Synthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

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Synthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis Kang-Yeoun Jung a,d, Jihyun Park b,d, Young-Sung Han a, Young Han Lee c, Soon Young Shin c,⇑, Yoongho Lim b,⇑ a b c

Department of Biochemical Engineering, Gangneung-Wonju National University, Gangwondo 210-702, Republic of Korea Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 143-701, Republic of Korea Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 20 September 2016 Revised 31 October 2016 Accepted 2 November 2016 Available online xxxx Keywords: Hesperetin Hesperetin-7-butyrate Apoptosis JNKs

a b s t r a c t A flavanone, hesperetin, has been known to exert antitumor activity by inducing apoptosis. To find hesperetin derivatives showing better antitumor activity, 12 derivatives were designed and synthesized. Their antitumor activities were measured using a long-term survival clonogenic assay. Among the compounds, K-5b, hesperetin-7-butyrate, showed the half-maximal cell growth inhibitory concentration three times as low as that of hesperetin. To compare the cytotoxicity of hesperetin and K-5b, the HCT116 human colon cancer cell line was treated with various concentrations of each compound. K-5b decreased the cell viability to a larger extent than hesperetin and triggered apoptosis more efficiently than hesperetin in an apoptosis detection assay using fluorescein isothiocyanate-labeled annexin V. Immunoblotting analysis showed that K-5b promoted caspase-mediated apoptosis more efficiently than hesperetin. Because hesperetin has been reported to induce apoptosis through the activation of the c-Jun N-terminal kinase (JNK) pathway, we tested whether K-5b activates JNKs. K-5b stimulated JNK1 and JNK2 more efficiently than hesperetin as shown by western blot analysis. In conclusion, hesperetin derivatives exerting better antitumor activity than hesperetin by inducing apoptosis were found. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Flavonoids are generally stored in plants as glycosides because they are more stable than their aglycones. However, glycosides are hydrolyzed to aglycones to be used because of their poor bioavailability.1 Flavonoid aglycones, including hesperetin, are more potent in their antiperoxidative action, such as nonenzymatic lipid peroxidation, than their corresponding glycosides.2 Hesperetin-7O-rutinoside, hesperidin, widely found in citrus fruits, including oranges, lemons, limes, and grapefruits, is metabolized to an aglycone, hesperetin, and a disaccharide, rutinose, by hesperidin6-O-a-l-rhamnosyl-b-d-glucosidase.3 Hesperetin, 30 ,5,7-trihydroxy-40 -methoxyflavanone, reduces the intracellular replication Abbreviation: GI50, half-maximal cell growth inhibitory concentration.

⇑ Corresponding authors at: Department of Biological Sciences, Konkuk University, Hwayang-Dong 1, Kwangjin-Ku, Seoul 143-701, Korea, (S.Y. Shin). Division of Bioscience and Biotechnology, Konkuk University, Hwayang-Dong 1, Kwangjin-Ku, Seoul 143-701, Republic of Korea (Y. Lim). E-mail addresses: [email protected] (S.Y. Shin), [email protected] (Y. Lim). d These authors contributed equally to this work.

of viruses, including herpes simplex virus type 1, poliovirus type 1, parainfluenza virus type 3, and respiratory syncytial virus,4 and stimulates trypsin-activated phosphorylase kinase.5 Besides, hesperetin inhibits receptor-mediated endocytosis of beta-hexosaminidase6 and myeloperoxidase release.7 Hesperetin itself has been reported to exert antitumor activity by inducing apoptosis in many cancer cells in vitro and in vivo.8 Because hesperetin belongs to polyphenols, various functional groups can be substituted for its phenyl groups. However, its derivatives have rarely been reported, but it has been shown that 7,30 -dimethoxy hesperetin induces apoptosis.9 In particular, the 5-hydroxy group forms a hydrogen bond with the ketone of the chroman-4-one moiety.10 Therefore, we tried to prepare compounds derivatized at 40 - and 7-positions. There are many methods to screen for antitumor activity, among which clonogenic assays are long-term cell survival assays. While the assays require a long experimental time, such as seven days, they can discriminate between cancer cells with similar proliferation rates. Because the compounds synthesized in this study contain hesperetin as a common moiety, a clonogenic assay can give reliable results on cell proliferation. Colon cancer is the third most common type of cancer in the world.

http://dx.doi.org/10.1016/j.bmc.2016.11.006 0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

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Hesperetin is known to have potent chemo-preventive and antitumor properties against intestinal carcinoma, probable due to direct exposure to transformed intestinal epithelium with much higher efficiency than other tissues.11 As tumor suppressor p53 plays an important role in hesperetin-induced apoptosis,12 we used HCT116 human colon carcinoma cells expressing wild-type p53. Half-maximal cell growth inhibitory concentration (GI50) values for the compounds were obtained from the clonogenic assay, and the relationships between cytotoxicities of the derivatives and their structures were elucidated. For the derivative showing the best GI50 value, K-5b, further biological experiments were performed to confirm that it induces apoptosis. To understand the mechanism by which K-5b triggers apoptosis, we tested whether K-5b activates c-Jun N-terminal kinases (JNKs) because hesperetin has been reported to induce apoptosis through the activation of the JNK pathway.13 The molecular binding modes among hesperetin, K-5b, JNK1, and JNK2 were elucidated using in silico docking. The goal of this study was to find hesperetin derivatives showing better biological activity than hesperetin itself. The title compound showed cytotoxicity three times as high as that of hesperetin. Analysis of the structures of the derivatives and their cytotoxicities may help us design a novel chemotherapeutic agent. 2. Materials and methods 2.1. General methods To determine the structures of the hesperetin derivatives synthesized in this study, nuclear magnetic resonance (NMR) experiments were performed on a Bruker Avance 400 NMR spectrometer (9.4 T; Karlsruhe, Germany). All derivatives were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and transferred into 2.5-mm NMR tubes. The concentrations of the samples were adjusted to approximately 100 mM. One-dimensional experiments, including 1H NMR, 13C NMR, and distortionless enhancement by polarization transfer, and two-dimensional experiments, including correlated spectroscopy, total correlated spectroscopy (TOCSY), nuclear Overhauser exchange spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bonded connectivities (HMBC), were carried out as described previously.14 To confirm the results obtained in the NMR experiments, high resolution mass spectrometry (HR/MS) was performed on an ultraperformance liquid chromatography–hybrid quadrupole-time-of-flight mass spectrometer (Waters Corp., Milford, MA, USA) with the help of Prof. Choong Hwan Lee at Konkuk University, Korea.15 2.2. Preparation of hesperetin derivatives Twelve hesperetin derivatives were synthesized containing substituents at the 30 - or/and 7-position (Table 1). Commercially available hesperetin (INDOFINE Chemical Company, Hillsborough Township, NJ, USA) was used with corresponding acid chlorides and triethylamine to synthesize the 12 derivatives. All of these 7- and 30 -O-acylated hesperetin derivatives, K-1a to Y-4a, were prepared by the following general procedure. A flame-dried round-bottom flask was charged under argon with hesperetin (1 equiv.) and acetone. To this solution, freshly distilled triethylamine (2 equiv.) was then added under argon. After the solution was stirred for 10 min at room temperature, the corresponding acid chloride (1.2–2.4 equiv.) was added quickly. The reaction mixture was stirred for 10 min at the same temperature and quenched with distilled water. This aqueous mixture was extracted with CH2Cl2, and combined organic extracts were dried over anhydrous MgSO4. After removal of the solvent, the crude product was purified by

