In Vitro Antibacterial Activity of Prenylated ... - ACS Publications

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In Vitro Antibacterial Activity of Prenylated Guanidine Alkaloids from Pterogyne nitens and Synthetic Analogues Aline Coqueiro,†,‡ Luis Octávio Regasini,† Paul Stapleton,‡ Vanderlan da Silva Bolzani,*,† and Simon Gibbons*,‡ †

Department of Organic Chemistry, Institute of Chemistry, São Paulo State University, Rua Prof. Francisco Degni 55, Araraquara14800-900, Brazil ‡ Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom S Supporting Information *

ABSTRACT: The present investigation deals with the antibiotic activity of eight natural guanidine alkaloids and two synthetic analogues against a variety of clinically relevant methicillin-resistant Staphylococcus aureus strains. Galegine (1) and pterogynidine (2) were the most potent compounds, with a minimum inhibitory concentration of 4 mg/L, to all tested strains. The preliminary chemical features correlating to anti-MRSA activity showed that the size of the side chain and the substitution pattern in the guanidine core played a key role in the antibacterial activity of the imino group. Guanidine alkaloids 1 and 2 are promising molecular models for further synthetic derivatives and, thus, for medicinal chemistry studies.

W

(Fabaceae, Caesalpinioideae) is one of these species and is catalogued in our database (NuBBEDB).8 Pterogyne nitens is a native tree of South America and is popularly known as “balsam”, “cocal”, “yvira-ró” , and “amendoinzeiro”. It bears attractive flowers and fruits and, therefore, has application as an ornamental plant.9 Although scientists have not frequently reported the medicinal uses of this species, the Paraguayan population has used aqueous preparations from the stem bark of P. nitens to treat ascariasis.10 Our previous phytochemical studies on the leaves, flowers, fruits, bark, and roots of P. nitens led to the isolation of guanidine alkaloids, which are unusual in plants, as well as flavones, flavonols, and terpenoids.11−15 Several articles have reported that these guanidine alkaloids possess different biological activities including cytotoxic, antimutagenic, antiangiogenic, pro-apoptotic, and antifungal actions.11,16−20 While guanidine alkaloids belonging to marine sources display a broad spectrum of actions,21 few studies have dealt with higher plants, where these compounds rarely occur. As part of the SisBiotaCNPq/FAPESP Biodiscovery and CIBIFar Programs, which aim to search for new antibacterial agents from the Brazilian flora, this investigation evaluated the antibacterial activity of eight guanidine alkaloids from P. nitens and two synthetic analogues using different multi-drug-resistant strains. In the present work, we tested a panel of Staphylococcus aureus strains with clinical relevance, possessing different

hen antimicrobials were made available for therapeutic use over 60 years ago, they were considered miracle drugs. However, their indiscriminate use and popularity have contributed to the appearance of many resistant bacterial strains, which has made antimicrobials progressively less effective over the past decade. Infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) currently constitute a major public health concern in hospitals and communities, and these bacteria have developed resistance to the most commonly used antibiotics.1 Indeed, S. aureus has acquired resistance to many classes of antibiotics including the glycopeptide family (such as vancomycin and teicoplanin), oxazolidinones (linezolid), and a combination of streptogramins (quinupristin and dalfopristin).2 Therefore, developing new classes of antibiotics to overcome the issue of drug resistance is mandatory.3 Natural products remain a superb source of chemical diversity and potential drugs. Thousands of natural products have been isolated, some of which exhibit great antimicrobial potential for medicinal use.4 Despite the potent antibacterial activity of some plant secondary metabolites2 and the ability of some of these compounds to modify the resistance associated with MDR strains5 and efflux pumps,6 plants are still an underexplored and underexploited source of antimicrobial agents. In our current research on biologically active natural products from Brazilian biodiversity, we have screened extracts obtained from many plant species collected mainly in the Cerrado and the Atlantic Forest, two biomes in the State of São Paulo seriously threatened by extinction.7 Pterogyne nitens Tul. © 2014 American Chemical Society and American Society of Pharmacognosy

