Antagonizing NOD2 Signaling with Conjugates of Paclitaxel and Muramyl Dipeptide Derivatives Sensitizes Paclitaxel Therapy and Significantly Prevents Tumor Metastasis

Article abstract of DOI:10.1021/acs.jmedchem.6b01704

A noncleavable paclitaxel (PTX) and N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) derivative conjugate, 22 (DY-16-43), and its analogues were prepared and characterized as antagonists of NOD2 signaling. This conjugate enhanced the antitumor and antimetastatic efficacy of PTX in Lewis lung carcinoma (LLC) tumor-bearing mice. This work first describes a molecular strategy that enables the sensitization of a chemotherapeutic response via antagonizing NOD2 inflammatory signaling and suggests NOD2 antagonist as potential adjunct in treating non-small-cell lung cancer (NSCLC).

Full text of DOI:10.1021/acs.jmedchem.6b01704

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Brief Article
Antagonizing NOD2 Signaling with Conjugates of Paclitaxel
and Muramyl Dipeptide Derivatives Sensitizes Paclitaxel
Therapy and Significantly Prevents Tumor Metastasis
Yi Dong, Suhua Wang, Chunting Wang, Zihua Li, Yao Ma, and Gang Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01704 • Publication Date (Web): 11 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
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Journal of Medicinal Chemistry
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Antagonizing NOD2 Signaling with Conjugates of Paclitaxel and Mu-
ramyl Dipeptide Derivatives Sensitizes Paclitaxel Therapy and Sig-
nificantly Prevents Tumor Metastasis
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,#
,#
, ,
,
Yi Dong† , Suhua Wang‡ , Chunting Wang†, Zihua Li†, Yao Ma† ‡ *, Gang Liu† *
†School of Pharmaceutical Sciences, Tsinghua University, Haidian Dist, Beijing 100084, P.R. China
‡Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 2A Nanwei Rd,
Xicheng Dist, Beijing 100050, P.R. China
ABSTRACT: A non-cleavable paclitaxel (PTX) and N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) derivative conjugate, 22
(DY-16-43), and its analogues were prepared and characterized as antagonists of NOD2 signaling. This conjugate enhanced the anti-
tumor and anti-metastatic efficacy of PTX in Lewis lung carcinoma (LLC) tumor-bearing mice. This work first describes a molecular
strategy that enables the sensitization of a chemotherapeutic response via antagonizing NOD2 inflammatory signaling and suggests
NOD2 antagonist as potential adjunct in treating non-small-cell lung cancer (NSCLC).
MDP analogues have been extensively designed and synthe-
■ INTRODUCTION
Pattern-recognition receptors (PRRs) activate signaling path-
sized.10 One drug in particular, namely, L-MTP-PE or mifamur-
tide (liposomal muramyl tripeptide phosphatidyl ethanolamine),
has been approved in Europe for the combination treatment of
osteosarcoma.11 Free L-MTP-PE, which can activate monocytes
or macrophages and increase the production of pro-inflamma-
tory cytokines, including tumor necrosis factor (TNF)-, inter-
leukin (IL)-1, IL-6 and IL-8, has been speculated to be an MDP
pro-drug via NOD2 stimulation.12 We have previously de-
scribed 1 (MTC-220), 13 which is a conjugate of PTX and an
MDP derivative that is currently under investigation as a new
drug (IND issued number: CXHL1502461, CHINA). Pharma-
cological studies have shown that 1 not only retained the ability
of PTX to inhibit tumor growth but also prevented tumor me-
tastasis in vivo by reversing the inflammatory TME.13 Pharma-
cokinetic studies have revealed that 1 exhibited a profile of high
plasma concentration and rapid clearance in rats after intrave-
nous administration, while only small amounts of its metabo-
lites (PTX and MDA-linker, Fig 1) were observed in rat
plasma.14 Further experiments demonstrated that 1 increased
the intratumoral concentration of PTX by nearly two fold in tu-
mor-bearing mice compared with PTX therapy alone (Table 1).
