NOVEL COMPOUNDS AND ANTIBACTERIAL COMPOSITION COMPRISING THE SAME

Abstract

The present disclosure relates to a compound represented by Chemical Formula 1 and an antibacterial pharmaceutical composition, an antibacterial food composition, an antibacterial cosmetic composition or an antibacterial feed composition comprising the compound as an active ingredient, and has excellent inhibitory activity against Mycoplasma, and thus may be usefully used to prevent or treat infectious diseases caused by Mycoplasma.

Claims

1. A compound of Chemical Formula 1 below, an optical isomer thereof or a pharmaceutically acceptable salt thereof: ##STR00007## In Chemical Formula 1, R.sub.1 is hydrogen, straight or branched C.sub.1-20 alkyl, straight or branched C.sub.1-20 alkenyl, straight or branched C.sub.1-20 alkynyl, C.sub.1-20 alkoxy, C.sub.1-20 thioalkoxy, C.sub.3-20 cycloalkyl, C.sub.3-20 heterocycloalkyl, C.sub.3-20 heteroaryl, phenyl, or halogen.

2. The compound of claim 1, wherein the compound of Chemical Formula 1 is a compound represented by Chemical Formula 2 below, an optical isomer thereof or a pharmaceutically acceptable salt thereof: ##STR00008## In Chemical Formula 2, R.sub.2 is hydrogen, straight or branched C.sub.1-13 alkyl, straight or branched C.sub.1-13 alkenyl, straight or branched C.sub.1-13 alkynyl, C.sub.1-13 alkoxy, C.sub.1-13 thioalkoxy, C.sub.3-13 cycloalkyl, C.sub.3-13 heterocycloalkyl, C.sub.3-13 heteroaryl, phenyl, or halogen.

3. The compound of claim 1, wherein the compound of Chemical Formula 1 is isolated from a microorganism deposited under Accession No. KCTC 12411BP.

4. The compound of claim 1, wherein the compound of Chemical Formula 1 is selected from the group consisting of compounds 1 to 3 shown in Table below: TABLE-US-00004 Compound Structure 1 2 3

5. A method for preparing a compound of Chemical Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, comprising isolating a compound of Chemical Formula 1 below, an optical isomer thereof, or a pharmaceutically acceptable salt thereof from a Bacillus subtilis 109GGC020 KCTC 12411BP strain or a culture thereof: ##STR00012## In Chemical Formula 1, R.sub.1 is hydrogen, straight or branched C.sub.1-20 alkyl, straight or branched C.sub.1-20 alkenyl, straight or branched C.sub.1-20 alkynyl, C.sub.1-20 alkoxy, C.sub.1-20 thioalkoxy, C.sub.3-20 cycloalkyl, C.sub.3-20 heterocycloalkyl, C.sub.3-20 heteroaryl, phenyl, or halogen.

6. An antibacterial composition comprising the compound according to any one of claims 1 to 4, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

7. The antibacterial composition of claim 6, wherein the compound has antibacterial activity against Mycoplasma.

8. The antibacterial composition of claim 7, wherein the compound has antibacterial activity against Mycoplasma hyorhinis.

9. The antibacterial composition of claim 6, wherein the composition is a pharmaceutical composition.

10. The antibacterial composition of claim 6, wherein the composition is a food composition.

11. The antibacterial composition of claim 6, wherein the composition is a cosmetic composition.

12. The antibacterial composition of claim 6, wherein the composition is a feed composition.

13. A method for preventing or treating infectious diseases caused by microorganisms to be antibacterial of the antibacterial composition, comprising administering the antibacterial composition according to claim 6 in a therapeutically effective dose to a subject other than a human.

14. The method for preventing or treating infectious diseases caused by microorganisms to be antibacterial of the antibacterial composition of claim 13, wherein the infectious diseases caused by the microorganisms to be antibacterial target of the antibacterial composition are at least one selected from the group consisting of polyserositis, arthritis, conjunctivitis, otitis, septicaemia, pneumonia, peritonitis, pleuritis, pericarditis, and porcine reproductive and respiratory syndrome (PRRS).

15. The method for preventing or treating infectious diseases caused by microorganisms to be antibacterial of the antibacterial composition of claim 13, wherein the subject is a pig.

16. A method for sterilization or bacteriostatic of microorganisms to be antibacterial of the antibacterial composition, comprising treating the antibacterial composition according to claim 6 in vitro.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 is a schematic diagram illustrating a process of fractionating Compounds 1 to 3.

[0061] FIG. 2 illustrates chemical structures of Compounds 1 to 3 [1: Compound 1/2: Compound 2/3: Compound 3].

[0062] FIG. 3 illustrates partial structures and major 2D NMR correlations of Compounds 1 to 3.

[0063] FIG. 4 illustrates results of HPLC analysis of amino acids in Compound 1 through total hydrolysis.

