KR101493800B1 - New compounds having antimicrobial activity produced by Marine Bacillus sp. - Google Patents

Abstract

[화학식 5]

The present invention relates to novel compounds represented by the following formulas (4) to (5) having antimicrobial activity, and these novel compounds are produced by microorganisms of the genus Bacillus.
[Chemical Formula 4]

[Chemical Formula 5]

Description

[0001] The present invention relates to a novel compound having an antimicrobial activity produced by microorganisms of the genus Bacillus sp.

The present invention relates to a microorganism belonging to the genus Bacillus that produces a novel compound and a novel compound produced from the microorganism of the genus Bacillus, and more particularly to a novel compound showing antimicrobial activity and a microorganism belonging to the genus Bacillus.

Marine microorganisms have been taxonomically diverse and have been genetically specific and have been recognized as producing good physiologically active substances. Recently, marine bacteria have attracted much interest due to their unique secondary metabolites, and their metabolites are quite different from those produced by terrestrial organisms. Since marine microorganisms survive in extreme conditions of life, they have a diverse and unique metabolic pathway, which is highly likely to find a good drug, but not much research.

While the discovery of new antibiotics is steadily declining, infectious diseases caused by drug-resistant pathogens are increasing day by day. Therefore, new antibiotics are urgently needed to treat such infectious diseases.

As a result of investigation of the prior patent relating to the present invention, it has been found that "a novel macrolactin compound having a peptide depo milia inhibitory and antibacterial activity" (Korean Patent Laid-Open Publication No. 2009-0030910), "a novel microorganism belonging to the genus Bacillus, (Korean Patent Laid-Open Publication No. 1998-017913), "a novel Bacillus strain inhibiting potato scab disease, an antibiotic substance produced by the strain, and a microorganism preparation containing the same" 1998-017913), "Macroactin A" produced by Bacillus polyfermenticus kayserase-2 strain (Korean Patent Laid-Open Publication No. 2008-0084479), "Macrolactone derivative" No. 2010-0031716). However, there was no compound having the same or similar structure as the novel compound of the present invention, and there was no invention effective for antimicrobial activity as in the present invention.

DISCLOSURE OF THE INVENTION In order to solve the above-mentioned problems, the inventors of the present invention have found a microorganism belonging to the genus Bacillus that produces a novel compound having antimicrobial activity, and completed the present invention. The present invention provides a microorganism belonging to the genus Bacillus that produces a novel compound having microbial activity and a novel substance produced from the microorganism of the genus Bacillus.

In order to accomplish the above object, the present invention provides a novel marine Bacillus sp. Microorganism deposited with deposit number KCTC 11975BP and a novel compound represented by the following formula (1) to (5) having antimicrobial activity generated from the new marine Bacillus sp. Microorganism to provide. Hereinafter, for convenience of explanation, the compounds represented by formulas (1) to (5) are referred to as compounds (1) to (5), respectively.

The present invention provides novel marine Bacillus sp. Microorganisms and novel compounds produced from such marine Bacillus sp. Microorganisms, and the novel compounds are effective for antimicrobial activity.

Figure 1 shows the HMBC and COZY correlation of the compounds of the present invention.
Figure 2 shows the ROESY correlation of the compounds of the invention.
Fig. 3 shows the results of the calculation of Δδ _H (Δδ _H = δ _S - δ _R ) obtained for the (S) - and - ( _R )
Figure 4 shows ¹ H NMR data of compounds of the present invention.
Figure 5 shows ¹³ C NMR data of compounds of the invention.
6 shows the relative arrangement of Compounds 4 and 5 based on Kishi's universal NMR database.
7 shows the chemical structure of the compounds of the present invention.

Hereinafter, the present invention will be described in detail. The following examples are only specific examples for carrying out the present invention and should not be construed as limiting the scope of the present invention.

The novel antimicrobial compound of the present invention was produced from a fermentation broth of marine Bacillus sp. 09ID194 isolated from marine sediments, and deposited with the deposit number KCTC 11975BP for this 09ID194.

