An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica

Accepted Manuscript An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica Erdenebileg Saruul, Toshihiro Murata, Erdenechimeg Selenge, Kenroh Sasaki, Fumihiko Yoshizaki, Javzan Batkhuu PII: DOI: Reference:

S0960-894X(15)00380-7 http://dx.doi.org/10.1016/j.bmcl.2015.04.048 BMCL 22631

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

12 February 2015 10 April 2015 15 April 2015

Please cite this article as: Saruul, E., Murata, T., Selenge, E., Sasaki, K., Yoshizaki, F., Batkhuu, J., An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.04.048

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Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

An antibacterial ortho-quinone diterpenoid and its derivatives from Caryopteris mongolica Erdenebileg Saruul a, Toshihiro Murata b, ∗, Erdenechimeg Selenge b,c, Kenroh Sasaki b, Fumihiko Yoshizaki b and Javzan Batkhuu a a

School of Engineering and Applied Sciences, National University of Mongolia, POB-617, Ulaanbaatar-46A, Mongolia Department of Pharmacognosy, Tohoku Pharmaceutical University, 4-1 Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, Japan c Monos University, Sonsgolon’s road – street 4/A, 20th khoroo, Songinokhairkhan district, Ulaanbaatar, Mongolia b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

To identify antibacterial components in traditional Mongolian medicinal plant Caryopteris mongolica, an ortho-quinone abietane caryopteron A (1) and three its derivatives caryopteron BD (2−4) were isolated from the roots of the plant together with three known abietanes demethylcryptojaponol (5), 6α-hydroxydemethyl cryptojaponol (6), and 14-deoxycoleon U (7). The chemical structures of these abietane derivatives were elucidated on the basis of spectroscopic data. Compounds 1−4 had C-13 methylcyclopropane substructures, and 2−4 had a hexanedioic anhydride ring C instead of ortho-quinone in 1. The stereochemistry of these compound was assumed from NOE spectra and ECD Cotton effects. Compounds 1 and 5−7 showed antibacterial activities against the Gram-positive bacteria Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, and Micrococcus luteus, being 1 the more potent.

Keywords: Caryopteris mongolica abietane diterpenoid Gram-positive bacteria ortho-quinone methylcyclopropane

2009 Elsevier Ltd. All rights reserved.

Caryopteris mongolica Bunge. (Lamiaceae) is a traditional herbal medicine from Mongolia, and the aerial parts of this plant are used for the treatment of aches, edema, and rheumatism.1 The presence of iridoid glycosides, alkaloids, diterpenoid glycoside, and flavone glycosides in the aerial parts of C. mongolica was reported.2-4 We examined the crude extract from the roots of C. mongolica for antibacterial activities using Staphylococcus aureus, Enterococcus faecalis, and Micrococcus luteus, which are Gram-positive bacteria, it showed activities (diameter of inhibition zone: S. aureus, 24.4 mm; E. faecalis, 26.3 mm; M. luteus, 25.0 mm; each sample was tested at 500 µg/disc). To identify antibacterial components in C. mongolica, the chemical constituents of the roots were investigated. Four diterpenoids (1−4) with three membered rings − spiro (methylcyclopropane) substructures − were obtained together with three known abietane diterpenoids (5−7). Similar diterpenoids with spiro-substructures have been isolated from Lamiaceae plants, such as from Coleus garckeanus,5 Plectranthus lanuginosus,6 and Anisochilus harmandii.7 In addition, their antibacterial and antiplasmodial activities were discussed and reported recently. 7,8 9

Roots of C. mongolica (285 g) were extracted with acetoneH2O (4:1), and a crude extract (24.6 g) was obtained. The extract was washed using H2O and the water insoluble residue (4.0 g) was dissolved in MeOH-H2O (1:1), passed through Diaion HP-