Received: March 28, 2014 Published: August 4, 2014 1972

dx.doi.org/10.1021/np500281c | J. Nat. Prod. 2014, 77, 1972−1975

Journal of Natural Products

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site compared to their parent compounds, demonstrating a potent effect.29 The guanidines are strong bases, and the reactivity of this chemical group can be attributed to the electron-donating nitrogen atoms that are present in a planar structure, which can participate in well-defined directional hydrogen bonds. Due to the similarities of the structures of the tested guanidine alkaloids, it was possible to suggest preliminary chemical features that were correlated to anti-MRSA activity. The most active compounds 1 and 2 possess one and two prenyl groups, respectively, and showed the same MIC values (4 mg/L) for all tested strains, but when the prenyl groups were substituted by one or two geranyl groups, the activity decreased (3, 4, and 6: MICs of 8−32 mg/L), being independent of which position the group was introduced. The same phenomenon was observed for compound 7, a cyclic guanidine, and 5, which possesses two methyl substituents in the guanidine core. Compounds 3−7 had poorer activity when compared to the prenylated guanidines (1 and 2). Interestingly, guanidine 8 (N-1,N-2,N-3-tri-isopentenylguanidine), the only compound possessing at least one substituent on each nitrogen, was the sole inactive compound. This information showed that the size of the side chain and the substitution pattern in the guanidine core appeared to play a key role in the antibacterial activity. This may be attributed to the fact that an increase in the size of the side chain decreased the electron donation ability of the nitrogen. When three of the nitrogen atoms in the guanidine core were substituted (e.g., 8), the steric effects could affect the reactivity and binding of these molecules. This spatial shielding effect makes potential interactions more difficult and less likely. The steric effect means that it would be more difficult to get close enough to allow attack by the lone pair of the nitrogen or to form hydrogen bonds. The side chains can also serve as modulators of lipid affinity and, therefore, cellular bioavailability; so when the size or number of side chains is increased, this enhances the membrane permeability but may result in poorer aqueous solubility, leading to a loss of activity.23 To confirm the importance of the imino group in the antimicrobial activity of the guanidines, we tested two analogues of nitensidine A that were synthesized by replacing the imino nitrogen atom in nitensidine A by sulfur (nitensidine AT) and oxygen (nitensidine AU). The antimicrobial activity of nitensidine A was reduced by replacing the imino nitrogen by sulfur (S), and the activity was extinguished when the imino group was substituted by oxygen (O). These results confirmed the requirement of the imino nitrogen atom for antibacterial activity. Nitensidine AU (10) did not show activity (MIC > 512 mg/L); however the oxygen atom can also participate in hydrogen bonds, and while they can contribute to the activity, they are not crucial. On the other hand, the basicity of the imino nitrogen in the guanidine core seems to play a key role in the antimicrobial activity. The steric effect was not high for galegine (1) and pterogynidine (2), demonstrating that one or two prenyl groups did not interfere in the interaction of the imino group and were optimal in this study in terms of lipophilicity and could explain the noteworthy activity of 1 and 2. These compounds were 8 times more potent than norfloxacin against the multi-drug-resistant strain that overexpresses the NorA efflux transporter (SA-1199B) and 32 times more potent than norfloxacin against the most resistant strain, EMRSA-16.

mechanisms of resistance. Among these, we tested strain SA1199B, a multi-drug-resistant strain that overexpresses the NorA efflux pump22 and also possesses a gyrase-encoding gene mutation that in addition to NorA confers a high level of resistance to some fluoroquinolones;23 a macrolide-resistant strain (RN4220);24 a clinical MRSA (XU212), resistant to tetracycline bearing the TetK efflux pump;25 a standard laboratory strain (ATCC25923);25 and the methicillin-resistant strains EMRSA-1526 and EMRSA-16.27 All of the natural guanidine alkaloids (1−8) and two synthetic analogues (9 and 10) were tested against the six S. aureus strains, and the minimum inhibitory concentration (MIC) was determined for the active alkaloids. All the guanidine alkaloids were tested at concentrations ranging from 512 to 0.5 mg/L.

With the exception of alchorneine (8), which was inactive (MIC > 512 mg/L), all of the natural alkaloids displayed activity stronger than or similar to norfloxacin against S. aureus SA-1199B, XU212, and EMRSA-16 strains. Galegine (1) and pterogynidine (2) were the most potent compounds among the investigated natural guanidines for all tested strains (Table 1). Previous work with synthetic guanidines has shown the antimicrobial potential of these compounds against drugresistant pathogens.28,29 Fair et al. showed that, by replacement of hydroxyl or amine groups with guanidine groups, the derivatives were superior binders of the bacterial ribosomal A1973



Note

Table 1. In Vitro Antibacterial Activity (MIC in mg/L and μM) of Guanidine Alkaloids 1−8 from Pterogyne nitens and the Synthetic Analogues 9 and 10 against Staphylococcus aureus Strains compound

