N otizen 1275
Two New Phloroglucinol Derivatives with Phosphodiesterase Inhibitory Activity from the Leaves of Eucalyptus robusta
Qi Cheng and John K. Snyder*
Department o f Chemistry, Boston University, 590 C om m onwealth Ave., Boston, M A 02215, U SA
Z. Naturforsch. 46b, 1 2 7 5 - 1277 (1991);
received February 14, 1991
Eucalyptus robusta, Phloroglucinols, Phosphodiesterase Inhibition
Tw o simple, new phloroglucinol derivatives have been isolated from the leaves o f Eucalyptus robusta Smith, used in the Chinese traditional medicine D a Ye An. These phenolic com pounds were purified using a phosphodiesterase inhibi
tion bioassay to guide the isolation. Both com pounds had moderate activity in this bioassay.
The leaves o f Eucalyptus robusta Smith (M yrta- ceae) are used in China to prepare the anti-m alari
al traditional medicine “Da Ye A n” [1], Previous w ork on the extract o f the leaves has yielded the bisphloroglucinol robustaol [21 as well as the robus- tadials A and B [3], The known phenols euglobal la, and Ia2, originally reported from E. globulus [4], were also isolated [3]. In a broad screening for biological activity, the crude ethanol extract of the leaves showed phosphodiesterase inhibitory activi
ty. Using this bioassay to guide the isolation, two simple, though to the best of our knowledge pre
viously unreported, phloroglucinols, 1 and 2, were isolated from the crude ethanol extract.
12 OH 0 12 OH 0
c h3>
/ t
JL s / 9 7
c h3 n
r t
7 c h3o'
KJ
-i 6
oIO
c h3o^
K a^OH c h3
13 3
1 13CH3 2
The high resolution mass spectrum o f 1 revealed the molecular formula C 13H 180 4 (m /z 238.1204, M + , calcd for C 13H lg0 4 238.1205). A hexasubsti- tuted arom atic com pound with a 1,3,5-trioxygen- ated ring pattern was immediately apparent from the 13C N M R spectrum (Table I). The 'H N M R spectrum readily accounted for five o f the six sub
stituents: one methoxyl group at ö 3.68, two aro-
* Reprint requests to Prof. J. K. Snyder.
Verlag der Zeitschrift für N aturforschung, D-7400 Tübingen 0932 - 0776/91 /0900 -1275/$ 01.00/0
matic methyl singlets at ö 2.09 and 2.10, and two phenolic hydroxyl groups, S 3.42 and 13.22, which exchanged with deuterium upon addition of D 20 . The 13C N M R spectrum indicated that the only oxygenated sp3 carbon was the methoxy methyl carbon. The shift o f the low field phenolic OH group was independent of concentration clearly indicating intram olecular hydrogen bonding. The remaining substituent was determined to be an acyl group by the low field signal at 210.6 in the l3C N M R spectrum o f a ketone carbonyl. The rel
atively low frequency carbonyl band in the IR spectrum o f carbonyl stretch, 1650 cm -1, was also best accounted for by conjugation with the aro matic ring as well as intram olecular hydrogen bonding. The presence of the phenolic hydroxyl group was also supported by the IR spectrum by a broad band centered at 3200 cm -1. Also present in the 'H spectrum was the six proton doublet of an isopropyl methyl groups coupled with a methine proton at 3 3.82. Thus 1 was thought to be an aryl isopropylketone.
W ith the 1,3,5-trioxygenated pattern as well as all six aryl substituents identified, the remaining problem was the location o f the aryl methyl groups and the acyl group relative to the methoxyl group.
The symmetric substitution pattern was estab
lished by a difference nOe experiment which showed nO e's between the methoxyl protons and both aryl methyl groups. Selective IN EPT experi
m ents [5] enabled assignment of all arom atic car
bons. Long rang ]H - 13C couplings from H - l2 to C-2 and C-3, from the low field hydrogen bonded phenolic - O H proton at S 13.22 to C-2, and from H - l3 to C-4 were observed. Thus, the loss of sym
m etry due to the hydrogen bonding must be a slow exchange phenom enon and the six arom atic car
bons appear as distinct, assignable resonances.
C om pound 2 had a molecular ion in the high resolution mass spectrum of m /z 252.1365, which required a m olecular form ula of C ,4H 20O4 (calcd for C 14H 20O4 252.1362). Inspection of the 'H and ,3C N M R data (Table I) indicated that 2 was a hom ologue o f 1. The sole difference was in the ali
phatic chain o f the ketone which contained an ad ditional carbon. An APT experiment indicated the four carbons o f the chain to be two methyls, a methylene and a methine. The COSY spectrum of 2 easily m apped out the spin system of a 2-methyl- butanoyl group as the acyl substituent. The substi
tution pattern o f the arom atic ring was established by nOe studies analogous to those for 1, and selec
tive IN E PT experiments enabled assignment of all arom atic carbons. The absolute stereochemistry of the stereocenter at C-8 was not defined.
