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Keywords:

  • antifungal agent;
  • molecular docking;
  • synthesis;
  • triazole

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

A series of triazole antifungal agents with piperidine side chains were designed and synthesized. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum. Moreover, a molecular model for the binding between compound 12 and the active site of CACYP51 was provided based on the computational docking results. The side chain of the compound 12 is oriented into substrate access channel 2 (FG loop) and forms hydrophobic and van der waals interactions with surrounding hydrophobic residues. The phenyl group of the side chain can interact with the phenyl group of Phe380 through the formation of π-π face-to-edge interaction.

Abbreviations:
CYP51

the cytochrome P450 14α-demethylase

MICs

the minimal inhibitory concentrations

C. alb

Candida albicans

C. par

Candida parapsilosis

C. tro

Candida tropicalis

Cry. neo

Cryptococcus neoformans

F. com

Fonsecaea compacta

T. rub

Trichophyton rubrum

M. gyp

Microsporum gypseum

A. fum

Aspergillus fumigatus

FCZ

fluconazole

ICZ

itraconazole

VCZ

voriconazole

Mp

melt point

1H NMR

hydrogen nuclear magnetic resonance

13C NMR

carbon nuclear magnetic resonance

IR

infrared spectrum

API-ES

atmospheric pressure electrospray ionization mass spectrometry

NCCLS

National Committee for Clinical Laboratory Standards

The incidence of systemic fungal infections such as Candidosis, Cryptococcosis, and Aspergillosis has been increasing in prevalence recently and is associated with the increase in the number of immunocompromised hosts. Opportunistic and invasive fungal infections have become the important causes of morbidity and mortality (1,2). In addition, the alarming rates of the growing emergence of antifungal resistance in hospitals are major concerns to the public health and scientific communities worldwide (3,4). Therefore, all these trends have emphasized the urgent need for new, more effective, and safe antifungal agents.

Among the attractive approaches to find novel antifungal agents, the structural modification or optimization of the existing agents has provoked special interest in the realm of medical chemistry. One kind of them used widely and efficiently is azoles that are increasing in number and diversity, such as fluconazole (FCZ), itraconazole (ICZ), ravuconazole (VCZ), and posaconazole. FCZ, the predominant azole agent, is a water-soluble triazole and also has a very low incidence of side effects. However, the widespread use of antifungal drugs and their resistance against fungal infections has led to serious health problems (5–7). In recent years, some of the current azole antifungal drugs are designed and used in clinic. But either the highly toxic or the low bioavailability restrains its usage, such as ICZ and VCZ. Therefore, the search for a novel, more effective antifungal agent with lower toxicity continues to be an area of investigation into medicinal chemistry.

Azoles exert antifungal activity through inhibition of the cytochrome P450 14α-demethylase (CYP51), which is crucial in the process of biosynthesis of ergosterol by a mechanism in which the heterocyclic nitrogen atom (N-4 of 1,2,4-triazole) binds to the heme iron atom (8). In our previous research, we have designed highly potent azole derivatives with different C-3 side chains (9–14). In the present study, we choose compound B1 [with the MIC80 value of 0.25 mg/mL against Candida albicans (C. alb)] as the lead compound (Figure 1) (15). We focused on modifying the side chain without loss of key interactions. As we all know the importance of the piperazinyl for the antifungal activity (12,14), we sought to investigate this further with heterocycles piperidinol, containing an oxygen atom (Figure 2). This replacement was based on the following considerations: (i) the oxygen atom could improve the flexibility of the molecule, making the side chain more easily lock its proper position; and (ii) the oxygen atom may interact with the amino acid residues through hydrogen bonding. We designed all the title compounds to keep the preferred combination of the piperidinol at C-3. Besides, we focused our attention on installing various substituted acids containing aromatic ring, such as benzenes, pyridines, and furans.

image

Figure 1.  Structure of the lead compound B1.

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image

Figure 2.  Structure and the modified position of the designed compound.

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Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

All reactions were monitored by thin-layer chromatography (Huanghai, Yantai, China). Compounds were visualized by UV light (Yukang, Shanghai, China). Melting points were measured on a YamatoMP-21 melting-point apparatus and are uncorrected. Infrared spectra were recorded in potassium bromide disks on a HITACHI270-50 spectrophotometer. NMR spectra were recorded on a Bruker AC-500P spectrometer at 500 MHz for 1H NMR and 125 MHz for 13C NMR with TMS as the internal standard. Chemical shifts are expressed in δ (ppm). Column chromatography was carried out using silica gel (300–400 mesh) (Huanghai, Yantai, China). Silica gel chromatography solvents were of analytical grade. Atmospheric pressure electrospray ionization mass spectrometry (API-ES) experiment was carried out in an API-3000 LC-MS spectrometer. All reaction solvents were dried prior to use according to standard procedures.

