Salutaxel, a Conjugate of Docetaxel and a Muramyl Dipeptide (MDP

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 1519−1540

Salutaxel, a Conjugate of Docetaxel and a Muramyl Dipeptide (MDP) Analogue, Acts as Multifunctional Prodrug That Inhibits Tumor Growth and Metastasis Xiaoming Wen,§,† Purong Zheng,§,† Yao Ma,‡ Yingye Ou,† Weixin Huang,† Shuo Li,† Shoujia Liu,† Xuan Zhang,† Ziyu Wang,† Qianli Zhang,† Wenming Cheng,† Ruwen Lin,† Hongzu Li,† Youyou Cai,† Chunyun Hu,† Ningbin Wu,† Long Wan,† Tingting Pan,† Jinlong Rao,† Xuelu Bei,† Weibin Wu,† Jian Jin,† Jie Yan,*,† and Gang Liu*,‡ †

Shenzhen Salubris Pharmaceuticals Co., Ltd., 1 Fenghuanggang Huabao Industrial District, Xixiang, Baoan District, Shenzhen 518102, China ‡ School of Pharmaceutical Sciences, Tsinghua University, Renhuan Building, Room 311, Beijing 100084, China S Supporting Information *

ABSTRACT: Salutaxel (3) is a conjugate of docetaxel (7) and a muramyl dipeptide (MDP) analogue. Docetaxel (7) has been recognized as a highly active chemotherapeutic agent against various cancers. MDP and its analogues are powerful potentiators of the antitumor actions of various tumor-necrotizing agents. This article documents the discovery of compound 3 and presents pharmacological proof of its biological function in tumor-bearing mice. Drug candidate 3 was superior to compound 7 in its ability to prevent tumor growth and metastasis. Compound 3 suppressed myeloid-derived suppressor cell (MDSC) accumulation in the spleens of tumor-bearing mice and decreased various serum inflammatory cytokines levels. Furthermore, compound 3 antagonized the nucleotide-binding oligomerization domain-like receptor 1 (NOD1) signaling pathway both in vitro and in vivo.

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INTRODUCTION

also providing support for the conjugate prodrug strategy, in general. Compound 1 suppresses myeloid-derived suppressor cell (MDSC) accumulation in the spleen and bone marrow of tumor-bearing mice and reduces the levels of inflammatory cytokines in tumor tissue. Characterization of compound 1 revealed that the conjugate relied on both tubulin inhibition by the parent compound paclitaxel and antagonism of the nucleotide-binding oligomerization domain-like receptor 2 (NOD2) signaling pathway by the intact prodrug before releasing the parent compound.5a The stronger preclinical efficacy and druggability profile for compound 1 encouraged us to explore other conjugates, such as conjugates of compound 7 and MDP analogues (Figure 1). The design, synthesis, and antitumor tests of a series conjugates of compound 7 and MDP analogues allowed us to obtain a lead compound, MDC-405 (compound 2),6 which boasted an exciting pharmacological profile against mice exhibiting 4T1 breast tumor growth and metastasis when the mice were treated weekly with the compound, but the compound has unsatisfactory physicochemical properties, such as very low solubility in water. The low water solubility pertained to the taxane-class compounds,

1

Cancer is one of the most common diseases in the world. Breast cancer is the most common cancer in women, and it is the second-most-frequent cause of cancer-related death.2 Metastasis is the main cause of mortality in patients with cancer; many patients with breast cancer die because tumor cells metastasize to other organs.3 Many researchers have focused on solving the problems of metastasis and drug resistance to cancer treatments. Cocktail treatments targeting multiple signaling pathways to attack cancer and produce a durable response for as long as possible are the current state-of-the-art treatments.4 Well-designed conjugate prodrugs are capable of initiating multiple signaling pathways for synergistically fighting cancer and possess precise advantages in some settings compared with combined dosing of individual drugs, such as improved physicochemical properties or pharmacodynamics/pharmacokinetics (PD/PK) profiles. The effects of paclitaxel and novel muramyl dipeptide (MDP) analogue conjugates as dual-functional prodrugs have been studied extensively, and these studies recently culminated in an anti-lung-cancer drug candidate with the status of an investigational new drug (IND), compound 1 (Figure 1).5a,b Using a cancer xenograft model and wild-type mice, IND-enabling studies demonstrated the effectiveness of compound 1, thereby © 2018 American Chemical Society

Received: September 25, 2017 Published: January 22, 2018 1519

DOI: 10.1021/acs.jmedchem.7b01407 J. Med. Chem. 2018, 61, 1519−1540

Journal of Medicinal Chemistry

Article

Figure 1. Conjugate of paclitaxel and MDP analogue 1 (MTC-220), conjugates of compound 7 and MDP analogues 2 and 3 (MDC-405 and salutaxel) and the peptides used in this article.

to yield the final pure conjugates. This protocol has been successfully used for pilot plant-scale preparation of compound 3 (salutaxel), thus enabling preclinical studies. Several follow-up studies in multiple prongs, including solubility, stability, formulation, metabolic profiles, efficacy, and safety, resulted in compound 3 (salutaxel) receiving IND status (SFDA issued No. CXHL1600182, China). In Vitro Metabolism and Stability of Compound 3. A set of P450 isoforms was screened against compound 3 in this study. Compound 3 specifically inhibited CYP2C8, with an IC50 value of 9.97 μM, but did not inhibit the other P450 isoforms, including CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP1A, CYP2B6, and CYP3A4. Compound 3 was metabolized into compounds 7 and 6 in mouse, rat, dog, monkey, and human hepatocytes (Scheme 3), and the amount of prototype 3 remaining was monkey < dog ∼ rat < human < mouse after a 120 min incubation at 37 °C. In mouse, rat, dog, monkey, and human plasma, the amount of prototype 3 remaining was mouse < human < rat ∼ monkey < dog after a 6 h incubation at 37 °C (data shown in the Supporting Information). Compound 3 Inhibits Tumor Growth in Vitro. The calculated IC50 was 16.3 nM for 3 in 14 human tumor cell lines (Table S1 and Figure S4). Compound 3 Inhibits the Growth of Multiple Tumors in Nude Mice. Four human tumor cell line xenograft models were utilized in this study, including MDA-MB-231 (breast), H1975 (lung), HCT116 (colon), and A549/T (lung for paclitaxel resistance). After implantation into the right flank of nude mice, tumors were allowed to grow to an average volume of 100−120 mm3 before the animals were treated with compound 3 or 7. The dosages were defined to yield equal molar ratios such as 10 mg/kg for compound 3, 5.7 mg/kg for compound 7,

which made it difficult to develop the formulation because paclitaxel must be formulated into an equivalent volume of Cremophor EL and ethanol. Compound 3, with a carboxylic acid at its C-terminus, was synthesized to improve the water solubility, and its pharmacological and physicochemical profiles were examined. Its methyl ester (9) and sodium and calcium salts (10 and 11) were also prepared as an alternative strategy when compound redesign was needed for property or PK/PD fine-tuning. Fortunately, compound 3 exhibited compelling efficacy and satisfactory druggability properties in the preclinical investigation and currently has the status of an IND.7 Here, we report the characterization of 3 as a novel anticancer drug candidate.

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RESULTS Chemistry. The standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS) method was used to obtain a muramyl tripeptide (MTP) derivative with primary amide (−CONH2, 5a)6 at the C-terminus of the peptide using a Rink amide resin or a carboxyl acid (−COOH, 5b)6 at the C-terminus using a Wang resin (Scheme 1). The methyl ester (5c)6 was directly gained in further reacting by 5b in the presence of MeOH and SOCl2. The 2′-OH of compound 7 and the amino group of the lysine residue in MTP derivatives were conjugated via a succinic linker. The 2′-OH of compound 7 first reacted with succinic anhydride to yield carboxylic acid 8. Compound 8 and HOSu were reacted in the presence of EDC·HCl to obtain the corresponding active ester in DMSO, which was directly treated with compound 5a, 5b, or 5c to obtain target products 2, 3, and 9 (Scheme 2). The corresponding sodium salt (10) and calcium salt (11) were carefully prepared by 3 with NaOH or Ca(OH)2, respectively. Crude compounds were further purified via preparative HPLC 1520



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Scheme 1. Fmoc-SPPS for Tripeptide Synthesisa

a Reagents and conditions: (a) (i) Fmoc-Lys(Boc)-OH, HOBt, DIC, DMF, rt, 3 h; (ii) Ac2O, pyridine, DMAP, DCM, rt, 12 h; (b) 20% Pip/DMF, rt, 15 min; (c) Fmoc-D-iso-Gln-OH, HOBt, DIC, DMF, rt, 3 h; (d) 20% Pip/DMF, rt, 15 min; (e) Fmoc-Ala-OH, HOBt, DIC, DMF, rt, 3 h; (f) 20% Pip/DMF, rt, 15 min; (g) E-3-(2-chloro-4-fluorophenyl)acrylic acid, HOBt, DIC, DMF, rt, 3 h; (h) TFA/H2O, rt, 1 h; (i) SOCl2, MeOH, rt, 12 h.

