CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201402027

Phytol Derivatives as Drug Resistance Reversal Agents Harish C. Upadhyay,[a] Gaurav R. Dwivedi,[b] Sudeep Roy,[b, c] Ashok Sharma,[b] Mahendra P. Darokar,*[b] and Santosh K. Srivastava*[a] Phytol was chemically transformed into fifteen semi-synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with nalidixic acid against E. coli strains CA8000 and DH5a. The pivaloyl (4), 3,4,5-trimethoxybenzoyl (9), 2,3-dichlorobenzoyl (10), cinnamoyl (11), and aldehyde (14) derivatives of phytol ((2E,7R,11R)3,7,11,15-tetramethyl-2-hexadecen-1-ol) were evaluated by using another antibiotic, tetracycline, against the MDREC-KG4 clinical isolate of E. coli. Derivative 4 decreased the maximal in-

hibitory concentration (MIC) of the antibiotics by 16-fold, while derivatives 9, 10, 11, and 14 reduced MIC values of the antibiotics up to eightfold against the E. coli strains. Derivatives 4, 9, 10, 11, and 14 inhibited the ATP-dependent efflux pump; this was also supported by their in silico binding affinity and down-regulation of the efflux pump gene yojI, which encodes the multidrug ATP-binding cassette transporter protein. This study supports the possible use of phytol derivatives in the development of cost-effective antibacterial combinations.

Introduction Bacterial diseases are one of the major causes of death in the entire world.[1] The diseases caused by Gram-negative bacteria, such as infections of the bloodstream, bones, joints, and urinary tract, as well as pneumonia, are of major concern in terms of their treatment.[2, 3] According to the United States National Healthcare Safety Network, Gram-negative bacteria are responsible for more than 30 % of hospital-acquired infections, of which 45 % are urinary tract infections, mainly caused by E. coli.[4] Development of drug resistance in bacteria is due to a number of strategies adapted by bacteria, such as enzyme inactivation, efflux pumps, target alteration, and permeability changes, which cause a wastage of 20–50 % of the antibiotics administered for treatment.[5, 6] Multidrug resistance in Gramnegative pathogenic bacteria is becoming a life-threatening problem, which is primarily caused by overexpression of efflux pumps that extrude unrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their attack at the intended targets.[7, 8] To combat the rapid spread of pathogens

[a] H. C. Upadhyay,+ Dr. S. K. Srivastava Medicinal Chemistry Department, P.O. CIMAP, Lucknow 226015 (India) E-mail: [email protected] [b] Dr. G. R. Dwivedi,+ Dr. S. Roy, Dr. A. Sharma, M. P. Darokar Biotechnology Division Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP) P.O. CIMAP, Lucknow 226015 (India) E-mail: [email protected] [c] Dr. S. Roy Present address: Department of Biomedical Engineering Faculty of Electronics and Communication Brno University of Technology Antonnsk 548/1, 601 90 Brno (Czech Republic) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402027: structures and detailed 13 C NMR (75 MHz) data for all phytol derivatives 1–15, along with an in silico interaction diagram.

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expressing resistance, recent research has focused on reducing the dose of antibiotics by using synergistic combinations.[9–11] There are many phytomolecules that do not possess significant antibacterial activity of their own, but when used in combination, they significantly enhance the antibiotic potential of the drug toward which the bacterium was resistant, that is, they act as drug resistance reversal (DRR) agents.[7] Due to lack of a general, suitable name and definite mechanism of action, these compounds are known by many names, such as helper molecules and bio-enhancers.[12–15] These compounds may be used to lower the dosage of antibiotics, leading to cost-effectiveness and shortening of the treatment course.[16] Phytol is a very common acyclic isoprenoid[17, 18] and a natural precursor of phytanic and pristanic acids, which work as ligands of the nuclear hormone receptor, peroxisome proliferator-activated receptor alpha (PPARa).[19] The semi-synthetic approach in drug development can generate numerous analogues by modifying the existing functional groups of a natural product, and most of the time, this strategy provides exciting results with high bioactivity and low toxicity.[20] Although antitubercular activity has been reported for phytol and its semisynthetic analogues,[21–23] the DRR activity has not been reported thus far. In continuation of our search for DRR agents,[24–26] the present study focused on the chemical transformation of phytol into several derivatives, followed by evaluation of their DRR potential in combination with nalidixic acid (NAL) against CA8000 and DH5a strains of E. coli. Significantly active derivatives were also evaluated for their DRR potential in the multidrug-resistant clinical isolate MDREC-KG4 of E. coli. To understand the possible molecular mechanism, potentially active combinations were further evaluated for time–kill kinetics, efflux pump inhibition, ATPase inhibition, real time expression analysis, and in silico docking studies.

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CHEMMEDCHEM FULL PAPERS Results and Discussions Chemistry For the semi-synthesis of phytol derivatives, the primary allylic hydroxy group of phytol was reacted with various acid chlorides. A total of eleven acyl/aryl derivatives (1–11) of phytol were synthesized following our previously published procedures (Scheme 1).[24, 27] The diphytyl oxalate derivative (12) was prepared by using the molar ratio of phytol to oxalyl chloride in a to 3:1 ratio, which gave the highest yield for derivative 12

www.chemmedchem.org there are many methods for preparing bioactive chalcones,[28, 29] in the present study, cross-aldol condensation of phytal 14 and acetophenone was carried out using boron trifluoride etherate (BF3·Et2O), which afforded derivative 15 in good yield.[30] Synthetic pathways for the preparation of derivatives 13–15 are depicted in Scheme 2. Biology

