Salutaxel, a Conjugate of Docetaxel and a Muramyl Dipeptide (MDP) Analogue, Acts as Multifunctional Prodrug That Inhibits Tumor Growth and Metastasis

Article abstract of DOI:10.1021/acs.jmedchem.7b01407

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 act

Full text of DOI:10.1021/acs.jmedchem.7b01407

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Article
Salutaxel, a conjugate of docetaxel and a muramyl dipeptide (MDP) analogue,
acts as multi-functional prodrug that inhibits tumor cell 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 Yao, Xuelu Bei, Wei-Bin Wu, Jian Jin, Jie Yan, and Gang Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01407 • Publication Date (Web): 22 Jan 2018
Downloaded from http://pubs.acs.org on January 23, 2018
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Journal of Medicinal Chemistry
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Salutaxel, a conjugate of docetaxel and a muramyl dipeptide
(MDP) analogue, acts as multi-functional prodrug that inhibits
tumor cell growth and metastasis
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Xiaoming Wen,1, Purong Zheng,1, Yao Ma2, Yingye Ou1, Weixin Huang1, Shuo Li1, Shoujia Liu1, Xuan Zhang1, Ziyu Wang1, Qianli
Zhang1, Wenming Cheng1, Ruwen Lin1, Hongzu Li1, Youyou Cai1, Chunyun Hu1, Ningbin Wu1, Long Wan1, Tingting Pan1, Jinlong
Yao1, Xuelu Bei1, Weibin Wu1, Jian Jin1, Jie Yan*,1, Gang Liu*,2
1. Shenzhen Salubris Pharmaceuticals Co., Ltd., 1 Fenghuanggang Huabao Industrial District, Xixiang, Baoan District, Shen-
zhen, China. Zip: 518102
2. School of Pharmaceutical Sciences, Tsinghua University, Renhuan Building, Rm 311, Beijing, CHINA, Zip: 100084
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 anti-tumor 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 recep-
tor 1 (NOD1) signaling pathway both in vitro and in vivo.
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INTRODUCTION
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Cancer is one of the most common diseases in the world.1 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 pro-
duce a durable response for as long as possible are the current state-of-the-art treatments.4
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Well-designed conjugate prodrugs are capable of initiating multiple signaling pathways for synergisti-
cally fighting cancer and possess precise advantages in some settings compared with combined dosing of
individual drugs, such as improved physicochemical properties or pharmacodynamics/pharmacokinetics
profiles. The effects of paclitaxel and novel muramyl dipeptide (MDP) analogue conjugates as dual-func-
tional 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 com-
pound 1, thereby also providing support for the conjugate prodrug strategy, in general. Compound 1 sup-
presses 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 com-
pound 1 revealed that the conjugate relied both on tubulin inhibition by the parent compound paclitaxel
and antagonism of the nucleotide-binding oligomerization domain-like receptor 2 (NOD2) signaling path-
way 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).
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The design, synthesis and anti-tumor tests of a series conjugates of compound 7 and MDP analogues
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allowed us to obtain a lead compound, MDC-405 (compound 2),6 which boasted an exciting pharmaco-
logical 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, 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 synthe-
sized to improve the water solubility, and its pharmacological and physicochemical profiles were exam-
ined. 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 pharmacokinetic/pharmacodynamics
(PK/PD) fine-tuning. Fortunately, compound 3 exhibited compelling efficacy and satisfactory druggabil-
ity 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 ob-
tain 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 corre-
sponding 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
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prepared by 3 with NaOH or Ca(OH)2, respectively. Crude compounds were further purified via prepara-
tive HPLC 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.
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Several follow-up studies in multiple prongs, including solubility, stability, formulation, metabolic pro-
files, efficacy, and safety, resulted in compound 3 (salutaxel) receiving IND status (SFDA issued No.
CXHL1600182, China).
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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.
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Scheme 1. Fmoc-SPPS for tripeptide synthesis
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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; and (i)
SOCl2, MeOH, rt, 12 h.