[0064] FIG. 5 is a partial hydrolysis flowchart of Compound 1.

[0065] FIG. 6 illustrates results of HPLC analysis of L-FDLA derivatives of P1 and P3 hydrolysates.

[0066] FIG. 7 illustrates results of HPLC analysis of L-FDLA derivatives of P2.

[0067] FIG. 8 illustrates a value (ppm) of ??.sub.H=?.sub.S-ester??.sub.R-ester for MTPA ester of 1a.

[0068] FIG. 9 is an .sup.1H NMR spectrum (600 MHz, CD.sub.3OH) of Compound 1.

[0069] FIG. 10 is a .sup.13C NMR spectrum (150 MHz, CD.sub.3OH) of Compound 1.

[0070] FIG. 11 is an HSQC spectrum of Compound 1 in CD.sub.3OH.

[0071] FIG. 12 is a COSY spectrum of Compound 1 in CD.sub.3OH.

[0072] FIG. 13 is a TOCSY spectrum of Compound 1 in CD.sub.3OH.

[0073] FIG. 14 is an HMBC spectrum of Compound 1 in CD.sub.3OH.

[0074] FIG. 15 is a NOESY spectrum of Compound 1 in CD.sub.3OH.

[0075] FIG. 16 is an HR-ESIMS spectrum of Compound 1.

[0076] FIG. 17 is an .sup.1H NMR spectrum (600 MHz, CD.sub.3OH) of Compound 2.

[0077] FIG. 18 is a .sup.13C NMR spectrum (150 MHz, CD.sub.3OH) of Compound 2.

[0078] FIG. 19 is an HSQC spectrum of Compound 2 in CD.sub.3OH.

[0079] FIG. 20 is a COSY spectrum of Compound 2 in CD.sub.3OH.

[0080] FIG. 21 is a TOCSY spectrum of Compound 2 in CD.sub.3OH.

[0081] FIG. 22 is an HMBC spectrum of Compound 2 in CD.sub.3OH.

[0082] FIG. 23 is a ROESY spectrum of Compound 2 in CD.sub.3OH.

[0083] FIG. 24 is an HR-ESIMS spectrum of Compound 2.

[0084] FIG. 25 is an .sup.1H NMR spectrum (600 MHz, CD.sub.3OH) of Compound 3.

[0085] FIG. 26 is a .sup.13C NMR spectrum (150 MHz, CD.sub.3OH) of Compound 3.

[0086] FIG. 27 is an HSQC spectrum of Compound 3 in CD.sub.3OH.

[0087] FIG. 28 is a COSY spectrum of Compound 3 in CD.sub.3OH.

[0088] FIG. 29 is a TOCSY spectrum of Compound 3 in CD.sub.3OH.

[0089] FIG. 30 is an HMBC spectrum of Compound 3 in CD.sub.3OH.

[0090] FIG. 31 is a ROESY spectrum of Compound 3 in CD.sub.3OH.

[0091] FIG. 32 is an HR-ESIMS spectrum of Compound 3.

DETAILED DESCRIPTION

[0092] Hereinafter, Examples of the present disclosure will be described in detail so as to easily implement those skilled in the art. However, the present disclosure may be embodied in many different forms and is limited to embodiments described herein.

[0093] In previous studies, cyclic lipopeptide-based bacilotetrins A and B having antibacterial activity were found from marine-derived Bacillus subtilis 109GGC020. Through additional research on an ethyl acetate (EtOAc) extract obtained from a culture of Bacillus subtilis 109GGC020, it was confirmed that three novel cyclic lipodepsipeptides, bacilotetrins C, D, and E (Compounds 1 to 3) were isolated, and these materials exhibited excellent antibacterial activity against Mycoplasma.

[0094] Hereinafter, the isolation, structural description, and antibacterial activity of bacilotetrins C, D, and E (Compounds 1 to 3), which were novel cyclic lipodepsipeptides, will be described.