The antibacterial potential of phytol and its derivatives was assessed by determining their minimum inhibitory concentrations (MIC) against the CA8000 and DH5a strains of E. coli. Although results showed slightly increased antimicrobial activity for most of the phytol derivatives [except benzoyl (7) and cinnamoyl (11)], none of the derivatives showed significant antimicrobial potential as per stringent activity criteria (Table 1).[31] Further, phytol and its various derivatives were evaluated for their DRR potential in combination with NAL against CA8000 and DH5a strains of E. coli. The MIC of NAL, in combination with 10 mg mL1 of phytol and its derivatives, are presented in Table 1. The antibiotic NAL alone Scheme 1. Synthesis of acyl/aryl derivatives 1–12 of phytol. Reagents and conditions: a) dry pyridine, RCOCl (each showed MIC values of 1.5 equiv per mol phytol), DMAP, RT, overnight (14–16 h), 96–68 %; b) dry pyridine, oxalyl chloride (3 equiv per mol 6.25 mg mL1 and 100 mg mL1 phytol), DMAP, RT, overnight (14–16 h), 64 %. against CA8000 and DH5a strains of E. coli, respectively, but when 10 mg mL1 of phytol was used in combination with NAL, the MIC value of NAL was reduced by half for both strains. Similar results were also observed for the acetyl (1), transcrotonyl (6), benzoyl (7), and manisoyl (8) derivatives of phytol. Similarly, lauroyl (2), palmitoyl (3), and bromide (13) derivatives showed a fourfold decrease in the NAL MIC value against DH5a and twofold decrease against Scheme 2. Synthesis of derivatives 13–15 of phytol. Reagents and conditions: c) dry pyridine, PBr3, 4 8C for 30 min, CA8000. On the other hand, dethen RT for 3 h, 86 %; d) dry CH2Cl2, PCC, reflux, 6 h; e) BF3·Et2O, acetophenone, reflux for 1 h, then overnight (14– 16 h) at 30–35 8C, 62 %. rivative 15 showed a fourfold decrease in the MIC value of NAL against both E. coli strains. (Scheme 1). The bromo derivative (13) was prepared by substiThe 3,4,5-trimethoxybenzoyl (9), 2, 3-dichlorobenzoyl (10), cintution of the allylic hydroxy group of phytol with bromine namoyl (11), and aldehyde (14) derivatives showed four- and through a SN2 reaction at the carbon using PBr3.[23] The phytol eightfold decreases in the MIC values of NAL against CA8000 and DH5a, respectively. Finally, among all of the compounds was oxidized to its aldehyde derivative, phytal 14, using pyriditested, pivaloyl derivative (4) was found to be the best DRR nium chlorochromate (PCC) as an oxidizing agent.[23] Although  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Table 1. In vitro drug resistance reversal activity of phytol derivatives against CA8000 and DH5a strains of E. coli. Compd NAL phytol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MIC [mm][a] CA8000 DH5a 0.027 3.378 1.479 1.046 0.936 1.316 NT[d] 1.374 2.500 1.163 0.510 1.066 2.347 NT[d] 0.696 0.850 1.263

0.431 3.378 1.479 0.523 0.936 1.316 – 1.374 2.500 1.163 0.510 1.066 2.347 – 0.696 1.700 1.263

MIC [mm] NAL + compd[b] CA8000 FR[c] DH5a FR[c] 0.027 0.013 0.013 0.013 0.013 0.003 – 0.013 0.013 0.013 0.007 0.007 0.007 – 0.013 0.007 0.007

– 2 2 2 2 8 – 2 2 2 4 4 4 – 2 4 4

0.431 0.216 0.216 0.108 0.108 0.027 – 0.216 0.216 0.216 0.054 0.054 0.054 – 0.108 0.054 0.108

– 2 2 4 4 16 – 2 2 2 8 8 8 – 4 8 4

[a] Minimum inhibitory concentration (MIC). [b] MIC values of nalidixic acid (NAL) in the presence of test compound (10 mg mL1). [c] Fold reduction (FR) in MIC of NAL. [d] Not tested due to paucity. MIC values are consistent across three independent experiments performed in duplicate.

agent, exhibiting eight- and 16-fold decrease in the MIC of NAL against both CA8000 and DH5a strains of E. coli, respectively. It is worth mentioning that the DRR potential of derivatives 4, 9, 10, 11, and 14 against DH5a (drug-resistant strain) was double that against CA8000 (drug-sensitive strain). As the clinical isolate MDREC-KG4 has been reported as the most resistant strain against various clinically used structurally and functionally different antibiotics,[25, 32] the phytol derivatives (4, 9, 10, 11 and 14) showing significant DRR potential in combination with NAL were further validated using another antibiotic tetracycline (TET), with results presented in Table 2. The minimum effective concentration (MEC) of derivatives 4, 9, 10, 11, and 14 was also deduced (Table 2). To determine the concentration-dependent antibacterial effect of TET and phytol derivatives alone as well as in combinations, MDREC-KG4 cells were treated with varying concentrations of TET and phytol derivatives. It was interesting to note that TET showed antibacterial activity at a very low concentration when used in combination with phytol derivatives 4, 9, 10, 11, and 14 (Figure 1). Accumulation and efflux of ethidium bromide are good indicators of the involvement of efflux pumps in a resistance mechanism, particularly in Gram-negative bacteria such as E coli.[33, 34] To evaluate a possible efflux pump inhibition mechanism for derivatives 4, 9, 10, 11, and 14, these compounds were subjected to a fluorescence-based ethidium bromide efflux assay using MDREC-KG4. In this assay, reserpine (RES) was used as a reference DRR agent. As shown in Figure 2, a significant decrease in fluorescence was observed in untreated control cells. In the presence of derivatives 4, 9, 10, 11, and 14, the loss of fluorescence was significantly re-

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Table 2. Potentiation of tetracycline (TET) against the multidrug-resistant clinical isolate of E. coli, MDREC-KG4, by phytol derivatives. Compd TET

MIC [mm]

Combinations Compd (c [mm]) TET[a]

1.801

4

0.658

9

0.510

10

1.066

11

1.174

14

0.850

RES

1.316

4 4 4 4 9 9 9 9 10 10 10 10 11 11 11 11 14 14 14 14 RES RES RES

– (0.263) (0.033) (0.016) (0.002) (0.213) (0.107) (0.053) (0.002) (0.213) (0.107) (0.053) (0.002) (0.235) (0.059) (0.029) (0.002) (0.340) (0.085) (0.043) (0.003) (0.164) (0.041) (0.001)

