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Jun 8, 2017 - (3-8) However, bufadienolides as Na+/K+-ATPase inhibitors exhibit significant cardiotoxicity, which limits their clinical usage and drug...
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Fibroblast Activation Protein α Activated Tripeptide Bufadienolide Antitumor Prodrug with Reduced Cardiotoxicity Li-Juan Deng,† Long-Hai Wang,† Cheng-Kang Peng, Yi-Bin Li, Mao-Hua Huang, Min-Feng Chen, Xue-Ping Lei, Ming Qi, Yun Cen, Wen-Cai Ye,* Dong-Mei Zhang,* and Wei-Min Chen* Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China S Supporting Information *

ABSTRACT: Bufadienolides are the major pharmacologic constituents of traditional Chinese medicine Chan’su, which is frequently used clinically for cancer treatment in China. Motivated by reducing or avoiding the cardiac toxicity of bufadienolides, we have designed, synthesized, and evaluated the fibroblast activation protein α (FAPα) activated tripeptide arenobufagin prodrugs with the purpose of improving the safety of arenobufagin (a representative bufadienolide). Among these FAPα-activated prodrugs, 3f exhibited the best hydrolytic efficiency by recombinant human FAPα (rhFAPα) and was activated in tumors. The LD50 of 3f was 6.5-fold higher than that of arenobufagin. We also observed that there are nonapparent changes in echocardiography, pathological section of cardiac muscle, and the lactate dehydrogenase activities (LDH) in 3f-treatment tumor-bearing mice, even when the dose reached 3 times the amount of parent drug arenobufagin that was used. Compound 3f also exhibits significant antitumor activity in vitro and in vivo. The improved safety profile and favorable anticancer properties of 3f warrant further studies of the potential clinical implications. Our study suggests that FAPα prodrug strategy is an effective approach for successful increasing the therapeutic window of bufadienolides.

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INTRODUCTION Bufadienolides, including arenobufagin, bufalin, and cinobufaginare, are C-24 steroids with a characteristic α-pyrone ring located at position C-17β.1,2 They are the major pharmacological constituents of traditional Chinese medicine Chan’su and several well-known Chinese patent medicines, such as Toad Venom Injection and Huachansu Injection which have been used clinically for cancer treatment in China.1,2 Currently, many experimental and clinical studies have suggested that bufadienolides have significant antitumor activities toward different types of human cancers, such as breast cancer, nonsmall-cell lung cancer, liver cancer, and gastric cancer.3−8 However, bufadienolides as Na+/K+-ATPase inhibitors exhibit significant cardiotoxicity, which limits their clinical usage and drug development.9−11 In recent years, numerous analogues of bufalin, cinobufagin, and resibufogenin have been prepared by chemical synthesis or biological transformation, and the cytotoxicities of these bufadienolide analogues have been evaluated in vitro.12−16 Unfortunately, these efforts have shown little evidence of reduced toxicity or cardiotoxicity of bufadienolide analogues. Consequently, new approaches are needed to extend investigations toward reducing or relieving the cardiotoxicity of bufadienolides when used as anticancer agents. Tumor-specific protease-activated prodrugs are a promising approach to reduction of the toxicity of anticancer agents.17 In © 2017 American Chemical Society

this prodrug strategy, coupling of the peptides to the toxic anticancer agents is accomplished to generate a low or nontoxic prodrug, which is a specific substrate of enzymes known to be expressed in tumor tissues. This prodrug can be activated by tumor specific proteases to kill tumor cells, while it is inactive in normal tissues. Fibroblast activation protein α (FAPα) is a tumor-specific protease, which is observed on the surface of carcinoma-associated fibroblasts (CAFs) in the stroma of >90% of solid tumors that have been examined but is generally absent from adjacent normal tissues and nonmalignant tumors.18−20 FAPα, a type II integral membrane serine protease, is distinguished from other proteases in the dipeptidyl deptidase (DPP) subfamily, as its activity is restricted to substrates containing the N-terminal benzyloxycarbonyl blocked Gly-Pro (Z-GP).21 The specific proteolytic activity and highly tumorrestricted distribution of FAPα makes it a very attractive target for the design of anticancer prodrugs. Motivated by reducing or avoiding the cardiac toxicity of bufadienolide agents, we have attempted to design and synthesize FAPα-activated bufadienolide prodrugs. Theoretically, FAPα-activated bufadienolide prodrugs are maintained in the inactive form in normal tissues including the heart but are only activated in tumors with high expression of FAPα. In this Received: December 1, 2016 Published: June 8, 2017 5320

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

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Figure 1. Design strategy of FAPα-activated tripeptide arenobufagin prodrugs: (a) A; (b) V; (c) L; (d) I; (e) F; (f) W; (g) p-aminobenzoic acid; (h) p-aminophenylacetic acid.

Figure 2. Synthetic route to FAPα-activated tripeptide arenobufagin prodrugs. Reagents and conditions: (i) N-Cbz-amino acid, N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), DMAP, DCM, rt, 8−16 h; (ii) Et3SiH, PdCl2, triethanolamine (TEA), DCM, rt, 20−50 min; (iii) N-Cbz-Gly-L-Pro, EDCI, HOBt, TEA, rt, 40−90 min.

breast cancer models. This FAPα prodrug strategy successfully reduces the cardiac toxicity and increases the therapeutic safety of arenobufagin, which leads to a significant benefit for the preclinical development and clinical usage of bufadienolide derivatives as cancer agents.

way, the cardiotoxicity induced by bufadienolides might be reduced or relieved. Previous reports have demonstrated that FAPα-activated prodrugs can result in significantly less toxicity when compared with the parent toxic compounds, including melittin, thapsigargin, and emetine.22−25 This further supports our hypothesis that FAPα-activated prodrugs might be an effective means with which to reduce the cardiac toxicity of bufadienolides. Arenobufagin (1, Figure 2), a representative bufadienolide, exerts broad-spectrum and potent antitumor activities independent of its cardiac toxicity as reported previously by our group. We have found that arenobufagin induces apoptosis and autophagy,4 intercalates with DNA leading to cell cycle arrest,5 and inhibits angiogenesis.26 In this report, we describe the design and synthesis of the FAPα-activated prodrugs by conjugating arenobufagin with FAPα-specific tripeptides to generate a series of prodrugs with different uncharged residues in the position of P1′ (Figure 1). Among the prodrugs, we found compound 3f could be specifically activated by recombinant human FAPα (rhFAPα) and FAPα-expressing tumor tissue. We also demonstrated that this FAPα-activated prodrug (3f) not only exhibits negligible cardiac toxicity at doses 3 times higher than those of arenobufagin but also maintains antitumor activity in vitro and in vivo against human

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RESULTS AND DISCUSSION Compounds Design. FAPα has a strict preference for substrates with L-Pro at P1 and Gly at P2,27 while it has broader tolerance at P1′ position of the cleavage site (Figure 1).28,29 It was demonstrated that uncharged residues are favored at the P1′ position of FAPα-specific substrates because the substratebinding site of FAPα is surrounding by multiple tyrosine residues and some negatively charged residues. 28 The structure−activity relationship (SAR) analysis revealed that the 3β-OH of arenobufagin can be modified without significant influence on its antitumor activity. Thus, hydrophobic amino acids including aromatic and nonaromatic amino acids were located at P1′ positions to produce prodrugs such as Z-GP-Xaaarenobufagin (Figure 1). To evaluate the affinities of prodrugs to FAPα, all the compounds were docked to FAPα by SYBYLX2.1.1. The docking scores were found to range between 4.7561 and 8.0692, indicating that the prodrugs are suitable 5321

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Figure 3. Structures of compounds 2a−h and 3a−h.

(3a), V (3b), L (3c), I (3d), F (3e), and p-aminophenylacetic acid (3h). FAPα tolerated the peptide bond between P1−P′ best if the P′ site is occupied by W (3f). Aggarwal et al. suggested that FAP prefers to cleave the peptide with GP in P2−P1 than A in P′1 (Figure 1), which is inconsistent with our data.31 As the substrate of FAP hydrolysis, recombinant human collagen I is a synthetic peptide chain.31 Our substrates 3a, 3e, and 3f are semisynthetic peptides with a compound arenobufagin in P2′ position instead of an amino acid (Figure 1). This suggests that the component in P2′ position may affect the FAP catalytic efficiency. We performed a docking analysis in an attempt to explain the interaction of 3a, 3e, or 3f with FAPα.32−35 The docking scores of 3a, 3e, and 3f are 5.10, 8.0322, and 8.0692, respectively. The number of hydrogen bonds between these substrates and the residues of FAPα are 3a < 3e, 3f (Figure 5). Importantly, 3e and 3f, when docked into the active site of FAPα characterized by the P1 of 3e and 3f, fit well with the cleavage site formed by five Y (Y124, Y541, Y625, Y656, and Y660), along with two E (E203 and E204) and one D (D703) (Figure 5), which contributes to P1−P1′ being cleavable by FAP.28 However, the P1 of 3a is apparently not in the cleavage site (Figure S1 in Supporting Information). These results above not only provide evidence that F and W of 3e and 3f are better tolerated in the P1′ position than A of 3a but also demonstrate that 3e and 3f have stronger binding ability to FAPα than 3a. In addition, 3f is predicted to form more hydrogen bonds with FAPα than 3e, indicating that 3f binds to FAPα more strongly than 3e. 3f Is Mainly Activated in Tumors. As previously reported, the expression of FAPα was observed on the surface of CAFs of solid tumors.18−20 Here, we also observed the expression of FAPα in MDA-MB-231 xenograft tissues (Figure 6A) but not in MDA-MB-231 cells (Figure 6B). These prodrugs were designed to be inactivated in the heart but specifically activated in tumors with FAPα expression. Since 3f is the most readily hydrolyzed of the prodrugs 3a−h, its hydrolytic efficiency in homogenates of heart and tumor from mice bearing MDA-MB231 xenografts was further examined. As shown in Figure 6C,

