Patent Application
20240122894
The invention relates to a method for making flavonoid dimers, trimers and oligomers, to flavonoid dimers and trimers, and their use in the treatment of diseases.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Flavonoids are a diverse class of plant secondary metabolites that have a range of bioactivity. The flavonoid core structure features a 15-carbon phenyl-chromone motif and is a privileged structure for drug discovery. The structural diversity of flavonoids stems from variable substituent positions of the phenyl, variable numbers and positions of phenol groups on the aromatic rings, number and degree of glycosylation, and formation of flavonoid dimers and oligomers.
While flavone monomers are relatively abundant in fruits and vegetables and can be extracted on an industrial scale from agri-food byproducts, flavonoid dimers and oligomers are minor components in plant biomass and are not economical to obtain in large quantities from natural sources. Synthetically, catalyzed C—C bond coupling reactions, such as Ullman reaction and Suzuki-Miyaura coupling, have been employed to make a limited number of biflavones with moderate overall yields. These high-temperature reactions suffered from major drawbacks due to their usage of toxic heavy metals, wasteful halogens and boronate by-products, and the requirement of protective groups for phenolic functional groups. The reactions also involve multiple steps which introduce additional complications and reduce yields. Therefore, these methods are not environmentally friendly and are unsuitable for large scale synthesis, or synthesis of food grade products. Furthermore, the lab synthesis of triflavonoids remains relatively unexplored.
There is therefore a need for a new method for producing flavone dimers, trimers and oligomers. Such methods may enable the production of entirely new compounds that may have useful therapeutic activity.
The invention solves the problems in the art by providing a method for coupling flavonoid compounds under advantageously mild conditions with environmentally friendly reagents. The reactions may be performed as one-pot syntheses with high yields and regioselectivity using simple bases under mild aqueous conditions, e.g. at room temperature. These conditions are compatible with a wide range of functional groups that may be present on the flavonoids, and can be used to obtain food-grade products in an environmentally friendly way. The method of the invention may be used to make a wide range of flavonoids that were previously impossible to prepare in a lab setting.
Compounds prepared according to the invention have useful activity as antifungal agents and inhibitors of starch hydrolase.
Thus, the invention provides the following numbered clauses.
or wherein the flavonoid-containing compound comprises a dimer or trimer thereof.
The word “comprising” refers herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa.
The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.
The invention provides a method for coupling a flavonoid-containing compound, said method comprising:
As used herein, a “flavonoid-containing compound” is a compound that contains a flavonoid carbon skeleton, which compound may be substituted with additional carbon-containing functional groups. A flavonoid carbon skeleton comprises a benzene ring fused to a 6-membered heterocyclic ring containing an oxygen atom, which heterocyclic ring is bonded to a phenyl ring by a C—C bond:
The carbon atoms of the 6-membered heterocyclic ring containing an oxygen atom that do not form part of the fused benzene ring may be connected by single or double bonds as shown in the structure above (provided that any one carbon atom in the heterocyclic ring forms part of only one double bond). The oxygen atom may be bonded to the non-aromatic carbon atom by a single or double bond. When this is a double bond, the oxygen atom will have a positive charge, and the compound will comprise a balancing negative charge or a counterion.
Flavonoids may contain a wide range of functional groups bonded to the above skeleton, for example one or more (e.g. one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one or two, or one) substituents selected from the group consisting of hydroxyl, methoxy, glycosyl, alkoxy, NO2, F, CN, SH, CF3, Cl, Br, I, ═O, ═CH2, C1-18 alkyl, C1-18 fluoroalkyl, C2-18 alkenyl, —O—C(O)—R (where R represents a C1-22 alkyl group), prenyl, phytyl, exocyclic C3-6 cycloalkyl, exocyclic C5-6 cycloalkenyl, phenyl, phenoxyl, C1-18 N-aklylaminyl, and C1-18 N,N-dialkylaminyl.
Specific examples of flavonoid backbones that may be present in flavonoid-containing compounds include the following:
and dimers and trimers thereof. As will be appreciated by a person skilled in the art, in this context a dimer or trimer thereof includes dimers/trimers formed from two/three of the same backbone, or formed from two/three different backbones.
In specific embodiments of the invention, the flavonoid may be selected from the group consisting of luteolin, apigenin, diosmetin, chrysin, wogonin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, a dimer formed from two of the foregoing, and a trimer formed from three of the foregoing. In other words, the flavonoid-containing compound may represent an optionally substituted flavonoid selected from the group consisting of luteolin, apigenin, diosmetin, chrysin, wogonin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, a dimer formed from two of the foregoing, and a trimer formed from three of the foregoing.
In specific embodiments of the invention, the flavonoid-containing compound may comprise a flavone backbone. In some embodiments of the invention the flavonoid-containing compound may be a monomer. In some embodiments of the invention the flavonoid-containing compound may be a dimer. In some embodiments of the invention the flavonoid-containing compound may be a trimer.
When the flavonoid-containing compound is a trimer, the coupling reaction of the invention may be an intramolecular reaction to provide a cyclic trimer. Alternatively (or in addition), the coupling reaction of the invention may couple a trimer with another flavonoid-containing compound, such as a monomer, to produce a tetramer.
The method involves contacting the flavonoid-containing compound with a base selected from the group consisting of a metal carbonate, a metal hydroxide and a base of the formula R4NOH, where each R independently represents H or C1-4 alkyl. Examples of bases that may be useful in embodiments of the invention include one or more of the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, caesium hydroxide, sodium carbonate, potassium carbonate, and alkaline water. As set out in the below Examples, the nature of the counterion of the base can have an effect on the yield provided by the reaction. Therefore, in some embodiments it may be preferable for the base to be selected from the group consisting of alkali metal hydroxides. In further embodiments, the base may be selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide and caesium hydroxide. In yet further embodiments, the base may be selected from the group consisting of sodium hydroxide and potassium hydroxide. In a specific embodiment, the base may be potassium hydroxide.
The base results in an alkaline reaction mixture. The reaction mixture may have a pH of from about 10 to about 14, such as from about 11 to about 13.
For the avoidance of doubt, it is explicitly contemplated that the end point of any range herein may be combined with any end point of another range for the same variable. Thus, for the above pH ranges, the invention explicitly contemplates the following pH ranges: from about 10 to about 11; from about 10 to about 13; from about 10 to about 14; from about 11 to about 13; from about 11 to about 14; and from about 13 to about 14.
Step (ii) of the method is performed in a sealed reaction vessel. This enables the amount of oxygen to which the reaction mixture is exposed to be controlled. If insufficient oxygen is present then the coupling reaction will not run to completion, and provide unsatisfactory yields. However, if too much oxygen is present then over-oxidation may occur. For this reason, the invention involves the use of a sealed reaction vessel, where from 10 to 95% of the volume of the reaction vessel is occupied by air (when assessed at 25° C. and a pressure of 101 kPa). In some embodiments of the invention, air may occupy from 30 to 90% (e.g. from 40 to 80%) of the volume of the reaction vessel at 25° C. and a pressure of 101 kPa. As will be appreciated by a person skilled in the art this temperature and pressure refer to the conditions for assessing the volume occupied by air in the reaction vessel, and do not limit the temperature or pressure at which the reaction may take place.
For similar reasons, the method of the invention may in some embodiments be performed without stirring (or without agitating the reaction mixture). Stirring the reaction mixture increases contact between the reaction mixture and air in the reaction vessel, increasing the rate at which additional oxygen is dissolved into the reaction mixture—and possibly leading to over-oxidation. The inventors surprisingly found that improved yields may be obtained when the method is performed in a sealed reaction vessel without stirring.
The reaction mixture comprises water. Water may advantageously be used as a solvent because it is environmentally friendly, cheap and safe. All of the reagents used in the method are stable in water, and the use of water allows the production of food-grade products. Nevertheless, a skilled person will understand that the method would be possible with other solvents.
Step (ii) of the method may be performed under a range of temperatures, for example from 0° C. to 50° C. (provided the reaction mixture does not freeze at 0° C.). In specific embodiments of the invention step (ii) of the method may be performed at a temperature of from 15 to 30° C., such as from 18 to 25° C. Step (ii) may also be performed for a varying length of time, for example from about 1 to about 20 hours, from about 5 to about 15 hours, such as about 8 to about 12 hours.
The invention provides certain flavonoid compounds selected from the group consisting of
and pharmaceutically acceptable salts thereof.
Such compounds may be prepared from their constituent monomers/dimers using the method of the invention. In the case of the cyclic trimers, they may be made from the acyclic trimers, which may themselves be made from their constituent monomers/dimers using the method of the invention.
The dimers and trimers made according to the invention may be useful in methods of medical treatment. Thus, the invention also provides:
In the above medical uses (1), (2) and (3) according to the invention, the compound may be a compound according to the invention. For example, the compound may be a compound selected from the group consisting of
and pharmaceutically acceptable salts thereof.
The compound may also be a compound selected from the group consisting of:
and pharmaceutically acceptable salts thereof.
In specific embodiments of the above medical uses (1), (2) and (3) according to the invention, the compound may be selected from dicranolamin, 3′″-Desoxydicranolomin, distichumtriluteolin, and pharmaceutically acceptable salts thereof. For example, the compound may be selected from dicranolamin, 3′″-Desoxydicranolomin, and pharmaceutically acceptable salts thereof. The compound may be dicranolamin or a pharmaceutically acceptable salt thereof.
The term “disorder or condition ameliorated by the inhibition of starch hydrolase” will be understood by those skilled in the art to include hyperglycaemia, diabetes and obesity.
Particular disorders or conditions that may be mentioned in relation to the aspects of the invention described hereinbefore include fungal infections (e.g. candida infection), hyperglycaemia, diabetes and obesity.
For the avoidance of doubt, in the context of the present invention, the term “treatment” includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.
The terms “patient” and “patients” include references to mammalian (e.g. human) patients. As used herein the terms “subject” or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
For the avoidance of doubt, references herein (in any aspect or embodiment of the invention) to flavonoid-containing compounds includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound disclosed herein with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound disclosed herein in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
Examples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric acids.
Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
As mentioned above, also encompassed by flavonoid-containing compounds are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of compounds of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.
For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
“Pharmaceutically functional derivatives” of flavonoid-containing compounds as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of the invention. A particular example of prodrugs of compounds of the invention that comprise at least one hydroxy group is an ester derivative, such as an acetate ester derivative.
Nevertheless, the term “prodrug” of a relevant compound disclosed herein includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
Prodrugs of compounds disclosed herein may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds disclosed herein wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound disclosed herein is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.
Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).
Compounds disclosed herein, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds disclosed herein”, or “compounds of the invention”.
Compounds disclosed herein may contain exocyclic double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
Compounds disclosed herein may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
Compounds disclosed herein may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
The term “glycosyl”, when used herein, refers to a group obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide, disaccharide, trisaccharide or oligosaccharide comprising 6 or fewer monosaccharide units. In particular cases, a glycosyl group may refer to a group obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide or disaccharide.
Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, acyclic or cyclic, saturated or hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Examples of “alkyl” groups include methyl, ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl, such as methyl. Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-6 cycloalkyl and, more preferably, C5-6 cycloalkyl.
The term “fluoroalkyl” refers to an alkyl group as defined above, where at least one hydrogen atom is replaced by a fluorine atom. In particular examples, a fluoroalkyl group may comprise from one to ten fluorine atoms, such as one to six, one to five, one to four, one to three, one to two, or one.
Further embodiments of the invention that may be mentioned include those in which the compounds disclosed herein are isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compounds disclosed herein are not isotopically labelled.
The term “isotopically labelled”, when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to “one or more positions in the compound” will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term “isotopically labelled” includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.
The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 18O, 35S, 18F, 37Cl, 77Br, 82Br and 125I).
When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive.
The invention is illustrated by the below Examples, which are not to be construed as limitative.
Materials
Sodium hydroxide, potassium hydroxide (KOH), lithium hydroxide, cesium hydroxide, potato Dextrose Agar (PDA) powder, dimethyl sulfoxide (DMSO) and ethanol were obtained from Merck & Co., Inc. Flavones (FL) including luteolin (Lu), apigenin (Ap), diosmetin (Dio), chrysin (Chry), wogonin (Wo), genistein (Ge), 5,6-dihydroxyflavone (“56”), 5,3′,4′-trihydroxyflavone (“534”), 6,3′,4′-trihydroxyflavone (“634”), 7,3′,4′-trihydroxyflavone (“734”) and 3′,4′-dihydroxyflavone (“34”) were obtained from Indofine Chemical Co., Inc., Hillsborough, NJ, USA. Hypochlorite acid (15%) was obtained from Merck & Co., Inc. Fluorescein disodium was obtained from Aldrich (Milwaukee, WI). Flavonoids compounds (7,8-dihydroxyflavone, baicalein, luteolin, scutellarein, fisetin, kaempferol, morin, myricetin, quercetin, 3,3′,4′-trihydroxyflavone, 3,5,7,8,3′,4′-hexahydroxyflavone, alpinetin, eriodictyol, liquiritigenin, hesperetin, naringenin, pinocembrin, ampelopsin, taxifolin, catechin, epicatechin, epigallocatechin) were obtained from Nanjing Plant Origin Biological Technology Co., Ltd. 96-well polystyrene microplates and the covers were purchased from VWR International Inc (Bridgeport, NJ). All aqueous solutions were prepared with 18.2 MΩ·cm ultrapure water obtained by a Millipore water purification system. Disodium tetraborate was obtained from Sigma.
