Synthesis and HPLC-ECD Study of Cytostatic Condensed O,N-Heterocycles Obtained from 3-Aminoflavanones

Racemic chiral O,N-heterocycles containing 2-arylchroman or 2-aryl-2H-chromene subunit condensed with morpholine, thiazole, or pyrrole moieties at the C-3-C-4 bond were synthesized with various substitution patterns of the aryl group by the cyclization of cis- or trans-3-aminoflavanone analogues. The 3-aminoflavanone precursors were obtained in a Neber rearrangement of oxime tosylates of flavanones, which provided the trans diastereomer as the major product and enabled the isolation of both the cis- and trans-diastereomers. The cis- and trans-aminoflavanones were utilized to prepare three diastereomers of 5-aryl-chromeno[4,3-b][1,4]oxazines. Antiproliferative activity of the condensed heterocycles and precursors was evaluated against A2780 and WM35 cancer cell lines. For a 3-(N-chloroacetylamino)-flavan-4-ol derivative, showing structural analogy with acyclic acid ceramidase inhibitors, 0.15 μM, 3.50 μM, and 6.06 μM IC50 values were measured against A2780, WM35, and HaCat cell lines, and apoptotic mechanism was confirmed. Low micromolar IC50 values down to 2.14 μM were identified for the thiazole- and pyrrole-condensed 2H-chromene derivatives. Enantiomers of the condensed heterocycles were separated by HPLC using chiral stationary phase, HPLC-ECD spectra were recorded and TDDFT-ECD calculations were performed to determine the absolute configuration and solution conformation. Characteristic ECD transitions of the separated enantiomers were correlated with the absolute configuration and effect of substitution pattern on the HPLC elution order was determined.


Introduction
The 3-aminoflavanone scaffold 1 is considered an efficient building block for the preparation of condensed chiral O,N-heterocycles 2-4 (Scheme 1), which contain a 2-arylchroman or

Introduction
The 3-aminoflavanone scaffold 1 is considered an efficient building block for the preparation of condensed chiral O,N-heterocycles 2-4 (Scheme 1), which contain a 2-arylchroman or 2-aryl-2Hchromene moiety fused at the C-3−C-4 bond with azine-or azole-type heterocycles. The 3aminoflavanone derivatives can be obtained by the Neber rearrangement [1] of the oxime tosylate derivative 5, readily available from flavanones 6 in two steps. The Neber rearrangement involves the conversion of the oxime tosylate of a ketone to a reactive 2H-azirine intermediate in the presence of an alkoxide base, the ring-opening of which produces an α-aminoketone [2,3]. Although oxime tosylate of flavanone rac-E were converted to 3-aminoflavanone in a Neber reaction as early as 1959 [4] and isolation of trans-3-aminoflavanone rac-C were reported [5,6], the synthetic potential of 3aminoflavanones for the preparation of condensed O,N-heterocycles has been underutilized (Scheme 1). Only the preparation of oxazoline-and imidazole-condensed derivatives rac-A and rac-B was reported with a few examples. Moreover, in this work the diastereoselectivity and side-products of the Neber reaction in the presence of an inherent C-2 chirality center of flavanones were studied further by modifying the reaction conditions and isolation of the diastereomeric products. Asymmetric organocatalytic Neber reactions of oxime tosylates producing optically active 2H-azirine derivatives have been recently reported, in which the reaction conditions are adjusted to stop the transformation at the stage of the 2H-azirine intermediates [7][8][9].
In this work, the synthesis of the target heterocycles 2-4 is carried out through the 3aminoflavanone derivatives and their antiproliferative activity was tested on WM35 melanoma and A2780 ovarian human cancer cell lines by MTT assay. Low micromolar IC50 values could be measured for several chiral racemic O,N-heterocyclic derivatives, which prompted us to separate the enantiomers with chiral HPLC, record the online HPLC-ECD spectra and determine the absolute configuration by TDDFT-ECD calculations.

Chemicals
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. The NMR spectra were recorded on Bruker Aspect 3000 ( 1 H: 360 MHz, 13 C: 90 MHz) and Bruker Avance II 400 ( 1 H: 400 MHz; 13 C: 100 MHz) spectrometers using TMS as internal standard. Chemical shifts were reported as ppm and 3 JH,H coupling constants in Hz. Chiral HPLC separation of rac-20a-g, rac-23a-g, rac-3a-g, and rac-4a-g were performed on a JASCO HPLC system with Chiralpak-IA column (5 μm, 150 × 4.6 mm, hexane/2-propanol 80:20, 90:10 eluent, respectively, 1 mL min −1 flow rate) or Chiralpak-IC column (5 μm, 250 × 4.6 mm, hexane/2-propanol 70:30 eluent, respectively, 1 mL min −1 flow rate) and HPLC-ECD spectra were recorded in stopped-flow mode on a JASCO J-810 electronic circular dichroism spectropolarimeter equipped with a 10 mm HPLC flow cell. ECD ellipticity (ϕ) values were not corrected for concentration. For an HPLC-ECD spectrum, three consecutive scans were recorded and averaged with 2 nm bandwidth, 1 s response, and standard sensitivity. The HPLC-ECD spectrum of the eluent recorded in the same way was used as background. The concentration of the injected sample was set so that the HT value did not exceed 500 V in the HT channel down to 230 nm. In this work, the synthesis of the target heterocycles 2-4 is carried out through the 3-aminoflavanone derivatives and their antiproliferative activity was tested on WM35 melanoma and A2780 ovarian human cancer cell lines by MTT assay. Low micromolar IC 50 values could be measured for several chiral racemic O,N-heterocyclic derivatives, which prompted us to separate the enantiomers with chiral HPLC, record the online HPLC-ECD spectra and determine the absolute configuration by TDDFT-ECD calculations.

