ES2948561A1 - COMPOUNDS FOR FLUORESCENT LABELING (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Fluorescent labeling compounds. The present invention refers to compounds with a BODIPY structure with good properties for the specific fluorescent labeling of C-nucleophiles, as well as its obtaining procedure that allows efficiently and economically the preparation of a great diversity of compounds directly, quickly. , and with good reaction yields. The application of these compounds includes, although not exclusively, their use as biological markers for the selective staining of living cells and microorganisms, as fluorescent probes in biosensors in bioimaging of living organisms or energy harvesters in solar cells, or as emitters in lasers. dye or as emitters in OLED systems.

Description

DESCRIPTION

Fluorescent labeling compounds

The present invention refers to compounds with a BODIPY structure with good properties for the specific fluorescent labeling of C-nucleophiles, as well as its obtaining procedure that allows efficiently and economically the preparation of a great diversity of compounds directly, quickly. , and with good reaction yields. The application of these compounds includes, although not exclusively, their use as biological markers for the selective staining of living cells and microorganisms, as fluorescent probes in biosensors in bioimaging of living organisms or energy harvesters in solar cells, or as emitters in lasers. dye or as emitters in OLED systems.

BACKGROUND OF THE INVENTION

The development of general, selective, efficient and highly versatile functionalization methods is currently one of the main challenges in the chemistry of 4-bora-3a,4a-diaza-s-indacenes (BODIPYs). Although methodologies have been described that allow these types of transformations to be carried out in all positions of BODIPY, the vast majority involve the use of multi-stage routes (substitutions in the pyrrole or in the precursor dipyrromethane) individually adapted to each final target dye. sought (Clarke, RG; Hall, MJ Adv. Heterocycl. Chem. 2019, 128, 181-261), with the disadvantages that this entails in terms of the greater synthetic effort required, overall loss of chemical performance and increased economic costs. For these reasons, there is great interest in the development of direct methods of functionalization of pre-formed BODIPYs, generally known as postsynthetic methods, that allow introducing the new functionality in a single and final reaction step, ideally.

In general, the postfunctionalization strategies of BODIPYs (Boens, N.; Verbelen, B.; Ortiz, MJ; Jiao, L.; Dehaen, W. Coord. Chem. Rev. 2019, 399, 213024) require prior introduction of a reactive atom or group (halogen, formyl, methyl, thioether, or even hydrogen) that facilitates the desired final functionalization. These reactive groups can be directly attached to the backbone of the chromophore (by boron atom or to the carbon atoms of the boradiazaindacene nucleus) or on peripheral positions, one or more bonds away from the chromophoric nucleus. Among the most versatile prefunctionalizations described for BODIPYs are (see: Boens, N.; Verbelen, B.; Ortiz, MJ; Jiao, L.; Dehaen, W. Coord. Chem. Rev.

2019, 399, 213024): 1) the very presence of fluorine atoms in boron that allows its substitution by C-, O- and N-substituents; 2) the introduction of halogens into the boradiazaindacene skeleton, which allows carrying out aromatic nucleophilic substitution and/or cross-coupling reactions catalyzed by transition metals; 3) the introduction of a thioalkyl group or a halogen in the (C8) position ( meso), which enables its direct substitution with heteronucleophiles (alcohols, amines, thiols) or cross-coupling reactions (Liebeskind-Srogl coupling); 4) the methyl groups directly attached to the chromophoric skeleton, which have a certain acidic character due to the electronic delocalization of the corresponding carbanion, which allows their activation with soft bases in Knoevenagel-type condensation reactions or electrophilic attack with halogenation reagents and subsequent nucleophilic substitution; 5) the absence of substituents in the C3 and C5 positions that enables the vicarious nucleophilic hydrogen substitution (VNS), its oxidative analogue (ONSH) and the CH functionalization catalyzed by transition metals or radicals, which in turn allow the direct introduction of C- and heterosubstituents in these positions. All of these methods present limitations in terms of the variety of substituents that can be introduced under the same reaction conditions (generally, carbonated and heteroatomic substituents require different reaction conditions), the type of “reactive” functionalization necessary that the new substituents must present. to participate in the reaction, which affects its price, availability and structural diversity, the chemical compatibility between the pre-existing functional groups in the BODIPY and in the new substituent under the reaction conditions and, of course, the chemical yield of the reaction post-functionalization.

Therefore, it is necessary to design new compounds with a BODIPY structure with good properties for specific fluorescent labeling and obtained in an efficient and economical manner, which allow the preparation of a great diversity of compounds directly, quickly, with good yields. reaction.

DESCRIPTION OF THE INVENTION

In the present invention, a new general, efficient and economical method of postfunctionalization of BODIPYs has been developed, using catalytic and mild reaction conditions, and that does not require prefunctionalization of the substituent to be incorporated, which allows the introduction of a great diversity of substituents. directly, quickly, with good yields and under similar reaction conditions.

In a first aspect, the invention relates to a compound of general formula (I) or any of its salts or isomers (hereinafter “compound of the invention”):

where:

Z is selected from a nitrogen atom (N) or a C(R?) group;

R6 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted _C2 -C18 alkenyl, substituted or unsubstituted _C2 -C18 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, -OR', -NR'R”, -CN, -COR', -COOR', -CONR'R”, -S¡R'R”R'” , -S¡R'R”(OR'” ) , -Si(OR') ₃ , -PR'R”, -P(=O)R'R”, -P(=O)(OR')(OR”), -P(=O)(OR' )(NR” R” '), -P(=O)(NR'R”)(NR” 'R” ”), -SR', -SOR', -SO ₂ R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O)R', -TeR', -Te(=O)R';

R2 to R5 are each independently selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted _C2 -C18 alkenyl, substituted or unsubstituted _C2 -C18 alkynyl, substituted or unsubstituted aryl, substituted heteroaryl or unsubstituted, halogen, -OR', -NR'R”, -N3, -NR'(C=O)R”, -NR'C(=O)OR”, -NR'C(=O)NR” R” ', -NR'(C=S)NR”R'” , -NR'S 02R”, -COR', -COOR', -CONR'R”, -SiR'R”R'” , -SiR'R ”(OR'” ), -Si(OR') ₃ , -PR'R”, -P(=O)R'R”, -P(=O)(OR')(OR”), -P( =O)(OR')(NR” R'” ), -P(=O)(NR'R”)(NR” 'R” ”), -SR', -SOR', -S02R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O )R', -TeR', -Te(=O )R';

R', R", R'" and R"" are each independently selected from hydrogen, C i-C 1s alkyl, C2-C18 alkenyl, C2-C18 alkynyl, aryl or heteroaryl;

R7 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, -CN, -CH ₂ OR', -CH ₂ 0(C= O )R', substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

R1 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, -CN, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, heteroaryl substituted, or unsubstituted or a group of formula (la):

where R2, R3, R4 and R6 are defined above and R5 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl substituted, substituted or unsubstituted heteroaryl, halogen, -OR', -NR'R”, -N3, -NR'(C=O )R”, -NR'C(=O )0 R”, -NR'C (=O )NR”R” ', -NR'(C=S)NR”R” ', -NR'S02R”, -COR', -COOR', -CONR'R”, -SiR'R”R ” ', -S¡R'R”(OR'” ), -Si(OR') ₃ , -PR'R”, -P(=O )R'R”, -P(=O )(0 R ')(0 R”), -P(=O )(0 R')(NR” R” '), -P(=O )(NR'R”)(NR” 'R” ” ), -SR ', -SOR', -SO ₂ R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O )R', -TeR' , -Te(=O )R' or a group of formula (la);

where each R2 to R6 of the groups (la) included in compound (I) and compound (I) are selected independently.

In a preferred embodiment of the compound of the invention Z is a C(R?) group, more preferably R7 is a C1-C6 alkyl group or a phenyl group, optionally substituted by at least one C1-C6 alkyl group, preferably R7 is a methyl group or a phenyl group. When R7 is a phenyl group it is preferably substituted by at least one methyl group.

In another preferred embodiment of the compound of the invention, R6 is a C1-C6 alkyl group, preferably it is a methyl.

In another preferred embodiment of the compound of the invention, R2 to R5 are each independently selected from a C1-C6 alkyl group. More preferably, R2 and Rs are an ethyl group and/or R3 and R4 are a methyl group.

In another preferred embodiment of the compound of the invention, the compound is of general formula (II):

where R1 has been described above.

In a more preferred embodiment, R1 is selected from a group -CH ₂ -CH ₃ , -CH ₂ -CH=CH ₂ , -CH(CO-CH ₃ ) ₂ , -CN, a phenyl group substituted by at least one -OH group or its corresponding tautomer where the group is a cyllohexadiene substituted by a =O group, azulyl substituted by at least one (C1-C4) alkyl group, a pyrrole group optionally substituted by at least one group of formula (la), an indole, and a group of formula (la).

In another preferred embodiment of the compounds of the invention R1 is selected from

the groups -CH ₂ -CH ₃ , -CH 2-CH=CH ₂ , -CH(CO-CH ₃ ) ₂ , -CN,

In another preferred embodiment of the compounds of the invention, Ri is a pyrrole group substituted by at least one group of formula (la):

where Z, R2 to R4 and R6 are described above and R5 is described above and preferably R5 is selected from hydrogen, C1-C6 alkyl or is a group of formula (la), where in turn Z and R2 to R6 have been described above and are independently selected from the same radicals of group (la) and compound (I).

In a preferred embodiment, the compounds are selected from:

The compounds of the invention are probes that can be used in biological labeling, in selective staining of cells and living microorganisms, as biosensors. in bioimaging of living organisms, in technological applications such as energy collectors in solar cells or as emitters in dye lasers or in OLED systems or similar. The developed methodology allows simple labeling in a single reaction step of biological molecules such as amino acids (tyrosine), resveratrol and guayazulene; probes with acetylacetone group for complexation of metal ions; or redox probes functionalized with a hydroquinone or quinone group, among others. The simplicity, generality and efficiency of the preparation method allows it to be extended to many other photonic and biophotonic applications.

Therefore, another aspect of the invention refers to the use of the compound of general formula (I) of the present invention, as a marker or fluorescent probe. More preferably as a biological marker for selective staining of living cells and microorganisms.

In a preferred embodiment, the compounds of the invention are fluorescent probes in biosensors for obtaining bioimaging of living organisms or cells.

In a preferred embodiment, the compounds of the invention are used as energy collectors in solar cells, as emitters in dye lasers or as emitters in OLED systems.

Obtaining the compounds of the invention is general, efficient and economical, where catalytic and mild reaction conditions are used and does not require prefunctionalization of the substituent to be incorporated into Ri, thus allowing the introduction of a great diversity of substituents directly, quickly, with good yields and under similar reaction conditions.