———

20 porous polymer gel (60 × 230 mm, Mitsubishi), and eluted with MeOH-H2 O (1:1) (1.55 g), MeOH-H2O (4:1) (290 mg), and MeOH (1.66 g). The MeOH fraction was chromatographed on an ODS packed column (Ultra Pack ODS-SM-50C-M, 37 × 100 mm, Yamazen) and subjected to preparative HPLC [TSK-gel ODS-120T, 21.5 × 300 mm, mobile phases: acetonitrile-H2O (3:2) and (7:3), Tosoh; Develosil C30-UG-5, 20 × 250 mm, mobile phases: acetonitrile-H2 O (3:2) and (7:3), Nomura Chemical] yielding 1 (13.4 mg), 2 (13.0 mg), 3 (5.5 mg), 4 (4.8 mg), 5 (24.6 mg), 6 (18.4 mg), and 7 (16.1 mg). Known abietane diterpenoids were identified based on spectroscopic data as demethylcryptojaponol (5),10 6α-hydroxydemethyl cryptojaponol (6),10 and 14-deoxycoleon U (7).11 Compounds 1−4 were obtained as amorphous powders, and their molecular formulae, estimated from mass spectra, suggested that they were diterpenoids. The 1H- and 13C-NMR spectra (Table 1) of 2−4 were recorded, and they showed very similar resonances. Compound 1 also had a moiety similar to 2−4; however, only 1 had two keto groups in the 13C-NMR spectrum. Caryopteron A (1)12 was determined to have the molecular formula C20H24O4 by HRFABMS (m/z 329.1754, calcd for C20H25O4, 329.1754). In the 1H NMR spectrum, a singlet methyl proton resonance at δH 1.36 (3H, H-20), two broad singlet proton resonances at δH 1.61 (3H, H-18) and 1.66 (3H, H-19), and

∗ Corresponding author. Tel.: +81-22-727-0086; fax: +81-22-727-0220; e-mail: [email protected]

doublet methyl proton resonances at δH 1.38 (3H, d, J = 6.5 Hz, H-17) were observed. Two protons (H-18 and 19) long range coupled with the olefinic carbons at δC 122.8 (C-4) and 127.3 (C3) in the HMBC spectrum. These resonances were observed instead of those of the dimethyl moiety (C-18 and 19) of known 5−7. The coupling system between H-5 (δH 2.77, br d, J = 15.0 Hz) and H-6 (δH 2.46, dd, J = 18.5, 15.0 Hz, 2.91, dd, J = 18.5, 4.0 Hz) was similar to that of 5. In the HMBC spectrum, H-20 correlated with another olefinic carbon resonance at δC 158.4 (C9), and H-6 correlated with a carbonyl carbon at δC 199.7 (C-7) and an olefinic carbon at δC 146.3 (C-8). The correlations from the above resonances showed two rings composed of C-1-10 and 18-20. The other carbon resonances, such as two carbonyl (δC 185.2, C-11; 195.5, C-12), an oxygenated (δC 63.7, C-14), and four aliphatic (δC 37.7, C-13; 22.7, C-15; 30.9, C-16; 13.6, C-17) carbons, and their corresponding proton resonances were similar to those of coleon P-Q, lanugones F-K, and their derivatives.5-8 These compounds are abietane diterpenoids with spiromethylcyclopropane moieties and a para- (p-) quinone moiety in the structure. The 13C-NMR spectrum of 1 suggested the presence of a diketone, and the chemical shifts in the 13C-NMR spectrum were shifted higher than those of coleons and lanugones. In many cases, the carbon resonances of ortho- (o-) quinones are shifted higher than those of p- quinones.13 This suggested that 1 had an o-quinones instead of p-quinones as found in coleons and lanugones. The HMBC correlations from δH 4.72 (s, H-14) to C-7, 8, -12, -13, -16 and from H-17 to C-13, -15, and -16 supported a structure with spiro-methylcyclopropane moieties and the oquinone. These results established that the planar structure of 1 was as shown in Figure 1.

3 was the same as that of 2. Therefore, 3 was determined to be a diastereomer of 2. The 1H- and 13C-NMR spectra of caryopteron D (4)16 were very similar to those of 3 except for resonances around C-2, -3, 4, -5, -18, and -19. These resonances were very similar to those of 1, which have a substructure with two methyl groups (δC 15.4, C-18; 19.3, C-19) attached to an olefinic double bond (δC 127.0, C-3; 123.0, C-4). The mass spectrum (HREIMS: m/z 344.1616, C20H24O5, calcd for C20H24O5, 344.1624) and HMBC correlations supported the planar structure of 4, as shown in Figure 1. The ECD Cotton effects of 517 were very similar to those of crytophyllone B.18 These curves showed the absolute stereochemistry of C-5 and C-10 of 5 were in S -configurations.18 Although the absolute stereochemistry of 1 could not be defined because the skeleton of 1 was different from that of 5, the curves in the ECD spectrum of 1 except for 220-250 nm were similar to those of 5. The fact that almost all abietanes from natural sources have the same configurations at C-5 and 10 supported the hypothesis which 1-7 had a same biosynthesis pathway. Based on the above data, the absolute stereochemistries of 1−4 were assumed as 5S and 10S. In the NOE spectra of 1−4, two types of key correlations were observed (Figure 2). All the compounds had weak NOE correlations between H-14 and H-20, and clear correlations from H-14 to H-16a (one of methylene proton at C16) and from H-14 to H-17. These correlations suggested that C14 was in the R-configuration, and the methyl group (C-17) turned in the direction of H-14. On the other hand, although 1 and 2 had no NOE correlation between H-14 and H-16b (the other proton at C-16), 3 and 4 had NOE correlations. These data suggested that both H-16a and H-16b were close to H-14, and C16 was β-orientation and C-15 was α-orientation at C-13 in 3 and 4. The drawn 3D structures referenced to the NOE correlations showed that C-13 of 1 and 2 was in the S-configuration and C-13 of 3 and 4 was in the R-configuration.

