found mainly in the immune system on mono-
cytes/macrophages,22 T cells,23 dendritic cells,24 neutrophils.21
One of the effects of adenosine acting through A2A receptors is
⇑
Corresponding author. Tel.: +48 58 347 20 54; fax: +48 58 347 26 94.
E-mail address: krydzier@pg.gda.pl (K. Dzierzbicka).
http://dx.doi.org/10.1016/j.bmcl.2014.05.043
0960-894X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Samsel, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.043

2
M. Samsel et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
OH
NH
7f–j and 1-O-Bn-MDP(L,D) 7k derivatives. The benzyl protective
NH
HO
HO
2
group was left untouched under the reaction conditions that
should not influence essentially the biological activity.33 Com-
pounds 7a–k were used without purification for the synthesis of
conjugates with adenosine. Derivatives 7e, 7f, 7i, 7j used for bio-
logical tests were purified by TLC and structures were confirmed
by MALDI-TOF mass analysis.
Initially, we attempted to couple adenosine directly through an
amide bond to the MDP derivative, but the reactivity of a purine
amino group at C-6 was insufficient. Therefore, we decided to use
a linker between the free carboxyl group of MDP derivatives 7a–k
and the adenosine moiety. 6-Chloropurine riboside 4 was used a
substrate to afford N6-(2-aminoethyl) adenosine 5 in reaction with
1,2-ethylenediamine as described by Brodelius et al. (Scheme 1).29
Next, the N-terminal amino group of 5 was coupled with 7a–k
by means of EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
ide) and HOBt (N-hydroxybenzotriazole) to yield conjugates
8a–k. The final products were purified with preparative TLC and
their composition was confirmed by 1H NMR and MALDI-TOF mass
analysis. The structures of compounds 8a and 8j were confirmed
additionally by 2D ROESY and TOCSY 1H NMR. A conjugate 8k
N
N
N
N
HO
O
O
O
NH
O
2
NH
NH
OH
O
OH OH
O
1
2
Figure 1. Structure of MDP 1 and adenosine 2.
the induction of T-cell anergy.25 Adenosine is quickly metabolized
by adenosine deaminase26 that limits its clinical usefulness.
The idea of synergistic activity of the two linked compounds
with different mode of action is very attractive in pharmacology.
On one side, drugs designed in this way are less toxic due to the
application of lower doses, on the other side, the efficacy is much
better due to the several mechanisms of activity. Unique suppres-
sive properties of MDP analogues applied in this study combined
with adenosine seems to be a model example of such conglomer-
ates, which can be used in immunosuppressive treatment.
MDP and nor-MDP derivatives 7a–k were synthesized
according to a procedure described previously.27–31 We started
with the synthesis of the dipeptides Cbz-D-X-D-isoGln(Ot-Bu)
containing L-valine was synthesized as a control. To check the
influence of the linker on a conjugate activity, we synthesized
compounds 11a–b (Scheme 2). Firstly, N-Cbz-6-aminohexanoic
anhydride was obtained from the condensation reaction of
N-Cbz-6-aminohexanoic acid with DCC (N,N0-dicyclohexylcarbodi-
imide) in dry dichloromethane. Next, the synthesis of N6-(N-ben-
3a–e (X = Ala, 2-ABA, Pro, Ser, Val).32 Cbz-
D-X were chosen as the
starting materials which reacted with
D-isoGln(Ot-Bu) using mixed
anhydride procedure with isobutyl chloroformate and NMM
(N-methylmorpholine) in dry DMF. The protected dipeptides Cbz-
zyloxycarbonyl)(6-aminohexanoyl)adenosine
9 was performed
according to a literature procedure.29 After removal of N-benzyl-
oxycarbonyl by catalytic hydrogenation (H2/Pd-C) an amide bond
between carboxyl group of MDP 7a or nor-MDP 7f derivatives
and primary amine of 10 was formed. DPPA (diphenyl phosphoro-
azidate) method allowed obtaining conjugates 11a–b in yields of
55–58%. 1H NMR and MALDI-TOF spectra were consistent with
the assigned structures.
