Design, Synthesis and Biological Evaluation of Lophanic Acid Derivatives as Antifungal and Antibacterial Agents

In order to discover more promising antifungal and antibacterial agents, a series of new derivatives were designed and synthesized by structure modification based on the naturally occurring antimicrobial compound lophanic acid. The structures of all the target compounds were well characterized by spectroscopic data. The stereochemistry of these compounds was further determined through the X-ray diffraction analysis of 6a. The synthetic compounds were evaluated for their antimicrobial activities against filamentous fungi (T. rubrum, T. mentagrophytes), yeasts (C. neoformans, C. albicans) and Gram-positive and Gram-negative bacteria (MRSA, S. mutans, S. sobrinus, and E. coli). Among them, 3d and 3i are found as the most promising leads that showed potent inhibitory effects against all the tested fungal and bacterial strains except for E. coli. The presence of the C-20 carboxylic ester groups and the free hydroxy group at C-13 was found to be essential for the antifungal and antibacterial activities of the lophanic acid derivatives.


Introduction
Tinea pedis is a chronic or recurring disease characterized by dermatophytic infection of the feet and toes, which can involve the interdigital web spaces or the sides of the feet, and it is often caused by anthropophiles, including Trichophyton rubrum sensu stricto, T. interdigitale and Epidermophyton floccosum [1]. Currently, the effective treatments for tinea pedis are topical or oral antifungals or a combination of both, such as terbinafine and clotrimazole [2,3]. Although synthetic chemical drugs have been used to treat tinea pedis, their overuse over the years has led to considerable concern for fungus resistance and other adverse effects on human health [4,5]. Thus, there is an urgent need to discover new promising alternatives from sustainable natural bioresources to effectively and selectively treat tinea pedis.
For decades, medicinal plants have been considered a rich source of lead compounds for drug discovery and development [6,7]. Abietane-type diterpenoid is one of the most prevalent classes of diterpenes widely found in the plants of the Isodon genus [8,9]. They have shown a variety of pharmacological activities, including anticancer, anti-inflammatory, and antivirus activities [10,11]. Meanwhile, abietane-type diterpenoids were also found to show exceeding antimicrobial activities. For example, our previous studies described that rubesanolide D ( Figure 1I) had antibacterial activity against biofilm formation of the dental bacterium Streptococcus mutans [12], kunminolide A ( Figure 1II) and fladin A ( Figure 1III) displayed inhibitory effects against the dental pathogens S. mutans, Porphyromonas gingivalis and Candida albicans and the athlete's foot fungus T. rubrum [13,14]. Abietane-type ( Figure 1III) displayed inhibitory effects against the dental pathogens S. mutans, Porphyromonas gingivalis and Candida albicans and the athlete's foot fungus T. rubrum [13,14]. Abietane-type diterpenoids appear to be attractive molecules for further structure modification to discover novel antifungal and antibacterial agents. Lophanic acid (1, Figure 1), a naturally occurring abietane-type diterpenoid, was found abundant in the medicinal plants I. flavidus [13] and I. lophanthoides [14]. Our study has demonstrated that the compound possesses potent biological activities against a wide range of fungi and bacteria, including T. rubrum, P. gingivalis, S. mutans, and S. albus [15]. However, little attention has been paid to the structural modification to synthesize the derivatives of 1 as antifungal and antibacterial agents. In our previous research on the bioactive compounds from Isodon plants, we obtained a rich amount of lophanic acid (1), which allowed us to carry out a synthetic study by using lophanic acid as a scaffold. Herein we report the design and synthesis of a series of new lophanic acid derivatives, and the antifungal and antibacterial activity evaluation of the synthetic derivatives against two filamentous fungi (T. rubrum, T. mentagrophytes), two yeasts (C. neoformans, C. albicans), and four Gram-positive and Gram-negative bacteria (MRSA, S. mutans, S. sobrinus, and E. coli).