Experimental Materials and Apparatus

[0095] UV spectrum was obtained using a UV-1650PC spectrophotometer (Shimadzu Co., Japan). IR spectrum was measured using an FT/IR-4100 spectrophotometer (JASCO Co., Japan). Specific optical rotation was measured using an Autopol III S2 polarimeter (Rudolph analytical Co., USA). NMR spectra were measured at 600 MHz for .sup.1H and 150 MHz for .sup.13C using a Bruker AVANCE III 600 spectrometer (Bruker BioSpin GmbH, Germany) and obtained using a 3 mm probe. Chemical shift values were based on solvent peaks (?.sub.H 3.31 and ?.sub.C 49.15) of CD.sub.3OH. LR-EIMS and Marfey's analysis were obtained using an Agilent 6100 single quadrupole mass spectrometer (Agilent Technologies, USA), and HR-ESIMS data were obtained using a SYNPT G2 Q-TOF mass spectrometer (Waters Co., USA) at the Korea Basic Science Institute (KBSI) in Cheongju, Korea. HPLC was used with a PrimeLine binary pump (Analytical Scientific Instruments, Inc., USA), a Shodex RI-101 refractive index detector (Shoko Scientific Co. Ltd., Japan), and an S3210 variable UV detector (Schambeck SFD GmbH, Germany), and Thermo Fisher Scientific UltiMate 3000 UHPLC (Thermo Scientific, Germany) was also used in the experiments. HPLC columns were used with YMC-ODS-A (250 mm?10 mm, 5 ?m and 250 mm?4.6 mm, 5 ?m), and YMC-Triart C.sub.18 (250 mm?10 mm, 5 ?m and 250 mm?4.6 mm, 5 ?m). As a filler for open column chromatography, reversed-phase silica gel (YMC-Gel ODS-A, 12 nm, S-75 ?m) was used. The organic solvents used in the experiment were HPLC-grade solvents purchased from Duksan (Korea) and Samchun (Korea). Distilled water and ultrapure water were obtained through a Milipore Mili-Q Direct 8 system (Milipore S.A.S., France).

Examples

Isolation and Culture of the Producing Strain

[0096] Bacillus subtilis 109GGC020 (KCTC 12411BP) was isolated from sponges collected from Gageo Reef, Korea in 2010.

[0097] For seed culture and mass cultures, a Bennett (BN) liquid medium (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 1.85% artificial sea salt, pH 7) was used. The seed culture was inoculated with cells in a 250 mL conical flask containing 100 mL of the BN liquid medium, and then cultured for 3 days in a shaking incubator at 28? C. and 120 rpm. For the mass culture, 70 L of the same liquid medium was prepared in a 100 L fermentor, a seed culture medium was inoculated in an aseptic condition, and then cultured at 28? C., 55 rpm, and airflow rate of 20 L/min (LPM) for 7 days. The culture medium was separated into cells and the culture medium using a high-speed centrifuge, and the separated culture medium was extracted twice with the same amount of ethyl acetate (EtOAc, 70 L?2).

[0098] Bacillus subtilis 109GGC020 was deposited on May 27, 2013 and was given accession number KCTC 12411BP.

Separation and Purification of Compounds

[0099] The ethyl acetate (EtOAc) extract prepared from the strain culture medium was concentrated under reduced pressure to obtain 28.4 g of a crude extract. 9.7 g of the crude extract was subjected to vacuum column chromatography using ODS-A gel (YMC Gel ODS-A, 12 nm, S 75 ?m). A solvent was eluted with a mixture of methanol and water stepwise (20, 40, 60, 80, and 100% MeOH in H.sub.2O). 2.3 g of the 100% methanol fraction was subjected to ODS-A vacuum column chromatography once more, and sequentially eluted with the solvent conditions of 80, 90, and 100% MeOH. Each fraction was divided into three subfractions, and the third subfraction (1.5 g) of 90% MeOH was separated and purified using reversed-phase HPLC (YMC ODS-A, 250?10 mm, 5 ?m, 86% MeOH, 2.0 mL/min, RI detector) to obtain Compound 1 (19.1 mg, t.sub.R 37 min). The first subfraction (200 mg) of 100% MeOH fraction was purified using reversed-phase HPLC (YMC Triart C.sub.18, 250?10 mm, 5 ?m, 90% MeOH, 2.0 mL/min, RI detector) to obtain a small subfraction containing Compounds 2 and 3. This small subfraction was again subjected to reversed-phase HPLC (YMC Triart C.sub.18, 250?4.6 mm, 5 m, 70% MeCN+0.010% TFA, 0.7 mL/min, UV detector: 224 nm) to separate and obtain pure compound 2 (3.7 mg, t.sub.R 49 min) and compound 3 (2.7 mg, t.sub.R 51 min).

[0100] Compound 1 (Bacilotetrin C (1)): amorphous solid; [?].sub.D.sup.25?50 (c 0.1, MeOH); IR(MeOH) ?.sub.max 3297, 2925, 1643, 1052 cm.sup.?1; .sup.1H and .sup.13C NMR data (Table 2); HR-ESIMS m/z [M+Na].sup.+717.4775 (calculated for C.sub.37H.sub.66N.sub.4O.sub.8Na, 717.4778).

[0101] Compound 2 (Bacilotetrin D (2)): amorphous solid; [?].sub.D.sup.25?70 (c 0.1, MeOH); IR(MeOH) ?.sub.max 3300, 2957, 1653, 1057 cm.sup.?1; .sup.1H and .sup.13C NMR data (Table 2); HR-ESIMS m/z [M+Na].sup.+731.4934 (calculated for C.sub.38H.sub.68N.sub.4O.sub.8Na, 731.4935).

