Synthesis and Biological Evaluation of Novel Furozan-Based Nitric Oxide-Releasing Derivatives of Oridonin as Potential Anti-Tumor Agents

To search for novel nitric oxide (NO) releasing anti-tumor agents, a series of novel furoxan/oridonin hybrids were designed and synthesized. Firstly, the nitrate/nitrite levels in the cell lysates were tested by a Griess assay and the results showed that these furoxan-based NO-releasing derivatives could produce high levels of NO in vitro. Then the anti-proliferative activity of these hybrids against four human cancer cell lines was also determined, among which, 9h exhibited the most potential anti-tumor activity with IC50 values of 1.82 µM against K562, 1.81 µM against MGC-803 and 0.86 µM against Bel-7402, respectively. Preliminary structure-activity relationship was concluded based on the experimental data obtained. These results suggested that NO-donor/natural product hybrids may provide a promising approach for the discovery of novel anti-tumor agents.


Introduction
Nitric oxide, a special gaseous molecular, is a key mediator involved in many physiological and pathological processes [1,2]. High concentrations of NO and its metabolic derivatives can modify functional proteins, leading to cell cycle arrest and apoptosis, particularly in tumor cells [3][4][5]. Indeed, some synthesized NO-releasing compounds have shown cytotoxic activity against human carcinoma cells in vitro and inhibit the growth and metastasis of cancers in vivo [6][7][8]. So, the NO-based anti-cancer agents have been investigating for cancer therapy at clinic [9,10]. Furoxans represent one class of NO donors that can produce high levels of NO and exhibit strong anti-cancer activity [11,12]. In the previous work by our group, several classes of NO-releasing compounds have been reported, which possess strong anti-proliferative activity against human carcinoma cells in vitro, inhibition of cancer cells growth in vivo and the ability to increase sensitivity of Pgp-mediated multidrug resistance (MDR) in solid tumors, separately [13][14][15][16][17]. These results motivated us to further design some novel NO-donor/natural product hybrids.
Oridonin (1, see Figure 1) is a commercially available natural ent-kaurene diterpenoid that has recently attracted much attention because of its anti-tumor activity with a mechanism of inhibition effect on nuclear factor κB (NF-κB) activation, induction of G 2 /M phase arrest and apoptosis [18]. Oridonin has been safely used for the treatment of hepatoma and promyelocytic leukemia in China for many years. In previous studies, we found that a series of novel 1-O-and 14-O-derivatives of oridonin exhibited stronger cytotoxicity against six cancer cell lines in vitro and some of them had stronger anti-tumor activity than the parent compound 1 and the positive control cyclophosphamide in mice with H22 liver tumor in vivo [19][20][21]. Hence, it may be a desired lead compound using for further design of novel furoxan-based NO-releasing derivatives for the development of anti-tumor agents. Therefore, a series of novel furozan-based nitric oxide-releasing derivatives of oridonin were designed and synthesized.

Synthesis of Furoxan-Based NO Donor
The substituted furoxans were prepared in five steps in the following way (Scheme 1). The starting material benzenethiol (2) was converted to 2-(phenylthio)acetic acid (4) by treatment with chloroacetic acid (3). Then, compound 4 was oxidized by 30% H 2 O 2 aqueous solution to give 2-(phenylsulfonyl) acetic acid (5) and fuming HNO 3 was added to obtain diphenylsulfonylfuroxan (6), which was then converted to various monophenylsulfonylfuroxans 7a-c by treatment with the corresponding diol. Finally, anhydrides were added and furoxan-based NO donors 8a-i were obtained. Scheme 1. Synthesis of compounds 8a-i.

Synthesis of Furoxan/Oridonin Hybrids
The resulting furoxans 8a-i were treated with oridonin to give the target compounds 9a-i, as shown in Scheme 2.

NO-Releasing Test of Hybrids 9a-i and 13a-i in Vitro
The levels of nitrate/nitrite in the lysates of target compounds 9a-i and 13a-i were determined at 100 μM by Griess assay over a duration of 0-60 min. As shown in Figure 2, variable levels of NO were produced by compounds 9a-i and 13a-i. The concentration of NO increased with time, and at the time point of 60 min, all tested compounds produced more than 15 μmol/L of NO. The amount of NO released by compounds 13a-i (13g with the lowest level of 16.88 μmol/L at the 60 min time point) was less than that of 9a-i (9d with the highest level of 45.44 μmol/L at the 60 min time point).

