A nucleoside, kumusine (1) from an Indonesian sponge Theonella sp. is 2-chloro-9-(5'-deoxy-2'-methyl-lS-D-ribofuranosyl)-adenine.Nucleosides are significant structural components of a broad spectrum of drugs. 4 Marine nucleosides are no exception to this observation. Two nucleosides, fLrst described by Bergmann and Burke, 5 in which arabinose replaces the conventional ribose, became after a long and meandering route spanning twenty-five years, the progenitor of the antiviral drug Ara-A (Vidabarine). 6 We now report the isolation and structural elucidation of kumusine (1), 7 a nucleoside constituted of 2-chloroadenine and a 2 methyl-5-deoxy ribose.Kumusine (1) was isolated from an Indonesian sponge, Theonella sp., a genus that is best known for structurally diverse complex constituents, among them swinholide, 8 onnamide A,9 and aurantoside A. 10 A MeOH extract of the sponge was partitioned between EtOAc and water. The organic phase yielded kumusine (1) as a colorless solid, mp 210-213 °C, by counter-current chromatography (EtOAc/H20, upper mobile phase). Two significant M + peaks at m/z 299 and 301 in a ratio of 3:1 indicated a chlorine atom, which was confirmed by HR-EIMS leading to a molecular formula of CIlH14C1N503 (m/z 299.0802, A -1.7 mmu).A diagnostic signal at 8 8.51 in the IH NMR spectrum of 1, coupled with five sp 2 carbon signals and five nitrogen atoms suggested a purine base. A UV maximum at 262 nm matched the value of 263 nm reported for synthetic 2-chloroadenosinell, 12 and excluded chloroguanosine (~-max 249-311) 11'13 derivatives.The structure of the ribose moiety was determined by 1D and 2D NMR studies. The 1H NMR spectrum indicated the presence of two methyl groups, one a singlet and the other a doublet. The carbon chemical shift at 8 92.7 indicated that CI' is an anomeric carbon. Proton decoupling experiments correlated MeS' to H4' and H3'. HMBC experiments showed two-bond correlations of C2' to H3', and three-bond correlations of C3' to HI' and C3' to Me6'. Furthermore, C2' has a two-bond correlations to HI' and MeG contrtrrning connectivity CI' through C6'. An ether linkage between CI' and C4' completed the methylated pentose moiety. Finally, HMBC correlations of C8 to HI', C4 to HI', and CI' to H8 proved that a ribose is linked to a chlorinated adenine through N9. Complete NMR data are summarized in Table I.| # | 13C | IH | # | 13C | 1H ||-------|---------|---------|----|------|------------------|| 2 | 154.8 b | | 1' | 92.7 | 6.64 s || 4 | 151.3 | | 2' | 79.6 | || 5 | 119.7 | | 3' | 80.5 | 4.32 d 8.7 || 6 | 158.3 b | | 4' | 79.0 | 4.63 dq 8.4, 6.3 || 8 | 139.8 | 8.51 s | 5' | 18.3 | 1.68 d 6.3 || 6-NH2 | | 9.01 br | 6' | 20.9 | 1.28 s |Table I. 1H and 13C NMR Data of 1 a a in pyridine-d5b assignments are interconvertibleThe relative stereochemistry was confirmed by NOE experiments of the monoacetyl derivative (2), which are depicted in Fig. 1. [3-Configuration of the pentose was established by NOE's of H8 to Me5' and H4' to HI'. An NOE between H8 and H3 indicates that the sugar is a ribose. Kumusine (1) therefore is 2 chloro-9-(5'-deoxy-2'-methyl- ~-ribofuranosyl) - adenine.Fig. I. NOE's and relative stereochemistry of 2.Our first attempt to determine the absolute stereochemistry of kumusine by Ohtani et aL's 14 improved Mosher method failed, presumably because of steric hindrance at (23'. In an effort to produce a crystalline derivative we prepared mono-, di-, and triacetyl derivatives but none crystallized. Encouraged by selective acetylation at C3' (2) we attempted to prepare a benzoate and ap-bromobenzoate without success. Reaction withp-bromophenylisocyanate yielded the desired carbamate (3) as a white amorphous solid.15 Its CD spectrum exhibited a negative Cotton effect (Fig. 2), indicating the D-configuration for the ribose. 16Fig. 2. CD spectrum of 3.While halogenated nucleosides are common synthetic intermediates, no other naturally occurring halogenated nucleosides have been reported so far. Similarly, the alkylated sugar also represents a new structural feature in a nucleoside.Kumusine (1) showed moderate immunosuppressive activity (MLR IC50 0.195 I.tg/mL, LcV IC50 5.0 ~tg/mL, potency >256), and cytotoxicity against P388 (IC50 5.0 lag/mL), A549 (IC50 2.5 gg/mL), HT29 (IC50 5.0 ttg/mL), and CV1 (IC50 2.5 ttg/mL). 17