– 0.113 0.113 0.225 0.450 0.225 0.225 0.450 0.450 0.225 0.225 0.450 0.901 0.225 0.225 0.450 0.450 0.225 0.225 0.450 0.450 0.901 0.901 1.801

FR[b] – 16 16 8 4 8 8 4 4 8 8 4 2 8 8 4 4 8 8 4 4 2 2 –

[a] Minimum inhibitory concentration (MIC) of tetracycline (TET) in combination with derivatives at various concentrations. [b] Fold reduction (FR) in MIC of TET. MIC values are consistent across three independent experiments performed in duplicate.

duced, reflecting a strong interference with ethidium bromide efflux. Further, to understand whether these compounds interfere with the ATP-dependent efflux pump, they were evaluated for ATPase inhibitory activity in MDREC-KG4. It was found that derivatives 4, 9, 10, 11, and 14 significantly inhibited ATPase activity in terms of liberated inorganic phosphate (Pi), indicating involvement of these compounds in the inhibition of ATP-dependent efflux pumps (Figure 3). The results are similar to those reported earlier for understanding the mechanism of action of known drugs such as reserpine, ouabain, and phenothiazine.[34] YojI is one of the important ATP-dependent multidrug ATP-binding cassette (ABC) transporter proteins in E. coli. TET is known to induce overexpression of different efflux pump genes.[35, 36] Derivatives 4, 9, 10, 11, and 14 not only enhanced the intrinsic susceptibility of MDREC-KG4 toward TET but also significantly decreased the expression of the yojI gene encoding a multidrug ATP-dependent efflux pump system when used alone or in combination with TET (Figure 4). Docking studies Molecular docking studies showed that derivatives 4, 9, 10, 11, and 14 have good binding affinity with the YojI receptor. The results are provided in Table S1 and Figure S1 (Supporting Information). ChemMedChem 2014, 9, 1860 – 1868

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www.chemmedchem.org Conclusions

Figure 1. Time–kill curves of MDREC-KG4 showing the dose-dependent bactericidal effect of a) tetracycline (TET) and b) TET in combination with phytol derivatives.

In silico ADME studies

Experimental Section

In silico ADME analysis revealed that all derivatives showed low blood–brain barrier permeability and are in the category of non-CNS drugs with moderate intestinal absorption (Table 3). In intestinal absorption parameters, derivative 4 showed moderate intestinal absorption. The plasma protein binding (PPB) level was less than 90 %, which suggests better distribution of drug into the blood. For orally administrated drugs, the fractional polar surface area (FPSA) value is a parameter for permeability, which should be less than 140 . In our case, all of the derivatives fell within permissible limits. All of the derivatives were non-hepatotoxic, while the control, reserpine, was found to be hepatotoxic (Table 3).

Chemistry

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The present study reports the semi-synthesis and DRR effect of phytol derivatives. A total of fifteen derivatives of phytol were prepared in good yields. Phytol and all of the derivatives prepared in this study possess DRR characteristics when used in combination with NAL. Among all of these, derivative 4 decreased the dose of NAL 16-fold, while derivatives 9, 10, 11, and 14 reduced the MIC of NAL up to eightfold against the drug-resistant DH5a strain of E. coli. Among the 15 derivatives, 4, 9, 10, 11, and 14 may be considered potent DRR agents. This DRR effect was made possible by the involvement of multiple mechanisms, such as ATP-dependent efflux pump inhibition and down-regulation of the efflux pump gene. In silico ADME analysis revealed that derivatives 4, 9, 10, 11, and 14 are non-CNS drugs with moderate intestinal absorption and are safe and nontoxic, which makes them suitable leads for further drug development. This study may be of great help in the development of inexpensive and doseeconomic combinatorial antimicrobial drug formulations from phytol, a very common and widely distributed phytomolecule.

All chemicals and reagents, including phytol (procured as cis-trans mixture), were obtained from Sigma–Aldrich Pvt. Ltd., India and were used without further purification. The solvents used in chromatographic separations were purchased from Merck India Pvt. Ltd. A 300 MHz NMR (Avance, Bruker, Switzerland) was used to record 1H and 13C NMR spectra with tetramethylsilane (TMS) as the internal standard. Hyphenated LC-PDA-MS (Prominence LC and mass MS-2010EV, Shimadzu, Japan) was used for mass spectrometry. IR spectra were recorded in CCl4 using a Spectrum BX FTIR spectrometer (PerkinElmer, USA). Reaction progress was monitored by thin layer chromatography on silica gel 60F254 (Merck, Germany) readymade aluminum sheets, first examined under UV illumination at 254 and 365 nm and then sprayed with vanillin-sulfuric acid (1:5, w/v) solution in EtOH, followed by heating at 95 8C for 5 min. ChroChemMedChem 2014, 9, 1860 – 1868

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www.chemmedchem.org tilled water and dried over anhydrous Na2SO4, and the solvent was removed under vacuum. The resulting crude products were separately purified by column chromatography (silica gel, 60–120 mesh, 24 g, column diameter 2  30 cm) to afford the respective derivatives, 1–12, in high purity (> 95 %).

Figure 2. Inhibition of ethidium bromide efflux by phytol derivatives.

