View PDF VersionPrevious ArticleNext Article
 
DOI: 10.1039/D4NJ02778G (Paper) New J. Chem., 2024, 48, 15590-15598

A self-assembling conjugate of SN38 with aminoguanidine for simultaneously suppressing proliferation and migration of breast cancer cells†

Yi Dai *ac, Yang Zhang b, Yupei Zhang a and Jiamiao Wang a
aCollege of Pharmaceutical Science, Anhui Xinhua University, Hefei 230088, China
bThe First Affiliated Hospital of University of Science and Technology of China, Hefei, 230031, China
cDepartment of Chemistry, University of Science and Technology of China, Hefei 230026, China. E-mail: daiyi@mail.ustc.edu.cn

Received 18th June 2024 , Accepted 9th August 2024

First published on 13th August 2024

Abstract

As an active metabolite of irinotecan, 7-ethyl-10-hydroxy-camptothecin (SN38) exhibits significantly stronger anticancer activity compared to irinotecan. However, its poor water solubility restricts its clinical use. Moreover, SN38 has only a weak effect on the metastasis of cancer cells. In the present work, a conjugate of SN38 with aminoguanidine was synthesized via a Cu-catalytic azide–alkyne click (CuAAC) reaction. As the sixth compound synthesized via the synthetic route, we here denoted the SN38-aminoguanidine conjugate as compound 6. Compound 6 is self-assembled into nanoparticles due to its amphiphilic structure. The resulting nanoparticles with a dynamic diameter of 134.05 ± 3.31 nm and a ξ of 1.27 ± 0.10 mV were irregular lumps of around 120 nm, as demonstrated by transmission electron microscopy (TEM), and significantly decreased the level of endogenous nitric oxide via inhibition of inducible nitric oxide synthase (iNOS). Compared with irinotecan, compound 6 showed much stronger cytotoxicity toward triple-negative breast cancer MDA-MB-231 cells while lower cytotoxicity against human normal breast epithelial MCF10A cells. More importantly, compound 6 significantly inhibited the migration of MDA-MB-231 cells while irinotecan did little at a dose of 0.5 μM. Once MDA-MB-231 cells were pretreated with a nitric oxide donor, the inhibitory effects of compound 6 on proliferation and migration of MDA-MB-231 cells both decreased, indicating that iNOS played an important role in proliferation and migration of breast cancer cells. In a molecular docking model, compound 6 formed two hydrogen bonds and one π–H interaction with topo1 in a different binding mode from camptothecin and SN38, while no interaction with the DNA of the topo1–DNA complex was observed. The lower docking score of compound 6 (−9.45), in contrast to that of SN38 (−8.82) suggested that topo1 was still an important target through which compound 6 exerted its antitumor activity. These findings indicated that the incorporation of aminoguanidine into SN38 was an effective strategy to simultaneously address the issue of poor water solubility and endow SN38 with antimigratory activity.

Introduction

SN38, an active metabolite of irinotecan, is one of the most potent camptothecin analogues. Similar to other camptothecin derivatives, SN38 functions by inhibiting DNA topoisomerase-1 (Topo-1).1–3 Nevertheless, SN38 has poor water solubility, which hinders its clinical application. Although irinotecan, a water-soluble drug, is approved by the US Food and Drug Administration as a prodrug of SN38 to treat a wide range of cancers, only 2–8% of the dosed irinotecan can be converted into SN38 to perform its therapeutic action.4 Therefore, improving the water solubility of SN38 merits further investigation. The strategy for enhancing the water solubility of SN38 mainly involves two methods: (1) introduction of a water-soluble group into SN38, and (2) nano-delivery of SN38. For example, water-soluble glucuronide, glucose and sulfonylamidine were introduced into SN38 to enhance water solubility.5–8 Increasing the water solubility of SN38 via the nano-delivery systems has been widely documented in many literature studies.9,10 These nanoparticles were mainly obtained by encapsulation of SN38 with carrier materials or conjugation of SN38 with carrier materials.11–16 Other nanoparticles were prepared by conjugation of SN38 with other small molecules, which represents a new concept of nano-delivery systems without any carriers. Since no carrier is required, this form of a nano-delivery system depends on the amphiphilicity of conjugates, such as the conjugate of SN38 with L-α-glycerophosphorylcholine.17

Cancer metastasis is the main cause of death in cancer patients, especially in breast cancer patients.18,19 However, SN38 alone has a limited effect on the metastasis of cancer cells. Hence, the combination of SN38 with other compounds to combat metastatic cancer is universally acknowledged as a feasible molecular design. For example, SN38 decorated with iRGD can suppress the migration and invasion of human umbilical vein endothelial cells.20 The conjugation of the E-selectin-binding peptide with SN38 can inhibit the metastasis of B16-F10 cells.21 However, SN38-based compounds capable of suppressing metastasis of cancer cells have rarely been reported in the literature, and those capable of simultaneously suppressing metastasis of cancer cells and enhancing water solubility have been rarely reported. Hence, developing novel SN38 derivatives capable of inhibiting cancer metastasis and enhancing water solubility is urgently needed.

