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Ester derivatives of Dictyostelium differentiation-inducing factors exhibit antibacterial activity, possibly via a prodrug-like function

BMC Research Notes volume 18, Article number: 40 (2025) Cite this article

Abstract

Objective

Dictyostelium differentiation-inducing factors 1 and 3 [DIF-1 (1) and DIF-3 (2), respectively], along with their derivatives, such as Ph-DIF-1 (3) and Bu-DIF-3 (4), demonstrate antibacterial activity in vitro against Gram-positive bacteria, including methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-sensitive Enterococcus faecalis (VSE), and vancomycin-resistant Enterococcus faecium [VRE (VanA)]. This study investigates the therapeutic potential of DIF compounds against these Gram-positive bacteria.

Results

In vitro tests revealed that the antibacterial activity of 3 and 4 was lost in the presence of human serum albumin (HSA), suggesting that HSA might inhibit their effectiveness. Further evaluation of less hydrophobic derivatives, DIF-1-NH2 (5) and NH2-Bu-DIF-3 (6), showed no antibacterial activity, even in the absence of HSA. However, ester derivatives Ph-DIF-1(AHA) (7) and Bu-DIF-3(2Ac) (8) exhibited antibacterial activity against the target bacteria in vitro, although this activity was also lost in the presence of HSA. We hypothesize that these ester derivatives may function as prodrugs, with their antibacterial activity possibly restored by hydrolysis through bacterial esterases. The results suggest that suitable ester modifications could enhance the in vivo antibacterial potential of DIF compounds, particularly if they can bypass HSA binding and be activated by bacterial enzymes.

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Introduction

Since the discovery of penicillin in 1928 and of streptomycin in 1943, microorganisms—such as fungi and actinomycetes—have contributed to humanity as resources for drug discoveries [1,2,3,4,5,6,7]. However, because bacteria that are resistant to existing antibacterial drugs have emerged and have become a major social problem, there is an urgent need to develop new antibacterial drugs or to discover and explore new drug discovery resources, but the hurdles in drug discovery are high [4,5,6,7,8,9,10,11,12].

In recent years, we have focused on cellular slime molds—a group of soil microorganisms—as untapped resources for drug discovery [13, 14]; the compounds that are currently most studied are differentiation-inducing factors 1 and 3 [DIF-1 (1) and DIF-3 (2), respectively] (Fig. 1A). DIFs were originally identified as stalk-cell differentiation-inducing factors in the cellular slime mold Dictyostelium discoideum [15, 16]. Most recently, however, we found that derivatives of DIFs such as Ph-DIF-1 (3) and Bu-DIF-3 (4) (Fig. 1A) have strong antibacterial activity in vitro against Gram-positive bacteria, such as methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-sensitive Enterococcus faecalis (VSE), and vancomycin-resistant Enterococcus faecium [VRE (VanA)], and E. faecalis [VRE (VanB)] [17].

Fig. 1
figure 1

Chemical structures of DIF compounds. A DIF-1 (1), 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one. DIF-3 (2), 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one. Ph-DIF-1 (3), 1-(3,5-dichloro-2,6-dihydroxy-4-phenoxyphenyl)hexan-1-one. Bu-DIF-3 (4), 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)hexan-1-one. DIF-1-NH2 (5), 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one. NH2-Bu-DIF-3 (6), 1-(4-(4-aminobutoxy)-3-chloro-2,6-dihydroxyphenyl)hexan-1-one. Note that 6 is a synthetic intermediate of BODIPY-DIF-3G [25]. B Ester derivatives of 3 and 4. Ph-DIF-1(AHA) (7), Ph-DIF-1–aminohexanoic acid ester, 2,4-dichloro-6-hexanoyl-5-hydroxy-3-phenoxyphenyl 6-aminohexanoate. Bu-DIF-3(2Ac) (8), Bu-DIF-3–acetic acid ester, 5-butoxy-4-chloro-2-hexanoyl-1,3-phenylene diacetate