substrates of FAPα. Accordingly, we synthesized these prodrugs for further investigations. Synthetic Chemistry. The synthetic route used is shown in Figure 2. First, an N-blocked amino acid is coupled to the 3βOH of arenobufagin by esterification. Next, the N-terminal amino acid is deprotected using Et3SiH/PdCl2 to give compound 2. Finally, the prodrug was obtained through the condensation between the terminal amino group of compound 2 and the carboxyl group of N-Cbz-glycine-L-proline. The active compounds 2a−h and synthetic tripeptide arenobufagin prodrugs 3a−h are shown in Figure 3. Evaluation of the Hydrolytic Efficiency of Prodrugs by rhFAPα. First, we determined if the eight synthetic prodrugs 3a−h (Figure 3) can be hydrolyzed by rhFAPα to release the corresponding active forms (2a−h). After incubation with rhFAPα buffer, there was no measurable active form (2g) in the rhFAPα buffer treated with prodrug 3g (Figure 4A), indicating that 3g was not a substrate of rhFAPα. We found that less than 40% of prodrugs 3b−3d and 3h were hydrolyzed by rhFAPα, whereas ∼80% of 3a was hydrolyzed by rhFAPα. And more than 95% of the prodrugs 3e and 3f were hydrolyzed by rhFAPα. Furthermore, pretreatment of 3e and 3f with talabostat (TAL), a selective inhibitor of FAPα, completely blocks their hydrolysis to release 2e or 2f (Figure 4B), demonstrating that 3e and 3f were specifically cleaved by rhFAPα. The kinetic parameters of 3a−f and 3h were also explored and were found to follow an allosteric sigmoidal model30 consistently (Figure 4C). The measured quantities S50 and Vmax are summarized in Table 1, and CLint, calculated from the ratio of Vmax to S50, was further used to compare the hydrolytic efficacy of these prodrugs. On the basis of CLint values, the enzymatic hydrolysis efficiency of these prodrugs by rhFAPα is as follows: 3f > 3e > 3a> 3c & 3h > 3b & 3d > 3g (Table 1), which almost agrees with the above results (Figure 4A). Docking Analysis. FAPα was unable to cleave 3g when a PABA is at the P1′ site. However, FAPα was able to cleave the peptide bond between P1−P′ if the P′ site is occupied by A 5322

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Figure 4. Prodrugs 3a−h are hydrolyzed by rhFAPα: (A) evaluation of the hydrolytic efficiency of 3a−h by rhFAPα; (B) TAL blocks the hydrolysis of 3e and 3f; (C) kinetic profiles for 3a−f and 3h reactions mediated by rhFAPα enzyme. Data are shown as the mean ± SEM from three independent experiments. Statistical analysis was conducted using GraphPad Prism 5 software.

the concentration of the activated form (2f) was about 19.82 ± 0.12 μmol/L after homogenates of tumors were incubated with the prodrug (3f). In addition, 2f in homogenates of tumors treated with 3f in the presence of TAL was about 20-fold less than that of homogenates incubated with 3f alone, suggesting that the release of 2f in tumor tissues was FAPα-dependent. It have been demonstrated that FAP-like activity is present multipotent bone marrow stromal cells (BMSCs)36,37 and in plasma or serum of several species.24,38,39 In this present study, approximately 5.3% (1.05 ± 0.24 μmol/L), 2.6% (0.51 ± 0.14 μmol/L), 14.77% (2.93 ± 0.60 μmol/L), and 23.46% (4.65 ±

0.53 μmol/L) of active form (2f) was detected in rat serum, BMSCs, 10% FBS, and homogenates of heart respectively when compared with tumor homogenates (19.82 ± 0.16 μmol/L). These results suggest that 3f is mainly hydrolyzed in tumor tissues. 2f was activated in homogenates of heart may be due to the hydrolysis of 3f by the small amount of blood in the heart. Previously, it was reported that some FAP-targeting strategies which were focused on targeting BMSCs with FAPreactive T cells or depletion of FAPα+ stromal cells induced cachexia and bone toxicity.36,37 It is noteworthy that our FAPactivated prodrug strategy only takes advantage of FAP’s post5323

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confidence limits of 3f are 44.036 and 40.717−49.597 μmol/ kg, respectively. The corresponding LD50 values for arenobufagin is 6.816 μmol/kg, and the 95% confidence limits were found to fall within the range 6.293−7.567 μmol/kg. The LD50 of 3f is 6.5-fold higher than that of arenoufagin, demonstrating that the toxicity of prodrug 3f toward mice is much lower than that of arenobufagin. After intravenous injection with 3f at 12 μmol/kg and arenobufagin at 4 μmol/kg for 10 days, no mice died. Consequently, 4 or 12 μmol/kg of 3f and 4 μmol/kg of arenobufagin respectively were selected as the doses for the in vivo antitumor assay. 3f Exhibits Potent Antitumor Effects in Vivo. The antitumor effect of 3f was examined in nude mice carrying an MDA-MB-231 tumor xenograft, with arenobufagin used as a reference. As shown in Figure 7A, compared with a control group, 3f treatment showed a tumor regression effect similar to that of arenobufagin. The tumor weight of 3f-treated (12 μmol/ kg) group was 0.43 ± 0.18 g, which is much lower than that in the control group (0.67 ± 0.14 g) (Figure 7B). We found that bufadienolide 3f at the concentrations of 4 and 12 μmol kg−1 day−1 failed to produce a dose-dependent in vivo antitumor activity. We think that this may be associated with the anticancer characteristics of bufadienolide (2f) and arenobufagin. We have found that 2f and arenobufagin at a concentration of 62.5−500 nmol/L did not show a dose-dependent antitumor effect in MDA-MB-231 cells in vitro (Figure S2B in Supporting Information). Nevertheless, the reasons for the phenomenon may be complex, and we intend to investigate it further in the future. Ki67 staining was utilized to determine the effect of the FAPα prodrug 3f on tumor proliferation, and as expected, in the 3f- or arenobufagin-treated groups, Ki67-positive cells were reduced by 45−50% when compared with the control group (Figure 7D). In addition, TUNEL-positive nuclei were observed ∼5 times more clearly in tumors treated with prodrug 3f when compared with those from the control group (Figure 7E). These results demonstrate a remarkable increase of apoptotic cells in the 3f-treated mice. Effects of 3f and Arenobufagin on Cardiac Function in Vivo. The cardiac function of mice was recorded by transthoracic echocardiography. As shown in Figure 8A, the interventricular septal dimension in systole (IVSs) and the interventricular septal dimension in diastole (IVSd) increased remarkably in the arenobufagin-treated group and the left ventricular internal dimension in systole (LVIDs) and left ventricular internal dimension in diastole (LVIDd) decreased. There were no apparent changes in echocardiography after treatment with 3f. Furthermore, pathological analysis demonstrated that the cardiac muscle in mice treated with arenobufagin presented extensive cytoplasmic vacuolization, whereas almost no vacuolization was observed in 3f-treated group (Figure 8B). Lactate dehydrogenase (LDH) being the main biochemical marker of myocardial damage, the activities of LDH in serum were determined. As shown in Figure 8C, the activities of LDH were significantly increased in the arenobufagin-treated group, while the activities of LDH were unchanged in the 3f-treated group. We also detected that the concentration of the activated form 2f was approximately 30fold less than the prodrug 3f in heart tissues (Figure S6 in Supporting Information), demonstrating that a very small amount of the prodrug 3f was activated by some other process in serum or heart in vivo. This result explains why the FAPactivated prodrug 3f might possess a reduced cardiotoxicity in

Table 1. Kinetic Parameters of Prodrugs Calculated by the Allosteric Sigmoidal Model compd

Vmax (μmol min−1 mg−1)

S50 (μmol/L)

CLint (mL min−1 mg−1)

3a 3b 3c 3d 3e 3f 3h

0.494 0.044 0.042 0.014 0.366 0.682 0.026

21.26 34.52 11.92 8.82 8.64 10.36 3.68

23.24 1.27 3.52 1.59 42.36 65.89 7.07

prolyl endopeptidase activity to activate the prodrug, and this can avoid the side effects caused by the alteration of FAP expression and enzymatic activity. Furthermore, although a small amount of prodrug 3f is activated by BMSCs, arenobufagin, 2f, and 3f at the concentration of 1000 nmol/L are only marginally toxic to BMSCs in vitro (Figure S2A in Supporting Information). The bone marrow toxicity reflected in the femur histology was not observed in 3f-treated mice (Figure S3 in Supporting Information). No abnormal pathological changes were observed in liver, spleen, lung, and kidney tissue morphology (Figure S3 in Supporting Information). In addition, mice maintained normal weight gain throughout the 3f treatment and did not show any abnormal food intake or behavior. These data indicate that prodrug 3f with reduced cardiotoxicity may be minimally toxic to nontarget tissues. 3f Exhibits a Potent Antitumor Effect in Vitro. The in vitro anticancer activities of 3f and its active form 2f or arenobufagin were determined using an MTT assay in either FAPα-negative cells (MDA-MB-231 and MCF-7)40 or wildtype FAPα-positive cells (MDA-MB-435, Figure S4 in Supporting Information). The prodrug 3f exhibited cytotoxicity comparable to that of arenobufagin in all tested cell lines. In FAPα-negative cells MDA-MB-231 and MCF-7, the cytotoxicity of the prodrug was approximately 6−10 times less than that of their corresponding hydrolysis products (Table 2). In contrast, the prodrug (3f) exhibits cytotoxicity comparable to that of its corresponding hydrolysis product in the FAPαpositive cells, MDA-MB-435. Similar results were observed in FAPα-transfected MDA-MB-231FAPα and MDA-MB-231NC cells (Figure 6B and Table 2). These results indicate that the prodrugs can be activated by FAPα. In order to further analyze whether there is something other than FAPα activating the prodrug 3f in the cytotoxicity assay in vitro, we determined the active form 2f level in 3f-treated MCF-7 and MDA-MB-231 (FAP-negative) and MDA-MB-435 (FAP-positive) cells by LC−MS. About 4.2% and 2.5% of 2f was detected in 3f-treated MCF-7 and MDA-MB-231 cells, respectively, whereas about 80% of 3f was hydrolyzed into 2f in 3f-treated MDA-MB-435 cells, indicating that 3f is activated selectively by FAP in the in vitro assay (Figure S5 in Supporting Information). These results are consistent with the results of the cytotoxicity evaluation. Acute Toxicities of 3f and Arenobufagin. Prior to the in vivo antitumor assay, acute toxicity tests of 3f and arenobufagin were conducted in KM mice. A single intravenous dose of 3f or arenobufagin at different concentrations, based on the predetermined LD0 and LD100 values, was administered. Toxic effects such as tachypnea, paralysis, muscle spasm, and arrhythmia are observed in all the groups, and all deaths occur less than 4 h after administration. The LD50 and 95% 5324

