Novel Nitric Oxide Donor Dinitroazetidine-Coumarin Hybrids as Potent Anti-Intrahepatic Cholangiocarcinoma Agents

Intrahepatic cholangiocarcinoma (iCC) is a serious liver cancer threatening human health. However, there are a few chemotherapeutic drugs for the treatment of iCC in the clinic. It is extremely urgent to develop new drugs for iCC. In this study, twenty dinitroazetidine and coumarin hybrids were synthesized and evaluated anti-iCC bioactivity as a new type of nitric oxide (NO) donors. Among them, compounds 2–5 and 21 showed a higher antiproliferative activity against RBE cell lines (human intrahepatic cholangiocarcinoma cell lines) and low cytotoxicity in nontumor cells (HOSEpiC and T29). The preliminary study of pharmacology mechanism indicated that compounds 2–5 and 21 could release effective concentration of NO in RBE cell lines, which leaded to inhibit the proliferation of RBE cell lines. The research results revealed that compound 3 inhibited the proliferation of RBE cell lines by inducing apoptosis and arresting cell cycle at G2/M phase. Additionally, compound 3 had acceptable metabolic stability. Therefore, compound 3 was merited to further explore for developing a desirable NO donor lead with anti-iCC activity.


Introduction
Cholangiocarcinoma (CCA) is a highly heterogeneous malignant tumor of the biliary tract that originates from the epithelial cells of the biliary duct and can occur anywhere in the biliary tree. Based on the anatomical location, CCA has three subtypes: intrahepatic, perihilar, and extrahepatic cholangiocarcinoma [1]. Among them, intrahepatic cholangiocarcinoma (iCC) is a primary liver cancer with high malignancy degree, difficult treatment, and poor prognosis, the incidence of which is second to hepatocellular carcinoma, accounting for about 10-15% of all primary liver cancer [2]. In recent decades, the incidence of iCC has been on the rise in all regions of the world [3].
Currently, therapies for iCC include surgical resection, ablation treatment, targeted treatment, and immunotherapy. Surgical resection has been a cornerstone in the management of iCC, which is an effective treatment for patients with early intrahepatic cholangiocarcinoma to achieve long-term survival. For patients with primary or recurrent iCC, percutaneous radiofrequency ablation (RFA) and percutaneous microwave ablation can be used [4]. The recurrence rate for iCC after surgical resection is as high as 40-80%, prompting a much greater need to develop and support strategies for adjuvant chemotherapeutic and targeted-agent therapeutics. National Comprehensive Cancer Network (NCCN) and Chinese Society of Clinical Oncology (CSCO) guidelines suggest that gemcitabine combining cisplatin (GP) and gemcitabine combining diageo (GS) are used as first-line

Chemistry
As shown in Scheme 1, the Mannich reaction of nitromethane, paraformaldehyde,

Chemistry
As shown in Scheme 1, the Mannich reaction of nitromethane, paraformaldehyde, and tert-Butylamine obtained intermediate 1a, which was hydrolyzed with 10% hydrochloric acid to generate 1b, further underwent the Mitsunobu reaction to get 1c in the present of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (Ph 3 P). Nitrification reaction of 1c with the NaNO 2 and K 3 Fe(CN) 6 of NaOH aqueous solution produced dinitroazetidine derivative 1d, which reacted with acetic anhydride using BF 3 ·Et 2 O as a catalyst to form amide 1e. Finally, the key intermediate 1 was synthesized via the hydrolyzation of 1e forming intermediate 1f in the present of 10% hydrochloric acid and NaHCO 3 aqueous solution, respectively.