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In this study, as a first step in estimating the in vivo antibacterial activity and identifying the therapeutic potential of DIF compounds (Fig. 1A), we examined their in vitro antibacterial activity [i.e., minimum inhibitory concentration (MIC)] against MSSA, MRSA, VSE, and VRE (VanA) in the presence or absence of a physiological concentration (5%) of human serum albumin (HSA) [18], to which drugs sometimes bind [19,20,21,22]. As anticipated, we found that the antibacterial activity of these compounds was abolished in the presence of HSA. We then assessed the MIC of DIF compounds in vitro, including DIF-1-NH2 (5) and NH2-Bu-DIF-3 (6), which are DIF derivatives with a hydrophilic group (Fig. 1A); and Ph-DIF-1(AHA) (7) and Bu-DIF-3(2Ac) (8), which are newly synthesized ester derivatives of DIFs (Fig. 1B). We demonstrated that 7 and 8 exert antibacterial activity, potentially by being degraded by bacterial esterases. This suggests that such ester derivatives of DIFs could be utilized as prodrugs, incorporating appropriate functional groups that can avoid adsorption to HSA.

Materials and methods

Bacterial species

The Gram-positive bacteria MSSA (ATCC29213 and 25923), MRSA (ATCC43300), VSE (ATCC29212), and VRE (VanA; ATCC700221) were used in this study.

DIF compounds and reagents

DIF-1 (1), DIF-3 (2), Ph-DIF-1 (3), Bu-DIF-3 (4) [23], DIF-1-NH2 (5) [24], and NH2-Bu-DIF-3 (6) [25] were synthesized as previously described and stored at − 20 °C as 10 mM solutions in dimethylsulfoxide (DMSO). Vancomycin was obtained from Sigma-Aldrich (St. Louis, MO, USA). The hydrophobic index (cLogP) of each compound was calculated using ChemDraw 16.0 software (PerkinElmer, Inc., Waltham, MA, USA).

Synthesis of DIF derivatives

Synthesis of 2,4-dichloro-6-hexanoyl-5-hydroxy-3-phenoxyphenyl 5-bromohexanoate (9) (Fig. 2A)

Fig. 2
figure 2

Synthetic routes of ester derivatives of 3 and 4, which were synthesized as described in “Materials and methods” section

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Triethylamine (63 μL, 0.452 mmol), 4-(dimethylamino)pyridine (2.0 mg, 16 μmol) and 6-bromohexanoyl chloride (27 μL, 180 μmol) were added to a solution of Ph-DIF-1 (3) (55.3 mg, 150 μmol) in dichloromethane (1.5 mL). The reaction mixture was stirred for 15 h at room temperature, poured into 1 M hydrochloric acid, and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over sodium sulfate, and evaporated under reduced pressure. The residue was chromatographed over a silica gel column eluted by hexane–ethyl acetate (9:1) to yield 9 (28.6 mg, 52.2 μmol, 35%). The data for 9: yellowish oil; 1H NMR (600 MHz, CDCl3) δ 7.32 (t, J = 7.5 Hz, 2H), 7.08 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 7.5 Hz, 2H), 3.41 (t, J = 6.7 Hz, 4H), 2.71 (t, J = 7.3 Hz, 2H), 2.59 (t, J = 7.4 Hz, 4H), 1.92–1.88 (m, 4H), 1.78–1.73 (m, 4H), 1.68–1.63 (m, 2H), 1.58–1.53 (m, 4H), 1.36–1.29 (m, 4H), 0.92 (t, J = 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 198.8, 169.7 (2C), 156.1, 149.3, 143.4 (2C), 129.8 (2C), 128.3, 123.1 (2C), 122.5, 115.1 (2C), 43.9, 33.5 (2C), 33.2 (2C), 32.3 (2C), 31.3, 27.5 (2C), 23.7 (2C), 23.2, 22.4, 13.9; HRESIMS m/z 743.0177 [M+Na]+ (743.0153 calculated for C30H36O679Br235Cl2Na) (Figures S1 and S2).

Synthesis of Ph-DIF-1(AHA) (7) (Fig. 2A)