A further aspect of the present invention relates to the chemical transformation of a compound of general formula (III) into the final compound of this invention also of general formula (I) by reaction with a molecule of general formula RiH where the carboxyalkyl group has been replaced by the Ri grouping, with H being a hydrogen atom or an unshared pair of electrons.

Therefore, another aspect of the invention refers to a procedure for obtaining the compounds of general formula (I) that comprises: reacting a compound CN-BODIPY, compound of formula (III), with an acetoxyalkyl group (OAc) in the C3 and/or C5 position with a Ri-H compound in the presence of an acid catalyst (preferably Bronsted or Lewis).

where R1 to R6 and Z are defined above.

In a preferred embodiment, the starting CN-BODIPYs are obtained by reacting the corresponding F-BODIPYs with trimethylsilyl cyanide (TMSCN) in the presence of SnCL at room temperature using dichloromethane as a solvent. The reaction proceeds quickly (approx. 30 min) and in most cases the CN-BODIPYs are obtained in quantitative yields after aqueous extraction of the reaction and removal of the solvent under reduced pressure, without the need for chromatographic purification.

The term "alkyl" refers, in the present invention, to saturated, linear or branched hydrocarbon chains, having from 1 to 18 carbon atoms, for example, methyl, ethyl, n-propyl, /-propyl, n-butyl , tert-butyl, sec-butyl, n-pentyl, n- hexyl, etc. Preferably the alkyl group has between 1 and 6 carbon atoms. The alkyl groups may be optionally substituted by one or more substituents such as alkynyl, alkenyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "alkenyl" refers, in the present invention, to unsaturated hydrocarbon chains, linear or branched, having 2 to 18 carbon atoms, preferably 2 to 6, and containing one or more carbon-carbon double bonds and which may optionally contain some triple bond, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2-butenyl, 1,3-butadienyl, etc. The alkenyl radicals may be optionally substituted by one or more substituents such as alkyl, alkynyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "alkynyl" refers to radicals with hydrocarbon chains, linear or branched, of 2 to 18 carbon atoms, preferably 2 to 6, and containing at least one or more carbon-carbon triple bonds and which may optionally contain some double bond, for example, ethylino, propynyl, butynyl, etc. The alkynyl radicals may be optionally substituted by one or more substituents such as alkyl, alkenyl, halo, hydroxyl, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "aryl" refers, in the present invention, to single or multiple aromatic rings that may be fused, and that have between 5 to 18 carbon atoms in the ring part, such as, but not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthril, azulyl, fluorenyl or anthracyl. Preferably the aryl group has 5 to 10 carbon atoms and more preferably the aryl group is a phenyl or azulyl. The aryl radicals may be optionally substituted in any of their positions by one or more substituents and are independently selected from such as alkyl, alkenyl, alkynyl, O-alkyl, O, halogen, hydroxyl, amino or acyl. In a preferred embodiment the aryl is a phenyl or azulyl, optionally substituted by at least one O, hydroxyl, O-alkyl or halogen group.

The term "heteroaryl" refers to an aryl, as defined above, that contains at least one atom other than carbon, such as S, N, ÓO, forming part of the aromatic ring. Examples of heteroaryl groups include, but are not limited to, pyrrole or indole. The heteroaryl is optionally substituted with one or more substituents and is independently selected from such as alkyl, alkenyl, alkynyl, O alkyl, O, halogen, hydroxyl, amino, acyl or a group of formula (la). In a preferred embodiment the heteroaryl is a pyrrole group, optionally substituted by at least one group of formula (la).

By "halogen" in the present invention is understood a bromine, chlorine, iodine or fluorine atom.

The term “isomer” includes enantiomers depending on their asymmetry or diastereoisomers, as well as tautomers when the two isomers differ only in the position of one functional group.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Normalized fluorescence absorption and emission spectra of the C-nucleophile derivatives 5 (with allyl substituent) and 11 (with guayazulene substituent) in different solvents: AcOEt (black dots), MeOH (gray) and PBS ( black).

Fig. 2. Fluorescence quenching phenomenon of compound 11 by photoinduced electron transfer (PET), modeled by density functional theory (DFT, B3LYP/6-311+G*) calculations.

Fig. 3. Specific subcellular staining. A) Staining of HeLa cells with 4 (prepared from diethylzinc) at a concentration of 100 nivi. White scale bar: 10 μm.

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

Example 1. Synthesis of starting compound 3

Compound 2

To a solution of commercial F-BODIPY 1 (300 mg, 0.94 mmol) in CH ₂ Cl ₂ (3 mL), added cyanotrimethylsilane (TMSCN) (0.84 mL, 6.6 mmol) and stannous chloride (SnCL) (0.056 mL, 0.47 mmol) at room temperature. After stirring the reaction mixture at room temperature for 30 min, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with an aqueous NaHC03 solution (0.1 M) and a saturated NaCl solution (20 mL), dried over _anhydrous sodium sulfate ( _Na2SO4 ), filtered and the solvent was removed under reduced pressure. , to obtain 2 (313 mg, 100%) as a red-orange crystalline solid of high purity.

Compound 3

To a stirred solution of lead acetate (Pb(OAc)4) (1.25 g, 2.81 mmol) in dichloromethane (CH ₂ Cl ₂ ) (5 mL), a solution of CN-BODIPY was added dropwise 2 (390 mg, 1.17 mmol) in acetic acid/acetic anhydride (ACOH/AC2O) mixture (20:1 v/v, 20 mL) at 0 °C under argon atmosphere. After stirring the reaction mixture at room temperature for 2.5 h, cold water (4 °C, 50 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 50 mL). The combined organic phases were washed with a sodium bicarbonate (NaHCO ₃ ) solution (0.1 M) and a saturated sodium chloride (NaCl) solution (20 mL), dried over anhydrous Na ₂ SO4 , filtered and removed. the solvent under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/ethyl acetate (AcOEt), 100:0 → 60:40), to obtain 3 (330 mg, 72%) as an orange solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 5.43 (2H, s, H1'), 2.70 (6H, s, CH ₃ 5 and CH38), 2.52 (2H, q, J = 7 .4 Hz, CH ₃ CH ₂ 2I. 2.48 (2H, q, J = 7.4 Hz, CH ₃ CH ₂ 6I. 2.41 (3H, s, CH ₃ 1), 2.40 (3H, s, CH ₃ 7), 2.16 (3H, s, H4'), 1.10 (3H, t, J = 7.4 Hz, CH ₃ CH ₂ 2I. 1.09 (3H, t, J = 7.4Hz, CH3CH26).

13C NMR (CDCl ₃ , 100 MHz): 5 = 170.9 (C3'), 157.8 (C5), 143.7 (C3), 142.9 (C8), 140.8 (C7), 136, 3 (C1), 136.2 (C6), 135.0 (C2), 132.3 (C7a), 130.6 (C8a), 127.1 (q, J _CB = 74 Hz, 2 x CN), 56.5 (C1'), 21.00 (C4'), 17.8 (CH ₃ CH26), 17.4 (CH ₃ CH ₂ 2/CH ₃ 8), 17.4 (CH ₃ 8/CH ₃ CH ₂ 2), 15.5 (CH ₃ CH ₂ 2), 15.1 (CH3CH ₂ 6), 14.7 (CH ₃ 7), 14.6 (CH ₃ 1), 14.1 (CH ₃ 5 ).

HRMS (API-ES+) m/z calculated for C ₂ 2H27BN4Na02 [M+Na]+ 413.2123; found 413.2138, Calculated for C22H31BN5O ₂ [M+NH4]+ 408.2569; found 408.2587.

Example 2. Synthesis of the compounds of the invention

The following scheme shows the synthesis of BODIPYs incorporating C-nucleophiles from the starting compound CN-BODIPY 3, using scandium trifluoromethanesulfonate Sc(OTf) ₃ (5-24) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (4) as catalyst, [a] cat: 0.025 equiv (5-9, 12-19, 23 and 24); 0.05 equiv (5, 6, 20 and 22); 0.08 equiv (21); 1.5 equiv (4). [b] Type of nucleophile: thymethylsilyl (TMS)Ri (5 and 7); diethylzinc (Et2Zn) (4); R1H (6, 8-24). [c] Equiv of R1H: 1 equiv (10, 23 and 24); 1.2 equiv (9); 1.4 equiv (11, 19); 1.5 equiv (4-8, 12-18); excess of 3 (1.5 equiv (20), 4.4 equiv (21) and 4.8 equiv (22)). [d] CH ₂ Cl ₂ (4-9 and 11-24); acetonitrile (MeCN) (10). [e] 10 required an increase in temperature, [f] Performance expressed as a mixture of regioisomers.

[g] They are formed by spontaneous oxidation of the corresponding hydroquinone [h] The products are obtained in the same reaction (14 and 15; 17 and 18; 23 and 24). [i] Ratio of isolated isomers 17:18 (1.4:1).

Hydroquinones (benzene-1,4-diols) were especially reactive nucleophiles under these conditions, also generating the C-alkylation product (13-17), instead of the phenyl ether product of O-alkylation, with very good yields and very fast way. Some of the hydroquinones thus alkylated were unstable to air/light, easily oxidizing to the corresponding quinone, which was sometimes the only product finally isolated from the reaction (13). The spontaneous oxidation reaction was faster the higher the degree of alkylation, that is, the more electronically rich the ring of the hydroquinone product, as expected.

Compound 4

To a stirred solution of 3 (16 mg, 0.041 mmol) in anhydrous CH ₂ Cl ₂ (1 mL), diethylzinc (15% w/w in hexane, 140.6 μL, 0.129 mmol) and TMSOTf (11.1 μL, 0.062 mmol) at room temperature under argon atmosphere (glove chamber). After stirring the reaction mixture at room temperature, under an argon atmosphere for 90 min, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/ethyl acetate (AcOEt), 95:5 → 60:40), to obtain 4 (7.3 mg, 49%) as an orange-red solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 3.00-2.90 (2H, m, H1'), 2.66 (3H, s, CH ₃ 5), 2.65 (3H, s, CH ₃ 8), 2.45 (2H, q, J= 7 .5 Hz, CH ₃ CH¿6), 2.46 (2H, q, J= 7 .5 Hz, CH ₃ CH¿2), 2, 38 (6H, s, CH ₃ 1 and CH ₃ 7), 1.86-1.72 (2H, m, H ₂ '), 1.16 (3H, t, J = 7.3 Hz, H3') , 1.11 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 2), 1.07 (3H, t, J= 7 .5 Hz, CH ₃ CH ₂ 6).