The method for producing the novel compound of the present invention can be applied to a known method for extracting a compound from a microorganism. That is, it may include a culture step, extraction with ethyl acetate, and separation by chromatography. Also, compounds were extracted by the method described in a previously published article (Mondol, MAM et al. Cyclic ether-containing macrolactins, antimicrobial 24-membered isomeric macrolactones from a marine Bacillus sp. J. Nat. Prod 74, 2582-2587, can do.

In the present invention, it is effective to perform the culture at low salt level. The salinity is 10 to 15 g / l, preferably 12 g / l.

The isolation, bulk fermentation and extraction of the production strains were carried out as described in the previous paper (Mondol, MAM et al. Cyclic ether-containing macrolactins, antimicrobial 24-membered isomeric macrolactones from a marine Bacillus sp. J. Nat. Prod 74, 2582-2587, 2011) . ≪ / RTI >

Purification was achieved by ODS column chromatography adsorption of a suspension of the extract (200 g), eluting with MeOH-H ₂ O (v / v) (1: 4, 2: 3, 3: 2, 4: 1 and 100: ) ≪ / RTI > according to a step gradient. The fractions eluted with MeOH-H ₂ O (3: 2, v / v) were again subjected to silica column chromatography and eluted with n-hexane-EtOAc (v / v) (100: Stepwise using EtOAc-MeOH (v / v) (4: 1, 3: 2, 2: 3, 1: 4, 0: 100) Lt; / RTI > The fractions eluted with EtOAc-MeOH (v / v) (4: 1) were separated by ODS HPLC for separation (H ₂ O-MeOH-MeCN: 3: 1: 1, flow rate: 1.5 ml / min, ) To obtain 11 fractions (Fr. 1-11). The compounds of formulas 1 to 5 (Fig. 7) were purified on silica HPLC from Fr.3, Fr.4, Fr.11, Fr.1 and Fr.1, respectively.

Determination of structure of compound

Determination of structure of compound 1

Compound 1 was isolated as an amorphous solid. The molecular formula was determined as C ₂₄ H ₃₄ O ₆ by high resolution Electrospray Ionization Mass Spectroscopy (HRESIMS) (m / z 441.2249 [M + Na] ⁺ ) and had eight unsaturations. The IR spectrum of Compound 1 showed characteristic absorption bands for hydroxy (3482 cm ^-1 ), carbonyl (1691 cm ^-1 ) and epoxide (1250 cm ^-1 ) functional groups. 232 and 261 nm, indicating the presence of an extended conjugated system in the molecule. This was supported by 10 sp ² carbon signals in the ¹³ C NMR spectrum. A total of 24 carbons were observed in the ¹³ C NMR spectrum, and as a result of HSQC spectrum analysis, one quaternary carbonyl, 10 sp ² Carbon, 6 oxymethines, 6 methylenes and 1 methyl carbons. One carbonyl and five double bonds showed six unsaturations and the remaining two unsaturations were due to the presence of two cyclic ring systems in compound one. ^{The 1} H and ¹³ C NMR signals clearly indicated that compound 1 belongs to the macrolactin family.

The structure of Compound 1 was determined based on ¹ H- ¹ H COZY, HSQC and HMBC spectroscopic data. ^{By 1} H- ¹ H COZY correlation one coupling sequence from H-2 of δ _H 5.55 (d, J = 11.5 Hz) to H ₃ -24 of δ _H 1.25 (d, J = 6.0 Hz) Could know. The oxymetidine hydrogen of H-23 (δ _H 4.93) showed an HMBC cross-peak with carbonyl carbon (C-1, δc 167.9), indicating the chemical shift of ¹ H NMR of this hydrogen As shown by the presence of an ester bond. The presence of the 15,16-epoxide group H ₂ -14 and H-17 were correlated with HMBC with C-15 and C-16, respectively.

