Nodulisporic Acids D-F: Structure, Biological Activities, and Biogenetic Relationships Sheo B. Singh,* John G. Ondeyka, Hiranthi Jayasuriya, Deborah L. Zink, Sookhee N. Ha, Arlene Dahl-Roshak, Joyce Greene, Jennifer A. Kim, McHardy M. Smith, Wesley Shoop, and Jan S. Tkacz Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065 Received May 3, 2004 Nodulisporic acids D, E, and F (7-12) are the newest members of a family of nontremorogenic indole- diterpenoids that are potent, orally bioavailable, antiflea agents derived from a fungus belonging to the genus Nodulisporium. The four members of the D series (7a-10a) are each devoid of an isoprene residue that is present at C-26 in the three nodulisporic acids described originally (the A series, 1a-3a). Nodulisporic acid E (11a) has a simpler structure, which lacks not only the isoprene residue at C-26 but also two that form the A/B rings. Nodulisporic acid F (12) is the simplest of all nodulisporic acids and is devoid of all three isoprene residues of the indole unit; as such, it represents the earliest biosynthetic intermediate in this series. A biogenetic grid based on mutation studies is proposed that encompasses all the known nodulisporic acids. Structure-activity relationships of the known natural nodulisporic acids have been elucidated. Within a series the most active compound possesses a dienoic acid chain, and overall, the end product of the biogenetic grid, i.e., nodulisporic acid A (1a), exhibits the most potent antiflea activity. Additionally, the stereochemistries of C-3′′ and C-4′′ of nodulisporic acid D 2 (9a) and therefore of nodulisporic acids A 2 (3a), B 2 (3b), and C 2 (6) have been assigned. As vectors of viral or bacterial pathogens, ectoparasites such as fleas represent a significant health hazard to companion animals and to the humans who maintain them. Although numerous insecticides are currently marketed for flea control, development of resistance is an issue that makes continued discovery of new chemical entities a necessity. Also, many of these antiflea drugs are applied topically, which can cause wide variability in efficacy as well as environmental problems. Therefore, new systemic drugs, preferably with novel mechanisms of action, are needed. Nodulisporic acid A (1a) is an indole-diterpenoid that was discovered in 1992 and was first reported by us in 1997 as a potent insecticidal agent. 1,2 It was purified from an endophytic fungus, Nodulisporium sp. (MF5954), by bio- assay-guided isolation using a mosquito-larval assay. Subsequent studies demonstrated that it was an effective systemic ectoparasiticidal agent against fleas on dogs with no apparent mammalian toxicity. 3 Like the avermectins and milbemycins, nodulisporic acid A modulates the glutamate-gated chloride channel in insects, but has no effect on helminths. 4-6 Nodulisporic acid A does not affect other related chloride channels present in both insects and mammals. To date, no report of nodulisporic acid-related vertebrate toxicity has appeared. While nodulisporic acid A exhibited good in vitro and in vivo activity against fleas, its potency and pharmacokinetic properties did not justify its development as a drug, so therefore a medicinal chemistry effort was initiated to optimize the profile of the lead. Chemical modifications were performed on most of the accessible sites, particularly the dienoic acid side chain, leading to a substantial improvement in activity; 7-13 the effort has been briefly reviewed. 14,15 In parallel with the medicinal chemistry efforts, we sought to find congeners and/or natural analogues of nodulisporic acid A, from the original producer as well as variants derived from it by chemical mutagenesis, in the hope of providing compounds that may not be accessible by the chemical derivatization. This process led to the discovery of nodulisporic acids A 1 (2a), A 2 (3a), 16 B(1b), B 1 (2b), B 2 (3b), 17 C(4), C 1 (5), and C 2 (6). 18 A continuation of this work with mutant Nodulisporium strains has led to the isolation of early biosynthetic intermediates named herein nodulisporic acids D (7a), D 1 (8a), D 2 (9a), D 3 (10), and E (11a) from ATCC74473, nodulisporic acid F (12) from MF6518, and A 4 (13), ∆ 23 -A 4 (14), and ∆ 23 -C 4 (15) from two other mutant cultures. Like nodulisporic acids of the B and C series, some of these compounds were detected in the extracts of the original producer but in quantities too low for purification. Details of the isolation, structure elucida- tion, and biological activities of these compounds are described in this paper. In addition, we propose a biogenetic grid of all known nodulisporic acids based on the products accumulated in various mutants of the original producer. Results and Discussion Isolation of Nodulisporic Acids. Mycelia from a filtered culture of Nodulisporium sp. mutant MF6227 (ATCC74473) were extracted with methyl ethyl ketone. The extract was subjected to chromatography on a reversed- phase HPLC, affording nodulisporic acids D (7a, 43 mg/ L), D 1 (8a, 70 mg/L), D 2 (9a, 80 mg/L), D 3 (10a, 30 mg/L), and E (11a, 130 mg/L) as pale powders. At acidic pH (in TFA), particularly at higher concentrations, the ∆ 18,19 -olefin tended to isomerize to ∆ 18,22 -olefin and thus complicated the purification. Methylation with diazomethane followed by purification by reversed-phase HPLC at neutral pH led to the isolation of the corresponding methyl esters 7b- 11b. This extract did not contain nodulisporic acids A-C (1-6). Nodulisporic acid F (12) was similarly isolated from fermentations of MF5954 and was the only nodulisporic acid produced by MF6518. Nodulisporic acid A 4 (13) was purified from other mutants MF6265 and MF6225. The MEK extraction of the mycelia of a mutant culture of Nodulisporium sp. followed by two successive reversed- phase HPLC steps afforded ∆ 23 -nodulisporic acid A 4 (14, 18 mg/L). Silica gel chromatography of the same extract * Corresponding author. Fax: (732) 594 6880. E-mail: sheo_singh@ merck.com. 1496 J. Nat. Prod. 2004, 67, 1496-1506 10.1021/np0498455 CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 07/31/2004