Design, Synthesis and Antitumor Activity of Quercetin Derivatives Containing a Quinoline Moiety

Quercetin is a flavonoid with significant biological and pharmacological activity. In this paper, quercetin was modified at the 3-OH position. Rutin was used as a raw material. We used methyl protection, Williamson etherification reactions, and then substitution reactions to prepare 15 novel quercetin derivatives containing a quinoline moiety. All these complexes were characterized by 1H NMR, 13C NMR, IR and HRMS. Of these, compound 3e (IC50 = 6.722 μmol·L−1) had a better inhibitory effect on human liver cancer (HepG-2) than DDP (Cisplatin) (IC50 = 26.981 μmol·L−1). The mechanism of the action experiment showed that compound 3e could induce cell apoptosis.

Research has shown that quercetin can treat and prevent various cancers including prostate cancer alone or in combination with other dietary natural products [12,13].The potential mechanism of its anti-proliferative effect on prostate cancer cells is related to its impacts on the cell cycle, apoptosis, and regulation of androgen receptors [14].Studies based on cells and animals, as well as clinical studies, have confirmed the medicinal value of quercetin as an anti-prostate cancer drug.However, in vivo and in vitro data demonstrated that its moderate potency hindered its further development [15].
Therefore, the chemical structure of quercetin should be modified.The literature shows that the introduction of one substituent group to the phenolic hydroxyl group of quercetin-especially lengthy and/or bulky groups-might be an effective strategy for the modification of quercetin as an anticancer drug [16].
Quercetin has a B ring with an o-diphenol structure.The A ring has an m-diphenol structure.The phenolic hydroxyl groups of the A and B rings of quercetin play an important role in anti-oxidation [17].The C ring contains enol and ketone structures, which gives quercetin some special biological activities.The derivatives obtained by modifying different groups have different biological activities and efficacies [18].3-OH is the unique hydroxyl group of quercetin, and in this study, chemical modifications are carried out at this position.
Quinoline is a nitrogen-containing heterocyclic aromatic compound with distinct biological activities such as antimalarial [19], antibacterial [20], analgesic [21], anti-inflammatory [22], We thus speculated that introducing hydroxyquinoline fragments into quercetin might generate novel lead compounds with greater biological activities.Thus, 15 derivatives of quercetin containing quinoline groups were synthesized by introducing quinoline active groups into the 3-OH group of quercetin through an active splicing method.The anti-tumor activities of the target compounds were tested to find compounds with good anti-tumor activities, which provide a theoretical basis for related work.

Chemistry
The synthetic routes are shown in Scheme 1. Rutin underwent methylation and deglycosylation steps to obtain intermediate 1. Midbody 2 was obtained by reacting 1,3-dibromopropane or 1,4-dibromobutane or 1,5-dibromopentane with 1.The target compound 3 was obtained by the substitution reaction of 2 with hydroxyquinoline under alkaline conditions.

Chemistry
The synthetic routes are shown in Scheme 1. Rutin underwent methylation and deglycosylation steps to obtain intermediate 1. Midbody 2 was obtained by reacting 1,3-dibromopropane or 1,4-dibromobutane or 1,5-dibromopentane with 1.The target compound 3 was obtained by the substitution reaction of 2 with hydroxyquinoline under alkaline conditions.

Anti-Tumor Activity In Vitro
The anti-tumor activity of all the target compounds 3a-3o was evaluated in vitro by an MTT assay against HepG-2, A549, and MCF-7 cell lines, with DDP as the positive control.Their inhibition rate and IC50 values are listed in Table 1.

Anti-Tumor Activity In Vitro
The anti-tumor activity of all the target compounds 3a-3o was evaluated in vitro by an MTT assay against HepG-2, A549, and MCF-7 cell lines, with DDP as the positive control.Their inhibition rate and IC 50 values are listed in Table 1.

Anti-Tumor Activity In Vitro
The anti-tumor activity of all the target compounds 3a-3o was evaluated in vitro by an MTT assay against HepG-2, A549, and MCF-7 cell lines, with DDP as the positive control.Their inhibition rate and IC50 values are listed in Table 1.The inhibitory effect of partially synthesized quercetin derivatives containing quinoline structures on HepG-2 cells, A549, and MCF-7 cell lines was higher than that of quercetin and DDP.The inhibitory effects of 3i, 3k, and 3e on HepG-2 cells were stronger than those of DDP and quercetin, with IC 50 values of 5.074 µmol•L −1 , 5.193 µmol•L −1 , and 6.722 µmol•L −1 , respectively.The inhibitory effects of 3a, 3e, and 3h on A549 cells were stronger than those of DDP and quercetin, with IC 50 values of 7.384 µmol•L −1 , 26.614 µmol•L −1 , and 31.678µmol•L −1 , respectively.3a had a stronger inhibitory effect on MCF-7 cells than DDP, with an IC 50 value of 1.607 µmol•L −1 .The inhibitory effect of 3e, 3i, 3b, and 3k on MCF-7 cells was stronger than that of quercetin, with IC 50 values of 3.004 µmol•L −1 , 6.464 µmol•L −1 , 6.793 µmol•L −1 , and 6.856 µmol•L −1 , respectively.Specifically, compound 3e had a higher inhibitory rate on HepG-2 and less toxicity to normal cells; thus, 3e was chosen as the lead compound for the next step of research.