D-X-D-isoGln(Ot-Bu) 3a–e were purified by crystallization from a
mixture of hexane and ethyl acetate. In the next step the Cbz group
was cleaved by hydrogenolysis (H2/Pd-C). This procedure was
carried out directly before coupling to the protected muramic or
nor-muramic acid by using a mixed anhydride method to afford
protected MDP(D,D) 6a–e, nor-MDP(D,D) 6f–j and MDP(L,D) 6k
derivatives. Their chemical structures were confirmed by 1H NMR
and MALDI-TOF mass analysis. The subsequent removal of the ben-
zylidene and tert-butyl groups was carried out using 90% TFA to
yield the desired 1-O-Bn-MDP(D,D) 7a–e, 1-O-Bn-nor-MDP(D,D)
Cytotoxic activities of compounds listed in Table 1 were
evaluated against lymphoid cell lines and activated peripheral blood
mononuclear cells (PBMC) as in vitro model of immunosuppression.
Cl
NH
2
HN
N
N
N
N
N
N
N
N
HO
a
O
HO
O
OH OH
4
OH OH
5
O
O
HO
HO
HO
HO
H C
O
O
5
6
O
X
OBn
NHAc
CO-X-D-isoGln(Ot-Bu)
OBn
NHAc
CO-X-D-isoGln
7a-k
OBn
c
b
O
O
NHAc
O
O
NH
2
R
R
O
R
NH
6a-k
O
NH
HN
f: R = H; X =
g: R = H; X =
h: R = H; X =
i: R = H; X =
j: R = H; X =
D
-Ala
-2-ABA
-Pro
-Ser
-Val
-Val
N
N
a: R = CH3; X =
b: R = CH3; X =
c: R = CH3; X =
d: R = CH3; X =
e: R = CH3; X =
D
-Ala
-2-ABA
-Pro
-Ser
-Val
N
D
D
N
HO
D
O
D
D
D
D
OH OH
D
k: R = CH3; X =
L
8a-k
Scheme 1. General synthesis of N6-(2-aminoethyl)adenosine 5 and the conjugates 8a–k. Reagents and conditions: (a) 1,2-ethylenediamine, water, 3 h; (b) 90% TFA, 0 °C,
30 min.; (c) 5, EDCI, HOBt, DMF, 0 °C, 2 h, then rt, 48 h.
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M. Samsel et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
O
NH
O
NH
N
HN
N
2
N
N
N
N
O
N
N
a
b
HO
HO
HO
HO
O
O
O
OCH Ph
2
OH OH
OH OH
NHAc
O
O
2
9
NH
2
O
N
X
O
R
NH
NH
2
HN
N
O
N
O
NH
N
N
HN
c
d
HO
N
N
O
N
HO
O
OH OH
11a: R = H; X =
D-Ala
10
11b: R = CH3; X =
D-Ala
OH OH
Scheme 2. General synthesis of compounds 11a–b. Reagents and conditions: (a) TMSCl, pyridine, 0 °C, 5 min., then rt, 2 h; (b) N-Cbz-6-aminohexanoic anhydride, rt, 17 h,
then 0 °C, water, concentrated ammonia solution; (c) H2/Pd-C, methanol; (d) 7a or 7f, DPPA, TEA, DMF, rt, 24 h.
Table 1
Table 2
IC50 (mM) values are mean SD of triplicate test
EC50 (mM) values are mean SD of triplicate test
⁄ The cell viability.
⁄ The highest concentration of drug used in the assay did not inhibit cell viability by
50% compare to the control.
# Proliferation in comparison to control for c = 0.5 mg/mL (7j: c = 0.215 mg/mL, 7e:
c = 0.25 mg/mL).
⁄⁄ The compound increased the cell viability as compared with DMSO; grey color
point the most active compounds.
C SI—MTT/[3H]TdR.
⁄⁄ The compound increased the cell viability as compared with DMSO; grey color
point the most selective compounds.
Two models of leukemia were used: the human T lymphocyte-based
Jurkat cell line and a mouse lymphocytic leukemia L1210. The MTT
colorimetric assay was used to measure cell viability, whereas the
[3H]-thymidine incorporation assay measured cell proliferation.
IC50 were given when a compound inhibited viability and were cal-
culated with MTT colorimetric test. EC50 values were calculated with
the [3H]-thymidine incorporation assay as a dose of a compound,
which reduces cells proliferation by 50% in regard to control sample.
Compounds were dissolved in DMSO due to poor solubility in water.
A mixture of adenosine and MDP derivative in the same ratio as in
the conjugate was insoluble in DMSO.
DMSO, the [3H]-thymidine incorporation assay revealed no signif-
icant change in cell proliferation in the presence of the conjugates.