Chemicals
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. The NMR spectra were recorded on Bruker Aspect 3000 ( 1 H: 360 MHz, 13 C: 90 MHz) and Bruker Avance II 400 ( 1 H: 400 MHz; 13 C: 100 MHz) spectrometers using TMS as internal standard. Chemical shifts were reported as ppm and 3 J H,H coupling constants in Hz. Chiral HPLC separation of rac-20a-g, rac-23a-g, rac-3a-g, and rac-4a-g were performed on a JASCO HPLC system with Chiralpak-IA column (5 µm, 150 × 4.6 mm, hexane/2-propanol 80:20, 90:10 eluent, respectively, 1 mL min −1 flow rate) or Chiralpak-IC column (5 µm, 250 × 4.6 mm, hexane/2-propanol 70:30 eluent, respectively, 1 mL min −1 flow rate) and HPLC-ECD spectra were recorded in stopped-flow mode on a JASCO J-810 electronic circular dichroism spectropolarimeter equipped with a 10 mm HPLC flow cell. ECD ellipticity (φ) values were not corrected for concentration. For an HPLC-ECD spectrum, three consecutive scans were recorded and averaged with 2 nm bandwidth, 1 s response, and standard sensitivity. The HPLC-ECD spectrum of the eluent recorded in the same way was used as background. The concentration of the injected sample was set so that the HT value did not exceed 500 V in the HT channel down to 230 nm. 2.3. General Procedure for Neber Rearrangement (rac-cis-1a-g, rac-trans-1a-g, 17a-g) Tosyl oxime derivatives 5a-g (8.895 mmol) were dissolved in anhydrous toluene (50 mL) under inert atmosphere and then 10.9 mL NaOEt (940 mg Na in 50 mL EtOH) was added dropwise to the solution. Stirring for 1 day at room temperature afforded an orange suspension. The suspension was filtered through cellite and washed with EtOH. Concentration of the filtrate under vacuum provided the crude product as an orange oil. Then it was dissolved in CH 2 Cl 2 and 3 N HCl solution (3 mL) was added to it. After 2 h stirring at room temperature, an orange suspension was obtained. Filtration and washing with acetone provided the cis product as white powder. Then the filtrate was thoroughly concentrated under reduced pressure and trituration with acetone afforded the trans product as off-white powder. The residue filtrate was purified after concentration by column chromatography using toluene/ethyl acetate 4:1 as eluent. The benzoxazole derivates 17a-g were obtained by this procedure.

General Procedure for the Synthesis of Flavan-4-ol Derivatives rac-19a-g and rac-22a-g
To the solution of the chloroacetamide derivatives (0.958 mmol) in MeOH (10 mL), NaBH 4 (1.150 mmol) was added and reaction mixture was stirred at room temperature. The reaction was completed in 10 min. The pH was adjusted to about 5 with 10% HCl solution and the mixture was concentrated and the residue was extracted with ethyl acetate and water. The combined organic phase was dried over MgSO 4 and the solvent was evaporated in vacuum. Column chromatography with CHCl 3 as eluent provided the pure product.    2.6. General Procedure for the Synthesis of 1,4-oxazin-3-one Derivatives rac-20a-g and rac-23a-e, g The flavan-4-ol derivative rac-19g or rac-22g (0.629 mmol) was dissolved in anhydrous THF (10 mL) under inert atmosphere. To the stirred solution, 60% dispersion of NaH (0.755 mmol) was added at room temperature. The reaction was quenched after 15 min with the addition of water. The pH was adjusted to about 5 with 10% HCl solution and then the mixture was extracted with CH 2 Cl 2 . The organic phases dried over MgSO 4 and concentrated under reduced pressure. Column chromatography using CHCl 3 as eluent provided the pure product.

General Procedure for the Synthesis of Condensed Morpholinee Derivatives
Under inert atmosphere, the condensed 1,4-oxazinone derivatives rac-20a-g or rac-23a-e, g (0.359 mmol) were dissolved in anhydrous dioxane (5 mL) and after heating the reaction mixture to 90 • C, 2 M LiAlH 4 solution in THF (216 µL) was added. The reaction was quenched after 15 min with the addition of ethyl acetate and water. The organic phase was collected and dried over MgSO 4 , then it was concentrated under reduced pressure. Procedure A: The product was obtained as the hydrochloride salt after stirring for 2 h at room temperature in a mixture of ethyl acetate (5 mL) and 3 N HCl solution (124 µL). Procedure B: The product was isolated as the amine base after column chromatography using CHCl 3 as eluent.            2.8. General Procedure for the Synthesis of Acetamide Derivatives rac-cis-24a-e,g and rac-trans-24a-g 3-Aminoflavanone hydrochloride salts rac-cis-1a-e, g or rac-trans-1a-g (0.655 mmol) were suspended in anhydrous THF (5 mL) under inert atmosphere. Under stirring, Et 3 N (230 µL, 1.64 mmol) was added to the suspension at room temperature or at 0 • C. After 10 min, acetyl chloride (56 µL, 0.786 mmol) was added dropwise to the reaction mixture and stirred for additional 10 min. Extraction with ethyl acetate and water, drying over MgSO 4 , and concentration under reduced pressure provided the crude product, which was purified by column chromatography using hexane/ethyl acetate 1:1 as eluent.

General Procedure for the Synthesis of Condensed Thiazole Derivatives 3a-g
Under inert atmosphere, acetamide derivatives rac-cis-24a-e, g or rac-trans-24a-g (0.355 mmol) and Lawesson's reagent (0.355 mmol) were dissolved in anhydrous toluene (5 mL). The mixture was stirred for 4 h at 70 • C. After cooling, toluene was evaporated and the crude product was purified by column chromatography using hexane/ethyl acetate 8:1 or toluene/ethyl acetate 8:1 as eluent, which provided the pure product.

General Procedure for the Knorr Reaction Affording the Pyrrole-Condensed Derivatives 4a-g
3-Aminoflavanone derivatives rac-cis-1a-e, g or rac-trans-1a-g (0.363 mmol) were dissolved in a mixture of 96% ethanol (4 mL) and water (2 mL). Under stirring, ethyl acetoacetate (55 µL, 0.436 mmol) and NaOAc x 3 H 2 O (300 mg, 2.178 mmol) were added to the reaction. The mixture was refluxed for 3 h and then water was added. Extraction with CH 2 Cl 2 , drying of the combined organic phases over MgSO 4 , and concentration under reduced pressure provided the crude product as orange oil. Column chromatography using toluene/ethyl acetate 4:1 with 0.1% Et 3 N as eluent and subsequent trituration in cold Et 2 O afforded the pure product.

X-Ray Diffraction Analysis
Single crystals of 17e have been obtained with the slow evaporation of its solution in chloroform. Data collection was carried out at 298 K using Mo-Kα radiation (λ = 0.71073 Å) with a Burker-Nonius MACH3 diffractometer equipped with point detector. The structure could be solved by SIR-92 program [20] and refined by full-matrix least-squares method on F 2 using the SHELX program [21]. Non-hydrogen atoms were refined anisotropically, hydrogen atoms were placed into geometric positions and methyl protons were fixed using the riding model. Publication material was prepared with the WINGX-suite [22] and publCIF software [23]. ORTEP view of the structure with selected geometric parameters are shown in Figure S342, other data are in the expected range. Further crystallographic information is compiled in Table S2. The structure is deposited in the Cambridge Crystallographic Data Centre under CCDC 2016874.

