Isolation and structure elucidation of tsugicolines A-D, novel protoilludane sesquiterpenes from Laurilia tsugicola1

Tetrahedron

Vol. 51, No. 48, pp. 13357-13364. 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved oo4s4020/95 $9.50+0.00

Pergamon 0040-4020(95)00848-9

Isolatio! and Structure Elucidation of Tsugicolines A-D, Novel Protollludane Sesquiterpenes from LawiZia tsugicola’ Albert0 Amone, Umberta Brambiir, Giaolucr Nasini’ and Orsu Vajna de Paw Diptimento di Chimicade1Politecniw,Centro&I CNRperle sostanze Organiche Naturali,Politecnicu di Milano,via MancineUi

7.120131

Iblilano, Italy

tsugicolines A-D (la. 2, 34 4). have been isolated from still (Basiimycetae). Their suuctwswweelucidatedbymeansof ad NhJR stud& and the relative cmfigurations were established through a seriesof NOEdifferencespectra.The absoluteco@gmtion of tsugicoline A la (3-epi-illudol-S-one)

Absfmd:

Four novelsesquiterpenes,

cuhres of the timgus chemical correlations

Lmdia

twgicoh

was determinedas 3S.6$7R.9R,13S by the ‘partial resolution’method of Horeau.Treatmenl of tsugicoline A la with trieiJ@amine in MeOHgavethe metabolite 4; a possible nwcbanism is reported. Tsu@coline A is inactive on bacteria and fungi but inhibits the @xmhation of the water cress Lepidium .wrivurn. introduction

The fungal genus Basidiomycetes is a rich source of new, interesting secondary metabolites of sesquiterpenictype. In particular, we have recently isolated a number of sesquiterpeneswith a protoilludane skeleton from Armillarid, Uitocybe3 and Luurilia sp~.~and a new classof compounds, the nor-iso-ill&lanes from Luurilia sulcata ’ In this paper we describe the isolation and structure elucidation of four protoilludane sesquiterpenes, named tsugiwlines A-D, 1a-4, produced by L. tsugicola (Echinoabntium tsugicoia} a decay agent on Tngue and Abies.The tingus was grown for one month in still liquid cultures (malt-peptone-glucose) and the metaboliteswere extracted with EtOAc. Results and Diicussion Tsugiwlme A la was isolated by filtration of the insoluble part of an Et20 washing of the extracts as white crystals(CH2C12-hexane),m.p. 16%17O’C; [a]D -156” (c 0.15, MeOH) and analysed for Cl5H2204 (h@, 266); chemical ionization mass spectroscopy(isobutane) gave a distinct peak at m/z 267 (hII-I+) and a fragment was found at mir 249 [(MH+)-H20; base peak] due to the ready loss of water; in addition, strong peaks were observed at m/z 23 1 [@@)-2H20] and 203 (23 l-28). The IR spectrum (KBr) exhibited a large absorption at 3350 and a band at 1730 cm- l, due to hydroxy and carbonyl groups respectively, and the UV spectrum [& 260 nm (E SSOO)] agreed with the presenceof a conjugated system. The broad-band lH-decoupled 13C NMR spectrum of tsugicoline A la (Table 1) revealed the presence of 15 signalsattributable to 3 s$- and 12 ~-hybridized carbon atoms on the basisof chemicalshift criteria and one-bond *H,13C HETCOR correiations.The s$ signalswere assignedto the carbonsof a fully-substituted u$ -unsaturated ketone moiety (C-2$-4 and C-5) while the $ signal s were assignedto three methyl (C-&C-14 and C-15), tiuee methylene (C-1$-10 and C-12, one of tbem oxygen-bearing), four methine (C-3$-6,C-9 and C-13, two of than oxy~bauing) and two quuernaty (C-7 and C-11) carbon atoms. The analysia of the carespadiae1HNMRspectnun~rMce2md3XucorroboratsdbyJdactivc~experiments, 13357

13358

A.