Scheme 2. Synthesis of Compound 3 and Its Analoguesa

a

Reagents and conditions: (a) succinic anhydride, TEA, THF, rt, 3 h, 95%; (b) (i) HOSu, EDC·HCl, DMSO, rt, 4 h. (ii) For 2: NMM, 5a, DMSO, rt, 2 h, 58%. For 3: NMM, 5b, DMSO, rt, 2 h, 55%. For 9: NMM, 5c, DMSO, rt, 2 h, 57%. (iii) For 10: NaOH, H2O, 0−5 °C, 5 min, 100%. For 11: Ca(OH)2, H2O, 0−5 °C, 5 min, 100%.

apparent side effects or significant body weight loss were observed with the doses administered. Compound 3 exhibited better potency than compound 7 in inhibiting the growth of several

4.53 mg/kg for compound 5b, and 4.43 mg/kg for compound 6. The results of each xenograft assay are summarized in Figure 2 and Table S2. The treatments were well tolerated, and no 1521



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Scheme 3. Compound 3 Was Metabolized into Compound 7 and a Peptide (6) in Vitro and in Vivo

Plasma Pharmacokinetic Study of Compound 3 in Rats and Dogs. A single dose of 2, 4, or 8 mg/kg of compound 3 was administered to rats via intravenous injection, and a single dose of 1, 2, or 4 mg/kg was administered to Beagle dogs via intravenous infusion. Compound 7 was administered to rats and dogs via the same route at a dose of 2.28 or 1.14 mg/kg, respectively. The pharmacokinetic parameters are listed in Table 1 and Tables S3 and S4. The clearance (CL) of compound 3 was low: 2.97−6.66 mL min−1 kg−1 in rats and 4.28−11.5 mL min−1 kg−1 in dogs. The CL represented 5.4−12.1% and 13.9−37.2% of the hepatic blood flow of rats (approximately 55 mL min−1 kg−1) and dogs (approximately 30.9 mL min−1 kg−1),9 respectively. The apparent steady-state distribution volumes (Vss) of compound 3 were low: 0.071−0.096 L/kg in rats and 0.080−0.169 L/kg in dogs. The Vss represented 10.6−14.3% and 13.3−28.2% of the bodily fluid of rats (approximately 0.67 L/kg) and dogs (approximately 0.60 L/kg),8 respectively, thus indicating that compound 3 was primarily distributed in the bodily fluid. The intersexual differences in the AUC were not significant. The plasma levels of compound 7, the main metabolite of compound 3, rapidly reached their peak concentrations, and the plasma levels of compound 7 were 0.79% and 0.23% of the levels of compound 3 in rats and dogs, respectively. The plasma levels of compound 7 generated from compound 3 were 45% (rats) and 0.8% (dogs) of the levels of directly injected compound 7. Tissue Distribution, Plasma Protein Binding, Metabolism, and Excretion of Compound 3 in Rats. After iv administration of 4 mg/kg compound 3 in rats, the concentrations of compound 3 in all tissues were less than the concentrations in plasma, and compound 3 was primarily distributed in the liver, kidney, and small intestine. The concentrations in tissues reached their peak values at 0.25 h, which was the first sampling time, and decreased rapidly. In contrast, after iv administration of compound 3, compound 7 from 3 was mainly distributed in the spleen, adrenal gland, kidney, and lung. The concentrations of compound 7 in tissues reached their peak values at 1 h. The concentrations of compound 7 in all tissues, with the exception of the brain, were greater than the concentrations in plasma. The concentrations of compound 7 decreased slowly and were still detectable at 45% of the peak value in all tissues, with the exception of the brain, 24 h after administration, in contrast with the concentrations of compound 3, which were less than the lower limit of quantitation (200 ng/g). In brain tissue, only a small amount of compound 3 and no compound 7 were detected, and the level of compound 3 was only 1% of the level in plasma, thus indicating that compounds 3 and 7 did not readily permeate the blood−brain barrier.

tumor types in vivo, particularly human breast (MDA-MB-231) and lung (H1975) tumors. Compound 3 Inhibits Tumor Growth and Lung Metastasis of 4T1 Tumors in Mice. A highly invasive and metastatic 4T1 mammary carcinoma model was further investigated to evaluate the ability of compound 3 to prevent tumor metastasis. The mammary fat pads of BALB/c mice were injected with 4T1 cells, and the mice were intravenously injected with saline (vehicle), compound 3 (5, 10, or 20 mg/kg), or compound 7 (5.7 mg/kg (with or without 5b dose, or compound 6) once per week for 4 weeks. As shown in Figure 3A, the body weights of animals treated with highest dose of compound 3 (20 mg/kg) decreased by approximately 8%. No discernible body weight loss was observed in any of the other treated groups. Treatments with compound 3 substantially inhibited tumor growth in a dosedependent manner: 12.0% (5 mg/kg), 37.0% (10 mg/kg), and 57.2% (20 mg/kg). In comparison, compounds 7 (5.7 mg/kg), (7 + 5b), and (7 + 6) inhibited tumor growth by 19.6%, 14.4%, and 15.4% inhibition, respectively. The 10 mg/kg dose of compound 3 was superior to compounds 7 (5.7 mg/kg), (7 + 5b), and (7 + 6) in suppressing tumor growth (Figure 3B and Figure 3C). At all doses administered, compound 3 significantly decreased the number of metastatic nodules in the mouse lungs compared with the vehicle control group (p < 0.001) (Figure 3D). Most interestingly, compound 3 was also significantly superior to compound 7 in preventing tumor metastasis (p < 0.05) (Figure 3D−F); however, no significant differences were observed between the compound 7-treated groups, including the (7 + 5b) and (7 + 6) groups, in terms of preventing tumor growth and metastasis. Coadministration of Compound 3 with Doxorubicin (DOX) Inhibits the Growth and Metastasis of 4T1 Breast Cancer Cells. The same invasive and metastatic 4T1 mammary carcinoma model was utilized for the coadministration of compound 3 and DOX. Significant toxicity was not observed, with the exception of a slight reduction in the body weights of all treated groups at the end of the experiment (Figure 4A). Significant reductions in these indicators were observed in all treated groups compared with those of the vehicle control group (Figure 4B−D). In addition, when combined with DOX, both compounds 7 and 3 exhibited more potent efficacies than when they were administered alone. Moreover, significant reductions in these indicators were observed in the (DOX + 3) group compared with those of the (DOX + 7) group; thus, compound 3 exhibited superior antitumor and antimetastasis effects compared with compound 7 when it was administered at the same molar dosage and combined with DOX. 1522



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Figure 2. Effects of compound 3 on nude mouse xenograft models: body weight (left); tumor weight (right); MDA-MB-231 (A); H1975 (B); HCT116 (C); A549/PTX (D). Tumors were established in nude mice. When the average tumor volume reached 100 mm3, compound 3 was intravenously (iv) administered once per week for 3 weeks. The iv injection of equal molar compound 7 was performed as a positive control. The values are expressed as the mean ± standard error of mean (SEM) (n = 8): (∗∗∗) p < 0.001 compared with the vehicle group, (##) p < 0.01, (###) p < 0.001 compared with the medium dose group of compound 3, as determined by analysis of variance (ANOVA) and Student’s t test.

High protein binding rates were observed for compound 3 in rat, dog, and human plasma: 99.3 ± 0.3%, 99.7 ± 0.0%, and 99.3 ± 0.2%, respectively. After iv administration in rats, compound 3 was mainly metabolized into compounds 7 and 6 in plasma, bile, feces, and

urine via cleavage of the ester bond, and the prototype was mainly excreted into bile. The cumulative excretion of the prototype in bile was 82.9% of the dosage from 0 to 48 h, and it was 69.9% and 9.38% in feces and urine, respectively, from 0 to 72 h. 1523



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Figure 3. Compound 3 inhibits tumor proliferation and lung metastasis of 4T1 tumors in mice: (A) body weight; (B) tumor volume; (C) tumor weight; (D) number of metastatic nodules in the lung; (E) lung metastasis score; (F) representative images of the lungs from mice in each group. The values are presented as the mean ± SEM (n = 10): (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 compared with the vehicle group and (#) p < 0.05, (##) p < 0.01, and (###) p < 0.001 compared with the 10 mg/kg compound 3 group, as determined by ANOVA and Student’s t test.

Pharmacokinetics and Tumor Distribution of Compound 3 in 4T1 Tumor-Bearing Mice. The pharmacokinetics and tumor distribution of compound 7 generated from compound 3 were analyzed to further demonstrate that compound 3 is superior to compound 7. The levels of compound 7 in plasma and tumor tissue were detected after a single iv administration of compounds 3 or 7, respectively, to the 4T1 tumor-bearing mice. The curves of the concentrations of compounds 3 and 7 in mouse plasma and tumor tissues over time are illustrated in Figure 5, and the corresponding pharmacokinetic parameters of compounds 3 and 7 determined by the noncompartmental analysis are listed in Table 2. Surprisingly, the concentrations of compound 7 in plasma and tumor tissue were much greater after administration of compound 3 than after administration of compound 7 alone, leading to larger Cmax and AUC values in both the tumor tissue and plasma. The Cmax values for compound 7 generated from compound 3 were 5.35- and 2.52-fold higher, and the AUC values were 3.76- and 3-fold higher than the values for compound 7 alone in plasma and tumor tissue, respectively. Moreover, compound 7 was still detected in tumor tissue 72 h after administration, resulting in a long t1/2 of more than 30 h. Although the t1/2 and mean residence time (MRT) values of compound 7 generated from compound 3 in plasma were lower than the values of compound 7 alone, the former values were at least 10 h longer than the latter in the tumor tissue. The t1/2 and

