Host-guest inclusion system of glycyrrhetic acid with polyamine-β-cyclodextrin: Preparation, characterization, and anticancer activity

Journal of Molecular Structure 1149 (2017) 155e161

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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Host-guest inclusion system of glycyrrhetic acid with polyamine-bcyclodextrin: Preparation, characterization, and anticancer activity Zhi Shen, Qi Qin, Xiali Liao, Bo Yang* Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2017 Received in revised form 28 July 2017 Accepted 29 July 2017 Available online 29 July 2017

The inclusion complexation behaviors of glycyrrhetic acid (CTA) with four polyamine-modified b-cyclodextrins (CDs) have been investigated by 1H and 2D NMR, thermal gravimetric analysis, X-ray power diffraction and scanning electron microscopy. The results showed that Glycyrrhetic acid was encapsulated into the cavity of cyclodextrin to form the complexes with 1:1 stoichiometry. The water solubility of GTA was significantly enhanced by inclusion complexation with polyamine-modified b-cyclodextrins. The calculated IC50 values indicated that the antitumor activities of inclusion complexes were better than that of GTA. Satisfactory aqueous solubility, along with high thermal stability of inclusion complexes will be potentially useful for their application on the formulation design of natural medicine. © 2017 Published by Elsevier B.V.

Keywords: Glycyrrhetic acid Polyamine-modified b-cyclodextrins Inclusion complex Solubilization Anticancer activity

1. Introduction Glycyrrhetic acid (GTA, Fig. 1), a 3b-hydroxy-1-oxo-18b, 20boleanane-12-acrylic, is a pentacyclic triterpenoid from the extracts of the root of plant Glycyrrhiza glabra L. It was widely used in Chinese medicinal preparations with various pharmacological and therapeutic properties, including anti-allergic [1], antiulcerative [2], antioxidant [3,4], anti-inflammatory [5] and antibacterial activities [6,7]. In addition, it has been clinically used as a remedy for the treatment of chronic hepatitis [8] and immunodeficiency virus infection [9]. Moreover, it is recently reported to inhibit the replication of the SARS-associated viruses [10] and induce cell apotheosis in the liver cancer cell line [11], and used as a ligand for liver targeting [12e18] because of the abundant receptors for GTA on hepatocyte membranes. Even though GTA has been used clinically for a long time, it is a lipophilic drug with a very low solubility in water, poor bioavailability. Furthermore, overconsumption of CTA results in mineralocorticoid-like side effects such as salt retention, hypokalaemic hypertension and aldosteronism [19,20]. It has presented a challenge in the development of formulations for oral administration and reducing the adverse effect. In previous study, great efforts have been made to improve its water solubility and

* Corresponding author. E-mail address: [email protected] (B. Yang). http://dx.doi.org/10.1016/j.molstruc.2017.07.104 0022-2860/© 2017 Published by Elsevier B.V.

reduce the adverse effect of GTA such as structural modification, micelle [21], or liposome formation [22,23]. Under the current situations, it will be of great significance to design a novel formulation to improve the biopharmaceutical properties and reduce manufacture for the mass production of GTA. It is well known that cyclodextrins (CDs) are truncated cone oligosaccharides mainly composed of 6e8 D-glucose monomers linked by a-1, 4-glycosidic bonds, referred to as a-, b- and g-CD, respectively. They have a hydrophobic central cavity and a hydrophilic outer surface, which enable them to form host-guest complexes with different kinds of guest molecules. Intensive studies revealed that it can enhance drug solubility in aqueous solutions, reduce the adverse effect of drug, and affect the chemical characteristics of encapsulated drug [24,25]. Those features of cavity render them a new function, successfully being utilized as drug carriers [26e29], enzyme mimics [30]. The applications of unmodified b-CD, however, are restricted by its relatively low aqueous solubility. Fortunately, chemically modified b-CD has been applied as a promising solution to this problem. Compared with native bCD, chemically modified b-CD with specific functional groups can significantly improve molecular-binding ability as well as water solubility [31,32]. Since GTA is an acidic triterpenoid, it is believed that the binding ability of polyamine-modified CDs to GTA could be higher than native b-CD. According to the previous work [33e36], we wish to report the preparation and characterization of the inclusion complexes formed by GTA and polyamine-modified CDs

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of CD), 4.98 (s, 7H, H-1 of CD). DETA-bCD, TETA-bCD and TEPA-bCD were prepared by the same approach.

O 29

OH

30 20

19 12

O 11

25 1 10

3

5

22 17

13

28

8 7

4

18

14

9

2

HO

26

H

21

16 15

The GTA/EN-bCD, GTA/DETA-bCD, GTA/TETA-bCD and GTA/ TEPA-bCD inclusion complex were prepared using a reported method [37]. The CDs (0.01 mmol) and GTA (0.03 mmol, 14.1 mg) were added to ultrapure water (10 mL), and the mixture was stirred for 72 h at room temperature. Then, the precipitate was removed by filtration, and the filtrate was lyophilized to dryness to obtain the GTA/CDs complexes. (1H NMR; 500 MHz, D2O): d 0.76 (s, 3H, H-24 of GTA), 0.98 (s, 3H, H-25 of GTA), 1.25 (s, 2H, H-29 of GTA), 1.4 (s, 3H, H-25 of GTA), 2.7e3.0 (m, 4H, CH2CH2), 3.3e3.6 (m, 14H, H-2, 4 of CD), 3.7e3.9 (m, 26H, H-3, 5, 6 of CD), 4.94 (s, 7H, H-1 of CD).

27

6 23

2.3. Preparation inclusion complexes

24 Fig. 1. Structure of glycyrrhetic acid (GTA).

2.4. Preparation of physical mixture The GTA and EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD physical mixtures were prepared [38]. By mixing GTA and CD derivatives thoroughly in an agate mortar, giving the 1:1 physical mixture. 2.5. Phase-solubility diagram

with different tethered chain lengths, including ethylenediamineb-cyclodextrin (EN-bCD), diethylenetriamine-b-cyclodextrin (DETA-bCD), triethylenetetramine-b-cyclodextrin (TETA-bCD), and Tetraethylenepentamine-b-cyclodextrin (TEPA-bCD). In this paper, the evaluation of the inclusion behaviors between GTA and polyamine-modified CDs have been studies by phasesolubility diagram. Additionally, the solid inclusion complexes have been characterized by means of 1H and 2D NMR spectra, powder X-ray diffraction, differential scanning calorimetry and scanning electron microscopy. In vitro cytotoxicity of these solid inclusion complexes was also measured with three cancer cell lines, HT26, HCT116, and SW480, as well as normal human lung fibroblast WI-38, to evaluate the potential of GTA in the form of inclusion complexes with polyamine-modified CDs, which would provide a useful approach to developing novel GTA products with high water solubility and bioavailability.

The phase-solubility diagram was investigated by using the Higuchi and Connors method [39]. An excess amount of GTA was suspended in ultrapure water containing increasing amounts of bCD, EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD (from 0.001 to 0.2 M). The mixtures were placed in an ultrasonic bath for 2 h at 25  C. After that, all suspensions were centrifuged and the supernatants were collected, and filtered over 0.45 mm millipore membranes. These experiments were repeated three times. The phasesolubility profiles were obtained by plotting the solubility of GTA versus the concentration of bCD, EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD. The apparent stability constant, Ks of GTA and bCD, ENbCD, DETA-bCD, TETA-bCD and TEPA-bCD complexes can be calculated from the slope and the intercept of the linear segment of the phase solubility line using the following equation:

Ks ¼ 2. Materials and methods 2.1. Reagents and materials Glycyrrhetinic acid (GA, MW ¼ 470.79, PC > 99%) was purchased from Sigma (USA). b-CD was purchased from Mengzhou Huaxin Biological Technology (Shanghai, China). Other chemicals and reagents were of analytical grade. All experiments were conducted by ultrapure water. 2.2. Synthesis of polyamine-b-cyclodextrin The method to synthesis EN-bCD was given in the succeeding text as a typical example. Mono-6-O-(tolylsulfonyl)-b-CD (3.0 g, 2.32 mmol) was dissolved in 20 mL ethanediamine solution, stirred at 85  C for 10 h under N2 atmosphere with a condense pipe. After that, the solution was poured into 300 mL of acetone. A yellow precipitate was formed and collected by filtration. Finally, the precipitate was dissolved in small amount of water and dropped in acetone again and a light yellow precipitate was formed and collected by filtration and dried under vacuum to yield a stable light yellow powder. (1H NMR; 500 MHz, D2O): d 2.6e2.8 (m, 4H, CH2CH2), 3.4e3.6 (m, 14H, H-2, 4 of CD), 3.7e3.9 (m, 26H, H-3, 5, 6

slope S0 ð1  slopeÞ

The value slope is found in the linear regression and S0 is the aqueous solubility of the drug at pH 7 (S0 ¼ 0.00632 mg/mL) in the absence of bCD, EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD. 2.6. X-ray powder diffraction The X-ray Powder diffraction (XRD) patterns were performed with a D/Max-3B diffractometer using Cu-Ka radiation (k ¼ 1.5460 Å, 40 kV, 100 mA), with a 5 /min scanning rate. Powder samples were mounted on a vitreous sample holder and scanned with a step size of 2q ¼ 0.02 between 2q ¼ 5e70 . 2.7. Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were analyzed using a STA449 F1 Jupiter (Netzch Corp, Germany). Samples were placed under an N2 flow in the temperature range from 25  C to 450  C at a heating rate of 10  C$min1. 2.8. Scanning electron microscopy The morphologies of the samples were performed on a scanning

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161

157

electron microscope (TESCAN, model VEGA3). Samples were distributed on metal stubs with double-sided adhesive tapes. Before examination, the samples were gold sputter-coated to render them electrically conductive, and the micrographs were obtained under reduced pressure. 2.9. Solubilization test The water solubility of the GTA/bCD, GTA/EN-bCD, GTA/DETAbCD, GTA/TETA-bCD and GTA/TEPA-bCD complex are assessed by preparation of its saturated solution [40]. An excess amount of the complex was placed in 2 mL water (pH 7.0), protected from light, and the mixture was stirred for 1 h at 25 ± 2  C. The solution was then filtered on a 0.45 mm Millipore membrane and the filtrate was lyophilized to dryness, and the resulting solid weighed. Fig. 2. Phase solubility diagram of Structure of glycyrrhetic acid (GTA) in water depending on the cyclodextrin (CD) concentration.

2.10. In vitro cytotoxicity studies The cytotoxicity tests for GTA and its inclusion complexes with EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD were evaluated in vitro for antitumor activity against human HT29, HCT116, SW480 cell lines and normal human lung fibroblast WI-38 as reference cell lines by the MTT cytotoxicity assay. The IC50 values that represented the concentration of drug required for 50% reduction of cellular growth have been calculated. Cells were cultured at 5  105 cells mL1 in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum at 37  C in a humidified atmosphere of 5% CO2 in air. Cells were seeded at 1  105 cells mL1 and treated with the indicated amounts of the GTA and its inclusion complexes. The cytotoxic activities of the GTA and its inclusion complexes were evaluated as cell survival after treatment. Cell viability was evaluated by a microculture tetrazolium reduction assay using 3-(4,5-dimethyltriazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). 3. Results and discussion 3.1. Phase-solubility Judging from the phase solubility diagram results (Fig. 2), the solubility of GTA increased as a linear function of the CD concentration. The regression equations are as follows:

[GTA] [GTA] [GTA] [GTA] [GTA]

¼ ¼ ¼ ¼ ¼

0.0162[b-CD] þ0.0295 0.0234[EN-bCD] þ0.0283 0.0290[DETA-bCD] þ0.0287 0.0416[TETA-bCD] þ0.0191 0.0529[TEPA-bCD] þ0.0167