Since the MDA-linker is not an agonist or antagonist of NOD2
signaling (Fig. 2A) and has no effect on either tumor growth or
the immune response (data not shown), we speculated that the
prototype 1 may account for the TME remodeling by affecting
NOD2 signaling, resulting in increased intratumoral PTX and
decreased lung metastasis in tumor-bearing mice compared
with PTX treatment alone.13
ways that induce various immune responses by recognizing
pathogen-associated molecular patterns (PAMPs) or damage-
associated molecular patterns (DAMPs).1 Toll-like receptors
(TLRs) are the most well-known PRRs, whereas NLRs are a
newly discovered family.2 NOD proteins NOD1 and NOD2 be-
long to the nodosome sub group of NLRs, which recognize bac-
terial peptidoglycan components, leading to activation of NF-
B and MAPK-mediated inflammatory response.3 In particular,
NOD1 recognizes L-Ala-c-D-Glu-meso-diaminopimel- ic acid
(Tri-DAP) present in gram-negative bacilli and certain gram-
positive bacteria, while muramyl dipeptide (MDP) in both
gram-positive and gram-negative bacteria is selectively recog-
nized by NOD2.4
Recent studies have revealed that TLR signals contribute to tu-
mor progression in the tumor microenvironment (TME).5
PAMPs derived from microbes and DAMPs derived from in-
jured tissue or/and necrotic cancer cells could activate TLRs ex-
pressed on immune cells and on cancer cells.6 TLR activation,7
especially that of TLR4 in tumor cells, can induce inflammatory
cytokine and chemokine production, increase tumor cell prolif-
eration and apoptosis resistance, promote invasion and metas-
tasis, and inhibit immune cell activity, resulting in tumor im-
mune escape.8 However, the roles of NLRs, especially NOD2,
in regulating tumor growth and metastasis remain largely un-
clear.9
Herein, we describe a molecular strategy that enables the sen-
sitization of a chemotherapeutic response via antagonizing
NOD2 inflammatory signaling and suggests NOD2 antagonist
as potential adjunct in treating non-small-cell lung cancer
(NSCLC).
Herein, we designed and synthesized a non-cleavable conjugate
of 1, 22 (DY-16-43) 15 (Fig. 1) and its derivatives, which were
first characterized as NOD2 antagonists by using an HEK-Blue
hNOD2-secreted alkaline phosphatase (SEAP) reporter cell line
(Fig. 2A). The low toxicity of 22 was further confirmed in
HEK-Blue hNOD2 cells using the established sulphorhodamine
B assay (Fig. S1 in supporting information).
■ RESULTS AND DISCUSSION
To extend the analysis of 22 beyond reporter gene assays and
immortalized cell lines, we measured the mRNA levels of an
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inhibited the mRNA expression of IL-6 induced by MDP
NF-B-inducible cytokine, IL-6, in human peripheral blood
mononuclear cell (PBMC)-derived macrophages. Again, 22
1
2
3
4
5
6
7
8
(Fig.2B). Since NOD2 stimulated pro-inflammatory cytokine
and chemokine release primarily via the activation of NF-κB
and MAPK pathways, the ability of 22 to inhibit both NF-κB
and MAPK pathways was further investigated via Western blot
for total IκBɑ and phosphorylated RIP2, p38, JNK and ERK
levels in 293/hNOD2 stable cell lines and human PBMC-
derived macrophages stimulated with MDP. As shown in Figs.
2C and S2, 22 antagonized the MDP-induced increase in p-
RIP2, p-ERK, p-p38 and p-JNK levels and reduced IκBɑ levels
in a concentration-dependent manner. Taken together, along
with the lack of RIP2 kinase inhibition (Fig. S3 in supporting
information), these results suggested that 22 targets NOD2 sig-
naling upstream of RIP2. To further characterize the molecular
target of 22, we examined the effects of 22 on NOD2 self-oli-
gomerization using co-immuno precipitation and immuno blot-
ting of His- and Flag-tagged NOD2 expressed in HEK293T
cells. Our results (Fig. 2D) showed that 22 inhibited NOD2 ol-
igomerization, which is one of the most upstream events of
NOD2 activation and signaling.2
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22
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Figure 1. Chemical structures of MDA, MDA-linker, 1 and 22
Table1. Pharmacokinetic parameters in tumor tissue of LLC tumor bearing mice after iv. injection of compound 1 or PTX at equimo-
lar dose (n=5)
1(30mg/kg)
PTX(18mg/kg)
PTX
Parameters
Unit
PTX
MDA-Linker
10.4±2.19
1.01±0.54
16.27±4.61
1
Tmax
Cmax
h
6.8±3.35
12**±6.93
5.83±1.41
1.05±0.62
4.85±0.74
46.62±3.05
nmol/g
nmol/g*h
15.39±4.15
211.02±56.08
AUC(0-t)
89.63*±40.34
*p < 0.05, **p < 0.01 vs. PTX group by student t test.