ARNONE

et al

extended the above evidence through the appearance of three tertiary methyl C(6)HOH moieties and a sequencelike that shown below:

groups,

a -C(l)H20H and a -

I -C( 12)H2-C( 13)H-C(9)H-C( 1O)H2 -C(3)HOH The presenceof three hydroxyl groups was confirmed by the formation of the triacetate lb. In a COLOC experiment optimized for the observation of two-and three-bond couplings of ca. 6 Hz, the cross-peaksobserved for both the 14- and 1Smethyl protons with the quaternary C-l 1 carbon and the C-10 and C-12 methylene carbons require the presence in tsugicoline A of a gemdimethyl group lied at C-l 1, it follows that the sequenceC-12, C-13, C-9, C-10 reported above is closed through C-l 1 to form a cyclopentanering. The long range couplings of 0.8 Hz observed between the 1S-methylprotons and both Ha- 10 and Ha-12 and that of 1.8 Hz observed between HP-10 and HP-12 gave further support. Moreover, additional correlations observed between the 8-methyl protons and C-4, C-6, C-7 and C-9 defined the formation of the four bonds between the quaternary C-7 carbon and C- 4, C-6, C-8 and C-9 while the coupling of 5 Hz observedbetween C-5 and H-6 suggestedthat C-S and C-6 are joined together. Evidencefor the liiage between C-4 and C-5, and not between C-2 and C-5, to form the a&unsaturated carbonyl moiety followed from the chemical shift values of 153.02 and 143.98 ppm exhibited by C-2 and C-4 which imply that these carbonsare in g- and in a-position with respectto the carbonyl grou~.~ OR1

H

OR2 ,5 la R1=R2=R3=H

‘,4

I,,

R1=R2=R3=,&

lc

R’-R2=CMe2,R3=H

1d R’ = R3 = QvG$~C-CO;

R2 = H

The relative configuration of the five chiral centresin tsugicoline A la was determined by the use of NOE experiments(Experimental). In particular, the mutual NOESobserved between H-9, assumedas B, and H-6, H13 and H3-15 require that these protons are on the same P-side of the molecule whereas the NOES observed between H-3 and H3 -8 and H3a-14 indicate that these protons are on the a-face. The absolute configuration of C-3 and C-6 was deduced as S for both carbons by application of the Horeau chirality rule to the acetonide derivative lc and to the 1,6-dipivalate Id, respective1y.7In fact, it can be reasonablyassumedthat in Id C-13 with the neighbouring groups is smaller than C- 2, and that in lc the carbonyl C-5 is larger than C-7. Tsugicoline A must therefore have the absolute contiguration as shown in structure la, i.e. 3S,6&7R,9&13R, and representsa new member of the protoilludane sesquiterpenesbeing idemiSed as 3spiilludol-5-one.8 The above findings together with the magnitude of the coupling constants of the protons of the cyclopentanering indicate that the tsugicoline A la assumesin solution the preferred conformation in which the cyclopentane and the cyclohexenerings adopts an exe-envelope and a boat-&e geometry, respectively; the same resuh was obtained by a molecular model depicted in the Figure buih by rnwu of molecular mechanisms calculations.The second metabolite, tsugicolme B 2, was obtained as an oil, [a]D -71.5 (c 4.5, CHCi3). It adped for C l5H22O3 and presentedUV and IR spectravery similarto those of compcmndla. Comparison of the 13C and ‘H NMR spectra of compound 2 with those of la (Tables 1,2 and 3) indicated a close similarity between the two metabolites, the only significant diB&nce being the presencein 2 of a C(6)H2 group (8~ 60.3 1; 6H 2.82 and 2.75) insteadof the C(6)HOH fragment (8~ 99.33; 6H 4.39 and 2.95).

13359

Tsugicolines A-D

Fig. - Model of tsugicoline A la, as derived by molecular mechanics

2 3a

R’=OH,

3b

R’ = H,

R2=H R’

4

=OH

Tsugicoline C 3a was obtained as white crystals, m.p. 74-76°C; [a] -39.6” (c 3, CHC13), and had an analysisconsistentwith its formulation as C15H2404; chemical ionisation mass spectroscopygave a molecular peak at m;‘r 269 (MI-l+) and a base peak at 233 due to the loss of two molecules of water. The 13C and ‘H NMR data of compound 3a were very similar to those exhibited by la, the only relevant difference beiig the presencein 3a of a C(5)HOH moiety (6C 70.90; SH 4.84 and 4.82) in place of a X(S)-0 group (SC 200.15). Definitive confirmation of the structure came from the reduction of la with NaBH4 in methanol to give a mixture of the two C-5 epimers 3a and 3b in a 5:95 ratio. NOE difference experiments allowed us to assignthe chirality of the C-5 carbons(Experimental). Speci!ically,irradiation of the %methyl protons led to enhancement of 5-H ( 10.5%) in compound 3b, thus indicating as S its absoluteconfiguration. Therefore the natural compound 3a, where this effect is not observed,must have 5R configuration. TsugicolineD 4 was isolated as a white solid, m.p. 107-l 10°C; [a]D +161.8” (c 0.1, CHCl3); elemental analysis and CI mass spectroscopy indicated the formula C15H2003; the IR spectrum (CHC13) exhibited absorption bands at 3400 (OH) and contained carbonyl bands at 1780 and 1680 cm-* indicative of the presence of a non conjugated and of a conjugated ketonic carbonyl groups; and the W spectrum was consistent with presenceof a conjugated carbonyl systemsinceit exhibited absorptionsat 215 and 240 run (E 6050 and 4800).

A. ARNONE et al.

13360

Table 1 ‘)C NMR data for compnmds l-4 la

2

3b

3s

b

Carbon

atom ‘JCH)ti 1 2 3 4 5 6 7 8 9 IO 11 12 13 14

15

%: 4145t 15302s 74.93 143.98 ZOO.15 90.33 42.70 14.66 47.02 42.50 40.89 47.51 50.90 29.79 27.23

‘J(C,H)/Hz 4. 1445 d s s d s q d t s t d q q

143.0

1465 127.5 131.0 127.0 128.5 134.0 124.5 124.5

6.

61.17 149.99 74.51 148.67 197.77 60.31 36.35 20.41 46.40 41.27 40.81 46.56 52.45

t s d s s t s q d i s t d 29.46q 26 82 q

59.43 t 139.59 s 74.16 d 141.03 s 70.9Od 76.92 d 51.03 s 15.48q 46.59d 41.82 t 40.50 s 47.70 t 51.58d 29.87q 27.35q

‘JG,H)IHz 140.5

‘J(C,HW

142.5 155 151.5 127 130 128 128 129 124 124

Me

in presence

SC

193.97 t 176.5 134.54 s 154.21 d 157.5 54.55 d 145 203.02 s 85.83 d 144 38.38 s 18.82q 127 43.02d 129 44.40 t 129 37.61 s 47.26 t 130 40.72d 130 32.OOq 125 31.61 q 125

6osot 144 136.55 s 74.44d 143 138.76 s 77.95 d 148 85.54d 150 42.49 s 15.88q 127 47.28d 131 42.14 t 127.5 40.14 s 47.20 t 127 50.16d 129 30.04q 124 27.60 q 124

Scheme -A powble mechamsmof formatmn ofmetabolite 4 from la

HO

%

ofE$N

Tsugicolines

Table 2 ‘H NMR

Proton”

la*

la lb 3 6 8 9 1Oa W 12a

4.46 4.42 4.23 4.36 1.00 2.54 1.51 1.55 1.30 1.86 2.32 1.13 1.01 3.82 4.57 5.16

w3 13 14 15 I-OR 3OR 6-oR

chemical

shifts for campowLs

(4.5# (4.57) (4.24) (4.39) (1.02) (2.51) (1.48) (1.62) (1.23) (1.88) (2.33) (1.15) (1.00) (1.55’) (2.15’) (2.95”)

lb’

lc*

4.86 4.72 5.43 5.12 0.99 2.65 I .42 1.67 121 1.65 2.48 1.10 0.99 2.12’ 2.14” 2.15’

4.51 4.44 4.26 4.36 1.00 2.54 1.53 1.57 1.30 1.80 2.33 1.12 1.01

13361

A-D

1-4

i 4.54 4.50 4.24 2.82 1.17 2.48’ 1.46 1.56 1.22 1.85 2.44’ 1.14 0.99 3.od 3.86

5.10

3a’ 4.28 4.26 4.15 3.70 1.04 2.30 1.35 1.39 1.17 1.78 2.25 1.08 0.97 3.97 4 50’ 3.95’

3bb

db

4.40 4.40 4.06 3.68 0.92 2.30 1.36 1.42 1.20 1.77 2.19 1.08 0.97 4.13’ 4.60* 5.15’

9.41 6.7l 4.71 1.13 2.64 1.07 1.59 1.72 2.10 3.16 1.08 1.01

5.14

“Compound lc exhibited hvo methyl protons at 1.45 and 1.33; compound 2 exhibited the remaining 6-H at 2.75; compound 38 exhibited 5-H and 5aH at 4.84 and 4.82; compound 3b exhiited 5-H and XXI at 4.65 and 4.60; and mmpound 4 exhibited 4-H at 3.28 p.b In [’ I$,] acetone. ‘In CD& dvalues in parentheses are chemical shifts in CDCl,.‘~‘Assignments within each mhuon may be interchauged.