1-O-Acetylphytol (1): Colorless liquid; yield: 642.5 mg, 95 %: IR (CCl4): n˜ max = 1654, 1742, 2867, 2927, 2954 cm1; 1H NMR (300 MHz, CDCl3): d = 0.83–0.92 (m, 12 H), 1.12 (m, 12 H), 1.32 (m, 2 H), 1.38 (m, 2 H), 1.52 (m, 1 H), 1.78 (brs, 3 H), 2.03 (m, 4 H), 2.21 (s, 3 H), 4.82 (d, J = 7.2 Hz, 2 H), 5.46 ppm (m, 1 H); 13 C NMR (75 MHz, CDCl3): d = 16.4, 19.6, 22.6, 22.8, 24.4, 24.8, 25.0, 27.9, 33.6, 33.8, 36.8, 36.9, 37.8, 39.3, 39.8, 61.9, 118.1, 142.8, 174.2 ppm; ESIMS: m/z: 339 [M + H] + . 1-O-Lauroylphytol (2): Viscous colorless liquid; yield: 898.5 mg, 94 %; IR (CCl4): n˜ max = 1654, 1745 cm1; 1H NMR (300 MHz, CDCl3): d = 0.82–0.95 (m, 15 H), 1.23 (brs, 16 H), 1.25 (m, 12 H), 1.32 (m, 2 H), 1.46 (m, 2 H), 1.65 (m, 1 H), 1.68 (m, 2 H), 1.74 (brs, 3 H), 1.99 (m, 4 H), 2.33 (t, J = 7.5 Hz, 2 H), 4.57 (d, J = 7.2 Hz, 2 H), 5.34 ppm (br, 1 H); 13C NMR (75 MHz, CDCl3): d = 14.6, 16.3, 19.5, 22.6, 24.7, 24.8, 25.0, 29.1, 29.5, 29.7, 32.6, 34.0, 34.6, 36.7, 37.3, 37.4, 39.1, 39.8, 61.4, 118.7, 142.6, 174.0 ppm; ESI-MS: m/z: 479 [M + H] + . 1-O-Palmitoylphytol (3): Viscous colorless liquid; yield: 1025.5 mg, 96 %; IR (CCl4): n˜ max = 1654, 1750, 2848, 2916, 2952 cm1; 1H NMR (300 MHz, CDCl3): d = 0.87 (d, J = 6.9 Hz, 15 H), 1.13 (m, 12 H), 1.25 (brs, 24 H), 1.38 (m, 2 H), 1.62 (m, 2 H), 1.65 (m, 1 H), 1.68 (m, 2 H), 1.74 (brs, 3 H), 2.31(m, 4 H), 2.33 (t, J = 7.5 Hz, 2 H), 4.59 (d, J = 7.2, 2 H), 5.35 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 14.4, 16.3, 19.7, 22.6, 22.7, 24.5, 24.7, 24.8, 25.0, 29.1, 29.5, 29.6, 29.7, 30.2, 32.6, 34.0, 34.4, 36.7, 37.3, 37.4, 39.3, 39.8, 61.2, 118.8, 142.6, 174.0 ppm; ESI-MS: m/z: 535 [M + H] + .

Figure 3. ATPase inhibitory activity of phytol derivatives.

matographic purifications were performed on silica gel (60– 120 mesh) procured from Merck (India). Purity of the derivatives was determined by HPLC. General procedure for synthesizing phytol derivatives 1–12: Phytol (592 mg, 2 mmol) was dissolved in dry pyridine, and the respective acyl/aryl chloride (3 mmol) was added (except for derivative 12, where 0.67 mmol oxalyl chloride was used). After adding a catalytic amount of 4-dimethylaminopyridine (DMAP), the reaction mixture was stirred at 30–35 8C for 14 h. After completion of the reaction, crushed ice was added, and the reaction mixture was extracted with chloroform (3  25 mL). The combined chloroform extract was washed with 6 % aqueous HCl solution to remove the pyridine. Finally, the combined CHCl3 extract was washed with dis 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1-O-Pivaloylphytol (4): Viscous colorless liquid; yield: 730 mg, 96 %; IR (CCl4): n˜ max = 1654, 1730, 2868, 2927, 2955 cm1; 1H NMR (300 MHz, CDCl3): d = 0.82 (d, J = 6.9 Hz, 12 H), 1.18 (brs, 9 H), 1.24 (m, 12 H), 1.62 (m, 2 H), 1.39 (m, 2 H), 1.67 (m, 1 H), 1.74 (brs, 3 H), 2.12 (m, 4 H), 4.56 (d, J = 7.2 Hz, 2 H), 5.30 ppm (br, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.3, 19.7, 22.6, 24.7, 24.8, 25.0, 27.2, 27.9, 32.3, 32.4, 36.7, 37.3, 37.4, 38.7, 39.3, 39.8, 61.3, 118.5, 142.6, 178.5 ppm; ESI-MS: m/z: 381 [M + H] + . 1-O-(3-Chloropropionyl)phytol (5): Colorless semisolid; yield: 557.5 mg, 72 %; IR (CCl4): n˜ max = 1654, 1768, 2955 cm1; 1H NMR (300 MHz, CDCl3): d = 0.86–0.94 (m, 12 H), 1.12 (m, 12 H), 1.15 (m, 2 H), 1.46 (m, 2 H), 1.62 (m, 2 H), 1.65 (m, 1 H), 1.76 (s, 3 H), 2.01(m, 2 H), 2.79 (t, J = 6.6 Hz, 2 H), 3.76 (t, J = 6.6 Hz, 2 H), 4.67 (d, J = 7.2 Hz, 2 H), 5.37 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.4, 19.7, 22.6, 22.7, 24.5, 24.8, 25.0, 28.0, 32.7, 32.8, 36.6, 37.3, 37.4, 39.4, 39.9, 61.5, 117.9, 142.9, 166.5 ppm; ESI-MS: m/z: 388 [M + H] + . ChemMedChem 2014, 9, 1860 – 1868

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Figure 4. Expression pattern of the yojI transcript of MDREC-KG4 in the presence of tetracycline (TET) and compounds 4, 9, 10, 11, and 14, both alone and in combination.

Table 3. In silico absorption, distribution, metabolism, and excretion (ADME) prediction for ligands 4, 9, 10, 11, 14, and reserpine (RES). Ligand 4 9 10 11 14 RES

BBB[a]

Abs.[b]

4 4 4 4 4 4

1 3 3 3 3 1

Solubility[c] No No No No No Yes

(1) (0) (0) (1) (1) (2)

Hepat.[d]

PPB[e]

FPSA[f]

0 0 0 0 0 1

2 2 2 2 2 2

26.23 26.23 26.23 26.23 17.3 115.519

[a] BBB (blood–brain barrier) level: 0!4, where 4 is very low. [b] Human intestinal absorption level: 1 = moderate, 2 = poor, 3 = very poor. [c] Aqueous solubility level: 0 = extremely low, 1 = very low, 2 = low. [d] Hepatotoxicity: 0 = nontoxic, 1 = toxic. [e] Plasma protein binding (PPB): 0!2, where 2 is tightly bound. [f] Fractional polar surface area (FPSA).