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Scheme 2. Synthesis of compound 3 and its analogues a
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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℃, 5 min, 100%; for 11: Ca(OH)2, H2O, 0-
5℃, 5 min, 100%.
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In vitro metabolism and stability of compound 3. A set of P450 isoforms was screened against
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compound 3 in this study. Compound 3 specifically inhibited CYP2C8, with an IC50 value of 9.97 M,
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).
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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 taxol 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 com-
pound 3, 5.7 mg/kg for compound 7, 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 toler-
ated, and no apparent side effects or significant body weight loss were observed with the doses adminis-
tered. Compound 3 exhibited better potency than compound 7 in inhibiting the growth of several 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),
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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 dose-dependent 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 sup-
pressing tumor growth (Figure 3B and C). 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, E, and 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.
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Co-administration of compound 3 with doxorubicin (DOX) inhibits the growth and me-
tastasis of 4T1 breast cancer cells. The same invasive and metastatic 4T1 mammary carcinoma
model was utilized for the co-administration of compound 3 and DOX. Significant toxicity was not ob-
served, 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. More-
over, 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 anti-tumor and anti-metastasis effects
compared with compound 7 when it was administered at the same molar dosage and combined with DOX.
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Scheme 3. Compound 3 was metabolized into compound 7 and a peptide (6) in vitro and in vivo
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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), and A549/PTX (D). Tumors were established in
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nude mice. When the average tumor volume reached 100 mm3, compound 3 was intravenously (iv) ad-
ministered once per week for 3 weeks. 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.
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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, and 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 <
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0.05, p < 0.01, and ###p < 0.001 compared with the 10 mg/kg compound 3 group, as determined by
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ANOVA and Student’s t test.
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Figure 4. Compound 3 inhibits the growth and metastasis of 4T1 breast cancer cells when administered
alone or co-administered with DOX. A body weight, B tumor volume, C tumor weight, D number of
metastatic nodules in the lung, E lung metastasis score, and 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 com-
pared with the vehicle control group; @@@p < 0.001, @p < 0.05 compared with the DOX (4 mg/kg)
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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.
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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 S3-S4. The clearance (CL) of compound 3 was low: 2.97-6.66 mL/min/kg in rats
and 4.28-11.5 mL/min/kg 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/kg) and dogs (approximately 30.9 mL/min/kg),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 com-
pound 3 was primarily distributed in the bodily fluid. The inter-sexual 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 com-
