Chrysosporazines A–E: P-Glycoprotein Inhibitory Piperazines from an Australian Marine Fish Gastrointestinal Tract-Derived Fungus, Chrysosporium sp. CMB-F214

Abstract

Chemical analysis of Chrysosporium sp. CMB-F214, yielded five new piperazines, chrysosporazines A–E (1–5), with structures assigned by spectroscopic and X-ray analyses and biosynthetic considerations. The chrysosporazines 2–5 exist as an equilibrium of major and minor N-acyl rotamers, while 1–3 incorporate an unprecedented hexahydro-6H-pyrazino­[1,2-b]­isoquinolin-6-one scaffold. The noncytotoxic chrysosporazines reverse doxorubicin drug resistance in P-glycoprotein overexpressing colon carcinoma cells (SW620 Ad300), with 2 delivering a comparable gain in sensitivity to the positive control, verapamil.

Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214

“…ROESY correlations between N-COCH 3 and Ha-1 in 3 permitted assignment of a Z configuration about the major acetamide rotamer (Figure 4), while excellent concordance among key 1 H NMR resonances in 3 (δ H 4.21,ddd,9.8,5.4,3.8 Hz,4.46,d,5.4 Hz,5.89,dd,5.7,1.3 Hz, and 12 (δ H 4.20,ddd,8.5,8.0,3.9 Hz,4.39,d,8.0 Hz,5.86,dd,5.5,1.3 Hz, supported a common relative configuration. This, together with biosynthetic considerations and the fact the absolute configuration for 12 had been prior confirmed by X-ray analysis [4], permitted assignment of the structure for azachrysosporazine B1 ( 3 1 and 2, Tables S4 and S12, Figures S19-S24) with 13 revealed many similarities, including the presence of major and minor acetamide rotamers (ratio 1:0.2), with key differences attributed to replacement of the C-3" to C-3 cyclised N-benzoyl moiety in 13, with a C-3" to C-3 cyclised N-nicotinoyl moiety in 4. The N-4" regiochemistry in 4 was evident from the contiguous nature and significant deshielding of H-5" (δ H 8.60, dd, 4.7, 1.8 Hz) and H-7" (δ H 8.18, dd, 7.8, 1.8 Hz) relative to H-6" (δ H 7.45, dd, 7.8, 4.7 Hz), as well as diagnostic 2D NMR correlations (Figure 4).…”

“…2,10.4,3.1 Hz,4.36,d,12.2 Hz, suggested a common diaxial re ration. These observations, together with biosynthetic considerations and t solute configuration for 11 had been prior confirmed by X-ray analysis [4] signment of the structures for azachrysosporazines A1 (1) and A2 (2) as sho HRESI(+)MS measurement on 3 returned a molecular formula (C28H25N 0.5) consistent with an aza analogue of chrysosporazine B (12) (C29H26N2O6 of the 1D NMR (DMSO-d6) data for 3 (Tables 1-2, Tables S3 and S11, Figures 12 revealed many similarities, including the presence of major and minor a mers (ratio 1:0.3), with key differences attributed to replacement of the C-3″ N-benzoyl moiety in 12, with a C-3″ to C-3 cyclised N-nicotinoyl moiety regiochemistry in 3 was evident from the contiguous nature and significa of H-5″ (δH 8.64, dd, 4.7, 1.8 Hz) and H-7″ (δH 8.18, dd, 7.8, 1.8 Hz) relative to dd, 7.8, 4.7 Hz), as well as diagnostic 2D NMR correlations (Figure 4). ROES between N-COCH3 and Ha-1 in 3 permitted assignment of a Z configura major acetamide rotamer (Figure 4), while excellent concordance among key 1 and 2, Tables S3 and S11, Figures S13-S18) with 12 revealed many similarities, including the presence of major and minor acetamide rotamers (ratio 1:0.3), with key differences attributed to replacement of the C-3" to C-3 cyclised N-benzoyl moiety in 12, with a C-3" to C-3 cyclised N-nicotinoyl moiety in 3.…”

“…ROESY correlations between N-COCH 3 and H-2 in 4 and 5 (in common with 13) permitted assignment of a E configuration for the major acetamide rotamers (Figure 4), with excellent concordance among key 1 H NMR resonances in 4 (δ H 3.88,ddd,11.4,10.0,3.8 Hz,4.49,d,10.0 Hz, H-3; 4.56, dd, 13.4, 1.1 Hz, Ha-1 ; 3.00, m, Hb-1 ; 4.25, m, H-2 ), 5 (δ H 3.91,ddd,11.2,11.0,3.9 Hz,4.46,d,11.2 Hz,4.56,dd,13.5,1.2 Hz,2.95,dd,13.5,4.1 Hz,4.27,m, and 13 (δ H 3.85,ddd,11.0,11.0,3.9 Hz,4.38,d,11.0 Hz,4.58,dd,13.5,1.2 Hz,2.95,dd,13.5,4.2,4.25,m, supporting a common relative configuration. This, together with biosynthetic considerations, permitted assignment of the structures for azachrysosporazines C1 (4) and C2 ( 5 S64 and Scheme S3) that co-eluted with an authentic sample of the piperazine 16 (Figure 5) previously prepared and fully characterised following acid hydrolysis of 14 [4]. These observations, together with biosynthetic considerations, permitted assignment of the structure for azachrysosporazine D1 (6) as shown.…”

“…These observations, together with biosynthetic considerations, permitted assignment of the structure for azachrysosporazine D1 (6) as shown. Scheme S3) that co-eluted with an authentic sample of the piperazine 16 (Figure 5) previously prepared and fully characterised following acid hydrolysis of 14 [4]. These observations, together with biosynthetic considerations, permitted assignment of the structure for azachrysosporazine D1 (6) as shown.…”

Methods in Microbial Biodiscovery

“…E. Fish: Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a Brisbane, Queensland, market-purchased fish, returned an new class of phenyl propanoid piperazine, including chrysosporazine F ( 11 ), exhibiting inhibitory activity against the multidrug efflux pump P-Glycoprotein with a potency > 2-fold that of the positive control verapamil ( Figure 2 ) [ 12 , 13 ].…”