Apr 22, 2019 - Quercetin has been found to possess diverse pharmacological properties including in different types of cancers. The application of quercetin in ...
Apr 22, 2019 - Quercetin has been found to possess diverse pharmacological properties including in different types of cancers. The application of quercetin in ...
Apr 22, 2019 - Cell Sciences (NCCS), Pune, India. Cell culture components including Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS) ...
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May 24, 2003 - Gause Institute of New Antibiotics, Russian Academy of Medical Sciences ... type 1 (HIV-1), HIV-2, and Moloney murine sarcoma virus at a 50%.
James W. McFarland,* Scott J. Hecker,â Burton H. Jaynes, Martin R. Jefson, Kristin M. ... Edward A. Glazer, Susan A. Froshauer, Shigeru F. Hayashi, Barbara J.
Repromicin Derivatives with Potent Antibacterial Activity against Pasteurella multocida. James W. McFarland,* Scott J. Hecker,â Burton H. Jaynes, Martin R.
... Scott J. Hecker, Burton H. Jaynes, Martin R. Jefson, Barbara J. Kamicker, Christopher A. Lipinski, Kristin M. Lundy, Catherine P. Reese, and Chi B. Vu.
Dipartimento di Scienze Molecolari Agroalimentari, Sezione di Chimica, ... Loana Musso, Lucio Merlini, Gabriella Morini, Sergio Penco, Claudio Pisano, Stella Tinelli, ... Sabrina Dallavalle, Anna Ferrari, Barbara Biasotti, Lucio Merlini, Sergio ...
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Article Cite This: ACS Omega 2019, 4, 7285−7298
Semisynthetic Quercetin Derivatives with Potent Antitumor Activity in Colon Carcinoma Ayan Mukherjee,†,§,⊥ Snehasis Mishra,‡,⊥ Naveen Kumar Kotla,†,⊥ Krishnendu Manna,‡ Swarnali Roy,† Biswajit Kundu,† Debomita Bhattacharya,† Krishna Das Saha,‡ and Arindam Talukdar*,† †
ACS Omega 2019.4:7285-7298. Downloaded from pubs.acs.org by 126.96.36.199 on 04/22/19. For personal use only.
Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology, 4 Raja Subodh Chandra Mullick Road, Kolkata, 700032 West Bengal, India ‡ Department of Cancer Biology and Inﬂammatory Disorders, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, West Bengal, India § Academy of Scientiﬁc and Innovative Research (AcSIR), Ghaziabad 201002, India S Supporting Information *
ABSTRACT: Quercetin has been found to possess diverse pharmacological properties including in diﬀerent types of cancers. The application of quercetin in the pharmaceutical ﬁeld is limited due to its poor bioavailability resulting from poor water solubility and poor permeability. We report a systematic chemical modiﬁcation of quercetin toward the development of semisynthetic derivatives through a selective synthetic methodology, which enables the installation of diﬀerent substitutions at C-3′ and C-5 positions of quercetin. The hypothesis of the present manuscript was to modulate the log D value and aqueous solubility of quercetin through the attachment of some facilitator moieties. The semisynthetic derivatives with an ideal log D value and improved aqueous solubility will possess a better cell-penetrating ability compared to quercetin. Representative compound 17 shows 96-fold increase in the cytotoxic activity in HCT-116 colon cancer cells as compared to quercetin. The in vivo treatment of 17 in CT-26 tumor-bearing mice in a colon cancer model resulted in a striking increase in the survival rate and reduction in tumor weight (60%) with respect to quercetin. We believe that the current study has an immense potential toward the systemic development of clinically approved quercetin semisynthetic derivatives.
increases the anticancer activity in colon cancer over twofold with respect to the native quercetin.13 To our surprise, studies related to the anticancer eﬃcacy of quercetin by chemical modiﬁcations were restricted to nonselective acylation or alkylation,8,14−17 which usually gives rise to the complex reaction mixture that requires extensive experimentation for identiﬁcation of the exact structure. Shi et al.17 showed that methyl substitution at C-3 and C-7 hydroxyl groups of quercetin increases the anticancer activity, whereas methylation at the C-5 position reduces its activity. It can be stated that careful manipulation of the diﬀerent hydroxyl groups on quercetin with speciﬁc substituents can be an eﬀective strategy to improve its cytotoxicity against cancer cells. However, the lack of eﬀort in such a direction may be attributed to coherent understanding and ﬁnding a generalized protocol for selective substitution of the hydroxyl group of quercetin. The present manuscript describes a synthetic methodology to selectively attach a diﬀerent substitution pattern at C-3′ positions of quercetin to modulate the solubility and Received: January 16, 2019 Accepted: April 2, 2019 Published: April 22, 2019 7285
in drug development.18,19 Addition of alkyl chains containing esters and weak base to quercetin increased the solubility and lipophilicity, which eﬀectively enhances the cell permeability. The novel semisynthetic derivatives with better physicochemical properties signiﬁcantly improve the anticancer activity against colon cancer. The strategy discussed in the present manuscript has the potential to transform the promiscuous quercetin into a privileged scaﬀold and will encourage similar research activities toward the systemic development of clinically approved quercetin semisynthetic derivatives. Notably, many marketed natural products derived from pharmaceuticals, such as artemisinin derivatives and pholcodine, a morphine derivative, have been developed through such single modiﬁcations of the parent natural product.20,21
Figure 1. X-ray crystal structure of compound 13. Atoms are colored white (hydrogen), gray (carbon), red (oxygen), and green (chlorine).
