Synthesis and Biological Evaluation of 3-Aryl-quinoxaline-2-carbonitrile 1,4-Di-N-oxide Derivatives as Hypoxic Selective Anti-tumor Agents

A series of 3-aryl-2-quinoxaline-carbonitrile 1,4-di-N-oxide derivatives were designed, synthesized and evaluated for hypoxic and normoxic cytotoxic activity against human SMMC-7721, K562, KB, A549 and PC-3 cell lines. Many of these new compounds displayed more potent hypoxic cytotoxic activity compared with TX-402 and TPZ in the tumor cells based evaluation, which confirmed our hypothesis that the replacement of the 3-amine with the substituted aryl ring of TX-402 increases the hypoxic anti-tumor activity. The preliminary SAR revealed that 3-chloro was a favorable substituent in the phenyl ring for hypoxic cytotoxicity and 7-methyl or 7-methoxy substituted derivatives exhibited better hypoxic selectivity against most of the tested cell lines. The most potent compound, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (9h) was selected for further anti-tumor evaluation and mechanistic study. It also exhibited significant cytotoxic activity against BEL-7402, HepG2, HL-60, NCI-H460, HCT-116 and CHP126 cell lines in hypoxia with IC50 values ranging from 0.31 to 3.16 μM, and preliminary mechanism study revealed that 9h induced apoptosis in a caspase-dependent pathway.


Introduction
Hypoxia is an inevitable circumstance in most solid tumors resulting from rapid tumor growth with an inefficient microvascular system. Tumor cells within these regions show resistance to radiotherapy and chemotherapy and present a tremendous challenge to cancer therapy [1,2]. Hypoxia also distinguishes solid tumor cells from physiologically normal cells and is marked as an attractive and exploitable therapeutic target. Five classes of chemical moieties (quinones, nitroimidazoles, aromatic N-oxides, aliphatic N-oxides and transition metal complexes) have been identified as hypoxic cytotoxins in recent years. These compounds were selectively activated by reductive enzymes within hypoxic environment and generated toxic metabolites causing cell death [3].
As classical aromatic N-oxides derivatives, tirapazamine (3-aminobenzotriazine-1,4-dioxide, 1, TPZ, Figure 1) and 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide (TX-402, 2, Figure 1) were extensively studied in the past years. TPZ was bioreductively activated through the one-electron reduction of the benzotriazine-l,4-di-N-oxide moiety by reductase to form hydroxyl and benzotriazinyl radicals that cause DNA damage. It had already been introduced into phase II and III clinical trials in combination with radiotherapy and chemotherapy for advanced head and neck cancers [4,5]. TX-402 also exhibited efficient hypoxic selective anti-tumor activities against various tumor cells with a similar DNA damage mechanism [6]. Although both of them exhibited poor extravascular transport, the unique one-electron reduction activation mechanism and encouraging antitumor profiles have stimulated in recent years intense research efforts in the design and synthesis of a variety of TPZ and TX-402 derivatives [7][8][9][10]. For example, benzotriazine-1,4-dioxide derivatives SN 29751 (3, Figure 1) and SN 30000 (4, Figure 1) were identified as the promising secondary generation TPZ analogues by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization [7,10]. Beatriz reported the synthesis and the biological evaluation of a series of 2-arylcarbonyl-3-trifluoromethylquinoxaline-1,4-di-N-oxide derivatives. The most potent compound 2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxaline 1,4-di-N-oxide (5, Figure 1) not only exhibited good cytotoxic activity against NCI 60 cell lines with mean GI 50 value of 0.07 μM, but also showed positive activity in an in vivo hollow fiber assay [8].
To overcome the poor extravascular transport of TPZ, our lab have synthesized and evaluated 3-aryl amino and 3-(alkoxymethylamino) benzotriazine-1,4-dioxide derivatives 6 and 7 ( Figure 1) through introduction of lipophilic groups into the C-3 amino of TPZ. Most of these compounds were more potent than TPZ in the tumor cell lines assay and some of them exhibited higher hypoxia selectivity. The preliminary SAR study revealed that the introduction of an aromatic group at the C-3 amino was favorable for hypoxic cytotoxic activity and the physico-chemical study showed a positive correlation between hypoxic activity and lipophilicity within a certain range [11][12][13].
Based on these research results, we have envisioned that replacement of the 3-amino moiety of TX-402 with a substituted aryl would be favorable for hypoxic anti-tumor activity. To find new lead compounds with enhanced potency and hypoxic selectivity, we report here the design, synthesis and evaluation of a series of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives 9a-t ( Figure 1) as hypoxic selective anti-tumor agents. Although compounds containing this skeleton have been reported as antimalarial agents [17][18][19], their hypoxic anti-tumor characteristic has never been disclosed. The main objective of present study was to investigate the effect of the replacement of the 3-amine moiety with a substituted 3-aryl moiety and the modification of substituent at the 7-position of TX-402 on anti-tumor activity and hypoxic selectivity. The study also has led to the identification of several new potent hypoxic selective anti-tumor compounds.

