Journal of Natural Proa'ucts VOI.54, NO.2, pp. 629-631, Mar-AP I991
629
THE STRUCTURE OF CLATHRODIN, A NOVEL ALKALOID ISOLATED FROM THE CARIBBEAN SEA SPONGE AGELAS CLATHRODES' JOSE
J . MORALES~ and ABIMAELD. RODRIGUEZ*
Department o f C h i s t r y , University ofPuerto Riro, Ria Piedra, Puerta Rico 00931 ABSTRACT.-A new alkaloid containing a nonbrominated pyrrole and a guanidine moiety, clathrodin, has been isolated from the MeOH extract of the Caribbean sea sponge Agefas rfatbroder. The structure of clathrodin was determined to be 1 on the basis of its spectral data.
Several C 1-N5 compounds containing a bromopyrrole and a guanidine moiety have been isolated from marine sponges belonging to the families Agelasidae and Axinellidae (1-1 1). However, not many related nonbrominated compounds are known from these families (12). During a recent expedition in search of physiologically active substances of marine organisms from Puerto Rico, we have examined the antimicrobial and cytotoxic actions of an MeOH extract of a sea sponge Agelas clathrodes (Schmitt) which was collected near Desecheo Island, Puerto Rico in March 1989. The material from the MeOH extract showed weak activity against Proteus vulgaris, Staphylococcusaureus, and Shigella fixneri (MIC = 1 mgl ml) using the standard disk assay. Similarly, the cytotoxicity assays inhibited the growth of SW-480 cells (ED,, = 5 3 Fglml). We report here the isolation
1 X=Y=H 2 X=Br,Y=H 3 X=Y=Br
'Presented at the XIV Senior Technical Meeting of the American Chemical Society, Puerto Rico Section, University of Puerco Rico, Rio Piedras, Puerto Rico, December 7 , 1990. *Graduate student sponsored by the NSFEPSCoR Program of F'uerto Rico.
and the structure elucidation of the cytotoxic constituent clathrodin 111. The MeOH extract of the sponge was suspended in H 2 0 and extracted with CHCI, ( 3 x 2 5 0 ml) and n-BuOH (2 X 250 ml), successively. After concentration, the n-BuOH-soluble portion (3.30 g) was chromatographed on a reversed-phase (C,8, 20 g) column with H 2 0 followed by a Si gel column (48 g) with CHC1,-MeOH (4: 1) saturated with NH,. Combination of like fractions on the basis of tlc analyses gave pure clathrodin 111 as a colorless semisolid (840 mg). The hrfabms of 1 showed an intense (M H}+ peak at rn/z 232.1198, indicating a molecular formula of Cl1H,,N5O (A 0.3 mmu) for the compound. The spectral data recorded indicated that clathrodin 111 was the 3-debromo derivative of hymenidin [2],a potent antiserotonergic constituent of the Okinawan marine sponge Hyrneniaczdon sp. (7). The 'H-nmr spectrum (Table 1) and the 'H-'H COSY spectrum revealed the partial structure -CO-NH-CH2-CH= CH- (trans). The signals for two aromatic protons at 6 6.96 (H-2 and H-4, overlapping multiplets) and another at 8 6.12 (H-3, complex multiplet) indicated the existence of a monosubstituted pyrrole ring (9). The connection of the pyrrole ring to the amide carbonyl at C-5 was argued on the presence of an intense cross peak between H-4 and H-7 in the 2D nOe spectrum (13,14). An aminoimidazole unit was suggested by the ',C-nmr signals at 6 127.35 (C-ll), 151.79 (C13), and 117.52 (C-15). Furthermore,
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Position ~
wol. 54, No. 2
Journal of Natural Products
630
~
6cb
&'(jinHz) ~
1. . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . 3. . . . . . . . . . . . . . 4 . . . . . . . . . . . . . . 5. . . . . . . . . . . . . . 6 . . . . . . . . . . . . . . 7 . . . . . . . . . . . . . . 8 . . . . . . . . . . . . . .