(B)
(A)
60
50
40
30
20
10
0
22
Figure 2. (A) Dose-dependent inhibitory response of MDP (5
µg/mL)-induced NOD2 activation by 22 and MDA-linker in HEK-
Blue hNOD2 cells. (B) 22 inhibits NOD2-mediatedIL-6 mRNA ex-
pression in human PBMC derived macrophages. (C) 22 inhibits
NOD2-mediated NF-κB and MAPK signaling in 293/hNOD2 cells.
(D) 22 inhibits MDP-induced self-oligomerization of NOD2 in
HEK293T cells. An NOD2 selective inhibitor (NOD2 Inh) was
used as positive control (Fig. S4 in supporting information)
80
60
40
20
0
+ MDP(5g/mL)
MDA-linker
22 (10μ M)
22 (1μ M)
NOD2 Inh(1μ M)
-10
-20
-10
-9
-8
-7
-6
-5
Log10 of sample concentration (Molar)
(C)
Next, we tested the efficacy of a 22 plus PTX combination ther-
apy in the LLC model, which recapitulates human NSCLC that
responds poorly to PTX. As shown in Fig. 3, the combination
with 22 markedly improved the therapeutic efficacy of PTX, not
only by reducing tumor weight but also by a two-fold increase
in tumor metastasis prevention. Interestingly, in the absence of
PTX, 22 showed no significant beneficial effects. Thereafter,
we speculated that NOD2 in tumor-bearing mice may be mainly
activated by DAMPs derived from chemotherapy, thus contrib-
uting to TME remodeling, chemo resistance and cancer metas-
tasis. Herein, we show that the inhibition of NOD2 inflamma-
tory signaling sensitizes chemotherapy, probably by preventing
the formation of an inflammatory TME induced by chemother-
apy.
1
+
1
+
10
MDP
(5μg/mL)
-
+
+
p-RIP2
RIP2
1
IκBα
p-ERK
ERK
p-JNK
JNK
p-p38
p38
β-actin
(D)
MDP
22
-
-
-
+
-
-
+
+
-
+
The chemical synthesis of 22 was retrosynthetically analyzed
(Scheme S2 in supporting information). The 22 and its deriva-
tives were fragment conjugated by a PTX linker and an MDA
peptide (Fig. 1) that is efficiently synthesized by employing a
-
NOD2 Inh
+
Flag-NOD2
IP: His IB: Flag
IP: His IB: His
His-NOD2
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solid-phase synthetic technology with an Fmoc protection strat- but no desired ester was produced (entry 9). When the substrate
1
2
3
4
5
6
7
8
egy. Therefore, the selective and efficient alkylation of 2’-OH
in the side chain of PTX to create the corresponding carboxylic
PTX linker (Scheme 1) is critical.
and benzyl but-3-enoate (linker 5) were treated with a Lewis
acid, i.e., N-(phenylseleno)phthalimde(N-psp)and boron triflu-
oride etherate in DCM and 0C to r.t., no reaction even occurred
(entry 10). In 2008, the gold(I)-catalyzed intermolecular hy-
droalkoxylation of allenes with alcohols to synthesize alkyl al-
lylic esters was reported by Widenhoefer et al.16 Inspired by
their synthetic strategy, allene (linker 6) was studied under the
same conditions. Unfortunately, no desired allylic ester was
produced in THF, DCM or toluene (entries 11~13). The hy-
droalkoxylation of alkenes under basic conditions was further
assayed, but no reactions were observed under these conditions
either (linker 7, entry 15). When linker 7 was treated with NaH
in THF from 0C to r.t., trace amounts of the desired addition
product could be isolated (entry 16). When treated with N-
methylmorpholine (NMM) but not pyridine in DCM, 71% of
the desired compound could be isolated (entries 17 and 18).
However, this condition suffered from PTX, as only trace
amounts of the desired alkylated product 2’-OH of PTX were
produced. Fortunately, the same conditions tested for alkynes
(linker 8) were completely successful in the presence of NMM
in DCM at r.t., with a yield of 96% of olefinated product from
the model reaction (entry 19) and a 90% yield of the PTX linker.
(A)
60
(B)
***
##
** ***
100
80
60
40
20
0
###
22 (30mg/kg)
PTX(12mg/kg)
22 + PTX
9
40
20
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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(C)
Lung metastasis
22
PTX
(12mg/kg)
Control
22+PTX
Table 2. Investigation of the Alkylation of OH in the Model
Reaction
(30mg/kg)
Figure 3. 22 sensitized the PTX treatment of LLC. (A) Tumor
weight inhibition rate. (B) Lung metastasis number inhibition rate.
