Synthesis and Antimicrobial Evaluation of Side-Chain Derivatives based on Eurotiumide A

Side-chain derivatives of eurotiumide A, a dihydroisochroman-type natural product, have been synthesized and their antimicrobial activities described. Sixteen derivatives were synthesized from a key intermediate of the total synthesis of eurotiumide A, and their antimicrobial activities against two Gram-positive bacteria, methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), and a Gram-negative bacterium, Porphyromonas gingivalis, were evaluated. The results showed that derivatives having an iodine atom on their aromatic ring instead of the prenyl moiety displayed better antimicrobial activity than eurotiumide A against MSSA and P. gingivalis. Moreover, we discovered that a derivative with an isopentyl side chain, which is a hydrogenated product of eurotiumide A, is the strongest antimicrobial agent against all three strains, including MRSA.


Introduction
Humans have always struggled against infectious diseases [1][2][3][4][5] and in relatively recent times have developed various antimicrobial therapies [6][7][8]. Since the discovery of penicillin [9], various natural products having antimicrobial activity have been discovered [10][11][12][13][14][15][16], and the majority of clinically used antibiotics are either natural products, semisynthetic derivatives, or compounds derived from them [17][18][19]. Despite the presence of many excellent antibiotics, multidrug-resistant bacterial pathogens have emerged all over the world [20][21][22], and the development of novel and effective antimicrobial agents against many kinds of pathogenic bacteria, including methicillinresistant Staphylococcus aureus (MRSA), should remain a continuous mission for medicinal chemists. In 2014, Wang and co-workers discovered eurotiumides, which are novel dihydroisocoumarin-type natural products, from a gorgonian-derived fungus, Eurotium sp. XS-200900E6 [23]. Among the series of eurotiumides, eurotiumide A (1), having cis configurations at H3/H4, exhibited potent antimicrobial activities against Staphylococcus epidermidis, Bacillus cereus, Vibrio anguillarum, and Escherichia coli. Based on that report, although 1 seems to be an attractive seed compound for antibiotics, further antimicrobial investigation and a structure-activity relationship study of 1 are needed. In particular, because there is a chance that modification of the side chain of the aromatic ring could improve antimicrobial activity and the spectrum, a structure-activity relationship study of the substituent effect of the aromatic ring is essential for discovering promising candidates for antimicrobial agents. Recently, we reported the first asymmetric total syntheses of (−)-eurotiumide A (1) and (+)-eurotiumide B and revised their reported structures [24]. In our synthetic route, the prenyl side chain of the aromatic ring was introduced in the late stage by the Stille coupling reaction with the key intermediate 2. Based on our previous results, we considered that a number of derivatives of 1, which have a variety of kinds of side-chain moiety, could be obtained from the common intermediate 2 and non-substituted compound 3 in the late stage of synthesis ( Figure 1). In this work, as part of our continuing research [24,25], we constructed a chemical library of the side-chain derivatives of eurotiumide A (1) to elucidate the effects of the side chains of the aromatic rings and to develop antimicrobial agents against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (both Gram-positive bacteria), as well as Porphyromonas gingivalis (a Gram-negative bacterium).