13C NMR (CDCl ₃ , 100 MHz): 5 = 156.3 (C3), 152.5 (C5), 141.0 (C8), 138.7 (C1/C7), 137.8 (C H C1) , 134.1 (C6), 133.8 (C2), 130.4 (C7a/C8a), 130.2 (C8a/C7a), 127.8 (q, J _CB = 74.8 Hz, 2 x CN ), 29.8 (C1'), 22.3 (C2'), 17.4 (CH ₃ CH ₂ 2/CH ₃ CH ₂ 6/CH ₃ 8). 17.4 (CH ₃ CH ₂ 6/CH ₃ 8/CH ₃ CH ₂ 2). 15.2 (CH ₃ CH ₂ 2/CH ₃ CH ₂ 6/CH ₃ 7/CH ₃ I/C 3'), 14.9 (CH ₃ CH26/CH ₃ 7/CH ₃ 1/C3'/CH ₃ CH ₂ 2), 14.9 (CH ₃ 7/CH ₃ I/C 37CH ₃ CH ₂ 2/CH ₂ CH ₂ 6), 14.8 (CH ₃ 1/C3'/CH ₃ CH ₂ 2/CH ₃ CH ₂ 6/CH ₃ 7), 13.6 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C ₂ 2H30BN4 [M+H]+ 361.2562; found 361.2573, Calculated for C ₂ 2H29BN4Na [M+Na]+ 383.2382; found 383.2391.

Compound 5

To a stirred solution of 3 (15 mg, 0.038 mmol) in anhydrous CH2Cl ₂ (2 mL) was added allyltrimethylsilane (9.2 pL, 0.058 mmol) and Sc(OTf) ₃ in anhydrous CH2Cl ₂ (96 pL of a 0 .01 M ^, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 2 h, water (10 mL) and CH2Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH2Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 80:20), to obtain 5 (12 mg, 84%) as a red-orange solid.1

1H NMR (CDCl ₃ , 400 MHz): 5 = 6.07-5.91 (1H, m, H3'), 5.22 (1H, dd, J= 1.2, 17.2 Hz, H4'frans ), 5.09 (1H, d, J = 10.2 Hz, H4'c/s), 3.09 (2H, m, H1'), 2.66 (6H, s, CH ₃ 5 and CH ₃ 8), 2.52 (2H, m, H ₂ '), 2.46 (4H, q, J ⁼ 7.5 Hz, CH ₃ CH72 and CH ₃ CH ₂ 6), 2.39 (6H, s, CH ₃ 1 and CH ₃ 7), 1.12 (3H, t, J= 7 .5 Hz, CH¿CH22), 1.07 (3H, t, J=7 .5 Hz, CH ₃ CH ₂ 6) .

13C NMR (CDCl ₃ , 100 MHz): 5 = 154.9 (C5), 153.0 (C3), 141.2 (C8), 138.5 (C7a), 138.2 (C8a), 137.2 (C3'), 134.3 (C7), 133.7 (C1), 130.5 (C6), 130.2 (C2), 127.7 (q, J _CB = 74 Hz, 2 x CN), 115.9 (C4'), 32.5 (C2'), 27.1 (C1'), 17.4 (CH ₃ CH22/CH ₃ CH26/CH ₃ 8), 17.4 (CH ₃ 8/CH ₃ CH72/CH ₃ CH76). 15.2 (CH ₃ CH72/CH ₃ CH76). 14.9 (CH ₃ CH76/CH ₃ CH ₂ 2), 14.9 (CH ₃ 7), 14.8 (CH ₃ 1), 13.7 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C ₂ 3H30BN4 [M+H]+ 373.2562; found 373.2556, Calculated for C ₂ 3H33BN5 [M+NH4]+ 390.2828; found 390.2826, Calculated for C ₂ 3H29BN4Na [M+Na]+ 395.2382; found 395.2367.

Compound 6

To a stirred solution of 3 (10 mg, 0.026 mmol) in anhydrous CH2Cl ₂ (2 mL), acetylacetone (4 mg, 0.038 mmol) and Sc(OTf) ₃ in anhydrous CH2Cl ₂ (64 μL of a 0. 01 M, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 24 h, water (10 mL) and CH2Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH2Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 80:20), to obtain 6 (10 mg, 90%) as an orange-red solid, almost equimolar mixture of the two tautomers. in CDCl ₃ at 25 0C (1H NMR).1

1H NMR (CDCl ₃ , 400 MHz): 5 = 4.82 (1H, t, J = 7.2 Hz, H ₂ b), 4.20 (2H, s, H1'b), 3.56 (2H , d, J = 7.2 Hz, H1b), 2.70 (3H, s, CH ₃ 5'/CH ₃ 8'), 2.70 (3H, s, CH ₃ 8'/CH ₃ 5') , 2.68 (6H, s, CH ₃ 5 and CH ₃ 8), 2.47 (4H, m, CH ₃ CH76 and CH ₃ CH76”). 2.41 (3H, s, CH ₃ I/CH ₃ I '), 2.40 (3H, s, CH ₃ 1'/CH ₃ 1), 2.38 (2H, m, CH ₃ CH72). 2.37 (3H, s, CH ₃ 7/CH ₃ 7'), 2.36 (3H, s, CH ₃ 7'/CH ₃ 7), 2.33 (2H, m, CH ₃ CH72'). 2.25 (6H, s, CH ₃ 4b and CH ₃ 6b), 2.19 (6H, s, 6H, s, CH ₃ 4'b and CH ₃ 6'b), 1.08 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 6'), 1.06 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 6), 1.01 (3H, t, J = 7.5 Hz , CH ₃ CH ₂ 2'), 0.91 (3H, t, J= 7 .5 Hz, CH¿CH22).

13C NMR (CDCl ₃ , 100 MHz): 5 = 202.9 (C3b and C5b), 192.1 (C3'b and C5'b), 155.0 (C5), 154.6 (C5'), 150 ,2 (C3'), 149.9 (C3), 141.9 (C8), 141.6 (C8'), 139.5 (C1'), 139.2 (C7), 139.1 (C7'), 138.0 (C1), 135.4 (C6), 135.2 (C6'), 134.9 (C2), 133.3 (C2'), 131.3 (C8a), 131.1 (C7a), 131 .0 (C7'a), 130.2 (C8'a), 128.3 (q, J _CB ⁼ 73.9 Hz, 2 x CN), 126.8 (q, J _CB = 73.9 Hz, 2 x CN'), 106.3 (C2'b), 65.1 (C2b), 30.7 (C4b and C6b), 28.6 (C1'b), 24.7 (C1b), 24.2 (C4'b and C6'b), 17.8 (CH38), 17.7 (CH ₃ 8'), 17.7 (CH ₃ CH¿2 '), 17.4 (CH3CH76 and CH ₃ CH76”). 17.2 (CH ₃ CH72I. 15.1 (CH ₃ 7), 15.0 (CH ₃ CH?21. 14.9 (CH ₃ 1 and CH ₃ 1'), 14.8 (CH ₃ CH ₂ 2 '), 14.7 (CH ₃ CH ₂ 6 v CH ₃ CH ₂ 6'), 14.5 (CH ₃ 7'), 13.9 (CH ₃ 5), 13.7 (CH ₃ 5'). HRMS (API-ES+) m/z calculated for C ₂ 5H35BN5O ₂ [M+NH4]+ 448.2883; found 448.2868, Calculated for C ₂ sH3iBN4Na02 [M+Na]+ 453.2437; found 453.2418, Calculated for C ₂ 5H32BN4O ₂ [M+H]+ 431.2617; found 431.2621.

Compound 7

To a stirred solution of 3 (20 mg, 0.051 mmol) in anhydrous CH2Cl2 (2 mL), TMSCN (9.6 μL, 0.077 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ (128 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 3 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 80:20), to obtain 7 (17 mg, 92%) as a red-orange solid.1

1H NMR (CDCl ₃ , 400 MHz): 5 = 4.14 (2H, s, H1'), 2.71 (3H, s, CH ₃ 5), 2.69 (3H, s, CH ₃ 8), 2.60 (2H, q, J= 7.6 Hz, CH ₃ CH¿2), 2.48 (2H, q, J= 7.6 Hz, CH ₃ CH¿6), 2.42 (3H, s, CH ₃ 7), 2.41 (3H, s, CH ₃ 1), 1.19 (3H, t, J= 7.6 Hz, CH3CH22), 1.09 (3H, t, J = 7, 6 Hz, CH ₃ CH ₂ 6).

13C NMR (CDCl ₃ , 100 MHz): 5 = 157.8 (C5), 142.9 (C3), 141.2 (C8), 137.3 (C7a), 136.9 (C8a), 136.3 (C7), 133.5 (C1), 132.0 (C6), 129.9 (C2), 126.3 (q, Jcb = 74 Hz, 2 x BCN), 114.5 (CN1'), 17 .6 (CH ₃ 8), 17.3 (CH ₃ CH72/CH ₃ CH76). 17.3 (CH ₃ CH76/CH ₃ CH72). 16.3 (C1'), 15.0 (CH ₃ CH72/CH ₃ CH76). 14.5 (CH ₃ CH76/CH ₃ CH ₂ 2), 14.4 (CH ₃ 1/CH ₃ 7), 14.4 (CH ₃ 7/CH ₃ 1), 13.9 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C ₂ 1H28BN6 [M+NH4]+ 375.2467; found 375.2479.

Compound 8

To a stirred solution of 3 (15 mg, 0.038 mmol) in anhydrous CH ₂ Cl ₂ (2 mL), phenol (5.4 mg, 0.058 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ (96 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 3 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ S04, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 75:25), to obtain 8 (11 mg, 80%) as a red-orange solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 7.09 (1H, br t, J= 7.5 Hz, H5'), 6.97 (1H, d, J= 7.5 Hz, H7') , 6.82 (1H, t, J= 7.5 Hz, H6'), 6.78 (1H, d, J=7.5 Hz, H4'), 5.20 (1H, s, OH3') , 4.46 (2H, s, H1'), 2.69 (3H, s, CH ₃ 8), 2.65 (3H, s, CH ₃ 5), 2.46 (2H, q, J= 7 ,6 Hz, CH ₃ CH76).

2.40 (3H, s, CH ₃ 7), 2.38 (3H, s, CH ₃ 1), 2.20 (2H, q, J = 7.6 Hz, CH ₃ CH72). 1.07 (3H, t, J = 7.6 Hz, CH ₃ CH76). 0.73 (3H, t, J=7 .6 Hz, CH ₃ CH ₂ 2).