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1276 N otizen
Position
Com pound 1
'H (ppm) 13C (ppm)
C om pound 2
'H (ppm) 13C (ppm)
1 — 158.5 (s) _ 158.1 (s)
2 - 107.8 (s) - 106.5 (s)
3 - 158.6 (s) - 158.2 (s)
4 - 108.4 (s) - 108.5 (s)
5 - 161.1 (s) - 161.5 (s)
6 - 106.5 (s) - 104.6 (s)
7 - 210.6 (s) - 210.7 (s)
8 3.82 (d, 8.1) 3 9.9(d ) 3.80 (m) 4 5 .3 (d )
9 1.19 (d, 8.1) 21.0 (q) 1.40 (m) 28.5 (t)
1.76 (m)
10 1.19 (d, 8.1) 21.0 (q) 0.89 (t, 7.9) 12.0 (q)
11 - - 1.15 (d, 6.4) 17.6 (q)
12 2.09 (s) 8.9 (q) 2.08 (s) 7-89 (q)
13 2.10 (s) 9.8 (q) 2.11 (s) 8.52 (q)
4-O C H , 3.68 (s) 63.7 (q) 3.67 (s) 62.8 (q)
2-OH 13.22 (s) - 13.15 (s) -
6-OH 3.42 (s) - 5.22 (s) -
Table I. 'H and I3C N M R data for 1 and 2 in CDCU.
Simple phloroglucinol derivatives are frequently encountered substituents o f Eucalyptus species [6-8]. The frequent co-occurrence of the iso- butanoyl group with higher hom ologues as the acyl group in phloroglucinol derivatives has been previously noted [9-11], In the PD E bioassay, the E D 50 o f 1 was 100 //g/ml, while th at of 2 was 125 //g/ml. In addition to 1 and 2, the well known Eucalyptus flavonoid eucalyptin [12, 13] and the 7-0-/7-D-glucoside of 5,7-dihydroxy-2-methyl- chrom one [14-16], which has been found in sever
al species, were also isolated.
Experimental General procedures
Plant collection and ethanol extraction are pre
viously described [3]. A voucher speciman is on deposit in the Chemistry D epartm ent at Boston University. The N M R spectra were recorded on a Varian XL-400 (93.93 kG, 400 M H z for ’H, 100 M Hz for l3C) in CDC13. Residual CHC13 (<5 7.24 ppm) and i3CD C l3 (J 77.0 ppm ) were used as internal references for ’H and 13C, respectively.
Assignment of “O H ” protons were confirmed by D 20 exchange. Mass spectra (medium and high resolution) were recorded on a Finnigan MAT-90 in the El mode (70 eV); IR spectra were recorded on a Perkin-Elm er 1800 F T IR spectrometer.
Isolation o f 1 and 2
The crude ethanol extract was partitioned be
tween equal volumes (11) of water and ethyl ace
tate, and the ethyl acetate fraction (700 mg) subse
quently partitioned between equal volumes (250 ml) of petroleum ether (3 0 -6 0 C) and 5%
aqueous m ethanol. The water fraction was like
wise partitioned between equal volumes of
«-butanol and water. The phosphodiesterase inhi
bition activity [17] was located in the aqueous m ethanol fraction (0.5 g) which was further frac
tionated by flash chrom atography on silica gel us
ing methylene chloride as eluant. Phenols 1 and 2 were purified from the active fraction by reverse phase H PLC (M icrosorb-C 18 Rainin, 5 //m, 4.6 X 250 mm; flow rate 1.4ml/min; UV detection, 254 nm) using 20% aqueous m ethanol as eluant.
Phenol 1
Colorless film: UV Amax (M eOH) 206 (e 28000), 280 (32000), 312 (11000); IR (CC14) cm “1 3600, 2950, 1650, 1630, 1590, 1550, 1461, 1410, 1350, 1320, 1150, 1110, 1000; HRM S 238.1204 (calcd for C 14H ,0O4 238.1205); EIM S m /z (% ) 238 (M + , 18), 195 (100), 180 (5), 152 (7); 'H and ,3C N M R (CDC13) see Table.
Phenol 2
Colorless film; UV ;.max (M eOH) 208 (e 21 000), 280 (28000), 312 (19000); IR (CC14) cm “1 3600, 2950, 1650, 1630, 1590, 1550, 1460, 1410, 1350, 1320, 1150, 1110, 1000; HRM S 252.1365 (calcd for C 14H ,0O 4 252.1362); EIMS m /z (% ) 252 (M + , 17), 195 (100), 180 (3), 152 (5); 'H and 13C N M R (CDC13) see Table.
We thank the Department o f Pharmacy, the Second Military M edical C ollege o f Shanghai, for providing the crude material, and Jie-Zhi W ang for performing the ini
tial ethanol extraction o f the E. robusta leaves. We also thank Schering-Plough Corporation for performing the phosphodiesterase bioassays, and for financial support.
N o tizen 1277
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[17] The phosphodiesterase bioassay was performed at Schering-Plough Corp., Bloomfield NJ. Details o f the procedure were not divulged.
