An NAD(P)H:Quinone Oxidoreductase 1 Responsive and Self

May 3, 2018 - 1 had considerable inhibitory activity toward Taxol-resistant. A549 cells. In comparison, 2, without the self-immolative linker, was rel...
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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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An NAD(P)H:Quinone Oxidoreductase 1 Responsive and SelfImmolative Prodrug of 5‑Fluorouracil for Safe and Effective Cancer Therapy Xian Zhang,†,§ Xiang Li,†,§ Zhihong Li,†,‡ Xingsen Wu,†,‡ Yue Wu,† Qidong You,*,† and Xiaojin Zhang*,†,‡ †

Sate Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China ‡ Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 211198, China S Supporting Information *

ABSTRACT: Tripartite prodrug 1, composed of an NAD(P)H:quinone oxidoreductase 1 (NQO1)-responsive trigger group, a self-immolative linker, and the active drug 5-fluorouracil (5-FU), was designed and synthesized for site-specific cancer therapy. Upon bioreductive activation by NQO1, 1 can release the parent drug 5-FU specifically in NQO1-overexpressing cancer cells. This prodrug exerts comparable antitumor activity and a more favorable safety profile compared with 5-FU both in vitro and in vivo.

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shortcomings, and considering the combination of NQO1 overexpression in cancer cells together with the Q3PA-based rapid chemical release upon NQO1 bioreduction, we rationally designed a tripartite prodrug (1) that was composed of an NQO1-responsive trigger group (Q3PA), a self-immolative linker, and the active drug 5-FU (Figure 1A). Utilizing the crystal structure of NQO1 (PDB ID: 3JSX),9 a molecular docking study was performed using GOLD 5.1 to elucidate the possible binding mode of 1 with NQO1. It was observed that 1 fit well with the catalytic pocket of NQO1 (Figure 1B, Figure S1). The quinone trigger group lay deep into the catalytic sites and was oriented above the bound cofactor FAD by π-stacking interaction with a reasonable distance of 3.38 Å between C(1)O and N(5)H for hydride transfer; meanwhile, the Hbond interaction between the other C(4)O and Tyr128 can probably facilitate the hydride transfer for reduction. The selfimmolative linker inserted into the pocket formed by Phe232, Phe236, Met154, Tyr128, and His161, whereas the 5-FU moiety was ideally extended toward the solvent region (Figure 1B). It was proposed that, in response to NQO1 bioreductive activation, the Q3PA trigger group of 1 would first undergo intramolecular cyclization, and then the linker could be eliminated via a well-known self-immolative 1,6-elimination reaction6,10 to spontaneously liberate the active agent 5-FU, leading to cancer cell-specific targeting (Figure 1C). In

he general goal in anticancer drug development is to achieve highly selective toxicity against cancer cells while simultaneously sparing normal cells. Prodrug strategies are an advanced research area in which nontoxic drugs are carried to cancer tissues and activated by overexpressing enzymes specifically, bringing about controllable drug release in target sites.1 NAD(P)H:quinone oxidoreductase 1 (NQO1) is such a cancer-specific cytosolic flavoenzyme that is highly elevated in numerous cancer cells,2 especially in nonsmall cell lung cancer (NSCLC), where it is constitutively overexpressed over 100fold greater than that in correlative normal tissues.2c NQO1 can catalyze the direct two-electron reduction of a broad range of substrates, including diverse antitumor quinones3 via a FADmediated hydride (H−) transfer from cofactor NAD(P)H to quinone substrate.4 Recently, McCarley and co-workers have identified the “trimethyl lock” containing quinone propionic acid (Q3PA) as an excellent NQO1-responsive trigger group5 and further developed Q3PA-based fluorescent probes for detection and imaging of NQO1 in cancer cells.6 Interestingly, the NQO1-mediated reduction of Q3PA ester or amide derivatives bearing a “trimethyl lock” is accompanied by spontaneous intramolecular cyclization to form lactone, which can lead to the rapid release of a chemical moiety.7 5-Fluorouracil (5-FU), an antimetabolic agent, is still in firstline chemotherapy for various cancers, including lung, breast, liver, colorectal, and pancreatic cancers. However, poor selectivity for cancer cells and high incidence of normal tissue toxicity limits its therapeutic utility.8 To overcome these © XXXX American Chemical Society