RESULTS AND DISCUSSION Chemistry. Schemes 1 and 2 illustrate the synthesis of monosubstituted and disubstituted derivatives of quercetin along with the reagents and reaction conditions. Benzylation of quercetin was carried out by following the reported procedure, which resulted in tribenzyl 2 (60%)- and tertrabenzyl 3 (33%)substituted derivatives.22 Meticulous treatment of 2 with 1.5 equiv of methyl bromoacetate and K2CO3 in acetone resulted in exclusive monomethyl ester substitution at the C-3′ position to yield product 4. Treatment of 2 with 3 equiv of methyl bromoacetate provided the dimethyl ester-substituted product 7.23 By applying a similar protocol, 1-bromo-3-chloropropane is installed in compound 13 (Scheme 2). Debenzylation of 4 using palladium hydroxide resulted in compound 5, and subsequent ester hydrolysis with 2 N NaOH in acetone yielded compound 6. Compound 2 upon treatment with excess methyl bromoacetate resulted in disubstitution at C-3′ and C-5 positions to yield the symmetrical product 7, which, upon debenzylation, gave 8, and subsequent ester hydrolysis yielded compound 9. Similarly, compound 3 upon benzylation provided compound 10, and following the similar strategy, compounds 11 and 12 were obtained. In Scheme 2, we have used the selective protocol to substitute the halide linker at the C-3′ hydroxyl of quercetin. Substitution with diﬀerent bases such as pyrrolidine and Nmethylpiperazine provided compounds 14 and 15, respectively. Global deprotection of the benzyl group yielded semisynthetic derivatives 16 and 17. Similar protocol was used for the synthesis of 18 by treating with excess 1-bromo-3chloropropane, which was converted to 19, and subsequent benzyl deprotection provided the symmetrical disubstituted product 20. Synthesis of 5-substituted derivative 23 was obtained by applying the same strategy used in the case of derivative 17. Regioselective Substitution. The ﬁve hydroxyl groups of quercetin (1) have diﬀerent reactivities.8 The regioselective substitution reaction was meticulously standardized by controlling the reaction time and equivalence of methyl bromoacetate or 1-bromo-3-chloropropane for exclusive C-3′ hydroxyl-substituted products (Scheme 1). Any variation would give rise to the complex reaction mixture, which requires tedious separation techniques for isolating a single product. We tried to crystallize several products, but with great diﬃculty, we could crystallize compound 13 and conﬁrmed the structure through X-ray crystallography. The X-ray structure of 13 (Figure 1) also provided the reason for lesser reactivity of the C-5 hydroxyl group as compared to the C-3′ hydroxyl
group, as there exists an acid weakening eﬀect of an intramolecular hydrogen bond between C-5 hydroxyl and ketone groups present at the C-4 position. To the best of our knowledge, compound 13 is the ﬁrst crystal structure of chemically modiﬁed nonsugar quercetin. Assessment of in Vitro Cytotoxicity. The synthesized library of semisynthetic quercetin derivatives were divided into two groups: monosubstituted (5, 6, 11, 12, 16, 17, and 23) and disubstituted symmetrical (7, 9, and 20) derivatives. The cell viability was gradually decreased in a dose-dependent manner when treated with diﬀerent concentrations of compounds for 24 h (Figure S1A,B, Supporting Information). The activity of quercetin in our assay system showed an IC50 value of 45.3 μM (Table 1), which is similar to the reported value.14 The aqueous solubility of quercetin at pH 7.4 is 0.1 μg/mL. To address the solubility issue, we initiated the SAR by introducing methyl ester functionality selectively in the C-3′ positions in compound 5. The C-5 substituted, monosubstituted ester derivative 11 was obtained from compound 3. To our great satisfaction, the IC50 value of 11 showed 0.34 μM, and that of 5 showed 3.03 μM in HCT-116 cells. It can be stated that the introduction of an ester group at the C-5 position concomitantly improved the solubility (∼380-fold) and cytotoxicity (∼150-fold) compared to quercetin in compound 11. Hydrolysis of respective ester derivatives resulted in compounds 6 and 12 with free carboxylic acid. Compound 6 showed a similar activity proﬁle as compared to its ester counterpart 5; when compared to quercetin, they exhibited ∼15-fold better cytotoxicity. In case of 12, there was a fourfold decrease in the activity compared to its ester derivative 11. Although the water solubility for both compounds are similar, notably, the log D (1.81) value for the ester derivative 11 is much better as compared to the negative log D value of 12 (−0.98), which can inﬂuence cell permeability.12 However, there was a loss of activity in both the disubstituted ester derivative 7 (IC50 value of 57.5 μM) and its hydrolysis derivative 9 (IC50 value of 7.34 μM), which signiﬁes that the disubstitution at both C-5 and C-3′ may not be desirable and the monosubstitution at either C-5 or C-3′ with ester and the corresponding free acid groups signiﬁcantly increase the cytotoxicity as compared to quercetin (Table 1). On a similar note in our next development, we introduced an aliphatic linker containing weak bases, such as Nmethylpiperazine and pyrrolidine, at C-5 and C-3′ positions. Monosubstitution at C-5 and C-3′ positions resulted in compounds 16, 17, and 23. Attachment of the pyrrolidine 7287
Table 1. SAR of the Semisynthetic Derivatives of Quercetina
ND: not determined. bThe IC50 values were calculated by MTT assay (mean ± SD, n = 3). cLog D value was experimentally determined.
In summary, by balancing the solubility and log D value in our semisynthetic derivatives, the cytotoxicity was improved to ∼100-fold against colorectal cancer cells as compared to quercetin. It is known that the catechol moiety in polyphenolic compounds can give rise to the electrophilic quinone moiety,24 which causes various cellular toxicities. Keeping this in mind, we selected the C-3′-substituted compound 17 over the C-5substituted 11 and 23 for further investigation. Moreover, the Caco-2 permeability assay showed that compound 17 is highly permeable with an eﬄux ratio of <2, which signiﬁes that 17 is not a P-gp substrate (Table 2). Compound 17 Promotes ROS Accumulation in HCT116 Cells. Quercetin is known to induce oxidative stress in
moiety at the C-3′ position in compound 16 increased the cytotoxicity (IC50 = 1.98 μM) by 22-fold and solubility by 325fold with respect to quercetin. Introduction of N-methylpiperazine at the C-3′ position in 17 resulted in a drastic improvement in the activity with an IC50 value of 0.48 μM (90-fold) with concurrent increase in ∼400-fold solubility as compared to quercetin. To our satisfaction, 17 did not show toxicity with peripheral blood mononuclear cells (PBMC) and HEK293 cells at a diﬀerent concentration, suggesting that 17 has selective toxicity toward cancer cells but not in primary immune cells (Figure S1D). Compound 17 showed a better log D value and solubility proﬁle as compared to 16. Switching the N-methylpiperazine moiety at the C-5 position in 23 resulted in similar potency with an IC50 value of 0.55 μM. We observed that, in the case of symmetrical disubstituted derivative 20 with substitution at both C-3′ and C-5 positions, the balance between the log D value and solubility is disturbed. The addition of two weak base pyrrolidines increased the water solubility (84.55 μg/mL), which in turn causes a poor log D value (0.65).
Table 2. Caco-2 Permeability of Compound 17 and Cytotoxicity in Normal Cell Papp (10−6 cm/s)
Figure 2. Determination of iROS, apoptosis/necrosis, and mitochondrial membrane potential upon the treatment of com-17 (0.47 μM) with/ without NAC for 6 and 12 h. Representative ﬂow cytometric dot plot and gating hierarchy used to deﬁne the (A) DCF + Ve and DCF − Ve cells and (B) viable cells (VB), early apoptotic cells (EA), late apoptotic cells (LA), and necrotic cells (NC).
