New muramyl dipeptide (MDP) mimics without the carbohydrate moiety as potential adjuvant candidates for a therapeutic hepatitis B vaccine (HBV)

Article abstract of DOI:10.1016/j.bmcl.2011.05.056

A series of new muramyl dipeptide (MDP) mimics were designed and synthesized via a solid-phase synthetic route. Their adjuvant activities were evaluated ex vivo for investigation of the synergism of the S_28-39 peptide, which is an MHC class I b

Full text of DOI:10.1016/j.bmcl.2011.05.056

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4292–4295
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Bioorganic & Medicinal Chemistry Letters
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New muramyl dipeptide (MDP) mimics without the carbohydrate moiety zas
potential adjuvant candidates for a therapeutic hepatitis B vaccine (HBV)
Nan Zhao a, , Yao Ma a, , Shengmei Zhang a, Xin Fang b, Zhenglun Liang b, Gang Liu a,
⇑
a Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2 Nanwei Rd., Xicheng District, Beijing 100050, PR China
b National Institute for the Control of Pharmaceutical and Biological Products, 2 Tiantan Xili Rd., Dongcheng District, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new muramyl dipeptide (MDP) mimics were designed and synthesized via a solid-phase
synthetic route. Their adjuvant activities were evaluated ex vivo for investigation of the synergism of
the S28–39 peptide, which is an MHC class I binding epitope of recombinant hepatitis B surface antigen
(HBsAg) for both humans and mice. Several compounds without the carbohydrate moiety exerted better
adjuvanticity than the MDP-C that has been reported by our laboratory previously. A primary screening
test revealed that compounds 6, 14 and 16 exhibited stronger adjuvanticity compared with other MDP
mimics.
Received 13 March 2011
Revised 10 May 2011
Accepted 18 May 2011
Available online 25 May 2011
Keywords:
MDP mimics
Solid-phase synthesis
HBV
Ó 2011 Elsevier Ltd. All rights reserved.
Adjuvanticity
Hepatitis B virus (HBV) infection affects about 350 million peo-
ple globally and is a leading cause of hepatocellular carcinoma and
mortality.1 Although the antiviral drugs currently available effi-
ciently decrease HBV load in the human serum, they fail to eradi-
cate infection because of the persistence of HBV covalently
closed circular DNA (cccDNA) in hepatocytes and of the emergence
of resistant viruses.2 A vaccine designed to break tolerance and
stimulate virus-specific T-cell responses in chronic HBV patients
offers an interesting alternative therapeutic approach. The eradica-
tion of infected liver cells by both humoral and cytotoxic T-cell re-
sponses would be required for effective therapeutic vaccination.3
However, alum-based vaccines can only be used to promote hu-
moral and Th2-biased immune responses.4 Therefore, the develop-
ment of novel classes of adjuvant for an HBV therapeutic vaccine is
an urgent clinical need.
The muramyl dipeptide (MDP, Fig. 1), which is the minimal
immunologically active component of the peptidoglycan molecule
of the bacterial cell wall,5,6 is a potent adjuvant for antigens and in-
duces both humoral and cell-mediated responses in animal mod-
els.7 However, MDP yielded significant concomitant side effects
in vivo, such as pyrogenicity,8 poor penetration of cell mem-
branes,9 and rapid elimination,10 which limit its use in clinical
applications.
low-toxicity immunostimulator with potential for immunothera-
peutic and prophylactic applications in diseases such as HBV and
Severe Acute Respiratory Syndromes Coronavirus (SARS-CoV).11,12
However, the structure of MDP-C is an integration with a muramic
acid moiety, which renders the synthetic route difficult, costly, and
time consuming. This Letter reported new MDP mimics (Fig. 2) in
which the carbohydrate moiety was replaced by an aromatic group
and which exhibited improved immunological activity.
The solid-phase synthetic route shown in Scheme 1 was em-
ployed for parallel preparation of novel MDP mimics. Rink Amide-
AM resin was selected as the solid carrier. Fmoc-D-Glu-OtBu, differ-
ent Fmoc-protected lipophilic amino acids, and o-nitro benzoic acid
derivatives were assembled onto the resin successively under mild
coupling conditions. The subsequent reduction of the aromatic ni-
tro group in the presence of Tin(II) chloride and the reductive
ammoniation steps were performed to produce resin-bound com-
pounds successfully (f). Finally, the target products (g) were cleaved
off the resin. Twenty new MDP mimics (Table 1) were obtained
with a satisfied yield and were characterized fully using 1H NMR
and HR MS (TOF).
The ability of the new MDP mimics to enhance the antigenicity
of the HBsAg MHC I restricted peptide (S28–39: IPQSLDSWWTSL)
was examined using an ELISPOT assay, to select promising com-
pounds for further evaluation. BALB/c mice were immunized sub-
MDP-C (Fig. 1), which is a novel compound reported previously
by our group, proved to be an apyrogenic, nonallergenic, and
cutaneously on day 0 and boosted on day 7 with S28–39 (100
in 100 L of PBS) and the various MDP mimics (100 g in 100
of PBS). MDP-C (100 g in 100 L of PBS) was used as a positive
lg
l
l
lL
l
l
⇑
Corresponding author. Tel.: +86 10 63167165; fax: +86 10 63165246.
E-mail address: gliu@imm.ac.cns (G. Liu).
control. Mice were sacrificed 7 days after the last immunization.
These authors contributed equally to this work.
Splenocytes were collected and incubated with or without the
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.056

N. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4292–4295
4293
O
O
OBn
O
O
OH
HO
HO
HO
HO
NHAc
NHAc
O
NH2
O
NH2
O
O
H
N
H
N
H
H
N
N
OH
N
H
NO2
N
H
O
O
O
O
O
O
NH2
MDP
MDP-C
Figure 1. Structures of MDP and MDP-C.
O
R1
O
MDP mimics exhibited the strongest adjuvanticity when R4 was a
phenylethyl group (e.g., 6, 14 and 16). It is worthy of note that der-
ivation of aromatic amino group (R4) of compound 15 using phen-
ylethyl group to generate compound 16 resulted in extremely
remarkable improvement of activity. Further introduction of a
strong electron-withdrawing group, such as a nitro group or fluo-
rine atom, onto phenylethyl group at R4 doesn’t lead to an apparent
improvement in activity compared with the control group (e.g., 9,
11 and 17). Similarly, it also doesn’t demonstrate dramatic effects
on the activity for derivation of the aromatic amino group with cer-
tain unsaturated or saturated carbon chains (R4, e.g., 18 and 20).
The new MDP mimics were composed of three building blocks.
H
R2
R3
N
N
NH2
H
O
NH
R4
O
OH
Figure 2. Structural diversity of MDP mimics.
HBsAg MHC I restricted peptide S28–39 (20
plates. The difference in the number of IFN-
tained in the presence or absence of HBsAg peptide S28–39 stimula-
tion was considered as an indicator of the HBsAg-specific cellular
response. As illustrated in Table 1 and 70% of MDP mimics syner-
l
g/mL) in ELISPOT
c
-secreting cells ob-
D
-Gln was used to replace
phore configuration. The lipophilic amino acids at the N terminus
of -Gln, such as -Val, -Ala, -Phe, -Leu, and -Ile, were then se-
D-isoGln, to guarantee the pharmaco-
gized the HBsAg peptide S28–39 to produce IFN-
c at significantly
higher levels ex vivo compared with S28–39 alone. Among all the
compounds tested, five (1, 2, 6, 14 and 16) exhibited significantly
better activity compared with MDP-C. Among them, compounds
6, 14, and 16 had the strongest adjuvanticity, as assessed using this
test.
D
L
L
L
L
L
lected to assist the penetration of the cell membrane by the com-
pounds. Herein, we first introduced o-amino benzoic acid or its
derivatives to replace the muramic acid moiety, which greatly sim-
plified the synthetic route and significantly improved the adjuvan-
ticity of MDP ex vivo, in synergy with the HBsAg S28–39 peptide.
Previous studies of the SARs of MDP, MDP derivatives and MDP
analogues revealed that the immunostimulating activity was spe-
Analysis of structure–activity relations (SAR) revealed that the
methyl and benzyl groups were more efficient R1 groups regarding
the enhancement of the immunological activity of MDP mimics
(e.g., 6, and 14). Specially, the comparison of compounds 4, 6 and
19 showed that increasing the lipophilicity of R1 decreased the
adjuvanticity, meanwhile, compound 4 basically lost its activity
possibly due to the influence of isopropyl group in terms of in-
creased lipophilicity or steric factors. The adjuvanticity of MDP
mimics (e.g., 6 and 14) was the strongest when R2 was a chloro
atom. Regarding the R3 group, it seems that replacement with an
electron-donating group, such as a methoxyl group, was unable
to contribute significantly to the activity of the compound (e.g.,
7, 8, 13, 18 and 20). The electron-withdrawing group was not in-
cluded in this Letter. Obviously, R4 is a critical factor that contrib-
utes to the immunological adjuvant activity of the whole molecule.
cifically connected with the
molecules. Only very limited variations are allowed regarding ami-
no acid type (e.g., -Ala to -Val, -isoGln to -Gln) are allowed, but
not regarding configuration changes (e.g., -isoGln to
-isoGln).13–15
This study further confirmed the necessity of the configuration of
glutamine, however, hints that -Gln is the basic requirement of
L-Ala-D-isoGln pharmacophore of the
L
L
D
D
D
L
D
D
MDP mimics to guarantee its adjuvanticity. Most interestingly,
the muramic acid moiety was fully replaced by an aromatic moiety
for the first time, which completely changed the chemophysical
properties of the molecule, from hydrophilicity to hydrophobicity.
Obviously, this diversification prompts us to synthesize and charac-
terize additional adjuvants in the near future.
O
O
R1
O
R1
O
O
H
N
H
N
H
N
H
N
I
II, III
II, IV
Fmoc
Fmoc
R2
R3
OH
N
N
N
H
Fmoc
N
N
H
H
H
H
O
d
O
O
Ot-Bu
O
Ot-Bu
b
NO2
O
Ot-Bu
O
Ot-Bu
a
c
R1
O
O
R1
O
O
R1
O
O
H
N
H
H
N
R2
R3
R2
R3
N
R2
R3
N
N
VI
VII
V
N
H
NH2
N
N
H
H
H
H
O
e
O
O
NH2
O
Ot-Bu
NH
R4
O
Ot-Bu
NH
R4
O
OH
f
g
= Rink Amide-Am Resin
Scheme 1. Reagents and conditions: (a) HOBt, DIC, and DMF, rt, 3 h; (b) 20% piperidine/DMF, rt, 20 min, two times; (c) Fmoc-protected amino acids, HOBt, DIC, and DMF, rt,
3 h; (d) o-nitro benzoic acid derivatives, HOBt, DIC, and DMF, rt, 3 h; (e) 2 M SnCl2, NMM, and DMF, rt, 12 h; (f) organic aldehyde, NaH3BCN, AcOH, and DMF; 40 °C, 36 h; (g)
95% TFA/H2O, rt, 1 h.