flash column chromatography to get the desired products. The synthetic process is summarized in Scheme 1. All derivatives except K1b are novel. The spectral data of the 12 hesperetin derivatives obtained from the NMR and HR/MS experiments are summarized as follows. 2.2.1. Hesperetin-30 ,7-dibenzoate (K-1a) 1 H NMR (400 MHz, DMSO-d6) d: 11.96 (s, 1H, 5-OH), 8.13 (dd, 2H, H-200 , H-600 , J = 1.8, 7.8 Hz), 8.10 (dd, 2H, H-2000 , H-6000 , J = 1.8, 7.8 Hz), 7.76 (ddd, 2H, H-400 , H-4000 , J = 1.8, 7.8, 7.8 Hz), 7.61 (dd, 2H, H-3000 , H-5000 , J = 7.8, 7.8 Hz), 7.60 (dd, 2H, H-300 , H-500 , J = 7.8, 7.8 Hz), 7.479 (dd, 1H, H-60 , J = 2.3, 9.1 Hz), 7.475 (d, 1H, H-20 , J = 2.3 Hz), 7.25 (d, 1H, H-50 , J = 9.1 Hz), 6.57 (d, 1H, H-8, J = 2.1 Hz), 6.53 (d, 1H, H-6, J = 2.1 Hz), 5.71 (dd, 1H, H-2, J = 2.9, 12.9 Hz), 3.79 (s, 3H, 40 -OCH3), 3.49 (dd, 1H, H-3, J = 12.9, 17.2 Hz), 2.92 (dd, 1H, H-3, J = 2.9, 17.2 Hz). 13C NMR (100 MHz, DMSO-d6) d: 197.9 (C-4), 164.0 (C-30 a), 163.5 (C-7a), 162.3 (C-5), 162.1 (C-9), 158.1 (C-7), 151.2 (C-40 ), 139.3 (C-30 ), 134.3 (C-4000 ), 134.1 (C-400 ), 130.7 (C-10 ), 129.8 (C-200 , C-2000 , C-600 , C-6000 ), 129.0 (C300 , C-3000 , C-500 , C-5000 ), 128.6 (C-100 ), 128.5 (C-1000 ), 125.8 (C-60 ), 121.7 (C-20 ), 112.9 (C-50 ), 106.0 (C-10), 103.0 (C-6), 101.9 (C-8), 78.1 (C-2), 56.0 (40 -OCH3), 42.2 (C-3). HRMS (m/z): calcd. for (M +H)+: 511.1393; found: 511.1382. 2.2.2. Hesperetin-7-benzoate (K-1b) 1 H NMR (400 MHz, DMSO-d6) d: 11.98 (s, 1H, 5-OH), 9.14 (s, 1H, 0 3 -OH), 8.10 (dd, 2H, H-2000 , H-6000 , J = 1.4, 7.4 Hz), 7.75 (ddd, 1H, H4000 , J = 1.4, 7.4, 7.4 Hz), 7.60 (dd, 2H, H-3000 , H-5000 , J = 7.4, 7.4 Hz), 6.96 (d, 1H, H-20 , J = 1.9 Hz), 6.95 (d, 1H, H-50 , J = 8.3 Hz), 6.91 (dd, 1H, H-60 , J = 1.9, 8.3 Hz), 6.53 (d, 1H, H-8, J = 2.1 Hz), 6.52 (d, 1H, H-6, J = 2.1 Hz), 5.59 (dd, 1H, H-2, J = 3.0, 12.6 Hz), 3.78 (s, 3H, 40 -OCH3), 3.39 (dd, 1H, H-3, J = 12.6, 17.2 Hz), 2.85 (dd, 1H, H-3, J = 3.0, 17.2 Hz). 13C NMR (100 MHz, DMSO-d6) d: 198.1 (C4), 163.7 (C-7a), 162.3 (C-5), 162.2 (C-9), 158.1 (C-7), 148.1 (C40 ), 146.5 (C-30 ), 134.3 (C-4000 ), 130.7 (C-10 ), 130.0 (C-2000 , C-6000 ), 129.0 (C-3000 , C-5000 ), 128.5 (C-1000 ), 117.9 (C-60 ), 114.2 (C-20 ), 112.0 (C-50 ), 106.0 (C-10), 102.8 (C-6), 101.9 (C-8), 78.7 (C-2), 55.7 (40 OCH3), 42.4 (C-3). HRMS (m/z): calcd. for (M+H)+: 407.1131; found: 407.1136. 2.2.3. Hesperetin-30 ,7-di-(4-methoxybenzoate) (K-2) 1 H NMR (400 MHz, DMSO-d6) d: 11.96 (s, 1H, 5-OH), 8.07 (d, 2H, H-200 , H-600 , J = 9.0 Hz), 8.04 (d, 2H, H-2000 , H-6000 , J = 9.0 Hz), 7.45 (dd, 1H, H-60 , J = 2.1, 8.4 Hz), 7.43 (d, 1H, H-20 , J = 2.1 Hz), 7.22 (d, 1H, H50 , J = 8.4 Hz), 7.11 (d, 2H, H-300 , H-500 , J = 9.0 Hz), 7.10 (d, 2H, H-3000 , H-5000 , J = 9.0 Hz), 6.53 (d, 1H, H-8, J = 2.1 Hz), 6.49 (d, 1H, H-6, J = 2.1 Hz), 5.69 (dd, 1H, H-2, J = 2.6, 13.0 Hz), 3.87 (s, 3H, 400 OCH3), 3.86 (s, 3H, 4000 -OCH3), 3.78 (s, 3H, 40 -OCH3), 3.48 (dd, 1H, H-3, J = 13.0, 17.1 Hz), 2.89 (dd, 1H, H-3, J = 2.6, 17.1 Hz). 13C NMR (100 MHz, DMSO-d6) d: 197.9 (C-4), 164.0 (C-7a), 163.8 (C4000 ), 163.7 (C-30 a), 163.3 (C-400 ), 162.3 (C-5), 162.1 (C-9), 158.3 (C-7), 151.4 (C-40 ), 139.4 (C-30 ), 132.3 (C-2000 , C-6000 ), 132.1 (C-200 , C-600 ), 130.7 (C-10 ), 125.8 (C-60 ), 121.8 (C-20 ), 120.7 (C-1000 ), 120.5 (C-100 ), 114.4 (C-300 , C-500 , C-3000 , C-5000 ), 112.8 (C-50 ), 105.9 (C-10), 103.0 (C-6), 102.0 (C-8), 78.2 (C-2), 56.0 (40 -OCH3), 55.70 (4000 OCH3), 55.66 (400 -OCH3), 42.2 (C-3). HRMS (m/z): calcd. for (M +H)+: 571.1604; found: 571.1616. 2.2.4. Hesperetin-30 ,7-di-(2-phenylacetate) (K-3) 1 H NMR (400 MHz, DMSO-d6) d: 11.93 (s, 1H, 5-OH), 7.40 (dd, 1H, H-60 , J = 2.1, 8.6 Hz), 7.37 (d, 4H, H-200 , H-300 , H-500 , H-600 , J = 7.0 Hz), 7.36 (d, 4H, H-2000 , H-3000 , H-5000 , H-6000 , J = 7.0 Hz), 7.295 (dd, 2H, H-400 , H-4000 , J = 7.0, 7.0 Hz), 7.292 (d, 1H, H-20 , J = 2.1 Hz), 7.17 (d, 1H, H-50 , J = 8.6 Hz), 6.37 (d, 1H, H-8, J = 2.0 Hz), 6.35 (d, 1H, H-6, J = 2.0 Hz), 5.65 (dd, 1H, H-2, J = 2.9, 13.1 Hz), 3.958 (s, 2H, H-30 b), 3.955 (s, 2H, H-7b), 3.77 (s, 3H, 40 -OCH3), 3.44 (dd,

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Table 1 The structures and numbering of the hesperetin derivatives, their mass data, and the half-maximal cell growth inhibitory concentrations (GI50) obtained in the clonogenic long-term survival assay using the HCT116 human colon cancer cell line and compound concentrations of 0, 5, 10, 20, and 40 lM. All HR/MS data were collected as positive molecular ions. OCH3

O R1

O

9

7

2

1'

R2 O

4

5 OH

O

Derivatives

R1

R2

Mass (calcd./found)

Name

GI50 /lM

Hesperetin K-1a

H

H

– 511.1393/511.1382

30 ,5,7-Trihydroxy-40 -methoxyflavanone Hesperetin-30 ,7-dibenzoate

36.1 46.0

H

407.1131/407.1136

Hesperetin-7-benzoate

16.5

H3CO

571.1604/571.1616

Hesperetin-30 ,7-di-(4-methoxybenzoate)

35.7

539.1706/539.1734

Hesperetin-30 ,7-di-(2-phenylacetate)