Figure 3. (A) High ΔΨ and low ΔΨ. Histogram showing (B) Bax and (D) Bcl2 expression. Bar graph showing relative ﬂuorescence intensities of (C) Bax-FITC, (E) Bcl2-PE, (F) caspase-3, and (G) caspase-9 activity in the diﬀerent experimental condition in HCT-116 cells. Values are represented as mean ± SEM (n = 5). The value of p < 0.05 was considered as signiﬁcant. Statistical comparison: *control versus com-17 (6 h); **control versus com-17 (12 h); ***control versus com-17 (12 h) + NAC; #control versus com-17 (12 h) + PFT-α; NS, nonsigniﬁcant.
Figure 4. (A) Representative ﬂow cytometric dot plot and gating hierarchy used to deﬁne the (A) p-PI3K-PE + Ve cells and p-PI3K-PE −Ve cells in the experimental condition. (B) Immunoﬂuorescence images showing the qualitative expression of p-AKT and p-mTOR. The nucleus was counterstained with DAPI (magniﬁcation: 60×). Intensity analysis of relative ﬂuorescence of p-AKT-FITC, p-mTOR-PE, and DAPI was done using ImageJ software from the corresponding immunoﬂuorescence micrographs.
cancer cells by the generation of ROS.25−27 A cell-permeant dye 2′,7′-dichloroﬂuorescein diacetate was used to detect the cellular level of ROS.14 The relative DCF ﬂuorescence was directly proportional to the amount of intracellular produced ROS. Compound 17-treated HCT-116 cells showed an overproduction of ROS (Figure 2A) by markedly increased DCF ﬂuorescence reﬂecting the DCF + Ve cells when treated for 6 h (59.1%) and 12 h (86.1%) and when compared with the control cells (0.14%). N-Acetyl cysteine (NAC), a potent scavenger of ROS, signiﬁcantly reduced DCF ﬂuorescence (59.8%) when treated with 17 for 12 h, suggesting that the
ROS production is directly associated with compound 17mediated cytotoxicity. The increase in the early (94.3%) and late (2.82%) apoptotic population with respect to the control cells (14.3% in the early apoptotic population and 0.11% in the late apoptotic population) was measured using Annexin VFITC/PI further supported the fact that 17-mediated cytotoxicity was guided through a ROS-dependent apoptotic pathway (Figure 2B). Compound 17 Regulates Mitochondrial Pathway Apoptosis in HCT-116 Cells. Disruption of mitochondria is an indicator of apoptotic cell death.28 Changes in the 7290
Figure 5. (A) Kaplan−Meier analysis of 30 day survival of CT-26 tumor-bearing mice post-administered with com-17 (12.5, 25, and 50 mg/kg). (B) Representative line diagram showing body weight change throughout the experimental period. (C) Representative image of tumor harvested after the completion of experimentation. (D) Bar graph showing tumor weight. Values are represented as mean ± SEM (n = 5). The value of p < 0.05 was considered as signiﬁcant. Statistical comparison: *CT-26 versus CT-26 + com-17 (25 mg/kg); **CT-26 versus CT-26 + com-17 (50 mg/ kg).
100% mortality was reached on the 22nd day of the CT-26 challenge. To our satisfaction, the maximum survival (66.67%) on the CT-26 tumor-bearing condition was evident with the treatment of 17 at 50 mg/kg of body weight on the 30th day of the CT-26 challenge. On the other hand, 83.33% of the total animals died when treated with 17 at 25 mg/kg of body weight. Eﬀect of Compound 17 on Tumor-Induced Weight Loss and Tumor Weight. The average body weight was reduced to 51% on the 22nd day of experimentation. After the treatment with 17 at 25 and 50 mg/kg, the tumor-induced body weight was markedly restored (85 and 88%), indicating that administration of 17 has helped maintain the body weight in tumor-bearing mice (Figure 5B). Administration of 17 at 25 and 50 mg/kg exhibited a signiﬁcant reduction (Figure 5C,D) in tumor weight (1.74 and 0.94 g) as compared to the untreated tumor mass (2.34 g). According to the previous report, treatment of quercetin at 50 mg/kg on CT-26 tumor-bearing mice did not show any signiﬁcant diﬀerence18 in the survival rate and tumor volume. Increasing the dose of quercetin to 100 mg/kg led to a slight improvement of the survival rate of CT-26 tumor-bearing mice, but on the 30th day of treatment, 100% mortality was observed. Comparison of the treatment with quercetin versus our semisynthetic quercetin derivative 17 has substantiated the hypothesis and established the fact that treatment of representative compound 17 resulted in a striking improvement in the survival rate of CT-26 tumor-bearing mice as compared to treatment with quercetin.
mitochondrial membrane potential (ΔΨm) due to the opening of the mitochondrial permeability transition pore (MPTP) is correlated with the early apoptotic fate.27 Compound 17 reduces ΔΨm (Figure 3A) when treated for 6 h (15.2%) and 12 h (42.7%) as compared with the control cells (7.06%), suggesting that 17-mediated cytotoxicity might be regulated through the mitochondrial pathway of apoptosis. Next, we evaluated the eﬀect of 17 on various mitochondria-associated apoptotic markers.29 Compound 17 augmented the expression of Bax as well as the inhibition of Bcl2 apoptotic marker (Figure 3B−E). The ﬁnal apoptotic fate was determined by the activity of caspase-9 and caspase-3, which was simultaneously enhanced with the treatment of 17. The result summarily conﬁrms that 17-mediated apoptosis might be regulated through the mitochondrial pathway of apoptosis. Compound 17 Dephosphorylated AKT/PI3K/mTOR Pathway in HCT-116 Cells. The PI3K/AKT/mTOR signaling pathway plays a vital role in cell proliferation and survival.30 Flow cytometry and immunoﬂuorescence evaluation show (Figure 4A,B) a marked down-regulation in the survival pathway upon treatment of 17 (6 and 12 h), which was further conﬁrmed in a study, where a speciﬁc inhibitor for particular survival proteins was used. Upon treatment with a speciﬁc inhibitor, such as LY294002,31 rapamycin also increased the cell viability18 (54.21 and 51.27%) over the control group. Our study suggests that compound 17 induces apoptosis in HCT116 cells through the suppression of PI3K/AKT/mTOR axis. Eﬀect of Compound 17 on Tumor-Induced Survival of Mice. The animal experiment (Figure 5A) demonstrates the survival rate of CT-26 tumor-bearing mice that were treated with three diﬀerent dosages (12.5, 25, and 50 mg/kg) of 17 compared to the untreated group. Intraperitoneal treatment (Figure S2, Supporting Information) of 17 for six alternate days to normal mice did not induce any death within the 30 day observational period. The ﬁrst death of CT-26 tumor-bearing mice was observed after the 15th day of a CT26 challenge due to the signiﬁcant symptoms including reduction of food/water intake, irritability, enhanced tumor burden, weight loss, lethargy, and roughening of hair. Results demonstrated that 50% of animals died within 17 days, and
