Rigidin E, a New Pyrrolopyrimidine Alkaloid from a Papua New Guinea Tunicate Eudistoma Species

A new pyrrolopyrimidine alkaloid, rigidin E (1) and the known metabolites rigidin (2) and 1-methylherbipoline (3) have been isolated from a Papua New Guinea tunicate Eudistoma sp. A combination of spectroscopic data were used to determine the structures of these metabolites.


Introduction
Tunicates belonging to the genus Eudistoma have been the sources of numerous novel and bioactive secondary metabolites. β-Carboline alkaloids have been the predominant structure class isolated from this particular genus to date [1][2][3][4][5], however macrolides [6], indoles [7,8] and aromatic polysulfides [9] have also been reported. In 1990 Kobayashi et al. reported the isolation of rigidin (2) from an Okinawan marine tunicate Eudistoma cf. rigida; this novel compound displayed calmodulin antagonistic activity and was the first pyrrolopyrimidine alkaloid to be isolated from a marine source [10]. More recently three new rigidin congeners, rigidins B-D (4-6) have been isolated from the tunicate Cystodytes sp. [11]. In our continuing search for unique secondary metabolites from marine tunicates [12,13], we investigated a previously undescribed tunicate Eudistoma sp. collected from Papua New Guinea. These chemical studies resulted in the isolation of a new pyrrolopyrimidine derivative, which we have named rigidin E (1) along with the known metabolites rigidin (2)
Rigidin E (1) was isolated as a stable dark green film. A molecular formula of C 20 H 14 N 3 O 5 was assigned to 1 by (-)-HRESMS analysis [m/z 376.0933 (M-H) -], which established that rigidin E was larger than rigidin (2) by a CH 2 unit. IR absorption bands for compound 1 at 3600-3000, 1690 and 1590 cm -1 were attributed to OH/NH and carbonyl group(s) respectively. UV absorptions at 276 (ε 7000) and 362 (ε 4000) suggested the presence of conjugated phenol chromophore(s) [14]. The presence of phenol(s) in 1 was further supported by the UV spectrum that underwent a bathochromic shift on addition of base.
All three compounds were tested for cytotoxicity against the p53 wildtype (p53 +/+ ) and p53 deficient (p53 -/-) HCT 116 human colon carcinoma cell lines, while 1 and 2 were also tested in the A431 human epidermoid carcinoma cell line. Only minimal growth inhibition at 100 µg/mL was identified for all metabolites, hence no further biological evaluations were pursued (see Table 1). Interestingly, rigidins B-D (4-6) have recently been shown to exhibit moderate cytotoxocity against murine leukemia L1210 cells. Preliminary biological results showed that at 10 µg/mL, compounds 4-6 inhibited leukemia tumor cell growth by 40%, 40% and 20% respectively [11].

Conclusions
We have isolated and spectroscopically characterized a new pyrrolopyrimidine alkaloid, which we have named rigidin E (1). The known metabolites rigidin (2) and 1-methylherbipoline (3) were also isolated during our chemical investigations of a Papua New Guinea tunicate Eudistoma sp. Vogelstein of Johns Hopkins University for generously donating the HCT 116 cell lines.

Experimental
General NMR spectra were recorded on a Varian Mercury 400 MHz spectrometer ( 1 H: 399.880 MHz, 13 C: 100.559 MHz) at 25 °C. 1 H and 13 C chemical shifts are reported in parts per million relative to the reference solvent peaks at δ 2.49 and 39.51 ppm for DMSO-d 6. FT-IR and UV spectra were recorded on a Jasco 420 spectrophotometer and a Hewlett Packard 8452A diode array spectrophotometer respectively, and LRESMS experiments were performed on a Micromass Quattro II Triple Quadrupole mass spectrometer. HRESMS were recorded on a Bruker BioAPEX 47e mass spectrometer equipped with a Bradford CT 06405 electrospray ion source. Gel permeation chromatography was performed on Lipophilic Sephadex LH-20 (45 × 730 mm). An Agilent 1100 quaternary solvent module equipped with a 7725i Rheodyne injector and an Agilent PDA detector was used for HPLC separations. A semipreparative Phenomenex Luna C 18 (2) 5 µm 100 Å (10 × 250 mm) column was used for HPLC. All solvents for HPLC, UV, and MS were Fisher HPLC grade and water used was Barnstead E-pure 0.2 µm filtered.

Animal Material
A specimen of the undescribed Eudistoma sp. [14] was collected during May of 2001 by SCUBA diving (-10 m) at Milne Bay (S 10° 14.278', E 150° 54.782'), Papua New Guinea, and kept frozen prior to freeze-drying and extraction. Taxonomic voucher specimen MZUSP15712 has been deposited at the Museu de Zoologia, Universidade Federal de São Paulo, São Paulo, Brazil.

Cells and Culture Conditions
All cell cultures were grown at 37 °C, 5% CO 2 and maximal humidity (by equilibration). All media reagents were purchased from Invitrogen Corporation (Carlsbad, CA). The HCT 116 strains were donated by Dr. Bert Vogelstein (Johns Hopkins University) and grown in McCoy's 5A media supplemented with 10% v/v heat inactivated fetal bovine serum (FBS), 1% v/v MEM (Mimimal Essential Media) sodium pyruvate, 1% v/v penicillin-streptomycin and 1% v/v L-glutamine. The A431 cell line was purchased from American Type Culture Collection (Manassas, VA) and grown in MEM supplemented with 10% v/v heat inactivated FBS, 1% v/v MEM sodium pyruvate, 1% v/v penicillin-streptomycin and 1% v/v L-glutamine. Cells were counted using a Beckman Coulter cell counter (Fullerton, CA) [16].

Cell Proliferation Assay
The ability of drug to inhibit cellular growth was determined using the MTT assay [16]. For the HCT 116 cell lines, 8000 cells per well were plated in 96-well plates in 200 µL of the appropriate media (see above) and the cells were allowed to grow overnight. Serial dilutions of drug were then added to wells in quadruplicate. Control wells were treated with vehicle (DMSO). After 72 h of incubation at 37 °C and 5% CO 2 , the media was removed and 100 µL fresh media was added along with 10 µL of 2.5 mg/mL aqueous 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The cells were then incubated for 4 h under the same conditions as above. The media was then removed and 100 µL DMSO added to each well. The amount of formazan resulting from MTT metabolism was measured by observing absorbance at 540 nm using a Labsystems Multiskan Plus plate reader. Using the absorbance in the vehicle treated lanes as 100% survival (1.00 fractional survival) the fractional survival of each treatment was observed. Because no growth inhibition was observed after most of the diluted treatments only the survival at 100 µg/mL is presented. The protocol using the A431 cells was essentially identical to the HCT 116 assays except that 16000 cell per well were initially plated and the cells were exposed to drug for 96 h. Both the HCT 116 and A431 cell lines responded typically when treated with laboratory standards. For example, the IC 50 of makaluvamine C towards HCT 116 cells was 0.37 µg/mL