63.9

371.1131/371.1132

Hesperetin-7-(but-2-enoate)

16.9

443.1706/443.1688

Hesperetin-30 ,7-dibutyrate

13.2

H

373.1287/373.1264

Hesperetin-7-butyrate

12.0

H

387.1444/387.1422

Hesperetin-7-pivalate

31.5

563.1706/563.1724

Hesperetin-30 ,7-dicinnamate

21.0

539.1706/539.1702

Hesperetin-30 ,7-di-(2-methylbenzoate)

48.7

7α

1'''

∗

1''

O

7α

∗

O

K-1b

1'''

3'α

∗

O

K-2

H3CO

7α

1'''

∗

3'α

1''

O

O

K-3

O

1''

∗

O

K-4

7γ

∗ O

3'γ

7α

7β

7δ

H3C

H3C

O

H3C

∗

∗

O

7γ

3'α

7α

7β

7δ

H3C

3'β

3'δ

∗

K-5b

7γ

∗

H

O

K-5a

7γ

3'α

3'β

7α

7β

7δ

H3C

Y-1b

O

7α

7β

1'''

∗

7α ∗

7β

H3C

CH3

Y-2

7β

1'''

7α

3'β

1''

∗

3'α 3'γ

7γ

O

O

Y-3a

1''' CH3

7α O

∗

∗

1'' CH3

3'α

∗

O

(continued on next page)

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K.-Y. Jung et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

Table 1 (continued) Derivatives

R1

Y-3b

1''' CH3

Y-4a

7α

R2

Mass (calcd./found)

Name

GI50 /lM

H

421.1287/421.1264

Hesperetin-7-(2-methylbenzoate)

22.9

H3C

539.1706/539.1712

Hesperetin-30 ,7-di-(4-methylbenzoate)

71.7

∗

O

H3C

1'''

7α O

∗

1''

3'α

∗

O

Scheme 1. The synthetic procedure to prepare the 12 hesperetin derivatives.

1H, H-3, J = 13.1, 17.1 Hz), 2.85 (dd, 1H, H-3, J = 2.9, 17.1 Hz). 13C NMR (100 MHz, DMSO-d6) d: 197.8 (C-4), 169.4 (C-30 a, C-7a), 162.3 (C-5), 162.1 (C-9), 158.0 (C-7), 151.2 (C-40 ), 139.3 (C-30 ), 133.9 (C-100 , C-1000 ), 130.7 (C-10 ), 129.5 (C-2000 , C-6000 ), 129.4 (C-200 , C-600 ), 128.5 (C-4000 ), 128.3 (C-400 ), 127.0 (C-3000 , C-5000 ), 126.6 (C-300 , C-500 ), 125.7 (C-60 ), 121.4 (C-20 ), 112.9 (C-50 ), 105.9 (C-10), 102.7 (C-6), 101.7 (C-8), 78.1 (C-2), 56.0 (40 -OCH3), 42.2 (C-3), 39.9 (C30 b, C-7b). HRMS (m/z): calcd. for (M+H)+: 539.1706; found: 539.1734. 2.2.5. Hesperetin-7-(but-2-enoate) (K-4) 1 H NMR (400 MHz, DMSO-d6) d: 11.94 (s, 1H, 5-OH), 9.14 (s, 1H, 30 -OH), 7.13 (dq, 1H, H-7c, J = 6.9, 15.5 Hz), 6.942 (d, 1H, H-50 , J = 8.3 Hz), 6.941 (d, 1H, H-20 , J = 1.9 Hz), 6.89 (dd, 1H, H-60 , J = 1.9, 8.3 Hz), 6.36 (d, 1H, H-8, J = 2.0 Hz), 6.34 (d, 1H, H-6, J = 2.0 Hz), 6.09 (dd, 1H, H-7b, J = 1.7, 15.5 Hz), 5.55(dd, 1H, H-2, J = 2.8, 12.6 Hz), 3.77 (s, 3H, 40 -OCH3), 3.35 (dd, 1H, H-3, J = 12.6, 17.2 Hz), 2.81 (dd, 1H, H-3, J = 2.8, 17.2 Hz), 1.93 (dd, 3H, H-7d, J = 1.7, 6.9 Hz). 13C NMR (100 MHz, DMSO-d6) d: 198.1 (C-4), 163.2 (C-7a), 162.23 (C-5), 162.19 (C-9), 158.0 (C-7), 148.9 (C7c), 148.1 (C-40 ), 146.5 (C-30 ), 130.7 (C-10 ), 121.1 (C-7b), 117.9 (C-60 ), 114.2 (C-20 ), 112.0 (C-50 ), 105.9 (C-10), 102.7 (C-6), 101.7 (C-8), 78.7 (C-2), 55.7 (40 -OCH3), 42.4 (C-3), 18.0 (C-7d). HRMS (m/z): calcd. for (M+H)+: 371.1131; found: 371.1132. 2.2.6. Hesperetin-30 ,7-dibutyrate (K-5a) 1 H NMR (400 MHz, DMSO-d6) d: 11.92 (s, 1H, 5-OH), 7.39 (dd, 1H, H-60 , J = 2.1, 8.5 Hz), 7.28 (d, 1H, H-20 , J = 2.1 Hz), 7.18 (d, 1H, H-50 , J = 8.5 Hz), 6.36 (d, 1H, H-8, J = 2.1 Hz), 6.34 (d, 1H, H-6, J = 2.1 Hz), 5.65 (dd, 1H, H-2, J = 2.9, 13.1 Hz), 3.78 (s, 3H, 40 OCH3), 3.44 (dd, 1H, H-3, J = 13.1, 17.2 Hz), 2.85 (dd, 1H, H-3, J = 2.9, 17.2 Hz), 2.543 (t, 2H, H-30 b, J = 7.3 Hz), 2.539 (t, 2H, H-7b, J = 7.3 Hz), 1.67 (tq, 2H, H-30 c, J = 7.3, 7.3 Hz), 1.63 (tq, 2H, H-7c, J = 7.3, 7.3 Hz), 0.98 (t, 3H, H-30 d, J = 7.3 Hz), 0.95 (t, 3H, H-7d, J = 7.3 Hz). 13C NMR (100 MHz, DMSO-d6) d: 197.8 (C-4), 171.0 (C-30 a), 170.8 (C-7a), 162.2 (C-5), 162.1 (C-9), 158.1 (C-7), 151.2

(C-40 ), 139.3 (C-30 ), 130.6 (C-10 ), 125.6 (C-60 ), 121.5 (C-20 ), 112.8 (C-50 ), 105.8 (C-10), 102.8 (C-6), 101.8 (C-8), 78.1 (C-2), 56.0 (40 OCH3), 42.2 (C-3), 35.3 (C-7b), 35.0 (C-30 b), 18.0 (C-30 c), 17.7 (C7c), 13.3 (C-30 d, C-7d). HRMS (m/z): calcd. for (M+H)+: 443.1706; found: 443.1688.

2.2.7. Hesperetin-7-butyrate (K-5b) 1 H NMR (400 MHz, DMSO-d6) d: 11.94 (s, 1H, 5-OH), 9.11 (s, 1H, 30 -OH), 6.943 (d, 1H, H-50 , J = 8.3 Hz), 6.940 (d, 1H, H-20 , J = 2.1 Hz), 6.89 (dd, 1H, H-60 , J = 2.1, 8.3 Hz), 6.33 (d, 1H, H-8, J = 2.0 Hz), 6.32 (d, 1H, H-6, J = 2.0 Hz), 5.55 (dd, 1H, H-2, J = 2.9, 12.6 Hz), 3.78 (s, 3H, 40 -OCH3), 3.36 (dd, 1H, H-3, J = 12.6, 17.1 Hz), 2.82 (dd, 1H, H-3, J = 2.9, 17.1 Hz), 2.54 (t, 2H, H-7b, J = 7.3 Hz), 1.63 (tq, 2H, H-7c, J = 7.3, 7.3 Hz), 0.95 (t, 3H, H-7d, J = 7.3 Hz). 13C NMR (100 MHz, DMSO-d6) d: 198.0 (C-4), 170.8 (C-7a), 162.22 (C-5), 162.17 (C-9), 158.0 (C-7), 148.1 (C-40 ), 146.5 (C-30 ), 130.7 (C-10 ), 117.8 (C-60 ), 114.1 (C-20 ), 112.0 (C-50 ), 105.9 (C-10), 102.7 (C-6), 101.7 (C-8), 78.6 (C-2), 55.7 (40 -OCH3), 42.3 (C-3), 35.3 (C-7b), 17.7 (C-7c), 13.3 (C-7d). HRMS (m/z): calcd. for (M+H)+: 373.1287; found: 373.1264.