Chemistry

The general synthetic methodology for the preparation of title compounds (637) is outlined in Scheme 1. As a key intermediate of our designed triazole antifungals, the oxirane compound 4 was synthesized by the reported procedure (16). The intermediate 5 was synthesized by ring-open reaction of oxirane 4 with 4-piperidinol. The title compounds were synthesized, and the good yield was obtained when the reaction was performed in CH2Cl2 in the presence of DMAP (4-dimethylaminopyridine) and EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl). All the new compounds (637) described previously were characterized by IR, API-ES, and NMR spectroscopic analysis.

image

Figure Scheme 1:.  Conditions: (a) ClCH2COCl, AlCl3, 50 °C, 5 h, in 87% yield; (b) C6H5CH3, NaHCO3, 1H-1,2,4-triazole, reflux, 5 h, in 47% yield; (c) C6H5CH3, (CH3)3SOI, NaOH, centylmethylammonium bromide, 60 °C, 3 h, in 56% yield; (d) CH3SO3H, 0 °C, 1 h, in 89% yield; (e) CH3CH2OH, Et3N, 4-piperidinol, reflux, 6 h, in 78% yield; (f) various acids, DMAP, EDCI, CH2Cl2, reflux, 8 h, in 67–81% yield.

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The procedure for the synthesis of the compound 5 1-[2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)-propyl]-piperidin-4-ol (5)

To a stirred mixture of 1-[2-(2,4-difluorophenyl)-2,3-epoxypropyl]-1H-1,2,4-triazole methanesulfonate (4) (1.65 g, 0.005 mol), C2H5OH (30 mL), and N(C2H5)3 (3 mL), 4-piperidinol (0.70 g, 0.006 mol) was added and refluxed for 6 h. The reaction was monitored by TLC. After filtration, the filtrate was evaporated under reduced pressure. Water (30 mL) was added to the residue, which was then extracted with ethyl acetate (80 mL × 3). The extract was washed with saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and evaporated. The residue was separated and purified readily by chromatography on silica gel to afford compound 5, 1.37 g.

General procedure for the preparation of the compounds 637

To a stirred mixture of 1-[2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)-propyl]-piperidin-4-ol (5) (0.338 g, 0.001 mol), DMAP (100 mg) and EDCI (200 mg) in 50 mL of dichloromethane under 0 °C and substituted acid (0.001 mol) were added. After 1 h, the reaction was heated and refluxed for 8 h. The reaction was monitored by TLC. After filtration, the filtrate was evaporated under reduced pressure. The residue was then extracted with ethyl acetate (60 mL × 3). The extract was washed with saturated NaCl solution (20 mL × 3), dried over anhydrous Na2SO4, and evaporated. The residue was crystallized from ethyl acetate to afford the title compounds, in 67.0–81.0% yield (Appendix S1).

Biological activity

The in vitro antifungal activities of all the title compounds were evaluated against eight human pathogenic fungi, C. alb, Candida parapsilosis (C. par), Candida tropicalis (C. tro), Cryptococcus neoformans (Cry. neo), Fonsecaea compacta (F. com), Trichophyton rubrum (T. rub), Microsporum gypseum (M. gyp), and Aspergillus fumigates (A. fum), which are often encountered clinically, and were compared with FCZ, ICZ, and VCZ. C. alb and Cry. neo were provided by Shanghai Changzheng Hospital; C. par, C. tro, F. com, T. rub, M. gyp, and A. fum were provided by Shanghai Changhai Hospital. C. alb and Cry. neo were purchased from ATCC, and other strains were clinic isolates. C. alb (ATCCY0109) and Cry. neo (ATCCBLS108) were used as the quality-controlled strains and tested in each assay. Intermediate 5 and compound B1 that served as the negative control were synthesized by us, while FLC, ICZ, and VCZ that served as the positive control were obtained from their respective manufacturers.