MTT Assay
The number of viable cells was indirectly determined by measuring the conversion of the tetrazolium salt MTT (3-{4,5-dimethilthiasol-2-il}-2,5-diphenyltetrasolium bromide, Sigma-Aldrich) to formazan by mitochondrial dehydrogenases. Cells were plated in 96-well multi-titer plates (10,000 cells per well density) in quadruplicates and were cultured for 3 days and treated by the compounds daily. Negative control group was treated with equal amount of vehicle solvent (DMSO) and positive control group was treated with 1 µg/mL doxorubicin. Cells were then incubated with 5 mg/mL MTT for 3 h, precipitated formazan crystals were dissolved in acidic isopropanol (10% 1 M HCl in isopropanol supplemented with 10% Triton X 100), and concentration of formazan was assessed colorimetrical way measuring absorbance at 565 nm.

MitoProbe TM DilC 1 (5) Assay and SYTOX Green Labeling
Decrease in mitochondrial membrane potential is an early hallmark of apoptosis and the disruption of the plasma membrane integrity is characteristic for cellular necrosis. These events were investigated simultaneously using MitoProbe TM DilC 1 (5) assay kit and SYTOX green (both from Molecular Probes/ThermoiFisher) staining, respectively. DilC 1 (5) (1,1 ,3,3,3 ,3 -hexamethylindodicarbo -cyanine iodide) is a fluorescent cyanine dye which penetrates cytoplasm with intact membrane and accumulates primarily in mitochondria depending on the mitochondrial membrane potential. Since decrease in mitochondrial membrane potential is an early marker of apoptosis, the DilC 1 (5) staining intensity is typically decreased in apoptotic cells. SYTOX Green is a nucleic acid stain impermeant to live cells with intact plasma membrane, but it penetrates the compromised membrane of necrotic, dead cells resulting in a bright green fluorescent nuclear staining.
Cells were plated in 96-well multi-titer plates (10,000 cells per well density) in quadruplicates and were incubated for 24 h with various concentrations of rac-19g. Negative control group was treated with equal amount of vehicle solvent (DMSO) and positive control groups were shortly treated with 50 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or lysis buffer (20 mM Tris HCl, 5 mM EDTA in H 2 O) to disrupt mitochondrial membrane potential or to lyse cells and disrupt membrane integrity, respectively. Then supernatant were removed and cells were incubated with DilC 1 (5) and SYTOX Green following the manufacturer's protocol. Finally, the excess of the dyes was removed, cells were gently washed in PBS, and fluorescence of DilC 1 (5) and SYTOX Green was measured at 630/680 nm and 490/520 nm (excitation/emission), respectively, using a FlexStation 3 (Molecular Devices) multimodal microplate reader.

CyQUANT ® Cell Proliferation Assay
CyQUANT assay assesses the cellular proliferation indirectly by directly determining the DNA content in a cell population using CyQUANT fluorescent dye. The dye exhibits strong fluorescence enhancement when bound to cellular DNA and the fluorescent intensity is proportional to the amount of bound DNA in a high dynamic range. Therefore, it is suitable to asses DNA synthesis associated with cellular proliferation. Cells were plated in 96-well multi-titer plates (10,000 cells per well density) in quadruplicates and were incubated for 24 h with various concentrations of rac-19g, then assayed for cellular proliferation following the manufacturer's protocol. Briefly, supernatants were gently removed and the plate was snap frozen and stored at −70 • C. Then plate was thawed, cells were lysed and incubated with CyQUANT dye. The excess of the dye was removed and fluorescence was measured at 490/520 nm (excitation/emission) using a FlexStation 3 (Molecular Devices) multimodal microplate reader.