CONCLUSIONS In conclusion, the present study describes a synthetic methodology for the selective substitution at the C-3′ position over the C-5 position of quercetin. The structure of the product and the reason for regioselective substitution were validated through X-ray crystallography. The strategic installation of facilitator moiety in semisynthetic quercetin derivatives resulted in improvement of aqueous solubility and lipophilicity, which are considered to be long-standing limiting factors for poor bioavailability of quercetin. To our achieve7291
(m, 16H), 7.31−7.23 (m, 4H), 6.96 (d, J = 6.0 Hz, 1H), 6.46 (d, J = 3.0 Hz, 1H), 6.43 (d, J = 3.0 Hz, 1H), 5.25 (s, 2H), 5.13 (s, 2H), 5.04 (s, 2H), 4.99 (s, 2H). 13C NMR (CDCl3, 100 MHz): δ 178.9, 164.5, 162.1, 158.1, 151.1, 148.3, 137.0, 136.7, 136.5, 135.5, 128.9, 128.8, 128.7, 128.6, 128.3, 128.0, 127.6, 127.4, 127.3, 123.5, 122.7, 115.3, 113.7, 106.3, 98.7, 93.1, 74.3, 71.2, 70.9, 70.5. MS (ESI) m/z: [M + Na] + 685.21. General Procedure A for Attaching Monomethyl Acetate. Methyl 2-(2-(Benzyloxy)-5-(3,7-bis(benzyloxy)-5hydroxy-4-oxo-4H-chromen-2-yl)phenoxy)acetate (4). Compound 2 (1.0 g, 1.7 mmol) and K2CO3 (0.24 g, 1.7 mmol) were taken in dry acetone in a nitrogen atmosphere, and then methyl bromoacetate (0.33 g, 2.09 mmol) was added slowly into the reaction mixture. Reaction was reﬂuxed for 6 h, then the reaction was ﬁltered, and column chromatography was performed to get pure compound 4 (70%, mp: 98 °C). 1H NMR (CDCl3, 400 MHz): δ 7.63−7.61 (m, 2H), 7.46−7.29 (m, 12H), 7.25−7.32 (m, 3H), 6.95 (d, J = 9.3 Hz, 1H), 6.46 (d, J = 2.7 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 5.06 (s, 2H), 4.57 (s, 2H), 3.72 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 178.9, 169.2, 164.4, 162.1, 156.8, 156.0, 151.1, 147.3, 137.6, 136.5, 136.3, 135.8, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 127.9, 127.6, 127.3, 123.9, 123.4, 115.9, 113.8, 106.3, 98.7, 93.2, 74.5, 71.1, 70.5, 66.6, 52.5. HRMS (ESI) m/z: (M + H) calculated for C39H33O9, 645.2125; found, 645.2118. General Procedure B for Hydrogenation. Methyl 2-(2Hydroxy-5-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenoxy)acetate (5). Compound 4 (0.1 g, 0.15 mmol) and palladium hydroxide on carbon (10 mol %) were taken in 1:1 THF/EtOH solution. The reaction was performed under a hydrogen atmosphere for 2 h, then the reaction mixture was ﬁltered through Celite, and reverse-phase chromatography was performed using a C18 gold column to obtain compound 5 (74%, mp: >250 °C). 1H NMR (DMSO-d6, 300 MHz): δ 12.5 (s, 1H), 7.72 (dd, J = 7.5 Hz, 0.9 Hz, 1H), 7.68 (d, J = 2.1, 1H), 6.98 (d, J = 9.3 Hz, 1H,), 6.44 (d, J = 2.7 Hz, 1H), 6.19 (d, J = 2.1 Hz, 1H), 4.83 (s, 2H), 3.73 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 176.4, 169.9, 169.4, 164.5, 161.2, 156.6, 146.8, 146.1, 136.5, 123.1, 122.4, 116.6, 103.5, 100.0, 98.7, 93.9, 66.3, 52.3. HRMS (ESI) m/z: (M + Na) calculated for C18H14NaO9, 397.0536; found, 397.0519. HPLC purity (95.2%). General Procedure C for Hydrolysis of Methyl Ester. 2-(2-Hydroxy-5-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenoxy)acetic acid (6). Compound 5 (0.1 g, 0.27 mmol) was taken in 1:1 acetone−water solution, and 2 N NaOH solution was added dropwise to it until pH was 9. Reaction mixture was stirred at room temperature for 1 h, then the solvent was removed, and the reaction mixture was puriﬁed by reversephase column chromatography using a C18 gold column to obtain compound 6 as a yellow solid (80%, mp: >250 °C). 1H NMR (CDCl3, 300 MHz): δ 12.5 (s, 1H), 9.37 (s, 1H), 7.80 (s, 1H), 7.76 (d, J = 2.1 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.45 (s, 1H), 6.18 (s, 1H), 4.13 (s, 2H). 13C NMR (CDCl3, 125 MHz): δ 176.4, 168.9, 165.2, 162.9, 158.4, 151.3, 145.9, 141.3, 125.2, 121.6, 116.2, 114.5, 106.6, 98.9, 95.0, 66.5. HRMS (ESI) m/z: (M + Na) calculated for C17H12NaO9, 383.0379; found, 383.0374. HPLC purity (95.98%). Methyl 2-(2-(Benzyloxy)-5-(3,7-bis(benzyloxy)-5-(2-methoxy-2-oxoethoxy)-4-oxo-4H-chromen-2-yl)phenoxy)acetate (7). Compound 2 (1.0 g, 1.7 mmol) and K2CO3 (0.24 gm., 1.7 mmol) were taken in dry acetone in a nitrogen
ment, the derivatives with improved physicochemical properties showed >130-fold increase in their cytotoxicity in HCT116 colon cancer cells as compared to quercetin. Compound 17 showed high permeability in the Caco-2 assay. The result corroborates that the representative compound 17 enhances the ROS level and induces cytotoxicity in cancer cells through DNA damage and an intrinsic mitochondria-mediated pathway. Furthermore, the in vivo experiment in CT-26 tumorbearing mice substantiated the fact that treatment of the representative compound 17 resulted in a striking increase in the survival rate and reduction in tumor volume as compared to treatment with quercetin. The representative terminal acid and halogen precursor provide a useful handle for further conjugation with diﬀerent structural moieties and thus may encourage the researchers from the diverse ﬁeld to explore diﬀerent applications of the modiﬁed quercetin.