Chemistry
As shown in Scheme 1, the Mannich reaction of nitromethane, parafor and tert-Butylamine obtained intermediate 1a, which was hydrolyzed with chloric acid to generate 1b, further underwent the Mitsunobu reaction to g present of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (Ph cation reaction of 1c with the NaNO2 and K3Fe(CN)6 of NaOH aqueous solutio dinitroazetidine derivative 1d, which reacted with acetic anhydride using B catalyst to form amide 1e. Finally, the key intermediate 1 was synthesized vi lyzation of 1e forming intermediate 1f in the present of 10% hydrochloric a HCO3 aqueous solution, respectively. The target compounds 2-21 were synthesized from coumarin derivatives and dinitroazetidine 1 using amide and carbon-chain as linkers, respectively. As depicted in Scheme 2, under the catalysis of NaH, the nucleophilic substitution of ethyl acetoacetate with various commercially available bromides 2-5a formed intermediates 2-5b. At room temperature, the derivates 2-5c bearing different substituents at 3-position of coumarin were prepared via the cyclization reaction of intermediates 2-5b with resorcinol in 70-75% sulfuric acid. Moreover, 7-hydroxy in the compounds 2-5c and 6a were alkylated with 2-chloroethanol to get 2-5d and 6b. With the catalysis of NaH, the nucleophilic substitution of tert-butyl bromoacetate with various coumarin derivatives 2-5d and 6b formed intermediates 2-5e and 6c, from which removed the tert-butyl to obtain 2-5f and 6d. Then, in the presence of 2-(7-azabenzotriazol-1-yl)-N, N, N , N -tetramethyluronium hexafluorophosphate (HATU) and N, N-diisopropylethylamine (DIPEA), compounds 2-5f and 6d with carboxyl acid side chain were condensed with intermediate 1 to synthesize the amide linker target compounds 2-6. The aliphatic carbon linker type target compounds 7-21 were synthesized via the etherification reaction of 7-hydroxycoumarin derivatives 2-5c and 6a with dibromide to produce the monobromide 7-21a, and the nucleophilic substitution of 7-21a with intermediate 1 in the present of K 2 CO 3 and NaI. hexafluorophosphate (HATU) and N, N-diisopropylethylamine (DIPEA), compounds 5f and 6d with carboxyl acid side chain were condensed with intermediate 1 to synthes the amide linker target compounds 2-6. The aliphatic carbon linker type target co pounds 7-21 were synthesized via the etherification reaction of 7-hydroxycoumarin d rivatives 2-5c and 6a with dibromide to produce the monobromide 7-21a, and the nuc ophilic substitution of 7-21a with intermediate 1 in the present of K2CO3 and NaI.

In Vitro Antiproliferation Activities
As Figure 2 and Table 1 show, twenty target compounds 2-21 were screened for cytotoxicity at the concentration of 10 µM against RBE cell lines and two nontumorigenic cell lines (HOSEpiC and T29) with RRx-001, paclitaxel (PTX) and doxorubicin (DOX) as references using the MTT assays. The results showed that five compounds 2-5 and 21 displayed more than 50% antiproliferation activity in RBE cell lines ( Figure 2a). Subsequently, they were further evaluated to figure out the values of IC 50 . As Table 1 described, four amide bond linker compounds (2-5) bearing 4-trifluoromethyl-benzyl, 4-cyanobenzyl, 4-fluorobenzyl and benzyl substituted at 3-position of coumarin and 4C-chain linker compound 21 without group at 3-position of coumarin exhibited stronger antiproliferation effects in RBE cell lines with the values of IC 50 ranging from 0.71 to 1.11 µM compared to RRx-001 with the 2.00 µM of IC 50 . Moreover, we evaluated the toxicity of the compounds 2-21 in HOSEpiC and T29. As Figure 2b,c shown, most of the target compounds did not exhibit significant cytotoxicity with cell viability higher than 95% in two nontumor cell lines, which indicated these newly synthesized dinitroazetidine-coumarin hybrids had a good safety.
The antiproliferation activities of individual compound to tumor cells were determined by the MTT assay.   The antiproliferation activities of individual compound to tumor ce mined by the MTT assay.

Nitric Oxide Releasing in RBE Cell Lines
As we known, anticancer activity of RRx-001 is relative to NO relea dinitroazetidine moiety [13]. Considering that compounds 2-21 were synth the combination of dinitroazetidine moiety from RRx-001 and coumarin that they showed better inhibitory activity in RBE cell lines compared wi then explored whether these compounds could also release NO comparab RBE cell lines. The nitric oxide release of RRx-001 and active compound RBE cell lines was determined using the fluorescent probe DAF-FM DA. A sented, compared to RRx-001, the exposure of RBE cells to compounds 2