MRT values of the prototype in the tumor tissue were also higher than the values in plasma, thus indicating that compound 3 may represent a tumor-targeting treatment. Evaluation of the Toxicity of Compound 3. In the pharmacological safety investigation, compound 3 had no significant toxic effects on the coordination and autonomic activities of HaM/ICR mice after a single intravenous administration of compound 3 at a dose of 5, 10, or 20 mg/kg. Significant toxic effects on the electrocardiogram index, respiratory frequency, and respiratory intensity of Beagle dogs were not observed after intravenous infusion of compound 3 at a dose of 1, 2, or 4 mg/kg. Blood pressure and body temperature decreased slightly during the infusion but recovered 1 and 4 h after administration, respectively. In the acute toxicity experiment, the LD50 value of a single intravenous administration in rats was 13.18 mg·kg−1, the 95% confidence interval was 12.03−14.43 mg·kg−1, and the MTD was 9.4 mg·kg−1. For dogs, the approximate lethal dose (ALD) range of a single intravenous administration was 33−50 mg/kg. In the long term toxicity test, rats and dogs were intravenously administered 2, 4, or 8 mg/kg or 3, 6, or 18 mg/kg compound 3, respectively, once per week in 30 days. The accompanying toxicokinetic parameters are listed in Tables S5−S8. Compound 3 was more toxic to male rats than female rats owing to the higher plasma levels of compounds 3 and 7 in males, but there were no obvious differences between male and female dogs. 1524



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Figure 4. Compound 3 inhibits the growth and metastasis of 4T1 breast cancer cells when administered alone or coadministered with DOX: (A) body weight; (B) tumor volume; (C) tumor weight; (D) number of metastatic nodules in the lung; (E) lung metastasis score; (F) representative images of the lungs from mice in each group. The values are presented as the mean ± SEM (n = 10): (∗∗∗) p < 0.001, (∗∗) p < 0.01 compared with the vehicle control group; (@@@) p < 0.001, (@) p < 0.05 compared with the DOX (4 mg/kg) group; (&) p < 0.05 compared with the compound 3 (10 mg/kg) group; and (#) p < 0.05 compared with the DOX (4 mg/kg) + 3 (10 mg/kg) group, as determined via ANOVA and Student’s t test.

Compound 3 Reduces the Serum Inflammatory Cytokine Levels in 4T1 Tumor-Bearing Mice. As shown in Figure 7, the serum levels of matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinases 1 (TIMP-1), interleukin-6 (IL-6), and S100 calcium-binding protein A9 (S100A9) in 4T1 tumor-bearing mice were significantly greater in the vehicle control group than in the naive group (p < 0.001). Treatments with compounds 3 (10 and 20 mg/kg), 7 (5.7 mg/kg), and (7 + 6) significantly reduced the MMP9 levels (p < 0.05). The elevated levels of TIMP-1 and IL-6 were significantly reduced by all doses of compounds 3, 7 (5.7 mg/kg), and (7 + 6) (p < 0.05). The compound 3 (20 mg/kg) treatment also decreased S100A9 secretion (p < 0.05). Compound 3 was superior to compound 7 in inhibiting MMP9 and TIMP-1 secretion (p < 0.05). Compound 3 Inhibited MMP9 Expression and Protected NOD1 from Degradation in the Spleens and Lungs of 4T1 Tumor-Bearing Mice. As shown in Figure 8, the matrix metalloproteinase 2 (MMP2) and MMP9 expression levels were significantly higher in the spleens and lungs of 4T1 tumor-bearing mice than in the naive mice. Significant differences in MMP2

Compounds 3 and 7 did not accumulate to significant levels in vivo after 5 administrations in both rats and dogs. The highest nonseverely toxic doses (HNSTD) were 4 and 2 mg/kg for female and male rats, respectively, and 6 mg/kg for dogs. In the acute and long-term studies, the main toxic effects on dogs were gastrointestinal effects (emesis and diarrhea), facial swelling, and general erythema. For rats, the principal toxic effect was irreversible testicular atrophy.9 All these toxic effects were similar to the effects of compound 7,9 thus indicating that the toxic effects of compound 3 may originate from its main metabolite, compound 7. Compound 3 Suppresses MDSC Accumulation in the Spleens of 4T1 Tumor-Bearing Mice. After 28 days of tumor growth, the accumulation of MDSCs in spleens was significantly increased in the mice of the vehicle group compared with the naive group (p < 0.001). Representative dot plots are shown in Figure 6. MDSCs represented 38.0% of the spleen cells in the untreated vehicle group but decreased significantly to 20.2% in the compound 3 (10 mg/kg)-treated group compared with the compound 7 (5.7 mg/kg)-treated group (36.4%) (p < 0.001). 1525



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Table 1. PK Parameters of Compound 3 in Rats and Dogs after iv Administrationa dose animal (mg/kg) rat

2

4

8

dog

1

2

4

a

Cmax

Tmax

sex

(h) male

0.083

AUC0−t

AUC0−∞

MRT0−∞

t1/2

(μg/mL)

(μg·h/mL)

(μg·h/mL)

(h)

(h)

15.8 ± 1.2

6.09 ± 1.11

6.12 ± 1.11

0.231 ± 0.037

0.150 ± 0.010

Vss

CL (mL min

−1

−1

kg )

5.58 ± 1.12

(L/kg) 0.076 ± 0.004

female

0.083

14.6 ± 1.3

4.32 ± 0.38

4.33 ± 0.38

0.144 ± 0.014

0.110 ± 0.006

7.73 ± 0.68

0.067 ± 0.008

all

0.083

15.2 ± 1.3

5.20 ± 1.22

5.23 ± 1.23

0.188 ± 0.054

0.130 ± 0.023

6.66 ± 1.44

0.071 ± 0.007

male

0.083

37.9 ± 5.2

20.7 ± 3.6

20.8 ± 3.6

0.375 ± 0.041

0.178 ± 0.016

3.28 ± 0.61

0.073 ± 0.006

female

0.083

30.5 ± 1.1

13.0 ± 0.8

13.0 ± 0.8

0.261 ± 0.028

0.174 ± 0.026

5.13 ± 0.30

0.080 ± 0.004

all

0.083

34.2 ± 5.2

16.9 ± 4.8

16.9 ± 4.8

0.318 ± 0.070

0.176 ± 0.019

4.21 ± 1.10

0.076 ± 0.006

male

0.083

61.9 ± 3.2

52.3 ± 6.0

52.8 ± 5.6

0.632 ± 0.083

0.356 ± 0.030

2.55 ± 0.26

0.096 ± 0.005

female

0.083

62.5 ± 1.3

39.3 ± 1.8

39.4 ± 1.7

0.470 ± 0.030

0.254 ± 0.104

3.39 ± 0.15

0.095 ± 0.003

all

0.083

62.2 ± 2.2

45.8 ± 8.1

46.1 ± 8.2

0.551 ± 0.105

0.305 ± 0.089

2.97 ± 0.50

0.096 ± 0.004

male

0.5b (0.5)c

3.74 ± 1.78

1.59 ± 0.69

1.63 ± 0.73

0.202 ± 0.058

0.227 ± 0.214

11.7 ± 5.2

0.134 ± 0.045

female

0.5b (0.5)c

4.02 ± 2.09

1.59 ± 0.63

1.64 ± 0.62

0.306 ± 0.109

0.483 ± 0.419

11.2 ± 4.3

0.204 ± 0.087

all

0.5 (0.5)

3.88 ± 1.74

1.59 ± 0.59

1.64 ± 0.60

0.254 ± 0.096

0.355 ± 0.329

11.5 ± 4.3

0.169 ± 0.073

male

0.5b (0.25−0.5)c

10.8 ± 3.4

4.84 ± 1.31

4.89 ± 1.35

0.219 ± 0.068

0.503 ± 0.267

7.13 ± 1.70

0.090 ± 0.012

female

0.5b (0.5)c

14.7 ± 3.1

6.97 ± 2.14

7.02 ± 2.14

0.253 ± 0.022

0.515 ± 0.119

5.04 ± 1.44

0.077 ± 0.024

all

0.5b (0.25−0.5)c

12.7 ± 3.6

5.90 ± 1.97

5.95 ± 1.98

0.236 ± 0.049

0.509 ± 0.185

6.08 ± 1.81

0.083 ± 0.018

male

0.5b (0.5)c

29.6 ± 6.9

15.2 ± 3.9

15.3 ± 4.0

0.275 ± 0.006

0.610 ± 0.212

4.56 ± 1.13

0.075 ± 0.019

female

c

0.5 (0.5)

30.8 ± 0.8

20.3 ± 11.1

20.3 ± 11.1

0.433 ± 0.201

0.563 ± 0.205

3.85 ± 2.10

0.087 ± 0.008

all

0.5b (0.5)c

30.1 ± 4.9

17.2 ± 6.8

17.3 ± 6.8

0.338 ± 0.133

0.591 ± 0.183

4.28 ± 1.37

0.080 ± 0.016

b

b

c

b

c

n = 6, half male and half female. Median. Range.