Computational Methods
Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl 3 [24]. Geometry reoptimizations were carried out at the B3LYP/6-31+G(d,p) level in vacuo, the CAM-B3LYP/TZVP [25] and the ωB97X/TZVP [26] levels with the PCM solvent model for CHCl 3 . TDDFT ECD calculations were run with various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set as implemented in the Gaussian 09 package with the same or no solvent model as in the preceding DFT optimization step [27]. ECD spectra were generated as sums of Gaussians with 1500-3000 cm −1 widths at half-height, using dipole-velocity-computed rotational strength values [28]. Ball-and-stick representations of the conformers were generated by using the Molekel software [29].
The oxime tosylates of cyclic ketones are common starting materials of the Neber rearrangement reaction, in which NaOEt or KOEt is generally used as a base in dry EtOH or benzene, followed by acidic hydrolysis to produce the hydrochloric salt of the α-aminoketone [2]. In enantioselective organocatalytic Neber rearrangements, in which the isolation of the optically active 2H-azirine derivative is needed, reactive oxime tosylates were reacted with thiourea [7,9] or cinchona organocatalysts [30] in the presence of an inorganic base and there was no subsequent acidic hydrolysis. In our experiments, we treated the oxime tosylates 5a-g with NaOEt base in dry toluene for one day at room temperature and performed the acidic hydrolysis with 3N HCl solution for two hours on the CH2Cl2 solution of the concentrated filtrate. This condition resulted in the formation of both the cis-(rac-cis-1a-e, g) and trans-3-aminoflavanone (rac-trans-1a-g) derivatives, which could be readily separated and isolated in the work-up procedure (Table 1). 2 -Hydroxyacetophenone (13) was reacted with seven different arenecarbaldehydes in a Claisen-Schmidt condensation reaction to produce the corresponding chalcones 15a-g, which were transformed to racemic flavanone analogues 6a-g in a biomimetic intramolecular oxa-Michael cyclization. The flavanones 6a-g were converted to the oximes 16a-g with NH 2 OH·HCl, which were tosylated to afford the oxime tosylates 5a-g.
The oxime tosylates of cyclic ketones are common starting materials of the Neber rearrangement reaction, in which NaOEt or KOEt is generally used as a base in dry EtOH or benzene, followed by acidic hydrolysis to produce the hydrochloric salt of the α-aminoketone [2]. In enantioselective organocatalytic Neber rearrangements, in which the isolation of the optically active 2H-azirine derivative is needed, reactive oxime tosylates were reacted with thiourea [7,9] or cinchona organocatalysts [30] in the presence of an inorganic base and there was no subsequent acidic hydrolysis. In our experiments, we treated the oxime tosylates 5a-g with NaOEt base in dry toluene for one day at room temperature and performed the acidic hydrolysis with 3N HCl solution for two hours on the CH 2 Cl 2 solution of the concentrated filtrate. This condition resulted in the formation of both the cis-(rac-cis-1a-e, g) and trans-3-aminoflavanone (rac-trans-1a-g) derivatives, which could be readily separated and isolated in the work-up procedure (Table 1).
After the acidic hydrolysis, the crude product was dissolved in CH 2 Cl 2 and 3 N HCl solution (3 mL) was added to it and the resultant orange suspension was filtered and the solid was washed with acetone that afforded the hydrochloride salt of the pure rac-cis-1a-e, g as white powder. The filtrate was concentrated and triturated with acetone to produce the hydrochloride salt of rac-trans-1a-g as off-white powder. The values of the 3 J 2-H,3-H coupling constants were found in the range of 5.2-5.6 Hz for rac-cis-1a-e, g, while in the range of 12.4-12.6 Hz for trans-1a-g. The residue was concentrated and purification by column chromatography provided the 2-styrylbenzoxazole side-products 17a-g with 10-20% yield, which were obtained by ring-opening of the γ-pyrone ring and intramolecular cyclization of the phenolic hydroxyl group on the intermediate of the Beckmann rearrangement. The planar structure of 17e was also confirmed by single crystal X-ray diffraction analysis (see Figure S332 and Table S2 for details). The formation of similar 2-styrylbenzoxazoles was also reported in the Beckmann reaction of flavanones through the trans-chalcone oximes [31]. The cis-(rac-cis-1a-e, g) and trans-3-aminoflavanones (rac-trans-1a-g) were isolated in 1:1 ratio with a C-2 phenyl substituent (1a) and with an approximate two-fold excess of the trans isomer with other C-2 aryl groups except for 1f, where only the rac-trans-1f was obtained (Table 1). In the reported Neber rearrangements of flavanones, the acidic hydrolysis step was performed for a longer period of time and surprisingly the trans isomer of 2-aminoflavanone could be isolated as the single product [5,6]. The formation of both cisand trans-3-amino-2-methylchroman-4-one was described in the Neber reaction of the O-(p-tolylsulfonyl)oxime of 2-methylchroman-4-one [32], and cis diastereoselectivity was reported in the Neber reaction of a 2-aryl-4-piperidone derivative [33]. Our finding suggested that the cis-3-aminoflavanone derivatives rac-cis-1a-e, g formed initially through the corresponding 2H-azirine (F → cis-G → cis-H) either diastereoselectively or together with the trans isomer (F → trans-G → trans-H) and then the acidic hydrolysis promoted the conversion of the cis isomer to the trans one by enolization-induced epimerization of the α-aminoketones 1a-g (Scheme 3). Table 1. Yields of the products obtained in the Neber reaction of 5a-g. i: (1) NaOEt, dry toluene, rt, 1 d.
The oxime tosylates of cyclic ketones are common starting materials of the Neber rearrangement reaction, in which NaOEt or KOEt is generally used as a base in dry EtOH or benzene, followed by acidic hydrolysis to produce the hydrochloric salt of the α-aminoketone [2]. In enantioselective organocatalytic Neber rearrangements, in which the isolation of the optically active 2H-azirine derivative is needed, reactive oxime tosylates were reacted with thiourea [7,9] or cinchona organocatalysts [30] in the presence of an inorganic base and there was no subsequent acidic hydrolysis. In our experiments, we treated the oxime tosylates 5a-g with NaOEt base in dry toluene for one day at room temperature and performed the acidic hydrolysis with 3N HCl solution for two hours on the CH2Cl2 solution of the concentrated filtrate. This condition resulted in the formation of both the cis-(rac-cis-1a-e, g) and trans-3-aminoflavanone (rac-trans-1a-g) derivatives, which could be readily separated and isolated in the work-up procedure (Table 1). 5f C-2 aryl: 1-naphthyl trans-1f (64) 17f (10) 0:1 7 5g C-2 aryl: 2-naphthyl cis-1g+trans-1g (69) 17g (11) 1:2.2 e a sum isolated yield of diastereomers cisand trans-1a-g, b isolated yield of the benzoxazole side-products 17a-g, c ratio of diastereomers cisand trans-1 as determined from the isolated yields, d not determined, since it could not be isolated as a single component by column chromatography, e determined by NMR.
If the acidic treatment was maintained for a long time, all the cis-3-aminoflavanones were transformed to the lower energy trans isomer by enolization at C-2 as reported in literature examples [5,6]. With bulky C-2 aryl group such as the 1-naphthyl one in 1f, only the trans product was isolated even with our procedure (Table 1). 5f C-2 aryl: 1-naphthyl trans-1f (64) 17f (10) 0:1 7 5g C-2 aryl: 2-naphthyl cis-1g+trans-1g (69) 17g (11) 1:2.2 e a sum isolated yield of diastereomers cis-and trans-1a-g, b isolated yield of the benzoxazole sideproducts 17a-g, c ratio of diastereomers cis-and trans-1 as determined from the isolated yields, d not determined, since it could not be isolated as a single component by column chromatography, e determined by NMR.
After the acidic hydrolysis, the crude product was dissolved in CH2Cl2 and 3 N HCl solution (3 mL) was added to it and the resultant orange suspension was filtered and the solid was washed with acetone that afforded the hydrochloride salt of the pure rac-cis-1a-e, g as white powder. The filtrate was concentrated and triturated with acetone to produce the hydrochloride salt of rac-trans-1a-g as off-white powder. The values of the 3 J2-H,3-H coupling constants were found in the range of 5.2-5.6 Hz for rac-cis-1a-e, g, while in the range of 12.4-12.6 Hz for trans-1a-g. The residue was concentrated and purification by column chromatography provided the 2-styrylbenzoxazole side-products 17a-g with 10-20% yield, which were obtained by ring-opening of the γ-pyrone ring and intramolecular cyclization of the phenolic hydroxyl group on the intermediate of the Beckmann rearrangement. The planar structure of 17e was also confirmed by single crystal X-ray diffraction analysis (see Figure  S332 and Table S2 for details). The formation of similar 2-styrylbenzoxazoles was also reported in the Beckmann reaction of flavanones through the trans-chalcone oximes [31]. The cis-(rac-cis-1a-e, g) and trans-3-aminoflavanones (rac-trans-1a-g) were isolated in 1:1 ratio with a C-2 phenyl substituent (1a) and with an approximate two-fold excess of the trans isomer with other C-2 aryl groups except for 1f, where only the rac-trans-1f was obtained (Table 1). In the reported Neber rearrangements of flavanones, the acidic hydrolysis step was performed for a longer period of time and surprisingly the trans isomer of 2-aminoflavanone could be isolated as the single product [5,6]. The formation of both cis-and trans-3-amino-2-methylchroman-4-one was described in the Neber reaction of the O-(ptolylsulfonyl)oxime of 2-methylchroman-4-one [32], and cis diastereoselectivity was reported in the Neber reaction of a 2-aryl-4-piperidone derivative [33]. Our finding suggested that the cis-3aminoflavanone derivatives rac-cis-1a-e, g formed initially through the corresponding 2H-azirine (F → cis-G → cis-H) either diastereoselectively or together with the trans isomer (F → trans-G → trans-H) and then the acidic hydrolysis promoted the conversion of the cis isomer to the trans one by enolization-induced epimerization of the α-aminoketones 1a-g (Scheme 3). If the acidic treatment was maintained for a long time, all the cis-3-aminoflavanones were transformed to the lower energy trans isomer by enolization at C-2 as reported in literature examples [5,6]. With bulky C-2 aryl group such as the 1-naphthyl one in 1f, only the trans product was isolated even with our procedure (Table 1).
The same synthetic scheme was also utilized for the preparation of stereoisomeric rac-2a-e, g target molecules starting from the rac-cis-1a-e, g. When the acylation reaction rac-cis-1a-e, g → rac-cis-21a-e, g was performed at room temperature (Scheme 5), partial epimerization occurred at C-3 and the thermodynamically more stable rac-trans-18a-e, g formed as the main product instead of the expected rac-cis-21a-e, g.
The α-amino-ketone moiety of rac-1a-g was also used to build condensed thiazole and pyrrole units at the C-3−C-4 bond, and the resultant heterocycles were tested for antiproliferative activities on human cancer cell lines. For the synthesis of thiazole-condensed heterocycles, the amino group of rac-cis-1a-e, g or rac-trans-1a-g was acetylated with acetyl chloride and the Lawesson reagent was utilized for the cyclization (Scheme 6). In the case of cis-1a-e, g, the acetylation reaction was carried out at 0 • C for 15 min, which could preserve the cis relative configuration of rac-cis-24a-e, g. Although the C-3 chirality center was lost during the cyclization, both the cis-1a-e, g and rac-trans-1a-g were cyclized separately to the thiazole-condensed derivatives rac-3a-g.
The cyclization of the trans-N-acetyl derivatives rac-trans-24a-g provided consistently higher yields (Table S1) than those of the corresponding rac-cis-24a-e, g.
units at the C-3−C-4 bond, and the resultant heterocycles were tested for antiproliferative activities on human cancer cell lines. For the synthesis of thiazole-condensed heterocycles, the amino group of rac-cis-1a-e, g or rac-trans-1a-g was acetylated with acetyl chloride and the Lawesson reagent was utilized for the cyclization (Scheme 6). In the case of cis-1a-e, g, the acetylation reaction was carried out at 0 °C for 15 min, which could preserve the cis relative configuration of rac-cis-24a-e, g. Although the C-3 chirality center was lost during the cyclization, both the cis-1a-e, g and rac-trans-1a-g were cyclized separately to the thiazole-condensed derivatives rac-3a-g. The cyclization of the trans-N-acetyl derivatives rac-trans-24a-g provided consistently higher yields (Table S1) than those of the corresponding rac-cis-24a-e, g.