(C) Representative images of lung metastases. Data shown as the
mean ± SEM (n=10); **p < 0.01, ***p < 0.001 vs. the 22 group;
##p < 0.01, ###p < 0.001 vs. the PTX group
Scheme 1. Method of Synthesizing 22 and Its Analogues
Yield
(%)
Entry
Linker
Conditions
1
2
Linker 1
Linker 1
K2CO3, THF, 0℃~r.t.
NaH, THF, 0℃~r.t.
0%
D
3
4
5
6
7
Linker 2
Linker 2
Linker 2
Linker 3
Linker 3
NaH, THF, 0℃~r.t.
NaH, DMF, 0℃~r.t.
BuLi, THF, 0℃~r.t.
NaH, THF, 0℃~r.t.
NaH, DMF, 0℃~r.t.
T
T
T
T
T
8
9
10
11
Linker 3
Linker 4
Linker 5
Linker 6
BuLi, THF, 0℃~r.t.
DIAD, PPh3, THF, r.t.
N-psp, BF3/Et2O, DCM
AgOtf, AuCl, THF
T
0%
NR
0%
12
13
14
15
16
17
18
19
Linker 6
Linker 6
Linker 6
Linker 7
Linker 7
Linker 7
Linker 7
Linker 8
AgOtf, AuCl, DCM
AgOtf, AuCl, toluene
NMM, DCM, r.t.
DBU, THF, r.t.
NaH, THF, 0℃~r.t.
NMM, DCM, r.t.
Pyd, DCM, r.t.
0%
0%
0%
NR
T
71%
0%
96%
To investigate the synthesis of the PTX linker, 2-BocNH alco-
hol of the PTX side chain was chosen as the model substrate for
optimizing the alkylation conditions of OH group (Table 2). In-
itial tests focused on the alkylation of OH group with benzyl
pentanoate halide [Br (linker 1) and I (linker 2)] under various
basic conditions, including those of inorganic bases (K2CO3,
NaH) and organic base (BuLi) in dry DMF or THF; however,
no or trace amounts of the desired compound were detectable
by LC-MS (entries 1~5). (E)-methyl 4-bromobut-2-enoate
(linker 3) was also tested under the same conditions, but only
trace amounts of the desired alkylated product were observed
(entries 5~7). The Mitsunobu reaction was then investigated by
using diisopropyl azodicarboxylate(DIAD), PPh3 and benzyl 4-
hydroxybutanoate (linker 4) in THF at room temperature (r.t.),
NMM, DCM, r.t.
Isolated yield. D = decomposed, T = trace, NR = no reaction,
DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, Pyd = pyridine.
Based on this successful selective olefination on the 2’-OH of
PTX, the gram-scale synthesis of 22 was performed according
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to the following procedure (Scheme 1). The olefinated PTX in- In conclusion, we first report the synthesis of an antagonist of
1
2
3
4
5
6
7
8
termediate was transformed into the corresponding PTX linker
with a quantitative yield under a Pd-catalytic hydrogenation
condition in ethyl acetate at 50C. The final amidation between
the PTX linker and MDA (or one of its analogues) was per-
formed by activating the PTX linker via 1-hydroxybenzotria-
NOD2, 22, which is a conjugate of PTX and MDP derivatives
via an ether bond linkage and is otherwise functionally different
from MDP and MTP-PE, agonists of NOD2. This antagonist
could significantly enhance the anti-tumor and anti-metastatic
efficacy of PTX in LLC tumor-bearing mice. This work de-
scribes a molecular strategy of antagonizing NOD2 inflamma-
tory signaling (or TME remodeling) that significantly sensitizes
the chemotherapeutic response, very likely via blocking
DAMPs activation. Collectively, these findings suggest that
NOD2 antagonists have potential as adjunct therapies in treat-
ing NSCLC.
zole(HOBt)
and
1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDCI) in DMSO overnight and sub-
sequently adding MDA under stirring in the presence of 4 equiv
NMM at r.t. for 30 min. The desired conjugate 22 or its ana-
logues were finally produced with good yields after purification
via a semi-preparative HPLC. PTX consists of two major frag-
ments, 10-deacetylbaccation III (10-DAB) and a side chain. To
explore which of the two fragments contributes the least to an-
tagonizing NOD2, we further synthesized three conjugates of
10-DAB with MDA at the 7-, 10-, and 13- positions, and at one
side chain of PTX with MDA, by a similar procedure (see sup-
porting information).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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40
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■ EXPERIMENTAL SECTION
General experiment. All biologically evaluated compounds
(2~28) were finally purified by a semi-preparative reversed
phase C18 column chromatography with H2O and MeOH until
≥95% detected by our UPLC-MS and 1H NMR.