Figure 1. Structure of compounds 1−7

Caryopteron B (2) 14 was suggested as a diterpenoid from the molecular formula of C20H26O5 by HRFABMS (m/z 347.1864, calcd for C20H27O5, 347.1859). Some of the 13C-NMR resonances were assigned to rings A and B of a cyclic diterpenoid (δC 33.3, C-1; 17.9, C-2; 41.2, C-3; 33.0, C-4; 51.7, C-5; 36.4, C-6; 196.1, C-7; 36.7, C-10; 32.8, C-18; 21.0, C-19; 16.9, C-20), which were similar to those of demethylcryptojaponol (5). The oxygenated carbon (δC 79.0, C-14) and the corresponding proton (δH 4.53, s, 3H, H-14) and resonances of spiro-methylcyclopropane moieties (δC 27.5, C-13; 24.8, C-15; 23.9, C-16; 14.2, C-17) were determined by HMBC correlations similarly to 1. On the other hand, it was suggested that 2 had two ester carbonyl carbons (δC 170.2, C-11; 178.6, C-12) instead of diketone carbons in 1. From its mass spectrum, 2 had an additional oxygen compared with 1. These data showed that the third ring of 2 is a cyclic hexanedioic anhydride moiety. Therefore, the planar structure of 2 was determined as shown in Figure 1. Caryopteron C (3)15 was determined to have the molecular formula of C20H26O5 by HREIMS (m/z 346.1779, calcd for C20H26O5, 346.1781), which was the same as 2. The 1H- and 13CNMR spectra and HMBC spectra showed the planar structure of

Figure 2. Key NOE correlations of compounds 1−4

In order to identify the antibacterial components in roots of C. mongolica, we estimated the anti-Gram-positive-bacterial activities of the seven isolated compounds against bacterial species S. aureus, S. epidermidis, E. faecalis, and M. luteus. Antibacterial tests were carried out using the disc diffusion method;19 100 µL of test microorganisms (106 colony-forming units, CFU/mL), grown in nutrient broth media for 18 h, were spread over the surface of meat peptone agar medium in Petri dishes. Kanamycin was used as a positive control. The results are shown in Table 2.

Abietane diterpenoids are of interest because of their various antibacterial activities including against methicillin-resistant Enterococcus (MRSA) and vancomycin-resistant Enterococcus (VRE).20-22 Actually, isolated abietane diterpenoids with an aromatic ring C (5−7) showed potent activities against the four bacterial species. 6-Hydroxysalvinolone (7) was confirmed as having strong inhibitory activity against MRSA. 23 Although 2−4 were not shown to have activity against three species of Grampositive bacteria, 1 showed the largest diameter values among the tested diterpenoids, and close to those of kanamycin. These data suggested that the oxidation of o-quinone in this type of diterpenoid reduced its antibacterial activities. The finding that quinone in terpenoids is an essential substructure for anti-Grampositive-bacteria activity has been reported previously.22,24 Our results also suggested the importance of o-quinone for antibacterial activity.