The antifungal potency was evaluated by means of the minimal inhibitory concentration (MICs), using the serial test in the broth microdilution modification method published by the National Committee for Clinical Laboratory Standard (NCCLS) method M27-A2 (17). The MIC80 was defined as the first well with an approximate 80% reduction in growth compared with the growth of the drug-free well. For assays, the title compounds to be tested were dissolved in dimethyl sulfoxide (DMSO), serially diluted in growth medium, and inoculated and incubated at 35 °C. Growth MIC was determined at 24 h for C. alb and at 72 h for Cry. neo. The results of assays are summarized in Table 1. The data point from the mean of replicates. All of our susceptibility tests were performed three times by each antifungal agent. After the antifungal test, none of the tested compounds were changed or hydrolyzed, which were detected by TLC, 1H NMR, and LC-MS.

Table 1.   Antifungal activities of the title compounds in vitro (MIC80, μg/mL)
CompoundC. alb Y0109C. par 0306392C. troC. neo BLS108F. comT. rub 0501124M. gyp 0310388A. fum 0504656
584166432168>64
60.00390.01560.01560.06250.250.250.06251
70.01560.01560.06250.06250.06250.250.06251
80.01560.06250.06250.0625110.251
90.00390.00390.01560.01560.250.06250.06251
100.01560.06250.062510.250.250.01564
110.00390.01560.01560.06250.06250.06250.06250.25
120.00390.01560.01560.01560.250.06250.06250.25
130.00390.01560.06250.250.250.06250.06254
140.06250.06250.06250.062510.250.25>64
150.06250.06250.250.06250.250.06250.251
160.01560.01560.06250.06250.06250.250.254
170.00390.01560.01560.06250.06250.06250.062516
180.00390.00390.01560.06250.250.250.06250.25
190.01560.250.06250.2510.06250.251
200.01560.06250.06250.0625110.251
210.06250.250.2511111
220.06250.250.06250.250.250.250.254
230.00390.01560.06250.06250.06250.06250.06251
240.01560.06250.06250.250.250.06250.06254
250.06250.01560.250.2511116
260.00390.06250.250.250.250.06250.254
270.00390.00390.01560.01560.01560.06250.06250.25
280.06250.00390.00390.00390.06250.06250.01560.25
290.01560.06250.06250.06250.250.250.06251
300.06250.06250.06250.06250.250.06250.06251
310.01560.250.250.015610.250.251
320.062510.250.2510.250.251
330.062510.25110.250.254
340.0625111410.254
350.062510.250.2510.250.251
360.06250.250.250.25410.251
370.01560.250.06250.250.250.250.251
B10.250.250.254444>64
FCZ0.250.25111641>64
ICZ0.06250.250.06250.250.250.06250.251
VCZ0.00390.01560.01560.01560.06250.01560.01560.25

Protocol of docking study

Based on the previous study (18), we constructed and modified the 3D model of CACYP51. All the molecular modeling calculations were performed using sybyl 6.9 version (Tripos, St. Louis, MO, USA). The structures of the compounds were assigned with Gasteiger–Hückle partial atomic charges. Energy minimization was performed using the Tripos force field, Powell optimization method, and MAXIMIN2 minimizer with a convergence criterion of 0.001 Kcal/mol. Simulated annealing was then performed. The system was heated to 1000 K for 1.0 ps and then annealed to 250 K for 1.5 ps. The annealing function was exponential; 50 such cycles of annealing were run, and the resulting 50 conformers were optimized using methods described previously. The lowest energy conformation was selected. All the other parameters were default values.

Results and Discussions

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

In vitro antifungal activity assay (Table 1) indicates that all the synthesized compounds (637) showed excellent activity and broad spectrum against all the tested fungal pathogens. The minimal inhibitory concentration (MIC80) value indicated that the title compounds showed excellent antifungal activities against C. alb, which is the most common cause of life-threatening fungal infections. Most of the compounds were more potent than compound B1, even more potent than FCZ and ICZ with their MIC80 value in the range of 0.0039–0.0625 μg/mL. Noticeably, the MIC80 value of compound 6,9,11,12,13,17,18,23,26, and 27 was 64 times lower than that of compound B1 (with the MIC80 value of 0.25 μg/mL) against C. alb in vitro and even the same as the positive control ICZ (with the MIC80 value of 0.0039 μg/mL). Moreover, these compounds also showed excellent inhibitory activity against other Candida spp., such as C. tro and C. par, with their MIC80 value in the range of 0.0039–1 μg/mL (compound B1 only showed 0.25 μg/mL against the two fungi). Cry. neo has a worldwide distribution and is the most common cause of life-threatening fungal infections. The inhibitory activity of the title compounds against Cry. neo was superior to that of compound B1. The MIC80 value of compound 28 (with the MIC80 value of 0.0039 μg/mL) was 1025 times higher than that of compound B1. Compound B1 was not effective against Aspergillus fumigates (A. fum), while our compounds showed moderate activity. The MIC80 value of compounds 11, 12, 18, 27, and 28 against A. fum was 0.25 μg/mL. Moreover, the designed compounds also showed good activity against dermatophytes (T. rub and M. gyp). For example, compounds 10 and 28 (with the MIC80 value of 0.0156 μg/mL) were 256 fold more potent against M. gyp than compound B1.