SA-1199B

RN4220

XU212

ATCC25943

EMRSA-15

EMRSA-16

1 2 3 4 5 6 7 8 9 10 norfloxacin

4/31.4 4/20.5 32/96.5 32/96.5 32/89.0 32/163.8 32/165.6 >512 128/367.5 >512 32/100.2

4/31.4 4/20.5 16/48.3 16/48.3 16/44.5 16/81.9 16/82.8 >512 128/367.5 >512 0.5/1.6

4/31.4 4/20.5 8/24.1 8/24.1 8/22.2 8/40.9 8/41.4 >512 128/367.5 >512 8/25.1

4/31.4 4/20.5 16/48.3 16/48.3 16/44.5 16/81.9 32−16/165.6−82.8 >512 128/367.5 >512 0.5/1.6

4/31.4 4/20.5 32−16/96.5−48.3 32/96.5 32/89.0 16/81.9 32/165.6 >512 >512 >512 0.5/1.6

4/31.4 4/20.5 16−8/48.3−24.1 8/24.1 8/22.2 8/40.9 16−8/82.8−41.4 >512 64−32/183.8−91.9 >512 128/400.8

RN4220, which is resistant to the macrolide erythromycin, was provided by Dr. J. Cove.24 EMRSA-1526 and EMRSA-1627 were provided by Dr. Paul Stapleton. Strain SA-1199B, which overexpresses the NorA MDR efflux pump, was the gift of Prof. Glenn W. Kaatz.23 Minimum Inhibitory Concentration. All of the strains were cultured on nutrient agar (Oxoid) and incubated for 24 h at 37 °C prior to MIC determination. An inoculum density of 1 × 106 cfu/mL of each S. aureus strain was prepared in normal saline (9 g/L) by comparison with a 0.5 MacFarland turbidity standard and appropriate dilution. Norfloxacin and the samples were dissolved in DMSO and diluted in cation-adjusted Mueller-Hinton broth (MHB) to give a starting concentration of 1024 μg/L. Using Nunc 96-well microtiter plates, 125 μL of MHB was dispensed into wells 1−11. Then, 125 μL of the test compound or the appropriate antibiotic was dispensed into well 1 and serially diluted across the plate, leaving well 11 empty for growth control. The final volume was dispensed into well 12, which was free of MHB or inoculum and served as the sterile control. Finally, the bacterial inoculum (125 μL) was added to wells 1−11, and the plate was incubated at 37 °C for 18 h. A DMSO control was also included. For MIC determination, 20 μL of a 5 mg/mL methanol solution of 3-[4,5-dimethylthiazol-2 yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) was added to each of the wells and incubated for 20 min. A color change from yellow to dark blue indicated bacterial growth. The MIC was recorded as the lowest concentration at which no growth was observed.22 Norfloxacin was used as positive control and was purchased from Sigma Chemical Co. Mueller-Hinton broth (Oxoid) was adjusted to contain 20 mg/L Ca2+ and 10 mg/L Mg2+. Minimum Bactericidal Concentration. The samples were prepared using the same protocol that was employed for the MIC assays and were added to the same inoculum density of the SA-1199B strain. The 96-well plates were incubated at 37 °C for 24 h. After incubation 10 μL of each solution at different concentrations of the tested compound were transferred to Petri dishes containing the corresponding drug-free culture medium (Mueller-Hinton agar). The Petri plates were then incubated for a further 24 h. The MBC was obtained by observing the growth of colonies on Petri dishes after 24 h of incubation; an observed 99.9% reduction in bacterial cell numbers from the starting inoculum was considered as the bactericidal end point. MBC values equivalent to or exhibiting no more than a 2-fold difference from the MIC of the agent would indicate a bactericidal drug. An MBC value of 8-fold or higher than the MIC would indicate a bacteriostatic drug.