Analytical Techniques
Nuclear Magnetic Resonance
1H and 13C NMR spectra were measured using a Bruker AVANCE I 400 or 500 NMR spectrometer. Chemical shifts were reported in ppm from the solvent resonance as the internal standard (DMSO-d6, δ=2.50). Spectra were reported as follows: chemical shift (δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, hept=heptet, m=multiplet), coupling constants (Hz), integration and assignment. Chemical shifts were reported in ppm from the solvent resonance as the internal standard (DMSO, δ=39.52).
Mass Spectroscopy
High resolution mass spectrometry (HRMS) was performed on a Thermo Scientific LCQ Fleet ion trap mass spectrometer in ES negative mode.
Electron Paramagnetic Resonance (EPR) Spectroscopy
The EPR spectra were recorded on X-band EPR spectrometer (JES-TE100, JEOL, Tokyo, Japan), which was equipped with WIN-RAD EPR Data Analyzer System (Radical Research, Inc., Hino, Tokyo).
The flavonoids solutions (5.0 mM) were loaded in a capillary tube plugged with sealing putty (TERUMO CORPORATION, Tokyo Japan). Then, the capillary tube was put into an EPR tube (WILMAD QUARTZ (CFQ), DIAM. 5 mm), before placing in the TE mode cavity. The ESR experiments were performed at room temperature with the following parameters: microwave frequency: 9.19 GHz, microwave power: 1 mW, centre magnetic field: 328.348 mT, field sweep width: ±5 mT, sweep rate: 0.67 mT/min, time constant: 0.03, field modulation frequency: 500 kHz, and field modulation width: 0.025 mT. EPR data acquisition was controlled by the WIN-RAD EPR Data Analyzer System. The spectra were simulated by JEOL IsoSimu/Fa Version 2.2.0 isotropic simulation program.
Fluorescence
A Synergy HT microplate fluorescence reader (Bio-Tek Instruments, Inc., Winooski, VT) was used with fluorescence filters for an excitation wavelength of 485±20 nm and an emission wavelength of 530±25 nm. The plate reader was controlled by software KC4 3.0 (revision 29). Sample dilution was accomplished by a Precision X automatic pipetting system managed by precision power software (version 1.0) (Bio-Tek Instruments, Inc.).
Thin-Layer Chromatography (TLC) and Column Chromatography
Merck F254 silica gel-60 plates were used for thin-layer chromatography. Silica gel-60 (230-400 mesh) was selected as the solid phase for column chromatography.
HPLC and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
The HPLC system (Waters Arc HPLC System) was equipped with a C18 column (Luna 5 μm C18(2) 100A, LC Column 250×4.6 mm). The Waters 2998 Photodiode Array (PDA) Detector was connected to the HPLC system with detection wavelengths from 190 and 800 nm.
Bruker AmaZon-X was applied for LC-MS and LC-MS/MS. The LC-MS system was equipped with a C18 column (Phenomenex, Luna 5u C18, 250×4.6 mm) guard column (4×3.0 mm). All mass spectra were acquired in both positive and negative ion mode using electrospray ionization. The parent ion was selected with a width of ±2.5 Da and fragmented with 50% setting.
Semi-Prep HPLC
The semi-prep HPLC instrument (Waters semiprep HPLC system) was equipped with a C18 column (Phenomenex, (Luna 5 μM C18(2) 100A, LC Column 250×10 mm)) with a PDA detector.
X-Ray Crystallography
The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. Data were corrected for absorption effects using the Multi-Scan method (SADABS).
Computational Studies
Geometry Optimization and Spin Density Distributions
Geometry optimization and spin density distributions were calculated using DFT at the UM062X/6-311+G(d,p) level of theory with Gaussian 09W software (M. Frisch et al., Gaussian 03, revision C. 02; Gaussian, Inc.: Wallingford, C T, 2004. The cube files for both the functions were generated using Multiwfn software (T. Lu & F. Chen, J. Comput. Chem. 2012, 33, 580-592) and isodensity surface plot for the non-covalent interaction studies were done using VMD visualization software (W. Humphrey, A. Dalke & K. Schulten, J. Mol. Graph. 1996, 14, 33-38). Molecular visualizations were created using CYLview (C. Legault, CYLview, 1.0b. Université de Sherbrooke 2009, 436, 437).
Gibbs Free Energies
The Gibbs free energies were calculated using DFT computations within the Gaussian 16 program (M. Frisch et al., Gaussian 03, revision C. 02; Gaussian, Inc.: Wallingford, C T, 2004.). Optimizations were done based on preliminary conformational searches with Schrödinger2 Maestro 10.6. The low-energy conformers that are with 5 kcal/mol of the global minimum were re-optimized at the level of M06-2X/6-31G(d) (Y. Zhao & D. G. Truhlar, Theor. Chem. Acc. 2008, 120, 215-241), with SMD (A. V. Marenich, C. J. Cramer & D. G. Truhlar, J. Phys. Chem. B 2009, 113, 6378-6396) solvation model for water. The vibrational frequency analyses were performed at the same level of theory to verify that minima have no imaginary frequencies and to evaluate its zero-point vibrational energy (ZPVE) and thermal corrections at 298 K. Single point energies were calculated using a larger basis set, 6-311+G(d,p), with the same solvation model.
General Procedure for the Characterization of Reaction Products with HPLC and LC-MS/MS
Isocratic elution method was applied for products analysis of luteolin homo-coupling. The LC-MS system was also applied in the analysis of the reaction products between flavonoids and HOCI. The samples (10 μL) were filtered through a 0.2 μm membrane (Merck Millipore, USA) before being injected into the HPLC system for analysis. Deionised (DI) water with 0.1% formic acid was selected as mobile phase A while ACN with 0.1% formic acid was applied for mobile phase B. The column was equilibrated with 71.5% mobile phase A for 10 min before isocratic elution of the same percentage mobile phase A from 0 to 35 min at a flow rate of 1.0 mL/min.
Bruker AmaZon-X was applied for LC-MS and LC-MS/MS to do characterization and fragments analysis of unknowns. The LC conditions for LC-MS analysis were similar to those mentioned above, except the detection wavelengths were 280 and 350 nm.
General procedure for the isolation of bioflavonoids using semi-prep HPLC The reaction solution was carefully acidified with 1 M HCl to neutral pH, then extracted with EA and purified by using the Semi-Prep HPLC instrument at a flow rate of 5 mL min−1. The injection volume was 500 μL. Compound 1a was obtained at retention time 20.4 min with CH3CN-water (45%:55%, 0.3% TFA v/v). Compound 5a was isolated at retention time 28.8 min eluted by CH3CN-water (20%:80%, 0.3% TFA v/v). Compounds 8a and 8b were purified by CH3CN-water (45%:55%, 0.3% TFA v/v) at retention time 36.6 min and 67.2 min, respectively. Compounds 10a, 10b and 10c were isolated with CH3CN-water (35%:65%, 0.3% TFA v/v) at retention time 30.5 min, 42 min and 54 min, respectively. Compounds 12a and 12b were purified by CH3CN-water (33%:67%, 0.3% TFA v/v), and were obtained at retention time 36.5 min and 53 min, respectively. The isolated yield was calculated from the mass obtained by semi-prep HPLC with C18 column.
Luteolin (1a, 143 mg, 0.5 mmol) was added to an aqueous KOH solution (0.1 M, 30 mL) in a 50 mL centrifuge tube. The resulting solution had a pH value of 11.5. The tube was capped tightly to seal the reaction vessel and kept at room temperature for 10 hours without stirring. The resulting solution was then acidified with concentrated HCl (1.0 mL, 10 M) to give a solution with a pH value of 1˜ 2. Then, the resulting solution was extracted with EA (3×50.0 mL), and the organic layers were combined. Removal of the volatiles in vacuo resulted in a crude solid, which was purified over semi-prep HPLC with automatic fraction collection system to give pure 2a, 2a′, and 3a.
68 mg, 48% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(2a)=19.045 min, concentration of 2a=1.0 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.15 (s, 1H), 12.79 (s, 1H), 10.73 (s, 1H), 10.68 (s, 1H), 10.13 (s, 1H), 9.91 (s, 1H), 9.42 (s, 1H), 8.45 (s, 1H), 7.48-7.40 (m, 2H), 7.19 (d, J=8.4 Hz, 1H), 6.95 (d, J=8.4 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.70 (s, 1H), 6.54 (s, 1H), 6.09 (d, J=2.1 Hz, 1H), 6.06 (s, 1H), 5.97 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 182.12, 181.76, 166.95, 164.45, 164.09, 161.76, 159.24, 157.85, 156.77, 150.12, 148.86, 146.20, 144.86, 124.19, 121.99, 120.70, 120.28, 119.44, 116.52, 114.70, 113.78, 108.53, 106.69, 103.86, 103.77, 103.33, 99.14, 93.79, 93.73. HRMS (ESI-TOF) calcd for C30H18O12=569.0725, found 569.0717.
Crystals of compound 2a suitable for X-ray analysis were obtained by slow evaporation from MeOH. A specimen of C30H18O12, approximate dimensions 0.072 mm×0.123 mm×0.146 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ=0.71073 Å). The total exposure time was 5.41 hours. The integration of the data using a triclinic unit cell yielded a total of 26930 reflections to a maximum θ angle of 28.31° (0.75 Å resolution), of which 7389 were independent (average redundancy 3.645, completeness=99.5%, Rint=3.10%, Rsig=2.95%) and 5796 (78.44%) were greater than 2σ(F2). The final cell constants of a=8.4524(4) Å, b=10.0982(4) Å, c=18.4738(8) Å, α=95.392(2°), β=101.343(2°), γ=102.776(2°), volume=1492.08(11) Å3, are based upon the refinement of the XYZ-centroids of 9938 reflections above 20 σ(I) with 5.070°<2θ<56.58°. The ratio of minimum to maximum apparent transmission was 0.920. The calculated minimum and maximum transmission coefficients (based on crystal size) were 0.6862 and 0.7457. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P−1, with Z=2 for the formula unit, C30H18O12. The final anisotropic full-matrix least-squares refinement on F2 with 543 variables converged at R1=6.80%, for the observed data and wR2=22.47% for all data. The goodness-of-fit was 0.848. The largest peak in the final difference electron density synthesis was 0.564 e−/Å3 and the largest hole was −0.685 e−/Å3 with an RMS deviation of 0.082 e−/Å3. On the basis of the final model, the calculated density was 1.515 g/cm3 and F(000), 712 e−. Crystallographic data have been deposited with the Cambridge Crystallograhic Data Centre (CCDC #2044714).
11 mg, 8% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(2a′)=30.572 min, concentration of 2a′=6.5 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.15 (s, 2H), 10.72 (s, 2H), 10.15 (s, 4H), δ 7.59 (d, J=7.7 Hz, 2H), 7.20 (d, J=8.3 Hz, 1H), 7.01-6.89 (m, 3H), 6.61 (s, 1H), 6.11 (d, J=2.0 Hz, 1H), 6.05 (s, 1H), 5.99 (d, J=2.0 Hz, 1H). 13C NMR (75 MHz, DMSO) δ 182.24, 181.68, 166.78, 164.38, 163.88, 163.42, 162.15, 161.67, 159.14, 157.77, 156.72, 151.09, 148.70, 148.41, 144.63, 124.11, 121.90, 120.76, 120.66, 120.11, 116.15, 114.71, 110.57, 108.35, 106.67, 103.83, 103.76, 103.65, 99.07, 93.73. HRMS (ESI-TOF) calcd for C30H18O12=569.0725, found 569.0715.
90 mg, 42% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(3a)=22.742 min, concentration of 3a=10 mM. 3a=3a*+3a**. Atropisomer 3a*: 1H NMR (500 MHz, Methanol-d4) δ 7.41 (m, 2H), 7.27 (d, J=8.4 Hz, 1H), 7.19 (d, J=8.3 Hz, 1H), 7.00 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.3 Hz, 1H), 6.59 (s, 1H), 6.58 (s, 1H), 6.57 (s, 1H), 6.23 (s, 1H), 6.17 (s, 1H), 6.13 (d, 1H), 6.04 (d, 1H), 6.01 (s, 1H). Atropisomer 3a**: 1H NMR (500 MHz, Methanol-d4) δ 7.41 (m, 2H), 7.27 (d, J=8.4 Hz, 1H), 7.19 (dd, J=8.3 Hz, 1H), 7.00 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.3 Hz, 1H), 6.59 (s, 1H), 6.58 (s, 1H), 6.57 (s, 1H), 6.19 (s, 1H), 6.09 (s, 1H), 6.13 (d, 1H), 6.04 (d, 1H), 6.01 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.21, 181.81, 181.72, 166.66, 166.56, 164.15, 164.09, 162.28, 161.75, 159.07, 157.87, 157.06, 156.81, 150.04, 148.72, 146.17, 144.64, 124.14, 122.09, 120.63, 120.29, 119.43, 116.50, 114.75, 113.86, 108.37, 108.29, 106.66, 103.89, 103.75, 103.64, 103.43, 99.27, 93.96, 93.87, 93.73. HRMS (ESI-TOF) calcd for C45H27O18=855.1192, found 855.1182.