Figure 5. Mean plasma (A) and tumor (B) concentration curves of compounds 3 and 7 after iv administration of compounds 3 (10 mg/kg) and 7 (5.7 mg/kg) (n = 6, mean ± SD).

also exhibited decreased metastasis and inflammation, but the changes were not as obvious as the group treated with an equal molar dose of compound 2 (10 mg/kg). Neutrophil and Thrombospondin-1 (TSP-1) Expression in the Lung Tissues of 4T1 Tumor-Bearing Mice Treated with the Conjugate. The attenuation of neutrophil infiltration in the mouse lung tissue was evaluated in 4T1 tumor-bearing mice treated with compound 2. The intense immunostaining of Ly-6G (a marker for neutrophil) in the lungs of the vehicle control group is shown in Figures 10A and S5A. Ly-6G expression in the lung tissue was significantly reduced in the compound 2-treated groups. A relative decrease in TSP-1 expression was observed in the vehicle control group compared with the naive group, as shown in Figures 10B and S5B. Groups treated with compound 2 exhibited TSP-1 levels similar to the levels in the naive group. Compound 3 Suppressed Neutrophil Accumulation in the Blood of 4T1 Tumor-Bearing Mice. The same invasive and metastatic 4T1 mammary carcinoma model was utilized for this experiment. Spontaneous metastasis occurred in 4T1 mice at

expression were not observed between the vehicle group and all treated groups. Treatment with compound 3 significantly reduced MMP9 expression in the spleen and lungs compared with the vehicle group (p < 0.05). NOD1 expression was significantly reduced in the spleen and lungs of the vehicle group. The compound 3 treatment increased NOD1 expression in a dose-dependent manner. No differences in MMP2, MMP9, and NOD1 expression in the tumor tissues from each groups were observed. Histopathological Changes in the Lungs of 4T1 TumorBearing Mice after Treatment with the Conjugate. Compound 2 exhibits a similar in vivo activity as the C-terminal amide analogue of compound 3 (Figure 1 and Figure S8).6 As shown in Figure 9, the lung tissues from the vehicle control group exhibited severe metastasis, obvious infiltration of inflammatory cells, and increased pulmonary alveolar wall thickness accompanied by edema, whereas the naive group presented a normal lung structure. Treatment with compound 2 clearly reduced spontaneous metastasis and ameliorated lung injuries in a dosedependent manner. The 7-, (7 + 5b)-, and (7 + 6)-treated groups 1526


0.50b ± (0.08−0.50)c 0.95 ± 0.09 49.10 ± 22.20 27.00 ± 0.90 73.30 ± 30.80 0.03 ± 0.00 8.09 ± 0.98 3.62 ± 1.22 2.24 ± 0.19 2.29 ± 0.58

plasma tumor

0.50b ± (0.08−0.50)c 2.39 ± 0.47 59.00 ± 17.10 81.00 ± 12.10 87.40 ± 25.30

tumor

0.25b ± (0.08−0.50)c 0.14 ± 0.03 1.56 ± _ 0.11 ± 0.06 2.20 ± _

plasma

0.03 ± 0.00 14.70 ± 2.20 0.69 ± 0.10 3.33 ± 0.21 0.45 ± 0.04

plasma

D19 after the 4T1 cells were injected.10 In this study, mice were selected and randomly divided into five groups at day 19 after the 4T1 cell injection and received iv administration of the vehicle control, compound 3 (20 mg/kg), or compound 7 (11.4 mg/kg) in addition to intraperitoneal injection of C12-iE-DAP (50 μg/ mouse) and MDP (100 μg/mouse) once per day for 2 days. The mice were then sacrificed after 6 h. Whole blood was collected, and the number of leukocytes in the peripheral blood was determined. As shown in Figure 11, 4T1 cell implantation increased the number of neutrophils in peripheral blood; however, the compound 3 treatment, but not the C12-iE-DAP and MDP treatments, significantly reduced the number of neutrophils in peripheral blood (p < 0.001). The compound 7-treated group also exhibited a significant decrease in the number of neutrophils. Compound 3 decreased the levels of the matrix metalloproteinase 8 (MMP8), MMP9, prokineticin 2 (PROK2), S100 calcium-binding protein A8 (S100A8), S100A9, and interleukin1β (IL-1β) mRNAs in the lung tissues of 4T1 tumor-bearing mice. The invasive and metastatic 4T1 mammary carcinoma model described above was utilized for this experiment. Nineteen days after implantation of the 4T1 cells, mice were selected, randomly divided into five groups, and received iv administration of the vehicle control, compound 3 (20 mg/kg), or compound 7 (11.4 mg/kg) in addition to intraperitoneal injection of C12iE-DAP (50 μg/mouse) and MDP (100 μg/mouse) once per day for 2 days. The mice were sacrificed after 48 h. Tumors and lungs were isolated for quantitative PCR (qPCR) analysis. Compound 3 was superior to compound 7 at decreasing the levels of the MMP8, MMP9, PROK2, S100A8, and S100A9 mRNAs in the lung tissues of 4T1 tumor-bearing mice (Figure 12). Compound 3 Antagonized NOD1 Signaling. A noncleavable prototype 3 analogue (4) was prepared using a previously reported method.5a Compound 4 was then characterized as an NOD1 antagonist using a HEK-Blue hNOD1-secreted alkaline phosphatase (SEAP) reporter cell line (Figure 13A). The NOD1 agonist C12-iE-DAP activates the NF-κB and MAPK pathways, leading to phosphorylation and nuclear translocation of the transcription factor NF-κB.11 Activation of NF-κB and MAPK signaling by C12-iE-DAP was evaluated via Western blots for the total inhibitor of NF-κB α (IκBα) and phosphorylated IκBα, p38, JNK, and extracellular regulated protein kinase (ERK) levels in human THP-1 cell lines and mouse peritoneal macrophages to further investigate the mechanisms of compound 3. The low toxicity of compound 4 was further confirmed in HEKBlue hNOD1 cells, THP-1 cell lines, and mouse peritoneal macrophages using the established sulforhodamine B assay (Figure S6). As shown in Figure 13B and Figure 13C, compound 4 inhibited the C12-iE-DAP-induced increases in the p-IκBα, p-ERK, p-p38, and p-JNK levels and reduced the IκBα level in a concentration-dependent manner. Activation of NF-κB, which is involved in the inflammatory response, upregulated proinflammatory cytokine production.12 MDP alone only weakly induces secretion of inflammatory cytokines such as IL-8 in the THP-1 cells but exhibits marked synergistic effects with LPS.13 C12-iE-DAP stimulation alone results in very low levels of IL-6 and TNF-α in these cells. The combination of C12-iE-DAP and LPS exhibited a synergistic stimulatory effect on peritoneal macrophages and induced the marked secretion of inflammatory cytokines, such as IL-6 and TNF-α. In contrast, compound 4 significantly decreased the IL-6 and TNF-α levels induced by LPS and C12-iE-DAP (p < 0.01; Figure 13D and Figure 13E). The effect of compound 4 on the NOD2 pathway was also evaluated. Compound 4 inhibited

0.03 ± 0.00 43.30 ± 4.10 2.97 ± 0.37 8.43 ± 0.48 0.88 ± 0.10

7 from 3

Article

n = 6, mean ± SD. bMedian. cRange.

Tmax Cmax t1/2 AUC0−t MRT

a

unit

h μmol/L or nmol/g h h·μmol/L or h·nmol/g h

parameter

3

Table 2. Pharmacokinetic Parameters of Compounds 3 and 7 in Mouse Plasma and Tumors after iv Administration of Compounds 3 and 7a

7 from 7

tumor


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Figure 6. Compound 3 suppresses MDSC accumulation in the spleens of 4T1 tumor-bearing mice. Cells from naive or tumor-bearing mice subjected to different treatments were examined for the expression of CD11b and Gr-1 to identify the MDSC population. Representative dot plots are shown. Numbers in the dot plots indicate the percentage of Gr-1+ CD11b+ cells. Bar graphs represent the average of eight mice per treatment group or five mice in the naive group (right). Data are presented as the mean ± SEM: (∗) p < 0.05 and (∗∗∗) p < 0.001 compared with the vehicle group and (#) p < 0.05 and (###) p < 0.001 compared with the compound 3 10 mg/kg group, as determined by ANOVA and Student’s t test.

MDP-induced NOD2 activation (IC50 = 13.12 μM) in HEK-Blue hNOD2 cells but exhibited weaker effects on the activation of NF-κB and MAPKs by the NOD2 ligand (Figure S7).

are to inhibit natural killer cells, B cells, dendritic cells, and cytotoxic T cells, induce regulatory T cells (Tregs), promote angiogenesis, and promote cancer cell invasion and metastasis, likely via MMP activity.15a,b MDSCs are present in most patients with breast, lung, prostate, kidney, head and neck and other types of cancer and in tumor-bearing experimental animals.16 MDSCs are another heterogeneous population of cells comprising immature myeloid progenitors for neutrophils, monocytes, and DCs. In mice, MDSCs are often, somewhat confusingly, termed CD11b+ Gr1+ cells. Tumor-bearing mice have markedly increased numbers of MDSCs (CD11b+ Gr1+) in their bone marrow, peripheral blood, and spleen compared with non-tumor-bearing mice.17 In the current study, large numbers of MDSCs accumulated in tumor-bearing mice and represented 38.0% of the spleen cells,

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DISCUSSION AND CONCLUSIONS Innate immune cells are largely responsible for inflammatory reactions. These cells include various myeloid-derived cells (e.g., macrophages, neutrophils, and mast cells). Myeloid cells regulate not only the immune response to cancer cells but also angiogenesis and metastasis.14 MDSCs are a poorly defined group of cells that share properties with both the macrophage and neutrophil lineages. As the tumor burden increases, MDSCs accumulate in the bone marrow, spleen, and peripheral blood and at the invasive tumor front.15a The reported functions of MDSCs 1528