In a previous study by Regasini et al., on the cytotoxicity of guanidine alkaloids, a larger number of prenyl groups elicited more potent activity.11 The authors found that 7 was the most active alkaloid against a number of cell lines; compounds 1, 2, and 6 were inactive against all of the human tested cell lines.11 On the basis of this information, we concluded that toxicity against bacteria was selective and that compounds 1 and 2, which did not harm tested human cell lines in a previous study, were the most active against prokaryotic cells. The minimum bactericidal concentration (MBC) assays were conducted on the most active alkaloidsgalegine (1) and pterogynidine (2)to verify whether they possessed a bactericidal or bacteriostatic effect. Ten microliters of sample from the wells of a plate used to determine the MIC values of compounds 1−7 against SA-1199B was removed where no growth was observed (i.e., the MIC and above) and plated out onto drug-free media. After 24 h incubation, no growth was observed at the MIC or higher, indicating that the compound had killed the organism, rather than just inhibiting its growth, suggesting that these agents had bactericidal activities; the MIC and MBC values were 4 mg/L. Guanidine alkaloids from P. nitens displayed antibacterial activity against multi-drug-resistant strains (MRSA). Galegine and pterogynidine were effective against different strains, mainly SA-1199B and MRSA-16, which were resistant to the standard drug norfloxacin. Therefore, guanidine alkaloids 1 and 2 are promising hits for further medicinal chemistry investigations to synthesize anti-MRSA drug leads. Further studies on these bactericidal compounds are necessary to determine the mechanism of action involved in the activity against MRSA.

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EXPERIMENTAL SECTION

Plant Material. Leaves and branches of Pterogyne nitens Tul., Fabaceae, were collected from the Botanic Garden of São Paulo (São Paulo, Brazil) by Dr. Maria C. M. Young in May 2003 and January 2005, respectively, and identified by Dr. Inês Cordeiro (IBt-SMA). Two voucher specimens (SP203419 and SP204319b) were deposited at the State Herbarium “Maria Eneida Kaufmann” of the Institute of Botany (São Paulo, Brazil). Test Compounds. The extraction, isolation, and the physicochemical and spectroscopy data of the guanidine alkaloids 1−8 from leaves and stem branches of P. nitens Tul. have been previously described by Regasini and co-workers.11,17 The synthesis, purification, and identification of nitensidines AT (9) and AU (10) were previously reported by Tajima et al.30 Bacterial Strains. A standard S. aureus susceptibility testing reference strain (ATCC 25923) and a clinical MRSA isolate (XU212) bearing the TetK efflux pump were obtained from Dr. E. Udo.25 Strain

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ASSOCIATED CONTENT

* Supporting Information S

The authors declare no competing financial interest. This material is available free of charge via the Internet at http:// pubs.acs.org. 1974



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(22) Kaatz, G. W.; Seo, S. M.; Ruble, C. A. Antimicrob. Agents Chemother. 1993, 37, 1086−1094. (23) Appendino, A.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.; Stavri, M.; Smith, E.; Rahman, M. J. Nat. Prod. 2008, 71, 1427−1430. (24) Ross, J. I.; Farrell, A. M.; Eady, E. A.; Cove, J. H.; Cunliffe, W. J. J. Antimicrob. Chemother. 1989, 24, 851−862. (25) Gibbons, S.; Udo, E. E. Phytother. Res. 2000, 14, 139−140. (26) Richardson, J. F.; Reith, S. J. Hosp. Infect. 1993, 25, 45−52. (27) Cox, R. A.; Conquest, C.; Mallaghan, C.; Maples, R. R. J. Hosp. Infect. 1995, 29, 87−106. (28) Hensler, M. E.; Bernstein, G.; Nizet, V.; Nefzi, A. Bioorg. Med. Chem. Lett. 2006, 16, 5073−5079. (29) Fair, R. J.; Hensler, M. E.; Thienphrapa, W.; Dam, Q. N.; Nizet, V.; Tor, Y. ChemMedChem 2012, 7, 1237−1244. (30) Tajima, Y.; Nakagawa, H.; Tamura, A.; Kadioglu, O.; Satake, K.; Mitani, Y.; Murase, H.; Regasini, L. O.; Bolzani, V. S.; Ishikawa, T.; Fricker, G.; Efferth, T. Phytomedicine 2014, 21, 323−332.

AUTHOR INFORMATION

Corresponding Authors

*(V. S. Bolzani) Tel: +55-16-3301-9660. Fax: +55-16-33222308. E-mail: [email protected]. *(S. Gibbons) Tel: +44-207-7535913. Fax: +44-207-7535964. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by grants 06/61187-7, 03/00886-7, and 2011/51313-3 from São Paulo Research Foundation (FAPESP), awarded to V.S.B., A.C., and L.O.R, as part of the BiotaFAPESP, the Biodiversity Virtual Institute Program (www. biotasp.org.br). We also acknowledge CNPq, CAPES, and FAPESP for researcher and student fellowships.

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REFERENCES

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In Vitro Antibacterial Activity of Prenylated ... - ACS Publications

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