2.2.8. Hesperetin-7-pivalate (Y-1b) 1 H NMR (400 MHz, DMSO-d6) d: 11.93 (s, 1H, 5-OH), 9.14 (s, 1H, 0 3 -OH), 6.94 (d, 1H, H-50 , J = 8.2 Hz), 6.93 (d, 1H, H-20 , J = 2.1 Hz), 6.88 (dd, 1H, H-60 , J = 2.1, 8.2 Hz), 6.32 (d, 1H, H-8, J = 2.1 Hz), 6.30 (d, 1H, H-6, J = 2.1 Hz), 5.56 (dd, 1H, H-2, J = 2.9, 12.5 Hz), 3.77 (s, 3H, 40 -OCH3), 3.35 (dd, 1H, H-3, J = 12.5, 17.2 Hz), 2.83 (dd, 1H, H-3, J = 2.9, 17.2 Hz), 1.27 (s, 9H, H-7c). 13C NMR (100 MHz, DMSO-d6) d: 198.0 (C-4), 175.6 (C-7a), 162.3 (C-5), 162.2 (C-9), 158.5 (C-7), 148.1 (C-40 ), 146.5 (C-30 ), 130.7 (C-10 ), 117.8 (C-60 ), 114.1 (C-20 ), 112.0 (C-50 ), 105.9 (C-10), 102.6 (C-6), 101.7 (C-8), 78.6 (C-2), 55.7 (40 -OCH3), 42.3 (C-3), 38.7 (C-7b), 26.6 (C-7c). HRMS (m/z): calcd. for (M+H)+: 387.1444; found: 387.1422.

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2.2.9. Hesperetin-30 ,7-dicinnamate (Y-2) 1 H NMR (400 MHz, DMSO-d6) d: 11.96 (s, 1H, 5-OH), 7.86 (d, 1H, H-30 c, J = 16.0 Hz), 7.85 (d, 1H, H-7c, J = 16.0 Hz), 7.81 (dd, 2H, H2000 , H-6000 , J = 1.8, 7.2 Hz), 7.79 (dd, 2H, H-200 , H-600 , J = 1.8, 7.2 Hz), 7.46 (dd, 4H, H-300 , H-3000 , H-500 , H-5000 , J = 7.2, 7.2 Hz), 7.452 (ddd, 2H, H-400 , H-4000 , J = 1.8, 7.2, 7.2 Hz), 7.445 (dd, 1H, H-60 , J = 2.1, 8.6 Hz), 7.39 (d, 1H, H-20 , J = 2.1 Hz), 7.22 (d, 1H, H-50 , J = 8.6 Hz), 6.89 (d, 1H, H-7b, J = 16.0 Hz), 6.85 (d, 1H, H-30 b, J = 16.0 Hz), 6.48 (d, 1H, H-8, J = 2.0 Hz), 6.45 (d, 1H, H-6, J = 2.0 Hz), 5.69 (dd, 1H, H-2, J = 2.7, 13.0 Hz), 3.80 (s, 3H, 40 -OCH3), 3.48 (dd, 1H, H-3, J = 13.0, 17.2 Hz), 2.89 (dd, 1H, H-3, J = 2.7, 17.2 Hz). 13C NMR (100 MHz, DMSO-d6) d: 197.9 (C-4), 164.3 (C-7a), 164.0 (C-30 a), 162.3 (C-5), 162.1 (C-9), 158.1 (C-7), 151.4 (C-40 ), 147.2 (C-30 c), 146.7 (C-7c), 139.2 (C-30 ), 133.8 (C-1000 ), 133.7 (C-100 ), 131.1 (C400 ), 131.0 (C-4000 ), 130.7 (C-10 ), 128.8 (C-300 , C-3000 , C-500 , C-5000 ), 128.7 (C-200 , C-2000 , C-600 , C-6000 ), 125.8 (C-60 ), 121.7 (C-20 ), 116.8 (C30 b), 116.7 (C-7b), 112.8 (C-50 ), 105.9 (C-10), 102.9 (C-6), 101.8 (C-8), 78.2 (C-2), 56.0 (40 -OCH3), 42.2 (C-3). HRMS (m/z): calcd. for (M+H)+: 563.1706; found: 563.1724. 2.2.10. Hesperetin-30 ,7-di-(2-methylbenzoate) (Y-3a) 1 H NMR (400 MHz, DMSO-d6) d: 11.98 (s, 1H, 5-OH), 8.05 (dd, 1H, H-6000 , J = 1.9, 7.5 Hz), 8.03 (dd, 1H, H-600 , J = 1.9, 7.5 Hz), 7.57 (ddd, 2H, H-400 , H-4000 , J = 1.9, 7.5, 7.5 Hz), 7.406 (d, 1H, H-300 , J = 7.5 Hz), 7.397 (dd, 1H, H-500 , J = 7.5, 7.5 Hz), 7.396 (d, 1H, H3000 , J = 7.5 Hz), 7.387 (dd, 1H, H-5000 , J = 7.5, 7.5 Hz), 7.48 (d, 1H, H20 , J = 2.0 Hz), 7.47 (dd, 1H, H-60 , J = 2.0, 8.9 Hz), 7.25 (d, 1H, H-50 , J = 8.9 Hz), 6.58 (d, 1H, H-8, J = 2.0 Hz), 6.54 (d, 1H, H-6, J = 2.0 Hz), 5.71 (dd, 1H, H-2, J = 2.6, 12.9 Hz), 3.82 (s, 3H, 40 OCH3), 3.50 (dd, 1H, H-3, J = 12.9, 17.2 Hz), 2.91 (dd, 1H, H-3, J = 2.6, 17.2 Hz), 2.58 (s, 3H, 2000 -CH3), 2.57 (s, 3H, 200 -CH3). 13C NMR (100 MHz, DMSO-d6) d: 197.9 (C-4), 164.8 (C-7a), 164.3 (C30 a), 162.3 (C-5), 162.1 (C-9), 158.2 (C-7), 151.3 (C-40 ), 140.4 (C200 ), 139.4 (C-2000 ), 139.3 (C-30 ), 133.2 (C-400 ), 133.0 (C-4000 ), 131.91 (C-3000 ), 131.86 (C-300 ), 130.91 (C-10 ), 130.74 (C-600 ), 130.68 (C-6000 ), 128.3 (C-1000 ), 127.8 (C-100 ), 126.3 (C-500 , C-5000 ), 125.8 (C-60 ), 121.8 (C-20 ), 112.9 (C-50 ), 106.0 (C-10), 103.1 (C-6), 102.1 (C-8), 78.2 (C2), 56.1 (40 -OCH3), 42.2 (C-3), 21.2 (200 -CH3), 21.1 (2000 -CH3). HRMS (m/z): calcd. for (M+H)+: 539.1706; found: 539.1702. 2.2.11. Hesperetin-7-(2-methylbenzoate) (Y-3b) 1 H NMR (400 MHz, DMSO-d6) d: 11.98 (s, 1H, 5-OH), 9.15 (s, 1H, 0 3 -OH), 8.04 (dd, 1H, H-6000 , J = 1.6, 7.5 Hz), 7.57 (ddd, 1H, H-4000 , J = 1.6, 7.5, 7.5 Hz), 7.40 (d, 1H, H-3000 , J = 7.5 Hz), 7.39 (dd, 1H, H5000 , J = 7.5, 7.5 Hz), 6.96 (d, 1H, H-20 , J = 1.7 Hz), 6.95 (d, 1H, H-50 , J = 8.2 Hz), 6.91 (dd, 1H, H-60 , J = 1.7, 8.2 Hz), 6.52 (d, 1H, H-8, J = 2.1 Hz), 6.51 (d, 1H, H-6, J = 2.1 Hz), 5.58 (dd, 1H, H-2, J = 2.7, 12.5 Hz), 3.78 (s, 3H, 40 -OCH3), 3.38 (dd, 1H, H-3, J = 12.5, 17.2 Hz), 2.84 (dd, 1H, H-3, J = 2.7, 17.2 Hz), 2.57 (s, 3H, 2000 -CH3). 13 C NMR (100 MHz, DMSO-d6) d: 198.1 (C-4), 164.3 (C-7a), 162.3 (C-5), 162.2 (C-9), 158.1 (C-7), 148.1 (C-40 ), 146.6 (C-30 ), 140.4 (C-2000 ), 133.3 (C-4000 ), 131.9 (C-3000 ), 130.9 (C-10 ), 130.7 (C-6000 ), 127.9 (C-1000 ), 126.3 (C-5000 ), 117.9 (C-60 ), 114.2 (C-20 ), 112.0 (C-50 ), 106.0 (C-10), 103.0 (C-6), 102.0 (C-8), 78.7 (C-2), 55.7 (40 -OCH3), 42.4 (C-3), 21.2 (2000 -CH3). HRMS (m/z): calcd. for (M+H)+: 421.1287; found: 421.1264. 2.2.12. Hesperetin-30 ,7-di-(4-methylbenzoate) (Y-4a) 1 H NMR (400 MHz, DMSO-d6) d: 11.96 (s, 1H, 5-OH), 8.01 (d, 2H, H-200 , H-600 , J = 8.8 Hz), 7.99 (d, 2H, H-2000 , H-6000 , J = 8.8 Hz), 7.47 (dd, 1H, H-60 , J = 2.0, 8.3 Hz), 7.45 (d, 1H, H-20 , J = 2.0 Hz), 7.404 (d, 2H, H-300 , H-500 , J = 8.8 Hz), 7.396 (d, 2H, H-3000 , H-5000 , J = 8.8 Hz), 7.23 (d, 1H, H-50 , J = 8.3 Hz), 6.55 (d, 1H, H-8, J = 2.1 Hz), 6.51 (d, 1H, H-6, J = 2.1 Hz), 5.70 (dd, 1H, H-2, J = 2.7, 13.0 Hz), 3.78 (s, 3H, 40 OCH3), 3.49 (dd, 1H, H-3, J = 13.0, 17.1 Hz), 2.90 (dd, 1H, H-3, J = 2.7, 17.1 Hz), 2.42 (s, 3H, 400 -CH3), 2.41 (s, 3H, 4000 -CH3). 13C