Scheme 2 lists all the prepared conjugates discussed in this pa-
per. Obviously, the complete structure of PTX is necessary for
maintaining antagonistic activity via MDP activating NOD2
signaling because compounds 24~28 presented much lower in-
hibitory percentages at the tested concentrations. Our previous
report indicated that conjugates of PTX with MDP (2-O-MTC-
01) as an agonist synergistically induced the production of
TNF- and IL-12 in murine peritoneal macrophages but did not
inhibit tumor metastasis in LLC-bearing mice.17 2-O-MTC-01
contains a natural muramic acid moiety of MDP; however, that
moiety was replaced by a cinnamic acid derivative in 1.13 In this
paper, the prototype 22 is first demonstrated as an antagonist of
NOD2 signaling that sensitizes PTX therapy and significantly
prevents tumor metastasis in tumor-bearing mice. The replace-
ment of muramic acid by cinnamic acid derivatives reversed the
molecule’s biological function as an antagonist and rendered it
a potential treatment for cancer superior to PTX treatment alone.
Various cinnamic acid derivatives were assembled onto the N-
terminal of a dipeptide (L-Ala-D-isoGln), which is a pharmaco-
phore of MDP.10 Among them, compounds 22 and 23 exhibited
the highest levels of antagonistic activity. We chose 22 as a
working molecule because it was more soluble in the various
tested condition.
Preparation of 22. 15mmol, 15g rink-amide resin (0.8 mmol/g)
and 100 mL DCM were added into a 200 mL solid-phasereac-
tion bottle to swelling resin for 30 min at r.t. DCM was then
removed, and the resin was washed again by 100 mL DMF. 50
mL piperidine solution (20% in DMF in volume)was added, and
the mixture was shaken 1h, then solvent was removed and the
resin was washed by 100mL DMF for three times. 7.03 g Fmoc-
Lys(Boc)-OH, 3.04 g HOBt, 3.49 mLDIC in 100 mL DMF were
added. The mixture was shaken, and the reaction was monitored
by ninhydrintest solution. When the reaction was completed,
DMF was removed and the resin was washed by 100 mL DCM
and 100 mL DMF for two times respectively, then 50mL 20%
piperidine in DMF was added again. The mixture was shaken
1h, DMF was removed and the resin was washed by DCM and
DMF for three times, respectively. After this step, the first
amino acid was introduced onto resin. The above steps to cou-
ple Fmoc-D-isoGln-OH, Fmoc-Ala-OH were subsequently re-
peated until the last 4-chlorocinnamic acid were successfully
assembled onto resin. The final peptide-resin was washed by
DMF and DCM for two times, respectively, after DMF was
drained. The peptide-resin was then completely dried under a
vacuum condition. Then the 50 mL mixed solution of trifluoro-
acetic acid (TFA) and DCM (v/v = 9:1) was added and the mix-
ture was shake for 2h at rt. The reaction mixture was filtered
and washed by DCM for two times. Then the filtrates were com-
bined and the solvent was removed under a reduced pressure.
500 mL cold Et2O was added at 0℃, white solid (MDA·TFA)
was precipitated. The white solid was used without further pu-
rification.
Scheme 2. Structure-Activity Relationship Analysis in MDP
(100ng/mL) Stimulated HEK-Blue hNOD2 Cells a
5 mmol, 4.27 g paclitaxel was dissolved in 50 mL DCM, 5.5
mmol NMM and 5.5 mmol benzyl propionate were then added,
and the reaction mixture was stirred at rt for 4h and purified by
silica gel column chromatography to afford 2’-OH olefinated
paclitaxelas a white solid (4.56 g, 90% yield). This intermediate
was then dissolved in 50 mL ethyl acetate, and 2.0 g Pd/C was
added, the mixture was stirred at 50C under hydrogen atmos-
phere overnight. The reaction mixture was filtered, and filtrate
was concentrated to afford 4.16 g paclitaxel-linker as a white
solid. This paclitaxel-linker was dissolved in 5 mL DMSO, 518
mg HOSu and 863 mg EDCI were added. The reaction mixture
was stirred overnight, then 2.8 g MDA·TFA and 1.98 mL N-
Methylmorpholine were added, the reaction mixture was stirred
a The Percentages Represent the Inhibitory Rates of the Com-
pound at 10M
■ CONCLUSIONS
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trib. Microbiol. 2008, 15, 45-60. (b) Lagiedo, M.; Sikora, J.; Kaczma-
rek, M. Damage-associated molecular patterns in the course of lung
cancer - a review. Scand. J. Immunol. 2015, 82, 95-101.