Results and Discussion
Hydroxyl group rich flavones (e.g. luteolin) are good reducing agents and have been well-known as potent dietary antioxidants in scavenging biologically relevant reactive oxygen species (N. Cotelle et al., Free Radic. Biol. Med. 1996, 20, 35-43). Moreover, under alkaline conditions, many weakly acidic flavones, including luteolin, undergo deprotonation to phenolates, which are sensitive to oxidation by molecular oxygen to their respective ortho-semiquinone radicals, as detected by ESR spectra (K. Kuwabara et al., Appl. Magn. Reson. 2018, 49, 911-924; and Ŝ. Rameŝová, R. S. J. Tarebek & I. Deganoca, Electrochim. Acta 2013, 110, 646-654). Nevertheless, the fates of these radicals were unknown. We envisioned that these electrons deficient semiquinone radicals may react with electron rich flavonoid anions by radical-nucleophile coupling. To verify this, we conducted HPLC analysis of the alkaline solution of luteolin (pH 11.5) and indeed found several products which were further characterized to be luteolin dimers and trimers by LC-MS. Herein, we report a novel catalyst-free oxidative coupling reaction of two sp2 C—H bonds of flavones mediated by dissolved molecular oxygen as a hydrogen atom acceptor (
20d
aThe reaction condition: luteolin 1a (0.045 mmol) were dissolved in 3 mL base solution, incubated in sealed tube 15 mL without stirring.
bUsing HPLC to calculate conversion ratio, isocratic elution method (71.5% of mobile phase A: DI water with 0.1% formic acid and 21.5% mobile phase B: ACN with 0.1% formic acid) was applied for products analysis of luteolin-luteolin cross-coupling. Standard curves were built using isolated products.
cAt 10 g scale.
dReaction conducted in dark.
With the success of homo cross coupling of luteolin, we pondered whether a similar homo cross coupling reaction could be extended to other flavones. Thus, Ap, Dio, Chry, Wo, 5,6-dihydroxyflavone, and Ge were dissolved in alkaline water (pH 11.5). However, no desired coupling products were detected under the same condition. Instead, only starting materials were recovered. No free radical signals were detected by EPR spectroscopy in the reaction solution, suggesting that they are insensitive to oxygen. These flavones lack catecholic groups preventing them from forming ortho-semiquinone radical anions.
By using the conditions in Example 1, the reaction was scaled up with 10 g of luteolin for the synthesis of 2a (42%, Lu-(2′-6)-Lu (This nomenclature was used to name the flavone dimers and oligomers. For example, Lu-(2′-6)-Lu represents luteolin (Lu) dimer linked by through the C(2′) of the first luteolin with the C(6) of the second luteolin), 2a′ (Lu-(2′-8)-Lu), 2a″ (Lu-(6′-6)-Lu) and 3a (Lu-(2′-6)-Lu-(2′-6)-Lu) in one-pot, where luteolin (10 g) was weighed and dissolved in KOH solution (0.05 M, 2.0 L) in four 1-liter plastic bottles with 500 mL per bottle, and the resulting solutions were neutralized with HCl (10 M) to give precipitates. The mixture was extracted with EA thrice (500 mL each). Then, the organic layers were combined and concentrated in vacuo to give the crude solid product, which was dissolved in methanol and purified over semi-prep HPLC with automatic fraction collection system to give pure 2a (4.2 g, 42%), 2a′ (0.12 g, 1.2%), 2a″ (0.10 g, 1.0%), and 3a (1.0 g, 10%).
3 mg, 2% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(2a″)=15.922 min, concentration of 2a″=3.14 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.07 (s, 1H), 12.72 (s, 1H), 10.14 (s, 3H), 9.62 (s, 3H), 7.29 (s, 1H), 7.12-6.97 (m, 2H), 6.83 (s, 1H), 6.76 (d, J=8.4 Hz, 1H), 6.61 (s, 1H), 6.31 (s, 1H), 6.08 (d, J=2.1 Hz, 1H), 5.98 (s, 1H), 5.81 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 182.29, 181.61, 166.41, 164.57, 164.29, 161.68, 160.65, 157.69, 154.31, 150.28, 148.75, 146.12, 145.54, 123.66, 123.48, 123.34, 121.80, 120.38, 119.04, 116.34, 116.06, 114.03, 107.51, 106.65, 103.91, 103.72, 102.88, 99.21, 98.93, 93.67. HRMS (ESI-TOF) calcd for C30H18O12=569.0725, found 569.0727.
Results and Discussion
2a, 2a′, 2a″, and 3a were successfully synthesized in one-pot (
Triluteolins such as 3a, have one B ring and one A ring on the terminal luteolin units, respectively, that are close to each other for intramolecular oxidative coupling (
Cyclotriluetolins (e.g. 4) were prepared from trimers (e.g. 3a) (50 mg, 0.06 mmol) in an aqueous KOH solution (10 mL, 0.1 M) in a 50 mL centrifuge tube, by an analogous protocol to Example 1 except the pH of the reaction mixture was adjusted to 12.5 with concentrated KOH before the tube was capped tightly and kept at room temperature for 10 h without stirring (
5 mg, 10% yield, white solid. HPLC (Luna 5 μm C18(2) 100 Å, LC Column 250×4.6 mm, ACN/Water=71.5:28.5, flow rate 1.0 mL/min, λ=300 nm), injection volume=10 μL, tr(4a)=9.045 min. 1H NMR (500 MHz, DMSO-d6) δ 12.74 (s, 1H), 12.70 (s, 1H), 12.69 (s, 1H), 10.56 (s, 1H), 10.49 (s, 1H), 9.96 (s, 1H), 9.59 (s, 1H), 9.39 (s, 1H), 8.45 (s, 1H), 6.94 (s, 1H), 6.89 (d, J=2.4 Hz, 4H), 6.63 (s, 1H), 6.30-6.24 (m, 3H), 6.06 (d, J=11.1 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 182.01, 181.98, 181.94, 168.03, 167.97, 167.67, 163.55, 163.46, 162.95, 158.62, 158.49, 158.46, 156.90, 156.67, 148.23, 148.19, 147.99, 145.20, 145.10, 145.00, 126.42, 126.38, 125.58, 123.87, 120.74, 120.60, 119.90, 119.79, 119.64, 116.96, 114.68, 111.40, 107.94, 107.80, 103.27, 103.10, 93.27. HRMS (ESI-TOF) calcd for C45H23O18=851.0890, found 851.0901.
4b was obtained by crystallisation of 4a. 1H NMR (500 MHz, DMSO-d6) δ 14.73 (s, 1H), 13.17 (s, 1H), 11.93 (s, 1H), 10.69 (s, 1H), 10.54 (s, 1H), 10.24 (s, 1H), 9.98 (s, 1H), 9.69 (s, 1H), 9.43 (s, 1H), 8.43 (d, J=12.2 Hz, 3H), 8.20 (s, 1H), 7.24 (d, J=8.5 Hz, 1H), 7.09 (s, 1H), 6.95-6.84 (m, 4H), 6.59 (s, 1H), 6.22 (s, 1H), 5.98 (s, 1H), 5.72 (s, 1H), 5.26 (s, 1H). 13C NMR (126 MHz, DMSO) δ 195.02, 183.13, 182.47, 169.26, 168.37, 165.97, 161.10, 160.11, 159.56, 158.24, 157.75, 157.01, 154.98, 154.58, 149.43, 148.15, 147.17, 145.73, 144.97, 144.74, 126.59, 123.16, 121.80, 121.47, 120.83, 120.08, 120.02, 118.56, 114.75, 114.63, 114.05, 112.68, 108.33, 108.29, 106.39, 106.36, 105.94, 105.81, 103.73, 102.11, 101.15, 95.19, 95.03, 92.67. HRMS (ESI-TOF) calcd for C45H23O18=851.0890, found 851.0882.
Results and Discussion
By dissolving 3a in alkaline water (pH 12.5) at room temperature overnight, three major cyclotriluteolins, 4a, 4a′, and 4a″ (
To confirm the structure of 4a prepared in Example 3, single crystals were grown from its methanolic solution and the molecular structure was determined.
Results and Discussion
Crystals of compound 4a suitable for X-ray analysis were obtained by slow evaporation from MeOH. A specimen of C45H24O18, approximate dimensions 0.248×0.247×0.168 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ=1.54178 Å). The integration of the data using a triclinic unit cell yielded a total of 33495 reflections. The final cell constants of a=10.4383(7) Å, b=16.9456(11) Å, c=18.4841(12) Å, α=116.927(3°), β=97.234(3°), γ=100.043(3°), volume=2791.1(3) Å3, are based upon the refinement of the XYZ-centroids of 4286 reflections above 20 σ(I) with 8.870°<2θ<133.1°. The calculated minimum and maximum transmission coefficients (based on crystal size) were 0.5865 and 0.7528. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P −1, with Z=2 for the formula unit, C45H24O18. The final anisotropic full-matrix least-squares refinement on F2 at R1=10.5%, for the observed data and wR2=30.28% for all data. The goodness-of-fit was 1.062. The largest peak in the final difference electron density synthesis was 1.044 e−/Å3 and the largest hole was −0.668 e−/Å3. On the basis of the final model, the calculated density was 1.285 g/cm3 and F(000), 1103 e−. Crystallographic data have been deposited with the Cambridge Crystallograhic Data Centre (CCDC #2044716).
O(2A)
O(8A)
O(5A)
The ORTEP plot (
Isomerization of Cyclotriluteolin
In solution, cyclobartramiatriluteolin exhibited one set of the 1H and 13C NMR spectral peaks for three luteolin units (H. Geiger et al., Phytochemistry 1995, 39, 465-467). Due to the C3 axis in 4a, its 1H NMR spectrum is in agreement with magnetically equivalent luteolin units C(sp2)-H at 25° C. (
One of 1a, 1h-k (0.045 mmol) was dissolved in KOH solution (0.1 M, 3 mL) in a 15 mL centrifuge tube. The pH was adjusted to 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 or 13.0 with concentrated KOH before the tube was sealed and kept overnight at room temperature without stirring. The resulting solution was then acidified with concentrated HCl (1.0 mL, 10 M) to give a solution with a pH value of 1˜ 2. Then, the resulting solution was extracted with EA (3×50.0 mL), and the organic layers were combined. Removal of the volatiles in vacuo resulted in a crude solid, which was purified over semi-prep HPLC with automatic fraction collection system to give pure 2a, and 2h-k.
Results and Discussion
Treating trihydroxyflavones containing catecholic B ring including 3′,4′-dihydroxyflavone, 3′,4′,5-trihydroxyflavone, 3′,4′,6-trihydroxyflavone and 3′,4′,7-trihydroxyflavone in alkaline water resulted in the formation of ortho-semiquinone radicals as detected by EPR spectra (
2b was prepared from 1a (0.25 mmol) and Ap (1b, 140 mg, 0.5 mmol) by following the protocol in Example 1 except the product was extracted with 100 mL EA.
43% yield (2b), 4% yield (2b′), 10% yield (3b). Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid=71.5:28.5), tr(2b)=31.758 min, tr(2b′)=25.601 min, tr(3b)=35.711 min.
Results and Discussion
When luteolin was mixed with excess Ap (1:1.5 molar ratio), (Lu-(2′-6)-Ap, 2b, was isolated in good yield (47%) together with a trace amount of Lu-(2′-8)-Ap, 2b′, a triflavone (Lu-(2′-6)-Lu-(2′-6)-Ap, 3b), and a trace amount of 2a. The structure of 2b was confirmed by single crystal X-ray diffraction analysis to be desoxydicranolomin (
aThe reaction condition: luteolin 1a (0.045 mmol) and apigenin 1b (Y equiv.) were dissolved in 3 mL KOH solution, incubated in sealed tube 15 mL without stirring.
bUsing HPLC to calculate conversion ratio, isocratic elution method (71.5% of mobile phase A: DI water with 0.1% formic acid and 21.5% mobile phase B: ACN with 0.1% formic acid) was applied for products analysis of luteolin-luteolin cross-coupling. Standard curves were built using isolated products.
cIsolated yield.
dpH 12.5 buffer (disodium tetraborate) was applied to take place of KOH solution.
Luteolin-flavone biflavones 2b-2k were prepared from luteolin (72 mg, 0.25 mmol) and a flavone (0.5 mmol) selected from Ap, Dio, Chry, Wo, 5,6-dihydroxyflavone (56), Ge, 5,3′,4′-trihydroxyflavone (534), 6,3′,4′-trihydroxyflavone (634), 7,3′,4′-trihydroxyflavone (734) and 3′,4′-dihydroxyflavone (34), by following the protocol in Example 1.