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NMR (100 MHz, DMSO-d6) d: 197.8 (C-4), 163.9 (C-30 a), 163.6 (C-7a), 162.2 (C-5), 162.1 (C-9), 158.2 (C-7), 151.3 (C-40 ), 144.9 (C-4000 ), 144.6 (C-400 ), 139.3 (C-30 ), 130.7 (C-10 ), 130.0 (C-200 , C-2000 , C-600 , C-6000 ), 129.8 (C-300 , C-3000 , C-500 , C-5000 ), 129.5 (C-100 , C-1000 ), 125.8 (C-60 ), 121.7 (C-20 ), 112.8 (C-50 ), 105.9 (C-10), 102.9 (C-6), 101.9 (C-8), 78.1 (C-2), 56.0 (40 -OCH3), 42.1 (C-3), 21.2 (400 -CH3, 4000 -CH3). HRMS (m/z): calcd. for (M+H)+: 539.1706; found: 539.1712.

2.3. Biological evaluation A long-term clonogenic assay was carried out as described previously.16 At seven days after the treatment, cells were treated with hesperetin derivatives (0, 5, 10, and 20 lM) for six days, followed by staining with 0.1% crystal violet. Inhibitory activities of the derivatives on clonogenicity were measured using densitometry (MultiGuage, Fujifilm, Japan), and GI50 values were computed using the SigmaPlot software (SYSTAT, Chicago, IL, USA). For a short-term cell viability assay, HCT116 cells seeded onto a 96-well plate (5  103 cells/well) were treated with hesperetin or K-5b (10, 20, 40, 80, and 100 lM) for 24 h. Cell viability was examined using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA), according to the manufacturer’s instructions. Absorbance was measured at 450 nm using an Emax Endpoint ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The detailed procedure for the cell viability assay followed the method reported previously.17 For an apoptosis assay, HCT116 cells were treated with hesperetin or K-5b (each 50 lM) for 24 h, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated annexin V and 10 lg/ mL propidium iodide (PI), according to the manufacturer’s instructions (ChemoMetec, Allerød, Denmark). Fluorescence intensities of annexin V-versus PI-positive cells were analyzed with a NucleoCounter NC-300 image cytometer (ChemoMetec). The detailed procedure for the apoptosis assay followed the method reported previously.18 Immunoblot analysis was performed as described previously.19 HCT116 cells were treated with hesperetin or K-5b (each 50 lM) for different times (0, 12, and 24 h). Antibodies specific to caspase-9, cleaved caspase-3, cleaved caspase-7, poly(ADP-ribose) polymerase (PARP), and phospho-JNK (Thr183/Tyr185) were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Signals were developed using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

2.4. In silico docking The three-dimensional (3D) structure of hesperetin was obtained from the PubChem compound database (PubChem ID: CID72281). The structure of K-5b was modified from the 3D structure of tri-O-acetylhesperetin (PubChem ID: CID69636311) using the Sybyl 7.3 program (Tripos, St. Louis, MO, USA) installed on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC. Among the 30 3D structures of JNK1 deposited in the Protein Data Bank (PDB), 2H96.pdb was selected because it contained the largest number of residues (7–182 and 186–364).20 Two crystallographic structures of JNK2 have been deposited in the PDB as 3NPC.pdb and 3E7O.pdb. Of them, 3NPC.pdb was chosen for in silico docking because of its larger number of residues (9–13, 15–202, 205–249, and 252–363).21 The detailed procedure for in silico docking followed the method reported previously.19

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2.5. Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons test using the GraphPad Prism software, version 7.0 (GraphPad Software, Inc., San Diego, CA, USA). The p-values of <0.05 were considered statistically significant.22

3. Results and discussion 3.1. Identification of hesperetin derivatives Of the 12 derivatives, seven derivatives contained the same substituents at the 7- and 30 -positions. Similar chemical shifts caused by the same substituents were assigned based on the interpretation of NOESY or TOCSY spectra. The procedure to assign the NMR data of derivative K-2 (Table 1) is explained as follows. The most deshielded 13C peak at 197.9 ppm in the 13C NMR spectrum should be the ketone carbon of the hesperetin moiety. There is only one methylene carbon in this derivative, and thus the 13C peak at 42.2 ppm was assigned to C-3. Of the methine carbons in this derivative, the most shielded 13C peak at 78.2 ppm was determined to be C-2. The 13C peak at 105.9 ppm, showing long-ranged couplings with H-3 (2.89, 3.48 ppm) in the HMBC spectrum, was assigned to C-10. Two 1H peaks at 6.49 and 6.53 ppm showed long-ranged couplings with C-10. They could be H-6 and/or H-8. Two 13C peaks at 103.0 and 102.0 ppm were directly attached to the two 1H peaks at 6.49 and 6.53 ppm in the HMQC spectrum. Because this derivative included one hydroxyl group, the 1H peak at 11.96 ppm was assigned to 5-OH. Among the two carbon peaks mentioned above, the 13C peak showing a long-ranged coupling with 5-OH should be C-6, which was observed at 103.0 ppm. As a result, the 13C peak at 102.0 ppm was C-8. Because the 13C peak at 162.3 ppm was long-range coupled to 5-OH, it was determined to be C-5. The standard long-range delay for the HMBC experiments was set to 70 ms in this study. This delay gives information about vicinal couplings between a carbon and proton.23 A shorter delay can give information on geminal couplings. In the HMBC spectrum obtained using a long-range delay of 40 ms, two longranged coupling peaks between H-8 (6.53 ppm) and two 13C peaks at 158.3 and 162.1 ppm and two long-ranged couplings between H-6 (6.49 ppm) and the 13C peaks at 158.3 and 162.3 ppm (C-5) were observed. As a result, the two 13C peaks at 158.3 and 162.1 ppm were assigned to C-7 and C-9, respectively, because H-8 could be long-range coupled to both C-7 and C-9, while H-6 could be coupled to C-5 and C-6. The chemical shifts and coupling constants of the protons and carbons contained in the chroman-4one moiety were determined completely. The 13C peak at 130.7 ppm was long-range coupled to H-3, and thus it was assigned to C-10 . Since the 1H peak at 7.2 ppm showed a long-ranged coupling with C-10 , it was determined to be H-50 . The 13C peak at 139.4 ppm, showing a long-ranged coupling with H-50 , was assigned to C-30 . The 13C peak at 121.8 ppm, showing a long-ranged coupling with H-2, was determined to be C-20 . The proton peak at 7.43 ppm was directly attached to C-20 , and thus it was H-20 . The 13 C peak at 151.4 ppm, showing a long-ranged coupling with H20 , was assigned to C-40 . Three methoxy protons were observed in the 1H spectrum. Of them, the 1H peak at 3.78 ppm was longrange coupled to C-40 , and thus it should be 40 -OMe. Among three 1 H peaks, at 7.22 (J = 8.4 Hz), 7.43 (J = 2.1 Hz), and 7.45 (J = 2.1, 8.4 Hz) ppm, the assigned proton at 7.45 ppm was determined to be H-60 . Derivative K-2 includes the same substituents, 4-methoxybenzoates, at C-30 and C-7. The chemical shifts and coupling constants of the protons and carbons contained in the methoxybenzene group were easily assigned based on the inter-