Chemistry
As shown in Scheme 1, fourteen C-20 ester derivatives (3a-3n) were first synthesized by reaction of lophanic acid (1) with the corresponding alkyl halides in the presence of K2CO3 at room temperature. The carboxyl group of 3a was further reduced to an alcohol group by LiAlH4 to obtain compound 4, which was substituted with different alkoxy groups at C-20 to produce 5a-5h. In addition, as outlined in Scheme 2, the hydroxyl groups of compounds 3a and 3d underwent an esterification reaction with acetyl chloride in the presence of pyridine to afford 6a and 6b. Interestingly, the acetyl group at C-13 of 6a was further acetylated to form acetylacetic ester (6c) through a C-C bond. The structure determination of 6c was evidenced by the compassion among the 1 H NMR spectra of 3a, 6a, and 6c as depicted in Figure 2 (Supplementary Materials). The stereochemistry of 6a was further confirmed by the X-ray crystallographic analysis ( Figure 3). Lophanic acid (1, Figure 1), a naturally occurring abietane-type diterpenoid, was found abundant in the medicinal plants I. flavidus [13] and I. lophanthoides [14]. Our study has demonstrated that the compound possesses potent biological activities against a wide range of fungi and bacteria, including T. rubrum, P. gingivalis, S. mutans, and S. albus [15]. However, little attention has been paid to the structural modification to synthesize the derivatives of 1 as antifungal and antibacterial agents. In our previous research on the bioactive compounds from Isodon plants, we obtained a rich amount of lophanic acid (1), which allowed us to carry out a synthetic study by using lophanic acid as a scaffold. Herein we report the design and synthesis of a series of new lophanic acid derivatives, and the antifungal and antibacterial activity evaluation of the synthetic derivatives against two filamentous fungi (T. rubrum, T. mentagrophytes), two yeasts (C. neoformans, C. albicans), and four Gram-positive and Gram-negative bacteria (MRSA, S. mutans, S. sobrinus, and E. coli).

Chemistry
As shown in Scheme 1, fourteen C-20 ester derivatives (3a-3n) were first synthesized by reaction of lophanic acid (1) with the corresponding alkyl halides in the presence of K 2 CO 3 at room temperature. The carboxyl group of 3a was further reduced to an alcohol group by LiAlH 4 to obtain compound 4, which was substituted with different alkoxy groups at C-20 to produce 5a-5h. In addition, as outlined in Scheme 2, the hydroxyl groups of compounds 3a and 3d underwent an esterification reaction with acetyl chloride in the presence of pyridine to afford 6a and 6b. Interestingly, the acetyl group at C-13 of 6a was further acetylated to form acetylacetic ester (6c) through a C-C bond. The structure determination of 6c was evidenced by the compassion among the 1 H NMR spectra of 3a, 6a, and 6c as depicted in Figure 2. The stereochemistry of 6a was further confirmed by the X-ray crystallographic analysis ( Figure 3).

In Vitro Antifungal Activity Evaluation against Filamentous Fungi and Yeasts
The antifungal activities of lophanic acid derivatives (3a-6c) were investigated against two filamentous fungi (T. rubrum, T. mentagrophytes) and two yeasts (C. neoformans, C. albicans) in vitro at 100 µg/mL with miconazole served as a positive control agent.
As shown in Table 1, compounds 3d and 3i exhibited a broad spectrum of antifungal activities against T. rubrum, T. mentagrophytes, and C. neoformans with inhibitory rates over 60%. On the other hand, some compounds were found to exhibit selective activities against filamentous fungi. For example, compound 3a inhibited the growth of T. rubrum by 81.61%, but it showed only a 49.1% inhibitory rate against C. neoformans. Compounds 3b, 3e, 5c, and 5h displayed mild antifungal effects against T. rubrum and C. neoformans. By analyzing the structure-activity relationship (SAR) of the lophanic acid derivatives, we observed that the C-13 hydroxy is essential for retaining their antifungal activities. For example, the antimicrobial inhibitory rates of compounds 3a and 3b were measured over 60%, whereas their corresponding derivatives 6a-6c showed almost no antimicrobial activities. Based on the antimicrobial activities of the lophanic acid derivatives (e.g., 3a, 3d, 5a, and 5b), the structural modification at C-20 could be performed to improve the bioactivity. On the other hand, when a phenyl ring was introduced with an electron-donating group, the corresponding compounds exhibited better activity potency than those with an electronwithdrawing group. For example, 3i showed growth inhibitory rates against T. rubrum, T. mentagrophytes, and C. neoformans at 74.91, 63.00, and 88.54%, respectively, while 3n displayed no antimicrobial activities against these fungi. Interestingly, no lophanic acid derivatives were found to show antifungal activities against C. albicans at the concentration of 100 µg/mL.