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Figure 5. Docking analysis of the interactions of 3a, 3e, or 3f with FAPα. 3a (A), 3e (B), and 3f (C) are docked with FAPα in a three-dimensional view (left). Red dashed lines: hydrogen bonds. Details of the interaction of 3a (A), 3e (B), or 3f (C) with FAPα in a two-dimensional view (right). Black dashed lines: hydrogen bonds. Green dashed lines with dots: π interactions including π−π stacking and π−cation interactions. Green full lines: hydrophobic contacts. The FAPα structure was obtained from the Protein Data Bank (PDB code 1Z68).

safety profile and favorable anticancer druglike properties of 3f warrant further studies of 3f as a drug candidate for cancer treatment. Our study suggests that FAPα prodrug strategy successfully increases the therapeutic window of bufadienolides and enhances the clinical benefit of bufadienolide derivatives. We also hope that this strategy could also be applied to the relief of the cardiac toxicity of the chemotherapeutic drugs frequently used in clinic. Further studies will be directed toward four interesting issues: (1) whether 3f is FAPα-selectively activated in vivo by developing a FAPα knockout transgenic mouse model; (2) how to increase the specificity and selectivity

tumor-bearing mice, even when the dose reaches 3 times that of arenobufagin.

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CONCLUSION In this study, we report on the design and synthesis of FAPαactivated prodrugs designed to overcome the cardiotoxicity of arenobufagin. It is clearly demonstrated that the arenobufagin analog (3f) is activated by rhFAPα and FAPα, enzymes that are expressed in tumor tissues. We also provide evidence that the prodrug 3f shows obvious reduced cardiac toxicity and exhibits significant antitumor activity in vitro and in vivo. The improved 5325

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Figure 6. (A) Expression of FAPα in MDA-MB-231 xenograft tissues. Immunofluorescence analysis was conducted with anti-FAPα antibody (green). Representative figure was shown. Original magnification: 40×. Scale bar: 20 μm. (B) The FAPα plasmids and the pCMV6-Entry vector were transfected into MDA-MB-231 cells. The expression of FAPα in these MDA-MB-231 cells was tested by Western blot with the indicated antibody. β-Actin was used as a loading control. (C) 3f was hydrolyzed by 10% FBS, 10% heat-inactivated FBS, rat serum, BMSCs, homogenates of heart and tumor. Each column represents the mean ± SEM of three independent samples. Statistical analysis was conducted using GraphPad Prism 5 software.

Table 2. Antiproliferative Activities of Areonbufagin, 2f, and 3f IC50 a (nM) compd

MDA-MB-435

MCF-7

MDA-MB-231

MDA-MB-231NC

MDA-MB-231FAPα

arenobufagin 2f 3f

22.45 ± 1.21 10.01 ± 3.68 10.64 ± 3.52

8.88 ± 4.41 1.06 ± 0.34 11.60 ± 4.03

15.67 ± 1.10 4.81 ± 0.54 27.00 ± 2.433

5.19 ± 1.19 23.97 ± 2.58

7.19 ± 3.74 11.72 ± 2.82

IC50 values are shown as the mean ± SEM from at least three independent experiments. Statistical analysis was conducted using GraphPad Prism 5 software.

a

and 5% NaHCO3. Finally, the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a residue, which was purified by flash column chromatography to yield compound 1. Method B. The solution of Et3SiH in 1 mL of anhydrous DCM was added dropwise to a stirred solution of compound 1, PdCl2, and TEA in anhydrous DCM under nitrogen at room temperature. Gas evolution was observed, and the mixture slowly turned black. The reaction was monitored by TLC and terminated after 20−50 min. The mixture was filtered through diatomite and extracted with DCM. The DCM layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a residue. The residue was then purified by flash column chromatography to yield compound 2. Method C. Compound 2, Z-Gly-Pro, EDCI, HOBt, and TEA were added to anhydrous DCM, and the mixture was stirred under nitrogen at room temperature. Brine was added after the reaction, monitored by TLC was complete. The organic phase was washed with brine and 5% NaHCO3 and dried over anhydrous Na2SO4. The organic was finally concentrated to obtain a residue, which was purified by flash column chromatography to yield compound 3. The purity of all biologically evaluated compounds 2a−h and 3a−h determined by HPLC is >95%.

of bufadienolide prodrugs for FAPα by structural modification; (3) why the similar antitumor efficacy at different doses of bufadienolides or prodrugs (3f) failed to produce a dosedependent in vivo antitumor activity; (4) exploring the effectiveness of FAP-activated arenobufagin in steroidogenesis and immune systems.

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EXPERIMENTAL SECTION

Docking Analysis. The FAPα structure was obtained from the Protein Data Bank (PDB code 1Z68). Docking experiments were performed by SYBYL-X2.1.1 to give the three-dimensional diagrams, and the details of ligand−protein interaction were performed by PoseView giving two-dimensional diagrams.32−35 Authors will release the atomic coordinates and experimental data upon article publication. Synthesis of Compounds. Method A. Arenobufagin, 4dimethylaminopyridine, EDCI, HOBt, and N-Cbz-amino acid in anhydrous DCM were stirred under nitrogen gas at room temperature. After the reaction, monitored by TLC, was terminated, brine was added. The organic phase was then separated and washed with brine 5326

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Figure 7. Anticancer activity of 3f and arenobufagin in a MDA-MB-231 xenograft model. (A) Growth curves of subcutaneous xenografts of MDAMB-231 (n = 6). (B) Tumor weights of MDA-MB-231 xenografts. (C) Stable body weight curves of treated mice (n = 6). (D) IHC was conducted with anti-Ki67 antibody. Representative figures of the Ki67 staining are shown (left), and quantitation of Ki67 indices was calculated using Image-Pro Plus 6.0 software (right). (E) IHC was conducted with Tunel staining. These stained mice tissue sections were examined by a pathologist who was blind to the treatment conditions. Representative figures were shown (left), and quantitation of apoptotic cells (%) was calculated using Image-Pro Plus 6.0 software (right). Original magnification: 40×. Scale bar: 100 μm. Each column represents the mean ± SEM (n = 6). Statistical analysis was conducted using GraphPad Prism 5 software with a one-way ANOVA with post hoc comparisons and Tukey’s test: (∗) P < 0.05 versus the 5% Solutol-HS15 (HS15) control; (∗∗) P < 0.01 versus the 5% Solutol-HS15 (HS15) control; (∗∗∗) P < 0.001 versus the 5% Solutol-HS15 (HS15) control. 1a. Synthesis of compound 1a with method A yielded a white foamy solid. Yield, 88%; mp 107−110 °C; 1H NMR (300 MHz, CDCl3) δ 7.76 (dd, J = 9.6, 1.7 Hz, 1H), 7.40 (s, 1H), 7.35 (s, 6H), 6.27 (d, J = 9.7 Hz, 1H), 5.47 (d, J = 7.5 Hz, 1H), 5.11 (s, 3H), 4.36 (dd, J = 19.0, 9.2 Hz, 2H), 4.14−4.05 (m, 1H), 3.86 (s, 1H), 3.47 (s, 2H), 2.45 (d, J = 13.7 Hz, 1H), 2.13−1.99 (m, 2H), 1.97−1.63 (m, 10H), 1.55 (d, J = 7.1 Hz, 1H), 1.49 (s, 1H), 1.43 (d, J = 7.1 Hz, 5H), 1.33 (d, J = 8.5 Hz, 5H), 1.18 (s, 3H), 0.91 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.77, 172.39, 162.36, 155.70, 150.09, 146.96, 136.20, 128.56, 128.21, 128.05, 120.92, 115.52, 85.44, 73.30, 71.94, 66.90, 61.97, 50.70, 49.89, 40.80, 40.75, 39.43, 38.61, 36.75, 32.76, 32.65, 30.64, 28.02, 26.30, 25.53, 23.44, 21.58, 18.82, 17.51. ESI-MS (m/z): 622.3 [M + H]+. HRESI-MS (m/z): 622.3011 [M + H]+; calcd for C35H43NO9 622.3011. 1b. Synthesis of compound 1b with method A yielded a white foamy solid. Yield, 90%; mp 226−228 °C; 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 9.7, 2.5 Hz, 1H), 7.40 (s, 1H), 7.32 (d, J = 19.6 Hz, 5H), 6.27 (d, J = 9.3 Hz, 1H), 5.38 (d, J = 8.2 Hz, 1H), 5.11 (s, 3H), 4.33 (d, J = 9.4 Hz, 2H), 4.10 (s, 1H), 3.86 (s, 1H), 2.63 (s, 1H), 2.46 (d, J = 13.1 Hz, 1H), 2.19 (s, 1H), 2.14−2.00 (m, 3H), 1.96−1.57 (m, 10H), 1.40 (dd, J = 39.4, 11.4 Hz, 6H), 1.18 (s, 3H), 0.98 (s, 4H), 0.91 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 213.75, 171.43, 162.33,

