Synthesis of Phidianidine B, a highly cytotoxic 1,2,4-oxadiazole marine metabolite

Phidianidine B ( 1 ), a natural 1,2,4-oxadiazole linking both an indole system and an aminoalkyl guanidine group that has been recently reported from a marine mollusk, has been synthesized in seven steps (14% total yield). The synthetic procedure, which is based on the coupling of 3-indolacetic acid methyl ester and the amino-alkyl hydroxy guanidine intermediate 2 , opportunely prepared, is of general application and allows the synthesis of analogues with either different alkyl chain length or substitution on the indole ring.


Introduction
Phidianidine B (1) (Figure 1) is a natural product recently isolated in our laboratory along with the corresponding 6-bromo-derivative, phidianidine A, from the opisthobranch mollusk Phidiana militaris. 1Phidianidines revealed to be highly cytotoxic against some tumor and non-tumor cell lines and exhibited specificity towards some cell types relative to others with IC50 values within the nanomolar range.
The structure of phidianidines is characterized by the presence of a 1,2,4-oxadiazole ring representing the first report of this scaffold in a marine natural product.3][4][5][6] In fact, 1,2,4-oxadiazole is extensively utilized in the design of compounds with improved physicochemical properties and bioavailability being a bioisostere of esters and amides and a dipeptide mimetic.][9] Among the known synthetic strategies to obtain 1,2,4-oxadiazoles, 2 one of the most common routes utilizes the cyclization of a suitable amidoxime derivative (i), which can be easily prepared by reaction of a nitrile (ii) with hydroxylamine followed by reaction with an activated carboxylic substrate (iii) (Scheme 1).
With the aim at confirming the proposed structures and getting phidianidines as well as their analogues in sufficient amounts for further investigating the promising biological activity, we have performed a synthesis of phidianidine B (1). 10 According to amidoxime cyclization strategy, our synthesis is based on the coupling of 3-indolacetic acid methyl ester and a suitable N-functionalized amino alkyl hydroxy-guanidine 2. 10 As we were preparing this manuscript, two papers by Snider et al. 11 and Lindsley et al. 12 reporting the synthesis of phidianidines appeared in the literature.Both synthetic approaches are similar to that we describe here but they present some critical aspects such as the use of very toxic reagents (i.e.cyanogen bromide) 11,12 and the formation of unstable intermediates. 11Our synthetic scheme seems to be simpler and easier to run by avoiding these inconveniences.

Results and Discussion
The synthesis (Scheme 2) was firstly planned by considering two subsequent steps: (i) the formation of a 5-indol substituted 3-amino-1,2,4-oxadiazole and (ii) the alkylation of the amino residue on the oxadiazole ring with a proper alkyl moiety linking a terminal protected amino group.Although the first step was easily accomplished by coupling 3-indolacetic acid methyl ester with hydroxy guanidine, the subsequent alkylation of the amino residue on the oxadiazole was unsuccessful, even conducted under different experimental conditions.In fact, a complex inseparable mixture of N-mono-and poly-alkylated products was formed, probably due to the presence of different competing nitrogen atoms with comparable nucleophilic reactivity.A different synthetic route was then planned (Schemes 3 and 4).The key intermediate of this strategy was an N-functionalized alkyl hydroxy guanidine (2) which was prepared starting from the commercial 5-amino-1-pentanol (3, Scheme 3). 10 Compound 3 was treated with hydrogen bromide (48% HBr) to obtain the aminobromo derivative 4. 13 The subsequent introduction of the tert-butyloxycarbonyl (BOC) group on the amino function of 4 was achieved by using di-tert-butyl dicarbonate and 10 mol% of I2 in a solvent free reaction, obtaining the protected derivative 5. 14 The following addition of a N,Ndimethyl formamide solution of cyanamide and sodium amide to compound 5 gave the corresponding 1-cyanamino derivative 6.This latter compound was treated with hydroxylamine hydrochloride and sodium methoxide in anhydrous methanol leading to the key intermediate Compound 2 was allowed to react under alkaline conditions (NaH/THF) with 3-indolacetic acid methyl ester (7), which was prepared by methylation of commercial 3-indolacetic acid (Scheme 4).The coupling product 8 containing the 1,2,4-oxadiazole nucleus was first deprotected by removing t-BOC group with trifluoroacetic acid, and then guanylated by 3,5dimethyl-1-pyrazolylformaminidium nitrate. 15The final product of these reactions resulted to be identical with natural phidianidine B (1) (see Experimental Section). 1 Scheme 4. Route 2: 1,2,4-oxadiazole formation and functionalization steps.
Starting from the commercially available 6-bromo-3-indolacetic acid, the same synthetic strategy here described could be used for the preparation of phidianidine A, the bromo derivative of 1.More generally, this methodology provides a general approach to the synthesis of phidianidine analogues differing in the alkyl chain length and/or in the indole substitution pattern (Scheme 5). 10  Considering the very promising biological activity showed by natural phidianidines, 1,11,12 the preparation of a library of phidianidine-based compounds could be of great interest for SAR studies aiming to deeply understand and optimize the mode of action of these unusual marine natural products.