Nitric oxide (NO) is the first gaseous signal molecule found in organisms and plays an important role in human physiology and pathophysiology, such as vasodilation, immune response to infection, cancer biology and pathology.22 Endogenous NO is generated by the oxidation of L-arginine catalysed by nitric oxide synthase (NOS). There are three isoforms of NOS: nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS), especially iNOS, which is highly expressed in cancer cells.23,24 The levels of NO (100–500 nM) mainly produced by iNOS in cancer cells promote the proliferation and metastasis of cancer cells.25–28 Hence, inhibiting iNOS to further decrease the level of NO in cells is a strategy to combat cancer. Aminoguanidine is an iNOS inhibitor with good water solubility.29,30 It is well known that the insertion of a guanidine moiety into hydrophobic molecules can improve their solubility and even facilitate the formation of nanoparticles via self-assembly due to the amphiphilicity of the resulting conjugates. Nano-medicines have a passive targeting ability to tumor tissue through the enhanced permeability and retention (EPR) effect to reduce systemic toxic side effects.31

Based on these findings, we designed and synthesized an amphiphilic SN38 derivative (compound 6) via the CuAAC reaction and selected breast cancer cells for biological evaluation due to the high morbidity and mortality rates of breast cancer in women. Amphiphilicity is an important prerequisite for the successful self-assembly of small molecule compounds. Due to the strong hydrophobicity of SN38, the design of target compound 6 was a dendritic compound, which contained three aminoguanidine moieties at the C20 position and facilitated its high solubility. Compound 6 could self-assemble into nanoparticles. Upon cellular internalization, the nanoconjugate simultaneously inhibited the proliferation and migration of the breast cancer cells (Scheme 1).


image file: d4nj02778g-s1.tif
Scheme 1 Schematic illustration of the anti-tumor and antimigratory actions of compound 6.

Results and discussion

Chemistry

Due to the strong hydrophobicity of SN38, coupling a single aminoguanidine molecule with one SN38 molecule proved insufficient to markedly enhance the water solubility of SN38. Hence, the designed target compound 6 contained three aminoguanidine moieties to facilitate its high solubility. In addition, it has been reported that the hydroxyl group at the C10 position of camptothecin derivatives contributes to the inhibition of topo 1.32 However, to ensure the structural integrity of SN38 and expose the hydroxyl group at the C10 position, there are not enough hydroxyl groups for one molecule of SN38 to link three aminoguanidine molecules via ester bonds. Hence, SN38-based dendritic compounds were designed to meet the requirement of linking the four molecules (SN38[thin space (1/6-em)]:[thin space (1/6-em)]aminoguanidine = 1[thin space (1/6-em)]:[thin space (1/6-em)]3). Compound 6 was synthesized, as shown in Scheme 2. First, the key intermediate, compound 1, was prepared (Scheme S2, ESI†). Subsequently, the hydroxyl group of SN38 at the C10 position was protected with di-tert-butyl decarbonate (BOC anhydride) to obtain compound 2. Esterification of compound 2 at the C20 position with compound 1 was performed to prepare compound 3. Then, compound 4 was synthesized by a Cu-catalytic azide–alkyne click (CuAAC) reaction between compounds 3 and 4-(prop-2-yn-1-yloxy)benzaldehyde. After the removal of the BoC of compound 4, compound 5 was obtained and reacted with aminoguanidine to prepare the final product, compound 6. These compounds were verified by NMR and ESI-MS (Fig. S1–S10, ESI†).
image file: d4nj02778g-s2.tif
Scheme 2 Synthetic route for compound 6.

Preparation and characterization of nanoparticles

The insertion of a guanidino moiety into some compounds can enhance the water solubility of the obtained derivatives.33,34 Here, the insertion of aminoguanidine into SN38 formed an amphiphilic molecule due to the hydrophilicity of the guanidino groups and the hydrophobicity of SN38. Compound 6 self-assembled into irregular and lumpy nanoparticles with a size of around 120 nm, as analysed by transmission electron microscopy (TEM) (Fig. 1A and B), while the average hydrodynamic diameters of the nanoparticles were 134.05 ± 3.31 nm. The distribution of the nanoparticles was narrow, with a polydispersity index (PDI) of 0.16–0.24. The surface of the nanoparticles was positively charged with a potential of 1.27 ± 0.10 mV, suggesting that compound 6 may be taken in effectively by cancer cells.35,36 Upon transferring the nanoparticles from the aqueous solution to a complete medium at room temperature, the size of the nanoparticles decreased. This phenomenon may be caused by an increase of repulsion among the particles in the buffer solution. Interestingly, after the nanoparticles in the complete medium were heated to 37 °C, their sizes were restored to 120 nm, which might be attributed to decreased repulsion among nanoparticles caused by the temperature. The stability of the nanoparticles was not affected (Fig. 1C). Time-dependent DLS measurements showed that the nanoparticle sizes did not significantly change in the complete medium over 51 h, indicating the high stability of the nanoparticles, which provided a guarantee for subsequent biological evaluation.
image file: d4nj02778g-f1.tif
Fig. 1 Characterization and stability of nanoparticles formed from compound 6. (A) DLS data of the nanoparticles. (B) TEM images of the nanoparticles. (C) Change in hydrodynamic diameters of nanoparticles in the complete medium over time.