pound 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.
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Table 1. PK parameters of compound 3 in rats and dogs after iv administration (n = 6, half male and half female)
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Animal
dose
Tmax
Cmax
AUC0-t
AUC0-∞
MRT0-∞
(h)
t1/2
CL
Vss
(mg/k sex
g)
(h)
(μg/mL)
(μg·h/mL) (μg·h/mL)
(h)
(mL/min/kg) (L/kg)
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rat
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8
male
0.083
0.083
0.083
0.083
0.083
0.083
0.083
0.083
0.083
0.5a
15.8 ± 1.2
14.6 ± 1.3
15.2 ± 1.3
37.9 ± 5.2
30.5 ± 1.1
34.2 ± 5.2
61.9 ± 3.2
62.5 ± 1.3
62.2 ± 2.2
6.09 ± 1.11 6.12 ± 1.11 0.231 ± 0.037
4.32 ± 0.38 4.33 ± 0.38 0.144 ± 0.014
5.20 ± 1.22 5.23 ± 1.23 0.188 ± 0.054
0.150 ± 0.010
0.110 ± 0.006
0.130 ± 0.023
0.178 ± 0.016
0.174 ± 0.026
0.176 ± 0.019
0.356 ± 0.030
0.254 ± 0.104
0.305 ± 0.089
5.58 ± 1.12
7.73 ± 0.68
6.66 ± 1.44
3.28 ± 0.61
5.13 ± 0.30
4.21 ± 1.10
2.55 ± 0.26
3.39 ± 0.15
2.97 ± 0.50
0.076 ± 0.004
female
all
0.067 ± 0.008
0.071 ± 0.007
0.073 ± 0.006
0.080 ± 0.004
0.076 ± 0.006
0.096 ± 0.005
0.095 ± 0.003
0.096 ± 0.004
male
female
all
20.7 ± 3.6
13.0 ± 0.8
16.9 ± 4.8
52.3 ± 6.0
39.3 ± 1.8
45.8 ± 8.1
20.8 ± 3.6
13.0 ± 0.8
16.9 ± 4.8
52.8 ± 5.6
39.4 ± 1.7
46.1 ± 8.2
0.375 ± 0.041
0.261 ± 0.028
0.318 ± 0.070
0.632 ± 0.083
0.470 ± 0.030
0.551 ± 0.105
male
female
all
dog
1
male
3.74 ± 1.78
4.02 ± 2.09
1.59 ± 0.69 1.63 ± 0.73 0.202 ± 0.058
1.59 ± 0.63 1.64 ± 0.62 0.306 ± 0.109
0.227 ± 0.214
0.483 ± 0.419
11.7 ± 5.2
11.2 ± 4.3
0.134 ± 0.045
0.204 ± 0.087
(0.5)b
0.5a
female
(0.5)b
0.5a
all
3.88 ± 1.74
10.8 ± 3.4
1.59 ± 0.59 1.64 ± 0.60 0.254 ± 0.096
4.84 ± 1.31 4.89 ± 1.35 0.219 ± 0.068
0.355 ± 0.329
0.503 ± 0.267
11.5 ± 4.3
0.169 ± 0.073
0.090 ± 0.012
(0.5)b
0.5a
2
male
7.13 ± 1.70
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1
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3
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7
8
(0.25-0.5)b
0.5a
female
all
14.7 ± 3.1
12.7 ± 3.6
29.6 ± 6.9
30.8 ± 0.8
30.1 ± 4.9
6.97 ± 2.14 7.02 ± 2.14 0.253 ± 0.022
5.90 ± 1.97 5.95 ± 1.98 0.236 ± 0.049
0.515 ± 0.119
0.509 ± 0.185
0.610 ± 0.212
0.563 ± 0.205
0.591 ± 0.183
5.04 ± 1.44
6.08 ± 1.81
4.56 ± 1.13
3.85 ± 2.10
4.28 ± 1.37
0.077 ± 0.024
0.083 ± 0.018
0.075 ± 0.019
0.087 ± 0.008
0.080 ± 0.016
(0.5)b
0.5a
9
(0.25-0.5)b
0.5a
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male
female
all
15.2 ± 3.9
15.3 ± 4.0
0.275 ± 0.006
(0.5)b
0.5a
20.3 ± 11.1 20.3 ± 11.1 0.433 ± 0.201
(0.5)b
0.5a
17.2 ± 6.8
17.3 ± 6.8
0.338 ± 0.133
(0.5)b
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a, b: median and range, respectively.
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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, com-
pound 7 was mainly distributed in the spleen, adrenal gland, kidney and lung. The concentrations of com-
pound 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 com-
pound 7 decreased slowly and were still detectable at 45% of the peak value in all tissues, with the excep-
tion 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 levels of compound 3 were only 1% of the levels in plasma,
thus indicating that compounds 3 and 7 did not readily permeate the blood-brain barrier. 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 com-
pounds 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
0-48 h, and it was 69.9% and 9.38% in feces and urine, respectively, from 0-72 h.
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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
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compounds 3 and 7 determined by the non-compartmental analysis are listed in Table 2. Surprisingly, the
concentrations of compound 7 in plasma and tumor tissue were much greater after administration of com-
pound 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.
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Figure 5. Mean plasma (A) and tumor (B) concentration curves of compounds 3 and 7 after iv admin-
istration of compounds 3 (10 mg/kg) and 7 (5.7 mg/kg) (n = 6, mean ± SD).