13C NMR (CDCl ₃ , 100 MHz): 5 = 153.6 (C3'), 153.3 (C3 and C5), 141.3 (C8), 138.7 (C7a), 138.3 (C8a), 134, 7 (C7), 134.4 (C1), 130.7 (C6), 130.5 (C7'), 130.3 (C2), 128.5 (C5'), 127.3 (q, J _CB = 74 Hz, 2 x CN), 123.8 (C2'), 121.0 (C6'), 115.7 (C4'), 28.0 (C1'), 17.5 (CH ₃ 8), 17, 4 8 (CH ₃ CH72/CH ₃ CH76). 17.3 (CH ₃ CH76/CH ₃ CH72). 14.9 (CH ₃ I/CH ₃ 7/CH ₃ CH ₂ 6), 14.9 (CH ₃ 7/CH ₃ CH ₂ 6/CH ₃ I), 14.8 (CH ₃ CH ₂ 6/CH ₃ I/CH ₃ 7), 14.1 (CH ₃ CH ₂ 2), 13.7 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C ₂ 6H33BN5O [M+NH4]+ 442.2777; found 442.2768, Calculated for C ₂ 6H29BN4NaO [M+Na]+ 447.2331; found 447.2322.

Compound 9

To a stirred solution of 3 (15 mg, 0.038 mmol) in anhydrous CH2Cl2 (1 mL), N-benzyloxycarbonyl-L-tyrosine methyl ester (15.5 mg, 0.046 mmol) and Sc(OTf) ₃ in CH ₂ Anhydrous Cl ₂ (96 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 5 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: CH2Cl2/MeOH, 100:0 → 90:10), to obtain 9 (19 mg, 75%) as a red-orange solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = (OH3' not observed) 7.34-7.24 (5H, m, H6”, H7”, H8”, H9” and H10”, 6.82 ( 1H, br d, H5'), 6.68 (1H, br d, H4'), 6.67 (1H, br s, H7'), 5.38 (1H, d, J = 9.2 Hz, H1”), 5.05 (1H, d, J = 12.4 Hz, H4”), 4.97 (1H, d, J = 12.4 Hz, H4”), 4.53 4.46 (1H , m, H9'), 4.40 (2H, m, H1'), 3.62 (3H, s, OCH ₃ 10'), 2.95 (2H, d, J = 4.8 Hz, H8' ), 2.67 (3H, s, CH ₃ 8), 2.63 (3H, s, CH ₃ 5), 2.45 (2H, q, J = 7.5 Hz, CH ₃ CH ₂ 2/CH ₃ CH ₂ 6), 2.39 (3H, s, CH ₃ 1/CH ₃ 7), 2.35 (3H, s, CH ₃ 7/CH ₃ 1), 2.18 (2H, q, J = 7.5Hz, CH ₃ CH76/CH ₃ CH72). 1.06 (3H, t, J = 7.5 Hz, CH ₃ CH72/CH ₃ CH76). 0.74 (3H, t, J = 7.5 Hz, CH ₃ CH76/CH ₃ CH72).

13C NMR (CDCl ₃ , 100 MHz): 5 = (2 x CN not observed) 172.3 (C10'), 156.1 (C2”), 153.4 (C3/C5), 152.9 (C5/ C3), 152.8 (C3'), 141.3 (C8), 138.6 (C1/C7), 138.5 (C7/C1), 136.7 (C5”), 134.7 (C2/ C6), 134.5 (C6/C2), 131.1 (C7'), 130.7 (C7a/C8a), 130.4 (C8a/C7a), 128.8 (C5'), 128.5 ( C6'/C6”/C7”/C8”/C9”/C10”), 128.0 (C6”/C7”/C8”/C9”/C10”/C6'), 127.8 (C7”/C8 ”/C9”/C10”/C6'/C6” ), 123.9 (C2'), 115.9 (C4'), 66.7 (C4”), 55.1 (C9'), 52.3 (OCH ₃ IO'), 37.1 (C8'), 27.6 (C1'), 17.5 (CH38), 17.4 (CH ₃ CH72/CH ₃ CH76I. 17.3 (CH ₃ CH76/ CH ₃ CH72I. 14.9 (CH ₃ I/CH ₃ 7/ CH ₃ CH76/CH ₃ CH22), 14.9 (CH ₃ 7/CH ₃ CH ₂ 6/CH ₃ CH ₂ 2/CH ₃ D. 14 .8 (CH ₃ CH ₂ 6/CH ₃ CH ₂ 2/CH ₃ I/CH ₃ 7), 14.2 (CH ₃ CH26/CH ₃ CH22), 13.7 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for Css→BNsNaOs [M+Na]+ 682.3178; found 682.3175, Calculated for C38H43BN5O5 [M+H]+ 660.3350; found 660.3358.

Compound 10

To a stirred solution of 3 (15 mg, 0.038 mmol) in anhydrous MeCN (1 mL), resveratrol (8.8 mg, 0.038 mmol) and Sc(OTf) ₃ in anhydrous MeCN (96 μL of a 0. 01 M, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 2 h, more Sc(OTf) ₃ in anhydrous MeCN (96 μL of a 0.01 M solution, 0.025 equiv) was added and heated at 50 °C for 3 h. After that time, the reaction was maintained at room temperature for an additional 12 h. Finally, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: CHCl/MeOH, 100:0 → 94:6), to obtain a mixture of regioisomers (12.5 mg, 58%). The major regioisomer 10 was isolated pure by crystallization from a hexane/AcOEt mixture as an orange-red solid.

1H NMR (CD3CN/CD3COCD3 mixture, 6:1, 500 MHz): 5 = 8.04 (1H, s, OH3'), 7.76 (1H, s, OH13'), 7.67 (1H, s, OH5'), 7.20 (2H, d, J = 8.6 Hz, H11' and H15'), 6.99 (1H, d, J = 16.0 Hz, H8'), 6.76 (1H , d, J= 16.0 Hz, H9'), 6.63 (1H, d, J = 2.4 Hz, H6'), 6.61 (2H, d, J = 8.6 Hz, H12' and H14'), 6.38 (1H, d, J = 2.4 Hz, H4'), 4.57 (2H, br s, H1'), 2.67 (3H, s, CH ₃ 5), 2.62 (3H, s, CH ₃ 8), 2.54 (2H, m, CH ₃ CH ₂ 2) 2.44 (3H, s, CH ₃ 7), 2.23 (3H, s, CH ₃ 1), 1.96 (2H, m, CH ₃ CH ₂ 6)1.09 (3H, m, CH ₃ CH ₂ 6), 0.64 (3H, t, J = 7.5 Hz, CH ₃ CH?21.

13C NMR (CD3CN/CD3COCD3 mix, 6:1, 125 MHz): 5 = 158.1 (C13'), 158.0 (C5'), 157.3 (C3'), 155.2 (C3), 152 .8 (C5), 143.4 (C8), 141.4 (C1), 141.2 (C7'), 139.9 (C7), 135.5 (C2), 135.3 (C6), 131 .6 (C9'), 131.3 (C7a and C8a), 130.4 (C10'), 129.2 (C11' and C15'), 124.8 (C8'), 116.3 (C12' and C14'), 113.6 (C2'), 105.3 (C6'), 102.6 (C4'), 26.8 (C1'), 18.2 (CH ₃ 8), 17.9 (CH ₃ CH ₂ 6), 17.7 (CH ₃ CH ₂ 21. 15.3 (CH¿CH26), 15.0 (CH ₃ 7), 14.7 (CH ₃ 1), 14.3 (CH¿CH22 ), 13.9 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C34H3sBN4Na03 [M+Na]+ 581.2700; found 581.2701.

Compound 11

To a stirred solution of 3 (16 mg, 0.041 mmol) in anhydrous CH ₂ Cl ₂ (1 mL), guayazulene (8.9 mg, 0.057 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ (103) were added. μL of a 0.01 M solution, 0.025 equiv). After stirring the reaction mixture at room temperature for 1 day, more Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ (103 μL of a 0.01 M solution, 0.025 equiv) was added again at room temperature. The reaction mixture was kept at room temperature for an additional 3 days. After that time, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 70:30), to obtain 11 (16.2 mg, 75%) as a purple solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 8.03 (1H, d, J = 2.0 Hz, H5'), 7.26 (1H, dd, J = 10.7, 2.0 Hz, H7'), 7.11 (1H, s, H3'), 6.87 (1H, d, J = 10.7 Hz, H8'), 5.14 (2H, s, H1'), 3.11 (3H, s, CH ₃ 9'), 3.06-2.94 (1H, m, C2H6CH6'), 2.72 (3H, s, CH ₃ 8), 2.64 (3H, s, CH ₃ 5), 2.51 (3H, s, CH ₃ 4'), 2.46 (2H, q, J = 7.6 Hz, CH ₃ CH26), 2.41 (6H, s, CH ₃ 1 and CH ₃ 7), 2.09 (2H, q, J = 7.6 Hz, CH ₃ CH¿2), 1.34 (6H, d, J = 6.9 Hz, C?HfiCH6'1. 1.07 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 6), 0.76 (3H, t, J= 7 .6 Hz, CH¿CH22).

13C NMR (CDCl ₃ , 100 MHz): 5 = (2 x CN not observed) 155.3 (C3), 152.9 (C5), 145.2 (C9'), 141.1 (C8), 139, 2 (C3'), 139.1 (C6'), 138.8 (C1), 138.0 (C4'), 137.9 (C7), 134.9 (C7'), 134.7 (C2) , 134.2 (C6), 133.7 (C5'), 133.3 (C9'a), 130.6 (C7a/C8a), 130.4 (C8a/C7a), 126.7 (C8') , 124.0 (C4'a), 121.3 (C2'), 37.8 (C ₂ H6CH6'), 30.7 (C1'), 27.8 (CH ₃ 9'), 24.7 ( C?HfiCH6'1. 17.5 (CH ₃ 8), 17.4 (CHaCH→), 17.3 (CHaCH→), 14.9 (CH ₃ 1 and CH ₃ 7), 14.8 (CH ₃ CH ₂ 6), 14.5 (CHaCH ₂ 21. 13.7 (CH ₃ 5), 13.0 (CH ₃ 4').

HRMS (API-ES+) m/z calculated for C3sH42BN4 [M+H]+ 529.3503; found 529.3506.