Biological evaluation

The effect of compound 6 on iNOS. Reduced levels of endogenous nitric oxide can inhibit the growth of cancer.37,38 The aminoguanidine moieties in target compound 6 were expected to inhibit iNOS. The inhibition of iNOS was reflected by the level of endogenous NO. We investigated the level of endogenous NO in MDA-MB-231 cells treated with compound 6via fluorescence imaging.39–41 After administration of MDA-MB-231 cells with different compounds over 24 h, these cells were stained with the NO probe DAF-FM DA. Fluorescence microscopy results showed that both compound 6 and aminoguanidine significantly decreased the levels of endogenous NO, while irinotecan had little effect on the level of endogenous NO, indicating that compound 6 could inhibit iNOS to decrease the level of endogenous NO, such as aminoguanidine (Fig. 2).
image file: d4nj02778g-f2.tif
Fig. 2 Levels of endogenous nitric oxide in MDA-MB-231 cells (A: control group, B: irinotecan group, C: aminoguanidine group and D: compound 6 group). MDA-MB-231 cells were co-incubated with 1 μM of the different compounds for 24 h, stained with the DAF-FM-DA probe and observed using a fluorescence microscope. Compound 6 was administered in the form of nanoparticles.
In vitro cytotoxicity. After validating its inhibitory effect on iNOS, the in vitro cytotoxicity of compound 6 against three human breast cancer cell lines (triple-negative breast cancer MDA-MB-231 cells, breast cancer SKBR3 cells and breast cancer MCF7 cells) and one normal cell line (human breast epithelial MCF10A cells) was evaluated using the MTT assay. Many studies have reported that the cytotoxicity of camptothecin derivatives is usually weaker than that of SN38.42–46 Here, SN38 and irinotecan were both selected as positive controls. The MTT results were as expected, which showed that among the tested compounds, SN38 still had the strongest cytotoxicity toward the three human breast cancer cell lines and normal MCF10A cells (Fig. 3 and Table 1). Encouragingly, although its cytotoxicity toward MCF7 and SKBR3 cells was comparable to that of irinotecan, compound 6 demonstrated more potent cytotoxicity against triple-negative breast cancer MDA-MB-231 cells than irinotecan. The IC50 value of compound 6 against MDA-MB-231 cells was 0.64 ± 0.33 μM, while that of irinotecan was 1.24 ± 0.63 μM. Notably, compound 6 showed lower cytotoxicity against MCF10A cells than irinotecan and SN38.
image file: d4nj02778g-f3.tif
Fig. 3 In vitro cytotoxicity of compound 6. Cancer cell viability under different concentrations of compounds was assayed using the MTT method (A: MDA-MB-231 cells, B: SKBR3 cells, C: MCF-7 cells, and D: MCF10A cells). Compound 6 was administered in the form of nanoparticles. Compound 6, irinotecan, and aminoguanidine were all dissolved in the medium; SN38 was first dissolved in DMSO, and then the DMSO parent solution was diluted with the medium. The final concentration of DMSO used for dissolving SN38 was less than 0.1%. After 48 h of treatment of cells with different compounds, cell viability was assayed.
Table 1 In vitro cytotoxicity of compound 6
Entry IC50 (μM)
MDA-MB-231 cells SKBR3 cells MCF-7 cells MCF10A cells Selective indexa
a The IC50 ratio of compounds against MCF10A/MCF-7.
Compound 6 0.64 ± 0.33 0.79 ± 0.29 0.44 ± 0.33 1.93 ± 0.80 4.39
Irinotecan 1.24 ± 0.63 0.63 ± 0.46 0.33 ± 0.16 0.98 ± 0.47 2.97
SN38 0.10 ± 0.10 0.35 ± 0.21 0.23 ± 0.10 0.17 ± 0.09 0.74
Aminoguanidine >750 >750 >750 11.19 ± 8.17

The selective indices were 4.39, 2.97 and 0.74 for compound 6, irinotecan and SN38, respectively (Fig. 3 and Table 1). Aminoguanidine showed only marginal cytotoxicity toward breast cancer cell lines, with IC50 values above 750 μM. Regrettably, aminoguanidine showed significant cytotoxicity against the normal MCF10A cells (Fig. 3 and Table 1). These results indicated that compound 6 had great potential for inhibiting the growth of breast cancer cells.

Antimigratory effect of compound 6. Since reduced levels of endogenous NO can inhibit the migration of breast cancer cells, compound 6 was expected to utilize aminoguanidine moieties to decrease the level of endogenous NO by the inhibition of iNOS. The antimigratory effect of compound 6 was evaluated with a scratch assay. The experimental results showed that the wound healing rates in the control group, aminoguanidine group, compound 6 group and irinotecan group were 100%, 92.6%, 62.7% and 91.8%, respectively (Fig. 4). Though SN38 also had low wound healing rate, it induced an amount of apoptosis at a dose of 0.50 μM due to ultra-strong cytotoxicity (Fig. S11, ESI†). The results of the scratch assay showed that compound 6 significantly inhibited cell migration. The incorporation of aminoguanidine endowed SN38 with antimigratory activity.
image file: d4nj02778g-f4.tif
Fig. 4 The effect of compounds on the migration of cancer cells measured with the scratch test. MDA-MB-231 cells were co-incubated for 24 h with drugs at a dose of 0.5 μM. Lateral migratory cells were analyzed by measuring the wound closure rate. Compound 6 was administered in the form of nanoparticles.
The effect of supplemental NO on antitumor activity of compound 6. To confirm the role of reduced nitric oxide in inhibiting the proliferation and migration of cancer cells, we investigated the effect of supplemental NO donors on the growth of cancer cells. After pre-treatment for 1 h with different concentrations of NO donors, the cells were co-incubated with 1 μM compound 6 for 48 h. The MTT results showed that the survival rate of MDA-MB-231 cells without pre-treatment with NO donors was around 21.91%, while that of MDA-MB-231 cells pre-treated with NO donors of concentration doubling decreased after an initial increase (Fig. 5A). These findings are consistent with those reported in the literature.25,26,47 Low concentrations of nitric oxide (100–500 nM) promote the growth of cancer cells, while high concentrations of nitric oxide (>500 nM) inhibit the growth of cancer cells.48 The survival rate of cells with pre-treatment of 0.5 μM NO donors reached 40%, indicating that NO supplements could resist the inhibition of iNOS to debilitate the antiproliferative effect of compound 6. Then, 0.5 μM and 1.0 μM of NO donors were selected to investigate the effect of supplemental NO on the antimigration of compound 6. A similar pattern was observed in a study on the migration of cells pre-treated with NO donors. Under the same dosage of compound 6, MDA-MB-231 cells pre-treated with NO donors had a higher wound healing rate than those without pre-treatment with NO donors (Fig. S12, ESI†). The statistical results shown in Fig. S12 (ESI†) indicate a one-fold increase in the wound healing rate in the 1.0 μM NO donors group (Fig. 5B). These findings indicated that aminoguanidine in compound 6 plays a key role in combating cancer cells through the inhibition of iNOS.
image file: d4nj02778g-f5.tif
Fig. 5 Effect of pretreatment with the NO donor on proliferation (A) and migration (B) of cancer cells treated with compound 6 using the MTT assay and scratch test. MDA-MB-231 cells were pretreated for 1 h with an NO donor and then co-incubated with compound 6 at a dose of 1 μM. The cell viability and wound healing rates were assayed. Compound 6 was administered in the form of nanoparticles. *p < 0.05, **p < 0.01, and ***p < 0.001.

