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Jan 11, 2017 - Antagonizing NOD2 Signaling with Conjugates of Paclitaxel and Muramyl Dipeptide Derivatives Sensitizes Paclitaxel Therapy and Significa...
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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

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†,‡,*, 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 antitumor 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).

■ INTRODUCTION Pattern-recognition receptors (PRRs) activate signaling pathways that induce various immune responses by recognizing pathogen-associated molecular patterns (PAMPs) or damageassociated 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 belong to the nodosome sub group of NLRs, which recognize bacterial 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 grampositive bacteria, while muramyl dipeptide (MDP) in both gram-positive and gram-negative bacteria is selectively recognized by NOD2.4 Recent studies have revealed that TLR signals contribute to tumor progression in the tumor microenvironment (TME).5 PAMPs derived from microbes and DAMPs derived from injured tissue or/and necrotic cancer cells could activate TLRs expressed 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 proliferation and apoptosis resistance, promote invasion and metastasis, and inhibit immune cell activity, resulting in tumor immune escape.8 However, the roles of NLRs, especially NOD2, in regulating tumor growth and metastasis remain largely unclear.9 Herein, we describe 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).

■ RESULTS AND DISCUSSION

MDP analogues have been extensively designed and synthesized.10 One drug in particular, namely, L-MTP-PE or mifamurtide (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-inflammatory cytokines, including tumor necrosis factor (TNF)-, interleukin (IL)-1, IL-6 and IL-8, has been speculated to be an MDP pro-drug via NOD2 stimulation.12 We have previously described 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). Pharmacological studies have shown that 1 not only retained the ability of PTX to inhibit tumor growth but also prevented tumor metastasis in vivo by reversing the inflammatory TME.13 Pharmacokinetic studies have revealed that 1 exhibited a profile of high plasma concentration and rapid clearance in rats after intravenous administration, while only small amounts of its metabolites (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 tumor-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 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). 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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Journal of Medicinal Chemistry NF-B-inducible cytokine, IL-6, in human peripheral blood mononuclear cell (PBMC)-derived macrophages. Again, 22

Figure 1. Chemical structures of MDA, MDA-linker, 1 and 22

inhibited the mRNA expression of IL-6 induced by MDP (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 PBMCderived macrophages stimulated with MDP. As shown in Figs. 2C and S2, 22 antagonized the MDP-induced increase in pRIP2, 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 signaling upstream of RIP2. To further characterize the molecular target of 22, we examined the effects of 22 on NOD2 self-oligomerization using co-immuno precipitation and immuno blotting of His- and Flag-tagged NOD2 expressed in HEK293T cells. Our results (Fig. 2D) showed that 22 inhibited NOD2 oligomerization, which is one of the most upstream events of NOD2 activation and signaling.2

Table1. Pharmacokinetic parameters in tumor tissue of LLC tumor bearing mice after iv. injection of compound 1 or PTX at equimolar dose (n=5) 1(30mg/kg) Parameters

PTX(18mg/kg)

Unit 1

PTX

MDA-Linker

PTX

Tmax

h

6.8±3.35

12**±6.93

10.4±2.19

1.05±0.62

Cmax

nmol/g

15.39±4.15

5.83±1.41

1.01±0.54

4.85±0.74

AUC(0-t)

nmol/g*h

211.02±56.08

89.63*±40.34

16.27±4.61

46.62±3.05

*p < 0.05, **p < 0.01 vs. PTX group by student t test. 60 50 40 30 20 10 0 -10 -20 -10

(B) 22 MDA-linker

+ MDP(5g/mL)

80

% inhibition

(A)

% inhibitory activity

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60 40 20

-9 -8 -7 -6 -5 Log10 of sample concentration (Molar)

0

(C)

MDP (5μg/mL)

-

+

10

1

1

+

+

+

p-RIP2 1

RIP2

IκBα p-ERK ERK p-JNK

JNK p-p38 p38 β-actin

(D)

MDP 22 NOD2 Inh

-

+ -

+ + -

+ +

Flag-NOD2

IP: His IB: Flag

His-NOD2

IP: His IB: His

22 (10μ M) 22 (1μ M) NOD2 Inh(1μ M)

Figure 2. (A) Dose-dependent inhibitory response of MDP (5 µg/mL)-induced NOD2 activation by 22 and MDA-linker in HEKBlue hNOD2 cells. (B) 22 inhibits NOD2-mediatedIL-6 mRNA expression 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)

Next, we tested the efficacy of a 22 plus PTX combination therapy 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 contributing to TME remodeling, chemo resistance and cancer metastasis. Herein, we show that the inhibition of NOD2 inflammatory signaling sensitizes chemotherapy, probably by preventing the formation of an inflammatory TME induced by chemotherapy. The chemical synthesis of 22 was retrosynthetically analyzed (Scheme S2 in supporting information). The 22 and its derivatives were fragment conjugated by a PTX linker and an MDA peptide (Fig. 1) that is efficiently synthesized by employing a