Table 3 ‘NMR

&J&H)”

coupling

constants

for mmpounds

1-4.

la’

lb

lc”

2’

3ah

3b*

4b

12q12p 12u.13

16.0 1.4 1.5 8.7 10.4 7.9 11.5 12.4 0.8 1.8 12.2 10.4

16.0 1.6 1.6 9.2 10.6 7.6 12.0 13.0 0.8 2.0 12.5 10.5

16.5 1.4 2.6 7.8 10.3 8.1 11.8 12.7 0.8 1.7 12.5 10.8

17.6 1.6 1.9 9.P 10.2d 8.3d 11.9d 12.6 0.8 1.8 12.3 11.3d

13.4 1.0 1.2 8.5 10.2 8.2 12.0 12.6 0.8 I6 I2 6 10.2

e 1 1 1.1 8.9 10.6 8.0 11.8 12 6 0.8 17 12.6 9.9

2.0 12.7 6.9 7.4 12.6 -0 -0 13.5 2.3

12a,15 12p,13

0.8 7.3

0.8 7.0

0.8 7.5

0.8 7.2

0.8 7.2

0.8 7.5

-0 9.2

la, lb la,3 lb,3 3,13 9.1Oa 9,lOP 9.13 10oop lOa,lS

mm

In compound la 9-H exhibitedJ = 0.6 with both 3- and 6-H and the OH protons presented J = 5.8, 5.8, 5.5 and 7.4 with la-, lb-, 3- and 6-H; in mmpound 3a 5-H exhibited J = 0.7, 0.7, 1.3 and 5.8 with la-, lb, 3- and 6-H; in compound 3b, 5-H exhibitedJ = 2. I. 2.1, 3.2 and 5.7 with la-, It+. 3- and 6-H; and in compound 4 4-H exhibited J = 1.4, 2.9 and 2.3 Hz with 3-, 6- and 13-H. and 6-H a J = 7.8 Hz with 6-OH. % [%J acetone. ‘In CDCl,.‘+In r21-k] benzene Wet assigned