Antiproliferative Activity
The antiproliferative activity of the morpholine-, thiazole-, and pyrrole-condensed derivatives and their precursors were evaluated initially against A2780 ovarian and WM35 melanoma cancer cell lines at 50 μM concentration by monitoring at 24 and 72 h (Figures S262 and S263). While morpholine and pyrrole units are common structural elements in cytotoxic compounds of synthetic or natural origin [35][36][37][38][39], there are fewer reports available on cytotoxic condensed thiazole derivatives [40,41]. The N-chloroacetyl-3-amino-flavan-4-ol derivatives rac-19a-g and 22a-g exhibited strong antiproliferative activity regardless the stereochemistry against both cell lines at 50 μM concentration, while the related N-acetyl derivatives rac-cis-24a-e, g or rac-trans-24a-g were inactive or they had much weaker activity. This suggested that the N-chloroacetyl derivatives act as alkylating agents and the chloroacetyl moiety is essential for the activity. The N-chloroacetyl-3-amino-flavanone derivatives rac-trans-18a-g and rac-trans-21a-g had usually weaker activity than the corresponding flavan-4-ol derivatives rac-19a-g and rac-22a-g, which suggested that the reduction of the C-4 carbonyl group to hydroxyl improved the antiproliferative activity. The IC50 value was determined for the most active N-chloroacetyl derivative rac-19g using the MTT assay, which was found 0.15 and 3.5 μM against A2780 and WM35 cancer cell lines ( Figures S264 and S265), respectively ( Table 2). Against the non-cancerous HaCaT human keratinocytes, 6.06 μM IC50 value was measured, which implies a remarkable 50-fold selectivity.