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solid-phase synthetic technology with an Fmoc protection strategy. 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. (A)

(B)

***

60

40

20

0

###

% Metastasis No. inhibition

** *** % Tumor weight inbition

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100

##

22 (30mg/kg) PTX(12mg/kg) 22 + PTX

80 60 40 20 0

(C)

Lung metastasis

Control

22 (30mg/kg)

PTX (12mg/kg)

22+PTX

but no desired ester was produced (entry 9). When the substrate and benzyl but-3-enoate (linker 5) were treated with a Lewis acid, i.e., N-(phenylseleno)phthalimde(N-psp)and boron trifluoride etherate in DCM and 0C to r.t., no reaction even occurred (entry 10). In 2008, the gold(I)-catalyzed intermolecular hydroalkoxylation of allenes with alcohols to synthesize alkyl allylic 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 hydroalkoxylation 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 Nmethylmorpholine (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. Table 2. Investigation of the Alkylation of OH in the Model Reaction

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

To investigate the synthesis of the PTX linker, 2-BocNH alcohol of the PTX side chain was chosen as the model substrate for optimizing the alkylation conditions of OH group (Table 2). Initial 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 4hydroxybutanoate (linker 4) in THF at room temperature (r.t.),

Entry

Linker

Conditions

1

Linker 1

2

Linker 1

K2CO3, THF, 0℃~r.t. NaH, THF, 0℃~r.t.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Linker 2 Linker 2 Linker 2 Linker 3 Linker 3 Linker 3 Linker 4 Linker 5 Linker 6 Linker 6 Linker 6 Linker 6 Linker 7 Linker 7 Linker 7 Linker 7 Linker 8

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. BuLi, THF, 0℃~r.t. DIAD, PPh3, THF, r.t. N-psp, BF3/Et2O, DCM AgOtf, AuCl, THF 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. NMM, DCM, r.t.

Yield (%) 0% D T T T T T T 0% NR 0% 0% 0% 0% NR T 71% 0% 96%

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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Journal of Medicinal Chemistry

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to the following procedure (Scheme 1). The olefinated PTX intermediate 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 performed by activating the PTX linker via 1-hydroxybenzotriazole(HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in DMSO overnight and subsequently adding MDA under stirring in the presence of 4 equiv NMM at r.t. for 30 min. The desired conjugate 22 or its analogues were finally produced with good yields after purification via a semi-preparative HPLC. PTX consists of two major fragments, 10-deacetylbaccation III (10-DAB) and a side chain. To explore which of the two fragments contributes the least to antagonizing 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 supporting information). Scheme 2 lists all the prepared conjugates discussed in this paper. Obviously, the complete structure of PTX is necessary for maintaining antagonistic activity via MDP activating NOD2 signaling because compounds 24~28 presented much lower inhibitory percentages at the tested concentrations. Our previous report indicated that conjugates of PTX with MDP (2-O-MTC01) 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 replacement 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 Nterminal of a dipeptide (L-Ala-D-isoGln), which is a pharmacophore 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. Scheme 2. Structure-Activity Relationship Analysis in MDP (100ng/mL) Stimulated HEK-Blue hNOD2 Cells a

a

The Percentages Represent the Inhibitory Rates of the Compound at 10M

■ CONCLUSIONS

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In conclusion, we first report the synthesis of an antagonist of 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 describes a molecular strategy of antagonizing NOD2 inflammatory 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 treating NSCLC.

■ 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. Preparation of 22. 15mmol, 15g rink-amide resin (0.8 mmol/g) and 100 mL DCM were added into a 200 mL solid-phasereaction 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 FmocLys(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 couple Fmoc-D-isoGln-OH, Fmoc-Ala-OH were subsequently repeated 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 trifluoroacetic acid (TFA) and DCM (v/v = 9:1) was added and the mixture was shake for 2h at rt. The reaction mixture was filtered and washed by DCM for two times. Then the filtrates were combined 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 purification. 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 atmosphere 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 NMethylmorpholine were added, the reaction mixture was stirred

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Journal of Medicinal Chemistry

at rt for 30 min and purified by reversed phase column chromatography [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 gradient 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). 1 HNMR (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), 7.537.61 (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 (M + H).calcd for C73H87O20N7Cl, 1416.5689, found: 1416.5649.

ASSOCIATED CONTENT 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, [email protected] *Y. Ma: phone, 86-10-62787371; e-mail, [email protected]

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-Hisplasmid.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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(17) (a) Li, X.; Yu, J.; Xu, S.; Wang, N.; Yang, H.; Yan, Z.; Cheng, G.; Liu, G. Chemical conjugation of muramyl dipeptide and paclitaxel to explore the combination of immunotherapy and chemotherapy for cancer. Glycoconjugate J. 2008, 25, 415-425. (b) China patent: ZL200510081265.X. August 11, 2010.

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