Received: May 3, 2018

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DOI: 10.1021/acs.orglett.8b01409 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

carbamate bond in the presence of triphosgene, giving designed prodrug 1. Meanwhile, compound 2, without the selfimmolative linker, was generated through condensation of 5FU with acid 5 using DCC. In addition, compound 3, missing the NQO1-responsive group, was synthesized by acylation of 5FU with benzyl carbonochloridate. Compounds 2 and 3 were designed as comparison controls for biological assessment. We first evaluated whether the prodrug could be efficiently reduced by NQO1 and could specifically release 5-FU. The initial NQO1 reductive rates of 1 and 2 under various concentrations were determined by NADPH assay.3b Then, the Michaelis−Menten curves for 1 and 2 were generated and their NQO1 kinetic parameters (kcat/KM), reflecting their catalytic efficiency, were calculated (Figure 2A). It was observed that Figure 1. (A) Rational design of NQO1-responsive prodrug 1. (B) Predicted binding mode of 1 in the NQO1 catalytic pocket. (C) Proposed process for NQO1-responsive drug release.

addition, the cyclized lactone is considered to be nontoxic in cells,11,11a whereas the formed iminoquinone methide causes toxicity just at a high concentration.11b We then synthesized prodrug 1 and further evaluated its capability to release 5-FU in response to NQO1, its stability in buffers or plasma, its selective antiproliferative activity toward NQO1-overexpressing cancer cells, and the antitumor effect as well as its short-term safety profile in vivo. The synthetic routes for prodrug 1 and its counterparts 2 and 3 are depicted in Scheme 1. The reaction between 2,3,5trimethylbenzene-1,4-diol and 3-methylbut-2-enoic acid in the presence of methanesulfonic acid provided 4, which was further treated with NBS to afford 5. Condensation of 5 with a TBSprotected linker followed by deprotection using TBAF provided 6. Consequently, 5-FU was incorporated into 6 by a Scheme 1. Synthetic Routes for Prodrug 1 and its Counterparts 2 and 3 Figure 2. (A) Michaelis−Menten curves for 1 and 2 with NQO1. (B) Percentage of 5-FU (as determined by HPLC) released from 1 (20 μM) and 2 (20 μM), respectively, as a function of time in the presence of NQO1 (14 μg/mL) and NADPH (100 μM) in PBS. (C) HRMS analyses for 1 (20 μM) after coincubation with NQO1 (14 μg/mL) and cofactor NADPH (100 μM) for 6 h.

both 1 and 2 were efficient substrates for NQO1, though 1 showed a better kcat/KM value as compared to 2; this suggests that the prodrug can bind to NQO1 catalytic sites and be reduced as expected. Subsequently, we set out to study the release profiles of 1 and 2 in response to NQO1 and cofactor NADPH using the HPLC assay (Figure 2B). As for prodrug 1, the time dependence of the prodrug’s release was observed. With increasing incubation time, the percentage of 5-FU increased steadily and reached a plateau in 48 h, coming up to ∼92%. However, for its counterpart 2 missing the linker in its structure, the release of 5-FU was extremely low even after 48 h incubation, indicating that the self-immolative linker in prodrug 1 is essential for 5-FU release. Furthermore, the anticipated release of the active agent 5-FU was also observed by HRMS (ESI) assay (Figure 2C). After prodrug 1 was coincubated with NQO1 and NADPH, the corresponding fragments including 5FU ([M + H]+ m/z = 131.0250) were detected, providing sufficient evidence for the release of 5-FU in the presence of NQO1. Importantly, 1 exhibited excellent stability at 37 °C in B

