A Facile Synthesis of Cytogenin (8-Hydroxy-3- hydroxymethyl-6-methoxyisocoumarin) Aamer Saeed * Department of Chemistry, Quaid-I-Azam University 45320, Islamabad Pakistan E-mail: aamersaeed@yahoo.com A facile and straightforward synthesis of cytogenin 1 (8-hydroxy-3-hydroxymethyl-6-methoxyiso- coumarin) metabolite of Streptoverticillium eurocidicum and Certocystis fimbriata has been achieved. Condensation of chloroacetyl chloride with 3,5-dimethoxyhomophthalic acid 2 directly furnished 3- chloromethyl-6,8-dimethoxyisocoumarin 3 which was hydrolyzed to 6,8-dimethoxy-3-hydroxymethyliso- coumarin 4 using 0.05% aqueous sodium hydroxide in THF. Regioselective demethylation of 4 yielded cyto- genin 1. In model experiments, 3-chloromethylisocoumarin was prepared and converted into 3-hydroxy- methyl isocoumarin. J. Heterocyclic Chem., 41, 975 (2004). Introduction. Cytogenin (8-hydroxy-3-hydroxymethyl-6-methoxy- isocoumarin) (1; Figure 1) is a natural isocoumarin first isolated in 1990 from Streptoverticillium eurocidicum MI43-37F11 strain identified as FERM BP-2783 and was given the name of antibiotic MI43-37F11 [1,2]. In 1994, it was reported as a metabolite of Certocystis fimbriata, a pathogenic ascomycete that causes black rot in sweet potato along with the related isocoumarin derivatives e.g. 6,8-dihydroxy-3-hydroxymethylisocoumarin and 6,8- dihydroxy-3-methylisocoumarin [3]. The latter are also metabolites of blue-stain fungi C. minor and C. ulmi, responsible for pine beetle disease and Dutch elm disease respectively [4]. Cytogenin is the first natural iso- coumarin to exhibit anticancer activity against experimen- tal tumor cells and human cancer cells: Leukemia L1210, mouse Leukemia P388 and Leukemia EL4, mouse IMC carcinoma, human lung cancer LX-1 cells and against Erhlich carcinoma in mice at 6.3 to 100 mg/kg/day, while exhibiting low cytotoxicity. It also possesses an activity to enhance the production of interleukin-1 in vivo in a mam- malian [1]. The antitumor and antirheumatoid arthritis effects possessed by cytogenin are due to its anti-angio- genic activity. Angiogenesis is the process for generation of new blood vasculature, involved in a variety of normal biological functions, as well as disease states such as rheumatoid arthritis, psoriasis, diabetic retinopathy, can- cer and solid tumors [5]. The inhibition of angiogenesis deprives the tumors of nutrients by inhibiting the genera- tion of microvasculature. Cytogenin is a novel oral antian- giogenic agent which suppresses angiogenesis induced by Sarcoma-180 tumor cells [6], reduced antitumor effector activity of spleen cells taken from tumor bearing mice in vitro [7] and was found to modify collagen-induced arthri- tis in mice [8,9]. It activates macrophages to produce monokines such as IL-1 alpha and stimulates proliferation and differentiation of T cells resulting in production of lymphokines such as IFN gamma and GM-CSF. Cytogenin has low cytotoxicity on murine and human tumor cells in vitro and a potent inhibitory effect on spon- taneous polyarthritis in MR/1 mice and pristine-induced arthritis in DBA/1J mice and its mode of action is differ- ent from those of NSAIDs [10]. Various 3-side chain modified analogues of cytogenin have been synthesized and tested among which NM-3 showed the best pharma- cological properties [8]. Biosynthetically, the carbon skeleton of cytogenin is derived from pentaketide intermediate due to head to tail condensation of five acetate units and methyl group of the 6-methoxyl is derived from methionine [11]. The great medicinal importance coupled with simple structure makes cytogenin an attractive target for syn- thesis. The patented synthesis by Hirano et al. [12] involved five steps starting from 8-hydroxy-6-methoxy- 3-methyl- isocoumarin, a natural product, itself synthe- sized through a number of steps. Taylor et al. [13] have recently reported an improved synthesis of cytogenin. In this approach, the benzylic anion of ethyl 2-ethoxymethoxy-4-methoxy-6-methylbenzoate gener- ated with LDA, was quenched with benzylglycolate. Proton transfer was a competing reaction, and the con- densation provided the α-hydroxyketone. TBS protec- tion of the primary hydroxyl followed lactonization under basic conditions. Finally, deprotection with hydrochloric acid provided the cytogenin. An efficient synthesis of cytogenin was undertaken as a continuation of our interest towards synthesis of anti- tumor metabolites [14] and naturally occurring iso- coumarins [15]. Nov-Dec 2004 975