156.34, 150.10, 146.95, 136.20, 128.57, 128.24, 128.07, 120.90, 115.53, 85.46, 73.31, 71.86, 67.03, 61.97, 59.15, 40.79, 39.43, 38.75, 36.76, 32.75, 31.43, 30.75, 28.03, 26.27, 25.61, 23.48, 21.57, 19.01, 17.52, 17.41. ESI-MS (m/z): 650.3 [M + H]+. HR-ESI-MS (m/z): 650.3324 [M + H]+; calcd for C37H48NO9 650.3244. 1c. Synthesis of compound 1c with method A yielded a white foamy solid. Yield, 91%; mp 95−97 °C; 1H NMR (300 MHz, CDCl3) δ 7.75 (dd, J = 9.7, 2.3 Hz, 1H), 7.41 (d, J = 1.7 Hz, 1H), 7.34 (d, J = 11.5 Hz, 5H), 6.28 (d, J = 9.7 Hz, 1H), 5.25 (d, J = 8.6 Hz, 1H), 5.09 (d, J = 13.3 Hz, 3H), 4.43−4.29 (m, 2H), 4.11 (dd, J = 9.3, 7.2 Hz, 1H), 3.85 (d, J = 3.5 Hz, 1H), 2.48 (s, 1H), 2.44 (s, 1H), 2.18 (s, 1H), 2.14−2.00 (m, 2H), 2.01−1.23 (m, 20H), 1.16 (d, J = 11.3 Hz, 3H), 0.97 (d, J = 5.0 Hz, 6H), 0.92 (s, 3H), 0.88 (d, J = 2.0 Hz, 1H), 0.86 (s, 1H), 0.84 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 213.71, 172.44, 162.26, 155.97, 150.10, 146.83, 136.19, 128.56, 128.22, 128.07, 120.80, 115.59, 85.50, 73.32, 71.78, 66.98, 61.92, 52.82, 41.89, 40.80, 40.75, 39.48, 38.67, 36.74, 32.81, 32.69, 31.59, 30.66, 28.01, 26.25, 25.56, 24.86, 23.48, 22.76, 22.12, 21.57, 17.51. ESI-MS (m/z): 664.6 [M + H]+. HR-ESI-MS (m/z): 644.3484 [M + H]+; calcd for C38H50NO9 644.3480. 5327

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

Journal of Medicinal Chemistry

Article

Figure 8. Effects of 3f and arenobufagin on cardiac function in the left ventricles of MDA-MB-231 tumor-bearing mice. (A) Analysis of cardiac morphology and function of mice by echocardiography after 22 days drug administration. Representative figures for echocardiography (top) and LVIDd, LVIDs, IVSd, and IVSs were calculated (n = 6) (bottom). (B) Representative images of morphological changes of myocardial cells observed by HE staining. Original magnification: 40×. Scale bar: 100 μm. (C) Effect of 3f and arenobufagin on serum levels of LDH. Each column represents the mean ± SEM. Statistical analysis was conducted using GraphPad Prism 5 software with a one-way ANOVA with post hoc comparisons and Tukey’s test: (∗) P < 0.05 versus the 5% HS15 control; (#) P < 0.05 versus the arenobufagin group alone (n = 6). These stained mice tissue sections were examined by a pathologist who was blind to the treatment conditions. 1d. Synthesis of compound 1d with method A yielded a white foamy solid. Yield, 89%; mp 104−106 °C; 1H NMR (300 MHz, CDCl3) δ 7.73 (dd, J = 9.8, 2.6 Hz, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.38−7.27 (m, 5H), 6.27 (d, J = 9.7 Hz, 1H), 5.33 (d, J = 9.1 Hz, 1H), 5.13 (s, 1H), 5.10 (s, 2H), 4.34 (dd, J = 14.1, 7.7 Hz, 2H), 4.14−4.06 (m, 1H), 2.46 (d, J = 14.1 Hz, 1H), 2.31 (s, 1H), 2.16 (d, J = 4.2 Hz, 1H), 2.05 (t, J = 10.8 Hz, 2H), 1.93 (s, 1H), 1.88 (s, 2H), 1.81 (d, J = 9.6 Hz, 1H), 1.79−1.60 (m, 6H), 1.46 (d, J = 14.0 Hz, 1H), 1.41−1.29 (m, 4H), 1.29−1.21 (m, 3H), 1.18 (s, 3H), 1.00−0.93 (m, 4H), 0.91 (s, 3H), 0.85 (dd, J = 8.1, 5.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ

213.65, 171.37, 162.19, 156.19, 150.10, 146.70, 136.22, 128.57, 128.23, 128.11, 120.70, 115.64, 85.53, 73.31, 71.80, 67.02, 61.89, 58.46, 40.81, 40.76, 39.53, 38.77, 38.22, 36.75, 32.84, 32.75, 30.75, 29.27, 27.99, 26.21, 24.97, 23.50, 21.57, 17.50, 15.59, 11.75. ESI-MS (m/z): 664.6 [M + H]+. HR-ESI-MS (m/z): 664.3485 [M + H]+; calcd for C38H50NO9 664.3480. 1e. Synthesis of compound 1e with method A yielded a white foamy solid. Yield, 86%; mp 118−120 °C; 1H NMR (300 MHz, CDCl3) δ 7.75 (dd, J = 9.7, 2.4 Hz, 1H), 7.37 (t, J = 2.8 Hz, 1H), 7.35−7.27 (m, 5H), 7.23 (t, J = 7.2 Hz, 3H), 7.15−7.09 (m, 2H), 6.24 (d, J = 9.7 Hz, 5328

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

Journal of Medicinal Chemistry

Article

δ 7.76 (dd, J = 9.7, 2.4 Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 6.21 (d, J = 9.7 Hz, 1H), 5.07 (s, 1H), 4.27 (d, J = 11.0 Hz, 1H), 4.05 (dd, J = 21.7, 14.3 Hz, 1H), 3.36 (s, 5H), 3.07 (q, J = 7.3 Hz, 2H), 2.38 (d, J = 13.7 Hz, 1H), 2.15−1.48 (m, 10H), 1.30−1.16 (m, 6H), 1.13 (s, 3H), 0.99−0.91 (m, 3H), 0.87 (d, J = 2.7 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 213.94, 173.65, 162.48, 149.98, 147.25, 121.13, 115.33, 85.16, 73.21, 71.53, 62.08, 59.41, 40.72, 40.66, 39.20, 38.76, 36.72, 32.74, 32.55, 31.75, 30.72, 28.05, 26.25, 25.58, 23.45, 21.51, 18.94, 17.58, 17.26. ESI-MS (m/z): 516.3 [M + H]+, 560.5 [M + HCOO]−. HR-ESI-MS (m/z): 516.2956 [M + H]+; calcd for C29H42NO7 516.2956. 2c. Synthesis of compound 2c with method B yielded a white foamy solid. Yield, 82%; mp 116−119 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 7.9 Hz, 1H), 7.39 (s, 1H), 6.28 (d, J = 9.7 Hz, 1H), 5.09 (s, 1H), 4.32 (d, J = 11.2 Hz, 1H), 4.08 (d, J = 7.4 Hz, 1H), 3.46 (s, 1H), 2.45 (d, J = 13.2 Hz, 2H), 2.04 (dd, J = 37.1, 27.5 Hz, 6H), 1.76 (d, J = 9.4 Hz, 18H), 1.59−1.22 (m, 14H), 1.18 (s, 4H), 0.96 (s, 1H), 0.94 (s, 3H), 0.91 (s, 5H), 0.85 (d, J = 6.4 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 213.75, 176.12, 162.21, 150.24, 146.68, 120.70, 115.81, 85.71, 73.47, 71.00, 61.98, 53.26, 44.51, 40.98, 40.89, 39.75, 38.85, 36.90, 33.03, 32.89, 30.89, 28.10, 26.39, 25.77, 25.09, 23.61, 22.97, 22.30, 21.73, 17.61. ESI-MS (m/z): 530.7 [M + H]+. HR-ESIMS (m/z): 530.3113 [M + H]+; calcd for C30H44NO7 530,3112. 2d. Synthesis of compound 2d with method B yielded a white foamy solid. Yield, 90%; mp 97−100 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.8, 2.6 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 6.28 (d, J = 9.7 Hz, 1H), 5.12 (s, 1H), 4.32 (d, J = 11.1 Hz, 1H), 4.10 (dd, J = 9.6, 7.2 Hz, 1H), 3.37 (d, J = 4.7 Hz, 1H), 2.45 (dt, J = 13.8, 2.7 Hz, 1H), 2.14−1.98 (m, 3H), 1.94 (d, J = 4.1 Hz, 1H), 1.92−1.57 (m, 16H), 1.52−1.21 (m, 10H), 1.19 (s, 3H), 0.97 (s, 2H), 0.95 (s, 2H), 0.93 (s, 1H), 0.91 (s, 3H), 0.91 (s, 2H), 0.85 (ddd, J = 11.3, 5.1, 2.6 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 213.72, 175.16, 162.22, 150.22, 146.65, 120.67, 115.84, 85.71, 73.47, 70.97, 61.96, 59.27, 40.94, 40.87, 39.74, 39.40, 38.90, 36.89, 33.03, 32.94, 30.93, 28.09, 26.33, 25.82, 24.76, 23.63, 21.71, 17.61, 15.95, 11.92. ESI-MS (m/z): 530.7 [M + H]+. HR-ESI-MS (m/z): 530.3112 [M + H]+; calcd for C30H44NO7 530.3112. 2e. Synthesis of compound 2e with method B yielded a white foamy solid. Yield, 78%; mp 97−99 °C; 1H NMR (300 MHz, CDCl3) δ 7.76 (dd, J = 9.7, 2.6 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.35−7.14 (m, 5H), 6.27 (d, J = 9.7 Hz, 1H), 5.06 (s, 1H), 5.06 (s, 1H), 4.27 (dd, J = 17.2, 10.7 Hz, 1H), 4.08 (dt, J = 17.0, 8.6 Hz, 1H), 3.82−3.68 (m, 1H), 2.98 (ddd, J = 33.4, 13.5, 6.8 Hz, 2H), 2.39 (d, J = 13.8 Hz, 2H), 2.05 (dd, J = 20.4, 9.6 Hz, 3H), 1.92−1.54 (m, 9H), 1.51−1.16 (m, 8H), 1.12 (s, 2H), 0.90 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 213.82, 174.61, 162.39, 150.14, 146.99, 137.21, 129.26, 128.62, 126.87, 120.94, 115.57, 85.49, 73.35, 71.30, 61.99, 55.92, 41.53, 40.83, 40.75, 39.47, 38.41, 36.73, 32.80, 30.63, 28.09, 26.37, 25.65, 23.40, 21.62, 17.56. ESI-MS (m/z): 564.3 [M + H]+. 608.6 [M + HCOO]−. HR-ESI-MS (m/z): 564.2955 [M + H]+; calcd for C33H42NO7 564.2956. 2f. Synthesis of compound 2f with method B yielded a light yellow foamy solid. Yield, 88%; mp 130−133 °C; 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.71 (dd, J = 9.8, 2.6 Hz, 1H), 7.64 (s, 1H), 7.62 (s, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.37 (s, 1H), 7.34 (s, 1H), 7.22−7.03 (m, 5H), 6.27 (d, J = 9.6 Hz, 1H), 5.05 (s, 2H), 4.26 (t, J = 9.0 Hz, 2H), 4.13−4.04 (m, 1H), 3.88 (t, J = 6.6 Hz, 2H), 3.81 (s, 1H), 3.48 (s, 1H), 3.15 (ddd, J = 21.8, 14.3, 6.6 Hz, 4H), 3.02−2.93 (m, 1H), 2.35 (d, J = 13.8 Hz, 2H), 2.11−1.92 (m, 6H), 1.91−1.48 (m, 25H), 1.41−1.10 (m, 17H), 1.05 (s, 4H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.79, 175.02, 162.27, 150.21, 146.75, 136.43, 127.62, 122.83, 122.28, 120.75, 119.63, 118.90, 115.77, 111.54, 111.39, 85.65, 73.41, 71.09, 61.95, 55.25, 40.86, 39.64, 38.41, 36.68, 32.95, 32.74, 31.50, 30.71, 28.08, 26.33, 25.66, 23.38, 21.69, 17.57. ESI-MS (m/z): 603.6 [M + H]+. HR-ESI-MS (m/z): 603.3074 [M + H]+; calcd for C35H43N2O7 603.3056. 2g. Synthesis of compound 2g with method B yielded a white foamy solid. Yield, 83%; mp 235−237 °C; 1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8.5 Hz, 3H), 7.75 (dd, J = 9.7, 2.5 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 6.63 (d, J = 8.5 Hz, 3H), 6.25 (d, J = 9.7 Hz, 1H), 5.23 (s, 2H), 4.35−4.26 (m, 2H), 4.08 (dd, J = 9.0, 7.4 Hz, 1H), 2.46 (d, J =