Compound 3e Induces HepG-2 Cell Apoptosis
Most anticancer drugs can kill tumor cells by inducing cell apoptosis, and thus, inducing cell apoptosis is considered one of the main mechanisms for killing tumor cells.Compound 3e was tested to clarify whether the inhibitory effects of these compounds on cell proliferation were related to apoptosis.HepG-2 cells were treated with DMSO or different concentrations of 3e for 48 h.Cells were stained with Annexin-V and PI, and the proportion of apoptotic cells was detected by flow cytometry.

Compound 3e Induces HepG-2 Cell Apoptosis
Most anticancer drugs can kill tumor cells by inducing cell apoptosis, and thus, inducing cell apoptosis is considered one of the main mechanisms for killing tumor cells Compound 3e was tested to clarify whether the inhibitory effects of these compounds on cell proliferation were related to apoptosis.HepG-2 cells were treated with DMSO or different concentrations of 3e for 48 h.Cells were stained with Annexin-V and PI, and the proportion of apoptotic cells was detected by flow cytometry.

Structure-Activity Relationship (SAR) Analysis
As indicated in Table 1, the anti-tumor activities of target compounds were greatly affected by structural variations.Comparing IC 50 for compounds 3a-3o, overall, increasing the length of the product alkane bridge was beneficial for enhancing activity.For instance, under the same conditions of R = 2-OH, the target compounds 3a (R = 2-OH, n = 5) had higher anti-tumor activity against MCF-7 than 3k (R = 2-OH, n = 4) and 3f (R = 2-OH, n = 3), with inhibition rates of 1.607 µmol•L −1 , 6.856 µmol•L −1 and >100 µmol•L −1 , respectively.In addition, for some target compounds, when the OH was substituted at the 2-position of quinoline, the compounds exhibited greater anti-tumor activity.For example, the target compounds 3k (R = 2-OH, n = 4) had higher anti-tumor activity against HepG-2 (IC 50 = 5.193 µmol•L −1 ) and MCF-7 (IC 50 = 6.856 µmol•L −1 ) than the products of OH substituted at other positions.However, on the contrary, there were also different situations.For example, the IC 50 value of target compound 3i (R = 6-OH, n = 3) on HepG-2 was 5.074 µmol•L −1 , which was superior to other substituent groups.

Discussion
Although quercetin demonstrates varied biological activities and pharmacological values, due to its molecular structure, it has poor water solubility and low bioavailability after entering the body, which affects the original efficacy of the drug and limits its application in the pharmaceutical field.Therefore, using quercetin as the lead compound to chemically modify its structure and search for high bioavailability and stronger activity precursor drugs has become a research hotspot in fields of medicine.3-OH is a unique hydroxyl group of quercetin, and introducing functional groups at this position often yields more active compounds.For instance, Rajaram et al. [15] and Al Jabban et al. [30] alkylated the 3-OH group of quercetin to obtain novel quercetin derivatives with higher anticancer activity.
The research suggested that 3-OH substitution of quercetin could significantly alter its anticancer activities.However, these reports introduced smaller volume groups into the 3-OH group of quercetin.Reports indicated that introducing larger active groups, such as quinazolinone and heterocycle, into the flavonol compounds would effectively enhance their antibacterial, anticancer, and other activities [31,32].In our study, we selected larger volume quinoline groups and bridged them with alkyl chains of different chain lengths to introduce them into the quercetin molecule.The results indicated that augmenting the length of the alkane bridge was beneficial for improving activity, consistent with the conclusion of Jiang et al. [33].

Chemistry
Melting points (M.p.) were determined on a Buchi-Tottoli apparatus and were uncorrected.IR spectra were recorded on a Tensor 27 (Bruker Optics, Ettlingen, Germany) spectrometer in KBr pellets. 1 H NMR spectra were obtained from a solution in DMSO-d 6 with Me 4 Si as the internal standard using a Bruker-400 spectrometer.HRMS analyses used a TOF-Q-MS analyzer (micro-TOF-QII, Bruker, Billerica, MA, US), and the values are expressed as [M + H] + .All starting materials were purchased from Saen Chemical Technology (Shanghai, China) Co., Ltd.The reaction courses and product mixtures were routinely monitored by TLC on silica gel (precoated F254 Merck plates).Organic solutions were dried over anhydrous Na 2 SO 4 .

General Synthesis Procedure for Intermediates 1 and 2
Rutin with a purity of 98% was used as a raw material.Intermediates 1 and 2 were synthesized by methods reported in the literature [32,34,35].
Scheme 1. Synthetic route of target compounds 3.

Figure 2 .
Figure 2. (A) The effect on cellular apoptosis after HepG-2 cells were incubated with compound 3e for 48 h; (B) statistic histogram indicated the percentages of apoptotic cells (compared with the con trol group, *** p < 0.001).

Figure 2 .
Figure 2. (A) The effect on cellular apoptosis after HepG-2 cells were incubated with compound 3e for 48 h; (B) statistic histogram indicated the percentages of apoptotic cells (compared with the control group, *** p < 0.001).

Table 1 .
Inhibition rate of target compound on proliferation of different tumor cells.

Table 1 .
Inhibition rate of target compound on proliferation of different tumor cells.

Table 1 .
Inhibition rate of target compound on proliferation of different tumor cells.