Compound 12

To a stirred solution of 3 (15 mg, 0.038 mmol) in anhydrous CH ₂ Cl ₂ (2 mL), indole (6.7 mg, 0.058 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ (96 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 1.5 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 70:30), to obtain 12 (15 mg, 88%) as a red solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 8.08 (1H, s, H4'), 7.66 (1H, d, J = 7.3 Hz, H9'), 7.32 (1H, d , J = 7.3 Hz, H6'), 7.18 (1H, t, J = 7.3 Hz, H7'), ), 7.14 (1H, t, J = 7.3 Hz, H8' ), 6.97 (1H, s, H3'), 4.55 (2H, s, H1'), 2.70 (3H, s, CH ₃ 5), 2.65 (3H, s, CH ₃ 8 ), 2.47 (2H, q, J = 7.5 Hz, CH ₃ CH76I. 2.41 (3H, s, CH ₃ 7), 2.36 (3H, s, CH ₃ 1), 2.20 (2H, q, J = 7.5 Hz, CH ₃ CH ₂ 2), 1.08 (3H, t, J =7.5 Hz, CH ₃ CH ₂ 6), 0.70 (3H, t, J =7 .5 Hz, 3H, CH ₃ CH ₂ 2).

13C NMR (CDCl ₃ , 100 MHz): 5 = 154.4 (C5), 152.8 (C3), 141.2 (C8), 138.6 (C8a), 138.0 (C7a), 134.6 (C7), 134.3 (C1), 130.5 (C6), 130.2 (C2), 127.6 (q, J ^cb = 74 Hz, 2 x CN), 127.5 (C10'), 123.3 (C3'), 122.1 (C7'), 119.7 (C8'), 118.7 (C9'), 111.3 (C6'), 111.1 (C2'), 24, 1 (C1'), 17.5 (CH ₃ 5), 17.4 (CH ₃ CH ₂ 6), 17.3 (CH ₃ CH ₂ 2), 14.9 (CH3CH ₂ 6), 14.8 ( CH ₃ 7), 14.8 (CH ₃ 1), 14.3 (CH ₃ CH72I. 13.7 (CH ₃ 8).

HRMS (API-ES+) m/z calculated for C ₂ 8H34BN6 [M+NH4]+ 465.2938; found 465.2953, Calculated for C ₂ 8H3oBNsNa [M+Na]+ 470.2491; found 470.2494.

Compound 13

To a stirred solution of 3 (14.8 mg, 0.038 mmol) in anhydrous CH2Cl2 (1 mL), 2,3,5-trimethylhydroquinone (8.7 mg, 0.057 mmol) and Sc(OTf) ₃ in CH ₂ were added. Anhydrous Cl ₂ (94 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 1 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 x 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 97:3 → 70:30), to obtain 13 (17.1 mg, 93%) as an orange-red solid.

1H NMR (CD3COCD3, 400 MHz): 5 = 4.37 (2H, s, H1'), 2.80 (3H, s, CH ₃ 8), 2.67 (3H, s, CH ₃ 5), 2 .56 (2H, q, J = 7.6 Hz, CH ₃ CH76I. 2.48 (3H, s, CH ₃ 7), 2.43 (3H, s, CH ₃ 1), 2.28 (2H, q, J = 7.6 Hz, CH3CH72I. 2.04 (6H, s, CH ₃ 4' and CH ₃ 5'), 1.98 (3H, s, CH ₃ 7'), 1.10 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 6), 0.87 (3H, t, J = 7.6 Hz, CHaCH?2).

13C NMR (CD3COCD3, 100 MHz): 5 = 187.5 (C6'), 186.9 (C3'), 153.9 (C5), 151.4 (C3), 143.9 (C8), 143, 4 (C2'/C7'), 141.5 (C47C5'), 141.4 (C5'/C4'), 140.4 (C7), 140.2 (C7'/C2'), 140.1 ( C1), 135.5 (C6), 134.1 (C2), 131.5 (C7a), 131.0 (C8a), 127.4 (q, J _CB = 73.7 Hz, 2 x CN) 27 .0 (C1'), 18.1 (CH ₃ CH ₂ 2), 18.0 (CH ₃ 8), 17.6 (CH ₃ CH26), 15.0 (CH ₃ CH ₂ 6), 14.8 (CH ₃ 7), 14.6 (CH ₃ 1), 14.5 (CH¿CH22), 13.6 (CH ₃ 5), 13.1 (CH ₃ 7'), 12.6 (CH ₃ 4 '/CH ₃ 5'), 12.4 (CH ₃ 5'/CH ₃ 4').

HRMS (API-ES+) m/z calculated for C29H37BN5O2 [M+NH4]+ 498.3040; found 498.3064, Calculated for C29H33BN4Na02 [M+Na]+ 503.2594; found 503.2615.

Compound 14y15

To a stirred solution of 3 (30 mg, 0.077 mmol) in anhydrous CH _{2 Cl} 2 (2 mL) was added 2,6-dimethylhydroquinone (15.9 mg, 0.12 mmol) and Sc(OTf) ₃ in CH ₂ Anhydrous Cl ₂ (192 μL of a 0.01 M solution, 0.025 equiv) under an argon atmosphere, in the absence of light and at room temperature. After stirring the reaction mixture at room temperature for 30 min, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 65:35), to obtain 14 (29.2 mg, 81%) and 15 (4.5 mg, 13%) as red solids.

1H NMR (CD3COCD3, 400 MHz): 5 = (CH ₃ CH ₂ 2 not observed), 7.97 (1H, s, OH3'), 6.66 (1H, s, OH6'), 6.63 (1H , s, H4'), 4.34 (2H, br s, H1'), 2.79 (3H, s, CH ₃ 8), 2.71 (3H, s, CH ₃ 5), 2.57 ( 2H, q, J = 7.6 Hz, CH ₃ CH ₂ 6I. 2.48 (3H, s, CH ₃ 7), 2.38 (3H, s, CH ₃ 1), 2.20 (3H, s , CH ₃ 5'), 2.10 (3H, s, CH ₃ 7'), 1.10 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 6I. 0.62 (3H, t, J = 7.6 Hz, CH¿CH22).

13C NMR (CD3COCD3, 100 MHz): 5 = (2 x CN not observed), 154.9 (C5), 152.4 (C3), 149.4 (C3'), 147.3 (C6'), 143 .1 (C8), 141.1 (C1), 139.2 (C7), 135.1 (C2/C6), 134.9 (C6/C2), 131.0 (C7a and C8a), 126.5 (C7'), 125.1 (C5'), 120.8 (C2'), 115.2 (C4'), 28.2 (C1'), 17.9 (CH ₃ 8), 17.7 ( CH ₃ CH ₂ 2/CH ₃ CH ₂ 6), 17.3 (CH ₃ CH ₂ 6/CH ₃ CH ₂ 2), 16.9 (CH ₃ 5'), 15.1 (CH ₃ CH ₂ 6) , 14.7 (CH ₃ 7), 14.5 (CH ₃ 1/CH ₃ CH ₂ 2), 14.2 (CH ₃ CH ₂ 2/CH ₃ 1), 13.53 (CH ₃ 5 and CH ₃ 7').

HRMS (API-ES+) m/z calculated for C28H33BN4Na02 [M+Na]+ 491.2594; found 491.2620, Calculated for C28H37BNs02 [M+NH4]+ 486.3040; found 486.3056.

1H NMR (CDCl ₃ , 500 MHz): 5 = 6.63 (1H, q, J = 1.5 Hz, H4'), 4.33 (2H, s, H1'), 2.68 (3H, s , CH ₃ 5/CH ₃ 8), 2.68 (3H, s, CH ₃ 8/CH ₃ 5), 2.47 (2H, q, J = 7.6 Hz, CH ₃ CH¿6), 2 .40 (3H, s, CH ₃ 7), 2.35 (3H, s, CH ₃ 1), 2.19 (2H, q, J = 7.6 Hz, CHsCH→), 2.07 (3H, d, J = 1.5 Hz, CH ₃ 5'), 2.03 (3H, s, CH ₃ 7'),1.07 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 6), 0 .86 (3H, t, J = 7.6 Hz, CH¿CH22).

13C NMR (CDCl ₃ , 125 MHz): 5 = 187.9 (C6'), 186.3 (C3'), 154.1 (C5), 150.4 (C3), 145.8 (C5'), 143.6 (C7'), 141.5 (C8), 139.9 (C2'), 138.8 (C7), 138.4 (C1), 134.9 (C6), 133.3 (C4' ), 133.2 (C2), 131.0 (C7a), 130.2 (C8a), 127.1 (q, J _CB = 74.3 Hz, 2 x CN), 26.4 (C1'), 17.8 (CH ₃ 8), 17.7 (CH ₃ CH¿2), 17.4 (CH ₃ CH?61. 16.1 (CH ₃ 5'), 14.9 (CH ₃ CH ₂ 6/ CH ₃ 7), 14.9 (CH ₃ 7/CH ₃ CH ₂ 6), 14.7 (CH ₃ 1), 14.3 (CH ₃ CH?21. 13.8 (CH ₃ 5), 13, 3(CH ₃ 7').

HRMS (API-ES+) m/z calculated for C28H35BNs02 [M+NH4]+ 484.2883; found 484.2881, Calculated for C28H3iBN4Na02 [M+Na]+ 489.2437; found 489.2428.

Compound 16

To a stirred solution of 3 (13 mg, 0.033 mmol) in anhydrous CH2Cl2 (1 mL), 2,3-dimethoxy-5-methyl-hydroquinone (9.2 mg, 0.050 mmol) and Sc(OTf) ₃ were added. in anhydrous CH ₂ Cl ₂ (94 μL of a 0.01 M solution, 0.025 equiv) at room temperature and in the absence of light. After stirring the reaction mixture at room temperature for 1 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 95:5 → 80:20), to obtain 16 (14.2 mg, 83%) as a red-orange solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 5.43 (1H, s, OH3'), 5.37 (1H, s, OH6'), 4.51 (2H, br s, H1'), 3 .92 (3H, s, OCH ₃ 5'), 3.88 (3H, s, OCH ₃ 4'), 2.71 (3H, s, CH ₃ 5), 2.67 (3H, s, CH ₃ 8), 2.47 (2H, q, J= 7.6 Hz, CH ₃ CH76). 2.39 (3H, s, CH ₃ 7), 2.30 (3H, s, CH ₃ 1), 2.13 (3H, s, CH ₃ 7'), 1.96 (2H, br q, CH3CH2 ). 1.08 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 6), 0.59 (3H, t, J = 7.6 Hz, CH ₃ CH ₂ 2).