Molecular docking study

Does the modification at the C20 position of SN38 with a new moiety disturb its binding to topo1? Lu et al. introduced a Hsp90 inhibitor (NVP-AUY922) at the C20 position of SN38. Western blot results showed that the synthesized conjugates, like SN38, could efficiently inhibit the activity of topo1.42 Another conjugate synthesized by the introduction of artemisinin at the C20 position of SN38 also showed a high inhibitory effect on topo1 in both in vivo and in vitro experiments.49 Here, we investigated the interaction between compound 6 and topo1 using molecular docking experiments.

Before investigating the putative binding mode of compound 6, camptothecin was re-docked to the binding site of the topo1–DNA complex (PDB:1T8I) to validate the reliability of the docking method. As shown in Fig. 6A, the binding model of camptothecin with the topo1–DNA complex was consistent with previous findings.6 Camptothecin formed three hydrogen bonds with Asp533, Lys532 and Arg364 of topo1, and strong π–π stacking interactions with DNA in the topo1–DNA complex. Then, SN38 was docked with the topo1–DNA complex, and a docking score of −8.82 was obtained. As depicted in Fig. 6B, apart from strong π–π stacking interactions with the DNA of the topo1–DNA complex, only one hydrogen bond was formed between the C10–OH of SN38 and Asn722 of topo1.


image file: d4nj02778g-f6.tif
Fig. 6 Putative bonding modes of camptothecin (A), SN8 (B) and compound 6 (C) with topo 1 (PDB: 1T8I).

Interestingly, the docking results for compound 6 revealed another binding model (Fig. 6C). The binding site of compound 6 was far from the binding pocket occupied by camptothecin, which was different from that of SN38. Compound 6 formed two hydrogen bonds with Gln318 and Asn237, and a π–H interaction with Lys321 of topo1. No interaction was observed between compound 6 and the DNA of the topo1–DNA complex. However, the docking score of −9.45 for compound 6 was lower than that of SN38, suggesting that topo1 is still an important target through which compound 6 exerts antitumor activity.

Conclusions

In summary, a novel hybrid of SN38 and aminoguanidine(compound 6) was synthesized via a click reaction. Compound 6 is an amphiphilic and dendritic compound with one SN38 moiety at one end and three aminoguanidine moieties at the other end. Compound 6 self-assembled into around 120 nm nanoparticles in water. Upon cellular internalization, compound 6 utilized the SN38 and aminoguanidine moieties to simultaneously exert anti-proliferative and antimigratory activities. In vitro fluorescence imaging of NO showed that compound 6 could significantly decrease the level of endogenous NO. The in vitro cytotoxicity assay demonstrated that compound 6 had more potent cytotoxicity toward MDA-MB-231 cells and lower cytotoxicity against normal MCF10A cells than irinotecan did. The wound-healing assay confirmed that compound 6 could inhibit the migration of breast cancer cells in vitro. Further mechanistic investigation revealed that supplemental NO could attenuate the anti-proliferative and antimigratory effects of compound 6, indicating that the inhibition of iNOS plays a key role in combating the proliferation and migration of breast cancer cells. Molecular docking showed that compound 6 could bind to the topo1–DNA complex with a different binding model compared to that of camptothecin and SN38. In a word, compound 6 not only enhanced the water solubility of SN38 but also endowed SN38 with antimigratory activity through the decoration of SN38 with aminoguanidine. This work also provides a reference for the structural modification of other hydrophobic compounds.

Experimental section

General information

SN38 and aminoguanidine were purchased from Energy Chemical Co., Ltd. Other chemical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. DMEM culture medium and FBS were purchased from Gibco Co., Ltd. MTT and DAF-FM-DA probes were purchased from Biyuntian Co., Ltd. Nitric oxide donors (furoxans) were synthesized, as described in the literature (Scheme S1, ESI†).50 NMR spectra were recorded on a Bruker DRX-400 NMR spectrometer. Fluorescence imaging was performed using an Olympus IX71 fluorescence microscope. The sizes and potential of nanoparticles were analysed on Particle Solutions v.3.0.