1H), 5.41 (d, J = 6.3 Hz, 1H), 5.06 (dd, J = 10.5, 5.0 Hz, 3H), 4.64 (dd, J = 14.2, 6.6 Hz, 1H), 4.28 (dd, J = 10.9, 2.4 Hz, 1H), 4.14−4.01 (m, 2H), 3.83 (d, J = 3.1 Hz, 1H), 3.19−2.98 (m, 2H), 2.79 (s, 1H), 2.34 (t, J = 14.2 Hz, 1H), 2.11−1.94 (m, 4H), 2.05−1.98 (m, 3H), 1.92−1.44 (m, 9H), 1.46−1.15 (m, 8H), 1.09 (s, 3H), 0.88 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 213.80, 171.34, 171.12, 162.40, 155.71, 150.09, 147.04, 136.17, 135.90, 129.23, 128.57, 128.25, 128.07, 127.06, 120.98, 115.49, 85.43, 73.28, 72.11, 66.98, 61.99, 60.48, 55.01, 40.78, 40.71, 39.36, 38.64, 38.34, 36.67, 32.71, 32.62, 30.57, 28.04, 26.31, 25.55, 23.32, 21.10, 17.52. ESI-MS (m/z): 698.3 [M + H]+. HR-ESIMS (m/z): 698.3324 [M + H]+; calcd for C41H48NO9 698.3324. 1f. Synthesis of compound 1f with method A yielded a white foamy solid. Yield, 90%; mp 138−140 °C; 1H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.72 (dd, J = 9.7, 2.5 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 1.7 Hz, 1H), 7.36−7.27 (m, 6H), 7.20−7.04 (m, 2H), 6.95 (d, J = 1.9 Hz, 1H), 6.25 (d, J = 9.7 Hz, 1H), 5.46 (d, J = 8.2 Hz, 1H), 5.11 (s, 1H), 5.09 (s, 1H), 5.01 (s, 1H), 4.75 (dd, J = 13.9, 6.1 Hz, 1H), 4.25 (dd, J = 11.0, 3.3 Hz, 1H), 3.81 (d, J = 3.5 Hz, 1H), 3.33− 3.25 (m, 1H), 2.53 (s, 1H), 2.31 (t, J = 11.9 Hz, 1H), 2.17 (s, 1H), 2.10−1.90 (m, 3H), 0.99 (s, 3H), 0.86 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.75, 171.61, 162.46, 155.89, 150.12, 147.02, 136.22, 128.58, 128.25, 128.11, 127.55, 122.74, 122.18, 120.96, 119.61, 118.70, 115.52, 111.33, 109.93, 85.46, 73.28, 71.93, 66.98, 61.92, 54.69, 40.77, 40.67, 39.38, 38.19, 36.55, 32.72, 32.51, 30.51, 28.54, 28.01, 26.18, 25.47, 23.21, 21.54, 17.47. ESI-MS (m/z): 737.7 [M + H]+. HR-ESIMS (m/z): 737.3433 [M + H]+; calcd for C43H49N2O9 737.3433. 1g. Synthesis of compound 1g with method A yielded a white foamy solid. Yield, 60%; mp 215−217 °C; 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 8.7 Hz, 2H), 7.73 (dd, J = 9.7, 2.5 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.43−7.32 (m, 6H), 7.18 (s, 1H), 6.26 (d, J = 9.7 Hz, 1H), 5.29 (s, 1H), 5.21 (s, 2H), 4.34 (d, J = 11.0 Hz, 1H), 4.10 (dd, J = 9.3, 7.0 Hz, 1H), 3.86 (s, 1H), 2.51 (d, J = 13.8 Hz, 1H), 2.07 (ddd, J = 12.7, 10.4, 3.4 Hz, 3H), 1.95 (s, 1H), 1.86 (t, J = 9.5 Hz, 4H), 1.76 (dd, J = 15.5, 12.0 Hz, 5H), 1.61 (d, J = 7.5 Hz, 1H), 1.57− 1.46 (m, 1H), 1.45−1.29 (m, 4H), 1.22 (s, 3H), 0.92 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.83, 165.66, 162.38, 153.09, 150.19, 146.88, 142.23, 135.78, 130.95, 128.78, 128.62, 128.46, 125.75, 120.88, 117.75, 115.71, 85.67, 73.49, 70.97, 67.43, 62.01, 40.93, 40.91, 39.68, 38.95, 36.99, 33.05, 31.00, 29.80, 28.11, 26.42, 25.94, 23.66, 21.75, 17.61. ESI-MS (m/z): 692.5 [M + Na]+. HR-ESI-MS (m/z): 670.2977 [M + H]+; calcd for C39H44NO9 670.3011. 1h. Synthesis of compound 1h with method A yielded a white foamy solid. Yield, 83%; mp 125−126 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.7, 2.4 Hz, 1H), 7.42−7.29 (m, 8H), 7.20 (d, J = 8.5 Hz, 2H), 6.96 (s, 1H), 6.25 (d, J = 9.7 Hz, 1H), 5.18 (s, 2H), 5.04 (s, 1H), 4.30 (t, J = 9.5 Hz, 1H), 4.07 (dd, J = 9.4, 7.1 Hz, 1H), 3.55 (s, 2H), 2.36 (d, J = 13.8 Hz, 1H), 2.04 (dd, J = 20.8, 9.0 Hz, 3H), 1.90−1.77 (m, 3H), 1.73 (dd, J = 11.4, 7.9 Hz, 4H), 1.66−1.55 (m, 3H), 1.42 (d, J = 3.8 Hz, 1H), 1.38−1.17 (m, 6H), 1.11 (s, 3H), 0.88 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.84, 171.25, 162.35, 153.50, 150.16, 146.90, 136.91, 136.12, 129.94, 129.55, 128.69, 128.43, 128.32, 120.89, 118.87, 115.63, 85.58, 73.44, 71.08, 67.07, 61.99, 41.38, 40.92, 40.81, 39.60, 38.48, 36.79, 32.89, 32.62, 30.73, 28.08, 26.31, 25.63, 23.45, 21.65, 17.57. ESI-MS (m/z): 706.5 [M + Na]+, 701.4 [M + NH4]+. HR-ESI-MS (m/z): 684.3168 [M + H]+; calcd for C40H46NO9 684.3167. 2a. Synthesis of compound 2a with method B yielded a white foamy solid. Yield, 85%; mp 117−119 °C; 1H NMR (300 MHz, CDCl3) δ 7.71 (dd, J = 9.7, 2.6 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 6.28 (d, J = 9.8 Hz, 1H), 5.09 (s, 1H), 4.32 (d, J = 11.1 Hz, 1H), 4.15−4.06 (m, 1H), 3.56 (dd, J = 13.7, 6.6 Hz, 1H), 2.45 (dt, J = 14.7, 2.7 Hz, 1H), 2.11− 1.93 (m, 3H), 1.92−1.59 (m, 11H), 1.53−1.28 (m, 7H), 1.25 (s, 2H), 1.19 (s, 1H), 0.92 (s, 3H), 0.88(s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.71, 162.16, 150.21, 146.56, 120.60, 115.89, 85.75, 73.45, 71.09, 61.93, 50.37, 40.95, 40.87, 39.78, 38.76, 36.91, 33.07, 32.86, 30.85, 29.83, 28.08, 26.40, 25.74, 23.58, 21.75, 20.93, 17.61. ESI-MS (m/z): 488.2 [M + H]+. HR-ESI-MS (m/z): 488.2643 [M + H]+; calcd for C27H38NO7 488.2643. 2b. Synthesis of compound 2b with method B yielded a white foamy solid. Yield, 86%; mp 75−76 °C; 1H NMR (300 MHz, CDCl3) 5329