Viability of L1210 cells was affected by compounds in the highest
examined concentrations. Conjugates 11a–b, 8i and control
MDP(D,D) increased viability in comparison to the solvent. Most sig-
nificant inhibitory effect was noted for nor-MDP derivative 7j
(IC50 = 0.17 0.037 mM) and the conjugate 8e, with IC50 = 0.161
0.019 mM (Table 1). Promising antiproliferative effects were noted
for MDP(D,D) and nor-MDP(D,D) derivatives 7e, 7f, 7i, 7j) (0.193–
0.363 mM) that were more active than the conjugates 8a–j
In the cultures of Jurkat cells there was no inhibitory effect on
the viability as compared with the solvent. Analysis of compounds
concentration greater than 0.05 mg/mL was impossible due to the
influence of DMSO on cell viability. Surprisingly, as compared with
(0.446–0.541 mM). The control conjugate 8k with the L-amino acid
also presented antiproliferative activity, however much weaker
than conjugates 8a–j (with exeption to 8i). Adenosine alone stimu-
lated cell proliferation. The same effect as for nucleoside was
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M. Samsel et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 2. Effect of examined compound on cell proliferation in the culture of PBMC from sample D and E. The data presents the mean SD of triplicate test. Compound 7j:
c = 0.021 mg/mL, 7e: c = 0.025 mg/mL, other compounds and DMSO: c = 0.05 mg/mL, #—there was significant difference (P = 0.05) compared with the respective value of
DMSO treated cells.
observed for the conjugates 11a–b and control MDP(D,D). Impor-
tantly, solvent did not inhibit cell proliferation.
Although we are far from the administration of these substances
as a ‘pill in the pharmacy’, it is a good introduction for in vivo exper-
iments in animal models. Preselection for such in vivo experiments
pave a way to more focused tests.
Technically, further in vitro development of this kind of com-
pounds should be probably based on carbonAnitrogen bond as
the most stable form. Any further experiments moving the com-
pounds to GMP/pharmaceutical grade should take it into account.
In summary, we found two compounds, marked here as 8f and
8g with low toxicity and preserved suppressive activity make them
good candidates for further studies and considering as immuno-
suppressive drugs.
EC50 values of MDP/nor-MDP derivatives were lower than of
other compounds, but some conjugates had better selectivity—
higher antiproliferative activity than cytotoxicity. Conjugates 8f,
8b, 8g had the highest selectivity indexes (SIc) with values 44.7,
33.8 and 19.6, respectively (Table 2). Standard SI measured as
IC50/EC50 was the highest for the compound 8g and scored 1.12.
Cell viability was assessed in two samples of PBMC from differ-
ent donors (PBMC A, PBMC B). In PBMC A all the conjugates 8a–j as
well as MDP(D,D) and nor-MDP(D,D) derivatives 7e, 7f, 7i, 7j did
not affect cell viability as compared with the solvent. On the other
hand, adenosine 2, the conjugates 8k and 11a–b caused an increase
in the viability. In case of PBMC B, IC50 values of conjugates 8a–i
were in the range of 0.212–0.420 mg/mL (Table 2). The most active
conjugate was 8e (IC50 = 0.234 0.008 mM). Antiproliferative
activity was measured in three samples of PBMC (PBMC C, PBMC
D, PBMC E). Activated lymphocytes were susceptible to conjugates
with aminoethyl linker. The most active were 8a, 8d, 8f and 8g. An
antiproliferative effect was noticed in concentration 0.05 mg/mL,
in higher concentration cell proliferation was affected by a solvent
action (Fig. 2).
Acknowledgments
This work was supported by the Polish National Science Center
(NCN) under the Grant No.
N N405 046440 and Medicinal
University of Gdansk (Grant ST49).
Supplementary data
Supplementary data (experimental part including synthetic
procedures, characterization of compounds and biological activity
investigations) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.05.043.
We have successfully synthesized conjugates of MDP(D,D) and
nor-MDP(D,D) derivatives with adenosine and evaluated for their
antiproliferative activity. Generally, MDP(D,D) derivatives 7e, 7f,
7i, 7j were more active than their conjugates with adenosine. Aden-
osine 2 alone increased cell proliferation. The conjugates 8f, 8b, 8g
had the highest values of the selectivity index. The nature of a spacer
influenced the conjugates activity. Both cell lines as well as PBMC
were resistant to conjugates 11a–b, with the 6-aminohexanoic
linker. We assume that an amide bond is less stable than a car-
bonAnitrogen bond, leading to adenosine release and its fast metab-
olism. In case of PBMC an antiproliferative activity was noticed in
two out of three samples of activated lymphocytes.
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