Nitric Oxide Releasing in RBE Cell Lines
As we known, anticancer activity of RRx-001 is relative to NO releasing level of its dinitroazetidine moiety [13]. Considering that compounds 2-21 were synthesized through the combination of dinitroazetidine moiety from RRx-001 and coumarin derivatives and that they showed better inhibitory activity in RBE cell lines compared with RRx-001, we then explored whether these compounds could also release NO comparable to RRx-001 in RBE cell lines. The nitric oxide release of RRx-001 and active compounds 2-5 and 21 in RBE cell lines was determined using the fluorescent probe DAF-FM DA. As Figure 3 presented, compared to RRx-001, the exposure of RBE cells to compounds 2-5 and 21 with the concentration of 2 µM for 2.5 h led to approximately same level of fluorescence intensity. This result implicated that these hybrids can release a relevant concentration of NO in RBE cells, which is closely related to their good inhibitory activities in RBE cell lines. Among them, compound 3 had the highest NO release concentration.
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Compound 3 Blocked Cell Cycle and Induced Apoptosis
The cell cycle of eukaryotic cells is the basic process of cell life action, in which DNA synthesis and cell division are the two main events [14]. Many reports showed that coumarin derivatives inhibited tumor cell proliferation through cell cycle arrest and inducing apoptosis [10,12,14]. In our previous study, furoxan-coumarin hybrids arrested A2780 cell cycle in G2/M phase [12]. Therefore, we performed the cell cycle arrest assay of these dinitroazetidine-coumarin hybrids in RBE cell lines. As Figure 4a shown, DMSO as a control, after treating RBE cell lines using compound 3 with the concentration of 1 μM, the mean percentage of cells in the G2/M phase increased from 18 to 23% and the percentages of cells in S and G0/G1 phase decreased concomitantly. This result implied that compound 3 was able to arrest the cell cycle at G2/M phase. Additionally, Western blotting analysis displayed that compound 3 apparently downregulated the expression of the antiapoptotic proteins PARP and Caspase-3 in a dose-dependent manner and cell cycle protein Cyclin B1, which is involved in the G2/M phase regulation [15] (Figure 4b). These results deduced that the antiproliferative ability of compound 3 might involve in the mechanism of arresting cell cycle in G2/M phase and inducing apoptotic pathway.

Compound 3 Blocked Cell Cycle and Induced Apoptosis
The cell cycle of eukaryotic cells is the basic process of cell life action, in which DNA synthesis and cell division are the two main events [14]. Many reports showed that coumarin derivatives inhibited tumor cell proliferation through cell cycle arrest and inducing apoptosis [10,12,14]. In our previous study, furoxan-coumarin hybrids arrested A2780 cell cycle in G 2 /M phase [12]. Therefore, we performed the cell cycle arrest assay of these dinitroazetidine-coumarin hybrids in RBE cell lines. As Figure 4a shown, DMSO as a control, after treating RBE cell lines using compound 3 with the concentration of 1 µM, the mean percentage of cells in the G 2 /M phase increased from 18 to 23% and the percentages of cells in S and G 0 /G 1 phase decreased concomitantly. This result implied that compound 3 was able to arrest the cell cycle at G 2 /M phase. Additionally, Western blotting analysis displayed that compound 3 apparently downregulated the expression of the antiapoptotic proteins PARP and Caspase-3 in a dose-dependent manner and cell cycle protein Cyclin B 1 , which is involved in the G 2 /M phase regulation [15] (Figure 4b). These results deduced that the antiproliferative ability of compound 3 might involve in the mechanism of arresting cell cycle in G 2 /M phase and inducing apoptotic pathway.

Metabolic Stability in Liver Microsomes
In this work, one of the purposes was to obtain new nitric oxide donor compou with improved metabolic stability compared to furoxan derivatives. Therefore, we ev ated the metabolic stability of compound 3 and furoxan-coumarin hybrid CY-14S-4A8 liver microsomes of human, rat, and mouse. The results showed that the newly syn sized dinitroazetidine-coumarin hybrid 3 has an obviously improved metabolic stab with 3.01, 9.44, and 6.48 of MF% (metabolic bioavailability) in the human, rat, and mo which were better than that furoxan-coumarin derivatives with lower than 0.5 of MF the same liver microsomes ( Table 2).