The relative arrangement of compound 1 was determined by coupling constant and ROESY data analysis. The arrangement of the double bonds of C-2, C-4, C-8 and C-10 are shown in FIG. 4, with their respective ¹ H coupling constants: 11.5, 15.5, 15.5 and 11.0 Hz E, and Z, respectively, based on the ROESY correlation (FIG. 2) between H-3, H-3 and H-5, H-9 and H-10, H-10 and H-11. The H-17 and H-18 hydrogens were superimposed at the CD ₃ OD and could not directly measure their coupling constants. However, the olefin sequence of C-17 / C-18 was found to be E by the ROESY correlation between H-16 and H-18 and between H-17 and H-19. The coupling constant (J = 5.0 Hz) between H-16 and H-15 indicated that the epoxide group was cis. The fact that the epoxide group is on the? Side proves that no ROESY correlation is observed between H-13 and H-15. The absolute structure of the hydroxyl group of Compound 1 was determined by the modified Mosher's method. Compound 1 was treated with (R) - (-) - and (S) - (+) - alpha -methoxy- alpha (trifluoromethyl) phenylacetyl chloride (MTPA-Cl) in anhydrous pyridine to give tris S) - and (R) -MTPA ester derivatives 1a and 1b (FIG. 3), respectively. All proton signals of these two triester derivatives were confirmed by the ¹ H- ¹ H COZY experiment and the ¹ H chemical shift of the Tris- (R) -MTPA ester (Ib) to the Tris- (S) -MTPA ester (1a ). ≪ / RTI > The Δδ _H (Δδ _H = δ _S -δ _R ) values are shown in FIG. As a result of analysis of Δδ _H values, it was found that the absolute arrangement structure of the cubic center at C-7, C-13 and C-19 is S. The ¹ H and ¹³ C resonances of H-23 (δ _H 4.93, m) and C-23 (δ _C 72.1) of compound 1 are very similar to those of macrolactin A, and compound 1 and macrolactin A share a common biosynthetic pathway Lt; / RTI > Thus, since all macrolactin sequences are generated by the same biosynthetic pathway and the common stereocenters between macrolactin A and its derivatives have the same absolute sequence structure, the absolute structure of C-23 in compound 1 is R .

Determination of Structure of Compound 2

Compound 2 was isolated as an amorphous solid by separation using an iterative chromatographic method from the EtOAc extract. The molecular formula of Compound 2 was determined to be C ₂₅ H ₃₈ O ₇ based on the HRESIMS measurement (m / z 473.2511 [M + Na] ⁺ ) and Compound 2 was found to have seven degrees of unsaturation. This degree of unsaturation was explained by strong UV absorption at 261 and 234 nm, which also indicated the presence of an extended conjugated system. The IR spectra of Compound 2 showed characteristic absorbances at 3311 and 1646 cm ^-1 , suggesting the presence of hydroxy and ester carbonyl groups, respectively. ^{The 13} C NMR spectra (in the CD ₃ OD) showed 25 well separated signals, consisting of 1 methyl, 1 methoxy, 10 sp ² methines, 6 methylenes, 6 oxymethines, and 1 estercar Lt; / RTI > carbonyls (Fig. 4). The linkage of all hydrogen and carbon atoms of compound 2 and the linkage of the carbon skeleton were determined by the interpretation of NMR spectroscopic data such as ¹ H- ¹ H COZY, HSQC and HMBC (FIGS. 1, 4 and 5) . Only one spin system was identified in the analysis of the ¹ H- ¹ H COZY spectrum, which allowed sequential connections from H-2 to H ₃ -24 (Fig. 1). The authorship shift values of H-23 represent ester linkages, identified by the HMBC correlation between H-23 and ester carbonyl carbon (C-1). a methoxy group (OCH ₃₎ δ _H 3.28 of hydrogen represent a carbon (C-18) and HMBC cross peaks resonance at δ _C 86.6, which indicates that the methoxy group is attached to C-18.