Synthetic procedures

Synthesis of compound 2. Compound 2 was synthesized in accordance with the literature.51 SN38 (1.00 g, 2.50 mmol), (Boc)2O (1.00 g, 4.50 mmol) and pyridine (5 mL) were dissolved in dichloromethane (50 mL). The mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, the mixture was washed three times with 1% HCl and water. The obtained dichloromethane fraction was dried with anhydrous sodium sulfate, followed by the removal of dichloromethane under vacuum to obtain compound 2, a faint yellow solid, with a yield of 86%. 1H-NMR (300 MHz, chloroform-d) δ 8.26 (d, J = 9.3 Hz, 1H), 7.91 (d, J = 2.7 Hz, 1H), 7.78–7.56 (m, 2H), 5.77 (d, J = 16.2 Hz, 1H), 5.44–5.16 (m, 3H), 3.84 (s, 1H), 3.18 (q, J = 7.8 Hz, 2H), 2.07–1.80 (m, 2H), 1.63 (s, 9H), 1.42 (t, J = 7.5 Hz, 3H), 1.05 (t, J = 7.5 Hz, 3H).
Synthesis of compound 3. To a solution of compound 1 (355 mg, 1.2 mmol) in dichloromethane (60 mL), compound 2 (518 mg, 1.0 mmol), EDCI (229 mg, 1.2 mmol) and DMAP (15 mg, 0.12 mmol) were added. Then, the resulting mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, the reaction solution was transferred to a separating funnel and washed three times with 1% HCl and brine. After the resulting organic fraction was dried with anhydrous sodium sulfate, dichloromethane was removed under vacuum to obtain compound 3, a faint yellow solid with a yield of 73%, and NMR verification was delayed to the next step.
Synthesis of compound 4. To a solution of 4-(prop-2-yn-1-yloxy) benzaldehyde (480 mg, 3 mmol) in t-BuOH/H2O mixture (2[thin space (1/6-em)]:[thin space (1/6-em)]1, 20 mL), sodium ascorbate (594 mg, 0.3 mmol) and CuSO4 (750 mg, 0.3 mmol) were added and stirred at room temperature under an argon atmosphere. Once the solution turned yellow, compound 3 (385 mg, 0.5 mmol) was added, and the reaction temperature was increased to 40 °C. TLC was used to monitor the reaction process. After the reaction was completed, the reaction solution was concentrated under vacuum and the obtained residue was lyophilized, followed by purification using silica gel column chromatography to obtain compound 4, a faint yellow solid with a yield of 49%. 1H-NMR (400 MHz, DMSO-d6) δ 9.85 (s, 3H), 8.16 (d, J = 9.0 Hz, 3H), 8.06 (d, J = 2.5 Hz, 1H), 7.90 (s, 1H), 7.85 (d, J = 8.3 Hz, 6H), 7.67 (dd, J = 9.1, 2.6 Hz, 1H), 7.27–7.10 (m, 7H), 5.60–5.00 (m, 10H), 4.82–4.39 (m,6H), 3.14 (d, J = 7.8 Hz, 2H), 2.99–2.83 (m, 1H), 2.71 (d, J = 17.5 Hz, 1H), 2.34 (d, J = 5.9 Hz, 2H), 2.13 (t, J = 8.2 Hz, 2H), 1.53 (s, 9H), 1.31–1.17 (m, 3H), 0.94 (t, J = 7.2 Hz, 3H).
Synthesis of compound 5. Compound 4 (700 mg, 0.52 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (10 mL) and stirred for 2 h at 25 °C. Then, ethyl acetate (60 mL) was added to the above reaction solution. The resulting solution was washed three times with a saturated solution of NaHCO3 and NaCl and dried with anhydrous sodium sulfate. After the removal of ethyl acetate and dichloromethane, the obtained product was further purified using silica gel column chromatography. The final product was a faint yellow solid with a yield of 53%.1H-NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.86 (s, 3H), 8.13 (s, 3H), 7.98 (d, J = 9.8 Hz, 1H), 7.92–7.80 (m, 6H), 7.36 (d, J = 7.4 Hz, 2H), 7.24–7.16 (m, 6H), 7.10 (s, 1H), 5.70–4.89 (m, 10H), 4.58 (dd, J = 54.4, 14.6 Hz, 6H), 3.02 (d, J = 7.7 Hz, 2H), 2.94–2.83 (m, 1H), 2.80–2.63 (m, 1H), 2.34 (dd, J = 8.7, 4.4 Hz, 2H), 2.21–2.07 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H), 0.95 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 191.77, 173.27, 172.64, 167.94, 163.34, 157.21, 156.98, 148.90, 147.15, 146.43, 143.97, 143.21, 142.14, 132.19, 131.84, 130.31, 128.57, 128.07, 127.39, 122.95, 117.84, 115.60, 105.23, 95.01, 76.86, 66.46, 61.65, 60.23, 59.45, 49.71, 49.24, 30.61, 30.28, 28.63, 22.69, 21.24, 14.55, 13.78, 8.15. ESI–MS, m/z: calcd. 1149.4 ([M−H]−); found 1149.5 ([M−H]−); calcd. 1195.4 ([M + HCOO]−); found 1195.6 ([M + HCOO]−); calcd. 1212.4 ([M + HCOO + H2O]−); found 1212.5 ([M + HCOO + H2O]−).
Synthesis of compound 6. Compound 5 (247 mg, 0.2 mmol) and aminoguanidine hydrochloride (79.2 mg, 0.72 mmol) were dissolved in ethanol (20 mL) and refluxed at 65 °C. The reaction process was monitored by TLC. After the reaction was completed, ethanol was removed from the reaction solution under vacuum. The residual solution was lyophilized, and a faint yellow solid was obtained. The crude product was washed with Na2CO3 solution and water and lyophilized again to obtain compound 6, a faint yellow solid with a yield of 47%. 1H-NMR (400 MHz, DMSO-d6) δ 8.06 (s, 3H), 7.95 (d, J = 2.8 Hz, 4H), 7.61 (d, J = 8.3 Hz, 6H), 7.36 (d, J = 7.6 Hz, 2H), 7.10 (s, 1H), 6.98 (d, J = 8.5 Hz, 6H), 6.05 (s, 6H), 5.71 (s, 6H), 5.54–5.35 (m, 2H), 5.27–4.95 (m, 8H), 4.56 (dd, J = 58.5, 14.6 Hz, 6H), 3.10–2.83 (m, 3H), 2.75–2.63 (m, 1H), 2.35 (d, J = 15.5 Hz, 2H), 2.23–2.01 (m, 2H), 1.35–1.16 (m,3H), 0.95 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6) δ 173.30, 172.59, 167.96, 160.18, 158.53, 158.46, 157.49, 157.04, 148.80, 147.19, 146.42, 143.91, 143.78, 143.01, 142.72, 131.79, 130.18, 128.61, 128.13, 128.06, 127.06, 123.10, 117.80, 115.22, 115.09, 105.24, 95.04, 76.82, 66.51, 61.31, 59.41, 49.74, 49.24, 39.62, 39.43, 30.64, 30.33, 28.70, 24.86, 22.69, 13.78, 8.15. ESI–MS, m/z: calcd. 1319.4 ([M+H]+); found 1320.0 ([M+H]+); ESI–HRMS, m/z: calcd. 1319.3748 ([M+H]+); found 1319.6442 ([M+H]+).

