New Substituted Isocoumarins and Dihydroisocoumarins and their Cytotoxic Activities

New isocoumarins were prepared in an efficient way from 2-iodobenzoic acid derivatives and hept-1-yne in a Sonogashira reaction, followed by spontaneous cyclization. Catalytic hydrogenation gave the corresponding dihydroisocoumarins. A 4-chloroisocoumarin was prepared on an alternative pathway. Some of the new compounds showed moderate cytotoxic activities against a human leukemia cell line (HL 60).


Introduction
Isocoumarins are a class of natural lactones with high structural diversity exhibiting a broad spectrum of biological activities [1]. Considerable work has been published over decades about their chemistry [2] and biology [3], and a number of natural and synthetic isocoumarins have been shown to exhibit significant cytotoxic and antitumor activities. Among these are numerous isocoumarins bearing various types of substituents (alkyl, alkenyl or aryl groups) at 3-position, as well as the dihydroisocoumarin AI-77-B (A, Figure 1). The latter one was first isolated from Bacillus pumilus, and shows in vitro cytotoxicity against human malignant A375-S2 and cervical cancer HeLa cells [4]. The paraphaeosphaerins (B) from cultures of Paraphaeosphaeria quadriseptana [5] are biogenetically related to the cytotoxic plant metabolites monocillin I and radicicol. NM-3 (C) Sci Pharm. 2011; 79: 21-30 is a synthetic analogue of cytogenin (E), and potentiates antineoplastic effects of other chemotherapeutic agents and inhibits angiogenesis [6]. This compound is in phase I clinical trials. The isocoumarin 185322 (D), an analogue of NM-3, is an inhibitor of microtubule assembly, and induces mitotic arrest and apoptosis of multiple myeloma cells [7]. Ochratoxin A (F), a mycotoxin from Aspergillus ochraceus shows nephrotoxic, hepatotoxic, carcinogenic and teratogenic properties in animals [8]; recently the European Pharmacopoeia introduced a limit test for ochratoxin A in plant material (Method 2.8.22). Scoparine A (G) was isolated from the North African medical plant Pituranthos scoparius [9].
The biological activities of the abovementioned and other isocoumarins and dihydroisocoumarins [10,11]   In a previous work [12] we prepared a number of isocoumarins and dihydroisocoumarins bearing alkyl and hydroxyalkyl residues at C-3 using a one-pot procedure originally described by Lia and Cheng [13]. Since these products showed only moderate cytotoxic activities, we assumed that additional functional groups at the bicyclic core should be of great importance. An isocoumarin with an isopentyl residue at C-3 was the most cytotoxic one in the previous investigations. This prompted us to investigate the influence of additional lipophilic substituents like halogens (cf.

Chemistry
The isocoumarins 2a-2d were prepared in poor to good yields in one pot reactions starting from commercially available 2-iodobenzoic acid derivatives 1a-d and hept-1-yne involving Sonogashira cross coupling reactions in the presence of zinc chloride [13]. The isocoumarins 2a, 2b [14] and 2d were further hydrogenated under palladium catalysis to give the corresponding dihydroisocoumarins 3a, 3b and 3d (Scheme 1). Hydrogenation of 2c did not appear promising due to the risk of reductive dechlorination.

Biology
The resulting isocoumarins and dihydroisocoumarins were tested for their cytotoxic activity in a MMT assay using the HL 60 leukemia cell line [18,19]. The results are shown in Table 1. Since we had found some correlation of the cytotoxic activities with lipophilicity in previous investigations on isocoumarins [12], we also calculated the log P values of the new compounds.

Discussion
Among the five isocoumarins and three dihydroisocoumarins described here, four compounds, the fluorinated isocoumarin 2b and the dihydroisocoumarins 3a, 3b and 3d, showed measurable, albeit very poor cytotoxic activity. No clear tendency for higher acitivity of one of the two structural subtypes (2 versus 3) can be deduced from these results. Since the log P values of all tested compounds are in the same range, influences of lipophilicity on cytotoxic activity cannot be deduced as well.
For obtaining isocoumarins with significant cytotoxicity the presence of polar groups at the ring system seems to be indispensible.

General procedure II: Preparation of dihydroisocoumarins 3a,b,d
Pd on charcoal (10%) was added to a solution of the isocoumarin in methanol (20 mL). The suspension was stirred for 12 h under H 2 atmosphere at ambient pressure, then the catalyst was filtered off, and the residue was washed with methanol. The combined organic layers were evaporated, and the residue was purified by FCC (n-hexane/ethyl acetate).