The arrangement of the double bonds at C-2, C-4, C-8, C-10 and C-16 was determined as Z, E, E, Z and E, , 15.3, 11.3 and 15.3 Hz) and the ROESY correlation (Figs. 2 and 4). H-18 and H-19 have a syn relation (J = 3.8 Hz), which was also confirmed by ROESY correlation (FIG. 2). The absolute arrangement structure of the hydroxyl groups of Compound 2 was determined by the method of Mosher, which was modified in a similar manner as Compound 1. Analysis of Δδ _H values obtained from the tetra- (S) and (R) -MTPA ester derivatives (2a and 2b) around the stereocenter at C-7 and C-19 revealed that C-7 and C- It was found that the absolute array structure is S. H-13 (+0.19), H 2 -14 (+ 0.02 / + 0.15) and the amount of _H Δδ values for the H-15 (+0.18) is fryer (Freire), etc. The anti-1,3-diol as reported by the same as the typical Δδ _H pattern to the die-esters of acids, C-13 and C-15 has S absolute configuration of each compound 2 And R, respectively. Macrolactines are thought to be produced by a common biosynthetic pathway and the ¹ H and ¹³ C resonances of H-23 (δ _H 4.95, m) and C-23 (δ _C 72.2) Respectively. Therefore, the absolute arrangement structure of C-23 in Compound 2 can be judged as R.

Determination of Structure of Compound 3

The molecular formula of Compound 3 was determined from the HRESIMS measurement as C ₂₉ H ₄₀ O ₈ and showed [M + Na] ⁺ ion at m / z 539.2617. The UV spectrum of compound 3 showed absorption bands at λ _max 227 and 256 nm, and these were identified as extended conjugated systems. IR spectroscopy at 3374 and 1733 cm ^{-1 indicated} that hydroxy (OH) and ester carbonyl groups (C = O) were present, respectively. The 1D and 2D NMR spectra of compound 3 were identical to those of 7-O-succinylmeclactin A except for the presence of an additional methoxy group, the methoxy group (delta _H 3.66) being C-4 '(delta _H 174.5 ) Was found to be located at C-4 'due to the HMBC correlation. The optical rotation value {[?] ²³ _D- 11 (c 0.05, MeOH)} of Compound 3 was similar to that of macrolactin A. Thus, compound 3 was identified as 4'-O-methyl-7-O-succinylmeclactin A.

Determination of Structure of Compound 4

The molecular formula of compound 4 was determined as C ₁₇ H ₃₀ O ₆ , which was determined from HRESIMS to represent [M + Na] ⁺ ion at m / z 353.1935 and was found to have three degrees of unsaturation. The IR spectrum of Compound 4 showed absorption bands at 1650 and 3317 cm ^-1 , indicating the presence of ester carbonyl and hydroxyl groups, respectively. The presence of a conjugated diene system was found from the fact that the absorption band of 230 nm was observed in the UV spectrum. ^{The 13} C NMR spectra (at CD ₃ OD) showed 17 well separated signals, which consisted of one ester carbonyl (C-1), two methyls (one methyl linked to oxygen), four sp ² Methine, 4 oxymethines and 6 methylene carbons. The ¹ H NMR spectrum of compound 4 taken with CD ₃ OD showed methyl, olefinic, methylene and methine signals. The linkage of all hydrogen to carbon atoms was done by interpreting ¹ H- ¹ H COZY, HSQC and HMBC NMR spectroscopic data (FIGS. 1, 4 and 5). One hydrogen sequence from H ₂ -2 (δ _H 2.48, m) to H ₃ -16 (δ _H 1.15, d, J = 6.0 Hz) was established by ¹ H- ¹ H COZY spectroscopic data analysis. The linkage of the OCH ₃ group to the carbonyl carbon C-1 was confirmed from the HMBC cross-peak of OCH ₃ hydrogen (δ _H 3.68, s) and C-1.

The double-substituted C-8 / C-9 and C-10 / C-11 double bonds of Compound 4 were H-8 / H-9 (J = 15.3 Hz) and H-10 / H- ) With a large vicinal coupling constant value. Based on the difference in chemical shift values of hydrogen atoms between the (S) - and (R) -MTPA esters (4a and 4b) of Compound 4 [Δδ _H = δ _S- δ _R ] , And the absolute arrangement structure of C-15 was determined as R (Fig. 3). There is no effective way to determine the absolute sequence structure of a 1,3,5-triol system. The relative arrangement of the 3,5,7-triol system of compound 5 was determined by considering the 1,3,5-triol powder using a Kishi universal NMR database (database ₃ at CD ₃ OD). 1,3,5-triol system of compound 4 compared to the value of δ _C 1,3,5-triol model characteristic value δ _C of the center of the system and the carbon C-5, C-3 / C- 5 and between C-5 / C-7 were found to be anti / anti (Figure 6). Based on all spectroscopic data (1D and 2D), the structure of compound 4 is (3R ^* , 5R ^* , 7S ^* , 8E, 10E, 15R) -methyl 3,5,7,15- tetrahydroxyhexadeca- 8,10-dienoate, and was designated leucydride A.