In Vitro Antifungal Activity Evaluation against Filamentous Fungi and Yeasts
The antifungal activities of lophanic acid derivatives (3a-6c) were in against two filamentous fungi (T. rubrum, T. mentagrophytes) and two yeasts (C. n C. albicans) in vitro at 100 μg/mL with miconazole served as a positive control a As shown in Table 1, compounds 3d and 3i exhibited a broad spectrum of activities against T. rubrum, T. mentagrophytes, and C. neoformans with inhibitory 60%. On the other hand, some compounds were found to exhibit selective against filamentous fungi. For example, compound 3a inhibited the growth of by 81.61%, but it showed only a 49.1% inhibitory rate against C. neoformans. Co   a Data were shown as mean ± SD. b No antimicrobial activity was found at the indicated concentration. c Miconazole (10 µg/mL) was served as the positive control agent in the antifungal assays. − no activity at the tested concentration.

In Vitro Antibacterial Activity Evaluation against Gram-Positive and Gram-Negative Bacteria
The antibacterial activities of compounds 3a-6c were further tested against Grampositive bacteria (MRSA, S. mutans, and S. sobrinus) and Gram-negative bacterium (E. coli) in vitro at the concentration of 100 µg/mL with tetracycline as the positive control agent ( Table 2). Compounds 3a, 3b, 3d, 3e, 3i, 5c, and 5h displayed more potent antibacterial activities against MRSA than they are against the other bacteria. Especially, 3b, 3d, 5c, and 5h were found to possess antibacterial activities with inhibitory rates greater than 90%. However, no antimicrobial activities were observed for the synthetic derivatives against S. mutans, S. sobrinus, and E. coli. Preliminary SAR analysis showed that the C-13 hydroxy plays an indispensable role in the antibacterial activities of the lophanic acid derivatives. For instance, compounds 3a and 3b showed strong activities with inhibitory rates of 84.43% and 95.89%, respectively. On the contrary, the introduction of an acyl group on the C-13 hydroxy group of 3a and 3b resulted in the loss of the antibacterial activities (e.g., 6a-6c). Furthermore, a proper ester or alkyloxy group substituted at C-20 was found important for retaining the antibacterial activity potency of a lophanic acid derivative (e.g., 3a, 3b, 3d, 5c, and 5h).  a Data were shown as mean ± SD. b No antimicrobial activity was found at the indicated concentration. c Tetracycline (10 µg/mL) was served as the positive control agent in the antibacterial assays. − no activity at the tested concentration.

Chemistry
All chemical reagents were purchased and utilized without further purification. Solvents were used directly or treated with standard methods before use. Melting points (m.p.) were determined on an X-6a digital melting point apparatus (Gongyi Tech Instrument Co., Ltd., Gongyi, China) and were uncorrected. Infrared spectra (IR) were recorded on a Bruker TENSOR 27 spectrometer. Proton nuclear magnetic resonance spectra ( 1 H NMR) and carbon nuclear magnetic resonance spectra ( 13 C NMR) were recorded in CDCl 3 on a Bruker Avance 400, 500, or 600 MHz instruments using tetramethylsilane (TMS) as the internal standard. High-resolution mass spectra (HRMS) were carried out with IonSpec 4.7 Tesla FTMS instrument. The purities of all the title compounds were determined on an UltiMate 3000 (Dionex, Sunnyvale, CA, USA) HPLC system and were of > 95% purity.
3.1.1. General Procedure for the Synthesis of Compound 3a-n Lophanic acid (1, 100 mg, 0.31 mmol) and potassium carbonate (86 mg, 0.62 mmol) were dissolved in N, N-Dimethylformamide (DMF, 5 mL), and the solution was stirred at room temperature. Then a solution of substituent haloalkane (0.62 mmol) in DMF (2 mL) was added dropwise for 10 min. When the reaction was complete, checked by thin-layer chromatography (TLC) analysis, pure water (30 mL) was added to the reaction, which was extracted with ethyl acetate (3 × 30 mL). The combined organic phase was dried over anhydrous Na 2 SO 4 , filtered, concentrated under reduced pressure, and purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate to afford compound 3a-n in 85-98%.