1-O-(trans-Crotonyl)phytol (6): Colorless semisolid; yield: 495 mg, 68 %; IR (CCl4): n˜ max = 1656, 1722, 2867, 2926, 2953 cm1; 1H NMR (300 MHz, CDCl3): d = 0.83–0.95 (m, 12 H), 1.23 (m, 12 H), 1.28 (m, 2 H), 1.35 (m, 2 H), 1.67 (m, 2 H), 1.72 (brs, 3 H), 1.82 (m, 1 H), 1.85 (d, J = 6.9 Hz, 3 H), 1.98 (m, 2 H), 4.60 (d, J = 7.2 Hz, 2 H), 5.33 (m, 1 H, m), 5.80 (d, J = 10.5 Hz, 1 H), 6.93 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.7, 18.3, 20.0, 23.0, 24.8, 25.2, 25.3, 28.3, 33.1, 33.2, 37.0, 37.3, 37.8, 39.8, 40.2, 61.8, 118.2, 122.8, 142.3, 144.2, 166.4 ppm; ESI-MS: m/z: 365 [M + H] + . 1-O-Benzoylphytol (7): Viscous liquid; yield: 736 mg, 92 %; IR (CCl4): n˜ max = 1654, 1686, 1721, 2867, 2926, 2953 cm1; 1H NMR  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

(300 MHz, CDCl3): d = 0.77–0.81 (m, 12 H), 1.19 (m, 12 H), 1.32 (m, 2 H), 1.41 (m, 2 H), 1.45 (m, 1 H), 1.48 (t, J = 7.5 Hz, 2 H), 1.69 (brs, 3 H), 1.95 (t, J = 7.2 Hz, 2 H), 4.77 (d, J = 7.5 Hz, 2 H), 5.40 (t, J = 6.0 Hz, 1 H), 7.28 (m, 2 H), 7.39 (m, 1 H), 7.97 ppm (d, J = 7.5 Hz, 2 H); 13 C NMR (75 MHz, CDCl3): d = 16.7, 19.7, 24.3, 24.7, 25.0, 25.5, 27.8, 32.6, 32.7, 36.6, 37.3, 37.4, 39.4, 39.8, 61.5, 118.6, 128.0, 129.6, 130.8, 142.1, 166.0 ppm; ESI-MS: m/z: 401 [M + H] + . 1-O-(m-Anisoyl)phytol (8): Viscous liquid; yield: 800 mg, 93 %; IR (CCl4): n˜ max = 1686, 1719, 2863, 2924 cm1; 1H NMR (300 MHz, CDCl3): d = 0.82–0.91 (m, 12 H), 1.12 (m, 12 H), 1.32 (m, 2 H), 1.38 (m, 2 H), 1.52 (m, 1 H), 1.78 (brs, 3 H), 2.03 (m, 4 H), 3.85 (s, 3 H), 4.82 (d, J = 7.2 Hz, 2 H), 5.46 (br, 1 H), 7.41 (d, J = 7.3 Hz, 1 H), 7.57 (s, 1 H), 7.64 (m, 1 H), 7.74 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.4, 19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.8, 36.8, 36.9, 37.8, 39.3, 39.8, 55.4, 61.9, 114.9, 118.1, 120.2, 122.8, 129.8, 131.8, 142.8, 159.8, 166.5 ppm; ESI-MS: m/z: 431 [M + H] + . 1-O-(3,4,5-Trimethoxybenzoyl)phytol (9): Viscous liquid; yield: 902 mg, 92 %; IR (CCl4): n˜ max = 1590, 1654, 1719, 2867, 2927, 2953 cm1; 1H NMR (300 MHz, CDCl3): d = 0.82–0.98 (m, 12 H), 1.12 (m, 12 H), 1.35 (m, 2 H), 1.38 (m, 2 H), 1.56 (m, 1 H), 1.68 (brs, 3 H), 2.15 (m, 4 H), 3.88 (s, 9 H), 4.89 (d, J = 7.2 Hz, 2 H), 5.08 (m, 1 H), 7.29 (brs, 1 H), 7.49 ppm (brs, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.4, 19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.8, 36.8, 36.9, 37.8, 39.3, 39.8, 55.6, 61.5, 69.2, 104.2, 118.1, 128.8, 141.4, 142.8, 153.6, 166.8 ppm; ESI-MS: m/z: 491 [M + H] + . 1-O-(2,3-Dichlorobenzoyl)phytol (10): Viscous liquid; yield: 613.5 mg, 72 %; IR (CCl4): n˜ max = 1578, 1636, 1717, 2862, 2926 cm1; 1 H NMR (300 MHz, CDCl3): d = 0.77–0.96 (m, 12 H, m), 1.17 (m, 12 H), 1.40 (m, 2 H), 1.46 (m, 2 H), 1.66 (m, 1 H), 1.72 (brs, 3 H), 2.05 (m, ChemMedChem 2014, 9, 1860 – 1868