Determination of Structure of Compound 5

Compound 5 is Positive and in ESIMS of the audio mode, each m / z 347 [M + Na ] + and 323 [MH] ^- pulled is that the molecules (quasimolecular) ions in, and these values were consistent with the molecular formula C ₁₈ H ₂₈ O _{5, 5} Dogs. This molecular formula was confirmed by HRESIMS and showed [M + Na] ⁺ ion at m / z 347.1826. The UV and IR spectra of Compound 5 were similar to Compound 4, indicating the presence of conjugated olefin powders, hydroxy groups and carbonyl groups. ¹ H- ¹ H COZY spectra, we could confirm the single-spin system from H-2 to H ₃ -18 (Fig. 1). The position of the carbonyl carbon was determined as C-1 (? _C 172.1) by HMBC cross peak between H-2 and C-1 and between H-3 and C-1 (Fig. The relative arrangement of the double bonds in C-2, C-4, C-10 and C-12 was found to be 15.2, 11.0, 14.8 and 14.7 Hz, E, and E based on the ROE between H-10 and H-12, H-11 and H-13, respectively (FIGS. 2 and 4). Due to the limited amount of sample, no data was available to determine the absolute sequence structure of compound 5. The relative arrangement of C-7 and C-9 stereocenters of Compound 5 was determined by considering the 1,3-diol powder using Kishy's universal NMR database (database 5 at CD ₃ OD). The ¹³ C NMR chemical shift values of C-7 / C-9 were consistent with the anti-sequence of the 1,3-diol model system (FIG. 6). The resonances of H-17 (δ _H 3.70, m) and C-17 (δ _C 68.4) were in agreement with H-15 (δ _H 3.72 m) and C-15 (δ _C 68.4) 4 and 5 will all be generated by the same biosynthetic pathway. Therefore, the absolute array structure of C-17 can be estimated as R. A number of spectroscopic data analyzes showed that the structure of Compound 5 was (2E, 4Z, 7S ^* , 9R ^* , 10E, 12E, 17R) -7,9,17-trihydroxyoctadeca-2,4,10,12- Tetraenoic acid (leuprolide B).

Physicochemical properties

Compound 1

Amorphous solid; [? ²³ ] _D -60 (c 0.05, MeOH); UV (MeOH)? _Max (log?) 200 (4.12), 232 (4.30) and 261 (4.09) nm; IR (MeOH) ν _max 3482 (br), 2922, 1691, 1250, 1195, 1038 cm ^-1 ; ¹ H and ¹³ C NMR data (CD ₃ OD) (FIGS. 2 and 4); HRESIMS m / z 441.2249 [M + Na] < ⁺ & gt ^; (C ₂₄ H ₃₄ O ₆ Na calculated, 441.2253).

Compound 2

Amorphous solid; [? ²³ ] _D -84 (c 0.05, MeOH); UV (MeOH)? _Max (log?) 200 (4.14), 234 (4.45) and 261 (4.25) nm; IR (MeOH) v _max 3311 (br), 2921, 1646, 1409, 1104, 1024 cm < ^{-1 &} gt ;; ¹ H and ¹³ C NMR data (CD ₃ OD) (FIGS. 2 and 4); HRESIMS m / z 473.2511 [M + Na] + (C 25 H 38 O 7 Na calcd, 473.2515).