Synthesis of Compound 4
To a suspension of compound 3a (100 mg, 0.30 mmol) in dry tetrahydrofuran (THF, 10 mL) at 0 • C under N 2 was added lithium aluminum hydride (56 mg, 1.5 mmol) in dry THF (2 mL) dropwise over 10 min. The resulting mixture was allowed to heat to 40 • C for 1 h. When the reaction was complete, pure water (30 mL) was added to the reaction, The solvent was removed and the residue was diluted by ethyl acetate (30 mL), washed with saturated brine (30 mL), dried over anhydrous Na 2 SO 4 , concentrated in vacuo, and purified by silica gel column chromatography to afford 4 in 83%. Data

In Vitro Antifungal Assay
To evaluate the antifungal activity against filamentous fungi, T. rubrum and T. mentagrophytes were separately cultured for 2 weeks at 28 • C on SDA to produce conidia. A mixed suspension of conidia and hyphae fragments was obtained by covering the fungal colonies with sterile saline (0.85%) and gently rubbing the colonies with the inoculation loop. Then, the suspension was filtered with four layers of sterile lens paper to remove the hyphae and centrifuged at 1000× g for 10 min to collect the conidia. Conidia were washed twice by agitation in sterile saline. The concentration of conidia or spore was adjusted with sterile saline to 1 × 10 4 cells/mL by hemocytometer counts. The antifungal susceptibility testing was performed as outlined in document M38-A2 and previous research, with minor changes [16][17][18][19]. The medium used was RPMI 1640 with L-glutamine buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) and was supplemented with 2% glucose (m/v). The 195 µL of prepared conidia or spore suspension was seeded on 96-well plates that had been previously added with 5 µL of tested agents in each well, and three replicates were used for each treatment. Miconazole was used as a positive control in the assay. The 96-well plates were then incubated at 28 ± 2 • C for 7 days. The optical density (OD) reading was measured by a microplate reader at 510 nm. The fungal growth inhibition is determined using the formula: Antifungal susceptibility testing of yeasts was performed by using a micro-broth dilution assay. The compounds were dissolved in DMSO at a stock concentration of 2-10 mg/mL and kept at 4 • C for the bioassays. Exponentially growing cultures of each strain were prepared from overnight cultures, and cultures were adjusted to the OD value of about 0.5 at 600 nm. Cultures were then diluted 1:1000 in broth (C. neoformans was directly used at OD600 = 0.05) and added to a 96-well plate (195 µL/well). Miconazole (Dalian Meilun, Dalian, China; 10 µg mL −1 ) was used as a positive control for C. albicans, and C. neoformans. Plates were read at 600 nm after incubation for 48 h. Inhibition was calculated by subtracting the absorbance of the blank wells, dividing by the average value for the DMSO-only wells, and multiplying by 100.

In Vitro Antibacterial Assay
Compounds were tested for planktonic microbial growth inhibition using the above micro-broth dilution assay [16]. The compounds and the standard drug were prepared in DMSO. Exponentially growing cultures of S. aureus, E. coli, S. mutans, and S. sobrinus were prepared from overnight cultures, and cultures were adjusted to the OD value of about 0.5 at 600 nm. Cultures were then diluted 1:1000 in broth and added to a 96-well plate (195 µL/well). Tetracycline (Sigma, St. Louis, MO, USA; 10 µg mL−1 in DMSO) was used as a standard drug. Plates were read at 600 nm after incubation for 24 h. Inhibition was calculated by the above calculation formula.

Conclusions
A series of novel lophanic acid derivatives have been prepared and evaluated for their antifungal and antibacterial activities. Among the derivatives, 3d is the only compound that showed > 70% inhibitory effects against three fungal and bacterial strains (T. mentagrophytes, C. neoformans, and MRSA), and 3b, 5c, and 5h were found to be able to inhibit the microbial growth of MRSA by over 90%. Through a structure-activity relationship analysis, we observed the presence of a C-20 carboxylic group and a free hydroxyl group at C-13 is essential to retain broad antimicrobial activities for the lophanic acid derivatives (e. g., 3a,  3b, 3d, and 3i). Without the C-20 carboxylic group, the inhibitory effects of the lophanic acid derivatives against T. rubrum and C. neoformans were much weakened (e.g., 5c and 5h). Our present study determined that the C-20 carboxylic group could be the key position for a structural modification to obtain lophanic acid analogs with broad-spectrum antimicrobial activity.