[0102] Compound 3 (Bacilotetrin E (3)): amorphous solid; [?].sub.D.sup.25?63 (c 0.1, MeOH); IR(MeOH) ?.sub.max 3297, 2961, 1650, 1057 cm.sup.?1; .sup.1H and .sup.13C NMR data (Table 2); HR-ESIMS m/z [M+Na].sup.+731.4937 (calculated for C.sub.38H.sub.68N.sub.4O.sub.8Na, 731.4935).

TABLE-US-00002 TABLE 2 1 2 3 ?.sub.C, ?.sub.H, mult. ?.sub.C, ?.sub.H, mult. ?.sub.C, ?.sub.H, mult. Position type (J in Hz) type (J in Hz) type (J in Hz) Glu 1 175.9 175.9, C 175.9, C 2 55.6, CH 4.11, td (7.3, 4.0) 55.5, CH 4.11, m 55.5, CH 4.11, m 3 27.3, CH.sub.2 1.95, q (14.9, 7.3) 27.3, CH.sub.2 1.95, q (14.9, 7.4) 27.3, CH.sub.2 1.94, q (14.8, 7.8) 4 31.1, CH.sub.2 2.42, m 31.1, CH.sub.2 2.43, m 31.1, CH.sub.2 2.42, m 5 176.3, C 176.3, C 176.3, C NH 8.39, d (4.7) 8.42, d (4.5) 8.41, d (4.5) Leu-1 1 173.9, C 173.9, C 173.9, C 2 55.0, CH 3.74, m 55.0, CH 3.75, m 55.0, CH 3.74, m 3 38.5, CH.sub.2 2.01, ddd (14.6, 11.0, 3.9) 38.5, CH.sub.2 2.01, m 38.5, CH.sub.2 2.01, m 1.80, o.1.sup.a 1.80, o.1.sup.a 1.80, o.1.sup.a 4 26.3, CH 1.61, m 26.3, CH.sub.2 1.60, o.1.sup.a 26.3, CH 1.60, o.1.sup.a 5 21.3, CH.sub.3 0.93, d (5.8) 21.3, CH.sub.3 0.93, d (5.4) 21.3, CH.sub.3 0.94, d (5.3) 6 24.0, CH.sub.3 0.95, d (5.8) 24.0, CH.sub.3 0.95, d (5.4) 24.0, CH.sub.3 0.95, d (5.3) NH 9.10, d (6.7) 9.13, d (6.6) 9.12, d (6.7) Leu-2 1 174.5, C 174.5, C 174.5, C 2 53.4, CH 4.43, m 53.3, CH 4.42, m 53.4, CH 4.42, m 3 40.4, CH.sub.2 1.81, o.1.sup.a 40.4, CH.sub.2 1.76, o.1.sup.a 40.4, CH.sub.2 1.78, o.1.sup.a 4 26.2, CH 1.70, m 26.2, CH 1.71, o.1.sup.a 26.2, CH 1.71, o.1.sup.a 5 21.2, CH.sub.3 0.90, d (6.4) 21.2, CH.sub.3 0.90, d (6.4) 21.2, CH.sub.3 0.91, d (6.4) 6 23.9, CH.sub.3 0.96, d (6.4) 23.9, CH.sub.3 0.96, d (6.4) 23.9, CH.sub.3 0.98, d (6.4) NH 7.74, d (8.6) 7.75, d (8.5) 7.74, d (8.8) Leu-3 1 172.9, C 173.0, C 173.0, C 2 51.6, CH 4.57, m 51.6, CH 4.57, m 51.6, CH 4.57, m 3 40.6, CH.sub.2 1.80, m 40.6, CH.sub.2 1.81, m 40.6, CH.sub.2 1.80, m 1.69, m 1.69, m 1.70, m 4 25.8, CH 1.65, o.1.sup.a 25.7, CH 1.65, o.1.sup.a 25.7, CH 1.66, m 5 21.8, CH.sub.3 0.89, o.1.sup.a 21.7, CH.sub.3 0.89, d (6.4) 21.7, CH.sub.3 0.90, d (6.5) 6 23.8, CH.sub.3 0.92, d (6.5) 23.7, CH.sub.3 0.92, d (6.4) 23.7, CH.sub.3 0.93, d (6.5) NH 7.76, d (9.5) 7.77, d (9.5) 7.77, d (9.4) ?-OH acid 1 173.3, C 173.3, C 173.0, C 2 41.5, CH.sub.2 2.72, dd (13.8, 4.6) 41.5, CH.sub.2 2.72, dd (13.8, 4.6) 41.5, CH.sub.2 2.72, dd (13.8, 4.7) 2.29, dd (13.8, 8.1) 2.29, dd (13.8, 8.1) 2.29, dd (13.8, 8.1) 3 73.8, CH 5.16, tt (7.8, 5.3) 73.8, CH 5.15, m 73.8, CH 5.15, m 4 35.5, CH.sub.2 1.80, o.1.sup.a 35.4, CH.sub.2 1.81, o.1.sup.a 35.4, CH.sub.2 1.81, o.1.sup.a 1.57, o.1.sup.a 1.56, o.1.sup.a 1.62, o.1.sup.a 5 26.3, CH.sub.2 1.28, o.1.sup.a 6 7 28.2- 1.29, o.1.sup.a 8 26.4- 30.6, CH.sub.2 9 30.8, CH.sub.2 1.29, o.1.sup.a 28.6- 1.28, o.1.sup.a 10 31.0, CH.sub.2 11 30.7, CH.sub.2 1.34, o.1.sup.a 1.13, o.1.sup.a 12 33.1, CH.sub.2 1.27, o.1.sup.a 35.7, CH 1.29, o.1.sup.a 40.3, CH.sub.2 1.16, m 13 23.8, CH.sub.2 1.30, o.1.sup.a 37.8, CH.sub.2 1.29, o.1.sup.a 29.2, CH 1.51, m 1.09, o.1.sup.a 14 14.5, CH.sub.3 0.89, o.1.sup.a 11.8, CH.sub.3 0.87, o.1.sup.a 23.1, CH.sub.3 0.87, d (6.4) 15 19.7, CH.sub.3 0.85, d (4.8) 23.1, CH.sub.3 0.87, d (6.4) .sup.aSignals were overlapped with other signals