4294
N. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4292–4295
Table 1
SAR investigation of twenty new MDP mimics
Compd.
R1
R2
Cl
R3
H
R4
H
IFN-
c
SFCs/4 Â 105 splenocytesa
1
212.5 19c,d
224.0 24c,d
164.0 26b
2
3
H
H
H
H
H
4
5
6
7
8
Cl
H
14.3
7
Cl
H
H
H
122.5 15
316.0 20c,d
186.8 47b
185.8 49b
Cl
H
CH3O
CH3O
CH3O
CH3O
9
H
H
H
H
102.0 37
173.3 21c
NO2
10
11
12
H
H
H
H
116.3 22
139.5 9b
O2N
H
H
13
14
CH3O
CH3O
144.3 30
Cl
H
H
H
317.0 46c,d
15
16
17
H
31.8 11
H
H
304.3 17c,d
130.3 10b
F
H
H
18
19
CH3O
CH3O
117.8
9
Cl
H
125.3 9b
125.3 29
20
CH3O
CH3O
a
HBsAg-specific IFN-
c
ELISPOT responses after immunization with various MDP mimics + HBsAg MHC I restricted peptide S28–39. BALB/c mice were immunized subcu-
g in 100 L of PBS) and the various MDP mimics (100 g in 100 L of PBS), or S28–39 alone. MDP-C was used as a
positive control. Mice were sacrificed 7 days after the last immunization and splenocytes were collected. Isolated splenocytes were tested for HBsAg MHC I restricted peptide
28–39-specific IFN- secretion using the ELISPOT assay. Data were expressed as mean SEM.
taneously on day 0 and boosted on day 7 with S28–39 (100
l
l
l
l
S
c
b
p <0.05 versus S28–39 alone.
p <0.01 versus S28–39 alone.
p <0.01 versus MDP-C.
c
d
In summary, we have designed and synthesized a class of new
MDP mimics that are composed of three building blocks and in-
clude the replacement of the carbohydrate with an aromatic moi-
ety. This was easily performed via the soliD-phase synthetic route.
All 20 compounds synthesized were primarily evaluated ex vivo
through the investigation of the synergism between new MDP
mimics and the S28–39 peptide. Compounds 6, 14, and 16 were the
MDP mimics that exhibited the relatively strongest adjuvanticity.
Since MDP and its mimics were reported to be NOD2 ligands16–19
or TLR2/TLR4 agonists,20 compounds 6, 14, and 16 may also act
on NOD2 or TLR2/TLR4 signaling. Further research is needed to
identify the target.

N. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4292–4295
4295
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