Sodium azide (8.2 mg, 126 μmol) was added to a solution of 9 (22.7 mg, 41.6 μmol) in DMSO (1 mL). The reaction mixture was stirred for 12 h at room temperature, poured into water, and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium chloride solution, dried over sodium sulfate, and evaporated under reduced pressure. The residue was dissolved in ethanol (1 mL), and palladium on carbon (10%; 1 mg) was added to the solution. The solution was stirred at room temperature for 5 h under a hydrogen atmosphere, and then the solution was filtered through a Celite pad, and the filtrate was concentrated in vacuo. The residue was chromatographed over silica gel eluted with chloroform–methanol (39:1) to yield 7 [1.1 mg, 2.3 μmol, 5% (two steps)]. The data for 7: yellowish oil; 1H NMR (600 MHz, CDCl3) δ 7.32 (t, J = 7.8 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 6.86 (d, J = 7.8 Hz, 2H), 2.83 (br. s, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.52 (t, J = 7.4 Hz, 2H), 1.85–1.80 (m, 2H), 1.66–1.63 (m, 2H), 1.52–1.40 (m, 8H), 0.96 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3/CD3OD = 19/1) δ 208.7, 167.0, 156.3, 156.0, 152.4, 150.7, 129.7 (2C), 123.0, 120.9, 115.1 (2C), 111.3, 108.1, 40.2, 36.7, 31.4, 31.1, 29.7, 29.5, 26.7, 23.6, 22.7, 13.8; HRESIMS m/z 464.1421 [M–H2O+H]+ (464.1395 calculated for C24H28NO435Cl2) (Figures S3 and S4).

Synthesis of Bu-DIF-3(2Ac) (8) (Fig. 2B)

Acetic anhydride (50 μL) was added to a solution of Bu-DIF-3 (4) (9.8 mg, 31.1 μmol) in pyridine (1 mL). The reaction mixture was stirred for 3 h at room temperature, diluted with 1 M hydrochloric acid, and then extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over sodium sulfate, and evaporated under reduced pressure. The residue was chromatographed over a silica gel column eluted with hexane–ethyl acetate (9:1) to yield 8 (10.9 mg, 27.3 μmol, 88%). The data for 8: colorless amorphous solid; 1H NMR (600 MHz, CDCl3) δ 6.64 (s, 1H), 4.02 (t, J = 6.5 Hz, 2H), 2.68 (t, J = 7.4 Hz, 2H), 2.31 (s, 3H), 2.26 (s, 3H), 1.84–1.80 (m, 2H), 1.65–1.60 (m, 2H), 1.54–1.48 (m, 2H), 1.32–1.25 (m, 4H), 0.98 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 199.7, 168.5, 167.5, 156.5, 146.7, 145.6, 121.5, 114.8, 105.4, 69.5, 43.9, 31.4, 30.8, 23.6, 22.4, 21.0, 20.5, 19.1, 13.9, 13.7; HRESIMS m/z 421.1416 [M+Na]+ (421.1394 calculated for C20H27O635ClNa) (Figures S5 and S6).

Measurement of the minimum inhibitory concentration (MIC)

The MIC was determined as described previously [17], where the Gram-positive bacteria in 0.1 mL Mueller–Hinton broth (5 × 105 CFU/mL) in 96-well plates (Corning, Corning, NY, USA) containing 0–5% (w/v) HSA were incubated for 20–24 h at 37 °C in the presence of DMSO (vehicle), various concentrations of serially diluted DIF derivatives, or vancomycin.

Results and discussion

Antibacterial activity of the DIF compounds in the presence of HSA

To estimate the in vivo antibacterial activity of the DIF derivatives, we evaluated the antibacterial effects of the DIF compounds 1–4 (Fig. 1A) against MSSA, MRSA, VSE, and VRE (VanA) in the presence or absence of 5% HSA, a concentration corresponding to albumin levels (approximately 35–50 g/L; 3.5–5%) in adult human blood [18]. Antibacterial agents are known to bind to proteins—primarily albumin—in vivo, and it is believed that antibacterial agents bound to albumin do not influence the infecting microorganisms in infected organs but that only their free form is active [19, 20]. As presented in Table 1, in the absence of HSA, the DIF compounds 1–4 exhibited antibacterial activity similar to our previous findings [17]. However, their activity was lost in the presence of 5% HSA, thereby indicating that DIF compounds may not be viable antibacterial agents in vivo (Table 1).

Table 1 Comparison of antimicrobial activity (MICa) of DIF derivatives against MSSA, MRSA, VSE, and VRE (VanA)
Full size table

Therefore, we examined the antibacterial activity of more hydrophilic DIF derivatives—DIF-1-NH2 (5) and the newly synthesized NH2-Bu-DIF-3 (6) (Fig. 1A)—against the same bacteria. Unfortunately, however, no antibacterial activity was observed in these compounds, even in the absence of HSA (Table 1).