Metabolic Stability in Liver Microsomes
In this work, one of the purposes was to obtain new nitric oxide donor compounds with improved metabolic stability compared to furoxan derivatives. Therefore, we evaluated the metabolic stability of compound 3 and furoxan-coumarin hybrid CY-14S-4A83 in liver microsomes of human, rat, and mouse. The results showed that the newly synthesized dinitroazetidine-coumarin hybrid 3 has an obviously improved metabolic stability with 3.01, 9.44, and 6.48 of MF% (metabolic bioavailability) in the human, rat, and mouse, which were better than that furoxan-coumarin derivatives with lower than 0.5 of MF% in the same liver microsomes (Table 2).

Metabolic Stability in Liver Microsomes
In this work, one of the purposes was to obtain new nitric oxide donor compounds with improved metabolic stability compared to furoxan derivatives. Therefore, we evaluated the metabolic stability of compound 3 and furoxan-coumarin hybrid CY-14S-4A83 in liver microsomes of human, rat, and mouse. The results showed that the newly synthesized dinitroazetidine-coumarin hybrid 3 has an obviously improved metabolic stability with 3.01, 9.44, and 6.48 of MF% (metabolic bioavailability) in the human, rat, and mouse, which were better than that furoxan-coumarin derivatives with lower than 0.5 of MF% in the same liver microsomes ( Table 2). Compounds CY-14S-4A83 and 3 (0.1 μM) were incubated with liver microsomes of different species (0.33 mg/mL) at 37 °C; then, the samples were analyzed by LC-MS/MS.

Metabolic Stability in Liver Microsomes
In this work, one of the purposes was to obtain new nitric oxide donor compounds with improved metabolic stability compared to furoxan derivatives. Therefore, we evaluated the metabolic stability of compound 3 and furoxan-coumarin hybrid CY-14S-4A83 in liver microsomes of human, rat, and mouse. The results showed that the newly synthesized dinitroazetidine-coumarin hybrid 3 has an obviously improved metabolic stability with 3.01, 9.44, and 6.48 of MF% (metabolic bioavailability) in the human, rat, and mouse, which were better than that furoxan-coumarin derivatives with lower than 0.5 of MF% in the same liver microsomes (Table 2). Compounds CY-14S-4A83 and 3 (0.1 μM) were incubated with liver microsomes of different species (0.33 mg/mL) at 37 °C; then, the samples were analyzed by LC-MS/MS. Compounds CY-14S-4A83 and 3 (0.1 µM) were incubated with liver microsomes of different species (0.33 mg/mL) at 37 • C; then, the samples were analyzed by LC-MS/MS.

General Information
Chemicals were purchased from the following suppliers: Sinopharm, Adamas, Merck, and Sigma Aldrich. Solvents were dried before use, if required. Air-and moisture-sensitive reactions were carried out under nitrogen atmosphere. Room temperature (r.t.) refers to 20-25 • C. The progress of a reaction was monitored by thin layer chromatography (TLC) using precoated TLC sheets purchased from Sinopharm. Detected spots were observed under UV light at λ 254 and 365 nm. Melting points were measured on a SGW X-4 microscopy melting point apparatus without correction. 1 H-NMR and 13 C-NMR spectral data (Supplementary Materials) were recorded with a Bruker DRX 600MHz spectrometer, both at 303 K using TMS as an internal standard. All chemical shifts are reported in ppm (δ) and coupling constants (J) are in hertz (Hz). Mass spectra were recorded on Agilent Technologies 1260 infinity LC/MS instrument. The chromatograms were conducted on silica gel (100-200 and 300-400 mesh) and visualized under UV light at λ 254 and 365 nm.