Antiproliferative Activity
The antiproliferative activity of the morpholine-, thiazole-, and pyrrole-condensed derivatives and their precursors were evaluated initially against A2780 ovarian and WM35 melanoma cancer cell lines at 50 µM concentration by monitoring at 24 and 72 h ( Figures S262 and S263). While morpholine and pyrrole units are common structural elements in cytotoxic compounds of synthetic or natural origin [35][36][37][38][39], there are fewer reports available on cytotoxic condensed thiazole derivatives [40,41]. The N-chloroacetyl-3-amino-flavan-4-ol derivatives rac-19a-g and 22a-g exhibited strong antiproliferative activity regardless the stereochemistry against both cell lines at 50 µM concentration, while the related N-acetyl derivatives rac-cis-24a-e, g or rac-trans-24a-g were inactive or they had much weaker activity. This suggested that the N-chloroacetyl derivatives act as alkylating agents and the chloroacetyl moiety is essential for the activity. The N-chloroacetyl-3-amino-flavanone derivatives rac-trans-18a-g and rac-trans-21a-g had usually weaker activity than the corresponding flavan-4-ol derivatives rac-19a-g and rac-22a-g, which suggested that the reduction of the C-4 carbonyl group to hydroxyl improved the antiproliferative activity. The IC 50 value was determined for the most active N-chloroacetyl derivative rac-19g using the MTT assay, which was found 0.15 and 3.5 µM against A2780 and WM35 cancer cell lines ( Figures S264 and S265), respectively ( Table 2). Against the non-cancerous HaCaT human keratinocytes, 6.06 µM IC 50 value was measured, which implies a remarkable 50-fold selectivity.
Following 24 hrs incubation, rac-19g decreased the mitochondrial membrane potential [DilC1 (5) staining], which is an early hallmark of apoptosis (Figure 2a), and inhibited proliferation-associated DNA synthesis (CyQUANT Assay) of A2780 ovarian carcinoma cells (Figure 2c). However, the integrity of the plasma membrane was found to be intact (negative SYTOX Green staining) arguing against necrotic cytotoxic effect of the compound (Figure 2b).  Figure 2. Effect of rac-19g on A2780 ovarian carcinoma cells. Cells were incubated with the indicated concentration of rac-19g and assayed to investigate (a) mitochondrial membrane potential using DilC1(5) staining, (b) plasma membrane integrity using SYTOX Green labeling, and (c) total DNA content using CyQUANT assay as described in the materials and methods. As positive control, CCCP and lysis were used to disrupt mitochondrial membrane potential and plasma membrane integrity, respectively. Data are presented as mean ± SEM, N = 4 at each data point presented.
Interestingly, rac-19g, containing a 1,3-oxygenated-2-N-chloroacetylaminopropane subunit as part of the flavanol scaffold, may be considered a cyclic analogue of irreversible acid ceramidase (AC) inhibitors such as SACLAC and SOCLAC (Figure 3) [42]. SACLAC and SABRAC were found to inhibit the growth of chemoresistant forms of prostate cancer [43] and to reduce the viability of acute myeloid leukemia cells with an EC50 of approximately 3 μM across 30 human cell lines [44]. Effect of rac-19g on A2780 ovarian carcinoma cells. Cells were incubated with the indicated concentration of rac-19g and assayed to investigate (a) mitochondrial membrane potential using DilC 1 (5) staining, (b) plasma membrane integrity using SYTOX Green labeling, and (c) total DNA content using CyQUANT assay as described in the materials and methods. As positive control, CCCP and lysis were used to disrupt mitochondrial membrane potential and plasma membrane integrity, respectively. Data are presented as mean ± SEM, N = 4 at each data point presented.
Interestingly, rac-19g, containing a 1,3-oxygenated-2-N-chloroacetylaminopropane subunit as part of the flavanol scaffold, may be considered a cyclic analogue of irreversible acid ceramidase (AC) inhibitors such as SACLAC and SOCLAC (Figure 3) [42].  Following 24 hrs incubation, rac-19g decreased the mitochondrial membrane potential [DilC1(5) staining], which is an early hallmark of apoptosis (Figure 2a), and inhibited proliferation-associated DNA synthesis (CyQUANT Assay) of A2780 ovarian carcinoma cells (Figure 2c). However, the integrity of the plasma membrane was found to be intact (negative SYTOX Green staining) arguing against necrotic cytotoxic effect of the compound (Figure 2b).  Figure 2. Effect of rac-19g on A2780 ovarian carcinoma cells. Cells were incubated with the indicated concentration of rac-19g and assayed to investigate (a) mitochondrial membrane potential using DilC1(5) staining, (b) plasma membrane integrity using SYTOX Green labeling, and (c) total DNA content using CyQUANT assay as described in the materials and methods. As positive control, CCCP and lysis were used to disrupt mitochondrial membrane potential and plasma membrane integrity, respectively. Data are presented as mean ± SEM, N = 4 at each data point presented.
Interestingly, rac-19g, containing a 1,3-oxygenated-2-N-chloroacetylaminopropane subunit as part of the flavanol scaffold, may be considered a cyclic analogue of irreversible acid ceramidase (AC) inhibitors such as SACLAC and SOCLAC (Figure 3) [42]. SACLAC and SABRAC were found to inhibit the growth of chemoresistant forms of prostate cancer [43] and to reduce the viability of acute myeloid leukemia cells with an EC50 of approximately 3 μM across 30 human cell lines [44]. SACLAC and SABRAC were found to inhibit the growth of chemoresistant forms of prostate cancer [43] and to reduce the viability of acute myeloid leukemia cells with an EC 50 of approximately 3 µM across 30 human cell lines [44].
From the morpholine-condensed derivatives, the rac-(4aS*,5R*,10bR*)-2b and rac-(4aS*,5R*,10bS*)-2d seemed to be the most active at 50 µM concentration (Figures S262 and S263) but their IC 50 values were found larger than 10 µM against both A2780 and WM35 cell lines. The thiazole-condensed derivative rac-3e containing a C-4 3,4,5-trimethoxyphenyl substituent had the best activities (2.74 and 2.14 µM IC 50 ) among the prepared condensed O,N-heterocycles ( Table 2). The type of the C-4 aryl substituent had significant effect on the antiproliferative activity, since the thiazole derivatives rac-3a,b,c,g had much weaker activities at 50 µM concentration. All the pyrrole-condensed derivatives rac-4a-g had distinct antiproliferative activity at 50 µM concentration and low micromolar IC 50 values were measured for 4b, 4c, and 4g in the range of 2.95-9.37 µM (Table 2). Similarly to compound 11 (Figure 1) [14], 4b, 4c, and 4g may be viewed as simplified analogues of cytotoxic lemallarins such as lemallarin C [17], in which there is a substituted pyrrole-condensed 2H-chromene unit instead of the pyrrole-condensed coumarine heterocyclic core of lemallarins. Although the condensed derivatives were effective in decreasing the viability of the A2780 and WM35 cancer cell lines, they did not display remarkable selectivity toward the cancer cell lines when compared their potency to non-cancerous HaCaT human keratinocytes ( Table 2).