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CHEMMEDCHEM FULL PAPERS 4 H), 4.85 (d, J = 7.2 Hz, 2 H), 5.12 (m, 1 H), 7.09 (m, 1 H), 7.13 (m, 1 H), 7.16 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 19.4, 20.1, 23.1, 24.8, 25.2, 27.5, 28.4, 33.1, 33.2, 37.4, 37.7, 37.8, 39.8, 62.5, 115.8, 128.3, 131.3, 132.2, 132.3, 134.2, 139.5, 142.1, 165.5 ppm; ESIMS: m/z: 427 [M + H] + . 1-O-Cinnamoylphytol (11): Viscous liquid; yield: 826 mg, 88 %; IR (CCl4): n˜ max = 1654, 1668, 1724, 2867 cm1; 1H NMR (300 MHz, CDCl3): d = 0.87 (m, 12 H), 1.15 (m, 12 H), 1.34 (m, 2 H), 1.38 (m, 2 H), 1.53 (m, 1 H), 1.75 (brs, 3 H), 2.04 (m, 4 H), 4.74 (d, J = 7.5 Hz, 2 H), 5.43 (t, J = 6.0 Hz, 1 H), 6.44 (d, J = 15.0 Hz, 1 H), 7.40 (m, 3 H), 7.54 (m, 2 H), 7.68 ppm (d, J = 16.2 Hz, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.4, 19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.7, 36.7, 36.9, 37.8, 39.3, 39.8, 61.5, 118.2, 128.0, 128.8, 130.4, 130.6, 134.4, 142.8, 144.6, 167.0 ppm; ESI-MS: m/z: 470 [M + H] + . Diphytyloxalate (12): Colorless oil; yield: 413.5 mg, 64 %; IR (CCl4): n˜ max = 1654, 1742, 2862, 2926 cm1; 1H NMR (300 MHz, CDCl3): d = 0.85–0.96 (m, 24 H), 1.26 (m, 24 H), 1.48 (m, 4 H), 1.55 (m, 4 H), 1.67 (m, 2 H), 1.74 (brs, 6 H), 2.37 (m, 8 H), 4.92 (d, J = 7.2 Hz, 4 H), 5.83 ppm (m, 2 H); 13C NMR (75 MHz, CDCl3): d = 16.3, 19.6, 22.6, 24.8, 25.0, 27.9, 33.2, 34.0, 37.1, 37.9, 39.8, 63.3, 121.9, 145.6, 164.2 ppm; ESI-MS: m/z: 647 [M + H] + . 1-Bromo-3,7,11,15-tetramethyl-2-hexadecene (13): PBr3 (3 mmol) was added to solution of phytol (2 mmol) in dry benzene. The reaction mixture was left at 4 8C for 30 min, followed by stirring at room temperature (30–35 8C) for 3 h. After completion, the solvent was removed by evaporation, and the residue was extracted with chloroform. The combined organic extracts were dried and evaporated to give crude products, which on purification by column chromatography on silica gel, afforded pure product 13 as a viscous dirty-white liquid in 86 % yield (619.5 mg) (purity > 95 %): IR (CCl4): n˜ max = 1664, 2928, 1460 cm1; 1H NMR (300 MHz, CDCl3): d = 0.83– 0.88 (m, 12H), 1.26 (m, 12 H), 1.38 (m, 2 H), 1.58 (m, 1 H), 1.62 (m, 2 H), 1.76 (brs, 3 H), 2.30 (m, 4 H), 3.57 (d, J = 6.8 Hz, 2 H), 5.25 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.2, 19.7, 22.6, 24.4, 24.8, 25.2, 27.9, 29.1, 32.7, 34.0, 36.8, 37.2, 37.8, 39.3, 39.8, 118.0, 141.1 ppm; ESI-MS: m/z: 359 and 361 [M + H] + , 381 [M + Na] + . 3,7,11,15-Tetramethyl-2-hexadecenal (14): PCC (3 mmol) was added to a solution of phytol (2 mmol) in dry CH2Cl2. The reaction mixture was heated at reflux for 6 h, then the solvent was removed by evaporation. The residue was extracted with chloroform in excess water. The combined organic extracts were dried and evaporated to give crude products which, after purification by column chromatography on silica gel, afforded pure product 14 as a viscous colorless liquid in 74 % yield (435.5 mg) (purity > 95 %): IR (CCl4): n˜ max = 1639, 1690, 2867, 2926, 2953 cm1; 1H NMR (300 MHz, CDCl3): d = 0.85 (m, 12 H), 1.14 (m, 12 H), 1.35 (m, 2 H), 1.37 (m, 2 H), 1.52 (m, 1 H), 1.67 (brs, 3 H), 1.97 (m, 4 H), 5.89 (d, J = 7.8 Hz, 1 H), 9.98 ppm (d, J = 7.8 Hz, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.4, 19.6, 22.6, 24.4, 24.8, 25.0, 27.9, 32.6, 32.7, 36.4, 36.6, 37.8, 39.3, 122.4, 142.8, 209.0 ppm; ESI-MS: m/z: 295 [M + H] + . 3,7,11,15-Tetramethyl-2’-phenyloctadec-1(1’),2(3)-diene-2’-one (15): BF3·Et2O (5 mmol) was gradually added to a stirred solution of 14 (2 mmol) and acetophenone (2 mmol). The reaction mixture was heated at reflux for 1 h and then left at room temperature overnight (14 h). After completion of the reaction, the mixture was diluted with diethyl ether (50 mL), followed by washing with distilled water (5  100 mL) to discharge the BF3·Et2O complex. The combined organic extract was dried over anhydrous Na2SO4 and evaporated to give crude products which, after purification by column chromatography on silica gel, afforded pure product 15 as a reddish viscous liquid in 62 % yield (491 mg) (purity > 95 %): IR  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org (CCl4): n˜ max = 1654, 1686, 1719, 2858, 2959 cm1; 1H NMR (300 MHz, CDCl3): d = 0.84–0.98 (m, 12 H), 1.18 (m, 12 H), 1.37 (m, 2 H), 1.52 (m, 1 H), 1.62 (m, 2 H), 1.77 (brs, 3 H), 2.02 (m, 4 H), 5.96 (d, J = 10.6 Hz, 1 H), 7.20 (d, J = 14 Hz, 1 H), 7.37 (m, 2 H), 7.39 (m, 1 H), 7.55 (m, 2 H, m), 7.59 ppm (m, 1 H); 13C NMR (75 MHz, CDCl3): d = 16.2, 20.2, 23.1, 24.1, 25.3, 25.6, 28.4, 32.3, 35.7, 37.9, 39.1, 39.8, 122.4, 125.5, 129.3, 131.3, 134.3, 135.2, 143.3, 146.6, 187.0 ppm; ESI-MS: m/z: 397 [M + H] + .