Synthesis
N-tert-butyl-5-hydroxymethyl-5-nitro-1,3-oxazine (1a): To aqueous solution of paraf ormaldehyde (24 g, 0.8 mol) and 40%NaOH (600 µL) in 120 mL of distilled water, nitromethane (10.5 mL, 0.195 mol) was added dropwise over 1 h at 40 • C. The reaction mixture was heated to 60 • C and stirred for 1 h. Then, the solution of tert-butylamine (20.3 mL, 0.262 mol) in distilled water (36 mL) was added dropwise slowly. The mixture was stirred for another 4 h, cooled to room temperature, and stirred for 1 h again. The precipitate was collected by vacuum filtration at room temperature, washed with distilled water, and vacuum freeze-dried to give 1a (yellow solid, 34 g, 78. N-tert-butyl-3,3-dinitroazetidine (1d): To a solution of 1c (1.35 g, 6 mmol) in distilled water (6 mL), NaOH aqueous solution (3 mL, 717 mg, 17.9 mmol) was added and was stirred for 3 h at room temperature. After cooling to 8 • C, cold NaNO 2 solution (4.5 mL, 1.65 g, 23.9 mmol) and K 3 Fe(CN) 6 (197 mg, 6 mmol) in distilled water were added slowly. Then, Na 2 S 2 O 8 (1.78 g, 7.5 mmol) was added. The yellow solution was stirred for another 1 h at room temperature and extracted with dichloromethane (150 mL). The organic layer was dried with MgSO 4 and the solvent was removed via vacuum evaporation to give 1d General Procedure for the Preparation of 2-5b. In an ice bath, a reaction solution of 60% NaH (680 mg, 17 mmol) in dry THF (40 mL) was stirred for 10 min. Ethyl acetoacetate (2.2 mL, 17 mmol) was added dropwise. After stirring the reaction for 30 min, starting materials 2-5a (15.3 mmol) were added and continued reaction at room temperature in the TLC monitor. After the reaction finished, the solids were filtered out, most of the solvents were removed, and water (100 mL) was added. The mixture was extracted with ethyl acetate, washed with saturated salt, dried over anhydrous sodium sulfate, and evaporated in vacuo to get compounds 2-5b. These products were directly used as the next reactants without any further purification. General Procedure for the Preparation of 2-5c. Resorcinol (1.35 g, 12.250 mmol) was added to a reaction flask containing 2-5b and 70% H 2 SO 4 (30 mL) in the ice bath. After 30 min, the ice bath was removed and the reaction continued at room temperature. After the reaction finished, the solution was slowly added into ice water (300 mL) and stirred for 30 min to precipitate a large number of solids. After extraction, filtration, and drying, compounds 2-5c were obtained. General Procedure for the Preparation of 2-5d and 6b. To a stirred solution of 2-5c and 6a (3.517 mmol) in DMF (15 mL) at room temperature, corresponding halo alcohol (10.553 mmol), NaI (1.05 mmol), and K 2 CO 3 (10.553 mmol) were added. The mixture was refluxed for 2-5 h and then poured into ice water (50 mL). After filtration, the residue was washed with water (3 × 10 mL) and dried to obtain 2-5d and 6b. General Procedure for the Preparation of 2-5e and 6c. To a stirred solution of 2-5d and 6b (4.54 mmol) in anhydrous DMF (15 mL) at 0 • C, NaH (60%, 363 mg, 9.08 mmol) was added. The mixture was stirred for 20 min and t-butylbromoacetate (1.34 mL, 9.08 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 1.5 h, and then poured into a saturated aqueous solution of NH 4 Cl (10 mL) and extracted with ethyl acetate (3 × 30 mL). The combined extracts were washed with brine, dried over Na 2 SO 4 , filtered, and evaporated in vacuo. The residue was purified by column chromatography on silica gel to give compound 2-5e and 6c. General Procedure for the Preparation of 2-5f and 6d. To a stirred solution of 2-5e and 6c (50 mg) in DCM (5 mL) at 0 • C was added TFA (100 µL). The reaction mixture was warmed to room temperature and stirred for 2 h. After the reaction finished, the solvent and unreacted TFA were evaporated in vacuo to get compounds 2-5f and 6d. General Procedure for the Preparation of 2-6. Substituted carboxylic acid 2-5f and 6d (0.2031 mmol) were dissolved in DCM (10 mL) and stirred for 30 min at room temperature. DIPEA (71 µL, 0.4062 mmol) and HATU (154 mg, 0.4062 mmol) were added to the solution and stirred for 40 min at room temperature. After being added intermediate 1 (60 mg, 0.4062 mmol), the mixture was further stirred at room temperature for another 4 h, then water was added. The mixture was extracted with ethyl acetate, washed with saturated salt, and dried over anhydrous Na 2 SO 4 to obtain crude product, which was purified by column chromatography on silica gel to give compounds 2-6.  13 (3) 13 167.60, 166.89, 158.92, 154.32, 144.95, 134.90, 129.57, 128.66, 128.07, 124.49, 114.96, 114.82, 107.43,  107.10, 105.59, 99.94, 69.40, 66.14, 60.42, 59.37, 58.95, 56.91, 33.87, 21.68.  13 7-(2-(2-(3,3-dinitroazetidin-1-yl)-2-oxoethoxy) 170.16, 161.26, 159.97, 154.57, 153.25, 126.35, 113.08, 112.22, 111.05, 107.68, 101.11,  69.54, 68.91, 67.37, 59.56, 56.77, 17.97.