~
11.52 ( l H , brs, exchangeable) 6.96(1H,m) 6.12 ( l H , m) 6.96(1H,m)
-
8.12(1H,t, 5.6,exchangeable) 3.98(2H,t, 5.5) 5.93(1H,dt,6.4, 15.6) 6.27(1H,d, 15.5)
9. . . . . . . . . . . . . . 10 . . . . . . . . . . . . . 11 . . . . . . . . . . . . . 12 . . . . . . . . . . . . . 13 . . . . . . . . . . . . . 15 . . . . . . . . . . . . . -NH2 . . . . . . . . . . .
nOe'
~
-
11.52(1H, brs, exchangeable) 6.49(1H, s) 5.41 (2H, brs, exchangeable)
12 1.86 (d) 109.04 (d) 1 10.34 (d) 130.93(s? 161.45(s) 4 1.39 (t) 120.78(d) 122.54 (d) 127.35 ( s ) ~ 15 1.79 (s) 117.52(d) -
H-3 H-7
H-4 H-9, H-10 H-4, H-8 H-8, H-15
H- 10
'Assignments were aided by 'H-'H COSY and 'H-"C COSY, spin splitting patterns, and NOESY experiments. The chemical shifts ace given in 6 units (ppm downfield from TMS). bAssignments were made on the basis of 'H-I3C COSY and APT experiments. The 6 values are in ppm and are referenced to the residual DMF signal (162.7 ppm). 'Obtained from the 2D nOe spectrum. dAssignments may be interchanged.
EXPERIMENTAL GENERALEXPERIMENTAL PROCEDURES.Ir and uv spectra were recorded on a Nicolet 600 m-IR and a Hewlett-Packard Chem Station 8452A spectrophotometer, respectively. 'H- and '3C-nrnr spectra were recorded on a General Electric Multinuclear QE-300; 'H chemical shifts are recorded with respect to TMS (6 0.0). Cc was performed on Analtech Si gel (35-75 mesh), and tlc analyses were carried out using Analtech glass packed precoated Si gel plates. All solvents used were either spectral grade or were distilled from glass prior to use.
CWTHRODIN [I].-White semisolid: ir (KBr) 3330 (broad), 1683, 1617, 1563, 1522, 1407, 1328, 1127, 1042, 960, 746 cm-'; uv (MeOH) 272 nm (E 24,500); 'H nmr (300 MHz, DMF-d,) see Table 1; 13Cnmr (75 MHz, DMFd,) see Table 1; hrfabms mlz 232.1198 [M HIf (55%) (CI,Hl4N5O requires 232.1195), 154 (88), 136 (85), 122 (35). 107 (45). 102 (100). In vitro screening data for pure clathrodin show significant cytotoxicity against CHO-K1 cells (ED5"= 1.33 Fg/ml). Clathrodin has also been found to possess potent blocking activity against cholinergic receptors on the isolated frog skeletal muscle at concentrations of 10-7-10-3 M. The
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Mar-Apr 199 11
Morales and Rodriguez:
Alkaloid from Sponge
pharmacological activities of clathrodin will be reponed elsewhere in detail. ACKNOWLEDGMENTS We are grateful to Dr. Vance Vicente for identification of the sponge and Dr. Gladys Escalona from the UPR Institute of Neurobiology for preliminary evaluation of the pharmacological p r o p erties of 1. We thank Mr. M.A. Medina, L.A. Fontan, and G. Cabrera for their kind assistance in collecting the marine sponge and Miss M. Ortiz for technical assistance. Special thanks are extended to Dr. A. Baez and Miss M.E. Pimentel for the CHO-KI cytotoxicity tests. This work was funded by grants from the National Science Foundation EPSCoR Program (Grant No. R118610677) and MRCE Program (Grant No. R 1 1-880296 1) and the University of Puerto Rico FIPI Program. High-resolution mass spectral determinations were performed by the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Facility (Grant No. CHE82 1 1 164).
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Received I October 1990