Stereochemical Analysis
The antiproliferative activity of our condensed chiral O,N-heterocycles prompted us to separate the enantiomers with HPLC using chiral stationary phase, measure the online HPLC-ECD spectra, and determine the absolute configuration (AC) by TDDFT-ECD calculations. The online HPLC-ECD approach aided with ECD calculations was proven an efficient method for the stereochemical analysis of scalemic or racemic mixtures of bioactive natural products [45,46] or synthetic derivatives [47,48]. The enantiomers of rac-3e and rac-4g were separated on Chiralpak IA column using hexane/2-propanol 80:20 as eluent and even partial separation of the enantiomers was sufficient to record mirror-image online HPLC-ECD spectra, which were not optimized further (Figure 4). From the morpholine-condensed derivatives, the rac-(4aS*,5R*,10bR*)-2b and rac-(4aS*,5R*,10bS*)-2d seemed to be the most active at 50 μM concentration ( Figures S262 and S263) but their IC50 values were found larger than 10 μM against both A2780 and WM35 cell lines. The thiazolecondensed derivative rac-3e containing a C-4 3,4,5-trimethoxyphenyl substituent had the best activities (2.74 and 2.14 μM IC50) among the prepared condensed O,N-heterocycles ( Table 2). The type of the C-4 aryl substituent had significant effect on the antiproliferative activity, since the thiazole derivatives rac-3a,b,c,g had much weaker activities at 50 μM concentration. All the pyrrolecondensed derivatives rac-4a-g had distinct antiproliferative activity at 50 μM concentration and low micromolar IC50 values were measured for 4b, 4c, and 4g in the range of 2.95-9.37 μM (Table 2). Similarly to compound 11 (Figure 1) [14], 4b, 4c, and 4g may be viewed as simplified analogues of cytotoxic lemallarins such as lemallarin C [17], in which there is a substituted pyrrole-condensed 2Hchromene unit instead of the pyrrole-condensed coumarine heterocyclic core of lemallarins. Although the condensed derivatives were effective in decreasing the viability of the A2780 and WM35 cancer cell lines, they did not display remarkable selectivity toward the cancer cell lines when compared their potency to non-cancerous HaCaT human keratinocytes ( Table 2).

Stereochemical Analysis
The antiproliferative activity of our condensed chiral O,N-heterocycles prompted us to separate the enantiomers with HPLC using chiral stationary phase, measure the online HPLC-ECD spectra, and determine the absolute configuration (AC) by TDDFT-ECD calculations. The online HPLC-ECD approach aided with ECD calculations was proven an efficient method for the stereochemical analysis of scalemic or racemic mixtures of bioactive natural products [45,46] or synthetic derivatives [47,48]. The enantiomers of rac-3e and rac-4g were separated on Chiralpak IA column using hexane/2propanol 80:20 as eluent and even partial separation of the enantiomers was sufficient to record mirror-image online HPLC-ECD spectra, which were not optimized further (Figure 4). Except for 4e (Chiralpak IC, hexane/2-propanol 70:30), the same HPLC condition was utilized to separate the enantiomers of the related 3a-d, f, g, and 4a-f, which afforded a base-line separation of ECD spectrum of (R)-3e had the best agreement ( Figure 5c) and thus (R) AC was determined for the first-eluting enantiomer of 3e. The HPLC-ECD spectra of the thiazole-condensed derivatives 3a-e had the same ECD profile ( Figures S311-S320), on the basis of which the AC of the separated enantiomers could be assigned. The configurational assignment was also confirmed by the TDDFT-ECD calculation of 3a ( Figures S340 and S341), which determined (R) AC for the first-eluting enantiomer of 3a. Interestingly, the enantiomers of 3c, containing a C-4 3,4-dimethoxyphenyl substituent, showed reversed elution order under the same HPLC condition, which was evident from the mirror-image HPLC-ECD spectrum of the first-eluting enantiomer ( Figure S316). TDDFT-ECD calculations were performed to determine the AC for the enantiomers of the thiazole-condensed derivatives 3f and 3g with 1-and 2-naphthyl substituents, which were expected to influence both the ECD spectra and the chiral separation. The 2-naphthyl group of (R)-3g adopted an axial orientation in all the three low-energy ωB97X/TZVP PCM/CHCl 3 conformers (Figure 5b) and the computed ECD spectra reproduced well the experimental HPLC-ECD spectrum of the first-eluting enantiomer, for which (R) AC was assigned (Figure 5d). The first-eluting enantiomer of 3f had completely different HPC-ECD spectrum from those of 3a-e, g with overlapping negative CEs and broad transitions in the range of 350-210 nm ( Figure S322). Although ECD calculations of (R)-3f could not produce a perfect agreement because of the improper estimation of the conformational ensemble ( Figures S342 and S343), the AC of the first-eluting enantiomer was determined as (R).
The DFT re-optimization of the initial 31 MMFF conformers of (R)-4g afforded 18 ωB97X/TZVP PCM/CHCl 3 low-energy conformers above 1% population, which differed in the orientation of the C-1 ethyloxycarbonyl and the C-4 2-naphthyl substituents (Figure 6a and Figure S344). The 2-naphthyl group had an equatorial arrangement in 13 computed conformers with a total population of 79.2%, while the axial conformer was represented by 5 conformers with 20.3% sum population ( Figure S344).
The computed ECD spectra of the equatorial and axial conformers were markedly different and the intense negative CE at 222 nm and the positive one at 207 nm derived from the axial conformers, since the equatorial conformers had different transitions in this region. The Boltzmann-weighted ECD spectra of (R)-4g reproduced well the experimental HPLC-ECD spectrum of the first-eluting enantiomer with negative CEs above 250 nm (Figure 6c), on the basis of which (R) AC was assigned to it. Similarly, (R) AC was deduced for the first-eluting enantiomer of 4f containing a 1-naphthyl group by the ECD calculation, although it could not reproduce well the 225 nm negative CE, which possibly derived from one of the equatorial conformers with underestimated population (Figures S345 and S346). The HPLC-ECD spectra of the first-eluting enantiomers of 4a-e were quite similar ( Figures S325-S334) and 4a was used as a reference compound to determine the AC for the separated enantiomers with aryl substitution pattern. In the ωB97X/TZVP PCM/CHCl 3 low-energy conformers of (R)-4a (Figure 6b and Figure S348), the equatorial conformers were the dominant with 83.2% total population (7 conformers), while the axial conformers had 16.7% sum population (3 conformers). The good agreement of the Boltzmann-weighted ECD spectrum of (R)-4a with that of the first-eluting enantiomer allowed assigning the AC of first-eluting enantiomer with negative CEs above 260 nm as (R) (Figure 6d and Figure S347). Since the first-eluting enantiomers of 4b-e had same HPLC-ECD profile ( Figures S327-334), their ACs were determined as (R), which means that the HPLC elution order did not change with the different substitution patterns of 4a-g.
Enantiomers of rac-20a-g and rac-23a-g, containing a morpholin-3-one residue condensed with a flavan moiety, were also separated on Chiralpak IA column using hexane/2-propanol as eluent, which provided base-line separation for most of the molecules (Figures S285-S310). Mirror-image HPLC-ECD spectra were recorded for the separated enantiomers, the long-wavelength 1 L b transition of which could be correlated with the absolute configuration and it could be used to check the validity of the chroman [50] or flavan helicity rule [51,52] on conformationally rigid condensed flavan derivatives with three chirality centers. The (4aR,5S,10bS)-20a was selected as a reference compound for the 20a-g series and the solution TDDFT-ECD protocol was performed on it to determine its absolute configuration independently from the helicity rule. The CAM-B3LYP/TZVP PCM/CHCl 3 re-optimization of the initial single MMFF conformer of (4aR,5S,10bS)-20a provided one low-energy conformer, in which the 4a-H, 5-H and 10b-H protons adopted axial orientation in agreement with the 9.2 and 10.4 Hz values for the 3 J 4a-H,10b-H and 3 J 4a-H,5-H coupling constants (Figure 7a). spectra reproduced well the experimental HPLC-ECD spectrum of the first-eluting enantiomer, for which (R) AC was assigned (Figure 5d). The first-eluting enantiomer of 3f had completely different HPC-ECD spectrum from those of 3a-e, g with overlapping negative CEs and broad transitions in the range of 350-210 nm ( Figure S322). Although ECD calculations of (R)-3f could not produce a perfect agreement because of the improper estimation of the conformational ensemble ( Figures S342 and  S343), the AC of the first-eluting enantiomer was determined as (R).
The condensed 2H-3,4-dihydropyran ring had a half-chair conformation with M-helicity as defined by the negative value (−41.0 • ) of the torsional angle ω C-6a, O-6, C-5,C-4a (Figure 7a,c). Positive CE was found for the highest wavelength ECD band in the CAM-B3LYP/TZVP PCM/CHCl 3 ECD spectrum of (4aR,5S,10bS)-20a, which agreed well with the positive 1 L b band [282sh (4.70, 274 (5.26)] observed in the HPLC-ECD spectrum of the second-eluting enantiomer (Figure 7b). Thus the positive 1 L b band CE of (4aR,5S,10bS)-20a derives from M-helicity of the condensed flavan chromophore, which corroborates well the flavan semi-empirical helicity rule. For the configurational assignment of the separated enantiomers of 20b-g, the sign of the long-wavelength 1 L b band CE was considered, since in contrast to the higher wavelength benzene transitions such as the 1 L a , this is not expected to change with the different substitution of the C-5 aryl group [51]. The first-eluting enantiomer of 20a,b,f, and g had negative 1 L b band CE, on the basis of which their ACs were assigned as (4aS,5R,10bR), while the positive 1 L b band CE of the first-eluting enantiomers of 20c-e derived from (4aR,5S,10bS) AC ( Figures S285-S298). The presence of a C-3 methoxy substituent, which is missing from 20a,b changed the elution order of the enantiomers on the Chiralpak IA column for 20c-e. from 20a,b changed the elution order of the enantiomers on the Chiralpak IA column for 20c-e.
The separated enantiomers of rac-23a-g had all cis relative configuration and hence they differed in the AC of C-4a from the corresponding 20a-g derivatives. Compound (4aR,5R,10bR)-23a was selected for TDDFT-ECD calculation. The CAM-B3LYP/TZVP PCM/CHCl3 re-optimization of the initial MMFF conformer of (4aR,5R,10bR)-23a provided only one low-energy conformer, in which the 5-H and 10b-H had axial orientation, while the 4a-H adopted equatorial one (Figure 8a).