2b was prepared from Ap. 61 mg, 44% yield, brown solid. HPLC (ACN/Water=71.5:28.5, A=300 nm), tr(2b)=30.447 min, concentration of 2b=1.66 mM. 1H NMR (500 MHz, DMSO-d6) b 13.15 (s, 1H), 12.80 (s, 1H), 10.34 (s, 5H), 7.95 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 1H), 7.02-6.86 (m, 3H), 6.77 (s, 1H), 6.54 (s, 1H), 6.09 (d, J=2.1 Hz, 1H), 6.03 (s, 1H), 5.98 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) b 182.13, 181.76, 167.11, 164.62, 164.43, 163.85, 161.75, 161.56, 159.22, 157.87, 156.86, 149.00, 145.11, 128.92, 128.92, 124.21, 121.72, 120.61, 120.47, 116.43, 116.43, 114.52, 108.77, 106.60, 103.85, 103.52, 103.26, 99.13, 94.09, 93.81. HRMS (ESI-TOF) calcd for C30H17O11=553.0776, found 553.0782.
Crystals of 2b suitable for X-ray analysis were obtained by slow evaporation from MeOH. A brown Block-like specimen of C30H18011, approximate dimensions 0.061 mm×0.063 mm×0.267 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. The total exposure time was 19.00 hours. The integration of the data using a triclinic unit cell yielded a total of 18025 reflections to a maximum θ angle of 67.11° (0.84 Å resolution), of which 5625 were independent (average redundancy 3.204, completeness=99.0%, Rint=6.56%, Rsig=6.46%) and 3788 (67.34%) were greater than 2σ(F2). The final cell constants of a=8.4765 Å, b=11.1018 Å, c=18.6315 Å, α=98.306°, R=95.037°, γ=109.024°, volume=1623.0 Å3, are based upon the refinement of the XYZ-centroids of 49 reflections above 20 σ(I) with 8.533°<2θ<40.75°. The ratio of minimum to maximum apparent transmission was 0.883. The structure was solved and refined using the Bruker SHELXTL Software Package, with Z=2 for the formula unit, C30H18O11. The final anisotropic full-matrix least-squares refinement on F2 with 491 variables converged at R1=4.71%, for the observed data and wR2=12.43% for all data. The goodness-of-fit was 1.029. The largest peak in the final difference electron density synthesis was 0.253 e−/Å3 and the largest hole was −0.240 e−/Å3 with an RMS deviation of 0.056 e−/Å3. On the basis of the final model, the calculated density was 1.434 g/cm3 and F(000), 720 e−. Crystallographic data have been deposited with the Cambridge Cystallograhic Data Centre (CCDC #2044715).
2b′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(2b′)=23.852 min, concentration of 2b′=8.3 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.01 (s, 1H), 12.75 (s, 1H), 10.76 (s, 3H), 10.33 (s, 2H), 7.53 (d, J=9.1 Hz, 2H), 7.33-7.22 (d, J=8.2 Hz, 1H), 7.03 (d, J=8.2 Hz, 1H), 6.81 (d, J=9.1 Hz, 2H), 6.76 (s, 1H), 6.28 (d, J=1.4 Hz, 1H), 6.08 (d, J=2.1 Hz, 1H), 6.04 (d, J=1.4 Hz, 1H), 5.76 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 207.07, 182.45, 181.71, 167.05, 164.43, 164.02, 162.11, 161.70, 161.51, 160.89, 157.71, 154.72, 148.99, 144.94, 128.59, 124.43, 121.73, 121.12, 119.26, 116.27, 115.08, 106.92, 104.00, 103.76, 103.58, 102.96, 99.17, 98.85, 93.63. HRMS (ESI-TOF) calcd for C30H17O11=553.0776, found 553.0782.
2c was prepared from Dio. 95 mg, 65% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(2c)=11.018 min, concentration of 2c=0.81 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.14 (s, 1H), 12.82 (s, 1H), 10.52 (s, 4H), 9.51 (s, 1H), 7.56 (dd, J=8.7, 2.3 Hz, 1H), 7.45 (d, J=2.4 Hz, 1H), 7.17 (d, J=8.3 Hz, 1H), 7.10 (d, J=8.7 Hz, 1H), 6.92 (d, J=8.3 Hz, 1H), 6.73 (s, 1H), 6.51 (s, 1H), 6.09 (d, J=2.1 Hz, 1H), 6.04 (s, 1H), 5.99 (d, J=2.1 Hz, 1H), 3.88 (s, 3H). 13C NMR (126 MHz, DMSO) δ 182.00, 181.77, 167.22, 164.43, 163.54, 161.75, 159.21, 157.87, 156.90, 151.52, 149.11, 147.27, 145.32, 124.21, 123.59, 120.57, 119.12, 114.42, 113.35, 112.68, 109.01, 106.52, 103.88, 103.85, 103.39, 99.12, 94.19, 93.81, 56.23. HRMS (ESI-TOF) calcd for C31H19O12=583.0882, found 583.0898.
2d was prepared from Chry. 62 mg, 46% yield, brown solid. HPLC (ACN/Water=60:40, λ=300 nm), tr(2d)=12.096 min, concentration of 2d=2.40 mM. 1H NMR (400 MHz, DMSO-d6) δ 13.01 (s, 1H), 12.79 (s, 1H), 11.14-10.09 (m, 4H), δ 8.16-8.05 (m, 2H), 7.68-7.53 (m, 3H), 7.20 (d, J=8.4 Hz, 1H), 7.00 (s, 1H), 6.95 (d, J=8.4 Hz, 1H), 6.62 (s, 1H), 6.09 (d, J=2.1 Hz, 1H), 6.07 (s, 1H), 5.98 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 182.38, 181.76, 166.90, 164.47, 163.46, 162.78, 161.77, 159.23, 157.85, 156.96, 148.84, 144.79, 132.47, 131.20, 129.63, 129.63, 126.91, 126.91, 124.19, 120.75, 120.13, 114.79, 108.75, 106.74, 105.68, 104.13, 103.86, 99.16, 93.97, 93.80. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0824.
2e was prepared from Wo. 121 mg, 85% yield, brown solid. HPLC (ACN/Water=60:40, λ=300 nm), tr(2e)=25.651 min, concentration of 2e=1.95 mM. 1H NMR (500 MHz, DMSO-d6) 12.80 (s, 1H), 12.73 (s, 1H), 10.73 (s, 1H), 10.29 (s, 1H), 10.18 (s, 1H), 8.53 (s, 1H), 8.18-8.05 (m, 2H), 7.68-7.55 (m, 3H), 7.22 (d, J=8.4 Hz, 1H), 7.06 (s, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.09 (d, J=2.1 Hz, 1H), 6.08 (s, 1H), 5.91 (d, J=2.1 Hz, 1H), 3.82 (s, 3H). 13C NMR (126 MHz, DMSO) δ 182.56, 181.77, 166.86, 164.44, 163.29, 161.76, 157.78, 155.48, 154.52, 149.05, 148.86, 144.75, 132.57, 131.28, 129.77, 127.91, 126.76, 124.07, 120.78, 119.76, 114.90, 108.89, 106.79, 105.69, 103.85, 103.82, 99.14, 93.75, 61.95. HRMS (ESI-TOF) calcd for C31H19O11=567.0933, found 567.0921.
Crystals of compound 2e suitable for X-ray analysis were obtained by slow evaporation from MeOH and DMSO. A specimen of C31H20O11, approximate dimensions 0.049 mm×0.122 mm×0.132 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ=1.54178 Å). The total exposure time was 17.13 hours. The integration of the data using a triclinic unit cell yielded a total of 8055 reflections to a maximum θ angle of 67.03° (0.84 Å resolution), of which 8055 were independent (average redundancy 1.000, completeness=97.6%, Rsig=10.51%) and 4577(56.82%) were greater than 2σ(F2). The final cell constants of a=8.6271(6) Å, b=10.2328(6) Å, c=17.4619(12) Å, α=102.701(4°), β=102.737(4°), γ=90.020(4°), volume=1464.87(17) Å3, are based upon the refinement of the XYZ-centroids of 4286 reflections above 20 σ(I) with 8.870°<2θ<133.1°. The ratio of minimum to maximum apparent transmission was 0.637. The calculated minimum and maximum transmission coefficients (based on crystal size) were 0.4799 and 0.7528. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P−1, with Z=2 for the formula unit, C31H20O11. The final anisotropic full-matrix least-squares refinement on F2 with 455 variables converged at R1=9.67%, for the observed data and wR2=32.15% for all data. The goodness-of-fit was 1.065. The largest peak in the final difference electron density synthesis was 0.495 e−/Å3 and the largest hole was −0.499 e−/Å3 with an RMS deviation of 0.104 e−/Å3. On the basis of the final model, the calculated density was 1.475 g/cm3 and F(000), 676 e−. Crystallographic data have been deposited with the Cambridge Crystallograhic Data Centre (CCDC #2044716).
2f was prepared from 5,6-dihydroxyflavone. 77 mg, 57% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(2f)=19.290 min, concentration of 2f=1.95 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.25 (s, 1H), 12.84 (s, 1H), 11.04 (s, 1H), 9.95 (s, 1H), 9.58 (s, 1H), 9.12 (s, 1H), 7.68-7.57 (m, 2H), 7.52-7.44 (m, 1H), 7.44-7.29 (m, 3H), 7.04 (s, 1H), 6.96-6.84 (m, 2H), 6.71 (s, 1H), 6.66 (d, J=8.9 Hz, 1H), 6.50 (s, 1H). 13C NMR (126 MHz, DMSO) δ 184.38, 182.45, 164.32, 164.10, 162.30, 161.40, 154.81, 150.11, 146.87, 146.39, 146.10, 141.03, 132.58, 131.18, 129.52, 126.42, 126.37, 121.82, 118.75, 116.04, 113.72, 111.16, 110.67, 104.86, 104.21, 103.06, 102.12, 99.19. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0837.
2f was prepared from 5,6-dihydroxyflavone. 8 mg, 6% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(2f)=25.948 min, concentration of 2f=2.50 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.52 (s, 1H), 12.73 (s, 1H), 9.51 (s, 4H), 7.82 (d, J=8.2 Hz, 2H), 7.53 (d, J=7.2 Hz, 1H), 7.48 (q, J=8.2, 7.2 Hz, 4H), 7.25 (s, 1H), 7.05 (s, 1H), 6.93 (s, 1H), 6.77 (s, 1H), 6.73 (s, 1H). 13C NMR (126 MHz, DMSO) δ 184.37, 182.28, 164.42, 164.23, 162.54, 159.59, 157.07, 150.30, 146.54, 146.27, 140.78, 132.59, 131.42, 129.66, 126.71, 126.36, 121.89, 119.59, 116.56, 113.87, 111.51, 111.05, 106.90, 104.85, 103.99, 103.38, 93.96. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0830.
2g was prepared from Ge. 39 mg, 28% yield, brown solid. HPLC (ACNA/Water=71.5:28.5, A=300 nm), tr(2g)=28.867 min, concentration of 2g=1.13 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.13 (s, 1H), 12.78 (s, 1H), 10.73 (s, 3H), 10.15 (s, 1H), 9.58 (s, 2H), 8.34 (s, 1H), 7.42-7.34 (m, 2H), 7.18 (d, J=8.4 Hz, 1H), 6.94 (d, J=8.4 Hz, 1H), 6.86-6.75 (m, 2H), 6.48 (s, 1H), 6.10 (d, J=2.1 Hz, 1H), 6.03 (s, 1H), 5.99 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 181.75, 180.73, 166.83, 164.48, 162.64, 161.77, 159.68, 157.85, 157.85, 157.07, 154.28, 148.81, 144.75, 130.67, 130.67, 124.19, 122.75, 121.74, 120.74, 120.19, 115.50, 115.50, 114.77, 108.59, 106.75, 104.62, 103.87, 99.16, 93.82, 93.47. HRMS (ESI-TOF) calcd for C30H17O11=553.0776, found 553.0781.
2g′ was prepared from Ge. 19 mg, 14% yield, brown solid. HPLC (ACN/Water=71.5:28.5, A=300 nm), tr(2g′)=29.696 min, concentration of 2g′=0.51 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.08 (s, 1H), 12.80 (s, 1H), 10.70 (s, 3H), 9.59 (s, 2H), 8.30 (s, 1H), 7.38-7.32 (m, 2H), 7.21 (d, J=8.4 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.84-6.77 (m, 2H), 6.30 (s, 1H), 6.11 (d, J=2.1 Hz, 1H), 6.08 (s, 1H), 5.85 (d, J=2.1 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 181.78, 180.87, 166.98, 164.49, 162.41, 161.77, 161.45, 157.84, 157.77, 155.50, 154.50, 149.08, 145.12, 130.57, 130.57, 124.42, 122.46, 121.66, 121.10, 119.26, 115.51, 115.51, 115.00, 106.92, 104.78, 103.83, 103.48, 99.18, 99.12, 93.74. HRMS (ESI-TOF) calcd for C30H17O11=553.0776, found 553.0776.