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

Journal of Medicinal Chemistry

Article

13.2 Hz, 3H), 2.04 (dd, J = 20.9, 9.1 Hz, 4H), 1.95−1.78 (m, 8H), 1.79−1.63 (m, 9H), 1.61−1.43 (m, 5H), 1.33 (dd, J = 20.0, 10.1 Hz, 6H), 1.19 (s, 5H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.96, 166.33, 162.58, 150.95, 150.14, 147.10, 131.58, 121.08, 120.43, 115.55, 113.89, 85.40, 73.44, 70.35, 62.08, 40.89, 40.83, 39.55, 38.93, 36.95, 33.12, 32.79, 31.02, 28.12, 26.44, 25.95, 23.60, 21.66, 17.57. ESIMS (m/z): 536.5 [M + H]+. HR-ESI-MS (m/z): 536.2649 [M + H]+; calcd for C31H38NO7 536.2643. 2h. Synthesis of compound 2h with method B yielded a white foamy solid. Yield, 86%; mp 85−87 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.7, 2.5 Hz, 1H), 7.38 (d, J = 1.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 2H), 6.63 (d, J = 8.2 Hz, 2H), 6.25 (d, J = 9.7 Hz, 1H), 5.02 (s, 1H), 4.29 (d, J = 11.1 Hz, 1H), 4.07 (dd, J = 9.3, 7.1 Hz, 1H), 3.48 (s, 2H), 2.36 (d, J = 13.8 Hz, 1H), 2.02 (dd, J = 12.2, 9.2 Hz, 2H), 1.91− 1.77 (m, 3H), 1.73 (dd, J = 9.9, 7.0 Hz, 4H), 1.67−1.54 (m, 4H), 1.47−1.34 (m, 2H), 1.33−1.18 (m, 7H), 1.13 (s, 3H), 0.88 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 213.87, 171.85, 162.37, 150.16, 146.94, 130.18, 124.50, 120.92, 115.62, 115.41, 85.57, 73.48, 70.82, 62.00, 41.20, 40.90, 40.78, 39.59, 38.45, 36.79, 32.87, 32.62, 30.72, 29.77, 28.08, 26.32, 25.63, 23.44, 21.64, 17.58. ESI-MS (m/z): 548.6 [M − H]+. HR-ESI-MS (m/z): 550.2799 [M + H]+; calcd for C32H40NO7 550.2799. 3a. Synthesis of compound 3a with method C yielded a white foamy solid. Yield, 80%; mp 135−136 °C; 1H NMR (300 MHz, CDCl3) δ 7.74 (dd, J = 9.8, 2.5 Hz, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.36−7.29 (m, 5H), 7.22 (d, J = 7.2 Hz, 1H), 6.26 (d, J = 9.6 Hz, 1H), 5.82 (d, J = 4.0 Hz, 1H), 5.09 (d, J = 7.5 Hz, 3H), 4.53 (d, J = 7.9 Hz, 1H), 4.45 (t, J = 7.2 Hz, 1H), 4.30 (d, J = 11.0 Hz, 1H), 4.15−4.04 (m, 2H), 4.01 (t, J = 4.0 Hz, 1H), 3.57 (dd, J = 16.5, 8.2 Hz, 1H), 3.40 (dd, J = 16.3, 8.6 Hz, 1H), 2.43 (d, J = 13.9 Hz, 1H), 2.34−2.21 (m, 1H), 2.03 (s, 3H), 2.00−1.53 (m, 12H), 1.50−1.21 (m, 12H), 1.16 (s, 3H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.81, 172.09, 170.56, 168.09, 162.28, 156.28, 150.07, 146.93, 136.32, 128.50, 128.13, 128.01, 120.88, 115.51, 85.40, 73.30, 71.82, 66.93, 61.98, 60.44, 60.13, 48.59, 46.38, 43.46, 40.78, 40.70, 39.39, 38.57, 36.74, 32.73, 30.61, 28.01, 27.89, 26.29, 25.52, 24.84, 23.40, 21.08, 18.19, 17.52, 14.19. ESI-MS (m/z): 776.8 [M + H]+. HR-ESI-MS (m/z): 776.3754 [M + H]+; calcd for C42H54N3O11 776.3753. 3b. Synthesis of compound 3b with method C yielded a white foamy solid. Yield, 79%; mp 123−125 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.8, 2.6 Hz, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.37−7.28 (m, 5H), 7.21 (d, J = 8.6 Hz, 1H), 6.28 (d, J = 9.7 Hz, 1H), 5.78 (s, 1H), 5.10 (d, J = 6.0 Hz, 3H), 4.59 (d, J = 6.3 Hz, 1H), 4.44 (dd, J = 8.5, 4.6 Hz, 1H), 4.31 (d, J = 11.0 Hz, 1H), 4.09 (dd, J = 12.2, 5.1 Hz, 2H), 4.02 (d, J = 9.7 Hz, 1H), 3.56 (t, J = 7.4 Hz, 1H), 3.42 (dd, J = 16.6, 9.0 Hz, 1H), 2.44 (d, J = 13.8 Hz, 1H), 2.32 (d, J = 9.3 Hz, 1H), 2.16 (dd, J = 11.5, 7.0 Hz, 2H), 2.06 (s, 3H), 2.04 (s, 2H), 1.98−1.60 (m, 11H), 1.52−1.21 (m, 7H), 1.17 (s, 3H), 0.91 (s, 1H), 0.90 (s, 3H), 0.88 (d, J = 2.4 Hz, 3H), 0.86 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 213.61, 170.96, 170.69, 168.16, 162.14, 150.02, 146.62, 136.23, 128.46, 128.09, 127.99, 120.61, 115.58, 85.48, 73.24, 71.63, 66.93, 61.82, 60.12, 57.50, 46.33, 43.36, 40.73, 40.67, 39.46, 38.61, 36.68, 32.77, 32.61, 31.23, 30.68, 27.91, 27.46, 26.18, 25.54, 24.83, 23.36, 21.51, 18.97, 17.50, 17.43. ESI-MS (m/z): 804.5 [M + H]+, 848.5 [M + HCOO]−. HR-ESI-MS (m/z): 804.4064 [M + H]+; calcd for C44H58N3O11 804.4066. 3c. Synthesis of compound 3c with method C yielded a white foamy solid. Yield, 90%; mp 122−125 °C; 1H NMR (300 MHz, CDCl3) δ 7.71 (dd, J = 9.7, 2.6 Hz, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.37−7.29 (m, 5H), 7.08 (d, J = 7.6 Hz, 1H), 6.27 (d, J = 9.8 Hz, 1H), 5.70 (t, J = 3.9 Hz, 1H), 5.11 (s, 2H), 5.09 (s, 1H), 4.56 (d, J = 6.5 Hz, 1H), 4.47 (dd, J = 14.4, 6.8 Hz, 1H), 4.30 (dd, J = 11.1, 3.4 Hz, 1H), 4.14−4.05 (m, 1H), 4.00 (dd, J = 7.4, 4.5 Hz, 2H), 3.81 (d, J = 3.5 Hz, 1H), 3.53 (dd, J = 11.8, 5.7 Hz, 1H), 3.40 (dd, J = 16.4, 8.9 Hz, 1H), 2.44 (d, J = 13.9 Hz, 1H), 2.15−1.96 (m, 6H), 1.83 (ddd, J = 22.4, 14.8, 6.9 Hz, 12H), 1.70−1.52 (m, 7H), 1.35 (ddd, J = 23.6, 12.8, 7.3 Hz, 7H), 1.17 (s, 3H), 0.93 (d, J = 6.0 Hz, 4H), 0.90 (s, 4H), 0.89 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 213.76, 172.08, 170.50, 168.27, 162.24, 156.34, 150.20, 146.72, 136.41, 128.65, 128.28, 128.18, 120.72, 115.78, 85.65, 73.43, 71.76, 67.11, 61.96, 60.19, 51.61, 46.46, 43.54, 41.46, 40.92,