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1
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3
4
Table 2. Pharmacokinetic parameters of compounds 3 and 7 in mouse plasma and tumors after iv administration of compounds 3 and
7 (n = 6, mean ± SD)
5
6
7
8
3
7 from 3
7 from 7
Parameters Unit
9
plasma
tumor
plasma
tumor
plasma
tumor
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11
12
13
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48
Tmax
Cmax
h
0.03 ± 0.00
14.70 ± 2.20
0.25a ± (0.08-0.50)b
0.14 ± 0.03
0.03 ± 0.00
43.30 ± 4.10
0.50a ± (0.08-0.50)b
2.39 ± 0.47
0.03 ± 0.00
8.09 ± 0.98
0.50a ± (0.08-0.50)b
0.95 ± 0.09
μmol/l or
nmol/g
h
t1/2
0.69 ± 0.10
3.33 ± 0.21
1.56 ± -
2.97 ± 0.37
8.43 ± 0.48
59.00 ± 17.10
81.00 ± 12.10
3.62 ± 1.22
2.24 ± 0.19
49.10 ± 22.20
27.00 ± 0.90
AUC0-t
h*μmol/l or
h*nmol/g
h
0.11 ± 0.06
MRT
0.45 ± 0.04
2.20 ± -
0.88 ± 0.10
87.40 ± 25.30
2.29 ± 0.58
73.30 ± 30.80
a, b: median and range, respectively.
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Evaluation of the toxicity of compound 3. In the pharmacological safety investigation, compound
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2
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 doses 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 doses of 1, 2, or 4 mg/kg. Blood pressure and body
temperature decreased slightly during the infusion but recovered 1 h and 4 h after administration, respec-
tively. 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 param-
eters are listed in Table 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. Compounds 3 and 7 did not accumulate to significant levels in vivo after 5 administra-
tions in both rats and dogs. The highest non-severely 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.
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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 naïve 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
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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).
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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 naïve 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).
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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 naïve mice. Significant differences in MMP2 expression were not observed be-
tween 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 tumor-bearing 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
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alveolar wall thickness accompanied by edema, whereas the naïve group presented a normal lung structure.
Treatment with compound 2 clearly reduced spontaneous metastasis and ameliorated lung injuries in a
dose-dependent manner. The 7-, (7+5b)- and (7+6)-treated groups 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).
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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 immunostain-
ing 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
naïve group, as shown in Figures 10B and S5B. Groups treated with compound 2 exhibited TSP-1 levels
similar to the levels in the naïve group.
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Figure 6. Compound 3 Suppresses MDSC accumulation in the spleens of 4T1 tumor-bearing mice. Cells
from naïve 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 naïve group (right). Data are presented as the mean ± SEM, p <
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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.
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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.
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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.
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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).
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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 naïve, vehicle-treated, and compound 2- or 7-treated groups, as determined by immunohistochemistry.
Light microscopy: 400× magnification.
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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 D19 after the 4T1 cells were injected10 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 two
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 num-
ber 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.
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Compound 3 decreased the levels of the matrix metalloproteinase 8 (MMP8), MMP9, prokineticin 2
(PROK2), S100 calcium-binding protein A8 (S100A8), S100A9 and interleukin-1β (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 C12-iE-
DAP (50 μg/mouse) and MDP (100 μg/mouse) once per day for two 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 non-cleavable prototype 3 analogue (4) was pre-
pared 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
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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 (IκBɑ) and phosphorylated
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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 HEK-Blue hNOD1 cells, THP-1 cell lines and mouse peritoneal
macrophages using the established sulforhodamine B assay (Figure S6). As shown in Figure 13B and 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 IκBɑ level in a concentration-dependent manner.
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Activation of NF-κB, which is involved in the inflammatory response, upregulated pro-inflammatory
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 E). The effect of com-
pound 4 on the NOD2 pathway was also evaluated. Compound 4 inhibited MDP-induced NOD2 activa-
tion (IC50: 13.12 μM) in HEK-Blue TM hNOD2 cells but exhibited weaker effects on the activation of NF-
κB and MAPKs by the NOD2 ligand (Figure S7).
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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).
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Page 29 of 63
Journal of Medicinal Chemistry
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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 pre-
*
**
sented 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
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