2h was prepared from 5,3′,4′-trihydroxyflavone. 60 mg, 43% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(2h)=14.195 min, concentration of 2h=4.29 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H), 12.64 (s, 1H), 9.76 (s, 5H), 7.53 (t, J=8.2 Hz, 1H), 7.48-7.40 (m, 2H), 7.25 (d, J=8.4 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.91 (d, J=8.2 Hz, 1H), 6.74-6.67 (m, 2H), 6.65 (d, J=8.4, 1H), 6.56 (s, 1H), 6.20 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.95, 182.16, 167.91, 164.19, 162.43, 160.24, 159.28, 156.83, 156.36, 150.19, 149.14, 146.22, 144.86, 136.16, 124.01, 121.94, 121.01, 120.36, 119.49, 116.54, 114.84, 113.78, 111.12, 110.09, 108.40, 107.34, 107.18, 103.87, 103.33, 93.71. HRMS (ESI-TOF) calcd for C30H19O11=553.0776, found 553.0735.
2h was prepared from 6,3′,4′-trihydroxyflavone. 51 mg, 37% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(2i)=9.532 min, concentration of 2i=6.99 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.12 (s, 1H), 10.70 (s, 1H), 10.06 (s, 1H), 9.92 (s, 1H), 9.45 (s, 1H), 8.43 (s, 1H), 8.14 (s, 1H), 7.50-7.39 (m, 2H), 7.26-7.08 (m, 4H), 6.96 (d, J=8.4 Hz, 1H), 6.91 (d, J=8.2 Hz, 1H), 6.69 (s, 1H), 6.54 (s, 1H), 6.05 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.16, 176.92, 165.67, 164.16, 163.51, 162.40, 159.31, 156.74, 155.05, 150.15, 149.90, 148.35, 146.21, 144.69, 125.02, 124.29, 123.24, 121.97, 120.42, 120.15, 119.55, 116.53, 114.77, 113.79, 108.57, 108.07, 107.81, 103.87, 103.33, 93.62. HRMS (ESI-TOF) calcd for C30H19O11=553.0776, found 553.0769.
2h was prepared from 7,3′,4′-trihydroxyflavone. 59 mg, 43% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(2j)=9.790 min, concentration of 2j=9.25 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.14 (s, 1H), 10.69 (d, J=8.2 Hz, 2H), 10.06 (s, 1H), 9.94 (s, 1H), 9.46 (s, 1H), 8.42 (s, 1H), 7.76 (d, J=8.6 Hz, 1H), 7.45 (d, J=8.6 Hz, 2H), 7.18 (d, J=8.3 Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 6.82 (dd, J=8.1, 2.1 Hz, 1H), 6.70 (s, 1H), 6.55 (s, 1H), 6.50 (d, J=2.1 Hz, 1H), 6.03 (s, 1H). 13C NMR (126 MHz, DMSO) δ 207.17, 182.21, 176.60, 165.55, 164.17, 162.85, 162.35, 159.29, 157.97, 156.76, 150.15, 148.31, 146.22, 144.67, 126.83, 124.93, 122.02, 120.47, 120.11, 119.49, 116.56, 116.22, 115.22, 114.80, 113.78, 108.63, 108.59, 103.90, 103.37, 102.36, 93.64. HRMS (ESI-TOF) calcd for C30H19O11=553.0776, found 555.0732.
2k was prepared from 3′,4′-dihydroxyflavone. 67 mg, 50% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(2k)=18.289 min, concentration of 2k=3.25 mM. 1H NMR (400 MHz, DMSO-d6) δ 13.14 (s, 1H), 9.69 (s, 5H), 7.92 (d, J=7.9 Hz, 1H), 7.68 (d, J=7.9 Hz, 1H), 7.47-7.35 (m, 3H), 7.25 (dd, J=16.9, 8.3 Hz, 2H), 6.97 (d, J=8.3 Hz, 1H), 6.90 (d, J=8.3 Hz, 1H), 6.68 (s, 1H), 6.54 (s, 1H), 6.13 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.14, 177.05, 166.20, 164.14, 162.64, 159.31, 156.78, 156.17, 150.17, 148.62, 146.22, 144.82, 134.46, 125.66, 125.15, 124.73, 123.41, 121.95, 120.57, 120.28, 119.48, 118.23, 116.53, 114.75, 113.77, 108.98, 108.61, 103.80, 103.30, 93.71. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0818.
21 was prepared from Ap. 73 mg, 54% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(21)=21.513 min, concentration of 21=15.80 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.19 (s, 1H), 12.63 (s, 1H), 10.81 (s, 1H), 10.40 (s, 1H), 10.26 (s, 1H), 8.55 (s, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.49 (d, J=10.1 Hz, 1H), 7.27 (d, J=8.0 Hz, 1H), 6.98 (m, 3H), 6.80 (s, 1H), 6.73-6.55 (m, 3H), 6.21 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.94, 182.28, 167.85, 164.11, 162.35, 161.66, 160.25, 159.31, 156.88, 156.37, 149.11, 144.85, 136.08, 128.96, 124.09, 121.62, 121.03, 120.38, 116.48, 114.90, 111.09, 110.10, 108.39, 107.41, 107.18, 103.96, 103.32, 93.85. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0821.
2m was prepared from Ap. 82 mg, 61% yield, brown solid. HPLC (ACN/Water=72.5:27.5, A=300 nm), tr(2m)=14.506 min, concentration of 2m=13.71 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.14 (s, 1H), 9.99 (s, 5H), 7.95 (d, J=8.2 Hz, 2H), 7.25-7.09 (m, 4H), 6.97 (dd, J=12.2, 8.2 Hz, 3H), 6.79 (s, 1H), 6.60 (s, 1H), 6.08 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.29, 177.02, 165.76, 164.10, 162.51, 161.66, 159.34, 156.84, 155.09, 149.96, 148.42, 144.75, 129.00, 125.09, 124.32, 123.31, 121.69, 120.49, 120.23, 119.60, 116.51, 114.83, 108.65, 108.13, 107.86, 103.92, 103.35, 93.83. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0821.
2m′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(2m′)=12.177 min, concentration of 2m′=2.32 mM. 1H NMR (500 MHz, DMSO-d6) δ 12.98 (s, 1H), 9.94 (s, 5H), 7.52 (d, J=8.5 Hz, 2H), 7.25 (d, J=8.2 Hz, 1H), 7.16 (d, J=3.0 Hz, 1H), 7.10-7.01 (m, 2H), 6.87 (d, J=8.2 Hz, 1H), 6.79 (d, J=8.5 Hz, 2H), 6.71 (s, 1H), 6.26 (s, 1H), 5.99 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.38, 176.80, 165.90, 163.89, 161.52, 160.85, 155.03, 154.73, 149.67, 148.73, 145.16, 128.61, 125.16, 124.17, 123.18, 121.69, 120.64, 119.44, 119.23, 116.23, 114.96, 108.09, 107.79, 103.94, 103.79, 102.83, 99.07. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0818.
2n was prepared from Ap. 65 mg, 48% yield, brown solid. HPLC (ACN/Water=72.5:27.5, A=300 nm), tr(2n)=14.399 min, concentration of 2n=22.53 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.13 (s, 1H), 10.32 (s, 5H), 7.96 (d, J=8.5 Hz, 2H), 7.75 (d, J=8.5 Hz, 1H), 7.18 (d, J=8.2 Hz, 1H), 7.03-6.87 (m, 3H), 6.82 (d, J=8.2 Hz, 2H), 6.58 (s, 1H), 6.50 (s, 1H), 6.02 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.28, 176.51, 165.49, 164.02, 162.85, 162.41, 161.62, 159.28, 157.97, 156.77, 148.31, 144.68, 128.97, 126.80, 124.92, 121.68, 120.41, 120.13, 116.46, 116.22, 115.19, 114.74, 108.65, 108.59, 103.88, 103.34, 102.37, 93.76. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0824.
2n′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(2n′)=12.139 min, concentration of 2n′=3.34 mM. 1H NMR (500 MHz, DMSO-d6) δ 12.98 (s, 1H), 10.42 (s, 5H), 7.70 (d, J=8.5 Hz, 1H), 7.52 (d, J=8.5 Hz, 2H), 7.23 (d, J=8.3 Hz, 1H), 7.02 (d, J=8.3 Hz, 1H), 6.78 (d, J=8.5 Hz, 3H), 6.72 (s, 1H), 6.26 (d, J=8.5 Hz, 1H), 6.25 (s, 1H), 5.95 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.38, 176.36, 165.70, 163.88, 162.78, 161.50, 160.81, 157.78, 154.71, 148.63, 145.08, 128.59, 126.76, 125.13, 121.71, 120.66, 119.37, 116.22, 116.08, 115.16, 114.93, 108.68, 103.93, 103.80, 102.85, 102.13, 99.03. HRMS (ESI-TOF) calcd for C30H17O10=537.0827, found 537.0822.
2o was prepared from Ap. 60 mg, 46% yield, brown solid. HPLC (ACN/Water=71.5:28.5, A=300 nm), tr(2o)=31.427 min, concentration of 20=10.06 mM. 1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H), 10.36 (s, 4H), 7.93 (t, J=8.6 Hz, 3H), 7.68 (t, J=8.0 Hz, 1H), 7.42-7.32 (m, 1H), 7.32-7.18 (m, 2H), 6.99 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.3 Hz, 2H), 6.79 (d, J=3.7 Hz, 1H), 6.60 (d, J=3.7 Hz, 1H), 6.15 (d, J=3.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 182.26, 177.04, 166.14, 164.06, 162.44, 161.65, 159.33, 156.81, 156.18, 148.58, 144.76, 134.42, 128.97, 125.63, 125.14, 124.78, 123.41, 121.64, 120.59, 120.27, 118.24, 116.46, 114.83, 109.04, 108.56, 103.91, 103.32, 93.81. HRMS (ESI-TOF) calcd for C30H17O9=521.0827, found 521.0871.
2h-2o were prepared from one of 1a-1b (1 equiv) and one of 1h-1k (1.5 equiv), by following the protocol in Example 1.
2h
2h was prepared from 1a and 1h. 43% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =64:36), t(2h)=14.273 min, t(2h′)=11.629 min.
2i
2i was prepared from 1a and 1i. 37/% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, 72.5:27.5), tr(2i)=9.744 min
2j was prepared from 1a and 1j. 43% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =72.5:27.5), tr(2j)=9.707 min.
2k
2k was prepared from 1a and 1k. 45% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =71.5:28.5), tr(2k)=21.725 min.
2l
2l was prepared from 1h and 1b. 74% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =64:36), tr(21)=17.840 min, t(21′)=16.807 min.
2m
2m was prepared from 1 i and 1 b. 86% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =72.5:27.5), tr2m)=14.695 min, tr2m′)=12.509 min.
2n
2n was prepared from 1j and 1b. 86% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =72.5:27.5), tr(2n)=13.003 min, tr(2n′)=11.015 min.
2o was prepared from 1k and 1b. 86% yield. Semi-prep HPLC (DI water with 0.1% formic acid/ACN with 0.1% formic acid, =71.5:28.5), tr(2o)=30.602 min, tr(20′)=28.932 min.
Results and Discussion
When luteolin was mixed with TFL with catecholic group on B ring (1h-1j) and 3′,4′-dihydroxyflavone (1k, DFL), luteolin became a nucleophile and 1h-1k was the radical precursor yielding corresponding biflavones FL-(2′-6)-Lu (2h-2k, FL=TFL and DFL,
3b-3k were prepared from 2a (143 mg, 0.25 mmol) and a flavone (0.375 mmol) selected from Ap, Dio, Chry, 5,6-dihydroxyflavone, Ge, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone and 3′,4′-dihydroxyflavone, by following the protocol in Example 1.
3b was prepared from Ap. 109 mg, 52% yield, brown solid. HPLC (ACN/Water=71.5:28.5, A=300 nm), tr(3b*)=32.142 min, tr(3b**)=33.539 min, concentration of 3b=0.89 mM. 3b=3b*+3b**. Atropisomer 3b*: 1H NMR (500 MHz, DMSO-d6) δ 7.90 (d, J=8.9, 2H), 7.22 (d, J=8.4 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 6.97 (d, J=8.5 Hz, 1H), 6.94 (d, J=8.9 Hz, 2H), 6.92 (d, J=8.5 Hz, 1H), 6.69 (s, 1H), 6.57 (s, 1H), 6.21 (s, 1H), 6.09 (d, J=7.8 Hz, 1H), 6.01 (s, 1H), 5.98 (d, J=7.8 Hz, 1H), 5.97 (s, 1H). Atropisomer 3b**: 1H NMR (500 MHz, DMSO-d6) δ 7.90 (d, J=8.9 Hz, 2H), 7.22 (d, J=8.4, 1H), 7.15 (d, J=8.4 Hz, 1H), 6.97 (d, J=8.5 Hz, 1H), 6.94 (d, J=8.9 Hz, 2H), 6.92 (d, J=8.5 Hz, 1H), 6.68 (s, 1H), 6.55 (s, 1H), 6.16 (s, 1H), 6.09 (d, J=7.8 Hz, 1H), 5.99 (s, 1H), 5.98 (d, J=7.8 Hz, 1H), 5.96 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.34, 181.82, 181.69, 166.65, 166.56, 164.53, 164.07, 162.21, 161.76, 161.57, 159.35, 159.09, 157.88, 156.93, 148.82, 144.74, 128.98, 124.18, 121.77, 120.63, 120.15, 116.42, 114.78, 108.41, 108.25, 106.73, 103.97, 103.83, 103.65, 103.45, 99.20, 94.03, 93.86, 93.78. HRMS (ESI-TOF) calcd for C45H25O17=837.1090, found 837.1097.
Crystals of compound 3b suitable for X-ray analysis were obtained by slow evaporation from MeOH. A brown Block-like specimen of C36H32O14, approximate dimensions 0.061 mm×0.063 mm×0.267 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. The total exposure time was 19.00 hours. The integration of the data using a triclinic unit cell yielded a total of 18025 reflections to a maximum θ angle of 67.110 (0.84 Å resolution), of which 5625 were independent (average redundancy 3.204, completeness=99.0%, Rint=6.56%, Rsig=6.46%) and 3788 (67.34%) were greater than 2σ(F2). The final cell constants of a=8.4765 Å, b=11.1018 Å, c=18.6315 Å, α=98.306°, p=95.037°, γ=109.024°, volume=1623.0 Å3, are based upon the refinement of the XYZ-centroids of 49 reflections above 20 a(I) with 8.533°<2θ<40.75°. The ratio of minimum to maximum apparent transmission was 0.883. The structure was solved and refined using the Bruker SHELXTL Software Package, with Z=2 for the formula unit, C36H32O14. The final anisotropic full-matrix least-squares refinement on F2 with 491 variables converged at R1=4.71%, for the observed data and wR2=12.43% for all data. The goodness-of-fit was 1.029. The largest peak in the final difference electron density synthesis was 0.253 e−/Å3 and the largest hole was −0.240 e−/Å3 with an RMS deviation of 0.056 e−/Å3. On the basis of the final model, the calculated density was 1.434 g/cm3 and F(000), 720 e−. Crystallographic data have been deposited with the Cambridge Cystallograhic Data Centre.
3c was prepared from Dio. 100 mg, 46% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3c*)=10.787 min, tr(3c**)=10.787 min, concentration of 3c=0.50 mM. 3c=3c*+3c**. Atropisomer 3c*: 1H NMR (500 MHz, DMSO-d6) δ 13.19 (s, 1H), 13.00 (s, 1H), 12.79 (s, 1H), 10.76 (s, 4H), 10.16 (s, 2H), 8.44 (s, 2H), 7.62 (d, J=8.6 Hz, 1H), 7.46 (m, 1H), 7.26 (d, J=8.4, 1H), 7.16 (d, J=8.7 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 6.98 (d, J=8.5, 1H), 6.93 (d, J=8.4 Hz, 1H), 6.78 (s, 1H), 6.60 (s, 1H), 6.28 (s, 1H), 6.12 (d, J=8.1 Hz, 1H), 6.09 (s, 1H), 6.02 (d, J=8.1 Hz, 1H), 5.96 (s, 1H), 3.87 (s, 3H). Atropisomer 3c**: 1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H), 12.96 (s, 1H), 12.76 (s, 1H), 10.76 (s, 4H), 10.16 (s, 2H), 8.44 (s, 2H), 7.62 (d, J=8.6 Hz, 1H), 7.46 (m, 1H), 7.26 (d, J=8.4, 1H), 7.16 (d, J=8.7 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 6.98 (d, J=8.5, 1H), 6.93 (d, J=8.4 Hz, 1H), 6.78 (s, 1H), 6.56 (s, 1H), 6.12 (d, J=8.1 Hz, 3H), 6.09 (s, 1H), 6.02 (d, J=8.1 Hz, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO) δ 182.33, 182.26, 182.18, 181.86, 181.75, 181.69, 181.63, 166.68, 166.63, 166.58, 166.54, 166.28, 164.67, 164.53, 164.48, 164.01, 163.87, 163.83, 162.27, 162.21, 162.07, 161.95, 161.77, 159.38, 159.32, 159.09, 157.89, 157.85, 157.80, 157.10, 156.93, 156.83, 151.61, 151.58, 151.55, 148.85, 148.79, 148.70, 147.26, 147.20, 144.73, 144.69, 144.63, 124.19, 124.06, 123.60, 123.57, 123.48, 120.83, 120.65, 120.19, 120.14, 120.10, 119.24, 119.20, 114.82, 114.74, 113.48, 113.42, 113.37, 112.65, 112.61, 112.55, 108.45, 108.32, 108.25, 108.18, 106.84, 106.76, 106.66, 104.24, 104.13, 104.06, 104.00, 103.95, 103.86, 103.82, 103.68, 103.62, 99.36, 99.31, 99.20, 94.40, 94.35, 93.90, 93.85, 93.80, 93.47, 93.29, 56.22, 56.17. HRMS (ESI-TOF) calcd for C46H27O18=867.1203, found 867.1202.
3d was prepared from Chry. 82 mg, 40% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3d*)=9.863 min, tr(3d**)=9.863 min, concentration of 3d=2.13 mM. 3d=3d*+3d**. Atropisomer 3d*: 1H NMR (500 MHz, DMSO-d6) δ 8.07-8.00 (m, 2H), 7.65-7.53 (m, 3H), 7.23 (d, J=8.4, Hz, 1H), 7.14 (dd, J=8.3, 1H), 6.99 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 6.85 (s, 1H), 6.65 (s, 1H), 6.24 (s, 1H), 6.11 (d, J=6.1 Hz, 1H), 6.06 (s, 1H), 6.02 (d, J=6.1 Hz, 1H), 5.96 (s, 1H). Atropisomer 3d**: 1H NMR (500 MHz, DMSO-d6) δ 8.07-8.00 (m, 2H), 7.65-7.53 (m, 3H), 7.23 (d, J=8.4 Hz, 1H), 7.14 (d, J=8.3 Hz, 1H), 6.99 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 6.85 (s, 1H), 6.63 (s, 1H), 6.16 (s, 1H), 6.11 (d, J=6.1 Hz, 1H), 6.02 (s, 1H), 6.01 (d, J=6.1 Hz, 1H), 5.94 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.48, 181.85, 181.69, 181.67, 166.67, 166.62, 166.32, 164.58, 163.57, 163.52, 162.20, 161.75, 159.32, 159.08, 159.04, 157.89, 157.08, 156.98, 156.93, 148.79, 148.70, 144.72, 144.62, 132.44, 131.28, 129.62, 126.93, 124.17, 120.64, 120.12, 120.08, 114.87, 114.77, 108.68, 108.54, 108.26, 108.20, 106.73, 105.77, 104.23, 103.82, 103.79, 103.65, 99.22, 93.89, 93.29. HRMS (ESI-TOF) calcd for C45H25O1s=821.1148, found 821.1135.
3e was prepared from Wo. 115 mg, 54% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3e*)=21.393 min, tr(3e**)=22.891 min, concentration of 3e=1.90 mM. 3e=3e*+3e**. Atropisomer 3e*: 1H NMR (500 MHz, DMSO-d6) δ 12.96 (s, 1H), 12.79 (s, 1H), 12.75 (s, 1H), 10.77 (s, 1H), 10.63 (s, 1H), 10.31 (s, 1H), 10.26 (s, 1H), 8.51 (s, 1H), 8.38 (s, 1H), 8.09 (d, J=7.7 Hz, 2H), 7.68-7.56 (m, 3H), 7.26 (d, J=8.4, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.04 (s, 1H), 6.98 (d, J=8.4, 1H), 6.91 (d, J=8.4, 1H), 6.15 (s, 1H), 6.12 (d, J=2.1 Hz, 1H), 6.04 (d, J=2.1 Hz, 1H), 6.01 (s, 1H), 5.95 (s, 1H). Atropisomer 3e**: 1H NMR (500 MHz, DMSO-d6) δ 12.96 (s, 1H), 12.77 (s, 1H), 12.74 (s, 1H), 10.77 (s, 1H), 10.57 (s, 1H), 10.27 (s, 1H), 10.12 (s, 1H), 8.51 (s, 1H), 8.35 (s, 1H), 8.09 (d, J=7.7 Hz, 2H), 7.68-7.56 (m, 3H), 7.26 (d, J=8.4, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.04 (s, 1H), 6.98 (d, J=8.4, 1H), 6.91 (d, J=8.4, 1H), 6.11 (s, 1H), 6.08 (d, J=2.1 Hz, 1H), 5.97 (s, 1H), 5.95 (d, J=2.1 Hz, 1H), 5.92 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.69, 182.59, 181.90, 181.78, 181.68, 181.64, 166.70, 166.59, 166.36, 164.47, 163.33, 162.21, 162.12, 161.76, 159.12, 159.00, 157.92, 157.84, 157.01, 156.78, 155.40, 154.58, 149.11, 148.88, 148.68, 144.76, 144.62, 132.52, 131.41, 129.75, 127.99, 127.87, 126.78, 124.30, 124.15, 124.01, 123.93, 120.74, 120.58, 120.14, 119.80, 114.91, 114.70, 108.86, 108.74, 108.20, 106.83, 106.71, 105.77, 103.90, 103.81, 103.64, 99.16, 93.93, 93.80, 93.38, 93.24, 61.97. HRMS (ESI-TOF) calcd for C46H27O17=851.1254, found 851.1248.
Crystals of compound 3e suitable for X-ray analysis were obtained by slow evaporation from MeOH and DMSO. A specimen of C35H26N2O11, approximate dimensions 0.049 mm×0.122 mm×0.132 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (λ=1.54178 Å). The total exposure time was 17.13 hours. The integration of the data using a triclinic unit cell yielded a total of 8055 reflections to a maximum θ angle of 67.03° (0.84 Å resolution), of which 8055 were independent (average redundancy 1.000, completeness=97.6%, Rsig=10.51%) and 4577(56.82%) were greater than 2σ(F2). The final cell constants of a=8.6271(6) Å, b=10.2328(6) Å, c=17.4619(12) Å, α=102.701(4°), β=102.737(4°), γ=90.020(4°), volume=1464.87(17) Å3, are based upon the refinement of the XYZ-centroids of 4286 reflections above 20 σ(I) with 8.870°<2θ<133.1°. The ratio of minimum to maximum apparent transmission was 0.637. The calculated minimum and maximum transmission coefficients (based on crystal size) were 0.4799 and 0.7528. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P−1, with Z=2 for the formula unit, C35H26N2O11. The final anisotropic full-matrix least-squares refinement on F2 with 455 variables converged at R1=9.67%, for the observed data and wR2=32.15% for all data. The goodness-of-fit was 1.065. The largest peak in the final difference electron density synthesis was 0.495 e−/Å3 and the largest hole was −0.499 e−/Å3 with an RMS deviation of 0.104 e−/Å3. On the basis of the final model, the calculated density was 1.475 g/cm3 and F(000), 676 e−. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre.
3f was prepared from 5,6-dihydroxyflavone. 154 mg, 75% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3f*)=9.731 min, tr(3f**)=9.731 min, concentration of 3f=3.50 mM. 3f=3f*+3f**. Atropisomer 3f*: 1H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J=7.1 Hz, 1H), 7.59 (d, J=7.1 Hz, 1H), 7.50 (dt, J=7.1, 3.8 Hz, 1H), 7.46-7.39 (m, 2H), 7.38 (d, J=6.9 Hz, 1H), 7.33 (d, J=7.1 Hz, 1H), 7.24 (s, 1H), 7.07 (s, 1H), 6.90 (d, J=3.8 Hz, 1H), 6.70 (s, 2H), 6.60 (d, J=6.9 Hz, 1H), 6.44 (d, J=7.1 Hz, 1H), 6.37 (s, 1H), 5.89 (s, 1H). Atropisomer 3f**: 1H NMR (400 MHz, DMSO-d6) δ 7.67 (d, J=7.1 Hz, 1H), 7.59 (d, J=7.1 Hz, 1H), 7.50 (dt, J=7.1, 3.8 Hz, 1H), 7.46-7.39 (m, 2H), 7.38 (d, J=6.9 Hz, 1H), 7.33 (d, J=7.1 Hz, 1H), 7.34 (s, 1H), 6.93 (s, 1H), 6.90 (d, J=3.8 Hz, 1H), 6.70 (s, 2H), 6.60 (d, J=6.9 Hz, 1H), 6.44 (d, J=7.1 Hz, 1H), 6.43 (s, 1H), 5.81 (s, 1H). 13C NMR (126 MHz, DMSO) δ 184.34, 184.25, 182.02, 181.89, 181.84, 166.66, 166.04, 164.17, 164.08, 164.00, 163.95, 162.39, 162.31, 162.26, 161.65, 161.30, 159.52, 158.94, 156.81, 156.70, 155.37, 155.31, 150.17, 150.11, 148.78, 148.75, 146.74, 146.68, 146.49, 146.45, 146.16, 144.66, 144.62, 141.05, 140.92, 132.66, 132.57, 131.22, 129.61, 129.53, 126.44, 126.41, 126.32, 126.12, 124.09, 123.87, 121.97, 121.92, 120.21, 120.18, 119.92, 119.49, 119.41, 116.48, 114.37, 113.78, 111.13, 111.08, 110.67, 110.56, 107.74, 107.47, 107.00, 106.81, 104.88, 103.97, 103.93, 103.84, 103.78, 103.41, 103.29, 102.23, 102.17, 98.99, 93.80, 93.48. HRMS (ESI-TOF) calcd for C45H25O16=821.1148, found 821.1133.