In Vitro Anti-Proliferative Assay
The in vitro antiproliferation of the chemical compounds was measured by the MTT reagent, as described in the literature. Briefly, 4000-6000 cells in 100 µL of medium per well were plated in 96-well plates. After incubated for 24 h at 37 • C, the cells were treated with different concentration of tested compound or DMSO (as negative control) for 48 h. At the same time, blank group without adding cells was set. Then, the medium per well was replaced with 150 µL of fresh medium containing 10% MTT (5 mg/mL in PBS) in each well and incubated at 37 • C for 4 h. Last, the MTT-containing medium was discarded and 150 µL of DMSO per well was added to dissolve the formazan crystals newly formed. Absorbance of each well was determined by a microplate reader (Synergy H4, Bio-Tek) at a 570 nm wavelength. The inhibition rates of proliferation were calculated with the following equation: The concentrations of the compounds that inhibited cell growth by 50% (IC 50 ) were calculated using GraphPad Prism, version 6.0.

Measurement of Intracellular NO
Intracellular NO was measured with 3-amino,4-aminomethyl-2 ,7 -diflfluorescein, diacetate (DAF-FM DA, Beyotime, Shanghai, China). In detail, RBE cells in the logarithmic growth phase were collected and spread on 6-well plate at a density of 150,000 per well overnight. The adherent cells were pretreated with 5 µM DAF-FM DA at 37 • C for 20 min and then incubated with RRx-001, compounds 2-6 and 21 for 2.5 h, followed by flow cytometer analysis (BD Accuri C6, Shanghai, China). Cells were washed three times with cold PBS between each step. The experiment was performed three times.

Cell Cycle Analysis
The experimental cells RBE were cultured and collected when the cells were in good growth state. After digesting with 0.25% trypsin (Beyotime), the cells were collected and centrifuged. The cells were inoculated in petri dishes (Φ = 6 cm) with an inoculation density of 500,000 cells per dish and 3 mL medium per dish. The cells were placed in the incubator, changed to serum-free RPMI 1640 medium after 12 h, left to continue to culture for 12 h, and then compounds were added. Compounds groups with the concentration of 1 µM and control group treated with DMSO were set. The original culture medium was removed, and the compounds were added. After incubation for 16h, the cells were collected and centrifuged at 1000 r for 5 min. The cells were rinsed twice with PBS, and the supernatant was removed by centrifugation. 75% ethanol (1 mL) was added and placed in a refrigerator at −20 • C overnight for cell fixation.
The fixed cells were centrifuged at 2000 r for 5 min, the supernatant was discarded, 1 mL of PBS solution was added, and the supernatant was resuspended and discarded. 0.5 mL of PI staining solution was added to each tube (staining buffer: 20 × PI staining solution: 50 × RNase A = 100:5:2) and stored at room temperature for 30 min in the dark for testing on the flow cytometry.

Conclusions
In this study, twenty novel NO donor compounds 2-21 were synthesized by coupling dinitroazetidine moiety and coumarin scaffold with amide and aliphatic carbon chain linkers, respectively. The antiproliferation activity evaluation of them showed that five compounds 2-5 and 21 had a strong inhibition activity in human intrahepatic cholangiocarcinoma cell lines RBE comparable with RRx-001 and displayed weak cytotoxicity to two normal cell lines HOSEpiC and T29. These five hybrids and RRx-001 all could release effective concentrations of NO in RBE cell lines, which supposed that high anticancer potency of these NO donors was positively associated with their intracellular NO release levels. The preliminary mechanism research revealed that compound 3 could arrest RBE cells cycle at G 2 /M phase and apparently downregulate the expressions of cell-cycle-and apoptosis-related proteins Cyclin B 1 , Caspase-3, and PARP. Moreover, compared to furoxan-coumarin hybrid CY-14S-4A83, dinitroazetidine-coumarin compound 3 showed an obviously improved metabolic stability in the tested liver microsomes. Overall, compound 3 was deserved further to study for developing an ideal lead compound with anticancer activity.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: