Antibacterial trichorabdal diterpenes from Rabdosia trichocarpa

Pergamon

Phpkxhrm~strg. Vol Cownahl

36. No. 5, pp. 1287 1291. 1594 0 1994 Elvv~cr Snena Lid Printed m-&cat Bntin. All n6hts reserved

0031-9422(94)E0165-0

rm-9422p4

ANTIBACTERIAL

TRICHORABDAL DITERPENES TRICHOCARPA

FROM

17.00+0.00

RABDOSZA

KENJI OSAWA, HIDEYUKI YASUDA, TAKASHI MARUYAMA, HIROSHI MORITA,* KOICHI TAKEYA* and HIDEJI ITOKAWA* Department

of Basic Research, Pharmacognosy,

Lotte Central Tokyo

Laboratory

Co., Ltd., Numakage

College of Pharmacy,

Horinouchi

(Receioed 4 January

Key Word Index-Rabdosia trichocarpu; Labiatae; oral microorganisms; conformation.

3-I-l.

Urawa,

1432-1, Hachioji,

Saitama

trichorabdal

type diterpene;

Four antibacterial diterpenes, trichorabdals A, B, C and H were isolated trichocurpa and the relationship between their conformations analysed by spectroscopic Their antibacterial activity is discussed.

In the course of our antibacterial studies [l] on natural sources against oral microorganisms which cause dental caries and periodontal disease, we found that the ethanolic extract of Ruhdosiu rrichocarpu showed antibacterial activity against cariogenic Streptococcus mutuns and periodontopathic Porphyromonus gingivulis. We have already reported the isolation, structure elucidation, and the antibacterial potency of several biological active diterpenes from R. trichocurpu [2]. Further chromatographic purification guided by antibacterial activity led us to isolate four trichorabdal type diterpenes, trichorabdais A, B, C and H. Trichorabdal type diterpenes are known to be potent antitumour agents against Ehrlich ascite carcinoma in mice [3]. Recently, the conformational analysis of these diterpenes has been reported. Some spirolactone type diterpenes are not rigid in the A ring conformation [4]. Fuji et al. [S] demonstrated the crystalline structure of trichorabdals B and C by X-ray crystallographic analysis, and pointed out that the A ring chair conformations of these two compounds differed significantly. The conformational feature of these flexible diterpenes is interesting but it is difficult to analyse the conformational peculiarity in solution only by local ‘H and 13C NMR analyses. In the present study, we further studied the conformation of trichorabdals A, B, C and H in solution by use of a NOE experiment and computational chemical methods (molecular dynamics and molecular mechanics calculation).

*Department

of

1994)

Abstract-

INTRODL’CHON

336, Japan,

Tokyo 192-03, Japan

antibacterial

activity;

from the leaves of Rubdosia and computational methods.

Streptococcus mutuns Ingbritt and Porphyromonus gingivulis ATCC 33277 by the paper disk assay method (inhibitory zones were formed at 5Opg disk-’ against both microorganisms). In a partition using n-hexane, ether, and ethyl acetate, the antibacterial activity was concentrated in the ether soluble fraction. The fraction was applied to silica gel column chromatography to separate several subfractions. From the biologically active ones, four diterpenes (l-4) were isolated by a repeated HPLC method and recrystallization. These four compounds had the same 6,7-secokaurene skeleton, and were identified as trichorabdals A (1). B (2), C (3) [6], and H (4) [7] by comparison of their physical and spectroscopic data with those in previous reports. In the ‘H and 13CNMR spectra of these compounds, several signals were broadened or disappeared at ambient temperature. Moreover, in a NOE experiment some inconsistent correlations were detected in the cases of trichorabdals A and B. These phenomena were probably due to the

RESULTS AND DISCLSSION

1. R,=R.pl+t-l,

The ethanolic soluble substances were extracted from the dried leaves of Rubdosiu trichocurpu (Labiatae). The extract showed appreciable antibacterial activity against

R&H,

R,=OAc

3. R,=RpH,

R&H,

R*=OAc

4. R&AC, 1287

R&3-l

2. R,=R2=H,

RP=R,=H,

R&H

K. OSAWA et al

1288

conformational conversion in solution, Initially, we investigated the possible conformation of these diterpenes by a computational chemical method. For the purpose of predicting or analysing complicated conformational features of this series of compounds, it is necessary to USCa computational method which can give us a result which is independent of the starting structure. As it is very difficult to cover all the possible ring structures, including conformational freedom in substituent groups on the ring, a molecular mechanics calculation is not efficient in this molecular system. The molecular dynamics (MD) calculation is much better for overcoming the local minima problem. However. it is still impossible to cover all possible conformers partitioned off by various energy barriers by MD calculation at low temperature. We have already reported that the possibility of using molecular dynamics techniques as a tool for simulated annealing has been tcstcd in the case of the molecules of tropoloisoquinoline alkaloids [6] and surfactins [8]. The method, applied to a broad class of problems, has also shown its practical utility in the case of conformational problems [9]. The starting geometries of trichorabdal B (2) for the simulation were modelled by X-ray crystallographic data [IO] and those of I,3 and 4 were modified from that of 2 by molecular modelling software SYBYL [I I, 121. A simulation was performed using a time step of I fsec and the structures were sampled every 90fsec. Each system was equilibrated for 5400 fsec with a thermal bath at 500 K and thereafter successively for 900 fsec with a thermal bath IO K lower in temperature until a final temperature of 50 K was obtained. Ten cycles were performed, giving a total simulation time of 63 psec, and each freezed conformation as sampled from the minimum temperature at 50 K. These computational analyses identified two important conformers, A and B. The minimized structures were ranked in order of increasing energy and low energy conformation involved in conformers A and B was finally minimized by use of a semiempirical MNDO calculation [ 133 which uses the neglect of diatomicdifferential overlap approximation in MOPAC ver. 5.0. Each of the minimized conformers A and B, and the relationship of NOE enhancement of these compounds are shown in Fig. I. Each heat of formation calculated by the MNDO method is shown in Table I.

Table

I.

Heat of

formation

of conformer

calculated Samples Trlchorabdal

A (I)

In the MNDO calculations, the relatively lower energy ring conformation of these four compounds are found in conformer B. In trichorabdals A (1) and B (2), however, the discrimination of heat of formation energy between conformers A and B is relatively small (A\E: 2.9495 and I.0391 kcal mol -.I). The NOE experiments of 1 and 2 showed the correlations which can be explained for both conformers A (trichorabdal A; H-9 and H-18, H-9 and H5, trichorabdal B; H-9 and H-18, H-6 and H-19) and B (trichorabdal A; H-5 and H-l I, H-9 and H-20, trichorabdal B; H-6 and H-18, H-5 and H-l I). For the reasons mentioned above, both conformers of I and 2 might be formed in solution. In trichorabdals C (3) and H (4). the energy of conformer B was considerably lower than that of conformer A (AE: 16.1870 and 5.4912 kcal mol. ‘). Actually, in the NOE experiments of 3 and 4, no data to explain the conformer A was obtained. Therefore, in these two compounds, the major conformer is B in solution. From the results of simulated annealing, MNDO calculation and NOE experiments, the conformational characteristics of trichorabdals A, B, C and H were clarified. The antibacterial potencies of these diterpenes isolated from R. trichocarpa against cariogenic S. mutans Ingbritt and S. sohrinus 6715, and periodontopathic P. gingivalis ATCC 33277 were estimated by the broth dilution method, and the results are summarized in Table 2. These four compounds had some inhibitory effect, but exhibited varying degrees of activity. Trichorabdal A and B had relatively strong antibacterial activity, these compounds inhibited S. mutans and S. sohrinus at concentrations of 100 and 25 pgrnl ‘. The activities of these compounds against S. mutans and S. sohrinus were a little higher than that of thymol, which is known as a wide-spectrum antibacterial substance. These two compounds possessed potent antibacterial activity against P. gingivalis; the bacterial growth was completely inhibited at 12.5 pegml- ’ and these two compounds showed appreciable inhibitory effect even at 3.13-6.25 pgml I. The inhibitory effect of these two compounds against P. gingivdis were stronger than that of thymol (MIC: 100 pg ml -I). Rabdosio diterpenes are known to have highly specific antibacterial activity against gram-positive bacteria, such as Bacillus suhtilis [ I43 but their inhibitory effects against gram-negative bacteria such as P. gingivalis have not been reported. In this study, we

A

and B of trichorabdals

by the MNDO

Conformer

Heat of formation

A

- 128.3134

B

- 131.2629

Trrchorabdal

B (2)

A

- 204.2239

B

- 205.2630

Trichorabdal

CZ(3)

A

- 192.2210

B

- 208.4080

Trlchorabdal

H (4)

A

- 187.8603

B

- 193.3515

A, B, C

and H

method (kcdl mol_ ‘)

A.E

2.9495 1.0391 16.1870 5.4912

Diterpenes

1289

from Rabdosiu trichocarpa

4

Conformer B

Conformer A Fig.

1. Two energetically

favourable

conformers

A and B in l-4.

found that the Rabdosia diterpenes had antimicrobial actions against not only gram-positive bacteria, but also gram-negative periodontopathic bacteria. Kubo et al. [lS] have reported that the structural requirement for the active group of Rabdosia diterpenes must be an exocyclic PHY 36:5-N

The arrows show the NOE

relationships.

methylene conjugated with a ketone. This active portion is highly reactive toward sulphydryl groups essential to biological function. In our previous study on Rabdosia diterpene (oridonin), we prepared dihydrooridonin by hydrogenation of the exomethylene, and confirmed

K. OSAWA er al.

1290

Table

2. Mmimum

inhibitory

concentration

(MIC)

of trichorabdals

S. rnutuns. S. sohrittus and P. ginqiculis S. nrutatrs Ingbritt

Samples

(I)

IO0(25)*

25 (6.25)

100 (25)

25 (6.25)

A

l‘richorahdal

R (2)

Trtchorahdal

C (3)

> 200 (200)

I cn,(50)

Trtchorabdal

H (4)

> 200 (200)

100 (‘5)

loo (50)

*The final dilute concentration

and H against

P. qinyit.alis 33277 12.5 (3.13) 12.5 (6.25) 200 (50) 50 (12.5) 100 (50)

50 (25)

possessing apparent

A. B. C

ml i)

s. .sohrrnus 67 I5

Trichorabdal

Thymol

(pg

antibactcrtal

ctTccl (the absorbance

IS less

than half the yaluc of the control at 660 nm).

that the strong antibacterial activity against cariogenic and periodontopathic bacteria disappeared completely with this slight modification of oridonin [2]. All of the four compounds isolated in this study have this structural moiety in the D-ring. Although, the existence of a D-ring alone is clearly not sufficient to explain the reduction of the biological activity of trichorabdals C and H. With regard to conformational stability, the highly active compounds, trichorabdals A and Bare more flexible than those of trichorabdals C and H. The reduction of the antibacterial activity of trichorabdals C and H is considered to be due to conformational factors. Further studies will be required to explain the relationship of the antibacterial activity and conformational characteristics with trichorabdals. The antibacterial activities of trichorabdals are not so strong as those of antibiotics such as tetracycline-HCI (bacterial growth was completely inhibited at 0.78 pg ml ’ for S. tnutan.s Ingbritt and 0.39 pg ml- ’ for P. yingidis ATCC 33277 under the same conditions). However, continuous use of antibiotics only for the prevention of oral disease should be avoided because resistant bacteria may appear. Our finding suggests that these diterpenes may be one of the most effective agents in preventing the growth of both cariogenic and periodontopathic bacteria from natural sourccs.

EXPERIMENTAl.