DOI: 10.1021/acs.orglett.8b01409 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters phosphate buffer (pH 4−11) with cofactor NADPH (Figure S2) as well as in rat or human plasma (Figure S3) even after 24 h incubation. The results suggest that 1 is relatively stable to provide enough exposure time under physiological conditions such that 1 can be specifically activated and release the cytotoxic drug in NQO1-overexpressing cancer tissues; these findings provide theoretical reference for further study in vivo. Next, we investigated the antiproliferative activity of 1, together with 2 and 3 as comparison controls, against the NQO1-overexpressing NSCLC cell line (A549), Taxol-resistant A549 cell line (A549/T), and NQO1-deficient normal human hepatocyte cell line (L02) using the MTT assay3d (Figure S4). The IC50 values obtained are given in Table 1. As compared to

Figure 3. In vivo effects of 1 on tumor growth against A549 xenografts in BALB/c nude mice (iv, 15 mg/kg). 5-FU was selected as control. (A) The tumor diameters were measured and used to calculate the tumor volumes. (B) After 20 days, mice were sacrificed, and the individual tumors were weighed. **p < 0.01; Student’s t test (n = 6).

Table 1. In Vitro Antiproliferative Activity, IC50 (μM) compd

A549

1 2 3 5-FU

6.6 ± 1.1 39.6 ± 1.1 >100 3.4 ± 1.1

A549/T

L02

A549+DIC

± ± ± ±

>100 >100 >100 7.6 ± 0.9

>100 >100 >100 4.5 ± 1.2

6.8 39.5 96.8 4.0

1.1 1.1 1.9 1.1

administration. Food intake in the group that received 1 was consistent with the control group, whereas the animals, both male and female, that received 5-FU consumed relatively less food (Figure S5A and B). As for body weight, no obvious differences were observed between the 1 group and the saline control group (Figure S5C and D). However, the weight of the mice in the 5-FU group significantly decreased compared with that of the control mice (Figure S5C and D). On the sixth day, all the mice in the 5-FU group had died, and following completion of the trial, all male mice died and the survival rate of female mice was only 20% (Figure 4). After the two week

5-FU, 1 exerted comparable antiproliferative activity toward both A549 and A549/T cancer cells with IC50 values of 6.6 and 6.8 μM, respectively. Importantly, 1 was almost nontoxic toward L02 cells (IC50 > 100 μM) as expected, whereas no such difference in selectivity was observed for 5-FU, indicating that prodrug 1 was selectively toxic to cancer cells that overexpress NQO1 while causing little damage to normal cells. In addition, 1 had considerable inhibitory activity toward Taxol-resistant A549 cells. In comparison, 2, without the self-immolative linker, was relatively less active toward A549 and A549/T cells with IC50 values of 39.6 and 39.5 μM, respectively; this may be attributed to its weak ability to release of 5-FU (Figure 2B). Furthermore, 3, missing the NQO1-responsive group in its structure, had no obvious antiproliferative activity against the NQO1-overexpressing cancer cells, demonstrating that 1 exhibited its therapeutic effect through the bioreduction of the Q3PA trigger group catalyzed specifically by NQO1. To further verify the role of NQO1 in the drug release process in the cells, we also performed MTT assays for A549 cells pretreated with dicoumarol (DIC), an inhibitor of NQO1. Upon pretreatment with DIC, the cell inhibition decreased significantly (Figure S4D), suggesting that the prodrug exerted its cytotoxicity depending on NQO1 bioreductive activation. Encouraged by the in vitro antitumor cell activity of 1, we further evaluated its in vivo effect against A549 xenografts in the BALB/c nude mice model; 5-FU was used as a comparison control. After tumor xenografts were established, the nude mice were randomly injected through the tail vein with vehicle, 1, and 5-FU at a dose of 15 mg/kg once every 2 days for three consecutive weeks. The results indicate that prodrug 1 is capable of effectively inhibiting tumor growth in vivo (Figure 3A). The tumor inhibition rate of 1 was 60.5%, which was comparable to that of 5-FU (68.9%) (Figure 3B). Consequently, we decided to study the short-term toxicity of 1 in vivo in normal mice to determine if prodrug 1 could cause toxicity under high doses. Thirty mice were randomly distributed into three groups (five female and five male in each group): group I, saline control group; group II, 100 mg/kg of 5-FU; group III, 100 mg/kg of 1. Mice were treated every other day by tail vein injection for 2 weeks; body weights and food intake were recorded every other day after drug