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Figure 7. Effect of compound 3 injection on the serum levels of MMP9 (A), TIMP-1 (B), IL-6 (C), and S100A9 (D). The values are presented as the mean ± SD (n = 6) and are representative of two independent experiments: (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.01 compared with the vehicle group and (#) p < 0.05 and (##) p < 0.01 compared with the compound 3 (10 mg/kg) group, as determined via an unpaired t test.

in various tumors. TIMP-1 specifically enhances the formation and growth of micrometastases in the liver. Although the role of TIMP-1 in cancer growth and metastasis is controversial, high TIMP-1 expression levels are always linked to poor prognoses in patients with lung cancer.22a,b Inhibitor of nuclear factor κB kinase 2 (IKK2) and NF-κB play essential roles in tumor cell proliferation, and TIMP-1 is one of the mediators of NF-κBinduced tumor proliferation.23 Compound 3 significantly suppressed MMP9 expression in the serum, spleen, and lungs of tumor-bearing mice and decreased the serum TIMP-1 levels to a greater extent than compound 7. Compound 3 inhibited the formation and growth of metastases in the lung, which might be associated with its effects on suppressing MMP9 and TIMP-1 expression. Metastasis is the main cause of mortality in patients with breast cancer. As demonstrated in a study by Wculek et al., neutrophils support the lung colonization of metastasis-initiating breast cancer cells.24 Patients with an elevated pretreatment neutrophil to lymphocyte ratio (NLR) exhibited worse disease-specific survival than patients without an elevated NLR.25 Clinical data correlate high neutrophil levels with a worse prognosis.26a,b Reduced NOD1 expression has been observed in dysfunctional neutrophils, and treatments that blocked the NOD1/NF-κB pathway inhibited neutrophil migration and their phagocytic killing capacity.27 Stimulation with synthetic NOD1 ligands induced neutrophil recruitment in vivo.28a,b Neutrophil counts in the C12-iE-DAP- or MDP-treated groups were much higher than the naive group and similar to those of the vehicle control group. Compound 3 suppressed neutrophil accumulation in the blood

whereas the normal percentage of MDSCs in the spleens of untreated mice is approximately 5%. Treatment with compound 3 significantly decreased MDSC accumulation in the spleens in a dose-dependent manner. The compound 7-treated group exhibited a high percentage of Gr-1+ CD11b+ MDSCs (36.4%), similar to the vehicle control group. MMPs are a group of zinc-dependent endopeptidases with different substrate specificities. These enzymes share many structural and functional properties, are known to participate in extracellular matrix remodeling, and are inhibited by TIMPs.18a MMPs are involved in extracellular matrix (ECM) degradation and basement membrane invasion. MMPs have long been associated with metastasis, and they have substantial functional contributions to the metastatic process. According to numerous studies, higher MMP expression levels in tumors indicate a more aggressive cancer.3,18a,b MMP2 and MMP9 are gelatinases that degrade type IV collagen, the major structural component of BMs, and are assumed to have important role in cellular invasion.19 The 5-year disease-free survival rate for a subgroup of patients with an estrogen receptor-negative and MMP9-positive tumor was only 37%, whereas patients with a tumor negative for both estrogen receptor and MMP9 had a 63% 5-year disease-free survival rate.18b MMPs are key regulators of tumor growth at both primary and metastatic sites. TIMPs are endogenous inhibitors of metalloproteinase activities.20 Most human malignancies are associated with increased matrix metalloproteinase (MMP) activity, and TIMPs modulate the invasion and metastasis of tumor cells.21 TIMP-1 is the prototypic inhibitor for most MMP family members and is expressed 1529



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Figure 8. Effects of compound 3 on MMP9, MMP2, and NOD1 expression in the spleens (A), lungs (B), and tumors (C) of tumor-bearing mice. β-Actin was used as the loading control. The bands represent the average of five mice per group and are representative of two independent experiments.

Figure 9. Compound 2 inhibited spontaneous metastasis and inflammation in the lungs of 4T1 tumor-bearing mice. Histopathological findings were obtained using hematoxylin and eosin (H&E) staining (light microscopy: 40× magnification).

neutrophils. The circulating S100A8/A9 levels in humans strongly correlate to the blood neutrophil counts.31 Distant primary tumors upregulate S100A8 and S100A9 expression in the premetastatic lung.32 S100A8 and S100A9 overexpression in various adenocarcinomas is considered a marker of poor prognosis.30,33 On the basis of our findings, neutrophils in the metastatic lung microenvironment induce S100A8 and S100A9 expression, and these proteins are essential for tumor cell migration and invasion. Treatment with compound 3 reduced the blood neutrophil counts, significantly decreased the PROK2, MMP8, MMP9, S100A8, and S100A9 mRNA levels in lung tissue in the premetastatic phase and decreased the serum S100A9 levels. To elucidate the mechanisms by which neutrophils promote metastasis, we focused on TSP-1, a multifunctional matricellular

of 4T1 tumor-bearing mice (Figure 11), and compound 2 suppressed neutrophil infiltration in the lung tissue (Figure 10A). Thus, compound 3 inhibited neutrophil recruitment, possibly by blocking the NOD1 pathway. PROK2, also known as Bv8, is produced by Ly6G+ Ly6C+ cells and critically involved in metastasis. A strong correlation between high PROK2 expression and metastatic potential in multiple tumor models has been reported. Increased PROK2 levels were measured in the premetastatic lungs of various cancer models.29 S100A8 and S100A9 (damage-associated molecular patterns (DAMPs) belonging to the S100 calgranulin family) preferentially form the S100A8/A9 heterodimer called calprotectin and might be critically involved in metastasis.30 S100A8 and S100A9 are constitutively expressed in myeloid cells, such as 1530



Article

Figure 10. Compound 2 attenuated Ly-6G expression and increased TSP-1 expression in the lung tissues of 4T1 tumor-bearing mice. (A) Neutrophil (Ly-6G) infiltration and (B) TSP-1 expression were detected in naive, vehicle-treated, and compound 2- or 7-treated groups, as determined by immunohistochemistry. Light microscopy: 400× magnification.

protein critical for lung homeostasis and inflammation.34 Increased numbers of neutrophils in inflamed lungs mediate TSP-1 proteolysis by secreting neutrophil elastase and cathepsin G and promote metastasis. TSP-1 deficiency extends inflammation and enhances metastasis. TSP-1 is a potent inhibitor of tumor angiogenesis and growth and contributes to the suppression of lung metastasis.35 Compound 2 suppressed neutrophil recruitment and increased TSP-1 expression, which may be associated with its antimetastasis effects. NOD1 and NOD2, which belong to the nodosome subgroup of NOD-like receptors (NLRs), play major roles in host defense against pathogen infections because they recognize bacterial peptidoglycan components, which leads to the activation of NF-κB and MAPK signaling, thereby resulting in an inflammatory response. However, this response is tightly regulated by numerous factors. Mutations in the NOD1 and NOD2 genes and ligand-elicited over activation have both been demonstrated to dysregulate NLRs function, which is linked to a variety of human diseases, including inflammatory disorders and cancer.12 NOD1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, controls tumor formation.36 NOD1 signaling has been demonstrated to play an important role in tumorigenesis. As reported in a study by Ozbayer et al., NOD1 gene variations are associated with an increased risk of lung cancer.37 In a study by Chen et al., a NOD1 deficiency increased the development of both colitis-associated and Apc tumor suppressor-related colon tumors.38 NOD1 expression decreased as OSCC progressed and was significantly correlated

with tumor differentiation, lymph node metastasis, and tumor size.39 NOD degradation is tightly controlled by the chaperone HSP90, which dissociates from activated NODs.40 Mifamurtide, also known as liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE), has been approved for the treatment of osteosarcoma in Europe. This drug is designed with an active component MDP linked to the phospholipid phosphatidyl ethanolamine (PE) via an alanine residue. Like MDP, MTP-PE is a specific ligand of the NOD2 receptor, an intracellular protein that is primarily expressed in monocytes, dendritic cells, and macrophages and induces nuclear factor NF-κB activation. Therefore, monocyte/macrophage activation by mifamurtide is thought to be mediated by NOD2.41a,b Compound 4 inhibited C12-iE-DAP-induced NOD1 activation (IC50 was 4.79 μM) in HEK-Blue hNOD1 cells and exhibited weaker inhibition of HEK-Blue hNOD2 cells (IC50 was 13.12 μM) (Figure S7). Compound 4 inhibited the activation of NF-κB and MAPKs by the NOD1 ligand. In the C12-iE-DAPactivated macrophages, compound 4 increased IκBα expression and potently suppressed C12-iE-DAP-stimulated IκBα, ERK, JNK, and p38 phosphorylation. Furthermore, compound 4 inhibited the C12-iE-DAP-induced secretion of inflammatory cytokines, such as IL-6 and TNF-α (Figure 13D and Figure 13E). NF-κB activation appears to promote tumor initiation and progression through mechanisms such as cell proliferation, apoptosis, angiogenesis, tumor metastasis, and reprogramming of metabolism. NF-κB homodimers or heterodimers are retained in the cytoplasm through a noncovalent interaction with an inhibitory 1531



Article

Figure 11. Compound 3 suppressed neutrophil accumulation in the blood of 4T1 tumor-bearing mice. The number of neutrophils/leukocytes was determined using a blood smear staining analysis (light microscopy: 1000× magnification).