Compared with the lead compound B1, the title compounds exhibit excellent antifungal activity against C. alb. Maybe the oxygen atom in the side chains could improve the flexibility of the molecule and make the side chain more easily lock its proper position. All the designed compounds esters are structurally flexible functional groups because rotation about the C–O–C bonds has a low barrier. Their flexibility and low polarity is manifested in their physical properties. Esters are more polar than ethers but less polar than alcohols. They participate in hydrogen bonds as hydrogen-bond acceptors. As the hydrogen bond and hydrophobic interactions of the ester, the title compounds can enhance the binding capacity with the active site of CYP51. As the hydrogen-bond acceptor, the title compounds esters have a good water solubility. It will be beneficial for the development of new drugs.

To validate the hypothesis, compound 12 was docked into the active site of CACYP51 (Figure 3). Literature reported that the chiral compounds were docked into the active site. The R isomer showed lower interaction energy with CYP51 than the S isomer, which indicated that the R isomer might have better antifungal activity than the S isomer (19). In our study, all the docked conformations refer to the R configuration of the compounds. The docking results revealed that the compound 12 binded to the active site of CACYP51 through the formation of a coordination bond with iron of heme group. The difluorophenyl group was located in the hydrophobic binding cleft lined with Phe126, Leu121, Tyr118, Met306, and Gly307. The side chain of the compound 12 was oriented into substrate access channel 2 (FG loop) and formed hydrophobic and van der waals interactions with Ala117, Tyr118, Phe228, Pro230, Leu376, Ile379, Phe380, and Met508. Moreover, the phenyl group attached to the piperidinol of the side chain interacted with the phenol group of Phe380 through the formation of π-π face-to-edge interaction. There are some differences between the molecular docking result of compound B1 (Figure 4) and compound 12. The difluorophenyl group of compound B1 could not locate the common hydrophobic binding cleft. Although the phenyl group of the side chain could interact with the phenol group of Phe380 through the formation of π-π face-to-edge interaction, the side chain could not embed into substrate access channel 2 (FG loop). All these affect the combination of compound B1 and the enzyme.

image

Figure 3.  The binding mode of compound 12 in the active site of CACYP51.

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image

Figure 4.  The binding mode of compound B1 in the active site of CACYP51.

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Besides, the negative control intermediate 5 showed poor activity. After forming ester, the activity of the title compounds was greatly improved. That might be because the short side chain could not orient into the substrate access channel 2. Besides, the intermediate 5 could not form π-π face-to-edge interaction. Meanwhile, compound 3135 showed less activity than others. Furthermore, the longer the alkyl side chain was, the poorer the activity was. That is because the long side chain might not be oriented into the substrate access channel properly. It can also affect the interaction of the other groups with the target enzyme. So the phenyl group of compound 3135 cannot form the π-π face-to-edge interaction with Phe380.

Conclusions

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

In summary, a series of triazole antifungal agents were synthesized, and their antifungal activities were screened for eight human pathogenic fungi, which were often encountered clinically. Compared with the lead compound B1, our present results clearly showed that the replacement of the piperazinyl side chain with the piperidinol side chain greatly enhanced the antifungal activity of these title compounds against Candida species and broadened the antifungal spectrum. This observation was explained by a molecular model resulting from the computational docking simulation. The side chain of the compound 12 was oriented into substrate access channel 2 (FG loop) and formed hydrophobic and van der waals interactions with surrounding hydrophobic residues. The phenyl group of the side chain can interact with the phenyl group of Phe380 through the formation of π-π face-to-edge interaction. Further evaluations are necessary to determine the antifungal activities of these title compounds in vivo and help us to optimize these new leading compounds, which are continuing in our laboratories.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

This work was supported by the National Natural Science Foundation of China (Grant No. 30300437), the Eleventh Five Year Military Medicine and Public Health Research Projects (Grant No. 06MB206), and by Shanghai Leading Academic Discipline Project (Project No. B906).

References

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results and Discussions
  5. Conclusions
  6. Acknowledgments
  7. References
  8. Supporting Information

Appendix S1. Supplementary material.

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