Biology In vitro antimicrobial and drug resistance reversal (DRR) assay: This assay used E. coli cultures, namely the NAL-resistant DH5a strain (MTCC 1652), procured from IMTEC (Chandigarh, India), and the NAL-sensitive strain, CA8000, which was a kind gift to CSIR-CIMAP by Dr. Sushil Kumar (Former Director, CIMAP, Lucknow, India). Multidrug-resistant clinical isolate KG4 (MDREC) was a kind gift from Dr. Mastan Singh and Dr. M. K. Gupta of K.G.M. University, Lucknow and was characterized previously.[25] The MIC values for antibacterial and DRR activities were determined by a twofold serial dilution broth assay and detected from the observatory data as per CLSI guidelines[37] using NAL and TET (Sigma–Aldrich) as positive controls. The MIC values reported were consistent across three independent experiments performed in duplicate. The DRR potential of various phytol derivatives was assessed by measuring the MIC value of NAL in combination with 10 mg mL1 of the derivatives against CA8000 and DH5a strains of E. coli, following procedures described in our earlier publications.[24, 25] In addition, synergy studies of most active combinations were also performed using the broth checkerboard method with TET against MDREC-KG4.[38] Cation-adjusted Mueller-Hinton broth (150 mL) was added to each well of a 96-well plate. The last four columns of wells served as controls for E. coli growth and plate sterility. The final concentrations ranged from 6.25 to 800 mg mL1 for tetracycline and from 0.78 to 100 mg mL1 for test compounds. Thus, each of the 64 wells had a unique combination of antibiotics and test compounds. The final bacterial inoculum in each well was 5  105 CFU mL1, except for the negative control. The plates were incubated at 37 8C for 24 h. The MIC was recorded as the last dilution without any turbidity, as per CLSI guidelines. Later, the MIC values were converted from mg mL1 to micromolar concentrations. Time–kill studies: Time–kill studies of TET alone and in combination with compounds 4, 9, 10, 11, and 14 against MDREC-KG4 strains was conducted at MIC, 2xMIC, and 4xMIC concentrations using a method described previously by Eliopoulos and Moellering.[39] Each analysis was done in triplicate with a control without test sample. Time–kill curves were derived by plotting log10 CFU mL1 against time (h). Time–kill kinetics were also studied in combinations of antibiotics and test compounds at the reduced concentrations at which maximum synergy was observed. Ethidium bromide efflux studies: Fluorometric determination of ethidium bromide (EB) efflux was performed as per reported methods.[40] To obtain metabolically active cells, MDREC-KG4 cultures were grown in 10 mL MHB (pH 7.3  0.2) with an optical density (OD) of 0.6 at 600 nm. The cells were collected by centrifugation at 16 060  g for 3 min and washed with phosphate-buffered saline (PBS). EB (25 mg mL1) was added to the bacterial suspension, which was then incubated for 60 min at 25 8C in the absence/presence of test compounds (4, 9, 10, 11, and 14) at their MECs. The EB-loaded bacterial suspensions were centrifuged at 16 060  g for 3 min, the supernatant was discarded, and the pellet was resuspended in cold PBS (1 ). The tubes were then placed on ice. AliChemMedChem 2014, 9, 1860 – 1868

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CHEMMEDCHEM FULL PAPERS quots (95 mL each) of the bacterial suspension were distributed in 0.3 mL 96-well plates. Loss of fluorescence was recorded for 30 min at regular intervals of 1 min at excitation and emission wavelengths of 530 nm and 585 nm, respectively, using a FLUOstar Omega spectrofluorimeter (BMG Labtech, Germany). ATPase inhibitory activity: The bacterial membrane protein was isolated by a previously described method.[25, 41] The ATPase assay was carried out in a 96-well microplate format using the Quantichrom ATPase Assay Kit (BioAssay Systems, USA). The initially optimal enzyme concentration was determined by a series of dilutions of enzyme (membrane protein containing ATPase) in assay buffer. The enzyme and inhibitor were first incubated for 5 min before adding the substrate. Reaction volumes of 40 mL were used, containing 20 mL assay buffer, 5 mL enzyme(20 mg mL1), 5 mL inhibitor, and 10 mL (4 mm) ATP, with controls without inhibitor in separate wells. At the end of the reaction, 200 mL malachite green reagent was added, and the mixture was incubated for 30 min at room temperature. The reagent forms a stable dark green color with liberated inorganic phosphate (Pi), which was measured spectrophotometrically on a plate reader (620 nm). qRT-PCR analysis: The transcriptional profile of the multidrug ABC transporter ATP-binding protein (YojI) gene was analyzed in the presence/absence of compounds 4, 9, 10, 11, and 14 in MDRECKG4 cells alone and in combinations. Cells were grown to mid-log phase in the presence of sub-inhibitory concentrations (25% of the MIC) of TET, 4, 9, 10, 11, and 14 alone and in combination. Realtime quantification of the RNA template was analyzed by SYBR GreenER qPCR supermix (Invitrogen, USA) using a 7900HT Fast Real-time PCR System (Applied Biosystems, USA). Observations were recorded in terms of log RQ after normalization of indigenous gene coding for d-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).[35]

In silico docking studies Docking studies were performed using Discovery Studio v 2.5. The three-dimensional structure of the YojI protein was modeled by the molecular threading methodology with the help of the I-Tasser server (Source: http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The ligands used included compounds 4, 9, 10, 11, 14, and RES [CID: 5770]. The scoring function employed in Discovery Studio v 2.5 calculates scores which includes LigScore1 and 2 (polar surface in receptor–ligand interactions), PLP1 and 2 (hydrogen bond formation), Jain (hydrophobic interactions), PMF (protein–ligand binding free energy), Ludi (degree of freedom), and Dock score. All of the derivatives and the positive control (RES) were docked into the active site of the receptors using the Ligand Fit option. The docked poses for compounds with lowest energy was recorded.

