Graduate Thesis Or Dissertation
 

Precursor-directed Biosynthesis and Total Synthesis of Aniline-containing Natural Products from the Endophytic Fungus Chalara sp. 6661

Public Deposited

Downloadable Content

Download PDF
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/wp988s72p

Descriptions

Attribute NameValues
Creator
Abstract
  • Chalaniline A, an unusual xanthone-like aminofulvene with modest cytotoxicity, and chalaniline B [1-anilino-2,8-dihydroxy-3-(hydroxymethyl)xanthone], an antibacterial aminoxanthone, were previously isolated by Loesgen and coworkers from vorinostat-treated Chalara sp. 6661. Of note, the anilino moiety found within both chalaniline structures originates from biotransformation of the HDAC inhibitor vorinostat (SAHA, suberanilohydroxamic acid) by the endophytic fungus. The phenomenon observed, in which an epigenetic agent ostensibly elicits the generation of new secondary metabolites while also providing a subunit of the same, warrants further investigation as do the novel bioactive chemotypes presented by chalanilines A and B. Accordingly, the work described herein concerned exploration of chemical and biological aspects of the chalanilines. The first part of the thesis (Chapter 2) describes further investigation of the Chalara sp. 6661 mediated biotransformation of vorinostat that leads to chalaniline A. Initially, the process was optimized for chalaniline A production and the range of metabolites resulting from it were analyzed in detail by LCMS; this work led to the identification of a cyclopentadienyl formaldehyde that is formally the result of hydrolysis of the enamine moiety of chalaniline A. Chalaniline A was submitted to the National Cancer Institute (NCI) for analysis against its standard 60 cancer cell line panel; no selective cytotoxic nor cytostatic activity was observed. In an effort to generate new chalaniline A analogs and to glean the scope of the biotransformation process, a range of modified vorinostat analogs with varied amido substituents were synthesized and incubated with Chalara sp. 6661. Vorinostat analogs [R1R2NC(=O)(CH2)6C(=O)NHOH] were successfully prepared from methylsuberate by carbodiimide (EDC) mediated amide formation with 19 different amines R1R2NH [XC6H4NH2: X = 3-F, 4-F, 4-Cl, 3-Br, 4-Br, 2-I, 3-OMe, 4-MeO, 3-CN, 3-CF3, 4-C2H; XYC6H3NH2: X,Y = 3,4-(MeO)2, 2,4-(MeO)2, 2-OMe-4-Cl, 2-Me-3-NO2, 2-OMe-6-Me; PhEtNH; 1-aminonaphthaline, propargylamine] followed by hydroxylaminolysis (60-75% yield, 2 steps). Precursor-directed biosynthesis using large scale (2 L) cultures of Chalara sp. 6661 from five of the vorinostat analogs prepared [ArNHC(=O)(CH2)6C(=O)NHOH: Ar = 3-FC6H4, 4-FC6H4, 3-MeOC6H4, 4-MeOC6H4, 1-naphthyl] resulted in wholly successful chalaniline A production with isolated yields in the range of 1-4 mg L–1. The molecular structures of the newly isolated chalaniline A analogs were verified in each case using a full range of spectrometric (HR ESIMS) and spectroscopic (UV, IR, 1H NMR, 13C NMR, COSY, HSQC, HMBC) analyses. Inhibition of cell viability by the five new chalaniline A analogs was evaluated using an MTT assay; however, no inhibition of viability was observed against colon (HCT-116) or melanoma (SK-MEL-5) cancer cell models. The same five compounds were also found to lack antimicrobial activity at a dose of 128 g mL–1 against four Gram-positive bacteria (S. aureus ATCC 25923; methicillin-resistant S. aureus ATCC BAA-41, multidrug-resistant S. aureus ATCC BAA-44, and E. faecium ATCC 49032), two Gram-negative bacteria (P. aeruginosa ATCC 15422, E. coli ATCC 8739) as well as the yeast Candida albicans (ATCC 90027). Finally, during the course of the aforementioned work, two new metabolites were isolated from the Chalara cultures: a desanilino chalaniline B analog [2,8-dihydroxy-3-(hydroxymethyl)xanthone] and a new isofusidienol-type compound. The second part of the thesis (Chapter 3) describes the development and successful execution of a total synthesis of chalaniline B via a route that is amenable to the potential generation of analogous substances. The synthetic approach is composed of two phases: (i) generation of a 2,8-dioxygenated-1,3-dihaloxanthone template, and (ii) regioselective conversion of the template into chalaniline B by amination (anilination) at C1 and hydroxymethylation at C3. An initial attempt to prepare the template via a conventional xanthone synthesis from fully oxidized precursors foundered when it proved impossible to effect Friedel-Crafts acylation of 3,5-dichloro- or 3,5-dibromo-4-methoxyphenol with 2,6-dimethoxybenzoyl chloride (related transformations, e.g., Fries rearrangement, also failed). A successful elaboration of 1,3-dibromo-2,8-dimethoxyxanthone was realized from 2-hydroxyxanthone by a series of regioselective oxidations, viz: bromination at C1 and C3 by classical electrophilic aromatic substitution (Br2, AcOH, rt, 74%) and then, following methyl ether formation at C2-OH (MeI, NaH, DMF, rt, 35%), the introduction of a hydroxyl group at C8 via ketone directed palladation/oxidation using palladium(II) trifluoroacetate in combination with iodosobenzene bis(trifluoroacetate) (DCE, 85 °C, 62%). Further to methylation of the newly introduced C8-OH (MeI, NaH, DMF, rt, 81%), various Pd(0)-catalyzed cross-coupling conditions were explored to introduce the requisite hydroxylmethyl group at C3 using Migita's reagent (MOMOCH2SnBu3), but these Stille-type processes uniformly favored the unwanted reaction at C1. The regioselectivity of a Buchwald-Hartwig-type reaction of 1,3-dibromo-2,8-dimethoxyxanthone with aniline was found to be ligand dependent and the desired C1 amination selectivity was realized using a combination of tetrakis(triphenylphosphine)-palladium as catalyst and cesium carbonate as base (PhMe, 100 °C, 16 h; isolated yield C1 anilino product 48%). The synthesis was concluded by demethylation of the two ether moieties (BBr3, CH2Cl2, rt, 79%) and Stille cross-coupling with (hydroxymethyl)tributylstannane [Pd(PPh3)4, PhMe, 80 °C] which yielded chalaniline B (22%) together with its deshydroxymethyl analog, 1-anilino-2,8-dihydroxyxanthone (44%). Antimicrobial activities for chalaniline B and 1-anilino-2,8-dihydroxy-xanthone were determined. Chalaniline B exhibited weak activity against a panel of Gram-positive bacteria when tested at a single dose of 128 μg mL–1, but its deshydroxymethyl analog showed potent activity against both methicillin-resistant S. aureus (ATCC# BAA-41) and Bacillus subtilis (ATCC# 49343), with MIC values of 8 μg mL–1 (25 μM) in each case.
License
Resource Type
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Rights Statement
Publisher
Peer Reviewed
Language
Embargo reason
  • Ongoing Research
Embargo date range
  • 2021-12-02 to 2024-01-03

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details KhoshbakhtMahsa2021.pdf 2021-12-02 Public Download