13C NMR (CDCl ₃ , 100 MHz): 5 = 153.6 (C3), 152.6 (C5), 141.1 (C3'), 140.8 (C8), 140.4 (C6'), 139 .2 (C1), 138.3 (C5'), 137.7 (C7), 137.0 (C4'), 134.2 (C2 and C6), 130.5 (C7a/C8a), 130.3 (C8a/C7a), 127.2 (q, J _CB = 74.6 Hz, 2 x CN), 119.5 (C7'), 117.4 (C2'), 61.1 (OCH ₃ 4'/ OCH ₃ 5'), 61.0 (OCH ₃ 5'/OCH ₃ 4'), 27.4 (C1'), 17.6 (CH ₃ 8), 17.4 (CH ₃ CH76I. 17.1 ( CH ₃ CH ₂ 2), 14.9 (CH3CH ₂ 6), 14.8 (CH ₃ 7), 14.6 (CH ₃ 1), 13.8 (CH ₃ CH ₂ 2), 13.7 (CH ₃ 5), 12.2 (CH ₃ 7').

HRMS (API-ES+) m/z calculated for C29H39BN5O ₄ [M+NH4]+ 532.3095; found 532.3078, Calculated for C29H3sBN4Na04 [M+Na]+ 537.2649; found 537.2630.

Compounds 17 and 18

To a stirred solution of 3 (50 mg, 0.128 mmol) in anhydrous CH ₂ Cl ₂ (1 mL), 2-chlorohydroquinone (24.7 mg, 0.192 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ were added. (320 μL of a 0.01 M solution, 0.025 equiv) at room temperature for 5 h. After that time, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 85:15 → 60:40), to obtain a mixture of the three possible regioisomers, from which the following can be isolated and characterized: 17 (13, 2 mg, 27%) and 18 (11.6 mg, 19%) as red-orange solids.

1H NMR (CD3COCD3, 400 MHz): 5 = 8.50 (1H, s, OH7'), 8.02 (1H, s, OH4'), 6.88 (1H, d, J = 8.7 Hz, H5'), 6.81 (1H, d, J = 8.7 Hz, H6'), 4.69 (2H, s, H1'), 2.78 (3H, s, CH ₃ 8), 2, 71 (3H, s, CH ₃ 5), 2.57 (2H, q, J = 7.6 Hz, CH ₃ CH ₂ 6I. 2.48 (3H, s, CH ₃ 7), 2.38 (3H , s, CH ₃ 1), 2.06 (2H, br q, CH ₃ CH ₂ 2), 1.10 (3H, t, J= 7 .6 Hz, CH ₃ CH ₂ 6), 0.65 ( 3H, t, J= 7.6 Hz, CH ₃ CH ₂ 2).

13C NMR (CD3COCD3, 100 MHz): 5 = 153.5 (C3), 152.8 (C5), 150.4 (C7'), 147.5 (C4'), 143.4 (C8), 140, 9 (C1), 139.5 (C7), 135.1 (C6), 134.7 (C2), 131.2 (C7a/C8a), 131.1 (C8a/C7a), 127.40 (q, J _CB = 74.0 Hz, 2 x CN), 123.4 (C2'/C3'), 123.2 (C37C2'), 116.5 (C5'), 115.1 (C6'), 29, 2 (C1'), 18.1 (CH ₃ 8), 17.8 (CH ₃ CH¿6), 17.6 (CH ₃ CH ₂ 2), 15.2 (CH ₃ CH ₂ 6), 14, 9 (CH ₃ 7), 14.6 (CH ₃ 1), 14.3 (CH¿CH22), 13.7 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C26H32BCIN5O ₂ [M+NH4]+ 492.2337; found 492.2303, Calculated for C26H ₂ sBCIN4Na02 [M+Na]+ 497.1891; found 497.1889, Calculated for C26H29BCIN4O ₂ [M+H]+ 475.2071; found 475.2070.

1H NMR (CD3COCD3, 400 MHz): 5 = 8.52 (1H, s, OH3'), 7.92 (1H, s, OH6'), 6.92 (1H, s, H7'), 6.52 (1H, s, H4'), 4.38 (2H, s, H1'), 2.83 (3H, s, CH ₃ 8), 2.65 (3H, s, CH ₃ 5), 2.56 (2H, q, J = 7.5 Hz, CH ₃ CH¿2/CH ₃ CH ₂ 6), 2.49 (3H, s, CH ₃ 1/CH ₃ 7), 2.47 (3H, s, CH ₃ 7/CH ₃ I), 2.30 (2H, q, J = 7.5 Hz, CH ₃ CH¿6/CH ₃ CH¿2), 1.10 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 2/CH ₃ CH ₂ 6), 0.82 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 6/CH ₃ CH ₂ 2).

13C NMR (CD3COCD3, 100 MHz): 5 = 153.6 (C3 and C5), 148.9 (C3'), 146.8 (C6'), 143.8 (C8), 140.2 (C1 and C7 ), 135.3 (C2/C6), 135.0 (C6/C2), 131.4 (C7a/C8a), 130.9 (C8a/C7a), 127.6 (q, J _CB = 74.6 Hz, 2 x CN), 124.8 (C2'), 119.0 (C5'), 118.7 (C4'), 116.5 (C7'), 26.4 (C1'), 17.8 (CH ₃ 8), 17.7 (CH 3CH¿2/CH 3CH¿6), 17.6 (CH ₃ CH26/CH ₃ CH ₂ 2), 15.1 (CH3CH ₂ 2/CH ₃ CH ₂ 6) , 14.8 (CH ₃ 1/CH ₃ 7), 14.8 (CH ₃ 7/CH ₃ 1), 14.4 (CH ₃ CH76/CH ₃ CH ₂ 2). 13.7 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C26H32BCIN5O ₂ [M+NH4]+ 492.2337; found 492.2349, Calculated for C26H28BCIN4Na02 [M+Na]+ 497.1891; found 497.1906.

Compound 19

To a stirred solution of 3 (10 mg, 0.026 mmol) in anhydrous CH2Cl ₂ (2 mL), pyrrole (2.6 mg, 0.038 mmol) and Sc(OTf) ₃ in anhydrous CH2Cl ₂ (96 pL of a solution 0.01 M, 0.025 equiv) at room temperature. After stirring the reaction mixture at room temperature for 2 h, water (10 mL) and CH2Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH2Cl ₂ (3 * 20 mL). The combined organic phases were washed with a saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under pressure. reduced. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 75:25), to obtain compound 19 (7 mg, 69%) as a red solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 8.78 (1H, br s, H3'), 6.69 (1H, s, 1H, H4'), 6.13 (2H, m, H5' and H6'), 4.45 (2H, s, H1'), 2.68 (3H, s, CH ₃ 5), 2.66 (3H, s, CH ₃ 8), 2.48 (2H, q, J = 7.5 Hz, CH ₃ CH76I. 2.42 (2H, q, J = 7.5 Hz, CH ₃ CH¿2), 2.41 (3H, s, CH ₃ 7), 2.36 ( 3H, s, CH ₃ 1), 1.09 (3H, t, J= 7 .5 Hz, CH ₃ CH ₂ 6), 0.73 (3H, t, J=7 .5 Hz, CH ₃ CH ₂ 2).

13C NMR (CDCl ₃ , 100 MHz): 5 = 153.3 (C5), 151.4 (C3), 141.7 (C8), 138.9 (C7a), 138.7 (C8a), 135.0 (C7 ), 134.6 (C1), 130.6 (C6), 130.0 (C2), 127.6 (q, J _CB = 73.9 Hz, 2 x CN), 125.8 (C2'), 118.2 (C4'), 108.6 (C5'), 108.3 (C6'), 26.7 (C1'), 17.5 (CH ₃ 5), 17.4 (CH ₃ CH76I. 17 .3 (CH ₃ CH ₂ 2), 14.9 (CH ₃ C7 and CH ₃ CH ₂ 6), 14.6 (CH ₃ 1), 14.1 (CH ₃ CH ₂ 2), 13.7 (CH ₃ 8 ).

HRMS (API-ES+) m/z calculated for C24H29BN5 [M+H]+ 398.2515; found 398.2526, Calculated for C24H28BNsNa [M+Na]+ 420.2334; found 420.2353.

Compound 20

To a stirred solution of 3 (13.9 mg, 0.038 mmol) in anhydrous CH ₂ Cl ₂ (2 mL), pyrrole (1.6 mg, 0.024 mmol) and Sc(OTf) ₃ (0.57 mg, 0.0012 mmol) at room temperature. After stirring the reaction mixture at room temperature for 2.5 h, water (10 mL) was added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 x 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 80:20), to obtain 20 (10 mg, 59%) together with the monosubstituted derivative 19 (1.5 mg, 15 %) and the trisubstituted 21 (4 mg, 15%), as red solids.

1H NMR (CDCl ₃ , 400 MHz): 5 = 8.76 (1H, s, NH), 5.98 (2H, d, J = 2.6 Hz, H3'), 4.36 (4H, s, H1'), 2.66 (6H, s, CH ₃ 8), 2.63 (6H, s, CH ₃ 5), 2.45 (4H, q, J = 7.6 Hz, CH ₃ CH ₂ 2 /CH ₃ CH ₂ 6), 2.38 (6H, s, CH ₃ 1/CH ₃ 7), 2.33 (6H, s, CH ₃ 7/CH ₃ 1), 2.31 (4H, q, J = 7.6 Hz, CH ₃ CH76/CH ₃ CH72I. 1.07 (6H, t, J = 7.6 Hz, CH ₃ CH72/CH ₃ CH76I. 0.76 (6H, t, J =7, 5Hz, CH ₃ CH76/CH ₃ CH72I.

13C NMR (CDCl ₃ , 100 MHz): 5 = 152.7 (C3/C5), 152.0 (C5/C3), 141.5 (C8), 139.1 (C1/C7), 138.2 ( C7/C1), 135.1 (C2/C6), 134.2 (C6/C2), 130.5 (C7a/C8a), 130.1 (C8a/C7a), 128.2 (q, J _CB = 74.0 Hz, 4 x CN), 125.4 (C2'), 108.7 (C3'), 27.0 (C1'), 17.5 (CH ₃ 8), 17.4 (CH ₃ CH ₂ 2/CH ₃ CH ₂ 6), 17.3 (CH ₃ CH76/CH ₃ CH72I. 14.9 (CH ₃ CH ₂ 2/CH ₃ CH ₂ 6), 14.8 (CH ₃ 1/CH ₃ 7 ), 14.7 (CH ₃ 7/CH ₃ 1), 14.1 (CH→CH ₂ 6/CH ₃ CH ₂ 2), 13.6 (CH ₃ 5).

HRMS (API-ES+) m/z calculated for C44H52B2N9 [M+H]+ 728.4541; found 728.4551, Calculated for C44H5iB2NgNa [M+Na]+ 750.4360; found 750.4377.