40.83, 39.66, 38.73, 36.83, 32.97, 32.76, 30.76, 28.08, 27.57, 26.33, 25.67, 25.15, 24.98, 23.54, 22.71, 22.40, 21.68, 17.60. ESI-MS (m/z): 818.8 [M + H]+. HR-ESI-MS (m/z): 818.4223 [M + H]+; calcd for C45H60N3O11 818.4222. 3d. Synthesis of compound 3d with method C yielded a white foamy solid. Yield, 80%; mp 125−127 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.7, 2.5 Hz, 1H), 7.38 (d, J = 1.7 Hz, 1H), 7.37−7.27 (m, 6H), 7.19 (d, J = 8.3 Hz, 1H), 6.26 (d, J = 9.7 Hz, 1H), 5.73 (s, 1H), 5.11 (s, 3H), 4.57 (d, J = 6.2 Hz, 1H), 4.49 (dd, J = 8.3, 4.3 Hz, 1H), 4.30 (dd, J = 10.9, 3.2 Hz, 1H), 4.08 (t, J = 8.1 Hz, 1H), 4.00 (dd, J = 8.6, 4.4 Hz, 2H), 3.81 (d, J = 3.4 Hz, 1H), 3.53 (d, J = 7.1 Hz, 1H), 3.40 (dd, J = 16.8, 8.4 Hz, 1H), 2.44 (d, J = 11.9 Hz, 1H), 2.31 (s, 2H), 2.16 (m, 4H), 2.09−1.57 (m, 19H), 1.41 (m, 10H), 1.17 (m, 4H), 0.90 (m, 4H), 0.89−0.81 (m, 7H). 13C NMR (75 MHz, CDCl3) δ 213.78, 171.01, 170.66, 168.19, 162.23, 156.31, 150.19, 146.76, 136.41, 128.62, 128.25, 128.15, 120.76, 115.72, 85.60, 73.42, 71.79, 67.07, 61.99, 60.24, 56.96, 46.45, 43.54, 40.91, 40.84, 39.62, 38.82, 37.99, 36.83, 32.92, 32.80, 31.05, 28.07, 27.56, 27.00, 26.30, 25.20, 24.99, 23.56, 21.66, 17.60, 15.71, 11.85. ESI-MS (m/z): 818.9 [M + H]+. HR-ESI-MS (m/z): 818.4221 [M + H]+; calcd for C45H60N3O11 818.4222. 3e. Synthesis of compound 3e with method C yielded a white foamy solid. Yield, 90%; mp 118−121 °C; 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 9.8, 2.5 Hz, 1H), 7.39 (s, 1H), 7.39−7.29 (m, 7H), 7.16 (ddd, J = 22.7, 14.6, 6.7 Hz, 6H), 6.27 (d, J = 9.8 Hz, 1H), 5.64 (s, 1H), 5.14 (d, J = 1.5 Hz, 2H), 5.10 (s, 1H), 4.81 (dd, J = 14.1, 6.6 Hz, 1H), 4.55 (d, J = 7.0 Hz, 1H), 4.32−4.23 (m, 1H), 4.09 (dd, J = 11.2, 7.1 Hz, 1H), 3.94 (d, J = 4.4 Hz, 1H), 3.84 (d, J = 4.0 Hz, 1H), 3.82−3.76 (m, 1H), 3.39−3.26 (m, 2H), 3.08 (ddd, J = 37.2, 14.1, 6.7 Hz, 3H), 2.38 (d, J = 13.8 Hz, 2H), 2.02 (dd, J = 19.3, 8.2 Hz, 7H), 1.90−1.48 (m, 15H), 1.44−1.17 (m, 10H), 1.12 (s, 4H), 0.88 (d, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 213.74, 170.86, 170.17, 168.23, 162.20, 150.19, 146.67, 136.46, 136.30, 129.33, 128.64, 128.39, 128.28, 128.19, 127.03, 120.68, 115.80, 85.67, 73.39, 72.09, 67.10, 61.94, 59.98, 53.48, 46.26, 40.92, 40.83, 39.66, 38.19, 36.79, 32.98, 30.72, 28.08, 27.03, 26.37, 25.66, 24.85, 23.36, 21.70, 17.59. ESI-MS (m/z): 852.3 [M + H]+. HR-ESI-MS (m/z): 852.4066 [M + H]+; calcd for C48H58N3O11 852.4066. 3f. Synthesis of compound 3f with method C yielded a light yellow foamy solid. Yield, 89%; mp 135−136 °C; 1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.70 (dd, J = 9.8, 2.6 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.47−7.27 (m, 8H), 7.10 (dt, J = 23.7, 7.3 Hz, 2H), 6.92 (s, 1H), 6.28 (d, J = 9.6 Hz, 1H), 5.49 (s, 1H), 5.15 (d, J = 6.4 Hz, 2H), 5.09 (s, 1H), 4.87 (dd, J = 13.3, 6.0 Hz, 1H), 4.52 (d, J = 7.3 Hz, 1H), 4.27 (dd, J = 11.0, 3.5 Hz, 1H), 4.13−4.05 (m, 1H), 3.80 (d, J = 3.5 Hz, 1H), 3.63 (dd, J = 60.8, 4.4 Hz, 1H), 3.43 (dd, J = 25.2, 5.4 Hz, 1H), 3.28 (dd, J = 35.3, 6.0 Hz, 2H), 3.19−3.11 (m, 1H), 3.10−3.03 (m, 1H), 2.37 (d, J = 14.8 Hz, 2H), 2.17 (s, 1H), 1.93 (ddd, J = 39.5, 23.3, 11.4 Hz, 9H), 1.72 (s, 6H), 1.61 (dd, J = 33.3, 11.5 Hz, 6H), 1.44−1.17 (m, 10H), 1.08 (s, 3H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.74 (d, J = 5.8 Hz), 171.34, 170.21, 168.30, 162.15, 156.41, 150.11, 146.55, 136.51, 136.02, 128.72, 128.41, 128.33, 127.95, 123.08, 122.15, 120.60, 119.48, 118.59, 115.82, 111.37, 110.08, 85.68, 73.41, 71.98, 67.16, 61.93, 59.96, 53.25, 46.12, 43.16, 40.92, 40.82, 39.67, 38.36, 36.77, 32.98, 32.53, 30.72, 29.82, 28.07, 27.44, 27.03, 26.31, 25.63, 24.83, 23.30, 21.70, 17.58. ESI-MS (m/z): 891.8 [M + H]+. HR-ESI-MS (m/z): 891.4175 [M + H]+; calcd for C50H59N4O11 891.4175. 3g. Synthesis of compound 3g with method C yielded a white foamy solid. Yield, 87%; mp 120−122 °C; 1H NMR (300 MHz, CDCl3) δ 9.57 (s, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.72 (dd, J = 9.8, 2.6 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 1.8 Hz, 1H), 7.38−7.29 (m, 9H), 6.28 (d, J = 10.2 Hz, 1H), 5.70 (s, 1H), 5.29 (s, 2H), 5.16− 5.05 (m, 4H), 4.76 (d, J = 7.3 Hz, 1H), 4.34 (d, J = 10.6 Hz, 1H), 4.06 (ddd, J = 18.1, 14.9, 6.9 Hz, 4H), 3.85 (s, 1H), 2.51 (d, J = 13.0 Hz, 3H), 1.85 (d, J = 9.8 Hz, 7H), 1.25 (s, 3H), 1.22 (s, 4H), 1.10 (t, J = 7.3 Hz, 2H), 0.92 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.83, 171.16, 162.27, 156.46, 150.17, 146.77, 137.12, 136.31, 129.82, 128.67, 128.35, 128.17, 120.78, 119.95, 115.73, 85.63, 73.44, 71.02, 67.23, 61.98, 61.18, 46.69, 43.58, 40.91, 40.83, 39.66, 36.80, 32.93, 32.66, 5330

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

Journal of Medicinal Chemistry

Article

to LC−MS/MS analysis. S50 and Vmax were calculated from the concentration plots with allosteric sigmoidal because Eadie−Hofstee plots of the data were indicative of autoactivation, and CLint was calculated from the ratio of Vmax to S50. Other prodrugs 3b−h were analyzed using the same experimental process as was used for 3a. Hydrolysis of Prodrug 3f by 10% FBS, 10% Heat-Inactivated FBS, BMSCs, Rat Serum, Homogenates of Tumor and Heart. Normal FBS was treated at 56 °C overnight to generate heatinactivated FBS. Tumor and heart tissues were weighed and homogenized on ice containing PBS (pH 7.4) by using a mechanical tissue grinder. Internal standard bufalin (2 μmol/L) was added to the homogenates. The standard curves were constructed by adding 2f (final concentration ranged from 0.625 to 40 μmol/L) to the corresponding reaction buffer. Prodrug 3f (40 μmol/L) was incubated with normal 10% FBS, 10% heat-inactivated FBS, BMSCs, rat serum, homogenates of tumor or heart. The reaction was stopped by adding acetonitrile. The reaction buffers were centrifuged at 18 000g for 30 min, and the supernatant was subjected to LC−MS/MS analysis. Generation of FAPα-Transfected Cells. The human FAPα cDNA clone (catalog SC117372) and pCMV6-Entry vector (catalog PS100001) were obtained from Origene Technologies (Rockville, MD). Lipofectamine LTX reagent with PLUS reagent (catalog 15338100) was obtained from Invitrogen (Carlsbad, CA). The human FAPα plasmids and the pCMV6-Entry vector were transfected into MDAMB-231 to generate FAP-positive cells MDA-MB-231FAPα and negative control cells MDA-MB-231NC respectively according to the manufacturer’s instructions. After 48 h, cells were lysed for Western blot analysis to test the transfected efficiency. Western Blotting. Total cellular protein lysate was prepared as described previously.5 Cell lysates were separated by SDS−PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked and immunoblotted with antibody against FAPα (1:500, abcam, ab53066). MTT Assay. 10% heat-inactivated FBS was used in in vitro cytotoxicity assays in order to rule out of the disturbance of FAP activity in FBS. The cytotoxic effects of these compounds were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as has been described previously.41 Acute Toxicity Assay. 100 Kunming mice (22.0−28.0 g), half male and half female, were randomly divided into 10 groups (n = 10) and intravenously injected with 3f (29.98, 34.07, 38.72, 44.00, and 50.00 μmol/kg) or arenobufagin (4.50, 5.27, 6.06, 6.96, and 8.00 μmol/kg) on day 1. The mice were observed continuously for 8 h after drug administration to detect any abnormal behavior, and the death rate of each group was recorded. Subsequently, the mice were observed intermittently for the next 2 weeks and sacrificed on the 14th day. The Bliss method was employed to calculate the LD50 values of 3f and arenobufagin. Tumor Xenograft in Nude Mice. MDA-MB-231 cells (1 × 107) in 200 μL of Matrigel−PBS buffer (v:v = 1:1) were inoculated subcutaneously into the backs of female BABL/C mice. Each day after day 5, animals were intravenously administered 3f dissolved in 5% HS15 at a concentration of 12 μmol/kg or arenobufagin dissolved in 5% HS15 at a concentration of 4 μmol/kg. Tumor size was assessed every 2 days by caliper. Tumor volume was calculated using the formula v = 0.5 × a × b2, where a refers to the longer diameter and b to the shorter diameter of the tumor. The experiment was terminated on day 22. Animals were anesthetized, and hearts were rapidly collected and fixed in 4% paraformaldehyde for 24 h. Tumors were also collected, weighed, and photographed, then fixed in 4% paraformaldehyde. Both heart and tumor tissues were further examined by histologically and immunohistochemically (IHC). Echocardiography. Cardiac function of animals were recorded by transthoracic echocardiography using a Technos MPX ultrasound system (ESAOTE, Italy) with a 21 MHz imaging transducer, as described previously.42 Briefly, animals were anesthetized with 1.5% isoflurane and placed in a supine position. The M-mode images were obtained from the parasternal short-axis view at the papillary muscle. The following parameters were measured: LVIDd, LVIDs, IVSd, IVSs. All the parameters were obtained from 6 samples per group.