Total Hydrolysis and Marfey's Analysis

[0103] Compound 1 (0.4 mg) was added with 6N HCl (300 ?L) and stirred at 110? C. for 12 hours. The completion of the reaction was confirmed by LR-LCMS analysis, and the reactant was cooled at room temperature and fractionated with water and hexane. A water layer was concentrated under reduced pressure, added with 600 ?L of 0.1% 1-fluoro-2,4-dinitro-phenyl-5-L-leucinamide (L-FDLA) dissolved in acetone and 120 ?L of 1 M NaHCO.sub.3 and then stirred at 40? C. for 1 hour. The mixture was cooled to room temperature, added with 120 ?L of 1N HCl to be neutralized, and then diluted with MeCN (420 ?L). Standard L- and D-amino acids were reacted with L-FDLA in the same manner. A Marfey's derivative of compound 1 was analyzed using LR-LCMS (YMC ODS-A, 250?4.6 mm, 5 ?m, 0.5 mL/min, UV: 340 nm) under a MeCNH.sub.2O (+0.02% TFA) solvent system with a gradient condition (40% MeCN 5 min, 40-80% MeCN 20 min, 80% MeCN 5 min), and then compared with retention times of standard amino acid derivatives. As a result, the composition of amino acids included in compound 1 was identified as L-Glu (16.9 min), L-Leu (23.6 min), and D-Leu (29.0 min). The retention times of standard amino acid derivatives bound with L-FDLA were L-Glu (16.9 min), D-Glu (17.8 min), L-Leu (23.6 min), and D-Leu (28.9 min).

Partial Hydrolysis and Marfey's Analysis

[0104] Compound 1 (2.0 mg) was added with 1 mL of 4N HCl:AcOH (1:1) and then reacted at 100? C. for 2 hours. The reactant was monitored using LR-LCMS, and the reactant for which partial hydrolysis was confirmed was concentrated with nitrogen gas and then fractionated using water and hexane. A concentrated water layer was eluted using LR-LCMS (YMC-ODS-A, 250?4.6 mm, 5 m, 0.5 mL/min, UV: 224 nm) under a MeCNH.sub.2O (+0.02% TFA) solvent condition with a gradient (20% MeCN 10 min, 20-100% MeCN 40 min, 100% MeCN 10 min), and separated into three partial structures P1: Glu-Leu. t.sub.R 7.6 min, m/z 261 [M+H].sup.+; P2: Leu-Leu, t.sub.R 23.0 min, m/z 245 [M+H].sup.+; P3: ?-OH acid-Leu, t.sub.R 28.6 min, m/z 358 [M+H].sup.+). Among them, P1 (Glu-Leu) and P3 (?-OH acid-Leu) were subjected to total hydrolysis once more, reacted with L-FDLA, and analyzed using LR-LCMS as described above, and as a result, leucine contained in these two substructures was confirmed as L-form (hydrolyzate of P1-L-FDLA: t.sub.R 23.7 min, hydrolyzate of P3-L-FDLA: t.sub.R 23.7 min). The remaining partial structure P2 (Leu-Leu) reacted with L-FDLA and then compared and analyzed for retention times with the standard reagents (L-Leu-D-Leu and D-Leu-L-Leu) reacted with L-FDLA. It was confirmed that Leu-Leu of P2 was a mixture of L-Leu-D-Leu and D-Leu-L-Leu (P2-L-FDLA: t.sub.R 26.1 min (major) and t.sub.R 31.6 min (minor), m/z 589 [M+H].sup.+; L-Leu-D-Leu-L-FDLA: t.sub.R 26.1 min, m/z 589 [M+H].sup.+, D-Leu-L-Leu-L-FDLA: t.sub.R 31.6 min, m/z 589 [M+H].sup.+).