Conclusions
The Neber rearrangement of seven oxime tosylates of flavanone analogues, containing a C-2 aryl substituent with different substitution pattern or a 1-or 2-naphthyl group, resulted in trans-3-aminoflavanones as the major product and the cis diastereomer as the minor one. The cis diastereomers could be obtained by simple filtration from the reaction mixture, while the trans isomers were isolated in pure form by trituration with acetone. The formation of 2-styrylbenzoxazol side-products was also observed, which were produced by ring-opening of the γ-pyrone ring and intramolecular cyclization of the Beckmann rearrangement intermediate. The cisand trans-2-aminoflavanones were utilized for cyclization reactions to condense the 2-aryl-chroman or -2H-chromene subunit with morpholine, thiazole, or pyrrole moieties at the C-3-C-4 bond. Three diastereomers of morpholine-condensed 2-aryl-chromans, containing three chirality centers, were prepared through the N-chloroacetyl derivatives in four steps. Seven thiazole-condensed derivatives with different C-2 substituents were produced by the cyclization of the N-acetyl derivatives with Lawesson's reagent and seven pyrrole-condensed one in the Knorr cyclization. Antiproliferative activities of condensed heterocycles and precursors were evaluated against A2780 and WM35 cancer cell lines at 50 µM concentration and IC 50 values were determined for the best ones with MTT assay. One of the 3-(N-chloroacetylamino)-flavan-4-ol derivatives, containing a C-2 2-naphthyl substituent and showing analogy with acid ceramidase inhibitors, had 0.15 µM IC 50 value against the A2780 cell line. This decrease in the viability is associated with an increase in apoptotic markers and decreased proliferation (DNA synthesis) but not with cellular necrosis. The IC 50 values against HaCat and WM35 cell lines were found to be 6.06 and 3.50 µM, respectively, which implies 50-and 20-fold selectivities compared to that against A2780. From the condensed heterocycles, the thiazole derivative containing a 3,4,5-trimethoxy substituent had the best activity with 2.72 and 2.14 µM IC 50 values against the A2780 and WM35 cell lines. Four pyrrole-condensed derivatives, which may be viewed as simplified analogues of natural lamellarins, had IC 50 values below 10 µM down to 2.95 µM. Enantiomers of the condensed heterocycles were separated by chiral HPLC, HPLC-ECD spectra were recorded and TDDFT-ECD calculations were carried out to determine the AC of the enantiomers. The configurational assignment may aid future stereoselective synthesis and exploration of stereochemistry-activity relationships.