13362

A. ARNONE et al.

Comparison of the 13C and 1H NMR spectraof compound 4 with la revealedthe presence in 4 of the a&unsaturated aldehydic moiety OHC(l)-C(2)-C(3)H in place of the HOC(I)H2-b(2)-&3)HOH grouping. The 1~ NMR spectrum of 4 showed signalsat 6 9.47 and 6.77 attributable to H-l and -3, which were correlated with the aid of an HETCOR spectrumto the correspondingcarbons resonating at 6 193.97 and 154.21, and o$' of 27 and 9.5 Hz observed in the fully one hydroxy proton due to OH-6 Furthermore, the 13C,lH couplings *H-coupled l3C NMR spectrum between C-2 and H-l and C-l and H-3 were indicative of a two-bond interaction between the aldehydicproton and the vinylic carbon and of a cis geometry between the aldehydic carbon and the vinylic proton, respectively.9Finally, the 1H NMR spectrum of 4 contained a signal at 6 3.28 which was assignedto H-4 sinceit presentedcoupling of 1.4 and 2.3 Hz with H-3 and -13 in accord with allylic and homoallylic relationships.Irradiation of H-4 in a NOE experiments (Experimental) led to enhancement of the I-methyl protons (1%) thus permitting to indicate as R the stereochemistryof C-4. Tsugicoline D was easilyobtained by reaction of la with MeOH/Et3N ; a possiblemechanismwhich may mimic the conversion in viw between the two products la and 4 is shown in the Scheme.Work is in progress to examine reactionsof la in the presenceof different nucleophilessincethe a&unsaturated carbonyl moiety is expected to behaveas Michael acceptor.10 Tsugicoline A la is the first reported sesquiterpeneof protoilludane origin with carrying two oxidated functions in the four-membered ring.8 It is therefore possible that it is a common intermediate in the biosynthesisof sesquiterpenessuch as 3-e~iilludol 511 (isolated from CIitocybe c&cm@ and melleolide 6l2 (from Armilhia mellea) via reduction processes. Tsugicoline A la is inactive against Bacillus subtilis, B. cereus, Sarcinea lutea, Ciadosporium chhporioides and Sacchomyces cerevisiaeat a concentration of 100 pg disc-1 but inhibited the growth of Lepiahm sativum;13 the sameactivity (allelopathic) was showed by 3,4dibydroxybenzaldehyde isolated in poor yield during the purification of the tsugicolines. Experimental General.-M.p.s were determined on a Kofler apparatus and are uncorrected; IR spectraon a Perk&Elmer 177 spectrophotometer; massspectra on a Finn&n-MAT-TSQ70 spectrometer; optical rotations on a JASCO500 DIP-181 polarimeter NMR spectra were recorded on a Bruker AC 250L spectrometer operating at 250.1 MHz for 1H and 62.9 MHz for 13C. Molecular modeIling was performed on a Silicon Graphics 4D-35GT, equipped with 16 Mb memory and 1 Gb hard disk, running Insight II & Discover package,version 2.10 (Biosyrn Technologies, San Diego, USA). A model was built by using standard bond lenghts and angles and by taking atomic potentials and charges as defined in the CVFF f?agment library; the molecular model was then energy optimized. Chemical shifis are in ppm (6) from SiMe4 as internal standard, and J-valuesare given in Hz. Flash column chromatography was performed with Merck silicagel (0.04-0.063 mm), and TLC and preparative TLC (pw) with Merck I-IF254 silicagel. Owing to the complexity of the purification procedure, we report the Rp vahies in hexane-EtOAc(1: 1) and CH2C12-MeOH (15: l), respectively. Isolation and Pwijication of Metaboiites la, 2, 3a and 4.- A strain of L.uurilia tsugicola @knn. and Shirai)[Echinodontium tsugicohJ(CBS 248.51) receivedfrom Centraal Bureau voor SchimmelCuhures, Baam, was maintained on MPGA (mah, peptone, glucose, agar, 20:4:20:15 g dmm3)slants and sub-cultured in 40 stationary Erlenmeyer tlasks (250 cm3) containing a liquid medium MPG (50 cm3) for 4 weeks at 24’C; the culture filtrates which were separated6om the mycelium were extracted twice with EtOAc and the extracts were dried (NySO4) and evaporated to yiekl a mixture (2.5 g) of sesquiterpenes.The mixture was treated with Et20 (50 cm3); the residue (0.8 g) is formed of pure tsugicoline A la and the mother liquid was chromatographed on a column of flash silicagel with hexane+EtOAc(2: 1) as eluent to give a mixture of tsugicoline B 2 and D 4 that was fbrther purified by PLC in CH2CI2 (15: 1) yielded the pure metabolites 2 (15 mg) and 4 (20 mg); with bexane-EtOAc(1:2) we obtained again compound la (300 mg) and finally with EtOAc we have tsugicolineD 3a (5 WI.

Tsugicolines A-D

13363

Tmgicohe A la. RfO. 1, 0.3 (Found: C, 67.5; H&3. Cl5H22O4 requires C, 67.64; H, 8.33%); 13C and lH NMR data are reported in Tables 1,2 and 3. SelectedNOE experiments([2H6] acetone + D20) : (H-3) enhancedH3 -8 (l%), Ha-12 (4%) and H-13 (0.5%); (H-6) enhancedH3-8 (0.5%) and H-9 (11.5%); {H-9} enhancedH-6 (lO.S%), Ha-10 (l%), HP-10 (4%), H-13 (6%) andH3-15 (1%); {H3-8 and -15 ) enhancedH-3 (9%), H-6 (O.S%),H-9 f6.5%), Ha-10 (6.5%), HP-10 (4%), HP-12 (5%) and H-13 (7%); (H-13) enhancedH3 (I%), II-9 (So/,),HP-12 (4%) and H3-15 (1%); {H3-14) enhancedH-3 (O.S%),Ha-10 (4.5%), HP-10 (3%), Ha-12 (3.5%) and Hg-12 (3%).

Acetylarion of Tsugicoline A la.- Tsugicoline A la (50 mg) was dissolvedin dry pyridme (0.5 cm3) and treated with Ac20 (1 cm3) overnight at 0°C. Standardwork-up followed by PLC on silicagel in hexane-EtOAc (2: 1) gave the triacetate derivative Ib (40 mg) as an oil ; [a]D -43.2 (c 0.1, CHCI3); vmax (fIIm)/cm-1 1750 (acetate) and 1670 (conj.CO) ; m/z (CI, &butane) 393 (h&)(100%), 333 [(MI-I+)-60](35), 273(50) and 23 l(30); 1H NMR data are reported in Tables 2 and 3. Acetoniak Derivative of Tmgicoline A la.- A solution of la (50 mg) in dry acetone (5 cm3) was treated with a trace of sulphuric acid during 2h at -20°C. The acetone was evaporated and ice was added. The mixture was then extracted with CHC13and the extracts were washedwith satd aqueousNaHC03 and dried (Na2S04). Evaporation of the soivent gave the acetonide lc (40 mg) as white crystals, m.p. 146-148°C; m/i (CI, isobutane), 307 (Iv@)(l4%) and 231(100); lH NMR data are reported in Tables2 and 3. Reaction of Tmgicobne A la with Pivuloyl Chfori&.- Tsugicoline A la (100 mg), pivaloyl chloride (0.2 cm3) and dry pyridine (1 cm3) were left at -30°C for 0 5 h. Standardwork-up followed by PLC on silicagel in hexane-EtOAc (4:l) afforded the dipivalate Id (20 mg) as the main product; m.p. 110-112°C; m/z (CT, isobutane), 435 @II@); 8~ (CDCl3) 5.06 (lH, br s, H-6), 4.94 and 4.83 (2H, br d, J = 13.0 Hz, H2-1), 4.10 (IH, br d,J= 8.7 Hz, H-3), 2.63 (lH, m, H-9), 2.30 (lH, m, H-13). 1.91, 1.69, 1.36 and 1.21 (4H, m, H2-10 and -12), 1.6 (lH, br signal,OH-3), 1.22 (H-18, s, 2 x tbu), and 1.12, 1.00 and 0.90 (9H, s, H3-8, -14 and -15). Reuction of the Acetonide lc with (f)-2-Phenylbutyric Anh+ide.The acetonide lc (40 mg) and (k )-2-phenylbutyric anhydride (50 mg) were dissolvedin dry pyridine (0.5 cm-3) and the solution was kept for 20 h at room temperature. (+)-2-phenylbutyric acid [a]D +3.5 (c 0.5, pyridine) was obtained upon work-up of the reaction mixture according to the literature method.7 Reaction of the dipivuhte Id with (f)-2-Phenylbutyric Anhydride.- Compound Id (90 mg) in dry pyridme (0.5 cm3) was treated with the anhydride; after normal work-up as above (-)-2-phenylbutyric acid [a], 3 1 (c 0.5, pyridine) was obtained.

TsugicolineB 2.- Rf0.5; 0.3 (Found: C, 71.6; H,8.7 Cl5H2203 requires C, 71.97; H, 8.86%); IJV: k max 254 run (E 5100); vmax (CHCl3Ycm-*, 1730 (conj.CO); miz (CI, &butane) 351 (MH+)(lOO%), 233 (MH+-18)(95), 215(38), 205(30), 203(22), 187(28) and 173(25); 1H and 13C NMR data are reported in Tables I, 2 and 3. kvgi~ohte c3a.- Rf0.2; 0.05 (Found: C, 66.9; H, 8.9. Cl5H2404 requiresC, 67.13; H, 9.02??); W: & 208sh (E 5600); vmax (CHCl3&m-*, 3400 (OH); 13C and 1H NMR data are reported in the Tables 1,2 and 3. SelectedNOE experiments ([2H6] acetone + D20) : (H3-8) enhanced H-3 (8.5%), H-6 (1.5%), H-9 (1%) and H2-10 (5%); (H3-14) enhancedH2-10 (5%) and HJ3-12(2.5%); (H3-15) enhancedH-9 (5.5%), HP10 (20/o),HP-12 (2.%) and H-13 (5%).

