Abstract

   Juglone has been extensively reported as a natural antitumor pigment.
   However, it is easy to be oxidized due to active hydroxy in the
   quinone. Here, we designed some new juglone derivatives, as the hydroxy
   was replaced by methyl (D1), allyl (D2), butyl (D3), and benzyl (D4)
   groups. Nuclear magnetic resonance spectra and mass spectrometry were
   applied to confirm the derivatives and oxidative products of juglone.
   U87 and U251 cell lines were used for tests in vitro, and primary human
   glioblastoma cells were applied for in vivo experiments. The CCK8 and
   EdU assay demonstrated the anti-tumor effect of the four derivatives,
   and IC50 for U87 was 3.99, 3.28, 7.60, and 11.84 μM, respectively. In
   U251, IC50 was 7.00, 5.43, 8.64, and 18.05 μM, respectively. D2 and D3
   were further selected, and flow cytometry showed that apoptosis rates
   were increased after D2 or D3 treatment via ROS generation. Potential
   targets were predicted by network pharmacology analysis, most of which
   were associated with apoptosis, cell cycle, and metabolism pathway.
   CDC25B and DUSP1 were two of the most likely candidates for targets.
   The orthotopic glioblastoma model was established to evaluate the
   anti-glioma effect and side-effect of juglone derivatives, and the in
   vivo experiments confirmed the anti-glioma effects of juglone
   derivatives. In conclusion, new derivatives of juglone were created via
   chemical group substitution and could inhibit glioma cell viability and
   proliferation and induce apoptosis rate via ROS generation.

   Keywords: glioblastoma, juglone, chemotherapy, ROS, apoptosis

Introduction

   Glioma is the most common primary malignant brain tumor, and
   glioblastoma (GBM) contributes 50–60% of them. Despite the advance in
   molecular research of GBM, the overall survival remains as poor as
   14.6 months even after comprehensive management ([44]Stupp et al.,
   2005; [45]Zhu et al., 2017). Temozolomide (TMZ) was demonstrated as a
   first-line chemotherapeutic agent through DNA alkylation by clinical
   trials, but GBM would resist TMZ when MGMT is unmethylated or when the
   tumor recurs ([46]Newlands et al., 1997; [47]Yung et al., 2000;
   [48]Stupp et al., 2001). Tumor treating fields (TTFields) also could
   partially benefit GBM patients ([49]Zhu et al., 2017). However, many
   endeavors such as anti-VEGFA ([50]Batchelor et al., 2014),
   anti-EGFRvIII ([51]Schuster et al., 2015), and anti-PDL1 ([52]Berghoff
   et al., 2015) all failed to meet the set goals. Therefore, there is
   still urgency to develop new therapeutic approaches for GBM.

   Juglone shows broad anti-cancer activity in traditional herbal medicine
   ([53]Sugie et al., 1998; [54]Xu et al., 2012; [55]Redaelli et al.,
   2015; [56]Zhang et al., 2015). It has been reported that juglone could
   exert its anti-glioma effect for its fat-soluble characteristics in
   vitro and in vivo in human GBM cells ([57]Wu et al., 2017) and also in
   C6 rat glioma cells ([58]Meskelevicius et al., 2016). It is also
   cytotoxic to human leukemia, cervical carcinoma, and pancreatic cancer
   cells ([59]Xu et al., 2012; [60]Zhang et al., 2012; [61]Avci et al.,
   2016). The potential mechanism includes the activation of the apoptotic
   caspase cascade and the accumulation of intracellular reactive oxygen
   species (ROS) ([62]Sajadimajd et al., 2016). Juglone is also taken as a
   PIN-1 (peptidyl-prolyl cis/trans isomerase 1) inhibitor for malignant
   solid tumors ([63]Xu et al., 2016).

   Although the anti-glioma effect was confirmed in our previous report
   ([64]Wu et al., 2017), there are still some concerning issues. The
   preservation of juglone is difficult due to its instability and
   susceptibility to oxidation, which could decrease the antitumor
   effects. Hence, new derivatives of juglone are designed to increase
   stability and lipophilicity, with the hydroxy-substituted by other
   chemical scaffolds, such as methyl, allyl, butyl, and benzyl group. The
   toxicity and potential mechanism of antitumor effects are also explored
   in this study both in vitro and in vivo.

Methods and Materials

Chemical Synthesis of New Juglone Derivatives

   New juglone derivatives (D1-D4) were prepared according to literature
   procedures ([65]Clive et al., 2004; [66]Mitchell et al., 2013; [67]Li
   and Shen, 2020). Ag[2]O (117 mg, 0.5 mmol) and alkyl halide (1.5 mmol)
   was added to the solution of juglone (174 mg, 1 mmol) in CH[2]Cl[2]
   (5 mL). The reaction mixture was stirred at room temperature for 24 h.
   After filtration through celite and removal of the solvent in vacuo,
   the residue was subjected to flash column chromatography on silica gel
   (230–400 mesh) using n-hexane/ethyl acetate as eluent to give the
   product D1-D4.

Identification of Derivatives With Nuclear Magnetic Resonance Spectra and
Oxidative Products With Mass Spectrometry

   All reactions were carried out in oven-dried glassware under an
   atmosphere of dry N[2] with the rigid exclusion of air and moisture
   using standard Schlenk techniques. Dichloromethane was freshly
   distilled from CaH[2] immediately before use. All other chemicals were
   purchased from either J&K Chemical Co. or used as received unless
   otherwise specified. ^1H and ^13C{^1H} NMR (Nuclear magnetic resonance)
   spectra were recorded on a Varian Inova 400 spectrometer at 400 and
   100 MHz, respectively. All signals were reported in ppm unit with
   references to the residual solvent resonances of the deuterated