In silico ADME analysis In-silico ADME was performed for all of the compounds using descriptors such as aqueous solubility, human intestinal absorption, plasma protein binding, blood–brain barrier penetration, cytochrome P450 inhibition, and hepatotoxicity. The study was performed using Discovery Studio v 2.5 software.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org Acknowledgements Financial support for this research by CSIR Network project BSC0203 and the University Grants Commission (UGC) for a fellowship to H.C.U. is gratefully acknowledged. Keywords: antibiotics · docking studies · drug resistance · efflux pumps · phytol [1] K. E. Jones, N. G. Patel, M. A. Levy, A. Storeygard, D. Balk, J. L. Gittleman, P. Daszak, Nature 2008, 451, 990 – 993. [2] R. Gaynes, J. R. Edwards, National Nosocomial Infections Surveillance System, Clin. Infect. Dis. 2005, 42, 848 – 854. [3] R. A. Weinstein, Am. J. Med. 1991, 91, 179S – 184S. [4] A. Y. Peleg, D. C. Hooper, N. Engl. J. Med. 2010, 362, 1804 – 1813. [5] J. Davies, Science 1994, 264, 375 – 382. [6] F. C. Tenover, Am. J. Med. 2006, 119, S3 – S10. [7] M. MuÇoz-Ochoa, J. I. Murillo-lvarez, L. A. ZermeÇo-Cervantes, S. Martnez-Diaz, R. Rodrguez-Riosmena, Eur. Rev. Med. Pharmacol. Sci. 2010, 14, 739 – 747. [8] H. Nikaido, J. Bacteriol. 1996, 178, 5853 – 5859. [9] M. Markowitz, B. Y. Nguyen, E. Gotuzzo, F. Mendo, W. Ratanasuwan, C. Kovacs, G. Prada, J. O. Morales-Ramirez, C. S. Crumpacker, R. D. Isaacs, L. R. Gilde, H. Wan, M. D. Miller, L. A. Wenning, H. Teppler, Protocol 004 Part II Study Team, J. Acquired Immune Defic. Syndr. 2007, 46, 125 – 133. [10] K. A. Marr, M. Boeckh, R. A. Carter, H. W. Kim, L. Corey, Clin. Infect. Dis. 2004, 39, 797 – 802. [11] P. B. Bloland, M. Ettling, S. Meek, Bull. World Health Organ. 2000, 78, 1378 – 1388. [12] M. Martins, S. G. Dastidar, S. Fanning, J. E. Kristiansen, J. Molnar, J.-M. Pages, Z. Schelz, G. Spengler, M. Viveiros, L. Amaral, Int. J. Antimicrob. Agents 2008, 31, 198 – 208. [13] Antibiotic pharmaceutical composition with lysergol as bio-enhancer and method of treatment : S. P. S. Khanuja, J. S. Arya, S. K. Srivastava, A. K. Shasany, T. R. Santhakumar, M. P. Darokar, S. Kumar, Int. PCT Pub. No. WO 2003080059 A1, US Pat. No. US 20030181425, Indian Pat. No. IN 233906, 2003. [14] S. P. S. Khanuja, J. S. Arya, R. S. K. Tiruppadiripulivur, D. Saikia, H. Kaur, M. Singh, S. C. Gupta, A. K. Shasany, M. P. Darokar, S. K. Srivastava, M. M. Gupta, S. C. Verma, A. Pal, Nitrile glycosides useful as a bio-enhancer of drugs and nutrients, process of its isolation from Moringa oleifera. US Patent 6,858,588, 2005. [15] J. Jia, F. Zhu, X. Ma, Z. W. Cao, Y. X. Li, Y. Z. Chen, Nat. Rev. Drug Discovery 2009, 8, 111 – 128. [16] G. Cottarel, J. Wierzbowski, Trends Biotechnol. 2007, 25, 547 – 555. [17] D. D. Raga, R. A. Espiritu, C.-C. Shen, C. Y. Ragasa, J. Nat. Med. 2011, 65, 206 – 211. [18] C. Y. Ragasa, E. S. C. Javier, I. G. Tan, Philipp. J. Sci. 2003, 132, 21 – 25. [19] J. Gloerich, D. M. van den Brink, J. P. N. Ruiter, N. van Vlies, F. M. Vaz, R. J. A. Wanders, S. Ferdinandusse, J. Lipid Res. 2007, 48, 77 – 85. [20] M. S. Butler, A. D. Buss, Biochem. Pharmacol. 2006, 71, 919 – 929. [21] J. P. Saludes, M. J. Garson, S. G. Franzblau, A. M. Aguinaldo, Phytother. Res. 2002, 16, 683 – 685. [22] M. S. Rajab, C. L. Cantrell, S. G. Franzblau, N. H. Fischer, Planta Med. 1998, 64, 2 – 4. [23] D. Saikia, S. Parihar, D. Chanda, S. Ojha, J. K. Kumar, C. S. Chanotiya, K. Shanker, A. S. Negi, Bioorg. Med. Chem. Lett. 2010, 20, 508 – 512. [24] H. C. Upadhyay, G. R. Dwivedi, M. P. Darokar, V. Chaturvedi, S. K. Srivastava, Planta Med. 2012, 78, 79 – 81. [25] A. Maurya, G. R. Dwivedi, S. K. Srivastava, M. P. Darokar, Chem. Biol. Drug Des. 2013, 81, 484 – 490. [26] V. K. Gupta, S. Verma, A. Pal, S. K. Srivastava, P. K. Srivastava, M. P. Darokar, Appl. Microbiol. Biotechnol. 2013, 97, 9121 – 9131. [27] H. C. Upadhyay, J. P. Thakur, D. Saikia, S. K. Srivastava, Med. Chem. Res. 2013, 22, 16 – 21. [28] M. L. Go, X. Wu, X. L. Liu, Curr. Med. Chem. 2005, 12, 481 – 499. [29] B. A. Bhat, K. L. Dhar, S. C. Puri, A. K. Saxena, M. Shanmugavel, G. N. Qazi, Bioorg. Med. Chem. Lett. 2005, 15, 3177 – 3180.

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Received: February 14, 2014 Revised: April 28, 2014 Published online on May 28, 2014

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