Synthesis of the Side-Chain Derivatives of Eurotiumide A
Our synthetic plan is shown in Figure 2. We planned to introduce three types of functional groups: a hydrocarbon group, including hydrogen, alkyl, and aromatic rings (Type A); a heteroatom and heteroatom-containing alkyl group (Type B); and halogen atoms group (Type C). The derivatives of groups A and B could be derived from 2 by the cross-coupling reaction and functional group transformation. The halogenated derivatives (Type C) would be obtained from 3 by direct introduction of the halogen atoms. Although Wang et al. isolated the natural eurotiumide A (1) as a racemic form, they evaluated the antimicrobial activities of its enantiomers after separation by chiral HPLC and revealed that there was no significant difference between the enantiomers [23]. From the viewpoint of the efficiency of compound supply, we decided to make racemic compounds. First, we initiated the syntheses of the derivatives of group A (Scheme 1). The non-substituted derivative 4 was obtained from 3 by deprotection of the diMOM group with aqueous 6 M HCl in methanol at 40 °C in 79% yield. Catalytic hydrogenation of eurotiumide A (1) gave the isopentyl derivative 6 in quantitative yield. Methyl and vinyl groups were introduced by the Stille coupling reaction with 2 to afford methyl derivative 5a and styrene derivative 7a in 83% and quantitative yields, respectively. Phenyl derivative 9a and biphenyl derivative 10a were obtained from 2 by the Suzuki-Miyaura cross coupling reaction with the corresponding boronic acids in 75% and 77% yields, respectively. Deprotection of the diMOM group of derivatives 5a, 7a, 9a, and 10a then gave the corresponding desired products (5, 7, 9, and 10). We tried to introduce the alkyne group by the Sonogashira coupling reaction; however, the desired alkyne product was obtained in only 12% yield. To improve the reaction yield, the Seyferth-Gilbert homologation using the Ohira-Bestmann reagent 21 was applied to the aldehyde derivative 12a (vide infra) and afforded the desired alkyne 8a in quantitative yield. After acidic treatment of 8a, the alkyne derivative 8 was obtained in 68% yield. With type A derivatives in hand, we turned our attention to preparing type B derivatives having heteroatom-containing side chains (Scheme 2). For the introduction of an alkyl group containing heteroatoms, we chose the styrene derivative 7a as a starting point. Ozonolysis of the alkene moiety of 7a afforded the diMOM-protected benzaldehyde 12a in excellent yield. Acidic treatment of 12a gave the desired deprotected benzaldehyde derivative 12 in 77%. On the other hand, reduction of the aldehyde moiety of 12a with sodium borohydride to give the benzyl alcohol 11a and the deprotection furnished the hydroxymethyl derivative 11 in moderate yield. To introduce a nitrogen group at the benzyl position of 11a, the primary alcohol moiety was converted to a mesyl group (22) and a nucleophilic substitution reaction with sodium azide afforded diMOM-protected azide 13a in good yield. Derivative 13a was treated with aqueous 6 M HCl in MeOH to furnish the desired dihydroxy azide derivative 13. We then tried to convert the azide into an amine functionality. After several attempts, we found that addition of triethylamine was crucial to keep the reaction clean and we succeeded to get 14a. Then, deprotection of the diMOM group gave the desired aminomethyl derivative 14. Next, a nitration reaction was conducted with non-substituted derivative 3 by adding HNO3 in AcOH to afford monoMOM-protected nitro derivative 15a as a crude product; then it was deprotected under acidic condition to give the nitro derivative 15 (Scheme 3). After that, hydrogenation with Adam's catalyst produced the aniline derivative 16 from 15. Finally, we tried to synthesize the halogenated derivatives (Scheme 4). Chloro and iodo groups were introduced to treat 3 with N-chlorosuccinimide and N-iodosuccinimide in DMF to afford the chloro derivative 18a and the iodo derivative 20a, respectively. The diMOM groups of 18a and 20a were then deprotected under acidic conditions to afford the desired 18 and 20. Bromo derivative 19 was obtained from 2 in 97% yield by acid treatment to cleave the diMOM group. However, despite several efforts to introduce fluorine to the aromatic ring from 3, we could not get the desired fluoro derivative 17. We also tried the Sandmeyer reaction with 16 but did not obtain the desired 17.

Antimicrobial Evaluation of Synthesized Derivatives