at rt for 30 min and purified by reversed phase column chroma-
1
2
3
4
5
6
7
8
tography [Heda ODS-2 C18 (25020mm) column] with H2O
and MeOH to afford 22 as a white solid (5.1 g).The running
solvents were A (MeOH) and B (H2O) with pH=2~3. The gra-
dient of purification with 5 mL/min was 5% A in B (0-5min), A
went to 30% (5-10 min), A went to 95% (10-80 min), A went
100% A (80-85 min) and finally A was back to 5% (85-90 min).
1HNMR (400MHz, DMSO-d6) δ1.00 (s, 3H), 1.03 (s, 3H),
1.12-1.24 (m, 2H), 1.23-1.33 (m, 5H), 1.40-1.46 (m, 1H), 1.50
(s, 3H), 1.57-1.61 (m, 2H), 1.62-1.68 (m, 1H), 1.70-1.76 (m,
2H), 1.80 (s, 3H), 1.95-2.05 (m, 1H), 2.11 (s, 3H), 2.16 (s, 5H),
2.26-2.34 (m, 1H), 2.35-2.41 (m, 2H), 2.80-3.00 (m, 2H), 3.60
(d, 1H, J = 6.4Hz), 3.70-3.85 (m, 2H), 3.97 (d, 1H, J = 7.8 Hz),
4.03 (d, 1H, J = 8.4Hz), 4.07-4.17 (m, 3H), 4.35-4.45 (m, 2H),
4.65 (brs, 1H), 4.87-4.94 (m, 2H), 5.31 (t, 1H, J =9.1Hz), 5.40
(d, 1H, J = 6.9Hz), 5.88 (t, 1H, J =8.8Hz), 6.31 (s, 1H), 6.76 (d,
1H, J = 15.9Hz), 6.94 (s, 1H), 7.08 (s, 1H), 7.16-7.24 (m,
1H),7.28 (s, 2H), 7.35-7.45 (m, 5H), 7.45-7.52 (m, 4H), 7.53-
7.61 (m, 3H), 7.62-7.67 (m, 2H), 7.70-7.76 (m, 1H), 7.78-7.90
(m, 4H), 7.95 (d, 2H, J =6.8Hz), 8.19 (d, 1H, J = 7.4Hz), 8.35
(d, 1H, J = 5.5Hz).8.97 (d, 1H, J =8.0Hz). 13CNMR (100 MHz,
DMSO-d6) δ 202.3, 173.9, 173.3, 172.3, 171.5, 171.0, 169.8,
169.3, 168.8, 166.2, 165.1, 164.7, 138.6, 138.2, 137.6, 134.5,
133.9, 133.8, 133.6, 133.5, 131.4, 129.9, 129.5, 129.2, 129.0,
128.7, 128.4, 128.3, 127.8, 127.7, 127.4, 122.7, 83.6, 81.3, 80.2,
76.6, 75.3, 74.7, 74.4, 70.4, 70.0, 66.6, 57.4, 55.3, 52.4, 52.1,
48.8, 46.1, 42.9, 38.4, 36.5, 35.8, 34.4, 31.7, 31.6, 28.8, 27.7,
26.3, 23.0, 22.8, 21.2, 20.7, 18.1, 14.3, 9.7.HRMS (ESI): m/z
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Supporting Information
Full details of the general methods and full characterization of the
individual compounds examined are included. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*G. Liu: phone, 86-10-62792820; e-mail, gangliu27@tsing-
hua.edu.cn
*Y. Ma: phone, 86-10-62787371; e-mail, mayao@imm.ac.cn
Author Contributions
Y. Dong and S. Wang contributed equally to this work.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge the funding support of grants from the
National Natural Science Foundation of China (Nos. 81273364 and
91213303). We thank Dr. Jijie Chai (Tsinghua University, Bei-
jing, China), who kindly provided PcDNA3.1-Nod2-Hisplas-
mid.We also thank Litao Zheng, Wenjun Yu and Xueyuan Li, who
provided assistance and produced intermediates and final products
on a large scale.
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novel anti-tumo ragent of conjugate of paclitaxel and muramyl dipep-
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