13364

A. ARNONE et al.

Rea’ucfionof TkugicolineA la.- TsugicoIiie A la (100 mg) was treated with NaBH4 (20 mg) in MeGH (5 cm3); usual work-up gave a 95:5 mixture of two compounds, which were purified by PLC in CH2C12-MeGH (15: 1) (3 runs) and identified astsugicoline C 3a (4 mg) and its C-5 epimer 3b (91 mg). 5-epi-Tsugicoline C 3b.-This compound was isolated as a solid, m.p. 85-87Y; [a] D -59 lo-ldeg cm2g-l (c 0.05, CHC13); m/z (CI, isobutane) 269 (MH+); -2 and ‘H NMR data are reported in Tables I,2 and 3. SelectedNOE experiments ([2I-I6] acetone + D20): (H3-8) enhanced H-3 (6%), H-S (10.5%), H-6 (I%), H-9 (1%) and H2-10 (3.5%); {H3-14) enhancedH2-10 (3%) and HP-12 (2%); {H3-15) enhanced H-9 (5%), HP-10 (2%) I@-12 (3.5%) and H-13 (5%). TsugicolineD 4.- Rf0.7; 0.3; (Found: C, 72.2; I-& 8.0. Cl5H20G3 requires C, 72.55; I-I, 8.12%); m/z (CI, isobutane) 249 (MH+)(SO%), 23 1 (MHe-18)(100), 220 (20) and 203 (25); 13C and lH NMR data are reported in Tables 1, 2 and 3. SelectedNOE experiments([2J-I6] acetone) : (H-l } enhancedH-3 (11.5%) and H-4 (1.5%); (H-3) enhancedH-1 (18%), Ha-12 (4.5%) and H-13 (4.5%); (H-4) enhancedH-l (1%) and H3-8 (1%); (H-6) enhancedH-9(3%)andH-13(10.5%); (H-13) e&ncedH-3 (3%),H-6(7%),H-9(4%)andHg12 (3.5%). Reaction of Tsugtcoiine A la with Triethykzmine.-Tsugicoline A la (50 mg) dissolvedin MeGH (3 cm3) was treated with ttiethylamine (0.1 cm3) at room temperature for 2 h; evaporation of the solvent and PLC with CH2Cl2-MeOH (15: 1) gave a compound (20 mg) identical with the natural one 4 (*H NMR, MS and TLC). Acknowledgements.Thanks are due to Dr. A. Mete for the molecular model calculations

Referencesand Notes 1. Part 51 in the series‘Secondary Mould Metabohtes”. For part 50 see, Amone, A.; Nasini G.; Vajna de Pava, 0.; J. Nat. Prod, in the press. 2. Arnone, A.; Cardillo, R; Nasini, G.; Meille, S.V.; J. Chem. SocPerkin. Trams.I, 1988, 503 and references therein. 3. Arnone, A.; Cardillo, R.; Nasini, G.; Vajna de Paw, 0.; J. Chem. Sot. Perkin Trans. I, 1991, 733 and referencestherein. 4. Amone, A.; Nasini, G., Assante,G; and Van Eijk, G.W.; Phytochemistry,1992,31,2047. 5. Arnone, A.; Nasini, 0. ; Vajna de Pava, 0.; Assante, G.; J. Chem. Sac.Perkin Trans. I, 1992,615. 6. Man, D.H.; Stothers, J.B.; J. Can. Chem.. 1965, 13, 596. 7. Horeau, A.; “Stereochemistry”,ed. H.B. Kagan, Thieme Stuttgart, 1977, Vol. 3, p.51. 8. Ayer, W.A.; Browne, L.M.; Tetrahe&an, 1981,37, 2199. 9. Vogeii, U., von Philipsborn,W.; Org. Magn. Reson., 1975,7,617. 10. Amone, A.; Nasini, G.; Vajna de Pava,0.; to be published, 11. Amone, A.; Cardillo, R.; Di Modugno, V.; Nasini, G.; J.Chem.Soc.PerkinTrans. 1, 1989, 1995. 12. Midland, S.L.; Izac, R.R.; Wing, R.M.; Zaki, AI.; Munecke, D.E.; Sims,J.J.; Tetrahebon Letters, 1982, 23, 2515. 13.Arnone, A. ; Assante,G.; Nasini, G.; Vajna de Pava,0.; Phytochemistry, 1990,29,613.

(Received in UK 11 September 1995; accepted 5 October 1995)