On the other hand, vancomycin exhibited strong antibacterial activity against the bacteria, both in the presence and absence of HSA, as indicated by the MIC values (Table 1), which were comparable to those in previous studies [22, 26, 27].

Antibacterial activity of ester derivatives of DIFs

Prodrugs are chemicals supplied in a modified (i.e., inactive) form that undergo enzymatic and chemical transformations in vivo to release the active parent drug, which produces the desired pharmacological effect [28, 29]. Since various types of esterases that hydrolyze ester bonds are believed to be widely present in almost all living cells, utilizing the hydrolytic ability of bacterial esterases could enable the modification of DIF compounds with various functional groups via ester bonds. This approach could allow ester derivatives of DIFs to be used as prodrugs [28]. Therefore, we synthesized two ester derivatives of DIFs—Ph-DIF-1(AHA) (7) and Bu-DIF-3(2Ac) (8) (Figs. 1B and 2)—and investigated whether they actually exhibited antibacterial activity against MSSA, MRSA, VSE, and VRE (VanA) (Table 2).

Table 2 Comparison of antimicrobial activity (MICa) of DIF derivatives against MSSA, MRSA, VSE, and VRE (VanA)
Full size table

In the absence of HSA, the two esters, 7 and 8, exhibited antibacterial activity at the same level as their original DIF derivatives 3 and 4, except that ester 7 showed relatively weak antibacterial activity against MSSA (Table 2). The MIC values for each bacterium with 3 and 7, as well as those with 4 and 8, were the same or within a twofold range, despite the considerable difference in the chemical structures of 3 and 7, and of 4 and 8. Although we do not exclude the possibility that esters 7 and 8 might have acted directly without being hydrolyzed, our results strongly suggest that 7 and 8 were hydrolyzed by bacterial esterase(s) to form 3 and 4, respectively, which suppressed bacterial growth (Fig. 3).

Fig. 3
figure 3

Schematic diagram of the expected mechanism of action of DIF esters

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To confirm whether the antibacterial activity of 7 against MSSA is generally low, we examined it against another MSSA strain and found that the antibacterial activity of 7 against the two MSSA strains and had a comparable result (Table 3). These results suggest that MSSA relatively rarely hydrolyzes 7, possibly due to the limited specificity of its esterase(s). In other words, Ph-DIF-1(AHA) (7) itself has little or no antibacterial activity.

Table 3 Comparison of antimicrobial activity (MICa) of DIF derivatives against two strains of MSSA
Full size table

To evaluate in detail the relationship between the HSA concentration and its inhibition of antibacterial activity and to obtain clues for the synthesis of new active compounds that are not inhibited by HSA, we examined next the antibacterial activity of 7 and 8 in the presence of a lower HSA concentration range. In the presence of 0.01–0.5% HSA, the antibacterial activity of 7 and 8 disappeared in a manner dependent on the HSA concentration (Tables 2 and 3). These results again suggest that 7 and 8, or their hydrolysis products 3 and 4, were adsorbed to HSA. Consequently, 7 and 8 cannot be used as prodrugs in vivo, likely due to their hydrophobicity. If this is the case, DIF derivatives with ester-bonded functional groups that can avoid adsorption to HSA would exhibit antibacterial activity in vivo.

DIFs as lead compounds for drug development

The chlorinated alkylphenones DIF-1 (1) and DIF-3 (2) were initially identified as stalk-cell differentiation-inducing factors in D. discoideum [15, 16]. Later, it was clarified that 1 is the primary physiological differentiation-inducing factor in D. discoideum, whereas 2 is a metabolite of 1. In fact, the differentiation-inducing activity of 2 was found to be only approximately 4% of that of 1 [30,31,32]. It was subsequently discovered that 1, 2, and their derivatives exhibit various biological activities in different cells beyond D. discoideum, including antitumor (antiproliferative and antimetastatic), glucose uptake-promoting, and immunoregulatory activities in mammalian cells, as well as anti-Trypanosoma, anti-Plasmodium, and antibacterial activities [14, 17, 23, 33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]. Interestingly, our analysis of these structure–activity relationships revealed that the potency of each biological activity does not always correlate with the chemical structure [14, 18, 35, 45, 46]. Therefore, it may be possible to differentiate these biological activities by designing side-chain modifications of DIF derivatives and developing various novel drugs using DIFs as leads [14, 23].