3g was prepared from Ge. 103 mg, 49% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3g*)=10.218 min, tr(3g**)=11.126 min, concentration of 3g=3.88 mM. 3g=3g*+3g**. Atropisomer 3g*: 1H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.39 (d, J=8.2 Hz, 2H), 7.25 (d, J=8.4 Hz, 1H), 7.18 (d, J=8.3 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 6.83 (d, J=8.2 Hz, 2H), 6.52 (s, 1H), 6.28 (s, 1H), 6.13 (d, J=2.1 Hz, 1H), 6.09 (s, 1H), 6.04 (d, J=2.1 Hz, 1H), 5.96 (s, 1H). Atropisomer 3g**: 1H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.42 (d, J=8.4 Hz, 2H), 7.25 (d, J=8.2 Hz, 1H), 7.17 (d, J=8.3 Hz, 1H), 6.97 (d, J=8.2 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 6.83 (d, J=8.2 Hz, 2H), 6.48 (s, 1H), 6.28 (s, 1H), 6.11 (d, J=2.1 Hz, 1H), 6.09 (s, 1H), 6.01 (d, J=2.1 Hz, 1H), 5.97 (s, 1H). 13C NMR (126 MHz, DMSO) δ 181.86, 181.75, 181.68, 180.85, 180.77, 166.67, 166.62, 166.60, 166.32, 164.51, 164.49, 162.42, 162.21, 162.17, 161.78, 161.75, 159.86, 159.75, 159.10, 157.90, 157.85, 157.83, 157.17, 157.09, 157.06, 156.91, 154.27, 148.82, 148.77, 148.71, 148.68, 144.70, 144.66, 144.64, 144.61, 130.73, 130.70, 130.63, 124.23, 124.17, 124.11, 122.84, 122.80, 121.86, 121.78, 120.66, 120.18, 120.12, 115.52, 115.49, 114.83, 108.51, 108.38, 108.22, 106.89, 106.79, 106.73, 104.77, 104.74, 103.86, 103.83, 103.68, 99.19, 93.89, 93.84, 93.62, 93.48, 93.27. HRMS (ESI-TOF) calcd for C45H25O17=837.1097, found 837.1087.
3h was prepared from 5,3′,4′-trihydroxyflavone. 88 mg, 42% yield, brown solid. HPLC (ACN/Water=64:36, λ=300 nm), tr(3h*)=11.579 min, t4(3h**)=12.618 min, concentration of 3h=3.43 mM. 3h=3h*+3h**. Atropisomer 3h*: 1H NMR (500 MHz, DMSO-d6) δ 7.46 (td, J=8.3, 3.8 Hz, 1H), 7.44-7.37 (m, 2H), 7.22 (d, J=8.3 Hz, 1H), 7.21 (d, J=8.2 Hz, 1H), 6.98 (d, J=8.3, 1H), 6.95 (d, J=8.2 Hz, 1H), 6.93 (d, J=8.2 Hz, 1H), 6.67 (d, J=3.8 Hz, 1H), 6.61 (s, 1H), 6.58 (s, 1H), 6.52 (d, J=8.3 Hz, 1H), 6.24 (s, 1H), 6.15 (s, 1H), 6.08 (s, 1H). Atropisomer 3h**: 1H NMR (500 MHz, DMSO-d6) δ 7.46 (td, J=8.3, 3.8 Hz, 1H), 7.44-7.37 (m, 2H), 7.22 (d, J=8.3, 1H), 7.21 (d, J=8.2 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.2 Hz, 1H), 6.93 (d, J=8.2 Hz, 1H), 6.67 (d, J=8.1 Hz, 1H), 6.60 (s, 1H), 6.57 (s, 1H), 6.46 (d, J=8.1 Hz, 1H), 6.19 (s, 1H), 6.15 (s, 1H), 6.02 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.91, 182.86, 182.25, 182.21, 181.84, 181.74, 167.83, 166.72, 166.46, 164.20, 164.16, 162.25, 162.19, 162.05, 160.20, 159.27, 159.05, 159.00, 157.07, 156.91, 156.85, 156.77, 156.24, 156.19, 150.13, 150.08, 149.05, 148.81, 148.73, 146.18, 144.72, 144.68, 136.03, 135.97, 124.27, 124.05, 123.87, 123.80, 122.05, 122.00, 121.06, 120.90, 120.67, 120.27, 120.21, 120.17, 119.47, 116.52, 114.82, 113.79, 111.10, 110.08, 110.03, 108.42, 108.34, 108.27, 108.22, 107.21, 107.13, 107.05, 106.97, 106.85, 106.60, 103.95, 103.65, 103.62, 103.44, 93.73, 93.37, 93.24. HRMS (ESI-TOF) calcd for C45H25O17=837.1097, found 837.1091.
3i was prepared from 6,3′,4′-trihydroxyflavone. 85 mg, 41% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(3i*)=10.218 min, tr(3i**)=11.126 min, concentration of 3i=3.73 mM. 3i=3i*+3i**. Atropisomer 3i*: 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J=8.5 Hz, 2H), 7.24 (d, J=8.4, 1H), 7.21 (d, J=8.3, 1H), 7.17-7.03 (m, 3H), 6.96 (d, J=8.4, 1H), 6.93 (d, J=8.3, 1H), 6.90 (d, J=8.5 Hz, 1H), 6.70 (s, 1H), 6.56 (s, 1H), 6.24 (s, 1H), 6.02 (s, 1H), 5.93 (s, 1H). Atropisomer 3i**: 1H NMR (400 MHz, DMSO-d6) δ 7.44 (d, J=8.5 Hz, 2H), 7.24 (d, J=8.4, 11H), 7.21 (d, J=8.3, 1H), 7.17-7.03 (m, 3H), 6.96 (d, J=8.4, 1H), 6.93 (d, J=8.3, 1H), 6.90 (d, J=8.5 Hz, 1H), 6.70 (s, 1H), 6.54 (s, 1H), 6.09 (s, 1H), 6.05 (s, 1H), 5.97 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.28, 182.23, 181.80, 181.72, 176.92, 176.89, 166.52, 166.31, 165.61, 164.24, 164.19, 162.33, 162.17, 161.97, 159.36, 159.32, 159.10, 159.06, 157.03, 156.89, 156.80, 155.05, 150.13, 150.09, 149.87, 149.83, 148.83, 148.77, 148.30, 146.19, 144.71, 144.68, 144.58, 124.91, 124.88, 124.28, 124.23, 124.18, 124.02, 123.25, 122.09, 122.06, 120.81, 120.65, 120.41, 120.19, 120.08, 120.04, 119.50, 119.44, 116.52, 114.81, 113.87, 108.50, 108.39, 108.28, 107.97, 107.92, 107.82, 107.79, 106.76, 106.58, 103.99, 103.62, 103.48, 93.80, 93.74, 93.38, 93.26. HRMS (ESI-TOF) calcd for C45H25O17=837.1097, found 837.1091.
3j was prepared from 7,3′,4′-trihydroxyflavone. 98 mg, 47% yield, brown solid. HPLC (ACN/Water=72.5:27.5, λ=300 nm), tr(3j*)=13.331 min, tr(3j**)=13.331 min, concentration of 3j=2.54 mM. 3j=3j*+3j**. Atropisomer 3j*: 1H NMR (400 MHz, DMSO-d6) δ 7.73 (td, J=8.7, 6.5 Hz, 1H), 7.49 (d, J=8.1 Hz, 2H), 7.24 (d, J=8.4 Hz, 1H), 7.14 (d, J=8.3 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 6.81 (dd, J=8.7, 6.5 Hz, 1H), 6.71 (s, 1H), 6.56 (s, 1H), 6.52 (d, J=6.5 Hz, 1H), 6.27 (s, 1H), 6.05 (s, 1H), 5.90 (s, 1H), 3.18 (s, 1H). Atropisomer 3j**: 1H NMR (400 MHz, DMSO-d6) δ 7.73 (td, J=8.7, 6.5 Hz, 1H), 7.49 (d, J=8.1 Hz, 2H), 7.24 (d, J=8.4 Hz, 1H), 7.14 (d, J=8.3 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 6.81 (dd, J=8.8, 6.5 Hz, 1H), 6.70 (s, 1H), 6.54 (s, 1H), 6.52 (d, J=6.5 Hz, 1H), 6.09 (s, 1H), 5.93 (s, 1H), 5.92 (s, 1H), 3.18 (s, 1H). 13C NMR (126 MHz, DMSO) δ 206.96, 182.26, 182.19, 181.80, 181.71, 176.37, 166.50, 166.20, 165.23, 165.15, 164.20, 164.16, 162.87, 162.28, 162.15, 161.95, 159.36, 159.31, 159.11, 157.97, 157.04, 156.90, 156.86, 156.77, 150.11, 150.07, 148.80, 148.74, 148.22, 148.18, 146.18, 144.68, 144.64, 144.52, 126.76, 126.74, 124.88, 124.20, 124.08, 122.08, 122.04, 120.27, 120.21, 120.06, 120.02, 119.47, 116.53, 116.18, 116.14, 115.19, 114.84, 113.89, 108.57, 108.40, 108.32, 108.21, 106.83, 106.66, 103.99, 103.96, 103.64, 103.45, 102.41, 93.85, 93.73, 93.30. HRMS (ESI-TOF) calcd for C45H25O17=837.1097, found 837.1086.
3k was prepared from 7,3′,4′-trihydroxyflavone. 88 mg, 43% yield, brown solid. HPLC (ACN/Water=71.5:28.5, λ=300 nm), tr(3k)=15.699 min, concentration of 3k=0.76 mM. 3k=3k*+3k**. Atropisomer 3k*: 1H NMR (500 MHz, DMSO-d6) δ 13.20 (d, J=13.1 Hz, 1H), 12.94 (d, J=20.1 Hz, 1H), 10.37 (s, 8H), 7.94 (d, J=8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.8 Hz, 1H), 7.13 (d, J=7.9 Hz, 1H), 6.93 (q, J=10.4 Hz, 3H), 6.78 (t, J=5.9 Hz, 2H), 6.53 (s, 1H), 6.44 (s, 1H), 6.22 (s, 1H), 5.98 (s, 1H), 5.86 (s, 1H). Atropisomer 3k**: 1H NMR (500 MHz, DMSO-d6) δ 13.20 (d, J=13.1 Hz, 1H), 12.94 (d, J=20.1 Hz, 1H), 10.37 (s, 8H), 7.94 (d, J=8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 1H), 7.22 (d, J=8.8 Hz, 1H), 7.13 (d, J=7.9 Hz, 1H), 6.93 (q, J=10.4 Hz, 3H), 6.78 (t, J=5.9 Hz, 2H), 6.53 (s, 1H), 6.44 (s, 1H), 6.12 (s, 1H), 5.91 (s, 1H), 5.89 (s, 1H). 13C NMR (126 MHz, DMSO) δ 182.23, 181.77, 176.41, 166.57, 166.38, 165.29, 163.97, 162.50, 161.55, 159.38, 159.28, 159.10, 157.99, 156.96, 148.81, 148.26, 144.76, 144.62, 128.96, 126.68, 126.67, 124.76, 124.20, 121.80, 120.55, 120.24, 120.12, 116.41, 114.64, 108.47, 108.38, 106.67, 106.66, 103.54, 103.39, 102.35, 102.32, 93.97, 93.37. HRMS (ESI-TOF) calcd for C45H25O1s=821.1148, found 821.1132.
Results and Discussion
The reaction in Example 1 can be extended to the synthesis of novel triflavonoids by reacting 2a (radical precursor) with nucleophilic flavones. These triflavones share the same type of interflavonyl bonds with the general formula of Lu-(2′-6)-Lu-(2′-6)-FL (
It is well-known that under alkaline conditions, flavonoids containing catechol moieties are sensitive to oxidation forming semiquinone radical intermediates (K. Kuwabara et al., Appl. Magn. Reson. 2018, 49, 911-924). However, the fates of these radicals were unclear and they are not harnessed for synthetic purposes, likely due to the formation of complex end-products. Our discovery is counter-intuitive and thus warrants an in-depth study on the key factors influencing the reaction outcome so that we can rationally maximize the yield and selectivity for synthetic use. These factors include pH, counter cations, and oxygen availability in the solution.
Determination of the pKa Values of Luteolin by 13C NMR Spectroscopy
Luteolin (200 mg) was added in the three-necks round bottle flask fitted with a pH meter under argon (
Where LH4, LH3−, LH22−, LH3− and L4− are the species of luteolin in fully protonated form, first deprotonated, second deprotonated, third deprotonated 100% deprotonated form.