pretation of the 1D and 2D NMR experiments. However, it was difficult to determine which one was attached to the C-30 or C-7 position. As shown in Fig. 1, proton–proton correlations between the proton peak corresponding to the protons of the 2/6-position of 4-methoxybenzoate at 8.04 ppm and H-6 (6.49 ppm) and H-8 (6.53 ppm) were observed in the TOCSY spectrum. One 4methoxybenzoate showed two proton chemical shifts of 7.10 and 8.04 ppm, and another 4-methoxybenzoate showed shifts of 7.11 and 8.07 ppm. As a result, the former should be attached to C-7, and the latter was attached to C-30 . To confirm the structure determined using NMR spectroscopy, HR/MS data was collected. The calculated mass (m/z) of derivative K-2 was 571.1604 (M+H), and the mass (m/z) found here was 571.1616. The other derivatives were identified similarly to derivative K-2. 3.2. Cytotoxicity in the clonogenic assay Cytotoxicity of hesperetin and its 12 derivatives for the HCT116 human colon cancer cell line was tested at 0, 5, 10, 20, and 40 lM using the clonogenic long-term survival assay (Fig. 2). The GI50 values ranged between 13.2 and 71.7 lM (Table 1 and Suppl. Fig. 1). While the GI50 of hesperetin was 36.1 lM, derivative K-5b showed the best GI50 value, which was 12.0 lM. 3.3. Biological evaluation of K-5b To further compare the cytotoxicity of hesperetin and K-5b, HCT116 cells were treated with various concentrations of each compound (0–100 lM) for 24 h, and then cell viability was measured using the CCK-8 assay. Fig. 3A shows that K-5b decreased the cell viability more efficiently than hesperetin; at the 80 lM concentration, hesperetin decreased the viability by approximately 15%, whereas K-5b decreased the viability by approximately 50% (p < 0.0001 by one-way ANOVA, followed by Sidak’s test). We next analyzed the effect of K-5b on the induction of apoptosis. It has been reported that phosphatidylserine (PS) in the inner layer of the plasma membrane is translocated to the external surface of the cell during apoptosis.24 To determine whether K-5b induces the translocation of PS, HCT116 cells were treated with 50 lM hesperetin or K-5b and then incubated with FITC-labeled annexin V, a PS-binding protein. Fig. 3B shows that the FITC-positive population increased from 10 to 44% after the K-5b treatment, while little effect was observed after the hesperetin treatment, suggesting that K-5b efficiently triggered apoptosis compared to hesperetin. Caspases are a family of endoproteases that play an essential role in the progression of apoptosis.25 Inactive pro-caspases are activated by cleavage. To address whether caspases participate in the K-5b-induced apoptosis, cleavage of caspases was analyzed by immunoblotting. We found time-dependent cleavages of caspase-9 and caspase-3 (Fig. 3C). In addition, PARP, a known substrate for activated caspase-3, was also effectively cleaved by the K-5b treatment. These data suggest that K-5b efficiently promotes caspase-mediated apoptosis compared to hesperetin. 3.4. Relationships between cytotoxicity of hesperetin derivatives and their structures Of the 12 hesperetin derivatives, eight derivatives showed better GI50 values than that of hesperetin (36.1 lM), while four had higher values. As mentioned above, five derivatives, including K-1b, K-4, K-5b, Y-1b, and Y-3b, do not contain substituents at C-30 . Their average GI50 value was 20.0 lM. Therefore, the substituents at the C-30 position can increase the cytotoxicity against the HCT116 human cancer cell line. Among the derivatives containing substituents at C-30 , derivatives including K-1a, K-3, Y-3a,

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Fig. 1. A partial TOCSY spectrum of K-2 collected in DMSO-d6, showing cross peaks between H-6, H-8, and H-2000 .

and Y-4a showed poor activities, with the GI50 values ranging between 46.0 and 71.7 lM. Their substituents are benzoate or phenylacetate, which includes an aromatic ring. Even though K5a contains a bulky substituent at C-30 , its GI50 was 13.2 lM. Therefore, bulky substituents at C-30 do not contribute to activity decrement, but the existence of an aromatic ring at C-30 decreases the activity. However, the GI50 value of Y-2, containing an aromatic ring at C-30 , was 21.0 lM. Even though cinnamate contains an aromatic ring, an ethene group exists between the aromatic ring and carboxyl group. As a result, the distance between C-30 and an aromatic ring is important for the activity. Since Y-2 with cinnamate at C-30 showed good activity and K-3 with phenylacetate at C-30 had poor activity, the flexibility of the aromatic ring at C-30 does not contribute to the activity. Of the five derivatives without any substituents at C-30 , Y-1b with pivalate at C-7 showed a worse GI50 value compared to the others. As a result, pivalate at C-7 contributed more to the decrement of the activity than flexible hydrocarbon chains such as enoate and butyrate. The 12 derivatives synthesized here have a common feature, the hesperetin moiety, and contain different substituents at C-7 and C-30 . As a result, the existence of an aromatic ring at C-30 is important for the activity and the substituents except pivalate at C-7 are not important. To elucidate why different substituents at C-7 and C-30 cause the changes in cytotoxicity against the HCT116 human cancer cell line, proteins targeted by hesperetin in the cells were investigated.

Fig. 2. Summary of the data obtained from the clonogenic long-term survival assay using the 12 hesperetin derivatives and hesperetin at five different concentrations (0, 5, 10, 20, and 40 lM).