30.73, 29.81, 28.08, 27.11, 26.32, 25.64, 25.06, 23.47, 23.39, 21.70, 17.60. ESI-MS (m/z): 824.3 [M + H]+. HR-ESI-MS (m/z): 824.3753 [M + H]+; calcd for C47H53N3O11 824.3753. 3h. Synthesis of compound 3h with method C yielded a white foamy solid. Yield, 80%; mp 127−128 °C; 1H NMR (300 MHz, CDCl3) δ 9.20 (d, J = 3.5 Hz, 1H), 7.71 (dd, J = 9.7, 2.0 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 1.8 Hz, 1H), 7.33 (d, J = 5.9 Hz, 5H), 7.19 (d, J = 8.3 Hz, 2H), 6.26 (d, J = 9.6 Hz, 1H), 5.76 (s, 1H), 5.11 (s, 2H), 5.03 (s, 1H), 4.72 (d, J = 7.5 Hz, 1H), 4.28 (d, J = 10.8 Hz, 1H), 4.03 (td, J = 17.1, 11.1 Hz, 3H), 3.80 (d, J = 3.0 Hz, 1H), 3.55 (s, 3H), 3.42 (dd, J = 16.5, 8.8 Hz, 1H), 2.48 (s, 1H), 2.41−2.31 (m, 1H), 2.18 (dd, J = 22.8, 15.7 Hz, 2H), 2.03 (d, J = 9.1 Hz, 3H), 1.93−1.51 (m, 13H), 1.42 (s, 1H), 1.25 (s, 4H), 1.12 (s, 2H), 1.09 (s, 2H), 0.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 213.83, 171.16, 162.27, 156.46, 150.17, 146.77, 137.12, 136.31, 129.82, 128.67, 128.35, 128.17, 120.78, 119.95, 115.73, 85.63, 73.44, 71.02, 67.23, 61.98, 61.18, 46.69, 43.58, 40.91, 40.83, 39.66, 36.80, 32.93, 32.66, 30.73, 29.81, 28.08, 27.11, 26.32, 25.64, 25.06, 23.47, 23.39, 21.70, 17.60. ESI-MS (m/z): 838.8 [M + H]+. HR-ESI-MS (m/z): 838.3909 [M + H]+; calcd for C47H56N3O11 838.3909. Cell Lines. Human breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-435 were obtained from American Type Culture Collection (ATCC, Rockville, MD). Breast cancer cell lines were incubated in DMEM medium with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin−streptomycin in a 5% CO2 humidified atmosphere at 37 °C. Animals. Four- to six-week-old female BALB/C mice were purchased from Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China). Kunming (KM) mice weighing 20−28 g were obtained from Guangdong Medical Experimental Animal Center (Guangzhou, China). All animal experiments were conducted in accordance with the ARRIVE guidelines and were approved by Experimental Animal Ethics Committee of Jinan University (Guangzhou, China). LC−MS/MS Methods. Quantitation of the active forms 2a−h were carried out by the Waters ACQUITY UPLC system equipped with a photodiode array detector (Milford, MA). Chromatographic separation was performed on an ACQUITY UPLC BEH column (2.1 mm × 100 mm, 1.7 μm; Waters). Elution was accomplished using a gradient of 0.1% formic acid in H2O (mobile phase A) versus MeOH (mobile phase B) at a flow rate of 0.40 mL/min. The gradient program was 5% B at 0−1.0 min, 25%−40% B at 1.0−2.0 min, 40%−70% B at 2.0−3.0 min, 70%−95% B at 3.0−3.5 min, 95%−90% B at 3.5−4.0 min, and 90%−5% B at 4.0−5.0 min. Data were collected in the positive mode using the electrospray ionization source (ESI) in full scan mode (200− 1200 Da). The desolvation gas (N2) was 800 L/h and cone gas was 30 L/h. The desolvation and source temperature were 350 and 100 °C, respectively. An external reference leucine enkephalin (lock spray, m/z 556.2771) at the concentration of 2 ng/mL was infused with a flow rate of 5 μL/min to ensure mass accuracy throughout an entire experiment. rhFAPα-Mediated Hydrolysis of Prodrugs in Vitro. Bufalin (2 μmol/L), as the internal standard, was added into the reaction buffer. The reaction buffer (0.2 mL per sample) was maintained at 37 °C and contained prodrug 2a (40 μmol/L) and rhFAPα (0.45 μg/mL) for 6 h. Acetonitrile (0.6 mL) was added to the reaction buffer to terminate the reaction. The buffer was centrifuged at 18 000g for 15 min, and the supernatant was subjected to UPLC−MS/MS analysis. A standard curve was produced by using the peak area ratio between various concentrations of 2a (0, 0.625−40 μmol/L) and bufalin (2 μmol/L). Peak areas of 2a released from 3a by rhFAPα were converted to concentrations, and the data were analyzed by GraphPad Prism. Other prodrugs 3b−h were analyzed using the same experiment process as 3a. Kinetic Analysis of rhFAPα Hydrolysis. Various concentrations of prodrug 3a (0.625−40 μmol/L) were incubated with rhFAPα (0.45 μg/mL) and an internal standard in 0.2 mL of phosphate buffer saline (PBS, pH 7.4) at 37 °C for 90 min. MeCN (0.6 mL) was added into the reaction buffer to terminate the reaction. The reaction buffer was centrifuged at 18 000g for 15 min, and the supernatant was subjected 5331

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

Journal of Medicinal Chemistry

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Immunofluorescence Analysis. The expression of FAPα in MDA-MB-231 xenograft tissues was assessed as described previously.24 Tumor samples were snap frozen in OCT compound for storage and further processing. Fresh cryosections were fixed and blocked, followed by incubation with antibody against FAPα (1:50, abcam, ab53066) and secondary antibody. The distributions of FAPα were imaged using confocal microscope (Zeiss LSM700, Germany) with a ×20 lens, at the excitation wavelength of 488 nm. Histopathological and Immunohistochemical Analysis. All IHCs were performed as described previously.43 After tissues were fixed for 24 h and then embedded in paraffin, tissue sections (5 μm thick) were mounted on glass slides. For histological examination, heart, liver, spleen, lung, kidney, and femur sections were stained with hematoxylin−eosin (H&E). For proliferative index analysis, sections were incubated with anti-Ki67. For apoptotic analysis, sections were examined using the in situ cell death detection kit (Roche Diagnostic). The stained mice tissue sections in the current study were examined by a pathologist who was blind to the treatment conditions. Images were collected by using an Olympus DP2-SAL microscope. Proliferative index (%) and apoptosis (%) were analyzed by Image-Pro Plus software. Assays for D-Lactate Dehydrogenase (D-LDH). Before sacrifice, the blood of mice was collected by cardiac puncture and centrifuged at 3000 rpm for 20 min at 4 °C to obtain serum. Enzyme activities of DLDH in serum were measured using an Amplite colorimetric D-lactate dehydrogenase assay kit (AAT Bioquest) in accordance with the manufacturer’s protocol. Statistical Analysis. Results were presented as the mean ± SEM. Statistical analysis was conducted using GraphPad Prism 5 software with a one-way ANOVA with post hoc comparisons and Tukey’s test. P value of ≤0.05 was considered to indicate significant differences.

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2013CXZDA006), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant 20124401110008), the Program for New Century Excellent Talents in University (D.-M.Z.), and the Project Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (D.-M.Z.).

ABBREVIATIONS USED

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REFERENCES

FAPα, fibroblast activation protein α; rhFAPα, recombinant human FAPα; CAF, carcinoma-associated fibroblast; Z-GP, Nterminal benzyloxycarbonyl blocked Gly-Pro; FBS, fetal bovine serum; PS, penicillin−streptomycin; DCM, dichloromethane; MeCN, acetonitrile; Me2CO, acetone; MeOH, methanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EtOH, ethanol; IC50, median inhibitory concentration; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; IHC, immunohistochemistry; IVSd, interventricular septal dimension in diastole; IVSs, interventricular septal dimension in systole; LVIDd, left ventricular internal dimension in diastole; LVIDs, left ventricular internal dimension; D-LDH, D-lactate dehydrogenase; HS15, SolutolHS15; EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; IHC, immunohistochemically; PVDF, polyvinylidene fluoride; PBS, phosphate buffer saline; TEA, triethanolamine; HOBt, 1-hydroxybenzotriazole

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01755. (1) General methods, (2) additional experimental results, (3) 1H NMR, 13C NMR, HRMS, and ESI-MS spectra of all compounds, and (4) HPLC data of 2a−h and 3a−h used in biological assays (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*W.-C.Y.: e-mail, [email protected]; phone, +86 20 85221559. *D.-M.Z.: e-mail, [email protected]; phone, +86 20 85222653. *W.-M.C.: e-mail, [email protected]; phone, +86 20 85224497. ORCID

Wen-Cai Ye: 0000-0002-2810-1001 Wei-Min Chen: 0000-0002-0292-7841 Author Contributions †

L.-J.D. and L.-H.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Science and Technology Program of China (Grant 2012ZX09103101-053), the National Science Foundation of China (Grant 81573455), Guangdong Province (Grants S20120100008130, S2013050014183, and 5332

DOI: 10.1021/acs.jmedchem.6b01755 J. Med. Chem. 2017, 60, 5320−5333

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