Methanolysis of Compound 1

[0105] Compound 1 (2.4 mg) was dissolved in 1.2 mL of 3 M methanolic HCl, and then refluxed for 2 hours. The completion of the reaction was confirmed through LR-LCMS analysis, and the reactant was concentrated with nitrogen gas and then fractionated with water and hexane. A hexane layer was concentrated to obtain fatty acid ester 1a (crude fatty acid ester).

Preparation of (S)- and (R)-MTPA Esters (1b and 1c)

[0106] Fatty acid ester 1a obtained by methanolysis was divided into two, and each solvent was removed with nitrogen gas. Each vial was added with some 4-dimethylaminopyridine (DMAP) and 80 ?L of anhydrous pyridine and then stirred at room temperature for 5 minutes. Then, R-(?) or S-(+)-?-methoxy-?-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) was added in 5 ?L each, and then stirred at room temperature for 16 hours. The reactant was concentrated with nitrogen gas at 40? C., dissolved in methylene chloride (MC), and washed with a 1N HCl solution, a saturated sodium bicarbonate (NaHCO.sub.3) aqueous solution, and brine. The MC layer was treated with anhydrous magnesium sulfate (MgSO.sub.4). The extract was concentrated under reduced pressure to obtain (S)-MTPA ester 1b (0.2 mg, t.sub.R 39.4 min) and (R)-MTPA ester 1c (0.3 mg, t.sub.R 39.6 min) using reversed-phase HPLC (YMC-Triart C.sub.18, 250?4.6 mm, 5 ?m, 1.0 mL/min, UV: 210 and 254 nm) and a MeCNH.sub.2O solvent system with a gradient (40% MeCN 5 min, 40-100% MeCN 30 min, 100% MeCN 10 min). .sup.1H chemical shift values around the stereogenic center of each MTPA ester were confirmed through .sup.1H and COSY spectra.

[0107] S-MTPA ester of 1a (1b): .sup.1H NMR (600 MHz, CDCl.sub.3) ?.sub.H 5.45 (m, H-3), 3.57 (s, OCH.sub.3), 2.62 (dd, J=15.9, 8.0 Hz, H-2a), 2.56 (dd, J=15.9, 5.0 Hz, H-2b), 1.72 (m, H-4a), 1.64 (m, H-4b); ESIMS m/z [M+Na].sup.+497.3.

[0108] R-MTPA ester of 1a (1c): .sup.1H NMR (600 MHz, CDCl.sub.3) ?.sub.H 5.45 (m, H-3), 3.64 (s, OCH.sub.3), 2.67 (dd, J=15.9, 8.3 Hz, H-2a), 2.60 (dd, J=15.9, 4.6 Hz, H-2b), 1.63 (m, H-4a), 1.58 (m, H-4b); ESIMS m/z [M+Na].sup.+497.2.

Determination of the Structures of Compounds

[0109] It was confirmed that bacilotetrin C (1) was an amorphous solid, and the molecular formula was C.sub.37H.sub.66N.sub.4O.sub.8 (unsaturation degree 7) through HR-ESIMS. NMR data of Compound 1 are shown in Table 2 above. In a .sup.1H NMR (CD.sub.3OH) spectrum, it was confirmed that there were four NH groups (?.sub.H 9.10, 8.39, 7.76 and 7.74). In .sup.1H and .sup.13C NMR and HSQC spectra, four ?-protons (?.sub.H 4.57, 4.43, 4.11, and 3.74), long fatty acids (?.sub.H 1.29), oxygen-bonded hydrogen (?.sub.H 5.16), seven methyl hydrogens (?.sub.H 0.96 to 0.89) and six carbonyl carbons (?.sub.C 176.3, 175.9, 174.5, 173.9, 173.3, and 172.9) were confirmed. Through detailed .sup.1H-.sup.1H COSY, TOCSY, and HMBC spectral analysis, the presence of three leucines (Leu), one glutamic acid (Glu), and ?-hydroxy fatty acid (?-OH acid) was confirmed. Based on the degree of unsaturation and molecular formula, the structure of Compound 1 was confirmed as a cyclic lipodepsipeptide (see FIG. 2). The linkage between each amino acid and fatty acid was determined by HMBC and NOESY spectral analysis. The linkage of the peptide was confirmed by HMBC signals (C-1 (?.sub.C 172.9) of Leu-3 in H-3 (?.sub.H 5.16) of ?-OH acid; C-1 (?.sub.C 173.9) of Leu-3 in NH (?.sub.H 7.76) and H-2 (?.sub.H 4.57) of Leu-3; C-1 (?.sub.C 173.9) of Leu-1 in NH (?.sub.H 7.74) of Leu-2; C-1 (?.sub.C 175.9) of Glu in NH (?.sub.H 9.10) and H-2 (?.sub.H 3.74) of Leu-1; and C-1 (?.sub.C 173.3) of ?-OH acid in NH (?.sub.H 8.39) of Glu). The linkage between amino acid residues and ?-OH acid was also supported by NOESY signals (NH (?.sub.H 7.76) of Leu-3 and H-2 (?.sub.H 4.43) of Leu-2; NH (?.sub.H 7.74) of Leu-2 and H-2 (?.sub.H 3.74) of Leu-1; NH (?.sub.H 7.74) of Leu-1 and H-2 (?.sub.H 4.11) of Glu; and NH (?.sub.H 8.39) of Glu and H-3 (?.sub.H 5.16) of ?-OH acid). The sequence of compound 1 was determined as cyclo-(?-OH acid-Glu-Leu-1-Leu-2-Leu-3) by the HMBC signal from the ?-proton and amide proton of each amino acid to the carbonyl carbon of adjacent amino acids or ?-OH acid and the NOESY spectrum between the ?-proton and amide proton of amino acids (see FIG. 3). The ?-OH acid chain was expected to be linear with one remaining methyl group (?.sub.C 14.5, ?.sub.H 0.89), and also consistent with chemical shift values (?.sub.C 14.4, ?.sub.H 0.90) of a methyl group of a terminal of linear ?-OH acid recently reported and the expectation was supported.