Nano-assembly study

Preparation and characterization of nanoparticles

The nanoparticles were prepared by nanoprecipitation. In brief, compound 6 (15 mg) was dissolved in 1 mL of DMSO. Then, the obtained solution was added dropwise to 20 mL of water under vigorous stirring. After the nanoparticles were formed, the resulting nanoparticle solution was dialyzed using a dialysis bag with a molecular weight cutoff of 2 kDa, followed by centrifugation at 1000 rpm for 5 min. The NP size and surface zeta potential were determined using a dynamic light scattering meter. The configuration of the samples was observed by TEM.

Stability tests of nanoparticles

The nanoparticles were dispersed in a complete medium and stored at room temperature in the dark, followed by the determination of nanoparticle size at different time points to investigate the stability of nano-assembly.

Biological evaluation

Cell lines and cell culture

Cell culture: Breast cancer cells, MDA-MB-231 cells, SKBR3 cells and MCF-7cells were purchased from the Cell Bank of Shanghai, Chinese Academy of Sciences (Shanghai, China). The cancer cells were cultured in DMEM containing 10% FBS at 37 °C in a humidified 5% CO2 incubator.

Fluorescence imaging of NO

MDA-MB-231 cells were dispersed in a complete medium and seeded in 6-well plates at a density of 50[thin space (1/6-em)]000 cells per well. After overnight incubation at 37 °C in 5% CO2, the cells were treated with different compounds at a dose of 1 μM and co-incubated for 24 h. Then, the medium containing compounds was removed and cells were rinsed with PBS three times, followed by staining with the DAF-FM-DA probe for 20 min at 37 °C. Finally, the cells were rinsed with PBS to remove the residual DAF-FM-DA probe and observed under a fluorescence microscope to detect the level of endogenous nitric oxide.

Cell viability assays

Cancer cells were seeded in 96-well plates at a density of 3000 cells per well and incubated overnight in a humidified 5% CO2 incubator at 37 °C. The culture medium was replaced with 100 μL of fresh medium containing different concentrations of the compounds. Then, the cells were co-incubated with the compounds for another 48 h. After that, the medium was sucked out, and 100 μL of fresh culture medium containing 1 mg mL−1 MTT was added, followed by incubation for another 4 h to ensure that viable cells could reduce the yellow tetrazolium salt (MTT) to dark blue formazan crystals. Finally, the medium containing residual MTT was replaced with 150 μL DMSO, followed by shaking for 15 min at 37 °C. Absorbance was measured at 490 nm using a Bio-Rad 680 microplate reader. Cell viability was calculated using the following formula, and IC50 values were obtained using GraphPad Prism software (version 8.0) based on data from three parallel experiments.
image file: d4nj02778g-t1.tif

Wound healing assay

MDA-MB-231 cells were seeded in 6-well plates at a density of 500[thin space (1/6-em)]000. The cells were incubated at 37 °C in 5% CO2 until they reached 95–100% confluency. Then, a linear wound was created using a 1-mL pipette plastic tip and washed with PBS to remove the cell debris. The cells were treated with different compounds at a dose of 1 μM and incubated for 24 h. Wound healing was analysed by photoimaging at 0 h and 24 h. The wound healing area (%) in individual groups was calculated according to the following formula:
image file: d4nj02778g-t2.tif

The effect of supplemental NO on antitumor activity of compound 6

The effect of NO donors on cytotoxicity caused by compound 6 was evaluated by the MTT assay. Briefly, MDA-MB-231 cells were seeded in 96-well plates and incubated overnight, followed by pre-treatment with 4, 2, 1, 0.5 or 0.25 μM NO donors for 1 h. Then, the medium was replaced with a fresh medium containing 1 μM compound 6 or irinotecan. After incubation for 48 h, the medium containing the drugs was replaced with medium containing MTT, followed by incubation of 4 h. Finally, the medium was sucked out, and 150 mL of DMSO was added to each well. The mixture was then shaken for 15 min at 37 °C. Absorbance was measured at 490 nm using a Bio-Rad 680 microplate reader. The cell viability was then calculated.

The effect of NO donors on antimigration caused by compound 6 was evaluated by the scratch test, as described in “The wound healing assay”. After a linear wound was created, the cells were pre-treated with 1 and 0.5 μM furoxan for 1 h. Then, the medium was replaced with fresh medium containing 1 μM compound 6. Wound healing was analysed by photoimaging at 0 h and 24 h. The wound healing area (%) in individual groups was calculated.

Molecular docking study

The interaction mode and binding energy of compound 6 were investigated using molecular docking simulations performed in a molecular operating environment (MOE, Version 2019). The crystal structure of topo 1 complexed with camptothecin (PDB code 1T8I) was obtained from the RCSB Protein Data Bank. The protein was optimized using the QuickPrep function of the MOE with default parameters. The 2D structures of the compounds were prepared using ChemDraw and imported into the MOE to minimize their energies. Then, docking between compounds and topo 1 was performed using the “General dock” mode of the MOE with default parameters. The docking results were analysed and presented in the form of 2D and 3D interaction diagrams using MOE. All docked conformations were ranked based on their docking scores.

Statistical analysis

Data were analysed statistically using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± SD of three individual experiments. Student's t-test and one-way ANOVA were used to analyse the differences between the groups; p < 0.05 was considered statistically significant.