Compound 3

White, amorphous solid; [? ²³ ] _D- 11 (c 0.05, MeOH); UV (MeOH)? _Max (log?) 200 (3.90), 227 (4.18) and 256 (3.83) nm; IR (MeOH) v _max 3374 (br), 2929, 1733, 1169, 990 cm < ^{-1 &} gt ;; ¹ H and ¹³ C NMR data (CD ₃ OD) (FIGS. 2 and 4); HRESIMS m / z 539.2617 [M + Na] < ⁺ & gt ^; (C ₂₉ H ₄₀ O ₈ Na calculated, 539.2621).

Compound 4

White, amorphous solid; [? ²³ ] _D -34 (c 0.05, MeOH); UV (MeOH)? _Max (log?) 201 (4.03), 230 (4.23) and 256 (3.28) nm; IR (MeOH) ν _max 3317 (br), 2925, 1650, 1413, 1017 cm ^-1 ; ¹ H and ¹³ C NMR data (CD ₃ OD) (FIGS. 2 and 4); HRESIMS m / z 353.1953 [M + Na] + (C 17 H 30 O 6 Na calcd, 353.1940).

Compound 5

White, amorphous solid; [? ²³ ] _D -62 (c 0.05, MeOH); UV (MeOH)? _Max (log?) 231 (4.26) and 259 (4.05) nm; IR (MeOH) v _max 3340 (br), 2928, 1623, 1456, 1039 cm < ^{-1 &} gt ;; ¹ H and ¹³ C NMR data (CD ₃ OD) (FIGS. 2 and 4); HRESIMS m / z 347.1826 [M + Na] + (C 18 H 28 O 5 Na calcd, 347.1834).

Compound 1 Tris - (S) - MTPA  The esters (1a)

Compound 1 (0.8 mg) were dissolved in 150 μl of pyridine and stirred at room temperature for 10 minutes. (R) - (-) -? - methoxy-? (Trifluoromethyl) -phenylacetyl chloride (MTPA-Cl) to prepare the tris- (S) -MTPA esters (1a) Was added to the reaction vial and the mixture was stirred at room temperature for 16 hours. The termination of the reaction was monitored using LC / MS. The reaction mixture was dried in vacuo, redissolved in EtOAc, washed with H ₂ O and purified on silica HPLC using 5% MeOH in CH ₂ Cl ₂ as eluent to give 1a (0.5 mg). All hydrogen signals of the triester derivatives (1a) were confirmed by ¹ H- ¹ H COZY experiments. 1a: amorphous solid; _{^{1 H NMR (CD 3 OD)}} δH 5.52 (d, J = 11.5Hz, H-2), 6.40 (t, J = 11.5Hz, H-3), 6.84 (dd, J = 15.5, 11.5Hz, H- 4), 5.76 (dt, J = 15.5,7.5Hz, H-5), 2.48 (m, H 2 -6), 5.55 (m, H-7), 5.66 (dd, J = 15.5, 7.5Hz, H J = 11.0, 5.0 Hz, H-11), 6.44 (dd, J = 15.5, 11.0 Hz, H- H-12a), 4.07 (m, H-13), 1.73 (m, H-14b), 2.58 (m, H-14a), 5.52 H-19), 1.52 (m, H), 4.55 (t, J = 4.5 Hz, H-16), 5.82 -20), 1.27 (m, H -21b), 1.30 (m, H-21a), 1.55 (m, H 2 -22), 4.50 (m, H-23), 1.20 (d, J = 6.0Hz, H ₃ -24), 3.47 (OCH ₃ , s), 3.53 (OCH ₃ , s), 3.54 (OCH ₃ , s), 7.34-7.56 (15H, m); ESIMS m / z 1089.90 [M + Na] < ⁺ >.