The chemical shift of the carbons in luteolin is dependent on the relative concentrations of the conjugate pair. For a solution at pH within pKa1 range, and the species in solution is ˜100% protonated (LH4), and the chemical shifts are that of the protonated species, δlow−1. For a solution at a higher pH the species in solution is first deprotonated (LH3−), and the chemical shifts are that of the deprotonated species, δhigh−1.
Equation 7-8 were applied to determine the mole fraction of conjugate pair from chemical shift of a specific carbon. The assignment of 13C NMR of luteolin was further confirmed by HSQC and HMBC methods with 2D correlation with 1H NMR.
Quantitative Measurement of Luteolin Radicals
The area under curve of luteolin radical EPR spectrum (AUCL) are in linear relationship with the concentration of luteolin radicals, then concentration of luteolin radical was calculated using manganese (inside sealing putty) as reference and TEMPO (25.17 mM) as standard (Equation 9).
pH Dependent EPR Spectrum of Luteolin Radicals
Luteolin (28.6 mg, 0.1 mmol) dissolved in 10 mL KOH of different concentrations, the EPR signal of aqueous luteolin was detected immediately after transferring into EPR tube. The pH of solutions was determined after EPR measurements. Acetone and methanol were introduced to improve solubility only for samples in alkaline solution with pH<10. Numerical data were statistically analyzed by using Origin 8.0 software package (Origin Lab, Northampton, MA).
Time-Dependent EPR Spectrum of Luteolin Radicals
Luteolin (28.6 mg, 0.1 mmol) was dissolved in KOH (10 mL, 0.02 M) to give luteolin aqueous solution with pH value of 11.17. The solution was divided into tube A and tube B equally. In tube A, the solution was under stirring all the time, while in tube B, there was no stirring. The EPR signal of aqueous luteolin in both tubes was recorded immediately every 5 min after transfer into EPR tube.
Luteolin Oxidative Coupling Reaction
Luteolin (214.5 mg 0.75 mmol) was added into a 250 mL round bottle flask. To the flask, alkaline water (50.0 mL KOH solution 0.03 M) was introduced to dissolve the luteolin and resulted in a solution with pH of 11.78. The reaction was real-time monitored in an air-tight system as shown in
Results and Discussion
Optimal Reaction pH
Oxidative cross-coupling of two luteolin occurred in a narrow pH range between 9.5 to 12.5, with optimal pH at 11.5 (
The first deprotonation occurred at C(7)-OH with pKa1 of 8.00. This value is about two units larger than literature values (˜6.0) (Ŝ. Rameŝová, R. S. J. Tarebek & I. Deganoca, Electrochim. Acta 2013, 110, 646-654). Our value agrees with the observation that luteolin has poor solubility in water or slightly basic aqueous solution. The pKa2 was found at 8.93 (C(4′)-OH). The pKa3 and pKa4 are close to each other at 12.78 (C(3′)-OH) and 13.03 (C(5)-OH), respectively (
To probe the presence of luteolin radical anions, we measured the EPR spectra of air-saturated luteolin solutions in different pH and found that oxidation of LuH22− only occurred significantly at pH above 9.5 (
LuH22−+O2→LuH2·−+O2·− (1)
HO−+LuH2·−→LuH·2−+H2O (2)
Thus, the pKa value of the unobserved intermediate [LuH2-] dictates the lower limit of the pH range of the reaction. From the EPR signal intensity plots against different pH, the pKa value of LuH2·− is estimated to be 9.65 (
The ortho semiquinone radicals of other flavones with catecholic B rings were detected and C2's also had the highest spin density (
Based on these observations, we proposed a coupling reaction mechanism (
Impact of Counter Cations in the Coupling Reaction
For two dianions (LuH·2− and LuH22−) to react, the charge repulsions have to be overcome possibly by ion pairing with countercation. Therefore, we examined the effects of different counter cations on the reaction and we found that tetramethylammonium (Me4N+, added as Me4NOH) gave the lowest yield (<20%). Lithium performed better but not as good as cesium, sodium and potassium (˜80%) (
Impact of Oxygen Availability
Although ortho-semiquinone radical of luteolin has been observed previously, the end-products were found to be complex not characterized (Ŝ. Rameŝová, R. S. J. Tarebek & I. Deganoca, Electrochim. Acta 2013, 110, 646-654), likely due to overoxidation by excessive oxygen in the solution. The positive outcome of our case is likely due to limiting oxygen and by conducting the reaction unstirred, which is counter-intuitive. We compared the reaction dynamics of alkaline luteolin solution in two test tubes; one tube was magnetically stirred vigorously (so that oxygen is in excess supply) while the other tube was not stirred (oxygen availability is dependent on the diffusion of the gas phase oxygen into solution). The coupled products in the stirred tube could not be detected after 4 hours, while in the unstirred tube, both 2a and 3a showed two major products after 10 hours (
It was suggested that, moss utilizes a large amount of bioresource in the synthesis of luteolin dimers and trimers, because of the need to defend against the microbial stress endured by the moss while growing on rotting wood in wet forests (H. Geiger et al., J. Hattori Bot. Lab. 1997, 83, 273-308). To test this hypothesis, we measured the antifungal activity of luteolin, 2a, and 3a using Aspergillus niger as a model fungus (
Antifungal Activity Study
The antifungal activity was evaluated against two fungi, Aspergillus niger ATCC 16888 and Botrytis cinerea ATCC 11542, isolated and identified with ITS gene (GenBank accession number AY373852, A. S. Taha et al., BioResources 2019, 14, 6025-6046). The bioassay was evaluated using the radial growth technique method (P. Wayne, Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. CLSI document M27-A2, 2002). Both fungal strains were cultivated aerobically on PDA medium at 25° C. for 14 days. The fungal spores were collected with sterile cotton swabs and suspended in sterile water, and the concentrations were adjusted according to the requirements of the antifungal assays to be performed.
Determination of In Vitro Antifungal Activity
Flavonoids oligomers were dissolved in DMSO (1.0 mg/mL), then transferred into sterilized warm PDA medium (40 to 45° C.) to achieve a final concentration of 1.0 mg/mL, 0.6 mg/mL and 0.4 mg/mL, before immediately pouring into 24-well sterile plastic microtitration plates containing flat-bottomed wells (Corning Incorporated, costar). After the plates were cooled to room temperature, 10 μL of freshly made A. niger suspension (1.25×10−6/mL) was inoculated onto the agar of each well. Drug-free agar with 1% DMSO was used as the negative control and amphotericin B was used as the positive control.
From a 7-day-old colony, the fungus with discs of 9 mm diameter was transferred to the center of the treated PDA plates and controls. All the plates were incubated at 26±1° C. for 7 days. All the tested concentrations, as well as positive and negative controls, were measured in triplicate.
High-Throughput Assay of Starch Hydrolase Inhibition Activity
The inhibitory activity of each fraction obtained on α-amylase and α-glucosidase were determined using the turbidity measurement according to a reported method (Liu, Song, Wang, & Huang, 2011, Journal of agricultural and food chemistry 2011 Vol. 59 Issue 18 Pages 9756-9762). Corn starch (20 mg/mL) was suspended in sodium phosphate buffer (0.1 M, pH 6.9) and gelatinized at 100° C. for 2.5 min. The inhibitor solution was diluted in buffer to appropriate concentrations. Acarbose was used as a positive control and a reference standard. α-amylase solution (2 U/mL in buffer, 20 μL) or α-glucosidase solution (1×10−2 U/mL in buffer) was pre-incubated with inhibitor solution (20 μL) with series of concentrations in a 96-well microplate and kept at 37° C. for 15 min. The reaction was initiated by injecting 60 μL of the gelatinized corn starch solution. The turbidity changes were recorded at 660 nm every minute for 2 h using a Synergy HT microplate reader (Biotek Instruments Inc., Winooski, VT, USA). The inhibition percentage was calculated using Equation 10.
Inhibition (%)=(AUCsample−AUCcontrol)/AUCsample×100% (Equation 10)
Where AUCsample is the area under the inhibitory curve and AUCcontrol is the area under the curve negative control. The IC50 can be defined as the concentration of an inhibitor that produces 50% inhibition of enzyme activity under a specified assay condition. It was obtained from interpolation of percentage of inhibition against inhibitor concentration curve. In order to avoid run-to-run error due to fluctuation of enzymatic activity, the acarbose equivalent (AE) was used to express the inhibitory activity of sample based on the following equation:
AE of a sample=IC50 of acarbose/IC50 of a sample (Equation 11)
Molecular Docking
Ligand preparation: All ligands including acarbose, luteolin, 2a, 2b and 3a were selected as the ligands and virtually constructed through their crystal structures. These ligand molecules were drawn and saved as 3D conformers in .cif format. The structure of these ligands was then converted into .pdb format via PyMol. Subsequently, the ligand molecules were uploaded as an input file using .pdb format onto Autodock, followed by output as .pdbqt format file.
Preparation of protein molecule: The active center of a mammalian alpha-amylase, 1ppi, was selected and retrieved in a .pdb format file from RCSB (M. Qian et al., Biochemistry 1994, 33, 6284-6294). The target amylase was loaded on the graphical user interface of Autodock (G. M. Morris et al., J. Comput. Chem. 1998, 19, 1639-1662; and R. Huey et al., J. Comput. Chem. 2007, 28, 1145-1152) in .pdb format. The amylase was prepared for docking by removal of the acarbose molecule, deleting water molecules, adding polar hydrogen atoms and adding Kollman charges to the macromolecule. Thereafter, the amylase was converted from a .pdb format to a .pdbqt format file. A grid box was selected and adjusted to specific dimensions of the docking site. The output file of the grid dimensions was saved as a .txt file.
Docking through AutoDock Vina and visualization using PyMOL: AutoDock Vina (O. Trott & A. J. Olson, J. Comput. Chem. 2010, 31, 455-461) is a virtual screening technique to predict the optimal bound conformations of ligands to a target protein of known structure. To conduct Autodock, the prepared ligands and amylase were used in .pdbqt format and a configuration file was set up in a .txt file. Docking through AutoDock Vina was executed using command prompt and the results of the docking were analysed through PyMOL. PyMOL is a molecular visualization program widely used for three-dimensional (3D) visualization of proteins and small molecules. The output .pdbqt file from AutoDock vina and amylase in .pdbqt format was loaded on the graphical interface of PyMOL. There, the docked structure was visualized under the “molecular surface” and “cartoon” option. The active site of the docking was shown using the “pockets” function of PyMOL.
Results and Discussion
2a and 3a could inhibit the growth of A. niger with IC50 of 0.86 μM and 0.96 μM, respectively, which is comparable to that of amphotericin B (IC50 of 0.50) in a dose-dependent manner. Notably, dimer 2a shows slightly higher activity than trimer (3a). Plant flavonoids protect the plant from being eaten by insects by inhibiting digestive enzymes such as alpha-amylase and alpha-glucosidase. We measured the activity of selected flavone dimers and trimer (2a-2f, 3-3f) in inhibiting α-amylase and α-glucosidase (
A 10% (w/v) starch solution was prepared by addition of distilled water to reagent grade corn starch and heating the solution it in a boiling water bath for about 15 minutes until it was completely gelatinized.
8-week-old male wild-type C57BL/6J mice were fasted overnight (free water intake), and weighed the next day. The tail of the mice was trimmed and the blood was taken for measurement of fasting blood glucose concentration with a Roche blood glucose meter.
Feeding method a, “YX2+Starch”: Luteolin dimer (2a) was gavaged as a 2 mg/mL water solution, at the dosage level of 20 mg/kg body weight of the animal. Thirty minutes later, the 10% (w/v) starch solution was gavaged at 1 g/kg body weight.
Feeding method b, “Starch (+YX2)”: Luteolin dimer (2a) was mixed with starch at 0.2% ratio by dried weight and mixed with water (1:9 by weight) to give a slurry labelled as Starch (+YX2) (
The blood glucose values were measured at 30, 60, 90, 120 minutes to make the blood glucose curve, GraphPad draws the blood glucose response curve after meal, and the area under the curve was calculated.
Results and Discussion
The glycemic response of the mice fed with different samples is shown in
From the results (
Feeding the mice only with luteolin dimer (YX2) at 20 mg/kg body weight did not lead to glycemic response.
If the YX2 was fed at 20 mg/kg dose 30 minutes before administration of starch at 1 g/kg body weight), there was no glycemic response (curve YX2+starch). In contrast, if the mice were fed with 0.2% mixture of YX2 in starch, the glycemic response was comparable to that of the mice fed only with same dose of starch (1 g/kg body weight) (curve: starch (+YX2).
Finally, if the mice were fed with 0.2% mixture of luteolin with starch, the glycemic response curve was almost the same as that of the mice fed only with same dosage of starch. This results suggested that for luteolin dimer 2a to be effect in suppressing glycemic response after consumption of starch, 2a needs to be taken 30 minutes before meal.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202012372V | Dec 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050779 | 12/10/2021 | WO |