protein, IκB. In response to an external signal, the IκB protein is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated, and degraded, leading to translocation of the NF-kB protein to the nucleus. TIMP-1 is one of the NF-κB target genes involved in tumor proliferation. Inhibition of NF-kB activation decreases TIMP-1 expression and impairs ERK phosphorylation. Selective inhibition of TIMP-1 in tumor cells affected their proliferation, and silencing TIMP-1 in tumor cells in vivo prolonged mouse survival.42 Compound 3 significantly decreased the serum TIMP-1 levels in the tumor-bearing mice, thus resulting in slower tumor progression and metastasis. In the pharmacological studies, compound 3 was much more potent than compound 7 when administered at the same molar dosage. However, when the peptide component of 3, i.e., 5b or 6, was coadministered with compound 7, improved efficacies were not observed in the various mice models assayed compared to compound 7 or the peptide treatment alone (Figures 2 and 3). Thus, compound 3 may have exerted its pharmacological effects on a pathway different from the pathway impacted by compound 7 alone or the combination of compound 7 with MDP analogues. The plasma levels of compound 7 generated from compound 3 were 45% (rats) and 0.8% (dogs) of the levels of compound 7 alone. However, in 4T1 tumor-bearing mice, the plasma and tumor levels of compound 7 generated from compound 3 were at least 3-fold higher than the levels of compound 7 alone; the higher levels of compound 7 in both the plasma and tumors of tumor-bearing mice after administration of compound 3 may be responsible for

the improved antitumor and antimetastasis efficacies of compound 3. In addition, the long t1/2 (over 50 h) and MRT (over 80 h) values for compound 7 generated from compound 3 in the tumor may be a good rationale for weekly administration of compound 3. Moreover, the t1/2 and MRT values of the prototype and compound 7 in the tumor were higher than the values in plasma, thus indicating that compound 3 had high affinity for tumor tissue. Moreover, the tissue distribution, protein binding rates, excretion, and toxicity were similar to those of compound 7,9 thus indicating that conjugation with the MDP analogue did not significantly alter the drug disposition and toxic properties of compound 7 but rather maintained or even enhanced its pharmacological efficacy. However, one question still remained: Why was treatment with compound 3 in addition to compound 7 not more toxic than treatment with compound 7 alone? One of major metabolites of compound 3, compound 6, was not a NOD1 agonist or antagonist and was ineffective at inhibiting tumor growth or metastasis in various mouse tumor models. Compound 6 did not increase the efficacy of compound 7. Thus, the side effects of compound 3 might be due to another major metabolite of compound 7. The tumor microenvironment, which consists of stromal cells (including fibroblasts, macrophages, regulatory T cells, myeloidderived suppressor cells, endothelial cells, pericytes, and platelets), the ECM (including inflammatory cytokines, chemokines, matrix metalloproteinases, integrins, and other secreted molecules), and exosomes (small extracellular vesicles loaded with molecules), 1532



Article

Figure 12. Compound 3 decreased the levels of the MMP8 (A), MMP9 (B), PROK2 (C), S100A8 (D), S100A9 (E), and IL-1β (F) mRNAs in the lung tissues of 4T1 tumor-bearing mice. The values are presented as the mean ± SEM: (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 compared with the vehicle control group; (#) p < 0.05, (##) p < 0.01, and (###) p < 0.001 compared with the compound 7 (11.4 mg/kg) group.

interactions and are therefore essential for signal transduction.12 NOD1 and NOD2 recognize the bacterial peptidoglycan derivatives iE-DAP and MDP, respectively, and undergo conformational changes and adenosine triphosphate (ATP)-dependent self-oligomerization of the NACHT domains, followed by downstream signaling leading to NF-κB activation.46 DY-16-43 (a conjugate of paclitaxel and MDP derivative) targets upstream NOD2 signaling and inhibits NOD2 oligomerization.5a In our study, compound 4 (a noncleavable prototype 3) inhibited NOD1 activation in vitro, and thus compound 3 is highly possible to protect NOD1 from degradation by inhibited NOD1 activation in vivo. MDP and its analogues have been reported to act as NOD2 ligands with the potential antitumor actions of various tumornecrotizing agents.47 Mifamurtide, also known as L-MTP-PE, has been successfully used in combination with ifosfamide to treat osteosarcoma.43 Free L-MTP-PE has been speculated to be a prodrug that could release free MDP and subsequently initiate innate NOD2 signaling in patients with cancer. Our recent studies using a noncleavable compound 1 analogue supplied an alternative approach for a combination treatment of cancer by inhibiting DAMP-induced activation of NOD2 signaling. In this paper, we demonstrated that in combination with a chemotherapeutic agent, antagonizing NOD1 signaling may also be an effective treatment for cancer. As demonstrated in this study, compound 3 is superior to compound 7 in its ability to prevent tumor growth and metastasis. Compound 3 was much more potent than compound 7 in reducing MDSC accumulation; blood neutrophil counts; serum MMP9, TIMP-1, and S100A9 levels; and the levels of the PROK2, MMP8, MMP9, S100A8, and S100A9 mRNAs in lung tissue. The strong beneficial effects of compound 3 might be associated with its effects on regulate the inflammatory tumor microenvironment. The antitumor and antimetastasis effects of compound 3 were significantly increased when it was combined

establishes an autocrine−paracrine communication circuit that reinforces tumor cell invasion and metastasis via reciprocal signaling. Recent studies have revealed the significant contribution of the tumor microenvironment to cancer metastasis.43 MDSCs inhibit natural killer cells, B cells, dendritic cells, and cytotoxic T cells; induce Tregs; promote angiogenesis; and promote cancer cell invasion and metastasis, likely via matrix metalloproteinase (MMP) activity.15a,b Innate immune signaling has been implicated in the pathogenesis of inflammatory bowel disease, and it has also been demonstrated to have a critical role in tumorigenesis.44 Two major classes of innate immune receptors, the Toll-like receptors (TLRs), which are located on the extracellular surface, and the cytoplasmic NOD-like receptors (NLRs), are involved in tumorigenesis and metastasis.45 These pattern recognition receptors (PRRs) recognize conserved microbial components known as pathogen-associated molecular patterns (PAMPs), such as peptidoglycan or LPS and DAMPs, and upon stimulation by their respective agonists activate inflammatory pathways.12 NF-κB is one of the key downstream effectors of PRR signaling. NOD1 stimulation activates both the NF-κB and MAPK pathways.38 A noncleavable form of compound 3, compound 4, blocked NOD1 activation by its natural ligand. On the basis of the pharmacokinetics study of compound 3 that the majority of prototype 3 was cleared and was metabolized into compound 7 in all tested animals, DAMPs resulted from chemotherapy, i.e., a metabolite of compound 7.5a We speculated that prototype 3 inhibited the DAMP-induced activation of NOD1, which effectively increased the potency of compound 7. NOD1 and NOD2 have a tripartite domain architecture composed of (i) a C-terminal sensor domain consisting of leucinerich repeats (LRRs) that are proposed to mediate ligand recognition; (ii) a centrally located nucleotide-binding domain, NACHT, that mediates self-oligomerization and activation; and (iii) N-terminal effector domains, consisting of one (NOD1) or two CARD domains (NOD2) that mediate protein−protein 1533



Article

Figure 13. Effects of compound 4 on the activated HEK-Blue hNOD1 cells (A). Compound 4 inhibited NOD1-mediated NF-κB and MAPK signaling in THP-1 cells (B) and mouse peritoneal macrophages (C). Compound 4 inhibited NOD1-mediated IL-6 (D) and TNF-α (E) secretion in mouse peritoneal macrophages. Data are presented as the mean ± standard deviation (SD) and are representative of three independent experiments: (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 compared with the LPS + C12-iE-DAP group. swelling and stirred gently for 15 min. Subsequently, the solvent was removed via vacuum filtration. Deprotection/Coupling Cycle. An amount of 100 mL of 20% piperidine/ DMF (v/v) was added, the solution was stirred gently for 15 min, and the solvent was removed by vacuum filtration. An amount of 100 mL of DMF was added, the solution was stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. An amount of 100 mL of DCM was added, the solution was stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. A solution of amino acid or other carboxylic acid (12.4 mmol), HOBt (1.68 g, 12.4 mmol), and DIC (1.90 mL, 12.4 mmol) in 100 mL of DMF was added to the resin, and the resin was stirred gently for 3 h. Upon completion, as indicated by the Kaiser test, the solvent was removed via vacuum filtration. An amount of 100 mL of DMF was added and stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. An amount of 100 mL of DCM was added and stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. Acetylation. A solution of acetic anhydride (3.90 mL), pyridine (3.4 mL), and DMAP (51 mg) in 100 mL DCM was added to the resin,

with DOX treatment. Thus, compound 3 is a novel agent that inhibits tumor growth and metastasis and has potential as a treatment for breast cancer and other types of cancers.