Chemistry
The synthetic route of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxides 9a-t is shown in Scheme 1. Refluxing of substituted benzoates 10a-e with acetonitrile in the presence of sodium methoxide provided arylacetonitriles 11a-e, followed by the classical Beirut reaction with 5-substituted benzofuroxans 12a-d in ethanol with catalytic amount of potassium carbonate at room temperature to yield target compounds 9a-t. The structures of all the newly synthesized compounds were confirmed by IR, 1 H-NMR and HRMS.

In Vitro Cytotoxic Activity
All the newly synthesized compounds were assayed for in vitro cytotoxicity against five human cancer cell lines, including SMMC-7721 (hepatoma), K562 (chronic myeloid leukemia), KB (epidermoid carcinoma of the nasopharynx), A549 (nonsmall cell lung carcinoma) and PC-3 (prostate cancer) under normoxic and hypoxic conditions. TX-402 and TPZ were employed as positive controls and the antiproliferative activity results are summarized in Table 1.
As shown in Table 1, many of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives showed higher or similar hypoxic cytotoxic activity and selectivity in comparison with those of TX-402 and TPZ against most of the tested cell lines, in particular for the SMMC-7721, K562 and KB cell lines.
The substituents on the 3-phenyl moiety affect the anti-tumor activity by changing the electronic and lipophilic properties of the entire molecule. Comparing the cytotoxic activity of 9c and 9h with that of 9a and 9f suggested that an electron-withdrawing 3-chloro group in the 3-phenyl moiety increased cytotoxicity against most tested cell lines, particularly for the SMMC-7721, K562 and KB cell lines. The substituents on the 7-position of the quinoxaline ring also have a significant impact on anti-tumor activity and hypoxia selectivity because of the disparity in the electronic properties of the resulting molecules. As shown in Table 1, 7-chloro derivatives 9p and 9q exhibited better hypoxic antiproliferative activity than the 7-unsubstituted derivative 9a and 9b in most tested cell lines. On the other hand, the introduction of electron-donating methyl or methoxy groups into the 7-position of the quinoxaline ring improved the hypoxic selectivity against most cell lines, in particular for the SMMC-7721, K562 and KB cell lines. For example, 7-methyl and 7-methoxysubstituted quinoxaline derivatives 9f and 9n showed very high hypoxic selectivity against SMMC-7721 cell line, with HCR values of 159 and 115, respectively, which were 23-and 16.7-fold more selective than TPZ (HCR = 6.90). The 7-methyl-substituted quinoxaline derivative 9i was the most hypoxic selective cytotoxin against the KB cell line (HCR value of 35.1), which is an 11.8-fold improvement compared with TPZ (HCR value of 2.97).
Among all the five tested cell lines, the SMMC-7721 was the most sensitive cell line to these newly synthesized quinoxaline derivatives, with IC 50 values in the 0.37-5.07 μM range and HCR values between 1.0 and 158.73. The A549 one was the most resistant cell line to the hypoxic cytotoxic effect of these derivatives, with IC 50 values in the range of 5.72-36.15μM and HCR values between 0.12 and 5.71. This result was consistent with that of 2-arylcarbonyl-3-trifluoromethylquinoxaline-1,4-di-Noxide derivatives [8], suggesting that these two series of quinoxaline-1,4-di-N-oxide derivatives may possess similar anti-tumor characteristics.
Compound 9h aroused our great interest because of its high hypoxic antiproliferative activity against all the five cell lines with IC 50 values range from 0.53 to 4.91 μM. It was further evaluated in other six tumor cell lines in hypoxia and in normoxia, including Human hepatoma BEL-7402, HepG2, Human promyelocytic leukemia HL-60, Human lung cancer NCI-H460, Human colon cancer HCT-116 and Human neuroblastoma CHP126. The results in Table 2 showed that 9h also exhibited significant cytotoxicity against all six tested human tumor cell lines with IC 50 values in the range of 0.31-3.16 μM in hypoxia. It also showed moderate to good hypoxia selectivity with HCR values between 1.52 and 17.8. These results suggest that 9h might be a promising candidate for further development as hypoxic selective anti-tumor agent.