EXPERIMENTAL SECTION General Experimental Conditions. All the reagents and solvents are purchased from a commercial source with analytical grade purity. Moisture- or air-sensitive reaction was performed under an ultrapure nitrogen gas atmosphere. Standard rotavapor instruments are used to remove solvent. NMR spectroscopy was done in Bruker 300 MHz, Bruker 600 MHz, and JEOL 400 MHz instruments. Deuterated NMR solvent was used according to the solubility of compounds to record the NMR spectra. All the HRMS analyses were done in an ESI-QTOF instrument. Reverse-phase column chromatography was performed in RediSep Rf “Gold” high-performance silica columns on a CombiFlash Rf instrument using water− acetonitrile as the mobile phase. Purity of the compound was checked by Hitachi chromaster HPLC using a Welch Xtimate C18 (5 μm, 4.6 × 150 mm). UV measurement was done in a Spectramax UV spectrometer, and LC−UV was done in a Shimadzu UFLC. The purity (HPLC) of all the compounds subjected to biological assay is >95% at a wavelength of 250 nm. 3,7-Bis(benzyloxy)-2-(4-(benzyloxy)-3-hydroxyphenyl)-5hydroxy-4H-chromen-4-one (2). A solution of quercetin 1 (5.00 g, 14.79 mmol), potassium carbonate (7.9 g, 51.77 mmol), and benzyl bromide (9.9 g, 51.77 mmol) were taken in DMF (10 mL) and stirred vigorously under nitrogen at 0 °C for 2 h. The reaction mixture was allowed to warm to room temperature over 2 h, and the stirring was maintained for further 12 h. The resulting mixture was diluted with water (100 mL) and extracted with EtOAc (2 × 500 mL). The organic layer was dried (Na2SO4) and evaporated, and the residue obtained was puriﬁed by ﬂash column chromatography using 7% EtOAc as an eluent to aﬀord product 2 as a yellow solid (60%, mp: 154 °C). 1H NMR (CDCl3, 300 MHz): δ 12.70 (s, 1H), 7.63−7.60 (m, 2H), 7.43−7.35 (m, 12H), 7.29−7.26 (m, 3H), 6.95 (d, J = 9.6 Hz, 1H,), 6.49 (d, J = 3.0 Hz, 1H), 6.45 (d, J = 2.1 Hz, 1H), 5.19 (s, 2H), 5.12 (s, 2H), 5.06 (s, 2H). 13 C NMR (CDCl3, 100 MHz): δ 178.9, 164.5, 162.0, 156.8, 147.9, 145.6, 136.5, 135.8, 135.7, 129.0, 128.9, 128.8, 128.7, 128.4, 128.3,127.9, 127.6, 124.0, 121.9, 114.5, 111.6, 106.3, 98.7, 93.1, 74.3, 71.2, 70.5. MS (ESI) m/z: [M + Na] + 595.64. 3,7-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-5-hydroxy-4H-chromen-4-one (3). From the above reaction, the ﬂash column chromatography elusion using 5% EtOAc as an eluent aﬀorded compound 3 as a yellow solid (33%, mp: 130 °C). 1H NMR (CDCl3, 300 MHz): δ 12.70 (s, 1H), 7.71 (d, J = 3.0 Hz, 1H), 7.55 (dd, J = 12.0 Hz, 3.0 Hz, 1H), 7.48−7.33 7292
7.4 buﬀer was prepared using one vial of Hank’s balanced salt (Sigma-H1387). The solution (10 μM) of test compound with HBSS buﬀer was prepared from the stock solution of test compound (10 mM) in DMSO. Revival of Caco-2 cells was performed as per SOP-BIO-IATCL-013-00. Subculturing of Caco-2 cells was performed as per SOPBIO-TCL-013-00. The basal compartment of 96-well multiscreen Caco-2 plate was ﬁlled with 250 μL of DMEM and seeded with 12,000 cells/well (0.16 × 106 cells/mL) in all the apical wells required and one well with only media as a blank without cells, and the Caco-2 plate was placed in the CO2 incubator at 37 °C for the proliferation of cells. On the day of assay, plate was washed twice with HBSS buﬀer. The medium was incubated with HBSS buﬀer for 30 min in an incubator, and wells with TEER values of >230 ohm cm2 were selected for the incubation. Two hundred ﬁfty microliters of HBSS buﬀer with 2% BSA was added to basal wells, and 75 μL of the test compound was added to apical wells. At 120 min, 25 μL of basal samples was collected and processed as mentioned below. Two hundred ﬁfty microliters of the test compound was added to basal wells, and 75 μL of HBSS buﬀer with 2% BSA was added to apical wells. Twenty-ﬁve microliters of apical samples was collected at 120 min and processed as stated below. The single-point calibration curve in HBSS buﬀer with 2% BSA was used. Donor and receiver samples were diluted with 1:1 HBSS containing 2% BSA and 1.1 mM. HBSS buﬀer, respectively. It was precipitated with 200 μL of acetonitrile containing an internal standard and vortexed for 5 min at 1000 rpm and centrifuged at 4000 rpm for 10 min. Finally, 100 μL of supernatant was diluted with 200 μL of water and submitted for LC−MS/MS analysis. Cell Lines and Chemicals. Human colorectal carcinoma (HCT-116) cell line was purchased from National Centre for Cell Sciences (NCCS), Pune, India. Cell culture components including Dulbecco’s modiﬁed Eagle medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin−neomycin (PSN) antibiotic cocktail, ethylenediaminetetraacetic acid (EDTA), and trypsin were obtained from Gibco (Grand Island, NY, USA). Other raw and ﬁne chemicals were procured from Merck (India), Sisco Research Laboratories (SRL), Mumbai, India. All biochemical assay kits and inhibitors were purchased from Calbiochem (Burlington, Massachusetts, USA). Antibodies were procured from Santa Cruz Biotechnology (Dallas, Texas USA), Abcam (U.K.), and eBioscience (San Diego, USA). Cell Culture. Brieﬂy, human colorectal carcinoma (HCT116) cells were cultured in DMEM containing 10% FBS with 1% antibiotic cocktail in a humidiﬁed condition under constant 5% CO2 at 37 °C. After 70−75% conﬂuence, cell seeding was completed with EDTA (0.52 mM) and trypsin (0.25%) in phosphate-buﬀered saline (PBS) and plated at a required density to allow them to re-equilibrate before the experimentation. Determination of Cell Viability. Determination of cell viability was done by MTT [(4,5- dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide] assay (Figure S1, Supporting Information). Cells were seeded at a required density (4 × 103 cells/well) in a 96-well plate. After 18−24 h of seeding, cells were treated with all the test compounds (1, 5, 6, 7, 11, 12, 16, 17, 20, and 23) with a wide range of concentrations (0−80 μM) followed by an initial screening for 24 h. Quickly after