Compound 1 Tris - (R) - MTPA  The ester (1b)

In a generally analogous manner, the Tris- (R) -MTPA ester 1b is obtained using (S) - (+) - alpha -methoxy- alpha - (trifluoromethyl) phenylacetyl chloride (MTPA-Cl) lost. 1b (0.4 mg): amorphous solid; _{^{1 H NMR (CD 3 OD)}} δ 5.54 (d, J = 11.5Hz, H-2), 6.51 (t, J = 11.5Hz, H-3), 6.97 (dd, J = 15.5, 11.5Hz, H- 4), 6.00 (dt, J = 15.5, 7.5Hz, H-5), 2.54 (m, H 2 -6), 5.66 (m, H-7), 5.50 (dd, J = 15.5, 7.5Hz, H J = 11.0 Hz, H-11), 6.40 (dd, J = 15.5, 11.0 Hz, H- (M, H-12b), 2.15 (m, H-12a), 4.07 (m, H-13), 1.68 H-18), 5.43 (m, H-19), 1.56 (m, H-17), 4.53 (t, J = 5.0 Hz, H-16) -20), 1.29 (m, H -21b), 1.43 (m, H-21a), 1.54 (m, H 2 -22), 4.77 (m, H-23), 1.88 (d, J = 6.0Hz, H ₃ -24), 3.49 (OCH ₃ , s), 3.50 (OCH ₃ , s), 3.54 (OCH ₃ , s), 7.36-7.60 (15H, m); ESIMS m / z 1089.82 [M + Na] < ⁺ >.

Compound 2 Tetra - (S) - and (R) - MTPA  The esters 2a and 2b,

Tetra- (S) - and (R) -MTPA ester derivatives of Compound 2 were prepared from (R) - (-) - and (S) - (+) - MTPA-Cl, respectively, according to the method described above. All ¹ H NMR signals were confirmed by ¹ H- ¹ H COZY experiments. 2a (0.5 mg): white, amorphous solid; _{^{1 H NMR (CD 3 OD)}} δH 5.51 (d, J = 11.5Hz, H-2), 6.46 (t, J = 11.5Hz, H-3), 7.18 (dd, J = 15.0, 11.5Hz, H- 4), 5.82 (dt, J = 15.0, 7.5Hz, H-5), 2.46 (m, H 2 -6), 5.58 (m, H-7), 5.70 (dd, J = 15.3, 5.5Hz, H J = 11.3 Hz, H-11), 6.46 (dd, J = 15.3, 11.3 Hz, H-9), 5.06 H-14b), 1.83 (m, H-14a), 5.42 (m, H-12b), 2.48 J = 15.3, 4.5 Hz, H-17), 3.80 (dd, J = 8.2, 3.8 Hz, H-18) ), 5.18 (m, H- 19), 1.48 (m, H-20b), 1.58 (m, H-20a), 2.23 (m, H 2 -21), 1.35 (m, H-22b), 1.40 ( m, H-22a), 5.00 (m, H-23), 1.16 (d, J = 6.5Hz, H 3 -24), 3.25 (OCH 3, s), 3.45 (OCH 3, s), 3.48 (OCH _{3, s), 3.50 (OCH} 3, s), 3.54 (OCH 3, s), 7.32 ~ 7.62 (20H, m); ESIMS m / z 1338.51 [M + Na] < ⁺ >. 2b (0.45 mg): white, amorphous solid; _{^{1 H NMR (CD 3 OD)}} δ H 5.56 (d, J = 11.5Hz, H-2), 6.55 (t, J = 11.5Hz, H-3), 7.30 (dd, J = 15.0, 11.5Hz, H -4), 6.04 (dt, J = 15.0, 7.5 Hz, H-5), 2.64 (m, H 2 -6), 5. 68 (m, H-7), 5. 68 (dd, J = 15.3 J = 11.3 Hz, H-9), 6.05 (t, J = 11.3 Hz, H-10), 5.23 (dt, J = 11.3, 6.5 Hz, H-14b), 1.68 (m, H-14a), 2.68 (m, (Dd, J = 15.3, 4.5 Hz, H-16), 5.44 (dd, J = 15.3, 4.5 Hz, H-17), 3.71 Hz, H-18), 5.21 (m, H-19), 1.73 (m, H-20b), 1.60 (m, H-20a), 2.29 (m, H 2 -21), 1.53 (m, H- 22b), 1.63 (m, H -22a), 5.95 (m, H-23), 1.18 (d, J = 6.5Hz, H 3 -24), 3.20 (OCH 3, s), 3.47 (OCH 3, s _{), 3.52 (OCH 3, s} ), 3.54 (OCH 3, s), 3.55 (OCH 3, s), 7.35 ~ 7.60 (20H, m); ESIMS m / z 1338.40 [M + Na] < ⁺ >.