Generd. Mps: uncorr. High-performance liquid chromatography (HPLC) was carried out on a Shimadzu LC8A system with Scnshu Pak silica-5251-S column as the normal phase and Senshu Pak ODS-5251~SH column as the reversed phase. Chemicals. The standard antibacterial sample thymol was purchased from Kokusan and tetracycline HCI was purchased from Sigma. Test hacrerud strains and culture media The two cariogenic bacteria S. nwans lngbritt and S. sobrinus B13 and the periodontopathic bacteria I’. gingil:alis ATCC 33277, were tested. S. mutans. and S. sohrinus were cultured on Brain Heart Infusion agar (BHI, DIFCO) at 37’. P. giny~culis was cultured on blood agar medium which consisted of Trypticase soy agar (BBL) supplemented with 5 pgrnl_ ’ hemin, 0.5 pgml ’ menadione,

and 10% dehbrinated horse blood at 37” in an anaerobic chamber with an atmosphere of 10% carbon dioxide, 10% hydrogen, and 80% nitrogen. Assay of untibucterial acticity. Two-fold serial dilutions of the test substances were prepared with EtOH and sterilized by passing through a filter (0.22 Ltm, Millipore). Then 100 ~1 of a given dilution of the test substance was mixed with 4.85 ml of liquid BHI broth (S. mufans and S. sohrinus) or Trypticasc soy broth containing 5 Icgmll ’ hemin and menadionc (P. gingicalis) in glass tubes (I5 mm in diameter). As a control, the culture medium was prepared by adding 100~1 of EtOH. An aliquot of 50 /tl of the bacterial suspension adjusted to an optical density (OD) of 0.8 (550 nm) was inoculated into media containing test substances. The culture test tubes were incubated at 37’ in aerobic (S. ~UIU~I.Slngbritt and S. sobrinus 6715) or anaerobic (P. @#a/is) atmospheres. Bacterial growth was monitored by measuring the absorbance increase (OD at 660 nm) by digital calorimeter (IMC). The minimum inhibitory concentration (MIC) of each test substance was judged when the absorbance grew 0.5 in the control tube for each bacterium. Extraction and isolation. EtOtl soluble substance was extracted from the dried leaves of Habdosia trichocurpu (I .9 kg) at room temp. for IO days. We obtained 60.21 g of ethanolic extract. The extract was fractionated into nhexane, Et,O, EtOAc and Hz0 soluble frs, respectively. The active fr. (Et,0 soluble fr.) was applied to silica gel CC. The column was eluted with CH,CI, MeOH (I :0-O: I) and separated into nine frs (A I) including active fr. B. Fr. B was separated by silica gel HPLC with CH,CI,-EtOAc-MeOH (470:30: 3) and ODS HPLC with MeOH-Hz0 (I I :9-l :0) to afford trichorabdal A (1). B (2). C (3), and H (4). Moleculur dynamics und molecular mechanics culculofions. Computer modelling and all calculations were performed using the molecular modelling software SYBYL (Tripos Associates. St. Louis, MO) on an IRIS 4D workstation. Molecular dynamics calculation was employed by standard SYBYL force held [ 1I]. The dielectric constant (c) was assumed to be proportional to interatomic distances (r) as 1:= r. Solvent molecules were not included in the calculations. The structures obtained by simulated

MNDO

annealing

program

(ver.