Compound 21 (0.5 g, 0.68 mmol) was taken in 1.5 mL of DMSO, then K 2 CO 3 (0.093 g, 0.68 mmol) and Nmethylpiperazine (0.135 gm, 1.35 mmol) were added to it, and the reaction was performed at 80 °C for 12 h. Then, workup was done using CHCl3 and water, and then the mixture was subject to column chromatography to obtain pure compound 22 as a white solid (75%, mp: 155 °C). 1H NMR (CDCl3, 6.00 MHz): δ 7.77 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.46−7.22 (m, 20H), 6.95 (d, J = 7.2 Hz, 1H), 6.57 (s, 1H), 6.47 (s, 1H), 5.22 (s, 2H), 5.14 (s, 2H), 5.0 (s, 2H), 4.90 (s, 2H), 4.25−4.24 (m, 2H), 3.39−3.28 (s, 2H), 2.78−2.76 (m, 4H), 2.37−2.32(m, 4H), 2.26 (s, 3H), 1.46−1.39 (m, 2H). 13C NMR (CDCl3, 150 MHz): δ 174.2, 163.3, 160.7, 158.6, 150.9, 148.3, 139.4, 136.4,136.9, 136.7, 135.5, 128.8, 128.6, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 127.6, 127.4, 127.2, 122.4, 115.0, 113.7, 97.7, 73.9, 71.1, 70.8, 70.7, 68.0, 55.3, 54.2, 52.7, 45.2, 39.8, 30.3. HRMS (ESI) m/z: (M + H) calculated for C51H50N2O7, 802.3623; found, 802.3672. 2-(3,4-Dihydroxyphenyl)-3,7-dihydroxy-5-(3-(4-methylpiperazin-1-yl)propoxy)-4H-chromen-4-one (23). Compound 22 (0.1 g, 0.125 mmol) and palladium hydroxide on carbon (10 mol %) were taken in 1:1 THF/EtOH solution, and the reaction was performed according to procedure B to get compound 23 as a gummy solid (73%). 1H NMR (CD3OD, 400 MHz): δ 7.67. (d, J = 2.8 Hz, 1H), 7.58. (dd, J = 8.6, 2.0 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H,), 6.42 (d, J = 2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 4.15−4.11 (m, 2H), 2.89−2.79 (m, 6H), 2.65−2.56 (m, 4H), 2.31 (s, 3H), 2.15−2.12 (m, 2H). 13 C NMR (DMSO-d6 + 1 drop of CDCl3, 150 MHz): δ 176.3, 164.4, 161.1, 156.6, 149.6, 147.0, 136.3, 132.2, 122.4, 116.2, 113.9, 103.4, 98.7, 94.0, 67.2, 54.0, 52.7, 52.5, 36.6, 16.2. HRMS (ESI) m/z: (M + H) calculated for C23H27N2O7, 443.1824; found, 443.1852. HPLC purity (95.31%). Method for Solubility Test at pH 7.4. Stock solution (20 mM) of the respective compounds was prepared in DMSO. Five microliters of 20 mM DMSO stock from the stock plate was added to 200 μL deep-well plate containing 495 μL of pH 7.4 pION buﬀer solution so that the concentration of sample become 200 μM. One hundred microliters of the sample from the storage plate was vacuum-ﬁltered using a ﬁlter plate after 18 h of incubation. This step wets the ﬁlters, and the ﬁltrate was discarded. Another 200 μL of the sample from deep-well plate was vacuum-ﬁltered into a new ﬁlter collection plate, and 75 μL of the ﬁltrate was transferred to a UV sample plate. Seventy-ﬁve microliters of acetonitrile was added to this UV plate. The solution was mixed, and the spectrum was read using a UV spectrophotometer at 240 nm. Ketoconazole, metoprolol, and propranolol are used as controls. Method for Log D Calculation at pH 7.4. Stock solution (10 mM) of the respective compounds was prepared in DMSO. In 2 mL deep-well plate, 500 μL of organic phase (1octanol) and 500 μL of phosphate buﬀer of pH 7.4 were taken, and 15 μL of the test substance was added. The plate was vortexed for 1 h on a plate shaker at 1200 rpm. After incubation, the samples were allowed to equilibrate for 20 min and then centrifuged at 4000 rpm for 30 min for complete phase separation and analyzed by LC−UV. Ketoconazole, metoprolol, and propranolol are used as experimental standards. Caco-2 Permeability Assay.32 Heat-inactivated fetal bovine serum of 385 mL of DMEM is mixed with 5 mL of 100 mM sodium pyruvate, 5 mL of 100× nonessential amino acids and 5 mL of pen−strep. One thousand milliliters of pH 7295
washing and incubation with respective ﬂuorophore-conjugated secondary antibodies (antimouse/rabbit FITC and PE) for 2 h. The slides were then counterstained with 6-diamidino2-phenylindole (DAPI) for 10 min and mounted with the ProLong antifade reagent (Molecular Probe, Eugene, OR, USA). Stained cells were examined using a confocal laser scanning microscope (FV 10i, Olympus, Japan). Measurement of Caspase-3 and Caspase-9 Activities. Caspase-3/9 activities were measured according to the manufacturer’s directions with commercially existing caspase3 and caspase-9 colorimetric assay kits (BioVision Research Products, Mountain View, CA). Absorbance was measured at 405 nm by an ELISA reader. Animals. Adult male BALB/c mice ranging from 8 to 10 weeks old, with initial body weights of 20−22 g were procured from the inbred institutional facility. The animals were kept under standard laboratory conditions of 21 ± 2 °C, relative humidity of 55%, and 12 h:12 h light/dark cycle maintained during the study. The animals were given standard rat pellets and tap water ad libitum. All experimental protocols were performed by the guideline of the Institutional Animal Ethics Committee, CSIR-Indian Institute of Chemical Biology and approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment, Forests and Climate Change, Government of India. In Vivo Experimentation. The schematic representation of the experimental design is included in Figure S2 (Supporting Information). On the ﬁrst day, the animals were shaved on the back ﬂank. In the shaved right ﬂank of each mouse, CT-26 (2 × 106) in PBS was injected subcutaneously. Eight days after tumor implantation, the animals were randomly divided into four groups as follows (ﬁve mice in each group): group I, served as a control and mice in this group were kept at a standard ambient temperature of 24 ± 2 °C and 60−70% relative humidity; group II, CT-26 tumorbearing mice treated with normal saline; groups III and IV, CT-26 tumor-bearing mice treated with com-17 (25 and 50 mg/kg) intraperitoneally for six alternate days. The resultant tumor length and weight were measured after the completion of the experimentation. In addition, the animal survival rate was evaluated up to 30 days. Statistical Analysis. All the values in the ﬁgures are expressed as mean values with their standard errors (mean ± SEM) of n observations, where n (n = 5) represents the number of animals studied. In the experiments involving histology and immunohistochemistry, the ﬁgures shown are representative of at least three sections of the tissues collected from all the animals in each group. The information obtained from each group was statistically processed according to the most suitable technique for each case. A statistical software, OriginPro version 8.0, was used for the data analysis, followed by the evaluation of statistical signiﬁcance using a one-way ANOVA through the Tukey method with the post hoc test. The critical signiﬁcance level was set at p < 0.05.