Compound21

To a stirred solution of 3 (20 mg, 0.051 mmol) in anhydrous CH2Cl ₂ (3 mL), pyrrole (1.0 mg, 0.015 mmol) and Sc(OTf) ₃ (0.57 mg, 0.0012 mmol) were added to room temperature. After stirring the reaction mixture at room temperature for 3 h, water (10 mL) and CH2Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH2Cl ₂ (3 x 20 mL). The combined organic phases were washed with a saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 30:70), to obtain compound 21 (11.5 mg, 72%), together with the disubstituted derivative 20 (1, 0 mg, 9%) and the tetrasubstituted 22 (3.5 mg, 17%) as red solids.

1H NMR (CDCl ₃ , 500 MHz): 5 = 8.46 (1H, s, NH), 5.88 (1H, s, H14), 4.41 (2H, s, H9/H9'/H9”) , 4.29 (2H, s, H9'/H9”/H9), 4.22 (2H, s, H9”/H9/H9'), 2.66 (3H, s, CH ₃ 5/CH ₃ 8 /CH ₃ 57CH ₃ 87CH ₃ 57CH ₃ 8” ), 2.66 (3H, s, CH ₃ 8/CH ₃ 5'/CH ₃ 8'/CH ₃ 5”/CH ₃ 8” / CH ₃ 5), 2.65 (3H, s, CH ₃ 5'/CH ₃ 8'/CH ₃ 5”/CH ₃ 8”/CH ₃ 5/CH ₃ 8), 2.63 (3H, s, CH ₃ 8'/ CH ₃ 5”/CH ₃ 8”/CH ₃ 5/CH ₃ 8/CH ₃ 5'), 2.62 (3H, s, CH ₃ 5”/CH _{3 8”/CH 3 5} /CH ₃ 8/ CH ₃ 5'/ CH ₃ 8'), 2.59 (3H, s, CH ₃ 8”/CH ₃ 5/CH ₃ 8/ CH ₃ 5'/CH ₃ 8'/CH ₃ 5” ), 2, 48-2.40 (8H, m, CH ₃ CH76/CH ₃ CH72/CH ₃ CH767CH ₃ CH727CH ₃ CH767CH ₃ CH72” '). 2.38 (6H, s, CH ₃ 1/CH ₃ 7/CH ₃ 17CH ₃ 77CH ₃ 17CH ₃ 7” ), 2.36 (3H, s, CH ₃ 7/CH ₃ 1'/CH ₃ 7'/ CH ₃ 1”/CH ₃ 7”/CH ₃ 1), 2.36 (3H, s, CH ₃ 17CH ₃ 77CH ₃ 17CH ₃ 77CH ₃ 1/CH ₃ 7), 2.35 (3H, s, CH ₃ 7'/CH ₃ 1”/CH ₃ 7”/CH ₃ 1/CH ₃ 7/CH ₃ 1'), 2.29 (3H, s, CH ₃ 1”/CH ₃ 7”/CH ₃ 1/CH ₃ 7/CH ₃ 1'/CH ₃ 7'), 2.26 (4H, q, J = 7.5 Hz, CH ₃ CH?2/CH ₃ CH76'/CH ₃ CH72'/CH ₃ CH76”/ CH ₃ CH72”/CH ₃ CH?61. 1.09-1.01 (9H, m, CH ₃ CH ₂ 2/CH ₃ CH ₂ 6/CH3CH72'/CH ₃ CH ₂ 6”/CH ₃ CH ₂ 2 ”/CH ₃ CH76”1. 0.93 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 6/CH ₃ CH ₂ 27CH ₃ CH ₂ 67CH ₃ CH ₂ 27CH ₃ CH ₂ 67CH ₃ CH ₂ 2 ), 0.84 (3H, t, J = 7.5 Hz, CH ₃ CH ₂ 2'/CH ₃ CH76”/CH ₃ CH72”/CH ₃ CH76”/CH ₃ CH ₂ 2/CH ₃ CH ₂ 6 ), 0.77 (3H, t, J = 7.5 Hz, CH ₃ CH767CH ₃ CH727CH ₃ CH767CH ₃ CH72/CH ₃ CH ₂ 6/CH ₃ CH ₂ 2').

13C NMR(CDCl ₃ , 125 MHz): 5= 155.2, 152.5, 151.7, 151.1, 141.8, 141.4, 141.0, 139.6, 139.3, 139, 1, 138.0, 137.9, 137.3, 135.5, 135.4, 134.0, 133.7, 130.6, 130.4, 130.2, 129.00-126.71( m, 6 x CN), 124.7, 122.4, 115.6, 110.2, 29.8, 27.11, 25.7, 25.6, 17.6, 17.5, 17.5 , 17.4, 17.2, 17.0, 15.0, 14.9, 14.8, 14.8, 14.7, 14.6, 14.5, 14.4, 13.6.

HRMS (API-ES+) m/z calculated for C64H74B3Ni3Na [M+Na]+ 1080.6389; found 1080.6427.

Compound 22

To a stirred solution of 3 (56 mg, 0.143 mmol) in anhydrous CH ₂ Cl ₂ (10 mL), pyrrole (2.0 mg, 0.030 mmol) and Sc(OTf) ₃ (0.72 mg, 0. 0015 mmol) at room temperature. After stirring the reaction mixture at room temperature for 1.5 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na3SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 30:70), to obtain compound 22 (33 mg, 80%) as a red solid.

1H NMR(CDCl ₃ , 500 MHz): 5 = 7.87 (1H, s, NH), 4.36 (d, J= 18.0 Hz, 2H), 4.19 (d, J = 17.9 Hz, 2H), 4.12 (d, J = 17.7 Hz, 2H), 4.01 (d, J = 17.3 Hz, 2H), 2.65 (12H, s), 2.58 ( 6H, s), 2.56 (6H, s), 2.53 (6H, s), 2.45-2.38 (14H, m), 2.33 (8H, s), 2.32 (6H , s), 2.29 (6H, s), 1.06-1.00 (18H, m), 0.95 (6H, t, J=7 .5 Hz).

13C NMR(CDCl ₃ , 100 MHz): 5= 153.3, 151.9, 151.1, 151.1, 142.2, 141.5, 140.7, 140.3, 137.7, 137, 4, 136.0, 135.8, 133.8, 133.5, 131.0, 130.7, 130.4, 130.4, 128.7-126.1 (m, 8 x CN), 122 .7, 115.1, 25.8, 25.7, 17.6, 17.5, 17.4, 17.3, 17.2, 15.0, 15.0, 14.9, 14.9 , 14.8, 14.7, 14.7, 14.5, 13.5.

HRMS (API-ES+) m/z calculated for C84H101B4N18 [M+NH4]+ 1405.8865; found 1405.8914.

Compound 23 and 24

To a solution of F-BODIPY 25 (Nepomnyashchii, AB; Broering, M.; Ahrens, J.; Bard, AJ J. Am. Chem. Soc. 2011, 133, 8633-8645) (204 mg, 0.563 mmol) in CH ₂ Cl ₂ (2 mL), TMSCN (479 μL, 3.76 mmol) and SnCL (32 μL, 0.268 mmol) were added at room temperature. After stirring the reaction mixture at room temperature for 30 min, water (30 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 50 mL). The combined organic phases were washed with an aqueous NaHCO3 solution (0.1 m) and a saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO4, filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 80:20), to obtain 26 (199 mg, 96%) as a red-orange solid.

1H NMR (CDCl ₃ , 400 MHz): 5 = 6.97 (2H, s, H3' and H5'), 6.14 (2H, s, H ₂ and H6), 2.73 (6H, s, CH ₃ 3 and CH ₃ 5), 2.34 (3H, s, CH ₃ 4'), 2.07 (6H, s, CH ₃ 2' and CH ₃ 6'), 1.42 (6H, s, CH ₃ 1 and CH ₃ 7).

13C NMR (CDCl ₃ , 100 MHz): 5 = 155.8 (C3 and C5), 143.8 (C7/C1/C7a/C8a), 143.0 8 (C8), 139.4 (C4'), 134.8 (C2' and C6'), 130.3 (C1'), 129.4 (C3' and C5'), 129.03 (C7a/C8a/C7/C1), 126.4 (q, J _CB = 74 Hz, 2 x CN), 122.4 (C2 and C6), 21.3 (CH ₃ 4'), 19.6 (CH ₃ 2' and CH ₃ 6'), 15.6 (CH ₃ 3 and CH ₃ 5), 13.8 (CH ₃ 1 and CH ₃ 7).

HRMS (API-ES+) m/z calculated for C24H26BN4 [M+H]+ 381.2249; found 381.2250, Calculated for C24H ₂ sBN4Na [M+Na]+ 403.2069; found 403.2060.

To a stirred solution of 3 (11 mg, 0.028 mmol) in anhydrous CH ₂ Cl ₂ (1 mL), 26 (10.7 mg, 0.028 mmol) and Sc(OTf) ₃ in anhydrous CH ₂ Cl ₂ were added (64 μL of a 0.01 M solution, 0.025 equiv) at room temperature. After stirring the mixture reaction at room temperature for 5.5 h, water (10 mL) and CH ₂ Cl ₂ (20 mL) were added, the organic phase was separated, and the aqueous phase was extracted with CH ₂ Cl ₂ (3 * 20 mL). The combined organic phases were washed with saturated NaCl solution (20 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The resulting crude oil was purified by flash column chromatography (linear gradient: hexane/AcOEt, 100:0 → 70:30), to obtain 23 (9.3 mg, 47%) and 24 (4.5 mg, 15%) as red-orange solids. Compound 22 (4.0 mg, 37%) was also recovered.

1H NMR (CDCl ₃ , 500 MHz): 5 = 6.97 (2H, s, H3” and H5” ), 6.14 (1H, s, H6'), 4.23 (br d, H9), 2 .72 (3H, s, CH ₃ 5'), 2.68 (3H, s, CH ₃ 5/CH ₃ 8), 2.68 (3H, s, CH ₃ 8/CH ₃ 5), 2.66 (3H, s, CH ₃ 3'), 2.47 (2H, q, J = 7.6 Hz, CH ₃ CH761. 2.41 (3H, s, CH ₃ 7), 2.33 (3H, s , CH ₃ 4” ), 2.32 (3H, s, CH ₃ 1), 2.06 (8H, m, CH ₃ CH72. CH ₃ 2” and CH ₃ 6” ), 1.42 (6H, s , CH ₃ 1' and CH ₃ 7'), 1.08 (3H, t, J= 7 .6 Hz, CH ₃ CH ₂ 6), 0.69 (3H, t, J=7 .6 Hz, CH ₃ CH ₂ 6).