were further minimized

5.0) distributed

with the

by Quantum

Diterpenes

from Rabdosia

Chemical Program Exchange (QCPE) and the convergence criterion PRECISE option. Trichorabdal A (1). Needles, mp 203-205’ [~]u - 60.8“ (EtOH;c 0.032); IR vkf:crn-‘: 3430, 2929, 1745, 1722, 1691, 1406, 1277, 1182, 1022; UV ,.:E” (log E):229.8 (3.78); EI-MSm/z(rel. int.): 346 CM]’ (27), 318 (22), 300(18), 151 (52), 91 (100); ‘H NMR (pyridine-d,): 60.97 (3H, s, H-18), l.O(3H,s, H-19), 244(1H, s, H-9), 2.91 (lH,d, 5=4.3, H5), 4.61 (lH, brs, H-11). 5.16, 4.92 (each lH, brs, H-20), 6.01,5.37(each lH,s,H-17), 10.06(1H,d,J=4.2Hz,H-6); ‘%NMR (pyridine-d,): 664.9 (d, C-l l), 117.9 (1, C-17). 150.7 (s, C-16), 171.3 (s, C-7), 205.5 (d, C-6). Trichorabdal E (2). Crystals, mp 158-162”, [z]u - 115.7’ (EtOH; ~0.021); IR vzf;crn-i: 3490,2944,1739, 1705, 1398, 1242, 1031; UV i!$” (log E): 229.6 (3.86); ElMS m/z (rel. int.): 404 [M] ’ (8) 344 (24), 298 (36) 225 (34). 161 (62), 105 (100); ‘H NMR (pyridine-d,): 6 1.16 (3H, s, H-18), 1.96 (3H, s, -OCOMe), 2.54 (lH, brs, H-9), 3.18 (lH, brs, H-5), 4.07 (2H, brs, H-19), 4.57 (lH, brs, H-11), 5.20,5.18 (each 1H, br s, H-20), 6.05.5.46 (each 1H, br s, H17) 10.21 (1 H, d, J = 3.6 Hz, H-6); 13C NMR (pyridine-d,): 620.4 (q, -OCOMe), 65.0 (d, C-l l), 118.1 (r, C-17), 150.7 (s, C-16) 171.2 (s, C-7), 203.7 (d, C-6). Trichorabdal C (3). Crystals, mp 148-150”, [a&, +25.3” (EtOH; ~0.029); IR vf$!!cm-‘: 3478, 2942, 1744, 1711, 1396, 1229, 1041,934; UV iz:” (log E): 231.4 (3.83); El-MS m/z (rel. int.): 404 [M] (6) 326 (18), 267 (36), 243 (30), 165 (35), ‘H NMR (pyridine-d,): 61.49 (3H, s, H-18), 1.96 (3H, s, -OCOMe), 2.68 (lH, dd, J= 12.7, 5.4, H-9), 3.25 (IH, d, J=3.4 Hz, H-5), 3.83 (lH, s, H-3), 4.55, 4.30 (eachlH,d,J=11.8Hz,H-19),5.01(1H,brd,J=ll.l Hz, H-20), 5.17 (lH, brs, H-20), 5.95, 5.32 (each lH, s, H-17), 10.15 (lH, d, J=3.2 Hz, H-6); ‘%NMR (pyridine-d,): 620.4 (y, -OCOMe), 24.2 (q, C-18). 55.6 (d, C-5), 68.9 (d, C-3), 69.7 (t, C-19) 118.0 (t. C-17), 150.7 (s, C-16), 170.2 (s, -OCOMe), 203.5 (d, C-6). 202.8 (s, C-15) 170.8 (s, C-7). Trichorabdal H (4). Crystals, mp 221-224, [x]u - 16.4’ (pyridine; c 0.055); IR vkz: cm-‘: 3428, 2943, 1754, 1710, 1642, 1267, 1055; UV j.::.“,” (loge): 230.8(3.88); EI-MSm!z(rel. int.):4W[M](55),386(52),344(72),316 (36), 214 (51) 193 (50), 149 (89). 105 (100); ‘HNMR (pyridine-d,): 60.84 (3H, s, H-18), 1.07 (3H, s, H-19), 2.14 (3H, s, -OCOMe), 2.42 (lH, s, H-9), 4.71 (lH, s, H-11), 5.46, 5.16(each lH, s, brd, 5=12.0, H-20) 5.74(1H,d,J

trichocarpa

1291

=7.4Hz,H-l),6.0,5.39(each1H,s,H-17),9.99(1H,s,H6). “CNMR (pyridine-d,): 621.3 (q, -QCOMe), 24.0 ((I, C19), 32.9(q,C-18),62.0(d, C-5), 63.5(d,C-11),77.9(d,C-1), 118.5 (t. C-17), 150.7 (s, C-16). 169.9 (s, -OCOMe), 170.7 (s, C-7), 202.0 (s, C-15), 204.4 (d, C-6).

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