Isatinones A and B, New Antifungal Oxindole Alkaloids from

Two new oxindole alkaloids isatinone A (1) and B (2) have been isolated from Isatis costata, along with the known trisindoline. Their structures have been assigned on the basis of spectroscopic techniques and chemical studies. Both new compounds showed significant antifungal activity.


Introduction
The genus Isatis, belonging to the family Brassicaseae, comprises 50 species, mainly distributed in the Irano-Turanian region.In Pakistan it is represented by seven species [1].Isatis tinctoria or woad is a common plant cultivated throughout the centuries to produce the blue dye indigo.Nowadays, woad is also used in Chinese folk and modern medicine [2]."Ban-Lan-Gen" is one of the most commonly used traditional Chinese medicines for antipyretic, anti-inflammatory, antiviral, antimicrobial and detoxifying purposes.Its original source was considered to be the dried roots of three plants, Isatis indigotica, Isatis tinctoria and Strobilanthes cusia [3][4].Now the roots of Isatis indigotica have been identified as the main source of "Ban-Lan-Gen" and recorded as such in Chinese Pharmacopoeia (1990 edn.) [5].The ethno-pharmacological importance of the genus Isatis prompted us to investigate the chemical constituents of Isatis costata, which is an annual or biennial herb, found in northern parts of Pakistan.Herein we report the isolation and structural elucidation of isatinones A (1) and B (2), along with the known trimeric oxindole alkaloid trisindoline [6].Both alkaloids 1 and 2 showed significant antifungal activity against various strains.
The 13 C-NMR spectrum (BB and DEPT) showed sixteen signals, comprising of one methyl, nine methine and five quaternary carbons.The downfield signals at δ 167.1 could be assigned to the carbonyl carbon of an amide.The olefinic carbons resonated at δ 148.7 and δ 103.1, respectively.The other 12 signals ranging from δ 144.0-115.1 were due to aromatic carbons, while the methoxy carbon resonated at δ 56.7.The above spectral data was consistent with an oxindole type alkaloid with additional phenyl and methoxyl moieties.Since the presence of a disubstituted indolic moiety has already been established, therefore the only location for these groups is the exocyclic olefinic carbon of a methylidene moiety.The structure was not only supported by 1 H-1 H COSY spectrum, but also by HMBC correlations (Figure 2) in which the H-4 proton at δ 7.54 showed 2 J correlations with C-3a (δ C 124.0) and 3 J correlations with C-7a (δ C 144.0) as well as C-3 (δ C 103.1).The proton at δ H 7.02 (H-3') showed 3 J correlations with C-1' (δ C 148.7) and 2 J correlations with C-2' (δ C 141.1).The methoxyl protons at δ H 3.87 showed 3 J correlation with C-1' (δ C 148.7).The geometry of the double bond was assigned on the basis of chemical shifts of the olefinic carbons.Thielke et al. [8] have reported that the olefinic carbon in the α-methylene lactam system is less shielded in the Z than in the E geometry because of a large paramagnetic anisotropy effect from the lactam carbonyl group.The values of olefinic carbons were consistent with theoretically calculated values for the E geometry and showed very close agreement to those of costinone B, reported in the literature [9].This was further confirmed by NOE correlation between δ H 7.54 (H-4) and protons of the methyl group at δ H 3.87 [10].The assignments of 13 C-NMR signals were facilitated by HMQC spectrum and found in complete agreement to the assigned structure of isatinone A (1) as 3-[(E)-methoxy (phenyl) methylidene]-1,3dihydro-2H-indol-2-one.
Isatinone B (2) was isolated as a pale yellow amorphous solid, mp 189-191°C.The molecular formula C 31 H 33 NO 4 was determined by negative ion HRFABMS, which showed a pseudomolecular ion peak at m/z 482.2328 (calc.for C 31 H 32 NO 4 : 482.2331).The UV and IR spectra were very similar to those of 1, except the presence of additional absorptions due to the ester moiety.The 1 H-and 13 C-NMR spectra were also found to be similar to those of 1, except for the replacement of the methoxyl group by a 2-ethylhexyl phenylacetic acid ester moiety.
The 13 C-NMR spectrum (BB and DEPT) showed thirty-one signals, comprising of two methyl, six methylene, fourteen methine and nine quaternary carbons.The signals at δ C 169.3 and 165.3 could be assigned to carbonyl carbons of ester and amide, respectively.The methylidene olefinic carbons resonated at δ C 149.4 and 103.9, respectively.The other signals ranging from δ C 144.4-115.0 were due to aromatic carbons.The oxymethylene carbon resonated at δ C 69.1, while signals of five methylene groups were observed from δ C 49.6-24.0.The two terminal methyl groups resonated at δ C 14.4 and 11.4,respectively.

Biological Activity
The antifungal activities of both 1 and 2 were determined by the agar tube dilution method and significant activity was observed against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.

Conclusions
In summary, the isolation of two novel antifungal oxindole alkaloids named isatinone A and B and the known alkaloid trisindoline from I. costata has been achieved and their structures elucidated with the help of spectroscopic techniques.

General
Optical rotations were recorded on a JASCO DIP-360 digital polarimeter.IR spectra were measured on a JASCO 302-A spectrophotometer in CHCl 3 .UV spectra was obtained on a Hitachi UV-3200 spectrophotometer.NMR spectra were run on an AMX-400 Bruker instrument.Chemical shifts δ are shown in ppm relative to TMS as internal standard and coupling constant J are given in Hz.EI-, FAB-, and HREIMS were recorded on a JEOL JMS-HX-110 and JMS-DA-500 mass spectrometers.Silica gel 230-400 mesh (E.Merck) was used for column chromatography.Silica gel plates (Si 60 F 254 , E. Merck) were used for TLC.

Plant Material
The whole plant material was collected in April 2004 from N.W.F.P Swat and identified as Isatis costata C. A. Mey by Dr. Ghosia Lutfullah, Centre of Biotechnology, University of Peshawar, Pakistan.A voucher specimen (BPU-105) is deposited in the Herbarium of the Department of Botany, University of Peshawar, Peshawar, Pakistan.

Extraction and Isolation
The shade-dried whole plant (17 kg) was chopped up and extracted three times with EtOH (60 L) at room temperature for 96 h.The ethanolic extract was evaporated in vacuo to give a dark greenish residue (400 g), which was partitioned between EtOAc and water.The aqueous fraction was made basic with 10% NH 4 OH and the liberated bases extracted with CH 2 Cl 2 .The CH 2 Cl 2 fraction (40 g) was subjected to column chromatography eluting with n-hexane-EtOAc mixtures in increasing order of polarity to afford six fractions F 1 -F 6 .Silica gel column chromatography of fraction F 2 (eluted with 7:3 n-hexane-EtOAc) and elution with mixtures of n-hexane-EtOAc provided fractions F 2A (7:3) and fraction F 2B (5:5), respectively.Slow evaporation of fraction F 2A deposited pale yellow crystals of isatinone A (1, 11 mg).The fraction F 2B was rechromatographed over silica gel, again eluting with n-hexane-EtOAc mixtures.The eluent obtained from 3:7 n-hexane EtOAc provided isatinone B (2, 17 mg).The fraction F 3 obtained from n-hexane-EtOAc (6:4) was rechromatographed over silica gel using mixtures n-hexane-EtOAc (8:2 → 3:7) as solvent to afford two successive fractions, the first of which, further on purification by column chromatography over silica gel and elution with 7:3 n-hexane-EtOAc afforded trisindoline (25 mg).[6].