Acknowledgments We thank Mr S. Sato and Mr T. Matsuki of Tohoku Pharmaceutical University, for assisting with the MS measurements. This work was supported by grants from Kanae Foundation for the promotion of medical science and JSPS KAKENHI Grant Number 26860068. This work was partially supported by a grant for Research Support Year at the National University of Mongolia and Honda Foundation of Japan. References and notes 1. Batkhuu, J.; Sanchir, C.; Ligaa, U.; Jamsran, T. Colored illustrations of Mongolian useful plants. Admon, Ulaanbaatar, Vol. 2, 2005, p.191 2. Zhang, Y. H.; Yang, L.; Cheng, D. L. Pharmazie 2000, 55, 845. 3. Dumaa, M.; Gerelt-Od, Y.; Puzhao, Z.; Yinggang, L.; Javzan, S.; Selenge, D.; Zhang, G. Mongolian J. Chem. 2012, 13, 41. 4. Murata, T.; Selenge, E.; Oikawa, S.; Ageishi, K.; Batkhuu, J.; Sasaki, K; Yoshizaki, F. J. Nat. Med. 2015, in press. 5. Miyase, T.; Yoshizaki, F.; Kabengele, N.; Ruedi, P.; Eügster, C. H. Helv. Chim. Acta 1979, 62, 2374. 6. Schmid, J. M.; Ruedi, P.; Eügster, C. H. Helv. Chim. Acta 1982, 65, 2136. 7. Lekphrom, R.; Kanokmedhakul, S.; Kanokmedhakul, K. Planta Med. 2010, 76, 726. 8. Simões, M. F.; Rijo, P.; Duarte, A.; Barbosa, D.; Matias, D.; Delgado, J.; Cirilo, N.; Rodríguez, B. Phytochem. Lett. 2010, 3, 221. 9. C. mongolica was collected in 10th August 2012 in Mongolia. A voucher specimen has been deposited at the herbarium of Tohoku Pharmaceutical University, No. 20130517. The plant was identified by Dr. C. Sanchir, Institute of Botany, Mongolian Academy of Sciences. 10. Rodríguez, B. Magn. Reson. Chem. 2003, 41, 741. 11. Fraga, B. M.; Díaz, C. E.; Guadaño, A.; González-Coloma, A. J. Agric. Food Chem. 2005, 53, 5200. 12. Caryopteron A (1): [α]25D +270 (c 0.74, MeOH); UV (MeOH) λ max (log ε) 244 (6.87), 286 (6.65); ECD (c 0.38, MeOH) nm ([θ]) 208

(4400), 224 (–10200), 236 (–5600), 247 (–7500), 270 (–300), 286 (–4000), 319 (17400) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR: (CDCl3 , 100 MHz) see Table 1 13. Berger, St.; Rieker, A. Tetrahedron 1972, 28, 3123. 14. Caryopteron B (2): [α]21D +56.6 (c 3.44, MeOH); UV (MeOH) λmax (log ε) 243 (6.91); ECD (c 0.41, MeOH) nm ([θ]) 201 (– 5500), 228 (10100), 268 (–13500), 361 (3300) nm 15. Caryopteron C (3): [α]25D +52.3 (c 0.52, MeOH); UV (MeOH) λ max (log ε) 252 (6.89); ECD (c 0.27, MeOH) nm ([θ]) 218 (13500), 255 (–10300), 296 (1300), 322 (500), 366 (2100) nm 16. Caryopteron D (4): [α]25D +56.3 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 245 (6.86); ECD (c 0.47, MeOH) nm ([θ]) 217 (17500), 257 (–2900), 284 (700), 310 (–800), 367 (2300) nm 17. Demethylcryptojaponol (5): ECD (c 0.47, MeOH) nm ([θ]) 214 (14500), 237 (–4300), 256 (700), 292 (–7000), 323 (7200) nm 18. Tian, X.; Min, Z.; Xie, N.; Lei, Y.; Tian, Z.; Zheng, Q.; Xu, R.; Tanaka, T.; Iinuma, M.; Mizuno, M. Chem. Pharm. Bull. 1993, 41, 1415. 19. Karaman, İ.; Şahin, F.; Güllüce, M.; Öğütçü, H.; Şengül, M.; Adıgüzel, A. J. Ethnopharmacol. 2003, 85, 231. 20. Yang, Z.; Kitano, Y.; Chiba, K.; Shibata, N.; Kurokawa, H.; Doi, Y.; Arakawa, Y.; Tada, M. Bioorg. Med. Chem. 2001, 9, 347. 21. González, M. A. European J. Med. Chem. 2014, 87, 834. 22. Tada, M.; Kurabe, J.; Yoshida, T.; Ohkanda, T.; Matsumoto, Y. Chem. Pharm. Bull., 2010, 58, 818. 23. Muchumi, F.; Samoylenko, V.; Yenesew, A.; Derese, S.; Midiwo, J. O.; Wiggers, F. T.; Jacob, M. R.; Tekwani, B. L.; Khan, S. I.; Walker, L. A.; Muhammad, I. Nat. Prod. Commun. 2010, 5, 853. 24. Shin, D. Y.; Kim, S. N.; Chae, J. H.; Hyun, S. S.; Seo, S. Y.; Lee, Y. S.; Lee, K. O.; Kim, S. H.; Lee, Y. S.; Jeong, J. M.; Choi, N. S.; Suh, Y. G. Bioorg. Med. Chem. Lett. 2004, 14, 4519.