Compound 5 Tetra - (S) - and (R) - MTPA  The esters (4a and 4b)

Tetra- (S) - and (R) -MTPA esters of compound 5 were prepared from (R) - (-) - and (S) - (+) - MTPA-Cl according to methods analogous to those described above. . 5a (0.2 mg): white, amorphous solid; _{^{1 H NMR (CD 3 OD)}} ( major 0 _{people) δ H 2.25 (m, H} 2 -12), 1.48 (m, H 2 -13), 1.58 (m, H 2 -14), 0.95 (d, J = 6.5Hz, H ₃ -16); ESIMS m / z 1217.34 [M + Na] < ⁺ >. 4b (0.2 mg): white, amorphous solid; _{^{1 H NMR (CD 3 OD)}} ( major 0 _{people) δ H 2.23 (m, H} 2 -12), 1.42 (m, H 2 -13), 1.28 (m, H 2 -14), 0.90 (d, J = 6.5Hz, H ₃ -16); ESIMS m / z 1217.35 [M + Na] < ⁺ >.

In conclusion, the structures of Compounds 1 to 5 were determined as described above, and these compounds were shown to be effective for antimicrobial activity. It was also found that these compounds are produced in low salt but not high salt culture medium.

Compounds 1 to 5 Antimicrobial  Active experiment

The MIC (minimum inhibitory concentration) of the compounds 1 to 5 was measured by using a conventional culture medium dilution method. The antimicrobial activity of the compounds 1 to 5 Three microorganism species, Bacillus subtilis (KCTC 1021), Escherichia coli (KCTC 1923) and Saccharomyces cerevisiae (KCTC 7913), were measured and the results are shown in Table 1 below. The antimicrobial and anti-yeast tests were conducted in nutrient broth (0.3% beef extract, 0.5% peptone and pH 7.2 before sterilization) and yeast maltose medium (1% dextrose, 0.3% beef extract, 0.5% peptone, 0.3% malt extract, PH 7.2). Two serial dilutions of each compound were prepared in a concentration range of 0.5-56 μg / ml using a 96-well microtiter plate. After culturing the culture of each strain overnight, the final microbial concentration of each culture was adjusted to 1.5 x ^{10 8} cfu / ml by comparing the turbidity of each culture with the 0.5 McFarland standard (McFarland standard). After the culture (30 μl) was added to each dilution of the compounds 1 to 5, the final amount of each well was adjusted to 200 μl using the respective culture medium. The plates were incubated for 24 hours at 37 ° C. for bacteria and in yeast Lt; 0 > C for 48 hours. The MIC is indicated by turbidity, which is the minimum concentration of sample in which the microorganism does not exhibit visible growth.

compounds MIC Bacillus subtilis Escherichia coli Saccharomyces cerevisiae One 16 16 64 2 16 32 64 3 16 32 64 4 64 64 128 5 64 32 128 Azithromycin (positive control) 4 4 - Amphotericin B (positive control) - - 2

-: not tested.

The results are shown in Table 1, which shows that the antimicrobial activity of the present invention is superior to that of the positive control Azithromycin (positive control).

Korea Biotechnology Research Institute KCTC11975BP 20110630

Claims (6)

A compound represented by the following formula (4).
≪ Formula 4 >

The method according to claim 1,
The compound represented by Formula 4 has an antimicrobial activity.
The method according to claim 1,
The compound represented by Chemical Formula 4 is produced from a marine Bacillus sp. Microorganism deposited with the deposit number KCTC 11975BP.
A compound represented by the following formula (5).
≪ Formula 5 >

5. The method of claim 4,
The compound represented by Chemical Formula 5 has an antimicrobial activity.
5. The method of claim 4,
The compound represented by Chemical Formula 5 is produced from a marine Bacillus sp. Microorganism deposited with the deposit number KCTC 11975BP.

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