After the initially set derivatives of eurotiumide A were synthesized, the first antimicrobial activity screening was conducted against the Gram-positive MSSA and MRSA as well as the Gramnegative P. gingivalis in 10 μM solutions of the synthesized derivatives to narrow down the promising  Figure 3. (+/−)-Eurotiumide A (1) exhibited mild antimicrobial activity against MSSA at this concentration ( Figure 3a). While most of the derivatives did not show antimicrobial activity against this strain, the isopentyl derivative 6 and the iodo derivative 20 exhibited more potent antimicrobial activity than 1. Next, we tested the same screening against MRSA (Figure 3b). Most of the derivatives that displayed good activity against MSSA showed no antimicrobial activity against MRSA. Even natural product 1 and the iodo derivative 20 also did not show good antimicrobial activity against MRSA. Surprisingly, only the isopentyl derivative 6, which was a reduced derivative of 1, was found to have good antimicrobial activity against MRSA. We also conducted antimicrobial screening against P. gingivalis (Figure 3c). Unlike the case with S. aureus, many derivatives, specifically eurotiumide A (1), isopentyl derivative 6, vinyl derivative 7, aniline derivative 16, and three halogenated derivatives (18,19,20), were effective against P. gingivalis. Since we acquired promising agents against all three strains, we determined the IC50 values of these candidates ( Table 1). The IC50 values of the isopentyl derivative 6 and the iodo derivative 20 against MSSA were 5.6 μM (2.0 μg/mL) and 9.0 μM (3.7 μg/mL), respectively. Moreover, the IC50 value of 6 against MRSA was 4.3 μM (1.5 μg/mL), which is the same level of activity against MSSA. The IC50 values of these seven candidates (1, 6, 7, 16, 18, 19, and 20) against P. gingivalis ranged from 2.0 to 7.0 μM. We also checked the cytotoxicity of three compounds (1, 6, and 20) against the A549 cell line, and these three compounds were non-toxic in 10 μM. In this study, we discovered that the isopentyl derivative 6, which is a one-point modified compound of natural product 1, and the iodo derivative 20 have superior antimicrobial activity to 1 against MSSA and P. gingivalis. Although 20 did not exhibit good efficacy against MRSA, 6 was found to maintain antimicrobial activity against these three strains, including MRSA. These results indicate that S. aureus is sensitive to changes in the side chain of the aromatic ring and that MRSA can distinguish the subtle difference between prenyl and isopentyl moieties. Moreover, the weak antimicrobial activity of 1 against MRSA suggests a binding affinity between 1 and the penicillin binding protein 2' [26], which is the main resistance mechanism of MRSA against antibiotics. The inhibition of cell wall synthesis seems to be the mode of action of 1, although a more detailed study is needed to clarify the mode of action of 6 and 20. On the other hand, we found that several compounds having alkyl and halogenated side chains well suppressed the increase in P. gingivalis.

General Procedure
All the reactions were carried out in a round-bottomed flask with an appropriate number of necks and side arms connected to a three-way stopcock and/or a rubber septum cap under an argon atmosphere. All vessels were first evacuated by rotary pump and then flushed with argon prior to use. Solutions and solvents were introduced by hypodermic syringe through a rubber septum. During the reaction, the vessel was kept under a positive pressure of argon. Dry THF was freshly prepared by distillation from benzophenone ketyl before use. Anhydrous CH2Cl2, DMF, ethanol, MeCN, methanol, pyridine, and toluene were purchased from Kanto Chemical Co. Inc. Infrared (IR) spectra were recorded on a JASCO FT/IR-4100 spectrophotometer using a 5 mm KBr plate. Wavelengths of maximum absorbance are quoted in cm −1 . 1H-NMR spectra were recorded on a JEOL ECA-400 (400 MHz), Bruker AV-400N (400 MHz), and Bruker AV-500 (500 MHz) in CDCl3. Chemical shifts are reported in parts per million (ppm), and signals are expressed as singlet (s), doublet (d), triplet (t), multiplet (m), broad (br), and overlapped. 13C-NMR spectra were recorded on a JEOL ECA-400 (100 MHz), Bruker AV-400N (100 MHz), and Bruker AV-500 (125 MHz) in CDCl3. Chemical shifts are reported in parts per million (ppm). High resolution mass (HRMS) spectra were recorded on a Thermo Scientific Exactive. All melting points were measured with a Yanaco MP-500D. Analytical thin layer chromatography (TLC) was performed using 0.25 mm E. Merck Silica gel (60F-254) plates. Reaction components were visualized phosphomolybdic acid or ninhydrin or panisaldehyde in 10% sulfuric acid in ethanol. Kanto Chem. Co. Silica Gel 60N (particle size 0.040-0.050 mm) was used for column chromatography.