[0110] The absolute structure of amino acids included in Compound 1 was determined using an advanced Marfey's method. As a result, it was confirmed that Compound 1 had two L-Leu, one D-Leu, and one L-Glu (see FIG. 4). To confirm the location of D-Leu, Compound 1 was partially hydrolyzed to obtain a dipeptide mixture. HPLC-MS was used to purify the mixture, and as a result, Leu-Glu, Leu-Leu, and ?-OH acid-Leu fragments were separated. Both the Leu-Glu and ?-OH acid-Leu parts were confirmed as L-form amino acids by Marfey's method (see FIG. 6). Leu-Leu (P2) was obtained by reacting the standard reagents L-Leu-D-Leu and D-Leu-L-Leu with 1-fluoro-2,4-dinitrophenyl-5-L-leucine amide (L-FDLA), respectively, and the retention times were compared and analyzed. As a result of HPLC-MS analysis of the Leu-Leu (P2) portion reacted with L-FDLA, both a large peak (t.sub.R 26.1 min, m/z 589 [M+H].sup.+) and a small peak (t.sub.R 31.6 min, m/z 589 [M+H].sup.+) appeared, which were consistent with L-Leu-D-Leu-L-FDLA (t.sub.R 26.1 min, m/z 589 [M+H].sup.+) and D-Leu-L-Leu-L-FDLA (t.sub.R 31.6 min, m/z 589 [M+H].sup.+) reacted with standard reagents, respectively (see FIG. 7). Accordingly, the Leu-Leu fragment (P2) was identified as a mixture of Leu-1-Leu-2 (major) and Leu-2-Leu-3 (minor) fragments, and the sequence of three leucines was determined to be L-D-L. To investigate the absolute configuration of ?-OH acid, compound 1 was subjected to methanolysis to obtain ?-OH acid methyl ester (1a) (see FIG. 5). Compound 1a was divided into two parts, respectively, and attached with (R)-(?)-?-Methoxy-?-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl) and (S)-(+)-?-Methoxy-?-(trifluoromethyl)phenylacetyl chloride ((S)-MTPA-Cl) to be converted to (S)-MTPA ester (1b) and (R)-MTPA ester (1c). .sup.1H NMR chemical shift values of Compounds 1b and 1c were confirmed by analysis of COSY spectra. Based on ??.sub.H values (?.sub.S-ester??.sub.R-ester), the absolute configuration of C-3 in ?-OH acid methyl ester was confirmed as R-form (see FIG. 8).