Anti-proliferative Activity in Vitro
The anti-proliferative activity of oridonin and its NO-donor hybrids 9a-i as well as 1-O-derivatives of oridonin (12) and its NO-donor hybrids 13a-i was evaluated against four human cancer cell lines (Bel-7402, K562, MGC-803, CaEs-17) by MTT assay. The results are shown in Table 1 [22]. All the target compounds 9a-i and 13a-i exhibited stronger anti-proliferative activity than their parent compounds 1 (oridonin) and 12 (1-oxo-oridonin), correspondingly. Among them, 13a-i released less NO ( Figure 2) and showed less potent anti-proliferative activity than 9a-i. For example, 13e with IC 50 value of 2.13 µM compared to 9e (1.33 µM) against Bel-7402 cells, 13g with IC 50 value of 2.45 µM compared to 9g (1.33 µM) against MGC803 cells, and so on. These results and our previous studies [13][14][15][16][17] indicated that the releasing of NO contributed to anti-proliferative activity and higher levels of NO releasing could produce stronger activity. Among the tested compounds, the series 9g-i and 13g-i with a o-C 6 H 4 linker (R 1 ) (compounds g-i) showed lower IC 50 values than the corresponding compounds a-f. Compared the IC 50 values of the compounds of series a-c with d-f in different cell lines, there was a decline with the extension of the length of R 1 . In general, when R 1 were aromatic groups (compounds g-i), the activity was stronger than those with alkyl groups. While R 1 were alkyl groups, IC 50 values decreased with lengthening of carbon chain. In almost all cases (except 13h), when the length of R 2 is three carbons, more potential anti-proliferative activity was observed than those of two and four carbons, correspondingly (for instance, 9b > 9a and 9c, 9e > 9d and 9f, 9h > 9g and 9i, 13b > 13a and 13c, 13e > 13d and 13f). This suggested that the length of the linker group R 2 with three carbons would be more suitable. In all the target synthetic hybrids, compound 9h (R 1 =o-C 6 H 4 ; R 2 =CH 2 CH 2 CH 2 ) exhibited the most potential anti-tumor activity against tested cell lines: IC 50 values of 0.86 µM against Bel-7402 (stronger than parent compound oridonin of 7.48 µM and positive control taxol of 1.89 µM), 1.82 µM against K562, 1.81 µM against MGC-803 (stronger than oridonin of 5.69 µM) and 4.56 µM against CaEs-17 (stronger than oridonin of 11.03 µM). Subsequent design and synthesis of novel NO releasing anti-tumor agents based on present SAR and more intensive biological studies were undertaking.

Chemistry
All commercially available solvents and reagents were used without further purification. Melting points were taken on XT-4 micro melting point apparatus and are uncorrected. Infrared (IR) spectra (KBr pellets) were recorded on a Nicolet Impact 410 instrument (Madison, WI, USA). 1 H-NMR spectra were recorded at 300 MHz with a Bruker AV-300 spectrometer (Karlsruhe, Germany) in the indicated solvents (TMS as internal standard): The values of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hz. Mass spectra were obtained using FTMS-2000 (Madison, WI, USA). HR-MS were obtained using an Agilent QTOF 6520 (Palo Alto, CA, USA). Compounds 2-4 were commercially available. Compounds 5, 6, 7a-c, 10, 11 and 12 were synthesized, as previously described [13,19,20].

General Procedure for the Preparation of 8a-i
Compound 7a-c (2 mmol) in pyridine (5 mL) was mixed with the corresponding anhydride (4 mmol) by stirring at room temperature for 6-12 h. The mixture was concentrated in vacuo, dissolved in H 2 O (15 mL) and extracted with CH 2 Cl 2 (15 mL × 3). The organic layers were combined, washed with water and saturated NaCl solution sequentially, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude products 8a-i used for the next step without further purification.

General Procedure for the Preparation of 9a-i and 13a-i
Compounds 8a-i (0.24 mmol) were dissolved in CH 2 Cl 2 (10 mL) and stirred at room temperature. Oridonin or its derivative 12 (0.2 mmol), EDCI (93 mg, 0.6 mmol) and DMAP (catalytic amount) were added. After 8-12 h, the reaction mixture was washed with water and saturated NaCl solution sequentially, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude products were purified by column chromatography (MeOH/CH 2 Cl 2 1:200 v/v) to give the title compounds.

In Vitro MTT Assay
The MTT assay was employed as an in vitro cytotoxicity assay, which was performed in 96-well plates. Test cells at the log phase of their growth cycle (5 × 10 4 cell/mL) were added to each well (100 µL/well), then treated in three replicates at various concentrations of the samples (0.39-100 µg/mL), and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 . After 72 h, 20 µL of MTT solution (5 mg/mL) per well was added to each cultured medium, which was incubated for further 4 h. Then, DMSO was added to each well (150 µL/well). After 10 min at room temperature, the OD of each well was measured on a Microplate Reader (BIO-RAD instruments Inc. No. 550, Hercules, CA, USA) at a wavelength of 490 nm. In these experiments, the negative reference was 0.1% DMSO, and taxol was used as the positive reference with the concentration of 10 µg/mL.

NO-Releasing Test: Nitrate/Nitrite Measurement in Vitro
The levels of nitrate/nitrite formed from individual compounds were determined by the colorimetric assay using the nitrate/nitrite colorimetric assay kit, in triplicate with 100 μM of individual compounds for 0-60 min according to the manufacturer's instructions (Beyotime, Shanghai, China). The lysates were mixed with Griess for 40 min and centrifugalized for 10 min, and then followed by measuring at 540 nm, similar as previously reported [13][14][15][16][17].

Conclusions
In summary, a series of novel furoxan/oridonin hybrids were synthesized and tested for antiproliferative activity against four human cancer cell lines by an in vitro MTT assay. Among them, compound 9h exhibited the most potential anti-tumor activity against all test cell lines. The preliminary SAR of the target compounds was discussed based on the experimental data obtained. Furthermore, more than 15 μmol/L NO were produced by all target compounds at the 60 min time point, and the results showed that higher levels of NO releasing produced stronger anti-proliferative activity, so high levels of NO release by these hybrids could play a role in growth inhibition activity. These results suggested that NO-donor/natural product hybrids may provide a promising approach for the discovery of novel anti-tumor agents. Further studies on the structure modification of these hybrids and the mechanism of the derivatives are currently in progress and will be reported in due course.