Data availability

All relevant data are included in the manuscript and its additional files.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Innovation and Entrepreneurship Training Program for College Students in the Anhui Province (No. S202212216011, S202212216012, S202212216013 and S202212216014), Scientific Research Team of Anui Xinhua University (No. kytd202211), Pharmaceutical Institute of Anui Xinhua University (No. yjs202107).

Notes and references

  1. E. Martino, S. Della Volpe, E. Terribile, E. Benetti, M. Sakaj, A. Centamore, A. Sala and S. Collina, Bioorg. Med. Chem. Lett., 2017, 27, 701–707 CrossRef CAS PubMed .
  2. N. Khaiwa, N. R. Maarouf, M. H. Darwish, D. W. M. Alhamad, A. Sebastian, M. Hamad, H. A. Omar, G. Orive and T. H. Al-Tel, Eur. J. Med. Chem., 2021, 223, 113639 CrossRef CAS PubMed .
  3. W. Bocian, B. Naumczuk, M. Urbanowicz, J. Sitkowski, A. Bierczynska-Krzysik, E. Bednarek, K. Wiktorska, M. Milczarek and L. Kozerski, Int. J. Mol. Sci., 2021, 22, 7471 CrossRef CAS PubMed .
  4. F. Innocenti, D. L. Kroetz, E. Schuetz, M. E. Dolan, J. Ramirez, M. Relling, P. X. Chen, S. Das, G. L. Rosner and M. J. Ratain, J. Clin. Oncol., 2009, 27, 2604–2614 CrossRef CAS PubMed .
  5. R. Walther, M. T. Jarlstad Olesen and A. N. Zelikin, Org. Biomol. Chem., 2019, 17, 6970–6974 RSC .
  6. Z.-L. Song, M.-J. Wang, L. Li, D. Wu, Y.-H. Wang, L.-T. Yan, S. L. Morris-Natschke, Y.-Q. Liu, Y.-L. Zhao, C.-Y. Wang, H. Liu, M. Goto, H. Liu, G.-X. Zhu and K.-H. Lee, Eur. J. Med. Chem., 2016, 115, 109–120 CrossRef CAS PubMed .
  7. R. M. Zhang, Y. Luo, C. H. Du, L. Wu, Y. K. Wang, Y. D. Chen, S. Q. Li, X. Jiang and Y. M. Xie, Bioorg. Med. Chem. Lett., 2023, 81, 129128 CrossRef CAS PubMed .
  8. C. Yang, A. J. Xia, C. H. Du, M. X. Hu, Y. L. Gong, R. Tian, X. Jiang and Y. M. Xie, Front. Pharmacol., 2022, 13, 1014854 CrossRef CAS PubMed .
  9. J. Si, X. Zhao, S. Gao, D. Huang and M. Sui, Int. J. Pharm., 2019, 568, 118499 CrossRef CAS PubMed .
  10. V. Bala, S. Rao, B. J. Boyd and C. A. Prestidge, J. Controlled Release, 2013, 172, 48–61 CrossRef CAS PubMed .
  11. H. Liu, H. Lu, L. F. Liao, X. M. Zhang, T. Gong and Z. R. Zhang, Drug Delivery, 2015, 22, 701–709 CrossRef CAS PubMed .
  12. S. Dimchevska, N. Geskovski, G. P. Sevski, M. Chacorovska, R. Popeski-Dimovski, S. Ugarkovic and K. Goracinova, Drug Dev. Ind. Pharm., 2017, 43, 502–510 CrossRef CAS PubMed .
  13. N. Karki, H. Tiwari, M. Pal, A. Chaurasia, R. Bal, P. Joshi and N. G. Sahoo, Colloids Surf., B, 2018, 169, 265–272 CrossRef CAS PubMed .
  14. C. Z. Gao, L. F. Zhang, M. H. Xu, Y. Luo, B. Wang, M. Y. Kuang, X. Y. Liu, M. Sun, Y. Guo, L. S. Teng, C. H. Wang, Y. Zhang and J. Xie, Eur. J. Pharm. Biopharm., 2022, 179, 156–165 CrossRef CAS PubMed .
  15. Y. H. Zhang, J. Wang, C. Liu, H. L. Xing, Y. H. Jiang and X. S. Li, J. Mater. Chem. B, 2023, 11, 2478–2489 RSC .
  16. Y. Huang, L. Wang, Z. Y. Cheng, B. Y. Yang, J. H. Yu, Y. Chen and W. Lu, J. Controlled Release, 2021, 339, 297–306 CrossRef CAS PubMed .
  17. Y. Du, W. Zhang, R. He, M. Ismail, L. Ling, C. Yao, Z. Fu and X. Li, Bioorg. Med. Chem., 2017, 25, 3247–3258 CrossRef CAS PubMed .
  18. M. Park, D. Kim, S. Ko, A. Kim, K. Mo and H. Yoon, Int. J. Mol. Sci., 2022, 23, 6806 CrossRef CAS PubMed .
  19. I. Meattini, N. Andratschke, A. M. Kirby, G. Sviri, B. V. Offersen, P. Poortmans and O. K. Person, Clin. Transl. Oncol., 2020, 22, 1698–1709 CrossRef CAS PubMed .
  20. H. Xie, X. Xu, J. Chen, L. Li, J. Wang, T. Fang, L. Zhou, H. Wang and S. Zheng, Chem. Commun., 2016, 52, 5601–5604 RSC .
  21. T. T. Hao, Y. Fu, Y. Yang, S. Y. Yang, J. Liu, J. J. Tang, K. A. Ridwan, Y. Teng, Z. Liu, J. Y. Li, N. Guo and P. Yu, Eur. J. Med. Chem., 2021, 219, 113430 CrossRef CAS PubMed .