3.5. In silico docking and western blot analysis of JNK JNK proteins, also known as stress-activated protein kinases, are members of the mitogen-activated protein kinase (MAPK) family. JNK proteins include three isoforms, JNK1 (MAPK8), JNK2 (MAPK9), and JNK3 (MAPK10). JNK1 and JNK2 are ubiquitously found in almost every cell, while JNK3 is expressed mainly in the brain, heart, and testes.26 The JNK proteins play pivotal roles in mediating anticancer drug-induced apoptosis through inactivation of the anti-apoptotic B-cell lymphoma 2 protein and activation of the p53 tumor suppressor protein.27–30

It has been reported that hesperetin induces apoptosis through the activation of the JNK pathway in breast cancer cells.13 To understand the mechanism by which K-5b triggers apoptosis, we tested whether K-5b activates JNKs. HCT116 cells were treated with different concentrations of hesperetin or K-5b, and then the JNK phosphorylation status was measured using western blotting. As shown in Fig. 4, hesperetin slightly increased phosphorylation of JNK1 and JNK2 after 12 h of treatment. In contrast, K-5b efficiently increased phosphorylation of JNK1 and JNK2 within 1 h, and the phosphorylation status was sustained up to 12 h of treat-

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Fig. 3. Comparison of cytotoxic activities of hesperetin and K-5b. (A) Cell viability assay using CCK-8. Error bars represent the mean ± SD (n = 9). ns, not significant; *p < 0.05; ** p < 0.001 compared to vehicle treatment; ***p < 0.0001. The p-values were analyzed by one-way ANOVA, followed by Sidak’s multiple comparisons test. (B) Apoptosis assay using FITC-labeled annexin V. HCT116 cells were treated with hesperetin or K-5b (each 50 lM) for 24 h and then stained with FITC-annexin V and propidium iodide (PI). Cells were harvested, washed, and analyzed for fluorescence intensity. Scatter plots (top panels) show the FITC-annexin V intensity versus that of PI. The lower left quadrant in each cytogram (PI- and FITC-double-negative) represents viable cells. The lower right quadrant (PI-negative, FITC-positive) represents early apoptotic cells, while the upper right quadrant (PI- and FITC-double-positive) shows late apoptotic and dead cells. Histograms (bottom panels) show the FITC-annexin V intensity of the cell counts (%). M1, live cells; M2, dead cells. (C) Immunoblot analysis. HCT116 cells were treated with hesperetin or K-5b (each 50 lM) for 0, 12, and 24 h. Total cell lysates were prepared and subjected to immunoblotting using antibodies against cleaved caspase-9, cleaved caspase-3, and PARP. The GAPDH antibody was used as an internal control.

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ment. These findings demonstrate that K-5b stimulates JNK1 and JNK2 more efficiently than hesperetin. To elucidate the molecular binding modes among K-5b, hesperetin, JNK1, and JNK2, in silico docking experiments were performed. The 3D crystallographic structure of JNK1, 2H96.pdb, consists of four polypeptide chains, A, B, F, and G. Chains F and G are JNK1-interacting protein 1, and chains A and B are a heterodimer of JNK1. Both chains A and B include the protein kinase domain of JNK1. Because human JNK1 is composed of 427 residues, the coverage of 2H96.pdb is 85%. Chain A was selected here for in silico docking because it contains one more residue than chain B. First, the apoprotein of 2H96.pdb (apo-2H96) was obtained by the Sybyl program. To obtain the solution structure, the apoprotein was subjected to energy minimization. Comparison of the minimized and crystallographic structures showed that their root mean square deviation was 0.39 Å. The flexible docking procedure was iterated 30 times so that 30 protein–ligand complexes were generated. The substrate-binding site was determined in a previous report31 and analyzed using the LigPlot program.32 2H96.pdb contained 5-cyano-N-(2,5-dimethoxybenzyl)-6-ethoxypyridine-2-carboxamide (named 893, Suppl. Fig. 2A) as its substrate. By docking the ligand, 893, into apo-2H96, 30 complexes were obtained. Their binding energies ranged from 13.55 to 12.44 kcal/mol. Based on the binding energy and pose, the second complex was selected. Its LigPlot analysis showed eight hydrophobic interactions (Ile32, Leu110, Met111, Ala113, Asn114, Gln117, Ser155, and Val158) and one hydrogen bond (H-bond, Asp112) (Suppl. Fig. 3). Likewise, among the 30 complexes generated by docking hesperetin into apo-2H96, whose binding energies ranged from 12.98 to 12.13 kcal/mol, the first complex showed the best binding energy and pose. Ten hydrophobic interactions (Ile32, Gly33, Gly35, Gly38, Val40, Val58, Ile86, Glu109, Leu110, and Leu168) and one H-bond (Met111) were observed by the LigPlot analysis (Suppl. Fig. 4). Three residues, Ile32, Leu110, and Met111, were found in both 893–2H96 and hesperetin–2H96 complexes. Hesperetin and ligand 893 have common features, which are marked in bold lines in Suppl. Fig. 2A and B. However, while hesperetin binds to the N-terminus, ligand 893 binds to the second helix of JNK1. The same docking procedure was carried out for K5b, and the binding energies of the complexes generated ranged from 9.98 to 5.98 kcal/mol. The binding energy between hesperetin and JNK1 is lower than that between K-5b and JNK1, but the reason why K-5b stimulates JNK1 more efficiently than hesperetin cannot be explained based on the binding energy. The LigPlot analysis of the first K-5b–2H96 complex demonstrated nine hydrophobic interactions (Ile32, Lys55, Ile86, Met108, Leu110, Asp112, Ser155, Val158, and Leu168) and two H-bonds (Val40 and Met111) (Suppl. Fig. 5). Six residues were common for both hesperetin–2H96 and K-5b–2H96 complexes. The number of residues neighboring the hesperetin–2H96 complex is the same as that neighboring the K-5b–2H96 complex. However, while the former forms one H-bond, the latter forms two H-bonds. A 3D image of the binding site of the hesperetin–2H96 complex, generated using the PyMOL program (PyMOL Molecular Graphics System, version 1.0r1, Schrödinger, LLC, Portland, OR, USA), was compared with that of the K-5b–2H96 complex (Fig. 5A and B). The butyrate group of K-5b forms hydrophobic interactions with Lys55 and Ser155, which are not observed in the hesperetin–2H96 complex. The results obtained from the LigPlot analysis and the 3D images generated by PyMOL may explain the reason why K-5b stimulates JNK1 more efficiently than hesperetin. Similar to JNK1, the molecular binding modes between JNK2 and K-5b or hesperetin were elucidated using in silico docking. Of the two crystallographic structures of JNK2 deposited in the PDB, 3NPC.pdb was chosen because its 3D structure contained more residues than that of 3E7O.pdb. 3NPC consists of two

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Fig. 4. Comparison of JNK-activating activity of hesperetin and K-5b. HCT116 cells were treated with different concentrations of hesperetin or K-5b (0, 50, 100, and 200 lM) for 1 and 12 h. Total cell lysates were prepared and subjected to immunoblotting using an antibody against phospho-specific JNK1/2 (Thr183/ Tyr185). The GAPDH antibody was used as an internal control.

polypeptide chains, A and B,21 including the same number of residues, and chain A was selected for in silico docking. Comparison of the minimized structure of apo-3NPC with the crystallographic structure, 3NPC.pdb, showed that their root mean square deviation was 0.29 Å. 3NPC.pdb contained 1-(5-tert-butyl-2-p-tolyl-2h-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)-naphthalen-1-yl]-urea (named BIRB796; Suppl. Fig. 6). The docking process was performed for BIRB796, hesperetin, and K-5b. Their binding energies ranged between 33.75 and 22.02, 18.43 and 6.18, and 11.31 and 4.72 kcal/mol, respectively. The residues participating in the binding site were analyzed using LigPlot. The BIRB796–3NPC complex showed 15 hydrophobic interactions (Glu37, Val40, Ala53, Lys55, Arg69, Arg72, Leu77, Ile85, Ile86, Met108, Glu109, Leu110, Leu142, Leu168, and Phe170) and three H-bonds (Glu73, Met111, and Asp169) (Suppl. Fig. 7). The hesperetin–3NPC complex showed nine hydrophobic interactions (Glu73, Leu76, Leu77, Ile86, Met108, Ile147, Leu168, Phe170, and Leu172) and two H-bonds (Lys55 and Asp169) (Suppl. Fig. 8). The K-5b–3NPC complex showed 11 hydrophobic interactions (Val40, Ala53, Glu73, Leu76, Leu77, Val80, Ile86, Met108, Leu142, Leu168, and Leu172) and two H-bonds (Lys55 and Asp169) (Suppl. Fig. 9). Seven residues, including Glu73, Leu76, Leu77, Ile86, Met108, Leu168, and Leu172, participating in hydrophobic interactions and two residues forming H-bonds were found in both hesperetin–3NPC and K-5b–3NPC complexes. More residues observed in the K-5b–3NPC complex may contribute to the lower binding energy. A 3D image of the binding site of the hesperetin– 3NPC complex, generated using the PyMOL program, was compared with that of the K-5b–3NPC complex (Fig. 6A and B). The butyrate group of K-5b forms hydrophobic interactions with Val40 and Ala53, which are not observed in the hesperetin–3NPC complex. Even though the binding energy of the K-5b–3NPC complex is not as good as that of the hesperetin–3NPC complex, the binding poses obtained from the LigPlot analysis and the 3D images generated by PyMOL may explain the reason why K-5b stimulates JNK2 more efficiently than hesperetin. Comparing the binding energy between K-5b and JNK1 (9.98 to 5.98 kcal/mol) with that between K-5b and JNK2 (11.31

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K.-Y. Jung et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

Fig. 6. 3D image of the JNK2 binding sites, generated using the PyMOL program: (A) the hesperetin–3NPC complex, where the yellow dot circle denotes the 7-position of hesperetin, and (B) the K-5b–3NPC complex where Val40 and Ala53 form hydrophobic interactions with the butyrate group of K-5b.