To a solution of bromo compound 3 (10.0 mg, 30.8 μmol) in MeOH (2.3 mL) was added 6 M aqueous HCl (0.77 mL) at 0 °C. After stirring for 30 min at 40 °C, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography (PTLC) (EtOAc:nhexane = 3:7) to give non-substituted derivative 4 (6.  (6) To a solution of eurotiumide A (1) (1.6 mg, 4.6 μmol) in MeOH (0.23 mL) was added Pd/C (1.6 mg, 100 w/w%) at room temperature. After stirring for 1.5 h under hydrogen atmosphere (balloon), the reaction mixture was passed through Celite and the organic solvent was removed under reduced pressure. The residue was purified with flash column chromatography (EtOAc:n-hexane = 2:3) to give isopentyl derivative 6 (1.  (7) To a solution of diMOM-protected methyl derivative 7a (13.7 mg, 34.7 μmol) in MeOH (2.6 mL) was added 6 M aqueous HCl (0.87 mL) at 0 °C. After stirring for 3 h at 40 °C, the reaction was quenched by adding saturated aqueous NaHCO3. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:n-hexane = 3:7) to give vinyl derivative 7 (8. To a solution of diMOM-protected methyl derivative 9a (7.4 mg, 16.8 μmol) in THF (1.0 mL) was added 6 M aqueous HCl (0.50 mL) at 0 °C. After stirring for 6 h at room temperature, the reaction was quenched by adding saturated aqueous NaHCO3. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:n-hexane = 3:7) to give phenyl derivative 9 (6.0 mg, 90%) as a yellow solid. To a solution of aldehyde 12a (5.4 mg, 13.6 μmol) in MeOH (0.14 mL) were added K2CO3 (5.7 mg, 40.9 μmol) and Ohira-Bestmann reagent (3.9 mg, 20.4 μmol) at room temperature. After stirring for 40 min at the same temperature, the mixture was concentrated under reduced pressure. The residue was purified with column chromatography (EtOAc:n-hexane = 1:4 to 1:1) to give diMOM alkyne derivative 8a (6. To a solution of diMOM alkyne derivative 8a (6.3 mg, 13.6 μmol) in MeOH (1.2 mL) was added 6 M aqueous HCl (0.40 mL) at room temperature. After stirring for 24 h at the same temperature, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with column chromatography (EtOAc:n-hexane = 1:4 to 1:1) to give alkyne derivative 8 (3.3 (11) To a solution of diMOM hydroxymethyl derivative 11a (7.2 mg, 24.1 μmol) in MeOH (1.8 mL) was added 6 M aqueous HCl (0.45 mL) at 0 °C. After stirring for 4 h at 40 °C, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:n-hexane = 1:1) to give hydroxymethyl derivative 11 (3.9  To a solution of diMOM mesylated derivative 22 (5.3 mg, 11.1 μmol) in DMF (55 μL) was added NaN3 (0.79 mg, 12.1 μmol) at room temperature. After stirring for 6 h at the same temperature, the reaction was quenched by adding water at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:n-hexane = 3:7) to give diMOM azide derivative 13a (3.7 mg, 79%) as a pale-yellow oil. 1H-NMR (500 MHz, CDCl3) δ 7.44 (1H, s), 5.26 (1H, d, J = 6.9 Hz), 5.25 (1H, d, J = 6.9 Hz), 5.13 (1H, d, J = 6.9 Hz), 5.11 (1H, d, J = 6.9 Hz), 4 (13) To a solution of diMOM azide derivative 13a (8.3 mg, 19.6 μmol) in MeOH (1.5 mL) was added 6 M aqueous HCl (0.49 mL) at room temperature. After stirring for 4 h at 40 °C, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:n-hexane = 3:7) to give nitro derivative 13 (  To a solution of diMOM amine derivative 14a (4.4 mg, 11.1 μmol) in MeOH (0.83 mL) was added 6 M aqueous HCl (0.28 mL) at 0 °C. After stirring for 5 h at room temperature, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with the mixture of MeOH and CH2Cl2 (MeOH:CH2Cl2 = 1:4) (×4) and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified with PTLC (MeOH:CHCl3 saturated with NH3 = 1:9) to give amiomethyl derivative 14 ( To a solution of 3 (28.9 mg, 89.1 μmol) in AcOH (0.50 mL) was added the mixture of AcOH and 70% HNO3 (0.80 mL:0.20 mL) at 0 °C. After stirring for 10 min at the same temperature, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 °C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was pathed through SiO2 plug and the resultant mixture of monoMOM nitro derivative 15a was used for the next reaction without further purification. To a solution of 15a mixture in MeOH (7.5 mL) was added 6 M aqueous HCl (2.4 mL) at 0 °C. After stirring for 5 h at 40 °C, the reaction was quenched by adding saturated

Conclusions
We constructed a chemical library of the side-chain derivatives of eurotiumide A, which is a dihydroisocoumarin-type marine natural product. The antimicrobial evaluation of these compounds was conducted against MSSA, MRSA, and P. gingivalis. We discovered several compounds to be effective against these strains; among them, the isopentyl derivative 6 is especially more active against all three strains than 1. Continuous research to clarify the modes of action of these derivatives is under way in our laboratory.