Figure 4. Survival rate curves of the various groups of male and female mice.

trial, mice were sacrificed by cervical dislocation and were dissected surgically for evaluation of possible pathological changes. Histological examination of heart, liver, spleen, lung, and kidney tissue samples was performed using standard hematoxylin-eosin staining protocols. Histologically, tissue damage, including myocardial cell injury, liver damage, and interstitial pneumonia, were observed only in mice that received 5-FU, whereas the 1 treatment group showed no histological damage (Figure 5). In addition, compared to the control group, no obvious abnormalities in organ coefficients of the 1 group were observed (Figure S6). These results indicate that, compared with the parent drug 5-FU, 1 had more favorable drug safety, exhibiting reduced toxicity to normal tissues. This prodrug would benefit from further research on its potential as an effective targeted therapy. Herein, we present a novel tripartite prodrug 1 composed of an NQO1-responsive trigger group, a self-immolative linker, and the active drug 5-FU. This prodrug can be bioreductively activated by NQO1, and following intramolecular cyclization and the 1,6-elimination reaction, it can effectively liberate the parent drug 5-FU. Prodrug 1 is metabolically stable under physiological conditions, which may assist in avoiding unexpected off-target drug release, and shows highly selective toxicity to cancer cells that overexpress NQO1, whereas no obvious damage to normal cells has been observed. Further in C

DOI: 10.1021/acs.orglett.8b01409 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Central Universities (2632017ZD03), and the “Qing Lan” Project of Jiangsu Province. Part of the work was supported by the National Major Science and Technology Project of China (Nos. 2015ZX09101032 and 2017ZX09302003), the Open Project of State Key Laboratory of Natural Medicines (SKLNMZZCX201803), and the College Students Innovation Project for the R&D of Novel Drugs (J1310032).

■ Figure 5. Representative images of heart, liver, spleen, lung, and kidney tissues stained by hematoxylin and eosin at 400×. First row: group I (saline control group) with no histological abnormality. Second row: group II (100 mg/kg of 5-FU) with tissue damage including myocardial cell injury, liver damage, and interstitial pneumonia. Third row: group III (100 mg/kg 1) with no histological abnormality as compared with group I.

vivo antitumor evaluation revealed that 1 can effectively inhibit tumor growth in the A549 xenograft nude mice model at a dose of 15 mg/kg. Notably, as compared to 5-FU, 1 exerts a remarkably improved safety profile; no cases of mortality and no obvious tissue damage were observed in normal mice under high-dose administration of 100 mg/kg. Thus, 1 has a wide therapeutic window and exhibits reduced toxicity to normal tissues; this prodrug would benefit from further research on its potential as a specific cancer therapy. Additionally, as an NQO1-responsive prodrug, 1 is expected to be promising in personalized chemotherapy for NQO1-overexpressing cancers when combined with NQO1 diagnostic reagents.6,12

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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.orglett.8b01409. Experimental procedures and compound characterization for all new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Qidong You: 0000-0002-8587-0122 Xiaojin Zhang: 0000-0002-1898-3071 Author Contributions §

Xian Zhang and Xiang Li contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 81603025 and 81773571), Jiangsu Province Funds for Excellent Young Scientists (BK20170088), the Fundamental Research Funds for the D

DOI: 10.1021/acs.orglett.8b01409 Org. Lett. XXXX, XXX, XXX−XXX