R2 R2 R2 R2 R2

¼ ¼ ¼ ¼ ¼

0.9922 0.9918 0.9901 0.9921 0.9939

According to Higuchi and Connors's theory [38], these five relationships are classified as a typical AL-type, suggesting that the stoichiometry of GTA in the b-CD, EN-bCD, DETA-bCD, TETA-bCD or TEPA-bCD inclusion complex was 1:1. To the best of our knowledge, several weak intermolecular forces, such as ion-dipole, van der Waals, electrostatic, hydrogen bond, and hydrophobic interactions, are cooperatively contribute to inclusion complexation. The apparent stability constant (Ks) value is usually at the range from 50 to 2000 M1. In our study (Table 1), the calculated Ks values of the b-CD, EN-bCD, DETA-bCD, TETA-bCD or TEPA-bCD complexes were 210, 604, 1047, 1191 and 1362 M1, respectively, showing that the polyamine-modified b-cyclodextrins have stronger binding ability

than the native b-CD. Besides, with the number of amino decreasing, the Ks value also decreases. These may be because that the native b-CD can slip onto the guest molecular chain like a bead [41], and the amino groups at the rim of CDs can reduce the effective cavity of the CDs and then hold the drugs more tightly [42]. 3.2. Inclusion mode To explore the possible inclusion mode of GTA/CDs inclusion complex, the 1H NMR and 2D NMR spectra of CDs, GTA or their inclusion complex were investigated. As shown in Fig. 3, protons of GTA displayed chemical shifts at d 0.5e2.0 ppm in the 1H NMR spectra of GTA/EN-bCD complex, which were obviously different from the CDs protons (d 3.0e5.0 ppm), indicating the formation of inclusion complex regarding the poor water solubility of GTA. After inclusion complexation with GTA, the H-3 and H-5 protons of CDs both shifted of 0.04 and 0.02 ppm, respectively. Given that both H-3 and H-5 protons are located in the interior of the CD cavity, which may indicate that GTA should be included in the CDs cavity. It is well know that spatial proximity between host and guest molecules can be identified by the nuclear Overhauser effect (NOE) cross-correlations between relevant protons through 2D NMR experiments, such as NOESY and ROESY. To further confirm the binding mode, a 2D ROESY spectrum of the GTA/EN-bCD inclusion complex was obtained (Fig. 4). It is apparent that the H-12, 27, 29 protons of GTA are strongly correlated with the H-3 protons of ENbCD, whereas, H-24, 25 protons of GTA is correlated with the H-5 protons of EN-bCD. Besides, correlations between ethylene protons of the amino side chain of EN-bCD and H-3, 5 protons of EN-bCD showed that the amino side chain was inserted into the cavity of EN-bCD. Based on these observations, together with the 1:1 inclusion stoichiometry, we deduced the possible inclusion modes

Table 1 Complex stability constant Ks, log Ks and Gibbs free energy change(-DG) for inclusion complexation of GTA guest with host b-CD, EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD at 25  C. Host

b-CD EN-bCD DETA-bCD TETA-bCD TEPA-bCD

Guest

Ks(L$mol1)

log Ks

-△G(KJ$mol1)

Glycyrrhetic acid

210 604 1047 1191 1362

2.32 2.78 3.04 3.08 3.13

13.2 15.8 17.39 17.45 17.93

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H-1 of EN- CD

H-3,6,5 of EN- CD H-2,4 of EN- CD

EN- CD 8

7

6

GTA

5

4

3

2

H-12 of GTA

8

7

6

1

0

CH3 of GTA

5

4

3

2

1

0

H-1 of EN- CD H-3,6,5 of EN- CD H-2,4 of EN- CD CH3 of GTA GTA/EN- CD H-12 of GTA 8

7

6

5

4

3

2

1

0

Fig. 3. Proton nuclear magnetic resonance spectra of EN-bCD (in D2O), GTA (in CDCl3), and GTA/EN-bCD (in D2O) inclusion complex.