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EXPERIMENTAL SECTION

Chemistry. All chemicals were purchased as reagent grade and used without further purification unless noted otherwise. Some reactions were monitored via analytical thin-layer chromatography on silica gel GF254 precoated on glass plates (10−40 μm, Yantai, China). Spots were detected under UV (254 nm). Solvents were evaporated under reduced pressure and below 35 °C (water bath). Column chromatography was performed on silica gel (200−300 mesh, Qingdao, China). Some reactions were monitored via HPLC using a Kromasil C18 column. The eluent was a mixture of acetonitrile and water. The UV detection wavelength was 220−254 nm. 1H NMR spectra were recorded at 25 °C using a 500 or 600 MHz spectrometer. 13C NMR spectra were recorded at 25 °C using a 500 or 600 MHz spectrometer. Unless noted otherwise, all compounds were determined to be >95% pure via analytical HPLC. General Synthesis of Compound 5. Resin Preparation. An amount of 100 mL of DCM was added to 10.0 g of dried resin for resin 1534



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Compound 9: white solid. 1H NMR (500 MHz, DMSO-d6): 8.42 (1H, d, J = 6.8 Hz), 8.20 (2H, t, J = 7.8 Hz), 7.98 (2H, d, J = 7.5 Hz), 7.90−7.60 (7H, m), 7.53 (1H, dd, J = 2.6, 8.8 Hz), 7.45−7.25 (6H, m), 7.18 (1H, t, J = 6.8 Hz), 7.09 (1H, s), 6.78 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.3 Hz), 5.40 (1H, d, J = 7.1 Hz), 5.09 (3H, s), 4.98 (1H, d, J = 7.1 Hz), 4.90 (2H, d, J = 10.4 Hz), 4.50−4.35 (2H, m), 4.25−4.10 (2H, m), 4.10−3.95 (3H, m), 3.64 (1H, d, J = 7.0 Hz), 3.61 (3H, s), 3.01 (2H, q, J = 5.9 Hz), 2.70−2.55 (2H, m), 2.38 (2H, t, J = 7.1 Hz), 2.35−2.20 (4H, m), 2.17 (2H, t, J = 8.0 Hz), 1.98 (1H, m), 1.85 (1H, m), 1.78−1.60 (6H, m), 1.60−1.55 (2H, m), 1.52 (3H, s), 1.45−1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: calcd for C71H87N6O22FCl [M − H]− 1429.5551. Found 1429.5549. Compound 10. A 0.002 mmol/mL NaOH aqueous solution (100 mL, 0.20 mmol) was added to a solution of compound 3 (283 mg, 0.20 mmol) in acetonitrile (25 mL) and H2O (25 mL) at −10 °C and incubated for 4 h at −20 °C. Lyophilization at 0 °C produced 290 mg of the title compound as a white solid. 1H NMR (500 MHz, DMSO-d6): 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90−7.80 (2H, m), 7.80−7.70 (2H, m), 7.70−7.60 (3H, m), 7.54 (1H, dd, J = 2.2, 8.7 Hz), 7.45−7.30 (6H, m), 7.18 (1H, t, J = 6.7 Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15−4.85 (6H, m), 4.50−4.38 (2H, m), 4.20−4.10 (2H, m), 4.10−3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70−2.55 (2H, m), 2.38 (2H, t, J = 6.9 Hz), 2.30−2.05 (6H, m), 1.98 (1H, m), 1.85 (1H, m), 1.78−1.60 (6H, m), 1.60−1.47 (5H, m), 1.45− 1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: calcd for C70H85N6O22FCl [M − Na]− 1415.5395. Found 1415.5380. Compound 11. A 0.01 mmol/mL Ca(OH)2 aqueous solution (20 mL, 0.20 mmol) was added to a solution of compound 3 (283 mg, 0.20 mmol) in acetonitrile (10 mL) and H2O (10 mL) at 0 °C and incubated for 4 h at −20 °C. Lyophilization at 0 °C yielded 260 mg of the title compound as a white solid. 1H NMR (500 MHz, DMSO-d6): 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90−7.80 (2H, m), 7.80−7.70 (2H, m), 7.70−7.60 (3H, m), 7.54 (1H, dd, J = 2.2, 8.7 Hz), 7.45−7.30 (6H, m), 7.18 (1H, t, J = 6.7 Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15−4.85 (6H, m), 4.50−4.38 (2H, m), 4.20−4.10 (2H, m), 4.10−3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70−2.55 (2H, m), 2.38 (2H, t, J = 6.9 Hz), 2.30−2.05 (6H, m), 1.98 (1H, m), 1.85 (1H, m), 1.78−1.60 (6H, m), 1.60−1.47 (5H, m), 1.45−1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: calcd for C70H85N6O22FCl [M − 1/2Ca]− 1415.5395. Found 1415.5386. Cell Culture. 4T1 cells were provided by Dr. Gang Liu. THP-1 cells were purchased from the Chinese Academy of Sciences Stem Cell Bank. All of the cells were grown in RPMI-1640 medium (Gibco, C22400500BT) supplemented with 10% fetal bovine serum (Gibco, Cibco-10099-141) and 1% penicillin/streptomycin (Gibco, 15140-122) at 37 °C in a 5% CO2 atmosphere. The HEK-Blue hNOD1 cells were a generous gift from Dr. Gang Liu and were maintained in the growth medium described below in the presence of Normocin (100 μg/mL) and two selective antibiotics, blasticidin (30 μg/mL) and zeocin (100 μg/mL). The HEKBlue hNOD1 cells were not passaged more than 20 times to maintain their efficiency. Reagents. Compound 7 was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China). Anti-mouse Ly-6G (Gr-1) FITC and anti-mouse CD11b PE were obtained from eBioscience (San Diego, CA). Antibodies against MMP9, MMP2 and enzyme-linked immunosorbent assay (ELISA) kits for mouse MMP9, TIMP-1, IL-6, and S100A9 were purchased from R&D Systems (Minneapolis, MN, USA). The anti-CARD4 (NOD1) antibody was obtained from Sigma (SAB3500543). C12-iE-DAP, muramyl dipeptide (MDP), HEK-Blue detection, QUANTI-Blue, Normocin, blasticidin, and zeocin were purchased from InvivoGen. RPMI-1640, DMEM, PBS, FBS, trypsin, and penicillin−streptomycin were purchased from Gibco. Unless specified otherwise, all other materials were purchased from Sigma (St. Louis, MO, USA) and were of the highest quality available. Mouse Xenograft Studies. The xenograft tumor model was established in BALB/c nu/nu mice by subcutaneously injecting human tumor cells into the right flanks of the animals; human breast (MDA-MB-231), colon (HCT116), and lung (H1975) tumor cell lines were used for this

stirred gently for 12 h, and then the solvent was removed via vacuum filtration. An amount of 100 mL of DMF was added and stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. An amount of 100 mL of DCM was added and stirred for 5 min, and then the solvent was removed via vacuum filtration. This step was repeated two times. Cleavage f rom the Resin and Side Chain Protecting Groups. A solution of H2O (5 mL) in TFA (45 mL) was added to the dried peptide resin and stirred gently for 1 h. The reaction mixture was filtered and added dropwise to 20 mL of cold methyl tert-butyl ether (MTBE) while stirring. After stirring for 15 min, the mixture was filtered to collect the white solid that yielded compound 5. Compound 5b: white solid. 1H NMR (500 MHz, DMSO-d6): δ12.59 (1H, br s), 8.45 (1H, d, J = 6.8 Hz), 8.23 (1H, d, J = 8.1 Hz), δ8.09 (1H, d, J = 7.9 Hz), 7.75 (1H, dd, J = 9.0,6.0 Hz), δ7.72 (3H, br s), 7.65 (1H, d, J = 15.7 Hz), 7.54 (1H, dd, J = 8.8,2.6 Hz), 7.32 (1H, m), 7.31 (1H, br s), 7.10 (1H, br s), 6.78 (1H, d, J = 15.7 Hz), 4.41 (1H, m), 4.15 (1H, m), 4.13 (1H, m), 2.77 (2H, m), 2.16 (2H, m), 1.96 (1H, m), δ1.73 (1H, m), 1.67 (1H, m), 1.55 (1H, m), 1.51 (2H, m), 1.32 (2H, m), 1.25 (3H, d, J = 7.0 Hz). HR-MS (ESI-TOF) m/z: calcd for C23H32N5O6FCl [M − CF3COO]+ 528.2020. Found 528.2023. Compound 5c. Thionyl chloride (0.75 mL, 10 mmol) was added dropwise to a solution of compound 5b (6.00 g, 9.4 mmol) in methanol (80 mL) at 0−5 °C and then incubated for 12 h at room temperature. The mixture was concentrated, and dichloromethane (80 mL) was added. The mixture was concentrated again to directly obtain a residual solid of compound 5c for the subsequent reaction. Synthesis of Compound 8. Triethylamine (14.0 g, 138 mol) was added dropwise to a solution of compound 7 (28.0 g, 35 mmol) in 520 mL of THF, and the reaction mixture was maintained at 2−6 °C. After the addition of triethylamine, a solution of succinic anhydride (5.20 g, 52 mmol) in 50 mL of THF was added dropwise, and the reaction mixture was maintained at 2−6 °C. Then, the reaction mixture was warmed to room temperature and stirred for 3 h. Upon completion, as indicated by HPLC, the reaction mixture was acidified to pH 3−5 with 2 M HCl and was added dropwise to 1.00 L of water. After a 1 h stirring, the mixture was filtered to collect the white solid compound 8, yield = 95%. 1H NMR (600 MHz, DMSO-d6): 12.26 (1H, br.s), 7.99 (2H, d, J = 7.6 Hz), 7.87 (1H, d, J = 8.8 Hz), 7.73 (1H, t, J = 7.4 Hz), 7.65 (2H, t, J = 7.6 Hz), 7.41 (2H, t, J = 7.5 Hz), 7.37 (2H, d, J = 7.5 Hz), 7.18 (1H, t, J = 7.2 Hz), 5.79 (1H, t, J = 8.7 Hz), 5.40 (1H, d, J = 7.1 Hz), 5.10−4.90 (6H, m), 4.44 (1H, br.s), 4.05 (1H, m), 4.02 (2H, m), 3.64 (1H, d, J = 7.0 Hz), 2.62 (2H, m), 2.53 (2H, m), 2.27 (1H, m), 2.23 (3H, s), 1.84 (1H, dd, J = 14.9, 9.5 Hz), 1.69 (3H, s), 1.65 (1H, m), 1.55 (1H, m), 1.52 (3H, s), 1.38 (9H, s), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: calcd for C47H56NO17 [M − H]− 906.3548. Found 906.3536. Synthesis of Compounds 3 and 9. A solution of EDC·HCl (0.77 g, 0.13 mmol) in 8.5 mL of DMSO was added dropwise to a solution of compound 8 (3.02 g, 0.10 mmol) and HOSu (0.42 g, 0.10 mmol) in 8.5 mL of DMSO, and the reaction mixture was maintained at 26−32 °C. The reaction mixture was stirred for 2 h. Upon completion, as indicated by HPLC, the reaction mixture was added to a solution of compound 5 (0.13 mmol) and NMM (1.70 mL, 0.13 mmol) in 18 mL of DMSO. The resulting solution was stirred for 2 h. Upon completion, as indicated by HPLC, the reaction mixture was acidified to pH 3−5 with 2 M HCl and was added dropwise to 360 mL of water. After a 1 h stirring, the mixture was filtered to collect the solid crude product, which was purified by preparative HPLC to obtain the final white solid product. Compound 3: white solid. 1H NMR (500 MHz, DMSO-d6): 12.49 (1H, br s), 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90−7.80 (2H, m), 7.80−7.70 (2H, m), 7.70−7.60 (3H, m), 7.54 (1H, dd, J = 2.2, 8.7 Hz), 7.45−7.30 (6H, m), 7.18 (1H, t, J = 6.7 Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15−4.85 (6H, m), 4.50−4.38 (2H, m), 4.20−4.10 (2H, m), 4.10−3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70−2.55 (2H, m), 2.38 (2H, t, J = 6.9 Hz), 2.30−2.05 (6H, m), 1.98 (1H, m), 1.85 (1H, m), 1.78−1.60 (6H, m), 1.60−1.47 (5H, m), 1.45−1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: calcd for C70H86N6O22FClNa [M + Na]+ 1439.5365. Found 1439.5358. 1535