  22. S. Habib and A. Ali, Indian J. Clin. Biochem., 2011, 26, 3–17 CrossRef CAS PubMed .
  23. H. Wang, L. Wang, Z. Xie, S. Zhou, Y. Li, Y. Zhou and M. Sun, Cancers, 2020, 12, 1881 CrossRef CAS PubMed .
  24. G. Pablo, S. Aliaa, M. W. Elaine, K. Nessa, W. Mark, M. K. Maccon, J. S. Francis, J. K. Michael, C. Grace, E. R. Aideen and A. G. Sharon, Oncotarget, 2017, 8, 80568–80588 CrossRef PubMed .
  25. D. Fukumura, S. Kashiwagi and R. K. Jain, Nat. Rev. Cancer, 2006, 6, 521–534 CrossRef CAS PubMed .
  26. H. Cheng, L. Wang, M. Mollica, A. T. Re, S. Wu and L. Zuo, Cancer Lett., 2014, 353, 1–7 CrossRef CAS PubMed .
  27. J. M. Fahey and A. W. Girotti, Cancers, 2019, 11, 231 Search PubMed .
  28. L. B. Vong and Y. Nagasaki, Antioxidants, 2020, 9, 791 CrossRef CAS PubMed .
  29. J. A. Corbett and M. L. McDaniel, Methods Enzymol., 1996, 268, 398–408 CAS .
  30. H. Ozgunes and S. Atasayar, Turk. Klin. Tip Bilimleri Derg., 2009, 29, 976–986 Search PubMed .
  31. H. Maeda, H. Nakamura and J. Fang, Adv. Drug Delivery Rev., 2013, 65, 71–79 CrossRef CAS PubMed .
  32. A. Tanizawa, K. W. Kohn, G. Kohlhagen, F. O. Leteurtre. and Y. Pommier, Biochemistry, 1995, 34, 7200–7206 CrossRef CAS PubMed .
  33. L. O. Calabretta, J. Y. Yang and R. T. Raines, J. Pept. Sci., 2023, 29, 3468 CrossRef PubMed .
  34. Y. Hamada, Bioorg. Med. Chem. Lett., 2016, 26, 1685–1689 CrossRef CAS PubMed .
  35. K. Hadidi, E. Wexselblatt, J. D. Esko and Y. Tor, J. Antibiot., 2018, 71, 142–145 CrossRef CAS PubMed .
  36. B. R. Vummidi, F. Noreen, J. Alzeer, K. Moelling and N. W. Luedtke, ACS Chem. Biol., 2013, 8, 1737–1746 CrossRef CAS PubMed .
  37. H. Wang, L. Y. Wang, Z. X. Xie, S. Zhou, Y. Li, Y. Zhou and M. Y. Sun, Cancers, 2020, 12, 1881 CrossRef CAS PubMed .
  38. J. H. Byun and M. Y. Kim, Life Science Journal-Acta Zhengzhou University Overseas Edition, 2012, 9, 2341–2346 Search PubMed .
  39. N. Sharma, V. Kumar and D. A. Jose, Dalton Trans., 2023, 52, 675–682 RSC .
  40. T. Beppu, K. Nishi, S. Imoto, W. Araki, I. Setoguchi, A. Ueda, N. Suetsugi, Y. Ishima, T. Ikeda, M. Otagiri and K. Yamasaki, Oncol. Rep., 2022, 48, 178 CrossRef CAS PubMed .
  41. A. Kumari, M. Bhatoee, P. Singh, V. C. Kaladhar, N. Yadav, D. Paul, G. J. Loake and K. J. Gupta, Curr. Protoc., 2022, 2, e420 CrossRef CAS PubMed .
  42. S. Zhu, Q. Shen, Y. Gao, L. Wang, Y. Fang, Y. Chen and W. Lu, J. Med. Chem., 2020, 63, 5421–5441 CrossRef CAS PubMed .
  43. Q. Zhu, X. Yu, Q. Shen, Q. Zhang, M. Su, Y. Zhou, J. Li, Y. Chen and W. Lu, Bioorg. Med. Chem., 2018, 26, 4706–4715 CrossRef CAS PubMed .
  44. L. Wang, S. Xie, L. Ma, Y. Chen and W. Lu, Eur. J. Med. Chem., 2016, 116, 84–89 CrossRef CAS PubMed .
  45. C. Jin, Q. Zhang and W. Lu, Eur. J. Med. Chem., 2017, 132, 135–141 CrossRef CAS PubMed .
  46. X. Y. Yang, H. Y. Zhao, H. Lei, B. Yuan, S. Mao, M. Xin and S. Q. Zhang, ChemMedChem, 2021, 16, 1000–1010 CrossRef CAS PubMed .
  47. J. R. Hickok, S. Sahni, Y. Mikhed, M. G. Bonini and D. D. Thomas, J. Biol. Chem., 2011, 286, 41413–41424 CrossRef CAS PubMed .
  48. S. Mocellin, V. Bronte and D. Nitti, Med. Res. Rev., 2007, 27, 317–352 CrossRef CAS PubMed .
  49. L. Botta, S. Filippi, C. Zippilli, S. Cesarini, B. M. Bizzarri, A. Cirigliano, T. Rinaldi, A. Paiardini, D. Fiorucci, R. Saladino, R. Negri and P. Benedetti, ACS Med. Chem. Lett., 2020, 11, 1035–1040 CrossRef CAS PubMed .
  50. I. T. Schiefer, L. VandeVrede, M. R. Fa, O. Arancio and G. R. J. Thatcher, J. Med. Chem., 2012, 55, 3076–3087 CrossRef CAS PubMed .
  51. S. X. Liu, Z. L. Hu, Q. M. Zhang, Q. W. Zhu, Y. Chen and W. Lu, ACS Omega, 2020, 5, 350–357 CrossRef CAS PubMed .

Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nj02778g
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2024