treatment, plates were placed in an incubator for 24 h at 37 °C in a humidiﬁed CO2-rich condition (5%). After 24 h of treatment, cells were washed with PBS; MTT solution (5 mg/ mL) was added in each well and kept in an incubator for 3−4 h to form a formazan salt, which was then solubilized by DMSO. The absorbance of the DMSO-solubilized intracellular formazan salt was recorded at 595 nm by an ELISA reader (Emax, Molecular Device, USA). The same protocol was followed with a narrow concentration range of 0−10 μM for compounds 5, 6, 11, 12, 16, 17, 20, and 23 for calculating the actual IC50. Measurement of Intracellular ROS (iROS). The level of intracellular reactive oxygen species (iROS) was measured using the oxidation sensor dye 2′,7′-dichloroﬂuorescein diacetate (H2DCFDA), wherein an increase in green ﬂuorescence intensity is used to quantify the generation of intracellular ROS in respect to the untreated control. The harvested renal cells (2 × 106) were resuspended in a complete medium containing H2DCFDA and incubated at 37 °C for 30 min, following the acquisition by a BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA) ﬂow cytometer using an argon laser at 488 nm. Quantiﬁcation of Apoptosis/Necrosis Using Flow Cytometry. Brieﬂy, treated cells were washed and stained with PE and Annexin V-FITC in accordance with the manufacturer’s instructions (Calbiochem, Merck Millipore, Burlington, Massachusetts, USA). The percentages of live, apoptotic (early and late), and necrotic cells were quantiﬁed using a ﬂow cytometer (BD LSRFortessa, San Jose, CA, USA). The acquired data were analyzed using the FlowJo (version 10.0) software. Determination of Mitochondrial Membrane Potential. Concisely, the treated cells were incubated with JC-1 according to the manufacturer’s instruction (BD Biosciences, San Jose, CA, USA). The data were analyzed using a ﬂow cytometer (BD LSRFortessa, San Jose, CA, USA). The change in ﬂuorescence from green (525 nm) to red (590 nm) allows to determine the percentage of depolarized and hyperpolarized mitochondria. Assessment of Protein Expression by Flow Cytometry. Treated cells (HCT-116 cells, isolated tumor, and colonic cells) were ﬁxed in paraformaldehyde (4%) in PBS (pH 7.4) for 20 min at room temperature followed by permeabilization (0.1% Triton X-100 in PBS) for 5 min with 0.1% FBS for 5 min. Then, the permeabilized cells were washed twice using PBS with FBS (3%) and incubated with respective primary antibodies (p-ATM, p-MDM2, p-p53, p21, Bax, Bcl2, p-PI3K, p-AKT, mTOR, and Ki67) overnight. After removing the unbounded primary antibody, cells were further incubated with a particular ﬂuorophore-tagged secondary antibody (anti-rabbit/mice/goat AF488, FITC, PE, and AF647) for 2 h on ice, and the stained cells were subjected to ﬂow cytometric analysis using a BD LSRFortessa ﬂow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with the FlowJo software. The mean ﬂuorescence intensities (MFI) were measured by the FlowJo (version 10.0) software. Immunoﬂuorescence. Brieﬂy, control/treated HCT-116 cells were washed twice for 10 min each in PBS (0.01 M) and incubated for 1 h in blocking solution containing 2% normal bovine serum and 0.3% Triton X-100 in PBS. After blocking, the cells were incubated overnight at 4 °C with the respective primary antibody (p-AKT, p-mTOR, and p-p65), followed by
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00143. 7296
(10) Kumari, A.; Yadav, S. K.; Pakade, Y. B.; Singh, B.; Yadav, S. C. Development of biodegradable nanoparticles for delivery of quercetin. Colloids Surf., B 2010, 80, 184−192. (11) Nam, J.-S.; Sharma, A.; Nguyen, L.; Chakraborty, C.; Sharma, G.; Lee, S.-S. Application of bioactive quercetin in oncotherapy: from nutrition to nanomedicine. Molecules 2016, 21, 108. (12) Zhao, L.; Shi, Y.; Zou, S.; Sun, M.; Li, L.; Zhai, G. Formulation and In Vitro evaluation of quercetin loaded Polymeric micelles composed of pluronic P123 and D-a-tocopheryl polyethylene glycol Succinate. J. Biomed. Nanotechnol. 2011, 7, 358−365. (13) Xu, G.; Shi, H.; Ren, L.; Gou, H.; Gong, D.; Gao, X.; Huang, N. Enhancing the anti-colon cancer activity of quercetin by selfassembled micelles. Int. J. Nanomed. 2015, 10, 2051−2063. (14) Khan, I.; Paul, S.; Jakhar, R.; Bhardwaj, M.; Han, J.; Kang, S. C. Novel quercetin derivative TEF induces ER stress and mitochondriamediated apoptosis in human colon cancer HCT-116 Cells. Biomed. Pharmacother. 2016, 84, 789−799. (15) Massi, A.; Bortolini, O.; Ragno, D.; Bernardi, T.; Sacchetti, G.; Tacchini, M.; De Risi, C. Research progress in the modification of quercetin leading to anticancer agents. Molecules 2017, 22, 1270. (16) Veverka, M.; Gallovič, J.; Š vajdlenka, E.; Veverková, E.; Prónayová, N.; Milácǩ ová, I.; Śtefek, M. Novel quercetin derivatives: synthesis and screening for anti-oxidant activity and aldose reductase inhibition. Chem. Pap. 2013, 67, 76−83. (17) Shi, Z.-H.; Li, N.-G.; Tang, Y.-P.; Shi, Q.-P.; Zhang, W.; Zhang, P.-X.; Dong, Z.-X.; Li, W.; Zhang, X.; Fu, H.-A.; et al. Synthesis, biological evaluation and SAR analysis of O-alkylated analogs of quercetin for anticancer. Bioorg. Med. Chem. Lett. 2014, 24, 4424− 4427. (18) Hashemzaei, M.; Far, A. D.; Yari, A.; Heravi, R. E.; Tabrizian, K.; Taghdisi, S. M.; Sadegh, S. E.; Tsarouhas, K.; Kouretas, D.; Tzanakakis, G.; et al. Anticancer and apoptosis-inducing effects of quercetin in vitro and in vivo. Oncol. Rep. 2017, 38, 819−828. (19) Castle, J. C.; Loewer, M.; Boegel, S.; de Graaf, J.; Bender, C.; Tadmor, A. D.; Boisguerin, V.; Bukur, T.; Sorn, P.; Paret, C.; et al. Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma. BMC Genomics 2014, 15, 190. (20) Medhi, B.; Patyar, S.; Rao, R. S.; Byrav Ds, P.; Prakash, A. Pharmacokinetic and toxicological profile of artemisinin compounds: an update. Pharmacology 2009, 84, 323−332. (21) Findlay, J. W. A. Pholcodine. J. Clin. Pharm. Ther. 1988, 13, 5− 17. (22) Bouktaib, M.; Lebrun, S.; Atmani, A.; Rolando, C. Hemisynthesis of all the O-monomethylated analogues of quercetin including the major metabolites, through Selective protection of phenolic functions. Tetrahedron 2002, 58, 10001−10009. (23) Wang, R. E.; Kao, J. L.-F.; Hilliard, C. A.; Pandita, R. K.; Roti Roti, J. L.; Hunt, C. R.; Taylor, J.-S. Inhibition of heat shock induction of heat shock protein 70 and enhancement of heat shock protein 27 phosphorylation by quercetin derivatives. J. Med. Chem. 2009, 52, 1912−1921. (24) Schweigert, N.; Zehnder, A. J. B.; Eggen, R. I. L. Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals. Environ. Microbiol. 2001, 3, 81−91. (25) Chen, T.-J.; Jeng, J.-Y.; Lin, C.-W.; Wu, C.-Y.; Chen, Y.-C. Quercetin inhibition of ROS-dependent and -independent apoptosis in rat Glioma C6 Cells. Toxicology 2006, 223, 113−126. (26) Cárdenas, M.; Marder, M.; Blank, V. C.; Roguin, L. P. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorg. Med. Chem. 2006, 14, 2966−2971. (27) Kim, G. T.; Lee, S. H.; Kim, J. I.; Kim, Y. M. Quercetin regulates the sestrin 2-AMPK-P38 MAPK signaling pathway and induces apoptosis by increasing the generation of intracellular ROS in a P53-independent manner. Int. J. Mol. Med. 2014, 33, 863−869. (28) Tait, S. W. G.; Green, D. R. Mitochondrial regulation of cell death. Cold Spring Harbor Perspect. Biol. 2013, 5, a008706.
X-ray crystallographic and structural reﬁnement parameters of compound 13, 1H NMR and 13C NMR spectra for synthesized compounds, MTT assay in PBMC and HCT-116 cell line, schematic representation of the in vivo experimental design, and photo of the harvest tumor (PDF)
A.M., S.M., and N.K.K. contributed equally to this work. A.T. conceptualized the study. A.T. and K.D.S. provided the methodology. A.M., N.K.K., S.R., B.K. and D.B. conducted the synthesis and crystallography. S.M. and K.M. carried out the biological investigation. K.M. took the photo in Figure S3. A.T. wrote, reviewed, and edited the manuscript. A.T. and K.D.S supervised the study.
The authors declare no competing ﬁnancial interest.
ACKNOWLEDGMENTS A.M., S.R., B.K. would like to acknowledge UGC for fellowship. S.M. wishes to thank UGC-DAE for a project fellowship. K.N.K. would like to acknowledge NIPER Kolkata. We thank Dr Ramalingam Natarajan, Senior Scientist CSIRIndian Institute of Chemical Biology for his help to solve the crystal structure. The authors gratefully acknowledge Central Instrumentation Facility (CIF), IICB for providing ﬂow cytometer, confocal microscope facilities, X-ray and Spectroscopy.
(1) Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 2016, 79, 629−661. (2) Erlund, I. Review of the flavonoids quercetin, hesperetin, and naringenin. dietary sources, bioactivities, bioavailability, and epidemiology. Nutr. Res. 2004, 24, 851−874. (3) Kuipers, E. J.; Grady, W. M.; Lieberman, D.; Seufferlein, T.; Sung, J. J.; Boelens, P. G.; van de Velde, C. J. H.; Watanabe, T. Colorectal cancer. Nat. Rev. Dis. Prim. 2015, 1, 15065. (4) Materska, M. Quercetin and its derivatives: chemical structure and bioactivity - a review. Polish J. Food Nutr. Sci. 2008, 58, 407−413. (5) Anand David, A. V.; Arulmoli, R.; Parasuraman, S. Overviews of biological importance of quercetin: a bioactive flavonoid. Pharmacogn. Rev. 2016, 10, 84−89. (6) Catanzaro, D.; Ragazzi, E.; Vianello, C.; Caparrotta, L.; Montopoli, M. Effect of quercetin on cell cycle and cyclin expression in ovarian carcinoma and osteosarcoma cell Lines. Nat. Prod. Commun. 2015, 10, 1365−1368. (7) Cai, X.; Fang, Z.; Dou, J.; Yu, A.; Zhai, G. Bioavailability of quercetin: problems and promises. Curr. Med. Chem. 2013, 20, 2572− 2582. (8) Shi, Z.-H.; Li, N.-G.; Tang, Y.-P.; Shi, Q.-P.; Tang, H.; Li, W.; Zhang, X.; Fu, H.-A.; Duan, J.-A. Biological evaluation and SAR analysis of O-methylated analogs of quercetin as inhibitors of cancer cell proliferation. Drug Dev. Res. 2014, 75, 455−462. (9) Rezaei-Sadabady, R.; Eidi, A.; Zarghami, N.; Barzegar, A. Intracellular ROS protection efficiency and free radical-scavenging activity of quercetin and quercetin-encapsulated liposomes. Artif. Cells, Nanomed., Biotechnol. 2016, 44, 128−134. 7297
(29) Zhang, J. Y.; Yi, T.; Liu, J.; Zhao, Z.-Z.; Chen, H.-B. Quercetin induces apoptosis via the mitochondrial pathway in KB and KBv200 cells. J. Agric. Food Chem. 2013, 61, 2188−2195. (30) Khan, K. H.; Yap, T. A.; Yan, L.; Cunningham, D. Targeting the PI3K-AKT-MTOR signaling network in Cancer. Chin. J. Cancer 2013, 32, 253−265. (31) Yang, L.; Liu, Y.; Wang, M.; Qian, Y.; Dong, X.; Gu, H.; Wang, H.; Guo, S.; Hisamitsu, T. Quercetin-induced apoptosis of HT-29 colon cancer cells via inhibition of the Akt-CSN6-Myc signaling Axis. Mol. Med. Rep. 2016, 14, 4559−4566. (32) Kundu, B.; Das, S. K.; Paul Chowdhuri, S.; Pal, S.; Sarkar, D.; Ghosh, A.; Mukherjee, A.; Bhattacharya, D.; Das, B. B.; Talukdar, A. Discovery and mechanistic study of tailor-made quinoline derivatives as topoisomerase 1 poison with potent anticancer activity. J. Med. Chem. 2019, 62, 3428−3446.