Supplementary Material Supplementary materials [1H (400 MHz), 13C (100 MHz), HMBC, and NOE NMR spectroscopic data (CDCl3) of compounds 1-4] and [Antibacterial activities of the isolated diterpenoids 1-7 against Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, and Micrococcus luteus] were attached.

Table 1. 1H (400 MHz), 13C (100 MHz) NMR spectroscopic data (CDCl3 ) of compounds 1-4 Posit

1

ion

δH (J = in Hz) 1

2 δH (J = in Hz)

δC

1.30, m

30.2

1.32, dd (14.5, 3.0)

2.37, m 2

3 δC 33.3

2.67, m

2.01, dd (18.0, 5.5) 2.25, m

1.65

3

17.9

a

41.2

1.55, m 4

δC

δH (J = in Hz)

δC

33.3

1.56, m

29.2

2.69, dd (13.0, 6.5)

1.34a

18.0

2.11, br dd (18.5, 7.0)

28.7

2.18a

1.34a

40.9

127.0

1.54, m

122.8

33.0

33.0

123.0 a

5

2.77, br d (15.0)

43.9

1.86, dd (13.5, 4.0)

51.7

1.87, dd (14.5, 3.0)

51.3

2.85

6

2.46, dd (18.5, 15.0)

37.0

2.54, dd (21.5, 13.5)

36.4

2.45, dd (18.0, 14.5)

36.2

2.39, dd (18.5, 16.5)

2.91, dd (18.5, 4.0)

2.57, dd (21.5, 4.0)

2.61, dd (18.0, 3.0)

45.3 38.6

2.82, dd (18.5, 6.5)

7

199.7

196.1

196.1

195.5

8

146.3

150.1

149.3

150.3

9

158.4

152.3

154.6

153.9

10

37.3

36.7

36.8

35.0

11

185.2

170.2

170.1

170.6

12

195.5

178.6

178.2

177.9

13

37.7

27.5

14

4.72, s

63.7

4.53, s

15

2.26a

22.7

16

0.96, dd (7.0, 3.5)

30.9

1.39a

a

1.29

a

1.72a

1.23a

127.3

δH (J = in Hz)

2.65, m

1.40a

29.0

4

26.7 4.54, s

2.00, m

24.8

1.91, m

24.5

1.86, m

24.2

1.04, dd (7.5, 7.0)

23.9

1.29, dd (7.5, 5.5)

23.8

1.26, dd (8.0, 5.0)

23.6

1.68, dd (13.5, 5.0)

78.3

26.8

79.0

1.68, dd (9.0, 5.5)

4.59, s

78.8

1.65a

17

1.38, d (6.5)

13.6

1.42, d (7.0)

14.2

1.36, d (6.5)

13.8

1.35, d (6.5)

13.8

18

1.61, br s

15.1

0.94, s

32.8

0.95, s

32.7

1.63, brs

15.4

19

1.66, br s

19.2

0.98, s

21.0

0.96, s

21.1

1.67, brs

19.3

20

1.36, s

16.9

1.27, s

16.9

1.27, s

17.8

1.14, s

16.2

Unclear signal pattern owing to overlapping signals

Table 2. Antibacterial activites of the isolated diterpenoids 1-7 against Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, and Micrococcus luteus Inhibition zone diameter around the disc (mm)b Dosea

Compounds

S. aureus

S. epidermidis

E. faecalis

M. luteus

1

5

12.9 ± 0.4

15.9 ± 0.1

23.2 ± 0.3

16.3 ± 0.9

2

500

N.A.c

N.A. c

9.6 ± 0.3

N.A.c

3 4

50 50

N.A.c N.A.c

N.A. c N.A. c

N.A. c N.A. c

N.A.c N.A.c

5

50

11.8 ± 0.2

13.7 ± 0.4

11.3 ± 0.0

9.0 ± 0.0

6

50

11.0 ± 0.0

11.5 ± 0.5

13.8 ± 0.7

9.1 ± 0.2

7

50

10.2 ± 0.2

10.2 ± 0.2

10.5 ± 0.5

9.0 ± 0.0

Kanamycin

50

−

−

17.9 ± 0.7

−

5

16.3 ± 0.8

20.5 ± 0.4

−

16.0 ± 0.6

a

The filter paper discs (8 mm in diameter) impregnated with 500, 50, 5 µg/disc concentrations of the isolated diterpenoids were placed on the surface of the Petri dishes. b

The treatments were replicated 3-5 times, mean ± S.E.M; Diameters were measured in millimeters after 24 h of incubation at 37oC. c

No activity

Graphical abstract