Mechanism Studies
To investigate the mechanism of these newly synthesized quinoxaline derivatives, compound 9h was further assayed for its effect on cell cycle progression and apoptosis-associated protein expression.
As shown in Figure 2A, spontaneous apoptosis (control) was seen in 8.42% of SMMC-7721 cells in normoxia and 9.78% in hypoxia. In normoxia, 9h (20 μM) did not induce obvious apoptosis (13.4%) relative to controls. However, in hypoxia, it caused apoptosis in 33.42% of SMMC-7721 cells at 48 h. These data clearly demonstrated that 9h exhibited a hypoxic-selective anti-tumor activity. Given that caspase signaling plays a critical role in stress induced apoptosis, we were thus encouraged to explore its role in 9h-induced SMMC-7721 cells apoptosis. As illustrated in Figure 2A, when SMMC-7721 cells were pretreated with pan-caspase inhibitor z-VAD-fmk (10.0 μM), 9h-induced apoptosis was significantly reduced from 33.42% to 16.83% at 48 h ( Figure 2A). Collectively, these results indicated that 9h serving as a potential hypoxic-selective compound and inducing apoptosis in a caspase-dependent pathway. In order to further validate our results, some proteins related to activation of caspase cascade were also detected. The expression of procaspase-3, and PARP and actin were measured in SMMC-7721 cells treated with 9h (20.0 μM, 48 h). As shown in Figure 2B, 9h decreased the protein levels of procaspase-3, and induce the cleavage of PARP in hypoxia. All these data further demonstrate the apoptosis triggered by 9h in hypoxia is mediated by caspase signaling.

General
Melting points were obtained on a B-540 Büchi melting-point apparatus and are uncorrected. IR spectra were performed on a Brüker VECTOR 22 FTIR spectrophotometer in KBr pellets (400-4000 cm −1 ). 1 H-NMR spectra were recorded on a Brüker AM 500 instrument at 500 MHz (chemical shifts are expressed as δ values relative to TMS as internal standard). Mass spectra (MS), ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. HRMS spectra were measured with an Agilent 6224 TOF LC/MS.

General Procedure for the Synthesis of Benzoylacetonitriles 11a-e
A mixture of ethyl benzoate 10a-e (11.7 mmol), sodium methoxide (1.08 g, 20 mmol) and acetonitrile (15 mL) was refluxed for 3 h. After cooling to room temperature, the formed white precipitate was filtered and dissolved in water (50 mL). Three mol/L HCl (10 mL) was added to the solution and the mixture was extracted with CH 2 Cl 2 (50 mL × 2). The combined organic layer was washed with brine and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure to give the crude product, which was recrystallized from CH 2 C1 2 -petroleum ether to provide pure benzoylacetonitrile.

General Procedure for the Synthesis of 3-Aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide Derivatives 9a-t
The substituted benzofuroxans 12a-d were synthesized according to a literature method and confirmed by melting point comparison [24]. To a solution of benzoylacetonitrile 11a-e (5.0 mmol) and benzofuroxan 12a-d (5.0 mmol) in ethanol (40 mL), a 1% amount of potassium carbonate was added and the mixture was stirred at room temperature for 3 h. The precipitate was filtered and washed with ethanol to give a yellow solid, followed by recrystallization from ethanol to yield pure product.   The SMMC-7721 cells were treated with 9h and/or the general caspase inhibitor, z-VAD-fmk (R&D Systems, Inc., Minneapolis, MN, USA), 9h (10 μM) alone for 24-48 h in 1% O 2 or 20% O 2 respectively; 9h (10 μM) +z-VAD-fmk (10.0 μM) for 48 h in hypoxia. Detection of apoptosis by FACS Calibur flow cytometer (Becton Dickinson, Lincoln Park, NJ, USA) was performed using the Propidium iodide (PI) apoptosis detection kit (BioVision, Mountain View, CA, USA).

Conclusions
In summary, a series of 3-aryl-2-quinoxalinecarbonitrile-1,4-di-N-oxide derivatives 9a-t have been synthesized and evaluated for their hypoxic and normoxic cytotoxic activity. Many of these 3-aryl-2quinoxalinecarbonitrile-1,4-di-N-oxide derivatives showed better hypoxic cytotoxic activity and higher hypoxic selectivity than that of TPZCN and TPZ against most tested cancer cell lines, in particular for the SMMC-7721, K562 and KB cell lines. The preliminary SAR study revealed that the 3-(3-chlorophenyl) moiety was favorable for hypoxic cytotoxicity and the 7-methyl or 7-methoxy moiety improved the hypoxic selectivity. Compound 9h decreased the protein levels of procaspase-3 and induced the cleavage of PARP in hypoxia, which suggested it induces apoptosis in a caspase-dependent pathway.