4. Conclusions

Fig. 5. 3D images of the JNK1 binding sites, generated using the PyMOL program: (A) the hesperetin–2H96 complex, where the yellow dot circle denotes the 7position of hesperetin, and (B) the K-5b–2H96 complex, where Lys55 and Ser155 form hydrophobic interactions with the butyrate group of K-5b.

and 4.72 kcal/mol), the case of JNK2 is better than that of JNK1. While the K-5b and JNK1 complex shows nine hydrophobic interactions (Ile32, Lys55, Ile86, Met108, Leu110, Asp112, Ser155, Val158, and Leu168) and two H-bonds (Val40 and Met111), the K-5b and JNK2 complex does 11 hydrophobic interactions (Val40, Ala53, Glu73, Leu76, Leu77, Val80, Ile86, Met108, Leu142, Leu168, and Leu172) and two H-bonds (Lys55 and Asp169). The lower binding energy between K-5b and JNK2 can be explained based on the difference of residues participating in hydrophobic interactions.

In summary, we prepared 12 hesperetin derivatives by treating commercially available hesperetin with corresponding acid chlorides and trimethylamine. The structures were identified using 2D NMR experiments, including NOESY and TOCSY, and additionally confirmed by HR/MS data. Of the 12 derivatives, seven were proven to be disubstituted with the same substituents at the 7and 30 -positions (K-1a, K-2, K-3, K-5a, Y-2, Y-3a, and Y-4a), while five derivatives were acylated only at the C-30 position (K-1b, K-4, K-5b, Y-1b, and Y-3b). The 12 derivatives were then evaluated for antitumor activity using a long-term survival clonogenic assay with the HCT116 human colon cancer cell line. The GI50 values obtained ranged between 13.2 and 71.7 lM. While the GI50 value of hesperetin was 36.1 lM, mono-substituted derivative K-5b, hesperetin-7-butyrate, showed the best GI50 value of 12.0 lM, i.e., a three times better inhibition effect on the cancer cell line than that of hesperetin. Further cytotoxicity comparison of hesperetin and

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K.-Y. Jung et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

K-5b was carried out by treating HCT116 cells with various concentrations of each compound, and the data showed decreased cell viability in the presence of K-5b and a better apoptosis-triggering effect of K-5b compared with that of hesperetin. Immunoblotting analyses showed that K-5b efficiently promoted caspase-mediated apoptosis. It has been reported that hesperetin induces apoptosis through the activation of the JNK pathway. To understand the mechanism of the K-5b apoptosis-triggering effect, we tested whether JNKs are activated by K-5b and found that K-5b stimulated JNK1 and JNK2 more efficiently than hesperetin. In silico docking experiments were performed to elucidate the molecular binding modes among K-5b, hesperetin, JNK1, and JNK2. The LigPlot analysis and the 3D images generated by PyMOL showed that the butyrate group of K-5b forms more hydrophobic interactions, which may explain why K-5b stimulates JNK1 and JNK2 more efficiently than hesperetin. This study thus provided valuable preliminary information on the effects of various C-7 and/or C-30 acyl substituents on the cytotoxicity profile of hesperetin. This, in turn, may facilitate the design of more potent hesperetin derivatives as antitumor leads.

Acknowledgments This work was supported by the Priority Research Centers Program (NRF, 2009-0093824) and the Agri-Bio Industry Technology Development Program (316028-3), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET). SY Shin was supported by the KU Research Professor Program of Konkuk University.

A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2016.11.006.

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References 1. Corcoran MP, McKay DL, Blumberg JB. J Nutr Gerontol Geriatr. 2012;31:176–189. 2. Ratty AK, Das NP. Biochem Med Metab Biol. 1988;39:69–79. 3. Neher BD, Mazzaferro LS, Kotik M, et al.. Appl Microbiol Biotechnol. 2016;100:3061–3070. 4. Kaul TN, Middleton E J, Ogra PL. J Med Virol. 1985;15:71–79. 5. Kyriakidis SM, Sotiroudis TG, Evangelopoulos AE. Biochim Biophys Acta. 1986;871:121–129. 6. Vladutiu GD, Middleton E J. Life Sci. 1986;39:717–726. 7. Ba TH, Ip Via Ching TR, Van Dijk H, Labadie RP. Chem Biol Interact. 1990;73:323–335. 8. Wu D, Zhang J, Wang J, Li J, Liao F, Dong W. Tumour Biol. 2016;37:3451–3459. 9. Li R, Cai L, Xie XF, Peng L, Li J. Phytother Res. 2010;24:1850–1856. 10. Harborne JB. The Flavonoids: Advances in Research since 1980. Chapman and Hall; 1988. 11. Kuntz S, Wenzel U, Daniel H. Eur J Nutr. 1999;38:133–142. 12. Devi KP, Rajavel T, Nabavi SF, et al.. Ind Crops Prod. 2015;16:582–589. 13. Palit S, Kar S, Sharma G, Das PK. J Cell Physiol. 2015;230:1729–1739. 14. Hwang D, Jo G, Hyun J, Lee SD, Koh D, Lim Y. Magn Reson Chem. 2012;50:62–67. 15. Jung Y, Ahn S, Jung H, Koh D, Lim Y. Magn Reson Chem. 2016;54:252–259. 16. Hoffman RM. J Clin Lab Anal. 1991;5:133–143. 17. Shin SY, Yoon H, Hwang D, et al.. Bioorg Med Chem. 2013;21:7018–7024. 18. Shin SY, Jung H, Ahn S, et al.. Bioorg Med Chem. 2014;22:1809–1820. 19. Shin SY, Yoon H, Ahn S, et al.. Bioorg Med Chem. 2013;21:4250–4258. 20. Zhao H, Serby MD, Xin Z, et al.. J Med Chem. 2006;49:4455–4458. 21. Kuglstatter A, Ghate M, Tsing S, et al.. Bioorg Med Chem Lett. 2010;20:5217–5220. 22. Jung Y, Shin SY, Yong Y, et al.. Chem Biol Drug Des. 2015;85:574–585. 23. Araya-Maturana R, Gavín-Sazatornil JA, Heredia-Moya J, Pessoa-Mahana H, Weiss-López B. J Braz Chem Soc. 2005;16:657–661. 24. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. J Immunol Methods. 1995;184:39–51. 25. Danial NN, Korsmeyer SJ. Cell. 2004;116:205–219. 26. Bode AM, Dong Z. Mol Carcinog. 2007;46:591–598. 27. Fuchs SY, Adler V, Pincus MR, Ronai Z. Proc Natl Acad Sci U S A. 1998;95:10541–10546. 28. Oleinik NV, Krupenko NI, Krupenko SA. Oncogene. 2007;26:7222–7230. 29. Srivastava RK, Mi QS, Hardwick JM, Longo DL. Proc Natl Acad Sci U S A. 1999;96:3775–3780. 30. Yamamoto K, Ichijo H, Korsmeyer SJ. Mol Cell Biol. 1999;19:8469–8478. 31. Yanagisawa M, Sugiya M, Iijima H, Nakagome I, Hirono S, Tsuda T. Mol Nutr Food Res. 2012;56:1783–1793. 32. Kramer B, Rarey M, Lengauer T. Proteins. 1999;37:228–241.

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