13C NMR (CDCl ₃ , 125 MHz): 5 = 156.0 (C5'), 155.1 (C3'), 154.5 (C5), 150.0 (C3), 144.0 (C7'/C7a '), 142.9 (C8'), 141.6 (C8), 141.0 (C1'/C8a'), 139.4 (C4”), 139.1 (C7), 138.9 (C1) , 135.0 (C6), 134.4 (C2” and C6” ), 133.6 (C2), 131.0 (C7a), 130.4 (C1” ), 130.0 (C8a), 129.6 ( C3” and C5”), 129.3 (C7a'/C7'), 128.4 (C8a'/C1'), 127.5 (C2'), 126.9 (q, J _CB = 74.0 Hz , 4 x CN), 122.5 (C6'), 24.7 (C9), 21.3 (CH ₃ 8” ), 19.7 (CH ₃ 2”/CH ₃ 6” ), 19.5 ( CH ₃ 6”/CH ₃ 2” ), 17.7 (CH ₃ 8), 17.4 (CH ₃ CH72/CH ₃ CH761. 17.4 (CH ₃ CH76/CH ₃ CH721.

15.6 (CH ₃ 5'), 15.0 (CH ₃ 7), 14.8 (CH ₃ CH76). 14.6 (CH ₃ 1), 14.4 (CH ₃ 3'), 14.2 (CH ₃ CH72).

13.9 (CH ₃ 7'), 13.8 (CH ₃ 5), 11.8 (CH ₃ 1').

HRMS (API-ES+) m/z calculated for C44H52B2N9 [M+NH4]+ 728.4541; found 728.4512, Calculated for C44H48B2N3Na [M+Na]+ 733.4095; found 733.4052.

1H NMR (CDCl ₃ , 500 MHz): 5 = 6.97 (2H, s, H3” and H5”), 4.23 (4H, br d, H9 and H9'” ), 2.68 (9H, s ), 2.68 (3H, s), 2.66 (6H, s), 2.47 (4H, q, J = 7.6 Hz), 2.41 (6H, s), 2.32 (9H , s, CH ₃ 2” , CH ₃ 6” ), 2.15-2.01 (10H, m), 1.41 (6H, s, CH ₃ 1' and CH ₃ 7'), 1.08 ( 6H, t, J = 7.6 Hz), 0.69 (6H, t, J= 7.5 Hz).

13C NMR(CDCl ₃ , 125 MHz): 5= 155.4, 154.4, 150.0, 142.8, 141.6, 141.1, 139.5, 139.1, 135.1, 133, 7, 131.1, 130.6, 130.0, 129.6, 128.7, 127.7, 29.9, 24.7, 21.4, 19.5, 17.7, 17.4, 14.9, 14.8, 14.6, 14.5, 14.2, 13.8, 11.9.

HRMS (API-ES+) m/z calculated for C64H75B3N13 [M+NH4]+ 1058.6569; found 1058.6567, Calculated for C64H7iB3Ni2Na [M+Na]+ 1063.6123; found 1063.6106.

Example 3. Photophysical study of representative compounds

With a view to their possible applications in biology, a preliminary study of the photophysical properties of some of the most representative compounds prepared in this chapter was carried out. The new BODIPYs were studied in three different solvents to determine the influence of the polarity of the medium: AcOEt, as a polar aprotic organic solvent with a lipophilic character; MeOH, as a protic polar organic solvent; and phosphate buffer saline (PBS), as an aqueous medium that will also be used in the fluorescence microscopy experiments described below.

All measurements were carried out under the same conditions, using an excitation wavelength of 490 nm and the commercial BODIPY 1 in methanol (0 = 0.91) as a reference for the estimation of quantum yields.

The photophysical properties of C-nucleophile derivatives are highly dependent of the nature of the introduced carbon system, as expected (Table 1). Compounds with acyclic alkyl systems (4-7) have properties very similar to those of the unsubstituted compound 2 , precursor of the entire series. However, in the case of derivatives with cyclic unsaturated systems, two general trends are observed. While the derivatives with a phenol ring (8, 9) are very similar to the previous ones, showing as the most significant difference only a small bathochromic shift of the absorption band, the guayazulene derivatives (11) (Fig. 1), indole (12) and pyrrole (19) have low or no fluorescence in the three solvents studied. This may be due to a photoinduced electron transfer (PET) process between the newly introduced, electron-rich ring and BODIPY, which quenches fluorescence. The contribution of this PET process has been theoretically modeled using DFT calculations in the case of the derivative with a guayazulene ring (11), which acts as a donor (reductive PET) and is located at a distance from the chromophore that allows interaction between both units. . Thus, the calculations carried out show that the lowest energy electronic transition occurs from its HOMO-1 to its LUMO, both located mainly in the core of BODIPY (Fig. 2). The calculations also indicate that the participation of an electron transfer (reductive PET) from the HOMO (located mainly in the guaiazulene system) to the HOMO-1, semi-vacant after excitation, is thermodynamically feasible. This process prevents the excited electron from returning to its own ground state, thus causing a non-radiative decay that is responsible for the dramatic deactivation of fluorescence observed for these compounds in all the solvents studied (Table 1). A similar effect on fluorescence has been described in other BODIPY derivatives with azulene or indole substituents.

The following table 1 shows the photophysical properties of the BODIPYs derived from C-nucleophiles in different solvents (Ab is the wavelength of the absorption maximum; £ is the molar absorption coefficient; A is the wavelength of the emission maximum fluorescence; 0 is the fluorescence quantum yield; ^t is the lifetime of the excited state).

[a] PBS is not soluble, [b] It has a shoulder at 557.0 nm,

Example 5. Microscopy studies

A preliminary screening of most of the new BODIPYs synthesized in this chapter has been carried out as fluorescent probes in live cell fluorescence microscopy, using two different cancer cell lines: HeLa and SCC38 (derived from human squamous cell carcinoma larynx). The Compounds have been incubated with live cells at three different concentrations (50 nM, 100 nM and 500 nM) for 30 minutes, before washing with phosphate buffered saline (PBS) to remove excess dye. The most suitable concentrations for visualization were 50 nM and 100 nM. In general terms, this screening has shown that most of the compounds can cross the cell membrane and present specific subcellular staining and no cytotoxicity.

It was observed that compound 4 (prepared from diethylzinc) selectively stains spherical vesicles without showing diffuse staining in the cell interior (Fig. 3).

Claims (15)

1. Compound of general formula (I)

where: Z is selected from a nitrogen atom (N) or a C(R?) group; R6 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, halogen, -OR ', -NR'R”, -CN, -COR', -COOR', -CONR'R”, -SiR'R”R'” , -S¡R'R”(OR” '), -Si( OR') ₃ , -PR'R”, -P(=O)R'R”, -P(=O)(OR')(OR”), -P(=O)(OR')(NR” R” '), -P(=O)(NR'R”)(NR” 'R” ” ), -SR', -SOR', -S02R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O)R', -TeR', -Te(=OR'); R2 to R5 are each independently selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl , halogen, -OR', -NR'R”, -N3, -NR'(C=O)R”, -NR'C(=O)OR”, -NR'C(=O)NR”R” ', -NR'(C=S)NR”R'” , -NR'S 02R”, -COR', -COOR', -CONR'R”, -S¡R'R”R'” , -SiR'R ”(OR'”), -Si(OR') ₃ , -PR'R”, -P(=O)R'R”, -P(=O)(OR')(OR”), -P( =O)(OR')(NR” R'” ), -P(=O)(NR'R”)(NR” 'R” ”), -SR', -SOR', -SO ₂ R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O)R', -TeR', -Te(=OR');R',R",R'" and R"" are each independently selected from hydrogen, Ci-C1s alkyl, C2-C18 alkenyl, C2-C18 alkynyl, aryl or heteroaryl; R ₇ is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, CH ₂ OR', -CH ₂₀ (C=O)R' , -CN, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and Ri is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, -CN, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, heteroaryl substituted or unsubstituted, or a compound of formula (la):

where R2, R3, R4 and R6 are defined above and R5 is selected from hydrogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl substituted, substituted or unsubstituted heteroaryl, halogen, -OR', -NR'R”, -N3, -NR'(C=O)R”, -NR'C(=O)OR”, -NR'C( =O)NR”R” ', -NR'(C=S)NR”R” ', -NR'S 02R”, -CN, -COR', -COOR', -CONR'R”, -SiR'R” R'” , -SiR'R”(OR'” ), -Si(OR') ₃ , -PR'R”, -P(=O)R'R”, -P(=O)(OR') (OR”), -P(=O)(OR')(NR” R” '), -P(=O)(NR'R”)(NR” 'R” ”), -SR', -SOR ', -SO ₂ R', -SO ₂ OR', -S02NR'R”, -S(=NR')R”, -SeR', -Se(=O)R', -TeR', -Te( =O)R' or a formula group (la); or any of its salts or isomers. 2. Compound according to claim 1, where Z is a C(R7) group. 3. Compound according to claim 2, wherein R ₇ is a C1-C6 alkyl group or a phenyl group substituted by at least one C1-C6 alkyl group, preferably the alkyl is a methyl. 4. Compound according to any of claims 1 to 3, where R6 is a C1-C6 alkyl group, preferably it is a methyl. 5. Compound according to any of claims 1 to 4, wherein R2 to R5 are each independently selected from a C1-C6 alkyl group. 6. Compound according to any of claims 1 to 5, where R2 and Rs are an ethyl group and/or R3 and R4 are a methyl group. 7. Compound according to any of claims 1 to 6, where the compound has general formula (II):

where R1 is described in claim 1. 8. Compound according to any of claims 1 to 7, where R1 is selected from a group -CH ₂ -CH ₃ , -CH ₂ -CH=CH ₂ , -CH(CO-CH ₃ ) ₂ , -CN, a phenyl group substituted by at least one -OH group or a cyclohexadiene group substituted by at least group, azulyl substituted by at least one (C1-C4) alkyl group, pyrrole, an indole, and a group of formula (la) . 9. Compound according to claim 8, where R1 is selected from the groups -CH ₂ -

10. Compound according to claim 8, where R1 is a pyrrole group substituted by at least one group of formula (la):

where Z, R2 to R4, King R ₇ are described in any of claims 1 to 7 and R5 is described in any of claims 1 to 7 or is a group of formula (la). 11. Compounds according to any of claims 1 to 10, where the compounds are:

12. Use of the compound of general formula (I) according to any of claims 1 to 11 as a marker or fluorescent probe. 13. Use according to claim 12, as a biological marker for the selective staining of living cells and microorganisms. 14. Use of the compound of general formula (I) according to any of claims 1 to 11 as fluorescent probes in biosensors in bioimaging of living organisms. 15. Use of the compound of general formula (I) according to any of claims 1 and 11, as energy collectors in solar cells, as emitters in dye lasers or as emitters in OLED systems.