H-3 of EN- CD H-5 of EN- CD

H-25 of GTA H-29 of GTA H-24 of GTA Ethylenediamine H-27 of GTA

H-12 of GTA

0.5

(A)

1.0 1.5 2.0 2.5 3.0

(C)

3.5 4.0

(A)

(B)

f1 (ppm)

(B)

4.5 5.0 5.5

(C) 6.0

5.5

5.0

4.5

4.0

6.0

3.5 3.0 f2 (ppm)

2.5

2.0

1.5

1.0

0.5

Fig. 4. ROESY of GTA/EN-bCD inclusion complex in D2O at 25  C.

between GTA and EN-bCD (Fig. 5). 3.3. X-ray powder diffraction The crystalline states of GTA, EN-bCD, DETA-bCD, and their inclusion complexes and physical mixtures were examined by X-

ray powder diffraction. As Fig. 6 depicted, GTA displayed series of characteristic peaks, which demonstrated its high degree of crystallinity. In contrast, their inclusion complexes displayed a very different amorphous halo pattern from the diffractogram, in which the feature diffraction peaks of GTA were completely disappeared, but different from their physical mixtures. These results

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161

H H

29

25

HN

H3C

12

NH2O

H

O 25

OH

H

OH

HO

H3 C

H

27

H-5

O

CH3

HO

24

29

H3 C

12

O

CH3

CH3

H-3

H-5

H-3

H-5

159

H 3C

CH3

H

27

24

H N H2N

H-5

H-3

H-3

Fig. 5. Possible inclusion mode of the GTA/EN-bCD complex.

5000

1

2500 0

Intensity [cps]

5000

0

20

40

0

20

40

0

20

40

0

20

2

60

2500 0 5000

3

60

2500 0 3000

4

60

2000 1000 0

2 [degree]

40

60

Fig. 6. XRD patterns of (1) GTA, (2) EN-bCD, (3) GTA/EN-bCD physical mixture (1: 1), and (4) GTA/EN-bCD inclusion complex.

further proved that the formation of GTA and CDs inclusion complexes.

3.4. Differential scanning calorimetry Thermal properties of GTA, GTA/EN-bCD inclusion complex were investigated by DSC. As showed in Fig. 7, the DSC curves of GTA and EN-bCD showed that the free GTA had obvious endothermic peak at ca 310  C (a), and the free EN-bCD displayed two broad endothermic bands between ca 100 and 400  C (b). Their physical mixtures DSC curve was nearly a combination of the two components (c). However, the DSC curve of GTA/EN-bCD inclusion complex was much different from their physical mixtures (d), indicating a different thermal stability from that of above. All these results showed that a new formation of GTA and EN-bCD, namely their inclusion complex.

3.5. Scanning electron microscopy From scanning electron microscopy analysis (Fig. 8), using GTA, EN-bCD, their physical mixture and inclusion complex for example. GTA was seen as claviform crystals that formed aggregates (a), while EN-bCD looked like irregularly 3D-shaped crystals (b). Their physical mixture was just like a simple overlay of their morphological characteristics (c). In contrast, their inclusion complex appeared similar to that of EN-bCD, which was distinct from the morphology of their physical mixture (d). That proved that the formation of GTA and EN-bCD inclusion complex once more. 3.6. Water solubility The water solubility of GTA/EN-bCD, GTA/DETA-bCD, and GTA/ TETA-bCD and GTA/TEPA-bCD inclusion complex was assessed by the preparation of its saturated solution. An excess amount of the

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3.0

3.0

H (mv/mg)

(a)

(b)

1.5

1.5

0.0

0.0

2

100

200

300

400

500

100

2

200

300

(c)

400

500

(d)

0

0

100

200

300

400

500

100

200

300

400

500

o

T ( C) Fig. 7. DSC curves of (a) GTA, (b) EN-bCD, (c) GTA/EN-bCD physical mixture (1: 1), and (d) GTA/EN-bCD complex.

complex was placed in 2 mL of water (ca pH 7.0), and the mixture was stirred vigorously at room temperature for 2 h. Subsequently, the insoluble substance was removed by filtration and then lyophilized to dryness, and the residue was dosed by the weighing method. The results show that the water solubility of this GTA (Table 2), compared with that of native GTA (ca. 0.00632 mg/mL), was markedly increased to approximately 2.34, 4.78, 7.15 and 8.33 mg/mL by the solubilizing effects of EN-bCD, DETA-bCD, TETAbCD, and TEPA-bCD, respectively. This confirmed the reliability of the obtained more satisfactory water solubility of GTA/CDs complexes, which would be beneficial for the utilization of this compound as medicine products. 3.7. In vitro cytotoxicity studies

Fig. 8. Scanning electron microscopy images of (a) GTA, (b) EN-bCD, (c) physical mixture of EN-bCD and GTA, and (d) GTA/EN-bCD complex.