[0111] Bacilotetrin D (2) and E (3) were separated as amorphous solids, respectively, and confirmed to have the same molecular formula as C.sub.38H.sub.68N.sub.4O.sub.8 (7 degrees of unsaturation) by HR-ESIMS data analysis. NMR data of compounds 2 and 3 were shown in Table 2 above. .sup.1H and .sup.13C NMR spectra of Compounds 2 and 3 were very similar to those of Compound 1, and there was a difference that only a terminal portion of ?-OH acid was different. A significant difference in the .sup.1H and .sup.13C NMR spectra of compounds 2 and 3 was in the chemical shift value of the methyl group present at the end of the ?-OH acid chain. Bacilotetrin D (2) was a signal of an anteiso-methyl group (?.sub.C 19.7/?.sub.H 0.85 and ?.sub.C 11.8/?.sub.H 0.87), whereas bacilotetrin E (3) was a signal of an iso-methyl group (?.sub.C 23.1/?.sub.H 0.87?2). In the HMBC spectrum of compound 2, the linkage to C-11 (?.sub.C 30.7) and C-12 (?.sub.C 35.7) in H-13 (?.sub.H 1.29 and 1.09) of ?-OH acid and the linkage to C-13 (?.sub.C 37.8) in H-14 (?.sub.H 0.87) and H-14 (?.sub.H 0.87) were shown. In these signals, it was confirmed that the terminal portion of the fatty acid of Compound 2 was an anteiso-type. Bacilotetrin E (3) was identified as a fatty acid having an iso-methyl terminal by showing a signal from H-14 and H-15 (?.sub.H 0.87) to C-13 (?.sub.C 29.2) of ?-OH acid and a signal from H-12 (?.sub.H 1.16) to C-14 (?.sub.C 23.1) and C-15 (?.sub.C 23.1) of ?-OH acid in an HMBC spectrum (see FIG. 2). In addition, the methyl signals of compounds 2 (anteiso-) and 3 (iso-) coincided with values reported in the literature (anteiso-type: ?.sub.C 19.6/?.sub.H 0.86 and ?.sub.C 11.8/?.sub.H 0.88; isotype: ?.sub.C23.1/?.sub.H 0.88). Thus, the sequences of compounds 1 to 3 were identified as cyclo-(R-?-OH acid-L-Glu-L-Leu-D-Leu-L-Leu).

[0112] The structures of compounds 1 to 3 were similar to surfactins. The surfactins are cyclic lipopeptides and consist of seven amino acids (L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu) and ?-OH acid having 13 to 15 carbon atoms. Compounds 1 to 3 were also cyclic lipopeptides consisting of four amino acids (L-Glu-L-Leu-D-Leu-L-Leu) and ?-OH acid having 14 or 15 carbon atoms, in a similar manner thereto. In the structural similarity, it was expected that compounds 1 to 3 were biosynthesized by a biosynthetic pathway (non-ribosomal peptide synthetase, NRPS) similar to surfactins. The cyclic lipopeptides, surfactins, were synthesized by three surfactin synthetase subunits SrfA-A, SrfA-B, and SrfA-C. Among these subunits, SrfA-A and SrfA-B each consisted of three modules, and SrfA-C consisted of one module, and each module was involved in the production of one amino acid. In the case of compounds 1 to 3, it is expected to be a structure generated by inactivating the three modules of the SrfA-B subunit.

Experimental Example

Measurement of Mycoplasma Inhibitory Activity

[0113] For compounds 1 to 3, the inhibitory activity against Mycoplasma hyorhinis was evaluated using a broth dilution assay. Briefly, an experimental strain Mycoplasma hyorhinis ATCC 17981 was cultured in a PPLO liquid medium at 37? C. in a 5% CO.sub.2 incubator. Compounds 1 to 3 were dissolved in DMSO, serially diluted 2-fold in the PPLO liquid medium to make a concentration of 500-1 ?g/mL, and the final DMSO concentration did not exceed 5%. 100 ?L of the culture medium, which made with the strain suspension at approximately 2?10.sup.4 CFU/mL, was added to each well of a 96-well plate. The plates were incubated for 7 days in a 37? C., 5% CO.sub.2 incubator. As the bacteria grew, the medium turned yellow. A minimum inhibitory concentration (MIC) was determined as the lowest concentration at which bacteria did not grow. BioMycoX? (CellSafe, Korea) was used as a positive control. The measurement results were shown in Table 3 below.

[0114] Referring to Table 3 below, it was found that the MIC values for Mycoplasma bacteria of compounds 1 to 3 were 31 ?g/mL. These results confirmed that the cyclic lipodepsipeptide structure played an important role in the inhibitory activity against M. hyorhinis, rather than the influence of the terminal portion of ?-OH acid.

TABLE-US-00003 TABLE 3 Compounds 1 2 3 BioMycoX?.sup.1 MIC (?g/mL) 31 31 31 62 .sup.1Positive control.

[0115] As described above, the present disclosure has been described through Examples. Those skilled in the art will be able to understand that the present disclosure may be easily executed in other detailed forms without changing the technical spirit or an essential feature thereof. Therefore, it is to be understood that the above-described embodiments are illustrative and not restrictive in all respects. The scope of the present disclosure is represented by claims to be described below rather than the detailed description, and it is to be interpreted that the meaning and scope of the claims and all the changes or modified forms derived from the equivalents thereof come within the scope of the present disclosure.

[Accession Number]

[0116] Depositary Authority Name: Korea Research Institute of Bioscience and Biotechnology

[0117] Accession number: KCTC 12411BP

[0118] Accession Date: 20130524