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at 4 °C to isolate the peritoneal macrophages.49 All procedures described here were performed under aseptic conditions, and the materials were previously sterilized and pyrogen-free. Western Blot Analysis. The spleens, tumors, and lungs from 4T1 tumor-bearing mice were cut into pieces, homogenized in RIPA lysis reagent (Beyotime, Shanghai, China), and incubated on ice for 30 min. After centrifugation at 12 000g for 30 min at 4 °C, the supernatants were collected, and the total protein concentrations were measured using the bicinchoninic acid assay (Beyotime, Shanghai, China). Samples were separated via SDS−PAGE and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% skim milk or 5% BSA (Biosharp-BS043A) for 1 h. Blots were probed with primary antibodies overnight at 4 °C. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1−2 h at room temperature. All signals were visualized with enhanced electrochemiluminescence (ECL) reagent. THP-1 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.05 mM β-mercaptoethanol (1000×, Gibco21985-023) at 37 °C in a 5% CO2 atmosphere. The THP-1 cells were seeded in a 6-well plate at a density of 2 × 106 cells per well and starved with 0.1% fetal bovine serum for 24 h. On the next day, the cells were incubated with compound 4 or a NOD1 inhibitor12 (NOD1I); 1 h later, C12-iE-DAP (100 ng/mL) was added. Cells were collected for the Western blot analysis described above at 15, 30, and 60 min. Approximately 8 × 106 mouse peritoneal macrophages were seeded in 60 cm2 cell culture dishes. After a 2 h incubation at 37 °C in a humidified atmosphere with 5% CO2, the cells were washed two times to remove the nonadherent cells. Similar to the THP-1 cells, the peritoneal macrophages were incubated with compound 4 or NOD1I; 1 h later, C12-iE-DAP (5 μg/mL) was added. Cells were collected for the Western blot analysis. Cytokine Secretion from Macrophages. The cells were resuspended with RPMI-1640 medium containing 10% heat-inactivated FBS and cultured in 96-well plates (2 × 105 cells/well). After a 2 h incubation at 37 °C in a 5% CO2 atmosphere, the cells were washed two times to remove the nonadherent cells. Cells were incubated with or without LPS (10 ng/mL) for 3 h.13 Subsequently, compound 4 or the NOD1I was added, and 3 h later, the cells were treated with C12-iEDAP (25 μg/mL) for 24 h at 37 °C in a 5% CO2 atmosphere. The culture media were collected to evaluate the levels of TNF-α and IL-6 using commercially available ELISA kits according to the manufacturer’s instructions. ELISA. Serum was collected (control, n = 8 mice; 4T1-injected, n = 19). The serum MMP9, TIMP-1, IL-6, and S100A9 levels were measured using ELISA kits according to the manufacturer’s instructions. Flow Cytometry. Spleens were isolated under aseptic conditions and homogenized by passing the tissue through a cell strainer (70 μm). RBCs were lysed by addition of RBC lysis buffer (eBioscience). The remaining nucleated cells (SP) were resuspended in RPMI-1640 medium. The SP population was used immediately after isolation. The suspension was washed twice with phosphate buffer. An amount of 100 μL of binding buffer, 0.125 μg of anti-mouse Ly-6G (Gr-1) FITC, and 0.125 μg of antimouse CD11b PE (Biolegend) were added and incubated in the dark at room temperature for 30 min. After two washes with buffer, an amount of 400 μL of binding buffer was added. The anti-mouse Ly-6G (Gr-1)and anti-mouse CD11b-positive cells were selected via flow cytometry. Histological Examination and Immunohistochemistry. Serial tissue sections (4 μm) were sliced from paraffin-embedded formalinfixed lung tissues and stained with hematoxylin and eosin (H&E). Immunohistochemical staining was performed using a previously described method.49 Briefly, tissue sections were deparaffinized, hydrated, subjected to antigen retrieval, and stained with specific primary antibodies and horseradish peroxidase (HRP)-conjugated avidin. Specific antibody interactions were detected with the chromogenic substrate 3,3′-diaminobenzidine (DAB). Subsequently, the sections were washed and counterstained with hematoxylin. The primary antibodies used in this study were rabbit polyclonal anti-TSP-1 (1:100; BOSTER, BA2130-1) and rat monoclonal anti-Ly6G (1:200 dilution; BD Biosciences, 551459). Quantitative Real-Time PCR (RT-PCR) and Cell Counts. The same invasive and metastatic 4T1 mammary carcinoma model was utilized

experiment. Then, tumors were maintained by regular monthly sc transplantations in BALB/c nu/nu mice. At each transfer, tumors were minced into 10 mm3 pieces and sc inoculated into the right flank using a trocar. When the tumor size reached approximate 100 mm3, compounds 3 and 7 (with or without 5b or 6) were iv injected into the animals once a week for 3 weeks; the control groups received saline. Tumor volumes were measured using Vernier calipers to determine two orthogonal axes. Tumor volumes (volume = 0.5 × long diameter × (short diameter)2)48 and body weights were monitored every 3 days. At the end of the experiment, tumor tissues were dissected and weighed. Tumor growth inhibition was calculated using the following formula: [(C − T)/C] × 100 (C, tumor weight of control group; T, tumor weight of the treated group). 4T1 Murine Mammary Carcinoma Model. Four-week-old female BALB/c mice were housed at 22 ± 2 °C on a 12 h light−dark cycle in pathogen-free conditions for at least 3 days before the experiment. All animal experiments were approved by the Animal Care and Use Committee of Salubris. The mice were provided standard laboratory chow and water ad libitum. After acclimatization, 4T1 cells (1.5 × 105 cells suspended in 0.1 mL of RPMI-1640 medium) were injected into the inguinal mammary fat pads of the animals. Thus, a 4T1 murine mammary carcinoma model was established and used in several experiments, which are described below. Experiment 1. Four days after the 4T1 cell injection, 70 mice were selected, randomly divided into seven groups, and received iv administration of the vehicle control or compound 3 (5 mg/kg), 3 (10 mg/kg), 3 (20 mg/kg), 7 (5.7 mg/kg), 7 (5.7 mg/kg) + 5b (4.53 mg/kg), or 7 (5.7 mg/kg) + 6 (4.43 mg/kg) once a week for 4 weeks (n = 10). Experiment 2. Seven days after the 4T1 cell implantation, the mice were randomly divided into six groups and received iv administration of the vehicle control, DOX (4 mg/kg), compound 7 (5.7 mg/kg), compound 3 (10 mg/kg, equimolar dose of 7), DOX (4 mg/kg) + compound 7 (5.7 mg/kg), or DOX (4 mg/kg) + compound 3 (10 mg/kg) once a week for 4 weeks. For the groups treated with a combination of agents, DOX was administered 4 h before the administration of the other drug (compound 7 or 3). The tumor sizes and body weights of the animals were monitored. Tumor volume was measured with calipers and calculated as (0.5 × long diameter × (short diameter)2) every 2 days. Twenty-four days after treatment, whole blood was collected. The tumors and lungs were removed from the mice and weighed. The lungs were fixed in Bouin’s solution to quantify the visible metastatic tumor nodules. The lung metastasis score = metastasis numbers (≥5 mm) × 4 + metastasis numbers (3−4.9 mm) × 3 + metastasis numbers (1−2.9 mm) × 2 + metastasis numbers (10 μM, between 1 μM and 10 μM, and

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