Table 3 IC50 (mM) of GTA and GTA/CDs complexes in various colon cancer cell lines.

Table 2 The water solubility of Glycyrrhetic acid/CDs complex at 25  C. Compound

GTA GTA/EN-bCD complex GTA/DETA-bCD complex GTA/TETA-bCD complex GTA/TEPA-bCD complex

The cytotoxicity of GTA and four GTA/CDs solid inclusion complex on human cancer cell lines (HT29, HCT116, and SW480) and normal human lung fibroblast WI-38 were individually evaluated using the MTT assay, using cisplatin (DPP) as a reference compound. The IC50 values that represented the concentration of a drug required for 50% reduction of cellular growth had been calculated and showed in Table 3. As seen, the IC50 values of GTA/CDs solid inclusion complex were better than that of the free GTA, but also as

Water Solubility Solubility (mg/mL)

Fold increase

0.00632 2.34 4.78 7.15 8.33

1.0 370 756 1132 1318

Samples

HT-29

HCT116

SW480

WI-18

EN-bCD DETA-bCD TETA-bCD TEPA-bCD DPP GTA GTA/EN-bCD complex GTA/DETA-bCD complex GTA/TETA-bCD complex GTA/TEPA-bCD complex

>100 >100 >100 >100 10.8 71.8 13.4 17.5 11.5 16.4

>100 >100 >100 >100 8.9 53.9 12.8 17.1 9.7 14.1

>100 >100 >100 >100 9.3 97.8 13.7 21.5 10.8 19.7

>100 >100 >100 >100 30.5 99.3 >100 >100 >100 >100

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161

well as the reference compound DPP, and GTA/TETA-bCD complex was the best one among them. Additionally, cytotoxicity of GTA/CDs solid inclusion complexes against normal human lung fibroblast WI-38, which indicates that, the EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD had no significant toxicity. In vitro toxicity study is essential to prove the safety of any drug carriers. These results would provide much useful information for the study of GTA in the future. 4. Conclusions In the present work, solid inclusion complexes between GTA and four polyamine-modified b-CDs were successfully prepared, and the characterization, inclusion complexation behavior, and binding ability of the inclusion complexes were investigated. The results showed that polyamine-modified b-CD dramatically enhanced the water solubility of GTA. The cytotoxicity assay showed that complexes of GTA with polyamine-modified b-CD exhibited higher anticancer activity on human cancer cell lines than that of free GTA. Considering the shortage of applications of GTA and the conveniently and environmentally friendly preparation of the GTA/CDs complex, inclusion complexation should be regarded as a useful approach to the design of a novel formulation of GTA for herbal medicine. Furthermore, high anticancer activity of inclusion complexes will be potentially applied to the cancer chemotherapy. Acknowledgments This work was supported by National Natural Science Foundation of China (NNSFC) (No. 21362016 and 21642001), which are gratefully acknowledged. References [1] H.Y. Park, S.H. Park, H.K. Yoon, M.J. Han, D.H. Kim, Arch. Pharm. Res. 27 (2004) 57. [2] S. Yano, M. Harada, K. Watanabe, et al., Chem. Pharm. Bull. 37 (1989) 2500. [3] H.E. Jian, Chin. J. Aesthet. Med. 10 (2001) 191. [4] K.H. Nwe, A. Hamid, P.B. Morat, B.A. Khalid, Steroids 65 (2000) 40. [5] D. Maitraie, C.F. Hung, H.Y. Tu, Y.T. Liu, et al., Bioorg. Med. Chem. 17 (2009) 2785. [6] D.R. Long, J. Mead, J.M. Hendricks, M.E. Hardy, J.M. Voyich, Antimicrob. Agents

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