As mentioned in “Introduction” section, the drug-resistant bacteria, such as MRSA and VRE, are becoming increasingly prevalent, thereby necessitating a search for new antibiotic molecules and bioresources that produce novel antibacterial agents [4,5,6,7,8,9,10,11,12]. Under these circumstances, DIF derivatives, such as Ph-DIF-1 (3) and Bu-DIF-3 (4), have demonstrated antibacterial activity against Gram-positive bacteria, including MRSA and VRE, in vitro (Table 1) [17]. Therefore, developing antibiotics based on DIF derivatives is considered a highly worthwhile endeavor.

Limitation

While this study might have demonstrated the potential of ester derivatives of DIFs as prodrugs, no derivatives capable of evading adsorption to HSA were found.

Conclusion

In the present study, we suggested that the ester derivatives of DIFs, 7 and 8, exhibit antibacterial activity, possibly due to degradation by bacterial esterases in vitro. This suggests their potential as prodrugs in vivo. Looking ahead, by designing ester derivatives of DIFs that can avoid interference by HSA, we aim to develop novel antibacterial drugs capable of eliminating bacteria that are resistant to current treatments by designing ester derivatives of DIFs that can avoid interference by HSA.

Availability of data and materials

The datasets used and/or analyzed during the current study were provided in the manuscript and supplementary information information files.

Abbreviations

DIF-1:

Differentiation-inducing factor 1, (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one)

DIF-3:

Differentiation-inducing factor 3, (1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one)

Ph-DIF-1:

Phenoxy-DIF-1, 1-(3,5-dichloro-2,6-dihydroxy-4-phenoxyphenyl)hexan-1-one

Bu-DIF-3:

Butoxy-DIF-3, 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)hexan-1-one

DIF-1-NH2 :

6-Amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one

NH2-Bu-DIF-3:

1-(4-(4-Aminobutoxy)-3-chloro-2,6-dihydroxyphenyl)hexan-1-one

Ph-DIF-1(AHA):

Ph-DIF-1-aminohexanoic acid ester, 2,4-dichloro-6-hexanoyl-5-hydroxy-3-phenoxyphenyl 6-aminohexanoate

Bu-DIF-3(2Ac):

Bu-DIF-3-acetic acid ester, 5-butoxy-4-chloro-2-hexanoyl-1,3-phenylene diacetate

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Funding

This work was supported by JSPS KAKENHI (Grant numbers 19K07139 and 24K08703 to YK, and 22K11759 to KT) and the Joint Research Program of Juntendo University, Faculty of Health and Sports Science (to YK).

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Authors and Affiliations

  1. Department of Medical Technology, Faculty of Health Science, Gunma Paz University, Takasaki, 370-0006, Japan

    Katsunori Takahashi, Hirotaka Ishigaki, Yusuke Miura & Ayuko Takahashi

  2. Division of Natural Medicines, Faculty of Pharmacy, Keio University, Tokyo, 105-8512, Japan

    Haruhisa Kikuchi & Takehiro Nishimura

  3. Laboratory of Health and Life Science, Graduate School of Health and Sports Science, Juntendo University, Inzai, 270-1695, Japan

    Yuzuru Kubohara

Authors
  1. Katsunori Takahashi
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  2. Haruhisa Kikuchi
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  3. Takehiro Nishimura
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  4. Hirotaka Ishigaki
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  5. Yusuke Miura
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  6. Ayuko Takahashi
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  7. Yuzuru Kubohara
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Contributions

KT, HK, TN, HI, YM, AT and YK performed the experiments and drafted the manuscript. HK and TN synthesized the compounds. HK and YK revised the manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Haruhisa Kikuchi or Yuzuru Kubohara.

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Competing interests

Patent related to this article were issued on February 15, 2019 (no. 6478378) in Japan. Juntendo University holds the patent; YK and HK are the inventors of the patent. KT, TN, HI, YM, and AT declare that they have no conflict of interest.

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Takahashi, K., Kikuchi, H., Nishimura, T. et al. Ester derivatives of Dictyostelium differentiation-inducing factors exhibit antibacterial activity, possibly via a prodrug-like function. BMC Res Notes 18, 40 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-025-07122-5

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BMC Research Notes

ISSN: 1756-0500

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